CN102041296A - In-vitro diagnostic reagent for homogeneous method of low-density lipoprotein cholesterol (LDL-C) of serum - Google Patents
In-vitro diagnostic reagent for homogeneous method of low-density lipoprotein cholesterol (LDL-C) of serum Download PDFInfo
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- CN102041296A CN102041296A CN2009102060190A CN200910206019A CN102041296A CN 102041296 A CN102041296 A CN 102041296A CN 2009102060190 A CN2009102060190 A CN 2009102060190A CN 200910206019 A CN200910206019 A CN 200910206019A CN 102041296 A CN102041296 A CN 102041296A
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Abstract
The invention relates to an in-vitro diagnostic reagent for a homogeneous method of low-density lipoprotein cholesterol (LDL-C) of serum, wherein the in-vitro diagnostic reagent is capable of being widely applied to the technical field of medicine and biochemistry and is characterized in that the in-vitro diagnosis is carried out by means of a method comprising the following steps of: step one, selectively cracking chyle particles (CM), very low density lipoprotein cholesterol (VLDL-C) and high density lipoprotein cholesterol (HDL-C) within the serum by using a group of surfactant comprising trimethyl-beta-cyclodextrin, ethylene oxide octadecyl amine, poloxamer F88 and Brij-58, then generating hydrogen peroxide (H2O2) during the catalytic reaction of cholesterol esterase (COE) and cholesterol oxidase (COD), and then discomposing the H2O2 by means of a chemiluminescence clearing system of hydrogen peroxide, wherein the LDL-C particles within the serum are still kept perfectly at the moment; step two, reacting the LDL-C by catalyzing with the COE and the COD under the effect of TritonX-100 so as to generate H202, then promoting a chemiluminescence quantitative system to produce chemiluminescence by catalyzing the H2O2 with POD, and quantitating the LDL-C after measuring luminous intensity. The measuring reagent provided by the invention has the advantages that the sensitivity is high, the capacity of resisting disturbance is strong, the purpose for detecting the LDL-C of serum in batch is realized on a microporous plate chemiluminescence apparatus by measuring chemiluminescence intensity, and the reagent is suitable for the application in clinical laboratory.
Description
Technical field
The present invention relates to the outer diagnostic reagent of the even phase body of laws of serum low-density LP cholesterol (LDL-C), can be widely used in medical science and technological field of biochemistry.
Background technology
LDL-C is the maximum a kind of lipoprotein of cholesterol level in the blood plasma, and particle is less, and its content of cholesterol (comprising cholesteryl ester and free cholesterol) is over half.The major physiological function of LDL-C is the cholesterol in the liver to be transported to each tissue through blood utilize, and promotes accumulating in a large number of cell inner cholesterol.Numerous epidemiological studies show that serum LDL-C and atherosclerosis (AS), coronary heart disease (CHD) are proportionate.
The reference method that the method for measuring LDL-C clinically has ultracentrifugation, Friedewald equation, various electrophoretic method and American NCEP to recommend is the Beta-quantification method, be called for short β Q method, the DirectLDL method that also has reports such as Mcnamara, the precipitator method and electrophoretic method trouble, time-consuming, computing method is influenced by several factors, the accuracy of β Q method is not good enough, although the DirectLDL method is called direct assay method, but still need the polystyrene latex immunity to separate pre-treatment, belong to heterogeneity phase assay method (heterogeneousassay).
