Different epitopes of the CD31 antigen identified by monoclonal antibodies: cell type-specific patterns of expression.
3 mAb-5A2.G5, B2B1 and 2BD4-all of IgG1 isotype were identified as belonging to the CD31 cluster by their binding to transfected murine cell lines expressing the CD31 antigen and by sequential immunoprecipitation experiments. Competitive binding experiments were carried out using the human myelomonocytic cell line RC-2A. mAb B2B1 and 2BD4 did not cross block. mAb 5A2.G5 partly inhibited binding of 2BD4 but not B2B1. Thus the epitopes identified by 5A2.G5 and 2BD4 appear to overlap but to be quite separate from that recognized by B2B1. All 3 antibodies bound to a 130 kDa species on Western blots after nonreducing polyacrylamide gel electrophoresis of platelet lysates. Culture of RC-2A cells in the presence of tunicamycin (after removal of surface antigens by pronase) blocked re-expression of the epitopes recognised by all 3 mAb suggesting that they involve N-linked glycosylation. Furthermore, treatment of platelet lysates with Endoglycosidase F prior to electrophoresis and Western blotting abolished the binding of the mAb but not a rabbit polyclonal antiserum to CD31. Nevertheless, neuraminidase treatment of RC-2A cells failed to affect mAb binding. The antibodies displayed typical properties of CD31 antibodies in that all 3 precipitated at 130 kDa cell surface protein and bound strongly to platelets, monocytes, neutrophils and vascular endothelium. All 3 antibodies were positive on hemopoietic progenitor cells which give rise to colonies in the CFU-C assay. However, differences in binding to peripheral blood lymphocytes and to certain human leukemic cell lines were noted. In particular, mAb 5A2.G5 bound weakly to lymphocytes and to the lymphoid cell line HSB-2 compared with the other 2 antibodies.
Ashman
L K
LK
Department of Microbiology and Immunology, University of Adelaide, South Australia.
Aylett
G W
GW
Cambareri
A C
AC
Cole
S R
SR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D002461:Cell Line, Transformed; D000939:Epitopes / Q000276:immunology; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007739:L Cells; D007938:Leukemia / Q000473:pathology; D051379:Mice; D014411:Neoplastic Stem Cells / Q000276:immunology; D009928:Organ Specificity; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011994:Recombinant Proteins / Q000276:immunology; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000276:immunology
D000911:Antibodies, Monoclonal; D015214:Antigens, Differentiation, Myelomonocytic; D000951:Antigens, Neoplasm; D000939:Epitopes; D007074:Immunoglobulin G; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011994:Recombinant Proteins