Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans
Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans
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DOI：
10.1128/jcm.02735-14
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发表时间：
2015-02-01
影响因子：
9.4
通讯作者：
Chang, Gwong-Jen J.
中科院分区：
文献类型：
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作者：
Chao, Day-Yu;Galula, Jedhan Ucat;Chang, Gwong-Jen J.
针对登革病毒（DENV）、西尼罗河病毒和日本脑炎病毒（JEV）的包膜蛋白（E）的IgM抗体捕获酶联免疫吸附测定（MAC/GAC-ELISA）被广泛用作用于推定确认病毒感染的血清学诊断试验。针对黄病毒非结构蛋白1（NS 1）的抗体已被提议作为接种人群中自然感染的血清学标志物。本研究的目的是优化IgM和IgG抗体捕获ELISA（MAC/GAC-ELISA）检测抗NS 1抗体，并与抗E MAC/GAC-ELISA进行比较。构建了表达登革病毒（DENV-1 ~ DENV-4）、西尼罗病毒（WNV）和日本脑炎病毒（JEV）等6种重要黄病毒的前膜/包膜（prM/E）或NS 1蛋白的质粒。这些质粒用于从COS-1细胞产生含有prM/E的病毒样颗粒（VLP）和分泌的NS 1（sNS 1）。将来自确诊DENV、JEV和WNV感染的患者的存档临床标本，沿着幼稚血清，在通过预吸收消耗抗prM/E抗体之前或之后，用或不用VLP进行NS 1-MAC/GAC-ELISA。来自先前确认的DENV感染的人血清标本在抗prM/E抗体耗尽后显示NS 1-MAC/GAC-ELISA的阳性与阴性（P/N）比率显著增强。新开发的NS 1-和VLP-MAC/GAC-ELISA之间的敏感性和特异性没有统计学差异。进一步应用于WNV和JEV感染的血清组显示了相似的结果。成功开发了一种用于检测NS 1抗体的MAC/GAC-ELISA的新方法，该方法具有区分由四价嵌合黄热病-17D/登革热疫苗或DENV感染引起的抗体的巨大潜力。
IgM antibody-and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1-and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV-and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection.