CN107449748B

CN107449748B - High-density lipoprotein cholesterol detection kit and use method thereof

Info

Publication number: CN107449748B
Application number: CN201710681745.2A
Authority: CN
Inventors: 郑佳; 孙文勇; 常俊骏; 梁芬
Original assignee: Neusoft Whitman Biotech Nanjing Co ltd
Current assignee: Neusoft Whitman Biotech Nanjing Co ltd
Priority date: 2017-08-10
Filing date: 2017-08-10
Publication date: 2020-08-28
Anticipated expiration: 2037-08-10
Also published as: CN107449748A

Abstract

The invention discloses a high-density lipoprotein cholesterol detection kit and a using method thereof, and relates to the field of biochemical detection. The high-density lipoprotein cholesterol detection kit is high in stability, low in toxicity, high in accuracy and high in precision, and the using method thereof is provided. The kit of the invention comprises a reagent 1: a surfactant; a polyvinyl sulfate salt; sodium lauryl sulfate; a composite stabilizer; calcium chloride; cholesterol oxidase; cholesterol esterase; ascorbic acid oxidase; a catalase; 4-aminoantipyrine; proclin series preservatives; ethylene glycol; sucrose; the rest is buffer solution; reagent 2: a surfactant; a polyoxyethylene alkylene tribenzyl group; ethylene glycol; sucrose; peroxidase, and the like. The kit adopts various mixed surfactants including poloxamer series, polyvinyl sulfate, sodium dodecyl sulfate and the like, and has better specificity and selectivity compared with polyanion adopted by the traditional method.

Description

High-density lipoprotein cholesterol detection kit and use method thereof

Technical Field

The invention relates to the field of biochemical detection, in particular to a kit for determining a material by means of determining the chemical and physical properties of the material.

Background

There are many methods for measuring high density lipoprotein cholesterol, and most of the methods in the market at present are homogeneous phase measurement methods, including PEG modified enzyme method, immune separation method, selective inhibition method and scavenging method. Among these, the elimination method is currently considered to be the most ideal and effective measurement method, and includes two types, i.e., a reaction accelerator peroxidase elimination method (SPD method) and a catalase elimination method (CAT method).

The principle of SPD is based on the difference in affinity between lipoproteins and surfactants. In the reagent 1, Chylomicron (CM) in serum is reacted with a reaction accelerator) Very Low Density Lipoprotein (VLDL) and Low Density Lipoprotein (LDL) form a soluble complex, and free cholesterol on the surface layer is converted into H by cholesterol oxidase (CHOD)₂O₂And is finally eliminated by Peroxidase (POD). The reagent 2 contains special surfactant, and acts on High Density Lipoprotein (HDL) to cleave it, release cholesterol in HDL, react with enzyme reagent and develop color.

The CAT method does not form a soluble complex during the reaction, but reacts cholesterol in CM, VLDL and LDL with an enzyme reagent to produce H under the action of a specific surfactant₂O₂，H₂O₂Is decomposed into H by Catalase (CAT)₂O and O₂When the HDL particles are cleared, sodium azide is added into the reagent 2 to inhibit the activity of CAT, and another surfactant dissociates HDL particles to expose cholesterol, and the HDL particles react with the cholesterol enzyme reagent to develop color.

Disclosure of Invention

The invention aims to solve the technical problem of providing a high-density lipoprotein cholesterol detection kit which has high stability, low toxicity, no generation of precipitate and high accuracy and precision and a using method thereof.

The invention relates to a high density lipoprotein cholesterol detection kit, the said kit includes reagent 1; the reagent 1 comprises: cholesterol oxidase and cholesterol esterase.

Preferably, the kit further comprises reagent 2;

the reagent 1 comprises:

the balance of GOOD' S buffer MOPS (3- (N-morpholine) propanesulfonic acid) (MOPS is the main component of the buffer), and the pH value of the buffer is 7.0;

the reagent 2 comprises:

the rest is GOOD' S buffer MOPS, and the pH value of the buffer is 7.0.

Preferably, the composite stabilizer comprises Tween 8010-13 g/L, glutamic acid 20-30 g/L, glutathione 1-3 g/L, trehalose 50-60 g/L, dodecyl dimethyl betaine 2-6 g/L, lecithin 3-6 g/L and buffer solution 100-120 mmol/L; it is a composite stabilizer of multiple antibodies of application No. 201610166107.2 and a composite stabilizer in a method of use thereof.

Preferably, the reagent 1 comprises:

the balance of GOOD' S buffer MOPS, wherein the pH value of the buffer is 7.0;

the reagent 2 comprises:

the rest is GOOD' S buffer MOPS, and the pH value of the buffer is 7.0.

The detection method related to the detection kit is an SPD clearing method, and the aim of directly detecting HDL-C is fulfilled by utilizing the reaction affinity difference between a surfactant and lipoprotein. The method mainly comprises two steps of reactions: the first reaction step is as follows: uses surfactant, polyanion and divalent cation with special affinity to HDL-C to form soluble complex with Low Density Lipoprotein (LDL), Very Low Density Lipoprotein (VLDL) and Chylomicron (CM) in serum sample, and uses the action of CHOD and CAT to make surface free cholesterol be converted into H₂O and O₂Is cleared; and a second reaction step: the other surfactant interacts with HDL to cause its cleavage, and generates peroxidation under the action of CHOD and CHERHydrogen, and HDL to C are measured by a color reaction in the presence of Peroxidase (POD) and a color-developing agent.

The invention also relates to a using method of the high-density lipoprotein cholesterol detection kit, which comprises the following steps:

the detection method of the detection kit is a two-point end point method;

(1) uniformly mixing 300 parts of

reagent

1 and 3 parts of samples to be detected, and incubating at 37 ℃ to obtain a reaction solution 1;

(2) then 100 parts of reagent 2 is added and mixed evenly to obtain reaction liquid 2, and the absorbance A1 of the reaction liquid 2 is measured;

(3) measuring the absorbance A2 of the reaction solution 2 again after 5min, and measuring the main wavelength to be 600nm and the sub-wavelength to be 700 nm;

(4) measuring the absorbance of the high-density lipoprotein cholesterol standard solution by the operations of the steps (1) to (3) to respectively obtain the absorbance A3 and the absorbance A4, wherein the main wavelength is 600nm, and the auxiliary wavelength is 700 nm;

(5) calculating the absorbance difference, and calculating the concentration of the high-density lipoprotein cholesterol according to formula 1;

formula 1:

high density lipoprotein cholesterol concentration ═ Δ a sample/Δ a standard solution × standard solution concentration;

Δ a sample ═ a2-a 1;

the standard solution of delta A is A4-A3;

the above parts are parts by volume.

The using method of the high-density lipoprotein cholesterol detection kit of the invention is different from the prior art in that:

1. the high-density lipoprotein cholesterol detection kit adopts various mixed surfactants, including poloxamer series, polyvinyl sulfate, sodium dodecyl sulfate and the like, and has better specificity and selectivity compared with polyanion adopted by the traditional method, and a sample can be kept clear in the detection process, thereby avoiding turbidity interference and having no risk of blocking the pipeline of an analysis instrument.

2. The GOOD' S buffer MOPS in the high-density lipoprotein cholesterol detection kit is a zwitterionic buffer, and has the main advantages of not participating and interfering in biochemical reaction processes and having no inhibiting effect on enzymatic reaction and the like. The ethylene glycol, the sucrose and each component in the composite stabilizer added into the reagent can play a role in keeping the stability of the enzyme in the reagent. The added protease inhibitor sodium azide can effectively inhibit the activity of catalase, and the added proclin preservative is a novel biological preservative, has good compatibility with various enzymes, and has good stability and low toxicity.

3. High density lipoprotein cholesterol consists of apolipoproteins, phospholipids, cholesterol and small amounts of fatty acids. The cholesterol oxidase (CHOD) of the present invention is combined with catalase in the first step of the reaction to react free cholesterol and finally produce H₂O and O₂In the process, CHOD plays a role in clearing, provides a premise for the next reaction of high density cholesterol (HDL-C), removes the interference of non-HDL-C, further improves the accuracy of a determination result, and is more critical to the next reaction; the second step of the reaction, the chromogenic reaction, is the hydrogen peroxide (H) required for this reaction₂O₂) Needs to be obtained by the reaction of HDL-C under the combined action of CHOD and CHER. In conclusion, cholesterol oxidase and cholesterol esterase play a crucial role in the reaction process.

Drawings

FIG. 1 is a graph showing the linear correlation between the high density lipoprotein cholesterol assay kit of the present invention and a control kit;

FIG. 2 is a graph showing the linear correlation between the control kit and the high density lipoprotein cholesterol assay kit of the present invention;

FIG. 3 is a test OD value scattergram of a control kit;

FIG. 4 is a test OD value scattergram of the high density lipoprotein cholesterol assay kit of the present invention.

Detailed Description

The use of the high density lipoprotein cholesterol assay kit of the present invention and its method of use are further illustrated by the following examples and confirmatory tests.

Example 1

The high-density lipoprotein cholesterol detection kit of the embodiment comprises the following raw materials in proportion:

the kit consists of a reagent 1 and a reagent 2;

reagent 1 was as follows:

the balance of GOOD' S buffer MOPS, wherein the pH value of the buffer is 7.0;

reagent 2 was as follows:

the rest is GOOD' S buffer MOPS, and the pH value of the buffer is 7.0.

Example 2

the kit consists of a reagent 1 and a reagent 2;

reagent 1 was as follows:

the balance of GOOD' S buffer MOPS, wherein the pH value of the buffer is 7.0;

reagent 2 was as follows:

the rest is GOOD' S buffer MOPS, and the pH value of the buffer is 7.0.

Example 3

the kit consists of a reagent 1 and a reagent 2;

reagent 1 was as follows:

the balance of GOOD' S buffer MOPS, wherein the pH value of the buffer is 7.0;

reagent 2 was as follows:

the rest is GOOD' S buffer MOPS, and the pH value of the buffer is 7.0.

Example 4

The detection of high density lipoprotein cholesterol in samples using the products prepared in examples 1-3 was carried out as follows:

(1) uniformly mixing 300 parts of

reagent

formula 1:

Δ a sample ═ a2-a 1;

the standard solution of delta A is A4-A3;

the above parts are parts by volume.

Verification test

The products of examples 1-3 were tested and compared with other companies of the same type, and the results are shown in Table 1.

TABLE 1 comparison of the parameters of the kit of the invention and the control kit

	Linear Range (mmol/L)	Accuracy of	Analytical sensitivity (mmol/L)
				Control kit	0.05～3.88	≦±10％	2.58
The kit of the invention	0～4	≦±10％	0.9

The contrast kit is a domestic commercial HDL-C detection kit, and comprises the following components in parts by weight: the reagent 1 comprises PIPES buffer solution, TOOS, ascorbic acid oxidase and Tween 80; the reagent 2 comprises PIPES buffer solution, 4-AAP, CHOD, CHER and POD.

Test 1

Preparing standard samples with different concentrations of high-density lipoprotein cholesterol, wherein the concentrations are respectively 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0mmol/L, and respectively measuring the concentrations by using the kit and the control kit. The results are shown in Table 2.

TABLE 2 Linear Range comparison

The results show that: when the sample concentration is not less than 3.0mmol/L, the control kit cannot be detected due to the small linear range, so that the linear range of the kit is wider.

Test 2

The control kit and the kit of the invention are used as materials, Landau calibrators are used for calibration, and Roche high-value and Roche low-value quality control is used for measurement, and the results are shown in Table 3.

TABLE 3 comparison of accuracy

The results show that: the test result of the kit is closer to the target value, and the accuracy is higher than that of a control kit.

Test 3

Internal precision: the measurement was repeated 10 times (n is 10) using high and low roche quality control samples, respectively, and the coefficient of variation (CV%) value was calculated. The details are shown in Table 4.

