CN1912590A - Chemiluminescence measuring method of adenosine deaminase in body fluid - Google Patents
Chemiluminescence measuring method of adenosine deaminase in body fluid Download PDFInfo
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- CN1912590A CN1912590A CN 200610114976 CN200610114976A CN1912590A CN 1912590 A CN1912590 A CN 1912590A CN 200610114976 CN200610114976 CN 200610114976 CN 200610114976 A CN200610114976 A CN 200610114976A CN 1912590 A CN1912590 A CN 1912590A
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- adenosine deaminase
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Abstract
A method for determining adenosine deaminase in body fluid by utilizing chemiluminescence includes catalyzing adenosine by adenosine deaminase to make adenosine hydrolysis for producing ammonia and inosine, measuring out produced ammonia by utilizing chemiluminescent matter using known adenosine deaminase concentration and measured light signal to work out dosage-reaction curve for calculating out adenosine deaminase 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 adenosine deaminase 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 1O
-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.
Adenosine deaminase (ADA) has important value in the transudate antidiastole that good pernicious difficulty is debated.Specificity and susceptibility to diagnosis tuberculosis transudate are better than biopsy and bacteriological method; Cerebrospinal fluid ADA detects the important indicator that can be used as the central nervous system disease diagnosis and differential diagnosis; Tubercular meningitis significantly increases, and ICT (intracranial tumor) and central nervous system leukemia are high slightly.
The method of adenosine deaminase is colourimetry in the mensuration body fluid that now generally uses, this method is to utilize the hydrolysis of adenosine deaminase catalysis adenosine, produce inosine and ammonia, measure the ammonia that is produced with the Podbielniak chromogenic reaction then, thereby calculate the concentration of the adenosine deaminase in the serum.The weak point of this method mainly contains:
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.Enzyme process is the depth than color, and chemiluminescence is to measure light signal, and sensitivity is high.
3, can only use instrumentation.Have plenty of the speed of measuring enzymatic reaction, very strict to time requirement, the equal difficulty of craft or semi-automatic operation reaches requirement, can only use fully-automatic equipment to carry out.
4, 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.
5, some the look source material in the enzyme process is harmful to, and easily causes environmental pollution, and is not fool proof.
Summary of the invention: technical matters to be solved by this invention is a kind of chemical luminescent detecting method that can overcome adenosine deaminase in above-mentioned defective and highly sensitive, that the range of linearity is wide, convenient, the safe body fluid that provides.Solve this technical problem the main technical schemes of employing: the chemical luminescent detecting method of adenosine deaminase in the body fluid, it is characterized in that the hydrolysis of adenosine deaminase catalysis adenosine, produce inosine and ammonia; Measure the ammonia that is produced with chemiluminescent substance then; With the adenosine deaminase of concentration known and the light signal of measuring, make dose-response curve; The adenosine deaminase content of unknown sample can come out from this curve calculating; Described chemiluminescent substance is a luminol; Described adenosine deaminase can be the adenosine deaminase in the various transudates; Described adenosine deaminase can be the adenosine deaminase in the serum; Described adenosine deaminase can be the adenosine deaminase in the cerebrospinal fluid; Described adenosine deaminase can be the adenosine deaminase in the urine.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 adenosine deaminase 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.The hydrolysis of adenosine deaminase catalysis adenosine produces inosine and ammonia; Measure the ammonia that is produced with chemiluminescent substance then; With the adenosine deaminase of concentration known and the light signal of measuring, make dose-response curve; The adenosine deaminase content of unknown sample can come out from this curve calculating; Described chemiluminescent substance is a luminol; Described adenosine deaminase can be the adenosine deaminase in the various transudates; Described adenosine deaminase can be the adenosine deaminase in the serum; Described adenosine deaminase can be the adenosine deaminase in the cerebrospinal fluid; Described adenosine deaminase can be the adenosine deaminase in the urine.This is the chemical luminescent detecting method of adenosine deaminase in the body fluid of the present invention.
Reaction principle of the present invention is expressed as follows with formula:
One, reagent preparation:
The adenosine damping fluid;
Mixed enzyme solution;
Adenosine deaminase standard (or substitute): 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 |
| Damping fluid | 20 | 20 |
| 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 adenosine deaminase from this curve.Or directly obtain by formula:
Adenosine deaminase 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, is suitable for large, medium and small medical institutions wide popularization and application.
Claims (6)
1, the chemical luminescent detecting method of adenosine deaminase in the body fluid is characterized in that the hydrolysis of adenosine deaminase catalysis adenosine, produces inosine and ammonia; Measure the ammonia that is produced with chemiluminescent substance then; With the adenosine deaminase of concentration known and the light signal of measuring, make dose-response curve; The adenosine deaminase content of unknown sample can come out from this curve calculating.
2, the chemical luminescent detecting method of adenosine deaminase in the body fluid according to claim 1 is characterized in that described chemiluminescent substance is a luminol.
3, the chemical luminescent detecting method of adenosine deaminase in the body fluid according to claim 1 is characterized in that described adenosine deaminase can be the adenosine deaminase in the various transudates.
4, the chemical luminescent detecting method of adenosine deaminase in the body fluid according to claim 1 is characterized in that described adenosine deaminase can be the adenosine deaminase in the serum.
5, the chemical luminescent detecting method of adenosine deaminase in the body fluid according to claim 1 is characterized in that described adenosine deaminase can be the adenosine deaminase in the cerebrospinal fluid.
6, the chemical luminescent detecting method of adenosine deaminase in the body fluid according to claim 1 is characterized in that described adenosine deaminase can be the adenosine deaminase in the urine.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200610114976 CN1912590A (en) | 2006-08-10 | 2006-08-10 | Chemiluminescence measuring method of adenosine deaminase in body fluid |
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| CN 200610114976 CN1912590A (en) | 2006-08-10 | 2006-08-10 | Chemiluminescence measuring method of adenosine deaminase in body fluid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101419238B (en) * | 2008-12-04 | 2012-06-27 | 山东莱博生物科技有限公司 | Hepatitis C virus core antigen chemiluminescence ELISA detection kit |
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2006
- 2006-08-10 CN CN 200610114976 patent/CN1912590A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101419238B (en) * | 2008-12-04 | 2012-06-27 | 山东莱博生物科技有限公司 | Hepatitis C virus core antigen chemiluminescence ELISA detection kit |
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