A cardiovascular marker quality control and a preparation method thereof

Abstract

The application belongs to the technical field of biological detection, and discloses a cardiovascular marker control article and a preparation method thereof. The cardiovascular marker control article comprises a serum matrix, antigens, a preservative and a stabilizer. The antigens include cardiac troponin I antigens, creatine kinase isoenzyme antigens, myoglobin antigens, amino-terminal brain natriuretic peptide precursor antigens, brain natriuretic peptide antigens, D-dimer antigens, heart-type fatty acid-binding protein antigens, myeloperoxidase antigens and lipoprotein phospholipase A2 antigens. The stabilizer includes at least one of D-trehalose, sucrose, glycine, inositol and polyethylene glycol. The cardiovascular marker control article can cover various cardiovascular disease markers, including heart failure, pulmonary embolism, vasculitis and atherosclerosis, myocardial injury, myocardial infarction and the like. The control article can be used as a third-party control article for indoor and inter-laboratory quality control of a cardiovascular disease screening and auxiliary diagnosis measurement system.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a cardiovascular biomarker control sample and its preparation method. Background Technology

[0002] Cardiovascular disease is one of the most prevalent major diseases worldwide, and early diagnosis and timely treatment are crucial for improving patient survival rates and quality of life. Cardiovascular biomarkers, such as cardiac troponin I (cTnI), creatine kinase isoenzyme antigen (CK-MB), myoglobin antigen (MYO), N-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), and lipoprotein phospholipase A2 antigen (Lp-PLA2), play important roles in the occurrence and development of cardiovascular diseases. However, due to the diversity and complexity of detection methods, ensuring the accuracy and consistency of test results has become a key issue in clinical laboratory testing.

[0003] Cardiac troponin I (cTnI) is a highly sensitive and specific biomarker for myocardial injury. It has become one of the "gold standards" for diagnosing myocardial injury in humans. Compared to other markers, cTnI has the advantages of early appearance and long duration in the blood, and it is specific to cardiomyocytes. Therefore, cardiac troponin I has broad clinical application prospects.

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

[0005] D-dimer (DD) is a specific degradation product of cross-linked fibrin, with a molecular weight of approximately 190 kDa. It has a half-life of >3 hours in vivo and is primarily excreted via the kidneys. A negative D-dimer value is an ideal predictor of pulmonary embolism; a negative result can essentially rule out pulmonary embolism, thus reducing the need for invasive examinations.

[0006] Myeloperoxidase (MPO), also known as peroxidase, is an important iron-containing lysosome found in azurophilic granules of myeloid cells (mainly neutrophils and monocytes), and is a specific marker of myeloid cells. MPO is involved in the development of various 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 45.4 kDa. Lp-PLA2 serves as a novel biomarker of inflammatory responses and plays a role in several key stages of atherosclerosis (AS).

[0008] Creatine kinase-MB (CK-MB) measurement is one of the most important indicators for diagnosing acute myocardial infarction (AMI), and it helps in the early diagnosis of AMI, as well as in assessing the severity of the condition and predicting prognosis.

[0009] Heart-type fatty acid-binding protein (H-FABP) has a protective effect on the myocardium. Under physiological conditions, H-FABP is absent or present in very low amounts in plasma and urine, and low concentrations can be detected in the blood of normal individuals. Numerous studies both domestically and internationally have found that in the early stages of acute myocardial infarction (AMI), due to the high sensitivity of cardiomyocytes to ischemia and hypoxia, fatty acids are mobilized for energy, leading to a rapid increase in intracellular H-FABP. Because H-FABP has a relatively small molecular weight, it can be rapidly released into the bloodstream, resulting in high levels of plasma H-FABP detectable even in the early stages of AMI.

[0010] Myoglobin (MYO) is an oxygen-binding heme protein with a molecular weight of 16.7 kDa. It is mainly distributed in cardiac and skeletal muscle tissue and is in a tightly packed spherical shape. MYO has a sensitivity of 83.3%, a specificity of 88.6%, and a negative predictive value of 99.5% for the diagnosis of acute myocardial infarction (AMI). Numerous studies have reported its clinical utility in the early diagnosis and monitoring of acute coronary syndrome (ACS). A negative MYO result helps rule out myocardial infarction. It can also serve as an early monitoring indicator for recurrence of myocardial injury or expansion of the infarct area.

[0011] With the rapid development of medical testing technology, the above-mentioned items are being applied more and more widely. However, some problems exist in the application process, such as the lack of internal quality control as required, which leads to large deviations in patient test results.

[0012] Given the significant clinical demand for these items, and the fact that they are often combined for testing, there is an urgent need for stable and high-performance composite quality control products to perform quality control. Summary of the Invention

[0013] This invention provides a cardiovascular biomarker control sample and its preparation method. The cardiovascular biomarker control sample has good uniformity and stability, and good real-time stability and reconstitution stability. It can be used as a third-party quality control sample for internal and external quality control of cardiovascular disease screening and auxiliary diagnostic measurement systems.

[0014] The present invention solves its technical problem by adopting the following technical solution:

[0015] A cardiovascular biomarker control product includes a serum matrix, antigen, preservative, and stabilizer;

[0016] The antigens include cardiac troponin I antigen, creatine kinase isoenzyme antigen, myoglobin antigen, N-terminal pro-brain natriuretic peptide antigen, brain natriuretic peptide antigen, D-dimer antigen, heart-type fatty acid-binding protein antigen, myeloperoxidase antigen, and lipoprotein phospholipase A2 antigen.

[0017] The stabilizer includes at least one of D-trehalose, sucrose, glycine, inositol, and polyethylene glycol.

[0018] The inventors of this invention discovered that the antigens have poor stability, especially myoglobin antigen (MYO), creatine kinase isoenzyme antigen (CK-MB), and cardiac troponin I antigen (cTnI), which are very unstable, resulting in substandard quality and inability to be stored for a long time.

[0019] The cardiovascular biomarker control products described in this invention cover a comprehensive range of detection items, encompassing all cardiovascular disease detection items (including those related to heart failure, pulmonary embolism, vasculitis and atherosclerosis, myocardial injury, and myocardial infarction). The quality control products described in this invention exhibit good homogeneity and stability, demonstrating excellent real-time stability and reconstitution stability. They can be used as third-party quality control products for internal and external quality control of cardiovascular disease screening and auxiliary diagnostic measurement systems.

[0020] The addition of the stabilizer to the antigen and serum systems described above effectively improves the stability and uniformity of the quality control products.

[0021] In a preferred embodiment of the present invention, the concentration of the cardiac troponin I antigen is 0.5~20 ng / mL;

[0022] The concentration of the creatine kinase isoenzyme antigen is 10~50 ng / mL;

[0023] The concentration of the myoglobin antigen is 50~250 ng / mL;

[0024] The concentration of the N-terminal pro-brain natriuretic peptide antigen is 200~25000 pg / mL;

[0025] The concentration of the brain natriuretic peptide antigen is 100~1000 pg / mL;

[0026] The concentration of the D-dimer antigen is 0.2~2 mg / L;

[0027] The concentration of the heart-type fatty acid-binding protein antigen is 10~50 ng / mL;

[0028] The concentration of the myeloperoxidase antigen is 200-2000 pmol / L;

[0029] The concentration of the lipoprotein phospholipase A2 antigen is 100~500 ng / mL.

[0030] In a preferred embodiment of the present invention, the concentration of the stabilizer is 0.1~20 g / L.

[0031] In a preferred embodiment of the present invention, the stabilizer comprises D-trehalose, inositol, sucrose and glycine, wherein the concentration of D-trehalose is 3-12 g / L, the concentration of inositol is 1-2 g / L, the concentration of sucrose is 0-0.5 g / L, and the concentration of glycine is 0-0.5 g / L.

[0032] As a preferred embodiment of the present invention, the preservative is at least one of Proclin 300, penicillin, streptomycin, gentamicin, and NaN3.

[0033] In a preferred embodiment of the present invention, the concentration of the preservative is 0.1~0.5wt%.

[0034] In a preferred embodiment of the present invention, the serum matrix includes at least one of human serum and animal serum.

[0035] This invention also provides a method for preparing a cardiovascular biomarker control product, comprising the following steps:

[0036] (1) Select serum matrix that is clear and free of precipitate;

[0037] (2) Add cardiac troponin I antigen to the serum matrix to the set concentration and let stand; add creatine kinase isoenzyme antigen, myoglobin antigen, N-terminal pro-brain natriuretic peptide antigen, brain natriuretic peptide antigen, D-dimer antigen, heart-type fatty acid-binding protein antigen, myeloperoxidase antigen and lipoprotein phospholipase A2 antigen to the set concentration.

