CN110456077A - 一种偶联抗EpCAM抗体的免疫磁珠富集检测CTCs的方法 - Google Patents
一种偶联抗EpCAM抗体的免疫磁珠富集检测CTCs的方法 Download PDFInfo
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Abstract
本发明为一种偶联抗EpCAM抗体的免疫磁珠富集检测CTCs的方法:1、抗EpCAM单克隆抗体与磁珠偶联;2.制备循环肿瘤细胞样品:取免疫磁珠,加入生物肿瘤细胞样本中混匀,然后用磁力分离器分离,PBS清洗,将磁珠吸附细胞悬浮于PBS溶液中,进行细胞计数,使用细胞计数板检测富集到的细胞;将纳米免疫磁珠加入1ml人工肿瘤细胞血液样品中磁力分离,PBS清洗,然后将磁珠吸附细胞重新悬浮于PBS中;取5ul样品,滴加于载玻片上,使用抗EpCAM‑FICT,进行孵育,使用流式细胞仪对富集的细胞进行检测,肿瘤细胞‑荧光标记EpCAM鉴定有效性。在荧光倒置显微镜下进行计数,肿瘤细胞株细胞混入外周血后的富集、鉴定同上。极大地提高了循环肿瘤细胞检测的敏感性和特异性。
Description
技术领域
本发明涉及免疫检测领域,具体为一种偶联抗EpCAM抗体的免疫磁珠富集检测CTCs的方法。
背景技术
检测循环肿瘤细胞有助于早期发现患者体内的肿瘤、监测患者术后体内是否出现肿瘤复发或转移、评估预后进而选择合适的治疗策略,尽早帮助患者治疗。近年来,检测循环肿瘤细胞的方法集中在免疫细胞疗法、逆转录聚合酶链反应、流式细胞术、免疫磁珠富集检测法等,但这些方法都存在不同方面的检测缺陷:
1、免疫细胞疗法:免疫细胞疗法的原理是将特异性抗体用显色剂标记,之后通过在组织细胞原位进行抗原抗体反应以及细胞进行化学呈色,对相应的目的抗原定位、定性以及定量检测分析的技术方法。免疫细胞疗法的缺点是:检测到的细胞数量很少,灵敏度仅为105-106。因为免疫细胞化学检测循环血液中的肿瘤细胞时,每个载玻片上可检测的细胞样品量仅为5×10 5个细胞。但是检测肿瘤细胞的理想方法应该能够检测来自2.5×108外周血有核细胞的单个肿瘤细胞。那么,从2.5×108个有核血细胞中筛选肿瘤细胞需要制备和分析约500个载玻片,因此免疫细胞化学检测率低,难以从外周血中的大量单核细胞中检测到非常少量的循环肿瘤细胞,简单的免疫细胞疗法不能用于肿瘤患者循环肿瘤细胞的临床检测。
2、逆转录聚合酶链反应:逆转录聚合酶链反应是在逆转录酶的作用下合成与标记的mRNA互补的cDNA,然后以cDNA为模板PCR周期扩增,进而制备大量的所需要的cDNA片段,大大提高了灵敏度。逆转录聚合酶链反应检测方法的缺点是:由于目的mRNA的非特异性表达、标本和PCR产物的污染,PCR扩增条件的不正确控制以及非特异性细胞的非法转录水平低,逆转录聚合酶链反应方法的检测结果具有高假阳性率。同时,逆转录聚合酶链反应的测试过程复杂,测试耗时较长。因此,逆转录聚合酶链反应在循环肿瘤细胞的检测中可能缺乏某些特异性,这限制了其临床诊断使用价值。
3.流式细胞术:流式细胞术是一项应用激光技术、电子物理技术、光电测量技术、计算机技术以及细胞荧光化学技术、单克隆抗体技术为一体的新型技术。流式细胞术检测循环肿瘤细胞的缺点是使用流式细胞术检测循环肿瘤细胞的价值在很大程度上取决于可以分析的细胞数量。通过流式细胞术检测的靶细胞的灵敏度仅为1/104,而外周血中的肿瘤细胞的数量通常小于1/106。
免疫磁珠技术是近年来国内外研究的一种新型免疫技术,因为纳米免疫磁珠具有良好的磁感应能力、超顺磁性、高分离性、高特异性且不会影响细胞活性等优点,因此近几年有更多研究者将纳米免疫磁珠技术应用到临床检测领域,特别是将纳米免疫磁珠技术联合其他检测技术使用检测循环肿瘤细胞更是成为热捧,国内外有应用纳米免疫磁珠检测卵巢癌、膀胱癌、前列腺癌、乳腺癌、食管癌、胃癌、大肠癌、肺癌、肝癌、恶性黑色素瘤等患者的外周血肿瘤细胞的文献报道。而本发明就是跟随临床检测潮流,将纳米免疫磁珠技术结合流式细胞术应用到检测循环肿瘤细胞上。
联合应用纳米免疫磁珠技术和流式细胞仪检测循环肿瘤细胞的原理是,利用纳米免疫磁珠的特异性富集和分离,通过流式细胞仪定量计数肿瘤细胞,从而获得更高的灵敏度和准确度和可靠性。Moreno等在37例转移性前列腺癌患者中,在7.5ml外周血肿瘤细胞中富集EpCAM抗体免疫磁珠,然后进行多指标流式细胞仪分析,其中核酸染色阳性,CK阳性,CD45阴性,定义为循环肿瘤细胞。结果发现,每7.5ml血液中前列腺癌细胞的数量从0到8586不等,若以每7.5ml血液中发现5个或更多个前列腺癌细胞作为阈值评估循环肿瘤细胞预测总体生存能力。在37名患者中,23名(62.0%)患有5个或更多癌细胞,平均总生存率为0.7年,而CTCs少于5个的患者总生存率超过4年。在单变量和多变量分析中,26例激素未控制的前列腺癌患者中存在CTCs是最重要的生存预测因子。结论是,在这项前瞻性研究中,每7.5ml血液中存在5个或更多癌细胞与转移性前列腺癌患者的总体存活率密切相关。
EpCAM作为肿瘤诊断和治疗的候选蛋白,其在肿瘤免疫治疗方面的作用将越来越显著。EpCAM可以通过wnt信号通路发挥其致癌效应,因此,以wnt信号通路中的酶为靶点来阻断EpCAM的信号转导将是肿瘤治疗的一个方向。
发明内容
针对现有技术上出现的灵敏度低、检测率低、测试过程复杂、测试耗时较长、假阳率高等问题,本发明提供一种偶联抗EpCAM抗体的免疫磁珠富集检测CTCs的方法,利用免疫细胞磁珠结合流式细胞术的方法检测循环肿瘤细胞可以大大改善这一系列检测缺陷。
本发明提供如下技术方案:一种偶联抗EpCAM抗体的免疫磁珠富集检测CTCs的方法,其特征在于,包括如下步骤:
S1.抗体与磁珠偶联:
1)抗EpCAM单克隆抗体偶联前,在4℃下,经过三次透析,过夜,储存在PBS中,将浓度分别调整到5μmol/L、10μmol/L、15μmol/L以及20μmol/L;
2)纳米免疫磁珠的制备:
取四份活化的羧基磁珠300μL,浓度2mg/mL,分别加入40μL浓度为5μmol/L、10μmol/L、15μmol/L、20μmol/L的抗EpCAM单克隆抗体,在1ml的离心管中,于室温下混合3h,用1ml的PBS清洗三次,加入300μL浓度为0.25mol/L甘氨酸混合25min,封闭剩余的醛基;再次用1ml的PBS清洗三次;加入含有质量百分比2.5%BSA的PBS溶液混合30min,封闭非特异性的吸附位点,之后用1ml的PBS清洗三次,加入300μL PBS缓冲液漩涡震荡,即得到浓度为2.0mg/ml的均匀的修饰有一抗的免疫磁珠;
S2.制备循环肿瘤细胞样品:
1)实验室培养各种肿瘤细胞样本,取各种肿瘤细胞,100μL,加入1.5mL离心管中,肿瘤细胞于外周血中制成人工样本,每种细胞为三管;
2)取5μL上述制备好的各种肿瘤细胞与5mL健康人血液混合,每种细胞制备三管;
3)取适量免疫磁珠,加入100ul生物肿瘤细胞样本中混匀,然后用磁力分离器分离,PBS清洗,将磁珠吸附细胞悬浮于PBS溶液中,进行细胞计数,使用细胞计数板检测富集到的细胞;
4)将适量纳米免疫磁珠加入1ml人工肿瘤细胞血液样品中磁力分离,PBS清洗,然后将磁珠吸附细胞重新悬浮于PBS中;取5ul样品,滴加于载玻片上,使用抗EpCAM-FICT,进行孵育,使用流式细胞仪对富集的细胞进行检测,肿瘤细胞-荧光标记EpCAM鉴定有效性;在荧光倒置显微镜下进行计数,肿瘤细胞株细胞混入外周血后的富集、鉴定同上。
作为优选,所述纳米免疫磁珠的制备的反应条件为:反应介质为0.01M PBS,PH=7.4,温度为室温,反应时间为3h,抗体和磁珠质量比为200μg:1000μg。
作为又一优选,所述肿瘤细胞为乳腺癌症(MCF-7),食道癌(EC109),非小细胞肺癌(A549),子宫颈癌(Hela),胃癌(NCI-N87)、结肠癌(HT29)。
