Monoclonal and anti-idiotypic anti-EBV/C3d receptor antibodies detect two binding sites, one for EBV and one for C3d on glycoprotein 140, the EBV/C3dR, expressed on human B lymphocytes.
Glycoprotein (gp) 140, the EBV/C3dR of B lymphocytes, is a membrane site involved in human cell regulation. To analyze the specificities of the binding sites for EBV and for C3d on the gp 140 molecule, two distinct approaches were used. First, anti-EBV/C3dR mAb were prepared against highly purified EBV/C3dR. Nine anti-EBV/C3dR mAb were obtained. Four of these anti-EBV/C3dR mAb inhibited C3d binding but not EBV binding on gp 140, whereas four others exerted an inverse effect. These differences could not be due to differences in isotype, antibody concentration, affinity constant, and number of molecules bound on cell surface, as these parameters were identical for the nine used mAb. Second, polyclonal anti-idiotypic antibodies (Ab2) were prepared against F(ab)'2 fragments of polyclonal anti-EBV/C3dR (Ab1). Ab2 recognized the variable portion of Ab1 as controlled by immunoblotting experiments. Ab2, which did not react with the cell surface, inhibited Ab1 binding on Raji cells. Ab2 mimicked the EBV/C3dR by its properties to bind to particle-bound C3d and EBV, preventing their binding on Raji cell surface. C3d binding specificities contained in Ab2 were isolated by affinity chromatography on C3b/C3bi-Sepharose. These specificities, being the internal image of C3d binding site of EBV/C3dR, reacted with Ab1 and inhibited particle-bound C3d binding on Raji cells but did not react with EBV. Taken together, these data support strongly that gp 140, the EBV/C3dR, carried two distinct binding sites, one for EBV and one for C3d.
Barel
M
M
Immunochimie des Antigènes de Membrane, INSERM U.23, Paris, France.
Fiandino
A
A
Delcayre
A X
AX
Lyamani
F
F
Frade
R
R
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000032:analysis / Q000502:physiology; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000502:physiology; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000378:metabolism*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002460:Cell Line; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000502:physiology; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D011951:Receptors, Complement / Q000276:immunology*; D017464:Receptors, Complement 3d; D011991:Receptors, Virus / Q000276:immunology*
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007130:Immunoglobulin Idiotypes; D008562:Membrane Glycoproteins; D011951:Receptors, Complement; D017464:Receptors, Complement 3d; D011991:Receptors, Virus