CN115093478A - 特异性结合基孔肯雅病毒e2蛋白的单克隆抗体及其应用 - Google Patents
特异性结合基孔肯雅病毒e2蛋白的单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明公开了一种能特异性结合基孔肯雅病毒E2蛋白的单克隆抗体，所述单克隆抗体为1B7‑H7，其轻链可变区的氨基酸序列如SEQ ID NO:1所示，重链可变区的氨基酸序列如SEQ ID NO:2所示；或者所述单克隆抗体为2A2‑E10，轻链可变区的氨基酸序列如SEQ ID NO:3所示，重链可变区的氨基酸序列如SEQ ID NO:4所示；或者所述单克隆抗体为4D3‑E8，轻链可变区的氨基酸序列如SEQ ID NO:5所示，重链可变区的氨基酸序列如SEQ ID NO:6所示；将上述单克隆抗体应用在制备特异性检测基孔肯雅病毒E2抗原试剂或试剂盒中，具有较高特异性和敏感度，本发明有利于基孔肯雅病毒的早期诊断。
Description
技术领域
本发明涉及一种能特异性结合基孔肯雅病毒E2蛋白的单克隆抗体及其用于基孔肯雅病毒血清学诊断的DAS-ELISA试剂盒，属于医学检验技术领域。
背景技术
基孔肯雅热是由基孔肯雅病毒（Chikungunya virus, CHIKV）引起的退行性关节炎的疾病。CHIKV是单股正链RNA病毒，属于披膜病毒科（Togaviridae），甲病毒属（Alphavirus），以埃及伊蚊和白纹伊蚊作为其主要传播媒介，人群普遍易感，感染急性期表现为突发性的高热（>39℃），并伴有红斑性斑丘疹、肌痛和关节痛，慢性期为持续性或复发性的关节炎，慢性期可长达几月到几年不等。据研究报道CHIKV感染80%-93%患者发展为慢性疾病，经济负担估计每名患者每年25000英镑。目前，没有针对于基孔肯雅病毒的疫苗及有效的治疗药物。
云南地区因特殊地理位置与生态环境极其适合白纹伊蚊的生存。此外，云南与老挝、缅甸等CHIKV高风险地区接壤，面临着CHIKV跨境输入的风险。基孔肯雅病毒、登革病毒、寨卡病毒均属于虫媒病毒，且流行区域存在交叉，存在共感染。此外，病毒感染初期具有相似的临床症状，仅根据临床症状无法准确地判断是何种病毒感染所致。因此，云南省的基孔肯雅疫情防控形势不容乐观。为防止疫情传入和扩散，需做好充分的技术储备，而快速检测是基础。
目前，检测基孔肯雅病毒感染的常规诊断方法通常包括病毒分离培养、血清学检测、核酸分子检测等方法。病毒分离培养、RT-PCR、RT-qPCR、多重PCR具有特异性高的特点，但受限于需要生物安全3级实验室、专业操作人员、检测时间长等因素，不利于快速诊断，也不适合用于基层检测机构进行CHIKV检测。基于血清学的抗原抗体检测，不仅检测时间短（2-3 h）、检测成本低、不需要专业的实验设备和操作人员，适用于海关等基层检测机构对CHIKV感染进行大规模筛查，也符合云南省乃至中国对CHIKV检测的需求。
发明内容
本发明提供了一种能特异性结合基孔肯雅病毒E2蛋白的单克隆抗体，所述单克隆抗体为1B7-H7，其轻链可变区的氨基酸序列如SEQ ID NO:1所示，重链可变区的氨基酸序列如SEQ ID NO:2所示；或者所述单克隆抗体为2A2-E10，轻链可变区的氨基酸序列如SEQ IDNO:3所示，重链可变区的氨基酸序列如SEQ ID NO:4所示；或者所述单克隆抗体为4D3-E8，轻链可变区的氨基酸序列如SEQ ID NO:5所示，重链可变区的氨基酸序列如SEQ ID NO:6所示。
本发明另一目的是将上述单克隆抗体应用在制备特异性检测基孔肯雅病毒E2抗原试剂或试剂盒中，所述试剂盒为特异性检测基孔肯雅病毒E2抗原的DAS-ELISA试剂盒，试剂盒包含上述单克隆抗体中的一个，以及其他用于DAS-ELISA检测的常规试剂。
本发明目的通过以下技术方案实现：
