CN116482369B - 一种癌胚抗原检测试剂盒 - Google Patents
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Abstract
本发明涉及到一种癌胚抗原检测试剂盒,磁微粒组分所使用的抗体与与癌胚抗原抗原具有比较好的亲和力与特异性,且该试剂盒的检测限能达到0.5ng/mL以下,有良好的应用前景。
Description
技术领域
本发明涉及体外诊断领域,具体是一种癌胚抗原检测试剂盒。
背景技术
癌胚抗原(CEA)是1965年Gold和Freedman最先从胎儿及结肠癌组织中发现的,故称癌胚抗原。癌胚抗原(CEA)是由一些分子量为175-200 KD的糖蛋白组成的,其编码基因位于19号染色体。一般情况下,CEA是由胎儿胃肠道上皮组织、胰和肝细胞合成,通常在妊娠前6个月内CEA含量增高,出生后血清中含量已很低下。CEA属于非器官特异性肿瘤相关抗原,分泌CEA的肿瘤大多位于空腔脏器,如胃肠道、呼吸道、泌尿道等。正常情况下,CEA经胃肠道代谢。而肿瘤状态时CEA则进入血液和淋巴循环。引起血清CEA含量异常增高。
持续监测CEA 水平在观察疾病过程方面是十分有用的。在手术切除癌组织后1到4个月内,CEA 水平通常恢复正常或接近正常水平。CEA水平的再升高可能是癌症复发的先兆,可能先于症状和其他体征。连续的CEA水平监测也可以帮助评估化学治疗或放射治疗的效果。CEA水平在治疗期间的持续上升可指示治疗无效或肿瘤转移的可能性。
肺癌的临床诊断方法一般包括:1)实验室一般检测;2)血清学肿瘤标志物检测;3)影像学检查,包括X线胸片、CT、MRI、超声、骨核素显像、PET-CT等;4)内镜及其他检查,包括支气管镜检查和超声支气管穿刺活检术、纵隔镜检查、胸腔镜或开胸肺活检、痰脱落细胞学检查;5)病理组织学检查。
胃癌的诊断方法包括:1)体征检查,早期胃癌,常无明显的体征,进展期乃至晚期胃癌患者出现上腹部深压痛、肿块、胃肠梗阻等表现;2)影像检查,包括X线气钡双重对比造影、超声检查、CT、MRI、PET-CT、ECT、胃镜检查;3)肿瘤标记物检查;4)病理学检查。
目前国内化学发光技术按标记物不同,主要包括辣根过氧化物酶(HRP)发光体系、碱性磷酸酶(AP)发光体系、及吖啶酯(AE)直接发光体系。随着国产化学发光技术全自动化,管式磁微粒方法已经成为最主要的方法,而由于吖啶酯直接放光较酶促发光有诸多优势,也成为最主要的发光体系。
吖啶酯-磁微粒体系有诸多优势:1)可有效节省成本:放免1mg抗体可以做十个左右试剂盒(100T),酶促板式发光可以做20-40个左右试剂盒,而基于吖啶酯-磁微粒体系的化学发光可以做约100个试剂盒;2)孵育时间短、操作简便:磁微粒体系反应速度快,15-30min即可出报告。板式发光一般在1小时左右,放免一般在1小时以上至2小时左右。所有操作步骤均由全自动仪器完成;3)试剂稳定、变异性小:作为作为标记的吖啶酯在特定环境下直接发光,干扰因素少。但目前国内很难找到特异性好、亲和力高的CEA单克隆抗体,主要依赖进口的产品,因此急需开发一种高亲和力高特异性的CEA单抗用于CEA检测试剂盒的研发。
发明内容
为此,本发明提供了一种效果理想的癌胚抗原检测试剂盒。
为达到上述目的,本发明提供如下方案:
所述剂盒包括磁微粒组分、吖啶酯标记组分;所述试剂盒的磁微粒组分所包被的单克隆抗体为1DW01,该单克隆抗体包括重链可变区上三个抗原互补决定区(CDR):CDR1、CDR2与CDR3和轻链可变区上的三个抗原互补决定区CDR4、CDR5、CDR6;6个抗原互补决定区CDR1、CDR2、CDR3、CDR4、CDR5和CDR6的氨基酸序列分别如说明书中ST.26标准氨基酸序列表中SEQ.ID.NO.3、SEQ.ID.NO.4、SEQ.ID.NO.5、SEQ.ID.NO.6、SEQ.ID.NO.7和SEQ.ID.NO.8所示。
