CN112251414A - 一种杂交瘤细胞株、其制备方法和应用 - Google Patents
一种杂交瘤细胞株、其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种杂交瘤细胞株、其制备方法和应用。所述杂交瘤细胞株的制备方法包括：以人SARS‑CoV‑2 S重组蛋白为免疫原免疫BALB/c小鼠，之后取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合，其后筛选出能够分泌新冠病毒表面S抗原单克隆抗体(简称“单抗”)的杂交瘤细胞。本发明还提供了利用所述杂交瘤细胞生产所述单抗的方法，所述单抗能够特异性结合新冠病毒表面S抗原，特异性强，效价高，有望实现新冠病毒的快速、敏感、准确、简便的检测，并能显著提高检测速度、检测灵敏度，大幅简化检测程序，而无需复杂漫长的PCR扩增过程和设备，尤其适合大量临床样本的快速筛查和即时检测，且所述单抗的制备方法产量高、稳定性好，生产成本低。
Description
技术领域
本发明具体涉及一种杂交瘤细胞株、其制备方法和应用，例如其在生产新冠病毒表面刺突糖蛋白S单克隆抗体中的用途，属于生物技术领域。
背景技术
新冠病毒属于传染性很强的RNA病毒，目前，病毒感染已经全球化，成为世界范围内高度重视的公共健康问题。为了更加有效的对新冠病毒感染进行控制，必须对新冠病毒进行快速、准确、有效的诊断。
目前常用的新冠病毒检测方法有核酸检测法和抗体检测法等。核酸检测法的优点是操作简便，快速，缺陷在于：核酸检测咽拭子采样有采集不到的风险，必要时需要重复采样；早期感染患者、咽喉部病毒滴度低的患者无法适用，容易假阴性；采样的工作人员容易暴露，风险大；需要增加提取核酸的操作，不熟练的技术人员容易提取失败造成假阴性，或者与他人的样本发生污染，造成假阳性。在疫情发生的早期，核酸检测法是唯一的选择。但是由于核酸法的局限性，只能将疑似患者和密切接触者进行隔离，而无法对他们进行直接检测(患病早期核酸法检测假阴性极高)。抗体检测方法的优点是灵敏度高，结果可靠，但抗体主要存在于体液循环系统中，取样和样本处理较繁琐，检测有滞后性，需要免疫细胞呈递抗原产生抗体，中间的时间窗口是很多致命性病毒大展宏图的好时机。
抗原检测法因具有采样准确(血液样本，不存在采集失败)、对早期感染者也能检测、测试速度快(胶体金试纸十几分钟到ELISA约一个小时)、方便快捷，弹性大，灵敏度高等优点，也有望应用于新冠病毒检测。实施抗原检测法的前提是需要获得特异性强的抗体，这正是业界亟待解决的难题。
发明内容
本发明的主要目的在于提供一种杂交瘤细胞株、其制备方法和应用，以克服现有技术中的不足。
为实现前述发明目的，本发明采用的技术方案包括：
本发明实施例提供了一种杂交瘤细胞株的制备方法，其包括：以人SARS-CoV-2S重组蛋白为免疫原免疫BALB/c小鼠，之后取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合，其后筛选出能够分泌新冠病毒表面S抗原单克隆抗体的阳性融合细胞，即为所述杂交瘤细胞。
在一些实施方式中，所述的制备方法具体包括：
以纯化的人SARS-CoV-2S重组蛋白为免疫原免疫8-12周龄BALB/c雌性小鼠，初次用弗氏完全佐剂与免疫原进行乳化，经皮下和腹腔多点免疫，剂量为100-150μg免疫原/只；隔两周加强免疫一次，共三次，佐剂为弗氏不完全佐剂，剂量为100-120μg免疫原/只/次；采用间接ELISA法检测小鼠尾血效价＞1∶104，融合前三天进行腹腔注射加强免疫100-120μg免疫原/只；
以及，取免疫后小鼠的脾细胞与骨髓瘤细胞SP2/0按照3-5∶1的比例混合后，以1000-1200rpm的转速离心10-15min，之后以RPMI-1640培养基重悬细胞，再次以1000-1200rpm的转速离心10-15min，并将沉淀细胞松散均匀且于37℃预热，在60s内加入预热的PEG1500，然后缓慢滴加RPMI1640培养基，再次以1000-1200rpm的转速离心10-15min，其后以HAT培养基重悬细胞，并置于37℃，5％CO2培养箱内培养。
在一些实施方式中，所述的制备方法具体包括：至少采用流式单细胞分选方法或有限稀释法对融合细胞进行筛选。
在一些实施方式中，所述的制备方法具体包括：采用Elisa法进行活性筛选，从而获得能够分泌新冠病毒表面S抗原单克隆抗体的阳性融合细胞。
在一些实施方式中，所述人SARS-CoV-2S重组蛋白的序列如SEQ ID NO：1所示。
本发明实施例还提供了由前述方法制备的杂交瘤细胞株。
本发明实施例还提供了一种新冠病毒表面刺突糖蛋白单克隆抗体，其由所述杂交瘤细胞株分泌。
