Anti-human CD45 monoclonal antibody hybridoma cell strain and secreted antibody as well as preparation method and application of secreted antibody

Abstract

The invention aims to provide an anti-human CD45 monoclonal antibody hybridoma cell strain, an anti-human CD45 monoclonal antibody secreted by the anti-human CD45 monoclonal antibody hybridoma cell strain, a related kit, a screening method, a preparation method and application, and belongs to the technical field of biology. The invention aims to solve the technical problems of insufficient specificity, low sensitivity and limited application effect in CTC enrichment and identification of the anti-human CD45 monoclonal antibody in the prior art. A CD45 extracellular domain conserved sequence antigen is designed and synthesized, a recombinant plasmid is constructed, the CD45 antigen is efficiently expressed and purified, mice are immunized, and specific hybridoma cells with the preservation number of CGMCC NO: 21414 are screened by adopting a double screening strategy that leukocyte-like cells are positive and tumor cell lines are negative through cell fusion. The antibody is applied to negative enrichment and identification of CTC, the leukocyte removal efficiency is greater than 95%, the CTC recovery rate is greater than 90%, and no non-specific binding exists. According to the anti-human CD45 monoclonal antibody and the preparation method, the efficiency and accuracy of CTC enrichment and identification are remarkably improved, and a powerful tool is provided for tumor diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibodies, and relates to anti-human CD45 monoclonal antibody hybridoma cell lines and secretory antibodies as well as preparation methods and applications thereof. Background Art

[0002] Circulating tumor cells (CTCs) refer to tumor cells that fall off from tumor lesions (primary or metastatic) and enter the peripheral blood circulation. Circulating tumor cells are a key link in the distant metastasis of tumors and are also one of the main materials for tumor liquid biopsy. CTCs are closely related to cancer metastasis, treatment efficacy, cancer recurrence, medication guidance and prognosis, and are therefore used as an important biomarker for early diagnosis and treatment of cancer metastasis.

[0003] The number of CTCs in peripheral blood is very small. 6 ~10 7 There is only one CTC in every leukocyte. Therefore, CTC detection first needs to be enriched according to its biological or physical properties, and then the enriched cells are identified according to the gene expression or functional characteristics of CTC. Affinity-based enrichment technology is currently the most widely used CTC enrichment method, which is mainly achieved by using unique antigens that are differentially expressed on CTCs and leukocytes, including positive enrichment using antigens expressed on CTCs but not on leukocytes [such as epithelial adhesion protein (EpCAM)], and indirect negative enrichment using antigens expressed on leukocytes but not on CTCs (such as CD45).

[0004] CD45 - protein tyrosine phosphatase receptor C (PTPRC), also known as leukocyte common antigen (LCA), is a type 1 transmembrane protein tyrosine phosphatase (PTPase) that is expressed in all hematopoietic stem cell lineage cells except mature red blood cells and platelets. It is an important regulator of T and B cell antigen receptor-mediated activation. CD45 is a large glycoprotein of 180-220 kDa, consisting of two intracellular phosphatase domains, a transmembrane domain, and an extracellular domain. The extracellular domain contains three membrane-proximal fibronectin type II repeats, a cysteine-rich region, and a variable n-terminal region. Due to the variability of the CD45 n-terminal region, antibodies against the CD45 protein either recognize all CD45 subtypes or only recognize a subset (CD45R). In CTC enrichment and detection, CD45 antibodies with strong specificity and recognition of all subtypes are urgently needed.

[0005] Among white blood cells, lymphocytes highly express CD45, monocytes express it at a moderate intensity, and granulocytes express it at a low level. CD45 antibodies can be used to separate and remove white blood cells from the blood to achieve negative separation of CTCs. In CTC detection technology, CD45 antibodies can be used to distinguish CTCs from normal white blood cells. However, in some cases, CD45 antibodies may cross-react with other proteins, thereby affecting the accuracy of the test.

