Reagent and kit for determining angiotensin converting enzyme and preparation method of reagent and kit

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 of the reagent and the kit. The reagent comprises a substrate and an enzyme activity regulator, the substrate can be catalyzed by angiotensin converting enzyme in a to-be-detected sample to react and generate a detectable signal, the strength 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, namely Zn < 2 + >, Co < 2 + > and Mn < 2 + >, and the concentration of the enzyme activity regulator is more than 10 times of the physiological concentration of metal ions in serum. By optimizing the reagent formula, the detection reaction rate is stabilized, and the analysis sensitivity, precision and accuracy are improved.

Description

Technical Field

[0001] This invention relates to an in vitro detection reagent, specifically to a reagent, kit, and preparation method for measuring angiotensin-converting enzyme. Background Technology

[0002] Angiotensin-converting enzyme (ACE), also known as kininase II or peptidyl-carboxypeptidase, is a zinc-containing membrane-bound glycoprotein with a molecular weight of 15,000. It is a crucial regulator of the renin-angiotensin-aldosterone system and the bradykinin system, influencing various physiological functions. ACE measurement is significant for the auxiliary diagnosis of lung injury and many other diseases. For example, serum ACE levels are elevated in patients with hyperthyroidism, viral hepatitis, cirrhosis, and diabetes, while serum ACE levels are decreased in patients with chronic obstructive pulmonary disease, asthma, adult respiratory distress syndrome, and lung cancer. Besides disease diagnosis, ACE measurement plays an important role in monitoring treatment efficacy. Many antihypertensive drugs (such as cilazapril and captopril) are ACE inhibitors. To reduce common adverse drug reactions, dosage reduction can be achieved, but this must be determined based on ACE concentration. Therefore, monitoring ACE activity is essential for controlling medication dosage in hypertensive patients.

[0003] Methods for measuring ACE activity in serum include radioisotope assays, fluorescence spectrometry, high-performance liquid chromatography (HPLC), and immunoassays. Spectrophotometry is currently the most commonly used method, including ultraviolet spectrophotometry (HGG substrate method), sodium trinitrobenzenesulfonate (TNBS) colorimetric method, enzyme-coupled assay, N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG) substrate method, and isotope dilution mass spectrometry. Among these, the FAPPGG substrate method is the most common for detecting ACE levels in serum. However, commercially available ACE test kits currently exhibit several issues in practical use, such as abnormal sensitivity enhancement curves, low analytical sensitivity, poor reagent precision, large fluctuations in test results, and a high rate of abnormal results, affecting the accuracy and reliability of clinical testing. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention adds an enzyme activity regulator to the angiotensin-converting enzyme assay kit to stabilize the detection reaction rate and improve analytical sensitivity, precision, and accuracy.

[0005] The first aspect of this invention provides a reagent for measuring angiotensin-converting enzyme. The technical solution is as follows:

[0006] A reagent for determining angiotensin-converting enzyme (ACE) includes a substrate capable of being catalyzed by ACE in a test sample to generate a detectable signal, the intensity of which is correlated with the ACE activity in the sample. The reagent kit further includes an enzyme activity regulator comprising Zn. 2+ Co 2+ and Mn 2+ The enzyme activity regulator contains at least one of three metal ions, and the concentration of the enzyme activity regulator exceeds 10 times the physiological concentration of the metal ion in serum.

[0007] In one embodiment, the concentration of the enzyme activity regulator is 1-50 mmol / L; preferably, the concentration of the enzyme activity regulator is 5-30 mmol / L.

[0008] In one embodiment, the enzyme activity regulator is a metal salt; optionally, the metal salt is selected from 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; preferably, the metal salt is one of the hydrochloride, sulfate, nitrate, and acetate of divalent zinc, divalent cobalt, or divalent manganese.

[0009] This invention is applicable to enzyme-catalyzed reaction-based detection methods. The rate of change in the intensity of the detectable signal is related to the reaction rate, which in turn is related to ACE activity. By detecting the relationship between the intensity of the detectable signal and time, and combining this with a standard curve, the ACE content in the sample can be obtained. This invention is not applicable to immunoreaction detection methods. In reaction rate-based detection methods, the uniformity of the intensity change of the detectable signal reflects the quality of signal linearity and sensitivity. This invention improves detection results by adding an excess of metal ions to the reaction system. Because these metal ions can competitively bind to ACE protein molecules, enzyme activity is reduced, the reaction rate is slowed down, and the linearity and sensitivity of the reaction are improved. This improvement mechanism differs from adding enzyme activity inhibitors such as ethylenediaminetetraacetic acid (EDTA), because EDTA inactivates the enzyme by binding to zinc ions at the active site of ACE, causing a conformational change. However, the concentration of ACE enzyme in typical samples is not high, making it difficult to control the amount of EDTA added. This invention uses an excess of metal ions to bind to the ACE protein, weakening but not inhibiting enzyme activity. Therefore, Zn is used as an enzyme activity regulator. 2+ Co 2+ and Mn 2+ It should exist in solution in a dissociated or weakly complexed state, so that it can competitively bind to ACE protein molecules.

[0010] Any substrate that can be catalyzed by ACE and produce a detectable signal related to the reaction rate in the detection system can be used. The detectable signal is preferably a light signal. The reaction rate can be estimated from the rate of change of the light signal intensity and the reaction time, thereby calculating the ACE concentration in the sample.

[0011] In one embodiment, the substrate is a chromogenic substrate or a fluorescent substrate; optionally, the substrate is hippuryl glycyl glycine (HGG), N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG) or sodium trinitrobenzenesulfonate (TNBS).

[0012] In one embodiment, the substrate concentration is 0.1~1.0 g / L.

[0013] In one embodiment, the reagent further includes a stabilizer to give the reagent an osmotic pressure 1 to 6 times that of physiological saline. A high osmotic pressure environment is beneficial for improving the stability of ACE in the sample, thereby maintaining its activity.

[0014] In one embodiment, the stabilizer is an inorganic salt, a sugar, or a polymer; optionally, the inorganic salt is a sodium or potassium salt, such as one of NaCl, KCl, CaCl2, MgCl2, Na2SO4, K2SO4, NaNO3, and KNO3; optionally, the sugar is sucrose, trehalose, or mannitol; optionally, the polymer is polyethylene glycol or dextran.

[0015] To create a highly permeable environment, the amount of stabilizer used is based on osmotic pressure, and its dosage can be flexibly adjusted according to the type of substance. Typically, the inorganic salt concentration is 10-50 g / L, the small molecule sugar concentration is 1-10% (w / v), and the polymer addition is 0.1-1% (w / v). The PEG molecular weight in the polymer can be selected from 500 to 6000, preferably 2000-4000; the dextran molecular weight should not exceed 10,000. Several stabilizers can be used in combination.

[0016] In one embodiment, the reagent further includes a buffer having a pH of 6.0-8.0, preferably 6.8-7.4; optionally, the buffer is tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), or 3-(N-morpholine)-2-hydroxypropanesulfonic acid (MOPSO); optionally, the concentration of the buffer is 50-200 mmol / L.

[0017] In one embodiment, the reagent further includes a preservative. Various preservatives are known to be commonly used in biochemical reagents, typically added at amounts of 0.5-1.5 g / L, such as sodium azide.

[0018] A second aspect of this invention provides a reagent kit. The technical solution is as follows: A reagent kit comprising the reagents described in any one of the above.

[0019] In one embodiment, the kit further includes at least one of calibrators and quality control products.

[0020] A third aspect of this invention provides a method for preparing the above-mentioned reagent. The technical solution is as follows: A method for preparing the above-mentioned reagent involves dissolving each component of the reagent in purified water according to the specified ratio, mixing them evenly, adjusting the pH to the target pH, and then making up the volume. Attached Figure Description

[0021] Figure 1 The original detection curve for detecting ACE in serum samples using the kit from Example 1 is shown. Figure 2 The original detection curve for detecting ACE in serum samples using the kit in Example 2; Figure 3 The original detection curve for detecting ACE in serum samples using the kit from Example 1; Figure 4 The test values ​​of the kit in Example 1 were compared with the theoretical values ​​to test a series of concentration control samples. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0024] In in vitro diagnostics, precision refers to the closeness of independent test results obtained under specified conditions. Sensitivity is the minimum amount of an analyte that an in vitro diagnostic reagent can detect in a sample, and it has two meanings: ① the minimum concentration or content at which a change in concentration or content can cause a significant change in the detection signal; ② the minimum concentration or content of the analyte that can be detected beyond zero with appropriate limits. Accuracy refers to the degree of agreement between the test result and the true value of the measured quantity.

