CN113929773A - 一种抗SARS-CoV-2 S1-RBD单克隆抗体及其应用 - Google Patents
一种抗SARS-CoV-2 S1-RBD单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明涉及一种抗SARS‑CoV‑2S1‑RBD单克隆抗体28D9,其重链为IgG1型,轻链为Kappa型,重链可变区的氨基酸序列如SEQ ID NO.1所示,轻链可变区的氨基酸序列如SEQ ID NO.2所示,该抗体具有天然构象表位,与SARS‑CoV‑2S1蛋白及SARS‑CoV‑2S1‑RBD均具有良好的反应原性,具有高特异性、高灵敏度、高效价等特性。本发明基于SARS‑CoV‑2S1蛋白抗原表位建立了一种双抗体夹心ELISA检测方法,可用于定量检测SARS‑CoV‑2灭活病毒及SARS‑CoV‑2S1蛋白,重复性好、精密度强、灵敏度高、显色背景低、线性良好。
Description
技术领域
本发明涉及生物医药领域,尤其涉及一种抗SARS-CoV-2 S1-RBD单克隆抗体及其应用。
背景技术
SARS-CoV-2是以前从未在人体中发现的冠状病毒新毒株,SARS-CoV-2 与另外两种密切相关的高致病性病毒SARS-CoV和MERS-CoV同属冠状病毒科β冠状病毒属。SARS-CoV-2可跨越物种屏障感染人类,可通过密切接触、呼吸道飞沫、高浓度气溶胶传播,引起以肺部病变为主的传染病,也可诱发包括神经系统和消化系统在内的全身性损伤,严重者可导致死亡。目前, SARS-CoV-2感染患者没有特定的治疗方法,早期诊断并及时管控是阻止疫情进一步播散和控制新感染线索的关键,因此,研发高灵敏度的检测技术和方法对SARS-CoV-2做出准确、快速的鉴定,对提高COVID-19的诊断和治疗效率、遏制其爆发流行起关键作用。
SARS-CoV-2的RNA基因组编码四种结构蛋白;刺突蛋白(S)、包膜蛋白(E)、膜蛋白(M)和核衣壳蛋白(N)。S蛋白是一种大型I型跨膜蛋白,包含两个亚基,S1和S2。S1主要包含一个受体结合域(receptor binding domain,RBD),负责识别细胞表面受体,即血管紧张素转换酶2 (ACE2)。S蛋白主要负责与ACE2的相互作用随后和病毒细胞膜融合,可通过RBD的基因重组或突变在不同宿主之间传递,导致较高的死亡率,在诱导中和抗体、T细胞应答以及保护性免疫中起着关键作用,是疫苗、治疗性抗体以及临床诊断的关键靶点。目前需要建立一个基于S蛋白抗原表位的检测方法,以实现SARS-CoV-2的快速检测。
目前已有多种SARS-CoV-2诊断技术,主要包括病毒分离培养、实时荧光RT-PCR、基因测序和胶体金技术等。但是,病毒分离、IFA及IMPA法操作繁琐耗费时间长、设备要求高、人为误差大,对操作者技术要求高; RT-PCR法人员操作不当、核酸提取失败、方法性能不佳等都会造成检测结果假阴性或假阳性,并且需要依靠昂贵的仪器,成本较高;基因测序所需时间较长,设备要求较高,不适于临床快速大批量诊断;免疫胶体金法的敏感度和特异性较低,假阳性率、假阴性率出现的概率高。因此,仍需要一种周期短、操作简便、灵敏度高、设备要求简单、能定性定量分析且可以实现大批量样本快速检测的SARS-CoV-2检测方法。
发明内容
为解决上述技术问题,本发明提供了一种抗SARS-CoV-2 S1-RBD单克隆抗体28D9,并应用此单克隆抗体建立了一种双抗体夹心ELISA定量检测方法,实现了SARS-CoV-2病毒或其S1蛋白的定量或定性检测。
本发明的一种抗SARS-CoV-2 S1-RBD单克隆抗体28D9,其特征在于,其重链可变区和轻链可变区的氨基酸序列为:
IgG1
EHGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVL QSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDV EVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPI EKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQ WNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHE GLHNHHTEKSLSHSPGLPLDETCAEAQDGELDGLWTTFTSSSALPGQGG YRAVA
Kappa
RRYRVCQPCLRKSLTGSVAVGQELLSHCTISTVEAEDVDFYYCMQQLEYPLTLGARTKLELKRADAAPTVSIFP。
本发明还提供一种抗SARS-CoV-2 S1蛋白单克隆抗体6B11,其重链可变区和轻链可变区的氨基酸序列为:
IgG2A
SSQVQAQQDCWELGLREISCKASGYSFTGYNMYWVKQSHRKSLEW IGYIDPYNGGTSYNQKSKGKATLTVDKSSSTAYMHLNSLTSEDSAIYNWC KK
Kappa
FTAILSIYMGEGHYVLQNQFKSKLHVLVPAEARIVSPALDLQGIQPIF WGPRQVQGAVDQGLIFIENQQEWRLKDLGIYFCLQVTHSRTRSEGDQAEI NAST
