Hybridoma cell line TNA1-6C3, monoclonal antibody, kit and application

Abstract

The present invention provides a hybridoma cell line TNA1-6C3, a monoclonal antibody, a kit and an application, which specifically belong to the technical field of anti-drug antibody monitoring. The hybridoma cell line ( Mouse muscle )TNA1‑6C3 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number CGMCC No. 46006. The monoclonal antibody TNA1‑6C3 produced by the hybridoma cell line TNA1‑6C3 of the present invention can realize the efficient monitoring of anti-adalimumab antibody, has strong anti-interference ability, high specificity, high sensitivity, simple and rapid, and high degree of automation.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-drug antibody monitoring, and specifically relates to a hybridoma cell line TNA1-6C3, a monoclonal antibody, a kit and an application. Background Art

[0002] Adalimumab is a recombinant human IgG1 monoclonal antibody directed against human tumor necrosis factor (TNF-α). Adalimumab is produced by expression in mammalian cells using recombinant DNA technology. Adalimumab specifically binds to (TNF-α) and blocks its interaction with p55 and p75 cell surface TNF receptors. Tumor necrosis factor is a naturally occurring cytokine that participates in normal inflammatory and immune responses. Elevated levels of TNF-a are found in the synovial fluid of rheumatoid arthritis, including juvenile idiopathic arthritis, and psoriasis. Tumor necrosis factor plays an important role in the pathological inflammation and joint destruction in patients with both arthritis and ankylosing spondylitis. Elevated levels of tumor necrosis factor are also found in psoriasis.

[0003] Secondary loss of response to adalimumab treatment is usually due to the production of anti-adalimumab antibodies (ADA), which can occur in any patient receiving adalimumab despite the observed full humanization of the drug. ADA primarily neutralize adalimumab to form immune complexes that are rapidly cleared from the system. ADA are often responsible for subtherapeutic adalimumab concentrations, with extreme concentrations of adalimumab <1µg / ml. Neutralizing ADA is a subset of total ADA and is generally defined as a class of anti-drug antibodies that block the binding of adalimumab to TNF-a. Therefore, the production of neutralizing ADA can have a significant negative impact on drug efficacy, and measurement of neutralizing ADA can help determine the best treatment strategy.

[0004] In neutralizing ADA assays, drugs are usually used as bridge assays or target protein competitive assays. The higher concentration of TNF-a that may exist in clinical samples will cause false positive results. Therefore, achieving anti-interference ability against high concentrations of TNF-a is a key factor in the development of neutralizing ADA assays.

[0005] At present, the blood concentration monitoring of neutralizing antibodies against adalimumab has basically not been carried out in domestic clinical practice. The liquid chromatography and LC-MS methods commonly used for small molecules are not suitable for this type of large molecule protein detection, and in the neutralizing ADA determination, very high anti-interference ability is required.

[0006] CN111024958A discloses a reagent for detecting monoclonal antibody drugs and anti-monoclonal antibody drug antibodies and its application. The patent describes the optimization of acid treatment to achieve drug tolerance of 1 μg / mL, but does not disclose the target tolerance. In addition, the reaction principle of the one-step target competition method will be affected by the target protein.

[0007] CN114740195A discloses an ADA instant detection test strip and kit for adalimumab, but the patent does not disclose any information on drug tolerance and target tolerance.

[0008] CN106164670A discloses an assay for detecting the presence or amount of anti-drug antibodies, which requires complex operating steps and overnight precipitation, is not conducive to automation, has low throughput and high cost, and cannot meet the extensive and rapid clinical needs.

[0009] In summary, the currently disclosed methods for monitoring anti-adalimumab neutralizing autoantibodies all require the use of adalimumab to participate in the reaction, which results in the endogenous TNF-a protein in the sample interfering with the detection reagent. Therefore, there is currently a lack of a highly specific, simple, rapid, and highly automated product for detecting the concentration of anti-adalimumab neutralizing autoantibodies. Summary of the invention

[0010] The purpose of the present invention is to provide a hybridoma cell line TNA1-6C3, a monoclonal antibody, a kit and an application. The monoclonal antibody TNA1-6C3 produced by the hybridoma cell line TNA1-6C3 of the present invention can realize the efficient monitoring of anti-adalimumab antibody, has strong anti-interference ability, high specificity, is simple and fast, and has a high degree of automation.

