CN111007266A - Chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma - Google Patents
Chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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- G01N2333/575—Hormones
- G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
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Abstract
The invention discloses a chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma, which comprises a reagent R1 containing streptavidin-coated magnetic particles, a reagent R2 containing acridinium ester labeled B-type natriuretic peptide antibody and a reagent R3 containing biotin labeled B-type natriuretic peptide antibody. The invention adopts the principle of a double-antibody sandwich method, utilizes an avidin-biotin system and adopts acridinium ester chemiluminescence, thereby greatly improving the detection accuracy of the kit, reducing the reaction time and reducing the reagent cost, the acridinium ester is easy to couple, the labeling process is simple, the stability is good, the influence on the luminescence performance after labeling is little, the kit has long effective period and relatively low cost, the luminescence is of a flash type, the luminescence is fast concentrated and strong, the rapid detection is convenient to realize, the detection sensitivity and precision are high, the requirement on instruments is simple, the full-automatic operation is convenient to realize, no reinforcing agent or catalyst is needed, the interference factor is few, the background is extremely low, and the signal-to-noise ratio is very high.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma, a preparation method and a detection method thereof.
Background
Type B Natriuretic Peptide (BNP) is an important natriuretic peptide family member discovered in recent years, and consists of 32 amino acids, and has a cyclic active structure consisting of 17 amino acids through a pair of disulfide bonds. BNP is a protease cleavage product of pro-natriuretic peptide, containing 134 amino acid residues, which is synthesized in cardiac muscle cells. The pro-natriuretic peptide (residues 1-26) is removed to form a natriuretic peptide (pro BNP) molecule (amino acid residues 27-134). proBNP (108 amino acid residues) forms two polypeptides in a convertase dependent reaction, namely BNP (amino acid residues 77-108) and the N-terminal part of proBNP (NT-proBNP, amino acid residues 1-76). BNP and NT-proBNP as well as pro BNP that is not degraded are secreted into the blood and circulate in the blood. Elevated concentrations are found in patients with Heart Failure (HF), Acute Coronary Syndrome (ACS), left ventricular hypertrophy, cardiomyopathy, heart valve disease, atrial fibrillation, and cardiac amyloidosis. The concentration of BNP is directly proportional to the severity of the disease. BNP assays are therefore widely used to assess the severity of heart failure.
Elevated BNP may also be found in a variety of diseases other than heart failure. The level of the Zhangjiang BNP is obviously increased in patients with acute coronary syndrome, and the BNP level is related to the myocardial infarction area, namely the higher the BNP level is, the larger the myocardial infarction area is, so that the level of the plasma BNP can be used for clinically evaluating the size of the myocardial infarction area; the lung and the patients with chronic obstructive pulmonary disease, pulmonary embolism and pulmonary arterial hypertension are also related to BNP, the level of plasma BNP is increased in patients with chronic obstructive pulmonary disease, but is lower than that of patients with acute exacerbation or heart failure, has certain identification significance on the causes of dyspnea, and is related to prognosis; the elevated level of BNP is a risk factor for acute kidney injury and death, and thus the occurrence and prognosis of acute kidney injury can be predicted by measuring the plasma BNP levels in critically ill patients. In patients with chronic kidney diseases, the increase of the level of the plasma BNP is positively correlated with the severity degree of the kidney injury, and the plasma BNP can be used as an effective biomarker molecule for detecting the progression of the kidney injury. In addition, the plasma BNP levels in patients with cirrhosis are also of interest.
Therefore, there is a need to develop a kit for detecting B-type natriuretic peptide in plasma. Chemiluminescence immunoassay (CLIA) is an emerging immunoassay technology developed after enzyme immunoassay, radioimmunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay. Chemiluminescence immunoassay is a rapid development in recent years, and has high specificity of immunoreaction and high sensitivity of luminous reaction, so that the chemiluminescence immunoassay is widely applied to monitoring and analysis of various hormones, special proteins and medicines in recent years by clinical laboratories and scientific research units at home and abroad. The method has the advantages of high sensitivity, strong specificity, wide linear range, simple operation, good reagent stability, simple operation and easy realization of automation, and is an ideal clinical trace biochemical test analysis means.
Disclosure of Invention
The invention aims to provide a chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma, which has the advantages of high sensitivity, good specificity, simple operation and the like.
