阅读说明：本技术 一种可消除羟苯磺酸钙、酚磺乙胺药物干扰的肌酐试剂盒的制备方法 (Preparation method of creatinine kit capable of eliminating interference of calcium dobesilate and etamsylate medicines ) 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羟苯磺酸钙、酚磺乙胺药物干扰的肌酐试剂盒的制备方法的制备方法,其技术方案要点是包括如下步骤：制备R1试剂：称取缓冲液→加入防腐剂→加入漆酶或铬酸盐或铁酸盐或五氧化二钒,搅拌1小时→以IN盐酸或氢氧化钠(或氢氧化钾)调至PH8.0→加入Trinder’s反应剂A→非离子表面活性剂→加入BSA重量比0.1％→搅拌1小时→放入2～8℃隔夜→再加入肌酸酶,肌氨酸氧化酶,过氧化物酶,抗坏血酸氧化酶搅拌1小时→检测后获得；所述的Trinder’s反应剂A为4－氨基安替比林；制备R2试剂；本发明克服现在各医院临床检验测定肌酐时,由于血清标本中含羟苯磺酸钙、酚磺乙胺造成的负偏差,使肌酐测定时不准确的缺陷,适用于全自动生化分析仪血清肌酐的检测。(The invention discloses a preparation method of a creatinine kit capable of eliminating interference of calcium dobesilate and etamsylate medicines, which adopts the technical scheme that the preparation method comprises the following steps: preparation of R1 reagent: weighing buffer solution → adding preservative → adding laccase or chromate or ferrite or vanadium pentoxide, stirring for 1 hour → adjusting pH to 8.0 by IN hydrochloric acid or sodium hydroxide (or potassium hydroxide) → adding Trinder's reactant A → nonionic surfactant → adding BSA IN a weight ratio of 0.1% → stirring for 1 hour → placing at 2-8 ℃ overnight → adding creatinase, sarcosine oxidase, peroxidase and ascorbate oxidase, stirring for 1 hour → detecting; the Trinder's reactant A is 4-aminoantipyrine; preparing an R2 reagent; 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.)

1. A preparation method of a creatinine kit capable of eliminating interference of calcium dobesilate and etamsylate medicines is characterized by comprising the following steps:

preparation of R1 reagent:

weighing buffer solution → adding preservative → adding laccase or chromate or ferrite or vanadium pentoxide, stirring for 1 hour → adjusting pH to 8.0 by IN hydrochloric acid or sodium hydroxide (or potassium hydroxide) → adding Trinder's reactant A → nonionic surfactant → adding BSA IN a weight ratio of 0.1% → stirring for 1 hour → placing at 2-8 ℃ overnight → adding creatinase, sarcosine oxidase, peroxidase and ascorbate oxidase, stirring for 1 hour → detecting; the Trinder's reactant A is 4-aminoantipyrine;

preparation of R2 reagent:

weighing buffer solution → adjusting pH to 8.0 with IN hydrochloric acid or sodium hydroxide (or potassium hydroxide), adding Trinder's reactant B → adding antiseptic → adding nonionic surfactant → serum albumin → stirring for 1 hour → cooling, adding creatinin at 2-8 deg.C overnight, mixing Trinder's reactant B with one or more of TOOS, DHBS, DAOS, HDAOS, TBHBA, TOPS, TODB and 4-chlorophenol, and detecting to obtain reagent;

the kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises buffer solution, creatinase, sarcosine oxidase, ascorbic acid oxidase, peroxidase, serum albumin, a nonionic surfactant, a preservative, a Trinder's reagent A, laccase or chromate or ferrite or vanadium pentoxide; the reagent R2 comprises buffer solution, serum albumin, creatinin, preservative, Trinder's reagent B; the Trinder's reactant A is 4-aminoantipyrine, and the Trinder's reactant B is one or a mixture of more than two of TOOS, DHBS, DAOS, HDAOS, TBHBA, TOPS, TODB and 4-chlorophenol.

