CN114480298A - 一株分泌抗tigit单克隆抗体的杂交瘤细胞株及其应用 - Google Patents
一株分泌抗tigit单克隆抗体的杂交瘤细胞株及其应用 Download PDFInfo
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Abstract
本发明提供了一株分泌抗TIGIT单克隆抗体的杂交瘤细胞株及其应用,所述分泌抗TIGIT单克隆抗体的杂交瘤细胞株命名为鼠抗人TIGIT单克隆抗体杂交瘤细胞系MJ12‑30,保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.45102,保藏日期为2022年02月24日。所述杂交瘤细胞分泌的抗TIGIT单克隆抗体与TIGIT蛋白具有较高的亲和力。以抗TIGIT单克隆抗体构建的TIGIT检测试剂盒灵敏度高、特异性好,在包括酶联免疫吸附、蛋白免疫印迹、免疫组织化学、流式细胞术和免疫荧光等在内的多种检测中应用效果较好。
Description
技术领域
本发明属于免疫化学技术领域,涉及一株分泌抗TIGIT单克隆抗体的杂交瘤细胞株及其应用。
背景技术
TIGIT的全称为T细胞免疫球蛋白和ITIM结构域蛋白(T-cell immunoreceptorwith Ig and ITIM domains),又名VSIG9或VSTM3。TIGIT是一种优先在NK、CD8+T、CD4+T细胞以及调节性T细胞(Treg cell,简称“Treg”)上表达的免疫共抑制受体,属于免疫球蛋白超家族的成员。TIGIT是一种跨膜蛋白,具有细胞外Ig可变结构域、一个跨膜结构域和胞内区,所述胞内区含有免疫受体酪氨酸抑制基序(ITIM)和免疫球蛋白尾部酪氨酸(ITT)样基序。TIGIT主要表达于淋巴细胞中,特别是在效应CD4+T细胞、调节性CD4+T细胞、滤泡辅助CD4+T细胞、效应CD8+T细胞和自然杀伤细胞(NK细胞)中高表达,同时在黑色素瘤、乳腺癌、非小细胞肺癌、结肠腺癌、胃癌、急性髓细胞性白血病和多发性骨髓瘤组织中异常高表达。
目前TIGIT已知的配体有CD155、CD112/PVRL2和CD113/PVRL3。其中TIGIT与CD155/PVR的亲和力最强,其次是CD113/PVRL3和CD112/PVRL2。CD155是一种免疫球蛋白超家族黏附分子,主要在树突状细胞、T细胞、B细胞和巨噬细胞的表面表达,也在很多肿瘤细胞上高表达。肿瘤表面高表达的CD155与NK和T细胞表面的TIGIT结合,可以减弱DC细胞的抗原呈递功能,向T细胞和NK细胞内部传递抑制性信号,从而在一定程度上抑制对肿瘤细胞的杀伤作用。总而言之,TIGIT信号通路在肿瘤细胞逃脱免疫监视的过程中发挥了重要作用,当其在调节T细胞上表达时,可以通过增强信号通路来调节T细胞的抑制功能,从而抑制多种免疫细胞的活性。
临床结果表明,TIGIT和CD155在结直肠癌组织中的表达量明显高于癌旁组织,且TIGIT和CD155的表达与肿瘤大小、淋巴结转移、肿瘤分化及病理分期具有显著的相关性,随着分期增高,TIGIT和CD155的表达水平显著上调。由此推断,TIGIT和CD155的表达水平与结直肠癌的进展显著相关,说明TIGIT和CD155可以作为评估结直肠癌预后的生物学标志物。
CD155和PD-L1在接受治疗的头颈部鳞状细胞癌(HNSCC)患者的骨髓源性抑制细胞(MDSCs)中高表达,与肿瘤CD3呈负相关。单用抗PD-L1治疗可上调MDSCs中CD155的表达,而抗TIGIT治疗可上调MDSCs中PD-L1的表达。
联合阻断TIGIT/CD155和PD-1/PD-L1信号对小鼠肿瘤生长有明显的抑制作用,增强了效应T细胞和细胞因子分泌的百分率,诱导了免疫记忆效应。
TIGIT和PD-1的表达量与肿瘤浸润性CD8+T细胞呈显著正相关,可能是影响胃癌患者预后的危险因素。TIGIT在胃癌肿瘤微环境中高表达可以抑制肿瘤浸润性CD8+T细胞的功能,导致胃癌细胞逃避免疫杀伤。联合阻断TIGIT、PD-1和T细胞免疫球蛋白粘蛋白-3(Tim-3),可增强Wilms肿瘤蛋白-1(WT-1)特异性CD8+T细胞的生长、增殖和细胞因子的产生,以恢复肿瘤诱导的T细胞功能障碍,并促进晚期胃癌患者的抗肿瘤CD8+T细胞反应。
免疫组织化学(Immunohistochemistry,IHC)是利用抗原-抗体高度特异性结合的特点来显示组织切片中抗原的分布和定位的原位检测技术。由于抗原与抗体的复合物是无色的,因此还必须借助组织化学的方法进行观察,如将酶偶联到抗体上,酶会催化底物在抗原位置产生有色沉淀,从而将抗原抗体结合的部位显示出来,以期达到对组织或细胞中的未知抗原进行定性、定位或定量的研究。为了提高免疫组织化学的检测效率和一致性,需要筛选出高特异性的单克隆抗体,且要求这些抗体在组化检测工作中的灵敏度、特异性和准确度等指标达到一定的标准,因此筛选出各项指标均较好的抗体对病理诊断具有重要的意义。
目前,市场上能够特异性并高灵敏度地检测TIGIT的抗体的数量极少,尤其是能够应用于免疫组织化学检测的抗TIGIT单克隆体。所以制备特异性强、灵敏度高、应用范围广的抗TIGIT单克隆抗体,具有重要的现实意义和应用价值。
发明内容
本发明的目的在于提供一株分泌抗TIGIT单克隆抗体的杂交瘤细胞株及其应用,所述杂交瘤细胞株分泌的抗TIGIT单克隆抗体与TIGIT蛋白具有较高的亲和力,能够特异性地识别TIGIT蛋白,在制备TIGIT检测试剂盒中具有重要的应用前景。以抗TIGIT单克隆抗体构建的TIGIT检测试剂盒灵敏度高、特异性好,在包括酶联免疫吸附、蛋白免疫印迹、免疫组织化学、流式细胞术和免疫荧光等在内的多种检测中应用效果较好。
