Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok, Thailand.
J Clin Virol. 2011 Apr;50(4):314-9. doi: 10.1016/j.jcv.2011.01.001. Epub 2011 Feb 1.
Dengue virus (DENV), which causes mosquito-borne disease dengue hemorrhagic fever (DHF), consists of four serotypes co-circulating in endemic areas. Currently, DENV serotypes can be identified by laborious virus isolation followed by immunofluorescent assay and sophisticated RT-PCR.
To establish a new assay designated as "serotyping-NS1-ELISA" to detect the NS1 protein and to identify DENV serotypes simultaneously.
The monoclonal antibodies (Mabs) against NS1 of each DENV serotype were produced and characterized for their serotype-specificity. To develop serotyping-NS1-ELISA, the selected serotype-specific anti-NS1 Mabs were applied to detect the NS1 antigen, which was previously captured by a flavivirus cross-reactive anti-NS1 Mab. Serotyping accuracy of the developed assay was validated with NS1 from DENV-infected cell culture supernatants and from well-characterized clinical specimens.
Of 30 anti-NS1 Mabs, 1 serotype-specific anti-NS1 Mab to each DENV serotype was selected based on NS1 capture ELISA results for developing the serotyping-NS1-ELISA. Using DENV-infected cell culture supernatants for validation, the selected antibodies were shown to be capable of differentiating four DENV serotypes. When acute phase plasma from DENV-infected patients was used for validation, 65 out of 85 specimens (76.5% overall sensitivity) were positive to one of the four serotypes developed in our assay. Interestingly, identification of DENV serotypes by our serotyping-NS1-ELISA was 100% accurate for DENV1, 3 and 4 and 82.4% for DENV2 as compared with standard RT-PCR. Assay specificity was 100% (90/90).
The developed serotyping-NS1-ELISA provides an alternative for simultaneous detection of DENV NS1 and identification of its serotype in acute patients' specimens. The assay would be applicable for dengue diagnosis and epidemiological studies.
登革病毒（DENV）引起蚊媒病登革热出血热（DHF），由四种血清型在流行地区共同循环。目前，DENV 血清型可通过费力的病毒分离，然后进行免疫荧光检测和复杂的 RT-PCR 来鉴定。
建立一种新的称为“血清型-NS1-ELISA”的检测方法，以检测 NS1 蛋白并同时鉴定 DENV 血清型。
针对各 DENV 血清型的 NS1 产生了单克隆抗体（Mabs），并对其血清型特异性进行了特征描述。为了开发血清型-NS1-ELISA，选择的血清型特异性抗-NS1 Mabs 用于检测先前由黄病毒交叉反应性抗-NS1 Mab 捕获的 NS1 抗原。该方法的血清型鉴定准确性已通过 DENV 感染细胞培养上清液和特征明确的临床标本中的 NS1 进行了验证。
在 30 种抗-NS1 Mab 中，根据 NS1 捕获 ELISA 结果，选择了针对每种 DENV 血清型的 1 种血清型特异性抗-NS1 Mab 用于开发血清型-NS1-ELISA。使用 DENV 感染的细胞培养上清液进行验证，结果表明所选抗体能够区分四种 DENV 血清型。当使用来自 DENV 感染患者的急性期血浆进行验证时，在我们的检测方法中开发的 4 种血清型中，有 65 种（总体敏感性为 76.5%）呈阳性。有趣的是，与标准 RT-PCR 相比，我们的血清型-NS1-ELISA 对 DENV1、3 和 4 的血清型鉴定准确率为 100%，对 DENV2 的准确率为 82.4%。该检测方法的特异性为 100%（90/90）。
所开发的血清型-NS1-ELISA 提供了一种替代方法，可用于在急性患者标本中同时检测 DENV NS1 并鉴定其血清型。该检测方法可用于登革热诊断和流行病学研究。
