New Product Launch - bioelisa HIV 1+2 Ag/Ab

Transcription

1 NEWS BK NEWS# 359 / BE/ MKT DATE : December 19, 2014 TITLE : New Product Launch - bioelisa HIV 1+2 Ag/Ab Dear customer, With the aim of continuous development of its products, Biokit now presents a new member of the retrovirus family: The bioelisa HIV 1+2 Ag/Ab is a fourth generation Elisa test for the detection of antibodies to HIV 1+2 and the HIV-1 p24 Antigen. The available presentations are the following: bioelisa HIV 1+2 Ag/Ab 96 Tests, Cod Tests, Cod The bioelisa HIV 1+2 Ag/Ab includes new features that improve the performance of the existing bioelisa HIV assay. - New HIV-1 gp41 recombinant antigen for better specificity - Two monoclonal antibodies for better sensitivity to HIV-1 p4ag - Two kit presentations for better adjustment to customer needs According to the internal and external evaluations, the new assay overcomes the performance of the today s state-of-the art HIV assays: Biorad Genscreen HIV1+2 Ultra and Abbott Architect HIV1+2 Combo The bioelisa HIV 1+2 Ag/Ab will substitute the bioelisa HIV It is important to point out that the bioelisa HIV will be available in Biokit during the time needed for registering the bioelisa HIV 1+2 Ag/Ab in the non-european Union countries. Public tenders already awarded with the bioelisa HIV will be supplied without restrictions. Please find enclosed a complete product information that we hope will help you in the promotion and introduction of bioelisa HIV 1+2 Ag/Ab.

2 bioelisa HIV 1+2 Ag/Ab Product features: Recombinant antigens and capture antibody bioelisa HIV 1+2 Ag/Ab uses a combination of two recombinant antigens: gp41 from HIV-1 and gp36 from HIV-2, in addition to a synthetic peptide for HIV-1 subtype O, together with a two monoclonal antibodies against HIV-1 p24 Ag. The new HIV antigens are able to detect HIV type 1 group M-O and HIV type 2 in human serum or plasma. Performance of bioelisa HIV 1+2 Ag/Ab in terms of sensitivity and specificity allowed biokit to achieve the Common Technical Specifications (CTS) requested for CE mark class IIA products. Fourth generation assay The bioelisa HIV 1+2 Ag/Ab is a fourth generation assay for total antibody detection and HIV-1 p24 antigen detection. HIV recombinant antigens and synthetic peptides are present in both solid phase and conjugate. If HIV antibodies (IgG, IgM or IgA) are present in the patient sample, they will bind to the HIV antigens on the plate coating and HIV antigens present in the conjugate. If p24 Antigen is present in the patient sample, it will bind to the capture monoclonal antibody present in the plate coating and capture antibody present in the sample diluent. Components MICROPLATE: 12 strips of 8 breakable wells coated with HIV specific gp36 and gp41 peptides and gp41 protein with two monoclonal antibodies specific to the HIV-1 p24 Ag. NEGATIVE CONTROL Ready-to-use control. It contains human serum negative for HIV antibodies and p24 antigen. POSITIVE HIV-1 CONTROL Ready-to-use control. It contains human serum positive to Anti- HIV 1. POSITIVE HIV-2 CONTROL Ready-to-use control. It contains human serum positive to Anti- HIV 2. POSITIVE HIV p24 CONTROL Ready-to-use control. It contains recombinant HIV-1 p24. CONJUGATE #1: Ready-to-use, blue colour-coded reagent. It contains a mixture of biotinylated antigens in phosphate buffer. CONJUGATE #2 CONC. 100x: 100x solution concentrate. It contains Horseradish Peroxidase conjugated with streptavidin in specific stabilizer reagent to be diluted with Conjugate # 2 Diluent. Page 2 of 15

3 CONJUGATE #2 DILUENT: Ready-to-use orange colour-coded reagent used for the dilution of Conjugate # 2 Concentrate 100x. SAMPLE DILUENT: Ready-to-use red-colour coded reagent. It contains active components and monoclonal p24 antigen antibodies. Used to dilute the sample. SUBSTRATE TMB: Ready-to-use component. It contains Tetra methyl benzidine and Hydrogen peroxide (H2O2) in a citrate buffer. STOP SOLUTION ready-to-use component. It contains 0,3 M H2SO4 solution. 25x concentrated solution. WASH BUFFER CONC. 25x: 25x concentrated solution. It contains saline phosphate buffer. ADHESIVE SEALS: To cover the microplate during incubation. Protocol: The bioelisa HIV 1+2 Ag/Ab uses in plate sample dilution: 100 µl of sample diluent and 100 µl of sample or control. The 1:2 sample dilution allows spectrophotometric monitoring of sample dispensing at 620 nm. The red colour of the sample diluent changes to a clear red dark colour. Definition of the OD limit must be validated customer by customer. Number of replicates controls have been reduced to allow the maximum well utilization: Negative control by triplicate and rest of Positive control for HIV-1, HIV-2 and p24 Ag, only by singlicate. The sample incubation time is to 60 minutes to reach the required sensitivity of fourth generation HIV assays. Conjugate incubation is split into two separate incubations of 30 minutes each because of the biotin streptavidin system. The substrate incubation is 30 minutes. The total incubation time is 2 hours and 30 minutes. Substrate is ready for use. STEP PROCEDURE Add 100 µl of Sample Diluent. Add 100 µl of sample/control to the diluent and pipette up & down for Sample step mixing. Test Negative control x3 Positive Controls x1. Incubate 60 minutes at 37 C. Wash step Perform wash step 5x. Conjugate #1 Add 200 µl of ready-to-use Conjugate #1 to wells of the microtiter plate. Incubate 30 minutes at 37 C. Wash step Perform wash step 3x. Conjugate #2 Add 200 µl of diluted Conjugate #2 to wells of the microtiter plate. Incubate 30 minutes at 37 C Wash step Perform wash step 5x. TMB Substrate Add 150 µl of TMB Substrate to the wells of the microtiter plate. Colour Development Incubate 30 minutes at C. Stopping Add 100 µl of Stop Solution and read OD at 450nm with reference wavelength at nm in an ELISA microplate reader. Page 3 of 15

4 Comparison with bioelisa HIV 1+2 Ag/Ab: Test name bioelisa HIV bioelisa HIV 1+2 Ag/Ab Code number Tests Tests Tests Method EIA/HRP/TMB EIA/HRP/TMB Format Solid phase 3 - step direct sandwich 12x8 Microtiter wells 3 - step direct sandwich 12x8 Microtiter wells Coating Recombinant antigens and synthetic peptides Recombinant antigens and synthetic peptides HIV-1 gp41 gp41 HIV-2 gp36 gp36 Anti p24 Ag One monoclonal antibody Two monoclonal antibodies Conjugate Substrate Specimen dilution Number of controls Performance: Conjugate #1: Biotinylated HIV Ag Ready-to-use Conjugate #2: Streptavidin HRP Concentrate Ready-to-use 150 µl 100 µl Sample diluent 100 µl Sample Blk, NC (x2), HIV1(x2), HIV2(x2), p24(x2): 9 wells in total Conjugate #1: Biotinylated HIV Ag Readyto-use Conjugate #2: Streptavidin HRP Concentrate Ready-to-use 150 µl 100 µl Sample diluent 100 µl Sample Blk, NC(x3), HIV1(x1), HIV2(x1), p24(x1) 7 wells in total Sample incubation 60 min / 37 C 60 min / 37 C Conjugate incubation Conjugate #1: 30 min / 37 C Conjugate #2: 30 min / 37 C Conjugate #1: 30 min / 37 C Conjugate #2: 30 min / 37 C Substrate incubation 30 min / RT 30 min / RT Cut-off Mean Neg Mean Neg Diagnostic Sensitivity Analytical Sensitivity 100% 1 IU/ml 100% 0.5 IU/ml Specificity Comments Fourth generation Nine wells per run One Kit presentation: 192 Tests Fourth generation Improved specificity and analytical sensitivity Only seven wells per run Two Kits presentation: 96/480Tests Sensitivity: Analytical Sensitivity (Limit of Detection HIV-1 p24 Ag) The analytical sensitivity of the test was determined using WHO International Standard HIV-1 p24 Antigen and the BIORAD HIV-1 Ag Standard. The WHO International Standard HIV-1 p24 Antigen and the BIORAD HIV-1 Ag Standard were serially diluted in negative human serum and analysed in duplicate with the test assay. Tables are showing the different dilutions of the HIV-1 Antigen standards, the absorbance results, and ABS/CO values have been prepared, from two different lots. The analytical sensitivity was determined as the concentration of the analyte in the highest dilution of the standards yielding an ABS/CO > 1.0. Lot 1 Lot 2 WHO Int std HIV-1 p24 OD/COV 2 IU/ml IU/ml IU/ml End point 0.32 IU/ml 0.32 IU/ml The sensitivity limit has been estimated at < 0.5 IU/mL Page 4 of 15

5 Analytical sensitivity AFSSAPS/Biorad standard Lot 1 Lot 2 Biorad HIV-1 Ag std OD/COV 100 pg/ml pg/ml pg/ml pg/ml pg/ml End point 8.3 pg/ml 8.3 pg/ml The sensitivity limit has been estimated at < 12.5pg/ml The analytical sensitivity for antibody detection was determined using the NIBSC International Standards: British Working Standard HIV-1, British Working Standard HIV-1 1 in 5 dilution and Monitor Sample for HIV-2. Lot 1 Lot 2 OD/COV British Working Std HIV British Working Std HIV-1 diluted 1: Monitor Sample HIV The HIV-1 p24 Ag diagnostic sensitivity was assessed by using the Seracare HIV p24 Antigen Mixed Titer Performance panel PRA204 All samples in this panel are positive for the presence of HIV-1 p24 Ag. In the next graphic it is showed the reactivity in terms of signal Page 5 of 15

6 OD/COV for the most difficult samples in comparison with other commercial assays. The bioelisa HIV 1+2 Ag/Ab presented the best score in all of them. Diagnostic Sensitivity The diagnostic sensitivity of the bioelisa HIV 1+2 Ag/Ab Kit was based on testing characterized HIV-1, HIV-2 and HIV-1 p24 Ag positive samples: - A total of 537 HIV-1 positive samples were evaluated with bioelisa HIV 1+2 Ag/Ab, among them: 28 HIV-1 fresh-same-day collected samples, 85 HIV-1 and 4 HIV-1 Subtype O samples in an European reference lab; 380 HIV -1 samples were evaluated at Biokit and 40 HIV-1 non B subtype samples (and at least 3 samples of each HIV-1 subtype) were evaluated in another European reference lab. bioelisa HIV 1+2 Ag/Ab detected all 537 samples. The sensitivity found was100% (537/537). - A total of 115 HIV-2 positive samples were evaluated with bioelisa HIV 1+2 Ag/Ab, among them: 27 HIV-2 samples in an European reference lab and 88 HIV-2 samples were evaluated at Biokit. bioelisa HIV 1+2 Ag/Ab detected all 115 samples. The sensitivity found was 100% (115/115). - A total of 61 HIV-1 p24 Ag positive samples were evaluated also with bioelisa HIV 1+2 Ag/Ab, among them: 15 samples in an European reference lab, 16 samples were evaluated in another European reference lab and 30 samples at Biokit. bioelisa HIV 1+2 Ag/Ab detected all 61 samples. The sensitivity found was 100% (61/61). - Additional sensitivity studies were carried out by testing a total of 31 seroconversion panels at Biokit and a European reference lab. Results obtained were comparable to the most sensitive commercial method for HIV 1+2 antigen and antibody detection. Page 6 of 15

7 - 16 HIV Seroconversion Panels from BBI/Seracare and Zeptometrix were evaluated at Biokit. bioelisa HIV-1+2 Ag/Ab identified correctly all the positive specimens. In the following table it can be observed the first sample detected as positive in each panel evaluated with bioelisa HIV-1+2 Ag/Ab in comparison with 4 th Generation assays (Ag/Ab Assays), 3 rd Generation assays (only Ab Assays), assays for HIV p24 antigen and NAT HIV RNA assays. bioelisa HIV-1+2 Ag/Ab showed excellent sensitivity to detect early antigen or antibodies in 16 HIV Seroconversion Panels evaluated. bioelisa HIV Ag/Ab HIV p24 Seroconversion HIV Ab Assay NAT HIV Ag/Ab Assay antigen Panel First sample detected positive in the panel BB1-PRB965-n= ZEPT n= ND 4 BBI-PRB954-n= BBI-PRB932-n=9 4 ND ZEPT-9017-n= ZEPT-9014-n= ZEPT-9012-n= ZEPT-9032-n= ZEPT-6243-n= ZEPT-9022-n= ZEPT-9018-n= BBI-PRB939-n=9 6 ND BBI-PRB956-n= BBI-PRB945-n= BBI-PRB955-n= BBI-PRB958-n= Bioelisa HIV-1+2 Ag/Ab detected the first positive sample at the same level as an Elisa for only HIV p24 detection and before than a commercial EIA assay for HIV1+2 Ag/Ab detection In the next graphic it is shown the aggregate score in seven seroconversion panels, in comparison with other commercial assays. The aggregate score means the addition of the first detection day in all panels. A lower aggregate value means earlier detection and higher sensitivity. Page 7 of 15

