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.
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: 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
multiple HRP-Antigen Conjugate binding sites. multiple HRP-Antigen Conjugate binding sites.
HRPHRP
HRHRPP
HRPHRP HRHRPP
HRPHRP
HRHRPP
IgMIgM
5 HRP-Antigen Conjugates
2 HIV-1 M peptidesHIV-1 M rec. p24HIV-1 O peptide
HIV-2 peptide
Rec. gp160 (HIV-1 M)
Pep. env(HIV-2)
Pep env(HIV-1 O)
4 Plate Antigens
Rec. p24 (HIV-1 M)
Earlier IgM Detection…a Benefit of the Earlier IgM Detection…a Benefit of the Antibody Sandwich FormatAntibody Sandwich Format
Seroconversion Panel PRB 940Seroconversion Panel PRB 940
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
0 5 10 15 20 25
Time (Days)
Sig
nal
/cu
toff
HIV-1/HIV-2 Peptide EIA GS HIV-1/HIV-2 PLUS O Abbott HIVAb
Antibody Sandwich:Antibody Sandwich:HIV-1/HIV-2 HIV-1/HIV-2 PLUS O PLUS O EIAEIA
Abbott HIVAb HIV-1/HIV-2Abbott HIVAb HIV-1/HIV-2
Indirect Antibody:Indirect Antibody:HIV-1/HIV-2 Peptide EIAHIV-1/HIV-2 Peptide EIA
CutoffCutoff
~Day 8~Day 8 ~Day ~Day 1414
Kit ComponentsKit 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’)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
AIDS (N=313) 313
(100.00%)
313
(100.00%)
Known HIV-1 Positive,
U.S. (N=490)
490
(100.00%)
490
(100.00%)
Known HIV-1 Positive,
Non-U.S. (N=199*)
199
(100.00%)
199
(100.00%)
TOTAL: 1002
(100.00%)
1002
(100.00%)* Australia, New S. Wales (N=36)
Central African Republic (N=40)
Ghana (N=5)
Kenya (N=3)
Nigeria (N=46)
Sierra Leone (N=40)
Thailand (N=21)
Zimbabwe (N=8)
Performance Results:Performance Results:HIV Variant/Low Titer SamplesHIV 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™ HIV-1/HIV-2 PLUS O EIA
130/130 (100.0%)
Performance Results:Performance Results:HIV-2 SensitivityHIV-2 Sensitivity
Results Obtained from Known Positive HIV-2 Samples with
Genetic Systems™ HIV-1/HIV-2 PLUS O EIA
Number Tested Initially Reactive Repeatedly ReactivePos. by HIV-2 Western blot *
302 302
(100.00%)
302
(100.00%)
302
(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:Performance Results:HIV-1 Group O SensitivityHIV-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(100.00%)
76**(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: Performance Results: SeroconversionsSeroconversionsReactivity with HIV-1 on
50 Commercial Seroconversion Panels
HIV-1/HIV-2 PLUS O Equivalent
HIV-1/HIV-2 PLUS O More Sensitive
HIV-1/HIV-2 PLUS O Less Sensitive
vs. HIV-1/HIV-2 Peptide EIA
12/46*
(26%)
34/46*
(74%)
0
(0%)
vs. Abbott HIVAb HIV-1/HIV-2
35/50
(70%)
9/50
(18%)
6/50
(12%)
vs. Licensed
Western Blot
13/50
(26%)
37/50
(74%)
0
(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.
Detection of Seroconversion Panel BCP 9017
4516
380 404619
2040
1199023590
62790 60180
25440
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
3 7 10 14 17 21 24 28 32 35
Days
OD
/Cu
toff
1
10
100
1000
10000
100000
Co
pie
s/m
l (lo
g s
cale
)
HIV-1/HIV-2 PLUS O Peptide EIA Abbott HIVAb Abbott Prism Chiron HIV-1 RNA
Performance Results: Performance Results: SeroconversionsSeroconversions
HIV-1/HIV-2 HIV-1/HIV-2 PLUS O: 21 Days EarlierPLUS O: 21 Days Earlier
cutoffcutoff
Performance Results: Performance Results: U.S. Blood DonorsU.S. Blood Donors
Results Obtained from Random Donors tested with
Genetic Systems™ HIV-1/HIV-2 PLUS O EIA
Sample TypeNumber Tested Non-Reactive
Initially Reactive
Repeatedly Reactive
HIV-2 EIA Repeatedly
Reactive
Pos. by HIV-1 Western blot
alone
Serum
(Sites 1, 2)
6103
(100.00%)
6097
(99.90%)
6
(0.10%)
6
(0.10%)0 0
Plasma
(Sites 2, 3)
5056
(100.00%)
5044
(99.76%)
12
(0.24%)
6
(0.12%)0 0
TOTAL 11,159
(100.00%)
11,141
(99.84%)
18
(0.16%)
12
(0.11%)0 0
Specificity 99.89%Specificity 99.89%(95% confidence interval 99.83-99.96)(95% confidence interval 99.83-99.96)
Subtypes/Variants Tested by Subtypes/Variants Tested by HIV-1 Western BlotHIV-1 Western Blot
Worldwide HIV Panel WWRB302(members 1-13)
All HIV+ samples in the panel All HIV+ samples in the panel (N=28) exhibited (N=28) exhibited
A450/A630>3.000A450/A630>3.000 in testing in testing with with
Genetic Systems™ Genetic Systems™ HIV-1/HIV-2 HIV-1/HIV-2 PLUS OPLUS O EIA EIA
G
G
HIV
-2H
IV-2
GG AA Neg
Neg
HIV
-2H
IV-2
CC AAPC
P
C
LP
CL
PC
NC
NC
Gro
up 0
Gro
up 0
AA GG GG AA
SUMMARYSUMMARY 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.