Glutamic acid determination kit and determination method

Abstract

The invention provides a glutamic acid determination kit and a determination method, and belongs to the technical field of in-vitro diagnosis. The kit comprises a reagent R1 and a reagent R2, the reagent R1 is composed of a first buffer solution, 4-aminoantipyrine, glutamate oxidase, ascorbic acid enzyme, a surfactant, a stabilizer and a preservative, and the reagent R2 is composed of a second buffer solution, a chromogenic substrate, peroxidase and a preservative. The preparation process of the kit is simple, the raw materials are easy to purchase, and the determination method using the kit has the advantages of being independent of large instruments, simple to operate, short in detection time, high in accuracy, good in precision and good in stability, is suitable for base layer detection, large-scale sample batch detection and the like, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a glutamate determination kit and a determination method. Background Art

[0002] Glutamic acid (Glu) is one of the basic amino acids for nitrogen metabolism in living organisms and is of great significance in metabolism. Glutamic acid is the main component of protein and glutamate is ubiquitous in nature. Glutamate is present in many foods and in the human body. It is both a structural amino acid of protein or peptide and a free amino acid. Glu is the most abundant excitatory amino acid in the central nervous system. During brain development, Glu can facilitate the metabolism of Ca 2+ The transport of Glu plays an important physiological role in the differentiation, migration, growth and survival of neurons. It is involved in a series of complex physiological processes such as learning, memory, and synaptic plasticity regulation, and is closely related to the death of nerve cells and the onset of mental and neurological diseases, such as epilepsy, schizophrenia, Parkinson's disease, and Alzheimer's disease. Studies have found that in pathological conditions such as cerebral ischemia, cerebral infarction, and migraine, while the concentration of Glu in the brain increases, Glu in the blood will also increase accordingly. Therefore, measuring the changes in Glu in the blood can reflect the progress of brain tissue damage. In addition, quantitative detection of Glu in the blood is also of great significance in health assessment and drug screening.

[0003] Conventional different analytical methods have been applied to the detection of glutamate, such as high performance liquid chromatography, capillary electrophoresis, fluorescence detection, and chemiluminescence, among which high performance liquid chromatography is the most commonly used. High performance liquid chromatography is an important branch of chromatography. It uses liquid as the mobile phase and a high-pressure infusion system to pump single solvents with different polarities or mixed solvents of different proportions, buffers and other mobile phases into a chromatographic column equipped with a stationary phase. After the components in the column are separated, they enter the detector for detection, thereby realizing the analysis of the sample. High performance liquid chromatography has the characteristics of high separation efficiency, high sensitivity, and a wide range of applications, but its disadvantage is the "extra-column effect". Between the injection and the detector, in any dead space other than the column (injector, column joint, connecting tube and detection cell, etc.), if the flow pattern of the mobile phase changes, any diffusion and retention of the separated substances will significantly lead to the broadening of the chromatographic peak and the reduction of column efficiency.

[0004] Therefore, it is of practical significance to find a new, efficient, accurate, simple and feasible method for determining glutamate content. Summary of the invention

[0005] In view of this, the present invention aims to provide a glutamate determination kit and a determination method, so as to achieve a simple, accurate and reliable detection of the glutamate content in a blood sample.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a glutamate determination kit, comprising R1 reagent and R2 reagent; wherein: The R1 reagent is composed of a first buffer, 4-aminoantipyrine (4-AAP), glutamate oxidase (L-glutamateoxidase, GLOD), ascorbic acid enzyme (ASO enzyme), a surfactant, a stabilizer and a preservative; the first buffer is selected from one or more of 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO), 3-morpholinopropanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), disodium hydrogen phosphate-sodium dihydrogen phosphate and disodium hydrogen phosphate-potassium dihydrogen phosphate, and its concentration is 50mmol / L~150mmol / L, and the pH is 6.5~7.5; R2 reagent consists of a second buffer, a chromogenic substrate, peroxidase (POD), and a preservative.

[0007] Preferably, in the above-mentioned glutamate determination kit, the R1 reagent consists of a first buffer, 0.1 g / L~2 g / L 4-aminoantipyrine, 1 KU / L~10 KU / L glutamate oxidase, 1 KU / L~6 KU / L ascorbic acid oxidase, 0.1%~0.5% (g / L) surfactant, 1 g / L~50 g / L stabilizer and 0.1%~1.0% (g / L) preservative.

