Ligand-binding characteristics of feline insulin-binding immunoglobulin G

Polyclonal immunoglobulin (Ig) G autoantibodies against insulin have beenidentified in sera of healthy cats. We purified and fractionated insulin-binding IgGs fromcat sera by affinity chromatography and analyzed affinity of insulin-binding IgGs forinsulin and their epitopes. Following the passing of fraction A, which did not bind toinsulin, insulin-binding IgGs were eluted into two fractions, B and C, by affinitychromatography using a column fixed with bovine insulin. Dissociation constant (KD) valuesbetween insulin-binding IgGs and insulin, determined by surface plasmon resonance analysis(Biacore™system), were 1.64e⁻⁴ M for fraction B (low affinity IgGs) and2e⁻⁵ M for fraction C (high affinity IgGs). Epitope analysis was conductedusing 16 peptide fragments synthesized in concord with the amino acid sequence of felineinsulin by an enzyme-linked immunosorbent assay. Fractions B and C showed higherabsorbance (affinity) of the peptide fragment of 10 amino acid residues at thecarboxyl-terminal of the B chain (peptide No. 19), followed by peptide fragments of 6 to15 amino acid residues of the B chain (peptide No. 8). Fraction C showed a higherabsorbance to 7 to 16 amino acid residues of the B chain (peptide No. 5) compared with theabsorbance of fraction B. Polyclonal insulin-binding IgGs may form a macromolecule complexwith insulin through the multiple affinity sites of IgG molecules. Feline insulin-bindingIgGs are multifocal and may be composed of multiple IgG components and insulin.