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

McKillop, Aine, McCluskey, Janie, Boyd, AC, Mooney, MH, Flatt, Peter and O'Harte, Finbarr (2000) Production and characterization of specific antibodies for evaluation of glycated insulin in plasma and biological tissues. JOURNAL OF ENDOCRINOLOGY, 167 (1). pp. 153-163. [Journal article]

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Previous studies have shown that glycation of insulin occurs in pancreatic beta -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-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 limper 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) (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/ mi (P<0.01), corresponding to 4 and 16% of total circulating insulin concentration respectively. Immunocytochemistry 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 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.

Item Type:Journal article
Faculties and Schools:Faculty of Life and Health Sciences
Faculty of Life and Health Sciences > School of Biomedical Sciences
Research Institutes and Groups:Biomedical Sciences Research Institute
Biomedical Sciences Research Institute > Diabetes
ID Code:3083
Deposited By: Professor Peter Flatt
Deposited On:13 Jan 2010 21:10
Last Modified:09 May 2016 10:48

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