CN111323576B - Method for enhancing antibody-fluorescent microsphere conjugate signal and application of method in troponin I detection - Google Patents
Method for enhancing antibody-fluorescent microsphere conjugate signal and application of method in troponin I detection Download PDFInfo
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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
- 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
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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Abstract
The invention discloses a method for enhancing an antibody-fluorescent microsphere conjugate signal, which comprises the steps of coupling an antibody with a fluorescent microsphere to obtain an antibody-fluorescent microsphere conjugate, and coupling the antibody-fluorescent microsphere conjugate with fluorescein to obtain the enhanced antibody-fluorescent microsphere conjugate. Before the antibody-fluorescent microsphere conjugate is coupled with fluorescein, inert protein is used for sealing the surface vacancies of the fluorescent microsphere. The method fully utilizes the carboxyl/amino groups of the protein on the surface of the fluorescent microsphere after the coupling is finished, and carries out covalent coupling of fluorescein again, so that the fluorescent signal is further enhanced, and the detection sensitivity is improved. The invention also discloses application of the method in troponin I detection.
Description
Technical Field
The invention relates to the technical field of fluorescence chromatography, in particular to a method for enhancing an antibody-fluorescent microsphere conjugate signal and application thereof in troponin I detection.
Background
The immunochromatography technology is taken as an important branch of Poct (Point of CARE TESTING, instant detection) and starts in the 80 th year of the 20 th century, wherein early pregnancy test paper is the most successful product due to the characteristics of convenience, rapidness and low price, and meanwhile, the immunochromatography technology is expanded to a plurality of detection fields, but compared with ELISA (enzyme-linked immunosorbent assay) and chemiluminescence methodologies, the immunochromatography test paper also has corresponding short plates, and is mainly characterized by poor precision and insufficient sensitivity; therefore, in the early twentieth century, various manufacturers change the colloidal gold into fluorescent substances for improving the performance of chromatographic products, the performance of the products is greatly improved, and the fluorescent chromatographic products gradually replace the colloidal gold to become the main stream of the market. The early fluorescence chromatography uses fluorescein, and is gradually changed into fluorescent microspheres, and the fluorescent microspheres contain more fluorescent substances, so that fluorescent signals can be effectively amplified, and the detection sensitivity is improved. In current fluorescence chromatography studies, it is critical to further improve the product performance to again increase the fluorescence signal of the conjugate.
The current antibody-fluorescent microsphere coupling generally adopts chemical coupling, firstly, the surface groups (generally carboxyl groups) of the microsphere are activated, the antibody is added, the amino groups of the antibody are combined with the activated carboxyl groups and fixed on the surface of the microsphere, and then inert proteins (generally BSA) are used for sealing the surface vacancies of the microsphere.
The sensitivity is determined by the strength of fluorescent substances captured on the detection line, and the existing antibody-fluorescent microsphere coupling technology generally corresponds to one fluorescent microsphere with a plurality of antibodies, so that when the detection line captures an antibody-fluorescent microsphere conjugate, a plurality of antibodies can be captured to have the fluorescence strength of one fluorescent microsphere.
Cardiac troponin is a regulator of cardiac contractility and is present on the filaments of cardiac contractility proteins. Early in myocardial injury, myocardial cells do not necrose, but cell membranes are destroyed, cTnI in free form enters the interstitial space and flows back into the blood via lymph. Serum cTnI rises at 6h, after which myofibrils are continuously disintegrated and destroyed, cTnI in a fixed form is continuously released, serum levels peak at 18-24h, and after 10 days, falls to normal. Troponin I (cTnI) has a high degree of myocardial specificity and sensitivity and is currently recognized as a relatively ideal myocardial infarction (AMI) marker. In order to more accurately predict cardiovascular risk, cTnI enters the hypersensitive age, and higher requirements are put on the sensitivity of fluorescence chromatography.
Disclosure of Invention
In order to solve the technical problems, the invention discloses a method for enhancing the signal of an antibody-fluorescent microsphere conjugate, which fully utilizes the carboxyl/amino groups of proteins on the surface of the fluorescent microsphere after the coupling is completed, and carries out covalent coupling of fluorescein again, so that the fluorescent signal is further enhanced, and the detection sensitivity is improved. The invention also discloses application of the method in troponin I detection.
The invention is realized by the following technical scheme:
A method for enhancing the signal of antibody-fluorescent microsphere conjugate comprises the steps of coupling an antibody with fluorescent microsphere to obtain an antibody-fluorescent microsphere conjugate, and coupling the antibody-fluorescent microsphere conjugate with fluorescein to obtain the enhanced antibody-fluorescent microsphere conjugate. Before the antibody-fluorescent microsphere conjugate is coupled with fluorescein, inert protein is used for sealing the surface vacancies of the fluorescent microsphere.
The key index of the fluorescent chromatography technology is sensitivity, and the sensitivity is determined by the number of fluorescent substances. The current antibody-fluorescent microsphere coupling generally adopts chemical coupling, firstly, the surface groups (generally carboxyl groups) of the microsphere are activated, the antibody is added, the amino groups of the antibody are combined with the activated carboxyl groups and fixed on the surface of the microsphere, and then inert proteins (generally BSA) are used for sealing the surface vacancies of the microsphere. The inventors have found that after the coupling is completed, the fluorescence of the conjugate comes only from the fluorescent microsphere itself, the microsphere surface is spread over the proteins (consisting of antibodies and BSA), and the large amount of amino/carboxyl groups contained in these proteins are underutilized.
After the coupling of the antibody and the fluorescent microsphere is completed, the antibody on the surface of the fluorescent microsphere and a large amount of available amino (-NH 2) on the blocking protein are used for coupling with fluorescein, so that the fluorescence carried by the single microsphere is increased, the fluorescence signal intensity of the whole conjugate is greatly enhanced, and the detection sensitivity is improved.
Use of a method for enhancing the signal of an antibody-fluorescent microsphere conjugate in the detection of troponin I, comprising the steps of:
(1) Coupling the anti-cTnI monoclonal antibody with fluorescent microspheres to obtain a cTnI antibody-fluorescent microsphere conjugate;
(2) Coupling the cTnI antibody-fluorescent microsphere conjugate with fluorescein to obtain the enhanced cTnI antibody-fluorescent microsphere conjugate.
Wherein, in the step (2), the surface vacancies of the fluorescent microspheres are blocked by inert proteins before the cTnI antibody-fluorescent microsphere conjugate is coupled with fluorescein.
Further, the fluorescein is fluorescein isothiocyanate, and the inert protein is bovine serum albumin.
Further, in the step (1), the preparation method of the cTnI antibody-fluorescent microsphere conjugate is as follows:
(11) Taking out the fluorescent microspheres, putting the fluorescent microspheres into a centrifuge tube, centrifuging, settling and removing supernatant;
(12) Adding a coupling buffer solution into the sediment, uniformly mixing, then adding an EDC solution and a sulfo-NHS solution, uniformly mixing, and incubating;
(13) Centrifuging the solution obtained in the step (12), settling, removing the supernatant, and adding a coupling buffer solution for uniform mixing;
(14) Adding anti-cTnI monoclonal antibody, mixing, and incubating at room temperature to obtain cTnI antibody-fluorescent microsphere conjugate.
Further, in the step (2), the preparation method of the enhanced cTnI antibody-fluorescent microsphere conjugate is as follows:
(21) Centrifuging, settling and removing the supernatant of the cTnI antibody-fluorescent microsphere conjugate solution in the step (14), adding a sealing solution, uniformly mixing, and incubating at room temperature;
(22) Centrifuging after incubation, settling, removing supernatant, adding 1% fluorescein isothiocyanate, mixing, and incubating at room temperature;
(23) And finally, centrifuging, settling, removing the supernatant, and adding the preservation solution for uniform mixing.
Further, cTnI detection is performed on a fluorescent chromatography detection test strip, which comprises a substrate and a sample pad, a marker pad, a chromatographic membrane and a water absorption pad which are sequentially connected on the substrate, wherein the marker pad is a glass fiber pad, and the chromatographic membrane is a nitrocellulose membrane.
Further, the glass fiber mat is treated as follows:
spraying the treatment liquid on one side of the glass fiber mat, wherein the spraying amount is 4ul/cm; and spraying the mixed solution of the enhanced cTnI antibody-fluorescent microsphere conjugate and the rabbit IgG-fluorescent microsphere conjugate on the other side of the glass fiber pad, and drying at 45 ℃.
Further, the nitrocellulose membrane is subjected to the following treatment:
Diluting the other anti-cTnI antibody to 1mg/mL by using a diluent, and scribing one side of the nitrocellulose membrane as a detection line; diluting goat anti-rabbit IgG to 0.5mg/mL by using a diluent, and marking on a nitrocellulose membrane as a quality control line; the film dividing amount is 1ul/cm, and the film is dried at 45 ℃.
The invention couples the marked cTnI antibody-fluorescent microsphere compound with fluorescein, fully utilizes a large amount of available amino (-NH 2) groups on the cTnI antibody and bovine serum albumin on the surface of the fluorescent microsphere to obtain the enhanced cTnI antibody-fluorescent microsphere compound, so that the same fluorescent microsphere can carry more fluorescent substances, the fluorescent signal intensity on a detection line is greatly improved, and the reagent sensitivity and the detection sensitivity are improved.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. After the antibody is coupled with the fluorescent microsphere, the antibody on the surface of the fluorescent microsphere and a large amount of available amino (-NH 2) on the sealing protein are utilized to be coupled with fluorescein, so that the fluorescence carried by a single microsphere is increased, the fluorescence signal intensity of the whole conjugate is greatly enhanced, and the detection sensitivity is improved;
2. The invention relates to an application of a method for enhancing antibody-fluorescent microsphere conjugate signals in troponin I detection, which is characterized in that a marked cTnI antibody-fluorescent microsphere complex is coupled with fluorescein, and a large amount of available amino (-NH 2) on the cTnI antibody and bovine serum albumin on the surface of the fluorescent microsphere is fully utilized to obtain an enhanced cTnI antibody-fluorescent microsphere complex, so that the same fluorescent microsphere can carry more fluorescent substances, the fluorescent signal intensity on a detection line is greatly improved, and the reagent sensitivity and the detection sensitivity are improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of embodiments of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:
FIG. 1 is a schematic representation of an enhanced cTnI antibody-fluorescent microsphere conjugate of the invention, in which The expression antibody, BSA is bovine serum albumin and FITC is fluorescein isothiocyanate.
FIG. 2 is a schematic diagram of the cTnI detection results of the present invention.
Detailed Description
For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present invention and the descriptions thereof are for illustrating the present invention only and are not to be construed as limiting the present invention.
Example 1
The invention relates to application of a method for enhancing antibody-fluorescent microsphere conjugate signals in troponin I detection, and a preparation method of an enhanced cTnI antibody-fluorescent microsphere conjugate, which comprises the following steps:
1. 500ul of fluorescent microspheres (1% concentration W/V, green fluorescence-excitation 475 nM-emission 525 nM) were removed and placed in a centrifuge tube.
2. Centrifuging (12000 rpm-20000rpm according to different particle sizes) for 10min, settling the microspheres, and removing the supernatant.
3. 500Ul of coupling buffer (50 mM MES ph 6.0) was added and mixed well.
4. 20Ul of EDC solution (200 mM), 20ul of sulfo-NHS solution (200 mM), were added, mixed well, and incubated on a rotating disk mixer for 30min.
5. Centrifuging (12000 rpm-20000rpm according to different particle sizes) for 10min, settling the microspheres, and removing the supernatant.
6. 500Ul of coupling buffer (50 mM MES ph 6.0) was added, and after mixing, 0.1mg of anti-cTnI monoclonal antibody was added, mixed, and incubated for 1h at room temperature using a rotating disk mixer.
7. Centrifuging (12000 rpm-20000rpm according to different particle sizes) for 10min, settling the microspheres, and removing the supernatant.
8. Blocking solution (1% BSA in water) was added, mixed well and incubated for 1h at room temperature using a rotating disk mixer.
9. Centrifuging (12000 rpm-20000rpm according to different particle sizes) for 10min, settling the microspheres, and removing the supernatant.
10. 1% Fluorescein isothiocyanate (FITC, aqueous solution) was added, mixed well and incubated with a rotating disc mixer for 1h at room temperature.
11. Centrifuging (12000 rpm-20000rpm according to different particle sizes) for 10min, settling the microspheres, and removing the supernatant.
12. 500Ul of preservative fluid (0.5% BSA,2% sucrose, tirs-HCl ph 8.0) was added and mixed well, the product was as shown in FIG. 1.
Example 2
The invention relates to application of a method for enhancing antibody-fluorescent microsphere conjugate signals in troponin I detection, and a preparation method of an enhanced cTnI antibody-fluorescent microsphere conjugate, which comprises the following steps:
1. 500ul of fluorescent microspheres (1% concentration W/V, green fluorescence-excitation 475 nM-emission 525 nM) were removed and placed in a centrifuge tube.
2. Centrifuging (12000 rpm-20000rpm according to different particle sizes) for 10min, settling the microspheres, and removing the supernatant.
3. 500Ul of coupling buffer (50 mM MES ph 6.0) was added and mixed well.
4. 20Ul of EDC solution (200 mM), 20ul of sulfo-NHS solution (200 mM), were added, mixed well, and incubated on a rotating disk mixer for 30min.
5. Centrifuging (12000 rpm-20000rpm according to different particle sizes) for 10min, settling the microspheres, and removing the supernatant.
6. 500Ul of coupling buffer (50 mM MES ph 6.0) was added, and after mixing, 0.1mg of anti-cTnI monoclonal antibody was added, mixed, and incubated for 1h at room temperature using a rotating disk mixer.
7. Centrifuging (12000 rpm-20000rpm according to different particle sizes) for 10min, settling the microspheres, and removing the supernatant.
8. Blocking solution (1% BSA in water) was added, mixed well and incubated for 1h at room temperature using a rotating disk mixer.
9. Centrifuging (12000 rpm-20000rpm according to different particle sizes) for 10min, settling the microspheres, and removing the supernatant.
10. 1% Fluorescein isothiocyanate (FITC, aqueous solution) was added, mixed well and incubated with a rotating disc mixer for 1h at room temperature.
11. Centrifuging (12000 rpm-20000rpm according to different particle sizes) for 10min, settling the microspheres, and removing the supernatant.
12. 500Ul of preservative fluid (0.5% BSA,2% sucrose, tirs-HCl ph 8.0) was added and mixed well.
Example 3
Preparation of troponin I (cTnI) fluorescent chromatography detection test strip:
the fluorescent chromatography detection test strip comprises a substrate and a sample pad, a marker pad, a chromatographic membrane and a water absorption pad which are sequentially connected on the substrate, wherein the marker pad is a glass fiber pad, and the chromatographic membrane is a nitrocellulose membrane.
The substrate adopts a PVC plate, the pasting parts among the sample pad, the marker pad, the chromatographic membrane and the water absorption pad are overlapped with each other by 2mm, and the sample pad, the marker pad, the chromatographic membrane and the water absorption pad are assembled and chopped to form a test strip with the width of 4mm, and the test strip is filled into a clamping shell and packaged in an aluminum foil bag.
The method comprises the following specific steps:
1. Preparing a glass fiber pad: glass fiber is cut into 3 x 3cm specification, and a treatment solution (10% of blocking agent, 2% of sucrose, 0.5% of Tween 20, 1% of anti-erythrocyte antibody and 50mM of PBS ph 7.2) is sprayed on one side of the glass fiber by using a metal spraying instrument, wherein the spraying amount is 4ul/cm. And spraying a mixed solution (20:1) of the enhanced cTnI antibody-fluorescent microsphere conjugate and the rabbit IgG-fluorescent microsphere conjugate on the other side of the glass fiber, and drying at 45 ℃ for 16 hours.
2. Preparation of nitrocellulose membrane: another anti-cTnI antibody was diluted to 1mg/mL using a diluent (2% sucrose, 50mm PBS ph 7.2), streaked on one side of nitrocellulose membrane as a detection line; sheep anti-rabbit IgG was diluted to 0.5mg/mL with diluent (2% sucrose, 50mM PBS ph 7.2), streaked onto nitrocellulose membrane as a quality control line; the film-dividing amount is 1ul/cm, and the film is dried for 16 hours at 45 ℃.
3. And (3) sticking a water absorption pad, a prepared glass fiber pad, a nitrocellulose membrane and a test strip which is cut into a width of 4mm on a PVC substrate, loading into a clamping shell, and packaging into an aluminum foil bag.
Example 4
This embodiment differs from embodiment 3 only in that: the marker adopts a cTnI antibody-fluorescent microsphere conjugate.
The samples with 4 different cTnI concentrations and the cTnI detection test strips are restored to room temperature, each sample is divided into two groups, and the cTnI detection test strips in the embodiments 3 and 4 are respectively adopted for detection, and the specific detection method is as follows:
75ul of the sample was added to 200ul of diluent (50 mM PBS ph 7.2), mixed well, 75ul was added to the test strip sample pad, and after 15min, the result was read using a fluorescence detector.
The results are shown in FIG. 2 and the following table:
As can be seen from the above table and FIG. 2, the test strip of example 3 has a much higher detection sensitivity than the test strip of example 4. Namely, the sensitivity of the enhanced antibody-fluorescent microsphere conjugate for detecting cTnI is obviously higher than that of the traditional antibody-fluorescent microsphere conjugate.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (5)
1. A method for enhancing the signal of an antibody-fluorescent microsphere conjugate is characterized in that the antibody is conjugated with a fluorescent microsphere to obtain an antibody-fluorescent microsphere conjugate, and the antibody-fluorescent microsphere conjugate is conjugated with fluorescein to obtain the enhanced antibody-fluorescent microsphere conjugate;
Before the antibody-fluorescent microsphere conjugate is coupled with fluorescein, the empty space on the surface of the fluorescent microsphere is blocked by using inert protein, and the antibody on the surface of the fluorescent microsphere and available amino groups on the inert protein for blocking are coupled with the fluorescein;
the preparation method of the enhanced antibody-fluorescent microsphere conjugate comprises the following steps:
(1) Coupling the anti-cTnI monoclonal antibody with fluorescent microspheres to obtain a cTnI antibody-fluorescent microsphere conjugate;
(2) Coupling the cTnI antibody-fluorescent microsphere conjugate with fluorescein to obtain an enhanced cTnI antibody-fluorescent microsphere conjugate;
before the cTnI antibody-fluorescent microsphere conjugate is coupled with fluorescein, the surface vacancy of the fluorescent microsphere is blocked by using inert protein;
In the step (1), the preparation method of the cTnI antibody-fluorescent microsphere conjugate is as follows:
(11) Taking out the fluorescent microspheres, putting the fluorescent microspheres into a centrifuge tube, centrifuging, settling and removing supernatant;
(12) Adding a coupling buffer solution into the sediment, uniformly mixing, then adding an EDC solution and a sulfo-NHS solution, uniformly mixing, and incubating;
(13) Centrifuging the solution obtained in the step (12), settling, removing the supernatant, and adding a coupling buffer solution for uniform mixing;
(14) Adding anti-cTnI monoclonal antibody, mixing uniformly, and incubating at room temperature to obtain cTnI antibody-fluorescent microsphere conjugate;
in the step (2), the preparation method of the enhanced cTnI antibody-fluorescent microsphere conjugate is as follows:
(21) Centrifuging, settling and removing the supernatant of the cTnI antibody-fluorescent microsphere conjugate solution in the step (14), adding a sealing solution, uniformly mixing, and incubating at room temperature;
(22) Centrifuging after incubation, settling, removing supernatant, adding 1% fluorescein isothiocyanate, mixing, and incubating at room temperature;
(23) And finally, centrifuging, settling, removing the supernatant, and adding the preservation solution for uniform mixing.
2. The method of claim 1, wherein the fluorescein is fluorescein isothiocyanate and the inert protein is bovine serum albumin.
3. The method for enhancing an antibody-fluorescent microsphere conjugate signal according to claim 1, wherein cTnI detection is performed on a fluorescent chromatography test strip, the fluorescent chromatography test strip comprises a substrate and a sample pad, a marker pad, a chromatographic membrane and a water absorption pad which are sequentially connected on the substrate, the marker pad is a glass fiber pad, and the chromatographic membrane is a nitrocellulose membrane.
4. A method of enhancing an antibody-fluorescent microsphere conjugate signal according to claim 3, wherein the glass fiber mat is treated as follows:
Spraying the treatment liquid on one side of the glass fiber mat, wherein the spraying amount is 4ul/cm; and spraying the mixed solution of the enhanced cTnI antibody-fluorescent microsphere conjugate and the rabbit IgG-fluorescent microsphere conjugate on the other side of the glass fiber pad, and drying at 45 ℃.
5. The method of claim 4, wherein the nitrocellulose membrane is treated by:
diluting the other anti-cTnI antibody to 1mg/mL by using a diluent, and streaking one side of the nitrocellulose membrane to be used as a detection line; diluting goat anti-rabbit IgG to 0.5mg/mL by using a diluent, and marking on a nitrocellulose membrane as a quality control line; the film dividing amount is 1ul/cm, and the film is dried at 45 ℃.
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