Hybridoma cell strain secreting anti-human Langerin protein monoclonal antibody, monoclonal antibody and application

Abstract

The invention provides a hybridoma cell strain secreting an anti-human Langerin protein monoclonal antibody, the monoclonal antibody and application, the hybridoma cell strain is OTI7A1, the preservation number of the hybridoma cell strain is CGMCC No.46247, the hybridoma cell strain is preserved in China General Microbiological Culture Collection Center, the preservation time is November 28, 2024, the preservation number is CGMCC No.46247, and the preservation number is CGMCC No.46247. The hybridoma cell strain can stably secrete the anti-human Langerin protein monoclonal antibody. The anti-human Langerin protein monoclonal antibody can be specifically combined with the human Langerin protein, so that the specificity, accuracy and reliability of Langerin protein immunodetection are remarkably improved, Langerin cells in tissues can be detected by an IHC method, the expressed Langerin protein in the cells can be obviously detected, and the anti-human Langerin protein monoclonal antibody can be well applied to immunohistochemical detection of the Langerin protein in the tissues.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoglobulins, and in particular relates to a hybridoma cell strain secreting anti-human Langerin protein monoclonal antibodies, monoclonal antibodies and applications. Background Art

[0002] Langerhans cells (LC) are immature dendritic cells (DC) derived from the bone marrow. They exist specifically in the skin and mucosal tissues and play an important role in the skin immune response, mainly in the uptake, processing and presentation of antigens, inducing T cell responses, etc. They are the main antigen presenting cells in the epidermis. Langerhans cells specifically express Birbeck granules, which are also called Langerhans granules or X-bodies. They are straight or curved rod-shaped membranous granules with a thickness of 40nm. The longitudinal section shows that there are two layers of unit membranes on each side and a longitudinal septum in the middle, so they are also called five-layer membrane granules. However, Birbeck granules can only be found in 2% to 69% of Langerhans cells. Therefore, Birbeck granules cannot be seen under an electron microscope and need to be determined in combination with cell morphology and immune phenotype under a light microscope.

[0003] Langerin (also known as CD207 protein) is located on chromosome 2p13, including 6 exons, with a protein molecular weight of 40kDa. It is a type II transmembrane C-type lectin associated with the formation of Birbeck granules in Langerhans cells. It is a specific mannose receptor, containing a sugar recognition domain (CRD) characterized by a glutamic acid-proline-asparagine (EPN) motif, and forms a trimer through the α-helix binding of the extracellular region. Each CRD can recognize mannose, but only the trimer can bind to the glycoprotein ligand. Langerin may participate in the formation of Birbeck granules through two different mechanisms, namely exocytosis and receptor-mediated endocytosis. Valladeau et al. used immunohistochemical studies to find that, except for Langerhans cells, the rest of the dendritic cells do not express Langerin, so it is distinguished from other subtypes of dendritic cells. Therefore, Langerin is a very useful specific molecular marker for studying the function of Langerhans cells and related diseases.

[0004] Langerhans cell histiocytosis (LCH) is a + / CD207 +It is a rare disease of the monocyte-macrophage system with abnormal proliferation of immature dendritic cells. Its histopathology is characterized by proliferation of reticuloendothelial cells. Its histopathology has similar characteristics, that is, a large number of Langerhans cells and their precursor cells proliferate, infiltrate and accumulate in the affected tissue. Langerhans cells can be seen under the microscope, and the lesions are mainly infiltrated by histiocytes or histiocytes and eosinophils. Eosinophil infiltration is more common in osteolytic lesions. LCH can occur at any age and is divided into single system LCH (single system-LCHSS-LCH), multipy system LCH (multipy system-LCHMS-LCH) and invasion of dangerous organs (RO) according to the site of the lesion and whether it infiltrates dangerous organs. + )MS-LCH(RO + MS-LCH). The clinical manifestations are complex and varied, and may vary depending on the site, scope and degree of involvement of the lesions. They can range from asymptomatic isolated skin or bone lesions to acute, diffuse, fulminant, life-threatening multi-system lesions. They may also present with fever, rash, anemia, thrombocytopenia, lymphadenopathy, hepatosplenomegaly, etc. There are many pitfalls or difficulties in pathological diagnosis, and it is often easy to misdiagnose. In order to avoid missed diagnosis and misdiagnosis, it is crucial to develop highly specific and sensitive monoclonal antibodies that recognize Langerin proteins for the diagnosis of diseases such as Langerhans cell histiocytosis. Summary of the invention

[0005] In view of this, the present invention aims to provide a hybridoma cell line secreting anti-human Langerin protein monoclonal antibody and monoclonal antibody and application. The monoclonal antibody can specifically bind to human Langerin protein, thereby realizing the detection of Langerhans cells in tissues by IHC method.

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

[0007] In a first aspect, the present invention provides a hybridoma cell line that secretes anti-human Langerin protein monoclonal antibodies, wherein the hybridoma cell line is OTI7A1, and its preservation number is CGMCC No.46247, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration (CGMCC), and the preservation time is November 28, 2024.

[0008] Furthermore, the deposit information of the hybridoma cell line is as follows:

[0009] Hybridoma cell lines used for deposit: Mouse anti-human Langerin monoclonal hybridoma cell line OTI7A1;

[0010] Deposit number: CGMCC No.46247

[0011] Deposit date: November 28, 2024;

[0012] Depository: China General Microbiology Center (CGMCC);

[0013] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] Furthermore, the hybridoma cell line is obtained by immunizing mice with Langerin protein, taking the spleen of the immunized mice, fusing the mouse spleen lymphocytes with myeloma cells, and screening and cloning. The hybridoma cell line can stably secrete anti-human Langerin protein monoclonal antibodies.

[0015] In a second aspect, the present invention provides an anti-human Langerin protein monoclonal antibody, the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1-3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; the heavy chain variable region comprises HCDR1-3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:10.

[0016] Further, the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:3, or comprises an amino acid sequence with more than 85% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminally modifying any one or more amino acids in the amino acid sequence shown in SEQ ID NO:3, for example, it can be an amino acid sequence with 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0017] Furthermore, the total length of the light chain variable region is 109 amino acids, the number of amino acids in the four domains of FR are 26, 17, 36 and 11 respectively, the number of amino acids in the three domains of LCDR are 11, 3 and 5 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-37aa, 55aa-57aa and 94aa-98aa respectively, and their amino acid sequences are: KSLLHSNGNTY (SEQ ID NO: 4), GAS (SEQ ID NO: 5), MQHLE (SEQ ID NO: 6).

[0018] Further, the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:7, or comprises an amino acid sequence with more than 85% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminally modifying any one or more amino acids in the amino acid sequence as shown in SEQ ID NO:7, for example, it can be an amino acid sequence with 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0019] Furthermore, the total length of the heavy chain variable region is 110 amino acids, the number of amino acids in the four domains of FR are 24, 17, 37 and 11 respectively, the number of amino acids in the three domains of HCDR are 8, 8 and 5 respectively, HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-57aa and 95aa-99aa respectively, and their amino acid sequences are: GYTFTDYN (SEQ ID NO: 8), IYPYNGNA (SEQ ID NO: 9), ASTAW (SEQ ID NO: 10), respectively.

[0020] Furthermore, the monoclonal antibody is secreted by the hybridoma cell line as described in the first aspect, and the hybridoma cell line is OTI7A1, whose deposit number is CGMCC No.46247, and it is deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration, and the address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit time is November 28, 2024.

[0021] In a third aspect, the present invention provides a polynucleotide encoding the monoclonal antibody as described in the second aspect.

[0022] In a fourth aspect, the present invention provides the use of the monoclonal antibody as described in the second aspect or the polynucleotide as described in the third aspect in labeling human Langerin protein in cells or tissues, preparing a reagent for labeling human Langerin protein in cells and living tissues, or preparing an immunohistochemical detection kit for human Langerin protein.

[0023] In a fifth aspect, the present invention provides an immunohistochemical detection kit for human Langerin protein, wherein the immunohistochemical detection kit comprises a primary antibody reagent, wherein the primary antibody reagent comprises the monoclonal antibody as described in the second aspect or the antibody or antibody fragment obtained by expressing the polynucleotide as described in the third aspect.

[0024] Furthermore, the kit also includes antigen repair solution, endogenous peroxidase blocker, ultrasensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated color developing solution, and hematoxylin staining solution.

[0025] Furthermore, the ultrasensitive secondary antibody reagent is an ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0026] Compared with the prior art, the hybridoma cell line secreting anti-human Langerin protein monoclonal antibody and the monoclonal antibody and application of the present invention have the following advantages:

[0027] (1) The anti-human Langerin protein monoclonal antibody of the present invention can specifically bind to human Langerin protein, thereby significantly improving the specificity, accuracy and reliability of Langerin protein immunoassay.

[0028] (2) The hybridoma cell line OTI7A1 described in the present invention can also stably secrete anti-human Langerin protein monoclonal antibodies.

[0029] (3) The anti-human Langerin protein monoclonal antibody and the kit containing the anti-human Langerin protein monoclonal antibody described in the present invention can detect Langerhans cells in tissues by IHC method, and can obviously detect Langerin protein expressed in cells, and are well applied to immunohistochemical detection of Langerin protein in tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0031] Figure 1 Schematic diagram of the Western blot detection results of the recombinant Langerin protein in Example 2 of the present invention, in which the expression of the recombinant Langerin protein in E. coli cells was detected by using anti-HIS antibody, wherein lane L is the detection result of the E. coli cell lysate transfected with an empty vector as the antigen, and lane R is the detection result of the E. coli cell lysate transfected with a pET23a-rLangerin plasmid as the antigen;

[0032] Figure 2 This is a schematic diagram of the results of SDS-PAGE of Langerin protein in Example 2 of the present invention. The recombinant Langerin protein was purified using a nickel affinity chromatography column, and the purified protein was subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining;

[0033] Figure 3 This is a schematic diagram of the results of IHC detection of tonsil tissue using the monoclonal antibody secreted by the hybridoma cell line OTI7A1 in Example 6 of the present invention;

[0034] Figure 4 This is a schematic diagram of the results of IHC detection of skin tissue using the monoclonal antibody secreted by the hybridoma cell line OTI7A1 in Example 6 of the present invention;

[0035] Figure 5 This is a schematic diagram of the results of IHC detection of Langerhans cell histiocytosis using the monoclonal antibody secreted by the hybridoma cell line OTI7A1 in Example 6 of the present invention. DETAILED DESCRIPTION

[0036] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The experimental methods in the following embodiments without specifying specific conditions are usually carried out according to conventional conditions, conditions described in laboratory manuals or conditions recommended by manufacturers.

[0037] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0038] Example 1 Construction of Langerin protein recombinant expression plasmid

[0039] The Langerin gene NM_015717.3 was selected from Genebank. According to the amino acid sequence characteristics of Langerin, the amino acid fragment of Langerin at positions 80-328 was designed as an immunogen (the corresponding nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2). Primers were designed, restriction endonuclease sites SgfI and MluI were introduced on both sides of the gene, respectively, and the gene was inserted into the expression vector pET23a-N-His to construct the recombinant expression plasmid pET23a-rLangerin.

