CN1912592A - Chemiluminescence investigating method of amylase in body fluid - Google Patents
Chemiluminescence investigating method of amylase in body fluid Download PDFInfo
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- CN1912592A CN1912592A CN 200610114991 CN200610114991A CN1912592A CN 1912592 A CN1912592 A CN 1912592A CN 200610114991 CN200610114991 CN 200610114991 CN 200610114991 A CN200610114991 A CN 200610114991A CN 1912592 A CN1912592 A CN 1912592A
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- diastase
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Abstract
A method for determining amylase in body fluid includes catalyzing a-1, 4-glucoside chain in starch molecule by amylase to make a-1, 4-glucoside chain be hydrilzed for producing glucose and maltose as well as dextrin of a-1, 6-glucoside, oxidizing glucose by glucose oxidase to generate perhydrol, acting perhydrol by peroxidase to make chemiluminescent matter be oxidized for lightening, making light signal be more stronger when starch concentration is more greater, using known amylase concentration and measured light signal to work out dosage-reaction curve for calculating out amylase content in unknown sample by utilizing said curve.
Description
Technical field: the present invention relates to a kind of medical agent detection method, particularly diastatic chemical luminescent detecting method in the body fluid.
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, be chemiluminescence immune assay (chemiluminescence immunoassay, CLIA) and immunochemiluminometry (Immunochemiluminometricassay, 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.
AMS in serum or the urine (AMY) can raise when acute pancreatitis, mumps; Can descend when hepatitis, obstructive jaundice, liver cancer, cirrhosis, pancreatic insufficiency etc.
The diastatic method of now generally using of mensuration is colourimetry, this method is to utilize α in the catalyzed by amylase starch molecule-1, the hydrolysis of 4-glycosidic linkage, produce glucose, maltose and contain α-1, the dextrin of 6-glycosidic linkage, the reaction back adds iodine liquid and unhydrolysed starch generates blue compound, judges diastatic concentration in the biological fluid by shade.The weak point of this method mainly shows:
1, the range of linearity is narrow.Because detection is so responsive unlike chemiluminescence to change in color resolution, 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.Colourimetry is the depth than color, and chemiluminescence is to measure light signal, and sensitivity is high.
3, full automatic biochemical apparatus costliness.The full automatic biochemical apparatus that uses at present almost is import entirely, and equipment is very expensive, mostly hundreds of thousands arrive millions of between, middle and small hospital is difficult to bear, universal use is restricted.
Summary of the invention: technical matters to be solved by this invention is that a kind of providing can overcome diastatic chemical luminescent detecting method in above-mentioned defective and highly sensitive, that the range of linearity is wide, convenient, the safe body fluid.Solve this technical problem the main technical schemes of employing: diastatic chemical luminescent detecting method in the body fluid, it is characterized in that utilizing α in the catalyzed by amylase starch molecule-1, the hydrolysis of 4-glycosidic linkage produces glucose, maltose and contains α-1, the dextrin of 6-glycosidic linkage; Glucose oxidase makes glucose oxidase and generates hydrogen peroxide; Hydrogen peroxide makes the chemiluminescent substance oxidation and luminous under the effect of peroxidase; The size of light signal and diastatic concentration are proportionate, and promptly the big more light signal that sends of diastase concentration is strong more; Write down this light signal and can infer diastatic concentration; With the diastase of concentration known and the light signal of measuring, make dose-response curve; The diastase content of unknown sample can come out from this curve calculating; Described chemiluminescent substance is a luminol; Described diastase can be the diastase in the serum; Described diastase can be the diastase in the urine; Described diastase can be the diastase in other body fluid.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.Can prepare diastatic commercially available reagent box in the corresponding quantitative measurement body fluid 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.Utilize α in the catalyzed by amylase starch molecule-1, the hydrolysis of 4-glycosidic linkage produces glucose, maltose and contains α-1, the dextrin of 6-glycosidic linkage; Glucose oxidase makes glucose oxidase and generates hydrogen peroxide; Hydrogen peroxide makes the chemiluminescent substance oxidation and luminous under the effect of peroxidase; The size of light signal and diastatic concentration are proportionate, and promptly the big more light signal that sends of diastase concentration is strong more; Write down this light signal and can infer diastatic concentration; With the diastase of concentration known and the light signal of measuring, make dose-response curve; The diastase content of unknown sample can come out from this curve calculating; Described chemiluminescent substance is a luminol; Described diastase can be the diastase in the serum; Described diastase can be the diastase in the urine; Described diastase can be the diastase in other body fluid; This is a diastatic chemical luminescent detecting method in the body fluid of the present invention.
Reaction principle of the present invention is expressed as follows with formula:
One, reagent preparation:
Soluble starch liquid;
Diastase standard: single or series concentration;
Substrate solution (containing GOD+HRP and luminol).
Two, mensuration program, according to the form below carries out, and application of sample unit is μ l.
| Standard | Sample | |
| Standard | 50 | |
| Sample | 50 | |
| Starch fluid | 50 | 50 |
| Mixing, room temperature or 37 ℃ of incubations 5 ~ 15 minutes | ||
| 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 diastatic concentration its serum from this curve.Or directly obtain by formula:
Diastase concentration=(sample RLU/ standard RLU) * normal concentration
Four, also available single stage method 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, is suitable for large, medium and small medical institutions wide popularization and application.
Claims (5)
1, diastatic chemical luminescent detecting method in the body fluid is characterized in that utilizing α in the catalyzed by amylase starch molecule-1, and the hydrolysis of 4-glycosidic linkage produces glucose, maltose and contains α-1, the dextrin of 6-glycosidic linkage; Glucose oxidase makes glucose oxidase and generates hydrogen peroxide; Hydrogen peroxide makes the chemiluminescent substance oxidation and luminous under the effect of peroxidase; The size of light signal and diastatic concentration are proportionate, and promptly the big more light signal that sends of diastase concentration is strong more; Write down this light signal and can infer diastatic concentration; With the diastase of concentration known and the light signal of measuring, make dose-response curve; The diastase content of unknown sample can come out from this curve calculating.
2, diastatic chemical luminescent detecting method in the body fluid according to claim 1 is characterized in that described chemiluminescent substance is a luminol.
3, diastatic chemical luminescent detecting method in the body fluid according to claim 1 is characterized in that described diastase can be the diastase in the serum.
4, diastatic chemical luminescent detecting method in the body fluid according to claim 1 is characterized in that described diastase can be the diastase in the urine.
5, diastatic chemical luminescent detecting method in the body fluid according to claim 1 is characterized in that described diastase can be the diastase in other body fluid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 200610114991 CN1912592A (en) | 2006-08-10 | 2006-08-10 | Chemiluminescence investigating method of amylase in body fluid |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610114991 CN1912592A (en) | 2006-08-10 | 2006-08-10 | Chemiluminescence investigating method of amylase in body fluid |
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| CN1912592A true CN1912592A (en) | 2007-02-14 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010888A (en) * | 2010-09-21 | 2011-04-13 | 烟台喜旺科技发展有限公司 | Method for detecting starch |
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2006
- 2006-08-10 CN CN 200610114991 patent/CN1912592A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010888A (en) * | 2010-09-21 | 2011-04-13 | 烟台喜旺科技发展有限公司 | Method for detecting starch |
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