WO2018120620A1 - 荧光免疫层析检测卡及其制备方法和应用 - Google Patents

荧光免疫层析检测卡及其制备方法和应用 Download PDF

Info

Publication number
WO2018120620A1
WO2018120620A1 PCT/CN2017/086069 CN2017086069W WO2018120620A1 WO 2018120620 A1 WO2018120620 A1 WO 2018120620A1 CN 2017086069 W CN2017086069 W CN 2017086069W WO 2018120620 A1 WO2018120620 A1 WO 2018120620A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
fluorescent
detection
treatment liquid
line
Prior art date
Application number
PCT/CN2017/086069
Other languages
English (en)
French (fr)
Inventor
孙宏浩
朱辉
周昌荣
Original Assignee
武汉纽康度生物科技股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 武汉纽康度生物科技股份有限公司 filed Critical 武汉纽康度生物科技股份有限公司
Priority to US16/336,042 priority Critical patent/US11959912B2/en
Publication of WO2018120620A1 publication Critical patent/WO2018120620A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

Definitions

  • the invention relates to the technical field of immunoassay, in particular to a fluorescent immunochromatographic detection card and a preparation method and application thereof.
  • methods for detecting disease markers include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (ECLIA), and POCT.
  • Radioimmunoassay often has large errors due to unstable samples or 125 I label instability, relatively low sensitivity and specificity, complicated measurement method, long time-consuming, and large sample size, which is not an ideal measurement method.
  • enzyme-linked immunosorbent assay ELISA
  • ELISA enzyme-linked immunosorbent assay
  • this method is difficult to prepare reagents, complicated in operation steps, time-consuming, and has many influencing factors. Control is difficult to guarantee.
  • the optical density of color is measured. Its precision and sensitivity are not as good as luminescent immune technology.
  • the detection of disease markers in serum mainly uses Roche Elesys electrochemiluminescence immunoassay system at home and abroad.
  • Chemiluminescence immunoassay has extremely high sensitivity and can quantitatively analyze the content of disease markers in samples, and the accuracy is high, but
  • the detection operation is more complicated than the POCT method, and the detection by the electrochemiluminescence immunofluorescence method is mostly for batch sampling, and is not suitable for small and medium-sized hospitals and individuals to detect the concentration of disease markers.
  • the expensive instrumentation and reagent prices required for detection limit the wide application of chemiluminescent immunoassays.
  • the utility model has the advantages of simple operation, low cost, quick result, small size and convenient carrying, but the sensitivity is not high. Therefore, it is necessary to develop a universal high-sensitivity disease marker detection card.
  • NT-proBNP amino-terminal B-type sodium urinary titanium
  • the concentration of NT-proBNP in serum is extremely low, in general, when the POCT method is used, the criterion is that the concentration of NT-proBNP is >300 ng/L, and the detection of heart failure markers prepared by the conventional immunofluorescence technique is stuck.
  • the sensitivity is low, and the extremely low concentration of NT-proBNP is difficult to be captured by the T line, which is difficult to detect. Therefore, it is very important to develop a high-sensitivity heart failure marker detection card for POCT detection of NT-proBNP.
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art, and provide a fluorescent immunochromatographic detection card with high sensitivity, high specificity and high stability, a preparation method thereof and application in rapid detection of disease markers .
  • the present invention adopts the following technical solutions:
  • a fluorescent immunochromatography detection card comprising a treatment liquid A, a treatment liquid B and a detection card, wherein the treatment liquid A contains an antibody 1 to be tested for antigen to be coupled with a fluorescent microsphere; and the treatment liquid B contains an even An antibody 2 for the antigen to be tested, the detection card comprising a detection line region and a quality control line region, wherein the detection line region is fixed with a streptavidin detection T line, and the quality control line region is fixed Antibody quality control C line.
  • the fluorescent microspheres form fluorescent microspheres by means of internal embedding of fluorescent molecules, and the T-line scribing substance is streptavidin, and the C-line
  • the line antibody is a goat anti-mouse antibody.
  • the antigen to be tested is NT-proBNP.
  • the antibody 1 is antibody 15C4 or antibody 5B6, and the antibody 2 is antibody 13G12 or antibody 15F11.
  • the present invention also provides a method for preparing a fluorescent immunochromatographic detection card, comprising the following steps:
  • the antibody 1 is added to the activated carboxyl fluorescent microsphere dispersion obtained in the step (1.1) to carry out a coupling reaction, and the precipitate is obtained after centrifugation to obtain a carboxyl fluorescent microsphere of the coupled antibody 1;
  • treatment solution B Adding antibody 2 to the biotin solution for coupling reaction, diluting and dialysis to remove unbound biotin, collecting samples to obtain treatment liquid B;
  • T-line is drawn in the detection line area of the detection card by streptavidin solution, and the C line is drawn in the quality control line area of the detection card by using the antibody solution.
  • the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and a carboxyl group The mass ratio of the fluorescent microspheres was 1:1, the mass ratio of the N-hydroxysuccinimide to the carboxy fluorescent microspheres was 1:1, and the activation time was 45 min.
  • the mass ratio of the antibody 1 to the carboxy fluorescent microsphere is 3:500, and the coupling reaction temperature is 4 ° C.
  • the time is 8h ⁇ 12h.
  • the molar ratio of the antibody 2 to the biotin is 1:50-200
  • the coupling reaction time is 4 h
  • dialysis The temperature was 4 ° C and the time was 48 h.
  • the concentration of the streptavidin solution is 10 ⁇ g/ ⁇ L, and the concentration of the antibody solution is 1 ⁇ g/cm.
  • the present invention also provides a fluorescent immunochromatographic test card prepared by the above fluorescent immunochromatographic test card or the above-mentioned method for preparing a fluorescent immunochromatographic test card, which is used for early diagnosis of diseases. .
