CN111896745B - Kit for jointly detecting cardiac marker by colloidal gold method and preparation method and application thereof - Google Patents
Kit for jointly detecting cardiac marker by colloidal gold method and preparation method and application thereof 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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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
- G01N33/54346—Nanoparticles
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
- G01N2333/9123—Phosphotransferases in general with a nitrogenous group as acceptor (2.7.3), e.g. histidine kinases
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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 belongs to the technical field of biological detection, and particularly relates to a kit for jointly detecting a cardiac marker by using a colloidal gold method, and a preparation method and application thereof. The kit comprises a detection card, wherein the detection card comprises a CK-MB test strip, a cTnI test strip and a MYO test strip; the detection test strip consists of absorbent paper, a nitrocellulose membrane, a sample pad, a colloidal gold pad, a PVC plate and a plastic card; coating goat anti-mouse IgG antibody on a nitrocellulose membrane C, and respectively coating mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies on a T line; the colloidal gold pad is sprayed with corresponding mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies marked by the colloidal gold. The kit prepared by the invention can obviously improve the detection sensitivity, and the capture capability is enhanced by pretreating the antibody, so that the missed diagnosis and misdiagnosis rate are reduced; the detection time is short, the requirement on the detection environment is low, and the specificity and the stability are good.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a kit for jointly detecting a cardiac marker by using a colloidal gold method, and a preparation method and application thereof.
Background
Cardiovascular diseases are serious diseases that harm human health and life, while acute myocardial infarction is the most common and dangerous number one killer that affects human health. The cardiac troponin is a regulatory protein of cardiac muscle contraction and consists of subunits of three different genes, and is usually used for laboratory diagnosis of cTnT and cTnI, and the cTnI has high sensitivity and longer window period for cardiac muscle injury due to high cardiac muscle specificity, is accepted by clinical and testing personnel, becomes a gold standard for diagnosing acute myocardial infarction, and becomes the most appropriate marker for judging the risk of cardiac muscle injury of patients with coronary syndrome; creatine kinase isoenzyme, CK-MB for short, is mainly present in cardiac muscle, and CK-MB is a valuable biochemical index for diagnosing myocardial damage. When the myocardial muscle is injured, CK-MB is released into blood, and rises after 3-8h, and can last for 3-5 days. Myo is a small molecular chromoprotein, which is formed by combining globin and methemoglobin, can be reversibly combined with oxygen, has the functions of transporting and storing oxygen in muscle cells, and can be dispersed from myocardial cells to enter blood circulation when the cardiac muscle is damaged, myoglobin is only present in the cardiac muscle and striated muscle, and other tissues including smooth muscle do not contain the myoglobin.
Early diagnosis and early treatment are the key to save the life of patients with acute myocardial infarction. The clinical symptoms of patients are not typical, and electrocardiograms may change nonspecifically, so the detection of cardiac markers becomes an important basis for early diagnosis. At present, the early diagnosis of acute myocardial infarction, including myocardial infarction, is often performed clinically by detecting specific markers. The colloidal gold immunochromatography method has good specificity and sensitivity when detecting the cardiac marker, but has higher misdiagnosis rate and missed diagnosis rate by statistical discovery.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a kit for jointly detecting a cardiac marker by using a colloidal gold method.
The invention also provides a preparation method of the kit.
The invention also provides an application of the kit.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a kit for jointly detecting a cardiac marker by using a colloidal gold method, which comprises a detection card, wherein the detection card comprises CK-MB, cTnI and MYO detection test strips; the detection test strip consists of absorbent paper, a nitrocellulose membrane, a sample pad, a colloidal gold pad, a PVC plate and a plastic card; coating goat anti-mouse IgG antibody on a nitrocellulose membrane C, and respectively coating mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies on a T line; the colloidal gold pad is sprayed with corresponding mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies marked by the colloidal gold. Further, the specific operation process of the mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies is as follows: dropwise adding ammonia water into the n-butyl titanate solution, after dropwise adding, centrifuging, washing precipitated titanium dioxide by using ethanol and deionized water, then adding polyglutamic acid and chitosan into the precipitate, adding the rat-resistant CK-MB, rat-resistant cTnI and rat-resistant MYO monoclonal antibody solution, and uniformly stirring to obtain the nano-titanium dioxide.
The volume ratio of the n-butyl titanate to the ammonia water is 1: 1.5; the volume fraction of the ammonia water is 28 percent; the mass ratio of the polyglutamic acid to the chitosan to the titanium dioxide is 0.1: 0.05: 2.
further, the volume ratio of the mouse anti-CK-MB antibody, the mouse anti-cTnI antibody, the mouse anti-MYO monoclonal antibody solution and the n-butyl titanate is 1: 0.2.
the invention also provides a preparation method of the kit, which comprises the following steps:
(1) soaking the polyester film in a colloidal gold solution at room temperature for 1-2h, drying, cutting into strips of 30cm multiplied by 0.7cm, spraying a colloidal gold marker working solution with the spraying amount of 2.0-2.5 muL/cm to uniformly spray the colloidal gold marker working solution on the treated polyester film, and then drying;
(2) respectively diluting the mouse anti-CK-MB, cTnI, MYO monoclonal antibodies and the sheep anti-mouse IgG antibodies to 0.08 mu L/mm by MPB buffer solution, then scribing the mouse anti-CK-MB, cTnI, MYO monoclonal antibodies and the sheep anti-mouse IgG antibodies at the positions of a T line and a C line of a nitrocellulose membrane by using a scribing instrument, coating the nitrocellulose membrane with the antibodies, and drying;
(3) soaking the glass fiber in the sample pad treatment solution at room temperature for 1-2h, and drying to obtain a sample pad;
(4) and (3) sticking the gold gel pad, the nitrocellulose membrane, the sample pad and the absorbent paper on a PVC plate to prepare a large plate, and then cutting the large plate to obtain the finished product.
The preparation process of the colloidal gold marker working solution used by the invention comprises the following steps: adding 0.1M potassium carbonate solution into colloidal gold solution, adjusting pH to 7.5, then respectively adding mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies, stirring at room temperature for 30min, continuously stirring with 1% BSA solution, sealing for 20min, respectively centrifuging at high speed for separation and purification, centrifuging at 2000r/min for 15min, taking precipitate, then centrifuging supernatant at 10000r/min for 45min, discarding supernatant, and diluting the precipitate to 6mL/100mL by using colloidal gold diluent.
Further, the colloidal gold solution is prepared by heating 0.01% chloroauric acid solution to boiling by a trisodium citrate reduction method, adding 1mL/100mL of 1% trisodium citrate solution, continuously heating and boiling for 15min, cooling to room temperature, and then recovering the volume to the original volume by using distilled water.
The invention also provides application of the kit in simultaneous detection of CK-MB, cTnI and MYO.
The colloidal gold solution used in the invention adopts a trisodium citrate reduction method, 0.01% chloroauric acid solution is heated to boiling, 1mL/100mL of 1% trisodium citrate solution is added, boiling is continued for 15min, distilled water is added after cooling to room temperature to restore the original volume, and the solution is stored at 4 ℃ for standby.
The invention has the beneficial effects that:
(1) the kit prepared by the invention can obviously improve the detection sensitivity, and the capture capability of the antibody can be enhanced by pretreating the antibody, so that the missed diagnosis and misdiagnosis rate are reduced;
(2) the kit prepared by the invention has short detection time, low requirement on detection environment and good specificity and stability.
Drawings
FIG. 1 is a schematic view of a test strip prepared according to the present invention.
Detailed Description
The technical solution of the present invention is further explained and illustrated by the following specific examples.
(1) The raw materials used in the invention are specifically required as follows:
murine anti-CK-MB monoclonal antibodies are described in Table 1.
TABLE 1
Murine anti-cTnI monoclonal antibodies are described in table 2.
TABLE 2
Murine anti-MYO monoclonal antibodies are shown in Table 3.
TABLE 3
(2) Internal positive reference product of creatine kinase isoenzyme CK-MB enterprise: collecting several parts of serum samples of acute myocardial infarction patients (with more than 3h of attack) or other patients with myocardial damage from hospitals, actually detecting CK-MB as positive by two patients, extinguishing fire at 60 ℃ for 1h, selecting several parts of strong positive, medium positive and weak positive serum from the samples, mixing each group, dividing the mixture into 3 parts according to the development intensity to form an internal positive reference substance of an enterprise, labeling, subpackaging according to 0.5 mL/branch, and storing at-20 ℃.
Internal negative reference product of creatine kinase isoenzyme CK-MB enterprise: collecting several parts of serum samples of non-polar myocardial infarction patients or normal persons from hospitals, inactivating at 60 deg.C for 1h, selecting several parts, mixing to 9 parts respectively to form enterprise internal negative reference substance, labeling, packaging at 0.5 mL/piece, and storing at-20 deg.C.
The internal detection limit reference product of the creatine kinase isoenzyme CK-MB enterprise: CK-MB standards (commercially available) were diluted to 40ng/mL, 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL with PBS +5% bovine serum, labeled, and dispensed at 0.5 mL/aliquot and stored at-20 ℃.
The internal precision reference product of creatine kinase isoenzyme CK-MB enterprise: selecting several acute myocardial infarction patients, carrying out two serum samples with CK-MB as median positive through actual detection, carrying out inactivation treatment at 60 ℃ for 1h, mixing to obtain CV value enterprise internal precision reference substances, and subpackaging according to 1 mL/branch at-20 ℃.
Internal specific reference product of creatine kinase isoenzyme CK-MB enterprise: 1000ng/mL creatine kinase isoenzyme MM (CK-MM) and 1000ng/mL creatine kinase isoenzyme BB (CK-BB) were dispensed at a rate of 0.5 mL/aliquot and stored at-20 ℃.
Internal positive reference of myocardial troponin I enterprise: collecting several parts of serum samples of acute myocardial infarction patients (with more than 3h of attack) or other patients with myocardial damage from hospitals, actually detecting cTnI as positive samples by two patients, extinguishing fire at 60 ℃ for 1h, selecting several parts of strong positive, medium positive and weak positive serum from the samples, mixing each group, dividing the mixture into 3 parts according to the development intensity to form an internal positive reference substance of an enterprise, labeling, subpackaging according to 0.5 mL/branch, and storing at-20 ℃.
Negative reference product in myocardial troponin I enterprise: collecting several parts of serum samples of non-polar myocardial infarction patients or normal persons from hospitals, inactivating at 60 deg.C for 1h, selecting several parts, mixing to 9 parts respectively to form enterprise internal negative reference substance, labeling, packaging at 0.5 mL/piece, and storing at-20 deg.C.
The internal detection limit reference product of the myocardial troponin I enterprise: cTnI standards (commercially available) were diluted to 64ng/mL, 16 ng/mL, 4ng/mL, 1 ng/mL, 2.5 ng/mL with PBS +5% bovine serum, labeled, and dispensed at 0.5 mL/aliquot and stored at-20 ℃.
The internal precision reference product of the myocardial troponin I enterprise: selecting several acute myocardial infarction patients, carrying out two actual detections on blood serum samples with the cTnI as the median positive, carrying out inactivation treatment at 60 ℃ for 1h, mixing to obtain CV value enterprise internal precision reference products, and subpackaging according to 1 mL/branch at-20 ℃.
Specific reference product in myocardial troponin I enterprise: 1000ng/mL cardiac troponin T (cTnT), 1000ng/mL cardiac troponin C (cTnC), 0.5 mL/branch, and storing at-20 deg.C.
Internal positive reference of myoglobin enterprise: collecting several parts of serum samples of acute myocardial infarction patients (with more than 3h of attack) or other patients with myocardial damage from hospitals, actually detecting Myo as positive by two patients, extinguishing fire at 60 ℃ for 1h, selecting several parts of strong positive, medium positive and weak positive serum from the samples, mixing each group, dividing the mixture into 3 parts according to the color development strength to form an internal positive reference substance of an enterprise, labeling, subpackaging according to 0.5 mL/branch, and storing at-20 ℃.
Internal negative reference product of myoglobin enterprise: collecting several parts of serum samples of non-polar myocardial infarction patients or normal persons from hospitals, inactivating at 60 deg.C for 1h, selecting several parts, mixing to 9 parts respectively to form enterprise internal negative reference substance, labeling, packaging at 0.5 mL/piece, and storing at-20 deg.C.
Internal detection limit reference products of myoglobin enterprises: myo standards (commercially available) were diluted to 64ng/mL, 400 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 25 ng/mL with 0.02M Tris +5% bovine serum, labeled, and then aliquoted at 0.5 mL/aliquot and stored at-20 ℃.
Internal precision reference products of myoglobin enterprises: selecting several acute myocardial infarction patients, carrying out two serum samples with Myo as median positive through actual detection, inactivating at 60 ℃ for 1h, mixing to obtain CV value enterprise internal precision reference, and subpackaging at 1 mL/branch and storing at-20 ℃.
Example 1
A kit for jointly detecting a cardiac marker by using a colloidal gold method comprises a detection card, wherein the detection card comprises a CK-MB test strip, a cTnI test strip and a MYO test strip; the detection test strip consists of absorbent paper, a nitrocellulose membrane, a sample pad, a colloidal gold pad, a PVC plate and a plastic card; coating goat anti-mouse IgG antibody on a nitrocellulose membrane C, and respectively coating mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies on a T line; the colloidal gold pad is sprayed with corresponding mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies marked by the colloidal gold. The specific operation process of the mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies is as follows: dropwise adding 1.5mL of ammonia water into 1mL of n-butyl titanate, after dropwise adding, centrifuging, washing precipitated titanium dioxide with ethanol and deionized water, and then adding a solvent according to a mass ratio of polyglutamic acid, chitosan and titanium dioxide of 0.1: 0.05: 2, adding the mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibody solution, and uniformly stirring to obtain the mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibody.
The preparation method comprises the following steps:
(1) the colloidal gold solution is prepared by heating 0.01% chloroauric acid solution to boiling by trisodium citrate reduction method, adding 1mL/100mL 1% trisodium citrate solution, heating and boiling for 15min, cooling to room temperature, and recovering to original volume with distilled water;
(2) the preparation process of the colloidal gold marker working solution comprises the following steps: adding 0.1M potassium carbonate solution into colloidal gold solution, adjusting pH to 7.5, then respectively adding mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies, stirring at room temperature for 30min, continuously stirring with 1% BSA solution, sealing for 20min, respectively centrifuging at high speed for separation and purification, centrifuging at 2000r/min for 15min, taking precipitate, then centrifuging supernatant at 10000r/min for 45min, discarding supernatant, and diluting the precipitate to 6mL/100mL by using colloidal gold diluent;
(3) soaking the polyester film in a colloidal gold solution at room temperature for 1-2h, drying, cutting into strips of 30cm multiplied by 0.7cm, spraying a colloidal gold marker working solution with the spraying amount of 2.0-2.5 muL/cm to uniformly spray the colloidal gold marker working solution on the treated polyester film, and then drying;
(4) respectively diluting the mouse anti-CK-MB, cTnI, MYO monoclonal antibodies and the sheep anti-mouse IgG antibodies to 0.08 mu L/mm by MPB buffer solution, then scribing the mouse anti-CK-MB, cTnI, MYO monoclonal antibodies and the sheep anti-mouse IgG antibodies at the positions of a T line and a C line of a nitrocellulose membrane by using a scribing instrument, coating the nitrocellulose membrane with the antibodies, and drying;
(5) soaking the glass fiber in the sample pad treatment solution at room temperature for 1-2h, and drying to obtain a sample pad;
(6) and (3) sticking the gold gel pad, the nitrocellulose membrane, the sample pad and the absorbent paper on a PVC plate to prepare a large plate, and then cutting the large plate to obtain the finished product.
In the kit prepared in example 1, the membrane width was 3.9. + -. 0.1mm, and the liquid moving speed was not less than 10 mm/min.
Comparative example 1
The preparation process and composition are basically the same as example 1, except that the antibody is not loaded by a titanium dioxide template, and the labeled antibody and the coated antibody are directly correspondingly labeled and coated.
Effect verification
(1) Negative reference product compliance rate
9 parts of myocardial troponin I enterprise internal negative reference substance, 9 parts of creatine kinase isoenzyme CK-MB enterprise internal negative reference substance and 9 parts of myoglobin enterprise internal negative reference substance, taking a same-batch test paper to detect corresponding detection substances, respectively adding 100 mu L of reference substance into a sample adding hole, reading results within 5-20min, wherein the detection results are all negative, and the coincidence rate is 100%.
(2) Positive reference compliance rate
9 parts of myocardial troponin I enterprise internal positive reference substance, 9 parts of creatine kinase isoenzyme CK-MB enterprise internal positive reference substance and 9 parts of myoglobin enterprise internal positive reference substance, taking the same batch of test paper to detect corresponding detection substances, and respectively adding 100 mu L of reference substance into the sample adding holes, wherein the detection results are all positive, and the coincidence rate is 100%.
(3) Minimum detection limit
The detection is carried out on 5 concentrations of cardiac troponin I, creatine kinase isoenzyme CK-MB and myoglobin detection limit reference substances respectively, 100 mu L of the detection result is added into a sample adding hole respectively, the result is read within 5-20min, the kit prepared by the invention meets the positive reaction, and S5 is negative, while the kit prepared by the comparative example 1 has less than 4 parts of positive reaction.
(4) Precision of
The kit prepared in example 1 randomly extracts 10 test cards of the same batch, adds 100 μ L of an internal precision reference product of an enterprise into a corresponding sample adding hole of each test strip, reads the result within 5-20min, and has consistent reaction in batches and uniform color development which are all positive.
(5) Specificity of
Randomly drawing 10 kits for detection, wherein the kit prepared in the example 1 has detection concentrations of 1000ng/mL of cardiac troponin T and 600 ng/mL of cardiac troponin C enterprise internal specific reference substances, and the results are negative; the CK-MM and CK-BB test results at 1000ng/mL are negative, while in comparative example 1, the cardiac troponin T shows a positive result for 1 time, and the CK-BB shows a positive result for one time.
The Myo test strip has no specific reference substance and does not carry out corresponding detection.
(6) Stability of
After the test paper is placed for 12 months at the temperature of 2-30 ℃, the test paper adopts internal reference products of enterprises for detection within one month, and the positive reference products, the negative reference products and the like of the test paper meet the requirements.
When 20 days of the to-be-detected product is placed at 37 ℃ and the kit is adopted for detection, all detections meet the requirements, while the kit prepared in the comparative example 1 is repeated for 10 times for detection, 2 negative results appear in a positive reference product, and false detection appears in specificity detection.
Claims (4)
1. A kit for jointly detecting a cardiac marker by using a colloidal gold method is characterized by comprising a detection card, wherein the detection card contains a CK-MB, cTnI and MYO detection test strip; the detection test strip consists of absorbent paper, a nitrocellulose membrane, a sample pad, a colloidal gold pad, a PVC plate and a plastic card; coating goat anti-mouse IgG antibody on a nitrocellulose membrane C, and respectively coating mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies on a T line; spraying corresponding mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies marked by colloidal gold on the colloidal gold pad respectively;
the specific preparation process of the mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibodies comprises the following steps: dropwise adding ammonia water into the n-butyl titanate solution, after dropwise adding, centrifuging, washing precipitated titanium dioxide by using ethanol and deionized water, then adding polyglutamic acid and chitosan into the precipitate, respectively adding mouse anti-CK-MB, mouse anti-cTnI and mouse anti-MYO monoclonal antibody solutions, and uniformly stirring to obtain the nano-titanium dioxide.
2. The kit according to claim 1, wherein the volume ratio of n-butyl titanate and ammonia water is 1: 1.5; the volume fraction of the ammonia water is 28%.
3. The kit according to claim 2, wherein the mass ratio of the polyglutamic acid, the chitosan and the titanium dioxide is 0.1: 0.05: 2.
4. the kit of claim 2 or 3, wherein the volume ratio of mouse anti-CK-MB, mouse anti-cTnI, mouse anti-MYO monoclonal antibody solution and n-butyl titanate is 1: 0.2.
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