1
EXHIBIT 10.15
AGREEMENT
Between
NEW YORK UNIVERSITY
and
CALYPTE BIOMEDICAL CORPORATION
License Agreement
2
080693 3573m
NYU/CALYPTE
LICENSE AGREEMENT
I N D E X
Section 1. Definitions page 2
Section 2. Effective Date page 4
Section 3. Title page 4
Section 4. Patents and Patent Applications page 5
Section 5. Grant of License page 7
Section 6. Payments for License page 8
Section 7. Method of Payment page 13
Section B. Development and Commercialization page 13
Section 9. NYU's "March-in" Rights and Obligations page 14
Section 10. Defense of NYU Patent page 18
Section 11. Infringement of NYU Patent page 20
Section 12. Liability and Indemnification page 25
Section 13. Security for Indemnification page 26
Section 14. Expiry and Termination page 28
Section 15. NYU's and CORPORATION's Agreements
with respect to HORL page 29
Section 16. Representations and Warranties by CORPORATION page 34
Section 17. Representations and Warranties by NYU page 35
Section 18. No Assignment page 36
Section 19. Use of Name page 36
Section 20. Confidentiality page 37
Section 21. Miscellaneous page 38
Appendix I - NYU Patents and Patent Applications
Appendix II - Correspondence and Documents Concerning Abbott
Appendix III - NYU-HORL Agreement and First Amendment
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080693
NYU/CALYPTE
LICENSE AGREEMENT
This Agreement, effective as of Aug 12th, (the "Effective Date"),
is by and between:
NEW YORK UNIVERSITY (hereinafter "NYU), a corporation organized and
existing under the laws of the State of New York and having a place of business
at 70 Washington Square South, New York, New York 10012
AND
CALYPTE BIOMEDICAL CORPORATION (hereinafter "CORPORATION"), a
corporation organized and existing under the laws of the State of California
having its principal office at 1440 Fourth Street, Berkeley, California 94710.
RECITALS
WHEREAS, NYU is the owner of certain inventions relating to the
detection of antibodies to human immunodeficiency virus (HIV) in urine, all as
more particularly described in the NYU Patents (as hereinafter defined)-.
WHEREAS, CORPORATION is engaged in the research, development,
manufacture, sale, use and distribution of products for the detection of
antibodies to HIV in urine; and
WHEREAS, subject to the terms and conditions hereinafter set forth,
NYU is willing to grant to CORPORATION and CORPORATION is willing to accept
from NYU the License (as hereinafter defined).
NOW, THEREFORE. in consideration of the mutual promises and agreements
contained herein, the parties hereto hereby agree as follows:
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1. Definitions.
Whenever used in this Agreement, the following terms shall have the
following meanings:
a. "Calendar Year" shall mean any consecutive period of twelve
months commencing on the first day of January of any year.
b. "Combination Product" shall mean a product containing Licensed
Product(s) combined or bundled with other non-Licensed
Product(s) in a single package.
c. "Corporation Entity" shall mean any company or other legal
entity which controls, or is controlled by, or is under common
control with, CORPORATION; control means the holding of fifty
and one tenth percent (50.1%) or more of (i) the capital stock
and/or (ii) the voting rights and/or OM the right to elect or
appoint directors.
d. "HORL" shall mean Home Office Reference Laboratory, Inc., a
corporation organized and existing under the laws of the State
of Delaware, having its principal offices at 10310 West 84th
Terrace, Lenexa, Kansas 66214, and shall include HORL
Corporation Entities (as such term is defined in the NYU-HORL
Agreement). The "NYU-HORL Agreement" shall mean the agreement
between NYU and HORL dated November 15, 1989 and amended in
April 1992 with respect to the practice of NYU Patents for use
in testing for insurance purposes, a copy of which agreement
and amendment, with financial terms redacted, is annexed
hereto as Appendix III.
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e. "License" shall mean the exclusive worldwide license under the
NYU Patents (as hereinafter defined) to make, have made, use,
sell or otherwise distribute the Licensed Products (as
hereinafter defined), during the term of this Agreement.
f. "Licensed Product(s)" shall mean any product for the analysis
of a human urine sample of an individual to assay HIV
antibodies in urine covered by one or more Valid Claims (as
hereinafter defined) of the NYU Patents.
g. "Net Sales" shall mean the total amount received in connection
with sales of the Licensed Products and/or performance of
Tests and/or Confirmatory Tests (as hereinafter defined),
after deduction of all the following to the extent applicable:
i) all trade, case and quantity credits, discounts,
refunds or rebates;
ii) allowances or credits for returns;
iii) sales commissions; and
iv) sales taxes (including value-added tax).
h. "NYU Patents" shall mean U.S. patents and patent applications
(including U.S. Patent No. 4,865,966, issued on September 12,
1989, and U.S. Patent No. 5,122,446, issued on June 16, 1992),
and foreign counterpart patent applications and patents
thereto; owned, assigned or assignable to NYU, and any
reissues, renewals, divisions, continuations,
continuationsin-part, substitutes, divisions-or extensions
thereof, and
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pending applications therefor, including those identified in
annexed Appendix I and forming an integral part hereof, which
are related to the detection of antibodies to HIV in urine.
i. "NYU Scientist" shall mean Dr. Alvin Friedman-Kien of NYU.
j. "Sublicensee shall mean any third party to whom a sublicense
is granted by CORPORATION as described in Section 5.c. below..
k. "Test" shall mean a test of a human urine sample to assay HIV
antibodies and which is covered by one or more Valid Claims
(as hereineafter defined) of the NYU Patents. "Confirmatory
Test" shall mean a Test (as defined in this subsection) used
to verify a result obtained by using a Licensed Product.
l. "Valid Claim" shall mean a claim of an issued NYU Patent which
has not expired, lapsed, become abandoned or dedicated to the
public or been declared or rendered invalid or unenforceable
by reason of reissue, reexamination, disclaimer or final
judgment by a court of competent jurisdiction or
administrative agency from which no appeal can be or is taken.
2. Effective Date.
This Agreement shall be effective as of the Effective Date and shall
remain in full force and effect until it expires or is terminated in
accordance with its provisions.
3. Title.
Subject to the terms and conditions of this Agreement, it is hereby
agreed that all right, title and interest, in and to the NYU Patents,
vests solely in NYU.
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4. Patents and Patent Applications.
a. CORPORATION shall, simultaneously with the signing of this
Agreement pay NYU the sum of
, being the amount of all costs and fees
incurred by NYU up to the Effective Date in connection with the
NYU Patents.
b. ALL applications and proceedings with respect to the NYU
Patents after the Effective Date shall be prosecuted and
maintained by NYU at the expense of CORPORATION. Against the
submission of detailed quarterly invoices, CORPORATION shall
reimburse NYU for all reasonable out of pocket costs and fees
incurred by NYU during the term of this Agreement, in connection
with the drafting, filing, maintenance, prosecution, and
issuance of the NYU Patents. Such reimbursable costs and fees
shall not include NYU overhead charges and/or internal costs.
NYU shall not be entitled to reimbursement for patent filing,
prosecution and maintenance expenses in excess of ten thousand
dollars (U.S. $10,000) per year with respect to the NYU Patents
unless NYU has obtained the prior written consent of CORPORATION
to- such expenses. In the event CORPORATION refuses to consent
to such expenditures on a particular patent or patent
application, NYU shall be free to pursue patent protection with
respect to that particular patent or application at NYU's
expense and all rights to that patent or application shall
revert to NYU and CORPORATION shall have no right to make, use,
sell, manufacture or have manufactured products which are
covered by a Valid Claim
Confidential portion has been omitted
and filed separately with the Commission
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of such patent or application. However, CORPORATION's
License under this Agreement shall continue with respect to
all other NYU Patents.
c. CORPORATION shall have the right to approve and comment on the
strategy and prosecution of the NYU Patents. NYU shall not
abandon any NYU Patents without first consulting with
CORPORATION and obtaining CORPORATION's consent for such
abandonment unless abandonment is in favor of a subsequent
patent application claiming the subject matter of the proposed
abandoned application and the patentability of the subject
matter is not negatively affected. Both parties agree to
cooperate with each other regarding the NYU Patents, and NYU
agrees to use its best efforts to obtain and maintain the NYU
Patents.
d. Nothing herein contained shall be deemed to be a warranty by
NYU that
i) NYU can or will be able to obtain any patent or
patents on any patent application or applications in
the NYU Patents or any portion thereof, or that any
of the NYU Patents will afford adequate or
commercially worthwhile protection, or
ii) that the manufacture, use, or sale of any Licensed
Product will not infringe any patent(s) of a third
party.
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5. Grant of License.
a. Subject to the terms and conditions hereinafter set forth, NYU
hereby grants to CORPORATION and CORPORATION hereby accepts
from NYU the License as defined in Section l.e.
b. The License granted to CORPORATION in Section 5.a. hereto
shall remain in force, if not previously terminated under the
terms of this Agreement, until the expiration date of the last
to expire of the NYU Patents.
c. CORPORATION shall be entitled to grant sublicenses under the
License to a Corporation Entity or to other third parties.
All sublicenses shall only be granted by CORPORATION under a
written agreement, copies of which shall be provided by
CORPORATION to NYU and HORL as soon as practicable after the
signing thereof. Each sublicense granted by CORPORATION.
hereunder shall be subject and subordinate to the terms and
conditions of this Agreement and shall contain (inter-alia)
the following provisions:
(1) the sublicense shall expire automatically on the
termination of the License;
(2) the sublicense shall not be assignable, in whole or
in part;
(3) the Sublicensee shall not grant further sublicenses;
(4) the sublicense agreement shall include i) an
obligation by the Sublicensee to obtain and maintain
insurance and to provide evidence thereof to NYU and
to indemnify NYU as described in Sections 12 and 13
of this Agreement and the
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sublicense agreement shall state that NYU is an
intended third party beneficiary of such sublicense
agreement for the purposes of enforcing such
indemnification and insurance provisions, and ii) the
text of Section 15.b. and 15.d. of this Agreement,
applicable to Sublicensee, and shall state that HORL
is an intended third party beneficiary of such
sublicense agreement for the purpose of enforcing
such provisions for so long as Section 15 is
applicable to this Agreement.
(5) the breach by any Sublicensee of its obligations to
CORPORATION shall not be deemed a breach by
CORPORATION and such breach shall not, in any way,
affect CORPORATION's rights and obligations under
this Agreement. However, in the event of a material
breach (including without limitation, a failure to
pay royalties due) by a Sublicensee, CORPORATION
shall promptly notify NYU of the breach and either
promptly terminate the sublicense agreement or shall
continue to be obligated for payment to NYU of all
royalties due from the Sublicensee.
(6) the right of NYU to audit Sublicensee's records at
its own expense.
6. Payments for License.
a. In consideration for the grant and during the term of the
License, CORPORATION shall pay to NYU:
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(1) on the Effective Date, a non-refundable,
non-creditable (except as provided in Section ll.j.)
license issue fee of
; and
(2) a royalty of percent of the Net Sales of
Licensed. Product(s) by CORPORATION, Corporation
Entity and Sublicensees in any country in which the
Licensed Product is covered by a Valid Claim(s); and
(3) a royalty of percent of the Net Sales for
each Test and Confirmatory Test by CORPORATION,
Corporation Entity, and Sublicensees in any country
in which the Test or Confirmatory Test is covered by
a Valid Claim; and
(4) a royalty of percent of Net
Sales of Licensed Products by CORPORATION,
Corporation Entity or Sublicensees in any country in
which an NYU Patent has not issued if such Licensed
Product was manufactured in a country in which the
Licensed Product is covered by a Valid Claim(s).
(5) If more than one of the royalty rates should be
applicable to any transaction or to any Licensed
Product, only a single royalty shall be due and that
royalty shall be computed at the highest applicable
rate. No royalties shall be due upon sales of
Licensed Products to and between Corporation Entities
or Sublicensee(s) for further sale; provided,
however, that royalties with respect to such sales
shall be payable upon a sale of such Licensed
Products to any person or entity that is not either a
'Corporation Entity or a Sublicensee.
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(6) If Licensed Product is sold as a Combination Product,
then the Net Sales attributable to such Licensed
Product shall be based upon the ratio of the list
price for the Licensed Product to the combined list
prices of the Licensed Product and the other
non-Licensed Product(s); provided that where there is
no list price for a component of the Combination
Product. the parties agree to negotiate the
appropriate ratio in good faith. If CORPORATION
desires to sell a Combination Product for which there
is no list price for components, CORPORATION shall
notify NYU and commence such .negotiations in good
faith. In such case, CORPORATION shall have no right
to sell such Combination Product-until and unless NYU
and CORPORATION shall have concluded a written
agreement with respect to the ratio of royalties to
be paid by CORPORATION with respect to such
Combination Product(s).
b. For the purpose of computing the royalties due to NYU
hereunder, the year shall be divided into four quarters ending
on March 31, June 30, September 30 and December 31'. Not
later than sixty (60) days after the end of each quarter in
each Calendar Year during the term of the License, CORPORATION
shall submit to NYU a full and detailed report of payments due
NYU under the terms of this Agreement for the preceding
quarter year (hereinafter "the Quarterly Report"), setting
forth the Net Sales and all royalties or other consideration
upon which such payments are computed and including at least
i) total sales of Licensed Products;
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ii) the deductions permitted under subsection 1.g. to
arrive at Net Sales of Licensed Products;
iii) total amount received with respect to Tests and
Confirmatory Tests;
iv) the deductions permitted under subsection 1.g. to
arrive at Net Sales of Tests and Confirmatory Tests;
and
v) the royalty computations on Licensed Products, Tests
and Confirmatory Tests.
The Quarterly Report shall separately state the total Net
Sales, Tests and Confirmatory Tests of CORPORATION.
Corporation Entity and Sublicensees with respect to Net Sales,
Tests and Confirmatory Tests to persons or entities engaged in
testing for insurance purposes.
If no royalties or other payments are due, a statement shall
be sent to NYU stating such fact. Payment of the full amount
of any royalties or other payments due to NYU for the
preceding quarter year shall accompany each Quarterly Report.
CORPORATION shall keep for a period of at least three (3)
years after the date of entry, full, accurate and complete
books and records consistent with sound business and
accounting practices and in such form and in such detail as to
enable the determination of the amounts due to NYU from
CORPORATION pursuant to the terms of this Agreement.
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As part of CORPORATION's normal annual audit or a special
audit if sooner, the payments and Quarterly Reports will be
verified for accuracy. In the event a correction needs to be
made regarding the payment and/or Report, CORPORATION will
make the ap propriate payment and send NYU a new Report within
sixty (60) days.
c. On reasonable notice and during regular business hours, NYU or
the authorized representative of NYU shall each have the right
to inspect the books of accounts, records and other relevant
documentation of CORPORATION or of Corporation Entity and of
Sublicensees insofar as they relate to the production,
marketing and sale of the Licensed Products or Tests, in order
to ascertain or verify the amount of royalties and other
payments due to NYU hereunder, and the accuracy of the
information provided to NYU in the aforementioned reports.
This inspection shall be at NYU's expense, provided, however,
that all information received as a result of the inspection
shall be maintained in confidence by NYU and its
representatives; provided, however, that NYU shall have the
right to use such information to enforce the terms of this
Agreement. NYU's right to inspect must be exercised within
three (3) years of NYU's receipt of the Report which NYU
desires to verify. In the event an audit conducted by NYU
demonstrates amounts due to NYU in excess of ten percent (10%)
of the total amount paid to NYU with respect to any Calendar
Year, CORPORATION shall reimburse NYU for the expenses of
NYU's audit.
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d. Beginning on January 1. 1994 and continuing thereafter until
this Agreement shall terminate or expire, CORPORATION agrees
that if the total amounts paid to NYU under subsection 6.a.
hereof do not amount to
in the 1994 Calendar Year,
in the 1995 Calendar Year,
in the 1996
Calendar Year and
in the 1997 Calendar Year and each Calendar Year
thereafter, CORPORATION will pay to NYU within ninety (90)
days after the end of each such Calendar Year, as additional
royalty, the difference between the amount of the total
royalties paid to NYU by CORPORATION in such Calendar Year and
the amount stated herein with respect to such Calendar Year
(hereinafter Minimum Annual Royalty), failing which NYU shall
have the right, upon written notice to CORPORATION, to convert
the License to a non-exclusive license, having the same
royalty rates as in Section 6.a. In the event the License has
been converted to a non-exclusive license, CORPORATION shall
no longer be obligated to pay the Minimum Annual Royalty of
this subsection.
7. Method of Payment.
Royalties and any other payments due to NYU hereunder shall be paid to
NYU in United States dollars. Any such royalties on or other payments
relating to transactions in a foreign currency shall be
Confidential portion has been omitted
and filed separately with the Commission
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converted into United States dollars based on the conversion rate for
the particular currency as listed in the Wall Street Journal on the
last business day of the quarter for which such royalty or other
payment is due. If restrictions on the transfer of currency exist in
any country such as to prevent CORPORATION from making payments in the
United States or in U.S. dollars, CORPORATION shall make payments due
in such country in local currency and/or deposit such payments in a
local bank designated by NYU.
8. Development and Commercialization.
a. CORPORATION agrees that it is developing at least one Licensed
Product(s), and will pursue reasonable activities necessary in
order to attempt to obtain the approval of the Food and Drug
Administration (FDA) or other appropriate authority, for the
production, use and sale of the Licensed Product(s) by
CORPORATION or its Corporate Entity.
b. CORPORATION undertakes to begin the regular commercial
production, use and sale of the Licensed Products in good
faith and as soon as practicable, subject to FDA or other
governmental agency license or approval.
9. NYU's "March-in" Rights and Obligations.
a. During the period commencing upon the Effective Date and
continuing for forty (40) months thereafter. CORPORATION
shall
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have no obligation to grant sublicenses under the License.
Following such initial 40-month period, CORPORATION undertakes
to negotiate and grant a sublicense under the License to any
interested party and in the event CORPORATION has not granted
a sublicense to said interested party after a six-month period
of, good faith negotiations with such party, NYU shall have
"march-in" rights to grant a non-exclusive license in and to
the NYU Patents directly to such third party. In the event
NYU grants a non-exclusive license with the interested party
which contains terms more favorable than those under Section
6.a.(2) (4) of this Agreement, NYU agrees that this Agreement
shall be deemed appropriately amended to provide such terms to
CORPORATION and its Sublicensees, effective immediately upon
execution of the non-exclusive license. Therefore,
CORPORATION and its Sublicensees shall have most favored
licensee status.
b. In the event NYU grants a non-exclusive license pursuant to
this Section 9, NYU shall provide CORPORATION with a copy of
the license agreement and pay to CORPORATION
of any monetary consideration
(including royalties on Net Sales) received by NYU under the
terms of, or as a consideration for the grant of, a
non-exclusive license of any rights in and to the NYU Patents.
Such non-exclusive license shall be in writing, shall not be
assignable in whole or in part and shall not include the right
to grant sublicenses under the non-exclusive license. Such
non-exclusive license shall contain terms substantially
identical to Section 15 of this Agreement, for so long as
Confidential portion has been omitted
and filed separately with the Commission
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Section 15 is applicable to this Agreement. A breach by the
non-exclusive licensee of its obligations shall not be deemed
a breach by NYU and such breach shall not, in any way, affect
NYU's rights and obligations under this Agreement. However,
in the event the non-exclusive licensee fails to pay royalties
due to NYU, and in which CORPORATION shares pursuant to this
Section, NYU shall notify CORPORATION and if requested by
CORPORATION, NYU shall promptly terminate the non-exclusive
license agreement. In the event the non-exclusive licensee
grants sublicenses in breach of the non-exclusive license with
NYU and CORPORATION demonstrates conclusively to NYU that such
sublicensing has occurred, NYU shall promptly terminate the
nonexclusive license agreement.
c. For the purpose of computing the payments due to CORPORATION
under this Section 9, the year shall be divided into four
quarters ending on March 31, June 30, September 30 and
December 31. Not later than sixty (60) days after the end of
each quarter in each Calendar Year during the term of the
non-exclusive license, NYU shall submit to CORPORATION a full
and detailed report of payments due CORPORATION under the
terms of the non-exclusive license for the preceding quarter
year, setting forth the payments due to CORPORATION, setting
forth the net sales and/or lump sum payments and all other
royalties or consideration upon which such payments are
computed and including at least the total sales of product,
the deductions permitted to arrive at the net sales and the
royalty computations.
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If no royalties or other payments are due, a statement shall
be sent to CORPORATION stating such fact. Payment of the full
amount due to CORPORATION for the preceding quarter year shall
accompany each report. NYU shall keep for a period of at
least three (3) years after the date of entry, full, accurate
and complete books and records consistent with sound business
and accounting practices and in such form and in such detail
as to enable the determination of the amounts due to
CORPORATION from NYU pursuant to the terms of this Agreement.
As part of NYU's normal annual audit, the payments and reports
will be verified for accuracy. In the event a correction
needs to be made regarding the payment and/or report, NYU will
make the appropriate payment and send CORPORATION a new report
within sixty (60) days.
d. On reasonable notice and during regular business hours,
CORPORATION or the authorized representative of CORPORATION
shall each have the right to inspect the books of accounts,
records and other relevant documentation of NYU insofar as
they relate to revenues from the non-exclusive license, in
order to ascertain or verify the amount of royalties and other
payments due to CORPORATION hereunder, and the accuracy-of the
information provided to CORPORATION in the aforementioned
reports. This inspection shall be at CORPORATION's expense,
provided, however, that all information received as a result
of the inspection shall be maintained in confidence by
CORPORATION and its representatives; provided, however that
CORPORATION
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shall have the right to use such information to enforce the
terms of this Agreement. CORPORATION's right to inspect must
be exercised within three (3) years of CORPORATION's receipt
of the report which CORPORATION desires to verify. In the
event an audit conducted by CORPORATION demonstrates amounts
due to CORPORATION in excess of five percent (5%) of the total
amount paid to CORPORATION with respect to any Calendar Year,
NYU shall reimburse CORPORATION for the expenses of
CORPORATION'S audit.
e. Payments due to CORPORATION shall be paid to CORPORATION in
United States dollars. Any payments relating to transactions
in a foreign currency shall be converted into United States
dollars based on the conversion rate for the particular
currency as listed in the Wall Street Journal on the last
business day of the quarter for which payment is due. If
restrictions on the transfer of currency exist in any country
such as to prevent NYU from making payments in the United
States or in U.S. dollars, NYU shall make payments due in such
country in local currency and/or deposit such payments in a
local bank designated by CORPORATION.
