Cardiovascular marker substance control product and preparation method thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly discloses a cardiovascular marker substance control product and a preparation method thereof, the cardiovascular marker substance control product comprises a serum matrix, an antigen, a preservative and a stabilizer; the antigens comprise a cardiac troponin I antigen, a creatine kinase isoenzyme antigen, a myoglobin antigen, an amino-terminal brain natriuretic peptide precursor antigen, a brain natriuretic peptide antigen, a D-dimer antigen, a heart-type fatty acid binding protein antigen, a myeloperoxidase antigen and a lipoprotein phospholipase A2 antigen; the stabilizer comprises at least one of D-trehalose, cane sugar, glycine, inositol and polyethylene glycol. The cardiovascular marker substance control substance disclosed by the invention can cover various cardiovascular disease markers including heart failure, pulmonary artery embolism, vasculitis, atherosclerosis, myocardial injury, myocardial infarction and the like. The quality control product disclosed by the invention can be used as a third-party quality control product for screening cardiovascular diseases and performing indoor and interventricular quality control on an auxiliary diagnosis and measurement system.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a cardiovascular marker substance control product and a preparation method thereof. Background Art

[0002] Cardiovascular disease is one of the most common major diseases worldwide. Its early diagnosis and timely treatment are crucial to improving the survival rate and quality of life of patients. Cardiovascular markers, such as cardiac troponin I (cTnI), creatine kinase isoenzyme antigen (CK-MB), myoglobin antigen (MYO), amino-terminal pro-brain natriuretic peptide antigen (NT-proBNP), brain natriuretic peptide antigen (BNP), D-dimer antigen (DD), heart-type fatty acid binding protein antigen (H-FABP), myeloperoxidase antigen (MPO), lipoprotein phospholipase A2 antigen (Lp-PLA2), etc., play an important role in the occurrence and development of cardiovascular disease. However, due to the diversity and complexity of detection methods, how to ensure the accuracy and consistency of test results has become a key issue in clinical testing.

[0003] Cardiac troponin I (cTnI) is a highly sensitive and specific marker of myocardial injury. In the diagnosis of human myocardial injury, cTnI has become one of the "gold standards". Compared with other indicators, cTnI has the advantages of appearing earlier in the blood, lasting longer, and being unique to myocardial cells, so cardiac troponin I has a wide range of clinical application prospects.

[0004] Brain natriuretic peptide (BNP) and amino-terminal pro-B type natriuretic peptide (NT-proBNP) have been widely used in clinical practice by hospitals and physicians at all levels and have become very useful biomarkers for the diagnosis of cardiovascular diseases, especially heart failure.

[0005] D-dimer (DD) is one of the specific degradation products of cross-linked fibrin, with a molecular weight of about 190 kDa, a half-life of >3 hours in the body, and is mainly excreted through the kidneys. A negative D-dimer value has an ideal negative predictive effect on pulmonary embolism. A negative result can basically rule out pulmonary embolism, thereby reducing invasive examinations.

[0006] Myeloperoxidase (MPO), also known as peroxidase, is an important iron-containing lysosome that exists in the azure granules of myeloid cells (mainly neutrophils and monocytes) and is a specific marker of myeloid cells. MPO is involved in the occurrence of many diseases, such as inflammation, multiple sclerosis, vasculitis and atherosclerosis.

[0007] Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a member of the phospholipase A2 (PLA2) superfamily. It is a serine lipase composed of 441 amino acid residues with a relative molecular mass of 45.4kD. As a new inflammatory response marker, Lp-PLA2 plays a role in several major stages of atherosclerosis (AS).

[0008] Creatine kinase isoenzyme (CK-MB) measurement is one of the most important indicators for diagnosing acute myocardial infarction (AMI). It helps in the early diagnosis of AMI, as well as the judgment of the severity of the disease and the prediction of prognosis.

[0009] Heart fatty acid binding protein (H-FABP) has a protective effect on the myocardium. Under physiological conditions, H-FABP is not contained in plasma and urine or is present in very low levels. Low concentrations of H-FABP can be detected in the blood of normal people. A large number of studies at home and abroad have found that in the early stages of AMI, due to the high sensitivity of myocardial cells to ischemia and hypoxia, fatty acids are mobilized for energy, leading to a rapid increase in H-FABP in myocardial cells. Since H-FABP has a small relative molecular mass and can be quickly released into the blood, high levels of plasma H-FABP can be detected in the early stages of AMI.

