An anti-il-10 monoclonal antibody and a preparation method and application thereof

Abstract

The application discloses an anti-IL-10 monoclonal antibody and a preparation method and application thereof, discloses the anti-IL-10 monoclonal antibody, the preparation method of the antibody, the application of the antibody in the preparation of drugs for various diseases, and further discloses the application of the antibody provided by the application in pairing use for detecting antigens.The anti-IL-10 monoclonal antibody of the application can be specifically combined with IL-10 antigens or sIL-10 antigens, lays a foundation for diagnosing or treating or preventing viral infections, tumors or inflammatory diseases by a genetic engineering method; the antibody can also be used in pairing, and the sIL-10 concentration in serum can be accurately measured and detected; in addition, the anti-IL-10 monoclonal antibody disclosed by the application has a specific antigen binding domain, can be targeted to combine with IL-10 proteins, and can also be used in immunohistochemical tests, ELISA tests and the like.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and genetic engineering technology, specifically relating to an anti-IL-10 monoclonal antibody, its preparation method, and its application. Background Technology

[0002] Interleukin-10 (IL-10) is a multifunctional negative class II cytokine, belonging to the homodimer secretion group. It consists of two subunits and has a molecular weight of 18 kDa. IL-10 is a multicellular, multifunctional regulatory factor that regulates cell growth and differentiation, participates in inflammatory and immune responses, and is currently recognized as an inflammatory and immunosuppressive factor. Almost all lymphocytes can synthesize IL-10, with monocytes / macrophages and T lymphocytes being the most important sources in the body. In addition, B cells, dendritic cells, NK cells, and mast cells can also synthesize IL-10.

[0003] IL-10 plays both immunosuppressive and immunostimulatory roles in both innate and adaptive immunity, thereby regulating the responses of various immune cell types. IL-10 can regulate the function of antigen-presenting cells (APCs) such as dendritic cells (DCs), Langerhans cells, and macrophages. IL-10 can enhance the phagocytic activity of monocytes and macrophages by increasing the expression of various receptors. These receptors can bind to and take up cytokines of pathogens, whether opsonin- or non-opsonin-bound. IL-10-stimulated monocytes can also enhance the expression of IgG-Fc receptors (such as CD64, CD32, and CD16) and CD14-like molecules. IL-10 can effectively inhibit the expression of pro-inflammatory cytokines and MHC class II molecules, and also has a significant inhibitory effect on the synthesis of factors such as IL-2 by T cells. Regulatory B cells can suppress the body's immune inflammatory response by producing IL-10. (Bibby JA et al., Cholesterol metabolism drives regulatory B cell IL-10. Nat Commun. 2020 Jul 8). On the other hand, IL-10 can enhance B cell survival, proliferation, and antibody production. In addition, there is an interdependence between IL-10 and CD4+ T cell-mediated neuroprotective mechanisms after axonal transection, and CD4+ T cells may enhance the central response to IL-10, while IL-10 signaling within CD4+ T cells is necessary for their ability to rescue the survival of axonal motor neurons (Runge EM et al., CD4+ T cell expression of the IL-10 receptor. J Neuroinflammation. 2020 Apr 17).

[0004] Abnormal IL-10 expression can lead to inflammation-related diseases. Tumor growth, lupus erythematosus, lymphoma, and skin cancer are examples of diseases with excessive IL-10 expression, while Crohn's disease, psoriasis, rheumatoid arthritis, and organ transplant reactions are examples of diseases with insufficient IL-10 expression. Therefore, serum IL-10 levels are crucial for controlling the progression of certain diseases, including inflammation, and their applications in clinical diagnosis and treatment need to be developed. Summary of the Invention

[0005] In view of this, the present invention aims to provide an anti-IL-10 monoclonal antibody, its preparation method and application. The provided anti-IL-10 monoclonal antibody can be used in pairs and can achieve accurate detection of recombinant IL-10 antigen and sIL-10 in clinical samples through a double antibody sandwich ELISA method.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] An anti-IL-10 monoclonal antibody, comprising a heavy chain variable region (V H ) and light chain variable region (V L );

[0008] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1;

[0009] The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:3.

[0010] Furthermore, the DNA sequence encoding the aforementioned anti-IL-10 monoclonal antibody includes DNA sequences encoding the heavy chain variable region and the light chain variable region;

[0011] The DNA sequence encoding the heavy chain variable region is shown in SEQ ID NO:2;

[0012] The DNA sequence encoding the variable region of the light chain is shown in SEQ ID NO:4.

[0013] Furthermore, another type of anti-IL-10 monoclonal antibody includes the heavy chain variable region (V... H ) and light chain variable region (V L );

[0014] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5;

[0015] The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:7.

[0016] Furthermore, the DNA sequence encoding the aforementioned anti-IL-10 monoclonal antibody includes DNA sequences encoding the heavy chain variable region and the light chain variable region;

[0017] The DNA sequence encoding the heavy chain variable region is shown in SEQ ID NO:6;

[0018] The DNA sequence encoding the variable region of the light chain is shown in SEQ ID NO:8.

[0019] Here, the aforementioned anti-IL-10 monoclonal antibodies can specifically bind to IL-10 antigen or sIL-10 antigen, laying the foundation for the diagnosis, treatment, or prevention of viral infections, tumors, or inflammatory diseases through genetic engineering methods; and the aforementioned anti-IL-10 monoclonal antibodies can be used in pairs to accurately measure and detect the concentration of sIL-10 in serum, which can quickly help doctors determine the patient's immune status.

[0020] The present invention also provides a method for preparing an anti-IL-10 monoclonal antibody, the method comprising the following steps:

[0021] 1) Rabbits were immunized with human IL-10 in vitro. After the immune response was elicited, the rabbits were sacrificed, the spleens were harvested, and spleen cells were isolated.

[0022] 2) Screening to obtain B cells that specifically bind to human IL-10;

[0023] 3) Subcloning B cells to obtain the variable region coding sequences of the antibody heavy and light chains;

[0024] 4) The obtained variable region coding sequence was recombined, transfected, and purified to obtain anti-IL-10 monoclonal antibody.

[0025] Here, the variable region coding sequence is an RNA sequence; in addition, the method of the present invention provides two rabbit-derived monoclonal antibodies for IL-10, enriching the types of antibodies.

[0026] Furthermore, the present invention also provides a pharmaceutical composition comprising the above-described anti-IL-10 monoclonal antibody and a pharmaceutically acceptable carrier.

[0027] Here, the pharmaceutical composition includes anti-IL-10 drugs, interferon, anti-IL-10 monoclonal antibodies, anti-IL-10 polyclonal antibodies, nucleoside analogs, DNA polymerase inhibitors, siRNA drugs, or therapeutic vaccines, etc.

[0028] Furthermore, the present invention also provides an expression vector containing the encoding DNA of the above-mentioned anti-IL-10 monoclonal antibody, which is used to express the above-mentioned anti-IL-10 monoclonal antibody.

[0029] Furthermore, the present invention also provides a prokaryotic or eukaryotic host cell containing the above-described expression vector.

[0030] Furthermore, the present invention also provides the use of anti-IL-10 monoclonal antibodies in the preparation of drugs for the treatment or prevention of human viral infections, tumors and inflammatory diseases.

[0031] Furthermore, the present invention also provides a kit for detecting IL-10 antigen or sIL-10 antigen, comprising the above-mentioned anti-IL-10 monoclonal antibody.

[0032] Here, the aforementioned anti-IL-10 monoclonal antibody has a specific antigen-binding domain, which can target and bind to the IL-10 protein, and can also be used in immunohistochemistry, ELISA and other experiments.

[0033] Furthermore, the present invention also provides a method for detecting IL-10 antigen or sIL-10 antigen, using the above-described kit for detecting IL-10 antigen or sIL-10 antigen, and performing detection by a double-antibody sandwich ELISA method.

[0034] The beneficial effects of this invention are as follows: 1) This invention provides an anti-IL-10 monoclonal antibody, its preparation method, and its application, which can specifically bind to IL-10 antigen or sIL-10 antigen, laying the foundation for the diagnosis, treatment, or prevention of viral infections, tumors, or inflammatory diseases through genetic engineering methods; 2) The anti-IL-10 monoclonal antibody provided by this invention can be used in pairs to accurately detect the concentration of sIL-10 in serum, helping doctors to quickly determine the patient's immune status; 3) This invention provides two rabbit-derived monoclonal antibodies for IL-10, enriching the types of antibodies; 4) The anti-IL-10 monoclonal antibody described in this invention has a specific antigen-binding domain, which can target and bind to IL-10 protein, and can also be used in immunohistochemistry, ELISA, and other tests. Attached Figure Description

[0035] Figure 1 This is a graph showing the serum titer test results of New Zealand white rabbits after immunization in Example 1 of the present invention;

[0036] Figure 2 This is a graph showing the specific binding of the purified monoclonal antibody to IL-10 in Example 3 of this invention.

