Contributions of Valence and Subclass to Antibody Binding the gamma DPGA Capsule of Bacillus anthracis
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Authors
Eckroth, Lauren
Issue Date
2012
Type
Thesis
Language
en_US
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Abstract
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.
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In Copyright(All Rights Reserved)