A composite quality control product for liver function biochemistry, its preparation method and application

Abstract

The present invention discloses a liver function biochemical composite quality control product and its preparation method and application. The quality control product includes 12 analyte raw materials and the following non-analyte raw materials: 5-12 g / L sodium chloride, 0.5-2 g / L casein sodium salt, 5-20 g / L mannitol, 0.05-0.2% Tween 20, 1-20 mg / L leupeptin, 10-60 g / L trehalose and 0.05-0.2% PC300; matrix liquid: 20-80% quality control product matrix liquid and 20-80% buffer. The liver function biochemical composite quality control product of the present invention has a high degree of complexity, contains 11 important liver function biochemical indicators, and performs well in terms of component homogeneity and stability, ensuring the precision of various indicators and meeting the needs of daily quality control.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical detection reagents, and in particular to a liver function biochemical composite quality control product and a preparation method and application thereof. Background Art

[0002] Clinical testing is the qualitative or quantitative analysis of specimens such as the patient's blood, body fluids, secretions, excretions and exfoliated materials through visual observation or the use of experimental methods such as physics, chemistry and biology to obtain objective data reflecting the body's functional state, pathological changes or causes of disease, and emphasizes the adoption of strict quality management measures throughout the entire testing process (before, during and after analysis) to ensure the quality of the test, thereby providing valuable experimental data for clinicians and patients.

[0003] Quality control products used in clinical practice are divided into two categories, namely single-item quality control products and composite quality control products. Single-item quality control products need to be used separately according to the test items, but the laboratory has many items, which increases the workload of quality control; composite quality control products contain target substances for multiple test items, and a single bottle controls multiple items, making the material procurement, storage, and use process more convenient, greatly improving work efficiency, and are more popular than single-item quality control products. However, the current preparation of composite quality control products has the following defects: (1) Composite quality control products need to consider the compatibility between multiple analytes and protective agents, and the requirements for the formula are high; (2) The stability conditions of different analytes are different. Most analytes are enzymes and proteins, which have high requirements for the production environment and storage environment. Therefore, in order to increase the stability of quality control products and extend the shelf life of the products, preservatives and stabilizers are often added. These complex ingredients will further increase the risk of enzyme degradation; (3) Various analytes naturally exist in healthy human serum. The basic concentration of some analytes is higher than the expected concentration of normal level quality control products, and cannot be used for the preparation of normal level quality control products. Although low-concentration quality control products can be prepared without using human serum, the matrix is ​​quite different from the actual clinical samples, which may cause matrix effects and lead to deviations in the analysis results.

[0004] In addition, since the biochemical detection items of liver function contain multiple enzymes and the storage conditions are relatively strict, it is quite difficult to keep the analytes of multiple detection items stable at the same time when preparing composite quality control. Therefore, most of the liver function quality control products used in clinical practice are single quality control products, or biochemical composite quality control products contain some liver function items. In the latter, on the one hand, the composite quality control products contain few types of liver function items and cannot meet the clinical quality control requirements for liver function items. On the other hand, since they contain other detection items, the cost of quality control products is increased. Therefore, providing a stable formula and preparation process for liver function biochemical composite quality control products can avoid the above defects and bring convenience to clinical use and management. Summary of the invention

[0005] In view of the defects in the prior art, the present invention proposes a liver function biochemical composite quality control product and a preparation method and application thereof.

[0006] The present invention uses human serum as a matrix to simulate clinical samples, and uses a verified non-analyte formula to blend multiple components together, thereby increasing the stability of the composite quality control product, and can prepare quality control products with normal and abnormal levels. In order to reduce the difficulty of storage, a freeze-drying process is used to prepare the quality control product into a powder, which is convenient for transportation and storage.

[0007] The present invention provides a liver function biochemical composite quality control product, comprising the following components:

[0008] (1) Matrix solution: 20~80% quality control matrix solution and 20~80% buffer solution;

[0009] (2) Analyte materials: 14~65 U / L alanine aminotransferase, 14~65 U / L aspartate aminotransferase, 21~97.5 U / L γ-glutamyl transferase, 84~520 U / L alkaline phosphatase, 2800~13000 U / L cholinesterase, 7~39 mol / L direct bilirubin, 7~39 mol / L indirect bilirubin, 21~91 g / L albumin, 49~130 g / L total protein, 21~104 U / L α-L-fucosidase and 17.5~78 U / L adenosine deaminase;

[0010] (3) Non-analyte materials: 5-12 g / L sodium chloride, 0.5-2 g / L casein sodium salt, 5-20 g / L mannitol, 0.05-0.2 v / v % Tween 20, 1-20 mg / L leupeptin, 10-60 g / L trehalose and 0.05-0.2 v / v % PC300.

