嗜肺军团菌快速检测胶体金试纸的研制  

Development of colloidal gold immunochromatography assay for the rapid detection of Legionella pneumophila

作　　者：张秋 李杰 王猛 王毅[2] 杨波[2] 胡征 ZHANG Qiu;LI Jie;WANG Meng;WANG Yi;YANG Bo;HU Zheng(School of Food and Biological Engineering,Hubei University of Technology,Wuhan 430068,Hubei,China;Key Laboratory of Fermentation Engineering,Ministry of Education,Wuhan 430068,Hubei,China;Hubei Collaborative Innovation Center for Industrial Fermentation,Wuhan 430068,Hubei,China)

机构地区：[1]湖北工业大学生物工程与食品学院,湖北武汉430068 [2]湖北工业大学发酵工程教育部重点实验室,湖北武汉430068 [3]工业发酵湖北省协同创新中心,湖北武汉430068

出　　处：《检验医学》2020年第7期726-733,共8页Laboratory Medicine

摘　　要：目的研制特异性检测嗜肺军团菌(Lp)的胶体金免疫层析试纸条。方法通过基因工程制备Lp-肽聚糖关联脂蛋白(PAL)重组蛋白并进行纯化,筛选分泌抗Lp-PAL重组蛋白单克隆抗体(简称单抗)的杂交瘤细胞株,采用间接酶联免疫吸附试验(ELISA)和免疫印迹法鉴定筛选到的Lp-1和Lp-2单抗的特异性,采用生物膜层干涉(BLI)技术鉴定抗原抗体反应的特异性。依据双抗体夹心原理制备胶体金免疫层析试纸条,并初步评价其检测性能(特异性、灵敏度及稳定性)。结果筛选到2株高效分泌抗Lp-PAL蛋白抗体的杂交瘤细胞株Lp-1和Lp-2,纯化得到2种单抗Lp-1和Lp-2。间接ELISA检测结果表明,Lp-1及Lp-2单抗只与Lp发生阳性反应,与其他10种呼吸道常见病原菌(肺炎链球菌、卡他莫拉菌、流感嗜血杆菌、肺炎支原体、金黄色葡萄球菌、肺炎克雷伯菌、产酸克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌、奇异变形杆菌)无交叉反应。免疫印迹法结果显示,Lp-1和Lp-2单抗与Lp-PAL天然蛋白和重组蛋白有强反应,与肺炎链球菌、流感嗜血杆菌和卡他莫拉菌无目的反应条带。BLI技术的验证结果显示,抗原抗体的相互作用力均较强,解离速率慢,Lp-1和Lp-2单抗识别的是同一抗原的不同抗原表位,且只与Lp有强反应,与其他10种细菌均无特异性结合反应。制备的胶体金免疫层析试纸条最低检出限为1×107 CFU/mL,与Lp常见4种血清型(Lp14、Lp12、Lp9和Lp6)均有特异性反应,与肺炎链球菌、卡他莫拉菌等10种常见呼吸道病原菌均无交叉反应,可在25℃环境下稳定6个月。结论 PAL蛋白具有高度的表面暴露性和较强的抗原性,可作为Lp的检测标志物,以此为基础制备的胶体金免疫层析试纸条具有快速、简便、灵敏的特点,适用于Lp感染的快速检测。Objective To establish a novel colloidal gold immunochromatography assay for the specific detection of Legionella pneumophila(Lp). Methods Lp-PAL recombinant protein was prepared and purified by genetic engineering,and hybridoma cell lines secreting anti-Lp-peptidoglycan-associated lipoprotein(PAL) recombinant protein monoclonal antibody were screened. The indirect enzyme-linked immunosorbent assay(ELISA) and immunoblotting were used to screen and identify the specificities of Lp-1 and Lp-2 monoclonal antibodies. The specificity of antigen-antibody response was identified using bio-layer interferometry(BLI) technology. A colloidal gold immunochromatography test strip was prepared based on double antibody sandwich principle,and its detection performance(specificity,sensitivity and stability) was initially evaluated. Results Totally,2 hybridoma cell lines that secreted anti-Lp-PAL protein antibodies were screened out,and 2 monoclonal antibodies,Lp-1 and Lp-2,were purified. Indirect ELISA showed that Lp-1 and Lp-2 monoclonal antibodies only positively reacted with Lp,and reacted with 10 other common respiratory pathogens(Streptococcus pneumoniae,Moraxella catarrhalis,Haemophilus influenzae,Mycoplasma pneumoniae,Staphylococus aureus,Klebsiella pneumoniae,Klebsiella oxytoca,Acinetobacter baumannii,Pseudomonas aeruginosa and Proteus mirabilis) had no crossreactivity. The results of western blotting showed that Lp-1 and Lp-2 monoclonal antibodies had strong reactions with natural PAL protein and Lp-PAL recombinant protein,and had no purposeful reaction bands with Streptococcus pneumoniae,Haemophilus influenzae and Moraxella catarrhalis. The verification results of BLI technology showed that the antigen-antibody interactions were strong,and the dissociation rate was slow. Lp-1 and Lp-2 monoclonal antibodies recognized different epitopes of the same antigen,and only had a strong reaction with Lp. They had no specific binding reaction with 10 other pathogens. The minimum detection limit of the prepared colloidal gold immun

关 键 词：嗜肺军团菌 单克隆抗体 肽聚糖关联脂蛋白 生物膜层干涉技术 胶体金免疫层析法 

分 类 号：R446.1[医药卫生—诊断学]