Preparation method and application of monoclonal antibody against rat interleukin 10

Abstract

The present application discloses a monoclonal antibody against rat interleukin 10, a preparation method and an application thereof, and belongs to the field of biotechnology. The monoclonal antibody can specifically bind to rat interleukin 10 protein, and therefore, the monoclonal antibody can be used to prepare a kit for detecting the level of rat interleukin 10 protein. In addition, since the monoclonal antibody against rat interleukin 10 has a high affinity, and the antibody is proved to bind to different antigenic determinants on the surface of rat interleukin 10 through testing, it can be used to establish a double antibody sandwich enzyme-linked immunosorbent assay method against rat interleukin 10 protein, which is of great significance in clinical diagnosis and scientific research applications.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for preparing a monoclonal antibody against rat interleukin 10 and its application. Background Art

[0002] Interleukin 10 (IL-10), also known as cytokine synthesis inhibitory factor (CSIF), is a pleiotropic cytokine that can exert immunosuppressive or immunostimulatory effects in various types of cells. IL-10 is mainly produced by helper T cells 2 (Th2), activated B cells, monocytes, and macrophages. IL-10 participates in the biological regulation of various cells such as immune cells, inflammatory cells, and tumor cells, and plays an important role in various diseases such as autoimmune diseases, severe infectious diseases, tumors, and transplant immunity.

[0003] IL-10 is an anti-inflammatory factor and an important regulator of immune response. IL-10 directly affects APC by downregulating the expression of MHC class II and co-stimulatory molecules on the surface of macrophages and monocytes. IL-10 inhibits the expression of many proinflammatory cytokines, chemokines and chemokine receptors, and mediates allergen tolerance in allergen-specific immunotherapy. IL-10 inhibits the production of IFN-g and IL-2 by TH1 lymphocytes, and inhibits the production of IL-4 and IL-5 by TH2 lymphocytes. IL-10 can also inhibit the expression of cell surface antigens, such as CD23, CD80, CD86, etc. This inhibition of auxiliary function is mainly the inhibition of TH1 and TH2 cytokine production. Recent research data show that IL-10 acts directly on T cells by inhibiting the expression of T cell co-stimulatory molecules CD28 and inducible T cell co-stimulatory factors, inhibiting the production of T cell-related factors, thereby regulating the threshold of T cell activation. IL-10 promotes the survival, proliferation and differentiation of human B cells and increases the production of IgG4.

[0004] Interleukin IL-10 is widely used in disease research. For example, IL-10 plays an important regulatory role in human allergic diseases. Studies have shown that the expression of IL-10 in the respiratory tract of healthy subjects is higher than that in the respiratory tract of patients with asthma and allergic rhinitis. In the test of knocking out IL-10 in mice, mice can develop chronic colitis. IL-10 has an inhibitory effect on eosinophil survival and IL-4-induced IgE synthesis; IL-10 is a growth auxiliary factor for cytotoxic T cells; the expression of cytokines related to cellular immunity and allergic inflammation will be inhibited by IL-10; IL-10 can also stimulate humoral responses. Summary of the invention

[0005] In order to solve or alleviate some of the above technical problems, the present application provides a rabbit monoclonal antibody against rat interleukin 10, which can specifically bind to rat interleukin 10 protein and has high affinity. The monoclonal antibody can be used to prepare a kit for detecting rat interleukin 10 protein levels to detect rat interleukin 10, and can be used in the detection of rat interleukin 10 in industry and the diagnosis and treatment of related diseases, and has important practical value.

[0006] To this end, this application discloses at least the following technical solutions:

[0007] (1) An antibody, wherein the first antibody specifically binds to rat interleukin 10, and the antibody comprises:

[0008] three complementarity determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VLCDR1 as shown in SEQ ID NO:3, VLCDR2 as shown in SEQ ID NO:4, and VLCDR3 as shown in SEQ ID NO:5;

[0009] and / or

[0010] Three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VHCDR1 as shown in SEQ ID NO:8, VHCDR2 as shown in SEQ ID NO:9, and VHCDR3 as shown in SEQ ID NO:10.

[0011] (2) An antibody that specifically binds to rat interleukin 10, comprising: a light chain as shown in SEQ ID NO: 1; and a heavy chain as shown in SEQ ID NO: 6.

[0012] (3) An antibody that specifically binds to rat interleukin 10, comprising: a light chain variable region (VL) as shown in SEQ ID NO: 2; and a heavy chain variable region (VH) as shown in SEQ ID NO: 7.

[0013] (4) An antibody, wherein the second antibody specifically binds to rat interleukin 10, and the antibody comprises:

[0014] three complementarity determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VLCDR1 as shown in SEQ ID NO: 13, VLCDR2 as shown in SEQ ID NO: 14, and VLCDR3 as shown in SEQ ID NO: 15;

[0015] and / or

[0016] Three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VHCDR1 as shown in SEQ ID NO:18, VHCDR2 as shown in SEQ ID NO:19, and VHCDR3 as shown in SEQ ID NO:20.

[0017] (5) An antibody that specifically binds to rat interleukin 10, comprising: a light chain as shown in SEQ ID NO: 11; and a heavy chain as shown in SEQ ID NO: 16.

[0018] (6) An antibody that specifically binds to rat interleukin 10, comprising: a light chain variable region (VL) as shown in SEQ ID NO: 12; and a heavy chain variable region (VH) as shown in SEQ ID NO: 17.

[0019] (7) An antibody derivative comprising the aforementioned antibody and a conjugate linked to the antibody.

[0020] (8) A kit for detecting rat interleukin 10, comprising the aforementioned antibody or the aforementioned antibody derivative.

[0021] (9) A method for detecting interleukin 10, comprising: obtaining the antibody; using the antibody as a capture antibody and the antibody labeled with biotin as a detection antibody to construct a double antibody sandwich enzyme-linked immunosorbent assay.

