Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.
Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.
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DOI：
10.1128/cvi.00209-16
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发表时间：
2016-08
期刊：
影响因子：
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通讯作者：
Morita K
中科院分区：
文献类型：
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作者：
Adungo F;Yu F;Kamau D;Inoue S;Hayasaka D;Posadas-Herrera G;Sang R;Mwau M;Morita K
黄热病（YF）是一种在非洲和南美洲由蚊子传播的急性出血性病毒感染。在非洲检测和确认YF疾病爆发方面的主要挑战是参考实验室有限，以及持续缺乏获得诊断测试的机会。我们利用野生型YF病毒序列在大肠杆菌表达系统中产生重组包膜蛋白。用重组蛋白和蔗糖梯度纯化的YF疫苗病毒17D （YF-17D）免疫BALB/c小鼠，产生单克隆抗体（mab）。建立了8个单抗，并采用间接酶联免疫吸附法（ELISA）、免疫印迹法（Western blot）和免疫荧光法（IFA）对其进行了系统表征。所建立的单克隆抗体与野生型YF病毒和重组蛋白具有较强的反应性，与登革热病毒和日本脑炎病毒无交叉反应。表位定位显示，3个单抗与包膜蛋白的1 ~ 51位氨基酸强结合，2个单抗与包膜蛋白的52 ~ 135位氨基酸强结合。其余3个单克隆抗体对包膜片段无反应性。所建立的单克隆抗体对野生型YF和17D病毒（两种病毒的滴度均<10）没有中和作用。采用IgM捕获ELISA进一步评价单克隆抗体8H3和3F4的适用性。共分析49份血清样本，其中12份阳性患者和接种者样本被正确鉴定。使用经2倍连续稀释的血清样本，IgM捕获ELISA能够检测到所有yf阳性样本。此外，基于单克隆抗体的抗原检测ELISA能够在含有约1,000个聚焦形成单位滴度的培养上清中检测病毒。
Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units.