Anti-CD3D antibody and application thereof

Abstract

The invention discloses an anti-CD3D antibody and application thereof.The antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of CDR-H1 of the heavy chain variable region is shown as SEQ ID NO: 4, the amino acid sequence of CDR-H2 of the heavy chain variable region is shown as SEQ ID NO: 5, and the amino acid sequence of CDR-H3 of the heavy chain variable region is GDL; the amino acid sequence of the CDR-L1 of the light chain variable region is as shown in SEQ ID NO: 6, the amino acid sequence of the CDR-L2 of the light chain variable region is as shown in SEQ ID NO: 7, and the amino acid sequence of the CDR-L3 of the light chain variable region is as shown in SEQ ID NO: 8. The anti-CD3D antibody is a rabbit monoclonal antibody, the obtained antibody is high in titer, clear in specific positioning in immunohistochemical staining and free of non-specific background staining, the antibody is wide in application, and the antibody can be used in pathological immunohistochemical staining experiments and can also be used in Western blot and flow cytometry experiments.

Description

Technical Field

[0001] The present invention belongs to the field of antibodies, and in particular relates to an anti-CD3D antibody and applications thereof. Background Art

[0002] Part of the TCR-CD3 complex, present on the surface of T lymphocytes, plays a crucial role in adaptive immune responses. When antigen-presenting cells (APCs) activate the T cell receptor (TCR), TCR-mediated signals are transmitted across the cell membrane via the CD3 chains CD3D, CD3E, CD3G, and CD3Z. All CD3 chains contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains. Upon TCR engagement, these motifs are phosphorylated by the Src family protein tyrosine kinases LCK and FYN, thereby activating downstream signaling pathways. In addition to its signal transduction role in T cell activation, CD3D also plays a crucial role in thymocyte differentiation. Indeed, it is involved in the proper intracellular assembly and surface expression of the TCR-CD3 complex. Without a functional TCR-CD3 complex, thymocytes cannot differentiate normally.

[0003] CD3D interacts with CD4 and CD8, thereby establishing a functional link between the TCR and the coreceptors CD4 and CD8, which is required for the activation and positive selection of CD4 or CD8 T cells.

[0004] CD3D is primarily expressed in immunohistochemistry on lymphocytes in the tonsils, lymph nodes, stomach, and intestines, as well as in T-cell lymphomas derived from them. Detection of CD3D can assess the body's immune function and provide clinical guidance. It is also crucial for the diagnosis of certain infectious and immune diseases, as well as tumors. Summary of the Invention

[0005] To address the above issues, the present invention provides an anti-CD3D antibody and its application. The antibody has high titer, clear specific localization in immunohistochemical staining, and no nonspecific background staining. It can also be widely used in Western blot analysis and flow cytometry.

[0006] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides an anti-CD3D antibody, which includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the complementarity determining region CDR-H1 of the heavy chain variable region is shown in SEQ ID NO: 4, the amino acid sequence of the complementarity determining region CDR-H2 is shown in SEQ ID NO: 5, and the amino acid sequence of the complementarity determining region CDR-H3 is: GDL; the amino acid sequence of the complementarity determining region CDR-L1 of the light chain variable region is shown in SEQ ID NO: 6, the amino acid sequence of the complementarity determining region CDR-L2 is shown in SEQ ID NO: 7, and the amino acid sequence of the complementarity determining region CDR-L3 is shown in SEQ ID NO: 8.

[0007] In some embodiments, the antibody is analyzed using abYsis online software for CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprising the following polypeptide sequences: (1) A CDR-H1 sequence of at least 5 amino acids; the sequence is SNVVS (SEQ ID NO: 4).

[0008] (2) A CDR-H2 of at least 16 amino acids; the sequence is VTKTGGRTYYANWAKG (SEQ ID NO: 5).

[0009] (3) CDR-H3 of at least 3 amino acid sequences; the sequence is GDL.

[0010] (4) A CDR-L1 of at least 13 amino acid sequences; the sequence is QSSPSVLGNNFLS (SEQ ID NO: 6).

[0011] (5) A CDR-L2 of at least 7 amino acid sequences; the sequence is TASSLSS (SEQ ID NO: 7).

[0012] (6) A CDR-L3 of at least 13 amino acids; the sequence is AGGYDCSSSDCNA (SEQ ID NO: 8).

[0013] In some embodiments of the present invention, the amino acid sequence of the full-length gene of the antibody heavy chain variable region is shown as SEQ ID NO: 2, and the amino acid sequence of the full-length gene of the light chain variable region is shown as SEQ ID NO: 3.

[0014] In some embodiments of the present invention, the antibody is a rabbit anti-human monoclonal antibody, and the rabbit immunogen comprises the amino acid sequence shown in SEQ ID NO:1.

[0015] In some specific embodiments of the present invention, a synthetic polypeptide fragment of aa158-aa171 DAQYSHLGGNWARNK (SEQ ID NO: 1) in the amino acid sequence of human CD3D protein (Uniprot ID: P04234) is selected as the immunogen sequence for preparing the antibody.

[0016] In some embodiments of the present invention, the rabbit immunogen is formed by cross-linking the amino acid sequence shown in SEQ ID NO: 1 with the macromolecule KLH to form a complete antigen.

[0017] A nucleic acid encoding the antibody described in the first aspect. By isolating the nucleic acid encoding the antibody described in the first aspect, the antibody can be produced in a recombinant manner, the nucleic acid is isolated and inserted into a replicable vector, and then further cloned or further expressed. Based on this, the second aspect of the present invention provides an expression vector comprising the nucleic acid described above. The sequence of the nucleic acid encoding the heavy chain variable region and light chain variable region of the present invention can be changed, and such changes include the addition, deletion or non-conservative / conservative substitution of nucleotides. The DNA encoding the antibody can be easily isolated or synthesized using conventional procedures. A variety of vectors are available, and the vector components generally include but are not limited to one or more of the following components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0018] Preferably, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0019] A third aspect of the present invention provides a host cell comprising the expression vector described above. In some embodiments of the present invention, the expression vector is a prokaryotic expression vector and the host cell is a prokaryotic cell; in other embodiments, the expression vector is a eukaryotic expression vector and the host cell is a eukaryotic cell. In a specific embodiment of the present invention, the expression vector is Escherichia coli and the host cell is a HEK293 cell.

[0020] The fourth aspect of the present invention provides use of the antibody described in the first aspect in preparing an immunoassay tool for detecting and identifying CD3D protein.

[0021] In some embodiments of the present invention, the immunoassay tool is a reagent, a kit, a chip or a test paper.

[0022] In some embodiments of the present invention, the immunodetection tool is used for at least one of immunohistochemistry, flow cytometry, and immunoblotting.

[0023] The fifth aspect of the present invention provides a kit for detecting and identifying CD3D protein, wherein the kit comprises the antibody described in the first aspect.

[0024] The present invention also provides a method for obtaining the above-mentioned antibody, which comprises: culturing the host cell described in the fourth aspect, and isolating and purifying the antibody from the cultured cells.

[0025] Beneficial effects of the present invention Compared to existing technologies, the present invention offers the following advantages: The anti-CD3D antibody provided herein is a rabbit monoclonal antibody, exhibits high titer, demonstrates clear specific localization in immunohistochemical staining, and exhibits no nonspecific background staining. This antibody can be used not only in pathological immunohistochemical staining experiments but also in Western blot, immunofluorescence, and flow cytometry assays, demonstrating its wide application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 ELISA binding curves of the antigens in Example 1 of the present invention are shown; Figure 2 The figures show the results of immunohistochemical staining of different tissue sections with anti-CD3D antibodies; Figure 3 The figure shows the results of Western blot detection of anti-CD3D antibody; Figure 4 The figure shows the detection results of anti-CD3D antibody in immunofluorescence; Figure 5 The graph shows the results of anti-CD3D antibody flow cytometry detection; the peak on the left indicates that the isotype control antibody does not recognize the CD3D protein on the Jurkat cell line, and the peak on the right indicates that the anti-CD3D antibody can recognize the CD3D protein on the Jurkat cell line. DETAILED DESCRIPTION

[0027] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0029] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0030] Example 1 Preparation of anti-CD3D monoclonal antibody The immunogen for this antibody preparation was a peptide fragment synthesized from aa158-aa171DAQYSHLGGNWARNK (SEQ ID NO: 1) within the amino acid sequence of the human CD3D protein (Uniprot ID: P04234). This peptide fragment was cross-linked with the macromolecule KLH to form a complete antigen.

[0031] The immunogen is used to immunize New Zealand white rabbits, and the steps are as follows: first, the polypeptide immunogen is coupled to the KLH macromolecular protein, and the New Zealand white rabbits are immunized after dialysis. For the first immunization, 1 mg / mL is used, and 0.5 mL is mixed with an equal amount of complete Freund's adjuvant for the first immunization; 2 weeks later, 0.5 mg / mL is used, and 0.5 mL is mixed with an equal amount of incomplete Freund's adjuvant for the second immunization, and the third and fourth immunizations are subsequently performed every 2 weeks according to the second immunization method.

[0032] After four immunizations, blood was collected from New Zealand white rabbits to test serum titers. B cells from qualified rabbits were cryopreserved. B cells were obtained using the method described in our patent: CN201610724209.1. The cells were then lysed using the method described in our patent: CN201910004299.0, and mRNA was extracted for RT-PCR. After RT-PCR, the resulting cDNA was subjected to PCR to obtain the antibody's light and heavy chain DNA. The light and heavy chain DNAs were ligated into the pBV vector and transformed into competent cells, which were then plated and cultured for 12 hours. Colonies were then picked and shake-cultured. After 24 hours of culture, the heavy and light chain plasmids were extracted. The extracted light and heavy chain plasmids were transfected into HEK293 cells and cultured for 6 days.

[0033] Six days after transfection, the cell culture supernatant was harvested and purified to obtain rabbit monoclonal antibodies, which were then sequenced.

[0034] The full-length amino acid sequence of the CD3D rabbit monoclonal antibody H chain (heavy chain variable region) (SEQ ID NO: 2) is as follows: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSNVVSWVRQAPGKGLEWIGVTKTGGRTYYANWAKGRFTISSTSTTVDLKIASPTTEDTATYFCTTGDLWGQGTLVTVS SGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPP PELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKA RGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK The full-length amino acid sequence of the CD3D rabbit monoclonal antibody L chain (light chain variable region) (SEQ ID NO: 3) is as follows: AQVLTQTASPVSAAIGGTVTINCQSSPSVLGNNFLSWYQQKPGQPPKRLISTASSLSSGVPSRFKGSGSGTQFTLTISDVQCDDAATYHCAGGYDCSSSDCNAFGGGT EVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC The obtained anti-CD3D antibodies were analyzed using abYsis online software; The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region are as follows: (1) a CDR-H1 sequence of at least 5 amino acids; the sequence is SNVVS (SEQ ID NO: 4); (2) a CDR-H2 of at least 16 amino acids; the sequence is VTKTGGRTYYANWAKG (SEQ ID NO: 5); (3) CDR-H3 of at least 3 amino acids; the sequence is GDL; The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region are as follows: (1) a CDR-L1 sequence of at least 13 amino acids; the sequence is QSSPSVLGNNFLS (SEQ ID NO: 6); (2) a CDR-L2 sequence of at least 7 amino acids; the sequence is TASSLSS (SEQ ID NO: 7); (3) A CDR-L3 of at least 13 amino acids; the sequence is AGGYDCSSSDCNA (SEQ ID NO: 8).

[0035] Example 2 Enzyme-linked immunosorbent assay of anti-CD3D monoclonal antibodies The antibody can specifically bind to the immunogen synthetic polypeptide sequence designed by human CD3D.

[0036] The procedure was carried out according to the conventional iELISA method. The steps were as follows: (1) Coating: Use immunogen synthetic peptides designed to specifically bind to human CD3D protein for coating, 1µg / mL, 50µL / well, and coat overnight at 4℃.

[0037] (2) Blocking: The next day, wash the plate three times with PBST, then add 5% skim milk for blocking, 100 µL / well, and block at 30°C for 1 h.

[0038] (3) Primary antibody incubation: After washing the plate, the obtained anti-CD3D rabbit monoclonal antibody was diluted 4-fold from 1 μg / mL, with a total of 8 gradients. The diluted antibody was added to the ELISA plate at 50 μL / well and incubated at 30°C for 1 h.

[0039] (4) Secondary antibody incubation: After washing the plate, add 1:30,000 diluted HRP-labeled goat anti-rabbit IgG (Jackson, Cat#111-035-144) to the ELISA plate at 50 µL / well and incubate at 30°C for 40-60 minutes.

[0040] Color development and termination: After washing, add TMB color development solution at 50µL / well and react at 30℃ in the dark for 15 minutes. After the reaction is completed, add 50µL 1M H2SO4 to each well to terminate the reaction and read the OD450 absorbance on a microplate reader. Prepare a standard curve based on the readings. ELISA binding curve is shown in Figure 1 .

[0041] The results showed that there were significant differences in the readings between the dilutions. The above antibodies could specifically bind to the immunogen synthetic peptide sequence designed for human CD3D. The ELISA binding curve of CD3D monoclonal antibody was as follows: Figure 1As shown, the EC50 was 1.01 ng / mL.

[0042] Example 3 Application of anti-CD3D monoclonal antibodies in pathological immunohistochemistry Normal tonsil, Hodgkin's lymphoma, spleen, colon, brain, esophagus and other tissue sections were selected for pathological immunohistochemical staining, and the primary antibody was CD3D monoclonal antibody.

[0043] The tissue sections were operated using conventional manual immunohistochemical procedures. After immunohistochemical staining was completed, the sections were placed under a microscope for reading and photographing. The immunohistochemical staining images taken under the microscope were as follows: Figure 2 shown.

[0044] The results show that: Figure 2 It can be clearly seen that the anti-CD3D rabbit monoclonal antibody has brown positive staining (indicated by the arrow) on T lymphocytes in normal tonsil tissue, Hodgkin's lymphoma tissue, and spleen tissue, and its subcellular localization is the cell membrane.

[0045] CD3D monoclonal antibody showed no nonspecific staining in brain tissue, multiple squamous epithelial tissues of the esophagus, glandular epithelium of the colon, etc. The expression spectrum of this antibody was consistent with the theoretical and practical immunohistochemical application standards.

[0046] Example 4 Application of anti-CD3D monoclonal antibody in Western blot (1) Jurkat whole cell lysate was used for polyacrylamide gel electrophoresis. The loading amount of Jurkat whole cell lysate was 20 μg / lane. The electrophoresis conditions were: 80 V, 40 min; 120 V, 80 min.

[0047] (2) Transfer: Remove the gel from the glass plate after electrophoresis in step (1), soak it in transfer buffer for 30 minutes, and then perform the transfer experiment. The transfer conditions are: 0.4A horizontal flow transfer for 20 minutes.

[0048] (3) After the transfer is completed, remove the PVDF membrane, rinse the PVDF membrane with ddH2O for 5 minutes, add 10mL of Ponceau red for staining, and wash the membrane with methanol for 5 minutes after staining. Dry it and set aside. At this time, the cell protein lysate on the PVDF membrane will appear a red area, indicating that the protein lysate on the cells has been transferred to the PVDF.

[0049] (4) Blocking: Take out the PVDF membrane prepared in step (3), activate it with methanol for 30 seconds, and then wash the membrane with ddH2O for 5 minutes. Then add 10 mL of 5% skim milk powder to block the nonspecific binding sites on the membrane and incubate at 60 rpm for 1 hour at room temperature.

[0050] (5) Primary antibody incubation: After step (4), add anti-CD3D monoclonal antibody diluted 1:10,000 and incubate at room temperature and 60 rpm for 1 h.

[0051] (6) Secondary antibody incubation: After step (5), add 1:4000 diluted HRP-conjugated goat anti-rabbit IgG polyclonal antibody and incubate at room temperature at 60 rpm for 1 h.

[0052] (7) Development and photography: After step (6), add the prepared ECL developer to the membrane. Place the PVDF membrane in a UV exposure instrument and take a photo.

[0053] (8) The result after the photo is inverted is as follows Figure 3 shown.

[0054] The results showed that the anti-CD3D rabbit monoclonal antibody recognized the CD3D protein in Jurkat whole-cell lysates, with an apparent molecular weight of 19 kD and no obvious miscellaneous bands, consistent with theoretical and literature reports. This indicates that this antibody can be used in Western blot experiments.

[0055] Example 5 Application of anti-CD3D monoclonal antibodies in immunofluorescence detection (1) Use Jurkat suspension cells in good culture condition, wash with PBS and adjust the cell density to 1M / mL.

[0056] (2) Add 1 mL of 4% paraformaldehyde to each cell volume, pipette to mix, and let it stand at room temperature for 30 minutes.

[0057] (3) After fixation, centrifuge at 215g for 5 minutes and discard the supernatant; add 10-20mL of PBS and gently pipette, centrifuge at 215g for 5 minutes and discard the supernatant; repeat once and discard the supernatant.

[0058] (4) Gently tap the centrifuge tube to break up the precipitate, add 1 mL of membrane permeabilization agent based on 1M cells, pipette to mix, and incubate at room temperature for 10 minutes; after membrane permeation, centrifuge at 215g for 5 minutes and remove the supernatant; add 10-20 mL of PBS, pipette gently, centrifuge at 215g for 5 minutes and remove the supernatant; repeat once and discard the supernatant.

[0059] (5) Resuspend the cells in PBS to 2M / mL. First, use an immunohistochemistry pen to draw 4 to 5 small grids of about 1 cm square from left to right on the slide (draw the corresponding number of small grids according to the project requirements), then rinse the slide with PBST once (to facilitate the flattening of cells), and slightly dry it. Add resuspended cells at 100 μL / grid and bake in a 60℃ oven for about 20 minutes until the slide is dry; add 10% goat serum at 100 μL / grid and incubate at room temperature for 30 minutes; after blocking, place the slide on a staining rack in a staining jar filled with sufficient PBS (the liquid level can cover the cells on the slide), rinse three times, remove the slide, and spin dry.

[0060] (6) Primary antibody incubation: Add negative control (PBS) sample and isotype control reagent (1ug / mL) and 1:500 diluted CD3D monoclonal antibody reagent (1ug / mL) at 100μL / grid, and incubate overnight at 4°C (water should be added to the incubation box to incubate in a wet box to avoid volatilization of the primary antibody).

[0061] (7) After the primary antibody incubation is completed, discard the primary antibody and place the slide on a staining rack in a staining jar filled with sufficient PBS (the liquid level can cover the cells on the slide). After rinsing three times, remove the slide and spin dry. Add green fluorescent secondary antibody (abcam, goat anti-rabbit IgG-Alexa Fluor®488, Cat#ab150077) at 100 μL / grid. The fluorescent secondary antibody dilution is 1:500 and incubate at room temperature in the dark for 1 hour. (8) After the secondary antibody incubation is completed, discard the secondary antibody and place the slide on a staining rack in a staining jar filled with sufficient PBS (the liquid level can cover the cells on the slide). After rinsing three times, remove the slide and spin dry. Use a 200μL pipette to add a drop of anti-fluorescence quenching mounting medium (containing DAPI) between every two grids on the slide, that is, 2 to 3 drops of mounting medium on each slide; cover with a coverslip, let it stand at room temperature, and dry it naturally in the dark before reading the slide.

[0062] (9) Observe and photograph the antibody staining results under a fluorescence microscope. Figure 4 shown.

[0063] The results showed that the CD3D rabbit monoclonal antibody produced green fluorescent membrane staining on Jurkat cells, consistent with experimental expectations. The anti-CD3D rabbit monoclonal antibody can be used for immunofluorescence staining.

