Production and characterization of a murine monoclonal IgM antibody to human C1q receptor (C1qR).
A hybridoma cell line that produces a monoclonal antibody (MAb) to cell surface C1q receptor (C1qR) has been produced by fusion of the P3 X 63-Ag8.653 mouse myeloma cell line with the spleen cells of a CD-1 mouse that had been hyperimmunized with viable Raji cell suspensions (5 X 10(7) cells/inoculum). This MAb, designated II1/D1, is an IgM antibody with lambda-light chain specificity. Radiolabeled or unlabeled, highly purified II1/D1 was used to determine that: this antibody competes for C1q binding sites on C1qR-bearing cells; the molecule recognized by this MAb is the C1qR; and cells that are known to bind C1q also bind II1/D1 in a specific manner. Western blot analysis of solubilized Raji, or U937 cell membranes, showed that the 125I-MAb detected a major protein band of approximately 85,000 m.w. in its unreduced state, indicating that the C1qR is similar, if not identical, in both types of cells. Analyses of 125I-II1/D1 binding experiments revealed that the antibody bound to Raji cells or U937 cells in a specific manner. Uptake of the antibody was saturable, with equilibrium virtually attained within 35 min. Scatchard analysis of the binding data using the intact MAb suggests that the affinity constant KD is 2.9 X 10(-10) M, and at apparent saturation, 24.6 ng of the antibody were bound per 2 X 10(6) cells, giving an estimated 7.8 X 10(3) antibody molecules bound per cell. That the II1/D1 antibody is specifically directed to the C1q was further evidenced by an ELISA in which the ability of C1qR-bearing cells to bind the MAb was abrogated by c-C1q in a specific and dose-dependent manner. These results indicate that the II1/D1 is a specific antibody directed against the C1q and can be a useful tool in studying the biologic interaction of human C1q with its receptors on a variety of cells.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D001666:Binding Sites, Antibody; D002352:Carrier Proteins; D002460:Cell Line; D003101:Collodion; D003166:Complement Activating Enzymes / Q000378:metabolism*; D015922:Complement C1q; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors; D006825:Hybridomas / Q000378:metabolism; D007075:Immunoglobulin M / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D008562:Membrane Glycoproteins; D051379:Mice; D024101:Mitochondrial Proteins; D011951:Receptors, Complement / Q000276:immunology*
D000911:Antibodies, Monoclonal; C087299:C1QBP protein, human; C490193:C1qbp protein, mouse; D002352:Carrier Proteins; D018960:Hyaluronan Receptors; D007075:Immunoglobulin M; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D011951:Receptors, Complement; C032402:complement 1q receptor; D015922:Complement C1q; D003101:Collodion; D003166:Complement Activating Enzymes