The Development Of One Step Innocuous Quantitative Detecting Techniques Of Zearalenone And Screening The Binding Peptide Of Zearalenoneby Phage Display Technology

Objective: According to a ZEN substitute monoclonal anti-idiotype antibody ofZearalenone (ZEN) and anti-ZEN monoclonal antibodies (mAb), we establish anon-toxic indirect competitive enzyme-linked immunosorbent assay (ELISA) methodand chemiluminescence enzyme-linked immunoassay (CLEIA). A sandwich ELISAmethod utilizing anti-ZEN monoclonal antibodies and the anti-ZEN peptide obtain byphage display technology.Methods:(1) To produce the ascitictype monoclonal anti-idiotype antibody ofZearalenone (ZEN) and anti-ZEN monoclonal antibodies (mAb) based on the positivehybridoma cell strain. The hybridoma cell lines which screening high titer and highaffinity antibodies were selected by cell cloning，and were used to produce ascitictypeMAbs, Then the non-toxic direct competitive ELISA for detecting ractopamine wasestablished.(2) based on the Luminol luminescence system, a direct competitiveCLEIA was established with the monoclonal anti-idiotype antibody of Zearalenone(ZEN) and anti-ZEN monoclonal antibodies (mAb).(3) Using the antigen-antibodycomplex as the target, the Ph.D-7Phage Display Peptide Library was biopanned for3times. The positive phage clones were selected and it can be used to establishthesandwich ELISA.Results:（1）The non-toxic direct competitive ELISA quantitative detectingtechnique for ZEN had been developed. The curve of this method wasy=-30.195x+61.622,(R2=0.9849)(y is the percentage inhibition of ZEN, x is thelogarithm of the concentrate of ZEN (ng/ml)). The lowest determined limit was0.114ng/ml, the IC50is2.426ng/mL and the linear range of the detecting method wasbetween0.25ng/mL and24.12ng/mL.(2) Direct competitive CLEIA had beenestablished, and the curve of the method was y=-40.205x+46.303（R2=0.9799）,the lowest determined limit was0.081ng/ml, the IC50is0.8ng/mL and the linearrange of the detecting method was between0.145ng/mL and4.51ng/mL.(3)40strainsof phage clones were identified by sandwich ELISA, the results showed that2strainsof phage clones could boundwith1G4-ZEN better than bound with1G4, but it had no specificity in sandwich ELISA to detected ZEN.Conclusions:(1)The anti-ZEN monoclonal antibodies (mAb) and the monoclonalanti-idiotype antibody of ZEN were successfully used to establish the non-toxicELISA and CLEIA quantitative detecting technique for ZEN.(2) We cannot get anystrain of phage clones which especially bound ZEN in different epitope between1G4.