Nunc MaxiSorp plates (Thermo Fisher Scientific) were coated overnight at 4°C with 2 μg/ml human fibrinogen (Sigma), washed 3× with PBS containing 0.1% Tween 20 (wash buffer), and blocked for 1 h at room temperature (RT) with 200 μl/well casein (Thermo Fisher). Following 3 washes, the plates were incubated for 1 h at room temperature with a mix of 50 μl AviTag ClfA221–559 (2 μg/ml) and serial dilutions of anti-ClfA MAb or BiSAb in a 100-μl final volume of PBS. In some assays, anti-ClfA IgG1 of BisAb was saturated with a 10 M excess of alpha-toxin (6.6 mM). After washes, bound ClfA was detected using horseradish peroxidase (HRP)-conjugated streptavidin (1:20,000; GE Healthcare) and then 100 μl 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL). The reaction was stopped after 10 min with 100 μl 0.2 M H2SO4. The OD450s on plates were read on a spectrophotometer.
The percent inhibition of ClfA binding to fibrinogen was calculated with the following formula: 100 − [100 × (ODClfA+MAb/ODClfA,no MAb)].
The percent inhibition of ClfA binding to fibrinogen was calculated with the following formula: 100 − [100 × (ODClfA+MAb/ODClfA,no MAb)].