抗脱氧雪腐镰刀菌烯醇单克隆抗体的制备及鉴定

山东大学学报(医学版) ›› 2010, Vol. 48 ›› Issue (12): 146-149.

抗脱氧雪腐镰刀菌烯醇单克隆抗体的制备及鉴定

赵丽1，温红玲1，侯霄煜2，蒲双双1，宋艳艳1，许洪芝1，李凤琴3

1.山东大学公共卫生学院微生物检验所，济南 250012； 2. 济南铁路疾病预防控制中心，济南 250012；
3.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:2010-10-10 发布日期:2010-12-16
通讯作者: 李凤琴（1963- ），女，研究员，博士生导师，主要从事微生物检验。 E-mail：lifengqin0224@gmail.com
作者简介:赵丽（1965- ），女，副教授，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 and characterization of monoclonal antibodies against deoxynivalenol

ZHAO Li1, WEN Hong-ling1, HOU Xiao-yu2, PU Shuang-shuang1, SONG Yan-yan1, XU Hong-zhi1, Li Feng-qin3

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

Received:2010-10-10 Published:2010-12-16

摘要/Abstract

摘要：

目的制备抗脱氧雪腐镰刀菌烯醇(DON)单克隆抗体（McAb）并对其进行初步鉴定。方法采用小剂量长周期的免疫方案，以DON-牛血清白蛋白（BSA）偶联物免疫雌性BALB/c小鼠，采用细胞融合的方法制备杂交瘤细胞株,酶联免疫吸附试验（ELISA）检测上清中McAb，对抗体分泌阳性的细胞株进行克隆化，直至抗体阳性率100%，用竞争抑制ELISA法进一步检测McAb的特异性，腹水诱生法制备大量McAb，并用饱和硫酸铵对其进行纯化，用抗体亚类鉴定试剂盒对该McAb亚类进行鉴定，BCA蛋白测定试剂盒测定蛋白含量，ELISA检测McAb的滴度、参考工作稀释度和亲和常数。结果 DON-BSA免疫的BALB/c小鼠血清效价为1∶256000，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，经3轮克隆化，建立了1株能稳定分泌抗DON-BSA McAb的杂交瘤细胞株3G5，腹水诱生法制备了大量的McAb。该McAb属IgG1,IgG含量为6.06g/L，抗体滴度为1∶500000，参考工作稀释度为1∶64000，抗体亲和常数为1.62×109。用间接竞争抑制ELISA测得校正曲线线性范围9.8～10000ng/mL，线性方程Y=-0.2726X+0.2259(r=0.9309)。结论获得了分泌抗DON-McAb的杂交瘤细胞株，该单克隆抗体灵敏度高，特异性强，可用于制备高质量国产DON-ELISA检测试剂盒。

关键词: 脱氧雪腐镰刀菌烯醇；单克隆抗体；特性鉴定；亲和常数

Abstract:

Objective To prepare monoclonal antibodies(McAbs) against deoxynivalenol（DON）and characterize their properties. Methods Using a low-dose and longcycle immunization scheme, female BALB/c mice were immunized with DON coupled to bovine serum albumin(DON-BSA). Hybridoma cell lines were obtained by cell fusion. McAbs in the supernatant were determined by ELISA. Hybridoma cells which secreted McAbs to DON-BSA were cloned again and again until the McAb-positive rate reached 100%, and the specificity of McAbs to DON was further confirmed by competitioninhibition ELISA, and a large amount of McAbs was made by ascites in liquid paraffinprimed mice and purified by saturated ammonium sulfate. The subtype of the McAb was tested by a subtype identification ELISA kit. The protein concentration was tested by a BCA kit. The titer, work concentration for reference and affinity constant of the McAb were measured by ELISA. Results The titer of antibodies in sera from Balb/c mice immunized by DON-BSAwas 1∶256000 and all had a strong cross-reaction with BSA. Hybridoma cells which secreted McAbs against DONBSA were screened by ELISA after cell fusion. A hybridoma cell line secreting McAb against DON-BSA was established by 3 rounds of cloning and named 3G5. A large amount of McAbs was obtained by ascites production. The subtype of the McAb belongs to IgG1, the concentration of IgG was 6.06g/L; the McAb titer was 1∶ 500000, the McAb work concentration for reference was 1∶64000 and the McAb affinity constant was 1.62×109. The linear range of the competitive inhibition ELISA calibration curve was from 9.8 to 10000ng/mL, and the linear regression equation was Y=-0.2726X+0.2259(r=0.9309). Conclusion The hybridoma cell line secreting McAbs against deoxynivalenol is obtained, and the prepared antibody showed high sensitivity and specificity to DON, so it can be used to produce a homemade DON-ELISA kit of high quality.

Key words: Deoxynivalenol; Monoclonal antibody; Characterization; Affinity constant

中图分类号:

R117

赵丽1，温红玲1，侯霄煜2，蒲双双1，宋艳艳1，许洪芝1，李凤琴3. 抗脱氧雪腐镰刀菌烯醇单克隆抗体的制备及鉴定[J]. 山东大学学报(医学版), 2010, 48(12): 146-149.

ZHAO Li1, WEN Hong-ling1, HOU Xiao-yu2, PU Shuang-shuang1, SONG Yan-yan1, XU Hong-zhi1, Li Feng-qin3. Preparation and characterization of monoclonal antibodies against deoxynivalenol[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2010, 48(12): 146-149.

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