综上所述,本发明的优点:纳米免疫磁珠偶联抗EpCAM抗体结合流式细胞术富集检测法是对常规检测技术缺陷的很好补充,极大地提高了循环肿瘤细胞检测的敏感性和特异性。
具体实施方式
实施例1:对EpCAM抗体标记纳米磁珠的反应条件进行优化,选择最佳的反应条件方法如下:
(1)分别取300μL纳米磁珠于7个离心管中,编号,在外加磁场的作用下,将1、2管中介质换成磷酸盐缓冲液,浓度依次为0.1M PB(pH=7.4)和0.01M PB(pH=7.4),第3-6管中介质分别为0.1M PBS(pH=6.0)、0.01M PBS(pH=6.0)、0.1M PBS(pH=7.4)和0.01M PBS(pH=7.4),第7管中介质为去离子水(pH=6.0),分别向管中加入40μL(5μmol)抗体,放到恒温振荡器上反应2.5h后(150r/min),分离上清液,在紫外分光光度计下测OD280nm,确定最佳的反应介质。
(2)羧基磁珠中加入最适浓度的抗体,分别采用室温(25℃)和37℃两种温度条件下反应3h,然后用0.01M PBS(pH=7.4)洗涤数次,恢复其原体积并测其上清中OD280nm值。
(3)取6个1.5ml离心管,依次编号1-6,向每管中加入300ul活化处理的纳米磁珠,PBS洗涤2-3次,加入40ul(5μmol)抗体,加入后立即测上清OD280nm值,然后每隔1h测OD280nm值,直至6h。各离心管OD值如表1所示。
(4)300ul 2mg/ml纳米磁珠分别用不同量的抗体5μmol,10μL、30μL、40μL、60ul,100μL,补足总体积为400ul,偶联于室温30分钟,加入后立即测上清OD280nm值,偶联完成后,再测一次OD280nm值。
表1抗体偶联磁珠不同偶联时间下OD280值
试验结论:最佳反应条件为反应介质为0.01M PBS(PH=7.4);温度为室温;反应时间为3h,抗体和磁珠质量比为200μg:1000μg。
实例2:采用300nm羧基磁珠与5μmol EpCAM抗体偶联,在荧光倒置显微镜下进行计数,统计回收肿瘤细胞占富集前细胞总数的比例,计算免疫磁珠富集血液样品中肿瘤细胞效率。
试验步骤:
1.抗体与磁珠偶联:
1)抗体偶联前,在4度下,经过三次透析,过夜,储存在PBS中,将浓度调整到5μmol。
2)纳米免疫磁珠的制备:
取活化的羧基磁珠300μL,2mg/mL,加入40μL 5μmol的抗EpCAM单克隆抗体,在1ml的离心管中,于室温下混合3h,用1ml PBS清洗三次,加入300μL0.25mol/L甘氨酸混合25min,封闭剩余的醛基;1ml PBS清洗三次;加入含有质量百分比2.5%BSA的PBS溶液混合30min,封闭非特异性的吸附位点,PBS清洗3次,加入300μL PBS缓冲液漩涡震荡,即得到浓度为2.0mg/ml的均匀的修饰有一抗的免疫磁珠。反应条件为:反应介质为0.01M PBS(PH=7.4);温度为室温;反应时间为3h,抗体和磁珠质量比为200μg:1000μg。
对上述羧基磁珠与EpCAM抗体偶联的磁珠分别对人乳腺癌症(MCF-7),食道癌(EC109),非小细胞肺癌(A549),子宫颈癌(Hela),胃癌(NCI-N87),结肠癌(HT29),进行捕获分离测试,检测捕获效果。
2.制备循环肿瘤细胞样品:
①实验室培养各种肿瘤细胞样本,取各种肿瘤细胞,100μL,加入1.5mL离心管中,形成肿瘤细胞在外周血中制成人工样本,每种细胞为三管;
②取5μL上述制备好的各种肿瘤细胞与5mL健康人血液混合,每种细胞制备三管;
③取适量免疫磁珠,加入100ul生物肿瘤细胞样本中混匀,然后用磁力分离器分离,PBS清洗,将磁珠吸附细胞悬浮于PBS溶液中,进行细胞计数,使用细胞计数板检测富集到的细胞;
