寨卡病毒NS1抗原的金标免疫层析检测试纸条研制

Development of an immunochromatographic strip for detection of Zika virus NS1 antigen

目的研制一种快速检测寨卡病毒(ZIKV)NS1抗原的胶体金免疫层析试纸条。方法以胶体金标记的抗寨卡病毒NS1单克隆抗体为特异性识别及信号产生的探针,抗寨卡病毒NS1单抗和羊抗鼠多克隆抗体IgG分别固定于硝酸纤维素膜,作为检测线和质控线以制备金标免疫层析试纸条。通过肉眼对检测线上条带进行判断、读取灰度值比等方式对试纸条结果进行分析。通过检测逐级稀释的寨卡病毒NS1抗原标准品,考察该试纸条的灵敏度。进一步通过检测分别感染了寨卡病毒、登革病毒、基孔肯雅病毒、日本脑炎病毒的细胞上清及血清样品,评价该试纸条检测的特异性。考察存储温度及放置时间对试纸条检测寨卡病毒NS1样品的影响,评价该试纸条的稳定性。结果所研制的试纸条在寨卡病毒NS1抗原标准品浓度为100 ng/mL时能够有效检测寨卡病毒培养上清,并与感染了登革病毒、基孔肯雅病毒以及日本脑炎病毒的细胞上清及血清样品均无交叉反应。此外,室温(22~25℃)或4℃储存条件下存放时间为36周的试纸条检测效果未受影响。结论本研究研制的金标免疫层析试纸条特异性好、稳定性高,快速、简便,该方法在即时检测、大样本疾病初筛等应用领域具备一定潜力。

Objective To develop a colloidal gold immunochromatographic strip-based method for the rapid detection of Zika virus(ZIKV)NS1 antigen.Methods The gold nanoparticles modified with the anti-ZIKV NS1 monoclonal antibody as the detection probe were coated on the glass-fiber pad.The anti-ZIKV NS1 monoclonal antibody and the goat anti-mouse polyclonal antibody were immobilized on a nitrocellulose membrane as the test line and the control line,respectively.In order to achieve critical results,the ratio of the optical density(OD)of the test line to that of the control line was compared.Serial diluted ZIKV NS1 standard antigen was applied to evaluate sensitivity of the immunoassay.The culture supernatant and serum samples for arboviruses(ZIKV,Dengue virus,Japanese encephalitis virus and Chikungunya virus)were utilized to demonstrate the specificity of the method.Results The detection result could read by naked eyes within 20 minutes.The visual cut-off level for the test strip was achieved at 100 ng/mL of the Zika virus NS1 standard antigen.No cross-reactions with Dengue virus,Japanese encephalitis virus and Chikungunya virus were observed.The strip could remain good stability within 36 weeks whether stored in 4℃or room temperature(22-25℃).Conclusion Apart from stability,the method was convenient,rapid and specific for ZIKV NS1 antigen,which showed a promising potential in the point of care test and the screening test.