EXHIBIT 10.3
DISTRIBUTION AGREEMENT
This Agreement ("Agreement") is entered into as of October 11, 2002
between bioMerieux, Inc., a Missouri corporation ("BMX"), and OraSure
Technologies, Inc., a Delaware corporation ("OSUR").
BACKGROUND
BMX, through its predecessor, Organon Teknika Corporation, a Delaware
corporation, and OSUR, through its predecessor, Epitope, Inc., an Oregon
corporation, previously entered into a Supply Agreement (the "Original Supply
Agreement") and Distribution Agreement (the "Original Distribution Agreement"),
each dated as of April 1, 1994. Pursuant to the Original Supply Agreement, BMX
agreed to supply all of OSUR's requirements of Antigen (as defined below) in
connection with the research and development, manufacture, use and sale of
Products (as defined below) to BMX under the Original Distribution Agreement.
Pursuant to the Original Distribution Agreement, OSUR appointed BMX and its
Affiliates (as defined below) as exclusive distributor of the Products within
the Territory (as defined below). BMX and OSUR desire to enter into this
Agreement in order to amend and restate the terms of the Original Supply
Agreement.
AGREEMENT
In consideration of the mutual covenants contained herein, and the
premises set forth above, the parties hereby amend and restate the Original
Distribution Agreement in its entirety, as follows:
1. Definitions. Capitalized terms not otherwise defined in this
Agreement shall have the meanings set forth in the Supply Agreement.
1.1 "Affiliate" shall mean any individual or entity that controls,
is controlled by, or is under common control with, the specified party. For
purposes of this definition, "control" shall mean direct or indirect beneficial
ownership of more than 50% of the voting stock, ownership interest or income
interest in an entity.
1.2 "Antigen" shall mean the Antigen, as that term is defined in
the Supply Agreement (as defined below), which is supplied to OSUR, pursuant to
the Supply Agreement, for the production of the appropriate Products.
1.3 "Territory" shall mean the entire world.
1.4 "Products" shall mean oral fluid confirmatory tests for HIV-1
manufactured from time to time during the Term by OSUR using the Antigen. For
reference purposes, a list of current Products is contained in Exhibit 1.4
attached hereto.
1.5 "Specifications" shall mean those specifications concerning
the Products as set forth in Exhibit 1.5, or such other specifications as may be
established pursuant to Article 7 hereof.
1.6 "Supply Agreement" shall mean that certain Supply Agreement
entered into by BMX and OSUR as of the date hereof, which amends and restates
the Original Supply Agreement.
1.7 "Term" shall have the meaning described in Section 13.1.
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1.8 "Transfer Price" shall mean the price per Test set forth on
Exhibit 1.8, subject to adjustment as provided in Section 3.1.
1.9 "Test" shall mean an individual confirmatory test strip for
HIV-1 contained in a Product kit for HIV-1 testing.
2. Distributorship.
2.1 OSUR hereby appoints BMX and its Affiliates as the exclusive
distributor to sell the Products within the Territory. "Exclusive" shall mean
that OSUR will not distribute or sell, or appoint any other person or entity to
distribute or sell, the Products. OSUR shall not be permitted to appoint any
other person or entity to distribute or sell the Products.
2.2 BMX hereby accepts such appointment and shall use its best
efforts to promote and sell Products. Notwithstanding the foregoing, if (a) at
any time BMX desires to market and sell a confirmatory test that can be used
with any specimen type other than oral fluid, or (b) after June 30, 2004, a new
confirmatory test becomes available for marketing and sale in the United States
that (i) simultaneously confirms the presence of HIV-1 plus HIV-2 or HIV-0 and
(ii) has received FDA approval for testing an oral fluid sample collected with
an FDA-approved oral fluid collection device, then, upon written notice to OSUR
from BMX stating that it desires to market and sell such a confirmatory test or
that such an oral fluid confirmatory test has become available, and in each case
identifying the specific confirmatory test, BMX may market and sell the
confirmatory test which it identified and, during the period in which BMX
markets and sells such confirmatory test, BMX shall be relieved of its
obligations to use best efforts to promote and sell Products and instead shall
use commercially reasonable efforts to sell Products as necessary to satisfy the
continuing demand for the Products for the remainder of the Term. BMX represents
that it is not aware of any confirmatory test intended for use in detecting any
strain of HIV in oral fluid or any oral fluid collection device intended for HIV
testing, that are currently in development.
2.3 The parties are independent businesses and neither has nor
will have any power, right, or authority, nor will either party represent that
it has any power, right, or authority, to bind the other or to assume or to
create any obligation or responsibility, express or implied, on behalf of the
other. Nothing stated in this Agreement shall be construed as constituting BMX
and OSUR as partners or as creating relationships of employer and employee,
master and servant, or principal and agent between the parties.
2.4 This Agreement gives BMX and its Affiliates the right to
purchase Products from OSUR and to resell them in accordance with the terms and
conditions of this Agreement. BMX's compensation, if any, will come solely from
the margin between the price it pays for Products and the price at which it
sells those Products. Except as may be provided elsewhere herein, BMX has no
right to any compensation from OSUR.
2.5 BMX shall maintain records of all sales of Products, which
records shall include the name and address of the purchaser, the date of
purchase, and quantity and lot number of the Products sold. If required by
regulatory authorities, BMX shall furnish, during normal working hours, such
information to said regulatory authorities upon OSUR's request at no charge. BMX
shall not remove, deface, or otherwise obliterate any labeling placed upon
Products by OSUR.
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2.6 As consideration for BMX agreeing to amend and restate the
Original Supply Agreement and Original Distribution Agreement and entering into
this Agreement and the Supply Agreement, OSUR shall pay an aggregate of $750,000
in fees to BMX, as follows:
2.6.1 $250,000 upon BMX's execution and delivery of this
Agreement and the Supply Agreement, $250,000 on or before December 31,
2002 and $250,000 on or before March 31, 2003.
2.6.2 Payment of the foregoing fees shall be made by wire
transfer by OSUR to an account designated in writing by BMX.
2.6.3 OSUR acknowledges and agrees that the payment of such
fees is an irrevocable commitment, and payment thereof is non-refundable
and may not be offset, reduced or delayed for any reason whatsoever. In
connection therewith, OSUR has agreed to execute and deliver to BMX a
Promissory Note in the form attached hereto as Exhibit 2.6.3, to evidence
its commitment to make the payments due December 31, 2002 and March 31,
2003.
3. Price; Payment.
3.1 BMX shall pay OSUR an amount equal to the Transfer Price for
the Products sold hereunder. Commencing on January 1, 2004 and on each January 1
thereafter during the Term, OSUR may increase the Transfer Price for Products
purchased during the calendar year beginning on such January 1, upon sixty (60)
days prior written notice to BMX, by an amount equal to the percentage change in
the Consumer Price Index published by the United States Bureau of Labor
Statistics of the United States Department of Labor during the twelve (12)
consecutive calendar months immediately prior to the date of the notice for
which data (either preliminary or final) is then available. Comparisons shall be
made using the index entitled U.S. City Average - All Items and Major Group
Figures for All Urban Consumers (1982-84 = 100), or the nearest comparable data
on changes in the cost of living if such index is no longer published. The
Transfer Price shall not include sales, use or similar taxes, and BMX shall be
responsible for payment of any such taxes.
3.2 BMX shall pay OSUR within thirty (30) days of the date of
OSUR's invoice, which invoice shall not be dated earlier than the date of
shipment of Products. Amounts not paid when due shall bear interest from the
invoice date at one (1) percent per month or, if less, the highest rate of
interest permitted under applicable law.
4. Purchase Orders; Forecasts.
4.1 All purchases of Products pursuant to this Agreement shall be
effected by BMX's issuance of purchase orders. Each purchase order shall contain
the following information: Product(s), quantity, delivery date(s) (in accordance
with the applicable forecast unless otherwise agreed), dating, routing
instructions, destination and confirmation of price. For accounting convenience,
each purchase order may bear a separate number having no numerical relationship
to this Agreement. No term or condition contained in any such purchase order
shall alter, amend, modify or supplement OSUR's obligations hereunder unless
specifically agreed to in writing by OSUR. OSUR shall accept purchase orders by
facsimile. BMX shall submit orders at least 90 days in advance of the requested
delivery date. OSUR may, but shall not be required to, accept orders placed less
than 90 days before the requested delivery date.
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4.2 By the last day of each calendar month, BMX shall provide OSUR
with a forecast of the quantity of Products that BMX expects to order for
shipment for each month during the next 12 months. The forecast shall constitute
a binding commitment by BMX to purchase not less than the quantity of Product
stated for each of the first three (3) months of the forecast. OSUR shall use
its best efforts to meet all delivery dates for Products up to the amounts
specified in the applicable forecast and shall use commercially reasonable
efforts to meet requested delivery dates for Products ordered by BMX in excess
of such quantities.
5. Product Assembly.
5.1 Each unit of Product shall consist of Tests in strips or other
components assembled and packaged together in a unit for sale, all as described
in the Products' package inserts attached to Exhibit 1.5. With respect to any
new Product, its package insert shall be added to and become a part of Exhibit
1.5. OSUR will assemble the Products for BMX and, subject to the provisions of
Article 10 hereof, agrees to label the Products with BMX's label in accordance
with text supplied by BMX and the Specifications. BMX will supply camera-ready
art for such labels. Said labeling shall also contain appropriate lot and kit
numbers and product expiration dates, which lot numbers and expiration dates
shall be provided by OSUR.
5.2 OSUR shall be responsible for boxing, crating, handling,
storage and other packing requirements prior to shipment. All Products shall be
packaged, marked and otherwise prepared for shipment in a manner which is (i) in
accordance with good commercial practice, (ii) acceptable to common carriers for
shipment at the lowest reasonable rate and (iii) adequate to insure safe arrival
of the Products. All such costs shall be paid by OSUR.
6. Delivery; Acceptance; Returns.
6.1 All Products shall be delivered EX WORKS (Incoterms 2000)
OSUR's facility. Products shall be shipped by air express or air freight. The
cost of shipping shall be borne by BMX. In the event any shipment exceeds OSUR's
validated transit time (currently 24 hours), BMX may refuse and return the
shipment to OSUR, at OSUR's expense. OSUR shall forward to BMX a Certificate of
Conformance with each lot of Product certifying that the Products conform to the
Specifications, and shall provide a copy of such Certificate to BMX's purchasing
agent within 24 hours after shipment. OSUR shall not deliver any Product more
than five (5) days in advance of BMX's requested delivery date, and BMX may
return any such Product to OSUR at OSUR's expense for subsequent delivery to BMX
in conformance with the applicable purchase order.
6.2 OSUR shall inspect all Products prior to shipment to BMX. All
Products shall be subject to inspection and acceptance by BMX. BMX shall inspect
incoming Product in accordance with the inspection procedures and criteria set
forth in Exhibit 6.2 hereto. Any Product which fails to pass the incoming test
or inspection requirements (based on the quality control specifications for each
Product) may be rejected by BMX. Unless otherwise mutually agreed, BMX shall
have 30 days from the receipt of Product to inspect such Product and notify OSUR
in writing of BMX's rejection of any Product. Inspection by and acceptance of,
or any failure to inspect or accept, any Product by BMX shall in no way relieve
OSUR of its obligation to deliver Product in accordance with the warranties set
forth in Article 8 or otherwise comply with this Agreement.
6.3 BMX is authorized upon reasonable notice to OSUR and during
normal business hours to inspect OSUR's manufacturing facilities and operations
and quality control records to review compliance with Product Specifications,
FDA Quality Systems Regulations and this Agreement. All such inspection and
review shall be subject to the obligations of confidentiality set forth in
Article 12 hereof. Any such inspection or right to inspect by BMX shall in no
way relieve OSUR of its obligation to deliver conforming Products and shall in
no way
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waive BMX's rights to inspect and accept or reject Products. OSUR shall advise
BMX promptly in writing (including a description of any and all observations or
notices made or given) of any inspection of its facilities by any governmental
or regulatory agency or authority and of any other governmental or regulatory
action, actual or threatened, or any other problem, condition or issue related
to the Product, which OSUR's management reasonably believes, or should
reasonably believe, may substantially and adversely affect the Products or
OSUR's performance of its obligations under this Agreement (including, without
limitation, OSUR's obligation to comply with applicable laws and regulations).
In addition, at BMX's request, OSUR shall make available for review by the FDA
the manufacturing and control documentation required in connection with any FDA
approvals, which may be accomplished in any manner acceptable to the FDA,
including the filing of a suitable master file.
6.4 OSUR shall retain 3 units from each lot of Product
manufactured and shall retain such units as samples for the stated shelf life of
the Product. All samples retained shall be stored in accordance with the
Specifications and shall be made available for inspection and testing by BMX.
6.5 BMX shall market the Products in accordance with their
intended purpose, as specified in the Specifications and any Product inserts.
BMX shall not instruct any of its customers to use the Products in any manner
inconsistent with such Product's intended purpose or any Product inserts.
7. Changes in Specifications. BMX may, at any time, make written
requests for changes in the Specifications as reasonably necessary to meet
market demands, provided that such change shall have no effect upon any FDA or
other governmental approval. If any requested change causes an increase or
decrease in the cost of or time required in the performance for an order, such
change shall not be implemented until an equitable adjustment in the price or
delivery schedule or both has been agreed upon. OSUR shall notify BMX in writing
at least 30 days prior to making any changes in Specifications, and shall not
make any changes that would adversely affect Product performance without BMX's
prior written approval, unless required by the FDA. Any change in Specifications
shall be subject to the warranties given by OSUR as set forth in Article 8
hereof.
8. Warranties.
8.1 OSUR warrants that it has full right and title to the Products
and has authority to sell the Products to BMX. OSUR further warrants to BMX
that, for the stated shelf-life of the Products, but not less than eight (8)
months, such Products will conform to the Specifications; that in the production
of the Products OSUR will comply with the FDA Quality System Regulations, as
well as the then current good manufacturing practices, good laboratory practices
and all other applicable requirements of the FDA, and with all other applicable
Federal, state and local laws and regulations; and that for the stated
shelf-life of the Products, but not less than eight (8) months, such Products
will be free from defects in materials and workmanship. The foregoing warranties
as to the quality of the Products shall not apply to any Products that have been
subjected to misuse, mishandling, storage in a manner inconsistent with
labeling, neglect, modification or unusual physical or chemical stress after
delivery to BMX or which are defective because of Antigen supplied by BMX which
does not meet the warranties therefore set forth in the Supply Agreement. THE
FOREGOING WARRANTIES ARE THE SOLE AND EXCLUSIVE WARRANTIES MADE BY OSUR AND ALL
OTHER WARRANTIES, EXPRESS OR IMPLIED, INCLUDING WARRANTIES OF
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MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE EXPRESSLY DISCLAIMED.
8.2 OSUR shall indemnify, defend and hold harmless BMX from all
claims, suits, liabilities, damages and expenses (including reasonable attorney
fees) incurred on account of any third party product liability claims or
recalls, resulting from, arising out of or connected with OSUR's breach of any
of the foregoing warranties or its other obligations under this Agreement;
provided, however, that the foregoing indemnity shall not extend to any claim,
suit, liability, damage or expense of any kind attributable to the negligent
conduct of BMX or to a defect in Products resulting solely from defective
Antigen supplied by BMX.
8.3 In the event OSUR or BMX, upon advice of legal counsel,
determines that a license from a third party or third parties is required to
enable OSUR to make and sell the Products without infringing patent rights of
any such parties, then OSUR shall use its best efforts to obtain such license.
If OSUR is unable or unwilling to obtain such a license within 6 months after
such determination is made, then BMX shall be free to negotiate with such third
party for a license. In the event neither party obtains a license from the third
party, either party shall be entitled to cancel any pending purchase commitments
and to terminate this Agreement.
8.3.1 Provided BMX has approved of the license agreement to
OSUR , OSUR may add the agreed net royalty which is due the third party
to the Transfer Price for each Test, which royalty shall be paid by BMX.
8.4 It shall be a condition to OSUR's indemnification obligation
that BMX follow the procedures set forth below:
8.4.1 BMX shall promptly notify OSUR upon learning of any
claim for which indemnification may be sought; provided however failure
to give such prompt notice shall only relieve OSUR of its obligation to
provide indemnification to the extent such failure has a materially
adverse effect which limits OSUR from making a proper defense of such
claim.
8.4.2 BMX shall permit OSUR to control the response to and
any settlement or defense of any claim as to which indemnification may be
sought, but may require that any settlement agreement impose no
obligation on BMX other than the payment of monetary damages for which
OSUR indemnifies BMX. BMX's written consent, not to be unreasonably
withheld, shall be required on any term other than the payment of such
damages. BMX shall have the right to participate in the response to and
any settlement or defense of the claim using its own counsel at its own
expense. If OSUR fails within a reasonable time to respond to and
undertake a settlement or defense of the claim, BMX shall have the right,
but not the obligation, to undertake such response, settlement, and
defense, at OSUR's expense and risk.
8.4.3 BMX shall cooperate fully with OSUR with respect to
any claim for which indemnification is sought, making available all
information and assistance that OSUR may reasonably request and that is
under BMX's control.
9. Regulatory Matters.
9.1 OSUR has obtained approval of a Pre-Market Approval
Application ("PMA") for the Products from the FDA. If any change to the
Specifications requires a supplement to the PMA, OSUR will use commercially
reasonable efforts to prepare, file and prosecute the necessary regulatory
submission. OSUR shall bear the cost of generating any
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supporting clinical data required in connection with the submission unless OSUR
is making the change at BMX's request, in which event BMX shall bear such cost.
9.2 No changes or amendments shall be made to OSUR's PMA if such
changes or amendments would involve changes to the Specifications or would
adversely affect the performance of the Products, unless BMX has given its prior
written approval. The parties acknowledge that OSUR intends to change the
manufacturing location for the Products from Beaverton, Oregon to Bethlehem,
Pennsylvania and nothing in this Agreement shall preclude OSUR from doing so, so
long as OSUR manufactures the Products in Bethlehem, Pennsylvania in accordance
with this Agreement.
9.3 BMX shall not market the Products in any country in which all
appropriate regulatory approvals have not been obtained.
10. Labeling; Trademarks.
10.1 All Products shall be labeled in accordance with BMX's chosen
format and specifications (e.g., under BMX's name and trademark(s)), which shall
be reasonably acceptable to OSUR and shall include, to the extent required by
the FDA or other applicable governmental authority, OSUR's name and other
information pertaining to OSUR. A copy of the current Product labeling which is
acceptable to BMX is attached as Exhibit 10.1. BMX shall be responsible for
ensuring that such labeling conforms to all applicable law in each jurisdiction
in which the Products are sold, and shall indemnify and hold harmless OSUR
against any and all claims, losses, or damages arising out of or in any way
resulting from BMX's failure to do so.
10.2 Neither party shall acquire any right, title or interest in
any trademark, trade name, logo or copyright of the other party by reason of
this Agreement, except for the rights hereby granted to BMX to use trademarks,
trade names, logos and copyrights of OSUR to distribute and promote the Products
during the Term. BMX's rights to use OSUR's trademarks, trade names, logos or
copyrights shall terminate upon the termination or expiration of this Agreement;
provided however, BMX may continue to use them for a reasonable time after such
termination or expiration in connection with the promotion and sale of any
remaining Products in inventory upon termination or expiration of this
Agreement. Each party shall be responsible for registering, as necessary, its
own trademarks, trade names, logos and copyrights.
11. Complaints; Recalls. Customer complaints and recalls shall be
handled pursuant to the criteria set forth in Exhibit 11.
12. Confidentiality.
12.1 Each party shall take such steps and, when necessary to
protect the rights of the other, shall cause its Affiliates to take such steps
as are reasonably required to protect and keep confidential, and shall not use,
publicize or otherwise disclose to third parties other than Affiliates,
Confidential Information (as defined below) of the other party (or its
Affiliates), which Confidential Information was acquired from the other party
(or its Affiliates) pursuant to this Agreement, including, without limitation,
following procedures designed to limit access to such Confidential Information
to those persons having the need to know it. The parties shall not disclose or
use such Confidential Information except as they may be entitled to do so under
this Agreement or if necessary pursuant to or in the performance of this
Agreement.
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12.2 The obligation of confidentiality and restriction on use
imposed by the foregoing Section 12.1 shall not apply to any particular item of
Confidential Information that:
12.2.1 is known or generally available, or subsequently
becomes known or generally available, to the public, or is otherwise
at the time of disclosure or subsequently becomes part of the public
domain, whether by printed publication or otherwise, through no
fault of the receiving party;
12.2.2 the receiving party can demonstrate by competent
evidence, based in substance upon writings and/or physical evidence,
(i) was known to the receiving party at the time of receipt or (ii)
is furnished to the receiving party without obligation of
confidentiality or nonuse by a third party, either before or after
the time of its disclosure by the disclosing party, which third
party is not restricted by confidential undertaking to the
disclosing party at the time of the disclosure;
12.2.3 the receiving party can demonstrate by competent
evidence, based in substance upon writings and/or physical evidence,
has been developed independently by the receiving party by persons
not having access to the Confidential Information; or
12.2.4 is the Confidential Information of the disclosing
party that the disclosing party discloses to a non-Affiliate without
restriction.
