Reaction solution for detecting trypsin-like transpeptidase activity and application thereof

Abstract

The present application belongs to the field of biomedical detection technology, and particularly relates to a reaction solution for detecting trypsin-like transpeptidase activity and application thereof. The present application uses BAPNA as a specific substrate, which can produce a detectable signal change in the transpeptidation reaction, and a high-concentration amino group-glycine amide is added as a receptor molecule to enhance the transpeptidation reaction efficiency. Therefore, the present application solves the technical problem that the enzyme activity cannot be detected by the immunization method, has high reaction efficiency, stable and reliable detection process, shortened operation time (within 15 minutes), simple operation, strong practicability, can be used for evaluating the severity of acute allergic reaction, providing scientific guidance for the diagnosis of mast cell-related airway inflammation and mast cell activation syndrome, monitoring the effect of allergic treatment, and has clinical value in airway inflammation typing, differential diagnosis and efficacy monitoring through induced sputum or BAL fluid trypsin activity determination.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a reaction solution for detecting trypsin transpeptidase activity and its application. Background Technology

[0002] Asthma is not a single phenotype, but a syndrome composed of multiple endotypes, including Th2 hyper-, Th2 hypo-, obesity-related, and neutrophilic endotypes. Airway inflammation can manifest as eosinophilic, neutrophilic, oligocytic, or mast-dominant endotypes, and the intensity of inflammation is highly dynamic with time, triggers, and location. Mast cells (MCs) trigger degranulation via IgE / FcεRI, transiently releasing tryptases. This directly damages the epithelial barrier and, through PAR-2 ​​activation of the IL-33 / TSLP axis, rapidly amplifies both type 2 and type 1 inflammatory networks. Because mast cells are distributed in a "patchwork" pattern on the airway walls rather than uniformly entering the bloodstream, serum tryptase often fails to reflect the local inflammatory burden; however, bronchoalveolar lavage fluid (BALF) and induced sputum (IS), sampled directly from the airway, can more accurately capture mast cell activation events. Therefore, conventional indicators such as blood eosinophil count, FeNO, serum total IgE, or total trypsin protein can only reflect the systemic or mast cell load and cannot capture the "transient activation" state of local mast cells in the airway in real time and accurately. However, trypsin activity can reflect its actual functional state.

[0003] Currently, clinical detection of trypsinoids mainly relies on immunological methods, such as ELISA / CLIA to measure total protein levels. However, these methods cannot distinguish between inactive precursor enzymes or inactivated enzymes neutralized by endogenous serine protease inhibitors (serpins), leading to false positives of "high concentration - low activity." In BALF / IS, mucins and surfactants create steric hindrance to capture antibodies, further amplifying the bias. Another method is the traditional hydrolysis activity assay, which uses BAPNA as a substrate and relies on trypsinoid enzymes to catalyze the hydrolysis of amide bonds to generate p-nitroaniline. Changes in absorbance reflect enzyme activity. During the reaction, water molecules act as the final nucleophile in the hydrolysis of the acyl-enzyme intermediate. However, these reactions have low catalytic efficiency, resulting in high background and weak signal in bronchoalveolar lavage fluid (BALF) or induced sputum (IS) rich in protease inhibitors and high viscosity, failing to meet the detection requirements for asthma or localized punctate activation foci.

[0004] Currently, there is an urgent need for a simple and efficient method to detect trypsin transpeptidase activity. Accurate detection of trypsin transpeptidase activity could confirm the presence of mast cell-associated airway inflammation, providing a basis for the auxiliary diagnosis of asthma. Summary of the Invention

[0005] The purpose of this invention is to provide a reaction solution for detecting trypsin transpeptidase activity and its application, which has the advantages of simple operation and high sensitivity.

[0006] The present invention provides a reaction solution for detecting trypsin transpeptidase activity, the reaction solution comprising: BAPNA, glycine amide and buffer.

[0007] Preferably, the concentration of BAPNA in the reaction solution is 0.5-5 g / L, and the concentration of glycine is 50-200 mM.

[0008] Preferably, the reaction solution further includes: a preservative;

[0009] The concentration of the preservative in the reaction solution is 0.5~2g / L.

[0010] Preferably, the buffer solution includes: Tris buffer, TAPS buffer, AMPSO buffer, CHES buffer, CAPSO buffer, AMP buffer, or borate-borax buffer;

[0011] The concentration of the buffer solution is 50~200 mM;

[0012] The preservatives include: sodium azide, PC300, KroVin series preservatives, IZU preservatives, FAD or sodium deoxyacetate.

[0013] This invention provides the application of the reaction solution described above in detecting trypsin transpeptidase activity and / or preparing products for detecting trypsin transpeptidase activity.

[0014] This invention provides a method for detecting trypsin transpeptidase activity, the method comprising the following steps:

[0015] The sample to be tested is mixed evenly with the reaction solution described in the above technical solution to obtain a mixture;

[0016] After the mixture is allowed to stand and react, a standing liquid is obtained.

[0017] The absorbance of the mixed solution and the standing solution was measured at a wavelength of 405 nm.

[0018] The degree of reactivity is determined based on the difference in absorbance between the static solution and the mixed solution.

[0019] Substitute the reactivity into the standard curve to determine the trypsin transpeptidase activity of the sample to be tested.

[0020] Preferably, the volume ratio of the sample to the reaction solution is 1:3-4.

[0021] Preferably, the conditions for the static reaction include: a time of 2-5 minutes and a temperature of 36-37°C.

[0022] Preferably, the standard curve is plotted using trypsin-like proteins as samples.

[0023] This invention provides a kit for detecting trypsin transpeptidase activity, the kit comprising: the reaction solution described in the above technical solution.

[0024] Beneficial effects:

[0025] This invention provides a reaction solution for detecting trypsin transpeptidase activity, comprising: BAPNA, glycine, and a buffer. This invention uses BAPNA as a specific substrate, which generates a detectable signal change during the transpeptidation reaction. A high concentration of glycine, an amino group, is then added as an acceptor molecule to enhance the transpeptidation efficiency. Therefore, the reaction solution prepared in this way can be used to detect trypsin transpeptidase activity.

[0026] Based on the above-mentioned technical advantages, the present invention also provides a method for detecting trypsin transpeptidase activity. The method involves reacting the reaction solution provided by the above-mentioned technical solution with the test solution, and quantifying the enzyme activity by monitoring the change in absorbance of the reaction product (p-nitroaniline) at 405 nm. This method solves the technical problem that the immunoassay method cannot detect enzyme activity. It has high reaction efficiency, stable and reliable detection process, and operation time is shortened to less than 15 minutes. It is simple to operate, highly practical, and can be used to assess the severity of acute allergic reactions, provide scientific guidance for the diagnosis of mast cell activation syndrome, and monitor the effect of allergy treatment.

[0027] The kit provided by this invention has the advantages of high sensitivity and high accuracy. The detection target is bronchoalveolar lavage fluid / induced sputum. The activity detection directly reflects the functional status of enzymes. The assay of trypsin activity in induced sputum or BAL fluid has clear clinical value in airway inflammation classification, differential diagnosis and efficacy monitoring. Specifically, it can be used as an auxiliary diagnosis of asthma and COPD. Trypsin activity can serve as common evidence of airway mast cell activation. Detailed Implementation

[0028] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1: Verification Experiment with Different Concentrations of Glycineamide

[0030] Group 1: Substrate reaction solution: using 50mM Tris-HCl buffer (pH 8.5) as solvent, consisting of 100mM glycine amide (Gly-Gly), 3.6g / L Nα-benzoyl-DL-arginine p-nitroaniline (BAPNA) and 0.5g / L sodium azide;

[0031] Group 2: Substrate reaction solution: using 50mM Tris-HCl buffer (pH 8.5) as solvent, consisting of 50mM glycine amide (Gly-Gly), 3.6g / L Nα-benzoyl-DL-arginine p-nitroaniline (BAPNA) and 0.5g / L sodium azide;

[0032] Group 3: Substrate reaction solution: using 50mM Tris-HCl buffer (pH 8.5) as solvent, consisting of 150mM glycine amide (Gly-Gly), 3.6g / L Nα-benzoyl-DL-arginine p-nitroaniline (BAPNA) and 0.5g / L sodium azide;

[0033] Group 4: Substrate reaction solution: using 50mM Tris-HCl buffer (pH 8.5) as solvent, consisting of 200mM glycine amide (Gly-Gly), 3.6g / L Nα-benzoyl-DL-arginine p-nitroaniline (BAPNA) and 0.5g / L sodium azide.

[0034] Group 5: Substrate reaction solution: using 50mM Tris-HCl buffer (pH 8.5) as solvent, consisting of 0mM glycine amide (Gly-Gly), 3.6g / L Nα-benzoyl-DL-arginine p-nitroaniline (BAPNA) and 0.5g / L sodium azide.

[0035] Test samples: Using recombinant trypsin as a positive control, the test samples were serially diluted 1-10 times - that is, 10 test samples were numbered sequentially as sample 1 (i.e., the sample diluted 1-fold), sample 2 (i.e., the sample diluted 2-fold) ... sample 10 (i.e., the sample diluted 10-fold); water was used as a blank control (ΔA≤0.003).

[0036] The testing process is as follows:

[0037] 500 μL of the sample to be tested was mixed with 2000 μL of different groups of substrate reaction solution to obtain a mixture. The absorbance of the mixture at a wavelength of 405 nm was immediately measured and recorded as A1.

[0038] After incubating the mixture at 37°C for 5 minutes, the absorbance of the mixture at a wavelength of 405 nm was measured again and recorded as A2.

[0039] Calculate the responsiveness ΔA = A2 - A1; record the ΔA (i.e. ΔOD405) value for each group, and determine the sensitivity of each treatment based on the magnitude of ΔA. The results are shown in Table 1.

[0040] Table 1. Reactivity (ΔOD405) of different treatments

[0041]

[0042] Based on the data in Table 1, when the difference between ΔA and the measured value of the blank control is ±0.001, it is considered that the sensitivity endpoint has been reached. The sensitivity endpoints of each group are statistically analyzed, and the results are shown in Table 2.

[0043] Table 2 Sensitivity endpoints for different treatments

[0044]

[0045] Based on the data in Table 1, the average value of the sample under test when diluted 1 time was calculated to determine the signal enhancement factor. The results are shown in Table 3.

[0046] Table 3 Signal enhancement factor for different processing methods

[0047]

[0048] Combining the detection results in Tables 1, 2, and 3, it can be seen that the sensitivity of the fifth group of positive samples was only 5-fold diluted; the signal intensity was 1 / 10 of that of the first group; there was a risk of weak positive false negatives; and the signal intensity was low (ΔA≈0.068), which could not meet the detection requirements for asthma or localized punctate activation foci. In contrast, the addition of 100 mM glycine (the first group) showed excellent performance in both sensitivity and signal intensity. Furthermore, in terms of cost, the molar amount of 100 mM glycine added was 33% less than that of 150 mM, significantly reducing reagent costs; its cost was similar to that of 50 mM and 200 mM, but its performance was superior. Therefore, it achieved an optimal balance in terms of economy and stability, providing an irreplaceable formulation basis for high-sensitivity detection of transpeptidase.

[0049] Example 2: Buffer and Preservative Validation Experiment

[0050] Based on the experimental results in Example 1, the substrate reaction solution was determined to consist of 100 mM glycine (Gly-Gly), 3.6 g / L Nα-benzoyl-DL-arginine p-nitroaniline (BAPNA), 50 mM buffer solution, and 0.5 g / L preservative. The types of preservatives and buffers in each substrate reaction solution were determined according to Table 4. The pH values ​​of buffer solutions Tris-HCl, TAPS, AMPSO, or boric acid-borax were all 8.5; the pH values ​​of buffer solutions CAPSO, CHES, or AMP were all 9.5.

[0051] 500 μL of recombinant trypsin solution was mixed with 2000 μL of substrate reaction solution of different groups to obtain a mixture. The absorbance of the mixture at a wavelength of 405 nm was immediately measured and recorded as A1.

[0052] After incubating the mixture at 37°C for 5 minutes, the absorbance of the mixture at a wavelength of 405 nm was measured again and recorded as A2.

[0053] Calculate the reactivity ΔA = A2 - A1; record the ΔA (i.e. ΔOD405) values ​​in different substrate reaction solutions, and the results are shown in Table 4.

[0054] Table 4 Reactivity of different treatments

[0055]

[0056] Based on the data in Table 4, it can be seen that rapid detection of samples can be achieved using Tris buffer, TAPS buffer, AMPSO buffer, CHES buffer, CAPSO buffer, AMP buffer, or borate-borax buffer as buffers and sodium azide, PC300, KroVin series preservatives, IZU preservatives, FAD, or sodium deoxyacetate as preservatives. However, the reagent composed of Tris-HCl buffer (pH 8.5), glycine (Gly-Gly), BAPNA, and sodium azide is the most effective.

[0057] Example 3 Clinical Sample Testing

[0058] 1. Preparation of substrate reaction solution:

[0059] The solution was prepared using 50 mM Tris-HCl buffer (pH 8.5) and consisted of 100 mM glycine (Gly-Gly), 3.6 g / L Nα-benzoyl-DL-arginine p-nitroaniline (BAPNA), and 0.5 g / L sodium azide (denoted as BAPNA+100 mM glycine).

[0060] The solution was prepared using 50 mM Tris-HCl buffer (pH 8.5) and consisted of 3.6 g / L Nα-benzoyl-DL-arginine p-nitroaniline (BAPNA) and 0.5 g / L sodium azide (denoted as BAPNA).

[0061] 2. Sample type

[0062] Forty bronchoalveolar lavage fluid (BALF) samples were collected (including 20 healthy controls and 20 cases diagnosed with asthma, which were respectively designated as BALF-01, BALF-02...BALF-40).

[0063] Forty induced sputum samples (IS samples) were collected (including 20 healthy controls and 20 confirmed asthma cases, which were respectively designated as IS-01, IS-02...IS-40).

[0064] 3. The testing process is as follows:

[0065] S1. After thawing the sample to be tested, centrifuge at 1000×g for 10 minutes and take the supernatant for later use.

[0066] S2. Take 500 μL of sample supernatant and add it to 2000 μL of substrate reaction solution, then mix well.

[0067] S3. Immediately measure the absorbance at a wavelength of 405nm and record it as A1;

[0068] S4, after incubation at 37°C for 5 minutes, the absorbance at 405 nm was measured again and recorded as A2;

[0069] S5. Calculate the reactivity ΔA = A2 – A1, record the ΔOD405 (i.e. ΔA) value for each group, and compare the reactivity of the two reaction solutions; substitute the ΔA of the substrate reaction solution BAPNA + 100 mM glycine into the standard curve (plotted with trypsin as the sample) to calculate the trypsin activity (U / mL).

[0070] The test results for each sample are shown in Tables 5 and 6.

[0071] Table 5. Detection results of bronchoalveolar lavage fluid samples

[0072]

[0073] Table 6 Detection results of induced sputum samples

[0074]

[0075] Combining the data in Tables 5 and 6, it can be seen that in the BALF sample (20 cases of confirmed asthma and 20 cases of healthy controls) and the IS sample (20 cases of confirmed asthma and 20 cases of healthy controls), the ΔOD405 value was very large when testing positive samples and very small when testing negative samples. Compared with the BAPNA single substrate method, the signal was amplified and the difference was widened. The results are consistent with the rules for judging positive and negative samples and are superior to the BAPNA single substrate method.

[0076] In summary, the substrate reaction solution prepared in this invention, namely the reagent composed of 50mM Tris-HCl buffer (pH 8.5), 100mM glycine (Gly-Gly), 3.6g / L BAPNA and 0.5g / L sodium azide, can be used for the detection of trypsin-like proteins in bronchoalveolar lavage fluid and induced sputum samples.

[0077] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A reaction solution for detecting trypsin transpeptidase activity, characterized in that, The reaction solution consists of BAPNA, glycine, buffer solution, and preservative. In the reaction solution, the concentration of BAPNA is 3.6 g / L, the concentration of glycine is 100 mM, and the concentration of preservative is 0.5 g / L.

2. The reaction solution according to claim 1, characterized in that, The buffer solution is: Tris buffer, Taps buffer, AMPSO buffer, CHES buffer, CAPSO buffer, AMP buffer, or boric acid. Borax buffer solution; The concentration of the buffer solution is 50~200mM; The preservatives are: sodium azide, PC300, KroVin series preservatives, IZU preservatives, FAD or sodium deoxyacetate.

3. The use of the reaction solution according to claim 1 or 2 in the preparation of products for detecting trypsin transpeptidase activity.

4. A kit for detecting trypsin transpeptidase activity, characterized in that, The kit includes the reaction solution as described in claim 1 or 2.

Images