Preparation and characterization of a monoclonal antibody that inhibits myoblast fusion of avian skeletal myoblasts.
To investigate the mechanism of myoblast fusion using quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells), we prepared monoclonal antibodies against a cell surface antigen involved in myogenic differentiation. For this, a Balb/c mouse was immunized with the membrane fraction of QM-RSV cells, and hybridomas producing monoclonal antibodies were raised by fusion of spleen cells from the immunized mouse with myeloma cells. By analysis of the hybridoma supernatants, we obtained a monoclonal antibody, termed H-145, that strongly inhibited myoblast fusion. H-145 inhibited myoblast fusion dose-dependently, and its effect was readily reversed by its removal. H-145 promoted biochemical differentiation of the cells until 48 h. It did not affect a fusion-commitment step to differentiation, but inhibited a later step. Indirect immunofluorescence and immunoblot analyses showed that the antigen reacting with H-145 was a glycoprotein with a molecular weight of approximately 116 kDa. This antigen is present throughout differentiation, but as differentiation progresses, its expression increases and its distribution on the cell surface changes. The antigen purified by H-145 affinity chromatography failed to react with beta 1-integrin, alpha 5-integrin, NCAM, or N-cadherin on immunoblotting. Thus, H-145 antigen differs from these components that are known to be associated with myogenic differentiation. Consequently, the results suggest that H-145 antigen may be a new cell surface antigen associated with cell differentiation.
Hyodo
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Institute of Molecular and Cellular Biology for Pharmaceutical Sciences, Kyoto Pharmaceutical University, Japan.
Kim
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J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000954:Antigens, Surface / Q000276:immunology; D002454:Cell Differentiation; D002459:Cell Fusion / Q000187:drug effects*; D002478:Cells, Cultured; D005260:Female; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D024510:Muscle Development; D009132:Muscles / Q000166:cytology* / Q000276:immunology; D011784:Quail; D013045:Species Specificity; D013234:Stem Cells / Q000166:cytology* / Q000276:immunology; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface