Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein.
Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein.
复制标题
DOI：
10.1371/journal.pntd.0004595
复制
发表时间：
2016-04
影响因子：
3.8
通讯作者：
Saijo M
中科院分区：
文献类型：
--
作者：
Fukuma A;Fukushi S;Yoshikawa T;Tani H;Taniguchi S;Kurosu T;Egawa K;Suda Y;Singh H;Nomachi T;Gokuden M;Ando K;Kida K;Kan M;Kato N;Yoshikawa A;Kitamoto H;Sato Y;Suzuki T;Hasegawa H;Morikawa S;Shimojima M;Saijo M
严重发烧伴血小板减少综合征（SFTS）是一种蜱传传染病，病死率很高，由SFTS病毒（SFTSV）引起。 SFTS 常见于中国、韩国和日本。已知 SFTS 患者血清中的病毒 RNA 水平与结果密切相关。疾病流行地区需要具有高敏感性和特异性的病毒学 SFTS 诊断。我们生成了针对 SFTSV 核衣壳 (N) 蛋白的新型单克隆抗体 (MAb)，并开发了一种夹心抗原 (Ag) 捕获酶联免疫吸附测定 (ELISA)，分别使用 MAb 和多克隆抗体作为捕获抗体和检测抗体来检测 SFTSV N 蛋白。 Ag 捕获系统能够从各种 SFTSV 菌株的培养上清液中检测至少 350–1220 TCID50/100 μl/孔。使用从日本疑似患有 SFTS 的患者采集的血清样本评估了 Ag 捕获 ELISA 在 SFTS 诊断中的功效。所有24份含有高拷贝数病毒RNA（>105拷贝/ml）的血清样本（100%）在Ag捕获ELISA中显示出阳性反应，而15份含有低拷贝数病毒RNA（<105拷贝/ml）的血清样本中的12份（80%）在Ag捕获ELISA中显示出阴性反应。在这些 Ag 捕获 ELISA 阴性的 12 个样本中，9 个 (75%) 的 SFTSV IgG 抗体呈阳性。新开发的 Ag 捕获 ELISA 可用于高水平病毒血症急性期患者的 SFTS 诊断。严重发烧伴血小板减少综合征（SFTS）是一种蜱传新发传染病，由一种新型布尼亚病毒 SFTS 病毒（SFTSV）引起。自 2011 年在中国首次发现以来，SFTSV 已在 SFTS 患者和蜱中检测到，其地理范围不断扩大，从中国到日本和韩国。 SFTS 传播到其他温暖或亚热带地区的可能性使其成为公众健康的严重问题。快速、特异性地检测SFTSV对于有效控制该病具有重要意义。对于病毒感染的诊断，夹心抗原（Ag）捕获ELISA检测病毒血症血清样本中的病毒核蛋白（N）已被广泛应用于检测试剂，因为它是最丰富的病毒抗原并且具有高度保守的氨基酸序列。在本研究中，使用针对 SFSTV-N 的新型单克隆抗体，开发了 Ag 捕获 ELISA 系统，并使用从 SFTS 疑似患者收集的血清对该系统进行了验证。我们的数据表明，Ag 捕获 ELISA 对于疾病急性期的 SFTS 患者的诊断很有用。这项研究展示了一种新的 SFTS 诊断方法，可能为有效控制该疾病提供有用的信息。
Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas. We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350–1220 TCID50/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>105 copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<105 copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV. The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia. Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne emerging infectious disease caused by a novel bunyavirus, SFTS virus (SFTSV). Since first discovered in China in 2011, SFTSV has been detected from SFTS patients and ticks with expanding geographic ranges from China to Japan and South Korea. The potential for SFTS spread to other warm or sub-tropical regions makes it a serious concern to the public health. It is of great importance to detect SFTSV rapidly and specifically for the effective control of the disease. For the diagnosis of viral infections, a sandwich antigen (Ag)-capture ELISA detecting viral nucleoprotein (N) in viremic serum samples has been widely applied to detect the agents, since it is the most abundant viral antigen and has highly conserved amino acid sequence. In this study, using the novel monoclonal antibodies raised against SFSTV-N, an Ag-capture ELISA system was developed, and the validation of this system was performed using sera collected from SFTS-suspected patients. Our data show that the Ag-capture ELISA was useful for the diagnosis of SFTS patients in the acute phase of the disease. This study shows a novel methodology for the diagnosis of SFTS, which may provide helpful information for the effective control of the disease.