CN111944872A - Reagent combination, reagent or kit for measuring creatinine content - Google Patents
Reagent combination, reagent or kit for measuring creatinine content Download PDFInfo
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- CN111944872A CN111944872A CN202010895328.XA CN202010895328A CN111944872A CN 111944872 A CN111944872 A CN 111944872A CN 202010895328 A CN202010895328 A CN 202010895328A CN 111944872 A CN111944872 A CN 111944872A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- 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
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- 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
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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- 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
- C12Q1/30—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving catalase
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- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
- C12Q2326/90—Developer
- C12Q2326/96—4-Amino-antipyrine
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90219—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- G01N2333/90222—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3) in general
- G01N2333/90225—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3) in general with a definite EC number (1.10.3.-)
- G01N2333/90235—Ascorbate oxidase (1.10.3.3)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
- G01N2333/9065—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on CH-NH groups of donors (1.5)
- G01N2333/90672—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3) in general
- G01N2333/90677—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3) in general with a definite EC number (1.5.3.-)
- G01N2333/90683—Sarcosine oxidase (1.5.3.1)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
Abstract
The invention relates to the technical field of biology, in particular to a reagent combination, a reagent or a kit for measuring creatinine content. The reagent combination provided by the invention comprises a reagent set 1 and a reagent set 2, wherein the reagent set 1 comprises creatinase, sarcosine oxidase, ascorbic acid oxidase, chromogen and catalase, and the reagent set 2 comprises creatinase, 4-aminoantipyrine and peroxidase, wherein the chromogen is selected from N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline or sodium salt thereof and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-5-dimethoxyaniline. The inhibitor is added into the reagent for determination, so that the generation of new creatine and creatinine in the determination process is blocked, and the interference existing in a sample in creatinine detection is reduced.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a reagent combination, a reagent or a kit for measuring creatinine content.
Background
Creatinine is a low molecular weight nitrogen-containing compound, having a molecular weight of 116000, and is produced primarily by muscle metabolism. In muscle, creatine slowly forms creatinine, primarily through an irreversible, non-enzymatic dehydration reaction, and is released into the blood and excreted with the urine. Under normal conditions, the creatinine content of the human body remains substantially stable. The upper normal limit for plasma creatinine is around 100 micromoles/liter. Creatinine is a small molecule substance that can be filtered through the glomerulus, so the change in serum creatinine concentration is mainly determined by the glomerular filtration ability. The creatinine concentration increases as the filterability decreases. The fact that the blood creatinine value is higher than the normal value mostly means that the kidney is damaged, and the high content of the urine creatinine can reflect that the filtering function of the kidney is damaged, so that the assay value of the creatinine can accurately reflect the condition of the kidney function.
Creatinine is the end product of creatine metabolism. Creatine is partly derived from food intake and partly produced in vivo, and its synthetic raw materials are glycine, arginine and methionine to provide methyl. Creatine is catalyzed by creatine kinase to generate creatine phosphate which is unstable and generates creatinine through dehydration and dephosphorylation.
The current medical clinical determination of plasma and urinary creatinine mainly adopts enzyme catalysis method and chemical determination method.
The enzymatic determination of creatinine is mainly of 3 types: creatininoaminohydrolase: a creatinine enzyme-creatine kinase-pyruvate kinase-lactate dehydrogenase-reduced coenzyme reaction system; sarcosine oxidase; a reaction system of creatininase-creatinase-sarcosine oxidase-peroxidase; ③ creatinine imidohydrolase); creatinine imidohydrolase-N-methyl hydantoin amidohydrolase-N amino formamide sarcosyl amidohydrolase-sarcosine oxidase-peroxidase reaction system. The enzymology method has high cost, high specificity and accurate result, is suitable for various automatic analyzers and can also be used for dry chemistry methods or electrochemical methods.
The principle of the sarcosine oxidase method is as follows: creatinine in the sample is hydrolyzed under the catalysis of creatininase to generate creatine. Creatine is hydrolyzed under the catalysis of creatinase to generate sarcosine and urea. Sarcosine is oxidized into glycine, formaldehyde and hydrogen peroxide under the catalysis of sarcosine oxidase, and is finally coupled with Trinder reaction and measured by a colorimetric method.
