A monoclonal antibody cAbD4 against hepatitis B virus core protein and its preparation and application

Abstract

The present invention discloses a monoclonal antibody cAbD4 for hepatitis B virus core protein and its preparation and application. cAbD4 recognizes the core protein HBc of hepatitis B virus HBV. The amino acid sequences of CDR1-H, CDR2-H and CDR3-H of its heavy chain variable region are SEQ ID NO.1-3, respectively, and the amino acid sequences of CDR1-L, CDR2-L and CDR3-L of its light chain variable region are SEQ ID NO.4, "LGS" and SEQ ID NO.5, respectively. The monoclonal antibody obtained by screening of the present invention has a strong binding ability to HBc. The present invention verifies the application of cAbD4 in various biochemical methods through examples, and verifies its ability to detect different HBV genotypes. The monoclonal antibody cAbD4 of the present invention has the characteristics of high efficiency, broad spectrum and versatility, and has broad application prospects in the field of HBV infection diagnosis.

Description

Technical Field

[0001] The present invention belongs to the medical technical field of molecular biology and cellular immunology, and specifically relates to a monoclonal antibody cAbD4 to hepatitis B virus core protein and its preparation and application. Background Art

[0002] Hepatitis B virus (HBV) infection remains a major global public health problem. While current antiviral drugs for chronic hepatitis B, such as nucleoside analogs (NAs) and interferon-α (IFN-α), can effectively inhibit HBV DNA replication and disease progression, functional cure rates are low. Therefore, there remains an urgent need to develop more effective strategies for the detection and treatment of HBV infection.

[0003] HBV is a hepatotropic DNA virus with a diameter of approximately 42 nm. Its inner capsid is composed of the core protein (HBc). In most HBV genotypes, the core protein consists of 183 amino acids (aa), including an N-terminal assembly domain (1-149 aa) for capsid formation and an arginine-rich C-terminal domain (150-183 aa) for nucleic acid binding and nuclear localization. HBc first forms dimers, and typically 120 dimers self-assemble into a capsid with T=4 icosahedral symmetry. The capsid encapsidates the viral RNA and polymerase, providing a closed environment for reverse transcription of the viral RNA and promoting nucleocapsid packaging and virion formation. HBc also regulates capsid transport into the nucleus and is associated with cccDNA formation during infection. In addition to serving as a structural protein and regulating multiple stages of the viral life cycle, HBc also plays a crucial role in the pathogenesis of HBV-related diseases by suppressing host immune responses or activating multiple signaling pathways, including the MAPK and Wnt / β-catenin pathways. These functions of HBc suggest that it could be an important target for HBV detection and intervention.

[0004] HBV-related research primarily assesses HBV infection titers indirectly through the detection of surface antigens, e-antigens, and HBV DNA in cell supernatants. Currently, there are few anti-HBc monoclonal antibodies available for the detection of multiple HBV genotypes and various biochemical assays, such as enzyme-linked immunosorbent assay (ELISA), Western blot, immunofluorescence assay (IFA), flow cytometry, immunospot assay, and immunohistochemistry (IHC). This makes direct detection of HBV-infected cells difficult. Therefore, the development of more sensitive and broad-spectrum anti-HBc monoclonal antibodies is crucial for the advancement of HBV-related research. Summary of the Invention

[0005] To enable the detection of multiple HBV genotypes and various biochemical assays, the present invention screened and obtained a monoclonal antibody against HBV core protein, which exhibits high efficacy, broad spectrum activity, and versatility. The present invention further provides a method for using a monoclonal antibody against HBc for the detection of HBV infection using various methods. This monoclonal antibody has strong specific binding to HBV core protein and can detect HBV core protein using various biochemical assays. This monoclonal antibody is expected to be a useful antibody for detecting live HBV infection, providing a powerful tool for antigen detection of multiple HBV genotypes.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention is to provide a monoclonal antibody cAbD4 against the core protein of hepatitis B virus, wherein the monoclonal antibody recognizes the core protein HBc of hepatitis B virus HBV.

[0008] Furthermore, the monoclonal antibody is a broad-spectrum human monoclonal antibody that can recognize HBc of all HBV genotypes, including HBV genotypes A, B, C, D, E, F, G, H, I, and J.

