Measurement of Antibodies to Insulin in Serum

I describe a method for measuring insulin antibodies in insulin-treated subjects. The antibodies are character-ized by maximum binding capacity and affinity constant. Endogenous and therapeutic insulin interferes in insulin antibody assay, and it is removed by dextran-charcoal treatment at pH 3.5. Endogenous total and free insulin may be measured by a simple extension of the method. Addftlonal Keyphrases: maximum binding capacity #{149}af-finity constant #{149}dextran-charcoal adsorption #{149}mea-surement of total or free insulin #{149}diabetes treatment #{149} antibody stability #{149}acid charcoal treatment #{149}insulin binding curve Antibodies to insulin develop in the serum of many patients who are receiving insulin treatment. The antibody-insulin complex does not precipitate, either in vivo or in vitro (1), and the serum antibodies are usually detected by their ability to combine with ra-dioactive insulin. Quantitation of such antibodies presents a number of difficulties. Thus the concentration of insulin-binding antibody in serum may lie in a wide range, 0.05 to 50 units of insulin per liter. Also such sera usually contain therapeutic insulin, which competes with radioactive insulin used in “in vitro ” tests of in-sulin binding. This interference is most marked for sera containing large amounts of antibody, because such sera also usually contain a large amount of insu-lin. Their antibody content is usually underestimated in methods that depend on percentage binding of ra-dioactive insulin at a fixed dilution of serum. This difficulty has been overcome by aging sera at 4 Cc (1), endogenous and therapeutic insulin being slowly destroyed, or by drawing blood some time after the last insulin therapy, when insulin concentration is as-sumed to be low (2, 3). However, insulin concentra-tion does not necessarily decrease to low values 24 h after insulin injection in patients whose sera contain a substantial amount of antibody (4). The procedures used to partition combined and free radioactive insu