CN113759115A - 用于膀胱癌检测的标志物及检测试剂盒 - Google Patents
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Abstract
本发明属于生物医学检测技术领域,具体涉及一种用于膀胱癌检测的标志物及检测试剂盒。本申请所述标志物包括核基质蛋白22(NMP22)、癌胚抗原(CEA)、***抗原(BTA)和细胞角蛋白19片段(CYFRA21‑1);所述检测试剂盒采用吖啶酯直接化学发光,用吖啶酯直接标记抗体(抗原),与待测标本相应的抗原(抗体)发生免疫反应后,形成包被抗体‑待测抗原‑吖啶酯标记抗体复合物,加入激发液,不需要催化剂即分解、发光。本发明建立了一种多肿瘤标志物的组合,进而通过多个指标的联合检测提高膀胱癌的检测特异性和敏感性,当对膀胱癌患者尿液检测时,具有较高的敏感度和特异度,可以对膀胱癌患者早期筛查、治疗效果和术后复发进行动态监测。
Description
技术领域
本发明属于生物医学检测技术领域,具体涉及一种用于膀胱癌检测的标志物及检测试剂盒。
背景技术
肿瘤是一类严重威胁人类健康,具有高发病率和高死亡率的疾病。膀胱癌是泌尿生殖***最常见的恶性肿瘤,早期症状隐匿,患者就诊时多处于中晚期。发病率位于泌尿***中恶性肿瘤首位,具有非常高的复发率和临床复杂性。在恶性肿瘤中,膀胱癌发病率排第六位,病死率排第四位。
目前***的诊断主要以尿液检测、影像学(KUB、IVP、CT、MRI、超声等)检查、膀胱镜检查等为主。***初次发现主要以表浅性的肿瘤为主,影像学检查对于表浅、初期的***诊断能力有限,膀胱镜检查为有创检查,有创检查费用高昂且还会造成泌尿***感染、出血等并发症,所以这些常用的诊断***的方法并不适用于***的筛查。尿液检测是目前较为理想的检测方法,为无创标本,可提供蛋白质、RNA及 DNA来源。因此,尿液是比较理想的用于检测肿瘤分子标志物的标本。
对于膀胱癌的筛查和复查,应尽量减少病人的痛苦,降低检测的成本,减轻患者的负担。肿瘤分子标志物是一种可以判断肿瘤发生、发展甚至预后的指示物。但单一的肿瘤标志物难以特异性地诊断膀胱癌。因此建立一种能够辅助,甚至替代尿脱落细胞学检测的无创、经济、高效、准确、快捷的方法尤为重要。
发明内容
本发明的目的在于提供一种用于膀胱癌检测的标志物及检测试剂盒,旨在建立一种多肿瘤标志物的组合,用于膀胱癌的早期筛查,通过多种膀胱癌肿瘤标志物的联合应用可以提高膀胱癌的诊断准确性。
为了实现上述目的,本发明采用的技术方案是:
一种用于膀胱癌检测的标志物,所述标志物包括核基质蛋白22 (NMP22)、癌胚抗原(CEA)、***抗原(BTA)和细胞角蛋白19 片段(CYFRA21-1)。
一种上述用于膀胱癌检测的标志物在制备用于膀胱癌检测的检测试剂或检测试剂盒中的应用。
优选的,所述检测试剂盒的检测方法为化学发光免疫分析方法,具体为双抗体夹心化学发光免疫分析法。
优选的,所述检测试剂盒包括:抗体包被的磁微粒、吖啶酯标记的抗体、校准品、激发液A、激发液B和洗涤液,所述抗体为标志物对应的抗体。
优选的,所述抗体包括:NMP22抗体、CEA抗体、BTA抗体、和 CYFRA21-1抗体;更优选的,所述抗体包括:核基质蛋白22(NMP22) 单克隆抗体、癌胚抗原(CEA)单克隆抗体、***抗原(BTA)单克隆抗体和细胞角蛋白19片段(CYFRA21-1)单克隆抗体。。
优选的,所述抗体包被的磁微粒包括:NMP22抗体包被的磁微粒、CEA 抗体包被的磁微粒、BTA抗体包被的磁微粒、和CYFRA21-1抗体包被的磁微粒;更优选的,所述的磁微粒的粒径为1-3μm;