[0006] The invention with publication number CN109507427A discloses a method for efficiently enriching circulating tumor cells based on high-affinity CD45 rabbit monoclonal antibody and its application, aiming to solve the problem that the existing CTCs enrichment method has too much residual white blood cells, resulting in low CTCs recovery rate, high identification difficulty and inability to obtain active CTCs. The invention provides a composition containing anti-CD45 monoclonal antibody, anti-CD16 monoclonal antibody, anti-CD19 monoclonal antibody and anti-CD235a monoclonal antibody, which is incubated with blood, combined with magnetic beads to remove white blood cells and enrich CTCs. Although the combination of multiple antibodies improves the enrichment effect, it also increases the complexity of the technology. Each antibody needs to be prepared, purified and quality controlled separately, which increases the production cost and time. In the combination of multiple antibodies, there may be potential interference between different antibodies, such as non-specific binding or competitive binding, which may affect the enrichment efficiency and specificity. The use of multiple antibody combinations increases production costs, which will affect the economic feasibility of the technology in clinical promotion and wide application.

[0007] Existing commercial CD45 antibodies are still insufficient in specificity, sensitivity and stability. The present invention obtains highly specific monoclonal antibodies by constructing a DNA sequence targeting the common conserved region of all CD45 subtypes and adopting a positive / negative cell double screening strategy. The specificity and sensitivity in CTC enrichment and identification are better than commercial antibodies. Summary of the invention

[0008] In view of this, one of the objects of the present invention is to provide an anti-human CD45 monoclonal antibody hybridoma cell line, the second object is to provide an anti-human CD45 monoclonal antibody, the third object is to provide a screening method for anti-human CD45 monoclonal antibody hybridoma cell lines, the fourth object is to provide a method for preparing anti-human CD45 monoclonal antibodies, the fifth object is to provide a kit containing anti-human CD45 monoclonal antibodies, the sixth object is to provide a method for enriching CTCs, the seventh object is to provide a method for enriching leukocytes, and the eighth object is to provide a method for identifying leukocytes.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention provides an anti-human CD45 monoclonal antibody hybridoma cell line, hybridoma cell line 4F9, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO: 21414, a deposit date of December 17, 2020, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and a classification name of hybridoma cell line;

[0011] Furthermore, an anti-human CD45 monoclonal antibody is provided, which is secreted and produced by the hybridoma cell line CGMCC NO:21414, and the anti-human CD45 monoclonal antibody is a mouse anti-human CD45 monoclonal antibody.

[0012] Furthermore, the present invention provides a kit, which contains the anti-human CD45 monoclonal antibody, and also includes diagnostic and detection reagents, enzymes and buffers. The kit can be used for various applications such as leukocyte separation, circulating tumor cell enrichment and leukocyte identification.

[0013] Furthermore, the present invention provides a method for screening the anti-human CD45 monoclonal antibody hybridoma cell line, comprising the following steps:

[0014] (1) Incubation: The antibodies secreted by the hybridoma cell line are incubated with negative and positive cells expressing human CD45 protein on the membrane (cells expressing CD45 on the membrane surface are positive cells, and cells not expressing CD45 protein are negative cells), wherein the positive cells are selected from at least one of Raji and Jurkat cells, and the negative cells are selected from at least one of Hela, Colo205, and A549 tumor cell lines;

[0015] (2) Detection: Detect the binding efficiency of antibodies to cells;

[0016] (3) Screening: Screening out the anti-human CD45 monoclonal antibody hybridoma cell line with high binding efficiency to positive cells and low binding efficiency to negative cells,

[0017] Furthermore, the present invention provides a method for preparing an anti-human CD45 monoclonal antibody, comprising the following steps:

[0018] (1) CD45 antigen sequence design and recombinant expression: design and synthesize the conserved region of the CD45 extracellular segment, the sequence of which is shown in SEQ ID NO: 1; construct a recombinant plasmid, transfect eukaryotic cells, express and purify the CD45 protein;

