赭曲霉毒素A单克隆抗体的制备及直接竞争酶联免疫吸附法的建立  

Development of monoclonal antibodies against ochratoxin A and its application in sensitive direct competitive ELISA assay

作　　者：石爽[1] 卢锋[1] 杨美华[2] 庄辉[1] 

机构地区：[1]北京大学医学部,北京100191 [2]中国医学科学院药用植物研究所

出　　处：《解放军医学杂志》2010年第12期1440-1442,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家科技部"十一五"重大新药创制专项(2009ZX09502-025);中医药行业科研专项基金项目(200807042)

摘　　要：目的建立快速且灵敏的赭曲霉毒素A(OTA)直接竞争酶联免疫吸附检测(cdELISA)方法。方法采用OTA与卵清蛋白(OVA)偶联作为抗原免疫BALB/c小鼠,取小鼠脾细胞与小鼠骨髓瘤细胞SP2/0进行细胞融合、克隆筛选,获得鼠抗OTA的杂交瘤细胞株;以ELISA方法测定抗体效价并鉴定抗体的IgG及亚类;用辣根过氧化物酶(HRP)标记OTA,建立cdELISA方法。结果获得了3株稳定分泌高效价抗OTA单克隆抗体的杂交瘤细胞株(S-17-8、S-212-7和S-225-11),3株重链均为IgG1亚型,轻链为κ亚型;建立了cdELISA检测方法,检测下限为0.5ng/ml。结论以抗OTA单克隆抗体和OTA-HRP为基础,建立了灵敏且省时的检测OTA的cdELISA方法,为进一步OTA快速筛查提供了条件。Objective To develop a sensitive direct competitive enzyme-linked immunosorbent assay（cdELISA）for detection of ochratoxin A（OTA）.Methods OTA was conjugated with ovalbumin（OVA）and keyhole limpet hemocyanin（KLH）by using the 1-ethyl-3-（3-dimethylaminopropyl）carbodiimide（EDC）method.BALB/c mice were immunized with the OTA-OVA conjugate.For hybridoma production,lymphocytes collected from the spleen of the immunized mice were fused with SP2/0myeloma cell line by using polyethylene glycol（PEG）.The hybridomas of interest were cloned by limiting dilution and expansion.The subcloning procedure for the selected clone was repeated 3times.The titer of monoclonal antibodies（mAbs）was assessed using ELISA,and a mouse mAb isotyping kit was used to determine the isotypes of the mAb.OTA that was conjugated with horseradish peroxidase （HRP）by using the carbonyldiimidazole（CDI）method was used in cdELISA.Results High-titer mAbs specific to OTA were produced by 3stable hybridoma cells（S-17-8,S-212-7,and S-225-11）derived from a BALB/c mouse immunized with the OTA-OVA conjugate.These mAbs belong to the IgG1heavy-chain subclass with aκlight chain.Thus,cdELISA was established;the detection limit of this method was 0.5ng/ ml.Conclusions The sensitive cdELISA assay involving mAb and OTA-HRP has been established in present study,which will provide a foundation for rapid screening of OTA.

关 键 词：赭曲霉毒素A 抗体 单克隆 酶联免疫吸附测定 

分 类 号：R392.116[医药卫生—免疫学]