本发明实施例还提供了一种新冠病毒表面刺突糖蛋白单克隆抗体，其包括单克隆抗体1S和/或单克隆抗体2S；所述单克隆抗体1S的重链VH CDR1、VH CDR2、VH CDR3序列分别如SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4所示，轻链VL CDR1、VL CDR2、VL CDR3序列分别如SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7所示；所述单克隆抗体2S的重链VH CDR1、VHCDR2、VH CDR3序列分别如SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10所示，轻链VL CDR1、VLCDR2、VL CDR3序列分别如SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13所示。
本发明实施例还提供了一种新冠病毒表面刺突糖蛋白单克隆抗体的制备方法，其包括：利用所述的杂交瘤细胞株，通过小鼠腹水法制备新冠病毒表面刺突糖蛋白单克隆抗体。
本发明实施例还提供了所述新冠病毒表面刺突糖蛋白单克隆抗体在制备检测新冠病毒检测试剂或试剂盒中的应用。
本发明实施例还提供了一种新冠病毒检测试剂，其包含所述新冠病毒表面刺突糖蛋白单克隆抗体。
本发明实施例还提供了一种新冠病毒检测试纸条，其包含所述新冠病毒表面刺突糖蛋白单克隆抗体。
本发明实施例还提供了一种新冠病毒检测试剂盒，其包含所述新冠病毒表面刺突糖蛋白单克隆抗体。
较之现有技术，本发明实施例提供的技术方案至少具有如下优点：
(1)成功构建了能够稳定持续分泌新冠病毒表面刺突糖蛋白单克隆抗体的杂交瘤细胞株；
(2)提供的新冠病毒表面刺突糖蛋白单克隆抗体能够特异性结合新冠病毒表面S抗原，特异性强，效价高，有望实现新冠病毒的快速、敏感、准确而又简便的检测，以显著提高检测速度，有效提高检测灵敏度，大幅简化检测程序，而无需复杂漫长的PCR扩增过程和设备，尤其适合大量临床样本的快速筛查和即时检测；
(3)提供的新冠病毒表面刺突糖蛋白单克隆抗体有助于大幅度提高新冠病毒诊断试剂盒的灵敏度和特异性，并可以在3分钟给出准确的检测结果，利于促进新冠病毒快速检测试纸的产业化，可用于诊断与治疗参考、流行病学调查、康复者血浆治疗重症患者时抗体含量测定以及有疫苗后的免疫评估等；
(4)提供的新冠病毒表面刺突糖蛋白单克隆抗体可以通过腹水法等方法制备，产量高、稳定性好，生产成本低，且所获新冠病毒表面刺突糖蛋白单克隆抗体经纯化后效价高，特异性强，亲和力高，还具备特异的抗体可变区序列，抗体轻重链的CDR1/2/3序列。
附图说明
为了更清楚地说明本发明实施方式的技术方案，下面将对实施方式中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1是本发明实施例中一种新冠病毒表面刺突糖蛋白单克隆抗体的制备工艺原理图；
图2是本发明实施例中新冠病毒表面刺突糖蛋白单克隆抗体1S、2S在纯化前后的考马斯亮蓝染色图；
图3a是本发明实施例中新冠病毒表面刺突糖蛋白单克隆抗体1S在不同稀释浓度下与S蛋白结合力的ELISA检测图；
图3b是本发明实施例中新冠病毒表面刺突糖蛋白单克隆抗体2S在不同稀释浓度下与S蛋白结合力的ELISA检测图；
图4是本发明实施例中新冠病毒表面刺突糖蛋白单克隆抗体1S、2S在稀释浓度1∶5000下WB检测S蛋白的图谱。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中所用试剂和原料均市售可得，而其中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。又及，除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORYMANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989and Thirdedition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Thirdedition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODSIN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
参阅图1所示，本实施例提供的一种新冠病毒表面刺突糖蛋白单克隆抗体(以下简称单抗)的制备方法包括如下步骤：
S1：动物免疫
以纯化的人SARS-CoV-2S重组蛋白(序列如SEQ ID NO：1所示，购自novoproteinCatalog#：DRA35)为免疫原，常规方法为免疫8-12周龄BALB/c雌性小鼠，初次用弗氏完全佐剂与抗原进行乳化，经皮下和腹腔多点免疫，剂量为100ug/只；隔两周加强免疫一次，共三次，佐剂为弗氏不完全佐剂，剂量为100ug/只/次；采用间接ELISA法检测小鼠尾血效价＞1∶104，融合前三天进行腹腔注射加强免疫100ug/只人SARS-CoV-2S抗原(即，前述S重组蛋白)。
S2：细胞融合
取免疫后小鼠的脾细胞与骨髓瘤细胞SP2/0按照5∶1的比例混合，1000rpm，离心10min，弃上清；用RPMI1640培养基重悬细胞，1000rpm，离心10min，弃上清；用吸管吸出残留液体，轻击管底，使沉淀细胞松散均匀，置于37℃水浴中预热，在60s内加入预热的PEG15001ml，然后缓慢滴加RPMI1640培养基至20ml，1000rpm，离心10min，弃上清。用HAT培养基重悬细胞，并置于37℃℃，5％CO2培养箱内培养。
