抗莱克多巴胺单克隆抗体杂交瘤细胞株的建立及其免疫学特性鉴定

doi:10.7506/spkx1002-6630-200903039

食品科学 ›› 2009, Vol. 30 ›› Issue (3): 175-179.doi: 10.7506/spkx1002-6630-200903039

抗莱克多巴胺单克隆抗体杂交瘤细胞株的建立及其免疫学特性鉴定

张海棠¹,王自良¹,邓瑞广²,钟华¹,范国英¹

1.河南科技学院动物科学学院 2.河南省动物免疫学重点实验室

收稿日期:2008-02-22 修回日期:2008-05-04 出版日期:2009-02-01 发布日期:2010-12-29
通讯作者: 王自良 E-mail:wangzl_2008@yahoo.com.cn
基金资助:
“十一五”国家科技支撑计划重大项目(2006BAK02A21/1)

Establishment of Hybridoma Cell Lines Secreting Anti-ractopamine Monoclonal Antibody and Identification of Their Immunological Properties

ZHANG Hai-tang1，WANG Zi-liang1,*，DENG Rui-guang2，ZHONG Hua1，FAN Guo-ying1

(1.College of Animal Science, Henan Institute of Science and Technology, Xinxiang 453003, China；
2.Henan Key Laboratory of Animal Immunology, Zhengzhou 450002, China)

Received:2008-02-22 Revised:2008-05-04 Online:2009-02-01 Published:2010-12-29
Contact: WANG Zi-liang1,* E-mail:wangzl_2008@yahoo.com.cn

摘要/Abstract

摘要：

莱克多巴胺(RAC)经化学修饰引入功能基团羧基，合成半抗原RAC- 戊二酸酐半醛化合物(RAC-SA)，用混合酸酐法(MA)将RAC-SA 偶联于牛血清白蛋白(BSA)，合成人工抗原BSA-RAC，用红外(IR)、紫外(UV)和凝胶电泳(SDS-PAGE)对其进行鉴定；用BSA-RAC免疫Balb/C 小鼠，细胞融合技术建立抗RAC的单克隆抗体(RAC mAb)杂交瘤细胞株，体内诱生腹水法制备RAC mAb，并鉴定其免疫学特性。结果表明，BSA-RAC 偶联成功，偶联率为24.5:1；筛选出3F10、3H12、4D8 共3 株杂交瘤细胞，其中最好的4D8 株间接ELISA 效价细胞培养上清为1:1.28 × 103，腹水为1:6.4 × 105，同种型为IgG1/κ，亲和常数(Ka)为1.65 × 1010L/mol，对RAC 的半数抑制浓度(IC50)为5.7ng/ml，与多巴酚丁胺的交叉反应率(CR)为22.3%，与其他化合物无CR。

关键词: 莱克多巴胺, 半抗原, 人工抗原, 单克隆抗体, 免疫学特性

Abstract:

The active group carboxyl was introduced to ractopamine (RAC) and formed RAC semialdehyde (RAC-SA) which has hapten structure by chemical modification. The method of mixed anhydride (MA) was used to conjugate RAC-SA to bovine serum albumin (BSA) and obtain artificial antigen BSA-RAC identified by infrared (IR), ultraviolet (UV) and SDS-PAGE. Balb/C mice were immunized with BSA-RAC and hybridoma lines that secrete monoclonal antibody against RAC (RAC mAb) were established through cell fusion and then screened using indirect ELISA. Blocking ELISA and the immunological traits of RAC mAb were identified. The results showed that BSA-RAC is synthesized successfully and its conjugation ratio between RAC and BSA is about 24.5:1. Three hybridoma lines of 3F10, 3H12 and 4D8 are screened out and the best one is 4D8 with indirect ELISA titers of 1:1.28×103 in supernatant and 1:6.4×105 in ascites. The isotype of the mAb is IgG1/κ and its affinity constant (Ka) is 1.65×1010L/mol. The mAb shows good sensitivity with an IC50 of 5.7 ng/m lto RAC, 22.3% cross-reactivity with dobutamine and little or no cross-reactivity with other compounds. Establishment of hybridoma lines that secrete RAC mAb makes it possible to develop immunoassay of RAC residues in animal food.

Key words: ractopamine, hapten, artificial antigen, monoclonal antibody, immunological properties

中图分类号:

S859.84

张海棠1，王自良1,*,邓瑞广2，钟华1，范国英1. 抗莱克多巴胺单克隆抗体杂交瘤细胞株的建立及其免疫学特性鉴定[J]. 食品科学, 2009, 30(3): 175-179.

ZHANG Hai-tang1，WANG Zi-liang1,*，DENG Rui-guang2，ZHONG Hua1，FAN Guo-ying1. Establishment of Hybridoma Cell Lines Secreting Anti-ractopamine Monoclonal Antibody and Identification of Their Immunological Properties[J]. FOOD SCIENCE, 2009, 30(3): 175-179.

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抗莱克多巴胺单克隆抗体杂交瘤细胞株的建立及其免疫学特性鉴定

Establishment of Hybridoma Cell Lines Secreting Anti-ractopamine Monoclonal Antibody and Identification of Their Immunological Properties

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