Alanine aminotransferase detection reagent

Abstract

The alanine aminotransferase detection reagent comprises the following components: a buffer solution, magnesium chloride, L-alanine, alpha-ketoglutaric acid, pyruvate oxidase, 4-AAP, a Trinder's reagent, peroxidase, FAD, TPP, an excipient, a stabilizer, a surfactant and a preservative. When the detection reagent is used for detection, the substrate alanine reacts with the alanine aminotransferase to generate pyruvic acid, then the pyruvic acid reacts with the pyruvate oxidase to generate hydrogen peroxide, the generated hydrogen peroxide further oxidizes chromogen pairs, and colored stable quinone compounds are generated through reaction. Compared with a dehydrogenase method, the method disclosed by the invention has the advantages that the linear range is wide, the accuracy and the sensitivity are high, and the repeatability is good; in addition, the excipient selected by the invention can ensure that the reagent has better morphology and remelting solubility when being prepared into the microspheres, and has higher stability.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic reagents, and particularly to a reagent for detecting alanine aminotransferase. Background Art

[0002] Alanine aminotransferase is widely present in the body and has high activity in the liver, skeletal muscle, kidney, and heart. The concentration of alanine aminotransferase in these tissues is much higher than that in serum. Therefore, when a small amount of tissue necrosis occurs, alanine aminotransferase in the necrotic tissue is released into the blood, increasing the concentration of alanine aminotransferase in the blood. Clinically, alanine aminotransferase is mainly used to assist in evaluating liver function and is a sensitive indicator reflecting liver injury.

[0003] Currently, clinically, the concentration of alanine aminotransferase in serum is usually measured by the dehydrogenase method. In the dehydrogenase method, reduced coenzyme I (NADH) in the reagent is converted into oxidized coenzyme I (NAD+). Based on the fact that the concentration of alanine aminotransferase is proportional to the decrease in the amount of reduced coenzyme I (NADH), the concentration of alanine aminotransferase is measured by monitoring the decrease in the amount of reduced coenzyme I (NADH). However, the activity of reduced coenzyme I (NADH) is easily interfered by various factors and is easily oxidized itself, directly affecting the measurement result of alanine aminotransferase, with low detection accuracy and a narrow linear range. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a reagent for detecting alanine aminotransferase to solve the problems of low accuracy and narrow linear range of existing detection reagents.

[0005] To solve the above problems, the present invention adopts the following solutions:

[0006] A reagent for detecting alanine aminotransferase, comprising the following components: buffer, magnesium chloride, L-alanine, α-ketoglutaric acid, pyruvate oxidase, 4-AAP (4-aminoantipyrine), Trinder's reagent, peroxidase, FAD (flavin adenine dinucleotide disodium hydrate), TPP (thiamine pyrophosphate), excipient, stabilizer, surfactant, and preservative.

[0007] Further, the concentration of the buffer is 50 - 200 mmol / L;

[0008] The concentration of the magnesium chloride is 1 - 20 mmol / L;

[0009] The concentration of the L-alanine is 10 - 100 mmol / L;

[0010] The concentration of the α-ketoglutaric acid is 1-30 mmol / L;

[0011] The concentration of the pyruvate oxidase is 10-100 KU / L;

[0012] The concentration of the 4-AAP is 2-20 g / L;

[0013] The concentration of the Trinder's reagent is 2-20 g / L;

[0014] The concentration of the peroxidase is 10-100 KU / L;

[0015] The concentration of the FAD is 0.1-1 g / L;

[0016] The concentration of the TPP is 0.1-1 g / L;

[0017] The concentration of the excipient is 10-100 g / L;

[0018] The concentration of the stabilizer is 1-10 g / L;

[0019] The concentration of the surfactant is 0.01-0.2 g / L;

[0020] The concentration of the preservative is 0.01-0.1 g / L.

[0021] Furthermore, the buffer solution includes at least one of Tris buffer solution, PBS buffer solution, imidazole buffer solution, and diethanol buffer solution.

[0022] Furthermore, the Trinder's reagent is one of MAOS (sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline monohydrate), TOOS (sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline), TOPS (sodium N-ethyl-N-(3-sulfopropyl)-3-methylaniline), and ADOS (sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline dihydrate).

[0023] Furthermore, the excipient includes at least one of sorbitol, mannitol, PEG 6000 (polyethylene glycol 6000), PEG 8000 (polyethylene glycol 8000), dextran 10,000, dextran 40,000, and dextran 50,000.

[0024] Furthermore, the stabilizer includes at least one of bovine serum albumin, trehalose, and sucrose.

[0025] Further, the surfactant includes at least one of Tween 20, Tween 80, Brij L23 (polyoxyethylene lauryl ether), Triton X-100, SDS (sodium dodecyl sulfate), and SDBS (sodium dodecyl benzene sulfonate).

[0026] Further, the preservative includes at least one of Kathon, Proclin 300, trichlorocarbanilide, and sodium azide.

[0027] The present invention adopts the above technical solutions and has the following advantages:

[0028] When the detection reagent of the present invention is used for detection, the substrate alanine reacts with alanine aminotransferase to produce pyruvic acid, which then reacts with pyruvate oxidase to generate hydrogen peroxide. The generated hydrogen peroxide further oxidizes the chromogenic pair, and a colored and stable quinone compound is formed by the reaction. Thus, the concentration of alanine aminotransferase can be quantitatively reflected by detecting the absorbance. Compared with the dehydrogenase method, the method of the present invention has a wide linear range, high accuracy and sensitivity, and good repeatability. In addition, the excipients selected in the present invention can ensure that the reagent has good morphology and re-melting solubility when prepared into microspheres, and has high stability. Description of the Drawings

[0029] Figure 1 It is the linear range diagram of the detection reagent in Example 1;

[0030] Figure 2 It is the linear range diagram of the detection reagent in Example 2;

[0031] Figure 3 It is the linear range diagram of the detection reagent in Example 3;

[0032] Figure 4 It is the correlation diagram of the detection reagent in Example 1;

[0033] Figure 5 It is the correlation diagram of the detection reagent in Example 2;

[0034] Figure 6 It is the correlation diagram of the detection reagent in Example 3. Detailed Embodiments

[0035] The following further illustrates the present invention through examples, but it does not limit the present invention. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

[0036] Example 1

[0037] An alanine aminotransferase detection reagent, comprising the following components: 50 mmol / L Tris buffer, 1 mmol / L magnesium chloride, 10 mmol / L L-alanine, 5 mmol / L α-ketoglutaric acid, 10 KU / L pyruvate oxidase, 20 g / L MAOS, 2 g / L 4-AAP, 10 KU / L peroxidase, 0.1 g / L FAD, 0.1 g / L TPP, 80 g / L PEG 6000, 5 g / L sucrose, 0.01 g / L Tween 20, 0.01 g / L sodium azide, pH 6.5.

[0038] Example 2

[0039] An alanine aminotransferase detection reagent, comprising the following components: 100 mmol / L PBS (phosphate buffer), 5 mmol / L magnesium chloride, 20 mmol / L L-alanine, 1 mmol / L α-ketoglutaric acid, 30 KU / L pyruvate oxidase, 2 g / L TOOS, 10 g / L 4-AAP, 40 KU / L peroxidase, 0.2 g / L FAD, 0.4 g / L TPP, 100 g / L mannitol, 5 g / L BSA (bovine serum albumin), 5 g / L trehalose, 0.05 g / L SDS, 0.03 g / L PC 300, pH 6.0.

[0040] Example 3

[0041] An alanine aminotransferase detection reagent, comprising the following components: 200 mmol / L PBS, 20 mmol / L magnesium chloride, 100 mmol / L L-alanine, 30 mmol / L α-ketoglutaric acid, 100 KU / L pyruvate oxidase, 15 g / L TOPS, 20 g / L 4-AAP, 100 KU / L peroxidase, 1 g / L FAD, 1 g / L TPP, 10 g / L dextran 10,000, 1 g / L BSA, 0.2 g / L Triton X-100, 0.1 g / L triclocarban, pH 5.2.

[0042] Example 4

[0043] The performance of the detection reagent prepared in the examples of the present invention was evaluated by detecting alanine aminotransferase.

[0044] The detection reagent of the present invention was prepared into microspheres and placed in a chip to form a detection chip. Alanine aminotransferase was detected by the detection chip. The preparation method of the detection reagent in microsphere form is as follows:

[0045] Buffer solution, magnesium chloride, L-alanine, α-ketoglutaric acid, pyruvate oxidase, chromogenic pair, peroxidase, FAD, TPP, excipient, stabilizer, surfactant and preservative are mixed in proportion with water as the solvent, adjusted to a preset pH value, and then dropped into liquid nitrogen in droplets to form ice balls, and the ice balls are freeze-dried to obtain detection microspheres.

