CEPHIA Consortium for the Evaluation and Performance of HIV Incidence Assays STANDARD OPERATING PROCEDURE

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1 CEPHIA Consortium for the Evaluation and Performance of HIV Incidence Assays STANDARD OPERATING PROCEDURE TITLE : SOP for (off board dilution) Less Sensitive Modified VITROS Enzyme Immunoassay CEPHIA DOCUMENT ID: 019A AUTHOR : Sheila Keating AUTHORIZER: Sheila Keating EFFECTIVE DATE: 7/11/12 ISSUED TO: CEPHIA GROUP SUMMARY The VITROS ECi/ECiQ Immunodiagnostic System is currently being used for the in vitro qualitative detection of antibodies to Human Immunodeficiency Virus types 1 and/or 2 (anti-hiv-1 and anti-hiv-2) in human serum and plasma (heparin, EDTA or citrate). The results of the VITROS Anti-HIV 1+2 assay, in conjunction with other serological evidence and clinical information, may be used as an aid in the diagnosis of infection with HIV-1 and/or HIV-2 in persons with signs or symptoms of, or at risk for, HIV infection. The identification of recently acquired HIV-1/2 infection provides important information on the dynamics of the HIV-1/2 epidemic, sexual or injecting drug use networks, patterns of transmitted drug-resistance as well as identifying potential candidates for clinical trials and vaccine strategies targeting early HIV-1/2 infection. This is the standard operating procedure for HIV-1/2 specific LS EIA Vitros Assay. This assay is used to discriminate between recently acquired (i.e. within the previous 120 days) and longstanding HIV-1/2 infection through the detection of anti HIV 1/2 antibodies in plasma quantified as a function of optical density. CEPHIA SOP 019A: LS-Vitros Page 1 of 5

2 SAFETY All human material should be treated as potentially infectious. Wear protective gloves and safety glasses whenever handling material of human origin and avoid contamination of equipment and surfaces. Ensure that Good Laboratory Practice is maintained. 1.0 Cross Reference 1.1 Manufacturer s kit insert 1.2 CEPHIA Worksheet 019B LS-VITROS Assay 2.0 Area of Application 2.1 Blood Systems Research Institute (BSRI), San Francisco 3.0 Personnel 3.1 Any medical or clinical microbiologist, or biomedical scientist or agency technician that has been trained in Level 2+ laboratories. 4.0 Principle An immunometric bridging technique is used; this involves a two-stage reaction. In the first stage HIV antibody present in the sample binds with HIV recombinant antigen coated on the wells. Unbound sample is removed by washing. In the second stage horseradish peroxidase (HRP)-labeled recombinant HIV antigens are added in the conjugate reagent. The conjugate binds specifically to any human anti-hiv-1 or anti-hiv-2 (IgG and IgM) captured on the well in the first stage. Unbound conjugate is removed by washing. The bound HRP conjugate is measured by a luminescent reaction. The amount of HRP conjugate bound is indicative of the level of anti-hiv-1 and anti-hiv-2 present. This assay contains four recombinant antigens (HIV-1 Env 13, HIV-1 Env 10, HIV-1 p24, and HIV-2 Env AL) derived from HIV-1 core, HIV-1 envelope and HIV-2 envelope. HIV-1 Env 13, envelope SOD fusion protein, contains regions from both gp 120 and gp 41 regions. HIV-1 Env 10, envelope SOD fusion protein, contains a gp41 region which extends beyond the C-terminus of Env 13. HIV-1 p24 is derived from full length core protein of HIV-1. HIV-2 Env AL, envelope SOD fusion protein, contains a region from gp 36 of HIV-2. These antigens detect antibodies to HIV-1 and antibodies to HIV-2 in the same test. Results are calculated as a normalized signal, relative to a calibrator cutoff value. During the calibration process, a lot-specific parameter, encoded on the lot calibration card, is used to determine a valid stored cutoff value for the VITROS ECi/ECiQ Immunodiagnostic System and results are given as Signal/Cutoff (S/C). CEPHIA SOP 019A: LS-Vitros Page 2 of 5

