CN112526140A - Magnetic particle chemiluminescence detection kit for determining content of hypersensitive troponin T - Google Patents
Magnetic particle chemiluminescence detection kit for determining content of hypersensitive troponin T Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- 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
- G01N33/54326—Magnetic particles
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- 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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Abstract
The invention relates to a magnetic particle chemiluminescence detection kit for determining the content of hypersensitive troponin T, which comprises: r1 reagent, R2 reagent, magnetic separation reagent, calibrator liquid series and chemiluminescent substrate liquid; the reagent R1 is a buffer solution, the magnetic separation reagent is a troponin T monoclonal antibody coated magnetic particle solution prepared by dissolving troponin T monoclonal antibody coated magnetic particles in a diluent of the magnetic separation reagent, the reagent R2 is an alkaline phosphatase-labeled troponin T monoclonal antibody solution prepared by dissolving alkaline phosphatase-labeled troponin T monoclonal antibody in a diluent of a reagent R2, and the chemiluminescent substrate solution is a chemiluminescent substrate solution catalyzed by alkaline phosphatase. The invention provides a more accurate, precise, convenient, rapid and simple method for clinically detecting troponin T in human serum.
Description
Technical Field
The invention relates to the field of medical diagnostic reagents, in particular to a magnetic particle chemiluminescence detection kit for detecting the content of hypersensitive troponin T by adopting a magnetic particle chemiluminescence technology and an antigen-antibody combination technology.
Background
Troponin is a regulatory protein of muscle contraction and is composed of three structurally distinct subunits, namely troponin t (tnt), troponin i (tni), and troponin c (tnc). Troponin relaxes actomyosin, allowing actomyosin to interact with sarcoplasmic globulin, causing muscle contraction. The T and I subunit structures in the cardiac muscle differ from those of other muscle tissues, and cardiac troponin T, I (cTnT, cTnI) rapidly increases in blood concentration after onset of disease due to its small molecular weight. cTnT is the polypeptide chain in the molecular structure of striated muscle with a relative molecular mass of 37X 103There are 4 troponin T isoforms in the human heart. Exist in two forms: the free form is present in the cytoplasm and accounts for 6-8%, and the bound form is present on the thin myofilaments of the myocardial contractile unit and accounts for 92-98%. Because the gene coding of cTnT is different from that of skeletal muscle TnT, there is no expression of cTnT in skeletal muscle. cTnT is 40% heterogenous with respect to the two skeletal muscle subtypes. The cTnT molecule is stable, hydrophilic and has good reactivity of specific antigenic determinant.
5% -8% of cTnT is distributed in the cytoplasm of the myocardial cells and is free cytoplasmic protein, and 92% -95% of cTnT is combined in the filaments and is combined structural protein; 3% of cTnI is distributed in the cytoplasm of the myocardial cells, and 97% is combined with myocardial structural protein. When myocardial cells are destroyed by ischemia, hypoxia, etc., free cTnI and cTnT can be first rapidly released from the cells into the blood, and then bound cTnI and cTnT bound to structural proteins of the myocardium are gradually decomposed and slowly released into the circulating blood, thereby explaining the disease manifested by the damage of the myocardial cells, such as Acute Myocardial Infarction (AMI), and the reason why cTnI and cTnT appear earlier in the circulating blood and last for a longer time. High levels of hypersensitivity cTnT can be well used in multifactorial assays to predict mortality in all cardiac diseases. The cTnT can improve the validity of AMI serological detection.
The cTnT detection can be used for the auxiliary diagnosis of acute coronary syndrome necrosis, such as acute myocardial infarction. And is also an index of risk stratification for patients with acute coronary syndrome and myocardial risk for patients with chronic renal failure. In addition, more effective treatments and interventions for increasing cardiac troponin T may be selected.
Troponin T (TnT) has high myocardial specificity and sensitivity, is a marker of myocardial necrosis, and is generally elevated in blood of patients with myocardial infarction, myocarditis, pericarditis, and the like, and the higher the value thereof, the worse the prognosis of patients.
The known troponin T detection methods include mainly RIA (radioimmunoassay) method, enzyme-linked immunosorbent assay (ELISA), colloidal Gold Immunochromatography (GICA), and chemiluminescence immunoassay (CLIA). However, these methods have certain defects, and the RIA method has great pollution and gradually exits from the field of clinical examination; the ELISA method has the defects of low automation degree, complex and fussy operation, low sensitivity, narrow linear range and the like; the GICA has the advantages of simple operation, high detection speed and the like, but also has the defects of low sensitivity, unstable reagent, poor repeatability, difficulty in quantification and the like; the CLIA method is an immunoassay technology developed on the basis of an enzyme-linked immunoassay method, and has the advantages of high sensitivity, wide detection linear range, simple operation, high automation degree and the like. The prior chemiluminescence immunoassay technology is widely applied due to the advantages, but most of chemiluminescence immunoassay kits in the prior art are imported closed full-automatic chemiluminescence detection systems, and expensive full-automatic chemiluminescence detectors are needed, so that the popularization and the use are limited.