[0022] (10) Use of the aforementioned antibody or the aforementioned antibody derivative or the aforementioned kit in detecting rat interleukin 10. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of constructing an expression vector containing the heavy chain constant region of a rabbit monoclonal antibody provided in the examples of the present application.

[0024] Figure 2 Schematic diagram of constructing an expression vector containing the rabbit monoclonal antibody light chain constant region provided in the examples of the present application.

[0025] Figure 3 This is an affinity curve diagram of the first anti-rat interleukin 10 antibody provided in the examples of the present application.

[0026] Figure 4 This is an affinity curve diagram of the anti-rat interleukin 10 secondary antibody provided in the examples of the present application.

[0027] Figure 5These are the EPassay test results of the anti-rat interleukin 10 primary antibody and secondary antibody provided in the examples of the present application.

[0028] Figure 6 This is a graph showing the ELISA test results of the first antibody and the second antibody provided in the examples of the present application for rat interleukin 10.

[0029] Figure 7 This is a graph of cross-protein recognition data of the first antibody and second antibody sandwich ELISA provided in the examples of the present application.

[0030] Figure 8 This is a statistical graph of the thermal stability test results of the first antibody and the second antibody provided in the examples of the present application. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail in conjunction with the embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional test methods and can be obtained from the prior art.

[0032] In this application, the term "antibody" should be interpreted in the broadest sense, including various antibody structures, including but not limited to Y-shaped antibodies, so-called full-length antibodies, antigen-binding portions of Y-shaped antibodies, and genetic or chemical modifications thereof. Among them, "antigen-binding portion" refers to one or more parts or fragments of the Y-shaped antibody that can retain the ability of the antibody to specifically bind to rat interleukin 10.

[0033] In this application, the term "monoclonal antibody" (mAb) includes a highly homogeneous antibody population that can recognize the same antigenic determinant. That is, in this antibody population, the individual antibodies are essentially the same, except for a small amount of mutations that may occur naturally. Monoclonal antibodies can show a single binding specificity and affinity for a specific epitope on an antigen. Compared with polyclonal antibodies that usually contain antibodies against different epitopes, each monoclonal antibody can be directed against the same or essentially the same epitope on the antigen. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous antibody population and cannot be interpreted as an antibody that needs to be made by any specific method. The antibody can be prepared by a variety of methods, including but not limited to hybridoma method, recombinant DNA method, phage antibody library and similar methods.

[0034] In the present application, the modifier "rabbit" in the term "rabbit antibody" or "monoclonal antibody against rat interleukin 10" or similar terms indicates that the complementary determining region (CDR) of the antibody is derived from a rabbit immunoglobulin sequence. In one embodiment, the rabbit monoclonal antibody against rat interleukin 10 may comprise CDRs and framework regions (FRs) from a rabbit immunoglobulin sequence. The term "rabbit monoclonal antibody against rat interleukin 10" may also include antibodies having amino acid residues not encoded by rabbit immunoglobulin sequences, for example, mutations introduced by random or site-specific mutations in vitro or by somatic mutations in vivo. However, the term "rabbit monoclonal antibody against rat interleukin 10" does not include antibodies whose CDR regions are derived from the germline of other mammals (such as mice).

[0035] In the present application, the term "antibody" refers to an immunoglobulin molecule composed of four heterologous polypeptide chains, of which the two chains with larger molecular weight are called heavy chains (H), and the two chains with smaller molecular weight are called light chains (L). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. The sequences of about 110 amino acids near the N-terminus of the heavy and light chains vary greatly, while the sequences of the other parts are relatively constant. Therefore, the regions of the light and heavy chains where the amino acid sequences near the N-terminus vary greatly are called variable regions (V), accounting for 1 / 4 and 1 / 2 of the heavy and light chains, respectively; the regions where the amino acid sequences near the C-terminus are relatively stable are called constant regions (C), accounting for 3 / 4 and 1 / 2 of the heavy and light chains, respectively.

[0036] The V regions of the heavy chain and light chain are called VH and VL, respectively. VH and VL each contain three regions with highly variable amino acid composition and arrangement order, called hypervariable regions (HVR) or complementarity determining regions (CDR), including HVR1 (CDR1), HVR2 (CDR2) and HVR3 (CDR3), among which HVR3 (CDR3) has a higher degree of variation. The three CDRs of VH and VL together constitute the antigen-binding site of the antibody, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen.

[0037] In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen to which it is directed. The intensity or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. In the present application, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between an antibody and an antigen.

[0038] In this application, the terms "double antibody sandwich enzyme-linked immunosorbent assay", "double antibody sandwich enzyme-linked immunosorbent assay", "double antibody sandwich enzyme-linked immunosorbent assay method", and "double antibody sandwich enzyme-linked immunosorbent assay" represent the same concept and can be used interchangeably; the terms "rat interleukin IL-10", "rat interleukin IL-10 protein", "Rat IL-10" and the like represent the same concept and can be used interchangeably.

[0039] The present invention successfully develops a pair of high-affinity anti-rat interleukin 10 protein rabbit monoclonal antibodies, and simultaneously develops a highly sensitive and specific double antibody sandwich enzyme-linked immunosorbent assay for rat interleukin 10 protein, which is of great significance in clinical diagnosis and scientific research applications.

[0040] The rabbit monoclonal antibody against rat interleukin 10 protein prepared in the examples of the present application includes a first antibody and a second antibody. The first antibody and the second antibody are verified by protein interaction non-label technology to be bound to different antigenic determinants on the surface of rat interleukin 10, and can be used to establish a double antibody sandwich enzyme-linked immunosorbent assay method for rat interleukin 10 protein.

[0041] The rat interleukin 10 double antibody sandwich enzyme-linked immunosorbent assay method provided in the embodiment of the present application is established based on the anti-rat interleukin 10 protein rabbit monoclonal antibody, the capture antibody is the first antibody, the detection antibody is the biotin-labeled second antibody, the standard sample is the rat interleukin 10 protein recombinantly expressed in vitro, the detection sensitivity is high, and the established method can be used for high-sensitivity detection of rat interleukin 10 protein.

