Anti-human tl1a monoclonal antibodies and related kits and detection methods

Abstract

The application provides an anti-human TL1A monoclonal antibody and a related kit and detection method thereof. The ELISA kit of the application can quantitatively detect the content of TL1A in various samples such as cell culture medium, is simple and convenient to operate, has high sensitivity, has good specificity, and can detect TL1A of different species, and has important significance for the research on the pathogenesis of TL1A related autoimmune diseases and drug models.

Description

Technical Field

[0001] This application belongs to the field of immunoassay technology, specifically relating to an anti-human TL1A monoclonal antibody and its related reagent kit and detection method. Background Technology

[0002] TL1A (tumor necrosis factor-like ligand 1A), also known as TNFSF15, is a member of the tumor necrosis factor superfamily. TL1A is a type 2 transmembrane protein that self-assembles into a stable trimer through interaction with the TNF homologous domain (THD). It is expressed in various immune cells, such as monocytes, macrophages, dendritic cells, and T cells, as well as non-immune cells, such as synovial fibroblasts and endothelial cells. TL1A interacts with death receptor 3 (DR3) to activate the TRADD (TNFR-associated death domain) signaling complex, initiating downstream TRAF2, RIP1, PI3K, MAPKs, and NFκB signaling pathways, leading to the release of pro-inflammatory cytokines. The strong interaction between TL1A and DR3 also induces the formation of the FADD (Fas-associated death domain) signaling complex, initiating downstream RIP1 / 3 and Caspase signaling pathways, regulating apoptosis and necrosis.

[0003] Studies have found that TL1A is abnormally expressed in autoimmune diseases and is involved in conditions such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. TL1A plays a crucial role in inflammatory responses due to its unique mechanism of action. Currently, TL1A has become a novel research target for diseases such as ulcerative colitis and Crohn's disease. Summary of the Invention

[0004] The purpose of this application is to provide anti-human TL1A monoclonal antibodies and related kits and detection methods.

[0005] Specifically, this application relates to the following aspects:

[0006] 1. An anti-human TL1A monoclonal antibody, wherein the monoclonal antibody comprises:

[0007] a) Antibody heavy chain complementarity-determining region, which includes: CDR-H1, CDR-H2, and CDR-H3, wherein:

[0008] CDR-H1 has the amino acid sequence of SEQ ID NO 1: SYAIN.

[0009] CDR-H2 has the amino acid sequence SEQ ID NO 2: IMTANLAHTTYYASWAKG, and CDR-H3 has the amino acid sequence SEQ ID NO 3: DYDGTTVYGDAFDP;

[0010] b) Antibody light chain complementarity-determining regions, which include: CDR-L1, CDR-L2, and CDR-L3, wherein:

[0011] CDR-L1 has the amino acid sequence of SEQ ID NO 4: QASQSISRYLA.

[0012] CDR-L2 has the amino acid sequence of SEQ ID NO 5: ETRSLAS.

[0013] CDR-L3 has the amino acid sequence SEQ ID NO 6: QQGYSSSNVDNI.

[0014] 2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody comprises an antibody heavy chain variable region (HCVR) and an antibody light chain variable region (LCVR), wherein:

[0015] HCVR has the sequence SEQ ID NO 13:

[0016] The amino acid sequence of QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAINWVRQAPGRGLEWIGIMT ANLAHTTYYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARDYDGTTV YGDAFDPWGPGTLVTVSS; and / or

[0017] LCVR has the sequence SEQ ID NO 14:

[0018] The amino acid sequence of AYDMTQTPASVSAGVGGTVTIKCQASQSISRYLAWYQQKPGQPPKLLIYE TRSLASGVSSRFKGSGSGTQFTLTINGVQCDDAATYYCQQGYSSSNVDNIFGG GTEVVVK.

[0019] 3. The monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein:

[0020] The amino acid sequence of the heavy chain is SEQ ID NO 17:

[0021] QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAINWVRQAPGRGLEWIGIMTANLAHTTYYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARDYDGTTVYGDAFDPWGPG TLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC The amino acid sequence of PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; and / or

[0022] The amino acid sequence of the light chain is SEQ ID NO 18:

[0023] AYDMTQTPASVSAGVGGTVTIKCQASQSISRYLAWYQQKPGQPPKLLIYETRSLASGVSSRFKGSGSGTQFTLTINGVQCDDAATYYCQQGYSSSNVDNIFGGGTEVVVKRT Amino acid sequence of VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0024] 4. An anti-human TL1A monoclonal antibody, wherein the monoclonal antibody comprises:

[0025] a) Antibody heavy chain complementarity-determining region, which includes: CDR-H1, CDR-H2, and CDR-H3, wherein:

[0026] CDR-H1 has the amino acid sequence of SEQ ID NO 7: SYAMG.

[0027] CDR-H2 has the amino acid sequence SEQ ID NO 8: IISSGNTYYASWAKG, and CDR-H3 has the amino acid sequence SEQ ID NO 9: DRYADDLVEFNL;

[0028] b) Antibody light chain complementarity-determining regions, which include: CDR-L1, CDR-L2, and CDR-L3, wherein:

[0029] CDR-L1 has the amino acid sequence of SEQ ID NO 10: QASESISSWLS.

[0030] CDR-L2 has the amino acid sequence of SEQ ID NO 11: RASTLAS.

[0031] CDR-L3 has the amino acid sequence of SEQ ID NO 12: QQGYIYGDVENV.

[0032] 5. The monoclonal antibody according to claim 4, wherein the monoclonal antibody comprises an antibody heavy chain variable region (HCVR) and an antibody light chain variable region (LCVR), wherein:

[0033] HCVR has the sequence SEQ ID NO 15:

[0034] The amino acid sequence of QSLEESGGRLVTPGTPMKLTCTVSGIDLSSYAMGWVRQAPGKGLEWIGIIS GSGNTYYASWAKGRFTISKTSTTVDLTFSSPTTGDTATYFCARDRYADDLVEFN LWGQGTLVTVSS; and / or

[0035] LCVR has the sequence SEQ ID NO 16:

[0036] The amino acid sequence of AIDMTQTPASVEAGVGGTITINCQASESISSWLSWYQQKPGQRPKLLIYRA STLASGVSSRFKGSGSGTQFTLTISDVECADAATYYCQQGYIYGDVENVFGGG TEVVVK.

