MS4A6C antigen epitope peptide, MS4A6C antigen, MS4A6C antibody, MS4A6C hybridoma cell strain and application

Abstract

The invention belongs to the field of immunology, and particularly relates to an MS4A6C antigen epitope peptide, an antigen, an antibody, a hybridoma cell strain and application. The amino acid sequence of the MS4A6C antigen epitope peptide is as shown in SEQ ID NO: 1. The MS4A6C antigen epitope peptide provided by the invention is good in specificity, and an antigen immune animal prepared from the MS4A6C antigen epitope peptide can generate an MS4A6C antibody with high specificity.

Description

Technical Field

[0001] The present invention belongs to the field of immunology, and specifically relates to a MS4A6C antigen epitope peptide, an antigen, an antibody, a hybridoma cell line and uses thereof. Background Art

[0002] MS4A6C (mouse homolog of human MS4A6A) is a member of the MS4A gene family of membrane molecules with four transmembrane domains. Studies have shown that MS4A6C is mainly expressed on the surface of immune cells such as macrophages. Macrophages are an important component of the body's innate immune system. It is now known that macrophages have different and environment-dependent functions in various physiological and pathophysiological environments. These functions include maintaining tissue homeostasis, sensing and clearance of immune responses during pathogen infection, and repair and remodeling during tissue development and tissue damage. In addition, after sensing various tissue-derived or environmental stimuli, macrophages are activated (or polarized) to different cell states and assume unique functions. For example, microbial stimulation activates macrophages to the M1 or inflammatory state, which is characterized by the production of proinflammatory cytokines and enhanced lethality to microorganisms. In contrast, during helminth and parasite infections, macrophages are activated to the M2 or another state involved in tissue repair and remodeling. At present, there are few reports on studies targeting MS4A6C, and it is worth exploring the potential mechanisms of MS4A6C. Therefore, it is of great significance to find suitable immunogenic MS4A6C antigen epitope peptides and prepare specific MS4A6C antigens for the study of macrophage membrane protein MS4A6C. Summary of the invention

[0003] In view of the above technical problems, the present invention provides the following technical solutions:

[0004] In a first aspect, the present invention provides an MS4A6C antigen epitope peptide, the amino acid sequence of the antigen epitope peptide is shown in SEQ ID NO:1.

[0005] In a second aspect, the present invention provides an MS4A6C antigen, which is obtained by coupling the antigen epitope peptide with a carrier protein.

[0006] Preferably, the carrier protein is KLH (keyhole limpet hemocyanin) or BSA (bovine serum albumin).

[0007] The third aspect of the present invention provides a use of the MS4A6C antigen as an immunogen in preparing anti-MS4A6C antibodies.

[0008] In a fourth aspect, the present invention provides a hybridoma cell line, which is prepared according to the following method:

[0009] Immunizing mice with the MS4A6C antigen to obtain immunized mice;

[0010] The spleen cells of the immunized mice were extracted, fused with mouse myeloma cells, and screened to obtain hybridoma cell lines.

[0011] In a fifth aspect, the present invention provides an MS4A6C antibody, which is a monoclonal antibody or a polyclonal antibody prepared using the MS4A6C antigen as an immunogen; or the MS4A6C antibody is a monoclonal antibody produced by the hybridoma cell line.

[0012] In a sixth aspect, the present invention provides a method for preparing MS4A6C antibody, which comprises inoculating the hybridoma cell line into the peritoneal cavity of mice, collecting ascites, purifying, and obtaining a monoclonal antibody.

[0013] In a seventh aspect, the present invention provides a use of the MS4A6C antibody in preparing an immunoassay tool for detecting the expression level of MS4A6C molecules.

[0014] Preferably, the immunodetection tool is a reagent, a kit, a test paper or a chip. The kit may include: a labeled compound or reagent capable of detecting MS4A6C protein in a biological sample; a method for determining the amount of MS4A6C protein in a sample; and a method for comparing the amount of MS4A6C protein in a sample with a standard. The compound or reagent may be packaged in a suitable container. The kit may further include instructions for using the kit to detect MS4A6C protein in a sample.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] 1. The MS4A6C antigen epitope peptide provided by the present invention has good specificity, and the antigen prepared by the peptide can produce highly specific MS4A6C antibodies in animals immunized with the peptide.

[0017] 2. The present invention provides a hybridoma cell line that can stably secrete MS4A6C antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the tertiary structure of MS4A6C protein;

[0019] Figure 2 It is a gene-specific analysis;

[0020] Figure 3 It is the conserved domain information of MS4A6C protein;

[0021] Figure 4 It is the conserved domain information of MS4A6C protein;

[0022] Figure 5 is the localization analysis of MS4A6C protein;

[0023] Figure 6 is the localization analysis of MS4A6C protein;

[0024] Figure 7 It is the analysis of protein hydrophobicity;

[0025] Figure 8 It is the MS4A6C antigen epitope prediction;

[0026] Fig. 9 is the peptide mass spectrum;

[0027] Fig.10 These are the WB verification results of the antibody effect; 1. Standard marker, 2. marker, 3. Protein extracted from mouse spinal cord, 4. Repeated protein extracted from mouse spinal cord. DETAILED DESCRIPTION

[0028] The present invention discloses a monoclonal antibody specifically binding to MS4A6C, a hybridoma cell line and its use. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0029] MS4A6C is a member of the MS4A gene family of membrane molecules with four transmembrane domains. Studies have shown that MS4A6C is mainly expressed on the surface of immune cells such as macrophages. Currently, there are few reports on studies targeting MS4A6C, and it is worth exploring the potential mechanism of MS4A6C. Therefore, it is of great significance to find suitable immunogenic MS4A6C epitope peptides and prepare specific MS4A6C antigens for the study of macrophage membrane protein MS4A6C.

[0030] Based on this, the present invention provides a MS4A6C antigen epitope peptide, the amino acid sequence of which is shown in SEQ ID NO: 1. The MS4A6C antigen epitope peptide has good specificity, and the antigen prepared by the MS4A6C epitope peptide can produce highly specific MS4A6C antibodies in animals.

[0031] 1. Amino acid sequence of MS4A6C protein

[0032] The protein sequence of MS4A6C is from the Gen Bank of the NCBI website. The amino acid sequence of mouse MS4A6C in the NCBI website was compared with the amino acid sequences of other members of the same family and analyzed. The amino acid sequence of MS4A6C is shown in SEQ ID NO: 2, which is as follows:

[0033] MIPQVVTNETITTISPNGINFPQKDESQPTQQRQDSLKKHLKAEIKVIVAIQIMC

[0034] AVTVLALGIILASVPPVPYFNSVFSVLLKSGYPFIGALFFIASGILSIITERKSTKPL

[0035] VDASLTLNILSVSFAFVGIIIISVSLAGLHPASEQCKQSKELSLIEHDYYQPFYNS

[0036] DRSECAVTKSILTGALSVMLIISVLELGLALLSAMLWLREGVLTSLRM

[0037] The structural characteristics of MS4A6C protein predicted by AlphaFold are as follows Figure 1 As shown. The AlphaFold system generates pLDDT (Per-residue confidence metric). When the pLDDT value is greater than 90, it means that the prediction of the position of an amino acid residue in the protein has a high degree of confidence; when the pLDDT value is greater than 70, it indicates that the prediction result is basically accurate.

[0038] 2. Peptide antigen design, synthesis and coupling

[0039] 2.1 Antigen design

[0040] Using the NCBI database and UniProt database, we analyzed the structural information (physical and chemical properties) of the MS4A6C protein, identified the higher-order structural region and surface region of the MS4A6C protein, designed possible MS4A6C polypeptide antigen sequences, and analyzed the physicochemical properties of the sequences. We analyzed the specificity of the amino acid sequence of the protein in the same species and analyzed the specificity of the gene in Mus musculus, and found that the specificity of the MS4A6C gene was average. Figure 2 The comparison result with the highest score.

[0041] Signal peptides are usually not considered in antigen design. Signal peptides usually show strong hydrophobicity and may limit antigen expression. Antigens should contain the entire domain. Analysis of the conserved domain of MS4A6C protein: Information such as Figures 3-4 The localization of MS4A6C protein was analyzed as shown. Figures 5-6 shown.

[0042] The analysis of MS4A6C peptide route is similar to the protein analysis process. The tertiary structure analysis is as follows: Figure 1 As shown, the secondary structure prediction is as follows: The analysis of the tertiary structure and the prediction of the secondary structure are helpful for the recognition analysis of B cell epitopes. Finally, the antigen epitope prediction understands the hydrophobicity of the protein, such as Figures 7-8 shown.

[0043] The final sequence of the MS4A6C protein selected the 149-165aa region as the antigen region selection, and the amino acid sequence of the screened polypeptide antigen was CKQSKELSLIEHDYYQP, as shown in SEQ ID NO: 1. The polypeptide fragment has good specificity in the same species, good predicted antigen epitope properties, and good hydrophilicity of the amino acids of the polypeptide.

[0044] 2.2. Peptide antigen synthesis and conjugation

[0045] The polypeptide CKQSKELSLIEHDYYQP was dissolved in 8M urea-PBS buffer solution, and the thiol group was coupled to KLH to obtain the polypeptide-KLH cross-linked immune antigen.

[0046] The peptide CKQSKELSLIEHDYYQP was dissolved in 8M urea-PBS buffer solution and thiol-coupled to BSA as a screening agent. The mass spectrum of the peptide is shown in Figure 9.

[0047] 3. Antigen immunization of mice and serum detection

[0048] 3.1. Peptide Antigen Immunization

[0049] The polypeptide-KLH cross-linked immune antigen was mixed with complete Freund's adjuvant and fully emulsified, and five female Balb / c mice aged 6 to 8 weeks were immunized, and multiple injections were made subcutaneously on the back of the neck of the mice. The first dose was 150 μg / mouse; booster immunization was performed every 21 days. For subsequent immunizations, the same dose of antigen as the first was emulsified with an equal amount of incomplete Freund's adjuvant and injected subcutaneously on the back of the neck of the mice at multiple points; after 6 immunizations, shock was performed 3 to 5 days later, 120 μg / mouse, the antigen was dissolved in physiological saline, and injected intraperitoneally; 7 days after the end of immunization, the mice were tail-cut and blood was collected to separate the antiserum.

[0050] 3.2. Determination of mouse serum MS4A6C titer by indirect ELISA

[0051] a. Antigen treatment: dilute the screening antigen to 2 μg / mL with PBS, add 100 μl / well to the ELISA plate, incubate at 4°C overnight, and wash the plate 3 times. Take 3% BSA blocking solution, add 200 μl / well to the ELISA plate, incubate at 37°C for 2 hours, and wash the plate;

[0052] b. Sample treatment: Take the antiserum and dilute it from 1:1000. Take 100μl and add it to each well of the ELISA plate in turn. Incubate at 37℃ for 1 hour and wash the plate 3 times. Add 100μl HRP-labeled goat anti-mouse Ig (H+L) to each well at a dilution ratio of 1:10000. Incubate at 37℃ for 1 hour. Wash the plate 3 times. Add 100μl TMB substrate. Color develop at room temperature in the dark for 5 minutes. Add 50μl stop solution.

[0053] c. Determination: OD value was determined using an ELISA reader (detection wavelength: 450 nm, reference wavelength: 630 nm).

[0054] Antiserum titer ELISA test (Table 1-2). Take the mouse with the highest titer for shock immunization: 3-5 days before fusion, take the polypeptide-KLH cross-linked immune antigen, and shock immunize the mice that have been immunized 6 times, and inject 120μg antigen into each mouse intraperitoneally.

[0055] Table 1 Results of serum ELISA test after the third immunization of mice with polypeptide antigens

[0056]

[0057] Table 2 Results of serum ELISA test after the sixth immunization of mice with polypeptide antigens

[0058]

[0059] Note: 1. * Indicates that equal volumes of blood are collected before mixed immunization; 2. ** Represents the antiserum collected from the animals with the specified numbers after the third immunization.

[0060] Negative judgment value: 0.05; positive judgment value: 0.25; antiserum Elisa titer: >1:128k

[0061] Analysis of results: AK361-M01 mice were selected for fusion, and AK361-M05 was used as an alternative.