Be applied to the existing report of the even phase measuring method of serum low-density LP cholesterol of automatic clinical chemistry analyzer, (Chinese laboratory medicine magazine such as Zhang Shijun, 2000,23 (5): 276-299.), Japan Daiichi Pure Chemicals Co., Ltd. (patent No.: 71950.28), change to give birth to and grind Co., Ltd.'s (patent No.: 7182112.7) all carried out LDL-C and removed the method methodological study, these methods are at first used the phospho-wolframic acid root, or single tensio-active agent, or the combination of phospho-wolframic acid root and single tensio-active agent and serum LDL-C form mixture, simultaneously non-LDL-C lipoprotein component in the serum carried out cracking and the final H that generates that removes with catalase (CAT)
2O
2, add tensio-active agent then with cracking of LDL-C component and colour developing, carry out the visible light colorimetric assay at last.When being detection, the shortcoming of these methods relies on automatic clinical chemistry analyzer, not only measurement sensitivity is low, and for satisfying the requirement that the automatic clinical chemistry analyzer parameter is provided with, even adopt the minimum sample amount of taking that instrument allowed, still exist the sample amount of taking excessive for the mensuration system, the too much problem of interfering substance with the LDL-C coexistence, phospho-wolframic acid root in addition, or single tensio-active agent, or the mixture that the combination of phospho-wolframic acid root and single tensio-active agent and LDL-C form has the trend of decomposition in removing the non-LDL-C process of serum, thereby can not get rid of the interference of non-LDL-C component in the serum fully.
Chemiluminometry is a kind of analytical procedure of setting up by the chemoluminescence phenomenon, this method does not need complicated instrument, do not need light source (thereby reducing or eliminating Rayleigh scattering and Raman scattering) and dispersion means, there are not the scattered light common in the visible light colorimetric analysis method and the interference of stray light, improved signal to noise ratio greatly, thereby have highly sensitive, the advantage that linearity range is wide, be widely used in life science in recent years, clinical medicine, environment measuring, every field such as food analysis and agricultural analysis, but be not applied to the quantitative analysis of serum LDL-C as yet.Be applied to the LDL-C quantitative analysis method of automatic clinical chemistry analyzer can't be applied to LDL-C chemoluminescence detection by quantitative reagent owing to contain the chemiluminescent material of inhibition such as catalase, sodium azide in the difference of buffer system buffer capacity and pH value, mensuration reagent foundation.
Summary of the invention
The mensuration reagent that the purpose of this invention is to provide a kind of chemoluminescence method detection by quantitative serum LDL-C is to overcome the shortcoming that existing measuring method and reagent exist.This mensuration reagent is put 2-8 ℃ of preservation and can be stablized 12 months at least, and the reagent that provides can be applied to present widely used microwell plate Chemiluminescence Apparatus, thereby reaches the requirement of extensive mensuration sample.
The feature of this mensuration reagent is: the first step, adopt chyle particle (CM), C-VLDL (VLDL-C), high density lipoprotein cholesterol (HDL-C) in one group of tensio-active agent selective splitting serum of forming by TM-, oxyethane octadecane amine, poloxamer F88, Bu Lijie-58, and under the catalyzed reaction of Sterol esterase (COE) and rCO (COD), generate H
2O
2, utilize the hydrogen peroxide chemoluminescence to remove the system decomposing H
2O
2, the LDL-C particle still is kept perfectly in the serum at this moment; In second step, under the TritonX-100 effect, COE and COD catalysis LDL-C reaction generate H
2O
2, H
2O
2Under POD catalysis, impel the quantitative system of chemoluminescence to produce chemoluminescence, after measuring luminous intensity on the microwell plate Chemiluminescence Apparatus, LDL-C is carried out quantitatively.Calculate LDL-C content in the serum according to following formula, calculation formula is: serum LDL-C (mmol/L)=Δ I
Serum/ Δ I
Calibration solution* calibration solution concentration wherein is Δ I
SerumThe test serum chemiluminescence intensity, Δ I
Calibration solutionBe LDL-C calibration solution chemiluminescence intensity.
Described hydrogen peroxide chemoluminescence is removed system and is made up of damping fluid, chemical luminous substrate, peroxidase, sanitas, and preferred damping fluid is pH6.75MOPSO-MOPSONa damping fluid, preferred chemical luminous substrate for to iodophenol, preferably sanitas is proclin300.The H that the quantitative system of described chemoluminescence is made up of reagent one and reagent two
2O
2-to the quantitative system of iodophenol-luminol,3-aminophthalic acid cyclic hydrazide, reagent one preferred damping fluid be pH6.75MOPSO-MOPSONa damping fluid, reagent two preferred damping fluids be pH9.00Tris-HCl damping fluid, reagent one and reagent two by preferred pH after 1: 1 mixed be 8.50, preferred sanitas is proclin300.
