CN1008403B - 免疫测定方法和装置 - Google Patents

免疫测定方法和装置

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CN1008403B
CN1008403B CN85104030A CN85104030A CN1008403B CN 1008403 B CN1008403 B CN 1008403B CN 85104030 A CN85104030 A CN 85104030A CN 85104030 A CN85104030 A CN 85104030A CN 1008403 B CN1008403 B CN 1008403B
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古纳斯·埃德温
瓦基塞
纽·科尔曼
欧文
菲利普·艾伦·莱文森
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Hybritech Inc
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Abstract

配体受体测定的装置和方法，该装置由膜或滤器为第一部件。它把配体的受体结合或从液样中提取带配体的细胞。第二部件由吸收材料组成，加入液样因第一部件，与第二部件接触，导致液体通过前者。测定时样品加到第一部件上表面，固定在其上的受体结合样品中的配体或提取与配体相关的细胞物质。先加样品后加待测抗原的标记抗体，洗去未结合标记受体；如第一部件上有标记抗体表示样品中有抗原。本发明以配体是抗原，受体是抗体为最好。

Description

本发明涉及配体-受体免疫测定，特别是免疫测定方法，尤其是使用单克隆抗体的免疫测定方法。另一方面，本发明涉及一种进行这些测定的装置。
近20年来，免疫测定方法已为体外检测与疾病或有临床意义的其它身体状况有关的各种抗原提供了灵敏的诊断工具。最初这些不同的测定法使用的是与固相结合的多克隆抗体制剂。在这些测定中，使标记抗原的溶液直接与待分析样品中的抗原竞争固相抗体，或将该溶液连续加入抗体中。标记抗原与固相结合或在液相中被检测出的程度可用来作为所分析样品中抗原的存在和其含量的量度。
后来又有了非竞争性免疫计量测定法（immunometric    assays）。在这些测定中也使用了与固相结合的多克隆抗体制剂。将含有可疑抗原的样品与固相接触以使该抗原与固相上的抗体结合。一般在保温步骤后把样品从固相中分离出来，然后洗涤固相，并将固相与另一些已用放射性核素、酶或荧光半分子等标记的多克隆抗体的溶液一起保温。
经此第二次保温后，将未结合的标记抗体与固相分开，测定液相中标记抗体的量，或者测定抗体-抗原-抗体夹层中与固相结合的标记抗体的量，作为所测试的样品中抗原的存在和/或其浓度的量度。
最近，免疫测定方法已得到改进而使用单克隆抗体。例如，美国专利4，376，110叙述了使用单克隆抗体对的两位点免疫计量测定法，其中一种抗体结合到固相上，另一种抗体被标记以便检测。使用能识别一个抗原上的不同抗原决定基位点的单克隆抗体对，能同时进行多 个免疫计量测定，其中抗原和标记抗体的保温过程无需先有技术方法中的中间洗涤步骤。
在前述各方法中，固相抗体一般结合到小珠或小颗粒上或被涂到表面上。所有这些方法都特征性地要求与固相和标记抗体都保温一段时间，因此，即使同时进行也很费时。事实上，完成一次测定通常需要几小时。此外，由于需要进行定时保温步骤并用量好的试剂进行多次洗涤，使这些方法主要限于在大医院和咨询临床实验室中应用，这些地方拥有训练有素的技术人员和复杂仪器设备可进行这些测定。因此这些方法并未满足需要。目前需要一种进行免疫测定的简单而迅速的方法，它应用较为简单的仪器，使这些测定方法能够在医生办公室中使用，甚至可供柜台销售给个人而用于家庭保健计划。
