Temperature-dependent immunoreactive assay to screen for digoxin-like immunoreactive factor(s).

Endogenous circulating digoxin-like immunoreactive factors (DLIF) are known to cross-react with antibodies to digoxin and to inhibit Na+/K(+)-transporting ATPase (Na+K+ATPase; EC 3.6.1.37). Moreover, increasing the immunoassay temperature from 4 to 37 degrees C markedly decreases DLIF from human cord serum. We tested several compounds, including hormonal steroids, bile salts, lipids, and methionine-enkephalin, for their ability to cross-react with two commercially available 125I digoxin RIAs, to inhibit porcine Na+K+ATPase, and to see whether they present the same incubation temperature dependence as human cord serum. Except for methionine-enkephalin, all compounds were inhibitors of Na+K+ATPase in the range of 1-10 mmol/L. Progesterone exhibited the highest cross-reactivity in the two RIAs. The apparent digoxin immunoreactivity for the majority of the cross-reacting steroids, bile salts, and linoleic acid was markedly decreased by increasing the incubation temperature from 4 to 37 degrees C, whereas estriol, pregnanediol, and nonspecific compounds (e.g., ethanol, human serum albumin) did not appear to be temperature-sensitive. Both lysophosphatidyl lipids gave an increased apparent digoxin concentration with increasing incubation temperature. Our data suggest that numerous weakly cross-reactive compounds can parallel the response of human cord serum. However, the temperature-dependent effect could be an additional criterion for identifying DLIF.