Human Proinsulin and Insulin: Antibody Production, Assay Development and Clinical Application

Production of high titre, high avidity, polyclonal antisera to insulin was attempted in a number of animal species but was only successful in guinea-pigs. In particular, the use of Strain 2 guinea-pigs resulted in a 100% success rate (Chapter 3). Ten murine monoclonal antibodies (McAbs) were produced using porcine insulin and human proinsulin as immunogens. Six reacted with insulin and proinsulin, one with C-peptide and proinsulin, and three with proinsulin only (Chapter 4). The detection of McAbs specific for proinsulin required the preparation of monoiodinated 125I-proinsulin. This involved the development of a reverse-phase high performance liquid chromatography (HPLC) technique to separate the various iodinated species of 125I-proinsulin (Chapter 5, Section 1.2). A two-site immunoradiometric assay (IRMA) for human insulin was developed using the guinea-pig anti-insulin in conjunction with an insulin McAb raised using porcine insulin as immunogen. Proinsulin cross-reacted in the assay to a degree dependent upon the form being measured and its concentration (Chapter 5, Section 2). Initial evaluation suggested that the avidity of the C-peptide McAb was too low to provide a proinsulin IRMA of the required sensitivity (Chapter 5, Section 4). Subsequent reassessment with the previously unavailable NIBSC proinsulin standard and a higher avidity insulin McAb suggested that a potentially useful assay could be developed (Chapter 5, Section 6.3). The highest avidity proinsulin-specific and insulin McAbs, raised using human proinsulin as immunogen, were used in a two-site IRMA to measure human proinsulin. The presence of insulin interfered with the initial assay developed (Chapter 5, Section 5) and the protocol was modified to abolish this effect (Chapter 5, Section 6). The assay measured intact, 65-66 split and des 64-65 proinsulin. Clinical validation of the assays developed was performed by measuring insulin and proinsulin concentrations in serum from normal adult subjects under a variety of conditions (Chapter 6, Section 1). The release of proinsulin relative to insulin was found to be delayed in response to an increase in blood glucose concentrations. Serum proinsulin and insulin concentrations were also measured in patients in various states of altered or patho-physiology. Measurement of fasting proinsulin compared to insulin concentrations provided improved discrimination in the diagnosis of insulinoma (Chapter 6, Section 2.3). The reported association between cirrhosis of the liver and glucose intolerance, hyperinsulinaemia and hyperproinsulinaemia was confirmed although the data suggested that only hyperinsulinaemia was truly secondary to the development of cirrhosis. The hyperinsulinaemia appeared to be due to reduced degradation rather than hypersecretion (Chapter 6, Section 2.5). Data is presented which supports the role of insulin resistance in the development of impaired glucose tolerance in pregnancy (Chapter 6, Section 2.2) and the high circulating concentration of insulin in a boy with Mendenhall's Syndrome (Chapter 6, Section 2.4). However, an abnormal pattern of insulin secretion in the basal state also appears to contribute to the hyperinsulinaemia in Mendenhall's Syndrome. The glucose intolerance has also been shown to be due to an abnormal pattern of insulin release in response to increased blood glucose concentrations in patients with non-insulin dependent diabetes mellitus (NIDDM) (Chapter 6, Section 2.1) and cirrhosis of the liver (Chapter 6, Section 2.5). From the clinical data presented in this thesis, it is hypothesised that two pools of insulin exist, only one of which contains intact proinsulin. It is hypothesised that the pattern of release of insulin from these pools varies in the basal and stimulated states, and that various pathophysiological conditions, including insulin and noninsulin dependent diabetes mellitus, exhibit altered patterns of secretion which are characteristic of the underlying disease mechanism