流感嗜血杆菌单克隆抗体免疫层析试纸的研制  

Development of a monoclonal antibody colloidal gold immunochromatographic test strip for the detection of Haemophilus influenzae

作　　者：郝惠文 陶冶 宋慧茹 杨波 王毅[1,2] 胡征 HAO Huiwen;TAO Ye;SONG Huiru;YANG Bo;WANG Yi;HU Zheng(School of Food and Biological Engineering,Hubei University of Technology,Wuhan 430068;Key Laboratory of Fermentation Engineering(Hubei University of Technology),Ministry of Education,Wuhan 430068,China)

机构地区：[1]湖北工业大学生物工程与食品学院,武汉430068 [2]湖北工业大学,发酵工程教育部重点实验室,武汉430068

出　　处：《生物学杂志》2020年第6期108-111,共4页Journal of Biology

基　　金：湖北省科技厅自然科学青年项目(2017CFB226)。

摘　　要：为研制快速检测流感嗜血杆菌胶体金免疫层析试纸,用P6重组蛋白免疫小鼠,通过单克隆抗体技术制备杂交瘤细胞,经腹水制备、亲和纯化得到单克隆抗体,利用双抗夹心原理研制免疫层析试纸。结果成功筛选并制备出两株抗P6重组蛋白单克隆抗体,其效价均达到1∶512000,经Western Blot验证均能特异性识别流感嗜血杆菌天然P6蛋白,两株单抗KD值分别为10-9和10-10,亲和力较好,建立的检测方法能在10 min内快速检测流感嗜血杆菌,且与常见9种呼吸道病原菌无交叉反应,经加速稳定实验测试,可在常温下保存使用1年,具有良好的重复性和稳定性。制备了抗流感嗜血杆菌P6的特异性单克隆抗体,并研制了以单克隆抗体为基础的胶体金免疫层析试纸,实现了人流感嗜血杆菌的快速、简便的检测。To establish a rapid and sensitive detection method for Haemophilus influenzae,mice were immunized with P6 recombinant protein,and hybridoma cells were prepared by monoclonal antibody technology.Monoclonal antibodies were obtained by ascites preparation and affinity purification.An immunochromatographic test paper was developed based on the double antibody sandwich principle.As a result,two monoclonal antibodies against P6 recombinant protein were successfully screened and prepared,and their titers reached 1∶512000.It confirmed that the monoclonal antibodies could specifically recognize the natural P6 protein of Haemophilus influenzae by Western Blot.The KD values of the two monoclonal antibodies were 10-9 and 10-10,respectively,and the affinity was good.By the established approach,Haemophilus influenzae was quickly and specifically detected in 10 min,and it had no cross-reaction with 9 common respiratory pathogens.After accelerated stability test,it could be stored and used at room temperature for one year,with good repeatability and stability.

关 键 词：流感嗜血杆菌 P6蛋白 单克隆抗体 胶体金免疫层析法 

分 类 号：R446.6[医药卫生—诊断学]