Colorimetric capture assay for human-immunodeficiency-virus-I reverse transcriptase activity

The development of a colorimetric capture assay for HIV-1 reverse transcrip tase (RT) activity is described. This assay consisted of three basic steps: enzyme purification, RT reaction and product detection, which were all per formed in the same microtitre plate. Mouse monoclonal anti-RT antibodies of subclass G2a were bound by polyclonal goat anti-(mouse IgG2a) immobilized in the wells of a microtitre plate. The monoclonal antibodies (mAbs) were s elected for their ability to bind HIV-1 RT without hampering the polymerase activity. The assay system first involved the RT's adherence to the immobi lized mAbs, Non-specific enzymes and other impurities were removed by a sim ple wash, after which an RT reaction mixture containing BrdUTP as nucleotid e substrate was added. After the RT reaction substrate and product had been separated by washing of the plate, the amount of BrdUMP-DNA in the wells w as finally detected with alkaline-phosphatase-conjugated mouse anti-BrdU an tibodies of subclass IgGI. The background signal in this system was similar to the signals obtained with control wells coated with BSA only. A detecti on limit of 1,2 mu-units of RT activity, corresponding to 0.3 pg of RT prot ein, was obtained for the capture assay when applying colorimetric product detection, The assay detected RTs from HIV-1 subtypes A and B and one of th e two D type isolates tested. None of the five non-HIV-1 RTs tested was fou nd positive. At least 50 mu l of human serum or plasma per sample could be included in the capture assay without adverse effects on the recovery of th e RT activity.