CN111323576A - Method for enhancing signal of antibody-fluorescent microsphere conjugate and application of method in troponin I detection - Google Patents
Method for enhancing signal of antibody-fluorescent microsphere conjugate and application of method in troponin I detection Download PDFInfo
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- CN111323576A CN111323576A CN202010125215.1A CN202010125215A CN111323576A CN 111323576 A CN111323576 A CN 111323576A CN 202010125215 A CN202010125215 A CN 202010125215A CN 111323576 A CN111323576 A CN 111323576A
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Images
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
- 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
- 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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- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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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
- G01N33/68—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
Abstract
The invention discloses a method for enhancing a signal of an antibody-fluorescent microsphere conjugate, which comprises the steps of coupling an antibody and a fluorescent microsphere to obtain the antibody-fluorescent microsphere conjugate, and coupling the antibody-fluorescent microsphere conjugate with fluorescein to obtain an enhanced antibody-fluorescent microsphere conjugate. Before the antibody-fluorescent microsphere conjugate is coupled with fluorescein, the vacant sites on the surface of the fluorescent microspheres are sealed by inert protein. The method fully utilizes the protein carboxyl/amino on the surface of the fluorescent microsphere after the coupling is finished, and performs the 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 a signal of an antibody-fluorescent microsphere conjugate and application of the method in troponin I detection.
Background
The immunochromatography technology as an important branch of Point (instant detection) starts in the 80 th 20 th century, wherein the early pregnancy test paper becomes the most successful product with the characteristics of convenience, rapidness and low price, and the chromatography technology is also expanded to a plurality of detection fields, but compared with the methodologies of ELISA (enzyme linked immunosorbent assay) and chemiluminescence, the immunochromatography technology also has corresponding short plates, and mainly shows that the precision is poor and the sensitivity is insufficient; therefore, in the beginning of the twentieth century, manufacturers replace colloidal gold with fluorescent substances in order to improve the performance of the chromatographic products, the performance of the products is greatly improved, and the fluorescent chromatographic products gradually replace the colloidal gold to become the mainstream of the market. The early fluorescence chromatography uses fluorescein, which is gradually changed into fluorescent microspheres at present, and the fluorescent microspheres contain more fluorescent substances, so that fluorescent signals can be effectively amplified, and the detection sensitivity is improved. In current fluorescent chromatography studies, the key to further improve product performance is to increase the fluorescence signal of the conjugate again.
The current antibody-fluorescent microsphere coupling generally adopts chemical coupling, firstly, a microsphere surface group (generally carboxyl) is activated, an antibody is added, an amino group of the antibody is combined with the activated carboxyl, the antibody is fixed on the microsphere surface, and then, an inert protein (generally BSA) is used for sealing the microsphere surface vacancy.
The sensitivity is determined by the intensity 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, the fluorescence intensity of one fluorescent microsphere can be obtained only by capturing a plurality of antibodies.
Cardiac troponin is a regulatory protein of cardiac contraction and is present on the thin filaments of cardiac contractile proteins. In the early stage of myocardial injury, myocardial cells are not necrotic, but the cell membrane is destroyed, and free form cTnI enters the interstitial space and flows back into the blood via lymph. The cTnI of the serum rises at 6h, then myofibrils are disintegrated and destroyed continuously, the cTnI of the fixed form is released continuously, the serum level reaches the peak at 18-24h, and the level drops to normal after 10 days. Troponin i (ctni) has high myocardial specificity and sensitivity, and is currently recognized as a more ideal myocardial infarction (AMI) marker. cTnI is now entering the hypersensitive era for more accurate prediction of cardiovascular risk, placing higher demands 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 protein carboxyl/amino on the surface of a 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.
The invention is realized by the following technical scheme:
a method for enhancing a signal of an antibody-fluorescent microsphere conjugate comprises the steps of coupling an antibody and a fluorescent microsphere to obtain the antibody-fluorescent microsphere conjugate, and coupling the antibody-fluorescent microsphere conjugate with fluorescein to obtain an enhanced antibody-fluorescent microsphere conjugate. Before the antibody-fluorescent microsphere conjugate is coupled with fluorescein, the vacant sites on the surface of the fluorescent microspheres are sealed by inert protein.
The key indicator of fluorescence chromatography is sensitivity, which is determined by the amount of fluorescent material. The current antibody-fluorescent microsphere coupling generally adopts chemical coupling, firstly, a microsphere surface group (generally carboxyl) is activated, an antibody is added, an amino group of the antibody is combined with the activated carboxyl, the antibody is fixed on the microsphere surface, and then, an inert protein (generally BSA) is used for sealing the microsphere surface vacancy. The inventors have found that after the coupling is complete, the fluorescence of the conjugate is derived only from the fluorescent microspheres themselves, with the proteins (consisting of antibody and BSA) distributed throughout the surface of the microspheres, and the large number of amino/carboxyl groups contained in these proteins is not fully utilized.
After the antibody and the fluorescent microsphere are coupled, the antibody on the surface of the fluorescent microsphere and a large number of available amino groups (-NH2) on the protein for blocking are coupled 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.
The application of the method for enhancing the signal of the antibody-fluorescent microsphere conjugate in troponin I detection comprises the following steps:
(1) coupling the anti-cTnI monoclonal antibody with a fluorescent microsphere to obtain a cTnI antibody-fluorescent microsphere conjugate;
(2) and coupling the cTnI antibody-fluorescent microsphere conjugate with fluorescein to obtain an enhanced cTnI antibody-fluorescent microsphere conjugate.
Wherein, in the step (2), before the cTnI antibody-fluorescent microsphere conjugate is coupled with the fluorescein, the vacant sites on the surface of the fluorescent microspheres are sealed by inert protein.
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, placing the fluorescent microspheres into a centrifugal tube for centrifugation, 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 in the step (12), settling, removing supernatant, adding a coupling buffer solution, and uniformly mixing;
(14) adding the anti-cTnI monoclonal antibody, mixing uniformly, and incubating at room temperature to obtain the 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 and settling the cTnI antibody-fluorescent microsphere conjugate solution obtained in the step (14), removing supernatant, adding confining liquid, mixing uniformly, and incubating at room temperature;
(22) centrifuging after incubation is finished, settling, removing supernatant, adding 1% fluorescein isothiocyanate, mixing uniformly, and incubating at room temperature;
(23) and finally, centrifuging, settling, removing supernatant, adding the preservation solution, and uniformly mixing.
Furthermore, the cTnI detection is carried out on a fluorescence chromatography detection test strip, the fluorescence 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, 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 treating fluid on one side of the glass fiber pad, wherein the spraying amount is 4 ul/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 was treated as follows:
diluting another 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 scratching 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 (-NH2) on the cTnI antibody on the surface of the fluorescent microsphere and bovine serum albumin 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 sensitivity of the reagent and the detection sensitivity are improved.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. after the antibody and the fluorescent microsphere are coupled, the antibody on the surface of the fluorescent microsphere and a large number of available amino groups (-NH2) on protein for blocking are coupled 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;
2. the invention relates to an application of a method for enhancing antibody-fluorescent microsphere conjugate signals in troponin I detection, which comprises the steps of coupling a marked cTnI antibody-fluorescent microsphere complex with fluorescein, fully utilizing a large amount of available amino (-NH2) on the cTnI antibody on the surface of a fluorescent microsphere and bovine serum albumin, and obtaining an enhanced cTnI antibody-fluorescent microsphere complex, so that one fluorescent microsphere can carry more fluorescent substances, the intensity of fluorescent signals on a detection line is greatly improved, and the sensitivity of a reagent and the detection sensitivity are improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 is a schematic diagram of an enhanced cTnI antibody-fluorescent microsphere conjugate of the present invention, in which
Antibody, BSA bovine serum albumin, FITC fluorescein isothiocyanate.
FIG. 2 is a diagram showing the detection result of cTnI according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example 1
The invention relates to an application of a method for enhancing a signal of an antibody-fluorescent microsphere conjugate in troponin I detection, wherein the preparation method of the enhanced cTnI antibody-fluorescent microsphere conjugate comprises the following steps:
1. 500ul of fluorescent microspheres (1% W/V, green fluorescence-excitation 475 nM-emission 525nM) were taken out and placed in a centrifuge tube.
2. Centrifuging (12000-20000 rpm according to different particle sizes) for 10min to allow the microspheres to settle, and removing supernatant.
3. 500ul of coupling buffer (50mM MESph 6.0) was added and mixed well.
4. 20ul EDC solution (200mM), 20ul sulfo-NHS solution (200mM) were added, mixed and incubated on a rotating disk mixer for 30 min.
5. Centrifuging (12000-20000 rpm according to different particle sizes) for 10min to allow the microspheres to settle, and removing supernatant.
