CN106868097A - The urea reagent and detection method of a kind of good stability - Google Patents
The urea reagent and detection method of a kind of good stability Download PDFInfo
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- CN106868097A CN106868097A CN201510915365.1A CN201510915365A CN106868097A CN 106868097 A CN106868097 A CN 106868097A CN 201510915365 A CN201510915365 A CN 201510915365A CN 106868097 A CN106868097 A CN 106868097A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/58—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving urea or urease
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
Abstract
The present invention relates to urea detection technique field, more particularly to a kind of urea detection reagent contains imidazole buffer, BSA in reagent R1, ɑ -one glutaric acids, sucrose, trehalose, GLDH, polyethylene glycol, DTAC, lauryl imidazolinium betaine, preservative；Contain Tris buffer solutions, BSA, urase, sucrose, trehalose, NADH, preservative in reagent R2.Using imidazole buffer, stabilizer BSA is added, sucrose, trehalose, polyethylene glycol, xanthans, mannitol greatly strengthen the stability of reagent；Using novel surfactant DTAC, lauryl imidazolinium betaine not only significantly improves the performance of measure, and enhances the stability of reagent.
Description
Technical field
The present invention relates to urea detection technique field, more particularly to a kind of good stability, sensitivity urea detection examination high
Agent, further relates to use the detection method of this detection reagent.
Background technology
Urea（Urea）It is the end-product of mammalian proteins catabolism.Circulated by ornithine in liver and synthesized,
Mainly by RE, freely can be leached from glomerulus.Because the molecular weight of urea is small and is easy to dissolving, extension is very big,
Therefore the urea concentration in cerebrospinal fluid, dropsy of serous cavity, saliva, sweat is basically identical.Plasma wrea concentration mainly receives renal function and egg
The influence of white matter intake and catabolism situation.According to relevant document report：When the content of human body urea has exceeded the suction of itself
After receipts ability, it will liver, kidney, alveolar to human body etc. cause harm.UREA contents increase visible following three situation：（1）
Kidney increases and sees acute nephritis, chronic nephritis, toxic nephritis, serious pyelonephritis, nephrophthisis, nephro-angiosclerosis disease, elder generation
The renal dysfunction that nature polycystic kindey and kidney neoplasms etc. cause.Especially there is special value to uremic diagnosis, it increases journey
Degree is directly proportional to disease severity.（2）Property increases and sees congestive heart failure, severe burn, shock, alimentary canal and go out greatly before kidney
Blood, dehydration, severe infections, diabetic ketoacidosis, hypoadrenocorticism, hepatorenal syndrome etc..（3）Property increases and sees after kidney
In nephridial tissue pressure is increased because of urinary obstruction, make glomerular filtration pressure drop low, such as the urethra caused by hypertrophy of the prostate, oncothlipsis
Obstruction or both sides ureteral calculi etc..Therefore, the urea in serum, blood plasma or urine is determined in clinical diagnosis with important
Meaning, therefore how to select a simplicity, quick, effective, accurate detection method, be in important in analysis method research
Hold.
At present, the assay method both at home and abroad to urea has a lot, and being summed up mainly has following several：Direct colo(u)rimetry,
Indirect colourimetry, chromatography, mid-infrared light spectrometry etc..But clinical labororatory determines urea most common method diacetyl-oxime
Method, o-phthalaldehyde method, enzyme coupling rate method etc..
Diacetyl monoxime method also makes the diacetyl monoxime method, the principle of the method to be：DAM is met acid and is hydrolyzed into diethyl
Acyl, under strong acid heating condition, there is the red former diazine of Fearon reaction generations in urea and diacetyl, reagent shade with
Urea content is directly proportional.The method agents useful for same raw material is easily obtained and low cost, but need to be heated and boiled in operation, and linear model
Enclose narrow, and agents useful for same has toxicity and corrosivity, easily pollutes environment.Laboratory practices behaviour is only applicable in this way
Carried out under work, be generally unsuitable for clinical sites quick detection.The principle of OPA colorimetric method is：In sour environment,
Urea acts on antipyrine generation yellow compound with the conjugate of OPA, is in deep after the latter and the conjunction of metavanadic acid root knot
Blueness, the color and luster depth is proportional with urea content.The method is linear up to 60mmol/L, and preci-sion and accuracy and correlation are all
Very well, stable reagent low cost, is capable of achieving automated analysis, and the coefficient of variation is small, and disturbing factor is few.But the sensitivity of the method
It is relatively low, it is not easy detection for urea micro in solution, the method has a foregone conclusion in the relatively low sample of detection urea concentration
It is sex-limited.Enzyme coupling rate method principle is that the ammonia that urase decomposing urea is produced is deposited in T- ketone valeric acid and DPNH (N ADH)
Under, through glutamte dehydrogenase (GLDH) catalysis generation glutamic acid.Meanwhile, N ADH are oxidized to NAD (N
AD).Wherein N ADH have absworption peak at 340nm wavelength, in speed and testing sample that its absorbance declines the content of urea into
Direct ratio.This method advantage is that specific high, quick, result is accurately accurate, is the first-selected chemical method for determining urea at present, but should
Method reagent stability is poor.
In consideration of it, the present invention is on the basis of enzyme coupling rate method, and optimizing reaction system, using imidazoles-HCL buffer solutions,
Addition plurality of stable agent, significantly improves the stability of reagent, is a kind of urea detection reagent of stabilization.
The content of the invention
Serum urea content is detected it is an object of the invention to provide a kind of reagent for detecting urea and using the reagent
Method.The kit uses enzyme coupling rate method, can be good, steady with correlation with the content of effective detection urea in serum
Qualitative good the advantages of.
General principle：
Urase
Urea+2H2O2NH4 + + 2HCO3 -
Glutamte dehydrogenase
KG+NH4 + + NADH Pidolidone+NAD+ + H2O。
What the present invention was obtained through the following steps：
A kind of urea detection reagent, including reagent R1 and reagent R2, the composition of the reagent R1 and reagent R2 are as follows：
Contain in reagent R1：
Imidazoles-HCL buffer solutions·······································100mmol/L,
BSA··················································1g/L,
ɑ-KG·················································8mmol/L,
Sucrose·················································5g/L,
Trehalose···············································2g/L,
Polyethylene glycol·············································5g/L,
Xanthans···············································2g/L,
Mannitol···············································4g/L,
Maltose···············································5g/L
GLDH·················································2KU/L,
Triton x-100········································2mL/L,
DTAC·································0.5g/L,
Lauryl imidazolinium betaine···································3ml/L,
Preservative···············································0.5g/L；
The component of reagent R2 is：
Tris-HCL buffer solutions·······································100mmol/L,
BSA················································ · ·1g/L,
NADH·················································3mmol/L
Sucrose·················································5g/L,
Trehalose···············································2g/L,
Urase·················································2KU/L,
Polyethylene glycol·············································5g/L,
Xanthans···············································2g/L,
Mannitol···············································4g/L,
Maltose···············································5g/L,
Preservative·················································0.5g/L。
Described urea detection reagent, buffer solution is 25 DEG C in reagent R1, and pH is 7.2 imidazoles-hydrochloride buffer.
Described urea detection reagent, buffer solution is 25 DEG C in reagent R2, and pH is 9.3 Tris- hydrochloride buffers.
Described urea detection reagent, the preservative is NaN3。
Described urea detection reagent detects the detection method of urea, using automatic clinical chemistry analyzer using it is fixed when
Between method be measured, detection dominant wavelength be 340nm.
The ratio of described detection method, R1 reagents and R2 reagents is 3:1.
Beneficial effects of the present invention：
1）Using new buffer system and stabilizer, the stability of reagent is significantly improved；
2）Using reagents such as sucrose, trehalose, xanthans and mannitol, the stability of reagent is effectively raised；
3）Using novel surfactant DTAC and lauryl imidazolinium betaine, not only significantly improve
The performance of measure, and enhance the stability of reagent；
4）The degree of accuracy of reagent and have good stability, it is cheap, it is easy to use, can meet clinical needs completely.
Brief description of the drawings
Fig. 1 is two kinds of correlation curve figures of reagent,
Fig. 2 is two kinds of reagent effect phase stability curve figures.
Specific embodiment
The present invention is further described with reference to specific embodiment：
Embodiment 1
The detection reagent of urea, including reagent R1 and reagent R2：
1）The composition of its R1 is：
Imidazoles-HCL buffer solutions············································100mmol/L,
BSA·······················································1g/L,
ɑ-KG······················································8mmol/L
Sucrose······················································5g/L,
Trehalose····················································2g/L,
Polyethylene glycol··················································5g/L,
Xanthans····················································2g/L,
Mannitol····················································4g/L,
Maltose····················································5g/L,
GLDH······················································2KU/L,
Triton x-100·············································2mL/L,
DTAC······································0.5g/L,
Lauryl imidazolinium betaine········································3ml/L,
Preservative····················································0.5g/L；
2) component of reagent R2 is：
Tris-HCL buffer solutions···········································100mmol/L,
BSA······················································1g/L,
NADH·····················································3mmol/L
Sucrose·····················································5g/L,
Trehalose···················································2g/L,
Urase·····················································2KU/L,
Polyethylene glycol·················································5g/L,
Xanthans···················································2g/L,
Mannitol···················································4g/L,
Maltose···················································5g/L,
Preservative···················································0.5g/L。
3）The application method of the present embodiment reagent：
The urea detection reagent of the present embodiment description, when in use using the automatic clinical chemistry analyzer with double reagent function,
Such as the fully-automatic analyzer of Hitachi 7180, is measured using fixed time.By R1 and R2 according to 3:It is right that 1 ratio is placed into
On the reagent position answered, distilled water, standard items and sample, operation such as table 1 are placed in the correspondence position of sample disc：
The reagent test method of 1 embodiment of table 1
Calculate：Urea content（mmol/L）=（Determine ÷ standards）× C standards.
Embodiment 2
Correlation is tested：Reagent preparation, the State Food and Drug Administration accreditation common with market are formulated using embodiment 1
The urea kit of certain company carry out control test, while have detected 20 clinical serum samples, the testing result such as institute of table 2
Show.And obtain two kinds of correlation curves of reagent（As shown in Figure 1）, shown by testing result, two correlations of kit
Coefficient is 0.9997, illustrates that both have great correlation.
The reagent of 2 embodiment of table 1 and market are common and urea that get the nod determines kit contrasting detection result
Embodiment 3
The effect phase stability contrast test of reagent：Uniform to dispense 13 groups to the reagent in embodiment 1, every group of amount of reagent R1 is
15mL, R2 are 5mL；And take 2 groups of urea reagents of certain company of the common State Food and Drug Administration's accreditation in market
Box is compared.It is placed into 2-8 DEG C of refrigerator, the group reagent of taking-up on the same day detection urea quality-control product monthly（Target value is
7.31mmol/L）, testing result is as shown in Fig. 2 more common than the market urea under 2-8 DEG C of condition of storage of the reagent of embodiment 1 is surveyed
Determine kit more to stablize.
By checking, this reagent is good with similar detection reagent contrast correlation, and clinical detection sample results are consistent, Neng Gouda
To market to the application requirement of product, be it is a kind of more stablize, good urea detection reagent.
Claims (6)
1. a kind of urea detection reagent, it is characterised in that including reagent R1 and reagent R2, the composition of the reagent R1 and reagent R2
It is as follows：
Contain in reagent R1
Imidazoles-HCL buffer solutions··············································100mmol/L,
BSA·························································1g/L,
ɑ-KG························································8mmol/L,
Sucrose························································5g/L,
Trehalose······················································2g/L,
Polyethylene glycol····················································5g/L,
Xanthans······················································2g/L,
Mannitol······················································4g/L,
Maltose······················································5g/L,
GLDH························································2KU/L,
Triton x-100···············································2mL/L,
DTAC········································0.5g/L,
Lauryl imidazolinium betaine·········································3ml/L,
Preservative······················································0.5g/L；
The component of reagent R2 is：
Tris-HCL buffer solutions··············································100mmol/L,
BSA·························································1g/L,
NADH························································3mmol/L
Sucrose························································5g/L,
Trehalose······················································2g/L,
Urase························································2KU/L,
Polyethylene glycol····················································5g/L,
Xanthans······················································2g/L,
Mannitol······················································4g/L,
Maltose······················································5g/L,
Preservative······················································0.5g/L。
2. urea detection reagent according to claim 1, it is characterised in that buffer solution is 25 DEG C in reagent R1, and pH is 7.2
Imidazoles-hydrochloride buffer.
3. urea detection reagent according to claim 1, it is characterised in that buffer solution is 25 DEG C in reagent R2, and pH is 9.3
Tris- hydrochloride buffers.
4. urea detection reagent according to claim 1, it is characterised in that the preservative is NaN3。
5. the urea detection reagent any one of a kind of usage right requirement 1-4 urinate in serum, blood plasma or urine to be detected
The detection method of element, it is characterised in that be measured using fixed time using automatic clinical chemistry analyzer, detects dominant wavelength
It is 340nm.
6. detection method according to claim 5, it is characterised in that the ratio of R1 reagents and R2 reagents is 3:1.
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Cited By (3)
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| CN110192452A (en) * | 2019-05-08 | 2019-09-03 | 东南大学 | A kind of catalysis of plant urase with kind is careless combines sand-fixation method |
| CN111657573A (en) * | 2019-03-08 | 2020-09-15 | 天津大学 | Local color-changing swimsuit with water quality detection function |
| CN113957121A (en) * | 2021-10-11 | 2022-01-21 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Urea determination kit |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111657573A (en) * | 2019-03-08 | 2020-09-15 | 天津大学 | Local color-changing swimsuit with water quality detection function |
| CN110192452A (en) * | 2019-05-08 | 2019-09-03 | 东南大学 | A kind of catalysis of plant urase with kind is careless combines sand-fixation method |
| CN113957121A (en) * | 2021-10-11 | 2022-01-21 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Urea determination kit |
| CN113957121B (en) * | 2021-10-11 | 2024-04-26 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Urea determination kit |
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