CN117368459A - 标记抗体与其相关生物材料及在新冠病毒检测中的应用 - Google Patents
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Abstract
本发明公开了标记抗体与其相关生物材料及在新冠病毒检测中的应用。所述应用为如下任一中的应用：(1)制备检测新型冠状病毒产品；(2)制备结合新型冠状病毒的S蛋白产品；(3)检测新型冠状病毒；(4)结合新型冠状病毒的S蛋白；所述新型冠状病毒为南非株或印度株。该标记抗体与传统HRP标记抗体相比，具有更高的检测灵敏度。
Description
技术领域
本发明属于生物技术领域，具体涉及标记抗体与其相关生物材料及在新冠病毒检测中的应用。
背景技术
以病毒抗原作为检测靶标，是病毒感染的重要检测方法。在实验室进行病毒分离培养、疫苗滴度测定及制备效果评价方面发挥着重要作用。但由于灵敏度低，通常不作为感染确诊的主要技术手段。
针对病毒抗原的检测主要采用双抗体夹心的模式，捕获抗体对样本中的病毒抗原进行捕获富集，然后加入检测抗体与病毒抗原相结合，通过检测抗体偶联物产生的发光信号或比色信号进行信号收集及结果判断。采用化学标记方法在抗体的赖氨酸残基上偶联辣根过氧化物酶(HRP)、碱性磷酸酶、生物素等是目前最主要的检测抗体标记方式，可通过酶促发光或化学发光进行结果检测，被目前的免疫学检测方法广泛采用，但这种标记方法对于抗体结合区有多个赖氨酸分布的抗体来说，标记后易影响抗体的结合活性，尤其是对于双抗体夹心的检测模式，检测抗体结合区存在多个标记酶分子，易形成空间位阻，检测抗体Fab双臂无法与病毒抗原充分结合，从而降低检测的灵敏性。
发明内容
本发明的目的之一在于提供抗体或荧光素酶标记抗体的应用。
本发明提供了抗体或荧光素酶标记抗体在如下任一中的应用，所述抗体或所述荧光素酶标记抗体的轻链可变区中的LCDR1、LCDR2和LCDR3的氨基酸序列依次如SEQ ID No.1中第27-36位，第54-56位以及第93-101位所示，所述抗体或所述荧光素酶标记抗体的重链可变区中的HCDR1、HCDR2和HCDR3的氨基酸序列依次如SEQ ID No.2中第25-48位，第50-57位以及第96-109位所示：
(1)制备检测新型冠状病毒产品；
(2)制备结合新型冠状病毒的S蛋白产品；
(3)检测新型冠状病毒；
(4)结合新型冠状病毒的S蛋白；
所述新型冠状病毒为南非株或印度株。
上述产品可以为试剂盒，具体可以为ELISA检测试剂盒或全自动磁微粒检测系统。
可选地，根据上述的应用，所述轻链可变区的氨基酸序列如SEQ ID No.1中第1-130位所示；所述重链可变区的氨基酸序列如SEQ ID No.2中第1-120位所示；所述荧光素酶的氨基酸序列如SEQ ID No.2中第477-646位所示。
可选地，根据上述的应用，所述抗体的轻链氨基酸序列如SEQ ID No.1所示；所述抗体的重链氨基酸序列如SEQ ID No.2中第1-470位所示；所述荧光素酶标记抗体的重链氨基酸序列如SEQ ID No.2所示。
本发明还提供了一种用于检测新型冠状病毒的试剂盒，包括上述的抗体或荧光素酶标记抗体。
可选地，根据上述的试剂盒，还包括捕获抗体标记磁珠、荧光素酶底物和/或没有新型冠状病毒的阴性对照。
所述捕获抗体可为MW06抗体。
所述捕获抗体标记磁珠的制备方法可包括：将羧基修饰磁珠置于摇床震荡使其充分分散开后，采用MES(0.015M，PH＝5.5)清洗200ul磁珠两次，定容到10mg/ml；涡旋加入EDC和NHS，震荡混匀后，置于37℃摇床孵育30min；磁分离弃上清，加入MES清洗1次后重悬至10mg/ml，涡旋加入40μg抗体，37℃摇床孵育4h；磁分离去上清；PBST(0.015M PBS，0.05％tween-20，PH＝7.4)清洗2次，磁分离，加入3％BSA-PBST至10mg/ml，37℃摇床孵育1h进行封闭；磁分离去上清，2％BSA-PBST定容至10mg/ml，获得捕获抗体标记磁珠(也被称为捕获抗体偶联的磁珠)。
本发明还提供了一种新冠病毒全自动磁微粒系统，包括上述的试剂盒和全自动磁微粒检测系统。
上述的荧光素酶标记抗体也属于本发明的保护范围之内。
本发明还提供了上述的荧光素酶标记抗体的相关生物材料，所述相关生物材料为下述任一种：
c1)编码上述荧光素酶标记抗体的核酸分子；
c2)含有c1)所述核酸分子的表达盒；
c3)含有c1)所述核酸分子的重组载体、或含有c2)所述表达盒的重组载体，例如下述实施例制备的pcDNA3.1-chC25L和/或pcDNA3.1-chC25H-Nluc；
c4)含有c1)所述核酸分子的重组微生物、或含有c2)所述表达盒的重组微生物、或含有c3)所述重组载体的重组微生物；
c5)含有c1)所述核酸分子的重组细胞、或含有c2)所述表达盒的重组细胞、或含有c3)所述重组载体的重组细胞。
c1)所述核酸分子可为如下任一种：
d1)核苷酸序列是SEQ ID No.3和/或SEQ ID No.4所示的DNA分子；
d2)与d1)限定的核苷酸序列具有90％或90％以上同一性，且编码上述标记抗体的DNA分子；
d3)在严格条件下与d1)或d2)限定的核苷酸序列杂交，且编码上述标记抗体的DNA分子。
本文中，同一性是指氨基酸序列或核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性，如NCBI主页网站的BLAST网页。
本文中，所述90％以上的同一性可为至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
所述严格条件可为在0.1×SSPE(或0.1×SSC)，0.1％SDS的溶液中，在65℃条件下杂交并洗膜。
上述的相关生物材料在如下任一中的应用也属于本发明的保护范围之内：
(1)制备检测新型冠状病毒产品；
(2)制备结合新型冠状病毒的S蛋白产品；
(3)检测新型冠状病毒；
(4)结合新型冠状病毒的S蛋白；
所述新型冠状病毒为南非株或印度株。
上述抗体或上述荧光素酶标记抗体的制备方法可包括：将含有编码上述抗体的基因的重组载体或c3)所述重组载体导入宿主细胞中获得重组细胞，培养所述重组细胞获得培养上清，所述培养上清中含有上述抗体或上述荧光素酶标记抗体。
上述方法中，所述宿主细胞可为真核细胞，如COS7细胞、CHO细胞，HEK293细胞，酵母细胞或昆虫细胞等。在本发明的一个实施例中，所述宿主细胞为COS7细胞。
本发明实施例采用生物标记的方法可将标记物分子定向重组在抗体基因恒定区末端，通过基因转染，细胞培养的方式，实现标记抗体的直接表达，从而完全避免标记物对抗体结合活性的影响，从而提高检测灵敏性，因此生物定向标记方法是一种更高效的标记抗体制备方法。利用Nano荧光素酶信号强度高、线性范围广的优势，本发明以Nano荧光素酶作为标记物，制备了一株检测新冠病毒的标记抗体Nluc-chC25，比传统HRP标记抗体，具有更高的检测灵敏度；并应用于全自动磁微粒检测系统中，建立了高灵敏检测新冠病毒的全自动磁微粒检测方法，与传统HRP标记的ELISA检测方法相比，灵敏度提高了10倍以上。
附图说明
图1为抗体表达载体构建示意图。
图2为Nluc-chC25抗体的制备及鉴定。
图3为实施例2检测结果。
图4为全自动磁微粒检测系统结构示意图。
图5为实施例3检测结果。
图6为实施例4检测结果。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，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。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
1质粒、细胞、蛋白、灭活病毒和临床样本
pcDNA3.1(+)质粒(Thermo Fisher Scientific)，COS7细胞(实验室保存，记载于蜱传脑炎病毒包膜糖蛋白胞外区的真核表达优化及其在血清学检测中的效果评价，霍耐凡，康晓平，户义，李裕昌，李靖，张雨，冉鑫，贾佳，曹雪峰，杨银辉，生物技术通讯，Vol.27No.3 May，2016，38-41)，MW06和MW05单克隆抗体；(上海迈威惠赠，记载于Wang S，Peng Y，Wang R，Jiao S，Wang M，HuangW，Shan C，Jiang W，Li Z，Gu C，Chen B，Hu X，Yao Y，Min J，Zhang H，Chen Y，Gao G，Tang P，Li G，Wang A，Wang L，Zhang J，Chen S，Gui X，Yuan Z，Liu D.Characterization of neutralizing antibody with prophylactic andtherapeutic efficacy against SARS-CoV-2in rhesus monkeys.Nat Commun，2020，11(1)：5752.doi：10.1038/s41467-020-19568-1.)，84号血清(实验室保存)，灭活新冠病毒(普通株BetaCoV/Beijing/IME-BJ05/2020 strain、南非株SARS-CoV-2 Variant BetaCSTR.16698.06.NPRC2.062100001 strain、印度株SARS-CoV-2 variant DeltaCSTR.16698.06.NPRC 6.CCPM-B-V-049-2105-6 strain，记载于Tang Y，Wang Y，Li Y，ZhaoH，Zhang S，Zhang Y，Li J，Chen Y，Wu X，Qin C，Jiang T，Kang X.An integrated rapidnucleic acid detection assay based on recombinant polymerase amplificationfor SARS-CoV-2.Virol Sin.2022 Feb；37(1)：138-141.doi：10.1016/j.virs.2022.01.006.Epub2022 Jan 13.PMID：35234627；PMCID：PMC8755414.)，其他病毒(风疹病毒(RV)，黄热病毒YF 170，副流感病毒1型，呼吸道合胞病毒，副流感病毒2型，腺病毒3型，流感病毒H1N1亚型，流感病毒H3N2亚型，记载于Tang Y，Wang Y，Li Y，Zhao H，ZhangS，Zhang Y，Li J，Chen Y，Wu X，Qin C，Jiang T，Kang X.An integrated rapid nucleicacid detection assay based on recombinant polymerase amplification for SARS-CoV-2.Virol Sin.2022 Feb；37(1)：138-141.doi：10.1016/j.virs.2022.01.006.Epub2022 Jan 13.PMID：35234627；PMCID：PMC8755414.)由实验室保存。
2试剂
Phusion超保真PCR试剂盒(New England Biolabs，货号#M0530L)
PureLinkTM Quick Gel Extraction Kit&PCR Purification Combo Kit(Invitrogen by Thermo Fisher Scientific，货号K220001)
限制性内切酶EcoR I(货号#R3101S)、Not l(货号#R3189S)、Afl II(货号#R0520V)(New England Biolabs)
T4 DNA Ligase(Promega，货号M1801)
DH5感受态细胞(博尔迈生物，货号BC102-01)
Spin Miniprep Kit(50)(QIAGEN，货号27104)
LipofectamineTM3000 Transfection Reagent(Thermol Fisher Scientific，货号L3000-015)
高灵敏快速考马斯亮蓝染色试剂盒protein stains H C510041(生工生物工程(上海)股份有限公司，货号C510041-0010)
预制胶Meilunbio Precast PAGE Gel(meilunbio，货号MA0296)
PageRulerPrestained Protein Ladder 26616(Thermo，货号26616)
PureProteome Protein A/G Mix Magnetic Beads(Millipore，CAT：LSKMAGAG10)
Nano-Glo Luciferase Assay，10ml(Promega，N1110)
高灵敏度化学发光检测试剂盒(康为世纪，CW0049M)
3仪器
电泳仪(Hoefer EPS 2A200)水平电泳槽
紫外凝胶透射仪(Gel DocTM XR+)
垂直电泳仪Mini-PROTEAN Tetra System(BIO-RAD)
全自动化学发光检测仪(华大基因)
GloMax微孔板发光检测仪(Promega)
实施例1、Nluc-chC25抗体的制备及鉴定
1.Nluc-chC25抗体重组表达质粒的构建：
以pcDNA3.1(+)作为表达载体，分别构建Nluc-chC25的轻链表达载体和重链表达载体。
人工合成IL-6信号肽序列和编码chC25抗体轻链序列融合基因，作为抗体轻链表达基因IL-6-chC25L，分别在IL-6-chC25L基因序列的5’端和3’加上AflII(CTTAAG)和EcoRI(GAATTC)酶切位点。人工合成IL-6信号肽序列、编码ch-2C5抗体重链序列、18个碱基的柔性肽和Nano荧光素酶基因的串联融合基因，作为抗体重链表达基因IL-6-chC25H-Nluc，分别在其的5’端和3’加上AflII(CTTAAG)和NotI(GCGGCCGC)酶切位点。利用限制性内切酶分别对IL-6-chC25L、IL-6-chC25H-Nluc和pcDNA3.1(+)质粒进行双酶切后，T4连接酶连接，转化进DH5α感受态细胞，获得重组质粒pcDNA3.1-chC25L和pcDNA3.1-chC25H-Nluc，并对质粒进行双酶切鉴定及测序鉴定。质粒构建的示意图如图1所示，左侧图为pcDNA3.1-chC25L，右侧图为pcDNA3.1-chC25H-Nluc。双酶切鉴定结果如图2中的A所示，泳道1为重组质粒pcDNA3.1-chC25L使用限制性内切酶Afl II和EcoRI双酶切得到IL-6-chC25L片段(810bp)，泳道2为质粒pcDNA3.1-chC25H-Nluc使用限制性内切酶Afl II和Not I双酶切得到IL-6-chC25-Nluc片段(1503bp)，泳道M1为DL2000 DNAmarker，泳道M2为DL15000 DNAmarker。双酶切及测序鉴定结果表明，pcDNA3.1-chC25L和pcDNA3.1-chC25H-Nluc是分别将IL-6-chC25L和IL-6-chC25H-Nluc插入pcDNA3.1(+)获得的载体。
pcDNA3.1-chC25L是将pcDNA3.1(+)AflII和NotI酶切位点之间的小片段替换为抗体轻链表达基因IL-6-chC25L所获得的载体。抗体轻链表达基因IL-6-chC25L的核苷酸序列如SEQ ID No.3所示，其中，第1-87位为IL-6信号肽序列，第88-93位为酶切位点，第94-810位为编码chC25抗体轻链序列。编码chC25抗体轻链序列中，第1-330位编码轻链可变区，第79-108位为LCDR1基因，第160-168位为LCDR2基因，第277-303位为LCDR3基因。该载体表达抗体轻链，其氨基酸序列如SEQ ID No.1所示，其中，第1-130位为轻链可变区，第27-36位为LCDR1，第54-56位为LCDR2，第93-101位为LCDR3。