[0019] (2) Preparation and screening of hybridoma cells: Immunizing mice with CD45 protein in step (1), obtaining hybridoma cells after cell fusion, and screening hybridoma cell lines using the anti-human CD45 monoclonal antibody hybridoma cell line screening method;

[0020] (3) Preparation of monoclonal antibodies: collecting antibodies secreted by hybridoma cell lines and purifying them to obtain the anti-human CD45 monoclonal antibodies;

[0021] Furthermore, the anti-human CD45 monoclonal antibody and / or the anti-human CD45 monoclonal antibody prepared by the preparation method of the anti-human CD45 monoclonal antibody are used in any of the following aspects:

[0022] (1) Application in separation of white blood cells (including granulocytes, lymphocytes, monocytes, etc.);

[0023] (2) Use in the preparation of reagents or kits for separating leukocytes;

[0024] (3) Application in enriching circulating tumor cells;

[0025] (4) Preparation of reagents or kits for enriching circulating tumor cells;

[0026] (5) Application of identification of leukocytes;

[0027] (6) Use in the preparation of reagents or kits for identifying leukocytes;

[0028] Furthermore, the present invention provides a method for separating leukocytes, the method comprising:

[0029] (1) incubating the sample with the anti-human CD45 monoclonal antibody, wherein the antibody is connected to a solid phase carrier (magnetic beads, agarose beads, nanoparticles, microspheres, etc.) (e.g., through protein A / G, biotin-streptavidin binding, etc.);

[0030] (2) After the incubation is completed, the solid phase carrier bound to the leukocytes is separated;

[0031] (3) Isolation and acquisition of white blood cells,

[0032] Furthermore, the present invention provides a method for enriching circulating tumor cells, the method comprising:

[0033] (1) incubating the sample with the anti-human CD45 monoclonal antibody, wherein the antibody is connected to a solid phase carrier (magnetic beads, agarose beads, nanoparticles, microspheres, etc.) (e.g., through protein A / G, biotin-streptavidin binding, etc.);

[0034] (2) After the incubation is completed, the solid phase carrier bound to the leukocytes is removed;

[0035] (3) Enrichment of circulating tumor cells. Further, the present invention provides a method for identifying leukocytes, the method comprising:

[0036] (1) incubating the sample with the anti-human CD45 monoclonal antibody, wherein the antibody is labeled with a signal system (fluorescence, nanozyme, colloidal gold or other signal systems);

[0037] (2) After the incubation is completed, the cells that emit signals from the signal system are identified as white blood cells.

[0038] The beneficial effects of the present invention are:

[0039] 1. Improved specificity: Positive cell screening is used to ensure that the antibody can specifically bind to the CD45 antigen on the cell surface, while negative cell screening is used to exclude antibody cell lines that may produce non-specific binding. Flow cytometry is used to detect the specificity of the antibody to leukocytes. The recognition rate of this antibody to leukocytes is significantly improved, with a coverage rate of nearly 100%. The non-specific binding rate of the antibody to tumor cells is less than 10%.

[0040] 2. Enhanced sensitivity: Antibodies with strong binding ability to the conserved region of CD45 are screened out through positive cells, and the antibody titer is better than commercial antibodies.

[0041] 3. Improved CTC enrichment efficiency and identification performance: The antibody is combined with magnetic beads to enrich CTCs in blood samples, and the leukocyte removal efficiency and CTC recovery rate are evaluated. The leukocyte removal efficiency is greater than 95%, and the CTC recovery rate is greater than 90%.

[0042] 4. Enhanced specificity of CTC identification: CD45 is labeled with fluorescent dye, and the cell lines, PBMCs and CTCs are specifically verified by immunofluorescence. There is no non-specific binding between tumor cells and CTCs, which is better than commercial antibodies.

[0043] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0045] Figure 1 It is the SDS-PAGE identification of CD45 protein expression and purification;

[0046] Figure 2 Flow cytometry was used to verify the specificity of the 4F9 antibody;

[0047] Figure 3 It was identified by SDS-PAGE of purified CD45 antibody;

[0048] Figure 4 It is the fluorescence identification after tumor sample enrichment;

[0049] Figure 5 It is a cell line to verify the fluorescence specificity of 4F9 antibody;

[0050] Figure 6 It is PBMC to verify the sensitivity of 4F9 antibody;

[0051] Figure 7 It is a simulated sample to verify the immunofluorescence identification performance of 4F9 antibody;

[0052] Figure 8 Tumor samples were used to verify the immunofluorescence identification performance of the 4F9 antibody. DETAILED DESCRIPTION

[0053] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0054] Among them, the drawings are only used for illustrative explanations, and they only represent schematic diagrams rather than actual pictures, and should not be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0055] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "rear", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0056] Example 1 CD45 antigen sequence design and recombinant expression

[0057] 1. Design and synthesis of recombinant protein CD45-mFc

[0058] The NCBI database was used to align the sequences of human CD45 isoforms (such as CD45RA, CD45RO, and CD45RB), and the common conserved region sequence with an extracellular segment length of 391 amino acids was selected. The C-terminal was connected to the coding sequence of 6×His and mouse IgG Fc region, respectively, to synthesize CD45-His and CD45-mFc fusion protein sequences and construct them into the pCMV3 vector.

[0059] The CD45 protein sequence is shown in SEQ ID NO:1.

[0060] 2. Protein expression and purification

[0061] Transfection: Mix the constructed expression vector (DNA plasmid) with Lipofectamine 3000 at a ratio of 1:1 and incubate at room temperature for 5 minutes. Slowly add the DNA-liposome complex to the cell culture shake flask and shake gently to mix. Add 5% feed 24 hours after transfection and harvest the cell culture supernatant after 5-7 days.

[0062] Protein purification: CD45 protein with His and Fc tags was purified by affinity chromatography with nickel column and Protein A resin. The column was first equilibrated with 5-10 column volumes of equilibration buffer (20mM PB+0.15M NaCl, pH 7.0). The sample was added to the column after centrifugation and filtration (0.22μm). After loading, the sample was continued to be eluted with equilibration buffer to the baseline. The elution was eluted with elution buffer (20mM citric acid, pH 3.0-4.0) and the effluent was collected. Immediately neutralize the collected antibody solution with alkaline buffer (1M Tris / HCl, pH 9.0) to a stable pH to avoid antibody inactivation and maintain the biological activity of the antibody.

[0063] 3. Protein identification and activity detection: SDS-PAGE was used to detect the molecular weight and expression level of CD45 protein. The target protein bands were around 100kd and 120kd, respectively, and the purity was high, without other mixed bands. The activity of CD45 antibody was detected by ELISA indirect method. CD45 protein was coated with 1ug / mL, blocked with 5% BSA, and rabbit anti-human CD45 antibody (10086-RP01, Sino Biological) was added. Incubated at 37℃ for 1h, and then sheep anti-rabbit HRP secondary antibody (M21002S, Abmart) was added. After incubation at 37℃ for 1h, the absorbance value was detected. The reactivity of CD45 protein and antibody was equivalent to commercial products.

[0064] Table 1 CD45-mFc protein activity

[0065]

[0066] Example 2 Preparation of CD45 monoclonal antibody

[0067] 1. Mouse immunization: Use purified CD45-mFc recombinant protein as an immunogen, dilute it at a concentration of 1 mg / mL and mix it with an equal amount of complete Freund's adjuvant (CFA) to prepare an emulsion. Each BALB / c mouse (6-8 weeks old, female) was injected with 50 μg of antigen for the first immunization by intraperitoneal injection. Reinforcement immunization is performed every 2 weeks, using incomplete Freund's adjuvant (IFA), 50 μg of antigen each time, for a total of 3 times. 3 days before the end of immunization, 50 μg of antigen is injected into the tail vein to ensure the production of high-titer antibodies. Collect the serum of immune mice, detect the antibody titer against CD45 antigen by ELISA, and screen mice with high antibody titer for cell fusion.

[0068] Table 2 Mouse serum titer detection

[0069] Serum dilution ratio 1 2 blank 0.085 0.087 Negative 1:10000 0.1 0.103 1∶10000 3.765 3.727 1∶20000 3.505 3.692 1：40000 2.699 2.798 1：80000 1.692 1.764 1：160000 1.002 1.071 1：320000 0.566 0.622

[0070] 2. Cell fusion: SP2 / 0 myeloma cell line was used as fusion cells, cultured in advance and adjusted to the logarithmic growth phase. The mice were killed after immunization, the spleen was separated under sterile conditions, and single cell suspension was obtained by grinding with PBS and adjusted to an appropriate concentration (2×10 7 cells / mL). Splenocytes and myeloma cells were mixed at a ratio of 5:1, and 50% PEG-1500 (P7181, Sigma) was added to induce cell fusion at 37°C. The fused cells were inoculated into 96-well plates with 200 μL HAT medium (H0262, Sigma) per well, cultured for 7 to 10 days, and the fused positive hybridoma cell lines were screened.

[0071] 3. Screening of hybridoma cell lines: Collect the culture supernatant, use CD45-His recombinant protein to coat the ELISA plate, detect the antibody titer in the culture supernatant, and select the clones with high titer in the positive wells for subclone screening. After three subclone screenings, 6 positive cell lines with high titers were obtained (the results are shown in Table 3, 6 cell lines with A450 values ​​greater than 1.5). The supernatants of the six cell lines were collected and divided into five groups. Positive cells expressing human CD45 protein on the membrane: Raji cells (human Burkitt's lymphoma cells), Jurkat cells (human peripheral blood leukemia T cells), and negative cells expressing human CD45 protein on the membrane: Hela cells (human cervical cancer cells), A549 (human non-small cell lung cancer cell line), and Colo205 (human colon cancer cells) were added respectively, and incubated. The binding efficiency of the antibody in the supernatant to the cells was detected by flow cytometry. Finally, a hybridoma cell line (4F9) with a high binding rate to Raji cells and Jurkat cells and a low binding rate to Hela cells, A549 and Colo205 was screened out. The results are as follows: Figure 2 As shown, the other five strains did not react with Raji cells and Jurkat cells.

[0072] Table 3 Hybridoma cell supernatant detection

[0073] Cell lines 5F8 2B4 4F9 2F3 4B5 5D6 4C6 3D5 A450 2.116 2.344 2.458 2.192 1.735 1.482 2.065 1.412 Negative control 0.056 0.052 0.047 0.058 0.061 0.053 0.053 0.057 Positive Control 1.07 1.12 1.188 1.153 1.048 1.115 1.093 1.095

[0074] 4. Antibody preparation and purification: Expand the cell lines stably expressing antibodies to the logarithmic growth phase and adjust the cell density to 0.5-1×10 7 , injected into the mouse peritoneal cavity to produce ascites. After 7 to 15 days, the ascites was extracted by abdominal puncture, centrifuged at 12,000 rpm for 15 minutes, the middle aqueous phase was aspirated, the ascites was filtered with a 0.22um filter membrane to remove impurities, and the ascites was purified with Protein A affinity chromatography filler. The purified antibody storage solution was replaced with PBS, and the purified antibody was identified by SDS-PAGE. The results are as follows Figure 3 shown.

[0075] 5. Antibody titer detection: CD45-His eukaryotic protein was coated at 1ug / mL and coated overnight at 2-8℃ for 16 hours. 5% BSA was added to block at 37℃ for 2 hours, and antibodies with concentrations of 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / mL, 3.125ng / mL, 1.56ng / mL, and 0ng / mL were added in sequence, and incubated at 37℃ for 1 hour. Sheep anti-mouse HRP (0.01M PBS diluted 1:8000) was added, incubated at 37℃ for 1 hour, TMB colorimetric solution for 10 minutes, and 2M H2SO4 was used to terminate the OD450 using an enzyme reader. The titer detection was better than commercial antibodies, and the results are shown in Table 4.

