Document Type

Thesis - Open Access

Award Date

1987

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

David A. Benfield

Abstract

The purpose of this study was to develop and characterize a panel of monoclonal ·antibodies specific for epitopes on the Nebraska strain of bovine coronavirus (BCV). The hybridomas were prepared by fusion of spleen cells from BALB/c mice hyperimmunized with BCV and from SP2/0Ml, NS-1, and P3-X63-Ag8.653 myeloma cell lines. Seven hybridoma clones were derived from the fusions. The immunoglobulin isotype IgGl was the most common antibody produced by the hybridomas. BALB/c mice were inoculated with hybridorna cells to produce ascitic fluids. Assays were performed on the ascitic fluids to determine which biological functions of the bovine coronavirus virions were identified by each of the seven monoclonal antibodies. One of the seven monoclonal antibodies neutralized BCV, one inhibited hemagglutination, while the remaining five reacted on indirect immunofluorescence and enzyme-linked immunosorbent assays. All monoclonal antibodies reacted with BCV on the indirect immunofluorescence assay and titers ranged from 1:10 to 1:3200. Three (71-44, 6-1, and 6-4) of the seven monoclonal antibodies also reacted on indirect immunofluorescence assays with hemagglutinating encephalomyelitis virus (HEV) of swine, which is antigenically related to BCV. These three monoclonal antibodies are directed against an epitope common to both BCV and HEV. Hybridoma 4-7 produced monoclonal antibody that inhibited hemagglutination of rat erythrocytes by BCV, and therefore, recognized epitopes common to the viral hemagglutinin. Clone 25-48 produced monoclonal antibody which neutralized BCV at titers ranging from 1:32 to 1:1024. The observation that Mabs which prevented hemagglutination independent of neutralization suggested that either these functions were on separate glycoproteins of the virion or the hemagglutinin and neutralization epitopes are separate regions on the same glycoprotein. The latter hypothesis was suggested by the immune electron microscopy results. When BCV was treated with bromelain, most of the outer row of surface projections were removed and the antigen (BCV) -antibody reaction observed on immune electron microscopy was enhanced. Since bromelain has been reported to remove all glycoproteins except the hemagglutinin, the enhanced reaction between Mab 25-48 and BCV on immune electron microscopy suggests that the neutralizing Mab 25-48 reacted with the viral hemagglutinin. Further characterization of the monoclonal antibodies obtained in this study by Western blotting or immunoprecipition will be required to clearly define the molecular weights of the proteins to which the monoclonal antibodies react. In summary, all of the monoclonal antibodies, 7/7, had immunofluorescent titers (IFA) against BCV, but 3/7 (71-44, 6-1, and 6-4) also reacted with HEV which indicates that these antigenically related viruses share a common viral protein.

Library of Congress Subject Headings

Cattle -- Diseases -- Prevention

Hybridomas

Monoclonal antibodies

Format

application/pdf

Number of Pages

82

Publisher

South Dakota State University

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