Anti-helicobacter pylori monoclonal antibody and preparation and application thereof

Abstract

The application discloses an anti-helicobacter pylori monoclonal antibody and a preparation and application thereof, and belongs to the technical field of medical detection. The antibody is prepared by using inactivated helicobacter pylori ATCC 43504 whole bacteria as an immunogen, immunizing a New Zealand white rabbit, and then performing single B cell sorting, limited dilution, gene cloning, plasmid construction and eukaryotic cell expression. The antibody has a specific CDR core sequence, high specificity and high affinity, can accurately recognize helicobacter pylori specific antigens, and effectively avoids cross reactions with other bacteria or tissue impurities. The antibody can be used for detecting helicobacter pylori, and provides a new tool and method for pathological diagnosis of helicobacter pylori infection and related digestive system diseases.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically relating to anti-Helicobacter pylori monoclonal antibodies and their preparation and application. Background Technology

[0002] Helicobacter pylori ( Helicobacter pylori , H. pylori (This is a Gram-negative, microaerophilic spiral-shaped bacterium that colonizes the gastric mucosa of the human body, with a global colonization rate of more than half of the population.) H. pylori It is a major pathogenic factor of chronic active gastritis and peptic ulcers, and is classified as a Group I carcinogen by the World Health Organization. It is closely related to the development and progression of gastric mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer; therefore, accurate and efficient detection is crucial. H. pylori Infections are of vital clinical significance for the prevention, early diagnosis, clinical treatment, and gastric cancer screening of related diseases.

[0003] at present, H. pylori Diagnostic techniques are broadly categorized into invasive and non-invasive methods, both with significant limitations. Among non-invasive tests, the urea breath test (UBT) is the clinical "gold standard," but its results are easily affected by proton pump inhibitors, antibiotics, and other drugs, and it cannot provide pathological information about the gastric mucosa. The fecal antigen test (SAT) is suitable for large-scale screening, but also lacks pathological information. Serological tests only reflect infection history and cannot determine current infection. Among invasive tests, the accuracy of the rapid urease test (RUT) is affected by bacterial load and sampling site, resulting in a high false-negative rate. Microbial culture is time-consuming (3-5 days) and technically demanding, making it only suitable for drug sensitivity testing. Molecular biological methods (such as PCR) are costly and require stringent laboratory conditions and personnel, making routine implementation difficult.

[0004] Traditional histological staining (such as HE staining and Giemsa staining) is fundamental to pathological diagnosis, allowing observation of the relationship between bacterial morphology and tissue. However, it suffers from low specificity, and false negative / false positive results are common when bacterial counts are low or morphology is atypical (e.g., spheroidization). It also places a high degree of reliance on the pathologist's experience. Immunohistochemistry (IHC), utilizing the principle of specific antigen-antibody binding, overcomes the specificity limitations of traditional staining and has become... H. pylori While IHC is an important detection method, existing antibodies used for IHC detection suffer from problems such as insufficient affinity, poor specificity, and limited detection sensitivity. Furthermore, the reagent formulations of the accompanying kits lack optimization, resulting in cumbersome detection procedures, poor color development, and excessive non-specific staining, making it difficult to meet the clinical demand for rapid and accurate detection.

[0005] Therefore, developing a highly specific and affinity anti-Helicobacter pylori antibody, as well as an immunohistochemical detection kit containing this antibody with optimized formulation and excellent detection performance, is a technical problem that urgently needs to be solved in the field of medical testing. Summary of the Invention

[0006] 1. Purpose of the invention

[0007] The primary objective of this invention is to provide a monoclonal antibody against Helicobacter pylori. This antibody is prepared by immunizing New Zealand white rabbits with inactivated whole Helicobacter pylori ATCC 43504 bacteria as an immunogen, followed by single B cell sorting, limiting dilution, gene cloning, plasmid construction, and eukaryotic cell expression. It has a specific CDR core sequence, high specificity and high affinity, and can accurately recognize Helicobacter pylori-specific antigens, effectively avoiding cross-reactions with other bacteria or tissue impurities.

[0008] Another objective of this invention is to provide an immunohistochemical detection kit containing the anti-Helicobacter pylori monoclonal antibody. The kit optimizes the precise formulation and working concentration of each reagent, enabling rapid detection, high sensitivity, and good color development, thus achieving accurate qualitative detection of Helicobacter pylori in gastric tissue.

[0009] Another object of the present invention is to provide the application of the antibody and kit for detecting Helicobacter pylori, providing new tools and methods for the pathological diagnosis of Helicobacter pylori infection and related digestive system diseases, and filling the gaps in existing detection technologies.

[0010] 2. Technical Solution

[0011] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0012] As a first aspect of the present invention, the present invention provides an anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, which can specifically recognize Helicobacter pylori-specific antigens, such as characteristic antigens like UreA (29 kDa), UreB (66 kDa), CagA (120 kDa), or VacA (95 kDa), without cross-reactivity, and can recognize Helicobacter pylori with low bacterial quantity and atypical morphology. It comprises three light chain CDRs: CDR-L1, CDR-L2, and CDR-L3; and three heavy chain CDRs: CDR-H1, CDR-H2, and CDR-H3; the CDRs are defined according to Kabat numbering; the amino acid sequences of each CDR are as follows:

[0013] CDR-L1: QSSESVYGNNRLA (SEQ ID NO.19);

[0014] CDR-L2: LASTLAS (SEQ ID NO. 20);

[0015] CDR-L3: AGGYSGGIGV (SEQ ID NO. 21);

[0016] CDR-H1: SNAMT (SEQ ID NO.22);

[0017] CDR-H2: TLYTSGGASYASWAKG (SEQ ID NO. 23);

[0018] CDR-H3: SLYAFDP (SEQ ID NO. 24).

[0019] Furthermore, the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein: the amino acid sequence of the light chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3.

[0020] Furthermore, the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment further includes a light chain constant region and a heavy chain constant region, wherein: the amino acid sequence of the light chain constant region is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.7.

[0021] Furthermore, the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment comprises a light chain and a heavy chain, wherein the amino acid sequence of the light chain is shown in SEQ ID NO.9 and the amino acid sequence of the heavy chain is shown in SEQ ID NO.11.

[0022] Furthermore, the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, the light chain and heavy chain of the antibody also include a leader sequence, as those skilled in the art know, the leader sequence is not related to the final antigen binding, but is used to be responsible for protein transport, etc., wherein: the amino acid sequence of the light chain leader sequence is as shown in SEQ ID NO.13, and the amino acid sequence of the heavy chain leader sequence is as shown in SEQ ID NO.14.

