Analysis method for detecting FGF21 anti-drug antibody in serum based on ELISA

Abstract

The invention discloses an analysis method for detecting an FGF21 anti-drug antibody in serum based on ELISA (Enzyme-Linked Immunosorbent Assay). The analysis method comprises the following steps: (1) carrying out detection through the ELISA; (2) a screening critical value SCPFloating is obtained, the SCPFloating is equal to MCF * Mean (NC), and an estimated multiplication correction factor MCF is equal to Median (S / N) + 1.645 * 1.4826 * MAD (S / N); and (3) a confirmation critical value CCPFloating is obtained, wherein CCPFloating is equal to Median (SIR%) + 2.326 * 1.4826 * MAD (SIR%), and CCPFloating is equal to Median (SIR%) + 2.326 * 1.4826 * MAD (SIR%). The method has the advantages of simple operation, accurate screening and confirmation, high sensitivity and appropriate drug tolerance concentration, and is suitable for analyzing the FGF21 anti-drug antibody in serum.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme-linked immunosorbent assay (ELISA), and in particular to an analysis method for detecting FGF21 anti-drug antibodies in serum based on enzyme-linked immunosorbent assay (ELISA). Background Art

[0002] Fibroblast growth factor FGF is an important growth factor, among which FGF21 is an important member, with hormone-like effects, playing an important role in regulating glucose and lipid metabolism. FGF21 is mainly synthesized by the liver, acts on adipose tissue through endocrine pathways, and regulates glucose and lipid metabolism. As a glucose and lipid metabolism regulatory molecule, FGF21 has been developed as a drug for the treatment of metabolic diseases.

[0003] Based on the extensive research results on the treatment of diseases with mesenchymal stem cells, the applicant innovatively introduced FGF21 into autologous adipose mesenchymal stem cells to obtain a cell therapy product for diabetes.

[0004] Since FGF21 is immunogenic as a therapeutic protein, anti-drug antibody safety evaluation and analysis are required for drugs using FGF21.

[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 FGF21 anti-drug antibodies in serum. Moreover, when ELISA is used to detect and analyze FGF21 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 FGF21 antidrug antibodies 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 FGF21 antidrug antibodies in serum.

[0008] The object of the present invention is to provide an analytical method for detecting FGF21 anti-drug antibodies in serum based on ELISA, comprising the following steps:

[0009] (1) Detection by ELISA,

[0010] (2) Obtaining the screening critical value SCP Floating ,

[0011] SCPFloating =MCF×Mean (NC) ,

[0012] Among them, the estimated multiplication correction factor MCF = Median (S / N) +1.645×1.4826×MAD (S / N)

[0013] Median (S / N) is the median value of the S / N values ​​of all valid blank individual matrices,

[0014] MAD (S / N) is the median absolute deviation of the S / N values ​​of all valid individual matrices,

[0015] Mean (NC) is the S / N value of all valid blank individual matrices, that is, the mean value of the duplicate wells of the valid blank individual matrix (S) divided by the mean value of the signal value of the negative control (NC) of the corresponding batch (N),

[0016] The blank individual matrix refers to individual serum that has not been treated with FGF21.

[0017] 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.

[0018] The negative control is a blank mixed serum, which refers to a mixture of individual sera that have not been treated with FGF21.

[0019] (3) Obtaining the confirmation critical value CCP Floating ,

[0020] CCP Floating =Median (SIR％) +2.326×1.4826×MAD (SIR％)

[0021] Among them, Median (SIR％) is the median value of the SIR% ​​values ​​of all valid blank individual matrices,

[0022] SIR%=(1-S 加FGF21后个体复孔均值 / S 未加FGF21个体复孔均值 )×100%

[0023] MAD (SIR％) is the median absolute deviation of the SIR% ​​values ​​of all valid blank individual matrices,

[0024] 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.

[0025] S 加FGF21后个体复孔均值 is the mean signal value of the blank individual matrix duplicate wells with FGF21 added,

[0026] S 未加FGF21个体复孔均值 It is the mean signal value of blank individual matrix duplicate wells without FGF21.

[0027] In a preferred embodiment, detection by ELISA comprises the following process:

[0028] (1-1-1) Coating: Dilute FGF21 with PBS solution to prepare coating solution, add the coating solution to each well of the multi-well plate, seal the plate with a sealing film, and place in a refrigerator for incubation overnight;

[0029] (1-1-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid;

[0030] (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 incubate in a constant temperature incubator.

[0031] (1-2-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid;

[0032] (1-3-1) Acidification: Mix the sample to be tested with CH3COOH solution and add it to a low adsorption plate. Seal the plate and place it on an ELISA plate shaker for acidification at room temperature.

[0033] (1-4-1) Neutralization: Add Tris solution, acidified sample and detection reagent or detection reagent containing FGF21 to the blocked multi-well plate, seal the plate and place it on an ELISA plate shaker for incubation at room temperature;

[0034] (1-4-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid;

[0035] (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;

[0036] (1-5-2) Washing the plate: shake off the liquid in the porous plate and wash the plate with washing liquid;

[0037] (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;

[0038] (1-7-1) Termination: Add stop solution to each well of the multi-well plate to terminate the reaction;

[0039] (1-8-1) Reading: After shaking using a microplate reader, read the sample at a wavelength of 450-560 nm.

[0040] In a further preferred embodiment, detection by ELISA comprises the following process:

[0041] (1-1-1) Coating: Dilute FGF21 to a concentration of 10.000 μg / mL with PBS solution to prepare a coating solution. Add the coating solution to each well of a multi-well plate, seal the plate with a sealing film, and place in a refrigerator (2-8°C) for incubation overnight (14-18 h);

[0042] (1-1-2) Washing: Shake off the liquid in the multi-well plate, add 300 μL of washing solution to each well, wash the plate three times, and pat dry on paper;

[0043] (1-2-1) Blocking: Add 285 μL of Casein in TBS solution to each well of the multi-well plate, seal the plate with a sealing film, and incubate in a constant temperature incubator at 25±1℃ for 3 to 3.5 hours.

[0044] (1-2-2) Washing: Shake off the liquid in the multi-well plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0045] (1-3-1) Acidification: Mix the sample to be tested with 300 mM CH3COOH solution at a volume ratio of 1:9 and add it to a low adsorption plate. Seal the plate and place it on an ELISA plate shaker for acidification at room temperature and 600 RPM for 30 ± 3 min.

[0046] (1-4-1) Neutralization: Add 15 μL / well of 1M Tris solution, 40 μL / well of acidified sample, and 60 μL / well of detection reagent or detection reagent containing FGF21 to the blocked multi-well plate, seal the plate and place on an ELISA plate shaker, incubate at room temperature at 600 RPM for 2 h±3 min;

[0047] (1-4-2) Washing: Shake off the liquid in the multi-well plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0048] (1-5-1) Connecting: dilute Streptavidin-HRP (streptavidin-horseradish peroxidase) 200 times with 1% BSA-PBS solution to prepare a connecting agent. Add 100 μL of the connecting agent to each well of the multi-well plate, seal the plate with a sealing film, and incubate at 25±1°C for 30±3 min.

[0049] (1-5-2) Washing: Shake off the liquid in the multi-well plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0050] (1-6-1) Color development: Add 100 μL of TMB single-component color development solution to each well of the multi-well plate and incubate at room temperature in the dark for 20 ± 3 min;

[0051] (1-7-1) Termination: Add 100 μL of stop solution to each well of the multi-well plate to terminate the reaction;

[0052] (1-8-1) Reading: After shaking for 5 seconds, read the sample at a wavelength of 450-560 nm using an ELISA reader.

[0053] In a preferred embodiment, the sample to be tested is a blank individual matrix, a negative control sample (NC) or a positive control sample (PC).

[0054] Particularly, the positive control sample / antibody is the anti-drug antibody produced in the serum of individual animals treated with FGF211, i.e., the anti-drug antibody produced by administering FGF21 protein to individual animals, such as rabbit anti-drug antibody, which is used at a concentration of, for example, 100,000 ng / mL.

[0055] In another preferred embodiment, the positive control sample is also tested at a high concentration such as 10000 ng / mL, a medium concentration such as 5000 ng / mL and a low concentration such as 500 ng / mL to analyze the sensitivity and drug tolerance of the method.

[0056] In a more preferred embodiment, the detection reagent is prepared by diluting biotin-labeled Protein G (Bio-Protein G) by 3001 times with Casein in TBS solution. Bio-Protein G is prepared by using EZ-Link TM Sulfo-NHS-LC-Biotin, No weight TM The recombinant protein G solution was obtained by labeling with Format reagent (Thermo Fisher Scientific Inc).

[0057] In a more preferred embodiment, when screening or titer analysis is performed, in the neutralization step (1-4-1), 15 μL / well of 1M Tris solution, 40 μL / well of acidified sample and 60 μL / well of detection reagent are added to the blocked multi-well plate.

[0058] In another more preferred embodiment, when confirmation is performed, in the neutralization of (1-4-1), 15 μL / well of 1M Tris solution, 40 μL / well of acidified sample and 60 μL / well of FGF21-containing detection reagent are added to the blocked multi-well plate, wherein the FGF21-containing detection reagent is obtained by diluting FGF21 to 200.000 μg / mL with the detection reagent.

[0059] In a preferred embodiment, the estimated multiplication correction factor MCF is 1.1464-1.2838, more preferably 1.1464 or 1.2838.

[0060] In a preferred embodiment, the confirmation critical value CCP is 31.5%-48.4%, preferably 31.5% or 48.4%.

[0061] In a preferred embodiment, the analytical method for detecting FGF21 anti-drug antibodies in serum based on ELISA further comprises the following steps:

[0062] (4) Obtaining the critical titer TCP Floating

[0063] TCP Floating =MCF Titration ×Mean (NC)

[0064] Among them, the titer multiplication correction factor MCF Titration =Mean (NC) +3.090×1.4826×MAD (S / N)

[0065] Mean (NC) is the S / N value of all valid blank individual matrices, that is, the mean value of the duplicate wells of the valid blank individual matrix (S) divided by the mean value of the signal value of the negative control (NC) of the corresponding batch (N),

[0066] MAD (S / N) is the median absolute deviation of the S / N values ​​of all valid individual matrices.

[0067] In a preferred embodiment, the titer threshold TCP Floating It is 1.5525.

[0068] In a preferred embodiment, the analytical method for detecting FGF21 anti-drug antibodies in serum based on ELISA further comprises the following steps:

[0069] (5) Obtaining screening sensitivity and confirmation sensitivity: The positive control antibody was diluted with blank mixed serum to the following series of concentrations as the positive control titer curve: 10000.000 ng / mL, 3333.333 ng / mL, 1111.111 ng / mL, 370.370 ng / mL, 123.457 ng / mL, 41.152 ng / mL, 13.717 ng / mL, 4.572 ng / mL, and FGF21 was added (FGF21 was diluted to 200.000 μg / mL with detection reagent) and without adding FGF21, respectively. ELISA was used for detection, with the sample concentration in the screening curve as the horizontal axis and the average of the replicate wells of the test object as the vertical axis. The screening sensitivity and confirmation sensitivity of the positive control curve were obtained by fitting with four parameters.

[0070] In a preferred embodiment, the screening sensitivity is ≤370.370 ng / mL, the confirmation sensitivity is ≤1111.111 ng / mL, and more preferably the confirmation sensitivity is ≤370.370 ng / mL.

[0071] In a preferred embodiment, the analytical method for detecting FGF21 anti-drug antibodies in serum based on ELISA further comprises the following steps:

[0072] (6) Obtaining drug tolerance: FGF21 was diluted with blank mixed serum to 350000.000 ng / mL, 116666.667 ng / mL, 38888.889 ng / mL, 12962.963 ng / mL, 4320.988 ng / mL, 1440.329 ng / mL, 480.110 ng / mL, and 0.000 ng / mL, and then mixed with positive control antibody (2000.000 ng / mL, 1000.000 ng / mL, and 500.000 ng / mL) diluted with blank mixed serum in a 1:1 ratio, and then detected and verified by ELISA.

