Isolation of lymphocyte membrane complement receptor type two (the C3d receptor) and preparation of receptor-specific antibody.
A glycoprotein binding complement component C3d was isolated from media used for culture of Raji human lymphoblastoid cells. Analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gas/liquid chromatography indicated that the C3d-binding glycoprotein consisted of a single polypeptide chain with extensive intrachain disulfide bonds, a molecular weight of 72,000, and several different bound carbohydrates. Several lines of evidence indicated that this medium-derived C3d-binding protein originated from membrane complement receptor type two (CR2, the C3d receptor), presumably shed during membrane turnover. The C3d-binding protein bound to sheep erythrocytes coated with C3d (EC3d) but not to sheep erythrocytes coated with C3b (EC3b). Antisera, prepared by immunization with the purified C3d-binding glycoprotein, inhibited lymphocyte rosette formation with EC3d but not with EC3b. Analysis by sodium dodecyl sulfate gel electrophoresis of the radiolabeled and solubilized lymphocyte antigens reactive with the anti-C3d-binding protein sera revealed a single-chain cell-surface protein of molecular weight 72,000 that was apparently identical to the isolated C3d-binding protein. Parallel assay of lymphocytes for Cr2 by direct immunofluorescence with F(ab')2 anti-C3d-binding protein (anti-CR2) and rosette formation with EC3d indicated that both assays had the same specificity and nearly the same sensitivity. With both systems CR2 expression was limited to B cells, and was undetectable on T cells, monocytes, or neutrophils.
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D006801:Humans; D007945:Leukemia, Lymphoid / Q000276:immunology; D008970:Molecular Weight; D011817:Rabbits / Q000276:immunology; D011951:Receptors, Complement / Q000276:immunology*; D017464:Receptors, Complement 3d; D012397:Rosette Formation
D000906:Antibodies; D000936:Antigen-Antibody Complex; D011951:Receptors, Complement; D017464:Receptors, Complement 3d