Electrochemiluminescence detection kit for amino-terminal brain natriuretic peptide precursor

Abstract

The invention discloses an electrochemiluminescence detection kit for an amino-terminal brain natriuretic peptide precursor. The kit comprises a magnetic microsphere reagent coated by an amino-terminal brain natriuretic peptide precursor capture antibody and an amino-terminal brain natriuretic peptide precursor 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 amino-terminal brain natriuretic peptide precursor capture antibody comprises magnetic microspheres coated with the amino-terminal brain natriuretic peptide precursor capture antibody and a diluent; the electrically neutral ruthenium compound labeled amino-terminal brain natriuretic peptide precursor detection antibody reagent comprises an electrically neutral ruthenium compound labeled amino-terminal brain natriuretic peptide precursor detection antibody and a diluent; the diluent comprises piperazine-1, 4-diethyl sulfonic acid or bis (2-ethoxyl) amino (trihydroxymethyl) methane. The reaction system of the electrochemical luminescence detection kit for the amino-terminal brain natriuretic peptide precursor does not contain streptavidin coated magnetic beads or biotinylated antibodies, so that the 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 an amino-terminal brain natriuretic peptide precursor electrochemiluminescence detection kit. Background Art

[0002] Brain natriuretic peptide (BNP) is a neurohormone substance with similar functions to atrial natriuretic peptide (ANP). It has the effects of promoting urinary sodium excretion and resisting sympathetic nerve activity. However, it is quite different from atrial natriuretic peptide in terms of tissue distribution and amino acid composition, so it is regarded as another substance and named BNP. BNP in the human body is obtained by cleavage of the C-terminus of BNP amino acid precursor protein (proBNP), while its N-terminus is cleaved to form NT-proBNP, which has only 76 amino acids, i.e., amino-terminal brain natriuretic peptide precursor. Amino-terminal brain natriuretic peptide precursor (NT-proBNP) is mainly synthesized by left ventricular myocardial cells. After being produced in the body, it is secreted into the blood. It can be used as an effective marker of heart failure and can independently predict the condition of increased ventricular pressure. It has important value in the auxiliary diagnosis and treatment evaluation and monitoring of cardiovascular-related diseases such as congestive heart failure and its induced dyspnea complications, essential hypertension and its evaluation of left ventricular dysfunction.

[0003] The chemiluminescence methods used to detect NT-proBNP 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, magnetic beads are coated with streptavidin and antibodies are coupled with biotin. Therefore, NT-proBNP detection is easily interfered by biotin in the sample. Summary of the invention

[0004] In order to solve the technical problem in the above-mentioned prior art that the detection of amino-terminal pro-brain natriuretic peptide (NT-proBNP) is easily interfered by biotin in the sample, the purpose of the present invention is to provide an amino-terminal pro-brain natriuretic peptide electrochemiluminescence detection kit to achieve high sensitivity, good repeatability, wide linear range, strong anti-interference ability, and effectively shorten the detection time.

[0005] The present invention provides an amino-terminal pro-brain natriuretic peptide electrochemiluminescence detection kit, which comprises a magnetic microsphere reagent coated with an amino-terminal pro-brain natriuretic peptide capture antibody and an amino-terminal pro-brain natriuretic peptide detection antibody reagent labeled with an electrically neutral ruthenium compound;

[0006] The general structural formula of the electrically neutral ruthenium compound (NRC) is as follows:

[0007]

[0008] Wherein, n=0~9;

[0009] The amino-terminal pro-brain natriuretic peptide capture antibody-coated magnetic microsphere reagent comprises amino-terminal pro-brain natriuretic peptide capture antibody-coated magnetic microspheres and a diluent;

[0010] The electrically neutral ruthenium compound labeled amino-terminal pro-brain natriuretic peptide detection antibody reagent comprises an electrically neutral ruthenium compound labeled amino-terminal pro-brain natriuretic peptide detection antibody and a diluent;

[0011] The diluent includes piperazine-1,4-diethanesulfonic acid or bis(2-hydroxyethyl)amino(trishydroxymethyl)methane.