[0011] The present invention provides a hybridoma cell line ( Mus musculus ) TNA1-6C3, the hybridoma cell line TNA1-6C3 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, and the deposit number is CGMCC No.46006.

[0012] The present invention also provides a monoclonal antibody TNA1-6C3, which is secreted and produced by the hybridoma cell line TNA1-6C3 described in the above technical solution.

[0013] The present invention also provides the use of the monoclonal antibody TNA1-6C3 described in the above technical solution in the preparation of an anti-TNF-α blocking reagent.

[0014] The present invention also provides the use of the monoclonal antibody TNA1-6C3 described in the above technical solution in preparing a reagent for inhibiting the binding of adalimumab monoclonal antibody to TNF-α protein.

[0015] The present invention also provides the use of the monoclonal antibody TNA1-6C3 described in the above technical solution in preparing a kit for detecting anti-adamimetic neutralizing autoantibodies.

[0016] The present invention also provides a kit for detecting anti-adalimum neutralizing autoantibodies, the kit comprising an anti-adalimum neutralizing autoantibody detection reagent and an anti-TNF-α blocking reagent; the anti-TNF-α blocking reagent is the monoclonal antibody TNA1-6C3 described in the above technical solution.

[0017] Preferably, when the kit is a double antigen sandwich detection kit, the anti-adalimumab neutralizing autoantibody detection reagent includes a magnetic particle working solution, an enzyme label working solution and an indicator solution; the magnetic particle working solution contains adalimumab monoclonal antibodies coupled to magnetic particles; and the enzyme label working solution contains enzyme-labeled adalimumab Fab antibodies.

[0018] Preferably, the enzyme in the enzyme-labeled adalimumab Fab antibody includes alkaline phosphatase; when the enzyme in the enzyme-labeled adalimumab Fab antibody is alkaline phosphatase, the indicator solution contains a color developing substrate of alkaline phosphatase.

[0019] Preferably, the amino acid sequence of the light chain of the adalimumab Fab antibody is as shown in SEQ ID NO.1; the amino acid sequence of the heavy chain Fd of the adalimumab Fab antibody is as shown in SEQ ID NO.2; when the enzyme in the enzyme-labeled adalimumab Fab antibody is alkaline phosphatase, the downstream of the heavy chain Fd of the adalimumab Fab antibody is connected to the alkaline phosphatase through a flexible amino acid connecting arm, and the sequence after connection is as shown in SEQ ID NO.3.

[0020] Preferably, an anti-TNF-α blocking agent is added to the magnetic particle working solution.

[0021] The present invention provides a hybridoma cell line TNA1-6C3. The monoclonal antibody TNA1-6C3 produced by the hybridoma cell line TNA1-6C3 of the present invention can specifically block the interference of the endogenous target protein TNF-a, and combined with the antibody fragment enzyme fusion expression scheme of the present invention, can realize the efficient monitoring of anti-adalimumab drug antibodies on a fully automatic magnetic microparticle enzyme immunoluminescence platform, has strong anti-interference ability, high specificity, simple and rapid, and high degree of automation, can be used for the development of clinical diagnostic reagents, and helps to determine the best treatment strategy.

[0022] The present invention also provides a kit for detecting anti-adalimumab neutralizing autoantibodies by a double antigen sandwich method. In the kit, adalimumab Fab antibody is combined with a target-specific blocking antibody to measure anti-adalimumab neutralizing autoantibodies, and can effectively resist >15000U / mL RF interference, achieve high-specificity detection, and target tolerance >100000pg / mL TNF-a.

[0023] Biological deposit information

[0024] Hybridoma cell lines ( Mus musculus )TNA1-6C3, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, referred to as CGMCC, on June 26, 2024. The address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.46006. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0026] Figure 1 The present invention provides an SDS-PAGE electrophoresis diagram of the adalimumab Fab fragment-alkaline phosphatase fusion protein. DETAILED DESCRIPTION

[0027] The present invention provides a hybridoma cell line ( Mus musculus ) TNA1-6C3, the hybridoma cell line TNA1-6C3 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, and the deposit number is CGMCC No. 46006. The monoclonal antibody TNA1-6C3 produced by the hybridoma cell line TNA1-6C3 of the present invention can be used to prepare an anti-TNF-α blocking reagent, inhibit the binding of adalimumab monoclonal antibody to TNF-α protein, and improve the anti-TNF-α interference ability when detecting anti-adalimumab neutralizing autoantibodies.

[0028] The present invention also provides a monoclonal antibody TNA1-6C3, which is secreted and produced by the hybridoma cell line TNA1-6C3 described in the above technical solution.

[0029] The present invention also provides the use of the monoclonal antibody TNA1-6C3 described in the above technical solution in the preparation of an anti-TNF-α blocking reagent.

[0030] The present invention also provides the use of the monoclonal antibody TNA1-6C3 described in the above technical solution in preparing a reagent for inhibiting the binding of adalimumab monoclonal antibody to TNF-α protein.

[0031] The present invention also provides the use of the monoclonal antibody TNA1-6C3 described in the above technical solution in preparing a kit for detecting anti-adamimetic neutralizing autoantibodies.

[0032] The present invention also provides a kit for detecting anti-adalimum neutralizing autoantibodies, the kit comprising an anti-adalimum neutralizing autoantibody detection reagent and an anti-TNF-α blocking reagent; the anti-TNF-α blocking reagent is the monoclonal antibody TNA1-6C3 (anti-TNF-a blocking antibody) described in the above technical solution. In the present invention, when the kit is a double antigen sandwich detection kit, the anti-adalimum neutralizing autoantibody detection reagent comprises a magnetic particle working solution, an enzyme label working solution and an indicator solution; the magnetic particle working solution contains an adalimum monoclonal antibody coupled to magnetic particles; the enzyme label working solution contains an enzyme-labeled adalimum Fab antibody. The use of the adalimum Fab antibody can enhance the anti-internal rheumatic factor (RF) interference ability of the kit of the present invention in detecting anti-adalimum neutralizing autoantibodies.

[0033] In the present invention, in the enzyme-labeled working solution, the mass concentration of the enzyme-labeled adalimumab Fab antibody is preferably 0.1-2 μg / mL, more preferably 1 μg / mL. In the present invention, the enzyme in the enzyme-labeled adalimumab Fab antibody preferably includes alkaline phosphatase; when the enzyme in the enzyme-labeled adalimumab Fab antibody is alkaline phosphatase, the indicator solution contains a chromogenic substrate for alkaline phosphatase. In the present invention, the chromogenic substrate is preferably an AMPPD substrate solution. The present invention does not specifically limit the source of the AMPPD substrate solution, which can be purchased from Beijing Deaoping Biology.

[0034]

[0035] In the present invention, the blocking agent of anti-TNF-α is added to the magnetic particle working solution. In the present invention, the preparation method of the magnetic particle working solution preferably comprises the following steps: mixing the magnetic particles with adalimumab monoclonal antibody, shaking reaction, and obtaining magnetic particles-antibodies; after obtaining the magnetic particles-antibodies, the present invention removes the supernatant by magnetic absorption, mixes the magnetic particles-antibodies with TBST for reaction, removes the supernatant by magnetic absorption, and mixes with TBST to obtain a magnetic particle working solution. In the present invention, the adalimumab monoclonal antibody is preferably Xiulemei. In the present invention, the magnetic particles preferably include magnetic beads, more preferably Dynal beads M280Tosyl magnetic beads. In the present invention, the reaction time is preferably 10~40h, more preferably 12h. In the present invention, the reaction temperature is preferably 20~50℃, more preferably 37℃. In the present invention, in the magnetic particle working solution, the mass concentration of the magnetic particles coated with antibodies is preferably 0.2~0.6mg / mL, more preferably 0.4mg / mL. The magnetic particle working solution of the present invention is preferably mixed with an anti-TNF-α blocking agent to prepare a magnetic particle working solution with a blocking function (referred to as the blocking magnetic particle working solution). In the present invention, the mass concentration of the anti-TNF-α blocking agent in the blocking magnetic particle working solution is preferably 10 to 1000 μg / mL, more preferably 200 μg / mL.