[0011] A certain amount of sodium chloride is added to the composite quality control product of the present invention, which helps to maintain the stability and activity of the composite enzyme and improve the ionic environment of the liquid, thereby preventing the aggregation or precipitation of proteins or enzymes. Casein sodium salt has a certain stabilizing effect and can bind to the enzyme in the analyte to prevent it from denaturing or inactivating.

[0012] In the composite quality control product of the present invention, mannitol is used as a protective agent to reduce the structural damage of the analyte caused by water loss or crystallization by maintaining the hydration state of the analyte. Mannitol also has antioxidant properties and can reduce the damage of oxidation to enzymes or proteins.

[0013] Alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, etc. in liver function analytes are usually susceptible to attack by proteases and degraded, resulting in loss of activity or function. The present invention inhibits the activity of these degrading enzymes by adding a certain amount of leupeptin, thereby reducing the risk of analytes being degraded, thereby improving their stability in quality control products. In addition, the addition of leupeptin can effectively inhibit the nonspecific degradation reaction of quality control products, ensure the stability of different analytes in the same system, and reduce the concentration changes of analytes caused by enzyme degradation.

[0014] Liver function biochemical detection items include more than ten kinds of enzymes, and the analytes contain different types of proteins and enzymes, which are easy to interact in the composite system and affect each other's stability. The liver function biochemical composite quality control product of the present invention is a stable combination as a whole, in which the various components complement each other. Under a specific range of concentrations, the liver function biochemical composite quality control product has excellent opening stability, freezing stability, accelerated stability, and good uniformity.

[0015] Furthermore, the control matrix fluid was negative in four tests: hepatitis B surface antigen (HBsAg), hepatitis C (HCV) antibody, human immunodeficiency virus (HIV 1 / 2) antibody and Treponema pallidum (TP).

[0016] Furthermore, the matrix liquid of the quality control product is human serum. Human serum contains many biologically active substances. Using human serum as a matrix liquid can provide a basis for the quality control product that is close to the real physiological environment, so that the stability of the quality control product and the test results are closer to the actual in vivo situation. The non-analyte raw material of the present invention can avoid the non-specific binding of the biochemical detection index using human serum as a matrix liquid, provide a protective effect, and reduce the denaturation or aggregation of the enzyme.

[0017] Furthermore, the liver function biochemical composite quality control product is a lyophilized powder. The quality control product of the present invention can be stored in the form of a lyophilized powder, and the biologically active components in the quality control product can be restored to a state close to that before lyophilization after reconstitution, maintaining its original activity. The lyophilized quality control product can be quickly and conveniently dissolved when needed to form a high-concentration solution.

[0018] Furthermore, the buffer is a phosphate buffer or a Tris-hydrochloric acid buffer.

[0019] The present invention also provides a method for preparing the liver function biochemical composite quality control product, comprising the following steps:

[0020] S1: Mix the quality control matrix solution and buffer solution in proportion to prepare the matrix solution;

[0021] S2: adding non-analyte raw materials and adjusting the pH to 7.2-7.4 to obtain a first mixed solution;

[0022] S3: adding analyte raw materials, adjusting the pH to 7.2-7.4, and obtaining a second mixed solution;

[0023] S4: Filter the second mixed solution, dispense into vials, and perform vacuum freeze-drying to obtain a liver function biochemical composite quality control product.

[0024] The present invention also provides a kit, which comprises the liver function biochemical composite quality control product.

[0025] The present invention also provides the use of the liver function biochemical composite quality control product in preparing a quality control product for liver function detection.

[0026] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0027] 1. The liver function biochemical composite quality control product of the present invention is highly complex, including 11 important liver function biochemical indicators, and excels in component homogeneity and stability, ensuring the precision of various indicators; using human serum as a matrix, it can simulate clinical samples, and using a verified non-analyte formula to blend multiple components together, thereby increasing the stability of the composite quality control product, and quality control products with normal and abnormal levels can be prepared.

