CN114002427B - 新型冠状病毒抗原检测的方法和试剂盒 - Google Patents
新型冠状病毒抗原检测的方法和试剂盒 Download PDFInfo
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Abstract
本发明提供了一种新型冠状病毒抗原检测的方法和试剂盒。试剂盒包括试剂条,试剂条包括底板,以及位于底板上的沿样品层析的方向依次相连的样品垫、胶体金垫、硝酸纤维素膜和吸水纸;胶体金垫上附着有胶体金标记的质控标记物和第二新型冠状病毒核衣壳蛋白抗体;硝酸纤维素膜上设有T线和C线,T线含有第一新型冠状病毒核衣壳蛋白抗体,C线包含与胶体金标记的质控标记物特异结合的配体;第一新型冠状病毒核衣壳蛋白抗体具有包含SEQ ID NO:1、2和3所示CDR序列的第一重链可变区和SEQ ID NO:4、5和6所示CDR序列的第一轻链可变区。所提供的试剂盒用于新型冠状病毒的体外检测,具有极高的灵敏度和特异性。
Description
技术领域
本发明涉及医药检测领域,具体涉及一种新型冠状病毒抗原检测的方法和试剂盒。
背景技术
新型冠状病毒(SARS-COV-2)其感染人体后会大概率导致新型冠状病毒肺炎(COVID-19),具有极强的传染性和致病性,被世界卫生组织认定为一种严重的流行性疾病,对人类社会造成了极大的影响。
新型冠状病毒疫情出现后,为实现疾病的诊断和阻断病毒传播链,多种新型冠状病毒体外诊断技术问世,主要方法包括新新型冠状病毒核酸检测,新新型冠状病毒抗体检测和新型冠状病毒抗原检测几大类型。抗体检测是疫情初期的主要检测手段,但其为间接检测,有着无法直接判断是否携带病毒且窗口期长等缺点,后续逐渐被淘汰。核酸检测由于其高灵敏度一直被作为金标准使用,但核酸检测需要专业的设备及操作人员,而且检测速度较慢,无法在缺乏这些资源的地区广泛使用。抗原快速检测由于其检测速度快、操作简便、结果准确率较高等特点,成为世界范围内新型冠状病毒检测最主要的手段。
新型冠状病毒抗原检测主要使用免疫学方法检测,其检测原理是由新型冠状病毒抗体特异识别样本中含有的微量新型冠状病毒抗原,并通过与抗体结合的信标物将检测信号放大,以实现检测的目的。该技术的关键原料为新型冠状病毒的抗体,其决定了检测的准确性。
但是针对新型冠状病毒的检测还存在一系列的问题。主要表现为:1)抗体与抗原结合的亲和力不够高,导致检测的灵敏度较低,在实际应用中容易出现漏检。2)抗体易收到样品中其他物质的干扰,导致检测的特异性较差,在实际应用中容易出现假阳性结果。有关新型冠状病毒的检测还需要进一步改进。
发明内容
本发明的目的是提供一种新型冠状病毒抗原检测的方法和试剂盒,其应用于新型冠状病毒的检测,具有极高的灵敏度和特异性。
具体而言,本发明提供了如下技术方案:
本发明的第一方面提供了一种试剂盒,包括试纸条,所述试剂条包括:
底板,以及位于底板上的样品垫、胶体金垫、硝酸纤维素膜和吸水纸;
沿样品层析的方向,所述样品垫、胶体金垫、硝酸纤维素膜和吸水纸依次相连;
所述胶体金垫上附着有胶体金标记的质控标记物和第二新型冠状病毒核衣壳蛋白抗体或抗原结合片段,
所述硝酸纤维素膜上设有T线(检测线)和C线(质控线),所述T线含有第一新型冠状病毒核衣壳蛋白抗体或抗原结合片段,所述C线上包含与所述胶体金标记的质控标记物特异结合的配体;
所述第一新型冠状病毒核衣壳蛋白抗体或抗原结合片段具有第一重链可变区和第一轻链可变区,
所述第一重链可变区包括SEQ ID NO:1、2和3所示的CDR序列,或者与SEQ ID NO:1、2和3所示的序列至少具有一个保守氨基酸取代的序列;
所述第一轻链可变区包括SEQ ID NO:4、5和6所示的CDR序列,或者与SEQ ID NO:4、5和6所示的序列至少具有一个保守氨基酸取代的序列。