12.3 The obligations of confidentiality and restriction on use
under this Article 12 shall continue to be binding upon the parties, for a
period of five years following termination or expiration of this Agreement.
12.4 Either party may also disclose Confidential Information
disclosed to it by the other party to the extent, and only to the extent, such
disclosure is necessary for such party to comply with applicable governmental
laws or regulations, including disclosures in any regulatory filings required in
connection with the Products. The party that desires to so disclose Confidential
Information shall give the other party reasonable advance notice of any such
proposed disclosure pursuant to such compliance with law or regulation, shall
use its best efforts to secure confidential treatment of the Confidential
Information thus disclosed, and shall advise the other party in writing of the
manner in which that was done.
12.5 For purposes of this Agreement, Confidential Information
shall mean: (a) data, inventions, information, processes, know-how, patent
applications, trade secrets and similar intellectual property rights of a party,
including, without limitation, the original and copies of all documents,
inventions, laboratory notebooks, drawings, specifications, devices, equipment,
prototype models and tangible manifestations embodying any technology disclosed
hereunder, (b) a party's customer lists and marketing, sales, costs, royalty and
similar information related to the manufacture or sale of Antigen, Vironostika
Assays or Products, and (c) any other information disclosed in writing and
marked as "Confidential Information" or, if disclosed orally, reduced to writing
and marked as "Confidential Information" and submitted within thirty (30) days
of the original oral disclosure.
13. Term; Termination.
13.1 The initial term of this Agreement shall commence on the date
first written above and shall continue until December 31, 2005 (the "Initial
Term"), unless terminated earlier as provided below; provided, however, that
this Agreement shall automatically renew for successive additional periods of
one year each (each a "Renewal Term" and together with the Initial Term, the
"Term") unless either party gives the other written notice of its election not
to
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renew this Agreement, which notice must be given not less than 180 days prior to
the expiration of the Initial Term or applicable Renewal Term. In the event FDA
approval for the use of BMX's HIV-O-TEK HIV-1 assay to detect HIV-1 in an oral
fluid sample collected with an OraSure(R) Oral Specimen Collection Device is
received on or before December 31, 2004, the Initial Term shall automatically be
extended to December 31, 2007 (notwithstanding either party giving notice of its
election not to renew).
13.2 Without waiving any other rights BMX may have hereunder, BMX
shall have the right to terminate this Agreement at any time within 90 days
following the occurrence of any of the following events:
13.2.1 OSUR fails on more than one occasion in any calendar
year to deliver Products within 20 working days after the delivery dates
established pursuant to Article 4 unless such failure results from BMX's
failure to supply Antigen or Vironostika Assays under the terms of the
Supply Agreement or unless BMX elects the remedy provided in Section 13.3
hereof;
13.2.2 The Products delivered do not conform to the
applicable Specifications or warranties contained in this Agreement,
provided such failure does not result from BMX's failure to supply
Antigen or Vironostika Assays which meet the specifications set forth in
the Supply Agreement, and OSUR does not provide conforming replacement
Products within 10 days after notice of the nonconformity;
13.2.3 OSUR is in material breach of any of the other
provisions of this Agreement or of any purchase order issued pursuant to
this Agreement and such breach is not cured within 30 days of written
notice thereof to OSUR;
13.2.4 OSUR becomes insolvent or files a voluntary petition
in bankruptcy; OSUR makes an assignment for the benefit of creditors; a
receiver, trustee in bankruptcy or similar officer is appointed to take
charge of all or part of OSUR's assets/property; or an involuntary
petition of bankruptcy is filed against OSUR and, in the case of any of
the foregoing, the same are not removed within 30 days; or
13.2.5 The Supply Agreement expires without being renewed or
is terminated other than for default of BMX.
13.3 In the event OSUR is in breach of Section 13.2.1 or Section
13.2.4 hereof and such breach is not promptly remedied, BMX may elect not to
terminate this Agreement but may instead assume the manufacture of, or may have
manufactured, the Products until such time as OSUR gives 60 days written notice
that it is able to resume production and delivery of the Products in accordance
herewith; provided, however, BMX may revoke such election at any time and
terminate this Agreement by giving written notice thereof to OSUR. If BMX makes
such an election, OSUR will provide BMX the know-how, data, documentation and
technical assistance, at BMX's expense, which is reasonably necessary for BMX to
manufacture the Products. During the time BMX is manufacturing the Products,
OSUR shall be relieved of its obligations under Articles 4, 5, 6, 8, 9, 10, 11,
and 14 hereof; provided however such obligations shall only be relieved with
respect to Products manufactured by BMX or others at BMX's request.
13.4 Without waiving any other rights OSUR may have, OSUR shall
have the right to terminate this Agreement at any time within 90 days following
the occurrence of any of the following events:
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13.4.1 BMX is in material breach of any of the provisions of
this Agreement and such breach is not cured within 30 days of written
notice thereof to BMX (15 days for breach of payment terms);
13.4.2 BMX becomes insolvent or files a voluntary petition
in bankruptcy; BMX makes an assignment for the benefit of creditors; a
receiver, trustee in bankruptcy or similar officer is appointed to take
charge of all or part of BMX's assets; or an involuntary petition of
bankruptcy is filed against BMX, and in the case of any of the foregoing,
the same are not removed within 30 days;
13.4.3 The Supply Agreement expires without being renewed or
is terminated other than for default of OSUR; or
13.4.4 The regulatory approvals necessary to allow OSUR to
manufacture and sell the Products for commercial purposes in the United
States are withdrawn, provided OSUR has used its best efforts to maintain
such approvals and to prevent such withdrawal.
13.5 Termination or expiration of this Agreement shall not
relieve any party from performance of any obligation then due nor affect any
rights accrued prior to the effective date of such termination or expiration.
14. Insurance. During the Term of this Agreement, each party shall
maintain comprehensive general liability insurance, including product liability
insurance, through a carrier reasonably satisfactory to the other, that will
adequately insure it against risks associated with the manufacture, use and sale
of Products in an amount not less than $4,000,000 per occurrence. Evidence of
such insurance shall be furnished to the other party upon request.
15. Taxes and Duties. BMX shall have the sole responsibility to pay all
import duties and fees, taxes and other charges levied by government authorities
upon or in connection with any transaction covered by this Agreement, including,
without limitation, taxes on sales, use, transactions or inventory, and value
added taxes.
16. Related Products.
16.1 HIV-O-TEK Assay.
16.1.1 OSUR will conduct, at its cost, the pre-clinical and
clinical trials required to obtain a claimed indication for BMX's new
HIV-O-TEK HIV-1 assay for testing of an oral fluid sample collected with
the OraSure(R) Oral Specimen Collection Device. BMX will provide all
HIV-O-TEK HIV-1 assays required for the trials. OSUR shall pay BMX for
50% of BMX's cost for up to the first 25,000 HIV-O-TEK HIV-1 assays
required for the pre-clinical and clinical trials, and shall pay BMX's
cost for all such additional HIV-O-TEK HIV-1 assays required therefore.
For purposes of this Section 16.1.1, BMX's cost shall not exceed $****
per test.
16.1.2 The parties have met and will continue to meet to
clarify what FDA submissions will be required and which party will
prepare and make such submissions. Subject to further discussions between
the parties and with the FDA, OSUR will use its best efforts to complete
all pre-clinical and clinical trials and prepare a report of the results
of such trials, in a format reasonably acceptable to the parties, by
August 31, 2003, so that whichever party must file the submission(s) may
do so by September 30, 2003. The foregoing best efforts obligation of
OSUR is subject to (i) BMX providing timely cooperation in developing a
regulatory filing strategy and clinical trial protocol that
-10-
is reasonably acceptable to the parties and acceptable to the FDA, (ii)
BMX providing sufficient quantities of its HIV-O-TEK HIV-1 assay in a
timely manner as required for the pre-clinical and clinical trials, (iii)
the HIV-O-TEK HIV-1 assays provided by BMX performing at a level required
to meet the FDA approval requirements for the detection of HIV-1 in an
oral fluid sample, and (iv) BMX receiving FDA approval of its current PMA
submission for the HIV-0-TEK HIV-1 assay on or prior to September 30,
2003.
16.1.3 OSUR shall be responsible for developing a protocol
for conducting the clinical trials in the most economical and expeditious
manner possible and BMX will cooperate in developing such protocol. The
parties shall also develop an action plan, specifying the respective
responsibilities of each party with respect to other requisite actions in
seeking regulatory approval. Further, the parties shall agree upon a
definition of "intended use" for purposes of presentation to the FDA.
Thereafter, the parties shall present the proposed protocol, action plan
and definition of intended use to the FDA for its review and concurrence
prior to beginning clinical trials. The parties shall use their
reasonable best efforts to meet with the FDA regarding these matters.
Such modifications as necessary to obtain FDA concurrence will be
negotiated between the parties. The party determined to be responsible
for preparing an FDA submission and each additional action item, based on
discussions between the parties and the FDA, shall use its best efforts
to file and prosecute such submission and complete the other actions in
as prompt a manner as is reasonably possible. The scope of the
pre-clinical and clinical trials shall be determined pursuant to the
protocol reasonably developed by the parties and approved by the FDA.
16.2 Vironostika Assay Availability. BMX will use commercially
reasonable efforts to continue to make available its Vironostika Assay on
commercially reasonable terms to its customers that purchase or use the
OraSure(R) Oral Specimen Collection Device until receipt of FDA approval for the
use of BMX's HIV-O-TEK HIV-1 assay to detect HIV-1 in an oral fluid sample
collected with an OraSure(R) Oral Specimen Collection Device. Notwithstanding
the foregoing, in the event the FDA submissions, contemplated by Section 16.1,
have not been filed by September 30, 2003, or the FDA approval for HIV-0-TEK
referred to in the preceding sentence is not received by December 31, 2004, and
in each such case such failure is not attributable solely to the acts or
omissions of BMX, then BMX shall have no further obligation pursuant to this
Section 16.2.
16.3 New Collection Device. OSUR does not currently plan to
commercialize its OraSure(R) II Oral Specimen Collection Device (the "OraSure(R)
II Device"). If, at the request of BMX, OSUR elects to seek FDA approval of
either the OraSure(R) II Device or an improved version of its OraSure(R) Oral
Specimen Collection Device for the detection of HIV-1 in oral fluid (it being
understood that such election shall be at OSUR's sole discretion), OSUR and BMX
each agree that either of BMX's Vironostika or another of BMX'sHIV-1 assays then
available on the market will be included in the clinical trials for
qualification with OSUR's new device, provided that (i) OSUR shall have no
obligation to make changes to its new device necessary for the Vironostika or
other assay to be so qualified; (ii) if the Vironostika or other HIV-1 assay
becomes qualified for use with OSUR's new device, BMX shall continue to make the
Vironostika or other HIV-1 assay used in such clinical trials available on
commercially reasonable terms to customers purchasing OSUR's new collection
device for a period of at least 2 years after receipt of FDA approval, (iii) BMX
provides all Vironostika or other HIV-1 assays required for the pre-clinical and
clinical trials and testing at no cost to OSUR and agrees to use best efforts to
file and prosecute, at its cost, any FDA submission which it must file in order
to qualify its assays for use with OSUR's new device, and (iv) the parties shall
agree in good faith to an equitable sharing of the costs of obtaining FDA
approval.
-11-
17. Miscellaneous.
17.1 Force Majeure. Neither party shall be liable for any delay
or default in such party's performance if such default or delay is caused by any
event beyond the reasonable control of such party, including, but not limited
to, acts of God; war; insurrection; civil commotion; labor disturbances,
epidemic, or destruction of production facilities or materials by earthquake,
fire, flood or storm, or other similar event. The party suffering such cause
shall immediately notify the other party of the cause and the expected duration
of such cause. Neither party shall be liable to the other for any such delay or
default.
17.2 Notices. Notices required or permitted hereunder shall be in
writing and shall be personally delivered or sent by registered or certified
mail, or facsimile (with confirmation by first class mail) or telex to the
addresses set forth below or to such other address in the United States that the
parties may hereafter specify, and shall be effective upon receipt:
If to BMX:
bioMerieux, Inc.
100 Rodolphe Street
Durham, NC 27712
Attn: President
Copy: General Counsel
Facsimile: (919) 620-2519
If to OSUR:
OraSure Technologies, Inc.
150 Webster Street
Bethlehem, PA 18015
Attn: President
Copy: General Counsel
Facsimile: (610) 882-2275
17.3 Governing Law. The rights of the parties under this
Agreement shall be governed by the laws of the State of North Carolina,
excluding choice of law rules and excluding the United Nations Convention on the
International Sale of Goods.
17.4 Assignment. This Agreement and the rights and obligations
arising hereunder may be assigned to an Affiliate, or to a third party, in whole
or in part, by either party with the prior written consent of the other. Such
consent shall not be unreasonably withheld. Notwithstanding the above, this
Agreement may be assigned by either BMX or OSUR to a third party which succeeds
to all or substantially all of the assigning party's business, whether by
merger, consolidation, sale or otherwise, without the consent of the
non-assigning party, except that, in the event of an assignment by OSUR to any
party reasonably deemed a competitor of BMX, as defined below, by BMX, BMX
retains the right to refuse such assignment. A competitor of BMX shall mean an
entity that competes with BMX in the manufacture, distribution or sale of
diagnostic products. Subject to the restrictions on assignment set forth herein,
this Agreement shall inure to the benefit of and bind the successors and
permitted assigns of each of the parties.
-12-
17.5 Entire Agreement. From the effective date hereof, this
Agreement (together with all Exhibits), the Supply Agreement and that certain
Release and Settlement Agreement of even date herewith between the parties (the
"Release"), set forth and constitute the entire agreement between the parties
with respect to the subject matter hereof, and supersede any and all other prior
agreements, understandings, promises and representations made by either party to
the other concerning the subject matter hereof; provided, however, that any
sales of Products which occurred under the Original Distribution Agreement or
which are pending as of the date of this Agreement, and the parties' rights and
obligations with respect thereto, shall continue to be governed by the terms of
the Original Distribution Agreement. This Agreement may not be released,
discharged, amended or modified in any manner except by an instrument in
writing, making specific reference to this Agreement, and signed by duly
authorized representatives of both parties.
17.6 Waiver. No waiver of any right under this Agreement shall be
deemed effective unless contained in writing and signed by the party charged
with such waiver, and no waiver of any right arising from any breach or failure
to perform shall be deemed to be a waiver of any future right or any other right
arising under this Agreement.
17.7 Survival. Articles 8, 11, 12, 17 and Sections 10.1
(indemnity only) and 10.2 shall survive expiration or termination of this
Agreement notwithstanding the delivery, acceptance or payment for Products.
17.8 Severability. If any provision of this Agreement is held
invalid by any law, rule, order or regulation of any government or by the final
determination of any state or federal court, such invalidity shall not affect
the enforceability of all other provisions of this Agreement not held to be
invalid.
17.9 Compliance with Law. Each party shall comply with all
applicable laws, rules and regulations, including FDA regulations, in its
performance under this Agreement.
17.10 Captions. Captions and section headings of this Agreement
are for convenience of reference only and shall not affect the interpretation or
meaning of this Agreement.
17.11 Attorney Fees. In the event suit or action or arbitration is
instituted to enforce or interpret the terms of this Agreement, the prevailing
party shall be entitled to recover from the other party such sum as the court or
arbitrator may adjudge reasonable as attorney fees at trial or arbitration, on
appeal, and on any petition for review, in addition to all other sums provided
by law; provided, however, such sums shall not exceed the damages awarded to the
prevailing party.
17.12 Press Release. Neither party shall make any public
disclosure (including press releases) of the terms of this Agreement without the
prior written consent of the other party, except to the extent required by
securities or other laws in the reasonable opinion of such party or its counsel.
If a party intends to issue a press release regarding this Agreement, it shall
provide the proposed release by facsimile or otherwise to the other party at
least twenty-four (24) hours before the release is issued and shall make any
changes reasonably requested by the other party before the release is issued.
17.13 Alternate Dispute Resolution. The parties shall attempt in
good faith to resolve promptly any dispute arising out of or relating to this
Agreement by negotiation. If the matter cannot be resolved in the normal course
of business, either party shall give the other party written notice of any such
dispute not resolved, after which the dispute shall be referred to senior
executives of both parties, who shall likewise attempt to resolve the
-13-
dispute. If the dispute has not been resolved by negotiation within forty-five
(45) days of the disputing party's written notice or if the parties fail to meet
within twenty (20) days from such notice, the parties shall endeavor to settle
the dispute by mediation under the supervision of and in accordance with the
Center for Public Resources ("CPR") Model Mediation Procedure for Business
Disputes. Unless otherwise agreed, both parties and either individual party may
request the CPR to appoint an independent mediator. The location of the
mediation shall be agreed upon by both parties and, in the event parties do not
timely agree, the location will be determined by the mediator. Any dispute not
settled by the mediation referenced above within sixty (60) days after
appointment of a mediator may, upon the request of either party, be submitted to
arbitration in accordance with the CPR Arbitration Rules and Commentary. A
single, impartial arbitrator mutually acceptable to the parties shall conduct
the arbitration. In the event the parties cannot agree on an arbitrator within
twenty-one (21) days after the end of the aforesaid sixty (60) days, either
party may have an arbitrator appointed by the CPR. The location of the
arbitration shall be agreed upon by both parties. As a condition of appointment
of the arbitrator, said arbitrator shall agree to use her/his best efforts to
conclude the proceeding within sixty (60) days. Said arbitrator shall further
have the authority to limit the volume of evidence and documents to be submitted
by the parties. Any court having jurisdiction thereof may enter judgment upon
the award rendered by the arbitrator. This Section 17.13 shall, however, not be
construed to limit or to preclude either party from bringing any action in any
court of competent jurisdiction for injunctive or other provisional relief as
necessary or appropriate.
17.14 Counterparts. This Agreement may be executed in more than
one counterpart, each of which shall be an original and together all such
counterparts shall constitute a single instrument. A facsimile transmission of a
signed counterpart shall be the same as delivery of an original.
IN WITNESS WHEREOF, the parties have executed this Agreement as of the
date and year first written above.
bioMerieux, Inc. OraSure Technologies, Inc.
By: /s/ Brian W. Armstrong By: /s/ Mike Gausling
-------------------------------- ------------------------------
Name: Brian W. Armstrong Name: Mike Gausling
------------------------------ ----------------------------
Title: CFO Title: CEO
----------------------------- ---------------------------
-14-
EXHIBIT 1.4
PRODUCTS
PRODUCT OSUR Part Number
ORASURE ORAL FLUID WESTERN BLOT KIT 501-0000
-15-
EXHIBIT 1.5
SPECIFICATIONS
-16-
HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1)
OraSure(R) HIV-1 Western Blot Kit
An Enzyme Immunoassay for the Detection of Antibodies
to Human Immunodeficiency Virus Type 1 (HIV-1)
in Human Oral Fluid Specimens Obtained with
OraSure(R) HIV-1 Oral Specimen Collection Devices
20 Tests
Store at 2-8(degrees)C
TABLE OF CONTENTS PAGE
NAME AND INTENDED USE .................................................. 1
SUMMARY AND EXPLANATION OF THE TEST .................................... 1
CHEMICAL AND BIOLOGICAL PRINCIPLES OF THE PROCEDURE .................... 2
KIT COMPONENTS SUPPLIED ................................................ 3
EQUIPMENT REQUIRED BUT NOT SUPPLIED .................................... 4
KIT STORAGE AND STABILITY .............................................. 4
CHEMICAL OR PHYSICAL INDICATIONS OF INSTABILITY ........................ 4
SPECIMEN COLLECTION, STORAGE, AND PREPARATION .......................... 5
PRELIMINARY PRECAUTIONS ................................................ 6
PROCEDURAL NOTES AND PRECAUTIONS ....................................... 6
ORASURE HIV-1 WESTERN BLOT TEST PROCEDURE .............................. 8
QUALITY CONTROL ........................................................ 12
INTERPRETATION OF RESULTS .............................................. 12
LIMITATIONS OF THE PROCEDURE ........................................... 14
PERFORMANCE CHARACTERISTICS ............................................ 14
REFERENCES ............................................................. 21
KIT AVAILABILITY ....................................................... 22
NAME AND INTENDED USE
The OraSure(R) HIV-1 Western Blot Kit is an in vitro qualitative assay for the
detection of antibodies to individual proteins of the Human Immunodeficiency
Virus Type 1 (HIV-1) in human oral fluid specimens obtained with the OraSure
HIV-1 Oral Specimen Collection Device. For convenience, the collection device
will hereafter be referred to as "OraSure". The OraSure HIV-1 Western Blot Kit
is intended for use as an additional, more specific test for HIV-1 antibodies in
OraSure specimens collected from individuals of unknown risk for HIV-1, which
are found to be repeatedly reactive by the Oral Fluid Vironostika(R) HIV-1
Microelisa System. The OraSure HIV-1 Western Blot Kit is intended for
professional use only.