本发明提供一种简单而迅速地进行配体-受体测定的方法，例如进行免疫测定和免疫计量测定以及利用核酸寡聚体杂交的测定，该方法使用简单的装置而不需长时间的保温步骤。本发明的装置包括一个作为第一部件的多孔部件，例如膜或滤器，该多孔部件上结合或固定有待测靶分析物（配体）的受体或抗受体，或者该多孔部件能够从待分析样品中分离出连有待测配体的细胞或细胞碎片，从而将该配体固定在多孔部件上。例如，在免疫测定和免疫计量测定中，如果配体是抗原，则把抗体（最好是单克隆抗体）结合在多孔部件上作为受体。该装置还包括一个作为第二部件的吸收部件，该部件中有许多毛细管通道，这些通道一般穿过该部件的上表面和下表面。此处所用的术语“毛细管”包括允许液体穿过吸收部件的毛细管或其他孔道或通道。第二部件与多孔第一部件以毛细管相通， 并且在选择第二部件时，要使毛细管具有一定的孔径，使得在用于测定的样品和后续的加入物的静水压不足以使液体流过第一部件时，第二部件能诱使液体流过第一部件而无需借用外部手段。第二部件还可以起支持第一部件的作用。
本发明的测定包括如下步骤：将液体样品加入多孔部件，从而在液体流过该部件的过程中，结合在多孔部件上的受体能够结合样品中溶解的或悬浮的配体，其结合速度明显高于不流过该部件时所观察到的速度；如果配体是在细胞材料的表面，则细胞材料或者被固定在多孔部件上的受体结合，或者在样品流过时被该部件捕获。在本发明的一个优选实施方案中，在加入样品后加入配体的另一种标记受体的溶液，以便进行检测。例如，在对抗原进行免疫计量测定时，使用一种抗体溶液，最好是一种单克隆抗体，它在不干扰抗原与第一受体结合的抗原决定基上与该抗原结合。优选标记物为酶，但也可以使用其他标记物，例如放射性核素或荧光标记。抗体与已通过结合抗体或细胞材料的捕获而从样品中提取出的抗原结合。加入标记抗体后可以立即进行一个洗涤步骤，也可以在短暂保温之后进行此步骤，除去未结合的标记抗体，以使抗原和标记抗体更强地结合，从而提高灵敏度。然后测定多孔部件上标记抗体的存在，作为样品中存在靶抗原的指示。如果是酶标记，则进行这一测定时是把生色底物的溶液加入多孔部件，使该底物与酶反应。
图1是进行本发明免疫测定的装置的截面图。
图2是用于本发明中从所测样品中除去抗原的多孔部件的上视图。
如前面所指出的，本发明的装置包括作为第一部件的多孔膜或 滤器，其上结合有一种配体的受体或结合有抗受体；或者，如果配体连在细胞材料上，该部件能够从所测样品中滤出细胞材料。在后一种情况下，选择膜或滤器时要使它具有能进行这一分离的孔径。可以使用多种过滤部件中的任一种，包括玻璃纤维滤器和各种合成或天然材料的滤器。
当多孔部件结合有受体时，选择该受体时应使其具有选择性地直接与靶配体结合的能力。例如，如果配体是一种抗原，则受体可以是一种抗体，最好是一种单克隆抗体。如果靶配体是一种抗体，受体则可以是一种抗原或抗抗体。如果配体是一种酶，则受体可以是该酶的受体。如果配体是一种核酸，如RNA或DNA，受体可以是DNA或RNA的互补寡聚体。在一个优选实施方案中，第一部件是一种共价结合有抗体制剂的膜或滤器。该抗体制剂最好是一种单克隆抗体，但也可以使用来自抗血清的多克隆抗体。单克隆抗体和多克隆抗体的制备技术已属公知技术，在此无需引述。
多孔部件的材料从能够结合受体或抗受体（如果使用的话）的材料中选择。如果受体或抗受体是蛋白质，例如抗体或抗原，则优选的材料是具有氨基残基或已用化学方法引入这类基团的尼龙，这种材料允许用公知的戊二醛法使其偶联蛋白质。抗体可以通过氨基硅烷与玻璃纤维偶联。也可以使用能够与受体或抗受体直接偶联或通过中间基团偶联的其他天然或合成材料。
前面强调了受体或抗受体与多孔部件的化学结合。但在适当的情况下，受体或抗受体也可以涂在多孔部件上，或者是一种能够被捕获在多孔部件缝隙内的颗粒。因此，此处所用的术语“结合”将 包括将受体或抗受体固定在多孔部件上的任何手段。
第二部件是一个吸收部件，它具有通常穿过上表面和下表面的毛细管通道。第二部件与第一部件的装配方式，是使第一部件的孔或间隙与第二部件的毛细管之间直接相通。这样，当把液体加到第一部件中并使其饱和时，液体就被吸入吸收部件中。所以，当把液体样品加到第一部件的上表面时，即使流体的静水压很低，以至于不加压迫其通过或减压驱其通过就不能自动流过第一部件，也能使液体样品流过第一部件。
