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Method and reagent for detecting small and dense low-density lipoprotein cholesterol in sample

A low-density lipoprotein, in-sample technology, which is applied in the field of measuring small and dense low-density lipoprotein cholesterol in samples, can solve the problems of many operation steps, unsuitable for routine detection, and low resolution.

Active Publication Date: 2017-12-08
深圳美康盛德医学检验实验室
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the ultracentrifugation method has accurate detection results, it requires special equipment and takes a long time to operate, so it is not suitable for routine clinical laboratory applications; equipment, and the resolution is not high, the test time is not short, so it is not suitable for routine testing

Method used

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  • Method and reagent for detecting small and dense low-density lipoprotein cholesterol in sample
  • Method and reagent for detecting small and dense low-density lipoprotein cholesterol in sample
  • Method and reagent for detecting small and dense low-density lipoprotein cholesterol in sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Reagents containing different ion selective agents have different reactivity to sdLDL-C and LLDL-C.

[0086] First, use ultracentrifugation to separate and purify sdLDL and LLDL. The specifics are as follows: Take the serum without turbidity and mix, add EDTA to a final concentration of 0.04%, and add NaN 3 The final concentration is 0.05%, and the density is adjusted to 1.019 with KBr. Centrifuge for 28h at 40,000 rpm and 20°C (centrifuge: Beckman Optima XE; rotor: Type 70Ti). After centrifugation, carefully discard the upper lipoprotein, including chyle. VLDL and IDL. The density of the lower layer was adjusted to 1.040 with KBr and centrifuged at 40,000 rpm and 20°C for 28 h. After centrifugation, the upper lipoprotein was carefully collected, that is, LLDL with a density of 1.019-1.040. Then adjust the density of the lower lipoprotein to 1.063 with KBr, and centrifuge at 40,000 rpm, 20°C for 28 hours. After centrifugation, carefully collect the upper lipoprotein, that ...

Embodiment 2

[0099] Through the above examples, it is found that as long as the conditions are suitable, a detection method for sdLDL-C can be established. Therefore, the following reagents are specially formulated for the detection of sdLDL-C.

[0100] R1

[0101]

[0102] R2

[0103] PIPES buffer (pH7.0) 50mM

[0104] Triton X-100 1.0%

[0105] TOOS 2mM.

[0106] Among them, the sample reagent ratio, sample: reagent R1: reagent R2 = 3:150:50. After incubating the sample with reagent R1 for 5 min, add R2, react for 5 min, and then measure the absorbance at 600 nm. Ion selective agents in R1 include ionic liquids and ionic polymers, wherein the cationic part of the ion selective agent is selected from the following types: imidazolium salt, pyridine, pyrrolidinium, quaternary phosphonium salt, ammonium base, sulfonium salt, anionic part It is selected from the following types: alkyl sulfate, tosylate, methanesulfonate, bis(trifluoromethylsulfonyl)imide, hexafluorophosphate, tetrafluoroborate, hali...

Embodiment 3

[0113] Influence of cholesterol esterase and cholesterol oxidase

[0114] It consists of the following R1 and R2 reagents, and uses the cholesterol oxidase (COO), cholesterol esterase (COE) and peroxidase (POD) in Table 3.

[0115] R1

[0116]

[0117] R2

[0118] PIPES buffer (pH7.0) 50mM

[0119] Triton X-100 1.0%

[0120] TOOS 2mM

[0121] table 3

[0122]

[0123]

[0124] It can be seen from Table 3 that as long as the content of cholesterol oxidase, cholesterol esterase, and peroxidase is sufficient, this method can effectively detect sdLDL-C. Therefore, cholesterol oxidase and cholesterol esterase can effectively detect sdLDL-C in the range of 0.1-50KU / L, preferably 1-5KU / L, while peroxidase is in the range of 0.1-10KU / L The sdLDL-C can be effectively detected within, preferably 1-5KU / L.

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Abstract

The invention discloses a method and reagent for detecting small and dense low-density lipoprotein cholesterol (sdLDL-C) in a sample. The method is characterized by comprising the steps of (1) eliminating cholesterol, except the sdLDL-C, in lipoproteins in the sample first in the presence of cholesterol esterase and an ion selective agent; and (2) conducting quantitative detection on remaining sdLDL-C obtained after treatment in step (1). The ion selective agent is used to selectively eliminate lipoproteins other than the sdLDL-C, so as to detect the sdLDL-C.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a method and reagent for measuring small and dense low-density lipoprotein cholesterol in a sample. Background technique [0002] As we all know, according to the classification of lipoproteins, lipoproteins can be divided into chylomicrons (CM), very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL) and high density lipoprotein ( HDL). Clinically, the cholesterol content of various lipoproteins is usually measured to guide diagnosis. Among them, low-density lipoprotein cholesterol is a risk factor for atherosclerosis and has been widely used in clinical testing. However, more and more studies have shown that low-density lipoproteins are heterogeneous. Some of the denser and smaller parts are called small and dense low-density lipoproteins (sdLDL), and the other part is less dense and larger. Some are called large and light low-densit...

Claims

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Application Information

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IPC IPC(8): C12Q1/60C12Q1/44C12Q1/26C12Q1/28
CPCC12Q1/26C12Q1/28C12Q1/44C12Q1/60C12Q2326/96
Inventor 邹炳德邹继华汪屹贾江花徐炜烽俞凤
Owner 深圳美康盛德医学检验实验室
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