[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2018120856A1 - 检测cTnI的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法 - Google Patents

检测cTnI的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法 Download PDF

Info

Publication number
WO2018120856A1
WO2018120856A1 PCT/CN2017/098114 CN2017098114W WO2018120856A1 WO 2018120856 A1 WO2018120856 A1 WO 2018120856A1 CN 2017098114 W CN2017098114 W CN 2017098114W WO 2018120856 A1 WO2018120856 A1 WO 2018120856A1
Authority
WO
WIPO (PCT)
Prior art keywords
ctni
test strip
time
antibody
resolved
Prior art date
Application number
PCT/CN2017/098114
Other languages
English (en)
French (fr)
Inventor
徐部灼
宋旭东
黄若磐
Original Assignee
广州瑞博奥生物科技有限公司
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 广州瑞博奥生物科技有限公司 filed Critical 广州瑞博奥生物科技有限公司
Publication of WO2018120856A1 publication Critical patent/WO2018120856A1/zh

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6408Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6486Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54386Analytical elements
    • G01N33/54387Immunochromatographic test strips
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the invention belongs to the technical field of medical testing, in particular to a time-resolved fluorescent immunochromatographic test strip, a kit and a preparation method thereof for quantitatively detecting cTnI.
  • Troponin is a regulatory protein of muscle contraction. Cardiac troponin (cTn) is composed of subunits of three different genes: cardiac troponin T (cTnT), cardiac troponin I (cTnI), and troponin C (TnC). Currently, cTnT and cTnI are used for ACS laboratory diagnosis. Since cTnI and heterogene in skeletal muscle are encoded by different genes, have different amino acid sequences, and have unique antigenicity, their specificity is significantly better than CK-MB isoenzyme. CK and CK-MB may increase when muscle tissue other than the myocardium is damaged or diseased, and cTnI does not exceed its critical value.
  • the determination of troponin mainly uses the immunological method of double antibody sandwich, and the detection methods include:
  • Double-antibody sandwich immunochemiluminescence method This method is simple in operation, strong in specificity and high in sensitivity.
  • gold standard method - the method has the characteristics of fast and easy, easy to observe, but the sensitivity is not high.
  • transmission immune turbidity method - the determination method is simple, fast, automatic, suitable for batch inspection
  • the methodology and clinical application of immunoturbidimetric turbidity need further verification.
  • troponin is generally a small fraction of troponin I and troponin T present in the fine form in free form, with further necrosis of cardiomyocytes, troponin I and troponin T on muscle fibers. It is constantly disintegrating into the peripheral cycle, which is about 4 hours.
  • cardiomyocytes are also constantly metabolizing, normal aging and apoptosis are inevitable, and the sub-health state of the body such as prolonged exercise, fever, thyroid dysfunction, impaired renal function can lead to myocardial micro-damage, transient reversible myocardial Ischemia, hypoxia such as coronary spasm, coronary heart disease and angina pectoris can cause a slight increase in troponin I and T in the blood.
  • the overly sensitive diagnostic indicators are not conducive to the diagnosis of myocardial infarction.
  • the traditional troponin assay cannot change the pathophysiological characteristics of troponin after a period of myocardial necrosis, and it is necessary to delay the release into the peripheral circulation. Therefore, the sensitivity of detection is continuously improved, and there is not much practicality. Troponin is unable to capture information about myocardial infarction at an earlier stage like an electrocardiogram.
  • POCT time-resolved immunochromatography
  • the currently used immunofluorescence chromatography device uses a reflection method to detect the fluorescent signal on the porous membrane, and the fluorescence detector captures a specific antibody modified by the fluorescent dye on the surface of the porous membrane, and the fluorescent signal inside the porous membrane is not detected, resulting in detection. The sensitivity is reduced.
  • the present invention provides a time-resolved immunochromatographic test strip, a kit and a preparation method thereof for quantitatively detecting cTnI, and the immunochromatographic test strip and kit can not only It provides high sensitivity and specificity, is easy to operate, meets the needs of clinical rapid testing, and reduces costs to meet the needs of the domestic market.
  • a time-resolved immunochromatographic test strip for detecting cTnI comprising a substrate, a sample pad, a bonding pad, a coating film and an absorbent paper sequentially disposed on the substrate, wherein the bonding pad is coated with fluorescent microspheres a labeled cTnI monoclonal detection antibody comprising a detection zone and a control zone arranged in parallel and spaced apart from each other, the detection zone being coated with a cTnI monoclonal capture antibody recognizing a single antigenic epitope, the control zone package A goat anti-mouse IgG antibody; the coating film is a chemically cross-linked nitrocellulose membrane having a transmittance of 10% or less at a wavelength of less than 450 nm and 95% or more at a wavelength of 500 nm or more Light transmittance of the material.
  • the polymer is a mixture of polystyrene acrylonitrile and polycarbonate in one or a different ratio. This material allows most of the visible light to pass through, and the photodetector can It captures the fluorescent signal on the surface and inside of the multilayer porous membrane, making the detection results more accurate.
  • the CTNI monoclonal capture antibody and the goat anti-mouse IgG antibody that recognize a single epitope are at a concentration of 1-1.5 mg/ml and 0.3-0.5 mg/ml, respectively, on the coating film.
  • the amount of coating is 1-1.5 ul / cm.
  • the bond pad is a nitrocellulose membrane that is capable of carrying a sufficient amount of fluorescent microspheres and that rapidly releases the microspheres upon encountering the sample.
  • the fluorescent microspheres are selected from Eu 3+ lanthanide fluorescent microspheres for labeling antibodies, and the microspheres have active groups on the surface thereof, which can be linked to biological substances such as proteins and sugars. Contains fluorescein.
  • the fluorescent microspheres have a diameter of from 290 nm to 350 nm.
  • the detection zone is adjacent to the bond pad
  • the control zone is adjacent to the absorbent paper
  • the detection zone and the control zone are spaced from 0.3 to 0.5 cm apart.
  • the invention also provides the preparation method of the above-mentioned time-resolved immunochromatographic test strip for detecting cTnI, comprising the following steps:
  • the method for preparing the fluorescently labeled cTnI monoclonal detection antibody in step (2) comprises the steps of:
  • the cTnI monoclonal detection antibody was dialyzed overnight at 4 ° C using a 0.02-0.05 mol/L phosphate buffer solution of pH 7.2-7.6, and the dialyzed cTnI monoclonal detection antibody was adjusted to a concentration of 2-4mg/ml;
  • the invention also provides a time-resolved fluorescent immunochromatography kit for detecting cTnI, the kit comprising a plastic cartridge, the above-mentioned test strip, and a buffer bag disposed in the plastic cartridge.
  • the buffer is a PBS buffer containing 0.5% BSA, 0.05% Tween 20, 0.1-1% reducing agent; the buffer is 0.01-0.1 based on the ordinary phosphate solution. % of a reducing agent used to reduce free peroxidase in the specimen.
  • the reducing agent is reduced glutathione or ascorbic acid.
  • the time-resolved fluorescent immunochromatography kit for detecting cTnI of the present invention is used, and after the blood sample to be tested is added to the sample-adding hole, the blood sample of the sample buffer is mixed and immersed on the sample pad through the needle-sucking buffer bag, and the sample pad is placed on the sample pad. After the sample reaches saturation, the sample is delivered to the bond pad by capillary action.
  • cTnI When cTnI is contained in a blood sample, cTnI forms an antigen-antibody complex with the antibody on the fluorescent microsphere. As the chromatography progresses, the complex moves forward to reach the cTnI monoclonal capture antibody that coats a single epitope.
  • an antibody-antigen-antibody sandwich complex is formed and aggregated at the detection zone T.
  • Rare earth ion microspheres (Eu 3 + lanthanides) that do not bind to the cTnI monoclonal antibody continue to advance, and when the control region C is reached, the goat anti-mouse IgG antibody and the murine monoclonal antibody on the rare earth ion microsphere (ie, cTnI single)
  • the clone detection antibody binds, and aggregation of rare earth ion microspheres occurs at the C line. The entire reaction was completed in 10 minutes and the card was read on the machine. The intensity of the fluorescence generated under the excitation light source is proportional to the content of the conjugate on the test strip.
  • the light source When the light source is irradiated onto the detection zone and the control zone of the test strip, the attached fluorescent substance is excited, and the emitted light is collected and converted into an electrical signal.
  • the strength of the electrical signal is related to the number of fluorescent molecules, and the detector calculates the content of the analyte in the sample.
  • the present invention has the following beneficial effects:
  • test strip of the invention adopts a special light-transmitting material, can not only achieve the quantitative analysis of the chemiluminescence method, but also achieve the rapid detection of the gold standard method, and ensures the accurate and reliable test results;
  • test strip of the invention introduces time-resolved immunochromatography into the quantitative detection of cTnI, and combines the time-resolved fluorescence detector to realize the single-quantity quantitative detection of cTnI, and has high sensitivity, intra- and inter-batch difference. Small, providing great convenience for clinical use;
  • test strip of the invention is simple and suitable for large-scale production, and has positive significance for the quantitative detection of cTnI.
  • a time-resolved immunochromatographic test strip for detecting cTnI of the embodiment comprising a substrate, a sample pad, a bonding pad, a coating film and an absorbent paper which are sequentially disposed on the substrate, and the bonding pad is coated a fluorescent microsphere-labeled cTnI monoclonal detection antibody (Raybiotech.), the coating membrane comprising a detection zone and a control zone disposed in parallel and spaced 0.5 cm apart from each other, the detection zone being adjacent to the binding pad, the control The region is adjacent to the blotting paper, and the detection zone is coated with a cTnI monoclonal capture antibody (produced by a conventionally known technique) that recognizes a single epitope, and the control region is coated with a goat anti-mouse IgG antibody. .
  • the coating film is a nitrocellulose membrane of a chemically crosslinked polycarbonate and a polystyrene acrylonitrile (polymer) having a wavelength of less than 450 nm at a wavelength of less than 450 nm.
  • the light transmittance below 100% has a light transmittance of 95% or more at a wavelength of 500 nm or more. This material allows most of the visible light to pass through, and the photodetector captures the fluorescent signals on the surface and inside of the multilayer porous membrane, making the detection results more accurate.
  • the bonding pad is a nitrocellulose membrane capable of carrying a sufficient amount of fluorescent microspheres and rapidly releasing the microspheres after encountering the sample.
  • the fluorescent microspheres are selected from Eu 3+ lanthanide fluorescent microspheres for labeling antibodies, and the microspheres have reactive groups on the surface thereof, which can be linked to biological substances such as proteins and sugars. Contains fluorescein.
  • the fluorescent microspheres have a diameter of 290 nm.
  • the time-resolved fluorescent immunochromatography kit for detecting CTNI of the present embodiment comprising: the test strip, the plastic card case and the buffer bag according to the embodiment 1; the test strip is mounted on the plastic card case
  • the buffer bag is located at a corner of the plastic card, adjacent to the sample pad of the test strip, and the surface of the buffer bag is provided with a round hole for needling.
  • the reagent strip was soaked in PBS buffer containing 0.5% BSA, 0.05% Tween 20, 0.1-1% reducing agent.
  • the buffer is a 0.01-0.1% reducing agent added to the ordinary phosphate solution for reducing the free peroxidase in the sample.
  • the reducing agent is reduced glutathione or ascorbic acid.
  • the time-resolved fluorescent immunochromatography kit for detecting cTnI of the present invention is used, and after the blood sample to be tested is added to the sample-adding hole, the sample buffer and the blood sample are mixed and immersed into the sample pad by the needle-punching buffer bag. After the sample on the pad is saturated, the sample is delivered to the bond pad by capillary action.
  • cTnI When cTnI is contained in a blood sample, cTnI forms an antigen-antibody complex with the antibody on the fluorescent microsphere. As the chromatography progresses, the complex moves forward to reach the cTnI monoclonal capture antibody that coats a single epitope.
  • an antibody-antigen-antibody sandwich complex is formed and aggregated at the detection zone T.
  • Rare earth ion microspheres (Eu 3 + lanthanides) that do not bind to the cTnI monoclonal antibody continue to advance, and when the control region C is reached, the goat anti-mouse IgG antibody and the murine monoclonal antibody on the rare earth ion microsphere (ie, cTnI single)
  • the clone detection antibody binds, and aggregation of rare earth ion microspheres occurs at the C line. The entire reaction was completed in 10 minutes and the card was read on the machine. The intensity of the fluorescence generated under the excitation light source is proportional to the content of the conjugate on the test strip.
  • the light source When the light source is irradiated onto the detection zone and the control zone of the test strip, the attached fluorescent substance is excited, and the emitted light is collected and converted into an electrical signal.
  • the strength of the electrical signal is related to the number of fluorescent molecules, and the detector calculates the content of the analyte in the sample.
  • Test Example 1 The cTnI was detected using the time-resolved immunochromatographic kit of Example 2.
  • the standard curve R 2 0.9919, which is linear, and the concentration of cTnI contained in the sample can be quantitatively analyzed by the standard curve.
  • the sample to be tested was added to the sample loading zone of the fluorescent immunochromatographic test strip of cTnI, and the membrane was subjected to a reaction for 10 minutes.
  • Turn on the fluorescence detection device read the standard curve in the IC card, insert the test strip into the card slot of the fluorescence detection device, run the instrument, and automatically calculate the cTnI concentration in the sample to be tested by the corresponding analysis software, according to the calibration
  • the information on the card takes the actual detected value into a preset standard curve to calculate the quantitative result.
  • the kit was tested for performance, including minimum detection limits, precision, sensitivity, specificity, and more.
  • Linear range Take the same batch of time-resolved immunochromatographic kits for six concentrations of cardiac troponin reference products (0ng/mL, 0.5mg/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50 ng / mL, 5 parallel samples per concentration), the detection range is 0 ng / mL ⁇ 50 ng / mL, calculate the correlation coefficient r, where r value should be ⁇ 0.99.
  • Intra-assay precision 10 times of time-resolved immunochromatographic kits of the same batch number were randomly selected, and the same concentration of cardiac troponin reference substance was detected, and the coefficient of variation CV (%) value was ⁇ 15%.
  • Inter-assay precision Randomly extract three consecutive batches of time-resolved immunochromatographic kits, and take 3 copies of each batch to detect the same concentration of cardiac troponin reference substance, and the coefficient of variation CV (%) value ⁇ 15%.
  • Minimum detection limit Take 10 copies of the same batch of time-resolved immunochromatography kit, test the matrix of the reference material, and calculate the average value of the sample. And standard deviation SD, where
  • Analytical specificity Select the same concentration of cardiac troponin reference substance to add cholesterol, triglyceride and bilirubin respectively, so that the final concentration of interfering substances is cholesterol 60mg/ml, triglyceride 40mg/ml, bilirubin 2mg/ Ml, each interference sample is repeatedly tested 3 times, and the mean and relative deviation of the sample detection results are calculated, wherein the relative deviation (B%) is within ⁇ 15%.

Landscapes

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

Abstract

一种检测cTnI的时间分辨免疫层析试纸条、试剂盒及其制备方法,试纸条包括底衬、以及依次设在底衬上的样品垫、结合垫、包被膜和吸水纸,结合垫上包被有荧光微球标记的cTnI单克隆检测抗体,包被膜包括平行设置、且相互间隔的检测区和对照区,检测区包被有识别单一抗原表位的cTnI单克隆捕获抗体,对照区包被有羊抗鼠IgG抗体;包被膜为结合聚合物的硝酸纤维膜,聚合物为在小于450nm波长具有10%以下透光率,在500nm波长以上具有95%以上的透光率的材料。试纸条可以快速定量检测,试验结果准确可靠,灵敏度高,制备方法简单,适合大规模生产,对cTnI定量检测有积极意义。

Description

检测cTnI的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法 技术领域
本发明属于医学检验技术领域,具体地说,本发明涉及一种定量检测cTnI的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法。
背景技术
肌钙蛋白是肌肉收缩的调节蛋白。心肌肌钙蛋白(cTn)是由三种不同基因的亚基组成:心肌肌钙蛋白T(cTnT)、心肌肌钙蛋白I(cTnI)和肌钙蛋白C(TnC)。目前,用于ACS实验室诊断的是cTnT和cTnI。由于cTnI与骨骼肌中的异质体分别由不同基因编码,具不同的氨基酸顺序,有独特的抗原性,故它的特异性要明显优于CK-MB同工酶。心肌以外的肌肉组织出现损伤或疾病时,CK和CK-MB可能会升高,而cTnI则不会超过其临界值。由于它们在正常血清中含量极微,在AMI时明显增高,且增高倍数一般都超过总CK和CK-MB的变化。cTnI由于分子量小,发病后游离的cTn从心肌细胞浆内迅速释放人血,血中浓度迅速升高,其时间和CK-MB相当或稍早。虽然肌钙蛋白半寿期很短(游离cTnI的半寿期据报道为2h~5d不等),但其从肌原纤维上降解的过程持续时间很长,可在血中保持较长时间的升高,故它兼有CK-MB升高较早和LD1诊断时间窗长的优点。故目前cTn已有逐渐取代酶学指标的趋势。
肌钙蛋白的测定主要采用双抗体夹心的免疫学方法,检测方法则包括:
1、双抗夹心免疫化学发光法——该法操作简便,特异性强,敏感性高。
2、金标法——该法具有快速简便,易观察的特点,但是灵敏度不高。
3、透射免疫浊度法——该测定方法简便、快速,可自动化,适合于批量检 测,但是免疫透射比浊的方法学及临床应用尚需做进一步的验证。
早期测得的肌钙蛋白一般是以游离形式存在于细浆中的很少部分的肌钙蛋白I和肌钙蛋白T,随着心肌细胞进一步坏死,肌纤维上肌钙蛋白I和肌钙蛋白T不断崩解排入外周循环,这个时间段大概要4个小时。
从生理角度出发心肌细胞也在不断新陈代谢,正常衰老和凋亡不可避免,同时机体亚健康状态比如长时间运动、发热、甲状腺功能异常、肾功能受损都可以导致心肌微损害,短暂可逆的心肌缺血、缺氧如冠脉痉挛、冠心病心绞痛这些因素都可以引起血液中肌钙蛋白I、T的微量升高,过于敏感的诊断指标并不利于心梗诊断。
传统肌钙蛋白检测方法无法改变心肌坏死后肌钙蛋白需要延迟一段时间才大量排入外周循环的病理生理学特征,所以不断提高检测敏感度,没有多少实用性。肌钙蛋白无法像心电图能够在更早期捕捉到心肌梗死的信息。
时间分辨免疫层析POCT最主要特点是强调出结果快速,大大缩短了实验结果周转时间。对于急诊治疗和抢救的病人,这些病人往往情况危急且病因不明,而传统的临床检验室测量时间一般要15分钟以上,POCT一般在5分钟以内即可完成测试,医生根据POCT提供的信息,对病人及时作出初步诊断并拟定救治方案,必将减少住院时间,降低发病率/死亡率,产生很大的社会效益和经济效益。同时对于一些需要长期监控的慢性病,如糖尿病的病人可以方便地按照医生的要求由病人自己或家属进行血糖和尿糖的监控。
目前POCT无可靠的质量保证。POCT每一个测试单元都是独立的,因此无法保证每个测试单元质量都是一样。其中光学法检测的仪器会受到标本中溶血和乳糜的干扰,化学发光法会受到外源性氧化还原物质的影响。基于免疫层析, 色谱和干化学技术的各种试纸条和仪器都会因温度,湿度和pH值的不同影响基质中微蛋白的活性,进而影响结果。部分POCT仪器方法学的缺陷,灵敏性和重复性欠佳,线性范围窄,只是急诊或者急求时用于参考,必要时还需送到检验科进行复查。
目前采用的免疫荧光层析装置采用反射法检测多孔膜上的荧光信号,荧光检测仪捕捉到的是多孔膜表面荧光染料修饰的特异性抗体,而检测不到多孔膜内部的荧光信号,导致检测灵敏度下降。
发明内容
基于此,为了克服上述现有技术的缺陷,本发明提供了一种定量检测cTnI的时间分辨免疫层析试纸条、试剂盒及其制备方法,该免疫层析试纸条和试剂盒不仅能够提供较高的灵敏度和特异性,操作简单,满足临床快速检验的需要,而且降低了成本,满足国内市场的需求。
为了实现上述发明目的,本发明采取了以下技术方案:
一种检测cTnI的时间分辨免疫层析试纸条,包括底衬、以及依次设在所述底衬上的样品垫、结合垫、包被膜和吸水纸,所述结合垫上包被有荧光微球标记的cTnI单克隆检测抗体,所述包被膜包括平行设置、且相互间隔的检测区和对照区,所述检测区包被有识别单一抗原表位的cTnI单克隆捕获抗体,所述对照区包被有羊抗鼠IgG抗体;所述包被膜为化学交联结合有聚合物的硝酸纤维膜,所述聚合物为在小于450nm波长具有10%以下透光率,在500nm波长以上具有95%以上的透光率的材料。
在其中一些实施例中,所述聚合物为聚苯乙烯丙烯腈和聚碳酸酯的一种或者不同比例的混合物。这种材料可以允许绝大多数的可见光透过,光检测器能 够捕捉多层多孔膜表面和内部的荧光信号,使检测结果更准确。
在其中一些实施例中,所述识别单一抗原表位的CTNI单克隆捕获抗体和羊抗鼠IgG抗体的浓度分别为1-1.5mg/ml和0.3-0.5mg/ml,在所述包被膜上的包被量均为1-1.5ul/cm。
在其中一些实施例中,所述结合垫为硝酸纤维膜,其能够载有足量的荧光微球,且遇样品后又能迅速释放微球。
在其中一些实施例中,所述荧光微球选用本领域公知的用于标记抗体的Eu3+镧系元素荧光微球,微球表面带有活性基团,可以连接蛋白、糖类等生物物质,内含荧光素。
在其中一些实施例中,所述荧光微球的直径为290nm-350nm。
在其中一些实施例中,所述检测区靠近所述结合垫,所述对照区靠近所述吸水纸,所述检测区和所述对照区的间隔为0.3-0.5cm。
本发明还提供了上述检测cTnI的时间分辨免疫层析试纸条的制备方法,包括以下步骤:
(1)、在包被膜上分别固定识别单一抗原表位的cTnI单克隆捕获抗体和羊抗鼠IgG抗体,形成检测区和对照区;
(2)、制备荧光微球标记的cTnI单克隆检测抗体,并喷涂在结合垫上;
(3)、在底衬上搭接粘贴样品垫、结合垫、包被膜和吸水纸,即得。
在其中一些实施例中,步骤(2)中所述荧光标记的cTnI单克隆检测抗体的制备方法包括如下步骤:
(1)、在4℃下,采用0.02-0.05mol/L的pH7.2-7.6的磷酸盐缓冲液将cTnI单克隆检测抗体透析过夜,再将透析后的cTnI单克隆检测抗体调整至浓度为 2-4mg/ml;
(2)、使用0.01-0.05mol/L的pH6.0的MES活化缓冲液洗涤微球,加入碳二亚胺和N-羟基琥珀酰亚胺,使微球终浓度为0.2mol/L,室温反应15-30分钟,充分洗涤微球,用0.02-0.05mol/LpH7.4-7.6的硼酸缓冲液复溶;
(3)、按cTnI单克隆检测抗体与微球的质量比为1:5-6的比例,在复溶后的微球中加入cTnI单克隆检测抗体,室温反应2小时,加入含有5%BSA的0.02mol/L的pH7.4-7.6的硼酸缓冲液,室温反应30分钟,洗涤,再用含有0.5%BSA,0.05%Tween-20的0.02mol/L的pH7.4-7.6的硼酸缓冲液复溶至原体积,使用定量喷膜仪以4ul/cm的量喷涂于玻璃纤维膜上,避光,烘干即得。
本发明还提供了一种检测cTnI的时间分辨荧光免疫层析试剂盒,所述试剂盒包括塑料卡壳、上述的试纸条、以及设置于塑料卡壳内的缓冲液袋。
在其中一些实施例中,所述缓冲液为含有0.5%的BSA、0.05%的吐温20、0.1-1%还原剂的PBS缓冲液;缓冲液是在普通磷酸盐液基础上加入0.01-0.1%的还原剂,用于还原标本中游离的过氧化物酶。
在其中一些实施例中,所述还原剂为还原型谷胱甘肽或抗坏血酸。
本发明的检测cTnI的时间分辨荧光免疫层析试剂盒在使用时,将待测血液样品加到加样孔后,通过针刺缓冲袋,样本缓冲液血液样本混合浸入到样品垫上,当样品垫上的样本达到饱和状态后,通过毛细管作用将样本输送到结合垫。当血液样本中含有cTnI时,cTnI与荧光微球上的抗体形成抗原-抗体复合物,随着层析作用,复合物向前移动,到达包被识别单一抗原表位的cTnI单克隆捕获抗体的检测区T处,形成抗体-抗原-抗体夹心复合物,聚集在检测区T处。未结合cTnI单克隆抗体的稀土离子微球(Eu3+镧系元素)继续前行,到达对照区C时, 羊抗鼠IgG抗体与稀土离子微球上的鼠源性单抗(即cTnI单克隆检测抗体)结合,在C线处出现稀土离子微球的聚集。整个反应在10分钟内完成,并进行上机读卡。在激发光源下产生的荧光强度与试纸条上的结合物含量成正比,当光源照射到试纸条的检测区和对照区时,激发附着的荧光物质,发射光收集并转化为电信号,电信号的强弱和荧光分子数量相关,检测仪计算样品中待测物的含量。
与现有技术相比,本发明具有以下有益效果:
(1)本发明的试纸条,采用特殊的透光材料,既可以达到化学发光法的定量分析,又能达到金标法的快速检测,且保证了试验结果的准确可靠;
(2)本发明的试纸条将时间分辨免疫层析技术引入cTnI的定量检测中,结合时间分辨荧光检测仪,实现了cTnI的单人份定量检测,且灵敏度高,批内、批间差小,为临床使用提供了极大便利;
(3)本发明的试纸条的制备方法简单,适合大规模生产,对于cTnI的定量检测有着积极的意义。
具体实施方式
以下通过具体实施例来详细说明本发明。
以下实施例中所使用的原料如无特殊说明,均来源于市售。
实施例1 检测cTnI的时间分辨免疫层析试纸条
本实施例的一种检测cTnI的时间分辨免疫层析试纸条,包括底衬、以及依次设在所述底衬上的样品垫、结合垫、包被膜和吸水纸,所述结合垫上包被有荧光微球标记的cTnI单克隆检测抗体(Raybiotech.),所述包被膜包括平行设置、且相互间隔0.5cm的检测区和对照区,所述检测区靠近所述结合垫,所述对照 区靠近所述吸水纸,所述检测区包被有识别单一抗原表位的cTnI单克隆捕获抗体(自产,采用现有公知技术制备获得),所述对照区包被有羊抗鼠IgG抗体。
本实施例中,所述包被膜为化学交联聚碳酸酯与聚苯乙烯丙烯腈(聚合物)的硝酸纤维膜,所述聚碳酸酯与聚苯乙烯丙烯腈聚合物在小于450nm波长具有10%以下透光率,在500nm波长以上具有95%以上的透光率。这种材料可以允许绝大多数的可见光透过,光检测器能够捕捉多层多孔膜表面和内部的荧光信号,使检测结果更准确。
本实施例中,所述结合垫为硝酸纤维膜,其能够载有足量的荧光微球,且遇样品后又能迅速释放微球。
本实施例中,所述荧光微球选用本领域公知的用于标记抗体的Eu3+镧系元素荧光微球,微球表面带有活性基团,可以连接蛋白、糖类等生物物质,内含荧光素。所述荧光微球的直径为290nm。
本实施例的检测cTnI的时间分辨荧光免疫层析试纸条的制备方法,包括以下步骤:
(1)、在包被膜上分别固定识别单一抗原表位的cTnI单克隆捕获抗体和羊抗鼠IgG抗体,形成检测区和对照区;具体方法为:使用含有5%蔗糖的0.02mol/L的pH为7.2-7.6的磷酸盐缓冲液,分别将识别单一抗原表位的cTnI单克隆捕获抗体和羊抗鼠IgG抗体稀释到1.5mg/ml的浓度,使用定量喷膜仪以1ul/cm的量将二者以0.5cm的间隔喷于硝酸纤维素膜上,50℃烘干5h,加入干燥剂封存备用;
(2)、制备荧光微球标记的cTNI单克隆检测抗体,并喷涂在结合垫上;具体方法为:(a)、在4℃下,采用0.02-0.05mol/L的pH7.2-7.6的磷酸盐缓冲液将 CTNI单克隆检测抗体透析过夜,再将透析后的cTNI单克隆检测抗体调整至浓度为2-4mg/ml;(b)、使用0.01-0.05mol/L的pH6.0的MES活化缓冲液洗涤微球,加入碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS),微球终浓度为0.2mol/L,室温反应15分钟,充分洗涤微球,用0.02mol/LpH7.4-7.6的硼酸缓冲液复溶;(c)、按cTNI单克隆检测抗体与微球的质量比为1:5的比例,在复溶后的微球中加入cTNI单克隆检测抗体,室温反应2小时,加入含有5%BSA的0.02mol/L的pH7.4-7.6的硼酸缓冲液,室温反应30分钟,洗涤,再用含有0.5%BSA,0.05%Tween-20的0.02mol/L的pH7.4-7.6的硼酸缓冲液复溶至原体积,使用定量喷膜仪以4ul/cm的量喷涂于玻璃纤维膜上,避光,在50℃烘干6小时,加入干燥剂封存备用;
(3)、在底衬上搭接粘贴样品垫、结合垫、包被膜和吸水纸,切割成宽度为0.42cm大小,即得。
实施例2 检测cTnI的时间分辨免疫层析试剂盒
本实施例的检测CTNI的时间分辨荧光免疫层析试剂盒,所述试剂盒包括:实施例1所述的试纸条、塑料卡壳、缓冲液袋;所述试纸条装于所述塑料卡壳内,所述缓冲液袋位于所述塑料卡壳的边角处,靠近所述试纸条的样品垫,所述缓冲液袋的表面留置圆孔用于针刺。
在本实施例中,所述试剂条采用含有0.5%的BSA、0.05%的吐温20、0.1-1%还原剂的PBS缓冲液浸溶。缓冲液是在普通磷酸盐液基础上加入0.01-0.1%的还原剂,用于还原标本中游离的过氧化物酶。所述还原剂为还原型谷胱甘肽或抗坏血酸。
本发明的检测cTnI的时间分辨荧光免疫层析试剂盒在使用时,将待测血液 样品加到加样孔后,通过针刺缓冲袋,样本缓冲液与血液样本混合浸入到样品垫上,当样品垫上的样本达到饱和状态后,通过毛细管作用将样本输送到结合垫。当血液样本中含有cTnI时,cTnI与荧光微球上的抗体形成抗原-抗体复合物,随着层析作用,复合物向前移动,到达包被识别单一抗原表位的cTnI单克隆捕获抗体的检测区T处,形成抗体-抗原-抗体夹心复合物,聚集在检测区T处。未结合cTnI单克隆抗体的稀土离子微球(Eu3+镧系元素)继续前行,到达对照区C时,羊抗鼠IgG抗体与稀土离子微球上的鼠源性单抗(即cTnI单克隆检测抗体)结合,在C线处出现稀土离子微球的聚集。整个反应在10分钟内完成,并进行上机读卡。在激发光源下产生的荧光强度与试纸条上的结合物含量成正比,当光源照射到试纸条的检测区和对照区时,激发附着的荧光物质,发射光收集并转化为电信号,电信号的强弱和荧光分子数量相关,检测仪计算样品中待测物的含量。
试验例1 利用实施例2的时间分辨免疫层析试剂盒检测cTnI
A、拟合标准曲线
在实施例1的时间分辨免疫层析试纸条的加样区加入不同浓度的cTnI标准品(取8个不同的浓度,分别为0ng/mL、0.1ng/mL、0.5ng/mL、1ng/mL、5ng/mL、10ng/mL、30ng/mL、50ng/mL,每个浓度做5个平行样),膜层析反应10分钟后,仪器读取质控线C、检测线T信号,以检测的样品的检测线荧光值与质控线的荧光值的比值为横坐标,cTnI标准品浓度为纵坐标,建立方程并拟合成标准曲线y=0.9333x+0.0831。
标准曲线R2=0.9919,线性较好,可以通过该标准曲线对样品中所含cTnI浓度进行定量分析。
B、样品检测
在cTnI的荧光免疫层析试纸条的加样区加入待测样品,膜层析反应10分钟。开启荧光检测设备,读取IC卡里的标准曲线,并将检测条插入荧光检测设备的插卡口,运行仪器,仪器通过相应的分析软件自动计算出待测样本中的cTnI浓度,根据定标卡上的信息将实际检测值带入预设的标准曲线中算出定量的结果。
试验例2 实施例2的试剂盒的性能测定
对试剂盒进行了性能方面的测定,包括最低检测限、精密度、灵敏度、特异性等。
1、线性范围:取同一批号的时间分辨免疫层析试剂盒分别对六个浓度的心肌肌钙蛋白参考品(0ng/mL、0.5mg/mL、1ng/mL、5ng/mL、10ng/mL、50ng/mL,每个浓度做5个平行样)进行检测,其检测范围是0ng/mL~50ng/mL,计算相关系数r,其中r值应≥0.99。
2、批内精密度:随机抽取同一批号的时间分辨免疫层析试剂盒10份,分别对同一浓度的心肌肌钙蛋白参考品进行检测,其变异系数CV(%)值≤15%。
3、批间精密度:随机抽取连续三个批号的时间分辨免疫层析试剂盒,每个批号取3份分别对同一浓度的心肌肌钙蛋白参考品进行检测,其变异系数CV(%)值≤15%。
4、准确度:用同一个批号的时间分辨免疫层析试剂盒分别测定三个水平浓度的心肌肌钙蛋白I参考品,计算样本测定结果均值和相对偏差,其中相对偏差(B%)在±15%内。
5、最低检测限:取同一批号的时间分辨免疫层析试剂盒10份,对配制参考品基质进行检测,计算样本测定结果均值和标准差SD,其中
Figure PCTCN2017098114-appb-000002
Figure PCTCN2017098114-appb-000003
6、分析特异性:选择同一浓度的心肌肌钙蛋白参考品分别加入胆固醇、甘油三酯、胆红素,使干扰物最终浓度胆固醇60mg/ml、甘油三酯40mg/ml、胆红素2mg/ml,各干扰样本重复检测3次,计算样本检测结果的均值和相对偏差,其中相对偏差(B%)在±15%内。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种检测cTnI的时间分辨免疫层析试纸条,其特征在于,包括底衬、以及依次设在所述底衬上的样品垫、结合垫、包被膜和吸水纸,所述结合垫上包被有荧光微球标记的cTnI单克隆检测抗体,所述包被膜包括平行设置、且相互间隔的检测区和对照区,所述检测区包被有识别单一抗原表位的cTnI单克隆捕获抗体,所述对照区包被有羊抗鼠IgG抗体;所述包被膜为结合聚合物的硝酸纤维膜,所述聚合物为在小于450nm波长具有10%以下透光率,在500nm波长以上具有95%以上的透光率的材料。
  2. 根据权利要求1所述的检测cTnI的时间分辨免疫层析试纸条,其特征在于,所述聚合物为聚苯乙烯丙烯腈和聚碳酸酯的一种或两种。
  3. 根据权利要求1或2所述的检测cTnI的时间分辨免疫层析试纸条,其特征在于,所述识别单一抗原表位的cTnI单克隆捕获抗体和羊抗鼠IgG抗体的浓度分别为1-1.5mg/ml和0.3-0.5mg/ml,在所述包被膜上的包被量均为1-1.5ul/cm。
  4. 根据权利要求1或2所述的检测cTnI的时间分辨免疫层析试纸条,其特征在于,所述结合垫为硝酸纤维膜,所述荧光微球为Eu3+镧系元素荧光微球。
  5. 根据权利要求4所述的检测cTnI的时间分辨免疫层析试纸条,其特征在于,所述荧光微球的直径为290nm-350nm。
  6. 根据权利要求1或2所述的检测cTnI的时间分辨免疫层析试纸条,其特征在于,所述检测区靠近所述结合垫,所述对照区靠近所述吸水纸,所述检测区和所述对照区的间隔为0.3-0.5cm。
  7. 权利要求1-6任一项所述的检测cTnI的时间分辨免疫层析试纸条的制备方法,其特征在于,包括以下步骤:
    (1)、在包被膜上分别固定识别单一抗原表位的cTnI单克隆捕获抗体和羊抗鼠IgG抗体,形成检测区和对照区;
    (2)、制备荧光微球标记的cTnI单克隆检测抗体,并喷涂在结合垫上;
    (3)、在底衬上搭接粘贴样品垫、结合垫、包被膜和吸水纸,即得。
  8. 根据权利要求7所述的检测cTnI的时间分辨免疫层析试纸条的制备方法,其特征在于,步骤(2)中所述荧光标记的cTnI单克隆检测抗体的制备方法包括如下步骤:
    (1)、在4℃下,采用0.02-0.05mol/L的pH7.2-7.6的磷酸盐缓冲液将cTnI单克隆检测抗体透析过夜,再将透析后的cTnI单克隆检测抗体调整至浓度为2-4mg/ml;
    (2)、使用0.01-0.05mol/L的pH6.0的MES活化缓冲液洗涤微球,加入碳二亚胺和N-羟基琥珀酰亚胺,使微球终浓度为0.2mol/L,室温反应15-30分钟,充分洗涤微球,用0.02-0.05mol/LpH7.4-7.6的硼酸缓冲液复溶;
    (3)、按cTnI单克隆检测抗体与微球的质量比为1:5-6的比例,在复溶后的微球中加入cTnI单克隆检测抗体,室温反应2小时,加入含有5%BSA的0.02mol/L的pH7.4-7.6的硼酸缓冲液,室温反应30分钟,洗涤,再用含有0.5%BSA,0.05%Tween-20的0.02mol/L的pH7.4-7.6的硼酸缓冲液复溶至原体积,使用定量喷膜仪以4ul/cm的量喷涂于玻璃纤维膜上,避光,烘干即得。
  9. 一种检测cTnI的时间分辨荧光免疫层析试剂盒,其特征在于,所述试剂盒包括塑料卡壳、权利要求1~6任一项所述的试纸条、以及设置于塑料卡壳内的缓冲液袋。
  10. 根据权利要求9所述的检测cTnI的时间分辨荧光免疫层析试剂盒,其 特征在于,所述缓冲液为含有0.5%的BSA、0.05%的吐温20、0.1-1%还原剂的PBS缓冲液;所述还原剂为还原型谷胱甘肽或抗坏血酸。
PCT/CN2017/098114 2016-12-28 2017-08-18 检测cTnI的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法 WO2018120856A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201611237917.9A CN108254563B (zh) 2016-12-28 2016-12-28 检测cTnI的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法
CN201611237917.9 2016-12-28

Publications (1)

Publication Number Publication Date
WO2018120856A1 true WO2018120856A1 (zh) 2018-07-05

Family

ID=62710176

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/098114 WO2018120856A1 (zh) 2016-12-28 2017-08-18 检测cTnI的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法

Country Status (2)

Country Link
CN (1) CN108254563B (zh)
WO (1) WO2018120856A1 (zh)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109884006A (zh) * 2019-02-15 2019-06-14 华东师范大学 时间分辨型荧光材料在检测抗坏血酸含量上的应用
CN110568188A (zh) * 2019-09-26 2019-12-13 天津华科泰生物技术有限公司 快速检测阿尔茨海默病相关神经丝蛋白AD7c-NTP的免疫层析检测卡及其制备方法
CN110702901A (zh) * 2019-10-10 2020-01-17 南京欧凯生物科技有限公司 一种检测心肌钙蛋白i的荧光免疫层析试纸
CN110736740A (zh) * 2018-07-18 2020-01-31 博阳生物科技(上海)有限公司 一种均相化学发光poct检测方法及利用该检测方法的装置
CN110954689A (zh) * 2019-11-25 2020-04-03 芜湖森爱驰生物科技有限公司 一种深部真菌感染快速诊断试剂盒及其制备方法
CN112083161A (zh) * 2020-10-14 2020-12-15 芜湖森爱驰生物科技有限公司 一种降钙素原(pct)荧光检测试剂盒及其制备工艺
CN112920272A (zh) * 2019-12-05 2021-06-08 菲鹏生物股份有限公司 抗cTnI的蛋白和检测cTnI的方法
CN114280312A (zh) * 2020-09-27 2022-04-05 河北特温特生物科技发展有限公司 一种用于免疫荧光层析检测的全血分离膜及其制备方法和应用
CN115856284A (zh) * 2022-12-15 2023-03-28 西交利物浦大学 一种具有仿生流道的层析试纸条及其应用
CN115902238A (zh) * 2022-11-11 2023-04-04 上海健康医学院 一种心肌肌钙蛋白i检测试纸条
CN116124753A (zh) * 2023-04-14 2023-05-16 北京芯迈微生物技术有限公司 基于荧光转化能力的微流控定量检测试剂盒及方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109239031A (zh) * 2018-09-10 2019-01-18 吉林大学 建立时间分辨荧光免疫层析法检测mybpc3试剂盒
CN111735941B (zh) * 2019-03-25 2022-02-18 中国科学院福建物质结构研究所 一种稀土钒酸盐纳米荧光标记材料及其制备方法和应用
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
CN117859050A (zh) * 2022-08-05 2024-04-09 柯正浩 快速筛检试纸中和抗体周期读取量测方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2358506A1 (en) * 1999-01-09 2000-07-20 Bernd Pevec Method and device for determining an analyte
CN102192983A (zh) * 2011-05-19 2011-09-21 博阳生物科技(上海)有限公司 时间分辨荧光免疫层析定量检测试纸条及其制备方法和应用
US8900881B2 (en) * 2008-12-30 2014-12-02 Jin Po Lee Quantitative analyte assay device and method
CN104569412A (zh) * 2014-05-22 2015-04-29 江苏金标世纪生物科技有限公司 一种心肌梗塞快速检测试剂盒及其制备方法
CN104730251A (zh) * 2014-11-28 2015-06-24 威海纽普生物技术有限公司 肌钙蛋白i检测试剂盒及检测方法
CN205246673U (zh) * 2015-12-15 2016-05-18 深圳市金准生物医学工程有限公司 快速定量检测cTnI和H-FABP的免疫层析试纸条及其试剂盒

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2495206C (en) * 2002-08-27 2012-10-30 Kimberly-Clark Worldwide, Inc. Membrane-based assays using time-resolved fluorescence
US20060019265A1 (en) * 2004-04-30 2006-01-26 Kimberly-Clark Worldwide, Inc. Transmission-based luminescent detection systems
AU2008209499A1 (en) * 2007-01-24 2008-07-31 Whatman Inc. Modified porous membranes, methods of membrane pore modification, and methods of use thereof
CN103308489B (zh) * 2012-01-02 2016-05-04 何爱民 利用时间分辨上转换发光技术的侧向流动免疫测定法
CN104422772B (zh) * 2013-09-10 2016-07-06 江苏省原子医学研究所 一种定量检测胃蛋白酶原i的时间分辨免疫层析试纸条及其制备方法
CN105277711B (zh) * 2014-07-21 2017-11-21 广州瑞博奥生物科技有限公司 一种用于检测he4的酶联免疫试剂盒
CN106153927A (zh) * 2016-04-12 2016-11-23 上海奥普生物医药有限公司 一种快速定量同时检测cTnI、CKMB、Myo的时间分辨荧光免疫层析试剂及制备方法
CN106248927A (zh) * 2016-07-21 2016-12-21 上海奥普生物医药有限公司 一种快速定量检测ck‑mb的时间分辨荧光免疫层析试剂及制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2358506A1 (en) * 1999-01-09 2000-07-20 Bernd Pevec Method and device for determining an analyte
US8900881B2 (en) * 2008-12-30 2014-12-02 Jin Po Lee Quantitative analyte assay device and method
CN102192983A (zh) * 2011-05-19 2011-09-21 博阳生物科技(上海)有限公司 时间分辨荧光免疫层析定量检测试纸条及其制备方法和应用
CN104569412A (zh) * 2014-05-22 2015-04-29 江苏金标世纪生物科技有限公司 一种心肌梗塞快速检测试剂盒及其制备方法
CN104730251A (zh) * 2014-11-28 2015-06-24 威海纽普生物技术有限公司 肌钙蛋白i检测试剂盒及检测方法
CN205246673U (zh) * 2015-12-15 2016-05-18 深圳市金准生物医学工程有限公司 快速定量检测cTnI和H-FABP的免疫层析试纸条及其试剂盒

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110736740A (zh) * 2018-07-18 2020-01-31 博阳生物科技(上海)有限公司 一种均相化学发光poct检测方法及利用该检测方法的装置
CN109884006B (zh) * 2019-02-15 2021-05-25 华东师范大学 时间分辨型荧光材料在检测抗坏血酸含量上的应用
CN109884006A (zh) * 2019-02-15 2019-06-14 华东师范大学 时间分辨型荧光材料在检测抗坏血酸含量上的应用
CN110568188A (zh) * 2019-09-26 2019-12-13 天津华科泰生物技术有限公司 快速检测阿尔茨海默病相关神经丝蛋白AD7c-NTP的免疫层析检测卡及其制备方法
CN110702901A (zh) * 2019-10-10 2020-01-17 南京欧凯生物科技有限公司 一种检测心肌钙蛋白i的荧光免疫层析试纸
CN110954689A (zh) * 2019-11-25 2020-04-03 芜湖森爱驰生物科技有限公司 一种深部真菌感染快速诊断试剂盒及其制备方法
CN112920272A (zh) * 2019-12-05 2021-06-08 菲鹏生物股份有限公司 抗cTnI的蛋白和检测cTnI的方法
CN114280312B (zh) * 2020-09-27 2023-09-15 河北特温特生物科技发展有限公司 一种用于免疫荧光层析检测的全血分离膜及其制备方法和应用
CN114280312A (zh) * 2020-09-27 2022-04-05 河北特温特生物科技发展有限公司 一种用于免疫荧光层析检测的全血分离膜及其制备方法和应用
CN112083161A (zh) * 2020-10-14 2020-12-15 芜湖森爱驰生物科技有限公司 一种降钙素原(pct)荧光检测试剂盒及其制备工艺
CN115902238A (zh) * 2022-11-11 2023-04-04 上海健康医学院 一种心肌肌钙蛋白i检测试纸条
CN115856284A (zh) * 2022-12-15 2023-03-28 西交利物浦大学 一种具有仿生流道的层析试纸条及其应用
CN115856284B (zh) * 2022-12-15 2023-11-28 西交利物浦大学 一种具有仿生流道的层析试纸条及其应用
CN116124753A (zh) * 2023-04-14 2023-05-16 北京芯迈微生物技术有限公司 基于荧光转化能力的微流控定量检测试剂盒及方法
CN116124753B (zh) * 2023-04-14 2023-07-25 北京芯迈微生物技术有限公司 基于荧光转化能力的微流控定量检测试剂盒及方法

Also Published As

Publication number Publication date
CN108254563A (zh) 2018-07-06
CN108254563B (zh) 2019-10-29

Similar Documents

Publication Publication Date Title
WO2018120856A1 (zh) 检测cTnI的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法
WO2018120854A1 (zh) 检测ck-mb的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法
WO2018120855A1 (zh) 检测myo的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法
Xu et al. Development of lateral flow immunoassay system based on superparamagnetic nanobeads as labels for rapid quantitative detection of cardiac troponin I
CN102323422B (zh) 半定量同时检测cTnI和Myo的免疫层析试纸条及其制备
US5989840A (en) Estimation of active infection by heliobacter pylori
KR100910982B1 (ko) 당화헤모글로빈의 정량분석을 위한 시스템 및 이를 이용한당화헤모글로빈 측정 방법
CN102662055B (zh) 一种快速定量检测肌钙蛋白i的免疫荧光试纸条组件、及其制成的检测卡组件和制备方法
US20080274565A1 (en) Method for the quantitative measurement of analytes in a liquid sample by immunochromatography
CN103954778A (zh) 心肌梗塞三联快速检测试剂盒及其制备方法
CN106248958A (zh) 一种定量检测cTnI的荧光免疫层析试剂及制备方法
CN106153927A (zh) 一种快速定量同时检测cTnI、CKMB、Myo的时间分辨荧光免疫层析试剂及制备方法
CN105723220A (zh) 能够测定广泛浓度范围的生物物质浓度的免疫层析条状传感器
CN106248927A (zh) 一种快速定量检测ck‑mb的时间分辨荧光免疫层析试剂及制备方法
CN102087293A (zh) 一种全程定量检测肌钙蛋白i的免疫层析试纸条及其制备方法
CN105891508A (zh) 一种快速定量检测h-fabp的时间分辨荧光免疫层析试剂及制备方法
WO2018068582A1 (zh) 一种免疫层析检测装置
CN107664700A (zh) 心肌肌钙蛋白i及肌酸激酶同工酶及肌红蛋白三合一检测试剂盒及其制备方法
CN109142758A (zh) 一种检测糖化血红蛋白的免疫层析试纸条、试剂盒及其制备方法
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
CN208060389U (zh) 一种用于心肌梗塞快速定量检测的多指标时间分辨荧光免疫层析试剂盒
CN107490699A (zh) 一种血液糖化血红蛋白荧光免疫检测方法
CN107505459B (zh) 定量检测人h-fabp的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法
CN101738476B (zh) 乙肝病毒前s1抗原快速诊断试剂盒及其制备方法
CN106645043A (zh) 快速定量检测小分子化合物的试剂盒和方法

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: 17886131

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 21/10/2019)

122 Ep: pct application non-entry in european phase

Ref document number: 17886131

Country of ref document: EP

Kind code of ref document: A1