[0012] The present invention uses an amino-terminal pro-brain natriuretic peptide capture antibody to coat magnetic microspheres, and uses an electrically neutral ruthenium compound to label an amino-terminal pro-brain natriuretic peptide detection antibody. The two reagent components undergo an immune binding reaction with the amino-terminal pro-brain natriuretic peptide 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 amino-terminal brain natriuretic peptide precursor 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 bis(2-hydroxyethyl)amino(trihydroxymethyl)methane) can not only maintain a stable pH value, but also provide the necessary ionic strength and ion composition, through which a stable reaction environment can be provided. The above two aspects work synergistically, so that the amino-terminal brain natriuretic peptide precursor 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 amino-terminal brain natriuretic peptide precursor electrochemiluminescence detection kit of the present invention does not contain streptavidin-coated magnetic beads or biotinylated antibodies when used for detection, which effectively avoids the interference of biotin that may exist in the sample on the test results, and can be widely used.

[0013] N-terminal pro-BNP capture antibody is an N-terminal pro-BNP antibody, used as a capture antibody for coating magnetic microspheres.

[0014] N-terminal pro-brain natriuretic peptide detection antibody is an N-terminal pro-brain natriuretic peptide antibody, used as a detection antibody for labeling with electrically neutral ruthenium compounds.

[0015] Preferably, n=2-5.

[0016] Preferably, the kit further comprises an amino-terminal pro-brain natriuretic peptide calibrator 1 and an amino-terminal pro-brain natriuretic peptide calibrator 2, wherein the concentration range of the amino-terminal pro-brain natriuretic peptide antigen in the amino-terminal pro-brain natriuretic peptide calibrator 1 is 100 to 150 pg / mL, and the concentration range of the amino-terminal pro-brain natriuretic peptide antigen in the amino-terminal pro-brain natriuretic peptide calibrator 2 is 4500 to 5500 pg / mL.

[0017] Further preferably, the kit also includes an amino-terminal pro-brain natriuretic peptide calibrator 1 and an amino-terminal pro-brain natriuretic peptide calibrator 2, wherein the concentration range of the amino-terminal pro-brain natriuretic peptide antigen in the amino-terminal pro-brain natriuretic peptide calibrator 1 is 120-130 pg / mL, and the concentration range of the amino-terminal pro-brain natriuretic peptide antigen in the amino-terminal pro-brain natriuretic peptide calibrator 2 is 4800-5200 pg / mL.

[0018] Preferably, in the amino-terminal pro-BNP capture antibody-coated magnetic microsphere reagent, the particle size of the magnetic microsphere is 1 to 5 μm, and the concentration range of the amino-terminal pro-BNP capture antibody-coated magnetic microsphere is 0.1 to 0.5 mg / mL.

[0019] Further preferably, in the amino-terminal pro-brain natriuretic peptide capture antibody-coated magnetic microsphere reagent, the particle size of the magnetic microsphere is 2 to 3 μm; and the concentration range of the amino-terminal pro-brain natriuretic peptide capture antibody-coated magnetic microsphere is 0.2 to 0.3 mg / mL.

[0020] Preferably, in the diluent, the concentration of piperazine-1,4-diethanesulfonic acid is 20 to 100 mmol / L, or the concentration of bis(2-hydroxyethyl)amino(trihydroxymethyl)methane is 20 to 100 mmol / L.

[0021] 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 bis(2-hydroxyethyl)amino(trihydroxymethyl)methane is 50 to 100 mmol / L.

[0022] Preferably, the pH value of the diluent is 5.5-7.5.

[0023] More preferably, the pH value of the diluent is 6.5.

[0024] Preferably, the diluent further comprises Tween-20 and bovine serum albumin (BSA).

[0025] 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.

[0026] Preferably, the preparation method of the amino-terminal pro-brain natriuretic peptide capture antibody-coated magnetic microsphere reagent is:

[0027] S1. Mix the magnetic microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution;

[0028] S2. Remove the supernatant by magnetic separation, add amino-terminal pro-brain natriuretic peptide capture antibody and mix well;

[0029] S3. Add sealing agent and mix well;

[0030] S4. After removing the supernatant by magnetic separation, add diluent and resuspend the magnetic microspheres to obtain the magnetic microsphere reagent coated with the amino-terminal pro-brain natriuretic peptide capture antibody.

[0031] Preferably, in the electrically neutral ruthenium compound labeled amino-terminal pro-brain natriuretic peptide detection antibody reagent, the concentration range of the electrically neutral ruthenium compound labeled amino-terminal pro-brain natriuretic peptide detection antibody is 0.25 to 2.5 μg / mL.

[0032] Further preferably, in the electrically neutral ruthenium compound labeled amino-terminal pro-brain natriuretic peptide detection antibody reagent, the concentration range of the electrically neutral ruthenium compound labeled amino-terminal pro-brain natriuretic peptide detection antibody is 0.5 to 2 μg / mL.

[0033] Preferably, in the amino-terminal pro-brain natriuretic peptide detection antibody labeled with the electrically neutral ruthenium compound, the molar binding ratio of the electrically neutral ruthenium compound to the amino-terminal pro-brain natriuretic peptide detection antibody is (2:1) to (10:1).

[0034] In the amino-terminal pro-brain natriuretic peptide detection antibody labeled with an electrically neutral ruthenium compound described in this scheme, the molar binding ratio of the electrically neutral ruthenium compound to the amino-terminal pro-brain natriuretic peptide detection antibody is (2:1) to (10:1), which can not only enhance the intensity of the luminescent signal, but also ensure that the amino-terminal pro-brain natriuretic peptide detection antibody labeled with an electrically neutral ruthenium compound can fully carry out an immune binding reaction with the amino-terminal pro-brain natriuretic peptide in the object to be detected.

[0035] Further preferably, in the amino-terminal pro-brain natriuretic peptide detection antibody reagent labeled with an electrically neutral ruthenium compound, the molar binding ratio of the electrically neutral ruthenium compound to the amino-terminal pro-brain natriuretic peptide detection antibody is (4:1) to (8:1).

[0036] Preferably, the preparation method of the amino-terminal pro-brain natriuretic peptide detection antibody reagent labeled with an electrically neutral ruthenium compound is:

[0037] S1. Mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, N-hydroxysulfosuccinimide solution, and electrically neutral ruthenium compound solution;

[0038] S2. Add amino-terminal pro-brain natriuretic peptide detection antibody for incubation;

[0039] S3. Remove the unbound electrically neutral ruthenium compound, add diluent, and obtain the amino-terminal brain natriuretic peptide precursor detection antibody reagent labeled with the electrically neutral ruthenium compound.

[0040] Preferably, the reaction volume ratio of the amino-terminal pro-brain natriuretic peptide capture antibody-coated magnetic microsphere reagent and the amino-terminal pro-brain natriuretic peptide detection antibody reagent labeled with an electrically neutral ruthenium compound is (50-100): (50-100).

[0041] Further preferably, the reaction volume ratio of the amino-terminal pro-BNP capture antibody-coated magnetic microsphere reagent to the amino-terminal pro-BNP detection antibody reagent labeled with an electrically neutral ruthenium compound is (75-85): (75-85).

[0042] Preferably, the incubation time for detection using the kit is 5 to 15 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the double antibody sandwich complex structure formed in the electrochemiluminescent immunoreaction of the present invention.

[0044] Figure 2 This is a calibration curve of the amino-terminal pro-brain natriuretic peptide calibrator of Example 1 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of NT-proBNP calibrator 1 is 125 pg / mL, and the assigned concentration of NT-proBNP calibrator 2 is 5000 pg / mL).

[0045] Figure 3 This is a calibration curve diagram of the amino-terminal pro-brain natriuretic peptide calibrator of Example 2 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of NT-proBNP calibrator 1 is 125 pg / mL, and the assigned concentration of NT-proBNP calibrator 2 is 5000 pg / mL).

[0046] Figure 4This is a calibration curve of the amino-terminal pro-brain natriuretic peptide calibrator of Example 3 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of NT-proBNP calibrator 1 is 125 pg / mL, and the assigned concentration of NT-proBNP calibrator 2 is 5000 pg / mL).

[0047] Figure 5 This is a calibration curve of the amino-terminal pro-brain natriuretic peptide calibrator of Example 4 on the YnY3030 fully automatic electrochemiluminescence immunoassay analyzer in the 5-minute incubation time detection procedure (the assigned concentration of NT-proBNP calibrator 1 is 125 pg / mL, and the assigned concentration of NT-proBNP calibrator 2 is 5000 pg / mL).

[0048] Figure 6 This is the calibration curve of the amino-terminal pro-brain natriuretic peptide 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 NT-proBNP calibrator 1 is 125 pg / mL, and the assigned concentration of NT-proBNP calibrator 2 is 5000 pg / mL).

[0049] Figure 7 This is the calibration curve of the amino-terminal pro-brain natriuretic peptide 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 NT-proBNP calibrator 1 is 125 pg / mL, and the assigned concentration of NT-proBNP calibrator 2 is 5000 pg / mL). DETAILED DESCRIPTION

[0050] 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.

[0051] NT-proBNP monoclonal antibody (capture antibody) was purchased from Chongqing Tansheng Technology Co., Ltd.; NT-proBNP monoclonal antibody (detection antibody) was purchased from Medix Biochemica.

[0052] 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:

[0053] 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.

[0054] A 100 mmol / L N-hydroxysulfosuccinimide (Sulfo-NHS) solution was prepared using 2-morpholineethanesulfonic acid (MES) buffer (pH=6.0).

[0055] Example 1

[0056] The preparation method of the amino-terminal pro-brain natriuretic peptide electrochemiluminescence detection kit of this embodiment is as follows:

[0057] The preparation of magnetic microsphere reagent coated with NT-proBNP monoclonal antibody (capture antibody) is as follows:

[0058] 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 solution (10 mg / mL) and rotate at room temperature for 30 minutes;

[0059] 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 NT-proBNP monoclonal antibody (capture antibody), and rotate and mix at room temperature for 4 hours;

[0060] 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;

[0061] S4. After the supernatant was removed by magnetic separation, 100 mL of a diluent containing 50 mmol / L bis(2-hydroxyethyl)amino(trihydroxymethyl)methane, 0.05% Tween-20 (w / v, g / mL), and 1% (w / v, g / mL) BSA (pH adjusted to 6.5) was added to resuspend the magnetic microspheres;

[0062] The concentration of S5.NT-proBNP monoclonal antibody (capture antibody) coated magnetic microspheres after dilution is 0.2 mg / mL.

[0063] The preparation of the NT-proBNP monoclonal antibody (detection antibody) reagent labeled with an electrically neutral ruthenium compound is as follows:

[0064] 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:

[0065]

[0066] S2. Add 5 μmol of NT-proBNP monoclonal antibody (detection antibody) to the above-mentioned electrically neutral ruthenium compound mixed solution and incubate at room temperature for 4 hours;

[0067] S3. removing unbound electrically neutral ruthenium compounds through a desalting column;

[0068] 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 NT-proBNP monoclonal antibody (detection antibody) in the NT-proBNP monoclonal antibody (detection antibody) labeled with the electrically neutral ruthenium compound was 6.5:1.

[0069] S5. Dilute the NT-proBNP monoclonal antibody (detection antibody) labeled with an electrically neutral ruthenium compound to 1.25 μg / mL using a diluent containing 50 mmol / L bis(2-hydroxyethyl)amino(trihydroxymethyl)methane, 0.05% Tween-20 (w / v, g / mL), and 1% (w / v, g / mL) BSA (pH adjusted to 6.5).

[0070] The amino-terminal pro-brain natriuretic peptide calibrator was prepared as follows:

[0071] Preparation of NT-proBNP calibrator 1: weigh 0.25 mL of NT-proBNP antigen stock solution (250 ng / mL), dilute to 500 mL with antigen diluent, the concentration of NT-proBNP calibrator 1 is 125 pg / mL, and lyophilize after aliquoting;

[0072] Preparation of NT-proBNP calibrator 2: Weigh 10 mL of NT-proBNP antigen stock solution (250 ng / mL), dilute to 500 mL with antigen diluent, the concentration of NT-proBNP calibrator 2 is 5000 pg / mL, and lyophilize after aliquoting.

[0073] Example 2

[0074] In the preparation method of the amino-terminal brain natriuretic peptide precursor electrochemiluminescence detection kit of this embodiment, the specific structural formula of the electrically neutral ruthenium compound used is as follows:

[0075] Other than that, the same as in Example 1.

[0076] Example 3

[0077] In the preparation method of the amino-terminal pro-brain natriuretic peptide electrochemiluminescence detection kit of this embodiment, except that bis(2-hydroxyethyl)amino(trihydroxymethyl)methane is replaced by piperazine-1,4-diethanesulfonic acid, the rest is the same as in Example 1.

[0078] Example 4

[0079] In the preparation method of the amino-terminal pro-brain natriuretic peptide electrochemiluminescence detection kit of this embodiment, except that the preparation method of the NT-proBNP monoclonal antibody (detection antibody) reagent labeled with an electrically neutral ruthenium compound is as follows, the rest is the same as in Example 1;

[0080] Preparation method of NT-proBNP monoclonal antibody (detection antibody) reagent labeled with electrically neutral ruthenium compound:

[0081] S1.5mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (40mmol / L), 5mL N-hydroxysulfosuccinimide solution (100mmol / L), 5mL electrically neutral ruthenium compound solution (5mmol / 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:

[0082]

[0083] S2. Add 5 μmol of NT-proBNP monoclonal antibody (detection antibody) to the above-mentioned electrically neutral ruthenium compound mixed solution and incubate at room temperature for 4 hours;

[0084] S3. removing unbound electrically neutral ruthenium compounds through a desalting column;

[0085] 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 NT-proBNP monoclonal antibody (detection antibody) in the NT-proBNP monoclonal antibody (detection antibody) labeled with the electrically neutral ruthenium compound was 3:1.

[0086] S5. Dilute the NT-proBNP monoclonal antibody (detection antibody) labeled with an electrically neutral ruthenium compound to 1.25 μg / mL using a diluent containing 50 mmol / L bis(2-hydroxyethyl)amino(trihydroxymethyl)methane, 0.05% Tween-20 (w / v, g / mL), and 1% (w / v, g / mL) BSA (pH adjusted to 6.5).

[0087] Comparative Example 1

[0088] In the preparation method of the amino-terminal brain natriuretic peptide precursor electrochemiluminescence detection kit of this comparative example, the specific structural formula of the electrically neutral ruthenium compound used is as follows:

[0089] Other than that, the same as in Example 1.

[0090] Comparative Example 2

[0091] In the preparation method of the electrochemiluminescence detection kit for amino-terminal brain natriuretic peptide precursor of this comparative example, the preparation method is the same as that of Example 1 except that bis(2-hydroxyethyl)amino(trihydroxymethyl)methane is replaced by 2-morpholineethanesulfonic acid.

[0092] Performance Testing

[0093] 5-minute incubation time test procedure - N-terminal pro-BNP calibrator calibration

[0094] S1. Pipette 80 μL of NT-proBNP monoclonal antibody (detection antibody) reagent labeled with an electrically neutral ruthenium compound and 40 μL of reconstituted calibrator (the solvent is purified water) into the test tube, mix for 3 to 5 seconds, pipette 80 μL of magnetic microsphere reagent coated with NT-proBNP monoclonal antibody (capture antibody) into the test tube, mix for 3 to 5 seconds, and incubate at 37°C for 5 minutes;

[0095] 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;

[0096] 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;

[0097] S4. The intensity of the light signal detected by the photomultiplier tube is positively correlated with the amount of NT-proBNP captured on the surface of the magnetic beads, thereby enabling quantitative analysis of NT-proBNP;

[0098] S5. Establish a calibration curve using the two calibrator concentrations and corresponding luminescent signal values;

[0099] S6.NT-proBNP calibrator was calibrated using the YnY 3030 fully automatic electrochemiluminescence immunoassay analyzer from Shenzhen Ansai Diagnostics Technology Co., Ltd.

[0100] The detection method of the amino-terminal pro-brain natriuretic peptide electrochemiluminescence detection kit comprises the following steps:

[0101] S1. Pipette 80 μL of NT-proBNP monoclonal antibody (detection antibody) reagent labeled with an electrically neutral ruthenium compound and 40 μL of sample into the test tube, mix for 3 to 5 seconds, pipette 80 μL of magnetic microsphere reagent coated with NT-proBNP monoclonal antibody (capture antibody) into the test tube, mix for 3 to 5 seconds, and incubate at 37°C for 5 minutes;

[0102] 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;

[0103] 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;

[0104] S4. The intensity of the light signal detected by the photomultiplier tube is positively correlated with the amount of NT-proBNP captured on the surface of the magnetic beads, thereby enabling quantitative analysis of NT-proBNP;

[0105] S5. The content of NT-proBNP in the sample is determined by the calibration curve.

[0106] Detection limit evaluation

[0107] The calibration curve was established by 5-minute incubation time detection procedure-NHT pro-BNP calibrator calibration (eg Figures 2 to 7 As shown in the table above, take 5 samples with a concentration close to the detection limit (10pg / mL) and test them with the kit from the same batch. Test each sample 5 times and the following requirements should be met: the number of test results with a value lower than the given blank limit (8pg / mL) should be less than or equal to 3.

[0108] Table 1 Detection limit evaluation results (unit: pg / mL)

[0109]

[0110] As shown in Table 1, the detection limit evaluation of the kits prepared in Example 1, Example 2, Example 3 and Example 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 evaluation of the kits prepared in Comparative Example 1 and Comparative Example 2 does not meet the requirements

[0111] Repeatability assessment

[0112] The calibration curve was established by 5-minute incubation time detection procedure-NHT pro-BNP calibrator calibration (eg Figures 2 to 7 As shown in the figure, the samples with concentrations between (125±25)pg / mL and (4000±800)pg / mL 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%.

[0113] CV=SD / M×100%

[0114] Table 2 Repeatability evaluation results (unit: pg / mL)

[0115]

[0116] 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.

[0117] Linear range assessment

[0118] The calibration curve was established by 5-minute incubation time detection procedure-NHT pro-BNP calibrator calibration (eg Figures 2 to 7 As shown in the figure, the high-value linear sample close to the upper limit of the linear interval (35000pg / mL) is diluted proportionally into 5 concentrations, among which the low-value linear sample should be close to the lower limit of the linear interval (10pg / mL). 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.

[0119] Table 3 Linear range evaluation results (unit: pg / mL)

[0120]

[0121] 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.

[0122] Biotin Interference Assessment

[0123] The calibration curve was established by 5-minute incubation time detection procedure-NHT pro-BNP calibrator calibration (eg Figures 2 to 7 As shown), add biotin to the sample with NT-proBNP concentration of (125±25) pg / mL to make its concentration reach 1000000 ng / mL. The addition ratio shall not exceed 1:9. Repeat the test for this sample 3 times. The interference deviation shall be within the range of ±10%.

[0124] Table 4 Biotin interference evaluation results (unit: pg / mL)

[0125]

[0126] 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.

[0127] Comparison with imported test kits:

[0128] Performance comparison with imported test kits

[0129] Table 5 Performance comparison of Examples 1 to 4 and imported kits

[0130]

[0131] As shown in Table 5, the kits prepared using Examples 1 to 4 are superior to the imported reagents in terms of incubation time and biotin interference, and both are significantly improved.

[0132] 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.

[0133] 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. An N-terminal pro-brain natriuretic peptide electrochemiluminescence detection kit, characterized in that: The kit comprises a magnetic microsphere reagent coated with an amino-terminal pro-brain natriuretic peptide capture antibody and an amino-terminal pro-brain natriuretic peptide 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 amino-terminal pro-brain natriuretic peptide capture antibody-coated magnetic microsphere reagent comprises amino-terminal pro-brain natriuretic peptide capture antibody-coated magnetic microspheres and a diluent; The electrically neutral ruthenium compound labeled amino-terminal pro-brain natriuretic peptide detection antibody reagent comprises an electrically neutral ruthenium compound labeled amino-terminal pro-brain natriuretic peptide detection antibody and a diluent; The diluent includes piperazine-1,4-diethanesulfonic acid or bis(2-hydroxyethyl)amino(trishydroxymethyl)methane.

