CN111707825A - 联合检测肿瘤标志物mct1和mct4的试剂盒及其制备方法、应用 - Google Patents
联合检测肿瘤标志物mct1和mct4的试剂盒及其制备方法、应用 Download PDFInfo
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Abstract
本发明公开了联合检测肿瘤标志物MCT1和MCT4的试剂盒及其制备方法、应用,所述试剂盒包括酶标二抗试剂、磁微粒试剂和化学发光底物,所述酶标二抗试剂由碱性磷酸酶标记的Anti‑MCT1‑2和碱性磷酸酶标记的Anti‑MCT4‑2组成,所述磁微粒试剂为包被有Anti‑MCT1‑1和Anti‑MCT4‑1的磁微粒。采用本发明所述试剂盒能够同时检测样本里的MCT1和MCT4,以提高肿瘤早期诊断的准确性。
Description
技术领域
本发明涉及磁微粒化学发光免疫诊断技术领域,具体涉及联合检测肿瘤标志物MCT1和MCT4的试剂盒及其制备方法、应用。
背景技术
肿瘤严重威胁人们的健康,全世界每年有700多万人死于恶性肿瘤。我国肿瘤诊治的国策是早发现,早诊断,早治疗。而实验诊断是肿瘤检查必不可少的手段之一,其中血清、体液、分泌物或排泄物中肿瘤细胞产生的糖蛋白、激素、酶类、代谢产物等肿瘤标志物(Tumor Markers,TM)的检测是目前肿瘤的诊断、疗效观察、预后等的重要手段。
目前对代谢途径的理解主要是基于分化组织中非增殖细胞的研究,在有氧条件下,大多数分化细胞主要通过三羧酸循环(TCA)将糖酵解产生的丙酮酸氧化为二氧化碳,并生成NADH,促进氧化磷酸化,最大限度地合成ATP,从而减少乳酸的产生。而在厌氧条件下,分化细胞会产生大量的乳酸,例如高强度剧烈运动时候。与之相反,大多数肿瘤细胞会产生大量的乳酸,而与氧气无关,因此,肿瘤细胞的代谢通常被称为“有氧糖酵解”,这一现象被称为瓦伯格效应(Warburg Effect)。糖酵解是肿瘤细胞获取能量的重要途径,其中一种重要的转运蛋白为单羧酸转运蛋白家族(Monocarboxylate Transporters,MCTs),其负责转运乳酸等单羧酸类化合物的跨膜转运,共有14个亚型,其中单羧酸转运蛋白1和4(MCT1、MCT4)主要通过介导乳酸的跨膜转运实现对糖酵解的调控。
现有的诊断技术导致肿瘤早期的诊断准确性较低,以肝癌为例,肝癌早期诊断的常规监测筛查指标主要包括甲胎蛋白(AFP)和肝脏超声检查(US)。综合国内外的报道和临床实际,AFP诊断肝癌的敏感性在60%左右,漏诊率为40%,特异性在80%左右,误诊率为20%,2010版美国肝病研究学会(AASLD)指南已经不再将AFP作为筛查指标。而超声检查对于大于20mm的结节,诊断符合率为84%,但对于肝内5-20mm的结节,超声的敏感性只有33%。因此,对于肿瘤的早期诊断仍具有很大的挑战。
发明内容
本发明的目的在于提供联合检测肿瘤标志物MCT1和MCT4的试剂盒,解决肿瘤早期的诊断准确性较低的问题。
此外,本发明还提供上述试剂盒的制备方法和应用。
本发明通过下述技术方案实现:
联合检测肿瘤标志物MCT1和MCT4的试剂盒,包括酶标二抗试剂、磁微粒试剂和化学发光底物,所述酶标二抗试剂由碱性磷酸酶标记的Anti-MCT1-2和碱性磷酸酶标记的Anti-MCT4-2组成,所述磁微粒试剂为包被有Anti-MCT1-1和Anti-MCT4-1的磁微粒。
本申请中的MCT1-1和MCT1-2是抗原,样本中的,Anti-MCT1-1和Anti-MCT1-2是抗体。
申请人发现:MCT1、MCT4在许多肿瘤细胞中呈现高表达状态,例如肝癌、肾癌、前列腺癌、口腔鳞状细胞癌、胰腺癌等,且与肿瘤的增殖、侵袭及预后密切相关。
