CN106771259B - 用于检测人血清中Pygo2蛋白含量的ELISA试剂盒、使用方法及用途 - Google Patents

用于检测人血清中Pygo2蛋白含量的ELISA试剂盒、使用方法及用途 Download PDF

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CN106771259B
CN106771259B CN201710129492.8A CN201710129492A CN106771259B CN 106771259 B CN106771259 B CN 106771259B CN 201710129492 A CN201710129492 A CN 201710129492A CN 106771259 B CN106771259 B CN 106771259B
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秦文英
唐景峰
周策凡
张毅
李舜尧
何文早
陈兴珍
胡婷
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Abstract

本发明公开了一种用于检测人血清Pygo2蛋白含量的ELISA试剂盒、使用方法及用途。基于Pygo2蛋白的双抗体夹心法ELISA试剂盒原料及配方为：包被抗Pygo2蛋白抗体的酶标板、Pygo2标准抗原、阴、阳性对照液、酶标抗体、PBST缓冲液、封闭缓冲液、底物液A、底物液B和终止液。本发明所提供的基于双抗夹心法检测人血清中Pygo2含量的酶联免疫试剂盒可实现对人血清中Pygo2含量的准确定量检测，该检测方法可以作为一种无创伤性、简便、迅速的诊断和监测结直肠癌、脑胶质瘤等疾病的病程发展。

