Immunoassay of Insulin: Two Antibody System: Plasma Insulin Levels of Normal, Subdiabetic and Diabetic Rats

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Abstract
A two-step reaction is used in this method. In the first, insulin forms a soluble complex with its specific antibody (AIS/hich is obtained from immunized guinea pigs. In the second, this soluble complex is precipitated by an antibody to guinea pig serum which is obtained from rabbits. When a tracer amount of 1-131 insulin is used, the per cent of radioactivity in the precipitate is dependent upon the concentration of insulin in the reaction mixture; with increasing concentrations of unlabeled insulin, the per cent of 1-131 insulin in the precipitate is decreased correspondingly. Experiments have been carried out to determine the optimal conditions for using the two antibody system. Dilutions of AIS-GP (1:5,000 to 1:20,000) are suitable for assaying small amounts of insulin (1 [mu]U to 100 [mu]U). Although the first step of the reaction may require several days to reach equilibrium, assays can be carried out within six hours; however, a 24-hour period is selected for convenience. Slight differences in the per cent of I-131 insulin precipitated were detectable when the reaction was carried out at 0[degree], 4[degree] or 23[degree]C; however, at 37[degree] the per cent precipitated was much less. As many as 16 [mu]U of I-131 insulin can be used as a tracer when assaying as few as 2[mu]U of insulin. Therefore, commercially available I-131 insulin with a specific activity of 20 to 30 mc./mg. can be used. Although an inhibitor of this immunoassay system is present in undiluted rat plasma, this inhibition can be minimized by using a 1:10 plasma dilution. Assay of plasma insulin levels (expressed as equivalent beef insulin units) were carried out in three groups of rats, ten rats in each group. The mean values obtained were as follows: rats, 104 [plus or minus] 27 [mu]U/ml.; subdiabetics, 56[plus or minus]19 [mu]U/ml.; and diabetics 30 [plus or minus] 12 /[mu]U/ml.