Purification And Partial Characterization Of Serum Immunoglobulin From Channel Catfish (Ictalurus Punctatus)

Serum immunoglobulin (Ig) from normal and Aeromonas sobria (A.s) immunized channel catfish (Ictalurus punctatus) were each purified by using ammonium sulfate precipitation followed by sepharose-4B gel filtration and DEAE-52 anion exchange chromatography. The purified Ig fractions were defined by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (MAF-13) before being pooled and accessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that normal channel catfish serum Ig were mainly present in the range of 35% - 45% ammonium sulfate precipitation, however, the serum Ig of immunized channel catfish were present in the range of 35%-55% ammonium sulfale precipitation. The immunoglobulin appeared in the first protein peak both in the Sepharose-4B gel filtration and DEAE-52 anion exchange chromatography. SDS-PAGE results showed that serum Ig had been further purified by each step of purification. The purified Ig were used as antigen to produce rabbit antiserum. Polyclonal and monoclonal antibodies both reacted with serum of European eel (Anguilla anguilla) and Japanese eel (Anguilla Japonica) in ELISA. Structure analysis of serum Ig were performed by Western blot under denatured and reduced, denatured but non-reduced, non-denatured and non-reduced conditions using monoclonal and polyclonal antibodies as probes. The results showed that the denatured and reduced serum Ig of normal channel catfish had two different heavy (H) chains with molecular weight about 72 0 00 and 55 000 respectively. For the immunized channel catfish, however, the molecular weight of heavy chain was predominantly determined to be 72 000, and three distinct light (L) chains were found with molecular weight of 21 000, 23 500, 26 000 respectively. The naive serum Ig was estimated to have a molecular weight about 900KD using 3%~12% gradient PAGE and western blot under non-reduced and non-denatured conditions. This polymeric Ig could dissociated into several subpopulations with molecular weight about 870 000,760 000,525 000,330 000 and 230 000 when analyzed in the present of denaturing solvent (SDS).