Method for detecting anti-peg-eop anti-drug antibody based on electrochemiluminescence and application thereof

Abstract

The application belongs to the technical field of detection and analysis, and particularly relates to a method for detecting anti-PEG-EPO drug-resistant antibodies based on electrochemiluminescence and application thereof. The application successfully detects anti-PEG-EPO drug-resistant antibodies in human serum by using electrochemiluminescence MSD technology for the first time. Compared with existing enzyme-linked immunosorbent assay methods, the detection method is simple, easier to operate, greatly shortens the detection time, significantly improves the experimental efficiency, has high detection sensitivity, strong specificity, good method robustness, and therefore has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of detection and analysis technology, and particularly relates to a method for detecting anti-PEG-EPO drug resistance antibody based on electrochemiluminescence and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of increasing the understanding of the background of the application without admitting that any of the information constitutes prior art.

[0003] Erythropoietin (EPO), also known as erythropoietic factor and erythropoietin, is an endogenous glycoprotein hormone in the human body, which can stimulate erythropoiesis. Hypoxia can stimulate the production of erythropoietin. Recombinant human erythropoietin has been used in clinical practice for a long time, and is used for the treatment of anemia associated with renal dysfunction, anemia caused by acquired immunodeficiency syndrome / acquired immunodeficiency syndrome itself or treatment, anemia associated with malignant tumors and rheumatic diseases, etc.

[0004] However, erythropoietin itself has the defects of short plasma half-life, easy degradation by proteases, and low utilization rate, which prevent them from achieving the maximum clinical efficacy. Therefore, obtaining long-acting erythropoietin has become a hot topic for research institutions and pharmaceutical companies to compete. The current commonly used methods include: ① changing part of the amino acids in the EPO peptide chain to increase the glycosylation site and thus increase the degree of glycosylation, thereby increasing the plasma half-life; ② obtaining polyethylene glycolized erythropoietin (PEG-EPO) by coupling EPO with PEG. Specifically, it is modified by covalently linking 1-3 low alkyl polyethylene glycol (PEG) groups to the EPO glycoprotein, which improves the circulating half-life and plasma retention time of EPO, reduces the clearance rate of EPO and improves its in vivo activity. Among them, PEG-EPO has become the most commonly used erythropoietin product due to its advantages.

[0005] However, similar to other protein drugs, the above-mentioned PEG-EPO may cause the production of drug resistance antibodies (ADA) during use due to drug factors or patient factors. These antibodies may be harmless, or reduce drug efficacy and drug half-life, or even be life-threatening. Therefore, it is very important to conduct immunogenicity tests on therapeutic protein products to understand the effect of drug resistance antibodies on a given drug. However, the inventors found that there are still few reports on the detection of anti-PEG-EPO drug resistance antibodies. SUMMARY

[0006] In view of the prior art, the present application provides a method for detecting anti-PEG-EPO anti-drug antibodies based on electrochemiluminescence and application thereof. Specifically, the present application successfully detects anti-PEG-EPO anti-drug antibodies in human serum by using electrochemiluminescence MSD technology, which is rapid, convenient, and has high sensitivity, specificity and robustness. Based on the above research results, the present application is completed.

[0007] To achieve the above technical purposes, the technical solutions adopted by the present application are as follows:

[0008] In a first aspect, the present application provides a method for detecting anti-PEG-EPO anti-drug antibodies based on electrochemiluminescence, which comprises: adding a biological agent solution to a SA-MSD plate (streptavidin-coated MSD plate) and fixing it in the MSD through the action of biotin and streptomycin; diluting the sample to be tested and adding it to the SA-MSD plate; capturing the antibody by the biological agent in the SA-MSD plate; adding Sulfo-Tag labeled PEG-EPO to the plate and combining it with the antibody; finally, exciting electrochemical light by Sulfo-Tag, capturing the signal value by MSD instrument, and the signal value intensity is proportional to the strength of anti-PEG-EPO anti-drug antibodies (ADA).

[0009] In a second aspect, the present application provides the application of the above detection method in PEG-EPO drug evaluation.

[0010] Specifically, the PEG-EPO drug evaluation is specifically PEG-EPO immunogenicity evaluation. The immunogenicity of drugs, especially protein drugs (such as PEG-EPO), affects the safety and effectiveness of the drugs, and even causes fatal new diseases to patients due to anti-drug antibodies and endogenous protein cross-reaction; and ADA is the main way of immunogenicity evaluation of the above protein drugs, so the detection of anti-PEG-EPO anti-drug antibodies can realize the evaluation of PEG-EPO immunogenicity.

[0011] The beneficial technical effects of the one or more technical solutions are as follows:

[0012] The above technical solution first successfully detects anti-PEG-EPO anti-drug antibodies in human serum by using electrochemiluminescence MSD technology, compared with the existing enzyme-linked immunosorbent assay, the detection method is simple and easy to operate, the detection time is greatly shortened, the experimental efficiency is significantly improved, and the detection sensitivity is high, the specificity is strong, and the method is robust, so it has good practical application value. DETAILED DESCRIPTION

[0013] It should be noted that the following detailed description is illustrative only, and is intended to provide further description in order to provide a thorough understanding of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains.

[0014] It is also important to note that the terms "preferably," "preferably," "preferred," and "desirably" are used herein to mean "desirable, but not necessarily essential." Although only a few embodiments of the present application have been described herein, it should be understood that the present application might be embodied in many other forms without departing from the spirit thereof. Therefore, it is desired that what is claimed should be understood to be within the true spirit and scope of the present application.

[0015] In one exemplary embodiment of the present application, a method for detecting anti-PEG-EPO anti-drug antibodies based on electrochemiluminescence is provided, which comprises: after a biological agent solution is added to a SA-MSD plate (a streptavidin-coated MSD plate), the biological agent is fixed in the MSD through the action of biotin and streptomycin; after the sample to be tested is diluted, it is added to the SA-MSD plate, the antibody is captured by the biological agent in the SA-MSD plate, and then Sulfo-Tag-labeled PEG-EPO is added to the plate to bind with the antibody; finally, Sulfo-Tag excites electrochemiluminescence, and the signal value is captured by the MSD instrument, and the signal value intensity is proportional to the strength of the anti-PEG-EPO anti-drug antibody (ADA).

[0016] In another exemplary embodiment of the present application, the biological agent is a biotin-modified PEG-EPO (Bio-PEG-EPO), and the concentration thereof is 0.1-1 μg / mL, preferably 0.5 μg / mL.

[0017] The step of "after the biological agent solution is added to the SA-MSD plate, the biological agent is fixed in the MSD through the action of biotin and streptomycin" specifically comprises: adding the biological agent solution to the SA-MSD plate, sealing the plate with a sealing film, incubating at 20-30°C (preferably 22°C) for 60-66 minutes, and shaking the plate at a speed of 500-1000 rpm, preferably 800 rpm.

[0018] The SA-MSD plate is blocked with bovine serum albumin before use, and then washed with a washing buffer; wherein the washing buffer can be a PBS buffer containing a surfactant, wherein the surfactant is Tween 20, and the content of the Tween 20 is 0.01-0.1% (v / v), preferably 0.05%.

[0019] The specific method for diluting the sample to be tested comprises: after the sample to be tested is completely melted and vortexed, the sample to be tested is added to a dilution plate, a sealing film is used to seal the plate, the plate is incubated at 20-30°C (preferably 22°C) for 10-30 minutes (preferably 20 minutes), and the rotation speed of a plate shaker is 500-1000 rpm, preferably 800 rpm.

[0020] In another specific embodiment of the present application, the sample to be tested is a blood sample of a subject, more specifically a serum sample of a subject; and the presence or absence and concentration of anti-PEG-EPO drug antibody in the blood (serum) sample of the subject are determined by the above-mentioned detection method.

[0021] The specific steps for adding the diluted sample to be tested to the SA-MSD plate comprise: the diluted sample to be tested is transferred from the dilution plate to the SA-MSD plate, a sealing film is used to seal the plate, the plate is incubated at 20-30°C (preferably 22°C) for 60-66 minutes, and the rotation speed of a plate shaker is 500-1000 rpm, preferably 800 rpm.

[0022] The minimum dilution ratio of the sample to be tested is 5 times.

[0023] The specific method for adding Sulfo-Tag-labeled PEG-EPO to the plate to bind with the antibody comprises:

[0024] The Sulfo-Tag-labeled PEG-EPO is transferred to the SA-MSD plate, the plate is incubated at 20-30°C (preferably 22°C) for 60-70 minutes, and the rotation speed of a plate shaker is 500-1000 rpm, preferably 800 rpm.

[0025] The excitation light wavelength of the electrochemical light excited by the Sulfo-Tag is 620 nm.

[0026] It should be noted that the present application also includes a positive quality control sample and a negative quality control sample, wherein the positive quality control sample (monoclonal mouse anti-erythropoietin) is divided into high, medium and low concentration positive quality control samples, and the concentrations thereof are 5000 ng / mL, 2500 ng / mL and 32.91 ng / mL respectively; and the concentration of the negative quality control sample is 0 ng / mL.

[0027] In another specific embodiment of the present application, the application of the above-mentioned detection method in the evaluation of PEG-EPO drugs is provided.

[0028] In still another specific embodiment of the present application, the PEG-EPO drug evaluation is specifically PEG-EPO immunogenicity evaluation. The immunogenicity of a drug, especially a protein drug (such as the above-mentioned PEG-EPO), affects the safety and effectiveness of the drug, and even causes a new fatal disease to the patient due to ADA and endogenous protein cross; and the anti-drug antibody (ADA) is the main way of immunogenicity evaluation of the above-mentioned protein drug, so that the evaluation of the immunogenicity of PEG-EPO is realized by detecting the above-mentioned anti-drug antibody against PEG-EPO.

[0029] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples. The raw reagents of the present application can be obtained by marketable means, wherein the PEG-EPO and EPO stock solution are provided by Huarun Angde Biological Pharmaceutical Co., Ltd., and the positive quality control monoclonal mouse anti-erythropoietin is provided by Novus.

[0030] Example

[0031] I. A method for detecting anti-drug antibodies against PEG-EPO based on electrochemiluminescence, the method is outlined as follows:

[0032]

[0033] In which, 0.5 μg / mL of Bio-PEG-EPO solution is added to the SA-MSD plate, sealed with a sealing film, incubated at 22°C for 60 minutes, and the shaking plate machine is rotated at 800 rpm; after the serum sample to be tested is completely melted and vortexed, it is added to the dilution plate, sealed with a sealing film, incubated at 22°C for 20 minutes, and the shaking plate machine is rotated at 800 rpm; after dilution (1:5) treatment, it is added to the SA-MSD plate, sealed with a sealing film, incubated at 22°C for 60 minutes, and the shaking plate machine is rotated at 800 rpm; the antibody is captured by Bio-PEG-EPO in the SA-MSD plate, and then Sulfo-Tag labeled PEG-EPO is transferred to the SA-MSD plate, incubated at 22°C for 60 minutes, and the shaking plate machine is rotated at 800 rpm; finally, Sulfo-Tag emits electrochemical light at 620 nm, and the signal value is captured by the MSD instrument, the signal value intensity is proportional to the intensity of anti-PEG-EPO anti-drug antibody (ADA), thereby realizing detection.

[0034] II. Method verification content and results:

[0035] 2.1 Threshold evaluation

[0036] 52 healthy human serum samples were tested in threshold evaluation analysis batches, each sample was tested at least 6 times in different analysis batches, and the same sample could only be analyzed once in a day by the same analyst. The threshold evaluation included statistics of screening threshold and titer threshold (statistical results are shown in Table 1), statistics of PEG-EPO and EPO CCP confirmation threshold (statistical results are shown in Table 2 and Table 3).

[0037] Table 1 Statistics of screening threshold and titer threshold

[0038]

[0039] Table 2 Statistics of PEG-EPO confirmation threshold

[0040]

[0041] Table 3 Statistics of EPO confirmation threshold

[0042]

[0043] 2.2 Sensitivity of screening and confirmation experiments

[0044] Each analysis batch included the following quality controls and samples, which were respectively incubated with (PEG-EPO) and without drug: 1) 3 sets of HPC (5000 ng / ml) and 3 sets of the following 6 concentrations of PC (PC1-PC6 in turn): 100.00, 50.00, 25.00, 12.50, 6.25, 3.13 ng / ml; 2) 3 sets of NC. The above experiment was repeated for 8 times. The average values of screening and confirmation sensitivity concentrations of all 24 analysis batches were calculated, which were the screening sensitivity and confirmation sensitivity of the analysis method respectively. The screening experiment sensitivity results are shown in Table 4, and the confirmation experiment sensitivity results are shown in Table 5.

[0045] Table 4 Screening sensitivity and sLPC

[0046]

[0047] sLPC = 27.88

[0048] Table 5 Confirmation sensitivity and cLPC

[0049]

[0050] cLPC = 32.91

[0051] sLPC = 27.88

[0052] cLPC / sLPC = 1.18 < 1.25

[0053] Conclusion: LPC = 32.91 ng / mL

[0054] 2.3 Titer Verification

[0055] 1) The signal value data of the positive controls PC1 to PC6 (without drug incubation) in the analysis batch was used for titer verification, PC1 as titer sample, PC1 to PC6 as serially diluted samples.

[0056] 2) A titer value was calculated for each analysis batch that passed the acceptance criteria, using the interpolation formula of Watson LIMS 7.6. If the signal value of PC6 was still greater than the titer threshold, the titer of this analysis batch was not calculated. A total of 24 valid titer values were obtained, and the mean of all titer values was calculated. The acceptance criteria for the titer value of PC1 in the subsequent experiments was: the calculated mean ± 3*SD.

[0057] The titer statistical process and results are shown in Table 6.

[0058] Table 6 Titer Statistics

[0059]

[0060] 2.4 Quality Control Acceptance Criteria

[0061] HPC Acceptance Criteria

[0062] The Mean-3*SD of all HPC signal values in the screening threshold experiment and sensitivity experiment was calculated, and the HPC signal value should be ≥ 504.44. The Mean-3*SD of all HPC % inhibition rates in the confirmation threshold experiment and sensitivity experiment was calculated, and the HPC % inhibition rate with PEG-EPO was ≥ 82.20%, and the HPC % inhibition rate with EPO was ≥ 80.28%. The HPC acceptance criteria were:

[0063] The statistical process and results of the HPC acceptance criteria are shown in Table 7.

[0064] LPC Acceptance Criteria

[0065] The signal value ≥ the screening threshold factor in the method validation * the median of NC (median of 6 wells), and the % Inhibition ≥ the confirmation threshold in the method validation.

[0066] Table 7 HPC Acceptance Criteria Statistics

[0067]

[0068] Conclusion: The signal value of HPC should be ≥ 504.44, the % inhibition rate with PEG-EPO should be ≥ 82.20%, and the % inhibition rate with EPO should be ≥ 80.28%.

[0069] NC Acceptance Criteria

[0070] The Mean+3*SD of all NC signal values in the screening threshold experiment and sensitivity experiment should be < 86.61; the NC inhibition rate should be < the confirmation threshold in the method validation. The NC acceptance criteria statistical process and results are shown in Table 8.

[0071] Table 8 NC acceptance criteria statistics

[0072]

[0073] Conclusion:

[0074] The signal value of the NC should be < 86.61.

[0075] 2.5 Precision evaluation

[0076] 1) The precision evaluation should be performed on 6 qualified analytical batches by 2 analysts in 2 days (excluding titer precision).

[0077] 2) Each analytical batch should be divided into drug-added and drug-free parts, each part including 6 sets of HPC, 6 sets of MPC (concentration: 2500 ng / mL, only for precision verification), 6 sets of LPC, and 6 sets of NC (12 wells).

[0078] 3) Calculate the Ratio and %Inhibition of all HPC, MPC, and LPC.

[0079] 4) In each analytical batch, the signal value and %Inhibition of at least 4 / 6 HPC, MPC, and LPC should meet the verified acceptance criteria and the duplicate well %CV acceptance criteria (MPC should be a positive result). Otherwise, the analytical batch is determined to be unqualified and is not included in the statistics.

[0080] 5) Calculate the intra-plate and inter-plate precision of HPC, MPC, and cLPC Ratio, which should be less than or equal to 20.0%; calculate the intra-plate and inter-plate precision of HPC, MPC, and cLPC %Inhibition, which should be less than or equal to 20.0%, and calculate the precision of NC signal value.

[0081] The precision evaluation statistical process and results are shown in Table 9.

[0082] Table 9 Precision evaluation

[0083]

[0084]

[0085] *outlier

[0086] Titer precision evaluation is as follows:

[0087] 1) Precision evaluation was performed with 3 qualified analytical batches.

[0088] 2) Each analytical batch included at least 3 sets of NC (6 wells), 1 set of HPC (duplicate wells), 3 sets of TPC curves (TPC1 concentration: 100.00 ng / mL, 2-fold serial dilution to 3.13 ng / mL for TPC2-TPC6, duplicate wells).

[0089] 3) Acceptance criteria: the signal value of high concentration positive control met: HPC > SCP, the titer value of TPC1 was calculated.

[0090] 4) The intra-plate and inter-plate precision of TPC1 titer value was calculated.

[0091] The statistical process and results of titer precision evaluation were shown in Table 10.

[0092] Table 10 Titer precision evaluation

[0093]

[0094] 2.6 Matrix effect (selectivity)

[0095] Matrix effect evaluation used 10 serum samples from healthy individuals, in which HPC, LPC concentrations and blank samples were prepared, each validation sample was detected once in the confirmation experiment. The detection results of 9 serum samples from healthy individuals met the acceptance criteria specified in the protocol: 1) positive control matrix effect sample was determined to be positive; 2) HPC signal value > LPC signal value and duplicate well signal value % CV ≤ 20.0%; 3) the blank sample of the corresponding batch was determined to be negative, and the duplicate well signal value % CV ≤ 20.0%. Therefore, the validated analytical method had no matrix effect in serum from healthy individuals. The detailed results of matrix effect evaluation were shown in Table 11.

[0096] Table 11 Matrix effect

[0097]

[0098]

[0099] 2.7 Hemolysis and high lipid

[0100] The hemolysis and high lipid samples were prepared as follows: 1 mixed 2% hemolysis and 1 mixed human serum with triglyceride content greater than 300 mg / dL were taken respectively, and 1 set of HPC, LPC (for confirmation test) and blank samples were prepared in each serum, and each sample was detected 3 times in the confirmation test. The hemolysis and high lipid results meet the following requirements: at least 2 / 3 of the positive control matrix effect sample is determined to be positive, and the %CV of the duplicate well signal value is ≤20.0%, the blank sample is determined to be negative, and the %CV of the duplicate well signal value is ≤20.0%. The detailed results of the hemolysis and high lipid evaluation are shown in Table 12.

[0101] Table 12 Hemolysis and high lipid evaluation

[0102]

[0103] *The sample was not prepared using the original whole blood at the time of preparation, and was not involved in the statistics together, but the original two hemolysis samples had passed, meeting the requirement of at least 2 / 3 passing, so it was determined that the verification item passed.

[0104] 2.8 Hook effect

[0105] A sample with a concentration of 100000.00 ng / mL was prepared in mixed healthy human serum, and the mixed healthy human serum was diluted 5 times in series to 160.00 ng / ml. Each was detected once, and the drug addition and non-drug addition incubation steps were performed respectively. The test results meet the acceptance criteria in the program: the signal value of the high concentration sample > the signal value of the low concentration sample > the screening threshold, the %Inhibition of the high concentration sample > the %Inhibition of the low concentration sample > the confirmation threshold, and the sample does not appear hook effect when the EPO antibody concentration is as high as 100000.00 ng / mL.

[0106] The detailed results of the hook effect evaluation are shown in Table 13.

[0107] Table 13 Hook effect evaluation

[0108]

[0109] 2.9 Drug tolerance

[0110] PEG-EPO was added to mixed healthy human serum to prepare 2-fold concentration drug samples with concentrations of 100.00, 50.00, 25.00, 12.50 and 0.00 ng / ml.

[0111] Positive antibodies were added to mixed healthy human serum to prepare 2-fold concentration ADA samples with concentrations of 500.00 and 200.00 ng / ml.

[0112] Mix 2x concentration drug sample with 2x concentration ADA sample 1:1 and incubate at room temperature for at least 1 hour. Each sample is tested 3 times in the screening experiment.

[0113] The results show that when the ADA concentration is 250.00 ng / mL and 100.00 ng / mL, the tolerable drug concentration is 50.00 ng / mL

[0114] The detailed results of drug tolerance evaluation are shown in Table 14.

[0115] Table 14 Drug tolerance evaluation

[0116]

[0117]

[0118] 2.10 Room temperature stability

[0119] Three sets of quality control samples (HPC and LPC) were taken out from the -80°C refrigerator, placed at room temperature for 49 hours, and analyzed using the analysis method, with each sample tested once. The test results meet the acceptance criteria specified in the protocol: 2 / 3 of the signal values of HPC and LPC meet the verified signal range and the %CV of the duplicate wells is ≤20%. Therefore, the verified quality control samples can be stably placed at room temperature for 49 hours. The detailed results of room temperature stability evaluation are shown in Table 15.

[0120] 2.112-8°C stability

[0121] Three sets of quality control samples (HPC and LPC) were taken out from the -80°C refrigerator, placed at 2-8°C for 49 hours, and analyzed using the analysis method, with each sample tested once. The test results meet the acceptance criteria specified in the protocol: 2 / 3 of the signal values of HPC and LPC meet the verified signal range and the %CV of the duplicate wells is ≤20%. Therefore, the verified quality control samples can be stably placed at 2-8°C for 49 hours. The detailed results of 2-8°C stability evaluation are shown in Table 15.

[0122] Table 15 Short-term stability

[0123]

[0124] In summary, the verification results of the method of the present application are as follows:

[0125]

[0126]

[0127] Finally, it should be noted that the above only for the preferred embodiments of the present application, and is not intended to limit the present application, although with reference to the foregoing embodiments of the present application has been described in detail, for those skilled in the art, it still can be modified, or part of the equivalent replacement of the technical solutions described in the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application, should be included within the scope of the present application. Although the above describes the specific embodiments of the present application, but not the limitation of the scope of protection of the present application, the skilled in the art should be clear that, on the basis of the technical solutions of the present application, the skilled in the art without the need for creative labor can make various modifications or deformation still within the scope of the present application.

Claims

1. A method for detecting anti-PEG-EPO anti-drug antibodies based on electrochemiluminescence, characterized in that, The method comprises the following steps: adding a biological agent solution to a streptavidin-coated MSD plate (SA-MSD plate), and fixing the biological agent in the MSD plate through the action of biotin and streptomycin; diluting a sample to be tested, and adding the diluted sample to the SA-MSD plate; capturing the antibody by the biological agent in the SA-MSD plate, and adding Sulfo-Tag-labeled PEG-EPO to the plate to combine with the antibody; and finally, exciting electrochemical light by the Sulfo-Tag, and the signal value intensity is proportional to the intensity of the anti-PEG-EPO antibody; the biological agent is biotin-modified PEG-EPO, and the concentration of the biological agent is 0.1-1 μg / mL.

2. The method of claim 1, wherein, The step of "adding a biological agent solution to a streptavidin-coated MSD plate, and fixing the biological agent in the MSD plate through the action of biotin and streptomycin" specifically comprises the following steps: adding the biological agent solution to the SA-MSD plate, sealing the plate with a sealing film, and incubating at 20-30 ℃ for 60-66 minutes, and the shaking speed of a plate shaker is 500-1000 rpm.

3. The method of claim 1, wherein, The SA-MSD plate is blocked with bovine serum albumin before use, and then washed with a washing buffer.

4. The method of claim 3, wherein, The washing buffer is a PBS buffer containing a surfactant, and the surfactant is Tween 20, and the content of the Tween 20 is 0.01-0.1% (v / v).

5. The method of claim 1, wherein, The specific method of diluting the sample to be tested comprises the following steps: completely melting and vortexing the sample to be tested, adding the sample to a dilution plate, sealing the plate with a sealing film, and incubating at 20-30 ℃ for 10-30 minutes, and the shaking speed of a plate shaker is 500-1000 rpm.

6. The method of claim 1, wherein, The sample to be tested is a blood sample of a subject, and more specifically, a serum sample of a subject.

7. The method of claim 1, wherein, The specific steps of adding the diluted sample to be tested to the SA-MSD plate comprise the following steps: transferring the diluted sample to be tested from the dilution plate to the SA-MSD plate, sealing the plate with a sealing film, and incubating at 20-30 ℃ for 60-66 minutes, and the shaking speed of a plate shaker is 500-1000 rpm. The minimum dilution multiple of the sample to be tested is 5 times.

8. The method of claim 1, wherein, The specific method of the step of "adding Sulfo-Tag-labeled PEG-EPO to the plate to combine with the antibody" comprises the following steps: transferring the Sulfo-Tag-labeled PEG-EPO to the SA-MSD plate, incubating at 20-30 ℃ for 60-70 minutes, and the shaking speed of a plate shaker is 500-1000 rpm. The excitation light wavelength of the Sulfo-Tag for exciting electrochemical light is 620 nm.

9. The method of claim 1, wherein, The method further comprises a positive quality control sample and a negative quality control sample, wherein the positive quality control sample is a high, medium and low concentration monoclonal mouse anti-erythropoietin sample, and the concentrations of the three samples are 5000 ng / mL, 2500 ng / mL and 32.91 ng / mL, respectively.

10. The use of the detection method according to any one of claims 1-9 in the evaluation of a PEG-EPO drug. The evaluation of the PEG-EPO drug is specifically the evaluation of the immunogenicity of PEG-EPO.

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