Using A SPR Biosensor For The Detection Of Maize Chlorotic Mottle Virus And Cry1f Protein

With the development of globalization trade and application oftransgenosis, international trade in the corn related areas become moreand more frequent in our country recently, which lead to the risk thatquarantine pests such as maize chlorotic mottle virus and GM proteinsmay be imported into China by maize seeds become more and moregreater. How to detect the quarantine virus from import corn and GMproteins from other products simply and efficiently has great significance.In this study, we use a SPR sensor for the research on detection of maizechlorotic mottle virus and GM insect resistance protein cry1F.This study are consist of three parts. Firstly, we utilize the healthmaize leaves as raw material for reproduction and purification of maizechlorotic mottle virus(MCMV) and get the purified MCMV monoclonalantibody through mice immunization, cell fusion, screening and cloningof hybridoma as well as preparation and purification of ascites antibody.Studies on potency test, identification, specifity and sensitivity of theantibody have also been done. After that researches on the detection ofpurified MCMV on the basis of surface plasma resonance biosensor have been achieved. Detection conditions have been optimized and study onsensitivity and specifity of the virus has been done. In addition, we detectactual maize sample carrying the virus by this method on the optimizedcondition. At last, we use a SPR biosensor for the detection of Bt cry1Fprotein. The main results are as follows:(1) Purified MCMV has been obtained with its concentrationbeing10mg/ml. A hybridoma cell strain(named1C5) which can secreteanti-MCMV monoclonal antibody stably has been acquired and itspotency is1×106as well the identification is IgG1. The concentration ofthe monoclonal antibody which has been purified by G protein SepharoseColumn is0.46mg/ml. The1C5monoclonal antibody has good specifityand the maximum fold of corn juice dilution carrying the MCMV that themonoclonal antibody can detect is6400folds.(2) SPR method for the detection of MCMV has been established.The optimized detection conditions have been entrenched: time ofantibody-fixed on SPR chips being3h, antibody-fixed concentration200μg/ml, antibody-fixed temperature37℃, antibody-fixed pH6,injection flow rate10μl/min, detection temperature20℃and detectionpH7.4. This SPR method has strong specifity as well as goodrepeatability and the limit of detection is1ng/ml. Besides, this methodcan be used for the detection of actual infected maize sample and thedetection time is15min. (3) SPR method for the detection of cry1F protein has beenestablished. This method has good specifity and repeatability and thelimit of detection is10ng/ml.