CN1690711B - Immune chromatographic test paper bar based on up conversion luminescence technology - Google Patents

Immune chromatographic test paper bar based on up conversion luminescence technology Download PDF

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CN1690711B
CN1690711B CN 200410034104 CN200410034104A CN1690711B CN 1690711 B CN1690711 B CN 1690711B CN 200410034104 CN200410034104 CN 200410034104 CN 200410034104 A CN200410034104 A CN 200410034104A CN 1690711 B CN1690711 B CN 1690711B
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ucp
pad
ml
sample
strip
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CN 200410034104
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CN1690711A (en
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周蕾
杨瑞馥
王津
郭兆彪
郑岩
侯秀洁
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中国人民解放军军事医学科学院微生物流行病研究所
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Abstract

The invention discloses an immune chromatography test paper based on luminous technique with upside conversion. Use UCP as biological mark, show the result with format of visible light signal on the exposure of infrared light, interpret the result by device, then realize the quantitative detection to object. The structure main comprises sample pad, combination pad or conjugate pad, analysis membrane, sopping pad and plastic backboard. The invention also discloses the preparation and application of the test paper in biological sample quantitative detection. According to the difference of immunological reaction ways of material, classify the test paper to sandwich mode, competition mode and indirect mode; all modes can take qualitative and quantitative detection to different material quicklyand acutely.

Description

基于上转换发光技术免疫层析试纸条 Converting luminescence technique based on the immunochromatographic strip

技术领域 FIELD

[0001] 本发明涉及一种免疫层析试纸条,特别是涉及采用上转换发光材料即UCP作为生物标记物的免疫层析试纸条。 [0001] The present invention relates to an immunochromatographic strip, particularly to the use of luminescent material i.e. conversion UCP immunochromatographic strip as biomarkers. 该种试纸条的结果在红外光的照射下以可见光光信号的形式表现出来,并可使用上换发光生物传感器进行判读,从而实现对目标被检物快速、灵敏的定性定量检测。 The results of this kind of strip under irradiation of infrared light shown in the form of visible optical signals out of, and may be used for the interpretation transducer luminescence biosensor, thereby achieving the qualitative and quantitative detection of the target analyte fast and sensitive.

背景技术 Background technique

[0002] 上转换发光材料(Up-Converting Phosphor, UCP)是一种可对能量进行上转的稀土金属合成物,即UCP可吸收低能量的(长波长)红外光,但却发射高能量的(短波长)可见光。 [0002] converting luminescent materials (Up-Converting Phosphor, UCP) on a rare earth metals may be energy transfer on the composition, i.e., can absorb low energy UCP (long wavelength) infrared light, but emitted high energy (short-wavelength) visible light. UCP是由几种稀土金属元素掺杂于某些晶体的晶格中构成的。 UCP is doped with some rare earth metal element constituting the crystal lattice in certain crystals. 在这种材料中有三种主要的成分:主基质(host matrix)、吸收子(absorber)和发射子(emitter)。 There are three major components in such materials: the host matrix (host matrix), absorber (absorber) and the sub emission (emitter). 作为主基质的晶体材料有:氧硫化物(如Y202S、GdO2S, La2O2S等)、氟化物(如YF3、GdF3> LaF3等)、镓酸盐(如YGa03、Y3Ga5O12等)以及硅酸盐(如YSi205、YSi3O7等)等;常用作吸收子的稀土金属离子有:镱离子(Yb3+)、铒离子(Er3+)、钐离子(Sm3+)等;常用作发射子的稀土金属离子有:铒离子(Er3+)、钦离子(Ho3+)、铥离子(Tm3+)、铽离子(Tb3+)等。 As the crystalline material has a host matrix: oxysulfide (e.g., Y202S, GdO2S, La2O2S, etc.), fluorides (e.g., YF3, GdF3> LaF3, etc.), gallates (e.g. YGa03, Y3Ga5O12 etc.) and silicates (e.g. YSi205 , YSi3O7 etc.); rare earth metal ion is commonly used as absorber are: ytterbium (Yb3 +), erbium ion (Er3 +), samarium ion (Sm3 +) and the like; used for transmitting the sub-rare earth metal ions: erbium ion (Er3 +) , Chin ion (Ho3 +), thulium ions (Tm3 +), Tb (Tb3 +) and the like. 吸收子和发射子这一离子对在主基质晶格内适宜的空间取向和距离,是产生上转换发光的基础。 This sub-sub-emission absorption and suitable ion pair in the main host lattice spatial orientation and distance, are generated on the basis of the conversion light emission.

[0003] 不同的吸收子、发射子、主基质结合可使UCP具有不同的光学性质。 [0003] Different absorber, the sub-emission, the host matrix UCP binding can have different optical properties. 如:(1)主基质相同的情况下,一系列UCP采用相同的吸收子,不同的发射子,则它可由相同波长的红外光激发,产生不同波长的发射光(如:980nm红外光激发吸收子-Yb3+-发射子-Er3+、吸收子-Yb3+—发射子-Tm3+,分别发射550nm绿色可见光、475nm蓝色可见光);采用不同的吸收子,相同的发射子,则虽经由不同的激发光,但却可产生相同的发射光。 Such as: (1) the same host matrix, the same series of UCP absorber, different sub emission, it may be infrared light of the same wavelength excitation, emit light of different wavelengths is generated (eg: excitation 980nm infrared absorption sub -Yb3 + - transmitted sub -Er3 +, absorber -Yb3 + - transmitted sub -Tm3 +, respectively emit green visible light 550nm, 475 nm blue visible light); different absorber, the same sub-emission, although the excitation light via different, but it can produce the same light emission. (2)主基质不同的情况下,UCP光谱特征也将改变(如:吸收子-Yb3+-发射子-Er3+,在主基质YF3、GdF3中,分别发射红色和绿色的可见光)。 (2) different host matrix case, the spectral characteristics of UCP will change (such as: absorber -Yb3 + - transmitted sub -Er3 +, in the host matrix YF3, GdF3, the red and green respectively emit visible light). 组成的多样性决定了UCP光谱(激发光谱、发射光谱)的多样性,这成为其使用中灵活性的基础。 Assemblages determine the UCP spectrum (excitation spectrum, the emission spectrum) diversity, which became the basis for its use flexibility. 本技术中所使用的UCP均由上海科炎光电技术有限公司提供。 This technology used by UCP Shanghai Branch Yan photoelectric Technology Limited.

[0004] UCP作为一种完全惰性的无机材料经过一系列的表面修饰与活化,其中包括:UCP表面硅化、UCP表面氨基化、UCP表面功能化、UCP表面连接抗体,便能与免疫层析技术相结合,在生物领域中充分的发挥其自身所具有的各种特性。 [0004] UCP as a completely inert inorganic material through a series of surface modification and activation, which comprises: UCP silicide surface, the surface of UCP amination, surface functionalization of UCP, surface attachment of UCP antibody will be able to immunochromatography combining a sufficient play has its own characteristics in various biological fields.

[0005]目前,免疫层析技术中所使用的标记物通常为酶、胶体金以及着色胶珠标记物,这几种标记物应用于免疫层析技术中有两个共同点:物理吸附交联法与通过颜色判断结果。 [0005] Currently, in immunochromatography is generally used as an enzyme label, colloidal gold and colored plastic beads marker, the marker is applied to these types of immunochromatography has two things in common: physical adsorption crosslinking method of determination results by the color. 其中物理吸附法(即通过疏水性以及静电吸附制备标记物)的脆弱性使得基于该种标记物的免疫层析对于反应的条件要求极为苛刻,从而一些有效的非特异去除试剂,如:吐温20 (Tween 20)、吹通100 (Triton 100)、十二烷基磺酸钠(SDS)等,由于对疏水性以及静电性的影响改变而只能在低浓度使用,由此便造成了基于该种标记物的免疫层析试纸假阳性率、假阴性率较高的必然结果;另外通过颜色判断结果在使用操作简便的同时必然受观察者主观影响大、灵敏度低,且只能停留在定性水平,而绝对无法实现精确定量。 Wherein the physical adsorption method (i.e. by electrostatic and hydrophobic adsorption Preparation marker) based on the vulnerability of such immunochromatography marker for this type of reactions the most demanding conditions, such that some non-specific removal of effective agents, such as: Tween 20 (Tween 20), blowing through 100 (Triton 100), sodium dodecyl sulfate (SDS) and the like, due to electrostatic and hydrophobic property changes can only be used in low concentrations, thereby causing it based this kind of immunochromatographic test strip marker false positive rate, high false negative rate corollary; Further the color determination result while using simple necessarily subjective observer by large impact, low sensitivity, and can only stay in the qualitative level, while not absolutely accurate quantification. 这些缺点大大的限制了免疫层析技术在生物检测中的应用范围。 These shortcomings greatly limits the application of immunochromatography in the bioassay.

[0006] 附图I中描述了UCP颗粒在生物检测领域中应用的原理示意图。 [0006] I of the accompanying drawings illustrates the principles applied in UCP particles bioassay art FIG. 将上转换发光颗粒与生物活性分子通过共价键相连接制备标记物,该标记物在层析的过程中通过免疫反应固定于固相载体的表面,并在红外光的激发下发射出可见光。 The upconversion emission particles and biologically active molecule through a covalent bond connected preparation marker, this marker immune response to a surface of the solid phase support is fixed by the process of chromatography, and emit visible light under excitation of infrared light. 由此实现上转换发光材料与免疫层析技术相结合对生物样品中的目标被检物进行检测。 Thereby achieving conversion luminescent material and immunochromatography binding pair in a biological sample with a target analyte to be detected.

[0007] 将上转换发光材料UCP与免疫层析技术相结合,为传统的免疫层析技术带来了突破性的变革: [0007] The UCP conversion luminescent materials in combination with immunochromatography, brings changes to breakthrough conventional immunochromatography:

[0008] A. UCP发光标记物的特点,使得以其作为标记物的免疫层析试纸条可与仪器结合,对目标被检物进行灵敏度极高的精确定量检测; [0008] A. UCP luminescent marker characteristics, such as its marker immunochromatographic strip can be combined with the instrument, the target subject was very high sensitivity detection of accurate quantification;

[0009] B. UCP所具有的多种特征光谱(激发光谱与发射光谱),使得基于上转换发光技术的免疫层析试纸可进行灵敏度极高的多重分析,即一次性的对生物样品中的多种目标被检物进行检测; [0009] B. UCP has various features spectrum (excitation and emission spectra), so that the test strip based on immunochromatography converting luminescence techniques may be multiple high sensitivity analysis, i.e., disposable biological sample more target subject was detected;

[0010] C. UCP独特的上转换发光现象,使得以其作为标记物的检测过程排除了由于待检测生物样品自发荧光造成干扰的可能,提高信噪比,从而提高了检测的灵敏度与稳定性; [0010] C. UCP upconversion unique phenomenon, ruled out the possibility that its biological sample to be detected due to the interfering autofluorescence as a marker detection process, improve the SNR, thereby improving the detection sensitivity and stability ;

[0011] D.通过共价方式交联生物活性分子,在保证检测灵敏度的前提下提高了系统的可靠性与稳定性。 [0011] D. bioactive molecules crosslinked by covalent way, to ensure improved reliability and stability of the system under the premise of detection sensitivity.

发明内容 SUMMARY

[0012] 为了对生物样品进行快速、灵敏、定量地检测,本发明提供了一种免疫层析试纸条,其采用上转换发光材料即UCP作为生物标记物,结果在红外光的照射下以可见光光信号的形式表现出来,并可使用上换发光生物传感器进行判读。 [0012] In order to biological samples rapid, sensitive and quantitative detection, the present invention provides an immunochromatographic test strip, which employs the conversion luminescent material i.e. as a biomarker UCP, results in the irradiation of infrared light in the form of visible optical signals manifested, and can be used for the interpretation transducer luminescence biosensor. 其结构包括: The structure includes:

[0013] a.样品垫; . [0013] a sample pad;

[0014] b.结合垫或结合物释放垫,其上固定有UCP标记的生物活性分子; [0014] b binding conjugate release pad, or pads, which are fixed on a biologically active molecule labeled UCP.;

[0015] c.分析膜,其上有检测带和质控带; . [0015] c analysis film, on which the detection zone and the control zone;

[0016] d.吸水垫,通过虹吸作用提供液体流过整个试纸条的动力; . [0016] d absorbent pad, there is provided a liquid flow throughout the test strip powered by a wicking action;

[0017] e.塑料背板,其中一面涂有粘胶; [0017] e plastic back sheet, one side coated with adhesive.;

[0018] 上述样品垫、结合垫、膜、吸水垫通过粘胶固定于塑料背板上。 [0018] The sample pad, conjugate pad, a membrane, an absorbent pad is fixed by adhesive to the plastic backing plate.

[0019] 上述各部分之间是相互重叠的,保证了液体在试纸条上流动的连续性。 [0019] The overlap between the various parts to each other, to ensure the continuity of liquid flow on the test strip. 当进行检测时,将样品滴加到样品垫上,样品通过渗透与虹吸作用进入结合垫,使其中的UCP结合物重新溶解游离,并在吸水垫的虹吸作用下,离开结合垫进入膜,在其内部向吸水垫的方向流动。 When detected, the sample was added dropwise to the sample pad, conjugate pad into the sample through the penetration and siphon action, which make UCP binding redissolved free, under the siphon action and the absorbent pad, conjugate pad into the membrane away, in which internal flows in the direction of the absorbent pad. 此过程中UCP结合物、目标被检物、检测带、质控带之间将特异地发生一定的免疫反应,并产生具有指示性的信号。 UCP process conjugate, the target analyte, detection with the certain specifically immunoreactive occur between the control line, and generates a signal indicative of. 依据在试纸条上所发生免疫反应方式的不同,可分为夹心模式、竞争模式与间接模式。 Depending on the way the immune response occurring in the strip, it can be divided into the sandwich mode, and indirect mode competition model.

[0020] A.夹心模式: [0020] A. sandwich mode:

[0021] 基于夹心模式的试纸条可用于检测样品中存在的病原体、微生物以及大分子抗原、抗体等。 [0021] Based on the test strip sandwich mode may be used to detect the presence of pathogens in a sample, microorganisms and macromolecular antigens, antibodies and the like. 其中,对病原体、微生物以及大分子抗原进行检测的模式为双抗体夹心,对抗体进行检测为双抗原夹心。 Wherein, pathogens, macromolecules and microbial antigens as detected by double antibody sandwich mode, the detection of antibodies to double antigen sandwich. 以下主要以双抗体夹心为例进行说明。 The following main double antibody sandwich as an example.

[0022] 在试纸条的制备过程中,首先将UCP与被检物特异性抗体A (即,可与被检物特异性结合的抗体,但其只可结合于被检物上的A位点)相结合,并固定于结合垫内;然后,将被检物特异性抗体B ( S卩,可与被检物特异性结合的抗体,但其只可结合于被检物上的B位点;注:此处所使用的抗体也可以为被检物特异性多克隆抗体。)固定于膜上作为检测带,将二抗(即,可与被检物特异性抗体A特异性结合的抗体)固定于膜上作为质控带,将变色范围5. 5-9. O的精密pH试纸贴于吸水垫上作为终点指示带。 [0022] In the preparation of the test strip, and the subject is first UCP specific antibody A (i.e., the antibody may be specifically bound the analyte, but can only be bound to a site on the analyte A point) combined, and is fixed to the pad binding; then, the subject-specific antibody B (S Jie, the antibody may be specifically bound the analyte, but can only be bound to the B-position of the detected object points; NOTE: as used herein, the antibody may be a polyclonal antibody specific for the analyte) is fixed on the membrane as a test line, the secondary antibody (i.e., the antibody may be specifically bound analyte a-specific antibody ) fixed to the membrane as a control line, the color range of 5. 5-9. O precise pH test paper affixed to the absorbent pad as an end point indicating band.

[0023] 附图2是生物样品中含有待测物,亦即阳性样品检测的示意图,样品中的被检物首先和结合垫中的UCP-抗体A相结合,即UCP-抗体A结合于被检物上的A位点。 [0023] Figure 2 is a biological sample containing analyte, i.e. a schematic diagram of the positive samples tested, the sample analyte and the first antibody binding pads UCP- A combination, i.e. UCP- antibody is bound to A a site on the analyte. 然后,二者形成的免疫复合物(UCP-抗体A-被检物)以及游离的UCP-抗体A结合物,与样品中的液体基质一起在吸水垫的带动下进入膜。 Then, immune complexes (UCP- A- subject antibody thereof) and free antibody UCP- A conjugate with the liquid sample matrix into the membrane formed in both driven together in the absorbent pad. 当其流过检测带时,检测带上的抗体B将与免疫复合物(UCP-抗体A-被检物)中被检物上的B位点相结合形成UCP-抗体A-被检物-抗体B复合物,并被固定于检测带上。 When it flows through the detection zone, the detection zone B antibody immune complexes (UCP- A- subject antibody composition) are combined with the B site on the subject to be formed UCP- A- analyte antibody - antibody B complex, and is fixed to the detection zone. 而游离UCP-抗体A不与抗体B结合,在吸水垫的带动下继续流动。 While the free antibodies UCP- A does not bind to the antibody B, continues to flow driven absorbent pad. 当通过质控带时,与其上的二抗(即,可与抗体A特异性结合的抗体)结合并被固定于质控带上。 When passing through the control line, the secondary antibody thereto (i.e., an antibody that specifically binds to the antibody A) and is fixed to the binding strip quality control. 残余的液体基质继续向吸水垫流动,并与其上PH试纸内的指示剂发生反应,使其由黄色变为绿色。 The residual liquid matrix to the absorbent pad to continue to flow, and on which the indicator reaction in PH test occurs, so that it changes from yellow to green. 终点指示带发生颜色变化的试纸条便可以进行结果判读。 The end of the test strip indicates the color change can occur with interpretation of the results. 使用上转换发光生物传感器(UPT生物传感器)对试纸条进行结果判读,阳性检测中试纸条的检测带与质控带上均有可见磷光产生,其所对应的信号强度分别为T与C。 Conversion using a luminescence biosensor (the UPT Biosensor) interpretation of the results of test strip, the test strip in a positive test and the control line are visible band phosphorescence is generated, its corresponding signal strength for the T and C, respectively . 检测带上磷光光强T与质控带上磷光光强C的比值,即T/C,与样品中含有的被检物浓度成正比。 Phosphorescence intensity detection zone T and quality control band phosphorescence intensity ratio C, i.e. T / C, proportional to the concentration of the analyte contained in the sample.

[0024] 附图3是生物样品中不含有待测物,亦即阴性样品检测时的示意图。 [0024] Figure 3 is a biological sample containing no analyte, schematic view of the negative samples i.e. detection. 由于样品中不含有被检物,因而重新溶解游离,并随样品的液体基质一起进入膜的只有UCP-抗体A。 Since the sample does not contain the test object, thus redissolved free, and with the liquid sample into the membrane matrix with only UCP- antibody A. 由此,只有在质控带上发生UCP-抗体A与二抗的免疫反应,并将UCP-抗体A固定其上。 Thus, only in the immune response UCP- second antibody Antibody A quality control band occurs, and UCP- antibody A is immobilized. 这样阴性检测中的试纸条在最终的UPT生物传感器判读下,只有质控带上有可见磷光产生。 Such negative test strip in UPT at the final interpretation of the biosensor, quality control strip only visible phosphorescence generated.

[0025] 将夹心模式系统中的抗体A与抗体B换为抗原A与抗原B,既可以双抗原夹心对某种病原体感染所产生的抗体进行检测。 [0025] The antibody in the sandwich mode of the system A and an antibody B to an antigen A and an antigen transducer B, may be some kind of double antigen sandwich antibody produced by pathogen infection is detected. 反应原理、结果指征与以上的双抗体夹心检测抗原相同。 The reaction principle, the results of the above indications the double antibody sandwich same antigen.

[0026] B.竞争模式: [0026] B. Competitive mode:

[0027] 基于竞争模式的试纸条可用于检测样品中存在的小分子抗原。 [0027] Based on the test strip can be used in competition model antigen detection of small molecules present in the sample.

[0028] 在试纸条的制备过程中,首先将UCP与被检物特异性抗体A相结合,并固定于结合垫内;然后,将与被检物相同的抗原(均含有抗体A可特异性结合的A位点)固定于膜上作为检测带,将二抗(即,可与被检物特异性抗体A特异性结合的抗体)固定于膜上作为质控带,将变色范围5. 5-9. O的精密pH试纸贴于吸水垫上作为终点指示带。 [0028] In the preparation of the test strip, and the subject is first UCP specific antibody A combination of the pad and fixed to the binding; then the subject with the same antigen (may contain an antibody specific for A binding the a site) is fixed on the membrane as a test line, the secondary antibody (i.e., the antibody may be specifically bound analyte a-specific antibody) is fixed on the membrane as a control line, the color range 5. 5-9. O precise pH test paper affixed to the absorbent pad as an end point indicating band.

[0029]当对阳性样品进行检测时,可以分为两种情况:被检物浓度高、被检物浓度较低。 [0029] When the positive samples tested, can be divided into two cases: a high analyte concentration, the analyte concentration is low.

[0030] 附图4为被检物浓度高时的竞争免疫反应的示意图。 [0030] 4 is a schematic diagram of the competitive immune response at high analyte concentrations the drawings. 当样品中被检物浓度高时,加样后样品中的被检物和结合垫中的UCP-抗体A全部结合,S卩UCP-抗体A结合于被检物上的A点。 When a high analyte concentration in the sample, the analyte and the sample application pads UCP- antibody binding in the sample A total binding, S Jie UCP- antibody binding to the A point on the subject object A is. 因而,在吸水垫的带动下,与样品中的液体基质一起进入膜的只有UCP-抗体A-被检物免疫复合物。 Thus, driven by the absorbent pad into the membrane with the liquid sample matrix is ​​only UCP- antibody immune complexes A- analyte. 当其流过检测带时,由于免疫复合物中UCP标记的抗体A已与被检物结合,所以不能与检测带上含有A位点的抗原结合。 When it flows through the detection zone, since the immune complex UCP A bound labeled antibody with the analyte, it is not the A site and the detection zone contains an antigen binding. UCP-抗体A-被检物免疫复合物继续流动,当流过质控带时发生免疫复合物中抗体A与二抗的免疫反应,并将UCP-抗体A-被检物免疫复合物固定其上。 A- UCP- antibody immune complexes detected object continues to flow, the immune response in an immune complex with antibody A secondary antibody occurs when the flow through the control line, and the test object A- UCP- antibody immune complex which is fixed on. 残余的液体基质继续向吸水垫流动,并与其上PH试纸内的指示剂发生反应,使其由黄色变为绿色。 The residual liquid matrix to the absorbent pad to continue to flow, and on which the indicator reaction in PH test occurs, so that it changes from yellow to green. 对变色后的试纸条进行传感器判读,在红外光的照射下只有质控带上有可见磷光产生。 The color of the test strip for sensor interpretation, quality control strip only visible phosphorescence generated in the irradiation of infrared light.

[0031] 附图5为被检物浓度低时的竞争免疫反应的示意图。 [0031] Figure 5 is a schematic diagram of the competitive immune response at low analyte concentrations. 当样品中被检物浓度较低时,加样后样品中的被检物首先和结合垫中的UCP-抗体A相结合。 When low analyte concentration in the sample, after loading the sample and the first analyte binding pads UCP- A combination of an antibody. 然后,二者形成的免疫复合物(UCP-抗体A-被检物)以及游离的UCP-抗体A结合物,与样品中的液体基质一起在吸水垫的带动下进入膜。 Then, immune complexes (UCP- A- subject antibody thereof) and free antibody UCP- A conjugate with the liquid sample matrix into the membrane formed in both driven together in the absorbent pad. 当其流过检测带时,由于免疫复合物中UCP标记的抗体A已与被检物结合,因而只有游离的UCP-抗体A结合物可与检测带上含有A位点的抗原结合,并被固定。 When it flows through the detection zone, since the immune complex UCP A labeled antibody has bound to the analyte, so that only the free UCP- A antibody conjugates may contain band A antigen site binding detection, and fixed. 剩余的UCP-抗体A-被检物免疫复合物继续流动,当流过质控带时发生免疫复合物中抗体A与二抗的免疫反应,并将UCP-抗体A-被检物免疫复合物固定其上。 The remaining test object A- UCP- antibody immune complex continues to flow, the immune response in an immune complex with antibody A secondary antibody occurs when the flow through the control line, and A- is UCP- antibody immune complexes analyte fixed on it. 这样试纸条在最终的红外光照射下,检测带与质控带上均有可见磷光产生,其所对应的信号强度分别为T与C。 Such a test strip in the final infrared light irradiation, and the control line are visible phosphorescence generated tape, it corresponds to the signal strength T and C. respectively 检测带上磷光光强T与质控带上磷光光强C的比值,即T/C,与样品中含有的被检物浓度成反比。 Phosphorescence intensity detection zone T and quality control band phosphorescence intensity ratio C, i.e. T / C, and is inversely proportional to the concentration of the analyte contained in the sample.

[0032] 附图6为对阴性样品进行检测时的竞争免疫反应的示意图。 [0032] 6 is a schematic view of the competitive immune response to a negative reference samples tested. 由于样品中不含有被检物,因而重新溶解游离,并随样品的液体基质一起进入膜的只有UCP-抗体A。 Since the sample does not contain the test object, thus redissolved free, and with the liquid sample into the membrane matrix with only UCP- antibody A. 其与检测带上含有A位点的抗原,质控带上的二抗均可结合。 A detection zone which contains antigen site, quality control can bring the secondary antibody binding. 这样试纸条在最终的红外光照射下,检测带与质控带上均有可见磷光产生。 Such a test strip in the final infrared light irradiation, and the control line are visible band phosphorescence generated. 但此时的T/C值与阳性检测低浓度时相比达到最强。 But this time the T / C values ​​when compared with the strongest positive detection of low concentration.

[0033] C.间接模式: [0033] C. Indirect mode:

[0034] 基于间接模式的试纸条可用于检测多种动物(包括人)受某种病原体感染后血清中的相应抗体。 [0034] Based on the test strip may be used to detect indirect mode a variety of animal (including human) subject to certain post-infection by a pathogen corresponding antibody in serum.

[0035] 在试纸条的制备过程中,首先将UCP与葡萄球菌蛋白A(SPA)相结合,并固定于结合垫内;然后,将某种病原体的表面抗原固定于膜上作为检测带,将羊IgG固定于膜上作为质控带,将变色范围5. 5-9. O的精密pH试纸贴于吸水垫上作为终点指示带。 [0035] In the preparation of the test strip, first UCP staphylococcal protein A (SPA) in combination, and the pad is fixed to the binding; then, some surface antigen of a pathogen as a detection band is fixed to the membrane, the goat IgG as a control line fixed on the membrane, the color range of 5. 5-9. O precise pH test paper affixed to the absorbent pad as an end point indicating band.

[0036] 附图7为对阳性血清样品进行检测时的间接免疫反应示意图。 [0036] BRIEF schematic reaction of indirect immunofluorescence positive serum sample 7 is detected. 加样后样品中的液体基质使UCP-SPA结合物重新溶解游离并与血清样品中存在的各种IgG(免疫球蛋白G,其中包括一部分病原体特异性抗体IgG)相结合。 After sample loading causes the liquid matrix UCP-SPA conjugate redissolved with various free and serum IgG present in the sample (immunoglobulin G, which comprises a portion of pathogen-specific IgG antibody) combination. 在吸水垫的带动下,UCP-SPA-IgG结合物与游离的UCP-SPA —起流动进入膜。 Driven absorbent pad, UCP-SPA-IgG conjugate and free UCP-SPA - from flowing into the membrane. 当流过检测带时,UCP-SPA-IgG结合物中的UCP-SPA-病原体特异性抗体IgG通过病原体特异性抗体IgG与检测带上的病原体抗原发生特异的免疫结合反应,并结合于其上。 While flowing through the detection zone, UCP-SPA-IgG conjugate of UCP-SPA- pathogens by pathogen-specific IgG antibodies and IgG antibodies specific detection zone pathogen antigen specific immune binding reaction occurs and the bound thereto . 游离的UCP-SPA和其它的一些UCp-SPA-IgG结合物(其中不含有UCP-SPA-病原体特异性抗体IgG)将继续流动,并在通过质控带时UCP-SPA与其上的羊IgG相结合。 UCP-SPA free and some other UCp-SPA-IgG conjugate (which does not contain a UCP-SPA- pathogen-specific antibody IgG) will continue to flow, and goat IgG UCP-SPA thereto through the control line on the phase combined. 残余的液体基质继续向吸水垫流动,并与其上pH试纸内的指示剂发生反应,使其由黄色变为绿色。 The residual liquid matrix to the absorbent pad to continue to flow, and on which the indicator reaction in the pH paper occurs, it changed from yellow to green. 对变色后的试纸条进行传感器判读,在红外光照射下质控带与检测带上均有可见磷光,其所对应的信号强度分别为T与C。 The color of the test strip for sensor interpretation, infrared light irradiation in the detection zone and the control line are visible phosphorescence, its corresponding signal strength are T and C. 检测带上磷光光强T与质控带上磷光光强C的比值,即T/C,与血清样品中病原体特异性抗体IgG的浓度成正比。 Phosphorescence intensity detection zone T and quality control band phosphorescence intensity ratio C, i.e. T / C, is proportional to the serum sample a pathogen specific IgG concentrations. (注:葡萄球菌蛋白A,即SPA,的特性是可以与多种动物的IgG发生结合。) (Note: staphylococcal protein A, i.e. SPA, the characteristics are more IgG binding can occur with the animals.)

[0037] 附图8为对阴性血清样品进行检测时的间接免疫反应示意图。 [0037] FIG 8 is a schematic diagram of the indirect immunofluorescence reaction serum sample negative detection. 由于样品中不含有病原体特异性抗体IgG,因而重新溶解游离,并随样品的液体基质一起进入膜的只有UCP-SPA与其它的一些UCP-SPA-IgG结合物(其中不含有UCP-SPA-病原体特异性抗体IgG)。 Since the sample does not contain pathogen-specific antibody IgG, thus re-dissolve free, and with the liquid sample matrix into the membrane UCP-SPA together only in combination with some other material UCP-SPA-IgG (containing no pathogens UCP-SPA- specific antibody IgG). 其中UCP-SPA与质控带上的羊IgG结合,而检测带上的病原体抗原未参加任何反应。 Wherein UCP-SPA binding belt and quality control sheep IgG, and the pathogen antigen detection zone did not participate in any reaction. 这样试纸条在最终的红外光照射下,只有质控带上有可见磷光产生。 Such a test strip in the final infrared light irradiation, visible only to bring a quality control to produce a phosphor.

[0038] 该试纸条结构中包括一外壳,所述外壳上包括加样孔,结果判读窗口和终点指示窗口。 [0038] The test strip comprises a housing structure, comprising a loading aperture of the housing, and the interpretation of the results window end indicator window. 通过加样孔,将生物样品添加到样品垫上;结果判读窗口对应于分析膜上的检测带和质控带;终点指示窗口对应于吸水垫上的终点指示带。 Through the loading aperture, the biological sample is added to the sample pad; interpretation of the results corresponding to the analysis window membrane detection zone and the control zone; end corresponding to the end point indicator window indicating band absorbent pad.

[0039] UCP所标记的生物活性分子,包括抗原、抗体、葡萄球菌蛋白A、外源凝激素、多聚核苷酸、受体配体、药物、细胞等。 [0039] UCP labeled biologically active molecule, including an antigen, an antibody, staphylococcal protein A, condensate exogenous hormones, polynucleotides, receptor ligand, drug, cells and the like.

[0040] UCP颗粒的直径为200_300nm。 [0040] The particle diameter of UCP 200_300nm. 并且,可以对UCP颗粒的表面进行了修饰。 Further, the surface may be modified UCP particles.

[0041] 当用于双抗体夹心免疫检测方法时,其中,结合垫上固定有UCP标记的第一特异性抗体,该特异性抗体可与待测物的一个位点相结合;分析膜的检测带上固定有与待测物的另一个位点相结合的第二特异性抗体,质控带上固定有可与第一特异性抗体发生特异性结合的抗体。 [0041] When used in double antibody sandwich immunoassay method, wherein the conjugate pad is fixed to a first labeled UCP specific antibody, the specific antibody may be combined with a site of the analyte; analytical detection film strip immobilized second specific antibody to another site of the analyte in combination, quality control strip is fixed antibody that specifically binds the first specific antibody may occur.

[0042] 当用于双抗原夹心免疫检测方法时,其中,结合垫上固定有UCP标记的抗原A ;分析膜的检测带上固定有抗原B,抗原A与抗原B可以结合于目标被检抗体的不同位点;质控带上固定有可与抗原A发生特异性结合的抗体。 [0042] When used in a double antigen sandwich immunoassay method, wherein the conjugate pad is fixed UCP A labeled antigen; analytical detection film strip is fixed B antigen, B antigen A and antigen can bind to the target of the test antibody different site; quality control strip is fixed antibody that specifically binds to the antigen may take place a.

[0043] 当用于竞争免疫检测方法时,其中,结合垫上固定有UCP标记的特异性抗体,该抗体可与待测物的一个位点相结合;分析膜的检测带上固定有与待测物具有相同的上述位点的抗原,质控带上固定有可与上述特异性抗体发生特异性结合的抗体。 [0043] When a competitive immunoassay method is used, wherein the conjugate pad is fixed UCP specific antibody labeled, the antibody may be combined with a site of the analyte; analytical detection film strip is fixed with a test having the same sites of an antigen, antibodies are immobilized to bring quality control specific binding can occur with the above-described specific antibody.

[0044] 当用于间接免疫检测方法时,其中,结合垫上固定有UCP标记的葡萄球菌蛋白A ;分析膜的检测带上固定有某种病原体的表面抗原,质控带上固定有IgG。 [0044] When used in the indirect immunoassay, wherein the fixed pad binding staphylococcal protein A labeled UCP; analytical detection film strip is fixed to a certain pathogen surface antigens, quality control strip is fixed to IgG.

[0045] 所述基于上转换发光技术的免疫层析试纸的制备方法为:采用上转换发光材料即UCP作为生物标记物对目标被检物进行定量检测;结合垫上固定有UCP标记的葡萄球菌蛋白A ;分析膜的检测带上固定有待测物的表面抗原,质控带上固定有IgG ;所述UCP保存液为pH = 7. 2 O. 03mol/L 磷酸盐缓冲液,含有O. 05% -O. 3% BSA,O. 01% -O. 1% Tween20,O. 01 % -O. I % NaN3 ;所述UCP生物活性分子的稀释液为pH = 7. 2的O. 03mol/L磷酸盐缓冲液,含有O. 5% -2%蔗糖,O. 5% -3% BSA ;所述样品垫封闭液为pH = 7. 2 O. 03mol/L磷酸盐缓冲液,含有1% -10% BSA,O. 05% -O. 2% Tween 20。 [0045] The preparation method of converting luminescence technique is based on immunochromatographic test strip: i.e., using the conversion luminescent materials UCP biomarker target subject was detected as quantitative; fixed pad binding staphylococcal protein labeled UCP a; analytical detection film strip is fixed to the surface antigen analyte, quality control IgG band is fixed; UCP preservation solution as the pH = 7. 2 O. 03mol / L phosphate buffer solution containing O. 05 ...% -O 3% BSA, O 01% -O 1% Tween20, O 01% -O I% NaN3;.. the diluent UCP biologically active molecule is a pH = 7. O. 2 03mol / L phosphate buffer containing O. 5% -2% sucrose, O 5% -3% BSA;. the sample pad blocking solution is pH = 7. 2 O. 03mol / L phosphate buffer, containing 1% -10% BSA, O. 05% -O. 2% Tween 20.

[0046] 本发明的另一个方面还涉及到所述试纸条的制备方法,其包括以下步骤: [0046] Another aspect of the present invention also relates to a method for preparing the test strip, comprising the steps of:

[0047] 1)UCP-生物活性分子的制备 Preparation UCP- biologically active molecule [0047] 1)

[0048] 对UCP颗粒进行表面修饰,与生物活性分子进行连接,在UCP保存液中进行保存; [0048] The UCP particles are surface modified with a biologically active molecule connection, save the UCP preservation solution;

[0049] 取一定量的保存于UCP保存液中的UCP-生物活性分子结合物,离心,并弃去上清液; [0049] A certain amount of UCP stored in the preservation solution UCP- biologically active molecule conjugate, centrifuged, and the supernatant was discarded;

[0050] 在沉降的UCP生物活性分子结合物中加入稀释液,并充分混匀,制备成悬浊液; [0050] UCP biologically active molecule is added in conjugate diluent settled in, and thoroughly mixed to prepare a suspension;

[0051] 2)样品垫的制备 [0051] 2) Preparation of the sample pad

[0052] 选用纤维素膜作为样品垫固相材料,剪切成具有一定规格的条带; [0052] The selection of the cellulose film is used as the solid phase material sample pad, was cut into strips with a certain specification;

[0053] 将样品垫放入样品垫封闭液中浸泡; [0053] The sample pad into the sample pad soaked in blocking solution;

[0054] 然后将样品垫从封闭液中取出,并烘干,使样品垫充分干燥; [0054] The sample pad was then removed from the blocking solution and drying, the sample was sufficiently dried pad;

[0055] 3)结合垫的制备 [0055] 3) Preparation of pad binding

[0056] 选用玻璃纤维素膜作为结合垫固相材料,剪切成具有一定规格的条带; [0056] use glass mat cellulose membrane as a solid phase bound material was cut into strips having a certain size;

[0057] 在该条带上加上UCP-生物活性分子结合物的悬浊液; [0057] In the strap suspension UCP- plus biologically active molecule conjugate;

[0058] 烘干该条带,使结合垫充分干燥; [0058] drying the strip so that sufficiently dried conjugate pad;

[0059] 4)分析膜的制备 Preparation of [0059] 4) Analysis of the film

[0060] 选用硝酸纤维素膜作为分析膜固相材料,剪切成具有一定规格的条带; [0060] The choice of a nitrocellulose membrane as a solid phase analytical membrane material is cut into strips having a certain strip specifications;

[0061] 在条带上的不同位置喷点生物活性分子,制成检测带和质控带; [0061] Dispensing the bioactive molecule at various positions on the strip, and the control is made with the detection zone;

[0062] 烘干该条带,使分析膜充分干燥; [0062] drying the strip, so that analysis of the film was sufficiently dried;

[0063] 5)装配该试纸条 [0063] 5) assembling the test strip

[0064] 将处理过的分析膜粘贴于塑料背板上,质控带向上,检测带向下; [0064] Analysis of the treated plastic film is attached to the back plate, the control line up, down detection zone;

[0065] 将处理过的结合垫粘贴于塑料背板上,上端压于分析膜的下端,并相互重叠; [0065] The treated plastic bonding pad attached to the back plate, the lower end of the upper end of pressure on the analysis of the film, and overlap one another;

[0066] 将处理过的样品垫粘贴子塑料背板上,下端与塑料背板平齐,上端压于结合垫的下端,并相互重叠; [0066] The treated sample pad attached on the sub-backplane plastic, plastic with a lower end of the back plate flush, pressure in the upper end of the lower end of the pad binding, and overlap one another;

[0067] 将吸水垫粘贴于塑料背板上,上端与塑料背板平齐,下端压于分析膜的上端,并与分析膜相互重叠; [0067] The absorbent pad attached to the plastic backing plate, the backing plate is flush with the plastic upper end, a lower end of the upper end of the membrane pressure on the analysis, and analysis and overlap film;

[0068] 将装配成形的条带剪切成一定规格的试纸条。 [0068] The assembly formed strips cut into a certain size of the test strip.

[0069] 由此制备成本发明的基于上转换发光技术免疫层析试纸条,该试纸条可以装入外壳中,在干燥条件下备用。 [0069] The invention is thus based on the preparation cost-converting luminescence technique immunochromatographic strip, the test strip can be contained in the enclosure, the standby under dry conditions. 该外壳可以是塑料的。 The housing may be plastic.

[0070] 其中,所述UCP保存液为pH = 7. 2 O. 03mol/L磷酸盐缓冲液(PB),含有O. I %BSA,O. 05% Tween20,0. 02% NaN3。 [0070] wherein, as the UCP preservation solution pH = 7. 2 O. 03mol / L phosphate buffer (PB), containing O. I% BSA, O. 05% Tween20,0. 02% NaN3.

[0071] 其中,所述UCP生物活性分子的稀释液为pH = 7.2 O. 03mol/L磷酸盐缓冲液(PB),含有1%蔗糖,1% BSA。 [0071] wherein said diluent is a biologically active molecule UCP pH = 7.2 O. 03mol / L phosphate buffer (PB), containing 1% sucrose, 1% BSA.

[0072] 其中,所述样品垫封闭液为pH = 7. 2 0.03mol/L磷酸盐缓冲液(PB),含有5%BSA, O. 1% Tween 20。 [0072] wherein said sample pad Blocking solution is pH = 7. 2 0.03mol / L phosphate buffer (PB), containing 5% BSA, O. 1% Tween 20.

[0073] 其中,可将变色范围5. 5-9. O的精密pH试纸贴于吸水垫作为终点指示带。 [0073] wherein the color range may be 5. 5-9. O precise pH test paper with an absorbent pad attached to an end point indicating band.

[0074] 本发明的另一个方面还涉及所述试纸条在检测生物样品中的应用。 [0074] Another aspect of the present invention further relates to the use of the test strip in the detection of a biological sample.

[0075]以经过表面修饰与活化的上转换发光材料UCP作为生物标记物,开发新型免疫层析试纸使其可以极高的稳定性、重现性及灵敏性应用于快速生物检测。 [0075] In the modification and conversion luminescent material activated surface-UCP as a biomarker to develop new immunochromatographic test strip so that it can be extremely high stability, reproducibility and sensitivity for rapid bioassays. 最终实现对病原体、病原体感染所产生的抗体、违禁药品、重大疾病(肿瘤、癌症和糖尿病等)标志物等多种目标被检物的定性、定量及多重检测,以满足生物恐怖剂检测、传染病和重大疾病的辅助诊断以及民用医疗保健的需求。 Ultimately is the qualitative and quantitative detection of multiple targets and multiple pathogens, the antibody produced by pathogen infection, illicit drugs, major diseases (tumors, cancer and diabetes) and the like markers analyte to meet bioterrorism agent detection, infection disease and diagnosis of major diseases and the needs of the civilian health care.

附图说明 BRIEF DESCRIPTION

[0076] 附图I :UCP颗粒用于生物检测领域的原理示意图 [0076] BRIEF I: UCP particles for biological detection principle schematic

[0077] 附图2 :夹心免疫反应为阳性的反应示意图 [0077] Figure 2: is a schematic view of a sandwich immunoreactivity positive reaction

[0078] 附图3 :夹心免疫反应为阴性的反应示意图 A schematic view of a sandwich immunoreactivity negative reaction: [0078] Figure 3

[0079] 附图4 :竞争免疫反应为阳性时,待测物为高浓度的反应示意图 [0079] Figure 4: The reaction was positive when competition immunoassay, the analyte is a schematic view of a high concentration of the reaction

[0080] 附图5 :竞争免疫反应为阳性时,待测物为低浓度的反应示意图 [0080] Figure 5: The reaction was positive when competition immunoassay, the analyte is a schematic view of a low concentration of reactive

[0081] 附图6 :竞争免疫反应为阴性的反应示意图 [0081] Figure 6: Competitive immunoreactive negative reaction scheme

[0082] 附图7 :间接免疫反应为阳性的反应示意图 [0082] Figure 7: is a schematic indirect immunoreactive positive reaction

[0083] 附图8 :间接免疫反应为阴性的反应示意图 [0083] Figure 8: Indirect immunofluorescence reactions negative reaction scheme

[0084] 附图9 =UPT生物传感器结果判读图(左为乙肝表面抗原阴性检测,右为阳性检测) [0084] FIG 9 = UPT biosensor interpretation of the results (left detection of hepatitis B surface antigen-negative, positive detection of Right)

[0085] 附图10 :乙肝病毒表面抗原检测标准工作曲线 [0085] Figure 10: hepatitis B virus surface antigen standard curve

[0086] 附图11 :UPT生物传感器结果判读图(左为SARS病毒阴性检测,右为阳性检测) [0086] FIG. 11: UPT biosensor interpretation of the results (left SARS virus is a negative test, a positive test is the right)

[0087] 附图12 =SARS病毒检测标准工作曲线 [0087] 12 = SARS virus detection reference standard curve

[0088] 附图13 :UPT生物传感器结果判读图(左为鼠疫FI-抗原阴性检测,右为阳性检测) [0088] FIG. 13: UPT biosensor interpretation of the results (left to plague FI- antigen-negative detection, detection of the right positive)

[0089] 附图14 :鼠疫FI-Ag检测标准工作曲线 [0089] Figure 14: Plague FI-Ag detection standard curve

[0090] 附图15 =UPT生物传感器结果判读图(左为安非他命阴性检测,右为阳性检测) [0090] FIG 15 = UPT biosensor interpretation of the results (left amphetamine is a negative test, a positive test is the right)

[0091] 附图16 :安非他命检测标准工作曲线图 [0091] Figure 16: Detection of amphetamine standard curve in FIG.

[0092] 附图17 :UPT生物传感器结果判读图(左为鼠疫感染抗体阴性检测,右为阳性检测) [0092] FIG. 17: UPT biosensor interpretation of the results (left to plague infection detection antibody negative, positive detection of Right)

[0093] 附图18 :鼠疫抗体检测标准工作曲线 [0093] Figure 18: plague antibody standard curve

[0094] 附图19 =UPT生物传感器结果判读图(左为SARS病毒感染抗体阴性检测,右为阳性检测) [0094] FIG 19 = UPT biosensor interpretation of the results (left infection is a SARS virus antibody negative detection, detection of the right positive)

[0095] 附图20 =SARS病毒抗体检测标准工作曲线 [0095] FIG 20 = SARS virus antibody standard curve

[0096] 附图21 :免疫层析试纸条的结构示意图 [0096] Figure 21: a schematic view of the structure of the immunochromatographic strip

[0097] 附图22 :装配试纸条时,各部分的粘贴示意图 [0097] Figure 22: assembling a test strip, a schematic view of the various parts of paste

[0098] 附图23 :试纸条的结构与尺寸 [0098] FIG. 23: the size and structure of the test strip

[0099] 附图24 :试纸条外壳的俯视图 [0099] BRIEF 24: a plan view of the housing of the test strip

[0100] 附图25 :试纸条外壳的内部结构,分为上片和下片 [0100] 25 Figures: an internal configuration of the housing strip, is divided into upper and lower pieces

[0101] 附图26 :上转换发光生物传感器的立体结构示意图 [0101] Figure 26: a perspective schematic view of the light emitting structure biosensor upconversion

[0102] 附图27 :上转换发光生物传感器包含激发光路光轴、试纸条法线和磷光图像接收光路光轴的平面结构示意图 [0102] Figure 27: a schematic view of a luminescence biosensor comprising a planar structure excitation light path of the optical axis, normal to the test strip and a phosphorescent light path of the optical axis of the image-receiving conversion

[0103] 附图28 :上转换发光材料的发射光谱曲线 [0103] Figure 28: Conversion of the emission spectrum of the luminescent material on the curve

[0104] 附图29 :滤光片的透过率曲线 [0104] Figure 29: filter transmission curve

[0105] 实验例I :双抗体夹心模式检测乙肝表面抗原HBs-Ag : [0105] Experimental Example I: double antibody sandwich detection pattern hepatitis B surface antigen HBs-Ag:

[0106] 一、UPT生物传感器结果判读: [0106] a, UPT biosensor Results Interpretation:

[0107] 附图9 :UPT生物传感器结果判读图(左为乙肝表面抗原阴性检测,右为阳性检测) [0107] FIG 9: UPT biosensor interpretation of the results (left detection of hepatitis B surface antigen-negative, positive detection of Right)

[0108] 二、标准工作曲线的绘制: [0108] Second, the standard curve drawing:

[0109] I.将提纯的乙肝表面抗原HBs-Ag标准品用I : 10稀释的正常人血清(以pH =7. 20. 03mol/L PB缓冲液稀释)作为稀释液配置系列浓度标准品,浓度为:0pg/ml、50pg/ml、100pg/ml、150pg/ml、200pg/ml、250pg/ml、300pg/ml、400pg/ml、500pg/ml、600pg/ml、700pg/ml、800pg/ml、900pg/ml、1000pg/ml、I100pg/ml、1200pg/ml、1300pg/ml、1400pg/ml、1500pg/ml、1600pg/ml 的20 份样品; [0109] I. The purified hepatitis B surface antigen HBs-Ag with standard I: 10 dilution of normal human serum (. Diluted to pH = 7 20. 03mol / L PB buffer) configured as a dilution series of concentration standards, concentration: 0pg / ml, 50pg / ml, 100pg / ml, 150pg / ml, 200pg / ml, 250pg / ml, 300pg / ml, 400pg / ml, 500pg / ml, 600pg / ml, 700pg / ml, 800pg / ml , 900pg / ml, 1000pg / ml, I100pg / ml, 1200pg / ml, 1300pg / ml, 1400pg / ml, 1500pg / ml, 1600pg / ml of the 20 samples;

[0110] 2.每个样品分别用10个UPT试纸条检测10次,10次检测中传感器判读得到的T值与C值分别取平均值,最终根据二者的比值得出与每个浓度对应的τ/c结果,列于下表I : [0110] 2. Each sample was detected 10 times with UPT strip 10, T 10 value and the C value obtained detection sensor interpretation are averaged, the final concentration obtained in accordance with each of the ratio of the two corresponding to τ / c results, listed in table I:

[0111] [0111]

[0112] 表I :乙肝病毒表面抗原检测标准工作曲线 [0112] Table I: hepatitis B virus surface antigen standard curve

[0113] 3.以Τ/C值作为X,以乙肝表面抗原HBs-Ag浓度作为Y绘制标准工作曲线,经统计拟和标准工作曲线的表达式为:Y = 268. 73Χ+103. 06,拟和系数的平方为:R2 = 0. 975 ;结果见附图10 :乙肝病毒表面抗原检测标准工作曲线。 [0113] 3. Τ / C value as X, HBs-Ag concentration to hepatitis B surface antigen standard curve plotted as a Y, and an expression proposed by the statistics for the standard curve:. Y = 268. 73Χ + 103 06, and squares fit coefficients is: R2 = 0. 975; see Figure 10 results: HBV surface antigen standard curve.

[0114] 4.乙肝表面抗原浓度的计算公式为: [0114] 4. The hepatitis B surface antigen concentration is calculated as:

[0115] 血清样品中含有的乙肝表面抗原HBs-Ag浓度(pg/ml) = 10Y =10X (268. 73X+103. 06) = 2687. 3X+1030. 6 [0115] HBs-Ag concentration (pg / ml) in serum samples containing HBsAg = 10Y = 10X (268. 73X + 103. 06) = 2687. 3X + 1030. 6

[0116] 三、实际检测结果: [0116] Third, the actual test results:

[0117] I.检测准确性: [0117] I. test accuracy:

[0118] 将50份购自生物制品鉴定所的乙肝病人血清盘(其中含13份阳性,37份阴性)同时用胶体金免疫层析试纸与本系统(UPT试纸条与传感器)进行双盲检测: [0118] 50 parts of hepatitis B patients sera obtained from the disc of Biological Products (which contains 13 parts of positive and negative 37 parts) simultaneously with GICA strip of the present system (the UPT test strip and sensor) in a double-blind detection:

[0119] 胶体金免疫层析试纸法——11份阳性,39份阴性(即,两份阳性漏检); [0119] --11 parts GICA positive test paper method, 39 parts of a negative (i.e., two positive missed);

[0120] UPT试纸条与传感器法一13份阳性,37份阴性,与实际结果完全吻合; [0120] UPT strip with 13 parts of a positive sensor method, 37 parts of a negative, fully consistent with the actual results;

[0121] 同时与胶体金免疫层析试纸的定性检测相对,UPT试纸条与传感器法给出了每份样品的最终准确浓度。 [0121] Also with GICA qualitative detection of the relative strip, UPT method test strip and the sensor gives accurately the final concentration of each sample.

[0122] 2.检测稳定性: [0122] 2. Stability detection:

[0123] 将一份乙肝病人血清用pH = 7. 20. 03mol/L PB缓冲液10倍稀释,用UPT试纸条与传感器重复检测10次,结果列于下表2 : [0123] An aliquot of serum hepatitis B patients with pH = 7. 20. 03mol 10-fold dilution / L PB buffer, with the sensor strip UPT Ten replicate measurements, the results are shown in Table 2:

[0124] [0124]

[0125] 同一份血清重复测量的变异系数(CV) = 2. 621% [0125] coefficient of variation (CV) for repeated measurements with a serum = 2.621%

[0126] 表2 :乙肝病毒表面抗原检测重复性 [0126] Table 2: Detection of HBsAg repeatability

[0127] 结论:在乙肝表面抗原HBs-Ag检测中,UPT试纸条与传感器法与胶体金免疫层析试纸法相比具有更高的灵敏度,且在实现精确定量检测的同时具有很好的稳定性。 [0127] Conclusion: hepatitis B surface antigen HBs-Ag in the detection, the UPT strip having higher sensitivity as compared with the sensor method and the method of gold immunochromatography strip, and having at the same time very accurate quantitative detection of stable sex.

[0128] 实验例2 :双抗体夹心模式检测冠状病毒(SARS病毒): [0128] Experimental Example 2: Double-antibody sandwich detection pattern coronavirus (SARS virus):

[0129] 一、UPT生物传感器结果判读: [0129] a, UPT biosensor Results Interpretation:

[0130] 附图11 :UPT生物传感器结果判读图(左为SARS病毒阴性检测,右为阳性检测) [0130] FIG. 11: UPT biosensor interpretation of the results (left SARS virus is a negative test, a positive test is the right)

[0131] 二、标准工作曲线的绘制: [0131] Second, the standard curve drawing:

[0132] I.将经过培养、记数并灭活的SARS病毒用pH = 7. 2 O. 03mol/L PB缓冲液作为稀释液配置系列浓度标准品,浓度为:Oorg/ml、IOOorg/ml、200org/ml、300org/ml、400org/ml、500org/ml、600org/ml、700org/ml、800org/ml、900org/ml、1000org/ml、IlOOorg/ml、1200org/ml>1300org/ml>1400org/ml>1500org/ml>1600org/ml>1700org/ml、1800org/ml>1900org/ml、2000org/ml 的21 份样品; [0132] I. After the culture, and counting with inactivated SARS virus pH = 7. 2 O. 03mol / L PB buffer was used as dilution series arranged standard concentration, concentration: Oorg / ml, IOOorg / ml , 200org / ml, 300org / ml, 400org / ml, 500org / ml, 600org / ml, 700org / ml, 800org / ml, 900org / ml, 1000org / ml, IlOOorg / ml, 1200org / ml> 1300org / ml> 1400org / ml> 1500org / ml> 1600org / ml> 1700org / ml, 1800org / ml> 1900org / ml, 2000org / ml of the 21 samples;

[0133] 2.每个样品分别用10个UPT试纸条检测10次,10次检测中传感器判读得到的T值与C值分别取平均值,最终根据二者的比值得出与每个浓度对应的τ/c结果,列于下表3 : [0133] 2. Each sample was detected 10 times with UPT strip 10, T 10 value and the C value obtained detection sensor interpretation are averaged, the final concentration obtained in accordance with each of the ratio of the two corresponding to τ / c results are shown in table 3:

[0134] [0134]

[0135] 表3 =SARS病毒检测标准工作曲线 [0135] TABLE 3 = SARS virus detection standard curve

[0136] 3.以Τ/C值作为X,以SARS病毒浓度作为Y绘制标准工作曲线,经统计拟和标准工作曲线的表达式为:γ = 337. 2Χ+216. 12,拟和系数的平方为:R2 = 0. 9631 ;结果见附图12 =SARS病毒检测标准工作曲线。 [0136] 3. Τ / C value as X, Y to the SARS virus concentration plotted as a standard curve, and expressions intended by the statistics for the standard curve:. Γ = 337. 2Χ + 216 12, fitting coefficients squared: R2 = 0. 9631; results are shown in figures 12 = SARS virus detection standard curve.

[0137] 4. SARS病毒浓度的计算公式为: [0137] 4. The SARS virus concentration is calculated as follows:

[0138]样品中含有的 SARS 病毒浓度(org/ml) = 10Y = 10X (337. 2X+216. 12)=3372X+2161.2 [0138] SARS virus concentration (org / ml) contained in a sample = 10Y = 10X (337. 2X + 216. 12) = 3372X + 2161.2

[0139] 三、实际检测结果: [0139] Third, the actual test results:

[0140] I.检测准确性: [0140] I. test accuracy:

[0141] 将30份待检样品(其中含6份阳性,24份阴性)用本系统(UPT试纸条与传感器)进行双盲检测: [0141] 30 parts of the sample to be tested (which contains 6 parts of positive and negative 24 parts) with the present system (the UPT test strip and sensor) double blind detection:

[0142] UPT试纸条与传感器法一6份阳性,24份阴性,与实际结果完全吻合; [0142] UPT method test strip and a sensor 6 samples were positive, 24 negative parts, fully consistent with the actual results;

[0143] 同时UPT试纸条与传感器法给出了每份样品的最终精确浓度。 [0143] Also with the sensor strip UPT method gives precise final concentration of each sample.

[0144] 2.检测稳定性: [0144] 2. Stability detection:

[0145] 将一份待检样品用pH = 7. 2 0. 03mol/L PB缓冲液10倍稀释,用UPT试纸条与传感器检测10次,结果列于下表4 : [0145] An aliquot of the sample to be = 7. 2 0. 03mol / L PB was diluted 10-fold with buffer pH, the strip sensor 10 detects times with UPT, the results are shown in Table 4:

[0146] [0146]

[0147] 同一份待检样品重复测量的变异系数(CV) = I. 03% [0147] with a coefficient of variation of the sample to be repeated measurements (CV) = I. 03%

[0148] 表4 =SARS病毒检测重复性 [0148] TABLE 4 = SARS virus detection repeatability

[0149] 结论:在SARS病毒检测中,UPT试纸条与传感器法具有很好的灵敏度与稳定性,且实现了精确定量。 [0149] Conclusion: the detection of SARS virus, test strip having the UPT good sensitivity and stability with the sensor method, and to achieve a precise quantification.

[0150] 实验例3 :竞争模式检测鼠疫FI-抗原(鼠疫FI-Ag): [0150] Experimental Example 3: Antigen competition mode detector FI- plague (Yersinia FI-Ag):

[0151] 一、UPT生物传感器结果判读: [0151] a, UPT biosensor Results Interpretation:

[0152] 附图13 :UPT生物传感器结果判读图(左为鼠疫FI-抗原阴性检测,右为阳性检测) [0152] FIG. 13: UPT biosensor interpretation of the results (left to plague FI- antigen-negative detection, detection of the right positive)

[0153] 二、标准工作曲线的绘制: [0153] Second, the standard curve drawing:

[0154] I.将提纯的鼠疫FI-Ag标准品用pH = 7. 2 O. 03mol/L PB缓冲液作为稀释液配置系列浓度标准品,浓度为:Opg/ml、IOOpg/ml、200pg/ml、300pg/ml、400pg/ml、500pg/ml、600pg/ml、700ng/ml、800pg/ml、900pg/ml、lOOOpg/ml、IlOOpg/ ml、1200pg/ml、1300pg/ml、1400pg/ml、1500pg/ml、1600pg/ml、1700pg/ml、1800pg/ml、1900pg/ml、2000pg/ml 的21 份样品; [0154] I. The purified plague FI-Ag with standard pH = 7. 2 O. 03mol / L PB buffer was used as dilution series arranged standard concentration, concentration: Opg / ml, IOOpg / ml, 200pg / ml, 300pg / ml, 400pg / ml, 500pg / ml, 600pg / ml, 700ng / ml, 800pg / ml, 900pg / ml, lOOOpg / ml, IlOOpg / ml, 1200pg / ml, 1300pg / ml, 1400pg / ml, 1500pg / ml, 1600pg / ml, 1700pg / ml, 1800pg / ml, 1900pg / ml, 2000pg / ml of the 21 samples;

[0155] 2.每个样品分别用10个UPT试纸条检测10次,10次检测中传感器判读得到的T值与C值分别取平均值,最终根据二者的比值得出与每个浓度对应的Τ/C结果,列于下表5 : [0155] 2. Each sample was detected 10 times with UPT strip 10, T 10 value and the C value obtained detection sensor interpretation are averaged, the final concentration obtained in accordance with each of the ratio of the two corresponding Τ / C results, set forth in table 5:

[0156] [0156]

[0157] 表5 :鼠疫FI-Ag检测标准工作曲线 [0157] Table 5: FI-Ag detection plague standard curve

[0158] 3.以Τ/C值作为X,以鼠疫FI-Ag浓度作为Y绘制标准工作曲线,经统计拟和标准工作曲线的表达式为:Y = -324. 63Χ+2058. 5,拟和系数的平方为:R2 = 0. 9993 ;结果见附图14 :鼠疫FI-Ag检测标准工作曲线。 [0158] 3. Τ / C value as X, to plague FI-Ag concentration standard curve plotted as a Y, and an expression proposed by the statistics for the standard curve:.. Y = -324 63Χ + 2058 5, Quasi and as the square of the coefficient: R2 = 0. 9993; see Figure 14 results: plague FI-Ag detection standard curve.

[0159] 4.鼠疫FI-Ag浓度的计算公式为: [0159] 4. formula plague FI-Ag concentration as follows:

[0160]样品中含有的鼠疫 FI-Ag 浓度(pg/ml) = IOY = IOX (-324. 63X+2058. 5)=-3246. 3X+20585 [0160] plague FI-Ag concentration (pg / ml) contained in a sample = IOY = IOX (-324 63X + 2058 5..) = -. 3246 3X + 20585

[0161] 三、实际检测结果: [0161] Third, the actual test results:

[0162] I.检测准确性: [0162] I. test accuracy:

[0163] 将32份待检样品(其中含19份阳性,13份阴性)用本系统(UPT试纸条与传感器)进行双盲检测: [0163] 32 parts of the sample to be tested (which contains 19 parts of positive and negative 13 parts) with the present system (the UPT test strip and sensor) double blind detection:

[0164] UPT试纸条与传感器法一19份阳性,13份阴性,与实际结果完全吻合; [0164] UPT method test strip and a sensor 19 were positive and 13 negative parts, fully consistent with the actual results;

[0165] 同时UPT试纸条与传感器法给出了每份样品的最终精确浓度。 [0165] Also with the sensor strip UPT method gives precise final concentration of each sample.

[0166] 2.检测稳定性: [0166] 2. Stability detection:

[0167] 将一份待检样品用pH = 7. 2 O. 03mol/L PB缓冲液10倍稀释,用UPT试纸条与传感器检测10次,结果列于下表6 : [0167] An aliquot of the sample to be = 7. 2 O. 03mol / L PB was diluted 10-fold with buffer pH, the strip sensor 10 detects times with UPT, the results are shown in Table 6:

[0168] [0168]

[0169] 同一份待检样品重复测量的变异系数(CV) = I. 034% [0169] with a coefficient of variation of the sample to be repeated measurements (CV) = I. 034%

[0170] 表6 :鼠疫FI-Ag检测重复性 [0170] Table 6: Detection reproducibility FI-Ag plague

[0171] 结论:在鼠疫FI-Ag检测中,UPT试纸条与传感器法具有很好的灵敏度与稳定性,且实现了精确定量检测。 [0171] Conclusion: FI-Ag in the detection of plague, the UPT test strip having a good sensitivity and stability with the sensor method, and to achieve a precise quantitative detection.

[0172] 实验例4 :竞争模式检测违禁药品(以检测安非他命为例进行说明) [0172] Experimental Example 4: Competitive mode detection of illicit drugs (amphetamine to detect as an example)

[0173] 一、UPT生物传感器结果判读: [0173] a, UPT biosensor Results Interpretation:

[0174] 附图15 =UPT生物传感器结果判读图(左为安非他命阴性检测,右为阳性检测) [0174] FIG 15 = UPT biosensor interpretation of the results (left amphetamine is a negative test, a positive test is the right)

[0175] 二、标准工作曲线的绘制: [0175] Second, the standard curve drawing:

[0176] I.将纯品安非他命用pH= 7. 2 O. 03mol/L PB缓冲液作为稀释液配置系列浓度标准品,浓度为:Opg/ml、10pg/ml、20pg/ml、30pg/ml、40pg/ml、50pg/ml、70pg/ml、90ng/ml、110pg/ml、130pg/ml、150pg/ml、170pg/ml、190pg/ml、210pg/ml、230pg/ml、250pg/ml、270pg/ml、290pg/ml、310pg/ml、330pg/ml 的20 份样品; [0176] I. The pure amphetamine with pH = 7. 2 O. 03mol / L PB buffer was used as dilution series arranged standard concentration, concentration: Opg / ml, 10pg / ml, 20pg / ml, 30pg / ml , 40pg / ml, 50pg / ml, 70pg / ml, 90ng / ml, 110pg / ml, 130pg / ml, 150pg / ml, 170pg / ml, 190pg / ml, 210pg / ml, 230pg / ml, 250pg / ml, 270pg / ml, 290pg / ml, 310pg / ml, 330pg / ml of the 20 samples;

[0177] 2.每个样品分别用10个UPT试纸条检测10次,10次检测中传感器判读得到的T值与C值分别取平均值,最终根据二者的比值得出与每个浓度对应的Τ/C结果,列于下表 [0177] 2. Each sample was detected 10 times with UPT strip 10, T 10 value and the C value obtained detection sensor interpretation are averaged, the final concentration obtained in accordance with each of the ratio of the two corresponding Τ / C results are shown in table

[0179] 表7 :安非他命检测标准工作曲线 [0179] Table 7: Detection of amphetamine standard curve

[0180] 3.以Τ/C值作为X,以安非他命浓度作为Y绘制标准工作曲线,经统计拟和标准工作曲线的表达式为:γ = -51. 985Χ+296. 45,拟和系数的平方为:R2 = 0. 9529 ;结果见附图.16 :安非他命检测标准工作曲线。 [0180] 3. Τ / C value as X, Y plotted as the concentration of amphetamines to the standard curve, the expression by standard curve fitting statistics as:.. Γ = -51 985Χ + 296 45, fitting coefficients squared: R2 = 0. 9529; results are drawings .16: detecting amphetamine standard curve.

[0181] 4.安非他命的浓度计算公式为: Concentration of the formula [0181] 4. amphetamine is:

[0182]样品中含有的安非他命浓度(pg/ml) = 10Y = 10X (-51. 985X+296. 45)=-519. 85X+2964.5 [0182] Amphetamine concentration (pg / ml) contained in a sample = 10Y = 10X (.. -51 985X + 296 45) = -. 519 85X + 2964.5

[0183] 三、实际检测结果: [0183] Third, the actual test results:

[0184] 1.检测准确性: [0184] 1. Detection Accuracy:

[0185] 将44份待检样品(其中含23份阳性,21份阴性)用本系统(UPT试纸条与传感器)进行双盲检测: [0185] 44 parts of the sample to be tested (which contains 23 parts of positive and negative 21 parts) with the present system (the UPT test strip and sensor) double blind detection:

[0186] UPT试纸条与传感器法一23份阳性,21份阴性,与实际结果完全吻合; [0186] UPT method test strip and a sensor 23 samples were positive, negative 21 parts, fully consistent with the actual results;

[0187] 同时UPT试纸条与传感器法给出了每份样品的最终精确浓度。 [0187] Also with the sensor strip UPT method gives precise final concentration of each sample.

[0188] 2.检测稳定性: [0188] 2. Stability detection:

[0189] 将一份待检样品用pH = 7. 2 0. 03mol/L PB缓冲液10倍稀释,用UPT试纸条与传感器检测10次,结果列于下表8 : [0189] An aliquot of the sample to be = 7. 2 0. 03mol / L PB buffer diluted 10-fold with the pH, the detection 10 times with the test strip and sensor UPT results are shown in Table 8:

[0190] [0190]

[0191] 同一份待检样品重复测量的变异系数(CV) = 0. 423% [0191] with a coefficient of variation of the sample to be repeated measurements (CV) = 0. 423%

[0192] 表8:安非他命检测重复性 [0192] Table 8: Detection amphetamine repeatability

[0193] 结论:在安非他命检测中,UPT试纸条与传感器法具有很好的灵敏度与稳定性,且实现了精确定量检测。 [0193] Conclusion: In the detection of amphetamines, the UPT test strip having a good sensitivity and stability with the sensor method, and to achieve a precise quantitative detection.

[0194] 实验例5 :间接模式检测鼠疫感染抗体(以检测兔感染鼠疫产生抗体为例进行说明) [0194] Experimental Example 5: Indirect detection mode plague infections antibody (to detect antibodies generated in rabbits infected with the plague as an example)

[0195] 一、UPT生物传感器结果判读: [0195] a, UPT biosensor Results Interpretation:

[0196] 附图17 :UPT生物传感器结果判读图(左为鼠疫感染抗体阴性检测,右为阳性检测) [0196] FIG. 17: UPT biosensor interpretation of the results (left to plague infection detection antibody negative, positive detection of Right)

[0197] 二、标准工作曲线的绘制: [0197] Second, the standard curve drawing:

[0198] I.将提纯的兔抗鼠疫IgG标准品用I : 10稀释的正常兔血清(以pH = 7. 2 [0198] I. The purified rabbit IgG anti-plague standard with I: 10 dilution of normal rabbit serum (to pH = 7. 2

0. 03mol/L PB缓冲液稀释)作为稀释液配置系列浓度标准品,浓度为:0ng/ml、2ng/ml、4ng/ml、6ng/ml、8ng/ml、10ng/ml、15ng/ml、20ng/ml、25ng/ml、30ng/ml、35ng/ml、40ng/ml、45ng/ml、50ng/ml、55ng/ml、60ng/ml、65ng/ml、70ng/ml、75ng/ml、80ng/ml 的20 份样品; 0. 03mol / PB buffer diluted L) arranged as a series of dilutions standard concentration, concentration: 0ng / ml, 2ng / ml, 4ng / ml, 6ng / ml, 8ng / ml, 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml, 35ng / ml, 40ng / ml, 45ng / ml, 50ng / ml, 55ng / ml, 60ng / ml, 65ng / ml, 70ng / ml, 75ng / ml, 80ng / 20 ml of the samples;

[0199] 2.每个样品分别用10个UPT试纸条检测10次,10次检测中传感器判读得到的T值与C值分别取平均值,最终根据二者的比值得出与每个浓度对应的T/C结果,列于下表9 : [0199] 2. Each sample was detected 10 times with UPT strip 10, T 10 value and the C value obtained detection sensor interpretation are averaged, the final concentration obtained in accordance with each of the ratio of the two corresponding to T / C the results, given in table 9:

[0200] [0200]

[0201 ] 表9 :鼠疫抗体检测标准工作曲线 [0201] Table 9: plague antibody standard curve

[0202] 3.以T/C值作为X,以鼠疫抗体浓度作为Y绘制标准工作曲线,经统计拟和标准工作曲线的表达式为:Y = 14. 129X-0. 3076,拟和系数的平方为:R2 = 0. 9749 ;结果见附图18 :鼠疫抗体检测标准工作曲线。 [0202] 3. T / C value as X, Y to plague antibody concentration plotted as a standard curve, the expression by standard curve fitting statistics as:. Y = 14. 129X-0 3076, fitting coefficients squared: R2 = 0. 9749; see Figure 18 results: plague antibody standard curve.

[0203] 4.鼠疫抗体浓度的计算公式为: [0203] 4. plague antibody concentration calculated as:

[0204] 兔血清中含有的鼠疫抗体浓度(ng/ml) = IOY = IOX (14. 129X-0. 3076)=141. 29X-3. 076 [0204] plague antibody concentration (ng / ml) contained rabbit serum = IOY = IOX (14. 129X-0. 3076) = 141. 29X-3. 076

[0205] 三、实际检测结果: [0205] Third, the actual test results:

[0206] I.检测准确性: [0206] I. test accuracy:

[0207] 将100份兔血清(其中含31份阳性,69份阴性)同时用酶联免疫吸附法(ELISA)与本系统(UPT试纸条与传感器)进行双盲检测: [0207] 100 parts of the rabbit serum (which contains 31 parts of positive and negative 69 parts) with simultaneous double blind detection system (the UPT and the sensor strip) by enzyme linked immunosorbent assay (ELISA):

[0208] ELISA法——23份阳性,77份阴性; [0208] ELISA method --23 parts positive, negative 77 parts;

[0209] UPT试纸条与传感器法——31份阳性(包括ELISA中的23份阴性,并从ELISA确定的77份阴性样品中又检出8份阳性),69份阴性,与实际结果完全吻合; [0209] UPT sensor strip and positive method --31 parts (including 23 parts of a negative in the ELISA, and 77 parts of the negative samples was determined by ELISA and the detection of positive 8 parts), 69 parts of negative results with actual full anastomosis;

[0210] 同时与ELISA的定性检测相对,UPT试纸条与传感器法给出了每份样品的最终精确浓度。 [0210] Meanwhile opposite, UPT method test strip and the sensor gives accurately the final concentration of each sample and qualitative detection of the ELISA.

[0211] 2.检测稳定性: [0211] 2. Stability detection:

[0212] 将一份感染鼠疫家兔的血清用pH = 7. 2 0. 03mol/L PB缓冲液10倍稀释,用UPT试纸条与传感器检测10次,结果列于下表10 : Serum with pH [0212] An aliquot of the infected rabbit plague = 7. 2 0. 03mol / L 10 PB-fold dilution buffer, twice with 10 UPT detected with the sensor strip, the results are shown in Table 10:

[0213] [0213]

[0214] 同一份血清重复测量的变异系数(CV) = I. 408% [0214] with a coefficient of variation of repeated measurements of serum (CV) = I. 408%

[0215] 表10 :鼠疫抗体检测标准工作曲线 [0215] Table 10: Antibody detection plague standard curve

[0216] 结论:在鼠疫感染抗体检测中,UPT试纸条与传感器法与ELISA法相比具有更高的灵敏度,且在实现精确定量的同时具有很好的稳定性。 [0216] Conclusion: In infected plague antibody detection, the UPT test strip having higher sensitivity as compared with the sensor method and the ELISA method, and has good stability at the same time achieving precise quantification.

[0217] 实验例6 :间接模式检测SARS病毒感染抗体: [0217] Experimental Example 6: Indirect detection of SARS virus antibody patterns:

[0218] 一、UPT生物传感器结果判读: [0218] a, UPT biosensor Results Interpretation:

[0219] 附图19 =UPT生物传感器结果判读图(左为SARS病毒感染抗体阴性检测,右为阳性检测) [0219] FIG 19 = UPT biosensor interpretation of the results (left infection is a SARS virus antibody negative detection, detection of the right positive)

[0220] 二、标准工作曲线的绘制: [0220] Second, the standard curve drawing:

[0221] I.将从SARS病人血清中提纯的人抗SARS病毒IgG标准品用I : 10稀释的正常人血清(以pH = 7. 2 0. 03mol/L PB缓冲液稀释)作为稀释液配置系列浓度标准品,浓度为:Ong/ml、lng/ml、2ng/ml、3ng/ml、4ng/ml、5ng/ml、6ng/ml、8ng/ml、IOng/ml、12ng/ml、14ng/ml、16ng/ml、18ng/ml、20ng/ml、22ng/ml、24ng/ml、26ng/ml、28ng/ml、30ng/ml、32ng/ml、34ng/ml 的21 份样品; [0221] I. sera from SARS patients a human anti-SARS virus purified IgG standard using I: 10 dilution of normal human serum (pH = 7. 2 0. 03mol / L PB in dilution buffer) was used as diluent configuration series of standard concentration, concentration: Ong / ml, lng / ml, 2ng / ml, 3ng / ml, 4ng / ml, 5ng / ml, 6ng / ml, 8ng / ml, IOng / ml, 12ng / ml, 14ng / ml, 16ng / ml, 18ng / ml, 20ng / ml, 22ng / ml, 24ng / ml, 26ng / ml, 28ng / ml, 30ng / ml, 32ng / ml, 34ng / ml of 21 samples;

[0222] 2.每个样品分别用10个UPT试纸条检测10次,10次检测中传感器判读得到的T值与C值分别取平均值,最终根据二者的比值得出与每个浓度对应的T/C结果,列于下表11 : [0222] 2. Each sample was detected 10 times with UPT strip 10, T 10 value and the C value obtained detection sensor interpretation are averaged, the final concentration obtained in accordance with each of the ratio of the two corresponding to T / C the results, set forth in table 11:

[0223] [0223]

[0224] 表11 =SARS病毒抗体检测标准工作曲线 [0224] TABLE 11 = SARS virus antibody standard curve

[0225] 3.以T/C值作为X,以SARS病毒抗体浓度作为Y绘制标准工作曲线,经统计拟和标准工作曲线的表达式为:Y = 5. 7365X+0. 8012,拟和系数的平方为:R2 = 0. 9841 ;结果见附图20 :SARS病毒抗体检测标准工作曲线。 [0225] 3. T / C values ​​as X, SARS virus antibody concentration as to draw the standard curve Y, the expression by standard curve fitting statistics as:. Y = 5. 7365X + 0 8012, fitting coefficients the squared: R2 = 0. 9841; results are drawings 20: SARS virus antibody standard curve.

[0226] 4. SARS病毒抗体浓度的计算公式为: [0226] 4. The formula for the SARS virus antibody concentration:

[0227]人血清中含有的 SARS 病毒抗体浓度(ng/ml) = IOY = IOX (5. 7365X+0. 8012)=57. 365X+8. 012 SARS virus antibody concentration (ng / ml) = IOY = IOX (5. 7365X + 0. 8012) = contained in [0227] human serum 57. 365X + 8. 012

[0228] 三、实际检测结果: [0228] Third, the actual test results:

[0229] I.检测准确性: [0229] I. test accuracy:

[0230] 将45份可能感染SARS病毒的病人血清(最终确诊其中含17份阳性,28份阴性)同时用酶联免疫吸附法(ELISA)与本系统(UP试纸条与传感器)进行双盲检测: [0230] 45 parts of the patient's serum may be infected with SARS virus (which contains 17 parts were finally diagnosed positive, negative 28 parts) the system simultaneously with the double-blind (UP test strip and sensor) by enzyme-linked immunosorbent assay (ELISA) detection:

[0231] ELISA法一17份阳性,28份阴性,与实际结果完全吻合,检测历时2小时左右; [0231] ELISA Method 17 parts of a positive and negative parts 28, completely consistent with the actual results, detected over about 2 hours;

[0232] UPT试纸条与传感器法一17份阳性,28份阴性,与实际结果完全吻合,检测历时半个小时左右; [0232] UPT method test strip and sensor 17 parts of a positive, negative 28 parts, completely consistent with the actual results, detected over about half an hour;

[0233] 与ELISA法相比,UPT试纸条与传感器法检测更为快速,且在ELISA法定性检测的基础上定量地给出了每份样品的最终精确浓度。 [0233] Compared with the ELISA assay, with the UPT strip sensor detected more quickly, and on the basis of ELISA methods for detection of a quantitative analysis of the final exact concentration of each sample.

[0234] 2.检测稳定性: [0234] 2. Stability detection:

[0235] 将一份SARS病人血清用pH = 7. 2 0. 03mol/L PB缓冲液10倍稀释,用UPT试纸条与传感器检测10次,结果列于下表12 : [0235] The sera from SARS patients with a pH = 7. 2 0. 03mol / L PB buffer was diluted 10 times, 10 times with UPT detected with the sensor strip, the results are shown in Table 12:

[0236] [0236]

[0237] 同一份血清重复测量的变异系数(CV) = 0. 819% [0237] coefficient of variation (CV) for repeated measurements with a serum = 0.819%

[0238] 表12 :SARS病毒抗体检测重复性 [0238] Table 12: SARS virus antibody test repeatability

[0239] 结论:在SARS病毒感染抗体检测中,UPT试纸条与传感器法与ELISA法相比更为快速、可实现精确定量检测,且稳定性很好。 [0239] Conclusion: SARS virus antibody in the detection, the UPT strip more rapidly as compared with the sensor method and ELISA method, quantitative detection can be accurate, and good stability.

具体实施方式 Detailed ways

[0240] 以下结合附图和实施例对本发明进行详细解释。 [0240] The present invention will be explained in detail in conjunction with the accompanying drawings and embodiments.

[0241] 附图21中显示了试纸条的一般结构包括:样品垫10 (Sample Pad),结合垫11(Conjugate Pad或结合物释放垫Conjugate Release Pad),分析膜12 (AnalyticalMembrane),吸水垫13 (fficking Pad)、塑料背板14 (其中一面涂有粘胶)(LaminatingCard)。 [0241] The general structure is shown in FIG. 21 of the test strip comprising: a sample pad 10 (Sample Pad), bond pads 11 (Conjugate Pad or conjugate pad Conjugate Release Pad), analytical membrane 12 (AnalyticalMembrane), an absorbent pad 13 (fficking Pad), a plastic back sheet 14 (one side coated with adhesive) (LaminatingCard). 样品垫10、结合垫11、分析膜12、吸水垫13通过粘胶15固定于塑料背板14上。 10 sample pad, conjugate pad 11, the analysis membrane 12, the absorbent pad 13 by the adhesive 15 is fixed to the back plate 14 of plastic.

[0242] a.样品垫10是使用过程中滴加被检样品的部位; . [0242] a sample pad portion 10 is a test sample is added dropwise during use;

[0243] b.结合垫11中固定有UCP-抗体、UCP-抗原等UCP-生物活性分子结合物,在加入检测样品后,其可在试纸条上发生免疫反应; . [0243] b binding UCP- pad 11 is fixed to an antibody, antigen, etc. UCP- UCP- biologically active molecule conjugate, after addition of the test sample, which may occur in immune response on the test strip;

[0244] c.分析膜12是层析试纸的核心部分,检测带17、质控带18均固定于分析膜12的某一具体位置; . [0244] c analysis film 12 is the core of the chromatography test strip, test line 17, control line 18 are fixed to a specific analysis of the position of the film 12;

[0245] d.吸水垫13在整个检测过程中,通过虹吸作用提供液体流过整个试纸条的动力。 [0245] d. Absorbent pad 13 over the entire course, there is provided a liquid flow throughout the power strip by siphon action.

[0246] 各个部分之间的重叠区域16保证了液体在试纸条上流动的连续性。 [0246] area of ​​overlap between the various portions 16 to ensure the continuity of liquid flow on the test strip. 当进行检测时,将样品滴加到样品垫10上,样品通过渗透与虹吸作用进入结合垫11,使其中的UCP结合物重新溶解游离,并在吸水垫13的虹吸作用下,离开结合垫11进入分析膜12,在其内部向吸水垫13的方向流动。 When detected, the sample was added dropwise to the sample pad 10, through penetration and siphon action sample into the bond pads 11, which enable the UCP binding redissolved free, and an absorbent pad 13 of the siphon action, leaving the bond pads 11 into the analytical membrane 12, the direction of flow of the absorbent pad 13 in its interior. 此过程中UCP结合物、目标被检物、检测带17、质控带18之间将特异地发生一定的免疫反应,并产生具有指示性的信号。 UCP process conjugate, the target analyte, detection of the belt 17, the control line will occur in certain specific immunological reaction between 18 and generates a signal indicative of.

[0247] 本发明中试纸条的装配,包括以下步骤: [0247] In the present invention, the test strip assembly, comprising the steps of:

[0248] A.将塑料背板14剪切成7. 4 X 30cm规格的条带; [0248] A. 14 cut into the plastic back plate 7. 4 X 30cm strip specifications;

[0249] B.将处理过的分析膜12粘于塑料背板14上21mm-46mm的位置,质控带18向上,检测带17向下; [0249] B. Analysis of the treated plastic film 12 adhered to the back plate 14 21mm-46mm position, the control line 18 upward, the detection zone 17 downwardly;

[0250] C.将处理过的结合垫11粘于塑料背板14上12mm的位置,上端压于分析膜12的下端,重叠Imm ; [0250] C. The treated plastic bonding pad 11 adhered to the back plate 14 12mm position, pressed against the upper end of the lower film 12 analysis, Imm, overlap;

[0251] D.将处理过的样品垫10粘于塑料背板14上,下端与塑料背板14平齐,上端压于结合垫11的下端,重叠3mm ; [0251] D. The treated sample pad 10 adhered to the back plate 14 of plastic, the plastic back plate 14 is flush with the lower end, the lower end of the upper end of the pressure pad 11 to the binding, overlapping 3mm;

[0252] E.将吸水垫13粘于塑料背板14上,上端与塑料背板14平齐,下端压于分析膜12的上端,重叠2mm ; [0252] E. The absorbent pad 13 adhered to a plastic backing plate 14, backing plate 14 is flush with the plastic upper, lower pressure on the upper end of the analytical membrane 12, overlapping 2mm;

[0253] F.将装配成形的条带用切条机剪切成4mm宽的试纸条; [0253] F. The assembly formed by the strip shear slitter into 4mm wide test strip;

[0254] G.将试纸条装入塑料外壳,干燥器中保存备用。 [0254] G. The test strip into plastic shell dryer to save backup.

[0255] 附图22中为粘贴时各部分之间的重叠关系。 Overlapping relationship between the portions of paste [0255] Figure 22 as.

[0256] 附图23为试纸条装配完毕后各部分的结构与尺寸,其中样品垫10重叠后的净暴露长度为15mm,结合垫11重叠后的净暴露长度为7mm,分析膜12重叠后的净暴露长度为22mm,吸水垫13重叠后的净暴露长度为30_。 After [0256] FIG 23 is a structure and dimensions of the parts of the assembled test strip, wherein after the net overlap sample pad 10 exposed length of 15mm, the net binding after the pad 11 exposed length of the overlap 7mm, analysis membrane 12 overlap net exposed length of 22mm, the net after the absorbent pad 13 exposed length of the overlap 30_. 装配完毕并经过剪切的试纸条便可以装入试纸条的外壳中。 Sheared and assembled test strip can mount the housing of the test strip.

[0257] 试纸条的外壳参见附图24,其包括加样孔20、结果判读窗口21和终点指示窗口22。 [0257] Referring to the drawings the housing of the test strip 24, which includes a sample hole 20, and the interpretation of the results window 21 indicating the end of window 22. 其中,结果判读窗口21中对应检测带17、质控带18。 Wherein the interpretation of the results corresponding to the window 21 in the detection zone 17, the control line 18. 所述终点指示窗口22中对应终点指示带19。 The end indicator window 22 indicating band 19 corresponding to the end point. 其中通过加样孔20将样品点加到试纸条上后,在吸水垫13的虹吸作用下液体样品依次经过检测带17、质控带18。 Wherein the test strip is added to the sample point 20 by the loading aperture 13 in the absorbent pad siphoning liquid sample 17 sequentially passes through the detection zone, the control line 18. 当终点指示带19变为绿色后,用生物传感器对外壳上结果判读窗口21中的各条带进行判读,就可以得到结果。 When the end turns green indicating band 19, interpretation of the results with a biosensor of the window 21 for interpretation of each strip on the housing, the results can be obtained.

[0258] 试纸条外壳分为上片和下片,参见附图25。23、24为咬合齿钉,将二者压紧便可以使试纸条外壳的上下片合为一体;25为挡板,26为倒钉,六个挡板25与三个倒钉26可将试纸条牢固地卡在外壳内,防止移动;27为压板,将上下片盖紧后,压板27的位置处于试纸条上样品垫10与结合垫11、结合垫11与分析膜12重叠处,以此来增加试纸条各部分的连接,保证检测中液体流动的连续性。 [0258] The test strip housing is divided into upper and lower pieces, see FIG 25.23,24 teeth to bite the staple, pressing both the test strip can cause upper and lower housing into one piece; stopper 25 plate, the nail 26 is inverted, six baffles 25 and 26 may be three reverse staple strip rigidly in the casing, preventing movement; platen 27, the upper and lower sheet tightly closed position of the platen 27 is in the test sample pad 10 and the bond pads 11 on the paper, in conjunction with the pads 11 and 12 overlap the film analysis, in order to increase the connection portions of the strip, to ensure continuity of the liquid flow detection.

[0259] 如图25所示,将试纸条放入外壳的下片,将上片与下片盖紧,便成为可以用于检测的成品。 [0259] As shown in FIG. 25, the test strip into the housing of the backsheet, the topsheet and backsheet tightly closed, it can be finished for detection.

[0260] 实施例I :上转换发光牛物传感器的结构 [0260] Example I: converting luminescence sensor structurally bovine

[0261] 参阅图26和图27。 [0261] Referring to FIGS. 26 and 27. 由图可知,本发明上转换发光生物传感器包括激发光路、磷光图像接收光路、图像处理系统,分别用于照明试纸条3、接收试纸条3发出的磷光图像、对试纸条3发出的磷光图像进行分析与处理,上转换发光生物传感器中试纸条结构示意图参阅图24。 The figure shows, the invention converts a luminescence biosensor includes an excitation light path, a phosphor image-receiving optical path, the image processing system, for illuminating the test strip 3, respectively, the image emitted from the phosphor 3 receiving a strip, a test strip 3 emitted phosphor image analysis and processing, converting luminescence biosensor test strip 24 a schematic structural diagram refer to FIG.

[0262] 在激发光路上,沿光轴依次设置红外激发光源I、一维聚焦镜2,其光轴为010。 [0262] In the optical path of the excitation, are sequentially disposed along the optical axis I of infrared excitation source, a two-dimensional focusing lens, the optical axis 010 thereof. 在磷光图像接收光路上,沿光轴依次设置前镜组5、滤光片6、后镜组7和图像接收器8,其光轴为002。 Phosphorescent image-receiving optical path, disposed along the optical axis, the front lens group 5, filter 6, 7 and the rear lens group of the image receiver 8, which is the optical axis 002. 磷光图像接收光路的物面与试纸条3的上表面重合,磷光图像接收光路的像面与图像接收器8的敏感面重合。 Surface of the test strip was a phosphor image-receiving surface of the light path coincides 3, the sensitive surface of the image plane with the phosphor pattern image receiver 8 receives light path coincides. 试纸条3安装于特制的外壳4中,其法线为003。 The test strip 3 is mounted in a special housing 4, which is a normal 003. 激发光路形成的焦线AA照明试纸条3,焦线AA与试纸条3的长边平行,且与试纸条3的对称线重合。 Focal line AA strip excitation light illumination path 3 is formed, and the focal line AA parallel to the long side of the test strip 3, and coincides with the line of symmetry 3 of the test strip. 激发光路的光轴010、试纸条3的法线003和磷光图像接收光路的光轴002位于同一个平面内,激发光路的光轴010与试纸条3的法线003的夹角为BI,磷光图像接收光路的光轴002与试纸条3的法线003的夹角为B2。 The angle between the normal to the optical axis 003 of the excitation light path 010, 003 normal to the optical axis of the test strip and a phosphorescent light path of the image receiving 3 002 lie in a common plane, the optical axis of the excitation light path 010 and the test strip 3 is BI , the angle between the normal to the optical axis 003 of the image receiving phosphorescent light path 002 and the test strip 3 is B2.

[0263] 所说的激发光路由红外激发光源I、一维聚焦镜2组成,其作用是产生一条强度为 [0263] The optical path of the infrared excitation of said excitation light source I, a 2-dimensional focusing lens, whose effect is to produce an intensity

0. 15瓦/平方厘米(W/cm2)的细长红外光焦线AA,照明试纸条3的所有功能带。 0.15 watts / square centimeter (W / cm2) of the elongated infrared focal line AA, with all the features of the lighting strip 3. 红外激发光源I通常是经过准直的半导体激光器,发出平行光束,其波长一般为980nm附近。 I infrared excitation light source is usually a collimated semiconductor laser, emits a parallel light beam with a wavelength typically near 980nm. 一维聚焦镜2可以是柱面透镜、棱镜或其他可以产生焦线的光学元件。 A 2-dimensional focusing mirror may be a cylindrical lens, a prism or other optical elements may produce focal line.

[0264] 所说的试纸条3上含有两个功能带,即检测带17、质控带18。 [0264] said test strip containing two functional band 3, i.e., detection zone 17, the control line 18. 其中检测带17将根据不同的检测模式与待检测样品中目标被检物以及UCP标记物发生特异性的免疫反应,其上结合UCP所产生的信号为T ;质控带18通过免疫反应结合UCP所产生的信号为C,T与C的比值,即T/C便是与不同浓度目标被检物对应的检测结果,其将与待检测样品中目标被检物的浓度呈一定的线形关系,同时C对于试纸条的生物学反应性能具有监控作用; Wherein the detection zone 17 will be different depending detection mode and a target to be detected in the sample and the analyte-specific marker UCP immune response, which in conjunction with the signal generated by UCP is T; the control line 18 by immunological binding reaction UCP the ratio of the generated signal is C, T and C, i.e. T / C is the target with varying concentrations corresponding to the detection result of the subject matter, which was certain to be a linear relationship with the concentration of the target sample to be tested for the analyte, C while having a performance monitoring function for the biological reaction of the test strip;

[0265] 所说的磷光图像接收光路由前镜组5、滤光片6、后镜组7和图像接收器8组成;磷光图像接收光路的物方孔径半角为U2。 [0265] The image receiving said phosphor front lens group optical path 5, filter 6, 7 and the rear lens group 8 composed of the image receiver; phosphor object side opening half-angle of the image-receiving optical path U2. 前镜组5将试纸条3上各功能带发出的磷光准直成平行光。 5 the front lens group 3 on the phosphor strip with the functions registration straight into parallel light emitted. 滤光片6滤除磷光信号中包含的杂光,以提高信噪比;它对磷光信号具有尽可能高的透过率(大于90% ),而对激发光具有尽可能低的透过率(小于10_5)。 6 phosphor was filtered off stray light filter included in the signal to noise ratio; phosphorescence signal having its transmittance as high as possible (greater than 90%), while the excitation light has a low transmittance as (less than 10_5). 后镜组7将滤除杂光后的磷光信号聚焦成像于图像接收器8的敏感面上。 The rear lens group 7 will filter phosphor focus imaging signal to the image-sensitive surface of the receiver 8 of the flare. 图像接收器8可以是一个线阵CXD摄像机,也可以是一个一维光电二极管阵列,其敏感元件的排列方向与试纸条3的细长方向一致。 The image receptor 8 may be a linear array camera CXD, may be a one-dimensional photodiode array, which array direction coincides with the sensitive element 3 in the elongated direction of the test strip. 所以图像接收器8可以对应地测量试纸条3上各功能带发出的磷光强度。 Therefore, the image receiver 8 may measure the phosphorescence intensity corresponding to each of the 3 test strip functional band emitted.

[0266] 所说的激发光路光轴010与试纸条3法线003的夹角为BI、以及磷光图像接收光路光轴002与试纸条3法线003的夹角为B2,且BI关B2,通常BI >B2-U2,或BI < B2-U2,U2为磷光图像接收光路的物方孔径半角。 [0266] an optical axis of said excitation light path 010 and the angle between the normal line 003 of the test strip 3 is BI, and a phosphor image-receiving optical path 002 and the angle between the optical axis of the test strip 3 is normal 003 B2, BI and off B2, typically BI> B2-U2, or BI <B2-U2, U2 is a phosphorescent light path of the image-receiving object side opening half-angle. 这种设计的目的是阻止照明光线的反射光进入磷光图像接收光路,以减小杂光。 The purpose of this design is to prevent light reflected illumination light enters a phosphor image-receiving optical path, in order to reduce stray light.

[0267] 与在先技术相比,本发明的特点在于:激发光路产生高强度红外激光焦线AA,同时将试纸条3上的各功能带照明;磷光图像接收光路对试纸条3上的各功能带同时成像;夹角BI与夹角B2不等,且满足BI > B2-U2,或BI < B2-U2。 [0267] Compared with the prior art, the present invention is characteristic in that: the excitation beam path to produce high-intensity infrared laser focal line AA, while the functional test strip illuminated on 3; an image on the phosphor receiving the light path of the strip 3 with the respective functions simultaneously imaged; BI angle and angle B2 unequal, and satisfy BI> B2-U2, or BI <B2-U2.

[0268] 上述特点使本发明具有判读效率高、判读灵敏度高、可进行多重定量检测等优点。 [0268] The above features of the present invention has the interpretation of high efficiency, high sensitivity interpretation may be made quantitative detection of multiple advantages.

[0269] 本发明上转换发光生物传感器的工作过程是:首先将安装于外壳4中的试纸条3放到判读位置。 [0269] converting luminescence biosensor of the present invention on the working process is: firstly attached to the housing 4 of the strip 3 position into interpretation. 由红外激发光源I发出的平行光束经一维聚焦镜2形成焦线AA,焦线AA位于试纸条3的上表面,且与试纸条3的细长方向平行,这样将试纸条3上的各功能带全部照明。 Parallel beam by the infrared excitation light I emitted by the one-dimensional focusing mirror 2 is formed focal line AA, AA focal line is located on the surface of the test strip 3 and parallel to the elongate direction of the strip 3, so that the test strip 3 each band on all of the illumination function. 由试纸条3上各功能带发出的磷光信号经前镜组5准直后变成平行光,经滤光片6滤除杂光后被后镜组7聚焦成像于图像接收器8的敏感面上。 Phosphorescent signal emitted by each functional belt 3 after the strip 5 collimated into a parallel light front lens group, the stray light filtered through the filter 6 after the rear focus imaging lens group 7 is sensitive to the image receiver 8 surface. 图像处理系统9对图像接收器8输出的试纸条磷光图像进行分析与处理,给出试纸条3上各功能带磷光信号的幅度,进而给出被检生物分子的属性和含量。 The test strip phosphorescent image output of an image processing system 9 on the image receiver 8 for analysis and processing, given the magnitude of the test strip with the functions of the phosphorescence signal 3, and then given attributes and content of the subject biomolecules.

[0270] 图26和图27是本发明的最佳实施例,其具体结构和参数叙述如下: [0270] FIG. 26 and FIG. 27 is a preferred embodiment of the present invention, the detailed structure and parameters are described below:

[0271] 激发光路中的红外激发光源I发出的平行光束的截面尺寸为4mmXlmm,中心波长为980nm,功率为30mW ;—维聚焦镜2为平凸柱面透镜,其焦距为20mm。 [0271] The excitation light path infrared excitation sectional dimension parallel beams emitted from the light source I is 4mmXlmm, a center wavelength of 980nm, power is 30mW; - 2-dimensional focusing lens plano-convex cylindrical lens, the focal length of 20mm. 焦线AA的尺寸为20mmX Imm,略小于试纸条3的结果判读窗口21尺寸。 Focal line AA size 20mmX Imm, slightly less than the results of test strip 21 3 interpretation window size. 结果判读窗口21中试纸条3上的两条带分别为:距终点指示窗一侧结果判读窗口21内沿12. 2mm处为检测带17、7. 2mm处为质控带18。 Results Interpretation window 21 in two bands on the test strip 3, respectively: a side away from the end indicator window 12. 2mm interpretation of the results in the window 21 to detect a band of 17,7 2mm at the control line 18. 研究中采用的上转换磷光材料是(YYbEr) F3 (氟化钇镱铒),在红外光激发下发出的磷光光谱如图28所示,其主峰值波长为541. 5nm。 Conversion phosphorescent material is used in the study (YYbEr) F3 (yttrium fluoride ytterbium erbium), phosphorescence spectrum of infrared light emitted by excitation at 28, the main peak wavelength of 541. 5nm. 磷光图像接收光路中的前镜组5的焦距为40mm,物方孔径半角U2 = 20° ;滤光片6的光谱透过率曲线如图29所示,在541. 5nm波长处的透过率大于90%,而在980nm波长处的透过率小于10'后镜组7的焦距为40mm,所以磷光图像接收光路的放大倍率为-I倍。 Focal length front lens group in the optical path of the image receiving phosphor 5 is 40mm, the object side opening half-angle U2 = 20 °; the spectral transmittance curve of the filter 6 shown in Figure 29, the transmittance at a wavelength of 541. 5nm more than 90%, while transmittance at a wavelength of 980nm is less than 10 'is the focal length of the rear lens group of 7 to 40mm, so the magnification of the image receiving phosphorescent light path is -I times. 图像接收器8是一个线阵CCD摄像机,其敏感面长度为22mm,共含有2200个像素,像素尺寸为lOiimXlOym。 8 is an image receiver array CCD line camera is sensitive surface length of 22mm, containing a total of 2200 pixels, pixel size lOiimXlOym. 所以图像接收器8对试纸条3的空间判读分辨率为10 um。 Therefore, the image receiver 8 pairs of strip 3 interpretation space resolution 10 um.

[0272] 最佳实施例对纯上转换磷光材料的判读灵敏度优于IU g/L,可同时检测4种以上的生物物质。 [0272] preferred embodiments of the conversion sensitivity interpretation phosphorescent material than pure IU g / L, can detect four or more biomass.