[0076] Table 4 CD45 antibody titer detection

[0077] Concentration (ng / mL) 4F9 Commercial Antibodies 100 3.727 3.692 50 3.513 3.134 25 3.143 2.138 12.5 2.064 1.408 6.25 1.248 0.905 3.125 0.718 0.618 1.5625 0.508 0.493 0 0.102 0.105

[0078] Example 3 Application of CD45 monoclonal antibody in leukocyte separation and circulating tumor cell enrichment

[0079] 1. Preparation of immunomagnetic beads: Add 1mL PBS to a centrifuge tube, take 36ul magnetic beads and add them to PBS and mix well, then place them on a magnetic rack for 2min, remove the liquid after magnetic adsorption, add 1mL PBS to wash twice, add 1.8ug CD45 antibody, vertically mix in 1mL PBS and react at room temperature for 1h, wash 3 times after the reaction is completed, add blocking solution (PBS+15mg / ml+10mg / ml glycine+10% PEG2000) to block for 2h. After blocking, wash the magnetic beads 3 times, add an appropriate amount of magnetic bead storage solution, and divide them into 6 parts for use.

[0080] 2. Leukocyte separation: Prepare 3 groups of positive cells expressing human CD45 protein on the membrane - Raji cells simulate leukocytes, and control the number of cells to 10 6 -10 7 3 equal volumes of immune magnetic beads were added, and the cells and magnetic beads reacted for 1 hour. The magnetic beads were separated and eluted, and the supernatant before and after the reaction and the cells separated by magnetic beads were counted. The leukocyte separation efficiency was >95%, and the results are shown in Table 5.

[0081] Table 5 Separation efficiency of leukocytes by CD45 antibody

[0082]

[0083]

[0084] 3. Enrichment of circulating tumor cells: A tube of healthy human whole blood was divided into three parts, and no more than 10 negative cells expressing human CD45 protein on the membrane - tumor cells colo205 (pre-stained with Celltracker Green) were put into each group, and equal volumes of immunomagnetic beads were added respectively. The reaction time between cells and magnetic beads was 1 hour, and the magnetic beads were removed. The number of tumor cells was recorded under a fluorescence microscope FITC before and after the reaction. The nonspecific adsorption of CD45 monoclonal antibody to tumor cells was <10%, and the results are shown in Table 6.

[0085] Table 6 Non-specific adsorption of CD45 antibody to tumor cells

[0086]

[0087] Example 4 Circulating Tumor Cell Enrichment Kit

[0088] Preparation of circulating tumor cell enrichment kit: Add 1mL PBS to a centrifuge tube, take 36ul magnetic beads and add them to PBS and mix well, then place them on a magnetic rack for 2min, remove the liquid after magnetic adsorption, add 1mL PBS to wash twice, add 1.8ug CD45 antibody, vertically mix in 1mL PBS and react at room temperature for 1h, wash 3 times after the reaction is completed, add blocking solution (PBS+15mg / ml+10mg / ml glycine+10% PEG2000) to block for 2h. After blocking, wash the magnetic beads 3 times, add an appropriate amount of magnetic bead storage solution, and divide them into 6 parts for use.

[0089] Tumor sample enrichment: 3 5 mL tumor (lung cancer) peripheral blood samples were collected, PBMCs were obtained after density gradient centrifugation, and counted. PBMCs were size filtered and then negatively enriched using a circulating tumor cell enrichment kit. Magnetic beads were separated and the supernatant was collected. The cell pellet was obtained by centrifugation at 800g for 5 minutes, which was the enriched circulating tumor cells. The enrichment results are shown in Table 7.

