CN114686443B - 杂交瘤细胞、抗血栓调节蛋白单克隆抗体及其制备方法和应用 - Google Patents
杂交瘤细胞、抗血栓调节蛋白单克隆抗体及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种杂交瘤细胞、抗血栓调节蛋白单克隆抗体及其制备方法和应用,该杂交瘤细胞分泌的单克隆抗体表位选择性好且全蛋白亲和力高。通过将该杂交瘤细胞所生产的抗TM单克隆抗体TM1应用于TM蛋白的体外检测例如制备化学发光检测试剂盒等,可对血液样本中的TM蛋白浓度准确定量,具有巨大的经济和社会效益。
Description
技术领域
本发明涉及细胞技术领域,特别是涉及一种杂交瘤细胞、抗血栓调节蛋白单克隆抗体及其制备方法和应用。
背景技术
血栓调节蛋白(Thrombomodulin,TM)为一单链的跨膜糖蛋白,相对分子质量为75kDa,降解二硫键后相对分子质量为105kDa。其基因定位于第20对常染色体,含有18个外显子,无内含子,长度为3.7kb,能转录碱基1725bp,其表达产物由575个氨基酸组成。其中,富含丝氨酸、苏氨酸区域或O2结合糖链区为肝素样多糖结构在TM分子上的附着部位,与TM抑制凝血酶的活性有密切关系,有可能是凝血酶在TM分子上的第二结合部位。由23个氨基酸组成的跨膜区主要由疏水氨基酸组成,无其他的已知受体蛋白的同源序列,而C端胞浆区与TM的降解和内吞作用有关。TM最初发现于血管内皮细胞。免疫组织化学染色证明,约99%以上的血管内皮细胞表达TM,每个内皮细胞有(0.3~1.0)× 105个TM分子。最近研究发现,TM亦存在于胎盘滋养层细胞、血小板、巨核细胞、单核细胞、中性粒细胞、滑液层细胞、角化细胞、脑膜细胞、平滑肌细胞、肿瘤细胞。
TM的表达由一系列机制来调控,TM仅存在于体内部分细胞中,这种组织特异性的表达可能由TM基因中特有的启动子控制,转录速度的改变和翻译的改变也影响量的表达。Cal研究发现,经血管内皮生长因子(VEGF)处理后的细胞TM抗原及TM mRNA水平能增加大约2.5倍,并将PC激活的效率提高50%~80%。同时,VEGF还能阻断IL21对TM抗原及mRNA表达的抑制,对抗TGF2β和脂多糖对TM表达的下调作用。1,25二羟基维生素D3及其衍生物也能抑制单核细胞TF的表达和上调TM的表达。TM有两种存在形式,即固定型(膜型)和溶解型(血液型)。前者存在于细胞表面,后者游离于血浆和尿液中。血浆及尿液中的TM相对分子质量大小不同。TM是近年来发现的存在于血管内皮细胞表面的跨膜糖蛋白,它在机体抗凝血机制中发挥着重要的作用。作为细胞黏附分子的一员,TM也参与调节肿瘤细胞的发生、发展及转移。因此,医学中TM已被视为内皮细胞损伤的标志物,用于临床疾病的诊断和鉴别,对疾病的诊断和衡量治疗状况具有重要指标性的意义。
发明内容
基于此,有必要提供一种可分泌表位选择性好且全蛋白亲和力高的抗血栓调节蛋白单克隆抗体的杂交瘤细胞。
本发明提供了一种可分泌抗血栓调节蛋白单克隆抗体的杂交瘤细胞,保藏号为CCTCC No:C202063。
本发明还提供了一种上述杂交瘤细胞分泌的抗血栓调节蛋白单克隆抗体,所述抗血栓调节蛋白单克隆抗体记为TM1。
本发明还提供了一种抗血栓调节蛋白单克隆抗体的制备方法,包括以下步骤:向小鼠腹腔注射上述杂交瘤细胞,采集腹水后分离纯化,得到所述抗血栓调节蛋白单克隆抗体。
在其中一个实施例中,在进行所述腹腔注射时,每只小鼠注射4×105~6× 105个所述杂交瘤细胞。
本发明还提供了一种上述杂交瘤细胞或上述抗血栓调节蛋白单克隆抗体在制备用于检测血栓调节蛋白的产品中的应用。
本发明还提供了一种血栓调节蛋白检测试剂盒,包括上述抗血栓调节蛋白单克隆抗体。
在其中一个实施例中,血栓调节蛋白检测试剂盒还包括保藏号为CCTCC No:C202064的杂交瘤细胞所分泌的抗血栓调节蛋白单克隆抗体TM2。
在其中一个实施例中,血栓调节蛋白检测试剂盒还包括磁微粒和碱性磷酸酶。
在其中一个实施例中,所述抗血栓调节蛋白单克隆抗体TM1包被于磁微粒上,且溶解于第一检测液中;所述抗血栓调节蛋白单克隆抗体TM2与碱性磷酸酶连接,且溶解于第二检测液中。
本发明还提供了一种上述杂交瘤细胞的制备方法,包括以下步骤:
取小鼠以血栓调节蛋白作为免疫原进行首次免疫,所述血栓调节蛋白的氨基酸序列如SEQ ID NO:1所示;
将经所述首次免疫的小鼠以血栓调节蛋白N末端片段作为免疫原进行至少两次加强免疫和一次冲击免疫,所述血栓调节蛋白N末端片段的氨基酸序列如 SEQ ID NO:2所示;
取经所述冲击免疫的小鼠的脾细胞与骨髓瘤细胞融合,并筛选得到稳定分泌抗血栓调节蛋白单克隆抗体TM1的杂交瘤细胞。
研究发现,TM在人体血浆中大部分以游离形式存在,其中一小部分会凝血酶结合,且存在不同分子量片段,因此若要通过免疫学对其准确定量,则需要筛选到表位选择性好且全蛋白亲和力高的单克隆抗体。然而,一般的TM单克隆抗体对表位选择性较差,亲和力也较低,无法准确地识别样本中以不同形式存在的血栓调节蛋白,定量准确性不高。本发明提供了一种筛选得到的杂交瘤细胞,该杂交瘤细胞分泌的单克隆抗体表位选择性好且全蛋白亲和力高。通过将该杂交瘤细胞所生产的抗TM单克隆抗体TM1应用于TM蛋白的体外检测例如制备化学发光检测试剂盒等,可对血液样本中的TM蛋白浓度准确定量,具有巨大的经济和社会效益。
附图说明
图1为实施例中免疫小鼠尾血效价检测结果图;
图2为实施例中TM1和TM2抗体的效价测定结果图;
图3为实施例中TM1、TM2和TM3抗体的灵敏度测定结果图;
图4为实施例中血栓调节蛋白的标准曲线;
图5为实施例中待测试剂与希森美康化学发光试剂检测100份临床样品相关性对比图。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述,并给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明一实施例的可分泌抗血栓调节蛋白单克隆抗体的杂交瘤细胞株TM1#,于2020年4月18日保藏在中国典型培养物保藏中心(CCTCC),保藏号为CCTCC No:C202063,地址为中国,武汉,武汉大学。该杂交瘤细胞可分泌出抗血栓调节蛋白单克隆抗体,记为杂交瘤细胞株TM1#,TM1可以作为血栓调节蛋白的检测抗体,应用于血栓调节蛋白的检测试剂盒或检测设备等产品的制备领域中。
本发明一实施例的抗血栓调节蛋白单克隆抗体TM1的制备方法,包括以下步骤:向小鼠腹腔注射上述杂交瘤细胞,采集腹水后分离纯化,得到抗血栓调节蛋白单克隆抗体TM1。优选地,在进行腹腔注射时,每只小鼠注射4×105~6 ×105个杂交瘤细胞。
本发明一实施例的血栓调节蛋白检测试剂盒,包括上述抗血栓调节蛋白单克隆抗体TM1。
在一个具体示例中,血栓调节蛋白检测试剂盒还包括保藏号为CCTCC NO:C202064的杂交瘤细胞分泌的单克隆抗体TM2。
