阅读说明：本技术 一种基于Trinder反应的检测试剂盒及其应用 (Trinder reaction-based detection kit and application thereof ) 是由 梁艳 赵畅 龚婷 吴年芬 凡速朋 舒芹 张雪娇 赵愿安 于 2020-06-15 设计创作，主要内容包括：本发明公开了一种基于Trinder反应的检测试剂盒及其应用,属于临床化学检测技术领域,由R1试剂和R2试剂组成,R1和R2试剂中包含催化待测物生成过氧化氢的酶,R1试剂中还含有缓冲液、防腐剂、过氧化物酶,R2试剂中含有缓冲液、防腐剂、过氧化物和色原底物,所述R1试剂中过氧化物酶的浓度为100～150KU/L,且R1试剂中还含有浓度为0.5～1g/L的4-氨基安替比林。其中R1试剂中的过氧化物酶和4-氨基安替比林可在Trinder反应前,去除待测物中可参与Trinder反应的临床药物,使得测定结果更加准确。本发明不需要在反应体系中额外加入其它物质,不会引入其它杂质；该方法简单高效,可广泛用于多种临床化学检测,避免临床药物的干扰,增强检测结果的准确性,同时还提高了试剂盒的功能灵敏度。(The invention discloses a Trinder reaction-based detection kit and application thereof, belonging to the technical field of clinical chemistry detection and comprising an R1 reagent and an R2 reagent, wherein the R1 reagent and the R2 reagent contain enzymes for catalyzing a substance to be detected to generate hydrogen peroxide, the R1 reagent also contains a buffer solution, a preservative and peroxidase, the R2 reagent contains a buffer solution, a preservative, peroxide and a chromogen substrate, the concentration of the peroxidase in the R1 reagent is 100-150 KU/L, and the R1 reagent also contains 4-aminoantipyrine with the concentration of 0.5-1 g/L. Wherein, the peroxidase and the 4-aminoantipyrine in the R1 reagent can remove clinical drugs which can participate in the Trinder reaction in a substance to be tested before the Trinder reaction, so that the determination result is more accurate. According to the invention, other substances are not required to be additionally added into the reaction system, and other impurities are not introduced; the method is simple and efficient, can be widely used for various clinical chemical detections, avoids the interference of clinical drugs, enhances the accuracy of detection results, and simultaneously improves the functional sensitivity of the kit.)

1. A Trinder reaction-based detection kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent and the R2 reagent contain enzymes for catalyzing a substance to be detected to generate hydrogen peroxide, the R1 reagent also contains a buffer solution, a preservative and peroxidase, and the R2 reagent contains a buffer solution, a preservative, peroxide and a chromogen substrate, and is characterized in that the concentration of the peroxidase in the R1 reagent is 100-150 KU/L, and the R1 reagent also contains 4-aminoantipyrine with the concentration of 0.5-1 g/L;

the detection kit can improve the capacity of resisting interference of clinical drugs, wherein the clinical drugs comprise: one or more of calcium dobesilate, etamsylate, methyldopa, levodopa and dopamine.

2. The Trinder reaction-based assay kit according to claim 1, wherein the R1 reagent further comprises a metal ion chelating agent and a surfactant.

3. The Trinder reaction-based detection kit according to claim 1, wherein the concentration of peroxidase in the R2 reagent is 25-30 KU/L.

4. The Trinder reaction-based assay kit according to claim 1, wherein the chromogen substrate is an aniline derivative, and the concentration of the aniline derivative is 1 g/L.

5. The Trinder reaction-based assay kit according to claim 4, wherein the aniline derivative is one or more of DHBS, TOOS, HDAOS, F-DAOS, DHBA, TOPS, ADOS, ADPS, ALPS, DAOS, MADB, MAOS, and TODB.