更优选的,所述抗体包被的磁微粒通过如下方法制备:取磁微粒用 EDC溶液活化后,加入相应抗体进行分子偶联;再用含牛血清白蛋白的 0.0.1mol/L磷酸盐缓冲液pH7.4进行封闭;将封闭后的抗体包被的磁微粒置于2~8℃保存备用。
优选的,所述吖啶酯标记的抗体包括:吖啶酯标记的NMP22抗体、吖啶酯标记的CEA抗体、吖啶酯标记的BTA抗体、和吖啶酯标记的 CYFRA21-1抗体;
更优选的,所述吖啶酯标记的抗体通过如下方法制备:将吖啶酯加入相应抗体溶液中,混匀,避光孵育;将反应液全部透析至0.02mol/L磷酸盐缓冲液pH 7.4中,于–20℃避光保存。
优选的,所述校准品为不同浓度的标志物校准品,包括NMP22校准品、 CEA校准品、BTA校准品、和CYFRA21-1校准品;更优选的,所述标志物的浓度为0、0.5ng/mL、5ng/mL、10ng/mL、50ng/mL、100ng/mL;更优选的,所述校准品是用校准品稀释液配制的不同浓度标志物的溶液;更优选的,所述标志物为相应的标准抗原。
优选的,所述激发液A:为0.5%的H2O2和0.1mol/L HNO3的溶液;所述激发液B:为0.35mmol/L的NaOH和3%Triton X-100的溶液;所述洗涤液为:含有1%Tween-20的20×PBS缓冲液(pH7.0-8.0),使用时按1: 20倍稀释使用。
优选的,所述检测试剂盒的检测包括如下步骤:
(1)加样:取检测样本和抗体包被的磁微粒于孵育杯内混匀并孵育;
(2)加入吖啶酯标记的抗体试剂:将吖啶酯标记的抗体加入步骤(1) 的孵育杯内继续孵育;
(3)洗涤:用洗涤液洗涤;
(4)发光:依次加入激发液A和激发液B,进行发光处理;
(5)读值:测定检测样本的相对发光强度,根据校准曲线,计算出检测样本中待测标志物的浓度。
优选的,所述检测试剂盒的检测包括如下步骤:
(1)加样:分别取50μl检测样本和50μl抗体包被的磁微粒于孵育杯内混匀,37℃孵育10min;
(2)加入吖啶酯标记的抗体试剂:取50μl吖啶酯标记的抗体,加入步骤(1)的孵育杯内,37℃孵育20min;
(3)洗涤:用洗涤液洗涤2遍;
(4)发光:依次加入50μl激发液A和激发液B,混匀;
(5)读值:测定检测样本的相对发光强度,根据校准曲线,计算出检测样本中待测标志物的浓度。
优选的,所述步骤(1)中的检测样本为校准品或待测尿液。
优选的,所述步骤(1)中的抗体包被的磁微粒为NMP22抗体包被的磁微粒、CEA抗体包被的磁微粒、BTA抗体包被的磁微粒、和CYFRA21-1 抗体包被的磁微粒的混合。
优选的,所述步骤(2)中的吖啶酯标记的抗体为吖啶酯标记的NMP22 抗体、吖啶酯标记的CEA抗体、吖啶酯标记的BTA抗体、和吖啶酯标记的CYFRA21-1抗体的混合。
核基质蛋白22(NMP22,nuclear matrix protein 22):是细胞核基质蛋白成员之一,特异性的存在于尿路移行细胞中,NMP22在恶性移行细胞中的含量约为正常细胞的80倍。NMP22主要参与DNA的复制及转录、 RNA合成和基因表达调控,可通过检测凋亡细胞释放出的蛋白量来测定其含量。NMP22含量与肿瘤大小、分期、分级呈正相关。NMP22是一种新型肿瘤标志物。
***抗原(BTA,bladder tumor antigen),又称***相关抗原(BTAA),又被称为补体因子H相关蛋白,***在发生生长的过程中会产生一种蛋白水解酶,它能够降解细胞基底膜的各种成分,进而分解形成各种糖蛋白、胶原片段和蛋白多糖,***抗原对于***检测的特异性和敏感性分别为77%和60%。
癌胚抗原(carcinoembryonic antigen,CEA)是一种参与细胞黏附的酸性糖蛋白,胚胎期主要存在于胎儿的胃肠道、胰腺和肝脏,出生后组织内含量很低。在正常人胃肠道、胰腺和肝脏中也有少量表达。当发生消化道肿瘤、乳腺癌、肺癌等疾病时,血清CEA水平可显著升高。因此CEA是一种广谱肿瘤标记物。但CEA在膀胱癌特异度比较高。因此尿及血清中CEA 的检测亦被应用于膀胱癌的诊断和监测。