基孔肯雅病毒E2蛋白是介导病毒入侵宿主的核心蛋白，具有较高免疫原性，是诱导机体产生广泛抗体的蛋白；本发明以云南省乃至中国的主要流行株东/中/南非型（Ross株）的E2蛋白（1-346aa）作为靶抗原，构建原核表达载体，通过Rosetta（DE3）菌株诱导表达重组蛋白。
本发明以原核表达的CHIKV-E2重组蛋白免疫BALB/c小鼠和新西兰白兔，获得三株特异性针对CHIKV-E2蛋白的杂交瘤细胞株（1B7-H7，2A2-E10，4D3-E8）和兔多抗血清；
本发明采用三株杂交瘤细胞株在小鼠体内接种制备单抗腹水，单克隆抗体纯化后，对单克隆抗体的纯度、抗体效价进行评估，用间接ELISA、IFA、Western Blot等实验对单克隆抗体的特异性进行分析。
本发明将单克隆抗体应用在制备具有高灵敏度和特异性的基孔肯雅病毒E2抗原检测试剂盒中，它可以直接检测CHIKV感染患者血清中的CHIKVE2抗原，实现基孔肯雅病毒感染的早期诊断；该试剂盒是基于双抗体夹心ELISA方法的检测试剂盒，其包括包被捕获抗体的微孔反应板、样品处理液、检测抗体、阳性对照物、阴性对照物、浓缩洗液、显色液、终止液，其中捕获抗体为兔多克隆抗体；检测抗体为单克隆抗体为1B7-H7、单克隆抗体为2A2-E10、单克隆抗体为4D3-E8中的一种与辣根过氧化物酶偶联获得的；所述样品处理液、浓缩洗液、显色液、终止液均是双抗体夹心ELISA方法中的常用试剂，阳性对照物是重组基孔肯雅病毒E2蛋白，阴性对照物是不含重组基孔肯雅病毒E2蛋白的空白对照物。
与现有技术相比，本发明具有如下优点和有益效果：
本发明提供的单克隆抗体能特异结合基孔肯雅E2蛋白，将单克隆抗体用于基于双抗体夹心ELISA方法的检测试剂盒，其中捕获抗体兔多克隆抗体和检测抗体是一组能够特异识别基孔肯雅病毒E2蛋白的不同表位的抗体，仅能特异性结合基孔肯雅病毒，因此该试剂盒能够准确、快速地检测出基孔肯雅病毒，而同其它虫媒病毒如登革病毒、寨卡病毒均无交叉反应，且对基孔肯雅病毒具有较高的敏感度，检测下限为1×106 PFU/mL，本发明有利于基孔肯雅病毒的早期诊断和虫媒监控。
附图说明
图1为基孔肯雅E2蛋白PCR扩增结果，其中A：凝胶电泳结果图，B：基因测序峰图，C：BLAST比对结果图；
图2为CHIKV-E2重组蛋白的诱导表达纯化后的蛋白检测结果，其中1：诱导前菌体，2：诱导后菌体，3：破碎上清，4：破碎沉淀，5-9：纯化蛋白（100-500mM咪唑洗脱）；
图3为单克隆抗体、多克隆抗体纯化SDS-PAGE检测结果；
图4为单克隆抗体、兔多克隆抗体ELISA检测结果；
图5 单克隆抗体、兔多克隆抗体Western Blot、IFA验证结果，其中A为WesternBlot检测结果，B为IFA检测结果；
图6为间接免疫荧光法检测单克隆抗体、兔多克隆抗体与常见虫媒病毒感染细胞免疫反应的结果；
图7为三株单克隆抗体标记HRP后抗体活性验证结果；
图8为优化最佳捕获抗体、检测抗体工作浓度结果；
图9为确定最佳封闭缓冲液实验结果；
图10为优化抗原孵育最佳温度、时间结果；
图11为DAS-ELISA的特异性验证，A为可视化结果，B为OD450nm值；
图12为临床样本检测结果，其中A图为10例CHIKV模拟临床样本，10例DENV阳性血清样本，10例阴性血清样本检测结果，B图为DAS-ELISA阳性或阴性结果的ROC曲线。
具体实施方式
以下结合实施例对本发明的具体实施作进一步说明，但本发明的实施和保护不限于此。需指出的是，以下若有未特别详细说明之过程，均是本领域技术人员可参照现有技术实现或理解的；所用试剂或仪器未注明生产厂商者，均为可以通过市售购买得到的常规产品。
实施例1：单克隆抗体、兔多抗的制备
1、免疫抗原的制备
1.1、pET-28a-CHIKV-E2原核表达载体的构建