本发明的28D9可特异性识别SARS-CoV-2 S1的RBD区域,28D9和 6B11均可识别SARS-CoV-2 S1蛋白,具有高特异性、高灵敏度、高效价等特性,两种单克隆抗体分别识别SARS-CoV-2 S1蛋白上不同的表位,可以应用于检测SARS-CoV-2病毒、SARS-CoV-2病毒S1蛋白、SARS-CoV-2 S1-RBD免疫诊断试剂的开发。
经研究发现,28D9或与6B11联用在双抗体夹心ELISA交叉实验中显示出较优结果,因此本发明基于SARS-CoV-2 S1蛋白抗原表位建立了一种双抗体夹心ELISA检测方法,可用于定量检测SARS-CoV-2病毒及 SARS-CoV-2 S1蛋白。
上述的SARS-CoV-2病毒或其S1蛋白ELISA定量检测方法的具体步骤为:
(1)以碳酸盐缓冲液为包被液稀释包被抗体(28D9或6B11),加入酶标板中,包被后弃去包被液,洗涤液洗涤,拍干;
(2)向酶标板中加入封闭液,孵育,洗涤液洗涤,拍干;
(3)将待测样品加入,同时加入使用封闭液梯度稀释的S蛋白S1亚基标准品或灭活SARS-CoV-2病毒疫苗标准品,并以封闭液设置对照,贴上封板贴,于37℃温箱中孵育,洗涤液洗涤,拍干;
(4)加入使用封闭液稀释的酶标抗体(HRP-28D9),贴上封板贴,孵育,洗涤液洗涤,拍干;
(5)加入显色液进行显色,于室温避光反应;
(6)加入ELISA终止液终止显色反应,轻微振板混匀,使用酶标仪读数;
(7)根据除去本底的标准品OD450吸光度值做出的标准曲线或标准品对应的除去本底的OD450吸光度值即可获得样品中待测物的含量。
进一步地,在步骤(1)中,当待测样品为S1蛋白时,包被抗体为6B11,其浓度为4-7μg/ml,优选为5μg/ml;当待测样品为灭活SARS-CoV-2病毒疫苗时,包被抗体为28D9,其浓度为2-3μg/ml,优选为2μg/ml。
进一步地,在步骤(3)中,当待检测样本为S1蛋白时,S1蛋白标准品梯度稀释的浓度为100000、50000、25000、12500、6250、3125、1562.5、 781.25、390.625pg/ml;当待检测样本为灭活SARS-CoV-2病毒疫苗时,先将SARS-CoV-2病毒疫苗用20%柠檬酸钠室温解离30min后,离心弃去沉淀以去除疫苗中的铝佐剂,然后将其梯度稀释为5、10、20、40、80、160、320、640、1280倍。
进一步地,在步骤(4)中,当待检测样本为S1蛋白时,酶标抗体的浓度为3-6μg/ml,优选为4μg/ml;当待检测样本为灭活SARS-CoV-2病毒疫苗时,酶标抗体的浓度为4-6μg/ml,优选为5μg/ml。
进一步地,在步骤(6)中,酶标仪读值时是在OD450nm。
进一步地,在步骤(7)中,当待检测样本为S1蛋白时,以S1蛋白标准品梯度稀释的浓度为横坐标,以对应的OD450吸光度值去除本底后为纵坐标作出标准曲线,将待测样品吸光度值代入标准曲线即获得样品中待测物的含量;当待检测样本为灭活SARS-CoV-2病毒疫苗时,需使用已知浓度的标准灭活SARS-CoV-2病毒疫苗,以待检测的灭活SARS-CoV-2病毒疫苗梯度稀释的倍数取倒数为横坐标,以对应的OD450吸光度值去除本底后为纵坐标作出标准曲线,将已知浓度的标准灭活SARS-CoV-2病毒疫苗对应的 OD450吸光度值代入标准曲线即可计算样品中灭活SARS-CoV-2病毒的相对含量。
为了实现本发明的高灵敏度,发明人还对其进行了一系列优化,包括:采用单因素变量法,分别对包被条件、封闭时间、酶标抗体孵育时间、显色时间进行比较,以P/N值最大为判定标准,最终确定在4℃下包被12-20h、在37℃下封闭2-3h、酶标抗体在37℃下孵育40-50min、显色20-30min条件下能得到较优结果,优选地,4℃包被20h、37℃封闭2h、酶标抗体37℃孵育45min,室温显色25min为最佳反应条件。
借由上述方案,本发明至少具有以下优点:
(1)本发明成功得到一种可用于双抗体夹心ELISA的单克隆抗体 28D9,该抗体具有天然构象表位,具有效价高、灵敏度高、特异性高等特点,与SARS-CoV-2 S1蛋白及SARS-CoV-2 S1-RBD均具有良好的反应原性。
(2)本发明利用单克隆抗体建立了一种双抗体夹心ELISA定量检测方法,并对该方法技术参数进行了一系列改进优化,实现了SARS-CoV-2病毒或其S1蛋白抗原定量或定性检测,且重复性好、精密度强、灵敏度高、显色背景低、线性良好。根据本发明的双抗体夹心ELISA检测方法,检测S1 蛋白的灵敏度达到254.764pg/ml,线性区间为390.625pg/ml-100000pg/ml,标准曲线的R2>0.99;检测灭活SARS-CoV-2病毒,线性区间为稀释倍数 5-1280倍,标准曲线的R2>0.99,ELISA读数结果和灭活SARS-CoV-2病毒有很强的线性关系,可以用来定量或定性检测SARS-CoV-2病毒。
(3)本发明方法与目前常用的病毒分离、IFA及荧光定量PCR等方法相比,检测周期短、操作简便、灵敏度高、主观因素误差小、设备简单、可以快速实现大批量样品的检测分析。