细胞角蛋白19片段(Cytokeratin-19-fragment CYFRA21-1,CK19), CK是构成上皮细胞细胞骨架的中间丝组成成分,具有高度的细胞和组织特异性,CK至少有20多种,其中与膀胱癌诊断、检测复发及预后关系最相关的主要有CK19、CK20。CYFRA21-1正常情况下其含量极低,当肿瘤产生时,蛋白酶的激活加速了细胞角蛋白的降解,大量细胞角蛋白片段得以释放,CYFRA21-1在血、尿中的含量均有所升高,CYFRA21-1可作为一种无创的膀胱癌诊断检测指标。
本发明建立一种多肿瘤标志物的组合,进而通过多个指标的联合检测提高膀胱癌的检测特异性和敏感性。
与现有技术相比,本发明的有益效果是:
(1)本发明提供了一种用于膀胱癌检测的标志物及检测试剂盒,该标志物及其试剂盒较为操作简便、廉价、高效,且属于无创的手段,可以用来诊断膀胱癌以及监测膀胱癌的复发。
(2)本发明提供的试剂盒灵敏度高、特异度强,并且便于群体性筛查的无创性检查,建立了一种方便、快速、敏感性高并且无创、普通人群较易接受的检测方法,对膀胱癌的及时发现、临床预后以及复发检测方面都具有重要的作用。
(3)本发明提供的试剂盒可以用于联合检测核基质蛋白22(NMP22)、***抗原(BTA)、癌胚抗原(CEA)、细胞角蛋白19片段(CYFRA21-1)。
(4)本发明提供的试剂盒采用吖啶酯直接化学发光,用吖啶酯直接标记抗体(抗原),与待测标本相应的抗原(抗体)发生免疫反应后,形成包被抗体-待测抗原-吖啶酯标记抗体复合物,加入激发液,不需要催化剂即分解、发光。收集、记录单位时间内所产生的光子能,这部分光的积分与待测抗原的量成正比,根据建立的标准曲线计算待测抗原的量。
(5)本发明提供的试剂盒当当对膀胱癌患者尿液NMP22、BTA、 CEA、CYFRA21四者联合检测时,具有较高的敏感度和特异度,可以对膀胱癌患者早期筛查、治疗效果和术后复发进行动态监测。
具体实施方式
下面通过具体实施例,对本发明的技术方案作进一步的具体说明。
实施例1
1.制备抗体包被的磁微粒
1.1鼠抗NMP22抗体包被的磁微粒制备
(1)取200μL磁珠溶液(50mg/mL),用0.01M PBS缓冲液(p H=7.4)清洗2次,最后用1m L PBS缓冲液重新悬浮磁珠;
(2)在步骤(1)得到的磁珠溶液中加入100μL的EDC溶液,涡旋混匀后于室温振荡反应2h;所述室温为15-40℃,本实施例中为25℃;EDC 溶液:0.01M MES((2-(N-吗啉代)乙烷磺酸))(PH=6.0)溶液配制5mg/ml 的EDC溶液(现配现用),避光备用;
(3)弃上清,用0.01M PBS缓冲液(p H=7.4)清洗3次;用1m L PBS缓冲液重新悬浮磁珠;
(4)加入纯化浓缩的鼠抗NMP22单克隆抗体50~800μg(本实施例中加入400μg),涡旋混匀后于室温振荡反应24h;
(5)用0.01M PBS缓冲液(p H=7.4)清洗3次,加入0.5mL 10%牛血清白蛋白(BSA)磷酸盐溶液,涡旋混匀,37℃振荡反应2h,封闭磁珠;
(6)弃上清,用0.0.1M PBS缓冲液pH7.4溶液清洗3遍,最后悬浮于20mL含有1%BSA、0.1%ProClin 300的PBS pH7.4溶液中,使磁珠的终浓度为0.5mg/mL,于4℃保存备用。
1.2鼠抗CEA抗体包被的磁微粒制备
方法同鼠抗NMP22抗体包被的磁微粒制备,替换其中抗体为鼠抗CEA 抗体。
1.3鼠抗BTA抗体包被的磁微粒制备
方法同鼠抗NMP22抗体包被的磁微粒制备,替换其中抗体为鼠抗BTA 抗体。