[0090] Table 7 Circulating tumor cell enrichment results

[0091] sample Cell number before enrichment The number of cells after enrichment Leukocyte depletion 1 16300000 12000 16288000 2 9500000 8430 9491570 3 11200000 9820 11190180

[0092] Fluorescence identification: The enriched CTCs were fixed with 4% paraformaldehyde and fluorescently stained with CD45-AF647 (CL647-65109, proteintech, CY5 channel), Pan-CK-AF488 (53-9003-82, Invitrogen, FITC channel) and Vimentin-AF568 (ab202504, abcam, TRITC channel) to identify the enrichment performance of CTCs. Obvious tumor cells and a small number of white blood cells were observed under the microscope. The results are as follows Figure 4 As shown. Among them, DAPI has a signal: it is identified as a cell; CY5 channel has a signal: it is identified as a leukocyte; FITC channel has a signal: it is identified as a tumor epithelial cell; TRITC channel has a signal: it is identified as a tumor stromal cell.

[0093] Example 5 Application of CD45 monoclonal antibody in cell identification

[0094] 1. CD45 antibody (4F9) labeling signal system: Prepare AF647 dye (D10157-1, Multifluorescent Biotechnology) stock solution with anhydrous DMSO at a concentration of 10 mg / mL and store at -80°C. The molar ratio of dye to labeled antibody is 5:1. Add 2ul fluorescent dye to 300ug antibody and react at room temperature in the dark for 3-4h. After the antibody labeling reaction is completed, dialyze with 500mL PBS at 4°C in the dark for 12h, replace PBS every 2-3h, and dialyze overnight, at least 3 times. After dialysis replacement, use Nano drop to measure the antibody concentration.

[0095] 2. Cell Identification

[0096] (1) Cell line verification of antibody specificity: Raji was selected as CD45-positive cells to simulate white blood cells, colo205 / hela was selected as CD45-negative cells, commercial antibody (CL647-65109, proteintech) was used as the control group, and the labeled CD45-AF647 (4F9) was used as the experimental group. The cell suspension was smeared on a slide (the cell density was controlled at 10 4 -10 5 ), fixed with 4% paraformaldehyde for 5 minutes, washed with PBS for 5 minutes, washed three times in succession, incubated with CD45-AF647 antibody at 37°C, reaction time 1 hour, washed with PBS for 5 minutes, washed three times in succession, and sealed with DAPI. 4F9 labeled antibody had no fluorescent staining in Colo205 and Hela cells, but had fluorescent signal in Raji cells, and its specificity was better than commercial antibodies. The results are shown in Figure 5 shown.

[0097] (2) PBMC validation of antibody sensitivity: 5 mL of healthy human whole blood was diluted with an equal volume of PBS, slowly spread on 4 mL of density gradient centrifugation solution, centrifuged at room temperature at 800 g for 20 min, and the speed was increased and decreased by 1 / 1. After centrifugation, PBMCs in the buffy coat layer were aspirated, washed with PBS, and sliced. CD45-AF647 antibody was incubated at 37°C for 1 h (commercial antibody was used as the control group, and 4F9 labeled in this case was used as the experimental group), washed with PBS for 5 min, and then sealed with DAPI after three consecutive washes. Under the same conditions, the fluorescent staining of 4F9 fluorescently labeled antibody was significantly stronger than that of commercial antibody, and there was no nonspecific staining. The results are as follows: Figure 6 shown.

[0098] (3) Simulated sample verification of antibody performance: 5 mL of healthy human whole blood was injected with colo205, hela, and colo205+hela tumor cells, and PBMCs were obtained by density gradient centrifugation. After filtration and negative enrichment, the PBMCs were prepared and stained, and co-incubated with antibodies CD45-AF647 (commercial antibodies were used as the control group, and the labeled 4F9 was used as the experimental group), Pan-CK-AF488 (53-9003-82, Invitrogen), and Vimentin-AF568 (ab202504, abcam). The 4F9 fluorescent antibody had no non-specific staining in other tumor cells, and its sensitivity in PBMCs was better than that of commercial antibodies. The results are shown in the figure. Figure 7 shown.

[0099] Example 6 Cell Identification Kit

[0100] The relevant reagents were prepared into a cell identification kit according to Example 5. Tumor samples were used to verify the detection performance.