[0042] Based on this, the present application discloses a first antibody that specifically binds to rat interleukin 10. The first antibody comprises three complementary determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VLCDR1, which consists of the sequence shown in SEQ ID NO: 3; VLCDR2, which consists of the sequence shown in SEQ ID NO: 4; and VLCDR3, which consists of the sequence shown in SEQ ID NO: 5.

[0043] Based on this, the present application also discloses a first antibody that specifically binds to rat interleukin 10. The first antibody comprises three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VHCDR1, which consists of the sequence shown in SEQ ID NO: 8; VHCDR2, which consists of the sequence shown in SEQ ID NO: 9; and VHCDR3, which consists of the sequence shown in SEQ ID NO: 10.

[0044] In certain embodiments, the first antibody comprises three complementarity determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VLCDR1, which consists of the sequence shown in SEQ ID NO:3; VLCDR2, which consists of the sequence shown in SEQ ID NO:4; VLCDR3, which consists of the sequence shown in SEQ ID NO:5; and three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VHCDR1, which consists of the sequence shown in SEQ ID NO:8; VHCDR2, which consists of the sequence shown in SEQ ID NO:9; VHCDR3, which consists of the sequence shown in SEQ ID NO:10.

[0045] Based on this, the embodiments of the present application also disclose a first antibody, which comprises: a light chain variable region (VL); the light chain variable region consists of the sequence shown in SEQ ID NO:2; and a heavy chain variable region (VH); the heavy chain variable region consists of the sequence shown in SEQ ID NO:7.

[0046] Based on this, the embodiments of the present application further disclose a first antibody, which comprises: a light chain (L), wherein the light chain consists of the sequence shown in SEQ ID NO:1; and a heavy chain (H), wherein the heavy chain (H) consists of the sequence shown in SEQ ID NO:6.

[0047] Based on this, an embodiment of the present application discloses a second antibody that specifically binds to rat interleukin 10, wherein the second antibody comprises three complementarity determining regions (CDRs) of a light chain variable region (VL) defined according to the Kabat numbering system: VLCDR1, which consists of the sequence shown in SEQ ID NO: 13; VLCDR2, which consists of the sequence shown in SEQ ID NO: 14; and VLCDR3, which consists of the sequence shown in SEQ ID NO: 15.

[0048] Based on this, the embodiments of the present application also disclose a second antibody that specifically binds to rat interleukin 10, and the second antibody comprises three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VHCDR1, which consists of the sequence shown in SEQ ID NO: 18; VHCDR2, which consists of the sequence shown in SEQ ID NO: 19; and VHCDR3, which consists of the sequence shown in SEQ ID NO: 20.

[0049] In certain embodiments, the second antibody comprises three complementarity determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VLCDR1, which consists of the sequence shown in SEQ ID NO:13; VLCDR2, which consists of the sequence shown in SEQ ID NO:14; VLCDR3, which consists of the sequence shown in SEQ ID NO:15; and three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VHCDR1, which consists of the sequence shown in SEQ ID NO:18; VHCDR2, which consists of the sequence shown in SEQ ID NO:19; VHCDR3, which consists of the sequence shown in SEQ ID NO:20.

[0050] Based on this, the embodiments of the present application also disclose a second antibody, which comprises: a light chain variable region (VL); the light chain variable region consists of the sequence shown in SEQ ID NO:12; and a heavy chain variable region (VH); the heavy chain variable region consists of the sequence shown in SEQ ID NO:17.

[0051] Based on this, the embodiments of the present application also disclose a second antibody, which comprises: a light chain (L), wherein the light chain consists of the sequence shown in SEQ ID NO:11; and a heavy chain (H), wherein the heavy chain (H) consists of the sequence shown in SEQ ID NO:16.

[0052] Based on this, the examples of the present application disclose a method for preparing the above-mentioned rabbit monoclonal antibody against rat interleukin 10. The monoclonal antibody of the present application can be prepared by various methods known in the art, such as obtaining it by genetic engineering recombination technology; obtaining a DNA molecule encoding the heavy chain and light chain genes of the antibody of the present application by chemical synthesis or PCR amplification, inserting the obtained DNA molecule into an expression vector, and then transfecting the host cell, and culturing the transfected host cell under specific conditions to express the antibody of the present application.

[0053] Based on this, the embodiments of the present application also disclose an antibody derivative, which comprises the aforementioned first antibody and a conjugate connected to the aforementioned first antibody, or comprises the aforementioned second antibody and a conjugate connected to the aforementioned second antibody.

[0054] In the present application, "conjugate" is an antigen binding portion produced by chemical modification. In one embodiment, the chemical modification can be chemical cross-linking. In one embodiment, one or more conjugates can be covalently attached to an antibody or non-covalently attached to an antibody. In one embodiment, the conjugate can be a molecular marker covalently attached to the antibody to facilitate detection of its antigen. The conjugate can be any suitable small molecule. The small molecule can include, but is not limited to, for example, biotin, streptavidin, and / or a fluorescent dye. The fluorescent dye can be any suitable fluorescent dye, including but not limited to AlexaFlour dye, aminocoumarin (AMCA), Atto dye, cyanine dye, DyLight dye, FITC, fluorescent probe 647H, rhodamine and Texas Red. The AlexaFlour dye includes but is not limited to AlexaFlour488, AlexaFlour555, AlexaFlour568, AlexaFlour594, AlexaFlour647 and AlexaFlour700. The Atto dye can include but is not limited to Atto390, Atto488, Atto565, Atto633 and Atto700. The cyanine dye may include but is not limited to Cy3, Cy5 and Cy5.5. The DyLight dye may include but is not limited to DyLight350, DyLight405, DyLight488, DyLight550, DyLight594, DyLight633, DyLight650, DyLight680, DyLight755 and DyLight800. In one embodiment, the conjugate may be a tandem dye with two covalently linked fluorescent molecules. In an embodiment, one fluorescent molecule serves as a donor and the other as an acceptor. In one embodiment, the donor has donor excitation characteristics, and the acceptor has acceptor emission characteristics, and the two can perform unique fluorescence excitation and emission reactions. The tandem dyes may include, but are not limited to, allophycocyanin-Cy5.5, allophycocyanin-Cy7, PE-Atto594, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, PE-AlexaFluor647, PE-AlexaFluor700, PE-AlexaFluor750, APC-AlexaFluor750 and PerCP-Cy5.5.

[0055] In some embodiments, the conjugate may also be a macromolecule. In one embodiment, the macromolecule may be an enzyme. The enzyme may include, but is not limited to, alkaline phosphatase (AP), glucose oxidase (Gox), horseradish peroxidase (HRP). In one embodiment, the macromolecule may be a fluorescent protein. The fluorescent protein may include, but is not limited to, allophycocyanin (APC), B-phycoerythrin (BPER-phycoerythrin (R-PE), PerCP, and R-phycocyanin (RPC). In one embodiment, the macromolecule may also be an antibody with different specificities of anti-SARS-CoV-2S1 rabbit monoclonal antibodies, forming a multivalent antibody with multiple specificities.

[0056] Based on this, the embodiments of the present application also disclose a kit for detecting rat interleukin 10, wherein the kit comprises the aforementioned first antibody and / or the aforementioned second antibody, or the aforementioned antibody derivative.

[0057] In certain embodiments, the kit comprises the aforementioned first antibody and the aforementioned second antibody that specifically recognize rat interleukin 10, wherein the first antibody is a capture antibody and the second antibody is a detection antibody.

[0058] Based on this, the embodiment also discloses a method for detecting interleukin 10, which comprises: obtaining the antibody; using the antibody as a capture antibody and the antibody labeled with biotin as a detection antibody to construct a double antibody sandwich enzyme-linked immunosorbent assay. In certain embodiments, the first antibody is used as a capture antibody and the second antibody labeled with biotin is used as a detection antibody to construct a double antibody sandwich enzyme-linked immunosorbent assay.

[0059] Based on this, the embodiments of the present application also disclose the use of the aforementioned first antibody or the aforementioned second antibody or the aforementioned antibody derivative or the aforementioned kit in detecting rat interleukin 10.

[0060] The present application is further described below in conjunction with more specific embodiments. Of course, the following embodiments should not be construed as limiting the present application.

[0061] 1. Preparation of monoclonal antibodies

[0062] In certain embodiments, the preparation method is a monoclonal antibody development technology based on single B lymphocyte screening and culture, and the preparation method comprises: immunizing New Zealand white rabbits with rat interleukin 10 protein (recombinant expression) as an immunogen; sorting B lymphocytes from spleen cells of white rabbits and culturing them; extracting RNA from positive cloned B lymphocytes and reverse transcribing them into cDNA; amplifying the cDNA by PCR to obtain naturally paired rabbit monoclonal antibodies; loading the heavy chain variable region (VH) gene and the light chain variable region (VL) gene of the naturally paired rabbit monoclonal antibody onto expression vectors, respectively, transfecting the vectors into host cells, culturing the host cells, and separating and purifying the monoclonal antibodies from the culture fluid of the host cells.

[0063] In some examples of the above implementation, the specific steps include:

[0064] (1) Preparation of immunogens: The immunogen used in the present application to prepare the Rat IL-10 rabbit monoclonal antibody is a recombinant rat interleukin 10 protein. The gene fragment corresponding to the Rat IL-10 (SwissProt: P29456) protein is constructed into the pYURK-secrecon-ChFC vector to express high-quality recombinant rat IL-10 mature protein with biological activity in a mammalian expression system.

[0065] (2) Animal immunization: Two New Zealand white rabbits were immunized with recombinant rat interleukin IL-10 protein as the immunogen; each rabbit was immunized with 200 μg of the immunogen. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant to prepare an emulsion, which was then injected subcutaneously at multiple points on the rabbit's abdomen and back; after the first immunization, 100 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant to prepare an emulsion every 3 weeks, which was then injected subcutaneously at multiple points on the rabbit's abdomen and back for two booster immunizations; after three immunizations, rabbit serum samples were collected, and their titers against Rat IL-10 were determined by ELISA. Rabbits with high serum titers were selected and booster immunized once with 200 μg of the immunogen injected subcutaneously at multiple points, and the spleen was collected three days later;

[0066] (3) Spleen cell separation: Take out a culture dish in a safety cabinet aseptically, add 30-40 ml of basal culture medium, put a cell sieve, take out the spleen and put it in the cell sieve, cut off the excess connective tissue and fat on the rabbit spleen tissue, cut the spleen tissue into pieces and put it into the cell sieve for grinding, take a clean grinding rod, and use the end of the pressing part to crush and grind the tissue. The cells in the membrane will slowly come out, pass through the cell sieve, and suspend in the culture dish solution; wash the cell sieve with 10 ml of basal culture medium, and collect the basal culture medium outside the cell sieve. Centrifuge at 400 g for 5 min at room temperature, remove the supernatant, retain the cells, add 13 ml of RBC lysis solution at room temperature (purchased from BioGems), gently blow away the cell clusters with a pipette, then time for 1 min to lyse the red blood cells, add 37 ml of basal culture medium, mix well, terminate the red blood cell lysis, centrifuge at 400 g for 5 min at room temperature, remove the supernatant, retain the cells, add 40 ml of basal culture medium placed at room temperature, gently blow away the cell clusters with a pipette, resuspend the cells, complete the first wash, centrifuge at 400 g for 5 min at room temperature, remove the supernatant, retain the cells, add 20 ml of basal culture medium placed at room temperature, gently blow away the cell clusters with a pipette, and resuspend the cells; filter the resuspended cells again through a cell sieve to remove the clumping cells, and then count the cells.

[0067] (4) B lymphocyte sorting: For the B lymphocyte screening method, please refer to Patent 201910125091.4 "Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells".

