Method for synchronously detecting content and activity of G6PD
Through the synchronous detection method of G6PD content and activity, the simultaneous content and activity determination of G6PD enzymes is achieved by using immune capture and enzyme labeling methods, solving the shortcomings of separate measurements in the prior art and improving the specificity and efficiency of detection.
Patent Information
- Application Number
- CN202411399464.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-09
- Publication Date
- 2024-12-10
AI Technical Summary
The existing G6PD detection methods can only determine the activity or content of the enzyme alone, and cannot be detected simultaneously, and there are specificity and interference problems.
A synchronous detection method for G6PD content and activity was adopted, and G6PD-specific antibodies and HRP-labeled antibodies were used through immune capture and enzymatic labeling methods, combining with enzymatic reactions, and the content and activity determination were achieved in one step, reducing the sample size and reagent use.
It improves the specificity of the detection, saves time and reagents, reduces interference from other proteins, and achieves the simultaneous determination of G6PD content and activity, improving detection efficiency.
Smart Images
Figure CN119104719A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of G6PD detection, and in particular to a method for synchronously detecting G6PD content and activity. Background Art
[0002] Glucose-6-phosphate dehydrogenase (G6PD) is one of the important enzymes in the human body, mainly playing a role in red blood cells. Detecting the content and activity of G6PD is very important for diagnosing diseases such as G6PD deficiency. Existing detection methods are generally simple enzymatic reactions (spot method) or enzyme labeling methods, which are single enzyme activity or enzyme content measurements.
[0003] Therefore, in view of this, the existing structure and defects were studied and improved, and a method for simultaneous detection of G6PD content and activity was proposed. Summary of the invention
[0004] In view of the deficiencies in the prior art, the present invention provides a method for synchronously detecting the content and activity of G6PD, which solves the problems raised in the above-mentioned background technology.
[0005] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for simultaneous detection of G6PD content and activity, the method for simultaneous detection of G6PD content and activity comprising the following steps: S1. Sample preparation: Whole blood was collected and red blood cells were separated, and red blood cells were lysed using lysis buffer to extract total protein; S2, immune capture: The G6PD-specific antibody was coated in a 96-well microplate at 4°C overnight, unbound sites were blocked with blocking solution, and incubated at room temperature for 1-2 hours. Then, the sample to be tested was added to the microplate wells and incubated at room temperature for 2 hours to allow G6PD to be captured by the antibody; S3, G6PD activity assay: Add the G6PD activity assay reaction solution to the microplate, incubate the microplate at 37°C for 10 minutes, and use a microplate reader to monitor the NADPH generation rate at a wavelength of 340 nm based on the reaction of G6PD catalyzing the oxidation of 6-phosphoglucose to 6-phosphogluconic acid and reducing NADP+ to NADPH. Calculate the G6PD activity based on the NADPH generation rate. S4, G6PD content determination: Wash the microplate 3-5 times, remove the reaction solution, add HRP-labeled G6PD-specific secondary antibody, incubate at room temperature for 1 hour, wash the microplate 3-5 times, add substrate TMB, color for 15-30 minutes in the dark, add stop solution to terminate the reaction, use a microplate reader to read the absorbance value at a wavelength of 450nm, and calculate the G6PD content in the sample according to the standard curve.
[0006] Furthermore, in step S1, the lysis solution is Triton X-100 lysis solution, and the total protein concentration in the sample is determined using the BCA method or the Bradford method.
[0007] Furthermore, in step S2, the G6PD-specific antibody refers to an antibody that can specifically recognize and bind to G6PD, and after G6PD is captured by the antibody, the microplate is washed 3-5 times with a washing solution to remove unbound proteins.
[0008] Furthermore, in step S3, the components of the G6PD activity assay reaction solution are as follows: Buffer: including Tris-HCl, HEPES, pH value between 7.4-8.0; MgCl2: activator of G6PD, promoting enzyme activity; NADP+: coenzyme of G6PD reaction, participating in the reaction as an electron acceptor; Glucose 6-phosphate is the substrate of the G6P:G6PD reaction.
