CN101526534A - Fluorescence immune chromatography test paper and preparing method and application thereof - Google Patents

Fluorescence immune chromatography test paper and preparing method and application thereof Download PDF

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CN101526534A
CN101526534A CN 200910047352 CN200910047352A CN101526534A CN 101526534 A CN101526534 A CN 101526534A CN 200910047352 CN200910047352 CN 200910047352 CN 200910047352 A CN200910047352 A CN 200910047352A CN 101526534 A CN101526534 A CN 101526534A
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antibody
fluorescein
fluorescence
particles
fluorescent
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CN 200910047352
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沈鹤柏
赵露晶
马经纬
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沈鹤柏
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Abstract

The invention relates to fluorescence immune chromatography test paper which is formed by mutually and sequentially overlapping a sample pad, a combination pad, an antibody carrying film and water absorbing paper on a lining board with adhesive, wherein the combination pad is coated with a fluorescence material antibody 1 composite, a fluorescence-marked material is fluorescein granules or fluorescence material converted by rare earth, the antibody carrying film is a cellulose nitrate film or a nylon film and is respectively connected with an antibody 2 and the T line and the C line of a secondary antibody, the fluorescence material is fluorescein granules or the fluorescence granules converted by the rare earth, and the fluorescein granules are selected from isosulfocyanic acid fluorescein or tetramethyl isosulfocyanic acid rhodamine or tetraethyl rhodamine, and the surface of the fluorescence-marked material is aminated and combined with the antibody by cross-linking reaction. The fluorescence quantitative measuring system is used for detecting the fluorescence strength of the areas of the T line and the C line, and the quantitative detection is completed by the standard curve of the antigen concentration. The invention is suitable for the immune detection of tumor marked objects of urinal fiber-connected protein, and the like.

Description

一种荧光免疫层析试纸及其制备方法和应用 A fluorescent immunochromatographic test strip and its preparation method and application

技术领域 FIELD

本发明属医学检验领域,特别是涉及一种荧光免疫层析检测试纸。 The present invention belongs to the field of medical testing, in particular, it relates to a fluorescent immunochromatographic test strip. 背景技术 Background technique

肿瘤是威胁人类健康的高发病率和高死亡率性疾病,大量研究和防治资料证实,早期诊断和早期治疗是防治肿瘤与降低死亡率的最有效办法。 Cancer is a threat to human health, morbidity and mortality of the disease, a large number of research and control data confirm that early diagnosis and early treatment is the prevention and treatment of cancer and most effective way to reduce mortality. 为此, 人们设想了多种措施和途径,对早期无症状肿瘤患者,肿瘤标记物常常作为能早期诊断肿瘤的方法之一。 To this end, it was envisaged various measures and ways of early symptoms of cancer patients, tumor markers often used as a method of early diagnosis of tumors can be one. 因此,不断寻求新的肿瘤标记物及其检测方法己经成为学术界研究的热点。 Thus, constantly looking for new tumor marker and detection method has become the focus of academic research.

目前,临床上主要还是通过免疫分析技术对肿瘤标记物进行检测,如酶 At present, mainly for clinical tumor marker is detected, such as by an enzyme immunoassay technique

联免疫法(ELISA)、放射免疫法(RIA)、化学发光法(CLIA)等,但是传 Linked immunosorbent assay (ELISA), radioimmunoassay (RIA), chemiluminescence (CLIA), etc., but the pass

统的免疫分析方法需要特殊的仪器设备,费用较贵。 Conventional immunoassay method requires special equipment, more expensive. 免疫层析试纸技术的发展一定程度上弥补了这一不足,它操作简单,准确直观,为个人、家庭常规快速诊断检测提供了可能。 Up for this shortfall to some extent the development of immunochromatographic test strip technology, it is simple, accurate and intuitive, offers the possibility for individuals, families conventional rapid diagnostic tests. 其以微孔膜作为发生免疫反应的固相载体,经渗透和毛细虹吸作用使待测样品中的抗体或抗原与膜上包被的抗原或抗体结合,通过标记物示踪反应结果。 Which is a microporous membrane as a solid support to immunoreact by diffusion and capillary siphon action so that the test sample with an antibody or antigen-film package to be bound antigen or antibody, the marker tracing the reaction results. 当前常见的标记材料主要包括放射性同位素、 酶、胶体金以及发光标记材料,其中发光标记材料又包括荧光、化学发光、 生物发光材料等。 Current common marking materials include radioisotopes, enzymes, colloidal gold and a luminescent marking material, wherein the luminescent marking materials and include fluorescent, chemiluminescent, bioluminescent materials and the like. 但是上述材料都有其局限性:放射性同位素易衰变,存在放射污染;酶自身性质不稳定,易受生物样品的干扰;胶体金难以进行多重、 定量分析;化学发光为瞬时发光,检测仪器昂贵;荧光材料则存在淬灭现象,使得时间分辨检测无法进行。 However, these materials have limitations: Easy radioisotopes decay, the presence of radioactive contamination; enzyme itself unstable, susceptible to interfere with the biological sample; colloidal gold is difficult to multiplex quantitative analysis; chemiluminescence instantaneous emission, expensive detection equipment; fluorescence quenching material there, so that the time resolved detection can not be performed.

因此,改进或寻找新的标记材料现已成为人们关注的焦点。 Therefore, to improve or find new marking materials has become the focus of attention. 常用的荧光染料存在光漂白现象,导致荧光信号不稳定,通过对其表面进行包裹不仅克服了它的淬灭现象,而且实现了荧光颗粒的表面功能化。 Presence of a conventional fluorescent dye photobleaching, resulting in a fluorescent signal is unstable, the surface thereof by wrapping it not only overcomes the quenching, and to achieve surface-functionalized fluorescent particles. Si02以稳定性高, 成键能力强,表面易功能化等优势自然成为首选。 Si02 with high stability and strong bonding ability, and easy surface functionalization advantages natural choice. 本专利制备了Si02包裹的荧光素颗粒-抗体复合物和荧光免疫层析试纸,并将其应用于荧光免疫层析技术检测肿瘤标志物,比免疫金层析试纸的灵敏度提到了10-100倍,进一步拓展了它在生物医学领域中的应用。 Si02 encapsulated particles of the present patent fluorescein prepared - antibody complex and the fluorescent immunochromatographic test strip, and applied to immunofluorescence assay for tumor markers, referred to 10-100 times more sensitive than in immunogold chromatographic test strips to further expand its application in the biomedical field.

