Purification of insulin-binding antibody by affinity chromatography using monocomponent insulin as ligand

Insulin-binding antibody (IBA) was purified by affinity chromatography using porcine monocomponent (MC) insulin as the ligand. The purity of the antibody was compared with that of the antibody extracted using porcine crystalline (Cr) insulin. Comparing the antibody solutions obtained with MC insulin (MC-lig-sol) or Cr insulin (Cr-lig-sol), the content of IBA in Cr-lig-sol was higher than in MC-lig-sol, but the content of proinsulin-binding antibody (PBA) in MC-lig-sol was very small and statistically lower than that in Cr-lig-sol (P less than 0.01). Adding native MC insulin to a competitive radioimmunoassay suppressed the IBA titer obtained with MC insulin more than that obtained with Cr insulin. By adding native proinsulin in a similar assay system, the PBA titer obtained with Cr insulin was suppressed more than that extracted with MC insulin. Scatchard analysis of the 2 solutions showed that the affinity constants of high affinity antibodies were almost identical, but that of low affinity antibody in MC-lig-sol was larger than in Cr-lig-sol. The binding capacity of low affinity antibody in Cr-lig-sol was 15 times as much as that in MC-lig-sol. Using MC insulin, instead of Cr insulin, as the ligand in affinity chromatography increased the purity of recovered IBA. chromatography increased the purity of recovered IBA.