Rabbit monoclonal antibody or antigen-binding fragment thereof against t cell surface glycoprotein cd1a, method of preparation and use thereof

Abstract

The application relates to the technical field of immunoglobulin, in particular to a rabbit monoclonal antibody or antigen binding fragment against T cell surface glycoprotein CD1a, a preparation method and application thereof. The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the VL of the rabbit monoclonal antibody against the T cell surface glycoprotein CD1a protein are respectively shown as SEQ ID NO. 4-6; the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the VH are shown as SEQ ID NO. 8-10. The application further provides an immunohistochemical detection kit containing the rabbit monoclonal antibody against the human CD1a protein, and application of the antibody or the kit in immunohistochemical detection. The rabbit monoclonal antibody against the human CD1a protein has high specificity and sensitivity, can be applied to labeling of CD1a protein in tissue cells, and significantly improves the specificity and reliability of immunodetection of the CD1a protein.

Description

Technical Field

[0001] This application relates to the field of immunoglobulin technology, specifically to rabbit monoclonal antibodies against T cell surface glycoprotein CD1a or their antigen-binding fragments, preparation methods, and applications. Background Technology

[0002] T cell surface glycoprotein (CD1a) is a transmembrane glycoprotein with a molecular weight of 43-49 kDa, belonging to the CD1 family of antigen-presenting molecules. It is primarily expressed in thymic cortical cells, Langerhans cells, and dendritic cells. The structure of CD1a is similar to that of MHC class I molecules, and it can form a heterodimer with β2-microglobulin. CD1a is unique in that its antigen-binding groove is hydrophobic, enabling it to specifically bind and present lipid and glycolipid antigens, thus establishing a crucial bridge between innate and adaptive immunity.

[0003] In clinicopathological diagnosis, CD1a is not only a diagnostic marker for Langerhans cell hyperplasia, but also helps in the differential diagnosis of precursor T-lymphoblastic lymphoma and other mature T-cell lymphomas. Studies have shown that the expression level of CD1a in dendritic cells within the tumor microenvironment is correlated with tumor differentiation and clinical stage.

[0004] However, current antibody reagents targeting CD1a still have room for improvement in terms of specificity and sensitivity, which limits their application in clinical pathological diagnosis and basic research. Developing monoclonal antibodies capable of highly specifically recognizing CD1a is therefore crucial. High-quality antibodies can not only improve the accuracy and reliability of diagnosing these diseases but also provide key tools for in-depth research into the mechanisms of CD1a-mediated immune responses, laying the foundation for developing new immunotherapeutic strategies. Summary of the Invention

[0005] In view of this, this application provides a rabbit monoclonal antibody against human CD1a protein, a preparation method thereof, and its application, to provide a monoclonal antibody that specifically binds to CD1a protein, as well as the preparation method and application of the antibody, providing strong support for the diagnosis of Langerhans cell hyperplasia, and at the same time contributing to basic and clinical research related to CD1a.

[0006] In a first aspect, this application provides a rabbit monoclonal antibody against human CD1a protein or an antigen-binding fragment thereof, comprising a light chain variable region VL and a heavy chain variable region VH. The light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is the sequence shown in SEQ ID NO.4 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.4. The amino acid sequence of LCDR2 is KVS (SEQ ID NO.5) or a variant of KVS (SEQ ID NO.5) with one or two amino acid substitutions, deletions, or additions. The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.6 or a variant with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO.6; The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is the sequence shown in SEQ ID NO.8 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.8. The amino acid sequence of HCDR2 is the sequence shown in SEQ ID NO.9 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.9. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.10 or a variant having one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.10; Furthermore, the rabbit monoclonal antibody against human CD1a protein or its antigen-binding fragment can specifically bind to anti-T cell surface glycoproteins.

[0007] Further, the light chain variable region VL comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.3, wherein any one or more amino acids have been substituted, deleted, and / or added, and / or terminally modified; the heavy chain variable region VH comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.7, wherein any one or more amino acids have been substituted, deleted, and / or added, and / or terminally modified.

[0008] Furthermore, amino acid sequences with 95%, 96%, 97%, 98%, or 99% identity.

[0009] Furthermore, the light chain variable region VL includes the amino acid sequence shown in SEQ ID NO.3, and the heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO.7.

[0010] Furthermore, the light chain variable region VL domain has a total length of 108 amino acids, with the four FR domains having amino acid numbers of 26, 21, 36, and 10, respectively, and the LCDR1-3 domains having amino acid numbers of 7, 3, and 5, respectively, which are 27-33 aa, 55-57 aa, and 94-98 aa.

[0011] Furthermore, the VH domain of the heavy chain variable region has a total length of 108 amino acids, and the number of amino acids in the four domains of its FR are 25, 17, 38 and 11, respectively, and the number of amino acids in the HCDR1-3 domains are 6, 6 and 5, respectively, which are 26-31 aa, 49-54 aa and 93-97 aa.

[0012] Furthermore, the antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.

[0013] Secondly, this application also provides a biomaterial selected from polynucleotides, carriers, or cells. The polynucleotide encodes the rabbit monoclonal antibody against human CD1a protein or its antigen-binding fragment as described in the first aspect; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the vector, or is capable of expressing a rabbit monoclonal antibody encoding the anti-human CD1a protein described in the first aspect, or an antigen-binding fragment thereof.

[0014] Thirdly, this application also provides a method for preparing a rabbit monoclonal antibody or antigen-binding fragment thereof encoding the anti-human CD1a protein described in the first aspect, comprising culturing cells as described in the second aspect, wherein the cells are prepared by transforming the cells with a polynucleotide encoding a rabbit monoclonal antibody or antigen-binding fragment thereof comprising the anti-human CD1a protein, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprising co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.

[0015] Furthermore, the cells are eukaryotic cells, preferably mammalian cells, more preferably 293 cells or CHO cells.

[0016] Fourthly, this application also provides the application of the rabbit monoclonal antibody against human CD1a protein as described in the first aspect or its antigen-binding fragment, or the biological material as described in the second aspect, wherein the application is selected from one or more of the following: 1) Detection and / or labeling of human CD1a protein for non-diagnostic and non-therapeutic purposes; 2) Prepare immunoassay products for detecting and / or labeling human CD1a protein; 3) Detection of Langerhans cell hyperplasia and / or T-cell lymphoma for non-diagnostic and non-therapeutic purposes; 4) Prepare immunoassay products for the detection of Langerhans cell hyperplasia and / or T-cell lymphoma.

[0017] In this application, the application is for non-diagnostic and non-therapeutic purposes. Specifically, it can be used to locate the spatial distribution of CD1a+ cells in tissue sections such as skin, tonsils, and lymph nodes through immunohistochemistry / immunofluorescence, and study their role in immune surveillance; it can be used to label CD1a+ cells with IHC / IF in tumor tissues such as cutaneous T-cell lymphoma, Langerhans cell hyperplasia, and cortical thymoma, and analyze the potential correlation between their infiltration density, location, and patient prognosis; it can also be used to construct molecules that guide T cells to attack specific tumor cells with CD1a as one of the targets, and use CD1a antibodies to verify target binding and function.

[0018] Fifthly, this application also provides an immunoassay product for human CD1a protein, the immunoassay product comprising the rabbit monoclonal antibody against human CD1a protein as described in the first aspect or its antigen-binding fragment or the biological material as described in the second aspect.

[0019] Furthermore, the immunoassay products are reagents or kits for detection such as enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), and immunoprecipitation.

[0020] Furthermore, the immunoassay product is an immunoassay kit, which includes a primary antibody reagent containing a rabbit monoclonal antibody against human CD1a protein or its antigen-binding fragment as described in the first aspect of this application.

[0021] Furthermore, the immunoassay kit 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] Sixthly, this application also provides a recombinant CD1a protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0023] Furthermore, the nucleotide sequence of the recombinant CD1a protein is shown in SEQ ID NO.2.

[0024] In a seventh aspect, this application also provides the use of the recombinant CD1a protein described in the sixth aspect in the preparation of rabbit monoclonal antibodies against human CD1a protein or antigen-binding fragments thereof as immunogens.

[0025] Furthermore, the application involves immunizing animals with purified recombinant CD1a protein as an immunogen, and isolating peripheral blood mononuclear cells (PBMCs) from the immunized animals; sorting the PBMCs using purified recombinant CD1a protein to obtain antigen-specific B cells; and culturing and screening the obtained B cells to obtain positive clones.

[0026] Compared to existing technologies, this application provides a rabbit monoclonal antibody that specifically binds to human CD1a protein, significantly improving the specificity, accuracy, and reliability of CD1a protein immunoassay. Furthermore, this application establishes an immunohistochemical detection method and kit based on this anti-human CD1a protein rabbit monoclonal antibody. Using this immunohistochemical detection kit, specific labeling of human CD1a protein can be achieved in tissues including the thymus, tonsils, skin, and Langerhans cell hyperplasia. It can also be used for chemical detection of immunohistochemical tissues and for identifying Langerhans cell hyperplasia associated with human CD1a protein expression, providing strong support for the accurate diagnosis of Langerhans cell hyperplasia. It can also be used for basic and clinical research related to CD1a. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of this application, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below.

[0028] Figure 1 The image shows the Western blot results of the recombinant CD1a protein described in Example 1. The expression of the recombinant CD1a protein in E. coli cells was detected using anti-HIS antibody. Lane L represents the detection results of E. coli cell lysate transfected with the empty vector as the antigen, and lane R represents the detection results of E. coli cell lysate transfected with pET23a-His-rCD1a plasmid as the antigen.

[0029] Figure 2 The image shows the SDS-PAGE results of the CD1a protein described in Example 1. The recombinant CD1a protein was purified using a nickel affinity chromatography column, and the purified protein was subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining.

[0030] Figure 3 This is a schematic diagram showing the results of immunohistochemical detection of thymus tissue using the rabbit monoclonal antibody OTIR4A6 against human CD1a protein described in Example 5.

[0031] Figure 4 This is a schematic diagram showing the results of immunohistochemical detection of tonsil tissue using the rabbit monoclonal antibody OTIR4A6 against human CD1a protein described in Example 5.

[0032] Figure 5 This is a schematic diagram showing the results of immunohistochemical detection of skin tissue using the rabbit monoclonal antibody OTIR4A6 against human CD1a protein described in Example 5.

[0033] Figure 6 This is a schematic diagram showing the results of immunohistochemistry detection of Langerhans cell hyperplasia using the rabbit monoclonal antibody OTIR4A6 against human CD1a protein described in Example 5. Detailed Implementation

[0034] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer. Unless otherwise specified, the test reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods unless otherwise specified.

[0035] Example 1: Preparation of recombinant CD1a protein 1. Synthesis and cloning of the gene encoding recombinant CD1a protein Based on the complete sequence of the human CD1a protein (accession number P06126) in the UniProt database, its homology with other proteins in the same family was compared using BLAST. Antigenicity, hydrophilicity / hydrophobicity, and secondary structure were analyzed using Protean software. By comprehensively evaluating its biological characteristics, antigenicity, hydrophilicity / hydrophobicity, amino acid distribution, and human-animal homology, a 284-amino acid region from positions 17 to 300 of the CD1a protein (SEQ ID NO. 1) was selected as the amino acid sequence for the synthesized protein. This region is the full-length extracellular region after removing the signal peptide, with a molecular weight of approximately 35 kDa. Based on this, the corresponding nucleotide sequence was designed and synthesized as shown in SEQ ID NO. 2. SgfI and MluI restriction enzyme sites were introduced upstream and downstream of the gene, respectively, and the gene was cloned into the pET23a-N-His expression vector, successfully constructing the CD1a recombinant expression plasmid.

[0036] 2. Expression and purification of recombinant CD1a protein The CD1a recombinant plasmid was transformed into BL21(DE3) competent cells, and after heat shock, the cells were plated on LB agar plates containing ampicillin. Single colonies were picked and cultured until the OD600 reached 0.6, and then IPTG was added and induced at 25°C for 16 hours. The bacterial cells were collected, sonicated, centrifuged, and Western blotting analysis showed a specific band at 35 kDa. Figure 1 This confirms the successful expression of the recombinant protein.

[0037] Purification was performed using a nickel affinity chromatography column. The lysate supernatant was filtered through a membrane and incubated with nickel agarose beads, followed by gradient elution with buffers containing 20 mM, 50 mM, and 250 mM imidazole. The collected protein solution was immediately adjusted to pH 7.4, and a protective agent was added to maintain stability.

