阅读说明：本技术 一种小而密脂蛋白的测定试剂、方法和试剂盒 (Reagent, method and kit for measuring small and dense lipoproteins ) 是由 王学忠 于 2018-07-20 设计创作，主要内容包括：本发明公开了一种用于清除脂蛋白样品中的乳糜微粒的组合物,所述组合物包括以下的组分：2.0g/L～16.0g/L聚乙二醇,0.145～1.6％表面活性剂A,0.1～2.0KU/L脂蛋白酯酶,所述表面活性剂A是花王公司Emulgen B66与陶氏公司Tergitol 15-S系列或NP系列的组合,所述％为质量百分比。公开了一种清除脂蛋白样品中的乳糜微粒的方法和上述的组合物在测定小而密脂蛋白中的应用。还公开了一种用于测定小而密脂蛋白的组合试剂和一种小而密脂蛋白的非疾病诊断的测定方法。所述的测定方法能够特定、专一地检测sdLDL。还公开了一种测定小而密脂蛋白的试剂盒,所述的试剂盒操作简便、效果良好。(The invention discloses compositions for removing chylomicron in a lipoprotein sample, which comprise 2.0-16.0 g/L of polyethylene glycol, 0.145-1.6% of surfactant A and 0.1-2.0 KU/L of lipoprotein esterase, wherein the surfactant A is a combination of Emulgen B66 of Huawang company and Tergitol 15-S series or NP series of Dow company, and the percentage is mass percent.)

A composition for removing chylomicrons from a lipoprotein sample, the composition comprising the following components:

2.0 g/L-16.0 g/L of polyethylene glycol,

0.145 to 1.6 percent of surfactant A,

0.1 to 2.0KU/L of a lipoprotein esterase,

the surfactant A is a combination of Emulgen B66 from Huawang company and Tergitol 15-S series or NP series from Dow company;

preferably, the polyethylene glycol is polyethylene glycol 6000, and the content of the polyethylene glycol is 5.0-15.0 g/L;

the content of Emulgen B66 in the surfactant A is 0.135-1.5%,

the Tergitol 15-S content is 0.01-0.10%,

the content of lipoprotein esterase is 0.15-1.0 KU/L;

more preferably, the content of the polyethylene glycol is 12.0g/L,

the content of Emulgen B66 in the surfactant A is 0.55 percent,

the Tergitol 15-S content is 0.05 percent,

the content of lipoprotein esterase is 0.2 KU/L; the percentage is mass percent.

A method of removing chylomicrons from a lipoprotein sample comprising using the composition of claim 1 in .

3. Use of the composition of claim 1 for the determination of small, dense lipoproteins.

4, reagent for the determination of small, dense lipoproteins, comprising the composition of claim 1.

5. The reagent of claim 4, further comprising the following components:

1.0 to 3.0mmol/L Trinder's chromogen compound,

200 to 4000U/L of heparin sodium,

2 to 50mmol/L of divalent metal ions,

1.0 to 4.8KU/L of cholesterol esterase,

1.0 to 5.0KU/L of cholesterol oxidase, and

1.0 to 5.0MU/L catalase,

the pH of the reagent is 6.40-6.70.

6. The reagent of claim 5,

1.5-2.0 mmol/L Trinder's chromogen compound; and/or the presence of a gas in the gas,

300-3500U/L heparin sodium; and/or the presence of a gas in the gas,

15-25 mmol/L of divalent metal ions, wherein the divalent metal ions are magnesium ions or manganese ions; and/or the presence of a gas in the gas,

1.5-2.8 KU/L cholesterol esterase; and/or the presence of a gas in the gas,

2.0-4.0 KU/L cholesterol oxidase; and/or the presence of a gas in the gas,

2.0-3.0 MU/L catalase; and/or the presence of a gas in the gas,

25-50 mmol/L buffer solution for adjusting pH, preferably MOPS buffer solution; and/or the presence of a gas in the gas,

the reagent further comprises a stabilizer which is or more of ascorbic acid oxidase, bovine serum albumin, sodium chloride or EDTA, preferably the content of the ascorbic acid oxidase is 3.5KU/L, preferably the content of the bovine serum albumin, the sodium chloride and the EDTA, the content of the bovine serum albumin is 1.0-5.0 g/L, preferably 2.0-4.0 g/L, the content of the sodium chloride is 5-150 mmol/L, preferably 25-55 mmol/L, and/or the content of the EDTA is 0.2 mmol/L;

preferably, the th reagent comprises the following components:

12.0g/L of polyethylene glycol 6000,

0.55％Emulgen B-66，

0.05％Tergitol 15-S-12,

0.2KU/L lipoprotein esterase,

2.0mM Trinder's chromogen compound,

3000U/L of heparin sodium is added,

15mM of magnesium sulfate,

2.0KU/L cholesterol esterase,

3.5KU/L cholesterol oxidase,

2.0MU/L of a catalase,

the concentration of sodium chloride was 55mM in the medium,

0.2mM EDTA，

3.5KU/L ascorbic acid oxidase,

2.5g/L bovine serum albumin, and

50mM MOPS buffer (pH6.5), wherein the% is mass percent.

