Preliminary Study of the Isolation and Characterization of the Immunoglobulins of the Harbor Seal, Phoca vitulina
In recent years, the importance of studying the humoral immune system of marine mammals from both a comparative and clinical standpoint has become evident. In this study, we attempted to isolate and characterize the immunoglobulins from serum of the harbor seal, Phoca vitulina, which was collected from several individuals at the Alaska SeaLife Center.
Assuming that the harbor seal has similar immunoglobulins to related mammals, we subjected the serum to various affinity column chromatography methods that have been previously successful in isolating immunoglobulins of other species. The Protein G Sepharose® 4 Fast Flow Affinity column (Pharmacia) contains a molecule from Streptoccoci that has been shown to bind the heavy chain of immunoglobulin G (IgG). SDS-PAGE analysis of the fraction that was bound to the column, and assumed to contain IgG, showed protein bands in locations comparable to where the heavy and light chains of IgG from other species would be found. SigmaGel placed the heavy chain weight at 57,386 Daltons. A Jacalin column (Pierce), which contains a lectin from jackfruit seeds, was used to isolate immunoglobulin A (IgA). SDS-PAGE analysis of the bound fraction showed a faint band in the location of where IgA would be found when IgA of another species is subjected to the same gel. SigmaGel placed the molecular weight of the heavy chain at 60,777 Daltons. Tests were done to determine if anti-dog antisera was cross-reactive with proteins in harbor seal serum. An Ouchterlony test showed that harbor seal serum produced a similar band to that of dog immunoglobulin M (IgM) when both were tested against the anti-dog antisera. Similarly, harbor seal serum and dog IgM produced similar arcs against anti-dog antisera when subjected to Grabar Williams immunoelectrophoresis. Anti-dog IgM was coupled onto an AminoLink® Plus Coupling Gel (Pierce) in an attempt to obtain harbor seal IgM. SDS-PAGE analysis of the bound fraction produced a faint band in the location where IgM is usually found in similar species. SigmaGel placed the weight of the heavy chain at 82,226 Daltons. The average weight of the light chains for these classes of immunoglobulins was shown to be 28,341 Daltons. All three methods have been successful; however, as noted, the methods to isolate IgA and IgM yielded small amounts. Other methods are being attempted in hopes of extracting higher amounts of IgA and IgM. Size exclusion and ammonium sulfate precipitation will be tested for successfulness in purifying IgM. Obtaining IgA from other sample types, such as saliva and feces of other species, has proven successful, we are collecting these sample types.
Another goal of this study will be to use these immunoglobulins in ELISA tests to determine normal baseline serum values. We will also inject isolated immunoglobulins into BALB/c mice in an attempt to produce monoclonal antibodies against harbor seal immunoglobulins that can then be used in diagnostic tests.