Glutamic acid determination kit and determination method

Abstract

Provided in the present invention are a glutamic acid determination kit and a determination method, belonging to the technical field of in vitro diagnosis. The kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent consists of a first buffer, 4-aminoantipyrine, glutamate oxidase, ascorbic acid oxidase, a surfactant, a stabilizer and a preservative, and the R2 reagent consists of a second buffer, a chromogenic substrate, a peroxidase and a preservative. The kit of the present invention involves simple preparation processes and readily available raw materials; in addition, the determination method using the kit has advantages such as independence from large instruments, simple operation, short test time, high accuracy, good precision and excellent stability and is thus suitable for community-level testing and large-scale batch testing of samples and exhibits a promising application prospect.

Description

A glutamate assay kit and assay method Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, specifically to a glutamate assay kit and assay method. Background Technology

[0002] Glutamic acid (Glu) is one of the essential amino acids for nitrogen metabolism in living organisms and plays a vital role in metabolism. Glutamic acid is a major component of proteins, and glutamate is ubiquitous in nature. It is found in many foods and in the human body; it is both a structural amino acid of proteins and peptides 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 calcium... 2+ Glutamate (Glu) transport plays a crucial physiological role in the differentiation, migration, growth, and survival of neurons. It participates in a series of complex physiological processes, including learning, memory, and synaptic plasticity regulation. Furthermore, it is closely related to nerve cell death and the pathogenesis 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, the concentration of Glu in the brain increases, and the Glu level in the blood also increases accordingly. Therefore, measuring changes in blood Glu can reflect the progression 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] Various conventional analytical methods have been applied to the detection of glutamic acid, such as high-performance liquid chromatography (HPLC), capillary electrophoresis, fluorescence detection, and chemiluminescence, with HPLC being the most commonly used. HPLC is an important branch of chromatography that uses a liquid as the mobile phase. A high-pressure delivery system pumps a single solvent or a mixture of solvents and buffer solutions with different polarities into a chromatographic column packed with a stationary phase. After separation within the column, the components are detected by a detector, thus enabling sample analysis. HPLC is characterized by high separation efficiency, high sensitivity, and wide applicability. However, its drawback is the "outside-column effect." In any dead space outside the column (injector, column connector, connecting tubing, and detection cell, etc.), any change in the flow pattern of the mobile phase between the injection point and the detector, any diffusion or retention of the separated substances will significantly lead to peak broadening and reduced column efficiency.

[0004] Therefore, finding new, efficient, accurate, and simple methods for determining glutamic acid content is of practical significance. Summary of the Invention

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

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a glutamate assay kit, comprising reagent R1 and reagent R2; wherein,

[0008] Reagent R1 consists of a first buffer, 4-aminoantipyrine (4-AAP), L-glutamate oxidase (GLOD), ascorbic acid oxidase (ASO), surfactant, stabilizer, and preservative; the first buffer is selected from one or more of 3-(N-morpholino)-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 50 mmol / L to 150 mmol / L, and its pH is 6.5 to 7.5;

[0009] The R2 reagent consists of a second buffer, a chromogenic substrate, peroxidase (POD), and a preservative.

[0010] Preferably, in the above-mentioned glutamate assay kit, reagent R1 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% (g / L) surfactant, 1 g / L to 50 g / L stabilizer, and 0.1% to 1.0% (g / L) preservative.

[0011] In the above-mentioned glutamate assay kit, the surfactant is selected from one or more of Triton X-100, Triton X-405, polyoxyethylene castor oil (Cremophor EL), propylene glycol block polyether (Pluranic L64), polyoxyethylene lauryl ether (Brij 35), Tween-80, and Tween-20.

[0012] In the above-mentioned glutamate assay kit, the stabilizer is one or more of bovine serum albumin (BSA), disodium ethylenediaminetetraacetate (EDTA-2Na), dipotassium ethylenediaminetetraacetate (EDTA-2K), sucrose, sorbitol, and mannitol.

[0013] Preferably, in the above-mentioned glutamate assay kit, reagent R2 consists of a second buffer, 1 g / L to 5 g / L chromogenic substrate, 10 KU / L to 20 KU / L peroxidase, and 0.1% to 1.0% (g / L) preservative; wherein the second buffer is selected from one or more of 4-hydroxyethylpiperazine ethanesulfonic 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 50 mmol / L to 150 mmol / L, and its pH is 7.5 to 8.5.

[0014] In the above-mentioned glutamate assay kit, the chromogenic substrate is selected from one or more of the following: N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS), sodium 3,5-dichloro-2-hydroxybenzenesulfonate (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).

[0015] Preferably, in the above-mentioned glutamate assay kit, the preservative is sodium azide and / or ProClin300.

