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

Previous studies have shown that glycation of insulin occurs in pancreatic beta -cells under conditions of hyperglycaemia and that the site of glycati on is the N-terminal Phe(1) of the insulin B-chain. To enable evaluation of glycated insulin in diabetes, specific antibodies were raised in rabbits a nd 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 gly cation and corresponding closely to the N-terminal sequence of the glycated human insulin B-chain. For immunization, the glycated peptides were conjug ated either to keyhole limper haemocyanin or ovalbumin using glutaraldehyde , m-maleimidobenzoyl-N-hydroxysuccinimide ester or 1-ethyl-3-(3-dimethylami no propyl) carbodiimide hydrochloride. Antibody titration curves, obtained using I-125-tyrosylated tracer prepared from glycated peptide A, revealed h igh-titre antisera in five groups of animals immunized for 8-28 weeks. The highest titres were observed in rabbits and guinea-pigs immunized with pept ide B coupled to ovalbumin using glutaraldehyde. Under radioimmunoassay con ditions, 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 negl igible 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 meas ured using rabbit 3 antiserum (1:10 000 dilution; sensitivity <19 pg/ml) wa s 0.08 +/- 0.01 and 1.5 +/- 0.6 ng/ mi (P<0.01), corresponding to 4 and 16% of total circulating insulin concentration respectively. Immunocytochemist ry studies of the pancreas of strep tozotocin-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 u sefulness in radioimmunoassays and immunocytochemistry. The demonstration o f glycated insulin in plasma and islets of animal models of diabetes suppor ts the view that glycation of insulin is involved in the pathogenesis of th is disease.