Therefore, a troponin T detection kit and a use method thereof with low pollution, high sensitivity, simple operation, high automation degree, good specificity and low cost are still needed.
Disclosure of Invention
The invention aims to develop a magnetic particle chemiluminescence detection kit for determining the content of hypersensitive troponin T, which has the advantages of high sensitivity, no pollution, simple operation, good specificity and low cost.
Based on the above purpose, the magnetic particle chemiluminescence detection kit for determining the content of hypersensitive troponin T provided by the invention comprises: r1 reagent, R2 reagent, magnetic separation reagent, calibrator liquid series and chemiluminescent substrate liquid; the method is characterized in that:
the R1 reagent is a buffer solution,
the magnetic separation reagent is a troponin T monoclonal antibody coated magnetic particle solution prepared by dissolving troponin T monoclonal antibody coated magnetic particles in a diluent of the magnetic separation reagent,
the R2 reagent is alkaline phosphatase-labeled troponin T monoclonal antibody solution prepared by dissolving alkaline phosphatase-labeled troponin T monoclonal antibody in the diluent of the R2 reagent,
the chemiluminescence substrate solution is a chemiluminescence substrate solution which is catalyzed by alkaline phosphatase to emit light.
The R1 reagent can be physiological saline, i.e., 0.9% (wt) aqueous sodium chloride solution, or can be in the form of a buffer solution near the R2 reagent or in the form of a buffer solution for magnetic separation of the reagents.
Dissolving a magnetic separation reagent, namely magnetic particles coated by a troponin T monoclonal antibody in a diluent of the magnetic separation reagent to prepare a troponin T monoclonal antibody coated magnetic particle solution of 0.8-1.2 mg/mL; the pH of the diluent of the magnetic separation reagent is 7.5-8.5, and the method comprises the following steps: tris, the concentration is 12.0-12.3 g/L; sodium azide with the concentration of 0.9-1.1 g/L; sodium chloride with the concentration of 5.7-5.9 g/L; bovine serum albumin with the concentration of 3-6 g/L; MgCl2The concentration is 0.8 to 1.2 mM; ZnCl2The concentration is 0.08-0.12 mM; the balance of deionized water.
Preferably, the magnetic separation reagent, namely the magnetic particles coated by the troponin T monoclonal antibody is dissolved in a diluent to prepare a magnetic particle solution coated by the troponin T monoclonal antibody with the concentration of 1.0 mg/mL; the buffer pH of the magnetic separation reagent is 8.0, and comprises: tris, the concentration is 12.04 g/L; sodium azide with the concentration of 1 g/L; sodium chloride with concentration of 5.79 g/L; bovine serum albumin with a concentration of 5 g/L; MgCl2The concentration is 1.0 mM; ZnCl2The concentration is 0.1 mM; the balance of deionized water.
The preparation method of the troponin T monoclonal antibody coated magnetic particles comprises the following steps: measuring magnetic particles, adsorbing by using a magnet, washing for 2-3 times by using a buffer solution with 0.1M Hepes pH7.5, and adding a diluent of a magnetic separation reagent to enable the final concentration of magnetic beads to be 8-12 mg/mL; and adding the antibody to make the concentration of the antibody be 0.05-0.2 mg/mL, uniformly mixing for 14-22 hours at 37 ℃, adding 5-15% BSA for sealing, uniformly mixing for 4-8 hours at 37 ℃, adsorbing by using a magnet, discarding the supernatant, cleaning for 3 times by using a cleaning solution consisting of 25mM Tris-HCl pH7.2, 0.15M NaCl and 0.05% Tween20, cleaning for one time by using a diluent of a magnetic separation reagent, and fixing the volume to make the final concentration of the magnetic beads be 8-12 mg/mL to obtain a concentrated solution.
Preferably, the preparation method of the troponin T monoclonal antibody coated magnetic particles comprises the following steps of measuring the magnetic particles, adsorbing the magnetic particles by using a magnet, washing the magnetic particles for 2-3 times by using 0.1M Hepes pH7.5, preferably, adding a diluent of a magnetic separation reagent to enable the final concentration of the magnetic particles to be 10mg/mL, adding an antibody to enable the concentration of the antibody to be 0.1mg/mL, uniformly mixing the magnetic particles at 37 ℃ for 18 hours, adding 10% BSA for sealing, uniformly mixing the magnetic particles at 37 ℃ for 6 hours, adsorbing the magnetic particles by using a magnet, discarding supernatant, washing the magnetic particles for 3 times by using 25mM Tris-HCl pH7.2, 0.15M NaCl and 0.05% Tween20, washing the magnetic particles once by using a diluent of a magnetic separation reagent and fixing the volume to enable the final concentration of the magnetic particles to be 10 mg;
the R2 reagent, namely the troponin T monoclonal antibody marked by alkaline phosphatase is dissolved in the diluent of the R2 reagent to prepare 0.2-0.5 mu g/mL alkaline phosphatase-marked troponin T monoclonal antibody solution;
the pH value of the diluent of the R2 reagent is 6.7-7.6, and the method comprises the following steps: tris, the concentration is 12.0-12.3 g/L; sodium azide with the concentration of 0.9-1.1 g/L; sodium chloride with the concentration of 5.7-5.9 g/L; bovine serum albumin with the concentration of 8-12 g/L; the trehalose concentration is 30-50 g/L; MgCl2The concentration is 0.8 to 1.2 mM; ZnCl2The concentration is 0.08-0.12 mM; the Brij 35 concentration is 0.01-1%; the balance of deionized water.
Preferably, the R2 reagent, namely the alkaline phosphatase-labeled troponin T monoclonal antibody is dissolved in a diluent of the R2 reagent to prepare a 0.3 mu g/mL alkaline phosphatase-labeled troponin T monoclonal antibody solution; the pH of the dilution of the R2 reagent was 6.8, including: tris, the concentration is 12.04 g/L; sodium azide with the concentration of 1 g/L; sodium chloride with concentration of 5.79 g/L; bovine serum albumin with a concentration of 10 g/L; the trehalose concentration is 50 g/L; MgCl2The concentration is 1.0 mM; ZnCl2The concentration is 0.1 mM; the Brij 35 concentration is 0.35%; the balance of deionized water.
In another embodiment, the chemiluminescent substrate solution is a chemiluminescent substrate solution catalyzed and luminous by alkaline phosphatase with a molar concentration of 0.1-0.3M, a Tris-HCl buffer solution with a molar concentration of 0.2M and a pH value of 8-10, and contains dioxane compound (APCL) with a concentration of 0.2-0.4 mg/mL.
In another embodiment, the chemiluminescent substrate solution is Tris-HCl buffer at a molar concentration of 0.2M and pH 9.3, and contains 0.3mg/mL of dioxane compound (APCL).
In another scheme, the kit further comprises a cleaning solution, wherein the cleaning solution is 0.1-0.2M, and 0.01-0.04% of Tween20 and 15-20% of sodium chloride in percentage by mass are added into a Tris-HCl buffer solution with the pH value of 8-9.
In another scheme, 0.02% of tween20 and 15% of sodium chloride in percentage by mass are added into a Tris-HCl buffer solution with the cleaning solution of 0.1M and the pH value of 8.
In another embodiment, the calibrator is Tris-HCl buffer containing troponin T antigen at different concentrations.
In another embodiment, the buffer of the calibrator liquid series comprises bovine serum albumin with a concentration of 20.0 g/L; sodium chloride with concentration of 8.5g/L, casein sodium with concentration of 2g/L, and sodium azide with concentration of 1 g/L. The buffer solution of the troponin T calibrator can ensure the stable existence of troponin and prolong the service life of the kit.
In another scheme, the concentration range of the calibrator liquid is 0-6000 pg/mL, and the pH value of the buffer liquid is 7.4.
Alternatively, the range of different concentrations of the calibrator liquid is 0pg/mL, 30pg/mL, 100pg/mL, 500pg/mL, 2000pg/mL, 6000 pg/mL.
A preparation method of a magnetic particle chemiluminescence detection kit for determining the content of hypersensitive troponin T comprises the following steps:
preparation of R1 reagent: the buffer composition was determined here as a buffer consisting of a preparation of physiological saline, 0.9% sodium chloride, and the balance deionized water.
Preparation of R2 reagent: 1) preparing a diluent according to the component content of the diluent of the R2 reagent, and adjusting the pH value; 2) preparing an alkaline phosphatase-labeled troponin T antibody; 3) the alkaline phosphatase-labeled troponin T antibody was diluted with a dilution of the R2 reagent.
Preparing magnetic particles: 1) preparing magnetic particles coated by the troponin T monoclonal antibody, namely concentrated solution; 2) the magnetic particles are diluted with a diluent of a magnetic separation reagent.
Preparing a calibrator: 1) preparing a buffer solution according to the content of the buffer solution components of the calibrator liquid series; 2) troponin T antigen was dissolved in calibrator buffer to be formulated at different concentrations.
Preparing a chemiluminescent substrate solution: 1) preparing 0.2M Tris-HCl buffer solution with the pH value of 9.3, and adding 0.3mg/mL dioxane; 2) the chemiluminescent substrate for which alkaline phosphatase catalyzes luminescence is dissolved in a buffer of chemiluminescent substrate.
The use method of the magnetic particle chemiluminescence detection kit for detecting the hypersensitive troponin T comprises the following steps:
(1) mixing and incubating a sample to be tested or a calibrator, the R1 reagent, the R2 reagent and the magnetic particles for 10min at 37 ℃;
(2) washing, removing the unbound antibody and impurities, and adding a luminescent substrate;
(3) adding a luminescent substrate, and measuring relative luminescence intensity (RLU) after ALP catalyzes the substrate to emit light;
in a certain range, the RLU is in direct proportion to the concentration of troponin T antigen, and the content of troponin T in the sample to be detected can be read from the standard curve by an interpolation method.
The data of methodology identification when the detection kit is used for determining the content of the hypersensitive troponin T can reach the following indexes:
the minimum detection limit-the minimum detection amount is 3pg/mL, and the minimum detection amount can reach 0.045pg/mL in the embodiment;
the linear detection range of the hypersensitive troponin T is 3-6000 pg/mL;
the precision-the intra-analysis precision is 2.32% on average (n is 10), the inter-analysis precision is 4.51% on average (n is 10), and the precision is far lower than the national requirement, which shows that the kit has good repeatability in the experimental process;
accuracy-serum with known troponin T concentration is diluted by adopting calibrator buffer solution in different proportions, and the recovery rate is 85-115%;
specificity-the crossing rates with cardiac troponin I, troponin C, skeletal troponin T were all less than 0.1%.
Compared with the prior art, the invention has the beneficial effects that:
the reaction mode of the sandwich method is adopted, the principle of combining the chemiluminescence detection technology with the magnetic particle immune separation technology is utilized, the troponin T content in the human serum sample is quantitatively detected, the detection sensitivity is ensured, the pollution is avoided, the specificity is strong, the operation is simple, the rapid high-throughput detection of a large batch of samples can be realized, and the application of clinical reagents is facilitated. The invention does not use a fluorescein coupling antibody process, but adopts a buffer solution with a specific composition in an antibody coupling magnetic bead process, reduces unnecessary interference during troponin T detection, can obviously improve the detection sensitivity by combining a magnetic particle preparation method, achieves the level of hypersensitivity, and can improve the specificity and stability of troponin T by using a diluent of an R2 reagent with a specific composition.
The invention provides a more accurate, precise, convenient, rapid and simple method for clinically detecting troponin T in human serum.
Drawings
FIG. 1 is a graph of concentration-luminescence of troponin T in a kit according to example 2 of the present invention;
FIG. 2 shows the comparison result between the test kit and the imported test kit in example 2 of the present invention.
Detailed Description
The present invention is further explained with reference to the following examples and drawings, but the scope of the present invention is not limited thereto.
The invention relates to a magnetic particle chemiluminescence detection kit for determining the content of hypersensitive troponin T, which comprises an R1 reagent, an R2 reagent, a magnetic separation reagent, a calibrator liquid series and a chemiluminescence substrate liquid.
The R1 reagent was 0.9% sodium chloride, the remainder being deionized water.
The R2 reagent includes: 1) r2 antibody: the concentration of the troponin T antibody marked by alkaline phosphatase is 0.2-0.5 mu g/mL; 2) diluting liquid: dilutions of the R2 reagent had a pH of 6.8, including: tris, the concentration is 12.04 g/L; sodium azide with the concentration of 1 g/L; sodium chloride with concentration of 5.79 g/L; bovine serum albumin with a concentration of 10 g/L; the trehalose concentration is 50 g/L; MgCl2The concentration is 1.0 mM; ZnCl2The concentration is 0.1 mM; the Brij 35 concentration is 0.35%; the balance of deionized water.
The magnetic separation reagent comprises: 1) magnetic particles: the concentration of the magnetic particles coated by the troponin T monoclonal antibody is 1.0 mg/mL;
2) the pH value of the diluent of the magnetic separation reagent is 8.0, and the method comprises the following steps: tris, the concentration is 12.04 g/L; sodium azide with the concentration of 1 g/L; sodium chloride with concentration of 5.79 g/L; bovine serum albumin with a concentration of 5 g/L; MgCl2The concentration is 1.0 mM; ZnCl2The concentration is 0.1 mM; the balance of deionized water.
The calibrator liquid comprises: 1) Tris-HCl buffer of troponin T antigen; 2) buffer solution: 0.1M Tris-HCl, bovine serum albumin, concentration 20.0 g/L; sodium chloride with concentration of 8.5 g/L; casein sodium, the concentration is 2 g/L; sodium azide at a concentration of 1 g/L. The pH of the buffer of the calibrator liquid series was 7.4. The calibrator liquid series were reagent series containing different concentrations (0pg/mL, 30pg/mL, 100pg/mL, 500pg/mL, 2000pg/mL, 6000 pg/mL).
The substrate solution is chemiluminescence substrate solution with the molar concentration of 0.1-0.3M and catalyzed luminescence by alkaline phosphatase, wherein the concentration of a dioxane compound (APCL) in the chemiluminescence substrate solution is 0.2-0.4 mg/mL, the buffer solution is Tris-HCl with the molar concentration of 0.2M, and the pH value is 8-10.
