Method and kit for determining concentration of d-2-hydroxyglutaric acid

Abstract

The present invention relates to the technical field of enzymatic detection. Disclosed in the present invention are a method and kit for determining the concentration of D-2-hydroxyglutaric acid. The determination method is not used for the diagnosis and treatment of diseases. D-2-hydroxyglutaric acid in a solid tissue section is detected by means of using a system comprising an FAD-dependent D-2-hydroxyglutarate dehydrogenase, resazurin and a PBS buffer as a working solution. During the preparation of a standard curve, D-2-hydroxyglutaric acid with different concentrations is added to an aqueous agarose solution to prepare standard sections. According to the method and kit for determining the concentration of D-2-hydroxyglutaric acid of the present invention, the concentration of D-2-hydroxyglutaric acid in a tissue sample can be detected after a simple treatment of the tissue sample, the detection result is consistent with that of a mass spectrometry detection method, and the accuracy is high. Detecting standard sections and preparing a sample section require 20 minutes, detecting the sample section requires about 3 minutes, and calculating the concentration of D-2-HG requires about 2 minutes, that is, only less than 5 minutes are required after the standard curve and sample section are prepared, which greatly shortens the detection time and reduces the detection cost.

Description

A method and kit for determining D-2-hydroxyglutaric acid concentration Technical Field

[0001] The invention relates to the technical field of enzyme detection, in particular to a method and a kit for determining the concentration of D-2-hydroxyglutaric acid. Background Art

[0002] Mutations in isocitrate dehydrogenase 1 (IDH1) or its paralog IDH2 (collectively referred to as IDHs) are prevalent in various cancer types, including low-grade gliomas and secondary glioblastomas, acute myeloid leukemia, cholangiocarcinoma, chondrosarcoma, undifferentiated sinonasal carcinoma, and angioimmunoblastic T-cell lymphoma. Approximately 80% of low-grade gliomas carry IDH1 mutations. Mutations in the active site of IDH1 significantly reduce isocitrate activity, leading to the production of a novel allosteric enzyme that catalyzes the reduction of α-ketoglutarate (α-KG) to D-2-hydroxyglutarate, resulting in D-2-HG accumulation in tumors. Although D-2-HG levels in IDH-mutated tumors are 50-100 times higher than in normal tissues, the presence of IDH-mutated tumors is not uncommon.

[0003] Furthermore, D-2-HG concentrations are associated with non-tumor diseases. For example, D-2-hydroxyglutarate dehydrogenase (D-2-HGDH), an enzyme present in human mitochondria, uses FAD as a coenzyme to catalyze the oxidative dehydrogenation of D-2-HG to α-KG, thereby maintaining a low intracellular 2-HG level (approximately <100 mM). Mutations in D-2-HGDH prevent the dehydrogenation of D-2-HG to α-KG, leading to the accumulation of D-2-HG. In patients with D-2-HGDH mutations, excessive 2-HG accumulation can lead to 2-hydroxyglutaric aciduria (2-HGA). For example, a study published in Science in 2022 demonstrated that elevated D-2-HG levels around IDH-mutated brain tumors reduced lymphocyte infiltration. D-2-HG, in turn, impacted the proliferation and metabolism of CD8+ T cells in the tumor microenvironment, thereby suppressing the immune system.

[0004] In many diseases, D-2-HG or L-2-HG are metabolic abnormalities that exist in urine, tissue fluid, and tissues and accumulate abnormally in the body. However, in practice, the D-2-HG content alone cannot be used to determine the type of disease. Other tests are needed to finally determine the type of disease.

[0005] Furthermore, the ability to quickly and accurately measure D-2-HG or L-2-HG concentrations is crucial for exploring pathogenic mechanisms, screening for appropriate drugs that reduce D-2-HG or L-2-HG production, and exploring the role of D-2-HG or L-2-HG metabolism in protein or polysaccharide synthesis. For example, invention patent CN201380011978.1 discloses tools and methods for measuring D-2-HG or (D)2-hydroxyadipic acid, and also develops a kit. The literature A High-Throughput Fluorimetric Assay for 2-Hydroxyglutarate Identification of Zaprinast as a Glutaminase Inhibitor developed a simple and low-cost fluorescence microplate method for detecting D-2-HG based on phosphoglycerate dehydrogenase (PHGDH), and used it to screen compounds that can inhibit cellular production of D-2-HG; the literature A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DhdR developed a biosensor for detecting D-2-HG, which was also used to determine bacterial D-2-HG concentration, demonstrating the important role of D-2-HG metabolism in lipopolysaccharide synthesis in Pseudomonas aeruginosa.

[0006] Currently, liquid chromatography-mass spectrometry (LC-MS) or gas chromatography-mass spectrometry (GC-MS) are commonly used to measure D-2-HG and L-2-HG concentrations. However, these methods require cumbersome and time-consuming sample pretreatment, and mass spectrometry equipment is expensive, significantly increasing the cost of testing.

[0007] Patent CN112592958B discloses a method for detecting D-2-hydroxyglutarate using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin. By designing a novel FAD-dependent D-2-hydroxyglutarate dehydrogenase, the method avoids the need for cofactors, coenzymes, or diaphorase, significantly improving detection sensitivity compared to conventional testing. However, this method is more suitable for testing liquid samples, such as serum, urine, and cerebrospinal fluid, which require only protein removal for detection in a fluorescence microplate reader. However, for testing solid samples, such as tissue, pretreatment such as pulverization is required to release the D-2-hydroxyglutarate in the solid sample before detection. Therefore, pretreatment can affect detection accuracy and efficiency.

[0008] Therefore, there is an urgent need for a rapid, accurate and efficient method for determining the concentration of D-2-hydroxyglutaric acid in solid test samples. Summary of the Invention

[0009] To solve the above problems, the present invention aims to provide a method and a kit for determining the concentration of D-2-hydroxyglutaric acid.

[0010] To achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions:

[0011] The coding gene sequence, amino acid sequence and preparation method of FAD-dependent D-2-hydroxyglutarate dehydrogenase (FAD-dependent D-2-HGBH) used in the present invention have been disclosed in CN112592958B and will not be repeated in this patent.

[0012] A method for measuring D-2-hydroxyglutaric acid concentration uses FAD-dependent D-2-hydroxyglutaric acid dehydrogenase, resazurin, and a PBS buffer system as a working solution to detect D-2-hydroxyglutaric acid in solid tissue sections. The method is intended for non-disease diagnosis and treatment purposes.

[0013] A method for determining the concentration of D-2-hydroxyglutaric acid comprises the following steps:

[0014] ① Prepare D-2-hydroxyglutaric acid standard sections: Add D-2-hydroxyglutaric acid to an aqueous agarose solution; heat and stir until clear and transparent, pour onto a gel plate to solidify, remove, and cut into rectangular blocks with a cross-sectional area of ​​0.1-1.5 cm × 0.1-1.5 cm. Place the block in the tissue tray of a cryostat. Cover the agarose gel block with OCT embedding medium. After the OCT embedding medium is frozen solid in the cryostat, slice the agarose gel to a thickness of 5-25 μm, place on a slide, and cut into rectangular blocks with a cross-sectional area of ​​0.1-1 cm × 0.1-1 cm. Store in a -20°C refrigerator to obtain a series of D-2-hydroxyglutaric acid standard sections of different concentrations for later use.

[0015] The ratio of D-2-hydroxyglutaric acid to agarose aqueous solution is 0~5.0µmol:1g;

[0016] Three parallel experiments were set up for each concentration of standard slices;

[0017] ② Slice processing of tissue samples to be tested

[0018] Remove the tissue to be tested from the -80°C freezer, place on ice, and place in the tissue tray of a cryostat. Add OCT freezing embedding medium to the tissue to be tested. After the OCT freezing embedding medium turns white, slice the tissue to be tested to a thickness of 5-25 μm, place on a non-shedding slide, and cut into rectangular blocks with a cross-sectional area of ​​0.1-1 cm × 0.1-1 cm to obtain the tissue sections to be tested and set aside.

[0019] Three parallel experiments were set up for each tissue sample slice to be tested;

[0020] ③ Prepare the staining solution: The staining solution is a buffer solution at pH 7.0-8.0 containing resazurin and FAD-dependent D-2-hydroxyglutarate dehydrogenase. The buffer can be Tris-HCl, PBS, or HEPES, with a 1 / 15 mol / L PBS buffer solution being more preferred. The resazurin concentration ranges from 0.5 μM to 200 μM. The FAD-dependent D-2-hydroxyglutarate dehydrogenase concentration ranges from 1.3 μg / ml to 520 μg / ml.

[0021] The preferred staining solution is a PBS buffer solution with a pH of 7.4 (mass concentration of 1 / 15 mol / L) containing 10 µM resazurin and 26 µg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase;

[0022] ④ Staining of tissue sections

[0023] Take 15 μl to 100 μl of the staining solution obtained in step 3 and completely cover the tissue section to be tested obtained in step 2. Use a fluorescence detector to detect the fluorescence value of the staining solution on the slide for 1 to 10 minutes;

[0024] ⑤ Standard product data testing and result processing

[0025] Place a series of D-2-hydroxyglutaric acid standard sections of different concentrations from step ① on a glass slide, take 15 μl to 100 μl of the staining solution obtained in step ③ to completely cover the slide, and immediately detect the staining solution using a fluorescence detector with an excitation wavelength of 540 nm and an emission wavelength of 590 nm. The fluorescence value of the staining solution on the slide is the value measured by the fluorescence detector 1 to 10 minutes later. The average of the fluorescence values ​​of each concentration detected by the fluorescence detector is used as the y value. The concentration of D-2-hydroxyglutaric acid contained is used as the x value. A scatter plot is drawn, and the standard curve y = ax + b is constructed using the scatter plot.

[0026] Calculate the D-2-hydroxyglutaric acid content in the tissue section to be tested based on the fluorescence value obtained in step ③.

[0027] Preferably, step ① is to prepare D-2-hydroxyglutaric acid standard sections: D-2-hydroxyglutaric acid is prepared into a series of agarose aqueous solutions with concentrations of 0-2.0 μmol / g; the agarose aqueous solution is heated until clear and transparent, poured onto a gel plate to solidify, removed, and cut into rectangular blocks with a cross-sectional area of ​​0.1-1.5 cm × 0.1-1.5 cm, placed in a tissue tray of a freezing microtome, and covered with OCT embedding medium on the agarose gel block (the amount of OCT embedding medium is preferably sufficient to evenly cover the agarose gel block, and the amount used is generally 350-450 μL). After the OCT embedding medium is frozen solid in the freezing microtome, the agarose gel is cut into a thickness of 5-25 μm, placed on a non-shedding slide, and cut into rectangular blocks with a cross-sectional area of ​​0.1-1 cm × 0.1-1 cm, and stored in a -20°C refrigerator to obtain a series of D-2-hydroxyglutaric acid standard sections of different concentrations for later use;

[0028] When cutting into rectangular blocks with a cross-sectional area of ​​0.1~1cm×0.1~1cm, you can first cut into larger rectangular blocks, and then remove the part where the edge contacts the OCT embedding medium to prevent the agarose gel block OCT embedding medium from affecting the test results.

[0029] Three parallel experiments were set up for each concentration of standard slices.

[0030] Preferably, the concentration of D-2-hydroxyglutaric acid in the standard slice is a gradient concentration in the range of 0-5.0 µmol / g.

[0031] Further preferably, the concentration of D-2-hydroxyglutaric acid in the standard slice is 0 µmol / g, 0.1 µmol / g, 0.25 µmol / g, 0.5 µmol / g, 1.0 µmol / g, 1.5 µmol / g and 2.0 µmol / g.

[0032] Preferably, after adding the staining solution to the standard sample slice and the tissue slice to be tested, the fluorescence detector detection time is the same, which is 1-3 minutes;

[0033] The further preferred fluorescence detector detection time is 3 minutes.

[0034] The present invention also includes a kit for detecting the concentration of D-2-hydroxyglutarate, comprising a buffer solution with a pH of 7.0 to 8.0, a resazurin mother solution, a FAD-dependent D-2-hydroxyglutarate dehydrogenase mother solution, and an agarose gel slide containing a series of D-2-hydroxyglutarate concentrations; the concentration of the resazurin mother solution in the kit ranges from 0.2 mM to 25 mM, and the concentration of the FAD-dependent D-2-hydroxyglutarate dehydrogenase mother solution in the kit ranges from 1.3 mg / ml to 26 mg / ml;

[0035] The buffer is Tris-HCl buffer, PBS buffer or HEPES buffer.

[0036] Preferably, the agarose gel containing a series of concentrations of D-2-hydroxyglutaric acid is placed on an anti-shedding slide; the concentration of D-2-hydroxyglutaric acid in the agarose gel ranges from 0 to 5.0 µmol / g.

[0037] Further preferably, the concentration of D-2-hydroxyglutaric acid in the agarose gel is 0 µmol / g, 0.1 µmol / g, 0.25 µmol / g, 0.5 µmol / g, 1.0 µmol / g, 1.5 µmol / g and 2.0 µmol / g.

[0038] Preferably, the size and thickness of the agarose gel containing a series of D-2-hydroxyglutaric acid concentrations are cut as needed. The thickness can range from 5 to 25 μm, and the cross-sectional area can be a rectangle of 0.1 to 1 cm x 0.1 to 1 cm. The size and thickness of the agarose gel can also be adjusted based on the size of the tissue to be tested.

[0039] The PBS concentration, pH, resazurin concentration, and enzyme concentration are not fixed concentrations for efficacy; they are optimized conditions based on the aforementioned patent. These conditions do not constitute a limitation for other concentrations.

[0040] The present invention also includes a method for using a kit for detecting the concentration of D-2-hydroxyglutaric acid, comprising the following steps:

[0041] Prepare staining solution: Add resazurin solution and FAD-dependent D-2-hydroxyglutarate dehydrogenase solution to PBS buffer solution to prepare staining solution;

[0042] The concentration range of the resazurin stock solution in the kit is 0.2mM-25mM, and the concentration range of the FAD-dependent D-2-hydroxyglutarate dehydrogenase stock solution is 1.3mg / ml-26mg / ml; when used, they need to be added to PBS buffer to prepare a staining solution;

[0043] The final prepared staining solution was a buffer solution with a pH of 7.0-8.0, containing 10 μM resazurin and 26 μg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase;

[0044] Prepare a standard curve: Completely cover the staining solution obtained in step A onto an agarose gel slide containing a series of D-2-hydroxyglutaric acid concentrations. Immediately measure the fluorescence value of the staining solution on the slide using a fluorescence detector. Measure the fluorescence value detected by the fluorescence detector 1 to 10 minutes later. Use the average fluorescence value of each concentration detected by the fluorescence detector as the y value. Use the concentration of D-2-hydroxyglutaric acid as the x value. Draw a scatter plot and use the scatter plot to create a standard curve y = ax + b.

[0045] When preparing the standard curve, the fluorescence detection time of the agarose gel slides of each concentration of D-2-hydroxyglutaric acid is as similar as possible, preferably 1 to 3 minutes; the characteristic excitation wavelength is 540 nm and the emission wavelength is 590 nm;

[0046] C. Remove the tissue to be tested from the -80°C freezer, place it on ice, and place it in the tissue tray of a cryostat. Add OCT freezing embedding medium to the tissue to be tested. After the OCT freezing embedding medium turns white, slice the tissue to be tested to the same thickness as the agarose gel slide, place it on a non-shedding slide, and cut it into rectangular parallelepipeds with the same cross-sectional area as the agarose gel slide to obtain the tissue sections to be tested and set aside.

[0047] Three parallel experiments were set up for each tissue sample slice to be tested;

[0048] Completely cover the tissue section to be tested with the staining solution obtained in step A, and use a fluorescence detector to detect the fluorescence value of the staining solution on the slide for 1 to 10 minutes; preferably, the detection time is the same as the detection time of the fluorescence detector used to prepare the standard curve; the detection environment and characteristics are the same as the detection time of the fluorescence detector used to prepare the standard curve;

[0049] D. Substitute the fluorescence value tested in step C into the standard curve in step B to calculate the D-2-hydroxyglutaric acid content of the tissue to be tested.

[0050] Compared with the prior art, the present invention has the following advantages:

[0051] The D-2-hydroxyglutaric acid concentration determination method of the present invention allows detection of D-2-hydroxyglutaric acid concentration in tissue samples after simple treatment. The test results are consistent with those of the "gold standard" detection method (mass spectrometry), with high accuracy. Conventional mass spectrometry methods require 5 to 6 hours from sample processing to calculation of D-2-hydroxyglutaric acid concentration. However, the D-2-hydroxyglutaric acid concentration determination method of the present invention allows the standard to be prepared and stored in a -20°C refrigerator in advance. Testing the standard and preparing sample slices takes 20 minutes, sample slice testing takes approximately 3 minutes, and calculation of D-2-HG concentration takes approximately 2 minutes. After preparing the standard curve and sample slices, the calculation takes less than 5 minutes, significantly shortening the detection time and reducing detection costs.

[0052] The kit for detecting the concentration of D-2-hydroxyglutaric acid of the present invention comprises a PBS buffer solution, a resazurin solution, a FAD-dependent D-2-hydroxyglutarate dehydrogenase solution, and an agarose gel slide containing a series of D-2-hydroxyglutaric acid concentrations, which can be used to prepare a standard curve. A user only needs to prepare tissue to be tested, thereby facilitating detection, greatly shortening detection time, reducing detection costs, and facilitating widespread use. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG1 is a schematic diagram showing the effect of using OCT embedding medium on enzyme activity reaction;

[0054] FIG2 is a schematic diagram showing the effect of using agarose as a medium on enzyme activity reaction;

[0055] FIG3 is a schematic diagram showing the effect of using PAGE as a medium on enzyme activity reaction;

[0056] FIG4 is a schematic diagram showing the effect of different buffers on enzyme reaction activity;

[0057] FIG5 is a schematic diagram showing the effect of different slice areas on the standard curve;

[0058] FIG6 is a schematic diagram showing the effect of different slice thicknesses on the standard curve;

[0059] Figure 7 shows the range of standards when testing standards at different time points;

[0060] FIG8 is a schematic diagram of a standard curve in Example 1;

[0061] FIG9 is a comparison diagram of data obtained by the detection method of the present invention and the traditional mass spectrometry detection method. DETAILED DESCRIPTION

[0062] The object of the present invention is to provide a method and a kit for determining the concentration of D-2-hydroxyglutaric acid, which are achieved by the following technical solutions:

[0063] The resazurin used in the examples of the present invention is a product purchased from Sigma.

[0064] The method for determining the concentration of D-2-hydroxyglutaric acid of the present invention is initially designed by selecting a medium for the D-2-HG standard. The available mediums include OCT embedding medium, agarose, and polyacrylamide (PAGE) solution. The process is as follows:

[0065] 1. Effect of different D-2-HG standard medium on reaction rate

[0066] (1) Preparation of D-2-HG standard medium

[0067] i. OCT embedding medium: Prepare 1 / 15M PBS solutions with concentrations of 0%, 0.1%, 0.2%, 0.5%, and 5% OCT embedding medium.

[0068] ii. Agarose: Prepare 1 / 15M PBS solutions with concentrations of 0%, 0.05%, 0.1%, and 0.2% agarose.

[0069] iii. PAGE solution: Prepare 1 / 15M PBS solutions with concentrations of 0% PAGE, 0.1% PAGE, 1% PAGE, and 5% PAGE.

[0070] (2) Enzyme reaction system

[0071]

[0072] (3) After the above solution is prepared, place it on a 96-well black plate and perform detection in a Biotek Cytation5 instrument with emission light at 540 nm and excitation light at 590 nm, setting the detection interval to 2 s for a total of 300 s.

[0073] The results are shown in Figures 1 to 3. Figure 1 shows that the OCT embedding medium significantly inhibits the enzymatic reaction and is unsuitable as a medium for the D-2-HG standard. Figure 2 shows that agarose has no inhibitory effect on the enzymatic reaction and can be used as a medium for the D-2-HG standard. Figure 3 shows that the components in the PAGE gel significantly inhibit the enzymatic reaction and are unsuitable as a medium for the D-2-HG standard. Therefore, we chose agarose gel as the medium for the D-2-HG standard.

[0074] The present invention has optimized and tested some experimental items and conditions as follows:

[0075] 2. Experiments with different buffers in the staining solution

[0076] (1) Enzyme reaction system

[0077]

[0078] The above buffer solution is 1 / 15 M PBS solution, 0.1 M Tris-HCl buffer or 0.1 M HEPES buffer;

[0079] (2) After the above solution is prepared, place it on a 96-well black plate and perform detection in a Biotek Cytation5 instrument with emission light at 540 nm and excitation light at 590 nm, setting the detection time to be once every 2 seconds for a total of 300 seconds.

[0080] (3) As shown in Figure 4, the fluorescence of 0.1M Tris-HCl buffer, 0.1M HEPES buffer, and 1 / 15M PBS buffer all increased with increasing D-2-hydroxyglutaric acid concentration. Among them, 1 / 15M PBS buffer had a higher fluorescence value at the same resazurin concentration, indicating better performance. Therefore, 1 / 15M PBS buffer was selected as the preferred buffer for subsequent experiments.

[0081] 3. Experimental Study of Different D-2-HG Standards and Tissue Sample Section Areas in Frozen Sections

[0082] 1. Fluorescence detection machine

[0083] The fluorescence detection machine equipment uses a customized instrument (BLP-FD-100CS) developed by Wave-Particle Photonics, which integrates excitation light, beam collimation, light homogenization, extinction, fluorescence collection, fluorescence detection, filtering, and signal shielding protection. It has an excitation wavelength of 540nm, an emission wavelength of 590nm, an optical density of OD5, a spot diameter of 6.5mm, an input voltage of 5V, an analog signal output of 0-3.3V, and a digital signal output.