A combined reagent for measuring small and dense lipoproteins, comprising the reagent of any one of claims 4 to 6 and a second reagent, wherein the second reagent comprises the following components:

2.0 to 10KU/L peroxidase,

0.2 to 1.0g/L of 4-aminoantipyrine,

0.1 to 0.2% sodium azide, and,

1.0-3.0% of surfactant Triton X-100,

the pH value of the reagent is 6.40-6.70, and the% is mass percentage.

8. The combination of claim 7, wherein said second agent comprises the following components:

2.5-6.5 KU/L peroxidase; and/or the presence of a gas in the gas,

0.3-0.6 g/L4-aminoantipyrine; and/or the presence of a gas in the gas,

1.2-2.5% Triton X-100; and/or the presence of a gas in the gas,

25-50 mmol/L buffer solution for adjusting pH; and/or the presence of a gas in the gas,

the second reagent further comprises a stabilizer, the content of the stabilizer is 0.2-6.5 g/L, and preferably, the stabilizer is bovine serum albumin; the percentage is mass percentage;

preferably, the second agent comprises the following components:

6.0KU/L of peroxidase,

0.5g/L of 4-aminoantipyrine,

5.0g/L of bovine serum albumin,

0.1 percent of sodium azide,

2% Triton X-100, and

30mM MOPS buffer (pH6.5), wherein the% is mass percent.

The method for determining the non-disease diagnosis of small and dense lipoproteins, comprising the steps of:

(1) mixing a sample with the reagent of claims 4-6 to obtain a reaction solution 1, wherein the volume ratio of the sample to the reagent is the conventional volume ratio in the art, preferably 1: 75-1: 100, more preferably 1: 75;

(2) mixing the reaction solution 1 obtained in the step (1) with a second reagent in the combined reagent of in any claim from 7 to 8 to obtain a reaction solution 2, wherein the volume ratio of the reaction solution 1 to the second reagent is a volume ratio conventional in the art, preferably 5:1 to 3:1, and more preferably 3: 1;

(3) reading the absorbance values of the reaction liquid 2 obtained in the step (2) at the wavelengths of 546nm and 700nm, and calculating the content of small and dense lipoprotein; preferably, the spline method calculates the content of small, dense lipoproteins.

kit for the determination of small, dense lipoproteins, comprising the combination of reagents according to claim 7 or 8, preferably the kit further comprises standard, the kit standard can be the standard of commercial kits used conventionally in the art, preferably Langdian blood lipid standard solution.

Technical Field

The invention relates to the field of biological medicine, in particular to a reagent, a method and a kit for measuring small and dense lipoproteins.

Background

Lipoproteins existing in blood are roughly classified into High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL), Very Low Density Lipoprotein (VLDL) and Chylomicron (CM)4 according to their specific gravity, wherein Low Density Lipoprotein (LDL) is composed of series of particles having various sizes, densities and chemical compositions generally designates LDL having smaller particles and higher Density among LDL subfractions as small dense Lipoprotein (small dense Lipoprotein, abbreviated as sdLDL), designates LDL having larger particles and lower Density LDL as large and light LDL, abbreviated as LgLDL, and a subgroup therebetween is classified as medium Density LDL.

The present methods for measuring sdLDL in clinical tests include electrophoresis, ultracentrifugation, and fractional precipitation, but these methods have a long test time, cannot be used for large-scale automated tests, and require expensive equipment, and thus cannot be applied to clinical medicine tests.

Disclosure of Invention

The invention aims to solve the technical problem of providing small and dense cholesterol determination reagents, methods and kits aiming at the defects of the existing small and dense lipoprotein detection method, which can eliminate the interference of chyle in a sample so as to obtain a more accurate detection result.

Through a large number of experimental studies, the present inventors found that interference of chylomicron in a lipoprotein sample can be excluded in the th reagent by using a combination of a polyanionic compound binding to lipoprotein, a surfactant, and a lipoprotein esterase, thereby achieving the purpose of measuring sdLDL.

To solve the technical problem, of the present invention is to provide compositions for removing chylomicron from a lipoprotein sample, which comprises the following components:

2.0 g/L-16.0 g/L of polyethylene glycol,

0.145 to 1.6 percent of surfactant A,

0.1 to 2.0KU/L of a lipoprotein esterase,

the surfactant A is a combination of the King Emulgen B66 and the Dow Tergitol 15-S series or NP series.

Preferably, the polyethylene glycol is polyethylene glycol 6000, and the content of the polyethylene glycol is 5.0-15.0 g/L;

the content of Emulgen B66 in the surfactant A is 0.135-1.5%,

the Tergitol 15-S content is 0.01-0.10%,

the content of lipoprotein esterase is 0.2-1.0 KU/L.

More preferably, the content of the polyethylene glycol is 12.0g/L,

the content of Emulgen B66 in the surfactant A is 0.55 percent,

the Tergitol 15-S content is 0.05 percent,

the content of lipoprotein esterase is 0.2 KU/L; the percentage is mass percent.

To solve the above technical problems, in the technical solution of the present invention is to provide methods for removing chylomicron from a lipoprotein sample, which is characterized in that the methods comprise using the above composition.

To solve the technical problem, of the technical scheme of the invention provides applications of the composition in determination of small and dense lipoproteins.

To solve the technical problem, of the technical scheme of the invention is to provide reagent, which comprises the following components:

2.0 g/L-16.0 g/L of polyethylene glycol,

0.145 to 1.6 percent of surfactant A,

0.1 to 2.0KU/L of a lipoprotein esterase,

the surfactant A is a combination of Emulgen B66 from Huawang company and Tergitol 15-S series or NP series from Dow company;

preferably, the polyethylene glycol is polyethylene glycol 6000, and the content of the polyethylene glycol is 5.0-15.0 g/L;

the content of Emulgen B66 in the surfactant A is 0.135-1.5%,

the Tergitol 15-S content is 0.01-0.10%,

the content of lipoprotein esterase is 0.2-1.0 KU/L;

more preferably, the content of the polyethylene glycol is 12.0g/L,

the content of Emulgen B66 in the surfactant A is 0.55 percent,

the Tergitol 15-S content is 0.05 percent,

the content of lipoprotein esterase is 0.2 KU/L; the percentage is mass percent.

Preferably, the th reagent further comprises the following components:

1.0 to 3.0mmol/L Trinder's chromogen compound,

200 to 4000U/L of heparin sodium,

2 to 50mmol/L of divalent metal ions,

1.0 to 4.8KU/L of cholesterol esterase,

1.0 to 5.0KU/L of cholesterol oxidase, and

1.0 to 5.0MU/L catalase,

the pH of the reagent is 6.40-6.70.

More preferably, the th reagent includes the following components:

1.5-2.0 mmol/L Trinder's chromogen compound; and/or the presence of a gas in the gas,

300-3500U/L heparin sodium; and/or the presence of a gas in the gas,

15-25 mmol/L of divalent metal ions, wherein the divalent metal ions are magnesium ions or manganese ions; and/or the presence of a gas in the gas,

1.5-2.8 KU/L cholesterol esterase; and/or the presence of a gas in the gas,

2.0-4.0 KU/L cholesterol oxidase; and/or the presence of a gas in the gas,

2.0 to 3.0MU/L catalase.

In the reagent of the present invention, the Trinder's chromogen compound is a Trinder's chromogen compound conventional in the art, preferably sodium 3- (N-ethyl-3-methylanilino) -2-hydroxypropanesulfonate (TOOS), N-ethyl-N- (3-sulfopropyl) -3-methylaniline (TOPS) or N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline (HDAOS), more preferably TOOS.

In the present invention, the reagent preferably further comprises a buffer solution, wherein the buffer solution is a buffer solution conventional in the art, preferably a MOPS buffer solution or a MOPSO buffer solution, more preferably a MOPS buffer solution, and the content of the buffer solution is a content conventional in the art, preferably 25 to 120mmol/L, more preferably 30 to 50 mmol/L.

In the invention, the th reagent further comprises a stabilizer or more of ascorbic acid oxidase, bovine serum albumin, sodium chloride or EDTA, preferably the content of ascorbic acid oxidase is 3.5KU/L, preferably bovine serum albumin, sodium chloride and EDTA, the content of bovine serum albumin is 1.0-5.0 g/L, preferably 2.0-4.0 g/L, the content of sodium chloride is 5-150 mmol/L, preferably 25-55 mmol/L, and/or the content of EDTA is 0.2 mmol/L.

In the present invention, it is more preferable that the th reagent comprises the following components:

12.0g/L of polyethylene glycol 6000,

0.55％Emulgen B-66，

0.05％Tergitol 15-S-12,

0.2KU/L lipoprotein esterase,

2.0mM Trinder's chromogen compound,

3000U/L of heparin sodium is added,

15mM of magnesium sulfate,

2.0KU/L cholesterol esterase,

3.5KU/L cholesterol oxidase,

2.0MU/L of a catalase,

the concentration of sodium chloride was 55mM in the medium,

0.2mM EDTA，

3.5KU/L ascorbic acid oxidase,

2.5g/L bovine serum albumin, and

50mM MOPS buffer (pH6.5), wherein the% is mass percent.

according to the technical scheme of the invention, second reagents for measuring small and dense lipoproteins are provided, and comprise the following components:

2.0 to 10KU/L peroxidase,

0.2 to 1.0g/L of 4-aminoantipyrine,

0.1 to 0.2% sodium azide, and,

1.0-3.0% of surfactant Triton X-100,

the pH value of the reagent is 6.40-6.70, and the% is mass percentage.

More preferably, the second agent comprises the following components:

2.5-6.5 KU/L peroxidase; and/or the presence of a gas in the gas,

0.3-0.6 g/L4-aminoantipyrine; and/or the presence of a gas in the gas,

1.2-2.5% Triton X-100; and/or the presence of a gas in the gas,

25-50 mmol/L buffer solution for adjusting pH; and/or the presence of a gas in the gas,

the second reagent further comprises a stabilizer, the content of the stabilizer is 0.2-6.5 g/L, and preferably, the stabilizer is bovine serum albumin; the percentage is mass percent.

More preferably, the second agent comprises the following components:

6.0KU/L of peroxidase,

0.5g/L of 4-aminoantipyrine,

5.0g/L of bovine serum albumin,

0.1 percent of sodium azide,

2% Triton X-100, and

30mM MOPS buffer (pH6.5), wherein the% is mass percent.

The second reagent of the present invention is in the form of a clear liquid.

In the present invention, it is preferable that or more of the buffer and the stabilizer are added before the pH is adjusted.

according to the present invention is a method for determining non-disease diagnosis of small and dense lipoproteins, comprising the steps of:

(1) mixing a sample with the reagent of claims 4-6 to obtain a reaction solution 1, wherein the volume ratio of the sample to the reagent is the conventional volume ratio in the art, preferably 1: 75-1: 100, more preferably 1: 75;

(2) mixing the reaction solution 1 obtained in the step (1) with a second reagent in the combined reagent of in any claim from 7 to 8 to obtain a reaction solution 2, wherein the volume ratio of the reaction solution 1 to the second reagent is a volume ratio conventional in the art, preferably 5:1 to 3:1, and more preferably 3: 1;

(3) reading the absorbance values of the reaction liquid 2 obtained in the step (2) at the wavelengths of 546nm and 700nm, and calculating the content of small and dense lipoprotein; preferably, the spline method calculates the content of small, dense lipoproteins.

The apparatus used in the above-mentioned assay method is an apparatus conventional in the art, preferably a fully automatic biochemical analyzer conventional in the art, more preferably a Hitachi 7180 fully automatic biochemical analyzer or a Beckmann series fully automatic biochemical analyzer.

according to the present invention provides kits for assaying small and dense lipoproteins, which include reagent and the second reagent.

In the invention, the kit preferably further comprises a standard substance, and the standard substance of the kit can be a commercial standard substance of the kit conventionally used in the field, and is preferably Langdian blood lipid standard solution.

The kit is simple and convenient to operate, and can efficiently and accurately detect small and dense lipoproteins specifically.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The reagent is simple and convenient to prepare, can be efficiently and accurately applied to the method for specifically detecting sdLDL, can specifically and specially detect small and dense lipoprotein (sdLDL) , has high reliability, low cost and high efficiency, and can automatically and specifically detect sdLDL in a large scale.

Drawings

FIG. 1 is a graph of a small and dense lipoprotein assay; wherein A denotes the method using the present invention, B denotes the method using the prior art CN201610139400.X, and 1 and 2 denote different specimen numbers, respectively.

Detailed Description

The invention is further illustrated by the following examples , but is not intended to be limited thereby within the scope of the examples.