CN116445419B - 11dhTxB2单克隆抗体及其制备方法和应用 - Google Patents
11dhTxB2单克隆抗体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种抗血栓素代谢物11dhTxB2杂交瘤细胞株,该抗血栓素代谢物11dhTxB2命名为杂交瘤细胞株NAT01,保藏号为CGMCC No.45457。该杂交瘤细胞株能够产生高特异性的抗血栓素代谢物11dhTxB2单克隆抗体,生产效率高。分泌的抗血栓素代谢物11dhTxB2单克隆抗体具有较高特异性、灵敏度和交叉反应率低。本发明提供的单克隆抗体可用于血栓素代谢物11dhTxB2免疫学检测方法的建立。
Description
技术领域
本发明属于生物技术领域,具体涉及一种11dhTxB2单克隆抗体及其制备方法和应用。
背景技术
心脑血管疾病是心脏血管和脑血管疾病的统称,泛指由于高脂血症、血液黏稠、动脉粥样硬化、高血压等所导致的心脏、大脑及全身组织发生的缺血性或出血性疾病。心脑血管疾病是由于长期动脉粥样硬化引起血小板功能异常,最终引起血栓或出血形式而发病,抗血小板药物是目前临床对血栓性疾病有效预防和治疗的主要措施。
阿司匹林(Aspirin),又名乙酰水杨酸,是一种有机化合物,化学式为C9H8O4,为白色结晶性粉末,溶于乙醇、乙醚,微溶于水,主要用作解热镇痛、非甾体抗炎药,抗血小板聚集药。阿司匹林是一种有效的血小板抑制剂,可抑制血栓素A2(TxA2)的合成,具有显著抑制血小板的粘附、聚集和释放的作用,从而可防止微血栓形成,防止粥样硬化和心肌梗死。已有研究证实,阿司匹林可显著降低心肌梗死的早期病死率及再梗死率。但近几年的研究显示并非所有的个体对于阿司匹林的反应都是一致的。大约30%的个体服药者对阿司匹林耐药,也就是常说的不起作用,因此监控阿司匹林疗效变得更加关键和重要。
11-脱氢血栓素B2(11-dehydro-thromboxaneB2,11-DH-TXB2)是血栓烷素A2(thromboxaneA2,TXA2)的终末代谢产物,主要经肾排出,在止血及心血管疾病的发生过程中均起到重要作用。花生四烯酸在前列腺素H合成酶1和2(COX-1和COX-2)的作用下生成前列腺素H。前列腺素H化学性质不稳定,可在异构酶的作用下转化为多种具有生物活性的前列腺素类物质,包括TXA2、前列环素I2、前列腺素D2、前列腺素E2及前列腺素F2α等。TXA2主要经COX-1途径在血小板中合成,新生的血小板同时表达COX-1和COX-2,而成熟血小板仅表达COX-1,COX-2则主要表达于单核细胞、内皮细胞等有核细胞中。TXA2有强烈的缩血管作用,还可通过结合血栓烷素血小板受体(thromboxaneplateletreceptor,TPR)激活血小板,促进其聚集,从而发挥促血栓形成作用。除TXA2外,凝血酶、胶原和腺苷二磷酸(adenosinediphosphate,ADP)亦能通过其他通路激活血小板。TXA2高度不稳定,会迅速被水解为较稳定的血栓烷素B2(thromboxaneB2,TXB2)。TXB2随后在肝中被转化为半衰期更长的11dhTXB2,并经尿液排出。TXA2则是花生四烯酸在环氧合酶(cyclooxygenase,COX)作用下产生的具有促血小板聚集、收缩血管等生物活性作用的物质,在血栓形成过程中有重要作用。阿司匹林能通过不可逆地抑制COX-1活性,减少TXA2合成,从而抑制血小板聚集及其在冠状动脉粥样硬化性心脏病(冠心病)发展和急性冠状动脉综合征(acutecoronarysyndrome,ACS)发生中的作用。
直接测定患者对阿司匹林的反应是指测定TxA2的循环水平,但TxA2的生物半衰期约为30秒,这样分析TxA2就非常困难,但是它会在酶的作用下分解成得11-脱氢血栓烷B2(11dhTxB2)被肾脏吸收,排泄在尿液里。11dhTxB2在尿液中非常稳定,它是一种生物非活性物质,是TxA2的下游代谢产物,其半衰期较长,不受体内血小板活性和其他分析变量的影响。有专家指出,检测尿中11dhTxB2水平可以更直接地反应阿司匹林对血小板聚集的抑制作用,检测无创,操作简单,耗费人力物力少,分析变异度小,使该方法优于其他的功能检测。目前有研究证实尿中11dhTxB2水平与阿司匹林低反应性及患者心血管事件的发生存在显著的相关关系,可用于评估阿司匹林的临床疗效。因此得到分泌高特异性和高灵敏度的11dhTxB2单克隆抗体的杂交瘤细胞株是应用于11dhTxB2免疫学检测的重要前提。