pcDNA3.1-chC25H-Nluc是将pcDNA3.1(+)AflII和NotI酶切位点之间的小片段替换为抗体重链表达基因IL-6-chC25H-Nluc所获得的载体。抗体重链表达基因IL-6-chC25H-Nluc的核苷酸序列如SEQ ID No.4所示，其中，第1-87位为IL-6信号肽序列，第88-93位为酶切位点，第94-1503位为编码chC25抗体重链序列，第1504-1509位为酶切位点，第1510-1521位为柔性肽编码基因，第1522-2034位为Nano荧光素酶基因。编码chC25抗体重链序列中，第1-360位编码重链可变区，第73-96位为HCDR1基因，第148-171位是HCDR2基因，第286-327位是HCDR3基因。该载体表达抗体重链，其氨基酸序列如SEQ ID No.2所示，其中，第1-470位为chC25抗体重链序列，第471-476位为柔性肽序列，第477-646位为Nano荧光素酶序列。chC25抗体重链序列中，第1-120位为重链可变区，第25-48位为HCDR1，第50-57位为HCDR2，第96-109位为HCDR3。
2.Nluc-chC25抗体的表达及纯化
使用10％FBS的DMEM培养COS7细胞，准备细胞长至80％-90％的24孔板，利用LipofectamineTM3000转染试剂将重组质粒pcDNA3.1-chC25L和pcDNA3.1-chC25H-Nluc瞬时转染COS7细胞，重组质粒采用两种方案进行转染。
方案一：重组质粒pcDNA3.1-chC25L和pcDNA3.1-chC25H-Nluc各0.25μg，P3000TM1μl，使用Opti-MEMTM培养基补至25μl。方案二：重组质粒pcDNA3.1-chC25L和pcDNA3.1-chC25H-Nluc各0.5μg，P3000TM2μl，使用Opti-MEMTM培养基补至25μl。接着将两种质粒预混液和25μl脂质体预混液(1.5μlLipofectamineTM3000试剂和25μlOpti-MEMTM培养基)混匀；重组质粒Nanoluc-pcDNA3.10.5μg，P3000 1μl，使用Opti-MEM培养基补充至25μl后，与25μl脂质体预混液(1.5μlLipofectamineTM3000试剂和25μl Opti-MEM培养基)混匀；空载体pcDNA3.10.5μg，P3000 1μl，使用Opti-MEM培养基补充至25μl后，与25μl脂质体预混液(1.5μlLipofectamineTM3000试剂和25μlOpti-MEM培养基)混匀；室温孵育15min后加入24孔板中，放入37℃5％CO2恒温孵箱中培养。重组质粒Nanoluc-pcDNA3.1作为阴性对照，空载体pcDNA3.1作为空白对照。
在转染后24h、48h、72h和96h分别收取细胞表达产物，细胞表达产物离心后取20微升上清加入50微升Nanoluc底物(Nano-Glo Luciferase Assay，Promega，货号N1110)，放入GloMax微孔板发光检测仪测荧光强度。结果如图2中的B所示，C25(0.5μg)为采用方案一转染的细胞表达产物荧光强度检测，C25(1.0μg)为采用方案二转染的细胞表达产物荧光强度检测，Nanoluc-pcDNA3.1为对照Nanoluc-pcDNA3.1的细胞表达产物荧光强度检测，pcDNA3.1为对照pcDNA3.1的细胞表达产物荧光强度检测，结果表明质粒转染24h后，荧光素酶强度即可达到108，已有高水平的抗体表达。
将20ml转染24h细胞表达产物，PBS稀释10倍后，加入20mg偶联ProteinA/G的磁珠(PureProteome Protein A/G Mix Magnetic Beads，Millipore，CAT：LSKMAGAG10)(10mg/ml)，80rpm转速的旋转混匀仪室温孵育60min，磁分离，收集上清液，PBST洗涤磁珠2次，加入2ml PH值为3.0的柠檬酸盐缓冲液洗脱，涡旋混匀5min，磁分离，收集上清液，加入1M Tris缓冲液将PH值调至7.0。
3.重组抗体的SDS-PAGE
准备10孔、0.75mm厚度、12.5％分离胶的蛋白质凝胶，样品(即未经过处理的细胞表达产物上清液和用protein A/G磁珠进行纯化后的细胞表达产物上清液)加入5×蛋白上样缓冲液，煮沸10min，冷却后上样，样品和标准蛋白Marker(分子量10～180kDa)上样量为10μl，电泳过程中，设定浓缩胶电压为80V，分离胶电压为120V，电泳结束后，蛋白凝胶用25％异丙醇洗2次，双蒸水洗两次，每次15min，将胶浸泡在Nanoluc底物中，使用化学发光成像仪曝光10min，采集胶片信息；接着将胶用20ml双蒸水，震荡清洗5min，重复3次，使用高灵敏快速考马斯亮蓝染色试剂盒染色30min，20ml脱色液震荡清洗15min，重复3次，用凝胶成像仪采集信息。
结果如图2中的C所示，泳道1为Nluc-chC25未纯化条带(即未经过处理的细胞表达产物上清液)，泳道2为Nluc-chC25纯化后的条带(即用protein A/G磁珠进行纯化后的细胞表达产物上清液)，M为Protein Ladder，10to 180kDa。结果表明重链及轻链蛋白条带大小分别为70KD及25KD，与预期目的条带相符，表明“2.Nluc-chC25抗体的表达及纯化”制备出Nluc-chC25抗体。
4.HRP-chC25抗体的制备
chC25抗体(其轻链序列如SEQ ID No.1所示，其重链序列如SEQ ID No.2第1-470位所示)使用EZ-LinkTM马来酰亚胺活化辣根过氧化物酶试剂盒(Thermo Scientific，货号31494)偶联辣根过氧化物酶。选择SATA方法将巯基添加到chC25抗体，取20μl SATA溶液加入到chC25抗体中，室温孵育30min，再加入200μl偶联缓冲液，室温下孵育2h，使用脱盐柱脱盐，收集流穿液。将流穿液与活化的HRP室温下孵育1h得到HRP-chC25抗体，抗体使用透析方法去除EDTA，再加入50％甘油储存在-20℃。
5.chC25-Nluc抗体的制备
根据本实施例“1-3”制备chC25-Nluc抗体(Nluc蛋白表达在抗体重链的N端)，chC25-Nluc抗体和Nluc-chC25抗体不同之处仅在于：chC25-Nluc抗体的抗体重链为IL-6-Nluc-chC25H，即其抗体重链依次为IL-6信号肽、Nano荧光素酶、柔性肽和chC25抗体重链。
chC25-Nluc抗体与新冠病毒蛋白进行结合检测实验，结果表明，相较于Nluc-chC25抗体((Nluc蛋白表达在抗体重链的C端)，chC25-Nluc抗体的结合活性显著低于Nluc-chC25抗体，推测是由于Nluc表达在N端，与抗体的结合表位较近，造成空间位阻从而影响了重组抗体与靶标抗原的结合。
实施例2、Nluc-chC25重组抗体与新冠病毒蛋白的结合检测
1.Nluc-chC25重组抗体与新冠病毒S1-RBD蛋白的结合活性的测定
采用直接ELISA，将2.0μg/mL的重组抗原(S1-RBD蛋白(义翘神州，北京)、JEV蛋白、TBE蛋白(这两个蛋白是本实验室自制的，记载于郑旸，康晓平，李裕昌等，乙型脑炎病毒E DIII蛋白的表达纯化及在Array-ELISA方法中的应用，中华微生物学和免疫学杂志2013年12月第33卷第12期，954-957)，3％BSA(索莱宝，北京))包被于白色96孔板，100ul/孔，4℃冰箱内过夜；然后用含3％BSA的PBS溶液进行封闭，37℃下孵育1h；其中重组抗原JEV，TBE和3％BSA作为无关抗原进行特异性分析。然后加入实施例1制备用protein A/G磁珠进行纯化后获得的Nluc-chC25重组抗体(浓度分别为10μg/ml，1μg/ml，100ng/ml)，Nano荧光素酶做阴性对照，0.1％BSA-PBST做空白对照，37℃孵育1.5h，加入50微升荧光素酶底物(Nano-GloLuciferase Assay，Promega，N1110)，GloMax微孔板发光检测仪中测值。
结果如图2中的D所示，可检测到Nluc-chC25与S1-RBD蛋白的特异结合，表明获得的Nluc-chC25与S1-RBD蛋白(即图中的S)结合活性良好。
2.重组抗体与S1-RBD蛋白的亲和力分析
使用GatorTM非标记分析系统(Gator Bio，CA，USA)在室温下测定，所有待测蛋白使用Q Buffer(PBS at pH 7.4with 0.02％Tween 20，0.2％BSA，and 0.05％NaN3)进行稀释，空白探针作为阴性对照，将S1-RBD蛋白稀释至385nM并预先包被在His探针上，2倍系列稀释的重组抗体(从118nM到1.88nM)与探针表面的S1-RBD蛋白结合，结束后，探针使用Gly-HCI缓冲液(PH＝1.5)再生，亲和力的计算通过Gator评估软件的1∶1(Rmax Local fit)Binding模型计算解离常数。
结果如图2的E-1和E-2所示，表明Nluc标记重组抗体和HRP标记重组抗体的亲和力解离常数KD值分别为1.51×10-11(M)和3.75×10-11(M)，表明在Nluc标记重组抗体与抗原的亲和力高于HRP标记重组抗体。
3.Nluc-chC25抗体和HRP-chC25抗体对新冠病毒S1-RBD蛋白、新冠病毒双抗体夹心模式的检测
采用双抗体夹心ELISA，浓度2μg/ml的MW06包被于白色酶标板上，37℃孵育2h，洗3次，3％BSA-PBST封闭1h，洗3次，加入4倍系列稀释的S1-RBD蛋白(1000ng/ml-0.06ng/ml，共8个浓度)，JEV蛋白、TBE蛋白和3％BSA做阴性对照，0.1％BAS-PBST做空白对照，加入Nluc-chC25(10ng/ml)或HRP-chC25(15.6ng/ml)，37℃孵育2h，分别加入100微升Nanoluc底物(Promega，N1110)和100微升化学发光底物(高灵敏度化学发光检测试剂盒，康为世纪，货号CW0049M)。
采用双抗体夹心ELISA，浓度2μg/ml的MW06包被于透明酶标板上，37℃孵育2h，洗3次，3％BSA-PBST封闭1h，洗3次，加入4倍系列稀释的灭活新冠病毒(普通株BetaCoV/Beijing/IME-BJ05/2020strain，10^5pfu/ml-39pfu/ml，共5个浓度)，50倍稀释的H7N9亚型流感病毒和3％BSA做阴性对照，0.1％BAS-PBST做空白对照，加入Nluc-chC25(10ng/ml)或HRP-chC25(15.6ng/ml)，37℃孵育2h，分别加入100微升Nanoluc底物(Promega，N1110)和100微升化学发光底物(康为世纪，货号CW0049M)。
以阴性对照组的平均值加上3倍的标准差作为cutoff值，分别计算两种标记抗体对不同浓度S1-RBD和新冠病毒的S/C值(S/C＝实验组luc值/cutoff值)，S/C＞1即为阳性。
结果如图3中的A，B所示。结果表明，对S1-RBD蛋白的检测，Nluc-chC25抗体(Nluc-2C5)可检测到S1-RBD蛋白的最低浓度为0.98ng/ml，HRP-chC25抗体(HRP-2C5)检测S1-RBD蛋白的最低浓度为3.91ng/ml；对灭活病毒的检测，Nluc-chC25抗体可检测到的最低浓度为6.25×10^3pfu/ml，HRP-chC25抗体可检测到的最低浓度为10^5pfu/ml，Nluc-chC25抗体的检测灵敏度显著高于HRP-chC25抗体。Nluc-chC25和HRP-chC25抗体中抗体蛋白序列完全一致，仅标记分子和标记物不同，该实验结果充分说明采用细胞内生物标记方法制备的抗体C端荧光素酶标记抗体Nluc-chC25，比HRP标记的HRP-chC25抗体可以更灵敏地检测病毒抗原。
实施例3、Nluc-chC25抗体在新冠病毒全自动磁微粒系统检测中的应用
全自动磁微粒检测系统，由北京华大吉比爱公司生产，主要用于进行磁微粒化学发光法检测，由试剂条和全自动化学发光检测仪组成。
该系统结果如图4所示，左侧为该系统内部结构，右侧为试剂条结构。该系统内置微电脑和磁力棒，磁力棒可按照运行程序设定，引导磁珠分别进入试剂条中的样本区、洗涤区、检测区进行样本检测。试剂条分为16个区(0号孔至15号孔)，1号孔装入200微升吸头，2号孔装入磁套，3号孔加入1.5ml洗液(PBST)，7号孔加入170微升标记抗体，14号孔加入100ul待测样本和5ul捕获抗体标记磁珠，15号孔加入150微升底物。
1.确定Nluc-chC25标记抗体合适的工作浓度
用于检测S蛋白和Nluc-chC25抗体结合能力的ELISA实验，S蛋白(SinoBiological Inc，Beijing，China)来自于新型冠状病毒普通株，通过昆虫真核表达系统表达，将稀释2μg/ml的S蛋白以及无关抗原(JEV，TBE，3％BSA)包被于透明酶标板，4℃过夜，用3％BSA-PBST(含0.05％Tween-20的PBS)封闭后，加入实施例1制备用protein A/G磁珠进行纯化后获得的Nluc-chC25抗体(10倍系列稀释，从10μg/ml到0.01ng/ml，共6个浓度)100μl每孔，37℃孵育1h，洗涤5次后，每孔加入荧光素酶底物50μl每孔，在GloMax微孔板发光检测仪中测值。
进行结果分析，以阴性对照组的平均值加上3倍的标准差作为cutoff值，对检测结果计算每个浓度下S/C值(S/C＝实验组luc值/cutoff值)。S/C值结果如图5中的A-1所示，荧光素酶强度如图5中的A-2所示。结果表明，Nluc-chC25抗体从10ug/ml至0.1ng/ml，均可与S蛋白特异结合，但在10ng/ml时，S/C值最高，表明结果最为特异，确定10ng/ml为用于病毒抗原检测的合适工作浓度。
2.捕获抗体的选择
分别采用抗新冠病毒多抗血清(编号84号)、两株新冠表达特异性抗体MW05和MW06作为捕获抗体，比较其对病毒抗原捕获效率的差异，筛选出结合活性更高的抗体作为新冠病毒检测的捕获抗体
将2μg/ml的84号、MW05、MW06包被于白色酶标板上，4度过夜，用3％BSA-PBST封闭后，加入10倍系列的灭活新冠病毒(10^5pfu/ml～10pfu/m1)、阴性对照稀释100倍JEV灭活病毒和3％BSA-PBST，37℃孵育1h，每孔加入Nluc-chC25抗体(10ng/ml)100μl，37℃孵育1h，洗涤5次后，每孔加入荧光素酶底物50μl，在GloMax微孔板发光检测仪中测值。
分别将新冠病毒特异性单抗MW05，MW06和免疫血清多抗84号作为候选捕获抗体，比较其捕获新冠病毒效果差异，以阴性对照组的平均值加上3倍的标准差作为cutoff值，对检测结果计算每个浓度下S/C值(S/C＝实验组luc值/cutoff值)，结果如图5所示，其中B-1为采用新冠病毒原始株作为捕获病毒，不同捕获抗体S/C值，B-2为采用新冠病毒不同株作为捕获病毒，MW05捕获抗体S/C值，B-3为采用新冠病毒不同株作为捕获病毒，MW06捕获抗体S/C值。采用新冠病毒原始株作为捕获病毒，MW05和MW06单抗比84号多抗血清具有更高的病毒结合活性；进而比较MW05和MW06对新冠病毒突变株的结合活性，结果表明尽管MW05对新冠原始株结合活性更高，但对于南非突变株及印度突变株，MW05结合活性较弱，表明MW06的结合表位较保守，可与多种新冠病毒株结合，是更理想的新冠病毒捕获抗体。
3.全自动磁微粒检测系统的检测程序的优化
全自动磁微粒检测系统由试剂条和全自动化学发光检测仪组成。试剂条分为16个区，1号孔装入200微升吸头，2号孔装入磁套，3号孔加入1.5ml洗液(PBST)，7号孔加入170微升标记抗体，14号孔加入100ul待测样本和5ul捕获抗体标记磁珠，15号孔加入150微升底物。
标记抗体为实施例1制备用protein A/G磁珠进行纯化后获得的Nluc-chC25抗体。待测样本为新冠感染者咽拭子样本，。捕获抗体为捕获抗体MW06偶联的磁珠。底物为Nanoluc底物(Promega，N1110)。
捕获抗体MW06偶联的磁珠制备方法具体如下。
将粒径3μm的羧基修饰磁珠(10mg/ml，MagnosphereTM MS300/Carboxyl，JSR LifeScience，货号MS300/CA)置于摇床(200rpm)震荡10分钟，充分分散开，取200ul磁珠至离心管中，用MES(0.015M，PH＝5.5)清洗两次，定容到10mg/ml；涡旋加入0.2mg EDC和NHS，震荡混匀后，置于37℃摇床孵育30min；磁分离弃上清，加入MES清洗1次后重悬至10mg/ml，涡旋加入40μg抗体，37℃摇床孵育4h；磁分离去上清；PBST(0.015M PBS，0.05％tween-20，PH＝7.4)清洗2次，磁分离，加入3％BSA-PBST至10mg/ml，37℃摇床孵育1h进行封闭；磁分离去上清，2％BSA-PBST定容至10mg/ml，获得捕获抗体偶联的磁珠，于4℃储存。
