Development of an Enzyme-Linked Immunosorbant Assay (ELISA) Using Anti-Human IgA Reagents to Determine IgA Levels in the Serum of Several Individuals of the Harbor Seal, Phoca vitulina
Studies of the components of the humoral immune system of marine mammals often involve taking advantage of homologies between the immunoglobulins of different species, specifically by using commercial reagents that are specific for immunoglobulins of another species but show cross-reactivity with immunoglobulins of the marine mammal species in question. These reagents are used until monoclonal antibodies that are species specific for marine mammal immunoglobulin isotypes are made or become commercially available.
Cross-reactivity was tested between a variety of commercial antisera (anti-dog, anti-human, and anti-mouse) and purified immunoglobulins (IgG, IgA, and IgM) of the harbor seal, Phoca vitulina. It was desirable to find antisera that were cross-reactive separately with each of the immunoglobulins and which were functional in enzyme-linked immunosorbant assays (ELISA's) to determine levels of each immunoglobulin in a series of individual serum samples taken from harbor seals at the Alaska SeaLife Center. Anti-human IgA was found to fit these criteria with respect to harbor seal IgA and an ELISA was developed to test the serum samples against a set of known harbor seal IgA concentrations. A total of 325 samples, representing 238 samples from captive animals and 87 samples from rehab (wild) animals, were tested by this assay. The sensitivity of the assay allowed IgA levels between 1.1 mg/ml and 100 mg/ml to be measured. Some of the samples had been previously diluted to concentrations that were below detectable levels using this assay (i.e., below 1.1 mg/ml). Altogether, 107 samples, representing 83 captive and 24 rehab animals, were measurable and the average IgA concentration in the serum of these test groups was found to be 6.62 mg/ml. IgA concentrations in most (approximately 80%) of the captive animals were in the 1-5 mg/ml range, with a few animals having measured concentrations as high as 59.4 mg/ml. In the rehab animals, concentrations ranged from 1.4-1.9 mg/ml, with an average of 1.6 mg/ml. The average IgA concentration for both groups combined was found to be 5.44 mg/ml. Further sample testing is needed to determine a normal average concentration for IgA in harbor seal serum. Monoclonal antibodies that have been developed in this lab against harbor seal IgA must be evaluated to determine if they are functional in the ELISA. This will provide insight into the validity of this assay by confirming these numbers or showing higher numbers due to possible increased specificity of the monoclonal.