The presence of HIV-1 is determined by detection of p24 viral antigen in an antigen capture assay or by detection of reverse transcriptase activity. The p24 assay is about 2000 fold more sensitive than previous RT assays. We have devised a DNA capture system which permits quantitative capture of RT DNA from 30 mu1 of the reaction instead of the 5 mu1 usually captured on DEAE paper. Overall the current RT assay is about 5 fold as sensitive as the p24 assay. This RT assay is a simple assay, is reproducible, and is more reliable and much less expensive than the p24 assay. Since RT activity is directly proportional to time of reaction, to TTP concentration in the range used and to virus concentration, the sensitivity desired can be selected and results from different laboratories can be equated. A method for successfully cloning 12D7 cells in Terasaki microwell plates with an efficiency of approximately 10% was devised. Individual cloned cell lines of 12D7 cells transfected with two different triple mutant HIV-1 constructs have been obtained. Both constructs have a gag-pol frame shift and an env deletion in addition to one of two RT mutations which render the transcribed RT RNase H negative. Tests are being conducted to determine if, as a result of infection of these transfected cells by competent HIV-1, a transdominant packaging of defective RT occurs.
