Human IL-13 antibody, ELISA kit and detection method thereof

Abstract

The invention discloses a human IL-13 antibody, an ELISA kit containing the human IL-13 antibody and a detection method of the human IL-13 antibody. Specifically, the ELISA kit provided by the invention comprises a pair of anti-human IL-13 monoclonal antibodies which are respectively used as a coating antibody and a detection antibody in a double-antibody sandwich ELISA method. The kit can be used for successfully completing quantitative detection of human IL-13, has relatively high detection sensitivity and good detection specificity, can be used for detecting a plurality of species of IL-13, and has good development and application prospects.

Description

Technical Field

[0001] The present application belongs to the technical field of immunoassay, and relates to an antibody binding to human interleukin 13 and an ELISA kit for quantitatively detecting human interleukin 13, as well as a method for quantitative detection using the kit. Background Art

[0002] Interleukin-13 (IL-13) is a Th2-type cytokine that can be secreted by a variety of cells, such as CD4+T cells, CD8+T cells, mast cells, basophils, eosinophils, natural killer cells, and type 2 helper T cells (Th2). IL-13 has a variety of biological effects on mononuclear macrophages, B lymphocytes, NK cells, and vascular endothelial cells, and is a regulatory mediator of the body's immune response. IL-13 receptors (IL-13R) include IL-4Rα, IL-13Rα1, and IL-13Rα2. IL-4Rα can form a heterodimer with IL-13Rα1, while IL-13Rα2 exists in monomeric form. IL-13 is first recruited by IL-13Rα1, and then the binary IL-13 / IL-13Rα1 complex combines with IL-4Rα to form a ternary signaling complex to exert its effect.

[0003] IL-13 is overexpressed and persists in atopic dermatitis (AD), and downstream genes induced by IL-13 are also upregulated. It plays a multifaceted role in the pathophysiology of AD, including the spread of inflammation, epidermal barrier dysfunction, and pruritus. IL-13 also plays an important role in the occurrence of asthma by inducing B cell proliferation and differentiation, promoting IgE synthesis, activating eosinophils, and inducing airway hyperresponsiveness.

[0004] Currently, there are few types of kits for measuring interleukin 13 content on the market, and they are expensive. The ELISA kit in this application can quantitatively detect the content of IL-13 in cell culture medium, is simple and convenient to operate, has high sensitivity, has good specificity, and can detect IL-13 of different species, which is of great significance to the study of the pathogenesis of IL-13-related autoimmune diseases and drug models. Summary of the invention

[0005] In order to overcome the deficiencies in the prior art, the present application provides a monoclonal antibody against human interleukin 13, and a human interleukin 13 ELISA kit comprising the antibody and developed based on a modified double antibody sandwich ELISA method, wherein the kit can be used to achieve quantitative detection of human interleukin 13.

[0006] The specific technical solutions of this application are as follows:

[0007] 1. An anti-human IL-13 monoclonal antibody, wherein the anti-human IL-13 monoclonal antibody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

[0008] The amino acid sequence of CDR-H1 (in this specification, CDR-H1 means heavy chain CDR1) is shown in SEQ ID No: 1 (SSAMS) or SEQ ID No: 7 (SYYMS);

[0009] The amino acid sequence of CDR-H2 (in this specification, CDR-H2 means heavy chain CDR2) is shown in SEQ ID No: 2 (AISASGTTYYASWVNG) or SEQ ID No: 8 (SIFPSGSTDYATWARG);

[0010] The amino acid sequence of CDR-H3 (in this specification, CDR-H3 means heavy chain CDR3) is shown in SEQ ID No: 3 (EDAGYIGDYFNL) or SEQ ID No: 9 (NPNYNFATGFNI);

[0011] The amino acid sequence of CDR-L1 (in this specification, CDR-L1 means light chain CDR1) is shown in SEQ ID No: 4 (QASQSIGSGFA) or SEQ ID No: 10 (QASQNIYSNLA);

[0012] The amino acid sequence of CDR-L2 (in this specification, CDR-L2 means light chain CDR2) is shown in SEQ ID No: 5 (AASNLAS) or SEQ ID No: 11 (KASTLES);

[0013] The amino acid sequence of CDR-L3 (in the present specification, CDR-L3 refers to light chain CDR3) is shown in SEQ ID No: 6 (QCTYYGSSYVDNA) or SEQ ID No: 12 (QNNYYSGSSSSSTYT).

[0014] 2. The anti-human IL-13 monoclonal antibody according to item 1, wherein

[0015] The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 1 (SSAMS);

[0016] The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 2 (AISASGTTYYASWVNG);

[0017] The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 3 (EDAGYIGDYFNL);

[0018] The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 4 (QASQSIGSGFA);

[0019] The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 5 (AASNLAS);

[0020] The amino acid sequence of the CDR-L3 is shown in SEQ ID No: 6 (QCTYYGSSYVDNA).

[0021] 3. The anti-human IL-13 monoclonal antibody according to item 1, wherein

[0022] The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 7 (SYYMS);

[0023] The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 8 (SIFPSGSTDYATWARG);

[0024] The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 9 (NPNYNFATGFNI);

[0025] The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 10 (QASQNIYSNLA);

[0026] The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 11 (KASTLES);

[0027] The amino acid sequence of the CDR-L3 is shown in SEQ ID No: 12 (QNNYYSGSSSSSTYT).

[0028] 4. The anti-human IL-13 monoclonal antibody according to any one of items 1 to 3, wherein the anti-human IL-13 monoclonal antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein:

[0029] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13

[0030] (QSVEESGGRLVTPGTPLTLTCTVSGFSLSSSAMSWVRQAPGKGLEWIG AISASGTTYYASWVNGRFTISKTSTTVDLKITSPTTEDTATYFCAREDAGY IGDYFNLWGPGTLVTVSS) or SEQ ID No:15

[0031] (QSLEESGGRLVTPGTPLTVTCTVSGIDLSSYYMSWVRQAPGKGLEWIG SIFPSGSTDYATWARGRFTISKTSTTVDLKMTSLTASDTATYFCATNPNY NFATGFNIWGPGTLVTVSS), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 13 or SEQ ID No: 15;

[0032] The amino acid sequence of the light chain variable region is as shown in SEQ ID No: 14

[0033] (DVVMTQTPASVSEPVGGTVTVKCQASQSIGSGFAWYQQKPGQPPSLLI YAASNLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQCTYYGSSYV DNAFGGGTEVVVK) or SEQ ID No:16

[0034] (DIVLTQTPSSVSAAVGGTVTINCQASQNIYSNLAWYQQKPGQRPKLLIY KASTLESGVPSRFKGSGSGTHYTLTISDLECADAATYYCQNNYYSGSSSS STYTFGGGTEVVVK), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No: 14 or SEQ ID No: 16.

[0035] 5. The anti-human IL-13 monoclonal antibody according to item 4, wherein

[0036] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13

[0037] (QSVEESGGRLVTPGTPLTLTCTVSGFSLSSSAMSWVRQAPGKGLEWIG AISASGTTYYASWVNGRFTISKTSTTVDLKITSPTTEDTATYFCAREDAGY IGDYFNLWGPGTLVTVSS), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No: 13;

[0038] The amino acid sequence of the light chain variable region is as shown in SEQ ID No: 14

[0039] (DVVMTQTPASVSEPVGGTVTVKCQASQSIGSGFAWYQQKPGQPPSLLI YAASNLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQCTYYGSSYVDNAFGGGTEVVVK), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No:14.

