Preparation method and application of monoamine oxidase treating fluid

Abstract

The invention relates to a preparation method and application of a monoamine oxidase treating fluid. The preparation method comprises the following steps: step 1, preparing a buffer solution; step 2, preparing an enzyme inclusion solution; and step 3, sequentially adding 0.25 g / L-0. 5 g / L of flavin adenine dinucleotide and 0.25 g / L-0. 5 g / L of a hydrophilic nonionic surfactant into the continuously stirred enzyme inclusion solution in an environment of 4 + / -1 DEG C, continuously stirring for 30 minutes, filtering, transferring into a dark brown glass bottle, and storing at 2-8 DEG C, thereby obtaining the product. The method disclosed by the invention has the beneficial effects that 1, the problem of too fast enzyme activity loss in the preparation process of the MAO quality control product is solved, and the enzyme activity loss is reduced from original 40% to below 10%; 2, the problem of short preservation period after the MAO quality control product is redissolved is solved, the preservation stability at 2-8 DEG C after the MAO quality control product is redissolved is improved to 7 days from the original 7 hours, and the frozen preservation at-20 DEG C is improved to 30 days from 10 days; and 3, the risk of too high production cost or patent infringement of reagent enterprises caused by MAO raw materials prepared by adopting patented technologies such as gene modification is avoided.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing a monoamine oxidase treatment solution and its application. Background Technology

[0002] Monoamine oxidase (MAO) (EC 1.4.3.4) is a membrane-bound mitochondrial enzyme that maintains homeostasis of neurotransmitters and other biogenic amines in biological systems through catalytic oxidation and deamination reactions. MAO dysfunction is closely related to many neurological and psychiatric diseases; therefore, monitoring MAO activity and expression levels is of great significance for a deeper understanding of its physiological functions and the clinical diagnosis of related diseases. Currently, commercially available in vitro diagnostic kits for MAO activity mainly utilize the method of MAO catalyzing the formation of ammonia from benzylamine. Ammonia, in the presence of α-ketoglutarate, NADH (or NADPH), and GLDH, generates glutamate, while NADH (or NADPH) is reduced to NAD+ (or NADP+), causing a decrease in absorbance at 340 nm. The activity of MAO in the sample can be determined by monitoring the rate of this decrease. This method is applicable to fully automated biochemical analyzers, is simple to operate, has high accuracy, and is relatively inexpensive, thus it is gradually being promoted in many primary hospitals.

[0003] However, as a flavin-containing dimeric enzyme, MAO's stability is threatened by multiple factors, especially the following three types of problems, which are particularly prominent in solution environments. This has resulted in a limited number of MAO quality control and calibrators available on the market that can be used with reagent kits, and these kits often suffer from poor reconstitution stability. Many reagent manufacturers have resorted to the K-factor method for testing, which significantly reduces the reliability of the test results and severely hinders the widespread use of MAO detection reagents:

[0004] a. The catalytic center of MAO contains numerous hydrophobic regions, allowing water molecules to easily penetrate and trigger hydration of the flavin adenine dinucleotide (FAD) microenvironment, leading to conformational changes and a rapid decline in enzyme activity per unit solution. A study published in the international journal *Bioorganic & Medicinal Chemistry Letters* showed that unprotected MAO aqueous solutions lost over 90% of their activity after 2 hours at 37°C.

[0005] b. The dimer structure of MAO is maintained by hydrophobic bonds and hydrogen bonds between subunits. Changes in solution ionic strength or interfacial tension disturbances (such as at the gas-liquid interface) can induce subunit dissociation. Experiments show that oscillation treatment can reduce MAO activity by 40%, mainly due to subunit separation.

[0006] The cysteine ​​residue at the active site of C.MAO is easily oxidized to sulfonic acid or disulfide bonds, resulting in irreversible deactivation. Trace metal ions (such as Cu) 2+ / Fe 2+) or light can accelerate this process, especially significantly when pH > 7.5.

