流感嗜血杆菌快速检测胶体金试纸的研制

Colloidal gold immunochromatographic strip for rapid detection of Haemophilus influenzae

摘要:

以流感嗜血杆菌外膜蛋白P 6为检测标志物，利用胶体金免疫层析技术建立一种快速、灵敏、准确检测流感嗜血杆菌的方法。对P6蛋白 (GenBank登录号：AGH02799) 进行生物信息学分析，获取其胞外结构域中抗原表位最丰富的肽段，预测其中的线性抗原表位P6Line (62–75 aa)，化学合成后经免疫、抗体纯化得到线性表位抗体，同时利用重组P6蛋白制备多克隆抗体，建立基于双抗体夹心免疫层析技术的流感嗜血杆菌快速检测方法，并对该方法的特异性、灵敏性、重复性和稳定性作出评价，同时对该方法进行临床模拟试验，平板培养法验证其准确性。建立的检测方法可在15 min内完成对样本的检测，检测敏感度高 (1×105 CFU/mL)、特异性强，与其他诸如肺炎链球菌、卡他莫拉菌、肺炎支原体、嗜肺军团菌等常见9种的呼吸道病原菌无交叉反应；试纸条在25 ℃保存具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90.5%。P6蛋白具有高度的表面暴露性和较强的抗原性，可作为流感嗜血杆菌检测标的物，胶体金免疫层析法具有快速、简便、灵敏的特点，适用于呼吸道感染的临床快速检测。

Abstract:

To establish a novel colloidal gold immunochromatography assay (GICA) for rapid, sensitive and accurate detection of Haemophilus influenzae infection by using the outer membrane protein?P6 as detection target. First, the linear antigen epitope located in the extracellular domain of the P6 protein (GenBank accession number: AGH02799) was predicted by bioinformatics analysis. The region (62–75 aa of the protein) with strong antigen specificity was chosen and synthesized. Two rabbits were then immunized by the polypeptides (14 aa) for production of polyclonal antibodies. Then, the recombinant P6 proteins were also obtained to produce polyclonal antibodies. Finally, based on the two antibodies, a novel colloidal GICA for detection of Haemophilus influenzae infection was established and the specificity, sensitivity, repeatability and stability of this method were evaluated. At the same time, the method was tested in clinical simulation, and the plate culture method was used to verify its accuracy. The test strip for Haemophilus influenzae infection was successfully prepared. The detection limit of the test strip was as low as 1×105 CFU/mL and the whole process can be completed within 15 minutes. The strip specifically recognized Haemophilus influenzae and did not react with nine of other common respiratory pathogens such as Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumonia, and Legionella pneumophila. And the strips could be stored at 25 °C for at least 6 months without losing sensitivity or specificity. The coincidence rate between the results of 200 clinical samples and the plate culture method was 90.5%. Haemophilus influenzae protein P6, which possessed a high degree of surface antigen accessibility and antigencity, could be used as a marker for Haemophilus influenzae detection. The immunochromatographic colloidal gold test strip which bears the features of rapidity, convenience and sensitivity provides a unique tool for the on-site surveillance and diagnosis of?Haemophilus influenzae infection in clinical test.