10. Defense of NYU Patent.
a. NYU has disclosed to CORPORATION that Abbott Laboratories,
Inc. (hereinafter "Abbott") has asserted a claim of ownership
rights with respect to the NYU Patents as contained in
correspondence and documents in Appendix II annexed hereto and
made an integral part of this Agreement. Throughout the term
of this Agreement,
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NYU shall notify CORPORATION in writing within ten (10)
business days every time NYU and/or the NYU Scientist is
contacted by or contacts (orally or in writing) Abbott
regarding the claim of ownership rights with respect to the
NYU Patent(s). NYU also shall provide copies to CORPORATION
of any correspondence it or the NYU Scientist receives or has
received from Abbott, or sends or has sent to Abbott in this
regard within ten (10) business days of receiving or sending.
b. Upon the Effective Date, CORPORATION shall pay to NYU the sum
of
to be held by NYU in a special interest-bearing
account and expended only for out-of-pocket legal defense fees
and costs in the event that a third party asserts a claim with
respect to the ownership and/or validity of the NYU Patents
("the Legal Defense Fund"). NYU shall not expend the Legal
Defense Fund (including the accumulated interest) for any
purpose except for reasonable out-of-pocket legal defense fees
and costs for six (6) years after the first sale of Licensed
Product or after the initiation of a lawsuit by the third
party, provided such lawsuit is initiated during such six-year
period, whichever is later. In the event that the Legal
Defense Fund is not expended during such period, the Legal
Defense Fund shall be the property of NYU without restriction;
however, any accumulated interest will be the property of
CORPORATION without restriction. In the event the Legal
Defense Fund (including the accumulated interest) is
exhausted, NYU shall have no further obligation to CORPORATION
Confidential portion has been omitted
and filed separately with the Commission
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with respect to defense against such ownership and/or validity
of lawsuit except as provided under Section 11. NYU shall not
settle such ownership and/or validity lawsuit without
providing CORPORATION with written notice at least thirty (30)
days in advance-of the settlement. In the event NYU decides
to settle such lawsuit, it shall not assign its entire
ownership rights of NYU Patents to a third party as part of
the settlement without CORPORATION's prior written approval.
c. Any expenses incurred by CORPORATION or NYU in conjunction
with the prosecution of any suit or the settlement thereof
relating to a third party assertion of a claim with respect to
the ownership and/or validity of the NYU Patents shall be
first paid for from the Legal Defense Fund (including the
accumulated interest), provided such lawsuit is initiated
during the six (6) year period described in Section 10.b.
11. Infringement of NYU Patent.
a. In the event a party to this Agreement acquires information
that a third party is infringing one or more of the NYU
Patents, the party acquiring such information shall promptly
notify the other party to the Agreement in writing of such
infringement.
b. In the event of an infringement of an NYU Patent, CORPORATION
shall have an exclusive right (but not the obligation) to
bring suit against the infringer for a period of six (6)
months after acquiring information that a third party is
infringing; provided,
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however, that CORPORATION shall not have the right to bring
suit against HORL or a HORL Corporation Entity or any third
party which has been granted immunity from claims of
infringement of the NYU Patents for the sole purpose of
providing Test kits exclusively for HORL or for HORL
Corporation Entities during the term of the NYU-HORL
Agreement. (This protection against an infringement action
shall not apply if the third party, which has been granted
immunity, itself sells Test kits or provides Test kits to any
entity other than to HORL and HORL Corporation Entities.)
Should CORPORATION elect to bring suit against an infringer
and NYU is joined as a party plaintiff in any such suit, NYU
shall have the right to approve the counsel selected by
CORPORATION to represent CORPORATION and NYU, which approval
shall not be unreasonably withheld. The expenses of such suit
or suits that CORPORATION elects to bring, including any
reasonable out-of-pocket expenses of NYU incurred in
conjunction with the prosecution of such suit or the
settlement thereof, shall be paid for entirely by CORPORATION
and CORPORATION shall hold NYU free, clear r and harmless from
and against any and all costs of such litigation, including
attorneys' fees.
c. In the event CORPORATION exercises the right to sue herein
conferred, it shall have the right to first reimburse itself
out of any sums recovered in such suit or in settlement
thereof for all costs and expenses of every kind and
character, including attorneys' fees, necessarily involved in
the prosecution of any
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such suit, and if after such reimbursement, any funds shall
remain from said recovery, CORPORATION shall promptly pay to
NYU an amount equal to five percent (5%) of such remainder and
CORPORATION shall be entitled to receive and retain the
balance of the remainder of such recovery.
d. In the event CORPORATION does not bring suit by the end of the
six (6) month period described in b. above, NYU shall have the
right (but not the obligation) to bring suit, after written
notice to CORPORATION of its intention. If CORPORATION is
joined as a party plaintiff in any such suit, CORPORATION
shall have the right to approve the counsel selected by NYU to
represent NYU and CORPORATION, which approval shall not be
unreasonably withheld. The expenses of such suit or suits
that NYU elects to bring, including any reasonable
out-of-pocket expenses of CORPORATION incurred in conjunction
with the prosecution of such suit or the settlement thereof,
shall be paid for entirely by NYU and NYU shall hold
CORPORATION free, clear and harmless from and against any and
all costs of such litigation, including attorneys' fees.
e. In the event NYU exercises-the right to sue herein conferred,
it shall have the right to first reimburse itself out of any
sums recovered in such suit or in settlement thereof for all
costs and expenses of every kind and character, including
attorneys' fees, necessarily involved in the prosecution of
any such suit, and if after such reimbursement, any funds
shall remain from
- 22 -
25
said recovery, NYU shall pay CORPORATION an amount equal to
five percent (5%) of such remainder and NYU shall be entitled
to receive and retain the balance of the remainder of such
recovery.
f. CORPORATION shall not have the right to grant cross-license(s)
to a third party in settlement of any infringement action,
except with the consent of NYU, and provided that the
following conditions are satisfied:
1) NYU is consulted beforehand and is reasonably
satisfied that the third party has a legal position
or right which does, or could, limit CORPORATION's
ability to market or to make, have made, sell or
distribute Licensed Products;
2) The rights received by CORPORATION under such
agreement cover only Licensed Products and are not
directed to other products;
3) NYU incurs no financial or legal liabilities under
such agreement, and the terms of such agreement would
not cause NYU to be subject to a claim of breach
under the NYU-HORL Agreement; and
4) NYU shall receive royalties in accordance with the
provisions of Section 6.a. with respect to Licensed
Products, Tests and Confirmatory Tests by such
crosslicensee.
g. In any suit with respect to the NYU Patents, the parties shall
cooperate fully, and upon the request and at the expense of
the party bringing suit, the other party shall make available
to the
- 23 -
26
party bringing suit all records, papers, information, samples,
specimens, individuals and the like which may be relevant.
h. Each party shall always have the right to be represented by
counsel of its own selection and at its own expense in any
suit for infringement of the NYU Patents instituted by the
other party to this Agreement under the terms hereof.
i. In the event a court of competent jurisdiction determines in a
final judgment from which no further appeal can or has been
taken that a claim of one or more NYU Patents is invalid or
unenforceable, no further payment with respect to Licensed
Products covered by said NYU Patent(s) shall be due or owing
hereunder and CORPORATION and HORL shall have a paid-up
license as to the affected NYU Patent(s).
j. In the event a court of competent jurisdiction determines in a
final judgment from which no further appeal can or has been
taken that NYU co-owns one or more NYU Patents with a third
party, or if there is a settlement approved by NYU to such
effect, this Agreement and the License granted hereunder shall
remain in effect; however, CORPORATION shall be credited in
the amount of with
respect to future royalties as of the date of such final court
determination or settlement and shall be immediately relieved
from CORPORATION's obligation to pay NYU Minimum Annual
Royalties.
Confidential portion has been omitted
and filed separately with the Commission
- 24 -
27
k. In the event a court of competent jurisdiction determines in a
final judgement that NYU does not own one or more NYU Patents,
or if there is a settlement approved by NYU to such effect, no
further payment with respect to Licensed Products covered by
said patent(s) shall be due or owing hereunder.
12. Liability and Indemnification.
a. CORPORATION shall indemnify, defend and hold harmless NYU and
its trustees, officers, medical and professional staff,
employees, students and agents and their respective
successors, heirs and assigns (the "Indemnitees"), against any
liability, damage, loss or expense (including reasonable
attorneys' fees and expenses of litigation) incurred by or
imposed upon the Indemnitees or any one of them in connection
with any claims, suits, actions, demands or judgments arising
out of the design, production, manufacture, sale, use in
commerce or in human clinical trials, lease, or promotion by
CORPORATION or by a Sublicensee, Corporation Entity or agent
of CORPORATION of any Licensed Product, process or service
relating to, or developed pursuant to, this Agreement.
b. CORPORATION's indemnification obligation under this Section 12
shall not apply to any liability, damage, loss or expense to
the extent that it is attributable to the negligent activities
or willful misconduct of any such Indemnitee.
c. CORPORATION agrees, at its own expense, to provide attorneys
reasonably acceptable to NYU to defend against any actions
brought
- 25 -
28
or filed against any Indemnitee with respect to the subject of
indemnity to which such Indemnitee is entitled hereunder,
whether or not such actions are rightfully brought.
13. Security for Indemnification.
a. At such time as any Licensed Product, process or service
relating to, or developed pursuant to, this Agreement is being
commercially distributed or sold (other than for the purpose
of obtaining regulatory approvals) by CORPORATION or by a
Corporation Entity, Sublicensee or agent of CORPORATION,
CORPORATION shall at its sole cost and expense, procure and
maintain policies of comprehensive general liability insurance
in amounts not less than five million dollars (U.S.
$5,000,000.00) per incident and five million dollars (U.S.
$5,000,000.00) annual aggregate and naming the Indemnitees as
additional insureds. Such comprehensive general liability
insurance shall provide (i) product liability coverage and
(ii) broad form contractual liability coverage for
CORPORATION's indemnification under Section 12 of this
Agreement. If CORPORATION elects to self-insure all or part
of the limits described above (including deductibles or
retentions which are in excess of two hundred fifty thousand
dollars (U.S. $250,000) annual aggregate) such self-insurance
program must be approved by NYU, which approval shall not be
unreasonably withheld. The minimum amounts of insurance
coverage required under this Section 13 shall not be
- 26 -
29
construed to create a limit of CORPORATION's liability with
respect to its indemnification under Section 12 of this
Agreement.
b. CORPORATION shall provide NYU with written evidence of such
insurance upon request of NYU. CORPORATION shall provide NYU
with written notice at least ninety (90) days prior to the
cancellation, non-renewal or material change in such
insurance, where possible, or ten (10) business days after
CORPORATION receives notice of such from the insurance
company; if CORPORATION does not obtain replacement insurance
providing comparable coverage within ninety (90) days of
notification, NYU shall have the right to terminate this
Agreement according to the provisions of this Agreement.
c. CORPORATION shall maintain such comprehensive general
liability insurance beyond the expiration or termination of
this Agreement during (i) the period that any product, process
or service, relating to, or developed pursuant to, this
Agreement is being commercially distributed or sold (other
than for the purpose of obtaining regulatory approvals) by
CORPORATION, Corporation Entity, a Sublicensee or agent of
CORPORATION and (ii) a reasonable period after the period
referred to in c(i) above which in no event shall be less than
seven (7) years after the term of this Agreement.
- 27 -
30
14. Expiry and Termination.
a. Unless earlier terminated pursuant to this Section 14 hereof,
this Agreement shall expire on the expiration of the period of
the License as set forth in Section 5.b. above.
b. At any time prior to expiration of this Agreement, either
party may terminate this Agreement forthwith for cause, as
"cause" is described below, by giving written notice to the
other party. Cause for termination by one party of this
Agreement shall be deemed to exist if the other party
materially breaches or defaults in the performance or
observance of any of the provisions of this Agreement and such
breach or default is not cured within ninety (90) days or, in
the case of failure to pay any amounts due hereunder, thirty
(30) days (unless otherwise specified herein) after the giving
of notice by the other party specifying such breach or
default, or if either NYU or CORPORATION or Corporation Entity
discontinues its business.
c. Any amount payable hereunder by one of the parties to the
other, which has not been paid by the date on which such
payment is due, shall bear interest from such date until the
date on which such payment is made, at the rate of one percent
(1%) per annum in excess of the prime rate prevailing at the
Citibank, N.A., in New York, during the period of arrears and
such amount and the interest thereon may be set off against
any amount due, whether in terms of this Agreement or
otherwise, to the party in default by any non-defaulting
party.
- 28 -
31
d. CORPORATION may terminate this Agreement without cause upon
thirty (30) days' written notice to NYU. CORPORATION
may, after the effective date of such termination, sell some
or all Licensed Products CORPORATION has in inventory at the
date of termination, provided it pays royalty thereon under
Section 6.a. of this Agreement.
e. Upon termination of this Agreement for any reason and prior to
expiration as set forth in Section 14.a. hereof, all rights in
and to the NYU Patents shall revert to NYU, and CORPORATION
shall not be entitled to make any further use whatsoever of
the NYU Patents.
f. Termination of this Agreement shall not relieve either party
of any obligation to the other party incurred prior to such
termination. In the event of termination during a Calendar
Year for which Minimum Annual Royalties would be due to NYU
pursuant to Section 6.d., CORPORATION shall pay such Minimum
Annual Royalties on a pro rata basis for such year.
g. Sections 3, 10, 12, 13, 14, 19 and 20 hereof shall survive and
remain in full force and effect after any termination,
cancellation or expiration of this Agreement.
15. NYU's and CORPORATION's Agreements with respect to HORL.
a. The provisions of this Agreement are subject to the provisions
of the NYU-HORL Agreement, as defined in Section l.d. hereof.
The provisions of this Section 15 shall be in effect until
such time as the NYU-HORL Agreement expires or is terminated.
During the
- 29 -
32
time the NYU-HORL Agreement is in effect, NYU agrees to
provide to CORPORATION with any further revisions or
amendments (with financial terms redacted) to the NYU-HORL
Agreement within ten (10) business days of their execution and
to provide to CORPORATION any information regarding the
NYU-HORL Agreement or relationship which alters or affects
CORPORATION's or NYU's obligations under the present
NYU-CORPORATION Agreement also within ten (10) business days
of NYU's knowledge of the information. If a revision or
amendment to the NYU-HORL Agreement would alter or affect
CORPORATION's or NYU's obligation under this Agreement, then
CORPORATION must provide its prior written consent to such
revision or amendment which consent shall not be unreasonably
withheld.
b. CORPORATION, Corporation Entity and Sublicensees shall offer
to HORL Licensed Products to perform Tests on terms no less
favorable than said Licensed Products are supplied to any
other person or entity performing testing for insurance
purposes; provided, however, HORL shall not be obligated to
purchase said Licensed Products from CORPORATION, Corporation
Entity or Sublicensee but shall be free to purchase said
Licensed Products from a third party.
c. NYU shall have the right to provide to HORL copies of
Quarterly and Annual Reports and other information which may
be obtained by NYU pursuant to Section 6.b-d., provided that
HORL has agreed in writing to maintain such Reports and
information in confidence and
- 30 -
33
not to disclose them to any third party except for the purposes
of enforcing HORL's rights pursuant to the NYU-HORL Agreement,
this Agreement, or both.
d. So long as the NYU-HORL Agreement is in effect, CORPORATION,
Corporation Entity and Sublicensees shall not bring any suit for
infringement of the NYU Patents against HORL or against a third
party which has been granted immunity from claims of
infringement of the NYU Patents for the sole purpose of
providing Test kits exclusively for HORL and HORL Corporation
Entities pursuant to Section 5.c. of the NYU-HORL Agreement.
CORPORATION, Corporation Entity and Sublicensees shall be
permitted to bring suit for infringement of the NYU Patents
against a third party which has been granted immunity if the
third party sells Test kits or provides Test kits to any entity
other than to HORL or HORL Corporation Entities.
e. In the event HORL purchases Test kits from a third party to
which immunity from claims of infringement of the NYU Patents is
granted pursuant to Section 5.c. of the NYU-HORL Agreement, NYU
shall pay CORPORATION
of any royalties and payments received by NYU pursuant
to Section 6.a. (2) of the NYU-HORL Agreement with respect to
Tests performed using such Test kits. NYU shall provide
CORPORATION with the HORL Agreement payment schedule. The
immunity contract to the third party shall not grant further
immunity to other parties. A breach by the third party of its
obligations shall not be deemed a breach by NYU of this
Agreement and such breach shall not, in any way, affect NYU's
rights and obligations under this Agreement. However,
Confidential portion has been omitted and filed separately with the Commission
- 31 -
34
in the event that such third party sells Test kits to any party
except HORL and/or HORL Corporation Entities in commercial
quantities and CORPORATION demonstrates conclusively to NYU
that such sales have occurred, NYU shall notify such third
party that it is in breach of the Immunity and shall demand
cure, failing which, NYU shall promptly terminate the immunity
contract.
f. For the purpose of computing the payments due to CORPORATION
under this Section 15, the year shall be divided into four
quarters ending on March 31, June 30, September 30 and December
31. Not later than sixty (60) days after the end of each
quarter in each Calendar Year during the term of the third
party immunity contract, NYU shall submit to CORPORATION a full
and detailed report of payments due CORPORATION under the terms
of the contract for the preceding quarter year, setting forth
the payments due to CORPORATION, setting forth the net sales
and/or lump sum payments and all other royalties or
consideration upon which such payments are computed and
including at least the total sales of product, the deductions
permitted to arrive at the net sales and the royalty
computations.
g. If no royalty or other payments are due, a statement shall be
sent to CORPORATION stating such fact. Payment of the full
amount due to CORPORATION for the preceding quarter year shall
accompany each report. NYU shall keep for a period of at least
three (3) years after the date of entry, full, accurate and
complete books and records consistent with sound business and
accounting practices and in such form and in such detail as to
enable the determination
- 32 -
35
of the amount due to CORPORATION from NYU pursuant to the terms
of this Agreement. As part of NYU's normal annual audit, the
payments and reports will be verified for accuracy. In the
event a correction needs to be made regarding the payment
and/or report, NYU will make the appropriate payment and send
CORPORATION a new Report within sixty (60) days.
h. On reasonable notice an during regular business hours,
CORPORATION or the authorized representative of CORPORATION
shall each have the right to inspect the books of accounts,
records and other relevant documentation of NYU insofar as they
relate to revenues from the third party immunity contract, in
order to ascertain or verify the amount of royalties and other
payments due to CORPORATION hereunder, and the accuracy of the
information provided to CORPORATION in the aforementioned
reports. This inspection shall be at CORPORATION's expense,
provided, however, that all information received as a result of
the inspection shall be maintained in confidence by CORPORATION
and its representatives; provided, however that CORPORATION
shall have the right to use such information to enforce the
terms of this Agreement. CORPORATION's right to inspect must be
exercised within three (3) years of CORPORATION's receipt of
the report which CORPORATION desires to verify. In the event
an audit conducted by CORPORATION demonstrates amounts due to
CORPORATION in excess of ten percent (10%) of the total amount
paid to CORPORATION with respect to any Calendar Year, NYU
shall reimburse CORPORATION for the expenses of CORPORATION's
audit.
- 33 -
36
Payments due to CORPORATION shall be paid to CORPORATION in
United States dollars. Any payments relating to transactions
in a foreign currency shall be converted into United States
dollars based on the conversion rate for the particular
currency as listed in the Wall Street Journal on the last
business day of the quarter for which payment is due. If
restrictions on the- transfer of currency exist in any country
such as to prevent NYU from making payments in the United
States or in U.S. dollars, NYU shall make payments due in such
country in local currency and/or deposit such payments in a
local bank designated by CORPORATION.
i. CORPORATION shall not perform Tests for insurance screening
purposes except that CORPORATION and/or Corporation Entity may
perform Confirmatory Tests.
j. HORL is an intended third party beneficiary of this Agreement
for the purpose of enforcing this Section 15.
16. Representations and Warranties by CORPORATION.
CORPORATION hereby represents and warrants to NYU as follows:
(1) CORPORATION is a corporation duly organized, validly-existing
and in good standing under the laws of the State of California.
CORPORATION has been granted all requisite power and authority
to carry on its business and to own and operate its properties
and assets. The execution, delivery and performance of this
Agreement have been duly authorized by the Board of Directors
of CORPORATION.
- 34 -
37
(2) There is no pending or, to CORPORATION's knowledge, threatened
litigation involving CORPORATION which would have any effect on
this Agreement or on CORPORATION's ability to perform its
obligations hereunder; and
(3) There is no indenture, contract, or agreement to which
CORPORATION is a party or by which CORPORATION is bound which
prohibits or would prohibit the execution and delivery by
CORPORATION of this Agreement or the performance or observance
by CORPORATION of any term or condition of this Agreement.
17. Representations and Warranties by HYU.
NYU hereby represents and warrants to CORPORATION as follows:
(1) NYU is a corporation duly organized,, validly existing and in
good standing under the laws of the State of New York. NYU has
been granted all requisite power and authority to carry on its
business and to own and operate its properties and assets. The
execution, delivery and performance of this Agreement have been
duly authorized by the Board of Trustees of NYU;
(2) There is no pending or, to NYU's knowledge, threatened
litigation involving NYU which would have any effect on this
Agreement or on NYU's ability to perform its obligations
hereunder;
(3) There is no indenture, contract, or agreement to which NYU
and/or the NYU Scientist is a party or by which NYU and the NYU
Scientist is bound which prohibits or would prohibit the
execution and delivery by NYU of this Agreement or the
- 35 -
38
performance or observance by NYU of any term or condition of
this Agreement; and
(4) Subject to the claim of ownership rights by Abbott as set forth
in Section 10.a. hereof and subject to the rights of HORL as
set forth in Section 15. hereof, NYU is the owner of the entire
right, title, and interest in and to NYU Patents and has the
sole right to grant licenses under such NYU Patents; and NYU
has not granted licenses thereunder to any other person or
entity except as set forth in Section 15 of this Agreement.
18. No Assignment.
Neither CORPORATION nor NYU shall have the right to assign, delegate or
transfer at any time to any party, in whole or in part, any or all of
the rights, duties and interest herein granted without first obtaining
the written consent of the other to such assignment, which consent
shall not be unreasonably withheld. However, CORPORATION shall have
the right to assign, delegate or transfer at any time, in whole or in
part, any or all of the rights, duties and interest herein granted to a
Corporation Entity provided written notice is given promptly to NYU and
Corporation Entity undertakes in writing to perform all the obligations
of CORPORATION pursuant to this Agreement.
19. Use of Name.
Without the prior written consent of the other party which consent
shall not be unreasonably withheld in accordance with the business
- 36 -
39
practices and policies of the party whose consent is sought, neither
CORPORATION nor NYU shall use the name of the other party or any
adaptation thereof or of any staff member, employee or student of the
other party, including without limitation, in any product labeling,
advertising or sales literature. However, in the event that disclosure
is in connection with any public or private offering, is in connection
with a lawsuit settlement involving NYU and/or Joint Inventions, is in
conjunction with any application for regulatory approval, or is
required by law, either party can make factual statements concerning
this Agreement or file copies of this Agreement so long as the other
party has an opportunity to review and comment on the statements. The
comment period shall be ten (10) working days from the receipt of the
statements unless the parties agree to an extension. Except as
provided herein, neither NYU nor CORPORATION will issue public
announcements about this Agreement.
20. Confidentiality.
Except to the extent expressly authorized in this Agreement, the
parties agree that, for the term of this Agreement plus six (6) years
thereafter, the receiving party of written information marked
confidential by the providing party, shall keep it confidential and
shall not publish, use, or otherwise disclose it except to the extent
the receiving party can establish that such information:
1. was already known to the receiving party, other than under an
obligation of confidentiality, at the time of disclosure by the
providing party;
- 37 -
40
2. was generally available to the public or otherwise part of the
public domain at the time of its disclosure to the receiving
party;
3. became generally available to the public or otherwise part of
the public domain after its disclosure and other than through
any act or omission of the receiving party in breach of this
Agreement; or
4. was subsequently lawfully disclosed to the receiving party by a
third party.
Each party may disclose the other's information to the extent such
disclosure is reasonably necessary in prosecuting or defending patents
and litigation, complying with applicable governmental regulations and
laws, conducting clinical trials, or negotiating with potential
sublicensees.
21. Miscellaneous.
a. In carrying out this Agreement the parties shall comply with
all applicable local, state and federal laws and regulations.
b. If any provision of this Agreement is determined to be invalid
or void, the remaining provisions shall remain in effect.
c. This Agreement shall be deemed to have been made in the State
of New York and shall be governed and interpreted in all
respects under the laws of the State of New York.
d. Any dispute arising under this Agreement shall be resolved in
an action in the courts of New York State or the federal courts
located in New York State, and the parties hereby consent to
personal jurisdiction of such courts in any such action.
- 38 -
41
e. All payments or notices required or permitted to be given under
this Agreement shall be given in writing and shall be effective
when either personally delivered or deposited, postage prepaid,
in the United States registered or certified mail, addressed as
follows:
To NYU: New York University Medical Center
550 First Avenue
New York, NY 10016
Attention: Isaac T. Kohlberg Vice President for
Industrial Liaison
and
Office of Legal Counsel
New York University
Bobst Library
70 Washington Square South
New York, NY 10012
Attention: Annette B. Johnson, Esq.
Associate General Counsel
To CORPORATION: Calypte Biomedical Corporation
1440 Fourth Street
Berkeley, California 94710
Attention: David J. Robison, Ph.D. President and
Chief Executive Officer
or such other address or addresses as either party may
hereafter specify by written notice to the other. Such notices
and communications shall be deemed effective on the date of
delivery or fourteen (14) days after having been sent by
registered or certified mail, whichever is earlier.
- 39 -
42
f. This Agreement (and the annexed Appendices) constitute the
entire Agreement between the parties with respect to the
subject matter contained herein and no variation, modification
or waiver of any of the terms or conditions hereof shall be
deemed valid unless made in writing and signed by both parties
hereto. This Agreement supersedes any and all prior
agreements or understandings with respect to the subject
matter contained herein, whether oral or written, between
CORPORATION and NYU.
g. No waiver by either party of any non-performance or violation
by the other party of any of the covenants, obligations or
agreements of such other party hereunder shall be deemed to be
a waiver of any subsequent violation or non-performance of the
same or any other covenant, agreement or obligation, nor shall
forbearance by ,any party be deemed to be a waiver by such
party of its rights or remedies with respect to such violation
or non-performance.
h. The descriptive headings contained in this Agreement are
included-for convenience and reference only and shall not be
held to expand, modify or aid in the interpretation,
construction or meaning of this Agreement.
i. It is not the intent of the parties to create a partnership or
joint venture or to assume partnership responsibility or
liability. The obligations of the parties shall be limited to
those set out herein and such obligations shall be several and
not joint.
- 40 -
43
j. In the event of a delay caused by inclement weather, fire,
flood, strike, or other labor dispute, act of God, act of
governmental officials or agencies, or any other cause beyond
the control of either party, such party shall be excused from
performance hereunder for the period of time attributable to
such delay.
IN WITNESS WHEREOF, the parties hereto have executed this Agreement
effective as of the date and year first above written.
NEW YORK UNIVERSITY
By:
--------------------------------
Isaac T. Kohlberg
Vice President for
Title: Industrial Liaison
-----------------------------
Date:
-------------------------------
CALYPTE BIOMEDICAL CORPORATION
By:
-------------------------------
Title:
-----------------------------
Date:
-----------------------------
3573/169m - 41 -
44
APPENDIX I
U.S. PATENTS
Method for Detecting Antibodies to Human Immunodeficiency Virus Patent No: 4,865,966
Granted: 9/12/89
Method for Detecting Antibodies to Human Immunodeficiency Virus Patent No: 5,122,446
Granted: 6/16/92
FOREIGN PATENTS AND APPLICATIONS
COUNTRY SERIAL NO, STATUS PATENT NO,
------- --------- ------ ---------
Australia 17182/88 Issued 617671
Canada 564,232 Pending
Japan 504037/88 Pending
Nigeria 59/88 Issued RP10207
South Korea 7016/88 Pending
Sri Lanka 9968 Issued 9968
45
United States Patent [19] [11] Patent Number: 4,865,966
Friedman-Kien et al. [45] Date of Patent: Sep. 12, 1989
------------------------------------------------------------------------------
[54] METHOD FOR DETECTING ANTIBODIES
TO HUMAN IMMUNODEFICIENCY VIRUS
[75] Inventors: Alvin E. Friedman-Kien; Yunzhen
Cao; William Borkowsky, all of New York, N.Y.
[73] Assignee: New York University, New York, N.Y.
[21] Appl. No.: 40,013
[22] Filed: Apr. 17, 1987
[51] Int. CL4........................ C12Q 1/68; GOIN 33/53
[52] U.S. CL................................... 435/5; 435/7;
435/810; 436/501; 436/513; 436/808; 436/811
[58] Field of Search 435/5, 7, 810; 436/501,
436/513, 808, 811
(56] References Cited
U.S. PATENT DOCUMENTS
4,464,474 8/1984 Coursaget et al. 436/513
4,725,669 2/1988 Essex et al. 530/395
OTHER PUBLICATIONS
Meryhew, N. L. et al., J. Rheumat., 10: 913-919, 1983.
Wrier, E. et al., J. Clin. Invest., 42:1340-1352, 1962.
Hanson, L. A. et al., J. Clin. Invest., 44: 703-715, 1963.
Rao, T. K. S. et al., New England J. Med. 310: 669-673, 1994.
Trinick. T. R. et al., Clinica Chimica Acta, 139:113-117, 1984,
Franklin, E. C., J Clin. Invest. 38: 2159-2167, 1959.
Lerner, A. M. et al., J Clin. Invest., 41: 805-815, 1962.
Remington, J. S. et al., Nature. 194: 408-409, 1962.
Primary Examiner - Christine M. Nucker
Attorney, Agent, or Firm - Darby & Darby
[57] ABSTRACT
Disclosed herein is a method of screening mammals for antibodies to viral
agents by collecting a urine sample from a mammal to be tested and assaying the
sample for antibodies directed against the specific viral agent.
20 Claims, 2 Drawing Sheets
46
U.S. PATENT Sep. 12, 1989 Sheet 1 of 2 4,865,966
FIG. 1
47
U.S. PATENT Sep. 12.1989 Sheet 2 of 2 4,865,966
FIG. 2
48
4,865,966
1
METHOD FOR DETECTING ANTIBODIES TO HUMAN IMMUNODEFICIENCY VIRUS
BACKGROUND OF THE INVENTION
The present invention relates to a method for detecting antibodies
directed against Human Immunodeficiency Virus which can be used for diagnosing
AIDS and related diseases, and identifying latent, asymptomatic carriers such
infections.
Acquired Immune Deficiency Syndrome (AIDS) was initially recognized and
reported in 1981. Since that time, clinical and epidemiological data have
revealed that the incidence of AIDS has reached epidemic levels
throughout the world. The causative agent of AIDS has been identified as an
RNA retrovirus, the Human T-Cell Leukemia Virus Type III (HTLV-III), also known
a Lymphadenopathy Associated Virus (LAV) and AIDS-associated retrovirus (ARV)
and recently renamed Human Immunodeficiency Virus (HIV). AIDS patients may
suffer from a broad spectrum of opportunistic infections such as Pneumocystis
carinii, Candida albicans, herpes simplex virus and cytomegalovirus, and are
also frequently afflicted with certain tumors, especially Kaposi's Sarcoma. It
has been estimated that the number of patients with AIDS in the United States
continues to double approximately every twelve months.
The putative AIDS virus, HIV, has been isolated from peripheral blood
mononuclear cells, cerebrospinal fluid, semen, neural tissue, saliva, tears and
rarely, urine. In order to determine the prevalence of HIV in the general
population, it has been suggested that mass screenings of the population for
the presence of antibodies directed against the AIDS virus be undertaken.
However, since antibody substances are generally found only in human blood and
serum, the proposed screening techniques involve obtaining a blood of serum
sample from the patient who is to be screened.
A variety of serological tests have been developed to detect the
presence of antibodies to HIV (indicative of exposure to HIV) in the blood of
patients with AIDS, AIDS-Related Complex (ARC) and healthy (i.e. asymptomatic
virus carriers. FDA-approved ELISA (enzyme-linked immunosorbent assay), as well
as experimental Western Blot kits for the measurement of antibodies against HIV
are now available. These include recent (but still experimental) ELISA assay
kits that detect specific antibodies directed against the viral envelope
protein (gp41 or ENV) and a viral core protein (p24 or CORE) as well as kits
utilizing Western Blot technology for detecting the major antigenic proteins of
HIV. In addition, methods have been recently developed for detecting these
viral antigens in tissue culture fluids of HIV-infected cells cultured in vitro
as well.
All of the current AIDS detection methods employ invasive procedures to
obtain a blood or serum sample to be analyzed for the presence of antibodies to
the HIV virus, i.e. the insertion of a hollow needle or other means for
withdrawing a fluid sample from a vein, artery or subcutaneous space. These
procedures involve some degree of risk to the health care personnel who are
involved in collecting and analyzing these samples as Acquired Immune
Deficiency Syndrome may possibly be contracted through inadvertent exposure to
a syringe or needle that has been employed to obtain a blood or serum sample
from a patient that is afflicted with the disease. Moreover, individuals who
are pres-
2
ently considered to be at a high risk of contacting AIDS, such as homosexual
men and intravenous drug users, and non-high risk individuals who should be
screened, often have unfounded fears that they can contract the disease while
being tested for it, and therefore avoid exposure to any test procedures which
involve withdrawing blood or serum using a needle.
These problems would be overcome by a non-invasive method for screening
for antibodies to HIV. Such a method should be suitable for use in mass
screenings and avoid the inherent drawbacks of the prior art invasive
serological techniques.
SUMMARY OF THE INVENTION
It has now been unexpectedly discovered that antibodies to HIV can be
detected in the urine of patients that have been exposed to, or infected with,
HIV. This is a particularly surprising discovery since heretofore it was
believed that antibodies could not be detected in human urine except in certain
individuals suffering from renal disease. The present invention discloses a
non-invasive method for determining whether a patient is infected with HIV virus
by detecting the presence of antibodies directed against HIV in the urine of a
patient to be screened for HIV.
It is therefore an object of the present invention to provide a
non-invasive method for screening for antibodies directed against infectious
agents.
In another aspect, the present invention provides a non-invasive method
for determining whether an individual has been exposed to a specific viral
agent. The method comprises detecting the presence of antibodies directed
against the specific viral agent in the urine of a patient who has not been
immunized against the specific viral agent.
These and other aspects of the present invention will be apparent to
those of ordinary skill in the art in light of the present description,
accompanying claims and appended drawings.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a representative Western Blot analysis of the concentrated
urine samples of the present invention.
FIG. 2 is a double-diffusion gradient immunometric analysis of HIV
antibodies present in the urine of an HIV-infected individual.
DETAILED DESCRIPTION OF THE INVENTION
It has now been unexpectedly discovered that antibodies to the AIDS
virus (HIV) are present in the urine of AIDS-patients and HIV-infected
individuals, including individuals, including individuals that are not
suffering from renal disorders. In addition, the present inventors have
directly identified these HIV antibodies as being members of the IgG and IgA
classes of immunoglobulins by immunodiffusion techniques. This discovery is
surprising because the prior art suggests that meaningful titers of antiviral
antibodies are only present in the urine of patients afflicted with kidney
disease, or those who have been immunized by vaccination with the poliovirus
vaccine.
Franklin. E. C. (J. Clin. Invest. 38:2159-2167, 1959) has disclosed
that proteins, including albumin, alpha, beta and gamma globulins, were present
in the concentrated urine of normal humans. However, these fragments were only
one sixth the size of mature immuno-
49
4,865,966
3
globulins and were thought to be natural breakdown products of antibody
molecules. Specific antibodies were not examined.
An article by Lerner, A. M. et al (J. Clin. Invest. 41: 805-815, 1962)
discloses that antibodies to poliovirus could be detected in the urine of
normal individuals who had been immunized with poliovirus vaccine. However,
all of the individuals with detectable urine antibodies had received at least 3
or more inoculations with the vaccine, and urines were analyzed shortly
thereafter. The presence of antiviral antibodies in the urine of non-immunized
individuals has not been previously reported in the literature. Indeed, the
present inventors were unable to detect antibodies directed against
cytomegalovirus (CMV) or hepatitis virus in the urine of individuals known to
be infected with such viral agents. In the case of CMV, AIDS patients with
serum titers of 1:1500 to 1:20,000 of anti-CMV antibodies did not have
detectable anti-CMV urinary antibodies, as assayed by ELISA.
Intact antibodies (or fragments of antibodies) directed against HIV
present in the urine of infected individuals have not heretofore been reported
in the literature. Urinary antibodies are usually found only in non-immunized
patients suffering from diseases of the kidney and/or liver such as nephrotic
syndrome, glomerulonephritis, hepatorenal syndrome or from those afflicted with
multiple myeloma, an immunoproliferative disorder.
The amount of HIV antibodies present in the urine is approximately 20
fold lower than that found in serum, and is below the limits of detection of
all currently available diagnostic techniques. Therefore, the urine must be
concentrated, i.e. its volume must be reduced relative to its initial void
volume must be reduced relative to its initial void volume, before assaying for
such antibodies. As more sensitive methods of antibody detection become
available, it is contemplated that the urine concentration step may be
eliminated.
The method of the present invention comprises obtaining a urine sample
voided by a patient to be screened for exposure to HiV, concentrating the urine
sample by reducing the volume of such sample at least about 20 fold in relation
to its initial (void) volume, and assaying the concentrated sample for the
presence of antibodies to HIV. The assay is conducted using techniques that
are well-known for detecting the presence of such antibodies in serum.
Although in theory as little as 1-5 ml of urine could be examined for the
presence of the antibodies sought to be detected, using currently available
methods for antibody detection, 40-100 mls of urine are desirably recovered
from the patient to be tested and used in the assay procedure. The
concentrated urine can be used immediately, stored for 24 hours or longer at 4
deg. C. before use and can be frozen (but deteriorates upon multiple
freezing and thawing).
Any of the numerous methods that are well known to those skilled in the
art can be used to concentrate (reduce the volume) of the urine sample to be
analyzed. Examples of techniques which can be used in practicing the method of
the present invention include, but are not limited to, air evaporation,
membrane dialysis, rotary evaporation, and preferably using a Minicon B15
concentrator (Amicon, Danvers, Mass.) with a 60,000 dalton membrane filter as
detailed in Example 1 below. Urine can also be concentrated by lyophilization,
but this requires larger volumes (e.g. at least 200 ml).
Once the urine sample has been substantially concentrated, it can be
assayed for the presence of antibodies to
4
HIV. As used herein with respect to urine volume reduction, substantially
concentrated means a volume reduction of at least 20 fold in relation to the
initial (void) urine volume, and preferably between about 40 fold and 200 fold
with respect to urine volume reduction, i.e. a reduction in volume from an
initial (void) volume of 40 ml to a final volume of 2 ml is a 20 fold
reduction. The sample thus obtained can be assayed for antibodies to HIV
proteins using standard antibody detection techniques including by way of
non-limiting example, ELISA, Western Blotting, ratio-immunoassay, and
immunodiffusion.
In a preferred embodiment of the present invention, the well-known
Western Blot Analysis method is employed for anti-HIV antibody detection. This
technique has been found to be the most reliable currently available method for
detecting HIV antibodies in the urine of mammals. The technique generally
comprises separating proteins (in this case, HiV proteins) by gel
electrophoresis on the basis of molecular weight, transferring the separated
proteins to a suitable solid support, (such as a nitrocellulose filter or
alternatively, a nylon filter), and incubating the serum (or urine) of an
HIV-infected individual with the separated proteins. This causes specific HIV
antibodies present in the serum (or urine) to bind to their respective
proteins. HIV antibodies are then detected using labeled anti-human HIV
antibodies. This method of detecting antibodies to HIV is preferred due to its
sensitivity and the fact that specific antibodies to viral proteins are
examined. The incidence of false positive results which are inherent when
employing ELISA assays is substantially reduced by using Western Blot Analysis.
An alternative embodiment of the present invention utilizes on Enzyme
Linked Immunosorbent Assay (ELISA) as a means for detecting antibodies specific
for HIV. ELISA assays for the detection of antibodies to the HIV virus can
either be competitive or non-competitive (as described by E. Engvall in Methods
in Enzymology, 70: 419439, incorporated herein by reference).
ELISA's are immunoassays used (in this case) to quantitate the amount
of antibodies present in a sample to be analyzed. The assays employ a
chromagen (a color producing substance) for detecting the antibody: antigen
complex formed. Antibodies used in ELISA are covalently coupled to these
chromogens, such as ortho-phenylendediamine or ortho-dianisidine, the formed
producing a tangerine-colored product in the presence of a peroxide (such as
hydrogen peroxide) and the latter a yellow-orange colored product in the
presence of a peroxide. These colors absorb light at specific wave-lengths
(ortho-phenylendediamine at 492nm and ortho-dianisidine at 400 nm) and are
detected and quantitated using a spectrophotometer.
Non-competitive ELISA tests employ an immobilized antigen in order to
capture any antibodies and an enzyme-labeled second antibody directed against
the species in which the test antibody has been elicited. If for example,
human antibodies are being measured, then goat or rabbit anti-human antibodies
are the labeled, second antibody. The amount of antibody present is directly
proportional to the amount of bound, labeled second antibody. Competitive
ELISA's comprise a reaction in which unlabeled (the biological sample to be
tested) and enzyme-labeled antibodies compete for a
50
4,865,966
5
limited, known amount of antigen. In this case, the reaction is performed
until equilibrium is reached, and the concentration of unknown antibody is
inversely proportional to the amount of bound, enzyme-labeled antibodies. For
example, if there are no antibodies in the unknown sample, all of the labeled
antibodies will bind to the antigen, and a high value, indicated by an increased
color, will be obtained when measuring the amount of enzyme label present.
Commercially-available ELISA assay kits which can be used in practicing
the alternative embodiment of the present invention are available from Abbott
Labs (Chicago, Ill.) under Catalog Number 1037 and as ENVACOR (Human T Cell
Lymphotropic Virus III, EIA, Catalogue No. 2791-22, Abbott International
Diagnostics, Weisbaden, West Germany). The 1037 assay utilizes a
non-competitive ELISA (as described in Example 3 below) whereas the ENVACOR
test uses a competitive ELISA (as described in Example 2 below). These kits
contain the components listed in Examples 2 and 3 below and are offered for use
in assaying serum (not urine) for antibodies to HIV.
One important advantage of the method of the present invention is that
the biological sample to be analyzed (urine) can be easily obtained by
non-invasive techniques, and therefore, the risk of transmitting an HIV
infection to laboratory and health care personnel (e.g. through accidental
puncture with a contaminated needle) is essentially eliminated.
The method of the present invention can be carried out rapidly, limited
only by the time it takes to concentrate the urine sample to be tested. In
broad terms, this can be done in a period of time of less than 90 minutes, when
the samples are to be concentrated 200 fold. Concentrating the urine 20-40 fold
can be accomplished in less than 60 minutes. The sample can then be analyzed
using the commercially-available HIV Serum testing kits.
The invention is described further below in specific working examples
which are intended to illustrate the invention without limiting its scope.
EXAMPLE 1:
Urine Concentration
Serum samples and 60-100 ml of urine were collected (and numerically
coded for patient confidentiality) from the following groups of patients: 28
AIDS-associated Kaposi's sarcoma (AIDS-KS) patients; 21
ARC patients; 48 asymptomatic HIV-infected high risk individuals including (37
homosexual men, 5 female intravenous drug users and 1 female transfusion
recipient); and 16 patients suffering from AIDS-related opportunistic
infections (AIDS-OI), such as Penumocystis carinii and cytomegalovirus
(Table I contains specific patient identification and assay results) In
addition, 17 non-AIDS disease patients' urine and serum, including patients
diagnosed as having cirrhosis and hepatoma (1), hepatitis (3), Lupus (3),
glomerulonephritis (3), nephrotic syndrome (1), Lupus-nephritis (1), heart
failure (1), Lepromatous leprosy (1), tuberculosis (2) and DM nephropathy (1),
plus samples from 30 apparently normal, healthy heterosexuals were obtained.
The urine samples obtained from all of the normal controls and AIDS patients'
displayed essentially normal controls and AIDS patients' displayed essentially
normal values for proteins, when assayed using standard urinalysis techniques
well known in the art. Proteinuria (the presence of abnormally high
concentrations of protein in the urine) is routinely detected using a
"dipstick" that regis-
6
ters the presence of high (over 150 mg) concentrations of
urinary protein. This is indicated by a color change on the dip stick, which
is then compared to standards for quantitation.
The collected urine specimens were centrifuged in conical tubes at 1500
RPM for 15 minutes at room temperature. The supernatant was decanted and saved
and the pellet was discarded.
The supernatant obtained above was concentrated between 20 and 200 fold
in relation to the volume of initial urine sample collected from the subject to
be tested using a Minicon B15 concentrator with a 60,000 dalton membrane
(Amicon, Danvers, Mass.). The concentrator operates by retaining any substance
greater than 60,000 daltons molecular weight on the membrane filter, while the
solution is evaporated. This takes approximately one hour. (To concentrate
the urinary volume 200 times (200X) from an initial or starting volume i.e to
1/200 of its starting volume using the Mincon apparatus takes approximately 90
minutes). The concentrated urine sample can be stored at 4 deg. C. for up to
30 days before use, or used immediately for assay as detailed in Examples 2-5
below. For Western Blot analysis or immunodiffusion studies, sample volumes
are preferably further concentrated 200 fold i.e. to 1/200 of the starting
volume. The amount each sample was concentrated is listed in Tables 1, 2 and
4 below.
EXAMPLE 2:
Elisa Assay Of Concentrated Urine Samples
The serum and concentrated urine samples collected in Example 1 were
assayed for the presence of antibodies to ENV and CORE HIV antigens as
described (Allain, J.P. et al, Lancet I: 1233-1236, 1986, incorporated by
reference) using an Abbott Envacore assay kit. This is a competitive ELISA in
which known amounts of HIV proteins and antibodies are added together with the
sample to be tested. The presence of antibodies in the ample is indicated by a
reduction in the binding of the control antisera with the control antibody.
The manufacturer's instructions were followed exactly as provided. In
addition, blood samples were collected and analyzed simultaneously.
The kit contains a specimen diluent (containing bovine and goat sera
and 0.1% sodium azide), enzyme-conjugated polyclonal antibody to HIV envelope
and core proteins, beads coated with recombinant gp41 or gp24 HIV proteins,
ortho-phenylenediamene (OPD) substrate, a positive and a negative HIV antibody
control and instructions for use.
Fifty microliters of the specimen to be tested, or control, were
incubated with 20 microliters of diluent supplied by the manufacturer and 200
microliters of enzyme-conjugated polyclonal human antibody to HIV envelope or
core proteins. Beads coated with recombinant gp41 or p24 HIV proteins were
added to separate wells containing either the HIV ENV or CORE antibodies.
After 16 to 22 hours incubation at room temperature, the beads were washed and
transferred to appropriate reaction tubes. Three hundred microliters of
ortho-phenylenediamine substrate was then added to each tube and the reaction
allowed to proceed for 30 minutes before addition of 1 ml 1N H(2)SO4 to stop the
reaction. Absorbance values of the solution were measured at 492 nm using a
spectrophotometer sold by Abbott Labs under the name Quantum II, number
3303-11. A positive result for the presence of urine or serum antibodies
51
4,865,966
7
to either the HIV p24 or gp41 proteins was defined as any specimen with
absorbance values equal to or less than 0.5 times the sum of the optical
density (0.D.) of mean negative control (provided by the manufacturer) optical
density (0.D.) plus the 0.D. of the mean positive
8
control (provided by the manufacturer) as measured in above.
A tabular identification of the patients screened with the present
invention and the assay results are presented in Tables 1 and 2 and summarized
in Table 3.
