An Immunochemical Assay of Total Extractable

For the precise assay of minute quantities of insulin in biological fluids, techniques using fibril formation (1) or paper chromatography (2), although specific for the insulin molecule, lack the required sensitivity. Methods based on the measurement in vivo of a fall in blood sugar in surgically modified rats (3) afford sensitivity, but lack both specificity and precision. In zvitro biological assays, in which glucose metabolism is measured in rat diaphragm or epididymal fat, while perhaps more precise than in vi*o methods, are subject to interference by other hormones such as somatotropin and epinephrine (4, 5), by unknown "insulin-like" substances whose extraction characteristics differ from those of insulin (6) and by numerous inhibitors of insulin activity (7-10). In contrast, an immunological method should be both chemically specific and, especially after application of an isotopic method for the immunologic quantitation, reasonably sensitive and precise. Berson and co-workers have originally reported (11) that when insulin-'131 is added to an excess of antibodies, it rapidly becomes bound. Furthermore, addition of unlabeled insulin in increasing quantities results in gradual saturation of the binding sites, reflected by an increase in the percentage of unbound insulin-I"'. We have subsequently confirmed these observations (12). The change in the percentage of unbound labeled insulin forms the basis of methods recently described for the measurement of injected insulin in rabbit plasma (13) and extracted insulin from mouse pancreas (14). Both of these methods utilized hydrodynamic flow techniques for the determination of the percentage of unbound insulin. The modification of this approach for the assay of circulating lhumiian insulin is the subject of this