[0037] 6. The monoclonal antibody according to claim 4 or 5, wherein the monoclonal antibody comprises a heavy chain and a light chain, wherein:

[0038] The amino acid sequence of the heavy chain is SEQ ID NO 19:

[0039] QSLEESGGRLVTPGTPMKLTCTVSGIDLSSYAMGWVRQAPGKGLEWIGIISGSGNTYYASWAKGRFTISKTSTTVDLTFSSPTTGDTATYFCARDRYADDLVEFNLWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP The amino acid sequence of CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; and / or

[0040] The amino acid sequence of the light chain is SEQ ID NO 20:

[0041] AIDMTQTPASVEAGVGGTITINCQASESISSWLSWYQQKPGQRPKLLIYRASTLASGVSSRFKGSGSGTQFTLTISDVECADAATYYCQQGYIYGDVENVFGGGTEVVVKRT Amino acid sequence of VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0042] 7. A kit for detecting TL1A, wherein the kit comprises a first antibody as a capture antibody and a biotin-labeled second antibody, wherein the first antibody is any one of claims 1 to 3, an anti-human TL1A monoclonal antibody.

[0043] 8. The kit according to item 7, wherein the second antibody is any one of items 4 to 6, an anti-human TL1A monoclonal antibody.

[0044] 9. The kit according to item 7 or 8, wherein the first antibody is immobilized on a solid-phase support.

[0045] 10. The kit according to item 9, wherein the solid-phase carrier is an enzyme-labeled plate.

[0046] 11. The kit according to any one of items 7 to 10, wherein the kit further comprises one or more of the following: horseradish peroxidase-labeled streptavidin, recombinant human TL1A protein standard, substrate, coating antibody diluent, washing buffer, blocking buffer / sample diluent, and stop solution.

[0047] 12. The kit according to any one of items 7 to 11, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0048] 13. The kit according to any one of items 7 to 12, wherein the kit is a modified double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0049] 14. A method for quantitatively detecting TL1A content, wherein a primary antibody and a biotin-labeled secondary antibody are used, and detection is performed using a modified double-antibody sandwich enzyme-linked immunosorbent assay (ELISA).

[0050] The first antibody is any one of items 1 to 3, which is an anti-human TL1A monoclonal antibody.

[0051] 15. The method according to item 14, wherein the second antibody is any one of items 4 to 6, an anti-human TL1A monoclonal antibody.

[0052] 16. The method according to item 14 or 15, wherein the method comprises the following steps:

[0053] The first antibody was immobilized onto a solid-phase support and then coated and blocked.

[0054] Add the sample to be tested, incubate it to allow it to bind to the coated first antibody, and then add the biotin-labeled second antibody for incubation;

[0055] Incubate with diluted horseradish peroxidase-labeled streptavidin;

[0056] Add the substrate colorimetric solution, incubate in the dark, then add the stop solution, measure the OD value, and prepare a standard curve;

[0057] The OD value of the sample to be tested is substituted into the equation to calculate the content of TL1A in the sample to be tested.

[0058] 17. The method according to any one of items 14 to 16, wherein the sample to be tested is a solution containing TL1A.

[0059] 18. The method according to item 17, wherein the sample to be tested is a cell culture medium containing TL1A.

[0060] The beneficial effects of this application are:

[0061] Both anti-human TL1A monoclonal antibodies provided in this application can specifically bind to TL1A, and they have different binding epitopes.

[0062] The ELISA kit of this application can quantitatively detect the content of TL1A in various samples such as cell culture medium. It is simple and convenient to operate, highly sensitive, and has good specificity. It can also detect TL1A in different species, which is of great significance for the study of the pathogenesis of TL1A-related autoimmune diseases and drug models. Attached Figure Description

[0063] Figure 1 This is a flowchart of the transient expression process of antibodies.

[0064] Figure 2 The binding status of anti-human TL1A monoclonal antibody 1# (primary antibody) and monoclonal antibody 2# (secondary antibody) to human TL1A.

[0065] Figure 3 The competitive inhibition of human TL1A binding sites by anti-human TL1A monoclonal antibody 1# (primary antibody) and monoclonal antibody 2# (secondary antibody).

[0066] Figure 4 This is the standard curve of human TL1A.

[0067] Figure 5 Results of the specificity study of the human TL1A ELISA kit.

[0068] Figure 6 The results of cross-species investigation of the human TL1A ELISA kit. Detailed Implementation

[0069] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0070] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0071] In this specification, tumor necrosis factor-like ligand 1A (TL1A), also known as TNFSF15, is a member of the tumor necrosis factor superfamily. TL1A is a type 2 transmembrane protein that self-assembles into a stable trimer through interaction with the TNF homologous domain (THD). It is expressed in various immune cells, such as monocytes, macrophages, dendritic cells, and T cells, as well as non-immune cells, such as synovial fibroblasts and endothelial cells. TL1A interacts with death receptor 3 (DR3) to activate the TRADD (TNFR-associated death domain) signaling complex, initiating downstream TRAF2, RIP1, PI3K, MAPKs, and NFκB signaling pathways, leading to the release of pro-inflammatory cytokines. The strong interaction between TL1A and DR3 also induces the formation of the FADD (Fas-associated death domain) signaling complex, initiating downstream RIP1 / 3 and Caspase signaling pathways, regulating apoptosis and necrosis. The two anti-human TL1A monoclonal antibodies of this application can specifically bind to TL1A, and their binding epitopes are different.

[0072] In this specification, "monoclonal antibody" means an antibody derived from a substantially homologous group of antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles), which are typically present in trace amounts. Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to this application can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies, which structurally comprise heavy and light chains, are described herein.

[0073] In this specification, CDR stands for complement determine region, also known as "hypervariant region." LCDR stands for light chain hypervariant region, and HCDR stands for heavy chain hypervariant region. Generally, both the heavy and light chains of an antibody have three CDRs, which together form the antigen-binding site of Ig. At this site, the antibody can form precise complementarity with the antigenic determinant in its spatial structure. In the following description, the use of the two monoclonal antibodies claimed in this application as "capture antibody" and "detection antibody," respectively, in the analytical technique to specifically bind to TL1A relies on the specific binding ability of the CDR regions defined herein to specific antigenic determinants on TL1A.

