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Contributions of Valence and Subclass to Antibody Binding the gamma DPGA Capsule of Bacillus anthracis
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Bacillus anthracis is a gram-positive spore-forming bacteria. The pathogen is surrounded by an anti-phagocytic polypeptide capsule that is entirely composed of poly- ?-D-glutamic acid (?DPGA). ?DPGA is an essential virulence factor of B. anthracis, and is a potential candidate for the development of vaccines and clinical diagnostics. Our laboratory has previously described the isolation of a panel of monoclonal antibodies (mAbs) that are reactive with ?DPGA. Here, we further examined mAb-binding as a function of mAb fractionation between two IgG subclasses, murine IgG3 and IgG1. These mAbs were truncated into Fab and F(ab’)2 fragments to study the overall contributions of mAb valence and subclass-specific constant regions (CH) to binding of capsular and synthetic ?DPGA. Checkerboard ELISA and SPR revealed that subclass-specific CH contributed to altered binding strengths between the two mAbs with respect to each other (IgG3>IgG1) and their fragments (IgG3 mAb>IgG3 F(ab)’2>IgG3 Fab and IgG1 F(ab)’2>IgG1 mAb>IgG1 Fab). DIC microscopy was used to visualize mAb interactions with the whole bacilli. The IgG3 mAb formed a “rim-type” capsule reaction with B. anthracis Ames cells, whereas binding by the IgG3 Fab resulted in a “spindle-type” pattern. Binding by all of the other antibody fragments, including the intact IgG1 mAb, formed a “puffy-type” capsular reaction. The unique binding capabilities of the IgG3 mAb, in comparison to its enzymatic fragments and intact IgG1, suggests that the antibody constant regions influence binding to ?DPGA independently of the variable region.