本发明的第二方面提供了一种新型冠状病毒抗原检测方法,包括:采用所述的试剂盒对待测样品进行检测。所述待测样品选自唾液、鼻拭子、鼻咽拭子、咽拭子、血液或尿液中的至少一种。
本发明所取得的有益效果为:本发明所提供的试剂盒,其含有的新型冠状病毒核衣壳蛋白抗体或抗原结合片段用于新型冠状病毒体外诊断,具有极高的灵敏度和特异性。
附图说明
图1是根据本发明的实施例提供的试纸条的结构示意图,其中标号1为样品垫,2为胶体金垫,3为硝酸纤维素膜,4为吸水纸,5为底板,6是检测线,7是质控线。
具体实施方式
下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。在对本发明描述的过程中,对于本文中有关的术语进行了解释和说明,这些解释和说明仅仅是为了方便对于方案的理解,并不能看做是对本发明保护方案的限制。
抗体
本文中,术语“抗体”是能够与特异性抗原结合的免疫球蛋白分子。包括两条分子量较轻的轻链和两条分子量较重的重链,重链(H链)和轻链(L链)由二硫键连接形成一个四肽链分子。其中,肽链的氨基端(N端)氨基酸序列变化很大,称为可变区(V区),羧基端(C端)相对稳定,变化很小,称为恒定区(C区)。L链和H链的V区分别称为VL和VH。
在可变区中某些区域氨基酸组成和排列顺序具有更高的变化程度,称为高变区(Hypervariable region,HVR),高变区为抗原和抗体结合的位置,因此也称为决定簇互补区(complementarity-determining region,CDR)。重链可变区和轻链可变区上均有三个CDR区。
本发明提供了一种试剂盒,包括试纸条,所述试剂条包括:底板,以及位于底板上的样品垫、胶体金垫、硝酸纤维素膜和吸水纸;沿样品层析的方向,所述样品垫、胶体金垫、硝酸纤维素膜和吸水纸依次相连;所述胶体金垫上附着有胶体金标记的质控标记物和第二新型冠状病毒核衣壳蛋白抗体或抗原结合片段;所述硝酸纤维素膜上设有T线(检测线)和C线(质控线),所述T线含有第一新型冠状病毒核衣壳蛋白抗体或抗原结合片段,所述C线上包含与所述胶体金标记的质控标记物特异结合的配体;所述第一新型冠状病毒核衣壳蛋白抗体或抗原结合片段具有第一重链可变区和第一轻链可变区,所述第一重链可变区包括SEQ ID NO:1、2和3所示的CDR序列,或者与SEQ ID NO:1、2和3所示的序列至少具有一个保守氨基酸取代的序列;所述第一轻链可变区包括SEQ ID NO:4、5和6所示的CDR序列,或者与SEQ ID NO:4、5和6所示的序列至少具有一个保守氨基酸取代的序列。“抗原结合片段”是指具有特异性结合新型冠状病毒核衣壳蛋白抗原能力的抗体片段。“保守氨基酸取代”指的是氨基酸被另一氨基酸发生生物学上、化学上或者结构上相似的残基所取代。生物学上相似的指的是该取代不破坏新型冠状病毒核衣壳蛋白抗体或者与新型冠状病毒核衣壳蛋白抗原的生物学活性。结构上相似指的是氨基酸具有相似长度的侧链,如丙氨酸、甘氨酸或丝氨酸,或具有相似大小的侧链。化学相似性指的是氨基酸具有相同的荷电或者都是亲水或者疏水的。例如疏水残基异亮氨酸、缬氨酸、亮氨酸或者甲硫氨酸相互取代。或者用极性氨基酸例如用精氨酸取代赖氨酸、谷氨酸取代天冬氨酸、谷氨酰胺取代天冬酰胺,丝氨酸取代苏氨酸等等。所提到的保守氨基酸取代可以是一个、两个或者三个氨基酸取代。
所示出的各序列如下所示,需要说明的是这些CDR序列来自于IMGT数据库(http://www.imgt.org)的结果。本领域技术人员应该知悉的是,选择分析的数据库发生改变时,来自于重链可变区和轻链可变区的CDR序列会稍有改变,这些改变也应包括在本发明的保护范围之内。
在至少一些实施方式中,所述第一重链可变区选自:(1)SEQ ID NO:7所示的序列,或者(2)与SEQ ID NO:7所示的序列相比,具有80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%以上同源性的序列。
在至少一些实施方式中,所述第一轻链可变区选自:(1)SEQ ID NO:8所示的序列,或者(2)与SEQ ID NO:8所示的序列相比,具有80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%以上同源性的序列。
在至少一些实施方式中,编码所述第一重链可变区为如SEQ ID NO:7所示的序列。
QSVEESGGRLVTPGTPLTLTCTASGFSPSSYGMSWVRQAPGKGLEWIGYINIEDYAYYAPWAKGRFTISKTSSTTVDLKVTSPTTEDTATYFCGRGGYAADIWGPGTLVTVSS(SEQ ID NO:7)
在至少一些实施方式中,编码所述第一轻链可变区为如SEQ ID NO:8所示的序列。
DVVMTQTASPVSAAVGGTVTISCQSSESVYTNNRLSWFQQKPGQRPKLLIYRASKLASGVPSRFSGSGSGTQFTLIISDVQCDDAATYYCAGVGSGGTDIAFGGGTKVEIK(SEQ ID NO:8)
所提供的抗体或抗原结合片段,除了上述提到的第一重链可变区和第一轻链可变区之外,还可以进一步包括重链恒定区和轻链恒定区。重链恒定区和轻链恒定区序列可以来自于人来源的序列。
在至少一些实施方式中,编码所述第一重链可变区的核苷酸序列选自:(1)SEQ IDNO:9所示的序列;或者(2)与SEQ ID NO:9所示的序列相比,具有80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%以上同源性的序列。在至少一些实施方式中,编码所述第一轻链可变区的核苷酸序列选自:(1)SEQ ID NO:10所示的序列;或者(2)与SEQ ID NO:10所示的序列相比,具有80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%以上同源性的序列。
在至少一些具体实施方式中,编码所述第一重链可变区的核苷酸序列如SEQ IDNO:9所示。
CAATCTGTAGAGGAGTCAGGTGGTAGACTTGTGACCCCCGGTACCCCACTTACTCTGACTTGTACGGCTAGTGGCTTCTCCCCGTCATCCTACGGGATGAGTTGGGTCCGACAGGCTCCCGGCAAGGGATTGGAATGGATAGGGTACATTAATATTGAAGACTACGCCTACTATGCCCCCTGGGCCAAGGGTCGATTCACCATCTCTAAAACTTCCAGCACGACTGTGGACTTGAAGGTAACATCACCAACAACTGAAGATACGGCAACATACTTCTGCGGACGCGGCGGATATGCGGCTGATATTTGGGGACCAGGAACCTTGGTTACCGTTTCTTCA(SEQ ID NO:9)
在至少一些具体实施方式中,编码所述第一轻链可变区的核苷酸序列如SEQ IDNO:10所示。
GATGTCGTTATGACACAGACCGCAAGTCCTGTGAGCGCAGCAGTAGGGGGCACGGTCACCATCAGTTGCCAAAGCTCTGAAAGCGTGTATACCAACAATAGGCTTTCTTGGTTTCAGCAGAAACCAGGGCAACGCCCTAAACTTCTTATCTACCGAGCGTCAAAGCTGGCTAGTGGCGTACCTAGCCGGTTCTCCGGTAGCGGAAGTGGGACCCAATTCACGCTCATTATCTCTGACGTACAATGCGACGACGCTGCCACATACTACTGTGCCGGGGTCGGGAGCGGTGGCACAGACATAGCTTTCGGTGGAGGCACGAAGGTAGAGATTAAA(SEQ ID NO:10)
在至少一些实施方式中,所述第二新型冠状病毒核衣壳蛋白抗体或抗原结合片段具有第二重链可变区和第二轻链可变区,所述第二重链可变区选自SEQ ID NO:11、12和13所示的CDR序列,或者与SEQ ID NO:11、12和13所示的序列至少具有一个保守氨基酸取代的序列;所述第二轻链可变区包括SEQ ID NO:14、15和16所示的CDR序列,或者与SEQ ID NO:14、15和16所示的序列至少具有一个保守氨基酸取代的序列。
所示出的各序列如下所示,需要说明的是这些CDR序列来自于IMGT数据库(http://www.imgt.org/)的结果。本领域技术人员应该知悉的是,选择分析的数据库发生改变时,来自于重链可变区和轻链可变区的CDR序列会稍有改变,这些改变也应包括在本发明的保护范围之内。
在至少一些实施方式中,所述第二重链可变区选自:(1)SEQ ID NO:17所示的序列,或者(2)与SEQ ID NO:17所示的序列相比,具有80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%以上同源性的序列。
在至少一些实施方式中,所述第二重链可变区为如SEQ ID NO:17所示的序列。
EVQLVESGGGLVKPGGSLKLSCAASGITFSDYYMYWVRQTPEKRLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCVRDKSMGFGAWFAYWGQGTLVTVSA(SEQ ID NO:17)
在至少一些实施方式中,所述第二轻链可变区选自:(1)SEQ ID NO:18所示的序列,或者(2)与SEQ ID NO:18所示的序列相比,具有80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%以上同源性的序列。
在至少一些实施方式中,所述第二轻链可变区为如SEQ ID NO:18所示的序列。
EIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQRPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGHSIPYTFGGGTKLEIK(SEQ ID NO:18)
所提供的抗体或抗原结合片段,除了上述提到的第二重链可变区和第二轻链可变区之外,还可以进一步包括第二重链恒定区和第二轻链恒定区。第二重链恒定区和第二轻链恒定区序列可以来自于人来源的序列。
在至少一些实施方式中,编码第二重链可变区的核苷酸序列选自:(1)SEQ ID NO:19所示的序列;或者(2)与SEQ ID NO:19所示的序列相比,具有80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%以上同源性的序列。在至少一些实施方式中,编码第二轻链可变区的核苷酸序列选自:(1)SEQ ID NO:20所示的序列;或者(2)与SEQ ID NO:20所示的序列相比,具有80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%以上同源性的序列。
在至少一些具体实施方式中,编码所述第二重链可变区的核苷酸序列如SEQ IDNO:19所示。
GAAGTCCAACTCGTTGAAAGTGGAGGCGGGTTGGTGAAACCAGGAGGGTCTCTCAAACTGAGCTGCGCTGCGAGCGGTATCACTTTCTCCGACTACTATATGTATTGGGTTAGACAAACTCCCGAGAAACGGCTGGAGTGGGTCGCAACTATCTCTGACGGAGGGAGTTACACGTACTATCCAGACTCCGTAAAGGGCAGGTTTACCATTAGCAGGGATAATGCCAAAAACAATCTTTATCTCCAAATGTCTTCCTTGAAGTCTGAGGATACTGCAATGTACTATTGTGTGCGCGACAAGTCAATGGGATTCGGGGCGTGGTTTGCTTATTGGGGACAGGGAACCCTTGTAACTGTCTCAGCC(SEQ IDNO:19)
