Development Of Non-toxic ELISA Quantitative Detecting Technique For Zearalenone And Screening The Analog Epitopes Of Deoxynivalenol By Phage Display Technology

Objective: This study could be divided into two parts. In the first part, to develop thenon-toxic ELISA immunological detecting technique, the anti-ZEN monoclonal antibody （McAb）and the monoclonal anti-idiotype antibody of Zearalenone （ZEN） which had been prepared fromour laboratory were used. In the second part, in order to obtain the mimic antigen epitopes ofdeoxynivalenol （Deoxynivalenol, DON）, phage display technology was applied.Methods:（1） A hybridoma cell strain1G4secreting anti-ZEN McAb was resuscitated andused to produce ascitictype McAb, then the anti-ZEN McAb was purified from ascites fluid.Based on the relationship between the internal image of antigen using the monoclonalanti-idiotype antibody of ZEN and unique type of toxin ZEN molecules, the non-toxic indirectcompetitive ELISA for detecting ZEN was established.（2） Using the anti-DON McAb as the target，the Ph.D-7Phage DisPlay PePtide Librarywas biopanned for4rounds. The phage clones were picked from plates and the specificity ofclones was assessed by indirect ELISA and indirect competitive ELISA. The DNA of thepositive clones which bond specifically to anti-DON McAb were extracted andthe PCR products were sequenced. Positive clones with different amino acid sequence wereselected and used to establish the competition curves for the substitution relationship betweenDON and phage.Results:（1） The non-toxic ELISA quantitative detecting technique for ZEN had beendeveloped. The curve regression equation of this method was y=1.284x^1.931（R²=1）（y:concentrate of ZEN ng/mL x: concentrate of monoclonal anti-idiotype antibody μg/mL）.Thelowest determined limit was1ng/mL and the linear range of the detecting method wasbetween1.041ng/ml and25.99ng/ml; the Coefficient of variation（CV） for within-run assaysand between-run assays were less than or equal to12%.（2）30strains of phage clones were identified by indirect ELISA and indirect competitiveELISA, results showed that28strains of phage clones could bond to anti-DON McAb,22strainsof them could be blocked by DON standard. The sequencing of the PCR products of the22strains of phage clones’ DNA showed that the amino acid sequences of21strains of phage clones were PFPNHPY, the other one was TPWTQHL.2strains of phage positive clones withdifferent amino acid sequence were selected to establish competition curves for the substitutionrelationship between DON and phage. Results showed that the8line competition curve waslinear in the range of29.2ng/ml to636ng/ml, IC50was169ng/m; the4line competition curvewas linear in the range of12.4ng/ml to271ng/ml, IC50was58ng/ml.Conclusion:1.The anti-ZEN McAb and the the monoclonal anti-idiotype antibody of ZENwere successfully used to establish the non-toxic ELISA quantitative detecting technique forZEN.2.Two mimic antigen epitopes of DON was obtained by using the tool of phage displaytechnology. The competition curves for the substitution relationship between DON and phagewere established respectively. Preliminary validation of that, the DON mimic epitope can replacethe DON toxin standard used for non-toxic immunological detection technology.