6. 500ul of coupling buffer (50mM MESph 6.0) was added, mixed well, 0.1mg of anti-cTnI monoclonal antibody was added, mixed well, and incubated for 1h at room temperature with a turntable mixer.
7. Centrifuging (12000-20000 rpm according to different particle sizes) for 10min to allow the microspheres to settle, and removing supernatant.
8. Add blocking solution (1% BSA in water), mix well, incubate for 1h at room temperature with a turntable mixer.
9. Centrifuging (12000-20000 rpm according to different particle sizes) for 10min to allow the microspheres to settle, and removing supernatant.
10. Add 1% fluorescein isothiocyanate (FITC, aq), mix well and incubate with a turntable mixer for 1h at room temperature.
11. Centrifuging (12000-20000 rpm according to different particle sizes) for 10min to allow the microspheres to settle, and removing supernatant.
12. 500ul of preservation solution (0.5% BSA, 2% sucrose, Tirs-HCl ph 8.0) was added and mixed well, and the product was shown in FIG. 1.
Example 2
The invention relates to an application of a method for enhancing a signal of an antibody-fluorescent microsphere conjugate in troponin I detection, wherein the preparation method of the enhanced cTnI antibody-fluorescent microsphere conjugate comprises the following steps:
1. 500ul of fluorescent microspheres (1% W/V, green fluorescence-excitation 475 nM-emission 525nM) were taken out and placed in a centrifuge tube.
2. Centrifuging (12000-20000 rpm according to different particle sizes) for 10min to allow the microspheres to settle, and removing supernatant.
3. 500ul of coupling buffer (50mM MESph 6.0) was added and mixed well.
4. 20ul EDC solution (200mM), 20ul sulfo-NHS solution (200mM) were added, mixed and incubated on a rotating disk mixer for 30 min.
5. Centrifuging (12000-20000 rpm according to different particle sizes) for 10min to allow the microspheres to settle, and removing supernatant.
6. 500ul of coupling buffer (50mM MESph 6.0) was added, mixed well, 0.1mg of anti-cTnI monoclonal antibody was added, mixed well, and incubated for 1h at room temperature with a turntable mixer.
7. Centrifuging (12000-20000 rpm according to different particle sizes) for 10min to allow the microspheres to settle, and removing supernatant.
8. Add blocking solution (1% BSA in water), mix well, incubate for 1h at room temperature with a turntable mixer.
9. Centrifuging (12000-20000 rpm according to different particle sizes) for 10min to allow the microspheres to settle, and removing supernatant.
10. Add 1% fluorescein isothiocyanate (FITC, aq), mix well and incubate with a turntable mixer for 1h at room temperature.
11. Centrifuging (12000-20000 rpm according to different particle sizes) for 10min to allow the microspheres to settle, and removing supernatant.
12. Adding 500ul of preservation solution (0.5% BSA, 2% sucrose, Tirs-HCl ph 8.0), and mixing.
Example 3
Preparation of troponin I (cTnI) fluorescence chromatography detection test strip:
the fluorescence 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 base plate adopts a PVC plate, the pasting parts among the sample pad, the marker pad, the chromatographic membrane and the water absorption pad are overlapped for 2mm, the sample pad, the marker pad, the chromatographic membrane and the water absorption pad are assembled and cut into test strips with the width of 4mm, and the test strips are loaded into a card shell and packaged in an aluminum foil bag.
The method comprises the following specific steps:
1. preparing a glass fiber mat: glass fibers were cut to a 3 × 3cm format, and the treatment fluid (10% blocking agent, 2% sucrose, 200.5% tween, 1% anti-erythrocyte antibody, 50mM PBS ph7.2) was sprayed onto one side of the glass fibers using a metal spraying instrument at a spray rate of 4 ul/cm. And (3) spraying the 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 for 16h at 45 ℃.
2. Preparation of nitrocellulose membrane: diluting another anti-cTnI antibody to 1mg/mL by using a diluent (2% sucrose, 50mM PBSph7.2), and scribing on one side of the nitrocellulose membrane to serve as a detection line; goat anti-rabbit IgG was diluted to 0.5mg/mL using diluent (2% sucrose, 50mM PBS ph7.2) and streaked on nitrocellulose membrane as a quality control line; the scratching amount is 1ul/cm, and the film is dried for 16h at the temperature of 45 ℃.
3. A water absorption pad, a prepared glass fiber pad and a nitrocellulose membrane are stuck on a PVC substrate, cut into test strips with the width of 4mm, put into a card shell and packaged into an aluminum foil bag.
