CN1912595A - Chemiluminescence investigating method of serum total cholesterol in blood - Google Patents
Chemiluminescence investigating method of serum total cholesterol in blood Download PDFInfo
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- CN1912595A CN1912595A CN 200610114994 CN200610114994A CN1912595A CN 1912595 A CN1912595 A CN 1912595A CN 200610114994 CN200610114994 CN 200610114994 CN 200610114994 A CN200610114994 A CN 200610114994A CN 1912595 A CN1912595 A CN 1912595A
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Abstract
A method for determining serum total cholesterol in blood by utilizing chemiluminescence includes hydrolyzing cholesterol ester by cholesterol esterase for obtaining free cholesterol, oxidizing free cholesterol by oxidase to generate perhydrol, making chemiluminescent matter be oxidized for lightening by perhydrol under action of peroxidase, making light signal be more stronger when serum total cholesterol concentration is more greater, using known serum total cholesterol concentration and measured light signal to work out dosage-reaction curve for calculating out serum total cholesterol content in unknown sample by utilizing said curve.
Description
Technical field: the present invention relates to a kind of medical agent detection method, particularly the chemical luminescent detecting method of serum total cholesterol in the blood.
Background technology: the ultramicro-determination method of utilizing modern biotechnology to develop has promoted the fast development of the every clinical and research work in biological and each field of medical science, greatly for outstanding contribution has been made in the mankind's cause of science and social progress.
Last century, the seventies was at first delivered the application luminol by Arakawa---the chemiluminescence reaction of peroxidase is carried out the technology that enzyme exempts to analyze, this technology since sensitivity up to 10
-18Mol can fast measuring go out the result in 20 minutes, can easy realization full automation, be valid up to more than 1 year, and "dead" and teratogen is beneficial to environmental protection and is well received, and is the detection technique the most rapidly of development at present.
At present, chemiluminescence has obtained quite universal degree in immunoassay, developed into the routine prison bed detection method that can detect multiple bioactivator, from immune response mode branch, this method has two kinds of fundamental types, i.e. chemiluminescence immune assay (CLIA) and immunochemiluminometry (ICMA); From illumination mode, the one, with the direct mark of luminescent substance on antibody or antigen, after the immune response, and luminous, another kind is with enzymic-labelled antibody or antigen under the effect of incipient reagent, after the immune response, add chemical luminous substrate, under the enzyme caloytic action and luminous.
Though this method has outstanding advantage, only limit to immunoassay at present, and still blank in biochemical analysis.
T-CHOL (TC) is present in the blood, one of main hazard factor that high T-CHOL mass formed by blood stasis is a coronary heart disease.High TC under the pathological state has two classes of former and secondary; Low TC mass formed by blood stasis also has former and secondary two classes.
The method of serum total cholesterol is the CHOD-PAP colourimetry in the mensuration blood that now generally uses, this method is with CEH (CEH) cholesteryl ester to be hydrolyzed into free cholesterol (CHOL), the latter is by its oxidase (CHOD) oxidation, and generation hydrogen peroxide, again through the effect of superoxide enzyme, make look source thing (PAP) become redness, TC content becomes positive correlation in absorbance and the sample, can carry out colorimetric estimation.The weak point of this method mainly contains:
1, the range of linearity is narrow.Owing to detect light absorption, its resolution is so responsive unlike chemiluminescence, and the range of linearity can not satisfy the needs of clinical detection fully, need measure after diluting the sample of high concentration, brings certain inconvenience.
2, sensitivity is not as chemiluminescence.Chemiluminescence is to measure light signal, and sensitivity absorbs high than photometry.
3, reagent composition complexity is operated loaded down with trivial detailsly, causes error easily, influences the accuracy of measurement result.
