Radioimmunoassay of insulin and glucagon with studies on the obese hyperglycaemic syndrome in mice

  • Peter Raymond Flatt

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Sensitive and precise radioimmunoassays for insulin and glucagon have been established. Although it was possible to employ similar precepts to the development of both hormone assays, the establishment of a reliable glucagon radioimmunoassay was complicated by the poor immunogenicity and
instability of the peptide. Thus, unlike insulin antisera which were prepared by monthly injection of guinea pigs with crystalline insulin emulsified in adjuvant, the successful production of glucagon antisera was accomplished by immunisation of rabbits and guinea pigs with glucagon covalently linked to bovine plasma albumin. The conventional chloramine-T iodination with purification by gel chromatography was only suitable for the production of labelled insulin. Quality tracer for use in the glucagon radioimmunoassay was prepared by trace iodination, with subsequent purification of monoiodinated glucagon by anion exchange chromatography. Separation of free and antibody bound moieties by coated charcoal was applicable to both hormone assays, and a computerised data processing system, relying on logit-log transformation, was used to analyse all assay results.
The assays were employed to evaluate the regulation of endocrine pancreatic function and the role of insulin and glucagon in the pathogenesis of the obese hyperglycaemic syndrome in mice. In the homozygous (ob/ob) condition, mice of the Birmingham strain were characterised by numerous abnormalities of glucose homeostasis, several of which were detected in heterozygous (ob/+) mice. Obese mice exhibited pancreatic alpha cell dysfunction and hyperglucagonaemia. Investigation of this defect revealed a marked insensitivity of an insulin dependent glucose sensing mechanism that inhibited glucagon secretion. Although circulating glucagon was of minor importance in the maintenance of hyperinsulinaemia, lack of suppression of alpha cell function by glucose and insulin contributed significantly to both the insulin insensitivity and the hyperglycaemia of obese mice.
Date of AwardDec 1977
Original languageEnglish
SupervisorTerence W. Atkins (Supervisor) & Clifford Bailey (Supervisor)

Keywords

  • Radioimmunoassay
  • insulin
  • glucagon
  • obese hyperglycaemic syndrome
  • mice

Cite this

Radioimmunoassay of insulin and glucagon with studies on the obese hyperglycaemic syndrome in mice
Flatt, P. R. (Author). Dec 1977

Student thesis: Doctoral ThesisDoctor of Philosophy