[0040] Example 2 Expression and purification of recombinant Langerin protein

[0041] (1) Transformation of E. coli cells: 100 μl of competent cells were placed on ice to melt, then recombinant expression plasmid DNA was added and gently mixed. After ice bathing for 30 minutes, heat shock was performed at 42°C for 90 seconds, and then ice bathed for 1-2 minutes. 500 μl of fresh antibiotic-free LB medium was added to the clean bench, and incubated on a shaker at 37°C for 45 minutes. After that, an appropriate amount of bacterial solution was evenly spread on the plate containing antibiotics, and the culture dish was inverted in a 37°C constant temperature incubator for overnight culture.

[0042] (2) Lysis of cells: Pick a single clone and place it in fresh culture medium. Cultivate at 37°C and 200 rpm until the OD value reaches 0.4-0.6. Add IPTG (final concentration 1 mM) and induce the culture for 7 hours. Collect the cells by centrifugation, then resuspend the cells in lysis buffer, ultrasonically disrupt for 20 minutes, and centrifuge at 12000 rpm at 4°C for 20 minutes to collect the supernatant. Take a small amount of supernatant protein and use anti-His antibody for WB identification. The results are as follows: Figure 1 shown.

[0043] Depend on Figure 1 The results showed that the E. coli cell lysate transfected with pET23a-rLangerin plasmid was located in lane R, with an obvious specific band at 29kD, while the control lysate lane L transfected with an empty vector had no band of the corresponding size, indicating that the cells specifically expressed the recombinant Langerin protein.

[0044] (3) Nickel affinity chromatography column purification: The nickel column was equilibrated with buffer, the supernatant was filtered through a 0.45 μm filter membrane, and then the sample was loaded and the outflow was collected. The unbound protein was removed by elution with buffer, and finally eluted with eluent containing different concentrations of imidazole. After collection, the eluted proteins that met the requirements were combined and 10% glycerol was added. The purified recombinant Langerin protein was identified by SDS-PAGE electrophoresis. The results are as follows: Figure 2 shown.

[0045] Depend on Figure 2 The results showed that the purified protein had an obvious specific band at 29kD in the SDS-PAGE electrophoresis gel image, which was consistent with the predicted molecular weight of the designed antigen amino acid sequence, indicating that the recombinant Langerin protein with good purity had been obtained.

[0046] Example 3 Preparation and Screening of OTI7A1 Secreted Anti-Human Langerin Protein Monoclonal Antibodies BALB / c mice (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were immunized with purified recombinant Langerin protein (hereinafter referred to as Langerin antigen) according to standard methods. The specific method is as follows:

[0047] (1) Animal immunization: The purified Langerin antigen was emulsified with complete Freund's adjuvant and immunized with 6-8 week old BALB / c mice by subcutaneous or intraperitoneal injection, with an immunization dose of 60 μg / mouse. Two weeks later, the second immunization was performed, emulsified with incomplete Freund's adjuvant, with an immunization dose of 30 μg / mouse. After the second immunization, tail blood was collected and serum titer was determined by gradient dilution using ELISA. The decision on whether to boost immunization was made based on the results, and the mouse with the highest antibody titer was selected for cell fusion.

[0048] (2) Cell fusion: Myeloma cells were sp2 / 0 from BALB / c, which were in the logarithmic growth phase at the time of fusion. The spleen of immunized mice was taken to prepare a single-cell suspension of lymphocytes. Mouse spleen lymphocytes and myeloma cells were mixed at a ratio of 1:5-1:10, 1 mL of 50% PEG (pH 8.0) preheated at 37°C was added dropwise, and incomplete culture medium and stop solution were added. After centrifugation and discarding the supernatant, HAT culture medium was added to suspend and mix. The MC volume was adjusted to 50 mL, and the suspension was dispensed into 3.5 cm culture dishes, placed in a humidified box, and cultured in a constant temperature incubator at 37°C and 5% CO2.

[0049] (3) Screening and cloning: Select hybridoma cell clones within 7-10 days of fusion and perform ELISA test using purified recombinant Langerin protein. Mark the cell line number. Perform limiting dilution on the cells in the positive wells, measure the ELISA value 5-6 days after each limiting dilution, and select the monoclonal wells with higher OD280 positive values ​​for limiting dilution until the ELISA result of the whole 96-well plate is positive. Select the monoclonal wells with high positive values ​​for final determination, and the corresponding fusion plate cell line is OTI7A1.

[0050] (4) Preparation and purification of monoclonal antibodies on cells: The hybridoma cell line OTI7A1 was cultured in a 10 cm culture dish using DMEM medium containing 15% serum and expanded to approximately 4×10 7 When the cells reached 3 × 10 cells, centrifuge at 800 rpm for 5 min, discard the supernatant and transfer the cells to a 2 L spinner bottle. Serum-free medium was added to make the cell density about 3 × 10 5 After 1-2 weeks of continuous culture, when the cell death rate reaches 60%-70% (the cell density is about 1-2×10 6 / mL), collect the cell suspension and centrifuge at 6000rpm for 20min, take the supernatant, purify the supernatant by affinity chromatography, select the corresponding column material for purification according to the antibody subtype (the subtype is IgG2a, and protein G column material is used for purification). The concentration of the purified anti-human Langerin protein monoclonal antibody is measured, lyophilized and packaged (100μg / tube), and finally stored at -20℃.

[0051] Example 4 Analysis of variable region genes and amino acid sequences of anti-human Langerin protein monoclonal antibodies

[0052] Purchased from Takara Bio USA RACE 5' / 3' kit uses 5'RACE (Rapid Amplification of cDNA Ends) technology to amplify the variable region light chain and heavy chain gene sequences of hybridoma cell functional antibodies. For detailed experimental procedures, please refer to Takara Bio USA RACE 5' / 3'Kit User Manual.

[0053] Since the antibody is of IgG2a subtype, specific gene primers pRace-H-GSP and pRace-K-GSP targeting the 3' end of its Ig and Kappa constant regions were designed. The primer sequences are as follows:

[0054] pRace-H-GSP: 5'-CATCDGTCTATCCACTGGCCCCTG-3' (SEQ ID NO: 11)

[0055] pRace-K-GSP: 5'-CTTCCCACCATCCAGTGAGCAGTT-3' (SEQ ID NO: 12)

[0056] mRNA was extracted from hybridoma cell line OTI7A1, reverse transcribed into cDNA, and RACE was used to amplify the DNA fragments of the heavy and light chains of the anti-human Langerin protein monoclonal antibody. The amplified light and heavy chains were respectively connected to the cloning vector PUC119 by restriction digestion, and positive clones were picked by blue-white staining, and the positive plasmids were purified for sequencing using the sequencer ABI 3730, and the sequencing primers were universal primers M13F and M13R.

[0057] M13F:5'-TGTAAAACGAGCGGCCAGT-3'(SEQ ID NO:13)M13R:5'-

[0058] CAGGAAACAGCTATGAC-3'(SEQ ID NO:14)

[0059] Using the Internet, IMGT / V-QUEST analysis software was used at http: / / www.imgt.org to perform data analysis on the sequencing results of the nucleotide sequences of the light chain and heavy chain of the anti-human Langerin protein monoclonal antibody, respectively, to obtain the amino acid sequence of the light chain variable region of the anti-human Langerin protein monoclonal antibody as shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:7.

[0060] The total length of the light chain variable region is 109 amino acids, the amino acid numbers of the four domains of FR are 26, 17, 36 and 11 respectively, the amino acid numbers of the three domains of LCDR are 11, 3 and 5 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-37aa, 55aa-57aa and 94aa-98aa respectively, and their amino acid sequences are: KSLLHSNGNTY (SEQ ID NO: 4), GAS (SEQ ID NO: 5), MQHLE (SEQ ID NO: 6).

