Anti-human cxcl13 rabbit monoclonal antibody, preparation method and application thereof

Abstract

This invention provides an anti-human CXCL13 rabbit monoclonal antibody, its preparation method, and its application, belonging to the field of immunoglobulin technology. This invention provides an anti-human CXCL13 rabbit monoclonal antibody that can specifically bind to the CXCL13 protein, significantly improving the specificity and sensitivity of immunoassay. It is suitable for preparing immunohistochemical detection kits and can accurately detect CXCL13 protein expression, providing a reliable tool for the diagnosis of diseases such as angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma.

Description

Technical Field

[0001] This invention belongs to the field of immunoglobulin technology, and particularly relates to an anti-human CXCL13 rabbit monoclonal antibody, its preparation method and its application. Background Technology

[0002] Chemokines are small, secreted proteins whose primary function is to stimulate cell migration, particularly that of leukocytes. This function is achieved through interactions with protein-coupled receptors on the cell surface. These chemokines participate in leukocyte recruitment and the control of the tumor microenvironment, thereby regulating tumor cell proliferation, metastasis, invasion, angiogenesis, and treatment resistance. Based on differences in the conserved cysteine ​​motif near the N-terminus, chemokines are classified into four families: CCL, CXCL, XCL, and CX3CL.

[0003] CXCL13 (CXC motif chemokine 13) is a chemokine that primarily acts on lymphocyte migration and localization. It plays a crucial role in the entry of B lymphocytes into the germinal center and can serve as a marker for helper T lymphocytes in the germinal center. CXCL13 is expressed in various immune cells, including follicular dendritic cells, histiocytes, and helper T lymphocytes. In pathological diagnosis, the expression pattern of CXCL13 helps differentiate between different types of lymphoid tissue tumors. It is highly expressed in angioimmunoblastic T-cell lymphoma (AITL) and can be used for the differential diagnosis between AITL and peripheral T-cell lymphoma. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a rabbit monoclonal antibody against human CXCL13, a preparation method thereof, and its application. Specifically, it provides a monoclonal antibody that specifically binds to the CXCL13 protein, as well as a preparation method and application of the antibody, which significantly improves the specificity and sensitivity of CXCL13 protein immunoassay and is suitable for labeling the CXCL13 protein.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: an anti-human CXCL13 rabbit monoclonal antibody, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in QSVYSNG (SEQ ID NO.3), LCDR2 comprises an amino acid sequence as shown in YTS (SEQ ID NO.4), and LCDR3 comprises an amino acid sequence as shown in LQYDD (SEQ ID NO.5); the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in GFTLSSYG (SEQ ID NO.7), HCDR2 comprises an amino acid sequence as shown in IYTGSDK (SEQ ID NO.8), and HCDR3 comprises an amino acid sequence as shown in ARFPYD (SEQ ID NO.9).

[0006] Further, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO.2, or an amino acid sequence with more than 90% homology obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO.2. Preferably, it is an amino acid sequence with 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0007] SEQ ID NO.2: DIVMTQSPSS LSVSAGEKVT MTCKSSQSVY SNGLAWYQQK PGQPPKLLIYYTSTRKSGVP 60 DRFTGSGSGT DFTLTISSVQ TEDLAVYYCL QYDDFGGGTE VVVK 104.

[0008] Furthermore, the light chain variable region has a total length of 104 amino acids, and the number of amino acids in the four domains of its FR are 26, 17, 36 and 10, respectively. The number of amino acids in the domains of LCDR1 to LCDR3 are 7, 3 and 5, respectively, which are 27aa-33aa, 51aa-53aa and 90aa-94aa.

[0009] Further, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO. 6, or an amino acid sequence with more than 90% homology obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO. 6. Preferably, it is an amino acid sequence with 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0010] SEQ ID NO.6: EVQLVESGGG LVQPKGSLKL SCAASGFTLS SYGAMNWVRQ APGKGLEWVARIYTGSDKIY 60 YADSVKDRFT ISRDDSQSMV YLQMNNLKTE DTAMYYCARF PYDWGQGTLV TVSA114.

