薏苡仁中呕吐毒素酶联免疫检测方法的建立

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for determination of vomitoxin (DON) in Coicis Semen

薏苡仁为药食同源的常用大宗药材,具有利水渗湿、健脾止泻的功效。其原植物薏苡为禾本科植物,在生长过程中由于气候原因易受禾谷镰刀菌Fusarium graminearum和黄色镰刀菌F.culmorum等产生呕吐毒素等多种真菌污染。《中国药典》2351真菌毒素测定法中呕吐毒素检测方法为高效液相色谱(HPLC)和液相色谱-串联质谱(LC-MS),检测周期长,费时、费力,不能满足药品的现场质量检测要求。通过将呕吐毒素进行化学衍生,与蛋白偶联后获得呕吐毒素的完全抗原。利用抗体制备技术,制备呕吐毒素的单克隆抗体,通过方法学考察,进而建立薏苡仁中呕吐毒素的酶联免疫检测方法(enzyme-linked immunosorbent assay, ELISA)。基于薏苡仁中呕吐毒素ELISA的IC_(50)为3.88μg·L^(-1),加样回收率为77.32%~93.73%,RSD为4.6%~9.7%。利用所建立的ELISA方法,测定了14批薏苡仁样品中的呕吐毒素残留量,并使用LC-MS进行验证,发现2种检测方法测定结果相关性为0.997 8,表明所建立的酶联免疫方法可用于薏苡仁中呕吐毒素残留量的定量检测,可以实现薏苡仁中呕吐毒素残留的快速定量检测。

Coicis Semen is a common bulk medicinal material used for both medicine and food, which has the effect of promoting diuresis, draining dampness, invigorating the spleen and checking diarrhea. It is derived from Coix lacryma-jobi var. ma-yuen of the family Poaceae, and is easily contaminated by fungi such as Fusarium graminearum and F. flavum due to climate reasons to produce vomitoxin. The guiding principles for determination of vomitoxin in Chinese medicinal materials in Chinese Pharmacopoeia are mainly HPLC and LC-MS, which have long detection period and are time-consuming and laborious, and thus cannot meet the requirements of on-site quality inspection of drugs. The complete antigen of vomitoxin-protein was obtained by chemical derivatization of vomitoxin. The monoclonal antibody against vomitoxin was prepared by classic monoclonal antibody preparation technology, and enzyme-linked immunosorbent assay(ELISA) method for detection of vomitoxin in Coicis Semen was established through methodological investigation. The IC_(50)based on the ELISA for vomitoxin in Coicis Semen was 3.88 μg·L^(-1), and the average recoveries and the RSD were 77.32%-93.73% and 4.6%-9.7%, respectively. With the established ELISA method, the vomitoxin residue in 14 batches of Coicis Semen samples were determined and validated by LC-MS, and the correlation between the two assays was found to be 0.997 8, indicating that the established ELISA method could be used for quantitative determination of vomitoxin residue in Coicis Semen and could achieve the rapid quantitative determination of the vomitoxin residue.