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Publication numberCN1879019 B
Publication typeGrant
Application numberCN 200480032754
PCT numberPCT/US2004/014000
Publication date17 Aug 2011
Filing date5 May 2004
Priority date21 Nov 2003
Also published asCN1879019A, EP1685403A1, US7640083, US7781172, US20040230352, US20050112780, WO2005057214A1
Publication number200480032754.X, CN 1879019 B, CN 1879019B, CN 200480032754, CN-B-1879019, CN1879019 B, CN1879019B, CN200480032754, CN200480032754.X, PCT/2004/14000, PCT/US/2004/014000, PCT/US/2004/14000, PCT/US/4/014000, PCT/US/4/14000, PCT/US2004/014000, PCT/US2004/14000, PCT/US2004014000, PCT/US200414000, PCT/US4/014000, PCT/US4/14000, PCT/US4014000, PCT/US414000
Inventors宋旭东
Applicant金伯利-克拉克环球有限公司
Export CitationBiBTeX, EndNote, RefMan
External Links: SIPO, Espacenet
Circulation test device and method for assaying presence or quantity of analyte of assay sample
CN 1879019 B
Abstract
A flow-through assay device for detecting the presence or quantity of an analyte residing in a test sample is provided. The device utilizes a detection zone and compensation zone within which are immobilized capture reagents. The present inventor has discovered that the presence of a compensation zone may enable the detection of an analyte over extended concentration ranges. In particular, the compensation zone facilitates the binding of probes that would otherwise bind within the interior of assay device or that would exhibit 'self-quenching'.
Claims(43)  translated from Chinese
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;ϋ)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ν)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。 A method for detecting the presence or amount of the test sample analyte, the method comprising: i) providing a flow test device comprising a porous membrane, said porous membrane and a calibration probe and detection probe needle communicating, the detection of a specific binding member of the probe binding with the analyte, the porous membrane defines a detection area, wherein a first immobilized capture reagent; compensation region, wherein the second capture reagent is immobilized; and Calibration Area wherein the third capture reagent immobilized, wherein the compensation area is located downstream of said detection zone; ϋ) the detection probes and said calibration probes contact the test sample containing the analyte is the binding; iii ) measuring the intensity of the detection signal generated in the detection area, the intensity of compensation signal generated in the compensation region, and the intensity of the calibration signal generated in the calibration area; iv) comparing the intensity of the detection signal and said compensation signal wherein said compensation signal is inversely proportional to the intensity and the intensity of the detection signal; and ν) of the detection signal calibrated by the intensity of the compensation signal and said calibration signal intensity compared, wherein with respect to said detection signal and said compensation signal intensity, the intensity of the calibration signal is substantially constant.
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。 2. As defined in claim 1, further comprising detecting in said excitation region and the compensating region of the binding of the detection probe, wherein the excitation causes said detection probes binding to transmit the detection signal and The compensation signal.
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。 3. As defined in claim 1, further comprising on a plurality of predetermined analyte concentrations were plotted by the ratio of the detection signal and said compensation signal calibrated by the intensity of the calibration signal to form a standard curve .
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。 The method according to any one of claims 1 to 3 4. The preceding claims, wherein the binding of the detection probe comprises a material which is selected from the spontaneous chromophore, catalysts, luminescent compounds, radioactive compounds, visible markers, liposomes and combinations thereof.
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。 The method according to any one of claims 1-3 5. preceding claims, wherein the binding of the detection probe comprising a light emitting compound.
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。 The method according to any one of claims 1-3 6. preceding claims, wherein said detection probes bound to contain visible marker.
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。 7. The method according to any one of 1 to 3 of the preceding claims, wherein the specific binding member is selected from antigens, haptens, aptamers, primary or secondary antibodies, biotin, and combinations thereof.
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。 The method according to any one of the preceding claims 1-3 8., wherein said first capture reagent is selected from antigens, haptens, protein A or G, neutral streptavidin, avidin, chain streptavidin protein CaptAvidin, primary or secondary antibodies and these complexes.
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。 9. The method according to any preceding claim 1-3 claims, wherein said second capture reagent is selected from antigens, haptens, protein A or G, neutral streptavidin, avidin, chain streptavidin protein, CaptAvidin, primary or secondary antibodies and these complexes.
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。 10. The method according to any one of 1 to 3 of the preceding claims, wherein said second capture reagent comprises a polyelectrolyte.
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。 11. The method of claim 10, wherein said polyelectrolyte has a net positive charge.
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。 12. The method of claim 11, wherein the polyelectrolyte is selected from polylysine, polyethylenimine, epichlorohydrin-functionalized polyamines or polyamidoamines, polydiallyldimethyl methyl - ammonium chloride, cationic cellulose derivatives, and combinations thereof.
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。 13. The method of claim 10, wherein said polyelectrolyte has a net negative charge.
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。 14. The method according to any one of the preceding claims 1-3, wherein said third capture reagent comprising an antigen, hapten, protein A or G, neutral streptavidin, avidin, streptavidin, avidin, CaptAvidin, primary or secondary antibodies or their complexes.
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。 15. The method according to any one of claims 1-3 of the preceding claims, wherein the test device is a sandwich-type test device.
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;ϋ)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ν)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。 16. A method for detecting the presence or amount of test sample analyte, the method comprising: i) providing a flow test device comprising a porous membrane, the porous membrane and the optical detector and optical probe To calibrate the probe in communication, the optical detecting probe and the analyte-specific binding of binding member, the porous membrane defines a detection area, wherein a first immobilized capture reagent; compensation region, wherein the second capture reagent is immobilized; and Calibration Area, which is fixed a third capture reagent, wherein the compensation area is located downstream of said detection zone; ϋ) the detection probe is the test sample containing the analyte with said optical coupling of the optical alignment and probe; iii) optically measuring the intensity of the detection signal generated in the detection area, the intensity of compensation signal generated in the compensation region, and the intensity of the calibration signal generated in the calibration area; iv) comparing said detection signal the intensity of the compensation signal, wherein the intensity is inversely proportional to the strength of said compensation signal with said detection signal; and ν) intensity of the detection signal calibrated by the intensity of the calibration signal and the compensation signal is compared, wherein with respect to the intensity signal and the compensation signal of the detector, the intensity of the calibration signal is substantially constant.
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。 17. As defined in claim 16, further comprising detecting in said excitation region and the compensating region of the binding of the detection probe, wherein the excitation causes said detection probes binding to said emitter of said detection signal and The compensation signal.
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。 18. The method of claim 16 to form a standard curve of defined requirements, further comprising on a plurality of predetermined analyte concentrations were plotted by the ratio of the detection signal and said compensation signal calibrated by the intensity of the calibration signal .
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I=IO 16-18 19. The method according to any one of the preceding claims, wherein the binding of the detection probe comprises a material which is selected from the spontaneous chromophore, catalysts, luminescent compounds, radioactive compounds, visible markers, Liposomes and group I = IO
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。 The method according to any one of claims 16-18 20. preceding claims, wherein the binding of the detection probe comprising a light emitting compound.
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。 The method according to any one of claims 16-18 21. preceding claims, wherein said detection probes bound to contain visible marker.
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。 The method according to any one of claims 16-18 22. preceding claims, wherein the specific binding member is selected from antigens, haptens, aptamers, primary or secondary antibodies, biotin, and combinations thereof.
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。 23. The method according to any one of the preceding 16-18 claims, wherein said first capture reagent is selected from antigens, haptens, protein A or G, neutral streptavidin, avidin, chain streptavidin protein, CaptAvidin, primary or secondary antibodies and these complexes.
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。 16-18 24. The method according to any of the preceding claims, wherein said second capture reagent is selected from antigens, haptens, protein A or G, neutral streptavidin, avidin, chain streptavidin protein, CaptAvidin, primary or secondary antibodies and these complexes.
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。 The method according to any one of claims 16-18 25. preceding claims, wherein said second capture reagent comprises a polyelectrolyte.
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。 26. The method of claim 25, wherein said polyelectrolyte has a net positive charge.
27.如权利要求沈所述的方法,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。 27. The method claimed in claim Shen described, wherein the polyelectrolyte is selected from polylysine, polyethyleneimine, epichlorohydrin-functionalized polyamines or polyamidoamines, polydiallyldimethyl methyl - ammonium chloride, cationic cellulose derivatives, and combinations thereof.
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。 28. The method of claim 25, wherein said polyelectrolyte has a net negative charge.
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。 29. The method according to any one of the preceding 16-18 claims, wherein said third capture reagent comprising an antigen, hapten, protein A or G, neutral streptavidin, avidin, streptavidin, avidin, CaptAvidin, primary or secondary antibodies or their complexes.
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。 The method according to any one of the preceding claims 16-18 30., wherein said test means is a sandwich-type test device.
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。 31. A method of detecting in a test sample analyte presence or amount of flow of the test apparatus for the flow test device comprising a porous membrane, the porous membrane with the detection probes and calibration probes communication, the detection probe needle with a specific binding member of the analyte binding, the porous membrane provides: detection region, in which a first capture reagent is fixed, the first capture reagent is configured to detect the binding of the probe so that it or by combining at least a portion of the probe with the analyte binding complex formed, to produce a detection signal having a certain strength; compensation region located downstream of the detection area, wherein the compensation region within a second capture reagent immobilized said second capture reagent is configured so that it will detect the binding of the probe or passes through the detection area bound by the probe bound with the analyte complex formed, to generate a certain intensity of compensating signal, which is inversely proportional to the intensity of the intensity of the compensation signal with said detection signal; and a measurement area, in which there is fixed a third capture reagent, said third capture reagent is configured so as to calibrate the probe with the binding to generate a calibration signal, with respect to the strength of the signal and the compensation signal of the detection, the intensity of the calibration signal is substantially constant; and wherein the number of the test sample analyte with the detection signal intensity with proportional to the ratio of the compensated signal strength, intensity calibration by said calibration signal.
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。 32. A flow test apparatus according to claim 31, wherein said detection probe comprises a binding substance, the substance selected from the spontaneous chromophore, catalysts, luminescent compounds, radioactive compounds, visual marker, liposomes and combinations.
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。 Flow test apparatus 31 according to claim 33., wherein said detection probe comprises a luminescent compound binding.
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。 Flow test apparatus 31 according to claim 34., wherein said detection probes bound to contain visible marker.
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。 Flow test apparatus 31 according to claim 35., wherein the specific binding member is selected from antigens, haptens, aptamers, primary or secondary antibodies, biotin, and combinations thereof.
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。 36. The flow test apparatus according to claim 31, wherein said first capture reagent is selected from antigens, haptens, protein A or G, neutral streptavidin, avidin, streptavidin, avidin , CaptAvidin, primary or secondary antibodies, and complexes of these.
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。 37. The flow test apparatus according to claim 31, wherein said second capture reagent is selected from antigens, haptens, protein A or G, neutral streptavidin, avidin, streptavidin, avidin , CaptAvidin, primary or secondary antibodies, and complexes of these.
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。 Circulation 38. The test apparatus according to claim 31, wherein said second capture reagent comprises a polyelectrolyte.
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。 Flow test apparatus 38 according to claim 39., wherein said polyelectrolyte has a net positive charge.
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。 40. The flow test apparatus according to claim 39, wherein the polyelectrolyte is selected from polylysine, polyethylenimine, epichlorohydrin-functionalized polyamines or polyamidoamines, poly diallyl dimethyl - ammonium chloride, cationic cellulose derivatives, and combinations thereof.
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。 41. The flow test apparatus according to claim 38, wherein said polyelectrolyte has a net negative charge.
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。 42. The flow test apparatus according to claim 31, wherein said third capture reagent comprising an antigen, hapten, protein A or G, neutral streptavidin, avidin, streptavidin, avidin, CaptAvidin, primary or secondary antibodies or their complexes.
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。 43. The flow test apparatus according to claim 31, wherein said test means is a sandwich-type test device.
Description  translated from Chinese