1.4鼠抗CYFRA21-1抗体包被的磁微粒制备
方法同鼠抗NMP22抗体包被的磁微粒制备,替换其中抗体为鼠抗 CYFRA21-1抗体。
2.吖啶酯标记的抗体的制备
2.1吖啶酯标记的鼠抗NMP22抗体的制备
取一定量的鼠抗NMP22单克隆抗体,用0.05mol/L pH 9.6PBS溶液调整鼠抗NMP22抗体浓度为1mg/mL,取一定量的吖啶酯,将NMP22抗体与吖啶酯按1:(5~20)摩尔比例进行混合(本实施例中摩尔比为1:10),室温避光震荡反应30min,将反应液移至透析袋,用0.02mol/L pH7.4的PBS 缓冲液4℃透析3次,去除游离吖啶酯,最后加入等体积甘油,放置于-20℃下保存待用。
2.2吖啶酯标记的鼠抗CEA抗体的制备
方法同吖啶酯标记的鼠抗NMP22抗体的制备,替换其中抗体为鼠抗 CEA抗体。
2.3吖啶酯标记的鼠抗BTA抗体的制备
方法同吖啶酯标记的鼠抗NMP22抗体的制备,替换其中抗体为鼠抗 BTA抗体。
2.4吖啶酯标记的鼠抗CYFRA21-1抗体的指标
方法同吖啶酯标记的鼠抗NMP22抗体的制备,替换其中抗体为鼠抗 CYFRA21-1抗体。
3.校准品的配制
校准品稀释液:配制含10%灭活新生牛血清的0.02mol/L pH7.4磷酸盐缓冲液。
3.1NMP22校准品的配制
将NMP22抗原用校准品稀释液稀释,分别配制浓度为:0、0.5ng/mL、 5ng/mL、10ng/mL、50ng/mL、100ng/mL的NMP22校准品。
3.2CEA校准品的配制
方法同NMP22校准品的配制。
3.3BTA校准品的配制
方法同NMP22校准品的配制。
3.4CYFRA21-1校准品的配制
方法同NMP22校准品的配制。
4.激发液A:为0.5%的H2O2和0.1mol/LHNO3的溶液;激发液B:为 0.35mmol/L的NaOH和3%Triton X-100的溶液;洗涤液为:含有 1%Tween-20的20×PBS缓冲液(pH7.0-8.0)。
5.检测步骤
(1)首先检测前准备工作:将试剂盒与待检测的样本从所储存的环境中取出,置于室温环境中大约30分钟;将尿液于通用离心机中2500rpm-3000rpm离心10-15min,吸取上清用于检测,得到待检测尿液样品;
(2)加样:分别取适量的NMP22抗体包被的磁微粒、CEA抗体包被的磁微粒、BTA抗体包被的磁微粒、CYFRA21-1抗体包被的磁微粒至 1×PBS缓冲液中,配制成混合磁微粒包被抗体的悬浮液。
分别取50μl检测样本(校准品和待测尿液)和50μl混合磁微粒包被的抗体溶液于孵育杯内混匀,37℃孵育10min;
(3)加入吖啶酯标记的抗体试剂:分别取适量的吖啶酯标记的NMP22 抗体、吖啶酯标记的CEA抗体、吖啶酯标记的BTA抗体、吖啶酯标记的 CYFRA21-1抗体至1×PBS缓冲液中,配制成混合的吖啶酯标记的抗体工作液,混合液中各吖啶酯标记抗体量均为:0.03μg/ml。
取50μl混合的吖啶酯标记的抗体工作液,加入步骤(2)的孵育杯内, 37℃孵育20min;
(4)洗涤:用洗涤液洗涤2遍;
(5)发光:依次加入50μl激发液A和50μl激发液B,混匀;
(6)读值:测定样本的相对发光强度,根据校准曲线,分别计算出样品中NMP22抗原、CEA抗原、BTA抗原、CYFRA21-1抗原的浓度,实验结果如下:
不同检测项目用于膀胱癌诊断的灵敏度、特异性
检测项目 | 灵敏度 | 特异性 |
NMP22单检 | 84.3% | 55% |
BTA单检 | 70% | 80.7% |
CEA单检 | 78.83 | 28.47 |
CYFRA21-1单检 | 76.6% | 85% |
NMP22+BTA | 71.9% | 64% |