A first reaction:
endogenous creatine present in the specimen is decomposed to form oxygen and water, avoiding an effect on the second reaction.
And (3) a second reaction:
the creatinine generates red quinone pigment under the action of various enzymes, and the content of the quinone pigment is in direct proportion to the creatinine in the sample, so that the content of the creatinine is calculated.
In general, the method for measuring the metabolite content does not dynamically consider the dynamic change of the metabolite content. The concentration of creatinine and creatine in the serum was taken as a static fixation. However, in the creatinine assay, the creatinine content is generally less than 110umol/L because of its low content. In such a trace state, the accuracy of the measurement data is easily affected by the amount of newly produced creatine. Therefore, it is of great practical significance to provide a reagent for assaying creatinine.
Disclosure of Invention
In view of the above, the present invention provides a reagent combination, a reagent or a kit for measuring creatinine content. The reagent provided by the invention is used for in vitro quantitative determination of creatinine content in human serum.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a reagent combination for measuring creatinine content, which comprises a reagent group 1 and a reagent group 2;
wherein, the reagent group 1 comprises creatinase, sarcosine oxidase, ascorbic acid oxidase, chromogen, catalase, biological enzyme stabilizer, inhibitor, preservative and surfactant;
the reagent group 2 comprises creatininase, 4-aminoantipyrine, peroxidase, surfactant and preservative;
the inhibitor comprises one or a mixture of more than two of guanidinoacetic acid analogues, methionine analogues or arginine analogues.
In some embodiments of the invention, the inhibitor comprises a combination of one or more of leucine, lysine, lactic acid, quercetin, luteolin, or MOPS (3-morpholinopropanesulfonic acid).
In some embodiments of the invention, the amount of creatinase in reagent group 1 is 20 to 60KU/L, preferably 30 to 50 KU/L; the dosage of the sarcosine oxidase is 8-30 KU/L, preferably 10-20 KU/L; the catalase is used for eliminating hydrogen peroxide generated by endogenous creatine so as to eliminate the interference of the endogenous creatine, the dosage is 100-500 KU/L, preferably 200-300KU/L, and too much catalase can compete for hydrogen peroxide generated by creatinine in the second step to cause a low measured value; the dosage of the ascorbic acid oxidase is 1-20 KU/L, preferably 2-10 KU/L.
In some embodiments of the invention, the chromogen in reagent set 1 comprises a combination of one or more of 3, 5-dichloro-2-hydroxybenzenesulfonic acid, N-ethyl- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxy-4-fluoroaniline, N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline; the concentration is 0.1-20 mmo1/L, preferably 1-5 mmo 1/L;
the biological enzyme protective agent comprises one or a composition of more than two of bovine serum albumin, sucrose, trehalose, propylene glycol or glycerol, the dosage of the biological enzyme protective agent is 1-10 g/L, preferably 3-5 g/L, and the combination effect of multiple protective agents is better;
the surfactant comprises one or more than two of TWEEN, TRITON and Emulgen series, and the dosage is 0.1-3 g/L, preferably 0.3-1 g/L;
the preservative comprises one or more than two of dichloroacetamide, sodium azide and Proclin series; the amount is 0.1-5 g/L, preferably 0.5-3 g/L.
In some embodiments of the invention, the amount of the creatinine enzyme in the reagent combination 2 is 200 to 600KU/L, preferably 300 to 400 KU/L;
the dosage of the peroxidase is 5-30 KU/L, preferably 10-20 KU/L.
In some embodiments of the invention, the amount of the 4-aminoantipyrine is 1 to 10mmol/L, preferably 3 to 5 mmol/L;
the surfactant comprises one or more than two of TWEEN, TRITON and Emulgen series, and the dosage is 0.1-3 g/L, preferably 0.3-1 g/L;
the preservative comprises one or more than two of dichloroacetamide, sodium azide and Proclin series; the dosage is 0.1-5 g/L, preferably 0.5-3 g/L.
In some embodiments of the invention, the reagent buffer is a zwitterionic buffer with a pH in the range of 6.5 to 8.5, preferably pH7.5 to 8.0; the concentration is 50 to 300mmol/L, preferably 100 to 200 mmol/L.
