Monoclonal antibody against ureB protein and its preparation method and application

Abstract

The application discloses an anti-UreB protein monoclonal antibody and a preparation method and application thereof, and relates to the technical field of monoclonal antibodies.The anti-UreB protein monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.5.The anti-UreB protein monoclonal antibody has excellent affinity effect on UreB protein, is used in combination with an anti-UreB protein antibody (8-B5-E6 antibody), can specifically recognize non-overlapping epitopes of the UreB protein, has no steric hindrance, and has a binding activity reaching an ng / mL level, can be used for double-antibody sandwich method detection, and has an excellent application prospect in the field of Helicobacter pylori detection.

Description

Technical Field

[0001] This application relates to the field of monoclonal antibody technology, and in particular to a monoclonal antibody against UreB protein, its preparation method and application. Background Technology

[0002] Helicobacter pylori (H. pylori) is a Gram-negative spirochete, a major pathogen of chronic gastritis and peptic ulcers, and is classified as a Group 1 carcinogen by the International Agency for Research on Cancer, closely related to the development of gastric cancer. With a global infection rate exceeding 50%, establishing efficient and convenient methods for detecting H. pylori is crucial for early screening and prevention. Currently, diagnostic methods for H. pylori are mainly divided into invasive and non-invasive categories: invasive methods such as endoscopic biopsy and rapid urease test, while considered the clinical gold standard, are invasive, have poor patient compliance, and are costly, making them unsuitable for large-scale screening; non-invasive methods such as urea breath test and fecal antigen test do not require endoscopy, but are affected by specialized equipment, diet, and gastrointestinal diseases, and both may produce false negative results due to recent antibiotic use. In recent years, the detection of Helicobacter pylori in saliva has provided a new sample source for the non-invasive detection of this bacterium. However, the abundance of Helicobacter pylori in saliva is extremely low, which places extremely high demands on the sensitivity of the detection method.

[0003] Related saliva-based detection technologies include conventional PCR, time-resolved fluorescence lateral flow immunoassay (TRFIA), and electrochemical immunosensor detection. While conventional PCR offers high sensitivity, it requires genomic DNA extraction, relies on specialized equipment and operation, and is time-consuming, making rapid on-site detection difficult. TRFIA primarily uses lanthanide-labeled antibodies, leveraging their long fluorescence lifetime (μs-ms level) combined with time-gated detection technology to effectively eliminate interference from short-lived background fluorescence such as serum autofluorescence. Electrochemical immunosensors rely on antigen-antibody specific binding and electrocatalytic signal amplification to detect target analytes. The latter two methods combine high sensitivity and ease of operation, but currently lack high-affinity antibody pairs against Helicobacter pylori-specific antigens, and the antibody recognition epitope characteristics are unclear, resulting in insufficient detection specificity and stability, limiting clinical applications. Urease B subunit (UreB), as a conserved outer membrane virulence factor of Helicobacter pylori, is an ideal antigen target for the detection of this bacterium. However, current antibody materials are difficult to meet the core requirements of high-sensitivity immunoassay platforms for affinity and pairing synergy.

[0004] Therefore, developing highly specific and high-affinity monoclonal antibody pairs targeting UreB, and constructing a portable, highly sensitive point-of-care testing platform adapted to saliva samples based on these pairs, is of great significance for meeting the medical needs of primary care clinics and large-scale screening. This will address the problems of insufficient sensitivity, complex operation, and reliance on large equipment in existing non-invasive testing methods, enabling non-invasive, rapid, and accurate detection of Helicobacter pylori. Summary of the Invention

[0005] In view of this, embodiments of this application provide a monoclonal antibody against UreB protein, its preparation method, and its application.

[0006] A first aspect of this application provides a monoclonal antibody against UreB protein or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

[0007] The heavy chain variable region contains complementarity-determining regions HCDR1, HCDR2 and HCDR3;

[0008] The amino acid sequence of HCDR1 is shown in SEQ ID NO.2;

[0009] The amino acid sequence of HCDR2 is shown in SEQ ID NO.3;

[0010] The amino acid sequence of HCDR3 is shown in SEQ ID NO.4;

[0011] The light chain variable region contains complementary determination regions LCDR1, LCDR2, and LCDR3;

[0012] The amino acid sequence of the LCDR is shown in SEQ ID NO.6;

[0013] The amino acid sequence of the LCDR is shown in SEQ ID NO.7;

[0014] The amino acid sequence of the LCDR is shown in SEQ ID NO.8.

[0015] In this application, the light chain variable region (VL) or heavy chain variable region (VH) of the monoclonal antibody consists of a "framework" region separated by three "complementarity-determining regions" or "CDRs". The framework regions are used to align the CDRs that specifically bind to the antigenic epitopes. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework (FR) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; and the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0016] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is any of the following sequences:

[0017] A1) The amino acid sequence as shown in SEQ ID NO.1;

[0018] A2) An amino acid sequence with the same function obtained by substituting and / or deleting and / or adding amino acid residues as shown in SEQ ID NO.1.

[0019] In some embodiments, the amino acid sequence of the light chain variable region of the monoclonal antibody is any of the following sequences:

[0020] B1) The amino acid sequence as shown in SEQ ID NO.5;

[0021] B2) An amino acid sequence with the same function obtained by substituting and / or deleting and / or adding amino acid residues as shown in SEQ ID NO.5.

[0022] In some embodiments, the antigen-binding fragment is selected from one or more of Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, and single-chain antibodies.

[0023] In this application, the "Fab fragment" is a heterodimer formed by the heavy chain Fd and the intact light chain linked by disulfide bonds, containing only one antigen-binding site. The aforementioned heavy chain Fd refers to approximately half of the H chain portion of Fab (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0024] In this application, the “Fab’ fragment” contains a light chain and a heavy chain portion that includes the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby enabling the formation of interchain disulfide bonds between the two heavy chains of the two Fab’ fragments to form the F(Fab’)2 molecule.

[0025] In this application, the “F(ab')2 fragment” contains two light chains and two heavy chains comprising portions of a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0026] In this application, "Fv fragment" refers to a vector containing VH and VL genes that can be constructed separately, co-transfected into cells to express them separately, and then assembled into a functional Fv antibody; alternatively, a stop codon can be set between VH and VL in the vector to express two small protein fragments separately, which can then be bound together by non-covalent bonds to form an Fv antibody (Fv fragment).

[0027] In this application, "single-chain antibody (ScFv)" refers to a polypeptide obtained by linking a light chain variable region and a heavy chain variable region. This polypeptide can spontaneously fold into its native conformation, maintaining the specificity and affinity of Fv.

[0028] In some embodiments, the monoclonal antibody or its antigen-binding fragment may further include a heavy chain constant region (CH) and a light chain constant region (CL). The heavy chain constant region may be selected from the heavy chain constant regions of IgG, IgA, IgM, IgD, and IgE. The heavy chain constant region may also be selected from CH1, Fc, and CH3 domains. The light chain constant region may be selected from Kappa (κ) and lambda (λ) type light chain constant regions. The heavy chain constant region and light chain constant region may be derived from humans or non-human mammals (such as mice, rats, guinea pigs, rabbits, sheep, camels, etc.).

[0029] In some embodiments, the monoclonal antibody is a human antibody, a mouse antibody, or a chimeric antibody. The monoclonal antibody of this application can be prepared as a bispecific or multispecific antibody.

[0030] The second aspect of this application provides biological material related to a monoclonal antibody against UreB protein or its antigen-binding fragment as described in any of the first aspects, wherein the biological material is any one of C1) to C5):

[0031] C1) A nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof of the anti-UreB protein as described in any one of the first aspects;

[0032] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0033] C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0034] C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3);

[0035] C5) A transgenic animal cell line containing the nucleic acid molecule described in C1), or a transgenic animal cell line containing the expression cassette described in C2), or a transgenic animal cell line containing the recombinant vector described in C3), or a transgenic animal cell line containing the recombinant microorganism described in C4).

[0036] In some embodiments, the recombinant vector may be selected from prokaryotic expression vectors (including but not limited to Escherichia coli expression vectors such as BL21 series expression cells, M15 expression cells, Top10 expression cells and Origamai series expression cells) and eukaryotic expression vectors (including but not limited to yeast expression vectors such as X33 cells, GS115 cells and SMD1168 cells, insect cell expression vectors such as Sf21 cells, Sf-9 cells and Hi-5 cells, mammalian cell expression vectors such as HET293 cells and CHO cells).

[0037] A third aspect of this application provides a composition for detecting UreB protein, comprising a detection antibody and a capture antibody, wherein the detection antibody is a monoclonal antibody against UreB protein as described in any of the first aspects or an antigen-binding fragment thereof.

[0038] In some embodiments, the capture antibody is an anti-UreB protein antibody or its antigen-binding fragment, wherein the anti-UreB protein antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0039] The heavy chain variable region contains complementarity-determining regions HCDR1, HCDR2 and HCDR3;

[0040] The amino acid sequence of HCDR1 is shown in SEQ ID NO.10;

[0041] The amino acid sequence of HCDR2 is shown in SEQ ID NO.11;

[0042] The amino acid sequence of HCDR3 is shown in SEQ ID NO.12;

[0043] The light chain variable region contains complementary determination regions LCDR1, LCDR2, and LCDR3;

[0044] The amino acid sequence of the LCDR is shown in SEQ ID NO.14;

[0045] The amino acid sequence of the LCDR is shown in SEQ ID NO.15;

[0046] The amino acid sequence of the LCDR is shown in SEQ ID NO.16.

[0047] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-UreB protein antibody is any of the following sequences:

[0048] D1) The amino acid sequence as shown in SEQ ID NO.9;

[0049] D2) An amino acid sequence with the same function obtained by substituting and / or deleting and / or adding amino acid residues as shown in SEQ ID NO.9.

[0050] In some embodiments, the amino acid sequence of the light chain variable region of the anti-UreB protein antibody is any of the following sequences:

[0051] E1) The amino acid sequence as shown in SEQ ID NO.13;

[0052] E2) An amino acid sequence with the same function obtained by substituting and / or deleting and / or adding amino acid residues as shown in SEQ ID NO.13.

[0053] The fourth aspect of this application provides the use of a monoclonal antibody against UreB protein or an antigen-binding fragment thereof as described in any of the first aspects, or a composition for detecting UreB protein as described in any of the third aspects, in the preparation of a Helicobacter pylori detection kit.

[0054] In some embodiments, the kits include, but are not limited to, chemiluminescent immunoassay kits, electrochemical immunosensor kits, enzyme-linked immunosorbent assay kits, immunoprecipitation assay kits, immunoblotting assay kits, immunochromatographic assay kits (such as time-resolved fluorescent microsphere lateral flow immunochromatographic assay kits), flow cytometry kits, immunohistochemical assay kits, colloidal gold immunoassay kits, or fluorescent immunoassay kits.

[0055] In some embodiments, the test samples of the kit include, but are not limited to, saliva samples, environmental samples, blood samples (such as whole blood, plasma, serum), tissue samples, cell samples, and fecal samples.

[0056] The fifth aspect of this application provides a time-resolved fluorescent microsphere lateral flow immunochromatographic kit, the raw materials of which include a detection probe, the detection probe comprising a monoclonal antibody against UreB protein as described in any of the first aspects or an antigen-binding fragment thereof.

[0057] In some embodiments, the time-resolved fluorescent microsphere lateral flow immunochromatographic assay kit includes a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, and a PVC base plate, wherein the conjugate pad is coated with the detection probe.

[0058] In some embodiments, the nitrocellulose membrane is coated with a capture antibody (as a detection line, i.e., a T line), the capture antibody being an anti-UreB protein antibody or its antigen-binding fragment, the anti-UreB protein antibody comprising a heavy chain variable region and a light chain variable region, wherein:

[0059] The heavy chain variable region contains complementarity-determining regions HCDR1, HCDR2 and HCDR3;

[0060] The amino acid sequence of HCDR1 is shown in SEQ ID NO.10;

[0061] The amino acid sequence of HCDR2 is shown in SEQ ID NO.11;

[0062] The amino acid sequence of HCDR3 is shown in SEQ ID NO.12;

[0063] The light chain variable region contains complementary determination regions LCDR1, LCDR2, and LCDR3;

[0064] The amino acid sequence of the LCDR is shown in SEQ ID NO.14;

[0065] The amino acid sequence of the LCDR is shown in SEQ ID NO.15;

[0066] The amino acid sequence of the LCDR is shown in SEQ ID NO.16.

[0067] In some embodiments, the nitrocellulose membrane is coated with goat anti-mouse IgG (as a control line, i.e., the C line).

[0068] A sixth aspect of this application provides an electrochemical immunosensor kit, the raw materials for which include detection antibodies and capture antibodies, wherein the detection antibodies include monoclonal antibodies against UreB protein as described in any of the first aspects or antigen-binding fragments thereof.

[0069] In some embodiments, the electrochemical immunosensor kit includes a capture antibody-modified electrode and a detection antibody conjugate, wherein:

[0070] The surface of the capture antibody-modified electrode is modified with an anti-UreB protein antibody or its antigen-binding fragment, wherein the anti-UreB protein antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0071] The heavy chain variable region contains complementarity-determining regions HCDR1, HCDR2 and HCDR3;

[0072] The amino acid sequence of HCDR1 is shown in SEQ ID NO.10;

[0073] The amino acid sequence of HCDR2 is shown in SEQ ID NO.11;

[0074] The amino acid sequence of HCDR3 is shown in SEQ ID NO.12;

[0075] The light chain variable region contains complementary determination regions LCDR1, LCDR2, and LCDR3;

[0076] The amino acid sequence of the LCDR is shown in SEQ ID NO.14;

[0077] The amino acid sequence of the LCDR is shown in SEQ ID NO.15;

[0078] The amino acid sequence of the LCDR is shown in SEQ ID NO.16.

[0079] In some embodiments, the electrode is a gold screen-printed electrode.

[0080] In some embodiments, the capture antibody modified electrode is prepared by sequentially treating a gold screen-printed electrode with cystamine solution, activating it with EDC / NHS, modifying it with capture antibodies, and blocking it with BSA.

[0081] In some embodiments, the detection antibody conjugate comprises an antibody portion and a conjugated portion of a monoclonal antibody against UreB protein as described in any of the first aspects or an antigen-binding fragment thereof.

[0082] In some embodiments, the coupling portion is a detectable tag.

[0083] In some embodiments, the detectable markers include, but are not limited to, enzymes (such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, etc.), chemiluminescent reagents (such as acridine esters, acridine sulfonamides, luminol and its derivatives, ruthenium derivatives, etc.), and fluorescent dyes (such as AMCA, FITC, CFSE, GFP, DAPI, 7-AAD, Hoechst 33342, Pacific Blue, PE, PE-TR, PE-Cy7, PE-Cy5, PI, PerCP-Cy5.5, APC, APC-CY7, APC-H7, V500, Alexa). 700, BV605, BV480, BV785, BV510, BV711, BV421, etc.), near-infrared dyes (such as cyanine dyes, BODIPY dyes, rhodamine dyes, squaric acid dyes, porphyrin dyes, etc.), radionuclides (such as 125I, 18F, 11C, 99mTc, 123I, etc.), biotin, nanoparticles for magnetic resonance imaging, quantum dots for magnetic resonance imaging, magnetic materials (such as magnetic beads, nanoparticles containing gadolinium complexes, superparamagnetic iron oxide nanoparticles), and colloidal gold, etc.

[0084] In some embodiments, the electrochemical immunosensor kit further includes at least one of the following: sample diluent, electrochemical detection buffer, TMB chromogenic solution, portable electrochemical workstation, positive control, and negative control.

[0085] This application includes at least the following beneficial effects:

[0086] This application describes the purification of high-purity Helicobacter pylori UreB recombinant protein through prokaryotic expression. Two hybridoma cell lines capable of secreting anti-UreB specific monoclonal antibodies were obtained through animal immunization, cell fusion, and subclonal screening. The optimal antibody combination was then screened through antibody pairing and molecular docking verification. This antibody combination specifically recognizes the non-overlapping epitopes of the UreB protein, has no steric hindrance, and exhibits high binding activity. All concentrations are at the ng / mL level, suitable for double-antibody sandwich assays. Furthermore, this antibody combination exhibits excellent sensitivity and specificity for Ureb detection, is compatible with various immunoassay platforms, and possesses well-defined CDR and complete variable region sequences, allowing for humanization and large-scale production, laying the foundation for subsequent product industrialization. Attached Figure Description

[0087] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0088] Figure 1 This is an SDS-PAGE image of the purified UreB protein from this application.

[0089] Figure 2 The results of the antibody-paired sandwich ELISA assay of this application are shown in Figure A, where A is a heatmap and B is the affinity test result of Ureb monoclonal antibody to Ureb protein.

[0090] Figure 3 This is a transmission electron microscope image of the TRFM-LFIA detection probe of this application. Scale bar: 100 nm.

[0091] Figure 4 The results show the physical performance characterization of the TRFM-LFIA detection probe in this application, where A is the particle size, B is the zeta potential, C is the fluorescence excitation spectrum, and D is the fluorescence emission spectrum.

[0092] Figure 5 The results show the sensitivity test results of the TRFM-LFIA kit in this application, where A is the test result and B is the standard curve.

[0093] Figure 6 This is the specific detection result of the TRFM-LFIA kit in this application.

[0094] Figure 7 The results are shown in the TRFM-LFIA kit for clinical sample testing, where A and B are the fluorescence signal detection results and their statistical results for each sample, and C is the AUC curve.

[0095] Figure 8 The figures show the electrode modification characterization of the electrochemical immunosensor of this application, where A-D are transmission electron microscope observations, E is a CV curve, and F is an EIS Nyquist plot.

[0096] Figure 9 The graphs show the analytical performance of the electrochemical immunosensor of this application, where A is the chronocurrent curve, B is the standard curve, C is the specific detection result, and D is the repeatability detection result.

[0097] Figure 10 The results are the clinical sample detection results based on the electrochemical immunosensor of this application, where A and B are the chronocurrent detection results and their statistical results for each sample, and C is the AUC curve. Detailed Implementation

[0098] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0099] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0100] Example 1: Preparation of monoclonal antibodies against Helicobacter pylori UreB protein

[0101] 1. Prokaryotic expression and purification of UreB recombinant protein

[0102] The cDNA gene sequence of Helicobacter pylori ureB was found on NCBI (NCBI reference sequence: NC_008086.1), and after codon optimization, it was inserted into the prokaryotic expression vector pColdII to construct the recombinant plasmid pColdII-ureB.

[0103] The recombinant plasmid was transformed into BL21 (DE3) competent cells containing the pTf16 molecular chaperone, and the expression of the 6×His-tagged UreB recombinant protein was induced by IPTG and L-arabinose; then the recombinant protein was purified using a Ni-NTA affinity chromatography column, and SDS-PAGE verified that the protein purity was above 95% (e.g., Figure 1 As shown in the figure, high-purity UreB antigen was obtained after dialysis and used for animal immunization.

[0104] 2. Animal immunization

[0105] The purified UreB recombinant protein was emulsified with Freund's complete adjuvant at a 1:1 volume ratio and injected subcutaneously / intradermally at multiple sites, with each mouse receiving 50 μg of antigen. A booster immunization was performed every 14 days, with Freund's incomplete adjuvant emulsified with the antigen at a 1:1 ratio, at a dose of 30 μg per mouse, for a total of 3 immunizations. Seven days after the second booster immunization, tail blood was collected from the mice, and serum titers were detected using indirect ELISA. Mice with the highest titers were selected for the final sprint immunization.

[0106] The experimental method for detecting serum titer using indirect ELISA is as follows:

[0107] (1) Coating: The purified Ureb protein was diluted to 2 μg / mL with 0.05M CBS buffer and added to a 96-well microplate at 100 μL / well. The plate was incubated at 4°C overnight to allow the antigen to be fully adsorbed.

[0108] (2) Washing: Discard the liquid in the ELISA plate, add 200 μL of PBST buffer to each well, and wash on a horizontal shaker for 5 minutes. Repeat this washing process 3 times to ensure complete removal of unbound antigen molecules. Finally, pat dry any remaining liquid on absorbent paper.

[0109] (3) Blocking: Add 100 μL of 5% skim milk blocking solution per well and block overnight at 4°C.

[0110] (4) Washing the plate: Same as step (2).

[0111] (5) Primary antibody incubation: Mouse tail blood was serially diluted with PBS at dilution ratios of 1:1000, 1:10000 to 1:320000, with three replicates for each dilution as positive controls; a negative control group (pre-immunization mouse serum) and a blank control group (PBS buffer) were also prepared and diluted in the same way. All samples were added to the ELISA plate and incubated at 37°C for 1 hour.

[0112] (6) Washing the plate: Same as step (2).

[0113] (7) Secondary antibody incubation: Horseradish peroxidase-labeled Goat Anti Mouse IgG (H+L) antibody was diluted in PBS at a ratio of 1:10000, and 100 μL / well was added to the microplate and incubated at 37°C for 60 minutes.

[0114] (8) Washing the plate: Same as step (2).

[0115] (9) Color development: Add 100 μL of color development solution to each well and react at room temperature in the dark for 10-15 minutes.

[0116] (10) Termination of reaction: Add 50 μL of 2M sulfuric acid to each well to terminate the reaction. Within 5 minutes after termination, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at a wavelength of 450 nm.

[0117] The test results showed that the mouse serum titer could reach 1:320,000; at each dilution gradient, the OD of positive serum was... 450 Values ​​and negative serum OD 450 The ratios (P / N) were all >2.1, meeting the positive criteria, indicating that the mice had produced high-titer, high-specificity anti-Ureb antibodies after immunization, which could be used for subsequent hybridoma fusion experiments.

[0118] 3. Cell fusion

[0119] Spleen Acquisition: 100 μg of the purified Ureb protein was dissolved in physiological saline and injected intraperitoneally into mice for immunization. Blood was collected from the eyeballs 72 hours later, and the mice were euthanized by cervical dislocation. The euthanized mice were sterilized by immersion in 75% alcohol for 5 minutes. The spleen was dissected and removed in a biosafety cabinet and placed in a 100 mm sterile culture dish. It was rinsed with RPMI-1640 medium to remove impurities and obtain the spleen.

[0120] Preparation of spleen cell suspension: The spleen was cut into pieces through a 200-mesh cell sieve, ground with sufficient culture medium, and the suspension was collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded. 3 mL of erythrocyte lysis buffer was added to the precipitate and the mixture was allowed to stand for 3 minutes. 10 mL of culture medium was added to stop the lysis and the mixture was centrifuged. The precipitate was washed twice with culture medium, centrifuged, and the supernatant was discarded.

[0121] Cell fusion: After counting, spleen cells and SP2 / 0 myeloma cells were mixed at a ratio of 5:1-10:1, centrifuged and the supernatant was discarded. The precipitate was dispersed by hand shaking. The precipitate was placed in preheated sterile water and incubated. 50% PEG1450 solution preheated to 37°C was slowly added dropwise (1 mL / min, 60±5 seconds). After gentle shaking and mixing, the mixture was allowed to stand for 1 minute. 1 mL of preheated culture medium was added dropwise at the same rate, followed by 10 mL over 2 minutes. After standing for 10 minutes, the mixture was centrifuged at 800 rpm for 10 minutes and the supernatant was discarded. The precipitate was resuspended in preheated 15% FBS complete culture medium containing 2×HAT and seeded into 96-well feeder cell plates. The cells were cultured in a 37°C, 5% CO2 incubator to obtain cell lines for subcloning.

[0122] 4. Positive cell subcloning

[0123] The subcloning procedure is as follows:

[0124] Cell lines to be subcloned were resuspended in the original culture medium and transferred to sterile 1.5 mL centrifuge tubes to prepare cell suspension. After cell counting, the cells were diluted to 150 cells / 10 mL using preheated HT (2×) RPMI 1640 complete medium and seeded at 100 μL / well into 96-well plates lined with feeder cells. Clones were labeled with their numbers and incubated at 37 ℃ in a 5% CO2 incubator.

[0125] After three rounds of cell screening and subcloning, four hybridoma cell lines that can stably secrete monoclonal antibodies against Ureb protein were successfully obtained and labeled as 2-C2-F5, 3-A5-B8, 7-E2-D6 and 8-B5-E6 hybridoma cell lines, respectively.

[0126] 5. Monoclonal antibody acquisition

[0127] Eight-week-old Balb / c mice were pre-sensitized by intraperitoneal injection of 0.5 mL of liquid paraffin. Seven days later, each mouse was injected intraperitoneally with 1 mL of 1×10⁻⁶ paraffin.6 The above hybridoma cell suspension was prepared at a concentration of cells / mL; mouse ascites was collected 7-10 days later as a crude extract of monoclonal antibodies.

[0128] The crude extract was purified using Protein G affinity chromatography to adapt the Fc fragment of the mouse monoclonal antibody. The specific steps were as follows: the collected hybridoma ascites was filtered through a 0.45 μm filter to remove impurities and then loaded onto a Protein G affinity chromatography column; the column was first equilibrated with PBS, and then the bound antibody was eluted with 0.1 M glycine buffer (pH 2.2), and the elution buffer was immediately neutralized with Tris-HCl (pH 9.0); finally, the neutralized antibody was dialyzed against 0.01 M PBS buffer, and the antibody concentration was determined by NanoDrop. The antibody was stored at -20 °C, and the purity was verified by SDS-PAGE.

[0129] Based on the purification method described above, monoclonal antibodies 2-C2-F5, 3-A5-B8, 7-E2-D6 and 8-B5-E6 were obtained respectively.

[0130] Example 2 Antibody Pairing and Affinity Detection

[0131] The four antibodies obtained above (2-C2-F5 antibody, 3-A5-B8 antibody, 7-E2-D6 antibody and 8-B5-E6 antibody) were combined in pairs to form detection antibodies / capture antibodies, and the affinity for 10 ng / mL UreB protein was detected by double-antibody sandwich ELISA.

[0132] Heatmap of antibody-paired double-antibody sandwich ELISA results is shown below. Figure 2 As shown in A and Table 1.

[0133] Table 1:

[0134]

[0135] The results showed that when 2-C2-F5 was the detection antibody and 8-B5-E6 was the capture antibody, the signal-to-background ratio was the highest, making it the optimal antibody pair.

[0136] Furthermore, the affinity test results of the two Ureb monoclonal antibodies for Ureb protein are as follows: Figure 2 As shown in B in the figure. The test results showed that the affinity of the 2-C2-F5 antibody to Ureb protein was -13.8 kcal / mol, and the affinity of the 8-B5-E6 antibody to Ureb protein was -12.1 kcal / mol.

[0137] The above results indicate that, based on the principle of the double-antibody sandwich method, using 2-C2-F5 antibody as the detection antibody and 8-B5-E6 antibody as the capture antibody can achieve the best detection effect. It can be applied to the preparation of reagents or kits for detecting Helicobacter pylori UreB, such as the time-resolved fluorescent microsphere lateral flow immunochromatography (TRFM-LFIA) kit and the electrochemical immunosensor kit.

[0138] Example 3 Sequencing analysis of monoclonal antibodies

[0139] 1. cDNA preparation

[0140] Total RNA was extracted from the two cell lines (2-C2-F5 and 8-B5-E6) determined in the above examples, and cDNA was obtained by reverse transcription using RNA as a template.

[0141] 2. Acquisition of light and heavy chain genes

[0142] Amplification was performed using specific primers designed based on the conserved sequences of the heavy chain variable region (VH) and light chain variable region (VL). The amplified antibody variable region fragments were then recovered. The recovered fragments were sequenced to obtain the nucleic acid sequences of the heavy and light chain variable regions of the two monoclonal antibodies. Analysis of the sequencing results shows the amino acid sequences of the heavy chain variable region and the light chain variable region of the 2-C2-F5 antibody (denoted as UrebmAb1) and the 8-B5-E6 antibody (denoted as Ureb mAb2), respectively, with the CDR regions underlined.

[0143] (1) 2-C2-F5 antibody (Ureb mAb1)

[0144] Heavy chain VH:

[0145] IELTQSPAFMSASPGEKVTMTC SGGDLVK MHWYQQKSDTSPKRWIY TYGMSWVRQT RFRGSGSGTSYSLTISNMEAYDAATYYC EWVATISNG FVYFGGGTKLEIK (SEQ ID NO. 1).

[0146] The amino acid sequences of the heavy chain variable regions HCDR1, HCDR2 and HCDR3 are as follows: SGGDLVK (SEQ ID NO.2), TYGMSWVRQT (SEQ ID NO.3), and EWVATISNG (SEQ ID NO.4).

[0147] Light chain VL:

[0148] VQYQESGAELVKPGASVKLSCTASGFNIKDTYMH GRFTIS LEWIGIDPANGNTKATIT ADTSSNTAYL QLSSLTSEDTA MYYCARHPAYYDAMDYW GWGAGTTVTVSS (SEQ ID NO. 5).

[0149] The amino acid sequences of the light chain variable regions LCDR1, LCDR2 and LCDR3 are as follows: GRFTIS (SEQ ID NO.6), ADTSSNTAYL (SEQ ID NO.7), and MYYCARHPAYYDAMDYW (SEQ ID NO.8).

[0150] (2) 8-B5-E6 antibody (Ureb mAb2)

[0151] Heavy chain VH:

[0152] IELTQSPAFMSASPGEKVTMTC SASDTA MHMHWYQQKSDTSPKRWIYRID PASGDTSYDP KFQGKLASGVPVRFRGSGSGTSYSLTISNMEAEDAATYYC ESPTYY FVYFGGGTKLEIK (SEQ ID NO. 9).

[0153] The amino acid sequences of the heavy chain variable regions HCDR1, HCDR2 and HCDR3 are as follows: SASDTA (SEQ ID NO.10), PASGDTSYDP (SEQ ID NO.11), ESPTYY (SEQ ID NO.12).

[0154] Light chain VL:

[0155] VQLQESGAELVKPGASVKLSCTASRS DQQLEYGNT YLEMHWVKQRPEQGLEW IGKVTAYLRFS KATITADTSSNTAYLQLSSLTSEDTA VQGVAESHVPAT WGAGTTVTVSS (SEQ ID NO. 13).

[0156] The amino acid sequences of the light chain variable regions LCDR1, LCDR2 and LCDR3 are as follows: DQQLEYGNT (SEQ ID NO.14), IGKVTAYLRFS (SEQ ID NO.15), and VQGVAESHVPAT (SEQ ID NO.16).

[0157] Example 4: Application in the preparation of a time-resolved fluorescent microsphere lateral flow immunochromatographic kit

[0158] 1. Preparation of Time-Resolved Fluorescent Microsphere Lateral Flow Immunochromatographic Kit

[0159] (1) Preparation of fluorescent microsphere-antibody complex:

[0160] 0.05% (w / w) time-resolved fluorescent microspheres were activated by EDC / NHS and then coupled with the screened detection antibody 2-C2-F5 at 37℃ for 2 hours. Non-specific binding sites were blocked with 1% BSA and 0.24% ethanolamine. After centrifugation and washing, the microspheres were resuspended in a storage buffer containing 5% trehalose and stored at 4℃ in the dark to obtain the detection probe.

[0161] Transmission electron microscopy image of the TRFM-LFIA detection probe as shown below. Figure 3 As shown, the results indicate that both TRFMs and TRFM-Ab1 exhibit excellent monodispersity and uniform particle size distribution.

[0162] Furthermore, dynamic light scattering (DLS) measurements showed that after conjugation with antibody Ab1, the particle size significantly increased from 255.3 nm to 396 nm. Figure 4 (A in the figure) confirmed that the antibody had been successfully immobilized on the TRFM surface. Zeta potential analysis showed that the surface charge of TRFM-Ab1 was significantly enhanced compared to the original TRFMs. Figure 4 The result of B in the image further corroborates the effective conjugation of the antibody. Spectral characterization results ( Figure 4 C in 4 and D in 4) indicate that the positions of the maximum excitation peak (360 nm) and emission peak (615 nm) of the material did not change after coupling, and still maintained a Stokes shift of up to 255 nm, indicating that its spectral characteristics were well preserved and that it could significantly reduce background interference by effectively separating the excitation light and the emission signal.

[0163] (2) Test strip assembly:

[0164] The detection antibody (detection probe) of the labeled time-resolved fluorescent microspheres was coated onto the conjugate pad. The sample pad was treated with Tris buffer containing 2% BSA and 5% trehalose and dried at 37°C. Then, the selected capture antibody 8-B5-E6 (1.5 mg / mL) and goat anti-mouse IgG (0.2 mg / mL) were coated onto the T and C lines of the nitrocellulose membrane, respectively, with a line spacing of 5 mm, and dried overnight at 37°C. The conjugate pad, sample pad, nitrocellulose membrane, and absorbent pad were overlapped by 2 mm and pasted onto a PVC base plate. The resulting strips were cut into 3 mm wide test strips, i.e., time-resolved fluorescent microsphere lateral flow immunochromatographic test strips (i.e., TRFM-LFIA test strips). The test strips were sealed in aluminum foil bags (containing desiccant) and stored at 25±2°C for later use.

[0165] (3) Kit components:

[0166] The time-resolved fluorescent microsphere lateral flow immunochromatographic kit of this embodiment includes the TRFM-LFIA test strip prepared above, sample diluent, positive control, negative control, and fluorescence reader.

[0167] 2. Sensitivity and specificity detection

[0168] (1) Sensitivity detection

[0169] Take 1 μL of UreB protein at different concentrations (0, 0.195, 0.39, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 ng / mL) and mix with 48 μL of sample dilution solution. Add 1 μL of fluorescent microsphere-detection antibody complex and incubate at room temperature for 3 min. Add 35 μL of reaction solution to the sample pad of the test strip and incubate at room temperature for 15 min. Detect the fluorescence intensity of the T line and C line using a fluorescence reader and calculate the UreB protein concentration according to the standard curve. If there is no fluorescence at the C line, the detection is invalid; if the fluorescence intensity of the T line is higher than the threshold, it is considered positive.

[0170] Test results as follows Figure 5 As shown, the results indicate that the detection limit of the TRFM-LFIA kit constructed based on this antibody is as low as 91 pg / mL.

[0171] (2) Specific detection

[0172] Take 1 μL of UreB protein, other Helicobacter pylori virulence factors (CagA, VacA) or common interfering substances in saliva (such as glucose, ascorbic acid, uric acid and cholesterol), and mix them with 48 μL of sample dilution solution. Add 1 μL of fluorescent microsphere-detection antibody complex and incubate at room temperature for 3 min. Take 35 μL of reaction solution and add it to the sample pad of the test strip. Incubate at room temperature for 15 min. Use a fluorescence reader to detect the fluorescence intensity of the T line and C line. Calculate the UreB protein concentration according to the standard curve. If there is no fluorescence in the C line, the detection is invalid. If the fluorescence intensity of the T line is higher than the threshold, it is considered positive.

[0173] Test results as follows Figure 6 As shown, the time-resolved fluorescent microsphere lateral flow immunochromatographic kit constructed based on the antibody of this invention has excellent specificity and no cross-reactivity with other virulence factors of Helicobacter pylori (CagA, VacA) and common interfering substances in saliva (glucose, ascorbic acid, uric acid, etc.).

[0174] 3. Clinical sample testing performance

[0175] In this case, 21 saliva samples were collected (11 of which were Helicobacter pylori positive and 10 were healthy controls). The TRFM-LFIA kit described above was used for testing to evaluate its detection performance. The specific method is as follows:

[0176] Mix 1 μL of saliva sample with 48 μL of sample diluent, add 1 μL of fluorescent microsphere-detection antibody complex, and incubate at room temperature for 3 min; add 35 μL of reaction solution to the sample pad of the test strip and incubate at room temperature for 15 min; use a fluorescence reader to detect the fluorescence intensity of the T line and C line, and calculate the UreB protein concentration according to the standard curve; if there is no fluorescence on the C line, the detection is invalid, and if the fluorescence intensity of the T line is higher than the threshold, it is determined to be positive for Helicobacter pylori.

[0177] Test results as follows Figure 7 As shown, the UreB protein of Helicobacter pylori is specifically detected only in positive saliva samples. Figure 7 A and Figure 7 (B in the text). Receiver operating characteristic (ROC) curve analysis showed excellent diagnostic performance, with an area under the curve (AUC) of 1.0 (…). Figure 7 The C in the figure indicates that the detection method can completely distinguish between Helicobacter pylori positive saliva samples and negative saliva samples.

[0178] The above results demonstrate that the time-resolved fluorescent microsphere lateral flow immunochromatographic assay kit constructed based on the antibody of this invention has excellent sensitivity and specificity, and is simple to operate and has good reproducibility, making it suitable for saliva sample detection.

[0179] Example 5: Application in the preparation of electrochemical immunosensor kits

[0180] 1. Preparation of Electrochemical Immunosensor Kit

[0181] (1) Electrode modification

[0182] The gold screen printed electrode (AuSPE) was ultrasonically cleaned with 75% ethanol and dried with nitrogen. 20 mM cystamine solution was added and incubated at room temperature for 1 h to form a self-assembled monolayer. After EDC / NHS activation, 50 μg / mL capture antibody 8-B5-E6 was added and incubated at 37℃ for 1 h. Non-specific sites were blocked with 2.5% BSA to obtain the capture antibody modified electrode.

[0183] Characterization diagram of the antibody-modified electrode surface as shown in the figure. Figure 8 As shown, the electrode surface modification layer is uniform, and CV and EIS verification confirm the successful electrode modification.

[0184] (2) Preparation of detection probe

[0185] The detection antibody 2-C2-F5 was conjugated with HRP to prepare the HRP-2-C2-F5 conjugate, which was stored at 4°C for later use.

[0186] (3) Kit composition

[0187] The electrochemical immunosensor kit of this embodiment includes the above-mentioned capture antibody modified electrode, HRP-labeled detection antibody, sample diluent, electrochemical detection buffer, TMB chromogenic solution, portable electrochemical workstation, positive control, and negative control.

[0188] 2. Specificity, sensitivity, and repeatability of the test

[0189] Take 1 μL of different concentrations of standard (UreB protein), mix with 49 μL of sample dilution buffer, and drop onto the surface of the capture antibody modified electrode. Incubate at 37°C for 1 h, and wash with PBST. Add HRP-labeled detection antibody, incubate at 37°C for 1 h, and wash with PBST. Add TMB chromogenic solution, and use a portable electrochemical workstation to detect the chronocurrent signal at -100 mV (vs. Ag / AgCl). Record the current value at 240 s. Calculate the UreB protein concentration according to the standard curve. A current value higher than the threshold is considered positive.

[0190] The detection results of the UreB standard based on the constructed electrochemical reagent kit using this antibody are as follows: Figure 9As shown, the linear range of this kit is 0.1-1000 ng / mL, R²=0.99, and the limit of detection is 38.3 pg / mL; the specificity detection interference signal is <2%, and there is no cross-reaction with other virulence factors of Helicobacter pylori (CagA, VacA) and common interfering substances in saliva (glucose, ascorbic acid, uric acid, etc.). Figure 9 As shown in C), the coefficient of variation (CV) of the detection results for the nine electrodes was less than 6%, indicating that the electrochemical kit has excellent repeatability and stability.