在初次的实验测试中，直接用磁微粒化学发光法运行程序1进行检测，整体结果偏低且未检测到特异性的阳性信号，推测由于样本与磁珠未充分结合所导致，因此设置运行程序2，在孵育及洗涤过程中增加了震荡步骤，且将第一步孵育后增加1次洗涤。
运行程序1如下：
试剂条放置仪器的卡槽内，仪器分液装置装上吸头，分液器将3号孔的液体分别分装至11、12、13号孔，150微升/次，每孔各加入300微升洗液。14号孔待测样本与捕获抗体标记磁珠37℃孵育30分钟。孵育结束后，磁力棒装磁套，吸附14号孔抗原-捕获抗体磁珠复合物释放到10号孔，37℃孵育30分钟，孵育结束后，磁力棒将磁珠分别移动到11、12、13号孔，洗涤3次，最后磁珠释放至15号孔，测值。
运行程序2如下：
试剂条放置仪器的卡槽内，仪器分液装置装上吸头，分装洗液：分液器将3号孔的液体分别分装至11、12、13号孔，150微升/次，每孔各加入300微升洗液。14号孔待测样本与捕获抗体标记磁珠37℃孵育45分钟，每15分钟装有吸头的分液器吹吸混匀10次。孵育结束后，磁力棒装磁套，吸附14号孔抗原-捕获抗体磁珠复合物移至11号孔洗涤1次，分液器将7号孔标记抗体吸取150微升转移至10号孔，在11号孔洗涤后抗原-捕获抗体磁珠复合物的磁珠释放到10号孔。10号孔磁珠与标记抗体37℃孵育45分钟，每15分钟装有吸头的分液器吹吸混匀10次。孵育结束后，磁力棒将磁珠分别移动到11、12、13号孔，洗涤3次，最后磁珠释放至15号孔，测荧光素酶强度值。
对两种运行程序的检测效果进行了比较，程序1未设置样本与磁珠、磁珠复合物与标记抗体两步孵育的混匀过程，导致磁珠不能与待测蛋白充分结合，并且磁珠与样本孵育后没有洗涤过程，使得阴阳性样本的结果没有差异，也没有高特异的阳性结果，通过以阴性对照组的平均值加上3倍的标准差作为cutoff值，对检测结果计算每个浓度下S/C值(S/C＝实验组luc值/cutoff值)，S/C值结果如图5中的C所示，程序1阳性样本都为阴性。程序2增加了混匀和洗涤过程后，阳性样本(S/C值＞1)检测出阳性且有高特异的阳性结果。
4.确定捕获磁珠的最适体积
试剂条内的3号孔的洗液分别为PBST，14号孔内分别加入200微升10倍系列稀释灭活新冠病毒，阴性对照为稀释100倍的JEV灭活病毒和3％BSA-PBST，空白对照为lipsbuffer，再分别加入5微升、10微升和20微升MW06抗体标记的磁珠(10mg/ml)，放置仪器内，并运行运行程序2。
磁珠的使用量对检测效果同样有显著影响，磁珠太少，将对样本中的病毒捕获少，若磁珠太多，洗涤不充分，会造成本底太高。标记磁珠的浓度为10mg/ml，对200ul样本，分别用5ul、10ul、20ul磁珠进行捕获，然后加入Nluc-chC25进行检测，以阴性对照组的平均值加上3倍的标准差作为cutoff值，对检测结果计算每个浓度下S/C值(S/C＝实验组1uc值/cutoff值)，结果如图5中的D所示。5ul磁珠表现出更高的S/C值，获得更好的检测效果。
5.新冠病毒全自动磁微粒系统检测
试剂条放置仪器的卡槽内，仪器分液装置装上吸头，分装洗液：分液器将3号孔的液体分别分装至11、12、13号孔，150微升/次，每孔各加入300微升洗液。14号孔待测样本与捕获抗体标记磁珠37℃孵育45分钟，每15分钟装有吸头的分液器吹吸混匀10次。孵育结束后，磁力棒装磁套，吸附14号孔抗原-捕获抗体磁珠复合物移至11号孔洗涤1次，分液器将7号孔标记抗体吸取150微升转移至10号孔，在11号孔洗涤后抗原-捕获抗体磁珠复合物的磁珠释放到10号孔。10号孔磁珠与标记抗体37℃孵育45分钟，每15分钟装有吸头的分液器吹吸混匀10次。孵育结束后，磁力棒将磁珠分别移动到11、12、13号孔，洗涤3次，最后磁珠释放至15号孔，测荧光素酶强度值。
捕获抗体标记磁珠为5ul MW06抗体标记的磁珠(10mg/ml)。待测样本为200ul，标记抗体为实施例1制备用protein A/G磁珠进行纯化后获得的Nluc-chC25抗体。底物为Nanoluc底物(Promega，N1110)。
可选地，步骤(A1)待测样本用量为200μl。步骤(A1)捕获抗体标记磁珠(10mg/ml)用量为5μl。步骤(A2)和步骤(A4)PBST为300μl。步骤(A3)标记抗体用量为150μl。
6.新冠病毒磁微粒化学发光检测方法
本实施例提供了一种新冠病毒检测方法，具体包括如下步骤。
(B1)混合阴性对照与捕获抗体标记磁珠37℃进行孵育45分钟获得阴性对照-捕获抗体磁珠复合物，为使抗体和病毒充分结合，每隔一段时间(例如15分钟)可对阴性对照与捕获抗体标记磁珠进行混匀(例如采用装有吸头的分液器吹吸混匀8-12次，具体可为10次)；
(B2)采用PBST洗涤阴性对照-捕获抗体磁珠复合物；
(B3)混合经步骤(B2)洗涤后的阴性对照-捕获抗体磁珠复合物和标记抗体37℃进行孵育45分钟获得标记抗体-阴性对照-捕获抗体磁珠复合物，为使充分结合，每隔一段时间(例如15分钟)可对阴性对照-捕获抗体磁珠复合物和标记抗体进行混匀(例如采用装有吸头的分液器吹吸混匀8-12次，具体可为10次)；
(B4)采用PBST洗涤标记抗体-阴性对照-捕获抗体磁珠复合物；
(B5)混合经步骤(B4)洗涤后的标记抗体-阴性对照-捕获抗体磁珠复合物和底物，测定荧光素酶强度值；
(B6)重复步骤(B1)-(B5)至少3次，根据(B5)获得的荧光素酶强度值计算cut off值，cut off值＝(B5)获得的荧光素酶强度值的平均值+3×标准差；
(A1)混合待测样本与捕获抗体标记磁珠37℃进行孵育45分钟获得抗原-捕获抗体磁珠复合物，为使充分结合，每隔一段时间(例如15分钟)可对待测样本与捕获抗体标记磁珠进行混匀(例如采用装有吸头的分液器吹吸混匀8-12次，具体可为10次)；
(A2)采用PBST洗涤抗原-捕获抗体磁珠复合物；
(A3)混合经步骤(A2)洗涤后的抗原-捕获抗体磁珠复合物和标记抗体37℃进行孵育45分钟获得标记抗体-抗原-捕获抗体磁珠复合物，为使充分结合，每隔一段时间(例如15分钟)可对抗原-捕获抗体磁珠复合物和标记抗体进行混匀(例如采用装有吸头的分液器吹吸混匀8-12次，具体可为10次)；
(A4)采用PBST洗涤标记抗体-抗原-捕获抗体磁珠复合物；
(A5)混合经步骤(A4)洗涤后的标记抗体-抗原-捕获抗体磁珠复合物和底物，测定荧光素酶强度值；
(A6)计算S/C值＝(A5)获得的荧光素酶强度值/cut off值，S/C值＞1，所述待测样本有或候选有新型冠状病毒，S/C值＜1，所述待测样本没有或候选没有新型冠状病毒。
阴性对照为3％BSA和无关病毒(即除新冠病毒以外的病毒，例如JEV病毒、风疹病毒、黄热病毒YF 17D、副流感病毒1型、呼吸道合胞病毒、副流感病毒2型、腺病毒3型、流感病毒H1N1亚型或流感病毒H3N2亚型)。标记抗体为实施例1制备用protein A/G磁珠进行纯化后获得的Nluc-chC25抗体。待测样本为新冠病毒感染者咽拭子临床样本。捕获抗体标记磁珠为“3.全自动磁微粒检测系统的检测程序的优化”中所制备的捕获抗体标记磁珠。底物为Nanoluc底物((Promega，N1110)。
可选地，步骤(A1)待测样本用量为200μl。步骤(A1)捕获抗体标记磁珠(10mg/ml)用量为5μl。步骤(A2)和步骤(A4)PBST为300μl。步骤(A3)标记抗体用量为150μμl。步骤(A4)可为洗涤3次。
可选地，步骤(B1)待测样本用量为200μl。步骤(B1)捕获抗体标记磁珠(10mg/ml)用量为5μl。步骤(B2)和步骤(B4)PBST为300μl。步骤(B3)标记抗体用量为150μμl。步骤(B4)可为洗涤3次。
7.新冠病毒磁微粒检测试剂盒
本实施提供了一种检测试剂盒，包括Nluc-chC25抗体、捕获抗体标记磁珠、底物和/或阴性对照。
实施例4、全自动磁微粒检测系统对不同新冠病毒株检测灵敏度及特异性
