The double-antibody immunoassay of insulin : A standardized second antibody reaction that eliminates spurious results with human serum.

1. Tracer(131)I-guinea-pig γ globulin provides a useful check of the efficiency of the insulin-antibody precipitation. It is employed (a) for the accurate preliminary standardization of the precipitating antibody; and (b) during assays to assess completeness of precipitation (in monitor tubes). 2. Similar assays of human serum frequently yield spuriously high results; two responsible factors are (a) suboptimal second antibody precipitation arising from accentuation by serum of the "prozone phenomenon", and (b) cross-reaction of rabbit anti-guinea-pig serum with human proteins. The former can be overcome by using ideal concentrations of reactants, prolonging incubation, and incorporating a standard amount of heparin, during the second reaction. The latter is minimized by using a rabbit anti-serum made to pure guinea-pig γ globulin. 3. The double antibody method, thus modified, has proved consistently reliable. In the assay of human serum samples diluted 1/10 (i.e. 0.1 ml volumes of serum) optimal precipitation always occurs. Undiluted serum samples (i.e. 1.0 ml volumes) can also be assayed satisfactorily, when the occasional sub-optimal precipitation can be identified and a correction derived from the monitor tubes to yield a valid reading.