Preparation method and application of stable multifunctional enzyme composite quality control product

Abstract

The invention relates to the technical field of medical examination, in particular to a preparation method and application of a stable multifunctional enzyme compound quality control product, and the preparation method comprises the following steps: washing pig lungs with purified water, removing vascular connective tissues, cutting the pig lungs into small blocks with a knife, weighing and carrying out freezing pretreatment to obtain a first mixed solution; adding homogenate into the first mixed solution according to a weight ratio, and homogenizing to obtain a second mixed solution; refrigerating the second mixed solution or staying overnight, and fully extracting to obtain a third mixed solution; centrifugally filtering the third mixed solution, adding a salting-out drug and a protein protective agent, removing impure protein, centrifuging, and taking supernatant to obtain a fourth mixed solution; continuously adding a salting-out medicine into the fourth mixed solution, centrifuging, taking a precipitate, adding a dialysate into the precipitate, and fully dissolving to obtain a fifth mixed solution; dialyzing the fifth mixed solution, and collecting a solution in a dialysis bag to obtain an enzyme extracting solution; preparing a composite quality control product by using the enzyme extracting solution, and carrying out freeze-drying treatment on the composite quality control product to obtain composite quality control product freeze-dried powder.

Description

Technical Field

[0001] The present invention relates to the field of medical testing technology, and in particular to a preparation method and application of a stable multifunctional enzyme composite quality control product. Background Art

[0002] Adenosine deaminase (ADA) is a nucleic acid metabolizing enzyme that is crucially involved in cellular immune activity. ADA activity is a sensitive indicator of liver damage and is primarily used to monitor and diagnose liver disease. Changes in serum angiotensin-converting enzyme (ACE) levels can be used as an important indicator to measure the extent of lung damage caused by various factors. Serum ACE levels are decreased to varying degrees in conditions such as pulmonary tuberculosis, asthma attacks, acute cardiogenic pulmonary edema, chronic obstructive pulmonary disease, spontaneous pneumothorax, pulmonary fibrosis, and adult respiratory distress syndrome. Monoamine oxidase (MAO) activity reflects the extent of liver fibrosis and is an important indicator for diagnosing liver cirrhosis. Therefore, it is primarily used clinically as an auxiliary diagnosis for liver fibrosis. Lactate dehydrogenase isoenzyme 1 (LDH1) is primarily derived from myocardial cells. Changes in LDH1 serum concentration can specifically reflect the overall extent of myocardial damage and is widely used in the clinical diagnosis of acute myocardial infarction.

[0003] The four test items of ADA, ACE, MAO, and LDH1 are commonly single-item matching quality control products, among which MAO and LDH1 quality control products are even rarer. The enzymes used in single quality control products are mostly pure enzymes, and their preparation process is complicated and expensive. In addition, the use of single-item quality control products will cause operational inconvenience. Composite quality control products can improve both efficiency and accuracy. However, due to the different performances of different enzymes, the extraction and purification processes are different, and their stability conditions are also different. For example, the LDH1 enzyme has poor anti-freezing ability in the mixed solution of the four items. Taking enzyme extraction and composite quality control product preparation as an example, CN2019108139 30.1 discloses a method for separating and purifying angiotensin-converting enzyme from pig lungs. This method uses strong acid precipitation, salting out, and ultrafiltration. Although the operation is simple, it only targets ACE enzyme. At the same time, the strong acid extraction system cannot meet the stability of multiple enzymes after compounding; CN202011013809.X discloses a liver function composite quality control product and its preparation method and application. This method extracts three enzymes, AFU, ADA, and 5-NT, from pig liver, and uses the extracted enzyme raw materials to prepare a composite quality control product. Although this method can extract high-purity and highly active ADA enzyme, it cannot extract ACE enzyme at the same time. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method and application of a stable multifunctional enzyme composite quality control product, aiming to solve the problem that existing methods cannot simultaneously extract ACE, ADA, and MAO enzymes.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing a stable multifunctional enzyme composite quality control product, comprising the following steps:

[0006] The pig lungs were washed with purified water, the vascular connective tissue was removed, the pieces were cut into small pieces with a knife, and the pieces were weighed and frozen to obtain a first mixed solution;

[0007] adding a homogenate solution to the first mixed solution according to a weight ratio, and homogenizing to obtain a second mixed solution;

[0008] refrigerate or store the second mixed solution overnight for full extraction to obtain a third mixed solution;

[0009] Centrifugally filter the third mixed solution, add salting-out drugs and protein protective agents to remove impurities, centrifuge, and collect the supernatant to obtain a fourth mixed solution;

[0010] Further adding the salting-out drug to the fourth mixed solution, centrifuging, taking the precipitate, adding the dialysate to the precipitate, and fully dissolving it to obtain a fifth mixed solution;

[0011] dialyzing the fifth mixed solution, collecting the solution in the dialysis bag, and obtaining an enzyme extract;

[0012] The enzyme extract is used to prepare a composite quality control product, and the composite quality control product is freeze-dried to obtain a composite quality control product freeze-dried powder.

[0013] Wherein, the weight ratio includes 1:3.

[0014] The step of refrigerating or overnighting the second mixed solution and fully extracting the third mixed solution comprises:

[0015] The second mixed solution was refrigerated at 2-8° C. for 5 h or overnight and fully extracted to obtain a third mixed solution.

[0016] The third mixed solution is centrifuged and filtered, salting-out drugs and protein protective agents are added to remove foreign proteins, centrifuged, and the supernatant is taken to obtain a fourth mixed solution, comprising:

[0017] The third mixed solution is centrifuged and filtered, and a salting-out drug and a protein protective agent are added, wherein the amount of the salting-out drug added is 10% to 20% of the weight of the mixed solution, and impurities are removed. The mixture is centrifuged and the supernatant is taken to obtain a fourth mixed solution.

[0018] The salting-out drug is further added to the fourth mixed solution, centrifuged, and the precipitate is taken. The dialysate is added to the precipitate and fully dissolved to obtain a fifth mixed solution, comprising:

[0019] Continue to add salting-out drugs to the fourth mixed solution, wherein the amount of the salting-out drugs added is 20% to 30% of the weight of the mixed solution, centrifuge, take the precipitate, add dialysate to the precipitate at a ratio of 1:3, fully dissolve, and obtain the fifth mixed solution.

[0020] The step of dialyzing the fifth mixed solution and collecting the solution in the dialysis bag to obtain an enzyme extract comprises:

[0021] The fifth mixed solution was dialyzed at 2-8° C. for 24 hours, and the solution in the dialysis bag was collected to obtain an enzyme extract.

[0022] Wherein, the composite quality control product includes a buffer system, a protective agent or stabilizer, an activator and the enzyme extract.

[0023] Wherein, the composite quality control product also includes bovine serum albumin and preservatives.

[0024] Wherein, the buffer solution is 0.1M to 1.0M buffer solution, the protective agent is 1g / L to 5g / L, the stabilizer is 20g / L to 70g / L, and the activator is 0.1g / L to 0.5g / L;

[0025] The buffer is PB buffer, and the pH value of the buffer is 7.4-8.0; the protective agent is one or more of mannitol, polyethylene glycol, sucrose, trehalose, and dextran; the activator is one or more of sodium chloride, magnesium chloride, and potassium chloride;

[0026] The concentration of the bovine serum albumin is 20 g / L to 50 g / L, and the concentration of the preservative is 0.05% to 0.2%.

[0027] In the second aspect, the present invention provides an application of a stable multifunctional enzyme composite quality control product, which is a freeze-dried powder of the composite quality control product prepared by the preparation method of the stable multifunctional enzyme composite quality control product described in the first aspect, and the freeze-dried powder of the composite quality control product is used for the clinical diagnosis of acute myocardial infarction.

[0028] The present invention provides a method for preparing a stable multifunctional enzyme composite quality control product, comprising the steps of washing pig lungs with purified water, removing vascular connective tissue, cutting into small pieces with a knife, weighing and freezing for pretreatment to obtain a first mixed solution; adding a homogenate according to a weight ratio to the first mixed solution, homogenizing to obtain a second mixed solution; refrigerating or overnight the second mixed solution, and fully extracting to obtain a third mixed solution; centrifuging the third mixed solution, adding a salting-out drug and a protein protective agent, removing foreign proteins, centrifuging, and taking a supernatant to obtain a fourth mixed solution; and continuing to add the salting-out drug to the fourth mixed solution. , centrifuge, take the precipitate, add dialysate to the precipitate, fully dissolve it, and obtain a fifth mixed solution; dialyze the fifth mixed solution, collect the solution in the dialysis bag, and obtain an enzyme extract; use the enzyme extract to prepare a composite quality control product, and lyophilize the composite quality control product to obtain a composite quality control product lyophilized powder. Through the above steps, a composite quality control product containing angiotensin-converting enzyme, adenosine deaminase, monoamine oxidase, and lactate dehydrogenase isoenzyme 1 is prepared, which solves the problem that the existing method cannot simultaneously extract ACE, ADA, and MAO enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 The present invention provides a flow chart of a method for preparing a stable multifunctional enzyme composite quality control product. DETAILED DESCRIPTION

[0031] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0032] See also Figure 1 In a first aspect, the present invention provides a method for preparing a stable multifunctional enzyme composite quality control product, comprising the following steps:

[0033] S1: washing the pig lung with purified water, removing the vascular connective tissue, cutting into small pieces with a knife, weighing and freezing the pieces to obtain a first mixed solution;

[0034] S2: adding a homogenate solution to the first mixed solution according to a weight ratio, and homogenizing to obtain a second mixed solution;

[0035] Specifically, the weight ratio includes 1:3.

[0036] S3 refrigerate or store the second mixed solution overnight for full extraction to obtain a third mixed solution;

[0037] Specifically, the second mixed solution is refrigerated at 2-8° C. for 5 hours or overnight and fully extracted to obtain a third mixed solution.

[0038] S4 centrifugally filters the third mixed solution, adds salting-out drugs and protein protective agents, removes impurities, centrifuges, and collects the supernatant to obtain a fourth mixed solution;

[0039] Specifically, the third mixed solution is centrifuged and filtered, and a salting-out drug and a protein protective agent are added, wherein the amount of the salting-out drug added is 10% to 20% of the weight of the mixed solution, and impurities are removed. The mixture is centrifuged and the supernatant is collected to obtain a fourth mixed solution.

[0040] S5: continuing to add the salting-out drug to the fourth mixed solution, centrifuging, taking the precipitate, adding the dialysate to the precipitate, and fully dissolving it to obtain a fifth mixed solution;

[0041] Specifically, the salting-out drug is further added to the fourth mixed solution, wherein the amount of the salting-out drug added is 20% to 30% of the weight of the mixed solution, centrifuged, and the precipitate is taken. Dialysis fluid is added to the precipitate in a ratio of 1:3 and fully dissolved to obtain the fifth mixed solution.

[0042] S6 dialyzing the fifth mixed solution, collecting the solution in the dialysis bag, and obtaining an enzyme extract;

[0043] Specifically, the fifth mixed solution is dialyzed at 2-8° C. for 24 hours, and the solution in the dialysis bag is collected to obtain an enzyme extract.

[0044] S7 uses the enzyme extract to prepare a composite quality control product, and freeze-dries the composite quality control product to obtain a composite quality control product freeze-dried powder.

[0045] Specifically, the composite quality control product includes a buffer system, a protective agent or stabilizer, an activator, and the enzyme extract. The buffer is 0.1M to 1.0M, the protective agent is 1g / L to 5g / L, the stabilizer is 20g / L to 70g / L, and the activator is 0.1g / L to 0.5g / L; the buffer is PB buffer, and the buffer pH is 7.4 to 8.0; the protective agent is one or more of mannitol, polyethylene glycol, sucrose, trehalose, and dextran; the activator is one or more of sodium chloride, magnesium chloride, and potassium chloride; the concentration of bovine serum albumin is 20g / L to 50g / L, and the preservative concentration is 0.05% to 0.2%.