本发明将MCT1和MCT4作为一种有效的靶点用于检测试剂盒,在酶标抗体和免疫磁珠中均含有MCT1和MCT4融合蛋白,采用本发明所述试剂盒能够同时检测血清/血浆样本里的MCT1和MCT4,以提高肿瘤早期诊断的准确性。
进一步地,所述化学发光底物为3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷二钠盐。
本发明采用全自动磁微粒化学发光检测方法,选取碱性磷酸酶(AP)-金刚烷(AMPPD)体系,具有稳定性好,灵敏度高,重复性好等优点。同时全自动化能大大缩短检测时间,完成一个测试所需的总时间在20分钟以内,而且操作简易方便,真正实现了检测全自动化。
进一步地,磁微粒的直径为0.5-2.7μm,具有超顺磁性。
进一步地,磁微粒试剂的浓度为0.05μg/mL。
进一步地,酶标二抗试剂的浓度为0.05μg/mL。
进一步地,还包括校准品和清洗浓缩液,所述校准品为MCT1和MCT4融合蛋白,用于制备校准曲线。
进一步地,磁微粒试剂的制备方法如下:
将生物素标记的Anti-MCT1-1和Anti-MCT4-1与链亲和素磁微粒进行结合获得磁微粒试剂。
具体地:构建的MCT1和MCT4融合蛋白,用0.05mol/L pH7.6的PBS缓冲液稀释,分别制备成400ng/mL,200ng/mL,50ng/mL,20ng/mL,5ng/mL,0ng/mL的梯度标准液,制备标准曲线,如图2所示。
进一步地,酶标二抗试剂的制备方法如下:
将碱性磷酸酶与Anti-MCT1-2和Anti-MCT4-2进行偶联获得酶标二抗试剂。
联合检测肿瘤标志物MCT1和MCT4的试剂盒的制备方法,包括以下步骤:
步骤S1、制备酶标二抗试剂:将碱性磷酸酶与Anti-MCT1-2和Anti-MCT4-2进行偶联获得酶标二抗试剂;
步骤S2、制备磁微粒试剂:将生物素标记的Anti-MCT1-1和Anti-MCT4-1与链亲和素磁微粒进行结合获得磁微粒试剂;
步骤S3、将酶标二抗试剂、磁微粒试剂和化学发光底物组装成试剂盒。
联合检测肿瘤标志物MCT1和MCT4的试剂盒的应用,所述试剂盒用于肿瘤标志物MCT1和MCT4的检测。
本发明试剂盒的检测原理(如图1所示):
采用将生物素标记的Anti-MCT1-1/MCT4-1与链亲和素磁微粒(SA-M)进行结合,最终获得一种浓度为0.05μg/mL磁微粒,然后加入样本孵育一定时间后清洗去除未结合的样本;然后加入R2试剂(酶标二抗试剂)孵育一定时间后清洗去除未结合的R2;最后加入底物进行发光并读取光子数,通过标准曲线实现定量检测。
本发明与现有技术相比,具有如下的优点和有益效果:
1、采用本发明所述试剂盒能够同时检测样本里的MCT1和MCT4,以提高肿瘤早期诊断的准确性。
2、本发明完成所有流程出结果时间约为20min,相比核酸检测大大缩短了反应时间。
3、发明采用碱性磷酸酶(AP)-金刚烷(AMPPD)系统,灵敏度高,相比核酸检测,极大提高了阳性检出率。
4、本发明为全自动封闭操作系统,可靠性高,稳定性好,检测结果重复性好。
5、本发明从稀释、加样、孵育、清洗以及检测步骤实现了全自动化,避免了人为操作带来的结果偏差。
附图说明
此处所说明的附图用来提供对本发明实施例的进一步理解,构成本申请的一部分,并不构成对本发明实施例的限定。在附图中:
图1为基于磁微粒化学发光免疫检测原理图;
图2为通过构建的MCT1和MCT4融合蛋白制备的标准曲线图;
图3为MCT1和MCT4的抗体通过蛋白免疫印迹检测图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施例和附图,对本发明作进一步的详细说明,本发明的示意性实施方式及其说明仅用于解释本发明,并不作为对本发明的限定。
实施例:
联合检测肿瘤标志物MCT1和MCT4的试剂盒,包括酶标二抗试剂、磁微粒试剂和化学发光底物,所述酶标二抗试剂由碱性磷酸酶标记的Anti-MCT1-2和碱性磷酸酶标记的Anti-MCT4-2组成,所述磁微粒试剂为包被有Anti-MCT1-1和Anti-MCT4-1的磁微粒,所述酶标二抗试剂的浓度为0.05μg/mL(R2试剂中含有浓度为0.05μg/mL的碱性磷酸酶标记的链霉亲和素),所述磁微粒试剂的浓度为0.05μg/mL,所述化学发光底物为3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷二钠盐,要成分为金刚烷(AMPPD),在碱性磷酸酶的催化下释放光子,所述磁微粒的直径为0.5-2.7μm,还包括校准品、质控品和清洗浓缩液,所述校准品为MCT1和MCT4融合蛋白,用于制备校准曲线。
本实施例所述试剂盒的组成如表1所示:
表1
本实施例所述试剂盒的制备方法,包括以下步骤:
步骤S1、制备酶标二抗试剂:利用已有成熟的偶联技术,将碱性磷酸酶(AP)与Anti-MCT1-2和Anti-MCT4-2进行偶联;最终获得一种酶标记的Anti-MCT1-2/Anti-MCT4-2浓度为0.05μg/mL的R2试剂:
取出1mg AP溶于0.5mL蒸馏水中,充分溶解,取0.125mL进行标记,加入新鲜配制的12mmol/L的过碘酸钠蒸馏水溶液0.125mL,混匀,2-8℃放置30min,加入32mmol/L的乙二醇蒸馏水溶液0.125mL中,混匀,室温避光放置30min以终止过碘酸钠的作用,将0.25mg的MCT1-2/MCT4-2抗体加入上述溶液中,混匀,装入透析袋用0.05mol/L碳酸钠缓冲液(PH为9.5)2-8℃透析16-24小时。最后将溶液取出,加入硼氢化钠溶液放置2h,用0.02mol/LPH7.4的PBS溶液透析,最后取出离心,测量体积加等体积的甘油保存。
步骤S2、制备磁微粒试剂:将生物素标记的Anti-MCT1-1和Anti-MCT4-1与链亲和素磁微粒(SA-M)进行结合,最终获得0.05μg/mL的磁微粒(M-SA-Biotin-Anti-MCT1-1/Anti-MCT4-1)试剂:
通过化学共沉淀方法制备,选用粒径为500nm的棕色圆形磁微粒,用0.05mol/L的MES缓冲液进行清洗,之后再加入1.25%的戊二醛和0.05mol/L PH6.0的PBS振荡反应1h;然后再用0.05mol/L的MES缓冲液进行清洗,得到活化磁珠,取出活化磁珠与MCT1-1/MCY4-1抗体,以1mg磁珠:250μg抗体的比例在25℃的条件下孵育1h,磁性分离后再加入2%的BSA封闭游离基,MES再次清洗得到免疫磁珠;
步骤S3、将酶标二抗试剂、磁微粒试剂和化学发光底物组装成试剂盒;
步骤S4、构建的MCT1和MCT4融合蛋白,用0.05mol/L pH7.6的PBS缓冲液稀释,分别制备成400ng/mL,200ng/mL,50ng/mL,20ng/mL,5ng/mL,0ng/mL的梯度标准液,制备标准曲线,如图2所示。
本实施例中配制校准剂和质控剂所用的缓冲液包括:浓度为0.1M的三羟甲基氨基甲烷缓冲液(Tris);所述三羟甲基氨基甲烷缓冲液(Tris)中含有质量分数为0.5%的牛血清白蛋白(BSA)、质量分数为0.1%的吐温20(Tween-20)和质量分数为1.0%的蔗糖。
以肝癌样本为例,使用本发明的检测方法对其进行检测,验证本试剂盒可以同时检测样本里的MCT1和MCT4,具体步骤如下:
验证所包被的MCT1和MCT4抗体可以同时与样本里的MCT1和MCT4反应,首先将磁珠拉下来的抗体通过磁力架吸附下来,再使用洗脱液将样本中的MCT1和MCT4脱离,最后使用MCT1和MCT4的抗体通过蛋白免疫印迹检测,如图3所示,MCT1为19KDa,MCT4为50KDa,并且同时检测出MCT1和MCT4的存在。说明了本试剂盒可以同时将MCT1和MCT4检测出来,提高了检测肿瘤的阳性率。
