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.