Hybridoma cell strain 6F92E10 as well as monoclonal antibody and application thereof

Abstract

The invention discloses a hybridoma cell strain 6F92E10 as well as a monoclonal antibody and application of the hybridoma cell strain 6F92E10. Belongs to the technical field of biology. The invention provides a hybridoma cell strain capable of secreting an anti-Ackermann muciniphila monoclonal antibody. The secreted antibody is only combined with AKK bacteria and has no cross reaction with other common intestinal bacteria. Besides, through an indirect immunofluorescence localization experiment, the problem of how to detect the adhesion and colonization condition of Ackermann muciniphila in the colon of the mouse by using the prepared monoclonal antibody is solved, and the application range of the antibody is expanded.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a hybridoma cell line 6F92E10 and its monoclonal antibody and application. Background Art

[0002] Akkermansia muciniphila (AKK), a Gram-negative anaerobic bacterium native to the human intestine, uses mucin as its sole source of carbon, nitrogen, and energy. It hydrolyzes mucin to release free sulfate, which is then fermented to produce acetic and propionic acids. AKK is a common colonizer of the human intestine, comprising approximately 0.5% to 5% of the human microbiome. Research has shown that AKK exhibits promising potential in improving metabolic diseases (such as obesity and diabetes), limiting inflammation, combating aging, and assisting in cancer immunotherapy. It is considered a promising candidate for second-generation probiotics.

[0003] However, Akkermansia muciniphila is difficult to isolate and culture, and rapid isolation and identification of AKK bacteria remains a research bottleneck. Furthermore, the lack of tools targeting specific functional molecules of AKK hinders in-depth research on its mechanisms of immune regulation and intestinal colonization. These technical bottlenecks all involve the preparation of monoclonal antibodies against AKK bacteria.

[0004] In summary, how to provide a monoclonal antibody against Akkermansia muciniphila is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a hybridoma cell line 6F92E10 and its monoclonal antibody and application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A hybridoma cell line 6F92E10, classified and named Hybridoma cell line 6F92E10, was deposited in the China Center for Type Culture Collection on June 30, 2025, with the deposit number CCTCC NO: C2025203, and the deposit address is Wuhan University, Wuhan, China.

[0008] The invention relates to the use of the hybridoma cell line in preparing monoclonal antibodies against Akkermansia muciniphila.

[0009] The hybridoma cell line is used to prepare a product for detecting Akkermansia muciniphila.

[0010] The invention relates to a monoclonal antibody against Akkermansia muciniphila, which is secreted by the hybridoma cell line.

[0011] Furthermore, the monoclonal antibody includes a light chain and a heavy chain, the light chain includes light chain LFR1, light chain CDR-L1, light chain LFR2, light chain CDR-L2, light chain LFR3, light chain CDR-L3 and light chain LFR4 as shown in the amino acid sequences of SEQ ID No.1 to SEQ ID No.7; the heavy chain includes heavy chain HFR1, heavy chain CDR-H1, heavy chain HFR2, heavy chain CDR-H2, heavy chain HFR3, heavy chain CDR-H3 and heavy chain HFR4 as shown in the amino acid sequences of SEQ ID No.8 to SEQ ID No.14.

[0012] Use of the above monoclonal antibody in preparing a product for detecting Akkermansia muciniphila.

[0013] A kit for detecting Akkermansia muciniphila comprises the above hybridoma cell line or the above monoclonal antibody.

[0014] Furthermore, ELISA reagents are also included.

[0015] It can be seen from the above technical solutions that, compared with the prior art, the present invention has the following beneficial effects:

[0016] ① Subtype identification: The monoclonal antibody obtained was identified by subtype identification using a monoclonal antibody subclass identification kit, which confirmed that the antibody type and subtype were IgG1, providing important information for the further application of the antibody.

[0017] ②Variable region sequence: The variable region sequence of the monoclonal antibody was obtained, which provided convenience for subsequent research.

[0018] ③Titer determination: The indirect ELISA method was used to detect the ascites titer of the monoclonal antibody. The results showed that the ascites titer of the monoclonal antibody was 4.5×10 -4 , indicating that the antibody has high activity.

[0019] ④Specificity: The specificity of the monoclonal antibody was tested by indirect ELISA using four different isolates of Akkermansia muciniphila and 12 strains of eight other common bacteria as test samples. The results showed that the monoclonal antibody had a strong positive immune response to the four strains of Akkermansia muciniphila, but not to the other bacteria, fully demonstrating the specificity of the antibody.

[0020] ⑤ Indirect immunofluorescence localization: Through indirect immunofluorescence localization experiments, the adhesion and colonization of Akkermansia muciniphila in the mouse colon were successfully detected, providing new methods and technical means for studying the mechanism of action of this bacterium in the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0022] Figure 1 The WB results of the monoclonal antibody and Akkermansia muciniphila in Example 4 of the present invention are shown, wherein lane 1 is Marker 250 / 150 / 100 / 70 / 50 / 40 / 35 / 25 / 20 / 15 / 10 (kDa), and lane 2 is the size result of the monoclonal antibody reaction protein;

[0023] Figure 2 This is the immunofluorescence result of mouse colon in Example 5 of the present invention. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0026] Example 1

[0027] Screening of hybridoma cell lines and preparation of monoclonal antibodies

[0028] 1. Materials

[0029] The strains used in this example and their sources are shown in Table 1.

[0030]

[0031] 2. Immunogen Preparation

[0032] The immunization strain A. muciniphila SDLYAKK8 was initially cultured anaerobically at 37°C for 72 h on BHI solid medium supplemented with 5% mucin and 0.25% L-cysteine ​​hydrochloride. After PCR verification, individual colonies were transferred to 200 μL of BHI liquid medium supplemented with 5% mucin and 0.25% L-cysteine ​​hydrochloride and incubated anaerobically at 37°C for 24 h in a 96-well plate. The culture was then transferred to 2 mL of BHI liquid medium supplemented with 5% mucin and 0.25% L-cysteine ​​hydrochloride and cultured until logarithmic phase. The culture was then transferred to 50 mL of BHI liquid medium supplemented with 5% mucin and 0.25% L-cysteine ​​hydrochloride and cultured until the end-logarithmic and early-stationary phase. The culture was then centrifuged at 12,000 rpm for 5 minutes at 4°C. The resulting pellet was then resuspended in 0.4% formaldehyde in PBS and incubated at 4°C for 24 hours. The pellet was then washed twice with PBS before use for immunization.

[0033] 3. Immunization of Animals

[0034] 8-week-old female SPF BALB / c mice were immunized to prepare monoclonal antibodies. Inactivated A. muciniphila SDLYAKK8 bacterial suspension (10 9 CFU / mouse) was mixed with an equal amount of Freund's complete adjuvant and emulsified, then injected subcutaneously into mice (primary immunization). Two weeks later, the same inactivated bacterial solution was mixed with Freund's incomplete adjuvant and used to immunize mice (secondary immunization). Two weeks later, the same inactivated bacterial solution was mixed with Freund's complete adjuvant and used to immunize mice (third immunization). Two weeks later, a booster immunization was performed with the same inactivated bacterial solution mixed with Freund's incomplete adjuvant and used to immunize mice (fourth immunization). After the fourth immunization, the same inactivated bacterial solution was mixed with PBS and injected intraperitoneally into the mice for a booster immunization.

[0035] 4. Cell Fusion

[0036] Three days after the last booster immunization, the eyes were removed and the animals were sacrificed. Positive control blood was collected, the spleen was removed, and a single-cell suspension was prepared. SP2 / 0 cells in the logarithmic phase were removed and treated, and then mixed with spleen cells at a cell number ratio of 1:10. 50% PEG1450 was added for 1 min, and the cells were diluted with basal medium DMEM. After low-speed centrifugation, the cells were gently suspended and mixed with HAT medium containing 20% ​​fetal bovine serum. The suspension was then diluted to 2×10 7 / Plate onto a pre-prepared feeder cell plate and culture at 37°C with 5% CO2.

[0037] 5. Screening and cloning of hybridoma cell lines

[0038] After the cells in the fusion plate have grown to about 10,000 cells, the test is started. After the ELISA quality control is qualified (i.e., negative control <0.2, positive control >1.0), the positive wells (OD 450 ≥0.5) for subcloning. Wells with high positive test values ​​in the fusion plate were selected for limiting dilution. 60% of the wells per plate were counted for subcloning. Each time, single clones with high positive test values ​​were selected for limiting dilution. ELISA testing was performed 5-7 days after each subcloning step until a monoclonal cell line that consistently secreted positive antibodies was identified for expansion. Cell lines that consistently secreted positive antibodies, identified during the subcloning phase, were expanded in 24-well plates. The supernatant was collected for antigen detection. The cells were then expanded in 10 cm culture dishes, and the supernatant was again collected and assayed for antibody titer. A hybridoma cell line specifically secreting monoclonal antibodies against Akkermansia muciniphila was identified and designated 6F92E10. The cell line was cryopreserved in liquid nitrogen.

[0039] 6. Deposit of Hybridoma Cell Line 6F92E10

[0040] The hybridoma cell line 6F92E10, which is classified and named Hybridoma cell line 6F92E10, was deposited in the China Center for Type Culture Collection on June 30, 2025, with the deposit number CCTCC NO: C2025203, and the deposit address is Wuhan University, Wuhan, China.

[0041] 7. Preparation and purification of monoclonal antibody ascites

[0042] First, liquid paraffin was injected into the mouse peritoneal cavity. One week later, hybridoma cell line 6F92E10 was inoculated into the mouse peritoneal cavity. Hybridoma cell line 6F92E10 was expanded and cultured in 10% fetal bovine serum medium. When the cell density reached 1×10 6 When the volume of the solution is 100 mL, centrifuge at 800 rpm, collect the precipitate, resuspend it in PBS, and inject it intraperitoneally into mice. After 7-10 days, collect the ascites. The collected ascites is pretreated and purified using a Protein G-agarose affinity chromatography column and stored at -20°C until use.

[0043] 8. Monoclonal Antibody Subtype Identification and Ascites Titer Determination

[0044] The subtype of the monoclonal antibody obtained was identified using a monoclonal antibody subclass identification kit. For specific steps, please refer to the instructions. The antibody subtype identification results showed that the type and subtype of the antibody secreted by the hybridoma cell line 6F92E10 were IgG IgG1 (Table 2). 6CFU / mL) was used as the coating antigen, and the ascites titer of the monoclonal antibody was detected by indirect ELSIA method. The analysis results showed that the ascites titer of the monoclonal antibody was 4.5×10 -4 .

[0045]

[0046] Example 2

[0047] Monoclonal antibody light and heavy chain amino acid sequence determination

[0048] 1. RNA Extraction from Hybridoma Cell Line 6F92E10

[0049] Remove the cryovial containing the hybridoma cell line 6F92E10 from the liquid nitrogen tank and immediately thaw it in 37°C water. Immediately centrifuge at 1000 rpm for 3 minutes. Remove the supernatant and quickly add 1 mL of DMEM cell culture medium to the cryovial to resuspend the cells. Transfer the tube to a cell culture flask containing 10 mL of DMEM cell culture medium.

[0050] After centrifugation, discard the supernatant. Wash the cultured cells twice with sterile 1× PBS and centrifuge to discard the PBS. Add 1 mL of Trizol to the centrifuged cells and mix thoroughly by pipetting. Add 0.2 mL of chloroform per 1 mL of Trizol and shake vigorously for 15 seconds. Let the tube stand at room temperature until distinct layers form in the EP tube. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Transfer the upper aqueous layer to a new EP tube and add an equal volume of isopropanol. Mix thoroughly and place the tube on ice for 30 minutes to precipitate RNA. Centrifuge at 12,000 rpm for 10 minutes at 4°C. Discard the supernatant and wash the RNA pellet with 1 mL of 75% ethanol. Centrifuge at 12,000 rpm for 5 minutes at 4°C. Discard the supernatant and retain the pellet. Add 50 µL of double-distilled water to the RNA pellet.

[0051] Purity was determined by agarose gel electrophoresis and quantification was performed. The dissolved RNA was divided into PCR tubes according to the reverse transcription system and the reverse transcription reaction program of the PCR instrument was set as 37°C for 15 minutes and 98°C for 5 minutes.

[0052]

[0053] Prepare the PCR reaction system (Table 5) and use the designed mouse IgG VH VL primers (Table 4) to amplify the above cDNAs respectively. Perform the PCR reaction under the following conditions: pre-denaturation at 98°C for 5 min; denaturation at 94°C for 30 s; annealing at 54°C for 30 s; extension at 72°C for 2 min; after 30 cycles, extend at 72°C for 6 min.

[0054]

[0055]

[0056] The monoclonal antibody light and heavy chain variable region genes were amplified separately. The monoclonal antibody variable region genes were ligated into the pMD-18T plasmid and then transformed into a DH5α strain. Colonies that tested positive by PCR were sent for sequencing. The monoclonal antibody gene sequences were then extracted by aligning the PCR primers with the sequence obtained. The gene sequences are shown in SEQ ID No. 1 to SEQ ID No. 14, respectively.

[0057] DIQMTQSPASSLSASVGETVTITC, SEQ ID No.1;

[0058] HSSENIFSYLA, SEQ ID No. 2;

[0059] WYRQKQGKSPQLLVS, SEQ ID No.3;

[0060] HENILAE, SEQ ID No. 4;

[0061] GVPSRFSASGSGTQFSLRINSLQPEDFGSFYC, SEQ ID No.5;

[0062] QHHYGFPWT, SEQ ID No. 6;

[0063] FGGGTKLEVK, SEQ ID No. 7;

[0064] QVQLKESGPGLVAPSQSLSITCTVSGFSLT, SEQ ID No.8;

[0065] SFGVS, SEQ ID No. 9;

[0066] WVRQPPGKGLEWLG, SEQ ID No.10;

[0067] VIWDDGNTNYNSTLIS, SEQ ID No.11;

[0068] RLSISKDNSKSQVFLKLKSLQTDDTATYYCAK, SEQ ID No. 12;

[0069] LGFYVWYFDV, SEQ ID No. 13;

[0070] WGAGTTVTVSS, SEQ ID No.14.

[0071] Example 3

[0072] Monoclonal antibody specificity detection

[0073] The specificity of the 6F92E10 monoclonal antibody was tested by indirect ELSIA using four different isolates of Akkermansia muciniphila (SDLYAKK5, SDLYAKK8, SDLYAKKC1, and ATTCBAA-835) and other common bacteria such as Escherichia coli (ATCC43895, ATCC25922), Salmonella typhimurium (CMCC50115), Pseudomonas aeruginosa (ATCC15442), Fusobacterium nucleatum (ATCC10953, ATCC25586), Enterococcus faecalis (E745, V583), Enterococcus faecium (E.980), Listeria monocytogenes (ATCC13932), and Enterobacter cloacae (ATCC23355, 002) as test samples. Negative serum was used as negative control, and Akkermansia muciniphila SDLYAKK8 was used as positive control.

[0074] 1. Coating

[0075] The above bacterial suspension (1×10 7 ELSIA plates were coated with 100 μL of negative serum (CFU / mL) or 300 μL of negative serum per well, with 3 replicates for each sample, and incubated at 37°C for 2 h or at 4°C overnight.

[0076] 2. Closed

[0077] After washing three times with PBST, the sections were blocked with PBST containing 1% BSA for 2 h.

[0078] 3. Primary Antibody Incubation

[0079] After washing three times with PBST, 100 μL / well of 1:5000 diluted monoclonal antibody was added and incubated at 37°C for 1 h.

[0080] 4. Secondary Antibody Incubation

[0081] After washing four times with PBST, 100 μL / well of 1:10,000 diluted HPR-labeled rabbit anti-mouse IgG secondary antibody was added and incubated at 37°C for 1 h.

[0082] 5. Color rendering

[0083] After washing four times with PBST, the cells were developed with TMB substrate for 20 min.

[0084] 6. Degree observation

[0085] After the stop solution is terminated, read the OD with a microplate reader 450 Samples with an OD value greater than 2.1 compared to negative OD values ​​were considered positive.

[0086] The results are shown in Table 6 . The monoclonal antibody had a strong positive immune reaction against 4 strains of Akkermansia muciniphila , but had no reaction against the other 8 species and 12 strains of bacteria.

[0087]

[0088] Example 4

[0089] Monoclonal antibody immunoblotting experiments

[0090] 1. Preparation of Polyacrylamide Gel

[0091] Prepare a 12% separation gel solution. Quickly add the separation gel solution to the gap between the two glass plates, leaving enough space for pouring the stacking gel. Carefully add 1 mL of isopropanol to the separation gel solution. After the separation gel is fully polymerized (approximately 30 minutes at 25°C), drain as much liquid from the gel as possible and absorb any remaining liquid with the edge of a filter paper. Prepare a 5% stacking gel solution (4 mL). Quickly pour the stacking gel directly onto the polymerized separation gel. Immediately insert a clean comb into the stacking gel solution. After the stacking gel is fully polymerized (approximately 30 minutes at 25°C), carefully remove the comb and add protein electrophoresis buffer to the electrophoresis tank.

[0092] 2. Sample Preparation

[0093] After lysis of Akkermansia muciniphila SDLYAKK8, take 30µL of the extracted sample lysate and add 5µL of 6X Loading buffer, mix well, boil in a boiling water bath for 5-10 minutes, and centrifuge at 2500 rpm for 2 minutes to centrifuge the tube wall solution.

[0094] 3. Electrophoresis

[0095] Use a pipette to slowly add the sample mixture to the sample reservoir. Add 10 µL of prestained marker. Turn on the power supply and run electrophoresis at 80V for 30 minutes. Then increase the voltage to 120V and run until the bromophenol blue loading buffer migrates to the bottom of the gel.

[0096] 4. Wet transfer

[0097] Remove the gel and immerse it in transfer buffer. Soak the PVDF membrane in methanol for 1 minute, then blot dry with filter paper. Transfer the membrane to transfer buffer and immerse the filter paper in transfer buffer. Prepare a transfer sandwich: 3 sheets of transfer filter paper (black side for the negative electrode) + gel + PVDF membrane + 3 sheets of transfer filter paper (white side for the positive electrode). Place the transfer sandwich in the transfer tank, black side facing black and white side facing red. Apply a constant current of 200mA for 60 minutes.

[0098] 5. Closed

[0099] Remove the membrane and wash once with TBS for 5 minutes each time (shake on a horizontal shaker). Remove the membrane and immerse it in blocking solution at 25°C for 1 hour or at 4°C overnight.

[0100] 6. Binding Antibodies

[0101] Remove the membrane and wash once with TBST (5 min each time, shaken horizontally). Remove the membrane and soak in primary antibody diluted in diluent at 25°C for 2 hours or overnight at 4°C. Remove the membrane and wash five times with TBST (5 min each time, shaken horizontally). Remove the membrane and soak in secondary antibody diluted in diluent at 25°C for 1 hour (goat anti-mouse-HRP 1:10,000).

[0102] 7. ECL exposure

[0103] Dilute and mix the A and B luminescent solutions in equal proportions (500µL each per membrane), place the membrane on plastic wrap, evenly drip the AB mixture onto the membrane, cover with plastic wrap, and react in the dark for 1 minute; place the membrane on filter paper to absorb the liquid (no liquid should flow out, the membrane should not be completely dry), transfer it to clean plastic wrap, wrap it (keep the front flat), and fix it in a dark box; place the dark box in a dark room, remove the film and quickly place it on the membrane inside the dark box, close the dark box, and expose it according to the observed fluorescence intensity (the exposure time is generally 1 minute, if the bands are weak, you can choose to press the film overnight); open the dark box, remove the film, and immediately and completely immerse it in the developer for 1 minute; remove the film, rinse with deionized water, and then immerse it in the fixer for 1 minute; remove the film, rinse with deionized water, and dry it, calibrate the marker, and analyze the experimental results.