[0038] The purified product showed a single, clear band after SDS-PAGE. Figure 2 This indicates the acquisition of high-purity recombinant CD1a protein. The final protein was quantified using the BCA method and aliquoted for storage at -80°C. This method can obtain recombinant CD1a protein with a purity exceeding 95% and a yield of approximately 50 mg / L, providing reliable material for subsequent antibody preparation and screening experiments.

[0039] Example 2: Preparation of rabbit monoclonal antibody against human CD1a protein 1. Animal immunization New Zealand white rabbits (2.0-2.5 kg) were immunized with the CD1a antigen described in step 1 using Freund's adjuvant emulsification. The initial immunization dose was 500 μg / rabbit, followed by a booster immunization of 250 μg two weeks later. Serum was collected 7-10 days after the third immunization, and antibody titers were detected by ELISA. IHC verification was performed when the titer reached 1:128,000 (OD450>1.0). Based on the staining intensity and specificity of CD1a in tissue sections, a decision was made regarding whether to collect peripheral blood mononuclear cells for monoclonal antibody screening or to administer subsequent immunizations.

[0040] 2. PBMCs isolation and specific single B cell sorting Peripheral blood was collected from New Zealand white rabbits with serum antibody titers meeting the target, and peripheral blood mononuclear cells (PBMCs) were isolated. The specific procedure was as follows: the animals were anesthetized and fixed, blood was collected via cardiac puncture, mixed with an equal volume of PBS, and slowly added to the supernatant of the lymphocyte separation medium. After density gradient centrifugation, the white membrane layer cells were collected. Residual components were thoroughly washed with PBS to remove them, obtaining high-purity PBMCs. Subsequently, antigen-specific B cells were sorted by flow cytometry, and after in vitro culture, B cell clones that specifically bind to the recombinant CD1a antigen were screened using ELISA.

[0041] 3. Construction of rabbit monoclonal antibody expression plasmid Based on the initial ELISA screening results, IHC verification was performed on the supernatant of single B cell culture. Total RNA was extracted from highly specific positive clones and reverse transcribed into cDNA. Naturally paired rabbit antibody light and heavy chain genes were obtained by PCR amplification, cloned separately into eukaryotic expression vectors, constructed recombinant plasmids, and verified by sequencing. The verified heavy and light chain plasmids were co-transfected into HEK293 cells. After culturing for 120 hours, the supernatant was collected to obtain rabbit monoclonal antibodies with CD1a binding activity.

[0042] 4. Preparation and purification of rabbit monoclonal antibodies Cell supernatant was collected after transfection, cell debris was removed by centrifugation, and the antibody was purified using a Protein A affinity chromatography column. High-purity rabbit monoclonal antibody OTIR4A6 targeting human CD1a was finally obtained. The concentration of the purified antibody was determined using the BCA method, yielding a rabbit monoclonal antibody OTIR4A6 with a concentration of 2.7 mg / mL targeting human CD1a.

[0043] 5. Determination of affinity constant The recombinant CD1a protein from step 1 was serially diluted with carbonate buffer to concentrations of 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, and 0.0625 μg / mL, and coated onto 96-well plates. The plates were incubated overnight at 4°C. After blocking with 5% skim milk powder, serially diluted primary antibody and horseradish peroxidase-labeled secondary antibody were added sequentially. The reaction was developed using the TMB substrate system, and the reaction was terminated with sulfuric acid. Readings were obtained using a microplate reader. Finally, by fitting dose-response curves at four different coating concentrations, the EC50 of the primary antibody corresponding to the half-maximum OD value was calculated using the formula Ka = (n-1) / [2(n[Ab']t - [Ab]t)] (where n is the ratio of adjacent coating concentrations, and [Ab]t and [Ab']t are the corresponding EC50 values). Based on this, the affinity constant (Ka) of the CD1a rabbit monoclonal antibody OTIR4A6 was calculated, and its affinity constant was determined to be 3.6 × 10⁻⁶. 9 M -1 .