[0084] 2. D-2-HG standard processing:

[0085] (1) Preparation of D-2-HG standard: Prepare 20 ml of 3% agarose solution containing 0 μmol / g, 0.1 μmol / g, 0.25 μmol / g, 0.5 μmol / g, 1 μmol / g, 1.5 μmol / g and 2.0 μmol / g of D-2-HG (for conversion to g / cm 3 The density of brain tumor tissue is based on the normal brain tissue density of 1.054 g / cm 3 Using a microwave, heat 3% agarose solutions containing various concentrations of D-2-HG on medium heat until clear and transparent. Pour the solution onto a gel plate and allow it to solidify after half an hour. Remove the solidified agarose and cut it into rectangular blocks with a cross-sectional area of ​​1.5 cm x 1.5 cm. Place the block in a tissue tray in a cryostat (Leica) and cover the agarose block with 400 µl of OCT embedding medium. After the OCT embedding medium solidifies in the cryostat, slice the agarose gel into 20 µm thicknesses and mount it on a slide. For each agarose concentration, divide the sample into three groups of tissue sections: 0.1 cm x 0.1 cm, 0.5 cm x 0.5 cm, and 1 cm x 1 cm. When sectioning, start by slicing the tissue into a larger area, then trim it into sections of 0.1cm*0.1cm, 0.5cm*0.5cm, or 1cm*1cm. This will remove any surface contamination with OCT and prevent the OCT embedding agent from affecting the test data. Once completed, store the sliced ​​tissue in a -20°C freezer.

[0086] (2) Staining of D-2-HG standard sections

[0087] i Staining solution: 500µl 1 / 15M PBS + 1µl 5mM resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase;

[0088] ii. At room temperature (25°C) and protected from light, add 50 µl of staining solution to the tissue to completely cover the D-2-HG standard. Detect the fluorescence of the staining solution on the slide using a fluorescence detector, measuring every 2 seconds for a total of 300 seconds.

[0089] 3. Preparation of standard curve

[0090] The D-2-HG concentrations in the standard slices with a slice area of ​​0.1 cm*0.1 cm of 0 µmol / g, 0.1 µmol / g, 0.25 µmol / g, 0.5 µmol / g, 1 µmol / g, 1.5 µmol / g, and 2.0 µmol / g D-2-HG were used as x values, and the fluorescence values ​​measured at the 180 s time point using the above method were used as y values. A scatter plot was drawn, and a linear fitting relationship of y=ax+b was made using the scatter plot, as shown in Figure 4.

[0091] The D-2-HG concentrations in the standard slices with a slice area of ​​0.5 cm*0.5 cm of 0 µmol / g, 0.1 µmol / g, 0.25 µmol / g, 0.5 µmol / g, 1 µmol / g, 1.5 µmol / g, and 2.0 µmol / g D-2-HG were used as x values, and the fluorescence values ​​measured at the 180 s time point using the above method were used as y values. A scatter plot was drawn, and a linear fitting relationship of y=ax+b was made using the scatter plot, as shown in Figure 4.

[0092] A scatter plot was plotted, with the D-2-HG concentration in 1 cm*1 cm sections of standard samples containing 0 µmol / g, 0.1 µmol / g, 0.25 µmol / g, 0.5 µmol / g, 1 µmol / g, 1.5 µmol / g, and 2.0 µmol / g D-2-HG as the x value and the fluorescence value measured at 180 s using the above method as the y value. A linear relationship was fitted to the scatter plot using the equation y = ax + b, as shown in Figure 5. The results showed a positive correlation between fluorescence value and D-2-HG for sections measuring 0.1 cm*0.1 cm, 0.5 cm*0.5 cm, and 1 cm*1 cm, respectively, and these sections could all be used as the cross-sectional area of ​​the standard sample sections.

[0093] 3. Experimentation with different D-2-HG standards and tissue sample thicknesses in frozen sections

[0094] 1. Fluorescence detection machine

[0095] The fluorescence detection equipment uses a customized instrument (BLP-FD-100CS) from Wave-Particle Photonics, with the same parameters as above.

[0096] 2. D-2-HG standard processing:

[0097] (1) Preparation of D-2-HG standards: Prepare 20 ml of each of 3% agarose solution containing 0 µmol / g, 0.1 µmol / g, 0.25 µmol / g, 0.5 µmol / g, 1 µmol / g, 1.5 µmol / g, and 2.0 µmol / g of D-2-HG (for conversion to g / cm 3 The density of brain tumor tissue is based on the normal brain tissue density of 1.054 g / cm 3 Heat the 3% agarose solution containing various concentrations of D-2-HG in a microwave oven over medium heat until clear and transparent. Pour the solution into a gel plate and allow it to solidify after half an hour. Remove the plate and cut the solidified agarose into rectangular blocks with a cross-sectional area of ​​1.5 cm x 1.5 cm. Place the block in the tissue tray of a cryostat (Leica) and cover the agarose block with a small amount of OCT embedding medium. Once the OCT embedding medium has solidified in the cryostat, cut the agarose into three thickness gradients of 5 µm, 20 µm, and 25 µm. Place the block onto a slide with a non-stick glass slide and use a knife to adjust the tissue sections to 0.5 cm x 0.5 cm. Store in a -20°C freezer.

[0098] (2) Staining of D-2-HG standard sections

[0099] i Staining solution: 500µl 1 / 15M PBS + 1µl 5mM Resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0100] ii. At room temperature (25°C) and protected from light, add 50 µl of staining solution to the tissue to completely cover the D-2-HG standard. Detect the fluorescence of the staining solution on the slide using a fluorescence detector, measuring every 2 seconds for a total of 300 seconds.

[0101] 3. Preparation of standard curve

[0102] A scatter plot was drawn with the D-2-HG concentrations of 0 µmol / g, 0.1 µmol / g, 0.25 µmol / g, 0.5 µmol / g, 1 µmol / g, 1.5 µmol / g, and 2.0 µmol / g in 5 µm thick standard slices as the X value and the fluorescence value measured at 180 s using the above method as the Y value. A linear fit of y = ax + b was performed on the scatter plot, and the results are shown in Figure 6.

[0103] A scatter plot was drawn with the D-2-HG concentrations of 0 µmol / g, 0.1 µmol / g, 0.25 µmol / g, 0.5 µmol / g, 1 µmol / g, 1.5 µmol / g, and 2.0 µmol / g in 20 µm thick standard slices as the X value and the fluorescence value measured at 180 s using the above method as the Y value. A linear fit of y = ax + b was performed on the scatter plot, and the results are shown in Figure 6.

[0104] A scatter plot was plotted using the D-2-HG concentrations of 0, 0.1, 0.25, 0.5, 1, 1.5, and 2.0 µmol / g in 25 µm thick standard sections as the X value and the fluorescence value measured at 180 s using the above method as the Y value. A linear relationship was fitted to the scatter plot using the equation y = ax + b, as shown in Figure 6. It can be seen that the fluorescence value and D-2-HG are positively correlated at section thicknesses of 5, 20, and 25 µm, respectively, and these can all be used as the section thickness for standard sections.

[0105] IV. Experiments with different concentrations of resazurin in the staining solution

[0106] 1. Fluorescence detection machine

[0107] The fluorescence detection machine equipment uses a customized instrument (BLP-FD-100CS) developed by Wave-Particle Photonics, which integrates excitation light, beam collimation, light homogenization, extinction, fluorescence collection, fluorescence detection, filtering, and signal shielding protection. It has an excitation wavelength of 540nm, an emission wavelength of 590nm, an optical density of OD5, a spot diameter of 6.5mm, an input voltage of 5V, an analog signal output of 0-3.3V, and a digital signal output.