[0007] Currently, domestic invention patents related to the detection of MAO activity are all limited to the field of detection kits. For example, "Monoamine Oxidase MAO Single Reagent Determination Kit" with the patent number disclosed as 101498662A, "An Anti-Interference and Stable Serum Monoamine Oxidase Determination Kit and Its Preparation Method and Application" with the patent number disclosed as CN116497084A, etc. There is no invention patent for the quality control products supporting the kits. The purpose of the present invention is to provide a preparation method of monoamine oxidase treatment solution and its application in the preparation of calibration products and quality control products, aiming to solve the performance defects of existing MAO calibration products and quality control products, and at the same time taking into account the applicability of MAO enzyme raw materials from different sources and mass production. Summary of the Invention

[0008] To sum up, in order to overcome the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a preparation method and application of monoamine oxidase treatment solution.

[0009] The technical solution of the present invention to solve the above technical problems is as follows: A preparation method of monoamine oxidase treatment solution includes the following steps:

[0010] Step 1, prepare the buffer solution: Weigh 80% of the total weight of purified water, and dissolve 2-hydroxypropyl-β-cyclodextrin at 100 g / L - 200 g / L, a protective agent at 100 g / L - 200 g / L, and Tris-hydrochloride at 7.88 g / L in sequence to obtain a mixed solution. After adjusting the pH of the mixed solution to 7.5 - 8.0, then quantitatively add purified water to 1000 g to obtain the buffer solution;

[0011] Step 2, prepare the enzyme inclusion solution: First, weigh 0.25 g / L - 0.5 g / L of freeze-dried MAO enzyme in an environment of 4 ± 1 °C and add it to the continuously stirred buffer solution, and continuously stir for 30 minutes to obtain a completely dissolved enzyme treatment solution; Secondly, ultrasonically treat the completely dissolved enzyme treatment solution for 10 - 20 minutes, and during this period, ensure that the temperature of the enzyme treatment solution changes within the range of 23 ± 5 °C; Finally, transfer the enzyme treatment solution to an environment of 4 ± 1 °C and balance for 1 h to obtain the enzyme inclusion solution;

[0012] Step 3, in an environment of 4 ± 1 °C, sequentially add 0.25 g / L - 0.5 g / L of flavin adenine dinucleotide and 0.25 g / L - 0.5 g / L of hydrophilic non-ionic surfactant to the continuously stirred enzyme inclusion solution, continuously stir for 30 minutes, filter and transfer it to a light-proof brown glass bottle and store it at 2 - 8 °C.

[0013] Based on the above technical solutions, the present invention can be further improved as follows:

[0014] Further, in step 1, the pH of the mixture is adjusted with 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution.

[0015] Further, the protective agent in step 1 is one or a combination of mannitol, sucrose, and trehalose.

[0016] [[ID=⑥]]Further, in step 2, the buffer solution is continuously stirred by a magnetic stirrer, and the rotation speed of the stirring bar is controlled within 300 - 500 rpm.

[0017] Further, the power of the ultrasonic treatment in step 2 is 200 W.

[0018] Further, in step 3, the buffer solution is continuously stirred by a magnetic stirrer, and the rotation speed of the stirring bar is controlled within 200 - 300 rpm.

[0019] Further, the hydrophilic non - ionic surfactant in step 3 is a polyoxyethylene polyoxypropylene ether block copolymer with an HLB value above 25, and its molecular formula is HO·(C2H4O)m·(C3H6O)n·H, which can be Pluronic L65 or Pluronic F68.

[0020] Further, in step 3, filtration is carried out with a polyvinylidene fluoride with a pore size of 0.45 μm.

[0021] This application also provides the use of the monoamine oxidase treatment solution prepared by the described preparation method in the preparation of a quality control product supporting a monoamine oxidase assay kit, including the following steps:

[0022] Step 1, determination of the value of the monoamine oxidase treatment solution: The monoamine oxidase treatment solution is analyzed and tested on a biochemical analyzer using a monoamine oxidase assay kit to obtain the MAO activity per unit volume.

[0023] Step 2, calculation of the weighed amount of the monoamine oxidase treatment solution: First, calculate the total activity feeding amount of MAO according to the theoretical target concentration and the preparation amount of the quality control product, and then calculate the amount of the monoamine oxidase treatment solution to be measured, denoted as Xml, based on the MAO activity per unit volume obtained in step 1.