[0009] Furthermore, the amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of the monoclonal antibody are SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively, and the amino acid sequences of CDR1-L, CDR2-L and CDR3-L of the light chain variable region VL of the monoclonal antibody are SEQ ID NO.4, "LGS" and SEQ ID NO.5, respectively.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region VH of the monoclonal antibody is as shown in SEQ ID NO.11 or an amino acid sequence having at least 80% identity thereto, and the amino acid sequence of the light chain variable region VL of the monoclonal antibody is as shown in SEQ ID NO.12 or an amino acid sequence having at least 80% identity thereto.

[0011] It is understandable that the heavy chain sequence and the paired light chain sequence constitute the above-mentioned monoclonal antibody, and their variable region determines the antibody's binding recognition and specificity to the antigen, and antibody specificity depends on the interaction between the antibody binding site and the antigenic determining region. The binding site of heavy chain and light chain is mainly composed of the residues of 3 complementary determining regions (CDRs), which are connected by framework regions (FRs) between the CDRs. Thus, those skilled in the art can easily determine the framework regions after knowing the amino acid sequence of the CDRs. That is, under the constant conditions of the CDR sequences of heavy chain and light chain, the function and application of monoclonal antibody cAbD4 of the present invention can be realized substantially. Therefore, the monoclonal antibody of the present invention, its specific sequence is not limited only to above specific heavy chain sequence variable region and light chain sequence variable region, and the variable region of above specific sequence is the monoclonal antibody sequence specifically adopted in a kind of implementation of the present invention.

[0012] A protein consisting of an amino acid sequence that is "at least 80% identical" to a reference sequence may contain mutations such as deletions, insertions and / or substitutions compared to the reference sequence. In the case of substitutions, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence derived from a species different from the reference sequence. "Amino acid substitutions" can be conservative or non-conservative. Preferably, the substitution is a conservative substitution, in which one amino acid is replaced by another amino acid with similar structure and / or chemical properties. Specifically, the sequence of the heavy chain or light chain variable region differs from the reference sequence only by conservative amino acid substitutions.

[0013] The second aspect of the present invention is to provide a biomaterial related to the monoclonal antibody described in the first aspect of the present invention, wherein the biomaterial is selected from one of the following (A) to (B):

[0014] (A) a nucleic acid molecule encoding the monoclonal antibody cAbD4 according to the first aspect of the present invention;

[0015] (B) Expression cassettes, recombinant vectors, and recombinant cell lines containing the nucleic acid molecule described in (A).

[0016] Furthermore, the expression cassette, recombinant vector, and recombinant cell line can be used to express the aforementioned heavy chain sequence, light chain sequence, or monoclonal antibody.

[0017] Furthermore, the nucleic acid sequence encoding CDR1-H shown in SEQ ID NO.1 is shown in SEQ ID NO.6;

[0018] The nucleic acid sequence encoding CDR2-H shown in SEQ ID NO. 2 is shown in SEQ ID NO. 7;

[0019] The nucleic acid sequence encoding CDR3-H shown in SEQ ID NO. 3 is shown in SEQ ID NO. 8;

[0020] The nucleic acid sequence encoding CDR1-L shown in SEQ ID NO.4 is shown in SEQ ID NO.9;

[0021] The nucleic acid sequence encoding CDR2-L is "TTGGGTTCT";

[0022] The nucleic acid sequence encoding CDR3-L shown in SEQ ID NO.5 is shown in SEQ ID NO.10.

[0023] Furthermore, the nucleic acid sequence encoding the VH shown in SEQ ID NO.11 is shown in SEQ ID NO.13;

[0024] The nucleic acid sequence encoding the VL shown in SEQ ID NO.12 is shown in SEQ ID NO.14.

[0025] It is understandable that the above specific nucleic acid sequence is only a specific nucleic acid sequence used in one implementation of the present invention. According to the degeneracy of the codons, while ensuring that the encoded amino acid sequence remains unchanged, in addition to the nucleic acid sequences defined above, several nucleic acid sequences that can encode the same heavy chain sequence or light chain sequence (for example, conservative nucleotide sequence variants are derived from genetic code degeneracy and silent variants, including nucleotide substitutions, deletions and additions) are all within the scope of protection of the present invention.

[0026] In the above-mentioned biological materials, the expression cassette containing the nucleic acid molecule encoding the antibody refers to a DNA capable of expressing the antibody in a host cell. The DNA may include not only a promoter for initiating transcription of the antibody gene, but also a terminator for terminating transcription of the antibody gene.

[0027] Among the aforementioned biological materials, a vector is a nucleic acid delivery vehicle that can insert a polynucleotide encoding a protein and enable protein expression. Vectors can transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within them. Vectors can be plasmids, cosmids, bacteriophages, or viruses.