8 Maximum number detection days by less sensitive HIV CE mark 3rd generation assay: #238. bioelisa HIV 1+2 Ag/Ab showed the best performance. - Another study was conducted to assess the sensitivity in 32 early seroconversion samples. These type of samples are characterized to be positive to the presence of HIVp24 Ag and/or positive by NAT (Nucleic Acid Testing), not detected by all HIV 3rd generation methods and to be negative or Indeterminate by Western-Blot methods. bioelisa HIV 1+2 Ag/Ab detected all 32 samples. The sensitivity found was 100% (32/32) cell culture supernatans from HIV cell cultures, including different HIV-1 subtypes and HIV-2 were evaluated in a European Reference Lab. bioelisa HIV 1+2 Ag/Ab detected all 50 samples. The sensitivity found was100% (50/50). - Finally a study was conducted in the Paul Ehrilch Institute in Germany, to assess the sensitivity of bioelisa HIV 1+2 Ag/Ab by comparing reactivity with other commercial assays in 10 seroconversion panels. bioelisa HIV 1+2 Ag/Ab showed the best performance. Maximum number detection days by less sensitive HIV CE mark 3rd generation assay: #471 Specificity The specificity was evaluated by testing a total of 6,465 unselected blood donors' samples at three different sites. - In the first site 3,045 unselected samples including 684 first donors were tested. From this total, 4 samples were reactive. The specificity obtained in this study was 99.87% (3,041/3,045). - In the second site 500 unselected samples from Blood Bank were tested. All 500 samples were negative. The specificity was 100% (500/500) Page 8 of 15

9 - An internal evaluation of 2,920 unselected samples from Blood Bank was performed. From this evaluation, 6 samples were reactive. The specificity obtained in this study was 99.79%. (2,914/2,920) The final specificity was found to be 6,455/6,465: 99.85% Evaluation 1 (3,045 sera) Initial Reactive Repeated Reactive False Positive Specificity % Serum Evaluation 2 (500 sera) Initial Reactive Repeated Reactive False Positive Specificity % Serum Internal Evaluation (1,000 sera + 1,920 plasmas) Initial Reactive Repeated Reactive False Positive Specificity % Plasma Serum Total In addition 200 samples from hospitalized patients have been tested and compared to a reference test. 194 samples were found negative by both tests and six samples were reactive. The six samples were also reactive with other ELISA tests. A specificity of 100% was obtained in this study (194/194). Precision Intra-assay reproducibility: The coefficient of variation obtained for the absorbance values of a HIV-1 positive sample assayed in a minimum of 40 replicates was 3.42%, 5.79% and 4.71% in three lots studied. The coefficient of variation obtained for the absorbance values of a HIV-2 positive sample assayed in a minimum of 40 replicates was 6.39%, 5.91% and 8.38% in three lots studied. The coefficient of variation obtained for the absorbance values of a HIV-1 p24 positive sample assayed in a minimum of 40 replicates was 4.08%, 3.44% and 3.71% in three lots studied. Inter-assay reproducibility: Four positive samples of different levels were tested in 15 different assays. The coefficients of variation obtained for the ratios absorbance/cut-off of the four samples were 4.87%, 5.37%, 7.49% and 7.54%. Interferences Interference by addition No interference has been found for haemoglobin (500 mg/dl), bilirubin (20 mg/dl), human Albumin (5 g/dl) and triglycerides (500 mg/dl). Page 9 of 15

10 Cross-reactivity To evaluate possible interferences, 125 potential cross-reacting specimens were analyzed. Among those samples, 4 samples (RF+), 4 samples positive for anti-nuclear antibodies (ANA), 5 samples Mononucleosis, 89 samples from other related infectious diseases and 23 samples from pregnant women. No evidence of cross reactivity was found in the samples evaluated. The 2 anti-toxoplasma IgM samples that tested positive were also positive by other HIV assays. Potential interfering samples = 125 RF (Rheumatoid factor) - 4 Elevated IgG - 4 ANA (anti-nuclear Antibodies) - 4 Elevated IgM - 5 Mononucleosis - 5 HSV 1 IgG (Herpes Simplex Virus) - 5 Pregnant women- 23 (including 7 multiparous) HSV 2 IgG (Herpes Simplex Virus) - 4 Syphilis Ab - 6 anti-cmv IgG (Cytomegalovirus) - 5 anti-ebv (Epstein-Barr Virus) - 3 anti-cmv IgM (Cytomegalovirus) - 7 anti-rubella (Rubella Virus) - 4 anti-htlv (Human T-lymphotropic Virus) - 4 anti-vzv IgG (Varicela Zoster Virus) - 2 anti-hcv (Hepatitis C Virus) - 8 anti-vzv IgM (Varicela Zoster Virus) - 2 anti-e.coli (Escherichia coli) - 5 anti-toxoplasma IgG (T. gondii) - 10 HBsAg (Hepatitis B Antigen) - 8 anti-toxoplasma IgM (T. gondii) - 7 Comparison vs. Competitors bioelisa HIV 1+2 Ag/Ab can be compared with the two Premium products on the market: The Biorad Genscreen ULTRA HIV and the Murex HIV Ag/Ab combination. The sensitivity and specificity of bioelisa HIV 1+2 Ag/Ab matches what until now was considered the reference assay in Elisa, the Biorad Genscreen assay. The biokit assay is even better in terms of analytical sensitivity or limit of detection of HIV-1 p24 Antigen. Specificity is also better because of the utilization of an improved gp41 protein. In terms of ease-of-use, the bioelisa HIV 1+2 Ag/Ab incorporates all liquid reagents, in comparison with the Biorad and Murex assays that use lyophilized conjugates. There is a unique disadvantage compared to the Biorad and Murex assay. The bioelisa HIV 1+2 Ag/Ab uses two independent incubations for the conjugate incubation step. This means the total incubation time increases by 30 minutes, but the specificity and background of the assay improves because of this extra wash cycle. The internal and external evaluations performed on the bioelisa HIV 1+2 Ag/Ab kit demonstrate better performance with seroconversion panels and the detection limit for p24 Ag. Page 10 of 15

11 Competitor s table Test name bioelisa HIV 1+2 Ag/Ab Genscreen ULTRA HIV Murex HIV Ag/Ab Ag/Ab Combination Code number G79-01 Method EIA/HRP/TMB EIA/HRP/TMB EIA/HRP/TMB Format 3 - step direct sandwich 2 - step direct sandwich 2 - step direct sandwich Solid phase 12x8 Microtiter wells 12x8 Microtiter wells 12x8 Microtiter wells Tests per kit /480 96/480 Coating Recombinant antigens Synthetic peptide Recombinant antigens Synthetic peptide Recombinant antigens Synthetic peptide HIV-1 gp41 Rec gp160 and Synthetic peptide HIV Group O ENV rec Ag and Synthetic peptide HIV Group O HIV-2 gp36 Synthetic peptide HIV2 ENV rec Ag Anti p24 Ag Two Monoclonal antibody One Monoclonal antibody One Monoclonal antibody Conjugate Conjugate #1: Biotinylated Conjugate #1: Biotinylated HIV Ag Ready to-use polyclonal Ab to p24 Ready to-use Conjugate #2: Streptavidin HRP Concentrate Conclusion Conjugate #2: Antigens labelled with lyophilized Streptavidin HRP A+B 80 µl 25 µl Conjugate #1 75 µl Sample Conjugate: Antigens and monoclonal antibodies conjugated with lyophilized HRP. Substrate Ready to-use 150 µl Ready to-use Specimen dilution 100 µl Sample diluent 25 µl Sample diluent 100 µl Sample 100 µl Sample Sample incubation 60 min / 37 C 60 min / 37 C 60 min / 37 C Conjugate incubation Conjugate #1: 30 min / 37 C Conjugate #2: 30 min / 37 C Conjugate: 30 min / 37 C Conjugate #2: 30 min / 37 C Substrate incubation 30 min / RT 30 min / RT 30 min / 37 C Cut-off Mean Neg Mean Neg Mean Neg Analytical sensitivity 0.5 IU/ml p24 Ag 0.85 IU/ml p24 Ag 28 pg/ml Biorad std pg/ml Biorad std pg/ml Biorad std Diagnostic Sensitivity 100% 100% 100% Specificity 99.87% 99.72% 99.78% Comments + Best analytical sensitivity + 2h incubation time +Reference assay in the + Best sensitivity - Lyophilized conjugate market for diagnostic + Very low background sensitivity - 2h 30 min incubation time + 2h incubation time - Lyophilized conjugate New features have been introduce in the bioelisa HIV 1+2 Ag/Ab, compared to the existing Biokit assay: Two new monoclonal antibodies and a new gp41 protein for HIV-1 detection. The new bioelisa HIV 1+2 Ag/Ab improves the performance of previous Biokit assay in terms of analytical sensitivity for p24 detection. Limit of detection is superior to the most important competitors: Murex-Diasorin and Biorad. Specificity has been also improved by means of the new gp41. The large evaluation with blood donor reported a level of specificity not overcome neither Murex-Diasorin nor Biorad. The two kit presentations: 96 and 480 Test adjust better to the customer needs As per the Biokit internal and external results, the bioelisa 1+2 Ag/Ab has to be considered as a new state-of-the-art assay for HIV detection. Page 11 of 15

12 Launch package: CE Mark Package insert External Evaluations Paul Erhlich Institute Germany Central tropical diseases Establiment Français du Sang (EFS) Page 12 of 15

13 CE MARK Page 11 of 13

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15 PACKAGE INSERT Page 12 of 13

16 bioelisa READ HIGHLIGHTED CHANGES bioelisa HIV-1+2 Ag/Ab x 96 TESTS x 96 TESTS (480) ELISA test for the detection of antibodies to Human Immunodeficiency Viruses (HIV) type 1 (group M-O) or type 2 and HIV-p24 antigen in human serum or plasma samples in clinical laboratories and as a first-line screening assay in blood centers. Summary Serological evidence of infection with HIV may be obtained by testing for presence of HIV antigens or antibodies in serum or plasma of individuals suspected for HIV infection. Antigen can generally be detected during both acute phase and the symptomatic phase of AIDS. The antibodies to HIV-1 and/or HIV-2 can be detected throughout virtually the whole infection period, starting at or shortly after the acute phase and lasting till the end stage of AIDS. HIV fourth generation ELISA allows the simultaneous determination of HIV p24 antigens and HIV-1/HIV-2 antibodies reducing therefore the diagnostic window and allowing an early diagnosis of acute HIV infection. Apart from sexual transmission, the principal route of infection with HIV is blood transfusion. HIV can be present in both cellular and cell-free fractions of human blood. Therefore, all donations of blood or plasma should be tested due to the risk of HIV transmission through contaminated blood. This can be effectively achieved by testing for antibodies to HIV and p24 antigen by using a highly sensitive ELISA test. Principle bioelisa HIV-1+2 Ag/Ab is a fourth generation enzyme immunoassay for the simultaneous qualitative detection of HIV p24 antigen and antibodies to HIV-1 (groups M and O) or HIV-2. Antigens representing epitopes of HIV-1 gp41 and HIV-2 gp36 are coated onto microplate wells together with monoclonal antibodies against HIV p24. In the first step of the immunoassay, serum or plasma samples are added to these wells along with the sample diluent containing biotinylated anti-p24 antibodies. If specific HIV-1 and/or HIV-2 antibodies are present in the sample, they will form stable complexes with the HIV antigens attached to the wells. If p24 antigen is present in the sample, it will bind simultaneously to the antibodies in the well and to the biotinylated antibodies present in the sample diluent. The microwells are then washed to remove unbound serum proteins. A set of biotinylated antigens are added to the wells, these antigens will bind to the specific captured antibodies. After a second wash to remove unbound conjugate, a second conjugate of Streptavidin- immunocomplex, the colourless Chromogen is oxidised by the bound conjugate to a blue coloured product. The blue colour changes to yellow after stopping the reaction with sulphuric acid. The intensity of colour can be measured and is proportional to the concentration of anti-hiv 1/2 antibodies or HIV p24 antigen present in the sample. Wells containing negative samples remain colourless. Components 1. MCPL MICROPLATE: 12 x 8 wells coated with antigens representing epitopes of HIV-1 gp41 and HIV-2 gp36 and monoclonal antibodies against p24. Plates are sealed into aluminum pouch with desiccant. 2. CONTROL + 1 HIV-1 POSITIVE CONTROL: Diluted human serum containing antibodies to HIV-1 (Heat inactivated). Contains 0.1% ProClin TM preservative. Ready to use. 3. CONTROL + 2 HIV-2 POSITIVE CONTROL: Diluted human serum containing antibodies to HIV-2 (Heat inactivated). Contains 0.1% ProClin TM preservative. Ready to use. 4. CONTROL + Ag HIV POSITIVE CONTROL p24: Diluted human serum spiked with recombinant HIV-1 p24 antigen. Contains 0.1% ProClin TM preservative. Ready to use 300 as 300 as 300 as 5. CONTROL NEGATIVE CONTROL: Diluted human serum negative for antibodies to HIV-1/2 and HIV p24 antigen. Contains 0.1% ProClin TM 300 as preservative. Ready to use. 6. DIL SAMP SAMPLE DILUENT: Biotinylated monoclonal antibodies against HIV p24 diluted in Tris-buffer. Contains 0.05% ProClin TM 300 as preservative and red dye. Ready to use. BIOKIT, S.A. - Can Malé s/n - - Barcelona - SPAIN _R00_ _eng.docx

17 bioelisa 7. CONJ 1 CONJUGATE READY TO USE: Phosphate buffer containing HIV-1 and HIV-2 biotinylated antigens. Contains 0.05% ProClin TM preservative and blue dye. Ready to use. 300 as 8. DIL CONJ 2 CONJUGATE DILUENT 2: Phosphate buffer containing protein stabilizers, 0.05% ProClin TM 300 as preservative and yellow dye. 9. CONJ 2 101X CONCENTRATE CONJUGATE 2 (101X): Contains peroxidase labelled streptavidin. To be diluted 1:101 in conjugate diluent 2 before use. 10. SUBS TMB SUBSTRATE-TMB: Contains 3,3', 5,5'-Tetramethylbenzidine (TMB) and hydrogen peroxide. Ready to use. 11. WASH SOLN 25x CONCENTRATE WASHING SOLUTION: Concentrate PBS buffer ph 7.4 (25x). Contains Tween-20 as detergent, <0.2% ProClin TM 300 as preservative To be diluted 1:25 in distilled or deionised water before use. 12. H 2 SO 4 0.8N STOPPING SOLUTION: 0.8N sulphuric acid. Ready to use. 13. SEALS ADHESIVE SEALS: To cover the microplate during incubations. 14. BAG RESEALABLE BAG: For storage of unused strips. Precautions bioelisa HIV-1+2 Ag/Ab is intended for IN VITRO diagnostic use. For professional use only. WARNING: The Negative Control, HIV-1 Positive Control, HIV-2 Positive Control, HIV p24 Positive Control, Sample Diluent, Conjugate 1, Conjugate Diluent 2 and Concentrate Washing Solution contain <0.2% ProClin TM 300 as preservative. Hazard statements H317: May cause an allergic skin reaction. Precautionary statements P280: Wear protective gloves/protective clothing/eye protection/face protection. P302+P352: IF ON SKIN: Wash with plenty of soap and water. P333+P313: If skin irritation or rash occurs: Get medical advice/attention. P363: Wash contaminated clothing before reuse. P501: Dispose of contents/container in accordance with local/regional/national/international regulations. WARNING: POTENTIALLY BIOHAZARDOUS MATERIAL. All human source material used in the preparation of this product was found to be negative for the presence of HCV antibodies, Treponema pallidum antibodies, hepatitis B surface antigen and HIV 1/2 antibodies (except for the Positive Controls 1 and 2), using a commercial licensed method. Nevertheless, because no test method can offer complete assurance of the absence of infectious agents, this product should be handled with caution: - Avoid contact of reagents with the eyes and skin. If that occurs, wash thoroughly with water. - Wear gloves. - Do not pipette by mouth. - Do not smoke. - Dispose all used materials in a suitable biohazardous waste container. Remains of samples, controls, aspirated reagents and pipette tips should be collected in a container for this purpose and autoclaved 1-hour at 121 C or treated with 10% sodium hypochlorite (final concentration) for 30 min before disposal. (Remains containing acid must be neutralised prior to the addition of sodium hypochlorite). BIOKIT, S.A. - Can Malé s/n - - Barcelona - SPAIN _R00_ _eng.docx

18 bioelisa Handling instructions: - Adjust washer to the plate used (flat bottom) in order to wash properly. - Do not mix reagents from different lots. - Do not use reagents after the expiration date. - Do not use the reagent if you observed any change in appearance of components included in the kit. - Extreme care should be taken to avoid microbial contamination and cross contamination of reagents. - Use a new pipette tip for each specimen and each reagent. - Do not exchange reagents from different lots or use reagents from other commercially available kits. The components of the kit are precisely matched for optimal performance of the tests. - CAUTION - CRITICAL STEP: Allow the reagents and specimens to reach room temperature (20-25 C) before use. Shake reagent gently before use. Return at 2-8 C immediately after use. - The enzymatic activity of conjugate 2 might be affected by dust and reactive chemical substances like sodium hypochlorite, acids, alkalis etc. Do not perform the assay in the presence of these substances. - It is very important to keep the Substrate-TMB solution in a well-sealed container and avoid light exposure. - Soaps and/or oxidising agents remaining in containers used for the Substrate-TMB solution can interfere with the reaction. If glass containers or re-used plastic containers are used, they should be washed with 1N sulphuric or hydrochloric acid, rinsed well with distilled water and dried before use. We recommend using disposable plastic containers. Storage and stability The components will remain stable through the expiration date shown on the label if stored at 2-8 C. The bag containing the microplate should be brought to room temperature before opening to avoid condensation in the wells. Once opened the bag, the microplate wells are stable for 3 months at 2-8 C in the plastic bag tightly sealed, along with the silicagel. Discard any microplate sealed without the silicagel. Once diluted, the washing solution is stable for 6 days only if stored at 2-8 C. Do not use diluted washing solutions if stored overnight at room temperature. Once diluted, the conjugate 2 is stable for 30 days at 2-8 C. Store the Substrate-TMB solution in the dark. Available packaging - 1 plate kit (1x96 tests), REF Contains: 1 plate, 1 x 2 ml negative control, 1 x 2 ml HIV-1 positive control, 1 x 2 ml HIV-2 positive control, 1 x 2 ml HIV-1 p24 positive control, 1 x 14 ml sample diluent, 1 x 25 ml conjugate 1,1 x ml conjugate 2 (101x), 1 x 25 ml conjugate 2 diluent, 1 x 20 ml substrate-tmb, 1 x 12 ml stopping solution, 2 x 50 ml washing solution (25x), 1 plastic bag and adhesive seals. - 5 plates kit (5 x 96 tests), REF Contains: 5 plates, 1 x 4 ml negative control, 1 x 4 ml HIV-1 positive control, 1 x 4 ml HIV-2 positive control, 1 x 4 ml HIV-1 p24 positive control, 1 x 70 ml sample diluent, 1 x 120 ml conjugate 1,1 x 1.2 ml conjugate 2 (101x), 1 x 120 ml conjugate 2 diluent, 1 x 100 ml substrate-tmb, 1 x 60 ml stopping solution, 3 x 100 ml washing solution (25x), 1 plastic bag and adhesive seals. Material required not provided - Distilled or deionised water. - Disposable gloves and timer. - Appropriate waste containers for potentially contaminated materials. - Disposable V-shaped troughs. - Dispensing system and/or pipette (single or multichannel) and disposable pipette tips. - Absorbent tissue or clean towel. - Dry incubator or water bath, 37 ± 1 C. - Microplate reader with a 450 nm filter. Reference filter of 620 nm or 630 is advisable. - Manual or automated wash system. Sample collection Use fresh serum with or without Serum separator tube (SST), EDTA plasma, Acid-citrate-dextrose (ACD) plasma, Lithium heparin plasma, Sodium heparin plasma, Sodium citrate plasma, Potassium oxalate/sodium fluoride plasma, CPD and CPDA plasma. Other anticoagulants should be evaluated before use. Samples can be stored at 2-8 C for 8 days. For longer periods, samples should be frozen (-20 C). Avoid repeated freezing and thawing (maximum 4 freeze-thaw cycles). Specimens showing visible particulate matter should be clarified by centrifugation. Serum or plasma samples should not be heat inactivated, since that may cause incorrect results. BIOKIT, S.A. - Can Malé s/n - - Barcelona - SPAIN _R00_ _eng.docx

19 bioelisa Automatic processing Automated or semi-automated assay may be used with different instruments. It is very important to validate any automated system to demonstrate that the results obtained for samples are equivalent to the ones obtained using the manual assay. It is recommended that the users validate periodically the instrument. If there is any difficulty in the programming and setting of Biokit automatic processors, please contact your distributor. PROCEDURE Previous operations Allow all the reagents to reach room temperature (20-25 C) before running the assay. Gently mix all liquid reagents before use. Check the washing solution concentrate for the presence of salt crystals. If crystals have formed, resolubilize by warming at 37 C until crystals dissolve. Use distilled or deionized water. Negative and Positive controls should be treated as the samples. Dilute the washing solution 1:25 in distilled water. If the entire plate is used, add 40 ml of concentrate washing solution (25x) to 960 ml of deionised water. If the entire plate is not used, prepare the proportional volume of solution. Dilute the concentrated conjugate 2 1:101 with the conjugate diluent 2 according to table 1. For the 1 plate packaging, if the entire plate is to be used, add 250 μl of concentrate conjugate directly to the bottle containing 25 ml of conjugate diluent. Mix gently. TABLE 1 Strips required Conjugate diluent 2 ml Concentrate conjugate 2 μl Assay procedure 1. Use only the number of strips required for the test. Reserve 7 wells for blank and controls. One well as Blank (e.g. A1), three wells as negative control (e.g. B1, C1, D1) and, at least, one well of each of the positive controls: positive control HIV-1 (e.g. E1), positive control HIV-2 (e.g. F1) and positive control HIV p24 (e.g. G1). 2. Add 100μL of sample diluent to all the wells except the Blank. 3. Add first 100 l of samples, and last 100 l of negative and each of the positive controls into their respective wells. 4. Cover the plate with an adhesive seal, mix gently and incubate at 37 C ± 1 C for 60 minutes with a tolerance range of minutes 5. Remove and discard the adhesive seal. Aspirate the contents of the wells and fill them completely (approximately 350 μl) with the diluted washing solution. Repeat the process of aspiration and washing 4 more times, 5 (five) cycles in total. Ensure that each column of wells soaks for at least 15 seconds before the next aspiration cycle. 6. Transfer 200 μl of conjugate 1 into each well, except the one reserved for the substrate blank. Avoid bubbles upon addition. 7. Cover the microplate with an adhesive seal and incubate at 37 C ± 1 C for 30 minutes with a tolerance range of minutes. 8. Remove and discard the adhesive seal. Aspirate and wash the wells as in the previous washing step for 3 (three) wash cycles. 9. Transfer 200 μl of working conjugate 2 into each well, except the one reserved for the substrate blank. Avoid bubbles upon addition. 10. Cover the microplate with an adhesive seal and incubate at 37 C ± 1 C for 30 minutes with a tolerance range of minutes. 11. Remove and discard the adhesive seal. Aspirate and wash the wells as in step 5, for 5 (five) wash cycles. BIOKIT, S.A. - Can Malé s/n - - Barcelona - SPAIN _R00_ _eng.docx

20 bioelisa 12. Add 150 μl of Substrate-TMB solution to each well, including the blank. 13. Incubate at room temperature (20-25 C) for 30 minutes with a tolerance range of minutes, protected from direct light exposure 14. Stop the reaction by adding 100 μl of stopping solution in the same sequence and time intervals as for the Substrate-TMB. 15. Blank the reader at 450 nm with the blank well and read the absorbance of each well within 30 minutes. It is recommended to read in bichromatic mode using a nm reference filter. Quality control Results of an assay are valid if the following criteria are accomplished: 1. The Absorbance value of the blank well must be The Absorbance value of the after subtracting the blank. If not the run should be repeated. 3. Each individual absorbance value of the negative control must after subtracting the blank. If one of the negative control values does not meet the Quality Control criteria, it should be discarded and the mean value calculated again using the remaining two values. If two or more negative control Absorbance values do not meet the Quality Control Range specifications, the test is invalid and must be repeated. Results 1. Subtract the blank from each of the valid negative controls. Calculate the mean absorbance of the negative control (NCx). Calculate the cut-off value by adding to the mean absorbance of the negative control. Cut-off = NCx Example: NCx = cut-off value = = Divide the absorbance of the sample by the cut-off value. Positive: ratio absorbance/cut-off 1.0 Negative: ratio absorbance/cut-off 0.9 Equivocal: ratio absorbance/cut-off 0.9 < 1.0 Interpretation of results Negative Results: Specimens with an absorbance to cut-off ratio lower than 0.9 are considered non reactive which indicates that no anti-hiv 1 and/or HIV-2 antibodies or HIV p24 antigen have been detected with bioelisa HIV-1+2 Ag/Ab kit. Positive Results: Specimens giving an absorbance equal to or greater than the cut-off value are considered initially reactive, which indicates that anti-hiv 1/2 antibodies or HIV p24 antigen have probably been detected using bioelisa HIV-1+2 Ag/Ab. All initially reactive specimens should be retested in duplicate to confirm the initial result. Equivocal: Specimens with an absorbance to cut-off ratio between 0.9 and 1.0 are considered equivocal and retesting of these specimens in duplicate is required to confirm the initial results. - If, after retesting the initially reactive samples, both wells report negative results (S/CO < 0.9), these samples should be considered as non-repeatable positive (or, false positive) and recorded as negative. - If, after retesting in duplicate, one or both wells report positive results, the final result from this ELISA test should be recorded as repeatedly reactive. Repeatedly reactive specimens can be considered positive for antibodies to HIV 1/2 or p24 antigen, therefore the patient is probably infected with HIV1 or HIV2. - After retesting in duplicate, samples with values close to the cut-off value should be interpreted with caution and considered as equivocal, or uninterpretable for the time of testing. - Follow-up, confirmation and supplementary testing of any positive specimen with other analytical system (e.g. WB, PCR) is required. Clinical diagnosis should not be established based on a single test result. It should integrate clinical and other laboratory data and findings. BIOKIT, S.A. - Can Malé s/n - - Barcelona - SPAIN _R00_ _eng.docx

