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/sup 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: a) this antibody competes for C1q binding sites on C1qR-bearing cells; b) the molecule recognized by this MAb is the C1qR; and c) 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 /sup 125/I-MAb detected a major protein band of approximately 85000 m.w. in its unreduced state, indicating that the C1qR is similar, if not identical, in both types of cells. Analyses of /sup 125/I-II/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 K/sub D/ is 2.9 x 10/sup -10/ M, and at apparent saturation, 24.6 ng of the antibody were bound per 2 x 10/sup 6/ cells, giving an estimated 7.8 x 10/sup 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 dose-dependent manner.
- Research Organization:
- State Univ. of New York, Stony Brook
- OSTI ID:
- 5459240
- Journal Information:
- J. Immunol.; (United States), Vol. 137:2
- Country of Publication:
- United States
- Language:
- English
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HYBRIDOMAS
IMMUNOGLOBULINS
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SPLEEN CELLS
TRACER TECHNIQUES
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MEMBRANE PROTEINS
NUCLEI
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PRIMATES
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551001* - Physiological Systems- Tracer Techniques