CN111733211B - Uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum and preparation method thereof - Google Patents

Uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum and preparation method thereof Download PDF

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Publication number
CN111733211B
CN111733211B CN202010821509.8A CN202010821509A CN111733211B CN 111733211 B CN111733211 B CN 111733211B CN 202010821509 A CN202010821509 A CN 202010821509A CN 111733211 B CN111733211 B CN 111733211B
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reagent
uric acid
trinder
calcium dobesilate
etamsylate
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CN111733211A (en
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俞永标
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Zhongshan Biaohong Biotechnology Co ltd
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Zhongshan Bgh Biotechnology Co ltd
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Priority to CN202011578733.5A priority Critical patent/CN112626170A/en
Priority to CN202010821509.8A priority patent/CN111733211B/en
Priority to CN202011578744.3A priority patent/CN112522365B/en
Priority to CN202011578366.9A priority patent/CN112662736B/en
Priority to CN202011578745.8A priority patent/CN112522366A/en
Priority to CN202011578743.9A priority patent/CN112522364B/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/62—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving uric acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
    • C12Q2326/90—Developer
    • C12Q2326/96—4-Amino-antipyrine
    • 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.)
    • 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)
    • 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)
    • G01N2333/90688—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on other nitrogen compounds as donors (1.7)
    • G01N2333/90694—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3), e.g. uricase (1.7.3.3)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to a uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate in serum and a preparation method thereof, and the key points of the technical scheme are as follows: the reagent comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises buffer solution, peroxidase, ascorbic acid oxidase, preservative, Trinder's reactant A, metalate or nonionic surfactant; the reagent R2 contains buffer, serum albumin, uricase, preservative, and Trinder's reagent B or a nonionic surfactant. According to the uric acid kit, one or more of the oxidation type metal acid salt with high oxidation-reduction potential, vanadium pentoxide or vanadium oxychloride is added into R1, calcium dobesilate and etamsylate are oxidized by using the metal acid salt, H2O2 generated by decomposing uric acid by uricase after R2 is added is prevented from being consumed, and an accurate uric acid measurement value is quickly obtained.

Description

Uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum and preparation method thereof
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 and a preparation method thereof.
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:
Figure DEST_PATH_IMAGE001
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, the reagent R1 contains peroxidase and Trinder's A reagent, and the addition of the peroxidase and the Trinder's A reagentAfter R2, R2 contains uricase and Trinder's reagent B, the uricase hydrolyzes uric acid into allantoin and H2O2, and H2O2 is subjected to color development in the presence of peroxidase in R1 and Trinder's A reagent for 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 such as cadmium and lead can cause the increase of blood uric acid concentration, and the blood uric acid content can be reduced due to the taking of medicines such as salicylic acid and purinol and the increase of renal excretion due to severe liver diseases. 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 bagContains buffer solution, 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, and if the specimen contains calcium dobesilate or etamsylate as a therapeutic drug, the oxidized metal acid salt or vanadium pentoxide or vanadium oxytrichloride in the reagent R1 can be oxidized firstly, 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 μ 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）
Figure DEST_PATH_IMAGE002
Sample preparation: 3 mu 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
Figure DEST_PATH_IMAGE003
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: s (sample size) =240:60: 3. mu.l
Primary/secondary wavelength: 505/660nm
2POINT END,INC.
Table 1 interference results measured by the existing two-reagent creatinine enzyme method kit: (unit: mu mol/L)
Control 1 2 3 Mean value of Relative deviation of
Mother liquor 347 347 348 347.3
Calcium dobesilate 8mg/L 336 334 335 335.0 ﹣3.5%
Calcium dobesilate 16mg/L 322 321 321 321.3 ﹣7.5%
Calcium dobesilate 32mg/L 308 308 308 308.0 ﹣11.3%
Calcium dobesilate 64mg/L 280 279 279 279.3 ﹣19.6%
Etamsylate 12.5mg/L 323 320 321 322.0 ﹣7.3%
Etamsylate 25mg/L 317 317 317 317.0 ﹣9.7%
Etamsylate 50mg/L 299 298 300 299.0 ﹣13.9%
Etamsylate 100mg/L 267 269 267 267.7 ﹣22.9%
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:
Figure DEST_PATH_IMAGE004
the creatinine reagents according to the present invention are described in detail as follows:
example 1:
reagent R1 component:
tris buffer 50mM pH8.0;
peroxidase 9 ku/L;
ascorbic acid oxidase 6 ku/L;
vanadium pentoxide is 1 mM;
4-AAP 3 mM；
TRITON X-100 0.2%；
borax 1%
Reagent R2 component:
tris buffer 50mM pH8.0
Uricase 9 ku/L
Bovine serum albumin 0.1%
TOOS 10 mM
1.0 percent of borax
TRITON 0.1%。
Example 2:
reagent R1 component:
HEPES buffer 50mM pH8.0
Peroxidase 7.2 ku/L
Ascorbic acid oxidase 6.4 ku/L
Sodium ferrate 1 mM
4-AAP 3 mM
TRITON X-100 0.2%
0.1% of gentamicin sulfate;
reagent R2 component:
HEPES buffer 50mM pH8.0
Uricase 11.2 ku/L
Bovine serum albumin 0.1%
TOOS 10 mM
0.1 percent of gentamicin sulfate
TRITON 0.1%。
Example 3:
reagent R1 component:
phosphate buffer 50mM pH8.0
Peroxidase 4.6 ku/L
Ascorbic acid oxidase 10 ku/L
Sodium metavanadate 2 mM
4-AAP 3mM
TRITON X-100 0.2%
0.1% of sodium azide;
reagent R2 component:
phosphate buffer 50mM pH8.0
Uricase 20 ku/L
Bovine serum albumin 0.1%
TOOS 10mM
Sodium azide 0.1%
TRITON X-100 0.1%。
Example 4:
reagent R1 component:
EPPS buffer 50mM pH8.0
Peroxidase 6.5 ku/L
Ascorbic acid oxidase 5.5 ku/L
Vanadium oxytrichloride 2 mM
4-AAP 3mM
Brij 0.1%
Proclin 300 0.1%；
Reagent R2 component:
EPPS buffer 50mM pH8.0
Uricase 16.8 ku/L
Bovine serum albumin 0.1%
TOPS 10 mM
Brij 0.1%
Proclin 300 0.1%。
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)
Measurement of 1 2 3 Mean value of Relative deviation of
Mother liquor 359 358 358 358.3
Calcium dobesilate 8ml/L 358 358 358 358 －0.1%
Calcium dobesilate 16ml/L 359 359 358 358.7 0.1%
32ml/L calcium dobesilate 358 360 359 359.0 0.2%
Calcium dobesilate 64ml/L 357 357 357 357.0 －0.4%
Etamsylate 12.5mg/L 358 358 358 358.0 ﹣0.1%
Etamsylate 25mg/L 358 359 359 358.7 0.1%
Etamsylate 50mg/L 359 360 360 359.7 0.4%
Etamsylate 100mg/L 359 360 359 359.7 0.4%
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 (8)

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.
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: the peroxidase is bound to uricase.
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:
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 1.5-9 ku/L;
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.
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 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.
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 reagent R1 and the reagent R2 further contain nonionic surfactant with the weight ratio of 0.01-0.3%, wherein the nonionic surfactant is one or two of BRIJ, EMULGEN, TRITON and TWEEN.
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: the preservative is one or two of Proclin, gentamicin sulfate, PARABEN, chloramphenicol, sodium azide and borax.
7. 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 reagent 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/L; the Trinder's reagent B content is 0.1-20 mM.
8. A preparation method of a uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum is characterized by comprising 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 oxidant → stirring and dissolving → adjusting pH to 8.0 → adding Trinder's reactant A and nonionic surfactant, mixing well → 2-8 ℃ overnight → adding ascorbic acid oxidase and peroxidase → stirring and dissolving → 2-8 ℃ overnight → detecting, and subpackaging; the Trinder's reactant A is 4-aminoantipyrine, and the oxidant comprises any one of vanadium pentoxide, sodium ferrate, sodium metavanadate and vanadium oxychloride;
preparation of reagent R2:
weighing buffer solution → adding preservative → stirring and dissolving → adjusting pH to 8.0 → adding Trinder's reactant B and nonionic surfactant → mixing evenly → 2-8 ℃ overnight → adding serum albumin, uricase → mixing evenly → 2-8 ℃ overnight → detecting and subpackaging, wherein the Trinder's reactant B is one or more than two of TOOS, DHBS, HDAOS, TBHBA, TOPS, TODB and 4-chlorophenol.
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CN202011578744.3A 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
CN202011578366.9A CN112662736B (en) 2020-08-15 2020-08-15 Preparation method of uric acid kit for rapidly and simply eliminating interference of calcium dobesilate and phenol sulfoethylamine drugs in serum
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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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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