Development Of Colorimetric Immunoassays For Bacillus Thuringiensis Cry1c Endotoxin

The Bacillus thuringiensis(Bt)Cry toxin transgenic crops have been rapidly cultivated since last decade.Cry1C is one of the emerging Bt toxin protein for the pest control in agriculture.Conventional detection of Cry toxins mainly depends on instrumental and chemometric analysis which are time taking and expensive.Therefore,due to growing Cry1C transgenics and potential health and environmental concerns regarding Cry toxins,development of an economical simple and robust detection method is need of time.In this work,a double antibody sandwich enzyme linked immunosorbent assay(DAS-ELISA)and Magnetic beads-based immunoassay was established to monitor Cry1C.Firstly,two cell lines C1 and C4that can produce Cry1C monoclonal antibodies were cultured and injected into the peritoneal cavity of mouse to produce large amounts of monoclonal antibodies.C1 antibodies were chosen as capture antibodies and C4 were labelled with Horseradish peroxidase(HRP)to be used as secondary antibody.Finally colorimetric reaction proceeded with addition of 3,3',5,5'-tetramethylbenzidine(TMB)and hydrogen peroxide(H2O2),the concentration was quantified by measuring absorption peak at OD₄₅₀ using colorimetric plate reader.Hence,our work contains the following sensors for Cry1C:(1)Sandwich ELISA:the detection limit was 10ng/m L(S/N=3.0),and the linear range was 10–120 ng/m L.The proposed ELISA exhibited good stability as relative standard deviation(RSD)of 4.43%was achieved;(2)Highly sensitive Magnetic beads-based Immunoassay:the limit of detection(LOD)was 1 ng/m L(S/N=3.0)with the working range of 1–13 ng/m L,this method showed excellent stability,the RSD comes out 2.75%.Furthermore,these methods were successfully validated by detecting Cry1C in transgenic rice seed and leaf samples.