CN104673880A - Method for detecting small and dense low-density lipoprotein cholesterin - Google Patents

Abstract

The invention provides a method for detecting small and dense low-density lipoprotein cholesterin (sdLDL-C). According to the method, double liquid reagents, including a reagent R1 and a reagent R2, are adopted; the reagent R1 comprises an MOPS buffer liquid (the pH value is 7.0), cholesterol esterase, cholesterol oxidase, phospholipase, catalase, polyoxyethylene alkyl phenyl ether (JK-14) as a surfactant A, polyoxyethylene benzyl styrene ether (A3PK) as a protecting agent, and N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-methylaniline sodium salt (TOOS); the reagent R2 comprises an MOPS buffer liquid (the pH value is 7.0), peroxidase, polyethylene glycol octylphenol ether (Triton X-100) as a surfactant, 4-amino antipyrine (4-AA), and sodium azide. The method provided by the invention is high in sensitivity, high in specificity, simple in reagent preparation, free of sample pretreatment, and worthy of popularization and application.

Description

A kind of sdLDL-C detects method for testing

Technical field:

The invention provides a kind of sdLDL-C (sdLDL-C) detection method, described detection method reagent used is liquid double reagent, comprise reagent R1 and R2, described reagent R1 is containing MOPS damping fluid (PH7.0) 20 ~ 80mmol/L, Sterol esterase 1400 ~ 1800U/L, rCO 2300 ~ 2600U/L, Phospholipid hydrolase 2500 ~ 3000U/L, catalase 1000 ~ 1300U/L, as polyalkylene glycol alkyl phenyl ether (JK-14) 1.0 ~ 3.0g/L of surfactant A, as protectant polyoxyethylene benzylstyrene ether (A3PK) 0.1 ~ 0.5g/L, N-ethyl-N-TOOS (TOOS) 0.3 ~ 0.7mmol/L, reagent R2 is containing MOPS damping fluid (PH7.0) 20 ~ 80mmol/L, peroxidase 2000 ~ 5000U/L, as Triton X-100 (Triton X-100) 5 ~ 18g/L of surfactant B, 4-AA (4-AA) 25 ~ 60mmol/L, sodium azide 0.05%.The inventive method is highly sensitive, high specificity, and preparation of reagents is simple, without the need to pre-treatment sample, is worth further genralrlization to use.

Background technology:

Low-density lipoprotein (LDL) has heterogeneity, the granulometric composition different by a series of size, density and chemical constitution.LDL can be divided into 3 kinds or more subfractions by application non denatured gradient gel electrophoresis or density gradient ultracentrifuge method, and its title is also different because of experimental technique.The particle of some LDL less (diameter < 26nm) in LDL subfraction, density comparatively large (1.04 ~ 1.06kg/L), be called small dense low-density lipoprotein (small dense Low-Density Lipoprotein, sdLDL), also known as Type B LDL; The particle comparatively large (diameter > 27nm) of part LDL, density less (1.02 ~ 1.03kg/L), is called light low-density lipoprotein (large, buoyant LDL, lbLDL) greatly, also known as A type LDL; Marginal subfraction claims osculant low-density lipoprotein.Recent research finds that sdLDL is compared with common LDL, its atherogenicity ability is stronger, with coronary heart disease (CHD) close relation, listed in one of newfound important cardiovascular risk factors by U.S.'s Cholesterol Education Program (National Cholesterol Education Program, NCEP) council's adult treatment group.

SdLDL-C measures by multiple methods such as Graded Density ultracentrifugation, gradient gel electrophoresis, chemical precipitation method, molecular sieve chromatography analytical method, capillary electrophoresis, nuclear magnetic resonance spectrometry, index evaluation method, dynamic light scattering method, homogeneous method.Graded Density ultracentrifugation, molecular sieve chromatography analytical method, capillary electrophoresis, dynamic light scattering method complex operation, require higher to laboratory condition and experiment operator, gradient gel electrophoresis is consuming time, poor specificity, chemical precipitation method measured value out of true, nuclear magnetic resonance spectrometry needs to be equipped with valuable experimental installation, and index evaluation method can only guestimate, can not its concentration of Accurate Determining.The maximum sdLDL-C detection method of current employing is homogeneous method.Special tensio-active agent is used optionally to be dissociated by non-LDL, its cholesteryl ester of discharging of dissociating is divided by Sterol esterase and rCO to be taken off, it is made not participate in color reaction, the lbLDL that phospholipid is rich on surface is decomposed by Phospholipid hydrolase, remaining sdLDL just can carry out chromogenic assay sdLDL-C by participating in complete Trinder reaction, has quick, special advantage.