[0064] Example 6 Application of anti-CD3D monoclonal antibodies in flow cytometry (1) Use cultured Jurkat cells, wash with PBS, and adjust the cell density to 1M / mL.

[0065] (2) Divide the 1M / mL cells into 3 tubes, 1 tube for negative control experimental cells, 1 tube for isotype control experimental cells, and 1 tube for anti-CD3D antibody experimental cells.

[0066] (3) Cell fixation: Add 1 mL of 4% paraformaldehyde fixative to each of the three tubes of cells and fix the cells for 30 minutes. Then add 1 mL of 1% BSA in PBS and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0067] (4) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0068] (5) Cell membrane disruption: Add 1 mL of 0.1% Triton X-100 permeabilization reagent to each of the three tubes of cells and permeabilize the cells for 10 minutes. Then add 1 mL of 1% BSA in PBS and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0069] (6) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0070] (7) Primary Antibody Incubation: Add 100 µL of 1% BSA in PBS to one tube of cells, 100 µL of 1 µg / mL isotype control antibody to another tube, and 100 µL of 1 µg / mL anti-CD3D rabbit monoclonal antibody to the final tube. Resuspend the cells and incubate at room temperature for 60 min. After incubation, add 1 mL of 1% BSA in PBS to each tube and centrifuge at 1400 rpm for 5 min at room temperature.

[0071] (8) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0072] (9) Secondary antibody incubation: Add 100 µL of 1:2000 diluted goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) to each tube, resuspend the cells, and incubate at room temperature for 60 min. After incubation, add 1 mL of 1% BSA in PBS to each tube and centrifuge at 1400 rpm for 5 min at room temperature.

[0073] (10) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0074] (11) Flow cytometry detection: Using the isotype control well as the standard, adjust the voltage for flow cytometry detection. The results are as follows Figure 5shown.

[0075] The results showed that in a flow cytometry experiment using an anti-CD3D rabbit monoclonal antibody on a Jurkat wild-type tumor cell line, the isotype control antibody did not recognize the CD3D protein on the Jurkat wild-type cell line (left curve), while the anti-CD3D rabbit monoclonal antibody did recognize the CD3D protein on the Jurkat wild-type cell line (right curve). This antibody can be used in flow cytometry applications.

[0076] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the present application.

Claims

1. An anti-CD3D antibody, characterized in that: It includes a heavy chain variable region and a light chain variable region, the amino acid sequence of CDR-H1 of the heavy chain variable region is shown in SEQ ID NO: 4, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR-H3 is: GDL; the amino acid sequence of CDR-L1 of the light chain variable region is shown in SEQ ID NO: 6, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 7, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

8.

2. The antibody according to claim 1, characterized in that: The amino acid sequence of the full-length gene of the antibody heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the full-length gene of the light chain variable region is shown in SEQ ID NO:

3.

3. The antibody according to claim 1 or 2, characterized in that: The antibody is a rabbit anti-human monoclonal antibody, and the rabbit immunogen comprises the amino acid sequence shown in SEQ ID NO:

1.

4. An expression vector, characterized in that: Comprising a nucleic acid encoding the antibody according to claim 1 or 2.

5. A host cell, characterized in that: Comprising the expression vector according to claim 4.

6. Use of the antibody according to any one of claims 1 to 3 in the preparation of an immunoassay tool for detecting and identifying CD3D protein.

7. The use according to claim 6, characterized in that: The immunoassay tool is a reagent, a test kit, a chip or a test paper.

8. The use according to claim 6, characterized in that: The immunodetection tool is used for at least one of immunohistochemistry, flow cytometry, immunofluorescence, and Western blotting.

9. A kit for detecting and identifying CD3D protein, characterized in that: Comprising the antibody of claim 3.

10. A method for obtaining the antibody according to claim 1 or 2, characterized in that: include: (1) cultivating the host cell according to claim 5, and (2) Isolating and purifying the antibody from the cultured cells.

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