CN112522366A - Uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum - Google Patents
Uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum Download PDFInfo
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Abstract
The invention relates to a uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum, which has the technical scheme that: the reagent comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises buffer solution, creatinase, sarcosine oxidase, ascorbic acid oxidase, peroxidase or catalase, serum albumin, a nonionic surfactant, a preservative, Trinder's reagent A, laccase or metallate; the reagent R2 contained buffer, serum albumin, creatinine, preservative, and Trinder's reagent B. The invention overcomes the defect that the creatinine measurement is inaccurate due to negative deviation caused by calcium dobesilate and etamsylate contained in serum samples when creatinine is measured in clinical examination of various hospitals at present, and is suitable for serum creatinine detection of a full-automatic biochemical analyzer.
Description
The present case is the divisional application, specifically is application number: 2020108215098, the name is: a uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum and a preparation method thereof are disclosed, and the application date is as follows: 2020.08.15.
Technical Field
The invention belongs to the technical field of medical examination, and relates to a uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum.
Background
Uric Acid (UA) is the end product of purine metabolism in vivo, and is weakly acidic. Uric acid can be obtained from the body or from the catabolism of purines in food. In vivo, uric acid is mainly produced in the liver, a small part of which is excreted through the liver along with bile, and the majority of the rest is excreted from the kidney. Uric acid has low solubility and is therefore likely to crystallize at a high in vivo concentration. Uric acid is considered to be closely related to the occurrence and development of gout, cardiovascular and cerebrovascular diseases, metabolic syndrome, ureteral calculus, kidney diseases, and the like. In recent years, the number of people suffering from hyperuricemia and gout has been on the rise with the improvement of living standard and the change of inclusion structure of people, especially the increase of food intake rich in protein and purine. Therefore, the clinical detection of the serum uric acid concentration has very important reference value and significance, and the detection principle is as follows:
the existing commercial detection kit is double reagents, the uricase-peroxidase coupling method based on Trinder reaction has the advantages of sensitivity, suitability for automatic biochemical analyzers and the like, a reagent R1 contains peroxidase and Trinder's A reagent, after R2 is added, R2 contains uricase and Trinder's reagent B, the uricase hydrolyzes uric acid into allantoin and H2O2, and H2O2 and the peroxidase in R1 and the Trinder's A reagent are jointly existed for color generation and determination. The reaction process utilizes the specificity of enzyme, but generates H2O2Is a strong oxidant, and is easily consumed by reducing drugs in serum to generate negative interference.
Uric acid is the final metabolite of purine, and when purine metabolism is abnormal or the excretion of uric acid by kidney is obstructed, the uric acid concentration in blood can be increased or reduced; gout, renal dysfunction, malignant tumor, heavy metal poisoning caused by cadmium, lead, etc., and medicines such as salicylic acid and purinolWhen the medicine is taken, the blood uric acid content is reduced due to serious liver diseases and increased renal excretion. Calcium dobesilate is used as a common medicine for protecting and treating capillaries, etamsylate is used as a common medicine for stopping bleeding, the medicines are strong reducing agents, and when a patient taking the medicines measures the content of uric acid in blood plasma, the reducing medicines and uricase decompose H generated by uric acid to generate2O2Direct reaction, consuming H2O2The negative deviation of the uric acid level can be serious, and the misjudgment of the doctor during diagnosis and treatment can be caused.
Disclosure of Invention
The invention discloses a uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum, wherein one or more of oxidation type metal acid salt with high oxidation-reduction potential, vanadium pentoxide or vanadium oxytrichloride are added into a reagent R1, and calcium dobesilate and etamsylate are oxidized by utilizing the oxidation characteristic of the metal acid salt, vanadium pentoxide or vanadium oxytrichloride, so that H2O2 generated by decomposition of uric acid by uricase after the reagent R2 is added is prevented from being consumed, the operation is simple, and accurate uric acid measurement value is quickly obtained.
The invention also discloses a uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises buffer solution, peroxidase, ascorbic acid oxidase, preservative, Trinder's reactant A and metal acid salt; the reagent R2 contains buffer, serum albumin, uricase, preservative, and Trinder's reagent B. The proper metal acid salt is selected as the oxidant to eliminate the negative deviation caused by the interference of calcium dobesilate and etamsylate in serum during the uric acid determination, simultaneously the stability of each component in the reagent R1 and the reagent R2 is not influenced, the calcium dobesilate and the etamsylate in the serum can be oxidized before the uricase (the reagent R2) is added, and the consumption of H generated during the uric acid determination is avoided2O2So that the uric acid measurement value is accurate.