[0011] Furthermore, the total length of the heavy chain variable region is 114 amino acids, with the number of amino acids in the four domains of its FR being 25, 18, 39 and 11, respectively, and the number of amino acids in the domains of HCDR1 to HCDR3 being 8, 7 and 6, respectively, which are 26aa-33aa, 52aa-58aa and 98aa-103aa.

[0012] Furthermore, the anti-human CXCL13 rabbit monoclonal antibody specifically binds to the CXCL13 protein and specifically recognizes the amino acid sequence shown in SEQ ID NO.1. The sensitivity and specificity of this monoclonal antibody were verified using immunoassay methods, such as immunohistochemistry (IHC).

[0013] SEQ ID NO. 1: LRKRSSSTLP VPVFKRKIP 19.

[0014] This invention also provides a method for preparing a rabbit monoclonal antibody against human CXCL13, comprising the following steps:

[0015] S1. Immunogen preparation: Select amino acids from position 91 to position 109 of the CXCL13 protein amino acid sequence (SEQ ID NO.1) and conjugate the carrier protein KLH at its N-terminus as an immunogen;

[0016] S2. Animal Immunization: The synthetic immunogen was used to immunize New Zealand white rabbits, which were divided into two groups with different immunization doses. After three immunizations, qualified animals were screened by ELISA and IHC.

[0017] S3. PBMCs Isolation and B Cell Sorting: Whole blood was collected from immunized animals, and peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. Antigen-specific B cells were sorted using CXCL13 naked peptide-conjugated magnetic beads, and positive clones were obtained by ELISA and IHC screening (the optimal clone was OTIR7C4).

[0018] S4. Antibody cloning and expression: RNA was extracted from positive B cell clones and reverse transcribed into cDNA. The variable region gene of the light and heavy chains was amplified by PCR, an expression vector was constructed and transfected into HEK293 cells. After culture, the antibody was purified by affinity chromatography, and the concentration was determined by BCA method and the specificity was verified by IHC.

[0019] This invention also provides an application of an anti-human CXCL13 rabbit monoclonal antibody, which is used to prepare an immunohistochemical detection kit, chip, or test strip for human CXCL13 protein, for the detection of CXCL13 protein.

[0020] Furthermore, the immunohistochemical detection kit for human CXCL13 protein includes a primary antibody reagent, which includes a rabbit monoclonal antibody against human CXCL13.

[0021] Furthermore, the immunohistochemical detection kit for human CXCL13 protein also includes antigen retrieval solution, endogenous peroxidase inhibitor, hypersensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated chromogenic solution, and hematoxylin staining solution; preferably, the hypersensitive secondary antibody reagent is a hypersensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0022] Since CXCL13 protein exhibits specific expression differences in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, the immunohistochemical detection kit for human CXCL13 protein can detect the expression level of CXCL13 protein in tissues, providing a basis for the diagnosis and differential diagnosis of the above diseases.

[0023] In summary, the anti-human CXCL13 rabbit monoclonal antibody provided by this invention can specifically bind to human CXCL13 protein, significantly improving the specificity, accuracy, and reliability of CXCL13 protein immunoassay. Furthermore, using the anti-human CXCL13 protein monoclonal antibody and the immunohistochemical detection kit for human CXCL13 protein of this invention, the expression of CXCL13 protein in cells can be detected by IHC. Therefore, the anti-human CXCL13 protein monoclonal antibody of this invention can be well applied to the immunohistochemical detection of CXCL13 protein in cells.

[0024] The beneficial effects of this invention are as follows:

[0025] (1) Excellent binding performance: It binds to CXCL13 protein with high specificity, recognizes only the target sequence shown in SEQ ID NO.1, has no cross-reactivity, and has an affinity constant of 3.18 × 10⁻⁶. 9 L / mol, which is a high-affinity antibody.

[0026] (2) Outstanding detection efficiency: Significantly improves the specificity, sensitivity and accuracy of CXCL13 protein immunoassay, the signal-to-noise ratio of IHC detection meets the clinical judgment requirements, and can accurately distinguish between positive and negative tissues.

[0027] (3) Diagnostic value: It can specifically label CXCL13 protein, providing a reliable tool for the diagnosis and differential diagnosis of angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, filling the performance gap of existing detection antibodies.

[0028] (4) Practical application scenarios: It is compatible with detection methods such as immunohistochemistry and ELISA, and can be made into kits and other tools. The kit components are optimized and compatible with fully automated staining systems. It is easy to operate and suitable for clinical pathological testing.

[0029] (5) Stable and reliable preparation: The immunogen is selected from amino acids 91-109 of CXCL13 (conjugated with KLH) with precision and the screening process is rigorous, which can stably obtain highly active antibodies and can be prepared on a large scale for clinical translation. Attached Figure Description

[0030] The present invention will be described in detail below with reference to the accompanying drawings and examples. The advantages and implementation methods of the present invention will become more apparent from this description. The accompanying drawings are for illustrative purposes only and do not constitute any limitation on the present invention. In the accompanying drawings:

[0031] Figure 1 This is a diagram showing the results of immunohistochemical detection of CXCL13 protein expression in tonsil, appendix, and gastric tissues in an embodiment of the present invention.

[0032] Figure 2 This is a diagram showing the results of immunohistochemical detection of CXCL13 protein expression in thyroid, pancreatic, and placental tissues in an embodiment of the present invention.

[0033] Figure 3 This is a diagram showing the results of immunohistochemistry detection of CXCL13 protein expression in angioimmunoblastic T-cell lymphoma in an embodiment of the present invention. Detailed Implementation

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the preferred embodiments.

[0035] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions, conditions described in the laboratory manual, or conditions recommended by the manufacturer.

[0036] Example

[0037] I. A method for preparing anti-human CXCL13 rabbit monoclonal antibody, comprising the following steps:

[0038] S1. Preparation of immunogen:

[0039] The immunogenicity, hydrophilicity, and three-dimensional structure of the CXCL13 sequence (GeneBank: NM_006419) were analyzed using websites such as DNAStar and Uniprot, as well as the homology between the human and rabbit sequences. Amino acids 91-109 (SEQ ID NO.1) of the synthesized CXCL13 protein were selected as detection materials for magnetic bead screening and ELISA screening. After being conjugated with KLH, it served as an immunogen. The immunogen sequence was synthesized by Zhongtai Biochemical Co., Ltd.

[0040] S2, Animal Immunization:

[0041] KLH-conjugated CXCL13 peptide was used as the immunogen for immunizing New Zealand white rabbits. Four experimental animals were divided into two groups according to different immunization doses. Group A received an initial immunization dose of 1000 μg / rabbit, followed by 500 μg / rabbit / immunization. Group B received an initial immunization dose of 800 μg / rabbit, followed by 400 μg / rabbit / immunization. After three immunizations, blood was collected from the ear vein, and serum titers were determined by ELISA and IHC. The serum was serially diluted. For indirect ELISA, an OD > 1.0 at 450 nm at a dilution of 1:128000 was considered immunization qualified. For IHC, moderate staining intensity in the germinal centers of tonsillar tissue at a dilution of 1:10000 was considered immunization qualified. The actual test results were used to determine whether the immunized animals produced an immune response and whether the titer reached the level required for collecting PBMCs.

[0042] Indirect ELISA analysis of the serum from the four immunized animals in the two groups showed absorbance values ​​of 3.654, 2.879, 3.450, and 3.063 at 450 nm at a dilution of 1:128000, indicating good immune responses in all cases.