[0028] In the aforementioned biological materials, host cells refer to cells into which the vector is introduced, including prokaryotic cells, fungal cells, insect cells, animal cells, and the like, such as Escherichia coli, yeast cells, S2 Drosophila cells, BHK cells, CHO cells, and HEK293 cells. The aforementioned expression cassettes, recombinant vectors, and recombinant cell lines can all be prepared using conventional methods in the art.

[0029] The third aspect of the present invention is to provide a method for preparing the monoclonal antibody cAbD4 described in the first aspect of the present invention, the method comprising: transfecting a host cell with a recombinant vector containing a nucleic acid molecule encoding the monoclonal antibody cAbD4; culturing the transfected host cells and collecting the supernatant, and purifying to obtain the monoclonal antibody cAbD4.

[0030] Furthermore, the recombinant vector is a pCDNA3.4 plasmid, and the host cell is a 293F cell.

[0031] The fourth aspect of the present invention is to provide the use of the monoclonal antibody cAbD4 described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, or the monoclonal antibody cAbD4 prepared by the preparation method described in the third aspect of the present invention in the preparation of products for detecting or diagnosing HBV.

[0032] The fifth aspect of the present invention is to provide a product for detecting or diagnosing HBV, which comprises the monoclonal antibody cAbD4 described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, or the monoclonal antibody cAbD4 prepared by the preparation method described in the third aspect of the present invention.

[0033] Furthermore, the above-mentioned product can be a diagnostic reagent (diagnostic kit), a detection reagent (detection kit), etc., which may also include other reaction reagents. The above-mentioned product can be used for ELISA, Western blot, flow cytometry, IFA, immunospot and immunohistochemistry methods to detect HBV core protein HBc.

[0034] The sixth aspect of the present invention is to provide a method for detecting HBV or its HBc for non-diagnostic purposes, the method comprising: mixing the product described in the fifth aspect of the present invention with a sample to be tested to detect the core protein HBc of HBV.

[0035] The above-mentioned samples to be tested cover various sample types obtained from subjects and can be used in diagnosis or detection, including but not limited to blood and other liquid samples of biological origin, solid tissue samples, clinical samples, cells in culture medium, cell supernatants, cell lysates, serum, plasma, biological fluids and tissue samples, etc.

[0036] Furthermore, detection methods include: ELISA, Western blot, flow cytometry, IFA, immunoblot and immunohistochemistry.

[0037] Compared with the prior art, the present invention adopts the above technical solution to achieve the following beneficial effects:

[0038] The present invention screened and obtained a monoclonal antibody, cAbD4, targeting the hepatitis B virus core protein, which has strong binding ability to HBc. The present invention demonstrates the application of monoclonal antibody cAbD4 in various biochemical methods and its ability to detect different HBV genotypes through examples. Monoclonal antibody cAbD4 is highly effective, broad-spectrum, and versatile, and has broad application prospects in the diagnosis of HBV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0040] Figure 1 This is a schematic diagram of the ELISA results of the binding activity of the monoclonal antibody cAbD4 to the genotype C HBV core protein in Example 2 of the present invention;

[0041] Figure 2 This is a schematic diagram of the results of the monoclonal antibody cAbD4 identifying HBc of different genotypes of HBV in Example 3 of the present invention;

[0042] Figure 3 This is a schematic diagram of the results of using the monoclonal antibody cAbD4 in Example 4 of the present invention to identify HBc in HBV-infected cells by Western blot;

[0043] Figure 4 This is a schematic diagram of the results of using the monoclonal antibody cAbD4 in Example 5 of the present invention to identify HBc in HBV-infected cells using the IFA method;

[0044] Figure 5 This is a schematic diagram of the results of using the monoclonal antibody cAbD4 in Example 6 of the present invention to detect HBc in HBV-infected cells by flow cytometry;

[0045] Figure 6 This is a schematic diagram of the results of using the monoclonal antibody cAbD4 in Example 7 of the present invention to detect HBc in HBV-infected cells using the immunospot method;

[0046] Figure 7 This is a schematic diagram of the results of using the monoclonal antibody cAbD4 in Example 8 of the present invention to detect intracellular HBc in HBV-infected liver tissue using the immunohistochemical method. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. It should be understood that the described embodiments are only some of the embodiments of the present invention, and are not intended to be exhaustive. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally measured in accordance with national standards. Experimental materials in the following examples where the sources are not specified are all commercially available. The equipment used in each step in the following examples is conventional equipment. Where there are no corresponding national standards, the procedures are carried out in accordance with generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise specified, all parts are by weight, and all percentages are by mass. Unless otherwise defined or indicated, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the methods of the present invention.

[0048] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0049] Example 1: Screening, expression and purification of antibodies

[0050] This study was approved by the Ethics Committee of Shenzhen Third People's Hospital (approval number: 2021-030). All participants provided written informed consent for subsequent sample processing and analysis. The required peripheral blood mononuclear cell (PBMC) samples and liver tissue sections were obtained from patients with chronic HBV infection. PBMCs were stored in liquid nitrogen, and liver tissue was embedded in paraffin blocks for preservation.

[0051] This example uses a PBMC sample, and the monoclonal antibody screening and purification steps are as follows:

[0052] (1) Monoclonal antibody screening

[0053] Resuscitation of frozen PBMCs: Wash twice with 10 mL of 1640 medium (72400047, Gibco) containing 10% fetal bovine serum. Take 20 μL of cell suspension, add 20 μL of AO / PI dye (RE010212, Countstar), and count the number of viable cells. The number of cells is 1×10 6Cells were aliquoted into 50 μL of staining buffer (PBS + 2% fetal bovine serum) containing Live / Dead dye (L34964, Invitrogen) and resuspended in peripheral blood mononuclear cells. The cells were stained at 4°C for 30 minutes. After washing twice with PBS, CD19-PE-Cy7, CD3-Pacific Blue, CD8-Pacific Blue, CD14-Pacific Blue, CD27-APC / Cy7, and IgG-FITC (557835, 558117, 558207, 558121, 560222, 555786, BD Pharmingen) were added to 50 μL of staining buffer and the peripheral blood mononuclear cells were resuspended and stained at 4°C for 30 minutes. After washing twice with PBS, peripheral blood mononuclear cells were resuspended in 50 μL of staining buffer containing genotype C HBc (P2301, Shenzhen Innocell Biotechnology Co., Ltd.) labeled with DyLight 550 (84531, Invitrogen) and DyLight 650 (84536, Invitrogen) and stained at 4°C for 30 minutes. After washing twice with PBS, HBc-specific IgG+ memory B cells were sorted using a BD FACSAria II sorting flow cytometer.

[0054] Single B cells were sorted into 96-well PCR plates containing lysis buffer. RT-PCR and nested PCR were then performed to amplify the variable regions of the heavy and light chains, respectively, according to the methods described in the literature (Liao HX, Levesque MC, Nagel A, Dixon A, Zhang R, Walter E, et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. Journal of virological methods. 2009; 158: 171-9.). The PCR amplification products were sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the obtained antibody heavy and light chain variable region sequences were synthesized by GenScript Biotech Co., Ltd. and constructed into the full-length IgG1 heavy and light chain expression vectors pCDNA3.4 (GenScript Biotech Co., Ltd.), respectively, to ultimately produce large quantities of antibody heavy and light chain plasmids.

[0055] (2) Antibody expression and purification

[0056] The monoclonal antibody in this example was expressed and purified from 293F cells. Taking 400 mL of 293F cells as an example, the specific steps are as follows:

[0057] 1. Prepare expression cells: 400 mL, 1.2 × 10 6 293F cells at 100 μg / mL were cultured in a shaker in an incubator at 8% CO2 and 37°C for 2 hours;

[0058] 2. Prepare transfection plasmid A: Take 200 μg each of antibody heavy chain plasmid and antibody light chain plasmid and dilute them into 10 mL of Opti-MEM (31985070, Gibco);

[0059] 3. Prepare transfection reagent B: Add 2 mL of 1 mg / mL PEI transfection reagent (24885-2, Polysciences) to 10 mL of opti-MEM and let it stand for 5 minutes;

[0060] 4. Prepare transfection mixture AB: add solution B to solution A, mix, and let stand for 20 minutes;

[0061] 5. Transfection: Add 22 mL of AB mixture dropwise to 293F cells, shaking well while adding;

[0062] 6. Collect cell supernatant: After culturing 293F cells for 5-6 days, centrifuge at 3500g for 30 minutes, collect the supernatant, and filter through a 0.45μm filter membrane;

[0063] 7. Antibody purification: Open the lid of the Protein A gravity column and add 5 column volumes of PBS solution when the ethanol solution has completely flowed out; then add the filtered cell supernatant, wash the gravity column with 3 column volumes of PBS solution, and finally elute with 5 volumes of 0.1 M glycine-hydrochloric acid solution (pH = 3.0); place the eluate in a 30KD ultrafiltration concentrator tube and centrifuge at 3500g at 4°C until the liquid in the concentrator tube is less than 1 mL; discard the waste liquid in the collection tube and fill the concentrator tube with PBS solution, centrifuge under the same conditions until the liquid in the concentrator tube is less than 1 mL, and measure the antibody concentration.

