Abstract A new method for affinity purification is described. A heterobifunctional ligand in a soluble form is added to the sample containing the substance to be purified. After binding of the substance with one of the functional groups of the ligand, the complex formed is isolated by passage through a sorption column having affinity for the second part of the heterobifunctional ligand. In a stepwise elution process, first the substance of interest is isolated, followed by the ligand which can then be reused. Model studies on the purification of lactate dehydrogenase using a heterobifunctional ligand containing Cibacron Blue and soy bean trypsin inhibitor are described. The affinity matrix used was trypsin immobilized on Sepharose.