| Zearalenone(ZEN) is a secondary metabolites produced by Fusarium, which has estrogenic agonistic. The pollution of food and feed by ZEN is wide. After intaking of contaminated food or feed, human and animal will be damaged. Therefore, it is important to monitor and detect the ZEN residues in food and feedstuffs. The matrix effect of samples has a certain impact on the quantitative test results. The ZEN immune magnetic microspheres, which can be used to efficiently enrich and capture the ZEN from samples and reduced the matrix effect, were prepared by the mediating system of polyethyleneimine-glutaraldehyde,. Then, with colloidal gold as a marker of material, this paper developed a colloidal gold immunochromatographic test strip which combined with HG-8 colloidal gold immunochromatographic test strip reader to realize the rapid and quantitative detection of ZEN residues in feedstuffs.ZEN-BSA was prepared by the method of EDC/NHS active ester. And it has been successfully compounded, then it was verifyed by TLC monitoring and UV wavelength scanning. The concentration of ZEN-BSA was 2.55 mg/m L.The purified antibody was obtained by the method of caprylic acid-ammonium sulfate. Meanwhile,the performances of antibody were evaluated: the measured titers of antibody was1:160000, the concentration of antibody was 5.36 mg/mL. Analysised by SDS-PAGE electrophoresis, the results showed that the purifiedl antibodies have a high purity.In order to ensure the capture efficiency, polyethyleneimine–glutaraldehyde mediated immune magnetic microsphere was prepared and the amount of anti-ZEN monoclonal antibody coupled to immune magnetic microspheres was optimized. In this study, 1 mg of PEI-magnetic microspheres was conjugated with 100 μg of antibody. The pH of immune magnetic enrichment capture buffer, the concentration of methanol, the acquisition time, the amount of microspheres, and the eluent type of conditions were optimized. The optimum conditions were as follows: 200 μg of PEI-immunomagnetic beads were added to 1 mL of PBS buffer(pH7.0) which contained 20% of methanol, the time of extraction was 10 min, 100 μL of 3%ammoniated methanol was selected to elution ZEN from magnetic microspheres aftermagnetic separation. The eluent dilution after redissolution was determined by ELISA kit. It showed that the recoveries increased gradually when the concentration of ZEN was from 50 to 250 μg/kg in the feed samples. When the concentration of ZEN was250 μg/kg, the recovery rate reached at 93.28%.In this paper, colloidal gold was used as a marker. The colloidal gold particle size, pH value, the concentration of labeled antibody, the concentration of antigen and donkey anti mouse IgG, the spray volume of gold labeled antibody and other parameters had been optimized. The optimal process conditions for quantitative detection of ZEN colloid gold immunochromatographic assay were as follows: 40 nm of colloidal gold particle was labeled with 4 μg/mL antibody under the condition of pH6.0; the amount of the colloidal gold labeled antibody is 2 μL/cm; the concentrations of ZEN-BSA and donkey anti mouse IgG on the NC membrane were0.8 mg/mL and 0.4 mg/mL, respectively. Thus, the quantitative detection of ZEN colloidal gold immunochromatographic test strip had been successfully developed.,The test strip had good specificity and stability while the detection time was 15 min.The limit detection was 2.559 μg/L while the IC50 was 9.09 μg/L. The recovery rate of the methd was 67.70%-104.48%. In addition, the ELISA assay kit was used to evaluate the accuracy of this method, the results of the two methods showed a good correlation. |
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