阅读说明：本技术 一种血管紧张素转化酶测定试剂盒、制备方法及用途 (Angiotensin converting enzyme assay kit, preparation method and application ) 是由 凡速朋 龚婷 张超 舒芹 赵愿安 于 2020-08-13 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,属于生物检测技术领域,所述试剂盒以单试剂构成,单试剂包含以下成分：缓冲液：50～100mmol/L、无机盐：200～500mmol/L、亚铁氰化钾：5～20mg/L、表面活性剂：0.1％～5.0％、保护剂：10～100mg/L、防腐剂：0.05％～0.1％、苯丙酰氨基双甘肽：0.5～2.0mmol/L；pH为8.20±0.05。该试剂盒与常规ACE检测试剂盒相比,具有更好的开瓶稳定性及抗胆红素干扰能力,同时试剂的重复性、线性范围、精密度、准确度均不受影响。本发明还提供了一种血管紧张素转化酶测定试剂盒的制备方法及用途。(The invention provides an angiotensin converting enzyme assay kit, which belongs to the technical field of biological detection, and the kit is composed of a single reagent, wherein the single reagent comprises the following components: buffer solution: 50-100 mmol/L, inorganic salt: 200-500 mmol/L, potassium ferrocyanide: 5-20 mg/L, surfactant: 0.1-5.0%, protective agent: 10-100 mg/L of preservative: 0.05-0.1%, phenylpropylamido diglycine: 0.5-2.0 mmol/L; the pH was 8.20. + -. 0.05. Compared with the conventional ACE detection kit, the kit has better bottle opening stability and bilirubin interference resistance, and the repeatability, linear range, precision and accuracy of the reagent are not affected. The invention also provides a preparation method and application of the angiotensin converting enzyme assay kit.)

1. The kit for measuring the angiotensin converting enzyme is characterized by comprising a single reagent, wherein the single reagent comprises the following components:

buffer solution: 50-100 mmol/L, inorganic salt: 200-500 mmol/L, potassium ferrocyanide: 5-20 mg/L, surfactant: 0.1-5.0%, protective agent: 10-100 mg/L of preservative: 0.05-0.1%, phenylpropylamido diglycine: 0.5-2.0 mmol/L; the pH was 8.20. + -. 0.05.

2. The angiotensin converting enzyme assay kit according to claim 1, wherein said single reagent comprises the following components:

buffer solution: 80mmol/L, inorganic salt: 400mmol/L, potassium ferrocyanide: 5mg/L, surfactant: 0.5%, protective agent: 100mg/L, preservative: 0.05%, phenylpropylamido diglycine: 1 mmol/L; the pH was 8.2.

3. The angiotensin-converting enzyme assay kit according to claim 1, wherein the buffer comprises any one of phosphate buffer, Tris-HCI buffer, borate buffer, sodium borate buffer, HEPES, TOPS, PIPES, and TAPSO.

4. The angiotensin-converting enzyme assay kit according to claim 1, wherein the inorganic salt comprises any one or more of sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, zinc sulfate and sodium sulfate.

5. The angiotensin converting enzyme assay kit according to claim 1, wherein said surfactant comprises any one of polyethylene glycol 6000, tween-20, Emulgen a90, tween-80, polyoxyethylene lauryl ether, Emulgen209, polyoxyethylene lauryl ether, glycerol, Emulgen430, castor oil, Tritox-405, Emulgen a60, PVP-40, triton x114, triton x-100, GENAPOLX-080, Emulgen B66, NP-1055, polyethylene glycol 2000, polyethylene glycol 4000 and Emulgen 709.

6. The angiotensin converting enzyme assay kit according to claim 1, wherein the protective agent comprises any one of sucrose, mannitol, trehalose, lactitol, sorbitol, glucose, dithiothreitol, and glutathione.

7. The angiotensin-converting enzyme assay kit according to claim 1, wherein the preservative is sodium azide.

8. A method of making the angiotensin converting enzyme assay kit of any one of claims 1-7 comprising:

preparing raw materials: buffer solution, inorganic salt, potassium ferrocyanide, surfactant, protective agent, preservative and phenylpropylamido diglycine;

preparing a solution from a buffer solution, an inorganic salt, potassium ferrocyanide, a surfactant, a protective agent, a preservative and the phenylamidodiglycine, and adjusting the pH to 8.20 +/-0.05 by an acid solution or an alkali solution to obtain the single reagent of claim 1.

9. The method for preparing an angiotensin-converting enzyme assay kit according to claim 8, wherein the acid solution is 17% hydrochloric acid and the alkaline solution is 20% Na0H solution.

10. Use of an angiotensin converting enzyme assay kit according to any of claims 1-7 in the detection of angiotensin converting enzyme.

Technical Field

The invention belongs to the technical field of biological detection, and relates to an angiotensin converting enzyme assay kit, a preparation method and application thereof.

Background

Angiotensin converting enzyme, ACE for short, is a membrane-bound glycoprotein containing zinc ions, has a molecular weight of 150KD, belongs to dipeptide carboxypeptidase, and can hydrolyze histidine and leucine residues at the C-terminal of peptide chain of angiotensin I to form angiotensin II with supercharging effect, which causes vasobronchoconstriction through the action with angiotensin smooth muscle.

Angiotensin converting enzyme has the function of reducing blood pressure, and extinguishes fire with polypeptide inflammatory substances such as bradykinin P substance, and Angiotensin Converting Enzyme (ACE) can also directly act on adrenal cortex to promote secretion of aldosterone, so the ACE is an important regulating factor of renin angiotensin aldosterone system and bradykinin system, and affects various physiological functions of human body.

Various Angiotensin Converting Enzymes (ACE) are mainly localized to capillary endothelial cells, as lung tissues have abundant vascular beds and Angiotensin (ACE) contained in the endothelial cells of the pulmonary capillary beds is located outside cells, the effect of promoting angiotensin I to be converted into angiotensin II is strong, the lung circulation is the only thing in vivo and does not make the content of angiotensin II the highest, the ACE of the vascular endothelial cells is considered to be closely combined with cell membranes to release ACE almost, and most of the ACE produced by macrophages and monocytes is released into blood, so when the ACE is increased, the ACE should be considered to be secreted in macrophage and monocyte systems to be hypersecretion.

At present, the basic principle of the method used by an angiotensin converting enzyme diagnostic kit is that ACE catalyzes phenylalanyl amido diglycine (FAPGG) to be hydrolyzed into phenylalamide and diglycine, the absorbance is in a descending trend at the wavelength of 340nm, the descending rate of the absorbance is in direct proportion to the activity of ACE in a sample in a certain range, and the activity of the ACE can be calculated by detecting the descending trend of the absorbance at the position of 340nm by a continuous monitoring method.

At present, most of angiotensin-converting enzyme assay kits (continuous monitoring methods) in the market generally have two defects, one is the problem of poor stability, and the bottle opening stability is difficult to meet the requirement of 30 days. Secondly, the detection result is easily interfered by bilirubin.

Disclosure of Invention

In order to solve the technical problem of poor stability of the conventional ACE detection kit, the invention provides an angiotensin converting enzyme assay kit, and compared with the conventional ACE detection kit, the kit has better bottle opening stability and bilirubin interference resistance, and the repeatability, the linear range, the precision and the accuracy of the reagent are not influenced.

The invention also provides a preparation method and application of the angiotensin converting enzyme assay kit.

The invention is realized by the following technical scheme:

an angiotensin converting enzyme assay kit, which is composed of a single reagent, wherein the single reagent comprises the following components:

buffer solution: 50-100 mmol/L, inorganic salt: 200-500 mmol/L, potassium ferrocyanide: 5-20 mg/L, surfactant: 0.1-5.0%, protective agent: 10-100 mg/L of preservative: 0.05-0.10%, phenylpropylamido diglycine: 0.5-2.0 mmol/L; the pH was 8.20. + -. 0.05.

Preferably, the single agent comprises the following components:

buffer solution: 80mmol/L, inorganic salt: 400mmol/L, potassium ferrocyanide: 5mg/L, surfactant: 0.5%, protective agent: 100mg/L, preservative: 0.05%, phenylpropylamido diglycine: 1 mmol/L; the pH was 8.2.

Further, the buffer solution includes any one of phosphate buffer, Tris-HCl buffer, boric acid buffer, sodium borate buffer, HEPES, TOPS, PIPES, and TAPSO.

Further, the inorganic salt comprises any one or more of sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, zinc sulfate and sodium sulfate.

Further, the surfactant comprises any one of polyethylene glycol 6000, tween-20, Emulgen A90, tween-80, polyoxyethylene lauryl ether, Emulgen209, polyoxyethylene lauryl ether, glycerol, Emulgen430, castor oil, Tritox-405, Emulgen A60, PVP-40, Tritonx114, TritonX-100, GENAPOLX-080, Emulgen B66, NP-1055, polyethylene glycol 2000, polyethylene glycol 4000 and Emulgen 709.

Further, the protective agent comprises any one of sucrose, mannitol, trehalose, lactitol, sorbitol, glucose, dithiothreitol and glutathione.

Further, the preservative is sodium azide.

A method for preparing an angiotensin converting enzyme assay kit comprises the following steps:

preparing raw materials: buffer solution, inorganic salt, potassium ferrocyanide, surfactant, protective agent, preservative and phenylpropylamido diglycine;

preparing a buffer solution, inorganic salt, potassium ferrocyanide, a surfactant, a protective agent, a preservative and the phenylalanyl amido diglycine into a solution, and adjusting the pH to 8.20 +/-0.05 by acid liquor or alkali liquor to obtain the single reagent.

Further, the acid solution is 17% hydrochloric acid, and the alkali solution is 20% NaOH solution.

An application of an angiotensin converting enzyme assay kit in angiotensin converting enzyme detection.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the kit for determining the angiotensin converting enzyme optimizes the ACE single reagent, and improves the open bottle stability of the ACE reagent by adding a proper protective agent into the reagent; and a certain amount of surfactant and potassium ferrocyanide are added, so that the anti-bilirubin interference capability of the ACE reagent is improved, and meanwhile, the repeatability, sensitivity, linear range, precision and accuracy of the ACE reagent are not influenced.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, 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. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

In order to solve the technical problems, the embodiment of the invention provides the following general ideas:

aiming at the problems of poor bottle opening stability and insufficient anti-bilirubin interference capability of the conventional angiotensin converting enzyme detection kit, the invention provides the angiotensin detection kit (colorimetric method) with good stability and strong anti-bilirubin interference capability, and compared with the conventional angiotensin converting enzyme detection kit, the kit has better stability and anti-bilirubin interference capability.

The invention provides an angiotensin converting enzyme assay kit, which optimizes an ACE single reagent, and improves the open bottle stability of the ACE reagent by adding a proper protective agent into an R reagent; and a certain amount of surfactant and potassium ferrocyanide are added, so that the anti-bilirubin interference capability of the ACE reagent is improved, and the repeatability, linear range, precision and accuracy of the ACE reagent are not influenced.

Specifically, the kit for measuring angiotensin converting enzyme is composed of a single reagent, wherein the single reagent comprises the following components:

buffer solution: 50-100 mmol/L, inorganic salt: 200-500 mmol/L, potassium ferrocyanide: 5-20 mg/L, surfactant: 0.1-5.0%, protective agent: 10-100 mg/L of preservative: 0.05-0.1%, phenylpropylamido diglycine: 0.5-2.0 mmol/L; the pH was 8.20. + -. 0.05.

The buffer solution comprises any one of phosphate buffer solution, Tris-HCl buffer solution, boric acid buffer solution, HEPES, TOPS, PIPES and TAPSO. The inorganic salt comprises any one or more of sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, zinc sulfate and sodium sulfate.

The surfactant comprises any one of polyethylene glycol 6000, Tween-20, Emulgen A90, Tween-80, polyoxyethylene lauryl ether, Emulgen209, polyoxyethylene lauryl ether, glycerol, Emulgen430, castor oil, Tritox-405, Emulgen A60, PVP-40, Tritonx114, TritonX-100, GENAPOLX-080, Emulgen B66, NP-1055, polyethylene glycol 2000, polyethylene glycol 4000 and Emulgen 709.

Further, the protective agent comprises any one of sucrose, mannitol, trehalose, lactitol, sorbitol, glucose, dithiothreitol and glutathione. The preservative is sodium azide.

According to the invention, the stabilizer, the sodium chloride and the preservative are added into the R reagent to form the composite stabilizer, and the components have synergistic effect to ensure that the reagent has excellent stability, so that the stability of the kit is effectively enhanced, the storage life of the reagent is prolonged, and the accuracy and linear range indexes of the reagent are not influenced.

The interference of bilirubin in the blood sample is eliminated by adding potassium ferrocyanide, and the result of inaccurate measuring value of the jaundice sample is avoided. However, the addition of potassium ferrocyanide alone to reagent R can eliminate bilirubin interference, but has an effect on the accuracy and linear range of the reagent. And by adding Emulgen series surfactants, the reduction of linearity and accuracy can be effectively avoided. The potassium ferrocyanide can effectively eliminate bilirubin interference, but can reduce the reaction rate of a substrate, while the Emulgen series surfactants play a protection effect on the substrate reaction, and the interaction of the surfactant and the substrate protects the bilirubin interference resistance of the reagent, and does not influence the accuracy and linear performance indexes.

An angiotensin converting enzyme assay kit according to the present application will be described in detail below with reference to examples, comparative examples and experimental data.