Purification of Subcomponents Clq, Cl[rbar] and Cl[sbar] of the First Component of Complement from Cohn Fraction I by Affinity Chromatography

Abstract A method is described for purification of Clq, Cl[rbar] and Cl[sbar] from Cohn Fraction I paste. Initial adsorption of the Cl complex is done by affinity chromatography on an IgG-Sepharose resin in which the IgG is attached to the agarose polymer by an azo-benzyloxyethylsulfono-ethoxy [-N=N-C6H4-CH2O-(CH2)2 -SO2-(CH2)2-O-] “arm”. The Cl[rbar] and Cl[sbar] are co-eluted from this resin by 100 mM EDTA, pH 7.4. The Cl[qbar] is eluted by a buffer containing 200 mM diaminopropane - 1 M NaCl - 200 H3BO3, pH 7.4. The Cl[rbar] and Cl[sbar] are then resolved by DEAE ion exchange chromatography. The Cl[sbar] is purified by rechromatography on DEAE, but the Cl[rbar] requires adsorption with an anti-Cl[sbar] antibody resin to remove the remaining Cl[sbar]. The Clq is purified in two further steps, affinity chromatography on IgG-Sepharose and gel filtration chromatography. Between 100 and 200 mg each of Clq, Cl[rbar] and Cl[sbar] are obtained in a high degree of purity from 8 to 10 kg of Cohn I paste. Biochem...