Development of an enzyme-linked immunosorbent assay to detect antibodies against Pasteurella multocida in infected rabbits Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/cr56n4370

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  • Enzyme-linked immunosorbent assays (ELISA) were developed to detect rabbit antibodies against Pasteurella multocida. Antigens for ELISA were prepared from 2.5% saline extracts of P. multocida by chromatographic methods. From a crude saline extract of a type 3, 4 isolate designated as R11146, two protein peaks were obtained by gel filtration with Sephadex G-200. The first peak produced a single precipitin line with rabbit anti-whole cell or anti-purified type 3 antigen sera. The first peak was further purified by DEAE-cellulose chromatography. Four protein peaks were obtained, and all of them contained a single identical antigen. The purified antigen contained two protein species of molecular weights of 44,000 and 25,500. The purified antigen was attached to 96 well microtiter plates, and an ELISA was developed by the use of alka-line phosphatase-conjugated sheep anti-rabbit IgG to detect anti bodies. Six Pasteurella-free rabbits were intranasally infected with R11146 isolate and antibody responses were measured by the ELISA. High titers ranging from 1:10³ to 1:10⁴ persisted for longer than 96 days after the infection, while the titers of all the preinoculation sera were lower than 1:10. A similar surface antigen was also purified from a type 12 isolate, R040. Three protein peaks were obtained from crude extract by gel filtration with Sephadex G-200. The first peak contained a major antigen. In some instances, crude extract was concentrated by ultracentrifugation prior to the gel filtration. A major protein component of molecular weights 42,000 and minor ones of 25,000 and 110,000 were present in t the first peak fraction. The antigen purified from a type 12 isolate showed antigenic cross-reaction with the purified antigen from a type 3, 4 isolate. An ELISA was developed with the purified type 12 antigen, and used for detecting antibodies in rabbits intranasally infected with the type 12 strain. Titers of ELISA ranging from 1:10⁴ to 1:10⁵ were obtained for longer than 77 days post inoculation. The preinoculation sera showed titers of less than 1:50. The results indicate that the purified antigen is useful to detect antibodies against P. multocida in rabbits infected with type 3 or type 12 strains.
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