bioelisa HIV-1+2 Ag/Ab Introduction

We announce the launch of a new member of the bioelisa family:

bioelisa HIV-1+2 Ag/Ab

Code 3000-1161 192 Tests Code 3000-1162 480 Tests

This is a 4th-generation assay for the simultaneous detection of antibodies to HIV-1 and HIV 2 as well as p24 antigen of HIV-1. The new assay is based on recombinant and synthetic peptides for antibody detection and monoclonal antibody specific to the p24 protein of HIV-1. Bioelisa HIV-1+2 Ag/Ab includes breakable wells and a system for sample and reagent addition monitoring. Bioelisa HIV-1+2 Ag/Ab is a CE mark product, manufactured by Adaltis Inc. and distributed by Biokit, S.A. under the biokit brand name. The performance and characteristics of Bioelisa HIV-1+2 Ag/Ab correspond to the Adaltis Detect-HIVTM v4 product. Product information is enclosed which we hope will help you in the launch of this new assay.

15 May 2007

236/MKT/E/30

Product Launch

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Index: Product information

- Introduction - Intended use - Assay principle - Assay description - Key features and benefits - bioelisa HIV 1+2 Ag/Ab vs competitors - Sensitivity compared to other HIV assays

Evaluations

- Internal evaluations - External evaluations

⋅ Institute of Tropical Medicine, Antwerp ⋅ BioMARIC N.V, Belgium ⋅ Institute National de la Transfusion Sanguine

Appendices

- CE mark certificate - Package insert - Biokit box label - Best 2000 protocol

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bioelisa HIV-1+2 Ag/Ab

Introduction

bioelisa HIV 1+2 Ag/Ab is a new assay based on recombinant antigens, synthetic peptide and monoclonal antibody to p24 Ag. Biokit SA has reached an agreement with Adaltis Inc. for the distribution of Detect-HIVTM v4 under the brand name bioelisa HIV 1+2 Ag/Ab. The product is manufactured in Canada by Adaltis Inc. This fact appears explicitly on the box label and in the package insert, since Adaltis Inc. is the legal manufacturer of the product. All product characteristics and evaluations mentioned in this dossier are related to the product named Detect-HIVTM v4. The bioelisa HIV 1+2 Ag/Ab characteristics and performance correspond exactly to the Detect-HIVTM v 4 assay. bioelisa HIV 1+2 Ag/Ab is a CE mark approved, Class II product with a dedicated CE mark certificate.

Explanation of the test The etiological agent of the AIDS infection has been identified as a retrovirus, human immunodeficiency virus, type 1 (HIV-1). A closely related but distinct type of immunodeficiency virus, designated HIV-2, has also been isolated. This virus causes a disease that is indistinguishable from AIDS. Serological cross-reactivity between HIV-1 and HIV-2 has proven to be highly variable from sample to sample. Such variability requires the inclusion of antigens to both HIV-1 and HIV-2 for the screening of antibodies to HIV-1 and HIV-2. In addition, antibodies directed against the HIV-1 p24 antigen are also included for detection of the pre-seroconversion stage of the infection. The presence of anti-HIV-1 and/or anti-HIV-2 in the blood indicates potential infection with HIV-1 and/or HIV-2. Consequently, this blood should not be used for transfusion or for the manufacturing of injectable products.

Intended use

The bioelisa HIV 1+2 Ag/Ab test kit is a solid-phase enzyme immunoassay which uses a mixture of antigens and antibodies for the in vitro diagnostic screening in human serum or plasma (ACD, heparin, EDTA, citrate, lithium) of antibodies to all known subtypes of HIV-1 and to HIV-2, and of the HIV-1 p24 antigen at the early stage of infection. This kit is a combined Ag/Ab assay and is not to be used for the detection of the HIV-1 p24 antigen alone.

Assay principle

bioelisa HIV 1+2 Ag/Ab is a ‘fourth-generation’ immunoassay, this designation referring to the ability of such assays to detect antibodies directed against HIV-1, including rare subtypes such as group O and HIV-2 as well as HIV-1 p24 antigen. The technology used to develop these assays evolved from the first-generation viral-lysate-based IgG tests, to the second-generation tests incorporating recombinant and/or synthetic peptide antigens, to the third-generation tests which detect all antibody isotypes (antigen sandwich techniques), and finally to the third-generation-plus assays which also belongs to bioelisa HIV-1 group O (Saville et al., 2001). Generally speaking, fourth-generation assays allow a significant reduction of the time between the initial infection and the laboratory diagnosis (diagnostic window). Comparative studies of third- and fourth-generation assays showed that, on the average, the latter assays

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reduced that diagnostic window by 2 to 4 days (Ly et al., 2004; Weber et al., 2003; WHO, 2004). Antigens representing immuno-dominant epitopes of HIV-1 gp41 and HIV-2 gp36 as well as antibodies against the antigen p24 are coated onto microplate wells. Antigens and antibodies have been carefully selected to ensure the screening of antibodies to all HIV-1 subtypes, including subtype O and HIV-2, and the detection of p24 antigens. Serum or plasma samples are added to these wells and, if p24 antigens and/or antibodies specific to HIV-1 and/or HIV-2 (IgG, IgM or IgA) are present in the sample, they form stable complexes with the HIV antigens or antibodies attached to the well. Antigen-antibody complexes are then identified through the successive addition of: (1) biotinylated antigens and antibodies, and (2) horseradish peroxidase (HRP) Streptavidin conjugate. The catalytic activity of horseradish peroxidase allows for the quantification of these antibody-antigen complexes. Peroxidase substrate solution is then added. During incubation, a blue colour will develop in proportion to the amount of anti-HIV-1/2 antibodies and/or p24 antigens bound to the well, thus establishing their presence or absence in the sample. Wells containing samples negative for anti-HIV antibodies and p24 antigens remain colourless. A stopping solution is added to each well and the resulting yellow colour is read on a microplate reader at 450 nm.

Assay description

Antigens on the solid phase (breakable wells):

- HIV-1: Recombinant protein representing the gp41 Synthetic peptide representing

immunodominant epitopes of different subtypes of gp41 (mosaic peptide)

- HIV-2: Synthetic peptide representing the gp36

- Anti-p24:

Monoclonal antibody specific to the p24 protein of HIV-1

Conjugates:

- Conjugate 1: Biotinylated antigens and ready-to-use antibodies.

Colour code: Green - Conjugate 2:

Ready-to-use peroxidase-labeled Streptavidin. Colour code: Blue.

Controls:

- Negative control - HIV-1 Calibrator (positive) - HIV-2 Positive control

Sample diluent:

- Colour code: red. Capability of changing colour after sample addition to orange.

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Substrate: - Ready-to-use TMB

Incubation time:

- Two hours and 30 minutes

Assay protocol: - 1 hour at 37°C sample incubation - 30 minutes at 37°C conjugate 1 incubation - 30 minutes at 37°C conjugate 2 incubation - 30 minutes at Room Temperature Substrate incubation

Cut-off calculation:

- Negative control + (HIV Calibrator/6)

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Key features and benefits

Immunoassay kits that can detect both anti-HIV antibodies and HIV-1 p24 antigen in a single test are now known as 4th-generation assays. This feature is very important in that it enables these kits to detect the HIV infection 5 to 7 days earlier than 2nd- or 3rd-generation assays. Essentially, 4th-generation kits can detect the infection before the appearance of any type of anti-HIV antibody. The only requirement is that the concentration of HIV p24 antigen (correlating with the number of HIV viral particles) should be beyond the limit of detection of the assay. This limit of detection currently ranges from 28 to 300 pg/mL amongst the various assays so, in theory (and in practice), an assay with a lower limit of detection will detect the infection sooner. bioelisa HIV 1+2 Ag/Ab shows one of the lowest detection limits on the market: 31 pg/mL

Sensitivity It is important to note that the foremost feature of HIV 4th-generation kits is sensitivity. In fact, sensitivity truly reflects the “plus” of 4th-generation kits, i.e. the capability of detecting positive patients, through detection of the HIV-1 p24 antigen BEFORE the appearance of any antibody, which acts as the only detection “vehicle” for traditional 3rd- generation kits. In other words, 4th-generation kits exist with the sole intention of increasing sensitivity and reducing the “Detection Window”, therefore anticipating detection of HIV-positive patients in any screening setting through the early detection of the HIV-1 p24 antigen. The more sensitive to HIV-1 p24 antigen a kit is, the better it represents the “spirit” of 4th-generation HIV kits. In this light, bioelisa HIV 1+2 Ag/Ab constitutes one of the “best” 4th-generation kits as it displays a higher sensitivity to HIV-1 p24 antigen when compared to international competitors, such as Abbott-Murex, Bio-Rad, Dade and Biomerieux 4th-Gen Kits.

