CN1378085A - 用于感染早期检测的丙型肝炎抗原－抗体联合测定 - Google Patents
用于感染早期检测的丙型肝炎抗原－抗体联合测定 Download PDFInfo
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Abstract
公开了一种检测样品中是否存在HCV的方法,该方法包括:将样品与固定在固相上的HCV抗原和抗HCV核心抗体接触,然后向样品中加入聚氧乙烯醚,通过加入被标记过的抗人IgG和被标记过的抗HCV核心抗体来检测被捕获的抗原和抗体,检测作为HCV的存在表征的放射信号。
Description
发明背景
据估计，全世界有1700万人已经感染了丙型肝炎病毒(HCV)。在未来的几年里，美国由HCV导致的肝疾病和癌症的死亡人数将超过由获得性免疫缺陷综合症(AIDS)导致的死亡人数。
一般来说，HCV的传染需要血液的接触。携带一个单链核糖核酸(RNA)的HCV只含有编码一个多蛋白的基因，该多蛋白能被剪切成至少10种功能蛋白。很明显，检测血液是否被HCV感染的能力是很重要的，而且，在HCV感染阶段越早时被检测出来越好。
因此，我们改进了一种用于检测HCV感染的ELISA技术。这种新的检测方法比现有的用于监测血液是否被HCV感染的方法能更早的检测到HCV的感染。现有技术是以检测被感染血液中的抗HCV抗体为基础的，通过含有HCV蛋白序列的重组蛋白或多肽来捕获这些抗体。
例如，用多抗原检测早期血清转化中的抗HVC抗体的Ortho HCV 3.0ELISA技术。但是，在病毒血症患者体内产生抗HCV抗体之前有一段较长的潜伏期，这段潜伏期能持续60天。在这期间内，即使患者已经被高度感染，用现有检测技术也不能检测出来。
发明概述
本发明的目的之一是提供一种用于诊断的检测方法，该检测方法通过捕获HCV核心抗原和抗体来检测上文所说的空白期(window period)的HCV的感染。我们在本发明中所描述的方法除了检测抗HCV抗体外，还检测HCV核心抗原，因此比现有的抗体检测技术能更早的检测到HCV感染，
本发明的另一个目的是提供一种检测样品中是否存在HCV的方法。本方法包括以下步骤：将样品与固定在固相上的HCV抗原和抗HCV核心抗体接触，然后向样品中加入聚氧乙烯醚，通过加入被标记过的人抗IgG和被标记过的抗HCV核心抗体来捕获其中的抗原和抗体，检测放射信号从而确定HCV的存在。
本发明的另一个目的是提供特定去污剂，该去污剂是通过破坏壳蛋白和/或脂层从病毒释放HCV核心抗原所必需的。发明的详细描述
本发明中，能够在联合测定中使病毒释放核心抗原而对HCV重组蛋白捕获抗HCV抗体的能力没有负作用的去污剂是优选的。在本发明的一个优选的实施方案中，聚氧乙烯醚类去污剂被用于此目的。这类去污剂一般包括：BRIJ30，BRIJ 35，56，58，92，96，98，700和MYRJ52，59，53，45。这些去污剂有助于从病毒中释放HCV核心抗原，但这些去污剂的存在对抗HCV的检测不会产生不利影响。某些去污剂能通过影响包被于固相的重组抗原或灭活样品中的抗HCV抗体从而影响抗体的检测。在Ortho HCV 3.0ELISA中的一些去污剂，如N-十二烷基肌氨酸，在用于HCV核心抗原的检测时就会破坏HCV重组蛋白c22-3，c200和NS5检测抗HCV抗体的能力。
本发明所用的“样品”，指的是任何含有目的分析物的物质。一种样品可以是生物流体，比如全血或包含有红细胞、白细胞、血小板、血清和血浆的全血组成成分、腹水、尿液、脑脊髓液及其他的含有目的分析物质的机体组成成分。
实施例
以下的实施例通过描述一系列的去污剂来证明本发明的优点和可实施性，这些去污剂都通过上文描述的检测方法进行评价。本领域的普通技术人员应该能认识到，HCV抗体和抗原的检测是可以分开或同时进行，也是可以同时进行的。以下实施例旨在证明，但不限制，本发明的保护范围和精神。
实施例1：抗原—抗体联合测定。
HCV抗原C200-3、NS-5和修饰过的抗原C22KSN 47、48或C22KSR47L与两种抗核心单克隆抗体(如鼠单克隆Pep10、12)一起在加有磷酸缓冲液的微孔中包被。抗原的修饰是通过修饰编码包含HCV1序列的重组蛋白C22-3的DNA克隆来实现的，此方法包含了合成寡核苷酸的定点突变(Sambrook、Fritsh、Maniatis，分子克隆实验指南第二版，冷泉港出版社，第15章，1989)。隔夜培养后去除含有包被蛋白的磷酸缓冲液，用包含有去污剂Tween 20的磷酸盐缓冲液洗涤微孔，然后往抗原/抗体包被的微孔中加入BSA/蔗糖溶液以抑制微孔中的所有蛋白结合位点，2-24小时后去除BSA/蔗糖溶液，微孔在空气中晾干，在有干燥剂的条件下保藏。