S3：细胞筛选及单克隆化
采用流式单细胞分选方法获得单细胞于96孔板，待单细胞扩增培养至合适密度后转移到大皿，收集上清置于4℃冰箱。
S4：采用间接ELISA法筛选与人SARS-CoV-2S重组蛋白特异性结合的阳性细胞
(1)抗原酶标板的制备：
用磷酸盐缓冲液将人SARS-CoV-2S重组蛋白稀释至2ug/ml，分别加入至96孔酶标板，100ul/well，4℃包被过夜；用PBST(10mM pH7.4PBS+0.05％Tween-80)洗涤2次，甩干后，用封闭液(10mM pH7.4PBS+3％BSA)37℃封闭2h后备用。
(2)细胞上清以及抗体ELISA检测
分别取待筛选的细胞上清100ul，加入至96孔已制备的抗原酶标板，37℃孵育1h，用PBST洗板6次。进一步，在每孔中加入1∶5000稀释的羊抗鼠-HRP 100ul，37℃孵育1h，用PBST洗板6次.最后加入底物TMB，100μl/well，避光显色10min，加入2M硫酸50μl/well中止反应，于酶标仪中测定450nm的吸光值。
(3)细胞冻存
细胞冻存液：90％胎牛血清+10％DMSO，将ELISA检测阳性杂交瘤细胞离心，重悬于预冷的细胞冻存液中，细胞密度为107cells/ml，1ml/支，分装至冻存管中，将冻存管放入程序降温盒中置于-80℃冰箱，次日放入液氮罐长期保存。
S5：杂交瘤细胞株制备出的能分泌新冠表面抗原特异性抗体的制备
(1)抗人SARS-CoV-2S重组蛋白单克隆抗体制备与纯化，包括：通过制备小鼠腹水法获得抗人SARS-CoV-2S重组蛋白的单克隆抗体，具体步骤为：
首先将8-12周龄BALB/c雌性小鼠致敏，将保存的杂交瘤细胞株注射入提前一周注射弗氏不完全佐剂的小鼠腹腔进行腹水生产(腹腔注射液体石蜡，0.5ml/只)；1周后腹腔接种上述获得的杂交瘤细胞(需经无血清培养基洗涤至少一次)，接种量106cells/mouse，待小鼠腹部可见明显膨胀，反复抽取腹腔腹水，4000rpm，离心30min，收集上清，分装，于-20℃备用。
将收集的腹水用滤纸、玻璃纤维与无纺布各两层进行过滤，再将得到的单抗腹水通过ProteinA亲和层析法进行纯化，具体为：
将过滤后腹水用A液(20mM pbs和0.3M NaCl 1∶1 pH值＝7.5)稀释；清洗proteinAbeads3000rpm离心3min 3次，1ml腹水加入1ml proteinAbeads；腹水与proteinAbeads共孵育2h；将混合物上吸附柱，用A液洗涤，并且收集清洗液，注射器加压清洗5次；之后用B液(0.1M甘氨酸-HCl缓冲液pH值＝4)进行洗脱，接样后立即用2M Tris-HCl缓冲液调节PH＝7.5，加入NaN3终浓度为万分之一；将得到的腹水定量、聚丙烯酰胺电泳、考马斯亮蓝染色、ELISAWB测活性功能验证；用20％无水乙醇保存纯化柱。
(2)抗体亲和力检测
利用BIAcore T-200生物分子相互作用仪(GE生命科学公司)进行抗体的亲和力测定，在测定S蛋白(刺突糖蛋白，spike glycoprotein)与其相应单域抗体间相互作用时，首先将S蛋白耦连于传感芯片上，然后以抗体作为流动相进行结合常数、解离常数与亲和力常数的测定。具体为：将S蛋白固定于CM5芯片，S蛋白耦连温度为25℃，缓冲液为HBS-T(10mMHEPES，150mM NaCl，3mM EDTA and 0.05％Tween20，pH值7.4)，程序模板为Immuobilization，选择CM5芯片通道2氨基偶联，配体为5μg/ml S蛋白，蛋白缓冲体系为pH5.5醋酸钠(NaAC5.5)，目标偶联量100RU，洗脱液为50mM NaOH。芯片激活剂50mmol/L的N-羟基丁二酰亚胺(NHS)与200mmol/L的3-(3-二甲基氨基丙基)卡二亚胺盐酸盐(EDC)激活芯片，封闭剂为1mol/L盐酸乙醇胺。结合时间180s，流速10μ l/min，解离时间为300s，再生洗脱液为Glycine-HCl PH＝2.5，再生液结合时间30s，流速30μl/min，稳定时间0s，抗体浓度系列稀释(3.125、6.25、12.5、25、50nM)。最后所得数据用Biacore Evaluation Software软件分析，计算结合常数(ka)，解离常数(kd)及亲和力常数(KD)。上述Biacore分析中所用芯片、试剂及缓冲液，均购自GE生命科学公司。KD值越低说明亲和力越强。请参阅图3a-图3b示出了本发明实施例所获新冠病毒表面刺突糖蛋白单克隆抗体1S、2S在不同稀释浓度下与S蛋白结合力的ELISA检测结果。图3a、图3b中横坐标所示1～8对应的稀释浓度如下：
进一步的，前述新冠病毒表面刺突糖蛋白单克隆抗体1S、2S与前述S蛋白相互作用的亲和力参数如下
| 抗体编号 | KD(M) | ka(1/Ms) | kd(1/s) |