[0025] This invention addresses the problems of abnormal signal intensity changes, low sensitivity, and poor detection accuracy in existing reagent kits based on enzyme-catalyzed reaction rate methods by optimizing reagents and improving detection performance.

[0026] A reagent for determining angiotensin-converting enzyme (ACE) includes a substrate capable of being catalyzed by ACE in a test sample to generate a detectable signal, the intensity of which is correlated with the ACE activity in the sample. The kit also includes an enzyme activity modifier comprising Zn. 2+ Co 2+ and Mn 2+ At least one of three metal ions, and the concentration of the enzyme activity regulator exceeds 10 times the physiological concentration of the metal ion in serum.

[0027] Since ACE is a zinc-dependent enzyme, under normal physiological conditions, zinc ions bind to ACE protein molecules in a complex structure, forming the enzyme's active site. Under physiological conditions, zinc ion concentrations are generally in the micromolar range; for example, serum zinc ion concentrations are approximately 10-20 μmol / L. In environments containing higher levels of zinc, cobalt, or manganese ions, these metal ions competitively bind to ACE protein molecules, thereby inhibiting enzyme activity. The enzymatic reaction proceeds at a more moderate but stable rate, facilitating the acquisition of a stable and varying detection signal intensity for calculating enzyme activity.

[0028] To ensure the reaction proceeds normally, the presence of chloride ions in the reaction system is detected.

[0029] The concentration of the enzyme activity regulator is 1–50 mmol / L, such as 1 mmol / L, 2 mmol / L, 5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, and 50 mmol / L. The concentration of the enzyme activity regulator can be flexibly adjusted according to different substrates and detection systems, with 5–30 mmol / L being more preferred. The enzyme activity regulator is added as a metal salt.

[0030] The substrate can be a chromogenic or fluorescent substrate, such as hippurylglycylglycine (HGG), N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG), or sodium trinitrobenzenesulfonate (TNBS). The substrate concentration is 0.1–1.0 g / L.

[0031] To further improve the reaction efficiency, the reagent also includes stabilizers to achieve an osmotic pressure 1 to 6 times that of physiological saline. A high osmotic pressure environment is beneficial for improving the stability of ACE in the sample, thereby maintaining its activity. Stabilizers can be inorganic salts, sugars, or polymers.

[0032] To maintain a suitable detection environment for the ACE catalytic reaction, the reagents are prepared using a buffer solution. There are no particular restrictions on the buffer, as long as it does not interfere with enzyme activity; the pH of the buffer solution should be 6.0-8.0.

[0033] Taking the FAPGG method reagent as an example, the solution of the present invention will be further explained.

[0034] (I) Reagents and their preparation Example 1 A detection reagent, the composition of which is shown in Table 1.

[0035] Table 1. Components of the kit

[0036] The reagent kit is prepared as follows.

[0037] Substrate purification process: Crude FAPGG was dissolved in methanol, and lithium hydroxide was dissolved in pure water and added to the reaction system. The reaction system slowly became clear. After the addition was complete, the reaction was allowed to proceed for 0.5 hours. The reaction was then checked for completeness, and the pH was adjusted to 6.0 with 0.2M dilute hydrochloric acid. 16 mL of water was added, and methanol was removed by rotary evaporation at 35°C. A white solid precipitated during rotary evaporation. The suspension was filtered, washed with pure water until the pH of the filtrate was between 6.0 and 7.0, dried under vacuum (using an oil pump) at 25°C for 0.5 hours, yielding 0.616 g of pure L-FAPGG. HPLC analysis confirmed a yield of 81.7% and a purity of 99.51%.

[0038] Reagent preparation: Weigh the preparation container, record the weight, and tare it. Add 0.9L of purified water required for preparation to the preparation container. Add buffer, enzyme activity regulator, stabilizer, substrate, and preservative in sequence. Adjust the pH to 7.0±0.05 (25 ℃) with sodium hydroxide. Filter the solution through a 0.45μm capsule filter into the reagent container and store at 2-8 ℃.

[0039] Example 2 The difference from Example 1 is that the stabilizer sodium chloride is not added.

[0040] Compare with Example 1 The difference from Example 1 is that sodium sulfate is used instead of the enzyme activity regulator ZnSO4.

[0041] (II) Verification of Testing Results Example 3: Detection effect of different reagents Testing was conducted using natural serum samples.

[0042] Detection principle: As shown in the reaction formula below, angiotensin-converting enzyme catalyzes the hydrolysis of phenylalanine diglypeptide (FAPGG) into furanoylphenylalanine (FAP) and diglypeptide. At a wavelength of 340 nm, the absorbance shows a decreasing trend. Within a certain range, the decrease in absorbance is directly proportional to the activity of angiotensin-converting enzyme in the sample.

[0043]

[0044] Angiotensin-converting enzyme (U / L) in the sample = CS × Δ AT / Δ AS (mg / L) In the formula: Δ AT is the absorbance value of the sample tube compared to the absorbance of the blank tube. Δ AS is the absorbance value of the calibration tube compared to the absorbance of the blank tube, and CS is the concentration of angiotensin-converting enzyme in the calibration solution.

[0045] The testing process is as follows: (1) The serum sample to be tested was mixed with reagent R1 of Example 1, Example 2 and Control Example 1 at a volume ratio of 20:160, and stirred until fully reacted; (2) The absorbance difference after the reaction was measured using a fully automated biochemical analyzer (Hitachi 7180) (main wavelength 340nm, secondary wavelength 450nm, instrument reading point 10-28). (3) Calculate the concentration of angiotensin-converting enzyme in the sample based on the absorbance change value.

[0046] Typical reaction original curves are as follows: Figures 1-3 As shown. Absorbance Δ AT Δ AS represents the change in absorbance of the test tube at the detection time relative to the initial time. For example... Figure 1 In Control Example 1, no enzyme activity regulators or stabilizers were added to the reagents. Initially, due to high substrate concentration and unconstrained enzyme activity, the reaction rate was excessively fast (non-linear). As the substrate was rapidly consumed, the reaction rate dropped sharply in the middle section (10-28 points), even approaching stagnation, but absorbance detection continued. Due to the inherent characteristics of the detection system, the measured values ​​fluctuated significantly. This "fast at the beginning, slow at the end" non-linear characteristic not only resulted in poor stability of the reaction curve (large fluctuations) but also directly led to: ① masking of signals from low-concentration samples, i.e., insufficient sensitivity; ② poor repeatability and low precision, and even the possibility of 0 or negative values ​​(false negatives) due to baseline drift.

[0047] like Figure 2In Example 2, an enzyme activity regulator was added to the reagent compared to the control example. The metal ions in this reagent can competitively inhibit or allosterically regulate enzyme activity by binding to the active site of ACE or the substrate, reducing the initial reaction rate and maintaining a relatively stable rate. This transforms the entire reaction process from "non-linear" to "linear," resolving the inconsistency of "fast in the early stage and slow in the later stage." However, although the reaction rate is relatively uniform, the reaction curve still fluctuates, indicating insufficient stability and room for improvement in precision. This is presumably due to the structural stability of the enzyme; the enzyme molecule may undergo conformational changes due to environmental fluctuations such as small changes in temperature and pH.

[0048] like Figure 3 The reagent in Example 1 contains both an enzyme activity regulator and a stabilizer. The high osmotic pressure environment can stabilize the spatial structure of ACE and protect the active site of the enzyme from being destroyed. It reduces the aggregation or denaturation of enzyme molecules, so that the enzyme activity remains consistent throughout the reaction, thereby making the reaction rate more stable and the reaction curve straighter.

[0049] Example 4: Accuracy and Precision Verification The kit from Example 1 was used to perform repeatability verification using two different levels of quality control samples (Zhongyuan Huiji Biotechnology Co., Ltd.). The test results are shown in Table 2. The relative deviation of the three measurements in each group was within 10%, and the intra-batch repeatability was within 5%.

