CN111778313B - Angiotensin converting enzyme assay kit, preparation method and application - Google Patents
Angiotensin converting enzyme assay kit, preparation method and application Download PDFInfo
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- CN111778313B CN111778313B CN202010811233.5A CN202010811233A CN111778313B CN 111778313 B CN111778313 B CN 111778313B CN 202010811233 A CN202010811233 A CN 202010811233A CN 111778313 B CN111778313 B CN 111778313B
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- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
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Abstract
The invention provides an angiotensin converting enzyme assay kit, which belongs to the technical field of biological detection, and the kit consists 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, 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.
Description
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 end of a peptide chain of angiotensin I to form angiotensin II with a pressure boosting 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, 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%, phenylalanyl diglycine: 1mmol/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, emulgen 209, 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 angiotensin converting enzyme assay kit provided by the invention optimizes an ACE single reagent, and improves the bottle opening 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 bilirubin interference resistance of the ACE reagent is improved, and 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 illustrative of 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 bottle opening 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, 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 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, emulgen 209, 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.
Examples
1. The invention is provided with 4 comparative examples and 1 example, the kit components of each comparative example and example are shown in table 1:
TABLE 1 kit Components of comparative examples 1-4 and example 1
2. Reagent evaluation
The performance indices of the reagents of comparative examples 1 to 4 and example 1 were evaluated according to the following methods:
(1) Accuracy: measuring the third-party quality control product, and calculating the deviation between the measured average value and the target value, wherein the deviation is less than or equal to 10 percent and meets the requirement;
(2) Linear range: measuring a sample in the range of 0-200U/L, calculating a correlation coefficient, wherein r is more than or equal to 0.9900, the absolute deviation does not exceed +/-4U/L in the range of (0, 20) < U >/L, and the relative deviation does not exceed +/-10% in the range of (20, 200) < U >/L;
(3) Precision: repeatedly measuring the same sample for 10 times, and calculating CV (%), wherein the CV is less than or equal to 5.0% to meet the requirement;
(4) The bottle opening stability is as follows: placing the reagent on a biochemical analyzer in an open bottle, measuring the value three times per day, calculating the average value and the deviation, and if the deviation exceeds 10%, the requirement is not met;
(5) Anti-bilirubin interference: 5 serum samples with different bilirubin concentrations are prepared respectively, and the deviation of the measured value is not more than 10% compared with the measured value of a blank sample, which indicates that the reagent resists the bilirubin interference at the concentration.
3. Evaluation results and conclusions
The performance indices of the reagents of comparative examples 1-4 and example 1 are shown in tables 2-7:
table 2 performance evaluation data
TABLE 3 precision evaluation data
| Repeatability of | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Example 1 |
| 1 | 41.6 | 42.1 | 55.6 | 42.5 | 41.3 |
| 2 | 41.4 | 41.5 | 56.3 | 40.5 | 40.5 |
| 3 | 40.9 | 40.3 | 55.1 | 41.8 | 42.5 |
| 4 | 41.4 | 40.8 | 55.2 | 41.8 | 41.3 |
| 5 | 40.4 | 41.5 | 56.9 | 41.1 | 40.8 |
| 6 | 41.5 | 42.2 | 54.8 | 40.3 | 41.2 |
| 7 | 41.0 | 41.6 | 54.3 | 41.2 | 41.3 |
| 8 | 41.8 | 41.8 | 55.8 | 40.5 | 41.2 |
| 9 | 41.6 | 41.8 | 55.2 | 40.8 | 40.5 |
| 10 | 42.1 | 42.1 | 54.9 | 41.1 | 40.1 |
| AVE | 41.37 | 41.57 | 55.41 | 41.16 | 41.07 |
| SD | 0.49 | 0.60 | 0.76 | 0.70 | 0.66 |
| CV | 1.18％ | 1.45％ | 1.38％ | 1.69％ | 1.60％ |
Table 4 accuracy evaluation data
TABLE 5 Linear Range assessment data
From table 5 it can be seen that: after the potassium ferrocyanide is added in the comparative example 3, the absolute deviation in the range of (0, 20) U/L exceeds +/-4 percent, and the linear relative deviation in the range of (20, 200) U/L exceeds +/-10 percent, and the linear range does not meet the requirement.
TABLE 6 evaluation data of decap stability
TABLE 7 evaluation data of bilirubin interference resistance
And (4) conclusion:
in comparison with comparative example 1, in comparative example 2 in which 100mg/L of dithiothreitol (DDT) was added to the reagent, the open bottle stability of comparative example 2 was greatly improved at 31 days, and that of comparative example 1 at 15 days, from which it was found that DDT functioned as a stabilizer in the reagent.
Compared with the comparative example 1, the potassium ferrocyanide and the Emulgen A90 are respectively added in more, the anti-bilirubin capability of the comparative example 3 and the comparative example 4 reaches 0.5g/L, and the anti-bilirubin capability is obviously enhanced; however, compared with comparative example 4, in comparative example 3, the surfactant Emulgen A90 is not added, and only potassium ferrocyanide is added, although the anti-bilirubin capability of the ACE reagent can be enhanced, the reagent accuracy is obviously influenced, and the factor of accuracy reduction can be eliminated by adding the Emulgen A90.
In example 1, the accuracy, the bottle-opening stability and the anti-bilirubin interference capability of the beverage are remarkably improved by respectively adding the stabilizer, potassium ferrocyanide and Emulgen A90.
Finally, it should be further noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (8)
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, preservative: 0.05-0.1%, phenylpropylamido diglycine: 0.5-2.0 mmol/L; the pH value is 8.20 +/-0.05;
the surfactant is Emulgen A90;
the protective agent comprises any one of sucrose, mannitol, trehalose, lactitol, sorbitol, glucose, dithiothreitol and glutathione.
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: 1mmol/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-HCl 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 the preservative is sodium azide.
6. A method for preparing the angiotensin converting enzyme assay kit according to any one of claims 1 to 5, 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.
7. The method of manufacturing an angiotensin-converting enzyme assay kit according to claim 6, wherein the acid solution is 17% hydrochloric acid, the alkali solution is 20% NaOH solution.
8. Use of the angiotensin converting enzyme assay kit according to any of claims 1-5 in angiotensin converting enzyme assays.
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| CN111041021A (en) * | 2019-12-27 | 2020-04-21 | 桂林优利特医疗电子有限公司 | Preparation method of angiotensin converting enzyme calibrator |
| CN113720836A (en) * | 2021-09-17 | 2021-11-30 | 北京安图生物工程有限公司 | Kit for detecting serum copper ions and preparation method thereof |
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| CN109609593A (en) * | 2018-12-17 | 2019-04-12 | 武汉艾迪康医学检验所有限公司 | A kind of kit of quantitative detection angiotensin converting enzyme |
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