[0028] 2. The present invention optimizes the components of non-analyte raw materials to avoid enzyme degradation caused by the addition of preservatives, stabilizers and other substances. The composite quality control product has good stability after opening the bottle and can maintain effective activity in the use environment, thereby reducing the cost and operational inconvenience caused by frequent replacement of quality control products and meeting the needs of daily diagnosis and monitoring.

[0029] 3. The liver function biochemical composite quality control product of the present invention has good freeze-thaw stability and can maintain the stability and activity of the product after undergoing the freezing or thawing process, ensuring that it can still meet the high-precision quality control requirements under different usage conditions. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0031] The present invention provides a liver function biochemical composite quality control product, which includes 11 important liver function biochemical indicators. The compatibility of the composite quality control product is ensured by compounding non-analyte raw materials such as leupeptin, sodium chloride (NaCl), casein sodium salt, mannitol and Tween 20. By optimizing the addition amount of each component of the quality control product, the stability and product shelf life of the composite quality control product are maximized to meet the needs of actual production.

[0032] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0033] Example 1 Preparation of liver function biochemical composite quality control product

[0034] The preparation process of the liver function biochemical composite quality control product in this example is as follows:

[0035] 50% of the quality control matrix solution and 50% of 50 mM Tris-HCl buffer were mixed to prepare the matrix solution and test the basic concentration of each analyte. The quality control matrix solution (human serum) was from Sera Care, USA.

[0036] By calculation, non-analyte raw materials were added in proportion, so that the concentration of non-analyte in the solution was as follows: 9 g / L NaCl, 1 g / L casein sodium salt, 10 g / L mannitol, 0.1% Tween 20, 15 mg / L leupeptin, 50 g / L trehalose, 0.15% PC300, and the pH was adjusted to 7.3±0.1.

[0037] By calculation, the analyte raw material (purchased natural purified raw material or recombinant protein) is added in proportion to make the analyte concentration in the solution as follows:

[0038] (1) Level 1: 20 U / L alanine aminotransferase, 20 U / L aspartate aminotransferase, 30 U / L γ-glutamyl transferase, 120 U / L alkaline phosphatase, 4000 U / L cholinesterase, 12 μmol / L ditaurobilirubin sodium (direct bilirubin), 12 μmol / L indirect bilirubin, 30 g / L albumin, 70 g / L total protein, 30 U / L α-L-fucosidase, and 25 U / L adenosine deaminase.

[0039] (2) Level 2: 50 U / L alanine aminotransferase, 50 U / L aspartate aminotransferase, 65 U / L γ-glutamyl transferase, 400 U / L alkaline phosphatase, 10,000 U / L cholinesterase, 30 μmol / L ditaurobilirubin sodium (direct bilirubin), 30 μmol / L indirect bilirubin, 70 g / L albumin, 100 g / L total protein, 80 U / L α-L-fucosidase, and 60 U / L adenosine deaminase.

[0040] Stir to prepare an aqueous solution, filter the solution into a clean container using a 0.2 μm filter membrane, and dispense into brown glass bottles with a specification of 3 mL / bottle.

[0041] Freeze-dry according to the freeze-drying curve in Table 1:

[0042] Table 1 Freeze-drying procedure for liver function biochemical composite quality control products

[0043]

[0044] Take out the freeze-dried liver function biochemical composite quality control product and store it at 2~8℃ away from light.

[0045] Example 2

[0046] The difference from Example 1 is that in the matrix solution preparation process in Example 1, the Tris-HCl buffer is replaced with PBS buffer.

[0047] Example 3

[0048] The difference from Example 1 is that in the non-analyte raw material feeding process in Example 1, the non-analyte concentrations are as follows: 9 g / L NaCl, 0.2 g / L casein sodium salt, 20 g / L mannitol, 0.1% Tween 20, 15 mg / L leupeptin, 40 g / L trehalose, and 0.15% PC300.

[0049] Example 4

[0050] The difference from Example 1 is that in the non-analyte raw material feeding process in Example 1, the non-analyte concentrations are as follows: 9 g / L NaCl, 1.5 g / L casein sodium salt, 10 g / L mannitol, 0.1% Tween 20, 10 mg / L leupeptin, 50 g / L trehalose, and 0.15% PC300.

[0051] Comparative Example 1

[0052] The difference from Example 1 is that in the matrix solution preparation process, 90% human serum and 10% 50 mM Tris-HCl buffer are mixed to prepare the matrix solution.

