A substrate for detecting human lipoprotein phospholipase A2 and a kit thereof

Abstract

The object of the present invention is to provide a reaction substrate of human lipoprotein-associated phospholipase A2 protein. Another object of the present invention is to provide a biochemical kit for detecting human lipoprotein-associated phospholipase A2 protein. In order to achieve the above-mentioned purpose, the present invention provides a reaction substrate for human lipoprotein-associated phospholipase A2 protein. The present invention prepares a substrate, dissolves the synthesized substrate 1-dodecyl-2 (4-nitrophenylglutaryl) phosphatidylcholine in TE buffer, and adds glycerol containing silicone oil as a stabilizer. In order to further improve the substrate stability, the present invention uses trifluoropropyl methyl silicone oil after testing. After testing, the substrate of this formula was accelerated to destroy stability at 55°C for 7 days, and the concentration of 4-nitrophenol was not increased after testing. After adding human lipoprotein-associated phospholipase A2 protein, the efficiency of the 4-nitrophenol concentration reaction curve was not decreased, and the stability of the reaction substrate of human lipoprotein-associated phospholipase A2 protein was improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a substrate for detecting human lipoprotein phospholipase A2 and a kit thereof. Background Art

[0002] Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), is a phospholipase secreted by inflammatory cells that can promote the hydrolysis of oxidized phospholipids. It is a member of the phospholipase A2 (PLA2) superfamily with a relative molecular mass of 45.4kD (441 amino acids). The basic function of Lp-PLA2 is to catalyze the hydrolysis of acyl bonds at the Sn-2 position of various oxidized phospholipids to produce free fatty acids and lysophospholipids. In addition, Lp-PLA2 can also hydrolyze inflammatory factors such as platelet-activating factor. Lp-PLA2 is one of the subtypes of the phospholipase superfamily, also known as platelet-activating factor acetylhydrolase, secreted by macrophages, T cells and mast cells in the vascular endothelium. Lp-PLA2 expression is upregulated in atherosclerotic plaques and is strongly expressed in macrophages in the fibrous cap of vulnerable plaques. Lp-PLA2 can hydrolyze oxidized phospholipids in oxidized low-density lipoprotein ox-LDL to generate lipid pro-inflammatory substances, such as lysolecithin and oxidized free fatty acids, which in turn produce a variety of atherogenic effects, including endothelial cell death and endothelial dysfunction, and stimulate the production of adhesion factors and cytokines. These substances can further produce a self-reinforcing cycle by chemotactic inflammatory cells to generate more pro-inflammatory substances. There are two methods for detecting Lp-PLA2: activity method and concentration method. The activity method mainly uses high-performance liquid chromatography, radioactivity determination method, and enzyme hydrolysis substrate method. High-performance liquid chromatography has low sensitivity and is easily interfered by various components in the blood. The radioactivity determination method has problems such as radioactive contamination of reagents, low accuracy, and poor repeatability. The commonly used substrates for the enzymatic hydrolysis substrate method are 1-decanoyl-2-(4-nitrophenylglutaryl)phosphatidylcholine, 1-myristoyl-2-(4-p-nitrophenol succinic anhydride)phosphatidylcholine, and 1-decanoyl-2-(4-nitrophenylglutaryl)phosphatidylcholine. The main reactions are hydrolysis of Sn-2 by lipoprotein-associated phospholipase A2 protein. 4-nitrophenylpentylphthalide and 4-p-nitrophenol succinic anhydride are produced at the 4-nitrophenol or p-nitrophenol positions. Since the hydrolysis product is unstable, it further decomposes to produce 4-nitrophenol or p-nitrophenol, which causes the absorbance of the corresponding wavelength of 4-nitrophenol or p-nitrophenol in the solution to change. At present, the above three substrates have the problem of poor stability and natural decomposition is fast. The substrate must be stored in an independent buffer liquid system until it is diluted and used before use. Otherwise, the substrate will decompose due to natural oxidation, resulting in a high background, thereby reducing the detection efficiency. Summary of the invention

[0003] The present invention aims to provide a reaction substrate of human lipoprotein-associated phospholipase A2 protein.

[0004] Another object of the present invention is to provide a biochemical kit for detecting human lipoprotein-associated phospholipase A2 protein.

[0005] In order to achieve the above-mentioned purpose, the present invention provides a reaction substrate for human lipoprotein-associated phospholipase A2 protein. The present invention prepares a substrate, dissolves the synthesized substrate 1-dodecyl-2 (4-nitrophenylglutaryl) phosphatidylcholine in TE buffer, and adds glycerol containing silicone oil as a stabilizer. In order to further improve the substrate stability, the present invention uses trifluoropropyl methyl silicone oil after testing. After testing, the substrate of this formula was accelerated to destroy stability at 55°C for 7 days, and the concentration of 4-nitrophenol did not increase after testing. After adding human lipoprotein-associated phospholipase A2 protein, the efficiency of the 4-nitrophenol concentration reaction curve was tested without decrease, and the stability of the reaction substrate of human lipoprotein-associated phospholipase A2 protein was improved.