[0016] Secondly, the present invention provides a method for preparing the above-mentioned glutamate assay kit, comprising the following steps:

[0017] Preparation of R1 reagent: Prepare a first buffer solution with a concentration of 50 mmol / L to 150 mmol / L and a pH of 6.5 to 7.5. Add the colorimetric reagent 4-AAP, GLOD, ASO enzyme, surfactant, stabilizer and preservative to the first buffer solution in sequence. After adjusting the volume, mix well to obtain the R1 reagent.

[0018] Preparation of R2 reagent: Prepare the second buffer solution by adding the chromogenic substrate, POD and preservative to the second buffer solution in sequence, and mix well after adjusting the volume to obtain R2 reagent.

[0019] Thirdly, the present invention provides a method for qualitative and / or quantitative determination of glutamic acid using the above-mentioned glutamic acid assay kit, comprising the following steps:

[0020] After incubating the sample with reagent R1, the absorbance A1 is measured; after incubating with reagent R2, the absorbance A2 is measured; the absorbance difference is calculated, and the content of glutamic acid in the sample is calculated according to the two-point calibration method.

[0021] In the above methods, the sample to be tested includes, but is not limited to, a serum sample.

[0022] In 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 produce α-ketoglutarate, ammonia, and hydrogen peroxide. The glutamate in the sample reacts with the glutamate oxidase. In the presence of the chromogenic oxygen acceptor 4-aminoantipyrine conjugate, the peroxidase catalyzes hydrogen peroxide to oxidize chromogen and generate a red quinone compound. The color intensity is directly proportional to the glutamate concentration. By comparing with a calibrator that has undergone the same treatment, the glutamate content in the sample can be calculated.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention utilizes the high stereoisomer selectivity and high catalytic efficiency of glutamate oxidase to catalyze reaction substrates to construct a novel glutamate assay kit and method. The kit is simple to prepare, uses readily available raw materials, and is low in cost. The method has advantages such as not relying on large instruments, simple operation, and short detection time, and is of great significance for grassroots testing and large-scale batch testing of samples.

[0025] Based on the catalysis of glutamate oxidase and the colorimetric principle of 4-aminoantipyrine, this invention, through extensive exploratory experiments, determines suitable concentrations of chromogenic substrate and colorimetric reagent within appropriate buffer concentrations and pH ranges, and adds suitable concentrations of surfactant. This significantly improves the accuracy, precision, and open-bottle stability of the prepared glutamate assay kit, achieving the goal of accurate and reliable detection of glutamate. Detailed Implementation

[0026] The embodiments of the technical solution of the present invention are described in detail below. These embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0028] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0029] Example 1

[0030] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 1.

[0031] Table 1. Composition of reagents R1 and R2 in the kit of Example 1

[0032] Example 2

[0033] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 2.

[0034] Table 2. Composition of reagents R1 and R2 in the kit of Example 2

[0035] Example 3

[0036] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 3.

[0037] Table 3. Composition of reagents R1 and R2 in the kit of Example 3

[0038] Example 4

[0039] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 4.

[0040] Table 4. Composition of reagents R1 and R2 in the kit of Example 4

[0041] Example 5

[0042] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 5.

[0043] Table 5. Composition of reagents R1 and R2 in the kit of Example 5

[0044] Example 6

[0045] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 6.

[0046] Table 6. Composition of reagents R1 and R2 in the kit of Example 6

[0047] Example 7

[0048] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 1.

[0049] Table 7 Composition of reagents R1 and R2 in the kit of Example 7

[0050] Example 8

[0051] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 1.

[0052] Table 8. Composition of reagents R1 and R2 in the kit of Example 8

[0053] Example 9

[0054] Based on the glutamate assay kit provided by this invention, this example provides a method for glutamate assay (endpoint method), which includes the following steps: Mix the sample to be tested with reagent R1, incubate at 37°C for 5 min, and measure the absorbance A1 at 546 nm (main wavelength, secondary wavelength 700 nm); add reagent R2, mix, incubate at 37°C for 5 min, and measure the absorbance A2; calculate ΔA = A2 - A1. Specific detection parameters are shown in Table 9.

[0055] Table 9. Operating parameters for glutamate determination Note: The reaction direction is forward, and the calibration method is two-point calibration.

[0056] Comparative Example 1

[0057] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 10.

[0058] Table 10 Composition of reagents R1 and R2 in Comparative Example 1 kit

[0059] Comparative Example 2

[0060] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 11.

[0061] Table 11 Composition of reagents R1 and R2 in Comparative Example 2 kit

[0062] Comparative Example 3

[0063] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 12.

[0064] Table 12 Composition of reagents R1 and R2 in Comparative Example 3 kit

[0065] Comparative Example 4

[0066] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 13.

[0067] Table 13 Composition of reagents R1 and R2 in Comparative Example 4 kit

[0068] Comparative Example 5

[0069] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 14.

