| Assay Method Information | |
| | HIV-1 RT Activity Assay |
| Description: | HIV-1 Reverse Transcriptase Activity was measured using the colorimetric photometric immunoassay kit provided by Roche. Estimation of RT activity is achieved by quantification of the DNA synthesized by RT in a known period of time. Incubation was performed for 2 h at 37°C. PolyA:oligodT RNA template:primer hybrid at a concentration of 2.2 μg/ml was incubated with 20 nMof purified recombinant HIV-1 reverse transcriptase in a 50 μL incubation mixture containing 50 mM Tris-HCl pH 7.8, 160 mM KCl, 11 mM MgCl2, 5.3 mM DTT, 0.5 mM EDTA, 0.3% Triton-X 100 and 10 μM dTTP (unless otherwise mentioned) in the presence of digoxigeninconjugated dUTP (dUTP-DIG) and biotin-conjugateddUTP (dUTP-biotin). In a following step, the synthesised biotin-DIG-labelled DNA was bound to the surface of streptavidin-coated ELISA-plate wells. After washing out the unbound material, an antibody to digoxigenin, conjugated to peroxidase (anti-DIG-POD), was added and bound to the digoxigenin-labelled nucleotides. After a washing step and addition of the appropriate substrate, peroxidase catalyzed the transformation of the substrate, ABTS, to a yellow coloured reaction product. 15 min after ABTS addition, the coloured product was measured using a microtitre plate (ELISA) reader. For the estimation of inhibitory action of the compounds, reverse transcriptase was incubated in the presence of the potential inhibitor for 45 min at room temperature before addition of its substrates (pre-incubation). |
| Affinity data for this assay | |
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