| 1S | 2.123E-8 | 2.556E+5 | 0.006228 |
| 2S | 3.156E-8 | 1.999E+5 | 0.008123 |
(3)抗体CDR序列检测
将稳定分泌前述单抗的杂交瘤细胞培养至状态良好，取5x105cell提取RNA，用预冷PBS清洗细胞两次，加入1ml trizol于1.5ml EP管并将细胞吹匀，室温裂解10min。加入200ul氯仿，上下颠倒混匀10次后于4℃离心机，12000g 15min。离心后管内呈现3层，最上层透明液体为RNA裂解液，中间层为蛋白质，最下面一层为有机溶剂。用200ul小枪头小心吸取最上层约500ul于新1.5ml ep管内，加入等体积异丙醇后静置3min。7500g 10min，可见沉淀为核酸RNA，弃上清，可将离心管倒扣通风橱内。加入1ml70％乙醇，将核酸沉淀吹散后离心7500g、5min，弃上清，将乙醇于室温挥干后，加入50ul DEPC H2O溶解核酸。测定浓度后在抗体可变区引物条件下进行抗体重轻链的扩增后送测序后比对的抗体轻重链CDR sequence。测序结果显示，本发明实施例获得的新冠病毒表面刺突糖蛋白单克隆抗体包括单克隆抗体1S、2S；其中，单克隆抗体1S的重链VH CDR1、VH CDR2、VH CDR3序列分别如SEQ ID NO：2、SEQID NO：3、SEQ ID NO：4所示，轻链VL CDR1、VL CDR2、VL CDR3序列分别如SEQ ID NO：5、SEQID NO：6、SEQ ID NO：7所示；其中，单克隆抗体2S的重链VH CDR1、VH CDR2、VH CDR3序列分别如SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10所示，轻链VL CDR1、VL CDR2、VL CDR3序列分别如SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13所示。
本发明实施例首先提供了一种杂交瘤细胞株，以序列如SEQ ID NO：1所示的特异性重组S蛋白为待测物，采用Elisa法进行活性筛选得到阳性克隆。基于所述杂交瘤细胞产生的新冠病毒表面刺突糖蛋白单克隆抗体的类型为IgG，具有良好的特异性和较高的效价。经检测，其在稀释1∶64000仍能通过ELISA检测到信号，且效价能达到1∶64000，在WesternBlot检测中，所述单克隆抗体仅识别目标位置的Spike特异性条带，且在稀释5000倍后仍显示良好的特异性。
本发明实施例中，以酶标仪测定450nm处各个孔的吸光度(A450)，计算阳性血清与阴性血清的吸光度之比(P/N)，当P/N＜1.5时为阴性，当P/N≥1.5而＜2.1为可疑，当P/N≥2.1为阳性，将P/N≥2.1时所对应的抗体最高稀释倍数作为抗体的效价。
应当理解，以上所描述的实施例是本发明一部分实施例，而不是全部的实施例。本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
序列表
<110> 中国科学院苏州纳米技术与纳米仿生研究所
<120> 一种杂交瘤细胞株、其制备方法和应用
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 671
<212> PRT
<213> 人工序列(人工序列)
<400> 1
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Gln Leu Glu Gln Pro Tyr Val Phe Ile Lys Arg Ser Asp Ala Arg Thr
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Ala Pro His Gly His Val Met Val Glu Leu Val Ala Glu Leu Glu Gly
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Ile Gln Tyr Gly Arg Ser Gly Glu Thr Leu Gly Val Leu Val Pro His
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Val Gly Glu Ile Pro Val Ala Tyr Arg Lys Val Leu Leu Arg Lys Asn
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Gly Asn Lys Gly Ala Gly Gly His Ser Tyr Gly Ala Asp Leu Lys Ser
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Phe Asp Leu Gly Asp Glu Leu Gly Thr Asp Pro Tyr Glu Asp Phe Gln