[0061] The total length of the heavy chain variable region is 110 amino acids, the amino acid numbers of the four FR domains are 24, 17, 37 and 11 respectively, the amino acid numbers of the three HCDR domains are 8, 8 and 5 respectively, HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-57aa and 95aa-99aa respectively, and their amino acid sequences are: GYTFTDYN (SEQ ID NO: 8), IYPYNGNA (SEQ ID NO: 9), ASTAW (SEQ ID NO: 10), respectively.

[0062] Example 5 Immunohistochemical Detection Kit Containing Anti-Human Langerin Protein Monoclonal Antibody

[0063] The immunohistochemical detection kit containing the anti-human Langerin protein monoclonal antibody of this embodiment includes antigen repair solution [1mM EDTA, 10mM Tris buffer (pH8.0)], the anti-human Langerin protein monoclonal antibody prepared and purified in Example 3, an endogenous peroxidase blocker (hydrogen peroxide), a supersensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer, a DAB substrate buffer, a DAB concentrated color developing solution, and a hematoxylin staining solution.

[0064] Example 6 Immunohistochemical detection using anti-human Langerin protein monoclonal antibody as primary antibody

[0065] The immunohistochemical detection kit in Example 5 was used to detect Langerin protein in tissues, and the specific steps were as follows:

[0066] (1) Formalin-fixed tonsil, skin, and Langerhans cell histiocytosis tissue blocks were paraffin-embedded and sliced ​​using a Leica tissue slicer with a tissue thickness of 4 μm.

[0067] (2) Dewaxing and hydration: Soak in analytical grade xylene for 10 min × 3 times, soak in anhydrous ethanol for 1 min × 3 times, soak in 95% ethanol for 1 min, soak in 85% ethanol for 1 min, soak in 75% ethanol for 1 min, and soak in deionized water for 2 min × 3 times.

[0068] (3) Antigen repair: Add antigen repair solution and perform high-pressure heat repair in a pressure cooker for 3 min. When the temperature of the pressure cooker drops to about 90°C, open the pressure cooker, take out the slices, and then cool them naturally to room temperature. Soak them in deionized water for 2 min × 3 times.

[0069] (4) Inactivation: Use 3% hydrogen peroxide to inactivate endogenous peroxidase in tissues, let stand at room temperature for 15 min, and soak in deionized water for 2 min × 3 times.

[0070] (5) Use an immunohistochemistry pen to draw a frame around the tissue and wash with 0.1% PBST for 2 min × 1 time.

[0071] (6) Incubation with primary antibody: Add 200 μl (1.2 μg / mL) of diluted anti-human Langerin monoclonal antibody secreted by hybridoma cell line OTI7A1 and place in a wet box, incubate at 37° C. for 60 min, and wash with 0.1% PBST for 2 min×3 times.

[0072] (7) Incubation with secondary antibody: Add 100 μl of secondary antibody PV-8000 and incubate at 37°C for 30 min. Wash with 0.1% PBST for 2 min × 3 times.

[0073] (8) DAB color development: Add 120 μl of DAB color development solution, let stand at room temperature for 5 min, rinse under tap water to stop color development, and rinse with tap water three times.

[0074] (9) Hematoxylin re-staining, differentiation and blueing: Let stand in the hematoxylin solution for 10-12 seconds, rinse with tap water three times to stop coloring, differentiate in 1% hydrochloric acid ethanol solution, rinse with tap water three times to stop, put in freshly boiled Tris-EDTA disodium solution at pH 8.0 to blue, then put in room temperature Tris-EDTA disodium solution at pH 9.0 for a few seconds, rinse with tap water three times. Observe the staining under a microscope. If it is normal, stop and recover the hematoxylin staining solution; if it is too differentiated, repeat the above steps until the staining is qualified.

[0075] (10) Dehydration and transparency: 75% ethanol for 1 min, 85% ethanol for 1 min, 95% ethanol for 1 min, 100% ethanol for 1 min × 3 times, xylene for 1 min × 3 times, and sealing with neutral gum.

[0076] (11) Microscopic examination, the results are as follows Figure 3-Figure 5 shown.