[0040] 6. The anti-human IL-13 monoclonal antibody according to item 4, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 15

[0041] (QSLEESGGRLVTPGTPLTVTCTVSGIDLSSYYMSWVRQAPGKGLEWIG SIFPSGSTDYATWARGRFTISKTSTTVDLKMTSLTASDTATYFCATNPNY NFATGFNIWGPGTLVTVSS), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 15;

[0042] The amino acid sequence of the light chain variable region is as shown in SEQ ID No: 16

[0043] (DIVLTQTPSSVSAAVGGTVTINCQASQNIYSNLAWYQQKPGQRPKLLIY KASTLESGVPSRFKGSGSGTHYTLTISDLECADAATYYCQNNYYSGSSSS STYTFGGGTEVVVK), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No:16.

[0044] 7. The anti-human IL-13 monoclonal antibody according to any one of items 1 to 6, wherein the anti-human IL-13 monoclonal antibody comprises a heavy chain and a light chain, wherein:

[0045] The amino acid sequence of the heavy chain is as shown in SEQ ID No: 17

[0046] (QSVEESGGRLVTPGTPLTLTCTVSGFSLSSSAMSWVRQAPGKGLEWIG AISASGTTYYASWVNGRFTISKTSTTVDLKITSPTTEDTATYFCAREDAGYIGDYFNLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK) or SEQ ID No:19

[0047] (As shown in (QSLEESGGRLVTPGTPLTVTCTVSGIDLSSYYMSWVRQAPGKGLEWIG SIFPSGSTDYATWARGRFTISKTSTTVDLKMTSLTASDTATYFCATNPNY NFATGFNIWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK));

[0048] The amino acid sequence of the light chain is as shown in SEQ ID No:18

[0049] (DVVMTQTPASVSEPVGGTVTVKCQASQSIGSGFAWYQQKPGQPPSLLI YAASNLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQCTYYGSSYVDNAFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) or SEQ ID No:20

[0050] (DIVLTQTPSSVSAAVGGTVTINCQASQNIYSNLAWYQQKPGQRPKLLIY KASTLESGVPSRFKGSGSGTHYTLTISDLECADAATYYCQNNYYSGSSSSSTYTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

[0051] 8. The anti-human IL-13 monoclonal antibody according to item 7, wherein the anti-human IL-13 monoclonal antibody is anti-human IL-13 monoclonal antibody 1#, whose heavy chain has the sequence as shown in SEQ ID No: 17 (QSVEESGGRLVTPGTPLTLTCTVSGFSLSSSAMSWVRQAPGKGLEWIG AISASGTTYYASWVNGRFTISKTSTTVDLKITSPTTEDTATYFCAREDAGYIGDYFNLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), the light chain has the amino acid sequence shown in SEQ ID No:18(DVVMTQTPASVSEPVGGTVTVKCQASQSIGSGFAWYQQKPGQPPSLLI YAASNLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQCTYYGSSYVDNAFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

[0052] 9. The anti-human IL-13 monoclonal antibody according to item 7, wherein the anti-human IL-13 monoclonal antibody is anti-human IL-13 monoclonal antibody 2#, whose heavy chain has the sequence as shown in SEQ ID No: 19 (QSLEESGGRLVTPGTPLTVTCTVSGIDLSSYYMSWVRQAPGKGLEWIGSIFPSGSTDYATWARGRFTISKTSTTVDLKMTSLTASDTATYFCATNPNYNFATGFNIWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), the light chain has the amino acid sequence shown in SEQ ID No:20(DIVLTQTPSSVSAAVGGTVTINCQASQNIYSNLAWYQQKPGQRPKLLIY KASTLESGVPSRFKGSGSGTHYTLTISDLECADAATYYCQNNYYSGSSSSTYTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

[0053] 10. A nucleic acid molecule encoding the anti-human IL-13 monoclonal antibody according to any one of items 1 to 9.

[0054] 11. Use of the anti-human IL-13 monoclonal antibody according to any one of Items 1 to 9, or the nucleic acid molecule according to Item 10, in the preparation of a kit for detecting a disease associated with IL-13 expression.

[0055] 12. A kit for detecting human IL-13, wherein the kit comprises:

[0056] A first antibody (coating antibody) that has been or is to be fixed to a solid phase carrier and a second antibody (detection antibody) labeled with biotin,

[0057] The first antibody and the second antibody are the anti-human IL-13 monoclonal antibodies according to any one of items 1 to 9.

[0058] 13. The kit according to item 12, wherein the solid phase carrier is an ELISA plate.

[0059] 14. The kit according to item 12, wherein the kit further comprises horseradish peroxidase-labeled streptavidin, a recombinant human IL-13 protein standard, a substrate, a coating antibody diluent, a washing solution, a blocking solution or a sample diluent, and a stop solution.

[0060] 15. The kit according to item 12, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0061] 16. The kit according to item 12, wherein the kit is a modified double antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0062] 17. The kit according to any one of items 12 to 16, wherein the first antibody is anti-human IL-13 monoclonal antibody 1#, and the second antibody is anti-human IL-13 monoclonal antibody 2#.

[0063] 18. Use of the kit described in any one of items 12 to 17 for detecting interleukin 13 in cell culture fluid or human serum.

[0064] 19. A method for detecting the content of IL-13 in a sample, wherein the method comprises using the anti-human IL-13 monoclonal antibody described in any one of items 1 to 9 to perform ELISA detection using a modified double antibody sandwich method.

[0065] 20. The method according to item 19, comprising the following steps:

[0066] Coating a solid phase carrier with a primary antibody;

[0067] Add the sample to be tested to the coated solid phase carrier and incubate;

[0068] Adding a biotin-labeled second antibody to the solid phase carrier and incubating;

[0069] diluting peroxidase-labeled streptavidin, adding it to the solid phase carrier, and incubating;

[0070] Add the substrate to the solid phase carrier, incubate in the dark, add the stop solution, and measure the OD value;

[0071] The standard curve was fitted using the OD value of the recombinant human IL-13 protein standard, and the OD value of the sample to be tested was substituted into the equation to calculate the content of human IL-13 in the sample to be tested;

[0072] Wherein, the first antibody and the second antibody are the anti-human IL-13 monoclonal antibodies described in any one of items 1 to 9, preferably, the first antibody is the anti-human IL-13 monoclonal antibody 1#, and the second antibody is the anti-human IL-13 monoclonal antibody 2#.

[0073] Effects of the Invention

[0074] The present application provides a novel anti-human IL-13 monoclonal antibody and a human IL-13 kit containing the antibody. The kit uses a modified double antibody sandwich enzyme-labeled immunosorbent assay to determine the level of interleukin-13 in a sample, has high detection sensitivity, good detection specificity, and can detect IL-13 of different species, and has good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 This is a flowchart of transient expression.

[0076] Figure 2 Schematic diagram of the binding activity of the anti-human IL-13 monoclonal antibody of the present application with human IL-13.

[0077] Figure 3 Schematic diagram of the competitive inhibition of the first antibody and the second antibody against the human IL-13 binding site.

[0078] Figure 4 This is a standard curve graph for human IL-13.

[0079] Figure 5 This is a schematic diagram of the specificity investigation of the human IL-13 ELISA kit of the present application.

[0080] Figure 6 This is a schematic diagram of species cross-check of the human IL-13 ELISA kit of the present application. DETAILED DESCRIPTION

[0081] The present application is further described in detail below in conjunction with specific implementation methods. The implementation methods given are intended to enable a more thorough understanding of the present application and to fully convey the scope of the present application to technicians in the field.

[0082] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0083] In the present application, "monoclonal antibody" means an antibody derived from a population of substantially homologous antibodies, that is, each antibody constituting the population is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody products), such variants are usually present in trace amounts. Different from polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody population, and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using all or part of a transgenic animal comprising a human immunoglobulin locus, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0084] In some embodiments of the present application, the antibody of the present application is a full-length antibody, which typically refers to an antibody composed of two "heavy chains" and two "light chains". A "heavy chain" is generally a polypeptide that consists of a heavy chain variable region (VH), a heavy chain constant region 1 (CH1), a hinge region (HR), an antibody heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3) in the N-terminal to C-terminal direction, abbreviated as VH-CH1-HR-CH2-CH3; in some embodiments, a "full-length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. A "full-length antibody light chain" is generally a polypeptide consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction, abbreviated as VL-CL.

[0085] In the present application, the light chain constant region and the heavy chain constant region can be any light chain constant region and heavy chain constant region. The light chain constant region (CL) can be κ (kappa) or λ (lambda). The heavy chain constant region can be any one of the heavy chain constant regions of IgG, IgM, IgA, IgE, and IgD.

[0086] It is well known to those skilled in the art that each heavy chain variable region can be composed of three complementary determining regions (CDRs) and four framework regions (FRs), and each light chain variable region can be composed of three complementary determining regions (CDRs) and four framework regions (FRs). The complementary determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. In some embodiments, from the N-terminus to the C-terminus, the heavy chain variable region and the light chain variable region both include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. There are two common definitions of the CDR sequence of the heavy chain variable region or the light chain variable region, namely the Kabat definition and the Chothia definition, for example, see Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, MD. (1991); Al-Lazikani et al., J Mol Biol 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). For the variable region sequence of a given antibody, the CDR sequence in the heavy chain variable region or the light chain variable region sequence can be determined according to the Kabat definition or the Chothia definition. In the embodiment of the present application, the Chothia definition is used to determine the CDR sequence.

[0087] In this application, the term "identity" may be defined as the percentage of identical residues in amino acid or nucleotide sequence variants after sequence alignment and introduction of gaps. Methods and computer programs for alignment are well known in the art. In this article, a reference to an amino acid sequence having a percentage identity with an amino acid sequence refers to a sequence having said percentage identity over the entire length of the referenced amino acid sequence.

[0088] In the present application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0089] In this application, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term may include nucleic acids containing analogs of natural nucleotides, which have binding properties similar to reference nucleic acids (e.g., sequence information is shown) and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, the sequence of a nucleic acid may include variants modified in a conservative manner thereof, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.

[0090] In the present application, human interleukin-13 (hIL-13) refers to a protein derived from humans. IL-13 is a Th2 type cytokine, which exerts its effects by binding to its receptor to form a ternary complex of IL-13 / IL-13Rα1 / IL-4Rα. IL-13 has a variety of biological effects on mononuclear macrophages, B lymphocytes, NK cells, and vascular endothelial cells, and is a mediator of the body's immune response. The monoclonal antibody of the present application is a neutralizing antibody that specifically recognizes IL-13 and can specifically bind to IL-13.

[0091] In the present application, "anti-human IL-13 monoclonal antibody" refers to a monoclonal antibody that is capable of binding to human interleukin 13 with sufficient affinity so that the monoclonal antibody can be used to identify / detect human interleukin 13.

[0092] The anti-human interleukin 13 (IL-13) monoclonal antibody of the present application does not bind to proteins unrelated to the target. Here, "unrelated proteins" refer to proteins other than human interleukin 13 as the target; here, "no binding" means: when the binding ability of the anti-human interleukin 13 (IL-13) monoclonal antibody of the present application to human interleukin 13 as its target is taken as 100%, the binding ability of the anti-human interleukin 13 (IL-13) monoclonal antibody of the present application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

[0093] In this application, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used in this specification refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D Affinity can be measured by common methods known in the art.

[0094] In the present application, "avidin" is a glycoprotein, each molecule of which is composed of four subunits and can bind closely to four biotin molecules. Streptavidin extracted from Streptomyces is more commonly used.

[0095] In this application, "biotin" is also called vitamin H. Biotin-hydroxysuccinimide (BNHS), a derivative made by chemical methods, can form biotinylated products with various types of large and small molecules such as proteins, carbohydrates and enzymes. Although the combination of avidin and biotin is not an immune reaction, it has strong specificity and affinity, and the two are extremely stable once combined. Since one avidin molecule has four binding sites for biotin molecules, more biotinylated molecules can be connected to form a complex similar to a lattice. Therefore, combining avidin and biotin with ELISA can greatly improve the sensitivity of ELISA.

[0096] The biotin-avidin system can be used in ELISA in various forms, which can be used for indirect coating or final reaction amplification. The enzyme-labeled antibody in conventional ELISA can also be replaced by a biotinylated antibody, which is then connected to an avidin-enzyme conjugate to amplify the reaction signal.

[0097] In this application, "enzyme-linked immunosorbent assay (ELISA)" refers to a detection method that utilizes the characteristic that antibody molecules can specifically bind to antigen molecules, combines free impurities with the target protein bound to a solid phase carrier, and uses special markers to conduct qualitative or quantitative analysis. The principle is: antigens or antibodies can be physically adsorbed on the solid surface and maintain their immune activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds while maintaining their respective immune activity or enzyme activity; after the enzyme conjugate is combined with the corresponding antigen or antibody, the occurrence of the immune reaction can be determined by the color reaction of the added substrate, and the depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen. Various types of detection methods can be designed according to the substance to be detected and the conditions for detection. The double antibody sandwich method is the most commonly used method for detecting antigens. The method is to adsorb the antiserum containing known antibodies into the small holes of the microtiter plate and wash it once; add the antigen to be tested. If the two are specific, they will bind to each other, and then wash away the excess antibody; add an enzyme-linked antibody that reacts specifically with the antigen to be tested to form a "sandwich"; add the substrate of the enzyme, and if a colored enzymatic hydrolysis product is seen, it indicates the presence of the corresponding antigen.

[0098] In the present application, the term "EC50 value" refers to the concentration for 50% of maximal effect (EC50), which refers to the concentration that can cause 50% of the maximum effect.

[0099] Specifically, in the first aspect, the present application provides an anti-human IL-13 monoclonal antibody, which comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of CDR-H1 is as shown in SEQ ID No: 1 (SSAMS) or SEQ ID No: 7 (SYYMS); the amino acid sequence of CDR-H2 is as shown in SEQ ID No: 2 (AISASGTTYYASWVNG) or SEQ ID No: 8 (SIFPSGSTDYATWARG); the amino acid sequence of CDR-H3 is as shown in SEQ ID No: 3 (EDAGYIGDYFNL) or SEQ ID No: 9 (NPNYNFATGFNI); the amino acid sequence of CDR-L1 is as shown in SEQ ID No: 4 (QASQSIGSGFA) or SEQ ID No: 10 (QASQNIYSNLA); the amino acid sequence of CDR-L2 is as shown in SEQ ID No:5 (AASNLAS) or SEQ ID No:11 (KASTLES); the amino acid sequence of CDR-L3 is shown in SEQ ID No:6 (QCTYYGSSYVDNA) or SEQ ID No:12 (QNNYYSGSSSSSTYT).

