CN111781374A - Magnetic particle chemiluminescence detection kit for hypersensitive troponin I and application thereof - Google Patents
Magnetic particle chemiluminescence detection kit for hypersensitive troponin I and application thereof Download PDFInfo
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- CN111781374A CN111781374A CN202010649243.3A CN202010649243A CN111781374A CN 111781374 A CN111781374 A CN 111781374A CN 202010649243 A CN202010649243 A CN 202010649243A CN 111781374 A CN111781374 A CN 111781374A
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Abstract
The invention discloses a magnetic particle chemiluminescence detection kit for hypersensitive troponin I and application thereof, and relates to the technical field of biology. The detection kit comprises a calibrator, a quality control product, a first anti-reagent, a second anti-reagent, a magnetic particle reagent and a luminescent substrate, wherein the first anti-reagent is a solution formed by mixing a first troponin I antibody marked by fluorescein isothiocyanate and a second troponin I antibody marked by fluorescein isothiocyanate according to the volume ratio of 1: 1; the second anti-reagent is a solution formed by mixing a third troponin I antibody marked by alkaline phosphatase and a fourth troponin I antibody marked by the alkaline phosphatase according to the volume ratio of 1: 1; the magnetic particle reagent is a magnetic microsphere coated by an anti-fluorescein isothiocyanate antibody. The invention has the advantages of accurate detection result, strong specificity, high sensitivity, wide linear range, short detection time and convenient use.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a magnetic particle chemiluminescence detection kit for hypersensitive troponin I and application thereof.
Background
Cardiac Troponin I (cTnI) is a marker highly correlated with Myocardial Injury (MI). At present, in the diagnosis of MI, because of the advantages of high sensitivity, good specificity and long duration, cTnI becomes the best marker for judging the MI risk of patients with Acute Coronary Syndrome (ACS), has replaced Creatine kinase isozyme MB (CK-MB), and becomes the gold standard for diagnosing Acute Myocardial Infarction (AMI). With the intensive research on myocardial infarction and troponin I, higher requirements are clinically put forward on the sensitivity and real-time property of troponin I detection.
In humans, there are three distinct subtypes of TnI, the cardiac, fast and slow skeletal muscle subtypes, respectively, with cTnI being highly correlated with cardiac muscle and having a molecular weight of about 29 KDa. Sequence homology of cTnI to fast and slow skeletal muscle subtypes is as high as 52% and 46%, respectively. Since hypersensitivity cTnI has higher requirements for cross-reaction of antibodies, if patient peripheral sTnI is elevated, false positive results may be caused due to high sensitivity of the reagent. Ensuring no cross-reactivity with both fast and slow skeletal muscle subtypes is therefore a factor that must be considered in designing a cTnI detection system.
When the cTnI molecule is released into the blood, it is rapidly decomposed into two molecules, i.e., cTnI-cTnC complex form and cTnT by proteolytic enzymes, and thus even if cTnI exists in the serum, it rarely exists in a free form, but remains mainly in the blood as a cTnI-cTnC complex. The central part of cTnI can interact strongly with cTnC, is not easily degraded by endogenous proteases, and has a significantly improved stability. Thus, the epitope in the center of cTnI is much more stable than the epitope at the ends. However, because of this, not all antibodies that specifically bind to the central portion of the cTnI molecule are able to recognize cTnI, because cTnC competitively interferes with the binding of the antibody to the cTnI molecule.
Due to the particularity of the molecular structure of cTnI, when patients are subjected to quantitative detection of cTnI in blood, various interference factors such as: phosphorylation, proteolysis, autoantibodies, etc. may all affect the detection result, and the effect is different according to the different epitope of the antibody used by the detection reagent. At present, in the detection system of the cTnI in the market, one or two pairs of antibodies capable of capturing the cTnI molecules are selected, and the detection system is susceptible to competitive influence of the cTnC when detecting the cTnI-cTnC compound in a serum sample, and is also susceptible to interference of phosphorylation, proteolysis and the like, so that the need for a cTnI detection system with higher sensitivity and more accurate detection result is very necessary.
Disclosure of Invention
The invention provides a magnetic particle chemiluminescence detection kit for hypersensitive troponin I, which has the advantages of accurate detection result, strong specificity, high sensitivity, wide linear range, short detection time and convenient use, and application thereof.