从-80℃冰箱取出保存的基孔肯雅病毒（CHIKV，Ross株）接种于BHK细胞，用含有2%血清的BHK培养基于细胞培养箱中培养48h，收集上清，12000g离心10分钟，弃沉淀，离心后上清用viral DNA/RNA Extraction Kit提取RNA，利用HiScript® II 1st Strand cDNASynthesis Kit反转录成cDNA，提取和反转录参照试剂盒说明书操作说明进行；
以基孔肯雅病毒的cDNA为模板进行PCR扩增，引物为如下：
CHIKV-E2-F：5'-CGCGGATCCAGTATTAAGGACCACTTCAATGTC-3'；
CHIKV-E2-R：5'-CCCAAGCTTTCATAACTGCGGCCAATACTTATAC-3'；
反应体系（20μL）如下：Premix Taq 10 μL、CHIKV-E2-F 1μL、CHIKV-E2-R 1μL、cDNA 2μL、ddH2O 6μL；扩增程序为：预变性：94℃，5min；变性：95℃，30s；退火：58℃，30s；延伸：72℃，2min，40个循环；延伸：72℃，10min。将PCR产物用2%琼脂糖凝胶进行验证，琼脂糖凝胶电泳结果显示扩增获得1038bp CHIKV-E2蛋白基因条带；将符合预期结果的目的条带用琼脂糖凝胶电泳DNA胶回收。
将胶回收的产物和pET28a用BamHⅠ和Hind Ⅲ分别在37℃下酶切12h，然后16℃连接8h，并转化至DH5α感受态细胞中；将菌液涂布于含卡那霉素的平板上，37℃培养过夜，挑取单菌落进行PCR扩增，琼脂糖凝胶电泳检测后，将与预期条带大小一致的PCR产物送昆明硕擎生物科技有限公司测序；测序结果为阳性菌落，BLAST序列比对一致；将测序正确的菌液进行扩大培养，然后用质粒小提取试剂盒提取质粒；随后将重组pET28a-CHIKV-E2质粒转化Rosetta（DE3）表达菌株，并挑取单克隆保存菌株，结果见图1，菌液PCR扩增得到了1038bp的条带，菌液PCR结果证明已经成功构建了CHIKV-E2蛋白重组表达载体，且测序峰图和NCBIBlast比对结果与目的片段相似度100%（图1 B、1C）。
1.2、CHIKV-E2蛋白的诱导、表达、纯化
为了表达重组CHIKV-E2蛋白（E2蛋白），我们将步骤1.1中获得的表达菌株“Z”型划线于LB平板上。大肠杆菌Rosetta（DE3）在37℃、50 g/mL卡那霉素存在的情况下过夜生长，培养过夜（约12h）后阳性转化子被转移至200mL LB液体培养基中，在震荡条件下37℃孵育，直到细菌培养OD值达到0.4-0.6，然后加入0.5mmol/L浓度的异丙基硫代-D-糖苷酶（IPTG）。每间隔4h取1mL菌液，共取4次，然后6000g (Sorvall, Thermo scientific)离心15分钟，在上样缓冲液中裂解，用12% SDS-PAGE进行电泳分析；观察是否诱导出E2蛋白。
将大量诱导表达E2蛋白的菌液在冰中超声10分钟（设置开启3秒脉冲，关闭3秒脉冲），使用设置为超声器频率为250Hz；待破碎结束后10000g离心30min，同时收集上清和微球（包涵体）。微球经8mol/L尿素4℃变性处理4h，再经过10000g离心30min处理，收集上清悬液并用0.45μm滤膜除杂；随后用ProteinIso Ni-NTA Resin亲和层析的方法进行纯化获得E2抗原，将E2蛋白纯化后，以SDS-PAGE检测纯化蛋白，结果见图2，经过IPTG的诱导得到了大量的E2蛋白表达。
2、抗CHIKV-E2兔多克隆抗体的制备
白兔免疫：取出已测好浓度的重组CHIKV-E2蛋白，按照每只兔子1mg蛋白的量进行
免疫；将1mg的蛋白溶于500μL的无菌PBS中然后加入等体积的佐剂，在漩涡震荡器上混匀1h，然后用注射器吹吸混匀后皮下多点注射；14天后重复该实验，多次免疫后，采取耳缘静脉血进行抗血清效价的检测；当抗血清的效价达到实验标准，进行一次加强免疫后颈部动脉大量取血；获得多抗血清用protein A Sepharose柱进行纯化。
