Structural and functional analysis of CR2/EBV receptor by means of monoclonal antibodies and limited tryptic digestion.
The receptor for the C3d fragment of the third component of complement, CR2, has recently been shown also to act as the receptor for the Epstein-Barr virus (EBV) and to be involved in the control of B-cell proliferation. In order to define functionally important domains on this molecule, we produced monoclonal antibodies to several distinct epitopes. CR2 was purified from a NP-40 lysate of human tonsils by a new method involving sequential chromatography on lentil lectin Sepharose 4B and DEAE-Sephadex and used to immunize mice. After fusion we obtained four stable hybridoma lines producing antibody to CR2. Specificity of these antibodies for CR2 was ascertained by immunofluorescence analysis on a panel of various cells known to possess CR2, by their reactivity in a recently described ELISA for C3 receptors, by Western blotting with purified CR2 and immunoprecipitation from 125I-labelled Raji cells. These four antibodies were found to recognize three distinct epitopes localized on the same fragments of 95,000, 72,000, 50,000, 32,000 and 28,000 MW obtained after mild tryptic digestion of CR2. The 72,000 MW fragment contains the binding site for C3d. Two monoclonal antibodies recognizing the same epitope did not inhibit the binding of C3d-coated sheep erythrocytes to Raji cells, whereas the other two antibodies against distinct epitopes did inhibit in the presence of a second antibody. All four monoclonal antibodies stimulated the proliferation of human peripheral blood B cells.
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology; D003176:Complement C3 / Q000276:immunology*; D000939:Epitopes / Q000032:analysis; D006801:Humans; D008970:Molecular Weight; D011951:Receptors, Complement / Q000032:analysis* / Q000276:immunology; D017464:Receptors, Complement 3d; D014357:Trypsin
D000911:Antibodies, Monoclonal; D003176:Complement C3; D000939:Epitopes; D011951:Receptors, Complement; D017464:Receptors, Complement 3d; D014357:Trypsin