柱状黄杆菌双抗体夹心ELISA检测方法的建立

中国畜牧兽医

柱状黄杆菌双抗体夹心ELISA检测方法的建立

吕娜¹^，2，殷晓平^{2，张虹茜²，张懋岚²，孙强²，赵国坤²，张捷¹}

（1.浙江万里学院生物与环境学院，浙江宁波 315100；2.吉林农业大学食品科学与工程学院，吉林长春 130118）

收稿日期:2013-09-09 出版日期:2014-03-20 发布日期:2014-05-15
通讯作者: 张捷，女，副教授，研究方向：病原菌免疫生物学检测。E-mail：zhangjie@zwu.edu.cn
作者简介:吕娜（1980—），女，吉林人，博士，副教授，研究方向：鱼类疾病快速检测。
基金资助:
2010年浙江省科技厅公益项目（2010C32066）；吉林农业大学博士科研启动基金(201308)。

Establishment of a Double Antibody Sandwich ELISA for the Detection of Flavobacterium columnaris

LV Na^{1, 2}, YIN Xiao-ping², ZHANG Hong-xi², ZHANG Mao-lan², SUN Qiang², ZHAO Guo-kun², ZHANG Jie¹

（1. College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China；2. College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China）

Received:2013-09-09 Online:2014-03-20 Published:2014-05-15

摘要/Abstract

摘要： 为快速检测柱状黄杆菌，本研究以纯化的柱状黄杆菌单克隆抗体为捕获抗体，以多克隆抗体为检测抗体，建立了双抗体夹心ELISA （DAS-ELISA）检测法。最佳的方案为用0.08 μg/孔纯化的单克隆抗体4 ℃包被过夜，30 g/L牛血清白蛋白37 ℃封闭90 min，多克隆抗体的工作浓度为0.11 μg/孔，检测抗原、多克隆抗体、酶标抗体均为37 ℃作用1 h，显色15 min后终止反应，当D_{492 nm}值≥0.776且P/N≥2.1时判定为阳性。该方法特异性强，与迟钝爱德华氏菌、大肠杆菌、嗜水气单胞菌、鳗弧菌、溶藻弧菌、副溶血弧菌、哈维氏弧菌等水产病原菌均无交叉反应。最低检测剂量为1×10³ CFU。本研究首次建立了检测柱状黄杆菌的双抗体夹心ELISA方法，经验证该方法特异性强、灵敏度高。

关键词: 柱状黄杆菌; 双抗体夹心ELISA; 单克隆抗体

Abstract: The aim of this study was to develop a rapid method for the detection of Flavobacterium columnaris based on a double antibody sandwich ELISA （DAS-ELISA）. Purified monoclonal antibody against Flavobacterium columnaris was used as the capture antibody, while polyclonal antibody was used as the detection antibody. The optimal conditions for the ELISA were as follows: monoclonal antibody with 0.08 μg per well was added to coat overnight at 4 ℃; the plate was blocked by 30 g/L bovin serum albumin for 90 min at 37 ℃; incubation concentration of polyclonal antibody was 0.11 μg per well; the incubation time for detection antigen, polyclonal antibody and enzyme labeled antibody was 1 h at 37 ℃ for each; the value of D_{492 nm} was obtained after 15 min coloration. Judging with P/N≥2.1 and D_{492 nm}≥0.776 as positive criteria. This method had no cross reaction with Edwardsiella tarda, E. coli, Aeromonas hydrophila, Vibrio anguillarum, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio harveyi. Its minimum detectable limit was 1×10³ CFU. Therefore, this study provided a specific and sensitive detection method for Flavobacterium columnaris for the first time.

Key words: Flavobacterium columnaris; double antibody sandwich ELISA; monoclonal antibody

吕娜，殷晓平，张虹茜，张懋岚，孙强，赵国坤，张捷. 柱状黄杆菌双抗体夹心ELISA检测方法的建立[J]. 中国畜牧兽医.

LV Na, YIN Xiao-ping, ZHANG Hong-xi, ZHANG Mao-lan, SUN Qiang, ZHAO Guo-kun, ZHANG Jie. Establishment of a Double Antibody Sandwich ELISA for the Detection of Flavobacterium columnaris[J]. .

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