[0077] In this example, the anti-human Langerin protein monoclonal antibody secreted by hybridoma cell OTI7A1 was used to detect tonsil, skin and Langerhans cell histiocytosis tissues by IHC method. Figure 3This is the result of IHC staining of the antibody on tonsil tissue. The Langerhans cells indicated by the arrow in the crypt epithelial layer are stained brown, which is considered positive expression. The squamous epithelial cells indicated by the triangle and the germinal center cells indicated by the asterisk are all negative. Figure 4 This is the result of antibody IHC staining on skin tissue. The arrow indicates the Langerhans cell with dendritic processes located between the spinous cells in the upper middle part of the epidermis. The result shows positive staining. The corneocytes and squamous layer cells do not show brown color and are marked as negative, that is, there is no expression of Langerin protein. Figure 5 This is the result of IHC staining of antibodies on Langerhans cell histiocytosis. The picture shows that this case contains a large number of inflammatory cells. The tumor cells are distributed in sheets, the nuclei are irregular, some cells show nuclear grooves, and the cytoplasm is granularly stained, which is consistent with the characteristics of Langerhans cell histiocytosis. The IHC staining pattern is consistent with the Langerin protein expression pattern. The staining results show that the anti-human Langerin protein monoclonal antibody secreted by OTI7A1 has high specificity. The concentration of the anti-human Langerin protein monoclonal antibody corresponding to the above experiment is 1.2μg / mL, showing that this antibody has a high detection sensitivity.

[0078] The above results show that the anti-human Langerin protein monoclonal antibody secreted by the hybridoma cell line OTI7A1 of the present invention has high specificity. Therefore, the anti-Langerin monoclonal antibody secreted by the hybridoma cell line OTI7A1 can be used for immunohistochemical detection to detect the expression of Langerin protein in tissues such as Langerhans cell histiocytosis, providing a basis for accurate diagnosis by clinicians.

[0079] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. A hybridoma cell line secreting anti-human Langerin protein monoclonal antibody, characterized in that: The hybridoma cell line is OTI7A1, and its preservation number is CGMCC No.46247. It is preserved in the General Microbiology Center of China National Microbiological Culture Collection Administration, and the preservation time is November 28, 2024.

2. An anti-human Langerin protein monoclonal antibody, characterized in that: The monoclonal antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1-3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; the heavy chain variable region comprises HCDR1-3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:8, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

10.

3. The anti-human Langerin protein monoclonal antibody according to claim 2, characterized in that: The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence with more than 85% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminal modification in the amino acid sequence shown in SEQ ID NO:

3.

4. The anti-human Langerin protein monoclonal antibody according to claim 2, characterized in that: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence with more than 85% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminal modification in the amino acid sequence shown in SEQ ID NO:

7.

5. The anti-human Langerin protein monoclonal antibody according to claim 2, characterized in that: The monoclonal antibody is secreted by the hybridoma cell line according to claim 1.

6. A polynucleotide, characterized in that The polynucleotide encodes the monoclonal antibody according to any one of claims 2 to 5.

7. Use of the monoclonal antibody according to any one of claims 2 to 5 or the polynucleotide according to claim 6 in preparing a reagent for labeling human Langerin protein in cells or tissues, or in preparing an immunohistochemical detection kit for human Langerin protein.

8. An immunohistochemical detection kit for human Langerin protein, characterized in that: The immunohistochemical detection kit comprises a primary antibody reagent, and the primary antibody reagent comprises the monoclonal antibody according to any one of claims 2 to 5 or the antibody or antibody fragment obtained by expression of the polynucleotide according to claim 6.

9. The kit according to claim 8, characterized in that The kit also includes antigen repair solution, endogenous peroxidase blocker, ultrasensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated color developing solution, and hematoxylin staining solution.

10. The kit according to claim 9, characterized in that The ultrasensitive secondary antibody reagent is an ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

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