Equilibrium Binding Studies of Insulin Antibodies in Diabetic Subjects

Equilibrium binding of insulin to human insulin antibodies from diabetic subjects has been studied by two methods. Results obtained by using a nondissociating gel filtration assay differed from those obtained by a conventional competitive binding method. Both methods yield data consistent with two classes of insulin binding site, having association constants of approximately i09 and i0 liter/mol. The principal differences are the higher asso-ciation constant and lower number of binding sites for the high-affinity binding component, observed with the gel filtration method. In the conventional competitive binding assay, damaged radioligand, dissociation of the binding complex, or limitations of ligand availability may be re-sponsible for the erroneous results. Data from nine diabetic patients indicate that insulin resistance and stability of diabetes cannot be explained by the binding properties of insulin antibodies alone. AddItIonalKeyphrases: inter-method comparison #{149}insulin resistance #{149}management of diabetes The development of radioligand assays has permitted the measurement of hormones, drugs, and other sub-stances at very low concentration in a wide variety of biological situations. These techniques have also been used to characterize the properties of binding proteins, including various antibodies and receptors (1, 2). Binding data of this type may be used to estimate pro-tein binding capacity and affinity. Accurate knowledge of the affinity constant (K) is important in the design of any radioligand assay, because this parameter de-termines sensitivity (3) and optimal tracer and antibody concentrations (4). Inaccuracies in the estimation of affinity and binding capacity may result from the use of high concentrations of binding protein and from disturbance of the binding equilibrium during the separation of bound and free ligand (5)