A bnp liquid calibrator diluent and a preparation method thereof

Abstract

The present application relates to a kind of BNP liquid calibration diluent and its preparation method, diluent is prepared from the following raw materials: MES buffer solution: 0.02-0.05 mol / L;Sodium chloride: 9g / L;Sugarcane sugar: 5-10g / L;Glycine: 5-10g / L;Bovine serum albumin: ≥10g / L;Glycerol: ≥150g / L;Non-ionic surfactant: 30g / L;Dithiothreitol: 1g / L;Proclin 300: 1g / L.The beneficial effects of the present application are: 1. The diluent provided by the present application can improve the stability of BNP calibration product under liquid condition, the signal retention rate is between 90%-110% under the condition of 37 DEG C for 7 days;Stored under the condition of 2 DEG C-8 DEG C lightproof seal, the effective period is 18 months.2. Stable performance, low cost, BNP calibration product prepared by the diluent provided by the present application does not need to be freeze-dried.3. The stability of the solution can be improved by adding bovine serum albumin in the diluent provided by the present application, and the stability can be further improved by adding sugarcane sugar, glycine, glycerol, non-ionic surfactant.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescence in vitro diagnostic reagent buffer technology and BNP detection, specifically to a BNP liquid calibrator diluent and its preparation method. Background Technology

[0002] Heart failure (HF) is a clinical syndrome caused by a variety of factors, including coronary artery disease, hypertension, valvular disease, and myocarditis. More precisely, heart failure refers to the gradual loss of the heart's ability to pump blood to the lungs and / or limbs. Common symptoms include shortness of breath, exercise-induced cough, edema of the extremities, and dizziness. Heart failure can be systolic, diastolic, or both. The New York Heart Association (NYHA) classifies cardiac function into four classes (I, II, III, and IV) based on the degree of limitation during exercise.

[0003] B-type brain natriuretic peptide (BNP) is a member of the natriuretic peptide family and was initially discovered by de Bold et al. Although BNP was initially isolated from pig brain tissue (and initially named brain natriuretic peptide), it was eventually confirmed that the heart is the primary source of BNP. When the heart experiences volume overload or other conditions that induce ventricular stretch, BNP synthesized by the body is released into the bloodstream, where it interacts with the renin-angiotensin-aldosterone system (RAAS) to regulate water and electrolyte balance. The pre-proBNP (134 amino acids) is synthesized in cardiomyocytes and converted into the proBNP (108 amino acids). The proBNP is then cleaved into the physiologically active BNP (32 amino acids) and the inactive N-terminal pro-BNP (NT-proBNP, 76 amino acids). BNP is cleared from the bloodstream via specific cellular receptors by neutral endopeptidases, with a half-life of approximately 23 minutes. Numerous studies have shown that BNP can be used for the diagnosis, prognosis, and treatment monitoring of patients with heart failure, as BNP levels are elevated in patients with heart failure. Plasma BNP levels can provide useful clinical information for the diagnosis and treatment of left ventricular dysfunction and heart failure, and can complement other diagnostic methods such as electrocardiography, chest X-ray, and echocardiography. Left ventricular ejection fraction or exercise tolerance assessments indicate that plasma BNP levels increase with the physiological decline in cardiac functional reserve. BNP levels can be used to assess the severity of heart failure, and BNP levels have a good correlation with NYHA functional classification. The European Society of Cardiology has included urinary natriuretic peptide (such as BNP) monitoring in its guidelines for the diagnosis or exclusion of heart failure.

[0004] Evidence suggests that BNP can be used for risk stratification in patients with heart failure and acute coronary syndrome (ACS). Elevated BNP levels in heart failure patients indicate disease progression and an increased incidence of complications and mortality. Furthermore, ACS patients with elevated BNP levels also have a higher probability of developing cardiac complications and a higher mortality rate after myocardial infarction.

[0005] Currently, commercially available BNP calibrators / control products are available in liquid and lyophilized powder forms, but their shelf life and effectiveness are not ideal. The BNP calibrator diluent of this invention solves these problems. The calibrator / control product can be stored for a long time in liquid form while maintaining good stability, thus meeting the requirements of manufacturers seeking to simplify production processes and reduce costs. Summary of the Invention

[0006] In summary, to overcome the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a BNP liquid calibrator diluent and its preparation method, which yields a BNP calibrator diluent with high storage stability and carrier stability, good thermal stability, simple preparation process and relatively low cost.