[0008] In the above-mentioned glutamate determination kit, the surfactant is selected from one or more of Triton X-100, Triton X-405, Cremophor EL, propylene glycol block polyether (Pluranic L64), polyoxyethylene lauryl ether (Brij 35), Tween-80 and Tween-20.

[0009] In the above glutamate determination kit, the stabilizer is one or more of bovine serum albumin (BSA), disodium ethylenediaminetetraacetate (EDTA-2Na), dipotassium ethylenediaminetetraacetate (EDTA-2K), sucrose, sorbitol, and mannitol.

[0010] Preferably, in the above-mentioned glutamate determination kit, the R2 reagent consists of a second buffer, 1g / L~5g / L chromogenic substrate, 10KU / L~20KU / L peroxidase and 0.1%~1.0% (g / L) preservative; wherein the second buffer is selected from one or more of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), tris(hydroxymethyl)aminomethane (Tris) and N,N-dihydroxyethylglycine (BICINE), and its concentration is 50mmol / L~150mmol / L, and the pH is 7.5~8.5.

[0011] In the above-mentioned glutamate determination kit, the chromogenic substrate is selected from one or more of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS), 3,5-dichloro-2-hydroxybenzenesulfonate sodium (DHBS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAOS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (HDAOS), 3,5-dichloro-2-hydroxybenzenesulfonic acid (DHBA) and N-ethyl (2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline (F-DAOS).

[0012] Preferably, in the above glutamate determination kit, the preservative is sodium azide and / or ProClin300.

[0013] In a second aspect, the present invention provides a method for preparing the above-mentioned glutamate determination kit, comprising the following steps: Preparation of R1 reagent: prepare a first buffer solution of 50 mmol / L to 150 mmol / L and a pH of 6.5 to 7.5, add a color developing agent 4-AAP, GLOD, ASO enzyme, a surfactant, a stabilizer and a preservative to the first buffer solution in sequence, and mix after constant volume to obtain R1 reagent; Preparation of R2 reagent: prepare the second buffer solution, add the color developing substrate, POD and preservative to the second buffer solution in sequence, make up to volume and mix well to obtain R2 reagent.

[0014] In a third aspect, the present invention provides a method for qualitatively and / or quantitatively determining glutamate using the above-mentioned glutamate determination kit, comprising the following steps: After the sample to be tested is incubated with the R1 reagent, the absorbance A1 is measured; after adding the R2 reagent for incubation, the absorbance A2 is measured; the absorbance difference is calculated, and the content of glutamate in the sample to be tested is calculated according to the two-point calibration method.

[0015] In the above method, the sample to be tested includes but is not limited to a serum sample.

[0016] In the scheme of the present invention, the glutamate oxidase used is a flavin protease with flavin adenine dinucleotide (FAD) as a cofactor, which can specifically oxidize glutamate to generate α-ketoglutaric acid, ammonia and hydrogen peroxide; the glutamate in the sample to be tested reacts with the glutamate oxidase, and in the presence of the chromogenic oxygen acceptor 4-aminoantipyrine-coupled phenol, the peroxidase catalyzes hydrogen peroxide to oxidize the chromogen to generate red quinone compounds, the color depth of which is proportional to the glutamate concentration, and the glutamate content in the sample can be calculated by comparing it with the calibrator that has been treated in the same way.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes the characteristics of glutamate oxidase having high stereoisomer selectivity for catalytic reaction substrates and high catalytic efficiency to construct a novel glutamate determination kit and determination method. The preparation process of the kit is simple, the raw materials are easy to purchase, and the cost is low. The method has the advantages of not relying on large instruments, simple operation, short detection time, etc., and is of great significance for grassroots detection, large-scale sample batch detection, etc.

[0018] Based on the above-mentioned glutamate oxidase catalysis and 4-aminoantipyrine color development principle, the present invention, on the basis of a large number of exploratory experiments, determines the appropriate concentration of the chromogenic substrate and the color developer within the appropriate buffer concentration and pH range, and adds the appropriate concentration of the surfactant, so that the accuracy, precision and opening stability of the prepared glutamate determination kit are significantly improved, thereby achieving the purpose of accurately and reliably detecting glutamate. DETAILED DESCRIPTION

[0019] The following embodiments of the technical solution of the present invention are described in detail. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the description and claims of the present invention and any variations thereof are intended to cover non-exclusive inclusions.