[0006] In order to better implement the above method, the present invention discloses a biochemical kit for detecting human lipoprotein-associated phospholipase A2 protein, the kit comprising: a dilution buffer R1 and a reaction substrate R2 of human lipoprotein-associated phospholipase A2 protein. The substrate in the above invention is used as a substrate, and the dilution buffer adopts a pH 7.4 phosphate buffer and adds 0.05% Tween-20. The above kit is further optimized to add a calibrator and a quality control product, and the main components of the calibrator and the quality control product are a human lipoprotein-associated phospholipase A2 protein solution or a serum containing human lipoprotein-associated phospholipase A2 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Attached Figure 1 .Determination of synthetic products.

[0008] Attached Figure 2 .OD405 change curve of samples with different concentrations.

[0009] Attached Figure 3 .40 times blank reagent detection concentration. DETAILED DESCRIPTION

[0010] The following examples are intended to explain the present invention in more detail, but the present invention should not be construed as being limited thereto.

[0011] Example 1: Synthesis of 1-dodecyl-2(4-nitrophenylglutaryl)-sn-glycero-3-phosphatidylcholine

[0012] The raw materials are lauramide, glycerylphosphatidylcholine, and 3-(4-nitrophenyl)glutaric acid, and the method is as follows:

[0013] 1. Add lauramide to the sn-3 position of glycerophosphatidylcholine;

[0014] 2. Chloroform extraction product 1-dodecyl-2-hydrogen-sn-glycero-3-phosphatidylcholine;

[0015] 3. Add 3-(4-nitrophenyl)glutaric acid to the above product 1-dodecyl-2-hydrogen-sn-glycero-3-phosphatidylcholine sn-2;

[0016] 4. Extract the product with chloroform and purify it with silica gel column.

[0017] Results analysis: A serum sample of human lipoprotein-associated phospholipase A2 protein with a concentration of 3 mmol / L was added to the aforementioned synthetic product 1-dodecyl-2 (4-nitrophenylglutaryl)-sn-glycerol-3-phosphatidylcholine and the OD405 change of the product was measured using a spectrophotometer. After testing, the OD405 continued to change after adding 1-dodecyl-2 (4-nitrophenylglutaryl)-sn-glycerol-3-phosphatidylcholine. The results are shown in the attached Figure 1 .The results of synthetic product determination show that the substrate can be well recognized and catalyzed by human lipoprotein-associated phospholipase A2 protein. The 3mmol / L serum sample was diluted to 2mmol / L, 1mmol / L, 0.5mmol / L, and 0mmol / L using pH7.0-pH8.0 phosphate buffer, and the OD change rate was determined using a spectrophotometer. The results are shown in the attached Figure 2 .The OD405 change curve of samples with different concentrations shows that the reaction rate is also positively correlated with the sample concentration, proving that the reaction rate of the substrate can reflect the activity and quantity of the enzyme in the sample, and can be used as a substrate to measure the concentration of human lipoprotein-associated phospholipase A2 protein in the sample.

[0018] Example 2: Preparation of reaction substrate buffer for human lipoprotein-associated phospholipase A2 protein.

[0019] Raw materials: 1-dodecyl-2 (4-nitrophenylglutaryl)-sn-glycero-3-phosphatidylcholine, pH 7.5~8.5 TE (Tris-EDTA) buffer, glycerol, trifluoropropylmethylpolysiloxane, polydimethylsiloxane, dimethyl sulfoxide.

[0020] Preparation method:

[0021] Dissolve 1-dodecyl-2 (4-nitrophenylglutaryl)-sn-glycero-3-phosphatidylcholine in pH 7.5-8.5 TE buffer to obtain component 1;

[0022] Glycerol was dissolved in dimethyl sulfoxide at a ratio of 5% by volume to obtain a mixed solvent 1;

[0023] Dissolve glycerol in dimethyl sulfoxide at a ratio of 10% by volume to obtain a mixed solvent 2;

[0024] Dissolve 1% trifluoropropylmethylpolysiloxane in the aforementioned mixed solvent 1 by mass ratio, and use ultrasonic emulsification and dispersion to obtain component 2;

[0025] Dissolve 1% polydimethylsiloxane in the mixed solvent 1, and disperse by ultrasonic emulsification to obtain component 3.

