CN110045120A - 检测生物样品中wisp1的浓度在制备用于乳癌筛检及侦测复发的检测试剂盒及其应用 - Google Patents

检测生物样品中wisp1的浓度在制备用于乳癌筛检及侦测复发的检测试剂盒及其应用 Download PDF

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CN110045120A
CN110045120A CN201810033799.2A CN201810033799A CN110045120A CN 110045120 A CN110045120 A CN 110045120A CN 201810033799 A CN201810033799 A CN 201810033799A CN 110045120 A CN110045120 A CN 110045120A
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江坤俊
叶俊男
庄宏亨
叶大森
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CHANGGENG UNIV
Chang Gung University CGU
Chang Gung Medical Foundation Chang Gung Memorial Hospital at Keelung
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Abstract

一种检测生物样品中WISP1的浓度在制备用于乳癌筛检及侦测复发的检测试剂盒及其应用，所述检测试剂盒包含：检测有效量WISP1浓度的试剂；选自下组的一种或多种物质：乙二胺四乙酸二钠、清洗液、生物素化抗体、链霉亲和素溶液、基质试剂和终止液，透过比较健康者及罹患乳癌患者血清样本中WISP1的浓度变化，以定义WISP1的浓度改变对于罹患乳癌风险高低之关联性，做为该试剂盒预测高罹患乳癌风险及复发侦测。

