A Method for Combined Immunoaffinity Purification and Assay of HIV-1 Reverse Transcriptase Activity Useful for Crude Samples

• Published in: Analytical biochemistry 1996-03, Vol.235 (2), p.141-152
• Main Authors: Lennerstrand, J., Rytting, A.-S., Örvell, C., Gronowitz, J.S., Källander, C.F.R.
• Format: Article
• Language: English
• Subjects: AIDS/HIV; Antibodies, Monoclonal; Cells, Cultured; Chromatography, Affinity - methods; Enzymes, Immobilized; HIV Reverse Transcriptase - analysis; HIV-1 - enzymology; Humans; Sensitivity and Specificity; Solubility; Templates, Genetic
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1006/abio.1996.0106

Detection of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity in crude specimens was greatly enhanced using a novel capture RT assay. Eighteen different monoclonal antibodies (Mabs) raised against purified HIV-1 RT were tested for their ability to bind 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 to 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 recovered on the bead. The Michaelis–Menten constants and the saturation velocity values for the nucleotide substrate were similar for free and immobilized RT. The reaction mechanism for the immobilized RT is discussed. When comparing the function of this assay with more conventional soluble RT assays for samples consisting of recombinant HIV-1 RT mixed with an extract of peripheral blood lymphocytes (PBL), an almost 100-fold higher sensitivity was found. The capture RT assay had the capacity to recover approximately 80% of the RT activity added to an extract of 1 × 107PBL cells/ml. A strong correlation (r= 0.947) between the results obtained with this assay and a HIV-1 p24 enzyme-linked immunosorbent assay was found, when samples from a collection of 16 HIV strains propagated in cell culture were analyzed.