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 Detecting in the test sample analyte presence or amount of flow test device and

方法 Method

[0001] 技术领域 [0001] Technical Field

[0002] 本发日月渉及用干、流诵i式骀装! [0002] The sun and the moon are re- quired dry hair, i-type flow chanting tired loaded! 1禾Π^τ^去。 1 Π ^ τ ^ Wo go.

[0003] 背景技术 [0003] BACKGROUND

[0004] 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。 [0004] The flow (flow-through) experiment generally apply various analytical procedures and means to determine the presence and / or concentration may be present in the test sample analyte. 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。 For instance, immunoassays utilize mechanisms of the immune system, in which the presence of the antigen and the antibody response generated, the antigen is a pathogenic organism or for that is foreign. 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。 These antibodies and antigens, i.e., immunoreactants, capable of binding to each other, thereby causing a highly specific reaction mechanism that can be used to determine the presence or concentration of a particular antigen in a biological sample.

[0005] 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。 [0005] There have been some well-known immunoassay methods, using the immunoreactant labeled with a detectable moiety, thereby analytically detected analyte. 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。 For example, "sandwich-type" test relates generally to the test sample with a detectable probe hybrid, a detectable probe such as dyed latex or a radioisotope, which specific binding member and the analyte binding. 结合的探针与被分析物形成复合物。 Bound probe with the analyte to form a complex. 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”。 These complexes then reach the area of immobilized antibody, where the antibody and the analyte binding occurs between the formed ternary "sandwich complexes." 夹层复合物被定位在用于检测被分析物的区域。 Sandwich composite is positioned in the region for detecting the analyte. 这种技术可用于获得定量的或半定量的结果。 This technique can be used to obtain quantitative or semi-quantitative results. 在GriAb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。 Describes some examples of such sandwich-type test in GriAb et al U.S. Patent No. 4,168,146, and Tom et al in U.S. Patent No. 4,366,241. 另一种供选择的技术是“竞争型”试验。 Another alternative technique is the "competitive-type" test. 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。 In the "competitive" assay, labeled generally labeled analyte or analyte any unlabeled analyte compete analogs, binding with antibody present in the sample. 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。 General application of the competition assay detecting analytes, such as haptens, each hapten is monovalent and binding only one antibody molecule. 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。 Illustrates an example of a competitive immunoassay device in Deutsch et al., U.S. Patent No. 4,235,601, Liotta in U.S. Patent No. 4,442,204 and Buechler et al., In U.S. Patent No. 5,208,535 .

[0006] 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。 [0006] While these devices are useful, but when exposed to relatively high analyte concentration, many conventional lateral flow test showed a significant inaccuracies. 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。 For example, at high analyte concentration tests rely on optical detection (e.g., fluorescence, reflected light, phosphorescence, etc.) are often inaccurate. 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。 In particular, the probes are typically not only be trapped in the surface of the film unit, and is captured inside the test apparatus. 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。 Unfortunately, most of the optical detection technique can not detect those probes deep inside the test apparatus in capture. 此外,相互间太靠近时,荧光探针有时展现出“自猝灭”现象。 Further, too close to each other, a fluorescent probe sometimes exhibit "self-quenching" phenomenon. 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。 Self-quenching is a known phenomenon, extinguish each other when two or more fluorescent fluorescent material interacting photochemically. 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。 Thus, at high analyte concentration, may begin to show by fluorescence quenching probe, which is actually cause a decrease in fluorescence intensity. 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。 These problems often limits the detection range, and produce inaccurate detection of analytes.

[0007] 针对这些问题已经提出了几种构想。 [0007] In response to these problems have been proposed several conception. 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。 For example, Ching of EP0462376 describes a test device comprising a solid phase having at least two predetermined continuous stream contacts and mark the detected sites. 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。 The first detection site is capture site to be immobilized with the competitive binding of the analyte capture reagent conjugate. 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。 The second detection site is conjugate recovery sites, comprising a capture reagent is different from the conjugate recovery reagent, for passing through the capture site or the conjugate complex binds. 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。 When the number of the test analyte in the sample was raised, conjugated binding sites thereof occupied by analyte molecules, the more free binding capture reagent conjugate less. 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。 Conversely, the analyte / conjugate complexes via capture sites and migrate to the conjugate recovery site. 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。 Comparative analysis of the number of markers each locus indicates the number of the test sample analyte.

[0008] 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。 [0008] Nevertheless, a need for a method of accurate, simple and cost-effective way to extend the dynamic detection range of the test apparatus.

[0009] 发明内容 [0009] SUMMARY

[0010] 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。 [0010] In accordance with one embodiment of the present invention, there is disclosed a method for detecting the presence in the test sample analyte presence or amount of flow of the test apparatus. 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。 The flow test device comprising a porous membrane in communication detection probes and calibration probes, said detection probes with the analyte-specific binding member binding. 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。 If desired, the detection probe bound (conjugated detectionprobes) may include a chromophore, catalysts, luminescent compounds (e.g., fluorescence, phosphorescence, etc.) selected from the spontaneous, radioactive compounds, visual marker, liposomes, and combinations of substances. 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。 The specific binding member may be selected from antigens, haptens, aptamers, primary or secondary antibodies, biotin, and combinations thereof.

[0011] 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。 [0011] The porous membrane defines a detection area, in which a first capture reagent is immobilized, disposed at least a portion of said first capture reagent binding it with the bound detection probe or a composite thereof, to produce a predetermined detection signal strength. 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。 In one embodiment, the first capture reagent is selected from antigens, haptens, protein A or G, neutral streptavidin (neutravidin), Avidin, Streptavidin, CaptAvidin, primary or secondary antibodies and their complexes. 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。 For example, the first capture reagent may be combined with the to-be formed between the analyte and detecting binding of the probe complex.

[0012] 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。 [0012] In order to further expand the dynamic detection range of a test apparatus, the porous membrane also defines a detection zone located downstream of the compensation zone. 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。 The second capture reagent immobilized within the compensation region is provided with a second capture reagent binding region of the detection by the detection probe binding or complex thereof, to generate a compensation signal having a certain strength. 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。 In one embodiment, the second capture reagent is selected from antigens, haptens, protein A or G, neutral streptavidin, avidin, streptavidin, avidin, CaptAvidin, primary or secondary antibody Its complex. 在另一个实施方式中, 所述第二俘获试剂包含聚电解质。 In another embodiment, the second capture reagent comprises a polyelectrolyte. 所述聚电解质可以是带正电的、带负电的、两亲性的等等。 The polyelectrolyte may be positively charged, negatively charged, amphipathic and the like. 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。 Whether a second material selected to capture reagent, the intensity is inversely proportional to the intensity of the detection signal with said compensation signal. 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。 Thus, the ratio of the signal intensity with the intensity of the compensation signal and the detection is proportional to the analyte concentration, and thus can be used to determine the amount of test sample analyte.

[0013] 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。 [0013] Self-correction techniques can be used to further improve the accuracy of the test under actual test conditions and compensation signal. 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。 In particular, the porous membrane further communication with calibration probes, and comprises a calibration zone, in which the capture reagent is fixed to the third, the third capture reagent is configured to engage with the calibration probe to generate a certain intensity of calibration signal. 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。 With respect to the signal intensity detection and compensation, on the strength of the calibration signal is substantially constant. 因而,所述校准信号可被用于校准所述检测和补偿信号。 Thus, the calibration signal may be used to calibrate the detection and compensation signal.

[0014] 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。 [0014] According to another embodiment of the present invention, there is disclosed a method for detecting the presence or quantity present in the test sample analyte is. 所述方法包括: The method comprising:

[0015] 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; [0015] i) providing a flow test device comprising a porous membrane, the porous membrane with the detection probes and calibration probes communication, detection probes with specific binding of said analyte binding member, the porous membrane specifies the detection region, the detection region is fixed a first capture reagent, the compensation area, the compensation area immobilized second capture reagent, and a calibration region, the calibration region a third capture reagent is immobilized, wherein said compensating zone located on the downstream of the detection zone;

[0016] 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; [0016] ii) a test sample containing the analyte is contacted with said detection probes and said calibration probes bound;

[0017] 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;[0018] 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 [0017] iii) measuring the intensity of the detection signal generated in the detection area, the intensity of compensation signal generated in the compensation region, and the intensity of the calibration signal generated in the calibration area; [0018] iv) comparing said detection signal the intensity of the compensation signal, wherein the intensity is inversely proportional to the strength of said compensation signal with said detection signal; and

[0019] ν)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。 [0019] ν) by comparing the intensity of the intensity of the calibration signal and the compensation signal detecting said calibration signal, wherein the intensity signal with respect to the intensity of the calibration signal and the detection of the calibration signal is substantially is constant. 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。 If desired, the method may further comprise by plotting the intensity of a plurality of predetermined analyte concentrations calibrated calibration signal, and the signal intensity ratio of the intensity of the compensation signal detection, to prepare a standard curve.