[0073] In a preferred embodiment, 1000.000 ng / mL of anti-drug antibody (positive control antibody / sample) tolerates 55555.556 ng / mL FGF21, and 500.000 ng / mL and 250.000 ng / mL of anti-drug antibody tolerate 6172.840 ng / mL FGF21;

[0074] More preferably, 1000 ng / mL, 500 ng / mL and 250 ng / mL of anti-drug antibody (positive control antibody / sample) tolerate 175000 ng / mL FGF21.

[0075] In a preferred embodiment, the analytical method for detecting FGF21 anti-drug antibodies in serum based on ELISA further comprises the following steps:

[0076] (7) Observation of hook effect: The positive control antibody was diluted with blank mixed serum to a series of concentrations, such as 50000.000 ng / mL, 10000.000 ng / mL, 1000.000 ng / mL, and 500.000 ng / mL. The assay was performed by ELISA. Each concentration point was measured in duplicate. The signal value was averaged to observe whether the hook effect occurred at each concentration point.

[0077] In a preferred embodiment, the mean signal value at a concentration point of 50000.000 ng / mL higher than HPC is higher than the mean signal value of HPC, and the mean signal value does not decrease significantly with the increase of the positive control antibody concentration, indicating that this method will not produce a hook effect that affects detection within 50000.000 ng / mL.

[0078] The analytical method for detecting FGF21 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 are suitable for the analysis of FGF21 anti-drug antibodies in serum. DETAILED DESCRIPTION

[0079] 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.

[0080] 1. The reagents and instruments used are as follows:

[0081]

[0082]

[0083] 2. Preparation of reagents

[0084] 1. Preparation of coating solution: Dilute FGF21 protein to a concentration of 10.000 μg / mL with PBS solution.

[0085] Preparation of 2.1% BSA-PBS solution: Weigh 1 g of BSA powder, add it to 100 mL of PBS, mix evenly until completely dissolved, prepare a 1% (w / v) BSA-PBS solution, and store it in a refrigerator (2-8°C).

[0086] 3. Preparation of detection reagents: using Blocker TM Dilute Bio-Protein G 3001-fold in TBS.

[0087] 4. Preparation of detection reagent containing protein FGF21: dilute protein FGF21 to 200.000 μg / mL with detection reagent and mix well.

[0088] 5. Preparation of Bio-Protein G: Using EZ-Link TM Sulfo-NHS-LC-Biotin, No weight TM Format reagent (Thermo Fisher Scientific Inc) was used by Blocker TM Casein in TBS and dilute Bio-Protein G 3001 times to create the Recombinant Protein G solution.

[0089] Preparation of Recombinant Protein G solution: Take one Recombinant Protein G (5 mg / vial), add 5 mL of sterile water for injection to dissolve, and shake manually.

[0090] 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.

[0091] 7. Preparation of 300 mM acetic acid solution (300 mM CH3COOH solution): Measure 17 mL of glacial acetic acid, add it to 983 mL of ultrapure water, mix well, and obtain 300 mM acetic acid (CH3COOH) solution.

[0092] 8. Preparation of 1M Tris solution: Weigh 24.2g Tris, dissolve in 200mL ultrapure water, mix well until completely dissolved to obtain 1M Tris solution, and store at room temperature.

[0093] 9. 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.

[0094] 10. Preparation of linker: Dilute Streptavidin-HRP 200 times with 1% BSA-PBS solution and mix well.

[0095] 11. 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.

[0096] 12. Preparation of positive control samples:

[0097]

[0098] 3. ELISA test process

[0099] (1-1-1) Coating: Dilute FGF21 to a concentration of 10.000 μg / mL with PBS solution to prepare a coating solution. Add the coating solution to each well of a multi-well plate, seal the plate with a sealing film, and place in a refrigerator (2-8°C) for incubation overnight (14-18 h);

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

[0101] (1-2-1) Blocking: Add 285 μL of Casein in TBS solution to each well of the multi-well plate, seal the plate with a sealing film, and incubate in a constant temperature incubator at 25±1℃ for 3 to 3.5 hours.

