Production and charactization of specific antibodies for evaluation of glycated insulin in plasma and biological tissues

Abstract

Previous studies have shown that glycation of insulin occurs in pancreatic β-cells under conditions of hyperglycaemia and that the site of glycation is the N-terminal Phe1 of the insulin B-chain. To enable evaluation of glycated insulin in diabetes, specific antibodies were raised in rabbits and guinea-pigs by using two synthetic peptides (A: Phe-Val-Asn-Gln-His-Leu-Cys-Tyr, and B: Phe-Val-Asn-Gln-His-Leu-Tyr-Lys) modified by N-terminal glycation and corresponding closely to the N-terminal sequence of the glycated human insulin B-chain. For immunization, the glycated peptides were conjugated either to keyhole limpet haemocyanin or ovalbumin using glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester or 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride. Antibody titration curves, obtained using I125-tyrosylated tracer prepared from glycated peptide A, revealed high-titre antisera in five groups of animals immunized for 8-28 weeks. The highest titres were observed in rabbits and guinea-pigs immunized with peptide B coupled to ovalbumin using glutaraldehyde. Under radioimmunoassay conditions, these antisera exhibited effective dose (median) (ED50) values for glycated insulin of 0.3-15 ng/ml and 0.9-2.5 ng/ml respectively, with negligible cross-reactivity against insulin or other islet peptides. The degree of cross-reaction with glycated proinsulin was approximately 50%. Glycated insulin in plasma of control and hydrocortisone-treated diabetic rats measured using rabbit 3 antiserum (1:10 000 dilution; sensitivity <19 pg/ml) was 0.08 ± 0.01 and 1.5 ± 0.6 ng/ml (P<0.01), corresponding to 4 and 16% of total circulating insulin concentration respectively. Immunocytochemistry studies of the pancreas of streptozotocin-treated diabetic rats using a 1:1000 dilution of guinea-pig 2 antiserum revealed clusters of fluorescent positively stained cells in islets. These studies document the successful production of polyclonal antisera specific for glycated insulin and their usefulness in radioimmunoassays and immunocytochemistry. The demonstration of glycated insulin in plasma and islets of animal models of diabetes supports the view that glycation of insulin is involved in the pathogenesis of this disease.