CN112834739A - Kit for determining amino-terminal brain natriuretic peptide precursor in human blood by magnetic particle chemiluminescence method - Google Patents

Kit for determining amino-terminal brain natriuretic peptide precursor in human blood by magnetic particle chemiluminescence method Download PDF

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CN112834739A
CN112834739A CN202011621615.8A CN202011621615A CN112834739A CN 112834739 A CN112834739 A CN 112834739A CN 202011621615 A CN202011621615 A CN 202011621615A CN 112834739 A CN112834739 A CN 112834739A
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probnp
fitc
antibody
kit
amino
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柳建敏
贺率
汪云峰
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Ningbo Haiyi Biotechnology Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

The invention belongs to the technical field of immunochemistry detection, and particularly relates to a kit for determining an amino-terminal brain natriuretic peptide precursor in human blood by a magnetic particle chemiluminescence method and a using method thereof. Comprises magnetic particles coated by an anti-FITC antibody, an NT-proBNP capture monoclonal antibody marked by FITC, an NT-proBNP detection monoclonal antibody marked by alkaline phosphatase and an NT-proBNP calibrator. The kit adopts a Fluorescein Isothiocyanate (FITC) -anti-FITC antibody system, has good correlation with the Roche NT-ProBNP reagent, effectively avoids biotin interference in human serum while improving the analysis sensitivity, and greatly reduces the production cost.

Description

Kit for determining amino-terminal brain natriuretic peptide precursor in human blood by magnetic particle chemiluminescence method
Technical Field
The invention belongs to the technical field of immunochemical detection, and particularly relates to a kit for determining an amino-terminal brain natriuretic peptide precursor in human blood by a magnetic particle chemiluminescence method and a method for determining the amino-terminal brain natriuretic peptide precursor in human blood.
Background
Heart failure (heart failure) is a severe and terminal stage of various heart diseases. According to statistics, the current heart failure prevalence rate in China is estimated to be about 1.3%, the number of patients with the current disease is about 1000 thousands, and China has become the world with the largest population of heart failure patients.
When myocardial cells are pulled or vascular transmural pressure is overloaded, a B-type pro-natriuretic peptide precursor (pre-proBNP) containing 134 amino acids is generated, and then a signal peptide sequence at the N terminal is cut off under the action of related enzymes to form BNP precursor (proBNP) containing 108 amino acids, and the proBNP precursor (proBNP) is cracked into an amino-terminal brain natriuretic peptide precursor (NT-proBNP) containing 76 amino acids and no bioactivity and B-type natriuretic peptide (BNP) containing 32 amino acids under the action of endonuclease.
NT-proBNP and BNP are the first-choice serum markers of heart failure recommended by heart failure guidelines at home and abroad. Compared with BNP, NT-proBNP has longer biological half-life in human body (about 1-2 h of NT-proBNP and about 20min of BNP), and relatively higher blood concentration (about 15-20 times of BNP). Therefore, the NT-proBNP has higher sensitivity for detecting early or mild heart failure, and the time for sending blood samples to a laboratory is more sufficient, so that the NT-proBNP is more suitable for clinical application.
At present, a double-antibody sandwich method is generally adopted to quantitatively detect the content of NT-proBNP, and the method mainly comprises a fluorescence immunochromatography method, an enzyme-linked immunosorbent assay and a chemiluminescence method. The fluorescence immunochromatography has more sample addition amount, and has poor batch precision and batch-to-batch repeatability; the enzyme-linked immunosorbent assay has the problems of low sensitivity, narrow linear range and the like; the chemiluminescence method has the advantages of high sensitivity, wide linear range, accurate detection result, high automation degree and wider prospect in clinical application.
For example, Chinese patent application (publication No. CN107656071A) discloses a NT-proBNP detection kit for a magnetic particle chemiluminescence method, which comprises a calibrator, a cleaning solution, a substrate solution, a pretreatment solution, an enzyme conjugate working solution and a magnetic bead conjugate working solution. However, in the method, the NT-proBNP antibody and the magnetic particles are directly coated, the minimum detection limit is 20pg/ml, the linear range is 20-50000 pg/ml, and the sensitivity is lower than that of an imported reagent.
The NT-proBNP reagent produced by Roche uses a biotin-streptavidin system, magnetic particles are coated by streptavidin, the two antibodies respectively mark biotin and terpyridyl ruthenium, the minimum detection limit is 5pg/mL, and the detection range is 5-35000 pg/mL. The biotin-avidin system is a very effective biological reaction amplification system, has high affinity and multi-stage amplification effect, and is currently generally used to improve the sensitivity of a reagent reaction. However, if free biotin exists in the sample to be detected, the binding of the free biotin and streptavidin in the partial sandwich complex is prevented, so that the capture signal is weakened, and the result is falsely reduced. Thus, biotin can interfere with certain tests, give inaccurate test results, and is not easily discovered. And the NT-proBNP reagent produced by Roche is an imported reagent, so the price is high and the detection cost is high.
Also, for example, Chinese patent application (publication No. CN 112067826A) discloses a NT-proBNP detection kit constructed based on high specific activity alkaline phosphatase, which comprises: an alkaline phosphatase labeled NT-proBNP detection antibody and a magnetic bead coated NT-proBNP coated antibody. The connection of enzyme and antibody, and magnetic bead and antibody in this scheme has been optimized: oxidizing a sugar chain on the Fc end of the antibody by using sodium periodate to generate an aldehyde group; treating with excessive cysteine hydrochloride and cyano sodium borohydride to make aldehyde group and cysteine hydrochloride produce aldehyde-amine condensation reaction to produce active mercapto; treating alkaline phosphatase with succinimidyl-4- (N-maleimide) cyclohexane-1-carboxylate (SMCC) to expose an active maleamide group; then mixed with antibody of Fc end active sulfhydryl to generate stable thioether bond. The protocol labeling process was somewhat cumbersome and the enzyme source was not sufficiently stable to mature using the non-commercial alkaline phosphatase CmAP.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provide a kit for determining the amino-terminal pro-brain natriuretic peptide in human blood by magnetic particle chemiluminescence with high sensitivity and low production cost.
The purpose of the invention can be realized by the following technical scheme: a kit for determining an amino-terminal brain natriuretic peptide precursor in human blood by magnetic microparticle chemiluminescence, the kit comprising: magnetic particles coated by an anti-FITC antibody, a FITC-labeled NT-proBNP capture monoclonal antibody, an alkaline phosphatase-labeled NT-proBNP detection monoclonal antibody and an NT-proBNP calibrator. The invention adopts a Fluorescein Isothiocyanate (FITC) -anti-FITC antibody system, improves the analysis sensitivity and effectively avoids the biotin interference in human serum. anti-FITC antibody is coated on the magnetic particles in advance, and the FITC-labeled NT-proBNP antibody is indirectly coupled to the magnetic particles through a FITC-anti-FITC antibody reaction. One anti-FITC antibody molecule can be combined with 3-4 FITC, the specificity and the affinity are strong, and the two are extremely stable once combined; not only can the amount of NT-proBNP antibody on the magnetic particle be increased, but also the binding sites of the antibody can be fully exposed by improving steric hindrance. The method has the advantages of high coupling efficiency, firm combination and stable process, and greatly reduces the product cost while improving the product performance.
In the kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method, the concentration of the magnetic particles coated by the anti-FITC antibody is 0.2-1.0 mg/mL; the concentration of the FITC-labeled NT-proBNP capture monoclonal antibody is 0.5-5.0 mug/mL; the concentration of the NT-proBNP detection monoclonal antibody marked by alkaline phosphatase is 0.2-2 mug/mL. If the concentration is too low, the sensitivity is low; high concentration is likely to result in high cost, false positive result due to high background, or missing detection due to poor signal-to-noise ratio. In the kit for measuring the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method, the preparation method of the magnetic particles coated by the anti-FITC antibody comprises the following steps:
diluting the magnetic particles with PBS buffer;
mixing the diluted magnetic particles with an anti-FITC antibody;
adding ammonium sulfate for reaction;
adding sealing liquid for sealing treatment.
Preferably, the mass ratio of the magnetic particles to the anti-FITC antibody is 1: 5-1: 50.
Preferably, the magnetic particles have a particle size of 1.0 to 3.0. mu.m.
Preferably, the amount of ammonium sulfate is 0.5 to 2 times the total mass of the magnetic particles and the anti-FITC antibody.
Further preferably, the preparation method of the anti-FITC antibody coated magnetic particle specifically comprises the following steps:
1) diluting the magnetic particles to 5-20 mg/mL by using PBS buffer solution;
2) mixing magnetic particles and an anti-FITC antibody according to a mass ratio of 1: 5-1: 50;
3) then adding ammonium sulfate, and carrying out suspension reaction for 4-24 hours;
3) after magnetic separation and cleaning, suspending and reacting for 6-24 hours by adopting a blocking solution containing BSA;
4) and (3) diluting the magnetic particles by using a blocking solution containing BSA to obtain the magnetic particles coated by the anti-FITC antibody, and storing the magnetic particles at the temperature of 2-8 ℃ for later use.
In the kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method, the preparation method of the FITC-labeled NT-proBNP capture monoclonal antibody comprises the following steps: dissolving FITC in Na2CO3-NaHCO3And adding the NT-proBNP capture monoclonal antibody into the buffer solution, stirring and reacting for 12-16 hours at the temperature of 2-8 ℃, and finally dialyzing to obtain the FITC-labeled NT-proBNP capture monoclonal antibody.
Preferably, the mass ratio of the FITC to the NT-proBNP is (3-5): 1.
further preferably, the preparation method of the FITC-labeled NT-proBNP capture monoclonal antibody comprises the following steps:
1) 2mg of FITC was dissolved in 1mL of 50mmol/L Na of pH 9.32CO3-NaHCO3In a buffer solution;
2) slowly dripping 0.5mg of NT-proBNP capture monoclonal antibody, and finishing the addition within 5-8 minutes;
3) stirring and reacting for 12-16 hours at the temperature of 2-8 ℃;
4) filling the reaction solution into a dialysis bag, dialyzing in 10mmol/L phosphate buffer solution with pH value of 7.4 overnight, replacing the dialysate for 3 times to obtain NT-proBNP capture monoclonal antibody labeled by FITC, and storing at-20 ℃ for later use.
In the kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method, the preparation method of the alkaline phosphatase-labeled NT-proBNP detection monoclonal antibody comprises the following steps:
activating the antibody: opening a disulfide bond between single-resistant heavy chains of the NT-proBNP detection by using a reducing agent to generate a sulfhydryl group;
activation of alkaline phosphatase: treating alkaline phosphatase with SMCC to expose a maleamide group;
addition reaction: and mixing the activated antibody and the activated alkaline phosphatase for reaction, and purifying to obtain the NT-proBNP detection monoclonal antibody marked by alkaline phosphatase.
Preferably, the reducing agent is Dithiothreitol (DTT) or tris (2-carbonylethyl) phosphate (TCEP), or both.
Preferably, the mass ratio of the activated antibody to the activated alkaline phosphatase is (0.8-1.5): 1.
preferably, the mixing of the activated antibody and the activated alkaline phosphatase further comprises adding a magnesium chloride solution.
Further preferably, the preparation method of the alkaline phosphatase-labeled NT-proBNP detection monoclonal antibody is as follows:
1) replacing 1mg of NT-proBNP detection monoclonal antibody with a buffer solution without amino and sulfhydryl groups by using dialysis or a PD-10 desalting column, and concentrating to 2-4 mg/mL by using a concentration tube;
2) adding 0.5-2.5 microliter of reducing agent per milliliter according to the volume of the antibody, and reacting for 10-30 minutes at room temperature;
3) adding 1M glycine pH7.3, reacting at room temperature for 5-10 min, replacing buffer solution with PD-10 desalting column, and concentrating to 2-4 mg/mL;
4) replacing 1mg of alkaline phosphatase with a buffer solution without amino and sulfhydryl groups by dialysis or a PD-10 desalting column, and concentrating to 2-4 mg/mL by using a concentration tube;
5) weighing SMCC, dissolving the SMCC into 5-8 mg/mL of DMF, adding the DMF into the mixture according to 2.5-7.5 per mill of the volume of alkaline phosphatase, and reacting the mixture for 10-30 minutes at room temperature;
6) 1M Glycine pH7.3 was added in the same volume as the amount of SMCC solution added. Reacting at room temperature for 10-20 min, replacing buffer solution by using a PD-10 desalting column, and concentrating to 2-4 mg/ml for later use;
7) mixing the activated antibody and alkaline phosphatase according to the mass ratio of 1:1, adding 0.1-0.5M magnesium chloride solution, and adding 1-5 thousandth milliliter of the volume of the total volume of the reaction. Reacting for 8-20 hours at the temperature of 2-8 ℃;
8) the ligation mixture was purified and stored at 2-8 ℃ for further use.
The alkaline phosphatase described in the present invention is commercial alkaline phosphatase BIAP.
In the kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method, a diluent in the NT-proBNP calibrator is a PBS buffer solution containing 5-10% of trehalose, 5-10% of glycerol, 5-15% of cyclodextrin and 3-8% of sorbitol, and the pH value is 6.5-7.4; the antigen in the NT-proBNP calibrator is NT-proBNP short peptide, and the linear structure is as follows: NT-proBNP capture antibody epitope peptide fragment-connecting peptide-NT-proBNP detection antibody epitope peptide fragment.
The invention also provides a method for determining the amino-terminal brain natriuretic peptide precursor in human blood by using the kit and adopting a magnetic particle chemiluminescence method.
The specific determination method comprises the following steps:
mixing the magnetic particles coated by the anti-FITC antibody and the NT-proBNP capture monoclonal antibody marked by FITC to form a magnetic bead-anti-FITC antibody-FITC-NT-proBNP capture monoclonal antibody compound;
incubating the magnetic bead-anti-FITC antibody-FITC-NT-proBNP capture monoclonal antibody compound, a sample to be detected and the NT-proBNP detection monoclonal antibody marked by alkaline phosphatase for 10min at 37 ℃ to form a magnetic compound suspension;
placing the magnetic compound suspension in a magnetic field, carrying out magnetic separation for 2min, and washing the magnetic compound;
and adding chemiluminescent substrate solution into the washed magnetic compound, and detecting the intensity of chemiluminescent photons.
Compared with the prior art, the invention has the following advantages:
1. the technical scheme provided by the invention adopts a Fluorescein Isothiocyanate (FITC) -anti-FITC antibody system, so that the analysis sensitivity is improved, and the biotin interference in human serum is effectively avoided. One anti-FITC antibody molecule can be combined with 3-4 FITC, so that the number of NT-proBNP antibodies on the magnetic particles can be increased, and the binding sites of the antibodies can be fully exposed by improving steric hindrance. The method has the advantages of high coupling efficiency, firm combination and stable process, and greatly reduces the product cost while improving the product performance. The process of coupling the magnetic beads with the anti-FITC antibody is simple, and the product can also be universally used for other projects.
2. The invention adopts an SMCC directional coupling method to mark alkaline phosphatase at the Fc end of an antibody: opening a disulfide bond between two heavy chains of the antibody by using a tris (2-carbonylethyl) phosphate (TCEP) reducing agent to generate an active sulfhydryl group; alkaline phosphatase was directionally conjugated to the antibody at a position intermediate between the two heavy chains after treatment with SMCC. The method is relatively simple to operate, does not generate steric hindrance on the binding site of the antibody, and is favorable for improving the sensitivity of the reagent.
3. The calibrator provided by the invention adopts the polypeptide antigen, and trehalose, glycerol, cyclodextrin, sorbitol and other components are introduced into the calibrator diluent, so that the stability of the calibrator is effectively improved. The natural NT-proBNP has small molecular weight and is easy to degrade. And the prokaryotic recombinant expressed NT-proBNP protein lacks glycosylation and has poorer stability than the natural protein. The artificially designed polypeptide antigen ensures the biological activity thereof, simultaneously selects more stable epitopes by rejecting unstable amino acid sequences, and connects the two epitopes by using stable connecting peptide, thereby obtaining more stable antigen. Meanwhile, chemical components added into the diluent can help maintain the spatial structure of the antigen, and the storage period of the calibrator is prolonged.
Drawings
FIG. 1 is a standard curve diagram of NT-proBNP calibrator.
FIG. 2 is a result chart showing the correlation between the detection results of the kit of the present invention and NT-proBNP detection kit of Roche.
Detailed Description
The following are specific embodiments of the present invention and are further described with reference to the accompanying drawings, but the present invention is not limited to these embodiments.
Example 1
A kit for determining an amino-terminal brain natriuretic peptide precursor in human blood by a magnetic particle chemiluminescence method comprises magnetic particles coated by an anti-FITC antibody, an NT-proBNP capture monoclonal antibody labeled by FITC, an NT-proBNP detection monoclonal antibody labeled by alkaline phosphatase, and an NT-proBNP calibrator.
The FITC antibody-coated magnetic particles are prepared by the following method:
1) magnetic particles 3.0 μ M in size were diluted to 10mg/mL with 0.01M PBS buffer, pH 7.4.
2) Magnetic particles by mass ratio: anti-FITC antibody was added at a ratio of 1: 10.
3) 3M ammonium sulfate was added in a volume equal to the total volume of the magnetic particles and the antibody.
4) The reaction was suspended at 37 ℃ for 18 hours.
5) After magnetic separation and washing, the reaction mixture was suspended in a blocking solution containing BSA at 37 ℃ for 18 hours.
6) The solution was diluted to 10mg/mL with the above blocking solution to obtain anti-FITC antibody-coated magnetic microparticles, which were stored at 4 ℃ until use.
7) When used, the solution is diluted to a final concentration of 0.5 mg/mL.
The FITC marked NT-proBNP capture monoclonal antibody is prepared by the following method:
1) 2mg of FITCDissolving in 1mL of 50mmol/L Na with pH value of 9.32CO3-NaHCO3In a buffer.
2) 0.5mg of NT-proBNP capture monoclonal antibody is slowly dripped into the mixture, and the addition is finished within about 5 minutes.
3) The reaction was stirred at 4 ℃ for 16 hours.
4) Filling the reaction solution into a dialysis bag, dialyzing in 10mmol/L phosphate buffer solution with pH value of 7.4 overnight, replacing the dialysate for 3 times to obtain NT-proBNP capture monoclonal antibody labeled by FITC, and storing at-20 ℃ for later use.
5) When used, the solution was diluted to a final concentration of 2.0. mu.g/mL.
The NT-proBNP detection monoclonal antibody marked by alkaline phosphatase is prepared by the following method:
1) 1mg of NT-proBNP detection monoclonal antibody is replaced by buffer solution without amino and sulfhydryl by dialysis or PD-10 desalting column, and is concentrated to 2mg/mL by a concentration tube.
2) Using tris (2-carbonylethyl) phosphate (TCEP), 1.thousandth of microliter per milliliter of the antibody volume was added and reacted at room temperature for 20 minutes.
3) Adding 1M glycine with the pH value of 7.3, reacting at room temperature for 5-10 min according to the volume of the added TCEP. Buffer was exchanged using PD-10 desalting column and concentrated to 2mg/mL for use.
4) 1mg of alkaline phosphatase was replaced with a buffer without amino groups and thiol groups by dialysis or a PD-10 desalting column, and concentrated to 2mg/mL by a concentration tube.
5) SMCC is weighed, dissolved to 5mg/mL by DMF, added according to 5 per mill of the volume of alkaline phosphatase and reacted for 20 minutes at room temperature.
6) 1M Glycine pH7.3 was added in the same volume as the amount of SMCC solution added. The reaction was carried out at room temperature for 20 min. Buffer was exchanged using a PD-10 desalting column and concentrated to 2mg/ml for use.
7) Mixing the activated antibody and alkaline phosphatase according to the mass ratio of 1:1, adding 0.2M magnesium chloride solution, and adding 2 thousandth milliliter of which the volume is equal to the total volume of the reaction. The reaction was carried out at 4 ℃ for 16 hours.
8) The ligation was purified and stored at 4 ℃ until use.
9) When used, the solution was diluted to a final concentration of 1. mu.g/mL.
The NT-proBNP calibrator is prepared by the following method:
1) a calibrator diluent was prepared as PBS buffer containing 5% trehalose, 5% glycerol, 5% cyclodextrin, 5% sorbitol, pH 7.0.
2) Selecting NT-proBNP short peptide antigen, the linear structure of which is: NT-proBNP capture antibody epitope peptide fragment-connecting peptide-NT-proBNP detection antibody epitope peptide fragment.
3) NT-proBNP short peptide antigen was added to the calibrator dilution at concentrations of 0pg/mL, 50pg/mL, 250pg/mL, 1000pg/mL, 5000pg/mL, and 35000pg/mL, respectively.
The method for determining the amino-terminal brain natriuretic peptide precursor in human blood by adopting the magnetic particle chemiluminescence method comprises the following steps:
mixing the magnetic particles coated by the anti-FITC antibody and the NT-proBNP capture monoclonal antibody marked by FITC to form a magnetic bead-anti-FITC antibody-FITC-NT-proBNP capture monoclonal antibody compound;
incubating the magnetic bead-anti-FITC antibody-FITC-NT-proBNP capture monoclonal antibody compound, a sample to be detected and the NT-proBNP detection monoclonal antibody marked by alkaline phosphatase for 10min at 37 ℃ to form a magnetic compound suspension;
placing the magnetic compound suspension in a magnetic field, performing magnetic separation for 2min, and washing the magnetic compound;
and adding chemiluminescent substrate solution into the washed magnetic compound, and detecting the intensity of chemiluminescent photons.
The detection was carried out by the method for the determination of the amino-terminal pro-brain natriuretic peptide in human blood described above using the kit of example 1.
The specific experimental contents are as follows:
1. standard curve
The calibrators in example 1 were tested in parallel at 0pg/mL, 50pg/mL, 250pg/mL, 1000pg/mL, 5000pg/mL, and 35000pg/mL to calculate the mean luminescence values, and a four parameter equation was used to fit the standard curve. The test luminescence values of the calibrator are shown in Table 1, and the standard curve is shown in FIG. 1.
Table 1: test luminescence value of calibrator
Figure BDA0002874094490000111
2. Blank limit and detection limit performance testing
1) Margin limit
And (3) parallelly measuring the luminescence value (RLU) of the zero-value calibrator for 20 times, performing two-point regression fitting according to the concentration-luminescence value (RLU) result between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation, and calculating the corresponding concentration of the zero-value calibrator (or the sample diluent), wherein at least 17 times of results are not higher than a blank limit (2 pg/mL). The results are shown in Table 2.
Table 2: margin test results
Number of tests RLU Fitting value Number of tests RLU Fitting value
1 7916 0.34 11 7977 0.46
2 8031 0.56 12 8068 0.63
3 8002 0.51 13 7925 0.36
4 7911 0.33 14 8151 0.79
5 8097 0.69 15 7918 0.35
6 7775 0.07 16 8012 0.53
7 8028 0.56 17 7783 0.09
8 8191 0.87 18 7820 0.16
9 8115 0.72 19 7996 0.50
10 8008 0.52 20 7843 0.20
2) Detection limit
And 5 low-value samples with approximate concentration detection limit (5pg/mL) are taken for detection, each sample is detected for 5 times, the detection result below the blank limit (2pg/mL) is less than or equal to 3, and no value above the detection limit (5pg/mL) exists. The results are shown in Table 3.
Table 3: detection limit test result
Number of tests Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
1 4.76 3.67 2.10 3.49 2.94
2 4.47 3.77 2.89 3.53 2.92
3 4.37 3.90 2.11 3.15 2.38
4 4.76 3.35 2.39 3.85 2.38
5 4.53 3.45 2.41 3.23 2.67
The results in tables 2 and 3 show that the test results of 20 times of zero-value calibrators are less than 2pg/mL, and the test results of 25 times of low-value samples are 2-5 pg/mL.
Therefore, the blank limit of the NT-proBNP kit is 2pg/mL, and the detection limit is 5 pg/mL.
3. Linear verification
Taking a high-concentration sample close to the upper limit of the linear range and a low-concentration sample close to the lower limit of the linear range, and mixing the high-concentration sample and the low-concentration sample into at least 5 diluted concentrations according to a certain proportion. Each dilution concentration was tested 3 times and the mean of the test results calculated. And (4) calculating a linear regression equation by taking the dilution concentration as an independent variable and the mean value of the detection result as a dependent variable, and calculating a correlation coefficient r. The results are shown in Table 4.
Table 4: results of the Linear test
Figure BDA0002874094490000121
The results in Table 4 show that the correlation coefficient r is greater than 0.99 in the concentration range of 5-35000 pg/ml.
Therefore, the detection range of the NT-proBNP kit is 5-35000 pg/ml.
4. Precision verification
And taking one portion of each of the high-value quality control product, the medium-value quality control product and the low-value quality control product, repeatedly testing for 10 times, and calculating the average value and the standard deviation. According to the formula: the coefficient of variation CV is (standard deviation/average value) × 100%, and the precision is calculated. The results are shown in Table 5.
Table 5: results of precision measurement
QCL QCM QCH
1 98.02 1023.82 9603.69
2 95.44 1029.28 9921.43
3 98.15 1044.52 10289.04
4 103.37 1005.81 10553.99
5 99.79 1033.55 9848.22
6 99.13 992.05 9724.63
7 98.34 1071.7 10262.16
8 101.69 1049.6 10182.02
9 90.69 971.91 9449.99
10 95.56 947.26 9932.38
X 98.02 1016.95 9976.76
SD 3.54 37.90 342.55
CV 3.62％ 3.73％ 3.43％
The results in Table 5 show that the CV of the high-value quality control product is 3.62%; the CV of the median quality control product is 3.73 percent; the CV of the low-value quality control product is 3.43 percent.
Therefore, the NT-proBNP kit disclosed by the invention is good in precision.
5. Stability of calibrator
The calibrator of example 1 was stored at 37 ℃ and 4 ℃ respectively for follow-up testing.
The calibrator stored at 37 ℃ was tested for two luminescence values (RLU) on days 0, 3, 7 and 9. The mean was calculated and the relative deviation from the day 0 results was calculated. The results are shown in Table 6.
Table 6: test result of 37 ℃ stability of calibrator
Figure BDA0002874094490000141
The calibrator stored at 4 ℃ was tested for two luminescence values (RLU) at month 0, month 6, month 9, month 12 and month 14. The mean was calculated and the relative deviation was calculated from the month 0 results. The results are shown in Table 7.
Table 7: 4 ℃ stability test result of calibrator
Figure BDA0002874094490000142
The results in tables 6 and 7 show that all relative deviations are < 15%.
Therefore, the NT-proBNP calibrator has good stability.
6. Sample test correlation verification
200 serum samples are taken, the correlation of the detection results of the kit of the embodiment 1 of the invention and the NT-proBNP detection kit of Roche is compared, and the sample test correlation test results are shown in FIG. 2. The results in fig. 2 show that the two reagent linear regression equations, Y, is 0.996X +13.374 with correlation coefficient R20.9888. It can be seen thatThe result of the NT-proBNP kit detection sample of the invention is basically consistent with the product result of Rogowski company.
In conclusion, the kit provided by the invention adopts a Fluorescein Isothiocyanate (FITC) -anti-FITC antibody system, so that the analysis sensitivity is improved, the sensitivity of the kit with the NT-proBNP reagent produced by Roche is achieved, the biotin interference in human serum is effectively avoided, and the production cost is greatly reduced.
The technical scope of the invention claimed by the embodiments herein is not exhaustive and new solutions formed by equivalent replacement of single or multiple technical features in the embodiments are also within the scope of the invention, and all parameters involved in the solutions of the invention do not have mutually exclusive combinations if not specifically stated.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims (10)

1. A kit for determining an amino-terminal brain natriuretic peptide precursor in human blood by magnetic particle chemiluminescence, the kit comprising: magnetic particles coated by an anti-FITC antibody, a FITC-labeled NT-proBNP capture monoclonal antibody, an alkaline phosphatase-labeled NT-proBNP detection monoclonal antibody and an NT-proBNP calibrator.
2. The kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method according to claim 1, wherein the concentration of the magnetic particles coated with the anti-FITC antibody is 0.2-1.0 mg/mL; the concentration of the FITC-labeled NT-proBNP capture monoclonal antibody is 0.5-5.0 mug/mL; the concentration of the NT-proBNP detection monoclonal antibody marked by alkaline phosphatase is 0.2-2 mug/mL.
3. The kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method according to claim 1, wherein the method for preparing the anti-FITC antibody coated magnetic particles comprises:
diluting the magnetic particles with PBS buffer;
mixing the diluted magnetic particles with an anti-FITC antibody;
adding ammonium sulfate for reaction;
and adding a sealing solution for sealing treatment to obtain the magnetic particles coated by the anti-FITC antibody.
4. The kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method according to claim 3, wherein the mass ratio of the magnetic particles to the anti-FITC antibody is 1: 5-1: 50.
5. The kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method according to claim 3, wherein the amount of ammonium sulfate is 0.5-2 times of the total mass of the magnetic particles and the anti-FITC antibody.
6. The kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method according to claim 1, wherein the method for preparing the FITC-labeled NT-proBNP capture monoclonal antibody comprises: dissolving FITC in Na2CO3-NaHCO3And adding the NT-proBNP capture monoclonal antibody into the buffer solution, stirring and reacting for 12-16 hours at the temperature of 2-8 ℃, and finally dialyzing to obtain the FITC-labeled NT-proBNP capture monoclonal antibody.
7. The kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method according to claim 6, wherein the mass ratio of FITC to NT-proBNP is (3-5): 1.
8. the kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method of claim 1, wherein the method for preparing the alkaline phosphatase labeled NT-proBNP detection monoclonal antibody comprises:
activating the antibody: opening a disulfide bond between single-resistant heavy chains of the NT-proBNP detection by using a reducing agent to generate a sulfhydryl group;
activation of alkaline phosphatase: treating alkaline phosphatase with SMCC to expose a maleamide group;
addition reaction: and mixing the activated antibody and the activated alkaline phosphatase for reaction, and purifying to obtain the NT-proBNP detection monoclonal antibody marked by alkaline phosphatase.
9. The kit for determining the amino-terminal brain natriuretic peptide precursor in human blood by the magnetic particle chemiluminescence method according to claim 1, wherein the diluent in the NT-proBNP calibrator is PBS buffer containing 5-10% trehalose, 5-10% glycerol, 5-15% cyclodextrin, and 3-8% sorbitol, and the pH is 6.5-7.4; the antigen in the NT-proBNP calibrator is NT-proBNP short peptide, and the linear structure is as follows: NT-proBNP capture antibody epitope peptide fragment-connecting peptide-NT-proBNP detection antibody epitope peptide fragment.
10. A method for determining an amino-terminal brain natriuretic peptide precursor in human blood, wherein the detection is performed by magnetic particle chemiluminescence using the kit of any one of claims 1 to 9.
CN202011621615.8A 2020-12-30 2020-12-30 Kit for determining amino-terminal brain natriuretic peptide precursor in human blood by magnetic particle chemiluminescence method Pending CN112834739A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114280311A (en) * 2021-11-10 2022-04-05 宏葵生物（中国）股份有限公司 Preparation method and detection application of nano-magnetic particle chemiluminescence detection kit for amino-terminal B-type natriuretic peptide precursor

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103592445A (en) * 2013-10-16 2014-02-19 北京利德曼生化股份有限公司 Kit for detecting procalcitonin
CN104820102A (en) * 2015-05-05 2015-08-05 南京格耀生物科技有限公司 Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof
WO2016127318A1 (en) * 2015-02-10 2016-08-18 深圳市新产业生物医学工程股份有限公司 Cardiac troponin i ultra-sensitive detection reagent kit, and ultra-sensitive detection method therefor
CN108362688A (en) * 2018-01-02 2018-08-03 北京利德曼生化股份有限公司 A kind of 25(OH)VD magnetic microparticle chemiluminescence detection kit
CN109748971A (en) * 2019-01-24 2019-05-14 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） A kind of ELISA antibody assay kit of duck tembusu virus and its application
CN111521816A (en) * 2020-04-21 2020-08-11 沈阳农业大学 Time-resolved fluorescence immunochromatographic assay test strip for echinococcosis granulosus of cattle and preparation method thereof
CN111781385A (en) * 2020-08-19 2020-10-16 武汉生之源生物科技股份有限公司 NT-proBNP detection kit and preparation method thereof
CN112014566A (en) * 2019-05-28 2020-12-01 上海奥普生物医药股份有限公司 Amino-terminal brain natriuretic peptide precursor detection kit, preparation method and application
CN112014575A (en) * 2020-09-03 2020-12-01 武汉生之源生物科技股份有限公司 CYFRA21-1 determination kit and preparation method thereof
CN112067826A (en) * 2020-09-15 2020-12-11 济南国科医工科技发展有限公司 NT-proBNP detection kit constructed based on high-specific-activity alkaline phosphatase and application thereof

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103592445A (en) * 2013-10-16 2014-02-19 北京利德曼生化股份有限公司 Kit for detecting procalcitonin
WO2016127318A1 (en) * 2015-02-10 2016-08-18 深圳市新产业生物医学工程股份有限公司 Cardiac troponin i ultra-sensitive detection reagent kit, and ultra-sensitive detection method therefor
CN104820102A (en) * 2015-05-05 2015-08-05 南京格耀生物科技有限公司 Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof
CN108362688A (en) * 2018-01-02 2018-08-03 北京利德曼生化股份有限公司 A kind of 25(OH)VD magnetic microparticle chemiluminescence detection kit
CN109748971A (en) * 2019-01-24 2019-05-14 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） A kind of ELISA antibody assay kit of duck tembusu virus and its application
CN112014566A (en) * 2019-05-28 2020-12-01 上海奥普生物医药股份有限公司 Amino-terminal brain natriuretic peptide precursor detection kit, preparation method and application
CN111521816A (en) * 2020-04-21 2020-08-11 沈阳农业大学 Time-resolved fluorescence immunochromatographic assay test strip for echinococcosis granulosus of cattle and preparation method thereof
CN111781385A (en) * 2020-08-19 2020-10-16 武汉生之源生物科技股份有限公司 NT-proBNP detection kit and preparation method thereof
CN112014575A (en) * 2020-09-03 2020-12-01 武汉生之源生物科技股份有限公司 CYFRA21-1 determination kit and preparation method thereof
CN112067826A (en) * 2020-09-15 2020-12-11 济南国科医工科技发展有限公司 NT-proBNP detection kit constructed based on high-specific-activity alkaline phosphatase and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
廖美琴;: "磁微粒化学发光法检测人血清中NT-proBNP方法的建立", 质量安全与检验检测, no. 04 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114280311A (en) * 2021-11-10 2022-04-05 宏葵生物（中国）股份有限公司 Preparation method and detection application of nano-magnetic particle chemiluminescence detection kit for amino-terminal B-type natriuretic peptide precursor

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