摘要
目的制备抗UspA1蛋白单克隆抗体,研制能快速、灵敏、准确检测卡他莫拉菌的胶体金免疫层析试纸,以期用于人卡他莫拉菌的临床检测。方法将重组UspA1-His蛋白作为抗原,免疫小鼠,并制备单克隆抗体;通过Western blot技术以及免疫荧光技术鉴定抗体的特异性;以双抗体夹心的原理研制胶体金免疫层析检测试纸条,并对其特异性、敏感性及稳定性进行评价分析。结果筛选出两株分泌特异性抗体的杂交瘤细胞株,经腹水制备纯化得到单克隆抗体,并验证了两种抗体均能特异性识别天然UspA1蛋白;利用双抗夹心原理制备的胶体金试纸能在10 min内快速检测卡他莫拉菌,检测灵敏度高(1×10~5 CFU/ml)、特异性强,与其他常见9种的呼吸道病原菌诸如肺炎链球菌、流感嗜血杆菌、肺炎支原体、嗜肺军团菌等无交叉反应;试纸条在37℃保存有良好的重复性和稳定性;200份临床样品检测结果与平板培养法的阳性符合率为93.3%。结论本研究制备了特异性强的抗UspA1蛋白单克隆抗体;以此研制的胶体金免疫层析试纸可快速、简便、灵敏的检测卡他莫拉菌,适用于呼吸道感染的临床快速检测。
Objective To prepare anti-UspA1 monoclonal antibodies and to develop colloidal gold immunochromatographic test paper for the rapid,sensitive,and accurate detection of Moraxella catarrhalis.Methods The recombinant protein UspA1-His was used as antigen to immunize mice and prepare monoclonal antibodies.The specificity of antibodies was identified using Western blotting and immunofluorescence techniques.A colloidal gold immunochromatography test strip was developed based on the principle of the double antibody sandwich.The specificity,sensitivity,and stability of test strips were evaluated.Results Two hybridoma cell lines secreting specific antibodies were obtained via fusion and screening.Monoclonal antibodies were obtained via preparation and purification of ascites,and both antibodies specifically recognized the native UspA1 protein.The detection limit of the test strip was as low as 1×10~5 CFU/mL and the whole process can be completed within 10 minutes.The strip specifically recognized M.catarrhalis and did not react to 9 other common respiratory pathogens such as Streptococcus pneumoniae,Haemophilus influenzae,Mycoplasma pneumoniae,and Legionella pneumophila;the test strips have good repeatability and stability at 37 C.For the detection of 200 clinical samples,the positive concordance rate between the test paper and the plate culture method was 93.3%.Conclusion Monoclonal antibodies prepared using the recombinant protein UspA1 have a high level of specificity.Colloidal gold immunochromatography is rapid,convenient,and sensitive;this technique is suitable for rapid clinical detection of a Moraxella catarrhalis infection.
作者
郝惠文
任萌
宋慧茹
程雨洁
杨波
王毅
胡征
HAO Hui-Wen;RENG Meng;SONG Hui-ru;CHENG Yu-jie;YANG Bo;WANG Yi;HU Zheng(School of Food and Biologicul Engineering,Hubei University of Technology,Key Laboratory of Fermentation Engineering(Hubei University of Technology)Wuhan,China 430068)
出处
《中国病原生物学杂志》
CSCD
北大核心
2020年第6期634-637,641,共5页
Journal of Pathogen Biology
基金
细胞调控与分子药物"111"引智基地青年学者国际合作研究基金项目(No.XBTK-2018005)。