[0036] The kit of the present invention adopts the fusion expression of antibody Fab fragment and alkaline phosphatase, which ensures the integrity of the antibody variable region structure and removes the Fc region, and significantly improves the endogenous RF interference ability in the sample. The added anti-TNF-a blocking antibody effectively eliminates the false positive caused by the bridging of the solid phase adalimumab and the adalimumab Fab enzyme conjugate by the multi-TNF-a protein.

[0037] To further illustrate the present invention, a hybridoma cell line TNA1-6C3, monoclonal antibody, kit and application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] Preparation of anti-TNF-a blocking antibodies.

[0040] (1) 0.2 mg TNF-a protein (Suzhou Nearshore Protein Technology Co., Ltd.; C008) was diluted to 1 mg / mL with 20 mM Pb7.4;

[0041] (2) Add 0.2 mL of Biolon QuickAntibody-Mouse5W adjuvant and gently pipette to mix;

[0042] (3) 50 μL / mouse was injected intramuscularly, once every 2 weeks, for a total of 3 immunizations;

[0043] (4) After the last tail vein injection of 10 μg TNF-a protein, the spleen was harvested 3 days later and fused with Sp2 / 0 cells to obtain the supernatant of fused cells with different clone numbers, as shown in the first column of Table 1.

[0044] (5) ELISA method was used to screen anti-TNF-a blocking antibodies.

[0045] TNF-a protein was diluted to 2 μg / mL with CB buffer (50 mM NaHCO3 pH 9.0), coated on ELISA plate overnight, and excess sites on the plate were blocked with 2% BSA. 50 μL 1 μg / mL adalimumab (Xulomec) was added, and 50 μL fusion cell supernatant was added to react for 1 hour, then the plate was washed, and 100 μL HRP-labeled goat anti-human IgG (Suzhou Biolong Technology Co., Ltd.) was added to react for 30 minutes, the plate was washed, TMB colorimetric solution was added to react, 0.1 M sulfuric acid was terminated, and OD was read. 450 The screening results are shown in Table 1 below.

[0046] Table 1 Screening results

[0047]

[0048] The negative control was obtained by replacing 50 μL of fused cell supernatant with 50 μL of cell culture medium.

[0049] Inhibition rate = (1-measured OD / negative control OD) * 100%

[0050] From the ELISA experiment, it can be seen that TNA1-6C3, TNA1-1B1, TNA1-2H4, and TNA1-3F10 can all inhibit the binding of adalimumab to TNF-a protein, indicating that there is a certain overlap in the epitopes. Among them, the TNA1-6C3 antibody showed the best inhibition efficiency and was used for subsequent reagent development. The hybridoma cell line corresponding to the antibody TNA1-6C3 was named hybridoma cell line ( Mus musculus )TNA1-6C3, deposited as a biological product with the deposit number CGMCC No.46006.

[0051] Example 2

[0052] Preparation of Adalimumab Fab-alkaline phosphatase fusion protein.

[0053] The heavy chain Fd downstream of the Fab sequence of the adalimumab drug is fused with alkaline phosphatase through a flexible amino acid linker for expression.

[0054] The amino acid sequence of adalimumab was retrieved from the Drug Bank database.

[0055] Adalimumab Fab antibody light chain sequence SEQ ID NO.1:

[0056] DIQMTQSPSSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0057] Adalimum Fab antibody heavy chain Fd sequence SEQ ID NO.2:

[0058] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV.

[0059] Connector arm SEQ ID NO.4: (GGGS)4.

[0060] Calf intestinal alkaline phosphatase from Uniprot P19111 SEQ ID NO.5:

[0061] LVPVEEEDPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQCKTTRGNEVTSVMNRAKKAGKSVGVVTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQMNGCQDIAAQLVNNMDIDVILGGGRKYMFPVGTPDPEYPDDASVNGVRKRKQNLVQAWQAKHQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLQEMTEVALRVVSRNPRGFYLFVEGGRIDHGHHDDKAYMALTEAGMFDNAIAKANELTSELDTLILVTADHSHVFSFGGYTLRGTSIFGLAPSKALDSKSYTSILYGNGPGYALGGGSRPDVNDSTSEDPSYQQQAAVPQASETHGGEDVAVFARGPQAHLVHGVEEETFVAHIMAFAGCVEPYTDCNLPAPTTAT。

[0062] Adalimumab Fab heavy chain Fd - alkaline phosphatase fusion sequence SEQ ID NO.3:

[0063] .