为达到此发明目的,本发明采用以下技术方案:
第一方面,本发明提供一株分泌抗TIGIT单克隆抗体的杂交瘤细胞株,所述分泌抗TIGIT单克隆抗体的杂交瘤细胞株命名为鼠抗人TIGIT单克隆抗体杂交瘤细胞系MJ12-30,保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.45102,保藏日期为2022年02月24日。
第二方面,本发明提供一种抗TIGIT单克隆抗体,所述抗TIGIT单克隆抗体由第一方面所述的鼠抗人TIGIT单克隆抗体杂交瘤细胞系MJ12-30分泌。
本发明中,所述抗TIGIT单克隆抗体以TIGIT蛋白为免疫原免疫动物制备获得。
优选地,所述抗TIGIT单克隆抗体的亚型为IgG1型。
优选地,所述抗TIGIT单克隆抗体的重链包括:SEQ ID No.3所示的CDR3、SEQ IDNo.4所示的CDR1和SEQ ID No.5所示的CDR2。
SEQ ID No.3:A。
SEQ ID No.4:GFTFHSFG。
SEQ ID No.5:ISSGGSTI。
优选地,所述抗TIGIT单克隆抗体的轻链包括:SEQ ID No.6所示的CDR3、SEQ IDNo.7所示的CDR1和SEQ ID No.8所示的CDR2。
SEQ ID No.6:FQGNHVP。
SEQ ID No.7:QSILHSGGNTY。
SEQ ID No.8:KVS。
优选地,所述抗TIGIT单克隆抗体的重链可变区包括SEQ ID No.9所示的氨基酸序列。
优选地,所述抗TIGIT单克隆抗体的轻链可变区包括SEQ ID No.11所示的氨基酸序列。
优选地,所述抗TIGIT单克隆抗体的重链全长包括SEQ ID No.10所示的氨基酸序列。
优选地,所述抗TIGIT单克隆抗体的轻链全长包括SEQ ID No.12所示的氨基酸序列。
SEQ ID No.9:
EVKLEQSGGVLVQPGGSRKLSCAASGFTFHSFGMHWVRQAPEKGLEWVAYISSGGSTIFYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCAGSDHGNLGYWGQGTTLTVSSTKTTP。
SEQ ID No.10:
EVKLEQSGGVLVQPGGSRKLSCAASGFTFHSFGMHWVRQAPEKGLEWVAYISSGGSTIFYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCAGSDHGNLGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。
SEQ ID No.11:
DIVLTQSPLSLSVSLGDQASISCRSSQSILHSGGNTYLDWFLQRPGQSPKLLIYKVSNRISGVPDRFSGSGAGTDFTLKISRVEAEDLGVYYCFQGNHVPPTFGGGTKLEIKRADAA。
SEQ ID No.12:
DIVLTQSPLSLSVSLGDQASISCRSSQSILHSGGNTYLDWFLQRPGQSPKLLIYKVSNRISGVPDRFSGSGAGTDFTLKISRVEAEDLGVYYCFQGNHVPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。
本发明中,以人TIGIT重组蛋白作为免疫原免疫小鼠,取免疫后的小鼠脾细胞与小鼠的骨髓瘤细胞融合形成杂交瘤细胞,筛选能分泌高灵敏度和高特异性的单克隆抗体的杂交瘤细胞株。
本发明中所述抗TIGIT单克隆抗体的抗原为人源TIGIT蛋白,所述人源TIGIT蛋白包括SEQ ID No.1所示的氨基酸序列,所述人源TIGIT蛋白的氨基酸序列见UniprotGenBank ID:Q495A1,相应的蛋白胞外区位于第Met22-Pro141位氨基酸,并在蛋白的C末端,添加6个组氨酸组成的标签。
SEQ ID No.1:
MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPHHHHHH。
在本发明中,所述鼠抗人TIGIT单克隆抗体杂交瘤细胞系MJ12-30分泌的单克隆抗体命名为单克隆抗体MJ12-30,在实际应用中,可根据需要采用已知的抗体改造方案对单克隆抗体MJ12-30进行结构和序列上的改造,以获得需要的性质,对单克隆抗体MJ12-30的改造包括:
(1)标记抗体
可对抗体进行标记以便于检测,标记方法可以是荧光标记、化学发光标记、放射性标记、酶联标记、生物素/亲和素标记、磁珠标记或纳米颗粒标记等。
(2)结构改造
可对抗体结构进行改造,构建成抗原亲和性类似但结构不同的分子,如Fab、F(ab’)2、Fab’、Fab’-SH、Fv片段、单链抗体(如scFv)、单域抗体、抗体偶联物、双功能抗体或多特异性抗体等。其中Fab’片段是指在重链CH1结构域的羧基端添加少量氨基酸残基(包括一个或多个来自抗体铰链区的半胱氨酸)的Fab,Fab’-SH是指在恒定区的半胱氨酸残基带有自由硫醇基的Fab’。
优选地,所述抗TIGIT单克隆抗体还含有修饰缀合物,所述修饰缀合物包括辣根过氧化物酶(HPR)、碱性磷酸酶、生物素、异硫氰酸荧光素、Cy3或Cy5中的任意一种或至少两种的组合。
