Method for efficiently detecting anti-autologous AchR antibody

Abstract

The invention relates to the field of anti-autologous AchR antibody detection equipment, in particular to a method for efficiently detecting an anti-autologous AchR antibody, which comprises the following operation steps: S1, antigen coating: selecting a proper microwell plate, diluting an AchR antigen to a proper concentration by using a carbonate buffer solution, adding 100-200 [mu] L of an antigen solution into each well, then placing the microwell plate in a refrigerator at 4 DEG C overnight, and taking the microwell plate out of the refrigerator; the antigen can be fully adsorbed on the surface of the microwell plate; s2, closing the microwell plate: after overnight coating, pouring away the antigen solution in the microwell plate, washing the microwell plate with a washing solution for three times, soaking for 3-5 minutes each time to remove uncombined antigens, then adding 200-300 microliters of a sealing solution into each hole, incubating for 1-2 hours at 37 DEG C, and after incubation is finished; according to the invention, the anti-autologous AchR antibody is detected based on the highly specific binding reaction of the antigen and the antibody, and the layer-by-layer specific binding mechanism enables the ELISA to have high specificity on the detection of the target antibody, so that the interference caused by non-specific binding can be effectively reduced.

Description

Technical Field

[0001] The invention relates to the field of anti-autologous AchR antibody detection equipment, and in particular to a method for efficiently detecting anti-autologous AchR antibodies. Background Art

[0002] Myasthenia gravis is an autoimmune disease with acquired neuromuscular transmission disorder mediated by autoantibodies. The main clinical manifestations are partial or systemic skeletal muscle weakness and fatigue. The symptoms worsen after activity and alleviate after rest. Acetylcholine receptor (AChR) antibodies are the most important pathogenic antibodies of MG. Anti-acetylcholine receptor (AchR) antibody detection is of great significance in the diagnosis and disease assessment of diseases such as myasthenia gravis. However, traditional detection methods are often cumbersome to operate. Therefore, we propose a method for efficient detection of anti-autologous AchR antibodies to solve the above-mentioned problems. Summary of the invention

[0003] The purpose of the present invention is to solve the shortcomings of the background technology and to propose a method for efficiently detecting anti-autologous AchR antibodies.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is: a method for efficiently detecting anti-autologous AchR antibodies, comprising the following steps: S1. Antigen coating: Select a suitable microplate, dilute the AchR antigen to an appropriate concentration with carbonate buffer, add 100-200 μL of antigen solution to each well, and then place the microplate in a 4°C refrigerator overnight to allow the antigen to be fully adsorbed on the surface of the microplate; S2. Block the microplate: After overnight coating, pour out the antigen solution in the microplate and wash the microplate 3 times with washing solution, soaking for 3-5 minutes each time to remove unbound antigens, then add 200-300 μL of blocking solution to each well and incubate at 37°C for 1-2 hours. After the incubation, wash again with washing solution 3 times in the same way as before; S3. Add patient serum: Dilute the patient serum appropriately with diluent. The dilution multiple is determined based on preliminary tests or experience, usually between 1:100-1:1000. Add 100-200 μL of diluted serum sample to each well. Set up positive control (serum known to contain anti-AchR antibodies), negative control (serum without anti-AchR antibodies) and blank control (only diluent). Incubate the microplate at 37°C for 1-2 hours to allow the antibody to fully bind to the antigen. Wash the microplate 3-5 times with washing solution to remove unbound serum components. S4. Add enzyme-labeled secondary antibody: Select appropriate enzyme-labeled secondary antibody according to the source of primary antibody (anti-AchR antibody). If the patient serum is human, select anti-human IgG enzyme-labeled secondary antibody. Dilute the enzyme-labeled secondary antibody to a suitable concentration with diluent, add 100-200 μL to each well, and incubate at 37°C for 30-60 minutes. After the incubation, wash the microplate 3-5 times with washing solution to remove unbound secondary antibody. S5. Color development reaction: If HRP-labeled secondary antibody is used, the commonly used substrate is tetramethylbenzidine (TMB). Add 100-200 μL of TMB substrate solution to each well and react at room temperature in the dark for 10-30 minutes. The color in the microwells can be observed to gradually change. If AP-labeled secondary antibody is used, p-nitrophenyl phosphate (p-NPP) is often used as a substrate. The reaction conditions and observation methods are similar. S6. Termination of reaction and detection: For TMB substrate, when the color change reaches the expected degree, generally 50-100 μL of 2M sulfuric acid solution is added to each well to terminate the reaction, and the color of the solution changes from blue to yellow. For p-NPP substrate, after adding the stop solution, the color change stops, and then use a microplate reader to read the absorbance value of each well at the corresponding wavelength (such as TMB substrate at 450nm wavelength, p-NPP substrate at 405nm wavelength). By comparing with the standard curve (made of anti-AchR antibodies of known concentrations), the content of anti-AchR antibodies in the patient's serum can be calculated.

[0005] Preferably, the microplate mentioned in step S1 may be a 96-well plate made of polystyrene.

[0006] Preferably, the carbonate buffer mentioned in step S1 may be a carbonate buffer with a pH of 9.6.

[0007] Preferably, the washing solution mentioned in step S2 can be a phosphate buffer containing 0.05% Tween-20.

[0008] Preferably, the blocking solution mentioned in step S2 may be a solution containing 1%-5% bovine serum albumin or skimmed milk powder.

[0009] Preferably, the diluent mentioned in step S3 may be a phosphate buffer containing 0.1% bovine serum albumin.

[0010] Preferably, the anti-human IgG enzyme mentioned in step S4 may be horseradish peroxidase (HRP) or alkaline phosphatase (AP).

[0011] Preferably, the stop solution mentioned in step S6 may be a 3M sodium hydroxide solution.

[0012] Preferably, during the S1 step, the purity and activity of the antigen need to be ensured. Impure antigens may introduce impure proteins, resulting in increased nonspecific binding and affecting the accuracy of the test results. If the antigen is mixed with other cell membrane proteins, it may cross-react with the secondary antibody in subsequent steps, so the antigen should be properly preserved.

[0013] Preferably, the serum sample processing and adding step in step S3 specifically includes the following steps: S3.1. Sample collection and storage: The collection of serum samples from patients should be standardized to avoid hemolysis, because hemoglobin released by ruptured red blood cells may interfere with the test results. The collected serum should be tested as soon as possible. If it cannot be tested in time, it should be stored at -20℃ or -80℃ and avoid repeated freezing and thawing. S3.2. Sample dilution: Serum samples need to be appropriately diluted. The dilution factor should be determined based on preliminary experiments or experience. If the dilution factor is too low, it may lead to enhanced nonspecific binding and false positive results; if it is too high, the antibody concentration may be too low and cannot be accurately detected, resulting in false negative results. S3.3. Accuracy of sample addition: When adding serum samples to the microplate, ensure that the sample amount is accurate and uniform, use a precision pipette, and calibrate regularly to prevent deviations in results due to sample addition errors. At the same time, avoid the generation of bubbles, which may affect the uniformity of the antigen-antibody reaction.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention detects anti-autologous AchR antibodies based on the highly specific binding reaction between antigen and antibody. In the experiment, the AchR antigen is fixed on the surface of the microplate, and only the anti-AchR antibody specifically corresponding to it can bind to it. The enzyme-labeled secondary antibody added subsequently is also highly specific and only binds to the bound anti-AchR antibody. This layer-by-layer specific binding mechanism makes ELISA highly specific for the detection of target antibodies and can effectively reduce the interference caused by non-specific binding.

[0015] 2. Generate detectable signals through the reaction of enzymes and substrates. Enzymes have catalytic amplification effects. One enzyme molecule can catalyze the reaction of multiple substrate molecules within a certain period of time, thereby amplifying the detection signal. For example, when the commonly used horseradish peroxidase (HRP)-labeled secondary antibody reacts with the substrate tetramethylbenzidine (TMB), one HRP molecule can catalyze a color reaction of multiple TMB molecules, so that even when the anti-AchR antibody content in the sample is low, a sufficiently strong signal can be generated to be detected. This signal amplification mechanism enables ELISA to detect low concentrations of anti-autologous AchR antibodies. For patients in the early stages of the disease, their anti-AchR antibody levels in the body may be low. The high sensitivity of ELISA helps to achieve early diagnosis when the antibody concentration is not high, which is of great significance for the timely detection and treatment of the disease.

[0016] 3. Using microplates as reaction carriers, the common 96-well plate format can process multiple samples at the same time. In the application scenario of large-scale screening of anti-autologous AchR antibodies, such as screening of suspected myasthenia gravis patient groups, serum samples of multiple patients can be tested simultaneously in the same batch of experiments, which greatly improves the detection efficiency. In addition to testing multiple samples at the same time, ELISA is also convenient for parallel experiments. In the experimental design, multiple replicate wells and control wells (including positive control, negative control and blank control) can be easily set up, so as to more accurately evaluate the experimental error and improve the reliability of the results. The mean and standard deviation can be calculated through the results of multiple replicate wells, which more accurately reflects the content of anti-AchR antibodies in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The present invention is a schematic diagram of the anti-autologous AchR antibody detection process of a method for efficiently detecting anti-autologous AchR antibodies. DETAILED DESCRIPTION

[0018] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0019] Embodiment 1: like Figure 1 A method for efficiently detecting anti-autologous AchR antibodies is shown, comprising the following steps: S1, coating antigen Principle: AchR antigen is fixed on the surface of the microplate to provide a basis for subsequent specific binding with antibodies; Operation details: Choose a suitable microplate. A 96-well plate made of polystyrene is commonly used. AchR antigen is usually diluted to an appropriate concentration with carbonate buffer (pH 9.6). Usually 100 μL of antigen solution is added to each well. Then the microplate is placed in a 4°C refrigerator overnight to allow the antigen to be fully adsorbed on the surface of the microplate. S2, closed microplate Principle: Fill the unbound antigen sites on the microplate with irrelevant proteins to reduce nonspecific binding in subsequent steps; Operation details: After overnight coating, pour out the antigen solution in the microplate and wash the microplate three times with washing solution (such as phosphate buffered saline containing 0.05% Tween-20, PBST), soaking for 3 minutes each time to remove unbound antigens, then add 200 μL of blocking solution (commonly used blocking solutions include 1% bovine serum albumin or skim milk powder solution) to each well, incubate at 37°C for 1 hour, and after the incubation, wash again with washing solution three times, using the same method as before; S3. Add patient serum Principle: If anti-AchR antibodies are present in the patient's serum, these antibodies will specifically bind to the AchR antigen coated on the microplate; Operation details: Dilute the patient's serum appropriately with diluent (such as phosphate buffer containing 0.1% bovine serum albumin). The dilution multiple is generally determined based on preliminary experiments or experience, usually between 1:100. Add 100 μL of diluted serum sample to each well, and set up a positive control (serum known to contain anti-AchR antibodies), a negative control (serum without anti-AchR antibodies) and a blank control (only diluent). Incubate the microplate at 37°C for 1 hour to allow the antibody to fully bind to the antigen. Afterwards, wash the microplate 3 times with washing solution to remove unbound serum components. S4. Add enzyme-labeled secondary antibody Principle: Enzyme-labeled secondary antibodies can specifically bind to anti-AchR antibodies that have been bound to the antigen, thereby introducing enzyme labels and preparing for subsequent color development reactions; Operation details: Select appropriate enzyme-labeled secondary antibody according to the source of primary antibody (anti-AchR antibody). For example, if the patient's serum is human, select anti-human IgG enzyme-labeled secondary antibody. Commonly used enzymes include horseradish peroxidase (HRP) or alkaline phosphatase (AP). Dilute the enzyme-labeled secondary antibody to a suitable concentration with diluent, add 100 μL to each well, and incubate at 37°C for 30 minutes. After the incubation, wash the microplate 3 times with washing solution to remove unbound secondary antibody. S5. Color reaction Principle: Under the catalysis of the enzyme, the substrate undergoes a chemical reaction, resulting in a color change. The depth of the color is related to the content of anti-AchR antibodies. Operation details: If HRP-labeled secondary antibody is used, the commonly used substrate is tetramethylbenzidine (TMB). Add 100 μL of TMB substrate solution to each well and react for 10 minutes at room temperature in the dark. The color in the microwells can be observed to change gradually. If AP-labeled secondary antibody is used, p-nitrophenyl phosphate (p-NPP) is often used as a substrate. The reaction conditions and observation methods are similar. S6. Termination of reaction and detection Principle: Stop the color reaction at an appropriate time point to stabilize the color reaction results, and read the absorbance value with an enzyme reader to quantitatively analyze the content of anti-AchR antibodies; Operation details: For TMB substrate, when the color change reaches the expected degree, 50 μL of 2M sulfuric acid solution is generally added to each well to terminate the reaction, and the solution color changes from blue to yellow. For p-NPP substrate, after adding the stop solution (such as 3M sodium hydroxide solution), the color change stops, and then a microplate reader is used to read the absorbance value of each well at the corresponding wavelength (such as 450nm for TMB substrate and 405nm for p-NPP substrate). By comparing with the standard curve (made of anti-AchR antibodies of known concentrations), the content of anti-AchR antibodies in the patient's serum can be calculated.

[0020] Among them, when the S1 step is carried out, the purity and activity of the antigen need to be ensured. Impure antigens may introduce impure proteins, resulting in increased nonspecific binding and affecting the accuracy of the test results. For example, if other cell membrane proteins are mixed in the antigen, cross-reactions may occur with the secondary antibody in subsequent steps. The antigen should be properly preserved.

[0021] The serum sample processing and adding step in step S3 specifically includes the following steps: S3.1. Sample collection and storage: The collection of serum samples from patients should be standardized to avoid hemolysis, because hemoglobin released by ruptured red blood cells may interfere with the test results. The collected serum should be tested as soon as possible. If it cannot be tested in time, it should be stored at -20°C and avoid repeated freezing and thawing. S3.2. Sample dilution: Serum samples need to be appropriately diluted. The dilution factor should be determined based on preliminary experiments or experience. If the dilution factor is too low, it may lead to enhanced nonspecific binding and false positive results; if it is too high, the antibody concentration may be too low and cannot be accurately detected, resulting in false negative results. S3.3. Accuracy of sample addition: When adding serum samples to the microplate, ensure that the sample amount is accurate and uniform, use a precision pipette, and calibrate regularly to prevent deviations in results due to sample addition errors. At the same time, avoid the generation of bubbles, which may affect the uniformity of the antigen-antibody reaction.

[0022] Embodiment 2: like Figure 1 A method for efficiently detecting anti-autologous AchR antibodies is shown, comprising the following steps: S1, coating antigen Principle: AchR antigen is fixed on the surface of the microplate to provide a basis for subsequent specific binding with antibodies; Operation details: Choose a suitable microplate. A 96-well plate made of polystyrene is commonly used. AchR antigen is usually diluted to an appropriate concentration with carbonate buffer (pH about 9.6). Usually 200 μL of antigen solution is added to each well. Then the microplate is placed in a 4°C refrigerator overnight to allow the antigen to be fully adsorbed on the surface of the microplate. S2, closed microplate Principle: Fill the unbound antigen sites on the microplate with irrelevant proteins to reduce nonspecific binding in subsequent steps; Operation details: After overnight coating, pour out the antigen solution in the microplate and wash the microplate 3 times with washing solution (such as phosphate buffered saline containing 0.05% Tween-20, PBST), soaking for 5 minutes each time to remove unbound antigens, then add 300 μL of blocking solution (commonly used blocking solutions include 5% bovine serum albumin or skimmed milk powder solution) to each well, incubate at 37°C for 2 hours, and after the incubation, wash again with washing solution 3 times, using the same method as before. S3. Add patient serum Principle: If anti-AchR antibodies are present in the patient's serum, these antibodies will specifically bind to the AchR antigen coated on the microplate; Operation details: Dilute the patient's serum appropriately with a diluent (such as phosphate buffer containing 0.1% bovine serum albumin). The dilution multiple is generally determined based on preliminary experiments or experience, usually between 1:1000. Add 200 μL of diluted serum sample to each well, and set up a positive control (serum known to contain anti-AchR antibodies), a negative control (serum without anti-AchR antibodies), and a blank control (only diluent). Incubate the microplate at 37°C for 2 hours to allow the antibody and antigen to fully bind. Afterwards, wash the microplate 5 times with a washing solution to remove unbound serum components. S4. Add enzyme-labeled secondary antibody Principle: Enzyme-labeled secondary antibodies can specifically bind to anti-AchR antibodies that have been bound to the antigen, thereby introducing enzyme labels and preparing for subsequent color development reactions; Operation details: Select the appropriate enzyme-labeled secondary antibody according to the source of the primary antibody (anti-AchR antibody). For example, if the patient's serum is human, select an enzyme-labeled secondary antibody against human IgG. Commonly used enzymes include horseradish peroxidase (HRP) or alkaline phosphatase (AP). Dilute the enzyme-labeled secondary antibody to an appropriate concentration with diluent, add 200 μL to each well, and incubate at 37°C for 60 minutes. After the incubation, wash the microplate 5 times with washing solution to remove unbound secondary antibody. S5. Color reaction Principle: Under the catalysis of the enzyme, the substrate undergoes a chemical reaction, resulting in a color change. The depth of the color is related to the content of anti-AchR antibodies. Operation details: If HRP-labeled secondary antibody is used, the commonly used substrate is tetramethylbenzidine (TMB). Add 200 μL of TMB substrate solution to each well and react at room temperature in the dark for 30 minutes. The color in the microwells can be observed to change gradually. If AP-labeled secondary antibody is used, p-nitrophenyl phosphate (p-NPP) is often used as the substrate. The reaction conditions and observation methods are similar. S6. Termination of reaction and detection Principle: Stop the color reaction at an appropriate time point to stabilize the color reaction results, and read the absorbance value with an enzyme reader to quantitatively analyze the content of anti-AchR antibodies; Operation details: For TMB substrate, when the color change reaches the expected degree, 100 μL of 2M sulfuric acid solution is generally added to each well to terminate the reaction, and the solution color changes from blue to yellow. For p-NPP substrate, after adding the stop solution (such as 3M sodium hydroxide solution), the color change stops, and then a microplate reader is used to read the absorbance value of each well at the corresponding wavelength (such as 450nm for TMB substrate and 405nm for p-NPP substrate). By comparing with the standard curve (made of anti-AchR antibodies of known concentrations), the content of anti-AchR antibodies in the patient's serum can be calculated.

[0023] Among them, when the S1 step is carried out, the purity and activity of the antigen need to be ensured. Impure antigens may introduce impure proteins, resulting in increased nonspecific binding and affecting the accuracy of the test results. For example, if other cell membrane proteins are mixed in the antigen, cross-reactions may occur with the secondary antibody in subsequent steps. The antigen should be properly preserved.

[0024] The serum sample processing and adding step in step S3 specifically includes the following steps: S3.1. Sample collection and storage: The collection of serum samples from patients should be standardized to avoid hemolysis, because hemoglobin released by ruptured red blood cells may interfere with the test results. The collected serum should be tested as soon as possible. If it cannot be tested in time, it should be stored at -80°C and avoid repeated freezing and thawing. S3.2. Sample dilution: Serum samples need to be appropriately diluted. The dilution factor should be determined based on preliminary experiments or experience. If the dilution factor is too low, it may lead to enhanced nonspecific binding and false positive results; if it is too high, the antibody concentration may be too low and cannot be accurately detected, resulting in false negative results. S3.3. Accuracy of sample addition: When adding serum samples to the microplate, ensure that the sample amount is accurate and uniform, use a precision pipette, and calibrate regularly to prevent deviations in results due to sample addition errors. At the same time, avoid the generation of bubbles, which may affect the uniformity of the antigen-antibody reaction.

