中国农业科学 ›› 2013, Vol. 46 ›› Issue (2): 417-423.doi: 10.3864/j.issn.0578-1752.2013.02.021

• 兽医 • 上一篇    下一篇

分泌抗春雷霉素单克隆杂交瘤细胞株的建立 及免疫学特性鉴定

 龚芳, 职爱民, 李青梅, 胡骁飞, 赵启发, 张小辉, 陆启玉, 张改平   

  1. 1.河南省农业科学院/河南省动物免疫学重点实验室,郑州 450002
    2.河南工业大学粮油食品学院,郑州 471001
  • 收稿日期:2012-05-11 出版日期:2013-01-15 发布日期:2013-01-08
  • 通讯作者: 通信作者张改平,E-mail:zhanggaiping2003@yahoo.com.cn
  • 作者简介:龚芳,Tel:13526573598;E-mail:gongfang870607@163.com
  • 基金资助:

    农业部公益性行业(农业)科研专项(201003008-09)

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

 GONG  Fang, ZHI  Ai-Min, LI  Qing-Mei, HU  Xiao-Fei, ZHAO  Qi-Fa, ZHANG  Xiao-Hui, LU  Qi-Yu, ZHANG  Gai-Ping   

  1. 1.Key Laboratory for Animal Immunology of the Ministry of Agriculture/ Henan Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002
    2.School of Food Science and Technology, Henan University of Technology, Zhengzhou 471001
  • Received:2012-05-11 Online:2013-01-15 Published:2013-01-08

摘要: 【目的】制备高灵敏、高特异性的抗春雷霉素单克隆抗体,并对其免疫学特性进行鉴定。【方法】采用EDC法将KSM分别偶联于载体蛋白BSA和OVA上,分别合成免疫原KSM-BSA和包被原KSM-OVA,经SDS-PAGE鉴定后,免疫Balb/c小鼠,免疫间隔4周,4次免疫后,利用间接ELISA和间接竞争ELISA测定其多抗血清效价和敏感性,选出效价高、敏感性好的小鼠进行细胞融合,经过多次亚克隆筛选出分泌高敏感的特异性抗KSM单克隆抗体的杂交瘤细胞株,体内诱生腹水法制备抗KSM的单克隆抗体并对其免疫学特性进行鉴定。【结果】SDS-PAGE鉴定结果显示KSM-BSA成功偶联,间接ELISA检测显示免疫的6只小鼠血清效价均达1:104以上,其中3号小鼠多抗血清敏感性最好,半数抑制浓度IC50为73.9ng•mL-1,融合后筛选出两株杂交瘤细胞株1G7和2C8,亚型均为IgG1型,其细胞上清效价分别为1:5.12×103 和1:1.28×103,腹水效价分别为1:5.12×105和1:2.56×105,抗KSM的单克隆抗体对春雷霉素的IC50为8.902ng•mL-1,与其它竞争物的交叉率均小于0.03%。【结论】本试验成功合成了春雷霉素人工抗原,并制备了敏感性高、特异性好的单克隆抗体,为春雷霉素免疫学快速检测方法的建立奠定了基础。

关键词: 春雷霉素 , 人工抗原 , 单克隆抗体

Abstract: 【Objective】 The aim of the study is to generate high sensitivity and high specificity monoclonal antibody against kasugamycin and identify its immunological characteristics.【Method】KSM was coupled with the carrier protein BSA and OVA by using EDC method and the immunogen KSM-BSA and coating antigen KSM-OVA were synthesized respectively, and SDS-PAGE was used to identify KSM-BSA. Balb/c mouse was immunized by using KSM-BSA. Immunization interval was 4 weeks. The titer of polyclonal antibody was detected by indirect ELISA and blocking ELISA after four times immunization. The high titer and high sensitivity mouse was selected for cell fusion. High sensitivity and high specificity monoclonal antibody was prepared after several times subcloning,and the immunological characteristics were characterized. The preparation of ascites was carried out using in vivo induction method. 【Result】 SDS-PAGE results showed that KSM-BSA artificial antigen was synthesized successfully.Indirect ELISA showed a high titer above 1:104 of the antiserum of all the six Balb/c mouse. The sensibility of antiserum of No.3 mice was the best, which IC50 was 73.9ng•mL-1. Hybridoma cell lines of 1G7 and 2C8 were screened after cell fusion, all the isotypes of the mAb were IgG1.Titers of the mAb were 1:5.12×103 and 1:1.28×103 in supernatant,1:5.12×105 and 1:2.56×105 in ascites. The IC50 of KSMmAb was 8.902 ng•mL-1 to kasugamycin. The rate of cross reaction of the mAb with other compounds was less than 0.03%.【Conclusion】KSM artificial antigens were synthesized successfully and the monoclonal antibody with high sensitivity and specificity was obtained. This study has laid a foundation for a KSM immunological fast detection method.

Key words: kasugamycin , artificial antigen , Monoclonal antibody