发明内容
为了解决上述问题,本发明提供了一种抗血栓素代谢物11dhTxB2杂交瘤细胞株,该抗血栓素代谢物11dhTxB2命名为杂交瘤细胞株NAT01,保藏号为CGMCC No.45457。该杂交瘤细胞株能够产生高特异性的抗血栓素代谢物11dhTxB2单克隆抗体,生产效率高。分泌的抗血栓素代谢物11dhTxB2单克隆抗体具有较高特异性、灵敏度和交叉反应率低。本发明提供的单克隆抗体可用于血栓素代谢物11dhTxB2免疫学检测方法的建立。
一方面,本发明提供了一种杂交瘤细胞株,其特征在于,所述的杂交瘤细胞株的保藏号为CGMCC No.45457。
具体地,所述的杂交瘤细胞株分泌抗血栓素代谢物11dhTxB2单克隆抗体。
具体地,所述的杂交瘤细胞株的制备方法包括将动物的免疫脾细胞与骨髓瘤细胞融合;筛选阳性杂交瘤细胞进行亚克隆,获得杂交瘤细胞株。
所述的免疫动物可以是小鼠,优选为雌性BALB/c小鼠。
所述的脾细胞与骨髓瘤细胞融合比例为5:1。
所述的亚克隆次数为2-5次,优选为3次。
另一方面,本发明提供了一种抗血栓素代谢物11dhTxB2单克隆抗体,所述的抗血栓素代谢物11dhTxB2单克隆抗体是由保藏号为CGMCC No.45457的杂交瘤细胞株产生。
另一方面,本发明提供了一种制备前述的抗血栓素代谢物11dhTxB2单克隆抗体的方法,包括培养前述的杂交瘤细胞株。
具体地,所述的方法包括以下步骤:
(1)将利要求1所述的杂交瘤细胞株注射至动物腹腔,培养7-10天,回收腹水;
(2)沉淀法结合亲和纯化法获得抗体洗脱液;
(3)将蛋白沉淀溶于缓冲液,4℃环境下缓冲液透析过夜;
(4)回收透析产物即得抗血栓素代谢物11dhTxB2单克隆抗体。
进一步具体地,所述的动物是经产鼠。
所述的沉淀法可以是硫酸铵沉淀法、辛酸-硫酸铵沉淀法、和聚乙二醇(PEG)沉淀法的一种或多种。
优选地,所述的沉淀法是辛酸-硫酸铵沉淀法。
所述的亲和纯化法可以是G蛋白亲和纯化。
所述的缓冲液可以是PBS缓冲液、TEN缓冲液和TBS缓冲液。
优选地,所述的缓冲液是磷酸盐缓冲液。
又一方面,本发明提供了前述的抗血栓素代谢物11dhTxB2单克隆抗体在制备评估阿司匹林作为血小板抑制剂的试剂或试剂盒中的应用。
又一方面,本发明提供了一种评估血小板抑制剂的试剂盒,所述的试剂盒包括前述的抗血栓素代谢物11dhTxB2单克隆抗体。
具体地,所述的抗血栓素代谢物11dhTxB2单克隆抗体的浓度为25-50ng/mL。
优选地,所述的抗血栓素代谢物11dhTxB2单克隆抗体的浓度为25ng/mL。
具体地,所述的试剂盒还包括酶标板、样品稀释液、标准品、酶结合物、检测抗体和洗涤液。
所述的酶标板可以包被羊抗鼠IgG。
所述的样品稀释液可以是三羟甲基氨基甲烷缓冲液、碳酸盐缓冲液、磷酸盐缓冲液、硼酸盐缓冲液、甘氨酸缓冲液和羟乙基哌嗪乙硫磺酸缓冲液其中的一种或多种;其pH9.0。
所述的洗涤液可以是三羟甲基氨基甲烷缓冲液、碳酸盐缓冲液、磷酸盐缓冲液、硼酸盐缓冲液、甘氨酸缓冲液和羟乙基哌嗪乙硫磺酸缓冲液其中的一种或多种;该洗涤液中还含有0.03-0.07%Tween-20V/V。
优选地,所述的洗涤液中含有0.05%Tween-20V/V。
具体地,所述的试剂盒还可以包括底物、终止液和封口膜。
本发明所取得的的技术效果:本发明提供的杂交瘤细胞株能够产生高特异性的抗血栓素代谢物11dhTxB2单克隆抗体,生产效率高。分泌的抗血栓素代谢物11dhTxB2单克隆抗体具有较高特异性、灵敏度和交叉反应率低。本发明提供的单克隆抗体可用于血栓素代谢物11dhTxB2免疫学检测方法的建立。
保藏说明:
杂交瘤细胞株NAT01已于2023年02月08日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.45457,分类命名为小鼠杂交瘤细胞。
附图说明
图1为抗体纯化SDS-PAGE电泳图;其中,1、Mark;2、纯化后腹水;3、饱和硫酸铵沉淀后上清液;4、正辛酸纯化后;5、腹水原液。
图2为11dhTxB2-ALP酶结合物AKTA纯化图;该图为由检测仪器直接输出获得,目的在于观察目标峰,当前图片已足以反映效果。
图3为11dhTxB2应用于ELISA法试剂盒检测范围;其中,Semi-log Fit:Y=slope*Log(X)+intercept;
20/50/80%:X=2840.722/601.861/127.516Y=-1.129/-0.722/-0.315;
intercept 0.958(+/-0.052),slope-0.605(+/-0.017);
chi2=0.016,RMS=0.044,r^2=0.993。
图4为标准曲线图;其中,Semi-log Fit:Y=slope*Log(X)+intercept;
20/50/80%:X=1786.075/351.151/69.038Y=0.267/0.655/1.043;
intercept 2.054(+/-0.117),slope-0.550(+/-0.040);
chi2=0.062,RMS=0.094,r^2=0.964。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1 11dhTxB2鼠源单克隆抗体制备及鉴定
1.1免疫原制备
将1mg 11dhTxB2乙酸甲酯溶液转移至反应管中,吹氮仪去除乙酸甲酯,用50μL二甲基亚砜将11dhTxB2稀释成20mg/mL溶液;10mM磷酸盐缓冲液(pH 7.4)分别将KLH和EDC稀释成20mg/mL溶液;依次向反应管中加入150μL 20mg/mL EDC,轻缓混匀,加入150μL 20mg/mL KLH,轻缓混匀,室温静置交联2小时;交联后产物用磷酸盐缓冲液(pH 7.4)补充至1mL,回收反应管中交联产物至透析袋(10000MW)中,透析液2L磷酸盐缓冲液,每间隔2小时更换透析液,更换4次;回收透析产物,利用10mM磷酸盐缓冲液调整透析产物浓度至1mg/mL,作为免疫抗原。
1.2小鼠免疫
免疫动物选择6周龄雌性BALB/c小鼠,使用11dhTxB2-KLH作为免疫抗原。首次免疫,免疫抗原与等体积的弗氏完全佐剂混匀,乳化30分钟,形成油包水乳白色粘稠状,乳化后抗原滴至水表面,水中3分钟不溶解视为有效乳化。将乳化后的抗原注射至小鼠腹部皮下,三点免疫,每点20μL抗原,形成绿豆粒大小皮下鼓包。二、三、四、五次免疫按照首免方式进行,佐剂更换为弗氏不完全佐剂,每次免疫间隔周期为2周。第五次免疫结束后2周,进行加强免疫,不需要佐剂乳化,小鼠腹部皮下直接接种抗原,加强免疫后三天进行细胞融合实验。
1.3细胞融合
融合准备工作:融合前3天小鼠追加免疫,用生理盐水溶解抗原,追加免疫后3天融合;融合前天,配好HAT选择性培养液;解剖小鼠用的手术器具进行高压灭菌;SP2/0骨髓瘤细胞进行传代处理,保持新传入细胞培养瓶中细胞密度约为60%最佳;取BALB/c小鼠腹腔细胞,用选择性培养液悬起,加入96孔细胞培养板中,100μL/孔,制备饲养层细胞。融合步骤:实验前,将PEG1450、HAT培养液和100mL培养基放入37℃水浴锅中孵育;小鼠眼球窦静脉丛采血,回收血清用于筛选参照,断颈处死,75%的酒精浸泡消毒5分钟,在无菌的条件下取出小鼠的脾脏,至于200目不锈钢筛网上,用注射器内芯研磨脾脏,回收脾细胞,计数约回收5×107个脾细胞;回收处于对数生产期的SP2/0细胞,利用无血清培养基洗涤细胞1次,计数约回收1×107个SP2/0细胞;将脾细胞和SP2/0细胞两者按照个数比为5:1的比例混合,无血清培养基洗涤一次,弃上清;加入预热的1mL PEG1450融合剂,加入过程中注意缓慢加入,并且边加边用吸管头轻轻拨动细胞团,边加边拨动,加完将融合细胞管37℃水浴静置1分钟;加入37℃预热的培养基,加入速度先慢后快,前3分钟内加入1mL,第四分钟内加入2mL,第五分钟内加入3mL,第六至第十分钟内加入10-15mL,将培养液补至50mL离心,1000转/分钟,10分钟,弃上清,用手指轻轻弹触管底,使细胞松动成糊状,加入100mL选择性培养液悬起融合后细胞;融合后细胞加入提前准备好的饲养层96孔细胞培养板中,100μL/孔,可铺10块96孔细胞培养板。
1.4筛选抗原制备
将1mg 11dhTxB2乙酸甲酯溶液转移至反应管中,吹氮仪去除乙酸甲酯,用50μL二甲基亚砜将11dhTxB2稀释成20mg/mL溶液;磷酸盐缓冲液(pH 7.4)分别将OVA和EDC稀释成20mg/mL溶液;依次向反应管中加入150μL 20mg/mL EDC,轻缓混匀,加入150μL 20mg/mLOVA,轻缓混匀,室温静置交联2小时;交联后产物用磷酸盐缓冲液(pH 7.4)补充至1mL,回收反应管中交联产物至透析袋(10000MW)中,透析液2L磷酸盐缓冲液,每间隔2小时更换依次透析液,更换4次;回收透析产物,利用磷酸盐缓冲液调整透析产物浓度至1mg/mL,作为筛选抗原。
1.5筛选方法建立
11dhTxB2与OVA偶联物作为筛选抗原,10mM磷酸盐缓冲液稀释至5μg/mL,100μL/孔,包被于96孔酶标板中,4℃包被过夜;次日,洗液(0.05%Tween20/10mM pBS)300μL/孔,洗涤两次,加入封闭液(2%BSA/10mM pBS),300μL/孔,37℃封闭2小时,拍干,制备成筛选酶标板;融合后细胞上清液加入筛选酶标板,100μL/孔,每块筛选酶标板要做阴性参照(选择性培养液)和阳性参照(选择性培养液稀释的鼠血清,稀释比例1:500),37℃反应1小时;弃酶标板孔中细胞上清液,洗液洗涤5次,300μL/孔;加入提前准备好的羊抗鼠HRP酶标二抗,100μL/孔,37℃反应1小时;弃酶标板孔中羊抗鼠HRP酶标二抗,洗液洗涤5次,300μL/孔;加入底物TMB,100μL/孔,避光显色15分钟;加入终止液,50μL/孔;酶标仪读取OD450nm数值。
1.6杂交瘤细胞筛选
融合后四至五天,准备筛选用检测酶标板。11dhTxB2与OVA偶联物作为筛选抗原,10mM磷酸盐缓冲液稀释至5μg/mL,100μL/孔,包被于96孔酶标板中,4℃包被过夜;次日,洗液(0.05%Tween20/10mM pBS)300μL/孔,洗涤两次,加入封闭液(2%BSA/10mM pBS),300μL/孔,37℃封闭2小时,拍干,制备成筛选酶标板。
融合后六天,根据已建立的筛选方法检测杂交瘤细胞上清液。每块酶标板均设立阴阳性对照,分别是选择性培养液和选择性培养液稀释的免疫鼠阳性血清,稀释比例1:500。利用已包被和封闭好的酶标板,加入待检测细胞上清液,100μL/孔,37℃反应1小时;弃酶标板孔中残液,洗液洗涤5次,300μL/孔;加入提前准备好的羊抗鼠HRP酶标二抗,100μL/孔,37℃反应1小时;弃酶标板孔中残液,洗液洗涤5次,300μL/孔;加入底物TMB,100μL/孔,避光显色15分钟;加入终止液,50μL/孔;酶标仪读取OD450nm数值。
按照OD450nm数值,由大到小原则,每块酶标板挑取约10个左右阳性酶标孔,显微镜观察记录阳性酶标板孔对应的细胞培养板孔中细胞克隆位置及估测每个克隆中细胞数量,回补选择性培养液。
次日,对初筛呈阳性孔者进行复筛,方法同初筛,两次筛选均呈强阳性者进行有限稀释法亚克隆。
1.7杂交瘤细胞亚克隆
两次杂交瘤细胞筛选均呈强阳性者,有限稀释法进行杂交瘤细胞克隆。显微镜下观察待克隆细胞,结合初筛后观察结果,进行有限稀释法亚克隆。克隆方法:待克隆细胞悬起力道均匀,避免细胞因机械外力造成死亡;待克隆细胞悬起后计数准确,若因工作量大,可目测估算待克隆细胞浓度;待克隆细胞浓度确定后,取出100个的待克隆细胞(个数=浓度×体积),移至装有21mL选择性培养液的离心管中,上下颠倒混匀,200μL/孔,转至新的96孔细胞培养板。若待克隆细胞数量少于100,全部取出待克隆细胞。第一次亚克隆后7-10天,根据细胞克隆大小进行克隆后筛选工作,阳性单克隆者进行第二次亚克隆,克隆方法同上。同样进行第三次亚克隆,经过3次亚克隆的杂交瘤细胞,其所有克隆均呈阳性,且该细胞株最后一次克隆,培养板中细胞呈现单克隆的孔数占含有细胞克隆的培养板孔数1/3以上,认定杂交瘤细胞株克隆成功,扩增杂交瘤细胞株,冻存备用。
