Date of Award

Spring 5-2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

School

Mathematics and Natural Sciences

Committee Chair

Sabine Heinhorst

Committee Chair Department

Chemistry and Biochemistry

Committee Member 2

Gordon Cannon

Committee Member 2 Department

Chemistry and Biochemistry

Committee Member 3

Faqing Huang

Committee Member 3 Department

Chemistry and Biochemistry

Committee Member 4

Bobby Middlebrooks

Committee Member 4 Department

Chemistry and Biochemistry

Committee Member 5

Robert Bateman Jr.

Committee Member 5 Department

Chemistry and Biochemistry

Abstract

The antigen-antibody interaction is known to be a high affinity and highly specific interaction that can readily be used for the detection and identification of biological and chemical agents. These studies were conducted to develop an efficient and cost-effective method of obtaining bacteria-specific antibody molecules for integration into a fielddeployable biosensor. Antigen-binding molecules were obtained both as full-length IgG molecules from a hybridoma cell line and as recombinant single-chain Fv (scFv) antibodies isolated from naïve and immunize libraries. Monoclonal and recombinant antibody systems were compared on the effectiveness of producing new, target-specific molecules; the efficiency of production and purification of these molecules; and the success/failure of integrating the molecules into the QCM biosensor for bacterial detection. Although selection of individual hybridoma cell lines was not conducted, monoclonal antibodies (mAbs) were obtained from an established cell line maintained in a hollow-fiber bioreactor. Recombinant antibodies, scFvs, capable of binding bacterial targets were isolated from libraries using a high-throughput phage display method of selection. Protocols were established for the purification of monoclonal antibodies from the bioreactor serum and scFvs from bacterial cell cultures to assess the efficiency of readily obtaining antibodies for integration into the biosensor. Finally, methods of immobilizing antibodies and scFvs to the gold electrode of a standard QCM crystal were explored to determine suitable procedures for consistent detection of target bacteria in aqueous samples to the lowest limit of detection.

ORCID ID

0000-0002-9436-3456

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