新西兰白兔第四次免疫3天后，大量的采取兔子血，分离血清，进行纯化；纯化后的抗体分别进行SDS-PAGE、ELISA、IFA验证；SDS-PAGE结果表明（图3）多克隆抗体已经被顺利纯化；ELISA检测结果表明（图4）纯化后的多克隆抗体能与重组CHIKV-E2蛋白反应；图5A是兔多抗血清检测重组蛋白表达的Western Blot结果，IFA结果表明（图5B）所制备的兔多克隆抗体能与天然的CHIKV-E2蛋白反应。
3、小鼠单克隆抗体的制备
3.1、免疫小鼠
取4-6周龄雌性BALB/c小鼠，免疫原为步骤1制得的原核表达的重组CHIKV E2蛋白，第一次采用弗氏完全佐剂与等体积CHIKV E2抗原混匀乳化，每只小鼠皮下多点注射30μg，以后每间隔14天以弗氏不完全佐剂与重组的CHIKV E2抗原免疫共4次，第三次、第四次免疫后3天，尾静脉取血，间接ELISA检测多抗血清效价；于融合前3天每只小鼠腹腔注射CHIKVE2抗原100μg进行加强免疫。
3.2、杂交瘤细胞的制备和鉴定
按1:10比例将骨髓瘤细胞SP2/0与免疫小鼠脾脏细胞均匀混合后加入50mL离心管中，加入37℃预热的PEG进行融合，融合产物加入50mL含15％胎牛血清的RPMI-1640培养基，室温800rpm离心10min，弃上清，用50mLHAT培养基（含20%FBS、2%HAT、RPMI-1640）悬浮细胞，将细胞悬液加入4块96孔培养板上，在37℃、5%CO2的二氧化碳培养箱中培养；融合后第五天半量更换HT培养基，在细胞融合后的第12-13天取培养上清，经间接ELISA、IFA双筛选仍为强阳性的克隆用有限稀释法进行克隆化，克隆化2-3次至阳性率达100%，选择分泌抗体效价高的细胞株扩增培养后置液氮保存，经过3轮亚克隆，成功筛选出3株抗CHIKV-E2的单抗细胞株，命名为1B7-H7、2A2-E10、4D3-E8。
3.3、单克隆抗体腹水制备和纯化
采用体内诱生法制备本发明单克隆抗体1B7-H7、2A2-E10和4D3-E8，即在小鼠体内分别接种杂交瘤细胞1B7-H7、2A2-E10、4D3-E8制备腹水；具体方法如下：每只小鼠腹腔内注射0.5mL弗氏不完全佐剂（Sigma公司），使瘤细胞能以腹水瘤形式在腹腔内生长；小鼠腹腔注射佐剂大约1-2周后，将约1×106个对数生长期的杂交瘤细胞悬浮于无血清RPMI 1640培养基中，注入小鼠腹腔；1-2周后，采集腹水并离心收集上清，存放于4℃备用，若长期储存，应置于-80℃冰箱；鼠单克隆抗体采用北京全式金生物有限公司ProteinA亲和纯化填料进行纯化，具体操作方法参照产品说明书进行；纯化后抗体SDS-PAGE检测，结果见图3，结果显示成功纯化出单克隆抗体；纯化抗体分别进行ELISA、Western Blot和IFA验证；ELISA结果见图4，3株单克隆抗体腹水均能与重组CHIKV-E2反应；Western Blot结果显示见图5A所制备的鼠单克隆抗体能与重组CHIKV-E2蛋白反应；IFA结果显示所制备的鼠单克隆抗体能与天然CHIKV-E2蛋白反应（图5B）；纯化后以BCA蛋白分析试剂定量，测定浓度后的抗体加入终浓度50%的甘油于-80℃保存。
3.4、间接免疫荧光鉴定单克隆抗体、兔多克隆抗体的特异性
分别用四种血清型的DENV病毒（DENV1、DENV2、DENV3、DENV4）、ZIKV病毒和CHIKV病毒感染BHK细胞，感染48h后，用预冷的1×PBS洗细胞二遍，然后加入固定液（甲醛:丙酮=1:1）固定20分钟，自然风干，以纯化后步骤3.3单克隆抗体或步骤2兔多克隆抗体为一抗，FITC标记的山羊抗小鼠IgG或山羊抗兔IgG为二抗，37℃孵育，并设立未感染病毒的BHK细胞作为阴性对照，细胞核用DAPI染色后，荧光显微镜下观察反应情况，结果见图6，结果显示3株单克隆抗体与其它虫媒病毒均无交叉反应，特异性100%。