[0023] Furthermore, the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment comprises a light chain containing a leader sequence and a heavy chain containing a leader sequence, wherein: the amino acid sequence of the light chain containing the leader sequence is as shown in SEQ ID NO. 15, and the amino acid sequence of the heavy chain containing the leader sequence is as shown in SEQ ID NO. 16.

[0024] Furthermore, the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab)'2 fragment, a single-chain Fv protein (scFv), or a disulfide-stabilized Fv protein (dsFv).

[0025] As a second aspect of the present invention, the present invention also provides a nucleic acid encoding the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment.

[0026] Furthermore, the nucleic acids encoding the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment include:

[0027] The nucleotide sequence shown in SEQ ID NO.2 encodes the light chain variable region of the above-mentioned anti-Helicobacter pylori monoclonal antibody, and the nucleotide sequence shown in SEQ ID NO.4 encodes the heavy chain variable region of the above-mentioned anti-Helicobacter pylori monoclonal antibody.

[0028] Furthermore, the nucleic acids encoding the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment include:

[0029] The nucleotide sequence shown in SEQ ID NO.10 encodes the light chain of the above-mentioned anti-Helicobacter pylori monoclonal antibody, and the nucleotide sequence shown in SEQ ID NO.12 encodes the heavy chain of the above-mentioned anti-Helicobacter pylori monoclonal antibody.

[0030] Furthermore, the nucleic acids encoding the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment include:

[0031] The nucleotide sequence shown in SEQ ID NO.17 encodes the light chain containing the leader sequence of the above-mentioned anti-Helicobacter pylori monoclonal antibody, and the nucleotide sequence shown in SEQ ID NO.18 encodes the heavy chain containing the leader sequence of the above-mentioned anti-Helicobacter pylori monoclonal antibody.

[0032] As a third aspect of the present invention, the present invention also provides a recombinant expression vector containing the nucleic acid encoding the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, which can express the anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment.

[0033] Furthermore, the recombinant expression vector includes a pcDNA3.4 plasmid containing the nucleic acid encoding the anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment.

[0034] As a fourth aspect of the present invention, the present invention also provides a recombinant expression cell comprising the above-described recombinant expression vector or the above-described nucleic acid encoding an anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, which can express the anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment.

[0035] Furthermore, the recombinant expression cells mentioned above include the 293 cell line or the CHO cell line.

[0036] As a fifth aspect of the present invention, the present invention also provides the use of the above-described nucleic acid, recombinant expression vector or recombinant expression cell encoding the anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment in the preparation of the anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment.

[0037] As a sixth aspect of the present invention, the present invention also provides a method for preparing the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, comprising:

[0038] Recombinant expression cells were obtained by transfecting cells with the above-mentioned recombinant expression vector and then culturing the recombinant expression cells; alternatively, the above-mentioned recombinant expression cells could be cultured directly. The supernatant was collected and purified to obtain an anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment.

[0039] As a seventh aspect of the present invention, the present invention also provides a horseradish peroxidase (HRP)-conjugated anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment thereof, which is prepared by conjugating horseradish peroxidase with the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment via an amino-conjugation method. The conjugated anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment retains its specific binding ability to Helicobacter pylori antigen, and can achieve efficient color development of the antigen-antibody binding site through the enzymatic reaction of HRP.

[0040] Furthermore, the above-mentioned horseradish peroxidase-conjugated anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment has a conjugation molar ratio of antibody:horseradish peroxidase = 1:(2~4).

[0041] As an eighth aspect of the present invention, the present invention also provides the use of the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, horseradish peroxidase-conjugated anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, nucleic acid encoding anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, recombinant expression vector or recombinant expression cell in detecting Helicobacter pylori or preparing products for detecting Helicobacter pylori.

[0042] Furthermore, the above-mentioned detection of Helicobacter pylori includes the use of any one or more of the following methods to detect Helicobacter pylori: immunohistochemistry, immunocytochemistry (immunofluorescence), flow cytometry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). All of the above detection methods rely on the binding of anti-Helicobacter pylori monoclonal antibodies or their antigen-binding fragments to Helicobacter pylori antigens.

[0043] Furthermore, the above-mentioned detection of Helicobacter pylori includes the detection of Helicobacter pylori in paraffin-embedded gastric tissue, including gastritis tissue, gastric cancer tissue, normal gastric mucosa tissue, and gastric mucosa intestinal metaplasia tissue.

[0044] Furthermore, the aforementioned products for detecting Helicobacter pylori include any one or more of the following: reagent kits, antibody conjugates, antibody chips, etc.

[0045] Furthermore, the above-mentioned kits include flow cytometry kits, immunocytochemistry kits, or immunohistochemistry kits (immunohistochemical kits).

[0046] As a ninth aspect of the present invention, the present invention also provides a kit for detecting Helicobacter pylori, the kit comprising the above-mentioned anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, horseradish peroxidase-conjugated anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, nucleic acid encoding the anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, a recombinant expression vector or recombinant expression cell, wherein the anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment binds to Helicobacter pylori antigen to detect Helicobacter pylori.

[0047] Furthermore, the above-mentioned kit for detecting Helicobacter pylori is an immunohistochemical detection kit for detecting Helicobacter pylori.

[0048] Furthermore, the aforementioned immunohistochemical detection kit for Helicobacter pylori also includes: blocking solution, endogenous peroxidase blocking solution, antigen retrieval solution, DAB chromogenic solution, hematoxylin counterstaining solution, and washing solution.

[0049] Furthermore, the steps for using the above-mentioned immunohistochemical detection kit for Helicobacter pylori detection include: slide preparation, dewaxing and hydration, antigen retrieval, endogenous peroxidase blocking, blocking, incubation with primary antibody (anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment or horseradish peroxidase-conjugated anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment), DAB staining, hematoxylin counterstaining, dehydration and mounting, and microscopic examination; wherein the slide preparation temperature is 63~65℃ for 1 h, the primary antibody incubation conditions are 37℃ for 1 h, and the DAB staining conditions are room temperature for 1~2 min.

[0050] 3. Technical Effects

[0051] This invention utilizes gene cloning and antibody engineering techniques to obtain a rabbit monoclonal antibody against Helicobacter pylori with a specific CDR core sequence, and develops an immunohistochemical detection kit containing this antibody. Compared with existing technologies, it has the following significant advantages:

[0052] (1) High antibody specificity and high affinity: The rabbit monoclonal antibody against Helicobacter pylori of the present invention has a clear CDR core protection sequence, which can specifically bind to one or more characteristic antigens of key proteins of Helicobacter pylori, without species cross-reaction and non-specific binding. It can effectively identify Helicobacter pylori with low bacterial quantity and atypical morphology (such as spherical deformation), and has high detection sensitivity, thus solving the technical problems of poor specificity and low sensitivity of existing antibodies.

[0053] (2) Optimized reagent kit formulation and excellent detection performance: The immunohistochemical detection kit of the present invention optimizes the precise formulation and working concentration of each reagent. The coupling ratio of the core HRP-conjugated anti-Helicobacter pylori rabbit monoclonal antibody has been screened. The ratio of the chromogenic solution to be prepared and used immediately can achieve efficient and rapid chromogenic development. The blocking solution and the blocking solution can effectively reduce background staining and non-specific reactions. The detection specificity and sensitivity of the kit are significantly better than those of existing similar products.

[0054] (3) Rapid detection and simple operation: The kit has a standardized detection process, and the entire detection process can be completed within 3 hours. Compared with traditional microbial culture (3-5 days), the detection time is greatly shortened. The operation steps are simple and do not require complicated experimental equipment. It is suitable for routine use in clinical laboratories and effectively reduces the detection cost.

[0055] (4) Objective and accurate interpretation of results: After the kit is tested, the brownish-yellow positive color signal contrasts sharply with the blue cell nucleus. Pathologists can directly observe and interpret the results through a microscope without relying on extensive clinical experience. This can effectively reduce human interpretation errors and make the test results objective and reliable.

[0056] (5) Preservation of tissue morphology information: While achieving accurate detection of Helicobacter pylori, the kit can completely preserve the morphological information of gastric tissue, allowing clear observation of the distribution, density and interaction of Helicobacter pylori with gastric mucosal cells, providing valuable pathological evidence for the etiology judgment, disease assessment and clinical treatment plan formulation of Helicobacter pylori infection-related diseases.

[0057] (6) Wide range of applications: The antibodies and kits of the present invention can be widely used for the detection of Helicobacter pylori in paraffin-embedded gastric tissue, as well as for the pathological diagnosis of Helicobacter pylori-related diseases such as chronic active gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer. They can also be used for early screening of high-risk groups for gastric cancer. They have important application prospects and promotional value in the fields of clinical pathological testing and diagnosis and treatment of gastroenterological diseases. Attached Figure Description

[0058] Figure 1 These are images showing the immunohistochemical detection results of rabbit immune serum from this invention, with the left image being F2671 and the right image being F2672.

[0059] Figure 2 This is an image showing the immunohistochemical detection results of the B cell phase of this invention, where the left image is human gastritis tissue (HP+) and the right image is human normal gastric tissue (HP-).

[0060] Figure 3 This is a diagram of the immunoblotting (WB) results of the small-transfer stage of this invention, where lane 1 is the P1 clone and lane 2 is the P3 clone.

[0061] Figure 4 These are images of the immunohistochemical detection results of the monoclonal recombination stage of this invention. The left image is human gastritis tissue (HP+), the middle image is human normal gastric tissue (HP-), and the right image is human colon tissue. All of these are detection results of P1 clone.

[0062] Figure 5 This is an SDS-PAGE gel image of the rabbit monoclonal antibody against Helicobacter pylori of this invention, in which lane M is the marker; lane R is the reduced lane; and lane NR is the non-reduced lane.

[0063] Figure 6 These are images of the immunohistochemical detection results of the kit of the present invention, wherein: the left image is human gastritis tissue (HP+), the middle image is human colon tissue, and the right image is normal human gastric mucosa tissue (HP-).

[0064] Figure 7 This invention provides a multi-normal tissue dot matrix pattern for detection using the kit.

[0065] Figure 8 This invention provides a multi-cancer tissue dot matrix pattern for detection using the kit.

[0066] Figure 9 This is a graph showing the immunoblotting (WB) specificity verification results of the kit of the present invention, wherein: Lane 1: Escherichia coli lysate, no band; Lane 2: Helicobacter pylori lysate, specific band.

[0067] Figure 10 This is a graph showing the specificity verification results of the enzyme-linked immunosorbent assay (ELISA) kit of the present invention.

[0068] Figure 11 These are stability test graphs of the present invention after storage for 1, 3, 6, 9, and 12 months, respectively. Detailed Implementation

[0069] The present invention will be further described below with reference to specific embodiments.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0071] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0072] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0073] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0074] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0075] Example 1

[0076] This embodiment provides the preparation of a monoclonal antibody against Helicobacter pylori.

[0077] The preparation process of anti-Helicobacter pylori (rabbit) monoclonal antibody includes immunogen preparation, animal immunization, B cell isolation and screening, clonal recombination, antibody expression and purification, and the specific steps are as follows:

[0078] (1) Immunogen preparation

[0079] Helicobacter pylori ATCC 43504 strain was cultured in a large-scale manner. After collecting the bacterial cells, they were inactivated by high temperature and high pressure at 121℃ to prepare (whole bacterial) immunogen. At the same time, Helicobacter pylori thermal lysis buffer was prepared: the bacterial cells were mixed with 1×HOT lysis buffer (10mM Tris-HCl pH 8 + 1% SDS + 1.0 mM Na-Orthovanadate) at a volume ratio of 1:3, boiled in a water bath until clear, and then sonicated in an ice-water bath (25% power, 3 s sonication, 5 s interval, for a total of 5 min). The supernatant was collected by centrifugation at 13300 rpm for 10 min at 4℃, and the protein concentration was determined by BCA method. The concentration was adjusted to 1 mg / mL and stored at -20℃ for subsequent antibody detection.

[0080] (2) Animal immunization

[0081] Two healthy 6-8 week old New Zealand White rabbits, designated F2671 and F2672, were selected. Each rabbit received five primary subcutaneous immunizations on its back and one booster immunization via ear vein. Each primary immunization dose was 2.5 × 10⁻⁶ mmol / L. 7 Inactivated bacteria + Freund's adjuvant, booster immunization dose is twice that of basal immunization without added adjuvant.

[0082] Rabbit serum was collected on day 10 after the fourth immunization. Serum titer was detected by ELISA, requiring an OD450nm value greater than 0.3 at a dilution of 1:64000. Simultaneously, the binding capacity of serum in Helicobacter pylori-infected gastritis tissue was detected by IHC method to screen rabbits with excellent serum titer and binding capacity.

[0083] The serum titer results detected by ELISA are shown in Table 1. Rabbit F2671 had an OD450nm value of 0.308 (lowest) at a dilution of 1:64000, which met the preset titer requirement; Rabbit F2672 had an OD450nm value of 0.146 (lowest) at the same dilution, which did not meet the standard.

[0084] Table 1. Serum titer ELISA test results

[0085]

[0086] IHC test results (such as) Figure 1 As shown in the figure, F2671 serum showed strong positive staining signal and little non-specific staining in Helicobacter pylori-infected gastritis tissue. F2671 was selected for subsequent B cell isolation and screening experiments.

[0087] (3) B cell isolation and screening

[0088] The selected rabbits (F2671) were given a booster immunization via the ear vein. Four days later, the spleen was aseptically harvested, and B cells secreting specific antibodies were isolated by antigen-coated adsorption method. The cells were then plated in six 96-well plates with a total of 576 wells, and the B cell supernatant was collected after culture.

[0089] After initial ELISA screening, supernatant from 120 wells of positive clones was collected. This supernatant was then subjected to IHC rescreening using Helicobacter pylori-infected gastritis tissue (supernatant diluted 10-fold), resulting in 4 strongly positive clones. The supernatant was further diluted 20-fold, and IHC was performed using a matrix of normal and cancerous tissue samples. Finally, one positive B-cell clone with optimal specificity and no nuclear localization errors was selected. Results are as follows: Figure 2 As shown, the supernatant of this clone was positive only in Helicobacter pylori-infected gastritis tissue, and no non-specific staining was observed in normal tissue.

[0090] (4) Clonal recombination (low-level expression)

[0091] Lysis-positive B cell clones were analyzed, and total RNA was extracted using the TurboCapture 96 mRNA Plate kit (QIAGEN, 72251). The RNA was then reverse transcribed into cDNA using a reverse transcription kit. Specific primers were designed to amplify the antibody heavy chain variable region (VH) and light chain variable region (VL) genes using cDNA as a template via PCR.

[0092] VH amplification program: 95℃ pre-denaturation for 5 min, 95℃ for 30 s, 70℃ for 30 s, 72℃ for 1 min, 35 cycles, 72℃ extension for 10 min;

[0093] VL amplification program: 95℃ pre-denaturation for 5 min, 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 35 cycles, 72℃ extension for 10 min.

[0094] After 1% agarose gel electrophoresis and gel recovery, the amplified products were transformed into TOP10 competent cells via homologous recombination with a mammalian cell expression vector (pcDNA3.4 plasmid, purchased from Addgene, catalog number 198249) containing genes encoding constant regions. The cells were cultured at 37°C for 12 h, and single clones were picked and sequenced. VH and VL expression plasmids that matched the sequence characteristics of rabbit-derived antibodies were screened using Snapgene software.

[0095] in:

[0096] The amino acid sequence of the light chain variable region (VL) is as follows:

[0097] DVVMTQTPSPVSAAVGSTVTISCQSSESVYGNNRLAWLQQKPGQPPKRLMYLASTLASGVSSRFKGSGSGTQFTLTISDLECDDAATYYCAGGYSGGIGVFGGGTEVVVK (SEQ ID NO.1);

[0098] The nucleotide sequence encoding VL (VL-DNA) is:

[0099] GACGTGGTGATGACCCAGACCCCTAGCCCTGTTAGCGCTGCCGTTGGAAGCACCGTGACCATCAGCTGCCAGAGCAGCGAAAGCGTGTACGGCAACAACAGGCTGGCCTGGTTACAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATGTATCTGGCCAGCACCCTGGCTAGCGGGGTCTCATCGCGATTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCCGGAGGATATTCTGGAGGCATCGGCGTGTTCGGCGGAGGAACAGAGGTGGTGGTGAAG (SEQ ID NO.2).

[0100] The amino acid sequence of the heavy chain variable region (VH) is:

[0101] QSLEESGGRLITPGGSLTLTCTVSGIDLSSNAMTWVRQAPGKGLEYIGTLYTSGGASYASWAKGRFTISKTSSTTVDLKMTSLTAADTATYFCGRSLYAFDPWGPGTVVTVSS (SEQ ID NO.3);

[0102] The nucleotide sequence encoding VH (VH-DNA) is:

[0103] CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGATAACGCCTGGAGGATCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGCAACGCTATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAACATTGTACACCAGCGGCGGAGCCAGCTATGCTTCCTGGGCCAAGGGACGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGTGGCAGAAGCCTGTACGCCTTCGACCCTTGGGGACCCGGAACAGTTGTGACCGTGAGCTCT (SEQ ID NO.4).

[0104] The amino acid sequence of the light chain constant region (CL) is as follows:

[0105] GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO.5);

[0106] The nucleotide sequence encoding CL (CL-DNA) is as follows:

[0107] GGAGATCCCGTTGCTCCTACCGTGCTGATCTTCCCTCCCGCCGCTGATCAGGTTGCTACCGGCACCGTTACCATCGTGTGTGTGGCCAACAAGTACTTCCCCGACGTGACCGTGACCTGGGAGGTGGACGGCACAACACAAACCACAGGCATCGAGAACAGCAAGACCCCTCAGAACAGCGCCGACTGCACCTACAACCTGAGCAGCACCCTGACCCTGACCTCTACCCAGTACAACAGCCACAAGGAGTACACCTGCAAGGTGACCCAGGGCACCACCAGCGTGGTGCAGAGCTTCAACAGGGGAGACTGCTGA (SEQ ID NO.6).

[0108] The amino acid sequence of the heavy chain constant region (CH) is as follows:

[0109] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO.7);

[0110] The nucleotide sequence encoding CH (CH-DNA) is:

[0111] GGACAGCCCAAGGCTCCTAGCGTGTTCCCCTTAGCTCCCTGCTGCGGAGACACACCCAGCAGCACCGTTACACTGGGCTGCTTGGTGAAGGGCTACCTGCCCGAGCCTGTGACCGTGACATGGAACAGCGGCACCTTGACCAACGGAGTGAGGACCTTCCCCAGCGTGAGGCAGTCTAGCGGCCTGTACAGCTTGAGCAGCGTGGTGAGCGTGACCAGCAGCAGCCAACCCGTGACATGCAACGTTGCCCACCCTGCTACCAACACCAAGGTGGACAAGACCGTGGCCCCTAGCACCTGCAGCAAGCCCACATGTCCTCCTCCTGAACTGCTGGGCGGACCTAGCGTGTTCATCTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCTGAGGTGACCTGCGTGGTGGTGGATGTTTCTCAGGACGACCCCGAGGTGCAGTTCACCTGGTACATCAACAACGAGCAGGTGAGGACCGCCAGGCCTCCCCTGAGGGAGCAACAGTTCAACAGCACCATCAGGGTGGTGAGCACCCTGCCCATCGCTCATCAGGACTGGCTGAGGGGAAAGGAGTTCAAGTGCAAGGTGCACAACAAGGCCCTGCCCGCCCCTATCGAGAAGACCATCAGCAAGGCTAGGGGACAACCCCTGGAGCCCAAGGTGTACACCATGGGCCCTCCTAGAGAGGAGCTGAGCAGCAGGAGCGTGAGCCTGACATGCATGATCAACGGCTTCTACCCCAGCGACATCAGCGTGGAGTGGGAGAAGAACGGCAAGGCCGAGGACAACTACAAGACCACACCCGCCGTGCTGGACTCTGACGGCAGCTACTTCCTGTACAGCAAGCTGAGCGTGCCCACATCTGAATGGCAGAGGGGCGACGTGTTCACCTGCAGCGTGATGCACGAGGCCTTGCATAACCACTACACCCAGAAGAGCATCAGCAGGAGCCCCGGCAAGTGA(SEQ IDNO.8)。