TABLE 1
--------------------------------------------------------------------------------
The Antibodies to HIV in the serum and urine of
AIDS-KS, AIDS-OI, ARC groups, and HR groups
-------------------------------------------
Presence in Presence in
Serum of Urine of
Patient ----------- -----------
Number Sex Code Concentration ENV Core ENV Core
---------------------------------------------------------------------------
1 M 1029 47X + + + -
2 M 993 100X + + - -
3 M 716 42X + - + -
4 M 818 33X + - + -
5 M 871 67X + + + -
6 M 894 33X + + + -
7 M 953 40X + - + -
8 M 5009 34X + + + -
9 M 5018 45X + - + -
10 M 5057 39X + + + -
11 M 5062 42X + + - -
12 M 1028 48X - + + -
13 M 828 42X + + + -
14 M 5061 37X + - + -
15 M 5059 35X + - - -
16 M 276 20X + + + -
17 M 1056 40X-200X + + - -
18 M 5098 40X + + - -
19 M 5013 35-200X + - - -
20 M 5050 20X + + + -
21 M 5074 42X + - - -
22 M 5080 42X + + - -
23 M 5051 40X + + - -
24 M 5083 41X + - + -
25 M 5097 40X + - + -
26 M 1083 42X + - + -
27 M 1081 40X + - - -
28 M 1084 40X + + - -
ARC Patients
------------
29 M 1022 56-200X + + - -
30 M 857 62-200X + + - -
31 M 920 47X + - + -
32 M 598 35X + + - -
33 M 1002 40X + + + -
34 M 1015 35X + - + -
35 M 1016 35X + + - -
36 M 1021 50X + + + -
37 M 949 40X + + + -
38 M 901 100X + - + -
39 M 898 100X + - + -
40 M 956 100X + + + -
41 M 839 47X + - + -
42 M 1039 41X + - + -
43 M HEN03 41X + - + -
44 M HEN04 42X + - + -
45 M HEN05 42X + - + -
46 M HEN06 42X + - + -
47 M HEN07 41X + - + -
48 M HEN08 42X + + + -
49 M HEN09 40X + + + -
---------------------------------------------------------------------------
ENV = Antibody to HIV envelope [ant-cav (gp41)]
Core = Antibody to HIV core [anti-gag (p24)]
TABLE 2
--------------------------------------------------------------------------------
Antibodies to HIV in the serum
and urine of the High-Risk group and AIDS-OI
--------------------------------------------
Serum Urine
Patient Fold ----------- -----------
Number Sex Code Concentration ENV Core ENV Core
---------------------------------------------------------------------------
50 M 1024 37-200X + - - -
51 M 1060 30X + - - -
52 M 1059 32X - - - -
53 M 1062 40X + - - -
54 M 1057 28X + + + -
55 M 1055 32X - - - -
56 M 1058 40X + + + -
52
4,865,966
9
TABLE 2 - continued
--------------------------------------------------------------------------------
Antibodies to HIV in the serum
and urine of the High-Risk group and AIDS-OI
--------------------------------------------
Serum Urine
Patient Fold ----------- -----------
Number Sex Code Concentration ENV Core ENV Core
---------------------------------------------------------------------------
57 M 1052 37X - - - -
58 M A(2)-014 50X + + + -
59 M A(1)-078 33X + - + -
60 M 0697550 40X + - + -
61 M 0820835 44X + + + -
62 M 1061 41X + + + -
63 M A(1)-029 40X + + - -
64 M A(1)-136 41X + + - -
65 M A(1)-138 39X + - - -
66 M A(1)-044 41X + + + -
67 M A(1)-045 40X + + + -
68 M A(1)-173 42X + + + -
69 M A(1)-003 42X + + + +
70 F 1065754 40X + + + -
71 M 0471024 40X + + + -
72 M A(2)-132 40X + + + -
73 M A(2)-115 40X + + + +
74 M A(1)-060 40X + + - -
75 M A(1)-178 43X + + - -
76 M A(2)-090 40X + + + -
77 M A(2)-097 40X + + + -
78 M A(2)-137 40X + + + -
79 F U16 41X + - + +
80 M 1091 42X - - - -
81 M 1093 42X + + + -
82 M 1110 40X + - + -
83 M 1121 40X + - + -
84 M 1080 41X - - - -
85 M 1070 40X + - + -
86 M 1111 40X + + - -
87 M 1107 40X + + + +
88 M 1116 49X + + + +
89 F 941151 46X + - + -
90 M 1098 41X + - + -
91 F U17 40X + - - -
92 F U19 42X + + + +
93 F U20 42X + + + -
94 M A(2)-036 42X + - + -
95 M A(1)-082 40X + + - -
96 M A(i)-177 42X + - - -
97 M 1073 40X + - - -
PATIENTS WITH OPPORTUNISTIC INFECTIONS
--------------------------------------
98 M 644 40X + + + -
99 M 1063 23X + - - -
100 M 155 43X + + - -
101 M 156 40X o/ / + -
102 M 157 40X + + - -
103 M 1108 40X + - + -
104 M 1112 40X + - - -
105 M 1113 42X + + - -
106 M 159 41X + - + -
107 F 160 41X + - + -
108 M 161 40X + + + -
109 F 162 42X + + + -
110 M 164 44X + + + -
111 M 165 40X + - + -
112 M 166 41X + - + -
113 M 473 33X + - + -
--------------------------------------------------------------------------------
o/ = not done
TABLE 3
--------------------------------------------------------------------------------
Summary of ELISA assay for HIV ENV antiodies
in the Concentrated Urine Samples
--------------------------------------------
SERUM/URINE SERUM/URINE SERUM/URINE % SERUM/URINE
PATIENT TYPE + - + + - - + +
-------------------------------------------------------------------------------------------
AIDS/KS 8 20 0 20/28 = 71.4%
ARC 4 17 0 17/21 = 81.0%
HIGH RISK 13 30 5 30/3 = 69.7%
OPPORTUNISTIC 5 11 0 11/16 = 68.8%
INFECTION
-------------------------------------------------------------------------------------------
TOTAL 30 78 5 78/113 = 72.3%
-------------------------------------------------------------------------------------------
Referring to Tables 1 and 2, it can be seen that 71.4% of the AIDS-induced
Kaposi's sarcoma patients, 81% of
10
the ARC patients, 69.7 of the patients in the high risk
53
4,865,966
11
group and 68.8% of patients suffering from opportunistic infections had
antibodies to the ENV protein (gp41) of HIV present in their urine and that
such antibodies were detected using the methods of the present invention. The
total percentage of patients with detectable HIV ENV antibodies was 72.2%. The
core antigen was only detected in the urine of 6 individuals, those being in
the asymptomatic infected high risk group. All of the non-AIDS disease
patients and the normal, healthy heterosexuals' urine and serum were negative
for antibodies to both HIV proteins.
EXAMPLE 3:
A subset of the urine samples analyzed in Example 1 were re-examined
using two commercially-available non-competitive HIV ELISA detection kits. KIT
I (HTLV III EIA kit, Lot No. 1590HR00, Abbott Laboratories, Chicago, Ill.) is
an FDA-approved clinical diagnostic kit; Kit II (EIA Clinical Diagnostic Kit,
Catalog No. 1037, Abbott Laboratories, Chicago, Ill.) is intended for
investigative use only.
Each of the kits contain HIV antigen-coated beads, goat anti-human
antibody conjugated to horseradish peroxidase, a positive control, a negative
control, specimen diluent containing bovine and goat sera, OPD and an OPD
diluent containing citrate-phosphate buffer and
12
0.02% hydrogen peroxide, reaction trays,, assay tubes and instructions for use.
The assay for the presence in the urine of a patient to be tested of
antibodies to HIV is performed as follows. 10 microliters of control or
diluted specimen is dispensed into preselected wells of the reaction tray.
Each well can hold up to 400 microliters of fluid. Two hundred microliters of
specimen diluent and one bead are added per well. The reactions are incubated
for about 1 hour at 40 leg. C. Thereafter, the supernatant (i.e. liquid) is
discarded and the bead washed three times with 4 to 6 ml of distilled or
deionized water. Two hundred microliters of labeled goat-anti-human antibodies
are then added and incubated at 40 deg. C. for about 2 hours. The supernatant
is removed and the bead is washed as above. The bead is transferred to an
assay tube, 300 microliters of OPD substrate solution is added, and the
solution is incubated for about 30 minutes. 1 ml of 1N sulfuric acid is added
and the absorbance of the solution determined at 492 nm in a standard Abbott
Labs spectrophotometer (Model 3303-11 available from Abbott Labs). A positive
value is indicated if a sample is within the rnge of 0.5 to 1.5 times the
positive control mean.
The results of these assays are presented below in Table 4.
TABLE 4
--------------------------------------------------------------------------------
KIT I KIT II
PATIENT FOLD URINE HIV URINE HIV ENVEL-
NUMBER PATENT CONCENTRATION ANTIBODY OPE ANTIBODY
--------------------------------------------------------------------------------
AIDS-KS
-------
1 1029 47X + +
3 716 42X + +
4 818 33X + +
5 871 67X - +
6 894 33X + +
7 953 40X - +
8 5009 34X + +
9 5018 45X - +
10 5057 39X - +
11 5062 42X - -
12 1028 48X + +
13 828 42X - +
14 5061 37X + +
16 276 20X - +
17 1056 40X-200X - +
19 5013 35X-200X - -
20 5050 20X - +
21 5074 42X + +
22 5080 42X - -
ARC
----
30 857 62x-200x - -
31 920 47X - +
32 598 35X - -
33 1002 40X + +
34 1015 35X - +
35 1016 35X - -
36 949 40X - +
40 956 100X - +
41 839 47X - +
HIGH-RISK
---------
52 1059 32X - -
53 1067 40X - -
54 1057 28X - +
55 1055 32X - -
57 1052 37X - -
58 A2-014 50X - +
59 A1-078 33X - +
60 0697550 40X - +
61 0820535 44X - +
62 1061 41X - +
63 A1-029 40X - -
64 A1-136 41X - -
65 A1-138 39X - -
66 A1-044 41X - +
67 A1-045 40X - +
54
4,865,966
13
TABLE 4-continued
-------------------------------------------------------------------------------
KIT I KIT II
Patient Fold Urine HIV Urine HIV
Number Patent Concentration Antibody Envelope Antibody
-------------------------------------------------------------------------------
68 A1-173 42X - +
69 A1-003 42X + +
PATIENTS WITH OPPORTUNISTIC INFECTION
--------------------------------------
98 644 40x + +
99 1063 23x - -
100 155 43x - -
101 156 40x + +
102 157 40x - -
-------------------------------------------------------------------------------
The results presented in Table 4 demonstrate that urine
antibodies to HIV are more readily detectable when employing a more sensitive
ELISA assay kit (Kit II). This is the same as for serum antibodies. A limited
number of these samples were further analyzed using the Western Blot technique
as described below in Example 4.
EXAMPLE 4:
Western Blot Analysis Of Concentrated Urine Samples
Western Blot analysis for the presence of antibodies to HIV was
performed on the urine and serum samples collected from 59 HIV sero-positive
(including 18 AIDS-KS, 27 High Risk individuals, 6 ARC and 8 O.I. patients
selected from those reported in Tables 1 and 2 above) and 30 non-AIDS disease
patients. A commercially-available kit (Biotech/DuPont HTLV-III Western Blot
Kit, available from E. I. DuPont de Nemours and Co., Inc., Wilmington, Del.) was
used in making the analysis. The kit contains precut nitrocellulose membrane
strips with immobilized viral antigens that have been separated by sodium
dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted
onto the membrane; control sera, including a negative control, a weak positive
control and a strong positive control; blotting buffer (Tris buffered saline
with 5% nonfat dry milk and also containing heat inactivated normal goat serum);
wash buffer (Tris buffered saline containing tween-20 detergent); biotinylated
goat anti-human IgG; avidin-horse-radish peroxidase; 4-chloro-1-naphthol in
solution; hydrogen peroxide; and an incubation tray. The kit was used exactly
according to the manufacturer's instructions as described below.
The assay comprises soaking the strips in 2 mls of wash buffer in a well
of the wash tray for 30 minutes
14
at room temperature; the liquid is then drained off and discarded. The strips
are then washed with 2 mls of blotting buffer for 5-10 minutes at room
temperature. Twenty microliters of a 200 fold concentrated urine sample are
added to the wells containing the strips and blotting buffer and the reactant
incubated overnight at room temperature. Thereafter, the mixture in the wells
is aspirated and discarded and the wells are washed once with 2 mls of wash
buffer. Two additional 2 ml washes (with wash buffer) are performed at room
temperature, allowing 5 minutes soaking between each wash and discarding the
wash afterwards.
The nitorcellulose strips are developed as follows. Two mls of
biotinylated goat anti-human IgG are added to each well and allowed to incubate
for 60 minutes at room temperature on a rocking or rotary apparatus. The strips
are then washed with 2 mls of wash buffer per strip for 5 minutes; this step is
then repeated 3 additional times at room temperature, discarding the wash after
each use. Two mls per strip of avidin-horseradish peroxidase are added and
incubated for 60 minutes at room temperature on a rocking or rotary apparatus.
The strips are washed 3 times as above. Two mls of a 50:50 mixture of
4-chloro-1-naphthol and hydrogen peroxide are then added and allowed to incubate
for 10-15 minutes or until the color develops at room temperature. The presence
of color on the strip indicates that the strip has been exposed to a biological
fluid (i.e. urine) containing antibodies to HIV.
The strips are scored for the presence of antibodies to HIV as negative
(-) i.e. no antibodies to HIV detected, weakly positive (+) or strongly positive
(+) using the controls supplied by the manufacturer as references. The results
are presented in Table 5 and FIG. 1.
TABLE 5
-----------------------------------------------------------------------------
Results of Western Blot analysis of concentrated urine samples
--------------------------------------------------------------
Patient
Number Code p17 p24 p31 p41 p51 p55 p66 p110/p120 p160
-----------------------------------------------------------------------------
AIDS-KS
-------
1 1029 - - - + - - - + +
7 953 - - + +- - - - + +
8 5009 - + + - + + - + +
9 5018 - - + + + - + + +
10 5057 - + + - + - + + +
12 1028 - - - + - - - + +
13 828 - - + - - - - + +
14 5061 - - + +- - - - + +
16 276 - + + + + + + + +
17 1056 - - - - +- +- +- + +
18 5098 - + - + - + - + +
21 5074 - - + - + - + + -
22 5080 - - - - - - - - -
23 5051 - - - - - - - - +
24 5083 - - - + - - - + +
25 5097 - - - + - - - + +
26 1083 - - - - - - - - -
27 1081 - - - + - + - - +
55
4,865,966
15
TABLE 5-Continued
------------------------------------------------------------------------------------------
Results of Western Blot analysis of concentrated urine samples
--------------------------------------------------------------
PATIENT
NUMBER CODE p17 p24 p31 p41 p51 p55 p66 p110/p120 p160
------------------------------------------------------------------------------------------
ARC
---
33 1002 - + + + + + + + +
40 956 - - + + + - + + +
42 1039 - + + + + + + + +
43 Hen03 - - - + - - + - -
43 Hen04 - - + + + - + + +
43 Hen05 + - + + + + + + +
Asymptomatic High-Risk homosexuals
----------------------------------
54 1057 - + + + - - + + +
58 A2-014 - + - + - + + + +
- - - -
59 A1-078 - + + + - + + + +
-
60 697550 - - + + + + + + +
61 820835 - + + * + + + + +
62 1061 - - - - - - - + +
66 A1044 - - + - - - - + +
67 A1-045 - + + + + + + + +
68 A1-173 + + + + + + + + +
69 A1-003 + + + + + + + + +
70 1065754 + + + + + + + + +
71 471024 - + - - - + + + +
72 A2-132 + + + + + + + + +
74 A2-060 - - - - - - - - +
75 A2-178 - + + + + + + + +
76 A2-090 - + - + - + + + +
77 A2-097 + + + + + + + + +
78 A2-137 + + + + + + + + +
79 U16 + + + + + + + + +
81 1093 - + - + - + + + +
82 1110 - + + + + + + + +
83 1121 - - - * - - - - +
85 1070 + + - + - + + + +
86 1111 - * + + + - + - +
87 1107 + + + + + + + + +
88 1116 + + + + + + + + +
90 1098 + + + + + + + + +
Opportunistic Infections
------------------------
98 644 - - - - - - - - -
99 1063 - - - - - - - - -
100 156 - - - + - - - + +
102 157 - * - - - - - - +
103 1108 + - + + + + + + +
104 1112 - - - + + - + + +
105 1113 - - + + + + + + +
113 473 + + + + + + + + +
-----------------------------------------------------------------------------------------
* = indeterminate results
TABLE 6
-------------------------------------------------------------------------------------------
Comparison of Results of Western Blot analysis
of concentrated urine and serum samples
----------------------------------------------
HIV AIDS-KS ARC HIGH-RISK O.L.
--------------- --------------- --------------- ---------------
Protein Serum Urine Serum Urine Serum Urine Serum Urine
-------------------------------------------------------------------------------------------
p17 18/18* 1/18 2/6 1/6 26/27 11/27 7/8 1/8
p24 18/18 5/18 6/6 2/6 25/27 23/27 7/8 1/8
p31 18/18 9/18 6/6 5/6 26/27 19/27 7/8 3/8
p41 18/18 11/18 6/6 6/6 26/27 23/27 8/8 5/8
p51 18/18 8/18 6/6 5/6 27/27 16/27 8/8 4/8
p55 18/18 7/18 6/6 3/6 27/27 21/27 8/8 3/8
p66 18/18 7/18 6/6 6/6 27/27 23/27 8/8 4/8
p120 18/18 15/18 6/6 5/6 27/27 24/27 8/8 5/8
p160 18/18 16/18 6/6 5/6 27/27 26/27 8/8 8/8
-------------------------------------------------------------------------------------------
*18 positives per 18 patients tested.
Referring to Table 5, p17 is a protein component produced upon cleavage
of p55 to p24 is the viral core protein, p31 is the viral endonuclease, p41 is
the mature envelope protein, p51 and p66 are components of the viral reverse
transcriptase, p55 is a precursor to the viral core protein and p160 is a
precursor to the envelope protein; p110/120 is a mixture of 2 proteins which
co-migrate in this gel system: p120 is a protein component produced upon
processing of the p160 pro-
16
tein to gp41; p110 is a protein component of the virus whose function is
currently unknown.
An example of a typical a Western Blot analysis of the concentrated
urine and serum samples obtained by practicing the method of the present
invention is shown in FIG. 1. Referring to FIG. 1, lane 22 is the negative
control; the numbers to the right represent the various HIV proteins discussed
above. Above lines 12-16 and 18-2 are the corresponding
56
4,865,966
17
patients as referred to in Table 5 above. For example, urine obtained from
patient No. 956 (FIG. 1, lane 16), suffering from ARC contained antibodies
directed against all of the HIV proteins except p24 (Core) and p17. The
significance of this finding is presently unknown.
As can be seen from the data in Table 5 and summarized in Table 6,
antibodies to HIV antigens can be readily found (when present) in the
concentrated urine samples using this more sensitive technique. In particular,
antibodies to p160 in the concentrated urine could be detected in 55 out of the
59 HIV-positive individuals tested (93.2%). These results also demonstrate
that the use of concentrated urine in this preferred embodiment of the present
invention does not lead to false positive, since specific viral proteins are
identified.
EXAMPLE 4:
Double-Diffusion Gradient Immunoelectrophoretic
Analysis Of HIV Antibody-Containing Urine Samples
Concentrated urine sample No. 956 (Table I), which tested positive for
HIV antigens using Western Blot analyses and antibodies directed against the
envelope protein using ELISA, was further concentrated 200 fold and analyzed by
double-diffusion gradient immuno-electrophoresis (DDG-IEB) as described by J.V.
Chuba, in J. App. Biochem., 1: 37-50, 1979, (incorporated by reference). Serum
samples from this patient were collected and analyzed in parallel to the patient
urine samples as a positive control.
Briefly, replicate samples of 3 microliters of urine and serum
respectively were electrophoresed in commercially prepared 0.5% agarose thin
layer gels (Paragon, Beckman Instruments Brea, Calif.). Troughs were placed at
right angles to the gels for the addition of antisera. Electrophoresis was
performed by subjecting the gels to 20 minutes of direct current on a slightly
modified Hyland Power Pack (Costa Mesa, Calif.) at the 40 mA setting. The
running buffer contained barbital buffer B-2 (0.075 M, pH 8.6; containing 0.2%
(w/v) sodium azide) mixed with an equal volume of 3.0 mM aqueous calcium
lactate solution.
After eletrophoresis, the antisera troughs were completed by removing
the corresponding segment of the gel between the precut slits, and conventional
parallel-trough immunodiffusion was performed. 7.5 microliters of anti-human
IgG, IgM and IgA (Behring Diagnostics, San Diego, Calif.) at a concentration of
5.5 micrograms per ml was added to the troughs and incubated for 20 minutes at
room temperature. Thereafter, the gels were stained and examined for the
development of precipitin lines. Anti-albumin antibodies were included in the
serum samples as a positive control.
As shown in FIG. 2, IgG (11) and IgA (13) immunoglobulins were
identified in the urine sample of patient No. 956. IgM, although present in
the serum did not appear in the urine (12). This is not surprising due to the
large size of this immunoglobulin (approximately 900,000 daltons).
Anti-albumin antibodies reacted with the serum samples, as expected (10),
forming a sharp percipitin line of identity (lane 1). These results confirm
the positive results obtained from the ELISA and Western Blot detection
procedures, and demonstrate that they were not due to artifacts caused by use of
concentrated urine samples.
The above invention has been described in terms of preferred
embodiments. It would be obvious to those of
18
ordinary skill in the art that many additions, deletions and substitutions
could be made without departing from the spirit and scope of the invention, as
claimed below.
What is claimed is:
1. A method for detecting antibodies to human immunodeficiency virus
which comprises the steps of:
collecting a void urine sample from a human subject to be tested for the
presence of antibodies to human immunodeficiency virus to form a
liquid specimen;
adjusting the volume of said liquid specimen to a level sufficient to
enable antibodies to human immunodeficiency virus present in
said specimen to be detected by assay; and
assaying a predetermined quantity of said liquid specimen for the
presence of said antibodies to human immunodeficiency virus.
2. The method of claim 1 wherein said adjusting step comprise reducing
the volume of said urine sample at least twenty-fold in relation to the void
volume of said urine sample.
3. The method of claim 1 wherein said assaying step comprises exposing
said urine specimen to an enzyme-linked immunosorbent assay for human
immunodeficiency virus.
4. The method of claim 1 wherein said assaying step comprises
conducting a western blot analysis of a portion of said urine specimen.
5. The method of claim 1 wherein said assaying step comprises
electrophoresing a predetermined quantity of said urine specimen using
an electrophoresis gel,
excising a portion of the electrophoresis gel, and
examining the excised gel portion for the presence of antibodies to
human immunodeficiency virus.
6. The method of claim 1 wherein said antibodies are directed against
viral proteins of human immunodeficiency virus.
7. The method of claim 6 wherein aid antibodies comprise antibodies
directed against human immunodeficiency virus viral protein p41.
8. The method of claim 1 wherein said antibodies comprise antibodies
directed against human immunodeficiency virus viral protein p24.
9. The method of claim 1 wherein said antibodies comprise antibodies
directed against human immunodeficiency virus viral protein p160.