[0038] (3) Add stabilizer and preservative, stir well, and let stand;

[0039] (4) Freeze-dry to obtain cardiovascular marker control products.

[0040] In a preferred embodiment of the present invention, the settling temperature in step (2) is 20~40℃ and the settling time is 24~48h.

[0041] In a preferred embodiment of the present invention, the settling temperature in step (3) is 2~8℃ and the settling time is 12~24h.

[0042] The beneficial effects of this invention are as follows: The cardiovascular biomarker control products described in this invention cover a comprehensive range of detection items, encompassing all cardiovascular disease detection items (including those related to heart failure, pulmonary embolism, vasculitis and atherosclerosis, myocardial injury, and myocardial infarction). The quality control products described in this invention exhibit good homogeneity and stability, demonstrating excellent real-time stability and reconstitution stability. They can be used as third-party quality control products for internal and external quality control of cardiovascular disease screening and auxiliary diagnostic measurement systems. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0045] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0046] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0047] Unless otherwise specified, all reagents or instruments used in this invention are commercially available products. Unless otherwise specified, the raw materials used in each comparative example and the parallel experiments of each embodiment are the same commercially available products.

[0048] Example

[0049] The types and concentrations of antigens, preservatives, and stabilizers in formulations 1-3 are shown in Table 1. The low and medium concentration levels of antigens are represented by C1 and C2, respectively.

[0050] The preparation methods for cardiovascular marker control products in formulations 1-3 all include the following steps:

[0051] (1) Select serum matrix that is clear and free of precipitate;

[0052] (2) Add cardiac troponin I antigen to the serum matrix to the set concentration and let it stand at 25°C for 24 hours; add creatine kinase isoenzyme antigen, myoglobin antigen, N-terminal pro-brain natriuretic peptide antigen, brain natriuretic peptide antigen, D-dimer antigen, heart-type fatty acid-binding protein antigen, myeloperoxidase antigen and lipoprotein phospholipase A2 antigen to the set concentration.

[0053] (3) Add stabilizer and preservative, stir well, and let stand at 5°C for 24 hours;

[0054] (4) Freeze-dry to obtain cardiovascular marker control products.

[0055] Table 1

[0056]

[0057] Comparison concentration level 1

[0058] Compared with concentration level 1 of formulation 1, the procedure of standing at 25°C for 24 hours in step S2 was cancelled, while the rest was the same as concentration level 1. The results showed that the stability of the quality control product did not meet the requirements.

[0059] Comparison concentration level 2

[0060] Compared to concentration level 2 of formulation 1, step S3, which involved allowing the quality control solution to stand at 5°C for 24 hours, was omitted. The results showed that the homogeneity of the quality control solution did not meet the requirements.

[0061] Test case

[0062] 1. Reconstitute the lyophilized powders prepared at various concentration levels to obtain control solutions of cardiovascular markers, and then conduct homogeneity performance testing. The requirement for homogeneity performance is: inter-bottle coefficient of variation (CV) < 10%.

[0063] Ten quality control samples were randomly selected from the overall sample for homogeneity testing and randomly numbered 1-10. The quality control samples were measured using a calibrated testing system. Each quality control sample was measured three times. Considering the random variation of the measurement system over time and other factors, the three measurements were performed in different orders, for example, 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.

[0064] Table 2 records the test results. The data processing and judgment criteria for the homogeneity test results refer to the People's Republic of China pharmaceutical industry standard YY / T1652-2019 "General Technical Requirements for Quality Control Materials for In Vitro Diagnostic Reagents". The test results are shown in Table 2.

[0065] Table 2

[0066]

[0067] As can be seen from the test results in Table 2, the homogeneity of the solution after reconstitution of the lyophilized powder is good.

[0068] 2. Test the stability of the lyophilized powder after reconstitution:

[0069] Stability performance requirements: The reconstituted quality control sample should be stable for 4 weeks when stored at 5°C in the dark, stable for 1 day at 25°C, and able to withstand at least 3 freeze-thaw cycles at -18°C. At the end of the stability period, the relative deviation between the quality control sample and the newly reconstituted quality control sample should be within ±10% of the labeled value.

