CN109628553B - A kind of low density lipoprotein cholesterol assay kit and its application - Google Patents
A kind of low density lipoprotein cholesterol assay kit and its application Download PDFInfo
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Abstract
The present invention relates to a kind of low density lipoprotein cholesterol assay kit and its applications, belong to technical field of biological.The kit is made of reagent R1 and reagent R2；Reagent R1 includes: surfactant a, cholesterol esterase, cholesterol oxidase, peroxidase, 4-AA, magnesium chloride and buffer；Reagent R2 includes: surfactant b, color developing agent, magnesium chloride and buffer.Reagent R2 further includes melezitose and lactulose.The kit is measured using direct method, has many advantages, such as that high sensitivity, accuracy are strong.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of low density lipoprotein cholesterol assay kit and
It is applied.
Background technique
Lipoprotein in human serum is broadly divided into according to density difference: chylomicron (chylomicrons, CM), extremely low
Density lipoprotein (very-low-density lipoprotein, VLDL), intermediated-density lipoprotein (intermediate-
Density lipoprotein, IDL), low-density lipoprotein (low-density lipoprotein, LDL) and high density rouge
Albumen (high-density lipoprotein, HDL).Wherein, cholesterol about 50% is contained in LDL particle, is that gallbladder is solid in blood
The most lipoprotein of alcohol content, therefore it is known as the lipoprotein rich in cholesterol, it is that tissue absorbs endogenous cholesterol and gallbladder
The main carriers of sterol ester.
Cholesterol is also known as cholesterine, deposits in the form of free cholesterol or in the form of the cholesteryl ester to combine with fatty acid
It is in blood lipoprotein, such as: low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), pole
Low density lipoprotein cholesterol (VLDL-C) etc..Baigent et al. is the study found that LDL-C is to cause the atherosclerotic heart
Important one of the risk factor of vascular diseases (atherosclerotic cardiovascular disease, ASCVD)
(Baigent C,Keech A,Kearney PM,et al.Efficacy and safety of cholesterol-
lowering treatment:prospective meta-analysis of data from 90056participants
in 14randomised trials of statins.Lancet,2005,366:1267-1278.).Therefore, clinically will
The height of LDL-C is as one of coronary heart disease and the auxiliary diagnostic index of atherosclerosis.
It is built according to the clinical lipid determination that China " Blood Lipid Abnormality in Chinese Adults guideline of prevention and treatment (revised edition in 2016) " proposes
View, the measurement of serum LDL-C mainly use even phase method, including to remove method, catalase clear for turbidimetry, surfactant
Division, soluble reaction method etc..For example, Chinese patent CN100365409C discloses a kind of measurement low density lipoprotein cholesterol
Reagent and preparation method, the reagent properties are stable, are applicable in the automatic clinical chemistry analyzer of various models；But contain in reagent
There are many preservatives, inhibit larger to the enzymes activity in reagent, sensitivity is poor.
For another example, Chinese patent CN107505272A discloses low density lipoprotein cholesterol detection kit and its user
Method, the kit remove the content of the LDL-C in method measurement serum using catalase.But the kit use it is more
Polyanion surfactant stability in alkaline aqueous solution is poor, and then influences the scavenging effect to non-LDL lipoprotein, therefore
And the kit is difficult to accurately carry out the assay of LDL-C.
Therefore, it is badly in need of the strong low density lipoprotein cholesterol detection kit of a kind of high sensitivity, accuracy.
Summary of the invention
The technical problem to be solved by the present invention is to the detecting step for current low density lipoprotein cholesterol is more numerous
Miscellaneous, especially the prior art has that reagent sensitivity is low, accuracy is poor.Low-density lipoprotein gallbladder provided by the invention is solid
Alcohol assay kit has easy to operate, detection efficiency and the high advantage of accuracy rate.
In order to realize that above-mentioned technical effect, the present invention adopt the following technical scheme that realization:
On the one hand, the present invention provides a kind of low density lipoprotein cholesterol assay kit, the kit includes
Reagent R1 and reagent R2；
The reagent R1 includes: surfactant a, cholesterol esterase, cholesterol oxidase, peroxidase, 4- ammonia
Base antipyrine, magnesium chloride and buffer；
The reagent R2 includes: surfactant b, color developing agent, magnesium chloride and buffer.
Wherein, the reagent R2 further includes melezitose and lactulose.
Preferably, in the low density lipoprotein cholesterol assay kit, the reagent R1 includes: surface-active
Agent a 80.0-120.0mmol/L, cholesterol esterase 1.0-6.0KU/L, cholesterol oxidase 1.0-10.0KU/L, peroxide
Enzyme 8.0-30.0KU/L, 4-AA 0.2-1.5mmol/L, magnesium chloride 5.0-15.0mmol/L, pH value be 7.0 it is slow
Fliud flushing 10.0-50.0mmol/L,；
The reagent R2 includes: surfactant b 50.0-150.0mmol/L, color developing agent 1.0-10.0mmol/L, chlorine
Change magnesium 5.0-15.0mmol/L, buffer 10.0-50.0mmol/L, melezitose 0.5-1.5g/L and the lactulose that pH value is 7.0
0.2-1.0g/L。
It is highly preferred that the reagent R1 includes: that surface is living in the low density lipoprotein cholesterol assay kit
Property agent a 90.0-110.0mmol/L, cholesterol esterase 1.5-3.0KU/L, cholesterol oxidase 3.0-6.0KU/L, peroxide
Enzyme 12.0-20.0KU/L, 4-AA 0.5-1.0mmol/L, magnesium chloride 8.0-12.0mmol/L and pH value are 7.0
Buffer 15.0-35.0mmol/L；
The reagent R2 includes: surfactant b 80.0-120.0mmol/L, color developing agent 2.0-6.0mmol/L, chlorine
Change magnesium 8.0-12.0mmol/L, buffer 15.0-35.0mmol/L, melezitose 0.4-0.8g/L and the lactulose that pH value is 7.0
0.4-0.7g/L。
Wherein, the surfactant a is one or more of sodium taurocholate, sulfonate and dextran sulfate；It is preferred that
Ground, the surfactant a are sodium taurocholate.
Wherein, the surfactant b is one or more of triton x-100, Tween-20 and Span-60；It is excellent
Selection of land, the surfactant b are triton x-100.
Wherein, the color developing agent be one of 2- hydroxyl -3- meta-aminotoluene propanesulfonate, phenol and 4- chlorophenol or
It is several；Preferably, the color developing agent is 2- hydroxyl -3- meta-aminotoluene propanesulfonate.
Wherein, the buffer is in 3- N-morpholinyl buffer, phosphate buffer and borate buffer solution
It is one or more of.Preferably, the buffer is 3- N-morpholinyl buffer.
It is further preferred that the reagent R1 includes: gallbladder in the low density lipoprotein cholesterol assay kit
Sour sodium 100.0mmol/L, cholesterol esterase 2.0KU/L, cholesterol oxidase 5.0KU/L, peroxidase 15.0KU/L, 4- ammonia
The 3- N-morpholinyl buffer 20.0mmol/ that base antipyrine 0.6mmol/L, magnesium chloride 10.0mmol/L and pH value are 7.0
L；
The reagent R2 includes: triton x-100 100.0mmol/L, 2- hydroxyl -3- meta-aminotoluene propanesulfonate
4.0mmol/L, magnesium chloride 10.0mmol/L, the 3- N-morpholinyl buffer 20.0mmol/L that pH value is 7.0, melezitose
1.0g/L and lactulose 0.5g/L.
It further include that low density lipoprotein cholesterol standard items freeze in the low density lipoprotein cholesterol assay kit
Dry powder 2g, need to be with using after physiological saline solution.
The present invention also provides a kind of application methods of low density lipoprotein cholesterol assay kit:
This kit is suitable for Du Pont AR, Hitachi 7080/7170/7600/7180, steps the auspicious CS600 of auspicious BS320, enlightening, Abbott Laboratories
C8000, Toshiba TBA40FR, Olympus AU2700, Beckman DXC800, Siemens ADVIA2400, Roche P800 etc. are complete certainly
Automatic Biochemical Analyzer.
