Fig. 3
Optimization of the newly developed sandwich-like FP screening assay. a Determination of an optimal working concentration of FP tracer in a FP screening assay. After addition of the FP tracer dilution at an indicated concentration in a black 384-well microplate, the mP value was measured. The dashed line in the presented figure represents the average of mP values. b The binding curve between FP tracer and avidin. A fixed amount of FP tracer was incubated with avidin at the indicated concentrations for 5 min at RT, and then the mP value was monitored. The maximal mP value (mPmax) indicated the arrival of the binding plateau, and an optimal amount of avidin was determined for screening. c, d The proteolytic reaction progression curves of Mpro at the indicated time. The mixture containing a fixed amount of FP tracer and increasing concentrations of Mpro was incubated at different time intervals. After quenching the proteolytic reaction by avidin, mP value was recorded to determine the EC50 and ΔmP values. e, f The proteolytic reaction progression curves of Mpro at the indicated temperature. The reaction mixture as described above was incubated for 20 min at the indicated temperature. After plotting the reaction curve according to the mP value, the EC50 and ΔmP values were compared separately to determine an optimal working temperature. g, h DMSO tolerance test in a FP screening assay. The proteolytic reaction curves of Mpro in the absence or presence of up to 10% DMSO were plotted. All experiments were independently repeated in triplicate. i Determination of an optimal concentration of Mpro at the optimized conditions in a FP screening assay. The reaction mixture was incubated for 20 min at RT, and then quenched by avidin. After measurement of mP value, the proteolytic reaction curve was plotted. The minimum mP value (mPmin) indicated an endpoint of proteolytic reaction, which can be regarded as an optimal working concentration of Mpro used in this FP screening assay