[0050] Table 2 Test Results of Quality Control Products

[0051] Different concentrations of test samples were prepared using high- and low-value quality control samples to validate the kit in Example 1. Two tests were performed: one from high to low and the other from low to high. Correlation analysis was conducted between the mean and estimated values ​​of each test group. The test results are as follows: Figure 4 As shown, the test results of the kit in Example 1 have good correlation and a wide linear range.

[0052] Example 5: Comparison of test results with commercially available products The kit from Example 1 was compared with five commercially available kits of the same type for clinical sample testing. The number of test cases with abnormal results was counted, and the abnormality rate of the test results was compared. Abnormal results are defined as test cases with clinical test results of 0 or negative values. As shown in Table 3, the abnormality rate of many commercially available products was above 2%, especially commercially available product 1, whose abnormality rate exceeded 5%, which greatly affected the accuracy of clinical testing. In contrast, the kit from Example 1 of this invention had a much lower probability of abnormality than these five kits, with an abnormality rate of only 0.07% in 4350 tests, demonstrating extremely high reliability.

[0053] Table 3 Comparison of the probability of anomalies in test results between the reagents of this invention and commercially available products.

[0054] In summary, this invention improves the uniformity of the reaction process and enhances the detection effect by adding metal ions to the reagent to regulate the linearity of the enzyme-catalyzed reaction rate. Furthermore, by using stabilizers to suppress the fluctuation of the reaction curve, the system is optimized from the aspects of "reaction rate uniformity" and "enzyme activity stability," completely solving the defects of zero / negative values ​​and poor precision in existing detection kits, meeting the requirements of clinical testing for accuracy and repeatability, and improving detection performance.

[0055] All data, reagents, and procedures described herein should be understood as illustrative rather than restrictive. Although the invention has been described in conjunction with the specific embodiments described above, many modifications and other variations will be apparent to those skilled in the art. All such modifications and other variations also fall within the scope of the invention.

Claims

1. A reagent for determining angiotensin-converting enzyme, comprising a substrate capable of being catalyzed by angiotensin-converting enzyme in a test sample to generate a detectable signal, the intensity of said detectable signal being correlated with the angiotensin-converting enzyme activity in the sample, characterized in that, The kit also includes an enzyme activity regulator, which comprises Zn. 2+ Co 2+ and Mn 2+ The enzyme activity regulator contains at least one of three metal ions, and the concentration of the enzyme activity regulator exceeds 10 times the physiological concentration of the metal ion in serum.

2. The reagent according to claim 1, characterized in that, The concentration of the enzyme activity regulator is 1~50 mmol / L; preferably, the concentration of the enzyme activity regulator is 5~30 mmol / L.

3. The reagent according to claim 1 or 2, characterized in that, 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; preferably, the metal salt is one of divalent zinc, cobalt, or manganese hydrochloride, sulfate, nitrate, or acetate.

4. The reagent according to claim 1 or 2, characterized in that, The substrate is a chromogenic substrate or a fluorescent substrate; optionally, the substrate is hippuryl glycyl glycine (HGG), N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG) or sodium trinitrobenzenesulfonate (TNBS).

5. The reagent according to claim 4, characterized in that, The substrate concentration is 0.1~1.0 g / L.

6. The reagent according to any one of claims 1 to 5, characterized in that, The reagent also includes a stabilizer to give the reagent an osmotic pressure 1 to 6 times that of physiological saline.

7. The reagent according to claim 6, characterized in that, The stabilizer is an inorganic salt, sugar, or polymer; optionally, the inorganic salt is one of NaCl, KCl, CaCl2, MgCl2, Na2SO4, K2SO4, NaNO3, and KNO3; optionally, the sugar is sucrose, trehalose, or mannitol; optionally, the polymer is polyethylene glycol.

8. The reagent according to claim 1 or 2, characterized in that, The reagent further includes a buffer having a pH of 6.0-8.0; optionally, the buffer is Tris-HCl, HEPES, or MOPSO; optionally, the buffer concentration is 50-200 mmol / L; optionally, the reagent further includes a preservative.

9. A reagent kit comprising the reagents as described in any one of claims 1 to 8; optionally, the reagent kit further comprising at least one of calibrators and quality control products.

10. A method for preparing the reagent according to any one of claims 1 to 8, characterized in that... The preparation process involves dissolving each component of the reagent in purified water according to the specified ratio, mixing them evenly, and then adjusting the concentration to the target level.

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