此外,近年来一类新型标记物一纳米上转换发光材料(UCP)的出现为免疫层析技术的发展提供了新的可能。 Further, in recent years, a new class of markers on a nano-conversion luminescent material occurs (of UCP) offers new possibilities for the development of immunochromatography techniques. UCP是由稀土金属元素掺杂于晶体的晶格中构成的纳米级颗粒。 UCP nanoscale particles doped in the crystal lattice constituted by rare earth metal element. 由于独特的结构,UCP可由红外光激发而发射波长远短于激发光的可见光,即上转发光。 Due to the unique structure, the infrared light of UCP can be excited to emit visible light shorter than the long-wave excitation light, i.e. the light forward. 这种绝无仅有的性质使UCP作为 Such unique properties make as UCP

标记物与传统标记物相比具有无本底干扰、无淬灭、使用安全、适合多重分 With conventional markers showed no marker has a background interference, non-quenched safe for multifractal

析和定量分析等显著优势,确保了UCP在检测过程中高度的敏感性、稳定性、 安全性和灵活性。 Analysis and quantitative analysis and other significant advantages to ensure a high degree of sensitivity UCP, stability, security and flexibility in the testing process. 中国食品卫生杂志2007, 19 (1) : 41-44公开了一种利用上转磷光免疫层析检测肠出血性大肠杆菌0157的方法,但是采用的UCP 颗粒未进行包裹,易发生荧光猝灭现象,且该检测为定性检测,不能够用于定量。 Chinese Journal of Food 2007, 19 (1): 41-44 discloses a method for transfer phosphorescent immunochromatographic detection of EHEC on utilizing 0157, but UCP particles used have no wrapping, prone to the phenomenon of fluorescence quenching , and the detection is qualitative detection, it can not be used for quantification. UCP材料应用于尿纤维连接蛋白肿瘤标志物的检测未见报道。 UCP material is applied to the detection of urinary Fibronectin tumor markers have not been reported.

发明内容 SUMMARY

所要解决的技术问题 The technical problem to be solved

本发明所要解决的技术问题是提供一种荧光免疫层析检测试纸,以克服现有技术采用的UCP颗粒未进行包裹,易发生荧光猝灭现象,且利用UCP The present invention is to solve the technical problem of providing a fluorescent immunochromatographic test strip, in order to overcome the prior art UCP particles employed have no wrapping, prone to the phenomenon of fluorescence quenching, and utilize UCP

颗粒的免疫检测为定性检测,不能够用于定量,且检测灵敏度低的缺陷。 Qualitative immunoassay for the detection of particles, can not be used for quantitative, and defect detection sensitivity is low. 技术方案 Technical solutions

本发明的技术方案之一是提供一种荧光免疫层析试纸,由依次在带有粘合剂的衬板上相互搭接的样品垫、结合垫、抗体承载膜、吸水纸组成,其中, 所述的结合垫上涂有荧光标记材料-抗体1复合物,所述的抗体承载膜为硝酸 One aspect of the present invention is to provide a fluorescent immunochromatographic test strip, successively from the liner having an adhesive on the mutually overlapping sample pad, conjugate pad, antibody carrier film, absorbent paper composition, wherein the binding said mat coated with a fluorescent material labeled - antibody complex, said antibody is a carrier film nitrate

纤维素膜或尼龙膜,抗体承载膜上分别为连接有抗体2和第二抗体的T线和C线,其特征在于,所述的荧光标记材料的表面经硅化包裹和氨基化,并通过交联反应与抗体结合;所述的T线和C线区域的荧光强度在检测中分别与标准抗原浓度的标准曲线相对应,完成定量检测。 Cellulose or nylon membrane, the membrane carries antibodies were connected to the antibody and the second antibody 2 T line and the C line, characterized in that the surface of the fluorescent marking material package and siliconized amination, and by cross- binding antibody linking reaction; the fluorescence intensity of T line and the C line region of the standard curve corresponding to the standard antigen concentration in the assay, respectively, to complete the quantitative detection.

上述的荧光免疫层析试纸的优选方案之一为,所述的荧光标记材料为荧光素颗粒或者稀土上转荧光材料。 One preferred embodiment described above for the fluorescent immunochromatographic test strip, the fluorescent material is fluorescein-labeled particles or transfected rare earth fluorescent material.

上述的荧光免疫层析试纸的优选方案之二为,所述的荧光素颗粒为异硫氰酸荧光素或四甲基异硫氰酸罗丹明或四乙基罗丹明。 The above-described second preferred embodiment of a fluorescent immunochromatographic test strip of the particles are fluorescein, fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate, or tetraethyl rhodamine.

上述的荧光免疫层析试纸的优选方案之三为,所述的稀土上转荧光颗粒为铕惨杂或铒掺杂或铥掺杂的碱土金属硫化物。 The above-described preferred embodiment of a fluorescent immunochromatographic test strip is three, miserable turn fluorescent particle europium or heteroaryl on the erbium-doped rare-earth or alkaline earth metal sulfides doped with thulium.

上述各荧光免疫层析试纸的优选方案为,所述的抗体1和抗体2均为甲胎蛋白或尿纤维连接蛋白的抗体。 Each of the above-described preferred embodiment of the fluorescent immunochromatographic test strip, said antibody 1 and antibody 2 are alpha-fetoprotein in urine or fibronectin.

本发明的技术方案之二是提供一种荧光免疫层析试纸的制备方法,依次包括如下步骤:1) 稀土上转荧光颗粒的表面硅化:取5-50mg稀土上转荧光颗粒,加入300-800mL 35-70%乙醇溶液,超声处理使反应体系混合均匀,在15-5CTC水浴中搅拌至平衡,再向其中加入0.1-1mL正硅酸乙酯 Aspect of the present invention is to provide a two fluorescent immunochromatographic test strip prepared, comprising the following steps in sequence: 1) the surface of the phosphor particles on the transfer silicide RE: Take turn phosphor particles rare 5-50mg added 300-800mL 35-70% ethanol solution, sonicated mixed reaction system, and stirred until equilibrium 15-5CTC water bath, and thereto was added ethyl orthosilicate 0.1-1mL

(TEOS),继续反应3-10小时,离心洗涤; (TEOS), reaction was continued for 10 hours, washed by centrifugation;

2) 稀土上转荧光颗粒的表面氨基化:取上述表面硅化的稀土上转荧光颗粒,加入30〜100mL 1:3〜3:1 (V/V)甲醇和丙三醇组成的混合溶液, 超声分散,然后向其中加入10-1000pLN- (2-氨基乙基)-3-氨基丙基三甲氧基硅垸,15-50。 2) the surface of the rare earth phosphor particles transfer amination: phosphor particles on the transfer surface to take the above rare earth silicide, adding 30~100mL 1: 3~3: a mixed solution of 1 (V / V) composed of methanol and glycerol, ultrasonic dispersion, then added 10-1000pLN- (2- aminoethyl) -3-aminopropyl trimethoxysilane embankment, 15-50. C恒温下搅拌5〜10小时,用乙醇离心洗涤, 干燥保存; C 5 to 10 hours under constant stirring, washed with ethanol and centrifugation, stored dry;

3) 稀土上转荧光颗粒表面连接抗体:将表面连接氨基的稀土上转荧光颗粒分散在磷酸缓冲液(PB)中,加入一定量戊二醛,反应2-5小时, 离心除去戊二醛,重新分散在磷酸缓冲液中,然后加入抗体1,反应1-5小时,离心洗涤,最终保存在含0.1。 3) a rare earth phosphor particle surfaces turn connected to the antibody: the surface amino groups turn connected phosphor particles dispersed rare earth phosphate buffer (PB), a certain amount of glutaraldehyde added, the reaction for 2-5 hours, centrifuged to remove glutaraldehyde, redispersed in phosphate buffer, followed by addition of an antibody, the reaction for 1-5 hours, washed by centrifugation, and finally kept in 0.1. /。 /. BSA和0.1。 BSA and 0.1. /。 /. Tween20的磷酸缓冲液中; Tween20 in phosphate buffer;

4) 荧光免疫层析试纸的组装:将稀土上转荧光颗粒-抗体1复合物涂覆在结合垫上,将抗体2和第二抗体分别以线状连接于抗体承载膜上形成T线和C线,并在带有粘合剂的衬板上相互搭接地粘合样品垫、结合垫、抗体承载膜、吸水纸。 4) The fluorescent immunochromatographic test strip assembly: the rare earth phosphor particles will turn - antibody complex coated on the conjugate pad, 2 antibodies and second antibodies are antibodies to the carrier film to the linear connecting line and a T-C line and the liner having an adhesive bonded to each other overlap sample pad, conjugate pad, antibody carrier film, absorbent paper.

上述的荧光免疫层析试纸的制备方法的优选方案为,所述的抗体1和抗体2均为甲胎蛋白或尿纤维连接蛋白的抗体。 The above-described preferred embodiment of the method of preparation of the fluorescent immunochromatographic test strip, said antibody 1 and antibody 2 are alpha-fetoprotein in urine or fibronectin.

本发明的技术方案之三是提供另一种荧光免疫层析试纸的制备方法,依次包括如下步骤:1) 荧光素颗粒的表面硅化:在乙醇体系中,分别加入0.1-10mg异硫氰酸荧光素或四甲基异硫氰酸罗丹明或四乙基罗丹明,和0.1-10pLN- Three aspect of the present invention to provide a method for preparing another fluorescent immunochromatographic test strip, comprising the following steps in sequence: 1) the surface of the silicide particles Fluorescein: ethanol system, were added fluorescein isothiocyanate 0.1-10mg biotin or tetramethyl rhodamine isothiocyanate or tetraethyl rhodamine, and 0.1-10pLN-

(2-氨基乙基)-3-氨基丙基三甲氧基硅烷,15-50。 (2-aminoethyl) -3-aminopropyl trimethoxy silane, 15-50. C恒温搅拌3〜10 小时,再依次加入0.1-10mL水、0.1-10mL 15。 C temperature was stirred for 3~10 hours, and then water were added 0.1 ml to 10, 0.1-10mL 15. /。 /. wt氨水、0.1-10mL 正硅酸乙酯(TEOS),继续反应3-10小时,洗涤获得表面硅化的荧光素颗粒; wt ammonia, 0.1 ml to 10 tetraethyl orthosilicate (TEOS), reaction was continued for 10 hours, washed with fluorescein obtained siliconized surface of the particles;

2) 荧光素的表面氨基化:取上述表面硅化的荧光素颗粒,加入10〜50mL 1:3〜3:1 (V/V)甲醇和丙三醇组成的混合溶液,超声分散,然后加入10-500|jLN- (2-氨基乙基)-3-氨基丙基三甲氧基硅烷,15-50。 2) Surface fluorescein amination: Take siliconized fluorescein of the surface particles, added 10~50mL 1: 3~3: a mixed solution of 1 (V / V) methanol and consisting of glycerol, ultrasonic dispersion, followed by addition of 10 -500 | jLN- (2- aminoethyl) -3-aminopropyl trimethoxy silane, 15-50. C恒温搅拌5〜10小时,用乙醇洗涤,干燥,获得表面氨基化的荧光素颗粒; C temperature was stirred for 5 to 10 hours, washed with ethanol and dried to obtain a surface of an amino fluorescein particles;

3) 荧光素颗粒表面连接抗体:将上述表面氨基化的荧光素颗粒分散在磷酸缓冲液中,加入戊二醛反应2-5小时,离心除去戊二醛,将荧光素颗粒重新分散在磷酸缓冲液中,加入抗体1,反应1-5小时,洗涤后获得荧光素颗粒-抗体1复合物,保存在含0.1% BSA和0.1% Tween 20的磷酸缓冲液中; 3) connected to the particle surfaces fluorescein antibody: The amination of the surface of fluorescein particles dispersed in phosphate buffer, glutaraldehyde was added the reaction for 2-5 hours, centrifuged to remove glutaraldehyde, fluorescein particles redispersed in phosphate buffered was added 1 antibody, the reaction for 1-5 hours, to obtain particles were washed with fluorescein - 1 antibody complex was stored in 0.1% BSA-containing phosphate buffer and 0.1% of Tween 20;

4) 荧光免疫层析试纸的组装:将荧光素颗粒-抗体1复合物涂覆在结合垫上,将抗体2和第二抗体分别以线状连接于抗体承载膜上形成T线和C线,并在带有粘合剂的衬板上相互搭接地粘合样品垫、结合垫、抗体承载膜、吸水纸。 4) The fluorescent immunochromatographic test strip assembly: Fluorescein particles - antibody complex coated on the conjugate pad, 2 antibodies and second antibodies are linearly formed on the film attached to the antibody carrier T line and the C line, and on the liner overlap each other with an adhesive adhered sample pad, conjugate pad, antibody carrier film, absorbent paper.

本发明的技术方案之四是提供一种利用上述的荧光免疫层析试纸定量检 Aspect of the present invention is to provide a four using the fluorescent immunochromatographic test strip quantitative detection

测抗原的方法,依次包括如下步骤: The method of measuring an antigen, comprising the steps of sequentially:

i. 将抗原标准品配制成2〜10倍梯度的浓度系列标准品;ii. 将上述浓度系列标准品分别用上述的试纸进行免疫层析,利用荧光 . I The standard antigen preparation of a concentration series of 2~10 times the standard gradient;. Ii above series of standard concentrations were above the immunochromatography test strip, using fluorescence

定量光谱仪分别检测T线和C线区域的荧光强度,制作抗原浓度 Quantitative spectrometer detect the fluorescence intensity of T line and the C-line region, making the concentration of antigen

的标准曲线; Standard curve;

iii. 待测抗原用上述的试纸进行免疫层析,利用上述的标准曲线求得抗 iii. immunochromatography test antigen by the above-mentioned paper, to obtain a standard curve using the anti-

原浓度。 The original concentration.

