TZM-bl Assay Description
Antibody-mediated neutralization of HIV, SIV and SHIV is measured as a function of reductions in Tat-regulated Firefly luciferase (Luc) reporter gene expression after a single round of infection in TZM-bl cells. This assay was first developed by Dr. George Shaw and colleagues at the University of Alabama, Birmingham and was later optimized and validated at Duke University. TZM-bl (also known as JC53BL-13) is a CXCR4-positive HeLa cell clone that was engineered to express CD4 and CCR5 (4). The cells were further engineered to contain integrated reporter genes for firefly luciferase and Escherichia coli b-galactosidase under control of an HIV long-terminal repeat sequence (5). TZM-bl cells are permissive to infection by a wide variety of HIV, SIV and SHIV strains, including primary HIV isolates and molecularly cloned Env-pseudotyped viruses. Assay stocks of Env-pseudotyped viruses are produced in 293T/17 cells by co-transfection with an Env expression plasmid and a second plasmid expressing the entire HIV-1 genome except Env. Only the latter env-minus plasmid replicates in 293T/17 cells; this plasmid is packaged by the pseudovirions for delivery of the tat gene to TZM-bl cells. Thus, co-transfection generates pseudovirus particles that are infectious but are unable to produce infectious progeny virions for subsequent rounds of infection. Reporter gene expression is induced in trans by viral Tat protein soon after single cycle infection. DEAE dextran is added to the medium to enhance infection and has been found to have no obvious effects on NAb activity. Luciferase activity is quantified as relative luminescence units (RLU) and is directly proportional to the number of infectious virus particles present in the initial inoculum over a wide range of values. Neutralization titers are the dilution at which RLU are reduced by 50% compared to virus control wells after subtraction of background RLUs. The assay is performed in 96-well plates for high throughput capacity, and utilizes well-characterized reference strains for uniformity across studies (6-9).