重金属镉单克隆抗体的制备与性质分析

doi:10.7506/spkx1002-6630-201321050

食品科学 ›› 2013, Vol. 34 ›› Issue (21): 248-253.doi: 10.7506/spkx1002-6630-201321050

重金属镉单克隆抗体的制备与性质分析

易翠平1，苏芳1，陈永发1，彭新凯2，朱向荣3

1.长沙理工大学化学与生物工程学院，湖南长沙 410114；2.长沙市食品质量安全监督检测中心，湖南长沙 410013；
3.湖南省食品测试分析中心，湖南长沙 410125

收稿日期:2013-06-28 修回日期:2013-10-11 出版日期:2013-11-15 发布日期:2013-10-28
通讯作者: 易翠平 E-mail:yicp963@163.com
基金资助:
湖南省科技计划重大专项(2011FJ1002-4)；国家自然科学青年基金项目(31301404)；
“十二五”国家科技支撑计划项目(2012BAD34B08)；长沙市科技计划项目(K1207176-21)

Preparation and Properties of Monoclonal Antibody against Heavy Metal Cd2+

YI Cui-ping1，SU Fang1，CHEN Yong-fa1，PENG Xin-kai2，ZHU Xiang-rong3

1. School of Chemical and Biomedical Engineering, Changsha University of Science and Technology, Changsha 410114, China；
2. Supervision and Inspection Center on Food Quality Safety in Changsha, Changsha 410013, China；
3. Hunan Food Test and Analysis Centre, Changsha 410125, China

Received:2013-06-28 Revised:2013-10-11 Online:2013-11-15 Published:2013-10-28
Contact: YI Cui-ping E-mail:yicp963@163.com

摘要/Abstract

摘要：

通过双功能螯合剂iEDTA螯合Cd2+并偶联卵白蛋白(OVA)和牛血清白蛋白(BSA)，合成免疫抗原Cd-iEDTAOVA和筛选抗原Cd-iEDTA-BSA；以Cd-iEDTA-OVA注射BALB/c小鼠，制备Cd2+单克隆抗体，以Cd-iEDTA-BSA通过酶联免疫吸附法(ELISA)进行筛选，并分析抗体性质。结果表明：以Cd2+和OVA含量分别为174.6230μg/L、1.7892mg/mL的Cd-iEDTA-OVA抗原免疫小鼠，Cd2+和BSA含量分别为48.1881μg/L、1.8065mg/mL的Cd-iEDTA-BSA抗原筛选，可获得7F4和7E8两株特异性较好的杂交瘤细胞；7E8E5、7E8G9、7F4B8、7F4D6和7F4H8共5株高纯度单抗，均属IgG1型，其中7E8E5效价最高，达1:512000，亲和常数也最高，达4.55×108 L/mol。建立了Cd2+间接竞争ELISA法，IC50为1150ng/mL，检测限为260ng/mL (R2 = 0.9916)；与Hg2+有较强交叉，与Zn2+、Cu2+、Ca2+交叉较弱，与Mg2+、Ni2+、Al3+、Pb2+、Ba2+几乎无交叉。

关键词: 镉, 抗原, 杂交瘤细胞, 单克隆抗体, 酶联免疫吸附法

Abstract:

Bifunctional chelator isotrhiocyanobenzyl-EDTA (iEDTA) was coupled with Cd2+ and conjugate with
ovalbumin (OVA) and bovine serum albumin (BSA) to obtain Cd-iEDTA-OVA as an immune-antigen and Cd-iEDTABSA
as a screening antigen, respectively. Cd-iEDTA-OVA was injected to BALB/c mice to produce mAb against Cd2+, and
Cd-iEDTA-BSA was utilized to screen the monoclonal antibodies (mAb) by enzyme-linked immunosorbent assay (ELISA).
Results showed that specific hybridoma cell lines 7F4 and 7E8 were obtained after immunizing mice with Cd-iEDTA-OVA containing
174.6230 μg/L Cd2+ and 1.7892 mg/mL OVA, and screening antibody with Cd-iEDTA-BSA containing 48.1881 μg/L Cd2+ and 1.8065 mg/mL
BSA. Five high purity mAbs were obtained, such as 7E8E5, 7E8G9, 7F4B8, 7F4D6 and 7F4H8, whose subtypes belong to
IgG1. Among them, the titer of 7E8E5 was the highest, which was 1:512000; the affinity constant (Ka) of 7E8E5 was
4.55 × 108 L/mol. As a result an indirect competitive ELISA method for Cd2+ was established, and the IC50 and detection
limit were 1150 ng/mL and 260 ng/mL (R2 = 0.9916), respectively. However, it showed strong cross reactivity with Hg2+,
weak cross reactivity with Zn2+, Cu2+ and Ca2+, and almost no cross reactivity with Mg2+, Ni2+, Al2+, Pb2+ or Ba2+.

Key words: cadium, antigen, hybridoma cell, monoclonal antibody (mAb), enzyme linked immunosorbent assay (ELISA)

中图分类号:

Q785

易翠平1，苏芳1，陈永发1，彭新凯2，朱向荣3. 重金属镉单克隆抗体的制备与性质分析[J]. 食品科学, 2013, 34(21): 248-253.

YI Cui-ping1，SU Fang1，CHEN Yong-fa1，PENG Xin-kai2，ZHU Xiang-rong3. Preparation and Properties of Monoclonal Antibody against Heavy Metal Cd2+[J]. FOOD SCIENCE, 2013, 34(21): 248-253.

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重金属镉单克隆抗体的制备与性质分析

Preparation and Properties of Monoclonal Antibody against Heavy Metal Cd2+

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