(D)-2-hydroxyglutarate assay kit

A technology of hydroxyglutaric acid and detection kits, which is applied in the measurement of color/spectral characteristics, material analysis by observing the influence on chemical indicators, and analysis by making materials undergo chemical reactions. Long cycle, high requirements and other issues, to achieve the effect of high-precision high-throughput inspection, strong specificity, and high precision

Inactive Publication Date: 2018-02-23
WAYEN BIOTECHNOLGIES SHANGHAI INC
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] At present, the methods for detecting the concentration of D-2-hydroxyglutaric acid at home and abroad are mainly through liquid chromatography-mass spectrometry (liquid chromatography-mass spectrometry, LC-MS) or gas chromatography-mass spectrometry (gaschromatography-mass spectrometry). spectrometry, GC-MS), but these two methods are complex in operation, long in cycle, expensive in equipment and high in requirements for operators, and are not suitable for high-throughput clinical sample detection

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D2

[0023] Embodiment 1D2HG detection kit components and preparation process

[0024] A. Sample treatment solution A: 11.26mL of 71% perchloric acid and deionized water to dilute to 100mL.

[0025] B. Sample treatment solution B: made by dissolving 30.8g potassium hydroxide in 100mL deionized water.

[0026] C. Reaction buffer: 1mol / L Tris-HCl buffer solution, adjust pH to 8.0 with saturated HCl solution.

[0027] D. Reaction substrate 1: Weigh 331.7 mg of nicotinamide adenine dinucleotide (NAD) powder, dissolve it in 5 mL of deionized water, and then dilute it 10 times to obtain a 10 mmol / L NAD solution.

[0028] E. Reaction substrate 2: Weigh 14.63 mg of tetrazolium salt (MTS) powder in the dark, dissolve it in 1 mL of DPBS buffer solution, and then dilute it 10 times to obtain a 3 mmol / L MTS solution.

[0029] F. Reaction substrate 3: Weigh 2.53 mg of N-methylphenazine methosulfate (PMS) powder in the dark, dissolve it in 1 mL of DPBS buffer solution, and then dilute it 10 ti...

Embodiment 2D2

[0032] Embodiment 2D2HG detection kit detection method

[0033] (1) Reagent preparation: Reaction buffer, reaction substrate 1 / 2 / 3, enzyme, standard, sample treatment solution A / B are dissolved at room temperature, mixed separately and placed on ice for later use.

[0034] (2) Preparation of D2HG standard substance: Dilute the standard substance to two concentrations of 128 μM and 96 μM with deionized water, 400 μL each. Then the 128 μM standard was diluted in half to 64 μM, 32 μM, 16 μM, 8 μM, and the 96 μM standard was diluted in half to 48 μM to form 200 μL of 0, 8, 16, 32, 48, 64, 96, and 128 μM D2HG standards respectively. Gradient concentration solutions were kept on ice for later use.

[0035] (3) Sample treatment: 50 μL of pre-cooled sample treatment solution A was added to 200 μL of serum sample, vortexed, placed on ice for 5 min, and then centrifuged at 14,000 g at 4°C for 8 min. Take 150 μL of supernatant to a new 1.5 mL EP tube, add 8.6 μL of pre-cooled sample tr...

Embodiment 3D2

[0040] Example 3 D2HG Detection Kit Precision and Accuracy Experiment

[0041] The D2HG standard was dissolved in the reaction buffer to prepare samples with concentrations of 0, 2, 10, 20, 40, 80, 100, and 120 μM, respectively. Each sample is divided into 5 parts, measured with a D2HG detection kit, and the average value and standard deviation are calculated to obtain the precision (CV%) and recovery (%) within the batch; meanwhile, the D2HG subpackaged samples are continuously measured for 5 day, calculate the mean value, standard deviation, and obtain the inter-assay precision (CV%) and recovery (%). As shown in the table below:

[0042]

[0043]

[0044] The results showed that the D2HG detection kit had good precision, and the intra-assay and inter-assay precision CVs were both less than 5.9%. The recovery rate of the sample test ranged from 86.1% to 107.75%, indicating that the D2HG detection kit has a high accuracy.

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Abstract

The invention provides a (D)-2-hydroxyglutarate (D2HG) assay kit, which is prepared from sample processing chemical A (perchloric acid), sample processing chemical B (potassium hydroxide), reaction buffer (Tris-HCl), reaction substrates (coenzyme NAD, methyl thiazolyl tetrazolium MTT and electron transfer agent PMS), (D)-2-hydroxyglutarate dehydrogenase, a standard substance, a 96-pore microporousplate, and a microplate sealer. The optimal assayed concentration range of the kit for D2HG is 5mu M to 120mu M (0.96mu g / ml to 23mu g / ml). The kit disclosed by the invention has the characteristicsof easiness in operation, quickness, accuracy and high specificity, is suitable for assaying the concentration of D2HG in serum sample, and provides beneficial guidance for the clinical early diagnosis of patients with tumors related to isocitrate dehydrogenase (IDH) mutation, the determination of therapeutic strategies and prognosis evaluation.

Description

technical field [0001] The invention belongs to the technical field of detection of (D)-2-hydroxyglutarate clinical samples, and in particular relates to a kit for detecting the concentration of (D)-2-hydroxyglutarate in in vitro serum. Background technique [0002] (D)-2-Hydroxyglutarate (D-2-Hydroxyglutarate, D2HG) is a dicarboxylic acid with five carbon atoms, the content in normal human cells and tissues is very low, but in some metabolic diseases and Levels were significantly elevated in cancer patients. In recent years, studies have found that isocitrate dehydrogenase (isocitrate dehydrogenase 1 / 2, IDH1 / 2) is found in cancer patients such as secondary glioblastoma, chondrosarcoma, cholangiocarcinoma and acute myeloid leukemia (AML). ) mutations, IDH1 / 2 mutations catalyze α-ketoglutarate (α-KG) into D2HG, resulting in a significant increase in the content of D2HG in cells, and D2HG competitively inhibits the activity of dioxygenases dependent on α-KG to promote cancer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78G01N21/31
Inventor 宋凯杨舟张庆华程磊
Owner WAYEN BIOTECHNOLGIES SHANGHAI INC
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