本发明中,所述抗TIGIT单克隆抗体应用于构建TIGIT检测试剂盒,或者直接应用于检测,所述抗TIGIT单克隆抗体可以修饰有缀合物或检测标记,例如荧光标记、酶标记、放射性标记、生物素标记或亲和素标记等。具体的缀合物或检测标记包括但不限于辣根过氧化物酶、碱性磷酸酶、生物素、异硫氰酸荧光素、Cy3或Cy5等,连接方式包括化学键偶联、静电吸附或亲疏水性吸附等。
第三方面,本发明提供一种第二方面所述的抗TIGIT单克隆抗体的制备方法,所述抗TIGIT单克隆抗体的制备方法包括:
培养第一方面所述的分泌抗TIGIT单克隆抗体的杂交瘤细胞株,纯化,获得所述抗TIGIT单克隆抗体。
本发明中,所述抗TIGIT单克隆抗体的制备方法包括以下步骤:
(1)应用真核表达平台,构建真核表达质粒,在293T细胞中瞬转表达,ELISA法证实蛋白能够表达,在293F细胞中扩大培养,瞬转表达,并纯化获得人源TIGIT蛋白。
(2)用得到的真核表达的人源TIGIT蛋白免疫小鼠,血清效价测定合格后,处理并获得免疫后的小鼠脾细胞,与小鼠骨髓瘤细胞SP2/0进行融合,经过培养、亚克隆分选和筛选后,获得杂交瘤细胞株,所述杂交瘤细胞株能分泌特异性识别TIGIT蛋白的单克隆抗体。
(3)培养杂交瘤细胞株,收集培养基的上清,并进行亲和纯化,获得单克隆抗体,ELISA测定单克隆抗体的活性。
第四方面,本发明提供一种第一方面所述的分泌抗TIGIT单克隆抗体的杂交瘤细胞株和/或第二方面所述的抗TIGIT单克隆抗体在制备TIGIT蛋白表达检测产品中的应用。
第五方面,本发明提供一种TIGIT免疫组织化学检测试剂盒,所述TIGIT免疫组织化学检测试剂盒包括第二方面所述的抗TIGIT单克隆抗体。
优选地,所述TIGIT免疫组织化学检测试剂盒还包括:免疫组织化学抗原修复缓冲液、封闭液、酶标二抗、显色剂或苏木素复染液中的任意一种或至少两种的组合。
本发明中,所述TIGIT免疫组织化学检测试剂盒中的抗TIGIT单克隆抗体与TIGIT蛋白具有高亲和力。
本发明中,在所述TIGIT免疫组织化学检测试剂盒中抗TIGIT单克隆抗体是指能够检测组织样本中TIGIT表达状态的抗体,其能够与TIGIT蛋白结合,在免疫组织化学实验中,抗TIGIT单克隆抗体作为一抗进行TIGIT表达状态的检测。
第六方面,本发明提供一种TIGIT免疫印迹检测试剂盒,所述TIGIT免疫印迹检测试剂盒包括第二方面所述的抗TIGIT单克隆抗体。
优选地,所述TIGIT免疫印迹检测试剂盒还包括:SDS-PAGE凝胶、聚偏二氟乙烯膜(PVDF膜)、封闭液或酶标二抗中的任意一种或至少两种组合。
第七方面,本发明提供一种TIGIT酶联免疫吸附检测试剂盒,所述TIGIT酶联免疫吸附检测试剂盒包括第二方面所述的抗TIGIT单克隆抗体。
优选地,所述TIGIT酶联免疫吸附检测试剂盒还包括:封闭液、酶标二抗、显色液或终止液中的任意一种或至少两种组合。
第八方面,本发明提供一种TIGIT免疫荧光检测试剂盒,所述TIGIT免疫荧光检测试剂盒包括第二方面所述的抗TIGIT单克隆抗体。
优选地,所述抗TIGIT单克隆抗体修饰有荧光基团,所述荧光基团为异硫氰酸荧光素、Cy3或Cy5中的任意一种。
第九方面,本发明提供一种TIGIT流式细胞术检测试剂盒,所述TIGIT流式细胞术检测试剂盒包括第二方面所述的抗TIGIT单克隆抗体。
发明中,在所述TIGIT流式细胞术检测试剂盒中抗TIGIT单克隆抗体是指能够检测表达TIGIT蛋白的细胞中TIGIT表达状态的抗体,抗TIGIT单克隆抗体能够与细胞膜上的TIGIT蛋白结合,在流式细胞术中,抗TIGIT单克隆抗体作为一抗进行表达TIGIT蛋白的细胞的检测。
相对于现有技术,本发明具有以下有益效果:
(1)本发明提供的抗TIGIT单克隆抗体能识别真核重组表达和原核重组表达的TIGIT蛋白,具有检测TIGIT蛋白的特性,具有广泛的用途。
(2)本发明提供的抗TIGIT单克隆抗体为IgG1亚型抗体,能够在免疫组化检测中特异性地识别肿瘤组织和正常组织中的TIGIT蛋白表达,且具有较高的亲和力。
(3)本发明提供的抗TIGIT单克隆抗体在免疫印迹检测、酶联免疫吸附检测和免疫组织化学检测中具有极高的灵敏度和特异性。
附图说明
图1为实施例1中人源TIGIT蛋白的SDS-PAGE凝胶电泳图,泳道1:蛋白Marker;泳道2:人TIGIT重组蛋白,还原型(R),上样量2 μg;泳道3:人TIGIT重组蛋白,非还原型(N),上样量2 μg。
图2为实施例3中杂交瘤细胞株产生的抗体与原核表达的TF-TIGIT蛋白间接ELISA法的检测结果图。
图3为实施例4中单克隆抗体MJ12-30的SDS-PAGE凝胶电泳图,泳道1:蛋白Marker;泳道2:牛血清白蛋白(BSA),上样量1 μg;泳道3:非还原状态下(N)的单克隆抗体MJ12-30,上样量1 μg;泳道4:还原状态下(R)的单克隆抗体MJ12-30,上样量1 μg。
图4为实施例8中商品化抗体E5Y1W的免疫组织化学检测结果图(放大倍数为100×)。
图5为实施例8中单克隆抗体MJ12-30的免疫组织化学检测结果图(放大倍数为100×)。
图6为实施例10中单克隆抗体MJ12-30与对照抗体E5Y1W的免疫印迹检测结果图。泳道1、泳道5和泳道9:蛋白Marker;泳道2和泳道6:真核表达的人TIGIT重组蛋白,上样量为0.2 μg;泳道3和泳道7:阴性对照BSA,上样量为0.2 μg;泳道4和泳道8:原核表达的TF-TIGIT蛋白,上样量为0.2 μg。
图7为实施例12中单克隆抗体MJ12-30的酶联免疫吸附检测结果图。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。
