Preparation method of special biochemical composite quality control product

Abstract

The invention belongs to the technical field of biochemical detection, and particularly relates to a preparation method of a special biochemical composite quality control product. The items of the composite quality control product comprise alpha-L-fucosidase (AFU), N-acetyl-beta-D-glucosaminidase (NAG), aspartate aminotransferase mitochondrial isoenzyme (mAST), lactic dehydrogenase isoenzyme 1 (LDH1), glycocholic acid (CG), glutathione reductase (GR) and creatine kinase MB isoenzyme (CK-MB), and the enzymes are rapidly released in a heating and pH adjusting mode, so that the quality control product is obtained. The extraction efficiency of the target enzyme is remarkably improved, the product is purified in a differential centrifugation mode, impurities are removed at a low rotating speed, the product is collected at a high rotating speed, most impurities are removed only through simple centrifugation, the enzyme purification work time and the material cost are greatly saved, the target enzyme is further purified by changing the salt concentration in the chromatography process, and the extraction efficiency of the target enzyme is greatly improved. And an accelerated test is combined to destroy the activity of impurity enzymes, so that the target enzyme with relatively high purity is obtained in a simple and low-cost manner.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical detection, and specifically relates to a preparation method of a special biochemical composite quality control product. Background Technique

[0002] α-L-fucosidase (AFU) is a lysosomal acid hydrolase, which is increased in the serum of patients with primary liver cancer and is one of the markers of primary liver cancer. N-acetyl-β-D-glucosidase (NAG) is a marker of renal tubular injury, and an increase in urinary NAG activity is a relatively sensitive indicator of kidney damage. The determination of NAG activity can be used for the early diagnosis of tubulointerstitial nephritis, urinary tract infection, diabetic nephrotic syndrome, hypertensive nephropathy, rejection after kidney transplantation, and nephrotic syndrome. Aspartate aminotransferase isoenzyme (mAST) accounts for 20% of human aspartate aminotransferase (AST). An increase in serum mAST can reflect the degree of damage to subcellular structures and become a sensitive indicator for diagnosing hepatocyte damage and myocardial infarction. In addition, it can also be used to evaluate the degree of damage to hepatocytes and cardiomyocytes, clinical efficacy, and prognosis judgment in clinical diagnosis. Lactate dehydrogenase isoenzyme 1 (LDH1) has important diagnostic significance in clinical practice and is mainly used for the determination of myocardial enzymes, especially for the diagnosis of acute myocardial infarction. Serum cholyglycine (CG) is one of the conjugated bile acids formed by the combination of cholic acid and glycine, and glycocholic acid is the main bile acid component in the serum in the third trimester of pregnancy. When hepatocytes are damaged, the ability of hepatocytes to uptake CG decreases, resulting in an increase in the content of CG in the blood. Glutathione reductase (GR) is one of the most important enzymes in the human redox system and is the main flavoenzyme that maintains the content of reduced glutathione (GSH) in cells. Its increase is generally related to liver diseases. Creatine kinase-MB isoenzyme (CK-MB) is mainly present in cardiomyocytes. It is released into the blood during myocardial injury and is therefore commonly used for the diagnosis and monitoring of myocardial diseases, especially myocardial infarction. The above seven items are different from conventional biochemical examination items and belong to special biochemical examination items.

[0003] Currently, single-item quality control products are generally available on the market, and the price of pure enzymes required for compound items is extremely expensive. Therefore, a preparation method that can reduce costs and accurately control the content of each item in the composite quality control product is proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of a special biochemical composite quality control product, so as to solve the problem that in the current market, generally single-item quality control products are available, and the price of pure enzymes required for composite items is extremely expensive as mentioned in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: A preparation method of a special biochemical composite quality control product, the items of the composite quality control product include: α-L-fucosidase (AFU), N-acetyl-β-D-glucosaminidase (NAG), mitochondrial isoenzyme of aspartate aminotransferase (mAST), lactate dehydrogenase isoenzyme 1 (LDH1), glycocholic acid (CG), glutathione reductase (GR), creatine kinase MB isoenzyme (CK-MB), and the preparation method of the composite quality control product is as follows:

[0006] Step 1 Pig liver treatment:

[0007] (1) Take fresh pig liver, chop or cut it into pieces and add it to the dissolution solution to make it reach 1 g of tissue per 10 ml of solution;

[0008] (2) Pour the mixed solution into a homogenizer for homogenization, starting from low speed, alternating between low and high speeds (low speed is 2000 - 4000 rpm, high speed is 10000 - 16000 rpm), 20 s each time, for a total of 100 s;

[0009] (3) Then put the mixed solution into a glassware to mix evenly, adjust the pH, adjust the pH of the mixed solution to 5.0 - 6.0 with glacial acetic acid, heat it to 30 - 40 °C, and maintain it for 0.5 - 2 h to release the target enzyme;

[0010] (4) Centrifuge the mixture at 3000 - 6000 RPM for 30 min, take the supernatant, and discard the precipitate;

[0011] (5) Add solid ammonium sulfate to the supernatant to make it reach 35% saturation, and let the solution stand overnight at 4 °C;

[0012] (6) Centrifuge and discard the precipitate, and adjust the supernatant to 50% saturation by adding solid ammonium sulfate, and let it stand overnight at 4 °C;

[0013] (7) Centrifuge the solution by differential centrifugation method, and take the precipitate of the second centrifugation;

[0014] (8) Dissolve the precipitate in the eluent, ultrafilter and desalt to obtain crude extract 1 containing AFU and NAG;

[0015] Step 2 Beef kidney treatment:

[0016] (9) Chop the bovine kidney tissue and add it to the eluent at 4°C to make the ratio reach 1:5 (mass:volume). Homogenize and let it stand overnight.

[0017] (10) Centrifuge the solution at 4°C for 30 min (3000 - 6000 RPM) and take the supernatant.

[0018] (11) Add solid ammonium sulfate to make it reach 50% saturation and let it stand overnight.

[0019] (12) Centrifuge at 4°C for 30 min (3000 - 6000 RPM), discard the precipitate, and collect the supernatant.

[0020] (13) Add solid ammonium sulfate to the supernatant to make it reach 80% saturation, and then let it stand overnight at 4°C.

[0021] (14) Centrifuge at 4°C for 30 min (3000 - 6000 RPM) and collect the precipitate.

[0022] (15) Redissolve the precipitate in the eluent and then perform ultrafiltration desalting to obtain the crude extract 2 containing mAST and LDH1.

[0023] Step 3 Purification of the crude extract:

[0024] (16) Pass the crude extract 1 and crude extract 2 through an anion separation column respectively. The crude extract 1 yields α-L-fucosidase under an eluent containing 50 mM NaCl with a volume of 4 column volumes, and then the eluent is changed to 100 mM NaCl, and N-acetylglucosaminidase is obtained after elution with a volume of 4 column volumes. The crude extract 2 yields mitochondrial aspartate aminotransferase isoenzyme under the elution of an eluent with a volume of 2 column volumes, and then the eluent is changed to the one containing 100 mM NaCl, and lactate dehydrogenase isoenzyme 1 is obtained after elution with a volume of 4 column volumes.

[0025] (17) Heat-treat the above four obtained substances for 3 days to obtain the final raw materials.

[0026] Step 4 Preparation of the quality control product:

[0027] (18) Mix human serum and glycerol in a ratio of 7:3 and add (3 - 10%) cryoprotectant, metal chelator, (0.5 - 2%) protein protectant, (0.1 - 0.2%) preservative to prepare the quality control product matrix solution, and add the above four self-extracted raw materials and the purchased raw materials for the three items of CG, GR, and CK-MB at the required concentrations to obtain the quality control product.

[0028] (19) Measure the items contained in the quality control product on a biochemical analyzer.

[0029] Preferably, the dissolution solution in step (1) is a mixed solution of sodium acetate and sodium chloride, with a concentration of 50 - 200 mM and a pH of 5.0 - 6.5.

[0030] Preferably, the differential centrifugation method in step (7) is to first centrifuge at 2000 - 4000 RPM for 20 min to remove the precipitate and retain the supernatant; then centrifuge the supernatant at 8000 - 16000 RPM for 20 min, and take the precipitate from the second centrifugation.

[0031] Preferably, the eluent is one of PB, Tris, and HEPES.

[0032] Preferably, the heating temperature in step (17) is 30 - 45 °C.

[0033] Preferably, the cryoprotectant in step (18) is one or both of trehalose and mannitol; the metal chelator is one of EDTA or EGTA; the protein protectant is one or both of BSA or casein; the preservative is one or both of Proclin300 or sodium azide.

[0034] Compared with the prior art, the beneficial effects of the present invention are:

[0035] 1) By heating and adjusting the pH, the enzyme is rapidly released, improving the enzyme extraction efficiency.

[0036] 2) The product is purified by differential centrifugation. The impurities are removed at low speed, and the product is collected at high speed. Most of the impurities are removed only by simple centrifugation, greatly saving the time and material costs of enzyme purification work.

[0037] 3) The target enzyme is further purified by changing the salt concentration in the chromatography process, and combined with the accelerated test to destroy the activity of the impurity enzyme, obtaining a target enzyme with higher purity in a simple and low-cost manner, significantly reducing the difficulty of accurately preparing the quality control product items. Description of the Drawings

[0038] Figure 1 It is a schematic diagram of the preparation process of the composite quality control product of the present invention. Detailed Embodiments

[0039] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0040] Example 1: Optimization Experiment on Enzyme Release Conditions

[0041] During the enzyme extraction process, the degree of enzyme release is crucial for the enzyme extraction efficiency. In this example, the temperature and pH in the enzyme release conditions are mainly optimized to enable a higher degree of release of the enzyme in the extraction tissue within a shorter time.

[0042] (1) Take fresh pig liver, chop or cut it into pieces and add it to the dissolution solution to make it contain 1 g of tissue per 10 ml of solution;

[0043] (2) Pour the mixed solution into a homogenizer for homogenization. Start from low speed and alternate between low and high speeds (low speed is 3000 rpm, high speed is 12000 rpm), 20 s each time, for a total of 100 s;

[0044] (3) Then put the mixed solution into a glassware to mix well, divide it into 5 groups, adjust the pH, and use glacial acetic acid to adjust the pH of the mixed solution to 4.5, 5.0, 5.5, 6.0, and 6.5 respectively. Heat it to 35 °C and maintain for 1 h to release the target enzyme;

[0045] (4) After the time is up, take appropriate samples from each group of solutions respectively, and use a professional biochemical analyzer to measure the concentrations of AFU (α-L-fucosidase) and NAG (N-acetylglucosaminidase) in each group. The results are as follows:

[0046] Table 1 (Example 1)

[0047] pH AFU (U / L) NAG (U / L) 4.5 70.62 82.63 5.0 88.21 125.61 5.5 85.31 132.45 6.0 91.13 128.34 6.5 72.43 95.45

[0048] It can be clearly seen from the data that when the pH value is in the range of 5.0 - 6.0, the concentrations of AFU and NAG are relatively high. This indicates that within this pH value range, the release rate of AFU and NAG can be significantly increased, providing a richer raw material basis for the subsequent extraction and purification of the enzyme. Through in-depth analysis of the experimental data, we can further understand the influence mechanism of the pH value on the enzyme release process, providing strong data support for optimizing the enzyme extraction process.