[0102] (1-2-2) Washing: Shake off the liquid in the multi-well plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0103] (1-3-1) Acidification: Mix the sample to be tested with 300 mM CH3COOH solution at a volume ratio of 1:9 and add it to a low adsorption plate. Seal the plate and place it on an ELISA plate shaker for acidification at room temperature and 600 RPM for 30 ± 3 min.

[0104] (1-4-1) Neutralization: Add 15 μL / well of 1M Tris solution, 40 μL / well of acidified sample, and 60 μL / well of detection reagent or detection reagent containing FGF21 to the blocked multi-well plate, seal the plate and place on an ELISA plate shaker, incubate at room temperature at 600 RPM for 2 h±3 min;

[0105] (1-4-2) Washing: Shake off the liquid in the multi-well plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0106] (1-5-1) Connecting: dilute Streptavidin-HRP (streptavidin-horseradish peroxidase) 200 times with 1% BSA-PBS solution to prepare a connecting agent. Add 100 μL of the connecting agent to each well of the multi-well plate, seal the plate with a sealing film, and incubate at 25±1°C for 30±3 min.

[0107] (1-5-2) Washing: Shake off the liquid in the multi-well plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper;

[0108] (1-6-1) Color development: Add 100 μL of TMB single-component color development solution to each well of the multi-well plate and incubate at room temperature in the dark for 20 ± 3 min;

[0109] (1-7-1) Termination: Add 100 μL of stop solution to each well of the multi-well plate to terminate the reaction;

[0110] (1-8-1) Reading: After shaking for 5 seconds, read the sample at a wavelength of 450-560 nm using an ELISA reader.

[0111] 4. Screening critical value

[0112] 32 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.

[0113] The data is shown in Table 1 below:

[0114]

[0115]

[0116]

[0117] The process of obtaining the screening critical value is as follows:

[0118] (1) First, exclude individuals with CV%>25.0%. This experiment did not have;

[0119] (2) Eliminate analytical outliers: The S / N of each analytical batch is subjected to Tukey test. The S / N greater than Q3+1.5×(Q3-Q1) or less than Q1-1.5×(Q3-Q1) is the analytical outlier, where Q3=75. th Percentile, Q1=25 th Percentiles, samples with outliers should be removed from the corresponding analysis batch. None in this test.

[0120] (3) Elimination of biological outliers: The S / N of the same individual in each analytical batch (after eliminating analytical outliers) is averaged. After Tukey's test, the S / N greater than Q3+1.5×(Q3-Q1) or less than Q1-1.5×(Q3-Q1) is a biological outlier, where Q3=75. th Percentile, Q1=25 th Percentiles, samples with biological outliers should be removed from each analytical batch. None for this test.

[0121] (4) After eliminating individuals and outliers that did not meet the CV% requirement (the number of individuals remaining after every 32 blank individuals were eliminated was not less than 24), the data distribution was tested using SPSS software. The Shapiro-Wilk test for data distribution showed that the skewness coefficient of the data distribution was in the range of [-1, 1], and the P value was <0.05, which met the robust parameter model acceptance criteria. Finally, all 96 test samples were valid and participated in the calculation of MCF.

[0122] MCF=Median (S / N) +1.645×1.4826×MAD (S / N) =0.9780+1.645×1.4826×0.1254=1.2838 (95% percentile)

[0123] Calculation of SCP for each analytical batch during the assay phase Floating =MCF×Mean (NC)

[0124] 5. Confirmation Critical Value

[0125] 32 blank individual sera were selected, and FGF21 (diluted to 200.000 μg / mL with the detection reagent solution) and no FGF21 were added, and the ELISA test process was followed.

[0126] The data is shown in Table 2 below:

[0127]

[0128]

[0129]

[0130] Notes: (1) The individual pre-drug results of each analytical batch are shown in Table 1 above.

[0131] (2) * indicates an outlier value that is not included in the CCP calculation.

[0132] (3) ** indicates biological outliers. Individuals with biological outliers are excluded from each analytical batch and do not participate in the calculation of CCP.

