Mapping of Antigenic Epitopes on CP4-EPSPS Protein and Detection Method Establishment of Rapid DAS-ELISA
HOU Ji-Chao1,2, LI Zhong-Peng1, LIANG Yu-Xin1, ZHANG Chun-Yu1, YU Han-Song2, LI Xiao-Yu1,*, WANG Yong-Zhi1,*
1 Jilin Academy of Agricultural Sciences, Gongzhuling 136100, China; 2 College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China
Abstract China imports a large number of transformed soybeans (Glycine max) from abroad every year, mainly cp4 epsps transformed soybean. It is necessary to establish a rapid-effective ELISA method for identification of cp4 epsps transformed soybean. This study, CP4-EPSPS protein was expressed in segments by synthetic peptides method, the antigenic epitopes recognized by 5 strains of CP4-EPSPS monoclonal antibodies were mapped. The monoclonal antibodies that recognized different epitope were paired by matrix approach, the working antibody and working concentration of antibody were determined by the maximum value of P/N. The optimal detection conditions were determined by controlling variable method, and the cp4 epsps transformed soybean rapid double antibody sandwich ELISA detection method was established. The performance of the established detection method was evaluated by specific test, repeatability test and positive determination value test, at the same time, 130 samples were detected by the established detection method and commercial kits, and compared their coincidence rates. The monoclonal antibodies 1D10 and 2D3 were paired and had the best matching detection effect by matrix approach. The monoclonal antibody 2D3 and 1D10 were determined as capture antibody and detection antibody, working concentration of 2D3 was 20 μg/mL, 1D10 was 10 μg/mL. The capture antibody coating condition was 37 ℃ 2 h, 4 ℃ overnight. The sample and detection antibody were added in ELISA plate at the same time, then co-incubated at 37 ℃ for 10 min. The sensitivity of this method was 160 (g/mL) times dilution for leaf and seed, the optimal dilution rate for soybean leaves and seeds were 10~80 and 10~40 (g/mL), respectively. The intra-plate and inter-plate variation coefficient was less than 25%, and the positive determination value was 1.41. 70 soybean leaves, 40 soybean seeds and 20 soybean milk samples were detected, the coincidence rate was 100%, and no cross react with other proteins. This detection method has good accuracy, repeatability and specificity. This testing program can be completed within 30 min, and is suitable for rapid qualitative detection for plant, seed and soybean milk.
HOU Ji-Chao,LI Zhong-Peng,LIANG Yu-Xin, et al. Mapping of Antigenic Epitopes on CP4-EPSPS Protein and Detection Method Establishment of Rapid DAS-ELISA[J]. 农业生物技术学报, 2021, 29(1): 159-168.
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