21 bioelisa Limitations of the procedure - Optimal assay performance requires strict adherence to the assay procedure described. Deviation from the procedure may lead to aberrant results. - As in all sensitive immunoassays, there is the possibility that non-repeatable positive results occur. - Positive results must be confirmed with another available method and interpreted in conjunction with the patient clinical information. - Negative results obtained with bioelisa HIV-1+2 Ag/Ab are considered negative for antibodies to HIV-1/2 and p24 antigen and further testing is not required. - - This assay cannot be used to test pooled (mixed) plasma. bioelisa HIV-1+2 Ag/Ab has been evaluated only with individual serum or plasma specimens. - bioelisa HIV-1+2 Ag/Ab is a qualitative assay and the results cannot be used to measure concentrations of antibodies to HIV or p24 antigen. This assay cannot distinguish between infections with HIV-1 and HIV-2. - A negative result does not exclude the possibility of exposure or infection with HIV. Expected results The number of positive results depends on the disease incidence in the geographic area and the type of tested population. In the world, the HIV incidence in people older than 15 years varies from 0.1% in Australia, New Zeeland and Asia Pacific, 0.3% in Western Europe, North Africa and Middle East, 0.5% in East Europe, Central Asia and Latin America, 0.6% in North America and South Asia and 9% in sub-saharan Africa. In the same country, incidence also varies enormously according to the tested population. In Western Europe, HIV antibodies prevalence in blood bank donations varies from 0 to 5 cases over 100,000 donations, while the incidence among prisoners, sex workers and drug abusers may easily reach 20% in these risk populations. Performance characteristics Analytical Sensitivity The limit of detection has been estimated below 0.5 IU/ml by testing the WHO HIV p24 Antigen, First International Reference NIBSC code 90/636 with two different lots during the internal evaluation. Additionally, the limit of detection has been also estimated below 12.5 pg/ml by testing BIORAD HIV-1 Antigen Standard with two different lots during the internal evaluation. Sensitivity - A total of 537 HIV-1 positive samples were evaluated with bioelisa HIV 1+2 Ag/Ab, among them: 28 HIV-1 fresh-same-day collected samples, 85 HIV-1 and 4 HIV-1 Subtype O samples in an European reference lab; 380 HIV -1 samples were evaluated at Biokit and 40 HIV-1 non B subtype samples (and at least 3 samples of each HIV-1 subtype) were evaluated in another European reference lab. bioelisa HIV 1+2 Ag/Ab detected all 537 samples. The sensitivity found was100% (537/537). - A total of 115 HIV-2 positive samples were evaluated with bioelisa HIV 1+2 Ag/Ab, among them: 27 HIV-2 samples in an European reference lab and 88 HIV-2 samples were evaluated at Biokit. bioelisa HIV 1+2 Ag/Ab detected all 115 samples. The sensitivity found was 100% (115/115). - A total of 61 HIV-1 p24 Ag positive samples were evaluated also with bioelisa HIV 1+2 Ag/Ab, among them: 15 samples in an European reference lab, 16 samples were evaluated in another European reference lab and 30 samples at Biokit. bioelisa HIV 1+2 Ag/Ab detected all 61 samples. The sensitivity found was 100% (61/61). - Additional sensitivity studies were carried out by testing a total of 31 seroconversion panels at Biokit and a European reference lab. Results obtained were comparable to the most sensitive commercial method for HIV 1+2 antigen and antibody detection HIV Seroconversion Panels from BBI/Seracare and Zeptometrix were evaluated at Biokit. bioelisa HIV-1+2 Ag/Ab identified correctly all the positive specimens. BIOKIT, S.A. - Can Malé s/n - - Barcelona - SPAIN _R00_ _eng.docx

22 bioelisa bioelisa HIV Ag/Ab HIV p24 HIV Ab Assay Seroconversion Panel HIV Ag/Ab Assay antigen NAT First sample detected positive in the panel BB1-PRB965-n= ZEPT n= ND 4 BBI-PRB954-n= BBI-PRB932-n=9 4 ND ZEPT-9017-n= ZEPT-9014-n= ZEPT-9012-n= ZEPT-9032-n= ZEPT-6243-n= ZEPT-9022-n= ZEPT-9018-n= BBI-PRB939-n=9 6 ND BBI-PRB956-n= BBI-PRB945-n= BBI-PRB955-n= BBI-PRB958-n= In the previous table it can be observed the first sample detected as positive in each panel evaluated with bioelisa HIV-1+2 Ag/Ab in comparison with 4 th Generation assays (Ag/Ab Assays), 3 rd Generation assays (Ab Assays), assays for HIV p24 antigen and HIV RNA NAT assays. bioelisa HIV-1+2 Ag/Ab showed excellent sensitivity to detect early antigen or antibodies in 16 HIV Seroconversion Panels evaluated. - Another study was conducted to assess the sensitivity in 32 early seroconversion samples. These type of samples are characterized to be positive to the presence of HIVp24 Ag and/or positive by NAT (Nucleic Acid Testing), not detected by all HIV 3rd generation methods and to be negative or Indeterminate by Western-Blot methods. bioelisa HIV 1+2 Ag/Ab detected all 32 samples. The sensitivity found was 100% (32/32). - Finally, 50 cell culture supernatans from HIV cell cultures, including different HIV-1 subtypes and HIV-2 were evaluated in a European Reference Lab. bioelisa HIV 1+2 Ag/Ab detected all 50 samples. The sensitivity found was100% (50/50). Specificity The specificity was evaluated by testing a total of 6465 unselected blood donors' samples at three different sites. - In the first site 3045 unselected samples including 684 first donors were tested. From this total, 4 samples were reactive. The specificity obtained in this study was 99.87% (3041/3045). - In the second site 500 unselected samples from Blood Bank were tested. All 500 samples were negative. The specificity was 100% (500/500) - An internal evaluation of 2920 unselected samples from Blood Bank was performed. From this evaluation, 6 samples were reactive. The specificity obtained in this study was 99.79%. (2914/2920) A detailed summary of these specificity studies is shown in the table below. BIOKIT, S.A. - Can Malé s/n - - Barcelona - SPAIN _R00_ _eng.docx

23 bioelisa Evaluation 1 (3045 sera) Initial Reactive Repeated Reactive False Positive Specificity % Serum Evaluation 2 (500 sera) Initial Reactive Repeated Reactive False Positive Specificity % Serum Internal Evaluation (1000 sera plasmas) Initial Reactive Repeated Reactive False Positive Specificity % Plasma Serum Total In addition 200 samples from hospitalized patients have been tested and compared to a reference test. 194 samples were found negative by both tests and six samples were reactive. The six samples were also reactive with other ELISA tests. A specificity of 100% was obtained in this study (194/194). Precision Intra-assay reproducibility: The coefficient of variation obtained for the absorbance values of a HIV-1 positive sample assayed in a minimum of 40 replicates was 3.42%, 5.79% and 4.71% in three lots studied. The coefficient of variation obtained for the absorbance values of a HIV-2 positive sample assayed in a minimum of 40 replicates was 6.39%, 5.91% and 8.38% in three lots studied. The coefficient of variation obtained for the absorbance values of a HIV-1 p24 positive sample assayed in a minimum of 40 replicates was 4.08%, 3.44% and 3.71% in three lots studied. Inter-assay reproducibility: Four positive samples of different levels were tested in 15 different assays. The coefficients of variation obtained for the ratios absorbance/cut-off of the four samples were 4.87%, 5.37%, 7.49% and 7.54%. Interferences Interference by addition No interference has been found for haemoglobin (500 mg/dl), bilirubin (20 mg/dl), human Albumin (5 g/dl) and triglycerides (500 mg/dl). Cross-reactivity To evaluate possible interferences, 125 potential cross-reacting specimens were analyzed. Among those samples, 4 samples (RF+), 4 samples positive for anti-nuclear antibodies (ANA), 5 samples Mononucleosis, 89 samples from other related infectious diseases and 23 samples from pregnant women. No evidence of cross reactivity was found in the samples evaluated. The 2 anti-toxoplasma IgM samples that tested positive were also positive by other HIV assays. Potential interfering samples = 125 RF (Rheumatoid factor) - 4 Elevated IgG - 4 ANA (anti-nuclear Antibodies) - 4 Elevated IgM - 5 Mononucleosis - 5 HSV 1 IgG (Herpes Simplex Virus) - 5 Pregnant women- 23 (including 7 multiparous) HSV 2 IgG (Herpes Simplex Virus) - 4 Syphilis Ab - 6 anti-cmv IgG (Cytomegalovirus) - 5 anti-ebv (Epstein-Barr Virus) - 3 anti-cmv IgM (Cytomegalovirus) - 7 anti-rubella (Rubella Virus) - 4 anti-htlv (Human T-lymphotropic Virus) - 4 anti-vzv IgG (Varicela Zoster Virus) - 2 anti-hcv (Hepatitis C Virus) - 8 anti-vzv IgM (Varicela Zoster Virus) - 2 anti-e.coli (Escherichia coli)-5 anti-toxoplasma IgG (T. gondii) - 10 HBsAg (Hepatitis B Antigen) - 8 anti-toxoplasma IgM (T. gondii)-7 BIOKIT, S.A. - Can Malé s/n - - Barcelona - SPAIN _R00_ _eng.docx

24 bioelisa bioelisa: Troubleshooting guide Problem Possible causes Solution 1. Controls out of validation. 2. No colour or only a light colour developed at the end of the assay. 1a. Incorrect temperature, timing or pipetting. 1b. Improper preparation of reagents, error of dilution, reagents not well mixed. 1c. Cross-contamination of controls. Check procedure. Repeat assay. Check procedure. Repeat assay. Pipette carefully. Do not interchange caps. Repeat assay. 1d. Incorrect reading filter. Check that the wavelenght of the filter used is 450 nm. If no reference nm is used, absorbance increases approximately e. Interference in the optical pathway. Check the reader. Clean or dry the bottom of microplate. Check for air bubbles. Repeat reading. 1f. Used components from different lots. Do not use components from different lots as they are adjusted for each batch released. 1g. Expired reagents. Check the kit expiry date. Use 2a. One or more reagents not added or added in wrong sequence. 2b. Inactive conjugate. Wrong dilution of concentrate conjugate 2. Improper conservation. 2c. Inactive microplate: Improper conservation. 2d. Inactive Sustrate-TMB: Improper conservation. The container used affects Substrate-TMB stability, cross-contamination with the stopping solution. a non- expired kit. Check procedure. Repeat assay. Check for contamination. Check procedure. Repeat assay. Always keep unused strips in the bag very well closed, with the desiccant inside. Repeat assay. Use disposable containers or wash with acid or ethanol and rinse with deionised water before re-use. Check procedure. Repeat assay. 2e. Too cold reagents. Let reagents reach room temperature before use. BIOKIT, S.A. - Can Malé s/n - - Barcelona - SPAIN _R00_ _eng.docx

25 bioelisa bioelisa: Troubleshooting guide Problem Possible causes Solution 3. Too much colour in all microplate wells. 4. Poor reproducibility or high number of non-repeatable reactive samples. 3a. Contaminated or oxidised Substrate-TMB solution. 3b. Contaminated or improperly prepared reagents. 3c. Contaminated washing solution (1x). 3d. Insufficient washing or washing not consistent: filling volume and/or aspiration insufficient or not uniform. Insufficient number of washing cycles, contaminated device. 3e. Using of a washing solution from other manufacturer. Check that substrate is colourless, discard if blue. Use acid or ethanol washed or disposable containers. Repeat assay. Check for contamination: turbid aspect. Check dilutions. Repeat assay. Check the quality of distilled or deionised water used for dilution. Repeat assay. Check the washing device. Fill wells with washing solution close to the top, aspirate completely. Increase the number of wash cycles. Use only the washing solution supplied with the kit. 4a. Washing problems. See 3c, 3d, 3e. 4b. Uncalibrated pipettes or tips not well fitted. Improper pipetting. 4c. Reagents too cold or not well mixed before using. 4d. Air currents over the microplate during incubations. 4e. Too long time for addition of samples and/or reagents. Inconsistency in time intervals. Air bubbles. 4f. Interference in the optical pathway. Use only calibrated pipettes, with well fitted tips and pipette carefully, without bubbles and splashing. Repeat assay. Equilibrate reagents to room temperature and mix thoroughly before using. Keep the microplate protected from air currents. Develop consistent and uniform technique. See 1e. BIOKIT, S.A. - Can Malé s/n - - Barcelona - SPAIN _R00_ _eng.docx

26 EXTERNAL EVALUATION Page 13 of 13

27 Prüflabor für In-vitro Diagnostika beim Paul-Ehrlich-Institut (PEI-IVD) Paul-Ehrlich-Straße D Langen Evaluation Report for bioelisa HIV-1+2 AG/AB Manufacturer: Biokit S.A. Can Malé s/n, Lliçà d Amunt, Barcelona Spain Dr. Sigrid Nick Prüflabor für In-vitro-Diagnostika Paul-Ehrlich-Institut Phone: Fax: Sigrid.Nick@pei.de

28 bioelisa HIV-1+2 AG/AB Purpose of the Evaluation: The purpose of the evaluation of the bioelisa HIV-1+2 AG/AB carried out by the Prüflabor for In-vitro Diagnostic Medical Devices at the Paul-Ehrlich-Institut (PEI-IVD) was to assess the clinical sensitivity of the device using 15 commercial HIV-1 seroconversion panels, 100 HIV-1 positive samples, including 25 fresh samples (<24 h of collection), a HIV-1 p24 antigen positive subtype panel (14 samples of HIV-1 subtypes A, B, C, D, E, F, G, H and O), 4 HIV-1 O samples and 25 HIV-2 samples. In addition, the analytical sensitivity was to be determined using the HIV-1 p24 reference material of the Paul-Ehrlich-Institut (PEI). The specificity was to be evaluated using 500 German blood donor sera to contribute to the performance requirements as specified in Table 1 of the Common Technical Specifications (CTS). Materials and Methods: Description of the Device The bioelisa HIV-1+2 AG/AB is a non-automated enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection of HIV-1 p24 antigen and IgM, IgA as well as IgG antibodies to HIV-1 and HIV-2 in human serum and plasma. The instructions for use specify that the assay is intended for the detection of HIV infection by clinical laboratories and for screening in blood donation centres. According to the labels and to the instructions for use, the kits contain the following components and show the following features: Table 1. Features of the bioelisa HIV-1+2 AG/AB Test Product name: Id.-no.: Manufacturer: bioelisa HIV-1+2 AG/AB (96 tests), (480 tests) Biokit S.A. Can Malé s/n, Lliçà d Amunt, Barcelona, Spain Instructions for Use (version): V.2 Intended use: Detection of HIV-1 p24 and HIV-1- and HIV-2-specific antibodies in human serum or plasma samples; blood screening assay Sample volume: 100 µl Number of determinations: 96 or 480 tests per kit Lot no.: J-1912 Expiry date: Storage temperature: 2-8 C Assay format: ELISA Kit components: - Breakable microtitre plates coated with anti-hiv-1 p24 antigen monoclonal mouse antibodies and recombinant HIV-1 (gp41) and HIV-2 (gp36) antigens PEI-IVD Page 2 of 34

