CN115856289A - Kit for early diagnosis of myocardial infarction - Google Patents
Kit for early diagnosis of myocardial infarction Download PDFInfo
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- CN115856289A CN115856289A CN202211192148.0A CN202211192148A CN115856289A CN 115856289 A CN115856289 A CN 115856289A CN 202211192148 A CN202211192148 A CN 202211192148A CN 115856289 A CN115856289 A CN 115856289A
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Abstract
The invention provides a kit for early diagnosis of myocardial infarction. The kit comprises a base plate and test paper arranged on the base plate, wherein the test paper comprises a sample pad, a combination pad, a reaction pad and a water absorption pad, two ends of the combination pad are respectively overlapped with the sample pad and the reaction pad, the reaction pad and the water absorption pad are overlapped, the combination pad is coated with ferrite colloid probe solution marked by an H-FABP monoclonal antibody and ferrite colloid probe solution marked by an hs-cTnI monoclonal antibody, the reaction pad comprises a nitrocellulose membrane, and H-FABP detection bands, an hs-cTnI detection band and a quality control band which are sequentially arranged on the nitrocellulose membrane in parallel at intervals, and the H-FABP detection band, the hs-cTnI detection band and the quality control band are respectively obtained by spray-printing anti-H-FABP polyclonal antibody solution, anti-hs-cTnI polyclonal antibody solution and goat anti-mouse IgG solution on the nitrocellulose membrane. The invention carries out joint synchronous detection on H-FABP and hs-cTnI by optimizing the detection marker, shortens the detection time and improves the accuracy and precision.
Description
Technical Field
The invention belongs to the technical field of in-vitro reagent detection, and particularly relates to a kit for early diagnosis of myocardial infarction.
Background
Acute myocardial infarction is acute in morbidity, high in lethality rate (more than 30 percent) and serious in sequelae, is one of diseases with the highest morbidity and lethality rate in the world, and seriously threatens the life health of human beings. Early diagnosis and early treatment are the key for improving the cure rate of the disease, the early effective treatment can cause the cardiac muscle to be damaged minimally, the fatality rate and the disability rate are greatly reduced, and the survival rate and the prognosis effect of a patient are improved. The research shows that: if acute myocardial infarction patients can be effectively treated within two hours after the attack, the fatality rate can be reduced to 5.6 percent; if it exceeds 4 hours, the lethality rises to 10.3%.
At present, the commonly used rapid detection kits mainly comprise an ELISA detection kit, a CLIA detection kit, a colloidal gold detection kit and the like. The ELISA detection kit has high sensitivity, but needs complicated operations such as separation, washing and the like, and takes a long time (more than 4 hours). The CLIA detection kit has high detection sensitivity, wide detection range and high detection speed (about 75 minutes), but needs a precise and expensive immunoassay system and professional laboratory technicians, is not suitable for rapid and timely detection in an emergency room or a primary hospital, and is more not favorable for self-detection outside the hospital of a patient. Although the colloidal gold detection kit is simple and convenient to operate and rapid (about 15 minutes), and does not need special instruments and equipment, the key component of the colloidal gold detection kit, namely colloidal gold, is complex to prepare and expensive, and is seriously dependent on foreign import, so that a large neck clamping risk exists. Meanwhile, common detection markers, such as Creatine Kinase (CK), creatine kinase isoenzyme (CK-MB), lactate Dehydrogenase (LD), cardiac troponin (cTn), glycogen phosphorylase isoenzyme BB (GPBB) and the like, can be released into the blood of a patient after 2 hours of attack, so that the detection of patients suffering from acute myocardial infarction is difficult to realize by the conventional means within two hours after the attack.
Therefore, a new detection means is needed to shorten the detection time and realize effective detection of the patient two hours after the disease.
Disclosure of Invention
The invention aims to provide a kit for early diagnosis of myocardial infarction, which uses ferrite colloid as a probe, can detect H-FABP or perform combined synchronous detection on the H-FABP and hs-cTnI, has the detection time of 10-30 minutes, greatly shortens the detection time, and has the advantages of high detection efficiency, high accuracy, high precision and the like.
In order to achieve the above object, the present invention provides a kit for early diagnosis of myocardial infarction, the kit comprising a base plate and a test paper disposed on the base plate, the test paper comprising a sample pad, a conjugate pad, a reaction pad and a water absorbent pad sequentially disposed along a first direction, both ends of the conjugate pad being respectively overlapped with a second end of the sample pad and a first end of the reaction pad, and a second end of the reaction pad being overlapped with a first end of the water absorbent pad, wherein,
the preparation method of the combined pad comprises the following steps:
1) Mixing ferrite colloid with a mixed solution of EDC and NHS as activating reagents, reacting for 30-240 min at 10-35 ℃, and separating to obtain functional ferrite colloid;
2) Respectively carrying out coupling reaction on an H-FABP monoclonal antibody solution and an hs-cTnI monoclonal antibody solution and the functional ferrite colloid to obtain an H-FABP monoclonal antibody marked ferrite colloid probe and an hs-cTnI monoclonal antibody marked ferrite colloid probe, and respectively adding the prepared H-FABP monoclonal antibody marked ferrite colloid probe and the prepared hs-cTnI monoclonal antibody marked ferrite colloid probe into a PBS solution to obtain an H-FABP monoclonal antibody marked ferrite colloid probe solution and an hs-cTnI monoclonal antibody marked ferrite colloid probe solution;
3) Providing a raw material combination pad and carrying out pretreatment to obtain a pretreated combination pad, firstly, uniformly coating a ferrite colloid probe solution marked by an H-FABP monoclonal antibody on the pretreated combination pad to form a first layer, then coating a ferrite colloid probe solution marked by an hs-cTnI monoclonal antibody on the first layer to form a second layer, and drying to obtain the combination pad;
the reaction pad comprises a nitrocellulose membrane, and H-FABP detection bands, hs-cTnI detection bands and quality control bands which are arranged on the nitrocellulose membrane in parallel at intervals along the first direction, wherein the H-FABP detection bands are positioned on one side close to the combination pad, the quality control bands are positioned on one side close to the water absorption pad, the hs-cTnI detection bands are positioned between the H-FABP detection bands and the quality control bands, and the H-FABP detection bands, the hs-cTnI detection bands and the quality control bands are obtained by respectively spray-printing an anti-H-FABP polyclonal antibody solution, an anti-hs-cTnI polyclonal antibody solution and a goat anti-mouse IgG solution on the nitrocellulose membrane.
