CN115677856B - 抗人IgM抗体及其应用 - Google Patents
抗人IgM抗体及其应用 Download PDFInfo
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- CN115677856B CN115677856B CN202110861408.8A CN202110861408A CN115677856B CN 115677856 B CN115677856 B CN 115677856B CN 202110861408 A CN202110861408 A CN 202110861408A CN 115677856 B CN115677856 B CN 115677856B
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Abstract
本发明属于抗体技术领域。具体而言,涉及抗人IgM抗体及其应用。本发明提供的抗人IgM抗体稳定性好,与人IGM的结合亲和力高,能够显著降低甚至消除内源性干扰,进而提高免疫检测的准确度,高效方便;因此,该抗体或其抗原结合片段、及其相关的核酸、载体或细胞能够广泛应用于免疫检测领域中。
Description
技术领域
本发明属于抗体技术领域。更具体地,涉及抗人IgM抗体及其应用。
背景技术
基于抗原抗体反应的免疫检测方法应用广泛,根据抗体标记物分为不同的检测方法,如:酶联免疫、放射免疫、化学发光等。在临床应用中,免疫检测结果的准确性往往在不同程度上受病人血清中干扰物的影响,从而导致错误的检测结果。血清中的干扰物可分为内源性干扰和外源性干扰,外源性干扰包括溶血、样本染菌、血液凝固不彻底、样本的稳定性与保存条件等;内源性干扰包括类风湿因子(RF)、嗜异性抗体(HA)、自身抗体、补体、黄疸、高脂等;其中,RF、HA是重要的干扰因素。研究证实,约3%-15%健康人群的体内含有内源性干扰因素,10%以上(30%-40%)的病人体内存在HA干扰,10%-40%的人群中存在HAMA干扰。在内源性干扰中,以RF、HA最为常见。因此,研究、发展降低甚至消除RF干扰、HA干扰的有效手段,是保证医学免疫检验结果可靠性、保障医患利益的重要课题。针对消除免疫诊断中的RF干扰、HA干扰,最简单、最有效的方法就是在检测***中添加阻断剂,直接阻断干扰物质与检测***中抗体或抗原的结合。
阻断剂是一种生物制剂,加入免疫检验体系中,可与内源性抗体反应,从而有效防止非分析物介导的抗体桥连。阻断剂可分为被动阻断剂和主动阻断剂。主动阻断剂是特异性针对人免疫球蛋白的,可以特异、主动、高效的中和干扰抗体的成分,从而阻断非预期结合的产生,如商品化试剂中的IIR、HBR等。这种制剂可排除各种异嗜性干扰,对造成干扰的异嗜性抗体具有特异性结合力,只需要低浓度即可高效阻断,将影响减到最低。IIR是一种由HA和HAAA为免疫原产生的混合的鼠McAb,它与HAAA有较高亲和力(109L/mol)。HBR是鼠抗人IgM的McAb。在主动阻断过程中,消除干扰的效果依赖于主动阻断剂对异嗜性抗体的亲和力。由于主动阻断剂的高亲和力,因此其在一些分析中的阻断能力强于被动阻断剂。
市场上的阻断剂产品虽然较多,但是均存在一定的性能缺陷,加之阻断剂本身用量非常大,而主流的阻断剂进口居多、价格偏高,所以市场亟需性能更优、成本更低的阻断剂。
发明内容
本发明要解决的技术问题是克服现有免疫阻断剂成本高、性能不好的缺陷和不足,提供抗人IgM抗体及其应用,所述抗人IgM抗体与人IgM具有很高的亲和力,稳定性好,成本低,能够应用于免疫检测领域中作为或制备免疫阻断剂,以及制备免疫检测试剂或试剂盒,以降低甚至消除内源性干扰,且其阻断效果显著好于市场阻断剂原料。
本发明的目的是提供抗人IgM抗体或其抗原结合片段,所述抗体含有以下重链互补决定区HCDRs:
氨基酸序列如SEQ ID NO.1所示的HCDR1,氨基酸序列如SEQ ID NO.2所示的HCDR2,氨基酸序列如SEQ ID NO.3所示的HCDR3。
本发明的另一目的是提供所述抗人IgM抗体或其抗原结合片段相关的核酸、载体或细胞。
本发明还提供了所述抗人IgM抗体或其抗原结合片段,及其相关的核酸、载体或细胞在免疫检测中的应用。
本发明还提供了所述抗人IgM抗体或其抗原结合片段,及其相关的核酸、载体或细胞在作为/制备免疫阻断剂中的应用。
本发明还提供了一种免疫阻断剂,含有所述抗人IgM抗体或其抗原结合片段、所述载体、所述核酸或所述细胞。
本发明还提供了一种降低/消除内源性干扰的方法,在免疫检测***中添加所述免疫阻断剂。
本发明还提供了一种免疫诊断试剂/试剂盒,其包含所述免疫阻断剂。
附图说明
图1是实施例1制备得到的Ig-M-9G1RMb1抗体的还原性SDS-PAGE结果图。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
本发明涉及抗人IgM抗体或其抗原结合片段,所述抗体含有以下重链互补决定区HCDRs:
氨基酸序列如SEQ ID NO.1所示的HCDR1,氨基酸序列如SEQ ID NO.2所示的HCDR2,氨基酸序列如SEQ ID NO.3所示的HCDR3。
在一些实施方式中,所述抗体还含有以下轻链互补决定区LCDRs:
氨基酸序列如SEQ ID NO.4所示的LCDR1,氨基酸序列如SEQ ID NO.5所示的LCDR2,氨基酸序列如SEQ ID NO.6或SEQ ID NO.7所示的LCDR3。
