Measurement of insulin in human sera using a new RIA kit. 2. Determination of free and total insulin--correlations to insulin antibody levels.

Using the micro-scale modification of a newly developed RIA kit for insulin, we established methods for the determination of free and total insulin in serum of insulin-treated diabetics. Precipitation with polyethylene glycol 6000 or acid alcohol extraction of sera was carried out to remove or to dissociate antibody-bound insulin. Both assays permit precise and accurate measurement of either serum insulin fraction. In 50 diabetic sera with tracer insulin binding of 0-97%, free (after equilibration of the sera at 37 degrees C) and total insulin levels as well as insulin antibody binding parameters were determined. There was a good correlation of free to total insulin levels with maximally 10-fold higher values of total insulin. Both free and total insulin were found to be correlated with the ability of the serum to bind insulin. In detail, binding affinities (i.e. the reciprocal of equilibrium dissociation constants) and binding site concentrations were evaluated which were shown to be positively correlated with free and total insulin levels as well. From these data we conclude that insulin antibodies in the serum may accumulate therapeutic insulin and function as a depot for delivering insulin in insulinopenic episodes (Keilacker et al., 1982 and 1986).