[0046] The addition of bovine serum albumin can effectively improve the matrix effect and excipient of the composite quality control product; the preservative has an antibacterial effect. The addition of the preservative is beneficial to ensure the stability of the quality control product, so that the quality control product can be stored for a long time without deterioration.

[0047] Beneficial effects:

[0048] The beneficial effects of the present invention are as follows: the present invention addresses the technical gap in the prior art of composite quality control products containing four different functional test items, namely angiotensin-converting enzyme, adenosine deaminase, monoamine oxidase, and lactate dehydrogenase isoenzyme 1, as well as the problems of complex and high cost of enzyme raw material extraction processes. The present invention provides a composite quality control product containing angiotensin-converting enzyme, adenosine deaminase, monoamine oxidase, and lactate dehydrogenase isoenzyme 1 and a preparation method thereof. By optimizing the enzyme raw material extraction process, enzyme inactivation during the preparation process is avoided. A freeze-drying process is further used to obtain a freeze-dried powder of the composite quality control product with better stability. The matrix liquid of the quality control product is optimized to enhance the thermal stability and post-reconstitution frozen stability of the freeze-dried powder of the composite quality control product (stability index: stability deviation of each item is controlled within ±10% after 5 days of heat load and 30 days of freezing after reconstitution). Thus, an enzyme composite quality control product with a simple and rapid preparation method, low cost, and high enzyme activity and stability is obtained. The composite quality control product can be used in conjunction with an enzyme detection kit to verify the kit or check the accuracy and stability of an analytical instrument or detection method.

[0049] Example 1

[0050] In the present embodiment, enzyme extraction is specifically as follows:

[0051] (1) Homogenate: Prepare PB buffer with a pH of 8.00 and a concentration of 50 mmol / L. Then add Triton X-100 to the buffer to a concentration of 1%, and dilute to volume to obtain a homogenate.

[0052] The preparation method of this embodiment includes:

[0053] S1. Take fresh pig liver, rinse it repeatedly with tap water to remove blood, remove connective tissue, cut the liver tissue into small pieces less than 1 cm3, and pre-cool it in a -20°C refrigerator for 10 minutes to obtain a first mixed solution;

[0054] S2. Add 3 times the amount of homogenate to the first mixed solution at a weight ratio of 1:3. Homogenize at low speed for approximately 5 minutes, pausing for 30 seconds every 40 seconds, for a total of 6-8 homogenizations. Keep the homogenization temperature as low as possible. Place the untreated homogenate in an ice bath to obtain the second mixed solution.

[0055] S3, refrigerating the second mixed solution at 2-8°C overnight and fully extracting to obtain a third mixed solution;

[0056] S4. Centrifuge the third mixed solution at 8000 rpm at 4°C for 30 min, filter with double-layer gauze, and take the filtrate and weigh it.

[0057] Add ammonium sulfate powder to the filtrate (the amount added is 20% of the weight of the supernatant), stir and dissolve for 1 hour, let it stand for 1 hour, centrifuge at 8000 rpm at 4°C for 20 minutes, collect the supernatant, and weigh it to obtain the fourth mixed solution;

[0058] S5. Add ammonium sulfate powder to the fourth mixed solution (the amount added is 25% of the weight of the supernatant), stir and dissolve for 1 hour, let it stand overnight, centrifuge at 8000 rpm at 4°C for 20 minutes, take the precipitate, weigh it, and add dialysate to the precipitate at a ratio of 1:3 to fully dissolve it to obtain a fifth mixed solution;

[0059] S6. Dialyze the fifth mixed solution at 2-8° C. for 24 h, and collect the solution in the dialysis bag as the enzyme extract.

[0060] Example 2

[0061] In order to improve the stability of the composite quality control product, this example, based on the preparation method described in Example 1, diluted the enzyme extract obtained in Example 1 with a matrix solution, and then obtained a composite quality control freeze-dried powder through a freeze-drying process, thereby significantly improving the stability of the product.

