Assay Method Information

Assay Name:  RT Assay
Description:  To a lysis buffer (20 μl), 4-6 ng recombinant HIV-1-RT was added in separate reaction tubes. Lysis buffer without HIV-1-RT was used as a negative control. Standards and test compound RT inhibitor (10 μg) were diluted with lysis buffer. This mixture was incubated for 1 h at 37 °C. For the number of micro plate modules to be used, enough foil bags were opened and micro plate modules were put into the frame in the correct orientation. The above samples were transferred to wells of microplate module. After addition it was covered with cover foil and incubated at 37 °C for 1 h. Solution was removed and rinsed five times with 250 μl of washing buffer per well for 30 s to each well. Then washing buffer was removed carefully. 200 μl of anti-DIG-POD working dilution was added to individual well and covered with cover foil and incubated at 37 °C for 1 h. Anti-DIG-POD working dilution solution was completely removed. Microplate module were washed five times for 30 s with washing buffer 250 μl and removed. 200 μl of ABTS substrate solution was added to individual well and incubated between 15 °C and 25 °C till (15−30 min) green color was obtained. Using ELISA reader absorbance of samples was measured at 405 nm (reference wavelength 490 nm).
Affinity data for this assay
 

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