[0064] Example 2: Enzyme-linked immunosorbent assay (ELISA) verification of the binding ability of monoclonal antibody cAbD4

[0065] One antibody was selected from the antibodies obtained in Example 1 for monoclonal antibody expression and related functional verification, and the monoclonal antibody was named cAbD4. The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of cAbD4 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, and the nucleic acid sequences encoding SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively. The amino acid sequences of CDR1-L and CDR3-L of the light chain variable region VL of cAbD4 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and CDR2-L is "LGS." The nucleic acid sequences encoding SEQ ID NO.4 and SEQ ID NO.5 are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively, and the nucleic acid sequence encoding CDR2-L is "TTGGGTTCT." The amino acid sequence of the heavy chain variable region VH of cAbD4 is shown in SEQ ID NO.11, and the nucleic acid sequence encoding the VH shown in SEQ ID NO.11 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region VL of cAbD4 is shown in SEQ ID NO.12, and the nucleic acid sequence encoding the VL shown in SEQ ID NO.12 is shown in SEQ ID NO.14.

[0066] The specific binding ability of the monoclonal antibody cAbD4 to the HBV core protein HBc was tested by ELISA. 2μg / mL, 100μL / well of genotype C HBc (P2301, Shenzhen Innocell Biotechnology Co., Ltd.) was coated in a 96-well ELISA plate and coated overnight at 4°C. A PBS solution with 0.05% Tween-20 (PBST) was prepared and the 96-well plate was washed five times. A blocking solution of 5% skim milk + 2% BSA (in PBS) was prepared at 200μL / well and blocked at room temperature for one hour. After washing three times, the cAbD4 antibody was diluted with blocking solution to a maximum concentration of 10μg / mL and then diluted five-fold in a total of seven dilutions. 100μL per well was added to the plate that had been washed three times with PBST and incubated at 37°C for one hour. The plate was washed five times with PBST and HRP-goat anti-human IgG (ZB-2304, Zhongshan Jinqiao) diluted 1:5000 in blocking buffer at 100 μL / well. The plate was incubated at 37°C for 1 hour. The plate was washed five times with PBST and a 1:1 mixture of colorimetric solution A and solution B (E661007, Sangon) was added at 100 μL / well. The plate was developed for 20 minutes at room temperature in the dark. The reaction was terminated by adding 50 μL of 2 mol / L H₂SO₄. The optical density was measured at 450 nm (OD) using a VarioskanLUX multimode microplate reader (Thermo Scientific).

[0067] The binding activity of monoclonal antibody cAbD4 to HBV core protein HBc was analyzed by ELISA. Figure 1 As shown in the figure, the monoclonal antibody cAbD4 can specifically bind to HBc of HBV genotype C with strong binding ability and half effective concentration EC 50 It is 2.66ng / mL.

[0068] Example 3: Verification of the broad-spectrum recognition ability of monoclonal antibody cAbD4 using flow cytometry

[0069] This example uses flow cytometry to verify that the monoclonal antibody cAbD4 recognizes HBc expressed in cells of different HBV genotypes. The specific experimental steps are as follows:

[0070] First, HBc protein expression plasmids of different HBV genotypes A, B, C, D, E, F, G, H, I and J were transfected into 293T cells respectively, and cells transfected with empty vector were used as controls. After 48 hours, the cells were digested with 0.5% trypsin and terminated by adding complete DMEM medium containing 10% FBS. The cells were gently blown with a pipette and observed under a microscope until more than 90% of the cells were in a single cell state. The single cell suspension was aspirated into a 15mL centrifuge tube and centrifuged at 400g for 5 minutes. The supernatant was discarded and 3mL of complete DMEM was added for culture. Take 20μL of cell suspension, add 20μL of AO / PI dye (RE010212, Countstar), count the number of live cells, and the number of cells was 5×10 5 cells / tube. The cells were fixed and permeabilized using a cell fixation / permeabilization kit (554714, BD Biosciences), centrifuged at 400g for 5 minutes, and the supernatant was discarded. 5μg / mL cAbD4 was then added to each flow cytometry tube and incubated at 4°C for 30 minutes. After washing, the cells were stained with Alexa Fluor 647-conjugated goat anti-human secondary antibody (A11013, Life Technologies) and incubated at 4°C for 30 minutes. After washing, the cells were stained with FACSymphony TM The data were collected by A3 flow cytometer (BD Biososciences), and analyzed by FlowJo software V10.9 (BD Biososciences). Figure 2 shown.