1.8抗血栓素代谢物11dhTxB2单克隆抗体纯化
扩增杂交瘤细胞株,回收细胞,无菌磷酸盐缓冲液洗涤细胞,计数调整细胞浓度至1×106个/mL,将细胞注射至经产鼠腹腔(经产鼠提前20-30天注射液体石蜡,0.5mL/只),1mL/只,约7-10天,经产鼠腹部膨胀,回收腹水。40mL醋酸缓冲液(60mM pH4.0)稀释10mL腹水,氢氧化钠(1M)调pH至4.5;室温下搅拌30分钟,期间逐滴加入1.7mL正辛酸;将样品转入高速离心管,20℃环境18000g离心10分钟;用滤纸过滤样品,滤液与2.5mL磷酸盐缓冲液(0.2MpH7.2)混合,氢氧化钠(1M)调pH至7.4;加入与滤液相同体积的饱和硫酸铵(pH7.4),冰浴搅拌30分钟,4℃静置沉淀3小时;4℃环境18000g离心15分钟,弃上清;将沉淀溶于5mL的磷酸盐缓冲液(0.01M,pH7.2),2L磷酸盐缓冲液(0.01M,pH7.0)4℃环境,透析过夜;回收透析产物,Nanodrop2000检测及调整蛋白浓度为2mg/mL。
1.9抗血栓素代谢物11dhTxB2单克隆抗体SDS-PAGE电泳
配制10mL 12%分离胶,迅速在两玻璃板之间灌注分离胶,上面用水密封,约30分钟后,待分离胶完全聚合,倒出覆盖胶上层的水,用滤纸吸干。配制5%浓缩胶,灌注在分离胶上层,之后插入塑料梳子,避免气泡产生。待浓缩胶凝固后,小心拔出梳子,用电泳缓冲液清洗孔洞。待测样品与5×上样缓冲液按比例混合于1.5mL离心管中,离心管煮沸10分钟,瞬离,移液器取Maker和样品20μL加入样品孔,加满电泳液,恒压80V,样品向下移动形成一条直线,调整电压至120V,直至溴酚蓝指示线到达分离胶底部,关闭电源。取出凝胶,考马斯亮蓝R250染色2小时,脱色至条带清晰可见,拍照,见图1。
实施例2
2.1 11dhTxB2抗体应用于ELISA方法酶结合物(11dhTxB2-ALP)制备及纯化
(1)取0.2mg 11dhTxB2于反应管中,通风橱吹氮仪吹干11dhTxB2稀释液乙酸甲酯;
(2)加入50μL DMF溶解附着于反应管内壁的11dhTxB2;
(3)加入50μL 10mM NHS,混匀;
(4)加入50μL 10mM DCC,混匀;
(5)室温过夜反应;
(6)加入100μL碱性磷酸酶(100KU/mL),混匀;
(7)加入1.5mL硼酸缓冲液(0.1M pH8.5),混匀;
(8)室温反应2小时;
(9)加入1.25mL 0.1M Tris缓冲液(pH7.2),混匀,得到产物约3mL;
(10)AKTA平台脱盐纯化步骤(脱盐柱体积26mL);
(11)52mL灭菌去离子水平衡脱盐柱,流速3mL/分钟;
(12)104mL翘液(0.1M Tris缓冲液)平衡脱盐柱,流速3mL/分钟;
(13)上样3mL,流速3mL/分钟;
(14)翘液继续平衡,流速3mL/分钟,约15mL翘液后出现紫外峰,收集样品约5mL;
(15)翘液继续平衡,流速3mL/分钟,约52mL翘液后无紫外峰;
(16)52mL灭菌去离子水平衡脱盐柱,流速3mL/分钟;
(17)52mL 20%酒精平衡脱盐柱,保存脱盐柱。
11dhTxB2-ALP酶结合物AKTA纯化见图2。
2.2 11dhTxB2抗体应用于ELISA方法主要成分组成
参考物质:congenix公司定标11dhTxB2高值尿液,36000pg/mL。
ELSIA方法校准品:congenix公司定标11dhTxB2高值标本,20000pg/mL。
ELSIA方法包被酶标板:羊抗鼠IgG(购自SIGMA)用10mM磷酸盐缓冲液稀释至5μg/mL,酶标板包被100μL/孔,4℃过夜包被。2%BSA封闭酶标板,300μL/孔,37℃封闭4小时。