[0064] The nucleotide sequences corresponding to SEQ ID NO.1 and SEQ ID NO.3 were constructed into pCDNA3.4 vector respectively, and the light chain and heavy chain Fd-ALP fusion proteins were co-expressed using the ExpiCHO transient transfection system of Thermo Fisher Scientific, and the target protein (adalimumab heavy chain Fab-alkaline phosphatase) was obtained by ProteinG purification. The fusion protein with a purity of more than 90% was confirmed by SDS-PAGE electrophoresis. Figure 1 As shown, there are two bands, which are light chain and heavy chain Fd-alkaline phosphatase, indicating that the adalimumab Fab-alkaline phosphatase fusion protein was successfully prepared.

[0065] Example 3

[0066] Establishment of a magnetic particle chemiluminescence method for detecting blood concentration of anti-adamimetic neutralizing antibodies.

[0067] 1) 50 mg Dynal beads M280 Tosyl magnetic beads were diluted in 2 mL 50 mM BB pH 8.0 buffer, and 1 mg adalimumab was added. After mixing, shake at 37 °C for 8 h.

[0068] The supernatant was removed by magnetic suction, and TBST (50 mM Tris, 0.9 wt% NaCl, 0.1 wt% TW20, pH 7.4) was added and reacted at 37 °C for 12 h.

[0069] The supernatant was removed by magnetic suction, and TBST was added to prepare a 10 mg / mL stock solution.

[0070] The above magnetic bead stock solution was diluted to 0.4 mg / mL with TBST, named as control magnetic particle working solution, and stored at 2-8°C for later use;

[0071] The magnetic bead mother solution was diluted to 0.4 mg / mL with TBST containing 200 μg / mL of TNA1-6C3 in Example 1, named as blocking magnetic particle working solution, and stored at 2-8°C for later use;

[0072] The adalimumab Fab-alkaline phosphatase obtained in Example 2 was diluted to 1 μg / mL with 50 mM MES 0.9% NaCl 5 mg / mL BSA 1 mM MgCl2, 0.1 mM ZnCl2, pH 6.7, and named as enzyme labeling working solution.

[0073] Example 4

[0074] Effect of anti-TNF-a blocking antibodies on improving target tolerance of agents.

[0075] Reaction procedure: 10μL sample + 100μL dissociation agent (50mM glycine pH2.5) react for 10 minutes, then add 50μL magnetic microparticle working solution (control magnetic microparticle working solution or blocking magnetic microparticle working solution) and react for 5 minutes, then wash, add 50μL enzyme label working solution, incubate at 37℃ for 5 minutes, wash, and add AMPPD luminescent solution for color development.

[0076] 0 pg / mL, 10 pg / mL, 100 pg / mL, 1000 pg / mL, 10000 pg / mL and 100000 pg / mL TNF-a were prepared in PBS solution containing 7.5% BSA, as well as the serum of patients who had not received any adalimumab-type drugs. The TNF-a concentration was determined using the TNF-a chemiluminescence immunoassay kit of Beijing DanDa Biological. The data in Table 2 were obtained by detection using Beijing DanDa fully automatic chemiluminescence immunoassay instrument DF200.

[0077] Table 2 Target tolerance level of anti-adamimetic neutralizing antibody blood concentration measured by magnetic particle luminescence method

[0078]

[0079] From the data, it can be seen that without anti-TNF-a blocking antibodies, as the TNF-a content in the samples without human-produced anti-adalimumab antibodies becomes higher and higher, a very significant false positive signal is generated through the bridging of the dimeric form of TNF-a, while the reagents added with anti-TNF-a blocking antibodies are not affected by TNF-a and show a target tolerance of at least >100,000 pg / mL.

[0080] Example 5

[0081] Effect of anti-TNF-a blocking antibodies on true signaling.

