Development Of Monoclonal Antibody Against Zearalenone

In this study, an artificial antigen for zearalenone(ZEN) was synthesized and used for Balb/c mice immunization. After immunization for three times, the SP2/0cell fusion was performed with50%PEG-1450, and the fusion cells were cultivated on semi-solid HAT medium. Three positive clones were screened from the fusion cells by indirect non-competitive ELISA and indirect competitive ELISA, and a cell line excreting specific antibody to zearalenone was screened and named3C4. The monoclonal antibody was charactered and an indirect competitive ELISA for ZEN detection was developed. This method laid a foundation for further ZEN immunoassay.1. O-(Carboxymethyl) hydroxylamine hemihydrochloride and ZEN were reacted in pyridine to generate ZEN oximation(ZENO), and then the ZENO was conjugated with bovine serum albumin(BSA) to form the complete antigen. The structure of the artificial antigen was verified by UV absorption spectroscopy, and the antibody titer to artificial antigen ZEN-BSA after three times immunization to Balb/c mice was1:16000. the protein content of ZEN-BSA is1.8mg/mL. All the results showed the artificial antigen synthesis was successful.2. After immunization for three times, the SP2/0cell fusion was performed with50%PEG-1450. and the fusion cells were cultivated on semi-solid HAT medium. Three positive cell lines were screened and these three positive cells were cultivated in liquid HT medium for further monoclonal antibody screening. At last, a cell line excreting specific antibody to zearalenone was screened and named3C4. The monoclonal antibody was charactered to be IgG2a isotype, and the IC5o of detection ZEN by indirect competitive ELISA was0.37ng/mL. and the cross reaction with AFG1、AFG2、AFB1、AFB2、DON、T-2、OTA were less than0.1%. The results showed that the antibody was very specific to ZEN.3. Based on monoclonal antibody3C4. an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for ZEN detection. According to the method of chequer board titration test, the ZEN-BSA coating dose was0.4μg/mL, and the antibody was1:4000dilμting in the ELISA. After factors such as blocking solution pH, ionic strength that influenced the assay performance were optimized, the method was characterized with IC500.322μg/L±0.042μg/Land detection range from0.016μg/L to3.08μg/L for ZEN detection. The work provided a powerful technical support for establishment of immunassy of ZEN in corn and cereals products.