10. The method of claim 1 wherein said antibodies comprise IgG.
11. The method of claim 1 wherein said antibodies comprise IgA.
12. A method for detecting the presence of antibodies to human
immunodeficiency virus in a human subject which comprises the steps of:
collecting a void urine sample from said human subject,
adjusting the volume of said void sample to a level sufficient to enable
antibodies to human immunodeficiency virus present in said
sample to be detected by assay, thereby forming a test specimen
and
assaying said test specimen for the presence of at least one antibody to
a human immunodeficiency virus protein selected from the group
consisting of human immunodeficiency virus viral protein p17,
and p24.
57
4,865,966
19
13. A method for determining whether a human subject has been infected
with human immunodeficiency virus (HIV) comprising the steps of:
collecting a void urine sample from said subject;
adjusting the volume of said void urine sample to a level sufficient to
enable antibodies to human immunodeficiency virus present in
said sample to be detected by assay, thereby forming a test
specimen;
assaying said specimen for the presence of antibodies to a HIV viral
protein;
comparing the results of said assay with those of the same assay
performed with urine from a HIV-free control human subject.
14. The method of claim 13 wherein said assaying step comprises
exposing said urine specimen to an enzyme-linked immunosorbent assay.
15. The method of claim 13 wherein said assaying step comprises
conducting a western blot analysis of a portion of said urine specimen.
16. The method of claim 13 wherein said antibodies comprise antibodies
directed against at least one HIV viral protein selected from the group
consisting of p41, p24, p160, and p17.
20
17. The method of claim 13 wherein said antibodies comprise antibodies
directed against at least one HIV viral protein selected from the group
consisting of p17 and p24.
18. The method of claim 13 wherein said antibodies comprise antibodies
of the IgG isotype.
19. The method of claim 13 wherein said antibodies comprise antibodies
of the IgA isotype.
20. A method for screening a human subject for exposure to human
immunodeficiency virus (HIV) comprising the steps of:
collecting a void urine sample from said subject;
adjusting the volume of said void urine sample to a level sufficient to
enable antibodies to HIV present in said sample to be detected
by assay, thereby forming a test specimen;
assaying said specimen for the presence in said specimen of at least one
antibody to an HIV viral protein; and
determining whether said subject has been exposed to HIV based on the
positive presence of said antibody in said sample.
* * * * *
58
UNITED STATES PATENT [19] [11] PATENT NUMBER: 5,122,446
FRIEDMAN-KIEN ET AL. [45] DATE OF PATENT: JUN. 16, 1992
--------------------------------------------------------------------------------
[54] METHOD FOR DETECTING ANTIBODIES TO HUMAN IMMUNODEFICIENCY VIRUS
[75] Inventors: Alvin Friedman-Kien; Cao Yunzhen, both of New York; William
Borkowsky, Brooklyn, all of N.Y.
[73] Assignee: New York University, New York, N.Y.
[21] Appl. No.: 204,871
[22] Filed: Jun. 10, 1988
RELATED U.S. APPLICATION DATA
[63] Continuation-in-part of Ser. No. 40,013, Apr. 17, 1987 Pat. No. 4,865,966.
[51] Int. CL(5) .................................................. G01N 33/569
[52] U.S. CL ................................................ 435/5; 435/7.92;
435/7.93; 435/7.94; 435/7.95; 435/974;
435/975; 436/501; 436/513; 436/808; 436/811
[58] Field of Search ................................435/5, 7, 810, 7.92-7.95,
435/974, 975; 436/501, 513, 808, 811
[56] REFERENCES CITED
U.S. PATENT DOCUMENTS
4,560,647 12/1985 Stocker ..................... 435/5
4,725,669 2/1988 Essex et. al ................ 530/395
4,865,966 9/1989 Freidman-Kien ............... 435.5
FOREIGN PATENT DOCUMENTS
0132170 1/1985 European Pat. Off.
WO86/02930 5/1986 World Int. Prop. O. .......... 435/5
88/07680 10/1988 World Int. Prop. O. .......... 435/5
OTHER PUBLICATIONS
Journal of Clinical Investigation, vol. 41, No. 4, 1962, pp. 805-815, M. Lerner
et al., "Neutralizing Antibody to Polioviruses in Normal Human Tissue".
Chemical Abstracts, vol. 77, Sep. 22, 1977, Abstract No. 87578.
Blood, vol. 67, No. 3, 1986, pp. 831-834, New York, D.W. Archibald et al.,
"Antibodies to Human T-Lymphotropic Virus Type III (HTLV-III) in Saliva of
Acquired Immunodeficiency Syndrome (AIDS) Patients and in Persons at Risk for
AIDS".
Clinica Chimica Acta, vol. 139, 1984, pp. 113-117, T.R. Trinick et al.,
"Measurement of Urinary Immunoglobulins G, A and M by an Enzyme Linked
Immunosorbent Assay (ELISA)".
Lancet, Apr. 9, 1988, pp. 831, 832, Cao et al., "IGG Antibodies to HIV-1 in
Urine of HIV-1 Seropositive Individuals".
AIDS Research and Human Retroviruses, vol. 5, No. 3, 1989, pp. 311-320, New
York, Y. Cao et al., "Antibodies to Human Immunodeficiency Virus Type I in the
Urine Specimens of HIV-1-Seropositive Individuals". Lerner et al.,
"Neutralizing Antibody to Poliovirus in Normal Human Urine", Virology, (1961),
14:383-5.
Meryhew, N.L. et al., J. Rheumat., 10:913-919, 1983.
Takayanagi et al., Clinicopathological Significance of the Analysis of Urinary
Antibody Activatives, Rinsho Byroi, The Japanese Journal of Clinical Pathology,
vol. 22 (Suppl.) Oct. 1974, p. 184, (English Translation).
Bulletino Dell Instituto Sieriterapico Milanese, vol. 51, No. 1, 1972, pp.
90-102, D. DeDonate et al., "Sugli Anticorporpi Antivirali Delle Urine",
(Chemical Abstracts, vol. 77, Sep. 22, 1977, Abstract No. 86578).
Primary Examiner -- Christine Nucker
Attorney, Agent, or Firm -- Darby & Darby
[57] ABSTRACT
Disclosed herein is a method of screening mammals for antibodies to viral
agents by collecting a urine sample from a mammal to be tested and assaying the
sample for antibodies directed against the specific viral agent.
13 CLAIMS, 2 DRAWING SHEETS
59
U.S. PATENT JUNE 16, 1992 SHEET 1 OF 2 5,122,446
[CHART]
FIG. 1
60
U.S. PATENT JUNE 16, 1992 SHEET 2 OF 2 5,122,446
[CHART]
FIG. 2
61
5,122,446
1
METHOD FOR DETECTING ANTIBODIES TO
HUMAN IMMUNODEFICIENCY VIRUS
This application is a continuation-in-part application of co-pending
U.S. patent application Ser. No. 040,013 filed Apr. 17, 1987 of Friedman-Kien
et al now U.S. Pat. No. 4,865,956.
The present invention related to a method for detecting antibodies
directed against Human Immunodeficiency Virus which can be used for diagnosing
AIDS and related diseases, and identifying latent, asymptomatic carriers of
such infections.
Acquired Immune Deficiency Syndrome (AIDS) was initially recognized and
reported in 1981. Since that time, clinical and epidemiological data have
revealed that the incidence of AIDS has reached epidemic levels throughout the
world. The causative agent of AIDS has been identified as an RNA retrovirus,
the Human T-Cell Leukemia Virus Type III (HTLV-III), also known as
Lymphadenopathy Associated Virus (LAV) and AIDS-associated retrovirus (ARV) and
recently renamed Human Immunodeficiency Virus (HIV). AIDS patients may suffer
from a broad spectrum of opportunistic infections such as Pneumocystis carinii,
Candida albicans, herpes simplex virus and cytomegalovirus, and are also
frequently afflicted with certain tumors, especially Kaposi's Sarcoma. It has
been estimated that the number of patients with AIDS in the United States
continues to double approximately every twelve months.
The putative AIDS virus, HIV, has been isolated from peripheral blood
mononuclear cells, cerebrospinal fluid, semen, neural tissue, saliva, tears and
rarely, urine. In order to determine the prevalence of HIV in the general
population, it has been suggested that mass screenings of the population for
the presence of antibodies directed against the AIDS virus be undertaken.
However, since antibody substances are generally found only in human blood and
serum, the proposed screening techniques involve obtaining a blood or serum
sample from the patient who is to be screened.
A variety of serological tests have been developed to detect the
presence of antibodies to HIV (indicative of exposure to HIV) in the blood of
patients with AIDS, AIDS-Related Complex (ARC) and healthy (i.e. asymptomatic)
virus carriers. FDA-approved ELISA (enzyme-linked immunosorbent assay), as
well as experimental Western Blot kits for the measurement of antibodies
against HIV are now available. These include recent (but still experimental)
ELISA assay kits that detect specific antibodies directed against the viral
envelope protein (gp41 or ENV) and a viral core protein (p24 or CORE) as well
as kits utilizing Western Blot technology for detecting the major antigenic
proteins of HIV. In addition, methods have been recently developed for
detecting these viral antigens in tissue culture fluids, of HIV-infected cells
cultured in vitro as well.
All of the current AIDS detection methods employ invasive procedures to
obtain a blood or serum sample to be analyzed for the presence of antibodies to
the HIV virus, i.e. the insertion of a hollow needle or other means for
withdrawing a fluid sample from a vein, artery or subcutaneous space. These
procedures involve some degree of risk to the health care personnel who are
involved in collecting and analyzing these samples as Acquired Immune
Deficiency Syndrome may possi-
2
bly be contracted through inadvertent exposure to a syringe or needle that has
been employed to obtain a blood or serum sample from a patient that is
afflicted with the disease. Moreover, individuals who are presently considered
to be at a high risk of contacting AIDS, such as homosexual men and intravenous
drug users, and non-high risk individuals who should be screened, often have
unfounded fears that they can contract the disease while being tested for it,
and therefore avoid exposure to any test procedures which involve withdrawing
blood or serum using a needle.
These problems would be overcome by a non-invasive method for screening
for antibodies to HIV. Such a method should be suitable for use in mass
screenings and avoid the inherent drawbacks of the prior art invasive
serological techniques.
SUMMARY OF THE INVENTION
It has now been unexpectedly discovered that antibodies to HIV can be
detected in the urine of patients that have been exposed to, or infected with,
HIV. This is a particularly surprising discovery since heretofore it was
believed that antibodies could not be detected in human urine except in certain
individuals suffering from renal disease. The present invention discloses a
non-invasive method for determining whether a patient is infected with HIV
virus by detecting the presence of antibodies directed against HIV in the urine
of a patient to be screened for HIV.
It is therefore an object of the present invention to provide a
non-invasive method for screening for antibodies directed against infectious
agents.
In another aspect, the present invention provides a non-invasive method
for determining whether an individual has been exposed to a specific viral
agent. The method comprises detecting the presence of antibodies directed
against the specific viral agent in the urine of a patient who has not been
immunized against the specific viral agent.
These and other aspects of the present invention will be apparent to
those of ordinary skill in the art in light of the present description,
accompanying claims and appended drawings.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a drawing showing a representative Western Blot analysis of
the concentrated urine samples of the present invention.
FIG. 2 is a drawing showing a double-diffusion gradient immunometric
analysis of HIV antibodies present in the urine of an HIV infected individual.
DETAILED DESCRIPTION OF THE
INVENTION
It has now been unexpectedly discovered that antibodies to the AIDS
virus (HIV) are present in the urine of AIDS-patients and HIV-infected
individuals, including individuals that are not suffering from renal disorders.
In addition, the present inventors have directly identified these HIV
antibodies as being members of the IgG and IgA classes of immunoglobulins by
immuno-diffusion techniques. This discovery is surprising because the prior
art suggests that meaningful titers of antiviral antibodies are only present in
the urine of patients afflicted with kidney disease, or those who have been
immunized by vaccination with the poliovirus vaccine.
62
5,122,446
3
Franklin, E. C. (J. Clin. Invest. 38:2159-2167, 1959) has disclosed that
proteins, including albumin, alpha, beta and gamma globulins, were present in
the concentrated urine of normal humans. However, these fragments were only one
sixth the size of mature immunoglobulins and were thought to be natural
breakdown products of antibody molecules. Specific antibodies were not
examined.
An article by Lerner, A. M. et al (J. Clin. Invest. 41: 805-815, 1962)
discloses that antibodies to poliovirus could be detected in the urine of
normal individuals who had been immunized with poliovirus vaccine. However,
all of the individuals with detectable urine antibodies had received at least 3
or more inoculations with the vaccine, and urines were analyzed shortly
thereafter. The presence of antiviral antibodies in the urine of non-immunized
individuals has not been previously reported in the literature. Indeed, the
present inventors were unable to detect antibodies directed against
cytomegalovirus (CMV) or hepatitis virus in the urine of individuals known to be
infected with such viral agents. In the case of CMV, AIDS patients with serum
titers of 1:1500 to 1:20,000 of anti-CMV antibodies did not have detectable
anti-CMV urinary antibodies, as assayed by ELISA.
Intact antibodies (or fragments of antibodies) directed against HIV
present in the urine of infected individuals have not heretofore been reported
in the literature. Urinary antibodies are usually found only in non-immunized
patients suffering from diseases of the kidney and/or liver such as nephrotic
syndrome, glomerulonephritis, hepatorenal syndrome or from those afflicted with
multiple myeloma, an immunoproliferative disorder.
The amount of HIV antibodies present in the urine is approximately 20
fold lower than that found in serum, and is often below the limits of detection
of currently available diagnostic techniques. Therefore, the urine must be
concentrated, i.e. its volume is preferably be reduced relative to its initial
void volume, before assaying for such antibodies. More sensitive methods of
antibody detection such as those of Example 5, permit the urine concentration
step to be eliminated.
The method of the present invention comprises obtaining a urine sample
voided by a patient to be screened for exposure to HIV, optionally
concentrating the urine sample by reducing the volume of such sample at least
about 20 fold in relation to its initial (void) volume, and assaying the
concentrated or unconcentrated sample as the casing for the presence of
antibodies to HIV. The assay is conducted using techniques that are well-known
for detecting the presence of such antibodies in serum. Although as little as
1-5 ml of urine could be examined for the presence of the antibodies sought to
be detected, using most currently available methods for antibody detection,
40-100 mls of urine are desirably recover from the patient to be tested and
used in the assay procedure. The urine can be used immediately, stored for 24
hours or longer at 4 deg. C. before use and can be frozen (but deteriorates
upon multiple freezing and thawing).
If it is determined that concentration of urine is desired, any of
the numerous methods that are well known to those skilled in the art can be used
to concentrate (reduce the volume) of the urine sample to be analyzed.
Examples of techniques which can be used in practicing the method of the
present invention include, but are not limited to, air evaporation, membrane
dialysis, rotary
4
evaporation, and preferably using a Minicon B15 concentrator (Amicon, Danvers,
MA) with a 60,000 dalton membrane filter as detailed in Example 1 below. Urine
can also be concentrated by lyophilization, but this requires larger volumes
(e.g. at least 200 ml).
Once the urine sample has been substantially concentrated, it can be
assayed for the presence of antibodies to HIV. As used herein with respect to
urine volume reduction, substantially concentrated means a volume reduction of
at least 20 fold in relation to the initial (void) urine volume, and preferably
between about 40 fold and 200 fold with respect to urine volume reduction, i.e.
a reduction in volume from an initial (void) volume of 40 ml to a final volume
of 2 ml is a 20 fold reduction. The samples can also by lyophilized and
resuspended (in, for example isotonic saline) in any volume desired, but as
mentioned above, this requires larger volumes.
The sample can be assayed for antibodies to HIV proteins using standard
antibody detection techniques including by way of non-limiting example, ELISA,
Western Blotting, radio-immunoassay, and immunodiffusion.
In a preferred embodiment of the present invention, the well-known
Western Blot Analysis method is employed for anti-HIV antibody detection. This
technique has been found to be the most reliable currently available method for
detecting HIV antibodies in the urine of mammals. The technique generally
comprises separating proteins (in this case, HIV proteins) by gel
electrophoresis on the basis of molecular weight, transferring the separated
proteins to a suitable solid support, (such as a nitrocellulose filter or
alternatively, a nylon filter), and incubating the serum (or urine) of an
HIV-infected individual with the separated proteins. This causes specific HIV
antibodies present in the serum (or urine) to bend to their respective
proteins. HIV antibodies are then detected using labeled anti-human HIV
antibodies. This method of detecting antibodies to HIV is preferred due to its
sensitivity and the fact that specific antibodies to viral proteins are
examined. The incidence of false positive results which are inherent when
employing ELISA assays is substantially reduced by using Western Blot Analysis.
An alternative embodiment of the present invention utilizes on Enzyme
Linked Immunosorbent Assay (ELISA) as a means for detecting antibodies specific
for HIV. ELISA assays for the detection of antibodies to the HIV virus can
either be competitive or non-competitive (as described by E. Engvall in Methods
in Enzymology. 70: 419439, incorporated herein by reference).
ELISA's are immunoassays used (in this case) to quantitate the amount of
antibodies present in a sample to be analyzed. The assays employ a chromogen (a
color producing substance) for detecting the antibody: antigen complex formed.
Antibodies used in ELISA are covalently coupled to these chromogens, such as
orthophenylenediamine or orthodianisidine, the former producing a
tangerine-colored product in the presence of a peroxide. These colors absorb
light at specific wavelengths (ortho-phenylenediamine at 492nm and
ortho-dianisidine at 400nm) and are detected and quantitated using a
spectrophotometer.
Non-competitive ELISA tests employ an immobilized antigen in order to
capture any antibodies and an enzyme-labeled second antibody directed against
the
63
5
species in which the test antibody has been elicited. If for example, human
antibodies are being measured, then goat or rabbit anti-human antibodies are
the labeled, second antibody. Competitive ELISA's comprise a reaction in which
unlabeled (the biological sample to be tested) and enzyme-labeled antibodies
compete for a limited, known amount of antigen. In this case, the reaction is
performed until equilibrium is reached, and the concentration of unknown
antibody is inversely proportional to the amount of bound, enzyme-labeled
antibodies. For example, if there are no antibodies in the unknown sample, all
of the labeled antibodies will bind to the antigen, and a high value, indicated
by an increased color, will be obtained when measuring the amount of enzyme
label present.
Commercially-available ELISA assay kits which can be used in practicing
the alternative embodiment of the present invention are available from Abbott
Labs (Chicago, Ill.) under Catalog Number 1037 and as ENVACOR (Human T Cell
Lymphotropic Virus III, EIA, Catalogue No. 2791-22, Abbott International
Diagnostics, Weisbaden, West Germany). The 1037 assay utilizes a
non-competitive ELISA (as described in Example 3 below) whereas the ENVACOR
test uses a competitive ELISA (as described in Example 2 below). These kits
contain the components listed in Examples 2 and 3 below and are offered for use
in assaying serum (not urine) for antibodies to HIV.
One important advantage of the method of the present invention is that
the biological sample to be analyzed (urine) can be easily obtained by
non-invasive techniques, and therefore, the risk of transmitting an HIV
infection to laboratory and health care personnel (e.g. through accidental
puncture with a contaminated needle) is essentially eliminated.
The method of the present invention can be carried out rapidly. In
cases where the urine is concentrated, the speed of carrying out the method of
the present invention limited only by the time it takes to concentrate the
urine sample to be tested. In broad terms, this can be done in a period of
time of less than 90 minutes, when the samples are to be concentrated 200 fold
Concentrating the urine 20-40 fold can be accomplished in less than 60 minutes.
The sample can then be analyzed using the commercially-available HIV Serum
testing kits.
The invention is described further below in specific working examples
which are intended to illustrate the invention without limiting its scope.
EXAMPLE 1
Urine Concentration
Serum samples and 60-100 ml of urine were collected (and numerically
coded for patient confidentiality) from the following groups of patients: 28
AIDS-associated Kaposi's sarcoma (AIDS-KS) patients; 21 ARC patients; 48
asymptomatic HIV-infected high risk individuals including (37 homosexual men, 5
female intravenous drug users and 1 female transfusion recipient); and 16
patients suffering from AIDS-related opportunistic infections (AIDS-OI), such
as Pneumocystis Carinii and cytomegalovirus (Table I contains specific patient
identification and assay results) In addition, 17 non-AIDS disease patients'
urine and serum, including patients diagnosed as having cirrhosis and hepatoma
(1), hepatitis (3). Lupus (3), glomerulonephritis (3), nephrotic
6
syndrome (1), Lupus-nephritis (1), heart failure (1), Lepromatous leprosy (1),
tuberculosis (2) and DM nephropathy (1), plus samples from 30 apparently
normal, health heterosexuals were obtained. The urine samples obtained from
all of the normal controls and AIDS patients' displayed essentially normal
values for proteins, when assayed using standard urinalysis techniques well
known in the art. Proteinuria (the presence of abnormally high concentrations
of protein in the urine) is routinely detected using a "dipstick" that
registers the presence of high (over 150 mg) concentrations of urinary protein.
This is indicated by a color change on the dipstick, which is then compared to
standards for quantitation.
The collected urine specimens were centrifuged in conical tubes at 1500
RPM for 15 minutes at room temperature. The supernatant was decanted and saved
and the pellet was discarded.
The supernatant obtained above was concentrated between 20 and 200 fold
in relation to the volume of initial urine sample collected from the subject to
be tested using a Minicon B15 concentrator with a 60,000 dalton membrane
(Amicon, Danvers, MA). The concentrator operates by retaining any substance
greater than 60,000 daltons molecular weight on the membrane filter, while the
solution is evaporated. This takes approximately one hour. (To concentrate
the urinary volume 200 times (200X) from an initial or starting volume i.e. to
1/200 of its starting volume using the mincon apparatus takes approximately 90
minutes). The concentrated urine sample can be stored at 4 deg.C for up to 30
days before use, or used immediately for assay as detailed in Examples 2-5
below. For Western Blot analysis or immunodiffusion studies, sample volumes are
preferably further concentrated 200 fold i.e. to 1/200 of the starting volume.
The amount each sample was concentrated is listed in Tables 1, 2 and 4 below.
EXAMPLE 2
Elisa assay of Concentrated Urine Samples
The serum and concentrated urine samples collected in Example I were
assayed for the presence of antibodies to ENV and CORE HIV antigens as
described (Allain, J. P. et al, Lancet I: 1233-1236, 1986, incorporated by
reference )using an Abbott Envacore assay kit. This is a competitive ELISA in
which known mounts of HIV proteins and antibodies are added together with the
sample to be tested. The presence of antibodies in the sample is indicated by
a reduction in the binding of the control antisera with the control antibody.
The manufacturer's instructions were followed exactly as provided. In
addition, blood samples were collected and analyzed simultaneously.
The kit contains a specimen diluent (containing bovine and goat sera
and 0.1% sodium azide), enzyme-conjugated polyclonal antibody to HIV envelope
and core proteins, beads coated with recombinant gp41 or gp24 HIV proteins,
orthophenylenediamene (OPD) substrate, a positive and a negative HIV antibody
control and instructions for use.