29 bioelisa HIV-1+2 AG/AB Measuring/ reference wave lengths: Control specifications: Blank HIV-1 positive control - Blank HIV-2 positive control - Blank HIV p24 positive control - Blank NC - Blank - CONTROL + 1: HIV-1 positive control (PC) - CONTROL + 2: HIV-2 positive control - CONTROL + Ag: HIV positive control p24 (recombinant purified HIV-1 p24 antigen diluted in human serum) - CONTROL - : Negative control (NC) - Sample diluent - CONJ 1 (biotinylated HIV-1 and HIV-2 antigens) - DIL CONJ 2: conjugate diluent 2 - CONJ 2 (101x concentrate; Peroxidase labelled streptavidin) - TMB substrate - Washing solution concentrate (25x) - Stopping solution - Adhesive seals - Re-sealable bag - Instructions for use 450 / nm Mean Absorbances (A): 0,050 0,500 0,500 0,500 Cut-off (CO): A NCmean + 0,170 0,100 (each individual value) Assay Performance Test kits were provided by Biokit, Spain. Testing was performed from December 2012 through January At the time of the investigation, the device was not yet CE-marked. Testing procedure of the bioelisa HIV-1+2 AG/AB strictly followed the instructions for use (IFU). In brief, test components and samples were allowed to reach room temperature. Washing solution and conjugate solution no. 2 were prepared in the required volumes. Sample diluent was pipetted to the wells except for the Blank well. Then 100 µl of the Controls or of the test samples were added to the wells of the microtitre plate and mixed. Thereafter, the plates were sealed and transferred to the incubator without delay. After incubation at 37 C for 1 h, plates were washed five times with 400 µl washing solution in the overflow modus using a HydroFlex washer (Tecan). Soak time was 30 sec. After the addition of 200 µl of the conjugate solution no. 1, plates were incubated for 30 min and washed again three times. Then 200 µl of the conjugate solution no. 2 were added to the plates and after sealing plates were incubated for another 30 min and thereafter washed again five times PEI-IVD Page 3 of 34

30 bioelisa HIV-1+2 AG/AB For colour development 150 µl of the TMB substrate were added, plates were incubated at room temperature as required and then the reaction was stopped with 100 µl sulphuric acid. Absorbance values (A) were read at dual wave lengths 450/620 nm using a Tecan sunrise photometer. All pipetting steps were performed manually. Anti-HIV-1 and anti-hiv-2 positive samples as well as the seroconversion panels were tested in single determination. In case of results that were discrepant from the known sample status or of unusual results of the test kit controls or the samples, the determination was repeated. Determination of Analytical Sensitivity The analytical sensitivity was determined using the PEI HIV-1 p24 reference material. The concentration range is indicated in Table 2 below. Each dilution step was tested in double determination and tested three times apart. Table 2. HIV-1 p24 Antigen Standard Preparations Reference Material Description Concentration Range PEI HIV-1 p24 reference material Recombinant HIV-1 p24 expressed in E. coli and provided by Roche Diagnostics, Germany 100 pg/ml 1,60 pg/ml Anti-HIV-1 Positive Samples Eighty-five Anti-HIV-1 positive samples were obtained from the HIV Center of the University hospital of Frankfurt, Germany, in September Samples were tested for HIV at the time of receipt. Comparative results were available from the Murex HIV Ag/Ab Combination (now DiaSorin), the ABBOTT PRISM HIV O Plus, the ABBOTT ARCHITECT HIV Ag/Ab or the AxSYM HIV-Ag/Ab Combo test. After pretesting, samples were stored at -20 C until use. In addition, 28 Anti-HIV-1 positive samples were tested at the day of receipt (within 24 h of sample collection). Anti-HIV-1 O Positive Samples To investigate the ability of the bioelisa HIV-1+2 AG/AB to detect HIV-1 O, 4 positive samples were tested. Three samples were collected in Cameroun and were tested neat. One sample (W 92005) originated from Germany. Sample W was tested in a 1:100 dilution. Anti-HIV-2 Positive Samples In total, 25 anti-hiv-2 positive samples were included in the investigation (Appendix 2). Samples obtained from SeraCare were collected in Guinea Bissau, West Africa. According to the information provided by SeraCare, these samples were positive by the Siemens ADVIA Centaur PEI-IVD Page 4 of 34

31 bioelisa HIV-1+2 AG/AB HIV Enhanced (EHIV) assay and yielded overflow results (index > 50). The HIV-1/-2 type differentiation was carried out by SeraCare with an Anti-HIV-1/2 rapid test (Genie Multispot HIV- 1/2, Bio-RAD). Final discrimination between HIV-1 and HIV-2 infection was performed at PEI- IVD using the CHIRON RIBA HIV-1/HIV-2 SIA supplemental assay. The latter investigation of PEI classified 21 samples as unequivocally HIV-2 positive, three samples exhibited equal reactivity with HIV-1 and HIV-2 antigens. For these 3 samples it remains unclear whether the results are due to cross-reactivity or to HIV-1 and HIV-2 double infection. Further two anti-hiv-2 positive PEI samples were included. One sample was collected in Germany; the second sample was obtained from SeraCare (former BBI). HIV-1 p24 Subtype Samples To investigate the ability to detect different HIV-1 p24 subtypes, 14 samples of the former Nationales Referenzzentrum (NRZ) für Retroviren, Virologisches Institut - Klinische und Molekulare Virologie of the Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany were tested. At the time of receipt, the NRZ HIV-1 subtype panel included 11 group M isolates (1 x A, 1 x B, 1 x C, 3 x D, 2 x CRF01_AE ( E ), 2 x F, 1 x G, 1 x H) and two group O isolates. Virus isolates were originally obtained via the Programme EVA, Centre for AIDS Reagents, National Institute for Biological Standards and Control (NIBSC), UK. These virus isolates were propagated on cells and supernatants were diluted in HIV-negative plasma to a concentration of about 100,000 HIV-1 RNA copies/ml. The isolates were quantified by both the branched DNA assay (Siemens) and different real time HIV-1 PCR assays (by Abbott or Siemens). Precise concentrations (copies/ml) are given in Table 3 below. Table 3. Characteristics of the HIV-1 p24 subtype panel No. HIV 1 Subtype Laboratory ID Production date Viral Load (NRZ) (copies/ml) 1 A A B B C C D D D D D D E E E E F F F F G G H H O O O O PEI-IVD Page 5 of 34

32 bioelisa HIV-1+2 AG/AB HIV-1 Seroconversion Panels In addition, 15 commercial seroconversion panels were tested (Table 4). Seroconversion panels were pre-selected for short bleeding intervals to allow for a good discrimination between sensitive and less sensitive HIV tests. The seroconversion panels were purchased from either SeraCare Life Sciences (37 Birch Street, Milford, MA 01757, USA) or from Zeptometrix Corporation (872 Main Street Buffalo, NY 14202, USA). Table 4. HIV-1 Seroconversion Panels Used for Testing of the bioelisa HIV-1+2 AG/AB Panel No. PRB 960 PRB 963 PRB 966 PRB 967 PRB 969 PRB 971 PRB 972 Donor No. Panel vendor Bleeds per panel HIV-1 p24 Agpositive panel members HIV Ag/Abpositive bleeds 1 st HIVantibody reactive bleed Results of confirmatory or supplemental tests Maximum no. of bleeds reactive by the most sensitive HIV assay n.a. SeraCare 9 8 to 9 8 to 9 n.a. n.a. 2 n.a. SeraCare 7 6 to 7 6 to 7 7 Western blot (WB) negative n.a. SeraCare 10 8 to 10 8 to indeterminate 3 n.a. SeraCare 6 4 to 6** 4 to 6 4** 5 indeterminate, 6 positive n.a. SeraCare 10 7 to 9** 8 to 10 8** 8 to 10 positive 3 n.a. SeraCare 4 3 to 4** 3 to 4 4** WB negative 2 n.a. SeraCare 6 5 to 6** 5 to 6 5** RIBA negative * ZeptoMetrix * (13) 7 to 12 7 to indeterminate; 11 to 13 positive Zeptometrix to to all negative ZeptoMetrix 10 7 to 10 7 to 10 10** all negative ZeptoMetrix 7 6 to 7 6 to 7 7 all negative ZeptoMetrix 11 9 to 11 9 to indeterminate ZeptoMetrix to to all negative** ZeptoMetrix to to all negative ZeptoMetrix to to all negative 3 *Panel 6240: members 1 and 2 could not be tested due to insufficient volume (VNS); ** information taken from the panel vendors sheets; n.a. not applicable; n.d. not determined The results obtained for the bioelisa HIV-1+2 AG/AB were compared to data of current CEmarked HIV-Ag/Ab combination tests with state-of the-art technology (Appendix 6). The HIV- Ag/Ab combination test I exhibits ELISA format. HIV-Ag/Ab combination test II is a chemiluminescence immunoassay; currently, this is the most sensitive test for which comparative data is available at the Testing Laboratory for IVD. Data from RNA detection by NAT (nucleic assay amplification techniques) were taken from the panel vendors sheets which are attached (Attachments 1 and 2). In addition, results from current CE-marked Anti-HIV-1/2 tests as well as confirmatory Western blots or immunoblots were taken into account. PEI-IVD Page 6 of

33 bioelisa HIV-1+2 AG/AB Specificity Specificity was tested using 500 sera from the German Red Cross Blood Donor Service Baden- Wuerttemberg Hessen, Institute for Transfusion Medicine and Immunohaematology Frankfurt/Main. Samples were routinely collected by the Red Cross one day and pretested negative by the Abbott Prism HIV Ag/Ab Combo test. Left-over samples were received that same day and tested by the bioelisa HIV-1+2 AG/AB within three days after receipt. Results Analytical Sensitivity Analytical sensitivity was determined with dilutions of the WHO HIV-1 p24 1 st International reference reagent on three days. Mean analytical sensitivities were 10,45 pg/ml and 9,81 pg/ml and 8,00 pg/ml (Appendix 1) with a grand overall mean of 9,42 pg/ml. Anti-HIV-1 Positive Samples and Anti-HIV-1 O Samples In total 85 samples collected in 2008 and stored at -20 C were tested by the bioelisa HIV-1+2 AG/AB test. A selection criterion for the samples was that results from current CE-marked devices with state-of-the-art performance were already available. All 85 samples were reactive by the bioelisa HIV-1+2 AG/AB (Appendix 2) and showed high S/CO ratios between 15,38 and 25,00. Neither was a false-negative result obtained, nor was any discrepancy from the predetermined sample status observed. In addition, 28 fresh samples that are obtained routinely in the laboratory were tested within 24 h after draw. All fresh samples were clearly reactive and showed S/CO ratios above 16 (Appendix 3). Sensitivity was thus 100% for anti-hiv positive specimens stored frozen or tested fresh. Four Anti-HIV-1 O samples were tested. Samples W 25290, W25291 and W yielded high S/CO ratios in the overflow range (Table 5). Sample W was tested in a 1:100 dilution and was positive, too. Table 5. HIV-1 Subtype O Samples PEI samples Test name bioelisa HIV-1+2 Ag/Ab Manufacturer Biokit S.A. ID-No Lot no. J-1912 Test date Sample No. HIV-1 subtype Origin s/co W O Cameroun 27,20 W O Cameroun 27,21 W O Cameroun 27,21 W * O Germany 1,52** *diluted 1:100 **mean of three determinations PEI-IVD Page 7 of 34

34 bioelisa HIV-1+2 AG/AB Anti-HIV-2 Positive Samples Twenty-five anti-hiv-2 positive samples collected in Guinea-Bissau (obtained from SeraCare) were tested. According to the vendor s data the samples are high positive and exhibited overflow reactivity (> 50 index) in the Siemens ADVIA Centaur HIV Enhanced (EHIV). In addition, the samples were confirmed HIV positive by the CHIRON RIBA HIV-1/HIV-2 SIA. Three samples were characterized anti-hiv-1 and anti-hiv-2 positive by the CHIRON RIBA HIV-1/HIV-2; thirty-six samples were classified anti-hiv-2 positive. All 25 samples were detected as reactive by the bioelisa HIV-1+2 AG/AB test and yielded high S/CO ratios (> 17,5) (Appendix 4). Results were thus compliant with the pre-determined sample status. Two further HIV-2 positive samples were tested. Sample 26 is a sample from India (obtained from BioClinical Partners, now ZeptoMetrix) and sample 27 was collected in Germany. Although both additional samples were already pre-diluted, they were also clearly reactive. Overall sensitivity on anti-hiv-2 positive specimens was 100%. HIV-1 p24 Antigen Subtype Samples To investigate the ability of the bioelisa HIV-1+2 AG/AB test to recognise different HIV-1 p24 antigen subtypes, 14 samples from the Institut für Klinische und Molekulare Virologie (National Reference Centre for Retroviruses from April 1996 to September 2012) were investigated. Samples originating from cell culture supernatants were spiked into normal human serum and then tested without further dilution in the bioelisa HIV 1+2 Ag/Ab test. Nine samples were reactive (S/CO ratio 1,0), one sample was in the grey zone (S/CO ratio 0,91) and four samples were negative (S/CO ratio < 0,9). The data is summarized in the table in Appendix 5. Early Seroconversion Sensitivity To assess early seroconversion sensitivity, the bioelisa HIV-1+2 AG/AB test was investigated using 15 commercial HIV seroconversion panels. Panels were selected for short bleeding intervals (generally 7 days). Test results are given in the table in Appendix 6. Results of the bioelisa HIV-1+2 AG/AB test were compared to data from HIV-1 p24 tests and to that of two current HIV-Ag/Ab combination tests: HIV Ag/Ab test-i and HIV Ag/Ab test-ii of different manufacturers. In summary, the bioelisa HIV-1+2 AG/AB test detected 44 samples as reactive (S/CO 1,0), while the CE-marked comparator HIV Ag/Ab combination test I detected only 42 samples as reactive. To enable a comparison for all 15 seroconversion panels, data of the comparator HIV Ag/Ab combination test I were taken from the vendor s data sheet for panel The bioelisa HIV-1+2 AG/AB test detected HIV infection either in the same bleed as HIV Ag/Ab test-i or one bleed earlier (panels 6240 and 6247). The HIV Ag/Ab test-ii is currently the most sensitive HIV Ag/Ab combination test for which comparative data is available at the PEI Testing Laboratory. The results of eleven panels can be compared to the HIV Ag/Ab test-ii. HIV Ag/Ab test-ii data is not yet available for the panels PRB 967, 969, 971 and 972. In summary, the bioelisa HIV-1+2 AG/AB test detected 34 PEI-IVD Page 8 of 34

35 bioelisa HIV-1+2 AG/AB samples as reactive while the comparator test detected only 33 samples as reactive. The reactivity of HIV Ag/Ab test-ii on panel member is considered an unspecific reaction. In conclusion, the sensitivity of the Biokit bioelisa HIV-1+2 AG/AB test is comparable to current CE-marked HIV Ag/Ab combination tests with high sensitivity in early infection. Specificity Specificity was assessed using 500 samples from the daily routine of the DRK Frankfurt, Germany. All samples were clearly negative upon the initial test. None of the samples was reactive nor grey zone reactive. Initial and repeat specificity thus were 100%. Mean S/CO value of the 500 samples was 0,23; the median value was 0,22. Minimum S/CO was 0,07 and the maximum S/CO ratio was 0,63 (Fig. 1, Table 6 and Appendix 7). Fig. 1. Distribution Histogram of S/CO Ratios of Blood Donor Samples. Table 6. Specificity Summary S/CO Mean 0,23 Median 0,22 SD 0,04 Mean + 3SD 0,35 Mean - 3SD 0,10 Min 0,07 Max 0,63 SD: Standard deviation PEI-IVD Page 9 of 34

36 bioelisa HIV-1+2 AG/AB Summary and Conclusions In the study, the clinical sensitivity and specificity of the bioelisa HIV-1+2 AG/AB test was evaluated with 113 anti-hiv-1 positive samples, 4 HIV subtype O and 27 anti-hiv-2 positive samples, 14 HIV-1 p24 antigen subtype samples, 15 commercial seroconversion panels and 500 blood donor samples. Results of the HIV-1 positive samples were compared to those obtained with CE-marked tests used for determining the sample status at the time of sample receipt. For the seroconversion panels two current CE-marked HIV-Ag/Ab combination tests and a sensitive CE-marked Anti-HIV-1/2 test were used for sample characterization. In addition, results from HIV-1 p24 tests and supplemental or confirmatory tests were taken into account for the characterization of the seroconversion panels. Overall sensitivity on the 113 anti-hiv-1 positive samples, on the four HIV-1 subtype O samples and on the 27 anti-hiv-2 positive samples was 100%. S/CO ratios were always high when samples were tested without dilution. There were no results discrepant from the initial sample status. Analytical sensitivity was evaluated using the PEI HIV-1 p24 reference material. The bioelisa HIV-1+2 AG/AB test exhibited a detection limit (9,42 pg/ml) for the PEI HIV-1 p24 reference material which is in a reasonable good range of current assays. Seroconversion panel results were in accordance with the good analytical sensitivity for HIV-1 p24 and demonstrate early detection of HIV infection similar to that of two current CE-marked HIV-Ag/Ab combination tests that exhibit either a good or an excellent sensitivity in early HIV infection. For the investigation of the detection of HIV-1 antigen subtypes 14 samples from the former Nationales Referenzzentrum (NRZ) für Retroviren, Virologisches Institut - Klinische und Molekulare Virologie in Erlangen, Germany were used. These samples constitute supernatants from cell cultures diluted into plasma. Overall, 9 samples were positive, one sample was in the grey zone and two samples showed elevated S/CO ratios (0,83 and 0,88). Sample 14 (subtype O) was negative. Specificity was evaluated on 500 blood donor sera originating from the DRK Hessen-Baden- Württemberg in Frankfurt. All samples tested initially negative and thus initial and repeat specificity was 100 % in this sample collection. Langen, 12/03/ (Dr. Sigrid Nick) PEI-IVD Page 10 of 34

37 bioelisa HIV-1+2 AG/AB Appendix 1 Results of PEI HIV-1 p24 Antigen Dilutions PEI HIV-1 p24 Ag [pg/ml] bioelisa HIV-1+2 Ag/Ab bioelisa HIV-1+2 Ag/Ab bioelisa HIV-1+2 Ag/Ab Lot: J-1912 J-1912 J-1912 B.-No.: 104s/12 104s/12 104s/12 Test date: S/CO S/CO Mean S/CO S/CO S/CO Mean S/CO S/CO S/CO Mean S/CO Grand Mean [pg/ml] 100,00 6,19 6,59 6,39 7,62 7,16 7,39 6,71 7,09 6,90 50,00 3,23 3,71 3,47 3,97 4,00 3,99 3,74 3,84 3,79 25,00 2,06 2,08 2,07 2,07 2,22 2,14 2,16 2,2 2,18 12,50 1,14 1,15 1,14 1,15 1,22 1,19 1,12 1,19 1,16 6,25 0,70 0,71 0,70 0,73 0,78 0,76 1,09 0,79 0,94 3,13 0,44 0,44 0,44 0,43 0,47 0,45 0,43 0,47 0,45 1,60 0,37 0,39 0,38 0,29 0,41 0,35 0,38 0,36 0,37 Dilution Matrix 0,30 0,40 0,35 0,24 0,35 0,29 0,35 0,38 0,37 Analytical Sensitivity [pg/ml] 10,45 9,81 8,00 9,42 PEI-IVD Page 11 of 34

38 bioelisa HIV-1+2 AG/AB Appendix 2 Results of Anti-HIV-1 Positive Samples Test: bioelisa HIV-1+2 Ag/Ab Murex HIV Ag/Ab Combination PRISM HIV O PLUS AxSYM HIV Ag/Ab Combo Lots: J-1912 L209510; L HN00; 70119HN LF00 Test dates: No. W.-No. Date of receipt S/CO S/CO S/CO S/CO ,85 18,10 n.d. n.d ,82 17,49 n.d. n.d ,92 17,43 n.d. n.d ,45 17,87 n.d. n.d ,90 17,41 n.d. n.d ,65 16,87 n.d. n.d ,97 17,47 n.d. n.d ,15 18,63 n.d. n.d ,22 18,19 n.d. n.d ,84 17,67 n.d. n.d ,51 18,98 n.d. n.d ,51 > max n.d. n.d ,97 > max n.d. n.d ,00 n.d. 99,55 22, ,25 n.d. 112,64 26, ,30 n.d. 95,17 n.d ,74 n.d. 104,98 n.d ,07 n.d. 107,99 n.d ,84 n.d. 90,46 n.d ,18 n.d. 111,73 n.d ,32 n.d. 120,46 n.d ,56 n.d. 113,86 n.d ,55 n.d. 123,10 n.d ,85 n.d. 103,23 n.d ,08 n.d. 109,88 n.d. PEI-IVD Page 12 of 34

39 bioelisa HIV-1+2 AG/AB Test: bioelisa HIV-1+2 Ag/Ab Murex HIV Ag/Ab Combination PRISM HIV O PLUS AxSYM HIV Ag/Ab Combo ,81 n.d. 114,31 n.d ,57 n.d. 101,45 n.d ,50 n.d. n.d. 29, ,68 n.d. n.d. 29, ,16 n.d. n.d. 10, ,94 n.d. n.d. 26, ,90 n.d. n.d. 27, ,66 n.d. n.d. 28, ,73 n.d. n.d. 22, ,16 n.d. n.d. 24, ,35 n.d. n.d. 20, ,73 n.d. n.d. 25, ,24 n.d. n.d. 23, ,54 n.d. n.d. 57, ,99 n.d. n.d. 19, ,70 n.d. n.d. 39, ,06 n.d. n.d. 51, ,58 n.d. n.d. 38, ,65 n.d. n.d. 45, ,60 n.d. n.d. 59, ,20 n.d. n.d. 23, ,47 n.d. n.d. 50, ,18 n.d. n.d. 24, ,39 n.d. n.d. 11, ,84 n.d. n.d. 23, ,18 n.d. n.d. 21, ,56 n.d. n.d. 27, ,46 n.d. 118,87 n.d ,77 n.d. 120,82 n.d ,65 n.d. 133,00 n.d ,26 n.d. 129,10 n.d ,51 n.d. 153,32 n.d. PEI-IVD Page 13 of 34

40 bioelisa HIV-1+2 AG/AB Test: bioelisa HIV-1+2 Ag/Ab Murex HIV Ag/Ab Combination PRISM HIV O PLUS AxSYM HIV Ag/Ab Combo ,96 n.d. 118,96 n.d ,12 n.d. 155,63 n.d ,24 n.d. 161,20 n.d ,89 n.d. 154,65 n.d ,96 n.d. 139,94 n.d ,48 n.d. 150,22 n.d ,83 n.d. 118,51 n.d ,38 16,64 n.d. 41, ,65 16,27 n.d. 64, ,49 16,47 n.d. 65, ,82 15,60 n.d. 65, ,59 16,35 n.d. 61, ,20 16,01 n.d. 56, ,38 15,56 n.d. 61, ,18 15,78 n.d. 50, ,18 17,33 n.d. 66, ,22 16,73 n.d. 44, ,78 16,55 n.d. 72, ,87 17,02 n.d. 53, ,70 16,40 n.d. 64, ,72 17,79 n.d. 59, ,06 n.d. n.d. 16, ,59 n.d. n.d. 53, ,07 n.d. n.d. 43, ,06 n.d. n.d. 69, ,13 n.d. n.d. 48, ,87 n.d. n.d. 54, ,78 n.d. n.d. 52,71 n.d. not determined PEI-IVD Page 14 of 34

41 bioelisa HIV-1+2 AG/AB Appendix 3 Results of Fresh Anti-HIV-1 Positive Samples bioelisa HIV-1+2 Ag/Ab Murex HIV bioelisa HIV-1+2 Ag/Ab Architect HIV Ag/Ab Combo Manufacturer: Biokit S.A. DiaSorin Biokit S.A. Abbott Lot: J-1912 D J LI00 Test date: No. W.-Nr. Date of Receipt S/CO S/CO S/CO S/CO ,33 13,87 n.d. n.d ,20 13,14 n.d. n.d ,64 13,37 n.d. n.d ,46 13,63 n.d. n.d ,58 12,50 n.d. n.d ,13 12,40 n.d. n.d ,98 13,02 n.d. n.d ,00 12,87 n.d. n.d ,76 13,21 n.d. n.d ,21 14,39 n.d. n.d ,30 14,02 n.d. n.d ,44 14,02 n.d. n.d ,06 13,75 n.d. n.d ,31 13,92 n.d. n.d n.d. n.d. 19,77 380, n.d. n.d. 18,77 66, n.d. n.d. 18,71 429, n.d. n.d. 18,98 501, n.d. n.d. 21,62 730, n.d. n.d. 19,43 416, n.d. n.d. 20,85 526, n.d. n.d. 18,03 151, n.d. n.d. 27,18 288, n.d. n.d. 27,19 328, n.d. n.d. 21,95 294, n.d. n.d. 21,81 638, n.d. n.d. 24,43 325, n.d. n.d. 20,72 545,44 n.d. not determined PEI-IVD Page 15 of 34

