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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 魏艳秋等 / 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 Dec. 25, 2018, 34(12): Chin J Biotech, All rights reserved 生物技术与方法 2025 魏艳秋 1*, 段勇成 2*, 毕玉海 1, 王萌 1,3, 李云龙 1, 王选 1, 林玮 5, 范文辉 1, 王晶刘文军 1,3 1, 杨利敏 ,,,,.., 2018, 34(12): Wei YQ, Duan YC, Bi YH, et al. A novel carbon nanoparticle probe-based ultrasensitive lateral flow assay for rapid detection of Ebola virus. Chin J Biotech, 2018, 34(12): 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 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 10 6 copies/ml), 远优于 ReEBOV 胶体金试纸条检出限 (10 μg/ml, 相当于 10 8 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 : 埃博拉病毒, 基质蛋白, 单克隆抗体, 胶体碳, 侧流免疫层析, 现场快检 Received: March 9, 2018; Accepted: May 11, 2018 Supported by: the National Key Program for Infectious Disease of China (No. 2016ZX ), the Key Research Program of the Chinese Academy of Sciences (No. ZDRW-ZS ). Corresponding author: Limin Yang. Tel/Fax: ; lmyang@im.ac.cn *These authors contributed equally to this study. (No. 2016ZX ) (No. ZDRW-ZS )

2 2026 ISSN CN /Q Chin J Biotech A novel carbon nanoparticle probe-based ultrasensitive lateral flow assay for rapid detection of Ebola virus Yanqiu Wei 1*, Yongcheng Duan 2*, Yuhai Bi 1, Meng Wang 1,3, Yunlong Li 1, Xuan Wang 1, Wei Lin 5, Wenhui Fan 1, Jing Wang 4, Wenjun Liu 1,3, and Limin Yang 1 1 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing , China 2 College of Animal Science and Technology, Guangxi University, Nanning , Guangxi, China 3 University of Chinese Academy of Sciences, Beijing , China 4 National Institute of Metrology, Beijing , China 5 Goodwe Biotechnology Taizhou Co., Ltd., Taizhou , Jiangsu, China 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 suspected cases and confirmed deaths in 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, 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 (10 6 copies/ml) which clearly higher than that of ReEBOV dipstick (10 8 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. Keywords: Ebola virus, matrix protein, monoclonal antibodies, colloidal carbon, lateral flow immunochromatography, POC RNA [1] EBOV 5 (Sudan Ebola virus SEBOV) (Zaire Ebola virus, ZEBOV) (Ta Forest Ebola virus TAFV) (Reston Ebola virus, REBOV) (Bundibugyo Ebola virus, BEBOV) [1] REBOV 4 ZEBOV SEBOV 53% 90% [2] 4 [3] EBOV EBOV

3 魏艳秋等 / 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 2027 WHO EBOV 4 (BSL-4) PCR EBOV [4] 5 d [5] EBOV EBOV [6-8] [9] (Point-of-care testing, POCT) 15 min (NIH) Corgenix EBOV (ReEBOV Antigen Rapid Test Kit) WHO FDA 10 8 copies/ml PCR RealStar Filovirus Screen RT-PCR Kit 1.0 (Altona Diagnostics GmbH) 91.8% 15.4% [10] EBOV ELISA 30 min [11] EBOV [12] VP40 Corgenix 100 1/ 材料与方法 1.1 主要试剂及仪器 IgG (H+L) M135 Millipore Alstrom 8964 Alstrom Whatman PVC Tris BSA AMRESCO XYZ HM3030 ZQ2000 CTD300 YK725 A/PR/8 (IVA/PR/8) (YFV-17D) 2 (DEN2) 1.2 病毒样颗粒的制备 [13-14] (GenBank KM ) (GenBank Z ) (GP) (VP40) (pcaggs/ebovgp pcaggs/ebovvp40 pcaggs/marvgp pcaggs/marvvp40) 293T 68 h g 4 h cjb@im.ac.cn

4 2028 ISSN CN /Q Chin J Biotech EBOV-VLP MARV-VLP (Western blotting) GP VP 重组蛋白及抗体的制备 2014 VP40 (GenBank KM ) pet28a Rosetta (DE3) VP40 3 Protein G VP40 BALB/c Protein G (Western blotting) (ELISA) VP40 EBOV 1.4 胶体碳试纸条的制备 r/min 10 min 3 min EBOV-VP mg/ml μl 5 μl 30 min 500 μl 30 min r/min 15 min 500 μl Alstrom 8964 (0.3% BSA 4% 0.2% Tween mol/l Tris-HCl ph 8.0) 1 min 37 XYZ 10 μl/cm 20 μl/cm 30 μl/cm 40 μl/cm μl 15 min T/C Alstrom 8964 (1% PVP-K30 1% Tritonx % BSA 4% 0.02 mol/l Tris-HCl ph 8.0) 1 min B7F9 Tris-HCl (0.02 mol/l ph 8.0) 2.0 mg/ml 1.0 mg/ml 0.5 mg/ml 2 μl/cm 2 μl/cm 1 mg/ml IgG 80 μl 15 min T/C B7F9 IgG XYZ 0.5 cm 1.0 cm 37 PVC 2 mm 4 mm ( 1) 图 1 胶体碳侧流层析试纸条结构示意图 Fig. 1 Schematic of colloidal carbon lateral flow test strip.