[0021] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0022] Example 1 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 1.

[0023] Table 1 Composition of R1 reagent and R2 reagent in the kit of Example 1

[0024] Example 2 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 2.

[0025] Table 2 Composition of R1 reagent and R2 reagent in the kit of Example 2

[0026] Example 3 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 3.

[0027] Table 3 Composition of R1 reagent and R2 reagent in the kit of Example 3

[0028] Example 4 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 4.

[0029] Table 4 Composition of R1 reagent and R2 reagent in the kit of Example 4

[0030] Example 5 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 5.

[0031] Table 5 Composition of R1 reagent and R2 reagent in the kit of Example 5

[0032] Example 6 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 6.

[0033] Table 6 Composition of R1 reagent and R2 reagent in the kit of Example 6

[0034] Example 7 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 1.

[0035] Table 7 Composition of R1 reagent and R2 reagent in the kit of Example 7

[0036] Example 8 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 1.

[0037] Table 8 Composition of R1 reagent and R2 reagent in the kit of Example 8

[0038] Example 9 Based on the glutamate determination kit provided by the present invention, this example provides a glutamate determination method (end point method), which includes the following process: mixing the sample to be tested with the R1 reagent, incubating at 37°C for 5 minutes, and measuring the absorbance A1 at 546nm (main wavelength, secondary wavelength is 700nm); adding the R2 reagent and mixing, incubating at 37°C for 5 minutes, and measuring the absorbance A2; calculating ΔA=A2-A1. The specific detection parameters are shown in Table 9: Table 9 Operating parameters for glutamate determination

[0039] Note: The reaction direction is positive and the calibration method is two-point calibration.

[0040] Comparative Example 1 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 10.

[0041] Table 10 Composition of R1 reagent and R2 reagent in the kit of Comparative Example 1

[0042] Comparative Example 2 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 11.

[0043] Table 11 Composition of R1 reagent and R2 reagent in the kit of Comparative Example 2

[0044] Comparative Example 3 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 12.

[0045] Table 12 Composition of R1 reagent and R2 reagent in the kit of Comparative Example 3

[0046] Comparative Example 4 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 13.

[0047] Table 13 Composition of R1 reagent and R2 reagent in the kit of Comparative Example 4

[0048] Comparative Example 5 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 14.

[0049] Table 14 Composition of R1 reagent and R2 reagent in the kit of Comparative Example 5

[0050] Comparative Example 6 This example provides a glutamate determination kit, including R1 reagent and R2 reagent, and the compositions of R1 reagent and R2 reagent are shown in Table 15.

[0051] Table 15 Composition of R1 reagent and R2 reagent in the kit of Comparative Example 6

[0052] From the above embodiments and comparative examples, it can be seen that the difference between comparative examples 1 and 2 relative to embodiment 1 is that the buffer concentration in the R1 reagent is different, the difference between comparative examples 3 and 4 relative to embodiment 1 is that the buffer pH in the R1 reagent is different, and the difference between comparative examples 5 and 6 relative to embodiment 1 is that the surfactant concentration in the R1 reagent is different; the difference between embodiment 2 relative to embodiment 1 is that the buffer pH in the R1 reagent is different, the difference between embodiment 3 relative to embodiment 1 is that the buffer concentration in the R1 reagent is different, the difference between embodiment 4 relative to embodiment 1 is that the buffer pH and concentration in the R2 reagent are different, the difference between embodiment 5 relative to embodiment 1 is that the concentrations of several substances in the R1 reagent are changed, the difference between embodiment 6 relative to embodiment 1 is that the concentrations of several substances in the R2 reagent are changed, the difference between embodiment 7 relative to embodiment 1 is that the types of buffers in the R1 reagent and the R2 reagent are changed, and the difference between embodiment 8 relative to embodiment 7 is that the types or concentrations of several substances in the R1 reagent and the R2 reagent are changed.

[0053] According to the detection parameters in Example 9, the kits provided in Examples 1-8 and Comparative Examples 1-6 were tested as follows: (1) Accuracy test

[0054] Test quality control products and calculate the deviation between the measured mean value and the target value. The relative deviation of inaccuracy should be ≤10%.

[0055] (2) Precision test

[0056] Under repeatability conditions, use the kit to test the same serum sample, repeat the test 10 times, and compare the coefficient of variation (CV). The CV should be ≤6%.

