一种超高灵敏的埃博拉病毒胶体碳侧流免疫层析检测方法的建立

A novel carbon nanoparticle probe-based ultrasensitive lateral flow assay for rapid detection of Ebola virus

摘要:

埃博拉出血热 (Ebola hemorrhagic fever, EHF) 由于其高感染性和高致死率特点，快速鉴别诊断并实施隔离是最有效的防止疫情扩散的措施。文中建立了一种可以快速、高灵敏筛查埃博拉病毒 (Ebola virus，EBOV)感染的现场检测技术，用碳纳米颗粒标记抗EBOV基质蛋白VP40兔多克隆抗体，组装成一种可在15 min内检测埃博拉病毒的胶体碳侧流免疫层析试纸条。将标记胶体碳颗粒的兔多抗喷涂于玻璃纤维素膜上制备碳标垫；以1 mg/mL的抗VP40单克隆抗体 (McAb，4B7F9) 和羊抗兔IgG，按照2 μL/cm的包被量印迹于硝酸纤维膜上，分别作为检测线与质控线，组装试纸条。该试纸条能够特异地检测EBOV重组VP40蛋白、EBOV病毒样颗粒(Virus-like particles，VLP) 和灭活EBOV，而与马尔堡病毒样颗粒 (MARV-VLP)、流感病毒A/PR/8 (IAV/PR/8)、黄热病毒 (YFV-17D)、登革热病毒2型 (DEN2) 无交叉反应，显示良好的特异性，对1 500份阴性血清进行检测，假阳性率为1.3‰，仅为WHO授权ReEBOV?胶体金试纸的1/100；该胶体碳试纸条检测灭活EBOV的最低检出限为100 ng/mL (相当于106 copies/mL)，远优于ReEBOV?胶体金试纸条检出限 (10 μg/mL，相当于108 copies /mL)。热稳定性评价显示试纸条可在室温稳定保存1年以上。文中建立的EBOV胶体碳免疫层析试纸条能够快速、超高灵敏、特异地检测EBOV，为现场快速筛查EBOV感染提供一种新方法。

Abstract:

Ebola virus (EBOV) is an extremely contagious pathogen first discovered in Africa associated with severe hemorrhagic disease in humans and nonhuman primates, which has resulted in at least 28 500 suspected cases and 11 300 confirmed deaths in 2014–2016 Ebola epidemic in West Africa. Rapid and sensitive detection of EBOV is the key to increasing the probability of survival and reducing infection rates in pandemic regions. Here, we report an ultrasensitive and instrument-free EBOV detection assay based on colloidal carbon immunochromatography. Carbon nanoparticle-labeled rabbit anti-EBOV-VP40 IgG were concentrated in the conjugate pad, monoclonal antibody (McAb, 4B7F9) against EBOV-VP40 and goat anti-rabbit IgG were immobilized on the nitrocellulose membrane with 2 μL/cm at a concentration of 1 mg/mL as test and control lines, respectively. Then the sample application pad, conjugate release pad, nitrocellulose membrane and absorbent pad were assembled into a lateral flow test strip. The test strip shows strong specificity against related viruses that share similar clinical symptoms and geographic range with EBOV, including marburg virus, influenza virus, yellow fever virus and dengue virus. In addition, 1 500 negative serums were tested with false-positive rate of 1.3‰ which significantly lower than that of ReEBOV? colloidal gold test kit recommended by World Health Organization (WHO). The sensitivity of this strip was analyzed using inactivated EBOV with detection limit of 100 ng/mL (106 copies/mL) which clearly higher than that of ReEBOV? dipstick (108 copies/mL). Furthermore, the strip showed excellent thermal stability characteristics in room temperature and could be as a point-of-care (POC), ultra-sensitive and specific promising candidate for EBOV serological screening in rural Africa or entry/exit ports.