CN1879019B - 检测测试样品中被分析物的存在或数量的流通试验装置和方法 - Google Patents
检测测试样品中被分析物的存在或数量的流通试验装置和方法 Download PDFInfo
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Abstract
提供了一种用于检测存在于测试样品中的被分析物的存在或数量的流通试验装置。所述装置利用了检测区域和补偿区域,在其中固定有俘获试剂。本发明人发现,补偿区域的存在可以容许检测扩展的浓度范围的被分析物。特别地,补偿区域便于探针的结合,不然将在试验装置的内部结合或将展现“自猝灭”。
Description
技术领域
本发明涉及用于流通试验装置和方法。
背景技术
流通(flow-through)试验中通常应用各种分析程序和装置来测定可能存在于测试样品中的被分析物的存在和/或浓度。举例来说,免疫测定利用了免疫系统的机制,其中抗体响应抗原的存在而产生,所述抗原是病原性的或对于该生物体而言是外来的。这些抗体和抗原,即免疫反应物,能相互结合,从而引起高度特异性反应的机制,可被用于测定生物样品中特定抗原的存在或浓度。
已有一些公知的免疫测定方法,使用了以可检测成分标记的免疫反应物,从而可以分析性地检测被分析物。例如,“夹层型”试验一般涉及将测试样品与可检测探针混合,可检测探针例如染色的胶乳或放射性同位素,其与被分析物的特异性结合部件结合。结合的探针与被分析物形成复合物。然后这些复合物到达固定化的抗体的区域,在这里抗体和被分析物之间发生结合形成三元的“夹层复合物”。夹层复合物被定位在用于检测被分析物的区域。这种技术可用于获得定量的或半定量的结果。在Grubb等人的美国专利No.4,168,146,和Tom等人的美国专利No.4,366,241中描述了这种夹层型试验的一些实例。另一种供选择的技术是“竞争型”试验。在“竞争型”试验中,标记一般是经标记的被分析物或被分析物类似物,与样品中存在的任何未标记的被分析物竞争结合抗体。一般应用竞争性试验检测的被分析物,例如半抗原,每个半抗原是单价的并仅能结合一个抗体分子。在Deutsch等人的美国专利No.4,235,601,Liotta的美国专利No.4,442,204和Buechler等人的美国专利No.5,208,535中描述了竞争性免疫测定装置的实例。
尽管这些装置是有益的,但当暴露于相对高的被分析物浓度时,许多常规的横向流动试验显示出显著的不准确。例如,在高的被分析物浓度下依靠光学检测的试验(例如,荧光、反射光、磷光等)常常变得不准确。特别地,探针通常不仅被俘获在膜装置的表面,而且被俘获在试验装置的内部。不幸地是,大多数光学检测技术不能检测俘获在试验装置内部深处的那些探针。此外,相互间太靠近时,荧光探 针有时展现出“自猝灭”现象。自猝灭是公知的现象,当两个或多个荧光材料光化学地相互作用时熄灭相互的荧光。因而,在高的被分析物浓度下,荧光探针可能开始展现出自猝灭,这实际引起荧光强度的下降。这些问题常常限制检测范围,并产生对被分析物的不准确的检测。
针对这些问题已经提出了几种构想。例如,Ching的EP0462376描述了一种试验装置,包括在连续的液流接触中具有至少两个规定的和标示出的检测位点的固相。第一个检测位点是俘获位点,固定有能与被分析物竞争结合共轭物的俘获试剂。第二个检测位点是共轭物回收位点,包括不同于俘获试剂的共轭物回收试剂,用于与通过所述俘获位点的共轭物或其复合物结合。当测试样品中被分析物的数量升高时,共轭物的结合位点被被分析物分子占用的越多,自由结合俘获试剂的共轭物越少。相反地,被分析物/共轭复合物通过俘获位点并迁移到共轭物回收位点。每个位点的标记数量的比较分析表明了测试样品中被分析物的数量。
尽管如此,仍然需要一种以精确、简单且低成本的方式来扩展试验装置的动态检测范围的方法。
发明内容
根据本发明的一个实施方式,公开了一种用于检测存在于测试样品中的被分析物的存在或数量的流通试验装置。所述流通试验装置包括连通检测探针和校准探针的多孔膜,所述检测探针与被分析物的特异性结合部件结合。如果需要,结合的检测探针(conjugated detectionprobes)可以包括选自发色团、催化剂、发光化合物(例如,荧光、磷光等)、放射性化合物、可见标记物、脂质体及其组合的物质。所述特异性结合部件可以选自抗原、半抗原、适体、初级或次级抗体、生物素及其组合。
所述多孔膜规定了检测区域,在其中固定有第一俘获试剂,设置所述第一俘获试剂使其与所述结合的检测探针或其复合物的至少一部分结合,来产生具有一定强度的检测信号。在一个实施方式中,所述第一俘获试剂选自抗原、半抗原、蛋白A或G、中性链亲和素(neutravidin)、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体及其复合物。举例来说,所述第一俘获试剂可以与在所 述被分析物和所述结合的检测探针之间形成的复合物结合。
为了进一步扩展试验装置的动态检测范围,所述多孔膜还规定了位于所述检测区域下游的补偿区域。第二俘获试剂固定化在所述补偿区域内,设置第二俘获试剂以与通过所述检测区域的结合的检测探针或其复合物结合,来产生具有一定强度的补偿信号。在一个实施方式中,所述第二俘获试剂选自抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体及其复合物。在另一个实施方式中,所述第二俘获试剂包含聚电解质。所述聚电解质可以是带正电的、带负电的、两亲性的等等。无论第二俘获试剂选择的材料如何,所述补偿信号的强度与所述检测信号的强度成反比。因此,所述检测信号强度与所述补偿信号强度的比值与被分析物浓度成比例,因而可用于测定测试样品中被分析物的数量。
可以使用自校正技术进一步提高在实际试验条件下检测和补偿信号的准确性。特别地,所述多孔膜还与校准探针连通,并包括校准区域,在其中固定有第三俘获试剂,所述第三俘获试剂被设置为与所述校准探针结合来产生具有一定强度的校准信号。相对于检测和补偿信号的强度,所述校准信号在强度上基本上是恒定的。因而,所述校准信号可被用于校准所述检测和补偿信号。
根据本发明的另一个实施方式,公开了一种用于检测存在于测试样品中的被分析物的存在或数量的方法。所述方法包括:
i)提供一种包括多孔膜的流通试验装置,所述多孔膜与检测探针和校准探针连通,所述检测探针与被分析物的特异性结合部件结合,所述多孔膜规定了检测区域,检测区中固定有第一俘获试剂,补偿区域,补偿区中固定有第二俘获试剂,和校准区域,校准区中固定有第三俘获试剂,其中所述补偿区域位于所述检测区域的下游;
ii)使含被分析物的测试样品与所述结合的检测探针和所述校准探针接触;
iii)测量在所述检测区域产生的检测信号强度,在所述补偿区域产生的补偿信号强度,和在所述校准区域产生的校准信号强度;
iv)比较所述检测信号与所述补偿信号的强度,其中所述补偿信号的强度与所述检测信号的强度成反比;和
v)用所述校准信号的强度校准所述检测信号和所述补偿信号的比 较的强度,其中相对于所述检测信号和所述校准信号的强度,所述校准信号的强度基本上是恒定的。如果需要,所述方法可进一步包括通过标绘由多个预定被分析物浓度的校准信号的强度校准的、所述检测信号强度与所述补偿信号强度的比值,来制备标准曲线。
在下文中更详细的讨论本发明的其他特征和方面。
附图说明
针对本领域的普通技术人员的、本发明的完整、有效的公开,包括其最佳的方式,结合附图在说明书的余下部分更为具体地阐述:
图1是本发明的流通试验装置的一个实施方式的透视图;
