Lactic dehydrogenase pretreatment method and application thereof in glutamic oxalacetic transaminase determination reagent

Abstract

The invention relates to the technical field of medical examination, in particular to a lactic dehydrogenase pretreatment method and application of the lactic dehydrogenase pretreatment method in an aspartate transaminase determination reagent, purified water is added into a mixing container, a biological buffer solution, polyethylene glycol, disodium ethylene diamine tetraacetate, polyoxyethylene lauryl ether and sodium cholate are weighed and stirred to be dissolved into the mixing container, and the mixture is stirred to be uniform; adjusting the pH value to obtain a pretreatment solution; the NAD dependent-D-lactic dehydrogenase is dissolved in the pretreatment solution, after stirring and standing balancing are conducted for 15 minutes through a magnetic stirrer, insoluble substances are filtered out, the lactic dehydrogenase solution treated through the method can stably coexist with a high-salt solution, the price is low, the production cost of manufacturers is greatly reduced, and the method is suitable for industrial production. The problems that under the alkaline condition, the activity of lactic dehydrogenase is low, the enzyme dosage has to be increased, and the production cost is too high due to the fact that more expensive NAD dependent type-L-lactic dehydrogenase is used are solved.

Description

Technical Field

[0001] The present invention relates to the field of medical testing technology, in particular to a lactate dehydrogenase pretreatment method and an application thereof in an aspartate aminotransferase determination reagent. Background Art

[0002] Lactate dehydrogenase (LDH, EC 1.1.1.27) comprises two major types: NAD-dependent lactate dehydrogenases (nLDHs) and NAD-independent lactate dehydrogenases (iLDHs). It is a commonly used biochemical reagent and clinical diagnostic enzyme. Currently, the most widely used diagnostic reagent is the NAD-dependent lactate dehydrogenase, which is divided into two major types: NAD-dependent L-lactate dehydrogenase (L-type LDH) and NAD-dependent D-lactate dehydrogenase (D-type LDH). In terms of stability, the L-type is more stable under neutral and alkaline conditions, while the D-type is more stable under neutral conditions. Both enzyme types are pH-sensitive, with an optimal pH of 6.8 to 7.6. In terms of price, the L-type is relatively expensive, so the L-type has a clear disadvantage in the production cost of diagnostic reagents.

[0003] Currently, LDH, as a key enzyme in diagnostic reagents, is most widely used in the preparation of assays for AST (aspartate aminotransferase) and ALT (alanine aminotransferase). The assay principle involves the use of a high-concentration substrate solution of aspartate or alanine, catalyzed by AST or ALT, to generate a secondary reactant. Malate dehydrogenase (MDH) and LDH, or LDH alone, catalyze the redox reaction of this secondary reactant with the reduced coenzyme NAD+ (NADH) to produce oxidized NAD+. The activity of AST or ALT in the sample is calculated by monitoring the rate of change in absorbance at 340 nm. Because LDH is a protease, it is unstable in high-salt solutions, prone to protein precipitation and co-precipitation with other reagent components, compromising proper reagent preparation and achieving a long-lasting effect. Furthermore, NADH, a strongly reducing substance, is extremely unstable in acidic or neutral pH solutions and prone to self-oxidative degradation, leading to substrate depletion during the reagent detection reaction. For these reasons, current reagent manufacturers all employ a dual-reagent approach, placing NADH and high-concentration amino acid salts separately in alkaline and neutral / acidic solutions. To avoid co-precipitation, proteases such as LDH are placed in a slightly alkaline NADH solution. However, this approach fails to address the low activity of lactate dehydrogenase under alkaline conditions, forcing manufacturers to increase enzyme dosage to achieve long-term stability. Furthermore, this approach also results in high production costs associated with using the more expensive NAD-dependent L-lactate dehydrogenase. Summary of the Invention

[0004] The purpose of the present invention is to provide a lactate dehydrogenase pretreatment method and its application in aspartate aminotransferase determination reagent, aiming to solve the problem of low LDH enzyme activity under alkaline conditions, which requires increasing the enzyme dosage and using the more expensive L-type LDH, resulting in excessively high production costs.

[0005] To achieve the above object, in a first aspect, the present invention provides a lactate dehydrogenase pretreatment method, comprising the following steps:

[0006] Purified water is added to a mixing container, and biological buffer, polyethylene glycol, disodium ethylenediaminetetraacetic acid, polyoxyethylene lauryl ether, and sodium cholate are weighed and dissolved in the mixing container respectively, and the pH value is adjusted to obtain a pretreatment solution;

[0007] NAD-dependent-D-lactate dehydrogenase was dissolved in the pretreatment solution, stirred with a magnetic stirrer, and allowed to stand for 15 minutes, and then insoluble matter was filtered off to obtain a pretreatment reagent.

[0008] Among them, the purified water accounts for about 80% of the total amount required for preparation, and the biological buffer is GOOD ，SThe biological buffer is one of 2-(N-morpholino)ethanesulfonic acid monohydrate, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, tris(hydroxymethyl)methylglycine and N-(2-hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid, and the polyethylene glycol is one of PEG2000, PEG3000, PEG4000, PEG6000 and PEG8000.

[0009] Wherein, the pH value is adjusted by using 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution, and the pH value is 6-7.

[0010] The NAD-dependent-D-lactate dehydrogenase activity concentration is 40-50 KU / L, the magnetic stirrer speed is 500-1000 rpm, the stirring time is 30-40 minutes, and the stirring temperature is 25-30°C.

[0011] In a second aspect, the present invention also provides the use of lactate dehydrogenase in an aspartate aminotransferase assay reagent, wherein the pretreatment reagent is prepared using the lactate dehydrogenase pretreatment method described in the first aspect, and the application of the pretreatment reagent includes the preparation of the aspartate aminotransferase assay reagent and verification of the precipitation phenomenon.

[0012] Wherein, the preparation of the aspartate aminotransferase assay reagent includes a first reagent and a second reagent;

[0013] The preparation of the first reagent comprises the following steps:

[0014] Add about 50% of the total reagent preparation volume of purified water to the mixing container, add and dissolve the full batch of trishydroxyaminomethane, disodium EDTA, sucrose, bovine serum albumin, and sodium azide one by one, stir thoroughly for at least 15 minutes, and dilute with purified water to 90% of the total preparation volume;

[0015] Use 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution to adjust the pH value to 9.1-9.3;

[0016] Sequentially add and dissolve the full batch of reduced coenzyme I disodium salt and malate dehydrogenase in a mixer, stir for at least 10 minutes, dilute with purified water to the total preparation volume, let it stand for 15 minutes, filter with a stainless steel filter and a 0.4μm nylon filter membrane, and store in a cold storage at 2-8°C.

[0017] The preparation of the second reagent comprises the following steps:

[0018] Add about 50% of the total reagent volume to the mixing container with purified water, then add and dissolve the full batch of trishydroxyaminomethane, L-aspartic acid, potassium hydroxide, disodium EDTA, and α-ketoglutaric acid one by one, and dilute with purified water to 80% of the total reagent volume;

[0019] Use 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution to adjust the pH value to the range of 7.0-7.4;

[0020] According to the enzyme activity conversion, add a sufficient volume of LDH treatment solution, stir for 15 minutes, dilute with purified water to the total preparation volume, let it stand for 30 minutes, filter with a stainless steel filter and a 0.4μm nylon filter membrane, and store in a cold storage at 2-8℃.

[0021] Among them, the pH value of the trihydroxyaminomethane buffer in the preparation of the first reagent is 9.1-9.3, 30 mmol / L, the disodium ethylenediaminetetraacetic acid, 2 g / L, the disodium salt of reduced coenzyme I ((NADH-2Na), 0.22 g / L, the malate dehydrogenase, 1.25-2 KU / L, the bovine serum albumin, 1 g / L, the sucrose, 5 g / L, the sodium azide, 1 g / L; the pH value of the trihydroxyaminomethane buffer in the preparation of the second reagent is 7.0-7.4, 260 mmol / L, L-aspartic acid, 500-600 mmol / L, potassium hydroxide, 50 g / L, α-ketoglutaric acid, 15 g / L, disodium ethylenediaminetetraacetic acid, 2 g / L, D-type LDH (treatment solution), 12-15 KU / L, sodium azide, 1 g / L.

[0022] The lactate dehydrogenase pretreatment method of the present invention comprises the following steps: adding purified water into a mixing container, weighing and stirring respectively a biological buffer solution, polyethylene glycol, disodium ethylenediaminetetraacetic acid, polyoxyethylene lauryl ether, and sodium cholate to dissolve in the mixing container, adjusting the pH value, and obtaining a pretreatment solution; dissolving NAD-dependent-D-lactate dehydrogenase in the pretreatment solution, stirring with a magnetic stirrer, equilibrating the solution for 15 minutes, and filtering out insoluble matter. The lactate dehydrogenase solution treated by the method can stably coexist with a high-salt solution and is low-priced NAD-dependent-D-lactate dehydrogenase, greatly reducing the production cost of manufacturers and solving the problem of low lactate dehydrogenase activity under alkaline conditions, which necessitates increasing the enzyme dosage and the excessively high production cost caused by using the more expensive NAD-dependent-L-lactate dehydrogenase. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 The present invention provides a flow chart of the lactate dehydrogenase pretreatment method.

[0025] Figure 2 This is a monitoring diagram of an appearance monitoring experiment of the application of lactate dehydrogenase in the aspartate aminotransferase determination reagent provided by the present invention. DETAILED DESCRIPTION

[0026] 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.

[0027] See also Figures 1 to 2 In a first aspect, the present invention provides a lactate dehydrogenase pretreatment method, comprising the following steps:

[0028] S1: adding purified water into a mixing container, weighing and stirring to dissolve biological buffer, polyethylene glycol, disodium ethylenediaminetetraacetic acid, polyoxyethylene lauryl ether, and sodium cholate into the mixing container, and adjusting the pH value to obtain a pretreatment solution;

[0029] Specifically, add about 80% of the total amount of purified water required for the preparation into the mixing container, accurately weigh and stir to dissolve the full batch of GOOD ,S Add biological buffer, polyethylene glycol, disodium ethylenediaminetetraacetic acid, polyoxyethylene lauryl ether BRIJ35, and sodium cholate to a mixing container. Continue stirring for 5 minutes after they are completely dissolved. Adjust the pH to 6-7 with 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution.

[0030] Among them, GOOD ,S The pH value of the biological buffer solution is 6-7, 20-50 mmol / L, polyethylene glycol, 6-15 g / L, disodium ethylenediaminetetraacetic acid, 0.5-1 g / L, polyoxyethylene lauryl ether BRIJ35, 0.5-1 g / L, sodium cholate, 0.1-0.5 g / L.

[0031] S2: dissolving NAD-dependent-D-lactate dehydrogenase in the pretreatment solution, stirring with a magnetic stirrer, and allowing the solution to stand for 15 minutes, and then filtering out insoluble matter.

[0032] Specifically, the D-type LDH derived from microorganisms is dissolved in the pretreatment liquid at an activity concentration of 40 to 50 KU / L, stirred at 500 to 1000 rpm with a magnetic stirrer at 25 to 30° C. for 30 to 40 minutes, and the solution is allowed to stand for 15 minutes for equilibrium, and the insoluble matter is filtered with a 0.2 micron pore size nylon filter membrane.

[0033] In a second aspect, the present invention also provides the use of lactate dehydrogenase in an aspartate aminotransferase assay reagent, a pretreatment reagent prepared using the lactate dehydrogenase pretreatment method, and the application of the pretreatment reagent includes the preparation of the aspartate aminotransferase assay reagent and verification of the precipitation phenomenon.

[0034] The preparation of the aspartate aminotransferase assay reagent includes a first reagent and a second reagent;

[0035] The first reagent is prepared by adding purified water accounting for approximately 50% of the total reagent preparation amount to a mixing container, sequentially adding and dissolving the full batch of trishydroxyaminomethane, disodium ethylenediaminetetraacetic acid, sucrose, bovine serum albumin, and sodium azide, stirring thoroughly for at least 15 minutes, and diluting with purified water to 90% of the total preparation amount. Adjusting the pH value to a range of 9.1 to 9.3 using a 6 mol / L hydrochloric acid solution or a 10 mol / L sodium hydroxide solution. Then, sequentially adding and dissolving the full batch of reduced coenzyme I disodium salt and malate dehydrogenase to the mixer, stirring for at least 10 minutes, diluting with purified water to the total preparation amount, allowing to stand for 15 minutes, filtering with a stainless steel filter and a 0.4 μm nylon filter membrane, and storing in a cold storage at 2 to 8°C.

[0036] Among them, the pH value of trishydroxyaminomethane buffer is 9.1-9.3, 30 mmol / L, disodium ethylenediaminetetraacetic acid, 2 g / L, disodium salt of reduced coenzyme I ((NADH-2Na), 0.22 g / L, malate dehydrogenase, 1.25-2 KU / L, bovine serum albumin, 1 g / L, sucrose, 5 g / L, sodium azide, 1 g / L.