[0037] Figure 3 This is an OD result graph of different concentrations of IL-10 detected by the double antibody sandwich method in Example 4 of the present invention. Detailed Implementation

[0038] To provide a more detailed understanding of the features and technical content of this invention, the implementation of the invention will be described in detail below with reference to the accompanying drawings. The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise stated, the technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise stated, the methods and materials of the embodiments described below are all commercially available conventional products. Those skilled in the art will understand that the methods and materials described below are merely exemplary and should not be considered as limiting the scope of the invention.

[0039] Example 1: Preparation of IL-10 specific monoclonal antibody

[0040] 1) New Zealand white rabbits were immunized with the extracellular region of recombinant human IL-10 to obtain an immune response against human IL-10.

[0041] The antigen used was a recombinant protein containing the extracellular domain of human IL-10 (IL-10). New Zealand white rabbits were subcutaneously immunized on day 0 with 400 μl of a 1:1 emulsion containing 400 μg of IL-10 protein in Sigma-Aldrich complete adjuvant. Subsequently, on days 7, 21, and 42, New Zealand white rabbits (#R1, #R2) were boosted with subcutaneous injections of a 1:1 emulsion containing 200 μg of IL-10 protein in Sigma-Aldrich incomplete adjuvant. The serum titer of the immunized New Zealand white rabbits reached 10 after three immunizations. 4 After and above. Figure 1 The image shows the serum titer test results of New Zealand white rabbits after immunization in Example 1 of this invention. Figure 1 As shown, the rabbit with the highest antibody titer (#R1) received a booster immunization with an intravenous injection of 200 μg IL-10 (without adjuvant).

[0042] 2) Select B lymphocytes that specifically bind to human IL-10 and perform subcloning.

[0043] IL-10 was labeled using the Lighting-Link R-Phycoerythrin (R-PE) Conjugation Kit (InnovaBiosciences). The IL-10 concentration was adjusted to no more than 1 mg / ml; 1 μl of LL-modifier reagent was added to 10 μl of IL-10 and mixed thoroughly; the mixture was added to the dry powder in the Lighting-Link mix and resuspended; the mixture was left to stand at room temperature for at least 3 hours or overnight; 1 μl of LL-quencher reagent was added to the mixture, and the R-PE-labeled IL-10 was ready for use after 30 minutes.

[0044] Spleen was extracted and homogenized to produce a single-cell suspension, and fluorescently labeled antibodies were added: ① 5 μl of PE-Cy7-labeled anti-rabbit IgG antibody; ② 5 μl of APC-labeled anti-rabbit MHCII antibody; ③ 2 μl of R-PE-labeled IL-10. The mixture was shaken to mix; MHCII-IgG+ cells were collected by flow cytometry, which identified B cells secreting IL-10 antibody.

[0045] 3) IL-10-specific B cell subclones.

[0046] RNA was extracted from B cells secreting IL-10 antibodies using the Neasy mini Kit (Qiagen). RT-PCR reactions were performed using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen). Primers for amplifying the full-length heavy and light chains of the rabbit monoclonal antibody were designed using PrimerPremier 5 software. The heavy chain primer sequences were RHC1 and RHC2, and the light chain primer sequences were RLC1 and RLC2. RNA from specific B cells was reverse transcribed into cDNA, and the full-length fragments encoding the antibody heavy and light chains were amplified.

[0047] The RT-PCR primer sequences are as follows:

[0048] RHC1: 5'-CCGTCCAAGCTTATGGAGACTGGGCTGCGCTGGC-3'

[0049] RHC2: 5'-CAACAAGGATCCCTATTTACCCGGAGAGCGGGAG-3'

[0050] RLC1: 5'-CCGTCCAAGCTTATGGACACGAGGGCCCCACTC-3'

[0051] RLC2: 5'-CAACAAGGATCCCTAACAGTCACCCCTATTGAAGC-3'

[0052] The reaction conditions were 50℃ for 30 min, 94℃ for 2 min, followed by 35 cycles of (94℃ for 30 s, 57℃ for 30 s, 68℃ for 1 min), with an extension at 68℃ for 5 min and 4℃ for 5 min. After PCR amplification, the PCR products were purified by agarose gel electrophoresis.

[0053] Example 2: Sequencing of the full-length heavy and light chains of IL-10 monoclonal antibody and production of recombinant antibodies

[0054] (1) Sequencing of the full-length heavy and light chains of the IL-10 monoclonal antibody.

