A test strip for detecting cardiac troponin I

Abstract

The present invention provides a cardiac troponin I detection test strip, which belongs to the technical field of cardiac troponin I detection, and comprises a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped on a bottom plate; the conjugation pad is coated with fluorescent nano-microspheres coupled with monoclonal antibodies of cardiac troponin I; the fluorescent nano-microspheres include 1 to 5 kinds, and different kinds of fluorescent nano-microspheres are respectively coupled with different monoclonal antibodies against cardiac troponin I; a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is coated with polyclonal antibodies against cardiac troponin I; the quality control line is coated with goat anti-mouse IgG. The test strip provided by the present invention has the advantages of high sensitivity, good stability, and low interference by natural fluorescence.

Description

Technical Field

[0001] The invention belongs to the technical field of cardiac troponin I detection, and in particular relates to a cardiac troponin I detection test strip. Background Art

[0002] The detection of cardiac troponin I is currently a common method for monitoring and diagnosing myocardial cell damage / necrosis. The detection of cardiac troponin I can be performed by immunological detection based on antigen-antibody reaction, or by solid phase immunochromatography for qualitative analysis. In some embodiments, the antibody can be a polyclonal antibody or a monoclonal antibody.

[0003] At present, cardiac troponin I detection methods are increasingly used in the diagnosis of myocardial injury / necrosis. The main methods used for detecting cInI in clinical practice include ELISA (enzyme immunosorbent assay), radioimmunoassay, colloidal gold immunochromatography, chemiluminescence, etc. Traditional cardiac troponin I detection methods have the defects of low sensitivity, low accuracy, long detection time, complicated operation, and high detection cost. For example, the ELISA method uses polyclonal antibodies, has many cross reactions, and the combination of antigen and antibody occurs on the reaction surface of the ELISA plate. The ELISA method has low sensitivity, low accuracy, and a long detection time. Solid phase immunochromatography technology is mainly used for bedside detection. Although it is convenient to operate and easy to use, it has low sensitivity and is expensive. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a test strip for detecting cardiac troponin I which is simple to operate and has high sensitivity.

[0005] The present invention provides a cardiac troponin I detection test strip, comprising a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped on a bottom plate; the conjugation pad is coated with fluorescent nano-microspheres coupled with a monoclonal antibody to cardiac troponin I;

[0006] The fluorescent nano-microspheres include 1 to 5 kinds, and different kinds of fluorescent nano-microspheres are respectively coupled with different monoclonal antibodies against cardiac troponin I;

[0007] The nitrocellulose membrane is provided with a detection line and a quality control line. The detection line is coated with polyclonal antibodies against cardiac troponin I; and the quality control line is coated with goat anti-mouse IgG.

[0008] Preferably, the fluorescent nanospheres are fluorescent polystyrene microspheres with a particle size of 80 to 100 nm.

[0009] Preferably, the volume ratio of the fluorescent nanospheres to the monoclonal antibody is (4-6):1; and the concentration of the monoclonal antibody is 0.08-0.15 mg / mL.

[0010] Preferably, when there are 2 to 5 kinds of fluorescent nanospheres, the fluorescent nanospheres are mixed in equal volumes.

[0011] Preferably, the concentration of the polyclonal antibody coated with cardiac troponin I on the test line is 0.5-1.5 mg / mL.

[0012] Preferably, the concentration of the goat anti-mouse IgG coated on the quality control line is 1.5-2.5 mg / mL.

[0013] Compared with the prior art, the present invention has the following beneficial effects: the cardiac troponin I detection test strip provided by the present invention detects cardiac troponin I in the sample by using the double antibody sandwich method; the cardiac troponin I in the sample forms a complex with the first antibody marked on the fluorescent microsphere, moves forward along the nitrocellulose membrane under the action of layer folding, and captures the complex with the cardiac troponin I polyclonal antibody coated on the nitrocellulose membrane in the detection area to form a T detection line; the fluorescent microspheres combined with cTnI cardiac troponin I will continue to move forward to the quality control area and combine with the sheep anti-mouse IgG coated on the nitrocellulose membrane to form a C detection line; during the detection process, under the action of the excitation light source, the fluorescent microspheres emit a 610-nanometer fluorescent signal, and the fluorescent immunoassay analyzer captures the fluorescent signal, which is automatically converted into quantitative data through signal conversion and the set standard curve, thereby calculating the cTnI concentration in the sample. The test strip provided by the present invention has the advantages of high sensitivity, good stability, and low interference from natural fluorescence. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a reaction schematic diagram of the cardiac troponin I detection test strip of the present invention;

[0015] Figure 2 The standard curve of the fluorescence signal of the test strip prepared in Example 1;

[0016] Figure 3 The standard curve of the fluorescence signal of the test strip prepared in Example 2;

[0017] Figure 4 This is the standard curve of the fluorescence signal of the test strip prepared in Example 3. DETAILED DESCRIPTION

[0018] The invention provides a cardiac troponin I detection test strip, comprising a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped on a bottom plate; the conjugation pad is coated with fluorescent nano-microspheres coupled with monoclonal antibodies to cardiac troponin I; the fluorescent nano-microspheres comprise 1 to 5 kinds, and different kinds of fluorescent nano-microspheres are respectively coupled with different monoclonal antibodies against cardiac troponin I; a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is coated with polyclonal antibodies to cardiac troponin I; and the quality control line is coated with goat anti-mouse IgG.

[0019] In the present invention, the conjugate pad is coated with fluorescent nanospheres coupled with monoclonal antibodies to cardiac troponin I; the fluorescent nanospheres are preferably fluorescent polystyrene microspheres, and the particle size of the fluorescent nanospheres is preferably 80-100 nm. In the present invention, the volume ratio of the fluorescent nanospheres to the monoclonal antibodies is preferably (4-6):1, further preferably (4.5-5.5):1, and further preferably 5:1; in the specific implementation of the present invention, 20 μL of monoclonal antibodies are preferably mixed and coupled with 100 μL of fluorescent polystyrene microspheres. In the present invention, the fluorescent polystyrene microspheres are preferably purchased from Shanghai Yipuri Biotechnology Co., Ltd.

[0020] In the present invention, the concentration of the monoclonal antibody is preferably 0.08-0.15 mg / mL, more preferably 0.10-0.13 mg / mL, and even more preferably 0.12 mg / mL. In the present invention, when the number of the fluorescent nano-microspheres is 2-5, the individual fluorescent nano-microspheres are mixed in equal volumes. In the present invention, the different types of fluorescent nano-microspheres are coupled with monoclonal antibodies targeting different binding epitopes of cardiac troponin I. The purpose of using more than two types of fluorescent nano-microspheres in the present invention is to improve the capture performance of the nano-microspheres, capture as much cardiac troponin I as possible in the sample to be detected, thereby improving the sensitivity of the detection; on the basis of maintaining a high sensitivity, cross-reactions are avoided to ensure the accuracy of the detection.

[0021] In the present invention, the concentration of the polyclonal antibody against cardiac troponin I on the test line is preferably 0.5-1.5 mg / mL, more preferably 0.6-1.2 mg / mL, and more preferably 0.8 mg / mL. In the present invention, the concentration of the goat anti-mouse IgG coated on the quality control line is preferably 1.5-2.5 mg / mL, more preferably 1.8-2.2 mg / mL, and more preferably 2.0 mg / mL.

[0022] In the present invention, the bottom plate is preferably a PVC bottom plate; the materials of the sample pad, the binding pad and the water absorbent pad are not particularly limited, and conventional materials in the art can be used.

[0023] The present invention also provides a method for preparing the cardiac troponin I detection test strip.

[0024] In the present invention, the method for preparing the fluorescent nanoparticles coupled with the first antibody to cardiac troponin I comprises: activating the fluorescent polypropylene microparticles, and coupling the first antibody to the activated fluorescent polypropylene microparticles.

[0025] The method for activating fluorescent polypropylene microspheres is as follows: disperse the purchased fluorescent polystyrene microspheres by ultrasonic treatment for 3 to 6 minutes, take 80 to 120 μL, add 650 to 750 μL 55 mM MES buffer thereto, ultrasonically mix, centrifuge, discard the supernatant, continue to add 550 to 650 μL 55 mM MES buffer, ultrasonically mix, centrifuge, collect the precipitate; add 650 to 750 μL 55 mM MES buffer, ultrasonic mixing, then adding ethanol solution containing NHS and ethanol solution containing EDC to the mixed mixture, ultrasonic reaction for 5 minutes, rotating and mixing in the dark, centrifuging and discarding the supernatant, then adding 1000 μL pH 6.5, 0.01M citric acid buffer, ultrasonic mixing, centrifuging, and obtaining activated fluorescent microspheres; wherein, the concentration of NHS in the ethanol solution containing NHS is 13-15 mg / ml; the concentration of EDC in the ethanol solution containing EDC is 13-151 mg / ml; ultrasonic mixing is performed by using 40KHz ultrasonic treatment for 2 minutes; centrifugation is performed at 16000 rpm for 15 minutes at 3°C; the rotation speed of rotating and mixing in the dark is 25 rpm, and the time is 50 minutes.

[0026] In the present invention, the method for coupling the first antibody with the activated fluorescent polypropylene microspheres is as follows:

[0027] The activated fluorescent polystyrene microspheres were mixed with 500 μL of MES buffer containing 0.5% glycerol, treated with 40 KHz ultrasound for 2 min, and then 20 μL of 0.08-0.15 mg / mL antibody solution was added, treated with 40 KHz ultrasound for 10 min, and then rotated and mixed at 35 rpm in the dark for 100 min.

[0028] The present invention performs blocking after the coupling, and the blocking is performed using BSA, and the final concentration of the BSA in the system is preferably 0.5%, and the blocking time is preferably 45 to 55 minutes, and more preferably 50 minutes. The present invention performs centrifugation after the blocking is completed to collect and obtain fluorescent nanoparticles coupled with the first antibody of cardiac troponin I; the centrifugal speed is 7000 to 9000 rpm, and more preferably 7500 to 8500 rpm, and the centrifugal time is 10 to 15 minutes, and more preferably 12 to 13 minutes.

[0029] Preparation of the conjugate pad: Dissolve the fluorescent nanoparticles coupled with the first antibody of cardiac troponin I in a PBS solution containing 1.2% BSA, 8% sucrose, and 0.2% PVP40; spray the conjugate pad pretreated with a gold film sprayer at a spray volume of 2 to 5 μL / cm; then place the sprayed pad in a 37°C oven, dry for 4 hours, and store. In the present invention, the pretreated conjugate pad is a conjugate pad soaked in a PBS solution containing 1.2% BSA, 8% sucrose, and 0.2% PVP40.

[0030] In the present invention, after diluting the cardiac troponin I polyclonal antibody with a PBS solution (pH7.4) containing 2% sucrose, a gold spray film streaking instrument is used to spray the test line on the nitrocellulose membrane, and the spraying amount is preferably 0.8-1.2 μL / cm, and more preferably 1 μL / cm. In the present invention, after diluting the sheep anti-mouse IgG with a PBS solution (pH7.4) containing 2% sucrose, a gold spray film streaking instrument is used to spray the quality control line on the nitrocellulose membrane, and the spraying amount is preferably 0.8-1.2 μL / cm, and more preferably 1 μL / cm. The test line and the quality control line are separated by 5 mm. In the present invention, after the nitrocellulose membrane is sprayed, it is dried and stored.

[0031] In the present invention, a sample pad (glass fiber cotton material), a binding pad, a nitrocellulose membrane and a blotting paper are overlapped and pasted on a PVC bottom plate by 2 mm in an interlaced manner, and cut into test strips. The present invention preferably assembles the test strips in a plastic shell formed by buckling a plastic upper shell and a plastic lower shell, and a sample addition hole and an observation window are provided on the plastic upper shell, the sample addition hole corresponds to the sample pad, and the result observation window corresponds to the detection line and the quality control line.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Sources of materials and reagents

[0034] Fluorescent polypropylene microspheres were purchased from Shanghai Yipuri Biotechnology Co., Ltd.

[0035] Cardiac troponin I monoclonal antibodies MAA478Hu21, MAA478Hu22, MAA478Hu25, and MAA478Hu27 were purchased from Wuhan Cloud-Clone Technology Co., Ltd.;

[0036] The cardiac troponin I polyclonal antibody PAA478Hu01 was purchased from Wuhan Cloud-Clone Technology Co., Ltd.

[0037] Goat anti-mouse IgG was purchased from Thermo Fisher Scientific.

[0038] Example 1

[0039] A cardiac troponin I detection test strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped on a bottom plate; the conjugation pad is coated with fluorescent nano-microspheres coupled with monoclonal antibodies to cardiac troponin I.

[0040] Activation of fluorescent nano-microspheres: Disperse the purchased fluorescent polystyrene microspheres after ultrasonic treatment for 5 minutes, take 90 μL, add 710 μL of 55 mM MES buffer thereto, ultrasonically mix, centrifuge, discard the supernatant, continue to add 610 μL of 55 mM MES buffer, ultrasonically mix, centrifuge, and collect the precipitate; add 610 μL of 55 mM MES buffer to the precipitate, ultrasonically mix, and then add 10 μL of ethanol solution containing 10 mg / ml NHS and 10 μL of ethanol solution containing 14 mg / ml EDC to the mixed mixture, ultrasonically react for 5 minutes, and rotate and mix at 25 rpm for 40 minutes in the dark; centrifuge and discard the supernatant, then add 1000 μL of pH 6.5, 0.01 M citric acid buffer, ultrasonically mix, and centrifuge to obtain activated fluorescent microspheres; centrifugation is centrifugation at 16000 rpm for 15 min at 4°C.

[0041] Monoclonal antibodies coupled to fluorescent nanospheres:

[0042] The activated fluorescent polystyrene microspheres were mixed with 500 μL of MES buffer containing 0.5% glycerol, and treated with 40 KHz ultrasound for 2 min. Then, 20 μL of 0.12 mg / mL monoclonal antibody MAA478Hu21 solution was added, and treated with 40 KHz ultrasound for 10 min. Then, the mixture was rotated and mixed at 35 rpm in the dark for 100 min.

[0043] After coupling, blocking is performed using BSA, the final concentration of BSA in the system is preferably 0.5%, and the blocking time is 50 minutes. Then centrifugation is performed at 8000 rpm for 10 minutes to collect the fluorescent nanoparticles coupled with the monoclonal antibody MAA478Hu21;

[0044] The preparation method of fluorescent nanoparticles coupled with monoclonal antibodies MAA478Hu22, MAA478Hu25, and MAA478Hu27 is the same as above.

[0045] The prepared fluorescent nanoparticles coupled to monoclonal antibodies MAA478Hu21, MAA478Hu22, MAA478Hu25, and MAA478Hu27 were mixed in equal volumes.

[0046] Preparation of conjugate pad:

[0047] Pretreatment of the conjugate pad: soak the conjugate pad in a PBS solution containing 1.2% BSA, 8% sucrose, and 0.2% PVP40 for 20 minutes, and then dry it at 37°C.

[0048] The mixed fluorescent nanospheres were mixed and dispersed with 600 μL of PBS solution containing 1.2% BSA, 8% sucrose and 0.2% PVP40; the gold film sprayer was used to spray the pretreated binding pad at a spray volume of 2 μL / cm; the sprayed pad was then placed in a 37°C oven and dried for 4 hours before storage.

[0049] After diluting the myocardial troponin I polyclonal antibody to 0.8 mg / mL with a PBS solution (pH 7.4) containing 2% sucrose, the test line is sprayed on the nitrocellulose membrane with a gold spray film streaking instrument, and the spraying amount is preferably 1 μL / cm. After diluting the sheep anti-mouse IGg to 2.0 mg / mL with a PBS solution (pH 7.4) containing 2% sucrose, the quality control line is sprayed on the nitrocellulose membrane with a gold spray film streaking instrument with a spraying amount of 1 μL / cm. The test line and the quality control line are separated by 5 mm. In the present invention, after the nitrocellulose membrane is sprayed, it is dried and stored.

[0050] Finally, the sample pad (made of glass fiber cotton), binding pad, nitrocellulose membrane and absorbent paper were pasted on the PVC base plate in sequence with an overlap of 2 mm and cut into test strips.

[0051] Example 2

[0052] A cardiac troponin I detection test strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped on a bottom plate; the conjugation pad is coated with fluorescent nano-microspheres coupled with monoclonal antibodies to cardiac troponin I.

[0053] Activation of fluorescent nano-microspheres: Disperse the purchased fluorescent polystyrene microspheres after ultrasonic treatment for 5 minutes, take 90 μL, add 710 μL of 55 mM MES buffer thereto, ultrasonically mix, centrifuge, discard the supernatant, continue to add 610 μL of 55 mM MES buffer, ultrasonically mix, centrifuge, and collect the precipitate; add 610 μL of 55 mM MES buffer to the precipitate, ultrasonically mix, and then add 10 μL of ethanol solution containing 10 mg / ml NHS and 10 μL of ethanol solution containing 14 mg / ml EDC to the mixed mixture, ultrasonically react for 5 minutes, and rotate and mix at 25 rpm for 40 minutes in the dark; centrifuge and discard the supernatant, then add 1000 μL of pH 6.5, 0.01 M citric acid buffer, ultrasonically mix, and centrifuge to obtain activated fluorescent microspheres; centrifugation is centrifugation at 16000 rpm for 15 min at 4°C.

[0054] Monoclonal antibodies coupled to fluorescent nanospheres:

[0055] The activated fluorescent polystyrene microspheres were mixed with 500 μL of MES buffer containing 0.5% glycerol, and treated with 40 KHz ultrasound for 2 min. Then, 20 μL of 0.12 mg / mL monoclonal antibody MAA478Hu21 solution was added, and treated with 40 KHz ultrasound for 10 min. Then, the mixture was rotated and mixed at 35 rpm in the dark for 100 min.

[0056] After coupling, blocking is performed using BSA, the final concentration of BSA in the system is preferably 0.5%, and the blocking time is 50 minutes. Then centrifugation is performed at 8000 rpm for 10 minutes to collect the fluorescent nanoparticles coupled with the monoclonal antibody MAA478Hu21;

[0057] The preparation method of fluorescent nanoparticles coupled to monoclonal antibody MAA478Hu22 is the same as above.

[0058] The prepared fluorescent nanoparticles coupled to monoclonal antibodies MAA478Hu21 and MAA478Hu22 were mixed in equal volumes.

[0059] Preparation of conjugate pad:

[0060] Pretreatment of the conjugate pad: soak the conjugate pad in a PBS solution containing 1.2% BSA, 8% sucrose, and 0.2% PVP40 for 20 minutes, and then dry it at 37°C.

[0061] The mixed fluorescent nanospheres were mixed and dispersed with 450 μL of PBS solution containing 1.2% BSA, 8% sucrose and 0.2% PVP40; the pre-treated binding pad was sprayed with a gold film sprayer at a spray volume of 2 μL / cm; the sprayed pad was then placed in a 37°C oven and dried for 4 hours before storage.

[0062] After diluting the myocardial troponin I polyclonal antibody to 0.8 mg / mL with a PBS solution (pH 7.4) containing 2% sucrose, a test line is sprayed on a nitrocellulose membrane using a gold spray film streaking instrument, and the spraying amount is preferably 1 μL / cm. After diluting the goat anti-mouse IGg to 2.0 mg / mL with a PBS solution (pH 7.4) containing 2% sucrose, a quality control line is sprayed on a nitrocellulose membrane using a gold spray film streaking instrument with a spraying amount of 1 μL / cm. The test line and the quality control line are spaced 5 mm apart. In the present invention, after the nitrocellulose membrane is sprayed, it is dried and stored.

[0063] Finally, the sample pad (made of glass fiber cotton), binding pad, nitrocellulose membrane and absorbent paper were pasted on the PVC base plate in sequence with an overlap of 2 mm and cut into test strips.

[0064] Example 3

[0065] A cardiac troponin I detection test strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped on a bottom plate; the conjugation pad is coated with fluorescent nano-microspheres coupled with monoclonal antibodies to cardiac troponin I.

[0066] Activation of fluorescent nano-microspheres: Disperse the purchased fluorescent polystyrene microspheres after ultrasonic treatment for 5 minutes, take 90 μL, add 710 μL of 55 mM MES buffer thereto, ultrasonically mix, centrifuge, discard the supernatant, continue to add 610 μL of 55 mM MES buffer, ultrasonically mix, centrifuge, and collect the precipitate; add 610 μL of 55 mM MES buffer to the precipitate, ultrasonically mix, and then add 10 μL of ethanol solution containing 10 mg / ml NHS and 10 μL of ethanol solution containing 14 mg / ml EDC to the mixed mixture, ultrasonically react for 5 minutes, and rotate and mix at 25 rpm for 40 minutes in the dark; centrifuge and discard the supernatant, then add 1000 μL of pH 6.5, 0.01 M citric acid buffer, ultrasonically mix, and centrifuge to obtain activated fluorescent microspheres; centrifugation is centrifugation at 16000 rpm for 15 min at 4°C.

[0067] Monoclonal antibodies coupled to fluorescent nanospheres:

[0068] The activated fluorescent polystyrene microspheres were mixed with 500 μL of MES buffer containing 0.5% glycerol, and treated with 40 KHz ultrasound for 2 min. Then, 20 μL of 0.12 mg / mL monoclonal antibody MAA478Hu21 solution was added, and treated with 40 KHz ultrasound for 10 min. Then, the mixture was rotated and mixed at 35 rpm in the dark for 100 min.