(4)本发明的单克隆抗体对新冠病毒相关疾病的治疗具有潜在开发前景。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明。
图1为28D9与6B11联用得到的双抗体夹心通过ELISA检测方法检测 S1蛋白建立的标准曲线;
图2为双抗体夹心ELISA检测方法检测灭活SARS-CoV-2病毒疫苗建立的标准曲线。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1鼠抗SARS-CoV-2 S1蛋白单克隆抗体的制备
本发明的单克隆抗体以SARS-CoV-2 S1重组蛋白为免疫原,由免疫小鼠得到的杂交瘤细胞株分泌得到,以下简称为28D9和6B11。其中,单克隆抗体28D9重链可变区的氨基酸序列如SEQ ID NO.1所示,轻链可变区的氨基酸序列如SEQ ID NO.2所示;单克隆抗体6B11重链可变区的氨基酸序列如SEQ ID NO.3所示,轻链可变区的氨基酸序列如SEQ ID NO.4所示。具体步骤如下:
(1)选用8周龄且健康的雌性纯种Balb/c小鼠5只,使用SARS-CoV-2 S1重组蛋白(购自北京义翘神州科技股份有限公司)进行抗原免疫。具体的,50μg抗原加弗氏不完全佐剂皮下多点注射(1ml,0.2ml/点)免疫,初次免疫后,每隔两周进行一次加强免疫,共进行三次加强免疫。
(2)最后一次加强免疫后3天进行眼球采血,分离血清,并用ELISA 检测血清滴度,确定其血清效价决定最优的免疫小鼠。
(3)将骨髓瘤细胞(SP2/0细胞系)在10%胎牛血清的RPMI-1640培养液中培养至细胞对数生长期,为与小鼠脾细胞融合做准备。选取最优的免疫小鼠,采用断颈法处死小鼠,在生物安全柜中将脾脏取出后注入0.5ml无血清培养液,刺破脾膜反复挤压得到脾细胞,并通过200目细胞筛,以2000r/min 离心5min,并使用RPMI-1640培养液清洗3次。将得到脾细胞与骨髓瘤细胞(SP2/0细胞系)1:10混合,以2000r/min离心5min,使用RPMI-1640培养液清洗3次,在90s内逐滴滴入1ml预温的PEG,轻轻吹匀,静置90s后,首次在120s内缓慢加入1ml预温的RPMI-1640培养液,接下来在120s内缓慢加入4ml预温的RPMI-1640培养液,最后较快的加入10ml预温的 RPMI-1640培养液。细胞悬液以2000r/min离心5min,使用RPMI-1640培养液清洗2次后在10%胎牛血清的RPMI-1640培养液中培养。
(4)将步骤(3)得到的融合细胞在5%CO2培养箱中37℃过夜培养,加入HAT选择性培养基筛选,筛选出可以在HAT选择性培养基生长的杂交瘤细胞继续培养,培养一周后收集细胞培养上清,通过ELISA法筛选出能与SARS-CoV-2 S1蛋白反应的阳性杂交瘤细胞,将初筛得到的阳性杂交瘤转到96孔板中继续培养,生长2天后,收集所有孔的上清,排除标签蛋白 (His-tag)杂交瘤细胞。
(5)通过有限稀释法进行亚克隆,得到单克隆杂交瘤细胞铺在96孔板, ELISA检测每个孔上清针对免疫抗原的反应,取OD值较高的孔进入下轮亚克隆,直至孔中细胞株阳性率100%,此时得到4株能稳定分泌SARS-CoV-2 S1蛋白抗体单克隆细胞株;
(6)将最后获得的4株单克隆细胞株,收集上清,用ProteinA纯化(白英生物科技有限公司提供抗体纯化服务),并用于后续检测;
(7)对获得的4株单克隆抗体(26D8、28D9、30G10、6B11)分别进行 SARS-CoV-2 S1重组蛋白、SARS-CoV-2 S1-RBD重组蛋白(购自北京义翘神州科技股份有限公司)ELISA验证,确定有效抗体并且进一步分析抗体的作用表位。分别将SARS-CoV-2 S1重组蛋白(带有His标签)、SARS-CoV-2 S1-RBD重组蛋白(带有His标签)包被在96孔酶标板,4℃孵育过夜20h;洗涤后,封闭液37℃封闭2h。洗涤后,待检测抗体(梯度稀释)37℃孵育 2h,同设置对照。洗涤后,加入兔抗鼠IgG-HRP 37℃孵育2h。洗涤后, TMB室温显色反应20min,酶标仪OD450读数。检测结果分别如下表1-1、表1-2所示。
表1-1 SARS-CoV-2 S1重组蛋白ELISA亲和力检测结果
抗体浓度 | 26D8 | 28D9 | 30G10 | 6B11 |
10 | 3.257 | 3.741 | 3.177 | 3.353 |
2.5 | 2.253 | 3.533 | 2.277 | 2.217 |
0.625 | 0.888 | 3.542 | 1.184 | 1.086 |
0.15625 | 0.283 | 3.352 | 0.39 | 0.356 |
0.0390625 | 0.104 | 3.072 | 0.122 | 0.12 |
0.009766 | 0.057 | 1.889 | 0.063 | 0.06 |
0.002441 | 0.051 | 0.659 | 0.044 | 0.043 |
0.00061 | 0.044 | 0.219 | 0.044 | 0.041 |
0.000153 | 0.044 | 0.075 | 0.04 | 0.043 |
3.81E-05 | 0.04 | 0.048 | 0.041 | 0.044 |
9.54E-06 | 0.037 | 0.04 | 0.04 | 0.04 |
