CN111521809A - Cardiac troponin T detection kit and preparation method and application thereof - Google Patents
Cardiac troponin T detection kit and preparation method and application thereof Download PDFInfo
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- 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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- 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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- G01N33/50—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
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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
- G01N33/53—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
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
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- 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
Abstract
The invention relates to a cardiac troponin T detection kit, a preparation method and application thereof. The kit comprises a first strain antibody coated on a magnetic microsphere and a second strain antibody marked with a tracer marker, wherein the first strain antibody and the second strain antibody are monoclonal antibodies for resisting cardiac troponin T, and specific binding sites of the cardiac troponin T recognized by the first strain antibody and the second strain antibody are different. The kit provided by the invention can be used for detecting the cTnT concentration of a sample with extremely high sensitivity, can obtain a detection result in about 10 minutes, can realize the rapid, sensitive and quantitative detection of the cTnT concentration of the sample, and can be used for diagnosing Acute Myocardial Infarction (AMI), unstable angina, postoperative myocardial damage and myocardial muscle damage as early as possible and accurately.
Description
Technical Field
The invention relates to the field of biochemical detection, in particular to a cardiac troponin T detection kit and a preparation method and application thereof.
Background
Troponin complexes are composed of three subunits, troponin I (TnI), troponin C (TnC) and troponin T (TnT), which play important regulatory roles in the contraction of skeletal and cardiac muscles. Wherein TnT is a tropomyosin binding subunit that modulates the interaction between troponin complexes and thin myofilaments. There are two (TnT) (sktnt) subtypes in human skeletal muscle, TnT has multiple subtypes in cardiac muscle, and subtypes in cardiac muscle are different from those in skeletal muscle. Cardiac troponin t (ctnt) can be used as a marker of myocardial cell death, and has been widely used for diagnosis of Acute Myocardial Infarction (AMI), unstable angina, post-operative myocardial injury, and diseases associated with myocardial muscle injury. cTnT is typically detected in the patient's blood within 3-6 hours after the onset of chest pain, and this detection will peak within 16-30 hours. Studies have shown that the increase in cTnT concentration in blood can continue even 5-8 days after the onset of symptoms, indicating that cTnT has some reference value for the late diagnosis of acute myocardial infarction. Meanwhile, the cardiac troponin has the advantages of rapid release, obvious peak value, strong tissue specificity, long window diagnosis period and the like when cardiac muscle is damaged, is used as a gold standard for diagnosing acute myocardial infarction, and is also called as an optimal marker for disease monitoring, curative effect observation, risk classification and prognosis evaluation of new-onset diseases.
Currently, there are many immunoassay methods for troponin, including Radioisotopic Immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), colloidal gold Immunochromatography (ICA), and chemiluminescence method (CLIA) which has been widely used in recent years.
The detection principle of Radioisotopic Immunoassay (RIA) is to perform a competitive binding reaction between an isotopically labeled antigen and an unlabeled antigen to be detected and an insufficient amount of a specific antibody, and then measure the radioactivity of the reaction to obtain the amount of the antigen to be detected. This method is an ultra-micro analysis technique that has been increasingly widely used since the 80 s.
The enzyme-linked immunosorbent assay (ELISA) detection principle, taking the double-antibody sandwich method as an example, is to coat a specific antibody on a solid phase carrier, add a sample to be detected, add another enzyme-labeled antibody to form a double-antibody sandwich compound, and then enable the instrument to quantitatively detect immunoreaction through the color development of a luminescent substrate.
The colloidal gold immunochromatography method as a POCT (point of care testing) test method has the advantages of small sample loading amount, simplicity, convenience and rapidness, and is suitable for bedside test. The principle is as follows, still taking the double antibody sandwich method as an example, when a sample containing an antigen is dropped into an absorption hole, the antigen is bound with an antibody originally labeled with colloidal gold to form an antigen-antibody complex, and then another antigen immobilized on a sample pad captures the complex to form a double antibody sandwich complex, and a color band is generated, so that the double antibody sandwich complex is detected.
Chemiluminescence is a product obtained by closely combining an immune reaction system and chemiluminescence technology, which is an immunoassay technique that has been developed in recent years. The chemiluminescence technology is a detection means which utilizes a luminescent substance, such as acridinium ester and the like, to form an excited intermediate after being oxidized by an oxidant and catalyzed by a catalyst, and then utilizes a corresponding measuring instrument to measure the yield of light quantum generated when the intermediate returns to the ground state. The immunoreaction system is to mark the luminous labeling matter on antigen or antibody and then to form antigen-antibody complex for detection. The technology has the advantages of strong specificity, high sensitivity, good precision, wide linear range, high flux and easy realization of automation.
Radioisotopic Immunoassay (RIA) has the problems of complex operation, long time consumption, poor result repeatability, nuclear pollution and the like, is not suitable for large-scale detection, and is not used basically at present.
Enzyme-linked immunosorbent assay (ELISA) has the defects of complex operation, low sensitivity, narrow linear range, long detection period and the like. And when the early AMI occurs, the content of troponin in the serum is often very low, and due to low sensitivity and long detection period, the method cannot quickly and accurately detect the early AMI, thereby further limiting the use of ELISA in myocardial detection.
The colloidal gold immunochromatography method as a POCT (point of care testing) test method has the advantages of small sample loading amount, simplicity, convenience and rapidness, and is suitable for bedside test. However, the sensitivity of the method is poor, so that the application of the method in the clinical detection of troponin is further limited.
At present, the common chemiluminescence method kits for detecting troponin on the market are mostly used for detecting cardiac troponin I (cTnI), wherein many kits have the defects of insufficient sensitivity, low precision, overlong detection time and the like, cannot meet the requirement of AMI early diagnosis, cannot be detected in surface healthy people basically, and can possibly cause delayed diagnosis and even misdiagnosis when ischemia symptoms or electrocardiogram changes uncharacteristically, and are not favorable for early diagnosis, risk assessment and prognosis judgment. Furthermore, cTnI has multiple forms, which leads to non-uniform troponin I detection standards and different cTnI results of different systems. In contrast, cTnT is of a single form, and it has been reported in the literature that cTnT is superior to cTnI in accuracy in late detection, post-cure detection, and prediction of post-cure mortality. Thus, the cTnT detection is clinically significant.
CN101226200A discloses a method for detecting human cTnT by chemiluminescence immunoassay, which uses acridinium ester and/or alkaline phosphatase as a marker, and the immune reaction adopts a double-monoclonal antibody sandwich method and/or a competition method. However, in the document, the incubation condition used for detecting the sample is 37 ℃ and 60 minutes, so that the problem of overlong detection time exists, and the high requirement of AMI early diagnosis cannot be met. Moreover, the document only mentions the sensitivity, i.e. the margin (LoB), but it is not a clinically reportable value, and the results become unreliable when the imprecision CV is > 20%. Most clinical institutions in the united states currently report with a limit of quantitation (LoQ), the limit of quantitation (LoQ) being the lowest concentration at which the CV is closest to 20%, detection of which is of great significance for performance testing of the kit.
Disclosure of Invention
Problems to be solved by the invention
Aiming at the defects of the prior art, the invention aims to provide a hypersensitive cardiac troponin T detection kit which has better sensitivity and specificity, can quickly obtain a detection result, effectively distinguishes normal persons from early AMI patients, and is favorable for quickly diagnosing patients with myocardial infarction and microminiature myocardial injury.
The invention also aims to provide a preparation method of the cardiac troponin T detection kit.
In addition, the invention also provides the application of the kit.
Means for solving the problems
In one technical scheme, the kit comprises a first strain of antibody coated on a magnetic microsphere and a second strain of antibody marked with a tracer marker, wherein the first strain of antibody and the second strain of antibody are monoclonal antibodies for resisting cardiac troponin T, and specific binding sites of the cardiac troponin T recognized by the first strain of antibody and the second strain of antibody are different.
In one embodiment, the first antibody recognizes the cardiac troponin T at amino acid position 171-190 and the second antibody recognizes the cardiac troponin T at amino acid position 132-152.
In another embodiment, the magnetic microspheres are selected from nanoscale Fe2O3And/or Fe3O4The composite of the magnetic fine particles and the polymer material of (3).
In another embodiment, the magnetic microspheres have an average particle size of 1 to 5 μm.
In another embodiment, the magnetic microsphere has a plurality of active groups on the surface, preferably, the active groups are-OH and/or-COOH.
In another embodiment, the tracer label is selected from one of acridinium ester, luminol, alkaline phosphatase, horseradish peroxidase and adamantane, and preferably, the tracer label is acridinium ester.
In another embodiment, the concentration of said first strain of antibody and said second strain of antibody is between 1 and 18. mu.g/ml, the concentration of said magnetic microspheres is between 0.1 and 1mg/ml, and the concentration of said tracer label is between 0.1 and 1. mu.g/ml.
In another embodiment, the mass ratio of the magnetic microspheres to the first strain of antibody is 100: 1-2; further preferably the mass ratio is 100: 1.5.
In another embodiment, the molar ratio of the tracer label to the second strain of antibody is 14-18: 1; further preferably, the molar ratio is 15: 1.
In another embodiment, the kit comprises a calibrator, a magnetic microsphere wash, and a chemiluminescent excitation fluid.
In another embodiment, the kit comprises a liquid calibrator, preferably the liquid calibrator comprises PBS buffer containing cardiac troponin T antigen, further preferably the liquid calibrator is supplemented with a preservative.
In another embodiment, the magnetic microsphere cleaning solution comprises a PBS buffer solution containing a surfactant, and preferably, the magnetic microsphere cleaning solution further comprises a preservative.
In another embodiment, the chemiluminescent excitation liquid comprises hydrogen peroxide and nitric acid solution.
In another embodiment, the present invention further provides a method for preparing the kit of the present invention, wherein the method comprises a step of coating a first strain of antibody with magnetic microspheres and a step of labeling a second strain of antibody with tracer labels.
In one embodiment, a 2- (N-morpholino) ethanesulfonic acid (MES) buffer is used in the first antibody-coated magnetic microsphere step.
In another embodiment, a PBS solution containing 1% casein is used as a blocking agent in the first strain of antibody-coated magnetic microspheres step.
In another embodiment, the coupling time of the first strain of antibody to the magnetic microspheres is 0.5 to 2 hours, preferably 0.5 hour.
In another embodiment, a Carbonate (CB) buffer is used in the second monoclonal antibody labeling tracer label step.
In another embodiment, the second strain of antibody is labeled with the tracer label for a labeling time of 1.8 to 2.5 hours; preferably, the labeling time of the second antibody labeled tracer label is 2 hours.
In another technical scheme, the invention also provides application of the kit in detecting the content of the cardiac troponin T.
In one embodiment, the kit of the present invention has the use of chemiluminescence immunoassay to detect the amount of cardiac troponin T.
In another embodiment, the present invention further provides a method for detecting the content of cardiac troponin T in a sample, wherein the method is performed by the cardiac troponin T detection kit provided by the present invention.
In one embodiment, the sample is obtained directly from serum, plasma and/or whole blood, or may be obtained by extracting a human blood sample and separating.
In another technical scheme, the invention further provides application of a first strain of antibody coated on the magnetic microspheres and a second strain of antibody marked with a tracer marker in preparation of a medicine or a kit for detecting cardiac troponin T, wherein the first strain of antibody and the second strain of antibody are monoclonal antibodies against cardiac troponin T, and specific binding sites of the cardiac troponin T recognized by the first strain of antibody and the second strain of antibody are different.
ADVANTAGEOUS EFFECTS OF INVENTION
On one hand, the kit provided by the invention can be used for detecting the concentration of cTnT of a sample with extremely high sensitivity, the incubation condition is 37 ℃ and 6 minutes, the detection result can be obtained within about 10 minutes of the total detection time, and the rapid, sensitive and quantitative detection of the concentration of cTnT of the sample can be realized. The invention can be used for diagnosing AMI as early as possible and accurately.
On the other hand, most of the calibrators in the common kits in the market are freeze-dried products, and the calibrators in the kit provided by the invention are liquid and have better thermal stability and are simpler and more convenient to use.
Drawings
Fig. 1 is a schematic diagram of the detection of the cTnT content in a sample using the cTnT detection kit provided by the present invention.
FIG. 2 is a graph of the coincidence rate of the detected serum samples in the antibody grouping screening.
FIG. 3 is a graph showing the linear correlation test of the kit prepared in example 1.
FIG. 4 is a graph showing the overall coincidence rate of 102 Roche samples detected by the kit prepared in example 1.
Detailed Description
Various exemplary embodiments, features and aspects of the present invention will be described in detail below with reference to the accompanying drawings. In the following detailed description, numerous specific details are set forth in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
< kit for detecting cardiac troponin T >
The invention provides a cardiac troponin T (cTnT) detection kit, which comprises a first strain of antibody coated on a magnetic microsphere and a second strain of antibody marked with a tracer marker, wherein the first strain of antibody and the second strain of antibody are monoclonal antibodies for resisting cardiac troponin T, and specific binding sites of the cardiac troponin T identified by the first strain of antibody and the second strain of antibody are different.
The method for detecting cTnT is a double-antibody sandwich method, which mainly uses two different specific monoclonal antibodies, wherein the binding site of the antibody marked on a tracer marker is different from the binding site of the antibody coated on a magnetic microsphere. The selection of these binding sites not only facilitates the labeling of the tracer or the coating of the magnetic microspheres, but also does not prevent the binding of the antibody to the antigen to form a sandwich complex, thus improving the specificity and sensitivity of the reaction. In one embodiment, in order to obtain better detection signals and sample coincidence rate of serum and plasma samples, preferably, the sequence of the amino acid site of cardiac troponin T recognized by the first antibody of the present invention is the sequence shown in SEQ ID No. 1, and the sequence of the amino acid site of cardiac troponin T recognized by the second antibody is the sequence shown in SEQ ID No. 3.
The tracer markers suitable for the present invention include luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester, adamantane, etc., among which acridinium ester is preferred because it has low background luminescence, high signal-to-noise ratio, fast and concentrated light release, high luminous efficiency, high luminous intensity, easy coupling with protein, no reduction in photon yield after coupling, stable marker (can be stored for several months at 2-8 deg.C), and good solubility in water, stability and uneasy hydrolysis.
The magnetic microspheres, referred to as magnetic beads or magnetic particles for short, suitable for use in the present invention may be magnetic beads commonly used in the art. The magnetic beads used in the invention are nanoscale Fe2O3And/or Fe3O4The magnetic particles are compounded with high molecular materials to form micron-sized solid phase microspheres with paramagnetism and extremely large protein adsorption quantity, the magnetic microspheres can be quickly magnetized under the action of an external magnetic field, and the remanence is zero after the magnetic field disappears. The kind of the polymer material used for the composition is not limited. In one embodiment, the magnetic microspheres used in the present invention have an average particle size ranging from 1 to 5 μm, and the magnetic microspheres may have a plurality of active groups added thereto by surface modification, including but not limited to-OH and-COOH.
In the present invention, the concentration of the antibody used is 1 to 18. mu.g/ml, more preferably 8 to 18. mu.g/ml, still more preferably 15. mu.g/ml for the first strain, 10. mu.g/ml for the second strain, 0.1 to 1mg/ml, still more preferably 0.6 to 1mg/ml, yet more preferably 1mg/ml for the magnetic microsphere, and 0.1 to 1. mu.g/ml, still more preferably 0.1 to 0.5. mu.g/ml, yet more preferably 0.1. mu.g/ml for the tracer. Because the concentration, especially the antibody concentration, has obvious influence on the signal of the test, the invention improves the sensitivity and the precision of the detection by accurately regulating and controlling the concentration. In one embodiment of the present invention, the mass ratio of the magnetic microspheres to the first strain of antibody is 100:1-3, and more preferably the mass ratio is 100: 1.3-2, and the mass ratio is more preferably 100: 1.5. In another embodiment of the invention, the molar ratio of the tracer label to the second strain antibody is 14-18: 1; further preferably, the molar ratio is 15: 1.
In one technical scheme, when the kit is used, the concentration of the magnetic microsphere-first strain antibody is diluted to be 0.01-0.1 mg/ml, preferably 0.03-0.07 mg/ml, and the concentration of the tracer marker-second strain antibody is 0.5-2 mug/ml, preferably 1 mug/ml.
In the invention, the kit also comprises a calibrator, a magnetic bead cleaning solution and a chemiluminescent excitation solution. Preferably, the kit comprises a liquid calibrator. The calibrator can be stably stored at 4 ℃ and has good thermal stability. In one embodiment, the liquid calibrator comprises a PBS buffer containing a cardiac troponin T antigen, and further preferably, a preservative is added to the liquid calibrator. The calibrator comprises a low-point calibrator and a high-point calibrator, wherein the low-point calibrator is obtained by diluting a cTnT working solution to 50-200 pg/ml with a PBS solution, and the high-point calibrator is obtained by diluting the cTnT working solution to 7000-12000 pg/ml with the PBS solution.
In another embodiment, the magnetic bead washing solution is PBS buffer containing a surfactant, preferably, a certain preservative is added thereto.
In another technical scheme, the chemiluminescence excitation liquid comprises a) excitation liquid A which is a solution containing hydrogen peroxide and nitric acid and b) excitation liquidAnd a hair dye B which is a mixed solution of polyethylene glycol octyl phenyl ether (Triton X-100) and NaOH. Wherein H2O20.01-5.0% of HNO3The concentration is 0.01-1.0mol/L, the mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1 mol/L. After the incubation wash, after addition of excitation solution a, excitation solution B was added again and immediately read.
The preservative suitable for use in the present invention may be one or two of preservatives commonly used in the art, such as potassium sorbate, sodium benzoate, sodium nitrite, sodium azide, proclin-300 (the main active ingredients are 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one), and antibiotics.
The surfactant suitable for use in the present invention may be one or two of surfactants commonly used in the art, such as the nonionic surfactants Triton X-100, Triton X-405, Tween 20 and Tween 80. The addition amount of the surface activity is 0.1-2 per mill, so that the dispersion of the magnetic microspheres is facilitated.
In one embodiment of the invention, the kit comprises the following components: 1) the first strain of antibody is coated on the magnetic microspheres, the concentration of the first strain of antibody is 15 mug/ml, and the concentration of the magnetic microspheres is 1 mg/ml; 2) a second antibody labeled with a tracer marker, wherein the concentration of the second antibody is 10 mug/ml, and the concentration of the tracer marker is 0.1 mug/ml; 3) the concentrations of the high-low point calibrator are 7000-12000 pg/ml and 50-200 pg/ml respectively; 4) a magnetic bead cleaning solution, a PBS buffer solution with the pH value of 7.0-9.0 and the concentration of 0.01-0.04mol/L, wherein the PBS buffer solution contains 0.1-1% of surfactant by mass fraction; 5) a chemiluminescent exciting liquid comprising a) an exciting liquid A which is H2O2And HNO3In which H is2O20.01-5.0% of HNO3The concentration is 0.01-1.0 mol/L; b) and the excitation liquid B is a mixed liquid of Triton X-100 and NaOH, wherein the mass fraction of the Triton X-100 is 0.01-2.0%, and the concentration of the NaOH is 0.05-1 mol/L.
< preparation method of kit for detecting cardiac troponin T >
The invention also provides a method for preparing the kit, which comprises the steps of coating the first strain of antibody on the magnetic microspheres and labeling the second strain of antibody with the tracer markers.
First strain antibody coated magnetic microsphere
The first strain of antibody-coated magnetic microspheres comprises the following steps:
(1) measuring magnetic microspheres (commercially available) and suspending in buffer solution, adsorbing and cleaning by using a magnet, and then adding the buffer solution and mixing uniformly;
(2) adding a first cTnT monoclonal antibody, and incubating at 30-40 ℃, preferably at 37 ℃.
(3) 1- (3-dimethylaminopropyl) -3-ethyldiimine hydrochloride (EDC) is added and incubated at 30 ℃ to 40 ℃ and preferably at 37 ℃.
(4) Blocking with blocking reagent.
(5) And adding a luminescence recovery solution into the closed magnetic bead suspension, and adsorbing and cleaning by a magnet.
(6) And (3) placing the prepared magnetic beads in a luminescence recovery solution, and storing at 2-8 ℃.
In one technical scheme, the mass ratio of the magnetic microspheres to the first cTnT monoclonal antibody is 100:1-3, and for stability and simplicity of operation, the mass ratio of the magnetic microspheres to the first cTnT monoclonal antibody is preferably 100: 1.5.
In another embodiment, the amino acid position of cardiac troponin T recognized by the first cTnT monoclonal antibody is 171-190.
The inventor finds that when the buffer solution is MES buffer solution, the detection signal is higher, and the sample coincidence rate is also better. In one embodiment of the invention 0.1M MES buffer is used.
With respect to the incubation time in the step (2), the present invention is not particularly limited, and the incubation time has little influence on the detection signal and the sample coincidence rate. For time saving, the preferred incubation or coupling time is 0.5 h.