[0007] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A BNP liquid calibrator diluent, prepared from the following raw materials:

[0008] MES buffer: 0.02–0.05 mol / L;

[0009] Sodium chloride: 9 g / L;

[0010] Sucrose: 5–10 g / L;

[0011] Glycine: 5–10 g / L;

[0012] Bovine serum albumin: ≥10g / L;

[0013] Glycerol: ≥150g / L;

[0014] Nonionic surfactant: 30 g / L;

[0015] Dithiothreitol: 1 g / L;

[0016] Proclin 300: 1g / L.

[0017] Based on the above technical solution, the present invention can be further improved as follows:

[0018] Furthermore, the nonionic surfactant is polyethylene glycol 20000.

[0019] Furthermore, the pH value is within the range of 6.0 ± 0.1.

[0020] This invention also provides a method for preparing a BNP liquid calibrator diluent, comprising the following steps:

[0021] Step 1, weigh out the following raw materials respectively:

[0022] MES buffer: 0.02–0.05 mol / L;

[0023] Sodium chloride: 9 g / L;

[0024] Sucrose: 5–10 g / L;

[0025] Glycine: 5–10 g / L;

[0026] Bovine serum albumin: ≥10g / L;

[0027] Glycerol: ≥150g / L;

[0028] Nonionic surfactant: 30 g / L;

[0029] Dithiothreitol: 1 g / L;

[0030] Proclin 300: 1g / L;

[0031] Step 2: Perform the operation at a temperature of 25±3℃. Weigh out 70% of the total weight of purified water and add the MES buffer weighed in Step 1 to the purified water and dissolve it.

[0032] Step 3: Adjust the pH of the solution after dissolving the MES buffer using 6 mol / L hydrochloric acid and 1 mol / L sodium hydroxide solution to stabilize the pH within the range of 6.0 ± 0.1.

[0033] Step 4: Add the sodium chloride, sucrose, glycine, glycerol, nonionic surfactant, dithiothreitol and Proclin 300 weighed in Step 1 to the solution with the pH value adjusted, and stir to make the components fully mixed.

[0034] Step 5: Add the bovine serum albumin weighed in Step 1 to the mixed solution and continue stirring until the bovine serum albumin is completely dissolved.

[0035] Step 6: Add purified water to the solution after dissolving bovine serum albumin to bring the solution volume to the total prepared volume, and then mix again.

[0036] Step 7: After the solution has been diluted to a certain volume and mixed, let it stand and then filter it to obtain the final product.

[0037] Based on the above technical solution, the present invention can be further improved as follows:

[0038] Furthermore, the nonionic surfactant is polyethylene glycol 20000.

[0039] Furthermore, the operation was carried out at a temperature of 25±3℃.

[0040] Further, in step 7, the solution after being brought to a constant volume and mixed is allowed to stand for 30 minutes, and then filtered using a nylon filter membrane with a pore size of 0.22 μm.

[0041] The beneficial effects of this invention are:

[0042] 1. The diluent provided by this invention can improve the stability of BNP calibrators under liquid conditions. After being placed at 37°C for 7 days, the signal retention rate is between 90% and 110%. When stored at 2°C to 8°C in a light-proof and sealed environment, the shelf life is 18 months.

[0043] 2. It has stable performance and low cost. The BNP calibrator prepared with the diluent provided by this invention does not require lyophilization.

[0044] 3. The present invention can improve the stability of the solution by adding bovine serum albumin to the provided diluent, and further improve the stability by adding sucrose, glycine, glycerol and nonionic surfactant. Detailed Implementation

[0045] The principles and features of the present invention are described below with reference to specific examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0046] A BNP liquid calibrator diluent is prepared from the following raw materials:

[0047] MES buffer: 0.02–0.05 mol / L;

[0048] Sodium chloride: 9 g / L;

[0049] Sucrose: 5–10 g / L;

[0050] Glycine: 5–10 g / L;

[0051] Bovine serum albumin (BSA): ≥10 g / L;

[0052] Glycerol: ≥150g / L;

[0053] Nonionic surfactant (polyethylene glycol 20000): 30 g / L;

[0054] Dithiothreitol (DTT): 1 g / L;

[0055] Proclin 300 (PC300): 1g / L.

[0056] The pH value of the BNP liquid calibrator diluent is in the range of 6.0 ± 0.1.