试剂盒的应用效果及性能测试:
通过收集肝癌样本和健康人样本进行性能验证:
1.阳性符合率:本发明试剂盒对34例临床诊断为肝癌早期的样本进行了测定。结果表明,本发明试剂盒阳性符合率97.1%,如表2所示,说明本试剂盒临床阳性检出率高。
表2检测试剂盒临床样本比对
2.阴性符合率:本发明试剂盒对50例随机健康人体检样本进行了测定。结果表明,本试剂盒阴性符合率100%(50/50),说明本试剂盒临床阴性符合率高。
3.精密度:分别对高(400ng/mL)、中(50ng/mL)、低(5ng/mL)值质控品进行测定,每天两次,分上下午检测,每次进行4个重复,共检测10天,每种浓度共测定80次,计算变异系数,检查试剂在高值、中值和低值标本时的精密度。如表3所示,结果表明变异系数都在8%以内,重复性好。
表3检测试剂盒精密度测试
AVERAGE | CV | |||||||||
5ng/mL | 156578 | 146485 | 135389 | 129647 | 152466 | 138975 | 159862 | 149987 | 146173.6 | 7.30% |
50ng/mL | 1334672 | 1445459 | 1398546 | 1535687 | 1369842 | 1469875 | 1293264 | 1520364 | 1420964 | 6.10% |
400ng/mL | 8859436 | 8209865 | 7834542 | 7636745 | 8423575 | 7998721 | 9064978 | 9265964 | 8411728 | 7.10% |
4.稳定性:本检测试剂盒(M,R2)37℃放置7天,测定高、中、低3种浓度质控的信号保留率。结果>95%,说明试剂盒稳定好,符合临床要求。
5.特异性:在含有高、中、低不同浓度待测物的血清中分别添加不同浓度的胆红素、血红蛋白、类风湿因子、脂、RF、HAMA进行检测验证,检测结果显示,上述6种物质对本试剂盒检测结果无干扰。
通过收集肾癌样本和健康人样本进行性能验证:
1.阳性符合率:本发明试剂盒对51例临床诊断为肝癌早期的样本进行了测定。结果表明,本发明试剂盒阳性符合率96.1%,如表4所示,说明本试剂盒临床阳性检出率高。
表4检测试剂盒临床样本比对
2.阴性符合率:本发明试剂盒对30例随机健康人体检样本进行了测定。结果表明,本试剂盒阴性符合率100%(30/30),说明本试剂盒临床阴性符合率高。
3.精密度:分别对高(400ng/mL)、中(50ng/mL)、低(5ng/mL)值质控品进行测定,每天两次,分上下午检测,每次进行4个重复,共检测10天,每种浓度共测定80次,计算变异系数,检查试剂在高值、中值和低值标本时的精密度。如表5所示,结果表明变异系数都在8%以内,重复性好。
表5检测试剂盒精密度测试
AVERAGE | CV | |||||||||
5ng/mL | 135578 | 148481 | 146989 | 129647 | 153966 | 138975 | 159862 | 137287 | 143848.125 | 7.10% |
50ng/mL | 1344672 | 1335459 | 1298516 | 1475687 | 1399842 | 1389875 | 1563264 | 1520754 | 1416008.625 | 6.70% |
400ng/mL | 8559436 | 7319865 | 8058454 | 8236745 | 8423575 | 9198721 | 9164978 | 8665964 | 8453467.25 | 7.20% |
4.稳定性:本检测试剂盒(M,R2)37℃放置7天,测定高(400ng/mL)、中(50ng/mL)、低(5ng/mL)3种浓度质控的信号保留率。结果>95%,说明试剂盒稳定好,符合临床要求。