[0020] 在下文中更详细的讨论本发明的其他特征和方面。 [0020] Other features and aspects discussed in the present invention in more detail hereinafter.

[0021] 附图说明 [0021] Brief Description

[0022] 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: [0022] For one of ordinary skill in the art, the present invention is a complete, effective disclosure, including the best way, with reference more particularly set forth in the remaining portions of the specification:

[0023] 图1是本发明的流通试验装置的一个实施方式的透视图; [0023] FIG. 1 is a perspective view of one embodiment of the flow test apparatus of the present invention;

[0024] 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; [0024] FIG. 2 is an antibody and the detection probe is covalently bound to one illustrated embodiment;

[0025] 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 [0025] Figure 3 is an embodiment of the invention illustrated in the relationship between analyte concentration and the detector and the signal strength between the compensation region; and

[0026] 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。 [0026] FIG. 4 is a mechanism used to sandwich the test of the present invention is an embodiment illustrated by FIG.

[0027] 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。 [0027] Reference symbols in the present specification and drawings is repeated using the same or similar features or elements are used to represent the present invention.

[0028] 具体实施方式 [0028] DETAILED DESCRIPTION

[0029] 定义 [0029] definition

[0030] 如在此使用的,术语“被分析物”泛指有待检测的物质。 [0030] As used herein, the term "analyte" refers to a substance to be detected. 举例来说,被分析物可包括抗原物质、半抗原、抗体及其组合。 For example, analytes may include antigenic substances, haptens, antibodies, and combinations thereof. 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。 Analytes include, but are not limited to, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs (including those administered for therapeutic purposes, as well as those administered for illicit purposes), drug intermediaries or byproducts, bacteria, virus particles and metabolites of or antibodies to any of the foregoing. 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(Iipocalins) ;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IgGCToxo-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(carmabinoids),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。 Some specific examples of analytes include ferritin; creatinine kinase MB (CK-MB); digoxin; phenytoin; Phenobarbital (phenobarbitol); carbamazepine; vancomycin; gentamicin; dimethyl xanthine base; valproic acid; quinidine; luteinizing hormone (LH); follicle stimulating hormone (FSH); estradiol, progesterone; C- reactive protein; lipocalin (Iipocalins); IgE antibodies; Cytokines; vitamin B2 microglobulin; glycated hemoglobin (Gly Hb.); cortisol; digoxigenin; N- acetyl procainamide (NAPA); procainamide; antibodies to rubella, such as rubella toxoplasmosis antibodies, such as toxoplasmosis IgGCToxo-IgG) and toxoplasmosis IgM (Toxo-IgM);; IgG and IgM rubella testosterone; salicylate; acetaminophen; hepatitis B virus surface antigen (HBsAg ); hepatitis B core antigen antibodies such as anti-hepatitis B core antigen IgG and IgM (Anti-HBC); human immunodeficiency virus 1 and 2 (HIV1 and 2 ,; human T-cell leukemia virus 1 and 2 (HTLV) ; hepatitis B e antigen (HBeAg); hepatitis B e antigen antibody (Anti-HBe); influenza virus; thyroid stimulating hormone (TSH); thyroxine (T4); total Triiodothyronine (TotalT3) ; free Triiodothyronine (FreeT3); carcinoembryonic antigen (CEA); lipoproteins, cholesterol, and triglycerides;. and alpha-fetoprotein (AFP) drug abuse and controlled substances include, but are not limited to, , amphetamine; methamphetamine; barbiturates, such as amobarbital, secobarbital, pentobarbital, phenobarbital and pentobarbital; benzodiazepines, such as chlordiazepoxide and stability; cannabinoid (carmabinoids), such as hashish and marijuana; cocaine; fentanyl; LSD; methaqualone; opiates, such as heroin, morphine, codeine, hydromorphone, hydrocodone, methadone, oxycodone, oxymorphone and opium; phencyclidine;. and propoxyphene Everhart et al., described in U.S. Patent No. 6,436,651, Tom et al., in U.S. Patent No. 4,366,241 Other potential analytes.

[0031 ] 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。 [0031] As used herein, the term "test sample ,, refers to material suspected of containing the analyte. For example, the test sample may include materials obtained directly from the source, and the use of some technology pretreated material such as, but not limited to, filtration, precipitation, dilution, distillation, mixing, concentration, passivation interference components, addition of reagents, etc. The test sample may be derived from a biological source, such as a physiological fluid, including, blood, interstitial fluid , saliva, eye lens fluid, cerebrospinal fluid, sweat, urine, milk, ascites, mucus, synovial fluid, peritoneal fluid, vaginal fluid, amniotic fluid, etc. In addition to physiological fluids, other liquid samples can be used, e.g., water , food products, etc. Further, the analyte is suspected of containing a solid material can also be used as a test sample.

[0032] 详细说明 [0032] Description

[0033] 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。 [0033] Reference will now be made in detail to various embodiments of the present invention, set forth below one or more embodiments. 提供每个实施例是为了说明本发明,而不是限制本发明。 Each example is provided to illustrate the invention and not to limit the present invention. 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。 In fact, apparent to those skilled in this art that the present invention may be various modifications and changes without departing from the scope or spirit of the invention. 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。 For example, a feature of an embodiment of the portion illustrated and described, can be used on another embodiment to yield a still further embodiment. 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。 Accordingly, the invention is intended to cover such modifications and variations fall within the appended claims and their equivalents range.

[0034] 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。 [0034] Generally, the present invention is directed to a method for detecting the presence in the test sample analyte presence or amount of flow of the test apparatus. 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。 The apparatus utilizes immobilized capture reagent in which the detection region and compensation zone. 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。 The present inventors have found that the presence in the compensation region can be extended to allow the detection of an analyte concentration range. 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。 In particular, signals may compensate for the loss of signal from the compensation region, the loss of signal from those probes too deeply embedded in the interior of the test device, or to show that quenching caused by the probe.

[0035] 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。 [0035] For example, in more detail with reference to Figure 1 describes one embodiment of a sandwich-type flow test device, to a sandwich-type flow test device 20 formed in accordance with the present invention. 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。 As shown, the apparatus 20 includes a porous membrane 23, 21 which may optionally be supported by a rigid material. 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。 Generally, the material of the porous membrane 23 may be a test sample can be adopted in which can be made of any kind. 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。 For example, the material used to form the porous membrane 23 may include, but are not limited to, natural, synthetic, or by synthesis of the modified natural materials, such as polysaccharides (e.g., cellulose materials such as paper and cellulose derivatives such as cellulose acetate hormone and nitrocellulose); polyethersulfone; polyethylene; nylon; poly vinylidene fluoride (PVDF); polyesters; polypropylene; siliceous; inorganic materials such as passivated alumina, diatomaceous earth, MgSO4, or other uniformly dispersed in a porous polymer matrix of finely divided inorganic polymeric materials, such as vinyl chloride, vinyl chloride - vinyl chloride copolymer and propylene - vinyl acetate copolymer; cloth, natural (e.g., cotton) and synthetic (e.g., nylon or rayon); porous gels such as silica gel, agarose, dextran, and gelatin; polymeric films, e.g., polyacrylamide and the like. 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。 In a particular embodiment, the porous membrane 23 is formed from nitrocellulose and / or polyether sulfone materials. 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。 It should be understood that the term "nitrocellulose" refers to cellulose nitrate, which may be nitrocellulose alone, or a mixed ester of nitric acid and other acids, other acids such as aliphatic carboxylic acid having 1-7 carbon atoms, .

[0036] 装置20也可含有灯芯垫(wicking pad) 28。 [0036] means 20 may also contain a wicking pad (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。 The wick pad 28 is generally accepted by the full liquid porous membrane 23 has been migrated. 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。 As is known in the art, and the wick pad 23 helps promote capillary action and fluid flow through the membrane 23.

[0037] 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。 [0037] In order to begin testing, the user can test the sample is applied directly to the test sample analyte to a portion of the porous membrane 23, which can then be on the porous film 23 indicated by the arrow 1 "L" Description direction. 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。 Alternatively, the test sample may first be applied to the sample pad (not shown) on the sample pad and the porous membrane 23 is in fluid communication. 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。 May be used to form some suitable materials include, but are not limited sample pads to, nitrocellulose, cellulose, porous polyethylene pads and glass fiber filter paper. 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。 If desired, the sample pad may also contain one or more test pretreatment reagent, spreading or non-dispersed adhered thereto.

[0038] 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。 [0038] In the embodiment exemplified, the test sample from the sample pad (not shown) to move to the conjugate pad 22, one end of the pad in combination with the sample pad in communication. 所述结合垫22由测试样品能够在其上流通的材料形成。 The bond pad 22 can flow from the test sample material formed thereon. 例如, 在一个实施方式中所述结合垫22由玻璃纤维形成。 For example, in one embodiment, the conjugate pad 22 is formed of glass fibers. 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。 Although only one conjugate pad is shown 22, it should be understood that, in the present invention may also be used other bond pad.

[0039] 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。 [0039] In order to facilitate the accurate detection of the test sample is analyzed for the presence or lack thereof, a predetermined number of the detection probe 20 is applied to a different location on the device. 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。 Generally produce material visually signals or any detectable by instrument means may be used as a detection probe. 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。 Various suitable substances can include chromophores; catalyst; luminescent compounds (e.g., fluorescence, phosphorescence, etc.); radioactive compound; visible markers include colloidal metal (e.g., gold) and particles, dye particles, non-metallic , enzymes or substrates, or organic polymer latex particles; liposomes or other signal-generating substance containing vesicles and so on. 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。 For example, discloses a number of enzymes suitable for use as a detection probe in Litman et al., U.S. Patent No. 4,275,149, as required, incorporated herein in its entirety by reference. 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。 One example of an enzyme / substrate system is the enzyme alkaline phosphatase and the substrate nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate, or derivative or analog, or substrate is 4-methylumbelliferyl (methylumbelliferyl) - phosphate. 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。 Describes other suitable detection probe in Jou et al., U.S. Patent No. 5,670,381, Tarcha et al., U.S. Patent No. 5,252,459, as required, incorporated herein in its entirety by reference.

[0040] 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。 [0040] In certain embodiments, the detection probe that produces a detectable signal can be a fluorescent compound. 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。 The fluorescent compounds may be fluorescent molecules, polymers, dendrimers, particles, and so on. 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。 For example, some examples of suitable fluorescent molecules include but are not limited to, fluorescein, europium chelates, phycobiliprotein, rhodamine and their derivatives and analogs.

[0041] 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。 [0041] The detection probe, as described above, may be used alone or in combination with microparticles (sometimes referred to as "beads" or "microbeads") used together. 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。 For example, microparticles can be natural, such as nuclear, mycoplasma, plasmids, plastids, mammalian cells (e.g., erythrocyte ghosts), unicellular microorganisms (e.g., bacteria), polysaccharides (e.g., agarose), and so on. 进一步的,也可以使用合成的微粒。 Further, synthetic microparticles may also be used. 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。 For example, in one embodiment, latex particles used in a fluorescent or colored dye marker. 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,酰胼衍生物形成。 Although any latex microparticle may be used in the present invention, latex particles are usually made from polystyrene, styrene-butadiene, styrene-acrylic vinyl terpolymer (styreneacrylic-vinyl terpolymer), poly (methyl methacrylate), poly A ethyl acrylate, styrene - maleic anhydride copolymer, polyvinyl acetate, polyvinyl pyridine, poly-divinylbenzene, polyethylene terephthalate, polybutylene terephthalate, acrylonitrile, vinyl chloride - acrylate, etc. or formed from their aldehyde, carboxyl, amino, hydroxy, acyloxy corpus derivatives. 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。 Other suitable microparticles are described in Jou et al., U.S. Patent No. 5,670,381, Tarcha et al., U.S. Patent No. 5,252,459, as required, incorporated herein in its entirety by reference. 可商购的适合的荧光颗粒的实例包括Molecularfrobes,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Molecularfrobesdnc.出售的。 Examples of suitable commercially available fluorescent particles include Molecularfrobes, Inc. Fluorescent carboxylated microspheres sold under the trade names "FluoSphere" (Red580 / 605) and "TransfluoSphere" (543/620), as well as "Texas Red" and 5 - and 6-carboxy-tetramethylrhodamine, which is also Molecularfrobesdnc sale. 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。 Further, examples of suitable commercially available colored latex particles include Bang 's Laboratory, Inc. of carboxylated latex beads sold.

[0042] 在使用时,颗粒的形状一般可以变化。 [0042] In use, the general shape of the particles can vary. 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。 In one particular embodiment, for example, the shape of the particles are spherical. 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。 However, it should be understood that other shapes are also included within the scope of the present invention, e.g., disc, rod-shaped, disc-shaped, cylindrical, tubular, irregular shape and the like. 此外,颗粒的大小也可以变化。 In addition, the size of the particles may also vary. 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。 For example, the average particle size (e.g., diameter) can be in the range from about 0.1 nanometers to about 1,000 microns, in some embodiments, from about 0.1 nanometers to about 100 microns, and in certain some embodiments, from about 1 nanometer to about 10 microns. 举例来说, “微米大小”的颗粒常常是期望的。 For example, "micron-sized" particles are often desired. 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。 When used, such a "micron-sized" particles may have from about 1 micron to about 000 microns average size, in some embodiments, from about 1 micron to about 100 microns, and in some embodiments in from about 1 micron to about 10 microns. 同样地,也可以使用“纳米大小”的颗粒。 Similarly, also can use the "nano-sized" particles. 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。 This "nano-sized" particles may have from about 0.1 to about 10 nm average size, in some embodiments from about 0.1 to about 5 nm, and in some embodiments, from about 1 to about 5 nm.

[0043] 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。 [0043] In some cases, it is desirable to modify the detection probes in some manner so that they can be more easily combined with the analyte. 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。 In this case, the detection probes may be attached to some of the specific binding member which is formed on the binding of the probe to be modified. 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。 Refers to a specific binding member specific binding pair member, i.e., two different molecules, wherein one molecule chemically and / or physically with a second molecule. 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。 For instance, immunoreactive specific binding members may include antigens, haptens, aptamers, those antibodies (primary or secondary), or composites thereof, including by recombinant DNA methods or peptide synthesis is formed. 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。 Antibodies may be monoclonal or polyclonal antibody, a recombinant protein or a fragment or a mixture thereof, and a mixture of an antibody and other specific binding members. 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。 Preparation of such antibodies and their suitability as details of the specific binding member is skilled in the art. 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。 Other common specific binding pairs include, but are not limited to, biotin and avidin (or derivatives thereof), biotin and streptavidin, avidin, carbohydrates and lectins, complementary nucleotide sequences (including probe and capture nucleic acid sequences used in DNA hybridization assays to detect a target nucleic acid sequence), complementary peptide sequences including those formed by recombinant methods, effector and receptor molecules, hormone and hormone binding protein, enzyme cofactors and enzymes , enzymes and enzyme inhibitors, etc. 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。 Further, a specific binding pair member may comprise analogs as the original specific binding member. 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。 For example, a derivative or fragment of the analyte, i.e., analyte analog, so long as it has the same analyte to at least one epitope.

[0044] 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。 [0044] Generally, the specific binding member may be used a variety of well known techniques is attached to the detection probe. 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。 For example, the specific binding member is attached to the detection probes (e.g., particles) covalently, through the use of carboxyl, amino, aldehyde, bromoacetyl, iodoacetyl, thiol, epoxy and other reactive or connecting functional groups, as well as residual free radicals and radical cations is achieved, which can be achieved by coupling reactive protein. 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。 Surface functional groups can be used as an auxiliary functional monomer incorporation, because the surface of the detection probe may contain a relatively high surface concentration of polar groups. 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。 In addition, although detection probes are often functionalized after synthesis, in certain cases, such as poly (thiophenol), the microparticles can be directly connected to the protein covalently without further modification. 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。 For example, with reference to Figure 2, illustrates one embodiment for covalently binding the detection probe containing particles of the present invention. 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。 As shown, the first step is to use a combination of a carbodiimide-activated carboxyl probe surface. 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。 In the second step, the activated carboxylic acid group of the amino-reactive with the antibody to form an amide bond. 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)。 Activation and / or antibody coupling can occur in a buffer, such as phosphate buffered saline (PBS) (e.g., pH7. 2) or 2- (N- morpholino) ethanesulfonic acid (MES) (e.g., 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。 As shown, may then be used to generate detection probes ethanolamine closed, for example, close any remaining activated sites. 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。 In general, the process of forming the bound detection probe, wherein the antibody is covalently attached to the probe. 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。 In addition to a covalent bond, in the present invention also may use other attachment techniques, such as physical adsorption.

[0045] 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。 [0045] Referring again to FIG. 1, the test apparatus 20 may also contain a detection zone 31, which is fixed with a detection probe capable of binding to the binding of the first capture reagent. 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。 For example, in certain embodiments, the first capture reagent may be a biological capture reagent. 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。 Such biological capture reagents are well known in the art, may include but are not limited to, antigens, haptens, protein A or G, neutral streptavidin, avidin, streptavidin, avidin, CaptAvidin, primary or secondary antibody (eg, polyclonal, monoclonal, etc.) and their complexes. 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。 In many cases, the desired specific binding member these biological capture reagent on the detection probe capable of occurring (e.g., an antibody) binding. 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。 The first capture reagent serves as a fixed binding site is formed between the analyte and detecting binding of the probe complex. 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)。 Specifically, analytes, such as antibodies, antigens, etc., typically have two or more binding sites (e.g., epitopes). 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。 In reaching the detection zone 31, one of these binding sites are specific probe binding binding member to occupy. 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。 However, the free binding site of the analyte can bind to the immobilized capture reagent. 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 When combined with the immobilized capture reagent, the probe complex to form a new ternary

夹层复合物。 Sandwich complexes.

[0046] 检测区域31—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。 [0046] 31--like detection region can provide any number of distinct detection regions so that a user can better test measurement within the sample of a specific concentration of the analyte. 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。 Each region may contain the same capture reagents, or may contain different capture reagents for capturing multiple analytes. 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)。 For example, the detection zone 31 may include two or more distinct detection regions (e.g., lines, dots, etc.). 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。 Detection zone can be arranged in the form of a straight line, substantially perpendicular to the test sample 20 in the direction of flow of the test apparatus. 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。 Similarly, in some embodiments, the detection zone can be arranged in the form of a straight line, substantially parallel to the direction of test sample flow 20 in the test apparatus.

[0047] 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。 [0047] Referring again to FIG 1, further comprising a porous membrane 23 is located downstream of the detection zone 31 of the compensation area 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。 Compensation region 35 generally provides a single distinct zones (e.g., lines, dots, etc.), but also a plurality of areas included within the scope of the invention. 例如,在例举的实施方式中,使用单个线。 For example, in the exemplified embodiment, a single line. 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。 Compensation region 35 may be arranged in the form of a straight line, substantially perpendicular to the test sample 20 in the direction of flow of the test apparatus. 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。 Similarly, in some embodiments, region 35 may be arranged in the form of a straight line, substantially parallel to the direction of test sample flow 20 in the test apparatus.