[0062] 4. Fusion screening

[0063] (1) Preparation of spleen cells: Anesthetize AK361-M01 immunized mice, disinfect with 75% alcohol, remove the spleen in a clean bench, place a nylon mesh on a culture dish containing lymphocyte separation medium and grind the spleen, transfer the cell suspension to a centrifuge tube containing 1640 culture medium, centrifuge until cell stratification is visible. Transfer the lymphocyte layer to a new 15 mL centrifuge tube, centrifuge, and then resuspend the cells in culture medium;

[0064] (2) Myeloma cells: One week before cell fusion, resuscitate SP2 / 0 mouse myeloma cells;

[0065] (3) Incomplete culture medium and PEG are prepared to a concentration of 50% PEG, and then sterilized by high pressure for later use;

[0066] (4) Cell fusion and culture: SP2 / 0 cells and spleen cells were mixed at a ratio of 1:4 and centrifuged at 1000 rpm. Preheated 50% PEG was added to promote the fusion of the two cells. The PEG effect was terminated by preheated incomplete culture medium, centrifuged at 1000 rpm, and 20% FBS HAT 1640 culture medium was added to gently resuspend the cells. The fused cells were evenly spread into 10 96-well plates. Cultured in a cell culture incubator at 37°C and 5% CO2. On the 4th, 6th, and 8th days, 50 μL of culture supernatant was aspirated from each well, and 50 μL of HAT culture medium was added. On the 10th day, HT culture medium was used for culture. When the cell density was observed to be 60% to 70%, the cell supernatant was collected and an ELISA experiment was performed. The fusion detection results are shown in Tables 3 to 12.

[0067] Table 3 Fusion detection results

[0068]

[0069] Table 4 Fusion detection results

[0070]

[0071] Table 5 Fusion detection results

[0072]

[0073] Table 6 Fusion detection results

[0074]

[0075] Table 7 Fusion detection results

[0076]

[0077] Table 8 Fusion detection results

[0078]

[0079] Table 9 Fusion detection results

[0080]

[0081] Table 10 Fusion detection results

[0082]

[0083] Table 11 Fusion detection results

[0084]

[0085] Table 12 Fusion detection results

[0086]

[0087] Note: ELISA screening agent: CKQSKELSLIEHDYYQP-BSA; coating concentration: 250ng / mL, 100ul / well; secondary antibody: Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) [Jackson ImmunoResearch, 115-035-003], secondary antibody dilution ratio: 1:10000.

[0088] Result analysis: A total of 40 positive cell lines were selected by fusion ELISA. The supernatant was tested and 10 cell lines (1G1, 4D9, 6D10, 7H8, 5G10, 6B5, 7D8, 8D2, 9E3, 10G2) were selected for subcloning.

[0089] 5. Subcloning

[0090] Hybridoma positive cells were transfected and expanded. Cell supernatants were subjected to ELISA test. Cell lines with good cell status and high affinity were selected for subcloning, and subcloning was performed twice. The results of the two subcloning tests are shown in Tables 13 to 20.

[0091] Table 13 Second cell subclone ELISA test results

[0092]

[0093] Table 14 Second cell subclone ELISA test results

[0094]

[0095] Table 15 Second cell subclone ELISA test results

[0096]

[0097] Table 16 Second cell subclone ELISA test results

[0098]

[0099] Table 17 Second cell subclone ELISA test results

[0100]

[0101] Table 18 Second cell subclone ELISA test results

[0102]

[0103] Table 19 Second cell subclone ELISA test results

[0104]

[0105] Table 20 Second cell subclone ELISA test results

[0106]

[0107] Note: ELISA screening agent: CKQSKELSLIEHDYYQP-BSA; coating concentration: 250ng / mL, 100ul / well; secondary antibody: Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) [Jackson ImmunoResearch, 115-035-003], secondary antibody dilution ratio: 1:10000.

[0108] Result analysis: Subcellular ELISA was performed and a total of 9 cell lines with 5 positive monoclonal clones (1G1E5E1, 1G1E5F3, 4D9G8F1, 4D9G8D3, 8D2D9F1, 8D2D9A4, 9E3B10F4, 9E3B10D7, and 10G2A9D10) were selected for expansion and establishment.

[0109] 6. Cell Line Establishment

[0110] Nine cell lines of 5 subcellular positive monoclonal clones were selected for re-examination, and the results are shown in Table 21.

[0111] Table 21 Retest results of 9 cell lines

[0112]

[0113]

[0114] According to Table 21, three cell lines were selected for cell identification: 1G1E5F3, 8D2D9F1, and 9E3B10F4; among them, 9E3B10F4 had the best titer and was used to prepare 2 mg of purified antibody.