Ease of use and assay procedure As is shown in detail in the rest of the document, the existing 4th-generation kits differ significantly as far as assay procedure and ease of use are concerned. While some features are common to all kits once used on any automated instruments (due to the fact that they are carried out automatically), other features are really differentiating since they need to be carried out by lab personnel. The fewer of these manual operations there are, the less likelihood there will be of laboratory errors and the less time will be required. Among the differentiating factors, there are few that are most important for lab personnel:

1. Presence in the kit of ready-to-use reagents versus reagents that either need to be reconstituted or diluted

2. Stability of the kit both once opened and unopened 3. Possibility of testing samples collected with the use of different anticoagulants

Ready-to-use reagents bioelisa HIV 1+2 Ag/Ab employs only ready-to-use reagents, thus increasing the service offered to customers. In fact “downtimes” are significantly reduced for lab personnel since, in contrast to any of the other competitor kits on the market, there is no need for any conjugate reconstitution or substrate preparation.

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Stability bioelisa HIV 1+2 Ag/Ab remains stable for 4 months once opened and correctly stored in a refrigerator in contrast to other competitors kits which are stable for a much shorter period. This also enables small-volume labs to fully benefit from the kit and not waste any well or strip due to expiration after the first opening. Furthermore, the shelf life (stability of the kit stored unopened at 2-8°C) of bioelisa HIV 1+2 Ag/Ab is 15 months. Anticoagulants bioelisa HIV 1+2 Ag/Ab can process serum as well as plasma specimens collected with a large number of anticoagulants: ACD, heparin, lithium, citrate and EDTA. No other competitor on the market can claim such a range of anticoagulants. Sample addition monitoring bioelisa HIV 1+2 Ag/Ab includes a sample diluent able to monitor the sample addition by a colour change. This process can be checked either visually or spectrophotometrically at different wave lengths. Conjugate #1, conjugate #2 and substrate can be also monitored by the colour code or the optical density.

OD Empty Strip

Sample Diluent

Conjugate #1

Conjugate #2 Substrate

Sample Diluent + Human Serum

Sample Diluent + Negative Control

Sample Diluent + Calibrator

Sample Diluent +

HIV 2 Positive Control

450 nm 0,034 1,621 0,269 0,072 0,081 1,613 1,893 1,951 1,951620 nm 0,032 0,085 0,088 0,253 0,073 0,604 0,222 0,258 0,258492 nm 0,034 0,734 0,110 0,070 0,076 1,199 0,927 0,972 0,972405 nm 0,036 1,244 0,277 0,105 0,069 1,505 1,619 1,663 1,663550 nm 0,034 0,276 0,068 0,103 0,115 1,908 0,706 0,773 0,773

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bioelisa HIV 1+2 Ag/Ab vs competitors

Competitor kits evaluated In addition to bioelisa HIV 1+2 Ag/Ab, other kits are commercialized throughout the world. The product characteristics and product performance mentioned in their respective package inserts have been evaluated.

- Murex Combination HIV Ag/Ab (Murex-Abbott) - Genscreen Plus HIV Ag-Ab (Bio-Rad) - Genscreen Ultra HIV Ag-Ab (Bio-Rad) - Enzygnost HIV Integral (Dade Behring) - Vironostika HIV Uni-Form II Ag-Ab (Biomerieux)

HIV-1 p 24 Antigen sensitivity declared in package insert - bioelisa HIV 1+2 Ag/Ab : 31 pg/ml - Vironostika Uni-Form II Ag/Ab: Not mentioned in package insert - Genscreen Plus HIV Ag-Ab: 140 pg/ml - Genscreen Ultra HIV Ag-Ab: 14 pg/ml - Enzygnost HIV Integral: 120 -150 pg/ml - Murex Combination HIV Ag-Ab : 28 pg/ml

Sensitivity vs seroconverters declared in package insert

- bioelisa HIV 1+2 Ag/Ab: 68 pos/132 specimens (on 24 panels) → 52% - Vironostika Uni-Form II Ag/Ab: 54 pos/132 specimens (on 24 panels) → 41% - Genscreen Plus HIV Ag-Ab: 67 pos/132 specimens (on 24 panels) → 51% - Genscreen Ultra HIV Ag-Ab: (90 panels tested) → data not available (better

than Genscreen Plus on 41 panels) - Enzygnost HIV Integral: 61 pos/132 specimens (on 24 panels) → 46% - Murex Combination HIV Ag-Ab : 75 pos/132 specimens (on 24 panels) → 57%

Specificity - bioelisa HIV 1+2 Ag/Ab 1st study: 4982/5006 → 99.52% - bioelisa HIV 1+2 Ag/Ab 2nd study: 1131/1132 → 99.91% - Vironostika Uni-Form II Ag/Ab: 4791/4796 → 99.90% - Genscreen Plus HIV Ag-Ab: 5578/5584 → 99.89% - Genscreen Ultra HIV Ag-Ab: 6035/6038 → 99.95% - Enzygnost HIV Integral: 8823/8851 → 99.82% - Murex Combination HIV Ag-Ab: 9269/9290 → 99.77%

Ready-To-Use reagents - bioelisa HIV 1+2 Ag/Ab: All reagents are ready-to-use. - Vironostika Uni-Form II S/Ab: Substrate solution needs to be prepared. - Genscreen Plus HIV Ag-Ab: Substrate and Conjugate 2 need to be prepared.

Lyophilised reagents. - Genscreen Ultra HIV Ag-Ab: Substrate and Conjugate 2 need to be prepared.

Lyophilised reagents. - Enzygnost HIV Integral: Substrate and Conjugate 1 need to be prepared - Murex Combination HIV Ag-Ab: Substrate and Conjugate 1 need to be

prepared.

Stability - bioelisa HIV 1+2 Ag/Ab: Stable for 4 months once opened. - Vironostika Uni-Form II Ag/Ab: Stable for 2 months once opened. - Genscreen Plus HIV Ag-Ab: Stable for 1 month once opened. - Genscreen Ultra HIV Ag-Ab: Stable for 1 month once opened. - Enzygnost HIV Integral: Stable for 1 month once opened. - Murex Combination HIV Ag-Ab: Stable for 1 month once opened.

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Type of specimen - bioelisa HIV 1+2 Ag/Ab: Serum or plasma collected with ACD or heparin or

lithium or citrate or EDTA. - Vironostika Uni-Form II S/Ab: Serum or plasma collected with heparin or citrate

or EDTA. - Genscreen Plus HIV Ag-Ab: Serum or plasma collected with heparin or citrate

or EDTA. - Genscreen Ultra HIV Ag-Ab: Serum or plasma collected with heparin or citrate

or EDTA. - Enzygnost HIV Integral: Serum or plasma collected with heparin or citrate or - EDTA. - Murex Combination HIV Ag-Ab: Serum or plasma collected with either citrate or

EDTA.