[0024] Step 3, preparation of the quality control product: Take 80% of the total weight of purified water, and sequentially add and dissolve GOOD at 20 mmol / L - 50 mmol / L ， It should be noted that "GOOD" in step 30 seems to be an incomplete or incorrect expression in the original text, but it is translated as it is according to the requirements.Mixed buffer was prepared by adding s buffer and 0.5 mmol / L-1 mmol / L complexing agent. The pH of the mixed buffer was adjusted to 7.5-8.0 with 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution. Then, 10 g / L-20 g / L protein protectant, 0.5 g / L-1 g / L preservative, and X ml of the monoamine oxidase treatment solution were added sequentially. The total volume was measured by weighing with purified water. After equilibration for 30 minutes, the solution was filtered through a 0.45-0.22 μm polyethersulfone membrane. Finally, the prepared liquid quality control sample was aliquoted and freeze-dried to obtain the lyophilized product, which is the quality control sample.

[0025] Furthermore, the GOOD ， The s buffer can be one of HEPES, MOPS, and Tris-hydrochloric acid buffer; the complexing agent can be one or more of EDTA, EGTA, and DTPA; the protein protectant can be one or more of bovine serum albumin, human serum albumin, and ovalbumin; and the preservative is sodium azide or Proclin 300.

[0026] The beneficial effects of this invention are:

[0027] 1. Solved the problem of excessively rapid enzyme activity loss during the preparation of MAO quality control products, reducing enzyme activity loss from 40% to below 10%;

[0028] 2. Solved the problem of short shelf life of MAO quality control products after reconstitution. The stability of the products after reconstitution at 2-8℃ has been improved from 7 hours to 7 days, and the stability of the products after freezing at -20℃ has been improved from 10 days to 30 days.

[0029] 3. Avoid the risk of excessively high production costs or patent infringement issues for reagent companies due to the use of MAO raw materials prepared with patented technologies such as genetic modification. Attached Figure Description

[0030] Figure 1 The chemical structural diagram of 2-hydroxypropyl-beta-cyclodextrin (HP-β-CD);

[0031] Figure 2 This is a chemical structural diagram of a polyoxyethylene-polyoxypropylene ether block copolymer. Detailed Implementation

[0032] The principles and features of the present invention are described below with reference to specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0033] A method for preparing a monoamine oxidase treatment solution includes the following steps:

[0034] Step 1, Preparation of buffer solution: Weigh 80% of the total weight of purified water, and dissolve 2-hydroxypropyl-beta-cyclodextrin (100g / L~200g / L), a protective agent (100g / L~200g / L), and Tris-hydrochloric acid (7.88g / L) sequentially to obtain a mixture. Adjust the pH of the mixture to 7.5-8.0 with 6mol / L hydrochloric acid solution or 10mol / L sodium hydroxide solution, and then measure to 1000g (density ρ=1.000g / cm3) with purified water to prepare the buffer solution. The 2-hydroxypropyl-beta-cyclodextrin has the molecular formula (C6H10O5)7•(C3H6O)n, where n=10~21; the protective agent is one or a combination of mannitol, sucrose, and trehalose.

[0035] The chemical structural formula of 2-hydroxypropyl-beta-cyclodextrin (HP-β-CD) is as follows: Figure 1 As shown.

[0036] Step 2, Preparation of enzyme inclusion solution: First, accurately weigh 0.25 g / L to 0.5 g / L of lyophilized MAO enzyme and add it to the continuously stirred buffer solution (prepared in Step 1) at 4±1℃. A magnetic stirrer is recommended, with the stir bar speed controlled between 300 and 500 rpm, and stirring continuously for 30 minutes. Next, sonicate the completely dissolved enzyme solution for 10 to 20 minutes (200W), ensuring the temperature of the enzyme solution remains within the range of 23±5℃ during this period. Finally, transfer the enzyme solution to a 4±1℃ environment for equilibration for 1 hour to prepare the enzyme inclusion solution.

