Transcript GENSCREEN PLUS HIV Ag-Ab

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
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.)
Group
O?
HIV-2
HIV-1
B
DE
BCE
ABD
F
BO
G
C
B
HIV-1: ABDEFGHIJ…O
HIV-2: ABCDEFG
BCF
C
DH
ABCD
BE
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
IgG
2 HIV-1 M peptides
HIV-1 M rec. p24
HIV-1 O peptide
HIV-2 peptide
Rec. p24
(HIV-1 M)
Rec. gp160
(HIV-1 M)
Pep. env
(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.
5 HRP-Antigen
Conjugates
2 HIV-1 M peptides
HIV-1 M rec. p24
HIV-1 O peptide
HIV-2 peptide
HRP
HRP
HRP
HRP
HRP
HRP
Rec. p24
(HIV-1 M)
Rec. gp160
(HIV-1 M)
Pep. env
(HIV-2)
Pep env
(HIV-1 O)
4 Plate Antigens
IgM
Earlier IgM Detection…a Benefit of the
Antibody Sandwich Format
Seroconversion 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
14.0
Signal/cutoff
12.0
10.0
~Day 8
8.0
~Day 14
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 Dispensing
Sample
OD630 > 0.150

Conjugate Dispensing
Conjugate
OD630 > 0.100

TMB

Stopping solution
After incubation
of a positive
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
Genetic Systems™ HIV-1/HIV-2 PLUS O EIA
Group
Licensed
HIV-1/HIV-2 EIA
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:
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™
HIV-1/HIV-2 PLUS O EIA
130/130 (100.0%)
Performance Results:
HIV-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 Reactive
Pos. 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:
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.
HIV-1/HIV-2
Peptide EIA
vs.
Abbott HIVAb
HIV-1/HIV-2
12/46*
(26%)
34/46*
(74%)
0
(0%)
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.
Performance Results:
Seroconversions
Detection of Seroconversion Panel BCP 9017
HIV-1/HIV-2 PLUS O: 21 Days Earlier
14.0
60180
11990
10000
4516
10.0
2040
1000
8.0
619
404
380
6.0
100
4.0
10
Copies/ml (log scale)
25440
23590
12.0
OD/Cutoff
100000
62790
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
Sample Type
Number
Tested
Serum
(Sites 1, 2)
HIV-2 EIA Pos. by HIV-1
Repeatedly Western blot
Reactive
alone
Non-Reactive
Initially
Reactive
Repeatedly
Reactive
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%
(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
G
HIV-2
G
A
Neg
HIV-2
C
A
PC
LPC
NC
Group 0
A
G
G
A
Genetic Systems™
HIV-1/HIV-2 PLUS O EIA
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.