For the selection of blocking reagents, 1% casein in PBS, 5% BSA in PBS, and 0.5% casein in PBS can be used. In terms of the intensity of the detection signal and the sample coincidence rate, a PBS solution containing 1% casein is preferably used as a blocking reagent. The concentration range of the blocking reagent is preferably 0.01-0.05M, and the dosage of the blocking reagent is 100-300 mu L.
In one technical scheme, the luminescence recovery solution used by the invention comprises 5g of bovine serum albumin, 400ml of 0.025M Tris-HCl buffer solution, 0.1-2 per thousand of Tween-80 and 100ml of newborn bovine serum. The luminescence recovery solution is selected to be beneficial to the dispersion of magnetic beads and the storage stability of the magnetic beads.
Labeling tracer marker with second antibody
The labeling of the tracer marker by the second antibody strain comprises the following steps:
(1) and (3) placing the second cTnT monoclonal antibody in a dialysis bag, dialyzing for 20-30h by using a buffer solution, and adding the antibody into an ultrafiltration centrifugal tube for concentration after dialysis is finished so that the antibody concentration is more than 5 mg/ml.
(2) Placing the concentrated cTnT monoclonal antibody into a centrifugal tube, adding a tracer marker solution to ensure that the molar ratio of the tracer marker to the cTnT monoclonal antibody is 14-18:1, uniformly mixing, reacting at room temperature (keeping out of the sun), adding a lysine solution, and reacting for a period of time.
(3) And (3) putting the reaction product obtained in the step (2) into a dialysis bag, dialyzing for 20-30h by using a citric acid-sodium citrate buffer solution, and separating the free tracer markers.
(4) The dialyzed tracer-labeled antibody was collected, added to BSA solution, followed by an equal volume of glycerol, and stored at-20 ℃.
In one embodiment, the amino acid position of cardiac troponin T recognized by the second strain of cTnT monoclonal antibody is 132-152.
In another technical scheme, the inventor finds that when the CB diluent is used as a buffer solution, the detection signal is high, and the sample coincidence rate is also good. In one embodiment of the invention 0.05M CB buffer (pH 9.6) is used.
The molar ratio of tracer label to cTnT monoclonal antibody in the invention is 14-18:1, if the ratio is lower than 14:1, the detection signal is influenced, and if the ratio is higher than 18:1, the background value of the detection kit is influenced. Further preferably, the molar ratio of tracer label to cTnT monoclonal antibody is 15: 1. In one embodiment of the invention, the tracer label is a solution of acridinedioic acid-NHS ester (NSP-SA-NHS) in DMF.
Regarding the selection of the labeling time (i.e., the time for the reaction at room temperature in a dark place) in step (2), the present inventors found that the detection signal was greatly increased when the labeling time was 2 h.
In one embodiment of the invention, the dialysis time is 24 hours and the fluid is changed four times in the middle to ensure that a relatively single component antibody or antibody containing a tracer label is obtained.
In another technical scheme of the invention, the method further comprises the steps of preparing a calibrator, preparing chemiluminescent excitation liquid and preparing magnetic microsphere cleaning liquid.
Preparation of calibrator
And (3) taking the cTnT working solution, and diluting the cTnT working solution to 7000-12000 pg/ml and 50-200 pg/ml of high-low point standard solution by using PBS (phosphate buffer solution) containing 0.5-3% bovine serum albumin.
Preparation of chemiluminescence exciting liquid
The chemiluminescence excitation liquid comprises an excitation liquid A and an excitation liquid B, and needs to be matched for use: the exciting liquid A is prepared by reacting H2O2And HNO3Mixing is carried out, wherein H2O20.01-5.0% of HNO3The concentration is 0.01-1.0 mol/L; the exciting liquid B is prepared by mixing Triton X-100 and NaOH, wherein the mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1 mol/L.
Preparation of magnetic microsphere cleaning solution
The magnetic microsphere cleaning solution uses phosphate Tween buffer solution (PBST solution), and the pH value is 7.0-9.0.
< use of the kit for detecting cardiac troponin T >
The cardiac troponin T detection kit disclosed by the invention is matched with a full-automatic chemiluminescence analyzer (obsidian cyclone) for use, and can be used for detecting the concentration of cTnT in a sample.
The sample to be detected of the invention is obtained by directly obtaining serum, plasma and whole blood, and can also be obtained by extracting a human blood sample and separating.
The specific detection process comprises the following steps: respectively diluting a first strain of antibody coated magnetic microspheres and a second strain of antibody marked by a tracer marker in the kit; adding the diluted reagent and the sample to be detected or/and the calibrator into the reaction cup in sequence, wherein the whole sample adding process is about 0.5 minute; after the solution in the reaction cup is fully and uniformly mixed, the mixture is incubated for 6 minutes at 37 ℃; then, the reaction cup is placed under a magnetic condition, and is cleaned for three times by using a magnetic microsphere cleaning solution, wherein the whole process needs 2 minutes; adding chemiluminescence excitation liquid into the reaction cup and detecting the value of the photons; according to the light intensity detected in the reaction cup, the instrument automatically calculates the concentration of cTnT in the sample to be detected, and the detection calculation process needs 1 minute. Therefore, the kit provided by the invention can obtain a detection result within 10 minutes.
Fig. 1 shows a schematic diagram of the detection of the cTnT content in a sample using the cTnT detection kit provided by the present invention in one embodiment of the present invention. The first strain of antibody coated magnetic microspheres (namely magnetic particle labeled antibodies), the acridinium ester labeled antibodies and the antigens to be detected are fully and uniformly mixed in a reaction cup to obtain the magnetic microsphere (namely magnetic particles) -antibody-antigen-acridinium ester compound. And then, cleaning, adding chemiluminescence excitation liquid, and detecting the intensity of optical signals to obtain the concentration of cTnT in the sample.
The kit provided by the invention can be used for detecting the cTnT concentration of a sample with extremely high sensitivity, the blank limit can reach 4.09pg/ml, the in-day precision can reach 5.09% and 3.84%, the linear correlation degree R is greater than 0.990, the recovery test result can reach 97.28%, the integral coincidence rate R value can reach 0.9962, and the quantitative limit (LoQ) can reach 16 pg/ml.
In addition, the kit provided by the invention is used for detecting the concentration of cTnT in a sample, the total detection time is short, the detection result can be obtained only in about 10 minutes, the full-automatic and rapid detection of the concentration of cTnT in the sample can be realized, and the AMI can be diagnosed as early as possible and accurately.
Examples
The technical solution of the present invention will be further described with reference to specific examples. It should be understood that the following examples are only for illustrating and explaining the present invention and are not intended to limit the scope of the present invention.
Example 1
The present example provides methods of making and using cardiac troponin T detection kits.
Preparation 1: first strain antibody coated magnetic microsphere
(1) Measuring 10mg of magnetic microspheres (average particle size of 1.5 μ M, purchased from Bangs Laboratories, Inc., solid content of 2.54%), suspending in 1mL of 0.1M MES buffer solution, carrying out magnetic adsorption for 5-10 min, discarding the supernatant, repeating the washing step for 3-5 times, adding 1mL of the buffer solution (0.1M MES buffer solution), and mixing by vortex.
(2) Adding 150 mu g of the first cTnT monoclonal antibody (the amino acid site of the cardiac troponin T identified by the first antibody is 171-190), leading the mass ratio of the magnetic microspheres to the antibody to be 100:1.5, evenly mixing by vortex, and incubating for 30min at 37 ℃.
(3) 10 μ L of 10mg/ml 1- (3-dimethylaminopropyl) -3-ethyldiimine hydrochloride (EDC) was added, vortexed and incubated at 37 ℃ for 1.5 h.
(4) Blocking was performed with 200. mu.L of 1% casein PBS (0.02M) for 2 h.
(5) Adding 1ml of luminescence recovery solution 1(5g of bovine serum albumin, 400ml of 0.025M Tris-HCl buffer solution for dissolving, 0.1-2 per mill of Tween-80 and 100ml of newborn bovine serum) into the sealed magnetic bead suspension, carrying out magnetic adsorption, removing supernatant, and repeating the cleaning step for 3-5 times.
(6) And (3) placing the prepared magnetic beads in 1ml of luminescence recovery solution 1, and storing at 2-8 ℃.
Preparation 2: second-strain antibody labeled acridinium ester
(1) 1mg of the second cTnT monoclonal antibody (the amino acid site of cardiac troponin T recognized by the second antibody: 132-152) was placed in a dialysis bag, and dialyzed with not less than 2L of 0.05M CB buffer (pH 9.6) for 24 hours, followed by four fluid changes in the middle. After dialysis, the antibody was concentrated in Millipore Amicon Ultra ultrafiltration tubes to a concentration of greater than 5 mg/ml.
(2) Placing the concentrated cTnT monoclonal antibody into a 0.5ml centrifugal tube, adding 100 mu L of 5mg/ml acridinedioic acid-NHS (NSP-SA-NHS) solution dissolved in DMF to ensure that the molar ratio of NSP-SA-NHS to the cTnT monoclonal antibody is 15:1, uniformly mixing, reacting at room temperature (in a dark place) for 2h, adding 100 mu L of lysine solution with the concentration of 100mg/ml, reacting for 30min, and stopping the reaction.
(3) The label was placed in a dialysis bag and dialyzed with not less than 2L of citric acid-sodium citrate buffer (10Mm/L, pH 4.5) for 24h, with four intermediate changes, to isolate free NSP-SA-NHS.
(4) The dialyzed label was collected, 5% BSA solution was added to make BSA a final concentration of 1%, followed by the addition of glycerol in equal volume and preservation at-20 ℃ to obtain a solution of cTnT monoclonal antibody labeled with acridinium ester.
Preparation 3: preparation of calibrator
The cTnT working solution was diluted to 9000pg/ml and 100pg/ml of the high and low point standard solutions using 1% bovine serum albumin in 0.02M PBS.
Preparation 4: preparation of chemiluminescence exciting liquid
The chemiluminescent exciting liquid A is H2O2And HNO3In which H is2O20.01-5.0% of HNO3The concentration is 0.01-1.0 mol/L.
The chemiluminescence excitation liquid B is a mixed liquid of Triton X-100 and NaOH, wherein the mass fraction of the Triton X-100 is 0.01-2.0%, and the concentration of the NaOH is 0.05-1 mol/L.
Preparation 5: preparation of magnetic microsphere cleaning solution
The magnetic microsphere cleaning solution is phosphate Tween buffer solution (PBST solution) with pH value of 7.0-9.0 and concentration of 0.02mol/L, wherein the phosphate Tween buffer solution contains Tween-20 with mass fraction of 0.5%.
And (3) coating the magnetic microspheres with the first strain of antibody obtained in the step (1), preparing acridinium ester labeled with the second strain of antibody obtained in the step (2), preparing a high-low point calibrator of the step (3), preparing a chemiluminescent excitation liquid of the step (4) and preparing a magnetic microsphere cleaning solution of the step (5) to prepare the cardiac troponin T detection kit.
The kit is matched with a full-automatic chemiluminescence analyzer for use, and the concentration of cTnT in a sample is detected.
Specifically, the cTnT concentration in the sample to be measured is measured by using a full-automatic chemiluminescence analysis instrument according to the following steps:
1. diluting the first strain of antibody coated magnetic microspheres prepared in the preparation 1 to 0.05mg/ml (reagent 1) according to the using concentration of the magnetic microspheres, and diluting the acridinium ester labeled second strain antibody prepared in the preparation 2 to 1 mu g/ml (reagent 2);
2. adding 50ul of reagent 1 and 50ul of reagent 2 into the reaction cup in sequence;
3. adding 50ul of sample to be detected or/and calibrator into the reaction cup, wherein the whole sample adding process needs 0.5 minute;
4. after the solution in the reaction cup is fully and uniformly mixed, the mixture is incubated for 6 minutes at 37 ℃;
5. placing the mixture under a magnetic condition, and cleaning the mixture for three times by using a magnetic microsphere cleaning solution, wherein the whole process needs 2 minutes;
6. adding 100ul of chemiluminescent exciting liquid A into the reaction cup, then adding 100ul of chemiluminescent exciting liquid B, and immediately detecting the photon value;
7. according to the light intensity detected in the reaction cup, the concentration of cTnT in the sample to be detected is automatically calculated by the instrument, and the whole detection process needs 1 minute.
Example 2
This example provides a process for screening antibody panels of a cardiac troponin T test kit.
Only the cTnT monoclonal antibody in preparation 1 and preparation 2 was changed compared to the preparation process of example 1. Wherein the content of the first and second substances,
the recognition amino acid site of the antibody 1 is 171-190 (the sequence of the recognition amino acid site is shown as SEQ ID NO: 1);
the recognition amino acid site of the antibody 2 is 119-138 (the sequence of the recognition amino acid site is shown as SEQ ID NO: 2);
the recognition amino acid site of the antibody 3 is 132-152 (the sequence of the recognition amino acid site is shown as SEQ ID NO: 3);
the recognition amino acid site of the antibody 4 is 139-152 (the sequence of the recognition amino acid site is shown as SEQ ID NO: 4).
Respectively coating the antibodies 1 and 2 on magnetic microspheres to form an antibody 1-magnetic microsphere suspension and an antibody 2-magnetic microsphere suspension, and respectively labeling the antibody 3 and the antibody 4 on acridinium ester to obtain the antibody 3-acridinium ester and the antibody 4-acridinium ester. The above antibodies were matched to obtain the group 1 (magnetic bead-antibody 2 and acridinium ester-antibody 3), the group 2 (magnetic bead-antibody 1 and acridinium ester-antibody 4) and the group 3, i.e., example 1 (magnetic bead-antibody 1 and acridinium ester-antibody 3), and the group 4 (magnetic bead-antibody 2 and acridinium ester-antibody 4) was excluded from the preliminary screening because all the samples were identical in detection because of no reactivity. The results of serum and EDTA plasma detection using the kits prepared in example 2 and example 1 in combination with a full-automatic chemiluminescence analyzer are shown in Table 1, Table 2, and FIG. 2, wherein Signal 1, Signal 2, and Signal 3 refer to light intensity.
Table 1 kit antibody grouping 43 serum sample screening results
TABLE 2 kit antibody grouping results of screening 5 EDTA plasma samples
The results in table 1 and fig. 2 show that the coincidence rates (R values) of the three matched groups of serum samples are similar, the signals of the matched group 3 (magnetic microsphere-antibody 1 and acridinium ester-antibody 3) are higher, and the measured values are distributed uniformly. Table 2 results of plasma detection show that the sample coincidence rate (R value) is better when only the group 3 (magnetic microsphere-antibody 1 and acridinium ester-antibody 3) is detected. The antibody preferably used in the preparation process of the kit of the present invention is ligand 3 (magnetic microsphere-antibody 1 and acridinium ester-antibody 3).
Example 3
This example provides a screening process for a buffer during the preparation of a first antibody-coated magnetic microsphere.
Example 3 substantially the same procedure of preparation 1 was used as in example 1. Example 3 mainly changes the buffer of step (1) in preparation 1, compared with the procedure of preparation 1 in example 1. Example 3 in preparation 1, step (1), 0.1M MES buffer, 0.02M acetate buffer or 0.05M 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer was used, respectively. Combining the three first antibody-coated magnetic microspheres prepared in the example 3 with the second antibody-labeled acridinium ester prepared in the example 1 and the preparation 3-5 to obtain three kits, and detecting the three kits by matching with a full-automatic chemiluminescence analyzer respectively, wherein the detection results are shown in Table 3.
TABLE 3 screening results of buffer solution in the preparation process of the first strain of antibody-coated magnetic microspheres
The results in Table 3 show that the detection signal is higher and the sample coincidence rate is better when MES buffer is used. MES buffer is preferably used in the preparation process of the kit of the invention.
Example 4
The embodiment provides a screening process of coupling time of a first cTnT monoclonal antibody and a magnetic microsphere in a preparation process of a first antibody-coated magnetic microsphere.
Example 4 substantially the same procedure of preparation 1 was used as in example 1. Example 4 mainly changed the incubation time of step (2) in preparation 1 compared to the preparation 1 procedure of example 1. Example 4 in step (2) of preparation 1, 0.5h or 1h or 1.5h, respectively, were used as incubation times. The three first antibody-coated magnetic microspheres prepared in the example 4 are combined with the second antibody-labeled acridinium ester prepared in the example 1 and the preparation 3-5 respectively to obtain three kits, and the three kits are respectively matched with a full-automatic chemiluminescence analyzer for detection, wherein the detection results are shown in a table 4.
TABLE 4 screening results of coupling time of the first strain of cTnT monoclonal antibody and magnetic microsphere
The results in table 4 show that the coupling time of the cTnT monoclonal antibody and the magnetic microsphere of the first strain has little influence on the detection signal and the sample coincidence rate. Therefore, in the preparation process of the first strain of antibody coated magnetic microspheres of the kit, 0.5h, 1h and 1.5h can be selected, but in order to save time, the preferred coupling time is 0.5 h.
Example 5
The embodiment provides a screening process of an antibody coating proportion in a preparation process of a first strain of antibody coated magnetic microsphere.
Example 5 substantially the same procedure of preparation 1 was used as in example 1. Example 5 mainly changes the mass ratio of the magnetic microspheres to the antibodies of step (2) in preparation 1, compared to the procedure of preparation 1 in example 1. Example 5 in step (2) of preparation 1, 100:1.5, 100:1 or 100: 2 as the mass ratio of the magnetic microspheres to the antibodies. The three first antibody-coated magnetic microspheres prepared in example 5 are combined with the second antibody-labeled acridinium ester prepared in example 1 to obtain three kits, and the three kits are then detected by a full-automatic chemiluminescence analyzer, wherein the detection results are shown in table 5.
TABLE 5 screening results of mass ratio of magnetic microspheres to antibody in the preparation of first strain antibody-coated magnetic microspheres
The results in table 5 show that when the mass ratio of the magnetic microspheres to the antibody was selected, 100: the ratio of 1.5 is higher in detection signal, and the sample coincidence rate is also better. When the mass ratio is 100: at 1, the signal is significantly lower, and the sample coincidence rate is also reduced. And when the mass ratio is 100: 2, the signal is equal to 100: the mass ratio of 1.5 is close and the sample coincidence rate is also reduced. For stability and simplicity of operation, the preferable mass ratio of the magnetic microspheres to the antibodies in the preparation process of the kit of the invention is 100: 1.5.
example 6
The embodiment provides a screening process of a blocking reagent in a preparation process of a first strain of antibody coated magnetic microsphere.
Example 6 substantially the same procedure of preparation 1 was used as in example 1. Example 6 mainly changes the blocking reagent of step (4) of preparation 1 as compared with preparation 1 of example 1. Example 6 in preparation 1, in step (4), 1% casein-containing PBS solution (0.02M), 5% BSA-containing PBS solution (0.02M), or 0.5% casein-containing PBS solution (0.02M) was used, respectively. The three first antibody-coated magnetic microspheres prepared in the example 6 are combined with the second antibody-labeled acridinium ester prepared in the example 1 and the preparation 3-5 respectively to obtain three kits, and the three kits are respectively matched with a full-automatic chemiluminescence analyzer for detection, wherein the detection results are shown in a table 6.
TABLE 6 screening results of blocking reagents in the preparation of the first strain of antibody-coated magnetic microspheres
The results in Table 6 show that the detection signal is higher and the sample coincidence rate is better when 1% casein-containing PBS solution is used as the blocking agent. The preferred blocking conditions for the preparation of the kit of the invention are 200. mu.L of 1% casein in PBS (0.02M).
Example 7
This example provides a screening procedure for the buffer used in the preparation of the second antibody labeled acridinium ester.
Example 7 substantially the same procedure of preparation 2 as in example 1 was employed. Example 7 mainly changes the kind of buffer of step (1) of preparation 2 as compared with the procedure of preparation 2 of example 1. Example 7 in preparation 2, step (1), 0.05MCB buffer (pH 9.6) or 0.05M tris (hydroxymethyl) aminomethane sulfonic acid (TAPS) buffer (pH 7.5) or 0.05MHEPES buffer (pH 8.5) was used, respectively. The three second-strain antibody labeled acridinium ester prepared in the example 7 are respectively combined with the first-strain antibody coated magnetic microspheres prepared in the example 1 and the preparation 3-5 to obtain three kits, and then the three kits are respectively matched with a full-automatic chemiluminescence analyzer for detection, wherein the detection results are shown in a table 7.
TABLE 7 screening results of buffer solution in preparation of second-strain antibody-labeled acridinium ester
The results in Table 7 show that the detection signal was high and the sample coincidence rate was good when the CB diluent was used as a buffer. The preferred buffer for the kit of the present invention in the preparation of the acridinium ester labeled antibody is 0.05M CB buffer (pH 9.6).
Example 8
This example provides a screening procedure for the marker time in the preparation of a second strain of antibody-labeled acridinium ester.
Example 8 substantially the same procedure of preparation 2 as in example 1 was employed. Example 8 varied mainly the room temperature (protected from light) reaction time of step (2) of preparation 2 compared to the procedure of preparation 2 of example 1. Example 8 in step (2) of preparation 2, the reaction time at room temperature (away from light) was set to 2 hours or 1 hour or 1.5 hours, respectively. The three second-strain antibody labeled acridinium ester prepared in the example 8 are combined with the first-strain antibody coated magnetic microspheres prepared in the example 1 and the preparation methods of the three second-strain antibody labeled acridinium ester and the three first-strain antibody coated magnetic microspheres are combined with the preparation methods of the three second-strain antibody coated magnetic microspheres and the preparation.
TABLE 8 screening results of the labeling time in the preparation of acridinium ester labeled with the second antibody
The results in Table 8 show that the detection signal is greatly improved when the mark time is 2 h. Therefore, the kit of the invention has a preferable labeling time of 2h in the process of preparing the acridinium ester labeled antibody.
Example 9
This example provides a screening procedure 1 for the molar ratio of acridinium ester to antibody in the preparation of a second strain of antibody-labeled acridinium ester.
Example 9 substantially the same procedure of preparation 2 as in example 1 was employed. Example 9 mainly changes the molar ratio of NSP-SA-NHS to cTnT monoclonal antibody of step (2) of preparation 2 compared to the preparation 2 process of example 1. Example 9 the molar ratio of NSP-SA-NHS to cTnT monoclonal antibody was set to 15:1, 12:1 or 10:1, respectively, in step (2) of preparation 2. The three second-strain antibody labeled acridinium ester prepared in the example 9 are combined with the first-strain antibody coated magnetic microspheres prepared in the example 1 and the preparation methods of 3-5 respectively to obtain three kits, and the three kits are respectively matched with a full-automatic chemiluminescence analyzer for detection, wherein the detection results are shown in a table 9.
TABLE 9 screening results of molar ratio of acridinium ester to antibody in preparation of second-line antibody-labeled acridinium ester 1
The results in table 9 show that the molar ratio of acridinium ester to the second strain antibody is 15:1, the detection signal is greatly improved, and the sample coincidence rate is better. Therefore, the kit of the invention preferably has a molar ratio of the acridinium ester to the second strain antibody in the process of preparing the acridinium ester labeled antibody of 15: 1.
example 10
This example provides a screening procedure 2 for the molar ratio of acridinium ester to antibody in the preparation of a second strain of antibody-labeled acridinium ester.
Example 10 substantially the same procedure of preparation 2 as in example 1 was employed. Example 9 mainly changes the molar ratio of NSP-SA-NHS to cTnT monoclonal antibody of step (2) of preparation 2 compared to the preparation 2 process of example 1. Example 9 in step (2) of preparation 2, the molar ratio of NSP-SA-NHS to cTnT monoclonal antibody was set to 30:1 or 15:1, respectively. The three second-strain antibody labeled acridinium ester prepared in the example 10 are respectively combined with the first-strain antibody coated magnetic microspheres prepared in the example 1 and the preparation steps 4 and 5 to obtain three kits, and then the three kits are respectively matched with a full-automatic chemiluminescence analyzer for detection, wherein the detection results are shown in a table 10.
TABLE 10 screening results of molar ratio of acridinium ester to antibody in preparation of second-line antibody-labeled acridinium ester 2
The results in table 10 show that the molar ratio of acridinium ester to the second strain antibody is 14:1 or 15:1 or 18: when 1, the coincidence rate of the low-value sample is better. This value affects the detection signal below 14:1 and the background value of the detection kit above 18: 1. And when the ratio is 30: when 1, the signals of the low-value samples are scattered, and the sample coincidence rate is poor. Therefore, the kit of the invention preferably has a molar ratio of the acridinium ester to the second strain antibody in the process of preparing the acridinium ester labeled antibody of not more than 30: 1.
performance testing
The kit prepared in example 1 was subjected to the following performance tests.
(1) Detection of white space limit
The detection method is carried out by referring to the test method of YY/T1233-.
The blank sample (sample dilution) was repeatedly tested 20 times, and the blank limit was calculated according to the test results using the formula, the results are shown in tables 10 and 11.
TABLE 10 examination results of initial calibration curves
| RLU | Concentration (pg/ml) | |
| S0 | 294 | 0 |
| S1 | 1124 | 32 |
| S2 | 31135.5 | 900 |
| S3 | 168634 | 3500 |
| S4 | 374582.5 | 8000 |
| S5 | 642595 | 10000 |
TABLE 11 detection results of blank limits
Two-point regression fitting is carried out according to the results of concentration-chemiluminescence (RLU) values between a zero-concentration calibrator (S0) and an adjacent calibrator (S1) to obtain a linear equation, then the RLU value corresponding to Mean +2SD obtained by detecting a blank sample (S0) with 20 holes is substituted into the equation, the obtained concentration value is the analysis sensitivity, the blank limit of the invention can reach 4.09pg/ml, and the method has great significance for early diagnosis of AMI.
(2) Detection of precision within a day
The high and low standards were repeatedly tested 10 times each, and then CV was calculated based on the test results, which are shown in Table 12.
TABLE 12 results of measurement of precision in days
| Number of tests | QCL signal | QCH signal | QCL concentration (pg/ml) | QCH concentration (pg/ml) |
| 1 | 3999 | 514967 | 113.129052 | 9778.8613 |
| 2 | 3983 | 506553 | 112.713493 | 9632.0963 |
| 3 | 4016 | 486553 | 113.570434 | 9282.427 |
| 4 | 4011 | 475627 | 113.440631 | 9090.9069 |
| 5 | 3615 | 460365 | 103.116561 | 8822.7749 |
| 6 | 3404 | 476370 | 97.578187 | 9103.9422 |
| 7 | 3731 | 467063 | 106.14997 | 8940.5378 |
| 8 | 3884 | 452854 | 110.139153 | 8690.5506 |
| 9 | 3784 | 462546 | 107.533336 | 8861.136167 |
| 10 | 4052 | 485269 | 114.504619 | 9259.938424 |
| MEAN | 109.1875 | 9146.317 | ||
| CV | 5.09％ | 3.84％ |
The precision in the day of the invention can reach 5.09% and 3.84%, which has great significance for the diagnosis accuracy of AMI.
(3) Detection of linear correlation
The linear samples were examined in duplicate wells and the linear correlation coefficient between the theoretical and measured values was calculated for each point, the results are shown in table 13 and fig. 3.