[0037] Step 3, Site Activation: At 4±1℃, add precisely weighed flavin adenine dinucleotide (FAD) (0.25 g / L–0.5 g / L) and a hydrophilic nonionic surfactant (0.25 g / L–0.5 g / L) sequentially to the continuously stirred enzyme inclusion solution (prepared in Step 2). A magnetic stirrer is recommended, with the stir bar speed controlled between 200 and 300 rpm, for 30 minutes. Filter through a 0.45 μm pore size polyvinylidene fluoride (PVDF) membrane and transfer to a light-proof brown glass bottle for storage at 2–8℃. The hydrophilic nonionic surfactant is a polyoxyethylene polyoxypropylene ether block copolymer with an HLB value of 25 or higher, and its molecular formula is HO·(C2H4O)m·(C3H6O)n·H, such as Pluronic L65 or Pluronic F68.

[0038] The chemical structure of polyoxyethylene polyoxypropylene ether block copolymer is as follows: Figure 2 As shown.

[0039] The effects of different grades of hydroxypropyl groups and different concentrations of HP-β-CD on the enzyme activity of monoamine oxidase treatment solutions are shown in Table 1 and Table 2, respectively.

[0040] Table 1. Effects of HP-β-CD of different hydroxypropyl groups on enzyme activity of monoamine oxidase in the treatment solution.

[0041]

[0042] Table 2. Effects of different concentrations of HP-β-CD on the enzyme activity of monoamine oxidase treated solution.

[0043]

[0044] Tables 1 and 2 show that the MAO activity retention rate varies with different grades and dosages of HP-β-CD when preparing enzyme treatment solutions. At a dosage of 100 g / L, HP-β-CD with 15-20 grades of hydroxypropyl groups exhibits the best MAO activity retention rate, while HP-β-CD with 15 grades of hydroxypropyl groups shows the best MAO activity retention rate at a dosage of 100-200 g / L.

[0045] The application of the monoamine oxidase treatment solution prepared by the method described in this invention in the preparation of quality control materials for a monoamine oxidase assay kit specifically includes the following steps:

[0046] Step 1, Monoamine oxidase treatment solution determination: The monoamine oxidase treatment solution prepared by the method described in this invention was analyzed and tested using a monoamine oxidase assay kit (continuous monitoring method) produced by Guilin Unitech Medical Electronics Co., Ltd. on a Unitech CA-2000 biochemical analyzer (using 10-fold dilution test) to obtain the MAO activity per unit volume (U / L).

[0047] Step 2, Calculation of Monoamine Oxidase Treatment Solution Volume: First, calculate the total MAO activity dosage based on the theoretical target concentration of the quality control sample and the preparation volume. Then, calculate the volume of monoamine oxidase treatment solution to be measured (denoted as X mL) based on the unit activity of the enzyme treatment solution (obtained in Step 1). Example: Given that the MAO activity of the monoamine oxidase treatment solution is 600 U / L, and 0.5 L of a quality control sample with an activity of 20 U / L is needed, the required volume of monoamine oxidase treatment solution is 0.5 × 30 × 1000 ÷ 600 = 25 mL.

[0048] Step 3, Preparation of quality control samples: Take 80% purified water by weight and add the dissolving agents in the following order.

[0049] GOOD ，A mixed buffer was prepared by mixing s buffer (20 mmol / L-50 mmol / L) and complexing agent (0.5 mmol / L-1 mmol / L). The pH of the mixed buffer was adjusted to 7.5-8.0 with 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution. Then, a protein-dissolving protectant (10 g / L-20 g / L), a preservative (0.5 g / L-1 g / L), and X mL of MAO enzyme treatment solution (step two) were added sequentially. The total volume was then measured using purified water by weighing. After equilibration for 30 minutes, the solution was filtered through a 0.45-0.22 μm polyethersulfone membrane. Finally, the prepared liquid quality control sample was aliquoted and freeze-dried to obtain the lyophilized product, which is the quality control sample. (The above is a GOOD...) ， The s buffer can be one of HEPES, MOPS, and Tris-hydrochloric acid buffer; the complexing agent can be one or more of EDTA, EGTA, and DTPA; the protein protectant can be one or more of bovine serum albumin, human serum albumin, and ovalbumin; and the preservative is sodium azide or Proclin 300.