[0108] 2. D-2-HG standard processing:

[0109] (1) Preparation of D-2-HG standard: Prepare 20 ml of 3% agarose solution containing 0 μmol / g, 0.1 μmol / g, 0.25 μmol / g, 0.5 μmol / g, 1 μmol / g, 1.5 μmol / g and 2.0 μmol / g of D-2-HG (for conversion to g / cm 3 The density of brain tumor tissue is based on the normal brain tissue density of 1.054 g / cm 3 Microwave ovens were used to heat 3% agarose solutions containing various concentrations of D-2-HG on medium heat until clear and transparent. The solutions were then poured onto gel plates. After half an hour, the solidified agarose solution was removed and cut into rectangular blocks with a cross-section of 1.5 cm x 1.5 cm. These blocks were placed in a tissue tray in a Leica cryostat and covered with 400 µl of OCT embedding medium. After the OCT embedding medium had solidified in the cryostat, the agarose gel was sliced ​​to a 20 µm thickness and mounted on a non-stick slide. Three different tissue sections were prepared for each agarose concentration, each with a 0.5 cm x 0.5 cm section. When sectioning, the tissue sections were first sliced ​​into larger sections and then trimmed to 0.5 cm x 0.5 cm. This method removes any surface contamination with OCT and prevents the OCT embedding medium from affecting the data. After the sections were completed, the cut tissue sections were stored at -20°C.

[0110] (2) Staining of D-2-HG standard sections

[0111] i The staining solution configuration is divided into the following 5 groups:

[0112] ①498.5µl 1 / 15M PBS + 0.5µl 0.5mM resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0113] ②498.5µl 1 / 15M PBS + 0.5µl 5mM resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0114] ③498µl 1 / 15M PBS + 1µl 5mM resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0115] ④489µl 1 / 15M PBS + 10µl 5mM resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0116] ⑤479µl 1 / 15M PBS + 20µl 5mM resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0117] ii. At room temperature (25°C) and protected from light, add 50 µl of staining solution to the tissue to completely cover the D-2-HG standard. Detect the fluorescence of the staining solution on the slide using a fluorescence detector, measuring every 2 seconds for a total of 300 seconds.

[0118] 3. Result processing

[0119] The five staining solutions described above each contained a different resazurin concentration, ranging from 0.5 μM to 200 μM. The final results showed that, at the same D-2-HG standard concentration, the fluorescence value of group ③ was greater than that of group ② and greater than that of group ①. The fluorescence values ​​of groups ④ and ⑤ were essentially equal to that of group ③. This indicates that the resazurin concentrations in groups ① and ② were insufficient, those in groups ④ and ⑤ were too high, and the resazurin concentration in group ③ was appropriate. All five staining solutions were suitable for D-2-hydroxyglutaric acid concentration determination, with group ③ being the optimal ratio.

[0120] 5. Experimental study on different concentrations of FAD-dependent D-2-hydroxyglutarate dehydrogenase in the staining solution

[0121] 1. Fluorescence detection machine

[0122] The fluorescence detection machine equipment uses a customized instrument (BLP-FD-100CS) developed by Wave-Particle Photonics, which integrates excitation light, beam collimation, light homogenization, extinction, fluorescence collection, fluorescence detection, filtering, and signal shielding protection. It has an excitation wavelength of 540nm, an emission wavelength of 590nm, an optical density of OD5, a spot diameter of 6.5mm, an input voltage of 5V, an analog signal output of 0-3.3V, and a digital signal output.

[0123] 2. D-2-HG standard processing:

[0124] (1) Preparation of D-2-HG standard: Prepare 20 ml of 3% agarose solution containing 0 μmol / g, 0.1 μmol / g, 0.25 μmol / g, 0.5 μmol / g, 1 μmol / g, 1.5 μmol / g and 2.0 μmol / g of D-2-HG (in order to convert to g / cm3, the density of brain tumor tissue is based on the normal brain tissue density of 1.054 g / cm3). Use a microwave to heat the 3% agarose solution containing various concentrations of D-2-HG on medium heat until it becomes clear and transparent. Pour it into a gel plate. After solidification for half an hour, remove it and cut the solidified agarose into a rectangular block with a cross-sectional area of ​​1.5 cm * 1.5 cm. Place it in the tissue tray of a freezing microtome (Leica) and cover the agarose gel block with 400 μl of OCT embedding medium. After the OCT embedding medium has solidified in a cryostat, slice the agarose gel to a 20µm thickness and place it on a slide. For each agarose concentration, divide the sample into three groups of 0.5cm x 0.5cm sections. When slicing, it's recommended to first slice the tissue into a larger area and then trim it to a 0.5cm x 0.5cm section. This will remove any surface contamination from the OCT embedding medium and prevent it from affecting the test data. After this procedure, store the sliced ​​tissue in a -20°C freezer.

[0125] (2) Staining of D-2-HG standard sections

[0126] i The staining solution configuration is divided into the following 5 groups:

[0127] ①498.5µl 1 / 15M PBS + 1µl 5mM resazurin + 0.5µl 1.3mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0128] ② 498.5µl 1 / 15M PBS + 1µl 5mM resazurin + 0.5µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0129] ③ 498µl 1 / 15M PBS + 1µl 5mM resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0130] ④489µl 1 / 15M PBS + 1µl 5mM resazurin + 10µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0131] ⑤479µl 1 / 15M PBS + 1µl 5mM resazurin + 20µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0132] ii. At room temperature (25°C) in a dark environment, add 50µl of staining solution to completely cover the D-2-HG standard. After 5 minutes, use a fluorescence detector to measure the fluorescence of the staining solution on the slide, measuring every 2 seconds for a total of 300 seconds.

[0133] 3. Result processing

[0134] The five staining solutions described above each contained a different concentration of FAD-dependent D-2-hydroxyglutarate dehydrogenase, ranging from 1.3µg / ml to 520µg / ml. The final results showed that, at the same D-2-HG standard concentration, the fluorescence value of group ③ was greater than that of group ② and greater than that of group ①. The fluorescence values ​​of groups ④ and ⑤ were essentially equal to that of group ③. This indicates that the concentrations of FAD-dependent D-2-hydroxyglutarate dehydrogenase in groups ① and ② were insufficient, those in groups ④ and ⑤ were too high, and the concentration in group ③ was appropriate. All five staining solutions were suitable for D-2-hydroxyglutarate concentration determination, with group ③ being the optimal concentration.

[0135] VI. Experiments with different concentrations of resazurin in the kit

[0136] 1. Fluorescence detection machine

[0137] The fluorescence detection machine equipment uses a customized instrument (BLP-FD-100CS) developed by Wave-Particle Photonics, which integrates excitation light, beam collimation, light homogenization, extinction, fluorescence collection, fluorescence detection, filtering, and signal shielding protection. It has an excitation wavelength of 540nm, an emission wavelength of 590nm, an optical density of OD5, a spot diameter of 6.5mm, an input voltage of 5V, an analog signal output of 0-3.3V, and a digital signal output.

[0138] 2. D-2-HG standard processing:

[0139] (1) Preparation of D-2-HG standard: Prepare 20 ml of 3% agarose solution containing 0 μmol / g, 0.1 μmol / g, 0.25 μmol / g, 0.5 μmol / g, 1 μmol / g, 1.5 μmol / g and 2.0 μmol / g of D-2-HG (for conversion to g / cm 3 The density of brain tumor tissue is based on the normal brain tissue density of 1.054 g / cm 3Microwave ovens were used to heat 3% agarose solutions containing various concentrations of D-2-HG on medium heat until clear and transparent. The solutions were then poured onto gel plates. After half an hour, the solidified agarose solution was removed and cut into rectangular blocks with a cross-section of 1.5 cm x 1.5 cm. These blocks were placed in a tissue tray in a Leica cryostat and covered with 400 µl of OCT embedding medium. After the OCT embedding medium had solidified in the cryostat, the agarose gel was sliced ​​to a 20 µm thickness and mounted on a non-stick slide. Three different tissue sections were prepared for each agarose concentration, each with a 0.5 cm x 0.5 cm section. When sectioning, the tissue sections were first sliced ​​into larger sections and then trimmed to 0.5 cm x 0.5 cm. This method removes any surface contamination with OCT and prevents the OCT embedding medium from affecting the data. After the sections were completed, the cut tissue sections were stored at -20°C.