所述重链可变区的氨基酸序列如说明书中ST.26标准氨基酸序列表中SEQ.ID.NO.1所示;所述轻链可变区的氨基酸序列如说明书中ST.26标准氨基酸序列表中SEQ.ID.NO.2所示。
本发明还提供一种所述单克隆抗体包被的磁微粒的方法。
具体方法为:
1)将100ul 10mg/ml的粒径为1.5μm的磁微粒与100μg权利要求1所述的单克隆抗体1DW01加入1ml的MES缓冲液中;
2)边涡旋边加入0.1ml 的浓度为10mg/ml的EDC溶液;
3)室温反应4h后,磁分离并加入1ml 0.2M 甘氨酸溶液;
4)室温反应1h后,磁分离并加入1ml 2%BSA ,4℃反应过夜;
5)磁分离并用1ml TBS-T缓冲液清洗三次,再次磁分离弃上清后加入1ml PBS缓冲液即制备获得包被了单克隆抗体1DW01的磁微粒。
有益效果:
本发明制备了一种癌胚抗原检测试剂盒,并且所述试剂盒的检测准确度高、检测限能达到1ng/mL以下,并且与CEA抗原具有比较好的亲和力与特异性,有良好的应用前景。
附图说明
图1 为所述试剂盒检测CEA标准品的标准曲线图。
图2 为所述试剂盒磁微粒组分所使用抗体的纯化后SDS-PAGE电泳图。
具体实施方式
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
1、癌胚抗原单克隆抗体的制备
该实施例中所使用的CEA抗原以购买的方式获得。将CEA抗原(50ug/只)与完全佐剂(50ug/只)溶于PBS中(0.5ml/只)。置于2mlEP管中震摇 至完全乳化,分装于注射器中,对小鼠进行腹腔注射。
第一次加强在初次免疫后两周进行,加强注射剂量与初次免疫相同或减半,强使用的佐剂为不完全福氏佐剂,加强其它流程与初次免疫完全一致,第一次加强后10天,给小鼠编号,采血检测血清效价,评价免疫效果:在每次加强后的第10天,用09-11mm内径的玻璃毛细管从小鼠眼角采血。将鼠血在室温搁置1-3小时后,3000转/分离心15分钟。收集血清,按照1:100、1:300、1:900、1:2.7k、1:8k、1:24k和1:72k的稀释比例溶于Blocking Buffer。另选取Blocking Buffer作为空白对照,进行ELISA检测。
第二次加强在第一次加强两周后进行,加强操作程序与第一次加强完全相同。
在最后一次加强后三周对小鼠进行终加强,终加强时抗原的剂量与初次免疫相同,终加强后3-5天,进行细胞融合。
将小鼠处死并取出脾脏置于细胞筛网中,将脾脏用剪刀剪碎并用5mLDMEM冲洗,用5mL注射器推头研磨脾脏至完全碾碎。用DMEM冲洗推头与滤网,将脾细胞溶液转入50ml离心管,补充DMEM至45mL。提前将CO2培养箱T-75细胞培养瓶取出,轻拍7-10下使细胞脱离瓶壁。移入50ml离心管,并向原培养瓶中补充含有10%FBS,1*HT的DMEM培养基,分别将脾细胞和sp2/0细胞离心管颠倒两次摇匀,1000转/分离心5分钟,弃上清,分别重悬脾细胞和sp2/0细胞,并分别加入45mLDMEM。取40μL计数细胞。将细胞颠倒两次摇匀,再次离心,同时细胞计数。
分别将脾细胞和sp2/0细胞重悬,加入30mlDMEM至脾细胞离心管中,10mlDMEM至sp2/0离心管中混匀,根据sp2/0细胞数量多少,按照sp2/0细胞:脾细胞1:5的比例将sp2/0细胞与脾细胞混合。再次离心后弃上清重悬并静置一分钟。用2ml移液管取预热15分钟的PEG1500进行细胞融合,按照1ml/1.0*108脾细胞的量然后将移液管口伸入细胞液中缓慢滴加,边加入边轻轻滑动移液管将细胞液搅拌均匀。将细胞静置30秒,用准确的1分钟时间匀速滴加1mlDMEM至离心管中,边加入边轻轻摇动离心管。同样的操作方法,在第二个1分钟内滴加2ml DMEM,在第三个1分钟内滴加5ml在第四个1分钟内滴加10ml。补充DMEM至50ml,离心1000转/分,5min。
