High-sensitivity cardiac troponin-T electrochemiluminescence detection kit

Abstract

The invention discloses a high-sensitivity cardiac troponin-T electrochemiluminescence detection kit. The kit comprises a magnetic microsphere reagent coated by a high-sensitivity cardiac troponin-T capture antibody and a high-sensitivity cardiac troponin-T detection antibody reagent labeled by an electrically neutral ruthenium compound, the structural general formula of the electrically neutral ruthenium compound is # imgabs0 # n = 0-9; the magnetic microsphere reagent coated with the high-sensitivity cardiac troponin-T capture antibody comprises a magnetic microsphere coated with the high-sensitivity cardiac troponin-T capture antibody and a diluent; the electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody reagent comprises an electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody and a diluent; the diluent is prepared from piperazine-1, 4-diethyl sulfonic acid or 3-(N-morpholinyl)-2-hydroxy propanesulfonic acid. When the high-sensitivity cardiac troponin-T electrochemiluminescence detection kit is used for detection, a reaction system does not contain streptavidin coated magnetic beads or biotinylated antibodies, so that interference of biotin in a sample is avoided.

Description

Technical Field

[0001] The invention belongs to the field of medical detection, and in particular relates to a high-sensitivity cardiac troponin-T electrochemiluminescence detection kit. Background Art

[0002] High-sensitivity cardiac troponin-T (cTnT) is a subunit of cardiac troponin, composed of 288 amino acids, with a molecular weight of about 39.7KDa. It originates from myocardial cells. When myocardial cells are damaged by ischemia, hypoxia and other factors, free and bound cTnT are continuously released into the circulating blood. For example, after acute myocardial infarction (AMI), the concentration of cardiac troponin-T in the blood increases rapidly, and this process continues for a long time. Since cTnT has a small molecular weight, it can be quickly released into the blood after myocardial damage and has unique antigenicity. Therefore, cardiac troponin T is a highly sensitive and specific marker of myocardial cell damage, and it plays an important reference role in the diagnosis of non-ST-segment elevation myocardial infarction. Cardiac troponin T is the preferred biomarker for the diagnosis, risk stratification, treatment and prognosis of acute coronary syndrome.

[0003] AACC / IFCC has established two standards for high-sensitivity troponin:

[0004] 1) cTn (cardiac troponin) can be stably and reliably detected in no less than 50% of healthy male and female populations, and the cTn concentration is equal to or higher than the minimum detection limit (LOD);

[0005] 2) At the upper reference limit of the 99th percentile value (99 th The total CV (coefficient of variation) at the concentration of 1:1 (URL) was ≤10%.

[0006] The chemiluminescence methods used to detect cTnT in human serum or plasma mainly include: enzyme-catalyzed magnetic microparticle chemiluminescence method, acridinium ester magnetic microparticle chemiluminescence method and traditional terpyridine ruthenium electrochemiluminescence method. Most of the above methodologies use the (streptavidin) avidin / biotin coupling method, that is, streptavidin is used to coat magnetic beads and biotin is used to couple antibodies. Therefore, cTnT detection is susceptible to interference from biotin in the sample, and this interference has a particularly serious impact on the detection of high-sensitivity troponin. Summary of the invention

[0007] In order to solve the technical problem that the high-sensitivity cardiac troponin-T detection in the above-mentioned prior art is easily interfered by biotin in the sample, the purpose of the present invention is to provide a high-sensitivity cardiac troponin-T electrochemiluminescence detection kit to achieve high sensitivity, good repeatability, wide linear range, strong anti-interference ability, and can effectively shorten the detection time.

[0008] The present invention provides a high-sensitivity cardiac troponin-T electrochemiluminescence detection kit, which comprises a magnetic microsphere reagent coated with a high-sensitivity cardiac troponin-T capture antibody and a high-sensitivity cardiac troponin-T detection antibody reagent labeled with an electrically neutral ruthenium compound;

[0009] The general structural formula of the electrically neutral ruthenium compound (NRC) is as follows:

[0010]

[0011] Wherein, n=0~9;

[0012] The high-sensitivity cardiac troponin-T capture antibody-coated magnetic microsphere reagent comprises high-sensitivity cardiac troponin-T capture antibody-coated magnetic microspheres and a diluent;

[0013] The electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody reagent comprises an electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody and a diluent;

[0014] The diluent includes piperazine-1,4-diethanesulfonic acid or 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid.

[0015] The invention uses a high-sensitivity cardiac troponin-T capture antibody to coat magnetic microspheres, and uses an electrically neutral ruthenium compound to label a high-sensitivity cardiac troponin-T detection antibody. The two reagent components undergo an immune binding reaction with the high-sensitivity cardiac troponin-T in the object to be detected, and finally a double antibody sandwich complex structure of magnetic microspheres·antibody-antigen-antibody*ruthenium compound (such as Figure 1 As shown). On the one hand, the electrically neutral ruthenium compound used in the present invention labels the high-sensitivity cardiac troponin-T detection antibody, which can reduce nonspecific interference in immunoassay and produce a strong luminescent signal; on the other hand, the diluent in the two reagent components (containing piperazine-1,4-diethanesulfonic acid or 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid) can not only maintain a stable pH value, but also provide the necessary ion strength and ion composition, through which a stable reaction environment can be provided. The above two aspects work synergistically, so that the high-sensitivity cardiac troponin-T electrochemiluminescence detection kit of the present invention can significantly improve the sensitivity, repeatability, linear range and anti-interference ability of the detection, and can effectively shorten the detection time. The reaction system of the high-sensitivity cardiac troponin-T electrochemiluminescence detection kit of the present invention when used for detection does not contain streptavidin-coated magnetic beads or biotinylated antibodies, which effectively avoids the interference of biotin that may exist in the sample on the test results, and can be widely used.

[0016] High-sensitivity cardiac troponin-T capture antibody is a high-sensitivity cardiac troponin-T antibody, used as a capture antibody for coating magnetic microspheres.

[0017] High-sensitivity cardiac troponin-T detection antibody is a high-sensitivity cardiac troponin-T antibody, used as a detection antibody for labeling with electrically neutral ruthenium compounds.

[0018] Preferably, n=2-5.

[0019] Preferably, the kit further comprises a high-sensitivity cardiac troponin-T calibrator 1 and a high-sensitivity cardiac troponin-T calibrator 2, wherein the concentration range of the high-sensitivity cardiac troponin-T antigen in the high-sensitivity cardiac troponin-T calibrator 1 is 10 to 30 ng / L, and the concentration range of the high-sensitivity cardiac troponin-T antigen in the high-sensitivity cardiac troponin-T calibrator 2 is 4000 to 5000 ng / L.

[0020] Further preferably, the kit also includes a high-sensitivity cardiac troponin-T calibrator 1 and a high-sensitivity cardiac troponin-T calibrator 2, the concentration range of the high-sensitivity cardiac troponin-T antigen in the high-sensitivity cardiac troponin-T calibrator 1 is 15 to 20 ng / L, and the concentration range of the high-sensitivity cardiac troponin-T antigen in the high-sensitivity cardiac troponin-T calibrator 2 is 4000 to 4500 ng / L.

[0021] Preferably, in the magnetic microsphere reagent coated with the high-sensitivity cardiac troponin-T capture antibody, the particle size of the magnetic microsphere is 1 to 5 μm, and the concentration range of the magnetic microsphere coated with the high-sensitivity cardiac troponin-T capture antibody is 0.1 to 0.4 mg / mL.

[0022] Further preferably, in the magnetic microsphere reagent coated with the high-sensitivity cardiac troponin-T capture antibody, the particle size of the magnetic microsphere is 2 to 3 μm; and the concentration range of the magnetic microsphere coated with the high-sensitivity cardiac troponin-T capture antibody is 0.2 to 0.3 mg / mL.

[0023] Preferably, in the diluent, the concentration of the piperazine-1,4-diethanesulfonic acid is 30 to 150 mmol / L, or the concentration of the 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid is 30 to 150 mmol / L.

[0024] More preferably, in the diluent, the concentration of the piperazine-1,4-diethanesulfonic acid is 50 to 100 mmol / L, or the concentration of the 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid is 50 to 100 mmol / L.

[0025] Preferably, the pH value of the diluent is 6.5-7.5.

[0026] More preferably, the pH value of the diluent is 6.8.

[0027] Preferably, the diluent further comprises Tween-20 and bovine serum albumin (BSA).

[0028] Preferably, in the diluent, the concentration of Tween-20 is 0.02-0.1% g / mL, and the concentration of bovine serum albumin (BSA) is 0.5-2% g / mL.

[0029] Preferably, the preparation method of the magnetic microsphere reagent coated with the high-sensitivity cardiac troponin-T capture antibody is:

[0030] S1. Mix the magnetic microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution;

[0031] S2. Remove the supernatant by magnetic separation, add high-sensitivity cardiac troponin-T capture antibody and mix well;

[0032] S3. Add sealing agent and mix well;

[0033] S4. After the supernatant is removed by magnetic separation, a diluent is added to resuspend the magnetic microspheres to obtain a magnetic microsphere reagent coated with a high-sensitivity cardiac troponin-T capture antibody.

[0034] Preferably, in the electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody reagent, the concentration range of the electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody is 0.25 to 2.0 μg / mL.

[0035] Further preferably, in the electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody reagent, the concentration range of the electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody is 1.0 to 2.0 μg / mL.

[0036] Preferably, in the high-sensitivity cardiac troponin-T detection antibody labeled with the electrically neutral ruthenium compound, the molar binding ratio of the electrically neutral ruthenium compound to the high-sensitivity cardiac troponin-T detection antibody is (2:1) to (8:1).

[0037] In the electrically neutral ruthenium compound-labeled high-sensitivity cardiac troponin-T detection antibody described in this scheme, the molar binding ratio of the electrically neutral ruthenium compound to the high-sensitivity cardiac troponin-T detection antibody is (2:1) to (8:1), which can not only enhance the intensity of the luminescent signal, but also ensure that the electrically neutral ruthenium compound-labeled high-sensitivity cardiac troponin-T detection antibody can fully carry out an immune binding reaction with the high-sensitivity cardiac troponin-T in the object to be detected.

[0038] Further preferably, in the electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody reagent, the molar binding ratio of the electrically neutral ruthenium compound to the high-sensitivity cardiac troponin-T detection antibody is (5:1) to (8:1).

[0039] Preferably, the preparation method of the electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody reagent is:

[0040] S1. mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, N-hydroxysulfosuccinimide solution and electrically neutral ruthenium compound solution;

[0041] S2. Add high-sensitivity cardiac troponin-T detection antibody for incubation;

[0042] S3. Remove the unbound electrically neutral ruthenium compound, add a diluent, and obtain a highly sensitive cardiac troponin-T detection antibody reagent labeled with the electrically neutral ruthenium compound.

[0043] Preferably, the reaction volume ratio of the magnetic microsphere reagent coated with the high-sensitivity cardiac troponin-T capture antibody to the high-sensitivity cardiac troponin-T detection antibody reagent labeled with an electrically neutral ruthenium compound is (50-100): (50-100).

[0044] Further preferably, the reaction volume ratio of the magnetic microsphere reagent coated with the high-sensitivity cardiac troponin-T capture antibody to the high-sensitivity cardiac troponin-T detection antibody reagent labeled with an electrically neutral ruthenium compound is (70-80): (70-80).

[0045] Preferably, the incubation time for detection using the kit is 5 to 15 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the double antibody sandwich complex structure formed in the electrochemiluminescent immunoreaction of the present invention.

[0047] Figure 2 This is a calibration curve diagram of the high-sensitivity cardiac troponin-T calibrator of Example 1 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of cTnT calibrator 1 is 20 ng / L, and the assigned concentration of cTnT calibrator 2 is 4500 ng / L).

[0048] Figure 3 This is a calibration curve diagram of the high-sensitivity cardiac troponin-T calibrator of Example 2 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of cTnT calibrator 1 is 20 ng / L, and the assigned concentration of cTnT calibrator 2 is 4500 ng / L).

[0049] Figure 4 This is a calibration curve diagram of the high-sensitivity cardiac troponin-T calibrator of Example 3 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of cTnT calibrator 1 is 20 ng / L, and the assigned concentration of cTnT calibrator 2 is 4500 ng / L).

[0050] Figure 5 This is a calibration curve diagram of the high-sensitivity cardiac troponin-T calibrator of Example 4 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of cTnT calibrator 1 is 20 ng / L, and the assigned concentration of cTnT calibrator 2 is 4500 ng / L).

[0051] Figure 6 This is a calibration curve diagram of the high-sensitivity cardiac troponin-T calibrator of comparative example 1 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of cTnT calibrator 1 is 20 ng / L, and the assigned concentration of cTnT calibrator 2 is 4500 ng / L).

[0052] Figure 7 This is a calibration curve diagram of the high-sensitivity cardiac troponin-T calibrator of comparative example 2 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of cTnT calibrator 1 is 20 ng / L, and the assigned concentration of cTnT calibrator 2 is 4500 ng / L). DETAILED DESCRIPTION

[0053] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.In addition, should be understood that after reading content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope of the appended claims limitation of the application.

[0054] cTnT monoclonal antibody (capture antibody) and cTnT monoclonal antibody (detection antibody) were purchased from MedixBiochemica.

[0055] The preparation methods of the freshly prepared 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution and N-hydroxysulfosuccinimide solution used in the following examples and comparative examples are as follows:

[0056] 10 mg / mL and 40 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solutions were prepared using 2-morpholineethanesulfonic acid (MES) buffer (pH=6.0), respectively.

[0057] A 100 mmol / L N-hydroxysulfosuccinimide (Sulfo-NHS) solution was prepared using 2-morpholineethanesulfonic acid (MES) buffer (pH=6.0).

[0058] Example 1

[0059] The preparation method of the high-sensitivity cardiac troponin-T electrochemiluminescence detection kit of this embodiment is as follows:

[0060] The preparation of magnetic microsphere reagent coated with cTnT monoclonal antibody (capture antibody) is as follows:

[0061] S1. Mix 1 mL of magnetic microspheres (20 mg / mL) with a particle size of 2.7 μm with 80 μL of freshly prepared 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution (10 mg / mL) and rotate at room temperature for 30 minutes;

[0062] S2. Remove the supernatant by magnetic separation, resuspend the magnetic microspheres in 1 mL of 25 mM 2-morpholineethanesulfonic acid buffer (MES buffer, pH 6.0), add 0.4 mg of cTnT monoclonal antibody (capture antibody), and rotate and mix at room temperature for 4 hours;

[0063] S3. Add an equal volume of 1% (mass / volume, g / mL) bovine serum albumin (BSA) solution and rotate and mix at room temperature for 30 minutes;

[0064] S4. After removing the supernatant by magnetic separation, 67 mL of diluent containing 75 mmol / L piperazine-1,4-diethanesulfonic acid, 0.05% Tween-20 (w / v, g / mL), and 1% (w / v, g / mL) BSA (pH adjusted to 6.8) was added to resuspend the magnetic microspheres;

[0065] The concentration of the magnetic microspheres coated with S5.cTnT monoclonal antibody (capture antibody) after dilution was 0.3 mg / mL.

[0066] The preparation of the cTnT monoclonal antibody (detection antibody) reagent labeled with an electrically neutral ruthenium compound is as follows:

[0067] S1.5mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (40mmol / L), 5mL N-hydroxysulfosuccinimide solution (100mmol / L), 5mL electrically neutral ruthenium compound solution (10mmol / L, solvent is purified water), mix at room temperature for 45 minutes to obtain an electrically neutral ruthenium compound mixed solution, the specific structural formula of the electrically neutral ruthenium compound is as follows:

[0068]

[0069] S2. Add 5 μmol of cTnT monoclonal antibody (detection antibody) to the above-mentioned electrically neutral ruthenium compound mixed solution and incubate at room temperature for 4 hours;

[0070] S3. removing unbound electrically neutral ruthenium compounds through a desalting column;

[0071] S4. The labeled antibody was quantified by bicinchoninic acid (BCA) protein assay. According to the extinction coefficient of the labeled molecule, the absorbance measurement of the electrically neutral ruthenium compound at 455 nm was related to the labeled amount. It was determined that the actual binding molar ratio of the electrically neutral ruthenium compound to the cTnT monoclonal antibody (detection antibody) in the cTnT monoclonal antibody (detection antibody) labeled with the electrically neutral ruthenium compound was 6.8:1.

[0072] S5. Dilute the cTnT monoclonal antibody (detection antibody) labeled with an electrically neutral ruthenium compound to 1.5 μg / mL using a diluent containing 75 mmol / L piperazine-1,4-diethanesulfonic acid, 0.05% Tween-20 (w / v, g / mL), and 1% (w / v, g / mL) BSA (pH adjusted to 6.8).

[0073] The high-sensitivity cardiac troponin-T calibrator was prepared as follows:

[0074] Preparation of cTnT calibrator 1: weigh 0.2 mL of cTnT antigen stock solution (50 ng / mL), dilute to 500 mL with antigen diluent, the concentration of cTnT calibrator 1 is 20 ng / L, and lyophilize after aliquoting;

[0075] Preparation of cTnT calibrator 2: Weigh 40 mL of cTnT antigen stock solution (50 ng / mL), dilute to 500 mL with antigen diluent, the concentration of cTnT calibrator 2 is 4000 ng / L, and lyophilize after aliquoting.

[0076] Example 2

[0077] In the preparation method of the high-sensitivity cardiac troponin-T electrochemiluminescence detection kit of this embodiment, the specific structural formula of the electrically neutral ruthenium compound used is as follows:

[0078] Other than that, the same as in Example 1.

[0079] Example 3

[0080] In the preparation method of the high-sensitivity cardiac troponin-T electrochemiluminescence detection kit of this embodiment, except that piperazine-1,4-diethanesulfonic acid replaces 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid, the rest is the same as in Example 1.

[0081] Example 4

[0082] In the preparation method of the high-sensitivity cardiac troponin-T electrochemiluminescence detection kit of this embodiment, except for the preparation method of the cTnT monoclonal antibody (detection antibody) reagent labeled with an electrically neutral ruthenium compound as follows, the rest is the same as in Example 1;

[0083] Preparation method of cTnT monoclonal antibody (detection antibody) reagent labeled with electrically neutral ruthenium compound:

[0084] S1.5mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (40mmol / L), 5mL N-hydroxysulfosuccinimide solution (100mmol / L), 5mL electrically neutral ruthenium compound solution (6mmol / L, solvent is purified water), mix at room temperature for 45 minutes to obtain an electrically neutral ruthenium compound mixed solution, the specific structural formula of the electrically neutral ruthenium compound is as follows:

[0085]

[0086] S2. Add 5 μmol of cTnT monoclonal antibody (detection antibody) to the above-mentioned electrically neutral ruthenium compound mixed solution and incubate at room temperature for 4 hours;

[0087] S3. removing unbound electrically neutral ruthenium compounds through a desalting column;

[0088] S4. The labeled antibody was quantified by bicinchoninic acid (BCA) protein assay. According to the extinction coefficient of the labeled molecule, the absorbance measurement of the electrically neutral ruthenium compound at 455 nm was related to the labeled amount, and the actual binding molar ratio of the electrically neutral ruthenium compound to the cTnT monoclonal antibody (detection antibody) in the cTnT monoclonal antibody (detection antibody) labeled with the electrically neutral ruthenium compound was determined to be 4:1;

[0089] S5. Dilute the cTnT monoclonal antibody (detection antibody) labeled with an electrically neutral ruthenium compound to 1.5 μg / mL using a diluent containing 75 mmol / L piperazine-1,4-diethanesulfonic acid, 0.05% Tween-20 (w / v, g / mL), and 1% (w / v, g / mL) BSA (pH adjusted to 6.8).

[0090] Comparative Example 1

[0091] In the preparation method of the high-sensitivity cardiac troponin-T electrochemiluminescence detection kit of this comparative example, the specific structural formula of the electrically neutral ruthenium compound used is as follows:

[0092] Other than that, the same as in Example 1.

[0093] Comparative Example 2

[0094] In the preparation method of the high-sensitivity cardiac troponin-T electrochemiluminescence detection kit of this comparative example, except that piperazine-1,4-diethanesulfonic acid is replaced by 4-hydroxyethylpiperazineethanesulfonic acid, the rest is the same as Example 1.

[0095] Performance Testing

[0096] 5-minute incubation time test procedure - calibration of high-sensitivity cardiac troponin-T calibrator

[0097] S1. Pipette 75 μL of cTnT monoclonal antibody (detection antibody) reagent labeled with an electrically neutral ruthenium compound and 50 μL of reconstituted calibrator (the solvent is purified water) into the test tube, mix for 3 to 5 seconds, pipette 75 μL of magnetic microsphere reagent coated with cTnT monoclonal antibody (capture antibody) into the test tube, mix for 3 to 5 seconds, and incubate at 37°C for 5 minutes;

[0098] S2. The reaction mixture after incubation is sucked into the measuring pool, and the magnet under the measuring pool fixes the magnetic beads in the reaction mixture on the electrode surface;

[0099] S3. Absorb the cleaning solution to clean other substances not fixed to the electrode surface, start the voltage, and the electrically neutral ruthenium compound undergoes an electrochemical reaction to form light emission;

[0100] S4. The intensity of the light signal detected by the photomultiplier tube is positively correlated with the amount of cTnT captured on the surface of the magnetic beads, so that cTnT can be quantitatively analyzed;

[0101] S5. Establish a calibration curve using the two calibrator concentrations and corresponding luminescent signal values;

[0102] S6.cTnT calibrator was calibrated using the YnY 3030 fully automatic electrochemiluminescence immunoassay analyzer from Shenzhen Ansai Diagnostics Technology Co., Ltd.

[0103] The detection method of the high-sensitivity cardiac troponin-T electrochemiluminescence detection kit comprises the following steps:

[0104] S1. Pipette 75 μL of cTnT monoclonal antibody (detection antibody) reagent labeled with an electrically neutral ruthenium compound and 50 μL of sample into the test tube, mix for 3 to 5 seconds, pipette 75 μL of magnetic microsphere reagent coated with cTnT monoclonal antibody (capture antibody) into the test tube, mix for 3 to 5 seconds, and incubate at 37°C for 5 minutes;

[0105] S2. The reaction mixture after incubation is sucked into the measuring pool, and the magnet under the measuring pool fixes the magnetic beads in the reaction mixture on the electrode surface;

[0106] S3. Absorb the cleaning solution to clean other substances not fixed to the electrode surface, start the voltage, and the electrically neutral ruthenium compound undergoes an electrochemical reaction to form light emission;

[0107] S4. The intensity of the light signal detected by the photomultiplier tube is positively correlated with the amount of cTnT captured on the surface of the magnetic beads, so that cTnT can be quantitatively analyzed;

[0108] S5. The cTnT content in the sample is determined using the calibration curve.

[0109] Detection limit evaluation

[0110] The calibration curve was established by 5-minute incubation time detection procedure-high-sensitivity cardiac troponin-T calibrator calibration (such as Figures 2 to 7 As shown in the figure, take 5 low-value samples with a concentration close to the detection limit (3ng / L), test them with the kit, and test each sample 5 times. The following requirements should be met: the number of test results with a value lower than the given blank limit (2ng / L) is less than or equal to 3.

[0111] Table 1 Detection limit evaluation results (unit: ng / L)

[0112]

[0113] As can be seen from Table 1, the detection limit assessment of the kits prepared using Examples 1, 2, 3 and 4 using the 5-minute incubation detection program of the YnY 3030 fully automatic electrochemiluminescence immunoassay analyzer of Shenzhen Ansai Diagnostics Technology Co., Ltd. all meets the requirements; the detection limit assessment of the kits prepared using Comparative Examples 1 and 2 does not meet the requirements.

[0114] Repeatability assessment

[0115] The calibration curve was established by 5-minute incubation time detection procedure-high-sensitivity cardiac troponin-T calibrator calibration (such as Figures 2 to 7 As shown in the figure, the samples with concentrations between (20±4.0) ng / L and (2000±400) ng / L were measured 10 times, and the mean (M) and standard deviation (SD) of the 10 measurement results were calculated to obtain the coefficient of variation (CV), which should satisfy CV≤8%.

[0116] CV=SD / M×100%

[0117] Table 2 Repeatability evaluation results (unit: ng / L)

[0118]

[0119] As can be seen from Table 2, the repeatability evaluation of the kits prepared using Example 1, Example 2, Example 3 and Example 4 in the 5-minute incubation detection program of the YnY 3030 fully automatic electrochemiluminescence immunoassay analyzer of Shenzhen Ansai Diagnostics Technology Co., Ltd. all met the requirements; the repeatability evaluation of the kits prepared in Comparative Examples 1 and 2 did not meet the requirements.

[0120] Linear range assessment

[0121] The calibration curve was established by 5-minute incubation time detection procedure-high-sensitivity cardiac troponin-T calibrator calibration (such as Figures 2 to 7 As shown in the figure, the high-value linear sample close to the upper limit of the linear interval (10000ng / L) is diluted proportionally into 5 concentrations, among which the low-value linear sample should be close to the lower limit of the linear interval (3ng / L). The sample of each concentration is measured 3 times, and the average value is calculated. The average value of the measured concentration and the theoretical concentration are linearly fitted using the least squares method, and the linear correlation coefficient r is calculated, which should meet r≥0.99.

[0122] Table 3 Linear range evaluation results (unit: ng / L)

[0123]

[0124] As can be seen from Table 3, the 5-minute incubation detection program of the YnY 3030 fully automatic electrochemiluminescence immunoassay analyzer of Shenzhen Ansai Diagnostics Technology Co., Ltd., and the linear range evaluation of the kits prepared using Example 1, Example 2, Example 3, Example 4, Comparative Example 1 and Comparative Example 2 all meet the requirements.

[0125] Biotin Interference Assessment

[0126] The calibration curve was established by 5-minute incubation time detection procedure-high-sensitivity cardiac troponin-T calibrator calibration (such as Figures 2 to 7 As shown), add biotin to the sample with cTnT concentration of (20±4.0) ng / L to make its concentration reach 1000000 ng / mL, and the addition ratio shall not exceed 1:9. Repeat the test for this sample 3 times, and the interference deviation shall be within the range of ±10%.

[0127] Table 4 Biotin interference evaluation results (unit: ng / mL)

[0128]

[0129] As can be seen from Table 4, the 5-minute incubation detection program of the YnY 3030 fully automatic electrochemiluminescence immunoassay analyzer of Shenzhen Ansai Diagnostics Technology Co., Ltd., and the biotin interference assessment of the kits prepared using Example 1, Example 2, Example 3, Example 4, Comparative Example 1 and Comparative Example 2 all meet the requirements.

[0130] Comparison with imported test kits:

[0131] Performance comparison with imported test kits

[0132] Table 5 Performance comparison of Examples 1 to 4 and imported kits

[0133]

[0134] As shown in Table 5, the kits prepared using Examples 1 to 4 are superior to imported reagents in terms of detection limit, incubation time, and biotin interference, and all have significant improvements.

[0135] The above contents are examples of specific embodiments of the present invention. Reagents, equipment, operating methods, etc. that are not described in detail should be understood as being implemented by using conventional reagents, equipment, operating methods, etc. available in the art.

[0136] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features disclosed herein.