3 The optimized version of the LS-VITROS assay uses a 1:400 dilution of HIV-positive sample in dilution buffer, with the assay run in duplicate. In the early stages of LSmodified assay development, the dilution of the HIV-positive plasma sample in dilution buffer B, this was prepared using a two-step (1:20 and 1:20) manual dilution. However, automation of this step is possible by transferring this dilution step onboard the VITROS platform after further development of system programming. After samples were diluted, the fully automated VITROS ECi robot took 49 minutes to run the each sample in the assay and to report the results as signal to cutoff (S/C) values. 5.0 Equipment and Reagents required 5.1 HIV negative human plasma donor or Ortho Clinical Diagnostics Diluent B Pipettors: 20 ul, 200 ul single and multi channel, tips 5.3 Ortho Clinical Diagnostics Vitros 5600 ECi plus software 5.4 Vitros Versa Tips Reference # Vitros Micro Sample Cups Ref # Universal Wash Reagent Ref # E & K Scientific Titer Tubes Ref # OCD Vitros Signal Reagent Ref # OCD Reagent Pack Anti HIV 1 & 2 Ref # OCD Anti HIV 1/2 Controls Ref # BioRad Positive/Negative Controls Ref # 02281A 5.12 OCD Universal Sample Tray, 10 slot 5.13 Laboratory Vortex Mixer 5.14 OCD Calibrator Kit/Magnetic Cards Ref # HIV positive specimens plasma (EDTA, Heparin, Citrated) or Serum 6.0 Assay Preparation and Procedure 6.1 Perform all routine maintenance and lot specific calibration as per OCD operators manual. Check universal wash volume and load all reagent/signal reagent packs as necessary. Remove all controls from 4C refrigerator and bring to room temperature before performing the assay. 6.2 Begin dilution by placing in rack 1.2 ml titer tubes in two separate rows, no spacing, with two tubes per sample. Samples should be run in duplicate. 6.3 Pipet 190 microliters room temperature normal donor human plasma or Ortho Clinical Diagnostics Diluent B into each titer tube. 6.4 Pipet 10 ul RT sample plasma into first titer tube for 1:20 dilution. Dispose tip. Repeat 10 ul of the same sample in next titer tube for sample duplicates. Dispose tip. Repeat for all samples in run. 6.5 Vortex all diluted samples. 6.6 Pipet 10 ul sample dilution into corresponding 190uL normal CEPHIA SOP 019A: LS-Vitros Page 3 of 5

4 donor human plasma tube in second row for a final 1:400 dilution. Repeat for all samples. This step may also be performed with a multi-channel pipet for larger sample runs. Save 1:20 dilutions until full assay complete and results returned. 6.7 Vortex all 1:400 sample dilutions. 6.8 Prepare 10-slot Universal Sample Tray with Versa Tips and Micro Sample Cups for all samples. Six trays may be loaded per run as necessary. 6.9 Transfer full volume of first 1:400 sample dilution to first sample cup position. Dispose sample tube into biohazard container. Repeat for duplicate sample into next sample cup position (#2). 5 samples may be loaded per sample tray Reconstitute Ortho Negative, Ortho HIV1 and Ortho HIV 2 controls in 1 ml DI water as necessary. These controls may be used for several runs assuming proper refrigeration after each use. Aliquot 200 ul of each control Ortho Anti HIV 1/2 Negative, HIV1 Positive and HIV2 positive controls into sample cups. Recent and l ong standing control samples can also be used 2. Controls may be run at any point during sample testing. Do not duplicate Load sample trays with barcode facing reader onto Vitros ECi carousel Using Vitros program interface enter first sample tray by pressing Sample Programming. Enter tray number. Hit enter. Select type of assay ( HIV, Hepatitis, Avidity ) and select Batch Save Enter sample ID s into corresponding tray positions 1-10 noting duplicates with A/B designation. Example 12345A, 12345B Allow machine to process samples and generate report with S/C readings 6.15 Enter these results onto the results reporting forms for entry into the CEPHIA database Enter printout into Excel format for easy analysis and transfer of test results. 7.0 Decontamination Dispose of all specimens and materials used to perform the test as though they contain an infectious agent. Disposal should comply with all applicable waste disposal requirements 8.0 Controls Kit Controls: The Positive and Negative Controls must be within the ranges specified in the package insert. External Controls: BioRad controls are used as external controls to monitor assay performance. CEPHIA SOP 019A: LS-Vitros Page 4 of 5

5 IQC Controls for Evaluation panel: IQC 1, 2 and 3 will be tested 10 times prior to evaluation, and confidence intervals established. This will be tested on every run of the assay and the CI will be used to determine whether or not assays perform within range. Confidence intervals for all sets of controls are quantified and performance of controls is determined to be within range based on these calculations. 9.0 Results Specimen Result: Specimen S/CO value as reported by the VITROS system. Retest Criteria: 1 Specimens with an S/CO value of 30 or lower were retested in duplicate. Qualitative Interpretation of Results: 1 Results below (or equal to) a threshold, e.g. 20, produce recent infection classifications. Results above the threshold produce non-recent infection classifications Decontamination Dispose of all specimens and materials used to perform the test as though they contain an infectious agent. Disposal should comply with all applicable waste disposal requirements SUMMARY OF REVISIONS DETAILS OF REVISION(S) No amendments. 1 For purposes of initial CEPHIA incidence assay characterisation, as published in Independent Assessment of Multiple HIV Incidence Assays on Specimens in the CEPHIA Repository a threshold of 20 was used that is, a final result below 20 was interpreted as indicating recent infection. The final result was the mean of the duplicate S/CO values if the specimen was retested. CEPHIA SOP 019A: LS-Vitros Page 5 of 5