  • the present invention provides a high-sensitivity antigen detection card and a preparation method thereof based on fluorescence immunochromatography and biotin-streptavidin labeling technology. Both streptavidin and biotin can be combined with proteins (including antigens, antibodies, enzymes, etc.), fluorescein and the like without affecting the biological activity of the latter, and are ideal labeling agents.
  • An antibody molecule can be coupled to dozens of biotin or streptavidin molecules, while streptavidin or biotin molecules can be combined with enzymes or fluorescein, using a very polar link between biotin and streptavidin
  • Strong affinity and streptavidin have multiple binding sites to form a biomagnification system that amplifies the signal and the biotin-streptavidin system (BAS) has high specificity and high
  • the sensitivity and high stability make the specificity and sensitivity of various tracer immunoassays further improved. Thereby, the rapid and sensitive detection of trace substances is realized, and the problem that the concentration of trace substances is too low to be difficult to detect is successfully solved.
  • fluorescent microspheres are formed by internal embedding using fluorescent molecules, and the formed fluorescent microspheres are stable, difficult to quench, strong in brightness, and high in sensitivity.
  • Streptavidin-coated microparticles are highly efficient, uniform, stable and versatile. The biotinylated antibody binds to the antigen in the sample, and is streaked with streptavidin, which is extremely strong between biotin-streptavidin.
  • Binding force promoting antibody 1 labeled with fluorescent microspheres and biotinylated antibody 2 capture antigen in the sample to form a sandwich-like immune complex, through biotin-labeled immune complexes and streptavidin on T-line
  • the fluorescence intensity on the T line (detection line) and the C line (control line) are detected separately, and the concentration of the disease marker in the sample is quantified by the ratio of the fluorescence intensity on the T line and the C line.
  • Streptavidin-biotin is the strongest and specific combination, ensuring a firm coating effect and specific test results.
  • the instrument and equipment required for the detection card of the invention are small in size, convenient to carry, easy to operate, easy to master, no professional operation, and low detection cost.
  • different disease markers can be detected by replacing the corresponding treatment liquids A and B for different detection items.
  • the invention is particularly suitable for the rapid detection of NT-proBNP, and the NT-proBNP detection card prepared by the biotin-streptavidin labeling technique and the immunofluorescence method can detect the trace amount of NT-proBNP in the sample more sensitively. Accurate and rapid diagnosis of heart failure patients, reasonable structure, using streptavidin stroke detection line, establish immunochromatography to detect the content of NT-proBNP in the sample.
  • the specificity is strong, the sensitivity is high, and the minimum detection concentration is up to 20 ng/ml; in addition, the antibody 15C4-labeled fluorescent microsphere prepared by the invention and the biotin-labeled antibody 13G12 are stored after lyophilization, have good stability, and are stored at room temperature for 12 months. Test card with 4 ° C The saved test results are consistent.
  • Figure 1 is a schematic view showing the structure of a fluorescent immunochromatographic test card of the present invention.
  • Example 2 is a fluorescence intensity fitting curve corresponding to the detection of different NT-proBNP concentrations by the NT-proBNP fluorescent immunochromatographic assay card according to Example 1 of the present invention.
  • the NT-proBNP fluorescent immunochromatographic test card of the present invention comprises a treatment liquid A, a treatment liquid B and a detection card, wherein the treatment liquid A contains the antibody 15C4 coupled with the fluorescent microsphere; the treatment liquid B contains the coupling Biotin antibody 13G12, the detection card includes a detection line region and a quality control line region, and the detection line region is fixed with a streptavidin detection T line, and the quality control line region is fixed with a goat anti-mouse antibody quality control C line.
  • step (1.3) adding 250 ⁇ L of bovine serum albumin (BSA) to the carboxy fluorescent microsphere of the coupled antibody 15C4 obtained in the step (1.2), Block at room temperature for 2 h to block the activated carboxyl site of unconjugated antibody, block and centrifuge (14400 rpm, 8 min), take a precipitate, wash once with 1000 ⁇ L of 10 mM PB, add 250 ⁇ L of antibody preservation solution, and store at 4 ° C to obtain treatment solution A. .
  • BSA bovine serum albumin
  • the treatment liquid A (antibody 15C4 containing the fluorescent microspheres coupled) prepared in the step (1) is mixed with a blood sample, and the antibody 15C4 forms an antigen-antibody complex with the NT-proBNP (antigen) in the blood sample, and then into the blood sample.
  • the treatment liquid B (containing biotin-labeled antibody 13G12) prepared in the step (2) was added, and the antibody 13G12 was combined with NT-proBNP to form a sandwich structure complex. Drip the mixture sample into the drop hole of the test card.
  • the schematic diagram of the detection principle is shown in Figure 1.
  • S2 Result determination: scanning with a quantitative fluorescence immunoassay analyzer to obtain a fluorescence intensity curve, which is analyzed and calculated by a specific algorithm to obtain a dimensionless ratio T/C (ratio of fluorescence intensity on the detection line and the control line), with reference to the standard.
  • the concentration curve can be used to obtain the concentration of NT-proBNP in the sample to be tested.
  • Table 1 and Figure 2 The test results are shown in Table 1 and Figure 2.
  • the NT-proBNP detection card prepared by the biotin-streptavidin labeling technique and the immunofluorescence method can detect the trace amount of NT-proBNP in the sample sensitively for the heart failure patient.
  • the structure is reasonable, and the detection line of streptavidin is used to establish the immunochromatographic method to detect the content of NT-proBNP in the sample.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Nanotechnology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

一种荧光免疫层析检测卡及其制备方法和应用，荧光免疫层析检测卡包括处理液A、处理液B和检测卡；处理液A中含有偶联荧光微球的针对待测抗原的抗体1；处理液B中含有偶联生物素的针对待测抗原的抗体2；检测卡包括检测线区域和质控线区域，检测线区域固定有链霉亲和素检测T线，质控线区域固定有抗体质控C线。制备方法包括：(1)配制处理液A；(2)配制处理液B；(3)检测卡划线。该荧光免疫层析检测卡具有高灵敏度高、高特异性、高稳定性等特点，可应用于疾病标志物的快速检测。

Description

荧光免疫层析检测卡及其制备方法和应用 技术领域
本发明涉及免疫测定技术领域，尤其涉及一种荧光免疫层析检测卡及其制备方法和应用。
背景技术
目前，检测疾病标志物的方法主要有放射免疫分析法(RIA)、酶联免疫分析法(ELISA)、电化学发光免疫分析法(ECLIA)和POCT法等。
放射免疫法因提取样本或125I标记不稳定常出现较大误差，灵敏度和特异性相对较低，测定方法复杂、耗时长，并且所需样本量也较大，不是理想的测定方法。近年来，酶联免疫分析法在疾病的临床诊断中取得了广泛的应用，但是此方法试剂制备困难，操作步骤复杂、耗时长，影响因素多，控制难以保证，最后测定的是颜色的光密度，其精密度和敏感性不如发光免疫技术。目前，血清中的疾病标志物检测国内外主要采用Roche Elesys电化学发光免疫分析系统，化学发光免疫分析法具有极高的灵敏度，可定量分析样本中疾病标志物的含量，且准确度高，但检测操作较POCT法复杂，且采用电化学发光免疫荧光法进行检测时多为批量采集样品进行检测，不适用于中小型医院及个人进行疾病标志物浓度的检测。除此之外，检测所需的昂贵的仪器和试剂价格限制了化学发光免疫分析法的广泛应用。采用POCT法使用现有的检测卡检测疾病标志物时，具有操作简单、成本低、能快速得到结果、仪器体积小方便携带等优点，但是灵敏度不高。因此有待开发一种通用的高灵敏度的疾病标志物检测卡。
例如，心力衰竭是各种心血管疾病发展的终末阶段，病情凶险，死亡率高，如不能早期识别并及时处理，往往预后不佳，因此，对心力衰竭患者特别是急诊送诊的患者进行快速诊断并给予尽早的治疗非常重要。众多研究表明，氨基末端B型钠尿钛原(NT-proBNP)是很好的心衰诊断标志物。NT-proBNP浓度的检测对于临床上诊断心衰具有重要意义，NT-proBNP是美国和欧洲心脏病学会推荐使用的目前最好的用于评价心力衰竭的实验室检测指标。由于血清中NT-proBNP浓度极低，通常情况下，采用POCT法时，判断标准为是NT-proBNP浓度＞300ng/L即为阳性，使用传统的免疫荧光技术制备的心衰标志物检测卡在检测NT-proBNP时，灵敏度低，浓度极低的NT-proBNP很难被T线捕捉，难以检测。因此开发一种高灵敏度的心衰标志物检测卡应用于POCT法检测NT-proBNP十分重要。
技术问题
本发明要解决的技术问题是克服现有技术的不足，提供一种高灵敏度高、高特异性、高稳定性的荧光免疫层析检测卡及其制备方法和在疾病标志物快速检测中的应用。
技术解决方案
为解决上述技术问题，本发明采用以下技术方案：
一种荧光免疫层析检测卡，包括处理液A、处理液B和检测卡，所述处理液A中含有偶联荧光微球的针对待测抗原的抗体1；所述处理液B中含有偶联生物素的针对待测抗原的抗体2，所述检测卡包括检测线区域和质控线区域，所述检测线区域固定有链霉亲和素检测T线，所述质控线区域固定有抗体质控C线。
上述的荧光免疫层析检测卡，优选地，所述荧光微球为荧光分子通过内部包埋的方式形成荧光微球，所述T线划线物质为链霉亲和素，所述C线划线抗体为羊抗鼠抗体。
上述的荧光免疫层析检测卡，优选地，所述待测抗原为NT-proBNP。
上述的荧光免疫层析检测卡，优选地，所述抗体1为抗体15C4或抗体5B6，所述抗体2为抗体13G12或抗体15F11。
作为一个总的发明构思，本发明还提供一种荧光免疫层析检测卡的制备方法，包括以下步骤：
(1)配制处理液A：
(1.1)将1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液和N-羟基琥珀酰亚胺溶液加入到羧基荧光微球中进行活化，活化后离心，收集沉淀，用磷酸盐缓冲液重悬，得到活化后的羧基荧光微球分散液；
(1.2)将抗体1加入到步骤(1.1)所得的活化后的羧基荧光微球分散液中进行偶联反应，离心后取沉淀物，得到偶联抗体1的羧基荧光微球；
(1.3)向步骤(1.2)所得的偶联抗体1的羧基荧光微球中加入牛血清白蛋白，封闭未偶联抗体的活化羧基位点，封闭后离心，取沉淀，加入抗体保存液，得到处理液A；
(2)配制处理液B：将抗体2加入到生物素溶液中进行偶联反应，稀释后透析，以去除未结合的生物素，收集样品，得到处理液B；
(3)检测卡划线：采用链霉亲和素溶液在检测卡的检测线区域内划T线，采用抗体溶液在检测卡的质控线区域内划C线。
上述的荧光免疫层析检测卡的制备方法，优选地，所述步骤(1.1)中，所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与羧基荧光微球的质量比为1∶1，所述N-羟基琥珀酰亚胺与羧基荧光微球的质量比为1∶1，活化时间为45min。
上述的荧光免疫层析检测卡的制备方法，优选地，所述步骤(1.2)中，所述抗体1与羧基荧光微球的质量比为3∶500，所述偶联反应温度为4℃，时间为8h～12h。
上述的荧光免疫层析检测卡的制备方法，优选地，所述步骤(2)中，所述抗体2与所述生物素的摩尔比为1∶50～200，偶联反应时间为4h，透析温度为4℃，时间为48h。
上述的荧光免疫层析检测卡的制备方法，优选地，所述步骤(3)中，所述链霉亲和素溶液的浓度为10μg/μL，所述抗体溶液的浓度为1μg/cm。
作为一个总的发明构思，本发明还提供一种上述的荧光免疫层析检测卡或上述的荧光免疫层析检测卡的制备方法所制备的荧光免疫层析检测卡在疾病的早期诊断中的应用。
有益效果
与现有技术相比，本发明的优点在于：
1、本发明基于荧光免疫层析法，同时结合生物素-链霉亲和素标记技术，提供了一种高灵敏度待测抗原检测卡及其制备方法。链霉亲和素与生物素都可与蛋白质(包括抗原、抗体、酶等)、荧光素等分子结合而不影响后者的生物活性，是理想的标记剂。一个抗体分子可偶联数十个生物素或链霉亲和素分子，而链霉亲和素或生物素分子又可与酶或荧光素结合，利用生物素和链霉亲和素之间极强的亲和力以及链霉亲和素具有多个结合位点的特性，从而组成一个生物放大系统，将信号多极放大，并且生物素-链霉亲和素系统(BAS)具有高特异性、高灵敏度、高稳定性的特点，使各种示踪免疫分析的特异性和灵敏度进一步提高。从而实现痕量物质快速灵敏的检测，成功地解决了痕量物质浓度太低造成难以检测的困扰。
2、进一步地，本发明的目的通过以下技术方案实现：使用荧光分子通过内部包埋的方式形成荧光微球，所形成的荧光微球稳定，不易淬灭，亮度强，灵敏度高。链霉亲和素包被微粒子高效、均一、稳定、通用，生物素化抗体与样品中的抗原结合，使用链霉亲和素划线，利用生物素-链霉亲和素之间极强的结合力，促进标记有荧光微球的抗体1和生物素化的抗体2捕捉样品中的抗原，形成夹心结构的免疫复合物，通过标记有生物素的免疫复合物与T线上的链霉亲和素相结合，分别检测T线(检测线)上和C线(控制线)上的荧光强度,通过T线和C线上荧光强度的比值来定量样品中疾病标志物的浓度。链霉亲和素-生物素是最牢固和特异的结合，保证了牢固的包被效果和特异的检测结果。
3、本发明的检测卡所需仪器设备体积小，携带如方便，操作简单易于掌握，无需专业人员操作，并且检测成本低。另外，只要针对不同的检测项目更换相应的处理液A和B，即可检测不同的疾病标志物。
4、本发明尤其适用于NT-proBNP的快速检测，利用生物素-链霉亲和素标记技术与免疫荧光法制备的NT-proBNP检测卡能够较灵敏地检测出样本中微量的NT-proBNP从而对心衰病人进行准确的快速诊断，结构合理，使用链霉亲和素划检测线，建立免疫层析法检测样品中NT-proBNP的含量。特异性强，灵敏度高，最低检测浓度达20ng/ml；另外，本发明制备的抗体15C4标记的荧光微球和生物素标记的抗体13G12冷冻干燥后进行保存，稳定性好，常温保存12个月检测卡与4℃ 保存的检测结果一致。
附图说明
图1为本发明的荧光免疫层析检测卡的结构原理示意图。
图2为本发明实施例1的NT-proBNP荧光免疫层析检测卡检测不同NT-proBNP浓度对应的荧光强度拟合曲线。
本发明的实施方式
以下结合说明书附图和具体优选的实施例对本发明作进一步描述，以下实施例描述了本发明的特殊实施例子，以便对本发明作进一步的说明，这些实施例只是说明而不表示本发明所有的可能性，本发明并不仅仅局限于这些实施例中的材料、反应条件或参数。任何在相关领域具备经验的人，都可以按照本专利的原理利用其他的材料或反应条件实现本发明所描述的生物素-链霉亲和素标记技术结合免疫层析技术制备疾病标志物检测卡。这些并不脱离本发明描述的基本概念，因此这些修改的或者不同的应该都在本发明覆盖的范围内。
