Wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice. Subcutaneous inoculation of the antigen is performed. The intra-peritoneal injection is completed 3 days before fusion with myeloma cell (SP2/0) via PEG-1500. The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay (ELISA). Positive hybrid cells are further verified three times by limit dilution of the culture cells. A hybridoma cell line is successfully obtained. The monoclonal antibody belongs to IgG1 subclass. In immunoblotting, the antibody binds to all HMW-GS of T. aestivum cultivars, but does not bind to other storage proteins in seeds of wheat. This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat. The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T. durum (durum wheat). Furthermore, it also binds to HMW storage proteins in Secale cereale (rye), Hordeum vulgare (barley). However, it never binds seed storage proteins in other cereals such as maize, oat, rice, foxtail millet, sorghum etc. The antigen determinant recognized by the antibody has been located within hexapeptide [PGQGQQ] or / and nonapeptide [GYYPTSPQQ] in the central repetitive region of HMW-GS.