实施例2：加入100mL PBS溶液以稀释100mL待测样品，该PBS溶液含有牛血清蛋白、过氧化物歧化酶、酵母抽提液、和1％的Brij58或Brij35或两者的混合物。用微量管把稀释过的样品移入HCV抗原/抗体包被的微孔中(见实施例1)，这些微孔在37℃培养90分钟，然后用含有0.5％Tween20的PBS溶液洗涤5次。往微孔中加入200ml辣根过氧化物酶(HRP)标记的鼠源抗人IgG和HRP标记的抗HCV核心单克隆抗体(Anti Pep 4)，培养30分钟后用含有Tween20的PBS溶液洗涤5次。再往微孔中加入邻苯二胺和过氧化氢，在黑暗环境中培养30分钟，最后加入50μl 4N硫磺酸终止反应。在495nM的光下检测微孔板，任何一个微孔中显现橙色光就表明被测试样品被HCV感染。
实施例3：聚氧乙烯醚对HCV抗原或抗体检测的影响
不同聚氧乙烯醚对HCV3.0抗HCV检测或HCV核心抗原的ELISA检测的不同影响如下表1A和1B所示。在HCV核心抗原ELISA检测中，用于商业HCV核心抗原的N-十二烷基肌氨酸被第一栏不同浓度的去污剂所替代。在HCV3.0抗HCV的检测中，用于检测的稀释样品中加入了去污剂。
表1A
| 试验用去污剂 | HCV3.0抗体ELISA | |||||
| 基质 | HCV抗体活性 | |||||
| 0.25％ | 0.125％ | 0.06％ | 0.25％ | 0.125％ | 0.06％ | |
| 0.125％ | 0.06％ | 0.03％ | 0.125％ | 0.06％ | 0.03％ | |
| 聚氧乙烯-2-油烯醚Brij92聚氧乙烯-8-硬脂酸盐Myrj45聚氧乙烯-5-十二烷基醚聚氧乙烯-50-硬脂酸盐Myrj53聚氧乙烯-8-十二烷基醚聚氧乙烯-9-十二烷基醚聚氧乙烯-10-十二烷基醚聚氧乙烯-10-十六烷基醚Brij56聚氧乙烯-10-油烯醚Brij97聚氧乙烯-100-硬脂酸盐Myrj59聚氧乙烯-20-十六烷基醚Brij58聚氧乙烯-40-硬脂酸盐Myrj52聚氧乙烯-100-十八烷基醚Brij700聚氧乙烯-20-油烯醚Brij98聚氧乙烯-23-十二烷基醚Brij35聚氧乙烯-7-十二烷基醚聚氧乙烯-4-十二烷基醚Brij30聚氧乙烯-3-十二烷基醚多聚醇F-127 | 0.056 0.035 0.0410.019 0.024 0.0250.033 0.046 0.0580.044 0.069 0.0830.064 0.070 0.0590.057 0.055 0.0610.049 0.059 0.0640.053 0.093 0.0590.031 0.056 0.0720.047 0.050 0.0450.058 0.046 0.0620.048 0.063 0.0670.043 0.045 0.0520.053 0.044 0.0500.067 0.055 0.0650.065 0.105 0.1000.093 0.058 0.1850.035 0.042 0.0750.025 0.030 0.035 | 1.543 1.432 1.4051.208 1.286 1.3131.168 1.274 1.3511.368 1.361 1.2621.223 1.219 1.2771.312 1.260 1.2251.192 1.069 1.1610.994 1.212 1.2091.316 1.341 1.5341.751 1.615 1.4181.568 1.507 1.4881.502 1.308 1.1941.600 1.482 1.5031.632 1.542 1.4931.492 1.415 1.4731.151 1.458 1.1900.867 1.135 1.1900.995 0.927 1.1561.521 1.327 1.271 | ||||
| 对照 | 0.075(3.0SD) | 1.358(3.0SD) | ||||
表1B
| 试验用去污剂 | HCV抗原原型 | |||||
| 基质 | HCV抗原活性 | |||||
| 0.25％ | 0.125％ | 0.06％ | 0.25％ | 0.125％ | 0.06％ | |
| 0.125％ | 0.06％ | 0.03％ | 0.125％ | 0.06％ | 0.03％ | |