[0068] (5) Cloning of genes encoding rabbit monoclonal antibodies: The supernatant of cultured B cells was used to identify positive clones using antigen-coated ELISA. After the cells of the positive clones were collected and lysed, RNA was extracted using the Quick-RNATM MicroPrep kit (purchased from ZYMO) and reverse transcribed into cDNA. Using cDNA as a template, the naturally paired rabbit monoclonal antibody light chain variable region (VL) and heavy chain variable region (VH) genes were amplified from the cDNA of the corresponding positive clones using the PCR method, and several clones were selected for sequencing. The sequencing work was completed by Jinkairui Biotechnology Co., Ltd.; wherein, the primer pair used to amplify the light chain variable region (VL) is shown in SEQ ID NO: 21 and SEQ ID NO: 22. The primer pair used to amplify the heavy chain variable region (VH) is shown in SEQ ID NO: 23 and SEQ ID NO: 24.

[0069] The PCR reaction system was as follows: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2× Gloria HiFi, 6.5 μL H O; 2× Gloria HiFi reagent was provided by Wuhan Abotek Biotechnology Co., Ltd.;

[0070] PCR amplification procedure: pre-denaturation at 98°C for 30 seconds; then 40 cycles of 98°C for 10 seconds, 64°C for 30 seconds, and 72°C for 30 seconds; finally, keep at 72°C for 5 minutes; the resulting reaction solution was stored at 4°C;

[0071] (6) The amplification results are sequenced to determine the sequence.

[0072] (7) Production and purification of monoclonal antibodies: In order to obtain multiple rabbit monoclonal antibodies that recognize Rat IL-10, the heavy chain and light chain genes of rabbit monoclonal antibodies were loaded on expression vectors respectively. The mammalian expression vector pBR322 used was Figure 1 and Figure 2 . Figure 1 and Figure 2 In the pRB322 origin and f1 origin are replication promoters in E. coli, Ampcillin is a plasmid resistance gene, CMV immearly promoter is a promoter in eukaryotes, and SV40 PAterminator is a tailing signal. Figure 1 The heavy chain constant is the nucleotide sequence of the heavy chain constant region of the rabbit monoclonal antibody. Figure 2 The light chain constant is the nucleotide sequence of the light chain constant region of a rabbit monoclonal antibody.

[0073] The plasmid was transfected into 293F cells; the culture supernatant was obtained 72 to 96 hours after transfection, and the recombinant rabbit monoclonal antibody that recognized Rat IL-10 was contained. The recombinant rabbit monoclonal antibody that recognized Rat IL-10 was purified from the culture supernatant after transfection using protein A affinity gel resin, and the antibody was subpackaged after qualified verification and stored at -20°C for future use.

[0074] Results: The present application example screened out two rabbit monoclonal antibodies that recognize rat interleukin 10, namely the first antibody (6E12) and the second antibody (11E3); wherein the light chain amino acid sequence of the first antibody is SEQ ID NO: 1; the heavy chain amino acid sequence is SEQ ID NO: 6; the amino acid sequence of the light chain variable region is SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region is SEQ ID NO: 7; the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 are SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0075] The light chain amino acid sequence of the second antibody is SEQ ID NO: 11 and the heavy chain amino acid sequence is SEQ ID NO: 16; the amino acid sequence of the light chain variable region is SEQ ID NO: 12; the amino acid sequence of the heavy chain variable region is SEQ ID NO: 17; the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 are SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0076] 2. Screening and identification of monoclonal antibodies

[0077] In the examples of the present application, after obtaining multiple rabbit monoclonal antibodies against rat interleukin 10, the antibodies were firstly preliminarily identified and screened, including the identification of antibody affinity and antigen recognition epitopes.

[0078] (1) Identification of antibody affinity

[0079] The obtained rabbit monoclonal antibody against rat interleukin 10 was preliminarily identified for antibody affinity using the Gator Biomolecular Interaction Analyzer (GatorPrime) of ProbeLife, using the Pro1 probe. The first antibody (6E12) and the second antibody (11E3) to be tested were immobilized on the Pro1 probe at a concentration of 3 μg / ml; then the first antibody and the second antibody were debinded with recombinant Rat IL-10 protein at two concentrations of 150 nM and 70 nM, respectively, to obtain affinity curves, as shown in FIG. Figures 3-4 shown.

[0080] Results: The affinity test results of the first antibody and the second antibody to rat interleukin 10 are shown in Table 1:

[0081] Table 1

[0082] <![CDATA[K off (1 / s)]]> <![CDATA[K on (1 / Ms)]]> KD(M) Primary antibody (6E12) <![CDATA[3.75×10 -4 ]]> <![CDATA[5.78×10 5 ]]> <![CDATA[6.49×10 -10 ]]> Secondary antibody (11E3) <![CDATA[6.25×10 -5 ]]> <![CDATA[3.7×10 5 ]]> <![CDATA[1.69×10 -10 ]]>

[0083] From Table 1, Figures 3-4 It can be seen that the dissociation coefficients of the first antibody (6E12) and the second antibody (11E3) against rat interleukin 10 are 3.75×10 -4 , 6.25×10 -5 , and the affinity constants were 5.78×10 5 , 3.7×10 5 The dissociation equilibrium constant is 6.49×10 -10 , 1.69×10 -10 ; It shows that both the first antibody and the second antibody have a high affinity for rat interleukin 10.

[0084] (2) Identification of antigen recognition epitopes

[0085] In the examples of the present application, the antigen recognition epitopes of the rabbit monoclonal antibody against rat interleukin 10 obtained in the present application and the rat interleukin 10 protein were identified by using a GatorPrime device, and the obtained antibody was paired with the probe HFC (Anti-HIgGFC) Probe to test the antigen epitope determinant cluster recognized by it; the material used was recombinant Rat IL-10 protein, the concentration of which was 5 μg / ml, the first antibody (6E12) was used as the first antibody, the concentration was 3 μg / ml, and the second antibody (11E3) was used as the second antibody, the concentration was 1 μg / ml; by analyzing the pairing data between the two antibodies, it was determined whether the antigen epitope determinants they recognized were the same.

