CN117030418A - N-terminal B-type pro-natriuretic peptide diluent, preparation method thereof and N-terminal B-type pro-natriuretic peptide calibrator - Google Patents
N-terminal B-type pro-natriuretic peptide diluent, preparation method thereof and N-terminal B-type pro-natriuretic peptide calibrator Download PDFInfo
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- 239000000692 natriuretic peptide Substances 0.000 title claims description 25
- 238000002360 preparation method Methods 0.000 title abstract description 10
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- 239000000427 antigen Substances 0.000 claims abstract description 32
- 102000036639 antigens Human genes 0.000 claims abstract description 32
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- 229940009714 erythritol Drugs 0.000 claims description 2
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 claims description 2
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- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims 1
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- HPNRHPKXQZSDFX-OAQDCNSJSA-N nesiritide Chemical compound C([C@H]1C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)CNC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CO)C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 HPNRHPKXQZSDFX-OAQDCNSJSA-N 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
-
- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Physics & Mathematics (AREA)
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- Urology & Nephrology (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a dilution of N-terminal B-type natriuretic peptide precursor, a preparation method thereof and a calibrator of the N-terminal B-type natriuretic peptide precursor, wherein the dilution of the N-terminal B-type natriuretic peptide precursor comprises the following components in concentration: 0.5-10% of protein protecting agent, 0.1-5% of reducing agent and 10-50 mmol/L of buffer solution; the pH of the buffer solution is 4.0-6.0. Through the synergistic effect of the buffer solution, the protein protecting agent and the reducing agent, the N-terminal B-type natriuretic peptide antigen is stably protected, the assistance of more components is not needed, and the stability of the reagent is improved under the condition that the performance of the reagent is in an optimal state.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a dilution of N-terminal B-type natriuretic peptide precursor, a preparation method thereof and a calibrator of the N-terminal B-type natriuretic peptide precursor.
Background
Heart failure is a symptom of heart circulatory disturbance caused by venous blood stasis and insufficient arterial blood perfusion due to failure of systolic or diastolic function of the heart to sufficiently discharge venous blood back to the heart. Heart failure is not an independent disease, but rather the final stage of heart disease progression. Wherein, after myocardial cells are stimulated, the pro-B-type natriuretic peptide (pre-proBNP) is secreted firstly, then the BNP precursor (proBNP) is formed, and the proBNP is cracked into the B-type natriuretic peptide (BNP, containing 32 amino acids) with biological activities such as sodium benefiting, urination, blood vessel expanding and the like and the N-terminal pro-B-type natriuretic peptide (NT-proBNP, containing 76 amino acids) without biological activities under the action of endoenzymes, the NT-proBNP has long half-life compared with the BNP, is more stable in vitro, the concentration of the NT-proBNP can reflect the release of newly synthesized BNP in a short time, the concentration in a heart failure patient is higher than that of the BNP, has high sensitivity and specificity, can be used as a sensitive index for heart failure diagnosis, and has important value in prognosis and curative effect monitoring of heart failure.
At present, the NT-proBNP calibrator is mainly a freeze-dried product, the re-dissolution of the freeze-dried product is troublesome, and in the re-dissolution process, the concentration of the re-dissolved calibrator and the target value have deviation due to water quality difference and manual operation errors, so that the clinical detection result is inaccurate, the re-dissolved calibrator is not easy to preserve, and the preservation time is short.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a diluent of N-terminal B-type natriuretic peptide precursor which does not need freeze-drying and has strong stability and long validity period, a preparation method thereof and a calibrator of the N-terminal B-type natriuretic peptide precursor.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a dilution of N-terminal pro-B-type natriuretic peptide comprising the following concentration components:
0.5-10% of protein protecting agent, 0.1-5% of reducing agent and 10-50 mmol/L of buffer solution; the pH of the buffer solution is 4.0-6.0.
The invention also provides a preparation method of the N-terminal B-type pro-natriuretic peptide diluent, which comprises the following steps:
providing the following concentration of components: 0.5-10% of protein protecting agent, 0.1-5% of reducing agent and 10-50 mmol/L of buffer solution;
and mixing the protein protecting agent, the reducing agent and the buffer solution, and regulating the pH value to 4.0-6.0 to obtain the diluent.
The invention also provides a calibrator of the N-terminal B-type pro-natriuretic peptide, which comprises the N-terminal B-type pro-natriuretic peptide antigen and a diluent; the diluent is the diluent or the diluent obtained by the preparation method.
The implementation of the embodiment of the invention has the following beneficial effects:
according to the embodiment of the invention, under an acidic buffer system with the pH value of 4.0-6.0, a protein protecting agent and a reducing agent are added to play a synergistic effect, so that N-terminal B-type pro-natriuretic peptide antigen is stably protected, the preservation time of N-terminal B-type pro-natriuretic peptide antigen is prolonged, wherein the protein protecting agent can enable a diluent matrix to be close to a serum matrix, ensure the biological state of N-terminal B-type pro-natriuretic peptide antigen, improve the stability of N-terminal B-type pro-natriuretic peptide antigen, the reducing agent can prevent proteins from being oxidized, and the buffer can provide a basic environment for the stable preservation of N-terminal B-type pro-natriuretic peptide antigen, so that the stability of the form and structure of N-terminal B-type pro-natriuretic peptide antigen is better maintained. The diluent provided by the embodiment of the invention does not need the assistance of more components, and improves the stability of the reagent under the condition that the performance of the reagent is in an optimal state. The calibrator provided by the invention does not need to be freeze-dried, so that the production period is shortened, the production cost is reduced, and meanwhile, a re-dissolution link is not needed, and uncontrollable factors possibly occurring in the re-dissolution process are avoided, so that the clinical detection result is more accurate.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention discloses a diluent of N-terminal B-type natriuretic peptide, which comprises the following components in concentration: 0.5-10% of protein protecting agent, 0.1-5% of reducing agent and 10-50 mmol/L of buffer solution; the pH of the buffer solution is 4.0-6.0.
Specifically, in the embodiment of the invention, under an acidic buffer system with the pH value of 4.0-6.0, a protein protecting agent and a reducing agent are added to play a synergistic role, so that N-terminal B-type natriuretic peptide antigen is stably protected, the preservation time of N-terminal B-type natriuretic peptide antigen is prolonged, wherein the protein protecting agent can enable a diluent matrix to be close to a serum matrix, ensure the biological state of N-terminal B-type natriuretic peptide antigen, improve the stability of N-terminal B-type natriuretic peptide antigen, the reducing agent can prevent proteins from oxidizing, a basic environment can be provided for the stable preservation of N-terminal B-type natriuretic peptide antigen by the buffer, the stability of the form and structure of N-terminal B-type natriuretic peptide antigen is better maintained, the diluent in the embodiment of the invention does not need the assistance of more components, and the stability of the reagent is improved under the condition that the reagent performance is ensured.
In a specific embodiment, the reducing agent comprises one or more of sodium thiosulfate, L-alanine and arginine.
In a specific embodiment, the buffer comprises one or more of acetic acid buffer, 2- (N-morpholinoethanesulfonic acid buffer, glycine-hydrochloric acid buffer, and citric acid buffer.
In a specific embodiment, the pH of the buffer includes, but is not limited to, 4.0, 4.5, 5.0, 5.5, 6.0, etc.
Specifically, the buffer solution ensures the stability of the reaction environment and ensures that the detection result is not fluctuated or affected.
In a specific embodiment, the protein protectant comprises one or more of recombinant human serum albumin, bovine serum albumin, casein, deoxyribose, proteoglycan, erythritol, and trehalose.
In one embodiment, the diluent further comprises an alcohol at a concentration of 5% to 20%.
Specifically, the alcohol substances can not only keep the surface of the N-terminal B-type natriuretic peptide antigen moist and prevent the N-terminal B-type natriuretic peptide antigen from being inactivated due to water loss, but also reduce molecular movement and prevent protein aggregation.
In one embodiment, the alcohol comprises glycerol and/or ethylene glycol.
In one embodiment, the diluent further comprises a preservative at a concentration of 0.05% to 0.15%.
