Anti-drug antibody analysis method for detecting GLP-1 in serum based on ELISA (Enzyme-Linked Immunosorbent Assay)

Abstract

The invention discloses an analysis method for detecting a GLP-1 drug-resistant antibody in serum based on ELISA (Enzyme-Linked Immunosorbent Assay), which is used for analyzing and verifying a screening critical value, a confirmation critical value, a titer critical value, sensitivity, drug tolerance, a hook effect and the like. A screening critical value, a confirmation critical value and a titer critical value are determined reasonably, the precision is high, the sensitivity is high, the drug tolerance is proper, the hook effect is avoided under high concentration, the selectivity is high, and the method is suitable for analyzing the anti-drug antibody of the GLP-1 in the serum.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme-linked immunosorbent assay (ELISA), and in particular to an anti-drug antibody analysis method for detecting GLP-1 in serum based on ELISA. Background Art

[0002] GLP-1 (insulin and glucagon-like polypeptide 1), a key factor in the regulation of glucose and lipid metabolism, has become the most important drug for the treatment of diabetes.

[0003] Based on the extensive research results on the treatment of diseases with mesenchymal stem cells, the applicant innovatively introduced GLP-1 into autologous adipose mesenchymal stem cells to obtain a cell therapy product for diabetes.

[0004] Since GLP-1 is an immunogenic therapeutic protein, it is necessary to conduct safety evaluation and analysis of anti-drug antibodies for GLP-1 drugs.

[0005] Enzyme-linked immunosorbent assay (ELISE) is a method of binding soluble antigens or antibodies to a solid phase carrier and utilizing the specific binding of antigens and antibodies to carry out immune reaction for determination / measurement.

[0006] However, it is not certain whether ELISA is suitable for detecting and analyzing GLP-1 anti-drug antibodies in serum. Moreover, when ELISA is used to detect and analyze anti-drug antibodies in serum, how to determine the analysis process also needs to be actively explored. Summary of the invention

[0007] In order to solve the above problems, the inventors tried to use enzyme-linked immunosorbent assay (ELISA) to detect anti-drug antibodies of GLP-1 in serum, analyzed the results, obtained the screening critical value and confirmation critical value, and obtained the sensitivity, drug tolerance and hook effect, etc. The screening and confirmation were accurate, the sensitivity was high, and the drug tolerance concentration was appropriate, which is suitable for analyzing GLP-1 anti-drug antibodies in serum.

[0008] The object of the present invention is to provide an analytical method for detecting GLP-1 anti-drug antibodies in serum based on ELISA, comprising the following steps:

[0009] (1) Obtaining the screening critical value SCP Floating ,

[0010] SCP Floating =MCF×Mean (NC) ,

[0011] Among them, the estimated multiplication correction factor MCF = Median (S / N) +1.645×1.4826×MAD (S / N)

[0012] Median (S / N) is the median value of the S / N values ​​of all valid blank individual matrices,

[0013] MAD (S / N) is the median absolute deviation of the S / N values ​​of all valid individual matrices,

[0014] Mean (NC) It is the mean S / N value of all valid blank individual matrices, that is, the mean of the duplicate wells of all valid blank individual matrices (S) divided by the mean signal value of the negative control (NC) of the corresponding batch (N).

[0015] The blank individual matrix refers to individual serum that has not been treated with GLP-1.

[0016] The effective blank individual matrix refers to the blank individual serum remaining after excluding individuals with CV%>25.0%, excluding analytical outliers, and excluding biological outliers.

[0017] The negative control is a blank mixed serum, which refers to a mixture of individual sera that has not been treated with GLP-1.

[0018] (2) Obtaining the confirmation critical value CCP Floating ,

[0019] CCP Floating =Median (SIR％) +2.326×1.4826×MAD (SIR％)

[0020] Among them, Median (SIR％) is the median value of the SIR% ​​values ​​of all valid blank individual matrices,

[0021] SIR%=(1-S 加GLP-1后个体复孔均值 / S 未加GLP-1个体复孔均值 )×100%

[0022] MAD (SIR％) is the median absolute deviation of the SIR% ​​values ​​of all valid blank individual matrices,

[0023] The effective blank individual matrix refers to the individual serum remaining after excluding individuals with CV%>25.0%, excluding analytical outliers, and excluding biological outliers.

[0024] S 加GLP-1后个体复孔均值 is the mean signal value of the blank individual matrix duplicate wells with GLP-1 added,

[0025] S 未加GLP-1个体复孔均值 It is the mean signal value of the blank individual matrix duplicate wells without GLP-1.

[0026] In a preferred embodiment, the estimated multiplicative correction factor MCF is 1.0626.

[0027] In a preferred embodiment, the confirmation cut-off CCP is 31.0%.

[0028] In a preferred embodiment, the detection based on ELISA comprises the following process:

[0029] (1-1-1) Coating: dilute the GLP-1 polypeptide with PBS solution to prepare a coating solution;

[0030] (1-1-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid;

[0031] (1-2-1) Blocking: Add Casein in TBS solution to each well of the multi-well plate, seal the plate with a sealing film, and place it in a constant temperature incubator for incubation;

[0032] (1-2-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid;

[0033] (1-3-1) Sample preparation: Mix the test substance and Casein in TBS solution (or Casein in TBS solution containing GLP-1) and add them to the low adsorption plate. Seal the plate and place it on an ELISA plate shaker and incubate at room temperature.

[0034] (1-4-1) Sample addition: Add 100 μL / well of the diluted sample to the blocked multi-well plate, seal the plate and place it on an ELISA plate shaker and incubate at room temperature;

[0035] (1-4-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid;

[0036] (1-5-1) Connecting: Prepare Streptavidin-HRP (streptavidin-horseradish peroxidase) as a connecting agent, add the connecting agent to each well of the multi-well plate, seal the plate with a sealing film, and incubate it statically;

[0037] (1-5-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid;

[0038] (1-6-1) Color development: Add color developing solution to each well of the multi-well plate and let it stand at room temperature away from light for color development;

[0039] (1-7-1) Termination: Add stop solution to each well of the multi-well plate to terminate the reaction;

[0040] (1-8-1) Reading: After shaking using a microplate reader, read the sample at a wavelength of 450-560 nm.

[0041] In a further preferred embodiment, the detection based on ELISA comprises the following process:

[0042] (1-1-1) Coating: Dilute the GLP-1 peptide to a concentration of 2.000 μg / mL with PBS solution to prepare the coating solution, which is ready for use. Add 100 μL of 2.000 μg / mL coating solution to each well of the 96-well plate, seal the plate with a sealing film, and place it in a refrigerator (2-8°C) for incubation overnight (14-18 hours);

[0043] (1-1-2) Washing the plate: shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0044] (1-2-1) Blocking: Add 285 μL of Casein in TBS solution to each well, seal the plate with sealing film, and incubate in a constant temperature incubator at 25±1℃ for 3 to 3.5 h;

[0045] (1-2-2) Washing the plate: shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0046] (1-3-1) Sample preparation: Mix the test substance and Casein in TBS solution (or Casein in TBS solution containing GLP-1) at a 50-fold (3 μL + 147 μL) concentration and add to the low adsorption plate. Seal the plate and place it on an ELISA plate shaker at room temperature at 600 RPM for 30 ± 5 min.

[0047] (1-4-1) Sample addition: Add 100 μL / well of the diluted sample to the blocked 96-well plate, seal the plate and place it on an ELISA plate shaker, incubate at room temperature at 600 RPM for 1 h±5 min;

[0048] (1-4-2) Washing the plate: shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0049] (1-5-1) Connecting: Dilute Streptavidin-HRP with Casein in TBS solution 200 times to prepare the connecting agent. Add 100 μL of the connecting agent to each well of the plate, seal the plate with a sealing film, and incubate at 25±1℃ for 30±3 min.