Example 4
This example differs from example 3 only in that: the label is cTnI antibody-fluorescent microsphere conjugate.
The method comprises the following steps of (1) recovering 4 samples with different cTnI concentrations and a cTnI detection test strip to room temperature, dividing each sample into two groups, and respectively adopting the cTnI detection test strips of the embodiments 3 and 4 to detect, wherein the specific detection method comprises the following steps:
adding 75ul of the sample into 200ul of diluent (50mM PBS ph7.2), mixing, adding 75ul of the sample into a test strip sample pad, and reading the result by using a fluorescence detector after 15 min.
The results are shown in figure 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 enhanced antibody-fluorescent microsphere conjugate is adopted to detect cTnI, and the sensitivity is obviously higher than that of the traditional antibody-fluorescent microsphere conjugate.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for enhancing a signal of an antibody-fluorescent microsphere conjugate is characterized in that the antibody and the fluorescent microsphere are coupled to obtain the antibody-fluorescent microsphere conjugate, and the antibody-fluorescent microsphere conjugate is coupled with fluorescein to obtain the enhanced antibody-fluorescent microsphere conjugate.
2. The method of claim 1, wherein the surface vacancies of the fluorescent microspheres are blocked with an inert protein before the antibody-fluorescent microsphere conjugate is conjugated with fluorescein.
3. The application of the method for enhancing the signal of the antibody-fluorescent microsphere conjugate in troponin I detection is characterized by comprising the following steps:
(1) coupling the anti-cTnI monoclonal antibody with a fluorescent microsphere to obtain a cTnI antibody-fluorescent microsphere conjugate;
(2) and coupling the cTnI antibody-fluorescent microsphere conjugate with fluorescein to obtain an enhanced cTnI antibody-fluorescent microsphere conjugate.
4. The use of the method for enhancing the signal of the antibody-fluorescent microsphere conjugate in troponin I detection according to claim 3, wherein in step (2), the surface vacancies of the fluorescent microspheres are blocked by inert proteins before the cTnI antibody-fluorescent microsphere conjugate is conjugated with fluorescein.
5. The method for enhancing the signal of the antibody-fluorescent microsphere conjugate according to claim 4, wherein the fluorescein is fluorescein isothiocyanate, and the inert protein is bovine serum albumin.
6. The method for enhancing the signal of the antibody-fluorescent microsphere conjugate in the troponin I detection according to claim 4, wherein the cTnI antibody-fluorescent microsphere conjugate is prepared by the following steps in step (1):
(11) taking out the fluorescent microspheres, placing the fluorescent microspheres into a centrifugal tube for centrifugation, 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 in the step (12), settling, removing supernatant, adding a coupling buffer solution, and uniformly mixing;
(14) adding the anti-cTnI monoclonal antibody, mixing uniformly, and incubating at room temperature to obtain the cTnI antibody-fluorescent microsphere conjugate.
7. The use of the method for enhancing the signal of the antibody-fluorescent microsphere conjugate in troponin I detection according to claim 6, wherein in the step (2), the enhanced cTnI antibody-fluorescent microsphere conjugate is prepared by the following steps:
(21) centrifuging and settling the cTnI antibody-fluorescent microsphere conjugate solution obtained in the step (14), removing supernatant, adding confining liquid, mixing uniformly, and incubating at room temperature;
(22) centrifuging after incubation is finished, settling, removing supernatant, adding 1% fluorescein isothiocyanate, mixing uniformly, and incubating at room temperature;
(23) and finally, centrifuging, settling, removing supernatant, adding the preservation solution, and uniformly mixing.
8. The method for enhancing the signal of the antibody-fluorescent microsphere conjugate in the troponin I detection of claim 3, wherein the cTnI detection is performed on a fluorescence chromatography detection test strip, the fluorescence 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 with the substrate, the marker pad is a glass fiber pad, and the chromatographic membrane is a nitrocellulose membrane.
9. The method for enhancing the signal of the antibody-fluorescent microsphere conjugate according to claim 8, wherein the glass fiber pad is treated by the following steps:
spraying the treating fluid on one side of the glass fiber pad, wherein the spraying amount is 4 ul/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 ℃.
10. The use of the method for enhancing the signal of the antibody-fluorescent microsphere conjugate in the troponin I detection according to claim 9, wherein the nitrocellulose membrane is treated by:
diluting another 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 scratching amount is 1ul/cm, and the film is dried at 45 ℃.
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