Anti-idiotypic VHH Phage Display-mediated Immuno-PCR For Ultrasensitive Determination Of Mycotoxin Zearalenone In Cereals

Zearalenone, a common mycotoxin in food, is bio-synthesized in Fusarium species, particularly F. graminearum, F. equiseti, F.crookwellense. Zearalenone has posed a great threat on human and animal's health, including teratogenicity,carcinogenicity, genotoxicity, hepatotoxicity, immunosuppression and reproductive problems. The quick quantitive method of mycotoxins in cereals and cereal product makes a significant sense in the control of food safety.In this research, zearalenone is used as the research object, using the method of solid phase biopanning and anti-zearalenone monoclonal antibody was coated on the microplates to select anti-idiotypic antibody from native alpacas phage display library,secondly combined with immune PCR technology, anti-ZEN anti-idiotypic antibody was employed to establish a more sensitive detection method in the form of phage clone; At last, prokaryotic expression system was used to express anti-ZEN anti-idiotypic antibody, after purification, the anti-ZEN anti-idiotypic antibody was then used to replace ZEN-BSA to construct a non-toxic ELISA detection method. The main research contents and results are as follows:1. Two kinds of unique sequence of nano antibody was successfully selected by means of the phage biopanning strategy, and were named Z1, Z6 respectively. With the method of indirect phage ELISA, the standards curves were established on the basis of Z1?Z6 and ZEN-BSA. The IC50 value was 0.25 ± 0.02 ng/mL, 0.49 ± 0.03ng/mL, 6.57 ± 0.04 ng/mL, respectively. The sensitivity of two phage clones was 26 and 13 times improved than that of the sensitivity of ZEN-BSA.2. The phage clone Z1 was selected and used as the template for PD-IPCR. Then a pair of primers was designed aimed at the VHH fragments. And the final limit of detection is 6.5 pg/mL and a linear range of 0.01~100 ng/mL. As for the reproducibility, there was an acceptable result in PD-IPCR according to the performance criterion established by EU with CV level ranging from 5.51 to 28.12%.The recovery rate was from 78% to 122.4%, which demonstrated that adequate accuracy and efficiency from cereals were achieved with VHH-based PD-IPCR.Specific measurement of the phage clone Z1 was performed by comparing IC50 with those of three other mycotoxins: AFB1, DON, and OTA. Negligible CR(<0.01%) was observed with the excellent selectivity of Z1. Then this method and LC-MS technology were used to test a total of 35 corn, wheat, rice, actual samples. Test3. results showed that the correlation between the two methods is good, with a linear correlation coefficient of R2 = 0.993.4. Taking advantage of the molecular biology techniques, the VHH fragments were obtained by enzyme the pHEN plasmid and then ligased to express vector pET25b(+). After that it was cloned to E.coli BL 21 and induced by 0.01 mM IPTG.After purification by nickel column, the nano antibody is harvested, namely the biosynthesis of antigen. Finally the non-toxin ELISA is constructed by BMP-Z1 and BMP-Z6 with the IC50 value of 2.52 ± 0.03 ng/mL, 4.08 ± 0.04 ng/mL, respectively.