Detect effect and enough stability in order to reach ideal, the mensuration reagent of chemiluminescence determination serum LDL-C provided by the invention can be divided into reagent one, 2 two components of reagent especially.The concrete composition of two components forms and optimal concentration can be expressed as follows.
Reagent one:
Reagent two:
During mentioned reagent was formed, Vitamin C oxidase was used for eliminating the interference of serum xitix; Bilirubin oxidase is used to eliminate the interference of serum mesobilirubin; The combination of tensio-active agent TM-, oxyethane octadecane amine, poloxamer F88, Bu Lijie-58 is used for the affine LDL-C of selectivity; The combination of sodium-chlor, ethylene glycol, sucrose, bovine serum albumin, proclin300 is used for stable cholesterol esterase, rCO, peroxidase, Vitamin C oxidase, bilirubin oxidase.Under the said determination condition, HDL-C＜4.50mmol/L, triglyceride level＜12.0mmol/L, bilirubin＜300mg/L, oxyphorase＜10g/L, xitix＜44mg/L do not disturb the mensuration of serum LDL-C.
Embodiment
Embodiment: the measuring method of employing is a microwell plate chemoluminescence end-point method.The first step, the sample diluent preparation: get serum specimen 10 μ l, be diluted to 1000 μ l with physiological saline, mixing is standby; In second step, l operates by table.
Table 1
20 routine serum LDL-C samples have been measured simultaneously with the LDL-C precipitator method mensuration reagent that this law and national Clinical Laboratory working specification (third edition) are recommended.Table 2 is present embodiment measured value and LDL-C precipitator method reagent measured value synopsis.The correlation coefficient r of the present embodiment and the LDL-C precipitator method is 0.9968, and both have shown fabulous dependency.
Table 2
| LDL-C precipitator method measured value (mmol/L) | Embodiment measured value (mmol/L) |
| 1.35 | 1.37 |
| 0.78 | 0.80 |
| 2.50 | 2.44 |
| 2.41 | 2.40 |
| 0.75 | 0.73 |
| 0.96 | 0.87 |
| 0.79 | 0.81 |
| 1.21 | 1.17 |
| 1.33 | 1.41 |
| 2.04 | 2.06 |
| 1.58 | 1.60 |
| 1.99 | 1.98 |
| 1.56 | 1.57 |
| 2.19 | 2.16 |
| 0.17 | 0.18 |
| 1.07 | 1.10 |
| 0.98 | 0.9 |
| 0.66 | 0.68 |
| 1.12 | 1.12 |
| 2.04 | 2.18 |
Claims (4)
1. a serum low-density LP cholesterol (LDL-C) is spared the outer diagnostic reagent of phase body of laws, it is characterized in that: the first step, adopt chyle particle (CM), C-VLDL (VLDL-C), high density lipoprotein cholesterol (HDL-C) in one group of tensio-active agent selective splitting serum of forming by TM-, oxyethane octadecane amine, poloxamer F88, Bu Lijie-58, and under the catalyzed reaction of Sterol esterase (COE) and rCO (COD), generate hydrogen peroxide (H
2O
2), utilize the hydrogen peroxide chemoluminescence to remove the system decomposing H
2O
2, the LDL-C particle still is kept perfectly in the serum at this moment; In second step, under the TritonX-100 effect, COE and COD catalysis LDL-C reaction generate H
2O
2, H
2O
2Under POD catalysis, impel the quantitative system of chemoluminescence to produce chemoluminescence, after measuring luminous intensity on the microwell plate Chemiluminescence Apparatus, LDL-C is carried out quantitatively.Calculate LDL-C content in the serum according to following formula, calculation formula is: serum LDL-C (mmol/L)=Δ I
Serum/ Δ I
Calibration solution* calibration solution concentration wherein is Δ I
SerumThe test serum chemiluminescence intensity, Δ I
Calibration solutionBe LDL-C calibration solution chemiluminescence intensity.
