Assay Method Information

Assay Name:  NS5B Polymerase Inhibition Assay (IC50) and HCV Replicon Assay (EC50)
Description:  Assays were performed in a 96-well streptavidin-coated Flash-Plate using enzyme, RNA substrate, and [alpha-33P]GTP/GTP with inhibitor concentration varied over a suitable range. The reaction was stopped by aspiration after 75 min incubation and the plate was washed. After washing and drying the plate, incorporated radioactivity was counted using a Microbeta scintillation counter. IC50 values were calculated relative to the uninhibited control and inhibition data were fitted to a 4-parameter IC50 equation. The estimated average standard deviation for IC50 data is 32% from the mean value. The exponentially growing HCV Huh-7/C24 replicon cells were seeded at 4500 cells/well in 96-well plates and 24 h later cells were treated with eight point half-log concentration of compound. After 72 h exposure, the cell monolayers were lysed, and each lysate was then analyzed by bDNA assay to determine EC50. The estimated average standard deviation for EC50 data is 56% from the mean value.
Affinity data for this assay
 

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