HIV-1 reverse transcriptase activity assays based on monoclonal antibodies to native enzyme : Applications on studies of antiviral substances and immunological variation of reverse transcriptase

Abstract: Specific detection of HIV-1 reverse transcriptase (RT) activity in crude materials was enhanced using a capture assay for immunopurification and subsequent activity assay. The purification step was achieved by anti-HIV-1 RT monoclonal antibodies (Mabs) immobilized onto a solid carrier. The polymerization reaction was performed in the same tube or microtitre plate well and terminated by washing away surplus substrate, while the newly synthesized DNA remained bound to the immobilized enzyme. The amount of synthesized DNA could either be measured as amount of incorporated radioactive nucleotide or calorimetrically by using alkaline phosphatase conjugated Mab to the nucleotide analogue 5-bromodeoxyuridine 5´-triphosphate(BrdUTP).The capture assay proved useful for: 1) Determination of HIV-1 RT activity in crude cell extracts. 2) Analysis of chain terminating capacity of RT from AZT resistant virus. 3) Screening for hybridoma cell lines producing RT reactive Mabs. 4) Evaluation of the isozyme specificity of RT reactive Mabs.Monoclonal antibodies to native HIV-1 RT were produced by immunization of mice with RT and the iscom matrix forming adjuvant Quil A. Twelve clones produced Mabs which inhibited at least 50% of the RT-activity, and 34 clones produced Mabs which immobilized catalytically active enzyme. This Mab panel was used to characterize RTs from HIV-1 isolates of different subtype identity.Surprisingly large immunological differences were found between RTs from different subtypes as well as from homologous subtype isolates.One of the monoclonal antibodies, reactive to an epitope in the RNase H region, was used for measuring RT protein binding to template/primer in presence of non-nucleoside RT inhibitors. This assay for RT protein is a part of an assay system to evaluate the mode of action of RT inhibitors.