Luo Miner, Tang Yong, Xiang Junjian, Zhang Xiaoli, Fu Qiangqiang, Wang Hong
Guangdong Key Laboratory of Molecular Immunology and Antibody Engineering, Jinan University, Guangzhou 510632, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Jul;29(7):729-33.
To prepare the monoclonal antibody against zearalenone (ZEN) toxin and preliminarily establish the colloidal gold immunochromatographic detection method for ZEN.
The artificial antigen ZEN-BSA and ZEN-OVA were prepared by active ester method. Mice were immunized with ZEN-OVA and monoclonal antibodies against ZEN were prepared by regular cell fusion and subcloning approach. The titer, subtype and specificity of the antibodies were identified by ELISA. To establish a method of colloidal gold immunochromatographic assay (GICA) for the determination of ZEN, colloidal gold binding cushion was coated with anti-ZEN monoclonal antibody-colloidal gold complex, and the synthetic ZEN-BSA and goat anti-mouse immunoglobulins were sprayed on cellulose nitrate film to form the test (T) band and control (C) band.
Identified by SDS-PAGE and UV spectroscopy, the artificial antigen ZEN-BSA and ZEN-OVA were successfully coupled respectively. One hybridoma line (1G4) which could secrete monoclonal antibody specifically against ZEN was obtained by three-time cloning. The ascites titer of this monoclonal antibody reached 1:1.6×10(5);. The antibody subtype was IgG2b. The IC50; to ZEN was 10.2 ng/mL, and the detction limit was 0.58 ng/mL. In addition, the mAb was proved highly specific for ZEN. ZEN metabolites α-zearalanol, β-zearalanol, zearalanone and other similar toxins deoxynivalenol, fumonisin B1, ochratoxin A showed very low or no cross-activity with the monoclonal antibody. The whole assay using the prepared colloidal gold immunochromatographic strip could be finished in 5 min, which could be read by naked eyes. The limit of detection was 100 ng/mL.
The anti-ZEN mAb was successfully prepared. And the gold immunochromatographic assay(GICA) for ZEN was preliminarily established with the limit of detection being 100 ng/mL.
制备抗玉米赤霉烯酮（ZEN）毒素的单克隆抗体，并初步建立ZEN的胶体金免疫层析检测方法。
采用活性酯法制备人工抗原ZEN-BSA和ZEN-OVA。用ZEN-OVA免疫小鼠，通过常规细胞融合和亚克隆方法制备抗ZEN的单克隆抗体。采用ELISA法鉴定抗体的效价、亚型和特异性。为建立一种测定ZEN的胶体金免疫层析分析（GICA）方法，用抗ZEN单克隆抗体-胶体金复合物包被胶体金结合垫，并将合成的ZEN-BSA和羊抗鼠免疫球蛋白喷在硝酸纤维素膜上形成检测（T）线和对照（C）线。
经SDS-PAGE和紫外光谱鉴定，人工抗原ZEN-BSA和ZEN-OVA分别成功偶联。经三次克隆获得一株能分泌特异性抗ZEN单克隆抗体的杂交瘤细胞株（1G4）。该单克隆抗体腹水效价达1:1.6×10⁵；抗体亚型为IgG2b。对ZEN的半数抑制浓度（IC50）为10.2 ng/mL，检测限为0.58 ng/mL。此外，该单克隆抗体对ZEN具有高度特异性。ZEN的代谢产物α-玉米赤霉醇、β-玉米赤霉醇、玉米赤霉酮以及其他类似毒素脱氧雪腐镰刀菌烯醇、伏马毒素B1、赭曲霉毒素A与该单克隆抗体的交叉活性很低或无交叉活性。使用所制备的胶体金免疫层析试纸条进行的整个检测可在5分钟内完成，且可肉眼读取结果。检测限为100 ng/mL。
成功制备了抗ZEN单克隆抗体，并初步建立了ZEN的胶体金免疫层析检测方法，检测限为100 ng/mL。
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