[0046] In an environment with an air humidity of 15-20%, the microspheres prepared by the detection reagent of the embodiment of the present invention are loaded into the chip body of the microfluidic chip, a detection sample is injected into the microfluidic chip, and then an automatic biochemical analyzer is used for detection to detect the change value of the absorbance at 37°C, and the change value is positively correlated with the concentration of alanine aminotransferase.

[0047] Combined with specific test experiments, the performance of the detection reagent prepared by the present invention is described, including linear range, correlation, accuracy, precision and stability.

[0048] 1. Linear range test

[0049] The test method is as follows. Use a low-concentration sample close to the lower limit of the linear range (0-20 U / L) and a high-concentration sample close to the upper limit of the linear range (900-1000 U / L). The low-concentration sample and the high-concentration sample are mixed in proportion to form 5 samples, and the mixing ratios of each sample are shown in Table 1.

[0050] Table 1 shows the mixing ratios of the high-concentration sample and the low-concentration sample in 5 samples

[0051] Sample number 1 2 3 4 5 High-concentration sample 0 20％ 40％ 60％ 100％ Low-concentration sample 100％ 80％ 60％ 40％ 0

[0052] The alanine aminotransferase concentrations in plasma samples with 5 dilution concentrations are respectively tested by the detection reagents of Examples 1-3 of the present invention. Each plasma sample with a dilution concentration is tested 3 times, and the average value (yi) of the measured concentration values of alanine aminotransferase in the plasma samples with 5 dilution concentrations is calculated respectively. Taking the concentration (xi) after dilution of each sample as the independent variable and the average value (yi) of the measured concentration value of each sample as the dependent variable, a linear regression equation is obtained. As Figures 1-3 shown, the upper limits of the linear ranges of the detection reagents of Examples 1-3 all reach above 1400 U / L, which is significantly improved compared with the dehydrogenase method.

[0053] 2. Correlation and accuracy test

[0054] Prepare 15 plasma samples with different concentrations of alanine aminotransferase (low value: 0 - 40 U / L, medium value: 40 - 150 U / L, high value: 150 - 1500 U / L). Use the microfluidic chips prepared with the detection reagents of Examples 1 - 3 to detect the concentration of alanine aminotransferase in each of the 15 samples. Also, use the wet reagents of Beckman AU480 biochemical analyzer to detect the concentration of alanine aminotransferase in each plasma sample as the target value. Calculate the deviation between the concentration measured by this method and the target value, and then the accuracy of this method can be analyzed (calculate the absolute deviation in the low-value range and the relative deviation in the medium and high-value ranges). The test results are shown in Table 2.

[0055] Table 2: Accuracy test results of Examples 1 - 3 of the present invention

[0056]

[0057] Compared with the test results of wet reagents, for the low-value samples detected by the detection reagents of Examples 1 - 3 of the present invention, the absolute deviation is less than 5 U / L. For the medium and high-value samples, the deviation of the measured values compared with the test results of wet reagents is less than 10%. The accuracy of this method is good. Taking the concentration measured by this method as the independent variable and the concentration of alanine aminotransferase in each plasma sample detected by the wet reagents of Beckman AU480 biochemical analyzer as the dependent variable, a linear regression equation is obtained. As Figures 4-6 shown, the correlation coefficient r is greater than 0.99, indicating good correlation of this method.

[0058] 3. Repeatability

[0059] Use the detection reagents of Examples 1 - 3 of the present invention with the same batch number to perform 10 repeated determinations on low, medium, and high-concentration plasma (low value: 0 - 40 U / L, medium value: 40 - 150 U / L, high value: 150 - 1500 U / L) respectively. Calculate the average value and the standard deviation (S) of the measured values, and calculate the within-batch coefficient of variation (CV). The results are shown in Table 3.

[0060] Table 3: Repeatability test results of samples with different concentrations

[0061]

[0062] As can be seen from Table 3, for the detection reagents provided in Examples 1 - 3 of the present invention, the CV of medium and high-value samples is < 3%, and that of low-value samples is < 8%, indicating good repeatability.

[0063] 4. Stability

[0064] High temperature stability: In an environment with a humidity of 15 - 20%, the microfluidic chips prepared with the detection reagents of Examples 1 - 3 of the present invention were sealed in bags and stored in the dark at 37°C for 0, 1, 3, 5, and 7 days. Using low, medium, and high concentration plasma (low value 0 - 40 U / L, medium value 40 - 150 U / L, high value 150 - 1500 U / L) as detection samples, the accuracy of the detection reagents of the examples of the present invention was tested, and the relative deviation should be within ±10.0%. The results are shown in Table 4.