在一个具体示例中,血栓调节蛋白检测试剂盒还包括磁微粒和碱性磷酸酶。具体地,血栓调节蛋白检测试剂盒包括第一检测液和第二检测液,第一检测液中含有磁微粒包被的抗血栓调节蛋白单克隆抗体TM1,第二检测液中含有碱性磷酸酶标记的抗血栓调节蛋白单克隆抗体TM2。可以理解,用于包被抗体的物质不限于磁微粒,用于标记抗体的物质也不限于碱性磷酸酶,其他常用的包被物和标记物均可。检测时,向待测样品中加入第一检测液和第二检测液,抗血栓调节蛋白单克隆抗体TM1和TM2分别针对不同的血栓调节蛋白表位,待测样品中的血栓调节蛋白能够夹心于抗体TM1和TM2之间,根据碱性磷酸酶的发光信号即可测定待测样品中血栓调节蛋白的含量,可避免血栓调节蛋白上的部分表位被屏蔽而导致检测准确性降低。
在一个具体示例中,第一检测液通过以下方法制备得到:将磁微粒、抗血栓调节蛋白单克隆抗体TM1和结合缓冲液混合,振荡培养过夜。孵育后除去上清液,用牛血清白蛋白(BSA)封闭磁微粒上的残留结合位点,洗涤后分散于检测缓冲液中即可。
在一个具体示例中,第二检测液通过以下方法制备得到:将碱性磷酸酶、抗血栓调节蛋白单克隆抗体TM2和水混合,加入含戊二醛的磷酸盐缓冲液,在黑暗中轻微振荡温育。然后加入单乙醇胺溶液并振荡温育,接着在低温下用磷酸盐缓冲液透析过夜,再与等体积的甘油和1%BSA混合即可。
本发明一实施例的上述杂交瘤细胞的制备方法,包括以下步骤S1~S3:
S1、取小鼠以血栓调节蛋白作为免疫原进行首次免疫,血栓调节蛋白的氨基酸序列如SEQ ID NO:1所示。
S2、将经首次免疫的小鼠以血栓调节蛋白N末端片段作为免疫原进行至少两次加强免疫和一次冲击免疫,血栓调节蛋白N末端片段的氨基酸序列如SEQ ID NO:2所示。
S3、取经冲击免疫的小鼠的脾细胞与骨髓瘤细胞融合,并筛选得到稳定分泌抗血栓调节蛋白单克隆抗体TM1的杂交瘤细胞。
在一个具体示例中,血栓调节蛋白、血栓调节蛋白N末端片段和血栓调节蛋白核心片段通过基因克隆、质粒构建、转染、宿主表达和分离纯化等步骤得到。
在一个具体示例中,脾细胞与骨髓瘤细胞融合的方式为电融合。
本发明另一实施例的可分泌抗血栓调节蛋白单克隆抗体的杂交瘤细胞株 TM2#,于2020年4月18日保藏在中国典型培养物保藏中心(CCTCC),保藏号为CCTCC No:C202064,地址为中国,武汉,武汉大学。该杂交瘤细胞可分泌出抗血栓调节蛋白单克隆抗体,记为杂交瘤细胞株TM2#,TM2可以作为血栓调节蛋白的检测抗体,应用于血栓调节蛋白的检测试剂盒或检测设备等产品的制备领域中。
本发明一实施例的抗血栓调节蛋白单克隆抗体TM2的制备方法,包括以下步骤:向小鼠腹腔注射上述杂交瘤细胞,采集腹水后分离纯化,得到抗血栓调节蛋白单克隆抗体TM2。优选地,在进行腹腔注射时,每只小鼠注射4×105~6 ×105个杂交瘤细胞。
本发明一实施例的血栓调节蛋白检测试剂盒,包括上述抗血栓调节蛋白单克隆抗体TM2。在一个具体示例中,血栓调节蛋白检测试剂盒还包括磁微粒和碱性磷酸酶。
本发明一实施例的上述杂交瘤细胞的制备方法,包括以下步骤S1~S3:
S1、取小鼠以血栓调节蛋白作为免疫原进行首次免疫,血栓调节蛋白的氨基酸序列如SEQ ID NO:1所示。
S2、将经首次免疫的小鼠以血栓调节蛋白核心片段作为免疫原进行至少两次加强免疫和一次冲击免疫,血栓调节蛋白核心片段的氨基酸序列如SEQ ID NO:3所示。
S3、取经冲击免疫的小鼠的脾细胞与骨髓瘤细胞融合,并筛选得到稳定分泌抗血栓调节蛋白单克隆抗体TM2的杂交瘤细胞。
研究发现,TM在人体血浆中大部分以游离形式存在,其中一小部分会凝血酶结合,且存在不同分子量片段,因此若要通过免疫学对其准确定量,则需要筛选到表位选择性好且全蛋白亲和力高的单克隆抗体。然而,一般的TM单克隆抗体对表位选择性较差,亲和力也较低,无法准确地识别样本中以不同形式存在的血栓调节蛋白,定量准确性不高。本发明提供了两株筛选得到的杂交瘤细胞,该杂交瘤细胞分泌的单克隆抗体表位选择性好且全蛋白亲和力高。通过将该杂交瘤细胞所生产的抗TM单克隆抗体TM1、TM2应用于TM蛋白的体外检测例如制备化学发光检测试剂盒等,可对血液样本中的TM蛋白浓度准确定量,具有巨大的经济和社会效益。
