Anti-human cd45 monoclonal antibody hybridoma cell strain and secreted antibody and preparation method and application thereof

Abstract

The present application aims to provide an anti-human CD45 monoclonal antibody hybridoma cell strain and an anti-human CD45 monoclonal antibody secreted by the hybridoma cell strain, and related kits, screening methods, preparation methods and applications, and belongs to the technical field of biotechnology. The present application 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. By designing and synthesizing a CD45 extracellular segment conserved sequence antigen, a recombinant plasmid is constructed and a CD45 antigen is efficiently expressed and purified, a mouse is immunized, and a specific hybridoma cell is screened by cell fusion and a double screening strategy of taking leukocyte-like cells as positive and tumor cell lines as negative. The hybridoma cell preservation number is CGMCC NO:21414. The antibody is applied to CTC negative enrichment and identification, the leukocyte removal efficiency is greater than 95%, the CTC recovery rate is greater than 90%, and there is no non-specific binding. The anti-human CD45 monoclonal antibody and the preparation method of the present application significantly improve the efficiency and accuracy of CTC enrichment and identification, and provide a powerful tool for tumor diagnosis and treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of monoclonal antibodies, and relates to an anti-human CD45 monoclonal antibody hybridoma cell strain and an antibody secreted by the cell strain, and a preparation method and application thereof. BACKGROUND

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

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

[0004] CD45, also known as protein tyrosine phosphatase receptor C (PTPRC), is also known as leukocyte common antigen (LCA), which is a type 1 transmembrane protein tyrosine phosphatase (PTPase) expressed in all hematopoietic stem cell lineage cells except mature red blood cells and platelets, and is an important regulator of T and B cell antigen receptor-mediated activation. CD45 is a large glycoprotein of 180-220 kDa, which is composed of two intracellular phosphatase domains, a transmembrane domain and an extracellular domain, and 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 n-terminal region of CD45, antibodies against CD45 protein either recognize all CD45 isoforms or only recognize a subset of them (CD45R). In CTC enrichment and detection, there is an urgent need for CD45 antibodies with strong specificity and recognizing all isoforms.

[0005] In white blood cells, lymphocytes highly express CD45, monocytes express it at moderate intensity, and granulocytes express it at low intensity. CD45 antibodies can be used to separate and remove white blood cells from blood, achieving 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 can cross-react with other proteins, affecting the accuracy of detection.

[0006] The application discloses a method for efficiently enriching circulating tumor cells based on high-affinity CD45 rabbit monoclonal antibodies and application thereof, and aims to solve the problems of excessive residual white blood cells in existing CTCs enrichment methods, low CTCs recovery rate, high identification difficulty and inability to obtain active CTCs. The application provides a composition containing anti-CD45 monoclonal antibodies, anti-CD16 monoclonal antibodies, anti-CD19 monoclonal antibodies and anti-CD235a monoclonal antibodies, which is used for incubating with blood and removing white blood cells by combining with magnetic beads to enrich CTCs. Although the multi-antibody combination 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, and in the multi-antibody combination, 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 multi-antibody combination increases the production cost, which affects the economic feasibility of the technology in clinical promotion and wide application.

[0007] The existing commercial CD45 antibodies still have deficiencies in specificity, sensitivity and stability. The application obtains a monoclonal antibody with high specificity by constructing a DNA sequence for the common conservative region of all subtypes of CD45 and adopting a positive / negative cell double screening strategy, and the specificity and sensitivity of the monoclonal antibody in CTC enrichment and identification are better than those of the commercial antibodies. SUMMARY

[0008] Therefore, one of the purposes of the application is to provide an anti-human CD45 monoclonal antibody hybridoma cell strain, the second purpose is to provide an anti-human CD45 monoclonal antibody, the third purpose is to provide a screening method of the anti-human CD45 monoclonal antibody hybridoma cell strain, the fourth purpose is to provide a preparation method of the anti-human CD45 monoclonal antibody, the fifth purpose is to provide a kit containing the anti-human CD45 monoclonal antibody, the sixth purpose is to provide a method for enriching CTCs, the seventh purpose is to provide a method for enriching white blood cells, and the eighth purpose is to provide a method for identifying white blood cells.

[0009] To achieve the above purposes, the application provides the following technical solutions.

[0010] The application provides an anti-human CD45 monoclonal antibody hybridoma cell strain, the hybridoma cell strain 4F9, which is preserved in the China General Microbiological Culture Collection Center, has a preservation number CGMCC NO: 21414, a preservation date of December 17, 2020, an address of No. 1, Beichen West Road, Chaoyang District, Beijing, and a classification name of hybridoma cell strain.

[0011] Further, an anti-human CD45 monoclonal antibody is provided, which is secreted by the hybridoma cell strain CGMCC NO: 21414, and is a murine anti-human CD45 monoclonal antibody.

[0012] Further, the application provides a kit containing the anti-human CD45 monoclonal antibody, and further comprising a diagnostic, detection reagent, enzyme and buffer, which can be used for white blood cell separation, circulating tumor cell enrichment, white blood cell identification and various applications.

[0013] Further, the application provides a screening method of the anti-human CD45 monoclonal antibody hybridoma cell strain, which comprises the following steps:

[0014] (1) Incubation: the antibody secreted by the hybridoma cell strain is incubated with positive and negative cells (cells expressing CD45 on the membrane surface are positive cells, and cells not expressing CD45 protein are negative cells) expressing human CD45 protein, 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: the binding efficiency of the antibody and the cells is detected;

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

[0017] Further, the application provides a preparation method of the anti-human CD45 monoclonal antibody, which comprises the following steps:

[0018] (1) CD45 antigen sequence design and recombinant expression: a conserved region of the extracellular segment of CD45 is designed and synthesized, the sequence is shown in SEQ ID NO: 1, a recombinant plasmid is constructed, eukaryotic cells are transfected, and CD45 protein is expressed and purified;

[0019] (2) Hybridoma cell preparation and screening: the mouse is immunized by using the CD45 protein in step (1), the hybridoma cell is obtained after cell fusion, and the screening method of the anti-human CD45 monoclonal antibody hybridoma cell strain is used to screen the hybridoma cell strain.

[0020] (3) Monoclonal antibody preparation: collecting the antibody secreted by the hybridoma cell strain, and purifying to obtain the anti-human CD45 monoclonal antibody;

[0021] Further, 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 separating white blood cells (including granulocytes, lymphocytes, monocytes, etc.);

[0023] (2) Application in preparing a reagent or kit for separating white blood cells;

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

[0025] (4) Application in preparing a reagent or kit for enriching circulating tumor cells;

[0026] (5) Application in identifying white blood cells;

[0027] (6) Application in preparing a reagent or kit for identifying white blood cells;

[0028] Further, the present application provides a method for separating white blood cells, which comprises:

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

[0030] (2) After the incubation is completed, the solid carrier combined with white blood cells is separated out;

[0031] (3) White blood cells are separated out,

[0032] Further, the present application provides a method for enriching circulating tumor cells, which comprises:

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

[0034] (2) After the incubation is completed, the solid carrier combined with white blood cells is removed;

[0035] (3) Enriching circulating tumor cells, Further, the present application provides a method for identifying white blood cells, which comprises:

[0036] (1) Incubate the sample with the anti-human CD45 monoclonal antibody, wherein the antibody labels a signal system (fluorescence, nanometer enzyme, colloidal gold, etc. signal system);

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

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

[0039] 1. Specificity improvement: The use of positive cell screening ensures that the antibody can specifically bind to the CD45 antigen on the cell surface, and the negative cell screening excludes antibodies that may produce non-specific binding. The use of flow cytometry to detect the specificity of the antibody to white blood cells significantly improves the recognition rate of the antibody to white blood cells, with a coverage rate close to 100%. The non-specific binding rate of the antibody to tumor cells is less than 10%.

[0040] 2. Sensitivity enhancement: The use of positive cell screening to select antibodies with strong binding ability to the conserved region of CD45 improves the titer of the antibody, which is better than that of commercial antibodies.

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

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

[0043] Other advantages, objects, and features of the present application will be in part apparent and in part pointed out hereinafter. The objects and other advantages of the present application can be achieved and obtained by means of the description that follows. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to make the purposes, technical solutions and advantages of the present application more clear, the preferred detailed description of the present application will be combined with the drawings as follows, wherein:

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

[0046] Figure 2 is the flow cytometry verification of 4F9 antibody specificity;

[0047] Figure 3 is the SDS-PAGE identification of CD45 purified antibody;

[0048] Figure 4 is the fluorescence identification of tumor samples after enrichment;

[0049] Figure 5 is the verification of 4F9 antibody fluorescence specificity by cell lines;

[0050] Figure 6 is the verification of 4F9 antibody sensitivity by PBMCs;

[0051] Figure 7 is the verification of 4F9 antibody immunofluorescence identification performance by simulation samples;

[0052] Figure 8 is the verification of 4F9 antibody immunofluorescence identification performance by tumor samples. DETAILED DESCRIPTION

[0053] The present application is further illustrated by the following specific examples that set forth in detail and together with the above description and the examples demonstrate various principles of the application. Other advantages and novel features of the application will be readily apparent to those skilled in the art from the following of the principles of the application. The application can be practiced by other variations of these procedures without departing from the spirit of the application, and the application is to be limited only by the claims and equivalents thereof.

[0054] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:

[0055] The same or similar reference numerals in the drawings denote the same or similar components. In the description of the application, it should be understood that the orientation or positional relationship indicated by terms such as "upper", "lower", "left", "right", "front", "back", and the like is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the application and simplifying the description, and does not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, therefore the terms describing the positional relationship in the drawings are only for illustrative purposes, and cannot be understood as a limitation of the application. For those skilled in the art, the specific meanings of the above terms can be understood according to the specific circumstances.

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

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

[0058] Using the NCBI database to sequence align human CD45 subtypes (such as CD45RA, CD45RO, CD45RB), select the common conserved region sequence with an extracellular length of 391 amino acids, and connect the coding sequences of 6xHis and mouse IgG Fc region at the C terminal, respectively, to synthesize CD45-His and CD45-mFc fusion protein sequences, and construct into pCMV3 vector.

[0059] The sequence of CD45 protein 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, incubate at room temperature for 5 minutes. Slowly add the DNA-liposome complex to the cell culture flask, and gently shake to mix. Add 5% feed 24 hours after transfection, and harvest the cell culture supernatant after 5-7 days.

[0062] Protein purification: Purify the CD45 protein with His and Fc tags by nickel column and Protein A resin affinity chromatography. First, equilibrate the chromatography column with 5-10 column volumes of equilibration buffer (20mM PB+0.15M NaCl, pH 7.0), then add the sample to the chromatography column after centrifugation and filtration (0.22μm), and continue to rinse with equilibration buffer until the baseline is reached. Elute with elution buffer (20mM citric acid, pH 3.0-4.0), and collect the effluent. Immediately neutralize the collected antibody solution to a stable pH with alkaline buffer (1M Tris / HCl, pH 9.0) to avoid inactivation of the antibody and maintain the biological activity of the antibody.

[0063] 3. Protein identification and activity detection: SDS-PAGE detects the molecular weight and expression level of CD45 protein, and there are protein bands of interest at around 100kd and 120kd, respectively, with high purity, and no other bands are seen. The indirect ELISA method is used to detect the activity of CD45 antibody, CD45 protein is coated at 1ug / mL, 5% BSA is blocked, then rabbit anti-human CD45 antibody (10086-RP01, Yiqiao God) is added, incubated at 37℃ for 1h, then goat anti-rabbit HRP secondary antibody (M21002S, Abmart) is added, incubated at 37℃ for 1h, and then the absorbance value is detected. The reaction activity of CD45 protein with antibody is comparable to that of commercial products.

[0064] Table 1 CD45-mFc protein activity

[0065]

[0066] Example 2 CD45 monoclonal antibody preparation

[0067] 1. Mouse immunization: Purified CD45-mFc recombinant protein was used as immunogen to prepare emulsion with 1 mg / mL concentration dilution and mixed with equal amount of complete Freund's adjuvant (CFA). BALB / c mice (6-8 weeks old, female) were injected with 50 μg antigen per mouse by intraperitoneal injection for primary immunization. Booster immunization was performed every 2 weeks with 50 μg antigen per mouse using incomplete Freund's adjuvant (IFA) for 3 times. Three days before the end of immunization, 50 μg antigen was injected into the tail vein to ensure the production of high titer antibodies. The serum of immunized mice was collected and tested for antibody titer against CD45 antigen by ELISA, and the mice with high antibody titer were selected 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, which was cultured in advance and adjusted to logarithmic growth phase. The mice at the end of immunization were sacrificed, and the spleen was isolated under sterile conditions. Single cell suspension was obtained by grinding with PBS and adjusted to an appropriate concentration (2 x 10 7 cells / mL). The spleen cells 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 in 96-well plates with 200 μL HAT medium (H0262, sigma) per well, and cultured for 7-10 days to screen the fused positive hybridoma cell lines.

