沙拉沙星单克隆抗体制备及鉴定

doi:10.7668/hbnxb.2016.02.015

华北农学报 ›› 2016, Vol. 31 ›› Issue (2): 87-91. doi: 10.7668/hbnxb.2016.02.015

沙拉沙星单克隆抗体制备及鉴定

刘儒彪¹, 胡骁飞², 张改平^2,3, 王慧¹, 王方雨², 刘璐¹, 刘玺¹, 邓瑞广², 宋照军¹

1. 河南科技学院食品学院, 河南新乡 453003;
2. 河南省农业科学院动物免疫学重点实验室, 河南郑州 450002;
3. 河南农业大学牧医工程学院, 河南郑州 450002

收稿日期:2016-02-26 出版日期:2016-04-28
通讯作者: 宋照军(1973-),男,河南新乡人,副教授,主要从事肉品加工与质量安全控制研究;邓瑞广(1960-),男,河南平顶山人,研究员,硕士,主要从事动物疫病及食品安全免疫学检测技术研究。
作者简介:刘儒彪(1987-),男,河南商丘人,在读硕士,主要从事农产品深加工技术研究。
基金资助:
2015年河南省科技攻关项目(152102310094);河南省高校科技创新团队支持计划项目(13IRTSTHN006)

Preparation and Identification of Monoclonal Antibody for Sarafloxacin

LIU Rubiao¹, HU Xiaofei², ZHANG Gaiping^2,3, WANG Hui¹, WANG Fangyu², LIU Lu¹, LIU Xi¹, DENG Ruiguang², SONG Zhaojun¹

1. College of Food Science and Technology, Henan Institute of Science and Technology, Xinxiang 453003, China;
2. Henan Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
3. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China

Received:2016-02-26 Published:2016-04-28

摘要/Abstract

摘要： 为制备敏感性好、亲和力高、特异性强的抗沙拉沙星(Sarafloxacin,SARA)单克隆抗体,采用碳二亚胺法合成SARA人工抗原,通过免疫BALB/c小鼠,选择血清效价高且敏感性好的小鼠,采用杂交瘤细胞技术进行细胞融合,筛选分泌抗SARA单克隆抗体(Monoclonal antibody,mAb)的杂交瘤细胞株;采用体内诱生腹水法制备SARA mAb,通过间接ELISA和间接竞争ELISA对SARA mAb的免疫学特性进行鉴定。结果表明:小鼠经免疫原免疫后,小鼠多抗血清效价均达到1:128000。选择敏感性较好的2号小鼠进行细胞融合,经多次亚克隆后,筛选出1G3、3H3两株杂交瘤细胞株,其中1G3所产SARA mAb效价较高,为1:512000,IC₅₀为6.34 ng/mL,亲和常数为8.55×10⁸ L/mol,与诺氟沙星的交叉反应率为1.06%,与其他药物交叉反应率均低于0.50%。

关键词: 沙拉沙星, 单克隆抗体, 杂交瘤细胞, 免疫学特性

Abstract: In order to preparation of sarafloxacin(SARA) monoclonal antibody with good sensitivity, high affinity and strong specificity, synthesized SARA artificial antigen by carbodiimide method.It was used to immunize BALB/c mice.The mice which possessed high serum titer and good sensitivity were selected for cell fusing through cell hybridoma technique.Then to filtrate the hybridoma cell strain which secreted SARA monoclonal antibody(mAb).Prepared SARA mAb by inducing ascites in vivo, and identificated the immunity characteristics of SARA mAb via indirect ELISA and indirect competitive ELISA.The result indicated that after mice were immune by immunogen, their serum antibody titer were all up to 1:128 000.Number 2 mouse with good sensitivity was chosen for cell fusion.After multiple sub cloning, 1G3 and 3H3 hybridoma cell strain were filtered.The titer of SARA mAb from 1G3 was 1:512 000, IC₅₀ was 6.34 ng/mL, affinity constant was 8.55×10⁸ L/mol, cross-reactivity with norfloxacin was 1.06%, cross-reactivity with other drugs were below 0.50%, higher than that of 3H3.

Key words: Sarafloxacin, Monoclonal antibody, Hybridoma cell, Immunological characteristics

中图分类号:

S859.84

刘儒彪, 胡骁飞, 张改平, 王慧, 王方雨, 刘璐, 刘玺, 邓瑞广, 宋照军. 沙拉沙星单克隆抗体制备及鉴定[J]. 华北农学报, 2016, 31(2): 87-91. doi: 10.7668/hbnxb.2016.02.015.

LIU Rubiao, HU Xiaofei, ZHANG Gaiping, WANG Hui, WANG Fangyu, LIU Lu, LIU Xi, DENG Ruiguang, SONG Zhaojun. Preparation and Identification of Monoclonal Antibody for Sarafloxacin[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(2): 87-91. doi: 10.7668/hbnxb.2016.02.015.

http://www.hbnxb.net/CN/Y2016/V31/I2/87

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