[0025] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for efficiently detecting anti-autologous AchR antibodies, characterized in that: The steps include: S1. Antigen coating: Select a suitable microplate, dilute the AchR antigen to an appropriate concentration with carbonate buffer, add 100-200 μL of antigen solution to each well, and then place the microplate in a 4°C refrigerator overnight to allow the antigen to be fully adsorbed on the surface of the microplate; S2. Block the microplate: After overnight coating, pour out the antigen solution in the microplate and wash the microplate 3 times with washing solution, soaking for 3-5 minutes each time to remove unbound antigens, then add 200-300 μL of blocking solution to each well and incubate at 37°C for 1-2 hours. After the incubation, wash again with washing solution 3 times in the same way as before; S3. Add patient serum: Dilute the patient serum appropriately with diluent. The dilution multiple is determined based on preliminary tests or experience, usually between 1:100-1:1000. Add 100-200 μL of diluted serum sample to each well. Set up positive control (serum known to contain anti-AchR antibodies), negative control (serum without anti-AchR antibodies) and blank control (only diluent). Incubate the microplate at 37°C for 1-2 hours to allow the antibody to fully bind to the antigen. Wash the microplate 3-5 times with washing solution to remove unbound serum components. S4. Add enzyme-labeled secondary antibody: Select appropriate enzyme-labeled secondary antibody according to the source of primary antibody (anti-AchR antibody). If the patient serum is human, select anti-human IgG enzyme-labeled secondary antibody. Dilute the enzyme-labeled secondary antibody to a suitable concentration with diluent, add 100-200 μL to each well, and incubate at 37°C for 30-60 minutes. After the incubation, wash the microplate 3-5 times with washing solution to remove unbound secondary antibody. S5. Color development reaction: If HRP-labeled secondary antibody is used, the commonly used substrate is tetramethylbenzidine (TMB). Add 100-200 μL of TMB substrate solution to each well and react at room temperature in the dark for 10-30 minutes. The color in the microwells can be observed to gradually change. If AP-labeled secondary antibody is used, p-nitrophenyl phosphate (p-NPP) is often used as a substrate. The reaction conditions and observation methods are similar. S6. Termination of reaction and detection: For TMB substrate, when the color change reaches the expected degree, generally 50-100 μL of 2M sulfuric acid solution is added to each well to terminate the reaction, and the color of the solution changes from blue to yellow. For p-NPP substrate, after adding the stop solution, the color change stops, and then use a microplate reader to read the absorbance value of each well at the corresponding wavelength (such as TMB substrate at 450nm wavelength, p-NPP substrate at 405nm wavelength). By comparing with the standard curve (made of anti-AchR antibodies of known concentrations), the content of anti-AchR antibodies in the patient's serum can be calculated.

2. A method for efficiently detecting anti-autologous AchR antibodies according to claim 1, characterized in that: The microplate mentioned in step S1 may be a 96-well plate made of polystyrene.

3. A method for efficiently detecting anti-autologous AchR antibodies according to claim 1, characterized in that: The carbonate buffer mentioned in step S1 may be a carbonate buffer with a pH of 9.

6.

4. A method for efficiently detecting anti-autologous AchR antibodies according to claim 1, characterized in that: The washing solution mentioned in step S2 can be a phosphate buffer containing 0.05% Tween-20.

5. A method for efficiently detecting anti-autologous AchR antibodies according to claim 1, characterized in that: The blocking solution mentioned in step S2 can be a solution containing 1%-5% bovine serum albumin or skimmed milk powder.

6. A method for efficiently detecting anti-autologous AchR antibodies according to claim 1, characterized in that: The diluent mentioned in step S3 can be a phosphate buffer containing 0.1% bovine serum albumin.

7. A method for efficiently detecting anti-autologous AchR antibodies according to claim 1, characterized in that: The anti-human IgG enzyme mentioned in step S4 may be horseradish peroxidase (HRP) or alkaline phosphatase (AP).

8. A method for efficiently detecting anti-autologous AchR antibodies according to claim 1, characterized in that: The stop solution mentioned in step S6 can be a 3M sodium hydroxide solution.

9. A method for efficiently detecting anti-autologous AchR antibodies according to claim 1, characterized in that: During the S1 step, the purity and activity of the antigen must be ensured. Impure antigens may introduce impure proteins, resulting in increased nonspecific binding and affecting the accuracy of the test results. For example, if other cell membrane proteins are mixed in the antigen, cross-reactions may occur with the secondary antibody in subsequent steps. Therefore, the antigen should be properly preserved.

10. A method for efficiently detecting anti-autologous AchR antibodies according to claim 1, characterized in that: The serum sample processing and adding step in step S3 specifically includes the following steps: S3.

1. Sample collection and storage: The collection of serum samples from patients should be standardized to avoid hemolysis, because hemoglobin released by ruptured red blood cells may interfere with the test results. The collected serum should be tested as soon as possible. If it cannot be tested in time, it should be stored at -20℃ or -80℃ and avoid repeated freezing and thawing. S3.

2. Sample dilution: Serum samples need to be appropriately diluted. The dilution factor should be determined based on preliminary experiments or experience. If the dilution factor is too low, it may lead to enhanced nonspecific binding and false positive results; if it is too high, the antibody concentration may be too low and cannot be accurately detected, resulting in false negative results. S3.

3. Accuracy of sample addition: When adding serum samples to the microplate, ensure that the sample amount is accurate and uniform, use a precision pipette, and calibrate regularly to prevent deviations in results due to sample addition errors. At the same time, avoid the generation of bubbles, which may affect the uniformity of the antigen-antibody reaction.

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