Application of HIV-1-green fluorescent protein (GFP) reporter viruses in neutralizing antibody assays.
We made reporter HIV-1 DNA constructs carrying green fluorescent protein (GFP) gene and exchangeable env of subtype E. The recombinant constructs were used to produce infectious reporter viruses, which induced infected cells to emit green fluorescent light and rendered them easily detectable at single cell level. Because the env in this construct can be easily exchanged, viruses with different antigenic epitopes can be made. We used these reporter viruses to set up a neutralizing antibody assay based on fluorescence reduction by flow cytometric measurement. The result of this new assay correlated with the standard infectivity reduction assay using primary isolates. Because this new assay is faster and much less costly than the standard assay using a p24 endpoint and can be performed in peripheral blood mononuclear cells (PBMC), it provides a useful tool for analysis of HIV-1 immune responses.
Auewarakul
P
P
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Paungcharoen
V
V
Louisirirotchanakul
S
S
Wasi
C
C
D023381:Endpoint Determination / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods; D017930:Genes, Reporter / Q000502:physiology; D005814:Genes, Viral / Q000502:physiology; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007202:Indicators and Reagents / Q000032:analysis*; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics*; D009500:Neutralization Tests / Q000379:methods; D012680:Sensitivity and Specificity; D013997:Time Factors; D017735:Virus Latency / Q000276:immunology
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins