CN103364468A - Reagent for detecting urea nitrogen by urease electrode method - Google Patents
Reagent for detecting urea nitrogen by urease electrode method Download PDFInfo
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- CN103364468A CN103364468A CN2013103081222A CN201310308122A CN103364468A CN 103364468 A CN103364468 A CN 103364468A CN 2013103081222 A CN2013103081222 A CN 2013103081222A CN 201310308122 A CN201310308122 A CN 201310308122A CN 103364468 A CN103364468 A CN 103364468A
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Abstract
The invention discloses a reagent for detecting urea nitrogen by a urease electrode method and is characterized in that the reagent comprises a buffer, urease, a stabilizing agent and an antiseptic. The reagent provided by the invention has good relativity, wide linear range of detection and more rapid detection speed. More importantly, the reagent is free from interference of endogenous substances, and results are more reliable.
Description
Technical field
The present invention relates to the reagent that a kind of urea Enzyme Electrode detects urea nitrogen.
Background technology
Urea nitrogen (BUN) be one to the test item of clinical important in inhibiting, this be since BUN be one of leading indicator of renal function, be widely used clinically, be in great demand.
BUN is the main end-product of human body protein metabolism.The amino acid deaminizating produces NH
3And CO
2, both are urea synthesis in liver, and every gram protein metabolism produces urea 0.3g.Nitrogen content is 28/60 in the urea, almost reaches half.Common kidney is for draining the major organs of urea, and urea all can heavily absorb at each section tubule behind glomerular filtration, but the faster heavily absorption of urine flow velocity is fewer in the renal tubule, has also namely reached maximal clearance.Early stage at kidney function damage, blood BUN can be in normal range, and when glomerular filtration rate(GFR drops to normal 50% when following, the concentration of blood BUN just raises rapidly.
The higher clinical manifestation of BUN content is: 1, the chronic renal failure due to the various primary glomerulonephritises of organic kidney function damage: a., pyelonephritis, interstitial nephritis, kidney neoplasms, the polycystic kindey etc.; B. the acute renal failure renal function is slight when impaired, and BUN can be unchanged, but glomerular filtration rate(GFR (GFR) drops to below 50%, and BUN can raise.Therefore measure can not be as the Renal function in early period index for blood BUN, but to chronic renal failure, and especially the degree that increases of uremic BUN is general consistent with coincident with severity degree of condition: the compensatory phase GFR of kidney failure drops to 50ml/min, blood BUN＜9mmol/L; Kidney failure is lost the compensatory phase, blood BUN〉9mmol/L; The kidney failure phase, blood BUN〉20mmol/L.2, property oliguresis before the kidney: the hypovolemia, the renal blood flow minimizing hypoperfusion that cause such as serious dehydration, a large amount of ascites, cardiac cycle MSOF, hepatorenal syndrome etc. cause oliguresis.This moment, BUN raise, but the creatinine rising is not obvious, BUN/Cr(mg/dl)〉10:1, be called prerenal azotemia.Increase through dilatation urine amount multipotency, but the BUN automatic descending.3, protein decompose or Excessive Intake such as acute infectious disease, high heat, massive bleeding from upper digestive tract, large-area burns, severe trauma, major operation after and thyroid function is high, high-protein diet etc., but serum creatinine does not generally raise.After above situation was corrected, blood BUN can descend.
The method that clinical labororatory's tradition is measured BUN is colourimetry, is broadly divided into two kinds.The 1st kind is enzyme process, with urase urea decomposition is become ammonium ion and carbonate first, and then with the growing amount of ammonium ion in Polaises reaction or glutamte dehydrogenase (GLDH) the assaying reaction process, the former is the growing amount of the blue indoxyl of monitoring under 560nm; The latter utilizes NADH to be oxidized to NAD, in the speed of 340nm wavelength place monitoring absorbance decline.The 2nd kind is chemical method, the acetyl group of diacetyl-oxime directly with urea condensation reaction, generation chromogen diazine (diazine).
Along with promoting the use of and the update of Biochemical Analyzer of large automatic Biochemical Analyzer, the assay method that is widely used in the BUN of clinical laboratory is taken as the leading factor from traditional colourimetry and is transitioned into gradually the situation that enzymic colorimetric and two kinds of methods of Enzyme Electrode are laid equal stress on.
Enzymic colorimetric is according to the enzymatic reaction of BUN under urase catalysis, generation has the material of characteristic absorption peak, under specific wavelength, measure the speed that absorbance changes, calculate the concentration of BUN in the solution, using is the NADH enzyme coupling method more widely, but there are two large major defects in the party's science of law, in the situation that the Blood Ammonia Concentration in the serum increases, can consume simultaneously GLDH and NADH, so that testing result is inaccurate; Endogenic enzyme and GLDH competitive oxidation NADH, thus the accuracy of enzymatic coupling system reduced.For this two large defective, the solution that there is no so far can be come correcting parameter according to diagnostic result clinically.
The urea Enzyme Electrode is according to electrochemical principle, utilizes the linear relationship of the logarithm of given ionic mobility in electromotive force and the solution, directly measures the concentration of effects of ion.On electrode film connects behind the biology enzyme, just the selectivity quick and enzymatic reaction of electrode method can be combined, make measurement result reliable, quick, and can avoid the endogenous material in the sample to exert an influence by contrast colors, guarantee the reliability of testing result, and then provide real data for clinical diagnosis.
On detection speed, Enzyme Electrode can go out the result in average 30 seconds, and traditional enzymic colorimetric is time-consuming more than 1 minute, and therefore, Enzyme Electrode more is applicable to emergency treatment, in time provides testing result for the clinician, strives for valuable treatment time.
In addition, other performance index of Enzyme Electrode such as the range of linearity, accuracy, are compared with traditional enzymic colorimetric and also to be met the methodology requirement, and have good correlativity, are fit to modern fast clinical examination demand.
But, still do not utilize Enzyme Electrode to detect the report of urea nitrogen content at present, more not applicable Enzyme Electrode detects the reagent of urea nitrogen content; Therefore, how to provide a kind of reagent that Enzyme Electrode is measured BUN content that is applicable to, become the technical matters that this area needs to be resolved hurrily.
Summary of the invention
The present invention is directed to the above-mentioned deficiency of prior art, a kind of good correlativity that has is provided, the wider detection range of linearity, detection speed is quicker, the more important thing is the interference that is not subjected to endogenous material, the reagent of more reliably urea Enzyme Electrode detection of result urea nitrogen.
For solving the problems of the technologies described above, concrete technical scheme of the present invention is: a kind of urea Enzyme Electrode detects the reagent of urea nitrogen, and this reagent comprises: damping fluid, urase, stabilizing agent and antiseptic.
The above-mentioned urea Enzyme Electrode of the present invention detects the reagent of urea nitrogen, and each component concentration of this reagent is:
Damping fluid (pH6.0): 50-500mmol/L,
Urase: 10-500KU/L,
Stabilizing agent: 0.1-10g/L,
Antiseptic: 0.1-10g/L.
In the above-mentioned component of the present invention, damping fluid is citric acid-trisodium citrate damping fluid, glycine buffer, Tris damping fluid or acetic acid-sodium-acetate buffer.
The above-mentioned stabilizing agent of the present invention is glycerine or octanol.
The above-mentioned antiseptic of the present invention is formaldehyde, Sodium azide or PC antiseptic.
It is as follows that the above-mentioned BUN of the present invention measures preparation method of reagent thereof: the composition of reagent described in the dilution is added to mix behind the distilled water according to formula rate stir evenly.
The cardinal principle of Enzyme Electrode of the present invention is, urea is by urase catalysis, hydrolysis generates ammonium ion, bicarbonate radical and hydroxide ion, transfer ion to by nonionic, the electric conductivity of solution changes, the speed that electrical conductivity of solution increases is directly proportional with the concentration of urea in the reaction cup, measures the concentration of BUN by the increment rate of measuring electrical conductivity of solution.
The key reaction equation is:
Advantage of the present invention and beneficial effect:
1. the invention provides the reagent that a kind of urea Enzyme Electrode detects urea nitrogen content, thereby successfully solved in the situation that the Blood Ammonia Concentration in the serum that traditional enzymic colorimetric exists increases, can consume simultaneously GLDH and NADH, so that testing result is inaccurate; Endogenic enzyme and GLDH competitive oxidation NADH, thus two large major defects of the accuracy of enzymatic coupling system reduced.
2. urea Enzyme Electrode provided by the invention detects the reagent of urea nitrogen content, is according to electrochemical principle, utilizes the linear relationship of the logarithm of given ionic mobility in electromotive force and the solution, directly measures the concentration of effects of ion.On electrode film connects behind the biology enzyme, just the selectivity quick and enzymatic reaction of electrode method can be combined, make measurement result reliable, quick, and can avoid the endogenous material in the sample to exert an influence by contrast colors, guarantee the reliability of testing result, and then provide real data for clinical diagnosis.On detection speed, Enzyme Electrode can go out the result in average 30 seconds, and traditional enzymic colorimetric is time-consuming more than 1 minute, and therefore, Enzyme Electrode more is applicable to emergency treatment, in time provides testing result for the clinician, strives for valuable treatment time.In addition, other performance index of Enzyme Electrode such as the range of linearity, accuracy, are compared with traditional enzymic colorimetric and also to be met the methodology requirement, and have good correlativity, are fit to modern fast clinical examination demand.
Description of drawings
The value (Y) that the value (X) that accompanying drawing reagent of the present invention records records HK method reagent is the linear as a result figure of regretional analysis.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment, but be not limited to following embodiment.
Embodiment 1
The citric acid of pH6.0-trisodium citrate damping fluid 200mmol/L,
Urase: 300KU/L,
Glycerine: 5g/L,
Sodium azide: 5g/L.
Test sample book, the BUNm module on Beckman LX20 automatic clinical chemistry analyzer is used mentioned reagent, and namely BUN electrode method reagent carries out test sample, and the CC on this Biochemical Analyzer partly carries out test sample with BUN enzyme coupling rate method reagent simultaneously.
Detect 40 parts of clinical serum samples with reagent of the present invention and enzyme coupling rate method reagent respectively, the former roughly finished whole tests in 15 minutes and goes out the result, and the latter roughly finished test in 40 minutes and goes out the result.Testing result shows that serum BUN value roughly is distributed in the 2.0-9.0mmol/L scope, and major part concentrates on 4.0-5.0mmol/L.The value (Y) that the value (X) that records with reagent of the present invention records HK method reagent is the linear as a result figure of regretional analysis and is seen Fig. 1, and the result is as follows: Y=0.9908X+0.0507, R2=0.9969.
The Tris damping fluid 50mmol/L of pH6.0,
Urase: 50KU/L,
Octanol: 0.5g/L,
Formaldehyde: 0.5g/L.
Test sample book, the BUNm module on Beckman LX20 automatic clinical chemistry analyzer is used mentioned reagent, and namely BUN electrode method reagent carries out test sample, and the CC on this Biochemical Analyzer partly carries out test sample with BUN enzyme coupling rate method reagent simultaneously.
Detect the sample that linear high value is pressed gradient dilution with reagent of the present invention and enzyme coupling rate method reagent respectively, the result shows that the former enough reaches 54.0mmol/L at its detection line performance, and the latter can reach 36.0mmol/L.
Embodiment 3
The acetic acid of pH6.0-sodium-acetate buffer 500mmol/L,
Urase: 500KU/L,
Glycerine: 10g/L,
PC300：10g/L。
Test sample book, the BUNm module on Beckman LX20 automatic clinical chemistry analyzer is used mentioned reagent, and namely BUN electrode method reagent carries out test sample, and the CC on this Biochemical Analyzer partly carries out test sample with BUN enzyme coupling rate method reagent simultaneously.
Detect the serum sample that adds interfering material with reagent of the present invention and enzyme coupling rate method reagent respectively, the result shows that the former reaches 30g/L to unconjugated bilirubin respectively, combined with bilirubin reaches 28.8g/L, vitamin C reaches 3g/L, haemoglobin reaches 500g/L, and chyle reaches 1450 turbidity and all do not produce interference; And the latter reaches 2.0g/L to unconjugated bilirubin respectively, and combined with bilirubin reaches 2.88g/L, and vitamin C reaches 0.3g/L, and haemoglobin reaches 50g/L, and chyle reaches 145 turbidity all can produce interference, and all is just to disturb.
Claims (5)
1. a urea Enzyme Electrode detects the reagent of urea nitrogen, and it is characterized in that: this reagent comprises: damping fluid, urase, stabilizing agent and antiseptic.
2. urea Enzyme Electrode according to claim 1 detects the reagent of urea nitrogen, and it is characterized in that: each component concentration of this reagent is:
Damping fluid (pH6.0): 50-500mmol/L,
Urase: 10-500KU/L,
Stabilizing agent: 0.1-10g/L,
Antiseptic: 0.1-10g/L.
3. urea Enzyme Electrode according to claim 2 detects the reagent of urea nitrogen, and it is characterized in that: described damping fluid is citric acid-trisodium citrate damping fluid, glycine buffer, Tris damping fluid or acetic acid-sodium-acetate buffer.
4. urea Enzyme Electrode according to claim 2 detects the reagent of urea nitrogen, and it is characterized in that: described stabilizing agent is glycerine or octanol.
5. urea Enzyme Electrode according to claim 2 detects the reagent of urea nitrogen, and it is characterized in that: described antiseptic is formaldehyde, Sodium azide or PC antiseptic.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108085279A (en) * | 2017-12-29 | 2018-05-29 | 重庆大学 | In a kind of calcareous sand urease-producing bacterium efficiently separate and identification method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0707074A1 (en) * | 1994-10-13 | 1996-04-17 | Kyowa Medex Co., Ltd. | Method for determination of urea nitrogen |
| CN101571488A (en) * | 2008-04-28 | 2009-11-04 | 北京华大吉比爱生物技术有限公司 | Method and kit for measuring urea nitrogen |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0707074A1 (en) * | 1994-10-13 | 1996-04-17 | Kyowa Medex Co., Ltd. | Method for determination of urea nitrogen |
| CN101571488A (en) * | 2008-04-28 | 2009-11-04 | 北京华大吉比爱生物技术有限公司 | Method and kit for measuring urea nitrogen |
Non-Patent Citations (3)
| Title |
|---|
| JOHN H.GLICK ET AL.: "Preparation of Reagents for Use with the Beckman Astra-8", 《CLINICAL CHEMISTRY》, vol. 26, no. 2, 31 December 1980 (1980-12-31), pages 358 - 359 * |
| NORBERT S.NORKUS ET AL.: "Four Commercial Urease Reagents and a Laboratory-Prepared Reagent Compared for Analysis of Blood Urea Nitrogen with the Beckman Analyzer", 《CLINICAL CHEMISTRY》, vol. 22, no. 5, 31 December 1976 (1976-12-31), pages 683 - 685 * |
| 刘宝琦等: "脉酶的制备及终点法检测尿素氮试剂盒的配制", 《吉林大学自然科学学报》, no. 3, 31 December 1993 (1993-12-31), pages 111 - 115 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108085279A (en) * | 2017-12-29 | 2018-05-29 | 重庆大学 | In a kind of calcareous sand urease-producing bacterium efficiently separate and identification method |
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