The OraSure HIV-1 Western Blot Kit is not intended for use with blood,
serum/plasma or urine specimens or for screening potential blood donors.
SUMMARY AND EXPLANATION OF THE TEST
Acquired Immunodeficiency Syndrome (AIDS) is caused by at least two etiologic
agents which are designated as Human Immunodeficiency Virus Type 1 (HIV-1) and
Human Immunodeficiency Virus Type 2 (HIV-2)./1,2/ Infections with HIV-2 are
found primarily in parts of West Africa./2/ Current data indicate that HIV
infections are transmitted by sexual contact, exposure to blood (including
sharing needles and syringes) or certain blood products, and perinatally by
mother to infant./3,4/ Published data have established that patients with AIDS
and individuals infected with HIV-1 produce antibodies against HIV-1 proteins.
Studies have shown that HIV-1 antibodies have been detected in saliva samples of
most HIV-infected patients./5-7/
A number of EIA kits are currently available for the screening of serum
specimens for HIV-1 antibodies. The Oral Fluid Vironostika HIV-1 Microelisa
System manufactured by bioMerieux, Inc. is available specifically for screening
OraSure oral fluid specimens. These specimens are obtained using the OraSure
HIV-1 Oral Specimen Collection Device which is designed to collect oral HIV-1
antibodies while minimizing problems inherent in saliva samples (namely in high
viscosity and instability). Samples found to be repeatedly reactive for HIV-1
antibodies are tested using additional, more specific tests, such as Western
blot or immunofluorescence assays. The Western blot assay, as described by Tsang
et al,/8/ is useful for elucidating the specificity of the antibody response to
HIV-1 (a summary of the principles of the assay is presented on page 2).
Clinical serum samples that are reactive in the screening assays but do not
contain HIV-1 antibodies have also been described./9/ Some of these samples
possess antibodies to certain Class II HLA histocompatibility antigens that are
found in some cell lines used to produce the virus. Other individuals, who have
had no known exposure to HIV-1, produce reactive results in the screening test
for unknown reasons. Such nonspecific results are found commonly when screening
tests are used in low risk populations. Since the psychosocial and medical
implications of a positive antibody test may be significant, it is recommended
that additional testing be performed on such samples to validate the presence of
antibodies specific to HIV-1./10/
The OraSure HIV-1 Western Blot Kit was developed in order to provide an
additional, more specific assay for HIV-1 antibody detection in OraSure
specimens found to be repeatedly reactive in the screening EIA. The OraSure
HIV-1 Western
Page 1
Blot Kit, when used as directed in this insert, will detect antibodies to HIV-1
when present in human oral fluid samples obtained with the OraSure HIV-1 Oral
Specimen Collection Device. The position of bands on the preblotted
nitrocellulose strips allows the antibody reactivity to be associated with
specific viral antigens. An OraSure sample that is reactive in both EIA
screening test and Western blot assay is presumed to be positive for antibodies
to HIV-1, indicating infection with this virus except in situations of passively
acquired antibody or experimental vaccination. Antibodies to HIV-2 may also
react with the protein antigens of HIV-1./11/ Therefore, individuals infected
with HIV-2 may have reactive tests in the HIV-1 Western blot assay. Usually,
however, the cross-reactivity is incomplete, resulting in an indeterminate test
result (see Interpretation of Results section). Absence of antibodies to HIV
cannot be taken as absolute proof that an individual is free of HIV-1 or
incapable of transmitting the virus. Individuals with positive tests should be
referred for medical evaluation.
CHEMICAL AND BIOLOGICAL PRINCIPLES OF THE PROCEDURE
The whole cell viral lysate used in the manufacture of the OraSure HIV-1
Western Blot Kit is manufactured by bioMerieux, Inc. (U.S. license number 956).
It is HIV-1 propagated in an H-9/HTLV-IIIB, T-lymphocyte cell line. It is
purified by ultracentrifugation and inactivated by treatment with nonionic
detergent and heat.
When used to manufacture the preblotted strips, inactivated and denatured
proteins of the HIV-1 virus are fractionated by SDS-polyacrylamide gel
electrophoresis. The resolved protein bands are electrophoretically transferred
to nitrocellulose sheets. These preblotted nitrocellulose sheets are cut into
strips.
OraSure HIV-1 specimens, diluted in Sample Buffer, are incubated with the
preblotted nitrocellulose strips. If antibodies to specific HIV-1 proteins are
present in a specimen, they bind to epitopes contained in the proteins banded on
the strip. Any antibody not bound is removed by washing. The conjugate, alkaline
phosphatase-labeled goat anti-human immunoglobulin, is then added to the strip
and allowed to incubate. It binds to antibodies already bound to viral proteins
on the strip. Excess conjugate is removed by washing. The strips are then
incubated with a substrate specific to the alkaline phosphatase. The color
reaction is stopped by aspiration and washing.
If antibodies to specific HIV-1 proteins (p) or glycoproteins (gp) are present
in the specimen in sufficient concentration, purple bands may be visible at one
or more of the following positions on the nitrocellulose strip: gp160, gp120,
p65, p55, p51, gp41, p31, p24, and p18 (number refers to apparent molecular
weight in kilodaltons).
Page 2
KIT COMPONENTS SUPPLIED
(20 Test Kit)
OraSure(R) HIV-1 Western Blot Strips 20 strips
Prenumbered nitrocellulose strips, preblotted with resolved HIV-1 proteins;
packed in a resealable plastic pouch between buffer-soaked absorbent paper;
buffer contains 0.1% sodium azide as a preservative.
Substrate 1 bottle; 22 mL
BCIP/NBT single reagent substrate in an organic base/TRIS buffer.
Powdered Milk 1 bottle; 30 g
Non-fat milk solids.
Sample Diluent Concentrate 1 bottle; 100 mL
Phosphate buffered saline with 3.0% Tween-20; contains 0.01% thimerosal as a
preservative.
Conjugate Concentrate 1 vial; 0.25 mL
Goat anti-human IgG (heavy and light chains) F(ab')\\2\\ fragment, labeled with
alkaline phosphatase; contains 0.1% sodium azide as a preservative.
OraSure(R) HIV-1 WB Negative Control 1 vial; 0.65 mL
Human serum or plasma, nonreactive for antibodies to HIV-1, in OraSure Control
Matrix; tested negative for HBsAg and antibodies to HCV; contains a proprietary
preservative.
OraSure(R) HIV-1 WB Low Positive Control 1 vial; 0.65 mL
Human serum or plasma, reactive for antibodies to HIV-1, in OraSure Control
Matrix; tested negative for HBsAg and antibodies to HCV; heat-inactivated to
render material noninfectious for HIV-1; contains a proprietary preservative.
OraSure(R) HIV-1 WB High Positive Control 1 vial; 0.65 mL
Human serum or plasma, reactive for antibodies to HIV-1, in OraSure Control
Matrix; tested negative for HBsAg and antibodies to HCV; heat-inactivated to
render material noninfectious for HIV-1; contains a proprietary preservative.
Reaction Trays 5 each
Eight lane disposable trays with lids.
Caution: The Negative Control is prepared from human plasma or serum found to be
nonreactive for HIV-1 antibodies and tested for Hepatitis B surface antigen
(HBsAg) and antibodies to HCV by FDA-licensed methods. The Positive Controls are
prepared from anti-HIV-1 positive human plasma or serum, which was
heat-inactivated to render it noninfectious for HIV-1. However, as no procedure
can offer complete assurance that infectious agents are absent, all specimens of
human origin should be considered potentially infectious and handled with
care./12/
Page 3
EQUIPMENT REQUIRED BUT NOT SUPPLIED
1. Centrifuge tube for each sample to be processed
2. Centrifuge
3. Refrigerator (2-8(degree)C)
4. 37(degree)C water bath
5. Graduated cylinders
6. Beaker or appropriate mixing vessel
7. Balance
8. Laboratory timer
9. Magnetic stir plate and stir bar
10. Test Tubes: glass, polypropylene, or polystyrene (12x75 mm) (optional)
11. Precision micropipets to deliver variable volumes from 5 to 1000 (*)L
12. Disposable pipet tips
13. Graduated pipets to deliver volumes to 25 mL
14. Scissors
15. Forceps for strip handling (plastic or Teflon-coated)
16. Transfer pipets
17. Rotary platform, capable of rotating at 50-60 rpm
18. Aspiration system
19. Repeating pipet to deliver 1-2 mL volumes
KIT STORAGE AND STABILITY
1. Store all components at 2-8(degree)C when not in use.
2. Expiration dates printed on the kit and kit components indicate the limits
of stability.
3. Stability of the components after reconstitution or dilution is as
follows:
a. Sample Buffer
Store at room temperature while performing assay. Discard excess buffer
at completion of assay.
b. Sample dilutions
Test tube sample dilutions must be applied to the strips within one
hour of dilution. The dilutions are stored at room temperature during
that time.
c. Conjugate dilution
Conjugate dilution must be prepared during the last five minute wash
and applied immediately after the wash is complete and the wash
solution is aspirated. Excess Conjugate dilution must be discarded.
d. All other kit components are supplied ready to use.
4. Keep strip pouch tightly sealed. Do not let strips dry out.
CHEMICAL OR PHYSICAL INDICATIONS OF INSTABILITY
Alterations in physical appearance of kit materials may indicate instability or
deterioration.
Note: Sample Diluent Concentrate may contain crystals. This will not affect
essay performance if crystals are dissolved before use (see page 8, step 1.a).
Substrate is pale yellow in color. A fine black precipitate may be observed but
its presence does not affect product performance.
(*) denote umalut
Page 4
SPECIMEN COLLECTION, STORAGE AND PREPARATION
Note: This test kit may not be used to assay blood specimens. This test kit may
only be used to assay OraSure HIV-1 oral fluid specimens obtained using the
OraSure HIV-1 Oral Specimen Collection Device.
A. Specimen Collection
1. Refer to the OraSure HIV-1 Oral Specimen Collection Device package
insert for instructions on collecting a specimen.
2. OraSure HIV-1 specimens must be transported to the laboratory in the
OraSure HIV-1 Specimen Vial.
3. OraSure HIV-1 specimens may be transported to the laboratory at ambient
temperature via courier, air freight, or regular mail. OraSure
specimens should be protected from impact, direct sunlight, and
temperatures exceeding 37(degree)C (98(degree)F). Federal, state and
local regulations regarding transportation of diagnostic specimens are
applicable to OraSure HIV-1 specimens.
B. Specimen Storage
1. After receipt at the laboratory, OraSure HIV-1 specimens (on or off the
collection pad) should be stored at 2-8(degree)C. Specimens may be
stored at 4(degree)C to 37(degree)C for a maximum of 21 days from the
time of collection, including the time for shipping and testing. If
testing of specimens cannot be completed within 21 days, OraSure HIV-1
specimens can be stored frozen at -20(degree)C for a maximum of six
weeks.
2. OraSure HIV-1 specimens frozen and thawed once must be tested within
the 21 days (see B.1 above). Specimens frozen and thawed twice must be
tested within 24 hours, or discarded.
C. Specimen Preparation
1. Record the specimen identification number from the OraSure HIV-1
Specimen Vial.
2. Ensure that the specimen is within acceptable dating for testing,
i.e., *21 days from collection. Note: All testing should be completed
within 21 days of specimen collection unless stored at -20(degree)C
(see B.1 above).
3. Hold the vial with the pointed tip up.
4. Move the pad away from the vial tip by gently tapping the vial.
5. Break the pointed tip of the vial off with the thumb.
6. Place a centrifuge tube over the vial and invert the tube and vial.
7. Centrifuge at 600-800 x g force for 15 minutes.
8. Determine that there is a minimum of 0.75 mL volume of specimen eluate.
If the volume of the centrifuged specimen is less than 0.75 mL, the specimen is
unsuitable for testing and a new specimen from the test subject must be
obtained. Notify the ordering physician if the volume of specimen is
insufficient.
* denotes less than
Page 5
PRELIMINARY PRECAUTIONS
1. Keep testing area separate from areas where blood or blood products for
transfusion are stored.
2. Do not pipet by mouth.
3. Do not smoke, eat, or drink while handling test materials.
4. Wear disposable gloves throughout the specimen processing and testing
procedure.
5. Handle all materials used in the test (including specimens, Sample Buffer,
reaction trays and pipets) as though capable of transmitting infectious
agents./12/
6. Consult a physician immediately in the event that contaminated materials
are ingested or come in contact with mucous membranes or breaks in the
skin.
7. Immediately clean up any spills containing potentially infectious material
with freshly prepared 1:10 dilution of ***5% sodium hypochlorite (bleach)
and dispose of the cleaning material by an appropriate method.
8. Dispose of all specimens and materials used to perform the test as if they
contain infectious agents. Prior to disposal, treat as follows:
----------------------------------------------------------------------------
Material Disposal Procedure
----------------------------------------------------------------------------
Reusable items Autoclave for 60 minutes at 121(degree)C.
----------------------------------------------------------------------------
Disposable items Incinerate.
----------------------------------------------------------------------------
Liquid waste Mix with bleach to yield a final ratio of one part
bleach to nine parts waste (1:10). Allow the mixture
to stand 30 minutes before flushing down the drain.
----------------------------------------------------------------------------
PROCEDURAL NOTES AND PRECAUTIONS
Note: This test kit may only be used to assay OraSure HIV-1 oral fluid specimens
obtained using the OraSure HIV-1 Oral Specimen Collection Device. Do not test
any specimens other than OraSure oral fluid specimens with this kit.
1. Do not interchange or combine any kit component, including strips, with
components of another kit lot.
2. The OraSure Low Positive and Negative Controls must be assayed with each
run. The OraSure High Positive must be assayed with the first run of every
package of strips, but is optional in subsequent runs. This High Positive
Control strip should be retained as a reference. It will be compared to
the test strips run from that package to determine band identification and
placement.
3. Do not perform the test in the presence of reactive vapors (e.g., from
acids, alkalis, or aldehydes), dust, or residual bleach or bleach fumes;
the enzymatic activity of the conjugate may be affected or reactivity may
be decreased.
4. Prepare an assay worksheet, ensuring that the patient sample and control
identification is linked to the number embossed on the nitrocellulose
strip.
5. For sample dilution and addition to nitrocellulose strips, two options are
offered (explained in detail on pages 8 and 9).
a. The first involves preparing the dilutions in test tubes and adding
them to the strips.
b. The second involves adding the sample directly into the trough which
contains the strip and Sample Buffer.
*** denote greater tnan equals to
Page 6
6. Avoid contamination of the strips and/or the buffer-soaked absorbent paper
in the resealable pouch during handling (this may cause false reactivity
in subsequent assays).
a. Prior to removing the strips from the pouch, clean the work surface and
forceps with isopropyl alcohol.
b. Change gloves prior to opening the pouch.
c. Always use clean forceps when handling strips.
d. For initial use of strips, cut pouch below the seal line, keeping upper
portion of pouch intact including seal line.
e. It is recommended that the lower portion of the strip pouch be cut on
the remaining two sides (dotted lines as shown in diagram to the
right).
f. Fold the plastic down, which acts as a protective barrier, to expose
the strips.
[DIAGRAM APPEARS HERE]
7. Place each prenumbered strip, with the green indicator line facing up, in
the reaction trays in numerical order. This facilitates band alignment,
for ease of reading results.
8. Do not allow strips to dry out prior to sample addition. If diluting the
samples in test tubes, place strips into trays only after dilutions have
been made.
9. As soon as the sample dilutions have been added to all strips in a tray,
cover the tray with a lid.
10. It is essential to avoid cross-contamination between troughs, especially
prior to and during sample incubation.
a. Add sample to the trough of the reaction tray, using a transfer pipet
for diluted samples or a pipet for undiluted samples.
b. It is suggested that an additional precaution be taken by positioning
the strips in every other trough of a tray.
c. Avoid delivering bubbles to the liquid in the troughs.
d. Be careful to avoid dislodging fluid from the troughs when transferring
trays.
e. Liquid in the troughs should not contact tray lids (if liquid should
contact tray lids, immediately remove the material with a lab wipe).
11. It is important that the items used to prepare and dispense Sample Buffer
be scrupulously clean (a repeating pipet is preferable for dispensing the
1-2 mL of Sample Buffer).
12. Prime the pipet tip when measuring samples or reagents.
13. A rotation speed of 50-60 rpm is recommended for each rotation step. 14.
Be certain that each strip is immersed in the liquid and moves freely;
however, liquid must not contact the tray lid during rotation.
15. Incomplete or ineffective washing will compromise the assay; it is
imperative to follow the wash procedure carefully.
16. Discard used disposable reaction trays as biohazardous waste. Reuse of the
trays and lids is not recommended.
17. Samples must be at room temperature (20-25(degree)C) before starting the
test.
Page 7
18. Reagents should be at room temperature (20-25(degree)C) before beginning
the assay except for Conjugate Concentrate and Substrate, which must both
remain refrigerated (2-8(degree)C) until just prior to use. Return all
reagents to 2-8(degree)C after use.
CAUTION: The Conjugate Concentrate and the buffer in the absorbent paper
surrounding the strips contain sodium azide. If discarding into the sewer
system, flush copiously with water. This helps prevent formation of metallic
azides which, when highly concentrated in metal plumbing, may be potentially
explosive. Decontaminate plumbing according to CDC guidelines./13/
ORASURE HIV-1 WESTERN BLOT TEST PROCEDURE
1. Prepare Sample Buffer as follows:
a. Check Sample Diluent Concentrate for crystals.
i. If crystals have formed, dissolve them by warming the entire
bottle and its contents in a 37(degree)C water bath for 10 minutes
or until crystals are completely dissolved.
ii. Allow the material to reach room temperature before use.
b. Determine the volume of Sample Buffer to be prepared and quantity of
each constituent required from the chart below.
- -------------------------------------------------------------------------------------
Total number mL of Sample mL of Deionized mL of Sample g of Powdered
of strips to be Buffer to H2O required** Diluent Conc. Milk
assayed+ prepare* required** required***
- -------------------------------------------------------------------------------------
3 - 6 200 180 20 6
- -------------------------------------------------------------------------------------
7 - 9 300 270 30 9
- -------------------------------------------------------------------------------------
10 - 13 400 360 40 12
- -------------------------------------------------------------------------------------
14 - 16 500 450 50 15
- -------------------------------------------------------------------------------------
17 - 20 600 540 60 18
- -------------------------------------------------------------------------------------
+ Include strips for controls.
* 30.0 mL of Sample Buffer is required for each strip assayed.
** Sample Diluent Concentrate is diluted 1:10 in deionized water.
*** 3 g of Powdered Milk is required for every 100 mL of diluted Sample
Diluent Concentrate.
c. Combine the required amounts of deionized water, Sample Diluent
Concentrate and Powdered Milk.
d. Mix the solution for a minimum of 15 minutes (ensure the Powdered Milk
is completely dissolved).
e. Store at room temperature while performing assay. Discard excess buffer
at completion of assay.
2. Prepare an assay worksheet, ensuring that the patient sample and control
identification is linked to the number embossed on the nitrocellulose
strip.
3. Both the OraSure Low Positive and Negative Controls must be assayed with
each run. The OraSure High Positive must be assayed with the first run of
every package of strips, but is optional in subsequent runs. This High
Positive Control strip should be retained as a reference. It will be
compared to the test strips run from that package to determine band
identification and placement.