2.38E-06 | 0.04 | 0.04 | 0.041 | 0.041 |
EC50 | 1.736 | 0.0091 | 1.475 | 2.169 |
表1-2 SARS-CoV-2 S1-RBD重组蛋白ELISA亲和力检测结果
抗体浓度 | 26D8 | 28D9 | 30G10 | 6B11 |
10 | 0.242 | 2.868 | 0.044 | 0.044 |
2.5 | 0.08 | 2.884 | 0.042 | 0.038 |
0.625 | 0.052 | 2.57 | 0.041 | 0.04 |
0.15625 | 0.045 | 2.22 | 0.046 | 0.043 |
0.0390625 | 0.046 | 2.026 | 0.042 | 0.043 |
0.009766 | 0.043 | 1.16 | 0.042 | 0.042 |
0.002441 | 0.044 | 0.466 | 0.041 | 0.043 |
0.00061 | 0.047 | 0.158 | 0.045 | 0.042 |
0.000153 | 0.044 | 0.066 | 0.044 | 0.043 |
3.81E-05 | 0.042 | 0.048 | 0.043 | 0.041 |
9.54E-06 | 0.045 | 0.044 | 0.044 | 0.043 |
2.38E-06 | 0.043 | 0.042 | 0.041 | 0.041 |
EC50 | 2847 | 0.0167 | ~0.000 |
表1-1的SARS-CoV-2 S1重组蛋白ELISA亲和力验证显示,抗体26D8、 28D9、30G10、6B11都可识别S1重组蛋白,EC50分别为1.736、0.0091、 1.475、2.169。表1-2的SARS-CoV-2S1-RBD重组蛋白ELISA亲和力验证显示,只有28D9可识别S1-RBD重组蛋白,EC50为0.0167。由此可见,抗体26D8、28D9、30G10、6B11具有高特异性、高灵敏度、高效价等特性,抗体26D8、28D9、30G10、6B11都可用于SARS-CoV-2 S1重组蛋白诊断试剂的研发,特别是其中的抗体28D9既可用于SARS-CoV-2 S1蛋白诊断试剂的研发,也可用于SARS-CoV-2 S1-RBD诊断试剂的研发。
实施例2双抗夹心ELISA法检测
为了获得适合定量检测SARS-CoV-2病毒或其S1蛋白的抗体对,进行如下双抗体夹心ELISA交叉实验,获取纯化的单克隆抗体26D8、28D9、 30G10、6B11,并将这4种抗体分别标记HRP(白英生物科技有限公司提供标记HRP服务),得到4种酶标单克隆抗体HRP-26D8、HRP-28D9、 HRP-30G10、HRP-6B11。将单克隆抗体26D8、28D9、30G10、6B11分别包被在96孔酶标板,4℃孵育过夜20h;洗涤后,封闭液37℃封闭2h。洗涤后,加入待检测SARS-CoV-2 S1重组蛋白(封闭液稀释为20000pg/ml) 或SARS-CoV-2灭活病毒疫苗(中国医学科学院医学生物学研究所赠送,封闭液稀释20倍),以封闭液为对照,37℃孵育2h。洗涤后,分别加入酶标抗体HRP-26D8、HRP-28D9、HRP-30G10、HRP-6B11两两配对,37℃孵育 45min。洗涤后,TMB室温显色反应25min,酶标仪OD450读数,并减去各自的本底,选择OD读数信号最强的配对为最佳配对。检测SARS-CoV-2 S1重组蛋白减去本底的OD450结果如表2-1所示,检测SARS-CoV-2灭活病毒疫苗减去本底的OD450结果如表2-2所示。
表2-1检测SARS-CoV-2 S1重组蛋白减去本底的OD450结果
HRP-26D8 | HRP-28D9 | HRP-30G10 | HRP-6B11 | |
26D8 | 0.0017 | 0.1318 | 0.01605 | 0.0246 |
28D9 | 0.00216 | 0.00031 | 0.01 | 0.02155 |
30G10 | 0.02165 | 0.0376 | 0.01723 | 0.01835 |
6B11 | 0.00045 | 0.3003 | 0.00025 | 0.00019 |
表2-2检测SARS-CoV-2灭活病毒疫苗减去本底的OD450结果
HRP-26D8 | HRP-28D9 | HRP-30G10 | HRP-6B11 | |
26D8 | 0.064 | 0.009 | 0.00025 | 0.0175 |
28D9 | 0.0625 | 0.3335 | 0.0055 | 0.00205 |
30G10 | 0.106 | 0.282 | 0.012 | 0.0017 |
6B11 | 0.1135 | 0.1735 | 0.0105 | 0.006 |
通过表2-1的检测SARS-CoV-2 S1重组蛋白双抗体夹心ELISA实验显示,6B11与HRP-28D9为最佳抗体对,在检测SARS-CoV-2 S1蛋白时有较高的信号强度,可用于建立检测SARS-CoV-2 S1蛋白的双抗体夹心ELISA 定量检测法。通过表2-2的检测SARS-CoV-2灭活病毒双抗体夹心ELISA 实验显示,28D9与HRP-28D9为最佳抗体对,在检测SARS-CoV-2灭活病毒时有较高的信号强度,可用于建立检测SARS-CoV-2灭活病毒的双抗体夹心ELISA定量检测法。