在至少一些具体实施方式中,编码所述第二轻链可变区的核苷酸序列如SEQ IDNO:20所示。
GAAATTGTCTTGACTCAGAGTCCCACCACAATGGCCGCTTCACCCGGCGAAAAGATAACTATTACGTGTTCTGCGTCCTCAAGTATCAGTAGCAATTATTTGCATTGGTATCAACAACGACCCGGTTTTTCCCCGAAACTTCTGATTTATCGAACCTCAAATCTCGCGTCAGGTGTTCCTGCGCGATTTAGCGGCTCTGGGAGTGGTACAAGCTACTCCCTTACAATTGGCACTATGGAGGCAGAGGACGTTGCCACCTATTATTGCCAACAAGGCCACTCAATCCCCTATACTTTCGGAGGTGGTACAAAGCTCGAAATAAAA(SEQ ID NO:20)
根据具体实施方式,所提到的质控标记物选自羊抗鸡IgY抗体、鸡IgY抗体、羊抗兔IgG抗体、兔IgG抗体、亲和素、生物素-BSA、兔抗鼠IgG、鼠IgG抗体、DNP抗体、DNP-BSA中的至少一种。根据具体实施方式,所提到的与所述胶体金标记的质控标记物特异结合的配体选自鸡IgY抗体、羊抗鸡IgY抗体、兔IgG抗体、羊抗兔IgG抗体、生物素-BSA、亲和素、鼠IgG抗体、兔抗鼠IgG、DNP-BSA、DNP抗体中的至少一种。
所提到的试剂盒除了上述提到的内容,还可以根据需要含有说明书以及样本采集工具。本领域技术人员可以根据说明书以及样本采集工具,采集样本并按照说明书的记载来进行新型冠状病毒抗原检测。
本发明还提供了一种新型冠状病毒抗原检测方法,包括:采用上述所述的试剂盒对待测样品进行检测。所述待测样品包括但不限于唾液、鼻拭子、鼻咽拭子、咽拭子、血液或尿液等。
本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1
应用B细胞单细胞克隆方法获得mAb#4抗体。具体步骤如下:
利用新型冠状病毒核衣壳蛋白作为抗原免疫兔子。收集兔子的全血并制备PBMCs。然后用流式细胞分析仪从PBMCs中分选抗原特异性记忆B血细胞。通过PCR方法扩增抗体可变区并克隆至pCMV_HC和pCMV_LC抗体表达载体上。最后进行抗体表达,并筛选抗原特异性高亲和力抗体。其中编号为mAb#4的抗体具有最强的活性。其重链可变区序列如SEQ ID NO:7所示,轻链可变区序列如SEQ ID NO:8所示。
通过脂质体法将pCMV_HC和pCMV_LC载体转染至CHO细胞系进行重组抗体的表达。用Protein A亲和柱分离纯化抗体,纯度达到95%以上。
通过杂交瘤的方法获得mAb#9抗体。具体步骤如下:
利用新型冠状病毒核衣壳蛋白作为抗原免疫小鼠。收集小鼠脾脏并制备脾细胞。将小鼠脾细胞与Myeloma cells融合并筛选稳定的杂交瘤细胞。然后通过ELISA的方法筛选产生抗原特异性的抗体。
对所获得高特异性抗体进行测序。其中一个抗体编号为mAb#9,其重链可变区序列如SEQ ID NO:17所示,轻链可变区序列如SEQ ID NO:18所示。
然后将该抗体的编码序列导入到pCMV_HC和pCMV_LC抗体表达载体上。通过脂质体法将pCMV_HC和pCMV_LC载体转染至CHO细胞系进行重组抗体的表达。用Protein A亲和柱分离纯化抗体,纯度达到95%以上。
实施例2