[0044] Example 3: Variable region gene and amino acid sequence analysis of rabbit monoclonal antibody against human CD1a protein Using the recombinant plasmid of the OTIR4A6 antibody as a DNA template, the light and heavy chain variable region genes of the functional antibody were amplified by 5'RACE technology, and sequenced for verification using M13F / R primers.

[0045] M13F: 5'-TGTAAAACGAGCGGCCAGT-3' (SEQ ID NO: 11) M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 12).

[0046] Bioinformatics analysis yielded the amino acid sequences of the light chain variable region (SEQ ID NO.3) and heavy chain variable region (SEQ ID NO.7) of the OTIR4A6 antibody.

[0047] The VL domain is 108 amino acids long. The four domains of its FR have amino acid numbers of 26, 21, 36, and 10, respectively, while the three domains of LCDR have amino acid numbers of 7, 3, and 5, respectively. The regions of LCDR1, LCDR2, and LCDR3 are 27-33 aa, 55-57 aa, and 94-98 aa, respectively. Their amino acid sequences are: QSVYKTY (SEQ ID NO.4), KVS (SEQ ID NO.5), and LNYDN (SEQ ID NO.6), respectively.

[0048] The VH domain is 108 amino acids long. The number of amino acids in the four domains of its FR are 25, 17, 38 and 11, respectively. The number of amino acids in the three domains of HCDR are 26-31 aa, 49-54 aa and 93-97 aa, respectively. HCDR1, HCDR2 and HCDR3 are 6, 6 and 5, respectively. Their amino acid sequences are: GIDLTS (SEQ ID NO.8), IDPAND (SEQ ID NO.9) and QARYG (SEQ ID NO.10).

[0049] Example 4: Immunohistochemical detection kit containing rabbit monoclonal antibody against human CD1a protein An immunohistochemical detection kit containing a rabbit monoclonal antibody against human CD1a protein includes antigen retrieval solution [1mM EDTA, 10mM Tris buffer (pH 8.0)]. Example 3 describes the preparation of purified rabbit monoclonal antibody against human CD1a protein (0.9 μg / mL), endogenous peroxidase inhibitor (hydrogen peroxide), ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer, DAB chromogenic solution, and hematoxylin staining solution.

[0050] Example 5 Immunohistochemical detection using anti-human CD1a rabbit monoclonal antibody as primary antibody 1. Experimental Methods Tonsils, thymus, skin, and paraffin-embedded tissues from Langerhans cell hyperplasia were selected and prepared into 4 μm sections. After dewaxing and hydration with xylene and graded ethanol, immunohistochemical detection was performed using the kit prepared in Example 4. Antigen retrieval was performed using the high-pressure heat retrieval method (antigen retrieval solution, pressure-cooked for 3 min, endogenous peroxidase blocked with 3% hydrogen peroxide, and tissue areas labeled with an immunohistochemical pen). 0.9 μg / mL of OTIR4A6-derived CD1a rabbit monoclonal antibody was added and incubated at 37°C for 60 min; subsequently, it was reacted with HRP-labeled goat anti-mouse / rabbit IgG polymer at 37°C for 30 min. Finally, after DAB staining, hematoxylin counterstaining was performed, followed by differentiation blueing treatment, graded ethanol dehydration, xylene clearing, and mounting with neutral resin. Microscopic examination was performed, and the results are shown in the figure. Figure 3-6 .

[0051] 2. Experimental Results Rabbit monoclonal antibody OTIR4A6 was used to detect CD1a distribution in thymus, tonsils, skin, and Langerhans cell hyperplasia tissues using intracellular chromatography (IHC). Microscopic examination showed that at an antibody concentration of 0.9 μg / mL, IHC staining produced strong and specific positive signals with a clean background and high signal-to-noise ratio, clearly and reliably displaying CD1a distribution and expression levels. Its affinity constant was determined to be 3.6 × 10⁻⁶. 9 M -1 It belongs to the category of high-affinity antibodies.

[0052] like Figure 3 As shown, CD1a expression in thymic tissue exhibits a characteristic distribution. Cells in the thymic cortex, indicated by the arrows, show clear linear positive staining of the cell membrane, while cells in the medullary region, marked with triangles, show a negative reaction. The staining results indicate that CD1a protein is specifically expressed only in thymic cortex cells, consistent with the typical expression pattern of CD1a.

[0053] Immunohistochemical staining results of CD1a in tonsil tissue are as follows: Figure 4 As shown, the dendritic cells in the interfollicular zone marked by arrow 1 and the Langerhans cells in the squamous epithelium marked by arrow 2 both showed significant positive cell membrane staining, while the germinal centers and mantle lymphocytes marked by triangles were completely negative and did not express CD1a protein.

[0054] CD1a expression in skin tissue as follows Figure 5 As shown, Langerhans cells of the epidermal spinous layer, marked by arrows, show typical positive staining of the cell membrane, with their dendritic processes clearly visible; in contrast, basal cells and dermal connective tissue marked by triangles 1 and 2 show negative reactions.

[0055] like Figure 6 As shown, in cases of Langerhans cell hyperplasia, tumor cells are distributed in nodular or sheet-like patterns. Figure b is an enlarged view of the area within the black box in Figure a. Under high magnification, round or oval tumor cells show strong positive staining of CD1a on the cell membrane, marked with arrows; while the lymphocytes surrounding the triangle markings are negative and do not express CD1a protein.

[0056] The above results demonstrate that the anti-human CD1a rabbit monoclonal antibody OTIR4A6 described in this application possesses high specificity and sensitivity, expressing only in thymic cortical cells, Langerhans cells, and some interfollicular dendritic cells. At a concentration of 0.9 μg / mL, all positive signals generated by the OTIR4A6 antibody exhibited a clear linear staining pattern on the cell membrane with significant staining intensity and no non-specific staining signals. Furthermore, the affinity constant of this antibody was experimentally determined to be 3.6 × 10⁻⁶. 9 M -1It belongs to the category of high-affinity antibodies, which provides a molecular-level explanation for its excellent performance in immunohistochemical detection.

[0057] Therefore, the OTIR4A6 rabbit monoclonal antibody can serve as a reliable tool for immunohistochemical detection of CD1a molecules, providing strong support for the pathological diagnosis and differentiation of diseases such as Langerhans cell hyperplasia, and can also be used for basic and clinical research related to CD1a.

[0058] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A rabbit monoclonal antibody against human CD1a protein or its antigen-binding fragment, characterized in that, Includes the light chain variable region VL and the heavy chain variable region VH. The light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is shown in SEQ ID NO.

4. The amino acid sequence of LCDR2 is KVS. The amino acid sequence of LCDR3 is shown in SEQ ID NO.6; The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO.

8. The amino acid sequence of HCDR2 is shown in SEQ ID NO.

9. The amino acid sequence of HCDR3 is shown in SEQ ID NO.

10.

2. The rabbit monoclonal antibody against human CD1a protein or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.

7.

3. A biomaterial, characterized in that, The biomaterial is selected from polynucleotides, carriers, or cells. The polynucleotide encodes a rabbit monoclonal antibody against human CD1a protein as described in any one of claims 1-2, or an antigen-binding fragment thereof; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the carrier, or is capable of expressing the rabbit monoclonal antibody against human CD1a protein as described in any one of claims 1-2, or its antigen-binding fragment.

4. A method for preparing the rabbit monoclonal antibody against human CD1a protein or its antigen-binding fragment according to any one of claims 1-2, characterized in that, The method includes culturing the cells as described in claim 3, wherein the cells are prepared by converting the cells with a polynucleotide encoding a rabbit monoclonal antibody or an antigen-binding fragment thereof comprising the anti-human CD1a protein, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, and the conversion comprising co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells.

5. The application of the rabbit monoclonal antibody against human CD1a protein as described in any one of claims 1-2, or its antigen-binding fragment, or the biomaterial as described in claim 3, characterized in that, The application is selected from one or more of the following: 1) Detection and / or labeling of human CD1a protein for non-diagnostic and non-therapeutic purposes; 2) Prepare immunoassay products for detecting and / or labeling human CD1a protein; 3) Prepare immunoassay products for detecting Langerhans cell hyperplasia.

6. An immunoassay product for human CD1a protein, characterized in that, The immunoassay product comprises a rabbit monoclonal antibody against human CD1a protein as described in any one of claims 1-2 or an antigen-binding fragment thereof, or the biological material as described in claim 3.

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