Preferably, the metalate comprises one or more of oxidation state nickelate, ferrite, cobaltate, chromate, manganate and vanadate, the vanadate is one or more of metavanadate, poly-vanadate, vanadium pentoxide or vanadium oxytrichloride, and the high oxidation-reduction potential metalate comprises sodium, potassium and ammonium salts containing the metal acid. In multiple experiments, it is found that the oxidation states nickelate, cobaltate, chromate, manganate, vanadate, metavanadate, polyvanadate, vanadic anhydride or vanadyl trichloride with high oxidation-reduction potential can be selected as oxidants to eliminate negative deviation caused by interference of calcium dobesilate and etamsylate in serum during uric acid determination by adopting 23, 24, 25, 26, 27 and 28 bits of metal element acid salts in the periodic table of elements, and the stability of each component in the reagent R1 and the reagent R2 is not influenced.
Preferably, the Trinder's reactant A is 4-Aminoantipyrine (4-Aminoantipyrine, 4-AAP for short). In a series of experiments, the invention discovers that 4-AAP can coexist with the metal acid salt or vanadium pentoxide and vanadium oxychloride as well as other components in the reagent R1 only by adding the reagent R1, and Trinder's reacts with another reagent phenol compound and uricase to coexist in the reagent R2, so that the uric acid kit can eliminate the interference of calcium dobesilate and phenolsulfoethylamine, can keep the stability of the kit for more than one year at the temperature of 2-8 ℃, and can effectively reduce the interference of ascorbic acid in blood samples on uric acid determination when the amount of the ascorbic acid oxidase is in the range.
Preferably, in the reagent R1, the concentration of a buffer solution is 5-200 mM, and the pH value is 6.5-9.5;
the content of the peroxidase is 1.0-10 ku/L;
1.0-10 ku/L ascorbic acid oxidase;
trinder's reagent A0.3-5 mM/L;
when the weight ratio of the preservative is 0.01-0.5%, a metalate oxidant or a vanadium pentoxide or a vanadium oxychloride oxidant with the content of 0.1-10 mM is adopted.
Preferably, the buffer solution of the reagent R1 is one or more of BES, BICINE, DIPSO, EPPS, PIPES, HEPES, MOPS, TAPS, TAPSO, TES, Tricine, Tris, glycylglycine and phosphate; the buffer solution of the reagent R2 is one or more of BES, BICINE, DIPSO, EPPS, PIPES, HEPES, MOPS, TAPS, TAPSO, TES, TRICINE, TRIS, diglycine and phosphate.
Preferably, the reagent R1 and the reagent R2 further contain a nonionic surfactant, the weight ratio of the nonionic surfactant is 0.01-0.3%, and the nonionic surfactant is specifically one or two of BRIJ, EMULGEN, TRITON and TWEEN.
Preferably, the preservative is one or two of Proclin, gentamicin sulfate, PARABEN, chloramphenicol, sodium azide and borax. The antiseptic can inhibit the action of biological enzyme in the reagent, so that the reagent can keep stable efficacy in a certain time, such as borax, sodium azide and the like.
Preferably, in the reagent R2, the concentration of a buffer solution is 10-200 mM, and the pH value is 6.5-9.0; the uricase content is 5-20 ku/liter; the Trinder's reagent B content is 0.1-20 mM.
Preferably, the Trinder's reagent B in the reagent R2 is one or more of TOOS, DHBS, DAOS, HDAOS, TBHBA, TOPS, TODB and 4-chlorophenol.
A preparation method of a uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum comprises the following steps:
preparation of reagent R1:
weighing buffer solution → adding preservative → stirring and dissolving → adjusting pH to 8.0 with sodium hydroxide or hydrochloric acid → adding metalate → stirring and dissolving → adjusting pH to 8.0 → adding Trinder's reactant A and nonionic surfactant, mixing uniformly → 2-8 ℃ overnight → adding ascorbic acid oxidase and peroxidase → stirring and dissolving → 2-8 ℃ overnight → detecting, and subpackaging;
preparation of reagent R2:
weighing buffer solution → adding preservative → stirring and dissolving → adjusting pH to 8.0 → adding Trinder's reactant B and surfactant → mixing evenly → overnight at 2-8 deg.C → adding serum albumin, uricase → mixing evenly → overnight at 2-8 deg.C → detecting and subpackaging.
The uric acid kit consists of a reagent R1 and a reagent R2, wherein the reagent R1 is firstly mixed with a specimen, for example, the specimen contains hydroxyphenyl which is a therapeutic drugWhen calcium sulfonate or etamsylate is used, the oxidized metal acid salt, vanadium pentoxide or vanadium oxytrichloride in the reagent R1 can be oxidized first, so that H generated by the reaction of uricase added into the reagent R2 is avoided2O2Consumed by the above-mentioned medicine, at the same time the reagent R2 contains Trinder's reactant B, i.e. phenol compound and H produced by reaction of 4-AAP in reagent R1 and uricase2O2Color is developed, the absorbance is measured, and the content is calculated.
The invention has the beneficial effects that:
the uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate in serum has the advantages of good stability, simplicity in operation and rapidness in detection reaction, particularly, peroxidase and uricase are respectively prepared in a reagent R1 and a reagent R2, and the kit is different from the existing kit components in the market, can prolong the stability of the kit within a period of time, continuously eliminates negative interference of calcium dobesilate and etamsylate, avoids misjudgment of doctors in clinical diagnosis, can accurately use medicines and treat, and is suitable for a full-automatic biochemical analyzer.
The preparation method of the uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate in serum has the advantages of good kit stability, simplicity and rapidness in operation, elimination of negative interference of calcium dobesilate and etamsylate, high accuracy and suitability for a full-automatic biochemical analyzer.
Detailed Description
The uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate in serum of the invention is further described by combining specific embodiments as follows:
the interference of calcium dobesilate and etamsylate is determined by a uricase method kit which is currently sold and approved by the national drug administration of China, and the results are shown in the table I:
the reagent composition is as follows in the specification:
[ major Components]From the reagent R1Reagent R2And a calibrator.
Reagent R1: dichlorohydroxybenzenesulfonic acid (DHBS), phosphate buffer;
reagent R2: uricase, peroxidase (in cross-substrate peroxidase in the component of R1), 4-aminoantipyrine.
Calibration products: the creatinine-containing aqueous solution with the concentration shown in the label can be traced to the Randox CAL3 calibrator.
[ measurement method ]
(1) The double reagents are not required to be prepared and are directly used.
(2) The test conditions are as follows: sample (S): 3 μ l reagent 1 (R1): 240 μ l reagent 2 (R2): temperature of 60. mu.l: 37 deg.C
The determination type is as follows: dominant wavelength of endpoint method: sub-wavelength of 505 nm: 660nm reaction direction: rise up
The method comprises the following steps: the standard or sample is mixed with R1, the reagent R2 is added after 5 minutes at 37 ℃, and then the reaction absorbance 5 minutes after the reagent R2 is added is measured.
Measurement of blank Absorbance (A)1) Measurement of reaction Absorbance (A)2)
Sample preparation: 3 μ l
R1：240μl R2：60μl
(3) And (3) calibration procedure: calibration is performed using a calibrator, which should be performed each time a reagent batch is changed. After calibration, each laboratory was verified with quality controls. If the quality control result is not in the acceptable range value, recalibration is needed.
(4) Quality control procedure: selecting proper quality control material for quality control. And establishing respective quality control frequency and acceptable range value by each laboratory. When the measurement result is out of the acceptable range, it is necessary to take corresponding measures.
(5) Computing
Measurement instruments and parameters:
unit: mu mol/L, normal value 90-420 mu mol/L
The measuring instrument: hitachi 7180
Measurement parameters are as follows: r1: r2: (sample size) 240:60: 3. mu.l
Primary/secondary wavelength: 505/660nm
2POINT END,INC.
Table 1 interference results determined by the existing two-reagent creatinine enzymatic kit: (unit: mu mol/L)
And (4) conclusion: the measurement result shows that calcium dobesilate and etamsylate both cause negative deviation on the uric acid measurement result.
Specifically, the effect of the uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate in serum is described in combination with examples 1 to 4:
table 2 reagent components for examples 1-4:
the creatinine reagents according to the present invention are described in detail as follows:
example 1:
reagent R1 component:
reagent R2 component:
example 2:
reagent R1 component:
reagent R2 component:
example 3:
reagent R1 component:
reagent R2 component:
example 4:
reagent R1 component:
reagent R2 component:
the preparation process of examples 1-4 comprises the following steps:
preparation of reagent R1:
weighing buffer solution → adding preservative → stirring and dissolving → adjusting pH to 8.0 with sodium hydroxide or hydrochloric acid → adding metalate → stirring and dissolving → adjusting pH to 8.0 → adding Trinder's reactant A and nonionic surfactant, mixing uniformly → 2-8 ℃ overnight → adding ascorbic acid oxidase and peroxidase → stirring and dissolving → 2-8 ℃ overnight → detecting, and subpackaging;
preparation of reagent R2:
weighing buffer solution → adding preservative → stirring and dissolving → adjusting pH to 8.0 → adding Trinder's reactant B and surfactant → mixing evenly → overnight at 2-8 deg.C → adding serum albumin, uricase → mixing evenly → overnight at 2-8 deg.C → detecting and subpackaging.
The determination method comprises the following steps:
placing the reagent R1 and the reagent R2 prepared in examples 1-4 at the corresponding reagent position of a full-automatic blood coagulation analyzer (Hitachi 7180), sucking the reagent R1210 μ l, the reagent R270 μ l, the calibration solution or the specimen 10 μ l, the calibration solution concentration 300 μ M, the main/auxiliary wavelength 540/660nm, the two-point end point method, the reaction direction: and INC, calibrating and measuring creatinine values of various samples containing calcium dobesilate or etamsylate with different concentrations by using the calibration solution.
TABLE 3 measurement results of example 1: (unit: mu mol/L)
| Measurement of | 1 | 2 | 3 | Mean value of | Relative deviation of |
| Mother liquor | 356 | 355 | 356 | 355.7 | |
| Calcium dobesilate 8ml/L | 357 | 355 | 356 | 356 | 0.1％ |
| Calcium dobesilate 16ml/L | 357 | 358 | 356 | 357 | 0.4％ |
| 32ml/L calcium dobesilate | 356 | 356 | 355 | 355.7 | 0.0％ |
| Calcium dobesilate 64ml/L | 349 | 348 | 349 | 348.7 | 2.0％ |
| Etamsylate 12.5mg/L | 357 | 358 | 358 | 357.7 | 0.6％ |
| Etamsylate 25mg/L | 357 | 357 | 357 | 357.0 | 0.4％ |
| Etamsylate 50mg/L | 357 | 356 | 355 | 356.0 | 0.1％ |
| Etamsylate 100mg/L | 358 | 357 | 358 | 357.7 | 0.6％ |
TABLE 4 measurement results of example 2: (unit: mu mol/L)
TABLE 5 results of the measurements of example 3: (unit: mu mol/L)
| Measurement of | 1 | 2 | 3 | Mean value of | Relative deviation of |
| Mother liquor | 360 | 360 | 362 | 361.0 | |
| Calcium dobesilate 8ml/L | 360 | 360 | 362 | 360.7 | －0.1％ |
| Calcium dobesilate 16ml/L | 361 | 361 | 361 | 361 | 0.0％ |
| 32ml/L calcium dobesilate | 360 | 360 | 359 | 359.7 | －0.4％ |
| Calcium dobesilate 64ml/L | 359 | 359 | 360 | 359.3 | －0.2％ |
| Etamsylate 12.5mg/L | 360 | 360 | 360 | 360.0 | －0.3％ |
| Etamsylate 25mg/L | 360 | 361 | 359 | 360.0 | －0.3％ |
| Etamsylate 50mg/L | 358 | 360 | 359 | 359 | －0.6％ |
| Etamsylate 100mg/L | 359 | 359 | 358 | 358.7 | －0.6％ |
TABLE 6 measurement results of example 4: (unit: mu mol/L)
| Measurement of | 1 | 2 | 3 | Mean value of | Relative deviation of |
| Mother liquor | 360 | 360 | 360 | 360.0 | |
| Calcium dobesilate 8ml/L | 361 | 360 | 361 | 360.7 | 0.2％ |
| Calcium dobesilate 16ml/L | 360 | 361 | 358 | 359.7 | -0.1％ |
| 32ml/L calcium dobesilate | 359 | 359 | 358 | 358.7 | -0.4％ |
| Calcium dobesilate 64ml/L | 358 | 357 | 357 | 357.3 | -0.8％ |
| Etamsylate 12.5mg/L | 361 | 360 | 361 | 360.7 | 0.2％ |
| Etamsylate 25mg/L | 361 | 360 | 359 | 360.0 | 0％ |
| Etamsylate 50mg/L | 359 | 359 | 360 | 359.3 | -0.2％ |
| Etamsylate 100mg/L | 359 | 357 | 359 | 358.3 | -0.5％ |
The results of the above examples 1-4 show that the uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate in serum of the present invention can effectively eliminate interference of calcium dobesilate and etamsylate in uric acid measurement by adding high oxidation-reduction potential oxidized ferrate, or poly-metavanadate, or vanadium pentoxide or vanadyl trichloride, and the kit has good stability, simple operation, and rapid detection reaction, and particularly, peroxidase and uricase are respectively prepared in reagents R1 and R2, which is different from the existing kit components in the market, can prolong the stability of the kit in a period of time, continuously eliminate negative interference of calcium dobesilate and etamsylate, and avoid misjudgment of doctors in clinical diagnosis, can accurately use drugs and treatments, and is suitable for full-automatic biochemical analyzers.