Fifty microliters of the specimen to be tested, or control, were
incubated with 20 microliters of diluent supplied by the manufacturer and 200
microliters if enzyme-conjugated polyclonal human antibody to HIV envelope or
core proteins. Beads coated with recombinant gp41 or p24 HIV proteins were
added to separate wells containing either the HIV ENV or CORE anti-
64
5,122,446
7
bodies. After 16 to 22 hours incubation at room temperature, the beads were
washed and transferred to appropriate reaction tubes. Three hundred
microliters of ortho-phenylenediamine substrate was then added to each tube and
the reaction allowed to proceed for 30 minutes before addition of 1 ml 1N H2SO4
to stop the reaction. Absorbance values of the solution were measured at 492
nm using a spectrophotometer sold by Abbott Labs under the name Quantum II,
number 3303-11. A positive result for the presence of urine or serum
antibodies
8
to either the HIV p24 or gp41 proteins was defined as any specimen
with absorbance values equal to or less than 0.5 times the sum of the optical
density (O.D.) of mean negative control (provided by the manufacturer) optical
density (O.D.) plus the O.D. of the mean positive control (provided by the
manufacturer) as measured in above.
A tabular identification of the patients screened with the present
invention and the assay results are presented in Tables 1 and 2 and summarized
in Table 3.
TABLE 1
--------------------------------------------------------------------------------
THE ANTIBODIES TO HIV IN THE SERUM AND URINE OF
AIDS-KS, AIDS-OI, ARC GROUPS, AND HR GROUPS
-----------------------------------------------
PRESENCE IN PRESENCE IN
SERUM OF URINE OF
PATIENT ------------ ------------
NUMBER SEX CODE CONCENTRATION ENV CORE ENV CORE
--------------------------------------------------------------------------------
AIDS-KS
--------
1 M 1029 47X + + + -
2 M 993 100X + + - -
3 M 716 42X + - + -
4 M 818 33X + - + -
5 M 871 67X + + + -
6 M 894 33X + + + -
7 M 953 40X + - + -
8 M 5009 34X + + + -
9 M 5018 45X + - + -
10 M 5057 39X + + + -
11 M 5062 42X + + - -
12 M 1028 48X - + + -
13 M 828 42X + + + -
14 M 5061 37X + - + -
15 M 5059 35X + - - -
16 M 276 20X + + + -
17 M 1056 40X-200X + + - -
18 M 5098 40X + + - -
19 M 5013 35-200X + - - -
20 M 5050 20X + + + -
21 M 5074 42X + - - -
22 M 5080 42X + + - -
23 M 5051 40X + + - -
24 M 5083 41X + - + -
25 M 5097 40X + - + -
26 M 1083 42X + - + -
27 M 1081 40X + - - -
28 M 1084 40X + + - -
ARC Patients
------------
29 M 1022 56-200X + + - -
30 M 857 62-200X + + - -
31 M 920 47X + - + -
32 M 598 35X + + - -
33 M 1002 40X + + + -
34 M 1015 35X + - + -
35 M 1016 35X + + - -
36 M 1021 50X + + + -
37 M 949 40X + + + -
38 M 901 100X + - + -
39 M 898 100X + - + -
40 M 956 100X + + + -
41 M 839 47X + - + -
42 M 1039 41X + - + -
43 M HEN03 41X + - + -
44 M HEN04 42X + - + -
45 M HEN05 42X + - + -
46 M HEN06 42X + - + -
47 M HEN07 41X + - + -
48 M HEN08 42X + + + -
49 M HEN09 40X + + + -
--------------------------------------------------------------------------------
ENV = Antibody to HIV envelope [anti-env (gp41)]
Core = Antibody to HIV core [anti-gag (p24)]
TABLE 2
--------------------------------------------------------------------------------
ANTIBODIES TO HIV IN THE SERUM AND URINE OF THE HIGH-RISK GROUP AND AIDS-OI
---------------------------------------------------------------------------
SERUM URINE
PATIENT FOLD ------------ ------------
NUMBER SEX CODE CONCENTRATION ENV CORE ENV CORE
--------------------------------------------------------------------------------
HIGH-RISK
---------
50 M 1024 37-200x + - - -
65
5,122,446
9
TABLE 2-continued
--------------------------------------------------------------------------------
Antibodies to HIV in the serum and urine of the High-Risk group and AIDS-O1
---------------------------------------------------------------------------
Serum Urine
Patient Fold -------------- --------------
Number Sex Code Concentration ENV Core ENV Core
--------------------------------------------------------------------------------
51 M 1060 30x + - - -
52 M 1059 32x - - - -
53 M 1062 40x + - - -
54 M 1057 28x + + - -
55 M 1055 32x - - - -
56 M 1058 40x + + + -
57 M 1052 37x - - - -
58 M A2-014 50x + + + -
59 M A1-078 33x + + + -
60 M 0697550 40x + - + -
61 M 0820835 44x + + + -
62 M 1061 41x + + + -
63 M A1-029 40x + + - -
64 M A1-136 41x + + - -
65 M A1-138 39x + - - -
66 M A1-044 41x + + + -
67 M A1-045 40x + + + -
68 M A1-173 42x + + + -
69 M A1-003 42x + + + -
70 F 1065754 40x + + + -
71 M 0471024 40x + + + -
72 M A2-132 40x + + + -
73 M A2-115 40x + + + -
74 M A1-060 40x + + - -
75 M A1-178 43x + + - -
76 M A2-090 40x + + + -
77 M A2-097 40x + + + -
78 M A2-137 40x + + + -
79 F U16 41x + - + -
80 M 1091 42x - - - -
81 M 1093 42x + + + -
82 M 1110 42x + + + -
83 M 1121 40x + - + -
84 M 1080 41x - - - -
85 M 1070 40x + - + -
86 M 1111 40x + + - -
87 M 1107 40x + + + +
88 M 1116 49x + + + +
89 F 941151 46x + - + -
90 M 1098 41x + - + -
91 F U17 40x + - - -
92 F U19 42x + + + +
93 F U20 42x + + + -
94 M A2-036 42x + - + -
95 M A1-082 40x + + - -
96 M A1-177 42x + - - -
97 M 1073 40x + - - -
PATIENTS WITH
OPPORTUNISTIC
INFECTIONS
-------------
98 M 644 40x + + + -
99 M 1063 23x + - - -
100 M 155 43x + + - -
101 M 156 40x ./ / + -
102 M 157 40x + + - -
103 M 1108 40x + - + -
104 M 1112 40x + - - -
105 M 1113 42x + + - -
106 M 159 41x + - + -
107 F 160 41x + - + -
108 M 161 40x + + + -
109 F 162 42x + + + -
110 M 164 44x + + + -
111 M 165 40x + - + -
112 M 166 41x + - + -
113 M 473 35x + - + -
--------------------------------------------------------------------------------
. / = not done
TABLE 3
--------------------------------------------------------------------------------
Summary of ELISA assay for HIV ENV antibodies in the Concentrated Urine Samples
-------------------------------------------------------------------------------
SERUM/URINE SERUM/URINE SERUM/URINE % SERUM/URINE
PATIENT TYPE + - + + - - + +
--------------------------------------------------------------------------------
AIDS/KS 8 20 0 20/28 = 71.4%
ARC 4 17 0 17/21 = 81.0%
HIGH RISK 13 30 5 30/3 = 69.7%
10
66
5,122,446
11
TABLE 3
--------------------------------------------------------------------------------
Summary of ELISA assay for HIV ENV antibodies in the Concentrated Urine Samples
-------------------------------------------------------------------------------
SERUM/URINE SERUM/URINE SERUM/URINE % SERUM/URINE
PATIENT TYPE + - + + - - + +
--------------------------------------------------------------------------------
OPPORTUNISTIC 5 11 0 11/16 = 68.8%
INFECTION -- -- -- --------------
TOTAL 30 78 5 78/113 = 72/2%
--------------------------------------------------------------------------------
Referring to Tables 1 and 2, it can be seen that 71.4% of the
AIDS-induced Kaposi's sarcoma patients, 81% of the ARC patients, 69.7% of the
patients in the high risk group and 68.8% of patients suffering from
opportunistic infections had antibodies to the ENV protein (gp41) of HIV
present in their urine and that such antibodies were detected using the methods
of the present invention. The total percentage of patients with detectable HIV
ENV antibodies was 72.2%. The core antigen was only detected in the urine of 6
individuals, those being in the asymptomatic infected high risk group. All of
the non-AIDS disease patients and the normal, healthy heterosexuals' urine and
serum were negative for antibodies to both HIV proteins.
EXAMPLE 3
A number of the urine samples analyzed in Example 1 were re-examined
using two commercially-available non-competitive HIV ELISA detection kits. KIT
I (HTLV III EIA kit, Lot No. 1590HR00, Abbott Laboratories, Chicago, Ill.) is
an FDA-approved clinical diagnostic kit., Kit II 9EIA Clinical Diagnostic Kit,
Catalog No. 1037, Abbott Laboratories, Chicago, IL) is intended for
investigative use only.
Each of the kits contain HIV antigen-coated beads, goat anti-human
antibody conjugated to horseradish peroxidase, a positive control, a negative
control, speci-
12
men diluent containing bovine and goat sera, OPD and OPD diluent containing
citrate-phosphate buffer and 0.02% hydrogen peroxide, reactron trays, assay
tubes and instructions for use.
The assay for the presence of the urine of a patient to be tested of
antibodies to HIV is performed as follows. 10 microliters of control or diluted
specimen is dispensed into preselected wells of the reaction tray. Each well
can hold up to 400 microliters of fluid. Two hundred microliters of specimen
diluent and one bead are added per well. The reactions are incubated for about
1 hour at 40 degrees C. Thereafter, the supernatant (i.e. liquid) is discarded
and the bead washed three times with 4 to 6 ml of distilled or deionized water.
Two hundred microliters of labeled goat-anti-human antibodies are then added
and incubated at 40 degrees C for about 2 hours. The supernatant is removed
and the bead is washed as above. The bead is transferred to an assay tube, 300
microliters of OPD substrate solution is added, and the solution is incubated
for about 30 minutes. 1 ml on IN sulfuric acid is added and the absorbance of
the solution is determined at 492 nm in a standard Abbott Labs
spectrophotometer (Model 3303-11 available from Abbott Labs). A positive value
is indicated if a sample is within the range of 0.5 to 1.5 times the positive
control mean.
The results of these assays are presented below in Table 4.
TABLE 4
--------------------------------------------------------------------------------
KIT I KIT II
Patient Fold Urine HIV Urine HIV Envelope
Number Patient Concentration Antibody Antibody
--------------------------------------------------------------------------------
AID-KS
------
1 1029 47 X + +
3 716 42 X + +
4 818 33 X + +
5 871 67 X - +
6 894 33 X + +
7 953 40 X - +
8 5009 34 X + +
9 5018 45 X - +
10 5057 39 X - +
11 5062 42 X - -
12 1028 48 X + +
13 828 42 X - +
14 5061 37 X + +
16 276 20 X - +
17 1056 40 X-200 X - +
19 5013 35 X-200 X - -
20 5050 20 X - +
21 5074 42 X + +
22 5080 42 X - -
ARC
-----
30 857 62 X-200 X - -
31 920 47 X - +
32 598 35 X - -
33 1002 40 X + +
34 1015 35 X - +
35 1016 35 X - -
36 949 40 X - +
40 956 100 X - +
41 839 47 X - +
HIGH-RISK
---------
52 1059 32 X - -
53 1067 40 X - -
67
5,122,446
13
TABLE 4-continued
--------------------------------------------------------------------------------
KIT URINE
PATIENT FOLD URINE HIV URINE HIV ENVELOPE
NUMBER PATENT CONCENTRATION ANTIBODY ANTIBODY
--------------------------------------------------------------------------------
54 1057 28x - -
55 1055 32x - -
57 1052 37x - -
58 A2-014 50x - +
59 A1-078 33x - +
60 0697550 40x - +
61 0820535 44x - +
62 1061 41x - +
63 A1-029 40x - -
64 A1-136 41x - -
65 A1-138 39x - -
66 A1-044 41x - +
67 A1-045 40x - +
68 A1-173 42x - +
69 A1-003 42x + +
PATIENTS WITH
OPPORTUNISTIC
INFECTION
-------------
98 644 40X + +
99 1063 23X - -
100 155 43X - -
101 156 40X + +
102 157 40X - -
--------------------------------------------------------------------------------
The results presented in Table 4 demonstrate that urine antibodies to
HIV are more readily detectable when employing a more sensitive ELISA assay kit
(Kit II). This is the same as for serum antibodies. A limited number of these
samples were further analyzed using the Western Blot technique as described
below in Example 4.
EXAMPLE 4
Western Blot analysis of Concentrated Urine Samples
Western Blot analysis for the presence of antibodies to HIV was
performed on the urine and serum samples collected from 59 HIV sero-positive
(including 18 AIDS-KS, 27 High Risk individuals, 6 ARC and 8 O.I. patients
selected from those reported in Tables 1 and 2 above) and 30 non-AIDS disease
patients. A commercially-available kit (Biotech/DuPont HTLV-III Western Blot
Kit, available from E. I. DuPont De Nemours and Co., Inc., Wilmington, Del.)
was used in making the analysis. The kit contains precut nitrocellulose
membrane strips with immobilized viral antigens that have been separated by
sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and
electroblotted onto the membrane; control sera, including a negative control, a
weak positive control and a strong positive control; blotting buffer (Tris
buffered saline with 5% nonfat dry milk and also containing heat inactivated
normal goat serum); wash buffer (Tris buffered saline containing tween-20
detergent); biotinylated goat anti-human IgG; avidin-horseradish peroxidase;
4-chloro-1-naphthol in solution; hydrogen peroxide; and an incubation tray.
The kit was used exactly according to the manufacturer's instructions as
described below.
The assay comprises soaking the strips in 2 mls of wash buffer in a
well of the wash tray for 30 minutes at
14
room temperature; the liquid is then drained off and discarded. The strips are
then washed with 2 mls of blotting buffer for 5-10 minutes at room temperature.
Twenty microliters of a 200 fold concentrated urine sample are added to the
wells containing the strips and blotting buffer and the reactant incubated
overnight at room temperature. Thereafter, the mixture in the wells is
aspirated and discarded and the wells are washed once with 2 mls of wash
buffer. Two additional 2 ml washes (with wash buffer) are performed at room
temperature, allowing 5 minutes soaking between each wash and discarding the
wash afterwards.
The nitrocellulose strips are developed as follows. Two mls of
biotinylated goat anti-human IgG are added to each well and allowed to incubate
for 60 minutes at room temperature on a rocking or rotary apparatus. The
strips are then washed with 2 mls of wash buffer per strip for 5 minutes; this
step is then repeated 3 additional times at room temperature, discarding the
wash after each use. Two mls per strip of avidin-horseradish peroxidase are
added and incubated for 60 minutes at room temperature on a rocking or rotary
apparatus. The strips are washed 3 times as above. Two mls of a 50:50 mixture
of 4-chloro-1-naphthol and hydrogen peroxide are then added and allowed to
incubate for 10-15 minutes or until the color develops at room temperature.
The presence of color on the strip indicates that the strip has been exposed to
a biological fluid (i.e. urine) containing antibodies to HIV.
The strips are scored for the presence of antibodies to HIV as negative
(-) i.e. no antibodies to HIV detected, weakly positive (plus or minus) or
strongly positive (+) using the controls supplied by the manufacturer as
references. The results are presented in Table 5 and FIG. 1.
TABLE 5
--------------------------------------------------------------------------------
RESULTS OF WESTERN BLOT ANALYSIS OF CONCENTRATED URINE SAMPLES
--------------------------------------------------------------
PATIENT
NUMBER CODE p17 p24 p31 p41 p51 p55 p66 p110/p120 p160
--------------------------------------------------------------------------------
AID-KS
------
1 1029 - - - + - - - + +
7 953 - - + +- - - - + +
8 5009 - + + - + + - + +
--------------------------------------------------------------------------------
68
TABLE 5
--------------------------------------------------------------------------------
RESULTS OF WESTERN BLOT ANALYSIS OF CONCENTRATED URINE SAMPLES
--------------------------------------------------------------
PATIENT
NUMBER CODE p17 p24 p31 p41 p51 p55 p66 p110/p120 p160
--------------------------------------------------------------------------------
AID-KS
------
9 5018 - - + + + - + + +
10 5057 - + + - - + - + +
12 1028 - - - + - - - + +
13 828 - - + - + + + + +
14 5061 - - + +- - - - + +
16 276 - + + + + + + + +
17 1056 - - - - +- +- +- + +
18 5098 - + - + - + - + +
21 5074 - - + - + - + + -
22 5080 - - - - - - - - -
23 5051 - - - - - - - - +
24 5083 - - - + - - - + +
25 5097 - + + + + + + + +
26 1083 - - - - - - - - -
27 1081 - - - + - + - + +
ARC
----
33 1002 - + + + + + + + +
40 956 - - + + + - + + +
42 1039 - + + + + + + + +
43 Hen03 - - - + - - + - -
44 Hen04 - - + + + - + + +
45 Hen05 + - + + + + + + +
Asymptomatic
High-Risk
homosexuals
------------
54 1057 - + + + - - + + +
58 A2-014 - +- - +- - +- +- + +
59 A1-078 - + + +- - + + + +
60 697550 - - + + + + + + +
61 820835 - + + o + + + + +
62 1061 - - - - - - - + +
66 A1-044 - - + - - - - + +
67 A1-045 - + + + + + + + +
68 A1-173 + + + + + + + + +
69 A1-003 + + + + + + + + +
70 1065754 + + + + + + + + +
71 471024 - + - - - + + + +
72 A2-132 + + + + + + + + +
74 A1-060 - - - - - - - - +
75 A1-178 - + + + + + + + +
76 A2-090 - + - + - + + + +
77 A2-097 + + + + + + + + +
78 A2-137 + + + + + + + + +
79 U16 + + + + + + + + +
81 1093 - + - + - + + + +
82 1110 - + + + + + + + +
83 1121 - - - o - - - - +
85 1070 + + - + - + + + +
86 1111 - o + + + - + - +
87 1107 + + + + + + + + +
88 1116 + + + + + + + + +
90 1098 + + + + + + + + +
Opportunistic
Infections
-------------
98 644 - - - - - - - - -
99 1063 - - - - - - - - -
100 156 - - - + - - - + +
102 157 - o - - - - - - +
103 1108 + - + + + + + + +
104 1112 - - - + + - + + +
105 1113 - - + + + + + + +
113 473 + + + + + + + + +
--------------------------------------------------------------------------------
o = indeterminate results
TABLE 6
--------------------------------------------------------------------------------
COMPARISON OF RESULTS OF WESTERN BLOT ANALYSIS
OF CONCENTRATED URINE AND SERUM SAMPLES
----------------------------------------------
HIV AIDS-KS ARC HIGH-RISK O.I.
-------------- -------------- -------------- --------------
PROTEIN SERUM URINE SERUM URINE SERUM URINE SERUM URINE
--------------------------------------------------------------------------------
p17 18/18* 1/18 2/6 1/6 26/27 11/27 ? ?
p24 18/18 5/18 6/6 2/6 25/27 23/27 ? ?
p31 18/18 9/18 6/6 5/6 26/27 19/27 8/8 ?
p41 18/18 11/18 6/6 6/6 26/27 23/27 8/8 ?
p51 18/18 8/18 6/6 5/6 27/27 16/27 8/8 4/8
p55 18/18 7/18 6/6 3/6 27/27 21/27 8/8 ?
--------------------------------------------------------------------------------
69
5,122,446
17
TABLE 6-continued
-------------------------------------------------------------------------------------------
Comparison of Results of Western Blot analysis
of concentrated urine and serum samples
----------------------------------------------
HIV AIDS-KS ARC HIGH-RISK O.L.
--------------- --------------- --------------- ---------------
Protein Serum Urine Serum Urine Serum Urine Serum Urine
-------------------------------------------------------------------------------------------
p66 18/18 7/18 6/6 6/6 27/27 23/27 8/8 4/8
p120 18/18 15/18 6/6 5/6 27/27 24/27 8/8 ?
p160 18/18 16/18 6/6 5/6 27/27 26/27 8/8 8/8
-------------------------------------------------------------------------------------------
*18 positives per 18 patients tested.
Referring to Table 5, p17 is a protein component produced upon cleavage
of p55 to p(greater than or equal to), p(greater than or equal to) is the viral
core protein, p31 is the viral endonuclease, p41 is the mature envelope protein,
p51 and p66 are components of the viral reverse transcriptase, p55 is a
precursor to the viral core protein and p160 is a precursor to the envelope
protein; p110/120 is a mixture of 2 proteins which co-migrate in this gel
system: p120 is a protein component produced upon processing of the p160 protein
to gp41; p110 is a protein component of the virus whose function is currently
unknown.
An example of a typical a Western Blot analysis of the concentrated
urine and serum samples obtained by practicing the method of the present
invention is shown in FIG. 1. Referring to FIG. 1, lane 22 is the negative
control, lane 23 the weakly positive control and lane 24 the strong positive
control; the numbers to the right represent the various HIV proteins discussed
above. Above lines 12-16 and 18-21 are the corresponding patients as referred
to in Table 5 above. For example, urine obtained from patient No. 956 (FIG. 1,
lane 16) suffering from ARC contained antibodies directed against all of the
HIV proteins except p24 (Core) and p17. The significance of this finding is
presently unknown.
As can be seen from the data in Table 5 and summarized in Table 6,
antibodies to HIV antigens can be readily found (when present) in the
concentrated urine samples using this more sensitive technique. In particular,
antibodies to p160 in the concentrated urine could be detected in 55 out of the
59 HIV-positive individuals tested (93.2%). These results also demonstrate
that the use of concentrated urine in this preferred embodiment of the present
invention does not lead to false positives, since specific viral proteins are
identified.
EXAMPLE 4
Double-Diffusion Gradient Immunoelectrophoretic
Analysis of HIV Antibody-Containing Urine Samples
Concentrated urine sample No. 956 (Table I), which tested positive for
HIV antigens using Western Blot analyses and antibodies directed against the
envelope protein using ELISA, was further concentrated 200 fold and analyzed by
double-diffusion gradient immuno-electrophoresis (DDG-IEP) as described by J. V.
Chuba in J.App. Biochem., 1:37-50, 1979, (incorporated by reference). Serum
samples from this patient were collected and analyzed in parallel to the patient
urine samples as a positive control.
Briefly, replicate samples of 3 microliters of urine and serum
respectively were electrophoresed in commercially prepared 0.5% agarose thin
layer gels (Paragon, Beckman Instruments Brea, Calif.). Troughs were placed at
right angles to the gels for the addition of antisera. Electrophoresis was
performed by subjecting the gels to 20 minutes of direct current on a slightly
modified Hyland Power Pack (Costa Mesa, Calif.) at the 40 mA setting. The
running buffer contained barbi-
18
tal buffer B-2 (0.075M, pH 8.6; containing 0.2% (w/v) sodium azide) mixed with
an equal volume of 3.0 mM aqueous calcium lactate solution.
After electrophoresis, the antisera troughs were completed by removing
the corresponding segment of the gel between the precut slits, and conventional
parallel trough immunodiffusion was performed. 7.5 microliters of anti-human
IgG. IgM and IgA (Behring Diagnostics, San Diego, CA) at a concentration of 5.5
micrograms per ml was added to the troughs and incubated for w0 minutes at room
temperature. Thereafter, the gels were stained and examined for the
development of percipitin lines. Anti-albumin antibodies were included in the
serum samples as a positive control.
As shown in FIG. 2, IgG (11) and IgA (13) immunoglobulins were
identified in the urine sample of patient No. 956. IgM, although present in the
serum did not appear in the urine (12). This is not surprising due to the
large size of this immunoglobulin (approximately 900,000 daltons).
Anti-albumin antibodies reacted with the serum samples, as expected (10),
forming a sharp percipitin line of identity (lane 1). These results confirm the
positive results obtained from the ELISA and Western Blot detection procedures,
and demonstrate that they were not due to artifacts caused by use of
concentrated urine samples.
