The use of a “hapten-analog” affinity column, lipoyl-Sepharose, is described for the purification of antibodies directed against biotin. The antibodies bind to biotin very tightly ( K d ⋍ 10 −8 m) ( M. Berger, 1975, Biochemistry 14, 2338–2342), and elution of antibody from biotin-Sepharose requires harsh conditions (1–1.5 m acetic acid) and results in a poor yield of antibody, which, upon storage, loses much of its capacity to inhibit the biotin-containing enzyme, transcarboxylase. With lipoyl-Sepharose, the antibody can be eluted in good yield and with high efficacy using 0.1 m acetic acid. In addition, antibody-biotin complexes or complexes of the antibody with biotin enzyme can be eluted directly from antibody-bound lipoyl-Sepharose using solutions of biotin or of biotin-containing enzyme.