[0071] The results showed that the cAbD4 antibody could clearly detect HBc expressed in cells and could widely identify HBc derived from different HBV genotypes including A, B, C, D, E, F, G, H, I and J.

[0072] Example 4: Verification of the function of monoclonal antibody cAbD4 using Western blot experiments

[0073] In this example, the monoclonal antibody cAbD4 was applied to the Western blot method to identify HBc in HBV-infected cells. The experimental steps are as follows:

[0074] This experiment was conducted in a certified biosafety level 2 laboratory. 6HepG2-NTCP cells were plated in 6-well plates coated with type I rat tail collagen (354236, Corning) and cultured for more than 18 hours. HBV virus (purified from HepAD38 supernatant) at 1800 HBV gene copies / cell was added to DMEM containing 4% PEG8000 (89510, Sigma), 2.5% DMSO, and 5% FBS, mixed well, and then added to the HepG2-NTCP cells. After 20 hours of HBV infection, the cells were diluted three times with PBS and cultured in DMEM containing 2.5% DMSO and 16% FBS. The medium was changed every two days. On the seventh day, HepG2-NTCP cells were washed twice with PBS and harvested by adding 300 μL of 1.5× Protein Loading Buffer (DL101-02, Quanshijin). Cell samples were treated at 100°C for 10 minutes, subjected to SDS-PAGE electrophoresis, and then transferred to a PVDF membrane and blocked with 5% skim milk powder for 1 hour at room temperature. After washing twice with TBST, 0.5 μg / mL cAbD4 was added and incubated at 4°C overnight. After washing five times with TBST, 1:5000 diluted HRP-goat anti-human IgG (ZB-2304, Zhongshan Jinqiao) was added and incubated at room temperature for 1 hour. After washing five times with TBST, the membrane was developed. The experimental results are shown in Figure 2. Figure 3 shown.

[0075] Western blot results showed that HBV-infected cells expressed the same β-actin reference band as uninfected cells, with similar total cell numbers. However, the HBc-specific band was expressed only in infected cells and could be identified by the monoclonal antibody cAbD4. This suggests that the monoclonal antibody cAbD4 can specifically bind to HBc in HBV-infected cells.

[0076] Example 5: Verification of the function of monoclonal antibody cAbD4 using immunofluorescence assay (IFA)

[0077] In this example, the monoclonal antibody cAbD4 was applied to IFA detection to identify HBc in HBV-infected cells. The experimental steps were as follows:

[0078] The HBV infection procedure was the same as that in Example 4. After 7 days of infection, HepG2-NTCP cells were washed twice with PBS and fixed with 4% paraformaldehyde solution at room temperature for 30 minutes. After washing once with PBS, the cells were permeabilized with PBS solution containing 0.2% Triton X-100 for 30 minutes and QuickBlock was added. TMImmunostaining blocking solution (P0260, Beyotime) was added at room temperature for 1 hour. After washing twice with PBS, 10 μg / mL cAbD4 antibody was added and incubated overnight at 4°C. After washing five times with PBS, Alexa Fluor was added at a ratio of 1:1000. TM 488-Goat anti-Human IgG (H+L) Secondary Antibody (A11013, ThermoFisher) was incubated at room temperature for 1 hour. After washing with PBS three times, DAPI staining solution (C1002, Biyuntian) was added for 10 minutes. After washing with PBS twice, the cells were photographed using a fluorescence microscope. The experimental results are shown in Figure 2. Figure 4 shown.

[0079] The results showed that strong HBc fluorescence intensity was observed under the field of view, indicating that the monoclonal antibody cAbD4 can clearly recognize HBc in the cytoplasm and nucleus of HBV-infected cells.

