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HIV-1/HIV-2 PLUS O

Genetic Systems ™ HIV-1/HIV-2 PLUS O EIA. HIV Subtypes and Variants Description of HIV-1/HIV-2 PLUS O EIA Format, components, QC criteria Detection of HIV-1 (M & O), and HIV-2 Detection of Seroconversions Specificity Confirmation. HIV-1/HIV-2 PLUS O.

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HIV-1/HIV-2 PLUS O

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  1. Genetic Systems™ HIV-1/HIV-2 PLUS O EIA • HIV Subtypes and Variants • Description of HIV-1/HIV-2 PLUS O EIA • Format, components, QC criteria • Detection of HIV-1 (M & O), and HIV-2 • Detection of Seroconversions • Specificity • Confirmation HIV-1/HIV-2 PLUS O To detect HIV Antibody Groups M & O

  2. HIV Subtypes and Variants HIV-1 Subtype B is predominant in the U.S., but non-B subtypes are found in 2% of HIV-Positive U.S. blood donors, and increasing. (Delwart et al., AIDS Research and Human Retroviruses 19:1065-1070, 2003.) BCE ABD Group O ? G HIV-2 F BO HIV-1 B C B BE DE HIV-1: ABDEFGHIJ…OHIV-2: ABCDEFG ABCD BCF C DH

  3. HIV Subtypes and Variants Most Problematic for Antibody Detection • HIV-2:HIV-2 testing mandated for blood screening June 1, 1992. 1st: Genetic Systems™ HIV-1/HIV-2 EIA (viral lysate) 2nd: Genetic Systems™ HIV-1/HIV-2 Peptide EIA • HIV-1 Group O:FDA asked U.S. manufacturers to modify HIV kits (7/31/96 and 7/30/97). 3rd: Genetic Systems™ HIV-1/HIV-2 PLUS O EIA licensed 8/5/03

  4. Principle of the Test: Direct Antibody Sandwich Rec. p24 (HIV-1 M) Rec. gp160 (HIV-1 M) Pep. env (HIV-2) Pep env (HIV-1 O) 4 Plate Antigens Immunoglobulins from the sample bind simultaneously to antigens (4 total) on the microplate and to similar HRP-conjugated antigens (5 total) in solution. 5 HRP-Antigen Conjugates 2 HIV-1 M peptides HIV-1 M rec. p24 HIV-1 O peptide HIV-2 peptide HRP IgG

  5. Principle of the Test: Direct Antibody Sandwich Rec. p24 (HIV-1 M) Rec. gp160 (HIV-1 M) Pep. env (HIV-2) Pep env (HIV-1 O) 4 Plate Antigens Binding of IgM to the microplate is stabilized by multiple attachments, and the signal is amplified by multiple HRP-Antigen Conjugate binding sites. HRP HRP 5 HRP-Antigen Conjugates 2 HIV-1 M peptides HIV-1 M rec. p24 HIV-1 O peptide HIV-2 peptide HRP HRP IgM HRP HRP

  6. Earlier IgM Detection…a Benefit of the Antibody Sandwich FormatSeroconversion Panel PRB 940 Antibody Sandwich: HIV-1/HIV-2 PLUS O EIA Abbott HIVAb HIV-1/HIV-2 Indirect Antibody: HIV-1/HIV-2 Peptide EIA ~Day 8 ~Day 14 Cutoff

  7. Kit Components • Three kit sizes: 480/960/4800 Tests • Direct Antibody Sandwich Format • Unique Kit Components: -R1 Microwell Strip Plates (8X12) -R3 Specimen Diluent -C0 Negative Control -C1 HIV-1 Positive Control -C2 HIV-2 Positive Control -C3 HIV-1 Group O Positive Control -R4 Conjugate Concentrate (11X) -R5 Conjugate Diluent • “Shared” Components: 30X Wash (R2), Substrate Buffer(R8), TMB Chromogen 11X (R9), Stopping Solution (R10)

  8. Procedure (60’ 30’ 30’) • Add 25µl Specimen Diluent + 75µl Control or Sample to each well.* • Cover and incubate60 +/- 5 min.at 37 +/- 2ºC. • Wash a minimum of 5 times with30-60 second soaks. • Add 100 µl Working Conjugate to each well.* • Cover and incubate 30 +/- 5 min. at 37 +/- 2ºC. • Wash a minimum of 5 times with30-60 second soaks. • Add 100 µl Working TMB to each well. • Cover and incubate 30 +/- 5 min. at RT. • Add 100 µl Stopping Solution to each well. • Read within 30 minutes at 450nm, with the 615-630nm • filter as a reference. • *Optional O.D. readings may be taken to verify addition of specimen or reagent. • Color indicates differences in the procedure from HIV-1/HIV-2 Peptide EIA.

  9. Procedure Monitoring • Sample Dispensing • Conjugate Dispensing • T M B • Stopping solution Before After Optional Verification Sample OD630> 0.150 Conjugate OD630> 0.100 After incubation of a positive sample

  10. Quality Control:Validation of Results • Kit Controls (6 wells total): C0 Negative Control (3 wells) Each value A450=0.001-0.150 (one may be discarded) C1 HIV-1 Positive Control (1 well) A450>0.700 C2 HIV-2 Positive Control (1 well) A450>0.700 C3 HIV-1 Group O Positive Control (1 well) A450>0.700 • Cutoff = xNC + 0.250 Color indicates differences in the procedure from the HIV-1/HIV-2 Peptide EIA.

  11. Performance Results:HIV-1 Group M Sensitivity

  12. Performance Results:HIV Variant/Low Titer Samples

  13. Performance Results:HIV-2 Sensitivity *HIV-2 samples were repeatedly reactive on an HIV-2 EIA, positive on an HIV-2 Western blot, and indeterminate or negative on an HIV-1 Western blot.

  14. Performance Results:HIV-1 Group O Sensitivity *Known HIV-1 Group O samples were obtained from individuals living in Cameroon (N=70), the United States (N=2), Spain (N=2) and France (N=3). **One initially reactive Group O specimen was not available in sufficient volume for repeat testing.

  15. Performance Results: Seroconversions

  16. Performance Results: Seroconversions HIV-1/HIV-2 PLUS O: 21 Days Earlier cutoff

  17. Performance Results: U.S. Blood Donors Specificity 99.89% (95% confidence interval 99.83-99.96)

  18. Subtypes/Variants Tested by HIV-1 Western Blot PC LPC NC Group 0 A G G A G HIV-2 G A Neg HIV-2 C A Worldwide HIV Panel WWRB302(members 1-13) All HIV+ samples in the panel (N=28) exhibited A450/A630>3.000 in testing with Genetic Systems™ HIV-1/HIV-2 PLUS O EIA

  19. SUMMARY • Dissemination of HIV-1 non-B subtypes and variants is a growing concern in the U.S. • Reliable detection of HIV-1 Group O or HIV-2 antibody requires the use of specific antigens in the test kit. • HIV-1/HIV-2 PLUS O EIA performance was demonstrated with human serum, plasma, and cadaveric serum samples. • 100% detection of known HIV-1 (N=1002), HIV-2 (N=302), and HIV-1 group O (N=77) specimens. • 100% detection of HIV-1 low titer samples and HIV subtypes of worldwide origin (N=130) from four panels. • Detection of HIV-1seroconversion panels (N=50) better than licensed competitors. • Specificity 99.89% (95% C.I. 99.83-99.96) in blood and plasma donors at three sites (N=11,159). • Current confirmatory algorithms will detect most HIV-1 group O samples; unique banding may assist identification.

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