[0086] Results: Figure 5 As shown, the probe after immobilizing the recombinant Rat IL10 protein can obviously bind to the second antibody (11E3) after binding to the first antibody (6E12), and the shift value is 0.233. The above results show that the epitopes of the first antibody and the second antibody binding to the Rat IL10 protein are inconsistent. Therefore, the first antibody and the second antibody can be combined into a rabbit monoclonal antibody pair against the Rat IL-10 protein.

[0087] 3. Establishment of double antibody sandwich enzyme-linked immunosorbent assay (sensitivity test) with the first antibody and the second antibody

[0088] In the embodiment of the present application, the first antibody is used as the capture antibody and the second antibody is used as the detection antibody to perform a double antibody sandwich ELISA test. The specific test steps include:

[0089] (1) Detection antibody-secondary antibody (11E3) biotin labeling

[0090] The anti-Rat IL-10 rabbit monoclonal secondary antibody (11E3) was prepared into a 1 mg / ml solution, and NHS-LC-biotin was prepared into a 60 mg / ml solution with DMSO; 200 μL of the 1 mg / ml anti-Rat IL-10 rabbit monoclonal secondary antibody (11E3) solution was taken, and 10 μL of the 60 mg / ml NHS-LC-biotin solution was added; after mixing, the mixture was placed at room temperature for 30 minutes, and then 50 μg of 500 mM Tris solution at pH 9.0 was added to terminate the reaction; a large amount of 1×PBS buffer at pH 7.4 was added, and the mixture was centrifuged using a centrifugal column with an exclusion limit of 30 KD to remove excess biotin molecules and balance the buffer system.

[0091] (2) Double antibody sandwich enzyme-linked immunosorbent assay

[0092] Coating: Dilute the primary antibody (6E12) to 1 μg / ml with 1× PBS, mix well with a vortexer, add 100 μl / well to a 96-well microplate, cover with a cover film, and incubate in a 4°C refrigerator for 16-20 hours;

[0093] Washing: After incubation, discard the liquid in the wells, wash the plate once with 1×PBST, add 300μl of sample, let it stand for 40s, then discard the liquid in the wells and pat dry on flat paper;

[0094] Blocking: Add blocking solution (1×PBS containing 2% BSA + 5% sucrose + 0.05% Tween20 + 0.1% proclin300, pH 7.2) into the plate at 200 μl / well, cover with cover film, block at 37°C for 2 h, discard the blocking solution after blocking, pat the ELISA plate dry, place it in a 37°C oven to dry for 0.5-2 h, and take it out for use;

[0095] Add protein: dilute the Rat IL10 protein with diluent (1×PBS containing 2% BSA + 0.05% Tween20 + 0.1% proclin300, pH 7.2) to the concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 0 pg / ml, then add to the ELISA plate at 100 μL / well, cover with cover film, and incubate at 37°C for 2 h;

[0096] Washing: After incubation, discard the liquid in the wells, wash the plate three times with 1×PBST, add 300μL of sample, let it stand for 40s, then discard the liquid in the wells and pat dry on flat paper;

[0097] Add detection antibody: dilute anti-Rat IL-10 rabbit monoclonal secondary antibody (11E3)-biotin to 0.025 μg / ml, add 100 μL / well to the ELISA plate, cover with cover film, and incubate at 37°C for 1 h;

[0098] Washing: After incubation, discard the liquid in the wells, wash the plate three times with 1×PBST, add 300μL of sample, let it stand for 40s, then discard the liquid in the wells and pat dry on flat paper;

[0099] Add SA-HRP: dilute 100×SA-HRP concentrate 100 times, add 100 μL / well to the ELISA plate, cover with cover film, and incubate at 37°C for 0.5 h;

[0100] Washing: After incubation, discard the liquid in the wells, wash the plate three times with 1×PBST, add 300μL of sample, let it stand for 40s, then discard the liquid in the wells and pat dry on flat paper;

[0101] Add TMB colorimetric solution: Add TMB colorimetric solution to the ELISA plate at 100 μL / well, cover with cover film, and incubate at 37°C for 15 min. After incubation, remove the ELISA plate, add 50 μL stop solution (1 mol / L HCL) to each well, and immediately read the plate at 450 nm using an ELISA reader.

[0102] The recombinant Rat IL-10 protein concentration was used as the horizontal axis and the corrected value of the absorbance value (OD450-OD630) was used as the vertical axis. The lowest recombinant Rat IL-10 protein concentration at which the average absorbance value was greater than three times the average absorbance value of the blank control was used as the sensitivity of the double antibody sandwich enzyme-linked immunosorbent assay.

[0103] The results are as follows Figure 6 As shown, the first antibody is used as the capture antibody and the second antibody is used as the detection antibody to perform ELISA detection on rat interleukin 10, which shows good linearity, so that the detection has good sensitivity and accuracy.

[0104] 4. Cross-reaction test between the primary antibody and the secondary antibody (specificity detection)

[0105] In the examples of the present application, the specific steps of the cross-reaction test of the anti-rat interleukin 10 protein rabbit monoclonal primary antibody and the secondary antibody include: using a variety of proteins similar to Rat IL-10 to detect the specificity of the anti-Rat IL-10 rabbit monoclonal primary antibody (6E12) and the rabbit monoclonal secondary antibody (11E3), and using the double antibody sandwich enzyme-linked immunosorbent assay method in the aforementioned examples for detection, wherein the concentration of the standard protein is 1000pg / ml.

[0106] like Figure 7 As shown, when the double antibody sandwich ELISA was performed on multiple proteins using the rabbit monoclonal first antibody (6E12) and the rabbit monoclonal second antibody (11E3) provided in the examples of the present application, the rabbit monoclonal first antibody (6E12) and the rabbit monoclonal second antibody (11E3) only bound to Rat IL-10 and did not produce any cross-reaction with other proteins; this indicates that the rabbit monoclonal first antibody (6E12) and the rabbit monoclonal second antibody (11E3) provided in the examples of the present application are highly specific to the Rat IL-10 protein.

