Critical Variables in the Radioimmunoassay of Serum Insulin Using the Double Antibody Technic

An adaptation of the double antibody radioimmunoassay for insulin originally presented by Morgan and Lazarow is described. These studies have confirmed the presence of an inhibitor in serum and plasma (heparin) which delays the rate at which the insulin-insulin antibody complex is rendered insoluble by rabbit serum containing antibodies to guinea pig globulin. Serum dilution reduces the effect of the inhibitor but increases dilutional error. Heparin similarly reduces the effect of the inhibitor, but excess heparin produces falsely low values for immunoreactive insulin (IRI). Data are presented for serum which show that if sufficient time (72 hrs.) is allowed to elapse after the addition of the precipitating antibody (rabbit, anti-guinea pig globulin, serum), the precipitating system reaches equilibrium. Employing this modification, additional data are presented, showing excellent recovery of human insulin added to serum, good duplication of respective serum IRI values in repeat assays, and constancy of fasting serum IRI in any one individual on repeated sampling. Normal adults exhibit fasting values of serum IRI from 1 to 20 μU./ml. with a mean of 8.4 μU./ml., and after rapid intravenous glucose, serum IRI usually reaches maximum levels in one to two minutes.