上述的荧光免疫层析试纸定量检测抗原的方法的优选方案为,所述的抗原为甲胎蛋白或尿纤维连接蛋白。 The above-described preferred embodiment of the chromatography strip fluorescence immunoassay method for quantitative detection of the antigen, the antigen is alpha-fetoprotein or urine fibronectin.

上述的荧光免疫层析试纸也可以用于抗原定性检测,方法如下: The fluorescent immunochromatographic test strip described above may also be used for qualitative detection of antigen, as follows:

将试纸条的样品垫端插入待测样品液,10-30min后取出,在紫外或红外光的激发下观察结果。 The test strip sample pad end into the test sample liquid taken out after 10-30min, observation under ultraviolet or infrared light excitation. 如果检测带与质控带处都有荧光,说明待测样品中含有目标物,为阳性结果,检测带上荧光越强目标物含量越高;如果只有质控 If the test and the control line at both fluorescence, indicating the test sample containing the object, a positive result, the fluorescence detection zone the stronger the higher the content of the object; if only quality control

带有荧光,说明待测样品中没有目标物,为阴性结果;如果检测带与质控带 With fluorescence, the test sample is not described object, a negative result; if it is detected and the control line

处都没有荧光,说明检测无效。 At no fluorescence, indicating detection of invalid.

有益效果 Beneficial effect

本发明通过制备表面进行硅化包裹的荧光标记材料-抗体复合物进而制备了荧光免疫层析试纸。 The present invention is prepared by wrapping siliconized surface of the fluorescent marker materials - antibody complex fluorescent immunochromatographic test strip further prepared. 对稀土上转换发光材料和荧光素颗粒表面进行硅化包裹,克服了荧光素容易淬灭的缺陷。 Rare earth conversion luminescent material particle surfaces and fluorescein silicided wrapped overcome easily quenched fluorescein defects. 应用荧光免疫层析技术定量检测肿瘤标志物,克服了金标试纸不能定量测定的缺点,而且该免疫层析试纸的灵敏度 Quantitative fluorescence immunochromatography detection of tumor markers overcomes the disadvantages of the gold standard test paper can not be quantitatively determined, and the sensitivity of the immunochromatographic test strips

达1-1.5ng/mL,较金标试纸提高了10-100倍。 Up 1-1.5ng / mL, compared with the gold standard test paper improves 10-100 times.

本研究通过荧光素颗粒或上转换发光材料标记与双抗夹心免疫层析技术相结合,检测肿瘤标记物的含量,具有灵敏性高,准确性好,可实现定量分析等优势。 In this study, fluorescein or luminescent materials particulate converter with labeled double-antibody sandwich immunochromatography combination, the content of tumor markers detected with high sensitivity, good accuracy can be realized and other advantages quantitative analysis. 并且,填补了对尿纤维连接蛋白(Fn)定量免疫检测的技术空白。 And fill the immunoassay quantitative urine fibronectin (Fn) technology gaps. 对于肿瘤的早期诊断、预后判断以及治疗过程的监控都具有深远的影响。 For early diagnosis, prognosis and monitoring of cancer treatment has a profound effect.

具体实施方式 Detailed ways

下面结合具体实施例,进一步阐述本发明。 The following embodiments with reference to specific embodiments, further illustrate the present invention. 应理解,这些实施例仅用于 It should be understood that these examples are for

说明本发明而不用于限制本发明的范围。 Illustrate the present invention but not to limit the scope of the present invention. 此外应理解,在阅读了本发明讲授 Furthermore, it should be understood that, after reading the teachings of the present invention

的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。 After, one skilled in the art can make various changes or modifications of the present invention, and these equivalents also fall within the scope of the appended claims of the present application as defined.

下列实施例中未注明具体条件的实验方法,通常按照常规条件,如:分子克隆操作手册,或按照制造厂商所建议的条件。 Experimental methods without specific conditions in the examples below, generally in accordance with conventional conditions, such as: Molecular Cloning Manual, or in accordance with the conditions recommended by the manufacturer. 所有无机化学试剂和有机 All organic and inorganic chemical reagents

溶剂购自上海化学试剂厂,FITC和AEAPS购自上海浩然生物技术有限公司,红外上转换荧光粉(UCP)购自上海科炎光电技术有限公司、上海华明高纳稀土新材料有限公司和上海科润光电材料有限公司,小鼠抗AFP单克隆抗体和小鼠抗FN单克隆抗体购自上海我武生物科技有限公司,羊抗鼠IgG 抗体为上海史瑞可生物科技有限公司产品。 Solvents were purchased from Shanghai Chemical Reagent Factory, FITC, and purchased from Shanghai AEAPS awe Biotechnology Co., Ltd., conversion phosphor (of UCP) infrared purchased from Shanghai Branch Yan Optoelectronic Technology Co., Shanghai Huaming Gauna Rare Earth New Materials Co., Ltd. and Shanghai Kerun photoelectric material Co., mouse anti-AFP monoclonal antibody and mouse anti-FN monoclonal antibody was purchased from Shanghai I-biological Technology Co., goat anti-mouse IgG antibody is available on Haishi Rui biotechnology Co., Ltd. products.

实施例1:制备核壳型FITC-甲胎蛋白(AFP)抗体复合物 Preparation of core-shell type FITC- alpha-fetoprotein (AFP) antibody complex: Example 1

荧光素颗粒的表面硅化:在乙醇体系中,分别加入0.1-10mg异硫氰酸荧光素FITC和0.1-10nLN-(2-氨基乙基)-3-氨基丙基三甲氧基硅烷,15-50°C 恒温搅拌3〜10小时,再依次加入0.1-10mL水、0.1-10mL15。 Fluorescein surface of the silicide particles: ethanol system, 0.1-10mg were added fluorescein isothiocyanate FITC and 0.1-10nLN- (2- aminoethyl) -3-aminopropyl trimethoxy silane, 15-50 3~10 ° C temperature was stirred for hours, then water were added 0.1-10mL, 0.1-10mL15. /。 /. wt氨水、 0.1-10mL正硅酸乙酯(TEOS),继续反应3-10小时,洗涤获得表面硅化的荧光素颗粒。 wt ammonia, 0.1 ml to 10 tetraethyl orthosilicate (TEOS), reaction was continued for 10 hours, washed with fluorescein obtained siliconized surface of the particles. 荧光素的表面氨基化:取上述表面硅化的荧光素颗粒,加入10〜50mL 1:3〜3:1 (v/v)甲醇和丙三醇组成的混合溶液,超声分散,然后加入10-500|jL N- (2-氨基乙基)-3-氨基丙基三甲氧基硅烷,15-50。 Fluorescein surface amination: Take siliconized surface of the phosphor particles in the element, was added 10~50mL 1: 3~3: 1 (v / v) mixed solution of methanol and consisting of glycerol, ultrasonic dispersion, and then added 10-500 | jL N- (2- aminoethyl) -3-aminopropyl trimethoxy silane, 15-50. c恒温搅拌5〜10小时, 用乙醇洗涤,干燥,获得表面氨基化的荧光素颗粒。 c thermostat 5 to 10 hours with stirring, washed with ethanol and dried to obtain a surface of an amino fluorescein particles.

