CN105044360A - Rbp4作为结直肠癌血清标记物的应用及诊断试剂盒 - Google Patents

Rbp4作为结直肠癌血清标记物的应用及诊断试剂盒 Download PDF

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CN105044360A
CN105044360A CN201510433047.1A CN201510433047A CN105044360A CN 105044360 A CN105044360 A CN 105044360A CN 201510433047 A CN201510433047 A CN 201510433047A CN 105044360 A CN105044360 A CN 105044360A
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colorectal cancer
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胡晓彤
何超
费伟强
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Affiliated Sir Run Run Shaw Hospital of School of Medicine Zhejiang University
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Abstract

RBP4作为结直肠癌血清标记物的应用及诊断试剂盒，属于生物技术领域。本发明一方面提供一种RBP4作为结直肠癌血清标记物的应用的技术方案，另一方面提供一种结直肠癌诊断试剂盒的技术方案。本发明的有益效果在于：1.提供的血清生物标志物的特异性为81.7%，敏感性为74.9%，具有较高特异性和敏感性。2.提供一种灵敏、可靠、可操作的方法，定量测定人血清中RBP4抗原水平，有助于辅助早期诊断结直肠癌。

Description

RBP4作为结直肠癌血清标记物的应用及诊断试剂盒
技术领域
本发明属于生物技术领域，具体涉及RBP4作为结直肠癌血清标记物的应用及诊断试剂盒。
背景技术
结直肠癌是最常见的恶性肿瘤之一，在全世界范围内，结直肠癌的死亡率高居前三，在中国结直肠癌的发生率呈不断上升之势，死亡率居第五。经过积极外科手术治疗和放化疗，结直肠癌患者5年总体生存率在66%左右。有数据表明，结直肠患者在I期甚至更早得到有效的诊断和治疗的话，生存率能够提高到90%以上。然而目前结直肠癌的早期诊断率只有37%左右，一旦到III期或者IV期才发现治疗，生存率则降低为10%-30%。可见提高结直肠癌的早期诊断率至关重要。目前结肠镜对结直肠癌检查的灵敏度和特异性最高，但由于费用较高、患者痛苦较大和操作要求高等原因降低了患者愿意做该项检查的程度，致使很多患者拖到晚期，一般的影像学检查确诊时患者常常已经处于癌症晚期，对提高患者生存率作用有限。因此，找到特异性和敏感性均佳的肿瘤标记物成了可行的方法。然而结直肠癌的发生发展是多种基因损伤的结果，目前还没找到理想的肿瘤标志物。
理想的肿瘤标志物应符合以下条件：（1）敏感性高；（2）特异性好；（3）肿瘤标志物浓度和肿瘤大小、转移、恶性程度有关，能监测肿瘤治疗效果和肿瘤的复发并预测预后；（4）半衰期短，有效治疗后浓度很快下降，能较快反映体内肿瘤的实际情况；（5）存在于体液，特别是血液中,易于检测。
现在应用于临床的肿瘤标记物大约有如下几种：CA-153，CA19-9，CA-125，AFP，CEA，PAS，Ferritin，CA724，CA242等。通过对临床600多例样本的统计分析，发现只有CEA在诊断结直肠癌时的敏感性在临床应用中能达到50%以上，其余都低于20%。
Retinol-bindingprotein4(RBP4)属于视黄醇运载蛋白,其血清水平常作为临床营养状况、肝肾疾病的早期诊断和疗效的评价指标。视黄醇结合蛋白4(RBP4)为一种新的脂肪源性信使,参与了胰岛素抵抗和II型糖尿病的发生。近年来，越来越多的研究将RBP4的表达量与肿瘤的发生发展联系起来，已有研究表明RBP4在上皮性软巢癌患者的血清中表达量下降，在胰腺癌中，RBP4的表达量则与NGAL相关。但目前尚未见有关于RBP4作为结直肠癌肿瘤标记物的报道。
发明内容
针对现有技术存在的问题，本发明的目的在于设计提供一种RBP4作为结直肠癌血清标记物的应用及诊断试剂盒的技术方案，来定性或定量检测人血清中的RBP4抗原水平，作为辅助结结直肠癌早期诊断的一种手段，大大提高结结直肠癌早期诊断的敏感性和特异性。
