Pretreatment method of drug-resistant antibody sample

Abstract

The invention provides an anti-drug antibody sample pretreatment method, and belongs to the technical field of biological detection. The anti-drug antibody sample pretreatment method provided by the invention is combined with a specific acidolysis step, meanwhile, the characteristics of large specific surface area and high capture efficiency of magnetic beads are utilized, the anti-drug antibody sample is pretreated, the drug resistance of the anti-drug antibody sample is effectively improved, and the detection efficiency is remarkably improved. The anti-drug antibody sample pretreatment method provided by the invention is further combined with and adopts a full-automatic multifunctional biological ligand separation and extraction instrument, so that the problems of low recovery rate, poor drug resistance and long consumed time in the existing method are avoided, the automation degree is higher, the pretreatment time of the anti-drug antibody sample is greatly shortened, and the detection efficiency is improved. The drug resistance and the detection effect of the sample are improved.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for pretreating anti-drug antibody samples. Background Art

[0002] Anti-drug antibodies (ADA) refer to antibodies produced by the human immune system that react to exogenous drugs (such as biological drugs). These antibodies may affect the efficacy and safety of drugs, so it is crucial to detect anti-drug antibodies. In addition to developing ADA detection methods with high sensitivity and specificity, it is also necessary to consider whether anti-drug antibodies can be detected when there is a high concentration of drug in the sample. Therefore, it is necessary to improve the drug tolerance of anti-drug antibodies during the development of bioanalytical methods, which is considered a major challenge in the field of analytical method development. In view of this, it is of great significance to develop a fully automatic acid dissociation pretreatment method to improve the drug resistance of anti-drug antibodies.

[0003] If there are a large amount of free drugs in the actual sample, the free drugs can combine with anti-drug antibodies, thereby affecting the detection of ADA and reducing the detection rate of ADA; the commonly used solution is to use the principle of acid dissociation, by acidifying the sample, the free drugs and anti-drug antibodies are dissociated, thereby achieving the purpose of detecting ADA.

[0004] Chinese patent CN117849327A discloses a sample pretreatment method and application for detecting secukinumab drug resistance antibodies. The sample is subjected to secondary acid hydrolysis treatment with an acid hydrolysis solution, which can effectively reduce the interference of free IL-17A in the sample and solve the problem that free IL-17A affects the detection of secukinumab drug resistance antibodies.

[0005] However, the drug resistance of anti-drug antibodies in existing technologies needs to be further improved. Summary of the invention

[0006] The purpose of the present invention is to provide a method for pretreating anti-drug antibody samples, which can effectively improve the drug resistance of anti-drug antibodies and optimize detection efficiency.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for pretreating an anti-drug antibody sample, comprising the following steps:

[0009] S1, coating the magnetic beads with biotin-labeled antigens to obtain coated magnetic beads;

[0010] S2, washing the coated magnetic beads to obtain washed magnetic beads;

[0011] S3, mixing the anti-drug antibody sample with the acidifier 1, and performing a first acid hydrolysis treatment to obtain an acidified sample;

[0012] S4, adding Tris-HCl to the acidified sample, and then mixing with the washed magnetic beads, and incubating to obtain magnetic beads bound to the anti-drug antibody sample;

[0013] S5, mixing the magnetic beads bound to the anti-drug antibody sample with the acidifier 2, performing a second acid hydrolysis treatment, and then removing the magnetic beads to obtain a treated anti-drug antibody sample;

[0014] The acidulant 1 contains acetic acid;

[0015] The acidulant 2 contains glycine.

[0016] Preferably, the final concentration of acetic acid in the acidifier 1 is 100-500 mM.

[0017] Preferably, the final concentration of glycine in the acidifier 2 is 0.1-0.5M.

[0018] Preferably, steps S3 to S5 are completed using a fully automatic multifunctional biological ligand separation and extraction instrument.

[0019] Preferably, the detergent used to wash the coated magnetic beads contains glycine.