[0062] The matrix solution is formulated as follows: 200 mmol / L phosphate buffer, 20 g / L bovine serum albumin, 2 g / L mannitol, 40 g / L trehalose, 1 g / L PEG8000, 0.1 g / L sodium chloride, and 0.1 g / L preservative. The pH is adjusted to 7.6 and the volume is fixed to obtain the matrix solution.

[0063] Example 3

[0064] The difference between this embodiment and embodiment 2 is that the formula of the matrix solution is: 100 mmol / L phosphate buffer, 30 g / L bovine serum albumin, 5 g / L mannitol, 50 g / L trehalose, 2 g / L PEG8000, 0.5 g / L sodium chloride, and 0.1 g / L preservative.

[0065] Example 4

[0066] The difference between this embodiment and embodiment 2 is that the formula of the matrix solution is: 50 mmol / L phosphate buffer, 40 g / L bovine serum albumin, 1 g / L mannitol, 60 g / L trehalose, 5 g / L PEG8000, 0.2 g / L sodium chloride, and 1 g / L preservative.

[0067] Evaluation plan

[0068] The thermal stability and post-reconstitution freezing stability of the composite quality control freeze-dried powder obtained in Examples 2-4 were evaluated.

[0069] The test kits used in the evaluation were angiotensin-converting enzyme assay kit, monoamine oxidase assay kit, lactate dehydrogenase isoenzyme 1 assay kit, and adenosine deaminase assay kit from Guilin Uni-Tech Medical Electronics Co., Ltd.

[0070] Thermal stability evaluation method: The composite quality control product obtained in each example was stored at 37°C for 5 days, and the content of each enzyme therein was detected using a kit on the 0th and 7th days of storage. Each example was measured 3 times, and the average value and the deviation (%) of the measured value on the 7th day compared with the 0th day were calculated. The deviation was within 10%, indicating good thermal stability. The results are shown in Table 1.

[0071] Evaluation method for frozen stability after reconstitution: The composite quality control product obtained in each example was reconstituted with purified water and frozen at -20°C for 30 days. The content of each enzyme therein was detected using a kit on the 0th, 8th, 16th, 24th and 30th day of storage. Each example was measured 3 times, and the average value and the deviation (%) of the measured value at each time point compared with the 0th day were calculated. The deviation was within 10%. At the same time, a trend analysis was performed, and the trend analysis should not be significant. The results are shown in Tables 2 to 4.

[0072] Table 1 Thermal stability test data

[0073]

[0074] Table 2 Freezing stability test data after reconstitution

[0075]

[0076]

[0077] Table 3 Freezing stability test data after reconstitution

[0078]

[0079] Table 4 Freezing stability test data after reconstitution

[0080]

[0081]

[0082] Based on the test results in Tables 1-4, the composite quality control products prepared according to the above three examples showed good thermal stability and post-reconstitution freezing stability, fully meeting the requirements of use. Furthermore, changes in the component content of the matrix solution have a certain impact on the stability of the reagent. Overall, after using the matrix solution described in Example 4, the LDH1 enzyme was more stable at -20 degrees freezing stability, and its deviation value was relatively low. In other words, the matrix solution described in Example 4 was the most effective in maintaining the stability of the LDH1 enzyme.

[0083] In the second aspect, the present invention provides an application of a stable multifunctional enzyme composite quality control product, which is a freeze-dried powder of the composite quality control product prepared by the preparation method of the stable multifunctional enzyme composite quality control product described in the first aspect, and the freeze-dried powder of the composite quality control product is used for the clinical diagnosis of acute myocardial infarction.