实施例2：DAS-ELISA检测方法的建立
1、单克隆抗体1B7-H7、2A2-E10、4D3-E8与HRP偶联制备抗体探针
实验中采用亚科因（Abbkine）生物公司的LinKineTM HRP Labeling Kit进行抗体标记，具体实验步骤如下：
（1）吸取200μg抗体于1.5mL EP管中，加入10μL HRP标记缓冲液，轻柔混匀；
（2）取出试剂盒中活化的HRP溶液，瞬时离心，吸取7.5μL活化的HRP溶液加入EP管中，轻柔混匀，再加入100μL的超纯水，轻柔混匀，将EP管用锡箔纸包裹避光室温（20-25℃）反应2h；
（3）向HRP quencher powder中加入1mL超纯水，振荡混匀制成浓度为100×的quencher溶液，使用时将其稀释100倍制成quencher工作液，注：100×quencher溶液室温保存，且在48h内使用；
（4）避光反应2h后，加入15μL quencher工作液，轻柔混匀置于4℃，避光反应2h，反应结束，加入一定体积的甘油，使标记抗体浓度为1mg/mL，-20℃保存使用；
三株单克隆抗体与HRP成功偶联制成抗体探针，采用直接ELISA检测3株单克隆抗体标记HRP后抗体活性，结果见图7，结果显示与HRP偶联的三株单克隆抗体均有活性，且2A2-E10活性最好。
2、单克隆抗体2A2-E10的DAS-ELISA检测方法的建立
（1）包被捕获抗体：用于捕获样本中特异性抗原的兔多抗被固定在ELISA板的小孔中，注意：多抗用包被液以2倍稀释法稀释，使其浓度为20μg/mL；按每个小孔100μL的量加入，在4℃条件下孵育过夜；
考虑到试剂盒的成本，分别对捕获抗体--兔多抗、标记抗体最佳工作浓度进行确定，兔多抗浓度范围设定为40μg/mL-0.3125μg/mL，DAS-ELISA检测结果显示确定捕获抗体的最佳工作浓度为20μg/mL（图8A），检测抗体2A2-E10（HRP）最佳稀释比例为1:250（图8B）；
（2）封闭：4℃包被过夜后，弃孔中液体。用含0.05%吐温20的PBS（PBST）清洗5次。ELISA微孔板用200μL 5%脱脂牛奶，于37℃，60 rpm条件下封闭2小时；
封闭液作为免疫学实验中的一个重要试剂，它可以用于非特异性结合位点的封闭，封闭剂可以有效地减少非特异性结合，这些非特异性结合主要来自于一抗或二抗与抗原的结合。因此，封闭有助于降低背景颜色，增强信噪比。在DAS-ELISA实验中，一种好的封闭液对于提高检测灵敏度和准确性非常重要；本实验中分别采用5%脱脂牛奶、1% BSA、10%FBS三种常见的封闭缓冲液进行实验；实验结果显示，使用5%脱脂奶进行封闭效果最佳，其P/N值相比于其他两种封闭缓冲液大（图9）；
（3）加入CHIKV-E2蛋白：封闭完成后，弃孔中液体，用含0.05%吐温的PBS（PBST）清洗5次，1% BSA稀释E2蛋白，按2μg/孔加入，于37℃，60 rpm条件下孵育2 h；
DAS-ELISA检测灵敏度的提高，有赖于捕获抗体与抗原的结合，抗体捕获抗原的能力强弱除了与抗体本身亲和力有关，还与孵育时间有关；本实验中排除抗体自身因素限制，只考虑抗原孵育的时间，并对其进行优化；分别在37℃、常温下孵育30 min、60 min、90min、120 min、150 min、180 min；实验结果所示，最佳孵育温度确定为37℃，最佳孵育时间确定为2h（图10）；
（4）加入酶标二抗：孵育2小时后，弃孔中液体，PBST清洗5次，用1% BSA将2A2-E10（HRP）抗体稀释成1:250的浓度，每孔加入100μL，将ELISA板置于37℃、60rpm恒温摇床孵育2小时；
（5）显色：2h后，PBST洗涤ELISA板5次，按100μL/孔加入可溶性TMB显色液，于37℃、60 rpm恒温摇床反应20 min；
（6）终止：使用2mol/L H2SO4终止反应，每孔加入50μL，使用酶标仪在450nm处测量光密度值（OD）。
3、DAS-ELISA试剂盒组成
a、浓缩清洗液（100mL）：含有5％Tween-20的10×PBS，即1L溶液中含有35.8gNa2HPO4·12H2O，2.7g KH2PO3，80g NaCl，121℃，20min高压灭菌后，加入5mL Tween-20搅匀，使用时10倍稀释；
b、样品稀释液（50mL）：1%BSA溶液；
c、特异性检测抗体（100μL）：使用时，用样品稀释液按1:250稀释使用；
d、阳性对照（1.5 mL）：CHIKV E2抗原（20μg/mL）；
e、阴性对照（1.5 mL）：1% BSA溶液；
f 、显色液(15 mL)：可溶型单组分TMB底物显色液，可直接使用；
g、终止液(15 mL)：2mol/L H2SO4；
h、微孔反应板（8×12）。
2、微孔反应板的制备方法如下：将实施例1制得的兔多克隆抗体用CBS缓冲液稀释至20μg/mL，按100μL/孔包被聚苯乙烯96孔微孔板，于4℃过夜，拍干后，每孔加入200μL 5%脱脂奶封闭液，于37℃封闭2h，以封闭非特异性结合位点。拍干板条，真空干燥12-24h，用铝膜袋真空包装4℃保存备用。
实施例3：DAS-ELISA检测方法的特异性、灵敏度评价
1、特异性实验
（1）样品选取及制备
采用空斑法测定CHIKV病毒、四个血清型DENV病毒（DENV1、DENV2、DENV3、DENV4）、ZIKV病毒的病毒滴度，病毒滴度分别为CHIKV: 3×1010 PFU/mL、DENV-1:3.16×109 PFU/mL、DENV-2: 3.16×109 PFU/mL、DENV-3: 1.58×109PFU/mL、DENV-4: 1.58×109 PFU/mL、ZIKV: 2.16×109 PFU/mL。
（2）检测方法
取上述待测病毒样品100μL加入包被兔多抗用于捕获抗原的微孔反应板中，同时设阴性对照（1% BSA溶液，不含CHIKV E2蛋白）和阳性对照（CHIKV E2抗原，浓度：20μg/mL），37℃温育2h，浓缩洗涤液10倍稀释后洗涤板条，200μL/孔洗板5次后；加入100μL/孔特异性检测抗体（2A2-E10-HRP），按1:250稀释，37℃温育2h；同上洗板5次后，加100μL/孔底物显色液，室温避光显色20min后，加入终止液，50μL/孔，终止反应，酶标仪测定OD450nm值。
（3）结果判定：以空白孔调零，于450nm波长测定吸光度(A值)，阳性对照平均值≥0.50，阴性对照平均值≤0.15，实验成立；样品A值≥阴性对照A值平均值×3，则判为阳性，反之为阴性。
采用本发明试剂盒检测灭活的CHIKV病毒液、四个血清型DENV、ZIKV病毒液，检测结果显示本发明试剂盒能特异性识别CHIKV病毒，与其余5种黄病毒属病毒无交叉反应（图11）。
2、灵敏度实验
（1）样品选取及制备
将CHIKV病毒灭活后，从1×1010 PFU/mL开始十倍稀释多个梯度进行DAS-ELISA检测，同时上述稀释样品取100μL利用RNAisoPlus(Ta Ka Ra,中国)提取RNA，同步进行RT-qPCR检测，确定检测方法灵敏度；
（2）检测方法同步骤1
采用本发明试剂盒检测梯度稀释的灭活CHIKV病毒液，结果显示DAS-ELISA检测灵敏度为1×106PFU/mL，结果下表所示；
本发明试剂盒检测倍比稀释CHIKV病毒培养液灵敏度
表注：本发明试剂盒的结果判定如下：样品检测值≥阴性对照平均值的3倍（计算为0.0628×3=0.1884）为阳性（+），反之为阴性（-）；基孔肯雅病毒核酸检测试剂盒结果判定如下：检测样品CT值≤36为阳性。