[0112] The amino acid sequence of the light chain (L) is as follows:

[0113] DVVMTQTPSPVSAAVGSTVTISCQSSESVYGNNRLAWLQQKPGQPPKRLMYLASTLASGVSSRFKGSGSGTQFTLTISDLECDDAATYYCAGGYSGGIGVFGGGTEVVV KGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ IDNO.9);

[0114] The nucleotide sequence encoding the light chain (L-DNA) is as follows:

[0115] GACGTGGTGATGACCCAGACCCCTAGCCCTGTTAGCGCTGCCGTTGGAAGCACCGTGACCATCAGCTGCCAGAGCAGCGAAAGCGTGTACGGCAACAACAGGCTGGCCTGGTTACAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATGTATCTGGCCAGCACCCTGGCTAGCGGGGTCTCATCGCGATTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCCGGAGGATATTCTGGAGGCATCGGCGTGTTCGGCGGAGGAACAGAGGTGGTGGTGAAGGGAGATCCCGTTGCTCCTACCGTGCTGATCTTCCCTCCCGCCGCTGATCAGGTTGCTACCGGCACCGTTACCATCGTGTGTGTGGCCAACAAGTACTTCCCCGACGTGACCGTGACCTGGGAGGTGGACGGCACAACACAAACCACAGGCATCGAGAACAGCAAGACCCCTCAGAACAGCGCCGACTGCACCTACAACCTGAGCAGCACCCTGACCCTGACCTCTACCCAGTACAACAGCCACAAGGAGTACACCTGCAAGGTGACCCAGGGCACCACCAGCGTGGTGCAGAGCTTCAACAGGGGAGACTGCTGA (SEQ ID NO.10).

[0116] The amino acid sequence of the heavy chain (H) is as follows:

[0117] QSLEESGGRLITPGGSLTLTCTVSGIDLSSNAMTWVRQAPGKGLEYIGTLYTSGGASYASWAKGRFTISKTSSTTVDLKMTSLTAADTATYFCGRSLYAFDPWGPGTVVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO.11);

[0118] The nucleotide sequence encoding the heavy chain (H-DNA) is:

[0119]

[0120] For ease of representation, the light and heavy chains also include a leader sequence.

[0121] The amino acid sequence of the light chain leader (L-leader) is as follows:

[0122] MDTRAPTQLLGLLLLWLPGARC (SEQ ID NO. 13);

[0123] The amino acid sequence of the heavy chain leader (H-leader) is as follows:

[0124] METGLRWLLLVAVLKGVQC (SEQ ID NO. 14).

[0125] The amino acid sequence of the light chain with a leader sequence (L with leader) is as follows:

[0126] MDTRAPTQLLGLLLLWLPGARCDVVMTQTPSPVSAAVGSTVTISCQSSESVYGNNRLAWLQQKPGQPPKRLMYLASTLASGVSSRFKGSGSGTQFTLTISDLECDDAATYYCAGGYSGGI GVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO.15);

[0127] The amino acid sequence of the heavy chain containing the leader sequence (H with leader) is as follows:

[0128] METGLRWLLLVAVLKGVQCQSLEESGGRLITPGGSLTLTCTVSGIDLSSNAMTWVRQAPGKGLEYIGTLYTSGGASYASWAKGRFTISKTSSTTVDLKMTSLTAADTATYFCGRSLYAFDPWGPGTVVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO.16).

[0129] The nucleotide sequence encoding the light chain with leader (L with leader - DNA) is as follows:

[0130] ATGGACACCAGGGCCCCTACCCAGCTGCTGGGCCTGCTGTTGTTATGGTTACCCGGAGCTAGGTGCGACGTGGTGATGACCCAGACCCCTAGCCCTGTTAGCGCTGCCGTTGGAAGCACCGTGACCATCAGCTGCCAGAGCAGCGAAAGCGTGTACGGCAACAACAGGCTGGCCTGGTTACAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATGTATCTGGCCAGCACCCTGGCTAGCGGGGTCTCATCGCGATTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTGCCGGAGGATATTCTGGAGGCATCGGCGTGTTCGGCGGAGGAACAGAGGTGGTGGTGAAGGGAGATCCCGTTGCTCCTACCGTGCTGATCTTCCCTCCCGCCGCTGATCAGGTTGCTACCGGCACCGTTACCATCGTGTGTGTGGCCAACAAGTACTTCCCCGACGTGACCGTGACCTGGGAGGTGGACGGCACAACACAAACCACAGGCATCGAGAACAGCAAGACCCCTCAGAACAGCGCCGACTGCACCTACAACCTGAGCAGCACCCTGACCCTGACCTCTACCCAGTACAACAGCCACAAGGAGTACACCTGCAAGGTGACCCAGGGCACCACCAGCGTGGTGCAGAGCTTCAACAGGGGAGACTGCTGA (SEQ ID NO.17);

[0131] The nucleotide sequence encoding the heavy chain with leader (H with leader - DNA) is:

[0132]

[0133] Light chain and heavy chain expression plasmids were paired at a 1:1 mass ratio and co-transfected to a cell density of 3 × 10⁶ cells / year. 6 Expi 293F cells per mL were cultured at 37°C and 8% CO2 for 3 days. The cell supernatant was then collected (small rotation). The two clones with qualified ELISA values ​​were labeled as P1 and P3.

[0134] Helicobacter pylori was lysed by boiling with HOT lysis buffer, followed by ultrasonic disruption and centrifugation to obtain the supernatant for protein quantification. After preparing the protein samples, SDS-PAGE electrophoresis was performed to separate the proteins according to molecular weight. The proteins were then transferred from the gel to a PVDF membrane, and non-specific sites were blocked with blocking buffer. The supernatants P1 and P3 obtained from the small transfection were incubated with goat anti-rabbit secondary antibody (Starter, catalog number S0B4002). The bands were obtained by chemiluminescence imaging in an imaging system. Figure 3 As shown in the image. The test results showed multiple bands of this antibody in Helicobacter pylori lysate, suggesting it may recognize characteristic protein bands such as UreA (29 kDa), UreB (66 kDa), CagA (120 kDa), and VacA (95 kDa). IHC testing showed a brownish-yellow positive result only in gastritis tissue (HP+), while normal gastric and colonic tissues were negative. Figure 4 As shown, the optimal clone P1 was selected and put into production.

[0135] (5) Antibody expression and purification

[0136] One day before transfection, HEK293 cells were adjusted to a density of 2 × 10⁻⁵ using OPM-293 CD05 Medium (OPM Biosciences, 81075-001). 6 Cells / mL were incubated at 37°C with 8% CO2 and shaking at 95 rpm; the cell density was adjusted to 3 × 10⁻⁶ cells / mL on the second day. 6 Units / mL are available for use.

[0137] Prepare the transfection mixture: 25 μg heavy chain plasmid + 25 μg light chain plasmid + 50 μL FectoPRO + 5 mL OPM-CDTrans293. After incubating at room temperature for 5 min, add 50 mL of the above HEK293 cells and culture at 37℃, 8% CO2, and 95 rpm with shaking. 24 h after transfection, add 3 mL OPM-293 ProFeed and continue culturing for 96 h. Collect the cell supernatant by centrifugation at 12000 rpm for 10 min.

[0138] The supernatant was filtered through a 0.45 μm filter, and Protein A packing material was added and incubated at room temperature for 1 h. The supernatant was then transferred to a 6 mL chromatography column. The packing material was washed sequentially with 15 column volumes of Wash Buffer A and 1×PBS until the OD280 of the post-column eluent was <0.01. Eluent was then eluted with Elute Buffer B, and the eluent was collected and neutralized with Neutralization Buffer C. The eluent was adjusted to a 1×PBS system by adding 10×PBS. The solution was then centrifuged at 4°C and 5000 rpm using an ultrafiltration tube to replace the buffer. The protein concentration was determined and adjusted to 0.5 mg / mL to obtain a high-purity rabbit monoclonal antibody against Helicobacter pylori, designated RMB1007013. The antibody was identified by SDS-PAGE electrophoresis, and the results are as follows: Figure 5 As shown, SDS-PAGE electrophoresis revealed a single antibody band with no impurities, indicating a purity >95%, which is suitable for subsequent conjugation and kit assembly.

[0139] Example 2

[0140] This embodiment provides an HRP-conjugated anti-Helicobacter pylori monoclonal antibody and its preparation.

[0141] The rabbit monoclonal antibody RMB1007013 (0.5 mg / mL) prepared in Example 1 was conjugated with horseradish peroxidase (HRP) using the amino-coupling method. The specific operation steps are as follows:

[0142] (1) HRP activation: Dissolve 10 mg HRP in 1 mL of 0.05 mol / L PBS (pH 7.4), add 0.2 mL of 0.1 mol / L NaIO4, stir magnetically for 30 min at room temperature in the dark, then add 0.1 mL of 0.16 mol / L ethylene glycol, continue stirring at room temperature for 1 h, put the mixture into a dialysis bag, and dialyze overnight in 0.05 mol / L PBS (pH 7.4) at 4°C to remove unreacted reagents;

[0143] (2) Antibody conjugation: The activated HRP solution and antibody solution were mixed at a molar ratio of 3:1, and 0.05 mol / L PBS (pH 7.4) was added to a total volume of 2 mL. The mixture was stirred at room temperature in the dark for 2 h, and 0.1 mL of 0.2 mol / L sodium borohydride was added. The mixture was allowed to stand at 4 °C for 2 h to terminate the conjugation reaction.

[0144] (3) Purification of the conjugate: The conjugate reaction solution was loaded onto a Sephadex G-200 gel chromatography column and eluted with 0.05 mol / L PBS (pH 7.4) at a flow rate of 0.5 mL / min. The first elution peak was collected, which was the HRP-conjugated anti-Helicobacter pylori antibody.

[0145] (4) Concentration adjustment and storage: The concentration of the conjugated antibody was measured using a protein concentration analyzer. The concentration was adjusted to 0.5 mg / mL using storage solution (PBS pH7.4 + 40% glycerol + 0.05% BSA + 0.03% Proclin 300). The solution was dispensed into 1 mL vials and stored at 2-8°C in the dark.

[0146] Example 3

[0147] This embodiment provides an immunohistochemical detection kit and its preparation.

[0148] In this embodiment, the immunohistochemical detection kit uses HRP-conjugated rabbit monoclonal antibody against Helicobacter pylori as the core detection component, along with blocking solution, endogenous peroxidase blocking solution, antigen retrieval solution, DAB chromogenic solution, hematoxylin counterstaining solution, and washing solution. The precise formulation and working concentration of each reagent have been optimized. The components and formulations of the kit are detailed in Table 2.

[0149] Table 2. Components, formulation, and function of immunohistochemical detection kits

[0150]

[0151] In this embodiment, the reagent components were prepared according to the reagent formulation in Table 2, and all components were individually sealed and packaged to assemble an immunohistochemical detection kit for rapid detection of Helicobacter pylori. The kit specifications are suitable for the detection of 100 paraffin sections, and the specific configuration is as follows:

[0152] HRP-conjugated anti-Helicobacter pylori monoclonal antibody: 1 mL / vial (0.5 mg / mL), 1 vial in total; the stock solution should be kept free from repeated freeze-thaw cycles and diluted after equilibration at room temperature for 10 min before use;

[0153] Blocking solution: 10 mL / vial, 1 vial total;

[0154] Endogenous peroxidase blocking solution: 5 mL / vial, 1 vial in total;

[0155] Antigen retrieval solution (EDTA type, pH 9.0): 50 mL / vial, 1 vial in total;

[0156] DAB colorimetric solution: 1 mL DAB stock solution / vial + 50 mL DAB solvent / vial, 1 vial of each;

[0157] Hematoxylin counterstain solution: 10 mL / vial, 1 vial in total;

[0158] Washing solution: 100 mL PBST buffer / vial + 100 mL 1×PBS buffer / vial, 1 vial each;

[0159] Reagent kit instruction manual: 1 copy, including operating procedures, precautions, and result interpretation criteria.