[0069] After coupling, blocking is performed using BSA, the final concentration of BSA in the system is preferably 0.5%, and the blocking time is 50 minutes. Then centrifugation is performed at 8000 rpm for 10 minutes to collect the fluorescent nanoparticles coupled with the monoclonal antibody MAA478Hu21;

[0070] Preparation of conjugate pad:

[0071] Pretreatment of the conjugate pad: soak the conjugate pad in a PBS solution containing 1.2% BSA, 8% sucrose, and 0.2% PVP40 for 20 minutes, and then dry it at 37°C.

[0072] Dilute the fluorescent nanoparticles coupled to the monoclonal antibody MAA478Hu21 with 150 μL of PBS solution containing 1.2% BSA, 8% sucrose, and 0.2% PVP40; spray them onto the pretreated binding pad using a gold sprayer at a spray volume of 2 μL / cm; then place the sprayed pad in a 37°C oven and dry it for 4 hours before storing.

[0073] After diluting the myocardial troponin I polyclonal antibody to 0.8 mg / mL with a PBS solution (pH 7.4) containing 2% sucrose, a test line is sprayed on a nitrocellulose membrane using a gold spray film streaking instrument, and the spraying amount is preferably 1 μL / cm. After diluting the goat anti-mouse IGg to 2.0 mg / mL with a PBS solution (pH 7.4) containing 2% sucrose, a quality control line is sprayed on a nitrocellulose membrane using a gold spray film streaking instrument with a spraying amount of 1 μL / cm. The test line and the quality control line are spaced 5 mm apart. In the present invention, after the nitrocellulose membrane is sprayed, it is dried and stored.

[0074] Finally, the sample pad (made of glass fiber cotton), binding pad, nitrocellulose membrane and absorbent paper were pasted on the PVC base plate in sequence with an overlap of 2 mm and cut into test strips.

[0075] Experimental example

[0076] The cardiac troponin I antigen was prepared into solutions of different concentrations, namely (8 concentrations in total, 50.0 ng / ml, 25.0 ng / ml, 10.0 ng / ml, 5.0 ng / ml, 2.0 ng / ml, 1 ng / ml, 0.5 ng / ml, 0.01 ng / ml, each concentration had three replicates, and all were prepared by diluting 1.0 mg / ml human cTnI antigen with sample buffer).

[0077] The standard products of 8 concentrations were respectively added dropwise to the test strips prepared in Examples 1 to 3, and chromatographed for 15 minutes; the results were detected by using the QFT9000 dry fluorescent immunoassay analyzer of Wuhan Mind Biotechnology Co., Ltd.

[0078] The concentration of cardiac troponin I calibrator was used as the X-axis and the fluorescence intensity was used as the Y-axis to establish an equation and fit it into a standard curve, such as Figures 2 to 4 As shown, the R values ​​are 0.9907, 0.9881, and 0.9966, respectively.

[0079] The test report shows that the minimum detection limits of the test strips prepared in Examples 1 to 3 are 0.01 ng / mL, 0.03 ng / mL, and 0.06 ng / mL, respectively, with good intra-batch and inter-batch repeatability.

[0080] It can be seen that the test strip provided by the present invention has the advantages of high sensitivity, good stability, and low interference from natural fluorescence.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A test strip for detecting cardiac troponin I, characterized in that: The method comprises a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped on a bottom plate; the conjugation pad is coated with fluorescent nano-microspheres coupled with monoclonal antibodies of cardiac troponin I; There are four kinds of fluorescent nano-microspheres, which are obtained by coupling activated fluorescent polystyrene microspheres with monoclonal antibodies MAA478Hu21, MAA478Hu22, MAA478Hu25 and MAA478Hu27 respectively; the four kinds of fluorescent nano-microspheres are mixed in equal volumes; The particle size of the fluorescent polystyrene microspheres is 80 to 100 nm; The nitrocellulose membrane is provided with a detection line and a quality control line, wherein the detection line is coated with polyclonal antibodies against cardiac troponin I; and the quality control line is coated with goat anti-mouse IgG; The volume ratio of the fluorescent nano-microspheres to the monoclonal antibody is (4-6):1; the concentration of the monoclonal antibody is 0.08-0.15 mg / mL.

2. The test strip according to claim 1, characterized in that: The concentration of the polyclonal antibody coated with cardiac troponin I on the detection line is 0.5-1.5 mg / mL.

3. The test strip according to claim 1, characterized in that: The concentration of the goat anti-mouse IgG coated on the quality control line is 1.5-2.5 mg / mL.

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