[0071] 3、Hybridoma cell line screening: Collect the culture supernatant, use CD45-His recombinant protein to coat the ELISA plate, detect the antibody titer in the culture supernatant, select the clone with high titer in the positive well for subcloning screening. After 3 times of subcloning screening, 6 positive cell lines with high titer were obtained (results as shown in Table 3, 6 cell lines with A450 value greater than 1.5). The supernatant of the 6 cell lines was collected and divided into 5 groups, and added to the positive cells expressing human CD45 protein: Raji cells (human Burkitt's lymphoma cells), Jurkat cells (human peripheral blood leukemia T cells), negative cells expressing human CD45 protein: Hela cells (human cervical cancer cells), A549 (human non-small cell lung cancer cell line), Colo205 (human colon cancer cells), incubated, and the binding efficiency of the antibody in the supernatant to the cells was detected by flow cytometry, and finally one hybridoma cell line (4F9) with high binding rate to Raji cells and Jurkat cells and low binding rate to Hela cells, A549 and Colo205 was screened out, and the results are shown in Figure 2 Table 3, and the remaining 5 strains do not react with Raji cells and Jurkat cells.

[0072] Table 3 Hybridoma cell supernatant detection

[0073] Cell line 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: The cell line stably expressing the antibody was expanded to the logarithmic growth phase, and the cell density was adjusted to 0.5-1 x 10 7 , injected into the abdominal cavity of mice to produce ascites. 7-15 days after injection, the ascites was extracted by abdominal puncture, centrifuged at 12,000 rpm for 15 min, and the middle water phase layer was collected. The ascites was filtered through a 0.22 um filter to remove impurities, and then purified by Protein A affinity chromatography packing. The purified antibody storage solution was replaced with PBS, and the purified antibody was identified by SDS-PAGE, and the results are shown in Figure 3 .

[0075] 5、Antibody titer detection: CD45-His eukaryotic protein was coated on the plate at 1 ug / mL, and the plate was coated at 2-8°C for 16 hours. 5% BSA was added for blocking at 37°C for 2 hours, and then 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.56 ng / mL and 0 ng / mL of the antibody were added in sequence, and incubated at 37°C for 1 hour. Goat anti-mouse HRP (0.01M PBS diluted at 1:8000) was added and incubated at 37°C for 1 hour, TMB color developing solution was added for 10 minutes, and 2M H2SO4 was added to terminate the reaction. The OD450 was detected by a microplate reader. The titer detection was better than that of the commercial antibody, and the results are shown in Table 4.

[0076] Table 4 CD45 antibody titer detection

[0077] Concentration (ng / mL) 4F9 Commercialized antibody 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 enrichment of circulating tumor cells

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

[0080] 2. Leukocyte separation: Prepare 3 groups of positive cells expressing human CD45 protein-Raji cell simulation leukocytes, with a cell number controlled at 10 6 -10 7 cells, add 3 parts of equal volume of immunomagnetic beads, and the reaction time of cells and magnetic beads is 1 h. Separate the magnetic beads and elute, and count the supernatant before and after the reaction and the cells separated by magnetic beads. The leukocyte separation efficiency is > 95%, and the results are shown in Table 5.

[0081] Table 5 CD45 antibody separation efficiency of leukocytes

[0082]

[0083]

[0084] 3. Enrichment of circulating tumor cells: Divide a tube of healthy human whole blood into 3 parts, each group of which is added with 10 or less negative cells expressing human CD45 protein-tumor cells colo205 (previously stained with Celltracker Green), and add equal volume of immunomagnetic beads, with a reaction time of cells and magnetic beads being 1 h, and remove the magnetic beads. Record the number of tumor cells before and after the reaction under the fluorescence microscope FITC. The non-specific adsorption of CD45 monoclonal antibody to tumor cells is < 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 Enrichment kit for circulating tumor cells

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

[0089] Tumor sample enrichment: Collect 3 cases of 5 mL tumor (lung cancer) peripheral blood samples, obtain PBMCs after density gradient centrifugation, and count. The PBMCs are filtered by size, and then subjected to negative enrichment using the circulating tumor cell enrichment kit. Separate the magnetic beads and collect the supernatant. Centrifuge at 800g for 5 min to obtain cell precipitate, which is the enriched circulating tumor cells. The enrichment results are shown in Table 7.