第二部件材料的选择并不重要，各种纤维过滤材料都可以使用。一种可用的材料是纤维取向与香烟滤嘴相同的乙酸纤维素纤维。本领域技术人员会认识到，由聚酯、聚烯烃或其他材料制成的其他吸收部件可用来代替乙酸纤维素。
参见图1，它显示了一个可用于本发明进行测定的装置的剖面图。图1给出了一个圆筒形容器10（但它也可具有任何适当的其他形状），它有一个由侧壁14限定的上开口12。该容器可由玻璃、塑料或合适的其他材料制成。如图1所示，容器10还有一个下开口16，其中插入了一个可拆卸的塞子18，使得能够插入多孔部件20（一个圆形膜或过滤片）和一个可有可无的部件21（其功能在下面叙述），这两个部件放在圆筒形吸收部件22上，部件22也是经过开口16插入的。
如图1所示，容器10的一部分在参考编号24处收缩而构成一个完整的漏斗，用于将样品导引至部件20，并确保能对加到部件20上的样品和其他加入物进行有效的洗涤。
在选择部件22的大小，也就是容器10缩颈以下部分的体积 时，最好使在测定过程中需加入装置中的所有液体都能被吸收部件22接收并保留在其中。在靠近容器10的底部有排放空气的装置（图1中未画出）如小排气孔，以使排出的空气逸出。根据需要，可以去掉容器10的底部，而使液体穿过部件20和22后从容器底部排出。但是，因为这一物件是要一次性使用并且是要便于以简单而卫生的方式处置样品，所以最好还是应用图1所示的结构。
如前面所指出的，部件20可以用来从样品中滤出细胞材料，也可以用作针对所测配体的结合受体的载体。在这两种情况下，都可以经开口12把液体样品加到部件20中。样品渗透进入部件20，液体被吸过和吸入吸收部件22之后，经开口12将标记受体的溶液加到部件20中。
这样，标记受体就与结合在部件20受体上的配体结合，或者与捕获在部件20表面上的细胞物质所结合的配体结合。在进行免疫计量测定时，如果部件20结合有单克隆抗体，则标记抗体也是一种单克隆抗体。如美国专利4，376，110和1981年6月6日提交的323，498号专利申请所述（其公开内容列为参考文献），选择这两种抗体时，要使它们分别与互不干扰的抗原结合位点结合。
最好用酶来标记可溶性受体，但在适当情况下也可以使用其它常规测定标记物。例如，还可以使用荧光标记（如荧光素或藻红素）或放射性核素。可用的标记物还包括载有有色染料或荧光颗粒的微球体。其他可用标记物中可以提到的有载有有色染料或荧光颗粒的脂质体或其他囊状物。
在标记受体溶液穿过部件20之后，在部件20中加入洗涤液，把部件20中未结合的标记受体冲洗到部件22中。壁24的倾斜 结构构成了一个完整的漏斗，有利于把洗涤液加到壁上而洗去附着的残余标记受体溶液。
在向部件20中加入标记受体溶液和洗涤液之前，可以进行短时间的保温，以使部件20表面或间隙中捕获的溶液中的受体或配体更充分地结合，从而提高测定的灵敏度。但是我们发现，这些保温步骤或者不需要，或者可以时间很短，即通常在60秒或更短的数量级。含有配体或标记受体的溶液流过部件20造成的结合速度，显著高于没有流动时所观察到的结合速度。
如果受体标记物是一种酶，则在从部件20中洗去未结合的受体后，在部件20中加入酶底物的溶液。如果靶配体与结合在部件20上的受体结合或者与部件20上的细胞材料结合，则该配体将结合有一部分标记受体。如果进行适当的选择，酶将使底物反应而产生可见的颜色变化。
我们发现，当用乙酸纤维素作吸收部件22的材料时，它会在上表面非特异性地结合标记受体。因此，恰在部件20之下的这一表面上会产生某种可见的颜色变化。为避免通过部件20时显现出这种颜色变化，最好在部件20和22之间置放一个多孔聚乙烯或其他材料的隔离部件（在图1中指定为21），这种材料不与受体非特异性地结合。
参见图2，图2显示了部件20的上视图。在一个优选实施方案中，虚线26代表结合有配体的区域28的外周线。此区的直径小于由壁24在其最窄点形成的缩颈的直径。这样，当用酶作受体标记物时，会产生下列结果：（1）如果在区域28显示出的颜色比部件20的周边强，即为阳性结果;（2）如果在部件20中未观察到显 色，则为阴性结果;（3）如果在洗涤后部件20中留有某些标记受体，则会产生均匀分布在整个可见表面上的中度颜色变化。这种结果也被解释为阴性结果。
前面是本发明装置和方法的一般性叙述。我们发现，它可用于在不到5分钟内进行从加入样品到得到阳性结果的免疫测定操作。例如，在一个具体实例中，利用戊二醛技术使一种针对人绒毛膜促性腺激素（HCG，一种在孕妇尿中含量较高的抗原）的单克隆抗体与多孔尼龙膜结合，将其放入如图1中的10那样的容器中，支持在乙酸纤维素吸收部件上但被多孔聚乙烯片隔开。
将尿样（4ml，含0和50mIU/ml    HCG）加到所述装置中，经部件20和21被吸入吸收材料22。然后加入3滴结合有碱性磷酸酶的抗HCG第二单克隆抗体的溶液。经过约1分钟的短时间保温使该结合物吸过部件20后，加入4ml水以除去部件20中未结合的抗体。加入后再加入3滴含磷酸吲哚酚（碱性磷酸酶的一种底物）的溶液。两分钟后，在用于测试不含HCG（0mIU/ml）样品的装置中未显示出颜色。50mIU/ml    HCG样品则在30秒内在圆片的中央显示出明显的蓝色，并在两分钟内变为深蓝色。在圆盘的周边没有显色。整个测定用了约5分钟。应该认识到，该测定的灵敏度可以通过改变体积或保温时间来调整。
虽然本发明已经以HCG的测定为例作了描述，但应该认识到，可以对其他抗原建立类似的测定方法。这些靶抗原太多，不能全部列出，但可以检测的抗原有IgE、前列腺酸磷酸酶、前列腺特异性抗原、α-胎球蛋白、癌胚抗原、促黄体生成激素、肌酸激酶MB，以及血清、血浆、尿或其他液体介质中的其他抗原。此外，可以利 用能捕获细胞的滤器或结合有抗原特异性抗体的滤器作为部件20，检测含有连有抗原的材料的液体样品，例如含有与下列材料相连的抗原：细菌、寄生虫、真菌或病毒，包括例如A组和B组链球菌、淋病奈氏球菌、阴道嘉氏菌（Gardnerella    Vaginalis）、阴道毛滴虫、白色念珠菌、沙眼衣原体、乙型肝炎病毒和细胞肥大病毒。例如，加入用酶标记的单克隆抗体溶液，将导致该抗体与抗原结合。洗涤并加入底物后，将导致与细胞上存在的标记抗体相关的颜色变化，这种变化可以目测或借助仪器检测。
如果使用非酶标记物，操作步骤可以不同。如果使用荧光标记，可以测量膜的荧光。如果使用放射性核素标记如125I，则可以取下膜进行计数。
前面强调了本发明应用于用单克隆抗体进行的连续免疫计量测定;即利用多孔部件上的第一单克隆抗体受体和标记的第二单克隆抗体受体的免疫测定。把样品加到多孔部件中，然后加入标记抗体。但也可以进行其他测定。例如，在进行免疫计量测定时，可以把标记抗体和样品在加入多孔部件之前进行混合。
本发明的装置还可用于测定抗体，即采用抗原作固相上的第一受体，并用标记的抗原或标记的抗抗体作第二受体。后者特别适于进行变态反应特异性测定，其中第一受体是一种结合在多孔部件上的变态反应原，第二受体是一种抗IgE抗体，最好是抗IgE单克隆抗体。在另一些情况下，可以类似地测定IgG对变态反应原的反应，即，用抗体如抗IgG单克隆抗体作第二受体。可以以这种方式进行的其他抗体测试包括抗疱疹、抗风疹、抗肝炎、抗细胞肥大病毒和抗HTL-Ⅲ的抗体的测试。
在本发明的另一个实施方案中，用所述装置来进行竞争性测定，即，在这些测定中配体受体结合在多孔部件上，样品中的配体与固定量的标记配体竞争该受体，标记配体加在样品溶液中或在加入样品后加入。利用抗体如单克隆或多克隆抗体制剂作为结合在固相上的受体，易于以上述方式进行竞争性免疫测定。可以在把样品加到多孔部件中之前把标记抗原加到样品中。也可以在加入样品之后或与加入样品同时加入标记抗原。
本发明的装置还可用于检测酶，方法是使酶的受体结合在多孔部件上作为测定受体。可以用针对该酶的标记抗体来检测多孔部件上形成的受体-酶复合物。
多孔部件还可以与一种核酸寡聚体结合而成为样品中核酸物质的探针受体。例如，该探针可以是互补于待测核酸中某段顺序的DNA寡聚体，在可能的情况下可用来结合作为配体的RNA或DNA。配体-受体复合物的检测，可以利用互补于待测核酸配体非干扰区的第二核酸寡聚体进行，第二寡聚体被标记以进行检测。
在本发明的另一个实施方案中，多孔部件可以结合有抗受体。此处所用术语“抗受体”意指能与配体-受体对中的受体选择性结合的试剂。例如，如果配体是一种抗原，受体是一种抗体如小鼠IgG抗体（最好是一种单克隆抗体），则抗受体可以是一种抗鼠IgG抗体，最好是一种单克隆抗体。在另一些情况下，受体可与能和抗受体选择性结合的半分子结合。例如，该半分子可以是一种半抗原，抗受体可以是一种针对该半抗原的抗体。优选的这种半抗原是荧光素。在另一些情况下，抗受体可以是抗生物素蛋白。在该情况下受体将结合有生物素。在另一些情况下，受体可以是一种核酸 寡聚体或结合有这样一种寡聚体，抗受体可以是一个互补于受体寡聚体一部分的核酸片段，该片段不影响受体与配体的结合。本领域技术人员可以由上述认识到，多以采用多种抗受体/受体组合。
使用抗受体时，样品可以以多种方式进行测定。例如，在“夹层测定”中，第一受体和标记的第二受体可以在加到多孔部件中之前与样品混合，再与配体结合。也可以把第一受体和样品在加到多孔部件中之前进行混合，或者按先第一受体后样品的顺序加入，而后加入标记受体。在这类夹层测定中，要选择结合第一受体而不结合标记受体的抗受体。
使用结合在多孔部件上的抗受体，有可能简化可用于配体-受体测定的多孔部件的设计和制备。例如，如果受体结合在多孔部件上，可能需要修改结合操作方法，以使一组测定中所要求的每个受体的结合达到最佳。但可以在多个测定中应用同一种结合在多孔部件上的抗受体。这样，如果这样一种“通用的”多孔部件是可能的，则设计工作和制造操作可以大大简化。

Claims (56)

1、一种用于为检测液体样品中的靶配体而进行的配体-受体测定方法的装置，该装置包括一个多孔的第一部件和一个由吸收材料体组成的第二部件，第一部件上结合有靶配体的受体或结合有抗受体，或能够从液体样品中分离出结合有配体的细胞材料，该第一部件具有上表面和下表面；该装置的特征在于，第一部件的下表面与第二部件上放置第一部件的表面以毛细管直接相通，第二部件内有毛细管穿过，其方向一般穿过放置第一部件的表面，这些毛细管适于吸引加到第一部件上表面并已渗入第二部件的毛细管中的液体。
2、一种根据权利要求1的装置，其特征在于，第一部件是一个结合有针对所述配体的受体的部件。
3、一种根据权利要求1的装置，其特征在于，第一部件是一种膜或滤器。
4、一种根据权利要求1、2或3的装置，其特征在于，配体选自抗原、抗体、酶和核酸寡聚体。
5、一种根据权利要求1、2或3的装置，其特征在于，多孔部件上结合有受体，该受体选自抗体、抗原、酶受体和核酸寡聚体。
6、一种根据权利要求5的装置，其特征在于，受体为核酸寡聚体。
7、一种根据权利要求6的装置，其特征在于，核酸寡聚体为DNA寡聚体。
8、一种根据权利要求5的装置，其特征在于，受体为一种抗体。
9、一种根据权利要求8的装置，其特征在于，受体为一种多克隆抗体制剂。
10、一种根据权利要求8的装置，其特征在于，抗体为一种单克隆抗体。
11、一种根据权利要求5的装置，其特征在于，受体为一种抗原，靶配体为针对该受体的抗体。
12、一种根据权利要求11的装置，其特征在于，抗原为一种变态反应原，抗体为针对该变态反应原的抗体。
13、一种根据权利要求12的装置，其特征在于，抗体为IgE。
14、一种根据权利要求12的装置，其特征在于，抗体为IgG。
15、一种根据权利要求1、2或3的装置，其特征在于，多孔部件由选自玻璃或尼龙的材料制成。
16、一种根据权利要求1的装置，其特征在于，多孔部件为一个能够从液体样品中分离出细胞材料的部件。
17、一种根据权利要求16的装置，其特征在于，多孔部件为一种膜或滤器。
18、一种根据权利要求1、2、3或16的装置，其特征在于，该装置还包括一个盛装第一部件和第二部件的容器，该容器有一个开口，允许将测定试剂加至第一部件的上表面。