[0037] Preparation of the second reagent: Add purified water accounting for approximately 50% of the total reagent preparation volume to a mixing container, add and dissolve the full batch of trihydroxyaminomethane, L-aspartic acid, potassium hydroxide, disodium ethylenediaminetetraacetic acid, and α-ketoglutaric acid in sequence, dilute with purified water to 80% of the total reagent preparation volume, adjust the pH value to a range of 7.0 to 7.4 with 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution, add a sufficient volume of LDH treatment solution based on enzyme activity conversion, stir for 15 minutes, dilute with purified water to the total preparation volume, let stand for 30 minutes, filter with a stainless steel filter and a 0.4 μm nylon filter membrane, and store in a cold storage at 2 to 8°C.

[0038] Among them, the pH value of trishydroxyaminomethane buffer is 7.0-7.4, 260mmol / L, L-aspartic acid, 500-600mmol / L potassium hydroxide, 50g / L, α-ketoglutaric acid, 15g / L, disodium ethylenediaminetetraacetic acid, 2g / L, D-type LDH (treatment solution), 12-15KU / L, sodium azide, 1g / L.

[0039] Aspartate aminotransferase assay reagent test:

[0040] The test temperature is 37°C and the test wavelength is 340nm

[0041] Table 1 Reagent test parameters

[0042]

[0043]

[0044] After mixing, place at 37°C. Measure the A1 value after 2 minutes, and the A2 value after another 2 minutes. Calculate ΔA / min. Use a blank tube to adjust to zero before measurement.

[0045] Calculation method:

[0046] Aspartate aminotransferase assay reagent test performance:

[0047] Store sealed and protected from light at 2°C to 8°C. The shelf life is 12 months. The analytical sensitivity is that the absorbance change rate (△A / min) is greater than 0.0002 when testing 1 U / L of aspartate aminotransferase. The linear correlation coefficient r in the range of [8, 600] U / L is ≥ 0.990. The reagent has no significant effect when endogenous interfering substances in the sample, such as bilirubin ≤ 400 mg / L, vitamin C ≤ 300 mg / L, and fat emulsion ≤ 5 g / L, are present.

[0048] Verification of precipitation phenomenon

[0049] The aspartate aminotransferase assay reagent II was prepared using untreated and pretreated D-type LDH, respectively, and was tested by an oscillation acceleration test (shaking at 200 rpm for 10 hours per day for 3 consecutive days at 37°C) and an appearance monitoring test of conventional storage samples (stored at 2°C to 8°C for 12 months).

[0050] Table 2 Reagent Appearance Monitoring Table

[0051]

[0052] In order to better understand the present invention, the following examples are provided for further explanation:

[0053] Example 1 Preparation of pretreatment solution

[0054] Table 3 Pretreatment liquid preparation table

[0055] drug Solution 1 Option 2 Option 3 <![CDATA[GOOD ,S Biological Buffers]]> HEPES (25mmol / L, pH 6.5) HEPPSO (30mmol / L, PH6.0) MES (45mmol / L, pH7.0) polyethylene glycol PEG8000 (6g / L) PEG4000 (8g / L) PEG2000 (12g / L) Disodium EDTA 1g / L 0.5g / L 0.5g / L Polyoxyethylene moon 0.5g / L 1g / L 0.5g / L Sodium cholate 0.2g / L 0.2g / L 0.5g / L D-type LDH 50KU / L (Roche, freeze-dried) 40KU / L((Toyobo, freeze-dried) 40KU / L((Lan Yi, freeze-dried)

[0056] Example 2 Preparation of Aspartate Aminotransferase Assay Reagent

[0057] The LDH pretreatment solution prepared in the three solutions of Example 1 was used to prepare the reagent and the performance of the reagent was tested.

[0058] Reagent 1 includes:

[0059]

[0060] Reagent 2 includes:

[0061]

[0062] Table 4 Performance verification of main reagents

[0063]

[0064] The above disclosure is merely a preferred embodiment of the lactate dehydrogenase pretreatment method of the present invention and its application in the aspartate aminotransferase assay reagent. Of course, this does not limit the scope of the present invention. A person skilled in the art will understand that implementing all or part of the processes of the above embodiments and making equivalent changes according to the claims of the present invention still fall within the scope of the invention.