In order to solve the technical problems, the invention provides the following technical scheme:
a hypersensitive troponin I magnetic particle chemiluminescence detection kit comprises a calibrator, a quality control product, a first anti-reagent, a second anti-reagent, a magnetic particle reagent and a luminescent substrate, wherein,
the first anti-reagent is a solution formed by mixing a first troponin I antibody marked by fluorescein isothiocyanate and a second troponin I antibody marked by the fluorescein isothiocyanate according to the volume ratio of 1: 1;
the second anti-reagent is a solution formed by mixing a third troponin I antibody marked by alkaline phosphatase and a fourth troponin I antibody marked by the alkaline phosphatase according to the volume ratio of 1: 1;
the magnetic particle reagent is a magnetic microsphere coated by an anti-fluorescein isothiocyanate antibody.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention combines the double antibody sandwich immunoassay principle with the superparamagnetic nano microsphere marking technology and the enzyme catalysis chemiluminescence immunoassay to quantitatively detect the product, and simultaneously combines the fluorescein isothiocyanate system with the magnetic particle system, thereby not only providing a reaction which is nearly homogeneous, but also avoiding the negative effect brought by steric hindrance when one or more antibodies are directly coupled on the magnetic microspheres, leading the reaction to have more specificity, and further laying a good foundation for the excellent performance of the kit;
2. in the invention, three antibodies which are respectively and specifically combined with different epitopes of troponin I antigen are selected, and meanwhile, the antibody which can be specifically combined with the cTnC epitope in the cTnI-cTnC compound is introduced on the basis, so that the advantage that the cTnI-cTnC compound can stably exist in serum is fully utilized, the competitive combination of the cTnC to the cTnI antibody is avoided, and the detection sensitivity can be obviously improved.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.
It should be noted that each reagent material used in the examples is commercially available, each apparatus used is also an existing apparatus in the art, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition according to the manufacturer's recommendation.
Example 1
The magnetic particle chemiluminescence detection kit for the hypersensitive troponin I comprises a magnetic particle reagent, a first anti-reagent, a second anti-reagent, a calibrator, a quality control material and a luminescent substrate.
1. The magnetic particle reagent was formulated as follows:
step 1: magnetic beads 0.5um in diameter were washed three times with 5mM MEST (containing 0.1% Tween 20) buffer at pH 5.0 and resuspended in MES buffer to obtain 10mg/mL of the first solution;
step 2: adding 25mg of EDC and NHS into the first solution, and coupling for 5 hours at room temperature to obtain a second solution;
and step 3: the second solution was washed three times with PBST (containing 0.1% Tween 20) buffer solution at pH 7.0 and a concentration of 10mM, and resuspended in PBS buffer solution, 50ug of goat anti-fluorescein isothiocyanate antibody was added, after coupling overnight at 20-25 ℃, left to stand in a magnetic field for 3min, the supernatant was aspirated, after which pH 7.4 containing 2.0% by mass volume BSA, phosphate buffer solution at a concentration of 0.01mol/L was added and blocked with stirring at 37 ℃ for 2 hours to obtain a third solution;
and 4, step 4: washing the third solution for 3 times by using a buffer solution, and preparing a 10mg/mL primary solution of the magnetic microspheres by using the buffer solution;
the buffer solution in the step is a phosphate buffer solution with the pH value of 7.4, the volume fraction of 0.01 percent of Tween-20, the mass volume fraction of 1.0 percent of casein and 0.01 percent of sodium azide.
And 5: and diluting the primary solution of the magnetic microspheres by using a buffer solution to prepare a magnetic particle reagent, wherein the concentration of the magnetic particle reagent is 1.0 mg/ml.
The buffer solution in the step is a phosphate buffer solution with the pH value of 7.4, the volume fraction of 0.01 percent of Tween-20, the mass fraction of 2.0 percent of bovine serum albumin, 1.0 percent of casein and 0.01 percent of sodium azide.
2. Preparation of anti-agents
1) The preparation method of the first anti-reagent comprises the following steps:
respectively dialyzing a first troponin I antibody and a second troponin I antibody to be coupled by using a carbonate buffer solution with the pH of 9.6 to prepare a first troponin I antibody solution and a second troponin I antibody solution;
wherein the first troponin I antibody specifically binds to a 20-30 epitope of the troponin I antigen and the second troponin I antibody specifically binds to a 40-50 epitope of the troponin I antigen.
Dissolving fluorescein isothiocyanate into 5mg/ml solution by using a buffer solution containing DMSO, preparing the solution at present, and keeping out of the sun to prepare the fluorescein isothiocyanate solution;
respectively mixing the first troponin I antibody solution and the second troponin I antibody solution with fluorescein isothiocyanate solution at a ratio of 100ug:1mg, reacting overnight in the dark, and adding NH4Stopping the reaction by Cl to respectively prepare a fluorescein isothiocyanate-first troponin I antibody mixed solution and a fluorescein isothiocyanate-second troponin I antibody mixed solution;
separating the fluorescein isothiocyanate-first troponin I antibody mixed solution and the fluorescein isothiocyanate-second troponin I antibody mixed solution by using a gel chromatography separation column respectively to obtain a fluorescein isothiocyanate-first troponin I antibody conjugate and a fluorescein isothiocyanate-second troponin I antibody conjugate;
the fluorescein isothiocyanate-first troponin I antibody conjugate and the fluorescein isothiocyanate-second troponin I antibody conjugate were diluted with an anti-reagent buffer, and the diluted fluorescein isothiocyanate-first troponin I antibody conjugate and the fluorescein isothiocyanate-second troponin I antibody conjugate were mixed at a ratio of 1:1, to obtain a first anti-reagent having a concentration of 1.5 μ g/ml.
2) The preparation method of the second anti-reagent comprises the following steps:
respectively dialyzing a third troponin I antibody to be coupled and a fourth troponin I antibody to be coupled by using a carbonate buffer solution with the pH of 9.6 to prepare a third troponin I antibody solution and a fourth troponin I antibody solution;
wherein the third troponin I antibody specifically binds to an epitope 83-93 of the troponin I antigen and the fourth troponin I antibody specifically binds to an epitope cTnC antigen in the cTnI-cTnC complex.
Dissolving alkaline phosphatase to 2mg/ml by using PBS buffer solution containing glutaraldehyde, and preparing the solution on site when the solution is used and keeping out of the sun;
respectively and uniformly mixing a third troponin I antibody solution and a fourth troponin I antibody solution with an alkaline phosphatase solution according to the molar ratio of 1:1, adding a lysine solution, and reacting overnight to prepare an alkaline phosphatase-third troponin I antibody mixed solution and an alkaline phosphatase-fourth troponin I antibody mixed solution;
separating the alkaline phosphatase-third troponin I antibody mixed solution and the alkaline phosphatase-fourth troponin I antibody mixed solution by adopting a gel chromatography separation column respectively to obtain an alkaline phosphatase-third troponin I antibody conjugate and an alkaline phosphatase-fourth troponin I antibody conjugate;
the alkaline phosphatase-third troponin I antibody conjugate and the alkaline phosphatase-fourth troponin I antibody conjugate were diluted with an anti-reagent buffer, respectively, and the diluted alkaline phosphatase-third troponin I antibody conjugate and alkaline phosphatase-fourth troponin I antibody conjugate were mixed in the following ratio of 1:1 to obtain a second anti-reagent with a concentration of 1.0. mu.g/ml.
3) The anti-reagent buffer solution comprises the following components in percentage by concentration:
final pH 8.0.
3. Preparation of calibrator and quality control
Troponin I was dissolved in the calibrator/quality control buffer to prepare calibrators and quality controls at target concentrations as shown in the following table.
The components and the concentrations of the calibrator buffer are as follows:
final PH 7.0.
4. The cleaning solution comprises the following components in percentage by weight:
5. the luminescent substrate is a buffer solution containing (adamantane) -1, 2-dioxyethane and derivatives thereof.
6. And subpackaging and sealing the magnetic particle reagent, the first anti-reagent, the second anti-reagent, the calibrator, the quality control product, the luminescent substrate and the cleaning solution to obtain the troponin I magnetic particle chemiluminescence detection kit.
Example 2
The magnetic particle chemiluminescence detection kit for the hypersensitive troponin I comprises a magnetic particle reagent, a first anti-reagent, a second anti-reagent, a calibrator, a quality control material and a luminescent substrate.
1. The magnetic particle reagent was formulated as follows:
step 1: the magnetic beads with a diameter of 0.2um were washed three times with PBST (containing 0.5% Tween 20) buffer solution at a concentration of 10mM at pH 5.5 and resuspended in PBS buffer solution to obtain a first solution of 5 mg/mL;
step 2: adding 10mg of EDC and NHS into the first solution, and coupling for 5 hours at room temperature to obtain a second solution;
and step 3: the second solution was washed three times with 15mM PBST (containing 0.5% Tween 20) buffer solution at pH 7.5 and resuspended in PBS buffer solution, 20ug of goat anti-fluorescein isothiocyanate antibody was added, after coupling overnight at 20-25 ℃, left to stand in a magnetic field for 2min, the supernatant was aspirated, after which phosphate buffer solution at pH 7.0 containing 1.0% by mass of BSA by volume at a concentration of 0.01mol/L was added and blocked with stirring at 37 ℃ for 2 hours to obtain a third solution;
and 4, step 4: washing the third solution for 3 times by using a buffer solution, and preparing a primary solution of 5mg/mL magnetic microspheres by using the buffer solution;
the buffer in this step is phosphate buffer with pH 7.0, tween-20 containing 0.05% by volume, casein containing 0.5% by mass by volume, and sodium azide containing 0.05%.
And 5: and diluting the primary solution of the magnetic microspheres by using a buffer solution to prepare a magnetic particle reagent, wherein the concentration of the magnetic particle reagent is 0.5 mg/ml.
The buffer in this step was phosphate buffer with pH 7.0, containing tween-20 in a volume fraction of 0.05%, bovine serum albumin in a mass volume fraction of 1.0%, 1.5% casein and 0.05% sodium azide.
2. Preparation of anti-agents
1) The preparation method of the first anti-reagent comprises the following steps:
respectively dialyzing a first troponin I antibody and a second troponin I antibody to be coupled by using a carbonate buffer solution with the pH of 9.0 to prepare a first troponin I antibody solution and a second troponin I antibody solution;
wherein the first troponin I antibody specifically binds to a 20-30 epitope of the troponin I antigen and the second troponin I antibody specifically binds to a 40-50 epitope of the troponin I antigen.
Dissolving fluorescein isothiocyanate into 10mg/ml solution by using a buffer solution containing DMSO, preparing the solution at present, and keeping out of the sun to prepare the fluorescein isothiocyanate solution;
respectively mixing the first troponin I antibody solution and the second troponin I antibody solution with fluorescein isothiocyanate solution at a ratio of 150ug:1mg, reacting overnight in the dark, and adding NH4Stopping the reaction by Cl to respectively prepare a fluorescein isothiocyanate-first troponin I antibody mixed solution and a fluorescein isothiocyanate-second troponin I antibody mixed solution;
separating the fluorescein isothiocyanate-first troponin I antibody mixed solution and the fluorescein isothiocyanate-second troponin I antibody mixed solution by using a gel chromatography separation column respectively to obtain a fluorescein isothiocyanate-first troponin I antibody conjugate and a fluorescein isothiocyanate-second troponin I antibody conjugate;
the fluorescein isothiocyanate-first troponin I antibody conjugate and the fluorescein isothiocyanate-second troponin I antibody conjugate were diluted with an anti-reagent buffer, and the diluted fluorescein isothiocyanate-first troponin I antibody conjugate and the fluorescein isothiocyanate-second troponin I antibody conjugate were mixed at a ratio of 1:1, to obtain a first anti-reagent having a concentration of 1.0 μ g/ml.
2) The preparation method of the second anti-reagent comprises the following steps:
respectively dialyzing a third troponin I antibody to be coupled and a fourth troponin I antibody to be coupled by using a carbonate buffer solution with the pH of 9.0 to prepare a third troponin I antibody solution and a fourth troponin I antibody solution;
wherein the third troponin I antibody specifically binds to an epitope 83-93 of the troponin I antigen and the fourth troponin I antibody specifically binds to an epitope cTnC antigen in the cTnI-cTnC complex.
Dissolving alkaline phosphatase to 6mg/ml by using PBS buffer solution containing glutaraldehyde, and preparing the solution on site when the solution is used and keeping out of the sun;
respectively and uniformly mixing a third troponin I antibody solution and a fourth troponin I antibody solution with an alkaline phosphatase solution according to the molar ratio of 1:1, adding a lysine solution, and reacting overnight to prepare an alkaline phosphatase-third troponin I antibody mixed solution and an alkaline phosphatase-fourth troponin I antibody mixed solution;
separating the alkaline phosphatase-third troponin I antibody mixed solution and the alkaline phosphatase-fourth troponin I antibody mixed solution by adopting a gel chromatography separation column respectively to obtain an alkaline phosphatase-third troponin I antibody conjugate and an alkaline phosphatase-fourth troponin I antibody conjugate;
the alkaline phosphatase-third troponin I antibody conjugate and the alkaline phosphatase-fourth troponin I antibody conjugate were diluted with an anti-reagent buffer, respectively, and the diluted alkaline phosphatase-third troponin I antibody conjugate and alkaline phosphatase-fourth troponin I antibody conjugate were mixed in the following ratio of 1:1, to obtain a second anti-reagent at a concentration of 0.5. mu.g/ml.
4) The anti-reagent buffer solution comprises the following components in percentage by concentration:
final pH 8.5.
3. Preparation of calibrator and quality control
Troponin I was dissolved in the calibrator/quality control buffer to prepare calibrators and quality controls at target concentrations as shown in the following table.
The components and the concentrations of the calibrator buffer are as follows:
final PH 7.4.
4. The cleaning solution comprises the following components in percentage by weight:
5. the luminescent substrate was the same as in example 1.
6. And subpackaging and sealing the magnetic particle reagent, the first anti-reagent, the second anti-reagent, the calibrator, the quality control product, the luminescent substrate and the cleaning solution to obtain the troponin I magnetic particle chemiluminescence detection kit.
Example 3
The magnetic particle chemiluminescence detection kit for the hypersensitive troponin I comprises a magnetic particle reagent, a first anti-reagent, a second anti-reagent, a calibrator, a quality control material and a luminescent substrate.
1. The magnetic particle reagent was formulated as follows:
step 1: magnetic beads with a diameter of 1um were washed three times with TrisT (containing 1.0% Tween 20) buffer solution at a concentration of 20mM at pH 6.0 and resuspended in Tris buffer solution to obtain a first solution of 15 mg/mL;
step 2: adding 50mg of toluene and NHS into the first solution, and coupling for 5 hours at room temperature to obtain a second solution;
and step 3: washing the second solution three times with 10mM TrisT (containing 1.0% Tween 20) buffer solution with pH 8.0, resuspending with Tris buffer solution, adding 100ug of goat anti-fluorescein isothiocyanate antibody, coupling overnight at 20-25 deg.C, standing in a magnetic field for 10min, aspirating the supernatant, adding 0.3mol/L phosphate buffer solution with pH 8.0 containing 1.5% BSA by mass volume, and blocking with stirring at 37 deg.C for 5 hours to obtain a third solution;
and 4, step 4: washing the third solution for 3 times by using a buffer solution, and preparing a 20mg/mL primary solution of the magnetic microspheres by using the buffer solution;
the buffer in this step is a phosphate buffer with pH 8.0, tween-20 containing 0.1% by volume, casein containing 1.5% by mass by volume, and sodium azide containing 0.1% by mass.
And 5: and diluting the primary solution of the magnetic microspheres by using a buffer solution to prepare a magnetic particle reagent, wherein the concentration of the magnetic particle reagent is 2.0 mg/ml.
The buffer in this step is a phosphate buffer with pH 8.0, containing tween-20 in a volume fraction of 0.1%, bovine serum albumin in a mass volume fraction of 2.0%, 1.0% casein and 0.1% sodium azide.
2. Preparation of anti-agents
1) The preparation method of the first anti-reagent comprises the following steps:
respectively dialyzing a first troponin I antibody and a second troponin I antibody to be coupled by using a carbonate buffer solution with the pH of 8.0 to prepare a first troponin I antibody solution and a second troponin I antibody solution;
wherein the first troponin I antibody specifically binds to a 20-30 epitope of the troponin I antigen and the second troponin I antibody specifically binds to a 40-50 epitope of the troponin I antigen.
Dissolving fluorescein isothiocyanate into a solution of 1mg/ml by using a buffer solution containing DMSO, preparing the solution at present, and keeping out of the sun to prepare a fluorescein isothiocyanate solution;
respectively mixing the first troponin I antibody solution and the second troponin I antibody solution with fluorescein isothiocyanate solution at a ratio of 200ug:1mg, reacting overnight in the dark, and adding NH4Stopping the reaction by Cl to respectively prepare a fluorescein isothiocyanate-first troponin I antibody mixed solution and a fluorescein isothiocyanate-second troponin I antibody mixed solution;
separating the fluorescein isothiocyanate-first troponin I antibody mixed solution and the fluorescein isothiocyanate-second troponin I antibody mixed solution by using a gel chromatography separation column respectively to obtain a fluorescein isothiocyanate-first troponin I antibody conjugate and a fluorescein isothiocyanate-second troponin I antibody conjugate;
the fluorescein isothiocyanate-first troponin I antibody conjugate and the fluorescein isothiocyanate-second troponin I antibody conjugate were diluted with an anti-reagent buffer, and the diluted fluorescein isothiocyanate-first troponin I antibody conjugate and the fluorescein isothiocyanate-second troponin I antibody conjugate were mixed at a ratio of 1:1 to obtain a first anti-reagent having a concentration of 2.0 μ g/ml.
2) The preparation method of the second anti-reagent comprises the following steps:
respectively dialyzing a third troponin I antibody to be coupled and a fourth troponin I antibody to be coupled by using a carbonate buffer solution with the pH of 8.0 to prepare a third troponin I antibody solution and a fourth troponin I antibody solution;
wherein the third troponin I antibody specifically binds to an epitope 83-93 of the troponin I antigen and the fourth troponin I antibody specifically binds to an epitope cTnC antigen in the cTnI-cTnC complex.
Dissolving alkaline phosphatase to 8mg/ml by using PBS buffer solution containing glutaraldehyde, and preparing the solution on site when the solution is used and keeping out of the sun;
respectively and uniformly mixing a third troponin I antibody solution and a fourth troponin I antibody solution with an alkaline phosphatase solution according to the molar ratio of 1:1, adding a lysine solution, and reacting overnight to prepare an alkaline phosphatase-third troponin I antibody mixed solution and an alkaline phosphatase-fourth troponin I antibody mixed solution;
separating the alkaline phosphatase-third troponin I antibody mixed solution and the alkaline phosphatase-fourth troponin I antibody mixed solution by adopting a gel chromatography separation column respectively to obtain an alkaline phosphatase-third troponin I antibody conjugate and an alkaline phosphatase-fourth troponin I antibody conjugate;
the alkaline phosphatase-third troponin I antibody conjugate and the alkaline phosphatase-fourth troponin I antibody conjugate were diluted with an anti-reagent buffer, respectively, and the diluted alkaline phosphatase-third troponin I antibody conjugate and alkaline phosphatase-fourth troponin I antibody conjugate were mixed in the following ratio of 1:1, to obtain a second anti-reagent at a concentration of 2.0. mu.g/ml.
5) The anti-reagent buffer solution comprises the following components in percentage by concentration:
final pH 9.0.
3. Preparation of calibrator and quality control
Troponin I was dissolved in the calibrator/quality control buffer to prepare calibrators and quality controls at target concentrations as shown in the following table.
The components and the concentrations of the calibrator buffer are as follows:
final PH 8.0.
4. The cleaning solution comprises the following components in percentage by weight:
5. the luminescent substrate was the same as in example 1.
6. And subpackaging and sealing the magnetic particle reagent, the first anti-reagent, the second anti-reagent, the calibrator, the quality control product, the luminescent substrate and the cleaning solution to obtain the troponin I magnetic particle chemiluminescence detection kit.
The method of using the test kit of examples 1-3 above is as follows:
(1) application method
a. Equilibrating the kit stored in a refrigerated environment at room temperature (over 15 minutes);
b. diluting the cleaning solution according to the proportion of 1:15, and shaking up for later use as an application cleaning solution;
c. placing a troponin I calibrator, a quality control product and a sample to be detected in corresponding reaction holes, and setting related measurement parameters in a full-automatic chemiluminescence apparatus;
(2) data processing
According to the luminous value of each standard solution, performing four-parameter Logistic fitting by means of analysis software of a full-automatic chemiluminescence immunoassay analyzer, and selecting X-Y coordinates to obtain a standard curve; a standard curve can also be obtained by adopting a double logarithmic Log (X) -Log (Y) linear analysis method. And finally, calculating the CTNI concentration in the sample according to the standard curve.
(3) Analysis of results
Normal human CTNI content: <0.04ng/mL
Clinical data:
1. minimum detection limit (sensitivity) test
For the reagent kit of example 1, 20-well parallel measurement sample diluents were used, the mean (M) and Standard Deviation (SD) of the measurement results were calculated, and the luminescence value corresponding to M +2SD was obtained and substituted into the dose-response curve to calculate the corresponding concentration value, which is the lowest detection limit of the reagent kit. The results of the detection of the minimum detection limit indicators of the kits of examples 1-3 are shown in tables 1-3:
TABLE 1 results of determination of the lowest detection limit of the kit of example 1
TABLE 2 determination of the lowest detection limit of the kit of example 2
TABLE 3 determination of the lowest detection limit of the kit of example 3
As can be seen from tables 1-3, the sensitivity of the kits of examples 1-3 is all <0.01ng/mL, and meets the requirements of national standards.
2. Linear correlation test
The calibrator (A. about. G, concentration of 0ng/mL, 0.02ng/mL, 0.05ng/mL, 0.5ng/mL, 2.0ng/mL, 10ng/mL and 50ng/mL) of the kit of example 1 was subjected to a double well assay, and the results of the assay were shown in tables 4 to 6, by performing fitting using four parameters:
table 4 results of linear correlation test of the dose-response curves of the kit of example 1
Table 5 results of linear correlation test of the kit dose-response curves of example 2
Table 6 results of linear correlation test of the kit dose-response curves of example 3
As can be seen from tables 4-6, the dose-response curves of troponin I determined by the kits of examples 1-3 all have linear correlation coefficients (r) greater than 0.9900, and meet the requirements of national standards.
3. Accuracy test
Calibrators were measured at concentrations of 0.02ng/mL and 0.05ng/mL using the kits of examples 1-3, respectively, 3 times for each concentration, and the mean M of the measurements was calculated, and the relative deviation (Bias%) of the measurements from the target value should not exceed 15.0%. The accuracy detection results of the kit are shown in table 7:
TABLE 7 accuracy test results of the kit
As can be seen from Table 7, the relative deviations of the actual concentrations of the calibrators measured by the kits of examples 1-3 at concentrations of 0.02ng/mL and 0.05ng/mL are both much less than 15%, and meet the national standards.
