In this study, we used a modified enzyme‐linked immunosorbent assay (ELISA)‐elution technique as a screening tool to select specific elution conditions. We examined 12 different elution conditions for the removal of antibodies from a complex on an ELISA plate; 0.2 mol/l glycine‐HCl (pH 2.5), 1.0 mol/l acetic acid (pH 2.5), 25% methanol (pH 2.5) and 3 mol/l NaSCN showed a higher elution efficiency. We conducted affinity chromatography with these four conditions for the purification of anti‐digoxin antibodies from hyperimmune sera with a digoxin‐specific column using ω‐aminoalkyl derivatives of Sepharose 4B, whose elution efficiency was similar to that of ELISA. We also monitored the relative specific activities during elution from the digoxin‐specific column. The optimum, general‐purpose dissociation reagent for this immunoaffinity system was identified as 25% methanol (pH 2.5) with an elution efficiency and relative specific activity of 88.40% and 62.25%, respectively. The high purity of the purified antibodies was demonstrated with sodium dodecyl sulphate‐polyacrylamide gel electrophoresis.