所述抗体或其抗原结合片段的一个重要优点在于其与人IGM具有高亲和力。
所述抗体或其抗原结合片段的一个重要优点在于其对假阳样本和RF样本等内源性干扰有显著的阻断效果,且其阻断效果甚至好于市场阻断剂原料,能够降低甚至消除内源性干扰。
在本发明中,术语“抗体”在最广义上使用,其可以包括全长单克隆抗体,双特异性或多特异性抗体,以及嵌合抗体,只要它们展示所需的生物学活性。术语“抗原结合片段”是包含抗体CDR的一部分或全部的物质,其缺乏至少一些存在于全长链中的氨基酸但仍能够特异性结合至抗原。此类片段具生物活性,因为其结合至抗原,且可与其他抗原结合分子(包括完整抗体)竞争结合至给定表位。此类片段选自Fab(由完整的轻链和Fd构成),Fv(由VH和VL构成),ScFv(单链抗体,VH和VL之间由一连接肽连接而成)或单域抗体(仅由VH组成)。此类片段可通过重组核酸技术产生,或可通过抗原结合分子(包括完整抗体)的酶裂解或化学裂解产生。在一些实施方式中,所述抗原结合片段选自Fab,Fab',F(ab')2,scFv,Fv,Fd,单链抗体,双价抗体或结构域抗体。在本发明具体实施方式中,抗人IgM抗体的抗原结合片段与人IGM具有高亲和力。在本发明具体实施方式中,抗人IgM抗体的抗原结合片段能够显著阻断假阳样本和RF样本等内源性干扰,降低甚至消除内源性干扰。
在本发明中,术语“互补性决定区”、“CDR”或“CDRs”是指免疫球蛋白的重链和轻链的高度可变区,指包含一种或多种或者甚至全部的对抗体或其抗原结合片段与其识别的抗原或表位的结合亲和力起作用的主要氨基酸残基的区域。在本发明具体实施方式中,CDRs是指抗人IgM抗体的重链和轻链的高度可变区。
在本发明中,重链互补决定区用HCDR表示,其包括HCDR1、HCDR2和HCDR1;轻链互补决定区用LCDR表示,其包括LCDR1、LCDR2和LCDR1。本领域常用的CDR标示方法包括:Kabat编号方案、Chothia和Lesk编号方案以及1997年Lefranc等人为免疫球蛋白超家族的所有蛋白质序列引入的新的标准化编号***。Kabat等人是第一个为免疫球蛋白可变区提出标准化编号方案的人。在过去的几十年中,序列的积累导致了KABATMAN数据库的创建,Kabat编号方案通常被认为是编号抗体残基广泛采用的标准。本发明采用Kabat注释标准标示CDR区,但其他方法标示的CDR区也属于本发明的保护范围。
在一些实施方式中,所述抗体还含有重链可变区和轻链可变区的至少之一;所述抗体的重链可变区的氨基酸序列如SEQ ID NO:8所示,所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:9或SEQ ID NO:10所示。
在一些实施方式中,所述抗体还含有重链恒定区和轻链恒定区;所述重链恒定区为IgG1、IgG2、IgG3、IgG4、IgA、IgD、IgE或IgM中的任一种或几种,所述轻链恒定区为κ链或λ链。
在一些实施方式中,所述重链恒定区和轻链恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在一些实施方式中,所述抗体的重链的氨基酸序列如SEQ ID NO:11所示,所述抗体的轻链的氨基酸序列如SEQ ID NO:12或SEQ ID NO:13所示。
本发明还涉及核酸,所述核酸编码所述抗体或其抗原结合片段。
核酸通常是RNA或DNA,核酸分子可以是单链或双链的。当将核酸与另一个核酸序列置于功能关系中时,核酸是“有效连接的”。例如,如果启动子或增强子影响编码序列的转录,那么启动子或增强子有效地连接至所述编码序列。当其连入载体时采用DNA核酸。
本发明还涉及载体,所述载体含有所述核酸。
本发明还涉及细胞,所述细胞含有所述核酸或所述载体。
所述抗体或其抗原结合片段、所述核酸、所述载体或所述细胞在免疫检测中的应用,及其在作为/制备免疫阻断剂中的应用,都应在本发明的保护范围之内。
本发明还涉及一种免疫阻断剂,含有所述抗体或其抗原结合片段、所述核酸、所述载体或所述细胞。
本发明还涉及一种降低/消除内源性干扰的方法,在免疫检测***中添加所述免疫阻断剂。在本发明具体实施方式中,所述内源性干扰为类风湿因子干扰或嗜异性抗体干扰。
本发明还涉及一种免疫诊断试剂/试剂盒,其包含所述免疫阻断剂。
在一些实施方式中,所述试剂盒为免疫层析检测试剂盒、酶免检测试剂盒、化学发光试剂盒或免疫比浊检测试剂盒。
在一些实施方式中,所述试剂盒可以包括测试条或测试卡,来自于所述受试者的所述液体样品放置到所述测试条上,或者ELISA测定板,所述ELISA测定板具有其中可放置来自于单个受试者的液体样品的孔。在一些实施方案中,所述试剂盒可以包括配置用于流式细胞仪、生物分析仪、生物传感器中的测试装置。
在一些实施方式中,所述试剂盒中包含的所述免疫阻断剂可为液体溶液形式、附着于固体支持物上的形式、或为干燥粉剂。当免疫阻断剂为一种液体溶液时,该液体溶液可以是水溶液。当免疫阻断剂是附着于固体支持物上的形式时,优选的固体支持物可以是层析介质如薄膜、测试条、塑料珠或平板、或显微镜栽玻片。当免疫阻断剂为一种干燥粉剂时,通过加入适当溶剂可重构粉剂。
本发明具有以下有益效果:
本发明提供了抗人IgM抗体及其应用,与市场阻断剂原料相比,该抗人IgM抗体稳定性好、成本低,与人IGM的结合亲和力高,对假阳样本和RF样本等内源性干扰有显著的阻断效果,使用时直接加入检测体系中即能够降低甚至消除内源性干扰,进而提高免疫检测的准确度,高效方便;因此,所述抗体或其抗原结合片段、及其相关的核酸、载体或细胞能够作为或制备免疫阻断剂,以及制备免疫检测试剂或试剂盒,广泛应用于免疫检测领域中。