所述的视黄醇运载蛋白RBP4作为检测结直肠癌的血清标记物的应用。
所述的视黄醇运载蛋白RBP4在制备结直肠癌诊断试剂盒中的应用。
所述的视黄醇运载蛋白RBP4在制备结直肠癌诊断试剂盒中的应用，其特征在于以常规方法制备抗视黄醇运载蛋白RBP4的抗体，建立检测视黄醇运载蛋白RBP4的定性或定量方法及配套的试剂盒。
所述的视黄醇运载蛋白RBP4在制备结直肠癌诊断试剂盒中的应用，其特征在于抗视黄醇运载蛋白RBP4的抗体是单克隆抗体或多克隆抗体。
所述的一种结直肠癌诊断试剂盒，包括固相、位于固相中的抗体、酶标记抗体，其特征在于所述的抗体为抗视黄醇运载蛋白RBP4的抗体。
所述的一种结直肠癌诊断试剂盒，其特征在于抗视黄醇运载蛋白RBP4的抗体是单克隆抗体或多克隆抗体。
本发明为结直肠癌的诊断和／或治疗提供一条全新的途径。
本发明的有益效果在于：1.提供的血清生物标志物的特异性为81.7%，敏感性为74.9%，具有较高特异性和敏感性。2.提供一种灵敏、可靠、可操作的方法，定量测定人血清中RBP4抗原水平，有助于辅助早期诊断结直肠癌。
附图说明
图1为实施例中的标准曲线图；
图2为实施例中ROC曲线图；
图3为实施例中RBP4点分布图。
具体实施方式
为了使本发明更加容易理解，下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围，下列实施例中未提及的具体实验方法，通常按常规实验方法进行。
实施例
1、血清样本的采集与制备：
采集结直肠癌患者和正常对照的全血标本，室温放置2小时内3000g离心5分钟，取上清即得到血清。得到的血清可立即用于检测，或者分装后于-80℃保存。
2、ELISA各种缓冲液及试剂的配置方法
（1）包被缓冲液：PH9.6、0.05M的碳酸盐缓冲液
（2）样品稀释液：PH7.4、0.01mol/L的PBS缓冲液
（3）洗涤缓冲液：PH7.4、0.01M的PBST缓冲液（含0.05%Tween-20）
（4）封闭液：含0.5%BSA的pH7.4PBS溶液。
（5）底物显色液：
显色液A
显色液B：（现配现用）
（6）终止缓冲液：2MH2SO4
（7）抗体（Anti-RBP4）由R&D公司提供，货号MAB33781，浓度为20mg/mL。
（8）标准品由R&D公司提供，浓度为200ng/mL。
（9）HRP酶标抗体浓度为0.1-1μg/mL。
3、双抗体夹心法ELISA测定血清中RBP4浓度，以辅助诊断结直肠癌：
具体操作步骤如下：
(1)包被：用PH9.6、0.05M碳酸盐缓冲液将抗体（Anti-RBP4）稀释至浓度为1～10μg／ml。96孔酶标板每孔加100μL上述抗体稀释液，37℃包被2h，弃去包被液，每孔加350μL洗涤缓冲液洗板3次，每次3分钟，拍干，得到捕捉抗体。
（2）封闭：加0.3ml封闭液于上述已包被的微孔中，置37℃封闭2小时。然后洗涤。
（3）样品稀释：每个血清样品按体积1:1000稀释。建议取20μL血清样品加入980μL样品稀释液，混匀后从中再取出25μL混合液加至475μL的样品稀释液中。
（4）标准品稀释：用样品稀释液将标准品稀释为100ng/ml,50ng/ml,25ng/ml,12.5ng/ml,6.25ng/ml,3.12ng/ml,1.56ng/ml和0ng/ml8个浓度梯度。
（5）加样：每孔加入20μL稀释后的血清样品和标准品，37℃孵育2小时；然后洗涤。
（6）酶标二抗：每孔加入200μLHRP酶标抗体，置于微型振荡器37℃孵育2小时，然后洗涤。
（7）显色：将显色液A和显色液B以1:1混合，每孔加入200μL，避光反应30分钟。
（8）终止：每孔加入50μL终止液，终止反应。5分钟内测量450nm处各孔吸光度值。
（9）标准曲线（如图1所示）：以标准品浓度为纵坐标，相应吸光度值为横坐标作标准曲线。根据标准曲线计算每个样品的浓度。
（10）结果判定：小于26.7为高风险，大于26.7为正常。
对400例肠癌病人血清，200例正常病人血清的ROC分析确立了以上参考值，其中ROC曲线如图2所示，RBP4点分布图（肠癌与正常对照比较）如图3所示。
对部分测定数据如下表：（深色标记为肿瘤患者，浅色标记为正常对照）：
本发明所提供的诊断结结直肠癌的特异性为81.7%，敏感度为74.9%。
以上公开的仅为本申请的几个具体实施例，但本申请并非局限于此，任何本领域的技术人员能思之的变化，都应落在本申请的保护范围内。