42 bioelisa HIV-1+2 AG/AB Appendix 4 Results of Anti-HIV-2 Positive Samples Product: Chiron RIBA HIV-1/HIV-2 SIA bioelisa HIV-1+2 Ag/Ab Manufacturer: Novartis Biokit SAU Lot: XA6518 / XA1371 J-1912 Dates of Test No. Lot No. Supplier ID ADVIA Centaur EHIV Index Genie Multispot HIV 1/2 Sample status as indicated by supplier HIV-1 gp120 HIV-1 gp41 HIV-2 Peptide HIV-1 p31 HIV-1/2 p24 / p26 Sample Status S/CO > 50 HIV-2 HIV-2 positive HIV-1/-2 positive 20, > 50 HIV-2 HIV-2 positive - +/ HIV-2 positive 21, > 50 HIV-2 HIV-2 positive HIV-2 positive 19, > 50 HIV-2 HIV-2 positive HIV-1/-2 positive 20, > 50 HIV-2 HIV-2 positive - +/ HIV-2 positive 21, > 50 HIV-2 HIV-2 positive - +/ HIV-2 positive 20, > 50 HIV-2 HIV-2 positive HIV-2 positive 21, > 50 HIV-2 HIV-2 positive HIV-2 positive 18, > 50 HIV-2 HIV-2 positive - +/ HIV-2 positive 18, > 50 HIV-2 HIV-2 positive HIV-2 positive 20, > 50 HIV-2 HIV-2 positive +/- +/ HIV-2 positive 19, > 50 HIV-2 HIV-2 positive HIV-2 positive 20, > 50 HIV-2 HIV-2 positive HIV-2 positive 20, > 50 HIV-2 HIV-2 positive +/- +/ HIV-2 positive 21, > 50 HIV-2 HIV-2 positive HIV-2 positive 20, > 50 HIV-2 HIV-2 positive HIV-2 positive 19, > 50 HIV-2 HIV-2 positive HIV-2 positive 17, > 50 HIV-2 HIV-2 positive - +/ HIV-2 positive 17, > 50 HIV-2 HIV-2 positive /- HIV-2 positive 17, > 50 HIV-2 HIV-2 positive HIV-1/-2 positive 18, > 50 HIV-2 HIV-2 positive +/- +/ HIV-2 positive 18,43 PEI-IVD Page 16 of 34

43 bioelisa HIV-1+2 AG/AB > 50 HIV-2 HIV-2 positive HIV-2 positive 18, > 50 HIV-2 HIV-2 positive HIV-2 positive 18, > 50 HIV-2 HIV-2 positive HIV-2 positive 17, > 50 HIV-2 HIV-2 positive HIV-2 positive 18,41 PEI samples PEI HIV-2 reference material 26 (1:1000) HIV-2 positive 5,28/ 5,34/ 4,97 27 W69169 HIV-2 positive 5,42/ 4,16/ 4,38 PEI-IVD Page 17 of 34

44 bioelisa HIV-1+2 AG/AB Appendix 5. HIV-1 p24 Subtype Panel No. HIV-1 Subtype HIV-1 Subtype (Laboratory ID) Production date (NRZ) bioelisa HIV-1+2 Genetic Systems Test: Ag/Ab Test: HIV-1 Ag HIV Ag/Ab Combo Test date: Test date: Viral Load copies/ml s/co Dilution factor (PEI) s/co s/co 1 A A ,78 3 1,62 2,39 2 B B ,16 1,5 2,09 1,96 3 C C ,90 1,5 2,47 3,18 4 D D ,24 1,5 2,46 2,73 5 D D ,91 3 1,82 2,47 6 D D ,29 1,5 2,11 1,78 7 E E ,83 3 2,30 3,72 8 E E ,18 2,5 2,09 3,25 9 F F ,48 3 2,17 1,84 10 F F ,01 2 1,22 1,18 11 G G ,42 3 2,11 1,36 12 H H ,88 3 2,07 3,32 13 O O ,85 3 8,69 21,12 14 O O ,57 3 0,75 8,21 PEI-IVD Page 18 of 34

45 bioelisa HIV-1+2 AG/AB Appendix 6. Commercial Anti-HIV-1 Seroconversion Panels No Panel Panel member Days since 1st Bleed Manufacturer BIO-RAD Biokit Chiron/ Novartis Test Genetic Systems HIV-1 Ag EIA bioelisa HIV-1+2 Ag/Ab HIV Ag/Ab Combination I HIV Ag/Ab Combination II HIV-1/2 Ab RIBA HIV-1/HIV- 2 SIA HIV RNA copies/ml* s/co s/co s/co s/co s/co HIV1 GP120 PRB < 50 0,13 0,30 0,25 0,12 0,72 n.a. 2 4 < 50 0,13 0,29 0,24 0,09 0,59 n.a. 3 7 < 50 0,12 0,28 0,25 0,09 0,58 n.a < 50 0,12 0,31 0,29 0,11 0,49 n.a < 50 0,07 0,31 0,26 0,11 0,83 n.a < 50 0,07 0,29 0,29 0,12 0,74 n.a ,9 x ,06 0,30 0,25 0,21 0,54 n.a ,8 x ,82 15,23 max 461,68 0,28 n.a ,1 x ,50 15,05 17,87 556,73 0,54 n.a. PRB < 50 0,17 0,67 0,26 0,11 0,45 n.d. 2 2 < 50 0,16 0,33 0,26 0,09 0,77 n.d. 3 7 < 50 0,15 0,29 0,26 0,12 0,65 n.d. 4 9 < 50 0,18 0,28 0,27 0,15 0,78 n.d ,8 x ,35 0,42 0,37 0,28 0,54 n.d ,6 x ,33 5,69 2,90 5,87 0,86 n.d ,2 x ,61 13,90 9,33 20,10 3,39 n.d. HIV1 GP41 HIV2 env Peptide HIV1 P31 HIV1/2 P24/26 Results PRB <50 0,13 0,26 0,29 0,14 0, /- - negative 2 2 <50 0,12 0,25 0,30 0,18 0, /- - negative 3 20 <50 0,13 0,21 0,28 0,16 0, /- - negative 4 22 <50 0,17 0,22 0,27 0,13 0, /- - negative 5 30 <50 0,06 0,22 0,27 0,21 0, /- - negative PEI-IVD Page 19 of 34

46 bioelisa HIV-1+2 AG/AB ,4x10 2 0,20 0,25 0,27 0,18 0, /- - negative ,9x10 3 0,22 0,32 0,30 0,30 0, /- - negative ,8x10 5 2,63 4,63 1,90 2,10 0, /- - negative ,8x10 4 1,04 5,27 4,02 2,10 2,66 - +/- - +/- - negative ,2x10 4 1,39 16,70 15,35 9,62 27, /- - indeterminate PRB <75 n.d. 0,25 0,27 n.d. n.d negative 2 3 <75 n.d. 0,32 0,25 n.d. n.d negative n.d. 0,53 0,43 n.d. n.d negative 4 17 > n.d. 15,97 >18,8 n.d. n.d. - +/ negative indeterminate 5 19 > n.d. 15,51 max n.d. n.d n.d. 16,14 16,45 n.d. n.d positive PRB <75 n.d. 0,24 0,31 n.d. n.d negative 2 29 <75 n.d. 0,24 0,26 n.d. n.d negative 3 48 <75 n.d. 0,24 0,29 n.d. n.d negative 4 53 <75 n.d. 0,33 0,25 n.d. n.d negative n.d. 0,37 0,29 n.d. n.d negative n.d. 0,44 0,44 n.d. n.d negative n.d. 0,57 0,60 n.d. n.d negative n.d. 15,94 8,70 n.d. n.d. +/ positive n.d. 16,47 10,26 n.d. n.d. +/ positive n.d. 17,46 7,10 n.d. n.d positive PRB n.d. 0,45 0,29 n.d. n.d negative n.d. 0,71 0,40 n.d. n.d negative n.d. 27,36 18,50 n.d. n.d negative 4 11 > n.d. 27,37 max n.d. n.d negative PRB n.d. 0,38 0,27 n.d. n.d negative n.d. 0,33 0,26 n.d. n.d negative n.d. 0,31 0,30 n.d. n.d negative n.d. 0,46 0,50 n.d. n.d negative PEI-IVD Page 20 of 34

47 bioelisa HIV-1+2 AG/AB n.d. 3,30 1,92 n.d. n.d. - +/ negative n.d. 5,18 4,27 n.d. n.d. - +/ negative ,39 VNS 0,23 0,16 0,17 VNS VNS VNS VNS VNS n.d ,40 VNS 0,23 0,18 0,17 VNS VNS VNS VNS VNS n.d ,37 0,21 0,22 0,06 0, negative ,38 0,25 0,26 0,16 0, negative ,35 0,25 0,24 0,13 0, negative ,35 0,22 0,23 0,11 0, negative ,12 1,83 0,66 0,79 0, negative ,13 12,40 4,26 6,40 0, negative x ,72 20,60 18,00 188,95 2, negative x ,51 17,69 max 270,68 16,63 - +/ negative indeterminate ,6x10 6 5,92 16,73 18,22 47,84 max / x10 4 1,09 17,99 17,56 11,30 max positive n.d. 0,70 19,82 18,26 15,50 max positive <400 0,14 0,25 0,47 0,12 0,42 negative 2 5 <400 0,18 0,21 0,25 0,14 0,71 negative 3 7 <400 0,16 0,24 0,26 0,13 0,38 negative 4 12 <400 0,15 0,20 0,31 0,11 0,42 negative 5 13 <400 0,16 0,20 0,24 0,19 0,43 negative 6 14 <400 0,15 0,19 0,27 0,12 0,68 negative 7 16 <400 0,22 0,22 0,22 0,21 0,52 negative 8 19 <400 0,17 0,26 0,24 0,17 0,65 negative 9 21 <400 0,17 0,22 0,23 0,17 0,42 negative <400 0,15 0,22 0,37 0,17 0,64 negative ,15 0,27 0,30 0,13 0,35 negative ,26 0,35 0,32 0,36 0,45 negative ,87 2,04 1,38 2,96 0,42 negative > ,82 18,43 17,28 68,87 0,90 negative > ,93 19,25 17,88 54,90 8,55 negative <500 0,22 0,28 0,32 0,20 0,07 n.d. n.d. 2 2 <500 0,22 0,26 0,36 0,15 0,08 n.d. n.d. 3 7 <500 0,21 0,30 0,37 0,13 0,07 n.d. n.d. PEI-IVD Page 21 of 34

48 bioelisa HIV-1+2 AG/AB 4 9 <500 0,22 0,29 0,30 0,17 0,08 n.d. n.d <500 0,20 0,28 0,33 0,15 0,08 n.d. n.d <500 0,18 0,29 0,31 0,18 0,09 n.d. n.d ,31 1,69 0,67 1,40 0,13 n.d. n.d ,49 8,33 3,55 8,14 0,09 n.d. n.d > ,44 16,58 max 166,88 0,08 negative > ,29 16,40 max 152,46 0,42 negative <500 0,24 0,29 0,23 0,07 0,06 negative 2 4 <500 0,22 0,30 0,23 0,11 0,09 negative 3 7 <500 0,20 0,29 0,25 0,12 0,09 negative 4 11 <500 0,20 0,27 0,25 0,09 0,09 negative ,24 0,35 0,28 0,14 0,12 negative ,59 4,55 1,62 3,56 0,09 negative 7 25 > ,66 17,13 18,91 883,52 16,01 negative <50 0,11 0,28 0,25 0,07 0,12 n.d. 2 2 <50 0,12 0,29 0,26 0,42 0,13 n.d <50 0,18 0,27 0,25 0,08 0,11 n.d <50 0,16 0,24 0,26 0,21 0,12 n.d <50 0,17 0,24 0,25 0,10 0,13 n.d <50 0,18 0,20 0,26 0,09 0,14 n.d <50 0,18 0,21 0,24 0,07 0,14 n.d ,20 0,27 0,30 0,08 0,12 n.d ,97 8,16 2,85 5,03 0,12 n.d ,16 13,20 2,27 2,29 1,16 +/- negative indeterminate ,88 15,16 4,79 3,14 max <50 0,40 0,23 0,21 0,23 0,07 n.d. 2 7 <50 0,38 0,27 0,25 0,35 0,08 n.d <50 0,46 0,29 0,22 0,19 0,07 n.d <50 0,40 0,26 0,22 0,20 0,07 n.d <50 0,36 0,26 0,22 0,22 0,06 n.d <50 0,38 0,27 0,22 0,18 0,10 n.d <50 0,41 0,27 0,22 0,22 0,09 n.d <50 0,37 0,28 0,24 0,22 0,07 n.d. PEI-IVD Page 22 of 34

49 bioelisa HIV-1+2 AG/AB 9 65 <50 0,36 0,23 0,22 0,20 0,06 n.d <50 0,36 0,26 0,23 0,21 0,07 n.d > ,60 10,24 6,50 10,17 0,08 n.d > ,51 18,58 19,80 140,09 2,17 n.d <50 0,17 0,25 0,25* 0,08 0,05 n.d. 2 4 <50 0,17 0,25 0,24 0,10 0,05 n.d. 3 7 <50 0,17 0,28 0,23 0,08 0,05 n.d <50 0,16 0,23 0,24 0,13 0,05 n.d <50 0,18 0,23 0,23 0,12 0,08 n.d <50 0,18 0,27 0,24 0,09 0,14 n.d <50 0,17 0,24 0,24 0,25 0,11 n.d <50 0,16 0,24 0,24 0,08 0,10 n.d <50 0,18 0,25 0,24 0,08 0,11 n.d <50 0,22 0,27 0,23 0,09 0,06 n.d <50 0,22 0,25 0,23 0,08 0,06 n.d ,18 0,25 0,23 0,08 0,06 n.d ,21 0,34 0,26 0,05 0,08 n.d ,07 10,09 4,70 8,67 0,09 n.d > ,03 17,62 11,43 22,78 0,56 n.d > ,18 25,20 17,87 60,41 max +/- negative <50 0,18 0,22 0,34 0,20 0,13 n.d. 2 2 <50 0,19 0,24 0,27 0,38 0,11 n.d. 3 7 <50 0,17 0,23 0,32 0,19 0,10 n.d. 4 9 <50 0,20 0,30 0,22 0,16 0,10 n.d <50 0,23 0,27 0,23 0,12 0,11 n.d <50 0,23 0,26 0,26 0,15 0,12 n.d <50 0,22 0,31 0,27 4,38 0,17 n.d <50 0,20 0,27 0,25 0,13 0,17 n.d <50 0,19 0,28 0,28 0,07 0,14 n.d ,17 0,29 0,25 0,06 0,16 n.d ,73 3,72 1,34 3,15 0,10 n.d > ,11 19,66 8,21 46,12 0,24 negative > ,20 20,35 17,69 72,89 7,35 negative *Data of HIV Ag/Ab Comnination test I are taken from the vendor s sheet for panel RNA copies/ml are taken from the panel vendors data sheets; n.d. not determined PEI-IVD Page 23 of 34