[0048] 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。 [0048] without regard to its configuration, the second capture reagent is immobilized on the compensation area 35 inside the membrane 35. 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。 The second capture reagent serves as the detection of any binding of the probe and / or analyte / binding site of the fixed probe complex, the complex is not bound at the detection zone 31 and the first capture reagent. 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。 Because the desired capture reagent and second detection probes complexed and uncomplexed binding are combined, the second capture reagent is usually different from the first capture reagent. 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)。 In one embodiment, the second capture reagent is different from the first capture reagent biological capture reagent (e.g., antigens, haptens, protein A or G, neutral streptavidin, avidin, streptavidin, avidin, primary or secondary antibody (eg, polyclonal, monoclonal, etc.) and complex). 例如,第一俘获试剂可以是单克隆抗体(例如,CRP Mabl),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)。 For example, the first capture reagent may be a monoclonal antibody (e.g., CRP Mabl), while the second capture reagent may be avidin (a highly cationic 66,000- dalton glycoprotein), Streptavidin (non-glycosylated 52,800- dalton protein), neutral streptavidin (the deglycosylated avidin derivative), and / or CaptAvidin (nitrated avidin derivative). 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。 In this embodiment, the second capture reagent may bind to biotin, which is biotinylated or contained on detection probes, the detection probes and the capture reagent monoclonal different from the first monoclonal antibody antibodies (e.g., CRP Mab2) binding.

[0049] 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。 [0049] In addition, for compensation region 35 of the second capture reagent, but also the use of various non-biological materials. 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。 In many cases, particularly desirable that such a non-biological capture reagent can better ensure that all remaining bound to the detection probes and / or analyte / bound probe complex is immobilized on the compensation area 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。 For example, in certain embodiments, the second capture reagent may include a polyelectrolyte.

[0050] 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。 [0050] polyelectrolytes may have a net positive or negative charge, and is generally a neutral net charge. 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(可以从乂. 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。 For example, some suitable examples have a net positive charge of polyelectrolytes include, but are not limited to, polylysine (available from qe Louis, Missouri the Sigma-Aldrich Chemical Co., Inc. available.), Polyethyleneimine; epichlorohydrin-functionalized polyamines and / or polyamidoamines, such as poly (dimethylamine - co - epichlorohydrin); poly diallyl dimethyl - ammonium chloride; cationic cellulose derivatives, As the water-soluble quaternary ammonium monomer grafted copolymer of cellulose or cellulose derivatives and the like. 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CelQuat SC-230M或H-100 (可从National Starch&Chemical, Inc.购得)。 In a particular embodiment, can be used cellulose derivatives containing a quaternary ammonium water-soluble monomer of CelQuat SC-230M or H-100 (available from National Starch & Chemical, Inc. commercially available). 此外,具有净负电荷的聚电解质的 In addition, having a net negative charge polyelectrolytes

12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。 12 Suitable examples include, but are not limited to, polyacrylic acid, such as poly (ethylene - co - methacrylic acid, sodium salt) and the like. 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)。 It should also be understood that it is also possible to use other polyelectrolyte, such as amphiphilic polyelectrolytes (ie, having polar and non-polar part). 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-Ν-甲基2-乙烯基碘化吡啶鐺和聚(苯乙烯_b-丙烯酸),两者都可以从Polymer Source, Inc. ofDorval, Canada获得。 For example, suitable amphiphilic polyelectrolytes Some examples include, but are not limited to, poly (styrene -b-Ν- methyl iodide, 2-vinyl pyridine pan and poly (styrene _b- acrylic acid), both of which can from Polymer Source, Inc. ofDorval, Canada obtained.

[0051] 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。 [0051] Although any polyelectrolyte may generally be used, but in specific applications, depending on the choice of the polyelectrolyte detection probes, the porous film of the changing nature. 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。 In particular, the charge distribution and polyelectrolyte allows it to combine with oppositely charged species. 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。 Thus, for example, has a net positive charge of the polyelectrolyte is usually better prepare electrical detection probe binding to negatively, and has a net negative charge of the polyelectrolyte is usually better prepare detection probes bind positively charged. 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。 Thus, in this case, the ion interaction between these molecules allows the required binding occurs within the compensation region 35. 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。 However, although the compensation region 35 of the main use ionic interactions to achieve the desired binding, also has been found that polyelectrolytes can bind to the detection probe having a similar charge.

[0052] 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。 [0052] Because the polyelectrolyte is designed in conjunction with the detection probes, the polyelectrolyte is generally desired substantially non disseminated fixed to the surface of the porous film 23. 不然,检测探针不会是使用者可容易地检测的。 Otherwise, the user can detect the probe will not be easily detectable. 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。 Thus, the polyelectrolytes can be applied to the porous membrane 23, even though they have not substantially diffuse into the porous membrane substrate 23 in. 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。 In particular, the presence of polyelectrolyte generally forming ionic bond and / or covalent bonds to the surface of the porous membrane 23 functional groups, such that they remain fixed thereon. 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。 Although not required, but is formed between the polyelectrolyte and the porous membrane 23 covalent bond is desirable, in order to be more permanently fixed to the polyelectrolyte thereon.

[0053] 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。 Monomers [0053] For example, in one embodiment, for forming the polyelectrolyte solution is first formed, then applied directly to the porous membrane 23. 可以使用各种溶剂(例如,有机溶剂、水等)来形成溶液。 May use various solvents (e.g., organic solvents, water, etc.) to form a solution. 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。 Once applied, using heat, electron beam radiation, free radical polymerization of the monomer to begin polymerization. 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。 In some cases, when the monomers are polymerized, they form a covalent bond with the porous film 23 of certain functional groups, thereby fixing the resulting polyelectrolyte thereon. 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。 For example, in one embodiment, an ethyleneimine monomer may be certain porous membrane (e.g., nitrocellulose) present on the surface of the carboxyl groups to form a covalent bond.

[0054] 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。 [0054] In another embodiment, the porous membrane 23 may be formed prior to applying to the polyelectrolyte. 如果需要,聚电解质可以首先使用有机溶剂、水等形成溶液。 If desired, the polyelectrolyte may first using an organic solvent, water and the like to form a solution. 此后,将聚电解质溶液直接施加到多孔膜23 上,然后干燥。 Thereafter, the polyelectrolyte solution was applied directly to the porous membrane 23, and then dried. 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。 When drying, the polyelectrolyte may be on the surface of the porous film 23 is present, having a polyelectrolyte of opposite charge of certain functional groups form ionic bonds. 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。 For example, in one embodiment, positively-charged polyethyleneimine can with certain porous membrane (e.g., nitrocellulose) present on the surface of the negatively charged carboxyl group to form an ionic bond.

[0055] 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。 [0055] It is also possible to use a variety of well-known techniques to the porous cross-linked polyelectrolyte film 23. 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。 For example, in some embodiments, epichlorohydrin - functionalized polyamines and / or polyamidoamines may be used as a crosslinkable, positively-charged polyelectrolyte. 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Kymene™商标由Hercules, Inc.,Wilmington, Del.出售。 Described in U.S. Patent No. 3,700,623 to Keim and 3,772,076 to Keim and 4,537,657 to Keim in the examples of these materials, according to need in its entirety incorporated herein by reference, it is believed that they According to Kymene ™ trademark sold by Hercules, Inc., Wilmington, Del.. 例如,Kymene™450 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。 E.g., Kymene ™ 450 and 2064 are epichlorohydrin-functionalized polyamines and / or polyamidoamines compound, containing epoxide rings and quaternary ammonium groups, in dealing with certain types of porous membranes (e.g., nitrated covalent bond and a porous film of cross-linked polymer backbone occurring fibers) is formed on the carboxy group. 在某些实施方式中,交联温度可以在约50C到约120C的范围,交联时间可以在约10到约600 秒的范围。 In certain embodiments, the crosslinking temperature may range from about 50 C to about 120 C, the crosslinking time may range from about 10 to about 600 seconds.

[0056] 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。 [0056] Although the above has been described for non-spread of the polyelectrolyte in a variety of techniques is fixed on the porous film 23, it should be understood that other techniques can be used in the present invention is a non-stationary polyelectrolyte dispersed compound used. 实际上, 上述的方法仅是可用于本发明中的技术的示例。 In fact, the above method is merely an example that can be used in the present invention technique. 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。 For example, in certain embodiments, some of the ingredients may be added to the polyelectrolyte solution, the component may substantially inhibit such polyelectrolytes dispersed into the porous membrane substrate 23 in.

[0057] 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。 [0057] irrespective of the material forming the second capture reagent, the compensation area 35 may improve the detection range of the analyte test device 20. 在图3中图示性说明了这个现象。 In Figure 3 diagrammatically illustrates this phenomenon. 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。 As shown, when the more analyte is captured in the detection region 31, the signal strength detection area 31 ("Idet") starts to increase. 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。 Ideally, the detection signal strength measurements for higher analyte concentration will continue to rise linearly. 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。 However, optical detection methods (e.g., fluorescence and reflected light) can not always be desirable to provide such measurements, particularly at relatively high concentration of the detection probe. 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。 In particular, at some point, the detection area 31 will not indicate a further accumulation detection probe. 结果,在检测区域31的信号将变平甚至降低。 As a result, a signal in the detection zone 31 will flatten or even reduced. 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。 For example, shown in Figure 3, when the analyte concentration is further increased, the analyte concentration is "Asat" Idet at the beginning to flatten.