实施例3
本发明的目的之一是将本发明提供的抗体获取的最佳抗体对应用于双抗体夹心ELISA,该方法可以定量检测SARS-CoV-2病毒或其S1蛋白,为了实现本发明的高灵敏度,对一系列实验进行了优化,技术方案如下:
(1)最佳抗体稀释浓度的确定
固定待测物浓度(当待测样品为S1蛋白时,浓度为25000pg/ml;当待测样品为灭活SARS-CoV-2病毒疫苗时,稀释倍数为30倍),将包被抗体 (当待测样品为S1蛋白时,包被抗体为6B11;当待测样品为灭活 SARS-CoV-2病毒疫苗时,包被抗体为28D9)和酶标抗体(HRP-28D9)分别设置不同浓度(1、2、3、4、5、6、7、8μg/ml)进行方阵ELISA实验,同时设置对照,以其中P/N值最大孔为最佳抗体稀释浓度。检测SARS-CoV-2 S1重组蛋白P/N值结果如表3-1所示,检测SARS-CoV-2灭活病毒疫苗P/N 值结果如表3-2所示。
表3-1检测SARS-CoV-2 S1重组蛋白P/N值结果
表3-2检测SARS-CoV-2灭活病毒疫苗P/N值结果
表3-1结果表明,当待测样品为S1蛋白时,包被抗体6B11浓度为 4-7μg/ml、酶标抗体浓度为3-6μg/ml时P/N值结果均较优,其中,包被抗体 6B11浓度为5μg/ml、酶标抗体浓度为4μg/ml时P/N值最大,为最佳抗体稀释浓度。表3-2结果表明,当待测样品为灭活SARS-CoV-2病毒疫苗时,包被抗体浓度为2-3μg/ml、酶标抗体浓度为4-6μg/ml时P/N值结果均较优,其中,包被抗体浓度为2μg/ml、酶标抗体浓度为5μg/ml时P/N值最大,为最佳抗体稀释浓度。
(2)孵育时间的优化
采用单因素变量法,分别对包被时间、封闭时间、酶标抗体孵育时间、显色时间进行比较优化,以P/N值最大为判定标准。
I、包被时间的优化
用50mM碳酸盐缓冲液将抗体6B11稀释为5μg/ml,包被在96孔酶标板,分别4℃包被12h、4℃包被16h、4℃包被20h、4℃包被16h、37℃包被2h、37℃包被3h;洗涤后,用封闭液(购自百英生物科技有限公司), 37℃封闭2h。洗涤后,加入用封闭液稀释到25000pg/ml的待检测 SARS-CoV-2 S1重组蛋白,以对应的封闭液为对照,37℃孵育2h。洗涤后,将酶标抗体HRP-28D9稀释为4μg/ml后加入,37℃孵育45min。洗涤后,TMB室温显色反应25min,酶标仪OD450读数,计算P/N值,选取P/N值最大的包被时间为最佳包被时间,结果如表4-1所示。
表4-1包被条件及其P/N值
包被条件 | P/N值 |
4℃,12h | 7.011846447 |
4℃,16h | 7.073066878 |
4℃,20h | 7.163672865 |
4℃,24h | 7.083735865 |
37℃,2h | 5.274201743 |
37℃,3h | 5.345481743 |
根据表4-1结果显示,可以发现在4℃包被12-20h的ELISA实验结果P/N值均较优,其中,4℃包被20h ELISA实验结果P/N值最高,所以采用 4℃包被20h作为最优包被条件。
II、封闭时间的优化
用50mM碳酸盐缓冲液将抗体6B11稀释为5μg/ml,包被在96孔酶标板,4℃包被20h;洗涤后,用封闭液(购自百英生物科技有限公司),分别37℃封闭1h、37℃封闭1.5h、37℃封闭2h、37℃封闭2.5h、37℃封闭3h;洗涤后,加入用封闭液稀释到25000pg/ml的待检测SARS-CoV-2 S1重组蛋白,以封闭液为对照,37℃孵育2h。洗涤后,将酶标抗体HRP-28D9稀释为 4μg/ml后加入,37℃孵育45min。洗涤后,TMB室温显色反应25min,酶标仪OD450读数,计算P/N值,选取P/N值最大的封闭时间为最佳封闭时间,结果如表4-2所示。
表4-2封闭条件及其P/N值
封闭条件 | P/N值 |
37℃,1h | 6.883816865 |
37℃,1.5h | 4.351672865 |
37℃,2h | 7.163672865 |
37℃,2.5h | 7.083735865 |
37℃,3h | 7.155016865 |
根据表4-2结果显示,可以发现在37℃下封闭2-3h的ELISA实验结果 P/N值均较优,其中,37℃封闭2h ELISA实验结果P/N值最高,所以采用 37℃封闭2h作为最优封闭条件。
III、酶标抗体孵育时间的优化
用50mM碳酸盐缓冲液将抗体6B11稀释为5μg/ml,包被在96孔酶标板,4℃包被20h;洗涤后,用封闭液(购自百英生物科技有限公司),37℃封闭2h;洗涤后,加入用封闭液稀释到25000pg/ml的待检测SARS-CoV-2 S1 重组蛋白,以封闭液为对照,37℃孵育2h。洗涤后,将酶标抗体HRP-28D9 稀释为4μg/ml后加入,分别37℃孵育30min、37℃孵育35min、37℃孵育 40min、37℃孵育45min、37℃孵育50min、37℃孵育55min、37℃孵育60min。洗涤后,TMB室温显色反应25min,酶标仪OD450读数,计算P/N值,选取P/N值最大的酶标抗体孵育时间为最佳酶标抗体孵育时间,结果如表4-3 所示。
