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Abstract <p>The present study demonstrates chromatography techniques for purifying immunoglobulin (Ig) from the serum of two fish species, <i>Clarias dussumieri</i> and <i>Etroplus suratensis</i>. 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 <i>C. dussumieri</i> showing a heavy chain of ~70 kDa and a light chain of ~25&#xa0;kDa, while <i>E. suratensis</i> 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 <i>C. dussumieri</i> and ~855&#xa0;kDa for <i>E. suratensis</i>. 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.</p>

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

  • 期刊论文

  • 发表日期：

    2025年4月18日

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  • S. G. Satkar,
  • A. Sudhagar,
  • T. R. Swaminathan,
  • N. Sood,
  • K. R. Saikrishnan,
  • C. Ravi,
  • U. K. Sarkar

摘要

Abstract

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.