[0058] 然而,根据本发明,可以测量补偿区域的信号强度(“I”)来解决检测区域31响应更高的被分析物浓度的欠缺。 [0058] However, according to the present invention, to measure signal intensity ("I ") to resolve compensation region 31 in response to the detection area is higher analyte concentration lacking. 当没有被分析物存在时,1_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。 When no analyte is present, will be at its maximum strength 1_, since all the detection probe will bind to the binding of the compensation area 35. 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,Icoffl同样减少。 When the analyte concentration, since the detection zone 31 retains a greater number of analyte / bound probe complex, Icoffl also reduced. 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。 As a result of an inverse relationship between the detection signal intensity as described above and between the compensation region, the present inventors have found that by comparing the detected signal strength and compensation region can be more effectively measure the concentration of analyte over an extended range. 特别地,检测探针的总数是预定的(例如,根据经验的)。 In particular, the total number of the detection probe is a predetermined (e.g., empirically). 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。 Because there is a predetermined number of detection probes, the number of compensation regions 35 and capture of the detection probe 31 is inversely proportional to the number of detection probe in the detection area. 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。 Thus, even when a large amount of the detection probe is captured at the detection zone 31, and this quantity of this detection probe value can not be accurately measured, it is possible to accurately measure the relative amount in the compensation area 35 of the detection probe . 例如,在一个实施方式中,被分析物的数量与Idrt对1 的比值成正比。 For example, in one embodiment, the analyte is directly proportional to the ratio of the number of objects on a Idrt of. 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。 Detection and compensation according to the intensity range of the gap region, can be analyzed to determine the general concentration range thereof. 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I。 If desired, relative to an analyte concentration plotted Idet and I. In the known range of the analyte concentration . m的比值,来产生强度曲线。 the ratio of m to generate intensity curve. 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。 In order to determine the number of unknown test sample analyte, according to the ratio of the signal intensity curve is converted into an analyte concentration. 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。 It should be noted, composite and uncomplexed capture efficiency of the detection probe bound for any given sample is generally the same. 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/ICOffl)而不是绝对信号强度。 Thus, changes in the capture efficiency is not considered to significantly interfere with the results of the sample and the sample, because the use of the intensity ratio (i.e., Idet / ICOffl) instead of absolute signal strength. 还应当注意到,可以相对于被分析物浓度标绘Idrt和I。 It should also be noted that, with respect to the analyte concentration and plotted Idrt I. . m之间的替换性的数学关系,来制备标准曲线。 alternative mathematical relationship between m, to prepare a standard curve. 例如,在一个实施方式中,可以相对于被分析物浓度标绘Idrt/(Idrt+I。。m)的值来产生强度曲线。 For example, in one embodiment, with respect to the analyte concentration plotted Idrt / (Idrt + I..m) is used to generate intensity curve.

[0059] 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。 [0059] Although the detection zone 31 and the compensation zone 35 may indicate the presence of analyte, under the actual test conditions is often difficult to accurately determine the relative concentration of the test sample analyte substance. 因而,试验装置20还可以包括校准区域32。 Thus, the test apparatus 20 may also include a calibration zone 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。 In this embodiment, the calibration zone 32 is formed on the porous film 23, disposed downstream of the detection zone 31 and the compensation zone 35. 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。 Alternatively, the calibration zone 32 can also be placed upstream of the detection zone 31 and / or the compensation regions 35.

[0060] 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。 [0060] Calibration region 32 having a third capture reagent capable of binding with the calibration probes 23 through the membrane length. 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。 To calibrate the probe and the detection probe may be formed by the same or different materials, which specific binding member as described above may be combined. 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。 Generally speaking, the calibration probes selected such, that they do not bind to the detection zone 31 and a first compensation region 35 or the second capture reagent. 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。 The third capture reagent can also be captured in the detection region with the compensation zone 31 or 35 using the same or different reagents. 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、CaptAvidiru初级或次级抗体或它们的复合物。 For example, in one embodiment, the capture reagent is a third biological capture reagent, e.g., antigens, haptens, protein A or G, neutral streptavidin, avidin, streptavidin, avidin , CaptAvidiru primary or secondary antibodies or their complexes. 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。 In addition, similar to the detection zone 31 and the compensation area 35, the calibration zone may also be provided by any of a plurality of different directions calibration area, so that a user can better determine the concentration of the specific test sample analyte.

[0061] 校准区域32可以提高检测的被分析物的精确性。 [0061] Calibration can improve the accuracy of the detection area 32 of the analyte. 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。 Calibration region 32 can be eliminated at different times under different conditions require independent measurement calibration inconvenience. 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。 Total number and a third capture reagent in the measurement area is a predetermined calibration probe. 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。 Thus, the number and the calibration signal generated by the calibration capture probe desirably according to the change of test conditions, such as temperature changes, in accordance with the case where the detection region 31 of the phase fluctuation occurs in a similar manner. 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。 Ideally, the third capture reagent and detection zone 31 having a first capture reagent similar degradation profile of. 校准探针也可以具有与检测探针类似的降解分布型。 Calibration probe and detection probe may have a similar distribution pattern degradation. 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。 With the signal fluctuations desirably changing conditions of the probe and the detection probe is the same or similar.

[0062] 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。 [0062] Thus, the calibration zone 32 may be used to calibrate the intensity under different experimental conditions the detection zone 31 and the compensation zone 35. 例如,再次参考图3,被分析物的数量与Idet对校准强度(“I。al”)和I。 For example, referring again to FIG. 3, the amount of the analyte and calibration Idet strength ("I.al") and I. . m的乘积的比值(即,W(Ical) (IeoJ))成正比。 the product of the ratio of m (i.e., W (Ical) (IeoJ)) proportional. 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。 If desired, relative to an analyte concentration standard curve plotted in a known range of concentrations of the analyte. 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。 To determine the number of the unknown test sample analyte, a standard curve can be converted into the signal ratio of the analyte concentration. 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。 It should also be noted that, with respect to the analyte concentration plotted alternative mathematical relationships, and to prepare the standard curve.

[0063] 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。 4, the use of a fluorescent probe to detect the presence of analyte the method of an embodiment will now be described in more detail [0063] with reference to FIG. 开始,将含有被分析物A的测试样品施加到样品垫上。 Initially, the test sample containing the analyte was applied to the sample A pad. 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。 Test sample from the sample pad by "L" direction is transferred to the conjugate pad 22, where the analyte A and the fluorescence detection probe bound fluorescent calibration probes 43 and 41 (which may be bound or unbound) mixture. 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。 Despite the use of fluorescence in this particular embodiment, it should be understood that other optical detection techniques, e.g., phosphorescence, reflected light, etc., is also suitable for the present invention. 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。 For example, in one embodiment, as known in the art, and can be read using a reflectance spectrophotometer to detect or show visible color probe (e.g., dyed latex particles) is present. 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。 For example, in Kaylor et al., U.S. Patent Application Publication No. 2003/0119202 describes a suitable reflection optical reader, according to need in its entirety incorporated herein by reference.

[0064] 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。 [0064] Nevertheless, in the embodiment illustrated in FIG. 4, A and analyte bound fluorescence detection probes 41 to form analyte / probe complexes 49 binding. 在检测区域31,这些复合物49被第一俘获试剂90俘获。 In the detection zone 31, the complexes 49 90 captured by the first capture reagent. 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)。 Then any fluorescent detection probe uncomplexed binding 41 and / or unbound analyte / binding probe complex 49 is moved to the compensation area 35, where they are combined with a second capture reagent (not shown). 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。 Finally, the calibration probes 43 through the fluorescence detection zone 31 and the compensation area 35 in the measurement area 32 and a third capture reagent (not shown) in combination.

[0065] 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。 [0065] Once captured, the fluorescence detection can be used to measure the fluorescent signal of the probe in the detection zone 31, the compensation area 35 and measurement area 32. 荧光是在某些荧光化合物中发生的三阶段过程的结果。 Fluorescence is the result of a three-stage process that occurs in certain fluorescent compounds. 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)。 In the first stage, energy is supplied from an external source, such as an incandescent lamp or a laser, a fluorescent compound and is absorbed, resulting in the excitation of electron singlet (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。 In the second stage, the excited state exists for a limited time, a fluorescent compound undergoes a conformational change in this period, but also by the impact of possible interactions with its molecular environment. 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。 At this time, part of the excitation energy states dissipate, produce the relaxed state, emits fluorescence emitted from the relaxed state. 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。 The third stage is the fluorescence emission stage, when energy is transmitted, back to the fluorescent compound to its ground state. 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。 Emitted energy is lower than its excitation energy (light or laser) and therefore have a longer wavelength. 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。 This aspect of energy or wavelength shift or tolerate differences detected are separated from the excitation energy and the transmitted energy.

[0066] 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。 [0066] Usually the use of fluorescence detection wavelength filtering to isolate the emission photons from the excitation photons, and with the detector, the detector emits photons and generate recording a recordable output, usually an electrical signal or a photographic image. 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。 There are four recognized types of detectors: fluorescent spectrophotometer and microplate readers; fluorescence microscopy; fluorescence scanners; and a flow cytometer. 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。 Used in the present invention is suitable fluorescence detector is FluoroLog III fluorescence spectrophotometer, which by SPEX Industries, Inc. Of Edison, NewJersey sale.

[0067] 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。 [0067] If desired, in the present invention can also be used is called "time-resolved fluorescence detection" technology. 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。 Time-resolved fluorescence detected by the use of certain fluorescent materials, such as europium fluorescence characteristics (Eu (III)) and terbium (Tb (III)) of the lanthanide chelate, to reduce scattering from the emission source or from (by excitation radiation scattering) background signal. 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。 After substantially shorter wavelength excitation chelates, which chelates can exhibit strongly red-shifted, narrow-band, long-lived emission. 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。 In general, since the chromophore next lanthanide in the molecule, the chelate possesses a strong ultraviolet absorption. 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。 After the light-absorbing chromophore, the excitation energy can be transferred from the excited chromophore to the lanthanide. 在这之后是以镧系元素为特征的荧光发射。 After this is characterized by lanthanide fluorescence emission. 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。 Using pulsed excitation and time-gated detection, combined with narrow-band emission filters, to allow specific detection only from the lanthanide chelate fluorescence, emission exclusion of other substances present in the sample, which is generally much shorter life or with a shorter emission wavelength. 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。 Disclosed for other time-resolved fluorescence measurement technique in U.S. Patent No. 5,585,279 to Davidson and Hemmila et al., U.S. Patent No. 5,637,509, as required, incorporated herein in its entirety by reference.

[0068] 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。 [0068] does not consider the technique for measuring fluorescence, the absolute amount of the analyte can be detected by the fluorescent signal the fluorescence signal of the fluorescent signal area compensation regions 31 and 35, optionally with the calibration region 32 will be determined by comparison. 例如,如上所指出的,被分析物的数量可以通过Idet/(I。al) (IcJ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。 For example, as noted above, the number of analytes may Idet / (I.al) (IcJ ratio by using standard curve pre-determined ratio is converted into the concentration of an analyte is determined.

