Biopanning Of Zearalenone Mimotope By Phage Displayed Peptide Library

Zearalenone(ZEN) is one of the most widely distributed mycotoxins produced by Fusarium species. It can be hepatoxic, teratogenic and immunosuppressive. The currently used methods to detect ZEN are high-performance liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay(ELISA) , and the ELISA is widely used. But ELISA needs to use ZEN standard which is expensive and harmful to the personnel .We used McAb 7G5 as target to biopanning ZEN mimotope. And ZEN mimotope was used as substitute of ZEN standard in ELISA. First, we synthesized artifical antigen of ZEN ,and then BALB/C mice were immunized with artifical antigen of ZEN. After the fusion between spleen cells got from immunized BALB/C mice and SP20/AG-14 cells by PEG, indirect ELISA and indirect competitive ELISA were used for screening the positive cell clones. Limiting dilution was used to screen the positive clones, and a clone producing antibody against ZEN, called 7G5, was obtained. The percentage of crossing reaction with Zearalanone, a-Zearalanol, β-Zearalanol, Citrinin, Deoxynivalenolwas3.8%, 1.9%, 23%, <0.01%, <0.01% respectively. With the standard curve of indirect competitive ELISA , the lowest limit of detection was 0.1ng/mL, the linearity range was 0.1-100 ng/mL, IC50 was 8ng/mL. The monoclonal antibodies against ZEN was used as target for biopanning from a heptapeptide Phage Display Peptide Library .We reduced the concentration of antibodies stepwise, and different blocking buffer was used between every round. After 3 rounds of biopanning , 20 clones were randomly selected for DNA sequencing, then detected by ELISA and ZEN competition experiments to indentify the postive clones. Ten sequences were obtained , two of them were proved to be positive , inhibition percentages were up to 90%, the amino acid sequences were DAVILLM, HHCHWWH respectively. With the standard curves of indirect competitive ELISA of phages, the lowest limit of detection was 0.1ng/mL, the linearity range was 0.1-10ng/mL.