5.特异性:在含有高、中、低不同浓度待测物的血清中分别添加不同浓度的胆红素、血红蛋白、类风湿因子、脂、RF、HAMA进行检测验证,检测结果显示,上述6种物质对本试剂盒检测结果无干扰。
综上,本发明所述试剂盒不仅能够同时检测肿瘤中标志物MCT1和MCT4,以提高不同种类的肿瘤检测的准确性,且具有重复性好、盒稳定好和特异性好的优点。
以上所述的具体实施方式,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施方式而已,并不用于限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、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.联合检测肿瘤标志物MCT1和MCT4的试剂盒,其特征在于,包括酶标二抗试剂、磁微粒试剂和化学发光底物,所述酶标二抗试剂由碱性磷酸酶标记的Anti-MCT1-2和碱性磷酸酶标记的Anti-MCT4-2组成,所述磁微粒试剂为包被有Anti-MCT1-1和Anti-MCT4-1的磁微粒。
2.根据权利要求1所述的联合检测肿瘤标志物MCT1和MCT4的试剂盒,其特征在于,所述化学发光底物为3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷二钠盐。
3.根据权利要求1所述的联合检测肿瘤标志物MCT1和MCT4的试剂盒,其特征在于,所述磁微粒的直径为0.5-2.7μm。
4.根据权利要求1所述的联合检测肿瘤标志物MCT1和MCT4的试剂盒,其特征在于,所述磁微粒试剂的浓度为0.05μg/mL。
5.根据权利要求1所述的联合检测肿瘤标志物MCT1和MCT4的试剂盒,其特征在于,所述酶标二抗试剂的浓度为0.05μg/mL。
6.根据权利要求1所述的联合检测肿瘤标志物MCT1和MCT4的试剂盒,其特征在于,还包括校准品和清洗浓缩液,所述校准品为MCT1和MCT4融合蛋白,用于制备校准曲线。
7.根据权利要求1所述的联合检测肿瘤标志物MCT1和MCT4的试剂盒,其特征在于,所述磁微粒试剂的制备方法如下:
将生物素标记的Anti-MCT1-1和Anti-MCT4-1与链亲和素磁微粒进行结合获得磁微粒试剂。
8.根据权利要求1所述的联合检测肿瘤标志物MCT1和MCT4的试剂盒,其特征在于,所述酶标二抗试剂的制备方法如下:
将碱性磷酸酶与Anti-MCT1-2和Anti-MCT4-2进行偶联获得酶标二抗试剂。
9.如权利要求1-8任一项所述的联合检测肿瘤标志物MCT1和MCT4的试剂盒的制备方法,其特征在于,包括以下步骤:
步骤S1、制备酶标二抗试剂:将碱性磷酸酶与Anti-MCT1-2和Anti-MCT4-2进行偶联获得酶标二抗试剂;
步骤S2、制备磁微粒试剂:将生物素标记的Anti-MCT1-1和Anti-MCT4-1与链亲和素磁微粒进行结合获得磁微粒试剂;
步骤S3、将酶标二抗试剂、磁微粒试剂和化学发光底物组装成试剂盒。
10.如权利要求1-8任一项所述的联合检测肿瘤标志物MCT1和MCT4的试剂盒的应用,其特征在于,所述试剂盒用于肿瘤标志物MCT1和MCT4的检测。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113552353A (zh) * | 2021-07-12 | 2021-10-26 | 江南大学 | 一种用于PCa及CRPC疾病诊断的磁微粒化学发光试剂盒 |
