CN1888862A - Low-density lipoprotein cholesterol quantitative determining method reagent and reagent kit - Google Patents
Low-density lipoprotein cholesterol quantitative determining method reagent and reagent kit Download PDFInfo
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- CN1888862A CN1888862A CN 200510080103 CN200510080103A CN1888862A CN 1888862 A CN1888862 A CN 1888862A CN 200510080103 CN200510080103 CN 200510080103 CN 200510080103 A CN200510080103 A CN 200510080103A CN 1888862 A CN1888862 A CN 1888862A
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Abstract
A reagent for the content of the low density lipoprotein cholesterol in the quantitative determining serum sample, consists of the reagent I and reagent II that places respectively, the reagent I contains the styrylphenol polyoxyethylene ether and the quantitative enzyme reagent; the reagent II contains the reaction accelerant. The invention provides a reagent box with the reagent I and the reagent II, and a method for determining the low density lipoprotein cholesterol content.
Description
Technical field
The present invention relates to method for quantitatively determining, reagent and the kit of LDL-C (LDL-C).
Background technology
Lipids such as cholesterol cooperate with apoprotein in serum and generate lipoprotein.According to its differences of physical properties, lipoprotein is divided into chylomicron (CM), very low density lipoprotein (VLDL) (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL) (HDL) etc.Because numerous epidemiology, science of heredity and clinical research confirmation, the incidence of serum low-density LP cholesterol (LDL-C) level and atherosclerotic, coronary heart disease is proportionate, usually with the primary pathogenesis of high LDL-C as coronary heart disease, for this reason, the clinicing aspect that is determined at of LDL-C is subjected to generally paying attention to day by day.Thus, there is important predicting function the cardiovascular disease morbidity aspect that is determined at of LDL-C.
Clinically, the laboratory conventional method of directly separating, measure LDL-C content comprises supercentrifugation, Friedewald computing method of formula, chemical precipitation method, immune partition method and even determination method mutually.
Wherein, supercentrifugation is to remove the VLDL component (behind the d＜1.006kg/L) by ultracentrifugation, measure the total cholesterol level of d＞1.006kg/L component, (d＞1.063kg/L) content obtains the content of LDL-C to deduct HDL-C, because this method needs expensive device, complicated operation, time-consuming and technical requirement is high, is difficult for carrying out in common lab.
The Friedewald computing method of formula is a method of using wider estimation LDL-C at present, and it forms the constant prerequisite that is assumed to be with VLDL, has advantages such as easy, direct, quick.If but a certain measured value error is bigger, then can causes result of calculation unreliable, thereby make its clinical practice be subjected to more challenge and restriction.
Chemical precipitation method precipitation agent commonly used is a polyanion, as phosphotungstic acid (PTA), dextran sulfate (DS), heparin, polyvinyl sulfuric acid salt (PVS), or detergent such as SDS, polyglycol and some bivalent cation (as the bivalent cation of magnesium, calcium, manganese) share.
The concrete steps that chemical precipitation method is measured LDL-C content are, add precipitation agent selectivity aggegation LDL-C in sample, and centrifuging removes, and measure the supernatant cholesterol then, deduct its supernatant cholesterol value with the T-CHOL value of sample and promptly obtain the LDL-C value.Because this method is used precipitation agent, need carry out operations such as precipitate and separate simultaneously, so the sample consumption is big, the link that easily produces error is more relatively, especially can't realize the full automation of whole analytical procedure, thereby has limited the use of this technology.
The immunity partition method has been saved the necessary centrifugation step of chemical precipitation method, but need to use special device, and the employed reagent of this method is unsuitable for applying, so its usable range also is subjected to suitable restriction.
Even phase determination method comprises the selectivity null method, modifies enzyme process, turbidimetry etc.Even phase determination method does not need precipitation process because of its sample consumption is few, and available automatic biochemistry analyzer is measured, and degree of accuracy and accuracy are suitably widely adopted by clinical.
Selectivity null method in the even phase determination method is by using two kinds of different surfactants and polyanion, according to the enzyme reaction selectivity of lipoprotein, directly measuring the LDL-C in the test serum sample.The employed reagent of this method generally includes two kinds of reagent I and reagent II.
When measuring, at first add reagent I, surfactant wherein can select to promote free cholesterol that the HDL particle in the blood serum sample dissociates, discharges and cholesterol ester immediately with the enzyme reagent reacting, the H of generation
2O
2Only be consumed under the situation of coupling agent but do not developed the color lacking.And this moment, the LDL particle did not dissociate.Subsequently, add reagent II, under the effect of another kind of surfactant, enzyme reagent decomposes LDL, produces H
2O
2And under the effect of developer, develop the color then, the depth of color and luster is directly proportional with LDL-C content.Therefore can not separate and directly measure the content of LDL-C.
A kind of method and reagent of direct mensuration LDL-C content are disclosed among the Chinese patent application 1219973A.
But the commercial reagent that is used for directly measuring LDL-C content at present is imported product, and is on the high side.Therefore, development is applicable to the selectivity null method of above-mentioned direct mensuration LDL-C content, and the reagent that performance is more good, price is more cheap is the direction that one of ordinary skill in the art make great efforts always.
Summary of the invention
We study by the direct assay method of the selectivity of cholesterol in to serum lipoprotein for many years, and the experiment back is found repeatedly, use special surface activating agent of the present invention as preferential selective agent, the structure of HDL is changed, thereby make the reaction of HDL-C elder generation and described enzyme reagent.The use of special surface activating agent of the present invention can be eliminated the influence of HDL for measurement result, and then measures the content of LDL-C in the blood serum sample.
One of purpose of the present invention is that a kind of reagent that is used for directly measuring blood serum sample LDL-C content is provided.Described reagent is made up of the reagent I and the reagent II that place respectively, and wherein, described reagent I contains styryl phenol polyoxyethylene (16) ether, quantitative enzyme reagent; Described reagent II contains reaction promoter.
Another object of the present invention is, the kit of LDL-C content in a kind of quantitative measurement blood serum sample is provided, and it is characterized in that wherein being equipped with the aforementioned agents I and the reagent II that place respectively.
A further object of the present invention provides the method for LDL-C content in a kind of quantitative measurement blood serum sample.
Embodiment
Particularly, the present invention's reagent of being used for directly measuring blood serum sample LDL-C content is made up of the reagent I and the reagent II that place respectively.
Described reagent I comprises preferential selective agent, quantitative enzyme reagent and is selected from the reagent of one of the amino antipyrine of 4-, developer.Wherein, the preferential selective agent among the reagent I of the present invention is styryl phenol polyoxyethylene (a 16) ether.
Described styryl phenol polyoxyethylene (16) ether is known surfactant, for example, can buy from the market with the trade name (production of chemical industry two factories of Jiangsu Jinling Petrochemical Co.) of farming breast No. 601 (Pesticide Emulsifier 601#).The working concentration of this surfactant can be according to the characteristic of the analyser that uses this reagent, and finishes the demand that detects LDL-C and decide.Its preferred working concentration scope is 1g/L-20g/L, and more preferred concentration range is 2g/L-8g/L.
Described quantitative enzyme reagent is selected from cholesteryl esterase, cholesterol oxidase and peroxidase.The preferred concentration range for of above-mentioned various enzymes is 0.1ku/L-100ku/L.
Reagent II of the present invention comprises reaction promoter and is selected from the reagent of one of the amino antipyrine of 4-, developer.
Wherein, the reaction promoter that the present invention uses can be commercially available TRITON X series class surfactant, preferred TRITON X-100.The concentration range of described reaction promoter is: 1g/L-50g/L, preferred 10g/L-30g/L.
The preferred phenol analog derivative developer of developer of the present invention, for example phenol, 4-chlorophenol, N-ethyl-N-(2 hydroxyls-3-sulfopropyl-)-meta-aminotoluene sodium (TOOS) or N-(2 hydroxyls-3-sulfopropyl-)-3,5-dimethoxyaniline sodium phenol analog derivatives such as (HDAOS).Wherein, preferred TOOS or HDAOS.
Described reagent I, II can also contain suitable pH damping fluid, remove agent interfering, antiseptic etc.Wherein, the pH damping fluid is selected from 2-morpholino ethyl sulfonic acid (MES), 3-morpholino propane sulfonic acid GOOD ' S series such as (MPOS), and the pH value is preferably 5-9, the preferred 20mmol/L-200mmol/L of buffering agent working concentration; Go agent interfering to be selected from potassium ferrocyanide, ascorbic acid oxidase etc.; Antiseptic is selected from sodium azide, and its concentration range is preferably 0.01g/L-5g/L.
Described developer should be selected a use in reagent I and II, its preferred concentration range is 0.1g/L-10g/L.
The amino antipyrine of described 4-should be selected a use in reagent I and II.Its preferred concentration range is 0.01g/L-1g/L.
The amino antipyrine of described developer and 4-is not present among reagent I or the reagent II simultaneously.
The mentioned reagent I and the reagent II that place respectively are housed in the kit of quantitative measurement LDL-C of the present invention.
The method of LDL-C content comprises in the direct mensuration blood serum sample of the present invention: at first add reagent I in blood serum sample, after constant temperature is regularly hatched, measure the absorbance A under certain wavelength
1In sample, add reagent II then, continue to hatch, under above-mentioned setted wavelength, measure absorbance A
2Use the same method and measure the absorbance A of calibration solution
1' value and A
2' value.LDL-C content can calculate by following formula:
LDL-C content=(A
1-A
2) ÷ (A
1'-A
2') * (calibration solution LDL-C concentration)
(mmol/L or mg/dL)
Calibration solution of the present invention be matching used with kit or reagent, please be the secondary standard (can be described as the calibration thing or proofread and correct thing Calibrator) of matrix with blood, the relation of the concentration of calibration solution and absorbance (OD) can be used for the calculating of testing sample concentration.
Method of the present invention only needs a few microlitre serum, and need not centrifugal or electrophoresis etc. separated processing, and be easy and simple to handle, can satisfy the requirement of automatical analysis.
Following embodiment is the specific descriptions to present patent application, and still, present patent application is not limited to these embodiment, and these embodiment can not be interpreted as the restriction to present patent application.
Embodiment 1:
One, prepare following reagent I of the present invention and reagent II according to following compositions and ratio:
Reagent I:
The MOPS damping fluid, pH7.0 30mM
The amino antipyrine 0.1g/L of 4-
Cholesterol esterase (CEH) 1520u/L
Cholesterol oxidase (COD) 1070u/L
Peroxidase (POD) 8000u/L
No. 601 5g/L of farming emulsion
NaN
3 1g/L
Reagent II:
The MOPS damping fluid, pH7.0 30mM
Developer TOOS 0.2g/L
NaN
3 1g/L
TRITON X-100 20g/L
Two, in sample hose 300 μ l reagent I and 3 μ l blood serum samples are mixed, hatched under 37 ℃ 5 minutes, use the HITACHI7060 automatic clinical chemistry analyzer, at predominant wavelength 600nm, commplementary wave length 700nm place measures absorbance A
1, in sample, adding 100 μ l reagent II then, mixing after hatching 5 minutes under 37 ℃, is measured the absorbance A under the identical wavelength
2Use the same method and measure the absorbance A of calibration solution with condition
1' value and A
2' be worth, press the LDL-C content of following formula calculation sample then:
LDL-C content=(A
1-A
2) ÷ (A
1'-A
2') * (calibration solution LDL-C concentration)
(mmol/L or mg/dL)
Blood serum sample to basic, normal, high LDL-C content is measured, and replication 20 times calculates the mean value (x) and the standard deviation (s) of measured value according to the following equation:
x＝∑x/n
Calculate coefficient of variation CV by following formula then:
CV％＝s/ x×100％
Wherein, described calibration solution is commercially available " directly LDL-C calibration solution " (lot number: middle life 240401, Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd.'s production)
The results are shown in following table 1.
Table 1
| High value | Intermediate value | Low value | |
| Multiplicity | 20 | 20 | 20 |
| Mean value (mg/dL) | 161.45 | 108.29 | 60.98 |
| s(mg/dL) | 1.07 | 1.43 | 0.58 |
| CV(％) | 0.66 | 1.32 | 0.95 |
Weigh a kind of precision of method by above repeated experiment, as the index of weighing, the coefficient of variation is more little accurate more with the coefficient of variation (CV), and general CV should be less than 5%.Listed test result all is close in 1% in the table 1, shows that this method has good precision.
Embodiment 2:
Use the embodiment of the invention 1 reagent, under embodiment 1 described condition, the LDL-C content among 11 parts of healthy human serum samples is measured.Same sample is used commercially available precipitation method kit (LDL-C (LDL-C) polyvinyl sulfuric acid salt precipitation method kit, lot number: middle life 210181, Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd.'s production) contrast property quantitative measurement simultaneously.The result is as shown in table 2 below.
Table 2
| Catalogue number(Cat.No.) | LDL-C(mg/dl) | |
| The inventive method | The precipitation method | |
| 1 | 85 | 87 |
| 2 | 79 | 70 |
| 3 | 111 | 112 |
| 4 | 97 | 93 |
| 5 | 130 | 118 |
| 6 | 88 | 89 |
| 7 | 73 | 70 |
| 8 | 253 | 263 |
| 9 | 120 | 129 |
| 10 | 143 | 133 |
| 11 | 80 | 91 |
By testing the accuracy of weighing this method with the correlativity of the precipitation method (reference method).Correlation coefficient r is represented the degree of correlation of two groups of numerical value, and r shows that near 1 two groups of correlation of data are good more more.It has been generally acknowledged that r greater than 0.9 o'clock correlativity for good.This method and the commercial reagent box precipitation method show that for correlation coefficient r=0.9893 of LDL-C Determination on content value the accuracy of this method is good.
The computing method of described related coefficient are as follows:
Wherein, the LDL-C content concn value of X for using the inventive method to record; X is the average of the above-mentioned X value that records;
The LDL-C content concn value of Y for using the precipitation method to record; Y is the average of the above-mentioned Y value that records.
We can draw such conclusion as a result according to said determination: reagent of the present invention all satisfies the requirement of clinical LDL-C content analysis at aspects such as stability, repeatability, accuracy, linearities, is applicable to the LDL-C content in direct mensuration fresh serum or the blood plasma.
Claims (13)
1. the reagent of LDL-C content in the quantitative measurement blood serum sample, this reagent is made up of the reagent I and the reagent II that place respectively, and wherein, described reagent I contains styryl phenol polyoxyethylene (16) ether, quantitative enzyme reagent; Described reagent II contains reaction promoter.
2. according to the reagent of claim 1, it is characterized in that the concentration range of styryl phenol polyoxyethylene (16) ether is 1g/L-20g/L among the described reagent I.
3. according to the reagent of claim 2, it is characterized in that the concentration range of styryl phenol polyoxyethylene (16) ether is 2g/L-8g/L among the described reagent I.
4. according to the reagent of claim 1, it is characterized in that the quantitative enzyme reagent among the described reagent I contains cholesteryl esterase, cholesterol oxidase and peroxidase.
5. according to the reagent of claim 4, it is characterized in that the concentration range of described three kinds of enzymes is respectively 0.1ku/L-100ku/L.
6. according to the reagent of claim 1, it is characterized in that the reaction promoter among the described reagent II is a TRITON X series of surfactants, concentration range is 1g/L-50g/L.
7. according to the reagent of claim 6, it is characterized in that described TX series of surfactants is TRITONX-100, its concentration range is 10g/L-30g/L.
8. reagent according to claim 1 is characterized in that, can also contain the pH damping fluid among described reagent I and the reagent II, removes agent interfering, antiseptic, and the reagent that is selected from one of the amino antipyrine of 4-, developer.
9. reagent according to Claim 8 is characterized in that, described pH damping fluid is selected from 2-morpholino ethyl sulfonic acid, 3-morpholino propane sulfonic acid; Go agent interfering to be selected from potassium ferrocyanide, ascorbic acid oxidase; Antiseptic is selected from sodium azide; Developer is selected from the phenol analog derivative developer; And amino antipyrine of 4-and described developer are not present among reagent I or the reagent II simultaneously.
10. according to the reagent of claim 9, it is characterized in that described developer is selected from phenol, 4-chlorophenol, N-ethyl-N-(2 hydroxyls-3-sulfopropyl-)-meta-aminotoluene sodium, N-(2 hydroxyls-3-sulfopropyl-)-3,5-dimethoxyaniline sodium.
11. according to Claim 8, one of 9,10 described reagent, it is characterized in that the concentration range of the amino antipyrine of described 4-is 0.01g/L-1g/L; The concentration range of described developer is 0.01g/L-10g/L.
12. the kit of LDL-C content is characterized in that in the quantitative measurement blood serum sample, and one of the claim 1-11 that places respectively described reagent I and reagent II wherein are housed.
At first add described reagent I 13. the method for LDL-C content in the quantitative measurement blood serum sample, this method comprise in blood serum sample, constant temperature is regularly hatched, working sample absorbance A under certain wavelength
1In sample, add reagent II then, continue to hatch the back and under above-mentioned wavelength, measure absorbance A
2Use the same method and measure the absorbance A of calibration solution
1' value and A
2' value; Calculate the LDL-C content of blood serum sample then by following formula:
LDL-C content=(A
1-A
2) ÷ (A
1'-A
2') * (calibration solution LDL-C concentration).
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Cited By (8)
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| CN102043043A (en) * | 2010-12-10 | 2011-05-04 | 张会生 | Multi-project biochemical test analysis integrated kit and method thereof |
| CN102323225A (en) * | 2011-06-09 | 2012-01-18 | 董理 | Method and reagent kit used for detecting low-density lipoprotein cholesterin |
| 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 |
| CN103235114A (en) * | 2013-05-09 | 2013-08-07 | 上海北加生化试剂有限公司 | Kit and method for detecting cholesterol concentration in lipoprotein remnant |
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| CN109580511A (en) * | 2018-12-06 | 2019-04-05 | 潍坊泽成生物技术有限公司 | A kind of detection method and detection kit of high-density lipoprotein cholesterol |
| CN114350744A (en) * | 2022-01-21 | 2022-04-15 | 广西康柏莱科技有限公司 | Small and dense low-density lipoprotein cholesterol detection kit |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05285382A (en) * | 1992-04-07 | 1993-11-02 | Toyobo Co Ltd | Low-specific gravity lipoprotein adsorptive material |
| JP3058602B2 (en) * | 1996-04-15 | 2000-07-04 | デンカ生研株式会社 | Determination method of cholesterol in low density lipoprotein |
| JP3193634B2 (en) * | 1996-05-29 | 2001-07-30 | 第一化学薬品株式会社 | LDL cholesterol determination method |
| EP2194142A1 (en) * | 2002-11-27 | 2010-06-09 | Daiichi Pure Chemicals Co., Ltd. | Method of measuring cholesterol in high density lipoproteins |
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2005
- 2005-06-29 CN CNB2005100801034A patent/CN100443884C/en active Active
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| CN102043043A (en) * | 2010-12-10 | 2011-05-04 | 张会生 | Multi-project biochemical test analysis integrated kit and method thereof |
| CN102323225A (en) * | 2011-06-09 | 2012-01-18 | 董理 | Method and reagent kit used for detecting low-density lipoprotein cholesterin |
| 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 |
| CN103235114A (en) * | 2013-05-09 | 2013-08-07 | 上海北加生化试剂有限公司 | Kit and method for detecting cholesterol concentration in lipoprotein remnant |
| CN106950095A (en) * | 2017-04-01 | 2017-07-14 | 合肥迪安医学检验所有限公司 | A kind of method for reducing the interference of piarhemia pattern detection |
| CN106950095B (en) * | 2017-04-01 | 2020-01-10 | 合肥迪安医学检验所有限公司 | Method for reducing detection interference of lipemia sample |
| CN107561297A (en) * | 2017-08-25 | 2018-01-09 | 美康生物科技股份有限公司 | Reagent for the detection of blood fat parting |
| CN107561297B (en) * | 2017-08-25 | 2019-12-10 | 宁波美康盛德医学检验所有限公司 | Reagent for blood fat typing detection |
| CN109580511A (en) * | 2018-12-06 | 2019-04-05 | 潍坊泽成生物技术有限公司 | A kind of detection method and detection kit of high-density lipoprotein cholesterol |
| CN114350744A (en) * | 2022-01-21 | 2022-04-15 | 广西康柏莱科技有限公司 | Small and dense low-density lipoprotein cholesterol detection kit |
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