表4-3酶标抗体孵育条件及其P/N值
酶标抗体孵育时间 | P/N值 |
37℃,30min | 6.464399942 |
37℃,35min | 6.335299942 |
37℃,40min | 7.055641865 |
37℃,45min | 7.146851865 |
37℃,50min | 7.017751865 |
37℃,55min | 6.946471865 |
37℃,60min | 6.534141865 |
根据表4-3结果显示,可以发现酶标抗体在37℃下孵育40-50min的 ELISA实验结果P/N值均较优,其中,37℃孵育45min条件下,ELISA实验结果P/N值最高,所以采用37℃孵育45min作为最优酶标抗体孵育时间。
IV、显色时间的优化
用50mM碳酸盐缓冲液将抗体6B11稀释为5μg/ml,包被在96孔酶标板,4℃包被20h;洗涤后,用封闭液(购自百英生物科技有限公司),37℃封闭2h;洗涤后,加入用封闭液稀释到25000pg/ml的待检测SARS-CoV-2 S1 重组蛋白,封闭液为对照,37℃孵育2h。洗涤后,将酶标抗体HRP-28D9稀释为4μg/ml后加入,37℃孵育45min;洗涤后,TMB室温分别显色反应10min、 15min、20min、25min、30min,酶标仪OD450读数,计算P/N值,选取P/N 值最大的显色时间为最佳显色时间,结果如表4-4所示。
表4-4显色时间及其P/N值
显色时间 | P/N值 |
10min | 3.747483117 |
15min | 4.668693117 |
20min | 6.486854565 |
25min | 7.199664565 |
30min | 6.486854565 |
根据表4-4结果显示,可以发现显色20-30min的ELISA实验结果P/N 值均较优,其中,室温显色25min条件下,ELISA实验结果P/N值最高,所以采用室温显色25min作为最优显色时间。
根据表4-1、4-2、4-3、4-4结果,最终确定4℃包被20小时、37℃封闭 2小时、酶标抗体孵育时间为37℃孵育45min、室温显色25min为此双抗体夹心ELISA检测方法的最佳反应条件。
实施例4双抗体夹心ELISA检测方法检测SARS-CoV-2 S1蛋白
(1)以50mM碳酸盐缓冲液为包被液稀释包被抗体(6B11,浓度为5 μg/ml),加入酶标板中,每孔100μL,于4℃中放置20小时过夜,弃去包被液,洗涤液洗涤3次,拍干;
(2)每孔加入200μL封闭液,于37℃温箱中孵育2小时,洗涤液洗涤 3次,拍干;
(3)将待测样品加入,同时加入使用封闭液梯度稀释的S1蛋白(梯度稀释的浓度分别为100000、50000、25000、12500、6250、3125、1562.5、 781.25、390.625pg/ml),并以封闭液设置对照,每孔100μL,贴上封板贴,于37℃温箱中孵育2小时,洗涤液洗涤3次,拍干;
(4)加入使用封闭液稀释的酶标抗体(HRP-28D9,浓度为4μg/ml),每孔100μL,贴上封板贴,于37℃温箱中孵育45分钟,洗涤液洗涤3次,拍干;
(5)加入北京梅科万德TMB显色液进行显色,每孔100μL,贴上封板贴,于室温避光反应25分钟;
(6)加入北京梅科万德ELISA终止液终止显色反应,每孔50μL,轻微振板混匀,使用酶标仪OD450读数;
(7)以S1蛋白标准品梯度稀释的浓度为横坐标,以对应的OD450吸光度值去除本底后为纵坐标作出标准曲线,将待测样品吸光度值代入标准曲线即获得样品中待测物的含量。
上述双抗体夹心ELISA检测方法检测S1蛋白建立的标准曲线如图1所示,标准曲线回归方程为Y=1.699e-0.05*X-0.004328。
实施例5双抗体夹心ELISA检测方法检测SARS-CoV-2灭活病毒
(1)以50mM碳酸盐缓冲液为包被液稀释包被抗体(28D9,浓度为2 μg/ml),加入酶标板中,每孔100μL,于4℃中放置20小时过夜,弃去包被液,洗涤液洗涤3次,拍干;
(2)每孔加入200μL封闭液,于37℃温箱中孵育2小时,洗涤液洗涤 3次,拍干;
(3)将待测样品加入,同时加入使用封闭液梯度稀释的灭活 SARS-CoV-2病毒疫苗标准品(先将SARS-CoV-2病毒疫苗标准品用20%柠檬酸钠室温解离30min后,离心弃去沉淀以去除疫苗中的铝佐剂,然后将其梯度稀释为5、10、20、40、80、160、320、640、1280倍),并以封闭液设置对照,每孔100μL,贴上封板贴,于37℃温箱中孵育2小时,洗涤液洗涤3次,拍干;
(4)加入使用封闭液稀释的酶标抗体(HRP-28D9,浓度为5μg/ml),每孔100μL,贴上封板贴,于37℃温箱中孵育45分钟,洗涤液洗涤3次,拍干;
(5)加入北京梅科万德TMB显色液进行显色,每孔100μL,贴上封板贴,于室温避光反应25分钟;
(6)加入北京梅科万德ELISA终止液终止显色反应,每孔50μL,轻微振板混匀,使用酶标仪OD450读数;