EXAMPLE 5
Analysis of Unconcentrated Urine Samples for HIV
Antibodies by Western Blot and Elisa
In example 4 above, 20 microliters of each concentrated urine sample
tested was diluted with 2 ml (i.e. a 1:100 dilution) of the diluted wash buffer
provided by the manufacturer before conducting the Western Blot analysis. Using
unconcentrated urine, the present inventors have increased this recommended
urine volume to be tested to 2 ml and no diluted wash buffer was added. The
Western Blot assay was then performed exactly as described in Example 4 above.
These samples were then compared with 200-fold concentrated urine samples and
serum samples obtained from HIV sero-positive individuals.
For the ELISA assay, the manufacturer's suggested assay procedure,
usually used for serum analysis, requires 10 microliters of each serum specimen
to be diluted with 200 microliters of the specimen diluent provided in the EIA
kit. 10 microliters of this diluted specimen is then further diluted with 200
microliters of the specimen diluent provided in each well. This is a 400-fold
dilution of the sample.
The above procedure (designed for serum analysis) was adapted and
modified for evaluation of detectable antibodies to HIV in the unconcentrated
urine specimens. The recommended first step dilution of the urine specimen with
the specimen diluent was eliminated. Instead, the volumes of unconcentrated
urine samples
70
5,122,446
19
tested were increased to 150 microliters, to which 50 microliters of specimen
diluent was added. The assay was then performed exactly as described in
Example 3 above.
The results of these assays is shown in Table 7.
TABLE 7
--------------------------------------------------------------------------------
Presence of antibodies to HIV-1 seropositive individuals
as determined by ELISA and Western Blot tests.
--------------------------------------------------------
Catalog Total Number of reactive (%) by Western Blot
of # ELISA -------------------------------------------------------------------------------------------------
Samples Tested No. (%) p17 p24 p31 gp41 p51 p55 p66 gp120 gp160
-----------------------------------------------------------------------------------------------------------------------------
Serum 100 100(100) 87(87.0) 96(96.0) 97(97.0) 100(100) 98(98.0) 99(99.0) 99(99.0) 100(100) 100(100)
Conc. 100 91/91 24/87 52/96 61/97 69/100 57/98 50/99 71/99 96/100 100/100
Urine (100) (27.6) (53.1) (62.9) (69.0) (58.2) (50.5) (71.7) (96.0) 100
200 X
Unconc. 100 93/100 27/87 56/96 67/97 79/100 58/98 48/99 73/99 99/100 100/100
Urine (93.0) (31.0) (58.3) (69.1) (79.0) (58.2) (48.5) (73.7) (99.0) (100)
using
*modified
pressure
--------------------------------------------------------------------------------
*See Method
From the results presented in Table 7 above, it can be seen that
unconcentrated urine can be used in both the Western Blot and ELISA assays.
The above invention has been described in terms of preferred
embodiments. It would be obvious to those of ordinary skill in the art that
man additions, deletions and substitutions could be made without departing from
the spirit and scope of the invention, as claimed below.
What is claimed is:
1. A method for identifying the members of a human patient population
that have been infected with Human Immunodeficiency Virus (HIV) which comprises:
contacting a quantity of urine voided by a member of said patient
population with an immunoreagent specific for detecting the presence of said
urine of an antibody to at least one HIV protein, to form a complex,
detecting the presence of said complex after said contacting step to
obtain a result,
comparing said result with a standard result which has been obtained by
contacting with said immunoreagent urine of at least one human subject know to
be free of HIV infection.
2. A method for determining whether a human subject has been infected
with Human Immunodeficiency Virus (HIV) comprising the steps of:
obtaining a urine sample from said human,
assaying said sample by contacting at least an aliquot of said sample
with an immunoreagent specific for detecting the presence of an antibody to at
least one HIV protein in said sample to form a complex,
detecting said complex after said contacting step to obtain a result,
comparing said result of said assay with those of the same assay
performed with urine from at least one HIV-free control human subject.
3. A method for screening a human subject for exposure to Human
Immunodeficiency Virus (HIV) comprising the steps of:
obtaining a urine sample from said subject;
assaying said sample by contacting at least an aliquot of said sample
with an immunoreagent specific for detecting the presence of an antibody to at
least one HIV protein to form a complex,
detecting said complex, and
20
determining whether said subject has been exposed to HIV based on the
positive presence of at least one antibody of HIV in said sample.
4. A method for detecting the presence of antibodies to Human
Immunodeficiency Virus (HIV) in a human subject comprising the steps of:
obtaining a urine sample from said human subject;
assaying said sample for the presence of at least one antibody to at
least one HIV protein by contacting at least an aliquot of said sample with an
immunoreagent specific for detecting the presence of said antibody, said
protein being selected from the group consisting of p17, p24 and combinations
thereof.
5. The method of any one of claims 1-4 wherein said antibodies are
directed against Human Immunodeficiency Virus viral protein p24, and said
immunoreagent comprises an antigen immunochemically reactive with said
antibodies.
6. The method of any one of claims 1-4 wherein said antibodies are
directed against HIV viral protein gp 160 and said immunoreagent comprises and
antigen immunochemically reactive with said antibodies.
7. The method of any one of claims 1-4 wherein said antibodies are
directed against HIV viral protein gp120 and said immunoreagent comprises an
antigen immunochemically reactive with said antibodies.
8. The method of any one of claims 1-4 wherein said urine sample is
less than one week old.
9. The method of any one of claims 1-4 wherein said immunoreagent
comprises an antigen immunochemically reactive with an antibody raised against
an HIV viral protein and specific for detecting antibodies to said protein.
10. The method of claim 9 wherein said antibodies are detected using
an enzyme-linked immunosorbent assay.
11. The method of claim 9 wherein said antibodies are detected using
Western Blot.
12. The method of claim 9 wherein said antibodies are detected using
immunodiffusion.
13. The method of claim 9 wherein said antibodies are members of the
group consisting of antibodies directed against HIV viral protein gp41
(anti-gp41), antibodies directed against HIV viral protein p24 (anti-p24) and
combinations thereof and said specific immunoreagent respectively comprises and
antigen selected from the group consisting of antigens immunochemically
reactive with anti-gp41, antigens immunochemically reactive with anti-p24 and
combinations of said antigens.
71
APPENDIX II
Brief Statement Relating to Rights To Licensed Patents
------------------------------------------------------
1. Patent Nos. 4,865,966 and 5,122,446 relate to a urine test for
antibodies to HIV. The three inventors, Drs. Friedman-Kien, Cao and Borkowsky,
are faculty members and employees of New York University (NYU). All have
assigned their rights in the inventions to NYU.
2. Abbott Laboratories has advised NYU that it claims "rights under
its agreements with Dr. Friedman-Kien." Abbott's claim is described in a
letter to NYU attached hereto. Without attempting to recite the full history
of the invention, Abbott appears to be referring to the following:
One of the inventors, Dr. Friedman-Kien, conducted research for Abbott
pursuant to an agreement signed by Abbott and Dr. Friedman-Kien which provided
that inventions made by Dr. Friedman-Kien as a result of conducting the study
were to be the sole property of Abbott. The protocol which defined the study
referred to tests on blood supplies with Abbott test kits for the presence of
HIV antigens. NYU received a payment for that work. Dr. Friedman-Kien signed a
second agreement on the same subject, but it was returned to Abbott by NYU with
a letter rejecting it and proposing revisions which were never agreed. (Abbott
also proposed an agreement to Dr. Friedman-Kien to test blood for the presence
of HIV antibodies, but that agreement was not signed.)
3. NYU maintains that it is the rightful owner of the patents, because
the inventions were assigned to NYU by the three
72
employee-inventors and NYU believes that the invention (relating ???? ??????
antibodies) was outside the scope of the Abbott agreement (relating to blood
antigens) with Dr. Friedman-Kien, among other reasons.
73
PATTERSON, BELKNAAP, WEBB & TYLER
A PROFESSIONAL CORPORATION
30 ROCKEFELLER PLAZA
NEW YORK, N.Y. 10110
(212) ???-????
October 30, 1989
Dr. A.E. Friedman-Kien
New York University Medical Center
Department of Microbiology
550 First Avenue
New York, NY 10016
New York Medical Center
School of Medicine
Office of Grants Administration
& Institutional Studies
550 First Avenue
New York, NY 10016
Re: Abbott Laboratories/
Method for Detecting Antibodies
to Human Immunodeficiency Virus
Dear Sirs:
On August 4, 1986, Dr. Friedman-Kien signed a contract with Abbott
Laboratories ("Abbott") pursuant to which he was retained by Abbott to conduct
a clinical study in relation to HTLV antigens. Pursuant to the terms of this
contact, Dr. Friedman-Kien, agreed, inter alia, as follows:
Any information, inventions or discoveries (whether patentable or not),
innovations, suggestions, ideas and reports, made or developed by you as a
result of conducting the Study shall be promptly disclosed to Abbott and shall
be the sold property of Abbott. You agree, upon Abbott's request and at
Abbott's expense, to execute such documents and to take such other actions as
Abbott deems necessary or appropriate to obtain patents in Abbott's name
covering any of the foregoing.
74
-2-
A further agreement, containing the same provision quoted above, was signed Dr.
Friedman-Kien on September 29, 1986.
We understand that Dr. Friedman-Kien has now obtained a patent for
inventions and discoveries that may be governed by the agreements referenced
above or are otherwise the result of work product to which Abbott has rights. We
further understand that Dr. Friedman-Kien and/or New York University have been
engaged in discussions with other parties concerning the possible commercial
development of inventions and discoveries governed by these agreements.
Abbott intends vigorously to protect any and all of its rights under
its agreements with Dr. Friedman-Kien.
In order for Abbott to be able to protect its rights under these
agreements, we ask you immediately to inform the undersigned, in writing, of
any and all agreements, or discussions that may lead to an agreement, involving
Dr. Friedman-Kien and/or New York University that relate in any way to Dr.
Friedman-Kien's research concerning HTLV antigens. We further ask that you
provide a copy of this letter to any and all persons or entities with whom Dr.
Friedman-Kien and/or New York University have entered into such agreements, so
that such persons or entities will be on notice of Abbott's rights concerning
Dr. Friedman-Kien's research.
Very truly yours,
/s/ FREDERICK T. DAVIS
-----------------------------
Frederick T. Davis
75
ABBOTT
Diagnostics Division
Abbott Laboratories
Abbott Park, Illinois 60064
July 14, 1986
Dr. A.E. Friedman Kien.
New York University Medical Center
Dept. of Microbiology
Medical Science Building - Rm 272
550 First Avenue
New York, NY 10076
Dear Dr. Friedman - Kien:
Abbott Laboratories ("Abbott") desires to retain you to conduct a clinical
study (Study") in relation to Abbott HTLV III Antigen EIA an the following
terms and conditions:
1. You agree to conduct the Study within the term of this Agreement, in
strict adherence to one or more protocols and related information to
be provided to you by Abbott. Abbott will provide you with a
sufficient quantity of Abbott HTLV III Antigen EIA to conduct the
Study, and may also, at its option, provide you with certain-other
Materials to be utilized in the Study. All such protocols,
information, Abbott HTLV III Antigen EIA kits, and other "materials"
shall remain Abbott's sole property. and you agree not to use
Materials for any purpose other than the study or give Materials to-
any third party without Abbott's prior written permission. You
further agree that you will assert no claim, patent or otherwise, to
the Materials, their use. or manufacture. Your Abbott contact will be
Sally Hojvat, of Abbott's Diagnostic Division, or whomever Abbott may
designate.
2. Within thirty (30) days following completion of the Study, you will
furnish Abbott with a written report, in such detail as Abbott may
specify, setting forth the results of the Study, and including all of
the data generated by the Study. Such report and data shall be the
sole property of Abbott.
3. It is understood and agreed that Abbott, in contracting for your
services hereunder, is contracting for the services of Dr. Friedman -
Kien, who will be personally responsible for your activities under this
Agreement. If such personal services are not available for any reason,
Abbott may terminate this Agreement immediately without any further
liability.
76
Dr. Friedman - Kien
July 14, 1986
Page Two
4. During the course of the Study, you agree that you will provide assay
results and patient histories where possible on the number of
specimens previously agreed to which will be assayed as per the
protocol These specimens will represent surplus specimens only, from
patients or subjects... in an ongoing program. Specimens must not be
taken specifically for the purpose of evaluating the Abbott HTLV III
Antigen EIA
5. At full consideration for your services hereunder and for your
agreement to the terms and conditions hereof, Abbott agrees to pay you
twenty dollars ($20.00) per specimen, not to exceed a total of fifteen
thousand dollars ($15,000.00), payable within forty-five (45) days
following completion of the Study.
6. During the term of this Agreement and thereafter, you will exercise
due care to prevent the unauthorized disclosure of Confidential
InformationConfidential Information shall include all information
concerning Abbott disclosed to you by Abbott or developed as a result
of conducting the Study and all Materials provided hereunder, except
any portion thereof which:
a. is known to you before receipt thereof under this Agreement,
as evidenced by your written records;
b. is disclosed to you after acceptance of this Agreement by a
third person who ha's a right to make such disclosure; or
c. is or becomes part of the public domain through no fault of
yours.
Further, during the term of this Agreement and thereafter. you
shall not use Confidential Information or Materials for any
purpose other than that indicated in this Agreement without
Abbott's prior written approval.
7. You agree not to use the name of Abbott in any publicity or
advertising without Abbott's prior written approval.
8. You will be free to publish the results of the Study, but with due
regard to the protection of Confidential Information. For that
purpose, you agree to provide Abbott with a copy of any proposed
publication relating to the results of the Study at least sixty (60)
days prior to submission thereof for publication. You further agree
not to submit any such proposed publication without Abbott's prior
written approval, which approval shall not be unreasonably withheld.
77
Dr. Friedman - Kien
July 14. 1986
Page Three
9. Any information, inventions or discoveries (whether patentable or
not), innovations, suggestions, ideas and reports, made or developed
by you as a result of conducting the Study shall be promptly disclosed
to Abbott. and shall be the sole property of Abbott. You agree, upon
Abbott's request and at Abbott's expense, to execute such suc
documents and to take such other actions as Abbott deems necessary or
appropriate to obtain patents in Abbott's name covering any of the
foregoing.
10. You agree not to disclose to Abbott any information which is
proprietary to a third party.
11. You warrant and represent that the terms of this Agreement are not
inconsistent with other contractual obligations you may have.
12. This Agreement shall be effective for the period beginning 07/21/86
and ending 12/21/86. Either party may terminate this Agreement
without cause upon thirty (30) days prior written notice to the other
party. Termination of this Agreement shall not affect any rights or
obligations which have accrued prior thereto.
13. Your status under this Agreement is that of an independent contractor,
and you have no authority to bind or act on behalf of Abbott. You may
not assign this Agreement to any third party, and any attempted
assignment shall be null and void.
14. This Agreement contains our entire understanding with respect to the
matters herein contained, and supersedes all previous agreements and
undertakings with respect thereto. This Agreement may be modified
only by written agreement signed by the parties.
15. This Agreement shall be governed by and construed In accordance with
the laws of the State of Illinois.
If the foregoing terms and conditions are acceptable, please sign and date the
enclosed copy of this letter In the space provided below, and return it to
Sally Hojvat.
Very truly yours,
ABBOTT LABORATORIES ACCEPTED:
/s/ WILLIAM STALL /s/ A.E. FRIEDMAN - KIEN
------------------------------- ------------------------------
William Stall Dr. Friedman - Kien
Manager, Hepatitis/AIDS
Quality and Scientific Support Date of Acceptance:
78
APPENDIX III
AGREEMENT
BETWEEN
HOME OFFICE REFERENCE LABORATORY
AND
NEW YORK UNIVERSITY
79
NYU/HORL
11 /I 6/89
I N D E X
Section 1. Definitions page 2
Section 2. Effective Date page 4
Section 3. Performance of the NYU Research Project page 4
Section 4. Funding of the NYU Research Project page 5
Section 5. Immunity from Claims of Infringement
by NYU page 6
Section 6. Payments for Immunity page 7
Section 7. Commercialization page 10
Section 8. Conversion to Non-Exclusive Immunity page 11
Section 9. Patents and Patent Applications page 12
Section 10. Infringement of NYU Patents
by Third Parties page 13
Section 11. Warranties by NYU page 16
Section 12. Tern, and Termination page 16
Section 13. Liability and Indemnification page 18
Section 14. Publication page 19
Section 15. Confidential Information page 20
Section 16. Representations and Covenants by Corporation page 21
Section 17. No Assignment page 22
Section 18. Use of Name page 22
Section 19. Miscellaneous page 23
80
AGREEMENT
This Agreement, made and effective as of ___________________, 198__,
("the Effective Date") is by and between:
NEW YORK UNIVERSITY a corporation organized and existing under the
laws of the State of New York and having a place of business at 550 First
Avenue, New York, New York 10016 (hereinafter "NYU")
AND
HOME OFFICE REFERENCE LABORATORY a corporation organized and existing
under the laws of the State of Delaware having its principal office at 10310
West 84th Terrace, Lenaxa, Kansas 66214 (hereinafter "HORL").
RECITALS
WHEREAS, Drs. Alvin Friedman-Kien, Yunzhen Cao, and William Borkowsky
of NYU have made certain inventions relating to methods and/or assays for the
detection of HIV antibodies in urine, which inventions have been assigned to
NYU, all as more particularly described in U.S. Patent no. 4,865,966, granted
on September 12, 1989, and a pending U.S. Patent Application no. 204,871 filed
on June 10, 1988 ("the C.I.P. Application") and counterpart foreign patent
application assigned to NYU, identified in annexed Appendix I forming an
integral PART hereof;
- 1 -
81
WHEREAS, NYU desires to conduct further research with respect to
detection of HIV antibodies in urine, with the hope of making advances in the
detection and prevention of the Acquired Immune Deficiency Syndrome ("A I DS"
all as more fully described with respect to the NYU Research Project (as
hereinafter defined);
WHEREAS, HORL is prepared to sponsor the NYU Research Project (as
hereinafter described);
WHEREAS, subject to the terms and conditions hereinafter set forth,
NYU is willing to grant to HORL and HORL is willing to accept from NYU an
assurance of immunity from infringement claims by NYU against HORL with respect
to HORL's use and practice of the inventions described in the NYU Patents (as
hereinafter defined);
NOW, THEREFORE, IT IS HEREBY DECLARED AND AGREED BETWEEN THE PARTIES
AS FOLLOWS:
1. Definitions
Whenever used in this Agreement, the following terms shall have the
following meanings:
a. "NYU Research Project" shall mean the investigations during
the Research Period (as hereinafter defined) with respect to
methods and/or assays for the detection of HIV antibodies in
urine under the supervision of Dr. Alvin Friedman-Kien
(hereinafter "the NYU Scientist") at NYU in accordance with
the research program described in annexed Appendix II which
forms an integral part hereof.
- 2 -
82
b. "NYU Research Period" shall mean the three year period
commenting upon January 1, 1990 and concluding on
December 31, l992.
c. "NYU Patents" shall mean all United States and foreign patents
and patent applications, and any divisions, continuations', in
whole or in part, reissues, renewals and extensions thereof,
and pending applications therefor:
(1) which are identified on annexed Appendix I; and
(2) any such patents and patent applications which claim
improvements to the patents and patent applications
described in Appendix I and which are conceived and
reduced to practice, by students or employees of NYU
during the term and in the course of the NYU Research
Project.
d. "HORL Corporation Entities" shall mean any company or other
legal entity which controls, or is controlled by, or is under
common control with, HORL; control means the holding of fifty
percent (50%) or more of (i) the capital and/or (ii) the
voting rights and/or (iii) the right to elect or appoint
directors.
e. "Test" shall mean a test of a urine sample of an individual
to. assay HIV antibodies in urine for use in the insurance
testing field and which is covered by a claim of any
unexpired NYU Patent which has not been disclaimed, or held
invalid by a court of competent jurisdiction (from which no
appeal can be taken).
- 3 -
83
f. "Calendar year 1. shall mean the consecutive period of twelve
months commencing on January I of each year and ending on
December 31 of such year.
2. Effective Date
This Agreement shall be effective as of the Effective Date and shall
remain in full force and effect until terminated in accordance with
Section 12 hereof.
3. Performance of the NYU Research Project
a. In consideration of the sums to be paid to NYU as set forth in
Section 4 below, NYU undertakes to perform the NYU Research
Project at NYU under the supervision of the NYU Scientist
during the the Research Period. If, during the Research
Period the NYU Scientist shall cease to supervise the NYU
Research Project, then NYU shall endeavor to find from among
the scientists of NYU a scientist or scientists acceptable to
HORL to continue the supervision of the NYU Research Project
in place of the NYU Scientist. Nothing herein contained,
however, shall be deemed to impose an obligation on NYU to
find a replacement for the NYU Scientist.
b. Nothing contained in this Agreement shall be construed as a
warranty on the part of NYU that any results or inventions
will be acnieved by the NYU Research Project, or that the NYU
Patents and/or any other results or inventions achieved by the
- 4 -
84
NYU Research Project, if any, are or will be commercially
exploitable and furthermore, NYU makes no warranties
whatsoever as to the commercial or scientific value of the NYU
Patents and/or as to any results which may be achieved in the
NYU Research Project.
c. Within sixty (60) days after the end of each half-year of the
Research Period, NYU shall prepare a written report
summarizing the results of the work conducted on the NYU
Research Project during the preceding half-year.
d. NYU will have full authority and responsibility for the NYU
Research Project. All students and employees of NYU who work
on the NYU Research Project will do so as employees or
students of NYU, and not as employees of HORL.
e. All right, title and interest, in and to any results or
inventions achieved by the NYU Research Project, and in and to
any drawings, plans, diagrams, specifications, and other
documents containing any such results or inventions shall vest
solely in NYU.
4. Funding of the NYU Research Project
a. As compensation to NYU for the performance of the NYU Research
Project during the Research Period, HORL will pay NYU a total
of one million one hundred and seventy five thousand dollars
($1,175,000) according to the following schedule:
- 5 -
85
(1) Upon the Effective date of this Agreement, HORL shall
pay to NYU three hundred ninety-one thousand six
hundred sixty seven dollars ($391,667.00).
(2) On January 1, 1991, HORL shall pay to NYU three
Hundred ninety-one thousand six hundred sixty-seven
dollars ($391,667.00).
(3) On January 1, 1992, HORL shall pay to NYU three
hundred ninety-one thousand six hundred sixty-six
dollars ($391,666.00).
b. Nothing in this Agreement shall be interpreted to prohibit NYU
(or the NYU Scientist) from obtaining additional financing or
research grants for the NYU Research Project from third
parties including government agencies, which grants or
financing may render ell or part of the NYU Research Project
and the results thereof subject to the patent rights of the
U.S. government and its agencies, as set forth 'IN Title 35
U.S.C. Section 200 et seq, or subject to the patent policies
of such third parties.
5. Immunity from Claims of Infringement by NYU
a. Subject to the terms and conditions hereinafter set forth, NYU
hereby grants to HORL and HORL Corporation Entities immunity
from claims of infringement of tne NYU Patents which, but for
the provisions of this Section 5, could lawfully be brought by
NYU ("Immunity"). The Immunity shall be exclusive with
respect to testing for insurance purposes and NYU shall not
grant
- 6 -
86
Immunity or a license to practice the NYU Patents to any other
person, company, or entity which provides testing services for
insurance purposes during the term of this Agreement except as
provided in Section 6. hereof.
b. The Immunity granted to HORL in Section 5.a. hereof shall
remain in force, if not previously terminated under the terms
of this Agreement, until the expiration date of the last to
expire of the NYU Patents.
c. Upon the written request of HORL, NYU shall extend the
Immunity to providers of Test kits which provide such kits
exclusively for HORL or for HORL Corporation Entities.