实施例1：
一种本发明的NT-proBNP荧光免疫层析检测卡，包括处理液A、处理液B和检测卡，其中，处理液A中含有偶联荧光微球的抗体15C4；处理液B中含有偶联生物素的抗体13G12，检测卡包括检测线区域和质控线区域，检测线区域固定有链霉亲和素检测T线，所述质控线区域固定有羊抗鼠抗体质控C线。
上述本实施例的NT-proBNP荧光免疫层析检测卡的制备方法，包括以下步骤：
(1)配制处理液A：
(1.1)吸取200μL羧基荧光微球(浓度为10mg/mL)，离心(14400rpm，10min)。取1000μL pH6.0MES缓冲液清洗，离心(14400rpm，8min)，重复该操作一次。称取0.0110g1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)溶于110μL的2-(N-吗啡啉)乙磺酸(MES)缓冲液中，称取0.0034gN-羟基琥珀酰亚胺(NHS)溶于34μLMES缓冲液中。向荧光微球中分别加入1250μLMES缓冲液、25μL上述EDC溶液和25μL上述NHS溶液，室温下搅拌(60rpm)，活化45min。取1000μL pH7.4的10mM磷酸盐(PB)缓冲液清洗，离心(14400rpm，10min)，重复该操作一次。清洗结束后，用1250μL PB缓冲液重悬，得到活化后的羧基荧光微球分散液。
(1.2)在步骤(1.1)所得的活化后的羧基荧光微球分散液中加入1.64μL抗体15C4(9.2mg/mL)，在4℃的条件下过夜进行偶联反应。离心(14400rpm，8min)后取沉淀物，得到偶联抗体15C4的羧基荧光微球。
(1.3)向步骤(1.2)所得的偶联抗体15C4的羧基荧光微球中加入250μL牛血清白蛋白(BSA)， 室温下封闭2h，以封闭未偶联抗体的活化羧基位点，封闭后离心(14400rpm，8min)，取沉淀，用1000μL 10mM PB清洗一次，加入250μL抗体保存液，4℃保存，得到处理液A。
(2)配制处理液B：
取100μg抗体13G12加入到生物素中进行偶联反应，抗体与生物素的摩尔比1:100，总体积50μL，室温下反应4h；反应完成后稀释溶液到250μL，4℃透析48h；以去除未结合的生物素，收集样品，得到处理液B；
(3)检测卡划线：
制备浓度10μg/μL的链霉亲和素溶液，浓度为1μg/cm的羊抗鼠IgG；分别使用上述浓度的链霉亲和素溶液在检测卡的检测线区域内划T线，羊抗鼠IgG在检测卡的质控线区域内划C线；4℃保存备用。
上述本实施例制备的NT-proBNP荧光免疫层析检测卡在检测NT-proBNP中的应用，包括如下步骤：
S1：将步骤(1)配制的处理液A(含偶联荧光微球的抗体15C4)与血液样品混合，抗体15C4与血液样品中的NT-proBNP(抗原)形成抗原抗体复合物，再向其中加入步骤(2)配制的处理液B(含生物素标记的抗体13G12)，抗体13G12与NT-proBNP结合，形成夹心结构复合物。将混合物样本滴入检测卡的滴样孔内。检测原理示意图如图1所示。
S2：结果判定：使用定量荧光免疫分析仪进行扫描，得到荧光强度曲线，经特定算法分析计算后得到一个无量纲的比值T/C(检测线上与质控线上荧光强度比值)，参照标准浓度曲线就可以得到待测样本中NT-proBNP的浓度。检测结果如表1和图2所示。
表1  不同NT-proBNP浓度对应的荧光强度比值
编号 T/C 抗原浓度
1 1.5960 2097
2 1.2951 1573
3 0.8872 1049
4 0.7091 699
5 0.6206 525
6 0.5162 393
7 0.4538 262
8 0.4134 233
9 0.3571 131
10 0.3231 78
11 0.3153 66
12 0.2956 33
13 0.2917 26
14 0.2887 20
15 0.2651 16
16 0.2412 0