实施例4：检测试剂盒在临床样本检测中的应用
1、样本收集
采用10份CHIKV阳性培养物、20个DENV阳性血清和20个正常对照进行临床检测分析；其中CHIKV阳性培养物由1×109PFU/mL的病毒培养液与正常人血清1:1混合制成；
2、样本的捕获
取上述待测样品100μL加入包被兔多抗用于捕获抗原的ELISA微孔反应板中，37℃温育2h，捕获样本中的特异性抗原；
3、加入2A2-E10-HRP
孵育2小时后，弃孔中液体，PBST清洗5次，用1% BSA（样品稀释液）将检测抗体2A2-E10（HRP）稀释成1:250的浓度，每孔加入100μL，将ELISA板置于37℃、60rpm恒温摇床孵育2h；
4、显色反应
2h后，PBST洗涤ELISA板5次，按100μL/孔加入可溶性TMB显色液，于37℃、60 rpm恒温摇床反应20 min；
5、终止反应
使用2mol/L H2SO4终止反应，每孔加入50μL，使用酶标仪在450nm处测量光密度值（OD）。
实验结果见图12，结果显示DAS-ELISA试剂盒在临床样本检测中具有良好的性能，特异性100%，图12 A显示除10例CHIKV阳性培养物检测呈阳性外，所有样本均为阴性。
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<110> 昆明理工大学
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Claims (5)
1.一种单克隆抗体，其特征在于：所述单克隆抗体为1B7-H7，其轻链可变区的氨基酸序列如SEQ ID NO:1所示，重链可变区的氨基酸序列如SEQ ID NO:2所示；或者所述单克隆抗体为2A2-E10，轻链可变区的氨基酸序列如SEQ ID NO:3所示，重链可变区的氨基酸序列如SEQ ID NO:4所示；或者所述单克隆抗体为4D3-E8，轻链可变区的氨基酸序列如SEQ ID NO:5所示，重链可变区的氨基酸序列如SEQ ID NO:6所示。
2.权利要求1所述的单克隆抗体在制备特异性检测基孔肯雅病毒E2抗原试剂中的应用。
3.权利要求1所述的单克隆抗体在制备特异性检测基孔肯雅病毒E2抗原试剂盒中的应用。
4.根据权利要求3所述的应用，其特征在于：试剂盒为特异性检测基孔肯雅病毒E2抗原的DAS-ELISA试剂盒。
5.根据权利要求5所述的应用，其特征在于：试剂盒包括包被兔多抗用于捕获抗原的微孔反应板、样品处理液、与标记物结合的单克隆抗体、阳性对照物、阴性对照物、浓缩洗液、显色液、终止液。
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|---|---|---|---|---|
| CN116621930A (zh) * | 2023-04-14 | 2023-08-22 | 深圳市第二人民医院(深圳市转化医学研究院) | 检测基孔肯雅病毒的多肽、试剂盒和方法 |
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2022
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116621930A (zh) * | 2023-04-14 | 2023-08-22 | 深圳市第二人民医院(深圳市转化医学研究院) | 检测基孔肯雅病毒的多肽、试剂盒和方法 |
| CN116621930B (zh) * | 2023-04-14 | 2024-02-20 | 深圳市第二人民医院(深圳市转化医学研究院) | 检测基孔肯雅病毒的多肽、试剂盒和方法 |
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