Abstract
In diabetics, insulin antibodies in the serum interfere with the action of injected insulin. The capacity of insulin to bind to insulin antibodies has been estimated by various methods according to the single and the double antibody system. In the present study, a quantitative determination of insulin binding capacity was devised at two points of the double antibody method of Andersen. First, free insulin in the patient serum was removed with dexran-coated charcoal, because insulin in the serum interfered with the insulin binding capaciy. Secondly, healthy human serum that had been proved not to contain insulin antibodies was used as the control, although in Andersen's method, buffer was used instead of human serum.
The procedure is as follows: 100 pl of serum that has been pretreated with dextran-coated charcoal and diluted, 100 μl of AIS-GP (anti-insulin serum of guinea pig), 100 μl of porcine 125Iinsulin (0.7-0.8 μU) and 500 μl of borate buffer containing 0.5% bovine albumin are mixed and incubated for 96 hours at 4°C. Then 100 μI of anti-guinea pig serum of rabbit (×4) and 100 μl of normal guinea pig serum (×100) are added to the solution and incubated for 24 hours at 4°C. During incubation, the test tubes are carefully shaken several times to mix the solution. The tubes are then centrifuged at 3000 r. p. m. for five minutes. The radioactivity of the precipitate is measured with a well type scintillation counter. The binding capacity of serum bo is calculated with the following formula: bo=a ([B] a/[B] s-1), where a is the binding capacity of AIS-GP that has been estimated by the method of dextran-coated charcoal.[B] a and [B] are the radioactivity of 125I-insulin in the precipitate of the healthy control serum and the patient serum, respectively.
This procedure for the determination of insulin binding capacity is simple. The insulin binding capacity is expressed as μU/ml insulin.
