Preparation Of The Monoclonal Antibody Against C₄-Pepcase And Establishment Of The Elisa Detection Method

Photosynthesis is the foundation of the biological yield. C4-plants have higher photosynthetic efficiency than C3-plants, because they have the Kranz type and a unique pathway of fixing atmospheric CO2. PEPCase is the primary enzyme in the C4-pathway, therefore, trials for introducing PEPC gene into rice have been carried out to improve the photosynthetic efficiency and the yield of crop. The present main methods for detecting genetically modified crops are biological, molecular and immunological methods. Because of the advantages of convenience, sensitivity, accuracy and lower expenditure, the immunological methods are better than others. To accelerate the process of breeding for high photosynthetic efficiency, we make the monoclonal antibodies against PEPCase and establish the ELISA examination method to detect the gene modified rice on the protein level.BALB/c mice were immunized with PEPCase, and the stimulated spleencytes were fused with myeloma cells. Three positive hybridomas secreting the McAb were obtained after subcloning4times by the technique of limiting dilution and screening by indirect ELISA, named3C11,1B9and4E5. The ascitic McAb were purified by the method of Octanoic acid-Ammonium sulfate. New Zealand White rabbits were immunized with PEPCase, and the PcAb was purified by the ammonium sulfate precipitation. The subtype of3C11and1B9were IgG1, while4E5was IgG2a. The ELISA titers of3C11,4E5,1B9and PcAb were1:128000,1:128000,1:4000and1:32000respectively. The protein concentration of3C11,1B9,4E5and PcAb were6.78mg/mL,7.62mg/mL,5.81mg/mL and21.48mg/mL respectively. The detection of SDS-PAGE and Western-blot showed that the McAb were highly specified for PEPCase. The affinity constant of4E5was2.21*10-8M through the competitive binding assay, suggesting that the McAb had good affinity with PEPCase.We established the double sandwich ELISA as the model of PcAb-sample-McAb. The results showed that the best concentration of PcAb and McAb were6.71μg/mL and7.26μ/mL, respectively, and the detection sensitivity was about5μg/mL. In addition, the optimal reaction time of sample, McAb, second antibody and substrate were1h,1.5h,1.5h and15min, respectively, and the best dilution of second antibody is1:10000. The total time of the entire process was only about5hours (without coating and blocking). The result of detecting PEPC GM rice using the double sandwich ELISA was quite well, which suggested the double sandwich ELISA was a rapid, sensitive, economic and specific method for detection of PEPCase to breed the PEPC gene modified rice.