Preparation And Evaluation Of Antibodies Against Zearalenone And Aflatoxin B₁

This work was supported by the China-Jilin Agricultural Product Quality andSafety Project (Grant No.2011-Z67).Zearalenone (ZON) and Aflatoxin B1(AFB1) are produced as a group ofsecondary metabolites by a number of fungal species. Both mycotoxins are naturallyoccurring by fungal that grow on grains, feeds and foods expecially corn and hayexposed to high moisture levels during storages. ZON shows oestrogenic and anabolicaction in serval animal species. Aflatoxin B1(AFB1), one of the most commonlyfound toxins in foods and feeds, has been classified as a Group1carcinogen byInternational Agency for Research on Cancer (IARC). It induces reproduction toxicity,immunotoxicity, cytotoxicity. Immunochemical methods, such as ELISA, becomemore and more popular due to its rapid, sensitive, low-cost features compared totraditional qualitative and quantitative analysis methods. Preparation of highlyspecific and strong sensitive antibody is the key to the success of ELISA.The article employed EDA-EDC method to prepare cationized protein. Thecationized protein shows a good immunological properties. Cationized protein wasthen conjugated to AFB1using AFB2amethod. ZON imunogeon was constructedthrough the coupling with the native protein by the DCC-NHS method. The ZON andAFB1imunogeon had been identified and analyzed by ultraviolet spectroscopy,thin-layer chromatography, gel electrophoresis and2,4,6-Trinitrobenzenesulfonic acidmethods. After immunized Balb/c mice with ZONO-BSA and AFB2a-cBSA completeantigens, respectively, polyclonal antisera were prepared. The tire, sensitivity anddetectability of the antisera were characterized and analyzed through indirect ELISAand indirect competitive ELISA. Meanwhile, the spleen cells from the ZONO-BSAimmunized mice were fused with the SP2/0cells to form the hybridomas. After the screening of the hybridomas, the monoclonal antibody was prepared. The antibodywas collected, purified and characterized through indirect ELISA and indirectcompetitive ELISA to identify its tire, sensitivity and detectability. In the end, AHPLC method for the detection of AFB1was established. Parameters such as recoveryand precision were calculated. The HPLC method was also used as an alternativevalidation for the ic-ELISA method that has been established earlier.The main resultswere as follows:(1) Preparation of cationized proteinIn the study, we introduced the primary amino groups through the reaction of EDAand EDC with the protein as described in Muckerheide[1].(2) Preparation of ZON immunogenTwo-step DCC-NHS active ester method was employed to construct theimmunogen of ZON. From UV spectrum of the artificial immunogen, a conclusioncould be made that the coupling of hapten to carrier protein was successful, and themolar ratio of nZONO:nBSAand nZONO:nOVAwere12.7:1and6.6:1, respectively.(3) Preparation of AFB1immunogenIn this study, two methods, AFB2areaction and Mannich-type reaction wereemployed to construct the AFB1immunogen. In the AFB2areaction, AFB1wastransformed into hemiacetal and then coupled to cBSA and cOVA to formAFB2a-cBSA and AFB2a-cOVA, respectively. In the Mannich-type reaction,AFB1-cBSA and AFB1-cOVA was constructed. From UV spectrum of the artificialimmunogen, a conclusion could be made that the coupling of hapten to carrier proteinby AFB2amethod was successful, and the molar ratio of nAFB1:ncBSA, nAFB1:ncOVAwere14.32:1and7.45:1, respectively.(4) Preparation and characterization of polyclonal antiserum and monoclonal antibodyagainst ZONAfter six times immunization of ZONO-BSA for the mice, the serum wascollected. Tires were identified through the indirect ELISA. The ELISA results showdthat all mice tires stayed in a relatively high level. The highest tire reached to1:64000. A conclusion could be made that IC50of the antiserum was74.69ng/mL while thelimit of the detection was23.24ng/mL from the indirect competitive ELISA.(5) Preparation and characterization of polyclonal antiserum against AFB1AFB1-cBSA was choosen as the immunogen to immunize the mice. IndirectELISA showed that the mice stayed in a relatively high level. The highest tire reachedto1:256000. IC50of the antiserum was90.00ng/mL and the limit of the detection was29.16ng/mL.(6) Preparation and characterization of ZON mAbZONO-BSA was choosen as immunogen to immunize the Balb/c mice. After theimmunization process, the spleen was extracted and used for cell fusion. A positivehybridoma named FG2was attatined after limited dilution cloning method. After theanalysis of indirect competitive ELISA, we know that the IC50of the cell supernatantwas9.21ng/mL and the limit of the detection was2.78ng/mL.(7) High performance liquid chromatography analysis of AFB1An effective and accurate HPLC method was established for the determination ofAFB1. Standard curve of AFB1was obtained. Based on the standard curve we get, aconclusion could be made that the linear regression equation of the curve isS=63187.94859*C+85195.63562. The correlation coefficient between the curve andthe actual detection value is0.99237. As you can see, the linear relationship was good.The average recovery of standard addition was89.32%while the RSD was3.83%.The comparision between HPLC method and ic-ELISA method for the detection ofAFB1showed that two methods had a relatively high correlation coefficient of0.83.This suggested that the ic-ELISA method for the detection of AFB1was credible.