CN-122012669-A - Reagent and kit for determining angiotensin converting enzyme and preparation method thereof

Abstract

The invention relates to an in-vitro detection reagent, in particular to a reagent and a kit for determining angiotensin converting enzyme and a preparation method thereof. The reagent comprises a substrate and an enzyme activity regulator, wherein the substrate can be catalyzed by angiotensin converting enzyme in a sample to be detected and generate a detectable signal, the intensity of the detectable signal is related to the activity of the angiotensin converting enzyme in the sample, the enzyme activity regulator comprises at least one of three metal ions Zn 2+ 、Co 2+ and Mn 2+ , and the concentration of the enzyme activity regulator exceeds the physiological concentration of the metal ions in serum by more than 10 times. By optimizing the reagent formula, the detection reaction rate is stabilized, and the analysis sensitivity, precision and accuracy are improved.

Inventors

• WANG XIN
• YANG KUNCHENG
• LI YUANLI
• WEN LUXIN
• RUI HAITAO

Assignees

• 中元汇吉生物技术股份有限公司

Dates

Publication Date

20260512

Application Date

20260313

Claims (10)

1. 1. A reagent for determining angiotensin converting enzyme comprising a substrate capable of being catalytically reacted by angiotensin converting enzyme in a sample to be tested and generating a detectable signal, the intensity of said detectable signal being related to the activity of angiotensin converting enzyme in said sample, characterized in that said kit further comprises an enzyme activity modifier comprising at least one of three metal ions Zn 2+ 、Co 2+ and Mn 2+ , and that the concentration of said enzyme activity modifier exceeds the physiological concentration of said metal ions in serum by a factor of more than 10.
2. 2. The reagent according to claim 1, wherein the concentration of the enzyme activity regulator is 1 to 50mmol/L, preferably 5 to 30mmol/L.
3. 3. The reagent according to claim 1 or 2, wherein the enzyme activity regulator is a metal salt, optionally the metal salt is selected from one of zinc chloride, zinc sulfate, zinc nitrate, zinc acetate, zinc gluconate, zinc citrate, zinc lactate, cobalt chloride, cobalt sulfate, cobalt nitrate, cobalt acetate, cobalt lactate, cobalt gluconate, manganese chloride, manganese sulfate, manganese nitrate, manganese acetate, manganese lactate, manganese gluconate, preferably the metal salt is one of hydrochloride, sulfate, nitrate, acetate of divalent zinc or cobalt or manganese.
4. 4. The reagent according to claim 1 or 2, wherein the substrate is a chromogenic substrate or a fluorogenic substrate, optionally the substrate is maleylglycine ‌ (HGG), N- [3- (2-furyl) acryloyl ] -L-phenylalanyl-glycyl-glycine (FAPGG) or sodium trinitrobenzene sulfonate (TNBS).
5. 5. The reagent according to claim 4, wherein the substrate concentration is 0.1 to 1.0 g/L.
6. 6. The reagent according to any one of claims 1 to 5, further comprising a stabilizer so that the reagent has an osmotic pressure 1 to 6 times that of physiological saline.
7. 7. The reagent of claim 6, wherein the stabilizer is an inorganic salt, sugar or polymer, optionally one of the inorganic salts NaCl、KCl、CaCl 2 、MgCl 2 、Na 2 SO 4 、K 2 SO 4 、NaNO 3 、KNO 3 , optionally the sugar is sucrose, trehalose, mannitol, optionally the polymer is polyethylene glycol.
8. 8. The reagent according to claim 1 or 2, further comprising a buffer having a pH of 6.0-8.0, optionally Tris-HCl, HEPES or MOPSO, optionally at a buffer concentration of 50-200mmol/L, optionally further comprising a preservative.
9. 9. A kit comprising the reagent according to any one of claims 1 to 8, optionally further comprising at least one of a calibrator and a quality control.
10. 10. The method for preparing the reagent according to any one of claims 1 to 8, wherein the preparation process comprises the steps of dissolving the components of the reagent in purified water according to a proportion, uniformly mixing, and adjusting to a target concentration.