[0160] After the kit is assembled, label it and store it at 2~8℃ in the dark, with a shelf life of 12 months.

[0161] The immunohistochemical detection kit in this embodiment is based on the dual core principles of antigen-antibody specific binding and enzyme-catalyzed colorimetric development, as detailed below:

[0162] The HRP-conjugated anti-Helicobacter pylori monoclonal antibody in the kit serves as the primary antibody, which can specifically bind to Helicobacter pylori antigens in gastric tissue sections to form a stable antigen-antibody complex, thereby achieving targeted recognition of Helicobacter pylori.

[0163] HRP conjugated to the primary antibody serves as an enzyme marker, catalyzing the decomposition of hydrogen peroxide to generate reactive oxygen species. These reactive oxygen species oxidize the colorless DAB substrate into a brownish-yellow insoluble precipitate, which is precisely deposited at the antigen-antibody binding site, enabling the chromogenic localization of Helicobacter pylori.

[0164] After hematoxylin counterstaining, the cell nuclei are stained blue, forming a sharp visual contrast with the brownish-yellow positive color signal. The distribution, density, and interaction with gastric mucosal cells of Helicobacter pylori can be clearly observed under an optical microscope, enabling qualitative detection of Helicobacter pylori while preserving the morphological information of the tissue.

[0165] Example 4

[0166] This embodiment provides the application and performance verification of the immunohistochemical detection kit in the detection of Helicobacter pylori.

[0167] (1) Detection of Helicobacter pylori

[0168] (a) Experimental sample

[0169] Clinical paraffin-embedded section samples were collected, all of which were pathologically confirmed: 10 cases each of Helicobacter pylori-infected gastritis tissue (HP+), normal gastric mucosa tissue (HP-), gastric cancer tissue, and colon tissue. All samples were prepared into 4 μm thick paraffin sections and attached to anti-detachment glass slides.

[0170] (b) Detection steps

[0171] The following standardized steps were followed when using the kit from Example 3:

[0172] Baking: Place the slices in an electric drying oven at 63~65℃ for 1 hour to ensure that the slices adhere tightly to the glass slide and prevent them from falling off;

[0173] Dewaxing and hydration: The sections were sequentially rinsed with xylene twice (7 min each time), 100% anhydrous ethanol twice (5 min each time), 90% ethanol twice (5 min each time), 75% ethanol once (3 min each time), and 50% ethanol once (3 min each time), and rinsed with distilled water three times for 5 min each time. All operations were performed in a fume hood.

[0174] Antigen retrieval: Place the slides in a staining box containing 0.05 mol / L EDTA retrieval solution (pH 9.0), place the staining box in an antigen retrieval autoclave, add 2 L of distilled water, and perform high-temperature and high-pressure retrieval for 40 min. After naturally cooling to room temperature, wash three times with 1×PBS for 3 min each time.

[0175] Endogenous peroxidase blocking: Place the sections in a 3% H2O2 aqueous solution and incubate at room temperature for 10 min to inactivate endogenous peroxidase in the tissue. Wash with 1×PBS 3 times, 5 min each time.

[0176] Blocking: Add blocking solution to the tissue section area to completely cover the tissue, incubate at room temperature for 30 min, pour off the blocking solution, no washing required;

[0177] Primary antibody incubation: Add 80 μL of HRP-conjugated anti-Helicobacter pylori monoclonal antibody diluted 1:500 to the tissue section area. Adjust the volume appropriately according to the tissue size. Place the sections in a humidified chamber and incubate at 37°C for 1 h. Wash with PBST 3 times for 5 min each time.

[0178] DAB staining: Prepare DAB staining solution at a volume ratio of 1:50, add the staining solution to the tissue section, and incubate at room temperature in the dark for 1.5 min. When the tissue turns light brownish-yellow, rinse thoroughly with tap water to stop the staining.

[0179] Hematoxylin counterstaining: Place the sections in a 0.5% hematoxylin aqueous solution for counterstaining for 3 min, rinse with distilled water for 5 min, then place them in PBS for destaining and blueing for 30 s, and rinse with distilled water for another 5 min;

[0180] Dehydration and mounting: The sections are sequentially mounted with 50%, 75%, and 90% ethanol for 5 min each, 100% anhydrous ethanol 3 times (5 min each time), and xylene 2 times (5 min each time). After the sections become transparent, 50-100 μL of neutral resin is added, and a coverslip is slowly placed on top to avoid air bubbles. The sections are then allowed to dry overnight.

[0181] Microscopic examination: Observe the staining results of the sections under an optical microscope (10×, 40× objective lens) and record the distribution and staining of Helicobacter pylori.

[0182] (c) Test results

[0183] The results are as follows Figure 6 As shown in the representative sections, in 10 cases of Helicobacter pylori-infected gastritis tissue (HP+) sections, obvious brownish-yellow positive staining signals were visible on the surface of the gastric mucosa and in the glands. Helicobacter pylori was clearly located and there was no background staining. In 10 cases of normal gastric mucosa tissue (HP-), 10 cases of gastric cancer tissue, and 10 cases of colon tissue sections, no brownish-yellow staining signals were observed. Only the cell nuclei were blue, and there was no non-specific staining. The concordance rate of the kit of this invention for the detection of Helicobacter pylori is 100%.

[0184] (2) Comprehensive validation of reagent kit performance

[0185] (a) Specificity verification

[0186] The kit of this invention was applied to IHC detection of multiple normal tissue fractions (13 types of normal tissues including liver, kidney, spleen, brain, testis, stomach, colon, myocardium, and skeletal muscle) and multiple cancer tissue fractions (14 types of cancer tissues including breast cancer, hepatocellular carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, cervical cancer, and gastric cancer). The statistical results after detection are as follows: Figure 7 and Figure 8 As shown, all normal and cancerous tissues that were not infected with Helicobacter pylori were negative;

[0187] Proteins were extracted from whole cultures of *E. coli* and *Helicobacter pylori*, and then analyzed using this kit via Western blot (WB). The results are as follows: Figure 9 As shown, the antibody in the kit showed no protein bands in E. coli lysate, but a specific band was detected in Helicobacter pylori lysate, which may recognize some of the key proteins UreA, UreB, CagA, and VacA.

[0188] The ELISA experiment also used Helicobacter pylori and Escherichia coli whole-cell lysates to coat microplates, respectively. The results were detected using this kit. Figure 10 As shown, the OD450nm value of the antibody binding to Helicobacter pylori lysate was significantly increased (S0B2252P), consistent with the positive control (PC), while the OD450nm value of the antibody binding to Escherichia coli lysate was consistent with the background (NC), indicating no specific binding.

[0189] The above results demonstrate that the kit of the present invention has extremely high specificity and no cross-reactivity.

[0190] (b) Sensitivity verification

[0191] The Helicobacter pylori lysate was serially diluted at 1:10, 1:100, 1:1000, 1:10000, and 1:100000. The binding ability of the kit was detected by ELISA. The results are shown in Table 3. The results show that the kit can still detect obvious positive signals at a dilution of 1:10000, with an OD450nm value > 0.2. This proves that the kit has high sensitivity and can detect trace amounts of Helicobacter pylori antigen. It can effectively identify Helicobacter pylori bacteria with small bacterial counts and atypical morphology.

[0192] Table 3. Sensitivity validation of the Helicobacter pylori detection kit

[0193]

[0194] (c) Stability verification

[0195] Store the kit at 2–8°C protected from light. Performance tests were performed at 1, 3, 6, 9, and 12 months. Results are as follows: Figure 11 As shown, the results at each time point were completely consistent with the initial results. Positive tissues showed clear color development and accurate localization, while negative tissues showed no non-specific staining. This demonstrates that the kit of the present invention has good stability and no performance degradation within its 12-month shelf life.

[0196] (d) Repeatability verification

[0197] Five tissue sections from Helicobacter pylori-infected gastritis cases were selected and independently tested by three researchers at different times using the kit of this invention, for a total of three tests.

[0198] Table 4. Reproducibility Validation of the Helicobacter pylori Detection Kit

[0199]

[0200] The results are shown in Table 4, indicating that all test results were consistent, with no significant difference in the intensity and location of the positive colorimetric signal. This demonstrates that the kit's test results have good repeatability and can effectively reduce human error. +, -, +, and ++ represent weak positive, positive, and strong positive, respectively.

Claims

1. A monoclonal antibody against Helicobacter pylori or its antigen-binding fragment, characterized in that, It contains three light chain CDRs: CDR-L1, CDR-L2, and CDR-L3, and three heavy chain CDRs: CDR-H1, CDR-H2, and CDR-H3; the CDRs are defined according to Kabat numbering; the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21, respectively; the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are shown in SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, respectively.

2. The anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, It includes a light chain variable region and a heavy chain variable region, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.1; The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.

3.

3. The anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment according to claim 2, characterized in that, It also includes a light chain constant region and a heavy chain constant region. The amino acid sequence of the light chain constant region is shown in SEQ ID NO.5; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.

7.

4. The anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment according to claim 3, characterized in that, It includes a light chain and a heavy chain, the amino acid sequence of which is shown in SEQ ID NO.9; the amino acid sequence of which is shown in SEQ ID NO.

11.

5. The anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment according to claim 4, characterized in that, The light chain and heavy chain also include leader sequences, the amino acid sequence of which is shown in SEQ ID NO.13 and the amino acid sequence of which is shown in SEQ ID NO.

14.

6. The anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment according to claim 5, characterized in that, The monoclonal antibody comprises a light chain containing a leader sequence and a heavy chain containing a leader sequence. The amino acid sequence of the light chain containing the leader sequence is shown in SEQ ID NO.15, and the amino acid sequence of the heavy chain containing the leader sequence is shown in SEQ ID NO.

16.

7. The anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment according to any one of claims 1-6, characterized in that, The antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab)'2 fragment, a single-chain Fv protein, or a disulfide-stabilized Fv protein.

8. A nucleic acid, characterized in that, The nucleic acid encodes the anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-7.

9. The nucleic acid according to claim 8, characterized in that, The nucleic acid is a nucleotide sequence as shown in SEQ ID NO.2 and a nucleotide sequence as shown in SEQ ID NO.

4.

10. The nucleic acid according to claim 8, characterized in that, The nucleic acid is a nucleotide sequence as shown in SEQ ID NO.10 and a nucleotide sequence as shown in SEQ ID NO.

12.

11. The nucleic acid according to claim 8, characterized in that, The nucleic acid is a nucleotide sequence as shown in SEQ ID NO.17 and a nucleotide sequence as shown in SEQ ID NO.

18.

12. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid as described in any one of claims 8-11.

13. A recombinant expression cell, characterized in that, The recombinant expression cells comprise the nucleic acid as described in any one of claims 8-11, or the recombinant expression vector as described in claim 12.

14. The use of the nucleic acid according to any one of claims 8-11, the recombinant expression vector according to claim 12, or the recombinant expression cell according to claim 13 in the preparation of anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment.

15. A method for preparing the anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment according to any one of claims 1-7, characterized in that, The method includes: Recombinant expression cells are obtained by transfecting cells with the recombinant expression vector of claim 12 and culturing the obtained recombinant expression cells or by culturing the recombinant expression cells of claim 13; the supernatant is collected and purified to obtain an anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment.

16. A horseradish peroxidase-conjugated anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment, characterized in that, Horseradish peroxidase is prepared by coupling horseradish peroxidase with any of the anti-Helicobacter pylori monoclonal antibodies or their antigen-binding fragments as described in any one of claims 1-7 via an amino-coupling method.

17. The horseradish peroxidase-conjugated anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment according to claim 16, characterized in that, The coupling molar ratio is 1:(2~4) for anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment: horseradish peroxidase.

18. The use of the anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-7, the nucleic acid as described in any one of claims 8-11, the recombinant expression vector as described in claim 12, or the recombinant expression cell as described in claim 13, or the horseradish peroxidase-conjugated anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment as described in claim 16 or 17 in the preparation of products for detecting Helicobacter pylori.

19. The application according to claim 18, characterized in that, The detection of Helicobacter pylori includes the use of any one or more of the following methods: immunohistochemistry, flow cytometry, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

20. A kit for detecting Helicobacter pylori, characterized in that, The kit comprises the anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-7, the horseradish peroxidase-conjugated anti-Helicobacter pylori monoclonal antibody or its antigen-binding fragment as described in claim 16 or 17, the nucleic acid as described in any one of claims 8-11, the recombinant expression vector as described in claim 12, or the recombinant expression cell as described in claim 13.

21. The kit for detecting Helicobacter pylori according to claim 20, characterized in that, The kit is an immunohistochemical detection kit for detecting Helicobacter pylori.

22. The kit for detecting Helicobacter pylori according to claim 21, characterized in that, The immunohistochemical detection kit also includes: blocking solution, endogenous peroxidase blocking solution, antigen retrieval solution, DAB chromogenic solution, hematoxylin counterstaining solution, and washing solution.

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