Summary of the invention: technical matters to be solved by this invention is a kind of chemical luminescent detecting method that can overcome serum total cholesterol in above-mentioned defective, highly sensitive, the blood that the range of linearity is wide, convenient, safe that provides.Solve this technical problem the main technical schemes of employing: the chemical luminescent detecting method of serum total cholesterol in the blood is characterized in that with CEH (CEH) cholesteryl ester being hydrolyzed into free cholesterol (Chol); Free cholesterol (Chol) is by its oxidase (CHOD) oxidation, and the generation hydrogen peroxide; Through the effect of superoxide enzyme, under the peroxidase effect, hydrogen peroxide makes the chemiluminescent substance oxidation and is luminous again; The size of light signal and the concentration of serum total cholesterol are proportionate, and promptly the big more light signal that sends of total cholesterol density of serum is strong more; Write down this light signal and can infer the concentration of serum total cholesterol; With the serum total cholesterol of concentration known and the light signal of measuring, make dose-response curve; The serum total cholesterol content of unknown sample can come out from this curve calculating; Described chemiluminescent substance is a luminol; Described cholesterol also can be a free cholesterol (Chol); Described cholesterol also can be a HDL-C (HDL-C); Described cholesterol also can be a LDL-C (LDL-C).The present invention compared with prior art has following advantage: replace the colour developing thing with chemiluminescent substance, reach sensitiveer, more stable, wide ranges, safer purpose; The commercially available reagent box that can prepare corresponding quantitative measurement serum total cholesterol with this method; Concrete advantage is: 1. sensitivity is higher.Classic method is to detect shade, and chemiluminescence is the photometry signal, photon counting one by one, and the minimum of being surveyed is littler, thereby sensitivity is higher.2. the range of linearity is wide.The resolution of the depth of survey color is more much lower than the precision of photon counting, and chemiluminescent sensing range is up to 10
53. safe and applicable.Chemiluminescent substance such as luminol etc. are very safe, and whole testing process does not have nuisance and occurs, and are that detection or processing of waste are all very safe, help environmental protection.4. be convenient to popularize.The kit that the present invention is prepared can be used for all automatic measurement, also can be used for craft or semi-automatic measuring, and the medical treatment of suitable large, medium and small each level and research institution use.5. huge social benefit.With chemiluminescence determination biochemical indicator or immune substance, can no longer need the biochemical instruments of import costliness together with a Chemiluminescence Apparatus, save substantial contribution, not only reduced cost, medical institutions, patient and society are all benefited.
Embodiment: now illustrate embodiments of the invention.With CEH (CEH) cholesteryl ester is hydrolyzed into free cholesterol (Chol); Free cholesterol (Chol) is by its oxidase (CHOD) oxidation, and the generation hydrogen peroxide; Through the effect of superoxide enzyme, under the peroxidase effect, hydrogen peroxide makes the chemiluminescent substance oxidation and is luminous again; The size of light signal and the concentration of serum total cholesterol are proportionate, and promptly the big more light signal that sends of total cholesterol density of serum is strong more; Write down this light signal and can infer the concentration of serum total cholesterol; With the serum total cholesterol of concentration known and the light signal of measuring, make dose-response curve; The serum total cholesterol content of unknown sample can come out from this curve calculating; Described chemiluminescent substance is a luminol; Described cholesterol also can be a free cholesterol (Chol); Described cholesterol also can be a HDL-C (HDL-C); Described cholesterol also can be a LDL-C (LDL-C).This is the chemical luminescent detecting method of serum total cholesterol in the blood of the present invention.This method can be used for measuring the content of T-CHOL (TC) in the serum, free cholesterol (Chol), HDL-C (HDL-C), LDL-C (LDL-C) etc.
One, reagent comprises:
Mixed enzyme solution;
T-CHOL standard: single or series concentration;
Substrate solution
Two, mensuration program:
According to the form below carries out, and application of sample unit is μ l.
| Standard | Sample | |
| Standard | 20 | |
| Sample | 20 | |
| Enzyme liquid | 50 | 50 |
| Substrate solution | 50 | 50 |
| Mixing, measure R LU behind room temperature or the 37 ℃ of incubations | ||
Three, make dose-response curve with the proper method processing, RLU value is per sample obtained the concentration of its serum total cholesterol from this curve.Or directly obtain by formula:
Total cholesterol concentration=(sample RLU/ standard RLU) * normal concentration
Four, also available double reagent is measured.
Sensitivity of the present invention is higher: because classic method is to detect shade, and the present invention is the photometry signal, photon counting one by one, and the minimum of being surveyed is littler, thereby sensitivity is higher.The range of linearity is wide: this is that chemiluminescence detection scope of the present invention is up to 10 because the resolution of the depth of traditional survey color is more much lower than the precision of photon counting
5Safe and applicable: chemiluminescent substance of the present invention such as luminol etc. are very safe, and whole testing process does not have nuisance and occurs, and are that detection or processing of waste are all very safe, help environmental protection.Be convenient to popularize: the kit that the present invention is prepared, can be used for all automatic measurement, also can be used for craft or semi-automatic measuring, the medical treatment of suitable large, medium and small each level and research institution use.Social benefit is huge: measure biochemical indicator or immune substance with chemical luminescent detecting method of the present invention, can no longer need the biochemical instruments of import costliness together with a Chemiluminescence Apparatus, save substantial contribution, not only reduced cost, medical institutions, patient and society have all been benefited.The inventive method is simple, and cost is low, is suitable for large, medium and small medical institutions wide popularization and application.
Claims (5)
1, the chemical luminescent detecting method of serum total cholesterol in the blood is characterized in that with CEH (CEH) cholesteryl ester being hydrolyzed into free cholesterol (Chol); Free cholesterol (Chol) is by its oxidase (CHOD) oxidation, and the generation hydrogen peroxide; Through the effect of superoxide enzyme, under the peroxidase effect, hydrogen peroxide makes the chemiluminescent substance oxidation and is luminous again; The size of light signal and the concentration of serum total cholesterol are proportionate, and promptly the big more light signal that sends of total cholesterol density of serum is strong more; Write down this light signal and can infer the concentration of serum total cholesterol; With the serum total cholesterol of concentration known and the light signal of measuring, make dose-response curve; The serum total cholesterol content of unknown sample can come out from this curve calculating.
2, the chemical luminescent detecting method of serum total cholesterol in the blood according to claim 1 is characterized in that described chemiluminescent substance is a luminol.
3, the chemical luminescent detecting method of serum total cholesterol in the blood according to claim 1 is characterized in that described cholesterol also can be a free cholesterol (Chol).
4, the chemical luminescent detecting method of serum total cholesterol in the blood according to claim 1 is characterized in that described cholesterol also can be a HDL-C (HDL-C).
5, the chemical luminescent detecting method of serum total cholesterol in the blood according to claim 1 is characterized in that described cholesterol also can be a LDL-C (LDL-C).
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| CN 200610114994 CN1912595A (en) | 2006-08-10 | 2006-08-10 | Chemiluminescence investigating method of serum total cholesterol in blood |
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| CN 200610114994 CN1912595A (en) | 2006-08-10 | 2006-08-10 | Chemiluminescence investigating method of serum total cholesterol in blood |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102798627A (en) * | 2012-08-01 | 2012-11-28 | 福建医科大学 | Method for determining cholesterol by using flow injection chemiluminescence with nano-copper oxide as catalyst |
| CN105353142A (en) * | 2015-12-07 | 2016-02-24 | 郑州兰森生物技术有限公司 | High-stability single reagent for serum total cholesterol detection |
-
2006
- 2006-08-10 CN CN 200610114994 patent/CN1912595A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102798627A (en) * | 2012-08-01 | 2012-11-28 | 福建医科大学 | Method for determining cholesterol by using flow injection chemiluminescence with nano-copper oxide as catalyst |
| CN102798627B (en) * | 2012-08-01 | 2015-07-15 | 福建医科大学 | Method for determining cholesterol by using flow injection chemiluminescence with nano-copper oxide as catalyst |
| CN105353142A (en) * | 2015-12-07 | 2016-02-24 | 郑州兰森生物技术有限公司 | High-stability single reagent for serum total cholesterol detection |
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