[0084] The above disclosure is only a preferred embodiment of the preparation method and application of a stable multifunctional enzyme complex quality control product of the present invention. Of course, this cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiments and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for preparing a stable multifunctional enzyme composite quality control product, characterized in that: The following steps are involved: The pig lungs were washed with purified water, the vascular connective tissue was removed, the pieces were cut into small pieces with a knife, and the pieces were weighed and frozen to obtain a first mixed solution; adding a homogenate solution to the first mixed solution according to a weight ratio, and homogenizing to obtain a second mixed solution; refrigerate or store the second mixed solution overnight for full extraction to obtain a third mixed solution; Centrifugally filter the third mixed solution, add salting-out drugs and protein protective agents to remove impurities, centrifuge, and collect the supernatant to obtain a fourth mixed solution; Further adding the salting-out drug to the fourth mixed solution, centrifuging, taking the precipitate, adding the dialysate to the precipitate, and fully dissolving it to obtain a fifth mixed solution; dialyzing the fifth mixed solution, collecting the solution in the dialysis bag, and obtaining an enzyme extract; The enzyme extract is used to prepare a composite quality control product, and the composite quality control product is freeze-dried to obtain a composite quality control product freeze-dried powder.

2. The method for preparing the stable multifunctional enzyme composite quality control product according to claim 1, wherein: The weight ratio includes 1:

3.

3. The method for preparing the stable multifunctional enzyme composite quality control product according to claim 2, wherein: The second mixed solution is refrigerated or left overnight for full extraction to obtain a third mixed solution, comprising: The second mixed solution was refrigerated at 2-8° C. for 5 h or overnight and fully extracted to obtain a third mixed solution.

4. The method for preparing the stable multifunctional enzyme composite quality control product according to claim 3, wherein: The third mixed solution is centrifuged and filtered, salting-out drugs and protein protective agents are added to remove foreign proteins, centrifuged, and supernatant is collected to obtain a fourth mixed solution, comprising: The third mixed solution is centrifuged and filtered, and a salting-out drug and a protein protective agent are added, wherein the amount of the salting-out drug added is 10% to 20% of the weight of the mixed solution, and impurities are removed. The mixture is centrifuged and the supernatant is taken to obtain a fourth mixed solution.

5. The method for preparing the stable multifunctional enzyme composite quality control product according to claim 4, wherein: The salting-out drug is further added to the fourth mixed solution, centrifuged, and a precipitate is obtained. The precipitate is added with a dialysate and fully dissolved to obtain a fifth mixed solution, comprising: Continue to add salting-out drugs to the fourth mixed solution, wherein the amount of the salting-out drugs added is 20% to 30% of the weight of the mixed solution, centrifuge, take the precipitate, add dialysate to the precipitate at a ratio of 1:3, fully dissolve, and obtain the fifth mixed solution.

6. The method for preparing the stable multifunctional enzyme composite quality control product according to claim 5, wherein: The step of dialyzing the fifth mixed solution and collecting the solution in the dialysis bag to obtain an enzyme extract comprises: The fifth mixed solution was dialyzed at 2-8° C. for 24 hours, and the solution in the dialysis bag was collected to obtain an enzyme extract.

7. The method for preparing the stable multifunctional enzyme composite quality control product according to claim 6, wherein: The composite quality control product comprises a buffer system, a protective agent or a stabilizer, an activator and the enzyme extract.

8. The method for preparing the stable multifunctional enzyme composite quality control product according to claim 7, wherein: The composite quality control product also includes bovine serum albumin and a preservative.

9. The method for preparing the stable multifunctional enzyme composite quality control product according to claim 8, wherein: The buffer solution is 0.1M to 1.0M buffer solution, the protective agent is 1g / L to 5g / L, the stabilizer is 20g / L to 70g / L, and the activator is 0.1g / L to 0.5g / L; The buffer is PB buffer, and the pH value of the buffer is 7.4-8.0; the protective agent is one or more of mannitol, polyethylene glycol, sucrose, trehalose, and dextran; the activator is one or more of sodium chloride, magnesium chloride, and potassium chloride; The concentration of the bovine serum albumin is 20 g / L to 50 g / L, and the concentration of the preservative is 0.05% to 0.2%.

10. Use of a stable multifunctional enzyme composite quality control product, the composite quality control product prepared by the preparation method of the stable multifunctional enzyme composite quality control product according to claim 9, characterized in that: The composite quality control product is used in the clinical diagnosis of acute myocardial infarction.

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