玉米赤霉烯酮单克隆抗体的制备及胶体金免疫层析法的建立

发布时间：2018-01-12 21:08

本文关键词：玉米赤霉烯酮单克隆抗体的制备及胶体金免疫层析法的建立　出处：《细胞与分子免疫学杂志》2013年07期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to prepare and identify zearalenone (ZEN) monoclonal antibody (mAb), and the initial establishment of colloidal gold immunochromatographic method. ZEN method using active ester preparation of artificial antigen of ZEN-OVA and ZEN-BSA, ZEN identified mAb.ELISA antibody titer, ZEN-OVA as immunogen to subtype specificity, etc..ZEN mAb- colloidal gold particles was coated with colloidal gold combined pad, check the original ZEN-BSA and Goat anti mouse IgG antibodies bind to nitrocellulose membrane as the test line (T line) and the control line (C line), colloidal gold immunochromatographic assay of ZEN was established. The results by UV spectrophotometry SDS-PAGE analysis and ZEN artificial antigen was synthesized successfully. After 3 times of cloning, screening of 1 hybridoma cell lines secreting ZEN mAb hybridoma cell line 1G4, titer of ascites was 1: 1.6 x 105; antibody subclass is IgG2b; the ZEN IC50 is 10.2 ng/mL, the detection limit is 0.58 ng/mL.mAb for ZEN high In addition to the degree of specificity, cross reactivity with beta - zearalenol rate was 12.8%, and the other ZEN metabolites alpha zearalanol, beta zearalanol zearalanone, and other similar toxin deoxynivalenol, fumonisins, ochratoxin A, almost no cross reaction. The preparation of colloidal gold immunochromatographic test strip within 5 min can be observed to the minimum detection limit for ZEN was 100 ng/mL. conclusion the successful preparation of the 1 strains of ZEN mAb, and established the detection method of the gold immunochromatographic assay, the detection limit was 100 ng/mL.

【作者单位】：暨南大学广东省分子免疫与抗体工程重点实验室;
【基金】：天河区科技计划项目(108ZH044) 暨南大学创新基金资助(11610312)
【分类号】：R392.11
【正文快照】： )玉米赤霉烯酮(zearalenone,ZEN)又称F-2毒素,是由多种镰刀菌产生的一种类雌激素样的真菌毒素。ZEN污染范围广泛,具有较强生殖毒性和致畸作用,可引起动物发生雌激素亢进症,导致动物不孕或流产,对猪、家禽、反刍动物影响较大,给畜牧业带来很大经济损失[1-2];此外,ZEN还具有细

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