阅读说明：本技术 一种高密度脂蛋白3的测定试剂、方法和试剂盒 (High-density lipoprotein 3 determination reagent, method and kit ) 是由 王贤理 池万余 王伊琳 苗准 邓慰 卢强 于 2020-12-16 设计创作，主要内容包括：本发明涉及生物医药领域。目的是提供一种HDL3胆固醇的直接测定试剂、方法和试剂盒,即在第一试剂中添加除HDL3以外的脂蛋白保护剂；在第二试剂添加针对水解HDL3-C的表面活性剂与酶的组合的方法。技术方案是：一种测定HDL3的第一试剂,第一试剂包括除HDL3以外的脂蛋白保护剂,还包括Trinder’s色原化合物、二价金属离子、胆固醇氧化酶、过氧化氢酶、缓冲液、稳定剂、防腐剂；其中所述的除HDL3以外的脂蛋白保护剂,是指作用于除HDL3以外的化合物组合。众所周知,血清样本中的HDL3是最致密的脂蛋白,与特定的脂蛋白相关的表面活性剂、环糊精、抗体、聚阴离子、二价金属离子等化合物的组合的反应性极低。(The invention relates to the field of biomedicine. Aims to provide a reagent, a method and a kit for directly measuring HDL3 cholesterol, namely, a lipoprotein protective agent except HDL3 is added into a first reagent; a method of adding a combination of a surfactant and an enzyme directed to hydrolyzing HDL3-C at a second reagent. The technical scheme is as follows: a first reagent for assaying HDL3, which comprises a lipoprotein protectant other than HDL3, a Trinder's chromogen compound, divalent metal ions, cholesterol oxidase, catalase, a buffer, a stabilizer, and a preservative; wherein the lipoprotein protective agent other than HDL3 is a compound combination acting on HDL 3. It is known that HDL3 in a serum sample is the most dense lipoprotein, and the reactivity of a combination of compounds such as a surfactant, cyclodextrin, an antibody, a polyanion, and a divalent metal ion, which are related to a specific lipoprotein, is extremely low.)

1. A reagent for measuring high-density lipoprotein 3; comprises the following components in percentage by weight:

a first reagent

0.3 to 20mM Trinder's chromogen compound,

2.0 to 50mM of divalent metal ions,

HDL3 lipoprotein protective agents other than HDL3,

1.0 to 5.0KU/L of cholesterol oxidase,

1.0 to 10.0MU/L catalase,

25 to 120mM MOPS buffer solution,

5 to 155mM sodium chloride, 0.1 to 2.0mM EDTA, 1.0 to 10.0g/L bovine serum albumin,

0.01 to 0.5 percent of Proclin-300 preservative,

the balance of water;

the HDL3 lipoprotein protective agent other than HDL3 comprises a surfactant other than HDL3, cyclodextrin other than HDL3, polyanion other than HDL3, and antibody other than HDL 3;

the non-HDL 3 surfactant comprising: emulgen A500, Emulgen LS-114, Emulgen 109P, Pluronic F68, polyoxyethylene lauryl ether Brij L23, polyoxyethylene (100) octadecyl ether Brij S100 and one or more of sodium dodecyl benzene sulfonate in any proportion;

the dosages of Emulgen A500, Emulgen LS-114 or Emulgen 109P are all 0.1-2.0%, the dosage of Pluronic F68 is 0.1-3.0%, and the dosages of Brij L12 and S100 are all 0.01-1.0%;

the cyclodextrin of the non-HDL 3 comprises dimethyl-beta-cyclodextrin or alpha-cyclodextrin sulfate, and the dosage of the cyclodextrin is 0.01-0.5%;

the polyanion of the non-HDL 3 comprises glucan and salt thereof, and the using amount is 0.05-1.0%;

the non-HDL 3 antibody comprises an anti-apolipoprotein B100 antibody or an anti-apolipoprotein B48 antibody, and the dosage is 1.0 g/L-10.0 mg/L;

second reagent

30 to 75mmol/L of a buffer solution,

0.5 to 10.0KU/L cholesterol esterase,

1.0 to 20.0KU/L peroxidase,

0.5 to 0.8g/L of 4-aminoantipyrine,

0.2-5 g/L bovine serum albumin,

0.1 to 0.3% of sodium azide,

0.02 to 2.0% of a surfactant,

the surfactant is one or a mixture of more of Emulgen B66, Emulgen A90, Tergitol NP-7, Triton X-100 or Pluronic L123 in any proportion,

the balance of water;

the percentages are mass percentages.