[0020] Preferably, the first acid hydrolysis treatment is performed for 20 to 40 minutes.

[0021] Preferably, the second acid hydrolysis treatment lasts for 20 to 40 minutes.

[0022] Preferably, the incubation time in step S4 is 40 to 80 minutes.

[0023] The present invention also provides the application of the above anti-drug antibody sample pretreatment method in the detection of anti-drug antibodies by electrochemiluminescence method;

[0024] Preferably, the anti-drug antibody is an anti-drug antibody against recombinant human growth hormone.

[0025] The present invention also provides a method for detecting anti-drug antibodies by electrochemiluminescence, comprising the following steps:

[0026] Pre-treating the anti-drug antibody sample using the above method to obtain a pre-treated anti-drug antibody sample;

[0027] Coating the biotin-labeled antigen onto the ELISA plate, washing and blocking;

[0028] Tris-HCl and pretreated anti-drug antibody samples were added, incubated and then washed;

[0029] Add SULFO-TAG labeled antigen, incubate and wash;

[0030] Add plate reading solution, read the plate, and record the ECL value.

[0031] Beneficial effects of the present invention:

[0032] The anti-drug antibody sample pretreatment method provided by the present invention combines a specific acid hydrolysis step and utilizes the characteristics of large specific surface area and high capture efficiency of magnetic beads to pretreat the anti-drug antibody samples, thereby effectively improving the drug resistance of the anti-drug antibody samples and significantly improving the detection efficiency.

[0033] The anti-drug antibody sample pretreatment method provided by the present invention is further combined with a fully automatic multifunctional biological ligand separation and extraction instrument, which avoids the problems of low recovery rate, poor drug resistance and long time consumption in the existing methods. It not only has a higher degree of automation, but also greatly shortens the pretreatment time of anti-drug antibody samples, and improves the drug resistance and detection effect of the samples. DETAILED DESCRIPTION

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Main reagent information and sample preparation method in the embodiments of the present invention:

[0036] Anti-rhGHAffinity Purified antibodies (Rabbit) were purchased from Beijing Tiancheng Biotechnology (Beijing) Co., Ltd.;

[0037] Recombinant human growth hormone was purchased from the China Food and Drug Administration with a product batch number of 140635-202206;

[0038] Biotin-Recombinant Human Growth Hormone:

[0039] The product is formed by labeling with Biotin (purchased from Japan Tongren Chemical, product model / specification LK03) and recombinant human growth hormone through conventional methods in the art.

[0040] SULFO-TAG-recombinant human growth hormone:

[0041] The recombinant human growth hormone was labeled by using SULFO-TAG (purchased from Meso Scale Discovery, USA, product model / specification R91AO-1) and conventional methods in the art.

[0042] Nano-SA magnetic beads were purchased from Beijing Xinaijin Biotechnology Co., Ltd.

[0043] Sample preparation:

[0044] 1) Negative control samples

[0045] Negative control sample: Mixed New Zealand rabbit blank serum as negative control sample (NC).

[0046] 2) Positive quality control samples

[0047] Positive quality control sample: Anti-rhGH Affinity Purifiedantibodies (Rabbit) with a concentration of 0.789 mg / mL was diluted to 10000.000 ng / mL (HPC), 1000.000 ng / mL (MPC) and 200 ng / mL (QC-L) with mixed New Zealand rabbit blank serum (MS).

[0048] Samples to be tested:

[0049] The samples to be tested are all derived from animal experiments, that is, after a certain dose of recombinant human growth hormone is given to New Zealand rabbits, whole blood is collected at different time points without anticoagulation, and centrifuged at 4°C and 1800×g for 10 minutes, and the supernatant is taken as the sample to be tested.