[0070] Table 14 Composition of reagents R1 and R2 in Comparative Example 5 kit

[0071] Comparative Example 6

[0072] This example provides a glutamate assay kit, which includes reagent R1 and reagent R2, and the composition of reagent R1 and reagent R2 is shown in Table 15.

[0073] Table 15 Composition of reagents R1 and R2 in Comparative Example 6 kit

[0074] As can be seen from the above examples and comparative examples, the only difference between Comparative Examples 1 and 2 and Example 1 is the concentration of the buffer solution in reagent R1; the only difference between Comparative Examples 3 and 4 and Example 1 is the pH of the buffer solution in reagent R1; the only difference between Comparative Examples 5 and 6 and Example 1 is the concentration of the surfactant in reagent R1; the only difference between Example 2 and Example 1 is the pH of the buffer solution in reagent R1; the only difference between Example 3 and Example 1 is the concentration of the buffer solution in reagent R1; the only difference between Example 4 and Example 1 is the pH and concentration of the buffer solution in reagent R2; the only difference between Example 5 and Example 1 is the change in the concentration of several substances in reagent R1; the only difference between Example 6 and Example 1 is the change in the concentration of several substances in reagent R2; the only difference between Example 7 and Example 1 is the change in the types of buffer solutions in reagents R1 and R2; and the only difference between Example 8 and Example 7 is the change in the types or concentrations of several substances in reagents R1 and R2.

[0075] The kits provided in Examples 1-8 and Comparative Examples 1-6 were tested as follows, according to the detection parameters in Example 9:

[0076] (1) Accuracy test.

[0077] For quality control samples, calculate the deviation between the measured average value and the target value. The relative deviation of the inaccuracy should be ≤10%.

[0078] (2) Precision test.

[0079] Under repeatability conditions, test the same serum sample 10 times with the kit and compare the coefficient of variation (CV). The CV should be ≤6%.

[0080] (3) Oscillation stability test.

[0081] The reagent was placed on a shaker at 42°C and shaken for 3 days. Then the accuracy and precision of the reagent after shaking were tested.

[0082] (4) Bottle opening stability test.

[0083] Open the reagent bottle at 2℃~8℃, test the quality control sample and the mixed serum sample at 2 concentration levels, take the average value after 3 measurements, test every few days, and compare the relative deviation with day 0. The deviation should be within ±10%.

[0084] The test results are shown in Tables 16 and 17:

[0085] Table 16 Results of accuracy, precision, and oscillation stability tests for each reagent kit

[0086] Table 17 Results of open-bottle stability tests for each reagent kit

[0087] The test results above show that: if the pH of the buffer solution in reagent R1 is not suitable or the concentration is too low, the stability of the reagent after opening will be reduced; while if the concentration of the buffer solution in reagent R1 is too high, the accuracy and precision of the reagent after shaking will be reduced; adding surfactant to reagent R1 can significantly improve the accuracy and precision of the reagent, but if the surfactant concentration is too high, it will reduce the accuracy to some extent. Therefore, the concentration of surfactant should be within a suitable range.

[0088] In summary, the glutamate assay kit and assay method provided by this invention can effectively quantify the glutamate content in blood, and have the advantages of high accuracy, good precision, and good stability.

[0089] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A glutamate assay kit, characterized in that, Including reagents R1 and R2; The R1 reagent consists of a first buffer solution, 4-aminoantipyrine, glutamate oxidase, ascorbic acid oxidase, surfactant, stabilizer, and preservative; the first buffer solution is selected from 3-(N-morpholino)-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 50 mmol / L to 150 mmol / L, and its pH is 6.5 to 7.5; The R2 reagent consists of a second buffer solution, a chromogenic substrate, a peroxidase, and a preservative.

2. The glutamate assay kit according to claim 1, characterized in that, The R1 reagent consists of a first buffer solution, 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 assay 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 assay kit according to claim 2, characterized in that, The stabilizer is one or more of bovine serum albumin, disodium EDTA, dipotassium EDTA, sucrose, sorbitol, and mannitol.

5. The glutamate assay kit according to claim 1, characterized in that, The second buffer solution is selected from 4-hydroxyethylpiperazine ethanesulfonic 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 50 mmol / L to 150 mmol / L, and its pH is 7.5 to 8.

5.

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

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

9. The application of the glutamate assay kit as described in any one of claims 1 to 8 in the detection of glutamate.

10. A method for determining glutamic acid, characterized in that, This method utilizes the glutamate assay kit according to any one of claims 1 to 8 for detection, and specifically includes the following operations: After incubating the sample with reagent R1, the absorbance A1 was measured; then, after incubating with reagent R2, the absorbance A2 was measured. Calculate the absorbance difference and determine the glutamic acid content in the sample using the two-point calibration method.

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