4. Add controls and patient specimens to nitrocellulose strips by one of the
following methods:
Page 8
a. METHOD I: Test Tube Dilution
i. Into appropriately labeled test tubes, add 150 (*)L of each
specimen or control to 1.0 mL of Sample Buffer and mix well. These
dilutions must be tested within an hour.
ii. Place one prenumbered strip with green indicator line facing up
into each trough as follows, ensuring the strips do not dry out:
a) For initial use of strips, cut pouch below the seal line,
keeping upper portion of pouch intact including seal line.
b) Cut the lower portion of the pouch on the two remaining sides
(see page 7, step 6.d).
c) Fold the packaging back to expose strips.
d) Beginning with the left side of the series (strip #1), remove
strips to be assayed and place them in numerical order.
e) Grasp the strip at the green indicator line with forceps.
f) Transfer the strips, avoiding contact with contaminated
surfaces, into the troughs of the reaction tray(s).
g) Place any remaining strips (still encased in moist blotting
paper and contained in the lower portion of the pouch) in the
upper portion of pouch, seal using zip closure, and return to
storage at 2-8(degree)C.
iii. Transfer the contents of each tube (~1.1 mL) into the
corresponding trough using a transfer pipet.
b. METHOD II: On-Strip Dilution
i. Add 1.0 mL Sample Buffer to each trough to be used.
ii. Place one prenumbered strip with green indicator line facing up
into each trough as follows, ensuring the strips do not dry out:
a) For initial use of strips, cut pouch below the seal line,
keeping upper portion of pouch intact including seal line.
b) Cut the lower portion of the pouch on the two remaining sides
(see page 7, step 6.d).
c) Fold the packaging back to expose strips.
d) Beginning with the left side of the series (strip #1), remove
strips to be assayed and place them in numerical order.
e) Grasp the strip at the green indicator line with forceps.
f) Transfer the strips, avoiding contact with contaminated
surfaces, into the troughs of the reaction tray(s).
g) Place any remaining strips (still encased in moist blotting
paper and contained in the lower portion of the pouch) in the
upper portion of pouch, seal using zip closure, and return to
storage at 2-8(degree)C.
iii. Add 150 (*)L of each OraSure specimen or control to the
corresponding trough.
5. Cover each tray with a lid and mix by gentle rotation (50-60 rpm) on a
rotator for 180 minutes (3 hours) at room temperature.
6. After incubation, completely aspirate the liquid from troughs (do not allow
the strips to dry).
(*) Umalut
Page 9
7. Wash the strips as follows:
a. Add 2.0 mL Sample Buffer to each strip.
b. Immediately aspirate all liquid from each trough.
c. Repeat steps a and b two more times.
d. Add 2.0 mL Sample Buffer to each strip and replace the lid(s).
e. Place the tray(s) on the rotator (at 50-60 rpm) for 5 minutes.
f. Aspirate the Sample Buffer completely.
g. Repeat steps d and e one more time.
8. Prepare diluted Conjugate during the final wash step (step 7.g) above):
a. Conjugate dilution must be prepared during the last five minute wash
and applied immediately after the wash is complete and the wash
solution is aspirated. Excess Conjugate dilution must be discarded.
b. Remove Conjugate Concentrate from the refrigerator.
c. Determine volumes required of Sample Buffer and Conjugate Concentrate
from the following chart:
- ---------------------------------------------------------------------------------------------------------
Total number mL of Sample (*)L of Conjugate Total number mL of Sample (*)L of Conjugate
of strips being Buffer Concentrate of strips being Buffer Concentrate
assayed+ required* required** assayed+ required* required**
- ---------------------------------------------------------------------------------------------------------
3 8 40 12 26 130
- ---------------------------------------------------------------------------------------------------------
4 10 50 13 28 140
- ---------------------------------------------------------------------------------------------------------
5 12 60 14 30 150
- ---------------------------------------------------------------------------------------------------------
6 14 70 15 32 160
- ---------------------------------------------------------------------------------------------------------
7 16 80 16 34 170
- ---------------------------------------------------------------------------------------------------------
8 18 90 17 36 180
- ---------------------------------------------------------------------------------------------------------
9 20 100 18 38 190
- ---------------------------------------------------------------------------------------------------------
10 22 110 19 40 200
- ---------------------------------------------------------------------------------------------------------
11 24 120 20 42 210
- ---------------------------------------------------------------------------------------------------------
(*) Umalut
+ Include strips for controls.
* Prepare 2.0 mL of diluted Conjugate for each strip assayed (a small
excess has been incorporated for pipetting ease).
** Conjugate Concentrate is diluted 1:201.
d. Prepare Conjugate dilution by combining required amounts of Conjugate
Concentrate and Sample Buffer, and mix well.
e. Return the remaining Conjugate Concentrate to storage at 2-8(degree)C.
9. Aspirate the Sample Buffer from the troughs and add 2.0 mL diluted
Conjugate to each strip.
10. Replace each lid and incubate on the rotator (at 50-60 rpm) for 45 minutes
at room temperature.
11. Repeat wash procedure from steps 6 and 7.
12. Completely aspirate the Sample Buffer and add 2.0 mL of deionized water to
each strip.
13. Replace each lid and place on rotator (at 50-60 rpm) for 5 minutes.
14. Remove Substrate from refrigerator during final wash (step 13).
15. Aspirate deionized water from each strip.
16. Add 1.0 mL of Substrate to each strip.
Page 10
17. Replace each lid and gently move trays back and forth 2-3 times by hand on
work surface to ensure strips are completely immersed in Substrate.
18. Incubate for exactly 10 minutes at room temperature without rotation.
Note: It is important not to exceed the 10 minute Substrate incubation
time.
19. Stop the color development of the strips as follows:
a. Aspirate the Substrate.
b. Add 2.0 mL of deionized water to each strip.
c. Immediately aspirate the contents of the tray(s).
d. Repeat steps b and c two more times.
e. Add 2.0 mL deionized water to each strip and replace the tray lid(s).
f. Place on rotator for 5 minutes.
g. Aspirate water completely.
20. Allow the developed strips to air dry in the tray(s).
21. Handle the strips carefully; use clean forceps to remove from troughs.
22. Read and interpret the dry strips as soon as possible, since developed
strips exposed to light may experience fading of bands. Store developed
strips in the dark at room temperature.
Page 11
QUALITY CONTROL
Both the OraSure Low Positive and Negative Controls must be assayed regardless
of the number of samples tested. The OraSure High Positive must be assayed with
the first run of every package of strips, but is optional in subsequent runs.
This High Positive Control strip should be retained as a reference. It will be
compared to the test strips run from that package to determine band
identification and placement.
The following conditions must be met for the assay to be considered valid:
1. Negative Control: No bands are observed on the strip.
2. Low Positive Control: Bands are present (P) at gp160, gp41, and p24.
(Weakly Reactive) Other bands may or may not be visible.
3. High Positive Control: Bands are present (P) at gp160, gp41 and p24. Bands
(Strongly Reactive) are visible at gp120, p65, p51, p31, and p18. The
p55 band may or may not be visible (see Figure 1,
page 12).
INTERPRETATION OF RESULTS
1. Band Identification
a. Correlate the band position of the OraSure High Positive Control strip
with Figure 1 on page 12 to identify the HIV-1 viral bands and their
positions.
b. Compare each test strip to the OraSure High Positive Control strip for
identification of reactive bands.
2. Band Intensity
a. Compare the bands of each strip and control to the gp41 band on the
OraSure Low Positive Control strip and assign a level of intensity as
follows:
---------------------------------------------------------------------------
Assignment Definition
---------------------------------------------------------------------------
Present (P) The band intensity is greater than or equal to the gp41
band on the OraSure Low Positive Control strip.
---------------------------------------------------------------------------
Indeterminate (I) The band is visible but intensity is less than the gp41
band on the OraSure Low Positive Control strip.
---------------------------------------------------------------------------
Absent (A) No reactivity is observed.
---------------------------------------------------------------------------
3. Strip Interpretation
a. Based on band position and reactivity, analyze the results and assign
each strip a final result.
---------------------------------------------------------------------------
Test Result Definition
---------------------------------------------------------------------------
Positive Any two of the three major bands of diagnostic
significance below must be Present.
gp160 and/or gp41 p24
gp120
Other bands may or may not be present.
---------------------------------------------------------------------------
Indeterminate Any visible band reactivity which does not meet the
criteria for a Positive result as described above.
---------------------------------------------------------------------------
Negative Band reactivity is Absent.
---------------------------------------------------------------------------
Page 12
Figure 1: Protein Band Identification on an OraSure(R) HIV-1 Western Blot Strip
On the left is a representation of an OraSure Western blot strip developed
with OraSure High Positive Control. The illustration is a reference for band
identification and position (see Interpretation of Results, page 11, step 1).
On the right is a representation of the HIV-1 virus. The bands correlate to
corresponding viral subpart origin.
[DIAGRAM]
Viral Origin of HIV-1 Associated Bands
------------------------------------------------
Virus Gene Gene Product and Description
------------------------------------------------
gp160 env protein precursor
env gp120 outer env protein
gp41 transmembrane protein
------------------------------------------------
p65 reverse transcriptase
pol p51 reverse transcriptase
p31 endonuclease
------------------------------------------------
p55 core protein precursor
gag p24 core
p18 core
-----------------------------------------------
Page 13
LIMITATIONS OF THE PROCEDURE
1. The assay must be performed in strict accordance with these instructions
to obtain accurate, reproducible results.
2. Although a Positive result may indicate infection with the HIV-1 virus, a
diagnosis of Acquired Immunodeficiency Syndrome (AIDS) can be made only if
an individual meets the case definition of AIDS established by the Centers
for Disease Control./10/ A repeat test on an independent sample should be
considered to control for sample mix-up or operator error, and to verify a
positive test result.
3. Individuals may present incomplete banding patterns due to the natural
history of AIDS or other immunodeficiency states, e.g.:
a. AIDS patients may lose antibody reactions to p24 and p31;
b. Infants born to HIV-1 infected mothers, but who are uninfected may
display incomplete patterns as passively acquired maternal antibodies
begin to disappear;
c. Individuals who have recently seroconverted may display incomplete band
patterns;
d. Infected patients with malignancies and individuals receiving
immunosuppressive drugs may fail to develop a Positive result;
e. Individuals infected with HTLV-I/II or HIV-2, may exhibit
cross-reactivity;
f. Individuals may develop incomplete patterns that reflect the composition
of experimental HIV sub-unit vaccines that they may have received.
4. A person who has antibodies to HIV-1 is presumed to be infected with the
virus, except that a person who has participated in an HIV vaccine study
may develop antibodies to the vaccine and may or may not be infected with
HIV. Clinical correlation is indicated with appropriate counseling, medical
evaluation and possibly additional testing to decide whether a diagnosis of
HIV infection is accurate.
5. Since reactivity of any degree with any of the proteins present on the
strip results in an indeterminate result, all samples interpreted as
Indeterminate should be repeated using the original specimen. In addition,
individuals with indeterminate results should be followed for up to six
months./14/
6. Do not use this kit as the sole basis of diagnosis of HIV-1 infection.
7. A Negative result does not exclude the possibility of HIV-1 infection.
8. The OraSure HIV-1 Western Blot Kit is a biological product which, although
highly consistent, does display variation from lot to lot. Examples of
these variations include bands which have a slightly wavy or slanted
appearance, small artifacts within the banding area, and a light smearing
pattern across a set of strips. These are considered normal assay
variations which infrequently affect assay interpretation. However, if they
do interfere with the assay interpretation, call the assay invalid and
repeat.
PERFORMANCE CHARACTERISTICS
The performance of the OraSure HIV-1 Western Blot Kit was evaluated by
comparison of OraSure results with those obtained from matched serum specimens
tested by a licensed HIV-1 Western blot. These specimens were collected
prospectively in a clinical study of low risk (n = 2,382), high risk (n = 698),
and AIDS (n = 242) populations. In addition, non-specificity specimens (n = 248)
were obtained from subjects with non-HIV-1 related medical conditions that might
result in antibodies cross-reactive with HIV-1 proteins. All of the high risk
and AIDS subjects, and 495 of the low risk subjects were tested by Western blot
irrespective of their EIA results. EIA testing of an additional 1,887 "screen
only" low risk subjects was carried out using the bioMerieux Oral Fluid
Vironostika HIV-1 Microelisa System in an effort to find EIA repeatedly reactive
samples (from uninfected individuals) with which to challenge the OraSure
Western blot. Testing of the 1,887 "screen only" subjects identified 14 OraSure
specimens as repeatedly reactive.
Page 14
These 14 OraSure specimens and their matching sera were also advanced to Western
blot testing. Thus, a total of 1,697 matched OraSure and serum specimens were
tested by Western blot at five testing laboratories throughout the United
States.
Low risk subjects, primarily normal blood donors, were persons with no known
risk factors. Of the 698 high risk specimens, 363 were from homosexuals, 116
from injection drug users (IDUs), 83 from persons with multiple heterosexual
contacts, and 44 from hemophiliacs. The remaining 92 high risk subjects included
bisexuals, prostitutes, and individuals with other acknowledged risk factors.
Specimens from 242 persons with clinically diagnosed AIDS were also tested.
The frequency of virus-specific bands and interpretation by risk group using the
OraSure HIV-1 Western Blot Kit are presented in Table 1.
Table 1. Frequency of Virus-Specific Bands ("Present" or "Indeterminate") and
Interpretation of Specimens Tested by the OraSure HIV-1 Western Blot Kit
- ------------------------------------------------------------------------------------------------------------
Low OraSure HIV-1 Band Specificities (# and % of samples)/g/ Non-
-----------------------------------------------------------------------
Risk/a/ WB Result gp160 gp120 p65 p55 p51 gp41 p31 p24 p18 Viral
- ------------------------------------------------------------------------------------------------------------
EIA POS 0 0 0 0 0 0 0 0 0 0 0
neg. (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
-----------------------------------------------------------------------------------------------
n=495 IND 100/b/ 0 0 0 0 0 5 0 13 2 87
(20.2) (0.0) (0.0) (0.0) (0.0) (0.0) (1.0) (0.0) (2.6) (0.4) (17.6)
- ------------------------------------------------------------------------------------------------------------
EIA POS 0 0 0 0 0 0 0 0 0 0 0
RR/h/ (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
-----------------------------------------------------------------------------------------------
n=14 IND 3/c/ 0 0 0 0 0 0 0 1 0 2
(21.4) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (7.1) (0.0) (14.3)
- ------------------------------------------------------------------------------------------------------------
- ------------------------------------------------------------------------------------------------------------
High OraSure HIV-1 Band Specificities (# and % of samples)/g/ Non-
-----------------------------------------------------------------------
Risk/d/ WB Result gp160 gp120 p65 p55 p51 gp41 p31 p24 p18 Viral
- ------------------------------------------------------------------------------------------------------------
EIA POS 0 0 0 0 0 0 0 0 0 0 0
neg. (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
-----------------------------------------------------------------------------------------------
n=264 IND 45/e/ 4 0 6 0 5 0 0 8 0 33
(17.0) (1.5) (0.0) (2.3) (0.0) (1.9) (0.0) (0.0) (3.0) (0.0) (12.5)
- ------------------------------------------------------------------------------------------------------------
EIA POS 429 429 427 417 160 412 427 394 410 238 1
RR/h/ (98.8) (98.8) (98.4) (96.1) (36.9) (94.9) (98.4) (90.8) (94.5) (54.8) (0.2)
-----------------------------------------------------------------------------------------------
n=434/i/ IND 3 3 2 2 0 2 1 0 0 0 1
(0.7) (0.7) (0.5) (0.5) (0.0) (0.5) (0.2) (0.0) (0.0) (0.0) (0.2)
- ------------------------------------------------------------------------------------------------------------
- ------------------------------------------------------------------------------------------------------------
AIDS/f/ OraSure HIV-1 Band Specificities (# and % of samples)/g/ Non-
-----------------------------------------------------------------------
WB Result gp160 gp120 p65 p55 p51 gp41 p31 p24 p18 Viral
- ------------------------------------------------------------------------------------------------------------
EIA POS 0 0 0 0 0 0 0 0 0 0 0
neg. (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
-----------------------------------------------------------------------------------------------
n=1 IND 1 1 0 0 0 0 0 0 1 0 0
(100.0) (100.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (100.0) (0.0) (0.0)
- ------------------------------------------------------------------------------------------------------------
EIA POS 236 236 236 211 35 207 236 190 189 87 0
RR/h/ (97.9) (97.9) (97.9) (87.6) (14.5) (85.9) (97.9) (78.8) (78.4) (36.1) (0.0)
-----------------------------------------------------------------------------------------------
n=241 IND 5 5 4 1 0 1 4 1 0 1 0
(2.1) (2.1) (1.7) (0.4) (0.0) (0.4) (1.7) (0.4) (0.0) (0.4) (0.0)
- ------------------------------------------------------------------------------------------------------------
a. Persons with no known risk factors; primarily normal blood donors.
b. 80 of 100 results Indeterminate due to non-viral bands only.
c. 2 of 3 results Indeterminate due to non-viral bands only.
d. Homosexuals, IDUs, and other accepted risk designations.
e. 32 of 45 results Indeterminate due to non-viral bands only.
f. CDC Classification; MMWR 1982; 31: 507-508.
g. Band patterns for negative samples do not appear in this table. By
definition, negative samples show no reactivity.
h. RR indicates repeatedly reactive OraSure EIA results.
i. Includes two EIA false positive individuals whose OraSure Western blots were
negative.
Page 15
Sensitivity Studies
The performance of the OraSure Western blot in seropositive subjects was
evaluated by comparing results to those obtained by testing matched serum
samples collected from individuals at high risk for HIV infection and from
clinically diagnosed AIDS patients. A comparison of OraSure and serum results
is presented in Table 2.
Table 2. OraSure and Serum Western Blot Results for
Confirmed Positive High Risk and AIDS Populations
- --------------------------------------------------------------------------------
Orasure Specimen Results Serum Results
----------------------------------------------------
Risk Confirmed EIA Western Blot Western Blot
----------------------------------------------------
Category Positives RR P I N P I N
- --------------------------------------------------------------------------------
AIDS 242 241/a/ 236 6/a,b/ 0 241 1 0
- --------------------------------------------------------------------------------
High Risk 431 431 429 2/c/ 0 431 0 0
- --------------------------------------------------------------------------------
Total 673 672 665 8 0 672 1 0
- --------------------------------------------------------------------------------
a. One OraSure sample was EIA false negative, Western blot indeterminate
(gp160+, p24+/-); the matching serum sample was EIA repeatedly reactive and
Western blot positive (gp160+, pg120+, p65+, p51+, p24+).
b. One of the six Western blot indeterminate OraSure samples was repeatedly
reactive on EIA and concordant on Western blot with an indeterminate result
for the matching serum specimen. The remaining five OraSure specimens were
discordant (indeterminate) with the matching serum specimens (positive) due
to the required cardinal bands being visible but of insufficient intensity
to be scored as Present.
c. One OraSure sample was discordant due to the intensity of bands on the
OraSure Western blot (gp160+, gp120+/-, p65+/-, p51+/-, gp41+/-). The
banding pattern of the second indeterminate OraSure sample was gp160+,
gp120+/-. The matching serum specimens' banding patterns were gp160+,
gp120+, p65+, p51+/-, gp41+, p24+/-, p18+/-, and gp160+, gp120+, p65+/-,
gp41+, p24+/-, p18+/-, respectively.
RR = Repeatedly Reactive; P = Positive; I = Indeterminate; N = Negative.
In this study, the sensitivity of the OraSure Western blot testing of oral
specimens from the 242 confirmed positive AIDS subjects was 97.5% (236/242)
with 2.5% (6/242) indeterminate, and from the 431 confirmed high risk subjects
was 99.5% (429/431) with 0.5% (2/431) indeterminate, with no OraSure Western
blot false negatives in either group. All OraSure indeterminate blots showed
the gp160 band as present and at least one additional cardinal band (gp120,
gp41, p24) as visible, but of insufficient intensity to be called present. One
of the OraSure indeterminate blots corresponded to the Western blot
indeterminate serum specimen.
Specificity Studies
The performance of the OraSure Western blot in documented seronegative subjects
was evaluated by testing specimens from 495 EIA negative subjects (using oral
fluid) at low risk for HIV-1 infection, 14 EIA repeatedly reactive specimens
found by screening 1,887 persons at low risk for HIV-1 infection, 248 subjects
with non-HIV related medical conditions (non-specificity subjects), and 267
specimens from high risk seronegative subjects. Thus, a total of 1,024 OraSure
HIV-1 Western blots and serum Western blots were performed on these
individuals. The results of this testing are presented in Table 3.
Page 16
Table 3. Comparative Study of Western Blot Results in Low Risk,
Non-Specificity, and High Risk, HIV-1 Negative Populations
- -----------------------------------------------------------------------------------------
EIA Result for OraSure HIV-1 Licensed Serum HIV-1 Western Blot
OraSure Western Blot Interpretation
-------------------------------------------
Specimens Interpretation Positive Ind. Negative Totals
- -----------------------------------------------------------------------------------------
EIA Positive 0 0 0 0
-------------------------------------------------------------------------
Negative Indeterminate 0 96 111 207
-------------------------------------------------------------------------
(n=1,007) Negative 0 295 505 800
- -----------------------------------------------------------------------------------------
EIA Positive 0 0 0 0
-------------------------------------------------------------------------
RR Indeterminate 0 3 1 4
-------------------------------------------------------------------------
(n=17) Negative 0 7 6 13
- -----------------------------------------------------------------------------------------
Totals 0 401 623 1,024
- -----------------------------------------------------------------------------------------
Seventeen OraSure specimens (14 low risk and 3 high risk) were EIA repeatedly
reactive. Thirteen of the 17 EIA false positive specimens were correctly
identified as negative by the OraSure HIV-1 Western blot. Thus 2,893 out of
2,897 subjects (99.9%) were correctly identified as HIV-1 antibody negative by a
combination of EIA and Western blot testing of OraSure samples. The four
remaining specimens were indeterminate by OraSure HIV-1 Western blot (two of the
four due to non-viral bands only). The indeterminate rate for uninfected persons
who are EIA repeatedly reactive by OraSure was 23.5% (4/17) as compared to 58.8%
(10/17) for serum.
Western blot was also performed on serum and OraSure specimens from 1,007 EIA
negative subjects (using oral fluid). This testing identified 20.6% of OraSure
specimens and 39.2% of sera as indeterminate. The overall concordance between
the two types of specimens was 59.6%. Differences were largely due to non-viral
bands that were present for one type of sample but not the other for individual
subjects.
Analytical Sensitivity
Titration of Matching OraSure and Serum Specimens
Fifteen randomly selected matching OraSure and serum repository specimens that
had been obtained from HIV-positive individuals were titrated. Titrated serum
specimens were tested with the licensed serum HIV-1 Western Blot Kit and
matching titrated OraSure specimens were tested in parallel with the OraSure
HIV-1 Western Blot Kit. The assay endpoint in this study was the last dilution
at which a positive Western blot result was observed for each specimen. The
results of this study are shown in Table 4.
Endpoints were obtained for all serum specimens and for 14 of the 15 OraSure
specimens tested. The one specimen not yielding an endpoint (ID# 19052) had an
indeterminate result when the neat OraSure specimen was tested. This assignment
was based on positive reactivity for the gp160 and gp120 bands, and an
indeterminate reactivity for the gp41 band. The corresponding serum specimen for
subject 19052 also had a comparatively low titer (1:4). A review of medical
records revealed that this individual was severely immunocompromised at the time
of specimen acquisition (CD4+ count = 18/mm/3/).
The average ratio of the serum endpoints to the OraSure endpoints was 5.7:1. The
difference in analytical sensitivity between the licensed serum Western Blot Kit
and the OraSure Western Blot Kit ranged from a ratio of 12.8 to 0.40.
Page 17
Table 4. Highest Dilution Yielding Positive
Western Blot Results for 15 Matching OraSure and Serum Specimens
- -------------------------------------------------------------------------------
Specimen Highest Highest
Number OraSure Dilution/a/ Serum Dilution/a/ Ratio/b/
- -------------------------------------------------------------------------------
19026 1:20 1:256 12.8
- -------------------------------------------------------------------------------
19027 1:20 1:256 12.8
- -------------------------------------------------------------------------------
19078 1:20 1:256 12.8
- -------------------------------------------------------------------------------
19095 1:20 1:256 12.8
- -------------------------------------------------------------------------------
19105 1:5 1:64 12.8
- -------------------------------------------------------------------------------
19079 1:10 1:64 6.4
- -------------------------------------------------------------------------------
19032 1:10 1:16 1.6
- -------------------------------------------------------------------------------
19059 1:10 1:16 1.6
- -------------------------------------------------------------------------------
19066 1:50 1:64 1.28
- -------------------------------------------------------------------------------
19069 1:50 1:64 1.28
- -------------------------------------------------------------------------------
19080 1:250 1:256 1.02
- -------------------------------------------------------------------------------
19044 1:5 1:4 0.8
- -------------------------------------------------------------------------------
19101 1:100 1:64 0.64
- -------------------------------------------------------------------------------
19033 1:10 1:4 0.40
- -------------------------------------------------------------------------------
Mean ratio 5.7
- -------------------------------------------------------------------------------
19052 Neatc 1:4 -
- -------------------------------------------------------------------------------
/a./ Beyond standard specimen dilution per assay protocol.
/b./ Ratio of serum endpoint dilution/OraSure endpoint dilution.
/c./ Specimen #19052 was indeterminate (with viral bands) when the undiluted
OraSure specimen was tested.
Titration of OraSure Seroconversion Specimens
Repository OraSure and plasma specimens from an earlier seroconversion study
were used to assess the analytical sensitivity of the OraSure HIV-1 Western
Blot.
OraSure specimens were diluted and each dilution was assayed by EIA and Western
blot. The objective of this study was to determine the highest dilution of the
OraSure specimen that would produce a repeatedly reactive EIA result, based on
product insert criteria, and would demonstrate viral band reactivity in the
OraSure HIV-1 Western Blot Kit.
Table 5 shows the results of this testing. For each of the four time points,
the OraSure Western Blot demonstrated viral bands at dilutions which produced
non-reactive EIA results, yielding an average of **10-fold enhanced sensitivity
over the EIA for OraSure specimens.
Table 5. Reactivity of OraSure HIV-1 Seroconversion Specimens in the Oral Fluid
Vironostika HIV-1 Microelisa System and the OraSure HIV-1 Western Blot Kit
- ------------------------------------------------------------------------------------------------
ID# Highest EIA Dilution Highest WB Dilution Blot to EIA Sensitivity
- ------------------------------------------------------------------------------------------------
01SC052591 Negative at Neat Positive at 1:10* ** 10
- ------------------------------------------------------------------------------------------------
01SC052691 RR at Neat Positive at 1:5* ** 5
- ------------------------------------------------------------------------------------------------
01SC052791 RR at Neat Positive at 1:5* ** 5
- ------------------------------------------------------------------------------------------------
01SC052991 RR at Neat Positive at 1:20* ** 20
- ------------------------------------------------------------------------------------------------
* Indicates the highest dilution tested RR = Repeatedly Reactive Reproducibility
** Less than or equal to
Reproducibility
The reproducibility of the OraSure HIV-1 Western Blot Kit was evaluated at
three separate test laboratories. The study included testing a three-member
panel of pooled OraSure specimens with the OraSure HIV-1 Western Blot Kit. The
OraSure reproducibility panel consisted of an HIV-1 antibody positive specimen,
an HIV-1 antibody negative specimen, and an HIV-1 Western blot indeterminate
specimen.
Page 18
The panel members were tested on three separate days, using three separate
OraSure HIV-1 Western Blot Kit production lots, resulting in a total of 27 test
results being generated for each panel member. The percentage of times each band
was scored reactive is presented in Table 6.
Table 6. Reproducibility of the OraSure HIV-1 Western Blot Kit
- --------------------------------------------------------------------------------
Specimen Specimen Percent Frequency of Visible Bands/c/
--------------------------------------------------------
# Reactivity gp160 gp120 p65 p55 p51 gp41 p31 p24 p18
- --------------------------------------------------------------------------------
1 Positive/a/ 100 100 100 0 100 100 100 100 18.5
- --------------------------------------------------------------------------------
2 Negative 0 0 0 0 0 0 0 0 0
- --------------------------------------------------------------------------------
3 Indeterminate/b/ 100 100 100 0 100 100 3.7 70.4 0
- --------------------------------------------------------------------------------
/a./ Positive specimen known banding pattern: reactivity for gp160, gp120, p65,
p51, gp41, p31, p24 and no reactivity for p55, p18.
/b./ Indeterminate specimen known banding pattern: reactivity for gp160, gp120,
p65, p51, gp41, p24; and no reactivity for p55, p31, p18.
/c./ Frequency of visible bands (either Indeterminate or Present).
The results demonstrate that for positive specimens, negative specimens, and
indeterminate specimens with known banding patterns, reproducibility is high.
Reactivity in Other Disease Conditions
Matching OraSure and serum specimens were obtained at three sites from 248
subjects who were enrolled in the clinical trial because they had non-HIV-1
medical conditions that might result in antibodies cross-reactive with HIV-1
proteins or other potentially interfering factors. Specimens studied included 89
from multiparous women, 69 from subjects with non-HIV viral infections, 50
receiving anticoagulation therapy, 26 with autoimmune diseases other than AIDS,
11 with oral pathology, and 3 with polyclonal or monoclonal gammopathy. Although
bands were present at viral band locations for four samples (1.6%), none of the
strips could be interpreted as positive. Results are presented in Table 7.
Table 7. Results of OraSure Western Blot Testing on Samples from Subjects with
Non-HIV Disease Processes or Other Potentially Interfering Factors
- ----------------------------------------------------------------------------------------
Band Specificities (# and % of samples)
-------------------------------------------------------------
OraSure HIV-1 Non-
WB Result gp160 gp120 p65 p55 p51 gp41 p31 p24 p18 viral
- ----------------------------------------------------------------------------------------
NEG 186 0 0 0 0 0 0 0 0 0 0
(75.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)
- ----------------------------------------------------------------------------------------
IND 62/a/ 0 0 0 0 0 2 0 2 1 58
(25.0) (0.0) (0.0) (0.0) (0.0) (0.0) (3.2) (0.0) (3.2) (1.6) (93.5)
- ----------------------------------------------------------------------------------------
Total 248 0 0 0 0 0 2 0 2 1 58
(100.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.8) (0.0) (0.8) (0.4) (23.4)
- ----------------------------------------------------------------------------------------
/a./ 58 of 62 results Indeterminate due to non-viral bands only.
Testing of specimens from this population revealed that the number of OraSure
indeterminates (62; 25.0%) was substantially less than the number of serum
indeterminates (121; 48.8%). The number of serum indeterminates due to the
presence of viral bands (27; 10.9%) was substantially greater than the number of
OraSure indeterminates due to the presence of viral bands (4; 1.6%).
Page 19
Summary
In this clinical trial using the recommended OraSure algorithm, 3,558/3,570
subjects received the correct HIV-1 antibody results from a single OraSure
sample the first time it was tested. In 11 of the remaining subjects, the
Western blot was indeterminate: for these 11, the algorithm would lead to
appropriate follow-up testing. Thus, in 3,569/3,570 (99.97%) subjects either the
correct result was reached or appropriate follow-up testing would be triggered.
It is concluded that OraSure testing is a highly accurate alternative to serum
testing.
Page 20
REFERENCES
1. Gallo RC, Salahuddin SZ, Popovic M, et al.: Frequent detection and
isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and
at risk for AIDS. Science 1984; 224: 500-503.
2. Montagnier L, et al.: A new type of retrovirus isolated from patients
presenting with lymphadenopathy and Acquired Immune Deficiency Syndrome:
Structural and antigenic relatedness with equine infectious anemia virus.
Ann Virol (Inst. Pasteur) 1984; 135E: 119.
3. Curran JW, Morgan WM, Hardy AM, et al.: The epidemiology of AIDS: Current
status and future prospects. Science 1985; 229: 1352-1357.
4. Centers for Disease Control: Antibodies to a retrovirus associated with
Acquired Immunodeficiency Syndrome (AIDS) in populations with increased
incidences of the syndromes. MMWR 1984; 33(27): 377-399.
5. Archibald DW, Zon LI, Groopman JE, et al.: Salivary antibodies as a means
of detecting human T-cell lymphotrophic virus type III
lymphadenopathy-associated virus infection. J Clin Micro 1986; 24: 873-875.
6. Parry JV, Perry KR and Mortimer PP: Sensitive assays for viral antibodies
in saliva: an alternative to tests on serum. Lancet 1987; 2: 72-75.
7. Major CJ, Read SE, Coates RA, et al.: Comparison of saliva and blood for
human immunodeficiency virus prevalence testing. J Infect Dis 1991; 163:
699-702.
8. Tsang VCW, Hancock K, Wilson M, et al.: Enzyme-linked
immuno-electrotransfer blot technique (EITB) (Western blot) for
HTLV-III/LAV antibodies. Developmental Centers for Disease Control,
Atlanta; 1985.
9. Watson-Martin P, Burger D, Caouette S, et al.: Importance of confirmatory
tests after strongly positive HTLV-III screening tests. N Engl J Med 1986;
314: 1577.
10. 1993 Revised classification system for HIV infection and expanded
surveillance case definition for AIDS among adolescents and adults. MMWR
1992; 41: No. RR-17.
11. Marlink RG, et al.: Clinical hematologic and immunologic cross-sectional
evaluation of individuals exposed to Human Immunodeficiency Virus Type-2
(HIV-2). AIDS Res. Human Retroviruses 1988; 4: 137-148.
12. OAR 437, Division 2, General occupational safety and health rules,
Subdivision Z: Toxic and hazardous substances: Bloodborne Pathogens
(1910.1030); 7/1/92:1-15.
13. Decontamination of laboratory sink drains to remove azide salts. Centers
for Disease Control: Safety Management No. CDC-22, Atlanta; 1976.
14. Interpretation and use of the Western blot assay for serodiagnosis of Human
Immunodeficiency Virus Type 1 Infections. MMWR 1989; 38: No. S-7.
Page 21
KIT AVAILABILITY
OraSure(R) HIV-1 Western Blot Kit
20 Tests
Product Number 501-0000
Manufactured by
OraSure Technologies, Inc.
8505 SW Creekside Place
Beaverton, Oregon USA 97008
Distributed by
bioMerieux, Inc.
Box 15969
Durham, North Carolina 27704-0969
For technical assistance,
contact bioMerieux Customer Support Services
at 1-800-682-2666
Printed in USA
101-0004-6
Page 22
Human Immunodeficiency Virus Type 1 (HIV-1)
Oral Fluid Vironostika(R) HIV-1 Microelisa System 43-01812
For in vitro diagnostic use. [LOGO OF BIOMERIEUX]
Store between 2-8(degrees)C.
INTENDED USE
Oral Fluid Vironostika(R) HIV-1 Microelisa System is an enzyme-linked
immunosorbent assay (ELISA) from bioMerieux, Inc. for the qualitative
determination of antibody to HIV-1 only with oral fluid specimens obtained using
the OraSure(R) HIV-1 Oral Specimen Collection Device manufactured by Epitope,
Beaverton, Oregon. The assay is intended to be used as an aid in the diagnosis
of HIV-1 infection and must not be used to qualify blood donors or units of
blood.
Note: See Warnings, Interpretations of results, Limitations and Performance
characteristics sections for information on:
1. Reduced sensitivity and specificity of testing with OraSure HIV-1 specimens
compared with testing blood specimens.
2. The need for follow up testing with a blood specimen when subjects have
repeatedly reactive ELISA results using OraSure HIV-1 specimens.
3. Reporting test results to the ordering physician or someone under the
supervision of the ordering physician.
SUMMARY AND EXPLANATION OF THE TEST
Available data indicate that the acquired immunodeficiency syndrome (AIDS) is
caused by a virus transmitted by sexual contact, exposure to blood (including
sharing contaminated needles and syringes) or certain blood products, or
transmitted from an infected mother to her fetus or child during the perinatal
period./1/ Human Immunodeficiency Virus Type 1 (HIV-1) has been isolated from
patients with AIDS and AIDS-related complex (ARC), and from healthy persons at
high risk for AIDS./2/ The incidence of antibodies specific for HIV-1 in AIDS
and ARC patients and persons at increased risk for AIDS is high. The prevalence
of HIV-1 infection in people not known to be at increased risk is not known.
The HIV-1 enzyme-linked immunosorbent assay (ELISA) was developed to detect
antibodies to HIV-1 and to identify potentially infectious units of donated
blood and plasma. Early experience using oral fluid for HIV-1 testing/3/
suggested that there was a problem of specimen instability and assay
insensitivity. Saliva is a complex mixture of parotid, submandibular, sublingual
and minor salivary gland secretions mixed with mucin, bacteria, leukocytes,
sloughed epithelial cells and gingival crevicular fluid. Gingival crevicular
fluid, or mucosal transudate, is the fluid derived from the passive transport of
serum components through the oral mucosa into the mouth. The OraSure HIV-1
Specimen Collection Device enhances the flow of mucosal transudate across the
mucosal surfaces onto an absorptive cotton pad. Antibodies are among the serum
components in mucosal transudate and OraSure HIV-1 specimens. Oral fluid
contains a number of enzymes (proteases) which degrade antibodies. The OraSure
HIV-1 device includes preservatives that are effective in protecting anitbodies
from degredation.
The ELISA was designed to be extremely sensitive in order to afford maximum
protection of the blood supply. The sensitivity of the ELISA test using OraSure
HIV-1 specimens is reduced compared with testing blood specimens (see
Performance Characteristics section). Non specific ELISA reactions may be seen
in specimens from some people who, for example, due to prior pregnancy, blood
transfusion, or other exposure, have antibodies to the human cells or media in
which the HIV-1 is grown for manufacture of the ELISA./4/ Because of nonspecific
reactions, it is appropriate to investigate specimens found to be reactive by
ELISA in a manner that gives improved predictability that HIV-1 antibody, in
fact, is present. When a specimen reacts in an initial test (is initially
reactive), the ELISA should be repeated in duplicate on the same specimen.
Reactivity in either or both of these duplicate tests (repeatedly reactive) is
highly predictive of the presence of HIV-1 antibody. Repeatedly reactive
specimens obtained from people at high risk for HIV-1 infection (e.g. homosexual
men, hemophiliacs, or intravenous drug users) are usually found to contain
antibodies by additional more specific, or supplemental, testing. Currently,
there is no supplemental assay that is FDA-licensed for use with OraSure HIV-1
specimens. When the ELISA is used to screen populations in which the prevalence
of HIV-1 infection is low, repeatedly reactive specimens may be found not to
contain antibodies to HIV-1 by additional more specific assays. The frequency of
nonspecific reactivity using OraSure HIV-1 specimens is increased compared with
using blood specimens (see Performance characteristics section). Information
about prevalence of HIV-1 infections in persons in various categories of risk,
as well as clinical and public health guidelines, are available in the
publication Morbidity and Mortality Weekly Reports.
Although for all clinical and public health applications of the ELISA, both the
degree of risk for HIV-1 infection of the person studied and the degree of
reactivity of the specimen may be of value in interpreting the test, these
correlations are imperfect. Therefore, in most settings it is appropriate to
investigate repeatedly reactive specimens by additional more specific, or
supplemental, tests. Due to the fact that, at this time, there is no
FDA-licensed additional more specific test for HIV-1 antibodies in an OraSure
HIV-1 specimen, subjects whose OraSure HIV-1 test result is repeatedly reactive
should be advised to have a blood specimen tested with a licensed screening test
for HIV-1 antibodies, and a licensed supplemental test when appropriate.
PRINCIPLE OF THE TEST
HIV-1 antigen is derived from HIV-1 virus propagated in T-lymphocyte culture as
reported by Popovic, et al./5/ The virus is purified by ultracentrifugation and
inactivated by disruption and is coated onto the microelisa wells contained in
the Oral Fluid Vironostika HIV-1 Microelisa System.
To process the oral fluid specimen, the OraSure HIV-1 Specimen Vial is
centrifuged and specimen is eluted from the collection pad. A minimum volume of
eluate is required to assess if an oral fluid specimen was collected. The eluate
is diluted and added to microelisa wells. During incubation, HIV-1 antibodies
in a test specimen form immune complexes by interacting with the HIV-1 antigens
of the solid phase. Following incubation, the specimen is aspirated and the well
is washed with buffer. Antibodies to human immunoglobulin (goat) conjugated with
horseradish peroxidase (HRP) are added and bind to the antibody-antigen complex
of the solid phase during a second incubation. Following a wash and incubation
with ABTS (2,2'-azino-di-[3-ethylbenzthiazoline-6-sulfonate]) substrate, a green
color is produced. The enzyme reaction is stopped by the addition of a fluoride
solution./6/ The amount of anti-HIV-1 present in the specimen is proportional to
color development.
2
REAGENTS
For in vitro diagnostic use.
Components in each Oral Fluid Vironostika(R) HIV-1 Microelisa System
192 Test 9600 tests
- -------- ----------
2 stripholders 100 stripholders HIV-1 Microelisa Strips - Eight per
holder, each containing 12 HIV-1 antigen
(inactivated) coated wells; contained in
a foil pack with silica gel desiccant.
1 vial 16 vials Diluent Concentrate, 5x - Normal goat
(50 ml) (50 ml ea.) serum in phosphate buffered saline;
contains 0.05% Thimerosal as a
preservative.
1 bottle 16 bottles Tween-20, 5% - Contains 0.1% Thimerosal
(2.5 ml) (2.5 ml ea.) as a preservative.
1 bottle 16 bottles Diluent Reagent Water.
(200 ml) (200 ml ea.)
1 vial 3 vials Positive Control Serum (Inactivated) -
Lyophilized human serum with protein
stabilizers; reactive for anti-HIV-1.
1 vial 8 vials Negative Control Serum - Lyophilized
human serum with protein stabilizers;
nonreactive for anti-HIV-1.