(1)灵敏度实验：
使用样本稀释液(含0.1BSA的PBS)将灭活新冠病毒普通株、南非株、印度株10倍系列稀释(从10^5pfu/ml到10pfu/ml)，全自动磁微粒检测系统进行检测，一组实验可以进行8个样本的检测。8个试剂条的14号孔中分别加入5微升MW06抗体偶联的磁珠(10mg/m1)和200微升待测样本，10倍系列稀释的新冠病毒毒株作为检测组(从10^5pfu/ml到10pfu/ml，共5个浓度)，灭活无关病毒(乙型脑炎病毒)，含3％BSA的PBS，样本稀释液作为阴性对照组。试剂条中其他孔中设置同实施例4中的5。运行程序同实施例4中的运行程序2。
以阴性对照组的平均值加上3倍的标准差作为cutoff值，通过计算每个待测样本的S/C值(S/C＝实验组luc值/cut off值)确定检测结果，以S/C值＞1作为阳性阈值，进行结果分析。
结果如图6中的A-1所示，可检测到的最低浓度为：原始株为104pfu/ml，南非株为104pfu/ml，印度株为103pfu/ml。
为了确定三株毒株的最低检出限，将原始株和印度株从104pfu/ml继续稀释，取5个浓度，分别为：104pfu/ml，5×103pfu/ml，2.5×103pfu/ml，1.25×103pfu/ml，103pfu/ml。，南非株从5×104pfu/ml浓度开始，稀释5个浓度，分别为：5×104pfu/ml，2.5×104pfu/ml，1.25×104pfu/ml，103pfu/ml，102pfu/ml，取采用上述方法进行检测，对检测结果计算每个浓度下S/C值(S/C＝实验组1uc值/cutoff值)。
结果如图6中的A-2、A-3和A-4所示，该检测体系对原始株的最低检出限为1.25×103pfu/ml、南非株的最低检出限为103pfu/ml，印度株的最低检出限103pfu/ml。
(2)特异性实验
取8种灭活病毒(风疹病毒(RV)，黄热病毒YF17D(YF-170)，副流感病毒1型(Pif-1)，呼吸道合胞病毒(RSV)，副流感病毒2型(Pif-2)，腺病毒3型(ADV3)，流感病毒H1N1亚型(H1N1)，流感病毒H3N2亚型(H3N2))(由本实验室保存)进行检测。200微升样本加上5微升MW06偶联的磁珠(10mg/ml)。阴性对照为100倍稀释的JEV病毒和3％BSA，空白对照为0.1％BSA-PBST，阳性对照为普通株BetaCoV/Beijing/IME-BJ05/2020strain。试剂条中其他孔中设置同实施例4中的5，检测程序同实施例4中的运行程序2。
以阴性对照组的平均值加上3倍的标准差作为cut off值，通过计算待测样本S/C值(S/C＝实验组luc值/cut off值)确定检测结果，以S/C值＞1作为阳性阈值，进行结果分析。
结果如图6中的B所示，S/C值均低于1，表明该体系特异性好，与其他病毒无交叉反应。
(3)比较实验
将Nluc-全自动磁微粒检测新冠病毒的灵敏性与以S蛋白作为检测靶标的新冠病毒ELISA检测试剂盒(北京万泰)进行比较。
待测样本为使用样本稀释液10倍系列稀释灭活病毒(普通株、南非株、印度株)(105pfu/ml-1pfu/ml，6个浓度)，阴性对照为稀释100倍的H3N2和3％BSA。
采用实施例4中“5.新冠病毒全自动磁微粒系统检测”检测待测样本和阴性对照。
采用新冠病毒ELISA检测试剂盒检测待测样本，具体为将待测样本对应的微孔板进行编号，设置0pfu/ml和空白对照各1孔，每孔先加入样本稀释液20μl，空白对照孔除外，分别在相应的孔中加入待测样本50μl，空白对照除外，轻轻振荡混匀，在0pfu/ml孔中加入样本稀释液50μl；置于37℃温育30min，洗涤5次；每孔加入酶标抗体试剂100μl，空白对照除外；37℃温育30min，洗涤5次；每孔加入显色剂A、B各50μl，轻轻振荡混匀，37℃避光显色15min；每孔加入终止液50μl，轻轻振荡混匀，10min内测定结果，设定酶标仪波长与450nm处，用空白孔调零点后测定各孔。
结果如下表所示，从结果可以看出普通株与南非株的磁微粒检测方法的最低检出限高于商用ELISA试剂盒，印度株两者结果相当，整体上Nluc-全自动磁微粒检测系统的灵敏性高于商用ELISA试剂盒。
表1多种方法灵敏度特异性比较
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
序列表
<110> 中国人民解放军军事科学院军事医学研究院
<120> 标记抗体与其相关生物材料及在新冠病毒检测中的应用
<130> GNCYZ220946
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 238
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asn Ile Val Leu Thr Gln Ser Pro Val Ser Leu Ala
20 25 30
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser
35 40 45
Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro
50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ile Ala Ser Asn Leu Glu Ser
65 70 75 80
Gly Val Pro Ala Arg Phe Ser Gly Gly Gly Ser Arg Thr Asp Phe Thr
85 90 95
Leu Thr Ile Asp Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110
Gln Gln Asn Tyr Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys Arg Ala Asp Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
130 135 140
Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
145 150 155 160
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
165 170 175
Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
180 185 190
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
195 200 205
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
210 215 220
Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 2
<211> 646
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Gly Trp Ser Cys Ile Met Phe Phe Leu Leu Ser Val Thr Ala Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Met Lys
20 25 30
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe
35 40 45
Ser Ser Tyr Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu
50 55 60
Glu Trp Ile Gly Gln Ile Phe Pro Gly Ser Gly Ser Ser Asn Tyr Asn
65 70 75 80
Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn
85 90 95
