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

Aine McKillop, Janie McCluskey, AC Boyd, MH Mooney, Peter Flatt, Finbarr O'Harte

Research output: Contribution to journalArticle

Abstract

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
LanguageEnglish
Pages153-163
JournalJournal of Endrocrinology
Volume167
Issue number1
Publication statusPublished - Oct 2000

Fingerprint

Insulin
Immune Sera
Antibodies
Ovalbumin
Glutaral
Rabbits
Guinea Pigs
Carbodiimides
Proinsulin
Hemocyanin
Peptides
Cross Reactions
Insulin-Secreting Cells
Hyperglycemia
Radioimmunoassay
Hydrocortisone
Immunization
Esters
glycosylated insulin
peptide A

Cite this

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title = "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 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",
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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; O'Harte, Finbarr.

In: Journal of Endrocrinology, Vol. 167, No. 1, 10.2000, p. 153-163.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Production and characterization of specific antibodies for evaluation of glycated insulin in plasma and biological tissues

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AU - Mooney, MH

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AU - O'Harte, Finbarr

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