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An immunochemical micromethod was designed to estimate total IgG and IgG sub-classes of anti-insulin antibodies in immunized diabetic patients. Insulin, immobilized on a solid phase, was allowed to react with serum samples containing anti-insulin antibodies. Bound anti-insulin IgG interacted with mouse monoclonal antibodies specific for total IgG or for each IgG isotype. The fixation of mouse monoclonal antibody was subsequently detected using a horseradish peroxidase-conjugated rabbit anti-mouse IgG in the presence of a chromogenic substrate. The test was standardized by an immunocapture assay utilizing coated rabbit anti-human IgG and known concentrations of purified human myelomatous proteins of each sub-class. Results of anti-insulin IgG and anti-insulin IgG sub-classes assay could therefore be expressed in ng equivalent myelomatous proteins per ml of serum. Analysis of serum samples from 24 insulin-immunized diabetic patients revealed a quasi absence of IgG2 anti-insulin antibodies and an increase of the relative abundance of the other three anti-insulin IgG isotypes. In our series, anti-insulin IgG1 was predominant, followed by IgG3 (in 17/24 patients) or IgG4 (in 7/24). Insulin immunization was deduced to be of polyclonal nature, the isotype pattern of which is not representative of the relative proportion of IgG sub-classes in whole normal serum.

diabetologia

Semi-quantitative assessment of anti-insulin total IgG and IgG sub-classes in insulin-immunized patients using a highly sensitive immunochemical micromethod