[0065] Table 4: Detection results of high temperature stability of the detection microspheres of Examples 1, 2, and 3 of the present invention

[0066]

[0067] After the detection reagents provided in Examples 1 - 3 of the present invention were stored in a high temperature (37°C) environment for 1, 3, 5, and 7 days, the absolute value of the relative deviation was still within 10%. Therefore, it has good thermal stability and can still ensure the accuracy of its detection results after being stored in a high temperature environment for many days.

[0068] Low temperature stability: In an environment with a humidity of 15 - 20%, the microfluidic chips prepared with the detection reagents of Examples 1 - 3 of the present invention were sealed in bags and stored in the dark at 2 - 8°C for 0, 3, 6, 12, and 18 months. Using plasma with different concentrations as detection samples, the accuracy of the microfluidic chips in the examples of the present invention was tested, and the relative deviation should be within ±10%. The results are shown in Table 5.

[0069] Table 5: Detection results of low temperature stability of the detection microspheres of Examples 1, 2, and 3 of the present invention

[0070]

[0071]

[0072] After the detection reagents provided in Examples 1 - 3 of the present invention were stored at 2 - 8°C for 0, 3, 6, 12, and 18 months, the absolute value of the relative deviation was still within 10%. Therefore, it has good real-time stability and can still ensure the accuracy of its detection results after being stored in the environment for a long time.

[0073] When the detection reagent of the present invention is used for detection, the substrate alanine reacts with alanine aminotransferase to produce pyruvate, which then reacts with pyruvate oxidase to generate hydrogen peroxide. The generated hydrogen peroxide further oxidizes the chromogenic pair, and the reaction generates a colored and stable quinone compound. Thus, the concentration of alanine aminotransferase can be quantified by detecting the increase in absorbance. Compared with the dehydrogenase method for determining the concentration of alanine aminotransferase, the detection reagent of the present invention has a wide linear range, high accuracy, and good repeatability. In addition, the excipient in the kit of the present invention can ensure that the microspheres have good morphology, re-dissolution solubility, and high stability.

[0074] When the detection reagent of the present invention is prepared into microspheres, it has good morphology, re-dissolution solubility, good accuracy, a wide linear range, high stability and precision.

[0075] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An alanine aminotransferase detection reagent, characterized in that, It includes the following components: buffer, magnesium chloride, L-alanine, α-ketoglutaric acid, pyruvate oxidase, 4-AAP, Trinder's reagent, peroxidase, FAD, TPP, excipient, stabilizer, surfactant and preservative.

2. The alanine aminotransferase detection reagent according to claim 1, characterized in that, The concentration of the buffer is 50 - 200 mmol / L; The concentration of the magnesium chloride is 1 - 20 mmol / L; The concentration of the L-alanine is 10 - 100 mmol / L; The concentration of the α-ketoglutaric acid is 1 - 30 mmol / L; The concentration of the pyruvate oxidase is 10 - 100 KU / L; The concentration of the 4-AAP is 2 - 20 g / L; The concentration of the Trinder's reagent is 2 - 20 g / L; The concentration of the peroxidase is 10 - 100 KU / L; The concentration of the FAD is 0.1 - 1 g / L; The concentration of the TPP is 0.1 - 1 g / L; The concentration of the excipient is 10 - 100 g / L; The concentration of the stabilizer is 1 - 10 g / L; The concentration of the surfactant is 0.01 - 0.2 g / L; The concentration of the preservative is 0.01 - 0.1 g / L.

3. The alanine aminotransferase detection reagent according to claim 1, characterized in that, The buffer includes at least one of Tris buffer, PBS buffer, imidazole buffer, and diethanol buffer.

4. The alanine aminotransferase detection reagent according to claim 1, characterized in that, The Trinder's reagent is one of MAOS, TOOS, TOPS, and ADOS.

5. The alanine aminotransferase detection reagent according to claim 1, characterized in that, The excipient includes at least one of sorbitol, mannitol, PEG 6000, PEG 8000, dextran 10,000, dextran 40,000, and dextran 50,000.

6. The alanine aminotransferase detection reagent according to claim 1, characterized in that, The stabilizer includes at least one of bovine serum albumin, trehalose, and sucrose.

7. The alanine aminotransferase detection reagent according to claim 1, characterized in that, The surfactant includes at least one of Tween 20, Tween 80, Brij L23, Triton X-100, SDS, and SDBS.

8. The alanine aminotransferase detection reagent according to claim 1, characterized in that, The preservative includes at least one of Kathon, Proclin300, triclocarban, and sodium azide.

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