Analysis of a system used to detect islet cell stimulating antibodies

Lunt, Helen (1992) Analysis of a system used to detect islet cell stimulating antibodies. University of Southampton, Doctoral Thesis.

Abstract

Several disorders of insulin/glucose homeostasis, such as insulinoma, islet cell hyperplasia, nesidioblastosis, insulin autoimmune syndrome and diabetes have aspects of their aetiology and pathogenesis that remain unexplained. In 1988, preliminary studies performed in the above department, suggested that functional antibodies may be responsible for some cases of the above-described conditions, in a manner analogous to thyroid stimulating and blocking immunoglobulins in autoimmune thyroid disease. This thesis analyses one aspect of these original experiments in detail, namely the ability of human immunoglobulin preparations to stimulate insulin release in a static rat islet culture system. On repeating this initial experiment, several methodological problems were identified which led to difficulties with interpretation of results. These included; i) a systematic variation in rat insulin ELISA results, ii) problems associated with immunoglobulin preparation, as samples contained less immunoglobulin than anticipated and also contained significant amounts of insulin autoantibody and human insulin, despite purification procedures, iii) inter-experiment variability of islet responsiveness to glucose stimulation, independent of islet size, which posed a problem in interpretation of results obtained over the course of several experiments, iv) difficulties in measuring rat insulin response using a polyclonal anti-insulin antibody in the presence of human insulin, v) the steric hindrance effect of the [(anti-insulin-antibody) - (human insulin) - (insulin autoantibody)] complex which interfered with measurement of insulin by ELISA, vi) use of immunoglobulin-free serum as a `negative' control, when this serum fraction displayed evidence of inhibitory activity compared to serum-free rat islet cultures. The predominant effect of these methodological problems was a tendency towards interpreting results as showing evidence of islet cell stimulatory activity, when in fact no evidence of stimulatory activity was seen on reanalysis of results. The final section of this thesis discusses other methodological problems likely to have been present in the original series of experiments and their effect on interpretation of results. In conclusion, the evidence to date suggests that the case for the existence of islet cell stimulating antibodies remains unproven and sensitive methods for detecting the presence of these putative antibodies have yet to be defined.

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