Claims (6)
1. A uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum is characterized in that: the reagent comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises a buffer solution, peroxidase, ascorbic acid oxidase, a preservative, Trinder's reactant A, an oxidant and a nonionic surfactant; the reagent R2 comprises buffer, serum albumin, uricase, preservative, Trinder's reagent B and non-ionic surfactant; the Trinder's reactant A is 4-aminoantipyrine, the oxidant comprises any one of vanadium pentoxide, sodium ferrate, sodium metavanadate and vanadium oxychloride, and the Trinder's reactant B is one or a mixture of more than two of TOOS, DHBS, HDAOS, TBHBA, TOPS, TODB and 4-chlorophenol; the peroxidase is bound to uricase.
2. The uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum according to claim 1, wherein the uric acid kit comprises:
in the R1, the concentration of a buffer solution is 5-200 mM, and the pH value is 6.5-9.5;
the content of the peroxidase is 1.5-9 ku/liter;
1-10 ku/L ascorbic acid oxidase;
trinder's reagent A0.3-5 mM/L;
0.01-0.5 percent of preservative by weight and 0.1-10 mM of oxidant.
3. The uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum according to claim 1, wherein the uric acid kit comprises: the buffer solution of the R1 is one or more of BES, BICINE, DIPSO, EPPS, PIPES, HEPES, MOPS, TAPS, TAPSO, TES, Tricine, Tris, glycylglycine and phosphate; the buffer solution of the reagent R2 is one or more of BES, BICINE, DIPSO, EPPS, PIPES, HEPES, MOPS, TAPS, TAPSO, TES, TRICINE, TRTS, glycylglycine and phosphate.
4. The uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum according to claim 1, wherein the uric acid kit comprises: the reagent R1 and the reagent R2 further contain nonionic surfactants, the weight ratio of the nonionic surfactants is 0.01-0.3%, and the nonionic surfactants are specifically one or two of BRIJ, EMULGEN, TRITON and TWEEN.
5. The uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum according to claim 1, wherein the uric acid kit comprises: the preservative is one or two of Proclin, gentamicin sulfate, PARABEN, chloramphenicol, sodium azide and borax.
6. The uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum according to claim 1, wherein the uric acid kit comprises: in the R2, the concentration of a buffer solution is 20-200 mM, and the pH value is 6.5-9.5; the uricase content is 5-20 ku/liter; the Trinder's reagent B content is 0.1-20 mM.
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| CN202011578745.8A CN112522366A (en) | 2020-08-15 | 2020-08-15 | Uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum |
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| CN202011578744.3A Active CN112522365B (en) | 2020-08-15 | 2020-08-15 | Preparation method of uric acid kit capable of eliminating interference of calcium dobesilate and phenol sulfoethylamine drugs in serum |
| CN202011578733.5A Pending CN112626170A (en) | 2020-08-15 | 2020-08-15 | Uric acid kit for quickly and simply eliminating drug interference and preparation method thereof |
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| CN112710609B (en) * | 2020-12-23 | 2022-03-04 | 中生北控生物科技股份有限公司 | Anti-chyle interference uric acid determination kit |
| CN114441516A (en) * | 2021-12-20 | 2022-05-06 | 苏州百源基因技术有限公司 | Uric acid detection kit and preparation method thereof |
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| CN106367472A (en) * | 2016-09-29 | 2017-02-01 | 四川迈克生物科技股份有限公司 | Kit and method for determining uric acid |
| CN108627654A (en) * | 2018-06-25 | 2018-10-09 | 武汉瀚海新酶生物科技有限公司 | Composition for eliminating Calcium Dobesilate drug to the detection interference of creatinine enzyme process |
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| IT1130252B (en) * | 1980-02-04 | 1986-06-11 | Elvi Spa | METHOD FOR THE ELIMINATION OF BILIRIBUNA INTERFERENCE IN THE DOSAGE OF HYDROGEN PEROXIDE THROUGH A MODIFIED TRINDER REACTION |
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| CN103278468B (en) * | 2013-05-24 | 2015-09-02 | 宁波美康生物科技股份有限公司 | A kind of creatinine detection reagent |
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| CN112662736A (en) | 2021-04-16 |
| CN112522364A (en) | 2021-03-19 |
| CN111733211B (en) | 2021-02-02 |
| CN112522365B (en) | 2023-12-26 |
| CN111733211A (en) | 2020-10-02 |
| CN112522364B (en) | 2023-09-08 |
| CN112626170A (en) | 2021-04-09 |
| CN112522365A (en) | 2021-03-19 |
| CN112662736B (en) | 2024-02-02 |
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