Degree Type

Dissertation

Date of Award

1991

Degree Name

Doctor of Philosophy

Department

Microbiology

First Advisor

Donald P. Durand

Second Advisor

Steven R. Bolin

Abstract

Epitopic analysis of the 53kd envelope glycoprotein of bovine viral diarrhea virus (BVDV) was performed. Monoclonal antibodies that neutralized virus infectivity were used in a competitive binding assay to determine the spatial orientation of epitopes. Results of the assay indicate that the panel of monoclonal antibodies defined at least two distinct antigenic domains: A, and B/C. The majority of monoclonal antibodies recognized overlapping epitopes in domain A. Antigenic domain B/C was defined by the binding of two monoclonal antibodies to gp53 from either of two viruses. The neutralizing activity of each monoclonal antibody was complement-independent. A dot-immunoblot assay, using denatured viral protein, determined the nature of each epitope. Endoglycosidase treatment indicated that the epitopes were located in or near the carbohydrate moiety of the glycoprotein;A novel method for rapid production of viral antigen was developed. Viral antigen (Singer isolate of cytopathic virus) was produced by two methods which were compared for ease of production and antigen yield. Virus was propagated in bovine turbinate (BT) cells. In one method, BVDV antigen was purified from infected BT cells by using glycerol-potassium tartrate density gradient centrifugation. In the second method, BVDV-infected BT cells were solubilized and antigen was released with the zwitterionic detergent CHAPS. Detergent was removed by chromatography and antigen was ready for immediate use, or could be stored for future use. Antigen yield by detergent solubilization was greater than gradient-purified antigen. Detergent-solubilized BVDV antigen could be detected by enzyme immunoassay at a sixteen-fold greater dilution than gradient-purified BVDV antigen. This antigen was used in an enzyme immunoassay to detect antibody to BVDV in fetal bovine sera. BVDV antigen obtained from one 150 cm[superscript]2 cell culture flask supplied sufficient antigen to assay 282 samples of fetal bovine serum. Peroxidase-labeled protein G was used to detect bovine antibody. A good correlation existed between results from enzyme immunoassay and those from viral neutralization tests.

DOI

https://doi.org/10.31274/rtd-180813-11347

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Robert Charles Unfer

Language

en

Proquest ID

AAI9126262

File Format

application/pdf

File Size

133 pages

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