Claims (6)

1.视黄醇运载蛋白RBP4作为检测结直肠癌的血清标记物的应用。
2.视黄醇运载蛋白RBP4在制备结直肠癌诊断试剂盒中的应用。
3.如权利要求2所述的视黄醇运载蛋白RBP4在制备结直肠癌诊断试剂盒中的应用，其特征在于以常规方法制备抗视黄醇运载蛋白RBP4的抗体，建立检测视黄醇运载蛋白RBP4的定性或定量方法及配套的试剂盒。
4.如权利要求2所述的视黄醇运载蛋白RBP4在制备结直肠癌诊断试剂盒中的应用，其特征在于抗视黄醇运载蛋白RBP4的抗体是单克隆抗体或多克隆抗体。
5.一种结直肠癌诊断试剂盒，包括固相、位于固相中的抗体、酶标记抗体，其特征在于所述的抗体为抗视黄醇运载蛋白RBP4的抗体。
6.如权利要求5所述的一种结直肠癌诊断试剂盒，其特征在于抗视黄醇运载蛋白RBP4的抗体是单克隆抗体或多克隆抗体。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109975547A (zh) * 2018-02-28 2019-07-05 中山大学 Rantes检测试剂在制备结直肠癌诊断剂方面的应用
CN112063719A (zh) * 2020-09-22 2020-12-11 中国科学院动物研究所 一种检测或辅助检测视网膜视锥细胞的标志物及其应用
CN113122625A (zh) * 2019-12-30 2021-07-16 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） Smco2基因作为标志物在肺癌、胃癌、结直肠癌、子宫内膜癌及卵巢癌中诊断及治疗的应用
CN116500256A (zh) * 2023-02-20 2023-07-28 郑州大学第五附属医院 一种快速检测血清视黄醇含量的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007134395A1 (en) * 2006-05-22 2007-11-29 Clinical Genomics Pty Ltd Detection method
US20090191575A1 (en) * 2006-06-05 2009-07-30 Makoto Watanabe Tumor marker and method for determination of the occurrence of cancerous disease
CN101967472A (zh) * 2009-07-27 2011-02-09 上海中科英沐生物科技有限公司 Rbp4杂交瘤细胞株及其制备方法和用途
WO2011031503A2 (en) * 2009-08-25 2011-03-17 Cytotech Labs, Llc Methods for treatment of a sarcoma using an epimetabolic shifter (coenzyme q10)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007134395A1 (en) * 2006-05-22 2007-11-29 Clinical Genomics Pty Ltd Detection method
US20090191575A1 (en) * 2006-06-05 2009-07-30 Makoto Watanabe Tumor marker and method for determination of the occurrence of cancerous disease
CN101967472A (zh) * 2009-07-27 2011-02-09 上海中科英沐生物科技有限公司 Rbp4杂交瘤细胞株及其制备方法和用途
WO2011031503A2 (en) * 2009-08-25 2011-03-17 Cytotech Labs, Llc Methods for treatment of a sarcoma using an epimetabolic shifter (coenzyme q10)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GERARD MLLANO ET AL.: "Serum Prealbumin, Retinol-Binding Protein, Transferrin, and Albumin Levels in Patients With Large Bowel Cancer", 《J NATL CANCER INST》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109975547A (zh) * 2018-02-28 2019-07-05 中山大学 Rantes检测试剂在制备结直肠癌诊断剂方面的应用
CN113122625A (zh) * 2019-12-30 2021-07-16 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） Smco2基因作为标志物在肺癌、胃癌、结直肠癌、子宫内膜癌及卵巢癌中诊断及治疗的应用
CN113122625B (zh) * 2019-12-30 2023-08-11 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） Smco2基因作为标志物在子宫内膜癌中诊断及治疗的应用
CN112063719A (zh) * 2020-09-22 2020-12-11 中国科学院动物研究所 一种检测或辅助检测视网膜视锥细胞的标志物及其应用
CN112063719B (zh) * 2020-09-22 2022-01-14 中国科学院动物研究所 一种检测或辅助检测视网膜视锥细胞的标志物及其应用
CN116500256A (zh) * 2023-02-20 2023-07-28 郑州大学第五附属医院 一种快速检测血清视黄醇含量的方法

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Application publication date: 20151111