50 bioelisa HIV-1+2 AG/AB Appendix 7: Blood Donor Samples Manufacturer Biokit Test bioelisa HIV-1+2 Ag/Ab Lot no. J-1912 Expiry date Testing date Plate No. W.-No. Sample Barcode No. OD s/co 1 n.a. Blank n.a. 0,025 n.a. n.a. NC n.a. 0,049 n.a. n.a. NC n.a. 0,048 n.a. n.a. NC n.a. 0,052 n.a. n.a. PC1 n.a. 3,077 n.a. n.a. PC2 n.a. 2,040 n.a. n.a. PC Ag n.a. 3,594 n.a. W Probe ,22 W Probe ,21 W Probe ,21 W Probe ,20 W Probe ,21 W Probe ,37 W Probe ,21 W Probe ,21 W Probe ,21 W Probe ,21 W Probe ,21 W Probe ,23 W Probe ,20 W Probe ,21 W Probe ,21 W Probe ,21 W Probe ,22 W Probe ,22 W Probe ,22 W Probe ,23 W Probe ,24 W Probe ,21 W Probe ,22 W Probe ,22 W Probe ,23 W Probe ,21 W Probe ,20 W Probe ,22 W Probe ,21 W Probe ,20 W Probe ,22 W Probe ,21 W Probe ,22 W Probe ,22 W Probe ,22 W Probe ,22 W Probe ,21 W Probe ,19 PEI-IVD Page 24 of 34

51 bioelisa HIV-1+2 AG/AB W Probe ,21 W Probe ,22 W Probe ,23 W Probe ,25 W Probe ,24 W Probe ,21 W Probe ,23 W Probe ,23 W Probe ,24 W Probe ,24 W Probe ,26 W Probe ,22 W Probe ,21 W Probe ,21 W Probe ,21 W Probe ,21 W Probe ,21 W Probe ,22 W Probe ,24 W Probe ,24 W Probe ,23 W Probe ,22 W Probe ,25 W Probe ,24 W Probe ,24 W Probe ,23 W Probe ,24 W Probe ,26 W Probe ,25 W Probe ,22 W Probe ,21 W Probe ,21 W Probe ,24 W Probe ,22 W Probe ,22 W Probe ,24 W Probe ,29 W Probe ,24 W Probe ,22 W Probe ,28 W Probe ,28 W Probe ,28 W Probe ,24 W Probe ,23 W Probe ,22 W Probe ,20 W Probe ,21 W Probe ,26 W Probe ,21 W Probe ,22 W Probe ,21 2 n.a. Blank n.a. 0,027 n.a. n.a. NC n.a. 0,058 n.a. PEI-IVD Page 25 of 34

52 bioelisa HIV-1+2 AG/AB n.a. NC n.a. 0,047 n.a. n.a. NC n.a. 0,048 n.a. n.a. PC1 n.a. 3,018 n.a. n.a. PC2 n.a. 2,096 n.a. n.a. PC Ag n.a. 3,611 n.a. W Probe ,23 W Probe ,23 W Probe ,22 W Probe ,22 W Probe ,21 W Probe ,20 W Probe ,21 W Probe ,22 W Probe ,21 W Probe ,22 W Probe ,16 W Probe ,20 W Probe ,21 W Probe ,20 W Probe ,21 W Probe ,07 W Probe ,20 W Probe ,21 W Probe ,23 W Probe ,24 W Probe ,23 W Probe ,22 W Probe ,21 W Probe ,21 W Probe ,23 W Probe ,20 W Probe ,19 W Probe ,20 W Probe ,22 W Probe ,19 W Probe ,24 W Probe ,21 W Probe ,25 W Probe ,21 W Probe ,21 W Probe ,20 W Probe ,36 W Probe ,20 W Probe ,22 W Probe ,20 W Probe ,26 W Probe ,21 W Probe ,25 W Probe ,21 W Probe ,23 W Probe ,24 W Probe ,24 W Probe ,24 PEI-IVD Page 26 of 34

53 bioelisa HIV-1+2 AG/AB W Probe ,27 W Probe ,23 W Probe ,20 W Probe ,20 W Probe ,20 W Probe ,21 W Probe ,21 W Probe ,22 W Probe ,23 W Probe ,24 W Probe ,24 W Probe ,21 W Probe ,22 W Probe ,47 W Probe ,24 W Probe ,24 W Probe ,24 W Probe ,26 W Probe ,22 W Probe ,21 W Probe ,23 W Probe ,23 W Probe ,22 W Probe ,22 W Probe ,26 W Probe ,21 W Probe ,22 W Probe ,23 W Probe ,23 W Probe ,24 W Probe ,23 W Probe ,23 W Probe ,26 W Probe ,21 W Probe ,21 W Probe ,21 W Probe ,24 W Probe ,21 W Probe ,22 W Probe ,23 W Probe ,23 3 n.a. Blank n.a. 0,020 n.a. n.a. NC n.a. 0,050 n.a. n.a. NC n.a. 0,046 n.a. n.a. NC n.a. 0,050 n.a. n.a. PC1 n.a. 3,247 n.a. n.a. PC2 n.a. 2,205 n.a. n.a. PC Ag n.a. 3,769 n.a. W Probe ,22 W Probe ,21 W Probe ,20 W Probe ,22 W Probe ,20 PEI-IVD Page 27 of 34

54 bioelisa HIV-1+2 AG/AB W Probe ,19 W Probe ,20 W Probe ,19 W Probe ,22 W Probe ,19 W Probe ,20 W Probe ,20 W Probe ,23 W Probe ,19 W Probe ,18 W Probe ,19 W Probe ,31 W Probe ,22 W Probe ,25 W Probe ,22 W Probe ,24 W Probe ,22 W Probe ,21 W Probe ,22 W Probe ,23 W Probe ,21 W Probe ,18 W Probe ,19 W Probe ,19 W Probe ,18 W Probe ,19 W Probe ,22 W Probe ,24 W Probe ,63 W Probe ,23 W Probe ,24 W Probe ,23 W Probe ,21 W Probe ,22 W Probe ,22 W Probe ,24 W Probe ,23 W Probe ,25 W Probe ,24 W Probe ,29 W Probe ,24 W Probe ,26 W Probe ,25 W Probe ,25 W Probe ,24 W Probe ,23 W Probe ,25 W Probe ,25 W Probe ,22 W Probe ,23 W Probe ,22 W Probe ,26 W Probe ,27 PEI-IVD Page 28 of 34

55 bioelisa HIV-1+2 AG/AB W Probe ,58 W Probe ,27 W Probe ,24 W Probe ,25 W Probe ,24 W Probe ,22 W Probe ,24 W Probe ,23 W Probe ,24 W Probe ,21 W Probe ,25 W Probe ,24 W Probe ,22 W Probe ,24 W Probe ,23 W Probe ,22 W Probe ,24 W Probe ,23 W Probe ,24 W Probe ,23 W Probe ,28 W Probe ,22 W Probe ,25 W Probe ,25 W Probe ,27 W Probe ,22 W Probe ,23 W Probe ,21 W Probe ,22 W Probe ,23 W Probe ,21 4 n.a. Blank n.a. 0,028 n.a. n.a. NC n.a. 0,056 n.a. n.a. NC n.a. 0,045 n.a. n.a. NC n.a. 0,046 n.a. n.a. PC1 n.a. 3,381 n.a. n.a. PC2 n.a. 2,537 n.a. n.a. PC Ag n.a. 4,923 n.a. W Probe ,38 W Probe ,22 W Probe ,22 W Probe ,21 W Probe ,23 W Probe ,22 W Probe ,23 W Probe ,22 W Probe ,27 W Probe ,27 W Probe ,24 W Probe ,25 W Probe ,24 W Probe ,23 W Probe ,22 PEI-IVD Page 29 of 34

56 bioelisa HIV-1+2 AG/AB W Probe ,22 W Probe ,27 W Probe ,23 W Probe ,22 W Probe ,25 W Probe ,23 W Probe ,22 W Probe ,26 W Probe ,23 W Probe ,29 W Probe ,23 W Probe ,23 W Probe ,24 W Probe ,22 W Probe ,24 W Probe ,24 W Probe ,24 W Probe ,27 W Probe ,25 W Probe ,26 W Probe ,24 W Probe ,23 W Probe ,25 W Probe ,22 W Probe ,24 W Probe ,25 W Probe ,27 W Probe ,26 W Probe ,27 W Probe ,24 W Probe ,27 W Probe ,27 W Probe ,23 W Probe ,27 W Probe ,25 W Probe ,22 W Probe ,23 W Probe ,23 W Probe ,22 W Probe ,24 W Probe ,26 W Probe ,25 W Probe ,27 W Probe ,25 W Probe ,23 W Probe ,56 W Probe ,25 W Probe ,24 W Probe ,23 W Probe ,23 W Probe ,24 W Probe ,29 W Probe ,24 PEI-IVD Page 30 of 34

57 bioelisa HIV-1+2 AG/AB W Probe ,24 W Probe ,24 W Probe ,22 W Probe ,22 W Probe ,24 W Probe ,24 W Probe ,27 W Probe ,24 W Probe ,27 W Probe ,27 W Probe ,27 W Probe ,27 W Probe ,26 W Probe ,24 W Probe ,22 W Probe ,24 W Probe ,24 W Probe ,24 W Probe ,23 W Probe ,22 W Probe ,22 5 n.a. Blank n.a. 0,022 n.a. n.a. NC n.a. 0,047 n.a. n.a. NC n.a. 0,047 n.a. n.a. NC n.a. 0,043 n.a. n.a. PC1 n.a. 2,995 n.a. n.a. PC2 n.a. 2,228 n.a. n.a. PC Ag n.a. 3,488 n.a. W Probe ,20 W Probe ,22 W Probe ,18 W Probe ,22 W Probe ,20 W Probe ,17 W Probe ,20 W Probe ,23 W Probe ,20 W Probe ,19 W Probe ,19 W Probe ,19 W Probe ,18 W Probe ,17 W Probe ,18 W Probe ,23 W Probe ,20 W Probe ,19 W Probe ,18 W Probe ,21 W Probe ,18 W Probe ,22 W Probe ,20 W Probe ,19 W Probe ,20 PEI-IVD Page 31 of 34

58 bioelisa HIV-1+2 AG/AB W Probe ,19 W Probe ,18 W Probe ,20 W Probe ,19 W Probe ,19 W Probe ,20 W Probe ,23 W Probe ,23 W Probe ,20 W Probe ,20 W Probe ,20 W Probe ,18 W Probe ,20 W Probe ,20 W Probe ,23 W Probe ,20 W Probe ,20 W Probe ,22 W Probe ,21 W Probe ,20 W Probe ,21 W Probe ,22 W Probe ,25 W Probe ,22 W Probe ,19 W Probe ,22 W Probe ,19 W Probe ,18 W Probe ,27 W Probe ,19 W Probe ,19 W Probe ,19 W Probe ,22 W Probe ,23 W Probe ,32 W Probe ,21 W Probe ,21 W Probe ,20 W Probe ,20 W Probe ,20 W Probe ,21 W Probe ,21 W Probe ,20 W Probe ,20 W Probe ,20 W Probe ,19 W Probe ,19 W Probe ,20 W Probe ,20 W Probe ,27 W Probe ,24 W Probe ,20 W Probe ,24 PEI-IVD Page 32 of 34

59 bioelisa HIV-1+2 AG/AB W Probe ,21 W Probe ,20 W Probe ,20 W Probe ,25 W Probe ,27 W Probe ,22 W Probe ,22 W Probe ,21 W Probe ,20 W Probe ,23 W Probe ,21 5 n.a. Blank n.a. 0,019 n.a. n.a. NC n.a. 0,047 n.a. n.a. NC n.a. 0,041 n.a. n.a. NC n.a. 0,042 n.a. n.a. PC1 n.a. 3,213 n.a. n.a. PC2 n.a. 2,677 n.a. n.a. PC Ag n.a. 3,602 n.a. W Probe ,27 W Probe ,22 W Probe ,20 W Probe ,23 W Probe ,22 W Probe ,22 W Probe ,23 W Probe ,22 W Probe ,30 W Probe ,18 W Probe ,19 W Probe ,18 W Probe ,19 W Probe ,19 W Probe ,19 W Probe ,20 W Probe ,21 W Probe ,22 W Probe ,20 W Probe ,23 W Probe ,24 W Probe ,20 W Probe ,21 W Probe ,19 W Probe ,23 W Probe ,19 W Probe ,20 W Probe ,21 W Probe ,24 W Probe ,19 W Probe ,23 W Probe ,22 W Probe ,29 W Probe ,19 W Probe ,20 PEI-IVD Page 33 of 34

60 bioelisa HIV-1+2 AG/AB n.a. not applicable W Probe ,21 W Probe ,23 W Probe ,19 W Probe ,21 W Probe ,20 W Probe ,27 W Probe ,23 W Probe ,26 W Probe ,23 W Probe ,23 W Probe ,31 W Probe ,24 W Probe ,23 W Probe ,22 W Probe ,22 W Probe ,22 W Probe ,23 W Probe ,23 W Probe ,23 W Probe ,24 PEI-IVD Page 34 of 34

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