Burkholderia pseudomallei 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 B. pseudomallei is a successful pathogen is that it is surrounded by a high molecular weight capsular polysaccharide (CPS) comprised of mannoheptopyranose 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 B. pseudomallei 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 B. pseudomallei 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 3 mAbs are best for diagnosing active infection. Isolating a high affinity IgG3 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™ Lateral Flow Immunoassay (AMD LFI) by replacing the original IgG3 mAb with a high affinity IgG1 mAb. This updated AMD LFI has increased sensitivity, is highly specific, and rapid; it can detect B. pseudomallei CPS in multiple sample types in 15 minutes or less.
|Advisor:||AuCoin, David P.|
|Commitee:||Kozel, Thomas R., Pari, Gregory S., Schegg, Kathleen M., Verma, Subash C.|
|School:||University of Nevada, Reno|
|Department:||Cell and Molecular Biology|
|School Location:||United States -- Nevada|
|Source:||DAI-B 76/06(E), Dissertation Abstracts International|
|Subjects:||Microbiology, Medicine, Immunology|
|Keywords:||Burkholderia pseudomallei, Capsular polysaccharide, Immunotherapeutic, Lateral flow immunoassay, Monoclonal antibody, Subclass switch|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be