Characterization of the humoral immune system of the Stellar sea lion, Eumetopias jubatus, and development of reagents and assays for quantitative measure of humoral immunity

Jennifer Ann Colvocoresses

Abstract

The Steller sea lion, Eumetopias jubatus , was listed as "threatened" on the endangered species list in 1990, and the western portion of the population was reclassified as "endangered" in 1997. Factors hypothesized as contributing to the decline of the species have included environmental changes, contamination and decline of the food supply, falling reproductive rates, increased pup mortality, predation, disease, anthropogenic activities, and impairment of immune function. A deterioration in immune function manifests as reduced immune responsiveness to infectious agents and disease by decreasing the function of crucial cellular components of the immune system. To investigate the contribution of impaired immune function, elements of the humoral immune system were identified, purified, and used to develop reagents to be used in immunoassays for evaluative measurement of humoral immunity. Affinity column matrices Protein G, Jacalin, and amino-linked anti-canine IgM were used to purify Steller sea lion immunoglobulin classes/subclasses IgG 1 , IgG2 , IgA, and IgM, respectively. The IgG subclass IgG2 was identified through the application of sequential chromatography. Purified Steller sea lion IgG and IgM were used as standards in capture ELISA's using anti-canine reagents to measure Steller sea lion IgG and IgM levels in 130 serum samples from three captive animals over a period of four years and from 688 individual wild animals representing the geographic range of the species. For approximately 1/3 of the samples received, data were provided concerning the gender, age and weight of the animals from which the samples were taken. For these samples, analysis of differences in immunoglobulin levels in relation to one or more of the parameters was performed. Mean immunoglobulin values for the captive animals were 38.3 mg/ml and 9.43 mg/ml for IgG and IgM, respectively. Mean IgG levels were 38.17 mg/ml for the Eastern population, 31.8 mg/ml for the Western population, and 14.51 mg/ml for the Russian population. Comparison of the three population mean IgM levels indicated significant differences by one way analysis of variance (p < 0.001) between the Eastern and Russian, and the Western and Russian, but not between the Eastern and Western populations. Purified Steller sea lion immunoglobulins were used as immunogens for the production of polyclonal and monoclonal Steller sea lion specific antibodies. Polyclonal antibodies against the immunoglobulin fraction of Steller sea lion serum, Steller sea lion IgG, and Steller sea lion gamma heavy chain were produced in New Zealand white rabbits. Monoclonal antibodies with specificities for Steller sea lion light chains, IgG, IgM and gamma heavy chains were produced by fusion of primed balb/c spleenocytes and a CRL 1580 cell line (ATCC). These monoclonal antibodies were characterized with regard to the mouse isotype of each.