1 Biopanning, Identification And Expression Of Phage Displayed Nanobody Against Immunocomplex Of Deoxynivalenol(DON) 2 Phage Displayed Nanobody Medicated Immuno-PCR For Ultrasensitive Detection Of Cry1Ac Protein

Part ?Compared with competitive immunoassay, the non-competitive immunoassay has several advantages such as simplicity of operation, high sensitivity, and wide detection range. In the case of small analytes, upon binding to a specific antibody most of its surface ends up buried in the antigen binding site of the antibody. This limits the possibility of reacting the analyte with a second antibody. To date, for the small analytes, competitive immunoassay type is more popular than noncompetive type. In this study, we intend to develop a noncompetive immunoassay for deoxynivalenol(as a model of small analytes) based on phage displayed nanobody against immunocomplex of deoxynivalenol(DON). Firstly, a naive phage displayed nanobody library was selected to biopaning the phage displayed nanobody against immunocomplex of DON. After four rounds of biopanning, the positive phage clones were identified by phage-ELISA and the amino acid sequences of nanobodies were analysed by DNA sequencing. The research results were summarized as below:1. Biopanning and identification of phage displayed nanobody against immunocomplex of DONThe anti-DON antibodies were coated in the microplate wells, then DON standard soultion was added to the coated wells to form the immunocomplex of DON. After that, a naive phage displayed nanobody library were inputted to the target wells, and four rounds of biopanning were performed. After phage-ELISA and DNA sequencing, four positive phage clones were indentified that can specifically bind to the immunocomplex of DON, these four phage clones were named as P-1, P-19, P-26, P-46, respectively. In addition, a reaction curve of noncompetive immunoassay for DON were developed based on the phage displayed nanobody(P-26). In the range of 0-500 ng/mL of DON, the OD values of the detecting wells were increased with the increase of DON concentration, which indicate that the phage displayed nanobody against immumocomplex can be applied to the noncompetive immnuassay format. 2. Expression of soluable nanobodies against immunocomplex of DONIn order to express soluable nanobodies, VHH genes extracted from P-1, P-19, P-26, P-46 were subclone to expression plasmid pET25b(+). Then the recombinant vectors(pET25b-VHH-P-1, pET25b-VHH-P-19, pET25b-VHH-P-26, pET25b-VHH-P-46) were transformed into E.coli Rosetta, respectively. After induced by 1%Glucose, 0.05 mM IPTG for 8 h, soluable nanobodies(N-1, N-19, N-26, N-46)were obtained and purified by Ni-chelating affinity chromatography. After that, the reaction curves of noncompetitive ELISA for DON based on nanobody(N-26) were established. In the range of 1-800 ng/mL of DON, the OD values of the detecting wells were increased with the increase of DON concentration, which indicate that the soluable nanobody against immumocomplex can be applied to the noncompetive immnuassay format. Part?Cry1Ac is a kind of common Bt protein. Now, Cry1 Ac is widely used in insect-resistant transgenic crops such as soybean, cotton, rice, wheat, etc. To date, lots of genetically modified crops have been commercialized and the number of transgenic plants is still increasing. Howevr, the gradually increasing culivation of transgenic plants has raised concerns about the enviroment and food safety in the public. Therefore, it is important to detect transgenic ingredients of food and ralated materials. So far, the detecting method for genetically modified ingredients(GMO) falls into two broad categories: immunoassay based on protein detection and PCR based on nucleic acid detection. Immunoassay has many advantages such as high specificity, simplicity of operation, and suitable for high-throughput screening. However, for the protein-based immunoassay, it still has limit incluing low sensitivity and high false positives. In this work, on the basis of anti-Cry1 Ac phage displayed nanobody, By combining the advantages of immunoassay and PCR method, we developed a phage displayed nanobody based immunoassay for ultrasensitive detection of Cry1 Ac. The research results were summarized as below:1. In order to remain the specificity of phage displayed nanobody against Cry1 Ac in the PCR amplification progress. The DNA region of CDR3 was selected as the amplification target, and amplifing primers were desinged and were applied to amplify the CDR3 region(98 bp).2. Based on anti-Cry1 Ac monoclonal antibodies(8A8) as capture antibody and anti-Cry1 Ac nanobody(P-72) as detecting antibody, immuno-PCR for ultrasensitive detection of Bt-Cry1 Ac was established, Its limit of detection(LOD) was 0.094 pg/mL and the liner range was 0.001~100 ng/mL, the linear correlation coefficient was 0.9968. Immuno-PCR based on nanobodies was highly specific to Bt-Cry1 Ac and with negligible cross reactivity with other types of proteins, the cross reaction rate with homologous protein(Cry1Ab protein) was 3.4%. Through recovery analysis of Cry1 Ac in spiked cereal samples, the recovery rate was 78.81~114%, CV(%) was 6.13~21.92. Furthermore, immuno-PCR for ultrasensitive detection of Bt-Cry1 Ac was reliable and reproducible.