Date of Award

12-1-2022

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (vi, 57 pages) : illustrations (chiefly color)

Dissertation/Thesis Chair

Nicholas J. Mantis

Committee Members

Janice D. Pata, David Vance, Klemen Strle

Keywords

Borrelia burgdorferi, Lyme Disease, mAbs, Nanobodies, Single Domain Antibodies, VHHs, Lyme disease, Antigenic determinants, Molecular immunology

Subject Categories

Biochemistry | Biology | Microbiology

Abstract

Epitope mapping a protein that enables pathogenesis is crucial for the development of therapies and prophylactics that can inhibit the pathogen’s function and its transmission of disease. The lipoprotein OspA enables Lyme Disease etiologic pathogen, Borrelia burgdorferi, to inhabit the tick midgut until transmission occurs. Anti-OspA mAbs and their smaller VHH counterparts are highly specific and tailored to bind proteins such as OspA, reproducibly, at established binding sites or epitopes. Previous studies found an array of mAbs that successfully bound OspA and have already been used in epitope mapping. To our knowledge, this is the first use of VHHs in epitope mapping OspA. Libraries were created by immunizing alpacas with recombinant OspA and the VHHs identified using phage display. The VHHs were then cloned, expressed, and purified for characterization. Subsequently, with the use of ELISAs, Bio-Layer Interferometry, and HXMS surface mapping, we established regional, or Bin allocations of VHH epitopes on OspA. By studying the competition between mAbs and VHHs on OspA, we established where the VHHs bind along the length of the protein. Finally, we compared the different Bins of VHHs in their ability to capture OspA. This thesis focuses on epitope mapping the Borrelia burgdorferi protein OspA with the use of a purified and characterized set of novel VHHs.

Download

Share

COinS