Description

检测生物样品中WISP1的浓度在制备用于乳癌筛检及侦测复 发的检测试剂盒及其应用
技术领域
本发明为一种检测乳癌的相关预测试剂盒及其应用，尤指一种检测生物样品中WISP1的浓度在制备用于乳癌筛检及侦测复发的检测试剂盒及其应用，可藉由测定生物样本中的WISP1的浓度多寡来检测乳癌筛检、疗效及复发与否。
背景技术
乳癌是女性最常被诊断的恶性肿瘤，即使接受了积极的治疗，大部分晚期的乳癌的受试者最后仍然会产生乳癌复发及转移，造成了愈后不佳。因此乳癌的早期诊断，复发的早期发现，以及对于乳癌治疗疗效的评估，是对于乳癌愈后改善的3大重点方向。然而到目前为止，并没有好的肿瘤指标可以满足上述的要求，现今最常用的乳癌肿瘤指标是CEA以及CA153，而这2者对乳癌的早期诊断及复发侦测的敏感度及特异度都不能让人满意。
根据上述缺失，先前技术仍有改善空间。
发明内容
本发明系提供一种检测生物样品中WISP1的浓度在制备用于乳癌筛检及侦测复发的检测试剂盒及其应用，所述WISP1系指可被Wnt-1诱导的蛋白质1(Wnt-1 inducedsecreted protein 1)，至少包括：该试剂盒检测源自检测者之血清样本中WISP1的浓度；比较健康者及罹患乳癌患者血清样本中WISP1的浓度变化，以定义WISP1的浓度改变对于罹患乳癌风险高低之关联性；预测参考值以健康者血清中WISP1的浓度平均值为631.5pg/ml，罹患乳癌患者血清中WISP1的浓度平均值为934.5pg/ml，因此，当检测者血清中WISP1的浓度等于或高于934.5pg/ml时，可做为该试剂盒预测高罹患乳癌风险及复发侦测。
其中WISP1的浓度低于934.5pg/ml时，表示乳癌患者手术、化疗或标靶治疗有效(预后良好)；WISP1的浓度等于或高于934.5pg/ml时，表示乳癌患者接受乳癌治疗后有复发及转移之高度风险(预后不良)，透过测量WISP1的浓度变化，可作为乳癌患者治疗反应和健康恢复状况的指标。
本发明之优点在于：
首先，本发明之WISP1能快速评估药物疗效，经由确认受试者WISP1之浓度即可确认，若WISP1浓度等于或高于934.5pg/ml则表示受试者为乳癌患者之高风险群，若WISP1浓度低于934.5pg/ml则表示受试者属于乳癌患者之低风险族群。
再者，本案之WISP1有高度的敏感度57％及特异性96％，藉由受试者的血清比较健康受试者血清中的WISP1浓度，即可评估是否为罹患乳癌肿瘤的高危险群，因此WISP1系为较佳评估乳癌肿瘤的指标。
接下来列举一较佳实施例，并配合图式及图号，对本发明做进一步的说明，期能使贵审查委员对本发明有更详细的了解，并使熟悉该项技术领域者能据以实施。
附图说明
图1：为本案之乳癌受试者及健康受试者血清中的WISP1浓度的中位数。
图2：为本案之乳癌受试者手术前后血清中WISP1、CEA及CA153浓度的平均值。
图3：为本案之乳癌受试者手术后血清中WISP1和健康受试者WISP1浓度的平均值。
图4：为本案之乳癌受试者及健康受试者中的WISP1之ROC分析曲线。
具体实施方式
参阅图1～图4，本发明揭露一种检测生物样品中WISP1的浓度在制备用于乳癌筛检及侦测复发的试剂盒中的应用。
其特征在于，所述生物样品选自于获自对象的血液或血清样本，所述检测血清样本的方法为酵素结合免疫吸附分析法(Enzyme-linked immunosorbent assay,ELISA)。
本发明另提供一种检测试剂盒，其包含：检测有效量WISP1浓度的试剂；选自下组的一种或多种物质：乙二胺四乙酸二钠、清洗液、生物素化抗体、链霉亲和素溶液、基质试剂和终止液。
其特征在于，当检测结果显示健康者血清中WISP1的浓度平均值为631.5pg/ml，罹患乳癌患者血清中WISP1的浓度平均值为934.5pg/ml，因此，当检测者血清中WISP1的浓度等于或高于934.5pg/ml时，可做为该试剂盒预测高罹患乳癌风险及复发侦测。
当WISP1的浓度低于934.5pg/ml时，表示乳癌患者手术、化疗或标靶治疗有效，表示预后良好；WISP1的浓度等于或高于934.5pg/ml时，表示乳癌患者接受乳癌治疗后有复发及转移之高度风险，表示预后不良，透过测量WISP1的浓度变化，可作为乳癌患者治疗反应和健康恢复状况的指标。
以下为实施WISP1浓度使用酵素结合免疫吸附分析法进行检测，其步骤如下：
1.从患者血液采集至含有乙二胺四乙酸二钠(ethylenediaminetetraaceticacid disodium salt dihydrate,EDTA-2Na)的容器，再进行3000g、15分钟的离心，接着收取100ul的上清液，此上清液即为血浆。
2.将上述100ul的血浆填入well(培养皿)中，并置放于震荡器上，以室温下反应2.5小时后，再将well内的液体去掉。
3.加入300ul的清洗液(1X wash buffer)至well中清洗三次，于第三次时将well中的液体完全去除。
4.加入100ul的生物素化抗体(biotinylated antibody)于well中，并置于震荡器上，在室温反应1小时候将well中的液体去除。
5.加入300ul的清洗液(1X wash buffer)至well中清洗三次，于第三次时将well中的液体完全去除。
6.加入100ul的链霉亲和素溶液(streptavidin solution)于well中，并置于震荡器上，在室温下反应45分钟后，将well中的液体去除。
7.再次加入300ul的1X清洗液(1X wash buffer)至well中清洗三次，于第三次时将well中的液体完全去除。
8.加入100ul的TMB一步骤基质试剂(TMB One-Step Substrate Reagent)于well中，并置于震荡器上，在室温下反应30分钟。
9.加入50ul的终止液(Stop solution)于well中。
10.接着将well置入酵素免疫分析测读仪中，以波长450nm读取结果。
本发明根据乳癌筛检结果进行治疗、追踪及评估如下：
乳癌筛检：当健康检查时，若个体血清中WISP1的浓度等于或高于934.5pg/mL，要怀疑有乳癌发生的可能性，应立即安排其它检查。
乳癌治疗后追踪:若乳癌患者在治疗后的追踪期间血液中WISP1的浓度等于或高于934.5pg/mL，要强烈怀疑复发或转移，马上安排进一步检查。
乳癌治疗疗效评估:对乳癌病人接受手术、化疗、标靶或其它治疗，若血清中WISP1的浓度降至934.5pg/mL以下，代表此治疗有疗效，若WISP1的浓度没有降至934.5pg/mL以下，建议立即改换其它治疗方式。
图1系将14位乳癌受试者及31位健康受试者血清中的WISP1浓度取中位数进行比较，31位的健康受试者血清中WISP1浓度之中位数是587.8pg/ml，而乳癌受试者血清中WISP1浓度之中位数是939.5pg/ml，明显可得到乳癌受试者血清中WISP1浓度之中位数高于健康受试者的WISP1浓度。
图2揭露乳癌受试者手术前后血清中WISP1、癌胚抗原(carcino-embryonicantigen，CEA)及癌症抗原15-3(Cancer Antigen CA15-3，CA153)浓度的变化。乳癌受试者手术前CA153的平均浓度是13.19U/ml，手术后降到12.66U/ml，由于血液中正常CA153的平均浓度是小于38U/mL，因此结果显示CA153对乳癌而言不是一个好的肿瘤指标。而乳癌受试者手术前的CEA平均浓度是2.12ng/ml，手术后降到1.57ng/ml，由于血液中正常CEA的浓度是小于10ng/ml，因此结果显示CEA对乳癌而言同样不是一个好的肿瘤指标。而在乳癌受试者血清中WISP1的浓度平均值从手术前934.54pg/ml(取四舍五入为934.5pg/ml)到手术后631.46pg/ml(取四舍五入为631.5pg/ml)，而从图3可知健康受试者血清中WISP1的平均浓度为675.91pg/ml，而乳癌受试者术后WISP1的浓度平均值会从934.54pg/ml降至631.46pg/ml，由于乳癌受试者WISP1的浓度平均值在术前以及术后的差距大，且乳癌受试者术后WISP1的浓度平均值又与健康受试者675.91pg/ml的数值接近，从此可说明WISP1是一个好的乳癌肿瘤指标。
图4中进一步使用ROC曲线来分析健康受试者和乳癌受试者血清中WISP1变化，AUC(Area under the Curve of ROC，在ROC底下的面积)为0.83，这表示WISP1是一个好的乳癌肿瘤指标。当把WISP1的临界值设在934.50pg/mL，对乳癌诊断的敏感度及特异性分别是57％及96％，经由上述实验可得知，乳癌受试者血清中WISP1的平均浓度高于健康受试者，而乳癌受试者在接受手术后血清中WISP1的浓度会回到和健康受试者接近之数值，因此可以判定WISP1是一良好的新乳癌肿瘤指标，可用来做乳癌的筛检及侦测复发乳癌机率。
需注意的是，上述实施例仅为例示性说明本发明之原理及其功效，而非用于限制本发明之范围。任何熟于此项技术之人均可在不违背本发明之技术原理及精神下，对实施例作修改与变化。因此本发明之权利保护范围应如后述之申请专利范围所述。