[0043] IHC testing uses tonsils as a quality control tissue. The specific testing procedure is as follows:

[0044] (1) Preparation of tissue sections: using tonsil tissue as quality control tissue, 4μm thick sections were cut with a microtome and collected for later use;

[0045] (2) Dewaxing and hydration: The sections were thoroughly dewaxed with xylene, hydrated with a series of ethanol gradients, and finally washed thoroughly with deionized water;

[0046] (3) Antigen repair: Antigen repair was carried out in EDTA repair solution (pH 8.0) using high pressure method, and then naturally cooled and washed with water after repair;

[0047] (4) Inactivation: Use 3% hydrogen peroxide to incubate at room temperature in the dark for 15 min to inactivate endogenous peroxidase, and then rinse thoroughly with deionized water;

[0048] (5) Incubation with primary antibody: Add serially diluted serum, place the slides in a humidified chamber and incubate at 37°C for 60 min, then wash thoroughly with 0.1% PBST as described above;

[0049] (6) Incubation with secondary antibody: Add hypersensitive HRP-labeled goat anti-mouse / rabbit IgG polymer, incubate at 37°C for 30 min, and then wash thoroughly with 0.1% PBST;

[0050] (7) DAB color development: Use freshly prepared DAB color development solution to develop color for 5 minutes, then stop the reaction and wash thoroughly.

[0051] (8) Hematoxylin staining: stain with hematoxylin for 12s, differentiate with 1% hydrochloric acid ethanol and then undergo blueing treatment with Tris-EDTA buffer (pH 8.0) according to the standard procedure, and rinse thoroughly with deionized water during the process.

[0052] (9) Dehydration and clearing: The sections were dehydrated by gradient ethanol, cleared by xylene, and finally mounted with neutral resin;

[0053] (10) Microscopic examination: Observe the staining results under a microscope.

[0054] After four immunizations, the serum of four New Zealand white rabbits in two groups was diluted 1:10000 and showed positive signals on the tonsil tissue. Animal A1 showed the best staining intensity, which was consistent with the results of indirect ELISA. Whole blood was collected from animals with this number for the separation and enrichment of PBMCs.

[0055] S3, PBMCs isolation, antigen-specific B cell sorting and antibody cloning:

[0056] Heart blood was collected from New Zealand white rabbit A1 and placed in a sterile centrifuge tube. Whole blood was separated by density gradient centrifugation strictly according to the lymphocyte separation reagent instructions. After centrifugation, the white mononuclear cell layer located between the plasma layer and the separation medium layer was aspirated and thoroughly washed with PBS to obtain rabbit peripheral blood mononuclear cells (PBMCs).

[0057] The naked CXCL13 peptide synthesized in step S1, coupled with magnetic beads, was used to sort antigen-specific B cells, which were then cultured. The B cell culture supernatant was screened using ELISA and IHC to identify positive clones recognizing the CXCL13 protein. The optimal clone was identified as OTIR7C4. The positive clone was lysed, and RNA was extracted and reverse transcribed into cDNA. Subsequently, the naturally paired light and heavy chain full-length sequences were amplified from the cDNA using PCR. Finally, a rabbit monoclonal antibody expression vector was constructed using clonal recombination technology.

[0058] S4. Preparation and purification of monoclonal antibodies:

[0059] The heavy and light chain genes of the rabbit monoclonal antibody OTIR7C4 were cloned into an expression vector, transfected into HEK293 cells, and cultured for 120–144 h. The supernatant was collected and purified using affinity chromatography to obtain a recombinant rabbit monoclonal antibody against human CXCL13 protein. Antibody concentration was determined by the BCA method, and its specificity was verified by immunohistochemistry (IHC).

[0060] II. Analysis of the variable region gene and amino acid sequence of the anti-human CXCL13 rabbit monoclonal antibody:

[0061] The 5' RACE technique was used to amplify the variable region gene sequences of the light and heavy chains of the rabbit monoclonal antibody OTIR7C4, using the recombinant plasmid as a template. The experiments were strictly performed in accordance with the instructions of the Takara Bio USA SMARTer® RACE 5' / 3' Kit.

[0062] Based on the antibody subtype, specific RACE primers targeting the 3' end of the constant region of its Ig heavy chain and Kappa light chain were designed and synthesized, named pRace-H-GSP and pRace-K-GSP, respectively. The primer sequences are as follows:

[0063] pRace-H-GSP: 5'-CATCDGTCTATCCACTGGCCCCTG-3' (SEQ ID NO. 10).

[0064] pRace-K-GSP: 5'-CTTCCCACCATCCAGTGAGCAGTT-3' (SEQ ID NO. 11).

[0065] The light and heavy chain DNA fragments obtained by RACE amplification were cloned into the pUC119 vector, and positive clones were initially obtained by blue-white screening. After purifying the plasmids of the positive clones, the sequences were determined using an ABI 3730 sequencer and universal primers M13F / M13R.

[0066] M13F: 5'-TGTAAAACGAGCGGCCAGT-3' (SEQ ID NO. 12).

[0067] M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO. 13).

[0068] Based on the IMGT / V-QUEST alignment analysis of the light and heavy chain nucleotide sequences, the variable region amino acid sequence of the anti-human CXCL13 rabbit monoclonal antibody was determined, with the light chain being SEQ ID NO.2 and the heavy chain being SEQ ID NO.6.

[0069] The light chain variable region, as shown in SEQ ID NO.2, has a total amino acid sequence of 104 amino acids. The number of amino acids in the four domains of its FR are 26, 17, 36 and 10, respectively. The number of amino acids in the three domains of LCDR are 7, 3 and 5, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-33aa, 51aa-53aa and 90aa-94aa, respectively. Their amino acid sequences are QSVYSNG (SEQ ID NO.3), YTS (SEQ ID NO.4) and LQYDD (SEQ ID NO.5), respectively.

[0070] The heavy chain variable region, as shown in SEQ ID NO:6, has a full-length amino acid sequence of 114 amino acids. The number of amino acids in the four domains of its FR are 25, 18, 39 and 11, respectively. The number of amino acids in the three domains of HCDR are 8, 7 and 6, respectively. HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 52aa-58aa and 98aa-103aa, respectively. Their amino acid sequences are: GFTLSSYG (SEQ ID NO.7), IYTGSDK (SEQ ID NO.8) and ARFPYD (SEQ ID NO.9).

[0071] III. Determination of the affinity constant of anti-human CXCL13 rabbit monoclonal antibody:

[0072] The non-competitive ELISA method for determining the antibody affinity constant (Ka) is as follows:

[0073] S1. Antigen coating: Dilute the CXCL13 synthetic peptide with carbonate buffer at gradient concentrations (5, 2.5, 1.25, 0.625 μg / mL), add 100 μL to each well of the ELISA plate, and coat overnight at 4°C.

[0074] S2, Blocking: Discard the coating solution, wash 3 times with PBS, add 200 μL of 5% skim milk powder to each well, and block at 37°C for 1 h.

[0075] S3, Primary Antibody Incubation: CXCL13 monoclonal antibody was serially diluted (7.00 × 10⁻⁶). -1 ~9.60×10 -4 100 μL of the solution (μg / mL) was added to each well of the coated plate and incubated at 37°C for 1 h.

[0076] S4. Secondary antibody incubation: After washing, add 100 μL of HRP-labeled goat anti-rabbit secondary antibody diluted 1:20000 to each well and react at 37℃ for 30 min.

[0077] S5. Color development and termination: After washing, add 50 μL of TMB color development solution to each well, react at 37℃ in the dark for 15 min, and then add 50 μL of 1mol / L H2SO4 to terminate the reaction.

[0078] S6. Absorbance detection: Use an ELISA reader to read the absorbance (OD value) of each well at a wavelength of 450nm.

[0079] S7. Data Analysis: Using the logarithm of antibody molar concentration as the x-axis and the corresponding OD value as the y-axis, an S-shaped curve was fitted, and the affinity constant Ka of the monoclonal antibody was calculated to be 3.18 × 10⁻⁶. 9 L / mol. This indicates that the anti-human CXCL13 rabbit monoclonal antibody OTIR7C4 prepared in this invention is a high-affinity antibody.

[0080] IV. Preparation of an immunohistochemical detection kit for detecting human CXCL13 protein:

[0081] An immunohistochemical assay kit for detecting human CXCL13 protein. This kit, with optimized component composition, can specifically locate and detect CXCL13 protein in formalin-fixed and paraffin-embedded tissue sections, exhibiting high specificity and high sensitivity.

[0082] (1) Immunohistochemical detection kit for human CXCL13 protein: Composition includes the following components:

[0083] Antigen retrieval solution: 1 mM ethylenediaminetetraacetic acid and 10 mM Tris buffer, pH 8.0;

[0084] Specific primary antibody: Anti-human CXCL13 rabbit monoclonal antibody with a concentration of 0.134 μg / mL, the preparation and purification methods of which are as described in Example 1;

[0085] Endogenous peroxidase inhibitor: 3% hydrogen peroxide solution;

[0086] Enzyme-labeled polymeric secondary antibody: Highly sensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer;

[0087] DAB colorimetric system: consists of DAB substrate buffer and DAB concentrated colorimetric solution;

[0088] Counterstaining solution: hematoxylin staining solution.

[0089] (2) Component Function Description:

[0090] The antigen repair solution is used for thermally induced epitope repair of antigen epitopes.

[0091] The endogenous peroxidase blocker is used to eliminate endogenous peroxidase activity in tissues;

[0092] The specific primary antibody binds specifically to the target protein CXCL13;

[0093] The enzyme-labeled polymer secondary antibody binds to the primary antibody and catalyzes the subsequent colorimetric reaction;

[0094] The DAB colorimetric system produces an insoluble brown precipitate under the catalysis of peroxidase, which is used to visualize and locate the target protein.

[0095] The hematoxylin staining solution is used to counterstain cell nuclei and provide a tissue morphology control.

[0096] V. Immunohistochemical detection kit for human CXCL13 protein: Protein expression detection in different tissues.

[0097] The expression of CXCL13 protein in different tissues was detected using the immunohistochemical kit for detecting human CXCL13 prepared in Example 4 on the UltraPATH fully automated staining system of Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. The specific steps are as follows:

[0098] S1. Preparation before staining:

[0099] Tissue section preparation: Select various tissues for verification, including tonsils, appendix, stomach, thyroid, placenta, pancreas, and angioimmunoblastic T-cell lymphoma. Use a Leica tissue microtome to cut sections with a section thickness of 4μm.

[0100] Instrument check: Confirm that the reagent rack, reaction tank, waste liquid tank, etc. of the staining machine are in normal working condition, and perform a self-test upon startup.

[0101] Reagent preparation: Detection system reagent kit. Load the reagents into the designated reagent slots on the instrument according to the staining requirements.

[0102] Sample loading: Place the slides in the slide holder in sequence, insert the slide holder into the instrument slide holder, and start scanning to load and verify information.

[0103] S2. Program Setup and Operation:

[0104] Program selection: Select a standardized staining program from the pre-stored program library that matches the antibody and tissue type for this test.

[0105] Parameter confirmation: Key parameters are as follows: Repair time: 20 min; Primary antibody incubation: 30 min at room temperature; Secondary antibody incubation: 15 min at room temperature; DAB color development: The color development time needs to be precisely controlled (usually 3~10 min), and should be monitored in real time according to the positive control to avoid over-development or under-development.

[0106] Operation monitoring: After starting the program, observe whether the initial operation is normal. If there are alarms or error messages, you need to check them immediately according to the instrument manual.

[0107] S3. Post-staining treatment:

[0108] After the staining process is complete, remove the slide and mount it.

[0109] S4. Observe the staining results under a microscope:

[0110] The detection of the above experiment is as follows Figures 1 to 3 As shown (the primary antibody used is the anti-human CXCL13 rabbit monoclonal antibody OTIR7C4 of this invention); Figure 1 a and Figure 1 b represents tonsils from two different cases. The staining results showed that follicular dendritic cells and follicular helper T cells in the germinal center showed brown staining, indicating the expression of CXCL13. However, squamous epithelial cells and endothelial cells only showed the color of hematoxylin and did not show positive brown staining, indicating that these types of cells did not express CXCL13. Figure 1 c and Figure 1 d represents appendices from two different cases. Figure 1 e represents gastric tissue. The staining results show that some cells in the lymphoid follicles are stained brown, while glandular epithelial cells, muscle cells, endothelial cells, and stromal cells are not positively stained.

