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 PDFInfo
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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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Classifications
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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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- 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
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- G—PHYSICS
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- 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
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
Description
Claims (10)
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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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