Purification of Immunoglobulin Isotypes G, A, and M from the Serum of the Steller Sea Lion (Eumetopias jubatus)
Department of Biological Sciences, The University of Southern Mississippi, Hattiesburg, MS, USA
There are two populations of Steller sea lions (Eumetopias jubatus) with the eastern population listed as threatened and the western population as endangered. Research projects have been developed to study the health of this species and to try to determine any factors that might be adversely affecting these populations. Several indicators can be used in the assessment of overall health and well-being of a species. The status of the humoral immune system is one such indicator that can be evaluated through immunoassays used to determine the concentrations of specific immunoglobulin isotypes within a sample. This approach has led to efforts in this lab to develop an ELISA to assess baseline immunoglobulin levels in Steller sea lions; however, it was first necessary to isolate the immunoglobulin isotypes (IgG, IgA, and IgM) of interest and to develop Steller sea lion specific reagents for the ELISA.
Purification of the immunoglobulins involved an initial purification step to remove non- immunoglobulin proteins from serum (leaving a mixture of all immunoglobulins present). Pooled serum was centrifuged in an Eppendorf centrifuge to remove any large particulate matter and the resulting supernatant was filtered through 0.45 µm and 0.2 µm filters, successively, to remove any small particulates or aggregates. The next step involved separating proteins in the filtrate based on molecular weights. Since the immunoglobulin isotypes being studied have molecular weights greater than 100,000 Dalton, the serum was further processed by centrifugation in a Millipore Ultra Free-15 centrifugal filter device with a molecular weight cutoff of 100,000 Dalton to remove the non-immunoglobulin proteins still present. In order to produce antisera containing antibodies against all Steller sea lion isotypes normally present (for use in evaluating the immunoglobulin purification techniques), serum processed through this initial partial purification step was used to immunize a rabbit.
The resulting serum fraction containing immunoglobulins was applied to selected affinity columns including Protein G Sepharose 4 fast flow (Amersham Pharmacia Biotech), Affi-gel Protein A (Bio-Rad), Immobilized Jacalin (Pierce), and Mannose Binding protein (Pierce) in 0.5 ml aliquots in order to separate individual isotypes based on binding affinities of each isotype to the selected matrices. Aliquots of the serum fractions were also applied to a gel filtration column, BioSep-SEC-S 4000 (Phenomenex), in order to separate the isotypes based on molecular weight differences. Protein peaks obtained from the various chromatography columns were analyzed by Grabar-Williams immunoelectrophoresis and SDS-PAGE. Grabar-Williams immunoelectrophoresis was performed using anti-human isotypes (Sigma), anti-canine isotypes (Bethyl Laboratories), and anti-Steller sea lion fractionated serum >100,000 Dalton to identify the protein(s) purified by the different affinity chromatography matrices. SDS-PAGE and gel filtration columns were used to verify molecular weights of the isotypes.