Publication Date

1-1-2002

Document Type

Dissertation/Thesis

First Advisor

Mitchell, John L. A.

Degree Name

B.S. (Bachelor of Science)

Legacy Department

Department of Biological Sciences

Abstract

Antibodies are proteins of the immune system that exhibit exquisite specificity to their binding partners, called antigens. Binding occurs at the hypervariable region (Fv), which is capable of discriminating even enantiomeric molecules. By targeting on the DNA sequences that code for Fv and expressing it as a protein, the binding properties of an antibody can be assessed. In this project, the heavy and light chains of the Fv region (Vh and V[_) of an anti-amino acid antibody (anti-phenylalanine 67.36) were linked using a short nucleotide linker and four primers designed from previously determined sequences of Vh and V|_. The resulting V|-H¡nker-V|_(HIL) construct - a single chain variable fragment (67.36 HIL scFv) - was inserted into a TOPO cloning vector and transformed into E. coli BL 21 expression cells. 1 L cultures were grown at 37°, 30° and 27 °C. Cells were collected, resuspended and lysed to release the protein of interest. Isolation and purification was performed using a metal (cobalt) affinity chromatography resin. Characterization by SDS-PAGE indicated a correlation between incubation temperature and extent of expression. Noncompetitive ELISA tests revealed a functional 67.36 HIL scFv. However, further tests e.g., competitive ELISA, must be carried out to confirm an enantiomeric preference.

Comments

Includes bibliographical references.

Extent

19 unnumbered pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type

Text

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