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr His Cys Ala Arg Trp Asp Gly Asn Leu Phe Tyr Tyr Ala Met Asp
115 120 125
Tyr Trp Gly Leu Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys
130 135 140
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
145 150 155 160
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
165 170 175
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
180 185 190
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
195 200 205
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
210 215 220
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
225 230 235 240
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
245 250 255
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
260 265 270
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
275 280 285
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
290 295 300
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
305 310 315 320
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
325 330 335
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
340 345 350
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
355 360 365
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
370 375 380
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
385 390 395 400
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
405 410 415
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
420 425 430
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
435 440 445
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
450 455 460
Ser Leu Ser Pro Gly Lys Glu Phe Gly Ser Ser Gly Val Phe Thr Leu
465 470 475 480
Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp
485 490 495
Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe Gln Asn Leu Gly
500 505 510
Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly Glu Asn Gly
515 520 525
Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Gly
530 535 540
Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val Val Tyr Pro Val
545 550 555 560
Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly Thr Leu Val Ile
565 570 575
Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly Arg Pro Tyr Glu
580 585 590
Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu
595 600 605
Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Asn Pro Asp Gly
610 615 620
Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly Trp Arg Leu
625 630 635 640
Cys Glu Arg Ile Leu Ala
645
<210> 3
<211> 810
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaactcct tctccacaag cgccttcggt ccagttgcct tctccctggg cctgctcctg 60
gtgttgcctg ctgccttccc tgccccaaag cttatggaga cagacacact cctgctatgg 120
gtgctgctgc tctgggttcc aggttccaca ggtaacattg tgctgaccca atctccagtt 180
tctttggctg tgtctctagg gcagagggcc accatatcct gcagagccag tgaaagtgtt 240
gatagttatg gcaatagttt tatgcactgg taccagcaga aaccaggaca gccacccaaa 300
ctcctcatct atattgcatc caacctagaa tctggggtcc ctgccaggtt cagtggcggt 360
gggtctagga cagacttcac cctcaccatt gatcctgtgg aggctgatga tgctgcaacc 420
tattactgtc agcaaaatta tgaggatccg tggacgttcg gtggaggcac caagctggaa 480
atcaaacggg ctgatgcggc gccatctgtc ttcatcttcc cgccatctga tgagcagttg 540
aaatctggta ccgctagcgt tgtgtgcctg ctgaataact tctatcccag agaggccaaa 600
gtacagtgga aggtggataa cgccctccaa tcgggtaact cccaggagag tgtcacagag 660
caggacagca aggacagcac ctacagcctc agcagcaccc tgacgctgag caaagcagac 720
tacgagaaac acaaagtcta cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc 780
acaaagagct tcaacagggg agagtgttag 810
<210> 4
<211> 2034
