Kit for measuring l-proline betaine in serum

Abstract

This invention relates to the field of medical reagent kit technology, and discloses a kit for determining L-proline betaine in serum. The kit is characterized by comprising reagent one, reagent two, and a standard substance solution. Reagent one comprises: tris, Prolin 300, potassium chloride, β-NAD+, and pH adjusted to 7.5 ± 0.05 with HCl or NaOH. Reagent two comprises: MOPSO, L-proline betaine dehydrogenase, magnesium acetate, Prolin 300, BSA, and ethylene glycol, with pH adjusted to 7.8. This invention uses enzymatic reagents and corresponding calibration and quality control for low-cost clinical testing, is simple to operate, and requires no sample preparation. It solves the problem that previous methods using liquid chromatography-tandem mass spectrometry (LC-MS / MS) required long processing times, complex sample preparation, and high costs, preventing the widespread clinical detection of PROB.

Description

Technical Field

[0001] This invention relates to the field of medical reagent kit technology, specifically a kit for determining L-proline betaine in serum. Background Technology

[0002] Currently, serum L-proline betaine detection is mainly used in large-scale epidemiological studies and high-level clinical research, and has not yet become a routine hospital test. However, with further research, it may become a powerful tool for personalized nutrition and early warning of disease risks. For example, if detection reveals active L-proline betaine / TMAO pathways in an individual, doctors may recommend reducing the intake of precursors such as red meat and egg yolks, or supplementing with probiotics / prebiotics to regulate gut microbiota. Current technology uses liquid chromatography-tandem mass spectrometry (LC-MS / MS), but the equipment is very expensive, the operation is complex, requires professional personnel, and the analysis time is relatively long. Summary of the Invention

[0003] The purpose of this invention is to provide a kit for determining L-proline betaine in serum, so as to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a kit for determining L-proline betaine in serum, characterized in that it comprises reagent one, reagent two, and a standard substance solution; The reagent one includes: tris 12.1±10% g / l Prolin300 1±10% g / l Potassium chloride 8±10% g / l β-NAD+ 4±5% g / l The pH was then adjusted to 7.5 ± 0.05 using HCl or NaOH. The second reagent includes: MOPSO 5.6±10% g / l L-proline betaine dehydrogenase 2±5% KU / l Magnesium acetate 0.42±10% g / l Prolin300 0.5±10% g / l BSA 1±10% g / l Ethylene glycol 3000±10% ml / l And adjust the pH to 7.8; The standard substance solution includes: NAH2PO4·2H2O 1.5±10% g / l NA₂HPO₄·12H₂O 14.5±5% g / l L-proline betaine Prolin300 0.5±10% g / l Then adjust the pH to 7.4.

[0005] Preferably, the amount of L-proline betaine in the 60 μmol / L L-proline betaine standard is 14.5 ± 5% g / L.

[0006] Preferably, the preparation of reagent one includes the following steps: weigh and tare a 1-liter beaker, add 1000 ml of purified water, and then add tris, Prolin300, glycine, and β-NAD+ in sequence, stir well, and adjust the pH to 7.5 to obtain reagent one.

[0007] Preferably, the preparation of reagent two includes the following steps: weigh and tare a 1-liter beaker, add 1000 ml of purified water, and then add MOPSO, L-proline betaine dehydrogenase, magnesium acetate, Prolin300, BSA, and ethylene glycol in sequence. Stir well and adjust the pH to 7.8 to obtain reagent two.

[0008] Preferably, the preparation of the standard substance solution includes the following steps: weigh and tare a 1-liter beaker, add 1000 ml of purified water, and then add NAH2PO4·2H2O, NA2HPO4·12H2O, L-proline betaine, and Prolin300 in sequence, stir well, and adjust the pH to 7.4 to obtain the standard substance solution.

[0009] The method for using the kit to determine L-proline betaine in serum includes the following steps: Step 1: Prepare reagent one and reagent two; Step 2: Add reagent one and sample to the reaction cup of the biochemical analyzer or spectrophotometer, mix well, incubate for 1-5 minutes, then add reagent two to start the enzymatic reaction; Step 3: Monitor the absorbance change at a wavelength of 340 nm and record the reaction rate; Step 4: Calculate the PROB concentration in the sample based on the standard curve.

[0010] Preferably, the biochemical analyzer or spectrophotometer in step 2 is set with detection parameters of wavelength 340nm and reaction direction: downward.

[0011] Compared with the prior art, the beneficial effects of the present invention are: In this invention patent, tris provides a buffer system, Prolin300 inhibits bacteria, potassium chloride protects the stability of β-NAD+, NMOPSO (3-(N-morpholino)-2-hydroxypropanesulfonic acid) serves as the buffer system, magnesium acetate protects the activity of L-proline betaine dehydrogenase, and BSA protects the long-term stability of L-proline betaine dehydrogenase. Using enzymatic reagents and corresponding calibration and quality control, this method is cost-effective for clinical testing, simple to operate, and requires no sample preparation. It solves the problem of the long testing time, complex sample processing, and high cost associated with previous liquid chromatography-tandem mass spectrometry (LC-MS / MS) methods, which prevented the widespread clinical detection of PROB. Attached Figure Description

[0012] Figure 1 This is a calibration curve diagram for the present invention. Detailed Implementation

[0013] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0014] 1. Reagent preparation Preparation of reagent 1: Take 1000 mL of purified water and add the following in sequence: Tris 12.1 g, Prolin300 1 g, potassium chloride 8.0 g, and β-NAD⁺ 4 g. Stir well and adjust the pH to 7.5±0.05 with HCl or NaOH.

[0015] Preparation of reagent 2: Take 1000 mL of purified water and add the following in sequence: 5.6 g of MOPSO, 2 KU of L-proline betaine dehydrogenase, 0.42 g of magnesium acetate, 0.5 g of Prolin300, 1 g of BSA, and 300 mL of ethylene glycol. Stir well and adjust the pH to 7.8.

[0016] Preparation of standard substance solutions (e.g., 60 μmol / L): Take 1000 mL of purified water and add the following in sequence: 1.5 g of NaH2PO4·2H2O, 14.5 g of Na2HPO4·12H2O, 10 mg of L-proline betaine, and 0.5 g of Prolin300. Stir well and adjust the pH to 7.4.

[0017] 2. Instrument Setup Use a biochemical analyzer or spectrophotometer equipped with a 340 nm wavelength. Set the detection parameters as follows: wavelength 340 nm, reagent one volume 240 µL, reagent two volume 80 µL, sample volume 30 µL, and reaction direction downward (absorbance decreases).

[0018] 3. Testing Steps Number the serum samples, standards, and quality controls separately. Add the following to a reaction vessel: 1240 µL of reagent, 30 µL of sample (serum / standard / quality control), mix well, and incubate for an appropriate time (e.g., 1-5 minutes). Add reagent 2:80 µL to initiate the enzymatic reaction. Monitor the absorbance change (ΔA) at 340 nm and record the reaction rate. Calculate the PROB concentration in the sample based on the standard curve.

[0019] 4. Establishment of Standard Curve The detection was performed using PROB standard substances of different concentrations (e.g., 8, 20, 40, 60 μmol / L).

[0020] Plot a standard curve with the concentration of the standard on the x-axis and ΔA on the y-axis (the linear range should cover the expected sample concentration).

[0021] 5. Result Verification Use quality control samples (such as 60 μmol / L and 8 μmol / L) to verify the accuracy and repeatability of the detection system. 6. Precautions Reagent 1 and Reagent 2 should be stored as specified (2℃~8℃ or room temperature).

[0022] No special pretreatment is required for the sample; serum can be used directly.

[0023] The reaction system should be kept away from strong light, high temperature and pollution.

[0024] II. Comparison of the detection results obtained by liquid chromatography-tandem mass spectrometry with those obtained in this application. 1. Main Instruments Liquid Chromatography System: Agilent Technologies 1200 series Mass spectrometry system: Agilent 6410A Triple Quad mass spectrometer (equipped with an electrospray ionization source) Centrifuge: Eppendorf-5415R High-Speed ​​Refrigerated Centrifuge Pure water system: Milli-Pore pH meter: Denver UB-10 2. Main reagents Standard: Betaine (Sigma-Aldrich) Isotope internal standard: d3-betaine (Isotec) Chromatographic reagents: Acetonitrile (Merck, chromatographic grade), ammonium formate (AlfaAesar) Other: Phosphate buffer 3. Testing Steps Step 1: Sample Collection and Processing Collect 3 mL of fasting venous whole blood in the morning. Centrifuge at 4℃ and 3000×g for 15 minutes within 30 minutes to separate the serum.

[0025] Dispense into EP tubes and store as required.

[0026] Step 2: Solution preparation Standard stock solution: Prepare a 20 mmol / L betaine stock solution with PBS and store at -20°C.

[0027] Standard application solution: Dilute the stock solution to a series of concentrations of 0.62–400 μmol / L with mixed serum diluted 10 times.

[0028] Internal standard solution (also used to remove protein): Prepare a 10 mmol / L d3-betaine stock solution with PBS, and dilute it to 10 μmol / L with acetonitrile before use.

[0029] Mobile phase: 15 mmol / L ammonium formate solution (pH 3.5), filtered and degassed before use.

[0030] Step 3: Sample pretreatment Take 30 μL of serum or standard working solution.

[0031] Add 90 μL of internal standard solution (acetonitrile containing internal standard) and vortex to mix.

[0032] Centrifuge at 4℃, 13,000×g for 10 minutes The supernatant was filtered through a centrifugal filter column (3000×g, 2 minutes).

[0033] Collect approximately 90 μL of filtrate, transfer it to a sample vial, and prepare it for instrumentation.