The invention also aims to provide a preparation method of the chemiluminescence quantitative detection kit for detecting the B-type natriuretic peptide in the blood plasma.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the invention provides a chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma, which comprises a reagent R1 containing streptavidin-coated magnetic particles, a reagent R2 containing acridinium ester labeled B-type natriuretic peptide antibody and a reagent R3 containing biotin labeled B-type natriuretic peptide antibody;
wherein:
the reagent R1 contains streptavidin-coated magnetic particles with the concentration of 0.01% -1%, the superparamagnetic particles are polymers with ferroferric oxide as an inner core and coated with active groups such as amino groups (NH2-), carboxyl groups (-COOH), Tosyl groups (Tosyl) or other groups easy to couple, and the particle size of the streptavidin magnetic particles is 0.05-3 mu m;
the reagent R2 contains the concentration of the acridinium ester labeled B-type natriuretic peptide antibody which is more than or equal to 0.1 mu g/mL, and the molar ratio of the B-type natriuretic peptide antibody to the acridinium ester label is 1: 3-1: 15;
the reagent R3 contains a biotin-labeled B-type natriuretic peptide antibody with the concentration of more than or equal to 0.5 mu g/mL, and the molar ratio of the B-type natriuretic peptide antibody to the coupling label biotin is 1: 3-1: 15.
Among them, the reagent R1 contains streptavidin-coated magnetic microparticles preferably at a concentration of 0.072%.
The reagent R2 preferably contains the acridinium ester-labeled B-type natriuretic peptide antibody at a concentration of 0.4. mu.g/mL.
The reagent R3 preferably contains biotin-labeled B-type natriuretic peptide antibody at a concentration of 2.0. mu.g/mL.
The chemiluminescence quantitative detection kit for detecting the B-type natriuretic peptide in the blood plasma further comprises a B-type natriuretic peptide quality control product, wherein the quality control product comprises B-type natriuretic peptide antigen solutions with the concentrations of 100pg/mL and 2000 pg/mL.
The chemiluminescence quantitative detection kit for detecting the B-type natriuretic peptide in the blood plasma further comprises a B-type natriuretic peptide calibrator, wherein the B-type natriuretic peptide calibrator is prepared by diluting a BNP pure product with a standard product to prepare B-type natriuretic peptide standard solutions with the concentrations of 0pg/mL, 20pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 2000pg/mL and 5000pg/mL respectively.
The chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma further comprises chemiluminescence excitation liquid, wherein the chemiluminescence excitation liquid comprises acidic excitation liquid A and alkaline excitation liquid B, the liquid A is hydrogen peroxide and nitric acid solution, and the liquid B is sodium hydroxide solution.
The invention also provides a preparation method of the chemiluminescence quantitative detection kit for detecting the B-type natriuretic peptide in the blood plasma, wherein,
the preparation method of the reagent R1 comprises the following steps: uniformly mixing a streptavidin magnetic particle solution and a TBST solution, placing the mixture on a magnetic separator until the supernatant is not turbid, removing the supernatant, reserving magnetic particles, and preparing an R1 reagent in a buffer solution after cleaning; the concentration of the streptavidin magnetic particle solution is preferably 50-100 mg/mL; the volume ratio of the streptavidin magnetic particle solution to the Tris solution is preferably (0.5-1): (5-10); the mixing time is preferably 10-15 min, and the buffer solution is 25mM Tris, 0.02% Tween-20, 0.05% Proclin300, pH7.2 or 50mM Bistirs, 0.02% Tween-20, 0.1% Proclin300, pH 6.0.
The preparation method of the reagent R2 comprises the following steps: taking the B-type natriuretic peptide antibody, replacing B-type natriuretic peptide antibody storage solution with 20mM PB buffer solution, wherein the replacement method adopts an ultrafiltration centrifugation method to effectively improve the replacement recovery rate and the labeling rate and shorten the working time, and then diluting the B-type natriuretic peptide antibody storage solution to 1mg/mL with 20mM PB buffer solution according to the molar ratio of the antibody to acridinium ester of 1: 3-1: adding acridine ester 15, incubating at 37 ℃ for 2-4 h for labeling, adding a confining liquid, incubating at 37 ℃ for 2-4 h, purifying by a protein purifier, collecting a protein peak value to obtain a B-type natriuretic peptide antibody labeled by the acridine ester, and preparing an R2 reagent by further adopting 50-100 mM Bistirs, 1-3% BSA, 0.1% BGG, 0.1% Tween-20, 0.5% sodium azide and a buffer solution with pH of 6.0, wherein the confining liquid is a 20mM PB buffer solution containing 2-5% BSA.
There are several common chemiluminescent systems, the most important of which are: HRP-luminol system, acridine ester system, electrochemical luminescence system, etc. Acridinium esters are an important class of chemiluminescent reagents with high quantum yield and high chemiluminescent efficiency, usually five or more times that of luminol. In addition, the acridinium ester has fast reaction in the chemiluminescence process and low background, and can emit light in the presence of sodium hydroxide and hydrogen peroxide. In the oxidation reaction process, the conjugate is decomposed, and the luminescence of free acridinium ester is not influenced; in addition, the acridinium ester chemiluminescence reagent has good stability and is easy to store. Acridinium esters are oxidized by hydrogen peroxide to N-methylacridone under alkaline conditions, releasing photons when returning from the excited state to the ground state. The electron-withdrawing group can facilitate the nucleophilic attack process and increase the light-emitting speed, thereby increasing the quantum yield. Therefore, the invention adopts acridinium ester as a chemiluminescence system.
The preparation method of the reagent R3 comprises the following steps: taking the B-type natriuretic peptide antibody, replacing the B-type natriuretic peptide antibody storage solution with 20mM PB buffer solution, wherein the replacement method adopts an ultrafiltration centrifugation method to effectively improve the replacement recovery rate and the labeling rate and shorten the working time, then diluting the solution with 20mM PB buffer solution to 1mg/mL, and performing the following steps according to the molar ratio of the antibody to biotin of 1: 3-1: adding a DMF solution of biotin, incubating at 37 ℃ for 2-4 h for labeling, adding a confining liquid (20 mM PB buffer solution containing 2-5% BSA), incubating at 37 ℃ for 2-4 h, purifying by a protein purifier, collecting a protein peak value to obtain a B-type natriuretic peptide antibody labeled by the biotin, and further preparing an R3 reagent by adopting 50-100 mM biospirs, 1-3% BSA, 0.1% BGG, 0.1% Tween-20, 0.5% sodium azide and a buffer solution with the pH value of 6.0.
In addition, the invention also provides a detection method of the chemiluminescence quantitative detection kit for detecting the B-type natriuretic peptide in the blood plasma, which comprises the following steps:
1) incubating 50 mu L of a sample to be detected with 50 mu L of an R2 reagent and 50 mu L of an R3 reagent for 10min, and carrying out immunoreaction on a B-type natriuretic peptide antigen in the sample, a biotin-B-type natriuretic peptide antibody-B-type natriuretic peptide antibody-acridinium ester compound by a B-type natriuretic peptide first antibody marked by biotin and a B-type natriuretic peptide second antibody marked by acridinium ester;
2) adding 40 mu L of R1 reagent, incubating for 10min, and combining streptavidin magnetic beads with biotin to form a magnetic bead-streptavidin-biotin-B type natriuretic peptide antibody-B type natriuretic peptide antibody-acridinium ester compound;
3) adding a washing buffer solution, and washing and removing unreacted solution;
4) adding an acidic excitation liquid to dissociate the acridinium ester marker; adding an alkaline excitation liquid to enable the acridinium ester to emit photons; the number of photons received by the photomultiplier tube is proportional to the amount of B-type natriuretic peptide.
Compared with the prior art, the invention has the technical effects that:
first, the biotin- (streptoenzyme) avidin system produces multi-stage signal amplification due to the property that one (streptoenzyme) avidin molecule can bind four biotin molecules, the reaction sensitivity can be significantly improved, and because of the good adaptability of the biotin (streptoase) avidin system, the labeling method is simple and the resistance to different environments is excellent, by virtue of the strong affinity between the (streptoase) avidin and the biotin, any biotinylated molecule can be easily connected by coupling (streptavidin) with other carriers such as polystyrene microwell plates, nano-particles and other suitable carrier materials as universal carriers, and further participate in further reaction, the application does not aim at signal amplification, but provides a universal carrier, so the method has good application prospect in the fields of large-scale production and automatic application. The invention utilizes (chain enzyme) avidin coupled superparamagnetic particles to form a universal carrier structure, and further is used for combining biotinylated antigen or antibody to develop a chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma.
Meanwhile, as a solid phase separation method, superparamagnetic particles can greatly improve the separation efficiency compared with the traditional microporous plate type immune reaction and are very convenient to realize automation, the surface area of magnetic particles greatly improved compared with microporous plates obviously increases effective binding sites, and the linear range of immunoassay can be greatly widened by combining the immunological liquid phase reaction, so that the clinical requirements can be better met, and the magnetic particles can adapt to the harsh reaction environment from acid to alkali due to the good environmental resistance, so that the reaction system has greater compatibility.
Moreover, the invention adopts the acridinium ester as a chemiluminescence system, and firstly, the acridinium ester is easy to couple, the labeling process is simple, the stability is good, the influence on the luminescence performance after labeling is less, the validity period of the kit is long, and the cost is relatively low; the light is flash, the light is fast, concentrated and strong, the fast detection is convenient to realize, the sensitivity and the precision of the detection are high, the requirement on the instrument is simple, and the full-automatic operation is convenient to realize; and secondly, the acridinium ester luminescent system is simple, the alkaline-hydrogen peroxide can directly emit light without a reinforcing agent or a catalyst, interference factors are few, the background is extremely low, and the signal-to-noise ratio is high.
In conclusion, the chemiluminescence quantitative detection kit for detecting the B-type natriuretic peptide in the blood plasma provided by the invention adopts the principle of a double-antibody sandwich method, and simultaneously utilizes superparamagnetic particles, an avidin-biotin system and acridinium ester chemiluminescence, so that the detection accuracy, sensitivity and linear range of the kit are greatly improved, the reaction time is shortened, and the reagent cost is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
FIG. 1 is a dose-response calibration curve for the kit for the determination of type B natriuretic peptide according to the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the accompanying drawings and examples. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.
Example 1
The embodiment provides a composition of a chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma, which comprises the following specific components:
(1) r1 reagent composition:
(2) r2 reagent composition:
(3) r3 reagent composition:
example 2
The embodiment provides a composition of a chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma, which comprises the following specific components:
(1) r1 reagent composition:
(2) r2 reagent composition:
(3) r3 reagent composition:
example 3
The embodiment provides a composition of a chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma, which comprises the following specific components:
(1) r1 reagent composition:
(2) r2 reagent composition:
(3) r3 reagent composition:
example 4
The embodiment provides a composition of a chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma, which comprises the following specific components:
(1) r1 reagent composition:
(2) r2 reagent composition:
(3) r3 reagent composition:
example 5
The embodiment provides a composition of a chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma, which comprises the following specific components:
(1) r1 reagent composition:
(2) r2 reagent composition:
(3) r3 reagent composition:
example 6 evaluation of the Performance of the kit
1. Drawing of standard curve
Preparation of type B natriuretic peptide calibrator: the BNP pure product is prepared into B-type natriuretic peptide standard solutions with the concentrations of 0pg/mL, 20pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 2000pg/mL and 5000pg/mL respectively by using the standard product diluent.
The method comprises the specific steps of preparing a calibrator buffer solution from 0.1mol/L PB, 0.15mol/L sodium chloride, 1% bovine serum albumin and 0.05% tween-20, storing the calibrator buffer solution at the pH value of 6.5 and the temperature of 2-8 ℃ for later use, preparing a certain amount of B-type natriuretic peptide into calibrators with the concentrations of 0pg/mL, 20pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 2000pg/mL and 5000pg/mL by using the calibrator buffer solution obtained in the step ①, and equivalently packaging into 7-bottle calibrators with the concentrations of 0pg/mL, 20pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 2000pg/mL and 5000pg/mL respectively.
The above calibrator was measured using the kit of example 2 by the following method:
1) incubating 50 μ L of the calibrator with 50 μ L of an R2 reagent and 50 μ L of an R3 reagent for 10min, and carrying out immunoreaction on a B-type natriuretic peptide antigen, a biotin-B-type natriuretic peptide antibody, a B-type natriuretic peptide antibody labeled by acridinium ester, and a B-type natriuretic peptide antibody-acridinium ester complex;
2) adding 40 mu L of R1 reagent, incubating for 10min, and combining streptavidin magnetic beads with biotin to form a magnetic bead-streptavidin-biotin-B type natriuretic peptide antibody-B type natriuretic peptide antibody-acridinium ester compound;
3) adding a washing buffer solution, and washing and removing unreacted solution;
4) adding an acidic excitation liquid to dissociate the acridinium ester marker; adding an alkaline excitation liquid to enable the acridinium ester to emit photons; the number of photons received by the photomultiplier tube is proportional to the amount of B-type natriuretic peptide.
5) Dose response curves were plotted.
The specific results of the test of 7 concentration calibrators by the B-type natriuretic peptide assay kit are shown in table 1, the dose response curve of the B-type natriuretic peptide assay kit is shown in fig. 1, the correlation coefficient r is 0.9926, the calibration is successful, the calibration curves of the kits of other embodiments are similar, and the correlation coefficients are all larger than or equal to 0.9900.
Table 1 calibrant test results
| Calibration point | Theoretical concentration (pg/mL) | Relative Light Unit (RLU) |
| Point 1 | 0.00 | 522 |
| Point 2 | 20.00 | 1516 |
| Point 3 | 50.00 | 3648 |
| Point 4 | 100.00 | 5913 |
| Point 5 | 200.00 | 48976 |
| Point 6 | 2000.00 | 214793 |
| Point 7 | 5000.00 | 660867 |
2. The following are the performance index evaluation results of the B-type natriuretic peptide chemiluminescence immunoassay kit prepared in example 2:
(1) evaluation of accuracy
The calibrator (concentration 100pg/mL) was used to perform the detection according to the conventional sample using the kit of example 2, each sample was measured 3 times, the test result was expressed as (Xi), the relative deviation (Bi) was calculated according to equation (1), and the relative deviation should not exceed ± 10%, and the results are shown in table 2:
Bi＝(Xi-T)/T×100％………………………(1)
in the formula:
bi-relative deviation;
xi-measured concentration;
t-calibration concentration.
TABLE 2 accuracy evaluation data
| Number of tests | Concentration of reference (pg/mL) | Relative deviation (%) |
| 1 | 96.09 | -3.91％ |
| 2 | 97.89 | -2.11％ |
| 3 | 96.61 | -3.39％ |
(2) Evaluation of linearity
Diluting the high value sample close to the upper limit of the linear range by at least 5 concentrations according to a certain proportion, wherein the low value sample is close to the lower limit of the linear range. The kit of example 2 is adopted to repeatedly detect each concentration sample for 3 times, the average value is calculated, the result average value and the dilution ratio are subjected to straight line fitting by a least square method, and a linear correlation coefficient r is calculated, wherein the result conforms to the linear range of 15 pg/mL-5000 pg/mL, and the linear correlation coefficient r is more than or equal to 0.9900. The results are shown in Table 3:
TABLE 3 Linear evaluation data
(3) Evaluation of reproducibility
The preparation method of the quality control product is the same as that of the calibrator, and the concentrations are 100pg/mL and 2000pg/mL respectively. The kit of example 2 is used to repeatedly detect samples with two concentrations of high value (2000pg/mL) and low value (100pg/mL) for 10 times, the average value M and standard deviation SD of 10 measurement results are calculated, the coefficient of variation CV is obtained according to the formula (2), and the result is equal to or less than 8.0 percent of the Coefficient of Variation (CV).
CV＝SD/M×100％………………………………(2)
In the formula:
CV — coefficient of variation;
SD-standard deviation of 10 measurements;
m-average of 10 measurements.
The results are shown in Table 4:
table 4 repeatability evaluation data
In conclusion, the evaluation result shows that the kit provided by the invention has a linear correlation coefficient r of more than or equal to 0.9900, namely the linear detection is qualified; the relative deviation of the accuracy evaluation should not exceed ± 10%; the repeatability detection result meets the requirement that the Coefficient of Variation (CV) is less than or equal to 8.0 percent, namely the repeatability detection is qualified. The kit has the characteristics of high sensitivity, wide linear range and good stability.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Accordingly, the drawings and description are illustrative in nature and should not be construed as limiting the scope of the invention.
Claims (10)
1. A chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in plasma is characterized by comprising a reagent R1 containing streptavidin-coated magnetic particles, a reagent R2 containing acridinium ester labeled B-type natriuretic peptide antibody and a reagent R3 containing biotin labeled B-type natriuretic peptide antibody;
wherein:
the reagent R1 contains streptavidin-coated magnetic particles with the concentration of 0.01-1%, and the particle size of the streptavidin magnetic particles is 0.05-3 mu m;
the reagent R2 contains the concentration of the acridinium ester labeled B-type natriuretic peptide antibody which is more than or equal to 0.1 mu g/mL, and the molar ratio of the B-type natriuretic peptide antibody to the acridinium ester label is 1: 3-1: 15;
the reagent R3 contains a biotin-labeled B-type natriuretic peptide antibody with the concentration of more than or equal to 0.5 mu g/mL, and the molar ratio of the B-type natriuretic peptide antibody to the coupling label biotin is 1: 3-1: 15.
2. The chemiluminescent quantitative detection kit for detecting B-type natriuretic peptide in blood plasma of claim 1, further comprising a B-type natriuretic peptide quality control substance, wherein the quality control substance comprises B-type natriuretic peptide antigen solution with concentration of 100pg/mL and 2000 pg/mL.
3. The chemiluminescent quantitative detection kit for detecting the B-type natriuretic peptide in the blood plasma as claimed in claim 1, further comprising a B-type natriuretic peptide calibrator prepared by diluting a BNP pure product with a standard diluent into B-type natriuretic peptide standard solutions with the concentrations of 0pg/mL, 20pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 2000pg/mL and 5000pg/mL respectively.
4. The chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma according to claim 1, further comprising chemiluminescence excitation liquid, wherein the chemiluminescence excitation liquid comprises acidic excitation liquid A and alkaline excitation liquid B, the liquid A is hydrogen peroxide and nitric acid solution, and the liquid B is sodium hydroxide solution.
5. The chemiluminescent quantitative detection kit for detecting B-type natriuretic peptide in blood plasma according to claim 1, wherein the preparation method of the reagent R1 comprises the following steps: uniformly mixing a streptavidin magnetic particle solution and a TBST solution, placing the mixture on a magnetic separator until the supernatant is not turbid, removing the supernatant, reserving magnetic particles, and preparing an R1 reagent in a buffer solution after cleaning; the concentration of the streptavidin magnetic particle solution is preferably 50-100 mg/mL; the volume ratio of the streptavidin magnetic particle solution to the Tris solution is preferably (0.5-1): (5-10); the mixing time is preferably 10-15 min, and the buffer solution is 25mM Tris, 0.02% Tween-20, 0.05% Proclin300, pH7.2 or 50mM Bistirs, 0.02% Tween-20, 0.1% Proclin300, and pH6.0.
6. The chemiluminescent quantitative detection kit for detecting B-type natriuretic peptide in blood plasma as claimed in claim 1 or 5, wherein reagent R1 contains streptavidin-coated magnetic microparticles at a concentration of 0.072%.
7. The chemiluminescent quantitative detection kit for detecting B-type natriuretic peptide in blood plasma according to claim 1, wherein the preparation method of the reagent R2 comprises the following steps: and (3) taking the B-type natriuretic peptide antibody, replacing the B-type natriuretic peptide antibody storage solution with 20mM PB buffer solution, and then diluting the B-type natriuretic peptide antibody storage solution to 1mg/mL with 20mM PB buffer solution according to the molar ratio of the antibody to the acridinium ester of 1: 3-1: adding acridine ester 15, incubating at 37 ℃ for 2-4 h for labeling, adding a confining liquid, incubating at 37 ℃ for 2-4 h, purifying by a protein purifier, collecting a protein peak value to obtain a B-type natriuretic peptide antibody labeled by the acridine ester, and preparing an R2 reagent by further adopting 50-100 mM Bistirs, 1-3% BSA, 0.1% BGG, 0.1% Tween-20, 0.5% sodium azide and a buffer solution with pH of 6.0, wherein the confining liquid is a 20mM PB buffer solution containing 2-5% BSA.
8. The chemiluminescent quantitative detection kit for detecting B-type natriuretic peptide in plasma according to claim 1 or 7, wherein reagent R2 contains acridinium ester labeled B-type natriuretic peptide antibody at a concentration of 0.4 μ g/mL.
9. The chemiluminescent quantitative detection kit for detecting B-type natriuretic peptide in blood plasma according to claim 1, wherein the preparation method of the reagent R3 comprises the following steps: and (3) taking the B-type natriuretic peptide antibody, replacing the B-type natriuretic peptide antibody storage solution with 20mM PB buffer solution, then diluting to 1mg/mL with 20mM PB buffer solution, and carrying out the following steps according to the molar ratio of the antibody to biotin of 1: 3-1: adding a DMF solution of biotin, incubating at 37 ℃ for 2-4 h for labeling, adding a confining liquid (20 mM PB buffer solution containing 2-5% BSA), incubating at 37 ℃ for 2-4 h, purifying by a protein purifier, collecting a protein peak value to obtain a B-type natriuretic peptide antibody labeled by the biotin, and further preparing an R3 reagent by adopting 50-100 mM Bistims, 1-3% BSA, 0.1% BGG, 0.1% Tween-20, 0.5% sodium azide and a buffer solution with the pH value of 6.0.
10. The chemiluminescent quantitative detection kit for detecting B-type natriuretic peptide in blood plasma according to claim 1 or 9, wherein reagent R3 contains biotin-labeled B-type natriuretic peptide antibody at a concentration of 2.0 μ g/mL.
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