阅读说明：本技术 一种保持脂蛋白相关磷脂酶a2试剂盒稳定的组合物及其制备方法 (Composition for keeping stability of lipoprotein-associated phospholipase A2 kit and preparation method thereof ) 是由 麻玉雯 于 2020-07-24 设计创作，主要内容包括：本发明提供了一种保持脂蛋白相关磷脂酶A2试剂盒稳定的组合物,包括：试剂R1和试剂R2,其中,所述试剂R1包括脂蛋白相关磷脂酶A2结构稳定剂、两性离子缓冲液、表面活性剂1和防腐剂；且所述试剂R2包括：两性离子缓冲液、果糖和包被脂蛋白相关磷脂酶A2的乳胶粒子；本发明所述的组合物,通过在组合物中添加合理配比的、特定的脂蛋白相关磷脂酶A2结构稳定剂(由选自表面活性剂、蛋白类物质、氧化类物质组成)以及特定表面活性剂和果糖,能够显著减少在试剂盒使用中外界对Lp-PLA2的影响,保持测试结果的稳定,且本发明的制备工艺简单,利于实施和工业化生产,具有良好的市场应用前景。(The invention provides a composition for keeping a lipoprotein-associated phospholipase A2 kit stable, which comprises: reagent R1 and reagent R2, wherein the reagent R1 comprises a lipoprotein-associated phospholipase A2 structure stabilizer, a zwitterionic buffer, a surfactant 1 and a preservative; and the reagent R2 comprises: a zwitterionic buffer, fructose and latex particles coated with lipoprotein-associated phospholipase A2; the composition provided by the invention has the advantages that the influence of the outside on Lp-PLA2 in the use of the kit can be obviously reduced and the stability of the test result is kept by adding the specific lipoprotein-related phospholipase A2 structure stabilizer (composed of surfactants, protein substances and oxidation substances) and the specific surfactant and fructose in a reasonable ratio into the composition, and the preparation process is simple, is beneficial to implementation and industrial production and has a good market application prospect.)

1. A composition for stabilizing a lipoprotein-associated phospholipase a2 kit, comprising: reagent R1 and reagent R2, wherein the reagent R1 comprises a lipoprotein-associated phospholipase A2 structure stabilizer, a zwitterionic buffer, a surfactant 1 and a preservative; and the reagent R2 comprises: zwitterionic buffer, fructose and latex particles coated with lipoprotein-associated phospholipase a 2.

2. The composition as claimed in claim 1, wherein the structure stabilizer of lipoprotein-associated phospholipase A2 is selected from surfactant 2, protein material, and oxidizing material.

3. The composition as claimed in claim 2, wherein the surfactant 2 is one or more selected from 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonate, linear secondary alcohol polyoxyethylene ether, polyoxyethylene derivative and polyoxyethylene alkylphenol ether; preferably, the 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid salt accounts for 0.1 to 0.5 percent of the weight of the composition; preferably, the linear secondary alcohol polyoxyethylene ether accounts for 0.1 to 0.5 percent of the composition by mass; preferably, the polyoxyethylene derivative accounts for 0.1 to 0.5 percent of the composition by mass; preferably, the polyoxyethylene alkylphenol ether accounts for 0.1 to 0.5 percent of the composition by mass percent.

4. The composition of claim 2, wherein the protein substance is one or more of a lipoprotein, a cholesterol esterase; preferably, the concentration of the lipoprotein in the composition is 1 KU/L-10 KU/L; preferably, the cholesterol esterase accounts for 1KU/L to 10KU/L of the total volume of the composition.

5. The composition of claim 2, wherein the oxidizing substance is one or more of sodium cholate and urea; preferably, the concentration of the sodium cholate in the composition is 1 mmol/L-10 mmol/L; preferably, the concentration of urea in the composition is 300mmol/L to 2000 mmol/L.

6. The composition of claim 1 or 2, wherein the concentration of the zwitterionic buffer in the composition is 25-150 mmol/L; more preferably, the zwitterionic buffer is 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid, and the pH value of the zwitterionic buffer is 6.5-7.5.