[0034] Step 4: Chromatographic and Mass Spectrometric Conditions Chromatographic column: Ultimate SiO2 column (2.1 mm × 100 mm, 5 μm) Mobile phase: Acetonitrile : 15 mmol / L ammonium formate buffer (pH 3.5) = 60 : 40 (isocratic elution) Flow rate: 0.2 mL / min Column temperature: 25℃ Injection volume: 5 μL Ion source: Electrospray ionization source (positive ion mode) Monitoring mode: Multiple response monitoring Major MRM ion pairs: Analyte precursor ion → Daughter ion (m / z) Betaine 118.0 → 59.3 d3-betaine (internal standard) 127.3 → 68.3 Step 5: Quantitative Analysis Simultaneously process and run the series of standard application solutions and samples. Plot a standard curve with the concentration of the standard on the x-axis and the ratio of the peak areas of the standard to the internal standard on the y-axis.

[0035] The concentration is calculated by substituting the peak area ratio of the sample into the standard curve.

[0036] Step 6: Methodological Validation and Stability Assessment Linearity and sensitivity: Linear range 0.62–400 μmol / L, limit of quantitation 0.62 μmol / L.

[0037] Precision: Intra-day RSD <10%, Inter-day RSD <15%.

[0038] Accuracy: Recovery rate is between 88.64% and 98.23%.

[0039] Stability recommendations Serum should be tested as soon as possible after separation, preferably within 3 hours.

[0040] If immediate testing is not possible, the sample can be treated with acetonitrile within 3 hours, stored at 4°C, and the determination completed within 24 hours.

[0041] Avoid repeated freeze-thaw cycles, especially with betaine.

[0042] The following is a comparison between the reagent kit of this application and liquid chromatography-tandem mass spectrometry, with the detection results as follows: Theoretical concentration of quality control standard 1: 60 μmol / L (1SD±10%), theoretical concentration of quality control standard 2: 8 μmol / L (1SD±10%) The test results show that both tandem mass spectrometry and the results of this invention are within the quality control range. The correlation coefficient between the two methods for testing serum samples is 0.9999, indicating that the test results of the two methods are equivalent, and the results of the enzyme method and the tandem mass spectrometry method are consistent.

[0043] In summary, the results of serum L-proline betaine (PROB) assay kit (enzymatic method) and tandem mass spectrometry are equivalent, and the enzymatic method can replace the tandem mass spectrometry method.

Claims

1. A kit for determining L-proline betaine in serum, characterized in that, This includes reagent one, reagent two, and standard substance solutions; The reagent one includes: tris 12.1±10% g / l Prolin300 1±10% g / l Potassium chloride 8±10% g / l β-NAD+ 4±5% g / l The pH was then adjusted to 7.5 ± 0.05 using HCl or NaOH. The second reagent includes: MOPSO 5.6±10% g / l L-proline betaine dehydrogenase 2±5% KU / l Magnesium acetate 0.42±10% g / l Prolin300 0.5±10% g / l BSA 1±10% g / l Ethylene glycol 3000±10% ml / l And adjust the pH to 7.8; The standard substance solution includes: NAH2PO4·2H2O 1.5±10% g / l NA₂HPO₄·12H₂O 14.5±5% g / l L-proline betaine Prolin300 0.5±10% g / l Then adjust the pH to 7.

4.

2. The kit for determining L-proline betaine in serum according to claim 1, characterized in that, The amount of L-proline betaine in the 60 μmol / L L-proline betaine standard is 14.5 ± 5% g / L.

3. The kit for determining L-proline betaine in serum according to claim 1, characterized in that, The preparation of the reagent includes the following steps: Weigh and tare a 1-liter beaker, add 1000 ml of purified water, and then add tris, Prolin300, glycine, and β-NAD+ in sequence. Stir well and adjust the pH to 7.5 to obtain reagent one.

4. The kit for determining L-proline betaine in serum according to claim 1, characterized in that, The preparation of reagent two includes the following steps: Weigh and tare a 1-liter beaker, add 1000 ml of purified water, and then add MOPSO, L-proline betaine dehydrogenase, magnesium acetate, Prolin300, BSA, and ethylene glycol in sequence. Stir well and adjust the pH to 7.8 to obtain reagent two.

5. The kit for determining L-proline betaine in serum according to claim 1, characterized in that, The preparation of the standard substance solution includes the following steps: Weigh and tare a 1-liter beaker, add 1000 ml of purified water, and then add NAH2PO4·2H2O, NA2HPO4·12H2O, L-proline betaine, and Prolin300 in sequence. Stir well and adjust the pH to 7.4 to obtain the standard substance solution.

6. The method of using the kit for determining L-proline betaine in serum according to claim 1, characterized in that, Includes the following steps: Step 1: Prepare reagent one and reagent two; Step 2: Add reagent one and sample to the reaction cup of the biochemical analyzer or spectrophotometer, mix well, incubate for 1-5 minutes, then add reagent two to start the enzymatic reaction; Step 3: Monitor the absorbance change at a wavelength of 340 nm and record the reaction rate; Step 4: Calculate the PROB concentration in the sample based on the standard curve.

7. The method of using the kit for determining L-proline betaine in serum according to claim 6, characterized in that, In step 2, the biochemical analyzer or spectrophotometer is set to the following detection parameters: wavelength 340nm, reaction direction: downward.

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