[0050] Example 1

[0051] Magnetic beads treatment:

[0052] Magnetic bead coating: Take 1 mL of SA magnetic beads and add 10 μL of Biotin-recombinant human growth hormone to it, and incubate at room temperature with shaking (300 rpm) for 60 min;

[0053] Magnetic bead washing: dilute 0.3M glycine 3 times with pure water as washing solution, place SA magnetic beads on the magnetic stand of the fully automatic separation and extraction instrument for 3 minutes, aspirate the liquid in the magnetic beads with a pipette, add an equal volume of washing solution, and mix with a pipette, and perform this procedure 3 times; place SA magnetic beads on the magnetic stand for 3 minutes, aspirate the liquid in the magnetic beads with a pipette, add an equal volume of 1% BSA-1×PBS, and mix with a pipette;

[0054] Sample processing:

[0055] Sample acidification: Take a 96-well dilution plate and add 120 μL of 300 mM acetic acid as the diluent. For the dilution of the drug-treated samples, the diluent is changed to 300 mM acetic acid containing 100 μg / mL recombinant human growth hormone. Then add 20 μL of each quality control and test sample; put it into the fully automatic multifunctional biological ligand separation and extraction instrument for shaking (300 rpm) and incubation for 30 minutes;

[0056] Neutralization and incubation: add 30 μL of 1M Tris-HCl to each well of the above dilution plate, then add 30 μL of washed magnetic beads, cover with film, put into the fully automatic multifunctional biological ligand separation and extraction instrument, shake (300 rpm) and incubate for 60 min;

[0057] Magnetic bead cleaning: place the dilution plate on the fully automatic multifunctional biological ligand separation and extraction instrument, place the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument into the deep-well plate, set the speed to 30, and the time to 5 min; after running twice, lift the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument, and then take a new dilution plate, add 300 μL of 1% BSA-1×PBS to each well and place it in the fully automatic multifunctional biological ligand separation and extraction instrument, place the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument into the deep-well plate, then take out the magnetic frame, and leave the magnetic cover in the deep-well plate. Because the magnetic beads lose their magnetism, they fall into the deep-well plate for cleaning.

[0058] This was done 3 times in total;

[0059] Magnetic beads were acidified by adding 120 μL of 0.3 M glycine to each well of the dilution plate, covering the membrane, and incubating at room temperature with shaking (300 rpm) for 30 min;

[0060] Post-treatment of magnetic beads after acidification: Place the dilution plate on the fully automatic multifunctional biological ligand separation and extraction instrument, place the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument into the deep well plate, set the speed to 30, and the time to 5 minutes; after running twice, lift the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument; the magnetic beads are adsorbed and removed by the magnetic frame, and the remaining sample is used for subsequent sample addition.

[0061] ELISA plate processing:

[0062] Coating: Biotin-recombinant human growth hormone was diluted 2000 times with coating solution (1×PBS), 100 μL / well was added to the ELISA plate (MSD GOLD 96-well Streptavidin Plate), coated, and incubated at 37°C for 120 min;

[0063] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0064] Blocking: Add blocking solution (5% BSA-1×PBS), 150 μL / well, cover the membrane, and incubate at 37°C for 60 min;

[0065] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0066] Neutralization and sample addition: 10 μL / well of 1M Tris-HCl was added to the ELISA plate, and then 40 μL / well of the acidified quality control and samples were added, the plate was covered, and incubated at room temperature with shaking (500 rpm) for 60 min;

[0067] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0068] Add SULFO-TAG: dilute SULFO-TAG-recombinant human growth hormone 4000 times with 1% BSA-1×PBS, 100 μL / well, add to ELISA plate, cover with film, and incubate at 37°C for 60 min;

[0069] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0070] (9) Plate reading: Dilute Read Buffer T (4×) with pure water to a final concentration of Read Buffer T (2×), add 150 μL / well to MSD GOLD 96-well Streptavidin Plate, and read the ECL value of the 96-well plate using MSD.

[0071] Example 2

[0072] Magnetic beads treatment:

[0073] Magnetic bead coating: Take 1 mL of SA magnetic beads and add 10 μL of Biotin-recombinant human growth hormone to it, and incubate at room temperature with shaking (300 rpm) for 50 min;

[0074] Magnetic bead washing: dilute 0.3M glycine 3 times with pure water as washing solution, place SA magnetic beads on the magnetic stand of the fully automatic separation and extraction instrument for 3 minutes, aspirate the liquid in the magnetic beads with a pipette, add an equal volume of washing solution, and mix with a pipette, and perform this procedure 3 times; place SA magnetic beads on the magnetic stand for 3 minutes, aspirate the liquid in the magnetic beads with a pipette, add an equal volume of 1% BSA-1×PBS, and mix with a pipette;

[0075] Sample processing:

[0076] Sample acidification: Take a 96-well dilution plate and add 120 μL of 300 mM acetic acid as the diluent. For the dilution of the drug-treated samples, the diluent is changed to 300 mM acetic acid containing 100 μg / mL recombinant human growth hormone. Then add 20 μL of each quality control and test sample; put it into the fully automatic multifunctional biological ligand separation and extraction instrument for shaking (300 rpm) and incubation for 25 minutes;

[0077] Neutralization and incubation: add 30 μL of 1M Tris-HCl to each well of the above dilution plate, then add 30 μL of washed magnetic beads, cover with film, put into the fully automatic multifunctional biological ligand separation and extraction instrument, shake (300 rpm) and incubate for 50 min;

[0078] Magnetic bead cleaning: place the dilution plate on the fully automatic multifunctional biological ligand separation and extraction instrument, place the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument into the deep-well plate, set the speed to 30, and the time to 5 min; after running twice, lift the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument, and then take a new dilution plate, add 300 μL of 1% BSA-1×PBS to each well and place it in the fully automatic multifunctional biological ligand separation and extraction instrument, place the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument into the deep-well plate, then take out the magnetic frame, and leave the magnetic cover in the deep-well plate. Because the magnetic beads lose their magnetism, they fall into the deep-well plate for cleaning.

[0079] This was done 3 times in total;

[0080] Magnetic beads were acidified by adding 120 μL of 0.3 M glycine to each well of the dilution plate, covering the membrane, and incubating at room temperature with shaking (300 rpm) for 25 min;

[0081] Post-treatment of magnetic beads after acidification: Place the dilution plate on the fully automatic multifunctional biological ligand separation and extraction instrument, place the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument into the deep well plate, set the speed to 30, and the time to 5 minutes; after running twice, lift the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument; the magnetic beads are adsorbed and removed by the magnetic frame, and the remaining sample is used for subsequent sample addition.

[0082] ELISA plate processing:

[0083] Coating: Biotin-recombinant human growth hormone was diluted 2000 times with coating solution (1×PBS), 100 μL / well was added to the ELISA plate (MSD GOLD 96-well Streptavidin Plate), coated, and incubated at 37°C for 100 min;

[0084] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0085] Blocking: add blocking solution (5% BSA-1×PBS), 150 μL / well, cover the membrane, and incubate at 37°C for 50 min;

[0086] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0087] Neutralization and sample addition: 10 μL / well of 1M Tris-HCl was added to the ELISA plate, and then 40 μL / well of the acidified quality control and samples were added, the plate was covered, and incubated at room temperature with shaking (500 rpm) for 50 min;

[0088] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0089] Add SULFO-TAG: dilute SULFO-TAG-recombinant human growth hormone 4000 times with 1% BSA-1×PBS, 100 μL / well, add to ELISA plate, cover with film, and incubate at 37°C for 50 min;

[0090] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0091] (9) Plate reading: Dilute Read Buffer T (4×) with pure water to a final concentration of Read Buffer T (2×), add 150 μL / well to MSD GOLD 96-well Streptavidin Plate, and read the ECL value of the 96-well plate using MSD.

[0092] Example 3

[0093] Magnetic beads treatment:

[0094] Magnetic bead coating: Take 1 mL of SA magnetic beads and add 10 μL of Biotin-recombinant human growth hormone to it, and incubate at room temperature with shaking (300 rpm) for 70 min;

[0095] Magnetic bead washing: dilute 0.3M glycine 3 times with pure water as washing solution, place SA magnetic beads on the magnetic stand of the fully automatic separation and extraction instrument for 3 minutes, aspirate the liquid in the magnetic beads with a pipette, add an equal volume of washing solution, and mix with a pipette, and perform this procedure 3 times; place SA magnetic beads on the magnetic stand for 3 minutes, aspirate the liquid in the magnetic beads with a pipette, add an equal volume of 1% BSA-1×PBS, and mix with a pipette;

[0096] Sample processing:

[0097] Sample acidification: Take a 96-well dilution plate and add 120 μL of 300 mM acetic acid as the diluent. For the dilution of the drug-treated samples, the diluent is changed to 300 mM acetic acid containing 100 μg / mL recombinant human growth hormone. Then add 20 μL of each quality control and test sample; put it into the fully automatic multifunctional biological ligand separation and extraction instrument for shaking (300 rpm) and incubation for 35 minutes;

[0098] Neutralization and incubation: add 30 μL of 1M Tris-HCl to each well of the above dilution plate, then add 30 μL of washed magnetic beads, cover with film, put into the fully automatic multifunctional biological ligand separation and extraction instrument, shake (300 rpm) and incubate for 70 min;

[0099] Magnetic bead cleaning: place the dilution plate on the fully automatic multifunctional biological ligand separation and extraction instrument, place the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument into the deep-well plate, set the speed to 30, and the time to 5 min; after running twice, lift the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument, and then take a new dilution plate, add 300 μL of 1% BSA-1×PBS to each well and place it in the fully automatic multifunctional biological ligand separation and extraction instrument, place the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument into the deep-well plate, then take out the magnetic frame, and leave the magnetic cover in the deep-well plate. Because the magnetic beads lose their magnetism, they fall into the deep-well plate for cleaning.

[0100] This was done 3 times in total;

[0101] Magnetic beads were acidified by adding 120 μL of 0.3 M glycine to each well of the dilution plate, covering the membrane, and incubating at room temperature with shaking (300 rpm) for 35 min;

[0102] Post-treatment of magnetic beads after acidification: Place the dilution plate on the fully automatic multifunctional biological ligand separation and extraction instrument, place the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument into the deep well plate, set the speed to 30, and the time to 5 minutes; after running twice, lift the magnetic frame (including the magnetic frame cover) of the fully automatic multifunctional biological ligand separation and extraction instrument; the magnetic beads are adsorbed and removed by the magnetic frame, and the remaining sample is used for subsequent sample addition.

[0103] ELISA plate processing:

[0104] Coating: Dilute Biotin-recombinant human growth hormone 2000 times with coating solution (1×PBS), add 100 μL / well to the ELISA plate (MSD GOLD 96-well Streptavidin Plate), cover with film, and incubate at 37°C for 140 min;

[0105] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0106] Blocking: Add blocking solution (5% BSA-1×PBS), 150 μL / well, cover the membrane, and incubate at 37°C for 70 min;

[0107] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0108] Neutralization and sample addition: 10 μL / well of 1M Tris-HCl was added to the ELISA plate, and then 40 μL / well of the acidified quality control and samples were added, the plate was covered, and incubated at room temperature with shaking (500 rpm) for 70 min;

[0109] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0110] Add SULFO-TAG: dilute SULFO-TAG-recombinant human growth hormone 4000 times with 1% BSA-1×PBS, 100 μL / well, add to ELISA plate, cover with film, and incubate at 37°C for 70 min;

[0111] Washing: Wash the plate three times with washing solution (0.05% PBST), 300 μL / well, and remove the residual liquid;

[0112] (9) Plate reading: Dilute Read Buffer T (4×) with pure water to a final concentration of Read Buffer T (2×), add 150 μL / well to MSD GOLD 96-well Streptavidin Plate, and read the ECL value of the 96-well plate using MSD.

[0113] Experimental example

[0114] 1. Effects of different acid dissociation methods on drug tolerance:

[0115] Two conventional acid hydrolysis treatment methods were used as control experiments of the present invention and compared with the scheme of Example 1:

[0116] The first method is to use acetic acid to perform conventional acid hydrolysis on the sample, then use a common ELISA plate to enrich the antidrug antibody sample, and finally acid hydrolyze and elute again for detection;

[0117] The second method is to use acetic acid to perform conventional acid hydrolysis on the sample and then directly perform the test.

[0118] The comparison results are shown in Table 1:

[0119] Table 1 Effects of different dissociation methods on drug tolerance

[0120]

[0121]

[0122] Note: The maximum concentration results that are greater than both SCP and CCP are resistance results.

[0123] The results showed that the drug tolerance results detected by this research method were better than those of the conventional method of only acid hydrolyzing the samples and enriching them with ordinary ELISA plates after acid hydrolysis, which showed that this method had good drug tolerance and higher sensitivity.

[0124] 2. Sensitivity

[0125] The positive control antibody (0.789 mg / mL Anti-rhGH Affinity Purified antibodies (Rabbit)) was diluted in series (concentrations: 781.250, 390.625, 195.313, 97.657, 48.829, 24.415, 12.208, 6.104 ng / mL) using mixed New Zealand rabbit blank serum to obtain sensitivity samples (A1-A8). Two replicate wells were set for each concentration (the OD value results were averaged) for detection; at the same time, a set of drug-treated sensitivity samples was set, namely: DTA1-DTA8, and the SIR value of each sample was calculated. At least 6 batches of independent and valid determinations of the above sensitivity samples were completed by at least 2 analysts in at least 3 days. The acceptance standard for sensitivity was ≤500.000 ng / mL.

[0126] The results showed that the screening sensitivity of the method of the present invention was 13.11 ng / mL, and the confirmation sensitivity was 16.48 ng / mL, indicating that the method provided by the present invention has excellent sensitivity.

[0127] The specific results are shown in Table 2:

[0128] Table 2 Sensitivity of electrochemiluminescence detection of anti-drug antibodies in New Zealand rabbit serum

[0129]

[0130]

[0131] 3. Drug tolerance

[0132] Positive control antibody (0.789 mg / mL Anti-rhGH Affinity Purified Antibodies (Rabbit)) were prepared into high concentration (20000ng / mL) and low concentration (2 times LPC concentration) positive quality control samples, and the drug was prepared into a series of samples with a concentration of 800.000, 400.000, 200.000, 100.000, 50.000, 25.000, and 12.500μg / mL using mixed New Zealand rabbit blank serum. Then, the high and low positive quality control sample solutions were mixed with equal volumes of the diluted drug samples of each concentration as drug tolerance samples. After mixing, the concentrations of high and low positive quality control samples were 10000ng / mL and LPC, respectively, and the drug concentrations were 400.000, 200.000, 100.000, 50.000, 25.000, 12.500, and 6.250μg / mL. The drug-resistant samples were placed at 37°C for at least 1 hour, and then stored at -60°C for at least 24 hours, and tested with 2 replicate wells (the ECL value results were averaged). At the same time, the drug tolerance confirmed by drug addition was investigated, that is, another set of drug-tolerant samples treated with drug addition was set up, and 2 replicate wells were tested to obtain the test results of each sample and calculate the SIR value.

[0133] The highest drug concentration corresponding to the drug tolerance sample test result being greater than or equal to SCP (screening) and CCP (confirmation) is the maximum drug concentration that the positive control antibody at that concentration can tolerate.

