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A New Preparative Method for Isolation of Human C3 with Affinity Chromatography¹

From the Laboratory of Immunochemistry from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands

Abstract

A new preparative method for isolation of highly purified human C3 was developed by using a reversed application of affinity chromatography. Antisera against contaminations usually present in crude C3 preparations were produced. These antibodies were irreversibly coupled to Sepharose 4B and as a whole used in column form. When crude C3 preparations were applied to these columns the impurities were bound and thus eliminated. The resulting C3 preparations were pure when tested in concentrations of 5 mg/ml against anti-human serum and when tested by immunizing rabbits. C3 appeared to be homogeneous in the analytical ultracentrifuge, in polyacrylamide gel electrophoresis, and according to N-terminal amino acid analysis. The recovery from 400 ml normal human serum was about 65 mg.

Footnotes

¹ This work was supported by financial aid of the Netherlands Organization for the Advancement of Pure Research (ZWO), The Hague, The Netherlands (Grant 91–5).