Purification of Complement Components from Human Serum and Standardization of Reagents Used in Their Assay

摘要

The nine known components of human complement and C'1 inactivator were purified. The report gives procedures involving (a) precipitation at strength, pH 7.5 and 5.5, (b) salting out with high concentrations of ammonium sulfate, (c) DEAE- and CM-cellulose column chromatography, and (d) gel filtration with Sephadex. Improved purification procedures were aimed at increasing production, especially of C'6 and C'7. Both solubilization chromatography and hydroxylapatite separation were explored (for C'2, 6, 7, and C'3 from C'5). Guinea pig serum was processed in starting quantities of 25 to 500 m1. Most of the human and guinea pig components, with the exception of C'4 hu, are stable for several months at -70C in the presence of 1% gelatin. Human C'4, as harvested, is extremely labile to freezing and storage of this component at 0C in the presence of 1% gelatin results in no loss of activity over prolonged periods of storage. An attempt was made to standardize and characterize antibodies to sheep erythrocytes, to prepare stable cellular intermediates for component assay (EAC'1, EAC'1,4, EAC'4 and EAC'1-7), and to develop a C'4-7 package. (Author)