A METHOD FOR COMBINED IMMUNOAFFINITY PURIFICATION AND ASSAY OF HIV-1 REVERSE-TRANSCRIPTASE ACTIVITY USEFUL FOR CRUDE SAMPLES

Detection of human immunodeficiency virus type 1 (HIV-1) reverse trans criptase (RT) activity in crude specimens was greatly enhanced using a novel capture RT assay, Eighteen different monoclonal antibodies (Mab s) raised against purified HIV-1 RT were tested for their ability to b ind to HIV-1 RT without affecting its activity. The anti-HIV-1 RT Mabs were immobilized on plastic macrobeads and used as solid carriers in the capture RT assay. The assay system first involved RT's adherence t o the immobilized Mabs, Nonspecific enzymes and other impurities were removed by a simple wash after which the RT reaction mixture was added , Substrate and product were finally separated by a wash of the beads, Practically all radioactivity incorporated into DNA (>98%) was recove red on the bead, The Michaelis-Menten constants and the saturation vel ocity values for the nucleotide substrate were similar for free and im mobilized RT. The reaction mechanism for the immobilized RT is discuss ed, When comparing the function of this assay with more conventional s oluble RT assays for samples consisting of recombinant HIV-1 RT mixed with an extract of peripheral blood lymphocytes (PBL), an almost 100-f old higher sensitivity was found, The capture RT assay had the capacit y to recover approximately 80% of the RT activity added to an extract of 1 X 10(7) PBL cells/ml. A strong correlation (r = 0.947) between th e results obtained with this assay and a HIV-1 p24 enzyme-linked immun osorbent assay was found, when samples from a collection of 16 HIV str ains propagated in cell culture were analyzed. (C) 1996 Academic Press , Inc.