| 聚氧乙烯-2-油烯醚Brij92聚氧乙烯-8-硬脂酸盐Myrj45聚氧乙烯-5-十二烷基醚聚氧乙烯-50-硬脂酸盐Myrj53聚氧乙烯-8-十二烷基醚聚氧乙烯-9-十二烷基醚聚氧乙烯-10-十二烷基醚聚氧乙烯-10-十六烷基脂Brij56聚氧乙烯-10-油烯醚Brij97聚氧乙烯-100-硬脂酸盐Myrj59聚氧乙烯-20-十六烷基脂Brij58聚氧乙烯-40-硬脂酸盐Myrj52聚氧乙烯-100-十八烷基醚Brij700聚氧乙烯-20-油烯醚Brij98聚氧乙烯-23-十二烷基醚Brij35聚氧乙烯-7-十二烷基醚聚氧乙烯-4-十二烷基醚Brij30聚氧乙烯-3-十二烷基醚多聚醇F-127 | 0.010 0.010 0.0320.028 0.021 0.0220.002 0.004 0.0070.014 0.040 0.0060.009 0.010 0.0140.008 0.008 0.0050.010 0.013 0.0200.004 0.001 0.0160.004 0.006 0.0210.019 0.021 0.0230.008 0.011 0.0280.009 0.005 0.0060.016 0.023 0.0190.008 0.011 0.0320.022 0.028 0.0290.009 0.009 0.016-0.001 0.001 0.0080.004 0.005 0.0130.013 0.013 0.012 | 0.609 0.847 0.5020.880 0.509 0.4330.195 0.243 0.2650.573 0.536 0.6590.326 0.384 0.3430.360 0.379 0.2120.495 0.556 0.3440.445 0.298 0.3900.371 0.419 0.4790.429 0.320 0.3690.546 0.898 0.5640.579 0.412 0.6610294 0.307 0.3190.557 0.718 0.5390.722 0.494 0.3090.228 0.289 0.3090.194 0.158 0.2120.106 0.168 0.1620.324 0.285 0.268 | ||||
| 对照 | 0.001(nLs) | 1.109(nLs) | ||||
实施例4：HCV核心抗体和/或抗HCV抗体的检测
检测平板上的HCV核心抗原或抗HCV抗体，该平板已被抗核心单克隆抗体C11-3、C11-7和HCV抗原C200-3、NS5及修饰过的核心抗原(含有带aa47和48缺失的核心序列aa10-99的SOD融合蛋白(C22KS(Δ47-48))包被。所用样品是获自商业血清转化板的4个连续血样。核心抗原用带有HRP标记的C11-4单克隆抗体来检测，抗HCV抗体用带有HRP标记人抗IgG来检测，两种抗体作用，积累产生可检测信号，显示于表2的被标记为COMBO的栏中。
表2
| 样品 | 抗IgG | 抗HCV核心抗原 | 联合(COMBO) |
| 阴性样品均值* | 0.045 | 0.036 | 0.094 |
| BCP6215 1BCP6215 2BCP6215 3BCP6215 4 | 0.0070.0090.0140.494 | 0.1350.1570.2310.197 | 0.1930.1910.2700.614 |
*表示HCV阴性样品检测到的信号实施例5：HCV核心抗体和/或抗HCV抗体的检测
检测平板上的HCV核心抗原或抗HCV抗体，该平板已被抗核心单克隆抗体C11-3、C11-7和HCV抗原C200-3、NS5及修饰过的核心抗原(含有第47位点处的精氨酸残基被亮氨酸残基取代的核心序列aa10-99的SOD融合蛋白(C22KS(R47L)))包被。用于检测的样品是获自商业血清转化板的4个连续血样，核心抗原用带有HRP标记的C11-4单克隆抗体来检测，抗HCV抗体用带有HRP标记抗人IgG来检测。在联合检测中，两种检测到的抗体、带有HRP标记的抗核心单克隆抗体和人抗IgG单克隆抗体作为一种混合物用于检测。
表3
| 样品 | 抗IgG | 抗HCV核心抗原 | 联合(COMBO) |
| 阴性样品均值* | 0.045 | 0.036 | 0.094 |
| BCP6215 1BCP6215 2BCP6215 3 | 0.0070.0090.014 | 0.1350.1570.231 | 0.1930.1910.270 |
| BCP6215 4 | 0.494 | 0.197 | 0.614 |
实施例6：抗IgG对照
血清转化板微孔用两种不同的HCV核心蛋白包被，比较这两种微孔中的抗HCV的活性，结果见表4
表4
| 样品 | SOD/C22KS(Δ47-48) | SOD/C22KS(R47L) |
| 阴性样品均值* | 0.045 | 0.076 |
| BCP6215 1BCP6215 2BCP6215 3BCP6215 4 | 0.0070.0090.0140.494 | 0.0150.0210.0280.498 |
实施例7：血清转化板微孔用两种不同的HCV核心抗原包被，比较这两种平板微孔中的核心抗原检测情况，结果见表5
表5
抗HCV核心抗原比较
| 样品 | SOD/C22KS(Δ47-48) | SOD/C22KS(R47L) |