Claims (6)

1.检测生物样品中WISP1的浓度在制备用于乳癌筛检及侦测复发的试剂盒中的应用。
2.如权利要求1所述的应用，其特征在于，所述生物样品选自于获自对象的血液或血清样本。
3.如权利要求1所述的应用，其特征在于，所述检测血清样本的方法为酵素结合免疫吸附分析法。
4.一种用于乳癌筛检及侦测复发的检测试剂盒，其包含：
检测有效量WISP1浓度的试剂；
选自下组的一种或多种物质：乙二胺四乙酸二钠、清洗液、生物素化抗体、链霉亲和素溶液、基质试剂和终止液。
5.如权利要求4所述的试剂盒，其特征在于，当检测结果显示健康者血清中WISP1的浓度平均值为631.5pg/ml，罹患乳癌患者血清中WISP1的浓度平均值为934.5pg/ml，因此，当检测者血清中WISP1的浓度等于或高于934.5pg/ml时，可做为该试剂盒预测高罹患乳癌风险及复发侦测。
6.如权利要求4所述的试剂盒，其特征在于，WISP1的浓度低于934.5pg/ml时，表示乳癌患者预后良好；WISP1的浓度等于或高于934.5pg/ml时，表示乳癌患者预后不良，有复发及转移之高度风险。
CN201810033799.2A 2018-01-15 2018-01-15 检测生物样品中wisp1的浓度在制备用于乳癌筛检及侦测复发的检测试剂盒及其应用 Pending CN110045120A (zh)

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