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Antibody interactions with the capsular polysaccharide of Burkholderia pseudomallei
AuthorDillon, Michael James
AdvisorAuCoin, David P
Biochemistry and Molecular Biology
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<italic>Burkholderia pseudomallei</italic> is an important human pathogen that causes melioidosis. Infection is highly lethal and notoriously difficult to diagnose and treat. As such, it has tremendous bioterror potential and has been classified as a Tier 1 select agent by the Centers for Disease Control and the Department of Health & Human Services. One reason that <italic>B. pseudomallei</italic> is a successful pathogen is that it is surrounded by a high molecular weight capsular polysaccharide (CPS) comprised of <italic>manno</italic>heptopyranose residues. CPS inhibits complement deposition, prevents phagocytosis, and greatly enhances virulence. Previous studies have indicated that antibodies targeting CPS have high therapeutic value and can be used to diagnose <italic>B. pseudomallei</italic> infection.The present work describes the development and characterization of 15 monoclonal antibodies (mAbs) in an effort to further the understanding of how antibodies interact with <italic>B. pseudomallei</italic> CPS. We have generated two complete Immunoglobulin G (IgG) subclass families; subclass families are antibodies that have identical variable regions, but different constant regions, and thus different effector functions. We have determined that some of these mAbs are protective in a murine model of pulmonary melioidosis in a subclass-independent manner. In this study, protection appears to be a function of mAb binding affinity. Additionally, we determined that non-IgG<sub>3</sub> mAbs are best for diagnosing active infection. Isolating a high affinity IgG<sub>3</sub> and generating a subclass-switch family yielded mAbs with low affinities that did not perform well in a diagnostic test format. Thus, immunization strategies should focus on eliciting alternative IgG immune responses. Using this information, we have updated a prototype Active Melioidosis Detect<super>TM</super> Lateral Flow Immunoassay (AMD LFI) by replacing the original IgG<sub>3</sub> mAb with a high affinity IgG<sub>1</sub> mAb. This updated AMD LFI has increased sensitivity, is highly specific, and rapid; it can detect <italic>B. pseudomallei</italic> CPS in multiple sample types in 15 minutes or less.