[0009] Furthermore, in step S3, the G6PD activity calculation formula is as follows:
[0010] in, : Absorbance change rate at 340nm; : total volume of the reaction system, in μL; : Sample volume, in μL; : The molar absorption coefficient of NADPH at 340 nm is 6.22×10^3L / mol·cm; : Molecular weight of G6PD.
[0011] Furthermore, in step S4, the addition of HRP-labeled G6PD-specific secondary antibody is specifically explained as follows: the secondary antibody refers to an antibody that can recognize and bind to the primary antibody, and is an antibody against the primary antibody host animal. This secondary antibody also specifically binds to horseradish peroxidase, which is HRP.
[0012] Furthermore, in step S4, TMB is tetramethylbenzidine, which is a substrate of HRP. When HRP encounters TMB, it catalyzes an oxidation reaction of TMB to produce a blue product.
[0013] Furthermore, in step S4, the stop solution is sulfuric acid, which can stop the enzymatic reaction of HRP and stabilize the blue product.
[0014] Furthermore, in step S4, the standard curve equation is as follows:
[0015] in, : absorbance value, : G6PD content.
[0016] The present invention provides a method for synchronously detecting the content and activity of G6PD, which has the following beneficial effects: 1. The G6PD content and activity simultaneous detection method adds the sample to be tested into the microplate wells, allowing G6PD to be captured by antibodies. Only one step of immune capture is required to simultaneously perform content and activity determination, saving time and reagents. In addition, immune capture can improve the specificity of G6PD detection and reduce interference from other proteins. In addition, only a small amount of sample is required to complete content and activity determination. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is a schematic diagram of the overall steps of a method for simultaneous detection of G6PD content and activity of the present invention; Figure 2 The figure is a schematic diagram of the specific operation flow of a method for synchronously detecting the content and activity of G6PD of the present invention. DETAILED DESCRIPTION
[0018] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.
[0019] like Figure 1-Figure 2 As shown, the present invention provides a technical solution: a method for simultaneous detection of G6PD content and activity, the method for simultaneous detection of G6PD content and activity comprising the following steps: S1. Sample preparation: Collect whole blood and separate red blood cells, and use lysis buffer to lyse red blood cells to extract total protein. The lysis buffer is TritonX-100 lysis buffer, and the total protein concentration in the sample is determined using the BCA method or Bradford method; S2, immune capture: The G6PD-specific antibody was coated in a 96-well microplate at 4°C overnight, unbound sites were blocked with blocking solution, and incubated at room temperature for 1-2 hours. Then, the sample to be tested was added to the microplate wells and incubated at room temperature for 2 hours to allow G6PD to be captured by the antibody; G6PD-specific antibodies refer to antibodies that can specifically recognize and bind to G6PD, and after G6PD is captured by the antibody, the microplate is washed 3-5 times with a washing solution to remove unbound proteins; S3, G6PD activity assay: Add the G6PD activity assay reaction solution to the microplate, incubate the microplate at 37°C for 10 minutes, and use a microplate reader to monitor the NADPH generation rate at a wavelength of 340 nm based on the reaction of G6PD catalyzing the oxidation of 6-phosphoglucose to 6-phosphogluconic acid and reducing NADP+ to NADPH. Calculate the G6PD activity based on the NADPH generation rate. The components of the G6PD activity assay reaction solution are as follows: Buffer: including Tris-HCl, HEPES, pH value between 7.4-8.0; MgCl2: activator of G6PD, promoting enzyme activity; NADP+: coenzyme of G6PD reaction, participating in the reaction as an electron acceptor; Glucose 6-phosphate is the substrate of the G6P:G6PD reaction; The calculation formula of G6PD activity is as follows:
[0020] in, : Absorbance change rate at 340nm; : total volume of the reaction system, in μL; : Sample volume, in μL; : The molar absorption coefficient of NADPH at 340 nm is 6.22×10^3L / mol·cm; : molecular weight of G6PD; S4, G6PD content determination: Wash the microplate 3-5 times, remove the reaction solution, add HRP-labeled G6PD-specific secondary antibody, incubate at room temperature for 1 hour, wash the microplate 3-5 times, add substrate TMB, color for 15-30 minutes in the dark, add stop solution to terminate the reaction, use a microplate reader to read the absorbance value at a wavelength of 450nm, and calculate the G6PD content in the sample according to the standard curve; The specific explanation of adding HRP-labeled G6PD-specific secondary antibody is as follows: secondary antibody refers to an antibody that can recognize and bind to the primary antibody, and is an antibody against the primary antibody host animal. This secondary antibody also specifically binds to horseradish peroxidase, which is HRP. TMB is tetramethylbenzidine, which is a substrate of HRP. When HRP encounters TMB, it will catalyze the oxidation reaction of TMB to produce a blue product. The stop solution is sulfuric acid, which can stop the enzymatic reaction of HRP and stabilize the blue product. Among them, after the secondary antibody is added to bind to the captured G6PD, it will specifically recognize and bind to the primary antibody that has bound to the G6PD protein. In this way, HRP is indirectly linked to the G6PD protein. The washing step is to remove the unbound secondary antibody and reduce background signal interference. When reading the absorbance value, the absorbance value of the blue product is proportional to the content of the G6PD protein. The standard curve equation is as follows:
[0021] in, : absorbance value, : G6PD content; For example, if the absorbance value of a sample to be tested is 0.8, its G6PD content can be calculated by substituting it into the standard curve equation:
[0022] Therefore, the G6PD content of the sample to be tested is about 80 ng / mL.
[0023] Based on the above description, the present invention can simultaneously perform content and activity determination with only one step of immunocapture, saving time and reagents. In addition, immunocapture can improve the specificity of G6PD detection, reduce interference from other proteins, and only a small amount of sample is needed to complete content and activity determination.
[0024] In summary, when using the G6PD content and activity simultaneous detection method, first collect whole blood and separate red blood cells, and use a lysis solution to lyse the red blood cells to extract total protein, the lysis solution is Triton X-100 lysis solution, and use the BCA method or Bradford method to determine the total protein concentration in the sample; The G6PD-specific antibody was coated in a 96-well microplate at 4°C overnight, and the unbound sites were blocked with a blocking solution. The cells were incubated at room temperature for 1-2 hours, and then the samples to be tested were added to the microplate wells and incubated at room temperature for 2 hours to allow G6PD to be captured by the antibody. The G6PD-specific antibody refers to an antibody that can specifically recognize and bind to G6PD, and after G6PD is captured by the antibody, the microplate is washed 3-5 times with a washing solution to remove unbound proteins. Add the G6PD activity assay reaction solution to the microplate, incubate the microplate at 37°C for 10 minutes, and use a microplate reader to monitor the NADPH generation rate at a wavelength of 340 nm based on the reaction of G6PD catalyzing the oxidation of 6-phosphoglucose to 6-phosphogluconic acid and reducing NADP+ to NADPH. Calculate the G6PD activity based on the NADPH generation rate. Wash the microplate 3-5 times, remove the reaction solution, add HRP-labeled G6PD-specific secondary antibody, incubate at room temperature for 1 hour, wash the microplate 3-5 times, add substrate TMB, color for 15-30 minutes in the dark, add stop solution to stop the reaction, use a microplate reader to read the absorbance value at a wavelength of 450nm, and calculate the G6PD content in the sample according to the standard curve; the secondary antibody refers to an antibody that can recognize and bind to the primary antibody, which is an antibody against the host animal of the primary antibody. This secondary antibody also specifically binds to horseradish peroxidase, which is HRP. TMB is tetramethylbenzidine, which is a substrate of HRP. When HRP encounters TMB, it will catalyze the oxidation reaction of TMB to produce a blue product. The stop solution is sulfuric acid, which can stop the enzymatic reaction of HRP and stabilize the blue product. Among them, after the secondary antibody is added to bind to the captured G6PD, it will specifically recognize and bind to the primary antibody that has been bound to the G6PD protein. In this way, HRP is indirectly connected to the G6PD protein. The washing step is to remove the unbound secondary antibody and reduce background signal interference. When reading the absorbance value, the absorbance value of the blue product is proportional to the content of the G6PD protein.