利用已经过表面氨基化的核壳型FITC与AFP抗体进行连接: It has been the use of core-shell type with FITC-AFP antibody surface amino groups of connections:

1、 取2mg氨基化的核壳型FITC加入到2mL0.03 mol/L的PB (pH=7.2)中, 充分混匀。 1, taken 2mg aminated FITC was added to the core-shell type 2mL0.03 mol / L of PB (pH = 7.2), and mix thoroughly.

2、 向上述体系中加入500pL25。 2, 500pL25 was added to the system. /。 /. 的戊二醛,室温下反应3小时。 Glutaraldehyde reacted at room temperature for 3 hours.

3、 离心洗去多余的戊二醛,重新分散在5mL 0.03 mol/L PB中。 3, the centrifugal wash off excess glutaraldehyde, redispersed in 5mL 0.03 mol / L PB in.

4、 再向其中加入100ijL小鼠抗AFP单克隆抗体,4"c反应3小时。 4, to which was added 100ijL mouse anti-AFP monoclonal antibody, 4 "c for 3 hours.

5、 4。 5, 4. c离心洗涤数次,保存于0.03 mol/L PB (含0.1。/。BSA, 0.1%Tween20)中。 c washed several times by centrifugation and stored at 0.03 mol / L PB (including 0.1./.BSA, 0.1% Tween20) in.

实施例2:制备双抗体夹心AFP抗原检测试纸 Example 2: Preparation of Double antibody sandwich antigen detection test AFP

1、 样品垫的制备:选用纤维素膜作为样品垫材料,剪成1.5x30.0cm规格的条带,将其放入样品垫封闭液(0.03mol/LPB (pH=7.2),含有5mg/mLBSA, 0.1%Triton X-100)中浸泡30min, 37。 1, the sample pad prepared: selection cellulose membrane as a sample pad material, cut into strips 1.5x30.0cm specifications, a sample pad into which a blocking solution (0.03mol / LPB (pH = 7.2), containing 5mg / mLBSA , 0.1% Triton X-100) immersed 30min, 37. c烘干备用。 c alternate drying.

2、 结合垫的制备:选用玻璃纤维素膜作为结合垫材料,将其剪成 2, the bond pads prepared: use glass as the bonding pads cellulose membrane material, which is cut

1.0x30.0cm规格的条带,将实施例1中制备的核壳型FITC-AFP抗体复合物离心,在沉降物中加入0.03mol/LPB (pH=7.2),含1%蔗糖,1%BSA), 充分混匀,加于该条带上,37。 1.0x30.0cm specifications strip, the embodiment centrifuged antibody complex prepared in Example 1, core-shell type FITC-AFP, added 0.03mol / LPB (pH = 7.2) in the sediment, containing 1% sucrose, 1% BSA ), mixed well, applied to the strap 37. c烘干。 c drying.

3、 NC的制备:将NC膜剪切成2.5x30.0cm规格的条带,用点样仪在NC膜上不同位置分别喷点AFP单抗和羊抗鼠IgG抗体,作为检测带和质控带,37'C烘干。 3, the prepared NC: NC membrane will be cut into strips 2.5x30.0cm specifications, respectively, with a spotter dots AFP antibody and goat anti-mouse IgG antibody at different positions NC membrane, a detection zone and the control band, 37'C drying.

4、 免疫层析试纸条的组装:将吸水纸、NC膜、结合垫、样品垫依次贴在带有粘合剂的底板上,并切成4cm宽的试纸条。 4, immunochromatographic strip assembly: the absorbent paper, NC membrane, conjugate pad, a sample pad attached to the bottom plate sequentially with adhesive, and cut into 4cm wide strip.

实施例3:分别以四甲基异硫氰酸罗丹明TRITC和四乙基罗丹明RB200替代实施例1-2中的异硫氰酸荧光素FITC制备双抗体夹心AFP抗原检测试纸。 Example 3: respectively tetramethyl rhodamine isothiocyanate and TRITC fluorescence tetraethyl rhodamine RB200 Alternative Preparation Example 1-2 isothiocyanate FITC embodiment double antibody sandwich element AFP antigen detection test.

实施例4:定量免疫检测AFP抗原 Quantitative immunoassay AFP antigen: Example 4

1 、 利用实施例1-3制成的荧光免疫层析试纸条定量检测20份临床血清AFP抗原样品: 1, using a fluorescent immunochromatographic test strip prepared in Example 1-3 embodiment quantitative detection of AFP antigen 20 clinical serum samples:

i. 将1mg/mL的AFP抗原标准品配制成10倍梯度的浓度系列标准 i. Set 1mg / mL of AFP antigen formulated into standard 10 times the concentration gradient series of standards

叫•, call•,

ii. 将上述浓度系列标准品分别用试纸进行免疫层析,利用荧光定量光谱仪分别检测T线和C线区域的荧光强度,制作抗原浓度的标准曲线; . Ii concentration series of the above standards were immunochromatography test strip, a standard curve using quantitative fluorescence spectroscopy to detect the fluorescence intensity of T line and the C line region, production of antigen concentrations;

iii. 将试纸条的样品垫端插入待测样品液,15min后取出,在紫外灯下观察结果,获得样品的定性检测结果:在20份样品血清中,临床 . Iii test strip sample pad end into the test sample solution, removed after 15min, observation under ultraviolet light, qualitative detection results obtained samples: 20 serum samples and clinical

符合率为95%; The rate was 95%;

iv. 用荧光检测仪检测20份样品血清的T线和C线区域的荧光强度, 利用上述的标准曲线求得抗原浓度,定量检测结果:在20份样品血清中,临床符合率为100%。 . IV detected by fluorescence detector 20 parts of the T line and the C-line region of the fluorescence intensity of the serum sample, the concentration of the antigen is obtained using the standard curve, quantitative results: the 20 serum samples, clinical accuracy was 100%. 2、利用AFP抗原标准品配制成10倍梯度的浓度系列,在免疫层析检测后用荧光检测仪分析本试纸监测系统的灵敏度,结果达1ng/mL。 2, using the standard AFP antigen formulated series of 10-fold concentration gradient, the analytical sensitivity of the present test strips monitoring systems for fluorescence detector after the immunochromatographic test results of 1ng / mL.

