CN1912589A - Chemiluminescence measuring method of urea in serum - Google Patents
Chemiluminescence measuring method of urea in serum Download PDFInfo
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- CN1912589A CN1912589A CN 200610114971 CN200610114971A CN1912589A CN 1912589 A CN1912589 A CN 1912589A CN 200610114971 CN200610114971 CN 200610114971 CN 200610114971 A CN200610114971 A CN 200610114971A CN 1912589 A CN1912589 A CN 1912589A
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- urea
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- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 239000004202 carbamide Substances 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 27
- 210000002966 serum Anatomy 0.000 title claims abstract description 17
- 239000000126 substance Substances 0.000 claims abstract description 24
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 18
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229910021529 ammonia Inorganic materials 0.000 claims abstract description 9
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 5
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 5
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 4
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 4
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 7
- 235000013922 glutamic acid Nutrition 0.000 claims description 7
- 239000004220 glutamic acid Substances 0.000 claims description 7
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims description 6
- 230000003647 oxidation Effects 0.000 claims description 6
- 238000007254 oxidation reaction Methods 0.000 claims description 6
- 210000002700 urine Anatomy 0.000 claims description 6
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 5
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 5
- 101710088194 Dehydrogenase Proteins 0.000 claims description 4
- 231100000673 dose–response relationship Toxicity 0.000 claims description 4
- HGBOYTHUEUWSSQ-UHFFFAOYSA-N pentanal Chemical compound CCCCC=O HGBOYTHUEUWSSQ-UHFFFAOYSA-N 0.000 claims description 4
- 210000002381 plasma Anatomy 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 abstract 2
- 230000001590 oxidative effect Effects 0.000 abstract 1
- 238000001514 detection method Methods 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 230000008901 benefit Effects 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 230000008676 import Effects 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 238000005375 photometry Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010018366 Glomerulonephritis acute Diseases 0.000 description 1
- 208000001940 Massive Hepatic Necrosis Diseases 0.000 description 1
- 206010037601 Pyelonephritis chronic Diseases 0.000 description 1
- 201000000660 Pyloric Stenosis Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 231100000851 acute glomerulonephritis Toxicity 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 201000006368 chronic pyelonephritis Diseases 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000028208 end stage renal disease Diseases 0.000 description 1
- 201000000523 end stage renal failure Diseases 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 235000008085 high protein diet Nutrition 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000003243 intestinal obstruction Diseases 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 201000008383 nephritis Diseases 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 231100000462 teratogen Toxicity 0.000 description 1
- 239000003439 teratogenic agent Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
A method for determining urea in serum by utilizing chemiluminescence includes reacting urea with urase to release ammonia, reacting on ammonia with some chemicals to produce glusate, oxidizing glusate by oxidase to generate perhydrol, acting perhydrol with peroxidase to make chemiluminescent matter be lightened, making light signal be more stronger when urea concentration is more greater, using known urea concentration and light signal to work out dosage-reaction curve for calculating out urea content in unknown sample by using said curve.
Description
Technical field: the present invention relates to a kind of medical agent detection method, particularly the chemical luminescent detecting method of urea in the serum.
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.
Serum urea or urea nitrogen are subjected to the influence of physiological and pathologic factor.Physiologic factor has: high-protein diet serum urea and urine discharge rate significantly increase; The serum urea male sex is higher than the women; Raise with the age increase; The gestation woman is lower than non-pregnant woman; Different time also changes in one day.
The pathologic blood urea increases: before the kidney there be property, dehydration makes the blood urea retention; As hyperemesis, pyloric stenosis, intestinal obstruction, long-term diarrhea etc.; Kidney: acute glomerulonephritis, End Stage Renal Disease, renal failure, chronic pyelonephritis, toxic nephritis etc.; Behind the kidney there be the property disease: prostate enlargement, lithangiuria, urinary tract are narrow, tumor of bladder etc.
The plain minimizing of blood urine is more rare, merges massive hepatic necrosis as hepatitis.
The method of urea is colourimetry in the mensuration serum that now generally uses, this method is to utilize urase to make urea emit ammonia, the latter and α-pentanone diacid and NADH urge at glutamte dehydrogenase and generate glutamic acid etc. down, and the fall off rate of NADH is directly proportional with content of urea, measures the concentration of urea with this.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.Colourimetry 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.
Summary of the invention: technical matters to be solved by this invention is a kind of chemical luminescent detecting method that can overcome urea in above-mentioned defective, highly sensitive, the serum 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 urea in the serum is characterized in that urea emits ammonia under the urase effect; Ammonia and α-pentanone diacid and NADH urge at glutamte dehydrogenase and generate glutamic acid down; And glutamic acid generates hydrogen peroxide through (GLOD) oxidation of its oxidase; Hydrogen peroxide makes the chemiluminescent substance oxidation and luminous under the effect of peroxidase; The size of light signal and the concentration of urea are proportionate, and promptly the big more light signal that sends of urea concentration is strong more; Write down this light signal and can infer the concentration of urea; With the urea of concentration known and the light signal of measuring, make dose-response curve; The urea content of unknown sample can come out from this curve calculating; Described chemiluminescent substance is a luminol; Described urea can be the urea in the blood plasma; Described urea also can be the urea in the urine.The present invention compared with prior art has following advantage: the method is characterized in that: 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 urea 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.Urea is emitted ammonia under the urase effect; Ammonia and α-pentanone diacid and NADH urge at glutamte dehydrogenase and generate glutamic acid down; And glutamic acid generates hydrogen peroxide through (GLOD) oxidation of its oxidase; Hydrogen peroxide makes the chemiluminescent substance oxidation and luminous under the effect of peroxidase; The size of light signal and the concentration of urea are proportionate, and promptly the big more light signal that sends of urea concentration is strong more; Write down this light signal and can infer the concentration of urea; With the urea of concentration known and the light signal of measuring, make dose-response curve; The urea content of unknown sample can come out from this curve calculating; Described chemiluminescent substance is a luminol; Described urea can be the urea in the blood plasma; Described urea also can be the urea in the urine; This is the chemical luminescent detecting method of urea in the serum of the present invention.This method can be used for measuring the urea content in blood plasma, serum or the urine.
One, reagent comprises:
Mixed enzyme solution;
Urea 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 | 100 | 100 |
| 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 urea or urea nitrogen from this curve.Or directly obtain by formula:
Urea concentration=(sample RLU/ standard RLU) * normal concentration
Urea nitrogen=urea concentration * 28
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 (4)
1, the chemical luminescent detecting method of urea in the serum is characterized in that urea emits ammonia under the urase effect; Ammonia and α-pentanone diacid and NADH urge at glutamte dehydrogenase and generate glutamic acid down; And glutamic acid generates hydrogen peroxide through (GLOD) oxidation of its oxidase; Hydrogen peroxide makes the chemiluminescent substance oxidation and luminous under the effect of peroxidase; The size of light signal and the concentration of urea are proportionate, and promptly the light signal that sends more very much of urea concentration is strong more; Write down this light signal and can infer the concentration of urea; With the urea of concentration known and the light signal of measuring, make dose-response curve; The urea content of unknown sample can come out from this curve calculating.
2, the chemical luminescent detecting method of urea in the serum according to claim 1 is characterized in that described chemiluminescent substance is a luminol.
3, the chemical luminescent detecting method of urea in the serum according to claim 1 is characterized in that described urea can be the urea in the blood plasma.
4, the chemical luminescent detecting method of urea in the serum according to claim 1 is characterized in that described urea also can be the urea in the urine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610114971 CN1912589A (en) | 2006-08-10 | 2006-08-10 | Chemiluminescence measuring method of urea in serum |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200610114971 CN1912589A (en) | 2006-08-10 | 2006-08-10 | Chemiluminescence measuring method of urea in serum |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102928468A (en) * | 2012-09-14 | 2013-02-13 | 北京体育大学 | Whole blood urea biosensing test paper |
| CN104388537A (en) * | 2014-11-28 | 2015-03-04 | 山东博科生物产业有限公司 | Reagent for detecting urea contained in blood serum |
| CN105277537A (en) * | 2015-10-12 | 2016-01-27 | 江苏三联生物工程有限公司 | Method for detecting enzyme activity and chemiluminescence reaction substrate performance |
| CN108918435A (en) * | 2018-04-17 | 2018-11-30 | 武汉景川诊断技术股份有限公司 | Determination of bilirubin method and kit |
-
2006
- 2006-08-10 CN CN 200610114971 patent/CN1912589A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102928468A (en) * | 2012-09-14 | 2013-02-13 | 北京体育大学 | Whole blood urea biosensing test paper |
| CN102928468B (en) * | 2012-09-14 | 2016-05-25 | 北京体育大学 | Whole blood urea bio-sensing test paper |
| CN104388537A (en) * | 2014-11-28 | 2015-03-04 | 山东博科生物产业有限公司 | Reagent for detecting urea contained in blood serum |
| CN105277537A (en) * | 2015-10-12 | 2016-01-27 | 江苏三联生物工程有限公司 | Method for detecting enzyme activity and chemiluminescence reaction substrate performance |
| CN105277537B (en) * | 2015-10-12 | 2019-04-16 | 江苏三联生物工程有限公司 | A method of detection enzymatic activity and chemiluminescence reaction substrate performance |
| CN108918435A (en) * | 2018-04-17 | 2018-11-30 | 武汉景川诊断技术股份有限公司 | Determination of bilirubin method and kit |
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