2 bottles 15 bottles Dilsim(TM) - Lyophilized specimen
inactivator medium; contains bovine
proteins, salt, and surfactant.
2 bottles 15 bottles Wash Concentrate 50X - Contains 2.5%
(60 ml ea.) (60 ml ea.) surfactant.
1 vial 32 vials Peroxidase Conjugated Goat Anti-human
Immunoglobulins (EnzAbody(R)-O) -
Lyophilized with protein stabilizers.
2 bottles 30 bottles ABTS Substrate - Lyophilized 2,2'-azino-
di-[3-ethylbenzthiazoline-6-sulfonate].
2 bottles 30 bottles ABTS Diluent - Contains hydrogen
(62 ml ea.) (62 ml ea.) peroxide in citric acid buffer.
1 bottle 15 bottles Stop Solution - Contains 0.28% sodium
(120 ml) (120 ml ea.) fluoride.
1 each 5 each Clamp and rod - Closure of foil pack.
10 sheets 300 sheets Plate sealers - Perforated, adhesive.
3
WARNINGS
1. Laboratories must successfully complete training and testing of the OraSure
HIV-1 Laboratory Qualification Panel prior to testing OraSure HIV-1 oral
fluid specimens.
2. A minimum of 0.75 ml volume of processed OraSure HIV-1 specimen is required
to assure that a specimen was collected.
3. HIV-1 antibody testing of OraSure HIV-1 specimens has reduced sensitivity
and specificity compared with HIV-1 antibody testing of blood specimens
(see Performance characteristics section for details).
4. Due to the possibility of false positive HIV-1 test results and the lack of
an FDA licensed supplemental test for HIV-1 antibodies in OraSure HIV-1
specimens, subjects whose OraSure HIV-1 specimen is repeatedly reactive on
the Oral Fluid Vironostika Microelisa System should be advised to have a
blood specimen tested by a licensed screening test and a licensed
supplemental test for HIV-1 antibodies, as appropriate.
PRECAUTIONS
For in vitro diagnostic use only.
Note: The HIV-1 Microelisa Strips are marked with the product-specific code, E1.
1. Caution: Handle all Oral Fluid Vironostika HIV-1 Microelisa System
biological materials though capable of transmitting infectious agents. The
antigen used to coat the microelisa wells has been inactivated and the
positive control serum has been treated for inactivation of HIV-1;
nevertheless, both should be handled as though they contain potentially
infectious agents. Other components prepared from human serum or plasma
have been tested using FDA-licensed methods and found to be nonreactive for
the presence of HIV-1 antibody, hepatitis B surface antigen (HBsAg) and
anti-HCV. However, as no test method can offer complete assurance that
infectious agents are absent, all materials of human origin should be
handled as though they contain infectious agents.
2. Handle specimens and materials contacting specimens as if potentially
infectious biological materials in accordance with "Universal Precautions
for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B
Virus, and other Bloodborne Pathogens in Health-Care Settings" (CDC, MMWR,
June 24, 1988). It has been reported that infectious HIV-1 can be isolated
from the oral fluid of some infected patients./7/ When detectable in oral
fluid specimens, infectious virus is present at low levels compared with
blood and may be inactivated by salivary inhibitors./8/
3. All test operators should adhere to the Occupational Safety and Health
Administration (OHSA) regulations (29 CFR 1910).
4. OraSure HIV-1 Specimen vials are breakable and should be handled with care.
5. Keep testing area separate from areas in which blood or blood products for
transfusion are stored.
6. Do not pipet any of the materials by mouth. Do not smoke, eat, or drink in
areas where specimens or kit reagents are handled.
7. Do not perform the test in the presence of reactive vapors (e.g., from
sodium hypochlorite, acids, alkalis, or aldehydes) or dust, because the
enzymatic activity of the conjugate may be affected.
8. Use disposable gloves and handle all materials used in the test including
specimens, wash solution, reaction trays, and pipets, cautiously as though
capable of transmitting infectious agents. Consult a physician immediately
in the event that contaminated materials are ingested or come in contact
with open lacerations, lesions, or other breaks in the skin.
9. Immediately clean up any spillage of materials containing antigen or
antibody with a 1:10 dilution of 5% sodium hypochlorite. Dispose of the
cleaning material by an acceptable method.
10. Treatment prior to disposal
a. Autoclave for 60 minutes at 121(degrees)C.
b. Incinerate disposable materials.
c. Mix liquid waste with 5% sodium hypochlorite solution so that
the final concentration is approximately 0.5% sodium
hypochlorite. Allow to stand 30 minutes before disposal.
11. Do not use reagents beyond their expiration date.
12. Do not mix microelisa plates, EnzAbody-O and serum control reagents from
different kit lots.
13. Use a clean pipet for dispensing specimens and reagents.
14. If a specimen is inadvertently not added in this assay, i.e., a well is
missed, the assay results will read negative.
15. Use accurately calibrated equipment.
16. Consider using dedicated equipment when cross contamination is a
possibility.
17. Inadequate adherence to package insert instructions may result in
erroneous results.
Reagent preparation
Prepare all reagents before beginning assay procedure. Reagents and specimens
should be at room temperature (15-30(degrees)C) before beginning the test and
can remain at room temperature during testing. Return reagents to 2-8(degrees)C
after use.
HIV-1 Microelisa Strips.
The foil packs should be brought to room temperature (15-30(degrees)C) before
opening to prevent condensation on the HIV-1 Microelisa Strips. After the
airtight foil pack has been opened, place unused strips back into the foil pack
and seal as shown in Figure 1. The Strips are stable for 4 weeks at
2-8(degrees)C if the foil pack is resealed with the clamp and rod provided. The
silicia gel bag must not be removed.
Figure 1: Foil Pack Closure.
[DIAGRAM]
Fold open end of foil pack over rod.
Apply clamp.
Diluent
1. check Diluent Concentrate for the presence of crystals. If crystals have
formed, resolubilize by warming at 37(degrees)C until crystals dissolve.
2. Add 1 vial of Diluent Concentrate and 1 bottle of Tween-20 to 1 bottle
of Diluent Reagent Water. Mix by inverting several times. Avoid
excessive foaming.
Positive and Negative Kit Controls
1. Pipet 500 (*)l prepared Diluent into each Positive and Negative Control
Serum vial to be reconstituted. Mix contents thoroughly.
2. Add 28 days to date of reconstitution and record date on vial label.
(*) denote umalut
Dilsim
1. Fill Dilsim bottle to the neck with prepared Diluent (60 ml). Cap bottle
tightly and warm at 40-50(degrees)C in a water bath or under a stream of
hot water for 20 30 minutes.
2. After warming, vortex bottle vigorously until cake is completely
solubilized.
3. Add 28 days to date of reconstitution and record date on bottle label.
Note: Reconstituted Dilsim has been shown to inactive HIV-1 in serum and
plasma preparations.
Wash Solution
1. Dilute the Wash Concentrate 1:50 with purified water in a clean
container. Prepare at least 50 ml of diluted Wash Solution for each
HIV-1 Microelisa Strip.
2. Label the container "Wash Solution." Add 28 days to date of preparation
and record date on container label.
EnzAbody(R)-O
Prepare the EnzAbody-O using one of the following methods.
If the entire EnzAbody-O will be used within one day following reconstitution:
Pipet 50 ml prepared Diluent to 1 vial EnzAbody-O. Mix by inverting several
times. Avoid excessive foaming. Add 1 day to the date and time of reconstitution
and record the date and time on the vial label.
If EnzAbody-O will be used for more than one day following reconstitution:
1. Pipet 5 ml prepared Diluent to 1 vial of EnzAbody-O. Mix by inverting
several times. Avoid excessive foaming. Label the vial as "concentrate."
Add 28 days to the date of reconstitution and record date and time on
the vial label.
2. On day of use, prepare a 1:10 dilution of the prepared EnzAbody-O
"concentrate" with Diluent. For each test strip used, prepare 2.0 ml of
EnzAbody-O working solution (0.2 ml EnzAbody-O concentrate to 1.8 ml
Diluent). Discard any unused working solution each day.
ABTS Substrate
1. Add ABTS Diluent (1 bottle) to 1 bottle of ABTS Substrate. Mix by
inverting several times.
Caution: To minimize auto-oxidation of ABTS, reconstitute with ABTS
Diluent that has been warmed to ambient temperature (15-30(degrees)C).
Do not add cold (2.8(degrees))C ABTS Substrate to the wells.
2. Add 14 days to the date of reconstitution and record date on bottle
label.
Note: Do not reconstitute second bottle of ABTS Substrate until first
has been depleted.
Stop Solution
Contains sodium fluoride. Avoid contact with skin. If contact is made, wash
area with water.
Kit storage instructions
Store all components at 2-8(degrees)C when not in use. Expiration date printed
on the kit indicates date beyond which product should not be used.
Chemical or physical indications of instability
Alterations in the physical appearance of test kit material may indicate
instability or deterioration. The expiration date shown on component labels
indicates the date beyond which product should not be used.
INSTRUMENTS
1. A microelisa plate reader is required which has a single wavelength
capacity for 405 nm with a linear absorbance range of 0 to 2.000, a drift
of less than 0.005% AU/hr and a bandwidth of 10 nm.
2. The aspiration/wash system must be capable of containing aspirated waste in
a closed system and capable of dispensing a 300 (*)l volume.
3. An incubator capable of maintaining 37 + 2(degrees)C.
-
4. A centrifuge capable of 600-800 x g force.
For any instrument, the manual provided by the manufacturer should be reviewed
for additional information regarding the following:
a) Installation and special requirements
b) Operation principles, instructions, precautions, and hazards
c) Manufacturer's specifications and performance capabilities
d) Service and maintenance information
SPECIMEN COLLECTION, STORAGE AND PREPARATION (ORASURE HIV-1 SPECIMEN)
Note: This test kit may not be used to assay blood specimens. This test kit may
only be used to assay OraSure HIV-1 oral fluid specimens obtained using the
OraSure HIV-1 Oral Specimen Collection Device manufactured by Epitope,
Beaverton, Oregon.
1. Refer to the OraSure HIV-1 Oral Specimen Collection Device package insert
for instructions on collecting a specimen.
2. OraSure HIV-1 specimens must be transported to the laboratory in the
OraSure HIV-1 Specimen Vial.
3. OraSure HIV-1 specimens may be transported to the laboratory at ambient
temperature via courier, air freight, or regular mail in accordance with
applicable Federal, state and local regulations which apply to the
transportation of OraSure HIV-1 specimens which may contain etiologic
agents (39 CFR 111).
4. OraSure HIV-1 specimens (on or off the collection pad) may be stored at
4(degrees)C to 37(degrees)C for a maximum of 21 days from the time of
collection, including the time for shipping and testing. If testing of
specimens cannot be completed within 21 days, OraSure HIV-1 specimens can
be stored frozen at -20(degrees)C for a maximum of six weeks.
5. OraSure HIV-1 specimens frozen twice and thawed a second time must be
tested within 24 hours, or discarded.
6. Record the specimen identification number from the OraSure HIV-1 Specimen
Vial.
7. Ensure that the specimen is within acceptable dating for testing, i.e. * 21
days from collection.
Note: All testing should be completed within 21 days of specimen collection
unless stored at -20(degrees)C (see Number 4 above).
8. Hold the vial upright with the pointed tip up.
9. Move the pad away from the vial tip by gently tapping the vial.
10. Break the pointed tip of the vial off with your thumb.
11. Place a tube over the vial and invert the tube and vial.
12. Centrifuge at 600-800 x g force for 15 minutes.
13. Determine that there is a minimum of 0.75 ml volume of specimen eluate.
If the volume of the centrifuged specimen is less than 0.75 ml, the
specimen is unsuitable for testing and a new specimen for the test subject
must be obtained. Notify the ordering physician if the volume of specimen
is insufficient.
* Denotes less than
ORAL FLUID VIRONOSTIKA HIV-1 MICROELISA SYSTEM TEST PROCEDURE
Materials provided
See Reagents Section
Additional materials required but not provided
Instruments/Equipment
Automated diluter/dispenser system or test tubes (12x75mm)
Aspiration/wash system connected to a waste trap and vacuum source
12-channel variable volume pipet system (50 to 300 (*)l) and suitable tips
Micropipet(s) capable of handling of 3(*)l, 75(*)l, and 500(*)l, and suitable
disposable tips
Graduated cylinder, 50 ml
Incubator, 37 + 2(degrees)C
-
Microelisa plate reader, single wavelength capacity for 405 nm with a linear
absorbance range of 0 to 2.000, with a drift of less than 0.005% AU/hr
and a bandwidth of 10 nm
Timer
Centrifuge capable of 600-800 x g force
Reagents/Disposables
Purified water, USP or equivalent
Absorbent paper
V-shaped troughs
Disposable gloves
Sodium hypochlorite solution (5%) or liquid bleach
Appropriate biohazard waste containers for materials potentially contaminated
with infectious agents
Tubes suitable for centrifuging OraSure HIV-1 specimen containers
OraSure HIV-1 Laboratory Qualification Panel (manufactured by Epitope, Inc.,
Beaverton, Oregon). This panel must be used to qualify a testing laboratory
prior to the initial use of the Oral Fluid Vironostika HIV-1 Microelisa
with OraSure Hiv-1 Specimens.
Materials available from bioMerieux
Incubator
Microelisa washer
Microelisa reader
Stripholder with uncoated well (Product No. 259576)
12-channel pipet and tips
Micropipet and tips
Microprocessor-controlled diluter/dispenser
OraSure HIV-1 Laboratory Qualification Panel (manufactured by Epitope, Inc.,
Beaverton, Oregon)
Procedural notes and precautions
1. HIV-1 Microelisa Strips, EnzAbody-O, and Controls used in an assay must
be of the same kit lot number and the HIV-1 Microelisa Strips and
Controls must be identified with a green sticker "For Oral Fluid
Vironostika HIV-1 Only." Materials should not be used after the
expiration date shown on the package label. Components and test specimens
should be at ambient temperature (15-30(degree)C) before testing begins.
Return the reagents to 2-8(degree)C after use.
2. Strips of the microelisa plate are removable. Remove Strips not needed
and replace with uncoated strips. Store unused Strips as described in the
Reagent preparation section under "HIV-1 Microelisa Strips." Before
testing begins, inspect the microelisa stripholder and ensure that all
wells are secure. Stripholders should be handled with care to ensure that
no Strip is dislodged during testing.
3. HIV-1 Microelisa Strips may be used only once.
4. Do not touch the top or bottom of strips, or the edge of wells with
fingers or pipet tips.
5. All reagents and specimens must be mixed well before use.
(*) denote umalut
8
6. Do not allow the microelisa wells to dry once the assay has begun. Fill the
wells with the next required reagent immediately after washing; if this is
not possible, fill the wells within 10 minutes.
7. All pipetting steps should be performed with the utmost care and accuracy.
Cross-contamination between reagents will invalidate test results. Use
micro-pipets for quantitative delivery of specimens and reagents. Avoid
microbial or any other contamination of reagents.
8. Refrain from opening the door of the incubator (37(degree)C) during the
incubation time.
9. Routine maintenance of the aspiration/wash system is strongly recommended
to prevent carryover from highly reactive specimens to nonreactive
specimens.
10. The aspiration/wash system should be flushed with copious amounts of water
upon completion of the final wash of the assay.
11. Dispose of all used materials as biohazardous waste.
12. Sensitivity and specificity using OraSure HIV-1 specimens are not
equivalent to serum or plasma.
Wash procedure
1. Incomplete washing will adversely affect the test outcome.
2. Aspirate well contents into a waste flask. Then fill the wells
(approximately 0.3ml) with diluted Wash Solution. Aspirate and fill the
wells four times.
3. After the last aspiration, invert stripholder and tap firmly on absorbent
paper taking not to dislodge any Strips. Remove any excess Wash Solution by
blotting with absorbent tissue. Note precaution Number 9 above.
Test procedure
Once the OraSure HIV-1 specimen is processed following the manufacturer's
procedures (see Specimen collection, storage and preparation), continue with the
steps listed below.
1. Fit stripholder with the required number of HIV-1 Microelisa Strips. If
less than eight Strips are needed, use uncoated strips to complete the
plate when using a 96-well washer.
2. Prepare dilutions of each control and OraSure HIV-1 specimen. Include three
Negative Controls and one Positive Control on each plate.
Caution: Use a clean tip for each specimen. Do not pipet specimen into an
empty well without Dilsim. Do not allow the microelisa wells to dry once
the assay has bagun.
Control dilution
A. Automated diluter/dispenser: Pipet 3 (*)l of Control with 225 (*)l
Dilsim into the designated microelisa well.
B. Premixed manual method: Pipet 5 (*)l Control into a clean test tube
containing 375 (*)l Dilsim. Mix well. Pipet 225 (*)l of the diluted
control into the designated microelisa well.
C. Direct manual method: Using a multichannel pipet, add 125 (*)l Dilsim
to each control microelisa well. Pipet 3 (*)l Control into the
designted wells. Using a multichannel pipet and clean tips, add 100
(*)l Dilsim to each control well and repeatedly aspirate and dispense
to mix.
OraSure HIV-1 specimen dilution
A. Automated diluter/dispenser: Pipet 75 (*)l of specimen with 75 (*)l
Dilsim into the designated microelisa well.
B. Direct manual Method: Pipet 75 (*)l of Dilsim into the designated
microelisa well followed by 75 (*)l of OraSure HIV-1 specimen. Mix
well by repeatedly aspirating and dispensing contents.
3. Cover the Strips with the adhesive plate sealer. Incubate at 37 + 2(degree)
C for 90 to 100 minutes.
4. Wash each well four times with diluted Wash Solution. Refer to "Wash
procedure."
(*) denote umalut
5. Pipet 150 (*)l of reconstituted EnzAbody-O solution into each well.
Caution: Do not allow EnzAbody-O to contaminate Substrate. If the same
equipment is used to add both reagents, new dispoable tips must be used.
6. Cover the strips with a new plate sealer. Incubate at 37 +/- 2(degree)C for
30 to 35 minutes.
7. Wash each well four times with diluted Wash Solution. Refer to "Wash
proecdure."
8. Pipet 150 (*)l of prepared ABTS Substrate into each well. Do not mix or
agitate. Strips should not be covered with a plate sealer.
9. Incubate at room temperature(15-30(degrees)C) for 10 to 12 minutes.
10. Stop reaction by adding 150 (*)l of Stop Solution to each well (maintain the
same sequence and time intervals used for Substrate solution addition) and
gently tap to mix. Plates should be read within 2 hours.
11. Blank the microelisa reader on air (without stripholder and Strips) and read
the absorbance of the solution in each well at 405 nm.
Quality control
Qualification of Negative Control (NC) values: Absorbance of each NC replicate
must be greater than or equal to 0.100 and less than or equal to 0.400.
Eliminate one outlier, if present, and calculate the NC mean (NCX). Absorbance
of NC must be less than 1.5 multiplied by NCX and greater than 0.5 multiplied by
NCX. If two or more values are outside range, the run is invalid and must be
repeated.
Qualification of Positive control (PC) value: Absorbance of PC must be greater
than or equal to 0.700. If PC is outside this limit, the run is invalid and
must be repeated.
Test validity: A test run is valid if the positive and negative control values
are qualified and
PC - NCX ** 0.500
If results do not meet this criterion, technique may be suspect and the run must
be repeated.
RESULTS
Calculations
Calculations must be made separately for each stripholder.
Cutoff value: If the test run is valid, calculate the cutoff value as follows:
NCX + 0.270
Determination of test specimen results: Compare the test specimen absorbance
value to the cutoff value.
A test specimen is nonreactive if specimen absorbance is less than the cutoff
value.
A test specimen is reactive if specimen absorbance is greater than or equal to
the cutoff value.
(*) denote umalut
** denote greater than equal to
Specimen calculations
Absorbance at 405 nm
NC = 0.175, 0.195, 0.225
NCX = 0.198
PC = 1.469
Acceptance criteria
Eliminate any control absorbance values not meeting the following criteria:
0.100***NC *** 0.400
NC* 1.5 (NCX) or 0.297
NC ** 0.5 (NCX) or 0.099
PC **** 0.700
None eliminated
Ensure that the following is within the specified acceptance criteria.
PC - NCX ** 0.500
1.469 - 0.198 = 1.271 Pass
Kit controls are within acceptable limits.
Calculate cutoff
Cutoff = NCX + 0.270 = 0.468
Interpretation of results
Note: Results of the test using an OraSure HIV-1 specimen must be reported only
to the physician who ordered the test or to a person under the supervision of
the ordering physician.
In providing test results to the physician, careful note must be taken of the
limitations of the procedure (see following section).
Specimens with absorbance values less than the cutoff value are considered
nonreactive by the criteria of Oral Fluid Vironostika HIV-1 Microelisa
System and may be considered negative for the antibody. Further testing is
not required.