实施例5:制备UCP-尿纤维连接蛋白(FN)抗体复合物 Preparation UCP- urinary fibronectin (FN) antibody complex: Example 5

稀土上转荧光颗粒(铕掺杂的碱土金属硫化物或上海华明高纳稀土新材料有限公司的上转化荧光粉)的表面硅化:取5-50mg稀土上转荧光颗粒,加入300-800mL 35-70%乙醇溶液,超声处理使反应体系混合均匀,在15-50。 Raising the rare earth phosphor particles (the alkaline earth metal sulfides doped with europium or Shanghai Huaming Gauna Rare Earth New Materials Co. phosphor conversion) the surface of the silicide: Take 5-50mg turn rare earth phosphor particles, added 300-800mL 35 70% ethanol solution, sonicated mixed reaction system, at 15-50. c 水浴中搅拌至平衡,再向其中加入0.1-1mL正硅酸乙酯(TEOS),继续反应3-10小时,离心洗涤; c water bath and stirred until equilibrium was added thereto 0.1-1mL orthosilicate (TEOS), reaction was continued for 10 hours, washed by centrifugation;

稀土上转荧光颗粒的表面氨基化:取上述表面硅化的稀土上转荧光颗粒, 加入30〜100mL 1:3〜3:1 (V/V)甲醇和丙三醇组成的混合溶液,超声分散, 然后向其中加入IO-IOOOijLN- (2-氨基乙基)-3-氨基丙基三甲氧基硅烷, 15-5CrC恒温下搅拌5〜10小时,用乙醇离心洗涤,干燥保存; Rare earth phosphor particles on the surface of the transfer amination: phosphor particles on the transfer surface to take the above rare earth silicide, adding 30~100mL 1: 3~3: a mixed solution of 1 (V / V) methanol and consisting of glycerol, ultrasonic dispersion, then added IO-IOOOijLN- (2- aminoethyl) -3-aminopropyl trimethoxysilane, 15-5CrC thermostat 5 to 10 hours under stirring, centrifugation and washed with ethanol, and dried to save;

利用己经过表面氨基化的UCP与FN抗体进行连接: Hexyl using surface-aminated UCP antibody and FN are connected:

1、 取5mg氨基化的UCP加入到3mL0.03 mol/L的PB (pH=7.2)中,充分混匀。 1, taken UCP was added 5mg of amino to 3mL0.03 mol / L of PB (pH = 7.2), the full mix.

2、 向上述体系中加入250iJL25。 2, 250iJL25 was added to the system. /。 /. 的戊二醛,室温下反应3小时。 Glutaraldehyde reacted at room temperature for 3 hours.

3、 离心洗去多余的戊二醛,重新分散在5mL 0.03 mol/L PB中。 3, the centrifugal wash off excess glutaraldehyde, redispersed in 5mL 0.03 mol / L PB in.

4、 再向其中加入15CHjL小鼠抗fn单克隆抗体,4"c反应3小时。 4, to which was added 15CHjL monoclonal antibody mouse anti-fn, 4 "C for 3 hours.

5、 4。 5, 4. c离心洗涤数次,保存于0.03 mol/L PB (含0.1。/。BSA, 0.1%Tween20)中。 c washed several times by centrifugation and stored at 0.03 mol / L PB (including 0.1./.BSA, 0.1% Tween20) in. 实施例6:制备双抗体夹心FN抗原检测试纸 Example 6: Preparation of Double antibody sandwich antigen detection test FN

1、 样品垫的制备:选用纤维素膜作为样品垫材料,剪成1.5x30.0cm规格的条带,将其放入样品垫封闭液(0.03mol/LPB (pH=7.2),含有5mg/mLBSA, 0.1。/。Triton X-100)中浸泡30min, 37。 1, the sample pad prepared: selection cellulose membrane as a sample pad material, cut into strips 1.5x30.0cm specifications, a sample pad into which a blocking solution (0.03mol / LPB (pH = 7.2), containing 5mg / mLBSA , the 0.1./.Triton X-100) immersed 30min, 37. C烘干备用。 C drying standby.

2、 结合垫的制备:选用玻璃纤维素膜作为结合垫材料,将其剪成 2, the bond pads prepared: use glass as the bonding pads cellulose membrane material, which is cut

1.0x30.0cm规格的条带,将实施例3中制备的110?- FN抗体复合物离心, 在沉降物中加入0.03mol/LPB (pH=7.2),含1%蔗糖,1%BSA),充分混匀,加于该条带上,37。 1.0x30.0cm specifications strips, 110 cases of the embodiment 3 prepared -? FN antibody complex by centrifugation, was added 0.03mol / LPB (pH = 7.2) in the sediment, containing 1% sucrose, 1% BSA), thoroughly mixed, applied to the strap 37. C烘干。 C drying.

3、 NC的制备:将NC膜剪切成2.5x30.0cm规格的条带,用点样仪在NC 膜上不同位置分别喷点FN单抗和羊抗鼠IgG抗体,作为检测带和质控带, 37。 3, the prepared NC: NC membrane will be cut into strips 2.5x30.0cm specifications, respectively, with a spotter dots FN monoclonal antibodies and goat anti-mouse IgG at different positions NC membrane, a detection zone and the control belt 37. C烘干。 C drying.

4、 免疫层析试纸条的组装:将吸水纸、NC膜、结合垫、样品垫依次贴在带有粘合剂的底板上,并切成4cm宽的试纸条。 4, immunochromatographic strip assembly: the absorbent paper, NC membrane, conjugate pad, a sample pad attached to the bottom plate sequentially with adhesive, and cut into 4cm wide strip.

实施例7:分别以铒掺杂或铥惨杂的碱土金属硫化物替代实施例5-6中的铕掺杂的碱土金属硫化物制备双抗体夹心FN抗原检测试纸。 Example 7: erbium-doped or thulium, respectively miserable heteroaryl alkaline earth metal sulfides Alternative Preparation of alkaline earth metal sulfides doped with europium embodiment 5-6 embodiment FN double antibody sandwich antigen detection test.