TABLE 13 detection results of linear correlation
| Concentration 1(pg/ml) | Concentration 2(pg/ml) | Measured value (pg/ml) | Theoretical value (pg/ml) | |
| Sample 1 | 14490.37 | 13110.33 | 13800.35 | 13000 |
| Sample 2 | 11336.4 | 10755.57 | 11046.33 | 10000 |
| Sample 3 | 8756.76 | 8584.85 | 8670.85 | 7508 |
| Sample 4 | 5463.42 | 5490.68 | 5477.05 | 5016 |
| Sample 5 | 2501 | 2556.21 | 2528.62 | 2524 |
| Sample 6 | 1002.58 | 989.92 | 996.25 | 1000 |
| Sample 7 | 41.83 | 31.84 | 36.87 | 32 |
| Sample 8 | 3.825 | 3.675 | 3.75 | 4 |
| Sample 9 | 0 | 0 | 0 | 0 |
| Linear dependence | 0.9990 |
The linear correlation of the present invention, R > 0.990. The complex aperture detects a linear sample (9 points), and calculates a linear correlation coefficient of a theoretical value and an actually measured value of each point. The theoretical concentration is not calculated by using a calibration curve, and the measured value is a sample value detected by using a Roche comparison machine. Figure 3 shows that the linear dependence is good.
(4) Detection of accuracy
The detection method is carried out by referring to a test method of YY/T1233-: 7. 1: 9 and 1: 11 and detecting after dilution.
The accuracy of the recovery test detection kit is adopted, and the detection results are shown in Table 14.
TABLE 14 test results of the recovery test
| Proportion of addition | Concentration 1(pg/ml) | Concentration 2(pg/ml) | Concentration mean (pg/ml) | Recovery rate |
| 1:7 | 1141.99 | 1114.13 | 1128.06 | 102.57％ |
| 1:9 | 860.00 | 859.33 | 859.66 | 97.28％ |
| 1:11 | 690.14 | 687.47 | 688.80 | 93.11％ |
| Serum sample A | 8766.24 | 8614.90 | 8690.57 | |
| Serum sample B | 16.10 | 15.56 | 15.83 |
The recovery test result can reach 97.28%, and the accuracy is good.
(5) Comparison of methodologies
After a part of the serum or plasma sample is detected using a Troponin T detection kit (electrochemiluminescence method) (Troponin T hsSTAT) produced by Roche diagnostics, the sample is detected using the kit of the present invention, and the detection results are shown in FIG. 4. 102 Roche samples are detected by using the kit, a scatter diagram is made of concentration values (pg/ml) detected by the Roche kit and detection signal values of the kit, the coincidence rate is found to be good, and the R value of the overall coincidence rate can reach 0.9962. Therefore, the kit has better correlation with the recognized Roche hypersensitivity cTnT kit at home and abroad, and can accurately screen the diseased individuals from the healthy people.
(6) Reference value
The reference values of the present invention are based on 86 samples derived from normal persons, and the test data are shown in Table 15.
TABLE 15 test results of normal persons
(7) Detection of quantitative limit (LoQ)
After the calibrator S1 in the kit was subjected to gradient dilution, sample 1(1/2S1), sample 2(1/4S1), sample 3(1/8S1), sample 4(1/16S1), sample 5(1/32S1), and sample 6(1/64S1) were obtained. The six samples were tested in duplicate for five days, each day for 4 days, and the overall CV was calculated, respectively, requiring the sample point with CV closest to 20% as the point of functional sensitivity. The results are shown in Table 16.
TABLE 16 detection results of quantitative limits (LoQ)
The results show that the limit of quantitation (LoQ) of the kit can reach 16 pg/ml.
(8) Thermal stability test of calibrator
The calibrator provided by the kit of the present invention was in a liquid state, and the calibrator in the kit prepared in example 1 was placed in a 37 ℃ incubator box and a 4 ℃ refrigerator for 7 days, respectively, and then the detection results were shown in table 17.
TABLE 17 thermal stability of calibrators
The results in Table 17 show that the liquid calibrator of the present invention has good thermal stability and the signal deviation at each point is less than 10%.
The above examples of the present disclosure are merely examples provided for clearly illustrating the present disclosure and are not intended to limit the embodiments of the present disclosure. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present disclosure should be included in the protection scope of the claims of the present disclosure.
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Claims (10)
1. The kit is characterized by comprising a first strain of antibody coated on a magnetic microsphere and a second strain of antibody marked with a tracer marker, wherein the first strain of antibody and the second strain of antibody are monoclonal antibodies for resisting cardiac troponin T, and the specific binding sites of the cardiac troponin T recognized by the first strain of antibody and the second strain of antibody are different.
2. The kit according to claim 1, wherein the sequence of the amino acid site of cardiac troponin T recognized by the first strain of antibody is the sequence shown in SEQ ID NO. 1, and the sequence of the amino acid site of cardiac troponin T recognized by the second strain of antibody is the sequence shown in SEQ ID NO. 3.
3. The kit according to claim 1 or 2, wherein the magnetic microspheres are selected from nanoscale Fe2O3And/or Fe3O4The magnetic fine particles of (2) and a polymer material, the average particle diameter of the magnetic fine particles being 1 to 5 μm; the tracer marker is selected from one of acridinium ester, luminol, alkaline phosphatase, horseradish peroxidase and adamantane; preferably, the tracer label is an acridinium ester.
4. The kit of any one of claims 1 to 3, wherein the molar ratio of the tracer label to the second strain of antibody is 14 to 18:1 and/or the mass ratio of the magnetic microspheres to the first strain of antibody is 100:1 to 2.
5. The kit of any one of claims 1 to 4, wherein the kit comprises a liquid calibrator; preferably, the liquid calibrator comprises a PBS buffer containing a cardiac troponin T antigen.
6. A method of making a kit as claimed in any one of claims 1 to 5, comprising the steps of coating the first strain of antibody with magnetic microspheres and labeling the second strain of antibody with a tracer label.
7. The method of claim 6, wherein 2- (N-morpholino) ethanesulfonic acid (MES) buffer and/or 1% casein in PBS is used in the first antibody-coated magnetic microsphere step.
8. The method according to claim 6 or 7, wherein a Carbonate (CB) buffer is used in the second antibody labeling tracer label step.
9. The method according to any one of claims 6 to 8, wherein the labeling time of the second antibody labeled tracer marker is 1.8 to 2.5 hours; preferably, the labeling time of the second antibody labeled tracer label is 2 hours.
10. The application of a first strain antibody coated on a magnetic microsphere and a second strain antibody marked with a tracer marker in preparation of a medicine or a kit for detecting cardiac troponin T is characterized in that the first strain antibody and the second strain antibody are monoclonal antibodies for resisting the cardiac troponin T, and specific binding sites of the cardiac troponin T recognized by the first strain antibody and the second strain antibody are different.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110129854A1 (en) * | 2009-12-02 | 2011-06-02 | Abbott Laboratories | Assay for diagnosis of cardiac myocyte damage |
| CN104634980A (en) * | 2015-02-10 | 2015-05-20 | 深圳市新产业生物医学工程股份有限公司 | Cardiac troponin I (cTn I) hypersensitive detection kit and hypersensitive detection method |
-
2019
- 2019-02-02 CN CN201910107801.0A patent/CN111521809A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110129854A1 (en) * | 2009-12-02 | 2011-06-02 | Abbott Laboratories | Assay for diagnosis of cardiac myocyte damage |
| CN104634980A (en) * | 2015-02-10 | 2015-05-20 | 深圳市新产业生物医学工程股份有限公司 | Cardiac troponin I (cTn I) hypersensitive detection kit and hypersensitive detection method |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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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