The improvement of specificity in radioimmunoassays

Abstract

The specificity of a radioimmunoassay for serum digoxin was investigated. The assay utilised [ 3H]digoxin and rabbit antiserum to a digoxin-albumin conjugate, with separation of antibody-bound and free tracer by adsorption of the latter on charcoal. In reaction mixtures where [ 3H]digoxin and the unlabelled steroid were mixed simultaneously with the antiserum, there was considerable cross reaction with levels of spironolactone, aldadiene, prednisone, prednisolone, and digitoxin which could be encountered in vivo. However, by exploiting the kinetics of dissociation of the steroid-antibody complex, digoxin could be distinguished from these other steroids. In the second assay system, the serum containing the unlabelled steroid was preincubated with the antiserum for 2 h before the addition of [ 3H]digoxin. During the 15-min incubation with the latter, complexes established between antibody and unlabelled digoxin during precipitation are broken to a considerably lesser extent than associations between antibody and steroids other than digoxin. Thus the comparison of apparent digoxin levels measured in the two types of assay based respectively on the principles of simultaneous addition and preincubation imposes a stringent specificity test in the assay of serum samples. The double assay principle is suggested for consideration in all radioimmunoassays utilising incompletely specific antisera.