[0025] The embodiments of the present invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.
Claims
1. A method for simultaneous detection of G6PD content and activity, Features: The G6PD content and activity simultaneous detection method comprises the following steps: S1. Sample preparation: Whole blood was collected and red blood cells were separated, and red blood cells were lysed using lysis buffer to extract total protein; S2, immune capture: The G6PD-specific antibody was coated in a 96-well microplate at 4°C overnight, unbound sites were blocked with blocking solution, and incubated at room temperature for 1-2 hours. Then, the sample to be tested was added to the microplate wells and incubated at room temperature for 2 hours to allow G6PD to be captured by the antibody; S3, G6PD activity assay: Add the G6PD activity assay reaction solution to the microplate, incubate the microplate at 37°C for 10 minutes, and use a microplate reader to monitor the NADPH generation rate at a wavelength of 340 nm based on the reaction of G6PD catalyzing the oxidation of 6-phosphoglucose to 6-phosphogluconic acid and reducing NADP+ to NADPH. Calculate the G6PD activity based on the NADPH generation rate. S4, G6PD content determination: Wash the microplate 3-5 times, remove the reaction solution, add HRP-labeled G6PD-specific secondary antibody, incubate at room temperature for 1 hour, wash the microplate 3-5 times, add substrate TMB, color for 15-30 minutes in the dark, add stop solution to terminate the reaction, use a microplate reader to read the absorbance value at a wavelength of 450nm, and calculate the G6PD content in the sample according to the standard curve.
2. A method for simultaneous detection of G6PD content and activity according to claim 1, Features: In step S1, the lysis solution is Triton X-100 lysis solution, and the total protein concentration in the sample is determined using the BCA method or the Bradford method.
3. A method for simultaneous detection of G6PD content and activity according to claim 1, Features: In step S2, the G6PD-specific antibody refers to an antibody that can specifically recognize and bind to G6PD, and after G6PD is captured by the antibody, the microplate is washed 3-5 times with a washing solution to remove unbound proteins.
4. A method for simultaneous detection of G6PD content and activity according to claim 1, Features: In step S3, the components of the G6PD activity assay reaction solution are as follows: Buffer: including Tris-HCl, HEPES, pH value between 7.4-8.0; MgCl2: activator of G6PD, promoting enzyme activity; NADP+: coenzyme of G6PD reaction, participating in the reaction as an electron acceptor; Glucose 6-phosphate is the substrate of the G6P:G6PD reaction.
5. A method for simultaneous detection of G6PD content and activity according to claim 1, Features: In step S3, the G6PD activity calculation formula is as follows: in, : Absorbance change rate at 340nm; : total volume of the reaction system, in μL; : Sample volume, in μL; : The molar absorption coefficient of NADPH at 340 nm is 6.22×10^3L / mol·cm; : Molecular weight of G6PD.
6. A method for simultaneous detection of G6PD content and activity according to claim 1, Features: In step S4, the addition of HRP-labeled G6PD-specific secondary antibody is specifically explained as follows: the secondary antibody refers to an antibody that can recognize and bind to the primary antibody, and is an antibody against the primary antibody host animal. This secondary antibody also specifically binds to horseradish peroxidase, which is HRP.
7. A method for simultaneous detection of G6PD content and activity according to claim 1, Features: In step S4, TMB is tetramethylbenzidine, which is a substrate of HRP. When HRP encounters TMB, it catalyzes an oxidation reaction of TMB to produce a blue product.
8. A method for simultaneous detection of G6PD content and activity according to claim 1, Features: In step S4, the stop solution is sulfuric acid, which can stop the enzymatic reaction of HRP and stabilize the blue product.
9. A method for simultaneous detection of G6PD content and activity according to claim 1, Features: In step S4, the standard curve equation is as follows: in, : absorbance value, : G6PD content.