以下为具体实施例。
1抗血栓调节蛋白单克隆抗体的制备
1.1抗原制备
1.1.1目的基因的克隆
从人全血中提取基因组DNA作为模板,分别以TM全序列、TM-N端区域及核心区域(EGF1~6)的相应引物克隆目标基因。测序正确后,将其连接到 pET28a(+)并转染到HD5α进行表达测试。确认表达后,以BL21为宿主,进行目标蛋白的大量表达,通过Ni-NTA亲和层析进行纯化,以获得高纯度蛋白。蛋白分别命名为TM-Total、TM-NT和TM-EGF,氨基酸序列分别如SEQ ID NO: 1~SEQ ID NO:3所示,-80℃保存备用。
1.2动物免疫方法及步骤
选取5~6周雌性BALB/c小鼠6只,以TM-Total完成首次免疫后,分两组,每组3只,分别以TM-NT和TM-EGF进行三次加强免疫,于三次免疫后的第七天,取尾血进行血清效价检测,结果见图1所示。选取效价最高的小鼠,进行冲击融合,免疫流程见表1所示。
表1蛋白免疫信息汇总
1.3脾细胞与骨髓瘤细胞的融合
融合前一周,采用常规的细胞复苏方法,复苏冻存的骨髓瘤细胞(SP2/0) 细胞,然后挑选状态好的SP2/0细胞,融合前一天换液。取免疫效价符合要求的小鼠的脾脏细胞,置于无菌筛网上。用注射器芯研磨脾脏,边研磨边滴加1640 不完全培养基,研磨至只剩白色的脾脏膜。收集脾细胞至50mL离心管,1200rpm 离心5min,弃上清,收集,重悬。SP2/0与脾细胞离心后分别用10mL不完全培养基重悬,稀释计数。最适宜比为1:1到1:4,按比例将两种细胞混合。离心1200rpm 5min,弃上清。用ECF Buffer重悬混匀,再1200r/min低速离心5min,去上清。重复ECF Buffer洗涤一遍。最后用6.4mL ECF Buffer重悬,备用。接通电融合仪电源,按“Ω”键,测电阻,电阻值应大于2KΩ。加入6.4ml ECF Buffer 重悬的细胞混合液。注意应缓慢滴加,以免产生气泡影响电阻。按“Ω”键,确认电阻值在0.8KΩ~2KΩ之间。无误后再按“Start”键,等待电融合提示完成。电击结束后将细胞混合液转移至12.8mL修复液中,修复破坏细胞膜。37℃(培养箱)静置10min。注意转移时吸头须伸到液面以下,再缓慢打出,800rpm离心5min,弃上清。用完全培养基(含20%胎牛血清、1%HAT和适宜比例饲养细胞)重悬细胞,转移至96孔板,于37℃细胞培养箱中进行培养。
1.4间接ELISA检测
采用免疫小鼠的免疫抗原用包被液稀释,加入96孔酶标板,每孔加入 100μL,4℃包被过夜,PBST洗涤孔板1次,然后用含3%吐温PBS封闭4℃包被过夜。PBST洗涤孔板1次后,将每只免疫小鼠的血清(或是融合培养后待检测的细胞上清及筛选的阳性克隆细胞)分别稀释103、104、105、106倍,加入反应孔中,以未免疫的小鼠血清作为阴性对照,放入37℃恒温箱内反应1h,采用辣根过氧化物酶(HRP)标记的羊抗鼠作为二抗进行检测,加入TMB显色底物反应后,酶标仪读取各反应孔OD450。
1.5抗体表位分析
以TM-Total、TM-EGF和TM-NT分别包被酶标板,检测所获得的单克隆抗体,并根据检测结果,将抗体分为两组,组1为核心区域组,命名为NT,组2 为非核心区域组,命名为EGF,如表2所示。
1.6抗体亚类的鉴定
