基于酶联免疫分析的中药薏苡仁玉米赤霉烯酮残留快速检测技术研究  

Rapid detection of zearalenone in Coicis Semen based on ELISA

作　　者：刘云翔 高鹏超 詹志来 康利平[1] 南铁贵[1] 袁媛[1] LIU Yun-xiang;GAO Peng-chao;ZHAN Zhi-lai;KANG Li-ping;NAN Tie-gui;YUAN Yuan(State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国中医科学院中药资源中心道地药材国家重点实验室培育基地,北京100700

出　　处：《中国中药杂志》2023年第11期2919-2924,共6页China Journal of Chinese Materia Medica

基　　金：中央级公益性科研院所基本科研业务费专项(ZZXT202106);中国中医科学院科技创新工程项目(CI2021A04005)。

摘　　要：玉米赤霉烯酮(zearalenone,ZEN)是由黄色镰刀菌、禾谷镰刀菌、三线镰刀菌等真菌产生的有毒代谢产物,具雌激素性特征。接触或摄入ZEN会导致生殖机能异常,妊娠期还会引起流产、死胎和畸胎,严重危害人类生命健康安全。《中国药典》2020年版中ZEN的检测方法为液相色谱(LC)和液相色谱-串联质谱(LC-MS),且规定每1000 g薏苡仁含ZEN不得过500μg。仪器检测方法虽可以实现薏苡仁中ZEN的定性和定量分析,但因其检测成本高、周期长,而无法满足大批量样本的快速筛查需求。该研究将ZEN半抗原通过与牛血清白蛋白(BSA)、卵清蛋白(OVA)偶联后获得ZEN的完全抗原。利用抗体制备技术,制备得到ZEN单克隆抗体4F6,该抗体与ZEN结构类似物玉米赤霉醇、玉米赤霉酮、α-玉米赤霉烯醇的交叉反应率分别为177.5%、137.1%、109.7%,与黄曲霉毒素等其他真菌毒素无交叉反应。通过方法学考察,建立了基于ZEN单克隆抗体4F6的薏苡仁中ZEN的直接竞争酶联免疫检测方法(direct competitive enzyme-linked immunosorbent assay,dcELISA),方法的IC50为1.3μg·L^(-1),检测范围为0.22~21.92μg·L^(-1)。加样回收率为83.91%~105.3%,变异系数为4.4%~8.0%。利用所建立的dcELISA方法,测定了9批薏苡仁样品中的ZEN残留,并使用LC-MS进行验证,2种检测方法测定结果相关性良好(R2=0.9939),表明所建立的dcELISA可用于薏苡仁中ZEN残留的快速检测。Zearalenone(ZEN)is a toxic metabolite produced by Fusarium culmorum,F.graminearum,F.tricinctum,and other fungi,with estrogenic characteristics.Exposure to or ingestion of ZEN during pregnancy can cause reproductive dysfunction,miscarriage,stillbirth,and malformation,and seriously endanger human life and health.The detection methods for ZEN in the Chinese Pharmacopoeia(2020 edition)are liquid chromatography(LC)and liquid chromatography-mass spectrometry(LC-MS),and it is stipulated that ZEN should not exceed 500μg in 1000 g of Coicis Semen.Although these detection methods by instruments can achieve the qualitative and quantitative analysis of ZEN in Coicis Semen,their high detection cost and long periods hinder the rapid screening of a large number of samples in the field.In this study,the synthesized ZEN hapten was conjugated with bovine serum albumin(BSA)and ovalbumin(OVA)to obtain the complete ZEN antigen.By virtue of antibody preparation techniques,ZEN monoclonal antibody 4F6 was prepared,which showed 177.5%,137.1%,and 109.7%cross-reactivity with ZEN structural analogs zearalanol,zearalenone,andα-zearalenol,respectively,and no cross-reactivity with other fungal toxins such as aflatoxin.Direct competitive enzyme-linked immunosorbent assay(dcELISA)based on ZEN monoclonal antibody 4F6 was developed for the determination of ZEN in Coicis Semen with an IC50 of 1.3μg·L^(-1) and a detection range of 0.22-21.92μg·L^(-1).The recoveries were 83.91%-105.3%and the RSD was 4.4%-8.0%.The established dcELISA method was used to determine the ZEN residuals in nine batches of Coicis Semen samples,and the results were validated by LC-MS.The correlation between the two detection methods was found to be 0.9939,indicating that the established dcELISA could be used for the rapid qualitative and quantitative detection of ZEN residuals in Coicis Semen.

关 键 词：薏苡仁 玉米赤霉烯酮 单克隆抗体 酶联免疫检测法 

分 类 号：R284.1[医药卫生—中药学]