1. Vironostika® HIV-1 Plus O Microelisa System
Main Features
Assay configuration and procedures for S/P and DBS
Solid Phase Antigens
Comparison to the current licensed Vironostika® HIV-1

2. Main Features
Vironostika® HIV-1 Plus O Mircoelisa System is an ELISA for the qualitative detection of antibodies to HIV-1 including Group O, in human specimens collected as serum, plasma, or dried blood spots on filter paper.
It is not intended for use in screening blood donors.
Solid phase contains HIV-1 viral lysate, viral envelope and Group O peptide.
The system contains Sample Addition Monitoring, Color Coded Reagents, liquid controls and Substrate, and an improved ease of use.
The assay is designed to improve the overall sensitivity while maintaining the specificity as compared to the current licensed Vironostika® HIV-1.

3. Kit Components

4. Overview of Vironostika® HIV-1 Plus O Microelisa System
WASH
WASH
WASH
WASH
STOP
STOP
Solid phase:
Solid phase:
Anti -
HIV in Sample
Goat anti -
human Ig
/HRP
conjugate
Chromogenic
substrate (ABTS)
NaF
substrate (ABTS)
Viral Lysate
Viral env
Group O Peptide
Sample diluent contains sample addition monitor
Incubation 37°C
One-step preparation
Incubation 37°C
Ready to use format
Incubation 15-30°C
Read (405nm)

5. Assay Procedures for Serum/Plasma (S/P) and Dried Blood Spots (DBS)
120 min, 37°C
60 min, 37°C
S/P
10µl SAMPLE
200µl DILSIM III
(1:21)
Conj
150µl
Wash
READ
At 405 nm
min, RT
DBS
Elute with 150µl
of DBS Elution Medium
25µl ELUTE
125µl DILSIM III
(1:6)
[S]
STOP

6. Sample Addition Monitoring

7. Solid Phase Antigen HIV-1 Viral Lysate Prepared from H9/HIV-1IIIB
Same viral lysate as in the current Vironostika HIV-1
Group O peptide
RRETLLQNQQLLSLWGCKGKLVCYT
Synthetic peptide of 23 amino acids with Arg-Arg added to the N-terminal
The sequence derived from ANT70 gp41 regions (Journal of Virology, 1990, )
Conjugated to BSA for plate coating
Native HIV-1 gp160

8. Development of Native HIV-1 gp160
Advanced Bioscience Laboratories developed a cell culture system that secreted gp160 which contained the gp41 moiety (minus a small truncation at the C-terminus).
The native gp160 is highly soluble and exists with carbohydrate moiety.
Kalyanaraman et al., AIDS Res. Hum. Retroviruses, 1990;
Kalyanaraman et al., Virology, 1990.
Shown to be an excellent diagnostic antigen.
Nair et al., J. Cln. Microbiol., 1994

9. Immunodominant Domains in HIV-1 env Proteins

10. Serum EIA Reactivity of HIV-1 gp16010
100
1000
False Positive
Very Weak
Positive
Medium Positive 1 10
100
1000
False Postive
Very Weak
Positive
Medium Positive
1.2
1.2
1.0
1.0
0.8
0.8
450
450
0.6
0.6 A A
0.4
0.4
0.2
0.2
0.0
0.0
gp160 (
gp160 ( ng ng
/well)
/well)

11. The Vironostika® HIV-1 Plus O Trial Summary
18 geographically diverse sites participated.
4 kit lots evaluated.
36,000+ tests performed (Serum/Plasma + DBS).
Studies included:
Reproducibility
Sensitivity
Known HIV-1 positive samples
High risk populations
Seroconversion panels
Dilution panels
Specificity
Low risk populations
Samples containing potentially interfering substances

12. Specificity in Low Specificity in Low - - Risk Populations (n=6019)
1000
2000
3000
4000
5000
< . 1 - 9 2 3 4 5 6 7 8
> =
FREQUENCY
SIGNAL TO CUTOFF RATIO
HIV
1 Plus O
VirHIV

13. Known HIV-1 Specimens DBS HIV-1 Clade A – 100% (10/10) Serum/Plasma
HIV-1 Clade B – 100% (12/12)
HIV-1 Clade B/D – 100% (1/1)
HIV-1 Clade C – 100% (10/10)
HIV-1 Clade C/E – 100% (1/1)
HIV-1 Clade D – 100% (10/10)
HIV-1 Clade E – 100% (7/7)
HIV-1 Clade E/A – 100% (3/3)
HIV-1 Clade E/C – 100% (1/1)
HIV-1 Clade E/F – 100% (1/1)
HIV-1 Clade F – 100% (10/10)
HIV-1 Clade G – 100% (4/4)
HIV-1 Clade H – 100% (2/2)
HIV-2 – 100% (20/20)
HIV-O – 100% (11/11)
Serum/Plasma
HIV-1 Clade A – 100% (10/10)
HIV-1 Clade B – 100% (12/12)
HIV-1 Clade B/D – 100% (1/1)
HIV-1 Clade C – 100% (10/10)
HIV-1 Clade C/E – 100% (1/1)
HIV-1 Clade D – 100% (10/10)
HIV-1 Clade E – 100% (7/7)
HIV-1 Clade E/A – 100% (3/3)
HIV-1 Clade E/C – 100% (1/1)
HIV-1 Clade E/F – 100% (1/1)
HIV-1 Clade F – 100% (10/10)
HIV-1 Clade G – 100% (4/4)
HIV-1 Clade H – 100% (2/2)
HIV-2 – 100% (20/20)
HIV-O – 100% (11/11)

14. Sensitivity
All serum/plasma specimens and dried blood spot specimens were repeatedly reactive with Vironostika® HIV-1 Plus O Mircoelisa System. Therefore, the sensitivity for both specimen types in this study was 100% (95% CI: – 100%).

