抗赭曲霉毒素A单克隆抗体的制备及其ELISA检测方法的建立

山东大学学报(医学版) ›› 2009, Vol. 47 ›› Issue (10): 60-63.

抗赭曲霉毒素A单克隆抗体的制备及其ELISA检测方法的建立

侯霄煜¹，温红玲¹，闫玉芬¹，宋艳艳¹，许洪芝¹，赵丽¹，李凤琴²

1. 山东大学公共卫生学院卫生检验研究所，济南 250012；
2. 中国疾病预防控制中心营养与食品安全所，北京 100052

收稿日期:2009-04-01 出版日期:2009-10-16 发布日期:2009-10-16
通讯作者: 赵丽（1965- ），女，副教授，博士，主要从事微生物检验。 Email：dlzhl@sdu.edu.cn
作者简介:侯霄煜（1979- ），男，技师，硕士研究生，主要从事微生物检验。
基金资助:
国家高技术研究发展计划（863计划）资助课题

（2007AA10Z423）。

Preparation of monoclonal antibody against orchratoxin A and 
establishment of ELISA to detect it

HOU Xiaoyu¹, WEN Hongling¹，　YAN Yu fen¹，　SONG Yanyan¹,　XU Hongzhi¹,　ZHAO Li¹,　LI Fengqin²

1. Department of Hygiene Detection, School of Public Health, Shandong University, Jinan 250012, China;
2. Institute of Nutrition and Food Hygiene, Chinese Center for Disease Control and Prevention, Beijing 100052, China

Received:2009-04-01 Online:2009-10-16 Published:2009-10-16

摘要/Abstract

摘要：

目的制备抗赭曲霉毒素A（OA）的单克隆抗体（McAb）并对其进行初步鉴定，在此基础上建立竞争抑制酶联免疫吸附试验（ELISA）用于OA的检测。方法采用小剂量长周期的免疫方案，以OA牛血清白蛋白（BSA）偶联物免疫雌性BALB/c小鼠，采用细胞融合法获得分泌抗OA的McAb的杂交瘤细胞株,用竞争抑制ELISA法进一步检测McAb的特异性，腹水诱生法大量制备McAb，以OA为竞争抗原，建立检测OA的竞争抑制ELISA。结果OABSA免疫的BALB/c小鼠血清效价为1:512?000，与BSA有强烈的交叉反应。细胞融合后，ELISA筛选抗体分泌阳性的杂交瘤细胞株，抗OABSA的McAb与BSA的交叉反应率仅为3.5%，对分泌抗OABSA特异的McAb的细胞株经3轮克隆化，抗体分泌阳性率达到100%，建立了1株能稳定分泌抗OABSA McAb的杂交瘤细胞株，竞争抑制法进一步证明了该抗体是特异针对OA的，腹水诱生法制备了大量的McAb。竞争抑制ELISA线性范围为0.24～125?ng/mL，线性方程y=-0.113?2logx+0.901?6，相关系数r=0.98，最低检出浓度为0.24?ng/mL。样品的加标回收率为97.07%～107.83%。结论获得了分泌抗OA的McAb的杂交瘤细胞株，建立了OA检测的简单、灵敏、高效的ELISA检测方法。

关键词: 赭曲霉犊素A, 单克隆抗体, 细胞融合, 酶联免疫吸附试验

Abstract:

To prepare and identify monoclonal antibody(McAb) against ochratoxin A（OA）and to establish a competitive inhibition ELISA method for detection of OA. MethodsUsing a lowdose and longcycle immunization scheme, female BALB/c mice were immunized with OA coupled to bovine serum albumin(OABSA). Hybridoma cell lines secreting McAbs against OABSA were obtained by cell fusion. The specifcity of McAbs to OA was further analyzed by a competition inhibition test. A large amount of McAbs was made by ascites in liquid paraffinprimed mice. A competition inhibition ELISA method using OA as a competitive antigen was established to monitor OA. ResultsTiter of McAbs in the sera from BALB/c mice immunized by OABSA was 1:512?000 and it had a strong cross reaction with BSA. Hybridoma cell lines which secreted McAbs against OABSA were screened by ELISA after cell fusion. The crossreactive rate of McAbs against OA and BSA was 3.5%. Hybridoma cell lines secreting McAbs against OABSA were completely established by 3 rounds of cell subcloning, and the specificity of McAbs for OA was further confirmed by competition inhibition ELISA. A large amount of McAbs was obtained by ascites production. The linear range of the competitive inhibition ELISA was 0.24125?ng/mL, the linear regression equation was y=-0.113?2logx+0.901?6, the linear correlation was 0.98, and the detection limit was 0.24?ng/mL. Recovery rate for the sample ranged from 97.07% to 107.8%. ConclusionHybridoma cell lines secreting McAbs against ochratoxin A were obtained and a simple and sensitive efficient method for OA determination was established.

Key words: Ochratoxin A； Monoclonal antibody； Cell fusion； ELISA

中图分类号:

R115

. 抗赭曲霉毒素A单克隆抗体的制备及其ELISA检测方法的建立[J]. 山东大学学报(医学版), 2009, 47(10): 60-63.

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