[0057] (3) Oscillation stability test.

[0058] The reagent was placed on a shaker at 42°C for 3 days, and then the accuracy and precision of the reagent after shaking were tested.

[0059] (4) Opening stability test.

[0060] Place the reagent at 2℃~8℃, open the bottle, test the quality control product and mixed serum samples at two concentration levels, measure three times and take the average value, test every few days and compare the relative deviation with day 0, which should be within ±10%.

[0061] The test results are shown in Table 16 and Table 17: Table 16 Accuracy, precision and oscillation stability test results of each kit

[0062] Table 17 Opening stability test results of each kit

[0063] From the above test results, it can be seen that: if the pH of the buffer solution in the R1 reagent is inappropriate or the concentration is too low, the stability of the reagent after opening the bottle will deteriorate; if the concentration of the buffer solution in the R1 reagent is too high, the accuracy and precision of the reagent after shaking will deteriorate; adding a surfactant to the R1 reagent can significantly improve the accuracy and precision of the reagent, but too high a concentration of the surfactant will deteriorate the accuracy to a certain extent, so the concentration of the surfactant should be within an appropriate range.

[0064] In summary, the glutamate determination kit and determination method provided by the present invention can effectively and quantitatively detect the glutamate content in blood, and have the advantages of high accuracy, good precision and good stability.

[0065] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A glutamate determination kit, characterized in that Including R1 reagent and R2 reagent; The R1 reagent is composed of a first buffer, 4-aminoantipyrine, glutamate oxidase, ascorbic acid enzyme, a surfactant, a stabilizer and a preservative; the first buffer is selected from 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid, 3-morpholinopropanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), disodium hydrogen phosphate-sodium dihydrogen phosphate and disodium hydrogen phosphate-potassium dihydrogen phosphate, and its concentration is 50mmol / L~150mmol / L, and the pH is 6.5~7.5; The R2 reagent consists of a second buffer, a color development substrate, peroxidase and a preservative.

2. The glutamate determination kit according to claim 1, characterized in that The R1 reagent consists of a first buffer, 0.1 g / L to 2 g / L 4-aminoantipyrine, 1 KU / L to 10 KU / L glutamate oxidase, 1 KU / L to 6 KU / L ascorbic acid oxidase, 0.1% to 0.5% surfactant, 1 g / L to 50 g / L stabilizer and 0.1% to 1.0% preservative.

3. The glutamate determination kit according to claim 2, characterized in that The surfactant is selected from Triton X-100, Triton X-405, polyoxyethylene castor oil, propylene glycol block polyether, polyoxyethylene lauryl ether, Tween-80 and Tween-20.

4. The glutamate determination kit according to claim 2, characterized in that The stabilizer is one or more of bovine serum albumin, disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, sucrose, sorbitol, and mannitol.

5. The glutamate determination kit according to claim 1, characterized in that The second buffer is selected from 4-hydroxyethylpiperazineethanesulfonic acid, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, tris(hydroxymethyl)aminomethane and N,N-dihydroxyethylglycine, and its concentration is 50mmol / L~150mmol / L, and the pH is 7.5~8.

5.

6. The glutamate determination kit according to claim 5, characterized in that The R2 reagent consists of a second buffer, 1 g / L to 5 g / L of a color developing substrate, 10 KU / L to 20 KU / L of peroxidase and 0.1% to 1.0% of a preservative.

7. The glutamate determination kit according to claim 6, characterized in that The chromogenic substrate is selected from N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, 3,5-dichloro-2-hydroxybenzenesulfonate sodium, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt, N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt, 3,5-dichloro-2-hydroxybenzenesulfonic acid and N-ethyl (2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline.

8. The glutamate determination kit according to claim 1, characterized in that The preservative is sodium azide and / or ProClin300.

9. Use of the glutamate determination kit according to any one of claims 1 to 8 in glutamate detection.

10. A method for determining glutamate, characterized in that: The method uses the glutamate determination kit according to any one of claims 1 to 8 for detection, and specifically comprises the following operations: After the sample to be tested is incubated with the R1 reagent, the absorbance A1 is measured; after adding the R2 reagent for incubation, the absorbance A2 is measured; the absorbance difference is calculated, and the content of glutamate in the sample to be tested is calculated according to the two-point calibration method.

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