5 魏艳秋等 / 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 试纸条性能评价 VP40 EBOV-VLP EBOV MARV-VLP IAV/PR/8 YFV-17D DENV2 80 μl 15 min T C PCR EBOV μg/ml 10 μg/ml 1 μg/ml 100 ng/ml 10 ng/ml ( PCR 10 9 copies/ml 10 8 copies/ml 10 7 copies/ml 10 6 copies/ml 10 5 copies/ml) (ReEBOV ) 15 min T C 结果与分析 2.1 重组抗原及抗体的制备 pet28a-ebov-vp40 SDS-PAGE 35 kda ( 2A) Protein G ( 2B) Western blotting VP40 EBOV ( 2C D) ELISA 图 2 分析鉴定重组 EBOV-VP40 蛋白及抗体 Fig. 2 SDS-PAGE and Western blotting analysis of rebov-vp40 and anti-ebov-vp40 antibody. (A) SDS-PAGE analysis results. 1: un-induced Rosetta-pET28a-EBOV-VP40; 2: post-induced Rosetta-pET28a-EBOV-VP40; 3: purified rvp40 protein. (B) 1: purified rabbit anti-ebov-vp40 IgG; 2: purified mouse anti-ebov-vp40 McAb (4B7F9). Western blotting analysis results: rvp40 protein (lane 1) and inactivated EBOV (lane 2) were analyzed by immunoblotting with rabbit anti-ebov-vp40 IgG (C) and mouse anti-ebov-vp40 McAb (D). Molecular mass markers are indicated on the left. ( 3) 4B7F9 2.2 胶体碳标记垫的制备 XYZ μl/cm 30 μl/cm ( 4) 2.3 检测线及质控线的制备 4B7F cjb@im.ac.cn

6 2030 ISSN CN /Q Chin J Biotech 1 mg/ml IgG 80 μl 15 min 1.0 mg/ml ( 5) 图 3 ELISA 鉴定不同 EBOV 单抗株与灭活 EBOV 相 互反应 Fig. 3 McAbs (4B7F9, 6E3F3 and 8B10F8) were assessed by Inactivated-EBOV-specific ELISA. 2.4 试纸条的组装与结果判定标准 80 μl 15 min (T) (C) (C) ( 6) 图 4 碳标记抗体最适喷涂量 Fig. 4 The optimal spraying quantity of carbon-labeled IgG. (A) Image of detection positive sample by strips which were sprayed with different amounts of carbon-labeled IgG. 1: 40 μl/cm; 2: 30 μl/cm; 3: 20 μl/cm; 4: 10 μl/cm. (B) The strips were scanned by lateral flow reader and signal intensity of control and test lines were compared. 图 5 单抗 4B7F9 的最适印迹量 Fig. 5 Concentration of immobilized McAb (4B7F9). (A) Image of detection positive sample by strips which were immobilized with different concentrations of McAb. 1: 2.0 mg/ml; 2: 1.0 mg/ml; 3: 0.5 mg/ml. (B) The strips were scanned by lateral flow reader and signal intensity of control and test lines were compared.

7 魏艳秋等 / 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 2031 图 6 EBOV 胶体碳侧流层析试纸条检测结果判定 Fig. 6 Detection criterion of EBOV colloidal carbon lateral flow test strip. 1: positive result; 2: negative result. T: test line; C: control line. 2.5 胶体碳试纸条性能评价 EBOV-VP40 EBOV-VLP EBOV MARV-VLP IAV/PR/8 YFV-17D DEN2 80 μl 15 min ( 7) RT-PCR 1.3 ReEBOV TM 15.4% [10] PCR EBOV ReEBOV TM ReEBOV 10 μg/ml (10 8 copies/ml) ( 8A) 100 ng/ml (10 6 copies/ml) ( 8B) ReEBOV TM ( 9) 图 7 EBOV 胶体碳侧流层析试纸条特异性评价 Fig. 7 Specificity of EBOV colloidal carbon lateral flow test strip. 1: EBOV-VP40; 2: inactivated-ebov; 3: DEN2; 4: YFV-17D; 5: EBOV-VLP; 6: MARV-VLP; 7: IAV/PR/8. 图 8 EBOV 侧流层析试纸条灵敏度评价 Fig. 8 Detection limit of EBOV colloidal carbon lateral flow test strip and ReEBOV test strip. Simulated clinical specimens spiked with different concentrations of inactivated-ebov were tested by colloidal carbon or colloidal gold test strip. (A) 1: 10 μg/ ml; 2: 1 μg/ ml; 3: 100 ng/ ml; 4: 10 ng/ ml. (B) 1: 100 μg/ ml; 2: 10 μg/ ml; 3: 1 μg/ ml. * indicates the limit of detection cjb@im.ac.cn

8 2032 ISSN CN /Q Chin J Biotech 图 9 EBOV 侧流层析试纸条稳定性评价 ( 试纸条分 别放置 25 和 37 并定期检测其最低检出限, 虚 线以上代表无法检出, 即试纸条已失效 ) Fig. 9 Thermal stability analysis of the colloidal carbon test strip. Strips were placed under different temperature conditions (25, 37 ) for 1 year and detection limit were tested per month. The test results above the dashed line mean that it cannot be detected, i.e., the test strip has expired. 3 讨论 [15-16] % PCR PCR 5 d [17] Corgenix EBOV EBOV ( 91.8%) ( 15.4%) EBOV copies/ml ReEBOV TM 10 8 copies/ml [18] EBOV EBOV VP40 VLP WHO FDA ReEBOV EBOV 100 ReEBOV TM 10 8 copies/ml WHO [10] 10 6 copies/ml EBOV copies/ml EBOV EBOV 5 d 1 d 80% 0 [5]

9 魏艳秋等 / 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 PCR PCR EBOV EBOV WHO FDA 100 1/ min PCR EBOV EBOV EBOV EBOV REFERENCES [1] Kuhn JH, Becker S, Ebihara H, et al. Proposal for a revised taxonomy of the family Filoviridae: classification, names of taxa and viruses, and virus abbreviations. Arch Virol, 2010, 155(12): [2] Towner JS, Sealy TK, Khristova ML, et al. Newly discovered ebola virus associated with hemorrhagic fever outbreak in Uganda. PLoS Pathog, 2008, 4(11): e [3] Wool-Lewis RJ, Bates P. Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines. J Virol, 1998, 72(4): [4] Cherpillod P, Schibler M, Vieille G, et al. Ebola virus disease diagnosis by real-time RT-PCR: a comparative study of 11 different procedures. J Clin Virol, 2016, 77: [5] Dhillon RS, Srikrishna D, Garry RF, et al. Ebola control: rapid diagnostic testing. Lancet Infect Dis, 2015, 15(2): [6] Kurosaki Y, Magassouba N, Oloniniyi OK, et al. Development and evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) assay coupled with a portable device for rapid diagnosis of ebola virus disease in guinea. PLoS Negl Trop Dis, 2016, 10(2): e [7] Kurosaki Y, Takada A, Ebihara H, et al. Rapid and simple detection of Ebola virus by reverse transcription-loop-mediated isothermal amplification. J Virol Methods, 2007, 141(1): [8] Xu CP, Wang HL, Jin HL, et al. Visual detection of Ebola virus using reverse transcription loop-mediated isothermal amplification combined with nucleic acid strip detection. Arch Virol, 2016, 161(5): [9] Ksiazek TG, West CP, Rollin PE, et al. ELISA for the detection of antibodies to Ebola viruses. J Infect Dis, 1999, 179 Suppl 1: S192 S198. [10] WHO. WHO emergency use assessment and listing for Ebola Virus Disease IVDs. Public report. Product: ReEBOV TM Antigen Rapid Test Kit. [ ] _reebov_antigen_rapid_test_public_report.pdf?u a=1. [11] Duan DM, Fan KL, Zhang DX, et al. Nanozyme-strip for rapid local diagnosis of Ebola. Biosens Bioelectron, 2015, 74: [12] Liu B, Wang LL, Tong B, et al. Development and comparison of immunochromatographic strips with three nanomaterial labels: colloidal gold, nanogoldpolyaniline-nanogold microspheres (GPGs) and colloidal carbon for visual detection of salbutamol. Biosens Bioelectron, 2016, 85: cjb@im.ac.cn

10 2034 ISSN CN /Q Chin J Biotech [13] Swenson DL, Warfield KL, Kuehl K, et al. Generation of Marburg virus-like particles by co-expression of glycoprotein and matrix protein. FEMS Immunol Med Microbiol, 2004, 40(1): [14] Warfield KL, Bosio CM, Welcher BC, et al. Ebola virus-like particles protect from lethal Ebola virus infection. Proc Natl Acad Sci USA, 2003, 100(26): [15] Zhu FC, Hou LH, Li JX, et al. Safety and immunogenicity of a novel recombinant adenovirus type-5 vector-based Ebola vaccine in healthy adults in China: preliminary report of a randomised, double-blind, placebo-controlled, phase 1 trial. Lancet, 2015, 385(9984): [16] Zhu FC, Wurie AH, Hou LH, et al. Safety and immunogenicity of a recombinant adenovirus type-5 vector-based Ebola vaccine in healthy adults in Sierra Leone: a single-centre, randomised, double-blind, placebo-controlled, phase 2 trial. Lancet, 2017, 389(10069): [17] WHO Ebola Response Team, Aylward B, Barboza P, et al. Ebola virus disease in West Africa--the first 9 months of the epidemic and forward projections. N Engl J Med, 2014, 371(16): [18] USFDA. ReEBOV TM Antigen Rapid Test (Ebolavirus VP40 antigen detection). Instructions for use. [ ]. Devices/Safety/EmergencySituations/UCM pdf. ( 本文责编郝丽芳 )