WO2018120854A1 - 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 - Google Patents

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 Download PDF

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WO2018120854A1
WO2018120854A1 PCT/CN2017/098111 CN2017098111W WO2018120854A1 WO 2018120854 A1 WO2018120854 A1 WO 2018120854A1 CN 2017098111 W CN2017098111 W CN 2017098111W WO 2018120854 A1 WO2018120854 A1 WO 2018120854A1
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test strip
detecting
time
antibody
monoclonal
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French (fr)
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徐部灼
宋旭东
黄若磐
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广州瑞博奥生物科技有限公司
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    • 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
    • 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
    • G01N33/543—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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/577—Immunoassay; 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

Definitions

  • the invention belongs to the technical field of medical examination, in particular to a time-resolved fluorescent immunochromatographic test strip, a kit and a preparation method thereof for quantitatively detecting CK-MB.
  • Creatine Kinase is commonly found in the cytoplasm and mitochondria of tissues such as the heart, muscles, and brain of animals. It is an important kinase directly related to intracellular energy function, muscle contraction, and ATP regeneration. It is reversible. It catalyzes the transphosphoryl reaction between creatine and ATP. Creatine kinase isoenzyme (CK-MB) is mainly distributed in the myocardium. When myocardial injury occurs, CK-MB is released into the bloodstream and begins to rise after 3 to 8 hours, which lasts for 3 to 5 days. The combined detection of troponin, myoglobin, and creatine kinase isoenzymes can detect myocardial damage as early as possible.
  • Creatine kinase isoenzyme is of great significance in clinical diagnosis. In various pathological changes including muscle atrophy and myocardial infarction, the level of creatine kinase in human serum is rapidly increased. It is currently considered that muscle is measured in the diagnosis of myocardial infarction. Acid kinase activity is more reliable than electrocardiography. In myocardial infarction, creatine kinase increased within 6 hours of onset, peaked at 24 hours, and returned to normal within 3-4 days, with the highest specificity of creatine kinase isoenzyme CK-MB. Creatine kinase has attracted extensive attention and in-depth research because of its important physiological functions and clinical application value.
  • the determination of myocardial creatine kinase isoenzyme mainly adopts 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.
  • the gold standard method - the method has the characteristics of fast and easy, easy to observe. However, qualitative detection is not sensitive.
  • transmission immune turbidity method - the determination method is simple, rapid, and can be automated, suitable for batch detection, but the method and clinical application of immunoturbidimetric turbidity needs further verification.
  • 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 CK-MB, and the immunochromatographic test strip
  • the kit not only provides high sensitivity and specificity, but also is easy to operate, meets the needs of rapid clinical testing, and reduces costs to meet the needs of the domestic market.
  • a time-resolved immunochromatographic test strip for detecting CK-MB 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 with fluorescence a microsphere-labeled CK-MB 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 CK-MB monoclonal capture antibody that recognizes a single antigenic epitope
  • the control zone is coated with a goat anti-mouse IgG antibody;
  • the coating film is a chemically crosslinked cyanocellulose membrane bonded with a polymer having a light transmittance of 10% or less at a wavelength of less than 450 nm at 500 nm A material having a light transmittance of 95% or more above the wavelength.
  • 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 captures the fluorescent signals on the surface and inside of the multilayer porous membrane, making the detection results more accurate.
  • 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, and the control zone is adjacent to the blotting paper.
  • the interval between the detection zone and the control zone is 0.3-0.5 cm.
  • the invention also provides a time-resolved fluorescent immunochromatography kit for detecting CK-MB, the test
  • the kit includes the above test strips.
  • the kit further includes a plastic cartridge, and a buffer bag disposed within 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 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 invention also discloses a preparation method of the above-mentioned time-resolved fluorescent immunochromatographic test strip for detecting CK-MB, comprising the following steps:
  • the concentration of the CK-MB monoclonal capture antibody and the goat anti-mouse IgG antibody that recognize a single epitope is 1-1.5 mg/ml and 0.3-0.5 mg/ml, respectively, in the package.
  • the coating amount on the film was 1-1.5 ul/cm.
  • the method for preparing the fluorescently labeled CK-MB monoclonal detection antibody in the step (2) comprises the following steps:
  • the CK-MB monoclonal detection antibody was dialyzed overnight at 4 ° C with 0.02-0.05 mol/L phosphate buffer pH 7.2-7.6, and the CK-MB monoclonal detection antibody after dialysis Adjusted to a concentration of 2-4 mg/ml;
  • the blood sample to be tested is added to the sample-adding hole, and the sample buffer is mixed with the blood sample and immersed in the sample pad through the needle-punching buffer bag. After the sample on the sample pad is saturated, the sample is delivered to the bond pad by capillary action.
  • CK-MB is contained in the blood sample, CK-MB forms an antigen-antibody complex with the antibody on the fluorescent microsphere. As the chromatography progresses, the complex moves forward to reach the CK-coated with a single epitope. At the detection zone T of the MB monoclonal capture antibody, an antibody-antigen-antibody sandwich complex is formed and aggregated at the detection zone T.
  • Rare earth ion microspheres (Eu3 + lanthanides) that do not bind to CK-MB monoclonal antibodies continue to advance, and when they reach control zone C, goat anti-mouse IgG antibodies and murine monoclonal antibodies on rare earth ion microspheres (ie CK-)
  • the MB monoclonal detection antibody binds and aggregates rare earth ion microspheres 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 present invention adopts a special light-transmitting material, which can meet the requirements of the chemiluminescence method.
  • quantity analysis can achieve the rapid detection of the gold standard method and ensure the accuracy and reliability of the test results;
  • test strip of the invention introduces time-resolved immunochromatography into the quantitative detection of CK-MB, and combines the time-resolved fluorescence detector to realize the single-agent quantitative detection of CK-MB, and the sensitivity is high, within the batch The difference between batches is small, which provides great convenience for clinical use;
  • test strip of the invention is simple and suitable for large-scale production, and has positive significance for quantitative detection of CK-MB.
  • a time-resolved immunochromatographic test strip for detecting CK-MB comprising a substrate, and a sample pad, a bonding pad, a coating film and an absorbent paper sequentially disposed on the substrate, the bonding pad a CK-MB monoclonal detection antibody (Raybiotech.) coated with fluorescent microspheres, 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 zone is adjacent to the blotting paper, and the detection zone is coated with a CK-MB monoclonal capture antibody (produced by a method known in the art) that recognizes a single antigenic epitope, and the control zone is coated with Goat anti-mouse IgG antibody.
  • CK-MB monoclonal detection antibody (Raybiotech.) coated with fluorescent microspheres
  • 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
  • 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.
  • a CK-MB monoclonal capture antibody and a goat anti-mouse IgG antibody that recognize a single epitope are respectively immobilized on the coating membrane to form a detection zone and a control zone; the specific method is: using 0.02 mol/containing 5% sucrose/ L-pH 7.2-7.6 phosphate buffer, respectively diluted CK-MB monoclonal capture antibody and goat anti-mouse IgG antibody recognizing a single epitope to a concentration of 1.5 mg / ml, using a quantitative spray film to 1ul The amount of /cm was sprayed on the nitrocellulose membrane at intervals of 0.5 cm, dried at 50 ° C for 5 h, and sealed with a desiccant for use;
  • the final concentration of the microspheres was 0.2 mol/L, and the reaction was carried out for 15 minutes at room temperature to fully wash the microspheres.
  • the ball is reconstituted with 0.02 mol/L borate buffer of pH 7.4-7.6; (c), the mass ratio of antibody to microsphere by CK-MB is 1:5, and the microsphere after reconstitution
  • Add CK-MB monoclonal detection antibody react at room temperature for 2 hours, add 0.02 mol/L borate buffer containing pH 7.4-7.6 containing 5% BSA, react at room temperature for 30 minutes, wash, and then Reconstituted to the original volume with a 0.02 mol/L borate buffer containing pH 7.4-7.6 containing 0.5% BSA, 0.05% Tween-20, and sprayed onto the glass fiber membrane at a dose of 4 ul/cm using a quantitative spray film apparatus.
  • the time-resolved fluorescent immunochromatography kit for detecting CK-MB of the present embodiment comprising: the test strip, the plastic card case, and the buffer bag described in Embodiment 1;
  • the buffer bag is located at a corner of the plastic card case, close 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 blood sample to be tested is added to the sample-adding hole, and the sample buffer is mixed with the blood sample and immersed in the sample pad through the needle-punching buffer bag. After the sample on the sample pad is saturated, the sample is delivered to the bond pad by capillary action.
  • CK-MB is contained in the blood sample, CK-MB forms an antigen-antibody complex with the antibody on the fluorescent microsphere. As the chromatography progresses, the complex moves forward to reach the CK-coated with a single epitope. At the detection zone T of the MB monoclonal capture antibody, 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 CK-MB monoclonal antibodies continue to advance, and when they reach control zone C, goat anti-mouse IgG antibodies and murine monoclonal antibodies on rare earth ion microspheres (ie The CK-MB monoclonal 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 CK-MB was detected using the time-resolved immunochromatographic kit of Example 2.
  • the standard curve R 2 0.9952, which is linear, can be quantitatively analyzed by the standard curve for the concentration of CK-MB contained in the sample.
  • the sample to be tested was added to the sampled area of the fluorescent immunochromatographic test strip of CK-MB, 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 CK-MB concentration in the sample to be tested by the corresponding analysis software.
  • the information on the calibration card brings the actual detected value into the 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 creatine kinase isoenzyme reference products (0ng/mL, 5ngm/L, 10ng/mL, 30ng/mL, 50ng/mL) 70 ng / mL, 5 parallel samples per concentration), the detection range is 0 ng / mL ⁇ 70 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 creatine kinase isoenzyme reference product was detected, and the coefficient of variation CV (%) value was ⁇ 15%.
  • Inter-assay precision Randomly extract three consecutive batches of time-resolved immunochromatographic kits. Each batch of 3 samples was tested for the same concentration of creatine kinase isoenzyme reference product, and its coefficient of variation was 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 ( +2SD) ⁇ 0.3 ng/mL.
  • Analytical specificity Select the same concentration of creatine kinase isoenzyme reference product 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 was repeatedly tested 3 times, and the mean and relative deviation of the sample detection results were calculated, wherein the relative deviation (B%) was within ⁇ 15%.

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Abstract

一种检测CK-MB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CK-MB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CK-MB单克隆捕获抗体，对照区包被有羊抗鼠IgG抗体；包被膜为结合有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CK-MB定量检测有积极意义。

Description

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 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。
背景技术
肌酸激酶(Creatine Kinase,CK)通常存在于动物的心脏、肌肉以及脑等组织的细胞浆和线粒体中，是一个与细胞内能量运转、肌肉收缩、ATP再生有直接关系的重要激酶，它可逆地催化肌酸与ATP之间的转磷酰基反应。肌酸激酶同工酶(CK-MB)主要分布于心肌中。当心肌损伤后，CK-MB释放到血液中，3～8h后开始升高，可持续3～5天。肌钙蛋白，肌红蛋白，肌酸激酶同工酶这三项联合检测能尽早检测出心肌是否受损。肌酸激酶同功酶在临床诊断中有十分重要的意义，在各种病变包括肌肉萎缩和心肌梗塞发生时，人的血清中肌酸激酶水平迅速提高，目前认为在心肌梗塞的诊断中测定肌酸激酶的活性比做心电图更为可靠。心肌梗死时，肌酸激酶在起病6小时内升高，24小时达高峰，3-4日内恢复正常，其中肌酸激酶的同工酶CK-MB诊断的特异性最高。肌酸激酶因其具有重要的生理功能和临床应用价值已引起人们广泛的重视和深入的研究。
心肌型肌酸激酶同功酶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。但是定性检测，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。
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CK-MB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CK-MB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CK-MB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CK-MB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。
本发明还提供了一种检测CK-MB的时间分辨荧光免疫层析试剂盒，所述试 剂盒包括上述试纸条。
在其中一些实施例中，所述试剂盒还包括塑料卡壳，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。
本发明还公开了上述检测CK-MB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CK-MB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CK-MB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，所述识别单一抗原表位的CK-MB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，步骤(2)中所述荧光标记的CK-MB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CK-MB单克隆检测抗体透析过夜，再将透析后的CK-MB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CK-MB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CK-MB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。
本发明的检测CK-MB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CK-MB时，CK-MB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CK-MB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CK-MB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CK-MB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CK-MB的定量检测中，结合时间分辨荧光检测仪，实现了CK-MB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CK-MB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CK-MB的时间分辨免疫层析试纸条
本实施例的一种检测CK-MB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CK-MB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CK-MB单克隆捕获抗体(自产，采用本领域公知方法制备获得)，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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB单克隆检测抗体透析过夜，再将透析后的CK-MB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CK-MB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CK-MB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CK-MB的时间分辨免疫层析试剂盒
本实施例的检测CK-MB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CK-MB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CK-MB时，CK-MB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CK-MB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CK-MB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 CK-MB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CK-MB
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CK-MB标准品(取6个不同的浓度，分别为0ng/mL、5ngm/L、10ng/mL、30ng/mL、50ng/mL、70ng/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，CK-MB标准品浓度为纵坐标，建立方程并拟合成标准曲线y＝1.0701x+2.6971。
标准曲线R2＝0.9952，线性较好，可以通过该标准曲线对样品中所含CK-MB浓度进行定量分析。
B、样品检测
在CK-MB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CK-MB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肌酸激酶同工酶参考品(0ng/mL、5ngm/L、10ng/mL、30ng/mL、50ng/mL、70ng/mL，每个浓度做5个平行样)进行检测，其检测范围是0ng/mL～70ng/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，分别对同一浓度的肌酸激酶同工酶参考品进行检测，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肌酸激酶同工酶参考品进行检测，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肌酸激酶同工酶参考品，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
Figure PCTCN2017098111-appb-000001
和标准差SD，其中(
Figure PCTCN2017098111-appb-000002
+2SD)≤0.3ng/mL。
6、分析特异性：选择同一浓度的肌酸激酶同工酶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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB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CK-MB单克隆检测抗体透析过夜，再将透析后的CK-MB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CK-MB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CK-MB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CK-MB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CK-MB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。
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