[0100] In some specific embodiments, the amino acid sequence of CDR-H1 of the anti-human IL-13 monoclonal antibody provided in the present application is shown in SEQ ID No: 1 (SSAMS), the amino acid sequence of CDR-H2 is shown in SEQ ID No: 2 (AISASGTTYYASWVNG), the amino acid sequence of CDR-H3 is shown in SEQ ID No: 3 (EDAGYIGDYFNL), the amino acid sequence of CDR-L1 is shown in SEQ ID No: 4 (QASQSIGSGFA), the amino acid sequence of CDR-L2 is shown in SEQ ID No: 5 (AASNLAS), and the amino acid sequence of CDR-L3 is shown in SEQ ID No: 6 (QCTYYGSSYVDNA).

[0101] In some specific embodiments, the amino acid sequence of CDR-H1 of the anti-human IL-13 monoclonal antibody provided in the present application is shown in SEQ ID No: 7 (SYYMS), the amino acid sequence of CDR-H2 is shown in SEQ ID No: 8 (SIFPSGSTDYATWARG), the amino acid sequence of CDR-H3 is shown in SEQ ID No: 9 (NPNYNFATGFNI), the amino acid sequence of CDR-L1 is shown in SEQ ID No: 10 (QASQNIYSNLA), the amino acid sequence of CDR-L2 is shown in SEQ ID No: 11 (KASTLES), and the amino acid sequence of CDR-L3 is shown in SEQ ID No: 12 (QNNYYSGSSSSSTYT).

[0102] In some specific embodiments, the anti-human IL-13 monoclonal antibody described in the present application comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13

[0103] (QSVEESGGRLVTPGTPLTLTCTVSGFSLSSSAMSWVRQAPGKGLEWIG AISASGTTYYASWVNGRFTISKTSTTVDLKITSPTTEDTATYFCAREDAGY IGDYFNLWGPGTLVTVSS) or SEQ ID No:15

[0104] (QSLEESGGRLVTPGTPLTVTCTVSGIDLSSYYMSWVRQAPGKGLEWIG SIFPSGSTDYATWARGRFTISKTSTTVDLKMTSLTASDTATYFCATNPNY NFATGFNIWGPGTLVTVSS), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 13 or SEQ ID No: 15; the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 14

[0105] (DVVMTQTPASVSEPVGGTVTVKCQASQSIGSGFAWYQQKPGQPPSLLI YAASNLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQCTYYGSSYV DNAFGGGTEVVVK) or SEQ ID No:16

[0106] (DIVLTQTPSSVSAAVGGTVTINCQASQNIYSNLAWYQQKPGQRPKLLIY KASTLESGVPSRFKGSGSGTHYTLTISDLECADAATYYCQNNYYSGSSSS STYTFGGGTEVVVK), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No: 14 or SEQ ID No: 16.

[0107] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-human IL-13 monoclonal antibody provided in the present application is as shown in SEQ ID No: 13

[0108] (QSVEESGGRLVTPGTPLTLTCTVSGFSLSSSAMSWVRQAPGKGLEWIG AISASGTTYYASWVNGRFTISKTSTTVDLKITSPTTEDTATYFCAREDAGY IGDYFNLWGPGTLVTVSS), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No: 13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 14

[0109] (DVVMTQTPASVSEPVGGTVTVKCQASQSIGSGFAWYQQKPGQPPSLLI YAASNLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQCTYYGSSYVDNAFGGGTEVVVK), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No:14.

[0110] In some specific embodiments, the amino acid sequence of the heavy chain variable region of the anti-human IL-13 monoclonal antibody provided in the present application is as shown in SEQ ID No: 15

[0111] (QSLEESGGRLVTPGTPLTVTCTVSGIDLSSYYMSWVRQAPGKGLEWIG SIFPSGSTDYATWARGRFTISKTSTTVDLKMTSLTASDTATYFCATNPNY NFATGFNIWGPGTLVTVSS), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 16

[0112] (DIVLTQTPSSVSAAVGGTVTINCQASQNIYSNLAWYQQKPGQRPKLLIY KASTLESGVPSRFKGSGSGTHYTLTISDLECADAATYYCQNNYYSGSSSS STYTFGGGTEVVVK), or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID No:16.

[0113] In some specific embodiments, the anti-human IL-13 monoclonal antibody described in the present application comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID No: 17

[0114] (QSVEESGGRLVTPGTPLTLTCTVSGFSLSSSAMSWVRQAPGKGLEWIG AISASGTTYYASWVNGRFTISKTSTTVDLKITSPTTEDTATYFCAREDAGYIGDYFNLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK) or SEQ ID No:19

[0115] (QSLEESGGRLVTPGTPLTVTCTVSGIDLSSYYMSWVRQAPGKGLEWIG SIFPSGSTDYATWARGRFTISKTSTTVDLKMTSLTASDTATYFCATNPNYNFATGFNIWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK) as shown; the amino acid sequence of the light chain is as SEQ ID No:18

[0116] (DVVMTQTPASVSEPVGGTVTVKCQASQSIGSGFAWYQQKPGQPPSLLI YAASNLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQCTYYGSSYVDNAFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) or SEQ ID No:20

[0117] (DIVLTQTPSSVSAAVGGTVTINCQASQNIYSNLAWYQQKPGQRPKLLIY KASTLESGVPSRFKGSGSGTHYTLTISDLECADAATYYCQNNYYSGSSSSSTYTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) as shown

[0118] In some specific embodiments, the anti-human IL-13 monoclonal antibody provided by the present application is anti-human IL-13 monoclonal antibody 1#, whose heavy chain has SEQ ID No: 17 (QSVEESGGRLVTPGTPLTLTCTVSGFSLSSSAMSWVRQAPGKGLEWIG AISASGTTYYASWVNGRFTISKTSTTVDLKITSPTTEDTATYFCAREDAGYIGDYFNLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), whose light chain has the amino acid sequence shown in SEQ ID No:18(DVVMTQTPASVSEPVGGTVTVKCQASQSIGSGFAWYQQKPGQPPSLLI YAASNLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQCTYYGSSYVDNAFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

[0119] In some specific embodiments, the anti-human IL-13 monoclonal antibody provided by the present application is anti-human IL-13 monoclonal antibody 2#, whose heavy chain has SEQ ID No: 19 (QSLEESGGRLVTPGTPLTVTCTVSGIDLSSYYMSWVRQAPGKGLEWIGSIFPSGSTDYATWARGRFTISKTSTTVDLKMTSLTASDTATYFCATNPNYNFATGFNIWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), whose light chain has the amino acid sequence shown in SEQ ID No:20(DIVLTQTPSSVSAAVGGTVTINCQASQNIYSNLAWYQQKPGQRPKLLIY KASTLESGVPSRFKGSGSGTHYTLTISDLECADAATYYCQNNYYSGSSSS STYTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

[0120] In a second aspect, the present application provides a nucleic acid molecule encoding any of the aforementioned anti-human IL-13 monoclonal antibodies.

