Presentation on theme: "Vironostika® HIV-1 Plus O Microelisa System"— Presentation transcript:
1 Vironostika® HIV-1 Plus O Microelisa System Main FeaturesAssay configuration and procedures for S/P and DBSSolid Phase AntigensComparison to the current licensed Vironostika® HIV-1
2 Main FeaturesVironostika® 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.
4 Overview of Vironostika® HIV-1 Plus O Microelisa System WASHWASHWASHWASHSTOPSTOPSolid phase:Solid phase:Anti-HIV in SampleGoat anti-humanIg/HRPconjugateChromogenicsubstrate (ABTS)NaFsubstrate (ABTS)Viral LysateViral envGroup O PeptideSample diluent contains sample addition monitorIncubation 37°COne-step preparationIncubation 37°CReady to use formatIncubation 15-30°CRead (405nm)
5 Assay Procedures for Serum/Plasma (S/P) and Dried Blood Spots (DBS) 120 min, 37°C60 min, 37°CS/P10µl SAMPLE200µl DILSIM III(1:21)Conj150µlWashREADAt 405 nmmin, RTDBSElute with 150µlof DBS Elution Medium25µl ELUTE125µl DILSIM III(1:6)[S]STOP
7 Solid Phase Antigen HIV-1 Viral Lysate Prepared from H9/HIV-1IIIB Same viral lysate as in the current Vironostika HIV-1Group O peptideRRETLLQNQQLLSLWGCKGKLVCYTSynthetic peptide of 23 amino acids with Arg-Arg added to the N-terminalThe sequence derived from ANT70 gp41 regions (Journal of Virology, 1990, )Conjugated to BSA for plate coatingNative 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
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:ReproducibilitySensitivityKnown HIV-1 positive samplesHigh risk populationsSeroconversion panelsDilution panelsSpecificityLow risk populationsSamples containing potentially interfering substances
12 Specificity in Low Specificity in Low - - Risk Populations (n=6019) 10002000300040005000<.1-92345678>=FREQUENCYSIGNAL TO CUTOFF RATIOHIV1 Plus OVirHIV
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/PlasmaHIV-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 SensitivityAll 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 PtsThe 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 TypePopulationNumber ofspecimensInitiallyReactiveRepeatedlyWestern BlotPositive1251161482513133500736842502827Serum orPlasmaTotal1,514130122116N/ADried BloodSpots75095
17 Reactivity of H&L vs Specific Conjugate Seroconversion Panels0.511.522.533.544.5PRB 924-4PRB 924-5PRB 924-6PRB 924-7PRB 924-8PRB 940-2PRB 940-3PRB 940-4PRB 940-5SV0241-CSV0241-DSV0241-ESV0341-CSV0341-DSV0341-ESV0341-FSV0321-ASV0321-BSV0321-CSV0321-DSV0321-ES/COH&L Specific
18 Dilution Panel Testing First dilutions with non-reactive test results for each dilution series.Sample IDClade TypeSERUMVironostika HIV-1Plus O SystemComparative Test 15805HIV-1 Clade B1:1,9201:240H6291:32,0001:8,000MD-OHIV-1 Group O1:25,6001:400302-11:1,600NR 2301-42HIV-1 Clade A1:12,800302-18HIV-1 Clade C1:6,0001:1,500301-24HIV-1 Clade D1:48,0001:3,000302-23HIV-1 Clade E1:24,000302-28HIV-1 Clade F302-171:4,0001:250( 1 ) The licensed Vironostika HIV-1 Microelisa System( 2 ) Non-reactive
19 Detection of HIV-1 Group O Specimens Vironostika HIV-1 Plus OSystemComparative Test1Specimen IDSampleDilutionMean SCR21:1006.12.01:504.00.437.41.618.104.22.1687.275.30.587.62.295.9101:53.9114.5Total Repeatedly Reactive11/117/11The licensed Vironostika HIV1 Microelisa SystemSCR = Signal to Cutoff Ratio
20 Detection of HIV-2 Positive Specimens Specimen IDSample DilutionMean SCR*11:103.325.831:5001.544.351:501.765.572.585.091:1003.7101:1,0001.8111:2001.3121.4131.914154.9163.617182.7192.020*Specimens with SCR equal to or greater than 1.0 are consideredreactive with the test.
21 ConclusionsVironostika® 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.