HIV-1/HIV-2 PLUS O To detect HIV Antibody Groups M & O Genetic Systems Genetic Systems ™ ™ HIV-1/HIV-2 HIV-1/HIV-2 PLUS O PLUS O EIA 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
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. - PowerPoint PPT Presentation
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HIV
-1/H
IV-2
PL
US
O
To detect HIV Antibody Groups M & O
Genetic SystemsGenetic Systems™™ HIV-1/HIV-2 HIV-1/HIV-2 PLUS OPLUS O EIA 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 Subtypes and VariantsHIV Subtypes and Variants
B
ABDBCE
B
C
C
BCF
F
ABCD
DE
Group O ?
BEHIV-1:
ABDEFGHIJ…O
HIV-2: ABCDEFG
DH
G
B
O
HIV-1 Subtype B is predominant in the U.S., but non-B subtypes are found in2% of HIV-Positive U.S. blood donors, and increasing.
(Delwart et al., AIDS Research and Human Retroviruses 19:1065-1070, 2003.)
HIV-2HIV-2HIV-1HIV-1
HIV Subtypes and VariantsHIV Subtypes and VariantsMost Problematic for Antibody Detection
HIV-2: HIV-2 testing mandated for blood screening June 1, 1992.
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: Principle of the Test: Direct Antibody SandwichDirect Antibody Sandwich
Immunoglobulins from the sample bind simultaneously to Immunoglobulins from the sample bind simultaneously to antigens (4 total) on the microplate and to similar antigens (4 total) on the microplate and to similar
HRP-conjugated antigens (5 total) in solution.HRP-conjugated antigens (5 total) in solution.
HRPHRP
IgGIgG
Rec. gp160 (HIV-1 M)
Pep. env(HIV-2)
Pep env(HIV-1 O)
4 Plate Antigens
Rec. p24 (HIV-1 M)
5 HRP-Antigen Conjugates
2 HIV-1 M peptidesHIV-1 M rec. p24HIV-1 O peptide
HIV-2 peptide
Principle of the Test: Principle of the Test: Direct Antibody SandwichDirect Antibody Sandwich
Binding of IgM to the microplate is stabilized by Binding of IgM to the microplate is stabilized by multiple attachments, and the multiple attachments, and the signal is amplifiedsignal is amplified by by
Procedure (60’ 30’ 30’)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 MonitoringProcedure Monitoring
Sample Dispensing
Conjugate Dispensing
T M B
Stopping solution
Before After
After incubationof a positive
sample
Sample Sample ODOD630630 >> 0.150 0.150
Conjugate Conjugate ODOD630630 >> 0.100 0.100
Optional Optional VerificationVerification
Quality Control:Quality Control:Validation of ResultsValidation 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 >0.700C2 HIV-2 Positive Control (1 well)
A450 >0.700C3 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:Performance Results:HIV-1 Group M SensitivityHIV-1 Group M Sensitivity
Reactivity in HIV-1 Known Positive SamplesResults Obtained with
Genetic Systems™ HIV-1/HIV-2 PLUS O EIA
Licensed
HIV-1/HIV-2 EIA
Group Number Repeatedly Reactive Number Repeatedly Reactive