[0050] (1-5-2) Washing: Shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0051] (1-6-1) Color development: Add 100 μL of TMB single-component color development solution to each well of the plate and incubate at room temperature in the dark for 15 ± 3 min;

[0052] (1-7-1) Termination: Add 100 μL of stop solution to each well to terminate the reaction;

[0053] (1-8-1) Reading: After shaking for 5 seconds, read the sample at a wavelength of 450-560 nm using an ELISA reader.

[0054] In the present invention, the sample to be tested is a blank individual matrix, a negative control sample (NC) or a positive control sample (PC).

[0055] In particular, the positive control sample / antibody is an anti-drug antibody produced in the serum of an individual animal treated with GLP1 polypeptide, that is, an anti-drug antibody produced by administering GLP-1 polypeptide protein to an individual animal, such as a polyclonal rabbit anti-drug antibody.

[0056] In a preferred embodiment, the detection reagent is prepared by combining biotinylated Protein G (Bio-Protein G) with a Blocker TM Casein in TBS solution was diluted 10,000 times. Bio-Protein G was prepared using EZ-Link TM Sulfo-NHS-LC-Biotin, No weight TM The Recombinant Protein G solution is obtained by labeling the Recombinant Protein G with Format reagent. The Recombinant Protein G solution is obtained by dissolving the Recombinant Protein G and 5 mL of sterile water for injection at a ratio of 1 mg:1 mL.

[0057] In the present invention, when screening or titer testing is performed, in (1-3-1) sample preparation, the test object is mixed with a Case in TBS solution; when confirmation is performed, in (1-3-1) sample preparation, the test object is mixed with a Case in TBS solution containing a GLP-1 polypeptide.

[0058] In a preferred embodiment, the analytical method for detecting GLP-1 anti-drug antibodies in serum based on ELISA further comprises the following steps:

[0059] (3) Obtaining the critical titer TCP Floating

[0060] TCP Floating =MCF Titration ×Mean (NC)

[0061] Among them, the titer multiplication correction factor MCF Titration =Mean (NC) +3.090×1.4826×MAD (S / N)

[0062] Mean (NC)It is the mean of the S / N values ​​of all valid blank individual matrices, that is, the mean of the duplicate wells of all valid blank individual matrices (S) divided by the mean signal value (N) of the negative control (NC) of the corresponding batch,

[0063] MAD (S / N) is the median absolute deviation of the S / N values ​​of all valid individual matrices.

[0064] In a preferred embodiment, the analytical method for detecting GLP-1 anti-drug antibodies in serum based on ELISA further comprises the following steps:

[0065] (4) Obtaining screening sensitivity and confirmation sensitivity: A series of concentrations of the positive control antibody were diluted with blank mixed serum as the positive control titer curve. GLP-1 was added and not added to the analysis for ELISA detection and verification. The sample concentration in the screening curve was used as the horizontal axis, and the average of the replicate wells of the test object was used as the vertical axis. The screening sensitivity and confirmation sensitivity of the positive control curve were obtained by fitting with four parameters.

[0066] In a more preferred embodiment, the screening sensitivity is less than 123.457 ng / mL.

[0067] In a preferred embodiment, the analytical method for detecting GLP-1 anti-drug antibodies in serum based on ELISA further comprises the following steps:

[0068] (5) Obtaining drug tolerance: GLP-1 was diluted with blank mixed serum to a series of concentrations, and then mixed with a positive control antibody diluted with blank mixed serum in a 1:1 ratio, and then tested and verified by ELISA.

[0069] In a further preferred embodiment, 1000.000 ng / mL of anti-drug antibodies tolerate 5555.556 ng / mL of GLP-1 polypeptide, 500.000 ng / mL of anti-drug antibodies tolerate 1851.852 ng / mL of GLP-1 polypeptide, and 250.000 ng / mL of anti-drug antibodies tolerate 617.284 ng / mL of GLP-1 polypeptide.

[0070] In a preferred embodiment, the analytical method for detecting GLP-1 anti-drug antibodies in serum based on ELISA further comprises the following steps:

[0071] (6) Observation of hook effect: The positive control antibody was diluted with blank mixed serum to a series of concentrations and tested by ELISA. Each concentration point was measured in duplicate and the signal value was averaged to observe whether the hook effect appeared at each concentration point.

[0072] In a preferred embodiment, the method of the present invention will not produce a hook effect that affects detection within 100000.000 ng / mL.

[0073] The analytical method for detecting GLP-1 anti-drug antibodies in serum based on ELISA provided by the present invention analyzes and verifies the screening critical value, confirmation critical value and titer critical value, sensitivity and drug tolerance, hook effect, etc., and finds that the screening critical value, confirmation critical value and titer critical value are reasonably determined, have high precision, high sensitivity, appropriate drug tolerance, no hook effect at high concentrations, high selectivity, and is suitable for the analysis of GLP-1 anti-drug antibodies in serum. DETAILED DESCRIPTION

[0074] The present invention is further described in detail below through preferred embodiments. Through these descriptions, the characteristics and advantages of the present invention will become clearer and more specific.

[0075] 1. The reagents and instruments used are as follows:

[0076]

[0077] 2. Preparation of reagents

[0078] 1. Preparation of GLP-1 solution: Take 1 vial of GLP-1, add 1 mL of ultrapure water to dissolve, shake manually to obtain a GLP-1 solution with a concentration of 5 mg / mL, and store in an ultra-low temperature refrigerator (-70 to -90°C).

[0079] 2. Preparation of coating solution: Dilute the GLP-1 solution with PBS solution to a concentration of 2.000 μg / mL.

[0080] 3. Preparation of Recombinant Protein G solution: Take one Recombinant Protein G (5 mg / vial), add 5 mL of sterile water for injection to dissolve, shake manually to obtain a Recombinant Protein G solution with a concentration of 1 mg / mL, and store in an ultra-low temperature refrigerator (-70 to -90°C).

[0081] 4. Preparation of Bio-Protein G: Using EZ-Link TM Sulfo-NHS-LC-Biotin, No-WeightTMFormat reagent labeled Recombinant Protein G solution, stored in ultra-low temperature refrigerator (-70~-90℃) away from light.

[0082] 5. Preparation of detection reagents: using Blocker TMDilute Bio-Protein G 100,000-fold by adding TBS.

[0083] 6. Preparation of blank mixed serum (negative control sample): Mix at least 10 blank individual sera and freeze them in an ultra-low temperature refrigerator.

[0084] 7. Preparation of washing solution (0.05% PBST): Measure 50 mL of 20×PBS and add it to 950 mL of ultrapure water or distilled drinking water, then add 0.5 mL of Tween 20 to prepare a washing solution containing about 0.05% (v / v) Tween 20.

[0085] 8. Preparation of connecting agent: use Blocker TM Dilute Streptavidin-HRP 200-fold in TBS and mix well.

[0086] 9. Preparation of stop solution (2M H2SO4): Slowly add 110 mL of sulfuric acid into 900 mL of ultrapure water, stirring while adding to mix well.

[0087] 10. Preparation of positive control samples:

[0088]

[0089] 11. Preparation of BlockerTM Casein in TBS (confirmation dilution solution) containing GLP-1: Dissolve 150 μL of GLP-1 solution in 7350 μL Blocker TM Dilute Casein in TBS and mix well.

[0090] 12. Preparation of positive control samples containing GLP-1: Mix the samples in 10 with the BlockerTM Casein in TBS (confirmation dilution solution) containing GLP-1 in 11 at a ratio of 50 times (5 μL+245 μL).

[0091] 3. ELISA test process

[0092] (1-1-1) Coating: Dilute the GLP-1 peptide to a concentration of 2.000 μg / mL with PBS solution to prepare the coating solution, which is ready for use. Add 100 μL of 2.000 μg / mL coating solution to each well of the 96-well plate, seal the plate with a sealing film, and place it in a refrigerator (2-8°C) for incubation overnight (14-18 hours);

[0093] (1-1-2) Washing the plate: shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0094] (1-2-1) Blocking: Add 285 μL of Casein in TBS solution to each well, seal the plate with sealing film, and incubate in a constant temperature incubator at 25±1℃ for 3 to 3.5 h;

[0095] (1-2-2) Washing the plate: shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0096] (1-3-1) Sample preparation: Mix the test substance and Casein in TBS solution (or Casein in TBS solution containing GLP-1) at a 50-fold (3 μL + 147 μL) concentration and add to the low adsorption plate. Seal the plate and place it on an ELISA plate shaker at room temperature at 600 RPM for 30 ± 5 min.

[0097] (1-4-1) Sample addition: Add 100 μL / well of the diluted sample to the blocked 96-well plate, seal the plate and place it on an ELISA plate shaker, incubate at room temperature at 600 RPM for 1 h±5 min;

[0098] (1-4-2) Washing the plate: shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0099] (1-5-1) Connecting: Dilute Streptavidin-HRP with Casein in TBS solution 200 times to prepare the connecting agent. Add 100 μL of the connecting agent to each well of the plate, seal the plate with a sealing film, and incubate at 25±1℃ for 30±3 min.

[0100] (1-5-2) Washing: Shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0101] (1-6-1) Color development: Add 100 μL of TMB single-component color development solution to each well of the plate and incubate at room temperature in the dark for 15 ± 3 min;

[0102] (1-7-1) Termination: Add 100 μL of stop solution to each well to terminate the reaction;

[0103] (1-8-1) Reading: After shaking for 5 seconds, read the sample at a wavelength of 450-560 nm using an ELISA reader.

[0104] 4. Screening critical value and confirmation critical value

[0105] Forty blank individual sera were selected and the ELISA test process was followed. The multiplication correction factor (MCF) was calculated by dividing the mean value (S) of the duplicate wells of the blank individual matrix by the mean value (N) of the signal value of the negative control (NC) of the corresponding batch, that is, S / N.

[0106] First, individuals with CV% greater than 25.0% were not included in the subsequent outlier statistics.

[0107] Secondly, the outliers were eliminated and the S / N of each individual in each analysis batch was subjected to Tukey test. The S / N greater than Q3+1.5×(Q3-Q1) or less than Q1-1.5×(Q3-Q1) was considered as an outlier, where Q3=75. th Percentile, Q1=25 th Percentiles, samples with analytical outliers should be removed from the corresponding analytical batch.

[0108] Finally, biological outliers were eliminated and the S / N of the same individual in each analytical batch (after eliminating analytical outliers) was averaged. After Tukey test, the S / N greater than Q3+1.5×(Q3-Q1) or less than Q1-1.5×(Q3-Q1) was a biological outlier, where Q3=75. th Percentile, Q1=25 th Percentiles, biological outlier samples should be removed from each analytical batch.

[0109] The results are shown in Table 1 below:

[0110]

[0111]

[0112] Note: “*”-outliers, not involved in statistical calculations.

[0113] 5. Drug tolerance

[0114] GLP-1 was diluted with blank mixed serum to the following concentration series: 200000.000ng / mL, 100000.000ng / mL, 20000.000ng / mL, 6666.666ng / mL, 2222.222ng / mL, 740.740ng / mL, 246.914ng / mL, 82.316ng / mL, 27.434ng / mL, 9.144ng / mL, and then mixed with different concentrations of positive control antibodies (2000.000ng / mL, 1000.000ng / mL, 500.000ng / mL) diluted with blank mixed serum in a 1:1 ratio and then tested. The data are shown in the table below:

[0115]

[0116] Note: Bold represents the highest drug concentration greater than the floating screening cut-off value for this analytical batch.

[0117] Results: 1000.000ng / mL of anti-drug antibodies tolerated 5555.556ng / mL of GLP-1 peptide, 500.000ng / mL of anti-drug antibodies tolerated 1851.852ng / mL of GLP-1 peptide, and 250.000ng / mL of anti-drug antibodies tolerated 617.284ng / mL of GLP-1 peptide.

[0118] 6. Hook Effect

[0119] Use blank mixed serum to dilute the positive control antibody to a series of concentrations for testing. Repeat the test at each concentration point. Investigate the signal values ​​at the above concentration points and take the average signal value as the result. The average signal value of the concentration point higher than HPC should be higher than the average signal value of HPC, and the signal value does not decrease significantly with the increase of concentration, indicating that there is no hook effect that affects the detection within the verified concentration range.

[0120] The present invention is described in detail above in combination with preferred embodiments and exemplary examples. However, it should be noted that these specific embodiments are only illustrative explanations of the present invention and do not constitute any limitation on the protection scope of the present invention. Without exceeding the spirit and protection scope of the present invention, various improvements, equivalent substitutions or modifications may be made to the technical content of the present invention and its embodiments, which all fall within the protection scope of the present invention. The protection scope of the present invention shall be subject to the attached claims.

Claims

1. An object of the present invention is to provide an analytical method for detecting GLP-1 anti-drug antibodies in serum based on ELISA, comprising the following steps: (1) Obtaining the screening critical value SCP Floating , SCP Floating =MCF×Mean (NC) , Among them, the estimated multiplication correction factor MCF = Median (S / N) +1.645×1.4826×MAD (S / N) , Median (S / N) is the median value of the S / N values ​​of all valid blank individual matrices, MAD (S / N) is the median absolute deviation of the S / N values ​​of all valid individual matrices, Mean (NC) It is the mean S / N value of all valid blank individual matrices, that is, the mean of the duplicate wells of all valid blank individual matrices (S) divided by the mean signal value (N) of the negative control (NC) of the corresponding batch. The blank individual matrix refers to individual serum that has not been treated with GLP-1. The effective blank individual matrix refers to the blank individual serum remaining after excluding individuals with CV%>25.0%, excluding analytical outliers, and excluding biological outliers. The negative control is a blank mixed serum, which refers to a mixture of individual sera that has not been treated with GLP-1. (2) Obtaining the confirmation critical value CCP Floating , CCP Floating =Median (SIR％) +2.326×1.4826×MAD (SIR％) , Among them, Median (SIR％) is the median value of the SIR% ​​values ​​of all valid blank individual matrices, SIR%=(1-S 加GLP-1后个体复孔均值 / S 未加GLP-1个体复孔均值 )×100%, MAD (SIR％) is the median absolute deviation of the SIR% ​​values ​​of all valid blank individual matrices, The effective blank individual matrix refers to the individual serum remaining after excluding individuals with CV%>25.0%, excluding analytical outliers, and excluding biological outliers. S 加GLP-1后个体复孔均值 is the mean signal value of the blank individual matrix duplicate wells with GLP-1 added, S 未加GLP-1个体复孔均值 It is the mean signal value of the blank individual matrix duplicate wells without GLP-1.

2. The method of claim 1, wherein: The estimated multiplicative correction factor MCF is 1.0626.

3. The method of claim 1, wherein: The confirmation critical value CCP was 31.0%.

4. The method of claim 1, wherein: ELISA-based testing involves the following steps: (1-1-1) Coating: dilute the GLP-1 polypeptide with PBS solution to prepare a coating solution; (1-1-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid; (1-2-1) Blocking: Add Caseinin TBS solution to each well of the multi-well plate, seal the plate with a sealing film, and place it in a constant temperature incubator for incubation; (1-2-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid; (1-3-1) Sample preparation: Mix the test substance and Caseinin TBS solution (or Caseinin TBS solution containing GLP-1) and add them to the low adsorption plate. Seal the plate and place it on an ELISA plate shaker and incubate at room temperature. (1-4-1) Sample addition: Add 100 μL / well of the diluted sample to the blocked multi-well plate, seal the plate and place it on an ELISA plate shaker and incubate at room temperature; (1-4-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid; (1-5-1) Connecting: Prepare Streptavidin-HRP (streptavidin-horseradish peroxidase) as a connecting agent, add the connecting agent to each well of the multi-well plate, seal the plate with a sealing film, and incubate it statically; (1-5-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid; (1-6-1) Color development: Add color developing solution to each well of the multi-well plate and let it stand at room temperature away from light for color development; (1-7-1) Termination: Add stop solution to each well of the multi-well plate to terminate the reaction; (1-8-1) Reading: After shaking using a microplate reader, read the sample at a wavelength of 450-560 nm.