NMP22+CEA | 81.75% | 20.95% |
NMP22+CYFRA21-1 | 82.8% | 35% |
NMP22+BTA+CEA | 90.3% | 80% |
NMP22+BTA+CYFRA21-1 | 93.7% | 55.2% |
NMP22+CEA+CYFRA21-1 | 96.13% | 50% |
NMP22+BTA+CEA+CYFRA21-1 | 98.4% | 80% |
结果表示,当对膀胱癌患者尿液NMP22、BTA、CEA、CYFRA21 四者联合检测时,具有较高的敏感度和特异度,可以对膀胱癌患者早期筛查、治疗效果和术后复发进行动态监测。
最后应说明的是:以上所述仅为本发明的优选实施例,不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种用于膀胱癌检测的标志物,其特征在于,所述标志物包括核基质蛋白22(NMP22)、癌胚抗原(CEA)、***抗原(BTA)和细胞角蛋白19片段(CYFRA21-1)。
2.一种权利要求1所述的用于膀胱癌检测的标志物在制备用于膀胱癌检测的检测试剂或检测试剂盒中的应用。
3.根据权利要求2所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述检测试剂盒包括:抗体包被的磁微粒、吖啶酯标记的抗体、校准品、激发液A、激发液B和洗涤液,所述抗体为标志物对应的抗体。
4.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述抗体包括:NMP22抗体、CEA抗体、BTA抗体、和CYFRA21-1抗体。
5.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述抗体包被的磁微粒包括:NMP22抗体包被的磁微粒、CEA抗体包被的磁微粒、BTA抗体包被的磁微粒、和CYFRA21-1抗体包被的磁微粒。
6.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述吖啶酯标记的抗体包括:吖啶酯标记的NMP22抗体、吖啶酯标记的CEA抗体、吖啶酯标记的BTA抗体、和吖啶酯标记的CYFRA21-1抗体。
7.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述校准品为不同浓度的标志物校准品,包括NMP22校准品、CEA校准品、BTA校准品、和CYFRA21-1校准品。
8.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述激发液A:为0.5%的H2O2和0.1mol/L的HNO3的溶液;所述激发液B:为0.35mmol/L的NaOH和3%TritonX-100的溶液;所述洗涤液为:含有1%Tween-20的20×PBS缓冲液,pH7.0-8.0。
9.根据权利要求3所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述检测试剂盒的检测包括如下步骤:
(1)加样:取检测样本和抗体包被的磁微粒于孵育杯内混匀并孵育;
(2)加入吖啶酯标记的抗体试剂:将吖啶酯标记的抗体加入步骤(1)的孵育杯内继续孵育;
(3)洗涤:用洗涤液洗涤;
(4)发光:依次加入激发液A和激发液B,进行发光处理;
(5)读值:测定检测样本的相对发光强度,根据校准曲线,计算出检测样本中待测标志物的浓度。
10.根据权利要求9所述一种用于膀胱癌检测的检测试剂盒,其特征在于,所述步骤(1)中的检测样本为校准品或待测尿液。
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