2. Specimens with absorbance values greater than or equal to the cutoff value
are considered reactive (initially reactive) by the criteria of Oral Fluid
Vironostika HIV-1 Microelisa System, but before interpretation, the
original specimen source should be retested in duplicate. If either
duplicate retest is reactive, the specimen is considered repeatedly
reactive and the OraSure HIV-1 specimen is considered positive in a
licensed screening test for HIV-1 antibodies.
3. Initially reactive specimens which do not react in either of the duplicate
repeat tests are considered negative for antibodies to HIV-1.
* Less than
** Greater than
*** Less than or equal to
**** Greater than or equal to
LIMITATIONS OF THE PROCEDURE
1. False negative results occur more frequently when testing OraSure HIV-1
specimens compared with testing blood specimens. See Performance
characteristics section for details.
2. False positive results occur more frequently when testing with OraSure
HIV-1 specimens compared with blood specimens. See Performance
characteristics section for details. At this time, there is no FDA licensed
additional, more specific test for HIV-1 antibodies in an OraSure HIV-1
specimen. The physician should be advised to arrange for follow-up testing
of a blood specimen from subjects whose OraSure HIV-1 specimen tests
repeatedly reactive.
3. False negative results (the subject is infected, but the OraSure HIV-1
Specimen tests negative) may be a result of antibody levels in oral fluid
which are below the sensitivity (lower limit of detection) of this
procedure which may occur, for example, during the early phase of
infection, or with inadequate processing of the specimen, or with
inadequate collection of the OraSure HIV-1 specimen.
4. False positive results may be obtained, for example, as a result of
nonspecific cross reacting antibodies, and not from an HIV-1 infection.
5. False results (either positive or negative) may occur as a result of
interfering substances, such as foreign matter in the mouth being collected
with the specimen.
The Oral Fluid Vironostika HIV-1 Microelisa System may be useful as an aid in
the diagnosis of infection with HIV-1. It is recommended that specimens that
are repeatedly reactive in an ELISA test be investigated by an additional, more
specific test to verify the presence of HIV-1 antibodies. However, in the case
of OraSure HIV-1 specimens, there is no licensed, additional, more specific test
currently available to demonstrate the presence of HIV-1 antibodies. Therefore,
for OraSure HIV-1 specimens that are repeatedly reactive, the follow up
investigation requires that a serum or plasma specimen be obtained from the same
person and tested in a licensed ELISA and an additional, more specific test, as
appropriate. When individuals are notified that their OraSure HIV-1 specimen is
repeatedly reactive in the ELISA test, the ordering physician should offer
appropriate counseling and medical evaluation and arrange for follow up testing
of a serum or plasma specimen.
ELISA testing alone should not be used to diagnose HIV-1 infection, and cannot
be used to diagnose AIDS, even if the recommended testing of reactive specimens
suggests a high probability that the antibody to HIV-1 is present. AIDS and
AIDS-related conditions are clinical syndromes and their diagnosis can only be
established clinically./9/ A negative test result at any point in the
investigation of individual patients does not preclude the possibility of
exposure to, or infection with, HIV-1. The risk of developing AIDS or
AIDS-related conditions in an asymptomatic person with a repeatedly reactive
ELISA is not known./10/
Data obtained from testing blood specimens of persons both at increased and at
low risk for HIV-1 infection suggest that repeatedly reactive specimens with
high absorbance by ELISA are more likely to demonstrate the presence of HIV-1
antibodies by additional testing./11/ Reactivity at, or only slightly above, the
cutoff value is more frequently nonspecific for OraSure HIV-1 specimens obtained
from persons at low risk for HIV-1 than from persons at risk based on follow up
testing of serum specimens.
The test procedure and the "Interpretation of results" must be followed closely
when testing for the presence of antibodies to HIV-1. Because the ELISA method
was designed to test individual units of blood or plasma, most data regarding
its interpretation were derived from testing individual specimens. Performance
characteristics for OraSure HIV-1 specimens were derived from individual
specimens. Insufficient data are available to interpret tests performed on other
body specimens, pooled specimens or products made from such pools, therefore,
results of testing such specimens may be inaccurate.
_______________
12
EXPECTED RESULTS
Performance characteristics of the test for OraSure HIV-1 specimens
Note: This assay was licensed for use with serum and plasma specimens at the
time of the clinical studies.
Sensitivity and specificity: At present, there is no recognized standard for
establishing the presence or absence of HIV-1 antibody in human oral fluid.
Therefore, sensitivity testing of OraSure HIV-1 specimens with the Oral Fluid
Vironostika HIV-1 Microelisa System was computed based on the clinical diagnosis
of AIDS and specificity was computed based on testing in low risk populations.
In addition, sensitivity and specificity of OraSure HIV-1 testing were computed
based on testing in high risk subjects using matched oral fluid/blood specimens
from the same subjects.
1. Sensitivity using OraSure HIV-1 specimens, based on an assumed 100%
prevalence of HIV-1 antibody in AIDS patients, is estimated in these
studies to be 98.6% (287/291). Sensitivity using OraSure HIV-1 specimens
was reduced compared with blood specimens in AIDS patients.
Sensitivity using OraSure HIV-1 specimens is estimated in these studies to
be 99.1% (546/551) in high risk subjects, based on the ability of the test
to detect HIV-1 antibody in matched oral fluid/blood specimens. Sensitivity
using OraSure HIV-1 specimens was reduced compared with blood specimens for
high risk subjects.
2. Specificity using OraSure HIV-1 specimens, based on an assumed zero
prevalence of HIV-1 antibody in low risk populations, is estimated in these
studies to be 99.6% (3991/4009). Specificity using OraSure HIV-1 specimens
was reduced compared with blood specimens for low risk subjects.
Specificity using OraSure HIV-1 specimens is estimated in these studies to
be 97.7% (837/857) for high risk subjects, based on the ability of the test
to detect HIV-1 antibody in matched oral fluid/blood specimens. Specificity
using OraSure HIV-1 specimens was reduced compared with blood specimens for
high risk subjects.
Clinical studies
Clinical studies of matched OraSure HIV-1 and serum specimens from AIDS
patients, high risk subjects and low risk subjects 18 years of age and older
were conducted at seven sites as shown in the following table.
AIDS High Risk Low Risk
Test Site Subjects Subjects Subjects
--------- -------- -------- --------
A 11 407 84
B 0 466 0
C 158 299 1,132
D 65 240 104
E 57 0 336
F 0 0 573
G 0 0 1,788
Total 291 1,412 4,017
Clinical sensitivity studies
Reactivity in AIDS patients and high risk populations
The sensitivity of testing OraSure HIV-1 specimens compared with matched serum
specimens using the Oral Fluid Vironostika HIV-1 Microelisa System in AIDS
patients and high risk subjects (38% intravenous drug users, 23% homosexuals,
17% sexual partners of individuals at risk, 6% prostitutes, 16% others with
acknowledged risk factors) is shown in the table that follows.
Number
No. of Nonreactive/a/ Reactive Confirmed Positive
Specimens No. No. with Serum
--------- --- --- ----------
AIDS Patients
OraSure HIV-1 291 4/b/ 287
Serum 291 1 290 291
High Risk Subjects
OraSure HIV-1 1,412 843/c/ 569/d/
Serum 1,412 858 554/e/ 551
/a/ Includes specimens that were nonreactive on the initial screening test
and specimens that were initially reactive, but not repeatedly reactive.
/b/ 4 matched serum specimens were positive (OraSure HIV-1 False Negative).
Screening tests for 2 patients were valid by ELISA kit criteria and the
matched serum specimens were positive with S/CO of 7.26 and 6.90, but
laboratory control reagents failed. Retests of these OraSure HIV-1 specimens
at the clinical site were positive for both specimens with S/CO values of
1.69 and 1.15.
/c/ 5 matched serum specimens were positive (OraSure HIV-1 False Negative).
/d/ 20 matched serum specimens were negative (OraSure HIV-1 False Positive);
3 OraSure HIV-1 specimens were IR and not retested and matched serum
specimens were negative (OraSure HIV-1 Unresolved).
/e/ 2 subjects were WB/neg/ (Blood False Positive); 1 subject was IR, not
retested and WB/ind/ (Unresolved).
ELISA = Oral Fluid Vironostika HIV-1 Microelisa System assay; IR = initially
reactive; RR = repeatedly reactive; WB = blood Western blot; pos = positive;
ind = indeterminate; neg = negative; RIPA = radioimmunoprecipitation assay;
S/CO = signal to cutoff
287 OraSure HIV-1 specimens (98.6%) and 290 matched serum specimens (99.7%) were
reactive in the Oral Fluid Vironostika HIV-1 Microelisa System screening test
out of the 291 AIDS patients studied. Of the four OraSure HIV-1 specimens that
were initially nonreactive, one was nonreactive when retested and three were
reactive when retested. One serum specimen that was initially nonreactive was
reactive when retested.
546 OraSure HIV-1 specimens (99.1%) and 551 matched serum specimens (100%) were
reactive in the Oral Fluid Vironostika HIV-1 Microelisa System screening test
out of the 551 high risk subject whose serum tested positive in additional, more
specific tests for HIV-1 antibodies (Western Blot, RIPA). Of the five OraSure
HIV-1 specimens that were initially nonreactive, two were repeatedly nonreactive
on ELISA retest, two were not retested and one was repeatedly reactive on
retest.
The sensitivity of testing OraSure HIV-1 specimens with the Oral Fluid
Vironostika HIV-1 Microelisa System compared with matched serum specimens was
reduced based on the reactivity in AIDS patients and high risk subjects.
Analytical sensitivity
The analytical sensitivity of testing OraSure HIV-1 specimens with the Oral
Fluid Vironostika HIV-1 Microelisa System was 1/1000th that of testing serum
specimens based on serial dilution of matched specimens from 13 HIV-1 antibody
positive subjects. At the dilutions recommended for testing, 1:2 for OraSure
HIV-1 specimens and 1:75 for serum specimens, the average sensitivity of testing
with OraSure HIV-1 specimens was 1/35th (range 1/7 to 1/107) that of testing
with serum specimens based on the highest dilution producing positive results as
shown in the following table.
Specimen OraSure HIV-1 Serum OraSure HIV-1
Number/a/ Titer/c/ Titer/c/ Hemoglobin/b/ (mg/dl)
-------- ------- ------- ---------------------
1 128 4,096
2 128 4,096
3 64 4,096
4 64 1,024
5 128 4,096
6 128 4,096
7 128 4,096
8 512 4,096
9 128 4,096
10 512 4,096
11 160 17,067 11.0
12 1,280 8,533 7.6
13 160 8,533 5.3
/a/ Matched specimens 1-10 were collected from analytical study. Matched
specimens 11-13 were collected as part of the clinical field trial.
/b/ Three OraSure HIV-1 specimens (#11-13) of 112 studies contained
hemoglobin (**** 5 mg/dl).
/c/ End-point titer indicates the maximum dilution of a specimen (prior to
dilution for ELISA testing) which produced positive Oral Fluid
Vironostika HIV-1 Microelisa System test results.
The analytical sensitivity of Oral Fluid Vironostika HIV-1 Microelisa System for
HIV-1 antibodies in three OraSure HIV-1 specimens studied was not enhanced by
the presence of blood in OraSure HIV-1 specimens.
**** Greater than or equal to
Reactivity in seroconversion
OraSure HIV-1 and serum specimens were obtained prospectively from one subject
undergoing seroconversion. Results of ELISA testing show that in this case
antibodies to HIV-1 were detected in serum specimens on day 8 and OraSure HIV-1
specimens on day 11 based on two consecutive positive determinations. Reactivity
of ELISA using serum and OraSure HIV-1 specimens and WB and HIV-1 antigen test
using serum is summarized in the following table.
OraSure Blood
HIV-1 Blood p24 Ag
Date (Day) S/CO S/CO (ng/ml) Blood Western blot band reactivity
- ---- ----- ---- ---- ------- ----------------------------------
5/14 (1) 0.36 0.40 13.64* none
5/15 (2) 0.57
5/16 (3) 0.31 0.40 20.36* none
5/17 (4) 0.52
5/21 (8) 0.49 2.92* 6.43* gp 160+/- p24+/*
5/22 (9) 1.07*
5/23 (10) 0.96 4.71* 1.92* gp 160+/- p24* gp41+/-*
5/24 (11) 1.75*
5/28 (15) 1.94* 5.26* 0 gp 160+/ p24+ gp41+/- gp120+/-*
5/29 (16) 2.05*
5/30 (17) 2.04* 5.27* 0 gp 160+/ p24+ gp41+/- gp120+/-*
5/31 (18) 2.04*
6/4 (22) 1.91* 5.44* 0 gp 160+/ p24+ gp41+/- gp120+/- p66+/- p55+/-*
*Asterisk indicates a reactive test result; S/CO = signal to cutoff; Ag =
HIV-1 antigen; + = positive reactivity; +/- = indeterminate reactivity
Reactivity in seropositive subjects with oral pathology
Oral examinations were carried out at a clinic site D on 65 AIDS subjects, 240
high risks subjects and 18 subjects of unknown risk. Of the 303 subjects found
to be seropositive, 29 (10%) had significant oral pathology. For these subjects
HIV-1 antibodies were detected in 96.6% (28/29) of the OraSure HIV-1 specimens
and 100% (29/29) of the serum specimens when tested using the Oral Fluid
Vironostika HIV-1 Microelisa System as shown in the following table.
Number Number OraSure Number Confirmed
Oral Pathology Tested HIV-1 ELISA Positive Positive with Serum
-------------- ------ -------------------- -------------------
Hairy Leukoplakia 10 10 10
Candida 10 9* 10
Gingivitis 5 5 5
Gingival Ulcer 1 1 1
Periodontitis 1 1 1
Hairy leukoplakia and candida 1 1 1
Hairy leukoplakia and gingivitis 1 1 1
Total 29 28 29
*Includes one OraSure HIV-1 false negative which was previously noted in
high risk population.
This study does not detect an increase in the frequency of false negative
results for OraSure HIV-1 specimens from subjects with the above oral
pathologies.
16
Clinical specificity studies
Specificity in low risk and high risk populations
The specificity of testing OraSure HIV-1 specimens with the Oral Fluid
Vironostika HIV-1 Microelisa System compared to testing serum specimens was
studied in 4017 low risk subjects (55% military inductees, 16% blood donors, 14%
insurance applicants, 11% students/hospital staff) and 1412 high risk subjects.
The results are shown in the table that follows:
Number
No. of Nonreactive/a/ Reactive Confirmed Positive
Specimens No. No. with Serum
--------- --- --- ----------
Low Risk Subjects
OraSure HIV-1 4,017 3,992 25/b/
Serum 4,017 4,008 9/c/ 4
High Risk Subjects
OraSure HIV-1 1,412 843 569/d/
Serum 1,412 858 554/e/ 551
/a/ Includes specimens that were nonreactive on the initial screen test and
specimens that were initially reactive, but not repeatedly reactive.
/b/ 18 matched serum specimens were negative (OraSure HIV-1 False Positive);
3 OraSure HIV-1 specimens were IR and not retested and matched serum
specimens were ELISA/neg/ and WB/ind/ (Unresolved).
/c/ 4 specimens were confirmed negative (Blood False Positive); 1 specimen
was RR, but not tested further (Unresolved).
/d/ 20 matched serum specimens were negative (OraSure HIV-1 False Positive);
3 matched serum specimens were negative and OraSure HIV-1 was IR and not
retested. 19/20 subjects with false positive OraSure HIV-1 results were
smokers.
/e/ 2 specimens were WB/neg/ (Blood False Positive); 1 specimen was ELISA IR
and WB/ind/ (Unresolved).
ELISA = Oral Fluid Vironostika HIV-1 Microelisa System assay; IR = initially
reactive; RR = repeatedly reactive; WB = blood Western blot; pos = positive;
ind = indeterminate; neg = negative; RIPA = radioimmunoprecipitation assay;
S/CO = signal to cutoff
There were 18 repeatedly reactive OraSure HIV-1 specimens (18/4009, 0.45%)
compared with 4 repeatedly reactive serum specimens (4/4009, 0.10%) in the low
risk subjects whose HIV antibody status was resolved to be negative by
additional testing. There were 20 repeatedly reactive OraSure HIV-1 specimens
(20/857, 2.3%) compared with 2 repeatedly reactive serum specimens (2/860,
0.23%) in the high risk subjects whose antibody status was resolved to be
negative by additional testing.
These results suggest that, compared with testing serum specimens, the incidence
of false positive test results using OraSure HIV-1 specimens is increased
4.5-fold in low risk populations and 10-fold in high risk populations.
17
Reactivity in seronegative subjects with oral pathology
Matched OraSure HIV-1 and serum specimens were obtained prospectively from 47
subjects with various forms of oral pathology. Results of testing OraSure HIV-1
and serum specimens with Oral Fluid Vironostika HIV-1 Microelisa System were
negative in all cases as shown in the following table.
Number Number Number
Oral Pathology Tested Positive Negative
-------------- ------ -------- --------
Periodontitis 21 0 21
Gingivitis 5 0 5
Multiple caries 5 0 5
Multiple caries and periodontitis 8 0 8
Multiple caries and gingivitis 6 0 6
Periodontitis and gingivitis 2 0 2
Total 47 0 47
This study did not detect differences in specificity among the OraSure HIV-1
specimens obtained from subjects with the oral pathology noted above when tested
in the Oral Fluid Vironostika HIV-1 Microelisa System.
Reactivity in other disease conditions
38 repository and 53 fresh matched OraSure HIV-1 and serum specimens from
patients with medical conditions other than HIV-1 infection were studied. ELISA
testing of OraSure HIV-1 and serum specimens were negative in all cases as shown
in the following table.
Number Number Number
Disease Tested Positive Negative
------- ------ -------- --------
Hepatitis A 12 0 12
Hepatitis B 5 0 5
Hepatitis C 18 0 18
Autoimmune 13 0 13
H. pylori 3 0 3
Lymphoid malignancy 16 0 16
Other neoplasia 24 0 24
Total 91 0 91
Additional performance studies
The testing of OraSure HIV-1 specimens collected by trained medical
professionals and trained non-medical individuals compared with testing of the
corresponding serum specimens were evaluated using the Oral Fluid Vironostika
HIV-1 Microelisa System. Matched OraSure HIV-1 and serum specimens were obtained
from 129 subjects prospectively and the results of testing are presented in the
table that follows.
Number Number
Trained Collector Specimen Negative Negative
----------------- -------- -------- --------
Medical professional OraSure HIV-1 94 35/a/
Non-medical OraSure HIV-1 95 34/b/
Medical professional Serum 96 35/c/
/a/ 3 matched sera were negative (OraSure HIV-1 False Positive)
/b/ 2 matched sera were negative (OraSure HIV-1 False Positive) and
coincided with those identified in footnote a above.
/c/ One serum was ELISA/RR/ and WB/ind/ with p65 reactivity (Unresolved).
Positive ELISA results for OraSure HIV-1 specimens collected by trained medical
professionals and non-medical individuals from 32 subjects were concordant with
serum results. Discordant results were obtained for one subject whose OraSure
HIV-1 specimen was ELISA repeatedly negative and serum specimen was ELISA
repeatedly reactive and Western blot indeterminate with p65 reactivity. There
was no follow up testing to resolve the true serostatus of this subject.
Three OraSure HIV-1 specimens collected by medical professionals and two OraSure
HIV-1 specimens collected by non-medical individuals were repeatedly reactive on
the ELISA test which matched serum specimens were negative on ELISA and WB tests
(OraSure HIV-1 false positive).
In this study, the performance of ELISA testing of OraSure HIV-1 specimens
collected by trained non-medical individuals was comparable to OraSure HIV-1
specimens collected by medical professionals.
Reproducibility
The reproducibility of testing, OraSure HIV-1 specimens in the Oral Fluid
Vironostika HIV-1 Microelisa System was studied and is summarized in the
following table.
- ------------------------------------------------------------------------------------
Mean Test Test Location
Specimen S/CO % CV n Days A B Epitope
- ------------------------------------------------------------------------------------
6.046 2.3 36
6.018 2.4 36
5.933 5.9 33
6.142 2.6 5
2.852 10.6 24 3 X X
1.639 29.6 10 3 X X
7 0.422 23.7 82 3 X X
8 0.299 14.4 88 3 X X
OraSure HIV-1 specimens from four seropositive subjects (specimens 1-4) and two
seronegative subjects (specimens 7 and 8) were collected at two test sites.
Specimens collected at one clinical site were tested on-site and exchanged with
the other site for testing, where indicated. OraSure HIV-1 specimens from two
seropositive subjects were collected and diluted at Epitope to provide on
intermediate (specimen 5) and one low positive specimen (specimen 6) for
testing. Specimens prepared at Epitope were sent to clinical sites for testing
and/or tested at Epitope as indicated.
