CN101393204A - 血清il-6在制备原发性肝细胞癌诊断试剂盒中的应用 - Google Patents
血清il-6在制备原发性肝细胞癌诊断试剂盒中的应用 Download PDFInfo
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Abstract
本发明涉及一种癌症诊断技术,特别是血清IL-6在制备原发性肝细胞癌诊断试剂盒制备中的应用。该试剂盒可以快速、准确地进行IL-6的检测,从而可以对各期(特别是早期)原发性肝细胞癌作出快速的辅助诊断,具有重要的临床应用价值。本发明包括建立了基于双抗体夹心原理的酶联免疫诊断系统。由于血清不用稀释,因此灵敏度大大提高。本发明有益的积极效果是:检测试剂盒操作简单、试剂稳定、重复性好、特异性强、敏感性高、操作简单,易于大范围推广应用,具有广阔的市场前景。
Description
[技术领域]
本发明涉及一种癌症诊断技术,特别是原发性肝细胞癌诊断试剂盒的制备。
[背景技术]
肝癌是人类最常见恶性肿瘤之一,其发病率在全世界范围内位居第五,而且在肿瘤致死率中位于第三。每年约新增500000病例,经年龄修正后的发病比率为5.5-14.9/10万人。肝细胞肝癌(Hepatocellular carcinoma,HCC)是原发性肝脏恶性肿瘤中的最主要类型,高发区域主要集中在东亚,东南亚,中西非等地区。从1990年开始,HCC在中国的肿瘤致死原因中高居第二,乙肝病毒、丙肝病毒感染,黄曲霉素B1的摄入和酗酒等被认为是主要的风险因子。肝细胞肝癌性肿瘤,也是中国华南地区常见恶性肿瘤之一,寻找HCC的早期血清学诊断标志物,对于肝癌的预防和早期治疗均具有重要意义。长期以来,AFP作为原发性肝癌的诊断指标,为临床作出了重大的贡献。但是,AFP的敏感性是有限的;特别是近年来,AFP阴性的HCC患者有上升趋势,因此,寻找AFP之外的肿瘤早期标志物是当务之急。
[发明内容]
自细胞介素-6(Interleukin-6,IL-6)是一种促炎症细胞因子,在宿主防疫机制中发挥重要作用。在正常生理状态时,IL-6的水平比较低;但是在创伤、炎症、肿瘤等各种病理情况下,IL-6的表达明显升高。
发明人通过检测正常人、乙肝病毒携带者和HCC患者血清中IL-6的含量,通过统计学分析,根据IL-6含量与HCC的相关性,希望为HCC的临床血清学早期诊断找到新的生物标记物。
本发明公开了一种原发性肝细胞癌诊断试剂盒及其制备方法与应用,该试剂盒可以快速、准确地进行IL-6的检测,从而可以对各期(特别是早期)原发性肝细胞癌作出快速的辅助诊断,具有重要的临床应用价值。本发明包括建立了基于双抗体夹心原理的酶联免疫诊断系统。由于血清不用稀释,因此灵敏度大大提高。本发明有益的积极效果是:检测试剂盒操作简单、试剂稳定、重复性好、特异性强、敏感性高、操作简单,易于大范围推广应用,具有广阔的市场前景。
[附图说明]
图1:正常人、HBV携带者、原发性肝细胞癌(HCC)患者血清IL-6浓度直方图。
图2:正常人、HBV急性感染者、HBV慢性感染者、小肝癌、中等大小肝癌及大肝癌患者血清IL-6浓度直方图。
图3:正常人、HBV急性感染者、HBV慢性感染者、AFP阴性肝癌患者及AFP>25μg/L肝癌患者血清IL-6浓度直方图。
[具体实施方式]
材料:
取血清样品,包括30份经临床实验室检测没有感染HBV、HCV、HIV,并没有肿瘤的正常体检人群血清样品;50份HBV携带者血清,其中大三阳患者22名,小三阳患者28名;84份原发性肝细胞癌患者血清样品,其中包括39份小肝癌(直径<5cm),27份中等大小肝癌(5≤直径≤10cm),18份大肝癌(直径>10cm)。并且,在全部检测的HCC样品中,AFP≤25μg/L占33份,AFP>25μg/L占51份。
试剂盒内设有:1、包被在固相96孔酶标板上的人IL-6重组单克隆抗体;2、酶结合物工作液为辣根过氧化物酶(HRP)标记的人IL-6重组单克隆抗体反应液;3、阳性对照为人IL-6标准蛋白,阴性对照为标准阴性血清;4、显色液ABTS底物:ABTS1mg+0.1M,pH5.0柠檬酸缓冲液2ml+3% H2O24μl(临用时配);5、洗涤液(0.05%Tween20-PBS);6、结合物稀释液:0.1%BSA-PBS(用于稀释标本、标准品和结合物)以及7、终止液(1~2MH2SO4)。
方法:
1.包被:包被缓冲液(0.05M,pH9.6 Na2 CO3-NaHCO3缓冲液)稀释IL-6单克隆抗体粗提蛋白至1μg/ml,加入96孔板,100μl/孔,放4℃,36小时。
2.封闭:洗涤液冲洗96孔板5次,加结合物稀释液200μl/孔,放37℃,2小时。
3.加待检样品:洗涤液冲洗96孔板5次,加待检样品及结合物稀释液稀释的标准品100μl/孔,放37℃,2小时。
4.加酶标抗体:洗涤液冲洗96孔板5次,结合物稀释液稀释酶标抗体至1∶800,加入96孔板,100μl/孔,放37℃,2小时。
5.显色:洗涤液冲洗96孔板5次,显色液ABTS底物,加入96孔板,100μl/孔,加终止液室温下或37℃显色15~30分钟。
6.标准曲线:根据标准品OD值绘制标准曲线,在标准曲线上查出待检标本的含量。
7.数据统计:所有的统计均使用SPSS 13.0软件包((Statisticat Package for theSocial Sciences,Chicago,IL,USA)。数据采用均值±S表示;所有P值均取两位小数,P<0.05被认为具有统计学意义,P<0.01被认为具有显著意义。
结果和讨论:
原发性肝细胞癌是一个在东亚和南亚地区高发的恶性肿瘤,每年大概有25万到100万人会得肝癌,每年因为肝癌而死亡的人口在20万左右。造成肝癌发病的原因很复杂,从环境影响和生活习惯角度说,摄入了黄曲霉污染的食物、酗酒、以及乙型、丙型肝炎的感染等等都会提高肝癌发生的可能性。
研究发现,在HIV、呼吸道合胞病毒、仙台病毒、HBV感染者等多种病毒感染者及肺癌、口腔癌、结直肠癌等肿瘤患者血清中IL-6的水平升高。较高水平的IL-6也反应了机体对内毒素的高度敏感性。
研究发现,HBV携带者血清中IL-6的水平虽然比正常对照组有轻微的升高,但是没有统计学意义(p>0.05);而HCC患者平均的IL-6水平明显高于正常对照组(p<0.01)和HBV携带者(p<0.01),其中小肝癌与正常对照组(p<0.05)及HBV组(p<0.05)也有统计学意义,提示HCC患者细胞大量分泌IL-6。此外,我们发现AFP≤25μg/L及AFP>25μg/L与正常对照组(p<0.01)和HBV组(p<0.01)比较,均有显著统计学意义,提示我们,在AFP阴性HCC患者中,IL-6也许是一个较好的检测指标。
由于肝细胞癌具有较高的异质性,同时检测2种或以上的肿瘤生物标记物被认为是肿瘤早期诊断的较好选择。我们的结果清楚表明,IL-6可以作为HCC的早期血清学诊断生物标志物,特别是当IL-6与经典标志物AFP相结合检测原发性肝细胞癌时,大大提高了HCC的早期诊断率,弥补了单纯检测AFP的不足。不仅如此,由于IL-6和AFP都与原发性肝细胞癌有高度相关性,这就使得两者联合检测,对原发性肝细胞癌具有早期诊断意义。
表1:正常人、HBV急性感染及携带者及原发性肝细胞癌患者血清中IL-6浓度及其统计学意义
急性感染:HBsAg +,HBeAg +,HBcAb +;慢性感染:HBsAg +,HBeAb +,HBcAb +;
小肝癌:直径≤5cm;中等大小肝癌:5cm<直径<10cm;大肝癌:直径≥10cm;
HBV患者:正常人p>0.05
HCC患者:正常人(或HBV携带者)◆p<0.01
其中,小肝癌患者:正常人(或HBV携带者)*p<0.05
中等大小或者大肝癌患者:正常人(或HBV携带者)◆p<0.01
表2:AFP相关的原发性肝细胞癌患者中血清IL-6浓度与正常人和HBV感染者的统计学比较
急性:HBsAg +,HBeAg +,HBcAb +;慢性:HBsAg +,HBeAb +,HBcAb +;*p<0.05 ◆p<0.01HCC患者:正常人(或HBV携带者)◆p<0.01
其中,不论AFP=25μg/L或者AFP>25μg/L肝癌患者:正常人(或HBV携带者)◆p<0.01。
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1、血清IL-6在制备原发性肝细胞癌诊断试剂盒中的应用。
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CN102782501A (zh) * | 2010-03-02 | 2012-11-14 | 弗·哈夫曼-拉罗切有限公司 | 在无症状患者中基于il-6检测的全身炎性应答综合征和败血症的早期诊断和预测 |
CN102782501B (zh) * | 2010-03-02 | 2015-07-22 | 弗·哈夫曼-拉罗切有限公司 | 在无症状患者中基于il-6检测的全身炎性应答综合征和败血症的早期诊断和预测 |
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CN102175868A (zh) * | 2011-01-07 | 2011-09-07 | 中国医学科学院肿瘤研究所 | 联合定量检测血清中IL-1β、IL-6、IL-10、IL-17、IL-23和TNF-α在肝癌早期预警中的应用 |
CN102175868B (zh) * | 2011-01-07 | 2014-07-02 | 中国医学科学院肿瘤研究所 | 联合定量检测血清中IL-1β、IL-6、IL-10、IL-17、IL-23和TNF-α在肝癌早期预警中的应用 |
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WO2015165357A1 (zh) * | 2014-04-29 | 2015-11-05 | 北京普恩光德生物科技开发有限公司 | 白介素6检测试剂盒 |
CN103969438B (zh) * | 2014-04-29 | 2016-03-30 | 北京普恩光德生物科技开发有限公司 | 白介素6检测试剂盒 |
CN108535494A (zh) * | 2018-06-29 | 2018-09-14 | 湖南师范大学 | 一种il-6elisa检测试剂盒及其使用方法 |
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