[0140] (2) Staining of D-2-HG standard sections

[0141] i The staining solution configuration is divided into the following 5 groups:

[0142] ① 474µl 1 / 15M PBS + 25µl 0.2mM resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0143] ② 489µl 1 / 15M PBS + 10µl 0.5mM Resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0144] ③ 496µl 1 / 15M PBS + 5µl 1mM Resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0145] ④ 498µl 1 / 15M PBS + 1µl 5mM resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0146] ⑤ 498.8µl 1 / 15M PBS + 0.2µl 25mM Resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0147] ii. At room temperature (25°C) and protected from light, add 50 µl of staining solution to the tissue to completely cover the D-2-HG standard. Detect the fluorescence of the staining solution on the slide using a fluorescence detector, measuring every 2 seconds for a total of 300 seconds.

[0148] 3. Result processing

[0149] 50µl of each of the five different staining solutions was added to a 20µm-thick section containing 0µmol / g, 0.1µmol / g, 0.25µmol / g, 0.5µmol / g, 1µmol / g, 1.5µmol / g, and 2.0µmol / g of D-2-HG. The D-2-HG concentration was designated as the X value, and the fluorescence value measured at the 180s time point using the above method was designated as the Y value. A scatter plot was plotted, and a linear relationship was fitted to the scatter plot using the equation y = ax + b. The standard curves produced by the five different staining solutions showed no significant differences (p>0.05), indicating that the resazurin concentration in the kit can range from 0.2mM to 25mM.

[0150] VII. Test of different concentrations of FAD-dependent D-2-hydroxyglutarate dehydrogenase in the kit

[0151] 1. Fluorescence detection machine

[0152] The fluorescence detection machine equipment uses a customized instrument (BLP-FD-100CS) developed by Wave-Particle Photonics, which integrates excitation light, beam collimation, light homogenization, extinction, fluorescence collection, fluorescence detection, filtering, and signal shielding protection. It has an excitation wavelength of 540nm, an emission wavelength of 590nm, an optical density of OD5, a spot diameter of 6.5mm, an input voltage of 5V, an analog signal output of 0-3.3V, and a digital signal output.

[0153] 2. D-2-HG standard processing:

[0154] (1) Preparation of D-2-HG standard: Prepare 20 ml of 3% agarose solution containing 0 μmol / g, 0.1 μmol / g, 0.25 μmol / g, 0.5 μmol / g, 1 μmol / g, 1.5 μmol / g and 2.0 μmol / g of D-2-HG (in order to convert to g / cm3, the density of brain tumor tissue is based on the normal brain tissue density of 1.054 g / cm3). Use a microwave to heat the 3% agarose solution containing various concentrations of D-2-HG on medium heat until it becomes clear and transparent. Pour it into a gel plate. After solidification for half an hour, remove it and cut the solidified agarose into a rectangular block with a cross-sectional area of ​​1.5 cm * 1.5 cm. Place it in the tissue tray of a freezing microtome (Leica) and cover the agarose gel block with 400 μl of OCT embedding medium. After the OCT embedding medium has solidified in a cryostat, slice the agarose gel to a 20µm thickness and place it on a slide. For each agarose concentration, divide the sample into three groups of 0.5cm x 0.5cm sections. When slicing, it's recommended to first slice the tissue into a larger area and then trim it to a 0.5cm x 0.5cm section. This will remove any surface contamination from the OCT embedding medium and prevent it from affecting the test data. After this procedure, store the sliced ​​tissue in a -20°C freezer.

[0155] (2) Staining of D-2-HG standard sections

[0156] i The staining solution configuration is divided into the following 4 groups:

[0157] ① 489µl 1 / 15M PBS + 1µl 5mM resazurin + 10µl 1.3mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0158] ②498µl 1 / 15M PBS + 1µl 5mM resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0159] ③498.33µl 1 / 15M PBS + 1µl 5mM resazurin + 0.67µl 19.5mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0160] ④498.5µl 1 / 15M PBS + 1µl 5mM resazurin + 0.5µl 26mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0161] ii. At room temperature (25°C) and protected from light, add 50 µl of staining solution to the tissue to completely cover the D-2-HG standard. Detect the fluorescence of the staining solution on the slide using a fluorescence detector, measuring every 2 seconds for a total of 300 seconds.

[0162] 3. Result processing

[0163] 50µl of the four different staining solutions containing 0µmol / g, 0.1µmol / g, 0.25µmol / g, 0.5µmol / g, 1µmol / g, 1.5µmol / g, and 2.0µmol / g of D-2-HG were added to 20µm-thick sections. The D-2-HG concentration was designated as the X value, and the fluorescence value measured at the 180s time point using the above method was designated as the Y value. A scatter plot was plotted, and a linear relationship was fitted to the scatter plot using the equation y = ax + b. The standard curves produced by the four different staining solutions showed no significant differences (p>0.05). This indicates that the concentration of FAD-dependent D-2-hydroxyglutarate dehydrogenase in the kit ranges from 1.3mg / ml to 26mg / ml.

[0164] 8. Test on the range of D-2-HG in standard sample slices and the detection time points in the standard curve

[0165] 1. Fluorescence detection machine

[0166] The fluorescence detection machine equipment uses a customized instrument (BLP-FD-100CS) developed by Wave-Particle Photonics, which integrates excitation light, beam collimation, light homogenization, extinction, fluorescence collection, fluorescence detection, filtering, and signal shielding protection. It has an excitation wavelength of 540nm, an emission wavelength of 590nm, an optical density of OD5, a spot diameter of 6.5mm, an input voltage of 5V, an analog signal output of 0-3.3V, and a digital signal output.

[0167] 2. D-2-HG standard processing:

[0168] (1) Preparation of D-2-HG standard: Prepare 20 ml of 3% agarose solution containing D-2-HG 0 umol / g, 0.1 umol / g, 0.25 umol / g, 0.5 umol / g, 1 umol / g, 1.5 umol / g, 2.0 umol / g, 3.0 umol / g, 4.0 umol / g, and 5.0 umol / g (for conversion to g / cm 3 The density of brain tumor tissue is based on the normal brain tissue density of 1.054 g / cm 3Microwave ovens were used to heat 3% agarose solutions containing various concentrations of D-2-HG on medium heat until clear and transparent. The solutions were then poured onto gel plates. After half an hour, the solidified agarose solution was removed and cut into rectangular blocks with a cross-section of 1.5 cm x 1.5 cm. These blocks were placed in a tissue tray in a Leica cryostat and covered with 400 µl of OCT embedding medium. After the OCT embedding medium had solidified in the cryostat, the agarose gel was sliced ​​to a 20 µm thickness and mounted on a non-stick slide. Three different tissue sections were prepared for each agarose concentration, each with a 0.5 cm x 0.5 cm section. When sectioning, the tissue sections were first sliced ​​into larger sections and then trimmed to 0.5 cm x 0.5 cm. This method removes any surface contamination with OCT and prevents the OCT embedding medium from affecting the data. After the sections were completed, the cut tissue sections were stored at -20°C.

