To produce the monoclonal antibodies （mAbs） against hygromycin B phosphotransferase （HPT） and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice （GM rice）. Methods ...To produce the monoclonal antibodies （mAbs） against hygromycin B phosphotransferase （HPT） and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice （GM rice）. Methods BALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants. Results Four hybridomas, named F1,D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 10×10^-4 to10×10^-5. Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected （80-150ng/mL）. But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay, Conclusion Anti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.展开更多
基金This work was supported by 973 National Fundamental Science Program Funds (2001CB109007 and 2001CB10901)and 863 National High-Tech Program Funds (2004AA212221 and 2002AA212041).
文摘To produce the monoclonal antibodies （mAbs） against hygromycin B phosphotransferase （HPT） and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice （GM rice）. Methods BALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants. Results Four hybridomas, named F1,D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 10×10^-4 to10×10^-5. Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected （80-150ng/mL）. But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay, Conclusion Anti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.
