Veterinary and Biomedical Sciences, Department of


First Advisor

Greg A. Somerville

Date of this Version



A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Veterinary Science, Under the Supervision of Professor Greg A. Somerville. Lincoln, Nebraska: May, 2018

Copyright 2018 Lauren M. Wilmes


Clostridium perfringens type C produces beta toxin, which is a primary virulence determinate that can cause necrotic enteritis in neonatal animals. Vaccines directed against C. perfringens toxins have shown to be efficacious in preventing disease. However, the production of a commercial vaccine requires not only an efficacious antigen, but also testing methods for quantifying antigens and potency on final product. This research was undertaken to fulfill a need for the reduction in small animal usage to produce and deliver vaccines. The current testing methods are performed in vivo according to the Code of Federal Regulations number 9 (9CFR) United States Department of Agriculture (USDA) and European Pharmacopeia (EP) monograph guidelines. With limited in vitro alternatives, the aim of this work was to develop an enzyme-linked immunosorbent assay (ELISA) to be able to measure C. perfringens type C beta toxoid, independent of laboratory animals. To accomplish this, investigational polyclonal and monoclonal antibody candidates were screened for their specificity to beta toxin and toxoid through western blot and ELISA. Additionally, through a neutralization assay, the capability to neutralize beta toxin was demonstrated. Through this process, one polyclonal antibody and one monoclonal antibody were selected and an in-process ELISA for quantifying beta toxoid was developed. By using the ELISA to quantify beta toxoid, the current practice of testing in animals could be reduced.

Advisor: Greg A. Somerville