Establishment And Application Of Immune Fast Detection Techniques For Zearalenone And Ochratoxin A

Mycotoxins were the secondary metabolites of fungus, which infectedcrops in the growth stage or grain in the storage stage. Once the mycotoxins enteredthe human body through the food chain, they may cause teratogenic, mutagenic,carcinogenic and other serious harm. So they were in the global food safety concern.In this research, zearalenone and ochratoxin A, the popular mycotoxins, werestudied by enzyme-linked immunosorbent assay (ELISA), and the results as follows:1）Zearalenone (ZEN) was coupled with bovin serum albumin (BSA) andovalbumin (OVA) to prepare immunogen ZEN-BSA and coating antigen ZEN-OVAby ester activation method. The two antigens were identified by UV Scan andSDS-PAGE. The spleen cells of BALB/c mice immunized by ZEN-BSA were fusedwith SP2/0myeloma cells, and hybridomas secreting antibodies against ZEN wereselected and cloned. A stable hybridoma cell line3D8that secret MAb of subclassesIgG1, kappa light chain were established. The titer of the MAb determined by indirectELISA was1︰2.048×106in ascites. Based on the ascites MAb, an indirectcompetitive ELISA (ic-ELISA) method was developed for the quantitative detectionof ZEN. The ic-ELISA had a good sensitivity with an IC50of22.89pg/mL, adetection limit of10.07pg/mL, and95%,8%,12%,6%,5%cross-reactivitives toα-zearalenol, β-zearalenol, zearalanone, α-zearalanol, and β-zearalanol respectively,and scarcely showed cross-reactivity to others. The spiked recovery of this methodranged from86.4%to104.8%in maize, barley, wheat and oat samples.2）A direct competitive ELISA(cd-ELISA) kit based on ochratoxin A(OTA)polyclonal antibodies was developed. In the0~10ng/mL range, the OTA ELISA kithad a good sensitivity with an IC50of1.09ng/mL and IC10of0.06ng/mL, showed6.28%,0.16%cross-reactivitives to ochratoxin B and C respectively, and scarcelyshowed cross-reactivity to others. The mean variation coefficient of intra-assay andinner-assay were2.21%and2.79%. The limit of detection in peanut, maize and maizeflour samples were1.71，1.26and1.85μg/kg, respectively, and spiked recoveryranged from83.80%to91.40%. Compared with HPLC, the correlation coefficients（R2）were0.94、0.88and0.90for peanut, maize and maize flour, respectively. Andthe detection time of the kit is20minutes. It is indicated that the OTA ELISA kit was available in rapid preliminary screening of OTA.In this study, monoclonal antibody-based ic-ELISA method for zearalenonedetection was established, which laid the foundation for the development of anzearalenone ic-ELISA kit, and the cd-ELISA kit for ochratoxin A also successfullydeveloped. This will provide valuable experience for the establishment of immunerapid detection technology for other small molecule mycotoxins in artificial antigensynthesis, antibody production and ELISA kit development.