In a specific embodiment, the temperature of the coupling reaction is 1-20 ℃, and the time of the coupling reaction is 3-24h.
In a specific embodiment, the mass ratio of the functionalized ferrite colloid to the H-FABP monoclonal antibody is 1: (0.05-20), wherein the mass concentration of the H-FABP monoclonal antibody solution is 0.1-1.0 mg/mL; the mass ratio of the functionalized ferrite colloid to the hs-cTnI monoclonal antibody is 1: (0.05-20), and the mass concentration of the hs-cTnI monoclonal antibody solution is 0.1-1.0 mg/mL.
In a specific embodiment, the anti-H-FABP polyclonal antibody solution, the anti-hs-cTnI polyclonal antibody solution and the goat anti-mouse IgG solution are all 0.1-5.0 mg/mL in mass concentration, and the jet printing amount is 0.1-5.0. Mu.L/cm.
In a specific embodiment, the sample pad is a phosphate buffer treated sample pad.
In a specific embodiment, the step of processing the sample pad comprises soaking and drying sequentially, wherein the soaking solution is 10mmol/L phosphate buffer solution, the soaking temperature is 20-40 ℃, and the soaking time is 30-240 min; the drying temperature is 30-40 ℃.
The invention also provides a kit for early diagnosis of myocardial infarction, which comprises a bottom plate and test paper arranged on the bottom plate, wherein the test paper comprises a sample pad, a combination pad, a reaction pad and a water absorption pad which are sequentially arranged along a first direction, two ends of the combination pad are respectively overlapped with a second end of the sample pad and a first end of the reaction pad, a second end of the reaction pad is overlapped with a first end of the water absorption pad, wherein,
the preparation method of the combined pad comprises the following steps:
1) Mixing ferrite colloid with a mixed solution of EDC and NHS as activating reagents, reacting for 30-240 min at 10-35 ℃, and separating to obtain functional ferrite colloid;
2) Carrying out coupling reaction on the H-FABP monoclonal antibody solution and the functional ferrite to obtain a ferrite probe marked by the H-FABP monoclonal antibody, and adding the prepared ferrite probe marked by the H-FABP monoclonal antibody into the PBS solution to obtain a ferrite probe solution marked by the H-FABP monoclonal antibody;
3) Providing a raw material combination pad, pretreating to obtain a pretreated combination pad, uniformly coating a ferrite colloid probe solution marked by an H-FABP monoclonal antibody on the pretreated combination pad, and drying to obtain the combination pad;
the reaction pad comprises a nitrocellulose membrane, and an H-FABP detection band and a quality control band which are arranged on the nitrocellulose membrane at intervals in parallel along the first direction, wherein the H-FABP detection band is positioned at one side close to the combination pad, the quality control band is positioned at one side close to the water absorption pad, and the H-FABP detection band and the quality control band are respectively obtained by spray-printing an anti-H-FABP polyclonal antibody solution and a goat anti-mouse IgG solution on the nitrocellulose membrane.
In a specific embodiment, the temperature of the coupling reaction is 1-20 ℃, and the time of the coupling reaction is 3-24h.
In a specific embodiment, the mass ratio of the functionalized ferrite colloid to the H-FABP monoclonal antibody is 1: (0.05-20), wherein the mass concentration of the H-FABP monoclonal antibody solution is 0.1-1.0 mg/mL.
In a specific embodiment, the mass concentration of the anti-H-FABP polyclonal antibody and the mass concentration of the goat anti-mouse IgG solution are both 0.1-5.0 mg/mL, and the jet printing amount is both 0.1-5.0 μ L/cm.
The beneficial effects of the invention at least comprise:
1. the invention optimizes the detection marker, takes the ferrite colloid probe marked by the H-FABP monoclonal antibody and the ferrite colloid probe marked by the hs-cTnI monoclonal antibody as probes, carries out combined synchronous detection on the H-FABP and the hs-cTnI, has the detection time of 10-30 minutes, greatly shortens the detection time, has the advantages of high detection efficiency, high accuracy, high precision and the like, can realize effective detection of acute myocardial infarction within two hours after a disease, enables the patient with the acute myocardial infarction to be effectively treated within a golden time window for treatment, greatly improves the cure rate and treatment effect, and greatly reduces the fatality rate and disability rate.
2. The detection marker is optimized, the ferrite probe marked by the H-FABP monoclonal antibody is used as the probe, the H-FABP is detected, the detection time is 10-30 minutes, the detection time is greatly shortened, and the method has the advantages of high detection efficiency, high accuracy, high precision and the like, and can realize effective detection of acute myocardial infarction within two hours after diseases.
3. The invention uses ferrite as a probe, integrates the dual functions of horseradish peroxidase catalysis and colloidal gold color development, does not need to use expensive materials such as horseradish peroxidase, biotin, colloidal gold and the like, has the dual advantages of convenience and rapidness of a colloidal gold detection kit and accuracy and reliability of a CLIA detection kit, does not need special detection equipment and technicians in the detection process, is convenient to operate, and is particularly suitable for testing in emergency departments, primary hospitals and families.
Drawings
Fig. 1 is a schematic perspective view of a kit for early diagnosis of myocardial infarction according to an embodiment of the present invention;
FIG. 2 is a transmission electron microscope image of the H-FABP monoclonal antibody labeled ferrite probe prepared in example 2;
FIG. 3 is a transmission electron microscope image of the hs-cTnI monoclonal antibody labeled ferrite colloid probe prepared in example 2;
FIG. 4 is a graph showing the test results of the kit prepared in example 2;
fig. 5 is a schematic perspective view of a kit for early diagnosis of myocardial infarction according to another embodiment of the present invention;
FIG. 6 is a transmission electron micrograph of the H-FABP monoclonal antibody-labeled ferrite probe prepared in example 8;
FIG. 7 is a graph showing the test results of the kit prepared in example 8.
Detailed Description
Example 1
Referring to fig. 1, the present invention provides a kit 100 for early diagnosis of myocardial infarction, where the kit 100 includes a bottom plate 11 and a test paper 12 disposed on the bottom plate 11, the test paper 12 includes a sample pad 121, a combination pad 122, a reaction pad 123 and a water absorption pad 124, which are sequentially disposed along a first direction, two ends of the combination pad 122 are respectively disposed to overlap a second end of the sample pad 121 and a first end of the reaction pad 123, and a second end of the reaction pad 123 is disposed to overlap a first end of the water absorption pad 124.
In this embodiment, the first direction refers to a direction from left to right as shown in fig. 1, it should be noted that, here, the left and right are relative concepts, and when the placing position of the reagent kit 100 is adjusted, the first direction is correspondingly adjusted.
Referring to fig. 1, first ends of the sample pad 121, the conjugate pad 122, the reaction pad 123, and the absorbent pad 124 are all at one end on the left side, and second ends of the sample pad 121, the conjugate pad 122, the reaction pad 123, and the absorbent pad 124 are all at one end on the right side.
It should be noted that the overlapping arrangement is not limited to the upper and lower relationship, for example, the second end of the reaction pad 123 is overlapped with the first end of the water absorption pad 124, that is, the second end of the reaction pad 123 is located on the upper side of the water absorption pad 124, and the orthographic projection of the second end of the reaction pad 123 to the water absorption pad 124 is overlapped with the first end of the water absorption pad 124; the first end of the absorbent pad 124 may be located on the upper side of the reaction pad, and the orthographic projection of the first end of the absorbent pad 124 to the reaction pad 123 coincides with the second end of the reaction pad 123.
In this embodiment, the second end of the sample pad 121 is located above the first end of the conjugate pad 121, the second end of the conjugate pad 121 is located above the first end of the reaction pad 123, and the first end of the absorbent pad 124 is located above the second end of the reaction pad 123.
The terms of orientation such as upper and lower are used herein with respect to the terms of orientation shown in fig. 1.
In this embodiment, the bottom plate 11 functions as a support for the test paper 12, and the bottom plate 11 is a PVC plate. The sample pad 121, the bonding pad 122, the reaction pad 123 and the absorbent pad 124 are assembled together to form the test paper 12, which is disposed on the base plate 10 and fixedly connected to the base plate 10.
The sample pad 21 is a pretreated sample pad, and the pretreatment method comprises the following steps: soaking the sample pad in the sample pad treatment solution at 20-40 deg.C for 30-120min, drying in a drier at 30-40 deg.C, completely drying, and sealing in a self-sealing bag; wherein the sample pad treatment solution is a 10mM phosphate buffer solution.
The method of making the conjugate pad 22 includes the steps of:
in the present invention, the ferrite colloid is nano Fe 3 O 4 Dispersed in a liquid to form a stable colloidal solution, having horseradish peroxidase-like enzymatic activity, catalytic efficiency and mechanism of action similar to horseradish peroxidase (HRP). The ferrite colloid used in the invention is prepared by adopting the technical scheme of preparing the nano water-based magnetic fluid by a one-pot method disclosed in the patent application number CN201610072048.2, and the mass concentration of the prepared ferrite colloid is 0.05-5.0 mg/mL.
In the embodiment, the mass concentration of EDC in the mixed solution is 0.2-10 mg/mL, and the mass concentration of NHS is 0.5-25 mg/mL;
the volume ratio of the ferrite colloid to the mixed solution is 1:1.
in this embodiment, the preparation method of the mixed solution is as follows: adding 2-100 mg of EDC and 5-250 mg of NHS into 10mL of deionized water, mixing and dissolving to obtain the composition.
The step 1 also comprises the steps of cleaning and volume fixing, wherein the separated functional ferrite colloid is cleaned by distilled water and PBS (phosphate buffer solution) with the pH value of 6.5-8.5 for 5 times respectively, then the functional ferrite colloid reacts for 30min at room temperature, and then the PBS with the pH value of 6.5-8.5 is added to be fixed to 1mL to obtain the PBS (phosphate buffer solution) of the functional ferrite colloid, wherein the mass concentration of the functional ferrite colloid is 0.05-5.0 mg/mL, and preferably 0.1-1.0 mg/mL.
Step 2, respectively carrying out coupling reaction on the H-FABP monoclonal antibody solution and the hs-cTnI monoclonal antibody solution with the functional ferrite colloid to obtain an H-FABP monoclonal antibody marked ferrite colloid probe and an hs-cTnI monoclonal antibody marked ferrite colloid probe, and respectively adding the prepared H-FABP monoclonal antibody marked ferrite colloid probe and the hs-cTnI monoclonal antibody marked ferrite colloid probe into a PBS solution to obtain an H-FABP monoclonal antibody marked ferrite colloid probe solution and an hs-cTnI monoclonal antibody marked ferrite colloid probe solution;
preferably, the temperature of the coupling reaction is 1-20 ℃, and the time of the coupling reaction is 3-24h, and more preferably, the time of the coupling reaction is 9-15 h.
Specifically, the preparation method of the ferrite colloid probe marked by the H-FABP monoclonal antibody comprises the following steps: adding the functionalized ferrite colloid into the H-FABP monoclonal antibody solution, uniformly mixing, standing overnight at 1-20 ℃, separating and washing after coupling is finished, then transferring the mixture into Tris buffer solution, separating and washing again, adding the ferrite colloid probe marked by the H-FABP monoclonal antibody into 1.2mL of PBS solution (containing 5 percent BSA) with the pH value of 6.2-8.2, and uniformly mixing for later use.
The mass ratio of the functionalized ferrite colloid to the H-FABP monoclonal antibody is 1: (0.05-20).
Preferably, the mass concentration of the H-FABP monoclonal antibody solution is 0.1-1.0 mg/mL.
In this embodiment, the mass concentration of the functionalized ferrite colloid is 0.05 to 5.0mg/mL, preferably 0.1 to 1.0mg/mL.
The preparation method of the ferrite colloid probe marked by the hs-cTnI monoclonal antibody comprises the following steps: adding the functionalized ferrite colloid into an hs-cTnI monoclonal antibody solution, uniformly mixing, standing overnight at 1-20 ℃, separating and washing after coupling is finished, then transferring into a Tris buffer solution, separating and washing again, adding the hs-cTnI monoclonal antibody marked ferrite colloid probe into 1.2mL of PBS solution (containing 5 percent BSA) with the pH value of 6.2-8.2, and uniformly mixing for later use.
The mass ratio of the functionalized ferrite colloid to the hs-cTnI monoclonal antibody is 1: (0.05-20).
Preferably, the mass concentration of the hs-cTnI monoclonal antibody solution is 0.1-1.0 mg/mL.
In this embodiment, the mass concentration of the functionalized ferrite colloid is 0.05 to 5.0mg/mL, preferably 0.1 to 1.0mg/mL.
Step 3, providing a raw material combination pad and carrying out pretreatment to obtain a pretreated combination pad, firstly, uniformly coating a ferrite colloid probe solution marked by an H-FABP monoclonal antibody on the pretreated combination pad to form a first layer, then coating a ferrite colloid probe solution marked by an hs-cTnI monoclonal antibody on the first layer to form a second layer, and drying to obtain the combination pad;
the pretreatment process of the raw material combination pad comprises the following steps: soaking the raw material bonding pad in 5% sucrose solution for 30min, and drying in a drier at 30 deg.C for 3-24 hr.
And (3) coating the ferrite colloid probe solution marked by the H-FABP monoclonal antibody and the ferrite colloid probe solution marked by the hs-cTnI monoclonal antibody on the binding pad, drying the binding pad at the temperature of 30 ℃ for 3-24 hours, completely drying, and then putting the binding pad into a self-sealing bag for sealing for later use.
The reaction pad 123 includes a nitrocellulose membrane 1231, and H-FABP detection bands 1232, hs-cTnI detection bands 1233, and a quality control band 1234 that are disposed in parallel on the nitrocellulose membrane at intervals in the first direction, the H-FABP detection band 1232 is located at a side close to the conjugate pad 122, the quality control band 1234 is located at a side close to the absorbent pad 124, the hs-cTnI detection band 1233 is located between the H-FABP detection band 1232 and the quality control band 1234, and the H-FABP detection bands 1232, hs-cTnI detection band 1233, and quality control band 1234 are obtained by respectively spray-printing an anti-H-FABP polyclonal antibody solution, an anti-hs-cTnI polyclonal antibody solution, and a goat anti-mouse IgG solution on the nitrocellulose membrane.
Preferably, the mass concentration of the anti-H-FABP polyclonal antibody solution is 0.1-5.0 mg/mL, and the jet printing amount is 0.1-5.0 muL/cm; the mass concentration of the anti-hs-cTnI polyclonal antibody solution is 0.1-5.0 mg/mL, and the jet printing amount is 0.1-5.0 muL/cm; the mass concentration of the goat anti-mouse IgG solution is 0.1-5.0 mg/mL, and the jet printing amount is 0.1-5.0 muL/cm.
The preparation method of the reaction pad comprises the following steps: pasting a nitrocellulose membrane on a bottom plate for membrane scribing, and then uniformly spraying and printing an anti-H-FABP polyclonal antibody solution (1 mg/mL), an anti-hs-cTnI polyclonal antibody solution (1 mg/mL) and a goat anti-mouse IgG solution (1 mg/mL) on the nitrocellulose membrane in a sub-region manner in an amount of 0.1-5.0 mu L/cm to respectively serve as an H-FABP detection zone, an hs-cTnI detection zone and a quality control zone; drying the nitrocellulose membrane at 35-40 deg.C for 1 hr, adding into 1% BSA solution, reacting at room temperature for 30-90min, and sealing; after the blocking, the nitrocellulose membrane was removed, washed 5 times with PBS buffer (pH 7.4), and dried at 35 ℃ for 3 hours after washing.
Assembling test boards: the sample pad 121, the combination pad 122, the reaction pad 123 and the absorbent pad 124 are adhered to the base plate 10, and then cut according to a preset width to obtain a test strip, which is the kit 100 for early diagnosis of myocardial infarction.
The chromatographic orientation of the kit 100 is from the sample pad 121 to the conjugate pad 122 to the reaction pad 123 to the bibulous pad 124.
The detection principle of the kit 100 is as follows:
when the sample to be detected contains the marker to be detected (H-FABP and/or hs-cTnI), the marker to be detected is firstly combined with the probe, moves under the action of chromatography, forms a compound when encountering a capture antibody and is enriched on a detection line, a precipitation line appears on the detection line, the excess probe continuously moves forwards and is combined with a secondary antibody of a quality control line, a quality control strip appears, and the qualitative detection is quickly carried out through color change. On the basis, the detection line substances are collected, a substrate is added for color reaction, and the light absorption value is measured after the reaction is finished, so that the quantitative detection can be realized.
The using method comprises the following steps: collecting the collected blood sample in a dry and clean container, placing the test strip on a table board, sucking 2 drops of blood sample by using a dropper, and dropping the blood sample on a sample pad. When the recording is started, the result is judged within 5-15 minutes.
The test results show that:
1. positive results: two or one black strip appears on the detection line, and a black strip appears on the quality control line, which indicates that H-FABP protein and hs-cTnI protein or one of the H-FABP protein and the hs-cTnI protein exist in the test sample and acute myocardial infarction occurs.
2. Negative results: only the quality control line shows a black band, and neither detection line shows a black band, which indicates that H-FABP protein and hs-cTnI protein do not exist in the test sample and the test sample does not suffer from acute myocardial infarction.
3. Invalid result: the absence of a black band in the control line indicates that this test is not effective.
Example 2
Functional modification of ferrite colloid: mixing and dissolving 2.0mg of EDC, 5.0mg of NHS and 10mL of deionized water to obtain a mixed solution; adding 1mL of mixed solution of EDC and NHS into 1mL of 1mg/mL ferrite colloid, and reacting at room temperature for 30min; separating, discarding supernatant, collecting functionalized ferrite colloid, washing with distilled water and PBS (pH 7.2) for 5 times, reacting at room temperature for 30min, adding PBS solution (pH 7.2) and diluting to 1mL.
Preparing a ferrite colloid probe marked by an FABP monoclonal antibody: adding 12 mu L of functional ferrite colloid (the mass concentration is 1 mg/mL) into an EP tube, adding 2 mu L of 0.4mg/mL H-FABP monoclonal antibody PBS solution (pH 7.2), uniformly mixing, and reacting for 8H at 4 ℃; separating, discarding supernatant, collecting the separated substance, washing with PBS solution (pH 7.2) for 5 times, and reacting in 50mM Tris buffer (pH 7.2) for 30min; after re-separation, the supernatant was discarded, and the collected probes were washed 5 times with PBS and added to 4. Mu.L of a PBS solution (containing 5% BSA) having a pH of 7.2.
The transmission electron micrograph of the FABP monoclonal antibody-labeled ferrite probe is shown in FIG. 2.