图2是抗体与检测探针共价结合的一个实施方式的图解说明;
图3是根据本发明的一个实施方式在被分析物浓度和所述检测和补偿区域的信号强度之间的关系的图解说明;和
图4是用于本发明的夹层试验形式的一个实施方式的机制的图解说明。
在本说明书和附图中重复使用的参考符号是用来代表本发明的同样的或类似的特征或元素。
具体实施方式
定义
如在此使用的,术语“被分析物”泛指有待检测的物质。举例来说,被分析物可包括抗原物质、半抗原、抗体及其组合。被分析物包括但不限于,毒素、有机化合物、蛋白、肽、微生物、氨基酸、核酸、激素、类固醇、维生素、药物(包括那些用于治疗目的施用的,以及那些用于非法目的施用的)、药物中间体或副产物、细菌、病毒颗粒和代谢产物,或对任何上述物质的抗体。一些被分析物的特定实例包括铁蛋白;肌酸酐激酶MB(CK-MB);地高辛;苯妥英;苯巴比妥(phenobarbitol);卡马西平;万古霉素;庆大霉素;二甲基黄嘌呤;丙戊酸;奎尼丁;促黄体生成激素(LH);促卵泡激素(FSH);雌二醇,黄体酮;C-反应蛋白;脂质运载蛋白(lipocalins);IgE抗体;细胞因子;维生素B2微球蛋白;糖化血红蛋白(Gly.Hb);皮质醇;毛地黄毒苷;N-乙酰普鲁卡因胺(NAPA);普鲁卡因胺;对风疹的抗体,例如风疹IgG和风疹IgM;弓形体病的抗体,例如弓形体病IgG(Toxo-IgG)和弓形体病IgM(Toxo-IgM);睾丸激素;水杨酸酯; 醋氨酚;乙型肝炎病毒表面抗原(HBsAg);乙型肝炎核心抗原的抗体,例如抗乙型肝炎核心抗原IgG和IgM(Anti-HBC);人类免疫缺陷病毒1和2(HIV1和2);人类T细胞白血病毒1和2(HTLV);乙型肝炎e抗原(HBeAg);乙型肝炎e抗原的抗体(Anti-HBe);流感病毒;促甲状腺激素(TSH);甲状腺素(T4);总三碘甲腺原氨酸(TotalT3);游离三碘甲腺原氨酸(FreeT3);癌胚抗原(CEA);脂蛋白,胆固醇,和甘油三酯;和甲胎蛋白(AFP)。滥用的药物和受控制的物质包括但不限于,安非他明;甲苯丙胺;巴比妥酸盐,例如异戊巴比妥、司可巴比妥、戊巴比妥、苯巴比妥和巴比妥;苯二氮,例如利眠宁和安定;大麻酯(cannabinoids),例如印度大麻和大麻;古柯碱;芬太尼;LSD;安眠酮;鸦片剂,例如海洛因,吗啡,可待因,氢吗啡酮,氢可酮,美沙酮,氧可酮,氧吗啡酮和鸦片;苯西克定;和丙氧芬。在Everhart等人的美国专利No.6,436,651,Tom等人的美国专利No.4,366,241中描述了其他潜在的被分析物。
如在此使用的,术语“测试样品”泛指被怀疑含有被分析物的材料。举例来说,测试样品可包括直接从来源获得的材料,以及使用一些技术预处理过的材料,例如但不限于,滤过、沉淀、稀释、蒸馏、混合、浓缩、干扰成分的钝化、添加试剂等等。测试样品可以来源于生物来源,例如生理性液体,包括,血液、组织间隙液、唾液、眼睛晶状体液、脑脊液、汗液、尿液、奶、腹腔积液、粘液、滑液、腹膜液、阴道液体、羊水等等。除生理性液体之外,可以使用其他液体样品,例如水、食物产品等等。此外,被怀疑含有被分析物的固体材料也可以被用作测试样品。
详细说明
现在将详细地参考本发明的各种实施方式,以下阐述其中的一个或多个实施例。提供每个实施例是为了说明本发明,而不是限制本发明。实际上,对本领域技术人员显而易见的是,可以对本发明进行各种修改和变化而不背离本发明的范围或精神。举例来说,作为一个实施方式的部分说明和描述的特征,可以用于另一个实施方式来产生更进一步的实施方式。因而,本发明意图覆盖这种修改和变化,归入附随的权利要求及其等同替代的范围之内。
总的说来,本发明是针对一种用于检测存在于测试样品中的被分 析物的存在或数量的流通试验装置。所述装置利用了在其中固定有俘获试剂的检测区域和补偿区域。本发明人发现,补偿区域的存在可以容许在扩展的浓度范围检测被分析物。特别地,来自补偿区域的信号可以补偿损失的信号,所述损失的信号是由过深地包埋在试验装置的内部的那些探针、或展现出自猝灭的那些探针引起的。
举例来说,参考图1更详细地阐述夹层型流通试验装置的一个实施方案,即可根据本发明形成的夹层型流通试验装置20。如所示,装置20含有多孔膜23,其可任选地由刚性材料21支持。一般地,所述多孔膜23可以由任何一种可使测试样品能够在其上通过的材料制成。例如,用于形成多孔膜23的材料可以包括但不限于,天然的、合成的、或经合成性改良的天然材料,例如多糖(例如,纤维素材料如纸和纤维素衍生物,例如醋酸纤维素和硝化纤维);聚醚砜;聚乙烯;尼龙;聚偏二氟乙烯(PVDF);聚酯;聚丙烯;硅质;无机材料,例如钝化的矾土、硅藻土、MgSO4或其他均一地分散在多孔聚合物基质中的无机的细分散的材料聚合物,例如氯乙烯、氯乙烯-丙烯共聚物和氯乙烯-醋酸乙烯共聚物;布,天然的(例如,棉花)和合成的(例如,尼龙或人造纤维);多孔的凝胶,例如硅胶,琼脂糖,葡聚糖和明胶;聚合的薄膜,例如聚丙烯酰胺等等。在一个特定的实施方式中,所述多孔膜23由硝化纤维和/或聚醚砜材料形成。应当理解的是,术语“硝化纤维”是指纤维素的硝酸酯,其可以是单独的硝化纤维,或硝酸和其他酸的混合酯,其他酸例如具有1到7个碳原子的脂肪族羧酸。
装置20也可含有灯芯垫(wicking pad)28。所述灯芯垫28一般地接受已经迁移通过完整多孔膜23的液体。如本领域中公知的,所述灯芯垫23有助于促毛细管作用和液体穿过膜23的流动。
为了开始进行测试样品中被分析物的检测,使用者可以直接将测试样品施加到所述多孔膜23的一部分上,然后其可以在多孔膜23上按图1中箭头“L”说明的方向移动。做为选择,测试样品可以首先施加到样品垫(未显示)上,所述样品垫与所述多孔膜23是液体连通的。可用来形成样品垫的一些适合的材料包括但不限于,硝化纤维、纤维素、多孔的聚乙烯垫和玻璃纤维滤纸。如果需要,所述样品垫也可以含有一种或多种试验预处理试剂,散布地或非散布地附着于其上。
在例举的实施方式中,测试样品从所述样品垫(未显示)移动到 结合垫22上,结合垫与样品垫的一端连通。所述结合垫22由测试样品能够在其上流通的材料形成。例如,在一个实施方式中所述结合垫22由玻璃纤维形成。尽管仅示出一个结合垫22,应当理解的是,在本发明中也可以使用其他结合垫。
为了便于精确的检测测试样品内被分析物的存在或缺乏,将预定数量的检测探针施加到所述装置20的不同位置上。一般地能产生视觉上或通过仪器装置可检测的信号的任何物质可被用作检测探针。各种的适合的物质可包括发色团;催化剂;发光化合物(例如,荧光,磷光等等);放射性化合物;可见标记物,包括胶体金属的(例如,黄金)和非金属的颗粒、染料颗粒、酶或底物,或有机聚合物胶乳颗粒;脂质体或其他含有信号产生物质的泡囊等等。举例来说,在Litman等人的美国专利No.4,275,149中公开了一些适合用作检测探针的酶,根据需要将其完整地引入本文作为参考。酶/底物系统的一个实例是酶为碱性磷酸酶,底物为硝基蓝四唑-5-溴-4-氯-3-吲哚基磷酸盐,或其衍生物或类似物,或底物为4-甲基伞形基(methylumbelliferyl)-磷酸盐。在Jou等人的美国专利No.5,670,381,Tarcha等人的美国专利No.5,252,459中描述了其他适合的检测探针,根据需要将其完整地引入本文作为参考。
在某些实施方式中,所述检测探针可含有产生可检测信号的荧光化合物。所述荧光化合物可以是荧光分子、聚合物、树状聚体、颗粒等等。举例来说,适合的荧光分子的一些实例包括但不限于,荧光素、铕螯合物、藻胆蛋白、罗丹明和它们的衍生物和类似物。
