Affinity-Column-MediatedlmmunoenzymometricAssays:Influenceof Affinity-

We describe an affinity-column-mediated, enzyme-linked im-munometric assay that is highly sensitive and adaptable to automation. Digoxin is the model test analyte. A comparison of digoxin with its analog, ouabain, for use as the immobilized ligand on the affinity column showed ouabain to be superior. We also report the effect of column elution rate. Antibody-enzyme conjugates prepared with the monovalent Fab’-fragment and the divalent F(ab’)2-fragment coupled to /3-galactosidase are compared in terms of their overall assay performance. Although the monovalent Fab’-/3-galacto-sidase conjugate yields a more sensitive assay and dose-response curves that are linear over a wider range, the divalent F(ab’)2-p-galactosidase conjugate provides an as-say with adequate sensitivity and extremely good precision, and is generally easier to synthesize reproducibly. This full