将融合好的细胞重悬,以脾细胞1.5-2*106/ml的浓度溶于含有30%FBS,1*B.S、2*H.A.T的DMEM 培养基中。100 μl/孔分装到饲养细胞板中。将分装好的细胞培养板放入CO2培养箱中培养。在细胞融合后第四天向培养板中补充50μl培养基,培养基成分为含有20%FBS、 1*PS、10%HCF的DMED培养基。在细胞融合后7-14天,当克隆生长到肉眼可以观测到大小时,ELISA筛选阳性克隆孔。
将阳性的杂交瘤细胞由小瓶转出或将一个冻存管复苏,转移到中瓶。用含有10%FBS,1*P.S的DMEM培养基培养3-5天,当细胞扩增到足以注射,1000转/分离心5分钟,弃上清。用DMEM洗两次,每次1000转5min离心,弃去上清。用DMEM重悬细胞,最终的细胞浓度为2-4x106cells/ml,在重悬后1小时内以1-2*106cells/0.5ml/mouse的剂量注入小鼠腹腔。所注射的小鼠需要在14-30天前免疫过液体石蜡,或是在7-32天之前免疫过不完全福氏佐剂。每天观察小鼠的腹部。在注射细胞后7-10天小凰腹部出现明显肿胀,用1.2#注射器针头每天连续采集腹水,直至小鼠死亡或停止产生腹水,每天将采集的腹水离心,4000转/分离心15分钟取上清存于4℃冰箱。
利用ELISA抗体效价检测试剂盒对抗CEA单抗进行检测,结果如表1所示。
表1 CEA单克隆抗体效价检测结果
当效价检测结果P/N>2时为阳性,从表1的结果可以看出1DW01单克隆抗体的效价达到了1:243000以上,表明该单抗具有较好的活力,可以用于后续实验。
2、单抗1DW01包被磁微粒的制备。
将2mL的EP管用MES缓冲液(PH=5.0)润洗三次,再在管中加入1ml的MES缓冲液(PH=5.0),取100μl磁微粒(JSR Life Science,MS160/Carboxyl,1%,10mg/ml,1.5μm)加入EP管中,涡旋振荡形成均一的磁微粒悬浮液,边震荡边加入100μLEDC溶液(10mg/ml),震荡反应45min,100μg的单克隆抗体1DW01,室温震荡反应4h,磁分离弃掉后加入1ml 0.2M的甘氨酸溶液,磁分离弃掉液体之后加入2% BSA,4℃封闭过夜。第二天磁分离后加入1mlTBS-T缓冲液清洗磁珠三次,最后一次磁分离后加入1ml PBS缓冲液(含有0.5%小鼠血清),4℃保存备用。
3、癌胚抗原检测试剂盒的检测效果。
利用磁微粒化学发光法对单克隆抗体的特异性与灵敏度。将上述包被了1DW01的磁微粒稀释40倍作为试剂盒的磁微粒溶液组分,将使用购买的抗CEA抗体连接吖啶酯制备的标记抗体作为试剂盒的吖啶酯标记组分,将 CEA抗原用20%胎牛血清配置为检测的校准品,浓度为0ng/ml、10ng/ml、30ng/ml、100ng/ml、200ng/ml、500ng/ml,以CEA浓度为横坐标,以CEA各浓度对应的发光值为纵坐标,用四参数法绘制标准曲线。发光值结果如表2所示,标准曲线如说明书附图1所示。
标准曲线的确定。
实验方法,对工作校准品的6个浓度点进行3次重复检测,然后求出平均值,以浓度值为横坐标,发光值平均值为纵坐标绘制标准曲线。
表2. 标准曲线发光值结果
如图1所示,标准曲线在四参数拟合之后r=0.9994。
准确度验证。
实验方法,将浓度约为100ng/mL(允许其浓度偏差为±20%)的CEA样本A,加入到浓度范围在2ng/mL-5ng/mL的血清B中,所加入CEA与血清B之间的体积比为1: 9,根据公式(R——回收率,V——加入样本A的体积,V——加入样本A的体积,C——样本A和样本B混液的检测浓度,C0——样本B液的浓度,CS——样本A液的浓度)计算回收率R, 回收率应在85%-115%范围内。