[0107] 5. Thermal stability test of primary and secondary antibodies

[0108] In the embodiment of the present application, the specific steps are as follows: the rabbit first antibody (6E12) is diluted to 2 μg / ml with 1×PBS, mixed with a vortex instrument, and then added to a 96-well microplate at 100 μl / well, covered with a cover film, and placed in a 4°C refrigerator for incubation for 16 to 20 hours; plate washing: after the incubation is completed, the liquid in the well is discarded, the plate is washed once with 1×PBST, 350 μl of sample is added, and the liquid in the well is discarded after standing for 40 seconds, and the liquid in the well is patted dry on a flat paper; blocking: the blocking solution is added to the plate well at 200 μl / well, the cover film is covered, and the plate is blocked at 37°C for 2 hours. After the blocking is completed, the blocking solution is discarded, the ELISA plate is patted dry, and then placed in a 37°C oven for drying for 0.5 to 2 hours; thermal destruction: the coated ELISA plate, freeze-dried protein, and 100X concentrated detection antibody are divided into two parts, sealed and stored at -20°C and 37°C, respectively, and taken out for testing after 7 days; protein addition: Rat IL-10 protein was diluted to 1000pg / ml with diluent, and then added to the ELISA plate stored at the corresponding temperature at 100μl / well, covered with a cover film, and incubated at 37℃ for 2h; Washing: After the incubation is completed, the liquid in the well is discarded, and the plate is washed three times with 1×PBST, 300μl of sample is added, and the liquid in the well is discarded after standing for 40s, and the liquid in the well is patted dry on a flat paper; Add detection antibody: 11E3-biotin stored under different conditions was diluted 100 times, and then added to the ELISA plate stored at the corresponding temperature at 100μl / well, covered with a cover film, and incubated at 37℃ for 1h; Washing: After the incubation is completed, the liquid in the well is discarded, and the plate is washed with 1×PBST for three times, and 300μl of sample is added. After standing for 40s, the liquid in the well is discarded, and the liquid in the well is patted dry on a flat paper; Add detection antibody: 11E3-biotin stored under different conditions was diluted 100 times, and then added to the ELISA plate stored at the corresponding temperature at 100μl / well, covered with a cover film, and incubated at 37℃ for 1h; Washing: After the incubation is completed, the liquid in the well is discarded, and Wash the plate three times with 1×PBST, add 300μl of sample, let it stand for 40s, then discard the liquid in the well, and pat the liquid in the well on a flat paper; Add SA-HRP: dilute the 100×SA-HRP concentrate 100 times, add 100μl / well to the ELISA plate in sequence, cover with a cover film, and incubate at 37°C for 0.5h; Wash the plate: After the incubation is completed, discard the liquid in the well, wash the plate three times with 1×PBST, add 300μl of sample, let it stand for 40s, then discard the liquid in the well, and pat the liquid in the well on a flat paper; Add TMB colorimetric solution: add TMB colorimetric solution to the ELISA plate in sequence at 100μl / well, cover with a cover film, and incubate at 37°C for 15min;

[0109] After the incubation is completed, the ELISA plate is removed, 50 μl of stop solution is added to each well, and the plate is immediately read at 450 nm using an ELISA reader.

[0110] The statistical results are as follows Figure 8 As shown in the figure, the results of the determination of rat interleukin IL-10 concentration after the rabbit monoclonal first antibody (6E12) and the rabbit monoclonal second antibody (11E3) were treated at -20°C. The standard curve equation established using the four parameters of the Logistic curve is: Y2 = 0.100 + 4.98X2 1.205 / (X2 1.205+297.82)(R 2 =0.9966); Y2 is the correction value of the absorbance value at -20°C (Y2=OD450nm-OD630nm), and X2 is the concentration of Rat IL-10.

[0111] The standard curve equation established after treatment at 37°C is: Y4=0.092+75.65X2 0.960 / (X2 0.960 +2591.89)(R 2 =0.9984); Y4 is the correction value of the absorbance value at 37°C (Y4=OD450nm-OD630nm); X2 is the concentration of Rat IL-10.

[0112] As shown in Table 2, the coefficient of variation of the Rat IL-10 protein concentration detected by the rabbit monoclonal first antibody (6E12) and the second antibody (11E3) treated at -20°C and 37°C, respectively, is less than 15%, indicating that the anti-Rat IL-10 rabbit monoclonal first antibody (6E12) and the second antibody (11E3) provided in the examples of the present application have strong thermal stability.

[0113] Table 2

[0114] Save benchmark values Single component (destruction of ELISA plate) value Single component (destruction standard) value Single component (destruction detection antibody) value Total component value 0.0809 0.07405 0.0737 0.057 0.0585 0.15825 0.1415 0.1412 0.1243 0.1422 0.21725 0.2981 0.1934 0.1778 0.36505 0.36755 0.32985 0.32205 0.2873 0.38725 0.626 0.5714 0.55325 0.5394 0.7466 1.11235 1.01785 0.9301 0.9288 1.06505 1.95435 1.8165 1.7069 1.8599 2.0021 3.2744 3.136 2.97715 2.7987 3.12335 Coefficient of variation CV (%) 8.12 8.73 13.85 11.30

[0115] 6. Sensitivity test of rabbit monoclonal primary antibody and secondary antibody against Rat IL-10

[0116] Specific steps of the embodiment of the present application: Coating: dilute rabbit monoclonal antibody 6E12 to 2 μg / ml with 1×PBS, mix on a vortexer, add 100 μl / well to a 96-well microplate, cover with a cover film, and incubate in a 4°C refrigerator for 16 to 20 hours; Wash: After the incubation is completed, discard the liquid in the well, wash the plate once with 1×PBST, add 350 μl of sample, let it stand for 40 seconds, then discard the liquid in the well, and pat dry the liquid in the well on a flat paper; Block: add blocking solution to the plate wells at 200 μl / well, cover with a cover film, and block at 37°C for 2 hours. After the blocking is completed, discard the blocking solution, pat the ELISA plate dry, place it in a 37°C oven to dry for 0.5 to 2 hours, and take it out for use; Add protein: Add Rat IL-10 protein was diluted with diluent to the concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, and 0 pg / ml, and then added to the ELISA plate at 100 μl / well, covered with a cover film, and incubated at 37°C for 2 h; Add blank wells: add the diluent to the ELISA plate at 100 μl / well, cover with a cover film, and incubate at 37°C for 2 h; Wash the plate: After the incubation is completed, discard the liquid in the well, wash the plate three times with 1×PBST, add 300 μl of the sample, let it stand for 40 seconds, then discard the liquid in the well, and pat the liquid in the well dry on a flat paper; Add detection antibody: dilute 11E34-biotin to 0.025 μg / ml, add 100 μl / well to the ELISA plate stored at the corresponding temperature, cover with a cover film, and incubate at 37°C for 1 h; Wash Plate: After incubation, discard the liquid in the well, wash the plate three times with 1×PBST, add 300μl of sample, let it stand for 40s, then discard the liquid in the well, and pat the liquid in the well on a flat paper; Add SA-HRP: Dilute the 100×SA-HRP concentrate 100 times, add 100μl / well to the ELISA plate in sequence, cover with a cover film, and incubate at 37°C for 0.5h; Wash the plate: After incubation, discard the liquid in the well, wash the plate three times with 1×PBST, add 300μl of sample, let it stand for 40s, then discard the liquid in the well, and pat the liquid in the well on a flat paper; Add TMB colorimetric solution: Add TMB colorimetric solution to the ELISA plate in sequence at 100μl / well, cover with a cover film, and incubate at 37°C for 15min; After incubation, take out the ELISA plate, add 50μl of stop solution to each well, and immediately read at 450nm with an ELISA reader.

[0117] Table 3 shows the sensitivity test results of the first and second rabbit monoclonal antibodies against Rat IL-10. It can be seen from Table 3 that the sensitivity of the antibody pair is 4.71 pg / ml.

[0118] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

[0119] Table 3

[0120]

[0121]

[0122] SEQ ID NO:1:"L-1"

[0123]

[0124] SEQ ID NO:2: "VL-1"

[0125]

[0126] SEQ ID NO:3: "VLCDR1-1"

[0127]

[0128] SEQ ID NO:4: "VLCDR2-1"

[0129]

[0130] SEQ ID NO:5: "VLCDR3-1"

[0131]

[0132] SEQ ID NO:6: "H-1"

[0133]

[0134] SEQ ID NO:7: "VH-1"

[0135]

[0136] SEQ ID NO:8: "VHCDR1-1"

[0137]

[0138] SEQ ID NO:9: "VHCDR2-1"

[0139]

[0140] SEQ ID NO:10:"VHCDR3-1"

[0141]

[0142] SEQ ID NO:11:"L-2"

[0143]

[0144] SEQ ID NO:12:"VL-2"

[0145]

[0146] SEQ ID NO:13:"VLCDR1-2"

[0147]

[0148] SEQ ID NO:14:"VLCDR2-2"

[0149]

[0150] SEQ ID NO:15:"VLCDR3-2"

[0151]

[0152] SEQ ID NO:16:"H-2"

[0153]

[0154]

[0155] SEQ ID NO:17:"VH-2"

[0156]

[0157] SEQ ID NO:18:"VHCDR1-2"

[0158]

[0159] SEQ ID NO:19:"VHCDR2-2"

[0160]

[0161] SEQ ID NO:20:"VHCDR3-2"

[0162]

[0163] SEQ ID NO:21:"VL-F"

[0164]

[0165] SEQ ID NO:22:"VL-R"

[0166]

[0167]

[0168] SEQ ID NO:23:"VH-F"

[0169]

[0170] SEQ ID NO:24:"VH-R"

[0171]

Claims

1. An antibody that specifically binds to rat interleukin 10, comprising: three complementarity determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VLCDR1 as shown in SEQ ID NO:3, VLCDR2 as shown in SEQ ID NO:4, and VLCDR3 as shown in SEQ ID NO:5; and Three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VHCDR1 as shown in SEQ ID NO:8, VHCDR2 as shown in SEQ ID NO:9, and VHCDR3 as shown in SEQ ID NO:

10.

2. An antibody that specifically binds to rat interleukin 10, comprising: The light chain shown in SEQ ID NO: 1; and The heavy chain is shown in SEQ ID NO:

6.

3. An antibody that specifically binds to rat interleukin 10, comprising: The light chain variable region (VL) as shown in SEQ ID NO: 2; and The heavy chain variable region (VH) is shown in SEQ ID NO:

7.

4. An antibody that specifically binds to rat interleukin 10, comprising: three complementarity determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VLCDR1 as shown in SEQ ID NO: 13, VLCDR2 as shown in SEQ ID NO: 14, and VLCDR3 as shown in SEQ ID NO: 15; and Three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VHCDR1 as shown in SEQ ID NO:18, VHCDR2 as shown in SEQ ID NO:19, and VHCDR3 as shown in SEQ ID NO:

20.

5. An antibody that specifically binds to rat interleukin 10, comprising: The light chain shown in SEQ ID NO: 11; and The heavy chain is shown in SEQ ID NO:

16.

6. An antibody that specifically binds to rat interleukin 10, comprising: The light chain variable region (VL) as shown in SEQ ID NO: 12; and The heavy chain variable region (VH) is shown in SEQ ID NO:

17.

7. An antibody derivative, wherein: The antibody derivative consists of the antibody according to any one of claims 1 to 6 and a conjugate connected to the antibody, wherein the conjugate is a molecular marker.

8. A kit for detecting rat interleukin 10, the kit comprising: The antibody according to any one of claims 1 to 6 or the antibody derivative according to claim 7.

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