所述检测探针,如以上描述的,可以单独使用或与微粒(有时称为“珠”或“微珠”)一同使用。举例来说,可以使用天然的微粒,例如核、支原体、质粒、质体、哺乳动物细胞(例如,红细胞血影)、单细胞微生物(例如,细菌)、多糖(例如,琼脂糖)等等。进一步的,也可以使用合成的微粒。例如,在一个实施方式中,使用以荧光或有色染料标记的胶乳微粒。尽管在本发明中可以使用任何胶乳微粒,胶乳微粒通常由从聚苯乙烯、丁二烯苯乙烯、苯乙烯丙烯酸乙烯基三聚物(styreneacrylic-vinyl terpolymer)、聚甲基丙烯酸甲酯、聚甲基丙烯酸乙酯、苯乙烯-马来酸酐共聚物、聚乙酸乙烯酯、聚乙烯基吡啶、聚二乙烯基苯、聚对苯二甲酸丁二醇酯、丙烯腈、氯乙烯-丙烯 酸酯等等,或由它们的醛、羧基、氨基、羟基,酰肼衍生物形成。在Jou等人的美国专利No.5,670,381,Tarcha等人的美国专利No.5,252,459中描述了其他适合的微粒,根据需要将其完整地引入本文作为参考。可商购的适合的荧光颗粒的实例包括MolecularProbes,Inc.出售的荧光羧化微球,商品名“FluoSphere”(Red580/605)和“TransfluoSphere”(543/620),以及“Texas Red”和5-和6-羧基四甲基罗丹明,其也是MolecularProbes,Inc.出售的。此外,商业上可获得的适合的有色胶乳微粒的实例包括Bang′s Laboratory,Inc.出售的羧化胶乳珠。
在使用时,颗粒的形状一般可以变化。在一个特定的实施方式中,举例来说,所述颗粒的形状是球形的。然而,应当理解的是,其他形状也包含在本发明的范围内,例如碟形、棒形、盘形、柱形、管形、不规则的形状等等。此外,颗粒的大小也可以变化。举例来说,颗粒的平均大小(例如,直径)可以在约0.1纳米到约1,000微米的范围,在某些实施方式中,从约0.1纳米到约100微米,和在某些实施方式中,从约1纳米到约10微米。举例来说,“微米大小”的颗粒常常是期望的。当使用时,这种“微米大小”的颗粒可具有从约1微米到约1,000微米的平均大小,在某些实施方式中,从约1微米到约100微米,和在某些实施方式中,从约1微米到约10微米。同样地,也可以使用“纳米大小”的颗粒。这种“纳米大小”的颗粒可具有从约0.1到约10纳米的平均大小,在某些实施方式中从约0.1到约5纳米,和在某些实施方式中,从约1到约5纳米。
在有些情况下,期望以某些方式修饰所述检测探针,使得它们能更容易地与被分析物结合。在这种情况下,所述检测探针可以用附着其上的某些特异性结合部件来修饰以形成结合探针。特异性结合部件泛指特异性结合配对的部件,即,两个不同的分子,其中分子之一化学地和/或物理地与第二个分子结合。举例来说,免疫活性特异性结合部件可包括抗原、半抗原、适体、抗体(初级或次级的)、或它们的复合物,包括由重组DNA方法或肽合成法形成的那些。抗体可以是单克隆的或多克隆的抗体、重组蛋白或其混合物或片段,以及抗体和其他特异性结合部件的混合物。这种抗体的制备和它们作为特异性结合部件的适用性的详细情况是本领域技术人员公知的。其他常见的特异性结合配对包括但不限于,生物素和抗生物素蛋白(或其衍生物)、 生物素和链霉抗生物素蛋白、碳水化合物和凝集素、互补的核苷酸序列(包括探针和俘获核酸序列,用于DNA杂交试验来检测目标核酸序列)、互补的肽序列,包括那些由重组方法形成的、效应物和受体分子、激素和激素结合蛋白、酶辅助因子和酶、酶抑制物和酶等等。此外,特异性结合配对可包括作为原始特异性结合部件的类似物的部件。例如,可以使用被分析物的衍生物或片段,即,被分析物类似物,只要它具有与被分析物相同的至少一个表位即可。
所述特异性结合部件一般地可以使用各种公知的技术附着于所述检测探针。举例来说,特异性结合部件共价附着到检测探针(例如,颗粒)上,可以通过使用羧基、氨基、醛、溴乙酰基、碘乙酰基、硫醇、环氧基和其他反应性的或连接功能基团,以及残余的自由基和自由基阳离子来实现,通过它们可以实现蛋白的偶联反应。表面功能基团也可以作为功能化的辅助单体来掺入,因为检测探针的表面可能含有极性基团的相对高的表面浓度。此外,尽管检测探针常常是在合成后功能化的,在某些情况下,例如聚(苯硫酚),微粒能够直接与蛋白共价连接而不需要进一步的修饰。例如,参考图2,说明了用于共价结合含颗粒的检测探针的本发明的一个实施方式。如所示,结合的第一个步骤是使用碳二亚胺活化探针表面的羧基。在第二个步骤中,活化的羧酸基团与抗体的氨基反应形成酰胺键。活化和/或抗体连结可以在缓冲液中发生,例如磷酸盐缓冲盐水(PBS)(例如,pH7.2)或2-(N-吗啉代)乙磺酸(MES)(例如,pH5.3)。如所示,然后可用乙醇胺封闭产生的检测探针,例如,封闭任何余下的活化位点。总的说来,这些过程形成结合的检测探针,其中抗体共价的附着于所述探针上。除了共价键之外,在本发明中也可以使用其他的附着技术,例如物理吸附。
再次参考图1,试验装置20还可以含有检测区域31,其中固定有能与所述结合的检测探针结合的第一俘获试剂。例如,在某些实施方式中,所述第一俘获试剂可以是生物学的俘获试剂。这种生物学的俘获试剂是本领域公知的,可以包括但不限于,抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体(例如,多克隆的、单克隆的等等)及其复合物。在许多情况下,期望这些生物学的俘获试剂能够与所述检测探针上存在 的特异性结合部件(例如,抗体)结合。所述第一俘获试剂充当了被分析物和结合的检测探针之间形成的复合物的固定结合位点。特别地,被分析物,例如抗体、抗原等,一般地具有两个或多个结合位点(例如,表位)。在到达检测区域31时,这些结合位点之一被结合探针的特异性结合部件占据。然而,被分析物的游离的结合位点可以与固定化的俘获试剂结合。在与固定化的俘获试剂结合时,复合的探针形成了新的三元夹层复合物。
检测区域31一般可以提供任何数量的不同检测区域,使得使用者可更好的测定测试样品内特定被分析物的浓度。每个区域可以含有相同的俘获试剂,或可以含有不同的俘获试剂用于俘获多种被分析物。例如,检测区域31可以包括两个或多个不同的检测区(例如,线、点等等)。检测区可以被排列成直线的形式,以基本上垂直于测试样品在试验装置20上流动的方向。同样地,在某些实施方式中,检测区可以被排列成直线的形式,以基本上平行与测试样品在试验装置20上流动的方向。
再次参考图1,多孔膜23还含有位于检测区域31下游的补偿区域35。补偿区域35一般地提供单个不同的区(例如,线、点等),但多个区的情况也包含在本发明的范围内。例如,在例举的实施方式中,使用单个线。补偿区域35可以被排列成直线的形式,以基本上垂直于测试样品在试验装置20上流动的方向。同样地,在某些实施方式中,区域35可以被排列成直线的形式,以基本上平行与测试样品在试验装置20上流动的方向。
不考虑它的配置,第二俘获试剂被固定在补偿区域35内部的膜35上。第二俘获试剂充当任何结合的检测探针和/或被分析物/结合的探针复合物的固定结合位点,所述复合物在检测区域31没有与第一俘获试剂结合。因为期望第二俘获试剂与复合的和未复合的结合的检测探针都结合,第二俘获试剂通常不同于第一俘获试剂。在一个实施方式中,第二俘获试剂是不同于第一俘获试剂的生物学的俘获试剂(例如,抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、初级或次级抗体(例如,多克隆的、单克隆的等等)及其复合物)。例如,第一俘获试剂可以是单克隆抗体(例如,CRP Mab1),而第二俘获试剂可以是抗生物素蛋白(高阳离子的66,000-道尔顿糖蛋 白)、链霉抗生物素蛋白(非糖基化的52,800-道尔顿蛋白)、中性链亲和素(去糖基化的抗生物素蛋白衍生物)和/或CaptAvidin(硝化的抗生物素蛋白衍生物)。在这个实施方式中,第二俘获试剂可以与生物素结合,其是生物素化的或被包含在检测探针上,所述检测探针与不同于第一俘获试剂的单克隆抗体的单克隆抗体(例如,CRP Mab2)结合。