TABLE 4 comparison of in-batch precision

From the results of the above table it can be seen that: the kit of the invention has higher batch precision than a control kit.

Test 4

The results of the measurement by using a Beckmann AU480 biochemical analyzer, the kit and the control kit as materials, the calibration by using Landau calibrators and the measurement of the HDL-C content of 40 clinical samples show that the test results of the kit have good correlation with the results obtained by using the control reagent, and are shown in Table 5.

TABLE 5 Linear correlation comparison

While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that these are by way of example only, and that the scope of the invention is defined by the appended claims. Various changes and modifications to these embodiments may be made by those skilled in the art without departing from the spirit and scope of the invention, and these changes and modifications are within the scope of the invention.

Claims

1. A high density lipoprotein cholesterol detect reagent box which characterized in that: the kit comprises a reagent 1, wherein the reagent 1 comprises: cholesterol oxidase and cholesterol esterase, and the kit also comprises a reagent 2;

the reagent 1 comprises:

1-3 g/L of surfactant poloxamer;

1-10 g/L of polyethylene sulfate;

1-5 g/L of sodium dodecyl sulfate;

1wt% of composite stabilizer;

30-50 g/L of calcium chloride;

5-10 KU/L of cholesterol oxidase;

cholesterol esterase 5-8 KU/L;

ascorbic acid oxidase 3-10 KU/L;

catalase 1-3 KU/L;

1-5 g/L of 4-aminoantipyrine;

0.01-5 g/L proclin series preservative;

15-25 ml/L of ethylene glycol;

10-30 g/L of sucrose;

the balance is GOOD 'S buffer solution, and the pH value of the GOOD' S buffer solution is 7.0;

the reagent 2 comprises:

the surfactant fatty alcohol-polyoxyethylene ether is 2-5 g/L in series;

1-8 g/L of polyoxyethylene alkylene tribenzyl;

15-25 ml/L of ethylene glycol;

10-30 g/L of sucrose;

1-4 KU/L of peroxidase;

1-5 g/L of color developing agent TOPS;

0.01-5 g/L of sodium azide;

the rest is GOOD 'S buffer solution, and the pH value of the GOOD' S buffer solution is 7.0.

2. The high-density lipoprotein cholesterol detection kit according to claim 1, characterized in that: the composite stabilizer comprises Tween 8010-13 g/L, glutamic acid 20-30 g/L, glutathione 1-3 g/L, trehalose 50-60 g/L, dodecyl dimethyl betaine 2-6 g/L, lecithin 3-6 g/L and buffer solution 100-120 mmol/L.

3. The high-density lipoprotein cholesterol detection kit according to claim 1, characterized in that:

the reagent 1 comprises:

the surfactant poloxamer is 2 g/L;

5.5g/L of polyethylene sulfate;

3g/L of sodium dodecyl sulfate;

1wt% of composite stabilizer;

40g/L of calcium chloride;

cholesterol oxidase 7.5 KU/L;

cholesterol esterase 6.5 KU/L;

ascorbic acid oxidase 6.5 KU/L;

catalase 2 KU/L;

3g/L of 4-aminoantipyrine;

proclin series preservative 2.5 g/L;

20ml/L of ethylene glycol;

25g/L of sucrose;

the reagent 2 comprises:

the surfactant fatty alcohol-polyoxyethylene ether series is 3.5 g/L;

4g/L of polyoxyethylene alkylene tribenzyl;

20ml/L of ethylene glycol;

20 g/L of sucrose;

peroxidase 2.5 KU/L;

3g/L of color developing agent TOPS;

2.5g/L of sodium azide;

4. The high-density lipoprotein cholesterol detection kit according to claim 1, characterized in that: the using method comprises the following steps:

(1) uniformly mixing 300 parts of reagent 1 and 3 parts of samples to be detected, and incubating at 37 ℃ to obtain a reaction solution 1;

formula 1:

high density lipoprotein cholesterol concentration =Δa sample/Δ a standard solution × standard solution concentration;

Δ a sample = a2-a 1;

Δ a standard = a 4-A3;

the above parts are parts by volume.