[0049] Example 2: Differential centrifugation effect comparison experiment

[0050] The purity of the original solution is particularly important during the process of column purification of enzymes. Through differential centrifugation, the target enzyme and impurities are preliminarily separated before column purification, providing a basis for column purification and greatly reducing the concentration of impurities in the final extract. In this example, the content of the target enzyme and impurities in differential centrifugation and direct centrifugation were compared. In Example 1, the mixed solution with pH adjusted to 6.0 was divided into 3 groups. Group 1 was directly centrifuged at 12000 RPM for 20 min. In Group 2, it was first centrifuged at 2000 RPM for 20 min to remove the precipitate, and then centrifuged at 16000 RPM for 20 min, and the precipitate was taken. In Group 3, it was first centrifuged at 4000 RPM for 20 min to remove the precipitate, and then centrifuged at 10000 RPM for 20 min, and the precipitate was taken. The precipitates of the 3 groups were redissolved with the same volume of eluent and measured on the machine. The results are as follows:

[0051] Table 2 (Example 2)

[0052]

[0053] It can be clearly seen from the data comparison that after differential centrifugation in Group 2 and Group 3, the content of impurity enzymes (such as CK-MB, etc.) is significantly lower than that in Group 1 with direct centrifugation. This fully shows that differential centrifugation can effectively remove impurity enzymes in the crude extract, provide a purer original solution for the subsequent column purification process, help improve the purity of the final extract, and thus enhance the quality of the composite quality control product. Through the detailed analysis of the results of different centrifugation methods, we further clarified the important role and advantages of differential centrifugation in the enzyme extraction process.

[0054] Example 3: Experiment on the effect of heating treatment on enzyme purity

[0055] The purity of the enzyme is particularly important for the preparation of the composite quality control. However, column purification cannot completely remove the impurity enzymes in the extract. Therefore, a heating treatment step is added after column purification to further remove the impurity enzymes. In this example, the enzymes obtained by four kinds of column purification were respectively divided into 3 groups and placed in an oven for heating treatment. The temperature of Group 1 was set at 30 °C, the temperature of Group 2 was set at 37 °C, and the temperature of Group 3 was set at 45 °C. After three days of treatment, the enzyme concentrations in the extracts before and after were compared. The results are as follows:

[0056] Table 3 (AFU extract in Example 3)

[0057]

[0058] Table 4 (NAG extract in Example 3)

[0059]

[0060] Table 4 (mAST extract in Example 3)

[0061]

[0062]

[0063] Table 5 (LDH1 extract of Example 3)

[0064]

[0065] By comparing the data before and after treatment, it can be found that after heating treatment at 30 - 45 °C, the content of impurity enzymes (such as CG, GR, CK - MB, etc.) is significantly reduced. This fully proves that the step of heating treatment after column purification can effectively further remove impurity enzymes, improve the purity of the enzyme, thus providing better raw materials for the preparation of composite quality control products and enhancing the performance and quality stability of composite quality control products. Through the detailed analysis of the heating treatment results of different extracts at different temperatures, we provide more comprehensive data basis for optimizing the enzyme purification process.

[0066] The above shows and describes the basic principles, main features and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above - mentioned exemplary embodiments, and without departing from the spirit or basic features of the present invention, the present invention can be implemented in other specific forms; therefore, from any point of view, the embodiments should be regarded as exemplary and non - restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention, and any reference signs in the claims should not be regarded as limiting the claims involved.

