摘要
目的建立快速检测哈维弧菌的免疫学方法。方法以甲醛灭活的哈维弧菌菌体为免疫抗原,对新西兰大白兔进行免疫得到哈维弧菌多克隆抗体;将多克隆抗体用间接ELISA法对创伤弧菌、副溶血性弧菌等进行特异性检测;将哈维弧菌多克隆抗体与磁珠结合,并将多克隆抗体标记在磁珠上形成特异性磁纳米探针,再分别以哈维弧菌多克隆抗体和山羊抗兔Ig G作为检测线T线和质控线C线,初步实现了双抗体夹心模式的磁性免疫层析试纸条,并对磁珠偶联抗体添加量进行优化。结果经ELISA法检测,哈维弧菌多克隆抗体效价为1∶80 000;其特异性良好,即此多克隆抗体与不同致病类型的食源性致病菌无交叉反应;最佳偶联抗体添加量为140μg;半定量检测时间为10 min,检测的准确性和灵敏度已达到最优,通过单纯的肉眼观察,可实现5×104CFU/ml哈维弧菌的检测。结论成功制备高效价哈维弧菌多克隆抗体并与纳米磁性材料进行了结合,为深入研究哈维弧菌的免疫原性与免疫学快速检测方法提供了基础。
Objective To establish an immunological method for rapid detection of Vibrio harveyi. Methods A Vibrio harveyi polyclonal antibody was obtained by immunizing New Zealand white rabbits with inactivated Vibrio harveyi. ELISA methods were applied in the current detection of the Vibrio harveyi polyclonal antibody. The multiresistance of the Vibrio harveyi was combined with magnetic beads. The Vibrio harveyi polyclonal antibody on the magnetic beads was taken as a specific magnetic nanoprobe. Magnetic immunochromatographic test strips were prepared using the Vibrio harveyi polyclonal antibody as a test line and goat anti-rabbit Ig G as a quality control line. Results The Vibrio harveyi polyclonal antibody of other common food-borne pathogens by ELISA method had a good specificity,and the agglutinin titer was 1 ∶ 80 000. The amount of the conjugated antibody was 120 μg. By simple visual observation,Vibrio harveyi of 5 × 104 CFU/ml could be detected in 10 min. Conclusion In this study,polyclonal antibodies against Vibrio harveyi are successfully prepared and combined with nano-magnetic materials,which will contribute to further study on the immunogenicity and rapid immunological detection of Vibrio harveyi.
作者
陈璐萍
李晓晖
宁喜斌
李环
袁静
卢瑛
CHEN Lu-ping;LI Xiao-hui;NING Xi-bin;LI Huan;YUAN Jing;LU Ying(College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306,China;Institute of Disease Control and Prevention of PLA,Beijing 100071,China)
出处
《军事医学》
CAS
CSCD
北大核心
2018年第4期279-283,共5页
Military Medical Sciences
基金
上海市科学技术委员会部分地方院校能力建设项目(15320502100)
上海市科技兴农重点攻关项目[沪农科攻字(2016)第4-4号]
关键词
哈维弧菌
多克隆抗体
磁性免疫层析试纸条
Vibrio harveyi
polyclonal antibody
magnetic immunochromatographic test strips