[0053] Comparative Example 2

[0054] The difference from Example 1 is that during the feeding process of non-analyte raw materials, the NaCl concentration is adjusted to 2 g / L.

[0055] Comparative Example 3

[0056] The difference from Example 1 is that during the feeding process of non-analyte raw materials, the concentration of casein sodium salt is adjusted to 1 g / L.

[0057] Comparative Example 4

[0058] The difference from Example 1 is that during the feeding process of the non-analyte raw materials, the concentration of mannitol was adjusted to 30 g / L.

[0059] Comparative Example 5

[0060] The difference from Example 1 is that during the feeding process of non-analyte raw materials, the concentration of Tween 20 was adjusted to 30 mg / L.

[0061] Comparative Example 6

[0062] The difference from Example 1 is that during the feeding process of non-analyte raw materials, aprotinin is not added.

[0063] Biochemical detection was performed on the Mindray platform, and the target value range is shown in Table 2:

[0064] Table 2 Target value range

[0065]

[0066] Test Example 1 Determination of the basic concentration of matrix solution

[0067] After preparing the matrix solution, the basic concentration of each analyte was tested, as shown in Table 3:

[0068] Table 3 Basic concentration of analytes in Examples 1-2 and Comparative Example 1

[0069]

[0070] As shown in Table 3, compared with the expected target value, the measured values ​​of Example 1 and Example 2 are lower, closer to the expected requirements, and can be used as raw materials for preparing composite quality control products. The predicted value of Comparative Example 1 is too high, in which alanine aminotransferase, aspartate aminotransferase, and albumin are all higher than the expected target value, and cannot be used as a raw material for preparing composite quality control products.

[0071] Test Example 2: Uniformity Test

[0072] The freeze-dried quality control products prepared in the examples and comparative examples were equilibrated at room temperature for 30 min, 3 mL of distilled water was added to each bottle to dissolve and mix, and the uniformity between the bottles was tested, that is, 10 bottles were randomly selected, each was tested once, and the CV value was calculated. The results are shown in Table 4:

[0073] Table 4 Homogeneity test results

[0074]

[0075] From the results in Table 4, it can be seen that the CV between bottles of Examples and Comparative Examples 2 to 5 is less than 3%, and the uniformity between bottles is good. The CV value of total bilirubin in Comparative Example 6 is greater than 3%, indicating that the uniformity of the quality control product without the addition of leupeptin is poor.

[0076] Test Example 3 2~8℃ stability test after opening the bottle

[0077] The liver function biochemical composite quality control freeze-dried powder prepared in the examples and comparative examples was equilibrated at room temperature for 30 min, 3 mL of distilled water was added to each bottle to dissolve and mix, and the deviation of the test results on the Mindray platform after being stored at 2-8°C in the dark for 7 days was shown in Table 5:

[0078] Table 5 Deviation of the test results of the quality control products after being stored at 2~8℃ and away from light for 7 days (%)

[0079]

[0080] After the dry powder was dissolved, it was stored at 2-8°C for 7 days. The test results of Examples 1-4 deviated from those of day 0 by less than 5%, while the deviation of the quality control products prepared in Comparative Examples 2-6 was greater than 5%, among which the deviation of the quality control products in Comparative Example 6 was mostly higher than 10%, which were higher than those in the examples, indicating that the input concentrations of NaCl, casein sodium salt, mannitol, Tween 20 and leupeptin had a great influence on the opening stability of the product, among which the addition of leupeptin had the greatest influence on the stability of the quality control product at 2-8°C.

[0081] Test Example 4 -20℃ stability test after opening the bottle

[0082] The liver function biochemical composite quality control freeze-dried powder prepared in the examples and comparative examples was equilibrated at room temperature for 30 minutes, 3 mL of distilled water was added to each bottle to dissolve and mix, and the deviation of the test results after being stored at -20°C and protected from light for 20 days compared with the test results on the Mindray platform on the same day is shown in Table 6:

[0083] Table 6 Stability test results of quality control products after storage at -20℃ in the dark for 20 days

[0084]

[0085] After the dry powder was dissolved, it was stored at -20°C for 20 days. The test results of Examples 1 to 4 deviated from those of 0 day by less than 5%, while the deviations of Comparative Examples 2, 3, 5, and 6 were significantly higher than those of the Examples, indicating that the input concentrations of NaCl, casein sodium salt, Tween 20, and leupeptin have a significant effect on the frozen storage stability of the product after opening the bottle.