[0121] In a third aspect, the present application provides a kit for detecting human IL-13, wherein the kit comprises: a first antibody (coating antibody) that has been or is to be fixed to a solid phase carrier and a second antibody (detection antibody) labeled with biotin.

[0122] In a specific embodiment, the first antibody and the second antibody in the kit are any one of the anti-human IL-13 monoclonal antibodies provided in the present application.

[0123] In some specific embodiments, the first antibody in the kit is the anti-human IL-13 monoclonal antibody 1# provided in the present application, whose heavy chain has the amino acid sequence shown in SEQ ID No: 17, and whose light chain has the amino acid sequence shown in SEQ ID No: 18.

[0124] In a specific embodiment, the first antibody is a coated antibody, which has been immobilized on a solid phase carrier or is to be immobilized on the solid phase carrier.

[0125] In some specific embodiments, the second antibody in the kit is the anti-human IL-13 monoclonal antibody 2# provided in the present application, whose heavy chain has the amino acid sequence shown in SEQ ID No: 19, and whose light chain has the amino acid sequence shown in SEQ ID No: 20.

[0126] In a specific embodiment, the second antibody is a detection antibody, which is labeled with biotin.

[0127] In a specific embodiment, the solid phase carrier is an ELISA plate.

[0128] In some preferred embodiments, the solid phase carrier is a 96-well ELISA plate, specifically a polystyrene plastic plate from Corning Coster, USA.

[0129] In a specific embodiment, the kit further includes devices or reagents necessary for detecting interleukin-13.

[0130] In some specific embodiments, the kit further includes horseradish peroxidase-labeled streptavidin, IL-13 standard, substrate, coating antibody diluent, washing solution, blocking solution or sample diluent, and stop solution.

[0131] In some specific embodiments, the peroxidase is preferably horseradish peroxidase (HRP), and the labeled streptavidin is streptavidin-horseradish peroxidase conjugate (SA-HRP).

[0132] In a specific embodiment, the IL-13 standard in the kit is a recombinant human IL-13 protein standard; the substrate is 3,3',5,5'-tetramethylbenzidine (TMB); the coating antibody diluent is phosphate buffered saline (PBS) pH7.4; the washing solution is phosphate buffered saline (PBS) containing 0.05% Tween20; the blocking solution / sample diluent is phosphate buffered saline (PBS) containing 0.5% BSA, 0.05% Tween20 and 0.05% Proclin300; and the stop solution is phosphoric acid.

[0133] In some specific embodiments, the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0134] In some specific embodiments, the kit is a modified double antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0135] In a preferred embodiment, the kit comprises the following components:

[0136] 1) Solid phase carrier: ELISA plate, 1 piece;

[0137] 2) Coating antibody: anti-human IL-13 monoclonal antibody 1# (i.e., primary antibody), 10 μg / tube, 1 tube;

[0138] 3) Detection antibody: anti-human IL-13 monoclonal antibody 2# (i.e., the second antibody) (labeled with biotin), 10 μg / tube, 1 tube;

[0139] 4) Standard: recombinant human IL-13 protein, 10 ng / tube, 1 tube;

[0140] 5) Streptavidin-horseradish peroxidase conjugate (SA-HRP), 500 ng / tube, 1 tube;

[0141] 6) Substrate: 3,3',5,5'-tetramethylbenzidine (TMB), 5 ml / tube, 1 tube each of solution A and solution B;

[0142] 7) Coating antibody diluent: phosphate buffered saline (PBS), pH 7.4, 50 ml / bottle, 1 bottle;

[0143] 8) Washing solution: phosphate buffered saline (PBS), containing 0.05% Tween20, 200 ml / bottle, 1 bottle;

[0144] 9) Blocking solution / sample diluent: phosphate buffered saline (PBS) containing 0.5% BSA, 0.05% Tween20 and 0.05% Proclin300, 200 ml / bottle, 1 bottle;

[0145] 10) Stop solution: 1M phosphoric acid, 10 ml / tube, 1 tube.

[0146] In a fourth aspect, the present application provides a method for detecting the content of interleukin-13 in a sample, specifically, comprising using the anti-human IL-13 monoclonal antibody provided by the present application to perform ELISA detection using a modified double antibody sandwich method.

[0147] In some specific embodiments, the method for detecting the content of interleukin-13 in a sample comprises the following steps:

[0148] Anti-human IL-13 monoclonal antibody 1# (i.e., the first antibody) was used to coat the ELISA plate;

[0149] Add the sample to be tested to the coated ELISA plate and incubate;

[0150] Add biotin-labeled anti-human IL-13 monoclonal antibody 2# (i.e., the second antibody) to the ELISA plate and incubate;

[0151] Dilute peroxidase-labeled streptavidin, add to the ELISA plate, and incubate;

[0152] Add the substrate to the ELISA plate, incubate in the dark, add the stop solution, and measure the OD value;

[0153] The OD value of the IL-13 standard was used to fit the standard curve, and the OD value of the sample was substituted into the equation to calculate the content of IL-13 in the sample.

[0154] In a specific embodiment, the ELISA kit provided in the present application can be used to quantitatively detect the content of IL-13 in the sample, which specifically includes the following steps:

[0155] (1) Coating: The coating antibody (anti-human IL-13 monoclonal antibody 1#, i.e., the primary antibody) was prepared into a coating antibody working solution with a concentration of 1 μg / ml using a coating antibody diluent (phosphate buffered saline (PBS), pH 7.4), and then added to an ELISA plate (96-well Coster ELISA plate) at a dosage of 50 μl / well and allowed to stand overnight at 2-8°C;

[0156] (2) Blocking: discard the antibody working solution in the ELISA plate, wash the plate three times with washing solution (phosphate buffered saline (PBS) containing 0.05% Tween20), then add blocking solution (phosphate buffered saline (PBS) containing 0.5% BSA, 0.05% Tween20 and 0.05% Proclin300) at a volume of 100 μl / well, and place on a shaker (120 rpm) at room temperature for 2 hours for blocking;

[0157] (3) Preparation of protein standards: Take 8 EP tubes and number them in sequence. Add 200 μl of sample diluent to each tube starting from the second tube and place them on the EP tube rack. Use the sample diluent to prepare the standard protein (recombinant human IL-13 protein standard) into a liquid with a concentration of 25 ng / ml. Pipet 400 μl and add it to the first EP tube. Then, pipette 200 μl of liquid from the first EP tube and add it to the second EP tube for double dilution. Repeat this process until the tenth EP tube, with the 25 ng / ml and 0.049 ng / ml points serving as anchor points.