[0273] 实施例2 :双抗体夹心检测乙肝表面抗原HBs-Ag : [0273] Example 2: Double antibody sandwich hepatitis B surface antigen HBs-Ag:

[0274] (I)免疫层析液相材料准备: [0274] (I) an immunochromatographic liquid material preparation:

[0275] A. UCP-抗体结合物: [0275] A. UCP- antibody conjugate:

[0276] a.利用已建立的表面修饰与活化方法对直径200_300nm的UCP颗粒进行表面修饰,并与抗乙肝表面抗原HBs-Ag单克隆抗体进行连接,在UCP保存液(pH = 7. 2 0. 03mol/LPB 缓冲液中,含0. 1% BSA,0. 05% Tween20、0. 02% NaN3)中以浓度lmg/mL,4°C保存备用; [0276] a. Surface modification and activation methods using established particle diameter of UCP 200_300nm the surface modification, and with an anti-HBsAg monoclonal antibody HBs-Ag connection, UCP preservation solution (pH = 7. 2 0 ... in 03mol / LPB buffer containing 0. 1% BSA, 0 05% Tween20,0 02% NaN3) at a concentration of lmg / mL, 4 ° C were stored;

[0277] b.将保存于UCP保存液中的lmg/mL UCP-抗体结合物6mL,12000r/min, 4°C,离心30min,尽量弃尽上清; . [0277] b stored in the UCP preservation solution lmg / mL UCP- antibody conjugate 6mL, 12000r / min, 4 ° C, centrifuged 30min, try to make the supernatant was discarded;

[0278] c.向离心管中的UCP-抗体结合物沉降,加入3mL结合物稀释液(pH =7. 20. 03mol/LPB缓冲液中,含I %蔗糖、I % BSA)涡旋充分混匀(终浓度:2mg/mL UCP-抗体结合物) [0278] c. UCP- antibody binding to sedimentation in a centrifuge tube, diluted conjugate was added 3mL solution (pH = 7. 20. 03mol / LPB buffer containing I% sucrose, I% BSA) mixed thoroughly vortexed homogenizer (final concentration: 2mg / mL UCP- antibody conjugate)

[0279] d.将悬浊液倒入试剂瓶中,4°C保存备用; . [0279] d The suspension was poured into a vial, 4 ° C were stored;

[0280] B.样品垫封闭液: [0280] B. Sample pad blocking solution:

[0281] a.用天平精确称量BSA(牛血清白蛋白)lg,放入小烧杯中; . [0281] a precisely weighed with a balance BSA (bovine serum albumin) lg, into a small beaker;

[0282] b.烧杯中加入pH = 7. 2 0. 03mol/L PB缓冲液20mL,玻璃棒搅拌充分混匀; . [0282] b beaker, pH = 7. 2 0. 03mol / L PB buffer 20mL, mix thoroughly stirred glass rod;

[0283] c. BAS溶液中加入Tween 20 (吐温20) 20 UL,玻璃棒搅拌充分混匀(终浓度:5%BAS, 0. 1% Tween 20); [0283] was added Tween 20 (Tween 20) 20 UL c BAS, a glass rod with stirring and mix well (final concentration: 5% BAS, 0. 1% Tween 20).;

[0284] d. 4°C保存备用; . [0284] d 4 ° C were stored;

[0285] C.检测带蛋白: [0285] C. Protein detection zone:

[0286] a.用天平精确称量纯化的兔抗HBs-Ag多克隆抗体2mg,置于I. 5mL的微离心管中; . [0286] a precisely weighed with a balance purified rabbit polyclonal antibody anti-HBs-Ag 2mg, placed in microcentrifuge tubes in I. 5mL;

[0287] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL); . [0287] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL);

[0288] c.分装为50 ii L每管,_20°C冻存备用; . [0288] c to 50 ii L aliquoted per tube, _20 ° C frozen for later use;

[0289] D.质控带蛋白; [0289] D. Protein control line;

[0290] a.用天平精确称量纯化的羊抗鼠IgG抗体2mg,置于I. 5mL的微离心管中; . [0290] a precisely weighed with a balance of purified goat anti-mouse IgG antibody 2mg, placed in microcentrifuge tubes in I. 5mL;

[0291] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL); . [0291] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL);

[0292] c.分装为50 ii L每管,_20°C冻存备用; . [0292] c to 50 ii L aliquoted per tube, _20 ° C frozen for later use;

[0293] (2)免疫层析固相载体材料准备: [0293] (2) an immunochromatographic solid support material preparation:

[0294] A.样品垫: [0294] A. sample pad:

[0295] a.选用纤维素膜(Cellulose Membrane)作为样品垫固相材料,将其剪切成 [0295] a. Selection cellulose membrane (Cellulose Membrane) As the sample pad solid phase material which was cut into

I. 5X30. Ocm规格的条带; . I. 5X30 Ocm specifications of the strip;

[0296] b.将样品垫放入长形平皿中,样品垫封闭液加于其上,常温浸泡30min ; . [0296] b The sample pads were placed in the elongate plate, blocking fluid on sample pad thereon, soak at room temperature for 30 min;

[0297] c.将样品垫由封闭液中取出,放于干净的平皿中; . [0297] c removed from the sample pad blocking solution, placed in clean petri dish;

[0298] d. 37°C烘干3小时,使样品垫充分干燥; . [0298] d 37 ° C drying for 3 hours, the sample was sufficiently dried pad;

[0299] e.封闭后的样品垫在干燥的环境中保存备用; [0299] e is closed after the sample pad in a dry environment to save backup.;

[0300] B.结合垫: [0300] B. conjugate pad:

[0301] a.选用玻璃纤维素膜(Glass Fiber)作为结合垫固相材料,将其剪切成1.0X30. Ocm规格的条带; . [0301] a cellulose membrane use glass (Glass Fiber) as a solid phase material conjugate pad, which was cut into strips 1.0X30 Ocm specifications.;

[0302] b.将4°C保存备用的2mg/mL UCP-抗体结合物(pH = 7.2 0. 03mol/L PB缓冲液,含1%蔗糖、1% BSA)超声IOs ; . [0302] b will alternate at 4 ° C 2mg / mL UCP- antibody conjugate (pH = 7.2 0. 03mol / L PB buffer containing 1% sucrose, 1% BSA) ultrasound IOs;

[0303] c.将结合垫放入长形平皿中,UCP-抗体结合物悬浊液加于其上; . [0303] c binding pads into elongated dish, UCP- antibody conjugate suspension was applied thereto;

[0304] d.将结合垫取出放于干净的平皿中; . [0304] d will be put out to the bonding pads clean dishes;

[0305] e. 37°C烘干2. 5小时,使结合垫充分干燥; . [0305] e 37 ° C 2.5 hours drying the bonding pad sufficiently dried;

[0306] f.处理后的结合垫在干燥的环境中保存备用; . [0306] f pad binding treated in a dry environment to save backup;

[0307] C.分析膜: [0307] C. Analysis of the film:

[0308] a.以孔径为12 iim的硝酸纤维素膜(Nitrocellulose Membrane)作为固相材料,将其剪切成2. 5 X 30cm规格的条带;; [0308] a. A nitrocellulose membrane (Nitrocellulose Membrane) to the aperture 12 iim as a solid phase material which was cut into a size of 2. 5 X 30cm strip ;;

[0309] b.用点样仪在2. 5cm宽的分析膜上,从下向上Icm处喷点2mg/mL兔抗HBs-Ag多克隆抗体,2 u L/cm,作为检测带; . [0309] b 2. 5cm wide analysis of the film, using a spotter spray upwardly from the point at Icm 2mg / mL rabbit polyclonal anti-HBs-Ag, 2 u L / cm, as a detection zone;

[0310] c.用点样仪在2. 5cm宽的分析膜上,从下向上I. 5cm处喷点2mg/mL羊抗鼠IgG抗体,2 u L/cm,作为质控带; . [0310] c 2. 5cm wide analysis of the film, using a spotter spray upwardly I. 5cm from the point at 2mg / mL goat anti-mouse IgG antibody, 2 u L / cm, as a control line;

[0311] d. 37°C烘干2小时,使分析膜充分干燥; . [0311] d 37 ° C and drying for 2 hours to sufficiently dry analysis membrane;

[0312] e.点样后的分析膜在干燥的环境中保存备用; [0312] e after spotting analytical membrane were stored in a dry environment.;

[0313] D.吸水垫: [0313] D. absorbent pad:

[0314] a.选用纤维素膜(Cellulose Membrane)作为吸水垫固相材料,将其剪切成3. OX 30cm规格的条带; . [0314] a selection cellulose membrane (Cellulose Membrane) as a solid phase absorbent pad material, which is cut into a size of 3. OX 30cm strip;

[0315] b.将变色范围5. 5-9. 0的精密pH试纸固定于吸水垫从下向上2. Ocm处,作为终点指示带; .. [0315] b the color range of pH 5. 5-9 0 precision test strip is fixed to the absorbent pad 2. Ocm from the bottom up, as the tape end indication;

[0316] c.吸水垫在干燥的环境中保存备用; . [0316] c absorbent pad in a dry environment to save backup;

[0317] (3)免疫层析试纸条检测 [0317] (3) detecting the immunochromatographic strip

[0318] A.将待检测血清样品用pH = 7. 2 0. 03mol/L PB缓冲液10倍稀释; [0318] A. serum sample to be detected is diluted with pH = 7. 2 0. 03mol / L PB buffer 10 times;

[0319] B. 100 u L稀释后的样品加于试纸条外壳上的加样孔中; [0319] After the sample B. 100 u L diluted in loading hole test strip housing;

[0320] C.待终点指示窗口中的终点指示带变为绿色后,便可以用传感器判读外壳上结果判读窗口中的检测带与质控带,以得出结果。 [0320] C. until the end of the window indicate the end turns green indicating the tape, can interpret results interpreted by a sensor and the control line with a window on the housing, in order to obtain the result.

[0321] 实施例3 :双抗体夹心检测冠状病毒(SARS病毒): [0321] Example 3: Double antibody sandwich coronavirus (SARS virus):

[0322] (I)免疫层析液相材料准备: [0322] (I) an immunochromatographic liquid material preparation:

[0323] A. UCP-抗体结合物: [0323] A. UCP- antibody conjugate:

[0324] a.利用已建立的表面修饰与活化方法对直径200_300nm的UCP颗粒进行表面修饰,并与纯化的兔抗SARS病毒抗体进行连接,在UCP保存液(pH = 7.2 0. 03mol/L PB缓冲液中,含0. 1% BSA,0. 05% Tween20,0. 02% NaN3)中以浓度lmg/mL,4°C保存备用; [0324] a. Surface modification and activation methods using established particle diameter of UCP 200_300nm the surface modification, and connected to the purified rabbit anti-SARS antibodies in UCP preservation solution (pH = 7.2 0. 03mol / L PB buffer containing 0. 1% BSA, the 0 05% Tween20,0 02% NaN3) at a concentration of lmg / mL, 4 ° C for use..;

[0325] b.将保存于UCP保存液中的lmg/mL UCP-抗体结合物6mL,12000r/min, 4°C,离心30min,尽量弃尽上清; . [0325] b stored in the UCP preservation solution lmg / mL UCP- antibody conjugate 6mL, 12000r / min, 4 ° C, centrifuged 30min, try to make the supernatant was discarded;

[0326] c.向离心管中的UCP-抗体结合物沉降,加入3mL结合物稀释液(pH = 7. 2 [0326] c. UCP- antibody binding to sedimentation in a centrifuge tube, diluted conjugate was added 3mL solution (pH = 7. 2

0.03mol/LPB缓冲液中,含1%蔗糖、1% BSA)涡旋充分混匀(终浓度:2mg/mL UCP-抗体结合物) 0.03mol / LPB buffer containing 1% sucrose, 1% BSA) vortex and mix well (final concentration: 2mg / mL UCP- antibody conjugate)

[0327] d.将悬浊液倒入试剂瓶中,4°C保存备用; . [0327] d The suspension was poured into a vial, 4 ° C were stored;

[0328] B.样品垫封闭液: [0328] B. Sample pad blocking solution:

[0329] a.用天平精确称量BSA(牛血清白蛋白)lg,放入小烧杯中; . [0329] a precisely weighed with a balance BSA (bovine serum albumin) lg, into a small beaker;

[0330] b.烧杯中加入pH = 7. 2 0. 03mol/L PB缓冲液20mL,玻璃棒搅拌充分混匀; . [0330] b beaker, pH = 7. 2 0. 03mol / L PB buffer 20mL, mix thoroughly stirred glass rod;

[0331] c. BAS溶液中加入Tween 20 (吐温20) 20 UL,玻璃棒搅拌充分混匀(终浓度:5%BAS, 0. 1% Tween 20); [0331] was added Tween 20 (Tween 20) 20 UL c BAS, a glass rod with stirring and mix well (final concentration: 5% BAS, 0. 1% Tween 20).;

[0332] d. 4°C保存备用; . [0332] d 4 ° C were stored;

[0333] C.检测带蛋白: [0333] C. Protein detection zone:

[0334] a.用天平精确称量纯化的羊抗SARS病毒抗体2mg,置于I. 5mL的微离心管中; . [0334] a precisely weighed with a balance of purified goat anti-SARS antibodies 2mg, placed in microcentrifuge tubes in I. 5mL;

[0335] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL); . [0335] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL);

[0336] c.分装为50 ii L每管,_20°C冻存备用; . [0336] c to 50 ii L aliquoted per tube, _20 ° C frozen for later use;

[0337] D.质控带蛋白; [0337] D. Protein control line;

[0338] a.用天平精确称量纯化的羊抗兔IgG抗体2mg,置于I. 5mL微离心管中; . [0338] a precisely weighed with a balance of purified goat anti-rabbit IgG antibody 2mg, I. 5mL placed in microcentrifuge tubes;

[0339] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL); . [0339] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL);

[0340] c.分装为50 ii L每管,-20°C冻存备用; . [0340] c is aliquoted per tube 50 ii L, -20 ° C frozen for later use;

[0341] (2)免疫层析固相载体材料准备: [0341] (2) an immunochromatographic solid support material preparation:

[0342] A.样品垫: [0342] A. sample pad:

[0343] a.选用纤维素膜(Cellulose Membrane)作为样品垫固相材料,将其剪切成 [0343] a. Selection cellulose membrane (Cellulose Membrane) As the sample pad solid phase material which was cut into

I. 5X30. Ocm规格的条带; . I. 5X30 Ocm specifications of the strip;

[0344] b.将样品垫放入长形平皿中,样品垫封闭液加于其上,常温浸泡30min ; . [0344] b The sample pads were placed in the elongate plate, blocking fluid on sample pad thereon, soak at room temperature for 30 min;

[0345] c.将样品垫由封闭液中取出,放于干净的平皿中; . [0345] c removed from the sample pad blocking solution, placed in clean petri dish;

[0346] d. 37°C烘干3小时,使样品垫充分干燥; . [0346] d 37 ° C drying for 3 hours, the sample was sufficiently dried pad;

[0347] e.封闭后的样品垫在干燥的环境中保存备用; [0347] e is closed after the sample pad in a dry environment to save backup.;

[0348] B.结合垫: [0348] B. conjugate pad:

[0349] a.选用玻璃纤维素膜(Glass Fiber)作为结合垫固相材料,将其剪切成1.0X30. Ocm规格的条带; . [0349] a cellulose membrane use glass (Glass Fiber) as a solid phase material conjugate pad, which was cut into strips 1.0X30 Ocm specifications.;

[0350] b.将4°C保存备用的2mg/mL UCP-抗体结合物(pH = 7.2 0. 03mol/L PB缓冲液,含1%蔗糖、1% BSA)超声IOs ; . [0350] b will alternate at 4 ° C 2mg / mL UCP- antibody conjugate (pH = 7.2 0. 03mol / L PB buffer containing 1% sucrose, 1% BSA) ultrasound IOs;

[0351] c.将结合垫放入长形平皿中,UCP-抗体结合物悬浊液加于其上; . [0351] c binding pads into elongated dish, UCP- antibody conjugate suspension was applied thereto;

[0352] d.将结合垫取出放于干净的平皿中; . [0352] d will be put out to the bonding pads clean dishes;

[0353] e. 37°C烘干2. 5小时,使结合垫充分干燥; . [0353] e 37 ° C 2.5 hours drying the bonding pad sufficiently dried;

[0354] f.处理后的结合垫在干燥的环境中保存备用; . [0354] f pad binding treated in a dry environment to save backup;

[0355] C.分析膜: [0355] C. Analysis of the film:

[0356] a.以孔径为12 iim的硝酸纤维素膜(Nitrocellulose Membrane)作为固相材料,将其剪切成2. 5 X 30cm规格的条带;; [0356] a. A nitrocellulose membrane (Nitrocellulose Membrane) to the aperture 12 iim as a solid phase material which was cut into a size of 2. 5 X 30cm strip ;;

[0357] b.用点样仪在2. 5cm宽的分析膜上,从下向上Icm处喷点2mg/mL羊抗SARS病毒抗体,2 u L/cm,作为检测带; . [0357] b 2. 5cm wide analysis of the film, using a spotter spray upwardly from the point at Icm 2mg / mL goat anti-SARS antibodies, 2 u L / cm, as a detection zone;

[0358] c.用点样仪在2. 5cm宽的分析膜上,从下向上I. 5cm处喷点2mg/mL羊抗兔IgG抗体,2 u L/cm,作为质控带; . [0358] c 2. 5cm wide analysis of the film, using a spotter spray upwardly I. 5cm from the point at 2mg / mL goat anti-rabbit IgG antibody, 2 u L / cm, as a control line;

[0359] d. 37°C烘干2小时,使分析膜充分干燥; . [0359] d 37 ° C and drying for 2 hours to sufficiently dry analysis membrane;

[0360] e.点样后的分析膜在干燥的环境中保存备用; [0360] e after spotting analytical membrane were stored in a dry environment.;

[0361] D.吸水垫: [0361] D. absorbent pad:

[0362] a.选用纤维素膜(Cellulose Membrane)作为吸水垫固相材料,将其剪切成3. OX 30cm规格的条带; . [0362] a selection cellulose membrane (Cellulose Membrane) as a solid phase absorbent pad material, which is cut into a size of 3. OX 30cm strip;

[0363] b.将变色范围5. 5-9. 0的精密pH试纸固定于吸水垫从下向上2. Ocm处,作为终点指示带; .. [0363] b the color range of pH 5. 5-9 0 precision test strip is fixed to the absorbent pad 2. Ocm from the bottom up, as the tape end indication;

[0364] c.吸水垫在干燥的环境中保存备用; . [0364] c absorbent pad in a dry environment to save backup;

[0365] (3)免疫层析试纸条检测: [0365] (3) Immunochromatographic strip tests:

[0366] A.将待检测血清样品用pH = 7. 2 0. 03mol/L PB缓冲液10倍稀释; [0366] A. serum sample to be detected is diluted with pH = 7. 2 0. 03mol / L PB buffer 10 times;

[0367] B. 100 UL稀释后的样品加于试纸条外壳上的加样孔中; Diluted sample [0367] B. 100 UL loading applied to the holes in the strip housing;

[0368] C.待终点指示窗口中的终点指示带变为绿色后,便可以用传感器判读外壳上结果判读窗口中的检测带与质控带,以得出结果。 [0368] C. until the end of the window indicate the end turns green indicating the tape, can interpret results interpreted by a sensor and the control line with a window on the housing, in order to obtain the result.

[0369] 实施例4 :鼠疫FI-Ag (鼠疫FI-抗原)检测: [0369] Example 4: Plague FI-Ag (FI- plague antigen) detection:

[0370] (I)免疫层析液相材料准备: [0370] (I) an immunochromatographic liquid material preparation:

[0371 ] A. UCP-抗体结合物: [0371] A. UCP- antibody conjugate:

[0372] a.利用已建立的表面修饰与活化方法对直径200_300nm的UCP颗粒进行表面修饰,并与纯化的兔抗鼠疫抗体进行连接,在UCP保存液(pH = 7. 2 0. 03mol/L PB缓冲液中,含0. 1% BSA,0. 05% Tween20、0. 02% NaN3)中以浓度lmg/mL,4°C保存备用; [0372] a. Surface modification and activation methods using established particle diameter of UCP 200_300nm the surface modification, and connected to the purified rabbit anti-plague antibody in UCP preservation solution (pH = 7. 2 0. 03mol / L PB buffer containing 0. 1% BSA, the 0 05% Tween20,0 02% NaN3) at a concentration of lmg / mL, 4 ° C for use..;

[0373] b.将保存于UCP保存液中的lmg/mL UCP-抗体结合物6mL,12000r/min, 4°C,离心30min,尽量弃尽上清; . [0373] b stored in the UCP preservation solution lmg / mL UCP- antibody conjugate 6mL, 12000r / min, 4 ° C, centrifuged 30min, try to make the supernatant was discarded;

[0374] c.向离心管中的UCP-抗体结合物沉降,加入3mL结合物稀释液(pH = 7. 2 [0374] c. UCP- antibody binding to sedimentation in a centrifuge tube, diluted conjugate was added 3mL solution (pH = 7. 2

0.03mol/LPB缓冲液中,含1%蔗糖、1% BSA)涡旋充分混匀(终浓度:2mg/mL UCP-抗体结合物) 0.03mol / LPB buffer containing 1% sucrose, 1% BSA) vortex and mix well (final concentration: 2mg / mL UCP- antibody conjugate)

[0375] d.将悬浊液倒入试剂瓶中,4°C保存备用; . [0375] d The suspension was poured into a vial, 4 ° C were stored;

[0376] B.样品垫封闭液: [0376] B. Sample pad blocking solution:

[0377] a.用天平精确称量BSA(牛血清白蛋白)lg,放入小烧杯中; . [0377] a precisely weighed with a balance BSA (bovine serum albumin) lg, into a small beaker;

[0378] b.烧杯中加入pH = 7. 2 0. 03mol/L PB缓冲液20mL,玻璃棒搅拌充分混匀; . [0378] b beaker, pH = 7. 2 0. 03mol / L PB buffer 20mL, mix thoroughly stirred glass rod;

[0379] c. BAS溶液中加入Tween 20 (吐温20) 20 UL,玻璃棒搅拌充分混匀(终浓度:5%BAS, 0. 1% Tween 20); [0379] was added Tween 20 (Tween 20) 20 UL c BAS, a glass rod with stirring and mix well (final concentration: 5% BAS, 0. 1% Tween 20).;

[0380] d. 4°C保存备用; . [0380] d 4 ° C were stored;

[0381] C.检测带蛋白: [0381] C. Protein detection zone:

[0382] a.用天平精确称量鼠疫FI-抗原2mg,置于I. 5mL的微离心管中; [0382] a 2mg accurately weighed with a balance plague FI- antigen, I. microcentrifuge tube was placed in 5mL.;

[0383] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL); . [0383] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL);

[0384] c.分装为50 ii L每管,_20°C冻存备用; . [0384] c to 50 ii L aliquoted per tube, _20 ° C frozen for later use;

[0385] D.质控带蛋白: [0385] D. Protein control line:

[0386] a.用天平精确称量纯化的羊抗兔IgG抗体2mg,置于I. 5mL的微离心管中; . [0386] a precisely weighed with a balance of purified goat anti-rabbit IgG antibody 2mg, placed in microcentrifuge tubes in I. 5mL;

[0387] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL); . [0387] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL);

[0388] c.分装为50 ii L每管,_20°C冻存备用; . [0388] c to 50 ii L aliquoted per tube, _20 ° C frozen for later use;

[0389] (2)免疫层析固相载体材料准备: [0389] (2) an immunochromatographic solid support material preparation:

[0390] A.样品垫: [0390] A. sample pad:

[0391] a.选用纤维素膜(Cellulose Membrane)作为样品垫固相材料,将其剪切成 [0391] a. Selection cellulose membrane (Cellulose Membrane) As the sample pad solid phase material which was cut into

1. 5X30. Ocm规格的条带; . 1. 5X30 Ocm specifications strip;

[0392] b.将样品垫放入长形平皿中,样品垫封闭液加于其上,常温浸泡30min ; . [0392] b The sample pads were placed in the elongate plate, blocking fluid on sample pad thereon, soak at room temperature for 30 min;

[0393] c.将样品垫由封闭液中取出,放于干净的平皿中; . [0393] c removed from the sample pad blocking solution, placed in clean petri dish;

[0394] d. 37°C烘干3小时,使样品垫充分干燥; . [0394] d 37 ° C drying for 3 hours, the sample was sufficiently dried pad;

[0395] e.封闭后的样品垫在干燥的环境中保存备用; [0395] e is closed after the sample pad in a dry environment to save backup.;

[0396] B.结合垫: [0396] B. conjugate pad:

[0397] a.选用玻璃纤维素膜(Glass Fiber)作为结合垫固相材料,将其剪切成1.0X30. Ocm规格的条带; . [0397] a cellulose membrane use glass (Glass Fiber) as a solid phase material conjugate pad, which was cut into strips 1.0X30 Ocm specifications.;

[0398] b.将4°C保存备用的2mg/mL UCP-抗体结合物(pH = 7.2 0. 03mol/L PB缓冲液,含1%蔗糖、1% BSA)超声IOs ; . [0398] b will alternate at 4 ° C 2mg / mL UCP- antibody conjugate (pH = 7.2 0. 03mol / L PB buffer containing 1% sucrose, 1% BSA) ultrasound IOs;

[0399] c.将结合垫放入长形平皿中,UCP-抗体结合物悬浊液加于其上; . [0399] c binding pads into elongated dish, UCP- antibody conjugate suspension was applied thereto;

[0400] d.将结合垫取出放于干净的平皿中; . [0400] d will be put out to the bonding pads clean dishes;

[0401] e. 37°C烘干2. 5小时,使结合垫充分干燥; . [0401] e 37 ° C 2.5 hours drying the bonding pad sufficiently dried;

[0402] f.处理后的结合垫在干燥的环境中保存备用; . [0402] f pad binding treated in a dry environment to save backup;

[0403] C.分析膜: [0403] C. Analysis of the film:

[0404] a.以孔径为12 iim的硝酸纤维素膜(Nitrocellulose Membrane)作为固相材料,将其剪切成2. 5 X 30cm规格的条带;; [0404] a. A nitrocellulose membrane (Nitrocellulose Membrane) to the aperture 12 iim as a solid phase material which was cut into a size of 2. 5 X 30cm strip ;;

[0405] b.用点样仪在2. 5cm宽的分析膜上,从下向上Icm处喷点2mg/mL鼠疫FI-抗原,2 UL/cm,作为检测带; . [0405] b 2. 5cm wide analysis of the film dots 2mg / mL antigen from Yersinia FI- upward at the lower Icm using a spotter, 2 UL / cm, as a detection zone;

[0406] c.用点样仪在2. 5cm宽的分析膜上,从下向上I. 5cm处喷点2mg/mL羊抗兔IgG抗体,2 u L/cm,作为质控带; . [0406] c 2. 5cm wide analysis of the film, using a spotter spray upwardly I. 5cm from the point at 2mg / mL goat anti-rabbit IgG antibody, 2 u L / cm, as a control line;

[0407] d. 37°C烘干2小时,使膜充分干燥; . [0407] d 37 ° C and drying for 2 hours and the membrane was dried sufficiently;

[0408] e.点样后的分析膜在干燥的环境中保存备用; [0408] e after spotting analytical membrane were stored in a dry environment.;

[0409] D.吸水垫: [0409] D. absorbent pad:

[0410] a.选用纤维素膜(Cellulose Membrane)作为吸水垫固相材料,将其剪切成3. OX 30cm规格的条带; . [0410] a selection cellulose membrane (Cellulose Membrane) as a solid phase absorbent pad material, which is cut into a size of 3. OX 30cm strip;

[0411] b.将变色范围5. 5-9.0的精密pH试纸固定于吸水垫从下向上2. Ocm处,作为终点指示带; . [0411] b the color range of pH 5. 5-9.0 precision test strip is fixed to the absorbent pad 2. Ocm from the bottom up, as the tape end indication;

[0412] d.吸水垫在干燥的环境中保存备用; . [0412] d absorbent pad in a dry environment to save backup;

[0413] (3)免疫层析试纸条检测: [0413] (3) Immunochromatographic strip tests:

[0414] A.将待检测样品用pH = 7. 2 0. 03mol/L PB缓冲液10倍稀释; [0414] A. The sample to be tested was diluted with pH = 7. 2 0. 03mol / L PB buffer 10 times;

[0415] B. 100 u L稀释后的样品加于试纸条外壳上的加样孔中; [0415] After the sample B. 100 u L diluted in loading hole test strip housing;

[0416] C.待终点指示窗口中的终点指示带变为绿色后,便可以用传感器判读外壳上结果判读窗口中的检测带与质控带,以得出结果。 [0416] C. until the end of the window indicate the end turns green indicating the tape, can interpret results interpreted by a sensor and the control line with a window on the housing, in order to obtain the result.

[0417] 实施例5 :违禁药品检泖I [0417] Example 5: illegal drugs subject Mao I

[0418] (I)免疫层析液相材料准备: [0418] (I) an immunochromatographic liquid material preparation:

[0419] A. UCP-抗体结合物: [0419] A. UCP- antibody conjugate:

[0420] a.利用已建立的表面修饰与活化方法对直径200_300nm的UCP颗粒进行表面修饰,并与纯化的兔抗违禁药品抗体或结合配基(违禁药品包括:安非他命、甲基安非他命、苯环己哌啶和鸦片酊等药物分子)进行连接,在UCP保存液(pH = 7. 2 0. 03mol/L PB缓冲液中,含0. 1% BSA,0. 05% Tween20、0. 02% NaN3)中以浓度lmg/mL,4°C保存备用; . [0420] a surface modification and activation methods using established particle diameter of UCP 200_300nm the surface modification, and with purified rabbit anti-illegal drugs antibody or binding ligand (including illegal drugs: amphetamine, methamphetamine, benzene piperidine and tincture of opium drug molecules) are connected, in UCP preservation solution (pH = 7. 2 0. 03mol / L PB buffer containing 0. 1% BSA, 0. 05% Tween20,0. 02% NaN3) at a concentration of lmg / mL, 4 ° C were stored;

[0421] b.将保存于UCP保存液中的lmg/mL UCP-抗体结合物6mL,12000r/min,4°C,离心30min,尽量弃尽上清; . [0421] b stored in the UCP preservation solution lmg / mL UCP- antibody conjugate 6mL, 12000r / min, 4 ° C, centrifuged 30min, try to make the supernatant was discarded;

[0422] c.向离心管中的UCP-抗体结合物沉降,加入3mL结合物稀释液(pH = 7. 2 [0422] c. UCP- antibody binding to sedimentation in a centrifuge tube, diluted conjugate was added 3mL solution (pH = 7. 2

0.03mol/LPB缓冲液中,含1%蔗糖、1% BSA)涡旋充分混匀(终浓度:2mg/mL UCP-抗体结合物) 0.03mol / LPB buffer containing 1% sucrose, 1% BSA) vortex and mix well (final concentration: 2mg / mL UCP- antibody conjugate)

[0423] d.将悬浊液倒入试剂瓶中,4°C保存备用; . [0423] d The suspension was poured into a vial, 4 ° C were stored;

[0424] B.样品垫封闭液: [0424] B. Sample pad blocking solution:

[0425] a.用天平精确称量BSA(牛血清白蛋白)lg,放入小烧杯中; . [0425] a precisely weighed with a balance BSA (bovine serum albumin) lg, into a small beaker;

[0426] b.烧杯中加入pH = 7. 2 0. 03mol/L PB缓冲液20mL,玻璃棒搅拌充分混匀; . [0426] b beaker, pH = 7. 2 0. 03mol / L PB buffer 20mL, mix thoroughly stirred glass rod;

[0427] c. BAS溶液中加入Tween 20 (吐温20) 20 UL,玻璃棒搅拌充分混匀(终浓度:5%BAS, 0. 1% Tween 20); [0427] was added Tween 20 (Tween 20) 20 UL c BAS, a glass rod with stirring and mix well (final concentration: 5% BAS, 0. 1% Tween 20).;

[0428] d. 4°C保存备用; . [0428] d 4 ° C were stored;

[0429] C.检测带蛋白: [0429] C. Protein detection zone:

[0430] a.用天平精确称量BSA-违禁药品分子复合物2mg,置于I. 5mL的微离心管中; . [0430] a precisely weighed with a balance BSA- illicit drugs molecular complex 2mg, placed in microcentrifuge tubes in I. 5mL;

[0431] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL); . [0431] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL);

[0432] c.分装为50 ii L每管,_20°C冻存备用; . [0432] c to 50 ii L aliquoted per tube, _20 ° C frozen for later use;

[0433] D.质控带蛋白: [0433] D. Protein control line:

[0434] a.用天平精确称量纯化的羊抗兔IgG抗体2mg,置于I. 5mL微离心管中; . [0434] a precisely weighed with a balance of purified goat anti-rabbit IgG antibody 2mg, I. 5mL placed in microcentrifuge tubes;

[0435] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL); . [0435] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL);

[0436] c.分装为50 ii L每管,_20°C冻存备用; . [0436] c to 50 ii L aliquoted per tube, _20 ° C frozen for later use;

[0437] (2)免疫层析固相载体材料准备: [0437] (2) an immunochromatographic solid support material preparation:

[0438] A.样品垫: [0438] A. sample pad:

[0439] a.选用纤维素膜(Cellulose Membrane)作为样品垫固相材料,将其剪切成 [0439] a. Selection cellulose membrane (Cellulose Membrane) As the sample pad solid phase material which was cut into

1. 5X30. Ocm规格的条带; . 1. 5X30 Ocm specifications strip;

[0440] b.将样品垫放入长形平皿中,样品垫封闭液加于其上,常温浸泡30min ; . [0440] b The sample pads were placed in the elongate plate, blocking fluid on sample pad thereon, soak at room temperature for 30 min;

[0441] c.将样品垫由封闭液中取出,放于干净的平皿中; . [0441] c removed from the sample pad blocking solution, placed in clean petri dish;

[0442] d. 37°C烘干3小时,使样品垫充分干燥; . [0442] d 37 ° C drying for 3 hours, the sample was sufficiently dried pad;

[0443] e.封闭后的样品垫在干燥的环境中保存备用; [0443] e is closed after the sample pad in a dry environment to save backup.;

[0444] B.结合垫: [0444] B. conjugate pad:

[0445] a.选用玻璃纤维素膜(Glass Fiber)作为结合垫固相材料,将其剪切成1.0X30. Ocm规格的条带; . [0445] a cellulose membrane use glass (Glass Fiber) as a solid phase material conjugate pad, which was cut into strips 1.0X30 Ocm specifications.;

[0446] b.将4°C保存备用的2mg/mL UCP-抗体结合物(pH = 7.2 0. 03mol/L PB缓冲液,含1%蔗糖、1% BSA)超声IOs ; . [0446] b will alternate at 4 ° C 2mg / mL UCP- antibody conjugate (pH = 7.2 0. 03mol / L PB buffer containing 1% sucrose, 1% BSA) ultrasound IOs;

[0447] c.将结合垫放入长形平皿中,UCP-抗体结合物悬浊液加于其上; . [0447] c binding pads into elongated dish, UCP- antibody conjugate suspension was applied thereto;

[0448] d.将结合垫取出放于干净的平皿中; . [0448] d will be put out to the bonding pads clean dishes;

[0449] e. 37°C烘干2. 5小时,使结合垫充分干燥; . [0449] e 37 ° C 2.5 hours drying the bonding pad sufficiently dried;

[0450] f.处理后的结合垫在干燥的环境中保存备用; . [0450] f pad binding treated in a dry environment to save backup;

[0451] C.分析膜: [0451] C. Analysis of the film:

[0452] a.以孔径为12 iim的硝酸纤维素膜(Nitrocellulose Membrane)作为固相材料,将其剪切成2. 5 X 30cm规格的条带;; [0452] a. A nitrocellulose membrane (Nitrocellulose Membrane) to the aperture 12 iim as a solid phase material which was cut into a size of 2. 5 X 30cm strip ;;

[0453] b.用点样仪在2. 5cm宽的分析膜上,从下向上Icm处喷点2mg/mL BSA-违禁药品分子复合物,2 u L/cm,作为检测带; . [0453] b 2. 5cm wide analysis of the film, using a spotter spray upwardly from the point at Icm 2mg / mL BSA- illicit drugs molecular complex, 2 u L / cm, as a detection zone;

[0454] c.用点样仪在2. 5cm宽的分析膜上,从下向上I. 5cm处喷点2mg/mL羊抗兔IgG抗体,2 u L/cm,作为质控带; . [0454] c 2. 5cm wide analysis of the film, using a spotter spray upwardly I. 5cm from the point at 2mg / mL goat anti-rabbit IgG antibody, 2 u L / cm, as a control line;

[0455] d. 37°C烘干2小时,使分析膜充分干燥; . [0455] d 37 ° C and drying for 2 hours to sufficiently dry analysis membrane;

[0456] e.点样后的分析膜在干燥的环境中保存备用; [0456] e after spotting analytical membrane were stored in a dry environment.;

[0457] D.吸水垫: [0457] D. absorbent pad:

[0458] a.选用纤维素膜(Cellulose Membrane)作为吸水垫固相材料,将其剪切成3. OX 30cm规格的条带; . [0458] a selection cellulose membrane (Cellulose Membrane) as a solid phase absorbent pad material, which is cut into a size of 3. OX 30cm strip;

[0459] b.将变色范围5. 5-9. 0的精密pH试纸固定于吸水垫从下向上2. Ocm处,作为终点指示带; .. [0459] b the color range of pH 5. 5-9 0 precision test strip is fixed to the absorbent pad 2. Ocm from the bottom up, as the tape end indication;

[0460] c.吸水垫在干燥的环境中保存备用; . [0460] c absorbent pad in a dry environment to save backup;

[0461] (3)免疫层析试纸条检测: [0461] (3) Immunochromatographic strip tests:

[0462] A.将待检测样品用pH = 7. 2 0. 03mol/L PB缓冲液10倍稀释; [0462] A. The sample to be tested was diluted with pH = 7. 2 0. 03mol / L PB buffer 10 times;

[0463] B. 100 u L稀释后的样品加于试纸条外壳上的加样孔中; [0463] After the sample B. 100 u L diluted in loading hole test strip housing;

[0464] C.待终点指示窗口中的终点指示带变为绿色后,便可以用传感器判读外壳上结果判读窗口中的检测带与质控带,以得出结果。 [0464] C. until the end of the window indicate the end turns green indicating the tape, can interpret results interpreted by a sensor and the control line with a window on the housing, in order to obtain the result.

[0465] 实施例6 :鼠疫感染抗体检泖丨: [0465] Example 6: Anti medical Mao Shu plague infections:

[0466] (I)免疫层析液相材料准备: [0466] (I) an immunochromatographic liquid material preparation:

[0467] A. UCP-SPA 结合物: [0467] A. UCP-SPA conjugate:

[0468] a.利用已建立的表面修饰与活化方法对直径200_300nm的UCP颗粒进行表面修饰,并与SPA (葡萄球菌蛋白A)进行连接,在UCP保存液(pH = 7.2 0. 03mol/L PB缓冲液中,含0. 1% BSA,0. 05% Tween20、0. 02% NaN3)中以浓度lmg/mL,4°C保存备用; [0468] a. Surface modification and activation methods using established particle diameter of UCP 200_300nm the surface modification, and is connected to the SPA (staphylococcal protein A), the UCP preservation solution (pH = 7.2 0. 03mol / L PB buffer containing 0. 1% BSA, the 0 05% Tween20,0 02% NaN3) at a concentration of lmg / mL, 4 ° C for use..;

[0469] b.将保存于UCP 保存液中的lmg/mL UCP-SPA 结合物6mL, 12000r/min, 4°C,离心30min,尽量弃尽上清; . [0469] b stored in the UCP preservation solution lmg / mL UCP-SPA conjugate 6mL, 12000r / min, 4 ° C, centrifuged 30min, try to make the supernatant was discarded;

[0470] c.向离心管中的UCP-SPA结合物沉降,加入3mL结合物稀释液(pH = 7. 2 [0470] c. Binding to UCP-SPA sedimentation centrifuge tube, diluted conjugate was added 3mL solution (pH = 7. 2

0. 03mol/LPB缓冲液中,含I %蔗糖、I % BSA)涡旋充分混匀(终浓度:2mg/mL UCP-SPA结合物) 0. 03mol / LPB buffer containing I% sucrose, I% BSA) vortex and mix well (final concentration: 2mg / mL UCP-SPA conjugate)

[0471] d.将悬浊液倒入试剂瓶中,4°C保存备用; . [0471] d The suspension was poured into a vial, 4 ° C were stored;

[0472] B.样品垫封闭液: [0472] B. Sample pad blocking solution:

[0473] a.用天平精确称量BSA(牛血清白蛋白)lg,放入小烧杯中; . [0473] a precisely weighed with a balance BSA (bovine serum albumin) lg, into a small beaker;

[0474] b.烧杯中加入pH = 7. 2 0. 03mol/L PB缓冲液20mL,玻璃棒搅拌充分混匀; . [0474] b beaker, pH = 7. 2 0. 03mol / L PB buffer 20mL, mix thoroughly stirred glass rod;

[0475] c. BAS溶液中加入Tween 20 (吐温20) 20 UL,玻璃棒搅拌充分混匀(终浓度:5%BAS, 0. 1% Tween 20); [0475] was added Tween 20 (Tween 20) 20 UL c BAS, a glass rod with stirring and mix well (final concentration: 5% BAS, 0. 1% Tween 20).;

[0476] d. 4°C保存备用; . [0476] d 4 ° C were stored;

[0477] C.检测带蛋白: [0477] C. Protein detection zone:

[0478] a.用天平精确称量鼠疫FI-Ag (鼠疫FI_Ag) 2mg,置于I. 5mL的微离心管中; . [0478] a precisely weighed with a balance plague FI-Ag (plague FI_Ag) 2mg, microcentrifuge tube was placed in I. 5mL;

[0479] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL 鼠疫FI-Ag); . [0479] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL plague FI-Ag);

[0480] c.分装为50 ii L每管,_20°C冻存备用; . [0480] c to 50 ii L aliquoted per tube, _20 ° C frozen for later use;

[0481] D.质控带蛋白: [0481] D. Protein control line:

[0482] a.用天平精确称量羊IgG 2mg,置于ImL的微离心管中; . [0482] a precisely weighed with a balance goat IgG 2mg, placed in ImL microcentrifuge tube;

[0483] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL 羊IgG); . [0483] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL goat IgG);

[0484] c.分装为50 ii L每管,_20°C冻存备用; . [0484] c to 50 ii L aliquoted per tube, _20 ° C frozen for later use;

[0485] (2)免疫层析固相载体材料准备: [0485] (2) an immunochromatographic solid support material preparation:

[0486] A.样品垫: [0486] A. sample pad:

[0487] a.选用纤维素膜(Cellulose Membrane)作为样品垫固相材料,将其剪切成 [0487] a. Selection cellulose membrane (Cellulose Membrane) As the sample pad solid phase material which was cut into

I. 5X30. Ocm规格的条带; . I. 5X30 Ocm specifications of the strip;

[0488] b.将样品垫放入长形平皿中,样品垫封闭液加于其上,常温浸泡30min ; . [0488] b The sample pads were placed in the elongate plate, blocking fluid on sample pad thereon, soak at room temperature for 30 min;

[0489] c.将样品垫由封闭液中取出,放于干净的平皿中; . [0489] c removed from the sample pad blocking solution, placed in clean petri dish;

[0490] d. 37°C烘干3小时,使样品垫充分干燥; . [0490] d 37 ° C drying for 3 hours, the sample was sufficiently dried pad;

[0491] e.封闭后的样品垫在干燥的环境中保存备用; [0491] e is closed after the sample pad in a dry environment to save backup.;

[0492] B.结合垫: [0492] B. conjugate pad:

[0493] a.选用玻璃纤维素膜(Glass Fiber)作为结合垫固相材料,将其剪切成1.0X30. Ocm规格的条带; . [0493] a cellulose membrane use glass (Glass Fiber) as a solid phase material conjugate pad, which was cut into strips 1.0X30 Ocm specifications.;

[0494] b.将4°C保存备用的2mg/mL UCP-SPA (pH = 7. 2 0. 03mol/L PB 缓冲液,含I %蔗糖、1% BSA)超声IOs ; . [0494] b will alternate at 4 ° C 2mg / mL UCP-SPA (pH = 7. 2 0. 03mol / L PB buffer containing I% sucrose, 1% BSA) ultrasound IOs;

[0495] c.将结合垫放入长形平皿中,UCP-SPA结合物悬浊液加于其上; . [0495] c binding pads into elongated dish, UCP-SPA conjugate suspension was applied thereto;

[0496] d.将结合垫取出放于干净的平皿中; . [0496] d will be put out to the bonding pads clean dishes;

[0497] e. 37°C烘干2. 5小时,使结合垫充分干燥; . [0497] e 37 ° C 2.5 hours drying the bonding pad sufficiently dried;

[0498] f.处理后的结合垫在干燥的环境中保存备用; . [0498] f pad binding treated in a dry environment to save backup;

[0499] C.分析膜: [0499] C. Analysis of the film:

[0500] a.以孔径为12 iim的硝酸纤维素膜(Nitrocellulose Membrane)作为固相材料,将其剪切成2. 5 X 30cm规格的条带;; [0500] a. A nitrocellulose membrane (Nitrocellulose Membrane) to the aperture 12 iim as a solid phase material which was cut into a size of 2. 5 X 30cm strip ;;

[0501] b.用点样仪在2. 5cm宽的分析膜上,从下向上Icm处喷点2mg/mL鼠疫Fl-Ag,2 u L/cm,作为检测带; . [0501] b 2. 5cm wide analysis of the film dots 2mg / mL plague Fl-Ag Icm at the lower upwardly from a spotter, 2 u L / cm, as a detection zone;

[0502] c.用点样仪在2. 5cm宽的分析膜上,从下向上I. 5cm处喷点2mg/mL羊IgG,2 u L/cm,作为质控带; . [0502] c 2. 5cm wide analysis of the film, using a spotter spray upwardly I. 5cm from the point at 2mg / mL sheep IgG, 2 u L / cm, as a control line;

[0503] d. 37°C烘干2小时,使分析膜充分干燥; . [0503] d 37 ° C and drying for 2 hours to sufficiently dry analysis membrane;

[0504] e.点样后的分析膜在干燥的环境中保存备用; [0504] e after spotting analytical membrane were stored in a dry environment.;

[0505] D.吸水垫: [0505] D. absorbent pad:

[0506] a.选用纤维素膜(Cellulose Membrane)作为吸水垫固相材料,将其剪切成3. OX 30cm规格的条带; . [0506] a selection cellulose membrane (Cellulose Membrane) as a solid phase absorbent pad material, which is cut into a size of 3. OX 30cm strip;

[0507] b.将变色范围5. 5-9. 0的精密pH试纸固定于吸水垫从下向上2. Ocm处,作为终点指示带; .. [0507] b the color range of pH 5. 5-9 0 precision test strip is fixed to the absorbent pad 2. Ocm from the bottom up, as the tape end indication;

[0508] c.吸水垫在干燥的环境中保存备用; . [0508] c absorbent pad in a dry environment to save backup;

[0509] (3)免疫层析试纸条检测: [0509] (3) Immunochromatographic strip tests:

[0510] A.待检测血清样品用pH = 7. 2 0. 03mol/L PB缓冲液10倍稀释; [0510] A. Serum samples to be detected is diluted with pH = 7. 2 0. 03mol / L PB buffer 10 times;

[0511] B. IOOii L稀释后的样品加于试纸条外壳上的加样孔中; [0511] diluted sample B. IOOii L is applied to the sample hole the test strip housing;

[0512] C.待终点指示窗口中的终点指示带变为绿色后,便可以用传感器判读外壳上结果判读窗口中的检测带与质控带,以得出结果。 [0512] C. until the end of the window indicate the end turns green indicating the tape, can interpret results interpreted by a sensor and the control line with a window on the housing, in order to obtain the result.

[0513] 实施例7 :SARS病毒感染抗体检测: [0513] Example 7: SARS virus antibody test:

[0514] (I)免疫层析液相材料准备: [0514] (I) an immunochromatographic liquid material preparation:

[0515] A. UCP-SPA 结合物: [0515] A. UCP-SPA conjugate:

[0516] a.利用已建立的表面修饰与活化方法对直径200_300nm的UCP颗粒进行表面修饰,并与SPA (葡萄球菌蛋白A)进行连接,在UCP保存液(pH = 7.2 0. 03mol/L PB缓冲液中,含0. 1% BSA,0. 05% Tween20、0. 02% NaN3)中以浓度lmg/mL,4°C保存备用; [0516] a. Surface modification and activation methods using established particle diameter of UCP 200_300nm the surface modification, and is connected to the SPA (staphylococcal protein A), the UCP preservation solution (pH = 7.2 0. 03mol / L PB buffer containing 0. 1% BSA, the 0 05% Tween20,0 02% NaN3) at a concentration of lmg / mL, 4 ° C for use..;

[0517] b.将保存于UCP 保存液中的lmg/mL UCP-SPA 结合物6mL, 12000r/min, 4°C,离心30min,尽量弃尽上清; . [0517] b stored in the UCP preservation solution lmg / mL UCP-SPA conjugate 6mL, 12000r / min, 4 ° C, centrifuged 30min, try to make the supernatant was discarded;

[0518] c.向离心管中的UCP-SPA结合物沉降,加入3mL结合物稀释液(pH = 7. 2 [0518] c. Binding to UCP-SPA sedimentation centrifuge tube, diluted conjugate was added 3mL solution (pH = 7. 2

0. 03mol/LPB缓冲液中,含I %蔗糖、I % BSA)涡旋充分混匀(终浓度:2mg/mL UCP-SPA结合物) 0. 03mol / LPB buffer containing I% sucrose, I% BSA) vortex and mix well (final concentration: 2mg / mL UCP-SPA conjugate)

[0519] d.将悬浊液倒入试剂瓶中,4°C保存备用; . [0519] d The suspension was poured into a vial, 4 ° C were stored;

[0520] B.样品垫封闭液: [0520] B. Sample pad blocking solution:

[0521] a.用天平精确称量BSA(牛血清白蛋白)lg,放入小烧杯中; . [0521] a precisely weighed with a balance BSA (bovine serum albumin) lg, into a small beaker;

[0522] b.烧杯中加入pH = 7. 2 0. 03mol/L PB缓冲液20mL,玻璃棒搅拌充分混匀; . [0522] b beaker, pH = 7. 2 0. 03mol / L PB buffer 20mL, mix thoroughly stirred glass rod;

[0523] c. BAS溶液中加入Tween 20 (吐温20) 20 UL,玻璃棒搅拌充分混匀(终浓度:5%BAS, 0. 1% Tween 20); [0523] was added Tween 20 (Tween 20) 20 UL c BAS, a glass rod with stirring and mix well (final concentration: 5% BAS, 0. 1% Tween 20).;

[0524] d. 4°C保存备用; . [0524] d 4 ° C were stored;

[0525] C.检测带蛋白: [0525] C. Protein detection zone:

[0526] a.用天平精确称量纯化的SARS病毒表面N蛋白2mg,置于I. 5mL的微离心管中; . [0526] a precisely weighed with a balance of purified surface SARS virus N protein 2mg, placed in microcentrifuge tubes in I. 5mL;

[0527] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL SARS病毒表面N抗原); . [0527] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL SARS virus surface antigen N);

[0528] c.分装为50 ii L每管,_20°C冻存备用; . [0528] c to 50 ii L aliquoted per tube, _20 ° C frozen for later use;

[0529] D.质控带蛋白: [0529] D. Protein control line:

[0530] a.用天平精确称量纯化的羊IgG 2mg,置于ImL的微离心管中; . [0530] a precisely weighed with a balance of purified sheep IgG 2mg, placed in ImL microcentrifuge tube;

[0531] b.微离心管中加入ImL pH = 7. 2 0. 03mol/L PB缓冲液,涡旋充分混匀(终浓度:2mg/mL 羊IgG); . [0531] b microfuge tube was added ImL pH = 7. 2 0. 03mol / L PB buffer, vortex mix well (final concentration: 2mg / mL goat IgG);

[0532] c.分装为50 ii L每管,_20°C冻存备用; . [0532] c to 50 ii L aliquoted per tube, _20 ° C frozen for later use;

[0533] (2)免疫层析固相载体材料准备: [0533] (2) an immunochromatographic solid support material preparation:

[0534] A.样品垫: [0534] A. sample pad:

[0535] a.选用纤维素膜(Cellulose Membrane)作为样品垫固相材料,将其剪切成 [0535] a. Selection cellulose membrane (Cellulose Membrane) As the sample pad solid phase material which was cut into

1. 5X30. Ocm规格的条带; . 1. 5X30 Ocm specifications strip;

[0536] b.将样品垫放入长形平皿中,样品垫封闭液加于其上,常温浸泡30min ; . [0536] b The sample pads were placed in the elongate plate, blocking fluid on sample pad thereon, soak at room temperature for 30 min;

[0537] c.将样品垫由封闭液中取出,放于干净的平皿中; . [0537] c removed from the sample pad blocking solution, placed in clean petri dish;

[0538] d. 37°C烘干3小时,使样品垫充分干燥; . [0538] d 37 ° C drying for 3 hours, the sample was sufficiently dried pad;

[0539] e.封闭后的样品垫在干燥的环境中保存备用; [0539] e is closed after the sample pad in a dry environment to save backup.;

[0540] B.结合垫: [0540] B. conjugate pad:

[0541] a.选用玻璃纤维素膜(Glass Fiber)作为结合垫固相材料,将其剪切成1.0X30. Ocm规格的条带; . [0541] a cellulose membrane use glass (Glass Fiber) as a solid phase material conjugate pad, which was cut into strips 1.0X30 Ocm specifications.;

[0542] b.将4°C保存备用的2mg/mL UCP-SPA 结合物(pH = 1.2 0. 03mol/L PB 缓冲液,含1%蔗糖、1% BSA)超声IOs ; . [0542] b will alternate at 4 ° C 2mg / mL UCP-SPA conjugate (pH = 1.2 0. 03mol / L PB buffer containing 1% sucrose, 1% BSA) ultrasound IOs;

[0543] c.将结合垫放入长形平皿中,UCP-SPA结合物悬浊液加于其上; . [0543] c binding pads into elongated dish, UCP-SPA conjugate suspension was applied thereto;

[0544] d.将结合垫取出放于干净的平皿中; . [0544] d will be put out to the bonding pads clean dishes;

[0545] e. 37°C烘干2. 5小时,使结合垫充分干燥; . [0545] e 37 ° C 2.5 hours drying the bonding pad sufficiently dried;

[0546] f.处理后的结合垫在干燥的环境中保存备用; . [0546] f pad binding treated in a dry environment to save backup;

[0547] C.分析膜: [0547] C. Analysis of the film:

[0548] a.以孔径为12 iim的硝酸纤维素膜(Nitrocellulose Membrane)作为固相材料,将其剪切成2. 5 X 30cm规格的条带; [0548] a nitrocellulose membrane (Nitrocellulose Membrane) to the aperture 12 iim as a solid phase material which was cut into 2. 5 X 30cm strip specifications.;

[0549] b.用点样仪在2. 5cm宽的分析膜上,从下向上Icm处喷点2mg/mL SARS病毒表面N蛋白,2 ii L/cm,作为检测带; . [0549] b using a spotter 2. 5cm wide analysis of the film dots 2mg / mL SARS virus upwardly from the surface of the N protein at a lower Icm, 2 ii L / cm, as a detection zone;

[0550] c.用点样仪在2. 5cm宽的分析膜上,从下向上I. 5cm处喷点2mg/mL羊IgG, 2 u L/cm,作为质控带; . [0550] c 2. 5cm wide analysis of the film, using a spotter spray upwardly I. 5cm from the point at 2mg / mL sheep IgG, 2 u L / cm, as a control line;

[0551] d. 37°C烘干2小时,使分析膜充分干燥; . [0551] d 37 ° C and drying for 2 hours to sufficiently dry analysis membrane;

[0552] e.点样后的分析膜在干燥的环境中保存备用; [0552] e after spotting analytical membrane were stored in a dry environment.;

[0553] D.吸水垫: [0553] D. absorbent pad:

[0554] a.选用纤维素膜(Cellulose Membrane)作为吸水垫固相材料,将其剪切成3. OX 30cm规格的条带; . [0554] a selection cellulose membrane (Cellulose Membrane) as a solid phase absorbent pad material, which is cut into a size of 3. OX 30cm strip;

[0555] b.将变色范围5. 5-9. 0的精密pH试纸固定于吸水垫从下向上2. Ocm处,作为终点指示带; .. [0555] b the color range of pH 5. 5-9 0 precision test strip is fixed to the absorbent pad 2. Ocm from the bottom up, as the tape end indication;

[0556] c.吸水垫在干燥的环境中保存备用; . [0556] c absorbent pad in a dry environment to save backup;

[0557] (3)免疫层析试纸条检测: [0557] (3) Immunochromatographic strip tests:

[0558] A.将待检测血清样品用pH = 7. 2 0. 03mol/L PB缓冲液10倍稀释; [0558] A. serum sample to be detected is diluted with pH = 7. 2 0. 03mol / L PB buffer 10 times;

[0559] B. 100 u L稀释后的样品加于试纸条外壳上的加样孔中; [0559] After the sample B. 100 u L diluted in loading hole test strip housing;

[0560] C.待终点指示窗口中的终点指示带变为绿色后,便可以用传感器判读外壳上结果判读窗口中的检测带与质控带,以得出结果。 [0560] C. until the end of the window indicate the end turns green indicating the tape, can interpret results interpreted by a sensor and the control line with a window on the housing, in order to obtain the result.

[0561] 上述实施例有助于理解本发明,但是并不是对本发明的限制。 [0561] The embodiments aid in understanding the present invention, but not limit the present invention. 本领域的普通技术人员可以根据上述实施例对本发明作出适当的修改和变动,均属于本发明的保护范围。 Those of ordinary skill in the art may make appropriate modifications and variations of the present invention, the above-described embodiments, all fall within the scope of the present invention.

Claims (2)

1. 一种基于上转换发光技术的免疫层析试纸的制备方法,其特征在于该方法包括以下步骤: UCP-生物活性分子的制备:对UCP颗粒进行表面修饰,与生物活性分子进行连接,在UCP保存液中进行保存;取一定量的保存于UCP保存液中的UCP-生物活性分子结合物,离心,并弃去上清液;在沉降的UCP生物活性分子结合物中加入稀释液,并充分混匀,制备成悬浊液;样品垫的制备:选用纤维素膜作为样品垫固相材料,剪切成具有一定规格的条带;将样品垫放入样品垫封闭液中浸泡;然后将样品垫从封闭液中取出,并烘干,使样品垫充分干燥;结合垫的制备:选用玻璃纤维素膜作为结合垫固相材料,剪切成具有一定规格的条带;在该条带上加上UCP-生物活性分子结合物的悬浊液;烘干该条带,使结合垫充分干燥;分析膜的制备:选用硝酸纤维素膜作为分析膜固相材料 1. A process for producing immunochromatographic strips upconversion Technology, characterized in that the method comprises the steps of: preparing a biologically active molecule UCP-: UCP particles to surface modification, the connection with the biologically active molecule, in UCP save storage solution; stored in a certain amount of biologically active molecule UCP- UCP binding preservation solution was centrifuged, and the supernatant was discarded; UCP diluted liquid biologically active molecule conjugate settled, and mix well to prepare a suspension; the sample pad prepared: selection cellulose membrane as a solid phase sample pad strip material cut to have a certain size; the sample pad into the sample pad soaked in blocking solution; then, the sample pad was removed from the blocking solution and drying, the sample was sufficiently dried pad; bond pads prepared: use glass mat cellulose membrane as a solid phase binding material is cut into strips having a certain size; tape in the article plus biologically active molecule UCP- suspension thereof; drying the strip so that sufficiently dried conjugate pad; preparation of membranes of: selection of nitrocellulose as the solid phase material film analysis ,剪切成具有一定规格的条带;在条带上的不同位置喷点生物活性分子,制成检测带和质控帯;烘干该条带,使分析膜充分干燥;装配该试纸条:将处理过的分析膜粘贴于塑料背板上,质控带向上,检测带向下;将处理过的结合垫粘贴于塑料背板上,上端压于分析膜的下端,并相互重叠;将处理过的样品垫粘贴于塑料背板上,下端与塑料背板平齐,上端压于结合垫的下端,并相互重叠;将吸水垫粘贴于塑料背板上,上端与塑料背板平齐,下端压于分析膜的上端,并与分析膜相互重叠;将装配成形的条带剪切成一定规格的试纸条;其中,所述UCP保存液为pH = 7. 20. 03mol/L磷酸盐缓冲液,含有O. I % BSA, O. 05% Tween20,0. 02% NaN3 ;所述UCP生物活性分子的稀释液为PH = 7. 20. 03mol/L磷酸盐缓冲液,含有I %蔗糖,1% BSA ;所述样品垫封闭液为pH=7. 20. 03mol/L 磷酸盐缓冲液,含 , Cut into strips having a certain size; biologically active molecule dots at different locations on the strip, the detection zone and the control is made Bands; drying the strip, so that analysis membrane was dried sufficiently; assembly of the test strip : analysis of the treated plastic film is attached to the back plate, the control line up, down detection zone; treated plastic bonding pad attached to the back plate, the lower end of the upper end of pressure on the analysis of the film, and overlap each other; and the treated sample pad back plate attached to the plastic, the plastic backsheet flush with the lower end, the lower end of the upper end pressed against the pad binding, and overlapped with each other; and an absorbent pad attached to the plastic backing plate, the upper end of the plastic backing plate flush, analysis of lower pressure on the upper end of the film, and overlapping and analysis film; forming the assembled strip was cut into test strips of a certain size; wherein the preservation solution of UCP pH = 7. 20. 03mol / L phosphate buffer containing O. I% BSA, O. 05% Tween20,0 02% NaN3;. the diluent is a biologically active molecule UCP PH = 7. 20. 03mol / L phosphate buffer, containing I% sucrose , 1% BSA; the sample pad blocking solution is pH = 7 20. 03mol / L phosphate buffer containing. 5% BSA, O. 1% Tween 20。 5% BSA, O. 1% Tween 20.
2.如权利要求I所述的免疫层析试纸的制备方法,其特征在于该方法还包括以下步骤:将变色范围5. 5-9. O的精密pH试纸贴于吸水垫作为终点指示带。 2. The production method of claim I immunochromatographic test strip as claimed in claim, characterized in that the method further comprises the step of: the color range of 5. 5-9 O precise pH test paper with an absorbent pad attached to an end point indicating band.
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