实施例中未标明具体技术或条件者,均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。所用试剂或未标注生产厂商者,均为可通过正规渠道采购获得的常规产品。
实施例1
本实施例提供一种人TIGIT重组蛋白,所述人TIGIT重组蛋白的制备过程如下:
在人TIGIT蛋白胞外区的C端添加6个组氨酸,在蛋白的N端添加适合于真核蛋白分泌的信号肽。利用同源重组的方法将目的基因与表达载体pcDNA3.4连接,构建表达克隆pcDNA3.4-TIGIT。
所述人源TIGIT蛋白的氨基酸序列如SEQ ID No.1所示,所述人源TIGIT蛋白的氨基酸序列见Uniprot GenBank ID:Q495A1,相应的蛋白胞外区位于第Met22-Pro141位氨基酸。
SEQ ID No.1:
MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPHHHHHH。
将表达克隆pcDNA3.4-TIGIT的质粒DNA在293T细胞中瞬转表达,48 h后收集细胞上清,通过ELISA进行验证,ELISA结果表明目的蛋白有表达。然后扩大培养,提取质粒并测序,测序正确后,将构建好的真核表达pcDNA3.4-TIGIT质粒DNA瞬时转染293F细胞,具体方法参见Thermofisher公司的Expi293™表达系统试剂盒(货号:A14635)的操作指南。在293F细胞中进行蛋白表达,在96 h时收集样品,并用ELISA法检测目的蛋白是否表达,收获细胞培养物,10000 rpm离心20 min,用孔径为0.45 μm的一次性无菌滤器过滤液体,将获得的上清液进行纯化,获得人TIGIT重组蛋白。
通过ELISA的检测结果可知,目的蛋白成功表达,所述人TIGIT重组蛋白的理论分子量为13.9 kDa。
采用AKTA蛋白纯化仪,利用Ni柱(NI sepharose High performance,GE,货号:17-5268-01)进行亲和纯化,对纯化后的蛋白进行SDS-PAGE凝胶电泳,检验所获得的蛋白的大小及纯度。
SDS-PAGE凝胶电泳的结果如图1所示。泳道1:蛋白Marker;泳道2:人TIGIT重组蛋白,还原型(R),上样量2 μg;泳道3:人TIGIT重组蛋白,非还原型(N),上样量2 μg。由结果可知,所述人TIGIT重组蛋白的C端带有6个组氨酸标签,所以纯化后的蛋白的分子量比理论分子量偏大,经ELISA确认表达获得的蛋白为人TIGIT重组蛋白,纯度>95%,满足实验要求。
实施例2
本实施例提供一种人TIGIT重组蛋白,所述人TIGIT重组蛋白的制备过程如下:
通过同源重组的方法将人TIGIT蛋白胞外区序列与表达载体pColdTMTF (品牌:Takara,Code No.3365)连接,构建表达克隆pCold TF-TIGIT,形成完整的TF-TIGIT融合蛋白,完整的融合蛋白氨基酸序列如SEQ ID No.2所示。
SEQ ID No.2:
MNHKVHHHHHHMQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQASAGLEVLFQGPSAGLVPRGSGGIEGRHMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP。
构建原核表达质粒,测序正确后,转化BL21(DE3)株,并涂布氨苄青霉素平板,37℃倒置培养12 h。挑菌并摇菌培养,采用终浓度为0.1 mM的IPTG进行诱导,破碎菌体,进行SDS-PAGE电泳,确定蛋白是否表达以及分子量,经验证蛋白在菌体破碎后上清液中,呈可溶性表达,分子大小符合预期。
将菌株接种在含有氨苄青霉素的LB培养基中,37℃,200 rpm培养,待菌液OD值升高至0.5,利用0.1 mM的IPTG进行低温诱导表达,诱导表达的温度为16℃,时间为18 h,诱导表达结束后在10000 rpm离心收集菌体。利用超声破碎仪进行破菌处理,10000 rpm离心30min,用0.45 μm的滤器过滤破菌上清液,获得待纯化样品,利用Ni柱(Ni sepharose Highperformance,GE,货号17-5268-01)进行亲和层析纯化获得用于制备TIGIT抗体的人源抗原,并通过ELISA验证蛋白有活性。
实施例3
本实施例提供一种杂交瘤细胞株的制备方法,所述制备方法包括如下步骤:
(1)免疫小鼠
取6周龄的雌性Balb/c小鼠,从眼眶采血20 µL,离心获得血清作为阴性对照。将实施例1纯化得到的人TIGIT重组蛋白在小鼠腹部皮下多点注射免疫;首次免疫剂量为每只小鼠120 µg,用生理盐水稀释至体积为200 µL,与等体积的弗氏完全佐剂混合,充分乳化后注射。隔14天皮下加强免疫一次,剂量为每只小鼠注射人TIGIT重组蛋白60 µg,用生理盐水稀释至体积为200 µL,与等体积的弗氏不完全佐剂混合,充分乳化后注射。第3次加强免疫后7天眼眶取血,用ELISA检测血清抗体效价,对达到要求的小鼠进行冲击免疫,免疫剂量为每只小鼠注射人TIGIT重组蛋白60 µg,3天后进行融合。
(2)融合
在融合的前一天,取一只未免疫的小鼠,采用腹腔灌洗方法获得腹腔细胞作为饲养层细胞,铺在96孔板板底。
