Evidence for the presence of inhibitor and some trials for its prevention in radioimmunoassay of plasma glucagon by two-antibody system

The radioimmuno assay for glucagon using two-antibody system was described in detail. The value of sensitivity ranged 0.06 to 0.60mμg on the standard curves obtained with crystalline glucagon (0 to 14mμg per ml) in buffer solution. The precision of the standard curves was 0.8 to 1.5 in per cent precipitated radioactivity for 0 to 14mμg per ml range of glucagon. Glucagon added to human plasma was recovered at the rate of 74.6 to 116.3 per cent. The glucagon level in fasting human plasma measured by this method showed a wide range of variety and even high values (2.2 to 7.1mμg/ml) were obtained in non-diabetics than in diabetics who gave the values of 1.5 to 5.6 mμg/ml. Human plasma globulin reduced the percentage of precipitated radioactivity, whereas human plasma albumin did not appear to interfere with the immunological reaction. Since human plasma globulin did not exhibit any effect upon the rate of bound radioactivity in paper chromatography, it is suggested that the human plasma globulin may inhibit the immunological reaction at the second reaction. The cross reaction between antirabbit serum goat serum (ARGS) and human plasma or plasma globulin was observed on the immunoelectrophoresis and this was regarded as one of the inhibitors. EDTA failed to reduce the inhibition. The inhibition could be minimized by the extraction of plasma glucagon using acid-ethanol acetone method, by absorption of cross reactant in ARGS by a small amount of human globulin and by the use of anti-rabbit serum globulin goat serum. Even in the assay using anti-rabbit serum globulin goat serum as the second antibody, however, the rate of precipitated radioactivity in the assay of the standard glucagon added to the pooled human plasma with the minimun glucagon was lower than expected. This suggests the presence of another inhibitor (s) in human plasma and the necessity of the use of glucagon-free human plasma in the preparation of the standard glucagon solution.