[0086] Test Example 5 Freeze-thaw stability test

[0087] The liver function biochemical composite quality control freeze-dried powder prepared in the examples and comparative examples was equilibrated at room temperature for 30 min, 3 mL of distilled water was added to each bottle to dissolve and mix, and freeze-thaw stability test was performed. The deviation of the test results on the Mindray platform after repeated freeze-thaw for 3 times and the non-freeze-thawed samples is shown in Table 7:

[0088] Table 7 Deviation value of composite quality control product after repeated freezing and thawing for 3 times (%)

[0089]

[0090] After the dry powder was dissolved, it was frozen and thawed three times repeatedly. The test results of Examples 1 to 4 deviated from the 0-day results by less than 5%, while the deviations of Comparative Examples 4, 5, and 6 were significantly higher than those of the Examples, indicating that the input concentrations of mannitol, Tween 20, and leupeptin have a significant effect on the freeze-thaw stability of the product after opening the bottle.

[0091] Test Example 6 Accelerated Stability Test

[0092] The liver function biochemical composite quality control freeze-dried powder prepared in the examples and comparative examples was placed at 37° C. for 7 days, 3 mL of distilled water was added to each bottle to dissolve and mix, and then tested. The results are shown in Table 8:

[0093] Table 8 Deviation of results after 7 days at 37°C (%)

[0094]

[0095] The lyophilized powder was treated at 37°C for 7 days. The test results of Examples 1 to 4 deviated from those at day 0 by less than 5%, while the deviations of Comparative Examples 3, 4, and 6 were significantly higher than those of the Examples, indicating that the concentrations of casein sodium salt, mannitol, and leupeptin have a significant effect on the accelerated stability of the product after opening the bottle.

[0096] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a liver function biochemical composite quality control product, characterized in that: The steps include: S1: Prepare matrix solution and test the basic concentration of each analyte; S2: adding non-analyte raw materials in proportion through calculation, adjusting the pH to 7.2-7.4, and obtaining a first mixed solution; S3: by calculation, adding analyte raw materials in proportion so that the analyte concentration in the solution is as shown in (1) or (2), adjusting the pH to 7.2-7.4, and obtaining a second mixed solution; (1) 20 U / L alanine aminotransferase, 20 U / L aspartate aminotransferase, 30 U / L γ-glutamyl transferase, 120 U / L alkaline phosphatase, 4000 U / L cholinesterase, 12 mol / L direct bilirubin, 12 mol / L indirect bilirubin, 30 g / L albumin, 70 g / L total protein, 30 U / L α-L-fucosidase and 25 U / L adenosine deaminase; (2) 50 U / L alanine aminotransferase, 50 U / L aspartate aminotransferase, 65 U / L γ-glutamyl transferase, 400 U / L alkaline phosphatase, 10,000 U / L cholinesterase, 30 mol / L direct bilirubin, 30 mol / L indirect bilirubin, 70 g / L albumin, 100 g / L total protein, 80 U / L α-L-fucosidase and 60 U / L adenosine deaminase; S4: filtering the second mixed solution, dispensing into vials, and performing vacuum freeze drying to obtain a liver function biochemical composite quality control product; Wherein, the matrix solution described in S1 is: 50 v / v% quality control matrix solution and 50 v / v% buffer solution; S2: non-analyte raw materials are: 5-12 g / L sodium chloride, 1-1.5 g / L casein sodium salt, 5-20 g / L mannitol, 0.05-0.2 v / v % Tween 20, 15 mg / L leupeptin, 10-60 g / L trehalose and 0.05-0.2 v / v % PC300; The liver function biochemical composite quality control product is a lyophilized powder; The quality control matrix liquid is human serum; The buffer is a phosphate buffer or a Tris-hydrochloric acid buffer.

2. The preparation method according to claim 1, characterized in that: The quality control matrix fluid was negative in four tests: hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus antibody and syphilis treponema.

3. A kit, characterized in that: A liver function biochemical composite quality control product obtained by the preparation method according to claim 1 or 2.

4. Use of the liver function biochemical composite quality control product obtained by the preparation method according to claim 1 or 2 in the preparation of quality control products for liver function testing.

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