阅读说明：本技术 一种血管紧张素转化酶测定试剂盒及其制备方法和应用 (Angiotensin converting enzyme assay kit and preparation method and application thereof ) 是由 刘安娜 王洪星 于 2020-03-20 设计创作，主要内容包括：本发明提供了一种血管紧张素转化酶测定试剂盒,试剂盒含有试剂R1和试剂R2,试剂R1包含以下组分：N-三(羟甲基)甲基-3-氨基丙磺酸、氯化钠、离子激活剂、聚甘油脂肪酸酯、4-羟基2,2,6,6-四甲基-1-氧-哌啶(AAQ-2)、防腐剂；试剂R2包含以下组分：N-三(羟甲基)甲基-3-氨基丙磺酸、FAPGG、稳定剂、防腐剂。本发明同时提供了该试剂盒的制备方法和应用,该试剂盒是一种抗干扰能力强、稳定性强、重复性好的液体试剂盒。(The invention provides an angiotensin converting enzyme assay kit, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components: n-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid, sodium chloride, an ion activator, polyglycerol fatty acid ester, 4-hydroxy-2, 2,6, 6-tetramethyl-1-oxy-piperidine (AAQ-2), and a preservative; reagent R2 contained the following components: n-tri (hydroxymethyl) methyl-3-aminopropanesulfonic acid, FAPGG, a stabilizer and a preservative. The invention also provides a preparation method and application of the kit, and the kit is a liquid kit with strong anti-interference capability, strong stability and good repeatability.)

1. An angiotensin converting enzyme assay kit, characterized in that the kit comprises a reagent R1 and a reagent R2:

reagent R1 contained the following components:

100 mmol/L of N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS buffer);

sodium chloride 200-400 mmol/L;

0.5-1 mmol/L of ion activator;

5-20 mg/L of polyglycerol fatty acid ester;

4-hydroxy-2, 2,6, 6-tetramethyl-1-oxo-piperidine (AAQ-2) 5-20 mg/L;

0.5-2 g/L of preservative;

the reagent R2 contains the following components:

100 mmol/L of N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS buffer);

FAPGG 0.7-1.5 mmol/L；

1-15 g/L of stabilizer;

0.5-2 g/L of preservative.

2. The angiotensin converting enzyme assay kit according to claim 1, wherein the pH of reagent R1 is 7.0 to 9.0; the pH value of the reagent R2 is 7.0-8.5.

3. The angiotensin converting enzyme assay kit according to claim 1, wherein the ion activator in reagent R1 is one or more of zinc chloride, zinc sulfate, zinc nitrate, and zinc phosphate.

4. The kit for measuring angiotensin-converting enzyme according to claim 1, wherein the stabilizer in reagent R2 is a mixture of chitosan oligosaccharide, glycerol, Surfynol-465, and the mass ratio of chitosan oligosaccharide, glycerol, Surfynol-465 is 2:3-5: 4-8.

5. The angiotensin converting enzyme assay kit according to claim 1, wherein said preservative in reagent R1 is one or more of sodium azide, PC300, MIT and gentamicin sulfate; the preservative described in the reagent R2 is one or more of sodium azide, PC300, MIT and gentamicin sulfate.

6. The angiotensin converting enzyme assay kit according to claim 1, wherein the volume ratio of the reagent R1 to the reagent R2 is 1-5: 1.

7. The angiotensin converting enzyme assay kit according to claim 1, wherein the volume ratio of reagent R1 to reagent R2 is 3: 1.

8. A method for preparing the angiotensin converting enzyme assay kit according to any one of claims 1 to 7, comprising the steps of:

(1) preparation of reagent R1: taking a proper amount of water, respectively and sequentially adding the raw materials shown in R1, stirring uniformly to dissolve one raw material, adding the next raw material, adjusting the pH value to 7.0-9.0 by using hydrochloric acid or sodium hydroxide, and fixing the volume to the required volume;

(2) preparation of reagent R2: adding appropriate amount of water into R2, stirring to dissolve one material, adding the next material, adjusting pH to 7.0-8.5 with hydrochloric acid or sodium hydroxide, and diluting to desired volume.

9. Use of a kit according to any one of claims 1 to 7 for the determination of the concentration of angiotensin converting enzyme in serum for non-disease diagnostic and therapeutic purposes.

Technical Field

The invention relates to the technical field of biochemical reagent determination, in particular to an angiotensin converting enzyme determination kit and a preparation method and application thereof.

Background

Angiotensin converting enzyme (ACE, EC3.4.15.1) is a ubiquitous Zn in mammalian tissues²+ dependent carboxypeptidase, a membrane-integrated single chain glycoprotein, is mainly present in pulmonary vascular endothelial cells, and can also be found in most tissues such as uterus, placenta, heart, kidney, brain, adrenal cortex, etc. By renin-angiotensinThe system RAS (rennin-angiotensin system) and kallikrein-kinin system, ACE plays an important regulatory role in blood pressure, electrolyte and fluid balance, cardiovascular system development and structural remodeling. In Zn²⁺In the presence of the enzyme, ACE can hydrolyze peptide bond between Phe8-His9 in decapeptide angiotensin I (angiotensin I, Ang I) to generate octapeptide angiotensin II (Ang II) and carboxyl-terminal dipeptide His-L eu. angiotensin II, which is one of the strongest vasoconstrictors found in the current research, acts on angiotensin receptor 1, constricts vascular smooth muscle, stimulates aldosterone secretion, and promotes Na pair of human kidney⁺、K⁺Reabsorption, which causes an increase in sodium storage and blood volume, resulting in an increase in blood pressure, has positive inotropic and chronotropic effects on the heart, and at the same time, ACE hydrolyzes the two amino acids at the carboxy terminus of bradykinin, a vasodilator, and inactivates its biological activity in dilating blood vessels, and is called kallikrein 2. in addition to the substrates angiotensin I and bradykinin, ACE hydrolyzes a variety of vasoactive peptides, such as enkephalin, substance P, neurotensin, etc., and has been shown in vitro to hydrolyze β amyloid overexpressed in Alzheimer's disease.

The level of ACE content in serum depends on the capacity of tissue cells to synthesize and release ACE. If the function of the cell is disordered, the dynamic balance of the ACE content between tissues and blood is influenced, the ACE content in serum is changed, the clinical diagnosis significance is provided for diseases such as lung diseases, liver diseases, diabetes, coronary heart diseases, myocardial infarction and the like, and the serum ACE can be used as an index for clinical relevant diagnosis.

In the early days, fluorescence photometry, biological radiation chromatography and spectrophotometry are mostly adopted for ACE determination, and high performance liquid chromatography, radioimmunoassay, enzyme coupling method, ACE gene determination method and the like are successively established along with the deepening of ACE research.

The enzyme coupling method is characterized in that ACE in serum acts on a substrate hippurate diglucoside to generate hippurate and diglucoside, gamma-glutamyltransferase is used as an indicator enzyme to catalyze enzyme coupling reaction, the absorbance is measured at 410nm, and the ACE activity is in direct proportion to the product amount. The coupling reaction is as follows:

radioimmunoassay using a combination of protease inhibitors L isinopril, Aprotinin and EDTA, but requires special equipment and is not widely used.

Spectrophotometry: hippuric acid-histidine-leucine is used as a reaction substrate of ACE and is decomposed into hippuric acid and histidine-leucine, hippuric acid and cyanuric chloride are combined to be light yellow, and the ACE activity can be calculated through a colorimetric method.

A continuous monitoring method: specific activity of ACE was measured using furylacryloylglycine (furylacryloyl-phe-Gly, fagg) as a substrate. Fagg, when hydrolyzed by ACE catalysis, produces a change in absorbance, measured at 340 nm. The sensitivity of the method is obviously improved, and the reference value range is also determined. The recent domestic and foreign data for determining the ACE activity of serum (plasma) or tissue fluid mostly adopts FAGG as a reagent of a substrate, and the method can be used for a full-automatic biochemical analyzer and is suitable for clinical routine examination. However, because the activity of angiotensin converting enzyme is easily inhibited by factors such as bilirubin, the common angiotensin converting enzyme detection reagent has the defects of poor repeatability, poor anti-interference performance and the like.

Disclosure of Invention

In order to solve the problems, the invention provides an angiotensin converting enzyme assay kit, a preparation method and application thereof.

