Development Of An ELISA For Detecting Cry1Ac Protein

Bt gene is one of the well known and most effective pesticidal genes in the genetically modified organisms (GMOs), and it has been applied in genetic engineering widely. There are 130 kinds of Bt genes have been found right now and new Bt genes are being developed and researched continuously. There are two kinds of Bt genes, including crystal protein gene(Cry) and cytolytic protein gene (Cyt). While the widely researching, developing and commercializing of GM food, researching and developing a method that could detect and determine the GM food sensitively and fast will strength the management of GMOs safety, guarantee the safety of human and animals, protect the ecological environment and promote the further study of GM technology. The detect target of this study is Bt Cry1Ac protein, through obtaining extreme pure Cry1Ac protein as the antigen (Ag), producing highly specific polyclonal antibodies (PcAb) and monoclonal antibodies (McAb), preparing HRP conjugate antibodies, comparing the sensitivity and linear detection range of three ELISA, an ELISA kit that could detect Cry1Ac protein sensitively and specificly is primarily developed. The main results of this study are as the followings:1. Purity and concentration of CrylAc proteinThe purity of CrylAc protein was studied by SDS-PAGE, it could be known from the result that the purity of CrylAc protein surpass 90%, the molecular weight was about 66.4KDa, the purity and molecular weight could meet call of the immune reaction. BCA result showed the concentration of Cry1Ac was 1.31mg/mL, and it met the concentration of immune reaction.2. Preparation of PcAbTwo rabbits (NO.3359, NO.3360) were immunized to obtain PcAb. After the fourth immunization, serum titer of NO.3360 rabbit was higher than NO.3359 rabbit, and the titer of NO.3360 surpassed 10000. So the serum containing anti-Cry1Ac PcAb was haversted and purified by Sepharose CL-4B Protain A. SDS-PAGE result showed there were not any other proteins except PcAb in the solution.3. Preparation of McAbThree rabbits (NO.82, NO.83, NO.84) were immunized to obtain McAb. In the research, NO.84 rabbit was chosen for cell fusion.17days after cell fusion,92 positive clones were primary-screened, then one week later,33 positive clones and 14 double positive clones were screened. Three clones (NO. zju-11-18, zju-11-71, zju-11-91) were chosen for sub-clone process, three clones (NO. zju-11-41, zju-11-47, zju-11-50) were for the backup of sub-clone process. Other eight clones (NO. zju-11-24, zju-11-28, zju-11-38, zju-11-61, zju-11-65, zju-11-70, zju-11-82, zju-11-84) were freezed. At last, one of the sub-clones (NO. zju-11-71) was chosen for producing McAb.McAb were purified by Sepharose CL-4B Protein A, and concentration of monoclonal was 1.78mg/mL. SDS-PAGE result showed there were not any other proteins in the McAb solution. The titer of McAb were 32000, and the affinity constant was Ka=2.69×109M-1. There was no abvious cross-reaction between McAb and CrylAb protein, and there was little cross-reaction when the concentration of Cry1c protein was 1000ng/mL.4. Preparation of HRP conjugate antibodiesHRP conjugate to antibodies through using improved sodium periodate method. Result of ELISA showed the process of conjugation was successful. The working concentration of HRP-PcAb was 1:200, HRP-McAb was 1:320.5. Determine the optimal ELISAIn this study, indirect ELISA, McAb-Ag-PcAb sandwich ELISA. PcAb-Ag-McAb sandwich ELISA were developed. The sensitivity and linear detection range were compared among these three ELISA methods. The limit of detection (LOD) of indirect ELISA was 50ng/mL, and the linear detection range was 100-400ng/mL. LOD of McAb-Ag-PcAb was 0.24ng/mL, the linear detection range was 0.975～62.5ng/mL. LOD of PcAb-Ag-McAb was 1.95ng/mL, the linear detection range was 3.9～62.5ng/mL. The sensitivity of McAb-Ag-PcAb ELISA was highest and the linear detection range was widest, so McAb-Ag-PcAb ELISA was chosen for further study.6. Method validationIn this study, for the optimal ELISA, assay sensitive, specificity, recovery and precision were evaluated.The Ab of this immunoassay method showed highly specific for Cry1Ac without significant cross-reaction with Cry1Ab, Cry1c proteins. This developed immunoassay method performed well with fortifying Cry1Ac protein in negative samples and the recoveries ranged from 89.2% to 121.92% with the mean value of 103.35%. Tissue protein extracts of the positive corn leaves were used to evaluate the precision of this developed ELISA showed that this immunoassay method could detect Cry1Ac of some positive crops qualitatively and quantitatively in some extent.All these results shows that this developed sandwich ELISA could meet the detection of Cry1Ac protein and have potential applications.