Specifically, the preservative can inhibit the growth of bacteria and fungi, improve the stability of the calibrator and prolong the effective period of the calibrator.
In a specific embodiment, the preservative comprises one or more of sodium azide, phenol, proClin150, proClin200, proClin300, and ProClin 5000.
The invention also provides a preparation method of the N-terminal B-type pro-natriuretic peptide diluent, which comprises the following steps:
1) Providing the following concentration of components: 0.5-10% of protein protecting agent, 0.1-5% of reducing agent and 10-50 mmol/L of buffer solution.
2) Mixing the protein protective agent, the reducing agent and the buffer solution, and regulating the pH value to 4.0-6.0 to obtain the diluent.
In one embodiment, the protein protectant, reducing agent and buffer are mixed with ultrapure water, and after dissolution, the pH is adjusted to 4.0-6.0, and the volume is fixed to 100ml to obtain a diluent.
In a specific embodiment, step 1) further comprises providing the following concentrations of components: 5 to 20 percent of alcohol substances and 0.05 to 0.15 percent of preservative.
In a specific embodiment, step 2) further comprises mixing an alcohol and a preservative with the protein protectant, the reducing agent and the buffer solution, and adjusting the pH value to 4.0-6.0 to obtain a diluent.
Specifically, in the embodiment of the invention, the protein protectant, the reducer, the preservative and the alcohol substances with the concentrations are combined and added into the diluent prepared by the buffer solution, and the synergistic effect of the components can effectively improve the stability of the N-terminal B-type natriuretic peptide antigen.
The invention also provides a calibrator of the N-terminal B-type pro-natriuretic peptide, which comprises the N-terminal B-type pro-natriuretic peptide antigen and a diluent; the diluent is the diluent according to any embodiment of the invention or the diluent obtained by the preparation method according to any embodiment of the invention.
Specifically, the N-terminal B-type pro-natriuretic peptide calibrator is prepared by mixing the N-terminal B-type pro-natriuretic peptide antigen with the diluent of any embodiment of the invention, and improves the stability of the reagent under the condition that the performance of the reagent is in an optimal state. The calibrator provided by the invention does not need to be freeze-dried, so that the production period is shortened, the production cost is reduced, a re-dissolution link is not needed, uncontrollable factors possibly occurring in the re-dissolution process are avoided, and the clinical detection result is more accurate.
The following are specific examples.
Example 1
The dilution of N-terminal pro-B-type natriuretic peptide in this example comprises the following concentrations of components: 50mmol/L glycine-HCl buffer (pH 5.0), 2% recombinant human serum albumin, 10% glycerol, 0.5% sodium thiosulfate and 0.1% Proclin300.
The preparation method of the diluent of the embodiment is as follows:
mixing the recombinant human serum albumin, glycerol, sodium thiosulfate, proclin300 and glycine-hydrochloric acid buffer solution, and regulating the pH value to 5.0 to obtain a diluent.
Comparative example 1
Comparative example 1 is different from example 1 only in that: does not contain recombinant human serum albumin, and is specifically as follows:
a dilution of N-terminal pro-B-type natriuretic peptide of this comparative example, comprising the following concentrations of components: 50mmol/L glycine-hydrochloric acid buffer (pH 5.0), 10% glycerol, 0.5% sodium thiosulfate and 0.1% Proclin300.
Comparative example 2
Comparative example 2 differs from example 1 only in that: does not contain sodium thiosulfate, and is specifically as follows:
a dilution of N-terminal pro-B-type natriuretic peptide of this comparative example, comprising the following concentrations of components: 50mmol/L glycine-HCl buffer (pH 5.0), 2% recombinant human serum albumin, 10% glycerol and 0.1% Proclin300.
Comparative example 3
Comparative example 3 differs from example 1 only in that: does not contain glycerol, and is specifically as follows:
a dilution of N-terminal pro-B-type natriuretic peptide of this comparative example, comprising the following concentrations of components: glycine-hydrochloric acid buffer (pH 5.0) at 50mmol/L, recombinant human serum albumin at 2%, sodium thiosulfate at 0.5% and Proclin300 at 0.1%
Test case
The dilutions prepared in example 1 and comparative examples 1 to 3 were each added with N-terminal pro-B-natriuretic peptide antigen at a concentration of 1000000pg/mL, and prepared into 4 concentration gradient calibrators (20 pg/mL, 200pg/mL, 2000pg/mL, and 35000 pg/mL), respectively.
The calibrators prepared in example 1 and comparative examples 1 to 3 were subjected to stability test, specifically as follows:
(1) Accelerated stability test of calibrator for N-terminal pro-B natriuretic peptide of example 1 and comparative examples 1-3:
after the calibrator obtained in example 1 and comparative examples 1 to 3 were split-packed into two portions, one portion was left at 37℃for 14 days, and the other portion was stored at 2℃to 8℃for accelerated stability verification of N-terminal type B pro-natriuretic peptide antigen in the diluent. The results of the real-time stability test for the calibrators of example 1 and comparative examples 1-3 are shown in Table 1.
Table 1 results of real-time stability testing of the calibrator of example 1 and comparative examples 1-3
From the test data in table 1, the acceleration stability of the calibrator of example 1 is better than that of the calibrators of comparative examples 1-3, the concentration degradation of the calibrator of example 1 is less than 10% after the calibrator of example 1 is placed for 14 days at 37 ℃, which indicates that the protein protectant and the reducer have synergistic effect, the stability of the form and structure of the N-terminal B-type natriuretic peptide antigen is better maintained, and the stability of the N-terminal B-type natriuretic peptide antigen can be improved.
(2) Real-time stability test of calibrator for N-terminal pro-B natriuretic peptide of example 1 and comparative examples 1-3:
the calibrants of example 1 and comparative examples 1 to 3 were stored at 2℃to 8℃and the content of N-terminal B-type natriuretic peptide antigen was measured using the brain natriuretic peptide test kit (electrochemiluminescence method) of Roche at month 0, month 3, month 6, month 9, month 12 and month 13, respectively, and the real-time stability of N-terminal B-type natriuretic peptide antigen in the diluted solution was verified, and the results of the real-time stability tests of the calibrants of example 1 and comparative examples 1 to 3 were shown in tables 2 to 5, respectively.
Table 2 results of the real-time stability test of the calibrator of example 1
Table 3 results of the real-time stability test of the calibrator of comparative example 1
Table 4 results of the real-time stability test of the calibrator of comparative example 2
Table 5 results of the real-time stability test of the calibrator of comparative example 3
From the test data in tables 2-5, the real-time stability of the calibrator in example 1 is better than that of the calibrator in comparative examples 1-3, and the calibrator in example 1 can be stably stored for 12 months under the airtight condition of 2-8 ℃, which indicates that the protein protectant and the reducer play a synergistic role in stabilizing and protecting N-terminal B-type natriuretic peptide antigen and prolonging the storage time of N-terminal B-type natriuretic peptide antigen.
Example 2
Example 2 differs from example 1 only in that: the components of the diluent are different in concentration, and the specific steps are as follows:
the dilution of N-terminal pro-B-type natriuretic peptide in this example comprises the following concentrations of components: glycine-hydrochloric acid buffer (pH 5.0) at 50mmol/L, recombinant human serum albumin at 0.5%, glycerol at 20%, sodium thiosulfate at 0.1% and Proclin300 at 0.05%.
The dilution of this example was taken, NT-proBNP antigen was added thereto at a concentration of 1000000pg/mL, and 4 concentration gradient calibrators (20 pg/mL, 200pg/mL, 2000pg/mL and 35000 pg/mL) were prepared, and stability test was performed on the calibrators obtained in this example, and the effect achieved by the dilution of this example was the same as in example 1.
Example 3
Example 3 differs from example 1 only in that: the components of the diluent are different in concentration, and the specific steps are as follows:
the dilution of N-terminal pro-B-type natriuretic peptide in this example comprises the following concentrations of components: 50mmol/L glycine-HCl buffer (pH 5.0), 10% recombinant human serum albumin, 20% glycerol, 5% sodium thiosulfate and 0.05% Proclin300.
The dilution of this example was taken, NT-proBNP antigen was added thereto at a concentration of 1000000pg/mL, and 4 concentration gradient calibrators (20 pg/mL, 200pg/mL, 2000pg/mL and 35000 pg/mL) were prepared, and stability test was performed on the calibrators obtained in this example, and the effect achieved by the dilution of this example was the same as in example 1.
Example 4
Example 4 differs from example 1 only in that: the components of the diluent are different in concentration, and the specific steps are as follows:
the dilution of N-terminal pro-B-type natriuretic peptide in this example comprises the following concentrations of components: 50mmol/L glycine-HCl buffer (pH 4.0), 2% recombinant human serum albumin, 10% glycerol, 0.5% sodium thiosulfate and 0.1% Proclin300.
The dilution of this example was taken, NT-proBNP antigen was added thereto at a concentration of 1000000pg/mL, and 4 concentration gradient calibrators (20 pg/mL, 200pg/mL, 2000pg/mL and 35000 pg/mL) were prepared, and stability test was performed on the calibrators obtained in this example, and the effect achieved by the dilution of this example was the same as in example 1.
Example 5
Example 5 differs from example 1 only in that: the components of the diluent are different in concentration, and the specific steps are as follows:
the dilution of N-terminal pro-B-type natriuretic peptide in this example comprises the following concentrations of components: 50mmol/L glycine-HCl buffer (pH 6.0), 2% recombinant human serum albumin, 10% glycerol, 0.5% sodium thiosulfate and 0.1% Proclin300.
The dilution of this example was taken, NT-proBNP antigen was added thereto at a concentration of 1000000pg/mL, and 4 concentration gradient calibrators (20 pg/mL, 200pg/mL, 2000pg/mL and 35000 pg/mL) were prepared, and stability test was performed on the calibrators obtained in this example, and the effect achieved by the dilution of this example was the same as in example 1.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A dilution of N-terminal pro-B-type natriuretic peptide, comprising the following concentrations of components:
0.5-10% of protein protecting agent, 0.1-5% of reducing agent and 10-50 mmol/L of buffer solution;
the pH of the buffer solution is 4.0-6.0.
2. The dilution of the N-terminal pro-B-type natriuretic peptide according to claim 1, wherein the reducing agent comprises one or more of sodium thiosulfate, L-alanine and arginine.
3. The dilution of the N-terminal pro-B-natriuretic peptide according to claim 1, wherein the buffer comprises one or more of an acetate buffer, a 2- (N-morpholino) ethanesulfonic acid buffer, a glycine-hydrochloric acid buffer, and a citric acid buffer.
4. The dilution of the N-terminal pro-B-type natriuretic peptide according to claim 1, wherein the protein protectant comprises one or more of recombinant human serum albumin, bovine serum albumin, casein, deoxyribose, proteoglycan, erythritol, and trehalose.
5. The diluent of N-terminal pro-B-natriuretic peptide according to any one of claims 1-4, wherein the diluent further comprises an alcohol at a concentration of 5-20%.
6. The dilution of N-terminal pro-B-natriuretic peptide of claim 5 wherein the alcohol comprises glycerol and/or ethylene glycol.
7. The dilution of the N-terminal pro-B-type natriuretic peptide according to claim 1, wherein the dilution further comprises a preservative at a concentration of 0.05-0.15%.
8. The dilution of N-terminal pro-B-type natriuretic peptide according to claim 7, wherein the preservative comprises one or more of sodium azide, phenol, proClin150, proClin200, proClin300 and ProClin 5000.
9. A method for preparing a dilution of N-terminal pro-B-type natriuretic peptide, comprising the steps of:
providing the following concentration of components: 0.5-10% of protein protecting agent, 0.1-5% of reducing agent and 10-50 mmol/L of buffer solution;
and mixing the protein protecting agent, the reducing agent and the buffer solution, and regulating the pH value to 4.0-6.0 to obtain the diluent.
10. A calibrator of N-terminal pro-B-type natriuretic peptide, comprising N-terminal pro-B-type natriuretic peptide antigen and a diluent; the diluent is the diluent according to any one of claims 1 to 8 or the diluent obtained by the production method according to claim 9.
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