在融合的当天,取血清效价合格的免疫小鼠,脱颈处死,在75%乙醇浸泡5 min消毒,放入无菌操作台摘取脾脏,除去脂肪和结缔组织,然后用DMEM(品牌:Hyclone,货号:SH30243.01B)基础培养基洗涤脾脏3遍,将其放在预先加入10 mL DMEM基础培养基的细胞培养皿中,用1 mL注射器吸入培养基吹打脾脏,吹出脾细胞,再进行充分研磨吹打,最后用40 µm网筛过滤除去粘性蛋白获得脾细胞,将脾细胞与SP2/0细胞按4:1的比例混合,1000rpm室温离心5 min,弃去上清,拍打离心管底部使细胞团均匀覆盖于离心管底部,然后将离心管放入37℃温水浴中,一边轻轻晃动离心管,一边逐滴加入1 mL聚乙二醇溶液(Sigma,P7181-5×5 mL),在90 s内加完。聚乙二醇溶液滴加完毕后,将15 mL预热的DMEM培养基倒入离心管中终止反应,37℃水浴内静置10 min,然后1000 rpm离心5 min,弃去上清。用含25% FBS的HAT选择培养基重悬细胞,并按100 μL/孔接种于96孔细胞培养板,在37℃、5%CO2的培养箱中静置培养。融合3天后,对细胞进行半量换液,吸取培养液160 μL,补加1×HAT选择培养基160 μL,融合后第8天,检测细胞上清中的抗体。
(3)筛选
细胞融合后约8天,融合孔中出现明显细胞团,此时,取培养基上清,利用间接ELISA法筛选分泌抗TIGIT单克隆抗体的杂交瘤细胞株。对所得阳性克隆株,采用有限稀释法进行杂交瘤细胞株的亚克隆分选。
间接ELISA法的操作步骤如下:取TF-TIGIT蛋白稀释至4 μg/mL,每孔加入100 μL,包被96孔板,37℃静置1 h,然后用PBST洗涤4次,用5% BSA封闭。封闭结束后,再次用PBST洗涤4次,取100 µL培养基进行一抗孵育,用免疫后具有抗体效价的小鼠血清按1:5000稀释作为阳性对照,用未免疫的小鼠血清作为阴性对照,37℃孵育1 h,用PBST洗涤4次。然后每孔加辣根过氧化物酶HRP标记亲和纯化山羊抗鼠IgG(H+L)F(ab’)2片段(品牌:Jackson,货号:115-036-003,稀释比例为1:5000),37℃孵育1 h,最后用TMB显色,终止显色后测定450 nm的OD值。OD450数值大于0.5的判定为可以进行亚克隆分选的阳性克隆。
(4)杂交瘤细胞株的构建
经过3次亚克隆和间接ELISA筛选,共得到31株针对人TIGIT重组蛋白且稳定分泌单克隆抗体的杂交瘤细胞株,克隆号分别为MJ12-1~MJ12-31,其产生的抗体在使用间接法ELISA检测的结果如图2所示。
在实验条件完全相同的情况下,由图2的结果可知,其中7株抗体的效价最好,分别由杂交瘤细胞株MJ12-9、杂交瘤细胞株MJ12-10、杂交瘤细胞株MJ12-25、杂交瘤细胞株MJ12-26、杂交瘤细胞株MJ12-27、杂交瘤细胞株MJ12-30和杂交瘤细胞株MJ12-31所产生,所述抗体的克隆号依次为MJ12-9、MJ12-10、MJ12-25、MJ12-26、MJ12-27、MJ12-30和MJ12-31。
图2中,N为阴性对照,P为阳性对照。结合免疫印迹检测实验(Western Blot)及免疫组化检测实验的结果,其中效价最高、特异性最强、用途最广泛的杂交瘤细胞株的克隆号为MJ12-30。
实施例4
本实施例提供一种抗TIGIT单克隆抗体MJ12-30,所述抗TIGIT单克隆抗体MJ12-30由实施例3所述的鼠抗人TIGIT单克隆抗体杂交瘤细胞系MJ12-30分泌,后续简称单克隆抗体MJ12-30。
(1)单克隆抗体腹水的制备与纯化
提前一周,将无菌石蜡油注射到小鼠体内使其致敏。取对数生长期的克隆号为MJ12-30的杂交瘤细胞,使用无血清培养基洗涤2遍,并用无菌PBS缓冲液重悬,重悬后调整细胞浓度为5×105个/mL,并确保细胞活率达到95%,再对致敏的小鼠进行腹腔注射。14天后利用注射器收集腹水,将取出的腹水于4℃、4000 rpm离心10 min。小心吸出腹水上清,转移至新的离心管中,4℃保存。
用Hi Trap rProtein G FF(GE Healthcare,货号:17061802)亲和层析填料及AKTA系统,参照说明书从腹水的上清中纯化单克隆抗体。纯化使用的平衡液为20 mM、pH7.0的磷酸盐缓冲液,洗脱液为100 mM、pH 2.7的甘氨酸,中和液为1 M、pH 8.5的Tris-HCl。
纯化的具体步骤为:首先取1 mL的纯化柱,用10 mL纯化水冲洗柱子,接着用10 mL平衡液冲洗柱子;进行样品上样;用10 mL平衡液冲洗柱子,用10 mL洗脱液进行抗体洗脱,并立即加入中和液,使其pH达到7.0,所有步骤的液体的流速均为1 mL/min。
用SDS-PAGE凝胶电泳鉴定纯度,并用Nanodrop测定浓度。SDS-PAGE凝胶电泳的结果如图3所示,从图3中可知,纯化后的单克隆抗体MJ12-30的条带大小位置正确(理论分子量150 kDa,由两条相同的重链和两条相同的轻链组成,重链轻链之间通过二硫键连接),纯化后的单克隆抗体MJ12-30的纯度>95%。其中,图3中,泳道1:蛋白Marker;泳道2:牛血清白蛋白(BSA),上样量1 μg;泳道3:非还原状态下(N)的单克隆抗体MJ12-30,上样量1 μg;泳道4:还原状态下(R)的单克隆抗体MJ12-30,上样量1 μg。
实施例5
本实施例通过ELISA法对实施例4中所述的单克隆抗体MJ12-30的亚型进行鉴定。
(1)单克隆抗体亚型鉴定
用100 mM PBS(pH 7.4)稀释包被所用的羊抗鼠IgG(杭州索莱尔博奥生物技术有限公司,SL200101)至1 µg/mL,每孔加入100 µL,在4℃孵育10 h。倾空液体,用含0.05%Tween-20的PBST清洗5次。每孔加入300 µL封闭液(质量分数为5% BSA),37℃封闭1 h,倾空液体,用PBST清洗5次。每孔加入100 µL单克隆抗体MJ12-30,37℃孵育1 h。倾空液体,用PBST清洗5次。用封闭液按1:5000稀释HRP标记的山羊抗鼠(IgM、IgA、IgG1、IgG2a、IgG2b和IgG3)抗体,每孔加入100 µL,37℃孵育1 h。倾空液体,用PBST清洗5次。每孔加入100 µL底物溶液,室温显色5 min,在450 nm波长下测OD值。结果如表1所示。
表1
根据阳性对照及样品检测的OD值,确定抗体的亚型,N为阴性对照,P为阳性对照,结果如表1所示,由此可知,本次融合获得的31株杂交瘤单克隆抗体的亚型均为鼠源IgG1,表1列出MJ12-30的亚型测定数据,证实MJ12-30亚型为IgG1。
所述单克隆抗体MJ12-30的重链CDR3的氨基酸序列如SEQ ID No.3所示,重链CDR1的氨基酸序列如SEQ ID No.4所示,重链CDR2的氨基酸序列如SEQ ID No.5所示。
SEQ ID No.3:A。
SEQ ID No.4:GFTFHSFG。
SEQ ID No.5:ISSGGSTI。
所述单克隆抗体MJ12-30的轻链CDR3的氨基酸序列如SEQ ID No.6所示,轻链CDR1的氨基酸序列如SEQ ID No.7所示,轻链CDR3的氨基酸序列如SEQ ID No.8所示。
SEQ ID No.6:FQGNHVP。
SEQ ID No.7:QSILHSGGNTY。
SEQ ID No.8:KVS。
所述单克隆抗体MJ12-30的重链可变区的氨基酸序列如SEQ ID No.9所示,重链全长的氨基酸序列如SEQ ID No.10所示。
SEQ ID No.9:
EVKLEQSGGVLVQPGGSRKLSCAASGFTFHSFGMHWVRQAPEKGLEWVAYISSGGSTIFYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCAGSDHGNLGYWGQGTTLTVSSTKTTP。
SEQ ID No.10:
EVKLEQSGGVLVQPGGSRKLSCAASGFTFHSFGMHWVRQAPEKGLEWVAYISSGGSTIFYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCAGSDHGNLGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。
所述单克隆抗体MJ12-30的轻链可变区的氨基酸序列如SEQ ID No.11所示,轻链全长的氨基酸序列如SEQ ID No.12所示。
SEQ ID No.11:
DIVLTQSPLSLSVSLGDQASISCRSSQSILHSGGNTYLDWFLQRPGQSPKLLIYKVSNRISGVPDRFSGSGAGTDFTLKISRVEAEDLGVYYCFQGNHVPPTFGGGTKLEIKRADAA。
SEQ ID No.12:
DIVLTQSPLSLSVSLGDQASISCRSSQSILHSGGNTYLDWFLQRPGQSPKLLIYKVSNRISGVPDRFSGSGAGTDFTLKISRVEAEDLGVYYCFQGNHVPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。
实施例6
本实施例对实施例3中所述的克隆号为MJ12-30的杂交瘤细胞株进行保藏,命名为鼠抗人TIGIT单克隆抗体杂交瘤细胞系MJ12-30,保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号,邮编为100101,保藏编号为CGMCCNo.45102,保藏日期为2022年02月24日。
实施例7
本实施例提供一种TIGIT免疫组织化学检测试剂盒,所述TIGIT免疫组织化学检测试剂盒包括实施例4所述的单克隆抗体MJ12-30、免疫组织化学抗原修复缓冲液、封闭液、酶标二抗、显色剂和苏木素复染液。
实施例8
本实施例使用实施例7中所述的TIGIT免疫组织化学检测试剂盒对样本进行检测。
(1)材料
本实施例中所用组织样本为阑尾、扁桃体和黑色素瘤组织样本,组织样本来源于迈杰转化医学研究(苏州)有限公司组织样本库,组织样本包括阑尾B882样本、扁桃体B516样本、黑色素瘤B531样本和黑色素瘤B774样本,组织样本是经福尔马林固定和石蜡包埋处理的人体组织样本,均经病理证实,并有患者知情同意书。其中黑色素瘤B531样本为经商业化抗体E5Y1W(公司:Cell Signaling Technology,商品名:TIGIT(E5Y1W)XP Rabbit mAb,货号#99567)验证为TIGIT表达阳性的组织,作为阳性对照。黑色素瘤B774样本为经商业化抗体E5Y1W验证为TIGIT表达阴性的组织,作为阴性对照。经文献验证和E5Y1W抗体验证证实,阑尾和扁桃体组织样本,均为TIGIT表达强阳性组织。
(2)实验步骤
样品经烤片后,在Leica BOND-MAX(国械备20140277)全自动组化系统上完成,其中一抗的用量均为抗体的最适使用浓度(3.5 μg/mL),具体程序如表2所示。
表2
(3)检测结果
免疫组织化学的检测结果如图4和图5所示,其中,图4为商品化抗体E5Y1W的免疫组织化学检测结果,图5为单克隆抗体MJ12-30的免疫组织化学检测结果。经过专业病理工作人员对结果进行判读,此次免疫组化实验结果显示,商品化抗体E5Y1W与本实施例4中的单克隆抗体MJ12-30在检测阑尾、扁桃体和黑色素瘤阳性组织中TIGIT表达时,呈现细胞膜阳性染色,这与人TIGIT表达在细胞膜上的结果相匹配;两种抗体在TIGIT表达呈阴性的黑色素瘤组织中,检测结果均为阴性。且实施例4中的单克隆抗体MJ12-30在组织细胞上的染色无特异性染色背景。