[0010] Myoglobin (MYO) is an oxygen-binding heme protein with a molecular weight of 16.7KD. It is mainly distributed in myocardial and skeletal muscle tissues and is in a compact spherical shape. The sensitivity of MYO for the diagnosis of AMI is 83.3%, the specificity is 88.6%, and the negative predictive rate is 99.5%. There are a lot of literature reports on the clinical utility of early diagnosis and monitoring of ACS. Negative MYO helps to exclude myocardial infarction. It can also be used as an early monitoring indicator for the recurrence of myocardial injury or the expansion of the myocardial infarction area.

[0011] With the rapid development of medical testing technology, the application scope of the above-mentioned projects has become increasingly wide. However, there are also some problems in the application process. For example, indoor quality control has not been carried out as required, resulting in large deviations in patient test results.

[0012] Since there is a large demand for the above-mentioned items in clinical practice, and combined testing is performed in most cases, it is necessary to conduct indoor quality control on the above-mentioned items at the same time. There is an urgent need for stable and high-performance composite quality control products for quality control. Summary of the invention

[0013] The present invention provides a cardiovascular marker substance control product and a preparation method thereof. The cardiovascular marker substance control product has good uniformity and stability, good real-time stability and re-dissolution stability, and can be used as a third-party quality control product for indoor and inter-laboratory quality control of cardiovascular disease screening and auxiliary diagnosis measurement systems.

[0014] The present invention solves the technical problem by adopting the following technical solutions: A cardiovascular marker substance control product, comprising a serum matrix, an antigen, a preservative and a stabilizer; The antigens include cardiac troponin I antigen, creatine kinase isoenzyme antigen, myoglobin antigen, amino-terminal pro-brain natriuretic peptide antigen, brain natriuretic peptide antigen, D-dimer antigen, cardiac fatty acid binding protein antigen, myeloperoxidase antigen and lipoprotein phospholipase A2 antigen; The stabilizer includes at least one of D-trehalose, sucrose, glycine, inositol and polyethylene glycol.

[0015] The inventors of the present invention have found that the stability of antigens is poor, especially myoglobin antigen (MYO), creatine kinase isoenzyme antigen (CK-MB), and cardiac troponin I antigen (cTnI), which are very unstable, resulting in unqualified quality and inability to be stored for a long time.

[0016] The cardiovascular marker substance control product described in the present invention contains comprehensive test items, which can cover all cardiovascular disease test items (including heart failure, pulmonary embolism, vasculitis and atherosclerosis, myocardial injury, and myocardial infarction). The quality control product described in the present invention has good uniformity and stability, good real-time stability and re-dissolution stability, and can be used as a third-party quality control product for indoor and inter-laboratory quality control of cardiovascular disease screening and auxiliary diagnosis measurement systems.

[0017] Adding the stabilizer to the above-mentioned antigen system and serum system effectively improves the stability and uniformity of the quality control product.

[0018] As a preferred embodiment of the present invention, the concentration of the cardiac troponin I antigen is 0.5-20 ng / mL; The concentration of the creatine kinase isoenzyme antigen is 10-50 ng / mL; The concentration of the myoglobin antigen is 50-250 ng / mL; The concentration of the amino-terminal pro-brain natriuretic peptide antigen is 200-25000 pg / mL; The concentration of the brain natriuretic peptide antigen is 100-1000 pg / mL; The concentration of the D-dimer antigen is 0.2-2 mg / L; The concentration of the heart-type fatty acid binding protein antigen is 10-50 ng / mL; The concentration of the myeloperoxidase antigen is 200-2000 pmol / L; The concentration of the lipoprotein phospholipase A2 antigen is 100-500 ng / mL.

[0019] As a preferred embodiment of the present invention, the concentration of the stabilizer is 0.1-20 g / L.

[0020] As a preferred embodiment of the present invention, the stabilizer includes D-trehalose, inositol, sucrose and glycine, the concentration of D-trehalose is 3~12g / L, the concentration of inositol is 1~2g / L, the concentration of sucrose is 0~0.5g / L, and the concentration of glycine is 0~0.5g / L.

[0021] As a preferred embodiment of the present invention, the preservatives Proclin300, penicillin, streptomycin, gentamicin, NaN 3 At least one of .

[0022] As a preferred embodiment of the present invention, the concentration of the preservative is 0.1-0.5 wt %.

[0023] As a preferred embodiment of the present invention, the serum matrix includes at least one of human serum and animal serum.