Claims

1. A high-sensitivity cardiac troponin-T electrochemiluminescence detection kit, characterized in that: The kit comprises a magnetic microsphere reagent coated with a high-sensitivity cardiac troponin-T capture antibody and a high-sensitivity cardiac troponin-T detection antibody reagent labeled with an electrically neutral ruthenium compound; The general structural formula of the electrically neutral ruthenium compound is as follows: Wherein, n=0~9; The high-sensitivity cardiac troponin-T capture antibody-coated magnetic microsphere reagent comprises high-sensitivity cardiac troponin-T capture antibody-coated magnetic microspheres and a diluent; The electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody reagent comprises an electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody and a diluent; The diluent includes piperazine-1,4-diethanesulfonic acid or 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid.

2. The high-sensitivity cardiac troponin-T electrochemiluminescence detection kit according to claim 1, characterized in that: The kit also includes a high-sensitivity cardiac troponin-T calibrator 1 and a high-sensitivity cardiac troponin-T calibrator 2, wherein the concentration range of the high-sensitivity cardiac troponin-T antigen in the high-sensitivity cardiac troponin-T calibrator 1 is 10 to 30 ng / L, and the concentration range of the high-sensitivity cardiac troponin-T antigen in the high-sensitivity cardiac troponin-T calibrator 2 is 4000 to 5000 ng / L.

3. The high-sensitivity cardiac troponin-T electrochemiluminescence detection kit according to claim 1, characterized in that: In the magnetic microsphere reagent coated with the high-sensitivity cardiac troponin-T capture antibody, the particle size of the magnetic microsphere is 1 to 5 μm, and the concentration range of the magnetic microsphere coated with the high-sensitivity cardiac troponin-T capture antibody is 0.1 to 0.4 mg / mL.

4. The high-sensitivity cardiac troponin-T electrochemiluminescence detection kit according to claim 1, characterized in that: In the diluent, the concentration of the piperazine-1,4-diethanesulfonic acid is 30 to 150 mmol / L, or the concentration of the 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid is 30 to 150 mmol / L.

5. The high-sensitivity cardiac troponin-T electrochemiluminescence detection kit according to claim 1, characterized in that: The preparation method of the magnetic microsphere reagent coated with the high-sensitivity cardiac troponin-T capture antibody is as follows: S1. Mix the magnetic microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution; S2. Remove the supernatant by magnetic separation, add high-sensitivity cardiac troponin-T capture antibody and mix well; S3. Add sealing agent and mix well; S4. After the supernatant is removed by magnetic separation, a diluent is added to resuspend the magnetic microspheres to obtain a magnetic microsphere reagent coated with a high-sensitivity cardiac troponin-T capture antibody.

6. The high-sensitivity cardiac troponin-T electrochemiluminescence detection kit according to claim 1, characterized in that: In the electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody reagent, the concentration range of the electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody is 0.25 to 2.0 μg / mL.

7. The high-sensitivity cardiac troponin-T electrochemiluminescence detection kit according to claim 1, characterized in that: In the electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody, the molar binding ratio of the electrically neutral ruthenium compound to the high-sensitivity cardiac troponin-T detection antibody is (2:1) to (8:1).

8. The high-sensitivity cardiac troponin-T electrochemiluminescence detection kit according to claim 1, characterized in that: The preparation method of the electrically neutral ruthenium compound labeled high-sensitivity cardiac troponin-T detection antibody reagent is as follows: S1. mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, N-hydroxysulfosuccinimide solution and electrically neutral ruthenium compound solution; S2. Add high-sensitivity cardiac troponin-T detection antibody for incubation; S3. Remove the unbound electrically neutral ruthenium compound, add a diluent, and obtain a highly sensitive cardiac troponin-T detection antibody reagent labeled with the electrically neutral ruthenium compound.

9. The high-sensitivity cardiac troponin-T electrochemiluminescence detection kit according to claim 1, characterized in that: The reaction volume ratio of the magnetic microsphere reagent coated with the high-sensitivity cardiac troponin-T capture antibody to the high-sensitivity cardiac troponin-T detection antibody reagent labeled with an electrically neutral ruthenium compound is (50-100): (50-100).

10. The high-sensitivity cardiac troponin-T electrochemiluminescence detection kit according to any one of claims 1 to 9, characterized in that: The incubation time for detection using the kit is 5 to 15 minutes.

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