FIG 2.

Establishment of an ELISA to evaluate the anti-pUL89-C activity of compounds. (A) Time course for an ELISA using different concentrations of pUL89-C and 10 nM the substrate. (B) Representative graph for the determination of substrate K_m values. Experiment were performed two independent times, and mean K_m values plus standard deviations are shown. ΔOD₄₀₅, change in the optical density at 405 nm. (C) Inhibitory effects of raltegravir on pUL89-C activity analyzed by an ELISA. Results included stopping the reaction at 15 or 60 min. (D) Inhibitory effects of raltegravir on pUL89-C activity analyzed by an agarose gel assay. Shown is linearized pUC18 in the absence (lane 1) or presence (lane 2) of pUL89-C. Lanes 3 to 10 show a range of concentrations of raltegravir with pUL89-C. Numbers above bands correspond to the fold change compared with the control. (E) Correlation between the inhibitory response in a gel assay and the inhibitory response in an ELISA (r² = 0.96). Intensities of gel bands were determined by using ImageJ and used to calculate percent activity in a gel assay. These values were plotted along with percent activity in an ELISA. The best-fit line and r² values were determined by using linear regression analysis with GraphPad Prism software.