[0024] The present invention also provides a method for preparing a cardiovascular marker control product, comprising the following steps: (1) Select a serum matrix that is clear in appearance and free of precipitates; (2) Add cardiac troponin I antigen to the serum matrix to the set concentration and let it stand; add creatine kinase isoenzyme antigen, myoglobin antigen, amino-terminal pro-brain natriuretic peptide antigen, brain natriuretic peptide antigen, D-dimer antigen, cardiac fatty acid binding protein antigen, myeloperoxidase antigen and lipoprotein phospholipase A2 antigen to the set concentration; (3) Add stabilizer and preservative, stir evenly and let stand; (4) Freeze-drying to obtain cardiovascular marker control products.

[0025] As a preferred embodiment of the present invention, in step (2), the standing temperature is 20-40° C. and the standing time is 24-48 hours.

[0026] As a preferred embodiment of the present invention, in step (3), the standing temperature is 2-8°C and the standing time is 12-24h.

[0027] Beneficial effects of the present invention: The cardiovascular marker substance control product of the present invention contains comprehensive test items, which can cover all cardiovascular disease test items (including heart failure, pulmonary embolism, vasculitis and atherosclerosis, myocardial injury, and myocardial infarction). The quality control product of the present invention has good uniformity and stability, good real-time stability and re-dissolution stability, and can be used as a third-party quality control product for indoor and inter-laboratory quality control of cardiovascular disease screening and auxiliary diagnosis measurement systems. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0030] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0031] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0032] The reagents or instruments used in the present invention without indicating the manufacturer are all conventional products that can be obtained through commercial purchase. Unless otherwise specified, the raw materials used in the comparative examples and the raw materials used in the parallel experiments of the embodiments are the same commercially available products.

[0033] Example The types and concentrations of antigens, preservatives and stabilizers in formulations 1-3 are shown in Table 1, and the low and medium concentration levels of antigens are represented by C1 and C2, respectively.

[0034] The preparation methods of the cardiovascular marker control products of Formulas 1 to 3 all include the following steps: (1) Select a serum matrix that is clear in appearance and free of precipitates; (2) Add cardiac troponin I antigen to the serum matrix to the set concentration and let stand at 25°C for 24 h; add creatine kinase isoenzyme antigen, myoglobin antigen, amino-terminal pro-brain natriuretic peptide antigen, brain natriuretic peptide antigen, D-dimer antigen, cardiac fatty acid binding protein antigen, myeloperoxidase antigen and lipoprotein phospholipase A2 antigen to the set concentration; (3) Add stabilizer and preservative, stir evenly, and let stand at 5°C for 24 hours; (4) Freeze-drying to obtain cardiovascular marker control products.

[0035] Table 1

[0036]

[0037] Comparative concentration level 1 Comparing concentration level 1 with concentration level 1 of formulation 1, the procedure of standing at 25°C for 24 hours in step S2 was cancelled, and the rest was the same as concentration level 1. It was found that the stability of the quality control product did not meet the requirements.

[0038] Comparative concentration level 2 Comparing the concentration level 2 with the concentration level 2 of formula 1, the step S3 of standing the control solution at 5°C for 24h was eliminated. It was found that the uniformity of the control solution did not meet the requirements.

[0039] Test Case 1. Redissolve the lyophilized powder prepared at each concentration level to obtain the cardiovascular marker control solution and perform a uniform performance test. The uniformity performance index requirement is: the coefficient of variation (CV) between bottles is less than 10%.

[0040] Ten bottles of quality control products are randomly selected from the sample population for uniformity inspection and randomly numbered 1-10. The quality control products are measured using a calibrated detection system. Each bottle of quality control product is measured three times. Considering the random variation of the measurement system caused by factors such as time, the three measurements are performed in different orders, such as 1, 3, 5, 7, 9, 2, 4, 6, 8, 10, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 1, 3, 5, 7, 9.

[0041] Table 2 records the test results. The data processing and judgment standards of the uniformity test results refer to the pharmaceutical industry standard of the People's Republic of China YY / T1652-2019 "General Technical Requirements for Quality Control Materials for In Vitro Diagnostic Reagents". The test results are shown in Table 2.

[0042] Table 2

[0043] It can be seen from the test results in Table 2 that the uniformity of the solution after reconstitution of the freeze-dried powder is good.

[0044] 2. Test the stability of the lyophilized powder after reconstitution: Stability performance index requirements: The reconstituted quality control product can be stable for 4 weeks at 5℃ and protected from light, and can be stable for 1 day at 25℃, and can be repeatedly frozen and thawed for no less than 3 times at <-18℃. At the end of the stability period, the relative deviation of the test results of the quality control product and the newly reconstituted quality control product should be within the range of ±10% of the labeled value.