[0115] 7. Preparation and titer determination of ascites antibodies

[0116] One week before the hybridoma cells were collected, female Balb / c mice in good condition were intraperitoneally injected with 0.5 mL of liquid paraffin. Hybridoma cells in good condition were selected and the precipitated cells were resuspended in serum-free medium and the density was adjusted to 1×10 6 / mL, 0.5mL was injected into the peritoneum of each mouse. After the injection, the health status of the mice and the degree of abdominal bulge were closely observed every day. After 7 to 14 days, the mice with abdominal bulge were killed, and the ascites were collected into a 15mL centrifuge tube. Centrifugation was performed to collect the light yellow liquid below the fat layer, which was the ascites antibody. The ascites antibody was purified by protein G affinity chromatography and ELISA was performed on the screening source. The results are shown in Table 22.

[0117] Table 22 Screening original ELISA test results

[0118]

[0119]

[0120] Result analysis: ELISA test was performed on the screening antigen, and the titer was qualified.

[0121] 8. Western Blot test antibody

[0122] Western blot analysis was performed using mouse spinal cord tissue lysate at a 1:500 dilution of the antibody:

[0123] 8.1. Mouse Collection

[0124] After the mice were deeply anesthetized with 1% sodium pentobarbital, the skin and muscle tissues were cut along the midline of the abdomen and sternum of the mice to expose the heart. The right auricle was cut with ophthalmic scissors, and the mice were perfused with 100 mL of 1×PBS buffer until the perfusion fluid flowing out of the right auricle became a clear liquid. The skin and muscle tissues on the back of the mice were cut to expose the spine of the mice. The spine was transected with surgical scissors at the mouth and tail of the injury center 5 mm. Fine ophthalmic scissors were used to remove the spinous processes and muscle tissues of the mice. The spinal canal was cleverly opened from the vertebrae at the tail end of the back of the spine to fully expose the spinal cord and remove the complete spinal cord tissue.

[0125] 8.2. Protein extraction from spinal cord tissue

[0126] Weigh 50 mg of spinal cord tissue, place it in a 2 mL thickened EP tube, add pre-cooled 1× PBS solution, cut the tissue into pieces with scissors, centrifuge at 3000g for 5 minutes, discard the supernatant, and repeat twice. Add RIPA lysis buffer (tissue mass: RIPA volume = 1:10), add 1 / 10 amount of protein / phosphatase inhibitors as needed, homogenize with a tissue homogenizer at 120Hz for 30s, and let stand on ice for 20 minutes after homogenization. Centrifuge at 12000rpm for 20 minutes at 4℃, and the supernatant is the tissue protein extract. Pipette 10μL and add 90ulddH2O to dilute for protein quantification, add 1 / 4 volume of 5*Loading buffer to the remaining protein volume, mix well, boil at 95℃ for 10min, centrifuge at 1000rpm for 3min, and store in a -80℃ refrigerator.

[0127] 8.3. Quantification of spinal cord tissue protein by BCA method

[0128] Follow the protein quantification kit operation instructions. Take 80μL of 5mg / mL protein standard and dilute it into 7 concentrations in sequence, and the last tube is the solvent ddH2O control. At the same time, dilute the extracted spinal cord tissue protein with 90μL ddH2O. Add protein quantification working solution to the 96-well plate, 200μL per well, then add 20μL of standard protein or dilution of the tissue protein to be tested to each well, and incubate at 37℃ in the dark for 30min. Under the 562nm parameter of the microplate reader, measure the absorbance of each well of the 96-well plate, draw the standard protein curve with the absorbance value, and then calculate the concentration of the spinal cord tissue protein to be tested.

[0129] 8.4 Protein electrophoresis

[0130] (1) Leak detection: Clean the glass plate for glue thoroughly, dry it at room temperature, install the glass plate, fill it with distilled water to check for leaks, ensure it is airtight, and then add glue.

[0131] (2) Glue preparation: Prepare 10% separation gel first, then prepare 5% concentrated gel according to the instructions of the gel preparation kit. Pour 4 mL of separation gel mixture between the glass plates, seal with anhydrous ethanol, make the gel surface level and accelerate gelation. When a clear refraction line is observed between the liquid surfaces, discard the anhydrous ethanol, pour 1 mL of concentrated gel on the separation gel, insert a comb, wait for 10 minutes, and store at 4°C after the gel solidifies.