WO2024017383A1 (zh) * | 2022-07-21 | 2024-01-25 | 佰舟生物科技(苏州)有限公司 | 抗mct1抗体及其用途 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050032135A1 (en) * | 2003-07-03 | 2005-02-10 | Xenoport, Inc. | Monocarboxylate transporters expressed in cancer cells |
CN104034892A (zh) * | 2014-06-23 | 2014-09-10 | 广西博士海意信息科技有限公司 | 一种肿瘤标志物afp磁微粒化学发光免疫分析试剂盒及其检测方法 |
CN106546732A (zh) * | 2015-09-17 | 2017-03-29 | 深圳迈瑞生物医疗电子股份有限公司 | 人类免疫缺陷病毒抗原和抗体联合检测试剂盒及其用途和检测方法 |
CN108008132A (zh) * | 2017-12-04 | 2018-05-08 | 北京润诺思医疗科技有限公司 | 一种联合检测卵巢癌肿瘤标志物he4和ca125的试剂盒及其制备方法和应用 |
CN110333352A (zh) * | 2019-08-07 | 2019-10-15 | 基因科技(上海)股份有限公司 | 肿瘤相关自身抗体及肿瘤标志物联合检测试剂盒 |
-
2020
- 2020-07-29 CN CN202010744902.1A patent/CN111707825A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050032135A1 (en) * | 2003-07-03 | 2005-02-10 | Xenoport, Inc. | Monocarboxylate transporters expressed in cancer cells |
CN104034892A (zh) * | 2014-06-23 | 2014-09-10 | 广西博士海意信息科技有限公司 | 一种肿瘤标志物afp磁微粒化学发光免疫分析试剂盒及其检测方法 |
CN106546732A (zh) * | 2015-09-17 | 2017-03-29 | 深圳迈瑞生物医疗电子股份有限公司 | 人类免疫缺陷病毒抗原和抗体联合检测试剂盒及其用途和检测方法 |
CN108008132A (zh) * | 2017-12-04 | 2018-05-08 | 北京润诺思医疗科技有限公司 | 一种联合检测卵巢癌肿瘤标志物he4和ca125的试剂盒及其制备方法和应用 |
CN110333352A (zh) * | 2019-08-07 | 2019-10-15 | 基因科技(上海)股份有限公司 | 肿瘤相关自身抗体及肿瘤标志物联合检测试剂盒 |
Non-Patent Citations (1)
Title |
---|
穆克飞等: "MCT1和MCT4在肿瘤发展中的作用及作为肾癌潜在治疗靶点的研究进展", 《泌尿外科杂志(电子版)》 * |
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CN113552353A (zh) * | 2021-07-12 | 2021-10-26 | 江南大学 | 一种用于PCa及CRPC疾病诊断的磁微粒化学发光试剂盒 |
CN113552353B (zh) * | 2021-07-12 | 2023-08-25 | 江南大学 | 一种用于PCa及CRPC疾病诊断的磁微粒化学发光试剂盒 |
WO2024017383A1 (zh) * | 2022-07-21 | 2024-01-25 | 佰舟生物科技(苏州)有限公司 | 抗mct1抗体及其用途 |
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