A highly sensitive affinity-column-mediated immunometric assay, as exemplified by digoxin.

We describe a highly sensitive heterogeneous enzyme-linked immunoassay in which digoxin is used as the model analyte. An excess of enzyme-labeled monovalent antibody is incubated with sample containing the analyte such that all analyte is rapidly and quantitatively bound. Excess antibody that does not acquire a antigen in its binding site is rapidly removed from the mixture by passage through a porous affinity column containing immobilized analyte (or analog), present in vast excess. Only the labeled monovalent antibody that possesses an antigen in its binding site elutes from the column in the unbound fraction. The label present in this fraction is then quantified. Such an assay is extremely sensitive and obviates the limitations imposed by antibody affinity constants on homogeneous and competitive heterogeneous immunoassays. This assay can be performed rapidly and is readily amenable to automation.