6. The Trinder reaction-based assay kit according to claim 1, wherein the kit comprises: the kit comprises a creatinine detection kit, a triglyceride detection kit, a total cholesterol detection kit, a free fatty acid detection kit, a glycated albumin detection kit, a high density lipoprotein cholesterol detection kit, a low density lipoprotein cholesterol detection kit, a uric acid detection kit, a glucose detection kit, an adenosine deaminase detection kit, a lipase detection kit and a 5' -nucleotidase detection kit.

7. A method for improving the anti-interference capability of Trinder reaction detection is characterized by comprising the following steps:

s1, adding peroxidase to a final concentration of 100-150 KU/L before the Trinder reaction, and simultaneously adding 4-aminoantipyrine to a final concentration of 0.5-1 g/L to remove clinical drugs in the object to be detected, wherein the clinical drugs can participate in the Trinder reaction;

s2, adding an enzyme capable of catalyzing the object to be detected to generate hydrogen peroxide during the Trinder reaction, and simultaneously adding peroxidase and aniline derivatives to the final concentrations of 25-30 KU/L and 1g/L respectively to perform the Trinder reaction to generate a color substance;

and S3, measuring the absorbance before and after the Trinder reaction to detect the concentration of the substance to be detected.

8. A creatinine assay kit, wherein said creatinine kit comprises R1 reagent and R2 reagent, wherein said R1 reagent comprises: 100mmol/L of Good's buffer solution, 20KU/L of sarcosine oxidase, 50KU/L of creatine amidinohydrolase, 10KU/L of ascorbic acid oxidase, 150KU/L of peroxidase, 1g/L of 4-aminoantipyrine, 0.5g/L, Proclin3001g/L, GENAPOLX-08010g/L of HEDTA, and pH is 7.5; the R2 reagent includes: good's buffer 50mmol/L, creatinine amidohydrolase 300KU/L, potassium ferrocyanide 0.05g/L, sodium azide 1g/L, peroxidase 30KU/L, DHBS1g/L, pH 7.5.

9. A free fatty acid detection kit, which is characterized by consisting of an R1 reagent and an R2 reagent, wherein the R1 reagent comprises: 50mmol/L phosphate buffer solution, 100KU/L peroxidase, 4g/L coenzyme A, 6g/L, Proclin3001g/L adenosine triphosphate, 0.5 g/L4-aminoantipyrine, and pH of 7.0; the R2 reagent includes: 50mmol/L phosphate buffer solution, 50KU/L fatty acyl-CoA oxidase, 5KU/L fatty acyl-CoA synthetase, 25KU/L peroxidase, 1g/L, HDAOS1 sodium azide, 1g/L, and pH 7.0.

10. A triglyceride test kit, which is characterized by consisting of an R1 reagent and an R2 reagent, wherein the R1 reagent comprises: HEPES buffer solution 50mmol/L, glycerol kinase 18KU/L, peroxidase 130KU/L, glycerol phosphate oxidase 18KU/L, sodium cholate 5mmol/L, PC-3001g/L, 4-aminoantipyrine 4mmol/L, and pH 6.5; the R2 reagent includes: HEPES buffer solution 50mmol/L, lipoprotein lipase 18KU/L, sodium azide 1g/L, adenosine triphosphate 0.15mmol/L, oxamic acid 0.05mmol/L, peroxidase 30KU/L, DHBS1g/L, pH 6.5.

Technical Field

The invention belongs to the technical field of clinical chemical detection, and particularly relates to a Trinder reaction-based detection kit and application thereof.

Background

The Trinder reaction is also called as coupled end point colorimetry, and the principle is that hydrogen peroxide (H) is generated by a substance to be detected through the action of enzyme₂O₂) In the presence of 4-aminoantipyrine (4-AAP) and Peroxidase (POD), red quinonimine compound can be generated, and the concentration of the substance to be detected can be calculated by measuring the absorbance difference before and after the reaction. The Trinder reaction is applied to many clinical biochemical detection projects, such as glucose, triglyceride, cholesterol, uric acid, high-density lipoprotein, low-density lipoprotein, free fatty acid and the like. As disclosed in Chinese patent CN106191213BThe reaction principle of the kit is that free fatty acid in human serum and coenzyme A react under the action of acetyl coenzyme A synthetase to generate fatty acyl coenzyme A, and fatty acyl coenzyme A generates H under the action of acetyl coenzyme A oxidase₂O₂Subsequently, a colored substance was generated by the Trinder reaction under the action of Peroxidase (POD) to determine the free fatty acid content.

However, with the rapid development of medicine, more and more medicines and test items are applied to clinic, and research shows that when a patient takes 1 kind of medicine, the percentage of the experiment interfered by the medicine is 7%, when taking 2 kinds of medicines, the percentage is 16.7%, when taking three or four kinds of medicines, the percentage is 66.7%, and when taking 5 kinds of medicines, the ratio of the interfered experiment is 100%. The influence of the drug on the detection result not only causes wrong interpretation and misdiagnosis of the detection result, but also increases some unnecessary examinations, so that the problem of drug interference in the detection is very important to solve. Clinically common drugs are: calcium dobesilate (used for treating microvascular diseases and the like), etamsylate (a hemostatic drug), methyldopa (an antihypertensive drug), levodopa (used for treating parkinsonism), dopamine (a nerve conduction substance) and the like, because of strong reducibility or similarity to a chromogen substrate structure in a Trinder reaction, hydrogen peroxide is consumed in the Trinder reaction, and the measured value of an object to be measured is low. And the drugs are stable in structure and are not easy to damage or degrade, so that the interference on the clinical chemical detection based on the Trinder reaction can be generated, and the determination result is influenced.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a Trinder reaction-based detection kit, which removes clinical drugs participating in Trinder reaction in an object to be detected by adding peroxidase and 4-aminoantipyrine in advance into an R1 reagent, thereby avoiding the interference of various clinical drugs on Trinder reaction detection in the chemical detection process.

In order to achieve the purpose, the invention adopts the technical scheme that:

a detection kit based on Trinder reaction comprises an R1 reagent and an R2 reagent, wherein the R1 reagent and the R2 reagent contain enzymes for catalyzing a substance to be detected to generate hydrogen peroxide, the R1 reagent also contains a buffer solution, a preservative and peroxidase, the R2 reagent contains a buffer solution, a preservative, peroxide and a chromogen substrate, the concentration of the peroxidase in the R1 reagent is 100-150 KU/L, and the R1 reagent also contains 4-aminoantipyrine with the concentration of 0.5-1 g/L;

the detection kit can improve the capacity of resisting interference of clinical drugs, wherein the clinical drugs comprise: one or more of calcium dobesilate, etamsylate, methyldopa, levodopa and dopamine.

The technical scheme of the invention also provides a method for improving the anti-interference capability of Trinder reaction detection, which comprises the following steps:

s1, adding peroxidase to a final concentration of 100-150 KU/L before the Trinder reaction, and simultaneously adding 4-aminoantipyrine to a final concentration of 0.5-1 g/L to remove clinical drugs in the object to be detected, wherein the clinical drugs can participate in the Trinder reaction;

s2, adding an enzyme capable of catalyzing the object to be detected to generate hydrogen peroxide during the Trinder reaction, and simultaneously adding peroxidase and aniline derivatives to the final concentrations of 25-30 KU/L and 1g/L respectively to perform the Trinder reaction to generate a color substance;

and S3, measuring the absorbance before and after the Trinder reaction to detect the concentration of the substance to be detected.

The technical scheme of the invention also provides the application of the Trinder reaction-based detection kit in clinical chemical detection.