[0158] (4) Preparation of quality control samples: Take 5 EP tubes and sample from 25 ng / ml respectively to prepare quality control samples of 12.5 ng / ml, 10 ng / ml, 5 ng / ml, 0.2 ng / ml, and 0.098 ng / ml;

[0159] (5) Sample addition: discard the blocking solution in the ELISA plate, wash the plate three times with washing solution, then add protein standard solution and quality control sample at a volume of 50 μl / well, and incubate at room temperature for 2 hours on a shaker (120 rpm);

[0160] (6) Adding detection antibody: discard the protein standard solution and the sample to be tested in the ELISA plate, wash the plate three times with a washing solution, prepare the detection antibody (anti-human IL-13 monoclonal antibody 2#, i.e., the second antibody, labeled with biotin) with a sample diluent to a detection antibody working solution with a concentration of 0.2 μg / ml, and then add 50 μl / well of the ELISA plate to the plate and incubate on a shaker (120 rpm) for 2 hours;

[0161] (7) Adding enzyme-labeled secondary antibody: discard the detection antibody working solution in the ELISA plate, wash the plate three times with washing solution, prepare the enzyme-labeled secondary antibody (streptavidin-horseradish peroxidase conjugate (SA-HRP)) with sample diluent to a concentration of 100 ng / ml enzyme-labeled secondary antibody working solution, then add 50 μl / well to the ELISA plate and incubate at room temperature on a shaker (120 rpm) for 1 hour;

[0162] (8) Color development: Mix equal volumes of substrate solution A and solution B (3,3',5,5'-tetramethylbenzidine (TMB), 5 ml / tube, 1 tube each of solution A and solution B); discard the enzyme-labeled secondary antibody working solution in the ELISA plate, wash the plate three times with washing solution, and then add the substrate at a volume of 50 μl / well, and react at room temperature in the dark for 5 to 10 minutes;

[0163] (9) Stop color development: Add 50 μl / well of stop solution (1 M phosphoric acid) to the ELISA plate to stop the reaction. Then use a microplate reader to measure the OD value of each well in the ELISA plate at a wavelength of 450 nm. Fit a standard curve based on the OD value of the standard. Substitute the OD value of the quality control sample into the equation to calculate the concentration of the quality control sample.

[0164] Another aspect of the present application also relates to the use of the anti-human IL-13 monoclonal antibody provided in the present application, the ELISA kit containing the anti-human IL-13 monoclonal antibody, and the method for detecting the IL-13 content in a sample to detect interleukin 13 in cell culture fluid and human serum.

[0165] Compared with the prior art, the human IL-13 ELISA kit provided in the present application not only has higher detection sensitivity and good detection specificity, but also can detect IL-13 of multiple species, such as crab-eating macaques, rhesus macaques and marmosets, and has good development and application prospects.

[0166] Example

[0167] The present application content will be described below in conjunction with specific examples, but the scope of the present application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following examples are all conventional reagents and instruments in this area and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the scheme and obtain corresponding results according to the embodiments.

[0168] Example 1: Screening and preparation of anti-human IL-13 monoclonal antibodies

[0169] Human IL-13 rabbit Fc fusion protein was used as an immunogen to immunize rabbits, and B cell cloning technology was used to screen monoclonal antibodies with inhibitory activity against human interleukin 13. First, the cell supernatant was detected by Binding ELISA to select clones that bind to hIL-13; then the reporter gene method was used to detect and select clones that can neutralize the binding of hIL-13 to its receptor, and the first antibody and the second antibody were obtained, and sequenced (commissioned to Sangon Biotechnology and General Biotechnology) to obtain the variable region sequence, and then according to the Kabat rule (reference: Kabat EA, Wu TT, Bilofsky H. Attempts to locate residues in complementarity-determining regions of antibody combining sites that make contact with antigen. Proc. Natl. Acad. Sci. USA. 1976; 73: 617–619.) to obtain its CDR sequence.

[0170] The first antibody and the second antibody are both anti-human IL-13 monoclonal antibodies, and the preparation method thereof is as follows: The heavy chain expression plasmid and the light chain expression plasmid of the first antibody (heavy chain sequence as shown in SEQ ID No: 17, and light chain sequence as shown in SEQ ID No: 18) and the second antibody (heavy chain sequence as shown in SEQ ID No: 19, and light chain sequence as shown in SEQ ID No: 20) obtained by the above screening are co-transfected into ExpiCHO-S cells. One day before transfection, the ExpiCHO-S cells were diluted to 3×10 6 On the day of transfection, the cell density was diluted to 6×10 6 cells / ml, 25ml cell suspension in a 125ml shake flask, waiting for transfection. Add 920μl OptiPRO SFM (purchased from Gibco) to well 1 and well 2 respectively, then add 12.5μg heavy chain and light chain plasmids to well 1, and add 150μl PEI reagent (purchased from Polysciences) to well 2. Transfer well 2 to well 1 and mix well, let stand for 15 minutes and then add to the cells. On the first and third days after transfection, add 2.5ml of feed medium OPM-CHOPFF05 (purchased from Aopuma). On the sixth day after transfection, harvest the culture supernatant and perform one-step purification with Protein A (purchased from Nanotech). The obtained antibodies are the first antibody (anti-human IL-13 monoclonal antibody 1#) and the second antibody (anti-human IL-13 monoclonal antibody 2#). See the transient expression process for details. Figure 1 The first antibody and the second antibody obtained above were both rabbit monoclonal antibodies and were not humanized.

[0171] The prepared anti-human IL-13 monoclonal antibodies 1# and 2# were further used in subsequent examples.

[0172] Example 2: Characterization of anti-human IL-13 monoclonal antibodies

[0173] 1. Binding experiment of anti-human IL-13 monoclonal antibody and human IL-13:

[0174] Coat the ELISA plate with human IL-13 overnight; after blocking, add anti-human IL-13 monoclonal antibodies 1# and 2# in gradient dilutions respectively, and incubate at room temperature for 2 hours; after washing the plate, add Mouse-anti-human IgG Fc (HRP) enzyme-labeled secondary antibody, and incubate at room temperature for 1 hour; finally, wash the plate, develop the color, and read the absorbance OD at 450nm 450 nm. The results are as follows Figure 2 As shown, it was shown that both anti-human IL-13 monoclonal antibodies 1# and 2# could bind to human IL-13, and the binding activities were comparable.

[0175] 2. Competitive inhibition experiment of antibody recognition antigen site:

[0176] Coat the ELISA plate with anti-human IL-13 monoclonal antibody 1#; after blocking, add equal volumes of gradient diluted anti-human IL-13 monoclonal antibodies 1#, 2# and a certain concentration of human IL-13 (his) in turn, and incubate at room temperature for 2 hours; after washing the plate, add Anti-his-HRP enzyme-labeled secondary antibody, and incubate at room temperature for 1 hour; finally, wash the plate, develop the color, and read the absorbance OD at 450nm 450 nm. The results are as follows Figure 3 As shown, it is shown that the anti-human IL-13 monoclonal antibodies 1# and 2# do not compete with the binding site of the human IL-13 antigen, and the anti-human IL-13 monoclonal antibody 2# can be used as the detection antibody of the human IL-13 kit.

[0177] 3. Biotin labeling of detection antibody (anti-human IL-13 monoclonal antibody 2#):

[0178] Biotinylation of the detection antibody was performed using EZ- Sulfo-NHS-LC-Biotin kit, specific steps are strictly carried out according to the kit instructions. After labeling, use ZebaTM Spin Deasling Colums to filter and remove free biotin, and the resulting solution is the biotin-labeled detection antibody.

[0179] Example 3: Composition of human IL-13 ELISA quantitative detection kit

[0180] The above-mentioned anti-human IL-13 monoclonal antibody 1# was used as the coating antibody and the anti-human IL-13 monoclonal antibody 2# (biotin-labeled) was used as the detection antibody to prepare an ELISA kit. The composition, specifications, source and storage conditions of the kit are shown in the following table.

