CN104865392B - A kind of LDL-C quantitative detecting method - Google Patents
A kind of LDL-C quantitative detecting method Download PDFInfo
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- CN104865392B CN104865392B CN201510227852.9A CN201510227852A CN104865392B CN 104865392 B CN104865392 B CN 104865392B CN 201510227852 A CN201510227852 A CN 201510227852A CN 104865392 B CN104865392 B CN 104865392B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- G—PHYSICS
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- G01—MEASURING; TESTING
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/323—Arteriosclerosis, Stenosis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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Abstract
The invention discloses a kind of by combining the detection method covering standard measure mensuration LDL C.This method uses liquid double reagent, and reagent 1 and reagent 2 by placing respectively form, and wherein, described reagent 1 is containing metal chloride, LDL C monoclonal antibody and quantitative enzyme reagent;Described reagent 2 is containing surfactant, developer, preservative, anti-interference material and reaction promoter.The present invention has that development properties is good, accuracy is high, agent formulations is stable, the feature being applicable to clinical various automatic clinical chemistry analyzer simple to operate.For detecting the content of LDL C in human serum.
Description
Technical field:
The invention discloses a kind of by combining the detection method covering standard measure mensuration LDL-C, belong to biotechnology neck
Territory.
Background technology:
Low density lipoprotein, LDL (LDL) be people blood in the most lipoprotein of content, the cholesterol (LDL-C) that it is entrained
Being considered as the main pathogenic of atherosclerosis and coronary heart disease, Clinical detection becomes more and more important.
Still there is no the reference method of the mensuration LDL-C of real meaning at present.CDC measures reference method tentative for LDL-C
Supercentrifugation (Betaquantification, β-quantitative method/BQ method), is also recommended by NCEP.The LDL-C that this method measures,
Actually include lipoprotein (a) [Lp (a)] and the cholesterol level of intermediated-density lipoprotein (IDL), be also to evaluate other detections
The basis of method correctness.This method needs the equipment of costliness, handles complicated, time-consuming and technology requirement height, is difficult in common lab
Carry out.Friedewald computing method of formula is the method for the estimation LDL-C that application is wider at present, is recommended as conventional determining by NCEP
Method.It is assumed to be condition with what VLDL formed constant (VLDL-C/TG=0.2, all in terms of mg/dl), have simplicity, directly,
The advantage such as quickly.Apply this formula calculating mountain LDL-C often to be made a variation by TC, TG and HDL-C to be affected, always make a variation up to 9.5%.But
[type III height fat when there is abnormal beta Lipoprotein in CM, serum TG > 4.52mmol/L (400mg/dl), serum is there is in serum
Mass formed by blood stasis (HLP)] time Friedewald equation should not be used to calculate.Chromatography and electrophoresis method are because of instrument, the high kind of Control requirements
Plant the most less application of reason clinical routine laboratory, be used for the research of lipoprotein.
Sample consumption is few, be not required to make centrifugation separating treatment, tool precision is high relevant to F equation because of it for even phase method
Property is good, can realize automatization and be attracted attention by clinical experiment.The key problem in technology of even phase method popularization and application is grinding of its detectable
Sending out, method is a lot, but its principle is similar, makes LDL-C determined in chromogenic reaction in reaction system selectively, but even phase
Method is incomplete owing to technical operation difficulty reason may cause shielding LDL-C, directly affects the accuracy of measurement result.Even phase method
Measuring LDL-C external (such as Japan, the U.S.) starting relatively early, technology is the most ripe, wherein with the commodity of surfactant method composition
Test kit is with Japanese first KCC, Britain's bright morals product as representative.Current domestic clinical laboratory uses even phase
The reagent that method measures LDL-C is many from Japan's import, the reagent that such as Japan one change and light are produced, or domestic employing foreign technology is raw
The reagent produced, such as Li get Man, the product of claim company, its price is higher.
Summary of the invention:
It is an object of the invention to, overcome the deficiency of above-mentioned technical background, it is provided that a kind of accuracy is high, reproducible, anti-
Interference performance is strong, substrate is stable, can be used for the features such as automatic clinical chemistry analyzer reach international standards for detecting LDL-C
Quantitative assay.Using liquid double reagent, reagent 1 and reagent 2 by placing respectively form, and wherein, described reagent 1 mainly contains
There are metal chloride, LDL-C monoclonal antibody, surfactant 1, cholesteryl esterase;Described reagent 2 mainly contains surface activity
Agent 2,4-AA, peroxidase, cholesterol oxidase.
