Detection method of adenosine deaminase content and application thereof

Abstract

The application provides an adenosine deaminase content detection method and application thereof. The adenosine deaminase content detection method comprises the following steps: using the intrinsic fluorescence of adenosine, using adenosine deaminase to hydrolyze adenosine, and detecting the content of adenosine deaminase by a fluorescence analysis method. Adenosine itself has strong fluorescence, and after being hydrolyzed by adenosine deaminase, the fluorescence of the obtained hydrolysis product is reduced, thereby causing the fluorescence intensity of the solution of the detection system to be reduced; the content of adenosine deaminase in the system is detected by the change of the fluorescence intensity before and after adenosine hydrolysis. The adenosine deaminase content detection method in the application is simple to operate, does not need large instruments and equipment, and is cheap and easy to obtain materials, can accurately and rapidly detect the content of adenosine deaminase in a sample to be detected, has good sensitivity and specificity, and has a relatively wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biochemical detection technology, specifically relating to a method for detecting adenosine deaminase content and its application. Background Technology

[0002] Adenosine deaminase (ADA) is a thiol enzyme belonging to the class of nucleic acid metabolic enzymes related to cellular immune function. ADA deficiency can lead to nucleic acid metabolism disorders, affecting thymus development and thus causing immune dysfunction. Severe combined immunodeficiency is associated with ADA deficiency.

[0003] ADA is distributed in many tissues of the human body, with the highest concentrations in the thymus, spleen, and other lymphatic tissues, and lower concentrations in the lungs, liver, kidneys, and skeletal muscles. Serum ADA primarily originates from the liver and is a sensitive indicator of liver damage, making it a routine component of liver function tests. Measuring serum ADA activity can be used to diagnose acute liver injury and residual disease, aid in the assessment of chronic liver disease, diagnose liver fibrosis, and differentiate between jaundice and amebic liver abscess. Detection of ADA in cerebrospinal fluid can serve as an important indicator for the differential diagnosis and treatment of central nervous system diseases.

[0004] Furthermore, ADA is also of significant value in the differential diagnosis of benign and malignant exudates. Therefore, measuring ADA levels in body fluids and blood is of great importance for the study of immune function and the identification, diagnosis, and treatment of related diseases. The development of simple, rapid, and sensitive ADA detection methods is receiving increasing attention.

[0005] As research into ADA has deepened, its detection methods have also continuously evolved. Currently, four generations of ADA detection methods have been developed on the market.

[0006] The first-generation ADA assay method resulted in excessively high absorbance due to high substrate concentrations, making it suitable only for adenosine concentrations below 40 μM. Such low substrate concentrations failed to achieve substrate saturation, leading to inaccurate ADA activity detection. Therefore, this method cannot meet clinical application requirements.

[0007] The second-generation ADA assay requires simple instruments and readily available reagents, but it suffers from low sensitivity, is susceptible to exogenous NH3, has an excessively high blank concentration, and cannot directly measure ADA activity in erythrocytes. Similarly, the ADA-coupled glutamate dehydrogenase (GLDH) reaction method, which calculates ADA activity by measuring the rate of decrease in NADPH absorbance at 340 nm, is also susceptible to interference due to the presence of ammonia in serum and non-specific oxidation caused by excessively high NADPH in the testing system.

[0008] The third-generation ADA testing method suffers from excessively high serum absorbance at 293 nm, hindering clinical application. While the fourth-generation ADA testing method overcomes these difficulties, its high reagent cost impedes practical clinical use. Given these shortcomings of existing technologies, a novel ADA detection method is urgently needed. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting adenosine deaminase content and its application. The method for detecting adenosine deaminase content in this invention is simple to operate, requires no large-scale instruments or equipment, uses inexpensive and readily available materials, and can accurately and rapidly detect the adenosine deaminase content in the sample, exhibiting good sensitivity and specificity, thus possessing significant application and promotion value.

[0010] To achieve this objective, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a method for detecting adenosine deaminase content, the method comprising the following steps:

[0012] Utilizing the intrinsic fluorescence of adenosine, adenosine was hydrolyzed using adenosine deaminase, and the content of adenosine deaminase was detected by fluorescence analysis.

[0013] This invention selects a suitable adenosine and simultaneously uses adenosine deaminase to hydrolyze adenosine, then detects the adenosine deaminase content using fluorescence analysis. The principle behind detecting the adenosine deaminase content is that adenosine has strong intrinsic fluorescence. When adenosine is hydrolyzed by adenosine deaminase to produce ammonia and inosine, the hydrolysis products release fluorescence of low intensity. Within a certain concentration range, the higher the concentration of adenosine deaminase, the more adenosine is consumed, and the weaker the fluorescence released by the detection system. The content of adenosine deaminase in the system is detected by observing changes in fluorescence intensity.

[0014] Preferably, the adenosine includes any one or a combination of at least two of adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate, and is preferably adenosine triphosphate.

[0015] Under the same conditions, adenosine triphosphate (ATP) exhibits more pronounced fluorescence than adenosine monophosphate (AMP) and adenosine diphosphate (ADP), making it more suitable for detection.

[0016] Preferably, the method for detecting adenosine deaminase content includes the following specific steps:

[0017] (1) Prepare a hydrolysis reaction mixture by mixing adenosine, adenosine deaminase standards and hydrolysis reaction solvent, carry out the hydrolysis reaction, and detect the fluorescence intensity after the reaction; simultaneously detect the fluorescence intensity of the test sample and blank control after the reaction.

[0018] (2) Based on the difference in fluorescence intensity between the adenosine deaminase standard and the blank control after the reaction, a standard curve is prepared, and the adenosine deaminase content in the sample to be tested is calculated based on the standard curve.

[0019] Preferably, in step (1), the hydrolysis reaction solvent includes HEPES buffer or water, preferably HEPES buffer.

[0020] Preferably, the concentration of the HEPES buffer is 8-12 mM, for example, it can be 8 mM, 9 mM, 10 mM, 11 mM or 12 mM, etc., and the pH value of the HEPES buffer is 7.2-7.6, for example, it can be 7.2, 7.4 or 7.6, etc.

[0021] Preferably, in step (1), the concentration of adenosine in the hydrolysis reaction mixture is 45-55 mM, for example, it can be 45 mM, 48 mM, 50 mM, 52 mM or 55 mM.

[0022] Preferably, in step (1), the concentration of the adenosine deaminase standard in the hydrolysis reaction mixture is 0-25 U / mL, for example, it can be 0 U / mL, 0.25 U / mL, 5 U / mL, 12.5 U / mL or 25 U / mL, etc.

[0023] Preferably, in step (1), the temperature of the hydrolysis reaction is 30-37℃, for example, it can be 30℃, 32℃, 35℃ or 37℃, etc., and the time of the hydrolysis reaction is 0.5-2h, for example, it can be 0.5h, 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h or 2h, etc.

[0024] Preferably, in step (1), the fluorescence detection parameters are: excitation wavelength of 270-280nm, for example, 270nm, 275nm or 280nm, and emission wavelength of 360-400nm, for example, 360nm, 380nm, 390nm or 400nm.

[0025] Preferably, in step (2), the vertical axis of the standard curve is the difference in fluorescence intensity between the adenosine deaminase standard and the blank control after the reaction, and the horizontal axis of the standard curve is the concentration of the adenosine deaminase standard solution.

[0026] As a preferred embodiment of the present invention, the method for detecting adenosine deaminase content includes the following steps:

[0027] (1) Prepare a hydrolysis reaction mixture by mixing adenosine, adenosine deaminase standard and hydrolysis reaction solvent. The concentration of adenosine in the hydrolysis reaction mixture is 45-55 mM, and the concentration of adenosine deaminase standard is 0-25 U / mL. Hydrolyze the mixture at 30-37℃ for 0.5-2 h. Detect the fluorescence intensity after the reaction. The fluorescence detection parameters are: excitation wavelength 270-280 nm and emission wavelength 360-400 nm. Simultaneously detect the fluorescence intensity of the test sample and the blank control after the reaction.

[0028] (2) Based on the difference in fluorescence intensity between the adenosine deaminase standard and the blank control after the reaction, a standard curve is prepared. The vertical axis of the standard curve is the difference in fluorescence intensity between the adenosine deaminase standard and the blank control after the reaction, and the horizontal axis of the standard curve is the concentration of the adenosine deaminase standard solution. The adenosine deaminase content in the sample to be tested is calculated based on the standard curve.

[0029] Secondly, the present invention provides a kit for detecting adenosine deaminase content, wherein the reagents in the kit include adenosine and adenosine deaminase standards.

[0030] Preferably, the adenosine includes any one or a combination of at least two of adenosine monophosphate, adenosine diphosphate, or adenosine triphosphate, and is preferably adenosine triphosphate.

[0031] Thirdly, the present invention provides a method for using the kit for detecting adenosine deaminase content as described in the second aspect, the method comprising the following steps:

[0032] (1) Fluorescence intensity detection: Adenosine, adenosine deaminase standards and hydrolysis reaction solvent were prepared into a hydrolysis reaction mixture, and the fluorescence intensity after the reaction was detected; the fluorescence intensity of the test sample and blank control after the reaction was detected simultaneously.

[0033] (2) Prepare a standard curve: Prepare a standard curve based on the difference in fluorescence intensity between the adenosine deaminase standard and the blank control after the reaction, and calculate the adenosine deaminase content in the sample to be tested based on the standard curve.

[0034] Fourthly, the present invention provides the application of the method for detecting adenosine deaminase content described in the first aspect, the kit for detecting adenosine deaminase content described in the second aspect, or the method of using the kit for detecting adenosine deaminase content described in the third aspect in the field of adenosine deaminase detection.

[0035] In this invention, the intrinsic fluorescence intensity of different nucleosides was investigated, including adenosine (AMP, ADP, and ATP), thymidine (TMP, TDP, and TTP), cytidine (CMP, CDP, and CTP), guanosine (GMP, GDP, and GTP), and uridine (UMP, UDP, and UTP). The fluorescence intensity detection results of different nucleosides (500 mM, 5 μL) at the same concentration in a 500 μL reaction system are shown in the figure below. Figure 1 As shown, from Figure 1 As can be seen from the data, adenosine (AMP, ADP, and ATP) exhibit the most pronounced intrinsic fluorescence intensity compared to other nucleosides. At an excitation wavelength of 280 nm, the fluorescence intensity of AMP reaches 1788, ADP reaches 3818, and ATP reaches 5986. Under the same conditions, adenosine triphosphate (ATP) shows a more pronounced fluorescence intensity than adenosine monophosphate (AMP) and adenosine diphosphate (ADP), making it more suitable for detection.

[0036] In this invention, the fluorescence intensity of different concentrations of ATP was also investigated. The fluorescence intensity detection results of different volumes of ATP (250 mM) in a 500 μL reaction system are shown in the figure below. Figure 2 As shown, from Figure 2 As the volume of ATP (250mM) gradually increases, the fluorescence of the reaction system first increases, then decreases after reaching a peak. The results indicate that the fluorescence released by the system is strongest when the volume of 250mM ATP is 10μL.

[0037] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] Most existing ADA detection methods have complex detection systems, high costs, and cumbersome operation procedures. The adenosine deaminase content detection method described in this invention overcomes these shortcomings, providing a simple, highly sensitive, selective, low-cost, and efficient ADA detection method. The detection method involves simple principles, uses inexpensive and readily available materials, and is quick to operate, exhibiting good sensitivity and specificity, thus possessing significant application and promotion value. Attached Figure Description

[0040] Figure 1 This is a graph showing the fluorescence intensity detection results of different nucleosides (500mM, 5μL) at the same concentration.

[0041] Figure 2 This is a graph showing the fluorescence intensity detection results of different volumes of ATP (250mM).

[0042] Figure 3 This is a graph showing the fluorescence intensity detection results after adding different volumes of ADA (250 U / mL) to a detection system consisting of ATP (250 mM, 10 μL).

[0043] Figure 4 The linear relationship between the fluorescence intensity change of the detection system composed of ATP (250 mM, 10 μL) and the volume of ADA (250 U / mL) is shown.

[0044] Figure 5 This is a graph showing the fluorescence intensity detection results after adding different types of enzymes to a detection system composed of ATP.

[0045] Figure 6 It is a detection system composed of ATP that reacts with different enzymes to change the fluorescence intensity. Detailed Implementation

[0046] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0047] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0048] The sources of the components used in the following examples and test cases are shown below:

[0049] Components factory CAS number Adenosine triphosphate Shanghai Yuanye Biotechnology Co., Ltd. 9000-83-3 adenosine monophosphate Shanghai Yuanye Biotechnology Co., Ltd. 61-19-8 Adenosine diphosphate Shanghai Yuanye Biotechnology Co., Ltd. 58-64-0 Adenosine deaminase standard Shanghai Yuanye Biotechnology Co., Ltd. 9026-93-1

[0050] Example 1

[0051] This embodiment provides a kit for detecting adenosine deaminase content, the kit consisting of adenosine triphosphate (ATP) and adenosine deaminase standards. The kit is used to detect adenosine deaminase content. The detection steps for adenosine deaminase content are as follows:

[0052] (1) Prepare a hydrolysis reaction mixture by mixing adenosine triphosphate (ATP), adenosine deaminase standard, and HEPES buffer (10 mM, pH = 7.4). The volume of the hydrolysis reaction mixture is 500 μL. The concentration of ATP in the hydrolysis reaction mixture is 50 mM. The concentrations of adenosine deaminase standard are 0 U / mL, 0.25 U / mL, 5 U / mL, 12.5 U / mL, and 25 U / mL, respectively. The hydrolysis reaction is carried out at 37 °C for 1 h. The fluorescence intensity after the reaction is detected. The fluorescence detection parameters are: excitation wavelength of 280 nm and emission wavelength of 380 nm. The fluorescence intensity of the test sample and the blank control after the reaction is detected simultaneously. The test sample contains 25 U / mL adenosine deaminase, and the blank control is water. The test sample detection experiment is performed in triplicate.

[0053] (2) Based on the difference in fluorescence intensity between the adenosine deaminase standard and the blank control after the reaction, a standard curve is prepared. The vertical axis of the standard curve is the difference in fluorescence intensity between the adenosine deaminase standard and the blank control after the reaction, and the horizontal axis of the standard curve is the concentration of the adenosine deaminase standard solution. The adenosine deaminase content in the sample to be tested is calculated based on the standard curve.

[0054] Test results as follows Figure 3 and Figure 4 As shown, where Figure 3 The graph shows the fluorescence intensity detection results after adding different volumes of ADA (250 U / mL) to the detection system composed of ATP (250 mM, 10 μL). Figure 3 It can be seen that the fluorescence regularity of the detection system decreases with the increase of ADA (250 U / mL) volume. Plotting the change in fluorescence intensity of the detection system against the volume of 250 U / mL ADA yields a straight line with a goodness of fit of 0.96, and the linear equation is y = 8.08 + 25.77x, R0. 2 The value is 0.96, as shown in the following figure. Figure 4 As shown, Figure 4 The linear relationship between the fluorescence intensity change of the detection system composed of ATP (250 mM, 10 μL) and the volume of ADA (250 U / mL) was established. Based on 3δ / s (δ is the standard deviation of 15 blank determinations, and s is the slope of the standard curve), the detection limit of the detection system for ADA was calculated to be 0.51 U / mL.

[0055] Example 2

[0056] This embodiment provides a kit for detecting adenosine deaminase content. The only difference between this kit and that of Example 1 is that the adenosine used is adenosine diphosphate (ATP). The detection procedure for adenosine deaminase content is the same as in Example 1.

[0057] Example 3

[0058] This embodiment provides a kit for detecting adenosine deaminase content. The only difference between this kit and that of Example 1 is that the adenosine used is adenosine monophosphate (ATP). The detection procedure for adenosine deaminase content is the same as in Example 1.

[0059] Example 4

[0060] This embodiment provides a kit for detecting adenosine deaminase content. The kit differs from that in Example 1 only in that the hydrolysis reaction solvent is water. The detection procedure for adenosine deaminase content is the same as in Example 1.

[0061] Example 5

[0062] This embodiment provides a kit for detecting adenosine deaminase content. The only difference from Example 1 is that, in the adenosine deaminase content detection step, the concentration of adenosine triphosphate in the hydrolysis reaction mixture is 30 mM. The detection step for adenosine deaminase content is the same as in Example 1.

[0063] Example 6

[0064] This embodiment provides a kit for detecting adenosine deaminase content. The only difference from Example 1 is that, in the adenosine deaminase content detection step, the concentration of adenosine triphosphate in the hydrolysis reaction mixture is 65 mM. The detection step for adenosine deaminase content is the same as in Example 1.

[0065] Example 7

[0066] This embodiment provides a kit for detecting adenosine deaminase content. The only difference from Example 1 is that the hydrolysis reaction time in the adenosine deaminase content detection step is 0.5 hours. The detection step for adenosine deaminase content is the same as in Example 1.

[0067] Example 8

[0068] This embodiment provides a kit for detecting adenosine deaminase content. The only difference from Example 1 is that the hydrolysis reaction time in the adenosine deaminase content detection step is 2 hours. The detection step for adenosine deaminase content is the same as in Example 1.

[0069] Example 9

[0070] This embodiment provides a kit for detecting adenosine deaminase content. The only difference from Example 1 is that the hydrolysis reaction time in the adenosine deaminase content detection step is 0.3 hours. The detection step for adenosine deaminase content is the same as in Example 1.

[0071] Example 10

[0072] This embodiment provides a kit for detecting adenosine deaminase content. The only difference from Example 1 is that the hydrolysis reaction time in the adenosine deaminase content detection step is 3 hours. The detection step for adenosine deaminase content is the same as in Example 1.

[0073] Example 11

[0074] This embodiment provides a kit for detecting adenosine deaminase content. The only difference from Example 1 is that, in the detection step of adenosine deaminase content, the fluorescence detection parameters are: excitation wavelength of 260 nm and emission wavelength of 380 nm. The detection step of adenosine deaminase content is the same as in Example 1.

[0075] Example 12

[0076] This embodiment provides a kit for detecting adenosine deaminase content. The only difference from Embodiment 1 is that, in the detection step of adenosine deaminase content, the fluorescence detection parameters are: excitation wavelength of 300 nm and emission wavelength of 380 nm. The detection step of adenosine deaminase content is the same as in Embodiment 1.

[0077] The detection results of three parallel detection experiments of the test samples (containing 25 U / mL adenosine deaminase) in Examples 1-10 are shown in Table 1.

[0078] Table 1

[0079] Reagent test kit Recovery rate (%) RSD (%) Example 1 97.5-105.8 1.2-3.6 Example 2 84.9-88.6 3.1-4.6 Example 3 85.3-87.7 3.4-4.7 Example 4 84.3-86.3 3.0-4.6 Example 5 91.1-93.6 1.7-4.3 Example 6 92.5-94.9 1.9-4.4 Example 7 93.4-95.2 2.5-4.2 Example 8 91.5-92.7 2.7-4.8 Example 9 88.4-91.2 4.3-5.1 Example 10 90.2-93.4 3.5-4.2 Example 11 82.6-84.9 3.6-5.8 Example 12 75.1-79.7 3.5-5.4

[0080] As shown in Table 1, the recovery rate in Example 1 was 97.5-105.8%, and the RSD was 1.2-3.6%. The detection method in Example 1 has good detection effect and high accuracy.

[0081] A comparison of Examples 1 and 2-3 shows that, under the same conditions, adenosine triphosphate (ATP) is more suitable for detection than adenosine monophosphate (ADP) and adenosine diphosphate (ADP).

[0082] A comparison between Example 1 and Example 4 shows that the detection effect is better when the hydrolysis reaction solvent is HEPES buffer. HEPES buffer provides a more suitable reaction condition for enzyme reaction, enabling the enzyme to exhibit its activity to the maximum extent.

[0083] A comparison of Examples 1 and 5-12 shows that changing the concentration of adenosine triphosphate, shortening or lengthening the reaction time, and changing the fluorescence detection parameters all affect the accuracy of the detection results.

[0084] Test Example 1

[0085] This test case is used to examine the specificity of the kit described in Example 1.

[0086] (1) Prepare a series of reaction systems with a total volume of 500 μL: Add ATP stock solution and different enzymes (including ADA, laccase, β-amylase, lipase, pepsin, β-glucosidase and glucose oxidase) to HEPES (10 mM, pH = 7.4) buffer respectively for reaction; the concentration of ATP in the reaction system is 50 mM; the concentration of the enzyme in the reaction system is 1 mg / mL. The reaction conditions are 37℃ for 1 h.

[0087] (2) Set the fluorescence measurement parameters as Ex (excitation wavelength) = 280 nm and Em (emission wavelength) = 380 nm. Use the blank group as the control (the blank group contains only ATP stock solution and HEPES buffer) to compare the changes in fluorescence intensity of the systems with different enzymes.

[0088] Test results as follows Figure 5 and Figure 6 As shown, Figure 5 The graph shows the fluorescence intensity detection results after adding different types of enzymes to the detection system composed of ATP. Figure 6 The fluorescence intensity changes of the ATP-based detection system after interaction with different enzymes. Figure 5 and Figure 6 It was found that the fluorescence of the detection system only decreased significantly upon the addition of adenosine deaminase. The results indicate that the method for detecting adenosine deaminase described above has good specificity, and laccase, β-amylase, lipase, pepsin, β-glucosidase, and glucose oxidase do not interfere with the detection.

[0089] In summary, the method for detecting adenosine deaminase content provided by this invention is simple to operate, has good specificity, does not require large-scale instruments and equipment, and uses inexpensive and readily available materials. It can accurately and quickly detect the content of adenosine deaminase in the sample to be tested, and exhibits good sensitivity and specificity, thus having broad application prospects.

[0090] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for detecting adenosine deaminase content, characterized in that, The method for detecting adenosine deaminase content includes the following steps: Utilizing the intrinsic fluorescence of adenosine, adenosine was hydrolyzed using adenosine deaminase, and the content of adenosine deaminase was detected by fluorescence analysis. The method for detecting adenosine deaminase content includes the following specific steps: (1) Prepare a hydrolysis reaction mixture by mixing adenosine, adenosine deaminase standards and hydrolysis reaction solvent, carry out the hydrolysis reaction, and detect the fluorescence intensity after the reaction; simultaneously detect the fluorescence intensity of the test sample and blank control after the reaction. (2) Based on the difference in fluorescence intensity between the adenosine deaminase standard and the blank control after the reaction, a standard curve is prepared, and the adenosine deaminase content in the sample to be tested is calculated based on the standard curve. The concentration of adenosine in the hydrolysis reaction mixture in step (1) is 5 mM; The adenosine mentioned in step (1) is adenosine triphosphate; The fluorescence detection parameters in step (1) are: excitation wavelength of 270-280 nm and emission wavelength of 360-400 nm.

2. The method for detecting adenosine deaminase content according to claim 1, characterized in that, In step (1), the hydrolysis reaction solvent includes HEPES buffer or water.

3. The method for detecting adenosine deaminase content according to claim 2, characterized in that, The solvent for the hydrolysis reaction is HEPES buffer solution.

4. The method for detecting adenosine deaminase content according to claim 3, characterized in that, The concentration of the HEPES buffer is 8-12 mM, and the pH value of the HEPES buffer is 7.2-7.

6.

5. The method for detecting adenosine deaminase content according to claim 1, characterized in that, In step (1), the concentration of the adenosine deaminase standard in the hydrolysis reaction mixture is 0-25 U / mL; In step (1), the temperature of the hydrolysis reaction is 30-37℃ and the time of the hydrolysis reaction is 0.5-2 h.

6. The method for detecting adenosine deaminase content according to claim 1, characterized in that, In step (2), the vertical axis of the standard curve is the difference in fluorescence intensity between the adenosine deaminase standard and the blank control after the reaction, and the horizontal axis of the standard curve is the concentration of the adenosine deaminase standard solution.

7. A kit for detecting adenosine deaminase content in the method for detecting adenosine deaminase content according to claims 1-6, characterized in that, The reagents in the kit for detecting adenosine deaminase content include adenosine and adenosine deaminase standards. The adenosine in question is adenosine triphosphate (ATP).

8. A method of using the kit for detecting adenosine deaminase content as described in claim 7, characterized in that, The method of use includes the following steps: (1) Fluorescence intensity detection: Adenosine, adenosine deaminase standards and hydrolysis reaction solvent were prepared into a hydrolysis reaction mixture, and the fluorescence intensity after the reaction was detected; the fluorescence intensity of the test sample and blank control after the reaction was detected simultaneously. (2) Prepare a standard curve: Prepare a standard curve based on the difference in fluorescence intensity between the adenosine deaminase standard and the blank control after the reaction, and calculate the adenosine deaminase content in the sample to be tested based on the standard curve.

9. The application of the method for detecting adenosine deaminase content according to any one of claims 1-6, the kit for detecting adenosine deaminase content according to claim 7, or the method of using the kit for detecting adenosine deaminase content according to claim 8 in the field of adenosine deaminase detection.

Images