Monoclonal antibody pair aiming at helicobacter pylori adhesin A and application thereof

Abstract

The invention belongs to the technical field of immunology, and particularly relates to a monoclonal antibody pair aiming at helicobacter pylori adhesin A and application of the monoclonal antibody pair. The invention discloses an anti-HpaA monoclonal antibody pair which is named as an MM07 antibody and an MM14 antibody respectively, and discloses a corresponding coding gene, and a test strip and a kit containing the monoclonal antibody pair. The result of the embodiment shows that the monoclonal antibody pair disclosed by the invention has good specificity and sensitivity aiming at the HpaA antigen.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, specifically relating to a monoclonal antibody pair against Helicobacter pylori adhesin A and its application. Background Technology

[0002] Helicobacter pylori adhesin A (HpaA) is mainly located on the outer membrane surface of Helicobacter pylori and plays a crucial role in mediating the adhesion of the bacteria to the host's gastric epithelial cells, making it one of the important factors in the pathogenicity of Helicobacter pylori. HpaA is a lipoprotein with a transmembrane signal peptide sequence that can anchor to the outer membrane surface. Its expression is closely related to the adhesion, colonization, and chronic infection of Helicobacter pylori. During infection, the immunostimulatory effect of HpaA is closely associated with the formation of chronic inflammatory responses, and HpaA-mediated host cell adhesion is key to the colonization of the gastric mucosa by Helicobacter pylori. HpaA has strong immunogenicity and can induce the host to produce specific antibodies; high titers of anti-HpaA antibodies can be detected in the serum of Helicobacter pylori-infected individuals.

[0003] Immunological detection methods for Helicobacter pylori, including serum ELISA and rapid diagnostic test strips, have the advantages of being easy to operate, low in cost, and non-invasive, making them suitable for large-scale epidemiological surveys and preliminary screening.

[0004] Immunological testing is based on antigen-antibody reactions; therefore, using highly conserved and specific antigens is crucial for the immunological diagnosis of Helicobacter pylori infection. Helicobacter pylori adhesin A (HpaA), cytotoxin-associated gene A protein (CagA), vacuoletoxin A (VacA), and urease are among the most researched and applied antigens. However, high-performance antibodies targeting these key antigens remain relatively limited. Developing more novel and efficient Helicobacter pylori-specific antibodies is of great significance to meet the growing demand for accurate testing. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody against Helicobacter pylori adhesin A and its application. The antibody provided by this invention has good specificity and sensitivity against HpaA.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a monoclonal antibody pair against Helicobacter pylori adhesin A, wherein the antibody pair is MM07 antibody and MM14 antibody; The heavy chain variable region of the MM07 antibody includes heavy chain CDR1-3, and the amino acid sequence of heavy chain CDR1-3 is shown in SEQ ID NO: 4-6; The light chain variable region of the MM07 antibody includes light chain CDR1-3, and the amino acid sequence of light chain CDR1-3 is shown in SEQ ID NO: 9-11; The heavy chain variable region of the MM14 antibody includes heavy chain CDR1-3, and the amino acid sequence of heavy chain CDR1-3 is shown in SEQ ID NO: 14-16; The light chain variable region of the MM14 antibody includes light chain CDR1-3, and the amino acid sequence of light chain CDR1-3 is shown in SEQ ID NO: 19-21.

[0007] Preferably, The heavy chain variable region of the MM07 antibody includes an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO: 3 and has the ability to bind to HpaA. The light chain variable region of the MM07 antibody includes an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO: 8 and has the ability to bind to HpaA. The heavy chain variable region of the MM14 antibody includes an amino acid sequence as shown in SEQ ID NO: 13, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO: 13 and has the ability to bind to HpaA. The light chain variable region of the MM14 antibody includes an amino acid sequence as shown in SEQ ID NO: 18, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO: 18 and has the ability to bind to HpaA.

[0008] The present invention also provides a nucleic acid molecule that encodes the monoclonal antibody pair described in the above technical solution.

[0009] Preferably, The sequence encoding the variable region of the heavy chain of the nucleic acid molecule for the MM07 antibody includes the nucleotide sequence shown in SEQ ID NO. 2; The sequence encoding the variable region of the light chain of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO. 7; The sequence encoding the variable region of the heavy chain of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO. 12; The sequence encoding the variable region of the light chain of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO. 17.

[0010] The present invention also provides a biomaterial expressing the monoclonal antibody pair described in the above technical solution; the biomaterial includes a carrier or cells.

[0011] The present invention also provides the application of the monoclonal antibody pairs described in the above technical solutions, or the monoclonal antibody pairs encoded by nucleic acid molecules described in the above technical solutions, or the monoclonal antibody pairs expressed by biological materials described in the above technical solutions, or the test strips or kits described in the above technical solutions in the detection of HpaA, or their application in the preparation of products for detecting HpaA.

[0012] Preferably, the application is in the detection of Helicobacter pylori, or in the preparation of products for detecting Helicobacter pylori.

[0013] The present invention also provides a test strip or kit, wherein the test strip or kit comprises the monoclonal antibody pair described in the above technical solution, or the monoclonal antibody pair encoded by the nucleic acid molecule described in the above technical solution, or the monoclonal antibody pair expressed by the biological material described in the above technical solution.

[0014] Preferably, the MM14 antibody is used as a coating antibody, and the MM07 antibody is used as a labeling antibody.

[0015] The present invention also provides the application of the test strips or kits described above in the detection of Helicobacter pylori.

[0016] Beneficial effects: The monoclonal antibody pair against HpaA of this invention is named MM07 antibody and MM14 antibody, respectively. Verification has shown that MM07 and MM14 antibodies have high titers and good specificity against the HpaA antigen, indicating high clinical potential for Helicobacter pylori infection detection. Furthermore, when this monoclonal antibody pair is applied to a fluorescent immunochromatographic test strip, with MM14 antibody as the coating antibody and MM07 antibody as the labeling antibody, it exhibits good specificity and sensitivity against HpaA. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a flowchart of the production process for monoclonal antibodies.

[0019] Figure 2 The image shows the results of antibody purity detection using sodium dodecyl sulfate polyacrylamide gel electrophoresis.

[0020] Figure 3 The results of Western blotting for the specificity of MM07 and MM14 antibodies are shown in Figure 1. (A) MM14 monoclonal antibody was used as the primary antibody, with lanes 1, 2, and 3 containing purified HpaA antigen, unpurified BL21 whole bacterial lysate containing HpaA, and 10% FBS, respectively. (B) MM07 monoclonal antibody was used as the primary antibody, with lanes 1, 2, and 3 containing unpurified BL21 whole bacterial lysate containing HpaA, purified HpaA antigen, and 10% FBS, respectively.

[0021] Figure 4 The graph shows the results of indirect ELISA detection of MM07 antibody titer.

[0022] Figure 5 The graph shows the results of indirect ELISA detection of MM14 antibody titer.

[0023] Figure 6 MM07 and MM14 antibodies were used in the detection of HpaA using fluorescent immunochromatographic test strips. (A) Fluorescent immunochromatographic test strips based on MM07 and MM14 antibodies were used to test with 10 μg / mL HpaA antigen, and a visible fluorescent signal appeared under ultraviolet light irradiation; (B) Preliminary application of the fluorescent immunochromatographic test strips was carried out to detect HpaA antigen protein at concentrations of 0, 0.5 ng / mL, 1 ng / mL, and 5 ng / mL, respectively. Detailed Implementation

[0024] This invention provides a monoclonal antibody pair against Helicobacter pylori adhesin A and a fluorescent immunochromatographic test strip, which can specifically target the HpaA antigen and provide effective application value for the clinical development of reagent kits for detecting Helicobacter pylori infection.

[0025] In this invention, the requirement of having at least 75% identity is preferably any value within the range of 75% to 99%, for example, having sequence identity of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%.

[0026] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0028] The various instruments, equipment, raw materials, or reagents used in the embodiments of this invention are not subject to any special restrictions on their source; they are all conventional products that can be purchased through legitimate commercial channels and can be prepared according to conventional methods well known to those skilled in the art. The flowchart for the production and preparation of monoclonal antibodies in the embodiments of this invention is shown below. Figure 1 As shown.

[0029] Example 1

[0030] This embodiment provides the preparation and screening process for hybridoma cell lines. After screening, a total of 13 hybridoma cell lines were obtained in this embodiment. The specific operation steps are as follows: 1. Animal immunization The immunization subjects were 8-week-old female BALB / c mice. The immunogen was HpaA expressed by Escherichia coli, consisting of 247 amino acids with a molecular weight of 27.94 kDa, and its sequence is shown in SEQ ID NO: 1.

[0031] MHHHHHHLEVLFQGPSPHIIETNEVALKLNYHPASEKVQALDEKILLLKPAFQYSDNIAKEYENKFKNQTTLKVEEILQNQGYKVINVDSSDKDDFSFAQKKEGYLAVAMNGEIVLRPDPKRTIQ KKSEPGLLFSTGLDKMEGVLIPAGFVKVTILEPMSGESLDSFTMDLSELDIQEKFLKTTHSSHSGGLVSTMVKGTDNSNDAIKSALNKIFASIMQEMDKKLTQRNLESYQKDAKELKNKRNR (SEQ ID NO: 1)

[0032] The immunization process includes the following steps: (1) First immunization: Immunogen HpaA was prepared into an emulsion with an equal volume of Freund's complete adjuvant (catalog number: F5881, Sigma), and injected subcutaneously at multiple points in the abdomen. The single immunization dose of antigen was 50 μg per mouse.

[0033] (2) Secondary immunization: Two weeks after the first immunization, the same dose of immunogen and an equal volume of incomplete Freund's adjuvant (catalog number: 263910, BD Company) were used to make an emulsion, which was then injected subcutaneously at multiple points in the abdomen.

[0034] (3) Three immunizations: Two weeks after the second immunization, the same dose of immunogen and an equal volume of incomplete Freund's adjuvant were mixed to make an emulsion, which was then injected subcutaneously into the abdomen at multiple points.

[0035] (4) Shock immunization: Blood was collected one week after three immunizations, and serum titers were determined using indirect ELISA. Mice with high titers were selected for shock immunization. Spleens of mice were collected 3-4 days after shock immunization for hybridoma fusion. Shock immunization refers to antigen injection without adjuvant.

[0036] 2. Cell fusion

[0037] Cell fusion begins 3-4 days after the initial immunization. Spleen cells from immunized mice are collected and mixed with mouse myeloma cells (SP2 / 0) at a 1:1 ratio. The mixture is then fused using an electrofusion method to obtain hybridoma cells.

[0038] 3. Hybridoma cell screening

[0039] The cell culture medium was changed twice, once on day 6 and again on day 8 after fusion, using selective medium. On day 10 after cell fusion, the supernatant from the master clone stage cells was collected for screening and testing. The specific procedures included the following steps: 3.1 Antibody Binding Detection Indirect ELISA was used to detect the binding of hybridoma cell culture supernatant, retaining clones that bound to the antigen. The procedure for indirect ELISA detection of hybridoma cell culture supernatant is as follows: (1) Coating: Take HpaA antigen, dilute it with coating buffer to 1 μg / mL, then add 100 μL to each well of a 96-well plate with a pipette, gently tap the plate to mix the sample, seal it with plastic wrap, and coat it overnight at 4°C.

[0040] (2) Blocking: After washing the plate, block the microplate with blocking buffer at 300 μL / well and block at room temperature for 1 h; (3) Sample addition: After washing the plate, add 50 μL of hybridoma cell supernatant to each well and incubate at room temperature for 1 h; (4) Secondary antibody incubation: After washing the plate, add 100 μL of enzyme-labeled antibody (catalog number: 50323-T16-H, Yiqiao Shenzhou) to each well and incubate at room temperature for 2 hours; (5) Color development: After washing the plate, add 200 μL of color development solution to each well and let stand at room temperature for 16 min; (6) Termination and detection: The reaction was terminated by adding 50 μL of the stop solution to each well, and then the reaction was detected by an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0041] 3.2 Limiting Dilution Method

[0042] The limiting dilution method and subclonal screening for positive monoclonal cells are performed as follows: (1) Using HT selective medium (product number: H0137-10VL, Sigma), each positive hybridoma cell obtained by screening was seeded into a 96-well cell culture plate at a cell density of 0.75 cells / well and cultured statically at 37°C with 5% CO2.

[0043] (2) After culturing for 7 days, the supernatant was collected for indirect ELISA detection to confirm that the antibody to be tested was positive for binding to the antigen. The indirect ELISA operation is performed according to the above procedure.

[0044] (3) Select ELISA-specific binding positive monoclonal cells and perform 1-2 rounds of limiting dilution to screen for stable positive monoclonal cells.

[0045] 3.3 Expanded cultivation

[0046] The selected positive monoclonal cells were then subjected to expansion culture, as follows: (1) Transfer 1 mL of hybridoma cells into a 100 mL culture flask. When the cells have multiplied to a certain density, discard the supernatant, replace with fresh culture medium and continue culturing for 7-9 days. Then collect the culture supernatant.

[0047] (2) The collected hybridoma cell supernatant was centrifuged using a benchtop centrifuge at 4000g for 30min. The cell supernatant was collected and filtered using a 0.45μm filter membrane.

[0048] 3.4 Antibody purification

[0049] Positive monoclonal cell culture supernatants were collected, and antibodies were purified using a protein A affinity chromatography column. The most prevalent form of mouse monoclonal antibodies is IgG. Utilizing the specific and reversible binding between biomolecules, protein A can bind with high specificity and high affinity to the Fc fragment of most IgG molecules. When cell culture supernatants containing mouse monoclonal antibodies flow through the chromatography column, antibody molecules are "captured" on the column due to the binding of their Fc fragments to protein A, while other impurities (such as host cell proteins, DNA, and culture medium components) are washed away directly through the column because they lack binding ability. Finally, by changing the elution conditions, the purified antibodies are dissociated from the column, thus achieving high-purity and high-recovery purification.

[0050] 3.5 Monoclonal Antibody Concentration and Purity Detection

[0051] (1) Detection of antibody concentration using a spectrophotometer

[0052] For the initial determination of monoclonal antibody concentration, the concentration of the sample is obtained by dividing the sample detection value by the extinction coefficient of IgG (1.414) using the absorption of aromatic amino acids in the protein at 280 nm.

[0053] (2) Antibody purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0054] Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates proteins in the sample based on their molecular weight, and then verifies the purity of the target antibody by staining with Coomassie brilliant blue.

[0055] 3.6 Monoclonal antibody binding screening

[0056] The selected monoclonal antibodies were subjected to paired ELISA detection, and the results are shown in Table 1.

[0057] Table 1 Summary of monoclonal antibody ELISA pairing results

[0058] The specific steps for ELISA paired testing are as follows: (1) The purified monoclonal antibody was conjugated with HRP in advance as an enzyme-labeled secondary antibody; (2) Coating: The purified monoclonal antibody was coated at 2 μg / mL, 100 μL / well, and 4℃ overnight; (3) Sealing: After washing the plate, seal with 2% BSA at 300 μL / well for 1 h at room temperature; (4) Sample addition: After washing the plate, dilute the HpaA antigen protein to 10 ng / mL and add 100 μL / well to the corresponding sample well. Incubate at room temperature for 2 h. (5) Addition of enzyme-labeled antibody: After washing the plate, add HRP-labeled monoclonal antibody at a rate of 0.5 μg / mL, 100 μL / well, and incubate at room temperature for 1 h; (6) Color development: After washing the plate, add TMB color development solution and incubate at room temperature for 20 min; (7) Termination: Add 50 μL of termination solution to each well and immediately measure the absorbance at a wavelength of 450 nm.

[0059] 4. Hybridoma cell screening

[0060] Hybridoma cell lines with well-matched antibodies and good cell condition were selected for subsequent large-scale production of monoclonal antibodies.

[0061] Example 2

[0062] This embodiment provides a process for preparing an anti-HpaA monoclonal antibody. The process includes the following steps: 1. Hybridoma cell culture (1) Revive hybridoma cells and adjust the cell density to 4×10⁻⁶. 5 Cells / mL were placed in T25 cell culture flasks, with culture medium of 90% RPMI 1640 + 10% FBS (RPMI 1640: catalog number C11875500BT-1, Gibco; FBS: catalog number Z7186FBS-500, Zeta Life), and culture conditions of 37℃, 8% CO2.

[0063] (2) After 2-3 days of resuscitation, the cells were well translocated and transferred to 125mL Erlenmeyer flasks for further culture, with the cell density adjusted to 4×10⁻⁶. 5 Cells / mL, culture medium was 90% RPMI 1640 + 10% FBS, culture conditions: 37℃, 8% CO2, 100 rpm / min.

[0064] 2. Mouse immunization

[0065] (1) Sensitization: Prepare 8-week-old female BALB / C mice and inject them with 0.4 mL / mouse of ascites-specific adjuvant (product number: KX0210048-10, Biodragon).

[0066] (2) Induction: 15 days after sensitization, the hybridoma cells in good culture condition were incubated at 1×10⁻⁶. 6 Cells / injected into the peritoneum of mice only.

[0067] (3) Ascites collection: About 7 days after cell injection, when the mice show obvious abdominal distension, difficulty eating, difficulty walking and dull fur, the mice are sacrificed and ascites is extracted once.

[0068] (4) Storage: Centrifuge the ascites at 3000 rpm for 10 min, take the middle colorless and transparent layer, and freeze it at -20℃.

[0069] 3. Monoclonal antibody purification

[0070] Antibody purification was performed using a protein A affinity chromatography column. The most prevalent form of mouse monoclonal antibody is IgG. Utilizing the specific and reversible binding between biomolecules, protein A can bind to the Fc fragment of most IgG molecules with high specificity and high affinity. The specific procedures are as follows: (1) Select a protein A affinity chromatography column to purify monoclonal antibodies in ascites fluid; (2) Equilibration: Equilibrate the chromatography column with 5 CV equilibration buffer (20 mM PB + 0.15 M NaCl, pH 7.0) until the effluent conductivity temperature and pressure are stable; (3) Sample loading: The ascites fluid sample was diluted 3 times with equilibration buffer before loading; (4) Washing: Wash the chromatography column with 5-10 CV of equilibration buffer at a flow rate of 0.5 mL / min; (5) System: Wash the chromatography column with 70% elution buffer (0.1 M glycine, pH 3.0) and 30% equilibration buffer. Collect 2 mL / tube, label the peak tube, and these are the monoclonal antibody elution tubes.

[0071] (6) Rinsing: Rinse the chromatography column with 5-10 CV pure water, then rinse with 5 CV alkaline solution (0.1M NaOH), rinse with 5-10 CV pure water to remove the alkaline solution, and finally fill the chromatography column with 5 CV column preservation solution (20% ethanol) before unloading and storing.

[0072] (7) Dialysis: The antibody solution obtained by elution was dialyzed three times in 1000 times the volume of PBS (pH=7.4) solution to obtain purified anti-HpaA monoclonal antibody.

[0073] 4. Monoclonal antibody concentration and purity detection

[0074] 4.1 Detection of antibody concentration using the BCA method

[0075] Monoclonal antibody protein concentrations were determined using a BCA protein assay kit (catalog number: KGB2101-250, Kaiji Biotechnology). The purified MM07 antibody concentration was 11.51 mg / mL, and the MM14 antibody concentration was 9.01 mg / mL.

[0076] 4.2 Antibody purity detection by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

[0077] Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates proteins in the sample based on their molecular weight, allowing them to be separated in the gel. The purity of the target antibody is then verified by staining with Coomassie brilliant blue. The detection results are as follows: Figure 2 As shown.

[0078] The specific operating procedure is as follows: (1) Prepare a 10% SDS-PAGE gel; (2) Adjust the amount of monoclonal antibody loaded to approximately 5 μg; (3) After running the stacking gel at a constant voltage of 80V, adjust the voltage to 120V and continue electrophoresis until the bottom of the separating gel is reached, then turn off the power. (4) Immerse the gel in Coomassie brilliant blue staining solution and incubate on a shaker at room temperature for 2 hours; (5) Recover Coomassie brilliant blue dye solution, decolorize with pure water, and then transfer it to the operating table for photography.

[0079] 5. Antigen-specific detection of monoclonal antibodies

[0080] Western blotting was used to detect the specificity of monoclonal antibodies. Purified antigen protein HpaA, unpurified BL21 whole-cell lysate containing HpaA, and irrelevant proteins in 10% FBS were subjected to electrophoresis, membrane transfer, and other procedures. The mixture was then incubated with specific monoclonal antibodies, followed by enzyme-labeled secondary antibody incubation and imaging. The specificity of the bands was then observed. The detection results are as follows: Figure 3 As shown.

[0081] The specific operating procedure is as follows: (1) Gum preparation: 10% separating gum and 5% stacking gum; (2) Sample loading: The sample includes 0.1 mg / mL HpaA antigen, unpurified BL21 whole bacterial lysate containing HpaA, and 10% FBS of irrelevant protein; (3) Electrophoresis: 80V constant voltage for stacking gel, 120V constant voltage for separating gel; (4) Transfer: PVDF membrane was used for transfer under the following conditions: constant current of 250mA for 60min; (5) Sealing: Use 5% skim milk powder and seal at room temperature for 2 hours; (6) Primary antibody incubation: Use purified monoclonal antibody solution (20 ng / mL) and incubate overnight on a shaker at 4°C; (7) Secondary antibody incubation: After washing the membrane, incubate at room temperature for 1 hour with RP-conjugated Goat Anti-Mouse IgG (H+L) solution (catalog number: SA00001-1, Proteintech); (8) Development: After washing the membrane, develop it using the BeyoECL Star ultrasensitive ECL chemiluminescence kit (catalog number: P0018AS, Beyotime) and take a picture.

[0082] 6. Monoclonal antibody titer detection

[0083] The titer of the purified monoclonal antibody pair was detected using an indirect ELISA. A positive result was defined as an OD450 > 0.1 in the test sample well and greater than twice that of the negative control well. The titer of the antibody was determined by the highest dilution yielding a positive reaction. The test results are as follows: Figure 4 , 5 Among them, the MM07 monoclonal antibody titer was 50 pg / mL, and the MM14 monoclonal antibody titer was 125 pg / mL.

[0084] The specific operating procedure is as follows: (1) Coating: Dilute the antigen or antibody with coating buffer to a final concentration of 5ug / mL, 100ul / well, and coat overnight at 4℃; wash the plate; (2) Blocking: Add 3% BSA blocking solution, 300 μL / well, incubate at 37℃ for 2 h; wash plate; (3) Sample addition: After diluting the monoclonal antibody with dilution buffer, add 100 μL / well to each microplate, incubate at room temperature for 1 h, and then wash the plate; (4) Add detection antibody: Dilute HRP-conjugated Goat Anti-Mouse IgG (H+L) detection antibody at a ratio of 1:5000, 100 μL / well, and incubate at 37°C for 30 min; (5) Color development: Add 100 μL of TMB substrate display solution to each well and incubate at room temperature in the dark for 15 min; (6) Termination: Add 50 μL of ELISA stop solution, gently shake to mix, and perform detection within 10 min.

[0085] Example 3

[0086] In this embodiment, the monoclonal antibodies obtained through the above screening were sequenced.

[0087] MM07 and MM14 cell lines were cultured on a large scale, and 5 × 10⁶ cells were collected. 6 Cells were placed in centrifuge tubes, the supernatant was aspirated, Trizol preservation solution was added, and the tubes were shipped on dry ice to Suzhou Genewiz Biotechnology Co., Ltd. for hybridoma cell sequencing. The sequencing results are as follows: 1. MM07 antibody (1) Heavy chain sequence information Heavy chain full-length base sequence: including the nucleotide sequence shown in SEQ ID NO: 2.

[0088] CAGGTCCAGCTGCAGCAGTCTGGGCCTGAGCTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGTTCCGGCTACACATTCACTGATTATGCTATACACTGGGTGAAGCAGAGTCATGCAAAGAGTCTAGAGTGGATTGGAATTATTAGTACTTACTATGGAAACACAAACTACAACCAGAA GTTTAAGGGCAAGGCCACAATGACTGTAGACAAATCCTCCAGCACAGCCTATATGGAACTTGCCAGATTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGAGGAGCCCCCAACTATAGGTACGACGACCCGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 2)

[0089] Heavy chain full-length amino acid sequence: including the amino acid sequence shown in SEQ ID NO: 3.

[0090] QVQLQQSGPELVRPGVSVKISCKGSGYTFTDYAIHWVKQSHAKSLEWIGIISTYYGNTNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGAPNYRYDDPAWFAYWGQGTLVTVSA (SEQ ID NO: 3)

[0091] The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. Heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 4. Heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 5. Heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

[0092] (3) Heavy chain CDR1: GYTFTDYA (SEQ ID NO: 4)

[0093] (4) Heavy chain CDR2: ISTYYGNT (SEQ ID NO: 5)

[0094] (5) Heavy chain CDR3: ARGAPNYRYDDPAWFAY (SEQ ID NO: 6)

[0095] (6) Light chain sequence information

[0096] Light chain full-length base sequence: including the nucleotide sequence shown in SEQ ID NO: 7.

[0097] GATATTGTGATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTGGAGAATCAGTTTCCATCTCCTGCAGGTCTACTAAGAGTCTCCTATAAGGATGGGAAGACATACTTGAATTGGTTTCTGCAGAGACCAGGACAATCTCCTCAGCTCCTGATCTATTTGATGTC CACCCGTGCATCAGGAGTCTCAGACCGGTTTAGTGGCAGTGGGTCAGGAACAGATTTCACCCTGGAAATCAGTAGAGTGAAGGCTGAGGATGTGGGTGTGTATTACTGTCAACAACTTGTTGATTTTCCGTACACGTTCGGATGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO:7)

[0098] Light chain full-length amino acid sequence: including the amino acid sequence shown in SEQ ID NO: 8.