(7)使用已知浓度的标准灭活SARS-CoV-2病毒疫苗,以待检测的灭活SARS-CoV-2病毒疫苗梯度稀释的倍数取倒数为横坐标,以对应的OD450 吸光度值去除本底后为纵坐标作出标准曲线,将已知浓度的标准灭活 SARS-CoV-2病毒疫苗对应的OD450吸光度值代入标准曲线即可计算样品中灭活SARS-CoV-2病毒的相对含量。
上述双抗体夹心ELISA检测方法检测灭活SARS-CoV-2病毒疫苗建立的标准曲线如图2所示,标准曲线回归方程为Y=5.760*X+0.01638。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
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<110> 国际遗传工程和生物技术中心泰州区域研究中心
<120> 一种抗SARS-CoV-2 S1-RBD单克隆抗体及其应用
<141> 2021-09-15
<160> 4
<170> SIPOSequenceListing 1.0
<210> 3
<211> 350
<212> PRT
<213> SARS-CoV-2
<400> 3
Glu His Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys
1 5 10 15
Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser
20 25 30
Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
35 40 45
Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp
50 55 60
Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr
65 70 75 80
Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys
85 90 95
Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys
100 105 110
Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val
115 120 125
Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe
130 135 140
Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu
145 150 155 160
Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His
165 170 175
Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala
180 185 190
Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg
195 200 205
Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met
210 215 220
Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro
225 230 235 240
Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn
245 250 255
Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val
260 265 270
Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr
275 280 285
Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu
290 295 300
Lys Ser Leu Ser His Ser Pro Gly Leu Pro Leu Asp Glu Thr Cys Ala
305 310 315 320
Glu Ala Gln Asp Gly Glu Leu Asp Gly Leu Trp Thr Thr Phe Thr Ser
325 330 335
Ser Ser Ala Leu Pro Gly Gln Gly Gly Tyr Arg Ala Val Ala
340 345 350
<210> 4
<211> 74
<212> PRT
<213> SARS-CoV-2
<400> 4
Arg Arg Tyr Arg Val Cys Gln Pro Cys Leu Arg Lys Ser Leu Thr Gly
1 5 10 15
Ser Val Ala Val Gly Gln Glu Leu Leu Ser His Cys Thr Ile Ser Thr
20 25 30
Val Glu Ala Glu Asp Val Asp Phe Tyr Tyr Cys Met Gln Gln Leu Glu
35 40 45