取细胞培养上清,按照Sigma公司的单克隆抗体亚类鉴定试剂盒对制备的单克隆抗体进行亚类分析,结果如表2所示,其中1号(即上述TM1)和8号 (即上述TM2)单克隆抗体性能较好。保藏号为CCTCC No:C202063的细胞系分泌的抗体即为TM1,保藏号为CCTCC No:C202064的细胞系分泌的抗体即为TM2。
表2抗体信息及表征汇总
1.7单克隆抗体的制备
挑选与TM-Total反应的细胞株,亚克隆至定株后,以5×105个细胞/只小鼠的密度注射到小鼠腹腔,于7~14天采集腹水,以Protein A进行亲和纯化,微量分光光度计测定蛋白浓度,SDS-PAGE测定抗体纯度。
1.8抗体的效价测定
间接ELISA法进行测定,将纯化后的抗体以PBS调整至1mg/mL,分别用含1%吐温PBST稀释到103、104、105、106倍。在ELISA检测仪上,于450nm (若以ABTS显色,则为410nm)处,以阴性对照孔调零后测各孔值,若OD 大于规定的阴性对照OD值的2.1倍,即为阳性,结果见图2(以筛选得到的性能最优的TM1和TM2为例),效价均可达1/105。
1.9抗体灵敏度测定
间接ELISA法进行测定,将TM-Total抗原用包被液从1μg/mL开始,进行 2倍稀释,共12个梯度,包被酶标板,将纯化后抗体以PBS调整至1mg/mL,最终用含1%吐温PBST稀释到1μg/mL,按传统简介ELISA实验流程操作。以阴性对照孔调零后测各孔值,若OD大于规定的阴性对照OD值的2.1倍,即为阳性,对应稀释度为该抗体的灵敏度。结果见图3(以筛选得到的性能最优的 TM1和TM2以及TM3为例),TM1和TM2两个抗体在该方法上,灵敏度均可达1ng/mL以下。
2化学发光TM检测试剂的建立
2.1抗TM单克隆抗体TM1包被磁微粒
首先,将20mg/mL的MPs置于2.0mL EP管中,用结合缓冲液洗涤MPs五次。在洗涤过程中,将EP管置于磁力浓缩器上并除去上清液。然后,将MPs 重悬于2mL结合缓冲液中,将抗体溶液加入到悬浮液中,在37℃下振荡培养过夜。孵育后,将EP管置于磁力浓缩器中以将它们与上清液分离。用3%牛血清白蛋白(BSA)封闭MPs上的残留结合位点,在37℃温育并轻微摇动2小时。洗涤5次后,将磁珠包被抗体(mAb-MPs)分散在2mL缓冲液中并在4℃下保存,备用。
2.2碱性磷酸酶标记抗TM单克隆抗体TM2的制备
首先,将碱性磷酸酶(AP)和识别另一个位点的抗TM单克隆抗体TM2悬浮在超纯水中并分别稀释至4mg/mL和8mg/mL。将250μL等份的4mg/mL AP 溶液转移至1.5mL EP管中并与250μL 8mg/mL抗TM单克隆抗体溶液混合。其次,向溶液中加入0.5mL含1%戊二醛的0.1mol/L磷酸盐缓冲液(pH7.4)。将所得混合物在37℃下于黑暗中轻微振荡温育4小时。第三步,向混合物中加入0.1mL 1mol/L单乙醇胺溶液,随后在室温下振荡温育2小时。将混合物在4℃下用PBS溶液透析过夜,透析后将酶标抗体转移至EP管中,并与等体积的甘油和1%BSA混合。最后,将酶标抗体(AP-mAbs)储存在-20℃,备用。
2.3基于MPs的全自动化学发光法检测TM
应用磁珠包被抗体(mAb-MPs)和酶标抗体(AP-mAbs)夹心反应模式在全自动化学发光仪上进行TM检测。首先,将50μL不同浓度的mAb-MP和CPP 样品或者标准品(30μL)分别移液到与仪器匹配的管中,并在37℃轻轻摇动孵育20分钟(捕获时间)。然后管子通过清洗站用洗涤液(含有0.05%Tween的 0.01mol/L PBS)洗涤3次,去除非特异性的结合。然后加入AP-mAbs,37℃轻轻摇动孵育10分钟。此时,夹心免疫复合物MPs-TM-AP形成。将形成的夹心免疫复合物磁分离,并通过洗涤除去过量的AP-mAbs。随后,将含有发光底物 AMPPD(200μL)的溶液加入夹心复合物。将所得混合物在免疫测定仪器中温育,并测量相对光单位(RLU)的值。