此外,对于补偿区域35的第二俘获试剂,还可利用各种非生物材料。在许多情况中,特别期望这种非生物学的俘获试剂能更好地确保所有余下的结合的检测探针和/或被分析物/结合的探针复合物被固定化在补偿区域35。例如,在某些实施方式中,第二俘获试剂可以包括聚电解质。
聚电解质可以具有净正电荷或负电荷,以及一般是中性的净电荷。例如,具有净正电荷的聚电解质的一些适合的实例包括但不限于,聚赖氨酸(可以从St.Louis,Missouri的Sigma-Aldrich Chemical Co.,Inc.购得),聚乙烯亚胺;表氯醇功能化的多胺和/或聚酰胺型胺类,例如聚(二甲胺-共-表氯醇);聚二烯丙基二甲基-氯化铵;阳离子纤维素衍生物,如嫁接了季铵水溶性单体的纤维素共聚物或纤维素衍生物等等。在一个特定的实施方式中,可以使用含有季铵水溶性单体的纤维素衍生物的SC-230M或H-100(可从National Starch&Chemical,Inc.购得)。此外,具有净负电荷的聚电解质的适合的实例包括但不限于,聚丙烯酸,例如聚(乙烯-共-异丁烯酸,钠盐)等。还应当理解的是,也可以使用其他聚电解质,例如两亲的聚电解质(即,具有极性和非极性部分)。例如,适合的两亲聚电解质的一些实例包括但不限于,聚(苯乙烯-b-N-甲基2-乙烯基碘化吡啶鎓和聚(苯乙烯-b-丙烯酸),两者都可以从Polymer Source,Inc.ofDorval,Canada获得。
尽管一般可以使用任何聚电解质,但在具体应用中选用的聚电解质取决于检测探针、多孔膜等的性质变化。特别地,聚电解质的分布电荷容许其与具有相反电荷的物质结合。因而,例如,具有净正电荷的聚电解质通常更好地预备与带负电的检测探针结合,而具有净负电荷的聚电解质通常更好地预备与带正电的检测探针结合。因而,在这种情况中,在这些分子之间的离子相互作用使所需的结合在补偿区域 35内发生。不过,尽管在补偿区域35中主要利用离子相互作用来实现期望的结合,也已经发现聚电解质可能与具有类似电荷的检测探针结合。
因为聚电解质被设计与检测探针结合,一般期望所述聚电解质基本上非散布性地固定在多孔膜23的表面上。不然,检测探针不会是使用者可容易地检测的。因而,可以这样将聚电解质施加到多孔膜23上,即使得它们基本上不扩散到多孔膜23的基质中。特别地,所述聚电解质一般与存在于多孔膜23的表面的功能基团形成离子健和/或共价键,使得它们保持固定于其上。尽管不是必须的,但聚电解质和多孔膜23之间形成共价键是期望的,以更为永久地将聚电解质固定于其上。
例如,在一个实施方式中,用于形成聚电解质的单体首先形成溶液,然后直接施加到多孔膜23上。可以使用各种溶剂(例如,有机溶剂、水等)来形成溶液。一旦施加了,使用热、电子束放射、自由基聚合等来开始单体的聚合作用。在有些情况下,在单体聚合时,它们与多孔膜23的某些功能基团形成共价键,从而将产生的聚电解质固定在其上。例如,在一个实施方式中,乙烯亚胺单体可以与某些多孔膜(例如,硝化纤维)表面上存在的羧基形成共价键。
在另一个实施方式中,可以在施加到多孔膜23之前形成聚电解质。如果需要,聚电解质可以首先使用有机溶剂、水等形成溶液。此后,将聚电解质溶液直接施加到多孔膜23上,然后干燥。在干燥时,聚电解质可以与多孔膜23的表面上存在的、具有与聚电解质相反电荷的某些功能基团形成离子键。例如,在一个实施方式中,带正电的聚乙烯亚胺可以与某些多孔膜(例如,硝化纤维)表面上存在的带负电羧基形成离子键。
此外,也可以使用各种公知的技术将聚电解质交联到多孔膜23上。例如,在某些实施方式中,表氯醇-功能化的多胺和/或聚酰胺型胺类可以用作可交联的、带正电的聚电解质。在Keim的美国专利No.3,700,623和Keim的3,772,076和Keim的4,537,657中描述了这些材料的实例,根据需要将其完整地引入本文作为参考,据信它们按照KymeneTM商标由Hercules,Inc.,Wilmington,Del.出售。例如,KymeneTM450和2064是表氯醇功能化的多胺和/或聚酰胺型胺类化合 物,含有环氧化物环和季铵基团,在处理时可以与某些类型的多孔膜(例如,硝化纤维)上存在的羧基形成共价键和与多孔膜的聚合物骨架交联。在某些实施方式中,交联温度可以在约50℃到约120℃的范围,交联时间可以在约10到约600秒的范围。
尽管以上已经描述了用于非散布性将聚电解质固定在多孔膜23上的各种技术,应当理解的是本发明中可以使用用于非散布性固定聚电解质化合物的其他技术。实际上,上述的方法仅是可用于本发明中的技术的示例。例如,在某些实施方式中,可以向聚电解质溶液中添加某些成分,所述成分可以基本上抑制这种聚电解质散布到多孔膜23的基质中。
不考虑形成第二俘获试剂的材料,补偿区域35可以改善试验装置20的被分析物检测范围。在图3中图示性说明了这个现象。如所示,当更多的被分析物被俘获在检测区域31上时,在检测区域31的信号强度(“Idet”)开始增加。理想地,测量的检测信号强度对于更高的被分析物浓度将持续线性地升高。然而,光学检测方法(例如,荧光和反射光)不总是能提供这样理想的测量,特别是在相对高的检测探针浓度下。特别地,在某个点上,检测区域31将不能指示检测探针的进一步的积累。结果,在检测区域31的信号将变平甚至降低。例如,如图3所示,当被分析物浓度进一步升高时,在被分析物浓度“Asat”处Idet开始变平。
然而,根据本发明,可以测量补偿区域的信号强度(“Icom”)来解决检测区域31响应更高的被分析物浓度的欠缺。当没有被分析物存在时,Icom将处在其最大强度,因为所有结合的检测探针将与补偿区域35结合。当被分析物浓度升高时,由于检测区域31保留了更多数量的被分析物/结合的探针复合物,Icom同样减少。作为如上所述检测和补偿区域信号强度之间的反比例关系的结果,本发明人发现,通过比较检测和补偿区域的信号强度,可以更有效地在扩展的范围上测量被分析物的浓度。特别地,检测探针的总数是预定的(例如,根据经验的)。因为存在预定数量的检测探针,在补偿区域35俘获的检测探针的数量与在检测区域31的检测探针的数量成反比。因而,即使当大量的检测探针在检测区域31被俘获、并且这种检测探针的这种数量的数值不能被精确地测量时,可以相对精确地测量在补偿区域35的检测探针的数 量。例如,在一个实施方式中,被分析物的数量与Idet对Icom的比值成正比。根据检测和补偿区域落差的强度范围,可以确定被分析物的一般浓度范围。如果需要,可以在已知的被分析物浓度的范围中相对于被分析物浓度标绘Idet与Icom的比值,来产生强度曲线。为了确定未知测试样品中被分析物的数量,可以根据强度曲线将信号比值转换成被分析物浓度。应当注意到,复合的和未复合的结合的检测探针的俘获效率对于任何给定样品一般是相同的。因此,在俘获效率上的变化不被认为显著地干扰样品和样品之间的结果,因为使用的是强度比值(即,Idet/Icom)而不是绝对信号强度。还应当注意到,可以相对于被分析物浓度标绘Idet和Icom之间的替换性的数学关系,来制备标准曲线。例如,在一个实施方式中,可以相对于被分析物浓度标绘Idet/(Idet+Icom)的值来产生强度曲线。
