Immunoprecipitation of 125I-insulin crosslinked receptor

Abstract

125I-insulin was covalently crosslinked to its receptor on human placental membrane fractions with disuccinimidyl suberate. The 125I-insulin crosslinked receptor was solubilized with Triton X-100 and used as a probe to determine autoantibodies to the insulin receptor in sera from patients with insulin resistance (type B syndrome). When the solubilized 125I-insulin: receptor complex was incubated with these sera and then with anti-human IgG serum, the complex was precipitated as a function of the amount of anti-receptor serum. Unlabelled insulin at concentrations less than or equal to 10 micrograms/ml did not affect immunoprecipitation of the complex, suggesting that a subpopulation of anti-receptor antibodies recognizes determinants outside the insulin binding region of the receptor molecule. 125I-insulin: receptor complex was precipitated also by the addition of anti-insulin antibodies. The effect of anti-insulin antibody was eliminated by the addition of excess unlabelled insulin. The immunoprecipitation assay using 125I-insulin crosslinked receptor was sensitive and convenient for detecting anti-receptor antibodies.