| 阴性样品均值* | 0.036 | 0.047 |
| BCP6215 1BCP6215 2BCP6215 3BCP6215 4 | 0.1350.1570.2310.197 | 0.2240.1890.3040.189 |
实施例8：血清转化板微孔用两种不同的HCV核心抗原包被，比较这两种平板微孔中联合反应的活性，结果见表6。
表6
HCV结合物的比较
| 样品 | SOD/C22KS(Δ47-48) | SOD/C22KS(R47L) |
| 阴性样品均值* | 0.094 | 0.144 |
| BCP6215 1BCP6215 2BCP6215 3BCP6215 4 | 0.1930.1910.2700.614 | 0.2470.2260.3280.658 |
Claims (1)
1、一种检测样品中是否存在HCV的方法，该方法包括：将样品与固定在固相上的HCV抗原和抗HCV核心抗体接触，然后向样品中加入聚氧乙烯醚，通过加入被标记过的抗人IgG和被标记过的抗HCV核心抗体来检测被捕获的抗原和抗体，检测作为HCV的存在表征的放射信号。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101196518B (zh) * | 2006-12-07 | 2011-11-02 | 北京科美东雅生物技术有限公司 | 丙型肝炎病毒抗体化学发光法诊断试剂盒及其制备方法 |
| CN103630690A (zh) * | 2013-12-17 | 2014-03-12 | 山东莱博生物科技有限公司 | 丙型肝炎病毒抗原抗体联合检测试剂盒及其检测方法 |
| WO2021134302A1 (zh) * | 2019-12-30 | 2021-07-08 | 深圳迈瑞生物医疗电子股份有限公司 | 用于检测hcv的免疫分析仪、方法及试剂盒 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US7101683B2 (en) * | 2001-06-26 | 2006-09-05 | Abbott Laboratories | Methods for the simultaneous detection of HCV antigens and HCV antibodies |
| US20030108563A1 (en) * | 2001-11-07 | 2003-06-12 | Chander Bahl | Reagents for the simultaneous detection of HCV core antigens and antibodies |
| US7332269B2 (en) * | 2001-11-11 | 2008-02-19 | Ortho-Clinical Diagnostics, Inc. | HCV core protein sequences |
| US7764416B2 (en) | 2006-12-04 | 2010-07-27 | 3M Innovative Properties Company | Electrochromic device based on layer by layer deposition |
| RU2380711C2 (ru) * | 2008-04-22 | 2010-01-27 | Федеральное государственное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФГУН ГНЦ ВБ "Вектор" Роспотребнадзора) | Способ изготовления рабочего стандарта сывороток, содержащих антитела к вирусу гепатита с |
| CN102378914B (zh) * | 2009-03-30 | 2014-12-10 | 生物梅里埃公司 | 用于hcv检测的固体支持物 |
| US20100297607A1 (en) * | 2009-05-20 | 2010-11-25 | Jian Zheng | Reagents For HCV Antigen-Antibody Combination Assays |
| FR2984328B1 (fr) | 2011-12-20 | 2016-12-30 | Bio-Rad Innovations | Procede de detection d'une infection par le virus de l'hepatite c |
| CN102955028A (zh) * | 2012-06-11 | 2013-03-06 | 郑州安图绿科生物工程有限公司 | 检测自身免疫性肝病相关抗体抗ama-m2型抗体的试剂盒及其检测方法 |
| CN104849460A (zh) * | 2015-05-29 | 2015-08-19 | 山东博科生物产业有限公司 | 一种稳定的丙型肝炎病毒核心抗原酶结合物稀释液 |
| MX2019005684A (es) | 2016-11-15 | 2019-10-30 | Quest Diagnostics Invest Llc | Métodos para detectar mutaciones de fibrosis quística mediante el uso de extracción con puntas mitra. |
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| US5616460A (en) * | 1995-06-07 | 1997-04-01 | Abbott Laboratories | Buffer composition for reagents for immunoassay |
| US6623921B2 (en) * | 1998-07-30 | 2003-09-23 | Advanced Life Science Institute, Inc. | Method for measurement of hepatitis C virus |
| WO2000007023A1 (fr) * | 1998-07-30 | 2000-02-10 | Advanced Life Science Institute, Inc. | Procede pour la determination de l'hepatite a virus de type c |
| DE60129598T2 (de) * | 2000-06-15 | 2008-04-17 | Novartis Vaccines and Diagnostics, Inc., Emeryville | Immunoassays für anti-hcv-antikörper |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101196518B (zh) * | 2006-12-07 | 2011-11-02 | 北京科美东雅生物技术有限公司 | 丙型肝炎病毒抗体化学发光法诊断试剂盒及其制备方法 |
| CN103630690A (zh) * | 2013-12-17 | 2014-03-12 | 山东莱博生物科技有限公司 | 丙型肝炎病毒抗原抗体联合检测试剂盒及其检测方法 |
| WO2021134302A1 (zh) * | 2019-12-30 | 2021-07-08 | 深圳迈瑞生物医疗电子股份有限公司 | 用于检测hcv的免疫分析仪、方法及试剂盒 |
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| JP2002365301A (ja) | 2002-12-18 |
| HRP20020263A2 (en) | 2002-12-31 |
| SG118120A1 (en) | 2006-01-27 |
| HU227950B1 (hu) | 2012-07-30 |
| PL353045A1 (en) | 2002-10-07 |
| AU785380B2 (en) | 2007-03-15 |
| BRPI0200986B1 (pt) | 2018-03-20 |
| AR033448A1 (es) | 2003-12-17 |
| AU2764102A (en) | 2002-10-03 |
| BRPI0200986B8 (pt) | 2021-07-27 |
| US20030049608A1 (en) | 2003-03-13 |
| KR20020077125A (ko) | 2002-10-11 |
| MXPA02003318A (es) | 2004-07-16 |
| EP1251353A2 (en) | 2002-10-23 |
| CZ305181B6 (cs) | 2015-06-03 |
| KR100844169B1 (ko) | 2008-07-04 |
| HUP0201087A2 (hu) | 2003-01-28 |
| HUP0201087A3 (en) | 2004-11-29 |
| HU0201087D0 (zh) | 2002-05-29 |
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