The Use of Affinity Chromatography for the Specific Purification of Antibodies and Antigens

Abstract

Our laboratory has been able to prepare highly efficient immunoadsorbents by coupling haptens or immunoglobulins to sepharose activated with cyanogen bromide (CNBr) (1–3).The general procedures reported by Cuatrecasas et al. (3) for selective enzyme purification by affinity chromatography are readily adapted to the specific purification of antibodies and antigens. These methods make the preparation of superior immunoadsorbents a remarkably easy task.Anti-hapten antibody purification. Sepharose activation is carried out as described (3); we have found it most satisfactory to add the sepharose slurry to a 25 mg/ml aqueous CNBr solution, immediately after adjusting the pH of the latter to 11.5, and the pH is maintained at 11.0 to 11.5 for the 8 to 10 min reaction time.Haptens with a free amino function are coupled to washed activated sepharose in 0.1 N sodium bicarbonate buffer, pH 9.0, at 4° to 5°C, also as previously described (3); we react 4 µmols of hapten/ml wet activated sepharose.