CN1742202A - 表层亲和色谱法 - Google Patents
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Abstract
本发明公开了一种亲和色谱分析系统，其包括含生物试剂的固定组分和含补充生物试剂的流动组分，其特征在于固定组分被支承在浸渍带或平面上，且流动组分适于沿高密度浸渍带向下流动。本发明还公开了一种进行亲和色谱分析的方法，其包括使用所述分析系统。
Description
免疫色谱法目前以两种主要方式来进行。第一种为当展开是通过被动扩散实现或由电化学诱发时，在凝胶中进行；第二种是在流体中进行。在使用凝胶的前种方式中，径向免疫扩散是最常用的系统，其具有通常位于圆盘中的实验凝胶，凝胶中存在中心孔，以及在环绕凝胶周围有另外的孔。抗血清或抗原被放在中心孔，抗原或抗血清被放在外围孔。在凝胶中发生被动扩散，由于抗体-抗原络合物的形成，在凝胶中可观察到免疫络合物的白色谱带。在电化学系统中，使用电流诱发凝胶中抗体/抗原的迁移。
在流动基的系统中，抗体或抗原通常被固定在放置到液体流动系统如流动注射分析系统中的柱体中。补充的抗原或抗体被注入并流过柱体，在柱体中发生专一的相互作用。被注入的组分通常携带可在下游被检测到的标记物，从而产生信号。可替换地，流动发生在平面上，如同侧流扩散免疫分析系统，流动是由膜浸润/毛细管效应而诱发的。
在新的方式下，固定组分和上述溶液中一种组分间在流体中发生等价免疫反应，但该反应发生在残留于缓冲溶液中浸渍带的表面上。在这种情况下，由于含一种免疫试剂的溶液具有较高密度，从而在带的表面上发生流动，其中免疫试剂最初存在于带的顶部，而带本身立于缓冲溶液中。由于所述带近乎竖直，该浓厚溶液沿着带的表面缓慢向下滚动，从而将流动相中的试剂带入浸渍带表面上固定的试剂中。
因此，根据本发明我们提供一种亲和色谱分析系统，其包括含生物试剂的固定组分和含补充生物试剂的流动组分，其特征在于固定组分被支承在浸渍带或其它平面上，且所述高密度流动组分适合沿着浸渍带向下流动。
为了能产生这种现象，必须满足一定的标准。首先，当其以层状从表面上经过时，浓厚溶液必须保持分离体的形式，而不是快速扩散到缓冲溶液的本体中。其次，浸渍带必须具有一定的性质，能够对滚动表层产生引力，从而使得该流动相保持整体性。为了实现第一个标准，申请人认真选择了滚动相的组成，以包括聚合性试剂如蛋白质和/或多糖，清洁剂和优化pH值的缓冲剂；对于第二个标准，我们使用了即具有憎水性又具有可润湿性的膜。
所述系统可被用作免疫色谱系统，且以竞争性或非竞争性免疫分析方式进行分析。在前一方式中，所述的固定色谱斑为抗体或抗原。对于固定抗体，使被标记的抗原沉积在纤维素方片中所述色谱斑上的条带中。将含抗原的一滴样品加到该方片上，经过适当间隔(1-10min)后，将整个条带浸入缓冲溶液。被标记和未被标记抗原的浓厚混合物流过抗体的色谱斑，发生结合竞争。若抗原固定在色谱斑处，那么被标记的抗体取代纤维素方片上的被标记的抗原，进行前述的分析。
当捕获抗体的色谱斑被固定在条带上时，所述系统也可以非竞争免疫分析方式进行。被标记的抗体沉积到第一个色谱斑上方的纤维素方片上。将所述条带放置在含抗原的样品溶液中，使得上部方片被浸入。发生诱导，在该过程中溶液内的抗原被色谱斑上的固定抗体捕获。同时，在将所述条带插入样品溶液5-10分钟后，被标记抗体在流过色谱斑的浓厚溶液中被再生。这种时间滞后使得抗原分子在到达含被标记抗体的表层之前被捕获到色谱斑上。该第二种抗体标记了在色谱斑表面被捕获的抗原。
另外，可使用任何标记物。若荧光标记物或有色标记物用于抗体或抗原，那么荧光或有色谱斑会在第一次诱导后形成，使得分析成为单步骤系统。
若使用酶标记物，那么在非竞争分析中使用调整顺序的步骤。其中，将酶标记的抗体加入到纤维素方片上，该方片固定在位于固定捕获抗体色谱斑下方条带的底部上。第二个纤维素方片也如前所述固定在该色谱斑上，但其含有用于酶的培养基加上生物高聚物如葡聚糖的干燥溶液。将所述条带放置到前述样品有限的体积中，从而不润湿上部纤维素方片。被标记抗体浓厚的再生浓溶液流过容器底部，并以分离层的形式停留在那儿。同时，溶液中的抗原被色谱斑上的抗体捕获。在该诱导步骤结束时(5-10分钟)使用浸渍带搅拌溶液。这使得被标记的抗体均匀分布在溶液中，然后抗体与色谱斑上被捕获的抗原结合。最后，增加容器体积以润湿上部纤维素方片。所述培养基被再生成流过色谱斑的浓厚溶液，当培养基发生转变时产生了有色谱斑。
根据本发明的另一个方面，我们提供了一种进行免疫色谱分析的方法，其包括使用前述的分析系统。
理论上，本发明新的表面色谱现象也可用作基因色谱法以分离其中组分对表面具有不同结合亲和力的被分析物混合物。例如，已知生物聚合物如蛋白质和DNA/RNA结合到硝酸纤维素上，从而其用在电泳后DNA-和蛋白质-粘吸作用中。我们还观察到抗体对表面的结合速度是pH敏感的[1]。因此应当将生物聚合物的混合物引入到硝酸纤维方片上方的纤维素条带上。所用缓冲剂的pH和密度应当这样，当条带被浸入第二种选择用来优化生物聚合物与表面的结合时，能够确保形成表层色谱。在这些条件下，当运动相层滚动经过表面时，在生物分子和表面间会发生不同的结合相互作用。这会导致展开相中组分的分离。所述条带然后可被除去，使用已知方法对膜进行处理以显现出所述混合物的固定组分。
