1 / 19

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.

cadman-mckinney
Download Presentation

HIV-1/HIV-2 PLUS O

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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.

More Related