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目的制备并鉴定玉米赤霉烯酮(ZEN)单克隆抗体(mAb),并初步建立ZEN的胶体金免疫层析检测方法。方法采用活泼酯法制备人工抗原ZEN-OVA和ZEN-BSA,以ZEN-OVA作为免疫原制备ZEN mAb。ELISA法鉴定抗体效价、亚类、特异性等。ZEN mAb-胶体金复合物包被于胶体金结合垫上,检测原ZEN-BSA和山羊抗小鼠IgG分别结合于硝酸纤维膜上作为检测线(T线)和质控线(C线),建立ZEN的胶体金免疫层析检测法。结果经紫外分光光度法和SDS-PAGE鉴定,ZEN人工抗原合成成功。经3次克隆化后,筛选出1株能稳定分泌ZEN mAb的杂交瘤细胞株1G4,腹水效价为1∶1.6×105;抗体亚类为IgG2b;对ZEN的IC50为10.2 ng/mL,检测限为0.58 ng/mL。mAb对ZEN具有高度的特异性,除与β-玉米赤霉烯醇的交叉反应率为12.8%外,与其他ZEN代谢物α-玉米赤霉醇、β-玉米赤霉醇、玉米赤霉酮及其他相似毒素呕吐毒素、伏马毒素、赭曲霉素A等几乎无交叉反应。制备的胶体金免疫层析试纸条在5 min内即可肉眼观察到结果,对ZEN的最低检测限为100 ng/mL。结论成功制备出1株ZEN mAb,并初步建立了其胶体金免疫层析检测方法,最低检测限为100 ng/mL。
Objective To prepare and identify the monoclonal antibody (mAb) of zearalenone (ZEN) and to establish a colloidal gold immunochromatographic assay for the determination of ZEN. Methods The artificial antigen ZEN-OVA and ZEN-BSA were prepared by active ester method. ZEN mAb was prepared by using ZEN-OVA as immunogen. ELISA method to identify antibody titer, subclass, specificity and so on. ZEN mAb-colloidal gold complex was coated on the gold-binding pad, and the original ZEN-BSA and goat anti-mouse IgG were respectively bound to the nitrocellulose membrane as a test line (T line) and a control line (C line) ZEN colloidal gold immunochromatographic assay. The results of UV spectrophotometry and SDS-PAGE identification, ZEN artificial antigen synthesis success. After three clones, one strain of hybridoma cell line 1G4 that can stably secrete ZEN mAb was screened. The ascites titer was 1: 1.6 × 105, the antibody subclass was IgG2b, and the IC50 of ZEN was 10.2 ng / mL. Limit 0.58 ng / mL. The mAbs have a high degree of specificity for ZEN with the exception of the other ZEN metabolites, alpha-zeranol, beta-zeranol, zeaxanthin, with a 12.7% cross-reactivity with beta-zearalenol And other similar toxins vomitoxin, fumonisin, ochratoxin A almost no cross-reaction. The results of colloidal gold immunochromatographic test strip prepared in 5 min can be visually observed, the detection limit of ZEN is 100 ng / mL. Conclusion A strain of ZEN mAb was successfully prepared and its colloidal gold immunochromatographic assay was established preliminarily. The detection limit was 100 ng / mL.