Liu Biing-Hui, Tsao Zih-Jay, Wang Jing-Jhih, Yu Feng-Yih
Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan.
Anal Chem. 2008 Sep 15;80(18):7029-35. doi: 10.1021/ac800951p. Epub 2008 Aug 13.
A monoclonal antibody (mAb) specific to ochratoxin A (OTA) was produced from a stable hybridoma cell line, 9C9H9, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with OTA-keyhole limpet hemocyanin. The 9C9H9 mAb belongs to the immunoglobulin G1 (kappa chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. The concentrations causing 50% inhibition of binding of OTA-horseradish peroxidase to the antibody by OTA, OTB, and OTC were found to be 0.32, 0.17, and 0.28 ng/mL, respectively, in the cdELISA. A sensitive and rapid mAb-based gold nanoparticle immunochromatographic strip was also developed using this mAb. This strip has a detection limit of 5 ng/mL for OTA and can be completed in 10 min. Analysis of OTA in coffee samples revealed that data obtained from immunochromatographic strip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunochromatographic strip assay established in this study were sensitive and accurate for rapid screening of OTA in coffee samples.
一种针对赭曲霉毒素A（OTA）的单克隆抗体（mAb）由稳定的杂交瘤细胞系9C9H9产生，该细胞系是通过将P3/NS1/1-AG4-1骨髓瘤细胞与从用OTA-钥孔戚血蓝蛋白免疫的BALB/c小鼠分离的脾细胞融合而生成的。9C9H9单克隆抗体属于免疫球蛋白G1（κ链）同种型。建立了竞争性直接酶联免疫吸附测定（cdELISA）和竞争性间接ELISA用于抗体表征。在cdELISA中，OTA、OTB和OTC导致OTA-辣根过氧化物酶与抗体结合抑制50%的浓度分别为0.32、0.17和0.28 ng/mL。还使用该单克隆抗体制备了一种灵敏且快速的基于单克隆抗体的金纳米颗粒免疫层析试纸条。该试纸条对OTA的检测限为5 ng/mL，10分钟内即可完成检测。对咖啡样品中OTA的分析表明，免疫层析试纸条获得的数据与cdELISA获得的数据高度一致。本研究建立的基于单克隆抗体的cdELISA和免疫层析试纸条检测方法灵敏且准确，可用于咖啡样品中OTA的快速筛查。