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Sensitivity compared to other HIV 4th- and 3rd-generation assays

Comparative sensitivity studies with all available HIV 4th- and HIV 3rd- generation assays have been conducted by the Microbiological Diagnostics Assessment Service (MDAS) in Colindale, United Kingdom. Cumulative results on 21 seroconversion panels produce a very objective score on sensitivity. bioelisa HIV-1+2 Ag/Ab is the third most sensitive assay, surpassed only by Axym HIV Ag/Ab combo and Genscreen ULTRA HIV Ag-Ab. It should be pointed out that a renamed Abbott Prism Ag/Ab combo assay is below bioelisa HIV-1+2 Ag/Ab in this ranking.

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Combined Seroconversion ranking (21 panels)

The number of samples detected by each assay has been reported based on a total of 126 seroconversion samples in BBI panels from PRB916 to 6240. The higher the number of samples detected, the better. The ranking has been determined from this number. bioelisa HIV-1+2 Ag/Ab as Detect HIV v.4 occupies the 3rd position in the ranking.

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Comparative timing of detection This is the delay in detecting primary HIV infection in Seroconversion panels compared to the earliest detection by a screening assay. Day Zero corresponds to the Axym HIV Ag/Ab Combo. bioelisa HIV-1+2 Ag/Ab (Detect HIV v.4) shows a delay of 1.5 days for the earliest detection. It is important to point out that Prism HIV Ag/Ab shows 3.5 days delay for the same set of seroconversion panels

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Internal evaluations Performance characteristics

Performance evaluation has been conducted in accordance with the Common Technical Specifications (CTS) 2002/364/EC (European Community) as required by Article 5 of IVD Directive 98/79/EC.

Diagnostic Sensitivity

Diagnostic sensitivity has been tested on specimens classified HIV-positive by a CE- approved kit. Positive specimens were collected from different geographical regions (including HIV-1 subtypes A, AE, B, C, D, E, F, F1, G, H, and O, and HIV-2). The five HIV-1 subtype O tested were found to be strongly positive. An overall diagnostic sensitivity of 100% (95% confidence interval: 99.5-100%) has been found in a study conducted on a total number of over 400 HIV-1 specimens and 100 HIV-2 specimens.

Seroconversion panel results

A total of 30 seroconversion panels have also been studied. The overall results of these 30 panels are represented in the table below. For each panel the first specimen detected by bioelisa HIV 1+2 Ag/Ab is described in the table in comparison with other 4th-generation assays, third-generation assays mentioned in the panel information and assays detecting only HIV antigen. It can be seen that bioelisa HIV 1+2 Ag/Ab matches the sensitivity of other 4th-generation assays and almost matches the sensitivity of dedicated p24 Ag assays for the majority of the panels.

Panel First positive specimen detected in the panel

bioelisa HIV 1+2 Ag/Ab

4th generation

assay

3rd generation

assay

HIV antigen assay

PRB927 2 Unknown 2 2 PRB928 2 Unknown 2 2 PRB929 4 Unknown 6 3 PRB930 2 Unknown 3 1 PRB931 6 Unknown 6 5 PRB933 2 Unknown 2 2 PRB934 1 Unknown 2 1 PRB935 6 Unknown 7 6 PRB938 1 Unknown 3 1

PRB939(E) 7 Unknown 9 6 PRB940 2 Unknown 3 2 PRB941 4 Unknown 4 3 PRB942 4 Unknown Not detected 4 PRB943 4 Unknown 6 3 PRB944 3 Unknown 5 3 PRB945 4 Unknown 4 4 PRB946 3 Unknown Not detected 3 PRB947 2 Unknown 2 2 PRB948 4 Unknown Not detected 4 PRB949 4 Unknown 5 4 PRB950 3 Unknown 4 2 PRB951 4 Unknown 6 3 PRB952 3 Unknown 4 3 PRB953 3 Unknown 4 3 PRB954 7 7 Not detected 7 PRB955 3 4 4 2 PRB956 4 5 Not detected 4 PRB957 6 6 Unknown 5 PRB958 4 4 Unknown 3 PRB959 2 2 Unknown 1

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Analytical sensitivity: p24 Antigen concentration detection

An analytical sensitivity of 31 pg/mL traceable to the French national standard approved by the AFSSAPS (Bio-Rad HIV-1 Antigen Cat. 72217), and of 1.17 U/mL of the HIV p24 Antigen 1st International Reference Reagent, NIBSC Code 90/636 has been found with bioelisa HIV 1+2 Ag/Ab.

Specificity

Study 1: Blood donor specificity A specificity of >99.50% (95% confidence interval: 99.3-99.7%) has been found in a study conducted on a total number of over 5 000 negative serum specimens collected from normal individuals and blood donors, classified HIV-negative with a CE-approved kit. Study 2: Blood donor specificity A specificity of 99.91% has been found in a study conducted on a total number of 1,132 negative serum specimens collected from normal individuals and blood donors, classified HIV-negative with a CE-approved kit. Study 3: Potential interferences A specificity of 100% (95% confidence interval: 98.1-100%) has been found in a study conducted on a total number of over 150 serum or plasma specimens with different viral (EBV, HAV, HBV, HCV, VZV, HTLV-I, HTLV-II) and non-viral pathologies (RF, monopar and multipar pregnant women, hemolyzed specimens, elevated triglyceride, elevated bilirubin), classified HIV-negative with a CE-approved kit. Study 4: Hospitalized patients specificity A specificity of 99.04% (95% confidence interval: 96.5-99.7%) has been found in a study conducted on a total number of more than 200 negative serum or plasma specimens collected from hospitalized patients, classified HIV-negative with a CE-approved kit. No difference in performance due to the method of specimen preparation (serum, ACD, heparin, EDTA, citrate and lithium) has been observed.

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External evaluations

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CE Mark certificate

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Package insert

3000-1161 / 1162 1

bioelisa HIV – 1+2 Ag/Ab

Fourth generation EIA test kit for in vitro diagnostic screening of antibodies to HIV-1 and HIV-2 and p24 antigen of HIV-1

3000-1161 / 1162 192/480

FOR IN VITRO DIAGNOSTIC USE ONLY 0459 Store at 2...8 °C

COMPLETELY READ THE INSTRUCTIONS BEFORE PROCEEDING

EN SYMBOLS USED ON LABELS EC REP Authorized Representative in Europe

Manufacturer

Attention. See Instructions For Use

IVD

In vitro diagnostic medical device (In vitro diagnostic use)

LOT Lot number REF Catalogue Code

YYYY-MM

Expiry date (Use by…)

Temperature limitation (store at 2...8°C)

Number of tests

Keep away from sunlight

Biological Risks

MT Plate Microplate CONTROL - Negative Control CAL Anti-HIV-1 HIV-1 Calibrator CONTROL + Anti-HIV-2 HIV-2 Positive Control DIL SPE Sample Diluent

3000-1161 / 1162 2

BUF WASH 25x Wash Buffer (25x) CONJ 1 Conjugate #1 CONJ 2 Conjugate #2 SUBS TMB Substrate H2SO4 1N Stop Solution 1N

1. COMPOSITION

KIT: The kit contains the general reagents required for the assay i.e. coated microstrips, sample diluent, antigens-Biotin, anti-p24-Biotin and HRP Streptavidin conjugates, buffers, substrate, and stop solution.

2. INTENDED USE

The bioelisa HIV 1+2 Ag/Ab test kit is a solid phase enzyme immunoassay utilizing a mixture of antigens and antibodies for the in vitro diagnostic screening in human serum or plasma (ACD, heparin, EDTA, citrate, lithium) of antibodies to all known subtypes of HIV-1 and to HIV-2, and of the HIV-1 p24 antigen at the early stage of infection. This kit is a combined Ag/Ab assay and is not to be used for the detection of HIV-1 p24 antigen alone. This kit is for in vitro diagnostic use by a health-care professional and will not be sold to the general public.

3. SUMMARY AND EXPLANATION OF THE TEST

Epidemiological evidence indicates that an infectious agent transmitted through intimate contact, intravenous drug use or use of infected blood or blood products leads to Acquired immunodeficiency Syndrome (AIDS). This disease affects T-cell mediated immunity, resulting in severe lymphopenia and a reduce subpopulations of helper T-lymphocytes. Destruction of this T-lymphocyte population by the virus causes an immune defficiency resulting in a reduced or deficient response to subsequent infections. Consequently infections become more severe and may cause death. At present, there is no successful treatment for AIDS.

The etiological agent has been identified as a retrovirus, human immunodeficiency virus type 1 (HIV-1). A closely related, but distinct type of immunodeficiency virus, designated HIV-2, has also been isolated. This virus causes a disease that is indistinguishable from AIDS. Serological cross-reactivity between HIV-1 and HIV-2 has been shown to be highly variable from sample to sample. This variability requires the inclusion of antigens to both HIV-1 and HIV-2 for the screening of antibodies to HIV-1 and HIV-2. In addition, antibodies directed against the HIV-1 p24 antigen are also included for detection of the pre-seroconversion stage of the infection. The presence of anti-HIV-1 and/or anti-HIV-2 in the blood indicates potential infection with HIV-1 and/or HIV-2 and consequently this blood should not be used for transfusion or for manufacture or injectable products.

4. PRINCIPLE OF THE TEST

bioelisa HIV 1+2 Ag/Ab is a ‘fourth-generation’ immunoassay, this designation referring to the ability of such assays to detect antibodies directed against HIV-1 (including rare subtypes such as group O) and HIV-2 as well as HIV-1 p24 antigen. The technology used to develop these assays evolved from the first-generation viral-lysate-based IgG tests, to the second-generation tests incorporating recombinant and/or synthetic peptide antigens, to the third-generation tests which detect all antibody isotypes (antigen sandwich techniques), and finally to the third-generation-plus assays which also detect HIV-1 group O (Saville et al., 2001). Generally speaking, fourth-generation assays allow a significant reduction of the time between the initial infection and the laboratory diagnosis (diagnostic window); comparative studies of third- and fourth-generation assays showed that in average, the latter assays reduced that diagnostic window by 2 to 4 days (Ly et al., 2004; Weber et al., 2003; WHO, 2004).

Antigens representing immuno-dominant epitopes of HIV-1 gp41 and HIV-2 gp36 as well as antibodies against the antigen p24 are coated onto wells of a microplate. Antigens and antibodies have been carefully selected to ensure the screening of antibodies to all HIV-1 subtypes, including subtype O and HIV-2, and the detection of p24 antigens. Serum or plasma samples are added to these wells and if p24 antigens and/or antibodies specific for HIV-1 and/or HIV-2 (IgG, IgM or IgA) are present in the sample, they form stable complexes with the HIV antigens or antibodies attached to the well. Antigen-antibody complexes are then identified through the successive addition of: (1) biotinylated antigens and antibodies and; (2) horseradish peroxidase (HRP) Streptavidin conjugate. The catalytic activity of horseradish peroxidase allows for the quantification of these antibody-antigen complexes.

Peroxidase substrate solution is then added. During incubation, a blue colour will develop in proportion to the amount of anti-HIV-1/2 antibodies and/or p24 antigens bound to the well, thus establishing their presence or absence in the sample. Wells containing samples negative for anti-HIV antibodies and p24 antigens remains colourless.

A stop solution is added to each well and the resulting yellow colour is read on a microplate reader at 450 nm.

3000-1161 / 1162 3

5. KIT COMPONENTS 5.1 MATERIAL SUPPLIES

# DESCRIPTION 192 tests 3000-1161

480 tests 3000-1162

1 Microplate (12 strips x 8 wells) 2 plates 5 plates 2 Negative Control 2.5 mL 6 mL 3 HIV-1 Calibrator 2.5 mL 6 mL 4 HIV-2 Positive Control 2.0 mL 5 mL 5 Sample Diluent 25 mL 75 mL 6 Wash Buffer (25X) 250 mL 2 x 250 mL 7 Conjugate #1 50 mL 125 mL 8 Conjugate #2 50 mL 125 mL 9 Substrate 50 mL 250 mL 10 Stop Solution (1N H2SO4) 30 mL 250 mL 11 Microplate Covers 8 20

5.2 MATERIAL SUPPLIED

1. Microplates: Code KMX-411A: Each bag contains a microplate of 12 strips by 8 wells. Each strip is breakable in 8 wells. The wells are coated with an optimized mixture of antigens and antibodies. Store the unused strips in the original releasable pouch.

2. Negative Control: Code KNX-912A, or KNX-915A: One vial of negative control containing human sera not reactive for the HIV-1 and HIV-2 peptides, or to the p24 HIV antigen. It contains also ProClin 300 preservative at 15 ppm active microbicide.

3. HIV-1 Calibrator: Code KPX-012A, or KPX-015A: One vial of calibrator containing antibodies against HIV-1 antigens. It contains also ProClin 300 preservative at 15 ppm active microbicide.

4. HIV-2 Positive Control: Code KPX-112A, or KPX-115A: One vial of positive control containing antibodies against HIV-2 antigens. It contains also ProClin 300 preservative at 15 ppm active microbicide. HIV-2 Positive Control is color coded orange.

5. Sample Diluent: Code KDX-412A, or KDX-415A: One bottle of ready-to-use sample diluent buffer containing ProClin 300 preservative at 15 ppm active microbicide. The sample diluent is colour-coded red and is used for specimen dilution.

6. Wash Buffer (25X): Code KWZ-910BP: One or two bottles of Wash Buffer (25X) containing ProClin 300 preservative at 15 ppm active microbicide.

Dilute 25 times (1 volume of Wash Buffer (25X) with 24 volumes of distilled or deionised water) before use to generate the wash solution. Mix well before use. Store this solution at 2…8°C if it is not to be used immediately. The wash solution is stable at room temperature (18…25°C) or one week.

Some crystal may appear in the Wash Buffer (25x). Dissolve the crystal by simply warming the bottle in a 30…40°C water bath before use.

7. Conjugate #1: Code KIX-412A, or KIX-415A: One bottle containing a ready-to-use conjugate. It contains also ProClin 300 preservative at 15 ppm active microbicide. Conjugate #1 is colour-coded green.

8. Conjugate #2: Code KJX-412A, or KJX-415A: One bottle containing a ready-to-use HRP streptavidin conjugate. It contains also ProClin 300 preservative at 15 ppm active microbicide. Conjugate #2 is colour-coded blue.

9. Substrate: Code KYZ-914B or KYZ-910B: One brown bottle of ready-to-use stabilized mixture of 3,3’,5,5’ tetra-methylbenzidine (TMB) substrate and hydrogen peroxide (H2O2).

10. Stop Solution: Code KSZ-912B or KSZ-910B: One bottle of 1N H2SO4 Stop Solution.

Warning, the Stop Solution contains Sulfuric Acid 1 N (4.9%)

11. Microplate Covers: Microplate plastic sheets cover with adhesive.

6. STORAGE AND STABILITY AFTER THE FIRST OPENING • Store kit components at 2...8oC and do not use after the expiry date on the box outer label. • Before use, all components should be allowed to warm up to ambient temperature (18...25oC). • Unused strips should be placed again in the bag and resealed, the bottle caps replaced and tightened and the kit stored

at 2...8oC. • The opened kit should be used within four months or before its expiry date, whichever comes first.

3000-1161 / 1162 4

7. MATERIALS AND EQUIPMENT REQUIRED BUT NOT PROVIDED • Disposable bench cover • Incubator (37°C) • Test tubes and racks or low-binding microplates • Adjustable micropipettes: 5 to 20 µL and 20 to 200 µL with disposable tips • Multi-pipettors: 100-200 µL and disposable tips or automated dispenser • Disposable pipettes: 1 mL, 5 mL, 10 mL • Measuring cylinders: 100 mL, 500 mL, 1 L and 4 L • Timer • Disposable gloves • Lab tissue • De-ionized or distilled water • Microplate washer/dispenser • Single or dual wavelength microplate spectrophotometer at 450 nm with 600-650 nm as reference (if available) • Autoclave • Refrigerator (2…8°C)

8. WARNING AND PRECAUTIONS • For use in in-vitro diagnostic procedures only. • Only experienced laboratory personnel should use this test and handling should be in agreement with Good Laboratory

Practices (GLP). • Do not pipette by mouth. • Do not smoke, eat or drink in areas in which specimens are handled. • Laboratory jacket, disposable gloves and proper safety glasses should be worn throughout the testing procedure. • Do not use the kit beyond its labelled expiry date. • The components of this kit have been tested as a unit. Do not interchange components from other sources or from

different lots. • Bring reagents to room temperature (18…25°C) approximately 30 minutes before use. Some crystals may appear in

the Wash Buffer (25x). Dissolve the crystals by simply warming the bottle in a 30…40°C water bath before use. • Immediately after use, each individual reagent component of this kit must be stored at 2…8°C. • When using automated dispensers, the tips must be adequately rinsed or replaced between each use. • The Negative Control has not been inactivated. This control includes human plasma negative for, HIV, HCV antibodies

and HBsAg. This does not ensure the absence of viable pathogens, and therefore, these sera should be handled as potentially biohazardous, following good laboratory practice.

• The HIV-1 Calibrator and HIV-2 Positive Control contain serum or plasma reactive with the HIV-1 and/or HIV-2 antigens, and were inactivated using β-propionolactone and UV. They may come from patients infected with HIV-1 and/or HIV-2 and should be considered as potentially infectious.

• After completion of the test, all materials used, including reagents and samples, should be disposed of in a manner that will inactivate human viruses, such as hepatitis viruses and HIV: • Solid Wastes: Autoclave 60 minutes at 121°C. • Liquid Wastes: Autoclave 60 minutes at 121°C or add sodium hypochlorite to a final concentration of 1.0% (v/v). The

waste should be allowed to stand a minimum of 30 minutes to inactivate viruses before disposal. • This kit requires the use of 1 N sulphuric acid. Do not combine acid with waste material containing sodium azide or

sodium hypochlorite. • The use of safety glasses and disposable plastic is strongly recommended when manipulating bio-hazardous or bio-

contaminated solutions. • Proper calibration of the equipment used with the test, such as the pipettes, incubators, microplate reader, is required. • The Substrate is light-sensitive: avoid a too long exposure to light. • The kit is a single use: do not reuse any components.

8.1 Sulphuric Acid Warning The Stop Solution contains sulphuric acid at a concentration of 4.9% w/w. Contact with skin, eyes, and other mucous membranes should be avoided. In case of accidental contact, rinse with plenty of water and seek medical advice. Risk Phrases R 36/38 Irritating to eyes and skin. Safety Phrases S 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice Please refer to the MSDS for further information.

9. SPECIMEN COLLECTION AND HANDLING • Handle all specimens as if capable of transmitting pathogens. • When possible, clear unhemolyzed specimens should be used. Specimens should be collected aseptically. Early

separation from the clot prevents hemolysis in serum. Use of heat inactivated serum or plasma is not recommended as this may lead to false positive results.

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• Undiluted specimens may be stored up to 25 days at 2…8°C or frozen if stored in suitable vials. Avoid multiple freeze-thaw cycles (not more than three).

• Mix the specimens before using them. • Diluted specimens kept at 2…8°C should be used within 24 hours of preparation. Bring to room temperature (18…25°C)

30 minutes before use.

10. ASSAY PROCEDURE 10.1 REAGENT PREPARATION • Bring all reagents to room temperature (18…25°C) at least 30 minutes before use. Take only the volume necessary for

the testing. Return the unused portion at 2…8°C. • Wash solution: Dilute the Wash Buffer (25X) with 24 volumes of distilled or de-ionized water. Wash solution is stable for

1 week at room temperature (18…25°C). • HIV Microplate: Bring to room temperature (18…25°C) before removing from pouch. Return unused strips to pouch and

seal it carefully. Store at 2…8°C. The opened kit should be used within four months or before its expiry date, whichever comes first.

10.2 PIPETTING AND INCUBATION STEPS

Placement of Calibrator and Controls on microtiter plate

Well Well A1 Negative Control A2 First specimen B1 Negative Control B2 Second specimen C1 Negative Control C2 Third specimen D1 HIV-1 Calibrator D2 Fourth specimen E1 HIV-1 Calibrator E2 Fifth specimen F1 HIV-1 Calibrator F2 Sixth specimen G1 HIV-2 Positive Control G2 Seventh specimen H1 HIV-2 Positive Control H2 Eighth specimen

10.3 NOTICE:

Strict adherence to the suggested timing of incubation and short periods between steps are necessary to obtain reliable test results.

10.4 IN PLATE DILUTION METHOD (IPD) A. Dispense 100 µL of the Sample Diluent (color-coded red, changing color upon sample addition) into as many wells as

there is calibrator, controls and samples to be tested (Wells A1, B1, C1, etc...). B. Dispense 100 µL of the Negative Control sample into each of 3 wells (A1, B1, C1), 100 µL of the HIV-1 Calibrator sample

into each of 3 wells (D1, E1, F1) and 100 µL of the HIV-2 Positive Control sample into each of 2 wells (G1, H1). Shake the plate or rinse the tip three times in order to mix the controls with Sample Diluent already present in the well

C. Directly add 100 µL of each test sample to the individual wells containing 100 µL of the Sample Diluent, using a separate pipette tip for each sample. Shake the plate or rinse the tip three times in order to mix the test sample with Sample Diluent already present in the well. Cover the microplate with a new adhesive microplate cover. Continue with step D in section 10.6.

10.5 OUT PLATE DILUTION METHOD (OPD) A. Into clean test tubes or low binding microplates, dilute 115 µL of each calibrator, control and sample to be tested with 115

µL Sample Diluent (color-coded red, changing color upon sample addition) (1:1 dilution) and mix well. Diluted specimens kept at 2…8°C should be used within 24 hours of preparation. Bring to room temperature (18…25°C) 30 minutes before use.

B. Dispense 200 µL of the 1:1 diluted controls and samples into individual wells (A1, B1, C1, etc...) using a clean pipette tip for each sample. Cover the microplate with a new adhesive microplate cover.

C. Continue with step D in section 10.6. 10.6 REMANING OF THE PROCEDURE

The following steps are used for both methods (IPD and OPD). D. Incubate the microplate at 37°C for 60 minutes without agitating. E. Remove the microplate cover. Aspirate and wash the plate 5 times with 350 µL/well of wash solution (Wash Buffer (25X)

diluted with 24 volumes of water, see section 10.1) with a soak time of 5-10 seconds. Automated washer should be adjusted to fill each well completely without overfilling. After the final wash, be sure all of the solution is removed from each well. Sharply tap the plate upside down on absorbent paper to remove the last remaining liquid, taking care not to dislodge strips from holder.

F. Dispense 200 µL of Conjugate #1 Solution (colour coded green) into each well of the microplate. Cover the microplate with a new adhesive microplate cover.

G. Incubate the plate for 30 minutes at 37°C. H. After removing the adhesive microplate cover, aspirate and wash the microplate 5 times as describe in step E. I. Dispense 200 µL of Conjugate #2 Solution (colour coded blue) into each well of the microplate. Cover the microplate with

a new adhesive microplate cover. J. Incubate the plate for 30 minutes at 37°C. K. After removing the adhesive microplate cover, aspirate and wash the microplate 5 times as describe in step E. L. Add 200 µL of Substrate Solution into each well. Cover the microplate. The Substrate is light-sensitive: avoid a too long

exposure to light.

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M. Incubate the substrate-filled plate for 30 minutes at room temperature (18…25°C). Start the timing within 3 minutes after the addition of the reagent to the first well

N. Remove the microplate cover. Stop the reaction by adding 100 µL of Stop Solution into each well, in the same order used for the addition of the Substrate

O. After adding the Stop Solution, the color developed may be read on a plate reader at 450 nm. The absorbance must be read within 30 minutes.

10.7 PROCEDURAL NOTES • Proper wash procedure is essential for good assay performance. Use of multi-pipettes is recommended for manual

addition of Conjugate #1 and #2 (step F and I), Substrate (step L) and Stop Solutions (step N). Manual single well additions may affect accurate timing at these points in the procedure.

• Bichromatic absorbance measurement with a reference wavelength of 600-650 nm is recommended when available. • Record the absorbance results on a data sheet. Include the kit lot number, date, operator name and any notes about the

run. A printed copy of the absorbance readings produced by the spectrophotometer should be attached to the data sheet.

11. CALCULATION OF RESULTS 11.1 VALIDITY OF THE ASSAY

Three (3) replicates of Negative Control, three (3) of HIV-1 Calibrator and two (2) of HIV-2 Positive Control must be included on each run. The results on the Controls and Calibrator must be examined before the sample results can be interpreted. • The Negative Control mean absorbance signal must be lower than 0.310. If the mean absorbance signal is equal or

greater than 0.310, the run should be repeated. • The HIV-1 Calibrator mean absorbance signal must be equal to or greater than 0.500. If the mean absorbance signal is

less than 0.500, the run should be repeated. • The HIV-2 Positive Control mean absorbance signal must be equal to or greater than 0.500. If the mean absorbance

signal is less than 0.500, the run should be repeated. Calculation of Negative Control Mean (NCx):

Negative Control example

Well No. Absorbance A1 0.195 B1 0.187 C1 0.203 Total 0.585

0.585 NCx = 3 = 0.195

Calculation of HIV-1 Calibrator Mean (HIV-1 CALx):

HIV-1 Calibrator example

Well No. Absorbance D1 2.622 E1 2.679 F1 2.655 Total 7.956

7.956 HIV-1 CALx = 3 = 2.652

Calculation of anti-HIV-2 Positive Control Mean (HIV-2 PCx):

HIV-2 Positive Control example

Well No. Absorbance G1 2.425 H1 2.405 Total 4.830

4.830 HIV-2 PCx = 2 = 2.415

11.2 CUT-OFF DETERMINATION

(HIV-1 CALx) Cut-off = NCx + 6

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Example:

NCx = 0.195

HIV-1 CALx = 2.652

(2.652) Cut-off = 0.195 + 6

= 0.637

11.3 FINAL TEST VALIDITY • After determining the cut-off, verify that all three (3) Negative Control replicates are below the cut-off. If all values are

below the cut-off, the run is valid. • If two (2) or more of the Negative Control replicates are equal or higher than the cut-off value, the run is invalid and

should be repeated. • If only one (1) Negative Control replicate is equal or higher than the cut-off, eliminate the value of the highest Negative

Control replicate and calculate a new cut-off with the two (2) remaining values. • If, after the new cut-off calculation, both Negative Control replicates used in the calculation of this new cut-off are below

the cut-off, the run is valid. • If, after the new cut-off calculation, one or more of the two Negative Control replicates used in the calculation of this new

cut-off are equal or higher than the new cut-off, the run is invalid and should be repeated. 11.4 INTERPRETATION OF SAMPLE RESULTS • If the absorbance value is less than the calculated cut-off value, then the sample is considered non-reactive (negative) for

HIV antibodies and HIV-1 p24 antigens. • If the initial absorbance value is equal to or greater than the cut-off, retest the sample in duplicate. • If the absorbance value of both retests is less than the calculated cut-off value, then the sample is considered

non-reactive (negative) for HIV antibodies and HIV-1 p24 antigens. • If the absorbance value of one or both retests is greater or equal than the cut-off, then the sample is considered reactive

or positive for HIV antibodies or HIV-1 p24 antigens by the criteria of this bioelisa HIV 1+2 Ag/Ab test.

12. LIMITATIONS OF THE PROCEDURE • The user of this kit is advised to carefully read and understand the instructions for use. Strict adherence to the protocol is

necessary to obtain reliable test results. In particular, correct sample and reagent pipetting, along with careful washing and timing of incubation steps is essential for accurate, reproducible detection of HIV-1 and HIV-2 antibodies and p24 antigens.

• If possible, use fresh serum or plasma samples. Sample degradation as well as multiple freeze-thaw cycles may cause erroneous results. Do not use heat-inactivated samples.

• Falsely reactive test results can be expected with a test kit of this nature. The proportion of reactives will depend on the sensitivity and specificity of the test kit and on the prevalence of HIV-1 and HIV-2 antibodies in the population to be screened.

• After the bioelisa HIV 1+2 Ag/Ab is performed, repeatedly reactive specimens should be submitted for additional testing using Western Blot (WB), Indirect Immunofluorescense Assay (IFA) or Radioimmunoprecipitation Assay (RIPA) tests. The determination that a person’s specimen contains antibodies to HIV and/or HIV antigens has extensive medical, social, psychological and economic implications. It is recommended that confidentiality, appropriate counselling and medical evaluation be considered an essential aspect of the testing sequence.

• AIDS and AIDS-related conditions are clinical diseases and their diagnosis can only be established clinically. EIA testing alone cannot be used to diagnose AIDS. A non-reactive test result at any point in the testing sequence does not preclude the possibility of exposure to or infection with HIV. The risk of an asymptomatic person, who is repeatedly reactive, of developing AIDS and/or AIDS-related conditions, is not known.

• The test result should be used in conjunction with all other clinical and diagnostic data. • Antibodies to HIV may occur due to voluntary participation in an HIV vaccine study. Interpretation of this diagnostic test

will depend on the type of vaccine given. Correlation with the medical history and additional testing may be necessary to accurately diagnose HIV in vaccine volunteers.

13. PERFORMANCE CHARACTERISTICS OF bioelisa HIV 1+2 Ag/Ab

Evaluation of Performance has been conducted in accordance with the Common Technical Specifications (CTS) 2002/364/EC as required by the article 5 of the IVD Directive 98/79/EC.

13.1 Diagnostic Sensitivity

The diagnostic sensitivity has been tested on specimens classified HIV-positive by a CE approved kit.

Positive specimens were collected from different geographical regions (including HIV-1 subtypes A, AE, B, C, D, E, F, F1, G, H, and O, and HIV-2). The five HIV-1 subtype O tested were found strongly positive.

An overall diagnostic sensitivity of 100% (95% confidence interval: 99.5-100%) has been found in a study conducted on a total number of more than 400 HIV-1 specimens and 100 HIV-2 specimens.

13.2 Sero-conversion Panels Results

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A total of 30 sero-conversion panels have also been studied. The overall results of these 30 panels are represented in the table below.

First specimen detected positive in the panel

Panel bioelisa HIV 1+2 Ag/Ab

4th generation

assay

3rd generation

assay

HIV antigen assay

PRB927 2 Unknown 2 2 PRB928 2 Unknown 2 2 PRB929 4 Unknown 6 3 PRB930 2 Unknown 3 1 PRB931 6 Unknown 6 5 PRB933 2 Unknown 2 2 PRB934 1 Unknown 2 1 PRB935 6 Unknown 7 6 PRB938 1 Unknown 3 1

PRB939(E) 7 Unknown 9 6 PRB940 2 Unknown 3 2 PRB941 4 Unknown 4 3 PRB942 4 Unknown Not detected 4 PRB943 4 Unknown 6 3 PRB944 3 Unknown 5 3 PRB945 4 Unknown 4 4 PRB946 3 Unknown Not detected 3 PRB947 2 Unknown 2 2 PRB948 4 Unknown Not detected 4 PRB949 4 Unknown 5 4 PRB950 3 Unknown 4 2 PRB951 4 Unknown 6 3 PRB952 3 Unknown 4 3 PRB953 3 Unknown 4 3 PRB954 7 7 Not detected 7 PRB955 3 4 4 2 PRB956 4 5 Not detected 4 PRB957 6 6 Unknown 5 PRB958 4 4 Unknown 3 PRB959 2 2 Unknown 1

13.3 Analytical Sensitivity

An analytical sensitivity of 31 pg/mL traceable to the French national standard approved by the AFSSAPS (Bio-Rad HIV-1 Antigen Cat. 72217) and of 1.17 U/mL of the HIV p24 Antigen 1st International Reference Reagent, NIBSC Code 90/636 has been found with bioelisa HIV 1+2 Ag/Ab. 13.4 Specificity

A specificity of >99.50% (95% confidence interval: 99.3-99.7%) has been found in a study conducted on a total number of more than 5 000 negative serum specimens collected from normal individuals and blood donors, classified HIV-negative with a CE approved kit.

A specificity of 100% (95% confidence interval: 98.1-100%) has been found in a study conducted on a total number of more than 150 serum or plasma specimens with different viral (EBV, HAV, HBV, HCV, VZV, HTLV-I, HTLV-II) and non viral pathologies (RF, monopar and multipar pregnant women. hemolyzed specimens, elevated triglyceride, elevated bilirubin), classified HIV-negative with a CE approved kit.

A specificity of 99.04% (95% confidence interval: 96.5-99.7%) has been found in a study conducted on a total number of more than 200 negative serum or plasma specimens collected from hospitalized patients, classified HIV-negative with a CE approved kit.

No difference in performance due to the method of specimen preparation (serum, ACD, heparin, EDTA, citrate and lithium) has been observed. 13.5 Precision

Precision was measured, according to the EP-5A guideline of the CLSI, on a panel of one negative specimen, three HIV-1 positive specimen (low, medium and high), three HIV-2 positive specimens (low, medium and high), and three p24 positive specimen (low, medium and high) but negative for HIV antibodies. Precision was performed on three lots of Adaltis bioelisa HIV 1+2 and in three different laboratories. The precision results can be found in the table below.

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Precision of bioelisa HIV 1+2 Ag/Ab

Precision (%CV) Specimens Repeatability

(Intra-Assay) Inter-Assay Intra-Day

HIV-1 6.3 6.9 HIV-2 8.8 5.6 p24 5.1 7.4

14. SUGGESTIONS FOR TROUBLESHOOTING PROBLEM POSSIBLE SOURCE TEST OR ACTION Invalid run (all negative) One or more reagents not added

or added in wrong sequence Recheck procedure Check for unused solutions. Repeat test.

Check for moisture in unused plate. (Silica gel desiccant must be pale yellow). Repeat test

Invalid run (all positive) Contamination of substrate Take new aliquot of substrate. Inadequate washing Ensure that wash apparatus works well Poor precision Incomplete washing of wells Ensure that wash apparatus works well Inadequate aspiration of wells Ensure that wash apparatus works well Pipetting error Check pipette function Reagent addition too slow Avoid drying of the plate after washing step.

Add reagents immediately Presence of bubbles Avoid air bubbles during pipetting. Optical pathway not clean Check instrument light source and detector

for dirt. Wipe bottom of plate with soft tissue. Inadequate color development Incorrect incubation times or

temperature Check for temperature control and time monitoring

Adhere to recommended instruction for use. Inadequate volume of substrate

added to the plate Check pipette function.

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15. BIBLIOGRAPHY 1. Alizon, M., Sonigo, P., Barré-Sinoussi, F., Chermann, J.-C., Tiollais, P., Montagnier, L. and Wain-Hobson, S., 1984.

Molecular Clloning of Lymphoadenopathy-Associated Virus. Nature 312:757-760. 2. Barré-Sinoussi, F., Chermann, J.-C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C.,

Vézinet-Brun, F., Rouzioux, C., Rozenbrum, W. and Montagnier, L. 1983. Isolation of a T-lymphotropic Retrovirus from a Patient at Risk for Acquired Immune Deficiency Syndrome (AIDS). Science 220:868-871.

3. Clavel, F., Mansinho, K., Chamaret, S. et al. 1987. Human Immunodeficiency Virus Type 2 Infection Associated with AIDS in West Africa. N. Engl. J. Med. 316:1180-1185.

4. Gallo, R.C., Salahuddin, S.Z., Popovic, M., Shearer, G.M., Kaplan, M., Haynes, B.F., Palker, T.J., Redfield, R., Oleske, J., Safal, B., White, G., Foster, P. and Markham, P.D. 1984. Frequent Detection and Isolation of Cytopathic Retroviruses (HTLV-III) from Patients with AIDS and at Risk for AIDS. Science 224:500-503.

5. Gold, J. and Dwyer, J., 1994. A Short History of AIDS. Med. J. Aust. 160:251-252. 6. Hahn, B.N., Shaw, G.M., Arya, S.K., Popovic, M., Gallo, R.C. and Wong-Staal, F., 1984. Molecular Cloning and

Characterization of the HTLV-III Virus Associated with AIDS. Nature 312:166-169. 7. IVD Directive 98/9/CE, Common Technical Specifications (CTS) – Annex II, List A. 8. Lin, H.J. 1995. Laboratory Tests for Human Immunodeficiency Viruses. J. Int. Fed. Clin. Chem. 7:61-65. 9. Luciw, P.A., Potter, S.J., Steimer, K., Dina, D. and Levy, J.A., 1984. Molecular Cloning of AIDS-Associated Retrovirus.

Nature 312:760-763. 10. Ly, T.D., Laperche, S., Brennan,C., Vallari, A., Ebel, A., Hunt, J., Martin, L., Daghfal, D., Schochetman, G. And Devare,

S. 2004. Evaluation of the sensitivity and specificity of six HIV combined p24 antigen and antibody assays. J. Virol. Meth. 122: 185-194.

11. Popovic, M., Sarngadharan, M.G, Read, E., and Gallo, R.C., 1984. Detection, Isolation, and Continuous Production of Cytopathic Retrovirus (HTLV-III) from Patient with AIDS and Pre-AIDS. Science 224:497-500.

12. Sarngadharan, M.G., Popovic, M., Bruch, L., Schüpbach, J. and Gallo, R.C., 1984. Antibodies Reactive with Human T-Lymphotrophic Retroviruses (HTLV-III) in the Serum of Patients with AIDS. Science 224:506-508.

13. Saville, R.D., Constantine, N.T., Cleghorn, F.R., Jack, N., Bartholomew, C., Edwards, J., Gomez, P. and Blattner, W.A. 2001. Fourth-generation enzyme-linked immunosorbent assay for the simultaneous detection of human immunodeficiency virus antigen and antibody. J. Clin. Microbiol. 39 (7): 2518-2524.

14. Sehulster, L.M., Hollinger, F.B., Dreesman, G.R. and Melnick, J.L., 1981. Immunological and Biophysical Alteration of Hepatitis B Virus Antigens by Sodium Hypochlorite Disinfection, App. and Environ. Microbiol. 42:762-767.

15. Sinicco, A., For a, R., Scalandra, M., Lucchini, A., Caramello, P. and Giovanni, P. 1993. Risk of Developing AIDS after Primary Accute HIV-1 Infection. J. Acquir. Immune. Defic. Syndr. 6:575-581.

16. Spire, B., Montagnier, L., Barré-Sinoussi, F. and Chermann, J.-C., 1984. Inactivation of Lymphoadenopathy Associated Virus by Chemical Dinsinfectants. Lancet: 889-901, Oct. 20.

17. Vézinet-Brun, F., Barré-Sinoussi, F., Salmot, A.G., Christol, D. Montagnier, L., Rouzioux, C., Klatzmann, D., Rozenbaum, W., Gluckmann, J.C. and Chermann, J.-C., 1984. Detection of IgG Antibodies to Lymphoadenopathy-Associated Virus in Patients with AIDS or Lymphoadenopathy Syndrome. Lancet: 1253-1256, June 9.

18. Weber, B., Thorstensson, R., Tanprasert, S., Schmitt, U. And Melchior, W. 2003. Reduction of the diagnostic window in three cases of human immunodeficiency-1 subtype E primary infection with fourth-generation HIV screening assays. Vox Sanguinis 85: 73-79.

19. World Health Organization. 2004. HIV assays: operational characteristics (Phase 1): report 15 antigen/antibody ELISAs. www.who.int/diagnostics_laboratory/publications/en/HIV_Report15.pdf.

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Manufactured by: For: Authorized Representative in Europe ADALTIS Inc. BIOKIT S.A. Adaltis Italia S.p.A. 10900 Hamon Street 08186 Llicà d’Amunt Via Cristoni 12 Montreal, Quebec Barcelona – Spain 40033 Casalecchio di Reno (BO) - Italy H3M 3A2 Canada tel. +39 051 6136511 Tel.: 514-335-9922 fax +39 051 575280 Fax: 514-335-9919 3000-1161 / 1162 Rev-01: 2007-03-01

Box Labels

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bioelisa HIV 1+2 Ag/Ab box label

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Best 2000 protocol

C:\..\BIOELISA HIV AG_AB.ASY Printed on 15/05/2007 at 10:34:48 Page 1 of 3

REVELATION DSX 5.15

Assay type : EndpointAssay title : bioelisa HIV Ag/Ab v1Password : Written by : Biokit, S.A.Prefix : Suffix : Report layout : Laboratory information

: Header information: Lot specific data: Removed outliers: Edited wells: Calculation mode: Blank mode: Q.C. equations: Data matrix: Ratio: Threshold

Header information : Filename, Date, Plate ID, Assay title, Page, Q.C. summaryFooter :

Dispense 100 uls of Sample Diluent HIV 4 to wells B1-H12, aspirate profile 1, dispense profile 4

Pipette Samples/Standards/Controls

Plate dispense time is not time critical

Pipette 100 ul of Sample to wells of type: Test (T)Preparation order: 1Tip to dispense into microtiter well does not have to be cleanFluid into microtiter well can be from a multiple shot dispense

Pipette 100 ul of NC HIV 4 to wells of type: NC1Preparation order: 2Fluid aspirate/dispense profile: 1 / 4Tip to dispense into microtiter well does not have to be cleanFluid into microtiter well can be from a multiple shot dispense

Pipette 100 ul of Std HIV 4 to wells of type: S1Preparation order: 3Fluid aspirate/dispense profile: 1 / 4Tip to dispense into microtiter well does not have to be cleanFluid into microtiter well can be from a multiple shot dispense

Pipette 100 ul of PC HIV 4 to wells of type: PC1Preparation order: always lastFluid aspirate/dispense profile: 1 / 4Tip to dispense into microtiter well does not have to be cleanFluid into microtiter well can be from a multiple shot dispense

Incubate for 60 minutes at 37,0 C

Longest Time: 66 minutesShake for 10 seconds at medium speed

Wash plate

Purge the washer with 4,00 mls of Wash Sol HIV 4 Perform a 5 cycle wash with constant timing For each strip perform the following operations: Dispense 350 uls of Wash Sol HIV 4 Do final aspirate cycle Clean the washer after use with 6,00 mls of DISTILLED WATER

Dispense 200 uls of Conjugate #1 to wells B1-H12, aspirate profile 1, dispense profile 4


Longest Time: 35 minutes

Wash plate


Dispense 200 uls of Conjugate #2 to wells B1-H12, aspirate profile 1, dispense profile 4



Wash plate

Dispense 100 uls of Sample Diluent HIV 4 to wells B1-H12, aspirate profile 1, dispense profile 4



Dispense 200 uls of Substrate HIV 4 to wells A1-H12, aspirate profile 1, dispense profile 4

Dispense Fluid is time critical. Lifetime is 60. Prep Time is 5.

Incubate for 30 minutes at ambient temperature


Dispense 100 uls of Stop Solution HIV 4 to wells A1-H12, aspirate profile 1, dispense profile 4

Reader

Test wavelength : 450 nmRef. wavelength : 620 nmInitial shake : 0 SecondsStart mode : : ImmediateCalculation mode : EndpointResults format : OD

1 2 3 4 5 6 7 8 9 10 11 12

A NC1s T1s T9s T17s T25s T33s T41s T49s T57s T65s T73s T81s

B NC1s T2s T10s T18s T26s T34s T42s T50s T58s T66s T74s T82s

C NC1s T3s T11s T19s T27s T35s T43s T51s T59s T67s T75s T83s

D S1s T4s T12s T20s T28s T36s T44s T52s T60s T68s T76s T84s

E S1s T5s T13s T21s T29s T37s T45s T53s T61s T69s T77s T85s

F S1s T6s T14s T22s T30s T38s T46s T54s T62s T70s T78s T86s

G PC1s T7s T15s T23s T31s T39s T47s T55s T63s T71s T79s T87s

H PC1s T8s T16s T24s T32s T40s T48s T56s T64s T72s T80s T88s

s indicates that a sample ID is required for this well location

Blank mode : IndividualQ.C. equations : NC1<0.310 (Negative Control too High)

: S1>=0.5 (Standard too Low): PC1>=0.5 (Positive Control too Low): NC1.i<NC1+(S1/6): Valid(NC1)>=3 (Invalid Results for Negative Control)

Full Q.C. Report : YesSuppress results : NoLot specific check : NoOutput format : MatrixMatrix options : Calculated data, Sample IDAverage replicates : NoMean : ArithmeticArea statistics : NoExport to file : ASCII Text,Table,tab separatedFile sorted on : Sample ID in ascending orderFile options : Reading date,Reading time,Assay title,

: Kit Lot Data,Reagent Lot Data,User name,: Area statistics,OD QC equations,Threshold Q.C.,: Threshold cutoffs,Curve fit Q.C.,Sample IDs,: Position,Well Label,OD Results,: Threshold,Curve Fit,Ratio,: Spreadsheet

Threshold

- equation : NC1+(S1/6)*0.9+ equation : NC1+(S1/6)No. of segments : 1- label : -0 label : IND+ label : POSHistogram : NoQ.C. equations : Full Q.C. Report : YesSuppress results : NoLot specific check : NoOutput format : No matrix, no tableAverage replicates : YesMean : Arithmetic


Ratio

Ratio equation : Sample/(NC1+(S1/6))Result units : Data conversion : Result units : Output format : No matrix, no tableAverage replicates : NoMean : Arithmetic

DYNEX TECHNOLOGIES

bioelisa HIV-1+2 Ag/Ab Introduction

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