title: 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.
authors.affiliation: Department of Veterinary Medicine, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Suzuki
authors.forename: Naohito
authors.initials: N
authors.lastname: Nishii
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takashima
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Matsubara
authors.forename: Atsushi
authors.initials: A
authors.lastname: Iwasawa
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Takeuchi
authors.forename: Kohei
authors.initials: K
authors.lastname: Tahara
authors.forename: Tatsuyuki
authors.initials: T
authors.lastname: Hachisu
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kitagawa
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D002415:Cats; D002846:Chromatography, Affinity; D005260:Female; D007074:Immunoglobulin G / Q000276:immunology*; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008297:Male; D011485:Protein Binding
chemical_list: D007074:Immunoglobulin G; D007328:Insulin; D008024:Ligands