Production and characterization 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 -cells under conditions of hyperglycaemia and that the site of glycation 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 and guinea-pigs by using two synthetic peptides (A: Phe-Val-Asn-Gln-His-Leu-Cys-Tyr, and B: Phe-ValAsn-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 I 125 -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) (ED 50 ) 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.

Demonstration of increased concentrations of circulating glycated insulin in human Type 2 diabetes using a novel and specific radioimmunoassay

“…Evidence includes the demonstration of increased concentrations of glycated insulin in the pancreas of various animal models of diabetes [1,8,9] and the time-and concentration-dependent glycation of insulin in both isolated mouse islets or clonal insulin secreting cell lines under hyperglycaemic conditions [1,2]. We have previously shown the impaired biological activity of glycated insulin using in vitro studies with isolated muscle preparations and in vivo studies using animal models or healthy human subjects subjected to hyperinsulinaemic euglycaemic glucose clamps [3,4,5,6].…”

“…Development of a specific RIA for glycated insulin has been described in detail elsewhere [8]. In brief, an N-terminally glycated synthetic insulin peptide, closely related to the amino-terminal sequence of the insulin B-chain (Phe-Val-Asn-Gln-His-Leu-Tyr-Lys) was used to raise specific antibodies in rabbits.…”

“…Assay sensitivity was less than 3 pmol/l with an intra-assay coefficient of variation of 9%. The glycated insulin antibody crossreacted 56% with glycated proinsulin but cross-reaction with non-glycated insulin, proinsulin and other pancreatic hormones was negligible [8]. Serum insulin was determined using a microparticulate enzyme immunoassay.…”

“…Previous studies have shown that glycated insulin has a reduced ability to regulate plasma glucose homeostasis in vivo and to stimulate adipose tissue lipogenesis or glucose uptake and oxidation by isolated diaphragm and abdominal muscle in vitro [3,4,5]. Studies in healthy human volunteers using the hyperinsulinaemic-euglycaemic glucose clamp technique suggest that glycated insulin could contribute to insulin resistance in Type 2 diabetes mellitus [6].The site of glycation of human insulin has now been identified by electrospray tandem mass spectrometry as the N-terminal Phe 1 of the B-chain [7], enabling the development of a sensitive and specific radioimmunoassay to measure concentrations of glycated insulin and to identify glycated insulin in pancreatic islets using immunohistochemistry [8]. High performance liquid chromatography techniques used previously were neither sensitive nor reliable Formation of advanced glycation-end products (AGEs) plays an important role in long-term metabolic consequences of diabetes including ophthalmic, renal and atherosclerotic vascular complications.…”

“…The site of glycation of human insulin has now been identified by electrospray tandem mass spectrometry as the N-terminal Phe 1 of the B-chain [7], enabling the development of a sensitive and specific radioimmunoassay to measure concentrations of glycated insulin and to identify glycated insulin in pancreatic islets using immunohistochemistry [8]. High performance liquid chromatography techniques used previously were neither sensitive nor reliable Formation of advanced glycation-end products (AGEs) plays an important role in long-term metabolic consequences of diabetes including ophthalmic, renal and atherosclerotic vascular complications.…”