由表1和图2可知，本发明利用生物素-链霉亲和素标记技术与免疫荧光法制备的NT-proBNP检测卡能够较灵敏地检测出样本中微量的NT-proBNP从而对心衰病人进行准确的快速诊断，结构合理，使用链霉亲和素划检测线，建立免疫层析法检测样品中NT-proBNP的含量。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例。凡属于本发明思路下的技术方案均属于本发明的保护范围。应该指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下的改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种荧光免疫层析检测卡，包括处理液A、处理液B和检测卡，其特征在于，所述处理液A中含有偶联荧光微球的针对待测抗原的抗体1；所述处理液B中含有偶联生物素的针对待测抗原的抗体2，所述检测卡包括检测线区域和质控线区域，所述检测线区域固定有链霉亲和素检测T线，所述质控线区域固定有抗体质控C线。
  2. 根据权利要求1所述的荧光免疫层析检测卡，其特征在于，所述荧光微球为荧光分子通过内部包埋的方式形成荧光微球，所述T线划线物质为链霉亲和素，所述C线划线抗体为羊抗鼠抗体。
  3. 根据权利要求1或2所述的荧光免疫层析检测卡，其特征在于，所述待测抗原为NT-proBNP。
  4. 根据权利要求3所述的荧光免疫层析检测卡，其特征在于，所述抗体1为抗体15C4或抗体5B6，所述抗体2为抗体13G12或抗体15F11。
  5. 一种如权利要求1～4任一项所述的荧光免疫层析检测卡的制备方法，包括以下步骤：
    (1)配制处理液A：
    (1.1)将1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液和N-羟基琥珀酰亚胺溶液加入到羧基荧光微球中进行活化，活化后离心，收集沉淀，用磷酸盐缓冲液重悬，得到活化后的羧基荧光微球分散液；
    (1.2)将抗体1加入到步骤(1.1)所得的活化后的羧基荧光微球分散液中进行偶联反应，离心后取沉淀物，得到偶联抗体1的羧基荧光微球；
    (1.3)向步骤(1.2)所得的偶联抗体1的羧基荧光微球中加入牛血清白蛋白，封闭未偶联抗体的活化羧基位点，封闭后离心，取沉淀，加入抗体保存液，得到处理液A；
    (2)配制处理液B：将抗体2加入到生物素溶液中进行偶联反应，稀释后透析，以去除未结合的生物素，收集样品，得到处理液B；
    (3)检测卡划线：采用链霉亲和素溶液在检测卡的检测线区域内划T线，采用抗体溶液在检测卡的质控线区域内划C线。
  6. 根据权利要求5所述的荧光免疫层析检测卡的制备方法，其特征在于，所述步骤(1.1)中，所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与羧基荧光微球的质量比为1∶1，所述N-羟基琥珀酰亚胺与羧基荧光微球的质量比为1∶1，活化时间为45min。
  7. 根据权利要求6所述的荧光免疫层析检测卡的制备方法，其特征在于，所述步骤(1.2)中，所述抗体1与羧基荧光微球质量比为3∶500，所述偶联反应温度为4℃，时间为8h～12h。
  8. 根据权利要求5所述的荧光免疫层析检测卡的制备方法，其特征在于，所述步骤(2)中， 所述抗体2与所述生物素的摩尔比为1∶50～200，偶联反应时间为4h，透析温度为4℃，时间为48h。
  9. 根据权利要求5所述的荧光免疫层析检测卡的制备方法，其特征在于，所述步骤(3)中，所述链霉亲和素溶液的浓度为10μg/μL，所述抗体溶液的浓度为1μg/cm。
  10. 一种如权利要求1～4任一项所述的荧光免疫层析检测卡或如权利要求5～9任一项所述的荧光免疫层析检测卡的制备方法所制备的荧光免疫层析检测卡在疾病的早期诊断中的应用。
PCT/CN2017/086069 2016-12-30 2017-05-26 荧光免疫层析检测卡及其制备方法和应用 WO2018120620A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/336,042 US11959912B2 (en) 2016-12-30 2017-05-26 Fluorescence immunochromatographic detection card and a preparation method therefor and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201611258692.5A CN106771168A (zh) 2016-12-30 2016-12-30 荧光免疫层析检测卡及其制备方法和应用
CN201611258692.5 2016-12-30

Publications (1)

Publication Number Publication Date
WO2018120620A1 true WO2018120620A1 (zh) 2018-07-05

Family

ID=58954536

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/086069 WO2018120620A1 (zh) 2016-12-30 2017-05-26 荧光免疫层析检测卡及其制备方法和应用

Country Status (3)

Country Link
US (1) US11959912B2 (zh)
CN (1) CN106771168A (zh)
WO (1) WO2018120620A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113970635A (zh) * 2021-10-27 2022-01-25 上海艾瑞德生物科技有限公司 一种免疫层析试纸及其制备方法和应用

Families Citing this family (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106771168A (zh) * 2016-12-30 2017-05-31 武汉纽康度生物科技股份有限公司 荧光免疫层析检测卡及其制备方法和应用
CN107782898B (zh) * 2017-09-22 2021-07-30 宁波瑞源生物科技有限公司 一种荧光免疫层析法检测试剂盒及其检测方法
CN107478829A (zh) * 2017-09-29 2017-12-15 亳州市新健康科技有限公司 一种基于双竞争免疫层析法的毒品检测试剂盒及其制备工艺
CN108226468A (zh) * 2017-12-28 2018-06-29 湖北工业大学 一种检测NT-proBNP的试纸条及其制备方法与应用
CN110133254A (zh) * 2018-02-02 2019-08-16 中国人民解放军军事科学院军事医学研究院 一种插拔式可实现信号二次放大的拉曼层析试纸
CN108732344B (zh) * 2018-04-12 2021-06-11 江苏维尔生物科技有限公司 一种用于快速检测紫杉醇的试纸及其制备方法、试剂盒
CN109358197A (zh) * 2018-11-29 2019-02-19 中国农业科学院油料作物研究所 一种基于桥联抗体偶联荧光微球的荧光检测器件及荧光检测方法
CN109799334A (zh) * 2019-02-06 2019-05-24 吉林双正生物工程有限公司 心脏标志物的荧光微球联合检测装置及其制备方法
CN109943663A (zh) * 2019-03-22 2019-06-28 安徽深蓝医疗科技股份有限公司 针对hpv 16和hpv 18基因型的重组酶扩增检测方法、试纸条及其应用
CN110596404A (zh) * 2019-09-20 2019-12-20 成都艾科斯伦医疗科技有限公司 一种il-6生物素-链霉亲和素免疫层析检测卡
CN110672840A (zh) * 2019-09-26 2020-01-10 科赫生物科技（北京）有限公司 一种夹心法抗体检测方法及试纸
CN110672841A (zh) * 2019-09-26 2020-01-10 科赫生物科技（北京）有限公司 一种夹心法抗原检测方法及试纸
CN111323576B (zh) * 2020-02-27 2024-04-26 四川新健康成生物股份有限公司 一种增强抗体-荧光微球偶联物信号的方法以及在肌钙蛋白i检测中的应用
CN111537752A (zh) * 2020-05-18 2020-08-14 巴迪泰（广西）生物科技有限公司 抗缪勒管激素检测试剂盒
CN111579800A (zh) * 2020-05-18 2020-08-25 巴迪泰（广西）生物科技有限公司 超敏降钙素原检测试剂及其制备方法及使用方法
CN111983243A (zh) * 2020-08-10 2020-11-24 深圳市宇诺生物技术有限公司 氨基末端脑利钠肽前体测定试剂盒、制备方法及检测方法
CN113834934A (zh) * 2021-11-29 2021-12-24 北京百普赛斯生物科技股份有限公司 免疫层析试纸条及其制备方法
CN114034854A (zh) * 2021-12-03 2022-02-11 广州达泰生物工程技术有限公司 定量检测cTnT/NT-proBNP/D-dimer的试剂盒及其应用
CN114113632A (zh) * 2021-12-03 2022-03-01 河南省农业科学院动物免疫学重点实验室 一种通用型动物疫病抗体检测免疫层析方法与应用
US20230042375A1 (en) * 2022-09-20 2023-02-09 Marvin Liu Membrane based chemiluminescence immunochromatography assay and its use
CN116400070B (zh) * 2023-06-07 2023-08-18 济南德亨医学科技有限公司 高通量定量检测过敏原特异性IgE抗体试剂的制备方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008056034A1 (en) * 2006-11-10 2008-05-15 Hytest Ltd. Stable standards for bnp immunoassays
CN102841207A (zh) * 2011-06-24 2012-12-26 北京乐普医疗科技有限责任公司 定量检测肌钙蛋白i的荧光免疫层析试纸条及其制备方法
CN103163297A (zh) * 2011-12-16 2013-06-19 王哲 多功能荧光免疫层析快速定量检测卡
CN103197074A (zh) * 2013-04-22 2013-07-10 北京润博福得生物科技发展有限公司 基于近红外荧光纳米微球标记物的免疫层析定量检测试剂
CN105198996A (zh) * 2015-11-10 2015-12-30 三诺生物传感股份有限公司 一种单克隆抗体制备方法及其试剂盒
CN105652008A (zh) * 2016-03-31 2016-06-08 广州市微米生物科技有限公司 人Lp-PLA2生物素-链霉亲和素荧光免疫层析检测卡及其制备方法
CN106771168A (zh) * 2016-12-30 2017-05-31 武汉纽康度生物科技股份有限公司 荧光免疫层析检测卡及其制备方法和应用