实施例2提供了一种试纸条,如图1所示。图1中标号1为样品垫(其为玻璃纤维素薄膜),2为胶体金垫,3为硝酸纤维素膜,4为吸水纸,5为底板,6是检测线(又称为T线),7是质控线(又称为C线)。试纸条中各部分制备方法如下:
1、制备硝酸纤维素膜
配制包被缓冲液:含0 .05M pH8.5 PBS缓冲液为包被缓冲液,采用0.22μm膜滤过,置4℃备用。其中缓冲液配方:NaCl 40g、KCl 1g、Na2HPO4·12H2O 14.5g、KH2PO4 1g,双蒸去离子水定容至1000mL。
制备硝酸纤维素膜:用包被缓冲液将实施例1获得的新型冠状病毒核衣壳蛋白重组抗体(mAb#4)稀释到0.5-2mg/mL,调整机器,划线为T线;用羊抗鸡IgY划线为C线,45℃烘干2~18小时,封装备用。
2、胶体金、胶体金标记单克隆抗体的制备
(1)准备如下试剂:
a. 配制氯金酸:用双蒸去离子水溶解氯金酸,配成0.04%溶液备用。氯金酸溶液配方为:0.4g氯金酸用双蒸去离子水定容至1000mL。
b. 配制柠檬酸三钠:用双蒸去离子水溶解柠檬酸钠,配成1%溶液备用。柠檬酸三钠溶液配方为:10g柠檬酸三钠用双蒸去离子水定容至1000mL。
c. 配制0.1M碳酸钾:用双蒸去离子水配制备用。0.1M碳酸钾溶液配方为:13.8g碳酸钾用双蒸去离子水定容至1000mL。
d. 配制5%BSA溶液:用0.02MPBS配制备用。5%BSA溶液配方为:BSA 5g用0.02MpH7.4 PBS定容至100mL。
(2)制备胶体金:
将0.04%氯金酸置于电炉煮沸,按每100mL 0.04%氯金酸加入6mL 1%柠檬酸三钠,继续煮沸,直到液体呈亮红色,继续加热15分钟即停止加热,冷却至室温。
(3)制备胶体金标记单克隆抗体:
用0.1M碳酸钾调胶体金的pH值至8.5,按15-40μg抗体/mL胶体金加入新型冠状病毒核衣壳抗体(mAb#9),磁力搅拌器混匀30min,搅拌下加入5% BSA至终浓度为1%持续搅拌1小时。13000rpm、4℃离心30min,弃上清,沉淀用用十分之一初始胶体金体积的5%BSA溶液将沉淀重悬,置4℃备用。
用0.1M碳酸钾调胶体金的pH值至8.5,按10-50μg抗体/mL胶体金加入鸡IgY,磁力搅拌器混匀30min,搅拌下加入5% BSA至终浓度为1%持续搅拌1小时。13000rpm、4℃离心30min,弃上清,沉淀用十分之一初始胶体金体积的5%BSA溶液将沉淀重悬,置4℃备用。
3、制备胶体金垫
a. 配制铺金液:
铺金液配方为:20g 酪蛋白,1g PEG20000,20mL Tween-20,20g海藻糖,用0 .02MpH7 .4 PBS溶液定容至1000mL。
b. 制备胶体金垫:
制备好的胶体金标记单克隆抗体和胶体金标记鸡IgY分别按1:5~1:20的稀释倍数与铺金液混匀,均匀的铺在玻璃纤维薄膜上,每毫升溶液铺20平方厘米,55℃烘干2~8小时备用。
c. 组装试纸条
将吸水纸、硝酸纤维素膜、胶体金垫、样本垫如图1所示贴合于塑料的底板上,并纵向裁切成3.6mm宽的小条,得到试纸条。
实施例3
实施例3采用实施例2的试纸条对新型冠状病毒的检测性能进行了验证:
选取经PCR确认阴阳性的临床鼻拭子样本435例,检测结果如下表1所示。其中PCR确认的新型冠状病毒阳性样本65例,采用实施例2提供的试纸条检出阳性60例,阴性5例;PCR确认的新型冠状病毒阳性样本370例,采用实施例2提供的试纸条检出阳性0例,阴性370例。具体检测结果如下表1所示。
表1 检测结果
计算试纸条的检测灵敏度和特异性结果如下,CI代表置信区间:
临床灵敏度: 真阳性结果量/阳性样本量*100%= 92.3% (95% CI: 83.0-97.5%)
临床特异性: 真阴性结果量/阴性样本量*100%=100% (95% CI: 99.0-100%)
总符合率:430/435= 98.9% (95% CI: 97.3-99.6%)