实施例8:检测尿纤维连接蛋白(FN)抗体复合物 Urinary fibronectin (FN) antibody complex: Example 8

1、 利用实施例5-7制成的UCP荧光免疫层析试纸条定量检测16份临床血清FN抗原样品: 1, using the procedure of Example 5-7 UCP fluorescent immunochromatographic test strip 16 made quantitative detection of clinical serum samples FN antigens:

i. 将200ng/mL的FN抗原标准品配制成2倍梯度的浓度系列标准口 i. Set 200ng / mL FN-antigen standard series of standards prepared to a concentration gradient of the mouth 2 times

叩5ii. 将上述浓度系列标准品分别用试纸进行免疫层析,利用荧光定量光谱仪分别检测T线和C线区域的荧光强度,制作抗原浓度的标准曲线; . Knock 5ii the above series of standard concentration were immunochromatography test strip, the use of quantitative fluorescence spectrometer fluorescence intensity were the T line and the C line region, antigen concentration standard curve;

iii. 将试纸条的样品垫端插入待测样品液,15min后取出,在紫外灯下观察结果,获得样品的定性检测结果:在16份样品血清中,临床符合率为94%; . Iii test strip sample pad end into the test sample solution, removed after 15min, observation under ultraviolet light to obtain qualitative detection results of samples: 16 serum samples, 94% of clinical compliance;

iv. 用荧光检测仪检测16份样品血清的T线和C线区域的荧光强度, 利用上述的标准曲线求得抗原浓度,定量检测结果:在16份样品血清中,临床符合率为100%。 . IV fluorescence detector for detecting fluorescence intensity of 16 parts of T line and the C line region of serum samples, determined antigen concentration using the standard curve, quantitative results: the 16 serum samples, clinical accuracy was 100%.

2、利用FN抗原标准品配制成2倍梯度的浓度系列,在免疫层析检测后用荧光检测仪分析本试纸监测系统的灵敏度,结果达1.5ng/mL。 2, by FN antigen formulated 2 times standard gradient concentration series, the analytical sensitivity of the present test strips monitoring systems for fluorescence detector after the immunochromatographic test, the result was 1.5ng / mL.

Claims (10)

