Preparation of Antibodies against CP4-EPSPS and Establishment of Its Quantitative DAS-ELISA

To rapidly and efficiently detect Cp4 epsps transgenic herbicide-tolerant soybeans, 5 strains of mouse anti-CP4-EPSPS protein monoclonal antibodies and 3 sheep anti-CP4-EPSPS protein serum were obtained by immunizing experimental animals with CP4-EPSPS protein. A double antibody sandwich ELISA for detecting CP4-EPSPS protein was established, which could also quantify CP4-EPSPS protein in different tissues from Cp4 epsps transgenic herbicide-tolerant soybeans at pod-setting stage. The results showed that the optimal detection conditions:The capture antibody was 1D12, work concentration was 1.25 μg/mL, coated with the ELISA plate, and stand overnight at 4℃. The test sample was incubated at 20℃ for 45 min, the detection antibody was 8092, work concentration was 2.5 μg/mL, incubated at 20℃ for 30 min.The minimum detection limit of CP4-EPSPS protein in simulated soybean environment was 3.6 ng/mL, the detection range was 7.5~125 ng/mL, and the repeatability coefficient of variation was less than 15%. Under the above detection conditions, the expression of CP4-EPSPS protein was detected in the roots, stems, leaves and seeds of the Cp4 transgenic epsps-resistant soybean, and the expression levels of various tissues were different. The expression of CP4-EPSPS protein was the highest in leaves, but decreased gradually in stem, seed and root, which was significantly lower than that in leaves.