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Glu Asn Trp Asn Thr Lys His Ser Ser Gly Val Thr Arg Glu Leu Met
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Arg Glu Leu Asn Gly Gly Ala Tyr Thr Arg Tyr Val Asp Asn Asn Phe
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Cys Gly Pro Asp Gly Tyr Pro Leu Glu Cys Ile Lys Asp Leu Leu Ala
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Arg Ala Gly Lys Ala Ser Cys Thr Leu Ser Glu Gln Leu Asp Phe Ile
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Asp Thr Lys Arg Gly Val Tyr Cys Cys Arg Glu His Glu His Glu Ile
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Ala Trp Tyr Thr Glu Arg Ser Glu Lys Ser Tyr Glu Leu Gln Thr Pro
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Phe Glu Ile Lys Leu Ala Lys Lys Phe Asp Ile Phe Asn Gly Glu Cys
245 250 255
Pro Asn Phe Val Phe Pro Leu Asn Ser Ile Ile Lys Thr Ile Gln Pro
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Arg Val Glu Lys Lys Lys Leu Asp Gly Phe Met Gly Arg Ile Arg Ser
275 280 285
Val Tyr Pro Val Ala Ser Pro Asn Glu Cys Asn Gln Met Cys Leu Ser
290 295 300
Thr Leu Met Lys Cys Asp His Cys Gly Glu Thr Ser Trp Gln Thr Gly
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325 330 335
Lys Glu Gly Ala Thr Thr Cys Gly Tyr Leu Pro Gln Asn Ala Val Val
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Lys Ile Tyr Cys Pro Ala Cys His Asn Ser Glu Val Gly Pro Glu His
355 360 365
Ser Leu Ala Glu Tyr His Asn Glu Ser Gly Leu Lys Thr Ile Leu Arg
370 375 380
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Gly Cys His Asn Lys Cys Ala Tyr Trp Val Pro Arg Ala Ser Ala Asn
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Asp Tyr Lys Ala Phe Lys Gln Ile Val Glu Ser Cys Gly Asn Phe Lys
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Val Thr Lys Gly Lys Ala Lys Lys Gly Ala Trp Asn Ile Gly Glu Gln
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Lys Ser Ile Leu Ser Pro Leu Tyr Ala Phe Ala Ser Glu Ala Ala Arg
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Val Val Arg Ser Ile Phe Ser Arg Thr Leu Glu Thr Ala Gln Asn Ser
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Val Arg Val Leu Gln Lys Ala Ala Ile Thr Ile Leu Asp Gly Ile Ser