Summary of the invention:

The object of the invention is to, collocation uses several special tensio-active agent, protective material and enzyme, carries out the preparation of sdLDL-C detection kit.

Technical scheme of the present invention: use a kind of special surfactant A and protective material; and Phospholipid hydrolase; optionally high-density lipoprotein (HDL) (HDL), vldl (VLDL) and chylomicron (CM) are dissociated; Phospholipid hydrolase decomposes the lbLDL that phospholipid is rich on surface; protective material protection sdLDL component not with Phospholipid hydrolase, Sterol esterase and cholesterol oxidation enzyme reaction, make that sdLDL is complete to be remained.The cholesteryl ester discharged after HDL, VLDL, CM are dissociated is divided by Sterol esterase and rCO to be taken off, and make it not participate in color reaction, lbLDL is decomposed by Phospholipid hydrolase.Remaining sdLDL, by surfactant B de-preservation, participates in complete Trinder reaction and carries out chromogenic assay sdLDL-C.

The Cleaning Principle of this detection method:

The preparation of sdLDL-C (sdLDL-C) detection reagent:

Comprise reagent R1 and R2, concrete composition following (following reagent place medicine and enzyme are commercially available):

The use of sdLDL-C (sdLDL-C) detection reagent:

1) detecting instrument: the Biochemical Analyzer with 546nm wavelength, 37 DEG C of thermostats.

2) sample to be tested: gather on an empty stomach, fresh not haemolysis serum, turbid without fat, 2-8 DEG C of Absorbable organic halogens seven days.

3) concrete trace routine:

4) calculation result: sdLDL-C (mmol/L)=Δ AT/ Δ AS × Cs

Δ AT: sample hose absorbancy

Δ AS: calibration tube absorbancy

Cs: calibration object concentration value

5) reference range: 0.07 ~ 0.59mmol/L.

6) precision: CV≤6% in batch; Relative extreme difference≤8% between batch.

7) accuracy: relative deviation controls in ± 10%.

8) linearity range: within the scope of 0.05 ~ 8mmol/L, correlation coefficient r >=0.990.

Accompanying drawing illustrates:

Fig. 1: adopt Olympus 400 automatic clinical chemistry analyzer, the reagent of 5 gradient concentrations is measured, and correlation analysis is carried out to measured value.What wherein X-axis represented is weaker concn, and what Y-axis represented is measured value average.Relation conefficient: r ²=0.9952, linear equation is: y=0.9445x+0.103.

Fig. 2: adopt reagent of the present invention and commercial reagent A respectively, adopts Olympus 400 automatic clinical chemistry analyzer, to 50 increments this (comprising normal and exceptional sample), measures, and carry out correlation analysis to measured value by each autoregressive parameter.The measured value of what wherein X-axis represented is reagent of the present invention, the measured value of what Y-axis represented is commercial reagent A.Relation conefficient: r ²=0.9930, linear equation is: y=0.99x+0.008.

Embodiment:

The preparation (1) of embodiment 1 sdLDL-C (sdLDL-C) detection reagent

Concrete composition following (following reagent place medicine and enzyme are commercially available):

1) precision measures: in same sample, continuous drawing measures for 20 times, calculates the mean of measured value, standard deviation and the variation coefficient,

$\mathrm{CV}=\frac{\mathrm{SD}}{\stackrel{\&OverBar;}{X}}\&times;100\%$

Table 1 precision detected result

Variation coefficient CV is generally used for the precision of a measurement measuring method, and CV value is less, represents that the result precision of this measuring method is better.For clinical chemistry test project, CV be less than 5% method precision generally acknowledge be acceptable.In table 1, CV value is less than 3%, shows that the inventive method has excellent precision.

2) accuracy determination: use same quality control product, replication 3 times, averages, should in ± 10% scope with quality control product target value relative deviation.

Table 2 accuracy detected result

Relative deviation CB=4.00% in table 2, be in ± 10% scope in, show that the inventive method has excellent accuracy.

3) linear determination: use deionized water reagent dilutions to be become 5 gradient concentrations, each gradient concentration detects 3 times, averages, and does regression analysis to measured value and desired value, calculates r value and relative deviation (the results are shown in Figure 1, unit mmol/L).

Table 3 linear correlation detection result

Relation conefficient is obtained: r by table 3 ²=0.9952, linear equation is: y=0.9445x+0.103, and result shows that this reagent dependency is good.

The preparation (2) of embodiment 2 sdLDL-C (sdLDL-C) detection reagent

Concrete composition following (following reagent place medicine and enzyme are commercially available):

Result shows precision, accuracy, the difference (data slightly) within the scope of nominal error between the data of reagent described in linear dependence and sensitivity for analysis and example 1.

The linear dependence of embodiment 3 reagent of the present invention and commercial reagent A measures:

Concrete composition following (following reagent place medicine and enzyme are commercially available):

Adopt reagent of the present invention and commercial reagent A respectively, adopt Olympus 400 automatic clinical chemistry analyzer, to 50 increments this (comprising normal and exceptional sample), measure by each autoregressive parameter, and correlation analysis is carried out to measured value (the results are shown in Figure 2, what X-axis represented is the measured value that the present invention tries, the measured value of what Y-axis represented is commercial reagent A).Relation conefficient: r ²=0.9930, linear equation is: y=0.99x+0.008, and result shows that this reagent and commercial reagent dependency are good.

Table 4 linear correlation detection result

Claims (12)

1. the invention provides a kind of sdLDL-C (sdLDL-C) detection method, described detection method reagent used is liquid double reagent, comprise reagent R1 and R2, described reagent R1 is containing MOPS damping fluid (PH7.0), Sterol esterase, rCO, Phospholipid hydrolase, catalase, as the polyalkylene glycol alkyl phenyl ether (JK-14) of surfactant A, as protectant polyoxyethylene benzylstyrene ether (A3PK), N-ethyl-N-TOOS (TOOS); Reagent R2 contains MOPS damping fluid (PH7.0), peroxidase, as the Triton X-100 (TritonX-100) of surfactant B, and 4-AA (4-AA), sodium azide.

2., according to claim 1 in reagent R1, the concentration range of MOPS damping fluid (PH7.0) is 20 ~ 80mmol/L.

3., according to claim 1 in reagent R1, the concentration range of the concentration range of Sterol esterase to be the concentration range of 1400 ~ 1800U/L rCO be 2300 ~ 2600U/L Phospholipid hydrolase is the catalatic concentration range of 2500 ~ 3000U/L is 1000 ~ 1300U/L.

4. according to claim 1 in reagent R1, surfactant A: the concentration range of polyalkylene glycol alkyl phenyl ether (JK-14) is 1.0 ~ 3.0g/L.

5. according to claim 1 in reagent R1, protective material: the concentration range of polyoxyethylene benzylstyrene ether (A3PK) is 0.1 ~ 0.5g/L.

6., according to claim 1 in reagent R1, the concentration range of N-ethyl-N-TOOS (TOOS) is 0.3 ~ 0.7mmol/L.

7., according to claim 1 in reagent R2, the concentration range of MOPS damping fluid (PH7.0) is 20 ~ 80mmol/L.

8., according to claim 1 in reagent R2, the concentration range of peroxidase is 2000 ~ 5000U/L.

9. according to claim 1 in reagent R2, surfactant B: the concentration range of Triton X-100 (Triton X-100) is 5 ~ 18g/L.

10., according to claim 1 in reagent R2, the concentration range of 4-AA (4-AA) is 25 ~ 60mmol/L.

11. according to claim 1 in reagent R2, and the concentration range of sodium azide is 0.05%.

12. according to claim 1, it is characterized in that the calculation formula of sdLDL-C in sample is:

sdLDL-C(mmol/L)＝ΔAT/ΔAS×Cs

Δ AT: sample hose absorbancy

Δ AS: calibration tube absorbancy

Cs: calibration object concentration value.