7. The composition according to claim 1 or 2, wherein the surfactant 1 is selected from one or two of pentapeptide-59, isooctanol polyoxyethylene ether; preferably, the pentapeptide-59 accounts for 0.3 to 1.0 percent of the composition by mass; preferably, the isooctyl alcohol polyoxyethylene ether accounts for 0.1-0.6% of the composition by mass percent.

8. The composition according to claim 1 or 2, wherein the fructose is an acyl polyfructose; furthermore, the fructose accounts for 0.05-0.5% of the composition by mass.

9. The composition of claim 1 or 2, wherein the latex particles coated with lipoprotein-associated phospholipase A2 are polystyrene particles.

10. A method for preparing a stable composition of the lipoprotein-associated phospholipase a2 maintenance kit of any of claims 1 through 9, comprising the steps of: the components are taken according to the proportion and are fully and uniformly mixed to obtain the composition.

Technical Field

The invention relates to the technical field of biology, in particular to a composition for keeping stability of a lipoprotein-associated phospholipase A2 kit and a preparation method thereof.

Background

Lipoprotein phospholipase A2(1 isoproteins-associated phospholipases A2, Lp-PLA2) is a phospholipase A2 superfamily, a novel inflammatory marker and an independent risk factor closely related to arteriosclerosis and ischemic cardiovascular and cerebrovascular diseases which have attracted much attention in recent years. It has a molecular weight of 45KD, is synthesized and secreted by mature macrophages and lymphocytes, and is regulated by inflammatory mediators. Lipoprotein phospholipase A2 has a pro-inflammatory effect, so the production and release of Lp-PLA2 from inflammatory cells can also be interpreted as an excellent indicator of pro-inflammatory response. Lp-PLA2 produces oxygenated molecules in the vessel wall, which are more likely to cause atherosclerosis and produce unstable plaques. Elevated levels of Lp-PLA2 are indicative of a significant risk of plaque formation and rupture, and are independent of other lipid and CRP levels. By detecting the Lp-PLA2 level in the circulatory system, the cardiovascular and cerebrovascular diseases can be independently predicted. The cardiovascular and cerebrovascular embolism disease determination kit-human plasma lipoprotein phospholipase A2(lipoprotein phospholipase A2, Lp-PLA2) quantitative determination kit can rapidly monitor and predict the cardiovascular and cerebrovascular embolism disease. Human plasma lipoprotein phospholipase A2(Lp-PLA2) is an important new clinical test for saving the life of patients as a suggestive clinical test index of cardiovascular and cerebrovascular embolism. By detecting the level of Lp-PLA2 in blood, the inflammation degree of atherosclerotic plaques in arteries can be effectively known, the occurrence and the morbidity degree of human cardiovascular and cerebrovascular embolic diseases are predicted, and the diagnosis and the prevention of the cardiovascular and cerebrovascular embolic diseases are facilitated.

The currently commonly used methods for measuring the lipoprotein phospholipase A2 protein include a fluorescence immunochromatography method, an enzyme-linked immunosorbent assay, an up-conversion luminescence method and a latex-enhanced immunoturbidimetry method. Most of the methods cannot accurately perform quantitative determination, and only the latex enhanced immunoturbidimetry can be used for accurate quantitative determination, and the basic principle is as follows: the antibody is coated on latex particles, after immunoreaction with corresponding antigen occurs, aggregated particles can be formed, and the turbidity formed by the aggregated particles has a proportional relation with the corresponding antigen under the condition of a certain wavelength, so that the content of the antigen to be detected in a sample can be measured by measuring the turbidity generated by the aggregated particles. The method for coating the anti-lipoprotein phospholipase A2 antibody on latex particles is a physical adsorption method or a random direction coupling method, which has poor stability, and the structure of lipoprotein-associated phospholipase A2(Lp-PLA2) is easily influenced by external conditions in practical application, so that a composition for stabilizing the Lp-PLA2 kit in a sample needs to be provided, the influence of the external environment on the Lp-PLA2 is reduced, the stability of a test result is kept, and the biological activity of the Lp-PLA2 in the sample can be truly reflected.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides the following technical scheme:

the invention provides a composition for keeping stability of a lipoprotein-associated phospholipase A2 kit, which comprises the following components in part by weight: the method comprises the following steps: reagent R1 and reagent R2, wherein the reagent R1 comprises a lipoprotein-associated phospholipase A2 structure stabilizer, a zwitterionic buffer (namely Good's buffer), a surfactant 1 and a preservative; the reagent R2 comprises: good's buffer, fructose and latex particles coated with lipoprotein-associated phospholipase a 2;

further, the structure stabilizer of the lipoprotein-associated phospholipase A2 is selected from one or more of surfactant 2, protein substances and oxidizing substances;

further, the surfactant 2 is selected from one or more of 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonate (CHAPS), linear secondary alcohol polyoxyethylene ether (TERGITOL 15-S-7), polyoxyethylene derivative (EMULGEN B-66) and polyoxyethylene alkylphenol ether (Tx-405); preferably, CHAPS accounts for 0.1-0.5% of the composition by mass; preferably, TERGITOL 15-S-7 accounts for 0.1 to 0.5 percent of the composition by weight; preferably, EMULGEN B-66 accounts for 0.1 to 0.5 percent of the composition by mass; preferably, Tx-405 accounts for 0.1% -0.5% of the composition by weight percent;

furthermore, the protein substance is one or more of lipoprotein and cholesterol esterase; preferably, the concentration of the lipoprotein in the composition is 1 KU/L-10 KU/L; preferably, the cholesterol esterase accounts for 1KU/L to 10KU/L of the total volume of the composition;

furthermore, the oxidizing substance is one or more of sodium cholate and urea; preferably, the concentration of the sodium cholate in the composition is 1 mmol/L-10 mmol/L; preferably, the concentration of urea in the composition is 300 mmol/L-2000 mmol/L;

further, the concentration of the zwitterionic buffer solution in the composition is 25-100 mmol/L; preferably, the zwitterionic buffer is prepared from 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO), and the pH of the zwitterionic buffer is 6.5-7.5;

further, the surfactant 1 is selected from one or two of pentapeptide-59 and isooctanol polyoxyethylene ether (namely CA-60); preferably, the pentapeptide-59 accounts for 0.3 to 1.0 percent of the composition by mass; preferably, the CA-60 accounts for 0.1 to 0.6 percent of the composition by weight;

further, the preservative is sodium azide; furthermore, the preservative accounts for 0.5 to 5.0 percent of the composition by mass;

further, the fructose is acyl polyfructose; furthermore, the fructose accounts for 0.05 to 0.5 percent of the composition by mass;

further, the latex particles coated with the lipoprotein-associated phospholipase A2 are polystyrene particles.

Another aspect of the present invention claims a method for preparing any one of the above compositions for stabilizing the lipoprotein-associated phospholipase a2 kit, comprising the steps of: the components are taken according to the proportion and are fully and uniformly mixed to obtain the composition.

Advantageous effects

Firstly, the composition for maintaining the stability of the lipoprotein-associated phospholipase A2(Lp-PLA2) kit can remarkably reduce the influence of the outside on the Lp-PLA2 in the use of the kit by adding a specific lipoprotein-associated phospholipase A2 structure stabilizer (consisting of a surfactant, a protein substance and an oxidation substance) and a specific surfactant and fructose into the composition in a reasonable ratio, maintain the stability of a test result, and truly reflect the biological activity of the Lp-PLA2 in a sample, wherein the test result is very stable from the experimental result of an example; secondly, the preparation conditions and the preparation process are simple, the implementation and the industrial production are facilitated, the inflammation degree of atherosclerotic plaques in arteries can be effectively known, the occurrence and the morbidity degree of human cardiovascular and cerebrovascular embolic diseases are predicted, the diagnosis and the prevention of the cardiovascular and cerebrovascular embolic diseases are facilitated, and the method has good market application prospect.

Detailed Description

Description of the drawings: the "%" in the following examples and comparative examples each means the mass% of the composition for stabilizing the lipoprotein-associated phospholipase A2 kit.