下面将结合实施例对本发明的实施方案进行详细描述。
以下实施例中,限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMARTTM RACE cDNA AmplificationKit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由Invitrogen公司完成。
实施例1抗人IgM抗体(Ig-M-9G1RMb1抗体)的制备
1、表达质粒构建
(1)Ig-M-9G1RMb1抗体基因制备
在分泌Ig-M-9G1RMb1抗体的杂交瘤细胞株中提取mRNA,通过RT-PCR方法获得DNA产物,该产物用rTaq DNA聚合酶进行加A反应后***到pMD-18T载体中,转化到DH5α感受态细胞中,长出菌落后分别取重链和轻链基因克隆各4个克隆,送基因测序公司进行测序。
(2)Ig-M-9G1RMb1抗体可变区基因的序列分析
将上述测序得到的基因序列放在IMGT抗体数据库中进行分析,并利用VNTI11.5软件进行分析。
经分析确定重链和轻链引物对扩增出的基因都是正确的;其中,轻链引物对扩增出的基因片段中,VL基因序列为396bp,属于VkII基因家族,其前方有57bp的前导肽序列;重链引物对扩增出的基因片段中,VH基因序列为432bp,属于VH1基因家族,其前方有57bp的前导肽序列。
(3)重组抗体表达质粒的构建
pcDNATM 3.4vector为构建的Ig-M-9G1RMb1抗体真核表达载体,该表达载体已经引入HindIII、BamHI、EcoRI等多克隆酶切位点,并命名为pcDNA3.4A表达载体,后续简称3.4A表达载体;根据上述Ig-M-9G1RMb1抗体可变区基因测序结果,设计Ig-M-9G1RMb1抗体的VL和VH基因特异性引物,两端分别带有HindIII、EcoRI酶切位点和保护碱基,通过PCR扩增方法扩出0.73KB的轻链基因片段和1.43kb的重链基因片段。
重链和轻链基因片段分别采用HindIII/EcoRI双酶切,3.4A载体采用HindIII/EcoRI双酶切,将片段和载体纯化回收后重链基因和轻链基因分别连接3.4A表达载体中,分别得到重链和轻链的重组表达质粒。
2、稳定细胞株筛选
(1)重组抗体表达质粒瞬时转染CHO细胞,确定表达质粒活性
Ig-M-9G1RMb1抗体表达质粒用超纯水稀释至40μg/100μL,调节CHO细胞1.43×107cells/mL于离心管中,100μL质粒与700μL细胞混合,转入电转杯,电转,第3、5、7天取样计数,第7天收样检测。
用包被液(主要成分为NaHCO3)稀释人IgM至1μg/mL,每孔100μL,4℃过夜;次日,洗涤液(主要成分为Na2HPO4+NaCl)清洗2次,拍干;加入封闭液(20%BSA+80%PBS),每孔120μL,37℃,1h,拍干;加入稀释后的细胞上清,100μL/孔,37℃,30min(部分上清1h)。同时以未加细胞上清作为空白对照;洗涤液清洗5次,拍干;加入羊抗鼠IgG-HRP,每孔100μL,37℃,30min;洗涤液清洗5次,拍干;加入显色液A液(50μL/孔,显色液A液的主要成分为柠檬酸、醋酸钠、乙酰苯胺和过氧化脲),加入显色液B液(50μL/孔,显色液B液的主要成分为柠檬酸、EDTA·2Na、TMB和浓HCl),10min;加入终止液(终止液的主要成分为EDTA·2Na和浓H2SO4),50μL/孔;酶标仪上450nm(参考630nm)处读OD值。
结果显示,细胞上清稀释1000倍后反应OD仍大于1.0,未加细胞上清孔反应OD小于0.1,表明质粒瞬转后产生的抗体对人IGM有活性。
(2)重组抗体表达质粒线性化
准备下述试剂:Buffer 50μL、DNA 100μg/管、PvuⅠ酶10μL、无菌水补至500μL,37℃水浴酶切过夜;先用等体积酚/氯仿/异戊醇(下层)25:24:1,再用氯仿(水相)依次进行抽提;0.1倍体积(水相)3M醋酸钠和2倍体积乙醇冰上沉淀,70%乙醇漂洗沉淀,去除有机溶剂,待乙醇挥发完全用适量的灭菌水进行复融,最后进行浓度的测定。
(3)重组抗体表达质粒稳定转染,加压筛选稳定细胞株
质粒用超纯水稀释至40μg/100μL,调节CHO细胞1.43×107cells/mL于离心管中,100μL质粒与700μL细胞混合,转入电转杯,电转,次日计数;25μmol/L MSX 96孔加压培养约25天。
显微镜下观察标记长有细胞的克隆孔,并记录汇合度;取培养上清,送样检测;挑选抗体浓度、相对浓度高的细胞株转24孔,3天左右转6孔;3天后保种批培,调整细胞密度0.5×106cells/mL,2.2mL进行批培养,细胞密度0.3×106cells/mL,2mL进行保种;7天6孔批培上清送样检测,挑选抗体浓度及细胞直径较小的细胞株转TPP保种传代。
3、Ig-M-9G1RMb1抗体制备
(1)细胞扩培
细胞复苏之后先在125mL规格的摇瓶中培养,接种体积为30mL,培养基为100%Dynamis培养基,放置于转速120r/min,温度为37℃,二氧化碳为8%的摇床中。培养72h,以50万cells/mL接种密度接种扩培,扩培体积根据生产需求进行计算,培养基为100%Dynamis培养基。之后每72h扩培一次。当细胞量满足生产需求时,严格控制接种密度为50万cells/mL左右进行生产。