通过下述实施例对本方进行阐述。
实施例1
在本发明新的免疫色谱系统中，该系统我们称之为表层免疫-色谱法(Surface Layer Immuno-Chromatography(SLLC))，通过已知方法使用专用于抗原如savinase的专一抗体对硝酸纤维素膜的小方片进行预处理，以形成固定试剂的色谱斑。该方片被粘到预处理后具有一个粘性表面的塑料条带的表面上。在其上方粘接了第二个用信息酶碱性磷酸酯酶标记的相同抗体的干燥溶液以及牛血清白蛋白(BSA)和Tween 20浸泡过的纤维素条带。该沉积过程所用的溶液是Tris(pH9.3)，使用体积为10μl。该溶液的组成为0.1％w/v BSA、0.1％w/v Tween20、在Tris缓冲液中以1∶1000比例稀释的酶标记的抗体。储存在室温干燥器中的这种形式的试剂稳定存在于条带上至少14天。
为了进行上述分析，将Tris缓冲液中的savinase(0.3ml)溶液放置在测试管如常规96孔微滴定盘中。当两个方片均被样品覆盖时，将浸渍带插入到孔中。润湿后，纤维素方片中的试剂流入溶液。由于其较高的密度，该溶液然后沿着条带表面以层状向下流动，并最后经过含固定抗体色谱斑的较低方片。在条带插入和流动相到达时的时间间隔内，本体溶液中的savinase分子已被较低色谱斑上的固定抗体捕获。在流动相到达后，当溶液流过色谱斑时，被标记的抗体将结合到被捕获的抗原分子上，如在常规夹心型ELLSA中。第一个诱导阶段通常为15分钟。
将所述条带从孔中除去，并放置在含用于碱性磷酸(酯)酶、溴氯吲哚基磷酸酯(BCIP)和硝基蓝四氮唑盐(NBT)的常用培养基混合物的孔中。1分钟诱导后成为紫/蓝色，其亮度与在第一个诱导步骤中色谱斑上被捕获的savinase的量直接相关，从而与样品中savinase的浓度相关。上部方片也被着色成紫/蓝色。对该被分析物得到的条带进行观察表明分析结果。应当注意的是我们使用了12个这种牛角状条带，这可通过使用单一微滴定板对多达96个样品/标准样(8×12)进行分析。还应当指出的是在零标点和5ng/ml标准件间可清晰观察到视觉差别。
实施例2
与实施例1相同，将具有专一抗体色谱斑的硝酸纤维素膜的方片固定到塑料条带的表面。在其下方放置第二个纤维素条带(被切成0.4×0.6cm的方片以快速硬化滤纸)。该方片用5μl与信号酶碱性磷酸酯酶标记的相同抗体在蓝色葡聚糖(3％w/v)、葡聚糖(25w/v)中的稀溶液进行浸泡，所有溶液均指在叠氮化物的Tris缓冲剂中，pH＝9.3(含0.1％Tween 20和0.15(w/v)牛血清白蛋白)。将所用溶液在室温下干燥30分钟。将第三个快速硬化的滤纸叠放在第一个方片上方，并用碱性磷酸酯酶的培养液进行浸泡。这通过加入15μl 1％(w/v)蓝色葡聚糖、1％(w/v)葡聚糖和BCIP-NBT原料溶液来制备。使其在环境温度下干燥60分钟，此时将方片粘到浸渍带上。
预制备的浸渍带然后被加入到含0.6ml标准液或样品的微型试管中。10分钟后在试管底部观察到蓝色溶液层。用所述条带搅拌试管中的内含物。从而使整个溶液形成均匀的蓝色。再诱导5分钟后，加入500μl的培养基缓冲溶液以覆盖在浸渍带上的顶部方片。最后诱导10分钟后，除去所述的条带并用水冲洗。
当使用兔的抗-savinase抗体作为捕获剂，并在savinase标准液存在下封存抗体，进行上述分析时可观察后续的条带。
得到的浸渍带如图1所示，其中
A＝零标点，B＝10ng/ml的savinase标准液
Claims (19)
1.一种亲和色谱分析系统，包括含生物试剂的固定组分和含补充生物试剂的流动组分，其特征在于固定组分被支承在浸渍带或平面上，流动组分适于沿高密度浸渍带向下流动。
2.如权利要求1的亲和色谱分析系统，其特征在于流动组分比本体溶液具有更高的密度。
3.如权利要求1的亲和色谱分析系统，其特征在于免疫试剂是抗原或抗体。
4.如权利要求1的亲和色谱分析系统，其特征在于流动组分保持为分离体。
5.如权利要求1的亲和色谱分析系统，其特征在于流动相的组分包括生物聚合物、清洁剂和具有优化pH的缓冲液。
6.如权利要求1的亲和色谱分析系统，其特征在于固定组分具有吸引流动组分的性质。
7.如权利要求6的亲和色谱分析系统，其特征在于吸引流动组分是通过膜实现的。
8.如权利要求7的亲和色谱分析系统，其特征在于所述膜既有憎水性又有可润湿性。
9.如权利要求3的亲和色谱分析系统，其特征在于所述分析是使用被标记的抗原或被标记的抗体以及所补充的未被标记的对应成分的适当组合，进行竞争性或非竞争性免疫分析。
10.如权利要求9的亲和色谱分析系统，其特征在于标记是荧光标记或有色标记。
11.一种进行亲和色谱分析的方法，其包括使用如权利要求1的分析系统。
12.如权利要求11的方法，其特征在于所述浸渍带基本上竖立于缓冲溶液中。
13.如权利要求11的方法，其特征在于流动组分分配于邻近浸渍带上部或下部处。
14.如权利要求11的方法，其特征在于所述方法包括分离分析物混合物。
15.如权利要求11的方法，其特征在于所述组分对表面具有不同的结合亲和力。
