Ligand-binding characteristics of feline insulin-binding immunoglobulin G

Abstract

Polyclonal immunoglobulin (Ig) G autoantibodies against insulin have been identified in sera of healthy cats. We purified and fractionated insulin-binding IgGs from cat sera by affinity chromatography and analyzed affinity of insulin-binding IgGs for insulin and their epitopes. Following the passing of fraction A, which did not bind to insulin, insulin-binding IgGs were eluted into two fractions, B and C, by affinity chromatography using a column fixed with bovine insulin. Dissociation constant (KD) values between insulin-binding IgGs and insulin, determined by surface plasmon resonance analysis (Biacore™system), were 1.64e−4 M for fraction B (low affinity IgGs) and 2e−5 M for fraction C (high affinity IgGs). Epitope analysis was conducted using 16 peptide fragments synthesized in concord with the amino acid sequence of feline insulin by an enzyme-linked immunosorbent assay. Fractions B and C showed higher absorbance (affinity) of the peptide fragment of 10 amino acid residues at the carboxyl-terminal of the B chain (peptide No. 19), followed by peptide fragments of 6 to 15 amino acid residues of the B chain (peptide No. 8). Fraction C showed a higher absorbance to 7 to 16 amino acid residues of the B chain (peptide No. 5) compared with the absorbance of fraction B. Polyclonal insulin-binding IgGs may form a macromolecule complex with insulin through the multiple affinity sites of IgG molecules. Feline insulin-binding IgGs are multifocal and may be composed of multiple IgG components and insulin.

Human Immunodeficiency Virus Proteins Mimic Human T Cell Receptors Inducing Cross-Reactive Antibodies

“…Half of the TCR-HIV antibody combinations yielded measurable binding constants ranging between 1 × 10 −7 to 2 × 10 −9 M. The rest of the combinations were deemed to be negative, since binding constants were either greater than 1 × 10 −6 or no evidence of binding was obtained. (For reference, the association constant of insulin antibody for insulin, which is a primary cause of type 1 diabetes mellitus, is about 1 × 10 −8 [ 55 , 56 ], whereas healthy human beings and cats have naturally circulating anti-insulin binding that binds in the range of 1 × 10 −5 and has no clinical effect [ 57 ]). Rabbit anti-M. tuberculosis antiserum also bound measurably to some of the TCR, but a guinea-pig anti-mycobacterial antiserum did not, suggesting some specificity in both the TCR and antibody involved in the binding combinations.…”