尽管检测区域31和补偿区域35可表明被分析物的存在,在实际测试条件下通常难以精确地测定测试样品内被分析物的相对浓度。因而,试验装置20还可以包括校准区域32。在这个实施方式中,校准区域32在多孔膜23上形成,置于检测区域31和补偿区域35的下游。可选择地,校准区域32也可置于检测区域31和/或补偿区域35的上游。
校准区域32具有第三俘获试剂,能与通过膜23的长度的校准探针结合。校准探针可以由与检测探针相同或不同的材料形成,其可以与如上所述的特异性结合部件结合。一般而言,这样选择校准探针,使得它们不与检测区域31和补偿区域35的第一或第二俘获试剂结合。第三俘获试剂也可以与在检测区域31或补偿区域35使用的俘获试剂相同或不同。例如,在一个实施方式中,所述第三俘获试剂是生物学的俘获试剂,例如抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体或它们的复合物。此外,类似于检测区域31和补偿区域35,校准区域也可以提供按任何方向的多个不同的校准区,使得使用者能更好地测定测试样品内特定被分析物的浓度。
校准区域32可以提高检测的被分析物的精确性。校准区域32也可以消除在不同的时间在不同的条件下需要进行独立的测量校准的不便。在校准区域上校准探针的总数和第三俘获试剂的总数是预定的。因而,俘获的校准探针的数量和产生的校准信号理想地根据试验条件 的变化,例如温度变化,按照与将在检测区域31发生的情况相类似的方式波动。理想地,第三俘获试剂具有与检测区域31的第一俘获试剂类似的降解分布型。校准探针也可以具有与检测探针类似的降解分布型。随着变化的条件检测探针和所述探针的信号波动理想地是相同或相似的。
因而,校准区域32可以用来校准不同的试验条件下检测区域31和补偿区域35的强度。例如,再次参考图3,被分析物的数量与Idet对校准强度(“Ical”)和Icom的乘积的比值(即,Idet/(Ical)(Icom)))成正比。如果需要,可以在已知的被分析物浓度的范围中相对于被分析物浓度标绘标准曲线。为了确定未知测试样品中被分析物的数量,可以根据标准曲线将信号比值转换成被分析物浓度。还应当注意到,可以相对于被分析物浓度标绘替换性的数学关系,来制备标准曲线。
参考图4,现在更详细地描述利用荧光探针检测被分析物的存在的方法的一个实施方式。开始,将含有被分析物A的测试样品施加到样品垫上。测试样品从样品垫按“L”方向转移到结合垫22,在此被分析物A与结合的荧光检测探针41和荧光校准探针43(可以是结合或不结合的)混和。尽管在这个特定实施方式中使用了荧光,应当理解的是其他光学检测技术,例如磷光、反射光等,同样适合于本发明。例如,在一个实施方式中,作为本领域公知的,可以使用反射分光光度计或读取器来检测展现出可见颜色的探针(例如,染色的胶乳微粒)的存在。例如,在Kaylor等人的美国专利申请公开No.2003/0119202中描述了一种适合的反射光读取器,根据需要将其完整地引入本文作为参考。
尽管如此,在图4说明的实施方式中,被分析物A与结合的荧光检测探针41结合形成被分析物/结合探针复合物49。在检测区域31,这些复合物49被第一俘获试剂90俘获。然后任何未复合的结合的荧光检测探针41和/或未结合的被分析物/结合探针复合物49移动到补偿区域35,在此它们与第二俘获试剂结合(未显示)。最后,荧光校准探针43穿过检测区域31和补偿区域35在校准区域32与第三俘获试剂(未显示)结合。
一旦被俘获,可以使用荧光检测来测量在检测区域31、补偿区域35和校准区域32的探针的荧光信号。荧光是在某些荧光化合物中发生 的三阶段过程的结果。在第一阶段,由外部来源提供能量,例如白炽灯或激光,并被荧光化合物吸收,产生激发的电子单线态(sigletstate)。在第二阶段,激发态存在有限的时间,在这期间荧光化合物经历构象变化,也受与它的分子环境的可能的相互作用的影响。在这个时候,激发态的能量部分地消散,产生松弛态,从松弛态发出荧光发射。第三阶段是荧光发射阶段,此时能量被发射,使荧光化合物回到它的基态。发射的能量低于它的激发能量(光或激光),因而有更长的波长。在能量或波长方面的这种偏移或差异容许检测出和从激发能量中分离出发射能量。
荧光检测一般利用波长过滤来从激发光子中分离发射光子,并利用检测器,检测器记录发射光子并产生可记录的输出,通常是电信号或摄影图像。存在着四种公认类型的检测器:荧光分光光度计和微量培养板读取器;荧光显微镜;荧光扫描器;和流动血细胞计数器。用于本发明的适合的荧光检测器是FluoroLog III荧光分光光度计,其由SPEX Industries,Inc.of Edison,NewJersey出售。
如果需要,在本发明中也可以使用被称为“时间分辨荧光检测”的技术。时间分辨荧光检测通过利用某些荧光材料,例如铕(Eu(III))和铽(Tb(III))的镧系螯合物的萤光特性,用来降低来自发射源或来自散射作用(由激发放射的散射引起)的背景信号。在实质上更短的波长激发螯合物后,这种螯合物可以展现出强烈地红移的、窄带的、长寿的发射。一般地,由于在分子中发色团紧挨着镧系元素,该螯合物具有强的紫外吸收。在发色团的光吸收之后,激发能量可以从激发的发色团转移到镧系元素。在这之后是以镧系元素为特征的荧光发射。使用脉冲式激发和时间门控的检测,结合窄带发射滤光器,容许特异性检测仅来自镧系元素螯合物的荧光,排除样品中存在的其他物质的发射,这一般是更短寿命的或具有更短的波长的发射。在Davidson的美国专利No.5,585,279和Hemmila等人的美国专利No.5,637,509中公开了用于测量荧光的其他时间分辨技术,根据需要将其完整地引入本文作为参考。
不考虑用于测量荧光的技术,被分析物的绝对数量可以通过将检测区域31的荧光信号与补偿区域35的荧光信号,任选的与校准区域32的荧光信号进行比较来确定。例如,如上所指出的,被分析物的数 量可以通过Idet/(Ical)(Icom)的比值、并使用预先确定的标准曲线将该比值转换成被分析物浓度来确定。
尽管在上文中已经描述了装置结构的各种实施方式,应当理解的是,本发明的装置一般地可具有任何期望的结构,不必含有如上所述的所有组件。例如,在Lambotte等人的美国专利No.5,395,754;Jou.等的5,670,381;和Malick等的6,194,220中描述了各种其他的装置结构,根据需要将其完整地引入本文作为参考。使用本发明的试验装置,也可以使用各种试验形式来测试被分析物的存在或缺乏。例如,在如上所述的实施方式中,使用了“夹层”形式。Grubb等人的美国专利No.4,168,146和Tom等的4,366,241描述了这种夹层型试验的其他实例,根据需要将其完整地引入本文作为参考。此外,也可以使用其他形式,例如“竞争”形式。在Deutsch等人的美国专利No.4,235,601,和Liotta的4,442,204,和Buechler等的5,208,535中描述了竞争性免疫测定装置的实例,根据需要将其完整地引入本文作为参考。
本发明人已经发现,在试验装置上补偿区域的存在可以允许以简单、有效、且低成本的方式在扩展的浓度范围上检测被分析物。特别地,补偿区域可补偿损失的信号,不然损失的信号将由光学检测技术的限制产生。
参考以下实施例可以更好的理解本发明。
实施例1
按以下方式形成结合的荧光检测探针。用铕螯合物封装羧基胶乳颗粒,具有0.20微米的颗粒大小、0.5%的固形物浓度,当在370纳米的波长激发时展现出615纳米的发射波长的荧光。从Molecular Probes,Inc.获得该颗粒,命名为“Eu-P”。
