Mohamed-Ali, Vidya; (1997) Developing sensitive and specific assays for human des 64, 65 and des 31, 32 proinsulin using antibodies raised in guinea-pigs. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The recent development of techniques to differentiate proinsulin and its intermediate conversion products, des 31, 32 and des 64, 65 proinsulin, from insulin have permitted a clearer understanding of the role of these molecules. Elevated concentrations of the des 31, 32 proinsulin may provide a marker for β-cell dysfunction and these molecules may also have a role in some of the abnormalities of cardiovascular risk in both diabetic and non-diabetic subjects. However, the sensitivity of currently available assays for des 64, 65 proinsulin is inadequate, and that for des 31, 32 proinsulin just adequate, to measure their levels in normal and diabetic subjects. Furthermore, even employing current two-site assays, substantial cross-reaction is observed with intact proinsulin. Lack of high affinity, specific antibodies to the two molecules have hindered the improvement of these assays. Polyclonal antibodies to des 31, 32 and des 64, 65 proinsulins were raised in guinea-pigs which showed high affinity and specificity. Attempts to produce guinea-pig x mouse heterohybridomas were made but proved unsuccessful as the guinea-pig spleen cells appeared to fuse to the mouse plasmacytoma cells but remained stagnant after fusion. Monoclonal antibodies with a wide range of specificities were produced by altering the route of immunisation and using forms of the propeptides not previously tried in mice, the des 31, 32 and des 64, 65 forms, prior to removal of lymphocytes for fusion. A combination of the polyclonal and monoclonal antibodies were then selected and used to develop a specific, 2-site assay for human des 64, 65 proinsulin, while the sensitivities of existing assays for intact and des 31, 32 proinsulin were improved. All the assays were set up as microplate immunoradiometric assays (IRMAs), where the 'capture' antibody was adsorbed onto microtitre plates and the detecting antibody was labelled with I125. The sensitivity of the assays for intact and des 31, 32 proinsulin, as ascertained by measuring the zero standard 20 times (mean cpm + 2 SD), was 0.12 pmol.1-1 for intact proinsulin and 0.49 pmol.1-1 for des 31, 32 proinsulin. There was no cross-reactivity with human insulin (up to 2500pmol.1-1), IGF-1 (up to 2500pmol.1-1) and C-peptide (up to 100nmol.1-1). There was no cross-reactivity with des 31, 32 proinsulin in the intact proinsulin assay, but 60% cross-reactivity of intact proinsulin in the des 31, 32 proinsulin assay. The concentration of des 31, 32 proinsulin was calculated as follows: Corrected counts for des 31, 32 proinsulin = (assayed counts for des 31, 32 proinsulin) - (0.60 x assayed counts for intact proinsulin). The corrected counts were then used in the standard curve for des 31, 32 proinsulin and the concentration of des 31, 32 proinsulin determined. Mean recovery of intact or des 31, 32 proinsulin added to human plasma (0.98–62.5 pmol.1-1) was 95.8% range (90.0–107.1%) and 94.8% range (90.0–100.7 %) respectively. The assay for des 64, 65 proinsulin used guinea-pig antibody coated plates (GP10) and showed only 25–30% cross-reaction with intact proinsulin which was reduced to less than 5% (at 250pmol.1-1) after adsorption of the polyclonal antibody with intact proinsulin, prior to use as the capture antibody. The assay showed no cross-reaction with des 31, 32 proinsulin (up to 250pmol.1-1) or insulin (up to 2500pmol.1-1). The sensitivity of the assay was 0.12 to 0.06pmol. 1-1. A minimum validation was carried out due to the limited amount of adsorbed antibody available. Recovery of des 64, 65 proinsulin added to human plasma or 5% BSA/ Tris buffer (n= 5) at 2.5 pmol.1-1 was 98.4% range 92–105%, at 7.6 pmol.1-1 was 89.0 range 84–95% and at 16.0pmol.1-1 was 102.8% range 98 to 104 %. The inter and intra-assay CVs at 3.0pmol.1-1 were 12.3 and 9.3%, at 8.5 pmol.1-1 were 7.4 and 8.2 % and at 15.6 pmol.1-1 were 6.9 and 11.0% respectively. In order to allow some speculation of a possible role for the proinsulins in the various physiological conditions their levels were determined in the following subject groups, under the following conditions and seeking the following relationships: i) non-diabetic subjects, ii) in subjects with non-insulin- dependent diabetes mellitus, iii) the effect of hypoglycaemic therapy, iv) in diabetic and non-diabetic cirrhotic subjects, v) their interaction with PAI-1, vi) their relationship to IGFs and IGFBPs and lastly vii) in subjects with insulinomas. These studies confirmed raised levels of the propeptides in subjects with NIDDM, and their concentrations showed statistically significant correlations with a range of established cardiovascular risk factors such as insulin resistance, lipids and fibrinogen. Significant statistical interactions were also seen with novel cardiovascular risk factors such as PAI-1 and IGFBP-1. Of course, statistical correlation is not proof of causation, so proinsulins cannot necessarily be implicated directly in determining abnormal levels of risk factors. The cross-reactivity of these assays with non-specific assays for insulin were previously thought to account for the hyperinsulinaemia seen in various conditions. However, these studies, using specific assays for insulin, intact proinsulin and des 31, 32 proinsulin, still demonstrate the presence of hyperinsulinaemia. In the last study all the insulin-like molecules, including des 64, 65 proinsulin, were measured in subjects with histologically proven insulinomas, where the levels of these proinsulin-like molecules are likely to be most abnormal, and found to not account for greater than 10% of all insulin-like molecules. This suggests that des 64, 65 proinsulin species may be of very little physiological relevance.

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