Immunoaffinity chromatography in antivenomics studies: Various parameters that can affect the results

• Several parameters of affinity chromatography in antivenomics study were investigated.
• These parameters involved the venom, antibody, affinity media preparation and eluents.
• Estimation of relative venom binding affinity gave useful data for antivenomics study.
• The findings can lead to improved chromatographic conditions and antivenomics results.

Antivenomics is a recently developed powerful method for the study of antivenom antibody profiles when bound to homologous and heterologous snake venoms. The information obtained is useful in gaining an understanding of venom protein immunogenicity, antivenom potency and also for the improvement of antivenom potency and paraspecificity. The preferred method used in this type of study is immunoaffinity chromatography of the venom proteins on an antivenom IgG (or F(ab′)2) column where the bound and unbound proteins can be separated and identified. However, there are some parameters of the immunochromatography that can significantly affect the binding of the proteins to the immunoaffinity matrix and lead to imprecise results in antivenom immunoprofiling. The present study demonstrated that the ligand density (mg IgG/ml of the matrix), the buffers used for binding and washing the venom proteins, the amount of venom loaded, the abundance of some venom protein(s) and the eluting buffers can significantly alter the binding of the proteins to the matrix and consequently the conclusions drawn from antivenomics studies. Furthermore, the immunochromatographic procedure can be extended to include the estimation of the relative affinity of venom protein-antibody interactions that can provide additional information useful to antivenomics study.