Effect of mAbs, directed against the repeat region of FbsA, on the interaction of S agalactiae 6313 or FbsA with human Fbg. (A) Concentration-dependent binding of the anti-FbsA mAbs 5H2 (▪) and 2B1 (⋄) to FbsA. Microtiter wells were coated overnight with 500 ng FbsA in 50 mM sodium carbonate, pH 9.5. The wells were treated for 1 hour at 22°C with 200 μL PBS containing 2% bovine serum albumin (BSA) and washed 5 times with PBS with 0.1% vol/vol Tween 20 (PBST). The plates were incubated with increasing amounts of mAbs 5H2 or 2B1 and the binding of each antibody was detected by incubating the wells with a 1:1000 dilution of rabbit antimouse peroxidase-conjugated polyclonal antibody. After washing, binding was quantitated using the substrate o-phenylenediamine dihydrochloride and measuring the absorbance at 490 nm. (B) The effect of mAbs 5H2 or 2B1 on the binding of fibrinogen to immobilized FbsA is reported. FbsA was immobilized onto microtiter wells (500 ng/well) and incubated with biotin-labeled human Fbg in the presence of the indicated concentrations of mAbs 5H2 or 2B1. After washing, binding of the ligand was quantitated by the addition of avidin-conjugated peroxidase and development with o-phenylenediamine. (C) To analyze the effect of the anti-FbsA mAbs on S agalactiae 6313 attachment to Fbg, microtiter wells were coated with Fbg (1 μg/well). Bacteria (5 × 10⁷/well) were preincubated with the indicated amounts of mAbs 5H2 or 2B1 and transferred to the Fbg-coated wells and the suspension was incubated for 2 hours. After extensive washes, 1 μg rabbit anti-GBS IgG was added to the wells, followed by an incubation for 90 minutes. Adherent cells were detected by peroxidase-conjugated goat anti–rabbit IgG and the plates were developed with o-phenylenediamine. All the data are expressed as percentage of bound Fbg or attached bacteria in the absence of mAbs. The bars show SDs of triplicate samples.