[0132] (3) Electrophoresis: Place the pre-made gel plate in the electrophoresis tank. Add the tissue protein sample to the gel wells in the order of loading. Add 5 μL marker to the reserved wells on the left and right sides. After the sample loading is completed, connect the electrophoresis tank to the electrophoresis instrument, red to red and black to black. Run the concentrated gel at 80V for 30 minutes, and then run the separation gel at 120V for 60 minutes.

[0133] (4) Transfer: Perform the transfer process immediately after the electrophoresis is completed. Pry open the small glass plate along the corners, cut off the upper gel layer, and carefully peel off the lower gel strip area. Cut the PVDF membrane. In the transfer solution, assemble the transfer clips in a sandwich shape. Put the clips into the transfer tank, so that the black clips face the black clips and the white clips face the red clips. Put an ice box on one side of the tank to cool down and add transfer buffer. Assemble the transfer equipment with red to red and black to black, and add ice cubes to reduce heat generation. Set the electrophoresis instrument parameters: constant current, 250mA, 300V transfer. The time is determined by the size of the target molecule, generally 1 min for 1kDa.

[0134] (5) Blocking: Cover the PVDF membrane with 10 mL of protein-free rapid blocking solution, incubate on a shaker at 45 rpm for 15 min, and wash the membrane with TBST for 5 min.

[0135] (6) Overnight incubation with primary antibody: Antibody dilution 1 / 500, buffer system: PBS, 50% glycerol, pH 7.3 to prepare primary antibody dilution solution. Place the washed PVDF membrane with the front side of the membrane on the inner wall of a drum containing the primary antibody dilution solution, and incubate on a shaker at 45 rpm at 4°C overnight.

[0136] (7) Secondary antibody incubation: PVDF membrane was incubated at 4°C overnight, and then rapidly washed with TBST on a shaker for 10 min × 3 times. Secondary antibody was incubated at room temperature for 1 h. Rapidly washed with TBST on a shaker for 10 min × 3 times.

[0137] (8) Exposure: Prepare chemiluminescent solution in a 1:1 ratio and mix thoroughly. Use a pipette to drop the luminescent solution onto the target protein surface of the PVDF membrane, gently spread it evenly with the tip of the pipette, and use a Bio-Ra luminometer to develop and take pictures.

[0138] 8.5 Results

[0139] The results are as follows Fig.10 As shown, the target protein was found to be around 25 KDa, and the buffer system was: PBS, 50% glycerol, pH 7.3.

[0140] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An MS4A6C antigen epitope peptide, characterized in that: The amino acid sequence of the antigen epitope peptide is shown in SEQ ID NO:

1.

2. An MS4A6C antigen, characterized in that: The antigen epitope peptide according to claim 1 is coupled with a carrier protein.

3. The MS4A6C antigen according to claim 2, characterized in that The carrier protein is KLH or BSA.

4. Use of the MS4A6C antigen according to claim 2 or 3 as an immunogen in preparing anti-MS4A6C antibodies.

5. A hybridoma cell line, characterized in that: It is prepared according to the following method: Immunizing mice with the MS4A6C antigen according to claim 2 or 3 to obtain immunized mice; The spleen cells of the immunized mice were extracted, fused with mouse myeloma cells, and screened to obtain hybridoma cell lines.

6. An MS4A6C antibody, characterized in that It is a monoclonal antibody or a polyclonal antibody prepared by using the MS4A6C antigen described in claim 2 or 3 as an immunogen.

7. An MS4A6C antibody, characterized in that It is a monoclonal antibody produced by the hybridoma cell line according to claim 5.

8. A method for preparing the MS4A6C antibody according to claim 7, characterized in that: The method comprises the steps of inoculating the hybridoma cell line into the peritoneal cavity of mice, collecting ascites, purifying the ascites, and obtaining monoclonal antibodies.

9. Use of the MS4A6C antibody according to claim 6 or 7 in preparing an immunoassay tool for detecting the expression level of MS4A6C molecules.

10. The use according to claim 9, characterized in that The immunoassay tool is a reagent, a test kit, a chip or a test paper.

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