Human IgG-Sepharose Research Articles

Studies of Fc gamma receptors of human B lymphocytes: phospholipase A2 activity of Fc gamma receptors.

The presence of phospholipase A2 activity within human B cell Fc gamma receptors was investigated. Lysate produced by detergent treatment of chronic lymphocytic leukemia cells that had 1% of the cells surface radioiodinated was subjected to affinity chromatography by using either rac-1-(9-carboxynonyl)-2-hexadecylglycero-3-phosphorylcholine-Sepharose (PC-Sepharose) or heat-aggregated human IgG-Sepharose 4B conjugate (IgG-Sepharose). The materials eluted from both adsorbants by ethylenediaminetetraacetate- or urea-containing buffer were further purified by gel filtration and isoelectric focusing in the presence of 6 M urea. Both isolated PC- and IgG-binding materials were homogeneous, when judged by gel filtration and isoelectric focusing, and had identical isoelectric points (pI = 6.5), peptide maps, and amino acid compositions. Furthermore, both preparations catalyzed equally the hydrolysis of phosphatidylcholine to release fatty acid from the 2 position. Optimal enzymatic activity depended on the presence of Ca2+, was maximal at pH 9.5, and was augmented by Fc gamma fragments. Both preparations specifically bound to the Fc portion of IgG and inhibited human antibody-coated erythrocyte rosette formation by peripheral mononuclear cells. Our data thus demonstrate the identity of PC- and IgG-binding materials and suggest that a functional activity of the human B cell Fc gamma receptor is the generation of phospholipase A2 activity within the plasma membrane.

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

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.