Tyr Pro Leu Thr Leu Gly Ala Arg Thr Lys Leu Glu Leu Lys Arg Ala
50 55 60
Asp Ala Ala Pro Thr Val Ser Ile Phe Pro
65 70
<210> 3
<211> 97
<212> PRT
<213> SARS-CoV-2
<400> 3
Ser Ser Gln Val Gln Ala Gln Gln Asp Cys Trp Glu Leu Gly Leu Arg
1 5 10 15
Glu Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Asn Met
20 25 30
Tyr Trp Val Lys Gln Ser His Arg Lys Ser Leu Glu Trp Ile Gly Tyr
35 40 45
Ile Asp Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Ser Lys Gly
50 55 60
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met His
65 70 75 80
Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Asn Trp Cys Lys
85 90 95
Lys
<210> 4
<211> 102
<212> PRT
<213> SARS-CoV-2
<400> 4
Phe Thr Ala Ile Leu Ser Ile Tyr Met Gly Glu Gly His Tyr Val Leu
1 5 10 15
Gln Asn Gln Phe Lys Ser Lys Leu His Val Leu Val Pro Ala Glu Ala
20 25 30
Arg Ile Val Ser Pro Ala Leu Asp Leu Gln Gly Ile Gln Pro Ile Phe
35 40 45
Trp Gly Pro Arg Gln Val Gln Gly Ala Val Asp Gln Gly Leu Ile Phe
50 55 60
Ile Glu Asn Gln Gln Glu Trp Arg Leu Lys Asp Leu Gly Ile Tyr Phe
65 70 75 80
Cys Leu Gln Val Thr His Ser Arg Thr Arg Ser Glu Gly Asp Gln Ala
85 90 95
Glu Ile Asn Ala Ser Thr
100
Claims (10)
1.一种抗SARS-CoV-2S1-RBD单克隆抗体28D9,其特征在于,所述抗SARS-CoV-2S1-RBD单克隆抗体28D9的重链为IgG1型,轻链为Kappa型,其重链可变区的氨基酸序列如SEQ IDNO.1所示,轻链可变区的氨基酸序列如SEQ ID NO.2所示。
2.权利要求1所述的抗SARS-CoV-2S1-RBD单克隆抗体28D9在SARS-CoV-2检测中的应用。
3.根据权利要求2所述的应用,其特征在于:所述抗SARS-CoV-2S1-RBD单克隆抗体28D9用于双抗夹心ELISA法检测SARS-CoV-2病毒。
4.根据权利要求2所述的应用,其特征在于:所述抗SARS-CoV-2S1-RBD单克隆抗体28D9与抗SARS-CoV-2S1蛋白单克隆抗体6B11联用于双抗夹心ELISA法检测SARS-CoV-2S1蛋白,其中,包被抗体为抗SARS-CoV-2S1蛋白单克隆抗体6B11;
所述抗SARS-CoV-2S1蛋白单克隆抗体6B11的重链为IgG2A型,轻链为Kappa型,其重链可变区的氨基酸序列如SEQ ID NO.3所示,轻链可变区的氨基酸序列如SEQ ID NO.4所示。
5.根据权利要求3或4所述的应用,其特征在于,双抗夹心ELISA法定量检测包括以下步骤:
(1)稀释包被抗体,包被,封闭;
(2)加入待测样品,同时加入梯度稀释的标准品,其中,检测SARS-CoV-2病毒时加入灭活SARS-CoV-2病毒疫苗标准品,检测SARS-CoV-2S1蛋白时加入S蛋白S1亚基标准品;
(3)加入稀释后的酶标抗体,孵育;
(4)进行显色反应,反应终止后得到OD450吸光度值,建立OD450吸光度值与标准品浓度的标准曲线,根据待测样品的OD450吸光度值计算出待测样品中待测物的含量。
6.根据权利要求5所述的应用,其特征在于:检测SARS-CoV-2病毒时,包被抗体的浓度为2-3μg/ml,酶标抗体的浓度为4-6μg/ml。
7.根据权利要求5所述的应用,其特征在于:检测SARS-CoV-2S1蛋白时,包被抗体的浓度为4-7μg/ml,酶标抗体的浓度为3-6μg/ml。
8.根据权利要求5所述的应用,其特征在于:在步骤(1)中,在4℃下包被12-20h,在37℃下封闭2-3h。
9.根据权利要求5所述的应用,其特征在于:在步骤(3)中,酶标抗体在37℃下孵育40-50min。
10.根据权利要求5所述的应用,其特征在于:在步骤(4)中,显色20-30min。
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