CN100476401C - High-density lipoprotein cholesterol quantitative determining method, reagent and reagent kit - Google Patents
High-density lipoprotein cholesterol quantitative determining method, reagent and reagent kit Download PDFInfo
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- CN100476401C CN100476401C CNB2005100801049A CN200510080104A CN100476401C CN 100476401 C CN100476401 C CN 100476401C CN B2005100801049 A CNB2005100801049 A CN B2005100801049A CN 200510080104 A CN200510080104 A CN 200510080104A CN 100476401 C CN100476401 C CN 100476401C
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Abstract
A reagent for the content of the high 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 heteropolymer anion compound, the divalency cation salt and the decentralized surfactants; the reagent II contains the styrylphenol polyoxyethylene ether. The invention provides a reagent box with the reagent I and the reagent II, and a method for determining the high density lipoprotein cholesterol content.
Description
Technical field
The present invention relates to method for quantitatively determining, reagent and the kit of HDL-C (HDL-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.The large-scale epidemiology survey of Frammingham in 1977 shows that it is the main hazard factor of ischemic heart disease that plasma hdl cholesterol (blood plasma HDL-C) descends, and henceforth, the mensuration of HDL-C is subjected to generally attention clinically day by day.High-density lipoprotein (HDL) (HDL) can be by the effect of enzyme and acceptor, and the cholesterol of surrounding tissue is moved to the liver degradation treatment, restrains cell combination and picked-up LDL-C simultaneously, stops the deposition of cholesterol at arterial wall.Therefore, HDL-C is considered to the atherosclerotic prevention factor.Just because of this reason, there is important predicting function the arteriosclerosis morbidity aspect that is determined at of HDL-C.
Clinically, the laboratory conventional method of directly separating, measure HDL-C content comprises chemical precipitation method, magnetic bead DS-Mg
2+Partition method and even determination method mutually.
Wherein, chemical precipitation method precipitation agent commonly used is a polyanion, and as phosphotungstic acid (PTA), dextran sulfate (DS), heparin, 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 HDL-C content are, add precipitation agent with the lipoprotein of aggegation except that HDL in the test serum sample, and agglutinator is removed in centrifuging, measures the serum cholesterol that only contains HDL that obtains by this separation then.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.
Magnetic bead DS-Mg
2+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 that selectivity suppresses method, PEG modifies enzyme process, removing method, immune partition method 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 inhibition 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 HDL-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.Wherein, reagent I contains polyanion compound, divalent metal salt and decentralized surfactant (also claiming reaction suppressor).CM in the test serum sample, VLDL, LDL condense in the presence of polyanion and divalent metal salt.Because the hydrophobic group of reaction suppressor and CM, VLDL, LDL has high affinity, so forming, its hdl particle surface that is adsorbed on cohesion covers circle, suppress the reaction of this surface free cholesterol, with other lipoprotein formation stabilized complex except that HDL.
Meanwhile, free HDL surface also is adsorbed with a spot of reaction suppressor, but since the affinity of the two a little less than, it is in conjunction with being reversible.Contain the high-affinity of soluble surfactant (also claiming reaction promoter), cholesteryl esterase (ChER), cholesterol oxidase (ChoD) and reaction solution agent have to(for) hydrophilic radical in the HDL particle subsequently among the reagent II of Jia Ruing.This promoter not only can displace the reaction suppressor of HDL surface adsorption to the absorption on HDL surface, and makes whole HDL form the solubility complexs, thereby makes the HDL-C can be directly and the enzyme reagent reacting, and color development under the effect of developer then.And cohesion after taking place with the reaction suppressor reaction and combining in CM, VLDL, LDL, therefore no longer have an effect, can not separate for test serum sample and directly measure the content of HDL-C through above-mentioned processing with cholesteryl esterase and cholesterol oxidase.
Disclose above-mentioned selectivity among the Chinese patent application 1268185A and suppressed method and the reagent that method is directly measured HDL-C content.
But the commercial reagent that is used for directly measuring HDL-C content at present is imported product, and is on the high side.Therefore, development is applicable to that the selectivity of above-mentioned direct mensuration HDL-C content suppresses method, 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
Our the direct assay method of the selectivity of cholesterol in to serum lipoprotein is studied and experiment back discovery repeatedly for many years, a kind of special surface activating agent, styryl phenol polyoxyethylene ether is as reaction promoter, can fully replace the reaction suppressor of HDL surface adsorption, this reaction promoter can combine with HDL and form dissolubility and reactive good solubility complex, guarantee the reaction of HDL-C and described enzyme reagent, and then guarantee the accurate mensuration of HDL-C content.
One of purpose of the present invention is that a kind of reagent that is used for directly measuring blood serum sample HDL-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 polyanion compound, divalent metal salt and decentralized surfactant; Described reagent II contains styryl phenol polyoxyethylene ether.
Another object of the present invention is, the kit of HDL-C content in a kind of quantitative measurement blood serum sample is provided, and it is characterized in that wherein being equipped with being placed with aforementioned agents I and reagent II respectively.
A further object of the present invention provides the method for HDL-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 HDL-C content is made up of the reagent I and the reagent II that place respectively.
Wherein, described reagent I contains polyanion compound, divalent metal salt and decentralized surfactant; Described reagent II contains styryl phenol polyoxyethylene ether.
The effect of described reagent I is to suppress cholesteryl esterase and the cholesterol oxidase reaction in CM, VLDL, LDL and second reaction reagent in the test serum sample.Described reagent I contains polyanion surfactant, divalent metal salt and decentralized surfactant.
The instantiation of described polyanion surfactant can comprise: cyclodextrin sulfate, heparin sulfate, dextran sulfate, sodium phosphotungstate, polyvinyl sulfuric acid salt.Described polyanion surfactant concentrations has no particular limits, and preferred concentration range is 0.1mmol/L-10mmol/L.
Kation in the described bivalent cation salt is selected from Mn
2+, Mg
2+And/or Ca
2+Described bivalent cation salt preferred concentration range is 0.1mmol/L-100mmol/L.
Described decentralized surfactant is selected from polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene-polyoxypropylene condensation product, polyoxyethylene alkyl ether sulfate salt and alkyl benzene sulfonate.Described decentralized surfactant preferred concentration range is 0.1g/L-50g/L.
Described reagent I can also contain following substances, and these materials comprise: the pH damping fluid, as the 2-morpholino ethyl sulfonic acid (MES) of GOOD ' S series, 3-morpholino propane sulfonic acid (MPOS) etc.; Remove agent interfering, as potassium ferrocyanide, ascorbic acid oxidase etc.; Antiseptic is as sodium azide etc.; Amino antipyrine of 4-and/or developer.Amino antipyrine of preferred 4-and developer are not stored among the reagent I simultaneously.
Can be used for developer of the present invention comprises: phenol, 4-chlorophenol, N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-methylaniline sodium (TOOS), N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium phenol analog derivative developers such as (HDAOS), and preferred concentration range is 0.01g/L-10g/L;
Reagent II of the present invention contains reaction promoter.
Described reaction promoter is a styryl phenol polyoxyethylene ether, described styryl phenol polyoxyethylene ether is known commercial 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 needs that HDL-C detects and decide.Generally, its working concentration scope is 1g/L-20g/L, and preferred concentration range is 2g/L-8g/L.
Described reagent II can also contain following substances, and these materials comprise: the pH damping fluid, as the 2-morpholino ethyl sulfonic acid (MES) of GOOD ' S series, 3-morpholino propane sulfonic acid (MPOS) etc.; Remove agent interfering, as potassium ferrocyanide, ascorbic acid oxidase etc.; Antiseptic is as sodium azide etc.; Amino antipyrine of 4-and/or developer.Amino antipyrine of preferred 4-and developer are not stored among the reagent II simultaneously.
Also need contain following quantitative enzyme reagent among reagent I of the present invention and the reagent II, these enzyme reagent comprise: cholesteryl esterase, cholesterol oxidase and peroxidase.Above-mentioned three kinds of enzymes can all be included among the reagent I, also can all be included among the reagent II, or be included in respectively among reagent I or the II.Its preferred concentration range is respectively 0.1ku/L-100ku/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 mentioned reagent I and the reagent II that place respectively are housed in the quantitative measurement HDL-C kit of the present invention.
The method of HDL-C content comprises in the direct mensuration blood serum sample of the present invention: at first add reagent I in the test serum sample, constant temperature is regularly hatched, and measures the absorbance A under certain wavelength
1In sample, add reagent II then, continue to hatch, 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.HDL-C content in the blood serum sample can calculate by following formula:
HDL-C content=(A
1-A
2) ÷ (A
1'-A
2') * (calibration solution HDL-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 (also 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
Sodium dextran sulfate 1g/L
MgCl
2·6H
2O 2mM
Developer TOOS 0.2g/L
NaN
3 1g/L
Cyclodextrin sulfate 0.5g/L
Reagent II:
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
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 same wavelength
2Use the same method and measure the absorbance A of calibration solution with condition
1' value and A
2' be worth, press the HDL-C content of following formula calculation sample then:
HDL-C content=(A
1-A
2) ÷ (A
1'-A
2') * (calibration solution HDL-C concentration)
(mmol/L or mg/dL)
Blood serum sample to basic, normal, high HDL-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 HDL-C calibration solution " (lot number: middle life 230401, 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) | 69.96 | 56.2 | 39.69 |
| s(mg/dL) | 0.59 | 0.36 | 0.27 |
| CV(％) | 0.84 | 0.64 | 0.67 |
* the coefficient of variation (CV) is generally used for weighing the precision of a method, and the coefficient of variation is more little, illustrates that this method is accurate more.CV is considered to acceptable less than the precision of 5% method.
CV all in the table 1 show that all less than 1% the inventive method has good precision.
Embodiment 2:
Use the embodiment of the invention 1 reagent, the HDL-C content among 11 parts of healthy human serum samples is measured according to embodiment 1 described method and condition.Same sample is used commercially available precipitation method kit (HDL-C (HDL-C) phosphotungstic acid-magnesium precipitate method kit simultaneously, lot number: middle life 19051, Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd. produces) quantitative measurement of contrast property, the result is as shown in table 2 below:
Table 2
By testing the accuracy of weighing this method with the correlativity of the precipitation method (contrast 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=0.9878 of HDL-C Determination on content value the accuracy of this method is good.
The computing method of described related coefficient are as follows:
Wherein, the HDL-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 HDL-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 HDL-C content analysis at aspects such as stability, repeatability, accuracy, linearities, is applicable to connect the HDL-C content of measuring in fresh serum or the blood plasma.
Claims (16)
1. the reagent of a quantitative measurement blood serum sample middle-high density lipoprotein cholesterol content, this reagent is made up of the reagent I and the reagent I I that place respectively, wherein, described reagent I contains polyanion compound, divalent metal salt and decentralized surfactant; Described reagent II contains styryl phenol polyoxyethylene ether.
2. reagent according to claim 1 is characterized in that, described polyanion compound is selected from: cyclodextrin sulfate, heparin sulfate, dextran sulfate, sodium phosphotungstate, polyvinyl sulfuric acid salt.
3. reagent according to claim 1 is characterized in that the kation of described divalent metal salt is selected from Mn
2+, Mg
2+And Ca
2+In one or more.
4. reagent according to claim 1 is characterized in that, described decentralized surfactant is selected from polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene-polyoxypropylene condensation product, polyoxyethylene alkyl ether sulfate salt and alkyl benzene sulfonate.
5. according to the described reagent of one of claim 2-4, it is characterized in that described polyanion compound concentrations scope is 0.1mmol/L-10mmol/L; The concentration range of divalent metal salt is 0.1mmol/L-100mmol/L; Decentralized surfactant concentrations scope is 0.1g/L-50g/L.
6. reagent according to claim 1 is characterized in that, the concentration range of described styryl phenol polyoxyethylene ether is 1g/L-20g/L.
7. reagent according to claim 6 is characterized in that, the concentration range of described styryl phenol polyoxyethylene ether is 2g/L-8g/L.
8. reagent according to claim 1 is characterized in that, described reagent I and reagent II also contain the pH damping fluid, remove agent interfering, in antiseptic, the amino antipyrine of 4-and the developer one or more.
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 phenol, 4-chlorophenol, N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-methylaniline sodium, N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium; And amino antipyrine of 4-and developer are not stored among reagent I or the reagent II simultaneously.
10. according to Claim 8 or 9 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 developer is 0.01g/L-10g/L.
11. reagent according to claim 1 is characterized in that, also contains quantitative enzyme reagent among described reagent I and the reagent II, described quantitative enzyme reagent is selected from cholesteryl esterase, cholesterol oxidase and peroxidase.
12. the reagent according to claim 11 is characterized in that, described three kinds of enzymes all are included among the described reagent I.
13. the reagent according to claim 11 is characterized in that, described three kinds of enzymes all are included among the described reagent II.
14. the reagent according to claim 11 is characterized in that, described three kinds of enzymes are included in respectively among described reagent I or the described reagent II.
15., it is characterized in that described three kinds of enzymes concentration range separately is respectively 0.1ku/L-100ku/L according to each reagent of claim 12-14.
16. the kit of a quantitative measurement blood serum sample middle-high density lipoprotein cholesterol content, it is characterized in that wherein being equipped with the reagent of each described quantitative measurement blood serum sample middle-high density lipoprotein cholesterol content of claim 1-15, this reagent is made up of the reagent I and the reagent II that place respectively.
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- 2005-06-29 CN CNB2005100801049A patent/CN100476401C/en active Active
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| CN108918895A (en) * | 2018-09-01 | 2018-11-30 | 山东博科生物产业有限公司 | A kind of high-density lipoprotein cholesterol detection reagent that accuracy is high |
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