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Lipoprotein phospholipase A2 detection reagent and preparation and usage method thereof

A technology for detecting reagents and phospholipases, which is applied in peptide preparation methods, chemical instruments and methods, ovalbumin, etc., can solve the problems of low sensitivity, complex conversion and weak specificity of detection reagents

Active Publication Date: 2019-06-21
苏州博源医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection reagents used in these immunological methods have low sensitivity and weak specificity, and the preparation process is complicated, especially the detection results are calculated by the substrate conversion speed: nm / min / ml, the conversion is complicated, and it is difficult to meet the needs of industry development

Method used

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  • Lipoprotein phospholipase A2 detection reagent and preparation and usage method thereof
  • Lipoprotein phospholipase A2 detection reagent and preparation and usage method thereof
  • Lipoprotein phospholipase A2 detection reagent and preparation and usage method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Synthesis of N-terminal tripeptide derivatives of lipoprotein phospholipase A2

[0057] The chemical structural formula of the amino-terminal tripeptide derivative of lipoprotein phospholipase A2 is shown in formula (Ⅲ):

[0058]

[0059] Formula (Ⅲ).

[0060] The synthesis route and preparation steps of the above-mentioned lipoprotein phospholipase A2 amino-terminal tripeptide derivatives are as follows:

[0061] .

[0062] Concrete synthetic steps are as follows:

[0063] (I) Synthesis of compound 3: Dissolve 41 g of compound 1 in 200 ml of dimethylformamide (DMF), add 64 g of tetramethylurea tetrafluoroborate (TBTU), and then stir the mixture at room temperature for 20 Minutes, after stirring, add 17 g of compound 2 and 45 ml of N-methylmorpholine (NMM), and stir the mixture at 0°C for 2 hours, then pour the above mixture into 500 ml of purified water, and then add 500 ml of ethyl acetate Ester extraction, washing the organic layer with 500 ml bri...

Embodiment 2

[0077] Example 2. Preparation of N-terminal tripeptide immunogen of lipoprotein phospholipase A2

[0078] The lipoprotein phospholipase A2 amino-terminal tripeptide immunogen in this example is formed by linking the lipoprotein phospholipase A2 amino-terminal tripeptide derivative represented by formula (III) with bovine serum albumin (BSA), and its structural formula is as follows Formula (I) shows:

[0079]

[0080] Formula (Ⅰ).

[0081] The specific steps of the synthesis method of the lipoprotein phospholipase A2 amino terminal tripeptide immunogen are as follows:

[0082] (1) Dissolve the carrier protein with a mass fraction of 1.0% in 0.2 mmol / L, pH=8.5 phosphate buffer to obtain a carrier protein solution;

[0083] (2) Dissolve 0.5% mass fraction of lipoprotein phospholipase A2 amino-terminal tripeptide derivative, 5.0% dimethylformamide, and 5.0% ethanol in 10 mmol / L, pH=5.0 potassium phosphate buffer , then add 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide wit...

Embodiment 3

[0085] Example 3. Preparation of anti-lipoprotein phospholipase A2 specific antibody

[0086] The lipoprotein phospholipase A2 amino-terminal tripeptide immunogen prepared in Example 2 was inoculated into experimental animal rabbits by conventional methods, and the antiserum was taken after booster immunization. The specific steps were as follows:

[0087] a. Dilute the lipoprotein phospholipase A2 amino-terminal tripeptide immunogen to 3.0 mg / ml with PBS buffer to obtain an immunogen solution, and then use 3.0 ml of the immunogen solution to mix with an equal amount of Freund's complete adjuvant. Experimental animal rabbits were injected;

[0088] b. After 2 weeks, mix 3.0 ml of the same immunogen solution with the same amount of Freund's incomplete adjuvant, inject once into the above-mentioned experimental animal rabbit, and then inject once every 3 weeks, a total of 5 injections;

[0089] c. Take blood from the immunized experimental animal rabbit, separate and purify to ...

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Abstract

The invention relates to a lipoprotein phospholipase A2 detection reagent and a preparation and usage method thereof. The detection reagent can be used to automatically determine a lipoprotein phospholipase A2 content on a fully-automatic biochemical analyzer. The lipoprotein phospholipase A2 content in a biological sample can be rapidly and accurately determined in a high-throughput mode. The reagent and the method have advantages of simple operation, high sensitivity, good specificity, an accurate result and the like. Lipoprotein phospholipase A2 detection cost is effectively reduced, and the reagent and the method are good for clinical wide popularization and usage.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a detection reagent for lipoprotein phospholipase A2 and a preparation and use method thereof. Background technique [0002] Lipoprotein phospholipase A2 (Lp-PLA2) is an important evaluation index for clinical evaluation and risk prediction of atherosclerosis and coronary heart disease. Lipoprotein phospholipase A2 is a calcium-independent serine lipase. Unlike phospholipases such as cPLA2 and sPLA2, lipoprotein phospholipase A2 is associated with low-density lipoproteins and weakly associated with high-density lipoproteins. Lipoprotein phospholipase A2 is produced by macrophages and other inflammatory cells, and its concentration is higher in the late stage of atherosclerotic lesions than in the early stage. Low-density lipoprotein oxidation is a critical step in the process of atherosclerosis. Lipoprotein phospholipase A2 participates in the degradati...

Claims

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Application Information

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IPC IPC(8): G01N33/573C07K16/40C07K14/77C07K14/765C07K14/795C07K14/47C07K1/34C07K5/083
Inventor 虞留明
Owner 苏州博源医疗科技有限公司
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