Abstract

The status of the humoral immune system can be assessed through the use of immunoassays that measure immunoglobulin concentrations in serum. Using species specific reagents in primary binding assays maximizes sensitivity and specificity; however, the availability of commercial antisera for non-domestic or exotic animals is often limited. Currently, this laboratory is developing species specific antisera for Steller sea lion immunoglobulin isotypes. In an effort to obtain preliminary results, canine (a common ancestral relation to pinnipeds) and human reagents (including purified immunoglobulins and antisera specific for the immunoglobulins) were checked for cross reactivity against Steller sea lion and harbor seal serum. The canine reagents were obtained from Bethyl laboratories and Sigma; the human reagents were from Sigma. Analysis of the pinniped serum proteins was performed through several immunoassays including Ouchterlony (double immunodiffusion) and Western blot/Immunostaining analysis.

The Ouchterlony assays revealed that anti-dog gamma, anti-dog mu, and anti-dog IgG (whole molecule) reagents have cross-reactivity with both harbor seal and Steller sea lion serum. Similar results were obtained with Western blot/Immunostaining analysis. The anti-human gamma had a weak interaction with Steller sea lion serum and no reaction with the harbor seal serum in the Ouchterlony procedure. Anti-human mu was cross-reactive with Steller sea lion serum in both immunoassays performed, but was cross-reactive with harbor seal serum only in the Ouchterlony procedure. While the results with Western blot/Immunostaining analysis were ambiguous, the Ouchterlony assay showed definitively that Steller sea lion serum was cross-reactive with anti-dog alpha, but not with anti-human alpha. Harbor seal serum did not react with either the anti-dog alpha or anti-human alpha antisera by either immunoassay methods.

The results indicate that anti-dog gamma, mu, and alpha, and anti-human mu will be used as reagents in a capture ELISA to measure concentrations of specific immunoglobulin isotypes in Steller secretion and harbor seal serum.