[0080] Example 6: Verification of the ability of monoclonal antibody cAbD4 to recognize HBV-infected cells using flow cytometry

[0081] In this example, the monoclonal antibody cAbD4 was applied to flow cytometry to verify its ability to recognize HBc in HBV-infected cells. The specific experimental steps are as follows:

[0082] The HBV infection steps in this experiment are the same as those in Example 4. After 7 days of infection, the cells were washed twice with PBS and digested with 0.5% trypsin at 37°C for 5 minutes. Then, 10% FBS DMEM medium was added to terminate the process. The cells were gently pipetted with a pipette until more than 90% of the cells were in a single cell state. The single cell suspension was aspirated into a 15 mL centrifuge tube and centrifuged at 300 g for 5 minutes. The supernatant was discarded and 3 mL of complete DMEM medium was added. 20 μL of cell suspension was mixed with an equal volume of AO / PI fuel and the viable cells were counted according to the 5×10 5 The subsequent staining and data collection steps were the same as in Example 3. The experimental results are shown in Figure 3. Figure 5 shown.

[0083] The results showed that the monoclonal antibody cAbD4 could specifically recognize HBc in HBV-infected cells and clearly distinguish between HBV-infected and non-infected cells.

[0084] Example 7: Validation of the monoclonal antibody cAbD4 for detecting HBV infection using live virus immunospot assay

[0085] In this example, the monoclonal antibody cAbD4 was used in an immunospot assay to detect HBc in HBV-infected cells. The specific experimental steps were as follows:

[0086] This experiment was conducted in a certified biosafety level 2 laboratory. 5 HepG2-NTCP cells were plated in 96-well plates coated with type I rat tail collagen (354236, Corning) and cultured for more than 18 hours. 1800, 600, 300, and 0 gene copies / cell of HBV virus were added to DMEM culture medium containing 4% PEG8000 (89510, Sigma), 2.5% DMSO, and 5% FBS, respectively, and the mixture was added to the HepG2-NTCP cells. After 20 hours of HBV infection, PBS was diluted three times and cultured with DMEM culture medium containing 2.5% DMSO and 16% FBS. The medium was changed every two days. On the 7th day, HepG2-NTCP cells were washed twice with PBS and fixed with 4% paraformaldehyde solution at room temperature for 30 minutes. After washing once with PBS, a PBS solution containing 0.2% Triton X-100 was added to permeabilize the cells for 30 minutes. QuickBlock was then added. TM Immunostaining blocking solution (P0260, Biyuntian) was used at room temperature for 1 hour. After washing twice with PBS, 10 μg / mL cAbD4 antibody was added and incubated at 4°C overnight. After washing 5 times with PBS, 1:5000 diluted HRP-goat anti-human IgG (ZB-2304, Zhongshan Jinqiao) was added and incubated at room temperature for 1 hour. After washing 5 times with PBS, KPL TrueBlue peroxidase substrate (Seracare Life Sciences) was used for color development. The cell imaging multifunctional microplate detection instrument Cytation 7 (BioTek) was used to take pictures and count the number of HBV-infected cells. The experimental results are shown in Figure 2. Figure 6 shown.

[0087] The results showed that cAbD4 could clearly identify HBV-infected cells and the number of spots decreased with the decrease of HBV infection virus amount, indicating that cAbD4 can be used for the detection of HBV core protein HBc in the immunospot experiment and is virus dose-dependent.

[0088] Example 8: Validation of the ability of monoclonal antibody cAbD4 to recognize HBV-infected liver tissue using immunohistochemistry

[0089] In this example, the monoclonal antibody cAbD4 was applied to IHC detection to verify its ability to recognize HBc in HBV-infected liver tissue. The specific experimental steps are as follows:

[0090] Liver tissues from two clinical chronic HBV patients, Donor 1 and Donor 2, were formalin-fixed, paraffin-embedded, and sectioned. The tissues were then deparaffinized with xylene and hydrated with varying concentrations of ethanol. After rinsing with tap water for 1 minute, 100 μL of endogenous peroxidase blocker (Kit-0014, Maixin Biotechnology Development Co., Ltd.) was dripped onto the tissues and incubated at room temperature for 10 minutes. After three washes with PBS, 100 μL of antibody diluent (ABD-0030, Maixin Biotechnology Development Co., Ltd.) was dripped onto the tissues and blocked at room temperature for 1 hour. After three washes with PBS, 100 μL of 20 μg / mL HRP-conjugated cAbD4 antibody (cAbD4-HRP) was dripped onto the tissues and incubated at room temperature for 2 hours. After five washes with PBS, the tissues were developed with DAB reagent (Kit-0014, Maixin Biotechnology Development Co., Ltd.) for 5-10 minutes at room temperature. After rinsing with tap water, the tissues were stained with hematoxylin for 20 seconds. After rinsing with tap water, the PBS solution returned to blue. After dehydration, transparency and sealing, the slides were photographed using Motic VM1000 (Motic Electric Group Co., Ltd). Figure 7 shown.