[0090] Table 7 Enrichment results of circulating tumor cells

[0091] Sample Number of cells before enrichment Number of cells after enrichment Number of leukocyte removed 1 16300000 12000 16288000 2 9500000 8430 9491570 3 11200000 9820 11190180

[0092] Fluorescence identification: The enriched CTCs were fixed with 4% paraformaldehyde, and were subjected to fluorescence staining 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 CTC enrichment performance. Under the microscope, obvious tumor cells and a small amount of white blood cells were observed, and the results are shown in Figure 4 . Among them, DAPI has signal: identified as cells; CY5 channel has signal: identified as white blood cells; FITC channel has signal: identified as tumor epithelial cells; TRITC channel has signal: identified as tumor interstitial cells.

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

[0094] 1. CD45 antibody (4F9) labeling signal system: AF647 dye (D10157-1, Multifluorescence) stock solution was prepared with anhydrous DMSO, with a concentration of 10 mg / mL, and stored at -80°C. The molar ratio of dye to labeled antibody was 5:1. 300 ug of antibody was added to 2 ul of fluorescent dye and reacted at room temperature in the dark for 3-4 h. After the antibody labeling reaction, the antibody was dialyzed against 500 mL of PBS at 4°C in the dark for 12 h, with the PBS being replaced every 2-3 h. The dialysis was overnight, and the PBS was replaced at least 3 times. The antibody concentration was measured using a Nano drop after dialysis.

[0095] 2. Cell identification

[0096] (1) Cell line verification of antibody specificity: Raji was selected as a CD45 positive cell to simulate white blood cells, and colo205 / hela was selected as a CD45 negative cell. Commercialized antibody (CL647-65109, proteintech) was used as a control group, and CD45-AF647 (4F9) labeled in this experiment was used as an experimental group. Cell suspension was smeared on a glass slide (cell density was controlled at 10 4 -10 5 ), and then fixed with 4% paraformaldehyde for 5 min. After PBS washing for 5 min, the cells were incubated with CD45-AF647 antibody at 37°C for 1 h. After antibody incubation, the cells were washed with PBS for 5 min, and then stained with DAPI. 4F9 labeled antibody showed no fluorescence staining in Colo205 and Hela cells, but showed fluorescence signal in Raji cells. The specificity was better than that of the commercialized antibody, and the results are shown in Figure 5 .

[0097] (2) PBMC verification of antibody sensitivity: 5 mL of whole blood from a healthy person was diluted with an equal volume of PBS, and then slowly spread on 4 mL of density gradient centrifugation solution. After centrifugation at 800 g for 20 min at room temperature, the PBMCs were collected from the white membrane layer, washed with PBS, and then smeared. The cells were incubated with CD45-AF647 antibody at 37°C for 1 h (commercialized antibody was used as a control group, and 4F9 labeled in this experiment was used as an experimental group). After PBS washing for 5 min, the cells were stained with DAPI. Under the same conditions, 4F9 labeled antibody showed significantly stronger fluorescence staining than the commercialized antibody, and no non-specific staining was observed. The results are shown in Figure 6 .

[0098] (3) Simulate sample to verify antibody performance: 5 mL of healthy human whole blood was respectively added with colo205, hela, and colo205+hela tumor cells, and PBMC was obtained by density gradient centrifugation. After filtration and negative enrichment of the PBMC, the sample was prepared, stained, and incubated with antibodies CD45-AF647 (4F9 as the experimental group and commercial antibody as the control 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 had better sensitivity than the commercial antibody in PBMC. The results are shown in FIG. 6. Figure 7

[0099] Cell identification kit

[0100] According to Example 5, the relevant reagents were prepared into a cell identification kit. The detection performance was verified by using tumor samples.

[0101] Two 5 mL tumor peripheral blood samples were collected, and PBMC was obtained by density gradient centrifugation. After filtration and negative enrichment of the PBMC, the sample was prepared, stained, and 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 shown in FIG. 7. Figure 8

[0102] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions, and they should all be covered in the scope of the claims of the present application.​​

Claims

1. Hybridoma cell line with anti-human CD45 monoclonal antibody, its accession number is CGMCC NO:21414.

2. An anti-human CD45 monoclonal antibody, characterized in that, The antibody is secreted by the hybridoma cell line CGMCCNO:21414 as 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 reagent kit, characterized in that, The kit contains the anti-human CD45 monoclonal antibody as described in any one of claims 2-3.

5. The use of the anti-human CD45 monoclonal antibody according to any one of claims 2-3 in any of the following aspects: (1) Application in the preparation of reagents or kits for isolating leukocytes; (2) Application in the preparation of reagents or kits for enriching circulating tumor cells; (3) Application in the preparation of reagents or kits for identifying leukocytes.

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