[0057] A method for preparing a BNP liquid calibrator diluent includes the following steps performed at a temperature of 25±3℃:

[0058] Step 1, weigh out the following raw materials respectively:

[0059] MES buffer: 0.02–0.05 mol / L;

[0060] Sodium chloride: 9 g / L;

[0061] Sucrose: 5–10 g / L;

[0062] Glycine: 5–10 g / L;

[0063] Bovine serum albumin (BSA): ≥10 g / L;

[0064] Glycerol: ≥150g / L;

[0065] Nonionic surfactant: 30 g / L, preferably polyethylene glycol 20000;

[0066] Dithiothreitol (DTT): 1 g / L;

[0067] Proclin 300 (PC300): 1g / L.

[0068] Step 2: Perform the operation at a temperature of 25±3℃. Weigh out 70% of the total weight of purified water and add the MES buffer weighed in Step 1 to the purified water to dissolve it.

[0069] Step 3: Adjust the pH of the solution after dissolving the MES buffer using 6 mol / L hydrochloric acid and 1 mol / L sodium hydroxide solution to stabilize the pH within the range of 6.0 ± 0.1.

[0070] Step 4: Add the sodium chloride, sucrose, glycine, glycerol, nonionic surfactant, dithiothreitol, and Proclin 300 weighed in Step 1 to the solution with the pH value adjusted, and stir to ensure that all components are fully mixed.

[0071] Step 5: Add the bovine serum albumin weighed in Step 1 to the mixed solution and stir continuously until the bovine serum albumin is completely dissolved.

[0072] Step 6: Add purified water to the solution after dissolving bovine serum albumin to bring the solution volume to the total prepared volume, and then mix again.

[0073] Step 7: After mixing and adjusting the volume, let the solution stand for 30 minutes, then filter the solution using a nylon filter membrane with a pore size of 0.22 μm.

[0074] The stability of the BNP calibrator prepared using the diluent provided in this invention was verified through experiments.

[0075] Experimental materials

[0076] Unitec Brain Natriuretic Peptide (BNP) Detection Kit (Magnetic Microparticle Chemiluminescence Method), substrate solution, washing solution, Unitec IA-260 fully automated chemiluminescence immunoassay analyzer, brain natriuretic peptide (BNP) antigen, 2-8℃ refrigerator, 37℃ incubator.

[0077] Test methods

[0078] BNP calibrators were prepared using the diluent provided in this invention. After being aliquoted and stored in a refrigerator at 2-8℃ and a constant temperature incubator at 37℃ for 7 days, they were detected using the Unit Brain Natriuretic Peptide (BNP) Detection Kit (Magnetic Microparticle Chemiluminescence Method) on the Unit IA-260 series fully automated chemiluminescence immunoassay analyzer, as shown in Table 1.

[0079] Table 1. Signal retention rate of BNP calibrators prepared with the diluent provided by this invention after storage at 2-8℃ / 37℃ for 7 days.

[0080]

[0081] Calculation method: .

[0082] The beneficial effects of the present invention will be illustrated through examples and comparative examples.

[0083] Example

[0084] Prepare BNP calibrator diluent R1 according to Table 2.

[0085] Table 2 Preparation of Diluent R1

[0086]

[0087] Prepare BNP calibrator diluent R2 according to Table 3.

[0088] Table 3. Preparation of Diluent R2

[0089]

[0090] Prepare BNP calibrator diluent R3 according to Table 4.

[0091] Table 4 Preparation of Diluent R3

[0092]

[0093] Comparison example

[0094] Prepare BNP calibrator comparison dilution C1 according to Table 5.

[0095] Table 5. Preparation of Comparative Diluent C1

[0096]

[0097] Prepare BNP calibrator comparison dilution C2 according to Table 6.

[0098] Table 6 Comparison of diluent C2 preparation table

[0099]

[0100] Comparison of test results

[0101] Accelerated stability testing: A series of BNP concentration levels 1-5 (P1-P5, concentration changing from low to high) and a 0 concentration point were prepared using diluents (R1, R2, R3) and control diluents (C1, C2, C3), respectively. These solutions were stored at 2-8℃ and 37℃ for 7 days in an accelerated stability test. No significant changes were observed in the appearance of the solutions. When tested according to the above-disclosed determination method of this invention, the signal retention rates of the BNP P1-P5 concentration points prepared with the diluents at 4℃ / 37℃ for 7 days met the requirements of 90% to 110%, as shown in Table 7.

[0102] Table 7 Comparison of signal retention rates of BNP after 7 days of treatment at 4℃ and 37℃

[0103]

[0104] Experimental conclusion: Adding sucrose, glycine 5-10 g / L, bovine serum albumin (BSA) ≥10 g / L, glycerol ≥150 g / L, nonionic surfactant PEG20000 or Triton X-100 3%, and dithiothreitol (DTT) 0.1% to the 0.02-0.05 mol / L MES buffer system can increase the stability of the diluent and prolong the storage time of BNP antigen under liquid conditions.