[0050] The beneficial effects of the present invention will be illustrated below through specific examples:

[0051] Example 1

[0052] I. Preparation of Monoamine Oxidase Treatment Solution

[0053] Step 1, Prepare buffer solution (0.5L): Weigh 400g of sufficient purified water, and dissolve 100g of 2-hydroxypropyl-beta-cyclodextrin (n: 15-20), 50g of trehalose, 50g of sucrose, and 3.94g of Tris-hydrochloric acid in sequence to obtain a mixture. Adjust the pH of the mixture to 7.5-8.0 with 6mol / L hydrochloric acid solution or 10mol / L sodium hydroxide solution, and then measure the volume to 500g (density ρ=1.0g / cm3) with purified water.

[0054] Step 2, prepare the enzyme inclusion solution: Equilibrate the treatment solution at 4±1℃ for 30 minutes, add 0.1g of lyophilized MAO enzyme powder to the continuously stirred buffer solution (prepared in Step 1) (magnetic stirrer speed at 350rpm), and continue stirring for 30 minutes; sonicate the solution for 15 minutes (200W), during which the temperature of the treatment solution must be kept within the range of 5±3℃. If the temperature rises rapidly during sonication, the temperature of the solution can be kept relatively constant by controlling the interval between each sonication; finally, transfer the enzyme treatment solution to an environment of 4±1℃ for 1 hour to equilibrate.

[0055] Step 3, Site Activation: Add 0.15 g flavin adenine dinucleotide (FAD) and 0.2 g Pluronic L65 sequentially to the continuously stirred enzyme inclusion solution at 4±1℃. Maintain the stir bar speed at 200 rpm and stir continuously for 30 minutes. Filter through a 0.45 μm pore size polyvinylidene fluoride (PVDF) membrane and transfer to a light-proof brown glass bottle for storage at 2–8℃.

[0056] II. Application: Preparation of quality control materials (0.5L, 18~35U / L)

[0057] Step 1, determination of monoamine oxidase treatment solution: The monoamine oxidase treatment solution prepared in this invention was analyzed and tested using the monoamine oxidase assay kit (continuous monitoring method) produced by Guilin Ulite Medical Electronics Co., Ltd. on the Ulite CA-2000 biochemical analyzer (using 10-fold dilution test), and the MAO activity per unit volume was found to be 661 U / L.

[0058] Step 2, Calculation of the amount of monoamine oxidase treatment solution: Given that the MAO activity of the monoamine oxidase treatment solution is 661 U / L, and 0.5L of a quality control sample with an activity of 28 U / L is required, the amount of monoamine oxidase treatment solution to be measured is approximately 0.5 × 28 × 1000 ÷ 661 ≈ 21 mL.

[0059] Step 3, Preparation of quality control sample: Take 400 mL of sufficient purified water, add 3.14 g MOPS and 0.15 g EGTA in sequence, adjust the pH to 7.5 ± 0.05 with 6 mmol / L sodium hydroxide, then add 15 g bovine serum albumin, 0.5 g Proclin 300 and 21 mL monoamine oxidase treatment solution in sequence, and weigh to make up the remaining purified water (ρ = 1.000 g / cm3). Filter using a 0.45 μm polyethersulfone filter membrane, and finally dispense and freeze-dry the prepared liquid quality control sample to obtain the lyophilized product.

[0060] Example 2

[0061] I. Preparation of Monoamine Oxidase Treatment Solution

[0062] Step 1, prepare buffer solution (0.5L): Weigh 400g of sufficient purified water, and dissolve 150g of 2-hydroxypropyl-beta-cyclodextrin (n: 10-15), 50g of trehalose, 25g of sucrose and 3.94g of Tris-hydrochloric acid in sequence to obtain a mixture; adjust the pH of the mixture to 7.5-8.0 with 6mol / L hydrochloric acid solution or 10mol / L sodium hydroxide solution, and then measure it to 500g with purified water (density ρ=1.0g / cm3).

[0063] Step 2, prepare the enzyme inclusion solution: Equilibrate the treatment solution at 4±1℃ for 30 minutes, add 0.15g of lyophilized MAO enzyme powder to the continuously stirred buffer solution (prepared in Step 1) (magnetic stirrer speed within 400rpm), and continue stirring for 30 minutes; sonicate the solution for 20 minutes (200W), during which the temperature of the treatment solution must be kept within the range of 5±3℃. If the temperature rises rapidly during sonication, the temperature of the solution can be kept relatively constant by controlling the interval between each sonication; finally, transfer the enzyme treatment solution to an environment of 4±1℃ for 1 hour to equilibrate.