[0111] Figure 2 a, Figure 2 b and Figure 2 c represents thyroid, pancreas, and placental tissues, respectively. The staining results showed that the follicular epithelium, vascular endothelium, and paracellular cells of the thyroid gland; the acinar epithelium, islet cells, and ductal epithelium of the pancreas; and the trophoblast cells, blood cells, and endothelial cells of the placenta were all without positive staining, indicating that CXCL13 was not expressed in these tissues and cells.

[0112] Figure 3 a, Figure 3 b and Figure 3 Image c shows tissue samples from three different cases of angioimmunoblastic T-cell lymphoma. The images reveal partial or complete destruction of follicles in normal lymph nodes, exhibiting structural loss or disorder. Dilated, irregular angiogenesis is visible, with perivascular cellular infiltration. Numerous small lymphocytes and plasmacytoid cells are visible in the background, structural characteristics consistent with angioimmunoblastic T-cell lymphoma. All three cases show moderate to strong staining with scattered distribution, and some locations show clustered or focal aggregations. Staining is primarily concentrated in lymphocytes and tumor cells, consistent with the expression pattern of CXCL13 in angioimmunoblastic T-cell lymphoma.

[0113] The above results are consistent with the theoretical expression pattern of CXCL13. The concentration of the monoclonal antibody used for the detection was 0.134 μg / mL, and the affinity constant of this antibody was 3.18 × 10⁻⁶. 9 L / mol is a high-affinity antibody match. Under these conditions, the staining results in various tissues show a signal-to-noise ratio that meets clinical requirements, indicating that the anti-human CXCL13 rabbit monoclonal antibody OTIR7C4 has good specificity and high sensitivity. It can be used for labeling CXCL13 protein for the diagnosis of diseases such as angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma.

[0114] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A rabbit monoclonal antibody against human CXCL13, characterized in that: It comprises a light chain variable region and a heavy chain variable region. The light chain variable region includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is shown as QSVYSNG, the amino acid sequence of LCDR2 is shown as YTS, and the amino acid sequence of LCDR3 is shown as LQYDD. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown as GFTLSSYG, the amino acid sequence of HCDR2 is shown as IYTGSDK, and the amino acid sequence of HCDR3 is shown as ARFPYD.

2. The anti-human CXCL13 rabbit monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.

2.

3. The anti-human CXCL13 rabbit monoclonal antibody according to claim 1, characterized in that: The light chain variable region has a total length of 104 amino acids, and the number of amino acids in the four domains of its FR are 26, 17, 36 and 10, respectively. The number of amino acids in the domains of LCDR1 to LCDR3 are 7, 3 and 5, respectively, and the amino acid positions are 27aa-33aa, 51aa-53aa and 90aa-94aa, respectively.

4. The anti-human CXCL13 rabbit monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

6.

5. The anti-human CXCL13 rabbit monoclonal antibody according to claim 1, characterized in that: The heavy chain variable region has a total length of 114 amino acids. The number of amino acids in the four domains of its FR are 25, 18, 39 and 11, respectively. The number of amino acids in the domains of HCDR1 to HCDR3 are 8, 7 and 6, respectively, and the amino acid positions are 26aa-33aa, 52aa-58aa and 98aa-103aa, respectively.

6. The anti-human CXCL13 rabbit monoclonal antibody according to claim 1, characterized in that: The anti-human CXCL13 rabbit monoclonal antibody specifically binds to the CXCL13 protein and specifically recognizes the amino acid sequence shown in SEQ ID NO.

1.

7. The use of the anti-human CXCL13 rabbit monoclonal antibody according to any one of claims 1 to 6 in the preparation of immunohistochemical detection kits, chips or test strips for detecting human CXCL13 protein.

8. The application according to claim 7, characterized in that: The immunohistochemical detection kit for human CXCL13 protein includes a primary antibody reagent, which includes a rabbit monoclonal antibody against human CXCL13.

9. The application according to claim 8, characterized in that: The immunohistochemical detection kit for human CXCL13 protein also includes antigen retrieval solution, endogenous peroxidase inhibitor, high-sensitivity secondary antibody reagent, DAB substrate buffer, DAB concentrated chromogenic solution, and hematoxylin staining solution.

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