[0067] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a special biochemical composite quality control product, the composite quality control product includes: α-L-fucosidase (AFU), N-acetyl-β-D-glucosaminidase (NAG), mitochondrial isoenzyme of aspartate aminotransferase (mAST), lactate dehydrogenase isoenzyme 1 (LDH1), glycocholic acid (CG), glutathione reductase (GR), creatine kinase MB isoenzyme (CK-MB), and the preparation method of the composite quality control product is as follows: Step 1: Pig liver processing: (1) Take fresh pig liver and chop or cut it into small pieces and add it to the dissolving solution so that 1g of tissue is contained in every 10ml of solution; (2) Pour the mixed solution into a homogenizer and homogenize, starting from low speed, alternating between low and high speed (low speed is 2000-4000 rpm, high speed is 10000-16000 rpm), each for 20 seconds, for a total of 100 seconds; (3) Then, the mixed solution is placed in a glass container and mixed, the pH is adjusted to 5.0-6.0 with glacial acetic acid, and the mixed solution is heated to 30-40° C. and maintained for 0.5-2 h to release the target enzyme; (4) Centrifuge the mixture at 3000-6000 RPM for 30 min, take the supernatant and discard the precipitate; (5) solid ammonium sulfate was added to the supernatant to reach 35% saturation, and the solution was allowed to stand at 4°C overnight; (6) centrifugation to discard the precipitate, adding solid ammonium sulfate to the supernatant to make it reach 50% saturation, and standing at 4°C overnight; (7) centrifuging the solution using differential centrifugation and taking the precipitate from the second centrifugation; (8) dissolving the precipitate in an eluent, and desalting by ultrafiltration to obtain a crude extract 1 containing AFU and NAG; Step 2: Processing of beef kidney: (9) Chop the bovine kidney tissue and add it to the 4°C eluent to make the ratio reach 1:5 (mass: volume), homogenize and let it stand overnight; (10) The solution was centrifuged at 4°C for 30 min (3000-6000 RPM) and the supernatant was collected; (11) adding solid ammonium sulfate to reach 50% saturation and allowing to stand overnight; (12) Centrifuge at 4°C for 30 min (3000-6000 RPM), discard the precipitate, and collect the supernatant; (13) adding solid ammonium sulfate to the supernatant to make the supernatant reach 80% saturation, and then standing at 4°C overnight; (14) Centrifuge at 4°C for 30 min (3000-6000 RPM) and collect the precipitate; (15) re-dissolving the precipitate in the eluent, and then performing ultrafiltration desalting treatment to obtain a crude extract 2 containing mAST and LDH1; Step 3: Purification of crude extract: (16) Crude extract 1 and crude extract 2 were respectively subjected to anion separation columns, and crude extract 1 was eluted with 4 times the column volume of 50 mM NaCl to obtain α-L-fucosidase, and then the eluent was changed to 100 mM NaCl, and N-acetylglucosidase was obtained after 4 times the column volume of elution; crude extract 2 was eluted with 2 times the column volume of eluent to obtain aspartate aminotransferase mitochondrial isozyme, and then the eluent was changed to 100 mM NaCl, and lactate dehydrogenase isozyme 1 was obtained after 4 times the column volume of elution; (17) The above four products were heated for 3 days to obtain the final raw material; Step 4: Preparation of quality control products: (18) Mix human serum and glycerol in a ratio of 7:3 and add (3-10%) cryoprotectant, metal chelator, (0.5-2%) protein protectant, and (0.1-0.2%) preservative to prepare a quality control matrix solution, and add the above four self-extracted raw materials and the purchased raw materials of the three items of CG, GR, and CK-MB at the required concentrations to obtain a quality control product; (19) Measure the items contained in the quality control product on the biochemical analyzer.

2. The method for preparing a special biochemical composite quality control product according to claim 1, characterized in that: The dissolving solution in step (1) is a mixed solution of sodium acetate and sodium chloride with a concentration of 50-200 mM and a pH of 5.0-6.

5.

3. The method for preparing a special biochemical composite quality control product according to claim 1, characterized in that: The differential centrifugation method in step (7) is to first centrifuge at 2000-4000RPM for 20 minutes to remove the precipitate and retain the supernatant; then centrifuge the supernatant at 8000-16000RPM for 20 minutes and take the precipitate from the second centrifugation.

4. The method for preparing a special biochemical composite quality control product according to claim 1, characterized in that: The elution buffer is one of PB, Tris, and HEPES.

5. The method for preparing a special biochemical composite quality control product according to claim 1, characterized in that: The heating temperature in step (17) is 30-45°C.

6. The method for preparing a special biochemical composite quality control product according to claim 1, characterized in that: The cryoprotectant in step (18) is one or both of trehalose and mannitol; the metal chelator is one of EDTA and EGTA; the protein protectant is one or both of BSA and casein; and the preservative is one or both of Proclin 300 and sodium azide.

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