[0070] Test method: After reconstitution, the quality control samples were stored and freeze-thawed under the conditions specified above. After the storage period, they were taken out for testing. Three replicates were performed for each reconstituted quality control sample under each condition, and three replicates were also performed for the newly reconstituted quality control sample as a control. After testing, the average values ​​of the test results after reconstitution and at the end of the stabilization period were calculated, and the relative deviation between the test results at the end of the stabilization period and the newly reconstituted sample was calculated. The test results are shown in Tables 3-5.

[0071] Table 3

[0072]

[0073] Table 4

[0074]

[0075] Table 5

[0076]

[0077] 3. Real-time stability test: The real-time stability requirements are: it should be stable for 18 months at 5℃, and the relative deviation between the test results of the quality control sample and the newly reconstituted quality control sample at the end of the stability period should be within ±10% of the labeled value.

[0078] Test method: After reconstitution upon opening, the quality control sample was tested three times. It was stored at 5°C in the dark, and tested three times at the end of the stability period for each quality control item. The average value of the test results after reconstitution and at the end of the stability period was calculated, and the relative deviation between the results at the end of the stability period and the newly reconstituted test results was also calculated. The test results are shown in Tables 6 to 8.

[0079] Table 6

[0080]

[0081] Table 7

[0082]

[0083] Table 8

[0084]

[0085] As can be seen from the test results in Tables 3 to 8, the quality control products of the present invention exhibit good reconstitution stability and real-time stability.

[0086] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A cardiovascular biomarker control product, characterized in that, It consists of serum matrix, antigen, preservative and stabilizer; The antigen is composed of cardiac troponin I antigen, creatine kinase isoenzyme antigen, myoglobin antigen, N-terminal pro-brain natriuretic peptide antigen, brain natriuretic peptide antigen, D-dimer antigen, heart-type fatty acid-binding protein antigen, myeloperoxidase antigen and lipoprotein phospholipase A2 antigen. The concentration of the cardiac troponin I antigen is 1 ng / mL; The concentration of the creatine kinase isoenzyme antigen is 10 ng / mL; The concentration of the myoglobin antigen is 60 ng / mL; The concentration of the N-terminal pro-brain natriuretic peptide antigen is 3000 pg / mL; The concentration of the brain natriuretic peptide antigen is 100 pg / mL; The concentration of the D-dimer antigen is 0.5 mg / L; The concentration of the heart-type fatty acid-binding protein antigen is 10 ng / mL; The concentration of the myeloperoxidase antigen is 500 pmol / L; The concentration of the lipoprotein phospholipase A2 antigen is 200 ng / mL; The stabilizer is composed of D-trehalose, inositol, sucrose and glycine, wherein the concentration of D-trehalose is 9 g / L, the concentration of inositol is 2 g / L, the concentration of sucrose is 0.5 g / L and the concentration of glycine is 0.5 g / L. The method for preparing the cardiovascular marker control product includes the following steps: (1) Select serum matrix that is clear and free of precipitate; (2) Add cardiac troponin I antigen to the serum matrix to the set concentration and let it stand at 25°C for 24 hours; add creatine kinase isoenzyme antigen, myoglobin antigen, N-terminal pro-brain natriuretic peptide antigen, brain natriuretic peptide antigen, D-dimer antigen, heart-type fatty acid-binding protein antigen, myeloperoxidase antigen and lipoprotein phospholipase A2 antigen to the set concentration. (3) Add stabilizer and preservative, stir well, and let stand at 5°C for 24 hours; (4) Freeze-dry to obtain cardiovascular marker control products.

2. The cardiovascular marker control product according to claim 1, characterized in that, The preservative is at least one of Proclin 300, penicillin, streptomycin, gentamicin, and NaN3.

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

4. 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.

5. The method for preparing the cardiovascular marker control product according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Select serum matrix that is clear and free of precipitate; (2) Add cardiac troponin I antigen to the serum matrix to the set concentration and let it stand at 25°C for 24 hours; add creatine kinase isoenzyme antigen, myoglobin antigen, N-terminal pro-brain natriuretic peptide antigen, brain natriuretic peptide antigen, D-dimer antigen, heart-type fatty acid-binding protein antigen, myeloperoxidase antigen and lipoprotein phospholipase A2 antigen to the set concentration. (3) Add stabilizer and preservative, stir well, and let stand at 5°C for 24 hours; (4) Freeze-dry to obtain cardiovascular marker control products.

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