[0191] 3. Clinical sample testing

[0192] Twenty-one saliva samples were collected (11 positive for Helicobacter pylori and 10 healthy controls), and the above-mentioned electrochemical immunosensor kit was used for detection. The specific method is as follows:

[0193] Mix 1 μL of saliva sample with 49 μL of sample dilution solution, add the mixture to the surface of the capture antibody-modified electrode, incubate at 37°C for 1 h, and wash with PBST; add HRP-labeled detection antibody, incubate at 37°C for 1 h, and wash with PBST; add TMB chromogenic solution, and use a portable electrochemical workstation to detect the chronocurrent signal at -100 mV (vs. Ag / AgCl), record the current value at 240 s, calculate the UreB protein concentration according to the standard curve, and determine the positive result of Helicobacter pylori if the current value is higher than the threshold.

[0194] The results are as follows Figure 10 As shown, A and B represent the chronoamperometry curve detection and statistical results, and C represents the specificity detection results. The electrochemical immunosensor kit showed a significant difference in current signal amplitude between the positive and negative saliva sample groups. Figure 10 In the A category, the response of positive saliva samples was significantly increased (p < 0.05). Figure 10 (B in the text). Receiver operating characteristic (ROC) curve analysis showed excellent diagnostic performance, with an area under the curve (AUC) of 1.0 (…). Figure 10 The C in the figure indicates that the detection method can completely distinguish between Helicobacter pylori positive saliva samples and negative saliva samples.

[0195] In summary, this invention provides a monoclonal antibody against UreB protein, its preparation method, and its application. The anti-Helicobacter pylori UreB monoclonal antibody (2-C2-F5 / 8-B5-E6) obtained by this invention exhibits high specificity and high affinity, specifically recognizing non-overlapping epitopes of the UreB protein, without steric hindrance, and with high binding activity. All concentrations are at the ng / mL level, suitable for double-antibody sandwich assays, helping to address the issues of insufficient antibody specificity and epitope overlap in existing technologies. Furthermore, the detection reagents based on this anti-Helicobacter pylori UreB monoclonal antibody pair (2-C2-F5 / 8-B5-E6) are compatible with various immunoassay platforms, exhibiting strong compatibility. The antibody's CDR region and complete variable region sequences are clearly defined, allowing for humanization and large-scale production, laying the foundation for subsequent product industrialization.

[0196] The above is a detailed description of the preferred embodiments of this application, but this application is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of this application, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A monoclonal antibody against UreB protein or its antigen-binding fragment, characterized in that, It includes a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region contains complementarity-determining regions HCDR1, HCDR2 and HCDR3; The amino acid sequence of HCDR1 is shown in SEQ ID NO.2; The amino acid sequence of HCDR2 is shown in SEQ ID NO.3; The amino acid sequence of HCDR3 is shown in SEQ ID NO.4; The light chain variable region contains complementary determination regions LCDR1, LCDR2, and LCDR3; The amino acid sequence of the LCDR is shown in SEQ ID NO.6; The amino acid sequence of the LCDR is shown in SEQ ID NO.7; The amino acid sequence of the LCDR is shown in SEQ ID NO.

8.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is any of the following sequences: A1) The amino acid sequence as shown in SEQ ID NO.1; A2) An amino acid sequence with the same function obtained by substituting and / or deleting and / or adding amino acid residues as shown in SEQ ID NO.

1.

3. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody is any of the following sequences: B1) The amino acid sequence as shown in SEQ ID NO.5; B2) An amino acid sequence with the same function obtained by substituting and / or deleting and / or adding amino acid residues as shown in SEQ ID NO.

5.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragment is selected from one or more of Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, and single-chain antibody.

5. Biomaterials relating to a monoclonal antibody against UreB protein as described in any one of claims 1 to 4, or an antigen-binding fragment thereof, characterized in that, The biomaterial is any one of C1) to C5): C1) A nucleic acid molecule encoding a monoclonal antibody against the anti-UreB protein as described in any one of claims 1 to 4, or an antigen-binding fragment thereof; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3); C5) A transgenic animal cell line containing the nucleic acid molecule described in C1), or a transgenic animal cell line containing the expression cassette described in C2), or a transgenic animal cell line containing the recombinant vector described in C3), or a transgenic animal cell line containing the recombinant microorganism described in C4).

6. A composition for detecting UreB protein, characterized in that, It includes a detection antibody and a capture antibody, wherein the detection antibody is a monoclonal antibody against UreB protein as described in any one of claims 1 to 4, or an antigen-binding fragment thereof.

7. The composition according to claim 6, characterized in that, The capture antibody is an anti-UreB protein antibody or its antigen-binding fragment, wherein the anti-UreB protein antibody comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region contains complementarity-determining regions HCDR1, HCDR2 and HCDR3; The amino acid sequence of HCDR1 is shown in SEQ ID NO.10; The amino acid sequence of HCDR2 is shown in SEQ ID NO.11; The amino acid sequence of HCDR3 is shown in SEQ ID NO.12; The light chain variable region contains complementary determination regions LCDR1, LCDR2, and LCDR3; The amino acid sequence of the LCDR is shown in SEQ ID NO.14; The amino acid sequence of the LCDR is shown in SEQ ID NO.15; The amino acid sequence of the LCDR is shown in SEQ ID NO.

16.

8. The use of the monoclonal antibody against UreB protein or its antigen-binding fragment as described in any one of claims 1 to 4, or the composition for detecting UreB protein as described in any one of claims 6 to 7, in the preparation of a Helicobacter pylori detection kit.

9. A time-resolved fluorescent microsphere lateral flow immunochromatographic assay kit, characterized in that, The raw materials for preparation include a detection probe, which comprises a monoclonal antibody against UreB protein as described in any one of claims 1 to 4, or an antigen-binding fragment thereof.

10. An electrochemical immunosensor kit, characterized in that, The raw materials for preparation include detection antibodies and capture antibodies, wherein the detection antibodies include monoclonal antibodies against UreB protein as described in any one of claims 1 to 4, or antigen-binding fragments thereof.

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