6. Payments for Immunity
a. In consideration for the grant of immunity hereunder, HORL
shall pay to NYU
(1) on the Effective Date, One Hundrea seventy-five
thousand dollars ($175,000.00) as reimbursement for
patenting costs and legal expenses incurred by NYU
with respect I to the NYU Patents; and
(2) a royalty for each Test performed by HORL and/or HORL
Corporation Entities, calculated upon the annual
total number of such Tests performed in each calendar
year, in accordance with the following schedule:
- 7 -
87
Tests performed in Royalty Payment
Calendar Year per lest
------------------ ---------------
0 - 500,000 $ 0.50
500,001 - 1,500,000 $ 0.35
1,500,001 - 2,500,000 $ 0.30
2,500,001 - 5,000,000 $ 0.25
5,000,001 and over $ 0.20
b. For the purpose of computing the royalties due to NYU
hereunder, each calendar year shall be divided into four
quarters, ending on March 31, June 30, September 30 and
December 31. HORL shall, within thirty (30) days from the end
of each quarter in each calendar year during the term of this
Agreement, submit to NYU a full and detailed report of
royalties due to NYU under the terms of this Agreement for the
preceding quarter (hereinafter "the Quarterly Report"),
setting forth the numbers of Tests performed by HORL and HORL
Corporation Entities upon which such royalties are computed.
If no royalties are due, a statement shall be sent to NYU
stating such fact. The full amount of any royalties due to
NYU for the preceding part of the year shall accompany each
such report. HORL shall keep and shall cause HORL Corporation
Entities to keep for a period of at least six (6) years after
the date of entry, full, accurate and complete books and
records consistent with sound business and accounting
practices
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88
and in such form and in such detail as to enable the
determination of the amounts due to NYU from HORL pursuant to
the terms of this Agreement.
c. Within thirty (30) days after the end of each calendar year,
commencing with the calendar year ending December 31, 1990,
HORL shall furnish NYU with a report (hereinafter "the Annual
Report"), verified BY an independent certified public
accountant, relating to the royalties due to NYU pursuant to
this Agreement in respect of the calendar year covered by the
said report and containing the same details as those specified
in subsection b. above in respect of the Quarterly Report.
d. On reasonable notice and during regular business hours, NYU or
the authorized representative of NYU shall each have the right
to inspect the books of accounts, records and other relevant
documentation of HORL or of HORL Corporation Entities insofar
as they relate to the Tests, in order to ascertain or verify
the amount of royalties due to NYU hereunder, and the accuracy
of the information provided to NYU in the aforementioned
reports.
e. Beginning on January 1, 1990 and continuing thereafter until
this Agreement shall terminate or expire, HORL agrees that if
the total royalties paid to NYU under subsection 6.a. (2)
nereof do not amount to two hundred thousand dollars
($200,000) for the first calendar year and two hundred fifty
thousand dollars ($250,000) for every calendar year
thereafter,
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89
HORL will pay to NYU within thirty days after the end of each
such calendar year, as additional royalty:
(1) for the first calendar year, the difference between
the amount of the total royalties paid to NYU by HORL
in the first calendar year and two hundred thousand
dollars ($200,000), failing which, NYU shall have the
right solely at its election, upon written notice to
HORL, to terminate this Agreement; and
(2) for the second calendar year and each calendar year
thereafter, the difference between the amount of the
total royalties paid to NYU by HORL in such calendar
year and two hundred fifty thousand dollars
($250,000), failing which, NYU shall have the right
solely at its election, upon written notice to HORL,
to terminate this Agreement.
7. Commercialization
a. HORL undertakes to begin the regular commercial use and
marketing of Tests for the life insurance testing field during
the first half of 1990, and thereafter during the remainder of
the term of this Agreement to continue such use and marketing
diligently.
b. In the event that a person, corporation or entity which
provides tests exclusively for HORL or for HORL Corporation
Entities or a person, corporation or entity acting pursuant to
written agreement with NYU undertakes to obtain approval in
the United States of America of the Food and Drug
Administration ("FDA")
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90
for use and marketing of Tests, HORL shall cooperate fully at
the written request of NYU, with such person, corporation or
entity, including by providing results of efficacy and clinical
tests, studies and other activities if any, undertaken by HORL
and HORL Corporation Entities with respect to Tests.
8. conversion to Non-Exclusive Immunity
a. In the event that the total Tests performed during the last
quarter of 1992 by HORL and HORL Corporation Entities, as
reported on the report due to NYU on January 30, 1993, does
not equal or exceed one million Tests or in the event that,
after such date, the total Tests performed by HORL and HORL
Corporation Entities, as reported to NYU on a quarterly basis
does not equal or exceed four million Tests for a calendar
year, NYU shall have the right to grant Immunity or a license
to third parties providing testing for insurance purposes,
provided that the terms of granting such Immunity or a license
to such third party shall De no more favorable to such third
party than the terms of this Agreement are to HORL.
b. In the event NYU exercises the right to grant Immunity or a
license to a third party providing testing for insurance
purposes as described in Section 8.a., NYU shall pay to HORL
fifty percent (50%) of the net royalties and fees (after
deduction of all costs and expenses, including reasonable
attorneys' fees, incurred by NYU in negotiating and concluding
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91
such agreement with such third party) paid to NYU by such third
party in consideration of such Immunity or license.
9. Patents and Patent Applications
a. NYU will promptly disclose to HORL in writing any inventions
which may constitute potential NYU Patents.
b. At the initiative of HORL or NYU, the parties shall consult
with each other regarding the filing of patent applications in
respect of any inventions which constitute potential NYU
patents, including but without limitation, the timing of the
filing of such applications, the jurisdiction within which
foreign counterparts of such applications should be filed and
other details pertaining to the prosecution and maintenance of
patent rights.
c. NYU will file, prosecute and maintain through patent counsel
selected by NYU, patent applications on any inventions which
constitute potential NYU Patents, and selected for patenting
by HORL pursuant to this Section 9 by written notice to NYU.
d. NYU and HORL shall assist, and cause their respective
employees and consultants to assist each other, in assembling
inventorship information and data for the filing and
prosecution of patent applications on inventions which
constitute potential NYU Patents. The-scope, content and
inventorship of such patent applications, and the prosecution
thereof, will be determined by NYU after consultation with
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92
HORL as set forth in Section 9.b. hereof.
e. All NYU Patents selected for patenting by HORL pursuant to
this Section 9 shall be filed, prosecuted and maintained by
NYU at the expense of HORL. Against the submission of
invoices, HORL shall reimburse NYU for all costs and fees
incurred by NYU during the tern, of this Agreement, in
connection with the preparation, filing, maintenance and
prosecution of the NYU Patents selected for patenting by HORL.
f. If at any time during the term of this Agreement HORL decides
that it is undesirable, as to one or more countries, to
prosecute or maintain any patents or patent applications
selected for patenting by HORL pursuant to this Section 9, it
shall give prompt written notice thereof to NYU, and upon
receipt of such notice HORL shall be released from its
obligations to bear all of the expenses to be incurred
thereafter as to such countries in conjunction with such
patent(s) or patent applications.
10. Infringement of NYU Patents by Third Parties
a. In the event a party to this Agreement acquires information
that a third party is infringing one or more of the NYU
Patents by using or marketing tests for insurance purposes,
the party acquiring such information shall promptly notify the
other party to this Agreement in writing of such infringement
setting forth in specific detail the name of the infringers
and the
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93
nature of the infringement. In such event, HORL shall be
entitled to request NYU to initiate suit for discontinuance of
said infringement, provided that such request is made in
writing by HORL and also provided that HORL submits evidence
reasonably acceptable to NYU of such infringement. Within 60
days after receipt of such written request and evidence
reasonably acceptable to NYU of such infringement, NYU shall
commence suit for discontinuance of said infringement. NYU
and HORL shall jointly select legal counsel. NYU shall manage
the conduct of litigation, in consultation with HORL, giving
consideration to HORL's preferences in such matters. The
expenses of such suit(s) that HORL requests NYU to bring,
including any expenses of NYU incurred in conjunction with the
prosecution of such suit(s) or the settlement thereof, shall
be paid for entirely by HORL and HORL shall hold NYU free,
clear and harmless from and against any and all costs and
expenses of such litigation, including attorneys fees
necessarily involved in the prosecution of any such suit(s),
provided, however, that HORL shall have the right to deduct
such costs and expenses from royalties as provided in Section
10.b. hereof. NYU shall not compromise or settle such
litigation Without the prior written consent of HORL, which
shall not be unreasonably withheld.
b. in the event HORL exercises the right to request NYU to sue
herein conferred, it shall have the right to deduct from
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94
royalties otherwise payable to NYU all costs and expenses of
every kind and character, including attorneys' fees,
necessarily involved in the prosecution of any such suit, and
paid by HORL, provided that such deductions shall not at any
time exceed fifty percent (50%) of the royalties payable to
NYU on each payment date.
c. Any sums recovered in such suit or settlement thereof shall be
disbursed as follows and in the following order:
(1) NYU shall be repaid any sums which were deducted from
royalties otherwise payable to NYU pursuant to
subsection b. above.
(2) HORL shall be reimbursed for sums paid by HORL for
costs and expenses of litigation which had not
previously been deducted from royalties otherwise
payable to NYU.
(3) Any remaining balance shall be divided equally
between NYU and HORL.
d. HORL agrees to cooperate fully with NYU at NYU's request,
including by giving testimony and producing documents lawfully
requested in the course of a suit prosecuted by NYU for
infringement of the NYU Patents. All reasonable expenses
(including attorneys' fees) incurred by HORL in connection
with such cooperation shall be an expense of litigation and
may be deducted from royalties as provided in subsection b.
above.
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95
11. Warranties by NYU
a. NYU has disclosed to HORL that Abbott Laboratories, Inc.
(hereinafter "Abbott") has asserted a claim of rights with
respect to NYU Patents. Subject to the claim of rights in the
NYU Patents which has been claimed by Abbott and which NYU has
disclosed to HORL, NYU warrants that NYU owns all rights and
title to the NYU Patents free and clear of any claim by a
third party and that NYU has all requisite authority to enter
into this Agreement.
b. Nothing herein contained shall be deemed to be a warranty by
NYU that
i) NYU can or will be able to obtain any patent or
patents on any patent application or applications in
the NYU Patents or any portion thereof, or that any
of the NYU Patents will afford adequate or
commercially worthwhile protection, or
ii) that the manufacture, use, practice or sale of any
element of the NYU Patents will not infringe any
patent(s) of a third party.
12. Term and Termination
a. This Agreement shall commence upon the effective Date and,
unless terminated pursuant to this Section 12 or Section 6.e.,
hereof, shall expire on the expiration of the last to expire
NYU Patents.
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96
b. Upon expiration of the NYU Research Period, HORL may terminate
this Agreement effective on February 1 of any calendar year by
giving sixty (60) days prior written notice to NYU of such
intended termination.
C. At any time prior to expiration either party may terminate this
Agreement for cause. Cause for termination of this Agreement
shall be deemed to exist when there has been a material breach by
either party of any of the terms of this Agreement, and when the
breaching party has failed to cure such breach within sixty (60)
days after receipt of written notice thereof from the
non-breaching party.
d. NYU may, upon giving written notice of termination, immediately
terminate this Agreement upon receipt of notice that HORL has
become insolvent or has suspended business or has filed a
voluntary petition or has filed an answer admitting the
jurisdiction of the U.S. Bankruptcy Court in the material
allegations of, or has consented to, an involuntary petition
purporting to be pursuant to any reorganization or insolvency law
of any jurisdiction, or has made an assignment for the benefit of
creditors or has applied for or consented to the appointment of a
receiver or trustee of a substantial part of its property.
e. Any amount payable hereunder by one of the parties to the other,
which has not been paid by its due date of payment shall bear
interest from its due date of payment until the
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97
date of actual payment, at the rate of two percent (2%) per annum
in excess of the Prime Rate prevailing at the Citibank, Inc.,
New York, New York, during the period of arrears and such amount
and the interest thereon may be set off against any amount due,
whether under terms of this Agreement or otherwise, to the party
in default by any non-defaulting party.
f. After termination of this Agreement for any reason, NYU shall be
entitled to assert claims of infringement of the NYU Patents
against HORL and/or HORL Corporation Entities for any use or
practice of the NYU Patents by HORL and HORL Corporation Entities
after the date of such termination.
g. Termination of this Agreement shall not relieve the parties of
any obligation occurring prior to such termination.
h. Sections 13 and 15 hereof shall survive and remain in full force
and effect after any termination, cancellation or expiration of
this Agreement.
13. Liability and Indemnification
a. HORL shall, at all times during the term of this Agreement and
thereafter, defend, indemnify and hold harmless NYU and its
trustees, officers, agents, employees, faculty and students from
and against any and all liability, loss, damages and expenses
(including attorneys' fees), they may suffer as the result of
claims, demands, costs or judgments which may be made or
instituted against them or any of them arising out of
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98
the performance of the Tests by HORL and/or HORL Corporation
Entities, or out of any representation made by HORL pursuant to
Section 16 of this Agreement. HORL's obligation to defend,
indemnify and hold harmless shall include, but not be limited to,
claims, demands, costs or judgments, whether for money damages or
equitable relief.
b. NYU agrees to notify HORL as soon as NYU becomes aware of a claim
or action for which indemnification may be sought pursuant to
this Section 13. At NYU's request, HORL shall provide attorneys
to defend against any claim or action with respect to the subject
of indemnity contained herein, whether or not such claims are
rightfully brought or filed.
14. Publication
a. Prior to submission of publication of a manuscript describing the
results of any aspect of the NYU Research Project, NYU shall send
HORL a copy of the manuscript to be submitted, and shall allow
HORL 30 days from the date of mailing to determine whether the
manuscript contains such subject matter for which patent
protection should be sought prior to publication of such
manuscript, for the purpose of protecting an invention which may
constitute NYU Patents. Should HORL believe the subject matter of
the manuscript contains a patentable invention, then prior to the
expiration of 30 days from the mailing date of such manuscript
to HORL by NYU, HORL shall
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99
give written notification to NYU of
i) its determination that such manuscript contains patentable
subject matter for which patent protection should be sought,
and
ii) the countries in which such patent protection should be
sought.
b. After the expiration of 30 days from the date of mailing such
manuscript to HORL, unless NYU has received the written notice
specified above from HORL, NYU shall be free to submit such
manuscript for publication to publish the disclosed research
results in any manner consistent with academic standards.
C. Upon receipt of such written notice from HORL, NYU will
thereafter delay submission of the manuscript for an additional
period of up to 60 days to permit preparation and filing of U.S.
patent application by NYU on the subject matter to be disclosed
in such manuscript. After expiration of such 60-day period, or
the filing of a patent application on each such invention,
whichever shall occur first, NYU shall be free to submit the
manuscript and to publish the disclosed results.
15. Confidential Information
a. Unless otherwise required by applicable law or regulations, HORL
and NYU shall maintain, in confidence, the terms of this
Agreement. Except as provided in Section 15.b. below, HORL shall
maintain any and all of the results or inventions
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100
achieved by the NYU Research Project and the NYU Patents in
confidence and shall not release or disclose any tangible or
intangible component thereof to any third party without first
receiving the prior written consent of NYU to said release or
disclosure. This obligation of confidentiality shall not apply to
any component of the results or inventions achieved by the NYU
Research Project and the NYU Patents which is part of the public
domain prior to the Effective Date of this Agreement or which
becomes a part of the public domain not due to some unauthorized
act by or omission of HORL after the Effective Date of this
Agreement or which is disclosed to HORL by a third party who has
the right to make such disclosure.
b. The provisions of Section 15.a. notwithstanding, HORL may
disclose the results or inventions achieved by the NYU Research
Project and the NYU Patents in confidence to third parties who
need to know the same in order to secure regulatory approval for
the Tests.
16. Representation and Covenants by HORL
HORL hereby represents and warrants to NYU as follows:
a. HORL is a corporation duly organized, validly existing and in
good standing under the laws of the State of Delaware. HORL has
been granted all requisite power and authority to carry on its
business and to own and operate its properties and assets. The
execution, delivery and performance of this Agreement have
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101
been duly authorized by the Board of Directors of HORL.
b. There is no pending or threatened litigation involving HORL which
would have any effect on this Agreement or on HORL's ability to
perform its obligations hereunder.
C. There is no indenture, contract, or agreement to which HORL is a
party or by which HORL is bound which prohibits or would prohibit
the execution and delivery by HORL of this Agreement or the
performance or observance by HORL of any term or condition of
this Agreement.
17. No Assignment
Subject to the terms of this Agreement, HORL shall not have the right to
assign, delegate or transfer at any time to any party, in whole or in
part, any or all of the rights, duties and interest herein granted
without first obtaining the written consent of NYU or such assignment,
which shall not be unreasonably withheld.
18. Use of Name.
Without the prior written consent of NYU, HORL shall not use the name of
NYU or of any NYU staff member, employee or student, or any adaptation
thereof;
i) in any advertising, promotional or sales literature;
ii) in connection with any public or private offering or in
conjunction with any application for regulatory approval,
unless disclosure is otherwise required by law, in which
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102
case HORL may make factual statements concerning the Agreement
or file copies of the Agreement after providing NYU with an
opportunity to comment and reasonable time within which to do so
on such statement in draft.
Except as provided herein, neither NYU or HORL will issue public
announcements about this Agreement or the status or existence of the NYU
Research Project without prior written approval of the other party.
19. Miscellaneous.
a. In carrying out this Agreement the parties shall comply with all
local, state and federal laws and regulations.
b. If any provision of this Agreement is determined to be invalid
or void, the remaining provisions shall remain in effect.
c. This Agreement shall be deemed to have been made in the State of
New York and shall be governed and interpreted in all respects
under the laws of the State of New York.
d. All payments or notices required or permitted to be given under
this Agreement shall be given in writing and shall be effective
when either personally delivered or deposited, postage prepaid,
in the United States registered or certified mail, addressed as
follows:
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To NYU: New York University Medical Center
550 First Avenue
New York, NY 10016
Attention: Isaac T. Kohlberg, Esq.
Vice President for
Industrial Liaison
and
Office of Legal Counsel
New York University
Bobst Library
70 Washington Square South
New York, NY 10012
Attention: Annette B. Johnson, Esq.
Associate General Counsel
To HORL: Home Office Reference Laboratory
10310 West 84th Terrace
Lenaxa, Kansas 66214
Attention: Kenneth A. Stelzer
President and Chief
Executive Officer
and
Hillix, Brewer, Hoffhaus, Whittaker
& Hormer, Esqs.
27th Floor
Commerce Towers
P.O. Box 13367
Kansas City, Missouri 64199-3367
Attention: R. Dennis Wright, Esq.
or such other address or addresses as either party may hereafter specify by
written notice to the other. Such notices and communications shall be deemed to
have been received by the addressee on the date of delivery or fourteen (14)
days after having been sent by registered mail.
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e. This Agreement (and the annexed Appendices) constitute the entire
Agreement between the parties, and no variation, modification or waiver
of any of the terms or conditions hereof shall be deemed valid unless
made in writing and signed by both parties hereto. This Agreement
supersedes any and all prior agreements or understandings, whether oral
or written, between HORL and NYU.
f. No waiver by either party of any non-performance or violation by the
other party of any of the convenants, obligations or agreements of such
other party hereunder shall be deemed to be a waiver of any subsequent
violation or non-performance of the same or any other covenant,
agreement or obligation, nor shall forbearance by any party be deemed to
be a waiver by such party of its rights or remedies with respect to such
violation or non-performance.
g. The descriptive headings contained in this Agreement are included for
convenience and reference only and shall not be held to expand, modify
or aid in the interpretation, construction or meaning of this Agreement.
h. It is not the intent of the parties to create a partnership or joint
venture or to assume partnership responsibility or liability. The
obligations of the parties shall be limited to those set out herein and
such obligations shall be several and not joint.
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IN WITNESS WHEREOF, the parties hereto have executed this Agreement the
date and year first above written.
NEW YORK UNIVERSITY
By: /s/ Isaac T. Kohlberg
-------------------------
Isaac T. Kohlberg
Witnessed By: Title: Vice President for
Industrial Liaison
/s/ Annette Johnson Date: November 17th, 1989
----------------------------- ------------------------
HOME OFFICE REFERENCE LABORATORY, INC.
By: /s/ Kenneth A. Stelzer
-------------------------
Kenneth A. Stelzer
Witnessed By:
Title: President and Chief Executive Officer
/s/ [illegible] Date: November 15, 1989
----------------------------- --------------------------
[Home Office Reference
Laboratory, Inc. Corporate Seal]
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106
4/8/92
FIRST AMENDMENT TO
NYU/HORL
AGREEMENT
This Agreement, effective as of November 15, 1989 ("the Effective
Date"), is by and between NEW YORK UNIVERSITY, a corporation organized and
existing under the laws of the State of New York and having a place of business
at 70 Washington Square South, New York, New York 10012 (hereinafter "NYU") and
HOME OFFICE REFERENCE LABORATORY a corporation organized and existing under the
laws of the State of Delaware having its principal offices at 10310 West 84th
Terrace, Lenaxa, Kansas 66214 (hereinafter "HORL").
RECITALS
WHEREAS, NYU and HORL have entered into an agreement, effective November
15, 1989 ("the Agreement") concerning a research project and rights to certain
intellectual property including certain intellectual property owned by NYU and
invented by Drs. Alvin Friedman-Kien, Yunzhen Cao, and William Borowsky relating
to methods and/or assays for the detection of HIV antibodies in urine and
identified in Appendix I of the Agreement ("the Pre-Existing Inventions", as
term is defined in the Agreement); and
WHEREAS, NYU and HORL desire to modify the Agreement with respect to the
Pre-Existing Inventions:
NOW, THEREFORE, the Agreement is hereby modified pursuant to Section
19(e) therein to amend Subsection 6e. as follows:
107
1. Section 6e. shall now read:
"Beginning on January 1, 1990 and continuing thereafter until this
Agreement shall terminate or expire, HORL agrees that if the total
royalties paid to NYU under subsection 6.a. (2) hereof do not amount to
two hundred thousand dollars ($200,000) for each of the first, second
and third calendar years and two hundred fifty thousand dollars
($250,000) for every calendar year thereafter, HORL will pay to NYU
within thirty days after the end of each such calendar year, as
additional royalty:
(1) for each of the first, second and third calendar years, the
difference between the amount of the total royalties paid to NYU
by HORL in each of the first, second and third calendar years and
two hundred thousand dollars ($200,000), failing which, NYU shall
have the right solely at its election, upon written notice to
HORL, to terminate this Agreement; and
(2) for the fourth calendar year and each calendar year thereafter,
the difference between the amount of the total royalties paid to
NYU by HORL in such calendar year and two hundred fifty thousand
dollars ($250,000), failing which, NYU shall have the right
solely at its election, upon written notice to HORL, to terminate
this Agreement.
Any term not defined herein is intended to have the meaning defined in
the Agreement. All Section numbers and Appendices herein refer to that
Agreement.
NEW YORK UNIVERSITY HOME OFFICE REFERENCE LABORATORY
By: /s/ Issac T. Kohlberg By: /s/ Kenneth A. Stelzer
-------------------------- ---------------------------
Isaac T. Kohlberg Kenneth A. Stelzer
Vice President for President and Chief Executive
Industrial Liaison Officer
Date: 4/8/92 Date: 4/24/92
------------ -----------