16.如权利要求11的方法，其特征在于所述方法包括单一步骤分析。
17.如权利要求11的方法，其特征在于所述方法包括分离生物聚合物。
18.如权利要求17的方法，其特征在于所述生物聚合物选自蛋白质和DNA/RNA。
19.一种亲和色谱分析系统，或一种基本如实施例所述的方法。
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| EP1718973B1 (en) * | 2004-02-09 | 2009-09-09 | Rapid Pathogen Screening Inc. | Method for the rapid diagnosis of targets in human body fluids |
| US8445293B2 (en) | 2005-02-09 | 2013-05-21 | Rapid Pathogen Screening, Inc. | Method to increase specificity and/or accuracy of lateral flow immunoassays |
| JP5057925B2 (ja) | 2007-10-18 | 2012-10-24 | 株式会社日立製作所 | デジタル情報再生方法 |
| US8815609B2 (en) | 2008-05-20 | 2014-08-26 | Rapid Pathogen Screening, Inc. | Multiplanar lateral flow assay with diverting zone |
| US8962260B2 (en) | 2008-05-20 | 2015-02-24 | Rapid Pathogen Screening, Inc. | Method and device for combined detection of viral and bacterial infections |
| US8609433B2 (en) | 2009-12-04 | 2013-12-17 | Rapid Pathogen Screening, Inc. | Multiplanar lateral flow assay with sample compressor |
| US20130196310A1 (en) | 2008-05-20 | 2013-08-01 | Rapid Pathogen Screening, Inc. | Method and Device for Combined Detection of Viral and Bacterial Infections |
| US9068981B2 (en) | 2009-12-04 | 2015-06-30 | Rapid Pathogen Screening, Inc. | Lateral flow assays with time delayed components |
| US20110086359A1 (en) * | 2008-06-10 | 2011-04-14 | Rapid Pathogen Screening, Inc. | Lateral flow assays |
| US10808287B2 (en) | 2015-10-23 | 2020-10-20 | Rapid Pathogen Screening, Inc. | Methods and devices for accurate diagnosis of infections |
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| US4235601A (en) * | 1979-01-12 | 1980-11-25 | Thyroid Diagnostics, Inc. | Test device and method for its use |
| EP0560411B1 (en) * | 1987-04-27 | 2000-07-26 | Unilever N.V. | Specific binding assays |
| US4943522A (en) * | 1987-06-01 | 1990-07-24 | Quidel | Lateral flow, non-bibulous membrane assay protocols |
| US5120643A (en) * | 1987-07-13 | 1992-06-09 | Abbott Laboratories | Process for immunochromatography with colloidal particles |
| US5013669A (en) * | 1988-06-01 | 1991-05-07 | Smithkline Diagnostics, Inc. | Mass producible biologically active solid phase devices |