开始,通过离心,500微升的该颗粒用1毫升的碳酸盐缓冲液洗涤一次,用2-(N-吗啉代)乙磺酸(MES)缓冲液(pH:6.1,20毫摩尔)洗涤两次。洗涤的颗粒重悬浮在250微升MES中。此后,将3毫克碳二亚胺(Polysciences,Inc.)溶于250微升MES中,添加到悬浮颗粒中。容许混合物在振荡器上在室温下反应30分钟。然后活化的颗粒用硼酸盐缓冲液(Polysciences,Inc)洗涤两次,重悬浮在250微升硼酸盐缓冲液中。然后将30微升C-蛋白单克隆抗体(CRP Mab1) (3.4毫克每毫升,来自BiosPacific,Inc.A#5811)添加到颗粒悬浮液中。容许混合物在翻滚振荡器上在室温下反应过夜。在反应期间,将悬浮液超声浴处理两次。然后收集颗粒,在250微升0.1摩尔乙醇胺(PolysciencesInc.)中温和摇动孵化15分钟。用hepes缓冲液(N-[2-羟乙基]哌嗪-N′-(2-乙磺酸)(20毫摩尔,pH:7.2)洗涤两次。洗涤的结合物悬浮在1毫升Hepes缓冲液中,保存在4℃。
实施例2
如实施例1中描述的形成结合的荧光校准探针,只是CRP Mab1被替换为兔抗山羊IgG(来自BiosPacific,Inc.Inc.,Cat#41-RG15)或山羊抗兔IgG(来自BiosPacific,Inc.Inc,Cat#41-GR30)。结合的荧光校准探针分别被命名为“Eu-P41-RG15”和“Eu-P41-GR30”。
实施例3
说明了形成有检测区域和补偿区域的横向流动试验装置的能力。将具有约30厘米长度的硝化纤维多孔膜(来自Millipore,Inc.的HF12002)叠盖到支撑卡上。将GoldlineTM(从British Biocell International获得的聚赖氨酸溶液)锚定(strip)到膜上以形成补偿区域。此外,将C-反应蛋白的单克隆抗体(CRP Mab2)(A#5804,可以从BiosPacific,Inc.获得,浓度为1毫克每毫升)固定到多孔膜上以形成检测区域。然后在37℃的温度下将膜样品干燥1小时。
如下所述制备结合垫。将实施例1的250微升的结合Eu-P CRP的荧光检测颗粒(在Hepes缓冲液中浓度2.5毫克每毫升)与375微升Tween20(2%,从Aldrich获得)和375微升蔗糖水(10%)混和。将混合物进行超声浴20分钟。然后将悬浮液加载到15厘米长的玻璃纤维结合垫(Millipore Co.)上。然后在37℃将玻璃纤维垫干燥2小时。
通过将900微升Tween20(0.5%)加载到15厘米长的玻璃纤维样品垫(Millipore Co.)上制备样品垫,然后将垫在37℃干燥2小时。然后将纤维素灯芯垫(Millipore Co.)、样品垫和结合垫层叠在多孔膜上。然后将层叠的完整卡片切割成4厘米宽的横向流动试验装置。
实施例4
说明了使用横向流动试验装置检测被分析物的存在的能力。特别地,测试了如实施例3描述制备的十一(11)个试验装置。将55微升稀释的人类血液(稀释100倍)掺杂十一(11)种不同的CPR浓度,0、0.2、0.5、1、2、10、40、100、200、500和2000纳克每毫升,施加到独立的样品垫上。容许装置展开30分钟。
测量检测区域和校准区域的荧光。特别地,在完成试验时,使用胶带将每个横向流动装置安装到Fluorolog III荧光分光光度计(从SAInstruments,Inc.获得)的样品夹上。检测和补偿区域各自符合座中的矩形孔,使得激发光束直接照射到区域上,而装置的其他部分相对于激发光束仍被遮蔽。使用时间分辨荧光技术。特别地,使用以下实验参数:(1)激发光束浴装置的表面法线的夹角是70℃;检测方式为正面;隙缝宽度5纳米;(4)扫描数是1;(5)激发波长370纳米;(6)在615纳米收集发射波长;(7)样品窗口是3毫秒(ms);(8)起始延迟是0.04ms;(9)每次闪光时间是50ms;和(10)闪光次数是10。
0、0.2、0.5、1、2、10、40、100、200、500和2000纳克每毫升的CRP浓度在检测区域的强度分别被测定为10.4K、12.4K、14.7K、15.8K、27.0K、61.7K、99.1K、145.8K、190.4K、214.5K、206.0K。0、0.2、0.5、1、2、10、40、100、200、500和2000纳克每毫升的CRP浓度在补偿区域的强度分别被测定为280.9K、216.3K、165.0K、187.5K、170.0K、123.7K、65.4K、56.0K、8.2K、3.9K、2.3K。检测区域的强度起初升高,但是在约200到500纳克每毫升的CRP浓度处变平。补偿区域的强度持续降低,甚至在CRP浓度高达2000纳克每毫升时也是。因此,检测区域的强度与补偿区域的强度的比值将更精确地呈现CRP浓度高于约200纳克每毫升的真实CRP浓度。
实施例5
说明了形成有检测区域、校准区域和补偿区域的横向流动试验装置的能力。将具有约30厘米长度的硝化纤维多孔膜(来自Millipore,Inc.的HF12002)叠盖到支撑卡上。将GoldlineTM(从British BiocellInternational获得的聚赖氨酸溶液)锚定到膜上以形成补偿区域。将C-反应蛋白的单克隆抗体(CRPMab2)(A#5804,可以从BiosPacific,Inc. 获得,浓度为1毫克每毫升)固定到多孔膜上以形成检测区域。此外,将兔抗促乳素抗体抗体(A#5804,可以从BiosPacific,Inc.获得,浓度为1.8毫克每毫升)固定到多孔膜上检测区域和补偿区域之间以形成校准区域。然后在37℃的温度下将膜样品干燥1小时。
将实施例1的250微升Eu-P CRP颗粒(在Hepes缓冲液中浓度2.5毫克每毫升)和实施例2的100微升Eu-P GR30(在Hepes缓冲液中2.5毫克每毫升)颗粒与300微升Tween20(2%,从Aldrich获得)和300微升蔗糖水(10%)混和。Eu-PCRP颗粒被用作检测探针,而EU-P GR30颗粒被用作校准探针。将混合物进行超声浴20分钟。然后将悬浮液加载到15厘米长的玻璃纤维结合垫(Millipore Co.)上。然后在37℃将玻璃纤维垫干燥2小时。
通过将900微升Tween20(0.5%)加载到15厘米长的玻璃纤维样品垫(Millipore Co.)上制备样品垫,然后将垫在37℃干燥2小时。然后将纤维素灯芯垫(Millipore Co.)、样品垫和结合垫层叠在多孔膜上。然后将层叠的完整卡片切割成4厘米宽的横向流动试验装置。
实施例6
说明了使用横向流动试验装置检测被分析物的存在的能力。特别地,测试了如实施例5描述制备的九(9)个试验装置。将50微升的Hepes缓冲液掺杂九(9)种不同的的CRP浓度0、5、20、100、500、1000、2000、5000、和10000纳克每毫升,施加到独立的样品垫上。容许装置展开30分钟。
