Research On Preparation And Application Of Immunological Analysis Of Anti-Bt CRy1Ac Monoclonal Antibodies And Nanobodies

Bt-Cry protein is a kind of prevalent insect-resistant transgenic protein and among them Cry1 Ac protein is the typical and widely studied Bt protein, which exists in many insect-resistant transgenic crops, such as corn, potatoes, cotton and rice etc.,and its output is increasing year by year. Ecological risks and environmental problems from genetically modified crops have drawn global attention and the safety and reliability of genetically modified foods are worrisome. It’s very important to monitor transgenic ingredients in food product. ELISA is the primary detecting technique for Bt protein and antibodies used in ELISA mostly were traditional monoclonal antibodies and polyclonal antibody. Genetically engineered antibodies were also used to detect Bt protein. Traditional antibodies are with high affinity and specificity and genetically engineered antibodies expecially for nanodies are also with a number of advantages, such as stability, smaller molecule weight and property that is easy to transform and expression.In this paper, Cry1 Ac protein was chosen as the target antigen to prepare anti-Cry1 Ac monoclonal antibodies and nobodies, by using the traditional techniques and phage display technology. And the analyzed these antibodies’ inmunological charactrerization, the results were as follows:(1) Preparation and application of immunological analysis of anti-Cry1 Ac monoclonal antibodiesPrepare monoclonal antibodies using traditional techniques. The mices were immunized by Cry1 Ac protein and its spleen B cells then fused with myeloma cells.After 3 to 5 times’ subcloning and non-competitive indirect ELISA identification,eight monoclonal anti-Cry1 Ac antibodies were selected ultimately. Antibody overlay ELISA preliminarily analyzed that monoclonal antibody 3A5 and 9F10 can recognize different antigen epitopes. With monoclonal antibody 8A8 as capture antibody, 9F10-HRP as detection antibody, a double-antibody sandwich ELISA was established and the linear range was 0.98 ~ 30 ng / mL, the lowest detection limit was0.1ng / mL.(2) Preparation and immunological analysis of anti-Cry1 Ac nanobodiesUsing phage display technology, three kinds of phages, which can specificly recoginized Cry1 Ac, with different sequences had been screened from the natural camel nanobodies library. Then we extracted the phages’ DNA and constructed pET25 b expression recombinant vectors which were transformed into E.coli Rosetta competent cells. The interest proteins were induced by IPTG and pured by Ni column.Finally, a double-antibody sandwich ELISA was taken to analyze muching degree between 8 monoclonal antibodies and 3 specific phages, 3 nanobodies and 3 specific phages, 3 nanobodies and 8 monoclonal antibodies. We chose the optimal combination to establish double-antibody sandwich ELISA standard curve. Tthe linear in the range was 0.1 ~ 3.1ng / mL, 0.1 ~ 6.25 ng / mL and 3.9 ~ 125 ng / mL respectively, and the lowest detection limit was 0.02 ng / mL, 0.08 ng / mL and 2.1ng /mL respectively.