Development of Monoclonal Antibody-based Sandwich ELISA for Detection of Neomycin Phosphotransferase(NPTⅡ)Protein in Genetically Modified Cotton(Gossypium sp.)
Abstract：In order to develop and verify a high sensitivity sandwich enzyme-linked immunosorbent assay (ELISA) for detection of neomycin phosphotransferase (NPTⅡ) as a novel method for the quantitative determination of genetically modified (GM) cotton(Gossypium sp.), NPTII protein was prepared as immunogen in vitro, the sandwich ELISA based on the rabbit(Lepus) polyclonal serum (as capture antibody) and Mus musculus monoclonal antibody (mAb) (as detecting antibody) was developed. The standard curve was established with series content NPTⅡ protein as the standard. Each index of the method was validated and the quantitative determination of NPTII in GM cotton was carried out by this method. The linear range of the sandwich ELISA was 3.370~108.125 ng∕mL with a detection limit of 1.97 ng/ml for NPTⅡ. The recoveries of NPTⅡ from non-GM cotton proteins ranged 96.11%~104.69%, respectively, with a coefficient of variation (CV) of 6.58%~7.33%. The sandwich ELISA method had no cross reaction with non-GM cotton endogenous proteins and other types of transgenic protein. After applying this method, we found that the NPTⅡ expression levels of various parts in transgenic cotton plant seedling were significantly different. The results showed that this sandwich ELISA can be used as a specific and reliable immunoassay for detecting NPTⅡ content in GM cotton, which can provide references for establishing the GM crops detection methods and biological safety evaluation, with good application value and prospects.