19
REFERENCES
1. Hardy AM, Allen JR, Morgan WM, et al: The Incidence Rate of Acquired
Immunodeficiency Syndrome in Selected Populations. JAMA 1985;253(2):
215-20.
2. Gallo RC, Salahuddin SZ, Popovic M, et al: Frequent Detection and
Isolation of Cytopathic Retroviruses (HTLV-III) from Patients with AIDS
and at Risk for AIDS. Science 1984; 224:500-503.
3. Archibald DW, Zon L, Groopman JE, McLane MF, and Essex M: Antibodies to
human T-lymphotrophic virus type III (HTLV-III) in saliva of acquired
immunodeficiency syndrome (AIDS) patients and in persons at risk for
AIDS. Blood 67:831-834, 1986.
4. Kuhnl P, Seidl S, Holzberger G: HLA DR4 Antibodies Cause Positive
HTLV-III Antibody ELISA Results. Lancet 1985; 1222-1223.
5. Popovic M, Sarngadharan MG, Read E, et al: Detection, Isolation, and
Continuous Production of Cytopathic Retroviruses (HTLV-III) from
Patients with AIDS and Pre-AIDS: Science 1984; 224:497-500.
6. Bartlett ML: Substrate Evaluation for the Horseradish Peroxidase Enzyme
Immunoassay. Am Soc Micro 79th Annual Meeting 1979; Abstract C25.
7. Ho DD, Byington RE, Schooley RT, et al: Infrequency of isolation of
HTLV-III virus from saliva in AIDS. N Engl. J. Med 313:1606, 1985.
8. Moore BE, Flaitz CM, Coppenhaver DH, et al: HIV recovery from saliva
before and after dental treatment: Inhibitors may have critical role in
viral inactivation. JADA 124:67-74, 1993.
9. Centers for Disease Control: Revision of the Case Definition of Acquired
Immunodeficiency Syndrome for National Reporting-United States. Ann
Intern Med 1985;103;402-403.
10. Hunter D, De Gruttola V: Estimation of Risk of Outcomes of HTLV-III
Infection. Lancet 1986;677-680.
11. Carlson JR, Bryant ML, Hinrichs SH, et al: AIDS Serology Testing in Low-
and High-Risk Groups. JAMA 1985;253(23):3405-3408.
AVAILABILITY
bioMerieux
Oral Fluid Vironostika(R) HIV-I Microelisa System
192 Test Kit Product number 259750
9600 Test Kit Product number 259748
For technical assistance, contact Customer Service at 1-800-682-2666.
Vironostika and EnzAbody are registered trademarks of bioMerieux in the USA and
other countries.
Dilsim is a trademark of bioMerieux, Inc.
OraSure is a registered trademark of Epitope, Inc.
[LOGO] Manufacturer: bioMerieux, Inc. bioMerieux, S.A.
BIOMERIEUX Box 15969 69280 Marcy-l'Etoile France
Durham, North Carolina 27704-0969 www.biomerieux.com
(C)BIOMERIEUX 1994 February 2002
Printed in USA U.S. License No. 1624
20
EXHIBIT 1.8
TRANSFER PRICE
PRODUCT Transfer Price
ORASURE ORAL FLUID WESTERN BLOT KIT ****************
-17-
EXHIBIT 2.6.3
$500,00.00 Durham, North Carolina
October ______, 2002
Promissory Note
For $1.00 and other valuable consideration, the receipt and sufficiency
of which is acknowledged, OraSure Technologies, Inc., a Delaware Corporation
("the undersigned"), promises to pay bioMerieux, Inc., with offices at 100
Rodolphe Street, Durham, NC ("BMX"), the principal sum of Five Hundred Thousand
Dollars ($500,000.00) pursuant to Section 2.6 of that certain Distribution
Agreement of even date herewith between the undersigned and BMX.
This note shall be due and payable as follows:
a) $250,000 on or before December 31, 2002
b) $250,000 on or before March 31, 2003
This note shall bear no interest if payments are made by the due date.
If the principal sum or any portion thereof is not paid on or before the due
date thereof, it shall thereafter accrue simple interest at the rate of nine
percent (9%) per annum.
It is agreed that if the undersigned shall fail to pay an installment
of this note, or any part thereof, promptly as the same shall become due, then
upon default in any one or more of the above respects and at any time before
such default is made good, the holder may declare this Promissory Note, together
with interest thereon, immediately due and payable. The undersigned agrees, in
the event of any default hereunder, to pay all collection and attorney fees
incurred by the holder hereof.
The makers and endorsers of this Promissory Note severally waive
presentment for payment, protest and notice of protest, and non-payment of this
note, and all defenses on the grounds of any extension of time for the payment
thereof which may be hereafter given by the holders hereof to the undersigned.
IN TESTIMONY WHEREOF, the undersigned has caused its authorized
representative to execute and deliver this Promissory Note, and to apply its
corporate seal hereto as of the date stated above.
Attest:__________________________ OraSure Technologies, Inc.
Corporate Secretary
By:
(Corporate Seal) Name:___________________________
Title:___________________________
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EXHIBIT 6.2
DISTRIBUTION AGREEMENT
[LOGO]
BIOMERIEUX CONTROLLED COPY
RELEASE A
- --------------------------------------------------------------------------------
Part Description: OraSure(R) HIV-1 Western Blot Kit Page: 1 of 5
- --------------------------------------------------------------------------------
Rev: RA-0 Effective Date: May 21, 2002 P/N: 259766
- --------------------------------------------------------------------------------
Part II
- --------------------------------------------------------------------------------
- --------------------------------------------------------------------------------
REFERENCE TITLE
================================================================================
P-17 Description
- --------------------------------------------------------------------------------
QA-IN.0004 Quality Assurance Inspection of Incoming Material
- --------------------------------------------------------------------------------
- --------------------------------------------------------------------------------
A. ACCEPTANCE REQUIREMENTS
================================================================================
1. Safety Considerations
a. Biohazards: Contains inactivated HIV-1 reactive human serum
and animal anti-serum (goat), handle as if
potentially infectious using NCCLS Universal
precautions.
b. Chemical Hazards: Sodium Azide: Use of rubber gloves and eye
protection is required during the course of all
handling operations.
- --------------------------------------------------------------------------------
2. Storage Requirements:
a. Upon receipt, store at 2 - 8(degrees)C
b. Kits may be inspected at room temperature.
- --------------------------------------------------------------------------------
3. Sampling Requirements
a. Refer to QA-IN.0004.
- --------------------------------------------------------------------------------
4. Visual Examination
a. Description:
1. Visually examine shipment for product identity.
b. Approved Supplier:
1. Orasure Technologies, Inc.
8505 SW Creekside Place
Beaverton, OR 97008
Catalog No. 501-0000
- --------------------------------------------------------------------------------
[LOGO]
BIOMERIEUX CONTROLLED COPY
RELEASE A
- --------------------------------------------------------------------------------
Part Description: OraSure(R) HIV-1 Western Blot Kit Page: 2 of 5
- --------------------------------------------------------------------------------
Rev: RA-0 Effective Date: May 21, 2002 P/N: 259766
- --------------------------------------------------------------------------------
Part II
- --------------------------------------------------------------------------------
- --------------------------------------------------------------------------------
A. ACCEPTANCE REQUIREMENTS (CONTINUED)
================================================================================
5. Labeling
a. Verify that carton label lists the following information: (Refer to
Appendix I)
Product Name
Part Number of kit label (located in the lower left margin)
Lot Number
- --------------------------------------------------------------------------------
6. Certificate of Conformance/Analysis
a. Certificate of Conformance/Analysis must be provided for the specific
lot received and must include a minimum of the following information:
OraSure Technologies, Inc. (company letterhead)
OraSure(R) HIV-1 Western Blot Kit
Epitope P/N 501-0000
BioMerieux P/N 259766
Lot Number (Specific to Lot)
Expiration Date: (Specific to Lot)
Number Shipped
BioMerieux Purchase Order Number
Summary of QA Results Statement
State of Human Testing
List of Component Information
Shipping date and statement that shipping time was met
Signature of person attesting to quality statement
- --------------------------------------------------------------------------------
7. Expiration Date
a. Minimum shelf life remaining at time of receipt must be 8 months.
b. Assign date according to the expiration date indicated on the
Certificate of Conformance.
- --------------------------------------------------------------------------------
8. Reassay: Inteval and Test
a. Not applicable.
- --------------------------------------------------------------------------------
[LOGO]
BIOMERIEUX CONTROLLED COPY
RELEASE A
- --------------------------------------------------------------------------------
Part Description: OraSure(R) HIV-1 Western Blot Kit Page: 3 of 5
- --------------------------------------------------------------------------------
Rev: RA-0 Effective Date: May 21, 2002 P/N: 259766
- --------------------------------------------------------------------------------
Part II
- --------------------------------------------------------------------------------
- --------------------------------------------------------------------------------
B. ANALYTICAL PROCEDURES
================================================================================
1. Not applicable.
- --------------------------------------------------------------------------------
- --------------------------------------------------------------------------------
C. INTERPRETATION OF RESULTS
================================================================================
1. Test results must meet the requirements stated.
- --------------------------------------------------------------------------------
[LOGO]
BIOMERIEUX CONTROLLED COPY
RELEASE A
- --------------------------------------------------------------------------------
Part Description: OraSure(R) HIV-1 Western Blot Kit Page: 4 of 5
- --------------------------------------------------------------------------------
Rev: RA-0 Effective Date: May 21, 2002 P/N: 259766
- --------------------------------------------------------------------------------
Part II
- --------------------------------------------------------------------------------
APPENDIX I
CARTON LABEL
[LOGO]
BIOMERIEUX CONTROLLED COPY
RELEASE A
- -----------------------------------------------------------------------------------------------------------
Part Description: OraSure(R) HIV-I Western Blot Kit P/N: 269766 Page: 5 of 5
- -----------------------------------------------------------------------------------------------------------
Rev: RA-0 Effective Date: May 21, 2002 Lot No.:
- -----------------------------------------------------------------------------------------------------------
Part II
- -----------------------------------------------------------------------------------------------------------
RESULTS REPORT FORM
- -----------------------------------------------------------------------------------------------------------
TEST SPECIFICATION RESULTS CONFORM
===========================================================================================================
Description OraSure(R) HIV-I Western Blot Kit, 20 test. Yes [_] or No [_]
- -----------------------------------------------------------------------------------------------------------
Material received must be from the following
supplier.
Approved Supplier Yes [_] or No [_]
OraSure Technologies, Inc.
- -----------------------------------------------------------------------------------------------------------
Verify that carton label lists the following
Labeling Information: (Refer to Appendix I)
Product Name Yes [_] or No [_]
Part Number of kit label (located in the
lower left margin) Yes [_] or No [_]
Lot Number Yes [_] or No [_]
- -----------------------------------------------------------------------------------------------------------
Certificate of
Conformance/Analysis Required for release. Yes [_] or No [_]
- -----------------------------------------------------------------------------------------------------------
Inspection Conditions Inspected at room temperature Yes [_] or No [_]
- -----------------------------------------------------------------------------------------------------------
Out of refrigerator: (am/pm)Time [_]NA
- -----------------------------------------------------------------------------------------------------------
In refrigerator
(Maximum 2 hours): (am/pm)Time [_]NA
- -----------------------------------------------------------------------------------------------------------
Inspected at refrigerator temperature: Yes [_] or No [_]
- -----------------------------------------------------------------------------------------------------------
Expiration Date Minimum shelf life remaining at time of receipt
must be 8 months. Yes [_] or No [_]
- -----------------------------------------------------------------------------------------------------------
- -----------------------------------------------------------------------------------------------------------
DOCUMENT SIGNATURE DATE
- -----------------------------------------------------------------------------------------------------------
Document and Data Results for this Part Number Inspector:
comply with Device Master Record.
-----------------------------------------------------
These Q.C./Q.A. test results were verified by: Authorization:
-----------------------------------------------------
Final Disposition: [_] Approved [_] Rejected
MDN#
- -----------------------------------------------------------------------------------------------------------
EXHIBIT 10.1
PRODUCT LABELING
-20-
201-3079-4
Label PN 201-3079-4
Exp.
Lot.
Powdered Milk 30 g
Store at 2-28(degree)C.
For in vitro diagnostic use.
Mfd. by:
OraSure Technologies, Inc.
Beaverton, OR 97008
201-3080-4
Label PN 201-3080-4
Exp.
Lot.
Sample Diluent Concentrate 100 mL
Contains 0.01% thimerosal.
Dissolve crystals prior to use.
Store at 2-8(degree)C. For in vitro diagnostic use.
Mfd. by:
OraSure Technologies, Inc.
Beaverton, OR 97008
201-3251-2
Lot
Exp.
Label PN 201-3251-3
Human Immunodeficiency Virus Type 1
OraSure(R)HIV-1 Western Blot Strips
20 strips
HIV-1 preblotted nitrocellulose strips.
For use in the OraSure(R) HIV-1 Western Blot
Assay to detect antibody to HIV-1 in oral
specimens collected with OraSure(R) HIV-1
Oral Specimen Collection Device.
For in vitro diagnostic use.
Store at 2-8(degrees)C. Keep bag tightly sealed.
Caution: Handle as if capable of transmitting
Infectious agents.
Contains 0.1% sodium azide as preservative.
Mfd. by OraSure Technologies, Inc.
Beaverton, OR 97008
201-3259-3
Lot
Exp.
Label PN 201-3259-3
Substrate
22 mL
Store at 2-8(degrees)C.
Protect from light.
For in vitro diagnostic use.
Mfd. by:
OraSure Technologies, Inc.
Beaverton, OR 97008
201-3261-3
Lot
Exp.
Label PN 201-3261-3
OraSure(R) HIV-1 WB
Negative Control
0.65 mL
Store at 2-8(degrees)C.
Caution: Handle as if capable of transmitting infectious agents.
For in vitro diagnostic use.
Mfd. by OraSure Technologies, Inc. Beaverton, OR 97008
201-3262-3
Lot
Exp.
Label PN 201-3262-3
OraSure(R) HIV-1 WB
Low Positive Control
0.65 mL
Store at 2-8(degrees)C.
HIV-1 inactivated.
Caution: Handle as if capable of transmitting infectious agents.
For in vitro diagnostic use.
Mfd. by OraSure Technologies, Inc. Beaverton, OR 97008
201-3263-3
Lot
Exp.
Label PN 201-3263-3
OraSure(R) HIV-1 WB
High Positive Control
0.65 mL
Store at 2-8(degrees)C.
HIV-1 inactivated.
Caution: Handle as if capable of transmitting infectious agents.
For in vitro diagnostic use.
Mfd. by OraSure Technologies, Inc. Beaverton, OR 97008
201-3264-3
Lot
Exp.
Label PN 201-3264-3
Conjugate
Concentrate
0.25 mL
Store at 2-8(degrees)C.
Goat antiserum to Human lgG (H&L)
F(ab')\\2\\ fragment phosphatase conjugate.
Contains 0.1% sodium azide
as preservative.
For in vitro diagnostic use.
Mfd. by OraSure Technologies, Inc. Beaverton, OR 97008
201-3265-3
Lot
Exp.
Label PN 201-3265-3
Human Immunodeficiency Virus Type 1 (HIV-1) 20 tests
OraSure(R) HIV-1 Western Blot Kit
Western Blot Assay for the detection of the antibody to Human Immunodeficiency
Virus Type 1 (HIV-1) in oral specimens collected with OraSure(R) HIV-1 Oral
Specimen Collection Devices.
Store at 2-8(degrees)C.
For in vitro diagnostic use.
Contains:
OraSure HIV-1 Western Blot Strips** 1 pkg., containing 20 HIV-1 preblotted nitrocellulose strips
Sample Diluent Concentrate* 1 bottle, 100 mL
Powdered Milk 1 bottle, 30 g
Conjugate Concentrate** 1 vial, 0.25 mL, goat antiserum to Human lgG (H&L) F(ab')\\2\\ fragment
phosphatase conjugate
Substrate 1 bottle, 22 mL
OraSure HIV-1 WB Negative Control 1 vial, 0.65 mL, human serum in OraSure matrix, negative for HIV-1 Ab
OraSure HIV-1 WB Low Positive Control 1 vial, 0.65 mL, human serum in OraSure matrix, positive for HIV-1Ab
OraSure HIV-1 WB High Positive Control 1 vial, 0.65 mL, human serum in OraSure matrix, positive for HIV-1 Ab
Disposable reaction trays with lids Five each
Read accompanying package insert for instructions. **Contains 0.1% sodium azide
Caution: Handle as if capable of transmitting infectious agents. *Contains 0.01% thimerosal
Warning: FDA has approved this test kit for use with OraSure oral specimens
only. Use of this test kit with specimens other than those specifically approved
for use with this kit may result in inaccurate test results.
Note: This test kit should be used to test OraSure specimens only.
OraSure specimens are not to be used to screen the blood supply.
[LOGO]
B I O M E R I E U X
Manufactured by: Distributed by:
OraSure Technologies, Inc. bioMerieux, Inc.
Beaverton, OR 97008 Box 15969
Made in USA Durham, NC 27704-0969
EXHIBIT 11
COMPLAINTS AND RECALLS
Customer communications and complaints and Product recalls shall be managed
in accordance with this EXHIBIT 11. BMX will have primary contact with
customers in North America.
11.1. If OSUR receives any communications from a customer that may
fit the definition of a complaint, it shall direct the person making the
complaint to BMX Customer Service.
11.1.1. All customer contacts to BMX involving Product
issues which require documentation, pursuant to pertinent laws or
FDA regulations or requirements, will be entered by BMX into BMX's
automated complaint tracking system. Each contact will be assigned a
contact code. Contacts that are considered complaints will be
investigated by BMX Customer Service, and to the extent possible,
resolved through troubleshooting between BMX Customer Service and
the complainant.
11.1.2. Any complaint that requires an investigation and
response from OSUR will be communicated by BMX to OSUR, in writing
within five (5) business days after receipt of the complaint, on a
BMX Supplier Corrective Action Request ("SCAR") form. In such case,
BMX will arrange for contact between OSUR and the customer if
needed. BMX may elect to participate in any communications between
OSUR and the customer.
11.1.3 OSUR will investigate each such complaint and
respond, in writing, to BMX on the SCAR form, or attachments
thereto. Such response shall include reasonable details relating to
the scope of the investigation and OSUR's findings and conclusions
regarding the complaint. OSUR will provide a completed complaint
response or, if it is not possible to provide a response, a progress
update within 20 business days after receipt of the SCAR from BMX.
Complaints which are Medical Device Reportable incidents will be
expedited via phone and fax.
11.1.4. BMX Quality Assurance will enter the complaint
response into the complaint system, and forward the complaint to BMX
Customer Service, who will evaluate the adequacy of the complaint
response.
11.1.5. BMX shall, using the information developed through
the foregoing procedures, communicate to the customer concerning the
complaint resolution within 10 business days of its receipt of a
completed SCAR.
11.1.6. Upon completion of all investigation activities,
technical evaluation of the complaint, and completion of a letter or
documented telephone call to the complainant, BMX shall close the
complaint in the complaint system.
-21-
BMX shall provide OSUR copies of all communications to the
complainant within 30 days of closing the complaint investigation.
11.1.7. BMX will provide OSUR with a regular weekly report
of all complaints received.
11.1.8 As needed, OSUR and BMX representatives will hold
teleconferences for the purpose of discussing complaint status and
to expedite the investigation of and closure of complaints.
11.1.9 OSUR and BMX shall maintain complaint files
containing the information required by 21 CFR 820.198.
11.1.10 All reports and other information required to be
provided to OSUR hereunder shall be provided to OSUR's Vice
President, Regulatory Affairs, or such other person designated by
OSUR.
11.2 If any regulatory authority issues or requests a recall or
takes similar action in connection with the Product, or if either party
determines an event, incident or circumstance has occurred that may result
in the need for a recall or market withdrawal, the party notified of or
determining the need for such recall or similar action shall, within 24
hours, advise the other party thereof by telephone or facsimile. Following
notification of a recall, within 48 hours, the parties shall discuss
whether or not to conduct a recall, and if so, the timing of the recall,
the breadth, extent and level of customer to which the recall shall reach,
the strategies and notifications to be used, and other related issues.
Upon reaching agreement as to the matters set forth above, BMX shall
prepare the customer distribution list and notify customers. The
notification letter will contain text agreed upon by BMX and OSUR, and
shall be distributed on OSUR letterhead. With OSUR's consent, BMX may
include a cover letter on BMX letterhead making reference to the
communication contained in the OSUR letter. Each party shall bear the
expenses of any recall resulting from a breach of its respective
obligations hereunder.
-22-