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Gln Tyr Ser Leu Arg Leu Ile Asp Ala Met Met Phe Thr Ser Asp Leu
565 570 575
Ala Thr Asn Asn Leu Val Val Met Ala Tyr Ile Thr Gly Gly Val Val
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Gln Leu Thr Ser Gln Trp Leu Thr Asn Ile Phe Gly Thr Val Tyr Glu
595 600 605
Lys Leu Lys Pro Val Leu Asp Trp Leu Glu Glu Lys Phe Lys Glu Gly
610 615 620
Val Glu Phe Leu Arg Asp Gly Trp Glu Ile Val Lys Phe Ile Ser Thr
625 630 635 640
Cys Ala Cys Glu Ile Val Gly Gly Gln Ile Val Thr Cys Ala Lys Glu
645 650 655
Ile Lys Glu Ser Val Gln Thr Phe Phe Lys Leu Val Asn Lys Phe
660 665 670
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Claims (10)
1.一种杂交瘤细胞株的制备方法，其特征在于包括：以人SARS-CoV-2S重组蛋白为免疫原免疫BALB/c小鼠，之后取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合，其后筛选出能够分泌新冠病毒表面S抗原单克隆抗体的阳性融合细胞，即为所述杂交瘤细胞。
2.根据权利要求1所述的制备方法，其特征在于具体包括：
以纯化的人SARS-CoV-2S重组蛋白为免疫原免疫8-12周龄BALB/c雌性小鼠，初次用弗氏完全佐剂与免疫原进行乳化，经皮下和腹腔多点免疫，剂量为100-150μg免疫原/只；隔两周加强免疫一次，共三次，佐剂为弗氏不完全佐剂，剂量为100-120μg免疫原/只/次；采用间接ELISA法检测小鼠尾血效价＞1∶104，融合前三天进行腹腔注射加强免疫100-120μg免疫原/只；
以及，取免疫后小鼠的脾细胞与骨髓瘤细胞SP2/0按照3-5∶1的比例混合后，以1000-1200rpm的转速离心10-15min，之后以RPMI-1640培养基重悬细胞，再次以1000-1200rpm的转速离心10-15min，并将沉淀细胞松散均匀且于37℃预热，在60s内加入预热的PEG1500，然后缓慢滴加RPMI1640培养基，再次以1000-1200rpm的转速离心10-15min，其后以HAT培养基重悬细胞，并置于37℃，5％CO2培养箱内培养。
3.根据权利要求1所述的制备方法，其特征在于具体包括：至少采用流式单细胞分选方法或有限稀释法对融合细胞进行筛选；和/或，所述人SARS-CoV-2S重组蛋白的序列如SEQID NO：1所示。
4.根据权利要求1所述的制备方法，其特征在于具体包括：采用Elisa法进行活性筛选，从而获得能够分泌新冠病毒表面S抗原单克隆抗体的阳性融合细胞。
5.由权利要求1-4中任一项所述方法制备的杂交瘤细胞株。
6.一种新冠病毒表面刺突糖蛋白单克隆抗体，其特征在于：所述单克隆抗体由权利要求6所述杂交瘤细胞株分泌。
7.一种新冠病毒表面刺突糖蛋白单克隆抗体，其特征在于包括单克隆抗体1S和/或单克隆抗体2S；所述单克隆抗体1S的重链VH CDR1、VH CDR2、VH CDR3序列分别如SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4所示，轻链VL CDR1、VL CDR2、VL CDR3序列分别如SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7所示；所述单克隆抗体2S的重链VH CDR1、VH CDR2、VH CDR3序列分别如SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10所示，轻链VL CDR1、VL CDR2、VL CDR3序列分别如SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13所示。
8.一种新冠病毒表面刺突糖蛋白单克隆抗体的制备方法，其特征在于包括：利用权利要求5所述的杂交瘤细胞株，通过小鼠腹水法制备新冠病毒表面刺突糖蛋白单克隆抗体。
9.权利要求6或7所述新冠病毒表面刺突糖蛋白单克隆抗体在制备检测新冠病毒检测试剂或试剂盒中的应用。
10.一种新冠病毒检测试剂，其特征在于包含权利要求6或7所述新冠病毒表面刺突糖蛋白单克隆抗体。
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