Immunoprecipitation of the Insulin Receptor: A Sensitive Assay for Receptor Antibodies and a Specific Technique for Receptor Purification*

Abstract

Autoantibodies that inhibit the binding of insulin to its receptors and elicit insulin-like biological responses are found in the sera of certain patients with severe insulin resistance. In the present study, five of these antireceptor sera inhibited binding of insulin to the Triton-solubilized insulin receptor at titers comparable to those for inhibition of binding to whole cells. Studies with the highest titer serum indicated that inhibition of insulin binding to the solubilized receptor was due to a reduction in the number of available binding sites, whereas with receptors in the intact membranes, the predominant effect of this antireceptor serum was to decrease receptor affinity. In addition to their ability to inhibit insulin binding, all of the sera were capable of completely precipitating the [¹²⁵I]insulin-receptor complex in an indirect immunoprecipitation assay; immunoprecipitation required the addition of either antihuman immunoglobulin G (IgG) or Staphylococcal protein A-Sepharose. Insulin antibodies in the sera could be measured independently in the assay. The titers for immunoprecipitation of receptor ranged from 1:50 to 1:6400 and were higher than the corresponding titers determined for inhibition of insulin binding. IgG prepared from the serum with the highest titer had a precipitating capacity of 24 mmol insulin-binding sites/mol IgG. Other hormones (epidermal growth factor, human GH, and ovine PRL) which were specifically bound to their receptors in the solubilized placental membranes were not precipitated using these sera. These findings 1) demonstrate the direct interaction of antireceptor antibodies with the insulin receptor; 2) show that immunoprecipitation is a sensitive assay for insulin receptor antibodies which may detect antibodies against determinants on the receptor that do not affect insulin binding; and 3) indicate the feasibility of isolating the insulin receptor using these antibodies as affinity reagents.