15. High-Risk Populations
Prison Inmates, STD, Hosp ER Pts, and HIV Outreach Clinic Pts
The specificity of the Vironostika® HIV-1 Plus O assay in this study of high-risk populations was calculated to be 1,392/1,398 = 99.57% (95% CI = 99.07% %).
Specimen Type
Population
Number of
specimens
Initially
Reactive
Repeatedly
Western Blot
Positive 1
251 16 14 8 2
513 13 3
500 73 68 4
250 28 27
Serum or
Plasma
Total
1,514
130
122
116
N/A
Dried Blood
Spots
750 95

16. Seroconversion Panel Testing
Panel ID
Sample Collection Days
Vironostika HIV-1 Plus O
Comparative Test1
924
8, 10, 26, 33, 35, 40 33 40
927
0, 28, 33, 35, 40
931
9, 15, 28, 33, 35, 42 28
932
0, 3, 13, 27, 34, 50, 78, 163, 194 34
NR2
940
0, 7, 11, 15, 18, 22, 25, 29 15 22
071
1, 3, 17, 22, 28 17
111
1, 2, 8, 16, 20, 22, 27 8 16
241
1, 7, 9, 15, 17, 22, 24
321
1, 8, 12, 15, 21 21
341
1, 7, 10, 21, 23, 28
351
1, 8, 11, 15 NR
361
1, 3, 9, 11, 16, 18 18
1The licensed Vironostika HIV-1 Microelisa System
2None of the specimens in this panel was reactive

17. Reactivity of H&L vs  Specific Conjugate
Seroconversion Panels
0.5 1
1.5 2
2.5 3
3.5 4
4.5
PRB 924-4
PRB 924-5
PRB 924-6
PRB 924-7
PRB 924-8
PRB 940-2
PRB 940-3
PRB 940-4
PRB 940-5
SV0241-C
SV0241-D
SV0241-E
SV0341-C
SV0341-D
SV0341-E
SV0341-F
SV0321-A
SV0321-B
SV0321-C
SV0321-D
SV0321-E
S/CO
H&L
 Specific

18. Dilution Panel Testing
First dilutions with non-reactive test results for each dilution series.
Sample ID
Clade Type
SERUM
Vironostika HIV-1
Plus O System
Comparative Test 1
5805
HIV-1 Clade B
1:1,920
1:240
H629
1:32,000
1:8,000
MD-O
HIV-1 Group O
1:25,600
1:400
302-1
1:1,600
NR 2
301-42
HIV-1 Clade A
1:12,800
302-18
HIV-1 Clade C
1:6,000
1:1,500
301-24
HIV-1 Clade D
1:48,000
1:3,000
302-23
HIV-1 Clade E
1:24,000
302-28
HIV-1 Clade F
302-17
1:4,000
1:250
( 1 ) The licensed Vironostika HIV-1 Microelisa System
( 2 ) Non-reactive

19. Detection of HIV-1 Group O Specimens
Vironostika HIV -
1 Plus O
System
Comparative Test 1
Specimen ID
Sample
Dilution
Mean SCR 2
1:100
6.1
2.0
1:50
4.0
0.4 3
7.4
1.6 4
5.6
1.1 5
7.7 6
7.2 7
5.3
0.5 8
7.6
2.2 9
5.9 10
1:5
3.9 11
4.5
Total Repeatedly Reactive
11/11
7/11
The licensed Vironostika HIV
1 Microelisa System
SCR = Signal to Cutoff Ratio

20. Detection of HIV-2 Positive Specimens
Specimen ID
Sample Dilution
Mean SCR* 1
1:10
3.3 2
5.8 3
1:500
1.5 4
4.3 5
1:50
1.7 6
5.5 7
2.5 8
5.0 9
1:100
3.7 10
1:1,000
1.8 11
1:200
1.3 12
1.4 13
1.9 14 15
4.9 16
3.6 17 18
2.7 19
2.0 20
*Specimens with SCR equal to or greater than 1.0 are considered
reactive with the test.

21. Conclusions
Vironostika® HIV-1 Plus O is intended for use as an aid in diagnosis of infection with HIV-1 and not for use in screening blood donors.
This assay uses three solid phase antigens; inactivated, purified HIV-1 viral lysate proteins, a purified viral envelope protein (native gp160), and a synthetic group O peptide.
Assay procedure similar to the current licensed Vironostika® HIV with added features to improve ease of use.
The assay design sought to improve overall sensitivity to Group M and Group O while maintaining the specificity compared to the current licensed Vironostika® HIV-1.
Detection of HIV-1 Group M antibodies was demonstrated with subtypes A, B, B/D, C, C/E, D, E, E/A, E/C, E/F, F, G and H.
The assay exhibited higher analytical sensitivity with dilutional panels from clinical specimens for both HIV-1 Group M and HIV-1 Group O specimens compared to the current licensed Vironostika® HIV-1.