The measuring method of the assay kit is Two point end assay, and the Direction of Reaction is to rise reaction:
(1) 300 μ L reagent R1 and 3 μ L samples to be tested are uniformly mixed, obtain reaction solution 1 after 37 DEG C of incubation 5min, measured
Absorbance A1, measurement dominant wavelength is 546nm, a length of 660nm of subwave；
(2) 100 μ L reagent R2 are added to be uniformly mixed, react 5min, obtain reaction solution 2, measure absorbance A2, measure main wave
The a length of 660nm of a length of 546nm, subwave；
(3) according to step (1)-(2), the absorbance of the low density lipoprotein cholesterol standard items of various concentration is surveyed
It is fixed, obtain absorbance ABlankAnd AStandard；
(4) with the absorbance change value Δ A (A of standard itemsStandard-ABlank) it is ordinate, it is cross with corresponding standard concentration c
Coordinate draws standard curve, obtains calibration curve equation；
(5) the absorbance change value Δ A (A of sample is calculated2-A1), according to calibration curve equation, obtain low-density in sample
Lipoprotein cholesterol concn.
Compared with prior art, the present invention the beneficial effects of the present invention are as follows:
(1) low density lipoprotein cholesterol assay kit component provided by the invention is simple, and each reagent component is with more excellent
Concentration is matched, and has the range of linearity up to 13.0mmol/L；
(2) present invention is by the way that melezitose and lactulose are added into reagent R2, so that the sensitivity of kit dramatically increases；
(3) in low density lipoprotein cholesterol assay kit provided by the invention, nonionic surfactant is in solution
Middle stability height, the not influence vulnerable to strong electrolyte inorganic salts and soda acid；Wherein: the nonionic surfactant (gallbladder in reagent 1
Sour sodium), LDL is protected to the property of can choose, non-LDL (HDL, VLDL, CM) is removed and reduces its interference to LDL-C assay；
Nonionic surfactant (triton x-100) in reagent 2 can open LDL, and LDL-C is made to react and show with cholesterol enzyme
Color；
(4) low density lipoprotein cholesterol assay kit accuracy provided by the invention is high, and theoretical concentration and actual measurement are dense
Linearly dependent coefficient r≤0.9950 of degree；
(5) low density lipoprotein cholesterol assay kit provided by the invention, required specimen amount is few, and (3 μ L serum are
Can), and do not influenced substantially by hemoglobin, triglycerides, bilirubin etc.；When hemoglobin, triglycerides, bilirubin are high respectively
When to 5g/L, 18.2mmol/L and 250 μm of ol/L, LDL-C measurement is had no significant effect.
Detailed description of the invention
Fig. 1 is low density lipoprotein cholesterol standard curve；
Fig. 2 is low density lipoprotein cholesterol detection kit range of linearity figure of the present invention.
Specific embodiment
In order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, tie below
Specific embodiment is closed, the present invention is furture elucidated, and however, the following embodiments are merely preferred embodiments of the present invention, and not all.
Based on the implementation example in the implementation mode, those skilled in the art's obtained other realities without making creative work
Example is applied, protection scope of the present invention is belonged to.Experimental method in following embodiments is unless otherwise specified conventional method,
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Test condition and method:
Instrument: 7080 automatic clinical chemistry analyzer of Hitachi
Parameter:
| Dominant wavelength | 546nm | Sample | 3μL |
| Secondary wavelength | 660nm | Reagent 1 | 300μL |
| Reaction temperature | 37℃ | Reagent 2 | 100μL |
| The Direction of Reaction | Rise reaction | Reaction type | End-point method |
Operating procedure:
Embodiment 1
The low density lipoprotein cholesterol assay kit component and its concentration of the present embodiment are as follows:
Reagent R1 includes: sulfonate 80.0mmol/L, cholesterol esterase 1.0KU/L, cholesterol oxidase 1.0KU/L, mistake
Oxide enzyme 8.0KU/L, 4-AA 0.2mmol/L, magnesium chloride 5.0mmol/L, the phosphate-buffered that pH value is 7.0
Liquid 10.0mmol/L；Reagent R2 includes: Tween-20 50.0mmol/L, phenol 1.0mmol/L, magnesium chloride 5.0mmol/L, pH value
For 7.0 phosphate buffer 1 0.0mmol/L, melezitose 0.5g/L and lactulose 0.2g/L.
Embodiment 2
The low density lipoprotein cholesterol assay kit component and its concentration of the present embodiment are as follows:
Reagent R1 includes: dextran sulfate 120.0mmol/L, cholesterol esterase 6.0KU/L, cholesterol oxidase
10.0KU/L, peroxidase 30.0KU/L, 4-AA 1.5mmol/L, magnesium chloride 15.0mmol/L, pH value are
7.0 borate buffer solution 50.0mmol/L；Reagent R2 includes: Span-60 150.0mmol/L, 4- chlorophenol 10.0mmol/
L, magnesium chloride 15.0mmol/L, borate buffer solution 50.0mmol/L, melezitose 1.5g/L and lactulose that pH value is 7.0
1.0g/L。
Embodiment 3
The low density lipoprotein cholesterol assay kit component and its concentration of the present embodiment are as follows:
Reagent R1 includes: sodium taurocholate 90.0mmol/L, cholesterol esterase 1.5KU/L, cholesterol oxidase 3.0KU/L, mistake
The 3- morpholine third that oxide enzyme 12.0KU/L, 4-AA 0.5mmol/L, magnesium chloride 8.0mmol/L and pH value are 7.0
Sulfonate buffer 15.0mmol/L；Reagent R2 includes: triton x-100 80.0mmol/L, 2- hydroxyl -3- the third sulphur of meta-aminotoluene
Sour sodium 2.0mmol/L, magnesium chloride 8.0mmol/L, the 3- N-morpholinyl buffer 15.0mmol/L that pH value is 7.0, melezitose
0.4g/L and lactulose 0.4g/L.
Embodiment 4
The low density lipoprotein cholesterol assay kit component and its concentration of the present embodiment are as follows:
Reagent R1 includes: sodium taurocholate 110.0mmol/L, cholesterol esterase 3.0KU/L, cholesterol oxidase 6.0KU/L, mistake
The 3- morpholine that oxide enzyme 20.0KU/L, 4-AA 1.0mmol/L, magnesium chloride 12.0mmol/L and pH value are 7.0
Propane sulfonic acid buffer 35.0mmol/L；Reagent R2 includes: triton x-100 120.0mmol/L, 2- hydroxyl -3- meta-aminotoluene third
Sodium sulfonate 6.0mmol/L, magnesium chloride 12.0mmol/L, the 3- N-morpholinyl buffer 35.0mmol/L that pH value is 7.0, pine three
Sugared 0.8g/L and lactulose 0.7g/L.
Embodiment 5
The low density lipoprotein cholesterol assay kit component and its concentration of the present embodiment are as follows:
Reagent R2 includes: sodium taurocholate 100.0mmol/L, cholesterol esterase 2.0KU/L, cholesterol oxidase 5.0KU/L, mistake
The 3- morpholine that oxide enzyme 15.0KU/L, 4-AA 0.6mmol/L, magnesium chloride 10.0mmol/L and pH value are 7.0
Propane sulfonic acid buffer 20.0mmol/L；Reagent R2 includes: triton x-100 100.0mmol/L, 2- hydroxyl -3- meta-aminotoluene third
Sodium sulfonate 4.0mmol/L, magnesium chloride 10.0mmol/L, the 3- N-morpholinyl buffer 20.0mmol/L that pH value is 7.0, pine three
Sugared 1.0g/L and lactulose 0.5g/L.
Comparative example 1
Kit described in Chinese patent CN102539731A.
Comparative example 2
Compared with Example 1, reagent R2 is free of melezitose and lactulose.
Comparative example 3
Compared with Example 1, reagent R2 is free of melezitose.
Comparative example 4
Compared with Example 1, reagent R2 is free of lactulose.
1 low density lipoprotein cholesterol assay kit standard curve of experimental example
Using low density lipoprotein cholesterol standard items, concentration is respectively 0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,
4.0mmol/L is obtained by taking experimental example 5 as an example according to said determination step, measures low density lipoprotein cholesterol assay kit
Standard curve (as shown in Figure 1).Wherein, the absorbance change value Δ A (A of standard itemsStandard-ABlank) it is ordinate, to mark accordingly
Quasi- product concentration c is abscissa, calibration curve equation: y=0.1004x+0.0025, R2=0.9987.
The test of the 2 low density lipoprotein cholesterol kit range of linearity of experimental example
Using physiological saline difference compound concentration be 2.0,4.0,6.0,8.0,10.0,12.0,13.0mmol/L it is low close
Lipoprotein cholesterol standard items are spent, the reagent described in embodiment 1-5 and comparative example 1 measure LDL-C content respectively, as a result such as table 1
It is shown:
The comparison of 1 range of linearity of table
The result shows that: in 0-13.0mmol/L concentration range, the test paper of kit of the present invention can reach theoretical value
And the two good relationship (Fig. 2), and comparative example concentration of specimens be 10.0mmol/L when, test result significantly lower than theory
Value, cannot meet the requirements, it can be seen that the range of linearity of this kit is wider.
3 low density lipoprotein cholesterol assay kit minimum detection limit of experimental example
By taking embodiment 5 as an example, using physiological saline as blank sample, continuous detection 20 times.Calculate the equal of 20 results
Value and standard deviation SD add twice of standard deviation to limit (+2SD) as the detection of kit using blank mean value.Test result passes through test number
According to obtaining (as shown in table 2), low density lipoprotein cholesterol of the present invention measures the minimum detection limit 0.015mmol/L of agent box.
The minimum detection limit of embodiment 1-4 is respectively 0.035,0.027,0.026,0.018 and 0.019mmol/L.
2 low density lipoprotein cholesterol of table measures agent box minimum detection limit
4 low density lipoprotein cholesterol assay kit sensitivity of experimental example
Blank solution and 3 difference LDL-C content samples are measured, each sample is surveyed 10 times, and absorbance values M and mark are calculated
Quasi- poor (SD) is greater than the sample concentration of blank absorbency+3SD as LDL-C assay kit most using sample absorbance -3SD
Low detection sensitivity.As a result, the sensitivity of kit is after adding melezitose and lactulose in reagent R2 according to table 3
0.10mmol/L or so, and the sensitivity for being not added with 2 kit of comparative example of melezitose and lactulose is 0.20mmol/L or more,
The sensitivity for only adding 4 kit of comparative example 3 and comparative example of melezitose or a kind of substance of lactulose is 0.15mmol/L.Thus
It is found that the present invention is creatively added to melezitose and lactulose, hence it is evident that improve the sensitivity of kit.
3 low density lipoprotein cholesterol of table measures the sensitivity of agent box
5 interference experiment of embodiment
A certain amount of hemoglobin, triglycerides, bilirubin are respectively added in normal serum, while isometric go is added
Ionized water using the LDL-C assay kit of embodiment 1-5, while measuring the concentration of sample as noiseless object serum.With dry
It is that measurement system endures limit to the highest of chaff interferent that the degree of disturbing, which is 5%, hemoglobin 5g/L or less, triglycerides in serum
18.2mmol/L or less and 250 μm of ol/L of bilirubin are not hereinafter, influence LDL-C measurement result.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (7)
1. a kind of low density lipoprotein cholesterol assay kit, it is characterised in that: the kit includes reagent R1 and examination
Agent R2；
The reagent R1 includes: that surfactant a 80.0-120.0mmol/L, cholesterol esterase 1.0-6.0KU/L, gallbladder are solid
Alcohol oxidase 1.0-10.0KU/L, peroxidase 8.0-30.0KU/L, 4-AA 0.2-1.5mmol/L, chlorination
Magnesium 5.0-15.0mmol/L, the buffer 10.0-50.0mmol/L that pH value is 7.0；
The reagent R2 includes: surfactant b 50.0-150.0mmol/L, color developing agent 1.0-10.0mmol/L, magnesium chloride
5.0-15.0mmol/L, buffer 10.0-50.0mmol/L, melezitose 0.5-1.5g/L and the lactulose 0.2- that pH value is 7.0
1.0g/L。
2. low density lipoprotein cholesterol assay kit according to claim 1, it is characterised in that: the reagent R1
It include: surfactant a 90.0-110.0mmol/L, cholesterol esterase 1.5-3.0KU/L, cholesterol oxidase 3.0-
6.0KU/L, peroxidase 12.0-20.0KU/L, 4-AA 0.5-1.0mmol/L, magnesium chloride 8.0-
The buffer 15.0-35.0mmol/L that 12.0mmol/L and pH value are 7.0；
The reagent R2 includes: surfactant b 80.0-120.0mmol/L, color developing agent 2.0-6.0mmol/L, magnesium chloride
8.0-12.0mmol/L, buffer 15.0-35.0mmol/L, melezitose 0.4-0.8g/L and the lactulose 0.4- that pH value is 7.0
0.7g/L。
3. low density lipoprotein cholesterol assay kit according to claim 2, it is characterised in that: the surface is living
Property agent a be one or more of sodium taurocholate, sulfonate and dextran sulfate.
4. low density lipoprotein cholesterol assay kit according to claim 2, it is characterised in that: the surface is living
Property agent b be one or more of triton x-100, Tween-20 and Span-60.
5. low density lipoprotein cholesterol assay kit according to claim 2, it is characterised in that: the color developing agent
For one or more of 2- hydroxyl -3- meta-aminotoluene propanesulfonate, phenol and 4- chlorophenol.
6. low density lipoprotein cholesterol assay kit according to claim 2, it is characterised in that: the buffering
Liquid is one or more of 3- N-morpholinyl buffer, phosphate buffer and borate buffer solution.
7. according to the described in any item low density lipoprotein cholesterol assay kits of claim 3-6, it is characterised in that: described
Reagent R1 include: sodium taurocholate 100.0mmol/L, cholesterol esterase 2.0KU/L, cholesterol oxidase 5.0KU/L, peroxide
The 3- N-morpholinyl that enzyme 15.0KU/L, 4-AA 0.6mmol/L, magnesium chloride 10.0mmol/L and pH value are 7.0
Buffer 20.0mmol/L；
The reagent R2 includes: triton x-100 100.0mmol/L, 2- hydroxyl -3- meta-aminotoluene propanesulfonate 4.0mmol/
L, magnesium chloride 10.0mmol/L, 3- N-morpholinyl buffer 20.0mmol/L, the melezitose 1.0g/L that pH value is 7.0 and newborn fruit
Sugared 0.5g/L.
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|---|---|---|---|---|
| CN1379234A (en) * | 2002-05-10 | 2002-11-13 | 肖洪武 | Process and reagent for measuring low-density lipoprotein and cholesterol |
| CN102539731A (en) * | 2012-01-09 | 2012-07-04 | 宁波天康生物科技有限公司 | Reagent and kit for quantitatively determining low-density lipoprotein cholesterol (LDL-C) in serum |
| WO2015158836A1 (en) * | 2014-04-16 | 2015-10-22 | Vital Solutions Swiss Ag | Mangifera indica as a sirtuin 1 activating agent |
| CN107505272B (en) * | 2017-08-10 | 2020-09-25 | 东软威特曼生物科技(南京)有限公司 | Low-density lipoprotein cholesterol detection kit and use method thereof |
| US20200392558A1 (en) * | 2017-09-01 | 2020-12-17 | Hitachi Chemical Diagnostics Systems Co., Ltd. | Method for measuring cholesterol in low-density lipoprotein, measurement reagent, and measurement kit |
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