表3.回收率实验结果
表3结果表明该试剂盒准确度符合标准。
最低检测限确认。
实验方法,用零浓度校准品作为样本进行检测,重复测定20次,得出20次测量结果的RLU值(相对发光值),计算其平均值(M)和标准差(SD),得出M+2SD所对应的RLU值,根据零浓度校准品与相邻校准品之间的浓度-发光值结果进行两点回归拟合得出一次方程,将M+2SD所对应的RLU值带入上述方程中,求出对应的浓度值,即为最低检测限,其结果应不大于0.5ng/mL。
表4.最低检测限实验结果
表4结果表明该试剂盒最低检测限为0.096ng/ml,符合最低检测限的标准。
重复性验证。
实验方法,分别用10ng/mL±2ng/mL和100ng/mL±20ng/mL的样本各重复检测10次,计算10次测量结果的平均值M和标准差SD,根据公式(CV——变异系数,SD——10次测量结果的标准差,M ——10次测量结果的平均值)得出变异系数CV,变异系数(CV)应不大于10%。
表5.重复性验证试验结果
由表5试验结果可知,该试剂盒重复性符合要求。
通过对试剂盒准确度、最低检测限、重复性、等性能指标进行检测,其结果均符合试验设计的要求,为该产品的进一步优化与后续生产提供了良好的基础。
Claims (3)
1. 一种能特异性检测人血清中癌胚抗原的检测试剂盒,其特征在于,所述试剂盒包括磁微粒组分、吖啶酯标记组分;所述试剂盒的磁微粒组分所包被的单克隆抗体为 1DW01,该单克隆抗体包括重链可变区上三个抗原互补决定区:CDR1、CDR2 与 CDR3 和轻链可变区上的三个抗原互补决定区 CDR4、CDR5、CDR6;6 个抗原互补决定区 CDR1、CDR2、CDR3、 CDR4、CDR5 和 CDR6 的氨基酸序列分别为SEQ.ID.NO.3:EYWMN、SEQ.ID.NO.4:RINTASGSVDYAPNFKG、SEQ.ID.NO.5:LDLYFSYFFMADY、SEQ.ID.NO.6:RTSENIVGSVA、SEQ.ID.NO.7:WTSNLAD 和 SEQ.ID.NO.8 :QQFPSNPWT;所述的癌胚抗原检测试剂盒检测的样本为全血或血清。
2.根据权利要求 1 所述的检测试剂盒,其特征在于,所述单克隆抗体的重链可变区的氨基酸序列为 SEQ.ID.NO.1:QVKLQQSLAGLKRPESVKLSCTASGFDFTEYWMNWVRQAPEQGLEWIGRINTASGSVDYAPNFKGKFTLSVDTSQSTAYLQMDSLTSEDTAIYYCARLDLYFSYFFMADYWGQGTTTVTSS;所述轻链可变区的氨基酸序列为 SEQ.ID.NO.2: DIQMTQSPASLSVSPGQRVTITCRTSENIVGSVAWYQQKQGSPHLLVYWTSNLADGVPSRFSGSGSGTSFSLTISSVEPEDIATYYCQQFPSNPWTFGGGTKLEIKR。
3.一种单克隆抗体包被磁微粒的方法,其特征在于,所述方法为: 1)将 100ul 10mg/ml 的粒径为 1.5μm 的磁微粒与 100μg 权利要求 1 所述的单克隆抗体 1DW01 加入 1ml的 MES 缓冲液中; 2)边涡旋边加入 0.1ml 的浓度为 10mg/ml 的 EDC 溶液; 3)室温反应 4h 后,磁分离并加入 1ml 0.2M 甘氨酸溶液; 4)室温反应 1h 后,磁分离并加入 1ml2%BSA ,4℃反应过夜; 5)磁分离并用 1ml TBS-T 缓冲液清洗三次,再次磁分离弃上清后加入 1ml PBS 缓冲液即制 备获得包被了单克隆抗体 1DW01 的磁微粒。
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