[0074] In this specification, CVR is an abbreviation for "variable domain," which is defined in contrast to "constant domain." The variable domains of both heavy and light chains include the three aforementioned "complementary determination regions."

[0075] In this specification, "identity" in the context of amino acid sequences refers to the number of identical residues in two sequences when aligned for maximum correspondence. Many different algorithms known in the art exist for measuring the percentage of amino acid identity (i.e., the Basic Local Alignment Tool or...). Unless otherwise specified, use the default parameters for the specific program or algorithm.

[0076] In this specification, "anti-human TL1A monoclonal antibody" refers to a monoclonal antibody that binds to human TSLP with sufficient affinity, enabling it to be used for the identification / detection of human TL1A and as an antibody drug, for example, as a treatment for autoimmune diseases. Therefore, the sandwich ELISA method established below using the kit of this application can be used as an analytical tool for pharmacokinetic studies of such antibody drugs.

[0077] In this specification, "affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity of a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K0). D Affinity can be measured using common methods known in the art.

[0078] In this specification, "Enzyme-Linked Immunosorbent Assay (ELISA)" refers to a detection method that utilizes the specific binding ability of antibody molecules to antigen molecules to bind free target proteins and target proteins bound to a solid-phase carrier, and then uses special labels for qualitative or quantitative analysis. The principle is as follows: antigens or antibodies can be physically adsorbed onto a solid-phase surface while maintaining their immunological activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds, while maintaining their respective immunological or enzymatic activities; after the enzyme conjugate binds to the corresponding antigen or antibody, the occurrence of an immune reaction can be determined by the color reaction after the addition of a substrate, and the intensity of the color reaction is directly proportional to the amount of the target antigen or antibody in the sample. Various types of detection methods can be designed depending on the substance to be detected and the available detection conditions; the sandwich method is also the most commonly used method for detecting antigens.

[0079] In this specification, a "standard curve" refers to the linear relationship between the content of the sample to be tested and the measurable value (e.g., absorbance value) of the analytical method, so as to achieve quantitative analysis of the sample to be tested.

[0080] This application provides a first anti-human TL1A monoclonal antibody, wherein the monoclonal antibody comprises: a) an antibody heavy chain complementarity-determining region comprising: CDR-H1, CDR-H2, and CDR-H3, wherein: CDR-H1 has the amino acid sequence of SEQ ID NO 1: SYAIN, CDR-H2 has the amino acid sequence of SEQ ID NO 2: IMTANLAHTTYYASWAKG, and CDR-H3 has the amino acid sequence of SEQ ID NO 3: DYDGTTVYGDAFDP; b) an antibody light chain complementarity-determining region comprising: CDR-L1, CDR-L2, and CDR-L3, wherein: CDR-L1 has the amino acid sequence of SEQ ID NO 4: QASQSISRYLA, CDR-L2 has the amino acid sequence of SEQ ID NO 5: ETRSLAS, and CDR-L3 has the amino acid sequence of SEQ ID NO 6: QQGYSSSNVDNI.

[0081] The monoclonal antibody comprises an antibody heavy chain variable region (HCVR) and an antibody light chain variable region (LCVR).

[0082] In one specific embodiment, HCVR has the amino acid sequence of SEQ ID NO 13: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAINWVRQAPGRGLEWIGIMTAN LAHTTYYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARDYDGTTVY GDAFDPWGPGTLVTVSS, or an amino acid sequence that is at least 90% identical to SEQ ID NO 13.

[0083] In one specific embodiment, the LCVR has the amino acid sequence of SEQ ID NO 14: AYDMTQTPASVSAGVGGTVTIKCQASQSISRYLAWYQQKPGQPPKLLIYETR SLASGVSSRFKGSGSGTQFTLTINGVQCDDAATYYCQQGYSSSNVDNIFGGG TEVVVK, or an amino acid sequence having at least 90% identity with SEQ ID NO 14.

[0084] In one specific embodiment, HCVR has the amino acid sequence shown in SEQ ID NO 13, and LCVR has the amino acid sequence shown in SEQ ID NO 14.

[0085] In one specific embodiment, the amino acid sequence of the monoclonal antibody heavy chain is SEQ ID NO 17: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAINWVRQAPGRGLEWI GIMTANLAHTTYYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARDYDGTTVYGDAFDPWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT The amino acid sequence of LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, or an amino acid sequence that is at least 90% identical to SEQ ID NO 17.

[0086] In one specific embodiment, the amino acid sequence of the monoclonal antibody light chain is the amino acid sequence of SEQ ID NO 18: AYDMTQTPASVSAGVGGTVTIKCQASQSISRYLAWYQQKPGQPPKL LIYETRSLASGVSSRFKGSGSGTQFTLTINGVQCDDAATYYCQQGYSSSNVDNIFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, or an amino acid sequence having at least 90% identity with SEQ ID NO 18.

[0087] In one specific embodiment, the amino acid sequence of the heavy chain of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO 17, and the amino acid sequence of the light chain is the amino acid sequence shown in SEQ ID NO 18.

[0088] In this specification, having at least 90% identity means having at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity.

[0089] This application also provides a second anti-human TL1A monoclonal antibody, wherein the monoclonal antibody comprises: a) an antibody heavy chain complementarity-determining region comprising: CDR-H1, CDR-H2, and CDR-H3, wherein: CDR-H1 has the amino acid sequence of SEQ ID NO 7: SYAMG, CDR-H2 has the amino acid sequence of SEQ ID NO 8: IISSGNTYYASWAKG, and CDR-H3 has the amino acid sequence of SEQ ID NO 9: DRYADDLVEFNL; b) an antibody light chain complementarity-determining region comprising: CDR-L1, CDR-L2, and CDR-L3, wherein: CDR-L1 has the amino acid sequence of SEQ ID NO 10: QASESISSWLS, CDR-L2 has the amino acid sequence of SEQ ID NO 11: RASTLAS, and CDR-L3 has the amino acid sequence of SEQ ID NO 12: QQGYIYGDVENV.

[0090] The monoclonal antibody comprises an antibody heavy chain variable region (HCVR) and an antibody light chain variable region (LCVR).

[0091] In one specific embodiment, HCVR has the amino acid sequence of SEQ ID NO 15: QSLEESGGRLVTPGTPMKLTCTVSGIDLSSYAMGWVRQAPGKGLEWIGIISGS GNTYYASWAKGRFTISKTSTTVDLTFSSPTTGDTATYFCARDRYADDLVEFNL WGQGTLVTVSS, or an amino acid sequence having at least 90% identity with SEQ ID NO 15.

[0092] In one specific embodiment, the LCVR has the amino acid sequence of SEQ ID NO 16: AIDMTQTPASVEAGVGGTITINCQASESISSWLSWYQQKPGQRPKLLIYRAST LASGVSSRFKGSGSGTQFTLTISDVECADAATYYCQQGYIYGDVENVFGGGT EVVVK, or an amino acid sequence that has at least 90% identity with SEQ ID NO 16.

[0093] In one specific embodiment, the HCVR has the amino acid sequence shown in SEQ ID NO 15, and the LCVR has the amino acid sequence shown in SEQ ID NO 16.

[0094] In one specific embodiment, the amino acid sequence of the monoclonal antibody heavy chain is SEQ ID NO 19: QSLEESGGRLVTPGTPMKLTCTVSGIDLSSYAMGWVRQAPGKGLE WIGIISGSGNTYYASWAKGRFTISKTSTTVDLTFSSPTTGDTATYFCARDRYADDLVEFNLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM The amino acid sequence of ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, or an amino acid sequence that is at least 90% identical to SEQ ID NO 19.

[0095] In one specific embodiment, the amino acid sequence of the monoclonal antibody light chain is the amino acid sequence of SEQ ID NO 20: AIDMTQTPASVEAGVGGTITINCQASESISSWLSWYQQKPGQRPKLL IYRASTLASGVSSRFKGSGSGTQFTLTISDVECADAATYYCQQGYIYGDVENVFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, or an amino acid sequence having at least 90% identity with SEQ ID NO 20.

[0096] In one specific embodiment, the amino acid sequence of the heavy chain of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO 19, and the amino acid sequence of the light chain is the amino acid sequence shown in SEQ ID NO 20.

[0097] This application also provides a kit for detecting TL1A, wherein the kit includes a first antibody as a capture antibody and a biotin-labeled second antibody.

[0098] In one specific embodiment, the first antibody is the first anti-human TL1A monoclonal antibody described above.

[0099] In one specific embodiment, the second antibody is the aforementioned second type of anti-human TL1A monoclonal antibody.

[0100] In one specific embodiment, the HCVR of the first antibody has the amino acid sequence SEQ ID NO 13: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAINWVRQAPGRGLEWIGIMTAN LAHTTYYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARDYDGTTVY GDAFDPWGPGTLVTVSS, and the LCVR has the amino acid sequence SEQ ID NO 14: AYDMTQTPASVSAGVGGTVTIKCQASQSISRYLAWYQQKPGQPPKLLIYETR SLASGVSSRFKGSGSGTQFTLTINGVQCDDAATYYCQQGYSSSNVDNIFGGG TEVVVK. The second antibody has the amino acid sequence SEQ ID NO 15: QSLEESGGRLVTPGTPMKLTCTVSGIDLSSYAMGWVRQAPGKGLEWIGIISGS GNTYYASWAKGRFTISKTSTTVDLTFSSPTTGDTATYFCARDRYADDLVEFNL WGQGTLVTVSS for HCVR and the amino acid sequence SEQ ID NO 16: AIDMTQTPASVEAGVGGTITINCQASESISSWLSWYQQKPGQRPKLLIYRAST LASGVSSRFKGSGSGTQFTLTISDVECADAATYYCQQGYIYGDVENVFGGGT EVVVK for LCVR.

[0101] In one specific embodiment, the first antibody is immobilized on a solid support. TL1A in the test sample can bind to the first antibody.

[0102] In one specific embodiment, the solid-phase carrier is an ELISA plate, preferably a 96-well ELISA plate, such as a transparent flat-bottomed non-sterile 96-well ELISA plate from Thermo.

[0103] In one specific embodiment, the kit further includes one or more of the following: horseradish peroxidase-labeled streptavidin, recombinant human TL1A protein standard, substrate, coating antibody diluent, washing buffer, blocking / sample diluent, and stop solution.

[0104] In one specific embodiment, the kit is an enzyme-linked immunosorbent assay (ELISA) kit. Preferably, the kit is a modified double-antibody sandwich ELISA kit.

[0105] In one specific implementation, the ELISA kit includes:

[0106] 1) Solid-phase carrier: ELISA plate, 1 piece;

[0107] 2) First antibody: The first antibody mentioned above;

[0108] 3) TL1A standard / quality control products;

[0109] 4) Secondary antibody: Biotin-labeled secondary antibody;

[0110] 5) Enzyme-labeled secondary antibody: horseradish peroxidase-labeled streptavidin (SA-HRP);

[0111] 6) Substrate: 3,3',5,5'-Tetramethylbenzidine (TMB), use after mixing equal volumes of solution A and solution B;

[0112] 7) First antibody dilution solution: phosphate buffer (1×PBS);

[0113] 8) Washing solution: a mixture of PBS solution and Tween 20 (V:V = 100:0.05);

[0114] 9) Diluent / Blocking Buffer: PBS solution containing 0.5% BSA, 0.05% Tween 20, and 0.05% Proclin 300;

[0115] 10) Termination solution: A mixture of water and phosphoric acid (3M).

[0116] This application also relates to a method for quantitatively detecting TL1A content, wherein a first antibody and a biotin-labeled second antibody are used for detection by a double-antibody sandwich enzyme-linked immunosorbent assay, wherein the first antibody is the first antibody mentioned above.

[0117] In one specific implementation, the second antibody is the second type of antibody described above.