如实施例4中描述的测量门控的荧光强度,不同的是延迟时间为0.04毫秒。0.5、20、100、500、1000、2000、5000和10000纳克每毫升的CRP浓度在检测区域的强度分别被测定为26.7K、39.0K、47.0K、109K、159K、186K、217K、219K、193k。0、5、20、100、500、1000、2000、5000和10000纳克每毫升的CRP浓度在校准区域的强度分别被测定为96.6K、136K、101K、119K、103K、88.7K、86.8K、88.1K、87.9K。0、5、20、100、500、1000、2000、5000和10000纳克每毫升的CRP浓度在补偿区域的强度分别被测定为123K、146K、93.6K、158K、131K、81.8K、69.3K、54.1K、34.0K。如所表明的,检测区域的强度起初升高,但是在约2000纳克每毫升的CRP浓度处然后变平, 而补偿区域的强度起初保持恒定,之后在约1000纳克每毫升的CRP浓度处开始降低。校准区域的强度保持相对恒定。因此,通过校准区域的强度校准的、检测区域的强度与补偿区域的强度的比值,将更精确地呈现2000纳克每毫升的CRP浓度或更高的真实CRP浓度。
实施例7
说明了形成有检测区域、校准区域和补偿区域的半横向流动试验装置的能力。将具有约30厘米长度的硝化纤维多孔膜(来自Millipore,Inc.的HF12002)叠盖到支撑卡上。将GoldlineTM(从British BiocellInternational获得的聚赖氨酸溶液)锚定到膜上以形成补偿区域。将C-反应蛋白的单克隆抗体(CRP Mab2)(A#5804,可以从BiosPacific,Inc.获得,浓度为1毫克每毫升,每毫升含1毫克海藻糖)固定到多孔膜上以形成检测区域。此外,将兔抗促乳素抗体抗体(A#5804,可以从BiosPacific,Inc.获得,浓度为1.8毫克每毫升)固定到多孔膜上检测区域和补偿区域之间以形成校准区域。然后在37℃的温度下将膜样品干燥1小时。
将80微升与山羊抗兔IgG结合的金颗粒(10纳米颗粒大小,来自Sigma)(“校准探针”)和50微升与CRP Mab1结合的金颗粒(40纳米颗粒大小,来自British Biocell International)(“检测探针”)与280微升的水和200微升的蔗糖水(10%)混和。然后将悬浮液加载到10厘米长的玻璃纤维结合垫(Millipore Co公司)上。然后在37℃将玻璃纤维垫干燥2小时。通过将300微升Tween20(0.5%)和1200微升水加载到10厘米纤维素垫(Millipore Co.)上制备样品垫,然后将垫在37℃干燥2小时。然后将纤维素灯芯垫(Millipore Co.)、样品垫和结合垫层叠在多孔膜上。然后将层叠的完整卡片切割成4厘米宽的横向流动试验装置。
实施例8
说明了使用横向流动试验装置检测被分析物的存在的能力。特别地,测试了如实施例7描述制备的十(10)个试验装置。将60微升的Hepes缓冲液掺杂十(10)种不同的的CRP浓度,0、5、10、20、50、100、200、500、1000和2000纳克每毫升,施加到独立的样品垫上。 容许装置展开30分钟。
使用反射光读取器测量反射光强度。0、5、10、20、50、100、200、500、1000和2000纳克每毫升的CRP浓度在检测区域的反射光强度分别被测定为0、0、0、0.0498、0.0806、0.4433、1.418、2.347、2.407和2.402。0、5、10、20、50、100、200、500、1000和2000纳克每毫升的CRP浓度在校准区域的反射光强度分别被测定为1.072、0.9650、0.9752、1.010、0.9993、0.8954、1.030、1.020、1.035和1.070。0、5、10、20、50、100、200、500、1000和2000纳克每毫升的CRP浓度在补偿区域的反射光强度分别被测定为1.414、1.167、1.345、1.312、1.045、1.241、1.331、0.843、0.6169和0.4608。如所表明的,检测区域的反射光强度起初升高,在约500纳克每毫升的CRP浓度处然后变平,而补偿区域的强度相对恒定,之后在约200纳克每毫升的CRP浓度处开始降低。校准区域的强度保持相对恒定。因此,通过校准区域的强度校准的、检测区域的反射光强度与补偿区域的强度的比值,将更精确地呈现500纳克每毫升的CRP浓度或更高的真实CRP浓度。
虽然已经根据其特定的实施方式详细描述了本发明,本领域技术人员应当理解的是,在通过上述内容了解了本发明之后,可以容易地构想这些实施方式的替换体、变体和等同体。因此,本发明的范围应当按照附随的权利要求和其任何的等同体来判定。
Claims (43)
1.一种用于检测测试样品中的被分析物的存在或数量的方法,所述方法包括:
i)提供一种包括多孔膜的流通试验装置,所述多孔膜与检测探针和校准探针连通,所述检测探针与被分析物的特异性结合部件结合,所述多孔膜规定了检测区域,其中固定有第一俘获试剂;补偿区域,其中固定有第二俘获试剂;和校准区域,其中固定有第三俘获试剂,其中,所述补偿区域位于所述检测区域的下游;
ii)使含被分析物的测试样品与所述结合的检测探针和所述校准探针接触;
iii)测量在所述检测区域产生的检测信号强度,在所述补偿区域产生的补偿信号强度,和在所述校准区域产生的校准信号强度;
iv)比较所述检测信号与所述补偿信号的强度,其中所述补偿信号的强度与所述检测信号的强度成反比;和
v)用所述校准信号的强度校准所述检测信号和所述补偿信号相比较的强度,其中相对于所述检测信号和所述补偿信号的强度,所述校准信号的强度基本上是恒定的。
2.如权利要求1中定义的方法,进一步包括在所述检测区域和所述补偿区域激发所述结合的检测探针,其中所述激发引起所述结合的检测探针发射所述检测信号和所述补偿信号。
3.如权利要求1中定义的方法,进一步包括就多个预定的被分析物浓度通过对经所述校准信号的强度校准的所述检测信号与所述补偿信号的比值进行绘制来形成标准曲线。
4.如前述权利要求1-3中任一项所述的方法,其中所述结合的检测探针包含一种物质,该物质选自发色团、催化剂、发光化合物、放射性化合物、可见标记物、脂质体及其组合。
5.如前述权利要求1-3中任一项所述的方法,其中所述结合的检测探针包含发光化合物。
6.如前述权利要求1-3中任一项所述的方法,其中所述结合的检测探针包含可见标记物。
7.如前述权利要求1-3中任一项所述的方法,其中所述特异性结合部件选自抗原、半抗原、适体、初级或次级抗体、生物素及其组合。
8.如前述权利要求1-3中任一项所述的方法,其中所述第一俘获试剂选自抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体及这些的复合物。
9.如前述权利要求1-3中任一项所述的方法,其中所述第二俘获试剂选自抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体及这些的复合物。
10.如前述权利要求1-3中任一项所述的方法,其中所述第二俘获试剂包含聚电解质。
11.如权利要求10所述的方法,其中所述聚电解质具有净正电荷。
12.如权利要求11所述的方法,其中所述聚电解质选自聚赖氨酸、聚乙烯亚胺、表氯醇、功能化的多胺或聚酰胺型胺类、聚二烯丙基二甲基-氯化铵、阳离子纤维素衍生物及其组合。
13.如权利要求10所述的方法,其中所述聚电解质具有净负电荷。
14.如前述权利要求1-3中任一项所述的方法,其中所述第三俘获试剂包含抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体或这些的复合物。
15.如前述权利要求1-3中任一项所述的方法,其中所述试验装置是夹层型试验装置。
16.一种用于检测测试样品中的被分析物的存在或数量的方法,所述方法包括:
i)提供一种包括多孔膜的流通试验装置,所述多孔膜与光学检测探针和光学校准探针连通,所述光学检测探针与被分析物的特异性结合部件结合,所述多孔膜规定了检测区域,其中固定有第一俘获试剂;补偿区域,其中固定有第二俘获试剂;和校准区域,其中固定有第三俘获试剂,其中,所述补偿区域位于所述检测区域的下游;