[0181] Table 1

[0182]

[0183] Example 4: Quantitative detection of IL-13 in cell culture medium using ELISA kit

[0184] The ELISA kit in Example 3 was used to quantitatively detect human IL-13 in the quality control sample.

[0185] The specific method is as follows:

[0186] 1. Sample processing:

[0187] The protein standard was diluted with sample diluent and cell culture medium, respectively, and the cell culture medium was RPMI1640 + 10% FBS. The protein standard diluted with cell culture medium was used as the sample to be tested. A standard curve was fitted based on the absorbance value of the protein standard diluted with sample diluent. The absorbance value of the sample to be tested was substituted into the standard curve to obtain the theoretical concentration value of human IL-13 in the sample to be tested, and then the recovery rate of the sample to be tested was calculated (recovery rate = theoretical concentration value / actual concentration value × 100), thereby analyzing the feasibility of the ELISA kit for quantitative detection of human IL-13 in cell culture medium.

[0188] 2. ELISA quantitative detection:

[0189] (1) Coating: Prepare the coating antibody working solution with a concentration of 1 μg / ml using coating antibody diluent, add 50 μl / well to the ELISA plate, and incubate at 2-8°C overnight;

[0190] (2) Blocking: Discard the antibody working solution in the ELISA plate, wash the plate three times with washing solution, add blocking solution at 100 μl / well, and place on a shaker (120 rpm) at room temperature for 2 hours;

[0191] (3) Preparation of protein standards: Take 8 EP tubes and number them in sequence. Add 100 μl of sample diluent to each tube starting from the second tube and place them on the EP tube rack. Use the sample diluent to prepare the standard protein into a liquid with a concentration of 500 pg / ml. Pipet 200 μl and add it to the first EP tube. Then pipette 100 μl of liquid from the first EP tube and add it to the second EP tube for multiple dilution. Repeat this process until the seventh EP tube (the concentration of the last tube is 0).

[0192] (4) Adding the sample to be tested and the standard: discard the blocking solution in the ELISA plate, wash the plate three times with the washing solution, add the protein standard solution and the sample to be tested at 50 μl / well, and incubate at room temperature for 2 hours on a shaker (120 rpm);

[0193] (5) Adding detection antibody: discard the protein standard solution and the sample to be tested in the ELISA plate, wash the plate three times with washing solution, prepare the detection antibody (biotin-labeled) into a detection antibody working solution with a concentration of 200 ng / ml using sample diluent, add 50 μl / well to the ELISA plate, and incubate on a shaker (120 rpm) for 2 hours;

[0194] (6) Adding enzyme-labeled secondary antibody: discard the detection antibody working solution in the ELISA plate, wash the plate three times with washing solution, prepare the enzyme-labeled secondary antibody working solution with a concentration of 100 ng / ml using sample diluent, add 50 μl / well to the ELISA plate, and incubate at room temperature on a shaker (120 rpm) for 1 hour;

[0195] (7) Color development: Mix equal volumes of substrate solution A and solution B; discard the enzyme-labeled secondary antibody working solution in the ELISA plate, wash the plate three times with washing solution, then add the substrate at a rate of 50 μl / well and react at room temperature in the dark for 10 to 15 minutes;

[0196] (8) Stop color development: Add 50 μl / well of the stop solution to the ELISA plate to stop the reaction. Then use a microplate reader to measure the OD value of each well in the ELISA plate at a wavelength of 450 nm. Fit a standard curve based on the OD value of the standard. Substitute the OD value of the sample to be tested into the equation to calculate the concentration of human IL-13 in the sample to be tested, thereby completing quantitative detection.

[0197] 3. Test results:

[0198] (1) The test data of human IL-13 standard is shown in Table 2 below:

[0199] Table 2

[0200]

[0201] (2) Curve equation: 4-P Fit: y = (0.029-1.995) / (1+(x / 0.234)^1.108+1.995, R2 = 0.999 (e.g. Figure 4 shown).

[0202] (3) The test data of the sample diluted with cell culture medium are shown in the following table:

[0203] Table 3

[0204]

[0205] The recovery rates at each concentration point were between 79% and 95%, indicating that the cell culture medium had no effect on the experimental results. The ELISA kit and the detection method can be applied to the determination of the content of human IL-13 in the cell culture medium system.

[0206] 4. Accuracy test:

[0207] (1) The detection experiment was carried out according to the method of Example 4. In 3 different experiments, three known concentrations of human IL-13 (400, 100, 25 pg / ml) diluted with sample diluent were used as test samples, and 2 replicate wells were set up respectively to perform quantitative detection of IL-13 and analyze the accuracy of the kit. Among them, the coefficient of variation CV% = SD / mean value × 100. The specific experimental results are shown in Table 4.

[0208] Table 4

[0209]

[0210]

[0211] The experimental results showed that the coefficient of variation CV values ​​of the three concentration levels were all <20%, indicating that the ELISA kit and detection method had good accuracy.

[0212] (2) The detection experiment was carried out according to the method of Example 4. In three different experiments, three known concentrations of human IL-13 (167, 50, 10 pg / ml) diluted with cell culture medium were used as test samples, and two replicate wells were set up respectively to perform quantitative detection of IL-13 to analyze the accuracy of the kit. The specific experimental results are shown in Table 5.

[0213] Table 5

[0214]

[0215] The above experimental results show that the coefficient of variation CV values ​​of the three concentration levels are all <20%, indicating that the ELISA kit and detection method have good accuracy.

[0216] 5. Specificity investigation:

[0217] The detection experiment was carried out according to the method of Example 4. Different concentration gradients of human IL-13, human IL-2, human IL-4, human IL-5, human GM-CSF and human M-CSF proteins (500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 0 pg / ml) were added to the ELISA detection wells pre-coated with anti-human IL-13 monoclonal antibody (1#), and the absorbance value of each protein at different concentrations was determined by the established ELISA detection method. The results are shown in FIG. Figure 5 shown.

[0218] The experimental results showed that the OD450 of the human IL-13 protein group showed a significant dose-dependent effect at different concentrations, and the OD450 absorbance value increased with the increase of protein concentration, while the absorbance values ​​of the human IL-2, human IL-4, human IL-5, human GM-CSF and human M-CSF protein groups were close to 0, and there was no dose-dependent effect. The experimental results showed that the ELISA kit and detection method had good specificity.

[0219] 6. Sensitivity test:

[0220] The detection experiment was carried out according to the method of Example 4, and different concentration gradients of human IL-13 protein (1000, 333.333, 111.111, 37.037, 12.346, 4.115, 1.372, 0.457, 0.152, 0.051, 0.017, 0 pg / ml) were added to the ELISA detection wells pre-coated with anti-human IL-13 monoclonal antibody (1#), and 3 duplicate wells were set up. The quantitative detection of IL-13 was performed using the established ELISA detection method, and the sensitivity of the kit was analyzed. The results are shown in Table 6 below.

[0221] Table 6

[0222]

[0223] The results showed that when the concentration was in the range of 333 to 0.152 pg / ml, the recovery rates at each point were between 70% and 112%, indicating that the sensitivity of the ELISA method could reach 0.152 pg / ml.