[0118] In one specific embodiment, the HCVR of the first antibody has the amino acid sequence SEQ ID NO 13: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAINWVRQAPGRGLEWIGIMTAN LAHTTYYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARDYDGTTVY GDAFDPWGPGTLVTVSS, and the LCVR has the amino acid sequence SEQ ID NO 14: AYDMTQTPASVSAGVGGTVTIKCQASQSISRYLAWYQQKPGQPPKLLIYETR SLASGVSSRFKGSGSGTQFTLTINGVQCDDAATYYCQQGYSSSNVDNIFGGG TEVVVK. The second antibody has the amino acid sequence SEQ ID NO 15: QSLEESGGRLVTPGTPMKLTCTVSGIDLSSYAMGWVRQAPGKGLEWIGIISGS GNTYYASWAKGRFTISKTSTTVDLTFSSPTTGDTATYFCARDRYADDLVEFNL WGQGTLVTVSS for HCVR and the amino acid sequence SEQ ID NO 16: AIDMTQTPASVEAGVGGTITINCQASESISSWLSWYQQKPGQRPKLLIYRAST LASGVSSRFKGSGSGTQFTLTISDVECADAATYYCQQGYIYGDVENVFGGGT EVVVK for LCVR.

[0119] In one specific embodiment, the method includes the following steps: immobilizing the first antibody onto a solid-phase carrier and coating it; adding the sample to be tested and incubating it to allow it to bind to the coated first antibody, then adding a biotin-labeled second antibody and incubating; adding diluted horseradish peroxidase-labeled streptavidin and incubating; adding a substrate chromogenic solution, incubating in the dark, adding a stop solution, measuring the OD value, and preparing a standard curve; and substituting the OD value of the sample to be tested into the equation to calculate the TL1A content in the sample to be tested.

[0120] The sample to be tested can be any solution containing TL1A, such as a common cell culture medium containing TL1A.

[0121] In one specific embodiment, the ELISA kit described in this application can be used to quantitatively detect the TL1A content in a sample, including the following steps:

[0122] (1) Coating: The first antibody was prepared into a working solution with a concentration of 1 μg / ml using the coating antibody dilution buffer. 50 μl was added to the microplate at 2-8℃ and incubated overnight.

[0123] (2) Blocking: Discard the working solution of the coated antibody in the microplate, wash the plate 3 times with washing buffer, add blocking solution at 100 μl / well, and place on a shaker (120 rpm) at room temperature for 2 hours;

[0124] (3) Preparation of protein standards: Take 10 EP tubes and number them sequentially. Starting from the second tube, add 120 μl of sample diluent to each tube and place them on the EP tube rack. Prepare the standard protein to a concentration of 4 ng / ml using the sample diluent. Take 240 μl of the solution and add it to the first EP tube. Then, take 120 μl of the solution from the first EP tube and add it to the second EP tube for serial dilution. Continue this process until the ninth EP tube (the last tube has a concentration of 0).

[0125] (4) Add the test sample and standard: Discard the blocking solution in the microplate, wash the plate 3 times with washing solution, add protein standard solution and test sample at 50 μl / well, and incubate at room temperature for 2 hours on a shaker (120 rpm);

[0126] (5) Add the second antibody: Discard the protein standard solution and the sample to be tested in the ELISA plate, wash the plate 3 times with washing buffer, prepare the second antibody (biotin-labeled) working solution with a concentration of 200 ng / ml using sample dilution buffer, add 50 μl / well to the ELISA plate, and incubate on a shaker (120 rpm) for 2 hours.

[0127] (6) Add enzyme-labeled secondary antibody: Discard the detection antibody working solution in the enzyme-labeled plate, wash the plate 3 times with washing buffer, prepare the enzyme-labeled secondary antibody working solution with a concentration of 100 ng / ml using sample dilution buffer, add 50 μl / well to the enzyme-labeled plate, and incubate at room temperature for 1 hour on a shaker (120 rpm).

[0128] (7) Color development: Mix equal volumes of substrate solution A and solution B; discard the enzyme-labeled secondary antibody working solution in the microplate, wash the plate 3 times with washing buffer, and then add substrate at a dosage of 50 μl / well. Incubate at room temperature in the dark for 10-15 minutes.

[0129] (8) Termination of color development: Add the stop solution to the microplate at a rate of 50 μl / well to terminate the reaction. Then, use a microplate reader to measure the OD value of each well in the microplate at a wavelength of 450 nm. Fit a standard curve based on the OD value of the standard and substitute the OD value of the sample to be tested into the equation to calculate the concentration of TL1A in the sample to be tested, thus completing the quantitative detection.

[0130] The kit and detection method of this application are applicable to the detection of TL1A content in various types of test samples, such as the content of TL1A in cell culture medium, and can also detect TL1A from different species. The kit and detection method of this application have high sensitivity and good specificity, and are of great significance for the study of the pathogenesis of TL1A-related autoimmune diseases and drug models.

[0131] Example

[0132] Example 1: Preparation of the first and second antibodies

[0133] Both the primary and secondary antibodies are anti-human TL1A monoclonal antibodies, and their preparation methods are as follows (e.g. Figure 1 (As shown): ExpiCHO-S cells were co-transfected with the correct sequenced heavy chain expression plasmids and light chain expression plasmids of the first and second antibodies. One day before transfection, ExpiCHO-S cells were diluted to 3 × 10⁻⁶ cells. 6 Cells were passaged at a density of 10 cells / ml before transfection. On the day of transfection, the cell density was diluted to 6 × 10⁻⁶ cells / ml. 6 Cells / ml, 25ml cell suspension in a 125ml shake flask, awaiting transfection. Add 920μl OptiPRO SFM (Gibco) to wells 1 and 2 respectively, then add 12.5μg heavy and light chain plasmids to well 1, and 150μl PEI reagent (Polysciences) to well 2. Transfer well 2 to well 1, mix well, let stand for 15 minutes, and then add to the cells. On days 1 and 3 post-transfection, add 2.5ml of OPM-CHO PFF05 feed medium (OPM). On day 6 post-transfection, harvest the culture supernatant and perform one-step purification using Protein A (NanoTech), obtaining the first antibody (monoclonal antibody 1#) and the second antibody (monoclonal antibody 2#). The first and second antibodies obtained in this application are rabbit monoclonal antibodies and have not undergone humanization. The complete heavy and light chain sequences of the first and second antibodies were obtained by sequencing (commissioned to Sangon Biotech). Then, their CDR sequences could be obtained according to Kabat's rule (reference: Kabat EA, Wu TT, Bilofsky H. Attemptsto locate residues in complementarity-determining regions of antibody combining sites that make contact with antigen. Proc. Natl. Acad. Sci. USA. 1976; 73:617–619.).

