铜绿假单胞菌胶体金免疫层析试纸的制备  

Preparation of colloidal gold immunochromatographic paper for Pseudomonas aeruginosa

作　　者：曹家敏 朱艳芳 华明明 刘书君 杨波[1,3,4] 胡征[1,3,4] CAO Jiamin;ZHU Yanfang;HUA Mingming;LIU Shujun;YANG Bo;HU Zheng(College of Life Sciences and Health Engineering,Hubei University of Technology,Wuhan 430068;Wuhan Anglo-Chinese High School,Wuhan 430068;Key Laboratory of Fermentation Engineering,Ministry of Education,Wuhan 430068;Hubei Collaborative Innovation Center for Industrial Fermentation,Wuhan 430068,China)

机构地区：[1]湖北工业大学生命科学与健康工程学院,湖北武汉430068 [2]武汉英中高级中学,湖北武汉430068 [3]发酵工程教育部重点实验室,湖北武汉430068 [4]工业发酵湖北省协同创新中心,湖北武汉430068

出　　处：《生物技术》2025年第1期95-101,共7页Biotechnology

摘　　要：[目的]筛选一对抗OprF蛋白单克隆抗体,制备快速检测铜绿假单胞菌的胶体金免疫层析试纸。[方法]将重组OprF蛋白免疫小鼠制备单克隆抗体,通过间接ELISA检测、BLI技术以及Western Blot鉴定抗体的特异性及结合力,以双抗体夹心法制备胶体金免疫层析试纸,对其灵敏度、特异性、稳定性进行分析测试。[结果]筛选出2株高亲和力抗体,均能特异性识别OprF蛋白,所制备胶体金免疫层析试纸检测铜绿假单胞菌的检出限达到2.5×10^(3) CFU/mL;试纸检测特异性强,与呼吸道常见病原菌如肺炎克雷伯菌、金黄色葡萄球菌、鲍曼不动杆菌、肺炎支原体、嗜肺军团菌、卡他莫拉菌、流感嗜血杆菌、肺炎链球菌、唾液链球菌、化脓链球菌均无交叉反应;试纸稳定性高,室温下保存1年仍具有良好的稳定性。[结论]所研制的免疫层析试纸在15 min内对铜绿假单胞菌快速检测,灵敏度为2.5×10^(3) CFU/mL,对呼吸道常见的10种病原体均无交叉反应,且在室温可稳定保存1年,满足临床应用的相关要求。[Objective]To screen a pair of monoclonal antibodies against OprF protein and prepare colloidal gold immunochromatographic test paper for rapid detection of Pseudomonas aeruginosa.[Method]Monoclonal antibody was prepared by immunizing mice with recombinant OprF protein.The specificity and binding force of the antibody were determined by indirect ELISA,BLI and Western Blot.The colloidal gold immunochromatographic test paper was prepared by double antibody sandwich method,and its sensitivity,specificity and stability were analyzed and tested.[Result]Two high-affinity antibodies were screened,both of which could specifically identify OprF protein.The detection limit of the prepared colloidal gold immunochromatographic test paper for Pseudomonas aeruginosa was 2.5×10^(3) CFU/mL.The test paper had strong specificity,and no cross reaction with common respiratrory pathogens such as K.pneumoniae,S.aureus,A.baumannii,M.pneumoniae,L.pneumophila,M.catarrhalis,H.influenzae,S.pneumoniae,S.salivarius and S.pyogenes,etc.The test paper demonstrate high stability and remain stable after being stored at room temperature for one year.[Conclusion]The immunochromatographic test paper prepared in this study can achieve rapid detection of P.aeruginosa within 15 min,with a sensitivity of 2.5×10^(3) CFU/mL no cross-reaction to 10 common respiratory pathogens,and can be statically stored at room temperature for more than one year,meeting the relevant requirements of clinical application.

关 键 词：铜绿假单胞菌 外膜蛋白OprF 单克隆抗体 生物膜干涉技术 胶体金免疫层析试纸 灵敏度 特异性 稳定性 

分 类 号：R446.5[医药卫生—诊断学]