[0064] Step 3, Site Activation: Add 0.2 g flavin adenine dinucleotide (FAD) and 0.5 g Pluronic F68 sequentially to the continuously stirred enzyme inclusion solution at 4±1℃. Maintain a stirrer speed of 250 rpm and stir continuously for 30 minutes. Filter through a 0.22 μm pore size polyvinylidene fluoride (PVDF) membrane and transfer to a light-proof brown glass bottle for storage at 2–8℃.

[0065] II. Application: Preparation of quality control material (500mL, 30-50U / L)

[0066] Step 1, Monoamine oxidase treatment solution determination: The monoamine oxidase treatment solution prepared in this invention was analyzed and tested using the monoamine oxidase assay kit (continuous monitoring method) produced by Guilin Ulite Medical Electronics Co., Ltd. on the Ulite CA-2000 biochemical analyzer (using 10-fold dilution test), and the MAO activity per unit volume was found to be 942 U / L.

[0067] Step 2, Calculation of the amount of monoamine oxidase treatment solution: Given that the MAO activity of the monoamine oxidase treatment solution is 942 U / L, and 0.5L of quality control solution with an activity of 40 U / L is required, the amount of monoamine oxidase treatment solution to be weighed is approximately 0.5 × 40 × 1000 ÷ 942 ≈ 21 mL.

[0068] Step 3, Quality Control Preparation: Take 400 mL of sufficient purified water, add 2.383 g HEPES and 0.1 g EDTA in sequence, adjust the pH to 7.8 ± 0.05 with 10 mmol / L sodium hydroxide, then add 10 g bovine serum albumin, 0.5 g Proclin 300 and 21 mL monoamine oxidase treatment solution in sequence, and weigh to make up the remaining purified water (ρ = 1.000 g / cm3). Filter using a 0.45 μm polyethersulfone filter membrane, and finally dispense and freeze-dry the prepared liquid quality control to obtain the lyophilized product.

[0069] Example 3

[0070] I. Preparation of Monoamine Oxidase Treatment Solution

[0071] Step 1, prepare buffer solution (0.5L): Weigh 400g of sufficient purified water, and dissolve 100g of 2-hydroxypropyl-beta-cyclodextrin (n: 10-15), 25g of trehalose, 50g of sucrose and 3.94g of Tris-hydrochloric acid in sequence to obtain a mixture; adjust the pH of the mixture to 7.5-8.0 with 6mol / L hydrochloric acid solution or 10mol / L sodium hydroxide solution, and then measure it to 500g with purified water (density ρ=1.0g / cm3).

[0072] Step 2, preparation of enzyme inclusion: Equilibrate the treatment solution at 4±1℃ for 30 minutes, add 0.15g of lyophilized MAO enzyme powder to the continuously stirred buffer (prepared in Step 1) (magnetic stirrer speed within 300rpm), and continue stirring for 30 minutes; Sonicate the completely dissolved enzyme treatment solution for 15 minutes (200W), during which the temperature of the treatment solution must be kept within the range of 5±3℃. If the temperature rises rapidly during sonication, the solution temperature can be kept relatively constant by controlling the interval between each sonication; Finally, transfer the enzyme treatment solution to an environment of 4±1℃ for 1 hour to equilibrate.

[0073] 3. Site activation: Add 0.15 g flavin adenine dinucleotide (FAD), 0.2 g Pluronic F68, and 0.1 g Pluronic L65 sequentially to the continuously stirred enzyme inclusion solution at 4 ± 1 °C. Maintain a stirrer speed of 300 rpm and stir continuously for 30 minutes. Filter through a 0.22 μm pore size polyvinylidene fluoride (PVDF) membrane and transfer to a light-proof brown glass bottle for storage at 2–8 °C.

[0074] II. Application: Preparation of quality control material (500mL, 60~83U / L)

[0075] Step 1, Monoamine oxidase treatment solution determination: The monoamine oxidase treatment solution prepared in this invention was analyzed and tested using the monoamine oxidase assay kit (continuous monitoring method) produced by Guilin Ulite Medical Electronics Co., Ltd. on the Ulite CA-2000 biochemical analyzer (using 10-fold dilution test), and the MAO activity per unit volume was found to be 908 U / L.

