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Genetic Systems™ HIV-1/HIV-2 PLUS O EIA HIV-1/HIV-2 PLUS O 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 To detect HIV Antibody Groups M & O 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? HIV-2 BO F HIV-1 G B B C DE BE HIV-1: ABDEFGHIJ…O HIV-2: ABCDEFG BCF ABCD C DH 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 Principle of the Test: Direct Antibody Sandwich 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 HRP 2 HIV-1 M peptides HIV-1 M rec. p24 IgG HIV-1 O peptide HIV-2 peptide Rec. p24 Rec. gp160 Pep. env (HIV-1 M) (HIV-1 M) (HIV-2) Pep env (HIV-1 O) 4 Plate Antigens Principle of the Test: Direct Antibody Sandwich 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 HRP HRP 2 HIV-1 M peptides HIV-1 M rec. p24 HRP HRP IgM HIV-1 O peptide HIV-2 peptide Rec. p24 Rec. gp160 Pep. env (HIV-1 M) (HIV-1 M) (HIV-2) Pep env (HIV-1 O) 4 Plate Antigens Earlier IgM Detection…a Benefit of the Antibody Sandwich Format Seroconversion Panel PRB 940 Antibody Sandwich: Indirect Antibody: HIV-1/HIV-2 PLUS O EIA HIV-1/HIV-2 Peptide EIA Abbott HIVAb HIV-1/HIV-2 14.0 12.0 Signal/cutoff 10.0 ~Day 8 ~Day 14 8.0 6.0 4.0 2.0 Cutoff 0.0 0 5 10 15 20 25 Time (Days) HIV-1/HIV-2 Peptide EIA GS HIV-1/HIV-2 PLUS O Abbott HIVAb 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) Procedure (60’ 30’ 30’) • Add 25µl Specimen Diluent + 75µl Control or Sample to each well.* • Cover and incubate 60 +/- 5 min. at 37 +/- 2ºC. • Wash a minimum of 5 times with 30-60 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 with 30-60 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. Procedure Monitoring Before After Optional Verification Sample Sample Dispensing OD630 > 0.150 Conjugate Conjugate Dispensing OD630 > 0.100 TMB After incubation of a positive Stopping solution sample 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. Performance Results: HIV-1 Group M Sensitivity Reactivity in HIV-1 Known Positive Samples Results Obtained with Licensed Genetic Systems™ HIV-1/HIV-2 PLUS O EIA HIV-1/HIV-2 EIA Group Number Repeatedly Reactive Number Repeatedly Reactive AIDS (N=313) 313 313 (100.00%) (100.00%) Known HIV-1 Positive, 490 490 U.S. (N=490) (100.00%) (100.00%) Known HIV-1 Positive, 199 199 Non-U.S. (N=199*) (100.00%) (100.00%) TOTAL: 1002 1002 (100.00%) (100.00%) * Australia, New S. Wales (N=36) Nigeria (N=46) Central African Republic (N=40) Sierra Leone (N=40) Ghana (N=5) Thailand (N=21) Kenya (N=3) Zimbabwe (N=8) Performance Results: HIV Variant/Low Titer Samples BBI Performance Panels (N=130 Positives) • Mixed Titer PRB203 (N=23 Positives) • Low Titer PRB105 (N=14 Positives) • African HIV Series AfrRB1 (N=46 Positives) • Worldwide WWRB301 (N=47 Positives) HIV-1/HIV-2 Peptide EIA 124/130 (95.4%) Abbott HIVAb HIV-1/HIV-2 128/130 (98.5%) Genetic Systems™ 130/130 (100.0%) HIV-1/HIV-2 PLUS O EIA Performance Results: HIV-2 Sensitivity Results Obtained from Known Positive HIV-2 Samples with Genetic Systems™ HIV-1/HIV-2 PLUS O EIA Pos. by HIV-2 Number Tested Initially Reactive Repeatedly Reactive Western blot * 302 302 302 302 (100.00%) (100.00%) (100.00%) *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. Performance Results: HIV-1 Group O Sensitivity Results Obtained from Known Positive HIV-1 Group O Samples with Genetic Systems™ HIV-1/HIV-2 PLUS O EIA Number Tested Initially Reactive Repeatedly Reactive 77* 77 76** (100.00%) (100.00%) *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. Performance Results: Seroconversions Reactivity with HIV-1 on 50 Commercial Seroconversion Panels HIV-1/HIV-2 PLUS O HIV-1/HIV-2 PLUS O HIV-1/HIV-2 PLUS O Equivalent More Sensitive Less Sensitive vs. 12/46* 34/46* 0 HIV-1/HIV-2 Peptide EIA (26%) (74%) (0%) vs. 35/50 9/50 6/50 Abbott HIVAb HIV-1/HIV-2 (70%) (18%) (12%) vs. Licensed 13/50 37/50 0 Western Blot (26%) (74%) (0%) • Four of the 50 seroconversion panels did not have test results with the licensed HIV-1/HIV-2 Peptide EIA and are no longer available for testing. Performance Results: Seroconversions Detection of Seroconversion Panel BCP 9017 HIV-1/HIV-2 PLUS O: 21 Days Earlier 14.0 100000 62790 60180 23590 25440 12.0 Copies/ml (log scale) 11990 10000 10.0 4516 2040 OD/Cutoff 1000 8.0 619 380 404 6.0 100 4.0 10 2.0 cutoff 0.0 1 3 7 10 14 17 21 24 28 32 35 Days HIV-1/HIV-2 PLUS O Peptide EIA Abbott HIVAb Abbott Prism Chiron HIV-1 RNA Performance Results: U.S. Blood Donors Results Obtained from Random Donors tested with Genetic Systems™ HIV-1/HIV-2 PLUS O EIA HIV-2 EIA Pos. by HIV-1 Number Initially Repeatedly Repeatedly Western blot Sample Type Tested Non-Reactive Reactive Reactive Reactive alone Serum 6103 6097 6 6 0 0 (Sites 1, 2) (100.00%) (99.90%) (0.10%) (0.10%) Plasma 5056 5044 12 6 0 0 (Sites 2, 3) (100.00%) (99.76%) (0.24%) (0.12%) TOTAL 11,159 11,141 18 12 0 0 (100.00%) (99.84%) (0.16%) (0.11%) Specificity 99.89% (95% confidence interval 99.83-99.96) Subtypes/Variants Tested by HIV-1 Western Blot 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 Group 0 HIV-2 HIV-2 LPC NC Neg PC G G G G A A A C A 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-1 seroconversion 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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