Technical scheme: in reagent 1, polyanion calcium chloride and LDL-C monoclonal antibody act on down jointly, make
In serum, free LDL-C produces coagulation effect and occurs antigen antibody reaction to form antigen antibody complex, reaches to combine and covers
The effect of LDL-C.In serum, HDL, VLDL and CM are then dissociated also under surfactant octyl phenyl polyoxyethylene ether effect
The micronized Chol molecule disengaged and cholesteryl esterase reagent produce incomplete TRINDER and react.When adding reagent 2,
Containing having the surfactant polyethylene 8000 of specific action can hydrolyze LDL LDL, discharge micronized Chol molecule, ginseng
React with complete TRINDER, the concentration of LDL-C in serum can be drawn at automatic clinical chemistry analyzer.
The present invention to be embodied as basic operation as follows:
1. the preparation of reagent 1:
In beaker, add 500ml-700ml purified water, add MOPSO8-10g, stir 3-5 minute under room temperature, add
MOPSO sodium salt 9-11g, stirs 3-5 minute under room temperature, adds sodium cholate 15-25g, stirs 3-5 minute under room temperature, adds chlorination
Calcium 0.4-0.5g, stirs 2-3 minute under room temperature, adds disodium EDTA 0.1-0.2g, stirs 2-3 and divide under room temperature
Clock, adds octyl phenyl polyoxyethylene ether 2-4ml, stirs 2-3 minute under room temperature, adds cholesteryl esterase 10-14KU, under room temperature
Stir 2-3 minute, add dextran sulfate 8-12g, stir 3-5 minute under room temperature, add apolipoprotein B monoclonal antibody 50-
150ml, stirs 3-5 minute under room temperature, is eventually adding N-ethyl m-toluidine-second hydroxypropyl azochlorosulfonate acid sodium 0.1-0.3g, room temperature
Lower stirring 2-3 minute, remaining supplies 1000ml by purified water.
2. the preparation of reagent 2:
In beaker, add 500ml-700ml purified water, add MOPSO8-10g, stir 3-5 minute under room temperature, add
MOPSO sodium salt 9-11g, stirs 3-5 minute under room temperature, adds PEG 8000 40-60g, stirs 3-5 minute, add under room temperature
Enter 4-AA 0.05-0.2g, stir 2-3 minute under room temperature, add peroxidase 1-2KU, under room temperature, stir 2-3
Minute, add cholesterol oxidase 0.4-0.8KU, stir 2-3 minute under room temperature, add sodium azide 0.01-0.02g, under room temperature
Stirring 2-3 minute, remaining supplies 1000ml by purified water.
3. utilize the use of the immue quantitative detection reagent box that the inventive method prepares
(1) detecting instrument: be applicable to all kinds of automatic clinical chemistry analyzer.
(2) test serum: not haemolysis serum, can stablize one week for 2-8 DEG C, it is to avoid fat is turbid.
(3) concrete detection program such as table 1 below:
Table 1 pattern detection operation sequence
Result of calculation: the LDL-C content (mmol/L) in sample=Δ AT/ Δ AS × calibration solution concentration
Reference range: in, old people the most about 2.7-3.1mmol/L
Detailed description of the invention:
Embodiment 1
1. the preparation of reagent 1:
2. the preparation of reagent 2:
Table 2 be this example 1 preparation LDL-C detection kit under conditions of external condition is identical, complete in Hitachi 7180
The result data of METHOD FOR CONTINUOUS DETERMINATION standard definite value serum 10 times on automatic biochemistry analyzer.
Table 2 accuracy determination result table
Measure number of times | Result (mmol/L) |
1 | 2.91 |
2 | 2.89 |
3 | 3.00 |
4 | 2.98 |
5 | 2.85 |
6 | 2.92 |
7 | 2.78 |
8 | 2.88 |
9 | 2.94 |
10 | 2.88 |
Average | 2.90 |
As known from Table 2, the LDL-C detection kit accuracy of this example 1 preparation is: 0.69%
Table 3 be this example 1 preparation LDL-C detection kit under conditions of external condition is identical, complete in Hitachi 7180
The result data of METHOD FOR CONTINUOUS DETERMINATION quality controlled serum 20 times on automatic biochemistry analyzer.