Description

用于检测人血清中Pygo2蛋白含量的ELISA试剂盒、使用方法 及用途
技术领域
本发明属于生物技术和医学领域，涉及一种用于检测人血清中Pygo2蛋白含量的ELISA试剂盒、使用方法及用途。
背景技术
Wnt信号通路广泛存在于无脊椎动物和脊椎动物中，是一类在物种进化过程中高度保守的信号通路。Wnt信号在动物胚胎的早期发育、器官形成、组织再生和其它生理过程中，具有至关重要的作用。如果这条信号通路中的关键蛋白发生突变或者异常表达，导致信号异常活化，就可能诱导癌症的发生。Wnt信号通路包括经典的Wnt信号通路与非经典的Wnt信号通路，在经典通路即Wnt-β-catenin信号通路中，Wnt因子通过激活细胞膜上的Frizzle/LRP5/6协同受体后抑制细胞内游离β-catenin蛋白的磷酸化和降解，细胞质中的β-catenin蛋白水平升高后将发生β-catenin蛋白的核移位，导致细胞核中β-catenin蛋白升高，胞核中β-catenin蛋白能够联合Pygo2、Bcl-9以及FoxM1蛋白共同与TCF/LEF-1转录因子家族形成复合体并激活Wnt信号通路下游靶基因的转录激活。
Pygo2蛋白是Wnt信号通路中β-catenin下游重要成员之一，目前越来越多的研究已经发现，在很多肿瘤中Pygo2蛋白均呈现高表达。
检测人血清中Pygo2蛋白的水平，可以快速诊断和监测结直肠癌、脑胶质瘤等疾病的病程发展。1971年瑞典学者Engvail和Perlmann，荷兰学者Van Weerman和Schuurs分别报道将免疫技术发展为检测体液中微量物质的固相免疫测定方法，即酶联免疫吸附测定法(enzyme-linked immunosorbent assay , ELISA)。它采用抗原与抗体的特异反应将待测物与酶连接，然后通过酶与底物产生颜色反应，用于定量测定。迄今为止，还没有利用ELISA检测Pygo2蛋白血清水平的试剂盒。
发明内容
本发明的目的是提供一种检测人血清Pygo2蛋白含量的酶联免疫试剂盒，本发明利用双抗体夹心法检测Pygo2蛋白的浓度,具有高灵敏度、简便快速、准确度高的特点。
本发明所提供的用于检测人血清中Pygo2蛋白含量的酶联免疫试剂盒，包含
Pygo2标准抗原、阴、阳性对照液、抗Pygo2蛋白抗体包被的酶标板、辣根过氧化物酶（HRP）标记的抗人Pygo2蛋白抗体溶液和辅助试剂。
在本发明中，包被酶标板的抗Pygo2蛋白抗体和HRP标记的抗Pygo2蛋白抗体可通过商业渠道获得。
在本发明的一个实施方案中，所述包被酶标板的制备过程为：将抗人Pygo2蛋白抗体用稀释液稀释后加入酶标板各孔，每孔300μL，置4℃条件下，孵育24小时；移去孔内液体，PBST洗后拍干，再用封闭液37℃封闭，PBST洗涤液洗3次，每次5分钟，洗后拍干，晾干，即获得抗Pygo2蛋白抗体包被的酶标板。
所述稀释液为0.05mol/L磷酸盐缓冲液，pH＝7.4，磷酸盐缓冲液是每升含有16gNaCl，0.4g KCl，0.4g KH2PO4，5.8g Na2HPO4的水溶液。
所述PBST为含有Tween 20体积百分比0.05％，pH为7.0的磷酸盐缓冲液。
所述封闭液为含5%BSA的PBST。
所述辣根过氧化物酶（HRP）标记的抗Pygo2蛋白抗体按1:2000稀释使用。
所述辅助试剂包括底物液A、底物液B、终止液。
所述底物液A为Na2HPO4 14.60g、柠檬酸9.33g、过氧化氢脲0.52g,溶于三蒸水,终定容至1000mL，调至pH5.0～5.4。
所述底物液B为四甲基联苯胺 20mg、无水乙醇8～10 mL，加双蒸水至1 000mL，过滤除菌。
所述终止液为1mol/L硫酸。
所述Pygo2标准抗原溶液浓度为200ng/mL，通过倍比稀释至终浓度为0、10、20、50、100、200ng/mL，进行标准曲线测定。
所述阴、阳性对照血清及待检血清做1∶100倍稀释使用。
所述试剂盒的检测步骤如下：
1、取包被酶标板，应用PBST洗涤ELISA反应板5次；
2、将阴、阳性对照血清及待检血清做1∶100倍稀释，按100μL加入酶标板反应孔中，37℃孵育1h后，应用PBST洗涤ELISA酶标板各反应孔5次；
3、封闭液稀释辣根过氧化物酶标记的的抗Pygo2蛋白抗体，加入到酶标板，37℃孵育，应用PBST洗涤ELISA酶标板各反应孔5次；
4、将底物液A和B各50μL加入ELISA反应板，避光室温显色10~15min，加入50μL终止液，立即在酶标仪450nm波长下读取OD值。
所述试剂盒的定性结果判定如下：
以P/N值(样品孔OD值/阴性孔OD值)大于或等于2.163为阳性；P/N值小于2.163，但大于1.541为可疑；P/N小于1.041为阴性。
定量结果分析：根据标准曲线可计算样品中Pygo2蛋白质含量。
所述酶联免疫试剂盒在检测人血清中Pygo2蛋白含量中的应用也属于本发明的保护范围。
所述酶联免疫试剂盒在脑胶质瘤、结直肠癌的辅助诊断中的应用也属于本发明的保护范围。
本发明提供的基于双抗夹心法检测人血清中Pygo2蛋白含量的ELISA试剂盒，不仅可以定性还可以定量的检测人血清中Pygo2蛋白质含量；具有高灵敏度、简便快速、准确度高的特点。检测方法可以作为一种无创伤性的诊断和监测结直肠癌和脑胶肿瘤病程发展的方法。
附图说明
图1是本发明试剂盒标准曲线图。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
【实施例1】利用ELISA试剂盒进行结直肠癌患者血清中Pygo2蛋白质的检测
1.样品处理：采集患者血液，置于离心管中，待血液凝固后，于2500rpm条件下离心10min，得到待测血清样本，于-20℃保存，避免反复冻融。
2.取包被酶标板，应用PBST洗涤ELISA反应板5次；将阴、阳性对照血清及待检血清做1：100倍稀释，按100μL加入酶标板反应孔中，每组样品复孔3次，在37℃孵育1h后，应用PBST洗涤ELISA酶标板各反应孔5次；封闭液稀释辣根过氧化物酶标记的的抗Pygo2蛋白抗体，加入到酶标板，37℃孵育，应用PBST洗涤ELISA酶标板各反应孔5次；将底物液A和B各50μL加入ELISA反应板，避光室温显色10～15min，加入50μL终止液，立即在酶标仪450nm波长下读取OD值；
3.结果分析：P/N=3.186>2.163,说明结直肠癌患者中Pygo2蛋白质含量偏高，为阳性。

Claims (1)

1.检测血清中Pygo2蛋白的试剂在制备结直肠癌诊断试剂中的应用。
CN201710129492.8A 2017-03-06 2017-03-06 用于检测人血清中Pygo2蛋白含量的ELISA试剂盒、使用方法及用途 Expired - Fee Related CN106771259B (zh)

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CN110540596B (zh) * 2018-12-20 2021-04-06 湖北工业大学 基于卡他莫拉菌表面蛋白抗体的卡他莫拉菌Elisa检测试剂盒及制备方法
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