The Preparation of a Reagent for the Titration of Human Complement Components C′8 and C′9

Abstract

When applied to human serum, the methods previously described by Nelson, Rommel and Stolfi for the preparation of a mixture of complement (C′) components from guinea pig serum suitable for the titration of C′8 and C′9 proved unsatisfactory. The reagents so obtained were contaminated with considerable quantities of C′8 and C′9 and were deficient in C′2. Three methods have been developed to separate a suitable human complement reagent mixture, using serum essentially free of C′1 as starting material. 1) Column chromatography on carboxymethyl (CM)-cellulose at 0.07 M relative salt concentration (RSC), pH 5.0, followed by diethylaminoethyl (DEAE)-cellulose at 0.16 M RSC, pH 7.5; 2) hydroxylapatite chromatography at 0.10 M PO4, pH 6.9, followed by Sephadex G-200 gel filtration; 3) DEAE-cellulose chromatography at 0.035 M RSC, pH 7.0, followed by G-200 gel filtration. These methods have yielded reagents containing sufficient C′4, C′2, C′3, C′5, C′6 and C′7 for the generation of the stable cellular intermediate (EAC 1423567).