Selection, Identification And Applications Of Anti-cry1Ac SCFV From A Phage Display Library By Magnetic Beads And Sandwich Elisa Immunoassay

Bacillus thuringiensis(Bacillus thuringiensis,Bt) can produce parasporal crystal toxinsknown as insecticidal crystal proteins(Icps) when spores appear. Icps have a stronginsecticidal activity. For a long time, Bt toxin is considered safe insect drugs, and Bt gene isone of the well known and most effective pesticidal gene in the genetically modifiedorganisms(GMOs), and it has been applied in genetic engineering widely. A large number ofstudies in recent years show that, the emergence of Bt transgenic crops constantly appearedand has been widely used, it has produced extremely significant economic benefits, but it alsohas brought ecological risk and potential security problems to the environment, human andother mammals. So it is necessary to establish a simple, sensitive and accurate detectionmethod, whether the environment residues or expression quantity of Bt transgenic crops.Cry1Ac is the most widely used, in recent years it is detected mainly by polymerase chainreaction(PCR),enzyme immunoassay(ELISA), bioassay, immune PCR, radioimmunoassaytime-resolved fluorescence immunoassay(TRFIA), western blotting, etc. Currently ELISAmethods for Cry toxins are based on monoclonal antibodies and polyclonal antibodies. Thereare few reports about the detection method which uses gene engineering-single chainantibodies(single chain antibodies, scFv) of high affinity and high specificity. Based ondomestic and international results,this research avoiding the traditional solid-phase screeningmethods make full use of the three-dimensional structure of the antigen exposurecharacteristics, to screen anti-Cry1Ac toxin scFv from human phage antibody library in orderto lay foundation of the detection of Cry1Ac toxin in genetically modified food. This researchmainly includes the following aspects.1. By using the biotin-avidin labeled magnetic beads screening strategy, this study aimsat selecting anti-Cry1Ac scFv from human phage antibody library by positive and negativescreening method with liquid beads.2. In this paper the purified anti-Cry1Ac scFv was used as the capture antibody, andanti-Cry1Ac-PAbs was used as the detection antibody, their optimal working concentrationwere1:125and1:1000by titration phalanx. The results show that the linear regressionequation was Y=0.4111X+0.0821(R2=0.9751), the limit of detection of Cry1Ac toxin was0.91ng∕mL, with the linear range from1.04ng∕mL to3.49μg∕mL.3. Cry1Ac and other toxins were used as antigens in order to detect the specificity of scFv, indicating that Cry1Ac toxin group show a significant positive reaction, and the ratio ofpositive–hole OD/negative-hole OD value was5.16. All Cry1Ac analogs such as Cry1Ab,Cry1C, Cry1B and Cry1F exhibited negative reaction, and the ratio of positive-hole OD/negative-hole OD value was2.1. Thus, anti-Cry1Ac single-chain antibody has a significantspecificity for Cry1Ac toxin.4. All CV values of the recoveries of Cry1Ac in five samples were less than15%afterstatistical analysis, showing that the test results were with good repeatability and accuracy. Sothis method can meet residue detection requirements of Cry1Ac toxin protein.