Description

Reagent and kit for determining angiotensin converting enzyme and preparation method thereof Technical Field The invention relates to an in-vitro detection reagent, in particular to a reagent and a kit for determining angiotensin converting enzyme and a preparation method thereof. Background Angiotensin converting enzyme (Angiotensin Converting Enzyme, ACE), also known as kallikrein II or peptidyl-carboxypeptidase, is systematically named peptidyl-dipeptidyl hydrolase, is a zinc-containing membrane-bound glycoprotein with a molecular weight of 15000, and is an important regulator of renin-angiotensin-aldosterone system and skin-slowing system, affecting various physiological functions of human body. Measurement of ACE is of great importance for the auxiliary diagnosis of lung injury diseases and other various diseases, such as hyperthyroidism, viral hepatitis, liver cirrhosis, and serum ACE increase in diabetics, while serum ACE decrease in patients suffering from chronic obstructive pulmonary disease, asthma, adult respiratory distress syndrome, lung cancer, etc. In addition to disease diagnosis, measuring ACE is also important for monitoring therapeutic effects. Various antihypertensive agents (e.g., cilazapril, captopril, etc.) are ACE activity inhibitors, and in order to reduce the occurrence of common adverse drug reactions, the amount of such agents can be controlled by reducing the amount of the agent, but the amount of the agent to be reduced must be determined depending on the concentration of ACE. Therefore, monitoring of ACE activity is essential for controlling the dosage of patients with hypertension. The method for measuring ACE activity in serum comprises radioisotope method, fluorescence photometry, high performance liquid chromatography, immunoassay method, etc., and more spectrophotometry is used before, including ultraviolet spectrophotometry (HGG substrate method), trinitrobenzene sodium sulfonate (TNBS) chromogenic method, enzyme coupling method, N- [3- (2-furyl) acryloyl ] -L-phenylalanyl-glycyl-glycine (FAPGG) substrate method, isotope dilution mass spectrometry, etc. The FAPGG substrate method is common in detecting the content of ACE in serum, but the existing ACE detection kit on the market has the problems of abnormal analysis sensitivity improving curve, low analysis sensitivity, poor reagent precision, large fluctuation of detection results and high abnormality rate of detection results in the actual use process, and influences the accuracy and reliability of clinical detection. Disclosure of Invention In order to solve the technical problems, the invention adds the enzyme activity regulator into the angiotensin converting enzyme detection kit to stabilize the detection reaction rate and improve the analysis sensitivity, precision and accuracy. In a first aspect, the invention provides a reagent for determining angiotensin converting enzyme. The technical proposal is as follows: A reagent for determining angiotensin converting enzyme comprising a substrate capable of being catalytically reacted by angiotensin converting enzyme in a sample to be tested and generating a detectable signal, the intensity of said detectable signal being related to the activity of angiotensin converting enzyme in said sample, characterized in that said kit further comprises an enzyme activity modifier comprising at least one of three metal ions Zn 2+、Co2+ and Mn 2+, and that the concentration of said enzyme activity modifier exceeds the physiological concentration of said metal ions in serum by a factor of more than 10. In one embodiment, the concentration of the enzyme activity regulator is 1-50 mmol/L, and preferably, the concentration of the enzyme activity regulator is 5-30 mmol/L. In one embodiment, the enzyme activity regulator is a metal salt, optionally, the metal salt is selected from one of zinc chloride, zinc sulfate, zinc nitrate, zinc acetate, zinc gluconate, zinc citrate, zinc lactate, cobalt chloride, cobalt sulfate, cobalt nitrate, cobalt acetate, cobalt lactate, cobalt gluconate, manganese chloride, manganese sulfate, manganese nitrate, manganese acetate, manganese lactate and manganese gluconate, and preferably, the metal salt is one of hydrochloride, sulfate, nitrate and acetate of divalent zinc, divalent cobalt or divalent manganese. The invention is suitable for a detection method based on enzyme catalytic reaction, the intensity change rate of a detectable signal is related to the reaction rate, the reaction rate is related to ACE activity, and the ACE content in a sample to be detected can be obtained by detecting the time-dependent change relation of the intensity of the detectable signal and combining a standard curve. The present invention is not applicable to an immunoreaction detection method. In the detection method based on the reaction rate, uniformity of intensity variation of the detectable signal is represented by the advantages of signal linearity and sensitivity. Ac