[0099] DIVITQDELSNPVTSGESVSISCRSTKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYCQQLVDFPYTFGWGTKLEIK (SEQ ID NO: 8)

[0100] The light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3. Light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 9. Light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 10. Light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 11.

[0101] Light chain CDR1: KSLLYKDGKTY (SEQ ID NO: 9)

[0102] Light chain CDR2: LMS (SEQ ID NO: 10)

[0103] Light chain CDR3: QQLVDFPYT (SEQ ID NO: 11)

[0104] 2. MM14 monoclonal antibody

[0105] (1) Heavy chain sequence information

[0106] The full-length base sequence of the heavy chain variable region includes the nucleotide sequence shown in SEQ ID NO: 12.

[0107] GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTTCTCCATCACCAGTGGTTATTCCTGGCACTGGATCCGGCAATTTCCAGGAAACAAACTGGAATGCATGGGCAACATACACTACGGTGGTAGCACTAACTAC AATCCGTCTCTCAAAAGTCGAATCTCTATCACTCTAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTCTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGGCCATGGTAACTACAATCCTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 12)

[0108] The full-length amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 13.

[0109] DVQLQESGPDLVKPSQSLSLTCTVTGFSITSGYSWHWIRQFPGNKLECMGNIHYGGSTNYNPSLKSRISITLDTSKNQFFLQLNSLTTEDTATYYCARGHGNYNPLDYWGQGTSVTVSS (SEQ ID NO: 13)

[0110] The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. Heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 14. Heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 15. Heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 16.

[0111] Heavy chain CDR1: GFSITSGYS (SEQ ID NO: 14)

[0112] Heavy chain CDR2: IHYGGST (SEQ ID NO: 15)

[0113] Heavy chain CDR3: ARGHGNYNPLDY (SEQ ID NO: 16)

[0114] (2) Light chain sequence information

[0115] The full-length base sequence of the light chain variable region includes the nucleotide sequence shown in SEQ ID NO: 17.

[0116] GACATTTGTGATGTCGCAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGATTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGTAATCAAAAGATCTACTTGGCCTGGTACCAGCGGAAACCAGGGCAGTCTCCTAAACTGCTGATTTATTGGGCA TCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGCAATATTATAGCTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 17)

[0117] The full-length amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 18.

[0118] DIVMSQSPSSLAVSVGEKITMSCKSSQSLLYSSNQKIYLAWYQRKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIK (SEQ ID NO: 18)

[0119] The light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3. Light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 19. Light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 20. Light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 21.

[0120] Light chain CDR1: QSLLYSSNQKIY (SEQ ID NO: 19)

[0121] Light chain CDR2: WAS (SEQ ID NO: 20)

[0122] Light chain CDR3: QQYYSYPYT (SEQ ID NO: 21)

[0123] Example 4

[0124] This embodiment provides a fluorescent immunochromatographic test strip for detecting HpaA antigen. The fluorescent immunochromatographic test strip is prepared using MM07 and MM14 antibodies. The immunofluorescence test strip is as follows... Figure 6 As shown in Figure A.

[0125] The preparation process of this test strip includes the following steps: 1. Sample pad treatment MM14 monoclonal antibody solution (0.5 mg / mL) and goat anti-mouse secondary antibody solution (0.5 mg / mL) were sprayed onto the NC membrane as the detection line (T line) and control line (C line), respectively. After spraying, the membrane was dried in an oven at 37°C overnight and then stored in a dry environment for later use.

[0126] 2. Bonding pad treatment

[0127] (1) Activation of fluorescent microspheres: Take a certain amount of time-resolved fluorescent microspheres (Europium) (product number: PE200AC, Maifu Micro-Nano) and resuspend them in coupling buffer, add carbodiimide (such as EDC) and N-hydroxysuccinimide (such as NHS), and incubate at room temperature in the dark for 30 minutes with shaking.

[0128] (2) Antibody conjugation: Mix the activated microspheres with an appropriate amount of MM07 antibody (antibody dosage: 50 μg / mg microspheres) and incubate at room temperature in the dark for 2 hours.

[0129] (3) Blocking: After centrifugation, blocking solution is added to the coupled fluorescent microspheres and incubated at room temperature in the dark for 1 hour to block the unreacted active groups on the microspheres.

[0130] (4) Spraying: Spray the prepared MM07 fluorescent microsphere conjugate onto the conjugate pad.

[0131] 3. Test strip assembly

[0132] (1) Attach each component to the PVC base plate in the order of sample pad, conjugate pad, NC membrane and absorbent pad. Ensure that there is a 1-2 mm overlap between each component to ensure the continuity of the chromatography process.

[0133] (2) Use a strip cutter to cut the test strips into 4mm wide strips.

[0134] (3) The cut test strips are put into plastic cartridges to prepare fluorescent immunochromatographic test strips.

[0135] 4. Detection of HpaA antigen using fluorescent immunochromatographic test strips

[0136] MM07 and MM14 antibodies were used in the detection of HpaA antigen using fluorescent immunochromatographic test strips. Preliminary results show that HpaA antigen concentrations as low as 0.5 ng / mL can be detected. Figure 6 B.

[0137] (1) Sample dilution: HpaA antigen protein was used as the detection target. HpaA was diluted with PBS to a concentration of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, and a blank control. (2) Sample loading: The diluted HpaA antigen solution was added to the fluorescent immunochromatographic test strip at a rate of 80 μL / test, and allowed to stand in the dark for 15 minutes. (3) Reading value: The fluorescence value is read when the fluorescence immunochromatographic test strip is loaded onto the dry fluorescence immunoassay analyzer. The excitation light is selected at 365nm and the filter is selected at 615±20nm to receive the emitted light.

[0138] As can be seen from the above embodiments, the monoclonal antibody provided by the present invention has good specificity and sensitivity against HpaA antigen.

[0139] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody pair targeting Helicobacter pylori adhesin A, characterized in that, The antibody pair is MM07 antibody and MM14 antibody; The heavy chain variable region of the MM07 antibody includes heavy chain CDR1-3, and the amino acid sequence of heavy chain CDR1-3 is shown in SEQ ID NO: 4-6; The light chain variable region of the MM07 antibody includes light chain CDR1-3, and the amino acid sequence of light chain CDR1-3 is shown in SEQ ID NO: 9-11; The heavy chain variable region of the MM14 antibody includes heavy chain CDR1-3, and the amino acid sequence of heavy chain CDR1-3 is shown in SEQ ID NO: 14-16; The light chain variable region of the MM14 antibody includes light chain CDR1-3, and the amino acid sequence of light chain CDR1-3 is shown in SEQ ID NO: 19-21.

2. The antibody pair according to claim 1, characterized in that, The heavy chain variable region of the MM07 antibody includes an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO: 3 and has the ability to bind to HpaA. The light chain variable region of the MM07 antibody includes an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO: 8 and has the ability to bind to HpaA. The heavy chain variable region of the MM14 antibody includes an amino acid sequence as shown in SEQ ID NO: 13, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO: 13 and has the ability to bind to HpaA. The light chain variable region of the MM14 antibody includes an amino acid sequence as shown in SEQ ID NO: 18, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO: 18 and has the ability to bind to HpaA.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody pair as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The sequence encoding the variable region of the heavy chain of the nucleic acid molecule for the MM07 antibody includes the nucleotide sequence shown in SEQ ID NO. 2; The sequence encoding the variable region of the light chain of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO. 7; The sequence encoding the variable region of the heavy chain of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO. 12; The sequence encoding the variable region of the light chain of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO.

17.

5. A biomaterial, characterized in that, The biological material is a biological material expressing the monoclonal antibody pair as described in claim 1 or 2; the biological material includes a carrier or cells.

6. The use of the monoclonal antibody pair of claim 1 or 2, or the monoclonal antibody pair encoded by the nucleic acid molecule of claim 3 or 4, or the monoclonal antibody pair expressed by the biological material of claim 5, in the detection of HpaA, or in the preparation of products for detecting HpaA.

7. The application according to claim 6, characterized in that, The application is in the detection of Helicobacter pylori, or in the preparation of products for detecting Helicobacter pylori.

8. A test strip or reagent kit, characterized in that, The test strip or kit includes the monoclonal antibody pair of claim 1 or 2, or the monoclonal antibody pair encoded by the nucleic acid molecule of claim 3 or 4, or the monoclonal antibody pair expressed by the biological material of claim 5.

9. The test strip or reagent kit according to claim 8, characterized in that, The MM14 antibody is used as the coating antibody, and the MM07 antibody is used as the labeling antibody.

10. The use of the test strip or kit according to claim 8 or 9 in the detection of Helicobacter pylori.

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