1.一种荧光免疫层析试纸,由依次在带有粘合剂的衬板上相互搭接的样品垫、结合垫、抗体承载膜、吸水纸组成,其中,所述的结合垫上涂有荧光标记材料-抗体1复合物,所述的抗体承载膜为硝酸纤维素膜或尼龙膜,抗体承载膜上分别为连接有抗体2和第二抗体的T线和C线,其特征在于,所述的荧光标记材料的表面经硅化包裹和氨基化,并通过交联反应与抗体结合;所述的T线和C线区域的荧光强度在检测中分别与标准抗原浓度的标准曲线相对应,完成定量检测。 A fluorescent immunochromatographic test strip, successively from the liner having an adhesive on the mutually overlapping sample pad, conjugate pad, antibody carrier film, absorbent paper composition, wherein said pad is coated with a fluorescent binding marking material - antibody complex, said antibody carrier film is a nitrocellulose membrane or a nylon membrane, the membrane carries antibodies were connected to the antibody and the second antibody 2 T line and the C line, characterized in that said surface of the fluorescent marking material package and siliconized amination, and bound to the antibody by a crosslinking reaction; T line and the C line region of the fluorescence intensity to a standard curve of a standard antigen concentration in the assay correspond, respectively, to complete the quantitative detection.
2. 根据权利要求1所述的荧光免疫层析试纸,其特征在于,所述的荧光标记材料为荧光素颗粒或者稀土上转荧光材料。 The fluorescent immunochromatographic test strip as claimed in claim 1, characterized in that said fluorescent marking material particle or fluorescein turn on rare earth fluorescent material.
3. 根据权利要求1所述的荧光免疫层析试纸,其特征在于,所述的荧光素颗粒为异硫氰酸荧光素或四甲基异硫氰酸罗丹明或四乙基罗丹明。 3. The fluorescent immunochromatographic test strip as claimed in claim 1, wherein said particles are fluorescein, fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate, or tetraethyl rhodamine.
4. 根据权利要求1所述的荧光免疫层析试纸,其特征在于,所述的稀土上转荧光颗粒为铕掺杂或铒掺杂或铥掺杂的碱土金属硫化物。 According to claim fluorescent immunochromatographic test strip of claim 1, wherein the phosphor particles doped with europium turn erbium-doped or thulium-doped alkaline earth metal or the rare earth sulfide.
5. 根据权利要求1-4之一所述的荧光免疫层析试纸,其特征在于,所述的抗体1和抗体2均为甲胎蛋白或尿纤维连接蛋白的抗体。 According to claim fluorescent immunochromatographic test strip according to one of 1-4, wherein the antibody of the antibody are both 1 and alpha-fetoprotein antibody or urine fibronectin.
6. —种荧光免疫层析试纸的制备方法,依次包括如下步骤:1)稀土上转荧光颗粒的表面硅化:取5-50mg稀土上转荧光颗粒,加入300-800mL 35-70%乙醇溶液,超声处理使反应体系混合均匀,在15-5CTC水浴中搅拌至平衡,再向其中加入0.1-1mL正硅酸乙酯,继续反应3-10小时,离心洗漆;2) 稀土上转荧光颗粒的表面氨基化:取上述表面硅化的稀土上转荧光颗粒,加入30〜100mL 1:3〜3:1 (V/V)甲醇和丙三醇组成的混合溶液, 超声分散,然后向其中加入10-1000[JLN- (2-氨基乙基)-3-氨基丙基三甲氧基硅垸,15-5CTC恒温下搅拌5〜10小时,用乙醇离心洗涤, 干燥保存;3) 稀土上转荧光颗粒表面连接抗体:将表面连接氨基的稀土上转荧光颗粒分散在磷酸缓冲液中,加入一定量戊二醛,反应2-5小时,离心除去戊二醛,重新分散在磷酸缓冲液中,然后加入抗体1,反应1-5小时,离心洗涤,最 6. - The method of producing a fluorescent immunochromatographic test strip, comprising the following steps in sequence: 1) the surface of the phosphor particles on the transfer silicide RE: Take turn phosphor particles rare 5-50mg, 300-800mL 35-70% ethanol solution was added, the reaction system was sonicated mixed, stirred in a water bath to balance 15-5CTC added thereto 0.1-1mL TEOS reaction was continued for 3-10 hours, centrifuged and removers; 2) transfer of rare earth phosphor particles surface amination: phosphor particles on the transfer surface to take the above rare earth silicide, adding 30~100mL 1: 3~3: a mixed solution of 1 (V / V) methanol and consisting of glycerol, ultrasonic dispersion, and then thereto was added 10- 1000 [JLN- (2- aminoethyl) -3-aminopropyl trimethoxysilane embankment, 15-5CTC thermostat 5 to 10 hours under stirring, washed with ethanol and centrifugation, stored dry; 3) on the transfer surface of the phosphor particles rare antibodies connection: the connection surface amino turn phosphor particles dispersed in the rare earth phosphate buffer, adding a certain amount of glutaraldehyde, the reaction for 2-5 hours, centrifuged to remove glutaraldehyde, redispersed in phosphate buffer, followed by addition of the antibody 1, the reaction for 1-5 hours, washed by centrifugation, most 终保存在含0.1% BSA和0.1% Tween 20的磷酸缓冲液中;4) 荧光免疫层析试纸的组装:将稀土上转荧光颗粒-抗体1复合物涂覆在结合垫上,将抗体2和第二抗体分别以线状连接于抗体承载膜上形成T线和C线,并在带有粘合剂的衬板上相互搭接地粘合样品垫、结合垫、抗体承载膜、吸水纸。 Finally stored in 0.1% BSA-containing phosphate buffer and 0.1% Tween 20 in; 4) a fluorescent immunochromatographic test strip assembly: the rare earth phosphor particles will turn - 1 antibody complex is coated in the pad binding, and antibody 2 diabodies linked to an antibody are linearly formed on the film carrier T line and the C line and overlap each other with the sample pad adhered to the adhesive backing, pad binding, antibody carrier film, absorbent paper.
7. 根据权利要求6所述的荧光免疫层析试纸的制备方法,其特征在于,所述的抗体1和抗体2均为甲胎蛋白或尿纤维连接蛋白的抗体。 7. A method of preparing a fluorescent immunochromatographic test strip according to claim 6, wherein the antibody of the antibody are both 1 and alpha-fetoprotein antibody or urine fibronectin.
8. —种荧光免疫层析试纸的制备方法,依次包括如下步骤:1)荧光素颗粒的表面硅化:在乙醇体系中,分别加入0.1-10mg异硫氰酸荧光素或四甲基异硫氰酸罗丹明或四乙基罗丹明,和0.1-10|jLN-(2-氨基乙基)-3-氨基丙基三甲氧基硅垸,15-5(TC恒温搅拌3〜10 小时,再依次加入0.1-10mL水、0.1國10mL15。/。wt氨水、0.1-10mL正硅酸乙酯(TEOS),继续反应3-10小时,洗涤获得表面硅化的荧光素颗粒;2) 荧光素的表面氨基化:取上述表面硅化的荧光素颗粒,加入10〜50mL 1:3〜3:1 (V/V)甲醇和丙三醇组成的混合溶液,超声分散,然后加入10-500MLN- (2-氨基乙基)-3-氨基丙基三甲氧基硅烷,15-5(TC恒温搅拌5〜10小时,用乙醇洗涤,干燥,获得表面氨基化的荧光素颗粒;3) 荧光素颗粒表面连接抗体:将上述表面氨基化的荧光素颗粒分散在磷酸缓冲液中,加入戊二醛反应2-5小时,离心除去戊 8. - preparation methods fluorescent immunochromatographic test strip, comprising the following steps in sequence: 1) the surface of the silicide particles Fluorescein: ethanol system, 0.1-10mg were added fluorescein isothiocyanate or tetramethyl isothiocyanate acid tetraethyl rhodamine or rhodamine, and 0.1-10 | jLN- (2- aminoethyl) -3-aminopropyl trimethoxysilane embankment, 15-5 (TC constant stirring 3~10 hours, and then successively of water was added 0.1 ml to 10, 0.1 State 10mL15./.wt ammonia, 0.1 ml to 10 tetraethyl orthosilicate (TEOS), reaction was continued for 10 hours, washed with fluorescein obtained siliconized surface of the particles; 2 surface amino) fluorescein of: take siliconized fluorescein of the surface particles, added 10~50mL 1: 3~3: a mixed solution of 1 (V / V) methanol and consisting of glycerol, ultrasonic dispersion, and then added 10-500MLN- (2- amino ethyl) -3-aminopropyl trimethoxysilane, 15-5 (TC thermostat 5 to 10 hours with stirring, washed with ethanol and dried to obtain a surface of the aminated particles fluorescein; 3) connected to the particle surfaces fluorescein antibody: the surface of the above-described amination fluorescein particles dispersed in phosphate buffer, glutaraldehyde was added the reaction for 2-5 hours, centrifuged to remove pentyl 醛,将荧光素颗粒重新分散在磷酸缓冲液中,加入抗体1,反应1-5小时,洗涤后获得荧光素颗粒-抗体1复合物,保存在含0.1% BSA和0.1% Tween 20的磷酸缓冲液中;4) 荧光免疫层析试纸的组装:将荧光素颗粒-抗体1复合物涂覆在结合垫上,将抗体2和第二抗体分别以线状连接于抗体承载膜上形成T线和C线,并在带有粘合剂的衬板上相互搭接地粘合样品垫、结合垫、抗体承载膜、吸水纸。 Aldehydes, fluorescein particles are redispersed in a phosphate buffer solution, the antibody was added, the reaction for 1-5 hours, to obtain particles were washed with fluorescein - 1 antibody complex was stored in 0.1% BSA containing phosphate buffer and 0.1% Tween 20 in solution; 4) a fluorescent immunochromatographic test strip assembly: fluorescein particles - antibody complex coated on the conjugate pad, 2 antibodies and second antibodies are antibodies to the carrier film to the linear connecting line and a T-C lines, and the liner having an adhesive bonded to each other overlap sample pad, conjugate pad, antibody carrier film, absorbent paper.
9. 一种利用权利要求1所述的荧光免疫层析试纸定量检测抗原的方法,依次包括如下步骤:i. 将抗原标准品配制成2〜10倍梯度的浓度系列标准品;ii. 将上述浓度系列标准品分别用权利要求1所述的试纸进行免疫层析,利用荧光定量光谱仪分别检测T线和C线区域的荧光强度, 制作抗原浓度的标准曲线;iii. 待测抗原用权利要求1所述的试纸进行免疫层析,利用上述的标准曲线求得抗原浓度。 A method of using a fluorescence claim 1 immunochromatographic strip quantitative detection of the antigen claim, comprising the following steps in sequence:.. I The standard antigen preparation of a concentration series of the standard gradient 2~10 times; ii above standard concentration series were used to test paper according to claim 1 immunochromatography using quantitative fluorescence spectroscopy to detect the fluorescence intensity of T line and the C line region, antigen concentration standard curve;. iii claimed in claim 1 with the antigen to be detected said strip for immunochromatography, antigen concentration obtained using the standard curve.
10.根据权利要求9所述的荧光免疫层析试纸定量检测抗原的方法,其特征在于,所述的抗原为甲胎蛋白或尿纤维连接蛋白。 Fluorescent immunochromatographic test strip 10. A method for the quantitative detection of the antigen according to claim 9, wherein said antigen is AFP or urine fibronectin.
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