实验结果表明,本发明所提供的新型冠状病毒核衣壳蛋白抗体用于体外诊断具有极高的灵敏度核特异性。
具体的临床数据附表:
表2 临床数据附表
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
SEQUENCE LISTING
<110> 北京健乃喜生物技术有限公司
<120> 新型冠状病毒抗原检测的方法和试剂盒
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Gly Arg Gly Gly Tyr Ala Ala Asp Ile
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Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
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Claims (11)
1.一种试剂盒,包括试纸条,其特征在于,所述试纸 条包括:
底板,以及位于底板上的样品垫、胶体金垫、硝酸纤维素膜和吸水纸;
沿样品层析的方向,所述样品垫、胶体金垫、硝酸纤维素膜和吸水纸依次相连;
所述胶体金垫上附着有胶体金标记的质控标记物和第二新型冠状病毒核衣壳蛋白抗体或抗原结合片段;
所述硝酸纤维素膜上设有T线和C线,所述T线含有第一新型冠状病毒核衣壳蛋白抗体或抗原结合片段,所述C线上包含与所述胶体金标记的质控标记物特异结合的配体;
所述第一新型冠状病毒核衣壳蛋白抗体或抗原结合片段具有第一重链可变区和第一轻链可变区,
所述第一重链可变区包括SEQ ID NO:1、2和3所示的CDR序列;
所述第一轻链可变区包括SEQ ID NO:4、5和6所示的CDR序列;
所述第二新型冠状病毒核衣壳蛋白抗体或抗原结合片段具有第二重链可变区和第二轻链可变区,
所述第二重链可变区选自SEQ ID NO:11、12和13所示的CDR序列;
所述第二轻链可变区包括SEQ ID NO:14、15和16所示的CDR序列。
2.根据权利要求1所述的试剂盒,其特征在于,所述第一重链可变区为:SEQ ID NO:7所示的序列。
3.根据权利要求1或2所述的试剂盒,其特征在于,所述第一轻链可变区为:SEQ ID NO:8所示的序列。
4.根据权利要求1所述的试剂盒,其特征在于,编码所述第一重链可变区的核苷酸序列为:SEQ ID NO:9所示的序列。
5.根据权利要求1所述的试剂盒,其特征在于,编码所述第一轻链可变区的核苷酸序列为:SEQ ID NO:10所示的序列。
6.根据权利要求1所述的试剂盒,其特征在于,所述第二重链可变区为:SEQ ID NO:17所示的序列。
7.根据权利要求1所述的试剂盒,其特征在于,所述第二轻链可变区为:SEQ ID NO:18所示的序列。
8.根据权利要求1所述的试剂盒,其特征在于,编码第二重链可变区的核苷酸序列为:SEQ ID NO:19所示的序列。
9.根据权利要求1所述的试剂盒,其特征在于,编码第二轻链可变区的核苷酸序列为:SEQ ID NO:20所示的序列。
10.根据权利要求1~8中任一项所述的试剂盒,其特征在于,所述质控标记物选自羊抗鸡IgY抗体、鸡IgY抗体、羊抗兔IgG抗体、兔IgG抗体、亲和素、生物素-BSA、兔抗鼠IgG、鼠IgG抗体、DNP抗体、DNP-BSA中的至少一种。
11.根据权利要求10所述的试剂盒,其特征在于,所述与所述胶体金标记的质控标记物特异结合的配体选自鸡IgY抗体、羊抗鸡IgY抗体、兔IgG抗体、羊抗兔IgG抗体、生物素-BSA、亲和素、鼠IgG抗体、兔抗鼠IgG、DNP-BSA、DNP抗体中的至少一种。
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