[0134] Specifically, the sequence information of monoclonal antibody 1# is as follows:

[0135] CDR-H1 has the amino acid sequence SEQ ID NO 1: SYAIN, CDR-H2 has the amino acid sequence SEQ ID NO 2: IMTANLAHTTYYASWAKG, CDR-H3 has the amino acid sequence SEQ ID NO 3: DYDGTTVYGDAFDP, CDR-L1 has the amino acid sequence SEQ ID NO 4: QASQSISRYLA, CDR-L2 has the amino acid sequence SEQ ID NO 5: ETRSLAS, and CDR-L3 has the amino acid sequence SEQ ID NO 6: QQGYSSSNVDNI.

[0136] HCVR has the amino acid sequence SEQ ID NO 13: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAINWVRQAPGRGLEWIGIMTAN LAHTTYYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARDYDGTTVYGDAFDPWGPGTLVTVSS.

[0137] LCVR has the amino acid sequence SEQ ID NO 14: AYDMTQTPASVSAGVGGTVTIKCQASQSISRYLAWYQQKPGQPPKLLIYETR SLASGVSSRFKGSGSGTQFTLTINGVQCDDAATYYCQQGYSSSNVDNIFGGG TEVVVK.

[0138] The amino acid sequence of the heavy chain is SEQ ID NO 17: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAINWVRQAPGRGLEWIGIMTANLAHTTYYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARDYDGTTVYGDAFDPW GPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK Amino acid sequence of AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0139] The amino acid sequence of the light chain is SEQ ID NO 18: AYDMTQTPASVSAGVGGTVTIKCQASQSISRYLAWYQQKPGQPPKLLIYETRSLASGVSSRFKGSGSGTQFTLTINGVQCDDAATYYCQQGYSSSNVDNIFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0140] The sequence information of monoclonal antibody #2 is as follows:

[0141] CDR-H1 has the amino acid sequence SEQ ID NO 7: SYAMG, CDR-H2 has the amino acid sequence SEQ ID NO 8: IISSGNTYYASWAKG, CDR-H3 has the amino acid sequence SEQ ID NO 9: DRYADDLVEFNL, CDR-L1 has the amino acid sequence SEQ ID NO 10: QASESISSWLS, CDR-L2 has the amino acid sequence SEQ ID NO 11: RASTLAS, and CDR-L3 has the amino acid sequence SEQ ID NO 12: QQGYIYGDVENV.

[0142] HCVR has the amino acid sequence SEQ ID NO 15: QSLEESGGRLVTPGTPMKLTCTVSGIDLSSYAMGWVRQAPGKGLEWIGIISGS GNTYYASWAKGRFTISKTSTTVDLTFSSPTTGDTATYFCARDRYADDLVEFNL WGQGTLVTVSS.

[0143] LCVR has the amino acid sequence SEQ ID NO 16: AIDMTQTPASVEAGVGGTITINCQASESISSWLSWYQQKPGQRPKLLIYRAST LASGVSSRFKGSGSGTQFTLTISDVECADAATYYCQQGYIYGDVENVFGGGT EVVVK.

[0144] The amino acid sequence of the heavy chain is SEQ ID NO 19: QSLEESGGRLVTPGTPMKLTCTVSGIDLSSYAMGWVRQAPGKGLEWIGIISGSGNTYYASWAKGRFTISKTSTTVDLTFSSPTTGDTATYFCARDRYADDLVEFNLWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA Amino acid sequence of KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0145] The amino acid sequence of the light chain is SEQ ID NO 20: AIDMTQTPASVEAGVGGTITINCQASESISSWLSWYQQKPGQRPKLLIYRASTLASGVSSRFKGSGSGTQFTLTISDVECADAATYYCQQGYIYGDVENVFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0146] Example 2: Characterization of anti-human TL1A monoclonal antibodies #1 and #2

[0147] 1. Binding assay of monoclonal antibody to human TL1A:

[0148] The microplates were coated with monoclonal antibodies #1 and #2, respectively; after blocking, serially diluted human TL1A was added and incubated at room temperature for 2 hours; after washing, Anti-his-HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour; finally, after washing, color development was performed, and the absorbance (OD) at 450 nm was read. 450nm .

[0149] The results are as follows Figure 2 As shown, both monoclonal antibodies 1# and 2# can bind to human TL1A.

[0150] 2. Competitive inhibition experiment of antibody recognition antigen site:

[0151] The microplate was coated with human TL1A overnight; after blocking, equal volumes of serially diluted monoclonal antibodies 1# and 2#, and a certain concentration of biotin-labeled monoclonal antibody 1# were added sequentially, and the plates were incubated at room temperature for 2 hours; after washing, SA HRP-labeled secondary antibody was added, and the plates were incubated at room temperature for 1 hour; after final washing, the plates were developed, and the absorbance OD at 450 nm was read. 450nm .

[0152] The results are as follows Figure 3 As shown, monoclonal antibodies 1# and 2# do not compete with the binding site of human TL1A antigen, and monoclonal antibody 2# can be used as the detection antibody in the human TL1A kit.

[0153] 3. Detection of biotinylation of antibody (monoclonal antibody 2#):

[0154] Biotin labeling for antibody detection is used The Sulfo-NHS-LC-Biotin kit was used, and the specific steps were strictly followed according to the kit's instructions. After labeling, Zeba was used. TM Spin Deasling Colums filtration removes free biotin, and the resulting solution is the biotin-labeled detection antibody (monoclonal antibody 2#).

[0155] Example 3: Human TL1A ELISA Quantitative Detection ELISA Kit

[0156] The composition, specifications, source, and storage conditions of this kit are shown in Table 1 below.