ELSIA方法样品稀释液:配制磷酸缓冲液,作为样品稀释液。
ELSIA方法洗液配制:配制磷酸缓冲液(0.05%Tween-20V/V),作为洗液。
ELSIA方法11dhTxB2-ALP结合物和抗体浓度棋盘滴定实验。
碱性磷酸酶稳定剂稀释AKTA纯化后11dhTxB2-ALP原液至50、100、200、400、800倍。
磷酸盐缓冲液稀释11dhTxB2抗体至:25ng/mL、50ng/mL、100ng/mL。
实验流程:
(1)已包被酶标板,依次加入100μL/孔样本稀释液(0pg/mL)和100μL/孔样本(36000pg/mL),加入50μL/孔11dhTxB2-ALP,再加入50μL/孔11dhTxB2抗体,室温振荡孵育2小时;
(2)洗板5次,350μL/孔;
(3)底物:商品化底物,200μL/孔,室温振荡孵育30分钟;
(4)终止液:终止反应,读取OD405nm,见表1和表2。
表1
表2
根据实验结果,选定11dhTxB2-ALP稀释100-200倍稀释使用,11dhTxB2抗体工作浓度25-50ng/mL。优选11dhTxB2-ALP 200倍稀释使用,11dhTxB2抗体工作浓度25ng/mL。
实施例3 11dhTxB2抗体性能评估
3.1 11dhTxB2抗体应用于ELISA法试剂盒检测范围
企业参考物质:congenix公司定标11dhTxB2高值尿液,36000pg/mL;
样品稀释液稀释至20000、15000、10000、7500、5000、2500、1250、625、312.5、156.3、78.1、39、19.5、9.75、0pg/mL。
实验流程:
(1)已包被酶标板,依次加入不同浓度标准品,100μL/孔,加入50μL/孔11dhTxB2-ALP(200倍稀释),再加入50μL/孔11dhTxB2抗体(25ng/mL),室温振荡孵育2小时;
(2)洗板5次,350μL/孔;
(3)底物:商品化底物,200μL/孔,室温振荡孵育30分钟;
(4)终止液:终止反应,读取OD405nm。
11dhTxB2-ALP(200倍稀释)和11dhTxB2(25ng/mL)抗体反应体系下不同浓度标准品OD405nm见表3。
表3 11dhTxB2抗体应用于ELISA法检测不同浓度样品OD405nm值
BIO-RAD酶标仪MPM 6软件分析见图3,Semi-log分析r^2=0.993>0.95,认为11dhTxB2抗体应用于ELISA法试剂盒检测11dhTxB2范围39-10000pg/mL。
3.2 11dhTxB2抗体应用ELISA方法灵敏度测试
检测体系设定空白孔,检测流程该孔只加底物。样本稀释液作为样本进行检测,重复测定20次,semi-log分析得出20次检测结果,计算浓度平均值M和浓度标准差SD,得M+2SD,即为最低检测限。测试样品布局见表4,测试结果见表5。
表4 11dhTxB2抗体应用ELISA方法测试样本稀释液OD405nm值
表5
BIO-RAD酶标仪MPM 6软件分析见图4,Semi-log分析r^2=0.964>0.95符合要求,样本稀释液20次检测结果,计算检测浓度平均值M=39.435pg/mL,标准差浓度SD=5.632pg/mL,11dhTxB2抗体应用于ELISA法检测灵敏度(最低检测限)M+2SD=50.699pg/mL。
试剂盒的检测灵敏度。
3.3 11dhTxB2抗体测试与11dhTxB2结构式类似物质交叉反应测试
实验方法:羊抗鼠IgG用10mM磷酸盐缓冲液稀释至5μg/mL,酶标板包被100μL/孔,4℃过夜包被。2%BSA封闭酶标板,300μL/孔,37℃封闭4小时。
11dhTxB2(11-脱氢-血栓烷B2)和与其结构式相似的化合物11-dehydro-2,3-dinor Thromboxane B2(11-脱氢-2,3-二恶烷B2)、Prostaglandin D2(前列腺素D2)、Thromboxane B2(血栓素B2)、6-keto Prostaglandin F1α(6-酮前列腺素F1α)、Prostaglandin E2(前列腺素E2)、13,14-dihydro-15-keto-tetranor Prostaglandin E2(13,14-二氢-15-酮前列腺素e2)、13,14-dihydro Prostaglandin F2α(13,14-二氢前列腺素F2α),利用稀释液稀释至浓度为10000pg/mL,化合物稀释液作为参照物。
封闭好的酶标板加入稀释好的化合物,100μL/孔;加入11dhTxB2-ALP酶结合物(1:200倍稀释),50μL/孔;再加入纯化后鼠源11dhTxB2单克隆抗体(25ng/mL),50μL/孔,微量振荡器振荡120分钟。洗涤液洗涤5次,300μL/孔,加入商品化pNPP底物,200μL/孔,微量振荡器振荡30分钟。加入终止液,100μL/孔,酶标仪读取OD405nm。
交叉率(%)=(11dhTxB2类似化合物稀释液OD405nm均值-11dhTxB2类似物10000pg/mL OD405nm)/(11dhTxB2化合物稀释液OD405nm-11dhTxB210000pg/mL OD405nm)×100%。实验结果见表6。
表611dhTxB2抗体测试与11dhTxB2结构式类似物质交叉反应结果
结果显示11dhTxB2鼠源单克隆抗体与11dhTxB2(11-脱氢-血栓烷B2)和与其结构式相似的化合物11-dehydro-2,3-dinor Thromboxane B2(11-脱氢-2,3-二恶烷B2)、Prostaglandin D2(前列腺素D2)、Thromboxane B2(血栓素B2)、6-keto Prostaglandin F1α(6-酮前列腺素F1α)、Prostaglandin E2(前列腺素E2)、13,14-dihydro-15-keto-tetranor Prostaglandin E2(13,14-二氢-15-酮前列腺素e2)和13,14-dihydroProstaglandin F2α(13,14-二氢前列腺素F2α)化合物交叉反应率分别为100%、100.09%、0.64%、0.55%、0.09%、0.83%、0.73%、0.92%。结果显示11dhTxB2鼠源单克隆抗体检测11-dehydro-2,3-dinor Thromboxane B2(11-脱氢-2,3-二恶烷B2)存在交叉反应;检测Prostaglandin D2(前列腺素D2)、Thromboxane B2(血栓素B2)、6-keto Prostaglandin F1α(6-酮前列腺素F1α)、Prostaglandin
E2(前列腺素E2)、13,14-dihydro-15-keto-tetranor Prostaglandin E2(13,14-二氢-15-酮前列腺素e2)和13,14-dihydro Prostaglandin F2α(13,14-二氢前列腺素F2α)化合物几乎没有交叉反应。说明11dhTxB2鼠源单克隆抗体可作为抗体原料应用于免疫学检测试剂的开发。
3.4 11dhTxB2抗体应用于ELISA方法抗干扰能力测试
检测体系设定空白孔,检测流程该孔只加底物。不同浓度4例尿液标本(S1-S4)添加和不添加干扰物质标本:乙酰氨基酚含量200mg/dL、乙酰水杨酸含量200mg/dL、抗坏血酸(维生素C)含量200mg/dL、咖啡因含量200mg/dL、龙胆酸含量200mg/dL、葡萄糖含量2000mg/dL、血红蛋白含量500mg/dL、总蛋白含量2000mg/dL、水杨酸含量200mg/dL、尿酸含量200mg/dL、大肠杆菌含量10000个/mL,重复检测均值M,按照公式计算相对偏差B%。
相对偏差(B%)=(添加干扰物质样品浓度–不添加干扰物质样品浓度)/不添加干扰物质样品浓度)×100%。实验结果见表7。
表7