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgaactcct tctccacaag cgccttcggt ccagttgcct tctccctggg cctgctcctg 60
gtgttgcctg ctgccttccc tgccccaaag cttatgggat ggagctgtat catgttcttc 120
ctcctgtcag taactgcagg tgtccactcc caggttcagc tgcagcagtc tggagctgag 180
gtgatgaagc ctggggcctc ggtgaagata tcctgcaagg cttctggcta catattcagt 240
tcctactgga tagagtggat aaagcagagg cctggacatg gccttgagtg gattggacag 300
atttttcctg gaagtggtag tagtaactat aatgagaagt tcaagggcaa ggccacattc 360
actgcagata catcgtccaa cacagcctac atgcagctca gcagcctgac atctgaggac 420
tctgccgtct atcactgtgc aagatgggat ggtaacctct tttactatgc tatggactac 480
tggggtctag gaacctcagt caccgtctcc tcagctagca ccaagggccc atcggtcttc 540
cccctggcac cctcctccaa gagcacctct gggggcacag cggccctggg ctgcctggtc 600
aaggactact tccccgaacc ggtgacggtg tcgtggaact caggcgccct gaccagcggc 660
gtgcacacct tcccggctgt cctacagtcc tcaggactct actccctcag cagcgtggtg 720
accgtgccct ccagcagctt gggcacccag acctacatct gcaacgtgaa tcacaagccc 780
agcaacacca aggtggacaa gaaagttgag cccaaatctt gtgacaaaac tcacacatgc 840
ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa 900
cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 960
agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 1020
gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 1080
accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 1140
gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1200
caggtgtaca ccctgcctcc atctcgggat gagctgacca agaaccaggt cagcctgacc 1260
tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1320
ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1380
tatagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1440
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1500
aaagaattcg gctcgagcgg cgtcttcaca ctcgaagatt tcgttgggga ctggcgacag 1560
acagccggct acaacctgga ccaagtcctt gaacagggag gtgtgtccag tttgtttcag 1620
aatctcgggg tgtccgtaac tccgatccaa aggattgtcc tgagcggtga aaatgggctg 1680
aagatcgaca tccatgtcat catcccgtat gaaggtctga gcggcgacca aatgggccag 1740
atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc atcactttaa ggtgatcctg 1800
cactatggca cactggtaat cgacggggtt acgccgaaca tgatcgacta tttcggacgg 1860
ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca ctgtaacagg gaccctgtgg 1920
aacggcaaca aaattatcga cgagcgcctg atcaaccccg acggctccct gctgttccga 1980
gtaaccatca acggagtgac cggctggcgg ctgtgcgaac gcattctggc gtaa 2034
Claims (9)
1.抗体或荧光素酶标记抗体在如下任一中的应用，所述抗体或所述荧光素酶标记抗体的轻链可变区中的LCDR1、LCDR2和LCDR3的氨基酸序列依次如SEQ ID No.1中第27-36位，第54-56位以及第93-101位所示，所述抗体或所述荧光素酶标记抗体的重链可变区中的HCDR1、HCDR2和HCDR3的氨基酸序列依次如SEQ ID No.2中第25-48位，第50-57位以及第96-109位所示：
(1)制备检测新型冠状病毒产品；
(2)制备结合新型冠状病毒的S蛋白产品；
(3)检测新型冠状病毒；
(4)结合新型冠状病毒的S蛋白；
所述新型冠状病毒为南非株或印度株。
2.根据权利要求1所述的应用，其特征在于：
所述轻链可变区的氨基酸序列如SEQ ID No.1中第1-130位所示；
所述重链可变区的氨基酸序列如SEQ ID No.2中第1-120位所示；
所述荧光素酶的氨基酸序列如SEQ ID No.2中第477-646位所示。
3.根据权利要求1或2所述的应用，其特征在于：
所述抗体的轻链氨基酸序列如SEQ ID No.1所示；
所述抗体的重链氨基酸序列如SEQ ID No.2中第1-470位所示；
所述荧光素酶标记抗体的重链氨基酸序列如SEQ ID No.2所示。
4.一种用于检测新型冠状病毒的试剂盒，其特征在于：包括权利要求1中所述的抗体或荧光素酶标记抗体。
5.根据权利要求4所述的试剂盒，其特征在于：还包括捕获抗体标记磁珠、荧光素酶底物和/或没有新型冠状病毒的阴性对照。
6.一种新冠病毒全自动磁微粒系统，其特征在于：包括权利要求4所述的试剂盒和全自动磁微粒检测系统。
7.权利要求1-3中任一所述的荧光素酶标记抗体。
8.权利要求7所述的荧光素酶标记抗体的相关生物材料，其特征在于：所述相关生物材料为下述任一种：
c1)编码权利要求7中所述荧光素酶标记抗体的核酸分子；
c2)含有c1)所述核酸分子的表达盒；
c3)含有c1)所述核酸分子的重组载体、或含有c2)所述表达盒的重组载体；
c4)含有c1)所述核酸分子的重组微生物、或含有c2)所述表达盒的重组微生物、或含有c3)所述重组载体的重组微生物；
c5)含有c1)所述核酸分子的重组细胞、或含有c2)所述表达盒的重组细胞、或含有c3)所述重组载体的重组细胞。
9.权利要求8所述的相关生物材料在如下任一中的应用：
(1)制备检测新型冠状病毒产品；
(2)制备结合新型冠状病毒的S蛋白产品；
(3)检测新型冠状病毒；
(4)结合新型冠状病毒的S蛋白；
所述新型冠状病毒为南非株或印度株。
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