(2)摇瓶生产及纯化
摇瓶参数:转速120r/min,温度为37℃,二氧化碳为8%。流加补料:在摇瓶中培养至72h时开始每天补料,HyCloneTM Cell BoostTM Feed 7a每天流加初始培养体积的3%,Feed 7b每天流加量为初始培养体积的千分之一,一直补到第12天(第12天补料)。葡萄糖在第六天补加3g/L。第13天收样。用proteinA亲和层析柱进行亲和纯化,即得到Ig-M-9G1RMb1抗体。取6.6μg Ig-M-9G1RMb1抗体进行还原性SDS-PAGE。
Ig-M-9G1RMb1抗体的还原性SDS-PAGE结果如图1所示,结果显示两条带,1条Mr为50KD(重链),另一条Mr为28KD(轻链)。
Ig-M-9G1RMb1抗体的HCDR1的氨基酸序列如SEQ ID NO.1所示,HCDR2的氨基酸序列如SEQ ID NO.2所示,HCDR3的氨基酸序列如SEQ ID NO.3所示;LCDR1的氨基酸序列如SEQID NO.4所示,LCDR2的氨基酸序列如SEQ ID NO.5所示,LCDR3的氨基酸序列如SEQ ID NO.6所示;
重链可变区的氨基酸序列如SEQ ID NO:8所示,轻链可变区的氨基酸序列如SEQID NO:9所示;
重链的氨基酸序列如SEQ ID NO:11所示,轻链的氨基酸序列如SEQ ID NO:12所示。
实施例2抗人IgM抗体(Ig-M-9G1RMb2抗体)的制备
对实施例1制备得到的Ig-M-9G1RMb1抗体的序列进行分析,通过常规氨基酸引入定点突变的方法,将Ig-M-9G1RMb1抗体的LCDR3第1位氨基酸I(IIe,异亮氨酸)突变为L(Leu,亮氨酸),并利用与实施例1的步骤1中的步骤(3)、步骤2和步骤3相同的方法,构建得到另外一株抗体(Ig-M-9G1RMb2抗体)。
Ig-M-9G1RMb2抗体的HCDR1的氨基酸序列如SEQ ID NO.1所示,HCDR2的氨基酸序列如SEQ ID NO.2所示,HCDR3的氨基酸序列如SEQ ID NO.3所示;LCDR1的氨基酸序列如SEQID NO.4所示,LCDR2的氨基酸序列如SEQ ID NO.5所示,LCDR3的氨基酸序列如SEQ ID NO.7所示;
重链可变区的氨基酸序列如SEQ ID NO:8所示,轻链可变区的氨基酸序列如SEQID NO:10所示;
重链的氨基酸序列如SEQ ID NO:11所示,轻链的氨基酸序列如SEQ ID NO:13所示。
实施例3抗人IgM抗体的亲和力分析
通过以下方法分析实施例1制备得到的Ig-M-9G1RMb1抗体、实施例2制备得到的Ig-M-9G1RMb2抗体和市场阻断剂原料的亲和力:
利用AMC传感器,Ig-M-9G1RMb1抗体和Ig-M-9G1RMb2抗体用PBST稀释到10μg/mL,人IGM用PBST进行梯度稀释;
运行流程:缓冲液1(PBST,主要成分为Na2HPO4+NaCl+TW-20)中平衡60s,抗体溶液中固化抗体300s,缓冲液2(PBST,主要成分为Na2HPO4+NaCl+TW-20)中孵育180s,抗原溶液中结合420s,缓冲液2中解离1200s,用10mM pH 1.69 GLY溶液及缓冲液3(PBST,主要成分为Na2HPO4+NaCl+TW-20)进行传感器再生,输出数据。
Ig-M-9G1RMb1抗体和Ig-M-9G1RMb2抗体的亲和力分析结果如表1所示,结果显示,本发明得到的Ig-M-9G1RMb1抗体和Ig-M-9G1RMb2抗体对人IGM的亲和力显著高于市场阻断剂原料。
表1
样品名称 | KD(M) | kon(1/Ms) | kdis(1/s) |
市场阻断剂原料 | 2.49E-10 | 8.15E+05 | 2.03E-04 |
Ig-M-9G1RMb1抗体 | 3.02E-12 | 9.41E+06 | 2.84E-05 |
Ig-M-9G1RMb2抗体 | 4.27E-12 | 1.42E+07 | 6.07E-05 |
注:表1中,KD表示平衡解离常数即亲和力;kon表示结合速率;kdis表示解离速率。KD值越低,代表亲和力越高。
实施例4抗人IgM抗体的阻断性能测定
1、在CTNI荧光平台验证阻断性能
在CTNI荧光平台配对检测中,实验组用实施例1制备得到的Ig-M-9G1RMb1抗体、实施例2制备得到的Ig-M-9G1RMb2抗体和市场阻断剂原料分别处理样品垫,空白对照组样品垫不作处理;分别检测样本(L1-L10)。
Ig-M-9G1RMb1抗体、Ig-M-9G1RMb2抗体在CTNI荧光平台的阻断效果如表2所示,结果显示,实验组对假阳样本有显著的消除作用;其中,Ig-M-9G1RMb1抗体、Ig-M-9G1RMb2抗体对假阳样本有显著的阻断效果,且其阻断效果显著好于市场阻断剂原料。
表2
表2中,T/C值说明:
待测样本加入到检测试剂卡的加样口中,在侧向毛细管作用下,待检样本先经过结合垫,与结合垫上的荧光基团标记抗体发生特异的免疫结合,各自结合形成抗原-抗体荧光复合物,从而被固定在T线中。C线上包被有与游离的荧光基团标记抗体发生反应的物质,当游离的荧光基团标记抗体经过C线时,能够与C线上的物质发生特异的免疫结合,从而被固定在C线中。荧光免疫分析仪检测出的两条带的荧光强度以峰面积体现,并通过仪器自身的计算软件计算T/C值。仪器读值T/C表示T峰面积与C峰面积比值,在质控样本和阳性样本下,T/C越高代表活性越高;在假阳样本下T/C越低,代表阻断效果越好;当T/C值<0.1时,即判断为阴性样本。