[0091] The results showed that cAbD4 could clearly recognize HBc in HBV-infected hepatocytes from both Donor 1 and Donor 2 liver tissues. HBc was distributed differently in the two hepatocytes, being present in both the cytoplasm and nucleus of Donor 1 and primarily in the cytoplasm of Donor 2. This suggests that cAbD4 can be used for clinical immunohistochemical detection of the core protein HBc in liver tissue from HBV patients.

[0092] It can be seen from the above examples that the monoclonal antibody cAbD4 obtained by screening in the present invention has a strong binding ability to the core protein HBc of hepatitis B virus, and can be used in a variety of detection methods to diagnose HBc of hepatitis B virus, showing good broad spectrum and sensitivity.

[0093] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody cAbD4 against hepatitis B virus core protein, characterized in that: The monoclonal antibody recognizes the core protein HBc of the hepatitis B virus HBV; the amino acid sequences of CDR1-H, CDR2-H and CDR3-H of the heavy chain variable region VH of the monoclonal antibody are SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively, and the amino acid sequences of CDR1-L, CDR2-L and CDR3-L of the light chain variable region VL of the monoclonal antibody are SEQ ID NO.4, LGS and SEQ ID NO.5, respectively.

2. The monoclonal antibody cAbD4 according to claim 1, wherein The amino acid sequence of the heavy chain variable region VH of the monoclonal antibody is shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region VL of the monoclonal antibody is shown in SEQ ID NO.

12.

3. A biomaterial related to the monoclonal antibody cAbD4 according to any one of claims 1 to 2, characterized in that: The biological material is selected from one of the following (A) to (B): (A) a nucleic acid molecule encoding the monoclonal antibody cAbD4 according to any one of claims 1 to 2; (B) Expression cassettes, recombinant vectors, and recombinant cell lines containing the nucleic acid molecule described in (A).

4. The biomaterial according to claim 3, characterized in that The nucleic acid sequence encoding CDR1-H shown in SEQ ID NO. 1 is shown in SEQ ID NO. 6; The nucleic acid sequence encoding CDR2-H shown in SEQ ID NO. 2 is shown in SEQ ID NO. 7; The nucleic acid sequence encoding CDR3-H shown in SEQ ID NO. 3 is shown in SEQ ID NO. 8; The nucleic acid sequence encoding CDR1-L shown in SEQ ID NO.4 is shown in SEQ ID NO.9; The nucleic acid sequence encoding CDR2-L is TTGGGTTCT; The nucleic acid sequence encoding CDR3-L shown in SEQ ID NO.5 is shown in SEQ ID NO.

10.

5. The biomaterial according to claim 3, characterized in that The nucleic acid sequence encoding the VH shown in SEQ ID NO.11 is shown in SEQ ID NO.13; The nucleic acid sequence encoding the VL shown in SEQ ID NO.12 is shown in SEQ ID NO.

14.

6. A method for preparing the monoclonal antibody cAbD4 according to any one of claims 1 to 2, characterized in that: include: The recombinant vector containing the nucleic acid molecule encoding the monoclonal antibody cAbD4 is transfected into the host cell; the transfected host cell is cultured and the supernatant is collected and purified to obtain the monoclonal antibody cAbD4.

7. The preparation method according to claim 6, characterized in that The recombinant vector is pCDNA3.4 plasmid, and the host cell is 293F cell.

8. Use of the monoclonal antibody cAbD4 according to any one of claims 1 to 2, the biomaterial according to any one of claims 3 to 5, or the monoclonal antibody cAbD4 prepared by the preparation method according to any one of claims 6 to 7 in the preparation of a product for detecting HBV.

9. A product for detecting HBV, characterized in that: The product comprises the monoclonal antibody cAbD4 according to any one of claims 1 to 2, the biomaterial according to any one of claims 3 to 5, or the monoclonal antibody cAbD4 prepared by the preparation method according to any one of claims 6 to 7.

10. A method for detecting HBV or HBc for non-diagnostic purposes, characterized in that: The method comprises: mixing the product according to claim 9 with a sample to be tested to detect the core protein HBc of HBV.

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