Table 3 precision measurement result table
Measure number of times | Result (mmol/L) |
1 | 2.51 |
2 | 2.50 |
3 | 2.50 |
4 | 2.49 |
5 | 2.48 |
6 | 2.51 |
7 | 2.48 |
8 | 2.47 |
9 | 2.48 |
10 | 2.48 |
11 | 2.49 |
12 | 2.47 |
13 | 2.50 |
14 | 2.53 |
15 | 2.51 |
16 | 2.52 |
17 | 2.52 |
18 | 2.51 |
19 | 2.49 |
20 | 2.53 |
Average | 2.49 |
SD | 0.0182 |
CV | 0.73% |
As known from Table 3, the LDL-C detection kit precision of this example 1 preparation is: 0.73%
Table 4 is that the LDL-C detection kit measured value of this example 1 preparation changes LDL-C detection kit mensuration with Japan one
The synopsis of value.
Table 4 measured value synopsis
Catalogue number(Cat.No.) | This example 1 measured value (mmol/L) | Japan one changes LDL-C detection kit measured value (mmol/L) |
1 | 3.02 | 3.10 |
2 | 2.70 | 2.73 |
3 | 2.89 | 2.98 |
4 | 2.89 | 2.97 |
5 | 2.77 | 2.82 |
6 | 2.48 | 2.53 |
7 | 2.82 | 2.90 |
8 | 2.70 | 2.77 |
9 | 2.52 | 2.55 |
10 | 2.68 | 2.75 |
11 | 2.44 | 2.47 |
12 | 2.56 | 2.63 |
13 | 2.71 | 2.75 |
14 | 2.81 | 2.87 |
15 | 2.82 | 2.88 |
16 | 2.46 | 2.51 |
17 | 2.57 | 2.62 |
18 | 2.85 | 2.88 |
19 | 2.72 | 2.77 |
20 | 2.54 | 2.56 |
21 | 2.46 | 2.46 |
22 | 2.69 | 2.77 |
23 | 2.81 | 2.90 |
24 | 2.34 | 2.34 |
25 | 2.67 | 2.70 |
26 | 2.49 | 2.51 |
27 | 2.89 | 2.93 |
28 | 2.35 | 2.39 |
29 | 2.51 | 2.57 |
30 | 2.51 | 2.55 |
31 | 2.72 | 2.77 |
32 | 2.17 | 2.19 |
33 | 2.39 | 2.43 |
34 | 2.33 | 2.36 |
35 | 2.23 | 2.23 |
36 | 2.42 | 2.43 |
37 | 2.60 | 2.62 |
38 | 2.39 | 2.44 |
39 | 2.75 | 2.79 |
40 | 2.70 | 2.71 |
As known from Table 4, the LDL-C detection kit measured value of this example 1 preparation changes LDL-C detection kit with Japan one
The coefficient R of measured value2Being 0.992, both show fabulous dependency.
Embodiment 2
1. the preparation of reagent 1:
2. the preparation of reagent 2:
Table 2 be this example 2 preparation LDL-C detection kit under conditions of external condition is identical, complete in Hitachi 7180
The result data of METHOD FOR CONTINUOUS DETERMINATION standard definite value serum 10 times on automatic biochemistry analyzer.
Table 2 accuracy determination result table
Measure number of times | Result (mmol/L) |
1 | 2.58 |
2 | 2.64 |
3 | 2.56 |
4 | 2.57 |
5 | 2.62 |
6 | 2.53 |
7 | 2.56 |
8 | 2.59 |
9 | 2.59 |
10 | 2.62 |
Average | 2.59 |
As known from Table 2, the LDL-C detection kit accuracy of this example 2 preparation is: 0.81%
Table 3 be this example 2 preparation LDL-C detection kit under conditions of external condition is identical, complete in Hitachi 7180
The result data of METHOD FOR CONTINUOUS DETERMINATION quality controlled serum 20 times on automatic biochemistry analyzer.
Table 3 precision measurement result table
Measure number of times | Result (mmol/L) |
1 | 2.37 |
2 | 2.36 |
3 | 2.40 |
4 | 2.39 |
5 | 2.38 |
6 | 2.34 |
7 | 2.37 |
8 | 2.37 |
9 | 2.33 |
10 | 2.37 |
11 | 2.36 |
12 | 2.36 |
13 | 2.41 |
14 | 2.35 |
15 | 2.34 |
16 | 2.39 |
17 | 2.38 |
18 | 2.39 |
19 | 2.34 |
20 | 2.37 |
Average | 2.37 |
SD | 0.0197 |
CV | 0.83% |
As known from Table 3, the LDL-C detection kit precision of this example 2 preparation is: 0.83%
Table 4 is that the LDL-C detection kit measured value of this example 2 preparation changes LDL-C detection kit mensuration with Japan one
The synopsis of value.
Table 4 measured value synopsis
Catalogue number(Cat.No.) | This example 2 measured value (mmol/L) | Japan one changes LDL-C detection kit measured value (mmol/L) |
1 | 2.85 | 2.90 |
2 | 2.55 | 2.55 |
3 | 2.62 | 2.67 |
4 | 2.61 | 2.68 |
5 | 2.71 | 2.78 |
6 | 2.45 | 2.46 |
7 | 2.73 | 2.74 |
8 | 2.20 | 2.23 |
9 | 2.83 | 2.88 |
10 | 2.13 | 2.19 |
11 | 2.61 | 2.61 |
12 | 2.51 | 2.52 |
13 | 2.98 | 2.98 |
14 | 2.62 | 2.67 |
15 | 2.57 | 2.63 |
16 | 2.42 | 2.48 |
17 | 2.78 | 2.82 |
18 | 2.39 | 2.39 |
19 | 2.98 | 3.07 |
20 | 2.48 | 2.55 |
21 | 2.90 | 2.93 |
22 | 2.79 | 2.84 |
23 | 2.55 | 2.57 |
24 | 2.84 | 2.91 |
25 | 2.84 | 2.89 |
26 | 2.55 | 2.62 |
27 | 2.23 | 2.27 |
28 | 2.95 | 3.03 |
29 | 2.27 | 2.30 |
30 | 2.97 | 3.04 |
31 | 2.77 | 2.82 |
32 | 2.33 | 2.38 |
33 | 2.79 | 2.85 |
34 | 2.45 | 2.48 |
35 | 2.61 | 2.65 |
36 | 2.40 | 2.41 |
37 | 2.51 | 2.53 |
38 | 2.63 | 2.64 |
39 | 2.53 | 2.55 |
40 | 2.18 | 2.23 |
As known from Table 4, the LDL-C detection kit measured value of this example 2 preparation changes LDL-C detection kit with Japan one
The coefficient R of measured value2Being 0.991, both show fabulous dependency.
Claims (8)
1. one kind is passed through to combine the detection method covering standard measure mensuration LDL-C, it is characterised in that: use liquid double reagent, by
Reagent 1 and the reagent 2 placed respectively form, and wherein, described reagent 1 is containing calcium chloride, LDL-C monoclonal antibody, octyl phenyl
Polyoxyethylene ether, cholesteryl esterase;Described reagent 2 is containing PEG 8000,4-AA, peroxidase, gallbladder
Sterin oxidase.
Detection method the most according to claim 1, it is characterised in that in described reagent 1, metal chloride can make to dissociate
There is coagulation in LDL-C, and forms antigen antibody complex with LDL-C monoclonal antibody generation antigen antibody reaction, reaches associating
The effect covered.
Detection method the most according to claim 1, it is characterised in that in described reagent 1, the concentration range of calcium chloride exists
0.4g/L-0.5g/L。
Detection method the most according to claim 1, it is characterised in that in described reagent 1, LDL-C monoclonal antibody is for carrying fat
Protein B monoclonal antibody, its concentration range is at 50ml/L-150ml/L.
Detection method the most according to claim 1, it is characterised in that octyl phenyl polyoxyethylene ether in described reagent 1
Concentration range is at 2ml/L-4ml/L.
Detection method the most according to claim 1, it is characterised in that in described reagent 1, cholesteryl esterase content is 10KU/
L-14KU/L。
Detection method the most according to claim 1, it is characterised in that the PEG 8000 concentration model in described reagent 2
It is trapped among 40g/L-60g/L.
Detection method the most according to claim 1, it is characterised in that in described reagent 2,4-AA content is
0.05-0.2g/L, cholesterol oxidase content are 0.4KU/L-0.8K U/L, peroxidase content is 1KU/L-2KU/L.
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CN1200113C (en) * | 1998-09-18 | 2005-05-04 | 协和梅迪克斯株式会社 | Methods for fractional quantification of cholesterol in lipoproteins and quantification reagents |
JP4478861B2 (en) * | 2003-11-05 | 2010-06-09 | 東洋紡績株式会社 | Method for measuring cholesterol in low density lipoprotein and its reagent |
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