Claims

1. Lactate dehydrogenase pretreatment method, characterized in that, The following steps are involved: Purified water is added to a mixing container, and biological buffer, polyethylene glycol, disodium ethylenediaminetetraacetic acid, polyoxyethylene lauryl ether, and sodium cholate are weighed and dissolved in the mixing container respectively, and the pH value is adjusted to obtain a pretreatment solution; NAD-dependent-D-lactate dehydrogenase was dissolved in the pretreatment solution, stirred with a magnetic stirrer, and allowed to stand for 15 minutes, and then insoluble matter was filtered off to obtain a pretreatment reagent.

2. The lactate dehydrogenase pretreatment method according to claim 1, wherein The purified water accounts for about 80% of the total amount required for preparation, and the biological buffer is GOOD ，S , is one of 2-(N-morpholino)ethanesulfonic acid monohydrate, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, tris(hydroxymethyl)methylglycine and N-(2-hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid, and the polyethylene glycol is one of PEG2000, PEG3000, PEG4000, PEG6000 and PEG8000.

3. The lactate dehydrogenase pretreatment method according to claim 1, wherein The pH value is adjusted using a 6 mol / L hydrochloric acid solution or a 10 mol / L sodium hydroxide solution, and the pH value is 6-7.

4. The lactate dehydrogenase pretreatment method according to claim 1, wherein The NAD-dependent-D-lactate dehydrogenase activity concentration is 40-50 KU / L, the magnetic stirrer speed is 500-1000 rpm, the stirring time is 30-40 minutes, and the stirring temperature is 25-30°C.

5. Application of lactate dehydrogenase in a reagent for determining aspartate aminotransferase, wherein the pretreatment reagent is prepared by the lactate dehydrogenase pretreatment method according to claim 1, characterized in that: The pretreatment reagent is used for the preparation of aspartate aminotransferase determination reagent and the verification of precipitation phenomenon.

6. The use of lactate dehydrogenase in a reagent for determining aspartate aminotransferase according to claim 5, wherein: The preparation of the aspartate aminotransferase assay reagent includes a first reagent and a second reagent; The preparation of the first reagent comprises the following steps: Add about 50% of the total reagent preparation volume of purified water to the mixing container, add and dissolve the full batch of trishydroxyaminomethane, disodium EDTA, sucrose, bovine serum albumin, and sodium azide one by one, stir thoroughly for at least 15 minutes, and dilute with purified water to 90% of the total preparation volume; Use 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution to adjust the pH value to 9.1-9.3; Sequentially add and dissolve the full batch of reduced coenzyme I disodium salt and malate dehydrogenase in a mixer, stir for at least 10 minutes, dilute with purified water to the total preparation volume, let it stand for 15 minutes, filter with a stainless steel filter and a 0.4μm nylon filter membrane, and store in a cold storage at 2-8°C. The preparation of the second reagent comprises the following steps: Add about 50% of the total reagent volume to the mixing container with purified water, then add and dissolve the full batch of trishydroxyaminomethane, L-aspartic acid, potassium hydroxide, disodium EDTA, and α-ketoglutaric acid one by one, and dilute with purified water to 80% of the total reagent volume; Use 6 mol / L hydrochloric acid solution or 10 mol / L sodium hydroxide solution to adjust the pH value to the range of 7.0-7.4; According to the enzyme activity conversion, add a sufficient volume of LDH treatment solution, stir for 15 minutes, dilute with purified water to the total preparation volume, let it stand for 30 minutes, filter with a stainless steel filter and a 0.4μm nylon filter membrane, and store in a cold storage at 2-8℃.

7. The use of lactate dehydrogenase in a reagent for determining aspartate aminotransferase according to claim 6, wherein: The pH value of the trihydroxyaminomethane buffer in the preparation of the first reagent is 9.1-9.3, 30 mmol / L, the disodium ethylenediaminetetraacetic acid, 2 g / L, the disodium salt of reduced coenzyme I ((NADH-2Na), 0.22 g / L, the malate dehydrogenase, 1.25-2 KU / L, the bovine serum albumin, 1 g / L, the sucrose, 5 g / L, and the sodium azide, 1 g / L; the pH value of the trihydroxyaminomethane buffer in the preparation of the second reagent is 7.0-7.4, 260 mmol / L, L-aspartic acid, 500-600 mmol / L, potassium hydroxide, 50 g / L, α-ketoglutaric acid, 15 g / L, disodium ethylenediaminetetraacetic acid, 2 g / L, D-type LDH (treatment solution), 12-15 KU / L, and sodium azide, 1 g / L.

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