[0134] The results showed that in New Zealand rabbit serum, when the concentration of the positive control antibody sample was 10000.000 ng / mL, the drug resistance concentration of recombinant human growth hormone was 1000.000 μg / mL, and the drug resistance ratio was 1:100; when the concentration of the positive control antibody sample was 30.000 ng / mL, the drug resistance concentration of recombinant human growth hormone was 3.125 μg / mL, and the drug resistance ratio was 1:104, indicating that the method provided by the present invention has good drug resistance. The specific results are shown in Table 3:

[0135] Table 3 Drug tolerance of anti-drug antibodies in New Zealand rabbit serum detected by electrochemiluminescence

[0136]

[0137]

[0138] 4) Selectivity

[0139] Select at least 10 New Zealand rabbit blank sera from different sources, and then take 5 blank hemolysis samples from different sources (simulated hemolysis samples obtained by adding 2% New Zealand rabbit whole blood to New Zealand rabbit blank sera) for investigation at the blank level (SE-NC) and the low-concentration positive control level (SE-LPC). At the same time, investigate the selective samples (SE-DTNC and SE-DTLPC) treated with drugs. The above samples are set with 2 replicates for detection within the same analysis batch, the detection results of each sample are obtained, and the SIR values of the selective samples treated with drugs are calculated and compared with the results of LPC and NC prepared with mixed blank matrix in the same analysis batch.

[0140] Acceptance criteria: The detection results of SE-LPC and SE-DTLPC samples prepared by at least 80% of individuals and the detection results of at least 80% of blank hemolysis samples are compared with the detection results of LPC and DTLPC samples respectively, meeting %CV≤20%, and all are positive (OD≥SCP, SIR≥CCP); the detection results of SE-NC and SE-DTNC samples of at least 80% of individuals and at least 80% of blank hemolysis samples are negative (OD<SCP, SIR<CCP), indicating that there is no interference from endogenous proteins and hemolysis in this detection method.

[0141] The results showed that when comparing the ECL values and SIR values of 10 New Zealand rabbit blank serum samples from different sources with those of the mixed serum samples, the blank serum samples met the requirements that the ECL value was less than the SCP value (67.38) of the detection batch and SIR<CCP (7.97%); at the same time, when comparing the ECL values and SIR values of the quality control samples prepared with 10 blank sera with those of the quality control samples prepared with mixed sera, the results were all positive (meeting ECL≥66.50, SIR≥7.97%), and %CV was between 0.56 and 6.84, meeting the predetermined selectivity criteria, suggesting that endogenous proteins in the samples had no interference with this detection method.

[0142] When comparing the ECL values and SIR values of 5 hemolysis blank samples from different sources with those of the mixed serum samples, the ECL values of 3 out of the 5 hemolysis blank samples were slightly greater than the SCP value (67.38) of the detection batch, so the SIR values of the 5 hemolysis blank samples were less than CCP (7.97%); at the same time, when comparing the ECL values and SIR values of the quality control samples prepared with 5 hemolysis blank sera with those of the quality control samples prepared with mixed sera, the results were all positive (meeting ECL≥66.50, SIR≥7.97%), and %CV was between 1.13 and 8.91, suggesting that sample hemolysis might interfere with the screening test, but had no interference with the confirmation test. It shows that this method has no endogenous interference and good selectivity.

[0143] The specific results are shown in Tables 4 - 5:

[0144] Table 4 Selectivity of electrochemiluminescence detection of anti-drug antibodies in New Zealand rabbit serum (I)

[0145]

[0146] Note: LPC (Mean) It is the average of the two sets of low quality controls of system adaptability for this analytical batch; “ / ” means no relevant information.

[0147] Table 5 Selectivity of electrochemiluminescence detection of anti-drug antibodies in New Zealand rabbit serum (II)

[0148]

[0149] Note: LPC (Mean) It is the average of the two sets of low quality controls of system adaptability for this analytical batch; “ / ” means no relevant information.