[0045] Test method: After the quality control product is opened and reconstituted, it is stored and frozen and thawed under the above-mentioned conditions. After the storage period, it is taken out for testing. The reconstituted quality control product under each condition is tested 3 times, and the newly reconstituted quality control product is also tested 3 times as a control. After the test is completed, the average value of the test results after reconstitution and at the end of the stable period is calculated, and the relative deviation between the end of the stable period and the newly reconstituted test results is calculated. The test results are shown in Tables 3 to 5.

[0046] Table 3

[0047] Table 4

[0048] Table 5

[0049] 3. Check the real-time stability performance: The real-time stability performance requirements are: stable for 18 months at 5°C. At the end of the stability period, the relative deviation of the test results of the quality control product and the newly reconstituted quality control product should be within ±10% of the labeled value.

[0050] Test method: After opening the bottle and reconstitution, the quality control product was tested three times, stored at 5°C in the dark, and tested three times at the end of the stable period of each quality control item. The average of the test results after reconstitution and at the end of the stable period was calculated, and the relative deviation between the test results at the end of the stable period and the newly reconstituted test results was calculated. The test results are shown in Tables 6 to 8.

[0051] Table 6

[0052] Table 7

[0053] Table 8

[0054] It can be seen from the test results of Tables 3 to 8 that the quality control product of the present invention has good re-dissolution stability and real-time stability.

[0055] Finally, it should be noted that the above embodiments are intended to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A cardiovascular marker substance control product, characterized in that: Includes serum matrix, antigens, preservatives and stabilizers; The antigens include cardiac troponin I antigen, creatine kinase isoenzyme antigen, myoglobin antigen, amino-terminal pro-brain natriuretic peptide antigen, brain natriuretic peptide antigen, D-dimer antigen, cardiac fatty acid binding protein antigen, myeloperoxidase antigen and lipoprotein phospholipase A2 antigen; The stabilizer includes at least one of D-trehalose, sucrose, glycine, inositol and polyethylene glycol.

2. The cardiovascular marker control product according to claim 1, characterized in that: The concentration of the cardiac troponin I antigen is 0.5-20 ng / mL; The concentration of the creatine kinase isoenzyme antigen is 10-50 ng / mL; The concentration of the myoglobin antigen is 50-250 ng / mL; The concentration of the amino-terminal pro-brain natriuretic peptide antigen is 200-25000 pg / mL; The concentration of the brain natriuretic peptide antigen is 100-1000 pg / mL; The concentration of the D-dimer antigen is 0.2-2 mg / L; The concentration of the heart-type fatty acid binding protein antigen is 10-50 ng / mL; The concentration of the myeloperoxidase antigen is 200-2000 pmol / L; The concentration of the lipoprotein phospholipase A2 antigen is 100-500 ng / mL.

3. The cardiovascular marker control product according to claim 1, characterized in that: The concentration of the stabilizer is 0.1-20 g / L.

4. The cardiovascular marker control product according to claim 1, characterized in that: The stabilizer comprises D-trehalose, inositol, sucrose and glycine, wherein the concentration of the D-trehalose is 3-12 g / L, the concentration of the inositol is 1-2 g / L, the concentration of the sucrose is 0-0.5 g / L, and the concentration of the glycine is 0-0.5 g / L.

5. The cardiovascular marker control product according to claim 1, characterized in that: The preservative is at least one of Proclin300, penicillin, streptomycin, gentamicin and NaN3.

6. The cardiovascular marker control product according to claim 1, characterized in that: The concentration of the preservative is 0.1-0.5wt%.

7. The cardiovascular marker control product according to claim 1, characterized in that: The serum matrix includes at least one of human serum and animal serum.

8. The method for preparing the cardiovascular marker control product according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Select a serum matrix that is clear in appearance and free of precipitates; (2) Add cardiac troponin I antigen to the serum matrix to the set concentration and let it stand; add creatine kinase isoenzyme antigen, myoglobin antigen, amino-terminal pro-brain natriuretic peptide antigen, brain natriuretic peptide antigen, D-dimer antigen, cardiac fatty acid binding protein antigen, myeloperoxidase antigen and lipoprotein phospholipase A2 antigen to the set concentration; (3) Add stabilizer and preservative, stir evenly and let stand; (4) Freeze-drying to obtain cardiovascular marker control products.

9. The method for preparing a cardiovascular marker control product according to claim 8, characterized in that: In the step (2), the standing temperature is 20-40° C. and the standing time is 24-48 hours.

10. The method for preparing a cardiovascular marker control product according to claim 8, characterized in that: In the step (3), the standing temperature is 2-8°C and the standing time is 12-24h.

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