The preparation method of the magnetic particle chemiluminescence detection kit for determining the content of the hypersensitive troponin T comprises the following steps:
preparation of R1 reagent: 0.9% sodium chloride, the balance being deionized water.
Preparation of R2 reagent: 1) preparing a diluent according to the component content of the diluent of the R2 reagent; 2) preparing an alkaline phosphatase-labeled troponin T antibody; 3) the alkaline phosphatase-labeled troponin T antibody was diluted with a dilution of the R2 reagent.
Preparing a magnetic separation reagent: 1) preparing a troponin T monoclonal antibody coated magnetic particle, measuring the magnetic particle, adsorbing by using a magnet, cleaning for 2-3 times by using 0.1M Hepes PH7.5, adding a diluent of a magnetic separation reagent to enable the final concentration of the magnetic particle to be 10mg/ml, adding an antibody to enable the concentration of the antibody to be 0.1mg/ml, uniformly mixing for 18 hours at 37 ℃, adding 10% BSA (bovine serum albumin) for sealing, uniformly mixing for 6 hours at 37 ℃, adsorbing by using the magnet, discarding the supernatant, cleaning for 3 times by using 25mM Tris-HCl PH7.2 and 0.15M NaCl 0.05% Tween20, cleaning once by using a diluent of the magnetic separation reagent and fixing the volume to enable the final concentration of the magnetic particle to be 10mg/ml, and obtaining a concentrated solution; 2) the concentrate was diluted with a diluent of the magnetic separation reagent.
Preparing a calibrator liquid series: 1) preparing a buffer solution according to the component content of the buffer solution of the calibrator; 2) dissolving troponin T antigens with different concentrations in the buffer solution of the calibrator respectively to prepare calibrator solutions with different concentrations.
Preparing a chemiluminescent substrate solution: 1) preparing Tris-HCl buffer solution, and adding dioxane compound (APCL); 2) a chemiluminescent substrate that catalyzes luminescence by alkaline phosphatase is dissolved in a buffer.
Determination method of magnetic particle chemiluminescence detection kit for hypersensitive troponin T content and standard curve drawing
(1) Firstly, 50 mul of calibrator series (with the concentration of 0, 30, 100, 500, 2000, 6000pg/mL respectively) and 50 mul of R1 reagent and 50 mul of R2 reagent are respectively added into a reaction tube in turn, and 50 mul of magnetic separation reagent is mixed and incubated for 10min at 37 ℃;
(2) washing with a washing solution 3 times to remove unbound antibodies and impurities;
(3) adding 150 μ l of luminescent substrate solution, catalyzing substrate luminescence with ALP, and measuring relative luminescence intensity (RLU) with a Lindman chemiluminescence detector to obtain troponin T concentration-luminescence value series numerical values.
(4) Fitting is carried out according to the troponin T concentration-luminous value series numerical values to obtain a troponin T concentration-luminous value standard curve.
(5) And calculating the concentration value of the sample to be measured according to the fitted troponin T concentration-luminous value standard curve.
In a certain range, the RLU is in direct proportion to the concentration of troponin T antigen, and the content of troponin T in the sample to be detected can be read from the standard curve by an interpolation method.
The data of methodology identification when the detection kit is used for determining the content of the hypersensitive troponin T can reach the following indexes:
the lowest detection limit is 3 pg/mL;
the linear detection range of the troponin T content is 3-6000 pg/mL;
the precision is 2.32 percent (n is 10) on average in analysis, the precision between analyses is 4.51 percent (n is 10) on average, and the precision is far lower than the national requirement, so that the kit has good repeatability in the experimental process;
accuracy, adopting serum with known troponin T concentration to dilute with de-hormone serum in different proportions to achieve 85-115% recovery rate;
the specificity, the crossing rate with cardiac troponin I, troponin C and skeletal muscle troponin T is less than 0.1%.
Example 1 a magnetic particle chemiluminescence assay kit for determining the content of hypersensitive troponin T comprises R2 reagent, magnetic separation reagent, calibrator liquid series, and substrate liquid.
In this example, R1 reagent: 0.9% sodium chloride, the balance being deionized water.
The R2 reagent includes: 1) r2 antibody: alkaline phosphatase-labeled troponin T antibody with the concentration of 0.3 mug/ml; 2) diluting liquid: dilutions of the R2 reagent were at pH 6.8 and included: tris, the concentration is 12.04 g/L; sodium azide with the concentration of 1 g/L; sodium chloride with concentration of 5.79 g/L; bovine serum albumin with a concentration of 10 g/L; the trehalose concentration is 50 g/L; MgCl2The concentration is 1.0 mM; ZnCl2The concentration is 0.1 mM; the Brij 35 concentration is 0.35%; the balance of deionized water.
In this exampleThe magnetic separation reagent comprises: 1) magnetic particles: the concentration of the magnetic particles coated by the troponin T monoclonal antibody is 1 mg/ml; 2) the pH value of the diluent of the magnetic separation reagent is 8.0, and the diluent comprises: tris, the concentration is 12.04 g/L; sodium azide with the concentration of 1 g/L; sodium chloride with concentration of 5.79 g/L; bovine serum albumin with a concentration of 5 g/L; MgCl2The concentration is 1.0 mM; ZnCl2The concentration is 0.1 mM; the balance of deionized water.
In this embodiment, the calibration liquid includes: 1) Tris-HCl buffer of troponin T antigen; 2) buffer solution: comprises 0.1M Tris-HCl and bovine serum albumin with the concentration of 20.0 g/L; casein sodium, the concentration is 2 g/L; sodium chloride, concentration 8.5g/L, sodium azide, concentration 1 g/L. The pH of the buffer of the calibrator liquid series was 7.4. The calibration lines were listed as containing different concentrations (0, 30, 100, 500, 2000, 6000pg/ml) of the actual line.
In this example, the substrate solution was a chemiluminescent substrate solution in which luminescence was catalyzed by alkaline phosphatase, wherein the concentration of the dioxane compound (APCL) in the luminescent substrate solution was 0.3mg/mL, the buffer solution was Tris-HCl with a molar concentration of 0.2M, and the pH was 9.3.
Example 2 preparation and determination methods of magnetic particle chemiluminescence assay kit for determining hypersensitive troponin T content
(1) First, 50. mu.l of the calibrator series (concentrations of 0, 30, 100, 500, 2000, 6000pg/ml, respectively) of example 1 and 50. mu.l of the R1 reagent, 50. mu.l of the R2 reagent, and 50. mu.l of the magnetic separation reagent of example 1 were sequentially added to a reaction tube, and mixed and incubated at 37 ℃ for 10 min;
(2) washing with a washing solution 3 times to remove unbound antibodies and impurities;
(3) after adding 150. mu.l of the luminescent substrate solution of example 1 and allowing the ALP-catalyzed substrate to emit light, the relative luminescence intensity (RLU) was measured using a Lindman self-developed chemiluminescence detector, and the results are shown in the following table:
TABLE 1
| Calibrator (pg/ml) | 0 | 30 | 100 | 500 | 2000 | 6000 |
| RLU(100000) | 0.015 | 0.825 | 2.716 | 13.783 | 46.068 | 92.948 |
(5) Fitting was performed according to the values of table 1 to obtain a troponin T concentration-luminescence value standard curve shown in fig. 1.
(6) Adding 50 μ l of sample to be tested, 50 μ l of R1 reagent and 50 μ l of R2 reagent in the example 1 into a reaction tube in sequence, and mixing and incubating for 10min at 37 ℃;
(7) washing with a washing solution 3 times to remove unbound antibodies and impurities;
(8) adding 150 μ l of luminescent substrate solution of example 1, and measuring relative luminescence intensity RLU of 5125412 with a Lindman self-developed chemiluminescence detector after ALP catalysis substrate luminescence;
(10) and (5) calculating the troponin T concentration value corresponding to the to-be-detected sample 5125412 to be 2304.41pg/mL according to the troponin T concentration-luminous value standard curve in the step (5).
The magnetic particle chemiluminescence detection kit for the content of the hypersensitive troponin T can be used together with a full-automatic chemiluminescence analyzer, the operation steps are greatly simplified, the detection speed and the detection flux are increased, the detection efficiency is improved, and errors caused by manual operation are avoided.
The kit of the invention has the advantages that the lowest detection limit reaches the hypersensitive level, and the result is shown in the table 2:
TABLE 2
The kit and the imported kit of the invention are used for comparing the measured values of clinical samples:
102 parts of human serum samples are simultaneously detected by using the kit provided by the invention and the imported kit. The detection result is shown in the attached figure 2, the cTnT concentration in the serum determined by adopting the imported kit is used as the abscissa, the determination result of the kit provided by the invention is used as the ordinate to perform regression analysis, and the correlation equation is as follows: y is 1.0249x-18.093 and the correlation coefficient r is 0.997.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to those examples; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Nothing in this specification is said to apply to the prior art.
Claims (10)
1. A magnetic particle chemiluminescence detection kit for determining the content of hypersensitive troponin T comprises: r1 reagent, R2 reagent, magnetic separation reagent, calibrator liquid series and chemiluminescent substrate liquid; the method is characterized in that:
the R1 reagent is a buffer solution,
the magnetic separation reagent is a troponin T monoclonal antibody coated magnetic particle solution prepared by dissolving troponin T monoclonal antibody coated magnetic particles in a diluent of the magnetic separation reagent,
the R2 reagent is alkaline phosphatase-labeled troponin T monoclonal antibody solution prepared by dissolving alkaline phosphatase-labeled troponin T monoclonal antibody in the diluent of the R2 reagent,
the chemiluminescence substrate solution is a chemiluminescence substrate solution which is catalyzed by alkaline phosphatase to emit light.
2. The kit of claim 1, wherein the R1 reagent is normal saline or the same as any one of the composition of a dilution of the R2 reagent and the composition of a dilution of the magnetic separation reagent.
3. The kit according to claim 1, wherein the magnetic separation reagent, namely the magnetic particles coated by the troponin T monoclonal antibody, is dissolved in a diluent of the magnetic separation reagent to prepare a magnetic particle solution coated by the troponin T monoclonal antibody of 0.8-1.2 mg/mL; the pH of the diluent of the magnetic separation reagent is 7.5-8.5, and the method comprises the following steps: tris, the concentration is 12.0-12.3 g/L; sodium azide with the concentration of 0.9-1.1 g/L; sodium chloride with the concentration of 5.7-5.9 g/L; bovine serum albumin with the concentration of 3-6 g/L; MgCl2The concentration is 0.8 to 1.2 mM; ZnCl2The concentration is 0.08-0.12 mM; the balance of deionized water.
4. The kit according to claim 3, wherein the magnetic separation reagent, i.e., the troponin T monoclonal antibody-coated magnetic microparticles, is dissolved in a diluent to prepare a troponin T monoclonal antibody-coated magnetic microparticle solution of 1.0 mg/mL; buffer for magnetic separation of reagentsThe pH value of the flushing liquid is 8.0, and the flushing liquid comprises: tris, the concentration is 12.04 g/L; sodium azide with the concentration of 1 g/L; sodium chloride with concentration of 5.79 g/L; bovine serum albumin with a concentration of 5 g/L; MgCl2The concentration is 1.0 mM; ZnCl2The concentration is 0.1 mM; the balance of deionized water.
5. The kit according to claim 1, characterized in that the preparation of the troponin T monoclonal antibody coated magnetic microparticles is carried out by: measuring magnetic particles, adsorbing by using a magnet, washing for 2-3 times by using a buffer solution with 0.1M Hepes pH7.5, and adding a diluent of a magnetic separation reagent to enable the final concentration of magnetic beads to be 8-12 mg/mL; and adding the antibody to make the concentration of the antibody be 0.05-0.2 mg/mL, uniformly mixing for 14-22 hours at 37 ℃, adding 5-15% BSA for sealing, uniformly mixing for 4-8 hours at 37 ℃, adsorbing by using a magnet, discarding the supernatant, cleaning for 3 times by using a cleaning solution consisting of 25mM Tris-HCl pH7.2, 0.15M NaCl and 0.05% Tween20, cleaning for one time by using a diluent of a magnetic separation reagent, and fixing the volume to make the final concentration of the magnetic beads be 8-12 mg/mL to obtain a concentrated solution.
6. The kit of claim 1, wherein the R2 reagent, namely the alkaline phosphatase-labeled troponin T monoclonal antibody, is dissolved in a diluent of the R2 reagent to prepare a 0.2-0.5 μ g/mL alkaline phosphatase-labeled troponin T monoclonal antibody solution;
the pH value of the diluent of the R2 reagent is 6.7-7.6, and the method comprises the following steps: tris, the concentration is 12.0-12.3 g/L; sodium azide with the concentration of 0.9-1.1 g/L; sodium chloride with the concentration of 5.7-5.9 g/L; bovine serum albumin with the concentration of 8-12 g/L; the trehalose concentration is 30-50 g/L; MgCl2The concentration is 0.8 to 1.2 mM; ZnCl2The concentration is 0.08-0.12 mM; the Brij 35 concentration is 0.01-1%; the balance of deionized water.
7. The kit of claim 6, wherein the R2 reagent, alkaline phosphatase-labeled troponin T monoclonal antibody, is dissolved in a diluent of R2 reagent to prepare a 0.3 μ g/mL alkaline phosphatase-labeled troponin T monoclonal antibody solution; the R2 testThe pH of the diluent of the formulation was 6.8, including: tris, the concentration is 12.04 g/L; sodium azide with the concentration of 1 g/L; sodium chloride with concentration of 5.79 g/L; bovine serum albumin with a concentration of 10 g/L; the trehalose concentration is 50 g/L; MgCl2The concentration is 1.0 mM; ZnCl2The concentration is 0.1 mM; the Brij 35 concentration is 0.35%; the balance of deionized water.
8. The kit according to claim 1, wherein the chemiluminescent substrate solution is a chemiluminescent substrate solution catalyzed and luminescent by alkaline phosphatase at a molar concentration of 0.1-0.3M, a Tris-HCl buffer solution at a molar concentration of 0.2M and having a pH value of 8-10, and contains dioxane compound (APCL) at 0.2-0.4 mg/mL; the calibrator liquid is Tris-HCl buffer solution containing troponin T antigens with different concentrations;
the buffer solution of the calibrator liquid series comprises: bovine serum albumin with a concentration of 20.0 g/L; sodium chloride with the concentration of 8.5g/L, casein sodium with the concentration of 2g/L, sodium azide with the concentration of 1 g/L;
the concentration range of the calibrator liquid is 0-6000 pg/mL, and the pH value of the buffer liquid is 7.4.
9. The kit according to claim 1, characterized in that the kit further comprises a cleaning solution, wherein the cleaning solution is a Tris-HCl buffer solution with a value of 0.1-0.2M, pH of 8-9, and 0.01-0.04% of Tween20 and 15-20% of sodium chloride in percentage by mass are added; preferably, 0.02 mass percent of tween20 and 15 mass percent of sodium chloride are added into a Tris-HCl buffer solution with the cleaning solution of 0.1M and the pH value of 8.
10. The kit according to any one of claims 1 to 9, wherein the minimum detection limit for assaying hypersensitive troponin T is not more than 3 pg/mL; the linear detection range of the hypersensitive troponin T is 3-6000 pg/mL; the intra-assay precision was 2.32% on average (n ═ 10), and the inter-assay precision was 4.51% on average (n ═ 10); diluting serum with known troponin T concentration by adopting a calibrator buffer solution according to different proportions, and then obtaining a recovery rate of 85-115%;
the crossing rate with cardiac troponin I, troponin C and skeletal troponin T is less than 0.1%.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113607962A (en) * | 2021-08-06 | 2021-11-05 | 三诺生物传感股份有限公司 | Preservation solution for cTnI antibody coated magnetic beads and preparation method thereof |
| CN114184603A (en) * | 2021-11-11 | 2022-03-15 | 宁波海壹生物科技有限公司 | Kit for determining copeptin by magnetic particle chemiluminescence method |
| CN114280307A (en) * | 2021-11-08 | 2022-04-05 | 宏葵生物（中国）股份有限公司 | Nano magnetic particle chemiluminescence detection kit for hypersensitive cardiac troponin I and preparation method and application thereof |
| CN115047183A (en) * | 2022-06-08 | 2022-09-13 | 广州康盛生物科技股份有限公司 | Detection reagent and preparation method and application thereof |
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| CN116298265A (en) * | 2023-04-12 | 2023-06-23 | 上海品峰医疗科技有限公司 | hs-cTnT chemiluminescent assay kit applying multistage signal amplification technology |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080102481A1 (en) * | 2006-10-26 | 2008-05-01 | Abbott Laboratories | Assay for cardiac troponin autoantibodies |
| CN105158483A (en) * | 2015-09-16 | 2015-12-16 | 北京九强生物技术股份有限公司 | Quantitative determination kit for hypersensitivity of Troponin I and detection method |
| US20180164306A1 (en) * | 2016-12-09 | 2018-06-14 | Abbott Point Of Care Inc. | Dual range cardiac troponin immunoassay devices and methods using immunosensor and magnetic immunosensor |
| CN109596835A (en) * | 2018-11-23 | 2019-04-09 | 深圳天辰医疗科技有限公司 | A kind of detection method of detection kit and preparation method thereof and troponin T |
| CN110082541A (en) * | 2019-05-14 | 2019-08-02 | 深圳天深医疗器械有限公司 | High quick Troponin I kit and sample treatment solution |
-
2020
- 2020-11-16 CN CN202011279944.9A patent/CN112526140A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080102481A1 (en) * | 2006-10-26 | 2008-05-01 | Abbott Laboratories | Assay for cardiac troponin autoantibodies |
| CN105158483A (en) * | 2015-09-16 | 2015-12-16 | 北京九强生物技术股份有限公司 | Quantitative determination kit for hypersensitivity of Troponin I and detection method |
| CN109580954A (en) * | 2015-09-16 | 2019-04-05 | 北京九强生物技术股份有限公司 | A kind of super quick quantitative determination reagent kit and its detection method of human troponin I |
| US20180164306A1 (en) * | 2016-12-09 | 2018-06-14 | Abbott Point Of Care Inc. | Dual range cardiac troponin immunoassay devices and methods using immunosensor and magnetic immunosensor |
| CN109596835A (en) * | 2018-11-23 | 2019-04-09 | 深圳天辰医疗科技有限公司 | A kind of detection method of detection kit and preparation method thereof and troponin T |
| CN110082541A (en) * | 2019-05-14 | 2019-08-02 | 深圳天深医疗器械有限公司 | High quick Troponin I kit and sample treatment solution |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113607962A (en) * | 2021-08-06 | 2021-11-05 | 三诺生物传感股份有限公司 | Preservation solution for cTnI antibody coated magnetic beads and preparation method thereof |
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