实验结果可知:尿液样本中含乙酰氨基酚含量200mg/dL、乙酰水杨酸含量200mg/dL、抗坏血酸(维生素C)含量200mg/dL、咖啡因含量200mg/dL、龙胆酸含量200mg/dL、葡萄糖含量2000mg/dL、血红蛋白含量500mg/dL、总蛋白含量2000mg/dL、水杨酸含量200mg/dL、尿酸含量200mg/dL、大肠杆菌含量10000个/mL时相对偏差均≤20%,不影响试剂盒检测结果,说明11dhTxB2抗体应用于ELISA方法测试,该抗体具备良好的抗干扰能力。
3.5 11dhTxB2抗体应用于ELISA法与进口试剂盒测试临床样品符合率测试
40例临床标本,赋值尿肌酐,分别用Congenix试剂盒(购自湖北惠康益生生物科技有限公司)和自我开发的NAT01杂交瘤细胞株生产的单克隆抗体应用ELISA方法同时检测临床标本40例,尿肌酐矫正,参照Congenix试剂盒阴阳性判断cut-off值1500pg/mg肌酐,分析得出Congenix试剂盒和NAT01杂交瘤细胞株生产的单克隆抗体应用ELISA法检测结果的临床符合率。结果显示:阳性符合率88%(22/25×100%)、阴性符合率93.33%(14/15×100%)、总体符合率90%[(14+22)/40×100%]。见表8。
表8Congenix试剂盒与NAT01-ELISA方法检测40例临床标本检测符合率
3.6 11dhTxB2抗体应用于ELISA法稳定性测试
测试11dhTxB2抗体稳定性,抗体分组经37℃加速制备不加速组、37℃加速3天、37℃加速7天、37℃加速12天,配制20ng/mL,ELISA法检测临床标本S,重复三次,测试其稳定性,计算组内变异系数和组间变异系数均<5%(表9),说明该抗体稳定性良好,适合后期作为原料生产体外诊断试剂。
组内变异系数(%)=(标准偏差SD/平均值Mean)×100%
组间变异系数(%)=(加速平均值Mean-不加速平均值Mean)/不加速平均值Mean×100%。
表9
临床标本S | 不加速组 | 37℃加速3天 | 37℃加速7天 | 37℃加速12天 |
S第一次测试 | 5645 | 5742 | 5549 | 5326 |
S第二次测试 | 5264 | 5369 | 5574 | 5745 |
S第三次测试 | 5279 | 5136 | 5126 | 5563 |
S均值 | 5396 | 5416 | 5416 | 5545 |
组内变异系数(%) | 3.3% | 4.6% | 3.8% | 3.1% |
组间变异系数(%) | 100% | 0.37% | 0.37% | 2.76% |
Claims (9)
1.一种杂交瘤细胞株,其特征在于,所述的杂交瘤细胞株的保藏号为CGMCC No.45457。
2.一种抗血栓素代谢物11dhTxB2单克隆抗体,其特征在于,所述的抗血栓素代谢物11dhTxB2单克隆抗体是由保藏号为CGMCC No.45457的杂交瘤细胞株产生。
3.一种制备权利要求2所述的抗血栓素代谢物11dhTxB2单克隆抗体的方法,其特征在于,包括培养权利要求1所述的杂交瘤细胞株。
4.根据权利要求3所述的方法,其特征在于,包括以下步骤:
(1)将利要求1所述的杂交瘤细胞株注射至动物腹腔,培养7-10天,回收腹水;
(2)沉淀法结合亲和纯化法获得抗体洗脱液;
(3)抗体洗脱液4℃环境缓冲液透析过夜;
(4)回收透析产物即得抗血栓素代谢物11dhTxB2单克隆抗体。
5.根据权利要求4所述的方法,其特征在于,所述的沉淀法为硫酸铵沉淀法、辛酸-硫酸铵沉淀法和聚乙二醇沉淀法的一种或多种;所述的亲和纯化法为G蛋白亲和纯化。
6.权利要求2所述的抗血栓素代谢物11dhTxB2单克隆抗体在制备评估阿司匹林作为血小板抑制剂的试剂或试剂盒中的应用。
7.一种评估血小板抑制剂的试剂盒,其特征在于,该试剂盒包括权利要求2所述的抗血栓素代谢物11dhTxB2单克隆抗体。
8.根据权利要求7所述的试剂盒,其特征在于,所述的抗血栓素代谢物11dhTxB2单克隆抗体的工作浓度为25-50ng/mL。
9.根据权利要求8所述的试剂盒,其特征在于,所述的抗血栓素代谢物11dhTxB2单克隆抗体的工作浓度为25ng/mL。
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