| US5731157A (en) * | 1993-12-30 | 1998-03-24 | The Procter And Gamble Company | Two-site allergen immunoassay |
| US5773234A (en) * | 1995-08-07 | 1998-06-30 | Quidel Corporation | Method and device for chlamydia detection |
| US6103536A (en) * | 1997-05-02 | 2000-08-15 | Silver Lake Research Corporation | Internally referenced competitive assays |
| US5895765A (en) | 1997-06-30 | 1999-04-20 | Bayer Corporation | Method for the detection of an analyte by immunochromatography |
| US6319466B1 (en) * | 1997-07-16 | 2001-11-20 | Charm Sciences, Inc. | Test device for detecting the presence of a residue analyte in a sample |
| DE69912904T2 (de) * | 1998-07-01 | 2004-11-04 | Nitto Denko Corp., Ibaraki | Markiertes Kojugat und Nachweismethode unter Verwendung desselben |
| JP2000019178A (ja) * | 1998-07-01 | 2000-01-21 | Nitto Denko Corp | 標識複合体 |
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| JP4223163B2 (ja) | 1999-10-25 | 2009-02-12 | パナソニック株式会社 | 免疫クロマトグラフィー試験片、及びクロマトグラフ分析方法 |
| EP1247094A4 (en) * | 2000-01-06 | 2006-10-04 | Biosite Diagnostics Inc | DOSAGE FOR THE DETECTION OF i BACILLUS ANTHRACIS / i |
| US6528325B1 (en) * | 2000-10-13 | 2003-03-04 | Dexall Biomedical Labs, Inc. | Method for the visual detection of specific antibodies in human serum by the use of lateral flow assays |
| US6723500B2 (en) * | 2001-12-05 | 2004-04-20 | Lifescan, Inc. | Test strips having reaction zones and channels defined by a thermally transferred hydrophobic barrier |
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| US7781225B2 (en) | 2010-08-24 |
| AU2004205779A1 (en) | 2004-08-05 |
| DE602004005158T2 (de) | 2007-11-22 |
| DK1585986T3 (da) | 2007-07-09 |
| US20060172434A1 (en) | 2006-08-03 |
| AU2004205779B2 (en) | 2008-10-16 |
| PT1585986E (pt) | 2007-06-19 |
| EP1585986B8 (en) | 2007-09-05 |
| CN100381821C (zh) | 2008-04-16 |
| JP2006515425A (ja) | 2006-05-25 |
| ATE356356T1 (de) | 2007-03-15 |
| DE602004005158D1 (de) | 2007-04-19 |
| EP1585986A1 (en) | 2005-10-19 |
| EP1585986B1 (en) | 2007-03-07 |
| ES2283973T3 (es) | 2007-11-01 |
| JP4658034B2 (ja) | 2011-03-23 |
| HK1079281A1 (en) | 2006-03-31 |
| GB0301225D0 (en) | 2003-02-19 |
| WO2004065962A1 (en) | 2004-08-05 |
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