4. Precision test of reagent kit
a. Internal precision of analysis: the quality control substances with the concentrations of 0.02ng/mL and 0.05ng/mL are repeatedly detected by using the kits of the examples 1 to 3, and the detection results are shown in Table 8:
TABLE 8 determination of precision in assay of kit
As can be seen from Table 8, the intra-analysis Coefficient of Variation (CV) scores of the 0.02ng/mL and 0.05ng/mL precision calibrators determined by the kit of examples 1-3 are both less than 8.0%, and meet the requirements of national standards.
b. Batch precision: the kits of examples 1-3 were tested with 3 lot kits for quality control at concentrations of 0.02ng/mL and 0.05ng/mL, respectively, each 10 times, and the test results are shown in tables 9-11:
TABLE 9 results of inter-batch precision detection of the kit of example 1
TABLE 10 results of inter-batch precision assay of example 2 kit
TABLE 11 test results of precision between lots of kit of example 3
As can be seen from tables 9-11, the inter-batch variation Coefficient (CV) of the quality control products with the concentrations of 0.02ng/mL and 0.05ng/mL determined by the kit of examples 1-3 is less than 15.0%, and meets the requirements of national standards.
5. Clinical sample testing
The contrast reagent is a mature troponin I detection kit approved by the drug administration for marketing, and a total of 72 serum samples collected from hospitals are used for parallel tests, and the detection results are shown in Table 12:
TABLE 12 results of clinical specimen examination using the kit of example 1
The kit has good correlation with a comparison kit on the market, wherein the correlation coefficient Y is 0.9315X-0.0371, and r is 0.9995, but in the detection process, the measured value of the comparison kit on the market is lower than that of the kit disclosed by the invention, and the false negative result appears in the measured value of the comparison kit combined with clinical diagnosis and analysis, so that the condition of missing detection exists.
In conclusion, the detection kit for the hypersensitive troponin I designed by the invention has leading positions on all quality parameters such as sensitivity, accuracy and precision.
In addition, the components such as S16 inert protein, protease inhibitor, glycerol and the like are added into the calibrator/quality control diluent, so that the stability of the cTnI calibrator is effectively maintained, and the accuracy of the detection result is powerfully guaranteed.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A hypersensitive troponin I magnetic particle chemiluminescence detection kit is characterized by comprising a calibrator, a quality control product, a first anti-reagent, a second anti-reagent, a magnetic particle reagent and a luminescent substrate, wherein,
the first anti-reagent is a solution formed by mixing a first troponin I antibody marked by fluorescein isothiocyanate and a second troponin I antibody marked by the fluorescein isothiocyanate according to the volume ratio of 1: 1;
the second anti-reagent is a solution formed by mixing a third troponin I antibody marked by alkaline phosphatase and a fourth troponin I antibody marked by the alkaline phosphatase according to the volume ratio of 1: 1;
the magnetic particle reagent is a magnetic microsphere coated by an anti-fluorescein isothiocyanate antibody.
2. The ultrasensitive troponin I magnetic particle chemiluminescent assay kit of claim 1 wherein the first troponin I antibody specifically binds to a 20-30 epitope of a troponin I antigen and the second troponin I antibody specifically binds to a 40-50 epitope of a troponin I antigen; the third troponin I antibody specifically binds to an epitope 83-93 of the troponin I antigen and the fourth troponin I antibody specifically binds to an epitope of the cTnC antigen in the cTnI-cTnC complex.
3. The magnetic particle chemiluminescence detection kit for hypersensitive troponin I according to claim 2, wherein the magnetic particle reagent is prepared by the following method:
step 1: washing magnetic beads with the diameter of 0.2-1.0 um by using a buffer solution, and carrying out heavy suspension by using the buffer solution to obtain a first solution with the concentration of 5-15 mg/mL;
step 2: adding EDC and NHS into the first solution, and coupling for 5 hours at room temperature to obtain a second solution;
and step 3: washing the second solution by using a buffer solution, carrying out heavy suspension by using the buffer solution, adding 50-200 ug of goat anti-fluorescein isothiocyanate antibody, coupling at 20-25 ℃ overnight, standing in a magnetic field for 3-5 min, sucking out supernatant, adding a phosphate buffer solution, stirring and sealing at 37 ℃ for 2-5 hours to obtain a third solution;
and 4, step 4: washing the third solution by using a buffer solution, and preparing a primary solution of 5-20 mg/mL magnetic microspheres by using the buffer solution;
and 5: and diluting the primary solution of the magnetic microspheres by using a buffer solution to prepare a magnetic particle reagent, wherein the concentration of the magnetic particle reagent is 0.5-2.0 mg/ml.
4. The ultrasensitive troponin I magnetic particle chemiluminescence detection kit according to claim 2, wherein the first anti-reagent is prepared by:
respectively dialyzing a first troponin I antibody and a second troponin I antibody to be coupled by using a carbonate buffer solution with the pH of 8.0-9.6 to prepare a first troponin I antibody solution and a second troponin I antibody solution;
dissolving fluorescein isothiocyanate into a solution of 1-10 mg/ml by using a buffer solution, preparing the solution at present and keeping out of the sun to prepare a fluorescein isothiocyanate solution;
respectively and uniformly mixing a first troponin I antibody solution, a second troponin I antibody solution and a fluorescein isothiocyanate solution according to the proportion of 100-200 ug:1mg, carrying out a dark reaction overnight, and adding NH4Stopping the reaction by Cl to respectively prepare a fluorescein isothiocyanate-first troponin I antibody mixed solution and a fluorescein isothiocyanate-second troponin I antibody mixed solution;
separating the fluorescein isothiocyanate-first troponin I antibody mixed solution and the fluorescein isothiocyanate-second troponin I antibody mixed solution by using a gel chromatography separation column respectively to obtain a fluorescein isothiocyanate-first troponin I antibody conjugate and a fluorescein isothiocyanate-second troponin I antibody conjugate;
diluting a fluorescein isothiocyanate-first troponin I antibody conjugate and a fluorescein isothiocyanate-second troponin I antibody conjugate by using an anti-reagent buffer solution, and mixing the diluted fluorescein isothiocyanate-first troponin I antibody conjugate and the diluted fluorescein isothiocyanate-second troponin I antibody conjugate according to the ratio of 1:1 to obtain the first anti-reagent with the concentration of 1.0-2.0 mu g/ml.
5. The ultrasensitive troponin I magnetic particle chemiluminescence detection kit according to claim 2, wherein the second anti-reagent is prepared by:
respectively dialyzing a third troponin I antibody to be coupled and a fourth troponin I antibody to be coupled by using a carbonate buffer solution with the pH of 8.0-9.6 to prepare a third troponin I antibody solution and a fourth troponin I antibody solution;
wherein the third troponin I antibody specifically binds to an epitope 83-93 of the troponin I antigen and the fourth troponin I antibody specifically binds to an epitope cTnC antigen in the cTnI-cTnC complex.
Dissolving alkaline phosphatase to 2-8 mg/ml by using a buffer solution, preparing the alkaline phosphatase at present and keeping out of the sun;
respectively and uniformly mixing a third troponin I antibody solution and a fourth troponin I antibody solution with an alkaline phosphatase solution according to the molar ratio of 1:1, adding a lysine solution, and reacting overnight to prepare an alkaline phosphatase-third troponin I antibody mixed solution and an alkaline phosphatase-fourth troponin I antibody mixed solution;
separating the alkaline phosphatase-third troponin I antibody mixed solution and the alkaline phosphatase-fourth troponin I antibody mixed solution by adopting a gel chromatography separation column respectively to obtain an alkaline phosphatase-third troponin I antibody conjugate and an alkaline phosphatase-fourth troponin I antibody conjugate;
the alkaline phosphatase-third troponin I antibody conjugate and the alkaline phosphatase-fourth troponin I antibody conjugate were diluted with an anti-reagent buffer, respectively, and the diluted alkaline phosphatase-third troponin I antibody conjugate and alkaline phosphatase-fourth troponin I antibody conjugate were mixed in the following ratio of 1:1 to obtain a second anti-reagent with the concentration of 0.5-2.0 mug/ml.
6. The magnetic particle chemiluminescence detection kit for hypersensitive troponin I according to claim 2, wherein the components and concentrations of the anti-reagent buffer are as follows:
7. the magnetic particle chemiluminescence detection kit for hypersensitive troponin I according to claim 1, wherein the preparation method of the calibrator and the quality control material comprises: dissolving troponin I antigen by adopting a calibrator/quality control buffer solution to obtain calibrators of 0ng/mL, 0.02ng/mL, 0.05ng/mL, 0.5ng/mL, 2ng/mL, 10ng/mL and 50ng/mL and quality control substances of 0.02ng/mL and 0.5ng/mL, wherein the calibrator and/or the quality control buffer solution comprises the following components in percentage by weight:
8. the troponin I magnetic particle chemiluminescence detection kit of claim 1, wherein the luminescent substrate is a buffer solution containing (adamantane) -1, 2-dioxyethane and derivatives thereof.
9. The troponin I magnetic particle chemiluminescence detection kit of claim 1, wherein the detection kit further comprises a wash solution, and the wash solution comprises the following components by weight:
10. use of a kit according to any one of claims 1 to 9 as an in vitro diagnostic reagent.
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| CN112649616A (en) * | 2020-12-30 | 2021-04-13 | 北京联众泰克科技有限公司 | Composition for detecting cardiac troponin I, application thereof, magnetic microsphere electrochemiluminescence immunoassay kit and detection method |
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| 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 |
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