CN103773833A - Creatinine measurement reagent - Google Patents
Creatinine measurement reagent Download PDFInfo
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- CN103773833A CN103773833A CN201310704186.4A CN201310704186A CN103773833A CN 103773833 A CN103773833 A CN 103773833A CN 201310704186 A CN201310704186 A CN 201310704186A CN 103773833 A CN103773833 A CN 103773833A
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- damping fluid
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Abstract
The invention discloses a creatinine measurement reagent which comprises a reagent I and a reagent II, wherein the reagent I comprises 10-200mmol/L Good's buffering solution with the PH of 7.0-9.0, 5-120KU/L creatine amidine hydrolase, 2-50KU/L sarcosine oxidase, 1-10ml/L surfactant, 1-100mmol/L stabilizing agent and 0.1-50ml/l preservative; the reagent II comprises 10-200mmol/L Good's buffering solution with the PH of 7.0-9.0, 0.1-5mml/L NAD<+>, 3-200KU/L creatinine amidohydrolase, 1-50KU/L formaldehyde dehydrogenase, 1-10ml/L surfactant, 1-100mmol/L stabilizing agent and 0.1-50ml/l preservative. According to the creatinine measurement reagent disclosed by the invention, the problem of interference is solved; and the reagent components are stable.
Description
Technical field
The present invention relates to clinical medicine assay technique field, particularly creatinine assay method and creatinine assay reagent.
Background technology
Creatinine is a kind of low molecule nitrogenous compound, and molecular weight 116000, is mainly produced by muscle metabolism.In muscle, creatine mainly gently forms creatinine by irreversible non-enzyme dehydration reaction, then is discharged in blood, with homaluria.Under normal circumstances, the creatinine content kept stable of human body.The upper limits of normal value of plasma creatinine is 100 micromoles per liter left and right.Creatinine is small-molecule substance, can pass through glomerular filtration, so the change in concentration of serum creatinine is mainly decided by the filtration ability of renal glomerulus.Filtration ability declines, and creatine concentration raises.Serum creatinine value exceeds normal value majority and means that kidney is impaired, and the content height of uric creatinine can reflect that kidney filtering function is impaired, and therefore the laboratory values of creatinine can be reacted the situation of renal function more accurately.
Clinical medicine mensuration blood plasma and uric creatinine mainly adopt enzyme catalysis method and chemical assay at present.Wherein enzyme catalysis method mainly comprises that creatine amidino groups lytic enzyme and creatinine imino-lytic enzyme are hydrolyzed two kinds of methods measuring creatinine.
The method that creatine amidino groups lytic enzyme is measured creatinine comprises two-step reaction, and concrete detailed step is as follows:
The first step:
Second step:
The method adopts double reagent method to remove creatine, and the creatine in the first step and second step is meeting colour generation successively, and the quinonimine that therefore the first step produces is blank node, the amount of the creatinine of the quinonimine producing take second step in the standard specimen that criterion calculation band is measured.The feature that the method developing sensitivity is high, linear response is wide, reagent stability is good, but be subject to vitamins C, haemolysis and bilirubinic interference and affect result.
The method steps of creatinine imino-lytic enzyme measurement creatinine is as follows:
Adopt the method to measure the fall off rate of L-glutamic acid absorbancy at 340nm, just can calculate the content of creatinine.When the method is used, bilirubin, chyle, vitamins C, haemolysis and common drug all do not disturb the mensuration of this method to creatinine.But reagent is stable not and creatinine imines lytic enzyme price used is high.
Summary of the invention
Main purpose of the present invention makes up the defect of existing two kinds of conventional creatinine assay methods, provides a kind of and changes by monitoring 340nm, can avoid vitamins C, haemolysis and bilirubinic interference, creatinine assay reagent in the blood plasma that reagent stability is good simultaneously, price is low.
The present invention proposes a kind of creatinine assay reagent, comprise reagent 1 and reagent 2; Wherein,
Described reagent 1 comprises:
Described reagent 2 comprises:
Preferably, described sanitas is ProClin series sanitas.
Preferably, described sanitas is ProClin300.
Preferably, described tensio-active agent and stablizer are trehalose.
Preferably, described reagent 1 comprises:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.4) 25mmol/L;
Described reagent 2 comprises:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.6) 100mmol/L;
Preferably, described reagent 1 comprises:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.4) 25mmol/L;
Described reagent 2 comprises:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.6) 100mmol/L;
Adopt creatinine assay reagent of the present invention, adopted formaldehyde dehydrogenase-NADH, eliminated creatine amidino groups lytic enzyme and measured creatinine method and be subject to the shortcoming of vitamins C, haemolysis and bilirubinic interference; And eliminate creatinine imino-lytic enzyme and measured the unsettled problem of reagent composition in creatinine method; And test result is accurately stable.
Embodiment
Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
The present invention proposes a kind of creatinine assay reagent, and this creatinine assay reagent comprises reagent 1 and reagent 2;
Wherein each composition of reagent 1 and concentration range are:
Each composition and the concentration range of reagent 2 are:
The principle that adopts above-mentioned creatinine assay reagent of the present invention to carry out creatinine content determination in blood sample mainly comprises " creatine amidino groups lytic enzyme, creatininase, sarcosine oxidase, formaldehyde dehydrogenase " these four kinds enzymatic the first step reactions and the second step reaction by reagent 1 and reagent 2 in above-mentioned creatinine assay reagent.
Wherein, the first step reaction principle is:
Second step reaction principle is:
The present invention adopts the continuous monitoring method of Creatininase-creatine amidino groups lytic enzyme coupling formaldehyde dehydrogenase reaction, the first step reaction object is to remove the impact in mensuration of endogenous creatine in blood plasma, first endogenous creatine in blood plasma is reacted, generate formaldehyde, generate after formaldehyde just and can reduce elimination by the material of reductibility; In second step reaction, creatinine produces creatine under the effect of Creatininase, and creatine produces formaldehyde by the catalysis of creatine amidino groups lytic enzyme, then by coupling formaldehyde dehydrogenase, by oxidized coenzyme (NAD
+) be converted into reduced coenzyme (NADH), by detecting gathering way of the NADH of 340nm place absorbancy, thereby just can calculate the content of creatinine.
And can find out from the component content of mentioned reagent 1 and reagent 2 and the content of the reaction of the above-mentioned the first step and second step reaction, the first step reaction is carried out catalysis by the enzyme in reagent 1, and second step reacts by the enzyme co-catalysis in reagent 1 and reagent 2.
Wherein, Good's damping fluid in above-mentioned creatinine assay reagent is the general designation of 12 kinds of Good's series buffered soln, those skilled in the art can obtain by the scribe's volume of teaching, this a series of damping fluid is mainly used in biological aspect, they have a series of general character, such as pH approaches neutrality, low ionic strength etc.Utilize in the present invention the damping fluid of Good's damping fluid as blood sample, first because its pH value approaches neutral, in use just in time approach the pH value of blood sample, surge capability is the strongest, and because its ionic strength is very low, so can there is not larger destruction in the steady potential combination for each material composition in blood sample, can not change the physical property in blood sample, is conducive to maintain the stability of blood sample.
In reagent composition, major part is organism, and long-term preservation has microbial reproduction growth, causes reagent to lose efficacy.In order to prevent this situation, in reagent of the present invention, contain the sanitas of aforementioned proportion, the principle of its role in being added on food is similar, guarantees that material is not by humic or corruption, further the stability of guaranteed reagent,G.R. makes detected result accurately and reliably.
Further in order to guarantee the homogeneity of solution of the present invention and the stability of contained enzymic activity, and further guarantee the activity of each component materials in diluent wherein in above-mentioned creatinine assay reagent, to be also added with tensio-active agent and stablizer.Further maintain the stability of each material composition.
Particularly, in use, foregoing preservatives of the present invention adopts ProClin series sanitas, especially preferably adopts ProClin300.ProClin series sanitas is as the sanitas adopting in the present invention, comparing other phenylformic acid, Sorbic Acid etc. as sanitas, itself is neutral, does not affect the pH value of blood sample, and its rot-resistant acid-base condition is also neutral, compares the required acidic conditions such as phenylformic acid, Sorbic Acid and want gentle.More importantly, ProClin series sanitas can not exert an influence to the association rate of the crucial enzyme-to-substrate in diagnostic reagent.
Further, in the above-described embodiment, the present invention preferably adopts trehalose as stablizer, and trehalose is by α, α-1, the nonreducing sugar that 1-glycosidic link forms by 2 glucose; Remove outside constitutionally stable factor, include multiple hydrophobic hydroxyls.More importantly, trehalose can form unique protective membrane at cell surface, and therefore protected protein matter molecule unchangeability inactivation effectively also has the protectant effect of excellent activity of enzyme in the use of mentioned reagent.Further, due to said protection film, therefore can further realize the effect of stablizer, achieve many things at one stroke.
Embodiment 1
In the present embodiment 1, adopt reagent 1 to comprise:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.4) 25mmol/L
Reagent 2 comprises:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.6) 100mmol/L
Test procedure is as follows: by sample (making sample with calibration object when calibration) 6ul, reagent 200ul mixes; After 5 minutes, add 60ul reagent 2, mix.Read 340nm absorbancy one time second at interval of 10-30, obtain the data of 10-20 point, calculate △ A
340/min.Detection calibration product under similarity condition, and record data calculate the creatinine content of sample.
Embodiment 2:
In the present embodiment 2, adopt reagent 1 to comprise:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.4) 25mmol/L
Reagent 2 comprises:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.6) 100mmol/L
Test procedure is as follows: sample 10ul, add in 400ul reagent 1, and 37 ℃ of temperature, react 5 minutes; Add reagent 2100ul to mix, after 1 minute, read absorbance A
1; 2 minutes, interval, reads absorbance A
2.Replace sample to measure equally with caliberator, and record data calculate the creatinine content of sample.
Embodiment 3:
Each composition and the concentration range of reagent 1 are:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH8.0) 10mmol/L;
Each composition and the concentration range of reagent 2 are:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH8.5) 10mmol/L;
Carry out creatinine measurement according to the above-mentioned step identical with embodiment 1.
Embodiment 4:
Each composition and the concentration range of reagent 1 are:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH8.0) 200mmol/L;
Each composition and the concentration range of reagent 2 are:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH8.5) 200mmol/L;
Carry out creatinine measurement according to the above-mentioned step identical with embodiment 2.
It is that 210-224 μ mol/L(is wherein added with the interfering substances such as 10% bilirubin, chyle, vitamins C in addition that the present invention selects target value) standard control liquid under the same conditions, according to the creatinine assay reagent of above-described embodiment 1-4 to the creatine concentration continuous detecting of same control liquid 4 times, wherein be spaced apart 5h with the time test of latter twice first twice, result is as shown in table 1:
As can be seen from Table 1, because Indicator Reaction of the present invention has adopted formaldehyde dehydrogenase-NADH, eliminated creatine amidino groups lytic enzyme and measured creatinine method and be subject to the shortcoming of vitamins C, haemolysis and bilirubinic interference.In the result of test and for embodying the result that affects of interfering substances such as being added with in addition 10% bilirubin, chyle, vitamins C.And in the test result of front twice and latter twice, result is substantially identical, therefore also can judge that having eliminated creatinine imino-lytic enzyme measures the reagent composition factors of instability in creatinine method.
The foregoing is only the preferred embodiments of the present invention; not thereby limit the scope of the claims of the present invention; every equivalent transformation that utilizes description of the present invention to do, or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (6)
1. a creatinine assay reagent, is characterized in that, comprises reagent 1 and reagent 2; Wherein, described reagent 1 comprises:
Described reagent 2 comprises:
2. creatinine assay reagent as claimed in claim 1, is characterized in that, described sanitas is ProClin series sanitas.
3. creatinine assay reagent as claimed in claim 2, is characterized in that, described sanitas is ProClin300.
4. creatinine assay reagent as claimed any one in claims 1 to 3, is characterized in that, described tensio-active agent and stablizer are trehalose.
5. creatinine assay reagent as claimed any one in claims 1 to 3, is characterized in that, described reagent 1 comprises:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.4) 25mmol/L;
Described reagent 2 comprises:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.6) 100mmol/L;
6. creatinine assay reagent as claimed any one in claims 1 to 3, is characterized in that, described reagent 1 comprises:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.4) 25mmol/L;
Described reagent 2 comprises:
N-tri-(methylol) methyl-3-aminopropanesulfonicacid acid damping fluid (PH7.6) 100mmol/L;
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310704186.4A CN103773833A (en) | 2013-12-19 | 2013-12-19 | Creatinine measurement reagent |
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| CN201310704186.4A CN103773833A (en) | 2013-12-19 | 2013-12-19 | Creatinine measurement reagent |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861631A (en) * | 2016-06-06 | 2016-08-17 | 天津市宝坻区人民医院 | Ultraviolet spectrophotometry determination method of creatinine in serum |
| CN106198509A (en) * | 2016-06-15 | 2016-12-07 | 四川迈克生物科技股份有限公司 | For measuring test kit and the method for creatinine |
| CN108181246A (en) * | 2017-12-19 | 2018-06-19 | 中国医学科学院阜外医院 | The kit of mortality risk after a kind of prediction heart infarction |
| CN108771961A (en) * | 2018-07-03 | 2018-11-09 | 珠海华泽生物科技有限公司 | A kind of biological enzyme removes formaldehyde agent and its application |
| US11022623B2 (en) | 2017-12-14 | 2021-06-01 | Maccura Medical Instrument Co., Ltd. | Sample transport method and apparatus, test instrument and computer-readable storage medium |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861631A (en) * | 2016-06-06 | 2016-08-17 | 天津市宝坻区人民医院 | Ultraviolet spectrophotometry determination method of creatinine in serum |
| CN106198509A (en) * | 2016-06-15 | 2016-12-07 | 四川迈克生物科技股份有限公司 | For measuring test kit and the method for creatinine |
| CN106198509B (en) * | 2016-06-15 | 2018-08-31 | 迈克生物股份有限公司 | Kit and method for measuring creatinine |
| US11022623B2 (en) | 2017-12-14 | 2021-06-01 | Maccura Medical Instrument Co., Ltd. | Sample transport method and apparatus, test instrument and computer-readable storage medium |
| CN108181246A (en) * | 2017-12-19 | 2018-06-19 | 中国医学科学院阜外医院 | The kit of mortality risk after a kind of prediction heart infarction |
| CN108771961A (en) * | 2018-07-03 | 2018-11-09 | 珠海华泽生物科技有限公司 | A kind of biological enzyme removes formaldehyde agent and its application |
| CN108771961B (en) * | 2018-07-03 | 2021-05-04 | 珠海华泽生物科技有限公司 | Biological enzyme formaldehyde removing agent and application thereof |
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Application publication date: 20140507 |