19、一种根据权利要求1、2、3或16的装置，其特征在于，所述开口还包括这样一个区段，该区段具有向内倾斜的侧面而构成一个漏斗，可将所加入的试剂导引至第一部件的上表面。
20、一种根据权利要求1、2、3或16的装置，其特征在于，容器的底部是密闭的，该容器有开口以使被所加入的试剂置换出的空气逸出。
21、一种根据权利要求1的装置，其特征在于，第一部件结合有抗受体。
22、一种根据权利要求21的装置，其特征在于，受体是一种抗体，抗受体是针对该受体的抗体。
23、一种根据权利要求21的装置，其特征在于，受体与一种半抗原结合，抗受体是针对该半抗原的抗体。
24、一种根据权利要求23的装置，其特征在于，半抗原是荧光素。
25、一种根据权利要求21的装置，其特征在于，抗受体是抗生物素蛋白，受体与生物素结合。
26、一种根据权利要求21、22、23或24的装置，其特征在于，抗受体是一种单克隆抗体。
27、一种根据权利要求26的装置，其特征在于，抗体为一种单克隆抗体。
28、一种根据权利要求1的装置，其特征在于，在第一部件和第二部件之间置放一个多孔分隔部件。
29、一种根据权利要求28的装置，其特征在于，分隔部件由聚乙烯制成。
30、一种用于检测液体样品中的靶配体的测定方法，该方法包括：将可能含有靶配体的液体样品加至权利要求1、2、或3的装置中第一部件的上表面，此样品中已加入或在它加到多孔部件中之后加入靶配体的第二受体，此受体经标记可供检测;然后分离未结合的标记受体，检测与结合在第一受体上的配体结合的标记第二受体（如果有的话）。
31、一种根据权利要求30的方法，其特征在于，通过洗涤来分离未结合的第二受体。
32、一种根据权利要求30的方法，其特征在于，与多孔部件结合的受体选自抗原、抗体、酶受体和核酸寡聚体。
33、一种根据权利要求31的方法，其特征在于，与多孔部件结合的受体和标记受体与配体在互不干扰的位点上结合。
34、一种根据权利要求32的方法，其特征在于，靶配体是一种核酸寡聚体，与多孔部件结合的受体和标记受体为互补的核酸寡聚体。
35、一种根据权利要求34的方法，其特征在于，受体为DNA寡聚体。
36、一种根据权利要求35的方法，其特征在于，靶配体为一种DNA寡聚体。
37、一种根据权利要求35的方法，其特征在于，靶配体为一种RNA寡聚体。
38、一种根据权利要求32的方法，其特征在于，靶配体为一种抗原，受体为单克隆抗体。
39、一种根据权利要求32的方法，其特征在于，靶配体为一种抗体，受体为抗原。
40、一种根据权利要求32的方法，其特征在于，靶配体为一种抗体，与多孔部件结合的受体是一种抗原，标记受体是针对靶抗体的抗体。
41、一种根据权利要求40的方法，其特征在于，抗原为一种变态反应原，靶配体为IgE。
42、一种根据权利要求40的方法，其特征在于，抗原为一种变态反应原，靶配体为IgG。
43、一种根据权利要求41的方法，其特征在于，标记受体为一种单克隆抗IgE。
44、一种根据权利要求42的方法，其特征在于，标记受体为一种抗IgG。
45、一种用于检测与细胞材料相连的靶配体的测定方法，该方法包括：将可能含有连在所述细胞材料上的配体的液体样品加至权利要求16的多孔部件的上表面，以从该样品中分离出细胞材料;该样品中已经加入或在它加至多孔部件中之后加入靶配体的受体，该受体经标记可供检测;然后分离出未结合的标记受体;检测由多孔部件从液体样品中分离出的细胞材料上的配体所结合的标记受体（如果有的话）。
46、一种根据权利要求45的方法，其特征在于，通过洗涤分离出未结合的标记受体。
47、一种根据权利要求45或46的方法，其特征在于，靶配体是一种抗原，受体是该抗原的抗体。
48、一种根据权利要求47的方法，其特征在于，抗体为一种单克隆抗体。
49、一种用于用权利要求21的装置来检测液体样品中的靶配体的测定方法，其特征在于：多孔部件结合有针对靶配体第一受体的抗受体;该方法包括，将第一受体、液体样品和第二标记受体加至多孔部件的上表面;然后分离出未结合的标记受体;检测与结合在第一受体上的配体结合的标记受体。
