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A highly sensitive enzyme immunoassay of anti-insulin antibodies in human serum is described. Serum samples were subjected to successive processes of incubation with insulin, dextran-charcoal treatment to remove free insulin, precipitation of insulin-anti-insulin antibodies by polyethylene glycol, acid-treatment of the precipitates to inactivate anti-insulin antibodies and measurement of insulin by sandwich enzyme immunoassay technique. The detection limit of anti-insulin IgG in human serum was 50 pg/assay or 450 ng/l of serum. This was 1,000- to 3,000-fold less than that obtained by a conventional enzyme immunoassay, in which an insulin-coated polystyrene ball was incubated with diluted serum and subsequently with (anti-human IgG gamma-chain) Fab'-horseradish peroxidase conjugate. By the present enzyme immunoassay, anti-insulin antibodies were demonstrated in most (89%) of serum samples from diabetic patients who had been treated with porcine insulin and porcine insulin plus bovine insulin for 0.6-10 mth, while only a small proportion (3%) of serum samples from the same patients was positive by the conventional enzyme immunoassay. Similar results were obtained with serum samples from diabetic patients who had been treated with human insulin for 0.5-8.2 mth. The present enzyme immunoassay may be useful for the measurement of antibodies not only for insulin but also other antigens which can be removed by dextran-charcoal treatment and are not precipitated by polyethylene glycol.
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