Compared with the prior art, the invention has the beneficial effects that: peroxidase and 4-aminoantipyrine are added into an R1 reagent before Trinder reaction to consume clinical drugs in a substance to be detected, so that the interference of the clinical drugs on the Trinder reaction detection is avoided, the determination result is more accurate, the excessive peroxidase and 4-aminoantipyrine can be directly used for subsequent Trinder reaction, and other substances are not required to be additionally added into a reaction system in the invention, so that other impurities are avoided from being introduced. The method is simple and efficient, can be widely used for various clinical chemical detections and preparation of corresponding clinical chemical detection kits, enhances the accuracy of detection results, and avoids drug interference.

Detailed Description

The invention provides a Trinder reaction-based detection kit, which consists of an R1 reagent and an R2 reagent, wherein the R1 reagent and the R2 reagent contain enzymes for catalyzing a substance to be detected to generate hydrogen peroxide, the R1 reagent also contains a buffer solution, a preservative and peroxidase, the R2 reagent contains a buffer solution, a preservative, peroxide and a chromogen substrate, the concentration of the peroxidase in the R1 reagent is 100-150 KU/L, and the R1 reagent also contains 4-aminoantipyrine with the concentration of 0.5-1 g/L;

the detection kit can improve the anti-interference capability of clinical medicines, and the clinical medicines comprise: one or more of calcium dobesilate, etamsylate, methyldopa, levodopa and dopamine.

The catalytic enzymes used in the detection kit are different for different analytes, and the catalytic reaction can be started only after the reagents R1 and R2 are mixed, so that the analytes are gradually catalyzed to generate hydrogen peroxide.

According to the detection kit, peroxidase and 4-aminoantipyrine are added into an R1 reagent to remove clinical drugs participating in Trinder reaction in a substance to be detected. Part of clinical drugs have strong reducibility or are similar to chromogen substrate structures in Trinder reaction, so that the clinical drugs can participate in the Trinder reaction and compete to consume hydrogen peroxide generated by a substance to be detected, and the measured value of the final substance to be detected is low. In order to avoid the influence of clinical drugs on the determination result, the invention adds excessive peroxidase and 4-aminoantipyrine into the R1 reagent to process a serum sample before the Trinder reaction, so that the clinical drugs react with the hydrogen peroxide in the serum, the peroxidase in the R1 reagent and the 4-aminoantipyrine, and the clinical drugs are consumed in advance, thereby ensuring more accurate determination result and avoiding the interference of the clinical drugs on the Trinder reaction detection.

Preferably, the buffer solution in the R1 reagent and the R2 reagent is any one of phosphate buffer solution, HEPES buffer solution, Good's buffer solution, MOPSP buffer solution, BICINE buffer solution and glycine buffer solution.

Preferably, the concentration of the buffer is 20-100 mmol/L.

Preferably, the preservative in the R1 reagent and the R2 reagent is one or more of sodium azide, dichloroacetamide, imidazolidinyl urea, thimerosal, gentamicin, isothiazolinone, potassium sorbate, sodium benzoate, paraben, Proclin series and KroVin series.

Preferably, the concentration of the preservative is 0.1-10 g/L.

Preferably, the R1 reagent also comprises a metal ion chelating agent and a surfactant for maintaining the stability of the peroxidase.

Preferably, in the R2 reagent, the concentration of the peroxidase is 25-30 KU/L.

Preferably, the chromogen substrate is an aniline derivative, the concentration of the aniline derivative is 1g/L, and the aniline derivative is used as the chromogen substrate, so that compared with the traditional phenol substance, the stability and the solubility of a chromogen group, the sensitivity of a product and the stability of color and luster are improved.

Preferably, the aniline derivative is one or more of DHBS, TOOS, HDAOS, F-DAOS, DHBA, TOPS, ADOS, ADPS, ALPS, DAOS, MADB, MAOS and TODB. Wherein DHBS is sodium 3, 5-dichloro-2-hydroxybenzenesulfonate, TOOS is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, HDAOS is N- (2-hydroxy-3-sulfopropyl) -3' 5-dimethoxyaniline sodium salt, F-DAOS is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxy-4-fluoroaniline, DHBA is 3, 4-dihydroxybenzylamine hydrobromide, TOPS is N-ethyl-N- (3-sulfopropyl) -3-toluidine sodium salt, ADOS is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline sodium salt, ADPS is N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline sodium salt, ALPS is N-ethyl-N- (3-sulfopropyl) aniline sodium salt, DAOS is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline sodium salt, MADB is N, N-bis (4-sulfopropyl) -3, 5-dimethylaniline sodium salt, MAOS is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylaniline sodium salt, TODB is N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt, and DHBS has a better color development effect.

Preferably, the kit comprises: the kit comprises a creatinine detection kit (enzyme method), a triglyceride detection kit (GPO-PAP method), a total cholesterol detection kit (COD-PAP method), a free fatty acid detection kit (enzyme method), a glycated albumin detection kit (enzyme method), a high-density lipoprotein cholesterol detection kit, a low-density lipoprotein cholesterol detection kit, a uric acid detection kit (enzyme method), a glucose detection kit (GPO-PAP method), an adenosine deaminase detection kit, a lipase detection kit, a 5' -nucleotidase detection kit and the like.

The invention also provides a method for improving the anti-interference capability of Trinder reaction detection by using the detection kit based on the Trinder reaction, which comprises the following steps:

s1, adding peroxidase to a final concentration of 100-150 KU/L before the Trinder reaction, and simultaneously adding 4-aminoantipyrine to a final concentration of 0.5-1 g/L to remove clinical drugs in the object to be detected, wherein the clinical drugs can participate in the Trinder reaction;

s2, adding an enzyme capable of catalyzing the object to be detected to generate hydrogen peroxide during the Trinder reaction, and simultaneously adding peroxidase and aniline derivatives to the final concentrations of 25-30 KU/L and 1g/L respectively to perform the Trinder reaction to generate a color substance;

and S3, measuring the absorbance before and after the Trinder reaction to detect the concentration of the substance to be detected.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The performance evaluation method for each kit in the embodiment of the invention is concretely as follows:

(1) precision: repeatedly measuring the same sample for 10 times by adopting each kit, calculating the coefficient of variation (CV,%) and recording the coefficient of variation as the precision, wherein the precision is less than or equal to 5 percent and is regarded as meeting the requirement;

(2) linear range: determining samples in a certain concentration range by using each kit, and calculating a linear correlation coefficient r, wherein r is more than or equal to 0.99 and is regarded as meeting the requirement;

(3) accuracy: measuring a third-party quality control product by using each kit, and calculating the deviation between the measured value of each kit and the target value of the third-party quality control product, wherein the deviation is less than or equal to 10 percent to meet the requirement;

(4) functional sensitivity: adopting each kit to measure a low-concentration sample, measuring and calculating the diurnal variation coefficient of each kit, wherein the average concentration of the corresponding detection sample when the diurnal variation coefficient is 20 percent is recorded as the functional sensitivity;

(5) stability: each kit was left at 37 ℃ for 7 days, and the above-mentioned indexes were evaluated: precision, linear range, accuracy, functional sensitivity;

(6) interference evaluation: the interfering substances were diluted with ultrapure water in a gradient, and the concentrations of the interfering substances were added to the blank control serum (i.e., normal human serum mixture containing no interfering substances) at a ratio of 1:9, respectively, to give final concentrations of the interfering substances as shown in the table in the examples. The absorbance values of the blank serum and the serum added with the interfering substances with different concentrations are respectively measured by using each kit, each serum sample is measured for 3 times to obtain a mean value, and the deviation between the mean value and the measured value of the blank control serum ((mean value-blank control)/blank control x 100%) is calculated, namely the interference degree. If the absolute value of the interference degree is less than 10%, recording as anti-interference, otherwise, recording as non-anti-interference. Wherein the interfering drugs comprise calcium dobesilate, etamsylate, methyldopa, levodopa, and dopamine; common interfering substances in serum are ascorbic acid, bilirubin, hemoglobin and triglycerides.