Active site radioimmunoassay for human urokallikrein and demonstration by radioimmunoassay of a latent form of the enzyme.
A radioimmunoassay specific for the active site in urokallikrein has been developed with a monospecific antibody that neutralizes the enzymatic activities of urokallikrein and a radioligand purified so as to maintain the active site. In order to favor the involvement of the antibody with high affinity for the active site in the competition between urokallikrein in biological fluids and radiolabeled urokallikrein, the radioligand was separated from denatured radiolabeled urokallikrein by affinity chromatography for the active site. The concentration of specific IgG used in the assay bound approximately 80% of the radioligand, which was displaced in a dose-related fashion by 0.2 to 2.5 ng of the unlabeled urokallikrein. When random urine samples from 21 healthy volunteers were assessed for endogenous urokallikrein by both active site radioimmunoassay and kinin generation, there was a linear relationship between the results of the two assays with a correlation coefficient of 0.89. A combined correlation plot of the results of the bioassay and the active site radioimmunoassay for the 21 urine samples before and after trypsin activation gave a linear regression line with a correlation coefficient of 0.91. The finding that trypsin activation increased the urokallikrein concentration of urine to a similar extent in both the radioimmunoassay and the bioassay means that latent urokallikrein was not detected until its active site was uncovered.