2. The electrochemiluminescence detection kit for amino-terminal pro-brain natriuretic peptide according to claim 1, characterized in that: The kit also includes an amino-terminal pro-brain natriuretic peptide calibrator 1 and an amino-terminal pro-brain natriuretic peptide calibrator 2, wherein the concentration range of the amino-terminal pro-brain natriuretic peptide antigen in the amino-terminal pro-brain natriuretic peptide calibrator 1 is 100 to 150 pg / mL, and the concentration range of the amino-terminal pro-brain natriuretic peptide antigen in the amino-terminal pro-brain natriuretic peptide calibrator 2 is 4500 to 5500 pg / mL.

3. The electrochemiluminescence detection kit for amino-terminal pro-brain natriuretic peptide according to claim 1, characterized in that: In the amino-terminal pro-brain natriuretic peptide capture antibody-coated magnetic microsphere reagent, the particle size of the magnetic microsphere is 1-5 μm, and the concentration range of the amino-terminal pro-brain natriuretic peptide capture antibody-coated magnetic microsphere is 0.1-0.5 mg / mL.

4. The electrochemiluminescence detection kit for amino-terminal pro-brain natriuretic peptide according to claim 1, characterized in that: In the diluent, the concentration of the piperazine-1,4-diethanesulfonic acid is 20 to 100 mmol / L, or the concentration of the bis(2-hydroxyethyl)amino(trihydroxymethyl)methane is 20 to 100 mmol / L.

5. The electrochemiluminescence detection kit for amino-terminal pro-brain natriuretic peptide according to claim 1, characterized in that: The preparation method of the amino-terminal brain natriuretic peptide precursor capture antibody-coated magnetic microsphere reagent is as follows: S1. Mix the magnetic microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution; S2. Remove the supernatant by magnetic separation, add amino-terminal pro-brain natriuretic peptide capture antibody and mix well; S3. Add sealing agent and mix well; S4. After removing the supernatant by magnetic separation, add diluent and resuspend the magnetic microspheres to obtain the magnetic microsphere reagent coated with the amino-terminal pro-brain natriuretic peptide capture antibody.

6. The electrochemiluminescence detection kit for amino-terminal pro-brain natriuretic peptide according to claim 1, characterized in that: In the electrically neutral ruthenium compound labeled amino-terminal pro-brain natriuretic peptide antibody detection reagent, the concentration range of the electrically neutral ruthenium compound labeled amino-terminal pro-brain natriuretic peptide antibody detection is 0.25-2.5 μg / mL.

7. The electrochemiluminescence detection kit for amino-terminal pro-brain natriuretic peptide according to claim 1, characterized in that: In the amino-terminal pro-brain natriuretic peptide detection antibody labeled with the electrically neutral ruthenium compound, the molar binding ratio of the electrically neutral ruthenium compound to the amino-terminal pro-brain natriuretic peptide detection antibody is (2:1) to (10:1).

8. The electrochemiluminescence detection kit for amino-terminal pro-brain natriuretic peptide according to claim 1, characterized in that: The preparation method of the amino-terminal pro-brain natriuretic peptide detection antibody reagent labeled with an electrically neutral ruthenium compound is as follows: S1. Mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, N-hydroxysulfosuccinimide solution, and electrically neutral ruthenium compound solution; S2. Add amino-terminal pro-brain natriuretic peptide detection antibody for incubation; S3. Remove the unbound electrically neutral ruthenium compound, add a diluent, and obtain the amino-terminal brain natriuretic peptide precursor detection antibody reagent labeled with the electrically neutral ruthenium compound.

9. The electrochemiluminescence detection kit for amino-terminal pro-brain natriuretic peptide according to claim 1, characterized in that: The reaction volume ratio of the amino-terminal brain natriuretic peptide precursor capture antibody-coated magnetic microsphere reagent and the amino-terminal brain natriuretic peptide precursor detection antibody reagent labeled with an electrically neutral ruthenium compound is (50-100): (50-100).

10. The electrochemiluminescence detection kit for amino-terminal pro-brain natriuretic peptide 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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