[0150] 5) Precision

[0151] This test includes screening, confirmation and titer precision. The positive control antibody is prepared into three concentration levels of high (HPC), medium (MPC) and low (LPC) positive control antibody samples (HPC: 10000.000ng / mL; MPC: 1000.000ng / mL; LPC is subject to the verification result) using mixed New Zealand rabbit blank serum, and then set up negative quality control NC (mixed New Zealand rabbit blank serum), a total of 6 sets as precision samples. In the same analytical batch, 6 sets of precision samples with drug addition treatment are set up at the same time, and 2 replicates are set up for each concentration (the ECL value results are averaged) for detection. At least 2 analysts are required to complete at least 3 independent and valid determinations for at least 3 days. The %CV of the ECL value within and between batches is calculated for each quality control sample.

[0152] Acceptance criteria: The average %CV of the ECL values ​​of positive quality control samples within and between batches is ≤20%, and the average %CV of the ECL values ​​of negative quality control samples is ≤30%, indicating that the method has good precision. The calculation method and standard for screening and confirmation precision are the same.

[0153] The results showed that two analysts conducted precision verification for five valid batches in five days. The intra-batch and inter-batch precision results of each positive quality control sample and negative quality control sample in the analysis batch met the requirements, indicating that the precision and reproducibility of this method are good. The specific results are shown in Table 6:

[0154] Table 6 Precision of electrochemiluminescence detection of anti-drug antibodies in New Zealand rabbit serum

[0155]

[0156]

[0157] Note: “ / ” in the table means there is no relevant information.

[0158] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preprocessing anti-drug antibody samples, characterized in that: The following steps are involved: S1, coating the magnetic beads with biotin-labeled antigens to obtain coated magnetic beads; S2, washing the coated magnetic beads to obtain washed magnetic beads; S3, mixing the anti-drug antibody sample with the acidifier 1, and performing a first acid hydrolysis treatment to obtain an acidified sample; S4, adding Tris-HCl to the acidified sample, and then mixing with the washed magnetic beads, and incubating to obtain magnetic beads bound to the anti-drug antibody sample; S5, mixing the magnetic beads bound to the anti-drug antibody sample with the acidifier 2, performing a second acid hydrolysis treatment, and then removing the magnetic beads to obtain a treated anti-drug antibody sample; The acidulant 1 contains acetic acid; The acidulant 2 contains glycine.

2. The method for pretreating anti-drug antibody samples according to claim 1, characterized in that: The final concentration of acetic acid in the acidifier 1 is 100-500 mM.

3. The method for pretreating anti-drug antibody samples according to claim 1, characterized in that: The final concentration of glycine in the acidifier 2 is 0.1-0.5M.

4. The method for pretreating anti-drug antibody samples according to any one of claims 1 to 3, characterized in that: The steps S3 to S5 are completed using a fully automatic multifunctional biological ligand separation and extraction instrument.

5. The method for pretreating anti-drug antibody samples according to claim 1, characterized in that: The detergent used to wash the coated magnetic beads contains glycine.

6. The method for pretreating anti-drug antibody samples according to claim 1, characterized in that: The first acid hydrolysis treatment lasts for 20 to 40 minutes.

7. The method for pretreating anti-drug antibody samples according to claim 1, characterized in that: The second acid hydrolysis treatment lasts for 20 to 40 minutes.

8. The method for pretreating anti-drug antibody samples according to claim 1, characterized in that: The incubation time in step S4 is 40 to 80 minutes.

9. Use of the anti-drug antibody sample pretreatment method according to any one of claims 1 to 8 in detecting anti-drug antibodies by electrochemiluminescence; Preferably, the anti-drug antibody is an anti-drug antibody against recombinant human growth hormone.

10. A method for detecting anti-drug antibodies by electrochemiluminescence, characterized in that: The following steps are involved: Pretreating the antidrug antibody sample using the method described in any one of claims 1 to 8 to obtain a pretreated antidrug antibody sample; Coating the biotin-labeled antigen onto the ELISA plate, washing and blocking; Tris-HCl and pretreated anti-drug antibody samples were added, incubated and then washed; Add SULFO-TAG labeled antigen, incubate and wash; Add plate reading solution, read the plate, and record the ECL value.

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