[0076] Step 2, Calculation of the amount of monoamine oxidase treatment solution: Given that the MAO activity of the monoamine oxidase treatment solution is 908 U / L, and 0.5L of a quality control sample with an activity of 75 U / L is required, the amount of monoamine oxidase treatment solution to be measured is approximately 0.5 × 75 × 1000 ÷ 908 ≈ 41 mL.

[0077] 3. Preparation of quality control samples: Take 400 mL of sufficient purified water, add 3.025 g Tris and 0.2 g DTPA in sequence, then adjust the pH to 8.0 ± 0.05 with 6 mol / L hydrochloric acid solution, then add 10 g bovine ovalbumin, 0.5 g sodium azide, and 41 mL monoamine oxidase treatment solution in sequence. Add the remaining purified water by weighing (ρ = 1.000 g / cm3), filter using a 0.45 μm polyethersulfone filter membrane, and finally dispense and freeze-dry the prepared liquid quality control sample to obtain the lyophilized product.

[0078] The comparison of detection results for different embodiments is shown in Tables 3 and 4.

[0079] Table 3. Significance analysis of the stability of quality control samples after reconstitution and storage at 2-8℃ for 7 days.

[0080]

[0081] As shown in Table 3, the stability analysis of the different concentration ranges of the quality control samples in the three examples after reconstitution and storage at 2-8℃ for 7 days showed no significant difference (P value ≥ 0.05).

[0082] Table 4. Significance analysis of the stability of quality control samples after reconstitution and storage at -20℃ for 32 days.

[0083]

[0084] As shown in Table 4, the stability analysis of the three embodiment schemes after reconstitution of the quality control samples at different concentration ranges and storage at -20℃ for 32 days showed no significant difference (P value ≥ 0.05).

[0085] The stability effect of MAO enzyme treatment before and after treatment is shown in Tables 5 and 6.

[0086] According to the quality control preparation method in Example 1, two levels of quality control samples were prepared using MAO before and after treatment under the same enzyme activity conditions. The changes of the quality control samples after reconstitution and storage at -20℃ for 32 days were compared and the stability significance was analyzed.

[0087] Table 5-1 Changes in enzyme activity of quality control samples prepared before MAO enzyme treatment and before freeze-drying.

[0088]

[0089] Table 5-2 Changes in enzyme activity of quality control samples prepared after MAO enzyme treatment before and after freeze-drying.

[0090]

[0091] Table 6-1 Analysis of the significant stability of MAO enzyme-treated quality control samples before and after treatment, after reconstitution and storage at -20℃ for 32 days.

[0092]

[0093] Table 6-2 Analysis of the stability of MAO enzyme-treated quality control samples before and after treatment, and after reconstitution and storage at -20℃ for 32 days.

[0094]

[0095] As shown in Tables 5 and 6, the enzyme activity of the quality control sample prepared before MAO enzyme treatment was about 40% lower than that after treatment. This indicates that the MAO treatment solution can significantly reduce the loss rate of MAO enzyme activity during the preparation of the quality control sample. At the same time, the stability analysis of the quality control sample after reconstitution and storage at -20℃ for 32 days showed that the test values ​​of the untreated group at 0 days and 32 days changed by nearly 20%, with a significant decreasing trend.

[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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a monoamine oxidase treatment solution, characterized in that, The steps include the following: Step 1, Prepare buffer solution: Weigh 80% of the total weight of purified water, and dissolve 100 g / L to 200 g / L of 2-hydroxypropyl-beta-cyclodextrin, 100 g / L to 200 g / L of protective agent and 7.88 g / L of Tris-hydrochloric acid in sequence to obtain a mixture. Adjust the pH of the mixture to 7.5-8.0, and then measure it to 1000 g with purified water to obtain the buffer solution. Step 2, preparation of enzyme inclusion solution: First, weigh 0.25 g / L to 0.5 g / L of lyophilized MAO enzyme at 4±1℃ and add it to a continuously stirred buffer solution. Stir for 30 minutes to obtain a completely dissolved enzyme solution. Next, sonicate the completely dissolved enzyme solution for 10 to 20 minutes, ensuring that the temperature of the enzyme solution changes within the range of 23±5℃ during this period. Finally, transfer the enzyme solution to an environment of 4±1℃ for equilibration for 1 hour to obtain the enzyme inclusion solution. Step 3: Add 0.25 g / L to 0.5 g / L flavin adenine dinucleotide and 0.25 g / L to 0.5 g / L hydrophilic nonionic surfactant sequentially to the continuously stirred enzyme inclusion solution at 4±1℃. Continue stirring for 30 minutes, filter, and transfer to a light-proof brown glass bottle for storage at 2 to 8℃ to obtain the monoamine oxidase treatment solution.