[0157] Table 1

[0158]

[0159]

[0160] Example 4: Quantitative detection of human TL1A in cell culture medium using an ELISA kit

[0161] 1. Sample preparation:

[0162] The protein (recombinant human TL1A protein) standard was diluted separately with sample diluent and cell culture medium (RPMI 1640 complete medium). The protein standard diluted with cell culture medium was used as the test sample. A standard curve was fitted based on the absorbance value of the protein standard diluted with sample diluent. The absorbance value of the test sample was substituted into the standard curve to obtain the theoretical concentration of human TL1A in the test sample. Then, the recovery rate of the test sample was calculated (recovery rate = theoretical concentration value / actual concentration value × 100%), thereby analyzing the feasibility of using this ELISA kit for the quantitative detection of human TL1A in cell culture medium.

[0163] 2. Quantitative detection procedure:

[0164] (1) Coating: The coating antibody was prepared into a working solution with a concentration of 1 μg / ml using the coating antibody dilution buffer. 50 μl was added to the microplate at 2-8℃ and incubated overnight.

[0165] (2) Blocking: Discard the working solution of the coated antibody in the microplate, wash the plate 3 times with washing buffer, add blocking solution at 100 μl / well, and place on a shaker (120 rpm) at room temperature for 2 hours;

[0166] (3) Preparation of protein standards: Take 10 EP tubes and number them sequentially. Starting from the second tube, add 120 μl of sample diluent to each tube and place them on the EP tube rack. Prepare the standard protein to a concentration of 4 ng / ml using the sample diluent. Take 240 μl of the solution and add it to the first EP tube. Then, take 120 μl of the solution from the first EP tube and add it to the second EP tube for serial dilution. Continue this process until the ninth EP tube (the last tube has a concentration of 0).

[0167] (4) Add the test sample and standard: Discard the blocking solution in the microplate, wash the plate 3 times with washing solution, add protein standard solution and test sample at 50 μl / well, and incubate at room temperature for 2 hours on a shaker (120 rpm);

[0168] (5) Add detection antibody: Discard the protein standard solution and the sample to be tested in the ELISA plate, wash the plate 3 times with washing buffer, prepare the detection antibody (biotin-labeled) working solution with a concentration of 200 ng / ml with sample dilution buffer, add 50 μl / well to the ELISA plate, and incubate on a shaker (120 rpm) for 2 hours;

[0169] (6) Add enzyme-labeled secondary antibody: Discard the detection antibody working solution in the enzyme-labeled plate, wash the plate 3 times with washing buffer, prepare the enzyme-labeled secondary antibody working solution with a concentration of 100 ng / ml using sample dilution buffer, add 50 μl / well to the enzyme-labeled plate, and incubate at room temperature for 1 hour on a shaker (120 rpm).

[0170] (7) Color development: Mix equal volumes of substrate solution A and solution B; discard the enzyme-labeled secondary antibody working solution in the microplate, wash the plate 3 times with washing buffer, and then add substrate at a dosage of 50 μl / well. Incubate at room temperature in the dark for 10-15 minutes.

[0171] (8) Termination of color development: Add the stop solution to the microplate at a rate of 50 μl / well to terminate the reaction. Then, use a microplate reader to measure the OD value of each well in the microplate at a wavelength of 450 nm. Fit a standard curve based on the OD value of the standard and substitute the OD value of the sample to be tested into the equation to calculate the concentration of human TL1A in the sample to be tested, thus completing the quantitative detection.

[0172] 3. Test Results:

[0173] (1) The detection data of human TL1A are shown in Table 2 below:

[0174] Table 2

[0175]

[0176] (2) Standard curve equation: 4-P Fit: y=(0.014-2.130) / (1+(x / 1.157)^1.018)+2.130, R 2 =0.999 (see Figure 4 ).

[0177] (3) The detection data of the cell culture medium diluted with the test sample are shown in Table 3 below:

[0178] Table 3

[0179]

[0180] The results showed that the recovery rates at each concentration point were between 101% and 114%, indicating that the cell culture medium had no effect on the experimental results. This ELISA method is applicable to the determination of human TL1A content in cell culture medium systems.

[0181] 4. Accuracy assessment:

[0182] (1) In three different experiments, three known concentrations of human TL1A (2.5, 0.2, and 0.02 ng / ml) diluted with sample diluent were used as test samples. Two replicates were set up for each sample to perform quantitative detection of human TL1A and to analyze the accuracy of the kit.

[0183] The results are shown in Table 4 below.

[0184] Table 4

[0185]

[0186] The results showed that the coefficient of variation (CV) was less than 20%, indicating that the detection method has good accuracy.

[0187] (2) In three different experiments, three known concentrations of human TL1A (2.5, 0.2, and 0.02 ng / ml) diluted with cell culture medium were used as test samples. Two replicates were set up for each sample to perform quantitative detection of human TL1A and to analyze the accuracy of the kit.

[0188] The results are shown in Table 5 below.

[0189] Table 5

[0190]

[0191] The results showed that the coefficient of variation (CV) was less than 20%, indicating that the detection method has good accuracy.

[0192] 5. Specificity assessment:

[0193] Different concentration gradients of human TL1A, human LIGHT, human RANKL, human TNFβ, human Fas Ligand, human CD27LTrimer, human CD40L, human TNFα, human Trail, and human BAFF proteins (100, 16.667, 2.778, 0.463, 0.077, 0.013, 0.002, 0 ng / ml) were added to ELISA wells pre-coated with anti-human TL1A monoclonal antibody (1#). The absorbance of each protein at different concentrations was measured using the established ELISA detection method.

[0194] The results are as follows Figure 5 As shown, the OD of the human TL1A proteome is displayed. 450nm It exhibits a significant dose-dependent effect across different concentrations, OD 450nm The absorbance increased with increasing protein concentration, while the absorbance values ​​of human LIGHT, human RANKL, human TNFβ, human FasLigand, human CD27L Trimer, human CD40L, human TNFα, human Trail, and human BAFF proteins were all close to 0, showing no dose-dependent effect. The experimental results indicate that this ELISA detection method has good specificity.

[0195] 6. Sensitivity test:

[0196] Different concentration gradients of human TL1A protein (4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 0 pg / ml) were added to ELISA wells pre-coated with anti-human TL1A monoclonal antibody (1#). Three replicates were set up, and the established ELISA detection method was used to quantitatively detect human TL1A. The sensitivity of the kit was analyzed.

[0197] The results are shown in Table 6 below.

[0198] Table 6

[0199]

[0200] The results showed that the recovery rates at each point were between 92% and 107% when the concentration ranged from 4000 to 15.625 pg / ml, indicating that the sensitivity of this ELISA method can reach 15.625 pg / ml.

[0201] 7. Cross-species and cross-genus investigation:

[0202] Different concentration gradients of cynomolgus monkey TL1A, rhesus monkey TL1A, and guinea pig TL1A (4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0 ng / ml) were added to ELISA wells pre-coated with anti-human TL1A monoclonal antibody (1#). The absorbance values ​​of TL1A at different concentrations from different species were measured using the established ELISA method. The results are as follows: Figure 6 As shown in Table 7.

[0203] Figure 6 The OD values ​​of cynomolgus monkey TL1A, rhesus monkey TL1A, and guinea pig TL1A were shown. 450nm It exhibits a significant dose-dependent effect across different concentrations, OD 450nm The absorbance value increased with increasing protein concentration. Experimental results show that this ELISA method can detect TL1A in cynomolgus monkeys, rhesus monkeys, and guinea pigs, demonstrating its wide applicability.

[0204] Table 7

[0205]

[0206] Table 7 shows that when the TL1A concentration in cynomolgus monkeys was in the range of 4–0.015625 ng / ml, the recovery rates at each point were between 88% and 110%, indicating that the sensitivity of this ELISA method for detecting TL1A in cynomolgus monkeys can reach 0.015625 ng / ml. When the TL1A concentration in rhesus monkeys was in the range of 4–0.25 ng / ml, the recovery rates at each point were between 92% and 108%, indicating that the sensitivity of this ELISA method for detecting TL1A in rhesus monkeys can reach 0.25 ng / ml. When the TL1A concentration in guinea pigs was in the range of 4–0.5 ng / ml, the recovery rates at each point were between 98% and 103%, indicating that the sensitivity of this ELISA method for detecting TL1A in guinea pigs can reach 0.5 ng / ml.

Claims

1. An anti-human TL1A monoclonal antibody, wherein, The monoclonal antibody comprises: a) an antibody heavy chain complementarity determining region comprising: HCDR1, HCDR2, HCDR3, wherein: HCDR1 is the amino acid sequence of sequence SEQ ID NO 1 : SYAIN, HCDR2 is the amino acid sequence of sequence SEQ ID NO 2: IMTANLAHTTYYASWAKG, HCDR3 is the amino acid sequence of sequence SEQ ID NO 3: DYDGTTVYGDAFDP; b) an antibody light chain complementarity determining region comprising: LCDR1, LCDR2, LCDR3, wherein: LCDR1 is the amino acid sequence of sequence SEQ ID NO 4: QASQSISRYLA, LCDR2 is the amino acid sequence of sequence SEQ ID NO 5: ETRSLAS, LCDR3 is the amino acid sequence of sequence SEQ ID NO 6: QQGYSSSNVDNI.

2. The monoclonal antibody of claim 1, wherein, The monoclonal antibody comprises an antibody heavy chain variable region HCVR and an antibody light chain variable region LCVR, wherein: HCVR is the amino acid sequence of sequence SEQ ID NO 13: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAINWVRQAPGRGLEWIGIMTANLAHTTYYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARDYDGTTVYGDAFDPWGPGTLVTVSS; and / or LCVR is the amino acid sequence of sequence SEQ ID NO 14: AYDMTQTPASVSAGVGGTVTIKCQASQSISRYLAWYQQKPGQPPKLLIYETRSLASGVSSRFKGSGSGTQFTLTINGVQCDDAATYYCQQGYSSSNVDNIFGGGTEVVVK.

3. The monoclonal antibody of claim 1 or 2, wherein, The monoclonal antibody comprises a heavy chain and a light chain, wherein: the amino acid sequence of the heavy chain is SEQ ID NO 17: the amino acid sequence of SEQ ID NO 18: AYDMTQTPASVSAGVGGTVTIKCQASQSISRYLAWYQQKPGQPPKLLIYETRSLASGVSSRFKGSGSGTQFTLTINGVQCDDAATYYCQQGYSSSNVDNIFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. The kit comprises a first antibody as a capture antibody and a biotin-labeled second antibody, the first antibody being the anti-human TL1A monoclonal antibody according to any one of claims 1-3.

4. A kit for detecting TL1A, wherein, The first antibody is immobilized on a solid support.

5. The kit of claim 4, wherein, The solid support is an enzyme-labeled plate.

6. The kit of claim 5, wherein, The kit further comprises one or more of the following: horseradish peroxidase-labeled streptavidin, a recombinant human TL1A protein standard, a substrate, a coating antibody diluent, a washing solution, a blocking solution, a sample diluent, and a termination solution.

7. The kit of claim 4, wherein, The kit is an enzyme-linked immunosorbent assay kit.

8. The kit of claim 4, wherein, The kit is a modified double-antibody sandwich enzyme-linked immunosorbent assay kit.

9. The kit of any one of claims 4-8, wherein, The detection is performed using a first antibody and a biotin-labeled second antibody by a modified double-antibody sandwich enzyme-linked immunosorbent assay, 10. A method of quantitatively detecting the amount of TL1A for non-diagnostic purposes, wherein, The first antibody is the anti-human TL1A monoclonal antibody according to any one of claims 1-3. ​ 11. The method of claim 10, wherein, The method comprises the following steps: The first antibody is fixed to a solid carrier for coating and blocking; The sample to be tested is added and incubated to combine with the coated first antibody, and then a biotin-labeled second antibody is added and incubated; Diluted horseradish peroxidase-labeled streptavidin is added and incubated; Substrate color developing liquid is added, and after incubation in the dark, stop solution is added, the OD value is determined, and a standard curve is prepared; The OD value of the sample to be tested is substituted into the equation to calculate the content of TL1A in the sample to be tested.

12. The method of claim 10 or 11, wherein, The sample to be tested is a solution containing TL1A.

13. The method of claim 12, wherein, The sample to be tested is a cell culture medium containing TL1A.

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