[0055] The PCR products encoding the full-length heavy and light chain genes obtained by cloning were ligated into the pcDNA3.1 (ThermoFisher Scientific) expression vector, and the ligation product was transformed into DH5α competent bacteria. The bacteria were cultured overnight at 37°C on plates containing ampicillin. Ten single colonies were randomly selected and amplified using RT-PCR primers. The RT-PCR primer sequences were as described in the example: RHC1, RHC2, RLC1, and RLC2. The reaction conditions were: 94°C pre-denaturation for 30 s, followed by 30 cycles of (94°C denaturation for 30 s, 57°C annealing for 30 s, and 68°C extension for 1 min), and a final extension at 68°C for 5 min. 5 μL of the PCR product was electrophoresed on a 1% agarose gel. Transformers containing both antibody heavy and light chains were identified among the positive transformants. These positive transformants were sent to Nanjing Qingke Company for sequencing, ultimately obtaining the unique nucleotide / protein sequences of 20G3 and 17E9. The sequence information is as follows:

[0056] 20G3 heavy chain variable region amino acid sequence: SEQ ID NO.1

[0057] METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFTISSYGVSWVRQAPGKGLEWIGIIGSSGDTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVR GIITAGIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVA PSTCSKPMCPPPELPGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIE KTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0058] 20G3 heavy chain variable region DNA sequence: SEQ ID NO.2

[0059]

[0060] 20G3 light chain variable region amino acid sequence: SEQ ID NO.3

[0061] MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTISCQSSQSVYMETWLSWYQQKLGQPPKLLIYEASKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCVGDYIS NIVTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDW

[0062] 20G3 light chain variable region DNA sequence: SEQ ID NO.4

[0063] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCGCAAGTGCTGACCCAGACTGCATCGTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGTGTTTATATGGAGACCTGGTTATCCTGGTATCAGCAGAAATTAGGGCAGCCTCCCAAGCTCTTGATCTACGAAGCATCCAAATTGGCATCTGGGGTCCCGCCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTGTAGGCGATTATATTAGTAATATTGTTACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGGTAG

[0064] Amino acid sequence of the variable region of the heavy chain of 17E9: SEQ ID NO.5

[0065] METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSTYSMSWVRQAPGKGLEWIGIISSSGTTIYASWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCARGDFYAGISHPYYFNIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0066] Variable region DNA sequence of the heavy chain of 17E9: SEQ ID NO.6

[0067]

[0068] 17E9 light chain variable region amino acid sequence: SEQ ID NO.7

[0069] MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAAVGGTVTINCQASQSLYNNKNLAWYQQKPGQPPKLLIYDASTLASGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCQGEFSCS SADCTAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0070] 17E9 light chain variable region DNA sequence: SEQ ID NO.8

[0071] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAACTGCCAGGCCAGTCAGAGTCTTTATAATAACAAAAATTTAGCCTGGTAT CAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACGATGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCGAATTTAGTTGTAGTAGT GCTGATTGTACTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACC ACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG

[0072] (2) Production and purification of IL-10 antibody

[0073] Plasmids expressing the heavy and light chains of the monoclonal antibody were co-transfected into 293F cells and cultured in shake flasks at 37°C for 4 days. The target antibody was then isolated and purified from the cell culture supernatant using a Protein A affinity chromatography column.

[0074] Example 3: ELISA detection of the binding ability of monoclonal antibodies 20G3 and 17E9 to IL-10 protein.

[0075] 100 μl of IL-10 protein (1 μg / mL) was coated in a 96-well plate (Costar, 42592) and incubated overnight at 4°C. The next day, the plate was washed five times with 200 μl of 0.1% Triton-X PBS buffer, blocked for 1 hour at room temperature in 200 μl of PBS-T (0.05% Tween) containing 5% milk powder, washed five times, and then 100 μl of serially diluted IL-10 antibody 20G3 or 17E9 was added and incubated for 1 hour at room temperature. After washing five times, 100 μl of goat anti-rabbit IgG coupled with horseradish peroxidase was added to each well and incubated for 1 hour at room temperature. The plate was washed five times with 0.1% Triton-X PBS buffer, and then TMB chromogenic solution was added to each well sequentially. After development for 3 minutes, 25 μl of stop solution was added to stop the reaction, and the plate was gently vortexed to mix. The fully automated multi-functional microplate reader was set to dual wavelengths of 450nm / 610nm. After zeroing the blank well, the OD value of each well was measured. Figure 2 This is a graph showing the specific binding of the purified monoclonal antibody to IL-10 in Example 3 of this invention. Figure 2 As shown, the purified monoclonal antibody can specifically bind to the IL-10 protein. The OD reading also shows a significant increasing trend with increasing antibody concentration.