[0105] In the diluent C1, R1, and R2 experiments, the signal retention rate of the BNP calibrator after 7 days of treatment at 4℃ / 37℃ was statistically analyzed. It was found that the BSA content has a significant impact on stability. When the BSA content is ≥10g / L, the stability of the calibrator can be further improved. Secondly, in the diluent C2, R1, and R3 experiments, the signal retention rate was statistically analyzed. When the glycerol content is ≥150g / L and the dithiothreitol (DTT) content is 0.1%, the stability of the calibrator is increased.

[0106] Aircraft stability: The applicant prepared BNP calibrators level 1 (S1) and level 2 (S2) using diluent 1, respectively, and placed them in an aircraft stability test at room temperature for 8 hours. The appearance of the calibrators did not change significantly. When tested according to the above-disclosed measurement method of the present invention, the signal retention rates at 0h, 3h, 6h and 8h all met the requirements of 90% to 110%, as shown in Table 8.

[0107] Table 8: Comparison of signal retention rates of BNP calibrators at room temperature for 0, 3, 6, and 8 hours.

[0108]

[0109] Storage stability: BNP calibrators level 1 (S1) and level 2 (S2) were prepared with dilution solution 1 and stored at 2-8℃ for 18 months. The appearance of the calibrators did not change significantly. When tested according to the above-disclosed test method of the present invention, the signal retention rates at 2 to 8℃ for 1 month, 5 months, 10 months, 15 months and 18 months can meet the requirements of 90% to 110%, as shown in Table 9.

[0110] Table 9. Signal retention rates of BNP calibrators at 2-8℃ for 1, 5, 10, 15, and 18 months.

[0111]

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A BNP liquid calibrator diluent, characterized in that, It is made from the following raw materials: MES buffer: 0.02–0.05 mol / L; Sodium chloride: 9 g / L; Sucrose: 5–10 g / L; Glycine: 5–10 g / L; Bovine serum albumin: ≥10g / L; Glycerol: ≥150g / L; Nonionic surfactant: 30 g / L; Dithiothreitol: 1 g / L; Proclin 300: 1g / L.

2. The BNP liquid calibrator diluent according to claim 1, characterized in that, The nonionic surfactant is polyethylene glycol 20000.

3. The BNP liquid calibrator diluent according to claim 1, characterized in that, pH value is within the range of 6.0±0.

1.

4. A method for preparing a BNP liquid calibrator diluent, characterized in that, The steps include the following: Step 1, weigh out the following raw materials respectively: MES buffer: 0.02–0.05 mol / L; Sodium chloride: 9 g / L; Sucrose: 5–10 g / L; Glycine: 5–10 g / L; Bovine serum albumin: ≥10g / L; Glycerol: ≥150g / L; Nonionic surfactant: 30 g / L; Dithiothreitol: 1 g / L; Proclin 300: 1g / L; Step 2: Perform the operation at a temperature of 25±3℃. Weigh out 70% of the total weight of purified water and add the MES buffer weighed in Step 1 to the purified water and dissolve it. Step 3: Adjust the pH of the solution after dissolving the MES buffer using 6 mol / L hydrochloric acid and 1 mol / L sodium hydroxide solution to stabilize the pH within the range of 6.0 ± 0.

1. Step 4: Add the sodium chloride, sucrose, glycine, glycerol, nonionic surfactant, dithiothreitol and Proclin 300 weighed in Step 1 to the solution with the pH value adjusted, and stir to make the components fully mixed. Step 5: Add the bovine serum albumin weighed in Step 1 to the mixed solution and continue stirring until the bovine serum albumin is completely dissolved. Step 6: Add purified water to the solution after dissolving bovine serum albumin to bring the solution volume to the total prepared volume, and then mix again. Step 7: After the solution has been diluted to a certain volume and mixed, let it stand and then filter it to obtain the final product.

5. The method for preparing the BNP liquid calibrator diluent according to claim 4, characterized in that, The nonionic surfactant is polyethylene glycol 20000.

6. The method for preparing the BNP liquid calibrator diluent according to claim 4, characterized in that, The operation is carried out at a temperature of 25±3℃.

7. The method for preparing the BNP liquid calibrator diluent according to claim 4, characterized in that, Step 7: After making up to a certain volume and mixing, let the solution stand for 30 minutes, and then filter the solution using a nylon filter membrane with a pore size of 0.22 μm.

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