[0101] 5 mL of peripheral blood samples from two tumors were collected and PBMCs were obtained by density gradient centrifugation. After filtration and negative enrichment, the PBMCs were prepared and stained, and co-incubated with antibodies CD45-AF647 (4F9), Pan-CK-AF488 (53-9003-82, Invitrogen) and Vimentin-AF568 (ab202504, abcam). The staining was observed under a microscope, and the white blood cells and tumor cells were clearly distinguished. The results are as follows Figure 8 shown.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. Anti-human CD45 monoclonal antibody hybridoma cell line, whose deposit number is CGMCC NO:21414.

2. An anti-human CD45 monoclonal antibody, characterized in that: The antibody is secreted and produced by the hybridoma cell line CGMCCNO:21414 described in claim 1.

3. The anti-human CD45 monoclonal antibody according to claim 2, characterized in that: The anti-human CD45 monoclonal antibody is a mouse anti-human CD45 monoclonal antibody.

4. A kit, characterized in that: The kit contains the anti-human CD45 monoclonal antibody according to any one of claims 2-3.

5. The method for screening anti-human CD45 monoclonal antibody hybridoma cell lines according to claim 1, characterized in that: The steps include: (1) The antibodies secreted by the hybridoma cell line are incubated with negative and positive cells expressing human CD45 protein on the membrane; (2) Detecting the binding efficiency of antibodies to cells; (3) Screening out the anti-human CD45 monoclonal antibody hybridoma cell line that has high binding efficiency with positive cells and low binding efficiency with negative cells.

6. The method for screening hybridoma cell lines according to claim 5, characterized in that: The positive cells are selected from at least one of Raji and Jurkat cells, and the negative cells are selected from at least one of Hela, Colo205 and A549 tumor cell lines.

7. A method for preparing an anti-human CD45 monoclonal antibody, characterized in that: The steps include: (1) CD45 antigen sequence design and recombinant expression: design and synthesize the conserved region of the CD45 extracellular segment; construct recombinant plasmids, transfect eukaryotic cells, express and purify CD45 protein; (2) Preparation and screening of hybridoma cells: using the CD45 protein in step (1) to immunize mice, obtaining hybridoma cells after cell fusion, and screening hybridoma cell lines using the method described in any one of claims 5 to 6; (3) Preparation of monoclonal antibodies: Antibodies secreted by hybridoma cell lines were collected and purified to obtain the anti-human CD45 monoclonal antibodies.

8. Use of the anti-human CD45 monoclonal antibody prepared according to any one of claims 2 to 3 and / or the method for preparing an anti-human CD45 monoclonal antibody according to claim 7 in any of the following aspects: (1) Application in separating leukocytes; (2) Use in the preparation of reagents or kits for separating leukocytes; (3) Application in enriching circulating tumor cells; (4) Preparation of reagents or kits for enriching circulating tumor cells; (5) Application of identification of leukocytes; (6) Use in the preparation of a reagent or kit for identifying leukocytes.

9. A method for separating leukocytes, characterized in that: The method comprises: (1) incubating a sample with the anti-human CD45 monoclonal antibody according to any one of claims 2 to 3, wherein the antibody is linked to a solid phase carrier; (2) After the incubation is completed, the solid phase carrier bound to the leukocytes is separated; (3) Isolate and obtain white blood cells.

10. A method for enriching circulating tumor cells, characterized in that: The method comprises: (1) incubating a sample with the anti-human CD45 monoclonal antibody according to any one of claims 2 to 3, wherein the antibody is linked to a solid phase carrier; (2) After the incubation is completed, the solid phase carrier bound to the leukocytes is removed; (3) Enrichment of circulating tumor cells.

11. A method for identifying leukocytes, characterized in that: The method comprises: (1) incubating a sample with the anti-human CD45 monoclonal antibody according to any one of claims 2 to 3, wherein the antibody labels a signal system; (2) After the incubation is completed, the cells that emit signals from the signal system are identified as white blood cells.

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