Advantages and pitfalls of radioimmune and enzyme linked immunosorbent assays of insulin antibodies

Abstract

Human sera were tested for insulin antibodies by fluid and solid phase assays. Radioimmune titres determined with 125-I Tyr A14 insulin were not correlated with those obtained using insulin coated microplates and enzyme linked immunodetection (n = 60). Several reasons for this lack of correlation were found. Iodine substitution on the A14 residue of insulin may significantly alter the avidity of some insulin antibodies for their ligand; hence, disclosing a heretofore unsuspected pitfall for antibody determination by radioimmunoassay. Specificity for bovine insulin was easily demonstrable in fluid phase by comparing the binding of monoiodinated bovine, porcine and human insulin. By contrast, in solid phase assay, titres obtained with microplates coated with bovine or human insulin were almost equal, regardless of the serum specificity for bovine insulin. This lack of specificity of the solid phase assay is not due to denaturation or unavailability of the bovine specific epitope because: bovine specificity could be demonstrated by competitive assay, after preincubation of the serum with insulin of the different species; and, coating with crosslinked insulin dimers or oligomers instead of monomers did not unmask bovine specificity. It is concluded that radioimmune methods are best suited to study specificity but may be biased by the presence of the radioiodine label whereas solid phase assay detects low avidity antibodies with great efficiency but is less appropriate to study specificity.