[0069] 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。 [0069] Although in the foregoing have been described various embodiments of the device structure, it should be understood that the apparatus of the present invention generally may have any desired structure, need not contain all of the components described above. 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。 For example, in Lambotte et al., U.S. Patent No. 5,395,754 Jou like 5,670,381;. 6,194,220 and Malick, etc. are described in a variety of other devices structures, as required in its entirety incorporated herein by reference. 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。 Test device of the present invention can also be used to test a variety of tests are stored in the form of an analyte or lack thereof. 例如,在如上所述的实施方式中,使用了“夹层”形式。 For example, in the embodiment described above, the use of a "sandwich" form. 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。 Grubb et al., Etc. U.S. Patent No. 4,168,146 and Tom 4,366,241 describes other examples of such sandwich-type test, as required in its entirety incorporated herein by reference. 此外,也可以使用其他形式, 例如“竞争”形式。 Alternatively, you can use other forms, such as "competition" form. 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。 Illustrates an example of a competitive immunoassay device in Deutsch et al., U.S. Patent No. 4,235,601, and Liotta's 4,442,204, 5,208,535 and the like Buechler, as required, in its entirety incorporated herein by reference.

[0070] 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。 [0070] The present inventors have found that, in the presence of the test apparatus compensation region may allow a simple, efficient, and cost-effective way over an extended concentration range of analyte detection. 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。 In particular, to compensate for the loss signal compensation area, or limiting the loss of the signal generated by the optical detection technique.

[0071] 参考以下实施例可以更好的理解本发明。 [0071] reference to the following embodiments of the present invention may be better understood.

[0072] 实施例1 [0072] Example 1

[0073] 按以下方式形成结合的荧光检测探针。 [0073] in the following manner to form a fluorescent detection probe bound. 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。 With europium chelate encapsulated carboxyl latex particles having a particle size of 0.20 microns, 0.5% of solid content, when the excitation wavelength of 370 nm exhibits 615 nm emission wavelengths of fluorescence. 从Molecular Probes, Inc.获得该颗粒,命名为“Eu_P”。 From Molecular Probes, Inc. to obtain the particles named "Eu_P".

[0074] 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(MEQ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 Mabl) (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(Polyscienceslnc.)中温和摇动孵化15分钟。用h印es缓冲液(N-[2-羟乙基]哌嗪-N' -(2-乙磺酸)QO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4C。 [0074] Start by centrifugation, 500 microliters of the pellets was washed with 1 ml of carbonate buffer, once with 2- (N- morpholino) ethanesulfonic acid (MEQ buffer (pH:. 6 1, 20 mmol) was washed twice. The washed particles were resuspended in 250 microliters of MES Thereafter, the 3 mg of carbodiimide (Polysciences, Inc.) was dissolved in 250 microliters of MES added to the suspended particles. permissible The reaction mixture on a shaker at room temperature for 30 minutes. Then the activated particles with borate buffer (Polysciences, Inc) were washed twice, resuspended in 250 microliters of borate buffer. Then 30 microliters of C - protein monoclonal antibody (CRP Mabl) (3. 4 mg per ml, from BiosPacific, Inc. A # 5811) added to the particle suspension mixture was allowed to roll on the oscillator overnight at room temperature during the reaction. The suspension was treated twice ultrasonic bath and then collect the particles, with gentle shaking incubator at 250 [mu] l 0.1 mole of ethanolamine (Polyscienceslnc.) for 15 min. with h printed es buffer (N- [2- hydroxyethyl] piperazine piperazine -N '- (2- ethanesulfonic acid) QO mmol, pH: 7. 2) conjugate was washed twice washed was suspended in 1 ml Hepes buffer and stored at 4 C..

[0075] 实施例2 [0075] Example 2

[0076] 如实施例1中描述的形成结合的荧光校准探针,只是CRP Mabl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. hc,Cat#41-GR30)。 [0076] The formation of a fluorescent probe calibration is described in Example 1 of the binding, only CRP Mabl is replaced by rabbit anti-goat IgG (from BiosPacific, Inc. Inc., Cat # 41-RG15) or goat anti-rabbit IgG (from BiosPacific, Inc. hc, 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”。 Calibration of the fluorescent probe binding were named "Eu-P41-RG15" and "Eu-P41-GR30".

[0077] 实施例3 [0077] Example 3

[0078] 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。 [0078] illustrate the ability of the detection area is formed and compensation region lateral flow test device. 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。 Nitrocellulose porous membrane (from Millipore, Inc. Of HF12002) having a length of about 30 cm overlap to the support card. 将Goldline™(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。 The Goldline ™ (available from British Biocell International polylysine solution) anchor (strip) to the membrane to form a compensation area. 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。 Further, the C- reactive protein monoclonal antibody (CRP Mab2) (A # 5804, available from BiosPacific, Inc. to obtain a concentration of 1 mg per mL) is fixed to the porous membrane to form a detection zone. 然后在37C的温度下将膜样品干燥1小时。 Then at a temperature of 37 C the film sample was dried for 1 hour.

[0079] 如下所述制备结合垫。 [0079] was prepared as follows conjugate pad. 将实施例1的250微升的结合Eu-P CRP的荧光检测颗粒(在H印es缓冲液中浓度2. 5毫克每毫升)与375微升Ίνθθη20(2%,从Aldrich获得)和375微升蔗糖水(10% )混和。 Example 250 [mu] l of Eu-P CRP binding fluorescent detectable particles 1 (concentration in buffer H printed es 2.5 milligrams per milliliter) and 375 microliters Ίνθθη20 (2%, obtained from Aldrich) and 375 micro- l sucrose (10%) mixture. 将混合物进行超声浴20分钟。 The mixture was subjected to an ultrasonic bath for 20 minutes. 然后将悬浮液加载到15厘米长的玻璃纤维结合垫(Millipore Co.)上。 The suspension was then loaded into 15 cm long glass fiber conjugate pad (Millipore Co.) on. 然后在37C将玻璃纤维垫干燥2小时。 Then at 37 C glass fiber mats to dry for two hours.

[0080] 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37C干燥2小时。 [0080] By 900 microliters TWeen20 (0. 5%) is loaded into the preparation of 15 cm long glass fiber sample pad (Millipore Co.) on the sample pad, then the pad at 37 C to dry for two hours. 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。 Then cellulose wick pad (Millipore Co.), the sample pad and conjugate pad laminated on the porous membrane. 然后将层叠的完整卡片切割成4厘米宽的横向流动试验 Then the complete card stacked cut into 4 cm wide lateral flow test

直ο Straight ο

[0081] 实施例4 [0081] Example 4

[0082] 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。 [0082] illustrates the ability of the presence of an analyte using lateral flow test device detection. 特别地,测试了如实施例3描述制备的十一(11)个试验装置。 In particular, the test eleven (11) a test apparatus as described in example 3 prepared. 将55微升稀释的人类血液(稀释100倍)掺杂-j^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 55 microliters of diluted human blood (diluted 100-fold) doped -j ^ a (11) different concentrations of CPR, 0,0. 2,0. 5,1,2,10,40,100,200, 500 and 2,000 nanograms per milliliter, Shi

加到独立的样品垫上。 Added to a separate sample pad. 容许装置展开30分钟。 Allow the device to start 30 minutes.

[0083] 测量检测区域和校准区域的荧光。 [0083] Fluorescence measurement and calibration of the detection zone area. 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SAhstruments,Inc.获得)的样品夹上。 In particular, at the completion of the test, using adhesive tape attached to each lateral flow device Fluorolog III fluorescence spectrophotometer (from SAhstruments, Inc. Obtained) on the sample holder. 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。 Detection and compensation region in line with the seat in the respective rectangular holes, so that the excitation beam is directly irradiated onto the area, while the rest of the plant with respect to the excitation beam still obscured. 使用时间分辨荧光技术。 Using time-resolved fluorescence techniques. 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70C ;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。 In particular, using the following experimental parameters: (1) the angle between the surface normal of the excitation beam bath apparatus is 70 C; detection method is a front; slit width of 5 nm; (4) the number of scan is 1; (5) an excitation wavelength of 370 nm; (6) at an emission wavelength of 615 nanometer collection; (7) the sample window is 3 milliseconds (ms); (8) the initial delay is 0. 04ms; (9) flash each time is 50ms; and (10) number of flashes is 10.

[0084] 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. OK,61. 7Κ、99. IK、145. 8Κ、190. 4Κ、 214. 5Κ、206 0Κ。 [0084] 0,0. 2,0. 5,1,2,10,40,100,200,500 and 2000 nanograms per milliliter of CRP concentration in the detection region, respectively, was determined to be the intensity of 10. 4K, 12. 4K, 14. 7K, 15. 8K, 27. OK, 61. 7Κ, 99. IK, 145. 8Κ, 190. 4Κ, 214. 5Κ, 206 0Κ. 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. 9Κ、216 3Κ、165 OK、187. 5Κ、170 OK、123. 7Κ、65 4Κ、56 OK、 8. 2Κ、3. 9Κ、2. I。 0,0 * 2,0 * 5,1,2,10,40,100,200,500 and CRP concentration of 2000 ng per milliliter was determined in intensity compensation region were 280. 9Κ, 216 3Κ, 165 OK, 187. 5Κ, 170 OK, 123. 7Κ, 65 4Κ, 56 OK, 8. 2Κ, 3. 9Κ, 2. I. 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。 Initially the intensity of the detection area increases, but at about 200-500 nanograms per milliliter of CRP concentration at the flattened. 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。 Intensity compensation region continues to decrease, even in CRP concentrations up to 2000 nanograms per milliliter, too. 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。 Thus, the ratio of the intensity of the intensity of the compensation region of a test zone will be more accurately represent CRP concentration of greater than about 200 nanograms per milliliter of the true concentration of CRP.

[0085] 实施例5 [0085] Example 5

[0086] 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。 [0086] illustrates the formation of a lateral flow test device capable of detection region, the calibration region and the compensation region. 将具有约30厘米长度的硝化纤维多孔膜(来自Millipore Jnc.的HF12002)叠盖到支撑卡上。 Nitrocellulose membrane (from Millipore Jnc. The HF12002) will have a length of about 30 cm overlap to the support card. 将Goldline™(从British BiocellInternational获得的聚赖氨酸溶液)锚定到膜上以形成补偿区域。 The Goldline ™ (available from British BiocellInternational polylysine solution) is anchored to the membrane to form a compensation area. 将C-反应蛋白的单克隆抗体(CRPMab2) (A#5804,可以从BiosI^acific,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。 The C- reactive protein monoclonal antibody (CRPMab2) (A # 5804, available from BiosI ^ acific, Inc. To obtain a concentration of 1 mg per mL) is fixed to the porous membrane to form a detection zone. 此外,将兔抗促乳素抗体抗体(A#5804,可以从Biosl^acific,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。 In addition, the rabbit anti-prolactin antibody antibody (A # 5804, can be obtained from Biosl ^ acific, Inc., A concentration of 1.8 milligrams per milliliter) is fixed to the porous membrane between the detection zone and the compensation area to form a calibration area . 然后在37C的温度下将膜样品干燥1小时。 Then at a temperature of 37 C the film sample was dried for 1 hour.

