THE ASSAY OF INSULIN WITH ANTI-INSULIN AND MOUSE DIAPHRAGM

Abstract

A method is described for assaying insulin in which anti-insulin is used as an insulin-neutralizing reagent and glycogen synthesis by mouse diaphragm as an indicator of free insulin. The test is thus analogous to the titration of a toxin with an antitoxin, using a biological indicator to determine the end point. The effect of using antiserum in the diaphragm test is to increase the slope of the dose–response curve about 30-fold, thus giving a test system which is capable of high precision with relatively few observations. This is reflected in the low λ-value of 0.015 for the diaphragm test with antiserum compared with 0.45 for the test without antiserum. Data are presented showing that the immunological test has good reproducibility and is capable of measuring the potency of unknown insulins with acceptable precision using only small numbers of animals. Sixteen samples of insulin in various stages of purification from seven mammalian species gave results by the new method in close agreement with mouse convulsion data. Two samples of fish insulin were found to be immunologically different from mammalian insulin and therefore could not be assayed with anti-ox-insulin sera.