阅读说明：本技术 一种腺苷脱氨酶测定试剂盒 (Adenosine deaminase assay kit ) 是由 马全新 吴艳芳 谢小丽 于 2020-07-26 设计创作，主要内容包括：一种腺苷脱氨酶测定试剂盒,由彼此独立的试剂R1和试剂R2组成；所述试剂R1的组分包括：缓冲液、防腐剂、保护剂、抗干扰剂、表面活性剂、反应增强剂、过氧化物酶、嘌呤核甘磷酸化酶、黄嘌呤氧化酶、4-氨基安替比林和水；所述试剂R2的组分包括：缓冲液、保护剂、反应增强剂、防腐剂、腺苷、色原物质和水。本发明分析灵敏度高,线性可高达300U/L以上,重复性好,抗干扰能力强。(An adenosine deaminase assay kit, which consists of a reagent R1 and a reagent R2 which are independent from each other; the components of the reagent R1 comprise: buffer solution, preservative, protective agent, anti-interference agent, surfactant, reaction enhancer, peroxidase, purine nucleoside phosphorylase, xanthine oxidase, 4-aminoantipyrine and water; the components of the reagent R2 comprise: buffer solution, protective agent, reaction enhancer, preservative, adenosine, chromogen substance and water. The invention has high analysis sensitivity, good repeatability and strong anti-interference capability, and the linearity can reach more than 300U/L.)

1. An adenosine deaminase assay kit, which is characterized in that: consisting of reagent R1 and reagent R2 independently of each other:

composition of reagent R1:

composition of reagent R2:

2. the adenosine deaminase assay kit according to claim 1, wherein: the phosphate is one or more of potassium phosphate, beta-sodium glycerophosphate and disodium hydrogen phosphate.

3. The adenosine deaminase assay kit according to claim 1, wherein: the reagent R2 further comprises glycerol, and the concentration of the glycerol is 0.5-30 wt%.

4. The adenosine deaminase assay kit according to claim 1, wherein: the preservative is one or more of sodium azide, ProClin300 and gentamicin sulfate.

5. The adenosine deaminase assay kit according to claim 1, wherein: the chromogen is one or more of TOPS, TOOS, 4-chlorophenol, TBHBA, MADB and EHSPT.

6. The adenosine deaminase assay kit according to claim 1, wherein: the buffer solution is one or more of Tris, PB, MOPSO/Na and Hepes buffer solutions.

7. The adenosine deaminase assay kit according to claim 1, wherein: the surfactant is one or more of TritonX-100, PEG6000, Tween20 and Tween 80.

8. The adenosine deaminase assay kit according to claim 1, wherein:

the reagent R1 comprises the following components in percentage by concentration: 100mmol/L Tris buffer, 1.2mol/L aspartic acid, 0.05mol/L glutamic acid, 0.2KU/L purine nucleotide phosphorylase, 0.8KU/L xanthine oxidase, 3KU/L peroxidase, 2 mmol/L4-AAP, 0.05 wt% Proclin300, 1.2KU/L ascorbic acid oxidase, 5mmol/L sodium metavanadate, 0.5 wt% Triton X-100, 0.2 wt% Tween80, 100mmol/L magnesium chloride, 50mmol/L potassium chloride, 20mmol/L beta-sodium glycerophosphate, 50mmol/L sodium benzoate, 20mmol/L mannitol, pH of the reagent R1 is 7.0;

the reagent R2 comprises the following components in percentage by concentration: 100mmol/L phosphate buffer, 20mmol/L adenosine, 5mmol/L LTOPS, 0.05 wt% proclin300, 20 wt% glycerol, 150mmol/L disodium hydrogen phosphate, 100mmol/L mannitol, the pH of the reagent R2 is 3.0.

Technical Field

The invention relates to the technical field of medicine and biochemistry, in particular to a kit for measuring adenosine deaminase.

Background

Adenosine Deaminase (ADA) is an important enzyme in glance sideways at Ringxib metabolism, and is a nucleic acid metabolic enzyme which has an important relation with the cellular immune activity of a body. The activity of the liver-protecting peptide is a sensitive index reflecting liver injury, can be used as one of routine liver function examination items, and can comprehensively reflect the enzymatic change of liver diseases together with liver enzymes such as ALT or GGT. The activity determination of ADA in serum can be used for judging acute liver injury and residual lesion, assisting in diagnosing chronic liver diseases, helping in diagnosing liver fibers and helping in identifying xanthosis. In addition, the lack of ADA activity is associated with severe combined immunodeficiency disease (SC1D), and as an enzyme important for nucleic acid metabolism, ADA deficiency may cause nucleic acid metabolism disorder, affecting the development of thymus, thereby causing immune function deficiency. Cerebrospinal fluid ADA detection can be used as an important index for diagnosis and differential diagnosis of central nervous system diseases. ADA also has important value in differential diagnosis of exudates with difficult differentiation of benign and malignant diseases. Therefore, the determination of ADA and its isozyme levels in blood and body fluids has been increasingly clinically important for the identification, diagnosis, treatment, and immune function of these diseases.

In recent decades, the detection method has been developed with the intensive research on ADA. At present, the colorimetric method has been developed for five generations.

First generation ADA test: ADA deaminates Adenosine (Adenosine) to produce Inosine (Inosine) and ammonia (NH 3). The activity of ADA was measured by dynamically measuring the rate of decrease in absorbance at 265nm of adenosine. However, the high substrate concentration of this method causes the absorbance to be too high, and is only suitable for adenosine concentrations below 40.00. mu. mol/L. Such low substrate concentrations do not meet substrate saturation requirements, resulting in distorted detection of ADA activity. Therefore, this method is not suitable for clinical application.

The second generation of ADA tests, the principle of which is that adenosine deaminase catalyzes the hydrolysis of adenosine to produce inosine and ammonia. Serum ADA activity units were then calculated by measuring the amount of ammonia produced during the reaction using a Berthelot color reaction. The reagent required by the method is easy to prepare, the instrument is simple, but the sensitivity is low, the reagent is easily influenced by exogenous NH3, the blank is too high, and the activity of the erythrocyte ADA cannot be directly measured.

For the same reason, ADA-coupled Glutamate Dehydrogenase (GLDH) reaction ADA activity was calculated by measuring the rate of decrease in absorbance of NADPH (nicotinamide adenine dinucleotide phosphate, reduced coenzyme II) at 340 nm. This method also fails to accurately calculate ADA activity due to serum ammonia interference and non-specific oxidation caused by excessive NADPH in the test system.

Third generation ADA test: continuous monitoring was performed by ADA coupling Purine Nucleoside Phosphorylase (PNP) and Xanthine Oxidase (XOD) reactions. ADA activity was calculated by measuring the rate of rise in absorbance of urate at 293 nm. However, the absorbance of serum at 293nm is too high, which causes inconvenience in clinical application.

Fourth generation ADA test: coupling PNP, XOD, catalase (Ca talase) and Aldehyde dehydrogenase (Aldehydedehydrogenase) by ADA with the aid of hydrogen peroxide (H)₂O₂) The rate of increase of NADPH absorbance at 334nm was measured in the reaction to estimate ADA activity. The method overcomes the difficulties of the previous ADA test, but the cost of the method is high, and the practical clinical use is hindered.

Fifth generation ADA test: adenosine (adenosine) is deaminated by ADA enzymatic hydrolysis to produce Inosine (Inosine), and then Hypoxanthine (Hypoxanthine) is produced by PNP action, and the Hypoxanthine (Hypoxanthine) is produced by the hypo enzymatic hydrolysis to produce Uric Acid (Uric Acid) and hydrogen peroxide (H) under the oxidation of XOD₂O₂) (ii) a Finally H under the action of Peroxidase (POD)₂O₂And reacting with N-ethyl-N- (2-aryl-3-sulfopropyl) -3-methylbenzene (EHSPT) and 4-aminoantipyrine (4-aminoantipyrine, 4-AAP) to form a purple-red colored eye (Ouinone dye). ADA activity was measured by dynamic measurement of the rate of absorbance increase at 550nm in the colored eye.

Most of the currently marketed kits suffer from the following problems: the analysis sensitivity is not enough, the linear range is narrow, the repeatability is poor, and the anti-interference capability is weak.

Disclosure of Invention

In order to solve the problems, the invention provides an adenosine deaminase assay kit which has high analysis sensitivity, good repeatability and strong anti-interference capability, and the linearity can reach as high as 300U/L.

The invention relates to an adenosine deaminase assay kit, which is characterized in that: consisting of reagent R1 and reagent R2 independently of each other:

composition of reagent R1:

composition of reagent R2:

further, the phosphate is one or more of potassium phosphate, beta-sodium glycerophosphate and disodium hydrogen phosphate.

Still further, the reagent R2 further comprises glycerol, and the concentration of the glycerol is 0.5-30 wt%.

Still further, the preservative is one or more of sodium azide, ProClin300 and gentamicin sulfate.

Further, the chromogen is one or more selected from TOPS, TOOS, 4-chlorophenol, TBHBA, MADB and EHSPT.

Further, the buffer solution is one or more of Tris, PB, MOPSO/Na and Hepes buffer solution.

Further, the surfactant is one or more of TritonX-100, PEG6000, Tween20 and Tween 80.

Furthermore, the reagent R1 comprises the following components and concentrations: 100mmol/L Tris buffer, 1.2mol/L aspartic acid, 0.05mol/L glutamic acid, 0.2KU/L purine nucleotide phosphorylase, 0.8KU/L xanthine oxidase, 3KU/L peroxidase, 2 mmol/L4-AAP, 0.05 wt% Proclin300, 1.2KU/L ascorbic acid oxidase, 5mmol/L sodium metavanadate, 0.5 wt% Triton X-100, 0.2 wt% Tween80, 100mmol/L magnesium chloride, 50mmol/L potassium chloride, 20mmol/L beta-sodium glycerophosphate, 50mmol/L sodium benzoate, 20mmol/L mannitol, pH of the reagent R1 is 7.0;

the reagent R2 comprises the following components in percentage by concentration: 100mmol/L phosphate buffer, 20mmol/L adenosine, 5mmol/L LTOPS, 0.05 wt% proclin300, 20 wt% glycerol, 150mmol/L disodium hydrogen phosphate, 100mmol/L mannitol, the pH of the reagent R2 is 3.0.

The invention has the beneficial effects that: the kit has high analysis sensitivity, a linear range of 300U/L, good repeatability and strong anti-interference capability.

Drawings

FIG. 1 is a line diagram of the adenosine deaminase assay kit according to the present invention.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and procedures disclosed herein may be performed using molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and techniques conventional in the art. These techniques are well described IN the literature, IN particular, see Sambrook et al, Mobile CLONING: A LABORATORY MANUAL, Second edition, Cold Spring harbor LABORATORY Press,1989AND Third edition, 2001: Ausubel et al, CURRENT PROTOCOLS INLECULAR BIOLOGY, John Wiley & Sons, New York,1987AND personal updates: the servers METHODS IN ENZYM, Academic Press, Sandsystem: Wolff, CHROMATINSTRUMENT FUNCTION, Third edition, Academic Press, San Diego, Sandsystem, Church.304, Church.P.S.A.Waorward, Wal.S.A.1999, AND primer, Press, Sandsystem, Church.P.119, AND B.S.S.S.A.B.A. AND B.S.B.C.C. 1999.