The invention is realized by the following technical scheme:

an angiotensin converting enzyme assay kit, comprising a reagent R1 and a reagent R2:

reagent R1 contained the following components:

the reagent R2 contains the following components:

preferably, the pH value of the reagent R1 is 7.0-9.0; the pH value of the reagent R2 is 7.0-8.5.

Preferably, the ionic activator in the reagent R1 is one or more of zinc chloride, zinc sulfate, zinc nitrate and zinc phosphate.

Preferably, the stabilizing agent in the reagent R2 is a mixture of chitosan oligosaccharide, glycerol and Surfynol-465, and the mass ratio of the chitosan oligosaccharide, the glycerol and the Surfynol-465 is 2:3-5: 4-8.

Preferably, the preservative in the reagent R1 is one or more of sodium azide, PC300, MIT and gentamicin sulfate; the preservative described in the reagent R2 is one or more of sodium azide, PC300, MIT and gentamicin sulfate.

The preparation method of the angiotensin converting enzyme assay kit comprises the following steps:

(1) preparation of reagent R1: adding appropriate amount of water into R1, stirring to dissolve one material, adding the next material, adjusting pH to 7.0-9.0 with hydrochloric acid or sodium hydroxide, and diluting to desired volume.

(2) Preparation of reagent R2: adding appropriate amount of water into R2, stirring to dissolve one material, adding the next material, adjusting pH to 7.0-8.5 with hydrochloric acid or sodium hydroxide, and diluting to desired volume.

The application of the angiotensin converting enzyme assay kit is used for determining the concentration of angiotensin converting enzyme in serum for the purposes of diagnosis and treatment of non-diseases.

The principle of the kit for measuring the angiotensin converting enzyme is that when FAGG is catalyzed and hydrolyzed by ACE, absorbance change is generated, measurement is carried out at 340nm, and the maximum absorption peak of FAGG is 340 nm.

The invention has the beneficial effects

1) The angiotensin converting enzyme assay kit is a liquid double reagent, does not need to be prepared by redissolution, can be directly used after being opened, and can be used for incubating a sample to remove interference by using a reagent R1, so that the stability of FAPGG can be maintained to the maximum extent by using a reagent R2.

2) The buffer solution in the angiotensin converting enzyme reagents R1 and R2 is TAPS buffer solution, can promote the catalytic action of angiotensin converting enzyme, and on the basis of adding sodium chloride, zinc metal salt is added at the same time, and the two are used as ion activators of the reaction, so that the capability of catalyzing substrates by enzyme is completely activated, and the specificity and the accuracy of the reaction are ensured.

3) The polyglycerol fatty acid ester in the angiotensin converting enzyme reagent R1 can remove the inhibition effect of bilirubin in serum on the activity of angiotensin converting enzyme, and the 4-hydroxy-2, 2,6, 6-tetramethyl-1-oxy-piperidine (AAQ-2) can be added to remove the interference effect of ascorbic acid on the reagent, thereby improving the anti-interference performance of the reagent.

4) According to the invention, by adding chitosan oligosaccharide, glycerol and Surfynol-465 into the reagent, FAPGG in the reagent can be protected in a synergistic manner, and Surfynol-465 is taken as a surfactant, so that the reaction system is uniform and stable, and the stability and the repeatability of the reagent are improved.

Drawings

FIG. 1 is a correlation curve for the reagents of example 1 and comparative example 1;

FIG. 2 is a correlation curve for the reagents of comparative example 1 and comparative example 2;

FIG. 3 is a graph showing the change in concentration of the sialic acid assay reagent provided in example 1 and comparative examples 1, 2, 5, 6 and 7 for stability test.

Detailed Description

The following is a description of specific embodiments of the present invention with reference to the drawings, and the technical solutions of the present invention will be further described, but the present invention is not limited to these embodiments.

The test conditions of the kit for measuring the angiotensin converting enzyme in the serum are as follows: the method comprises the following steps: a rate method; dominant wavelength: 340 nm; temperature: 37 ℃; the correction type is as follows: linearity; the calibration method comprises the following steps: calibrating at two points; the reaction direction is as follows: downwards.

Procedure for the preparation of the

TABLE 1 angiotensin converting enzyme assay reagent protocol

Calculation method

Sample requirements:

1. fresh serum.

2. Sample stability: the specimen can be stored stably for 30 days at the temperature of 2-8 ℃ and for 6 months at the temperature of-20 ℃.