ii)使含被分析物的测试样品与所述光学结合的检测探针和所述光学校准探针接触;
iii)光学地测量在所述检测区域产生的检测信号强度,在所述补偿区域产生的补偿信号强度,和在所述校准区域产生的校准信号强度;
iv)比较所述检测信号与所述补偿信号的强度,其中所述补偿信号的强度与所述检测信号的强度成反比;和
v)用所述校准信号的强度校准所述检测信号和所述补偿信号相比较的强度,其中相对于所述检测信号和所述补偿信号的强度,所述校准信号的强度基本上是恒定的。
17.如权利要求16中定义的方法,进一步包括在所述检测区域和所述补偿区域激发所述结合的检测探针,其中所述激发引起所述结合的检测探针发射所述检测信号和所述补偿信号。
18.如权利要求16中定义的方法,进一步包括就多个预定的被分析物浓度通过对经所述校准信号的强度校准的所述检测信号与所述补偿信号的比值进行绘制来形成标准曲线。
19.如前述权利要求16-18中任一项所述的方法,其中所述结合的检测探针包含一种物质,该物质选自发色团、催化剂、发光化合物、放射性化合物、可见标记物、脂质体及其组合。
20.如前述权利要求16-18中任一项所述的方法,其中所述结合的检测探针包含发光化合物。
21.如前述权利要求16-18中任一项所述的方法,其中所述结合的检测探针包含可见标记物。
22.如前述权利要求16-18中任一项所述的方法,其中所述特异性结合部件选自抗原、半抗原、适体、初级或次级抗体、生物素及其组合。
23.如前述权利要求16-18中任一项所述的方法,其中所述第一俘获试剂选自抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体及这些的复合物。
24.如前述权利要求16-18中任一项所述的方法,其中所述第二俘获试剂选自抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体及这些的复合物。
25.如前述权利要求16-18中任一项所述的方法,其中所述第二俘获试剂包含聚电解质。
26.如权利要求25所述的方法,其中所述聚电解质具有净正电荷。
27.如权利要求26所述的方法,其中所述聚电解质选自聚赖氨酸、聚乙烯亚胺、表氯醇、功能化的多胺或聚酰胺型胺类、聚二烯丙基二甲基-氯化铵、阳离子纤维素衍生物及其组合。
28.如权利要求25所述的方法,其中所述聚电解质具有净负电荷。
29.如前述权利要求16-18中任一项所述的方法,其中所述第三俘获试剂包含抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体或这些的复合物。
30.如前述权利要求16-18中任一项所述的方法,其中所述试验装置是夹层型试验装置。
31.一种用于检测测试样品中的被分析物的存在或数量的流通试验装置,所述流通试验装置包括多孔膜,所述多孔膜与检测探针和校准探针连通,所述检测探针与被分析物的特异性结合部件结合,所述多孔膜规定:
检测区域,在其中固定有第一俘获试剂,所述第一俘获试剂被配置以使其与所述结合的检测探针或由结合的探针与被分析物形成的复合物的至少一部分结合,来产生具有一定强度的检测信号;
位于所述检测区域下游的补偿区域,其中在所述补偿区域内固定有第二俘获试剂,所述第二俘获试剂被配置以使其与穿过所述检测区域的所述结合的检测探针或由结合的探针与被分析物形成的复合物结合,来产生具有一定强度的补偿信号,其中所述补偿信号的强度与所述检测信号的强度成反比;和
校准区域,在其中固定有第三俘获试剂,所述第三俘获试剂被配置以使其与所述校准探针结合,来产生校准信号,相对于所述检测信号和所述补偿信号的强度,所述校准信号在强度上是基本上恒定的;
其中测试样品中的被分析物的数量与所述检测信号强度同所述补偿信号强度的比值成正比,由所述校准信号强度校准。
32.如权利要求31所述的流通试验装置,其中所述结合的检测探针包含一种物质,该物质选自发色团、催化剂、发光化合物、放射性化合物、可见标记物、脂质体及其组合。
33.如权利要求31所述的流通试验装置,其中所述结合的检测探针包含发光化合物。
34.如权利要求31所述的流通试验装置,其中所述结合的检测探针包含可见标记物。
35.如权利要求31所述的流通试验装置,其中所述特异性结合部件选自抗原、半抗原、适体、初级或次级抗体、生物素及其组合。
36.如权利要求31所述的流通试验装置,其中所述第一俘获试剂选自抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体及这些的复合物。
37.如权利要求31所述的流通试验装置,其中所述第二俘获试剂选自抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体及这些的复合物。
38.如权利要求31所述的流通试验装置,其中所述第二俘获试剂包含聚电解质。
39.如权利要求38所述的流通试验装置,其中所述聚电解质具有净正电荷。
40.如权利要求39所述的流通试验装置,其中所述聚电解质选自聚赖氨酸、聚乙烯亚胺、表氯醇、功能化的多胺或聚酰胺型胺类、聚二烯丙基二甲基-氯化铵、阳离子纤维素衍生物及其组合。
41.如权利要求38所述的流通试验装置,其中所述聚电解质具有净负电荷。
42.如权利要求31所述的流通试验装置,其中所述第三俘获试剂包含抗原、半抗原、蛋白A或G、中性链亲和素、抗生物素蛋白、链霉抗生物素蛋白、CaptAvidin、初级或次级抗体或这些的复合物。
43.如权利要求31所述的流通试验装置,其中所述试验装置是夹层型试验装置。
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CN1240027A (zh) * | 1996-10-30 | 1999-12-29 | 莫克里诊断公司 | 被校准的分析物化验系统 |
US6509196B1 (en) * | 2000-01-04 | 2003-01-21 | Response Biomedical Corp. | Compensation for non-specific signals in quantitative immunoassays |
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CN104870652A (zh) * | 2012-10-05 | 2015-08-26 | 加州理工学院 | 用于微流体成像和分析的方法和系统 |
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WO2005057214A1 (en) | 2005-06-23 |
US7640083B2 (en) | 2009-12-29 |
KR101066707B1 (ko) | 2011-09-21 |
US20040230352A1 (en) | 2004-11-18 |
EP1685403A1 (en) | 2006-08-02 |
KR20060126964A (ko) | 2006-12-11 |
CN1879019A (zh) | 2006-12-13 |
US20050112780A1 (en) | 2005-05-26 |
US7781172B2 (en) | 2010-08-24 |
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