2. The reagent for measuring high-density lipoprotein 3 according to claim 1, which is characterized in that:

the first reagent comprises the following components in percentage by weight:

1.25 to 2.5mM Trinder's chromogen compound,

6 to 10mM of a divalent metal ion,

HDL3 lipoprotein protective agents other than HDL3,

1.0 to 3.0KU/L of cholesterol oxidase,

1.0 to 3.0MU/L catalase,

30 to 50mM MOPS buffer solution,

25-55 mM sodium chloride, 2.0mM EDTA, 1.0-5.0 g/L BSA, stabilizer;

0.02% of Proclin-300, wherein the percentage is mass percentage;

+ the balance of water;

surfactants other than HDL3 lipoprotein protective agents other than HDL3 other than HDL3, comprising:

emulgen A500, Emulgen LS-114 and Emulgen 109P with the dosage of 0.3-1.5%; pluronic F68 with the dosage of 0.5 to 2.0 percent; brij L12 and S100 with the dosage of 0.03-0.50 percent;

the cyclodextrin other than HDL3, other than HDL3 lipoprotein protective agent, other than HDL3, is non-HDL 3 cyclodextrin, including dimethyl-beta-cyclodextrin or alpha-cyclodextrin sulfate with an amount of 0.1-0.2%;

polyanions other than HDL3 in HDL3 lipoprotein protective agents other than HDL3, including dextran sulfate; dextran sulfate has a molecular weight of 5 or 50 ten thousand; the dosage is 0.2 to 0.7 percent;

the non-HDL 3 antibody in HDL3 lipoprotein protective agent other than HDL3 comprises anti-apolipoprotein B100 antibody or anti-apolipoprotein B48 antibody in an amount of 1.0 g/L-5.0 mg/L;

the second reagent comprises the following components in percentage by weight:

30 to 50mmol/L of a buffer solution,

1.05 to 5.0KU/L of cholesterol esterase,

2.0 to 10.0KU/L peroxidase,

0.5 to 0.8g/L of 4-aminoantipyrine,

0.2-5 g/L bovine serum albumin,

0.1 percent of sodium azide,

0.5 to 1.5% of a surfactant,

the balance of water;

the percentage is mass percentage.

3. The method for preparing a reagent for measuring high-density lipoprotein 3 according to claim 1, comprising the steps of:

1) a first reagent: adding a chromogen compound, divalent metal ions, a buffer solution and a stabilizer into water, stirring until the chromogen compound, the divalent metal ions, the buffer solution and the stabilizer are completely dissolved, adjusting the pH value to 6.50-7.50, and then adding an HDL3 lipoprotein protective agent, an HDL3 lipoprotein protective agent, cholesterol oxidase and catalase except HDL3 to obtain a first reagent;

2) a second reagent: adding 4-aminoantipyrine, a surfactant, bovine serum albumin, a buffer solution and sodium azide into water, stirring until the mixture is completely dissolved, adjusting the pH value to 6.50-7.50, and then adding peroxidase and cholesterol oxidase to obtain a second reagent.

4. A kit comprising the reagent for measuring high-density lipoprotein 3 according to claim 1, wherein: the kit comprises the first reagent and the second reagent.

5. The kit consisting of reagents for assaying high-density lipoprotein 3 according to claim 4, characterized in that: and also comprises a standard substance.

6. A method for measuring high density lipoprotein 3 using the reagent for measuring high density lipoprotein 3 according to claim 1, comprising the steps of:

(1) mixing a sample with the first reagent for reaction to obtain a reaction solution 1; the volume ratio of the sample to the first reagent is 1: 75-1: 120;

(2) mixing the reaction solution 1 obtained in the step (1) with the second reagent for reaction to obtain a reaction solution 2; the volume ratio of the reaction solution 1 to the second reagent is 5: 1-3: 1;

(3) and (3) reading the absorbance values of the reaction liquid 2 obtained in the step (2) at the wavelengths of 600nm and 800nm, and calculating the content of the high-density lipoprotein 3.

7. The method for measuring high-density lipoprotein 3 according to claim 6, wherein:

the volume ratio of the sample to the first reagent in the step (1) is 1: 100; the volume ratio of the reaction solution 1 to the second reagent in the step (2) is 3: 1.

8. The method for measuring high density lipoprotein 3 according to claim 7, which is used for non-disease diagnosis or treatment purposes.

Technical Field

The invention relates to the field of biomedicine, in particular to a reagent, a method and a kit for determining the content of cholesterol (HDL 3-C) in high-density lipoprotein 3(HDL 3).

Background

Lipoproteins present in blood are roughly classified into High Density Lipoproteins (HDL), Low Density Lipoproteins (LDL), Very Low Density Lipoproteins (VLDL), and Chylomicrons (CM) according to their specific gravity. Wherein the High Density Lipoprotein (HDL) is composed of a series of particles of varying size, density and chemical composition. HDL with smaller particles and larger density in HDL subfractions is generally called high density lipoprotein 3(HDL3), and the density range is 1.125-1.210 g/mL; HDL with larger particles and smaller density is called high density lipoprotein 2(HDL2), and the density range is 1.063-1.125 g/mL. Recent studies have found that HDL3 plays an important role in the exacerbation or recovery of arteriosclerosis. Clinical practice shows that standard tests for cholesterol, HDL, LDL and triglycerides usually detect only about 20% of patients with cardiovascular disease, and the remaining about 80% of patients can only be identified by differentiating sub-fractions and further lipid tests. Further studies have demonstrated that HDL3 is directly associated with an increased risk factor for myocardial infarction disease.

Currently, in clinical applications, methods for determining HDL3 include electrophoresis, ultracentrifugation, and fractional precipitation. However, these methods have long detection time, complicated operation steps, and are not suitable for large-scale automated detection, and expensive instruments and equipment and specialized technicians are required, so that they cannot be applied to general clinical medical detection.

In the field of application of full-automatic biochemical analyzers, methods for measuring HDL3 include a clearing method, a masking method, which removes or masks the reaction of non-HDL 3 or all lipoproteins with cholesterol lipase by using a characteristic compound, but the clearing method easily removes part of HDL3, and the masking method cannot completely mask cholesterol ester, thereby making the measurement inaccurate.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a reagent, a method and a kit for directly measuring HDL3 cholesterol, aiming at the defect of lack of a detection method specially aiming at HDL3 at present, namely, a lipoprotein protective agent except HDL3 is added into a first reagent; a method of adding a combination of a surfactant and an enzyme directed to hydrolyzing HDL3-C at a second reagent. The measuring method can specifically and exclusively detect the HDL3, has high reliability, low cost and high efficiency, can realize large-scale automatic detection, and is superior to a shielding method. The kit is simple and convenient to operate.

Through extensive and intensive studies, the invention finds that the reaction of a serum sample with a first reagent which comprises a surfactant with a proper concentration, a lipoprotein protective agent except HDL3 and other components can more completely protect lipoprotein cholesterol except HDL 3; and then reacting with a second reagent with the components and the content thereof specially selected, thereby further accurately determining the content of HDL3 in the serum sample.

One technical scheme of the invention is to provide a first reagent for determining HDL3, wherein the first reagent comprises a lipoprotein protective agent except HDL3, a Trinder's chromogen compound, divalent metal ions, cholesterol oxidase, catalase, a buffer solution, a stabilizer and a preservative; wherein the lipoprotein protective agent other than HDL3 is a compound combination acting on HDL 3. It is known that HDL3 in a serum sample is the most dense lipoprotein, and the reactivity of a combination of compounds such as a surfactant, cyclodextrin, an antibody, a polyanion, and a divalent metal ion, which are related to a specific lipoprotein, is extremely low. Through extensive selectivity studies, the first process solution of the present invention comprises such a combination; this combination is either unreactive or very unreactive with HDL 3. The combination of the present invention enables the measurement of high-density lipoprotein 3 by classification without the need for a preliminary separation treatment in the measurement of lipoproteins in a serum sample.

By "protect" herein is meant reducing and breaking down or preventing and breaking down specific lipoprotein cholesterol in the first agent and its reaction with cholesterol esterase.

The lipoprotein protective agent other than HDL3 comprises surfactant other than HDL3, cyclodextrin other than HDL3, polyanion other than HDL3, and antibody other than HDL 3;

the surfactant acting on non-HDL 3 in the first reagent may, for example, be Emulgen A500, Emulgen LS-114, Emulgen 109P, Pluronic F68, polyoxyethylene lauryl ether Brij L23, polyoxyethylene (100) stearyl ether Brij S100, sodium dodecylbenzenesulfonate, or the like. The surfactants may be used alone or in combination of a plurality of them. The dosage of the surfactant, Emulgen A500 is 0.1-2.0%, preferably 0.3-1.5%; pluronic F68 is 0.1% -3.0%, preferably 0.5% -2.0%; brij L12 and S100 are 0.01% -1.0%, preferably 0.03% -0.50%.

The cyclodextrin that acts on the non-HDL 3 in the first agent includes dimethyl- β -cyclodextrin, α -cyclodextrin sulfate. The amount of the cyclodextrin is not particularly limited as long as it protects non-HDL 3 lipoproteins, and is preferably 0.01% to 0.5%, more preferably 0.1 to 0.2%.

The polyanion acting on non-HDL 3 in the first agent is dextran and its salts, preferably dextran sulfate. The molecular weight of the dextran sulfate is not particularly required, and is usually 5 ten thousand or 50 ten thousand; the preferred amount is 0.05% -1.0%, more preferably 0.2% -0.7%.

The antibodies that act on non-HDL 3 in the first agent include anti-apolipoprotein B100 antibody, anti-apolipoprotein B48 antibody, simply anti-apolipoprotein B antibody. The amount of the antibody is not particularly limited as long as it protects non-HDL 3 lipoprotein, and is preferably 0.10g/L to 10.0g/L, more preferably 1.0g/L to 5.0 mg/L.

In the first 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. The content of Trinder's chromogen compound is 0.3-20 mmol/L, preferably 0.75-5.0 mmol/L, more preferably 1.25-2.5 mmol/L.

In the first reagent of the present invention, the divalent metal ion is a metal ion (such as magnesium sulfate) commonly used in the art, and the dosage of the divalent metal ion is 2.0 to 50mmol/L, preferably 5 to 20mmol/L, and more preferably 6 to 10 mmol/L.

In the first reagent of the present invention, the content of the cholesterol oxidase is 1 to 5KU/L, preferably 1.0 to 3.0 KU/L.

In the first reagent of the present invention, the content of catalase is 1-10 MU/L, preferably 1-3 MU/L.

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

In the present invention, preferably, the first reagent further comprises a stabilizer. The stabilizer is conventional in the art, and is preferably selected from one or more of ascorbate oxidase, bovine serum albumin, sodium chloride or EDTA, more preferably one or more of bovine serum albumin, sodium chloride or EDTA, and most preferably bovine serum albumin, sodium chloride and EDTA. The content of the stabilizer is the content conventional in the field, wherein the content of the ascorbic acid oxidase is preferably 1-10 KU/L; the content of the bovine serum albumin is preferably 0.1-10 g/L, more preferably 1-5 g/L; the content of the sodium chloride is preferably 5 to 155mmol/L, more preferably 25 to 55 mmol/L; preferably, the content of EDTA is 0.1-2 mmol/L.

In the present invention, preferably, the first agent further comprises a preservative. The preservative is conventional in the art, preferably Proclin-300. The content of the preservative is the content conventional in the field, preferably 0.01-0.5%, and the percentage is mass percent.

In the present invention, preferably, the first reagent comprises the following components:

1.25 to 2.5mM Trinder's chromogen compound,

3.0 to 10mM of divalent metal ions,

HDL3 lipoprotein protective agents other than HDL3,

1.0 to 3.0KU/L of cholesterol oxidase,

1.0 to 3.0MU/L catalase,

30 to 50mM MOPS buffer solution,

154mM sodium chloride, 2.0mM EDTA, 1.0 g/L-6.0 g/L BSA and a stabilizer;

0.02% Proclin-300 preservative,

the balance of water;

the percentage is mass percentage;

the invention also provides a preparation method of the first reagent, which comprises the following steps: adding the above reagent components into water, stirring to dissolve completely, adjusting pH to 6.50-7.50, adding HDL3 lipoprotein protectant except HDL3, HDL3 lipoprotein protectant, cholesterol oxidase and catalase to obtain first reagent.

In the present invention, preferably, one or more of the buffer, the stabilizer and the preservative are added before the pH is adjusted.

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

The second technical scheme of the invention provides a second reagent for measuring high-density lipoprotein 3, which comprises the following components:

30 to 50mmol/L of a buffer solution,

0.5 to 10.0KU/L cholesterol esterase,

1.0 to 20.0KU/L peroxidase,

0.5 to 0.8g/L of 4-aminoantipyrine,

0.2-5 g/L bovine serum albumin,

0.1 to 0.3% of sodium azide,

0.02 to 2.0% of a surfactant,

the balance of water;

the percentage is mass percentage.

In the second reagent of the present invention, the content of the peroxidase is 1.0 to 20KU/L, preferably 2.0 to 10KU/L, and more preferably 2.5 to 5 KU/L.

In the second reagent of the present invention, the surfactant is one or more of Emulgen B66 and Emulgen A90 from Kao corporation, Tergitol NP-7 from Dow corporation, Triton X-100, and Pluronic L123 from Pasteur corporation. The content is 0.02-2.0%, preferably 0.1-1.5%.

The second reagent of the invention also comprises a buffer solution; the buffer is a buffer conventional in the art, preferably a MOPS buffer or a MOPSO buffer, more preferably a MOPS buffer. The content of the buffer solution is conventional in the art, preferably 30 to 75mmol/L, and more preferably 30 to 50 mmol/L.

In the second reagent of the present invention, the action of cholesterol esterase is not particularly limited, and any reagent can be used as long as it can hydrolyze HDL3 together with the surfactant. The content of the cholesterol esterase is 0.5 to 10KU/L, preferably 1.0 to 5.0 KU/L.

In the second reagent of the present invention, the content of the sodium azide is 0.1 to 0.3%, preferably 0.1%, and the percentage is mass percent.

In the present invention, preferably, the second reagent further comprises a stabilizer. The stabilizer is conventional in the art, and is preferably bovine serum albumin. The content of the stabilizer is the content conventional in the field, and preferably 0.2-5 g/L.

In the present invention, the active ingredient is selected from the group consisting of surfactants acting on HDL3, and includes Emulgen B66 and Emulgen A90 from Kao corporation, Tergitol NP-9 and Triton X-100 from Dow corporation, and Pluronic L123 from Pasteur corporation, for example. The surfactants can be used alone or in combination, and the concentration range is 0.2% -2.0%, preferably 0.5% -1.5%. The percentage is mass percentage.

Through the combined action of the first reagent and the second reagent, the technical scheme disclosed by the invention can effectively detect the HDL3 in the serum sample.

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

The invention also provides a preparation method of the second reagent, which comprises the following steps: adding 4-aminoantipyrine, a surfactant, bovine serum albumin, a buffer solution and sodium azide into water, stirring until the mixture is completely dissolved, adjusting the pH value to 6.50-7.50, and then adding peroxidase and cholesterol oxidase to obtain a second reagent.

In the present invention, preferably, one or more of the buffer, the stabilizer and the preservative are added before the pH is adjusted.

The third technical means of the present invention is to provide a method for measuring high-density lipoprotein 3, comprising the steps of:

(1) mixing and reacting a sample with the first reagent to obtain a reaction solution 1;

(2) mixing the reaction solution 1 obtained in the step (1) with the second reagent for reaction to obtain a reaction solution 2;

(3) and (3) reading the absorbance values of the reaction liquid 2 obtained in the step (2) at the wavelengths of 600nm and 800nm, and calculating the content of the high-density lipoprotein 3.

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.

Wherein the step (1) is as follows: mixing the sample with the first reagent for reaction to obtain a reaction solution 1. Wherein the mixing reaction time is the conventional condition in the field. The temperature of the mixing reaction is a temperature conventional in the art, preferably 37 ℃. The volume ratio of the sample to the first reagent is conventional in the art, and is preferably 1:75 to 1:120, and more preferably 1: 100.

The step (2) is as follows: and (2) mixing the reaction solution 1 obtained in the step (1) with the second reagent for reaction to obtain a reaction solution 2. Wherein the mixing reaction time is the conventional condition in the field. The temperature of the mixing reaction is a temperature conventional in the art. The volume ratio of the reaction solution 1 to the second reagent is a conventional volume ratio in the art, and is preferably 5:1 to 3:1, and more preferably 3: 1.

The step (3) is as follows: and (3) reading the absorbance values of the reaction liquid 2 obtained in the step (2) at the wavelengths of 546nm and 660nm, and calculating by using a standard liquid sample calibrated by the same parameters, so as to obtain the content of the high-density lipoprotein 3. Wherein the calculation method is a conventional calculation method in an automatic biochemical analyzer in the field, preferably, a spline method.

The step (1) is the first step; the step (2) is the second step;

preferably, the assay is one for non-diagnostic or therapeutic purposes.

The fourth technical scheme of the invention provides a kit for measuring high-density lipoprotein 3, which comprises the first 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 be used for efficiently and accurately detecting the high-density lipoprotein 3 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 positive progress effects of the invention are as follows: the measuring method of the invention can specifically and exclusively detect high-density lipoprotein 3(HDL3), has high reliability, low cost and high efficiency, and can automatically and specifically detect HDL3 in a large scale. The reagent for measuring the high-density lipoprotein 3 is simple and convenient to prepare, and can be efficiently and accurately applied to the method for specifically measuring the HDL 3. The kit is simple and convenient to operate.

Drawings

FIG. 1 is a graph showing the comparison between example 1 of the present invention and the test results obtained by the precipitation method.

FIG. 2 is a graph showing comparison between example 2 of the present invention and the test results obtained by the precipitation method.

FIG. 3 is a comparison of the results of the test conducted in example 3 of the present invention and the results of the test conducted by the masking method.

FIG. 4 is a comparison of the results of the test conducted in example 4 of the present invention and the results of the test conducted by the masking method.

FIG. 5 is a comparison of the results of the test conducted in example 5 of the present invention and the results of the test conducted by the masking method.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The buffers described in the examples were purchased from Beck Biotechnology, Inc. Suzhou, Trinders chromogen from Dong ren chemical technology (Shanghai), all enzymes from Asahi Kasei corporation, 4-aminoantipyrine from Sigma Aldrich, USA, and other reagents from Shanghai Chemicals.

Example 1

A first reagent:

adding MOPS buffer solution, magnesium sulfate, sodium chloride, EDTA, BSA, TOOS, polyethylene glycol 6000, Eumlgen LS-114 and dimethyl-beta-cyclodextrin into water, stirring until completely dissolving, adjusting pH to 6.50-7.50, then adding anti-apolipoprotein B antibody, cholesterol oxidase and catalase, and enabling the substances to reach the following concentrations:

35mM of a MOPS buffer solution,

15mM of magnesium sulfate,

0.20％Emulgen LS-114，

0.07% of dimethyl-beta-cyclodextrin,

2.5g/L polyethylene glycol 6000

3.0g/L of an anti-apolipoprotein B antibody,

3.5KU/L cholesterol oxidase,

1.0MU/L of a catalase,

the concentration of sodium chloride was 51mM,

0.02％Proclin-300

0.2mM EDTA, and 5.0g/L BSA,

the percentage is mass percentage.

A second reagent:

adding MOPS buffer solution, 4-aminoantipyrine, bovine serum albumin, sodium azide, Eumlgen B66 and Emulgen A90 into water, stirring until completely dissolving, adjusting pH to 6.50-7.50, adding cholesterol esterase and peroxidase into water, and mixing to obtain the following concentrations:

on a Hitachi 7180 full-automatic biochemical analyzer, 150 mu L of the first reagent reacts with 2.0 mu L of clinical human serum samples for 5 minutes, then 50 mu L of the second reagent is added, and a two-point end point method is adopted at the wavelength of main/auxiliary 600nm/800nm to read 14-34 points. And see Kostner GM, Molinari E, Pichler P.evaluation of a new HDL2/HDL3 quantification method based on precipitation with polyethylene glycol. Clin Chim Acta 1985; 146: 139-47.) control, as shown in FIG. 1, correlation coefficient R determined in example 1²0.9437, it was demonstrated that the high-density lipoprotein 3 can be efficiently detected by using the first reagent and the second reagent described in example 1.

Example 2

A first reagent:

adding MOPS buffer solution, magnesium sulfate, sodium chloride, EDTA, BSA, TOOS, dextran sulfate sodium salt 50, Emulgen 109P, alpha-cyclodextrin sulfate, anti-apolipoprotein B antibody and Proclin-300 into water, stirring until completely dissolving, adjusting pH to 6.50-7.50, then adding cholesterol oxidase and catalase, and enabling the substances to reach the following concentrations:

70mM of MOPS buffer solution is added,

20mM of magnesium sulfate, sodium sulfate,

0.05 percent of alpha-cyclodextrin sulfate,

0.30％Emulgen 109P，

3.0g/L dextran sulfate sodium salt 50

2.50g/L of an anti-apolipoprotein B antibody,

3.5KU/L cholesterol oxidase,

2MU/L of a catalase enzyme,

the concentration of 100mM of sodium chloride was determined,

0.2mM EDTA, and 5.0g/L BSA,

the percentage is mass percentage.

A second reagent:

MOPS buffer, 4-aminoantipyrine, BSA, sodium azide, TritonX-100 and Pluronic L23 were added to water, stirred until completely dissolved, the pH was adjusted to 6.50-7.50, then cholesterol esterase and peroxidase were added to the solution to dissolve and bring the following concentrations:

on a Hitachi 7180 full-automatic biochemical analyzer, 150 mu L of the first reagent reacts with 2.0 mu L of clinical human serum samples for 5 minutes, then 50 mu L of the second reagent is added, and a two-point end point method is adopted at the wavelength of main/auxiliary 600nm/800nm to read 14-34 points. And compared with precipitation, as shown in FIG. 2, the correlation coefficient R determined in example 2²0.957, indicating that the high-density lipoprotein 3 can be efficiently detected by using the first reagent and the second reagent described in example 2.

Example 3

A first reagent:

MOPS buffer, magnesium sulfate, sodium chloride, Proclin-300, EDTA, BSA, TOOS, dextran sulfate sodium salt, Pluronic F68, alpha-cyclodextrin sulfate, were added to water, stirred to complete dissolution, the pH was adjusted to 6.50-7.50, then cholesterol oxidase, anti-apolipoprotein B antibody, and catalase were added and the following concentrations were achieved:

80mM of MOPS buffer solution is added,

10.0mM of magnesium sulfate, in the form of magnesium sulfate,

2.55g/L dextran sulfate sodium salt,

0.5g/L alpha-cyclodextrin sulfate

1.0％Pluronic F68，

3.5g/L of an anti-apolipoprotein B antibody,

3.5KU/L cholesterol oxidase,

4MU/L of a catalase enzyme,

the concentration of sodium chloride was 51mM,

0.03％Proclin-300

0.2mM EDTA, and 5.0g/L BSA,

the percentage is mass percentage.

A second reagent:

MOPS buffer, 4-aminoantipyrine, BSA, sodium azide and Tergitol NP-9 were added to water, stirred until completely dissolved, pH adjusted to 6.50-7.50, then 2.0KU/L cholesterol esterase was added and peroxidase was mixed in water to achieve the following concentrations:

on a Hitachi 7180 full-automatic biochemical analyzer, 150 mu L of the first reagent reacts with 2.0 mu L of clinical human serum samples for 5 minutes, then 50 mu L of the second reagent is added, and a two-point end point method is adopted at the wavelength of main/auxiliary 600nm/800nm to read 16-34 points. And compared with the masking method, as shown in FIG. 3, the correlation coefficient R measured in example 3²0.9437, the first reagent and the second reagent described in example 3 were usedHigh-density lipoprotein 3 can be efficiently detected.

Example 4

A first reagent:

adding MOPS buffer solution, magnesium sulfate, sodium chloride, Proclin-300, EDTA, BSA, TOOS, Brij-L123, dextran sulfate sodium salt and dimethyl-beta-cyclodextrin into water, stirring until completely dissolved, adjusting pH to 6.50-7.50, adding anti-apolipoprotein B antibody, cholesterol oxidase and catalase, and allowing the above substances to reach the following concentrations:

30mM of MOPS buffer solution is added,

5.5mM of magnesium sulfate,

3.50g/L dextran sulfate sodium salt,

0.3g/L of dimethyl-beta-cyclodextrin,

0.35％Brij-L123，

2.2g/L of an anti-apolipoprotein B antibody,

3.5KU/L cholesterol oxidase,

3MU/L of a catalase enzyme,

the concentration of sodium chloride was 51mM,

0.03％Proclin-300

0.2mM EDTA, and 5.0g/L BSA,

the percentage is mass percentage.

A second reagent:

adding MOPS buffer solution, 4-aminoantipyrine, BSA, sodium azide, Emulgen B66 and Tergitol NP-9 into water, stirring until complete dissolution, adjusting pH to 6.50-7.50, adding cholesterol esterase and peroxidase into water, mixing, and allowing the substances to reach the following concentrations:

on a Hitachi 7180 full-automatic biochemical analyzer, 150 mu L of the first reagent reacts with 2.0 mu L of clinical human serum samples for 5 minutes, then 50 mu L of the second reagent is added, and a two-point end point method is adopted at the wavelength of main/auxiliary 600nm/800nm to read 16-34 points. And contrasted with the masking method, as shown in FIG. 4The correlation coefficient R measured in example 4²0.9422, it was demonstrated that the high-density lipoprotein 3 can be efficiently detected by using the first reagent and the second reagent described in example 4.

Example 5

A first reagent:

adding MOPS buffer solution, magnesium sulfate, sodium chloride, Proclin-300, EDTA, BSA, TOOS, dimethyl-beta-cyclodextrin, dextran sulfate sodium salt and Brij-S100 into water, stirring until completely dissolved, adjusting pH to 6.40-6.60, adding anti-apolipoprotein B antibody, cholesterol oxidase and catalase, and allowing the above substances to reach the following concentrations:

24mM of a MOPS buffer solution,

13.0mM of magnesium sulfate,

2.80g/L dextran sulfate sodium salt,

0.27g/L dimethyl-beta-cyclodextrin

0.20％Brij S100

3.0g/L of an anti-apolipoprotein B antibody,

3.5KU/L cholesterol oxidase,

4MU/L of a catalase enzyme,

the concentration of sodium chloride was 51mM,

0.03％Proclin-300

0.2mM EDTA, and 5.0g/L BSA,

the percentage is mass percentage.

A second reagent:

adding MOPS buffer solution, 4-aminoantipyrine, BSA, sodium azide, Emulgen B66 and Tergitol NP-9 into water, stirring until complete dissolution, adjusting pH to 6.50-7.50, adding cholesterol esterase and peroxidase into water, mixing, and allowing the substances to reach the following concentrations:

on Hitachi 7180 full-automatic biochemical analyzer, 150 μ L of the first reagent reacts with 2.0 μ L of clinical human serum sample for 5 min, and then 50 μ L of the second reagent is addedAnd reading 16-34 points of the reagent by adopting a two-point end point method at the main/auxiliary 600nm/800nm wavelength. And compared with the masking method, as shown in FIG. 5, the correlation coefficient R measured in example 5²0.9495, it was demonstrated that high-density lipoprotein 3 can be efficiently detected by using the first reagent and the second reagent described in example 5.

The above examples clearly demonstrate that HDL3 in serum samples can be accurately determined using the methods of the present invention. The description is only for the purpose of illustrating preferred embodiments of the present invention and is not to be construed as limiting the scope of the claims, and other substantially equivalent alternatives, which may be apparent to those skilled in the art, are intended to be within the scope of the present invention.