因此,由实验结果可知,所述单克隆抗体MJ12-30能够特异性地检测人TIGIT在阑尾、扁桃体、黑色素瘤等组织中的表达。可以进一步地将所述单克隆抗体MJ12-30的性能进行优化和开发,以单独抗体或以试剂盒的形式应用于免疫组织化学实验,用于相关组织样本的检测。
所述单克隆抗体MJ12-30能够特异性地检测人体组织中TIGIT蛋白的表达,且灵敏度高、特异性强。
实施例9
本实施例提供一种TIGIT免疫印迹检测试剂盒,所述TIGIT免疫印迹检测试剂盒包括实施例4中所述的单克隆抗体MJ12-30、SDS-PAGE凝胶、PVDF膜、封闭液和酶标二抗。
所述SDS-PAGE凝胶的浓度为12%,所述封闭液为含5%脱脂奶粉的PBST封闭液,所述酶标二抗为HRP-山羊抗小鼠IgG。
实施例10
本实施例使用实施例9中所述的TIGIT免疫印迹检测试剂盒对样本进行检测。
(1)实验步骤
将真核表达的人TIGIT重组蛋白(分子量:13.9 kDa)及原核表达的TF-TIGIT蛋白(分子量:65.3 kDa)上样于12%的SDS-PAGE凝胶,并以相同上样量的真核商品化蛋白作为阳性对照;在100 V下电泳,电泳结束后,将凝胶在1×转移液中浸泡10 min;剪取PVDF膜用甲醇处理后,与凝胶一起在电压为100 V的条件下转膜60 min;然后将PVDF膜置于含5%脱脂奶粉的PBST封闭液中于37℃封闭1 h;纯化后的单克隆抗体MJ12-30及对照抗体E5Y1W用1%脱脂奶粉稀释后与PVDF膜室温孵育60 min;HRP-山羊抗小鼠IgG按1:3000稀释,室温孵育60min,用ChemiDoc™ MP成像系统曝光。
(2)实验结果
实验结果如图6所示,泳道1、泳道5和泳道9:蛋白Marker;泳道2和泳道6:真核表达的人TIGIT重组蛋白,上样量为0.2 μg;泳道3和泳道7:阴性对照BSA,上样量为0.2 μg;泳道4和泳道8:原核表达的TF-TIGIT蛋白,上样量为0.2 μg;泳道2、泳道3和泳道4:实施例4中的单克隆抗体MJ12-30,一抗孵育的浓度为0.2 µg/mL,二抗为HRP-山羊抗小鼠IgG,稀释比例为1:3000;泳道6、泳道7和泳道8:对照抗体E5Y1W,一抗孵育浓度为0.2 µg/mL,二抗为HRP-山羊抗兔IgG,稀释比例为 1:3000。
由实验结果可知,所述单克隆抗体MJ12-30与对照抗体E5Y1W在免疫印迹实验中均能够检测不同表达来源的TIGIT蛋白,免疫印迹的条带大小正确、特异性好、无非特异性背景。在相同的稀释比例和实验条件下,所述单克隆抗体MJ12-30与抗原的反应强度优于对照抗体E5Y1W。
实施例11
本实施例提供一种TIGIT酶联免疫吸附检测试剂盒,所述TIGIT酶联免疫吸附检测试剂盒包括实施例4中所述的单克隆抗体MJ12-30、封闭液和酶标二抗;所述封闭液为5%BSA;所述酶标二抗为HRP-山羊抗小鼠IgG。
实施例12
本实施例使用实施例11所述的TIGIT酶联免疫吸附检测试剂盒对样本进行检测。
(1)实验步骤
取纯化后的真核293F细胞表达的人TIGIT重组蛋白和原核表达的TF-TIGIT蛋白,进行ELISA实验,采用间接ELISA法测试单克隆抗体MJ12-30的特异性,阳性对照抗体为E5Y1W。
具体的操作步骤如下:用1 µg/mL真核表达的人TIGIT重组蛋白、2 µg/mL原核表达的人TF-TIGIT重组蛋白在37℃下包被检测板1 h,包被的体积均为100 µL。再用PBST洗涤4次,用5% BSA 37℃封闭1 h。接着进行一抗孵育,用1% BSA稀释单克隆抗体MJ12-30和对照抗体E5Y1W至浓度为0.5 µg/mL,阴性对照为1% BSA。用PBST洗涤4次,最后每孔加入对应的稀释比例为1:5000的辣根过氧化物酶HRP标记亲和纯化山羊抗鼠IgG(H+L)F(ab’)2片段各100 µL,最后测定450 nm的OD值。
ELISA实验的结果如图7所示,N阴性对照,一抗为抗体稀释液,P为阳性对照抗体E5Y1W,单克隆抗体MJ12-30与阳性对照抗体E5Y1W能够特异性地识别真核表达及原核表达的人TIGIT蛋白,结果均为强阳性。其中,单克隆抗体MJ12-30对真核293F细胞表达的人TIGIT蛋白的反应的OD值更高。
实施例13
本实施例对实施例4中所述的单克隆抗体MJ12-30进行亲和常数测定。
取实施例1中制备的人TIGIT重组蛋白,包被浓度为2 μg/mL,100 μL/孔,37℃包被1 h,PBST洗3次。每孔加200 μL封闭液4℃封闭过夜,PBS-T洗3次。将实施例4中纯化的单克隆抗体MJ12-30从1:200开始2倍梯度稀释,共稀释15个梯度,1孔留空白对照,37℃孵育1 h,PBST洗3次,所述单克隆抗体MJ12-30的初始浓度为0.8 mg/mL。HRP标记的羊抗鼠IgG二抗1:20000稀释,每孔100 μL,37℃孵育1 h,PBS-T洗3次。每孔加入100 μL TMB显色液(厂家:Solarbio,货号:PR1210),37℃显色10 min,加50 μL 0.5 M硫酸溶液,终止反应。用酶标仪测定波长450 nm的吸光值,吸光值检测结果如表3所示。找到最大结合OD值的一半时对应的稀释倍数A,利用以下公式计算亲和常数,亲和常数的计算公式如下所示:亲和常数=(150000×A)/抗体原始浓度。
表3
利用上述公式计算出本发明中所述单克隆抗体MJ12-30的亲和常数约为9.6×109。
