The measurement of digitoxin in human serum by radioimmunoassay

Abstract

A sensitive, specific, and relatively simple immunoassay permitting measurement of pharmacological levels of digitoxin in human serum has been developed. The assay involves binding of (125)I-labeled tyrosine-digitoxigenin (specific activity > 400 mc/mg) by rabbit antibody to digitoxin. Antibody-bound radioactivity is precipitated by addition of a second antibody (goat anti-rabbit gamma globulin), and precipitate radioactivity is measured. Unlabeled digitoxin can be determined by the extent to which it competes with (125)I-labeled digitoxigenin and thus reduces precipitation of radioactivity. Before the assay, unlabeled digitoxin is extracted from serum with chloroform, and the chloroform solution is evaporated to dryness. Quantitation is accomplished by reference to a standard curve in which known amounts of digitoxin are added to normal serum. As little as 1 mmug of digitoxin per ml of serum produces significant reduction in precipitate radioactivity. The sera of 5 patients were analyzed before and after digitalization. A highly significant reduction in precipitate counts in the postdigitalization sera was observed (P < 0.001). Serum digitalis levels were measured in 19 patients receiving no digitalis and in 19 patients taking digitoxin or digitalis leaf. Little of no digitalis-like activity was detected in control sera, whereas serum levels averaged 27 mmug/ml in those on digitalis (range 4-60 mmug/ml, P < 0.001). Patients judged clinically to show digitals toxicity in general had higher levels than those without signs of toxicity. Patients receiving digoxin had little or no detectable digitalis in their serum with this method. In addition to the assay itself, other potential uses of the antidigitalis antibody include treatment of digitalis toxicity and studies on the tissue localization of digitalis.