[0133] The process of obtaining the confirmation critical value is as follows:

[0134] (1) First, we excluded individuals with CV%>25.0%, which was not included in this study;

[0135] (2) Eliminate analytical outliers: The SIR% ​​of each analytical batch was subjected to Tukey test. The SIR% ​​greater than Q3+1.5×(Q3-Q1) or less than Q1-1.5×(Q3-Q1) was considered an analytical outlier, where Q3=75. thPercentile, Q1=25 th Percentiles, samples with outliers should be excluded from the corresponding analysis batch. In this test, individuals 12, 27, 29, 30 and 10, 26 were excluded.

[0136] (3) Elimination of biological outliers: The SIR% ​​of the same individual (after eliminating analytical outliers) in each analytical batch was averaged and, after Tukey's test, the SIR% ​​greater than Q3+1.5×(Q3-Q1) or less than Q1-1.5×(Q3-Q1) was considered a biological outlier, where Q3=75. th Percentile, Q1=25 th Percentiles, samples with biological outliers should be removed from each analysis batch. Individuals 06, 26, and 27 were removed from this test.

[0137] (4) After eliminating individuals and outliers that did not meet the CV% requirement (the number of individuals remaining after every 32 blank individuals were eliminated was not less than 24), the data distribution was tested using SPSS software. The Shapiro-Wilk test for data distribution showed that the skewness coefficient of the data distribution was in the range of [-1, 1], and the P value was <0.05, which met the robust parameter model acceptance criteria. In the end, 83 of the 96 test samples were valid and participated in the calculation of CCP.

[0138] The inhibition rate (SIR%) of the mean signal value of the blank individual serum duplicate wells with FGF21 added to the mean signal value of the blank individual serum duplicate wells without FGF21 was statistically calculated and the final CCP was set as follows:

[0139] IR 个体 =S 加FGF21后个体复孔均值 / S 未加FGF21个体复孔均值

[0140] SIR% 个体 =(1-S 加FGF21后个体复孔均值 / S 未加FGF21个体复孔均值 )×100%

[0141] The SIR% ​​of all individuals who meet the requirements were calculated using Excel software, and the CCP was determined using the 99% percentile of the one-sided normal distribution of SIR%. Individual data were not logarithmically transformed, and CCP = Median (SIR％) +2.326×1.4826×MAD (SIR％) =12.9%+2.326×1.4826×5.4%=31.5% (99% percentile).

[0142] Median: The median value of the SIR% ​​values ​​of all valid individual matrices in method validation.

[0143] MAD: Median absolute deviation of the SIR% ​​values ​​of all valid individual matrices for method validation.

[0144] VI. Titer Critical Value

[0145] TCP Floating The 99.9% percentile was calculated using the same individual data as the screening cutoff.

[0146] The specific formula is as follows:

[0147] MCF Titration =Median (S / N) +3.090×1.4826×MAD (S / N) =0.9780+3.090×1.4826×0.1254=1.5525 (99.9% percentile)

[0148] TCPF for the analytical batch used for titer testing loating =MCF Titration ×Mean (NC) .

[0149] VII. Method Sensitivity

[0150] The positive control antibody was diluted with blank mixed serum to an appropriate series of concentrations as the positive control titer curve. The concentrations were: 10000.000 ng / mL, 3333.333 ng / mL, 1111.111 ng / mL, 370.370 ng / mL, 123.457 ng / mL, 41.152 ng / mL, 13.717 ng / mL, and 4.572 ng / mL. FGF21 was added (FGF21 was diluted to 200.000 μg / mL with detection reagent) and without FGF21 for detection. The sample concentration in the screening curve was the horizontal axis, and the average of the replicate wells of the test object was the vertical axis. Four parameters were used to fit and provide relevant parameters of the positive control curve.

[0151] The data is shown in Table 3 below:

[0152]

[0153] Note: (1) Bold indicates that the mean signal value of each titration curve is higher than SCP Floating Or SIR% ​​is higher than the lowest point corresponding to CCP, that is, screening sensitivity or confirmation sensitivity.

[0154] (2) Titer precision (including intra-batch or inter-batch precision) is the ratio of the maximum titer to the minimum titer and should be less than the square of the dilution.