[0026] Dissolve trifluoropropylmethylpolysiloxane in the aforementioned mixed solvent 2 at a mass ratio of 5%, and use ultrasonic emulsification and dispersion to obtain component 4;

[0027] Dissolve polydimethylsiloxane in the aforementioned mixed solvent 2 at a mass ratio of 5%, and use ultrasonic emulsification and dispersion to obtain component 5;

[0028] Component 1 and component 2 are taken separately, and mixed in a volume ratio of 1:1 to obtain reaction substrate 1;

[0029] Component 1 and component 3 are taken separately, and mixed at a volume ratio of 99:1 to obtain reaction substrate 2;

[0030] Take component 1 and component 4 respectively, and mix them in a volume ratio of 1:1 to obtain reaction substrate 3;

[0031] Component 1 and component 5 are taken separately, and mixed at a volume ratio of 99:1 to obtain reaction substrate 4;

[0032] Test: Take the above reaction substrates 1 to 4 respectively, divide them into two parts, take one part and put it in 55℃ high temperature accelerated destruction, and put the other part in 2~8℃ refrigerator, take it out after one week, use spectrophotometer to test the substrate OD405 under two storage conditions, the results are shown in Table 1. Determination of reaction substrates, comparison of accelerated destructive experiments, that is, the above formula can be stably stored for more than 1 year with a decomposition rate of no more than 2%, use a concentration of 3mmol / L human lipoprotein-associated phospholipase A2 protein serum sample, add the above reaction substrates 1 to 4, and use a spectrophotometer to measure the change of product OD405 after reaction. After testing, the results of continuous change of OD405 are shown in Table 1. Determination of reaction substrates. The results show that all 4 reaction substrates have good reactivity, and can still guarantee more than 98% potency after accelerated destructive experiments.

[0033] Table 1. Reaction substrates assayed.

[0034]

[0035] Example 3: Human lipoprotein-associated phospholipase A2 protein detection kit

[0036] The kit contains components R1 and R2. R1 is a sample diluent, and any commonly used buffer can achieve the purpose of dilution. In order to better implement this experimental invention, the present invention specifically uses a phosphate buffer of pH 6.0 to pH 8.0, and adds 0.01% to 0.1% Tween 20. R2 is a reaction substrate. R1 and R2 can be used in a biochemical analyzer or other instruments with the ability to detect a wavelength of 405nm, such as a spectrophotometer, a specific protein analyzer with a wavelength of 405, and an enzyme marker with a wavelength of 405 to obtain the substrate decomposition rate by measuring the OD405 absorbance of the reaction substrate, thereby obtaining the activity and concentration of the human lipoprotein-related phospholipase A2 protein in the sample. In order to better implement the invention, quality control products and calibrators can be optionally included in the kit to provide calibration and quality control for the machine. The test was performed using a BECKMANCOULTER AU5400 machine, and the reagent of the present invention was used to perform the OD405 absorbance change rate method. The results showed that the blank change of the reagent of the kit was less than 2% after 30 days on the machine, the linear range of the detection was 5-3000 IU / L, and the detection limit was 0.5 IU / L.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention.

[0038] The term "sample diluent" used in this specification refers to those known to those skilled in the art and is included in the prior art.

Claims

1. A reaction substrate system for human lipoprotein-associated phospholipase A2 protein, comprising substrate 1, a buffer, a protective agent, and a stabilizer. Features: The substrate 1 is 1-dodecyl-2(4-nitrophenylglutaryl)-sn-glycerol-3-phosphatidylcholine.

2. The reaction substrate system of human lipoprotein-associated phospholipase A2 protein according to claim 1, Features: 1) The buffer is stored in TE buffer at pH 7.5 to 8.5; 2) The protective agent is silicone oil; 3) The stabilizer is glycerol dimethyl sulfoxide solution.

3. The reaction substrate system of human lipoprotein-associated phospholipase A2 protein according to claim 1 or 2, Features: The protective silicone oil is trifluoropropylmethylpolysiloxane.

4. The reaction substrate system of human lipoprotein-associated phospholipase A2 protein according to claim 3, Features: The concentration of the protective agent is 0.01% to 0.05%.

5. The reaction substrate system of human lipoprotein-associated phospholipase A2 protein according to claim 3, Features: The stabilizer is 5% to 10% glycerol dimethyl sulfoxide solution.

6. The reaction substrate system of human lipoprotein-associated phospholipase A2 protein according to claim 5, Features: The stabilizer accounts for 1% to 50% of the reaction substrate by volume.

7. The reaction substrate system of human lipoprotein-associated phospholipase A2 protein according to claim 3, Features: The protective agent is emulsified in a dimethyl sulfoxide solution in glycerol and then added into the buffer solution.

8. A biochemical kit for detecting human lipoprotein-associated phospholipase A2 protein, comprising components R1 and R2, Features: Wherein R1 is a sample dilution buffer, R2 is a reaction substrate system of human lipoprotein-associated phospholipase A2 protein, and the reaction substrate system is any one of claims 1 to 7.

9. The biochemical kit for detecting human lipoprotein-associated phospholipase A2 protein according to claim 8, Features: The sample dilution buffer is composed of a phosphate buffer of pH 6.0 to pH 8.0, with 0.01% to 0.1% Tween 20 added. The kit also includes a calibrator and a quality control product, which are a human lipoprotein-associated phospholipase A2 protein solution of a certain concentration or a serum containing human lipoprotein-associated phospholipase A2 protein.

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