Identification of the antigen recognized by the monoclonal antibody 31D8.
The monoclonal antibody (mAb) 31D8 has previously been described to bind more avidly to functionally active neutrophils and proved to be useful as a differentiation marker of neutrophils. However, attempts to further characterize the antigen recognized by 31D8 have not been successful. Studying the altered Fcgamma-receptor expression of human neutrophils induced by granulocyte colony-stimulating factor (G-CSF) in vivo, we could demonstrate a parallel decrease in the expression of 31D8 and the CD16 antigen. Furthermore, 31D8 showed a binding pattern on leukocyte subsets similar to that of clustered CD16 antibodies, exhibiting identical cells in double-staining experiments. Preincubation of neutrophils with 31D8 resulted in a dose-dependent inhibition of the binding of immune complexes. A decreased expression of the 31D8 antigen was found on the same cell clones to the same extent as found for the 3G8 antigen on neutrophils from patient with paroxysmal nocturnal hemoglobinuria (PNH). Treatment of polymorphonuclear leukocytes (PMN) with PIPLC resulted in a dose-dependent decrease of mAb 31D8 binding, showing that the 31D8 antigen is phosphatidylinositolglycan (PIG)-anchored. Moreover, 31D8 competed with the binding of antibodies (such as mAb 3G8) directed against the binding site of FcgammaRIII for the Fc-part of IgG. However, this mAb did not influence the binding of a CD16 antibody (mAb B73.1) which recognizes an epitope elsewhere on the CD16 antigen. We conclude from our experiments that the mAb 31D8 binds with high avidity to fcgammaRIII (CD16 antigen). Furthermore, our data indicate that its binding site is most probably located at the binding site for the Fc-part of IgG.
Spiekermann
K
K
Department of Hematology and Oncology, University of Göttingen; Germany.
Roesler
J
J
Elsner
J
J
Lohmann-Matthes
M L
ML
Welte
K
K
Malech
H
H
Gallin
J I
JI
Emmendoerffer
A
A
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000378:metabolism; D001667:Binding, Competitive; D002478:Cells, Cultured; D000939:Epitopes / Q000276:immunology*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006457:Hemoglobinuria, Paroxysmal / Q000276:immunology; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology; D007962:Leukocytes / Q000276:immunology; D009504:Neutrophils / Q000276:immunology; D017452:Receptors, IgG / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D017452:Receptors, IgG; D016179:Granulocyte Colony-Stimulating Factor