④将适量纳米免疫磁珠加入1ml人工肿瘤细胞血液样品中磁力分离,PBS清洗,然后将磁珠吸附细胞重新悬浮于PBS中;取5ul样品,滴加于载玻片上,使用抗EpCAM-FICT,进行孵育,使用流式细胞仪对富集的细胞进行检测,肿瘤细胞-荧光标记EpCAM鉴定有效性。
试验结果:
(1)300nm羧基磁珠5μmol抗体浓度富集人工样本肿瘤细胞1,统计结果如表2所示:
表2 300nm羧基磁珠5μmol抗体浓度富集人工样本肿瘤细胞
(2)300nm羧基磁珠5μmol抗体浓度富集人工样本肿瘤细胞2,统计结果如表3所示:
表3 300nm羧基磁珠5μmol抗体浓度富集人工样本肿瘤细胞2
(3)300nm羧基磁珠5μmol抗体浓度富集人工样本肿瘤细胞3,统计结果如表4所示:
表4 300nm羧基磁珠5μmol抗体浓度富集人工样本肿瘤细胞3
(4)300nm羧基磁珠5μmol抗体浓度富集人工样本肿瘤细胞3,统计结果如表5所示:
表5 300nm羧基磁珠5μmol抗体浓度富集人工样本肿瘤细胞4
表6各肿瘤细胞平均捕获率汇总
试验结论:如表6所示,将各人工样本肿瘤细胞的平均捕获率进行比较,EpCAM抗体在300nm磁珠与5μmol抗体偶联下,通过六种肿瘤细胞捕获检测,对乳腺癌的效果最佳,其次非小细胞肺癌,食道癌,胃癌,子宫颈癌和结肠癌。
Claims (3)
1.一种偶联抗EpCAM抗体的免疫磁珠富集检测CTCs的方法,其特征在于,包括如下步骤:
S1.抗体与磁珠偶联:
1)抗EpCAM单克隆抗体偶联前,在4℃下,经过三次透析,过夜,储存在PBS中,将浓度分别调整到5μmol/L、10μmol/L、15μmol/L以及20μmol/L;
2)纳米免疫磁珠的制备:
取四份活化的羧基磁珠300μL,浓度2mg/mL,分别加入40μL浓度为5μmol/L、10μmol/L、15μmol/L、20μmol/L的抗EpCAM单克隆抗体,在1ml的离心管中,于室温下混合3h,用1ml的PBS清洗三次,加入300μL浓度为0.25mol/L甘氨酸混合25min,封闭剩余的醛基;再次用1ml的PBS清洗三次;加入含有质量百分比2.5%BSA的PBS溶液混合30min,封闭非特异性的吸附位点,之后用1ml的PBS清洗三次,加入300μL PBS缓冲液漩涡震荡,即得到浓度为2.0mg/ml的均匀的修饰有一抗的免疫磁珠;
S2.制备循环肿瘤细胞样品:
1)实验室培养各种肿瘤细胞样本,取各种肿瘤细胞,100μL,加入1.5mL离心管中,肿瘤细胞于外周血中制成人工样本,每种细胞为三管;
2)取5μL上述制备好的各种肿瘤细胞与5mL健康人血液混合,每种细胞制备三管;
3)取适量免疫磁珠,加入100ul生物肿瘤细胞样本中混匀,然后用磁力分离器分离,PBS清洗,将磁珠吸附细胞悬浮于PBS溶液中,进行细胞计数,使用细胞计数板检测富集到的细胞;
4)将适量纳米免疫磁珠加入1ml人工肿瘤细胞血液样品中磁力分离,PBS清洗,然后将磁珠吸附细胞重新悬浮于PBS中;取5ul样品,滴加于载玻片上,使用抗EpCAM-FICT,进行孵育,使用流式细胞仪对富集的细胞进行检测,肿瘤细胞-荧光标记EpCAM鉴定有效性;在荧光倒置显微镜下进行计数,肿瘤细胞株细胞混入外周血后的富集、鉴定同上。
2.根据权利要求1所述的一种偶联抗EpCAM抗体的免疫磁珠富集检测CTCs的方法,其特征在于,所述纳米免疫磁珠的制备的反应条件为:反应介质为0.01M PBS,PH=7.4,温度为室温,反应时间为3h,抗体和磁珠质量比为200μg:1000μg。
3.根据权利要求1所述的一种偶联抗EpCAM抗体的免疫磁珠富集检测CTCs的方法,其特征在于,所述肿瘤细胞为乳腺癌症(MCF-7),食道癌(EC109),非小细胞肺癌(A549),子宫颈癌(Hela),胃癌(NCI-N87)、结肠癌(HT29)。
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