Preparing a ferrite colloid probe marked by an hs-cTnI monoclonal antibody: add 12. Mu.L of functionalized ferrite colloid into EP tube, add 2. Mu.L of 0.4mg/mL hs-cTnI monoclonal antibody PBS solution (pH 7.2), mix well, react for 8h at 4 ℃. Separating, discarding supernatant, collecting isolate, washing with PBS (pH 7.2) for 5 times, and reacting in 50mM Tris buffer solution (pH 7.2) for 30min; after separation again, the supernatant was discarded, and the collected probes were washed 5 times with PBS and added to 4. Mu.L of a PBS solution (containing 5% BSA) having a pH of 7.2.
FIG. 3 shows the transmission electron microscope image of the hs-cTnI monoclonal antibody labeled ferrite colloidal probe.
Preparation of sample pad, conjugate pad and reaction pad:
soaking the sample pad in 10mM phosphate buffer solution for 30min, and drying in a drier at 30 deg.C until completely dried;
soaking the bonding pad in 5% sucrose solution for 30min, drying in a 30 deg.C dryer, layering H-FABP monoclonal antibody labeled ferrite colloidal probe and hs-cTnI monoclonal antibody labeled ferrite colloidal probe in sequence, and uniformly coating on the bonding pad, and drying in the 30 deg.C dryer until completely dried;
pasting a nitrocellulose membrane on a bottom plate (a membrane of 30cm is pasted on the bottom plate) for membrane scratching, then uniformly spraying and printing an anti-H-FABP polyclonal antibody solution (1 mg/mL), an anti-hs-cTnI polyclonal antibody solution (1 mg/mL) and a goat anti-mouse IgG solution (1 mg/mL) on the nitrocellulose membrane to respectively serve as an H-FABP detection band, an hs-cTnI detection band and a quality control band (the spraying amount is 1 muL/cm), drying for 1 hour at 35 ℃, adding the mixture into a 1 BSA solution for room temperature reaction for 30min for sealing, taking out the nitrocellulose membrane after sealing, washing for 5 times by using a PBS buffer solution (pH 7.4), cleaning, and drying for 3 hours at 35 ℃ to obtain the reaction pad.
Assembling and cutting the test strip: and (3) alternately sticking a reaction pad of 3.0cm, a combination pad of 0.6cm, a sample pad of 1.8cm and a water absorption pad of 1.4cm on the bottom plate in a manner of 2.5mm in sequence, assembling the test paper plate, and cutting the assembled test paper plate into a width of 5mm by using a slitter to obtain the test paper strip.
Test strip assay
The test paper prepared in example 2 was used as an experimental subject, and 5 μ g/L of H-FABP solution (a), 0.2 μ g/L of hs-cTnI solution (B), and a mixed solution of H-FABP and hs-cTnI (C) with final concentrations of 5 μ g/L and 0.2 μ g/L, respectively, were prepared, and purified water was used as a control (D) to measure the detection performance of the test paper prepared in the present invention. The specific operation is as follows: placing the solution in a dry and clean container, placing the test strip on a table, sucking 2 drops of the solution by using a dropper, and dripping the solution on a test strip sample pad. After the completion of the dropwise addition, the mixture was left standing and started to be timed, and the results were observed for 15 minutes.
The test result of the test strip is shown in FIG. 4, T in FIG. 4 1 Is a H-FABP detection line, T 2 The test strip prepared in the embodiment 2 has a good detection effect on hs-cTnI and H-FABP after 15 minutes, and can generate dark detection strips on the detection line, and the detection line is specifically as follows: FIG. 4 is a detection diagram of the solution A, the solution B, the solution C and the solution D from left to right in sequence, wherein the H-FABP detection line and the quality control line in the solution A become deep, and H-FABP positivity is displayed; the hs-cTnI detection line and the quality control line in the solution B become deep, and the hs-cTnI is displayed to be positive; H-FABP detection line, hs-cTnI detection line and quality control line in the solution C are all deepened, and H-FABP and hs-cTnI double positive are displayed; the solution D only has a deepened quality control line, and no band appears on a detection line, thereby showing double negatives of H-FABP and hs-cTnI.
Examples 3 to 6
The preparation methods and measurement experiments of examples 3 to 6 were substantially the same as those of example 2, except that when the FABP monoclonal antibody-labeled ferrite probe and the hs-cTnI monoclonal antibody-labeled ferrite probe were prepared, the functionalized ferrite used in example 2 was 12. Mu.L, the functionalized ferrite used in example 3 was 16. Mu.L, the functionalized ferrite used in example 4 was 8. Mu.L, the functionalized ferrite used in example 5 was 4. Mu.L, and the functionalized ferrite used in example 6 was 0.6. Mu.L. The test results of the test strips prepared in examples 3 to 6 are shown in fig. 4.
Example 7
Referring to fig. 5, the present invention provides a kit 200 for early diagnosis of myocardial infarction, where the kit 200 includes a base plate 21 and a test paper 22 disposed on the base plate 21, the test paper 22 includes a sample pad 221, a combination pad 222, a reaction pad 223 and a water absorption pad 224, which are sequentially disposed along a first direction, two ends of the combination pad 222 are respectively disposed to overlap a second end of the sample pad 221 and a first end of the reaction pad 223, and a second end of the reaction pad 223 is disposed to overlap a first end of the water absorption pad 224.
In this embodiment, the first direction refers to a direction from left to right as shown in fig. 5, it should be noted that, of course, the left and right are relative concepts, and when the placement position of the reagent kit 200 is adjusted, the first direction is correspondingly adjusted.
Referring to fig. 1, first ends of the sample pad 221, the conjugate pad 222, the reaction pad 223, and the absorbent pad 224 are all ends located at the left side, and second ends of the sample pad 221, the conjugate pad 222, the reaction pad 223, and the absorbent pad 224 are all ends located at the right side.
It should be noted that, the overlapping arrangement is not limited to the upper and lower relationship, for example, the second end of the reaction pad 223 overlaps the first end of the water absorption pad 224, that is, the second end of the reaction pad 223 is located on the upper side of the water absorption pad 224, and the orthographic projection of the second end of the reaction pad 223 to the water absorption pad 224 coincides with the first end of the water absorption pad 224; the first end of the absorbent pad 224 may be located on the upper side of the reaction pad, and the orthographic projection of the first end of the absorbent pad 224 to the reaction pad 223 coincides with the second end of the reaction pad 223.
In this embodiment, the second end of the sample pad 221 is located above the first end of the binding pad 221, the second end of the binding pad 221 is located above the first end of the reaction pad 23, and the first end of the absorbent pad 224 is located above the second end of the reaction pad 223.
The terms of orientation such as upper and lower are used herein with respect to the terms of orientation shown in fig. 1.
In this embodiment, the bottom plate 21 functions to support the test paper sheet 22. The sample pad 221, the combination pad 222, the reaction pad 223 and the absorbent pad 224 are assembled together to form a test board 22 disposed on the bottom plate 21 and fixedly connected to the bottom plate 21.
The sample pad 221 is a pretreated sample pad, and the pretreatment method comprises the following steps: soaking the sample pad in the sample pad treatment solution at 20-40 deg.C for 30-120min, drying in a drier at 30-40 deg.C, completely drying, and sealing in a self-sealing bag; wherein the sample pad treatment solution is a 10mM phosphate buffer solution.
The method of making the conjugate pad 222 includes the steps of:
in this embodiment, the ferrite colloid is nano-Fe 3 O 4 Dispersed in a liquid to form a stable colloidal solution, having horseradish peroxidase-like enzymatic activity, catalytic efficiency and mechanism of action similar to horseradish peroxidase (HRP). The ferrite colloid used in the invention is prepared by adopting the technical scheme that the nano water-based magnetic fluid is prepared by a one-pot method disclosed in the patent application number of 201610072048.2, and the mass concentration of the prepared ferrite colloid is 0.05-5.0 mg/mL, preferably 0.1-1.0 mg/mL.
Preferably, the mass concentration of EDC in the mixed solution is 2-100 mg/mL, and the mass concentration of NHS is 5-250 mg/mL.
In this embodiment, the volume ratio of the ferrite colloid to the mixed solution is 1:1.
in this embodiment, the preparation method of the mixed solution is as follows: adding 2-100 mg of EDC and 5-250 mg of NHS into 10mL of deionized water, mixing and dissolving to obtain the composition.
The step 1 also comprises the steps of cleaning and volume fixing, wherein the separated functional ferrite colloid is cleaned for 5 times by distilled water and PBS solution with the pH value of 6.5-8.5 respectively, then the reaction is carried out for 30min at room temperature, and then the PBS solution with the pH value of 6.2-8.2 is added to fix the volume to 1mL.
Step 2, respectively carrying out coupling reaction on the H-FABP monoclonal antibody solution and the hs-cTnI monoclonal antibody solution with the functional ferrite colloid to obtain an H-FABP monoclonal antibody marked ferrite colloid probe and an hs-cTnI monoclonal antibody marked ferrite colloid probe, and respectively adding the prepared H-FABP monoclonal antibody marked ferrite colloid probe and the hs-cTnI monoclonal antibody marked ferrite colloid probe into a PBS solution to obtain an H-FABP monoclonal antibody marked ferrite colloid probe solution and an hs-cTnI monoclonal antibody marked ferrite colloid probe solution;
preferably, the temperature of the coupling reaction is 1-20 ℃, and the time of the coupling reaction is 3-24h, and more preferably, the time of the coupling reaction is 9-15 h.
Specifically, the preparation method of the ferrite colloid probe marked by the H-FABP monoclonal antibody comprises the following steps: adding the functional ferrite colloid into a H-FABP monoclonal antibody solution, uniformly mixing, standing overnight at 1-20 ℃, separating and washing after coupling is finished, then transferring the mixture into a Tris buffer solution, separating and washing again, adding the ferrite colloid probe marked by the H-FABP monoclonal antibody into 1.0mL of PBS solution (containing 5 percent BSA) with the pH value of 6.2-8.2, and uniformly mixing for later use.
The mass ratio of the functionalized ferrite colloid to the H-FABP monoclonal antibody is 1: (0.0.5-20).
In this example, the H-FABP monoclonal antibody solution has a mass concentration of 0.1 to 1.0mg/mL.
Step 3, providing a raw material combination pad, carrying out pretreatment to obtain a pretreated combination pad, uniformly coating a ferrite colloid probe solution marked by an H-FABP monoclonal antibody on the pretreated combination pad, and drying to obtain the combination pad;
the pretreatment process of the raw material combination pad comprises the following steps: soaking the raw material bonding pad in 5% sucrose solution for 30min, and drying in a drier at 30 deg.C for 3-24 hr.
Drying the binding pad coated with the H-FABP monoclonal antibody labeled ferrite colloidal probe solution at 30 ℃ for 3-24H, completely drying, and sealing in a self-sealing bag for later use.
The reaction pad 223 comprises a nitrocellulose membrane 2231, and H-FABP detection bands 2232 and quality control bands 2233 which are sequentially arranged on the nitrocellulose membrane 2231 in parallel at intervals along the first direction, wherein the H-FABP detection bands 2232 are located on one side close to the combination pad 222, the quality control bands 2233 are located on one side close to the absorbent pad 224, and the H-FABP detection bands and the quality control bands are obtained by respectively spraying and printing an anti-H-FABP polyclonal antibody solution and a goat anti-mouse IgG solution on the nitrocellulose membrane.
Preferably, the mass concentration of the anti-H-FABP polyclonal antibody solution is 0.1-5.0 mg/ml, and the jet printing amount is 0.1-5.0 muL/cm; the mass concentration of the goat anti-mouse IgG solution is 0.1-5.0 mg/ml, and the jet printing amount is 0.1-5.0 muL/cm.
The preparation method of the reaction pad comprises the following steps: pasting a nitrocellulose membrane on a bottom plate for membrane scratching, and then spraying and printing an anti-H-FABP polyclonal antibody solution (0.1-5.0 mg/mL) and a goat anti-mouse IgG solution (0.1-5.0 mg/mL) on the nitrocellulose membrane in a uniform amount of 0.1 muL/cm-5.0 muL/cm in different areas to respectively serve as an H-FABP detection band and a quality control band; drying the nitrocellulose membrane at 35-40 deg.C for 1 hr, adding into 1% BSA solution, reacting at room temperature for 30-90min, and sealing; after the blocking, the nitrocellulose membrane was removed, washed 5 times with PBS buffer (pH 7.4), and dried at 35 deg.C for 2-3 hours.
Assembling test boards: the sample pad 221, the combination pad 222, the reaction pad 223 and the absorbent pad 224 are adhered to the base plate 10, and then cut according to a preset width to obtain a test strip, which is the kit 200 for early diagnosis of myocardial infarction.
The direction of the flow of the kit 200 is from the sample pad 221 to the conjugate pad 222 to the reaction pad 223 to the absorbent pad 224.
The detection principle of the kit 200 is as follows:
when a sample to be detected contains a marker (H-FABP 2) to be detected, the marker to be detected is firstly combined with the probe and moves under the action of chromatography, when a capture antibody is encountered, a complex is formed and enriched on a detection line, a precipitation line appears on the detection line, the surplus probe continuously moves forwards and is combined with a secondary antibody of a quality control line, a quality control strip appears, and rapid qualitative detection is carried out through color change. On the basis, the detection line substances are collected, a substrate is added for color reaction, and the light absorption value is measured after the reaction is finished, so that the quantitative detection can be realized.
The using method comprises the following steps: collecting the collected blood sample in a dry and clean container, placing the test strip on a table board, sucking 2 drops of blood sample by using a dropper, and dropping the blood sample on a sample pad. The time is started to be recorded, and the result is judged within 5-15 minutes.
The test results show that:
1. positive results: the appearance of two black bands, both the examination line and the quality control line, indicates the presence of H-FABP protein in the test sample and acute myocardial infarction.
2. Negative results: only the quality control line has a black band, and the detection line has no black band, which indicates that no H-FABP protein exists in the test sample and the test sample has no acute myocardial infarction.
3. Invalid result: the control line shows no black band, indicating that the test is invalid.
Example 8:
functional modification of ferrite colloid: 20mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide), 50mg of NHS (N-hydroxysuccinimide) and 10mL of deionized water were mixed and dissolved to obtain a mixed solution. Adding 1mL of mixed solution of EDC and NHS into 1mL of 1mg/mL ferrite colloid, and reacting at room temperature for 30min; separating, discarding supernatant, collecting functionalized ferrite colloid, washing with distilled water and PBS buffer (pH 7.2) for 5 times, reacting at room temperature for 30min, and adding PBS solution (pH 7.2) to reach volume of 1mL.
Preparing a ferrite colloid probe marked by an H-FABP monoclonal antibody: adding 5 mu L of functional ferrite colloid (the mass concentration is 1 mg/mL) into an EP tube, adding 2 mu L of 0.4mg/mL H-FABP monoclonal antibody PBS solution (pH 7.2), uniformly mixing, and reacting for 8H at 4 ℃; separating, discarding supernatant, collecting isolate, washing with PBS (pH 7.2) for 5 times, and reacting in 50mM Tris buffer (pH 7.2) for 30min (4 deg.C); after re-separation, the supernatant was discarded, and the collected probes were washed 5 times with PBS and added to 4. Mu.L of PBS solution (containing 5% BSA).
FIG. 6 shows an SEM image of a ferrite probe labeled with FABP monoclonal antibody.
Preparation of sample pad, conjugate pad and nitrocellulose membrane:
the sample pad was soaked in 10mM phosphate buffer for 30min and then dried in a 30 ℃ desiccator.
Soaking the combined pad in 5% sucrose solution for 30min, drying in a 30 deg.C dryer, uniformly coating the H-FABP monoclonal antibody labeled ferrite colloid probe solution on the combined pad after completely drying, and drying in a 30 deg.C dryer.
Pasting a nitrocellulose membrane on a bottom plate (30 cm multiplied by 6 cm) for membrane scribing, then uniformly spraying and printing an anti-H-FABP polyclonal antibody solution (1 mg/mL) and a goat anti-mouse IgG solution (1 mg/mL) on the nitrocellulose membrane to be used as an H-FABP detection band and a quality control band (1 mu L/cm), drying at 35 ℃ for 1 hour, adding the dried antibody solution into a 1-percent BSA solution for reaction at room temperature for 30min for sealing, taking out the nitrocellulose membrane after sealing is finished, washing 5 times by using a PBS buffer solution (pH7.4), cleaning, and drying at 35 ℃ for 3 hours.
Assembling and cutting the test strip: and 3.0cm nitrocellulose membranes, 0.6cm combination pads, 1.8cm sample pads and 1.4cm water absorption pads are sequentially adhered to the bottom plate in a staggered manner by 2.5mm, so that the test paper board is assembled. The assembled test paper board is cut into test paper strips with the width of 5mm by using a strip cutting machine.
Test strip test experiment
The test paper prepared in this example was tested for its detection performance by preparing 5 μ g/L of each of the H-FABP solutions (a) and using purified water as a control (B). The specific operation is as follows: the solution was placed in a dry, clean container, the test strip was placed on the table top, 2 drops of the solution were pipetted and dropped onto the test strip sample pad. After the completion of the dropwise addition, the mixture was left standing and started to be timed, and the results were observed for 15 minutes.
The experimental result is shown in fig. 7, wherein T is a detection line, C is a quality control line, and fig. 7 is a detection graph of the solution a and the solution B from left to right; as is obvious from the results, the test strip prepared by the embodiment has a good detection effect on H-FABP after 10 minutes, and H-FABP detection lines and quality control lines in the solution A become deep, so that H-FABP positivity is displayed; only the control line in the control group became deep, and no band appeared on the detection line, indicating that H-FABP was negative.
Examples 9 to 13
Examples 9 to 13 were substantially the same as in example 8 except that the H-FABP monoclonal antibody-labeled ferrite colloid probe 9 was prepared in such a manner that the volume of the functionalized ferrite colloid added in example 8 was 5. Mu.L, the volume of the functionalized ferrite colloid added in example 9 was 10. Mu.L, the volume of the functionalized ferrite colloid added in example 10 was 15. Mu.L, the volume of the functionalized ferrite colloid added in example 11 was 2. Mu.L, and the volume of the functionalized ferrite colloid added in example 12 was 1. Mu.L. The test results of the test strips prepared in examples 9 to 13 are shown in FIG. 7.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, numerous and varied simplifications or substitutions may be made without departing from the spirit of the invention, which should be construed as falling within the scope of the invention.