[0169] (2) Staining of D-2-HG standard sections

[0170] i Staining solution: 500µl 1 / 15M PBS + 1µl 5mM Resazurin + 1µl 13mg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase

[0171] ii. At room temperature (25°C) and protected from light, add 50 µl of staining solution to the tissue to completely cover the D-2-HG standard. Detect the fluorescence of the staining solution on the slide using a fluorescence detector, measuring every 2 seconds for a total of 300 seconds.

[0172] 3. Result processing

[0173] 50 µl of staining solution was added to 20 µm thick sections containing 0 µmol / g, 0.1 µmol / g, 0.25 µmol / g, 0.5 µmol / g, 1 µmol / g, 1.5 µmol / g, 2.0 µmol / g, 2.5 µmol / g, 3.0 µmol / g, 4.0 µmol / g, and 5.0 µmol / g of D-2-HG. The D-2-HG concentration was designated as the X value, and the fluorescence values ​​measured at 60 s, 120 s, and 180 s using the above method were designated as the Y value. A scatter plot was plotted, and a linear relationship was fitted to the scatter plot using the equation y = ax + b. The results are shown in Figure 7. Because the fluorescence values ​​of the sections in the standard curve increase with increasing reaction time after the staining solution is added, which can reach the upper limit of fluorescence detection, it is important to select an appropriate time range for the standard curve. Therefore, when the values ​​detected at the 60s time point are used to fit the standard curve, the detectable range of the standard curve can reach 0µmol / g-5.0 umol / g; when the values ​​detected at the 120s time point are used to fit the standard curve, the detectable range of the standard curve can reach 0µmol / g-3.0 umol / g; when the values ​​detected at the 180s time point are used to fit the standard curve, the detectable range of the standard curve can reach 0µmol / g-2.0 umol / g. However, as shown in Figure 7, the standard curve fitting R 2 Standard curve fitting R<120s 2 Standard curve fitting R<180s 2 Therefore, although the detection range of the standard curve is smaller at 180s, the standard curve accuracy is higher. When using the kit, the user can select the standard curve value time node according to their own needs.

[0174] The present invention is further described below with reference to specific embodiments.

[0175] Eight patient-derived tumor tissues (whose acquisition had been approved by the unit's ethics committee) were obtained and numbered #1, #2, #3, #4, ..., #7, and #8. Each numbered sample was divided into two equal parts, one for detection using the method in Example 1, and the other for detection using the mass spectrometry method in the comparative example. Example 1

[0176] ① Preparation of D-2-hydroxyglutaric acid standard sections: Prepare a series of D-2-hydroxyglutaric acid concentrations of 0µmol / g, 0.1µmol / g, 0.25µmol / g, 0.5µmol / g, 1µmol / g, 1.5µmol / g, and 2.0µmol / g in agarose solution (20ml each). Heat the agarose solution until clear and transparent, pour it onto a gel plate to solidify, remove it, cut it into rectangular blocks with a cross-sectional area of ​​1.0cm × 1.0cm, and place it in the tissue tray of a freezing microtome. Cover the agarose gel block with 400µl of OCT embedding medium. After the OCT embedding medium is frozen solid in the freezing microtome, slice the agarose gel to a thickness of 20µm, place it on a slide, and cut it into rectangular blocks with a cross-sectional area of ​​0.5cm × 0.5cm. Store it in a -20°C freezer to obtain a series of D-2-hydroxyglutaric acid standard sections of different concentrations for later use.

[0177] Three parallel experiments were set up for each concentration of standard slices;

[0178] The concentration of agarose in the agarose aqueous solution is 3%, and the density of brain tumor tissue is based on the normal brain tissue density of 1.054 g / cm 3 ;

[0179] ② Slice processing of tissue samples to be tested

[0180] The tissue to be tested was removed from the -80°C freezer, placed on ice, and placed in the tissue tray of a cryostat. 400 µl of OCT freezing embedding medium was added to the tissue to be tested. After the OCT freezing embedding medium turned white, the tissue to be tested was sliced ​​to a thickness of 20 µm, placed on a non-shedding slide, and cut into rectangular blocks with a cross-sectional area of ​​0.5 cm × 0.5 cm to obtain the tissue sections to be tested and set aside.

[0181] Five parallel experiments were set up for each tissue sample slice to be tested;

[0182] ③ Prepare staining solution: add 1µl of resazurin and 1µl of FAD-dependent D-2-hydroxyglutarate dehydrogenase to 500µl of 1 / 15mol / L PBS buffer solution to obtain staining solution;

[0183] ④ Staining of tissue sections

[0184] At room temperature (25°C) and in a dark environment, take 50 μl of the staining solution obtained in step ③ and completely cover the tissue section to be tested obtained in step ②. Use a fluorescence detector to detect the fluorescence value of the staining solution on the slide, measuring once every 2 seconds for a total of 300 seconds.

[0185] ⑤ Standard product data testing and result processing

[0186] A series of D-2-hydroxyglutaric acid standard sections of varying concentrations from step ① were placed on a glass slide and completely covered with 50 μl of the staining solution from step ③ at room temperature (25°C) in a dark environment. The average fluorescence value of each concentration detected by a fluorescence detector over 180 s was taken as the y value, and the concentration of D-2-hydroxyglutaric acid contained was taken as the x value. A scatter plot was drawn using the scatter plot to create a standard curve y = ax + b; the results are shown in FIG8 .

[0187] Calculate the D-2-hydroxyglutaric acid content in the tissue section to be tested based on the fluorescence value obtained in step ③.

[0188] Comparative Example

[0189] Detection of D-2-HG content in pathological tissues by mass spectrometry

[0190] The detection process is as follows:

[0191] 1. Sample preparation:

[0192] (1) Prepare 80% methanol solution and pre-cool it in a 4°C refrigerator;

[0193] (2) Take 100 mg of tissue (8 samples from the same tissue as in the example) and place them in 1 ml of 80% methanol solution. Place two steel balls in each sample and use a tissue disruptor to disrupt the tissue at 20 times / second. Homogenize for 30 min.

[0194] (3) Centrifuge at 14,000 rpm, 4°C, for 30 min, filter the supernatant through a 100 µm sieve, and take a 750 µl volume of supernatant;

[0195] (4) The sample was completely dried in a vacuum concentrator (60°C) for 2.5 h, and resuspended in 300 µl of pure water. Centrifuged at 13,000 g for 5 min, and the supernatant was transferred to a new tube and stored in a -80°C freezer.

[0196] 2. LC-MS / MS Mass Spectrometry

[0197] (1) Standard curve (D-2-HG internal standard method): DL-2-hydroxyglutarate disodium salt (13C5, 99%) (purchased from Cambridge Isotope Laboratory, USA) was prepared into standards with concentrations of 100 nM, 300 nM, 700 nM, 1 µM, 5 µM, 10 µM, 50 µM, and 100 µM.

[0198] (2) The instruments used were a high-performance liquid chromatography LC-20A (Shimadzu, Japan) coupled with a nano-resolution combined liquid chromatography-mass spectrometer (Thermo Fisher, USA). The mobile phase consisted of methanol and 0.1% triethylamine, adjusted to pH 4.5 using 5% acetic acid. The run time was set to 15 min, and 20 µl of sample was added to a chiral column (Chirobiotic R column, 250 x 4.6 mm) at a flow rate of 0.5 ml / min. Standards and samples were tested on the instrument. The mass-to-nuclear ratio of D-2-HG was monitored as 147.0 m / z → 129.0 m / z and the internal standard of D-2-HG was monitored as 150.0 m / z → 132.0 m / z.