[0082] Test whether the kit containing anti-TNF-a blocking antibody (magnetic microparticle liquid working solution, enzyme label working solution, AMPPD luminescent solution) will affect the reagent measurement of the true positive signal. 50mM glycine pH2.5 is used as a dissociation agent. The reaction procedure of the reagent is the same as that of Example 4.

[0083] Anti-adalimumab rabbit polyclonal antibody (purchased from Beijing DanDa Biotechnology Co., Ltd.) was prepared at different concentrations using human serum not treated with adalimumab, and the reagents containing and not containing TNA1-6C3 blocker were tested simultaneously. The results are shown in Table 3.

[0084] Table 3 Effect of anti-TNF-a blocking antibodies on true signal

[0085]

[0086] From the results in Table 3, it can be concluded that the anti-TNF-a blocking antibody prepared by the present invention has no significant interference with the positive results produced by the reagent detecting autoantibodies (rabbit polyclonal antibody against adalimumab).

[0087] Example 6

[0088] Performance of the Adalimumab Fab-alkaline phosphatase fusion protein test on clinical samples.

[0089] The anti-interference ability of adalimumab Fab-alkaline phosphatase fusion protein on endogenous rheumatoid factor (RF) in real clinical samples was tested. The reaction procedure of the reagents was the same as that of Example 4.

[0090] The results are shown in Table 4.

[0091] Table 4 Performance of Fab-alkaline phosphatase fusion protein

[0092]

[0093] From the results in Table 4, it can be concluded that the reagent prepared by the adalimumab Fab-alkaline phosphatase fusion protein prepared by the present invention has no significant difference in the results when the sample contains 16272 IU / mL of RF, indicating that the anti-interference ability to RF is extremely strong.

[0094] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A hybridoma cell line ( Mus musculus ) TNA1-6C3, characterized in that The hybridoma cell line TNA1-6C3 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number being CGMCC No.46006.

2. A monoclonal antibody TNA1-6C3, characterized in that: The monoclonal antibody TNA1-6C3 is secreted and produced by the hybridoma cell line TNA1-6C3 according to claim 1.

3. Use of the monoclonal antibody TNA1-6C3 according to claim 2 in the preparation of an anti-TNF-α blocking agent.

4. Use of the monoclonal antibody TNA1-6C3 according to claim 2 in the preparation of an agent for inhibiting the binding of adalimumab monoclonal antibody to TNF-α protein.

5. Use of the monoclonal antibody TNA1-6C3 according to claim 2 in the preparation of a kit for detecting anti-adamimetic neutralizing autoantibodies.

6. A kit for detecting anti-adamimetic neutralizing autoantibodies, characterized in that: The kit comprises an anti-adamimetic neutralizing autoantibody detection reagent and an anti-TNF-α blocking reagent; the anti-TNF-α blocking reagent is the monoclonal antibody TNA1-6C3 described in claim 2.

7. The kit according to claim 6, characterized in that When the kit is a double antigen sandwich detection kit, the anti-adalimumab neutralizing autoantibody detection reagent includes a magnetic particle working solution, an enzyme label working solution and an indicator solution; the magnetic particle working solution contains adalimumab monoclonal antibodies coupled to magnetic particles; and the enzyme label working solution contains enzyme-labeled adalimumab Fab antibodies.

8. The kit according to claim 7, characterized in that The enzyme in the enzyme-labeled adalimumab Fab antibody includes alkaline phosphatase; when the enzyme in the enzyme-labeled adalimumab Fab antibody is alkaline phosphatase, the indicator solution contains a color developing substrate of alkaline phosphatase.

9. The kit according to claim 7 or 8, characterized in that The amino acid sequence of the light chain of the adalimumab Fab antibody is shown in SEQ ID NO.1; the amino acid sequence of the heavy chain Fd of the adalimumab Fab antibody is shown in SEQ ID NO.2; when the enzyme in the enzyme-labeled adalimumab Fab antibody is alkaline phosphatase, the downstream of the heavy chain Fd of the adalimumab Fab antibody is connected to the alkaline phosphatase through a flexible amino acid connecting arm, and the sequence after connection is shown in SEQ ID NO.

3.

10. The kit according to claim 6, characterized in that The anti-TNF-α blocking reagent is added to the magnetic particle working solution.

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