On the basis of the research, the invention also provides application of the reagent combination in preparing a reagent or a kit for measuring creatinine in serum.
The invention also provides a reagent for measuring creatinine in blood serum, which comprises the reagent combination and acceptable auxiliary agents.
The invention also provides a kit for measuring creatinine in serum, which comprises the reagent combination and acceptable auxiliary agents and/or carriers.
The invention adopts a sarcosine oxidase method; the creatinine in a sample is hydrolyzed under the catalysis of the creatinase to generate creatine. Creatine is hydrolyzed under the catalysis of creatinase to generate sarcosine and urea. Sarcosine is oxidized into glycine, formaldehyde and hydrogen peroxide under the catalysis of sarcosine oxidase, and is finally coupled with Trinder reaction and measured by a colorimetric method. The kit comprises a reagent group 1 and a reagent group 2, wherein the reagent group 1 comprises creatinase, sarcosine oxidase, ascorbic acid oxidase, chromogen and catalase, the reagent group 2 comprises creatininase, 4-aminoantipyrine and peroxidase, wherein the chromogen is selected from N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline or sodium salt thereof, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, 2,4, 6-tribromo-3-hydroxybenzoic acid, and N, N-di (4-sulfobutyl) -3-methylaniline disodium salt. The inhibitor is added into the reagent for determination, so that the generation of new creatine and creatinine in the determination process is blocked, and the interference existing in a sample in creatinine detection is reduced.
Detailed Description
The invention discloses a reagent combination, a reagent or a kit for measuring the creatinine content, and a person skilled in the art can appropriately modify process parameters by referring to the contents. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The related terms are interpreted.
TES is a biological buffer, the chinese name: 2- [ (tris (hydroxymethyl) methyl) amino ] -1-ethanesulfonic acid,
the name of English: 2- [ Tris (hydroxymethy) methylamino ] -1-ethanesulfonic acid
TOOS is Trinder's color reagent; the name of Chinese: N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt;
the name of English: sodium 3- (N-ethyl-3-methylilanilino) -2-hydroxyproline sulfonate
An inhibitor is a substance used to retard or reduce the rate of a chemical reaction.
The invention is the serum creatinine determination method, can use in the enzyme method determination creatinine content reagent, make the measured value more accurate. The kit comprises a reagent group 1 and a reagent group 2, wherein the reagent group 1 comprises creatinase, sarcosine oxidase, ascorbic acid oxidase, chromogen, catalase, biological enzyme stabilizer, inhibitor, preservative and surfactant, and the reagent group 2 comprises creatinin, 4-aminoantipyrine, peroxidase, surfactant and preservative.
A reagent buffer solution, which is a zwitterionic buffer solution, and has a pH range of 6.5-8.5, preferably a pH of 7.5-8.0; the concentration is 50 to 300mmol/L, preferably 100 to 200 mmol/L;
the amount of the 4-aminoantipyrine is 1-10 mmol/L, preferably 3-5 mmol/L.
The chromogen is N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline or sodium salt thereof, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, 2,4, 6-tribromo-3-hydroxybenzoic acid, N-di (4-sulfobutyl) -3-methylaniline disodium salt and the like; the concentration may be in the range of 0.1 to 20mmo1/L, preferably 1 to 5mmo 1L.
The dosage of the creatinase is 20-60 KU/L, preferably 30-50 KU/L, the dosage of the creatinase oxidase is 8-30 KU/L, preferably 10-20 KU/L, the catalase in the reagent 1 is used for eliminating hydrogen peroxide generated by endogenous creatine so as to eliminate the interference of the endogenous creatine, the dosage is 100-500 KU/L, preferably 200-300KU/L, and too much catalase competes for the hydrogen peroxide generated by creatinine in the second step, so that the measured value is low.
The inhibitor in the agent is a substance including analogues of guanidinoacetic acid, analogues of methionine and arginine; creatine biosynthesis is formed from glycine, arginine, methionine. Such as: leucine, lysine, lactic acid, but not limited to such things, such as: quercetin (quercetin), luteolin (luteolin), MOPS (3-morpholine propanesulfonic acid) also have good effects. The dosage of the inhibitor is 1-20 g/L, preferably 5-10 g/L;
the dosage of the creatininase in the reagent 2 is 200-600 KU/L, preferably 300-400 KU/L, and the dosage of the peroxidase is 5-30 KU/L, preferably 10-20 KU/L;
the reagent also comprises a biological enzyme protective agent comprising; bovine serum albumin, sucrose, trehalose, propylene glycol or glycerol; the dosage is 1-10 g/L, preferably 3-5 g/L, and the combination effect of various protective agents is better;
the surfactant comprises; TWEEN. TRITON and Emulgen, in an amount of 0.1-3 g/L, preferably 0.3-1 g/L,
the preservative comprises: dichloroacetamide, sodium azide and Proclin series; the amount is 0.1-5 g/L, preferably 0.5-3 g/L.
1. Enzymatic analysis of creatinine is a fundamental approach to address non-specific interferences in creatinine assays. The enzyme method of creatininase coupled with sarcosine oxidase is commonly used in the analysis of creatininase.
2. In order to eliminate the interference of creatine in a sample, the creatinase is added into a first reagent by using the characteristic of a two-reagent method in automatic analysis, and the interference of endogenous creatine can be eliminated by a two-step reaction.
3. The creatinine enzyme method has good specificity, and its reference value is slightly lower than that of picric acid rate method. Foreign data (Tietz Clinical Guide to Laboratory Test (third edition) report the creatinine amidohydrolase-sarcosine oxidase method, adult males are 55-96 mu mol/L, females are 44-66 mu mol/L, domestic reference intervals related to the creatinine enzyme method are less reported, Yangchang et al report that males are 60.6-136.2 mu mol/L, females are 44.8-110.8 mu mol/L, and it is recommended that laboratories best establish the reference intervals of the local area.
4. Creatininase is coupled with sarcosine oxidase, and Trinder reaction is used as an indication system. The sensitivity of different chromogens varies greatly and various reagent manufacturers compete to study and use new types of sensitive chromogens. The chromogens commonly used at present are: 3, 5-dichloro-2-hydroxybenzenesulfonic acid; n-ethyl- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxy-4-fluoroaniline; n- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline, and the like.
The Trinder reaction is interfered by bilirubin and vitamin C, and can be eliminated by adding potassium ferrocyanide (or potassium nitrosoferricyanide) and ascorbate oxidase to reagent I.
6. Heparin, citric acid, ethylene diamine tetraacetic acid, sodium fluoride were not interfered with the assay at conventional dosages.
The enzyme for eliminating creatine is added into a reagent 1, a reagent 2 is added after the reaction is carried out for 4 to 5 minutes, creatinine generates red quinone pigment under the action of various enzymes, and the content of the quinone pigment is in direct proportion to creatinine in a sample, so that the content of the creatinine is calculated. Human serum, however, contains various enzymes and metabolites of human metabolism, and creatinine and creatine are present in dynamic equilibrium. Creatine, an amino acid derivative naturally produced in the human body, can be synthesized by the human body itself, and can also be ingested from food. The invention adds inhibitor to block the creatine to reach accurate determination.
The reagent combination, the reagent or the raw materials and the reagents used in the kit for determining the creatinine content provided by the invention are all available in the market.
The invention is further illustrated by the following examples:
example 1
Reagents 1 and 2 were formulated at the concentrations shown in the following table:
reagent 1:
reagent 2:
comparative example 1
Reagents 1 and 2 were prepared at the concentrations shown in the table below.
Reagent 2:
effect example 1
20 samples were collected, which required no hemolysis and no lipemia, and the values should be 0-1500. mu. mol/L. The reagents prepared in example 1 and comparative example 1 were tested on a fully automated biochemical analyzer, and the two groups of reagents were calibrated with creatinine calibrator (460 μmol/L) from Beading bioengineering (Beijing) Ltd, and the test parameters were:
TABLE 1
| Method of producing a composite material | Two-point end point method | Reaction temperature | 37℃ |
| Dominant wavelength | 548nm | Sub-wavelength | 700nm |
| Sample size | 4μL | Reaction time | 10 minutes |
| Reagent 1 | 240μL | Reagent 2 | 80μL |
The existing method adopts a liquid chromatogram-isotope dilution mass spectrometry method, and the quantitative data is as follows:
TABLE 2
The reagent of example 1 has an added inhibitor, and the reagent of comparative example has no inhibitor; the reagents and comparative methods in example 1 deviate less. The addition of inhibitors enables more accurate measurements.
Effect example 2 Linear section detection
The reagents of example 1 were tested by mixing samples of approximately 1500. mu. mol/L concentration and samples of approximately 5. mu. mol/L concentration into 5 samples of 5 concentrations, and the measurement was repeated 3 times for each concentration to obtain the average of the measurement results. And (4) calculating a linear regression equation by taking the dilution concentration as an independent variable and the mean value of the detection result as a dependent variable. The correlation coefficient (r) of the linear regression is calculated.
TABLE 3
It can be seen that the correlation coefficient (r) between the measured value and the sample concentration value should be greater than 0.990, and meet the industry standard of the existing creatinine kit.
Effect example 3 detection of precision
Under repetitive conditions, high, medium and low 3 levels (high value 300. mu. mol/L)450. mu. mol/L, median 40. mu. mol/L to 80. mu. mol/L, low 10. mu. mol/L to 30. mu. mol/L) samples the reagents of example 1 were tested, the assay was repeated 10 times, and the average of the measurement results was calculated
And standard deviation (S). The Coefficient of Variation (CV) was calculated.
TABLE 4
The precision of the reagent can meet the industry standard of the existing creatinine reagent kit.
Effect example 4 analytical sensitivity
Samples of known concentration Ci (. mu. mol/L) (concentration in the linear interval) were measured 3 times using the reagent of example 1, and the mean change in absorbance (. DELTA.A) was calculated. The change in absorbance was converted to a concentration of 100. mu. mol/L according to the formula (1).
TABLE 5
The analysis sensitivity of the visible reagent meets the industry standard of the existing creatinine kit.
Example 2
Reagents 1 and 2 were formulated at the concentrations shown in the following table:
reagent 1:
reagent 2:
comparative example 2
Reagents 1 and 2 were prepared at the concentrations shown in the table below.
Reagent 2:
effect example 5
20 samples were collected, which required no hemolysis and no lipemia, and the values should be 0-1500. mu. mol/L. The reagents prepared in example 2 and comparative example 2 were tested on a fully automated biochemical analyzer, and the two sets of reagents were calibrated with creatinine calibrator (460 μmol/L) from Beading bioengineering (Beijing) Inc., and the test parameters were:
TABLE 6
| Method of producing a composite material | Two-point end point method | Reaction temperature | 37℃ |
| Dominant wavelength | 548nm | Sub-wavelength | 700nm |
| Sample size | 4μL | Reaction time | 10 minutes |
| Reagent 1 | 240μL | Reagent 2 | 80μL |
The existing method adopts a liquid chromatogram-isotope dilution mass spectrometry method, and the quantitative data is as follows:
TABLE 7
Example 3
Reagents 1 and 2 were formulated at the concentrations shown in the following table:
reagent 1:
reagent 2:
comparative example 3
Reagents 1 and 2 were prepared at the concentrations shown in the table below.
Reagent 2:
effect example 6
20 samples were collected, which required no hemolysis and no lipemia, and the values should be 0-1500. mu. mol/L. The reagents prepared in example 3 and comparative example 3 were tested on a fully automated biochemical analyzer, and the two sets of reagents were calibrated with creatinine calibrator (460 μmol/L) from Beading bioengineering (Beijing) Inc., and the test parameters were:
TABLE 8
| Method of producing a composite material | Two-point end point method | Reaction temperature | 37℃ |
| Dominant wavelength | 548nm | Sub-wavelength | 700nm |
| Sample size | 4μL | Reaction time | 10 minutes |
| Reagent 1 | 240μL | Reagent 2 | 80μL |
The prior method adopts liquid chromatography-isotope dilution mass spectrometry to determine the value data as follows:
TABLE 9
From the above examples, it can be seen that the reagent assay with the addition of inhibitors more closely approximates the prior art method. Liquid chromatography-isotope dilution mass spectrometry is the standard reference method for creatinine determination. Although the reagent without inhibitor is only 3-5% higher; however, the addition of inhibitors can bring the assay closer to the true value. This is also determined by the detection principle, the detection process and the serum characteristics. Creatine is eliminated in the first 5 minutes of the measurement; there was 5 minutes after addition of reagent 2 to the end of the test. The first 5 minutes eliminate creatine, destroying the creatine balance; there was a small amount of creatine produced during the second 5 minutes. Resulting in slightly higher creatinine assay results.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The reagent combination for measuring the creatinine content is characterized by comprising a reagent group 1 and a reagent group 2;
wherein, the reagent group 1 comprises a reagent buffer solution, creatinase, sarcosine oxidase, ascorbic acid oxidase, chromogen, catalase, a biological enzyme stabilizer, an inhibitor, a preservative and a surfactant;
the reagent group 2 comprises reagent buffer solution, creatinin, 4-aminoantipyrine, peroxidase, surfactant and preservative;
the inhibitor comprises one or a mixture of more than two of guanidinoacetic acid analogues, methionine analogues or arginine analogues.
2. The combination of claim 1, wherein the inhibitor comprises a combination of one or more of leucine, lysine, lactic acid, quercetin, luteolin, or MOPS (3-morpholinopropanesulfonic acid).
3. The reagent combination according to claim 1 or 2, wherein the amount of creatinase in reagent group 1 is 20 to 60 KU/L; the dosage of the sarcosine oxidase is 8-30 KU/L; the dosage of the catalase is 100-500 KU/L; the dosage of the ascorbic acid oxidase is 1-20 KU/L, preferably 2-10 KU/L.
4. A combination of reagents according to any one of claims 1 to 3, wherein the chromogen in set 1 comprises one or a combination of two or more of N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline or its sodium salt, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, 2,4, 6-tribromo-3-hydroxybenzoic acid, N-bis (4-sulfobutyl) -3-methylaniline disodium salt; the concentration is 0.1-20 mmo 1/L;
the biological enzyme protective agent comprises one or a composition of more than two of bovine serum albumin, sucrose, trehalose, propylene glycol or glycerol, and the dosage of the biological enzyme protective agent is 1-10 g/L;
the surfactant comprises one or more than two of TWEEN, TRITON and Emulgen series, and the dosage is 0.1-3 g/L;
the preservative comprises one or more than two of dichloroacetamide, sodium azide and Proclin series; the dosage is 0.1-5 g/L.
5. The reagent combination of any one of claims 1 to 4, wherein the amount of creatinine enzyme used in the reagent combination 2 is 200 to 600 KU/L;
the dosage of the peroxidase is 5-30 KU/L.
6. The reagent combination according to any one of claims 1 to 5, wherein the 4-aminoantipyrine is used in an amount of 1 to 10 mmol/L;
the surfactant comprises one or more than two of TWEEN, TRITON and Emulgen series, and the dosage is 0.1-3 g/L;
the preservative comprises one or more than two of dichloroacetamide, sodium azide and Proclin series; the dosage is 0.1-5 g/L.
7. The reagent combination of any one of claims 1 to 6, wherein the reagent buffer is a zwitterionic buffer, having a pH in the range of 6.5 to 8.5; the concentration is 50 to 300 mmol/L.
8. Use of a reagent combination according to any one of claims 1 to 7 in the preparation of a reagent or kit for the determination of creatinine in serum.
9. Reagent for the determination of creatinine in serum, characterized in that it comprises a combination of reagents according to any one of claims 1 to 7, and acceptable auxiliary agents.
10. Kit for the determination of creatinine in serum, characterized in that it comprises a combination of reagents according to any one of claims 1 to 7 and acceptable auxiliaries and/or carriers.
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| CN115201134A (en) * | 2022-09-15 | 2022-10-18 | 吉林大学第一医院 | Creatinine detection kit resistant to piceatannol interference and application thereof |
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| CN115201134B (en) * | 2022-09-15 | 2022-12-27 | 吉林大学第一医院 | Creatinine detection kit resistant to piceatannol interference and application thereof |
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