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 125I-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 125I-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 mμg of digitoxin per ml of serum produces significant reduction in precipitate radioactivity.
George C. Oliver Jr., Brent M. Parker, Daniel L. Brasfield, Charles W. Parker