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Affinity chromatography purification of diphtheria toxin

NAD was covalently linked to Sepharose‐4B using a 6 carbon spacer. Sterile, dialyzed spent culture medium containing 100 Lf/ml of diphtheria toxin or material concentrated by (NH4)2SO4 precipitation containing 1500 Lf/ml, was chromatographed on a column of NAD–Sepharose. Ultraviolet absorbing material which did not flocculate with diphtheria antitoxin was eluted with 0.02M phosphate buffer. When the elation buffer was changed to one containing 0.5M NaCl, purified toxin was eluted off the column.