[0087] 将实施例1的250微升Eu-P CRP颗粒(在!fepes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。 [0087] Example 1 250 l of Eu-P CRP particles (in! Fepes buffer concentration of 2.5 milligrams per milliliter) of Example 2 and 100 microliters of Eu-P GR30 (in Hepes buffer 2 5 milligrams per milliliter) particles with 300 microliters Tween20 (2%, obtained), and 300 l of sucrose (10%) mixed from Aldrich. Eu-PCRP颗粒被用作检测探针,而EU-P GR30颗粒被用作校准探针。 Eu-PCRP particles are used as the detection probe, and EU-P GR30 particles are used as a calibration probe. 将混合物进行超声浴20分钟。 The mixture was subjected to an ultrasonic bath for 20 minutes. 然后将悬浮液加载到15厘米长的玻璃纤维结合垫(Millipore Co.)上。 The suspension was then loaded into 15 cm long glass fiber conjugate pad (Millipore Co.) on. 然后在37C将玻璃纤维垫干燥2小时。 Then at 37 C glass fiber mats to dry for two hours.

[0088] 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37C干燥2小时。 [0088] By 900 microliters TWeen20 (0. 5%) is loaded into the preparation of 15 cm long glass fiber sample pad (Millipore Co.) on the sample pad, then the pad at 37 C to dry for two hours. 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。 Then cellulose wick pad (Millipore Co.), the sample pad and conjugate pad laminated on the porous membrane. 然后将层叠的完整卡片切割成4厘米宽的横向流动试验 Then the complete card stacked cut into 4 cm wide lateral flow test

直ο Straight ο

[0089] 实施例6 [0089] Example 6

[0090] 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。 [0090] illustrates the ability of the presence of an analyte using lateral flow test device detection. 特别地,测试了如实施例5描述制备的九(9)个试验装置。 In particular, tested as nine (9) test device prepared as described in Example 5. 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。 Fifty microliters of Hepes buffer doped nine (9) different concentrations of CRP 0,5,20,100,500,1000,2000,5000, and 10,000 nanograms per milliliter, the sample is applied to a separate pad.

容许装置展开30分钟。 Allow the device to start 30 minutes.

[0091] 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。 [0091] The measurement gated fluorescence intensity as described in Example 4, except that the delay time of 0.04 milliseconds. 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. 7Κ、39 0Κ,47. OK、109K、159K、186K、217K、219K、193k。 0.5, 20,100,500,1000,2000,5000 and 10,000 nanograms per milliliter of CRP concentration in the detection region, respectively, was determined to be the intensity of 26. 7Κ, 39 0Κ, 47. OK, 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、 119Κ、103Κ、88. 7Κ、86 8Κ、88 IK,87. 9K。 0,5,20,100,500,1000,2000, 5000 and 10 000 nanograms per milliliter of CRP concentration in the calibration of the intensity of each area was determined to be 96. 6K, 136K, 101K, 119Κ, 103Κ, 88. 7Κ, 86 8Κ, 88 IK, 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. 6Κ、158Κ、131Κ、81. 8Κ、 69. 3Κ.54. 1Κ、34. 0Κ。 0,5,20,100,500,1000,2000,5000 and 10,000 nanograms per milliliter of CRP concentration in the compensation area of strength were measured as 123K, 146K, 93. 6Κ, 158Κ, 131Κ, 81. 8Κ, 69 . 3Κ.54. 1Κ, 34. 0Κ. 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。 As indicated, the intensity of the first detection area increases, but at a concentration of approximately 2000 nanograms per milliliter of CRP and then flattened, but the intensity compensation region is initially kept constant, after concentration at about 1000 nanograms per milliliter of CRP in began to decrease. 校准区域的强度保持相对恒定。 Intensity calibration area remains relatively constant. 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。 Therefore, by calibrating the intensity calibration area, the ratio of the intensity of the intensity compensation region detection area, more accurately represent the concentration 2000 ng CRP or CRP concentration higher real per milliliter.

[0092] 实施例7 [0092] Example 7

[0093] 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。 [0093] illustrates the ability to form a semi-lateral flow test device detection area, calibration and compensation of regional areas. 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。 Nitrocellulose porous membrane (from Millipore, Inc. Of HF12002) having a length of about 30 cm overlap to the support card. 将Goldline™(从British BiocellInternational获得的聚赖氨酸溶液)锚定到膜上以形成补偿区域。 The Goldline ™ (available from British BiocellInternational polylysine solution) is anchored to the membrane to form a compensation area. 将C-反应蛋白的单克隆抗体(CRP Mab2) (A#5804,可以从BiosI^acif ic, 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。 The C- reactive protein monoclonal antibody (CRP Mab2) (A # 5804, available from BiosI ^ acif ic, Inc. to obtain a concentration of 1 milligram per milliliter, containing 1 mg per ml trehalose) is fixed to the porous membrane to the formation of the detection area. 此外,将兔抗促乳素抗体抗体(A#5804,可以从BiosI^acificJnc.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。 In addition, the rabbit anti-prolactin antibody antibody (A # 5804, available from BiosI ^ acificJnc. To obtain a concentration of 1.8 milligrams per milliliter) is fixed to the porous membrane between the detection zone and the compensation area to form a calibration region. 然后在37C的温度下将膜样品干燥1小时。 Then at a temperature of 37 C the film sample was dried for 1 hour.

[0094] 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 Mabl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。 [0094] 80 microliters of goat anti-rabbit IgG conjugated gold particles (10 nm particle size, from Sigma) ("Calibration probe"), and 50 microliters of CRP Mabl conjugated gold particles (40 nm particle size, from British Biocell International) ("detection probe") with 280 microliters of water and 200 microliters of sucrose in water (10%) mixture. 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。 The suspension was then loaded into 10 cm long glass fiber conjugate pad (Millipore Co Co.). 然后在37C将玻璃纤维垫干燥2小时。 Then at 37 C glass fiber mats to dry for two hours. 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37C干燥2小时。 By loading 300 l of Tween20 (0.5%) and 1200 l of water to 10 cm cellulose pad (Millipore Co.) sample was prepared on the pad, the pad was then dried at 37 C for 2 hours. 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。 Then cellulose wick pad (Millipore Co.), the sample pad and conjugate pad laminated on the porous membrane. 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。 Then the complete card stacked cut into 4 cm wide lateral flow test device.

[0095] 实施例8 [0095] Example 8

[0096] 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。 [0096] illustrates the ability of the presence of an analyte using lateral flow test device detection. 特别地,测试了如实施例7描述制备的十(10)个试验装置。 In particular, the test ten (10) test apparatus as described in Example 7 prepared. 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 The 60 microliters of Hepes buffer doped with different concentrations of CRP ten (10) species, 0,5,10,20,50,100,200,500,1000 and 2000 nanograms per milliliter, the sample is applied to a separate pad

上。 On. 容许装置展开30分钟。 Allow the device to start 30 minutes.

[0097] 使用反射光读取器测量反射光强度。 [0097] using a reflective optical reader measure the reflected light intensity. 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、 0,5,10,20,50,100,200,500,1000 and 2000 nanograms per milliliter of CRP concentration in the detection region of the reflected light intensity were measured as 0,0,0,0. 0498,0. 0806 , 0. 4433,1. 418,2. 347,2. 407 and 2. 402.0,5,10,20,50,100,200,500,1000 and 2,000 nanograms per milliliter of CRP concentration in the calibration area The reflected light intensity were measured as 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、 0. 9993,0. 8954,1. 030,1. 020,1. 035 and 1. 070.0,5,10,20,50,100,200,500,1000 and 2,000 nanograms per milliliter of CRP concentration In reflected light intensity compensation region are respectively determined to be 1. 414,1. 167,1. 345,1. 312,

1. 045、1. 241,1. 331,0. 843,0. 6169和0. 4608。 1. 045,1. 241,1. 331,0. 843,0. 6169 and 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。 As indicated, the intensity of the reflected light detection region initially increases from about 500 nanograms per milliliter at the CRP concentration is then flattened, while the strength of the compensation region is relatively constant, then at about 200 nanograms per milliliter of CRP concentration in began to decrease. 校准区域的强度保持相对恒定。 Intensity calibration area remains relatively constant. 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。 Thus, the ratio of the intensity of the reflected light intensity compensation region detection area, more accurately represent 500 ng CRP concentration or higher CRP concentration per milliliter of real intensity calibration by the calibration region.

[0098] 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。 [0098] While the present invention has been described in detail according to a particular embodiment thereof, the skilled artisan will appreciate that, in the above description, after understanding the present invention, can be easily replaced Conception of these embodiments, variants and equivalents. 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。 Accordingly, the scope of the invention should be according to the appended claims and any equivalents to determine.

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Referenced by
Citing PatentFiling datePublication dateApplicantTitle
CN104870652A *4 Oct 201326 Aug 2015加州理工学院Methods and systems for microfluidics imaging and analysis
Classifications
International ClassificationG01N33/543, B64D45/00, G07C5/08, G06F17/00
Cooperative ClassificationG07C5/0891, B64D2045/0035, G07C5/0883, G01N33/54393, B64D45/0015, G08B13/19663, G07C5/085, G08B13/1965, G08B13/19656, G08B13/19669, G08B13/19641, G01N33/54366, G08B13/19634
European ClassificationG01N33/543M, G01N33/543K, B64D45/00H, G08B13/196P, G08B13/196S2, G08B13/196L3A, G07C5/08R4B, G07C5/08R2, G08B13/196E, G07C5/08R4C, G08B13/196N1, G08B13/196L1
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