Detection Of Genetically Modified Maize By Monoclonal Antibodies Based Sandwich ELISA Targeting The PAT Protein

The safety and environmental risk of genetically modified organisms and food wasincreasingly arousing people’s concern, although the extensive application of transgenictechnology and commercial cultivation of genetically modified plants brought out hugeeconomic and social benefits. In order to track，monitor, assess risk and identify thegenetically modified crops and their products and to ensure the smooth implementation oflaws and regulations of genetically modified crops and food effectively, it is necessary toestablish accurate, efficient, stable and rapid detection technology.A monoclonal antibodies based sandwich enzyme-linked immunosorbent assay (ELISA)for detection of phosphinothricin acetyltransferase (PAT) was developed and evaluated as anovel method for the determination of genetically modified (GM) maize.The coding sequence of the blalaphos resistance gene (bar) was amplified by PCR fromthe plasmid P3301and cloned into the plasmid pET-28a at the Ecol I/Hind Ⅲ site and whichwas transformed into E. Coli BL21(DE3) and expressed under induction of IPTG in solubleform and analyzed by SDS-PAGE. The target protein purified by Ni-IDA Resin was identifiedby Western-blot. MAbs against PAT had been prepared purified recombinant PAT asimmunogen. After identification by Western-blot, purification and labeling of HRP, usingmAbs4D5and3E8, double-antibody sandwich ELISA method was develop.The standard curve of mAbs based sandwich ELISA was estimated with two-folddilution of the purified PAT protein in PBS buffer (0.1M, pH=7.4) from800μg/mL to0.78μg/mL. In order to evaluate the validation and reliability of the mAbs-based sandwich ELISA,the assay was performed at various times of three days.According to the standard curve whichhad a linear coefficient of R2＞0.99to determine the working range was about3.25～50ng/mL with a detection limit of1.88ng/mL for sandwich ELISA. Results of three repeatedtesting showed that the coefficient of variation of each concentration of standard sampledetection results is less than5%, demonstrating an acceptable reproducibility of this assay. Using the sandwich ELISA developed in this study, we could determined4GM maizeBt176samples out of36corn samples analyzed according to the thresh old value of0.210;PCR targeting the bar gene showed the same presence of4transgenic bar maize samples outof36maize samples. The agreement rate of the two detection methods is100%.Our results showed that, from the point of view of immunology, the exogenous proteinof transgenic maize can be used as the immunogen and target to prepare mAbs against thePAT protein. In addition, the sandwich ELISA established in the study can serve as a reliableand accurate method for detecting the presence of the PAT protein in herbicide toleranttransgenic maize Bt176and its derivatives.