2.4免疫分析试剂的优化及性能评估
一系列稀释的AP-mAbs(1:50,1:100,1:200,1:00,)和mAb-MPs(1:20,1:50,1:100,1:200,1:500)与TM的标准阳性校准品(S3,10TU/mL)和阴性样品(S0,0TU/mL)反应,以两者的最大RLU比(RLUS3/RLUS0)确定最佳稀释度。以最佳组合为基础,绘制标准曲线如图4所示,确定其灵敏度及线性区间,并与希森美康化学发光试剂对比分析100例临床样品,以考察新开发的免疫测定方法的可行性。结果如表3和图5所示所示,待测试剂阳性符合率:92.86%,阴性符合率:97.73%,总符合率:95%。
表3临床测试
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 广州万孚生物技术股份有限公司
<120> 杂交瘤细胞、抗血栓调节蛋白单克隆抗体及其制备方法和应用
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Claims (9)
1.一种可分泌抗血栓调节蛋白单克隆抗体的杂交瘤细胞,其特征在于,保藏号为CCTCCNo:C202063。
2.一种权利要求1所述的杂交瘤细胞分泌的抗血栓调节蛋白单克隆抗体,其特征在于,所述抗血栓调节蛋白单克隆抗体记为TM1。
3.一种抗血栓调节蛋白单克隆抗体的制备方法,其特征在于,包括以下步骤:向小鼠腹腔注射权利要求1所述的杂交瘤细胞,采集腹水后分离纯化,得到所述抗血栓调节蛋白单克隆抗体。
4.根据权利要求3所述的制备方法,其特征在于,在进行所述腹腔注射时,每只小鼠注射4×105~6×105个所述杂交瘤细胞。
5.权利要求1所述的杂交瘤细胞或权利要求2所述的抗血栓调节蛋白单克隆抗体在制备用于检测血栓调节蛋白的产品中的应用。
6.一种血栓调节蛋白检测试剂盒,其特征在于,包括权利要求2所述的抗血栓调节蛋白单克隆抗体。
7.根据权利要求6所述的血栓调节蛋白检测试剂盒,其特征在于,还包括保藏号为CCTCC No:C202064的杂交瘤细胞所分泌的抗血栓调节蛋白单克隆抗体TM2。
8.根据权利要求7所述的血栓调节蛋白检测试剂盒,其特征在于,还包括磁微粒和碱性磷酸酶。
9.根据权利要求7所述的血栓调节蛋白检测试剂盒,其特征在于,所述抗血栓调节蛋白单克隆抗体TM1包被于磁微粒上,且溶解于第一检测液中;所述抗血栓调节蛋白单克隆抗体TM2与碱性磷酸酶连接,且溶解于第二检测液中。
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CN1665526A (zh) * | 2002-05-01 | 2005-09-07 | 舍林股份公司 | 作为抗凝剂的新的靶向组织因子的血栓调节蛋白融合蛋白 |
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CN1665526A (zh) * | 2002-05-01 | 2005-09-07 | 舍林股份公司 | 作为抗凝剂的新的靶向组织因子的血栓调节蛋白融合蛋白 |
CN102858978A (zh) * | 2010-04-30 | 2013-01-02 | 旭化成制药株式会社 | 高纯度可溶性血栓调节蛋白及其制造方法 |
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