A Comparative Analysis of Chromatography Approaches for the Purification of Immunoglobulin from Fish Serum for Sero-diagnostic Application

The present study demonstrates chromatography techniques for purifying immunoglobulin (Ig) from the serum of two fish species, Clarias dussumieri and Etroplus suratensis. Four purification techniques including BSA-affinity chromatography, protein-A affinity chromatography, protein-L affinity chromatography, and ammonium sulfate precipitation followed by ion-exchange chromatography were evaluated. Subsequently, the purified proteins were analysed using SDS-PAGE, gel chromatography and ESI-nano-LC-MS/MS. BSA-affinity chromatography stands out as a versatile and highly effective technique for purifying Ig from both the fish species. Despite the successful isolation of serum Ig using ammonium sulfate precipitation followed by ion-exchange chromatography for both fish species, SDS-PAGE analysis revealed non-specific protein bands and protein-A and protein-L affinity chromatography failed for purification. The purified Ig displayed distinctive profiles, with C. dussumieri showing a heavy chain of ~70 kDa and a light chain of ~25 kDa, while E. suratensis exhibited a heavy chain of ~72 kDa and a light chain of ~28 kDa. Gel filtration chromatography indicated native molecular weights of Ig approximately ~803 kDa for C. dussumieri and ~855 kDa for E. suratensis. Further characterization of heavy chain bands through ESI-nano-LC-MS/MS confirmed homology matches with Ig proteins. This study enhances fish Ig purification protocols, laying the groundwork for future research to refine and expand methodologies essential for sero-diagnostic applications in aquaculture.