50、一种根据权利要求49的方法，其特征在于，在加入第一受体之后加入液体样品。
51、一种根据权利要求49的方法，其特征在于，使第一受体和液体样品在加至多孔部件前进行混合。
52、一种根据权利要求50或51的方法，其特征在于，在加入液体样品之后将标记受体加至多孔部件中。
53、一种根据权利要求50或51的方法，其特征在于，在将液体样品加至多孔部件之前将标记受体加至液体样品中。
54、一种根据权利要求49、50或51的方法，其特征在于，抗受体为一种抗体。
55、一种根据权利要求52的方法，其特征在于，抗受体为一种抗体。
56、一种根据权利要求53的方法，其特征在于，抗受体为一种抗体。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1314956C (zh) * 2000-10-18 2007-05-09 克拉里蒂技术公司 用于稀释流体和检测在稀释流体中的分析物的方法和装置
CN101111603B (zh) * 2004-11-24 2012-02-15 技术实验室有限公司 用于检测被分析物的设备和方法

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DK170352B1 (da) 1995-08-07
DK11186A (da) 1986-01-10
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FI91566B (fi) 1994-03-31
ES543068A0 (es) 1986-12-16
JPS61502214A (ja) 1986-10-02
US4632901A (en) 1986-12-30
FI910057A0 (fi) 1991-01-04
DE3586983D1 (de) 1993-02-25
AU6307594A (en) 1994-07-14
JPH0734016B2 (ja) 1995-04-12
EP0180638A4 (en) 1988-09-28
NO168388C (no) 1992-02-12
FI91565B (fi) 1994-03-31
CN85104030A (zh) 1986-11-19
IN163952B (zh) 1988-12-17
US4727019A (en) 1988-02-23
ES8800436A1 (es) 1987-10-16
FI91565C (fi) 1994-07-11
AU601357B2 (en) 1990-09-13
DK170353B1 (da) 1995-08-07
ATE84619T1 (de) 1993-01-15
FI860112A (fi) 1986-01-10
EP0180638A1 (en) 1986-05-14
NZ212049A (en) 1989-01-06
AU6805090A (en) 1991-05-02
ES556866A0 (es) 1987-10-16
DK124994A (da) 1994-10-28
DK11186D0 (da) 1986-01-10
ZA853583B (en) 1986-04-30
FI91566C (fi) 1994-07-11
CA1257542A (en) 1989-07-18
NO168388B (no) 1991-11-04
MX161338A (es) 1990-09-10
NO860057L (no) 1986-03-10
WO1985005451A1 (en) 1985-12-05
EP0180638B1 (en) 1993-01-13
DE3586983T2 (de) 1993-04-29
AU4401785A (en) 1985-12-13
AU673907B2 (en) 1996-11-28
FI860112A0 (fi) 1986-01-10

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