阪崎肠杆菌单克隆抗体制备及ELISA检测方法的建立

›› 2013, Vol. 40 ›› Issue (12): 52-55.

阪崎肠杆菌单克隆抗体制备及ELISA检测方法的建立

汪琳¹, 李勐伟^1,2, 刑佑尚¹, 柏亚铎¹, 尹羿¹, 蒲静¹, 乔彩霞¹, 高志强¹, 魏学良², 李朝明², 李琴²

1. 北京出入境检验检疫局, 北京 100026;
2. 西南大学, 重庆 400715

收稿日期:2013-05-06 出版日期:2013-12-20 发布日期:2014-02-11
作者简介:汪琳（1974- ），女，四川人，博士，研究方向：动物检疫；李勐伟（1988- ），男，四川人，硕士生，研究方向：动物医学。汪琳和李勐伟对本文具有同等贡献，并列为第一作者。
基金资助:
“十二五”国家科技支撑计划（2011BAK10B02）。

Preparation of Monoclonal Antibody against Enterobacter sakazakii and Establishment of an ELISA Detection Method

WANG Lin¹, LI Meng-wei^1,2, XING You-shang¹, BAI Ya-duo¹, YIN Yi¹, PU Jing¹, QIAO Cai-xia¹, GAO Zhi-qiang¹, WEI Xue-liang², LI Chao-ming², LI Qin²

1. Beijing Entry-exit Inspection and Quarantine Bureau, Beijing 100026, China;
2. Southwest University, Chongqing 400715, China

Received:2013-05-06 Online:2013-12-20 Published:2014-02-11

摘要/Abstract

摘要： 试验旨在制备抗阪崎肠杆菌的单克隆抗体，初步建立其ELISA检测方法。以灭活的阪崎肠杆菌全菌体为抗原免疫BALB/c小鼠，筛选血清效价高的小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合，制备杂交瘤细胞，并用间接ELISA法选取阳性杂交瘤细胞，扩大培养后测定单克隆抗体的效价，进行特异性及抗体间的配对，使用mAb亚类检测试剂盒鉴定单克隆抗体的亚型，并利用得到的抗体建立双抗体夹心ELISA检测方法。本试验得到3株具有良好特异性能稳定分泌单克隆抗体的阳性细胞株5C10、2B6和Ab02，经两两配对，据阳性D_{450 nm}值及P/N值选择1：20000稀释的5C10作为包被抗体，1：40000稀释的Ab02作为酶标二抗，建立ELISA检测法，应用建立的方法与荧光定量PCR方法检测动物实验室保存的20份进出口送检奶粉样品，结果显示试验结果一致。本试验成功制备阪崎肠杆菌的单克隆抗体并建立其ELISA检测法，为大批量快速检测阪崎杆菌奠定了基础。

关键词: 阪崎肠杆菌; 单克隆抗体; 杂交瘤细胞; ELISA检测

Abstract: The assay was aimed to prepare the monoclonal antibody against Enterobacter sakazakii and establish the ELISA detection method. The splenocytes from immunized mice were fused with myeloma cells SP2/0, and hybridoma cells were screened by indirect ELISA.Then culturing more monoclonal antibodies, ascites titers were identified with indirect ELISA.Subclass was determinated by mouse isotype. Double-antibody sandwich ELISA detection method was established by using the antibodies. Three hybridoma cell strains that could secret monoclonal antibodies against Enterobacter sakazakii stably were obtained,named as 5C10, 2B6 and Ab02, paired 5C10 as coating antibody and Ab02 as HRP-secondary antibody, to establish ELISA detection method. We compared with fluorescence PCR method to test 20 portion imports of milk powder, ELISA detection method had the same consequence. The monoclonal antibody against Enterobacter sakazakii and ELISA detection method had been successfully established, which laid the foundation for rapid detection of Enterobacter sakazakii in larger number.

Key words: Enterobacter sakazakii; monoclonal antibody; hybridoma cell; ELISA detection

中图分类号:

S852.4^＋3

汪琳, 李勐伟, 刑佑尚, 柏亚铎, 尹羿, 蒲静, 乔彩霞, 高志强, 魏学良, 李朝明, 李琴. 阪崎肠杆菌单克隆抗体制备及ELISA检测方法的建立[J]. , 2013, 40(12): 52-55.

WANG Lin, LI Meng-wei, XING You-shang, BAI Ya-duo, YIN Yi, PU Jing, QIAO Cai-xia, GAO Zhi-qiang, WEI Xue-liang, LI Chao-ming, LI Qin. Preparation of Monoclonal Antibody against Enterobacter sakazakii and Establishment of an ELISA Detection Method[J]. China Animal Husbandry & Veterinary Medicine, 2013, 40(12): 52-55.

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