[0199] The test results are shown in Table 1, and a comparison chart is shown in Figure 9. As can be seen from the results in Table 1 and Figure 9, the results obtained by this experimental method and the mass spectrometry detection method for D-2-hydroxyglutaric acid are quite consistent, demonstrating the high accuracy of this experimental method. The mass spectrometry method requires 5-6 hours from sample processing to calculating the D-2-HG concentration result. The experimental method used in this application can prepare D-2-HG standards in advance and store them in a -20°C refrigerator. The process from sample processing to calculating the D-2-HG concentration result takes a maximum of 15 minutes, significantly reducing the time required.

[0200] Alternatively, the kit for detecting the concentration of D-2-hydroxyglutaric acid of the present application can be used by simply using PBS buffer solution, resazurin solution, and FAD-dependent D-2-hydroxyglutarate dehydrogenase solution (the concentration range of the resazurin solution is 0.2 mM-25 mM, and the concentration range of the FAD-dependent D-2-hydroxyglutarate dehydrogenase solution is 1.3 mg / ml-26 mg / ml) as staining agents to stain an agarose gel slide containing a series of D-2-hydroxyglutaric acid concentrations. The fluorescence value on the slide is then detected on a fluorescence detector to create a standard curve. The sample is then stained with the stain, and the fluorescence value on the slide is detected on a fluorescence detector to determine the concentration of D-2-hydroxyglutaric acid in the sample. This kit saves customers the time of preparing stain and agarose gel slides containing D-2-hydroxyglutaric acid. Instead, they only need to cut the thickness and cross-sectional area of ​​the agarose gel containing D-2-hydroxyglutaric acid according to the thickness and cross-sectional area of ​​the sample to ensure that the thickness and cross-sectional area of ​​the sample and the agarose gel containing D-2-hydroxyglutaric acid are consistent.

[0201] Table 1 Test results

[0202]

[0203] .

Claims

1. A method for determining the concentration of D-2-hydroxyglutaric acid, characterized in that: Using FAD-dependent D-2-hydroxyglutarate dehydrogenase, resazurin and PBS buffer system as working solution, D-2-hydroxyglutarate in solid tissue sections can be detected. The specific steps include: ① Make D-2-hydroxyglutaric acid standard slices: add D-2-hydroxyglutaric acid to agarose aqueous solution; heat and stir until clear and transparent, pour on a gel plate to solidify, take out, cut into a rectangular block with a cross-sectional area of ​​0.1~1.5cm×0.1~1.5cm, place it in the tissue tray of the freezing microtome, cover the agarose gel block with OCT embedding agent, and after the OCT embedding agent is frozen solid in the freezing microtome, cut the agarose gel to a thickness of 5~25μm, put it on an anti-shedding slide, cut into a rectangular block with a cross-sectional area of ​​0.1~1cm×0.1~1cm, and store it in a -20℃ refrigerator to obtain a series of D-2-hydroxyglutaric acid standard slices with different concentrations for later use; The ratio of D-2-hydroxyglutaric acid to agarose aqueous solution is 0~5.0µmol:1g; Three parallel experiments were set up for each concentration of standard slices; ② Slice processing of tissue samples to be tested The tissue to be tested is taken out of the -80℃ refrigerator, placed on ice, and placed in the tissue tray of the cryostat. The OCT freezing embedding medium is added to the tissue to be tested. After the OCT freezing embedding medium turns white, the tissue to be tested is sliced ​​to a thickness of 5-25μm, placed on an anti-shedding slide, and cut into a rectangular block with a cross-sectional area of ​​0.1-1cm×0.1-1cm, which is the same as the cross-sectional area of ​​the standard slice, to obtain the tissue slice to be tested and set aside; Three parallel experiments were set up for each tissue sample slice to be tested; ③Prepare staining solution: the staining solution is a buffer solution with a pH of 7.0-8.0, containing resazurin and FAD-dependent D-2-hydroxyglutarate dehydrogenase; the buffer solution is Tris-Hcl buffer solution, PBS buffer solution or HEPES buffer solution, the resazurin concentration range is 0.5uM-200 uM, and the FAD-dependent D-2-hydroxyglutarate dehydrogenase concentration range is 1.3µg / ml-520µg / ml; ④ Staining of tissue sections Take 15 μl to 100 μl of the staining solution obtained in step ③ and completely cover the tissue section to be tested obtained in step ②. Use a fluorescence detector to detect the fluorescence value of the staining solution on the slide for 1 to 10 minutes. ⑤ Standard product data testing and result processing Place the series of D-2-hydroxyglutaric acid standard samples of different concentrations in step ① on a glass slide, take 15 μl to 100 μl of the staining solution obtained in step ③ to completely cover the slide, and immediately detect it with a fluorescence detector. The characteristics of the fluorescence detector are an excitation wavelength of 540 nm and an emission wavelength of 590 nm. The fluorescence value of the staining solution on the glass slide is the value detected by the fluorescence detector for 1 to 10 minutes. The average value of the fluorescence values ​​of each concentration detected by the fluorescence detector is taken as the y value, and the concentration of D-2-hydroxyglutaric acid contained is taken as the x value. Draw a scatter plot, and use the scatter plot to make a standard curve y=ax+b; Calculate the content of D-2-hydroxyglutaric acid in the tissue section to be tested according to the fluorescence value obtained in step ③.

2. The method for determining the concentration of D-2-hydroxyglutaric acid according to claim 1, characterized in that: The concentration of D-2-hydroxyglutaric acid in the standard slice is a gradient concentration in the range of 0-5.0 µmol / g.

3. The method for determining the concentration of D-2-hydroxyglutaric acid according to claim 1, characterized in that: The staining solution is a PBS buffer solution with a pH of 7.4, which contains 10 µM resazurin and 26 µg / ml FAD-dependent D-2-hydroxyglutarate dehydrogenase.

4. The method for determining the concentration of D-2-hydroxyglutaric acid according to claim 1, characterized in that: After the staining solution is added to the standard sample slices and the tissue slices to be tested, the fluorescence detector detection time is the same, which is 1-3 minutes.

5. A kit obtained by the method for determining the concentration of D-2-hydroxyglutaric acid according to claim 1, characterized in that: The kit comprises a buffer solution with a pH value of 7.0 to 8.0, a resazurin mother solution, a FAD-dependent D-2-hydroxyglutarate dehydrogenase mother solution, and an agarose gel slide containing a series of concentrations of D-2-hydroxyglutaric acid; the concentration range of the resazurin mother solution in the kit is 0.2 mM to 25 mM, and the concentration range of the FAD-dependent D-2-hydroxyglutarate dehydrogenase mother solution in the kit is 1.3 mg / ml to 26 mg / ml; and the buffer solution is a Tris-Hcl buffer solution, a PBS buffer solution, or a HEPES buffer solution.

6. The kit for detecting the concentration of D-2-hydroxyglutaric acid according to claim 5, characterized in that: The agarose gel containing a series of concentrations of D-2-hydroxyglutaric acid is placed on an anti-shedding slide; the concentration range of D-2-hydroxyglutaric acid in the agarose gel is 0-5.0 µmol / g.

7. The kit for detecting the concentration of D-2-hydroxyglutaric acid according to claim 6, characterized in that: The concentrations of D-2-hydroxyglutarate in agarose gel were 0µmol / g, 0.1µmol / g, 0.25µmol / g, 0.5µmol / g, 1.0µmol / g, 1.5µmol / g, and 2.0µmol / g.

8. The kit for detecting the concentration of D-2-hydroxyglutaric acid according to claim 5, characterized in that: The thickness of the agarose gel is 5~25μm, and the cross-sectional area is a rectangle of 0.1~1cm×0.1~1cm. The size and thickness of the agarose gel are adjusted according to the size of the tissue to be tested.

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