C1q: Isolation from Human Serum in High Yield by Affinity Chromatography and Development of a Highly Sensitive Hemolytic Assay

Abstract

C1q, a subcomponent of the first component of complement, has been isolated from human serum in fully hemolytically active form by affinity column chromatography and gel filtration with Bio-Gel A-5M. The affinity column was prepared by covalent coupling of purified human IgG to CNBr-activated Sepharose 4B. Final yields of C1q ranged from 25 to 40% with 650- 890-fold purification based on recovery of hemolytic activity. The preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide gel electrophoretic and immunochemical criteria. The final C1q preparations were also devoid of any demonstrable C1q-inhibitor activity. A C1q-depleted reagent (C1qD) was obtained from the nonabsorbed protein containing fractions of the human IgG-Sepharose 4B affinity column and utilized in conjunction with sensitized sheep erythrocytes (EA) for the detection and quantitation of C1q hemolytic activity. Employing optimal quantities of C1qD in the hemolytic assay mixture, the highly purified C1q preparations contained 0.5 to 1 x 10(13) effective molecules/mg and 0.5 to 1 x 10(12) effective C1q molecules/ml of human serum. This assay would therefore reproducibly detect less than 1 ng of C1q hemolytic activity.