综上,本发明提供的抗TIGIT单克隆抗体与TIGIT蛋白具有较高的亲和力,能够特异性地识别真核表达的TIGIT蛋白,在制备TIGIT检测试剂盒中具有重要的应用前景。
申请人声明,以上所述仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,所属技术领域的技术人员应该明了,任何属于本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,均落在本发明的保护范围和公开范围之内。
序列表
<110> 迈杰转化医学研究(苏州)有限公司
<120> 一株分泌抗TIGIT单克隆抗体的杂交瘤细胞株及其应用
<130> 2022
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<170> PatentIn version 3.3
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115 120 125
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
130 135 140
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
145 150 155 160
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
180 185 190
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
195 200 205
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215
Claims (11)
1.一株分泌抗TIGIT单克隆抗体的杂交瘤细胞株,其特征在于,所述分泌抗TIGIT单克隆抗体的杂交瘤细胞株命名为鼠抗人TIGIT单克隆抗体杂交瘤细胞系MJ12-30,保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.45102,保藏日期为2022年02月24日。
2.一种抗TIGIT单克隆抗体,其特征在于,所述抗TIGIT单克隆抗体由权利要求1所述的鼠抗人TIGIT单克隆抗体杂交瘤细胞系MJ12-30分泌。
3.根据权利要求2所述的抗TIGIT单克隆抗体,其特征在于,所述抗TIGIT单克隆抗体的亚型为IgG1型;
所述抗TIGIT单克隆抗体的重链包括:SEQ ID No.3所示的CDR3、SEQ ID No.4所示的CDR1和SEQ ID No.5所示的CDR2;
所述抗TIGIT单克隆抗体的轻链包括:SEQ ID No.6所示的CDR3、SEQ ID No.7所示的CDR1和SEQ ID No.8所示的CDR2;
所述抗TIGIT单克隆抗体的重链可变区包括SEQ ID No.9所示的氨基酸序列,所述抗TIGIT单克隆抗体的轻链可变区包括SEQ ID No.11所示的氨基酸序列;
所述抗TIGIT单克隆抗体的重链全长包括SEQ ID No.10所示的氨基酸序列;所述抗TIGIT单克隆抗体的轻链全长包括SEQ ID No.12所示的氨基酸序列。
4.根据权利要求2所述的抗TIGIT单克隆抗体,其特征在于,所述抗TIGIT单克隆抗体还含有修饰缀合物,所述修饰缀合物包括辣根过氧化物酶、碱性磷酸酶、生物素、异硫氰酸荧光素、Cy3或Cy5中的任意一种或至少两种的组合。
5.一种权利要求2~4中任一项所述的抗TIGIT单克隆抗体的制备方法,其特征在于,所述抗TIGIT单克隆抗体的制备方法包括:
培养权利要求1所述的分泌抗TIGIT单克隆抗体的杂交瘤细胞株,纯化,获得所述抗TIGIT单克隆抗体。
6.权利要求1所述的分泌抗TIGIT单克隆抗体的杂交瘤细胞株和/或权利要求2~4中任一项所述的抗TIGIT单克隆抗体在制备TIGIT蛋白表达检测产品中的应用。
7.一种TIGIT免疫组织化学检测试剂盒,其特征在于,所述TIGIT免疫组织化学检测试剂盒包括权利要求2~4中任一项所述的抗TIGIT单克隆抗体;
所述TIGIT免疫组织化学检测试剂盒还包括:免疫组织化学抗原修复缓冲液、封闭液、酶标二抗、显色剂或苏木素复染液中的任意一种或至少两种的组合。
8.一种TIGIT免疫印迹检测试剂盒,其特征在于,所述TIGIT免疫印迹检测试剂盒包括权利要求2~4中任一项所述的抗TIGIT单克隆抗体;
所述TIGIT免疫印迹检测试剂盒还包括:SDS-PAGE凝胶、聚偏二氟乙烯膜、封闭液或酶标二抗中的任意一种或至少两种组合。
9.一种TIGIT酶联免疫吸附检测试剂盒,其特征在于,所述TIGIT酶联免疫吸附检测试剂盒包括权利要求2~4中任一项所述的抗TIGIT单克隆抗体;
所述TIGIT酶联免疫吸附检测试剂盒还包括:封闭液、酶标二抗、显色液或终止液中的任意一种或至少两种组合。
10.一种TIGIT免疫荧光检测试剂盒,其特征在于,所述TIGIT免疫荧光检测试剂盒包括权利要求2~4中任一项所述的抗TIGIT单克隆抗体;
所述抗TIGIT单克隆抗体修饰有荧光基团,所述荧光基团为异硫氰酸荧光素、Cy3或Cy5中的任意一种。
11.一种TIGIT流式细胞术检测试剂盒,其特征在于,所述TIGIT流式细胞术检测试剂盒包括权利要求2~4中任一项所述的抗TIGIT单克隆抗体。
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