2. The preparation method of the creatinine kit capable of eliminating interference of calcium dobesilate and etamsylate drugs according to claim 1, wherein the preparation method comprises the following steps: the laccase is a laccase of product number SAE0050 of Sigma-Aldrich company.

3. The preparation method of the creatinine kit capable of eliminating interference of calcium dobesilate and etamsylate drugs according to claim 1, wherein the preparation method comprises the following steps:

in the R1, the concentration of a buffer solution is 5-200 mM, and the pH value is 7.0-9.0;

the content of creatinase is 5-50 ku/liter;

sarcosine oxidase content is 5-50 ku/liter;

the content of the peroxidase is 1-10 ku/L;

1-10 ku/L ascorbic acid oxidase;

1-2 g/L of serum protein;

trinder's reagent A0.5-5 mM/L;

when the weight ratio of the preservative is 0.005-0.2%, the laccase content is 0.1-10 ku/L, or chromate, ferrite or vanadium pentoxide with the content of 0.01-10 mM is adopted.

4. The preparation method of the creatinine kit capable of eliminating interference of calcium dobesilate and etamsylate drugs according to claim 1, wherein the preparation method comprises the following steps: 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 preparation method of the creatinine kit capable of eliminating interference of calcium dobesilate and etamsylate drugs according to claim 1, wherein the preparation method comprises the following steps: the reagents R1 and R2 also contain a surfactant, the weight ratio content of the surfactant is 0.01-0.3%, and the surfactant is a nonionic surfactant, and specifically is one or two of TRITON, TWEEN, BRIJ and EMULGEN.

6. The preparation method of the creatinine kit capable of eliminating interference of calcium dobesilate and etamsylate drugs according to claim 1, wherein the preparation method comprises the following steps: the preservative is one or two of Proclin, gentamicin sulfate, PARABEN, chloramphenicol and sodium azide.

7. The preparation method of the creatinine kit capable of eliminating interference of calcium dobesilate and etamsylate drugs according to claim 1, wherein the preparation method comprises the following steps: in the R2, the concentration of a buffer solution is 10-200 mM, and the pH value is 6.5-9.0; the content of Trinder's reactant B is 0.1-20 mM; the amount of creatinine is 50 to 1000 ku/liter.

[ technical field ] A method for producing a semiconductor device

The invention belongs to the technical field of medical examination, and relates to a preparation method of a creatinine kit capable of eliminating interference of calcium dobesilate and etamsylate medicines.

[ background of the invention ]

Creatinine is a main index and a detection item of renal function in clinical examination, and two methods for measuring serum creatinine mainly exist: one is the alkaline picric acid rate method (Jaffe method), and one is the sarcosine oxidase method; the Jaffe method is narrow in linear range and poor in specificity, is easily interfered by other substances (such as ketone bodies, ascorbic acid, bilirubin, cephalo, dopamine and the like) in a sample, has detection deviation and gradually exits the market. Currently, most of the enzymes used in the market are sarcosine oxidase methods, and the method is gradually eliminated by various hospitals and is abandoned by changing the detection of the sarcosine oxidase method, and the detection principle is as follows:

the existing commercial detection kit is a double reagent, the reagent R1 contains creatinase, and the creatinase in serum is firstly decomposed by the creatinase to generate H₂O₂Then using peroxidase and Trinder's reagent andformation of H₂O₂Reacting to produce colorless compound, or eliminating serum creatine-producing H with catalase₂O₂H to be generated₂O₂Decomposing into water and oxygen, adding R2 reagent, adding R2 reagent containing creatininase and Trinder's reagent, and sodium azide, creatininase hydrolyzing creatininase into creatine, and color-developing by the coexistence of creatinase, sarcosine oxidase in R1, 4-AAP of peroxidase and Trinder's reagent, and phenol compound in Trinder's reagent, reacting the catalase contained in R1 with sodium azide in R2, wherein the reaction process utilizes the specificity of enzyme, but the generated H₂O₂Is a strong oxidant, and is easily consumed by reducing drugs in serum to generate negative interference.

Creatinine is used as the most important index of renal function, and the common calcium dobesilate medicine for treating and protecting microangiopathy caused by nephropathy has very strong reducibility₂O₂Direct reaction, consuming H₂O₂(ii) a In addition, the common medicine of the etamsylate for preventing and treating excessive surgical hemorrhage also has strong reducibility, and the two medicines consume H generated in the conventional creatinine enzyme method determination in hospitals₂O₂The creatinine value has serious negative deviation, which causes misjudgment of doctors in diagnosis and treatment.

Negative interference of calcium dobesilate and etamsylate on the sarcosine oxidase assay for creatinine causes significant inconvenience to patients administered both drugs. For example, renal insufficiency is often associated with diabetic patients, and negative interference of calcium dobesilate administered with creatinine oxidase detection may cause incorrect renal function assessment, masking and delaying the condition in diabetic patients. For surgical patients, however, negative interference of administered etamsylate with the sarcosine oxidase assay for creatinine may render it impossible to properly assess its renal function, resulting in postoperative complications that affect the postoperative recovery of the patient.

[ summary of the invention ]

The invention discloses a creatinine kit capable of eliminating creatinine determination negative deviation interference caused by calcium dobesilate and etamsylate medicines in serum, which overcomes the defect that when creatinine is determined by clinical examination in various hospitals at present, the creatinine determination is inaccurate due to the negative deviation caused by the calcium dobesilate and the etamsylate contained in a serum sample, so that misjudgment in the treatment process of doctors is caused.

The invention also discloses a preparation method of the creatinine kit capable of eliminating negative bias interference of creatinine determination caused by calcium dobesilate and etamsylate drugs in serum.

The creatinine kit capable of eliminating negative bias interference of creatinine determination caused by calcium dobesilate and etamsylate medicines in serum comprises an independent 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 metal acid salt; the reagent R2 comprises buffer, serum albumin, creatininase, preservative, optionally containing non-ionic surfactant, and Trinder's reagent B. The laccase can oxidize calcium dobesilate and etamsylate into benzoquinone compounds by using oxygen (O2); the metal acid salt or vanadium pentoxide or vanadium oxychloride can be used as an oxidant to oxidize calcium dobesilate and etamsylate, so that H2O2 generated in creatinine determination is prevented from being consumed. In the above R1 reagent, laccase or oxidation state metal acid salt or vanadium pentoxide or vanadium oxychloride component can also be stripped from the reagent R1, and added into the reagent R1 before being used separately.

Preferably, the reagent R1 may also contain ascorbate oxidase in an amount in the range of 5-10KU/L, preferably 5.5-10KU/L, which is effective to reduce the interference of ascorbic acid with creatinine measurement, and in some embodiments, the amount of ascorbate oxidase may be 5.5KU/L, 6.4KU/L, 7.5KU/L, 8.8KU/L, 8.6KU/L, 6KU/L, 7.2KU/L, 9.2KU/L, 9.5KU/L or 10 KU/L.

Preferably, the laccase is a laccase of product number SAE0050 from Sigma-Aldrich. The laccase is a copper-containing polyphenol oxidoreductase, can oxidize various phenol compounds by using oxygen molecules in water to generate quinone compounds, and the oxygen molecules are reduced into water, so that the laccase is used for eliminating the phenol compounds in sewage by using an environment-friendly enzyme commonly used in sewage treatment. Calcium dobesilate and etamsylate are reducing agents containing a phenol ring structure, and laccase can oxidize and eliminate the reducing agents; however, laccase generally has the highest activity and better stability at pH 2-4, while the pH value of the creatinine enzyme reagent needs to be 7-9, after testing laccase from different sources, the laccase with product number SAE0050 of Sigma-Aldrich company is finally screened out, and the laccase has good activity and stability at pH 7.0-9.0, and can be added into a creatinine R1 reagent to eliminate the interference of calcium dobesilate and phenolsulfoethylamine.

Preferably, the metal acid salt comprises oxidation state nickelate, ferrite, cobaltate, chromate, manganate and vanadate, and the vanadate comprises poly-vanadate, metavanadate, vanadium pentoxide or vanadium oxychloride, or a mixture of more than one of poly-vanadate, metavanadate, vanadium pentoxide and vanadium oxychloride. The invention adopts 23, 24, 25, 26, 27 and 28 bits of metal element acid salt in the periodic table of elements, and specifically selects oxidation state nickelate, cobaltate, chromate, manganate and vanadate with high oxidation-reduction potential, wherein the vanadate comprises metavanadate, poly-vanadate, vanadic anhydride or vanadyl trichloride all can be used as an oxidant to eliminate negative deviation caused by interference of calcium dobesilate and etamsylate in serum during creatinine determination, and simultaneously does not influence the stability of each component in the reagent R1 and the reagent R2.

Preferably, the concentration of the buffer solution in the R1 is 5-200 mM, and the pH is 7.0-9.0; 5-50 ku/L of creatinase, 5-50 ku/L of sarcosine oxidase, 1-10 ku/L of ascorbic acid oxidase, 1-10 ku/L of peroxidase, or 100-500 ku/L of catalase, 1-2 g/L of serum protein and 0.5-5 mM/L of Trinder's reactant A [ the Trinder's reactant A is 4-Aminoantipyrine (4-Aminoantipyrine, 4-AAP for short) ]; the preservative is 0.005-0.2 wt%, and if laccase is adopted to eliminate the negative interference of calcium dobesilate and etamsylate, the laccase content is 0.1-10 ku/L; if the above-mentioned metalate oxidant or vanadium pentoxide or vanadium oxytrichloride oxidant is used, the content of the single or mixed two or more is 0.01-10 mM; the high oxidation-reduction potential metal acid salt comprises sodium, potassium and ammonium salts containing the metal acid.

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 surfactant, the weight ratio of the surfactant is 0.01-0.3%, and the surfactant is a nonionic surfactant, specifically one or two of TRITON, TWEEN, BRIJ and EMULGEN.

Preferably, the preservative is one or two of Proclin, gentamicin sulfate, PARABEN, chloramphenicol and sodium azide.

Preferably, in the R2, the concentration of a buffer solution is 10-200 mM, and the pH value is 6.5-9.0; the content of Trinder's reactant B is 0.1-20 mM; the amount of creatinine is 50 to 1000 ku/liter.

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. These components are mixed with Trinder's reagent various phenol or aniline derivatives to form chromogen components, Trinder's reagent A must be in reagent R1, Trinder's reagent B must be in reagent R2.

Reagent R1 and reagent R2, wherein reagent R1 includes eliminating hydroxyl benzene sulfonic acid calcium and phenol sulfoethylamine interference laccase, can be before adding creatinine enzyme (reagent R2) hydroxyl benzene sulfonic acid calcium and phenol sulfoethylamine oxidation in serum avoid its consumption in creatinine determination of H2O2, and add sodium azide in R2 reagent, reagent R2 after reagent R1 and serum complete the component reaction, kill laccase, also kill catalase, has avoided the influence on creatinine determination after adding creatinine enzyme.

A preparation method of a creatinine kit capable of eliminating negative bias interference of creatinine determination caused by calcium dobesilate and etamsylate drugs in serum comprises the following steps:

preparation of R1 reagent:

weighing buffer solution → adding preservative → adding laccase or metalate, stirring for 1 hour, adjusting pH to 8.0 with IN hydrochloric acid or sodium hydroxide (or potassium), adding Trinder's reactant A → nonionic surfactant → adding BSA with the weight ratio of 0.1% and stirring for 1 hour → adding creatinase, sarcosine oxidase, peroxidase or catalase, ascorbic acid oxidase → stirring for 1 hour → detecting; the metalate is one or more of oxidation state nickelate, ferrite, cobaltate, chromate, manganate and vanadate, and the Trinder's reactant A is 4-aminoantipyrine;

preparation of R2 reagent:

weighing buffer solution → adjusting pH to 8.0 with IN hydrochloric acid or sodium hydroxide (or potassium → adding Trinder's reagent B → adding antiseptic → adding nonionic surfactant → serum albumin → stirring for 1 hr → cooling, and adding creatininase at 2-8 deg.C overnight → detecting.

The reagent R1 and the reagent R2 are mixed with the specimen with the reagent R1, except that creatine in the specimen is eliminated firstly, for example, when the specimen contains calcium dobesilate or etamsylate, urushiol or the above-mentioned oxidation state metal salt or vanadic oxide or vanadyl trichloride in the reagent R1 can be oxidized firstly, H generated by series reactions of creatininase contained in the reagent R2 is avoided₂O₂Consumed by the above-mentioned medicine, at the same time the R2 reagent contains Trinder's reactant B, i.e. phenol compound and 4-AAP in reagent R1 and H produced by series reaction of creatinase₂O₂Color is developed, the absorbance is measured, and the content is calculated.

The invention has the beneficial effects that:

the creatinine kit capable of eliminating negative bias interference in creatinine determination caused by calcium dobesilate and etamsylate medicines in serum adopts double reagents and Trinder's reaction for creatinine determination in all sarcosine oxidase methods in the market at present, and the reagents R1 contain phenol or aniline compounds commonly used as Trinder's reactants, such as 4-chlorophenol, TOOS, DHBS, DAOS, HDAOS, TBHBA, TOPS, TODB in the R2 reagent contains 4-AAP, the invention finds in a series of experiments that 4-AAP must be added in the R1 reagent to coexist with laccase or the above-mentioned metallate or vanadic oxide and vanadyl trichloride and other components in the R1 reagent, Trinder's reaction another reagent, a phenolic compound must coexist with creatinin in R2, the creatinine kit can not only eliminate the interference of calcium dobesilate and etamsylate, but also keep the stability of the creatinine kit for more than one year at 2-8 ℃.

The creatinine kit capable of eliminating negative bias interference of creatinine determination caused by calcium dobesilate and etamsylate medicines in serum can enable creatine in a serum sample to be firstly generated by creatinase and creatinase oxidase in a reagent R1 under the condition that the reagent R1 contains catalase or peroxidase and 4-AAP₂O₂The interference of creatine in serum is firstly decomposed and eliminated, the R1 reagent also contains ascorbic acid oxidase, the ascorbic acid in the serum is firstly eliminated, the interference caused by the ascorbic acid is avoided, the obtained kit has good stability and simple and rapid operation, the negative interference of calcium dobesilate and etamsylate is eliminated, the accuracy is high, the misjudgment of doctors in clinical diagnosis is avoided, the kit can accurately use medicines and treat the diseases, and the kit is suitable for a full-automatic biochemical analyzer, is loaded on a machine by double reagents, and is simple to operate, safe and reliable.

The preparation method of the creatinine kit capable of eliminating negative bias interference of creatinine determination caused by calcium dobesilate and etamsylate drugs in serum is unique, simple and convenient in process and suitable for batch preparation operation.

[ detailed description ] embodiments

The creatinine kit for eliminating negative bias interference of creatinine determination caused by calcium dobesilate and etamsylate drugs in serum according to the present invention will be further described with reference to the following embodiments:

the interference of calcium dobesilate and etamsylate is determined by currently marketed creatinine enzyme method kit approved by the national drug administration, and the results are shown in the following table:

the reagent composition is as follows in the specification:

[ major Components]From the reagent R₁Reagent R₂And a calibrator.

Reagent R₁: tris buffer, creatine amidinohydrolase, sarcosine oxidase, ascorbic acid oxidase, sodium 2-hydroxy-3-m-toluidine propanesulfonate (TOOS), catalase, surfactant;

reagent R₂: tris buffer, creatinine amidohydrolase, 4-aminoantipyrine and peroxidase.

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): 10 μ l, reagent 1 (R1): 210 μ l, reagent 2 (R2): 70 μ l, temperature: 37 ℃;

the determination type is as follows: end point method, dominant wavelength: 546nm, secondary wavelength: 660nm, reaction direction: rising;

the method comprises the following steps: the sample was mixed with R1, the R2 reagent was added 5 minutes after 37 ℃, and then the reaction absorbance 5 minutes after the addition of R2 was measured.

(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.

Measurement instruments and parameters:

unit: mu mol/L, normal value is less than or equal to 70 mu mol/L

The measuring instrument: hitachi 7180

Measurement parameters are as follows: r1: r2: s (sample size) 210:70: 10. mu.l

Primary/secondary wavelength: 540/660nm

2POINT END,INC.

Table 1 interference results determined by the existing two-reagent creatinine enzymatic kit:

control	1	2	3	Mean value of	Relative deviation of
						Mother liquor	139	139	139	139
Calcium dobesilate 8mg/L	121	121	121	121.00	﹣12.9％
						Calcium dobesilate 16mg/L	103	102	102	102.3	﹣26.4％
Calcium dobesilate 32mg/L	82	81	82	81.7	﹣41.2％
						Calcium dobesilate 64mg/L	45	46	45	45.3	﹣67.4％
Etamsylate 12.5mg/L	114	114	113	113.70	﹣18.2％
						Etamsylate 25mg/L	90	91	91	90.3	﹣35.0％
Etamsylate 50mg/L	60	60	60	60.00	﹣56.8％
						Etamsylate 100mg/L	46	46	45	45.70	﹣67.1％

And (4) conclusion: the measurement result shows that calcium dobesilate and etamsylate both cause serious negative deviation on the creatinine measurement result.

The effect of the creatinine kit capable of eliminating negative bias interference of creatinine determination caused by calcium dobesilate and etamsylate drugs in serum is described by specifically combining with examples 1-4:

table 2 reagent components for examples 1-4:

the creatinine reagent according to the present invention has the following specific components (the percentages in the examples are by weight):

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:

specific preparation methods for examples 1-4 are as follows:

preparation of R1 reagent:

weighing buffer solution → adding preservative → adding laccase or metallate, stirring for 1 hour → adjusting pH to 8.0 by IN hydrochloric acid or sodium hydroxide (or potassium → adding Trinder's reactant A → nonionic surfactant → adding BSA with weight ratio of 0.1% → stirring for 1 hour → placing at 2-8 ℃ overnight → adding creatinase, sarcosine oxidase, peroxidase or catalase, and ascorbate oxidase, stirring for 1 hour → detecting;

preparation of R2 reagent:

weighing buffer solution → adjusting pH to 8.0 with IN hydrochloric acid or sodium hydroxide (or potassium), adding Trinder's reactant B → adding antiseptic → adding nonionic surfactant → serum albumin → stirring for 1 hr → cooling, adding creatininase at 2-8 deg.C overnight, and detecting.

The determination method comprises the following steps:

the reagents R1 and R2 obtained in examples 1 to 4 were placed in the corresponding reagent positions of a fully automatic blood coagulation analyzer (Hitachi 7180), and then, the reagent R1210. mu.l, the reagent R270. mu.l, the calibrator or the specimen 10. mu.l, the calibrator concentration 300. mu.M, the primary/secondary 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	139	140	139	139.30
Calcium dobesilate 8ml/L	141	137	138	138.70	﹣0.95％
						Calcium dobesilate 16ml/L	141	141	140	140.7	1.0％
32ml/L calcium dobesilate	141	141	139	140.30	0.13％
						Calcium dobesilate 64ml/L	139	138	138	138.3	﹣0.017％
Etamsylate 12.5mg/L	140	137	139	138.70	﹣0.15％
						Etamsylate 25mg/L	141	140	139	140.00	0.15％
Etamsylate 50mg/L	139	140	138	144.00	﹣0.2％
						Etamsylate 100mg/L	139	138	137	138	﹣1.0％

TABLE 4 measurement results of example 2: (unit: mu mol/L)

Measurement of	1	2	3	Mean value of	Relative deviation of
						Mother liquor	148	146	147	147.01
Calcium dobesilate 8ml/L	149	148	147	148.01	0.7％
						Calcium dobesilate 16ml/L	149	147	148	148.01	0.7％
32ml/L calcium dobesilate	148	148	149	151.01	1.0％
						Calcium dobesilate 64ml/L	147	148	148	152.01	0.5％
Etamsylate 12.5mg/L	147	146	147	146.70	﹣0.2％
						Etamsylate 25mg/L	148	147	147	147.30	0.2％
Etamsylate 50mg/L	147	148	148	147.70	0.5％
						Phenol sulfonic acid ethylAmine 100mg/L	147	148	146	147.0	0.0％

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	152	152	152	152.01
Calcium dobesilate 8ml/L	154	153	152	153.01	0.7％
						Calcium dobesilate 16ml/L	152	152	152	152.01	0.0％
32ml/L calcium dobesilate	152	152	151	151.7	-0.2％
						Calcium dobesilate 64ml/L	152	151	151	151.3	-0.5％
Etamsylate 12.5mg/L	153	153	153	153.01	0.7％
						Etamsylate 25mg/L	155	153	153	153.7	1.1％
Etamsylate 50mg/L	155	153	153	153.7	1.1％
						Etamsylate 100mg/L	153	154	153	153.3	0.9％

TABLE 6 measurement results of example 4: (unit: mu mol/L)

Measurement of	1	2	3	Mean value of	Relative deviation of
						Mother liquor	116	115	116	115.7
Calcium dobesilate 8ml/L	116	116	115	115.7	0
						Calcium dobesilate 16ml/L	116	115	115	115.3	-0.3％
32ml/L calcium dobesilate	115	115	115	115.0	-0.6％
						Calcium dobesilate 64ml/L	115	115	114	114.7	-0.9％
Etamsylate 12.5mg/L	115	115	115	115.0	-0.6％
						Etamsylate 25mg/L	115	116	116	115.30	-0.3％
Etamsylate 50mg/L	114	114	115	114.30	-1.0％
						Etamsylate 100mg/L	114	114	113	113.7	-1.7％

The above results of examples 1-4 show that the creatinine kit capable of eliminating negative bias interference in creatinine determination caused by calcium dobesilate and etamsylate in serum of the present application comprises reagent R1 and reagent R2, wherein reagent R1 is first mixed with the specimen, except that creatine in the specimen is first eliminated, for example, when the specimen contains calcium dobesilate or etamsylate as a therapeutic drug, urushiol or the above mentioned oxidized form metal acid salt or vanadium pentoxide or vanadium oxychloride in reagent R1 can be oxidized first, and laccase or oxidized form high-vanadate, polymetavanadate and metavanadate with high redox potential can be used to effectively eliminate creatinine ferriteThe interference of calcium dobesilate and the etamsylate during the determination is used for obtaining an accurate creatinine determination value, and H generated by serial reactions of creatininase added into a reagent R2 is avoided₂O₂Consumed by the above-mentioned medicine, at the same time the R2 reagent contains Trinder's reactant B, i.e. phenol compound and 4-AAP in reagent R1 and H produced by series reaction of creatinase₂O₂Coloring, measuring the absorbance, and calculating the content;

in the presence of catalase or peroxidase and 4-AAP in the reagent R1, creatine in the serum sample can be first converted from H produced by creatinase and sarcosine oxidase in the reagent R1₂O₂The kit has the advantages of good stability, simple and rapid operation, elimination of negative interference of calcium dobesilate and etamsylate, high accuracy, avoidance of misjudgment of doctors in clinical diagnosis, accurate medication and treatment, suitability for full-automatic biochemical analyzers and machines with double reagents, and simple operation, safety and reliability. Laccase or ferrate, poly-metavanadate and metavanadate with high oxidation-reduction potential in an oxidation state are adopted, so that the interference of calcium dobesilate and etamsylate during creatinine determination can be effectively eliminated, and an accurate creatinine determination value is obtained.