2. The method for preparing monoamine oxidase treatment solution according to claim 1, characterized in that, In step 1, the pH of the mixture is adjusted using 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution.

3. The method for preparing monoamine oxidase treatment solution according to claim 1, characterized in that, The protective agent mentioned in step 1 is one or a combination of mannitol, sucrose, and trehalose.

4. The method for preparing monoamine oxidase treatment solution according to claim 1, characterized in that, In step 2, the buffer solution is continuously stirred using a magnetic stirrer, and the stir bar speed is controlled within 300-500 rpm.

5. The method for preparing monoamine oxidase treatment solution according to claim 1, characterized in that, The power of the ultrasonic treatment in step 2 is 200 W.

6. The method for preparing monoamine oxidase treatment solution according to claim 1, characterized in that, In step 3, the buffer solution is continuously stirred using a magnetic stirrer, and the stirring speed is controlled within 200-300 rpm.

7. The method for preparing monoamine oxidase treatment solution according to claim 1, characterized in that, The hydrophilic nonionic surfactant mentioned in step 3 is a polyoxyethylene polyoxypropylene ether block copolymer with an HLB value of 25 or higher, and its molecular formula is HO·(C2H4O)m·(C3H6O)n·H, which can be Pluronic L65 or Pluronic F68.

8. The method for preparing monoamine oxidase treatment solution according to claim 1, characterized in that, In step 3, polyvinylidene fluoride with a pore size of 0.45 μm is used for filtration.

9. The use of the monoamine oxidase treatment solution prepared by the method according to any one of claims 1-8 in the preparation of quality control materials for a monoamine oxidase assay kit, characterized in that, Includes the following steps: Step 1, Monoamine oxidase treatment solution determination: The monoamine oxidase treatment solution was analyzed and tested on a biochemical analyzer using a monoamine oxidase assay kit to obtain the MAO activity per unit volume. Step 2, Calculation of the amount of monoamine oxidase treatment solution to be weighed: First, calculate the amount of total MAO activity to be fed based on the theoretical target concentration and preparation amount of the quality control sample. Then, calculate the amount of the monoamine oxidase treatment solution to be weighed based on the MAO unit volume activity obtained in Step 1, and record it as X ml. Step 3, Preparation of quality control samples: Take 80% of the total weight of purified water, and add 20 mmol / L-50 mmol / L of dissolved GOOD solution in sequence. ， Mixed buffer with 0.5 mmol / L to 1 mmol / L complexing agent to obtain a mixed buffer; Adjust the pH of the mixed buffer solution to 7.5-8.0 using 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution. Then, add 10 g / L-20 g / L of protein protectant, 0.5 g / L-1 g / L of preservative, and X ml of the monoamine oxidase treatment solution sequentially. Weigh the solution to the prepared total volume using purified water. After equilibration for 30 minutes, filter the solution through a 0.45-0.22 μm polyethersulfone membrane. Finally, dispense and freeze-dry the prepared liquid quality control sample to obtain the lyophilized product, which is the quality control sample.

10. The application of the monoamine oxidase treatment solution prepared by the method according to claim 9 in the preparation of quality control materials for a monoamine oxidase assay kit, characterized in that, The GOOD ， The s buffer can be one of HEPES, MOPS, and Tris-hydrochloric acid buffer; the complexing agent can be one or more of EDTA, EGTA, and DTPA; the protein protectant can be one or more of bovine serum albumin, human serum albumin, and ovalbumin; and the preservative is sodium azide or Proclin 300.

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