5. The method of claim 4, wherein: ELISA-based testing involves the following steps: (1-1-1) Coating: Dilute the GLP-1 polypeptide to a concentration of 2.000 μg / mL with PBS solution to prepare a coating solution. Add 100 μL of 2.000 μg / mL coating solution to each well of a 96-well plate, seal the plate with a sealing film, and place in a refrigerator (2-8°C) for incubation overnight (14-18 h); (1-1-2) Washing: Shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper; (1-2-1) Blocking: Add 285 μL of Caseinin TBS solution to each well, seal the plate with sealing film, and incubate in a constant temperature incubator at 25±1℃ for 3 to 3.5 h; (1-2-2) Washing the plate: shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper; (1-3-1) Sample preparation: Mix the test substance and Caseinin TBS solution (or Caseinin TBS solution containing GLP-1) at a 50-fold (3 μL + 147 μL) concentration and add to the low adsorption plate. Seal the plate and place it on an ELISA plate shaker at room temperature at 600 RPM for 30 ± 5 min. (1-4-1) Sample addition: Add 100 μL / well of the diluted sample to the blocked 96-well plate, seal the plate and place it on an ELISA plate shaker, incubate at room temperature at 600 RPM for 1 h±5 min; (1-4-2) Washing: Shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper; (1-5-1) Connecting: Dilute Streptavidin-HRP with Caseinin TBS solution 200 times to prepare the connecting agent. Add 100 μL of the connecting agent to each well of the plate, seal the plate with a sealing film, and incubate at 25±1℃ for 30±3 min. (1-5-2) Washing: Shake off the liquid in the plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper; (1-6-1) Color development: Add 100 μL of TMB single-component color development solution to each well of the plate and incubate at room temperature in the dark for 15 ± 3 min; (1-7-1) Termination: Add 100 μL of stop solution to each well to terminate the reaction; (1-8-1) Reading: After shaking for 5 seconds, read the sample at a wavelength of 450-560 nm using a microplate reader.

6. The method of claim 1, further comprising the steps of: (3) Obtaining the critical titer TCP Floating , TCP Floating =MCF Titration ×Mean (NC) , Among them, the titer multiplication correction factor MCF Titration =Mean (NC) +3.090×1.4826×MAD (S / N) , Mean (NC) It is the mean of the S / N values ​​of all valid blank individual matrices, that is, the mean of the duplicate wells of all valid blank individual matrices (S) divided by the mean signal value (N) of the negative control (NC) of the corresponding batch, MAD (S / N) is the median absolute deviation of the S / N values ​​of all valid individual matrices.

7. The method of claim 1, further comprising the steps of: (4) Obtaining screening sensitivity and confirmation sensitivity: A series of concentrations of the positive control antibody were diluted with blank mixed serum as the positive control titer curve. GLP-1 was added and not added to the analysis for ELISA detection and verification. The sample concentration in the screening curve was used as the horizontal axis, and the average of the replicate wells of the test object was used as the vertical axis. The screening sensitivity and confirmation sensitivity of the positive control curve were obtained by fitting with four parameters.

8. The method of claim 1, wherein: The screening sensitivity was lower than 123.457 ng / mL.

9. The method of claim 1, further comprising the steps of: (5) Obtaining drug tolerance: GLP-1 was diluted with blank mixed serum to a series of concentrations, and then mixed with a positive control antibody diluted with blank mixed serum in a 1:1 ratio, and then tested and verified by ELISA.

10. The method of claim 1, further comprising the steps of: (6) Observation of hook effect: The positive control antibody was diluted with blank mixed serum to a series of concentrations and tested by ELISA. Each concentration point was measured in duplicate and the signal value was averaged to observe whether the hook effect appeared at each concentration point.

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