Monoclonal antibodies directed against protoplasts of soybean glycine max cells generation of hybridomas and characterization of a monoclonal antibody reactive with the cell surface
Villanueva, M.A.; Metcalf, T.N.IIi; Wang, J.L.
Planta (Heidelberg) 168(4): 503-511
ISSN/ISBN: 0032-0935 Accession: 005923616
Splenocytes, derived from mice that had been immunized with protoplasts prepared from suspension cultures of root cells of Glycine max (L.) Merr. (SB-1 cell line), were fused with a murine myeloma cell line. The resulting hybridoma cultures were screened for the production of antibodies directed against the soybean protoplasts and were then cloned. One monoclonal antibody, designated MVS-1, was found to bind to the outer surface of the plasma membrane on the basis of several criteria: (a) agglutination of the protoplasts; (b) binding of fluorescence-labeled immunoglobin on protoplasts yielding a ring staining pattern with prominent intensity at the edges; and (c) saturable binding by protoplasts of 125I-labeled Antibody MVS-1. The antigenic target of Antibody MVS-1, identified by immunoblotting techniques, contained a polypeptide of relative molecular mass (Mr) approx. 400,000 under both reducing and non-reducing conditions. When the antigenic target of Antibody MVS-1 was chormatographed in potassium phosphate buffer, the position of elution corresponded to that of a high-molecular-weight species (Mr 400,000). These results provide the protein characterization required for the analysis of the mobility of Antibody MVS-1 bound to the plasma membrane of SB-1 cells.