| Recently, fungaltoxin contamination has become one of the main contaminations in the food contamination, especially the contaminations caused by zeranol(ZER) which cannot be neglected. Zeranol and its metabolite product have a certain function of estrogens, once they enter into the life system along the food chain, they could be induce growth retardation of human beings and animals, and could have an impact on the secondary sex characteristic. The ministry of agriculture in our country had been expressly prohibiting ZER used as one kind of growth promoter in livestock breeding as early as the year 2002. At present, the main detection methods for ZER in foods and feeds include thin layer chromatography(TLC), high performance liquid chromatography(HPLC) and enzyme-linked immune-sorbent assay(ELISA). TLC can be used to qualitative analysis only; HPLC can be used in a limit extent due to its disadvantages, such as complex pre-treatment process, demanding operation skills and massive use of toxic organic solvent. Instead, ELISA has several advantages which can make up for those disadvantages of the above methods, so ELISA is already successfully used for fungaltoxin in foods and feeds.1. Synthesis of ZER artificial antigens and preparation of murine polyclonal antiserumsBy modifying the sixteenth hydroxyl of ZER, the hapten of ZER, ZER-(16-hydroxypropyl butyl ether) was designed and synthesized successfully. Firstly, by modifying the sixteenth hydroxyl of ZER, the hapten of ZER, ZER-(16-hydroxypropyl butyl ether) was designed and synthesized successfully. Then, modified hapten were coupled with carrier proteins BSA and OVA by using mixed anhydrides method and Carbodiimide method respectively, thus we obtained the artificial antigen ZER-BSA and ZER-OVA. Identified by SDS-PAGE, the result showed that coupled proteins had shorter migration distances in TLC plates than carrier proteins, which means coupling was successful. After animal immunization, polyclonal antiserums with titer of 1:2.56×104 and IC50 of 15.77 ng/m L was prepared successfully.2. Preparation and character identification of monoclonal antibody against ZERAfter obtained high-efficiency polyclonal antiserums of ZER, based on monoclonal antibody technique and cell fusion technique, spleen cells of the immunized mice were fused with SP/20 myeloma cell, and subcloning several times, three hybridoma cell lines which secreted ZER-m Ab stably were obtained, 6B2E6, 6B2E11and12B10A7 with the titers of 1:2.56×104、 1:5.12×104 and1:2.56×104 respectively. Ascites by intraperitoneal injection of 12B10A7 has the titer of 1:6.4×105. The obtained monoclonal antibody was identified as Ig G1, the affinity constant(Ka) was 4.7×1011L/mol, standard inhibition curve was y =- 0.3796 x + 0.7746 with R2=0.9885,IC50=0.529ng/ml. The rates of cross reaction with other kinds of toxins, such as Aflatoxin B1(AFB1) and fumonisins B1(FB1), were lower than 0.5%. The cell line could secret antibodies stably either subcultivation in vitro or resuscitation from cryopreservation.3. Establishment of Indirect competitive ELISA for ZER and application of ZER residue detection in animal food productsThrough repeated tests, we obtained the optimum concentration of coating antigen was 1:2000 and the best working concentration of antibody was 1:10000, thus indirect competitive ELISA method for ZER was developed. This method was used to detect ZER residue in animal food products, the results were as follows: standard inhibition curve was y=-0.3582x+0.5949 with R2=0.9925, IC50=0.184ng/ml; The recoveries were above 85%; the coefficient variation was below 10%. The results demonstrated that Indirect competitive ELISA for ZER based on the study has strong sensitivity and high accuracy, can detect ZER residues in foods effectively. |
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