[0104] The results showed that the monoclonal antibody had a specific band at 60 kDa for Akkermansia muciniphila, but no bands at other positions ( Figure 1 ). This indicates that the monoclonal antibody specifically binds to Akkermansia muciniphila and can be used for immunoblotting experiments on this bacterium.

[0105] Example 5

[0106] Indirect immunofluorescence localization of Akkermansia muciniphila adherence and colonization in mouse colon

[0107] 1. Oral administration of Akkermansia muciniphila SDLYAKK8 into mice

[0108] Sixteen 8-week-old BALB / c mice were randomly divided into two groups (experimental group and control group), with 8 mice in each group. After 1 week of acclimatization, each mouse was gavaged with a combination of antibiotics for 5 days, followed by antibiotic drinking water for 3 days. After a 2-day interval, each mouse in the experimental group was gavaged with a bacterial concentration of 1×10 9 The control group was gavaged with 200 μL of PBS in the same manner.

[0109] 2. Cryosection Preparation

[0110] On the fifth day, mice were dissected, and the colons were removed and fixed in 4% paraformaldehyde for 24 hours. The tissues were then cryoprotected by incubation in a 30% sucrose solution, allowing them to sink to the bottom of the tube. Excess sucrose was removed from the tissues, and the tissues were placed in the center of a mold filled with OCT. The tissue blocks were then frozen on dry ice and ready for sectioning.

[0111] Cut the tissue into slices with a thickness of 5 to 20 microns, use a small brush to flatten the slices, and quickly attach the frozen tissue slices to a warm glass slide that has been coated (usually Poly-L-Lysine). Store the slices in a freezer at -80°C.

[0112] 3. Staining and Observation of Frozen Sections

[0113] Remove frozen tissue sections from the refrigerator and allow to stand at room temperature for 10-20 minutes. Rinse three times with PBS for 3 minutes each. Add blocking solution to the slides and block at room temperature for 1 hour. Add primary antibody diluted in blocking solution to the slides and incubate overnight at 4°C. Rinse three times with PBS for 5 minutes each. Add secondary antibody diluted in blocking solution and incubate at room temperature for 45 minutes. Rinse three times with PBS for 5 minutes each. Apply antifade mounting solution to the slides, cover with a coverslip, and observe under a fluorescence microscope.

[0114] The results are as follows Figure 2 As shown, this method can visually show the colonization location of Akkermansia muciniphila in mouse colon.

[0115] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0116] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A hybridoma cell line 6F92E10, characterized in that: It is classified and named Hybridoma cell line6F92E10, and was deposited in the China Center for Type Culture Collection on June 30, 2025, with the deposit number CCTCC NO: C2025203, and the deposit address is Wuhan University, Wuhan, China.

2. Use of the hybridoma cell line according to claim 1 in preparing monoclonal antibodies against Akkermansia muciniphila.

3. Use of the hybridoma cell line according to claim 1 in preparing a product for detecting Akkermansia muciniphila.

4. A monoclonal antibody against Akkermansia muciniphila, characterized in that The protein is secreted by the hybridoma cell line according to claim 1.

5. The monoclonal antibody according to claim 4, wherein The monoclonal antibody includes a light chain and a heavy chain, wherein the light chain includes a light chain LFR1, a light chain CDR-L1, a light chain LFR2, a light chain CDR-L2, a light chain LFR3, a light chain CDR-L3 and a light chain LFR4 as shown in the amino acid sequences of SEQ ID No.1 to SEQ ID No.7; and the heavy chain includes a heavy chain HFR1, a heavy chain CDR-H1, a heavy chain HFR2, a heavy chain CDR-H2, a heavy chain HFR3, a heavy chain CDR-H3 and a heavy chain HFR4 as shown in the amino acid sequences of SEQ ID No.8 to SEQ ID No.

14.

6. Use of the monoclonal antibody according to any one of claims 4 to 5 in the preparation of a product for detecting Akkermansia muciniphila.

7. A kit for detecting Akkermansia muciniphila, characterized in that: The method comprises the hybridoma cell line according to claim 1 or the monoclonal antibody according to any one of claims 4 to 5.

8. The kit according to claim 7, wherein Also includes ELISA reagents.

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