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001013120A1 (en) * 1999-08-17 2001-02-22 Luminex Corporation Microparticles with multiple fluorescent signals and methods of using same
DE10355731A1 (de) * 2003-11-28 2005-06-30 Roche Diagnostics Gmbh Analytischer Sandwichtest zur Bestimmung von NT-proBNP
WO2007022530A2 (en) * 2005-08-16 2007-02-22 The Children's Mercy Hospital Quantification of microsphere suspension hybridization and uses thereof
GB2436616A (en) * 2006-03-29 2007-10-03 Inverness Medical Switzerland Assay device and method
US20100291709A1 (en) * 2009-05-13 2010-11-18 Abbott Laboratories Human nt-pro b-type natriuretic peptide assay having reduced cross-reactivity with other peptide forms
EP2795328B1 (en) * 2011-12-23 2019-05-01 Abbott Point of Care Inc. Integrated test device for optical and electrochemical assays
US9738717B2 (en) * 2013-05-15 2017-08-22 The Translational Genomics Research Institute Hybridoma clones, monoclonal antibodies, and methods of use
CN103940798B (zh) * 2014-05-05 2016-08-17 武汉纽康度生物科技有限公司 一种实体荧光纳米微球及其制备方法和应用
CN204228723U (zh) * 2014-11-13 2015-03-25 江苏达骏生物科技有限公司 定量检测n末端脑钠肽前体的试剂条和试剂卡
CN104714025B (zh) * 2014-11-28 2017-05-17 威海纽普生物技术有限公司 NT‑proBNP检测试剂盒及检测方法
CN105424922B (zh) * 2015-12-09 2018-01-19 北京乐普医疗科技有限责任公司 基于磁珠包被抗体的微流控芯片及捕获心肌标志物的方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008056034A1 (en) * 2006-11-10 2008-05-15 Hytest Ltd. Stable standards for bnp immunoassays
CN102841207A (zh) * 2011-06-24 2012-12-26 北京乐普医疗科技有限责任公司 定量检测肌钙蛋白i的荧光免疫层析试纸条及其制备方法
CN103163297A (zh) * 2011-12-16 2013-06-19 王哲 多功能荧光免疫层析快速定量检测卡
CN103197074A (zh) * 2013-04-22 2013-07-10 北京润博福得生物科技发展有限公司 基于近红外荧光纳米微球标记物的免疫层析定量检测试剂
CN105198996A (zh) * 2015-11-10 2015-12-30 三诺生物传感股份有限公司 一种单克隆抗体制备方法及其试剂盒
CN105652008A (zh) * 2016-03-31 2016-06-08 广州市微米生物科技有限公司 人Lp-PLA2生物素-链霉亲和素荧光免疫层析检测卡及其制备方法
CN106771168A (zh) * 2016-12-30 2017-05-31 武汉纽康度生物科技股份有限公司 荧光免疫层析检测卡及其制备方法和应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113970635A (zh) * 2021-10-27 2022-01-25 上海艾瑞德生物科技有限公司 一种免疫层析试纸及其制备方法和应用
CN113970635B (zh) * 2021-10-27 2024-05-10 上海艾瑞德生物科技有限公司 一种免疫层析试纸及其制备方法和应用

Also Published As

Publication number Publication date
US11959912B2 (en) 2024-04-16
CN106771168A (zh) 2017-05-31
US20190219569A1 (en) 2019-07-18

Similar Documents

Publication Publication Date Title
WO2018120620A1 (zh) 荧光免疫层析检测卡及其制备方法和应用
CN104634980B (zh) 心肌肌钙蛋白i超敏检测试剂盒及超敏检测方法
Yorde et al. Competitive enzyme-liked immunoassay with use of soluble enzyme/antibody immune complexes for labeling. I. Measurement of human choriogonadotropin.
WO2016127318A1 (zh) 心肌肌钙蛋白i超敏检测试剂盒及其超敏检测方法
CN102998467B (zh) β人绒毛膜促性腺激素磁微粒化学发光免疫定量检测试剂盒及其制备方法
CN103033619B (zh) 一种综合检测肺癌标志物的蛋白质芯片试剂盒及方法
CN104730247A (zh) 一种适于利用双标记时间分辨荧光免疫法快速检测amh和inhb的试剂盒及其使用方法
JPH01503405A (ja) 二種の標識マーカーを使用するアナライト濃度の測定
CN102901810B (zh) 包被有前列腺特异性抗原抗体的胶乳颗粒的制备方法及psa胶乳增强免疫比浊法检测试剂盒
CN102692504A (zh) D-二聚体荧光免疫定量测定试纸条及其制备方法
CN108445222A (zh) 一种定量检测心型脂肪酸结合蛋白的试剂盒及制备方法
CN101819206A (zh) 产前筛查afp测定试剂盒(时间分辨荧光免疫法)及其制备方法
CN106959372A (zh) 血清淀粉样蛋白a和c‑反应蛋白二合一测定试剂盒及制法
CN105891463A (zh) 一种基于纳米磁微粒时间分辨荧光的β-HCG定量检测试剂盒
CN107796803A (zh) 一种游离β人绒毛膜促性腺激素的磁微粒化学发光检测试剂盒及制备方法
KR920000057B1 (ko) 특이적으로 결합가능한 물질의 측정 방법 및 시약
CN106443018A (zh) 一种肌红蛋白单克隆抗体酶标记复合及其制备方法和检测试剂盒
JP7267211B2 (ja) 高濃度の分析物等、分析物を測定するための用量反応曲線の減少信号部分を使用したサンドイッチ型アッセイ
CN106771239A (zh) 血清淀粉样蛋白a/降钙素原/c‑反应蛋白三合一测定试剂盒及制法
CN107677808A (zh) 一种甲状腺球蛋白的定量检测试剂盒及其制备方法和检测方法
CN106645756A (zh) 一种检测nmp22的试剂盒及其制备方法
JP7503505B2 (ja) 自己抗体の直接イムノアッセイ測定法
CN114487442A (zh) 一种鼠单抗包被的磁珠、制备方法及测定高敏心肌肌钙蛋白ⅰ的试剂盒
CN202631543U (zh) D-二聚体荧光免疫定量测定试纸条
CN106872716A (zh) 血清淀粉样蛋白a和降钙素原二合一测定试剂盒及制法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17885821

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17885821

Country of ref document: EP

Kind code of ref document: A1