2、在GRP化学发光平台验证阻断性能
在GRP化学发光平台配对检测中,实验组浓度分别将浓度为100μg/mL、30μg/mL、10μg/mL和5μg/mL的实施例1制备得到的Ig-M-9G1RMb1抗体、实施例2制备得到的Ig-M-9G1RMb2抗体和市场阻断剂原料分别加入到包被体系中,空白对照组包被体系中不加,分别检测RF样本1和RF样本2。
Ig-M-9G1RMb1抗体、Ig-M-9G1RMb2抗体在GRP化学发光平台的阻断效果如表3所示,结果显示,实验组对RF样本有显著的消除作用;其中,Ig-M-9G1RMb1抗体、Ig-M-9G1RMb2抗体在非常低的浓度(如5μg/mL)下即可对RF样本有显著的阻断效果,且其阻断效果显著好于市场阻断剂原料。
表3
表3中的数值为化学发光免疫分析仪读取的OD值,OD值越低,代表检测信号越弱,说明阻断效果越好。
实施例5抗人IgM抗体的稳定性考核
将实施例1制备得到的Ig-M-9G1RMb1抗体和实施例2制备得到的Ig-M-9G1RMb2抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天,取7天、14天、21天样品进行状态观察,并对21天样品进行活性检测(利用酶免检测OD结果考核样品的活性)。
Ig-M-9G1RMb1抗体稳定性测试结果如表4所示,结果显示,三种考核条件下抗体放置21天均未见明显蛋白状态变化,活性也未随考核温度的升高呈下降趋势,说明本发明制备得到的抗人IgM抗体的稳定高。
表4
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 东莞市朋志生物科技有限公司
<120> 抗人IgM抗体及其应用
<130> 2021
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Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe Gln
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Gly
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Ala Arg Glu Pro Leu Pro His Tyr Tyr Ala
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Arg Ala Ser Gln Glu Ile Ser Gly Phe Leu Ser
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Ala Ala Ser Thr Leu Asp Ser
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Ile Gln Tyr Thr Ser Phe Pro Leu
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Leu Gln Tyr Thr Ser Phe Pro Leu
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Ser Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Ser Ile Lys Asp Ala
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Tyr Ile His Trp Val Lys Gln Arg Pro Lys Gln Gly Leu Glu Trp Ile
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Gly Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe
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Gln Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
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Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
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Gly Thr Ser Val Thr Val Ser Ser
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Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Phe
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Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
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Ser Thr Ser Gly Ser Asp Tyr Arg Leu Lys Ile Ser Ser Leu Glu Ser
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Glu Asp Phe Ala Asp Tyr Tyr Cys Ile Gln Tyr Thr Ser Phe Pro Leu
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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
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Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
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Tyr Ile His Trp Val Lys Gln Arg Pro Lys Gln Gly Leu Glu Trp Ile
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Gly Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe
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Gln Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
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85 90 95
Ala Arg Glu Pro Leu Pro His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln
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Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val
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Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr
130 135 140
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Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys
210 215 220
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225 230 235 240
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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
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Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Phe
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Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
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Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
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Ser Thr Ser Gly Ser Asp Tyr Arg Leu Lys Ile Ser Ser Leu Glu Ser
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Glu Asp Phe Ala Asp Tyr Tyr Cys Ile Gln Tyr Thr Ser Phe Pro Leu
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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala
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Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
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Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
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Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
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Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
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Ser Thr Ser Gly Ser Asp Tyr Arg Leu Lys Ile Ser Ser Leu Glu Ser
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Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Thr Ser Phe Pro Leu
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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala
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Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
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Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
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Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
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Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
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Claims (15)
1.抗人IgM抗体或其抗原结合片段,其特征在于,所述抗体或其抗原结合片段包含重链可变区和轻链可变区,所述重链可变区含有:
氨基酸序列如SEQ ID NO.1所示的HCDR1,氨基酸序列如SEQ ID NO.2所示的HCDR2,氨基酸序列如SEQ ID NO.3所示的HCDR3;
所述轻链可变区含有:氨基酸序列如SEQ ID NO.4所示的LCDR1,氨基酸序列如SEQ IDNO.5所示的LCDR2,氨基酸序列如SEQ ID NO.6或SEQ ID NO.7所示的LCDR3。
2.抗人IgM抗体或其抗原结合片段,其特征在于,所述抗体或其抗原结合片段还含有重链可变区和轻链可变区;所述重链可变区的氨基酸序列如SEQ ID NO:8所示,所述轻链可变区的氨基酸序列如SEQ ID NO:9或SEQ ID NO:10所示。
3.根据权利要求1~2任一项所述抗体或其抗原结合片段,其特征在于,所述抗体或其抗原结合片段还含有重链恒定区和轻链恒定区;所述重链恒定区为IgG1、IgG2、IgG3、IgG4、IgA、IgD、IgE或IgM中的任一种或几种,所述轻链恒定区为κ链或λ链。
4.根据权利要求3所述的抗体或其抗原结合片段,其特征在于,所述重链恒定区和轻链恒定区的种属来源为牛、马、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅或人。
5.根据权利要求4所述的抗体或其抗原结合片段,其特征在于,所述重链恒定区和轻链恒定区的种属来源为乳牛。
6.根据权利要求4所述的抗体或其抗原结合片段,其特征在于,所述重链恒定区和轻链恒定区的种属来源为火鸡或斗鸡。
7.抗人IgM抗体或其抗原结合片段,其特征在于,所述抗体或其抗原结合片段还包含重链和轻链,所述重链的氨基酸序列如SEQ ID NO:11所示,所述轻链的氨基酸序列如SEQ IDNO:12或SEQ ID NO:13所示。
8.核酸,其特征在于,所述核酸编码权利要求1~7任一所述抗体或其抗原结合片段。
9.载体,其特征在于,所述载体含有权利要求8所述核酸。
10.细胞,其特征在于,所述细胞含有权利要求8所述核酸或权利要求9所述载体。
11.权利要求1~7任一所述抗体或其抗原结合片段、权利要求8所述核酸、权利要求9所述载体或权利要求10所述细胞在制备免疫检测试剂或试剂盒中的应用。
12.权利要求1~7任一所述抗体或其抗原结合片段、权利要求8所述核酸、权利要求9所述载体或权利要求10所述细胞在制备免疫阻断剂中的应用。
13.一种免疫阻断剂,其特征在于,含有权利要求1~7任一所述抗体或其抗原结合片段、权利要求8所述核酸、权利要求9所述载体或权利要求10所述细胞。
14.一种降低/消除内源性干扰的方法,其特征在于,在免疫检测***中添加权利要求13所述免疫阻断剂。
15.一种免疫诊断试剂/试剂盒,其包含如权利要求13所述免疫阻断剂。
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CN108712908A (zh) * | 2016-01-08 | 2018-10-26 | 梅迪托普生物科学有限公司 | 自交联抗体 |
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