2. the outer diagnostic reagent of the even phase body of laws of serum low-density LP cholesterol according to claim 1 (LDL-C), it is characterized in that: the described hydrogen peroxide chemoluminescence of the first step is removed system and is made up of damping fluid, chemical luminous substrate, peroxidase, sanitas, and preferred damping fluid is pH6.75MOPSO-MOPSONa damping fluid, preferred chemical luminous substrate for to iodophenol, preferably sanitas is proclin300.
3. the outer diagnostic reagent of the even phase body of laws of serum low-density LP cholesterol according to claim 1 (LDL-C) is characterized in that: the H that the quantitative system of the second described chemoluminescence of step is made up of reagent one and reagent two
2O
2-to the quantitative system of iodophenol-luminol,3-aminophthalic acid cyclic hydrazide, reagent one preferred damping fluid is that pH6.75MOPSO-MOPSONa damping fluid, reagent two preferred damping fluids are pH9.00Tris-HCl damping fluid, reagent one and reagent two by l: preferred pH is 8.50 after 1 mixed, preferred sanitas is proclin300.
4. the outer diagnostic reagent of the even phase body of laws of serum low-density LP cholesterol according to claim 1 (LDL-C) is characterized in that reagent is formed and optimal concentration is:
Reagent one:
Reagent two:
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| CN104195222A (en) * | 2014-08-18 | 2014-12-10 | 苏州康铭诚业医用科技有限公司 | Compound stabilizer for total cholesterol measurement kits |
| CN106282313A (en) * | 2016-08-19 | 2017-01-04 | 威海威仕泰医疗科技有限公司 | A kind of stabilizer measuring reagent for low-density lipoprotein cholesterol |
| 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 |
| CN109097436A (en) * | 2018-08-10 | 2018-12-28 | 山东博科生物产业有限公司 | A kind of low density lipoprotein cholesterol detection reagent of the single agents of precise and high efficiency |
| CN109312387A (en) * | 2016-06-14 | 2019-02-05 | 剑桥显示技术有限公司 | For analyzing method, composition and the sensor of analyte detection |
| CN110042146A (en) * | 2011-11-11 | 2019-07-23 | 安讯希特技术有限公司 | The assay method of blood sample |
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| CN110042146A (en) * | 2011-11-11 | 2019-07-23 | 安讯希特技术有限公司 | The assay method of blood sample |
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| CN104195222B (en) * | 2014-08-18 | 2018-09-07 | 苏州康铭诚业医用科技有限公司 | A kind of compound stabilizer for total cholesterol assay kit |
| CN104195222A (en) * | 2014-08-18 | 2014-12-10 | 苏州康铭诚业医用科技有限公司 | Compound stabilizer for total cholesterol measurement kits |
| CN109312387A (en) * | 2016-06-14 | 2019-02-05 | 剑桥显示技术有限公司 | For analyzing method, composition and the sensor of analyte detection |
| CN106282313B (en) * | 2016-08-19 | 2020-05-26 | 威海威仕泰医疗科技有限公司 | Stabilizer for low-density lipoprotein cholesterol determination reagent |
| CN106282313A (en) * | 2016-08-19 | 2017-01-04 | 威海威仕泰医疗科技有限公司 | A kind of stabilizer measuring reagent for low-density lipoprotein cholesterol |
| 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 |
| CN110736846A (en) * | 2018-07-20 | 2020-01-31 | 上海练佰生物技术中心 | Reagent, method and kit for measuring small and dense lipoproteins |
| CN110736846B (en) * | 2018-07-20 | 2023-07-14 | 上海微鸿企业管理有限公司 | Small and dense lipoprotein determination reagent, method and kit |
| CN109097436A (en) * | 2018-08-10 | 2018-12-28 | 山东博科生物产业有限公司 | A kind of low density lipoprotein cholesterol detection reagent of the single agents of precise and high efficiency |
| CN111647641A (en) * | 2020-06-15 | 2020-09-11 | 宁波瑞源生物科技有限公司 | Small and dense low-density lipoprotein cholesterol detection kit and detection method thereof |
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Application publication date: 20110504 |