A general method for screening mAbs specific for G-protein coupled receptors as exemplified by using epitope tagged BLR1-transfected 293 cells and solid-phase cell ELISA.
Monoclonal antibodies (mAb) against G-protein coupled receptors are rare. In this study we describe a cell ELISA-based screening system for monoclonal antibodies specific for the G-protein coupled receptor BLR1 (Eur. J. Immunol. 1992. 22:2795) using human embryonic kidney 293 cells transfected with a modified human BLR1 cDNA directing the synthesis of an epitope tagged BLR1 protein. Lou/C rats were immunized with BLR1 transfected, tagged 293 cells and after fusion of spleen cells with X63 Ag8.653 myeloma cells supernatants were tested for BLR1 specific antibodies by comparing the binding to BLR1 transfected 293 cells and to untransfected control cells immobilised on poly-L-lysine coated microtiter plates. Cells were fixed with 2% paraformaldehyde and permeabilized using digitonin in order to allow binding of mAb directed against intracellular epitopes. This mild fixation retained excellent morphology of 293 cells and allowed reliable binding to the trays. Screening of approximately 2500 supernatants identified 19 antibodies binding to BLR1 transfected 293 cells but not to control 293 cells. One of these mAb specifically bound to the G-protein coupled receptor BLR1.
Förster
R
R
Institut für Biochemie der LMU München, Martinsried, FRG.
Emrich
T
T
Voss
C
C
Lipp
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D004622:Embryo, Mammalian; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007668:Kidney; D051381:Rats; D011919:Rats, Inbred Strains / Q000276:immunology; D054380:Receptors, CXCR5; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D018013:Receptors, Neuropeptide / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; C095085:CXCR5 protein, human; D000939:Epitopes; D054380:Receptors, CXCR5; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine; D018013:Receptors, Neuropeptide; D019204:GTP-Binding Proteins