[0155] result:

[0156] Screening sensitivity: ≤370.370ng / mL in serum

[0157] Confirmed sensitivity: ≤370.370ng / mL in serum

[0158] 8. Drug tolerance

[0159] FGF21 was diluted with blank mixed serum to 350000.000 ng / mL, 116666.667 ng / mL, 38888.889 ng / mL, 12962.963 ng / mL, 4320.988 ng / mL, 1440.329 ng / mL, 480.110 ng / mL, 0.000 ng / mL, and then mixed with positive control antibody (2000.000 ng / mL, 1000.000 ng / mL, 500.000 ng / mL) diluted with blank mixed serum at a ratio of 1:1 (after mixing, the concentration became half of the prepared concentration) for verification. The data are shown in Table 4 below:

[0160]

[0161] Note: Bold represents the highest concentration of FGF21 in serum that is greater than the floating screening cut-off value of this analytical batch.

[0162] The results are shown in Table 5:

[0163]

[0164]

[0165] 9. Hook Effect

[0166] The positive control antibody was diluted with mixed serum to a series of concentrations (50000.000 ng / mL, 10000.000 ng / mL, 1000.000 ng / mL, 500.000 ng / mL) for detection. Each concentration point was measured in duplicate, and the average signal value was taken as the result, as shown in Table 6 below. The average signal value of the concentration point higher than HPC was higher than the average signal value of HPC, and the average signal value did not show a significant decrease with the increase of the positive control antibody concentration, which proved that this method would not produce a hook effect that affected the detection within 50000.000 ng / mL.

[0167]

[0168] 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 analytical method for detecting FGF21 anti-drug antibodies in serum based on ELISA, comprising the following steps: (1) Detection by ELISA, (2) 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) is the S / N value of all valid blank individual matrices, that is, the mean value of the duplicate wells of the valid blank individual matrix (S) divided by the mean value of the signal value of the negative control (NC) of the corresponding batch (N), The blank individual matrix refers to individual serum that has not been treated with FGF21. 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 FGF21. (3) 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FGF21后个体复孔均值 / S 未加FGF21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FGF21后个体复孔均值 is the mean signal value of the blank individual matrix duplicate wells with FGF21 added, S 未加FGF21个体复孔均值 It is the mean signal value of blank individual matrix duplicate wells without FGF21.

2. The method of claim 1, wherein: Testing by ELISA involves the following steps: (1-1-1) Coating: Dilute FGF21 with PBS solution to prepare coating solution, add the coating solution to each well of the multi-well plate, seal the plate with a sealing film, and place in a refrigerator for incubation overnight; (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) Acidification: Mix the sample to be tested with CH3COOH solution and add it to a low adsorption plate. Seal the plate and place it on an ELISA plate shaker for acidification at room temperature. (1-4-1) Neutralization: Add Tris solution, acidified sample and detection reagent or detection reagent containing FGF21 to the blocked multi-well plate, seal the plate and place it on an ELISA plate shaker for incubation 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.

3. The method of claim 2, wherein: Testing by ELISA involves the following steps: (1-1-1) Coating: Dilute FGF21 to a concentration of 10.000 μg / mL with PBS solution to prepare a coating solution. Add the coating solution to each well of a multi-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 multi-well plate, add 300 μL of washing solution to each well, wash the plate three times, and pat dry on paper; (1-2-1) Blocking: Add 285 μL of Casein in TBS solution to each well of the multi-well plate, seal the plate with a sealing film, and incubate in a constant temperature incubator at 25±1℃ for 3 to 3.5 h; (1-2-2) Washing: Shake off the liquid in the multi-well plate, add 300 μL of washing solution to each well and wash the plate three times, then pat dry on paper; (1-3-1) Acidification: Mix the sample to be tested with 300 mM CH3COOH solution at a volume ratio of 1:9 and add it to a low adsorption plate. Seal the plate and place it on an ELISA plate shaker for acidification at room temperature and 600 RPM for 30 ± 3 min. (1-4-1) Neutralization: Add 15 μL / well of 1M Tris solution, 40 μL / well of acidified sample, and 60 μL / well of detection reagent or detection reagent containing FGF21 to the blocked multi-well plate, seal the plate and place on an ELISA plate shaker, incubate at room temperature at 600 RPM for 2 h±3 min; (1-4-2) Washing: Shake off the liquid in the multi-well 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 (streptavidin-horseradish peroxidase) 200 times with 1% BSA-PBS solution to prepare a connecting agent. Add 100 μL of the connecting agent to each well of the multi-well plate, seal the plate with a sealing film, and incubate at 25±1°C for 30±3 min. (1-5-2) Washing: Shake off the liquid in the multi-well 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 multi-well plate and incubate at room temperature in the dark for 20 ± 3 min; (1-7-1) Termination: Add 100 μL of stop solution to each well of the multi-well plate 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.

4. The method of claim 3, wherein: When screening or titer analysis is performed, in (1-4-1) neutralization, 15 μL / well of 1 M Tris solution, 40 μL / well of acidified sample, and 60 μL / well of detection reagent are added to the blocked multi-well plate.

5. The method of claim 3, wherein: When confirming, in the neutralization of (1-4-1), 15 μL / well of 1M Tris solution, 40 μL / well of acidified sample and 60 μL / well of FGF21-containing detection reagent are added to the blocked multi-well plate, wherein the FGF21-containing detection reagent is obtained by diluting FGF21 to 200.000 μg / mL with the detection reagent.

6. The method of claim 1, wherein: The estimated multiplication correction factor MCF is 1.1464-1.2838, more preferably 1.1464 or 1.2838; The confirmation critical value CCP is 31.5%-48.4%, preferably 31.5% or 48.4%.

7. The method of claim 1, further comprising the steps of: (4) 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) is the S / N value of all valid blank individual matrices, that is, the mean value of the duplicate wells of the valid blank individual matrix (S) divided by the mean value of the signal value of the negative control (NC) of the corresponding batch (N), MAD (S / N) is the median absolute deviation of the S / N values ​​of all valid individual matrices. In a preferred embodiment, the titer critical value TCP Floating It is 1.5525.

8. The method of claim 1, further comprising the steps of: (5) Obtaining screening sensitivity and confirmation sensitivity: diluting the positive control antibody with blank mixed serum to the following series of concentrations as the positive control titer curve: 10000.000 ng / mL, 3333.333 ng / mL, 1111.111 ng / mL, 370.370 ng / mL, 123.457 ng / mL, 41.152 ng / mL, 13.717 ng / mL, 4.572 ng / mL, respectively adding FGF21 (diluting FGF21 with detection reagent to 200.000 μg / mL) and not adding FGF21, and detecting by ELISA, with the sample concentration in the screening curve as the horizontal axis and the average of the replicate wells of the test object as the vertical axis, and fitting by four parameters to obtain the screening sensitivity and confirmation sensitivity of the positive control curve; Preferably, the screening sensitivity is ≤370.370 ng / mL, the confirmation sensitivity is ≤1111.111 ng / mL, and more preferably the confirmation sensitivity is ≤370.370 ng / mL.

9. The method of claim 1, further comprising the steps of: (6) Obtaining drug tolerance: FGF21 was diluted with blank mixed serum to 350000.000 ng / mL, 116666.667 ng / mL, 38888.889 ng / mL, 12962.963 ng / mL, 4320.988 ng / mL, 1440.329 ng / mL, 480.110 ng / mL, and 0.000 ng / mL, and then mixed with positive control antibody (2000.000 ng / mL, 1000.000 ng / mL, and 500.000 ng / mL) diluted with blank mixed serum at a ratio of 1:1, and then tested and verified by ELISA; Preferably, 1000.000 ng / mL of anti-drug antibodies tolerated 55555.556 ng / mL FGF21, and 500.000 ng / mL and 250.000 ng / mL of anti-drug antibodies tolerated 6172.840 ng / mL FGF21; More preferably, 1000 ng / mL, 500 ng / mL and 250 ng / mL of anti-drug antibodies tolerate 175000 ng / mL of FGF21.

10. The method of claim 1, further comprising the steps of: (7) 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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