[0076] Example 4: Detection of IL-10 concentration using a double-antibody sandwich ELISA method

[0077] (1) Preparation of biotin-labeled antibodies

[0078] Antibody 17E9 was labeled and detected using the EZ-Link™ Sulfo-NHS-LC-Biotin kit (Thermo Scientific). The specific method was as follows: The sulfo-NHS-LC-biotin was removed from the refrigerator and allowed to equilibrate to room temperature. 26.6 μl of biotin-labeled reagent was added to each 1 mg of IgG antibody, and the mixture was placed on ice for 2 hours. After labeling, excess unlabeled biotin was removed by dialysis.

[0079] (2) Double antibody sandwich ELISA test

[0080] 100 μl of the captured antibody 20G3 (2 μg / mL) diluted with carbonate buffer (pH=9.4) was added to a 96-well plate (Costar) and incubated overnight at 4°C. The next day, the plate was washed 5 times with 200 μl of 0.1% Triton-X PBS washing buffer; and blocked in 200 μl of PBS-T (0.05% Tween) containing 5% milk powder at room temperature for 1 hour, followed by 5 washes; 100 μl of serially diluted recombinant protein IL-10 was added and incubated at room temperature for 1 hour; then the plate was washed 5 times with washing buffer, and the biotin-labeled detection antibody 17E9 (1 μg / mL) from (1) was added to each well and incubated at room temperature for 1 hour; then the plate was washed 5 times with washing buffer, and 100 μl of horseradish peroxidase-streptavidin (Jackson Immuno Lab) was added to each well and incubated at room temperature for 1 hour. Wash the plate five times with washing solution, then add TMB chromogenic solution to each well sequentially, incubate for 3 minutes, add 25 μL of stop solution to stop the reaction, and gently shake to mix. Set the dual wavelengths of the fully automated multi-functional microplate reader to 450 nm / 610 nm, zero the blank well, and measure the OD value of each well.

[0081] Figure 3 This is an OD result graph of the detection of different concentrations of IL-10 using the double antibody sandwich method in Example 4 of the present invention. Figure 3 It is known that the anti-IL-10 monoclonal antibody that can be used in pairs provided by the present invention can detect the concentration of recombinant protein IL-10 as low as 2.1 ng / ml using the double antibody sandwich method, with high detection accuracy.

[0082] The specific part numbers and models of the reagents and instruments mentioned above are not limited or described in detail, as they are common knowledge and can be understood by those skilled in the art.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An anti-IL-10 monoclonal antibody, characterized in that, Including heavy chains and light chains; The amino acid sequence of the heavy chain is shown in SEQ ID NO:1; The amino acid sequence of the light chain is shown in SEQ ID NO:

3.

2. A DNA encoding an anti-IL-10 monoclonal antibody, characterized in that, The DNA sequence encoding the anti-IL-10 monoclonal antibody as described in claim 1 comprises DNA sequences encoding the heavy chain and the light chain; The DNA sequence encoding the heavy chain is shown in SEQ ID NO:2; The DNA sequence encoding the light chain is shown in SEQ ID NO:

4.

3. An anti-IL-10 monoclonal antibody, characterized in that, Including heavy chains and light chains; The amino acid sequence of the heavy chain is shown in SEQ ID NO:5; The amino acid sequence of the light chain is shown in SEQ ID NO:

7.

4. A DNA encoding an anti-IL-10 monoclonal antibody, characterized in that, The DNA sequence encoding the anti-IL-10 monoclonal antibody as described in claim 3 comprises DNA sequences encoding the heavy chain and the light chain; The DNA sequence encoding the heavy chain is shown in SEQ ID NO:6; The DNA sequence encoding the light chain is shown in SEQ ID NO:

8.

5. An expression carrier, characterized in that, The DNA comprising the encoding of the anti-IL-10 monoclonal antibody as described in claim 2 or 4 is used to express the anti-IL-10 monoclonal antibody as described in claim 1 or 3, respectively.

6. A prokaryotic or eukaryotic host cell, characterized in that, It includes the expression vector as described in claim 5.

7. A kit for detecting IL-10 antigen, characterized in that, Including the anti-IL-10 monoclonal antibody as described in claim 1 or 3.

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