[0224] (2) A commercially available hIL-13 kit (R&D, Cat: DY213, Lot: P349842) was used to perform the detection experiment according to the instructions and analyze the sensitivity of the kit. The results are shown in Table 7 below.

[0225] Table 7

[0226]

[0227] The results showed that when the concentration was in the range of 6000 to 5.859 pg / ml, the recovery rate at each point was between 80% and 105%, indicating that the sensitivity of the ELISA method could reach 93.75 pg / ml.

[0228] According to the above experimental results, the sensitivity of the ELISA kit and detection method of the present application to human IL-13 is much higher than that of the R&D kit.

[0229] 7. Species Cross-investigation:

[0230] The detection experiment was carried out according to the method of Example 4. Different concentration gradients of human IL-13, cynomolgus monkey IL-13, rhesus monkey IL-13 and marmoset IL-13 (500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 0 pg / ml) were added to the ELISA detection wells pre-coated with anti-human IL-13 monoclonal antibody (1#), and the absorbance values ​​of different species of IL-13 at different concentrations were determined by the established ELISA detection method.

[0231] The experimental results show that the OD values ​​of human IL-13, cynomolgus monkey IL-13, rhesus monkey IL-13 and marmoset IL-13 are 450 There was a significant dose-dependent effect between different concentrations. 450 The absorbance value increases with the increase of protein concentration (e.g. Figure 6 The results show that the ELISA kit and detection method of the present application can detect cynomolgus monkey IL-13, rhesus monkey IL-13 and marmoset IL-13, and have a wide range of applications.

[0232] Table 8

[0233]

[0234] When the concentration of cynomolgus monkey IL-13 and marmoset IL-13 was in the range of 500-7.8125 pg / ml, the recovery rates of each point were between 92% and 130%, indicating that the sensitivity of the ELISA method for detecting cynomolgus monkey IL-13 and marmoset IL-13 could reach 7.8125 pg / ml. When the concentration of rhesus monkey IL-13 was in the range of 500-31.25 pg / ml, the recovery rates of each point were between 98% and 113%, indicating that the sensitivity of the ELISA method for detecting rhesus monkey IL-13 could reach 31.25 pg / ml.

[0235] It can be seen from the above embodiments that the ELISA kit of the present application has high accuracy, high precision and high sensitivity, is superior to the IL-13 detection kit in the prior art, and can detect multiple species of IL-13, and has good development and application prospects.

[0236] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. An anti-human IL-13 monoclonal antibody, wherein: The anti-human IL-13 monoclonal antibody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: The amino acid sequence of CDR-H1 is shown in SEQ ID No: 1 or SEQ ID No: 7; The amino acid sequence of CDR-H2 is shown in SEQ ID No: 2 or SEQ ID No: 8; The amino acid sequence of CDR-H3 is shown in SEQ ID No: 3 or SEQ ID No: 9; The amino acid sequence of CDR-L1 is shown in SEQ ID No: 4 or SEQ ID No: 10; The amino acid sequence of CDR-L2 is shown in SEQ ID No: 5 or SEQ ID No: 11; The amino acid sequence of CDR-L3 is shown in SEQ ID No:6 or SEQ ID No:

12.

2. The anti-human IL-13 monoclonal antibody according to claim 1, wherein The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 1; The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 2; The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 3; The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 4, The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 5; The amino acid sequence of the CDR-L3 is shown in SEQ ID No:

6.

3. The anti-human IL-13 monoclonal antibody according to claim 1, wherein The amino acid sequence of the CDR-H1 is shown in SEQ ID No: 7; The amino acid sequence of the CDR-H2 is shown in SEQ ID No: 8; The amino acid sequence of the CDR-H3 is shown in SEQ ID No: 9; The amino acid sequence of the CDR-L1 is shown in SEQ ID No: 10; The amino acid sequence of the CDR-L2 is shown in SEQ ID No: 11; The amino acid sequence of the CDR-L3 is shown in SEQ ID No:

12.

4. The anti-human IL-13 monoclonal antibody according to any one of claims 1 to 3, wherein The anti-human IL-13 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 13 or SEQ ID No: 15, or is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No: 13 or SEQ ID No: 15; The amino acid sequence of the light chain variable region is as shown in SEQ ID No:14 or SEQ ID No:16, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID No:14 or SEQ ID No:

16.

5. The anti-human IL-13 monoclonal antibody according to any one of claims 1 to 4, wherein The anti-human IL-13 monoclonal antibody comprises a heavy chain and a light chain, wherein: The amino acid sequence of the heavy chain is shown in SEQ ID No: 17 or SEQ ID No: 19; The amino acid sequence of the light chain is shown in SEQ ID No: 18 or SEQ ID No: 20; Preferably, the anti-human IL-13 monoclonal antibody is anti-human IL-13 monoclonal antibody 1#, whose heavy chain has the amino acid sequence shown in SEQ ID No: 17, and whose light chain has the amino acid sequence shown in SEQ ID No: 18; or The anti-human IL-13 monoclonal antibody is anti-human IL-13 monoclonal antibody 2#, whose heavy chain has the amino acid sequence shown in SEQ ID No: 19, and whose light chain has the amino acid sequence shown in SEQ ID No:

20.

6. A nucleic acid molecule encoding the anti-human IL-13 monoclonal antibody according to any one of claims 1 to 5.

7. Use of the anti-human IL-13 monoclonal antibody according to any one of claims 1 to 5, or the nucleic acid molecule according to claim 6, in the preparation of a kit for detecting diseases associated with IL-13 expression.

8. A kit for detecting human IL-13, wherein: The kit comprises: A first antibody (coating antibody) that has been or is to be fixed to a solid phase carrier and a second antibody (detection antibody) labeled with biotin, The first antibody and the second antibody are anti-human IL-13 monoclonal antibodies according to any one of claims 1 to 5, Preferably, the solid phase carrier is an ELISA plate; Preferably, the kit further comprises horseradish peroxidase-labeled streptavidin, recombinant human IL-13 protein standard, substrate, coating antibody diluent, washing solution, blocking solution or sample diluent, and stop solution; Preferably, the first antibody is anti-human IL-13 monoclonal antibody 1#, and the second antibody is anti-human IL-13 monoclonal antibody 2#.

9. Use of the kit according to claim 8 for detecting interleukin 13 in cell culture fluid and human serum.

10. A method for detecting the content of IL-13 in a sample, wherein: The method comprises using the anti-human IL-13 monoclonal antibody according to any one of claims 1 to 5, and performing ELISA detection using a modified double antibody sandwich method. Preferably, the method comprises the following steps: Coating a solid phase carrier with a primary antibody; Add the sample to be tested to the coated solid phase carrier and incubate; adding a biotin-labeled second antibody to the solid phase carrier and incubating; diluting peroxidase-labeled streptavidin, adding it to the solid phase carrier, and incubating; Add the substrate to the solid phase carrier, incubate in the dark, add the stop solution, and measure the OD value; The standard curve was fitted using the OD value of the recombinant human IL-13 protein standard, and the OD value of the sample to be tested was substituted into the equation to calculate the content of human IL-13 in the sample to be tested; Wherein, the first antibody and the second antibody are the anti-human IL-13 monoclonal antibodies according to any one of claims 1 to 5, preferably, the first antibody is the anti-human IL-13 monoclonal antibody 1#, and the second antibody is the anti-human IL-13 monoclonal antibody 2#.

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