Claims (10)
1. A kit for early diagnosis of myocardial infarction is characterized by comprising a bottom plate and test paper arranged on the bottom plate, wherein the test paper comprises a sample pad, a combination pad, a reaction pad and a water absorption pad which are sequentially arranged along a first direction, two ends of the combination pad are respectively overlapped with a second end of the sample pad and a first end of the reaction pad, a second end of the reaction pad is overlapped with a first end of the water absorption pad, wherein,
the preparation method of the combined pad comprises the following steps:
1) Mixing the ferrite colloid with a mixed solution of an activating reagent EDC and NHS, reacting for 30-240 min at 10-35 ℃, and separating to obtain a functional ferrite colloid;
2) Respectively carrying out coupling reaction on an H-FABP monoclonal antibody solution and an hs-cTnI monoclonal antibody solution with the functional ferrite to obtain an H-FABP monoclonal antibody labeled ferrite colloid probe and an hs-cTnI monoclonal antibody labeled ferrite colloid probe, and respectively adding the prepared H-FABP monoclonal antibody labeled ferrite colloid probe and the hs-cTnI monoclonal antibody labeled ferrite colloid probe into a PBS solution to obtain an H-FABP monoclonal antibody labeled ferrite colloid probe solution and an hs-cTnI monoclonal antibody labeled ferrite colloid probe solution;
3) Providing a raw material combination pad and carrying out pretreatment to obtain a pretreated combination pad, firstly, uniformly coating a ferrite colloid probe solution marked by an H-FABP monoclonal antibody on the pretreated combination pad to form a first layer, then coating a ferrite colloid probe solution marked by an hs-cTnI monoclonal antibody on the first layer to form a second layer, and drying to obtain the combination pad;
the reaction pad comprises a nitrocellulose membrane, and H-FABP detection bands, hs-cTnI detection bands and quality control bands which are arranged on the nitrocellulose membrane in parallel at intervals along the first direction, wherein the H-FABP detection bands are positioned on one side close to the combination pad, the quality control bands are positioned on one side close to the water absorption pad, the hs-cTnI detection bands are positioned between the H-FABP detection bands and the quality control bands, and the H-FABP detection bands, the hs-cTnI detection bands and the quality control bands are obtained by respectively spray-printing an anti-H-FABP polyclonal antibody solution, an anti-hs-cTnI polyclonal antibody solution and a goat anti-mouse IgG solution on the nitrocellulose membrane.
2. The kit for early diagnosis of myocardial infarction according to claim 1, wherein the temperature of the coupling reaction is 1 to 20 ℃, and the time of the coupling reaction is 3 to 24 hours.
3. The kit for early diagnosis of myocardial infarction according to claim 2, wherein the mass ratio of the functionalized ferrite colloid to the H-FABP monoclonal antibody is 1: (0.05-20), wherein the mass concentration of the H-FABP monoclonal antibody solution is 0.1-1.0 mg/mL; the mass ratio of the functionalized ferrite colloid to the hs-cTnI monoclonal antibody is 1: (0.05-20), and the mass concentration of the hs-cTnI monoclonal antibody solution is 0.1-1.0 mg/mL.
4. The kit for early diagnosis of myocardial infarction according to claim 1, wherein the anti-H-FABP polyclonal antibody solution, the anti-hs-cTnI polyclonal antibody solution and the goat anti-mouse IgG solution are all at mass concentrations of 0.1-5.0 mg/mL, and are all at inkjet printing amounts of 0.1-5.0 μ L/cm.
5. The kit for early diagnosis of myocardial infarction according to claim 1, characterized in that the sample pad is a phosphate buffer solution-treated sample pad.
6. The kit for early diagnosis of myocardial infarction according to claim 5, characterized in that the sample pad treatment step comprises sequential immersion and drying, the immersion solution is 10mmol/L phosphate buffer solution, the immersion temperature is 20-40 ℃, and the immersion time is 30-240 min; the drying temperature is 30-40 ℃.
7. A kit for early diagnosis of myocardial infarction is characterized by comprising a bottom plate and test paper arranged on the bottom plate, wherein the test paper comprises a sample pad, a combination pad, a reaction pad and a water absorption pad which are sequentially arranged along a first direction, two ends of the combination pad are respectively overlapped with a second end of the sample pad and a first end of the reaction pad, a second end of the reaction pad is overlapped with a first end of the water absorption pad, wherein,
the preparation method of the combined pad comprises the following steps:
1) Mixing ferrite colloid with a mixed solution of EDC and NHS as activating reagents, reacting for 30-240 min at 10-35 ℃, and separating to obtain functional ferrite colloid;
2) Carrying out coupling reaction on the H-FABP monoclonal antibody solution and the functional ferrite to obtain a ferrite probe marked by the H-FABP monoclonal antibody, and adding the prepared ferrite probe marked by the H-FABP monoclonal antibody into the PBS solution to obtain a ferrite probe solution marked by the H-FABP monoclonal antibody;
3) Providing a raw material combination pad, pretreating to obtain a pretreated combination pad, uniformly coating a ferrite colloid probe solution marked by an H-FABP monoclonal antibody on the pretreated combination pad, and drying to obtain the combination pad;
the reaction pad comprises a nitrocellulose membrane, and an H-FABP detection band and a quality control band which are arranged on the nitrocellulose membrane at intervals in parallel along the first direction, wherein the H-FABP detection band is positioned at one side close to the combination pad, the quality control band is positioned at one side close to the water absorption pad, and the H-FABP detection band and the quality control band are respectively obtained by spray-printing an anti-H-FABP polyclonal antibody solution and a goat anti-mouse IgG solution on the nitrocellulose membrane.
8. The kit for early diagnosis of myocardial infarction according to claim 7, wherein the temperature of the coupling reaction is 1 to 20 ℃, and the time of the coupling reaction is 3 to 24 hours.
9. The kit for the early diagnosis of myocardial infarction according to claim 8, wherein the mass ratio of the functionalized ferrite colloid to the H-FABP monoclonal antibody is 1: (0.05-20), wherein the mass concentration of the H-FABP monoclonal antibody solution is 0.1-1.0 mg/mL.
10. The kit for early diagnosis of myocardial infarction according to claim 7, wherein the mass concentrations of the anti-H-FABP polyclonal antibody and the goat anti-mouse IgG solution are both 0.1-5.0 mg/mL, and the jet printing amount is both 0.1-5.0 μ L/cm.
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| CN202211192148.0A CN115856289A (en) | 2022-09-28 | 2022-09-28 | Kit for early diagnosis of myocardial infarction |
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| CN202211192148.0A CN115856289A (en) | 2022-09-28 | 2022-09-28 | Kit for early diagnosis of myocardial infarction |
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