阅读说明：本技术 一种可消除血清中羟苯磺酸钙，酚磺乙胺药物干扰的尿酸试剂盒及其制备方法 (Uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate medicines in serum and preparation method thereof ) 是由 俞永标 于 2020-08-15 设计创作，主要内容包括：本发明的一种可消除血清中羟苯磺酸钙,酚磺乙胺药物干扰的尿酸试剂盒及其制备方法,其技术方案要点是：包括试剂R1和试剂R2,所述试剂R1中包含缓冲液、过氧化物酶、抗坏血酸氧化酶、防腐剂、Trinder’s反应剂A、金属酸盐或非离子表面活性剂；所述试剂R2包含缓冲液、血清白蛋白、尿酸酶、防腐剂、及Trinder’s反应剂B或非离子表面活性剂。本发明的尿酸试剂盒,在R1中加入高氧化还原电位之氧化型上述金属酸盐或五氧化二钒或三氯氧钒之一种或一种以上混合,利用所述金属酸盐将羟苯磺酸钙及酚磺乙胺氧化,避免了在加入R2后尿酸酶分解尿酸产生的H2O2被消耗掉,迅速得到准确的尿酸测定值。(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.)

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 buffer solution, peroxidase, ascorbic acid oxidase, preservative, Trinder's reactant A, metalate and nonionic surfactant; the reagent R2 comprises buffer solution, serum albumin, uricase, preservative, Trinder's reagent B and non-ionic surfactant; the Trinder's reactant A is 4-aminoantipyrine, and the metallate comprises one or more of oxidation state nickelate, ferrite, cobaltate, chromate, manganate and vanadate.

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 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 wt% of preservative, and 0.1-10 mM of metalate oxidant or vanadium pentoxide or vanadium oxychloride 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 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.

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 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.

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 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.

8. 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 Trinder's reactant B in the reagent R2 is one or more of TOOS, DHBS, DAOS, HDAOS, TBHBA, TOPS, TODB and 4-chlorophenol.

9. A preparation method of a uric acid kit capable of eliminating interference of calcium dobesilate and etamsylate in serum is characterized by comprising the following steps:

preparation of R1 reagent:

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 evenly → 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 metallate comprises one or more of oxidation state nickelate, ferrite, cobaltate, chromate, manganate and vanadate;

preparation of R2 reagent:

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.

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 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:

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 the R2 reagent is added, the R2 reagent contains uricase and Trinder's reagent B, the uricase hydrolyzes uric acid into allantoin and H2O2, and the H2O2 and the peroxidase in R1 and the Trinder's A reagent are jointly existed for color development 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 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 H₂O₂Cause severe negative deviation of uric acid value, and causeThe physician can make a diagnosis of misjudgment during treatment.

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 oxychloride are added into R1, and calcium dobesilate and etamsylate are oxidized by utilizing the oxidation characteristic of the metal acid salt, vanadium pentoxide or vanadium oxychloride, so that H2O2 generated by decomposition of uric acid by uricase after 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 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 must be added into an R1 reagent to coexist with the metallate or vanadium pentoxide and vanadium oxychloride and other components in an R1 reagent, Trinder's reaction and another reagent, namely a phenol compound must coexist with uricase in R2, so that the uric acid kit can eliminate the interference of calcium dobesilate and etamsylate, can keep the stability of the kit at the temperature of 2-8 ℃ for more than one year, and can effectively reduce the interference of ascorbic acid in a blood sample on uric acid determination when the amount of the ascorbic acid oxidase is in the range.

Preferably, 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 1.0-10 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 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.

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 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 in serum comprises the following steps:

preparation of R1 reagent:

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 evenly → 2-8 ℃ overnight → adding ascorbic acid oxidase and peroxidase → stirring and dissolving → 2-8 ℃ overnight → detecting, and subpackaging;

preparation of R2 reagent:

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, and at the same time the R2 reagent 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 reagents R1 and R2, the kit is different from the existing kit components in the market, the stability of the kit can be prolonged within a period of time, negative interference of calcium dobesilate and etamsylate can be continuously eliminated, misjudgment of doctors in clinical diagnosis is avoided, accurate medication and treatment can be realized, and the kit 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, simple and rapid 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 is further described by combining specific embodiments in the following:

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 R2 reagent is added after 5 minutes at 37 ℃, and then the reaction absorbance 5 minutes after the addition of R2 is measured.

Measurement of blank Absorbance (A)₁) Measurement of reaction Absorbance (A)₂）

Sample preparation: 3 mu l

R₁：240 µl R₂：60 µl

(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: 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 1-4:

table 2 reagent components for examples 1-4:

the creatinine reagents according to the present invention are described in detail as follows: