A monoclonal antibody cAbA1 against hepatitis B virus core protein and its preparation and application

Abstract

The present invention discloses a monoclonal antibody cAbA1 against the core protein of hepatitis B virus, and its preparation and application. cAbA1 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, "GVS," and SEQ ID NO.5, respectively. The monoclonal antibody obtained by screening in the present invention has a strong ability to bind to HBc. The present invention verifies the application of cAbA1 in various biochemical methods through examples, and verifies its ability to detect different HBV genotypes. The monoclonal antibody cAbA1 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] This invention belongs to the field of molecular biology and cellular immunology in the field of pharmaceutical technology, specifically relating to a monoclonal antibody cAbA1 of the core protein of hepatitis B virus and its preparation and application. Background Technology

[0002] Hepatitis B virus (HBV) infection remains a major global public health problem. Currently, while antiviral drugs such as nucleoside / acid analogs (NAs) and interferon-alpha (IFN-α) can effectively inhibit HBV DNA replication and disease progression in chronic HBV, the functional cure rate is very low. Therefore, there is still an urgent need to develop more effective strategies for the detection and treatment of HBV infection.

[0003] HBV is a hepatotropic DNA virus, approximately 42 nm in diameter. Its internal capsid is assembled from a core protein (HBc). In most HBV genotypes, the core protein consists of 183 amino acids (aa), including an N-terminal assembly domain (1-149 a.a.) for capsid formation and an arginine-rich C-terminal domain (150-183 a.a.) 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 encapsulates the viral RNA and polymerase, providing a closed environment for viral RNA reverse transcription and promoting nucleocapsid packaging and viral particle formation. Furthermore, HBc regulates capsid transport into the nucleus and is associated with cccDNA formation during infection. Besides functioning as a structural protein and playing a regulatory role in multiple stages of the viral life cycle, HBc also plays a crucial role in the pathogenesis of HBV-related diseases by inhibiting the host immune response or activating multiple signaling pathways, such as the MAPK and Wnt / β-catenin pathways. These functions suggest that HBc could be an important target for HBV detection and intervention.

[0004] HBV-related research primarily assesses HBV infection titers indirectly by detecting surface antigens, e antigens, and HBV DNA in cell supernatants. However, there are currently very few anti-HBc monoclonal antibodies available for detecting multiple HBV genotypes and performing various biochemical assays such as enzyme-linked immunosorbent assay (ELISA), Western blot, immunofluorescence assay (IFA), flow cytometry, immunospot assay, and immunohistochemistry (IHC). This poses a challenge for directly detecting HBV-infected cells. Therefore, developing more sensitive and broader-spectrum anti-HBc monoclonal antibodies is crucial for the advancement of HBV-related research. Summary of the Invention

[0005] To enable detection of multiple HBV genotypes and various biochemical methods, this invention screened and obtained a monoclonal antibody targeting the HBV core protein, which is characterized by high efficiency, broad spectrum, and versatility. This invention further provides a monoclonal antibody targeting HBc for the detection of HBV infection using multiple methods. This monoclonal antibody exhibits strong specific binding ability to the HBV core protein and can be detected through various biochemical experiments. This monoclonal antibody holds promise as a detection antibody for live HBV virus infection, providing a powerful tool for antigen detection of multiple HBV genotypes.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention is to provide a monoclonal antibody cAbA1 for the core protein of hepatitis B virus (HBV), said monoclonal antibody recognizing the core protein HBc of HBV.

[0008] Furthermore, the monoclonal antibody is a broad-spectrum human monoclonal antibody that can recognize HBc of all HBV genotypes, including 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, “GVS”, 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 has at least 80% similarity to it, 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 has at least 80% similarity to it.

[0011] It is understood that the heavy chain sequence and the paired light chain sequence constitute the aforementioned monoclonal antibody, and their variable regions determine the antibody's binding recognition and specificity to the antigen. Antibody specificity depends on the interaction between the antibody binding site and the antigen-determining region. The binding sites of both the heavy and light chains are primarily composed of residues in three complementarity-determining regions (CDRs), which are linked by frame regions (FRs). Therefore, those skilled in the art can easily determine the frame regions after knowing the amino acid sequences of the CDRs. That is, without altering the CDR sequences of the heavy and light chains, the function and application of the monoclonal antibody cAbA1 of this invention can be essentially achieved. Therefore, the specific sequence of the monoclonal antibody of this invention is not limited to the specific variable regions of the heavy chain and light chain sequences mentioned above; the variable regions of the above specific sequences are merely a specific monoclonal antibody sequence used in one implementation of this invention.

[0012] Proteins composed of amino acid sequences that are “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 substitution, proteins composed of amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence may correspond to homologous sequences derived from species different from the reference sequence. “Amino acid substitution” can be conserved or non-conserved. Preferably, the substitution is a conserved substitution, where one amino acid is replaced by another amino acid having similar structure and / or chemical properties. Specifically, the sequence of the variable region of the heavy or light chain differs from the reference sequence only in the conserved amino acid substitution.

[0013] A second aspect of the present invention is to provide biological materials related to the monoclonal antibodies described in the first aspect of the present invention, said biological materials being selected from one of (A) to (B) below:

[0014] (A) A nucleic acid molecule encoding the monoclonal antibody cAbA1 described in the first aspect of the present invention;

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

[0016] Furthermore, the expression cassette, recombinant vector, and recombinant cell line can be used to express the above-mentioned 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 as shown in SEQ ID NO.2 is shown in SEQ ID NO.7;

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

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

[0021] The nucleic acid sequence encoding CDR2-L is “GGTGTCTCC”;

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

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

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

[0025] It is understood that the specific nucleic acid sequences mentioned above are only the specific nucleic acid sequences used in one implementation of the present invention. Based on the degeneracy of the codon, 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 (e.g., conserved nucleotide sequence variants derived from genetic code degeneracy and silence variants, nucleotide substitutions, deletions and additions are also included) are all within the protection scope of the present invention, provided that the encoded amino acid sequence remains unchanged.

[0026] In the aforementioned biological materials, the expression cassette containing a nucleic acid molecule encoding an antibody refers to DNA capable of expressing the antibody in a host cell. This DNA may include not only a promoter that initiates transcription of the antibody gene, but also a terminator that terminates transcription of the antibody gene.

[0027] Among the aforementioned biological materials, a vector is a nucleic acid delivery tool that allows the insertion of polynucleotides encoding a protein, thereby enabling the protein to be expressed. Vectors can be transformed, transduced, or transfected into host cells, allowing the genetic material elements they carry to be expressed within the host cells. Vectors can be plasmids, granules, bacteriophages, or viral vectors.

[0028] In the aforementioned biological materials, the host cell refers to the cell into which the vector is introduced, including prokaryotic cells, fungal cells, insect cells, animal cells, etc., 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 this field.

[0029] A third aspect of the present invention is to provide a method for preparing the monoclonal antibody cAbA1 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 cAbA1; culturing the transfected host cell and collecting the supernatant; and purifying the supernatant to obtain the monoclonal antibody cAbA1.

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

[0031] The fourth aspect of this invention is to provide the use of the monoclonal antibody cAbA1 described in the first aspect of this invention, the biological material described in the second aspect of this invention, or the monoclonal antibody cAbA1 prepared by the preparation method described in the third aspect of this 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, the product comprising the monoclonal antibody cAbA1 described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, or the monoclonal antibody cAbA1 prepared by the preparation method described in the third aspect of the present invention.

[0033] Furthermore, the above-mentioned products can be diagnostic reagents (diagnostic kits), detection reagents (detection kits), etc., and may also include other reaction reagents. These products can be used in ELISA, Western blot, flow cytometry, IFA, immunospot, and immunohistochemistry methods to detect the core protein HBc of HBV.

[0034] A 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 aforementioned samples to be tested cover a variety of sample types obtained from subjects and can be used for diagnosis or testing, including but not limited to blood and other biological liquid samples, solid tissue samples, including clinical samples, cells in culture media, cell supernatant, cell lysate, serum, plasma, biological fluids and tissue samples, etc.

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

[0037] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects:

[0038] The monoclonal antibody cAbA1, obtained through screening in this invention, targets the core protein of hepatitis B virus (HBV) and exhibits strong binding affinity to HBc. This invention verifies the application of cAbA1 in various biochemical methods and its ability to detect different HBV genotypes through examples. cAbA1 is characterized by high efficiency, broad spectrum, and versatility, and has broad application prospects in the diagnosis of HBV infection. Attached Figure Description

[0039] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings:

[0040] Figure 1 This is a schematic diagram of the ELISA results of the monoclonal antibody cAbA1 binding to the core protein of genotype C HBV in Example 2 of the present invention.

[0041] Figure 2 This is a schematic diagram showing the results of the monoclonal antibody cAbA1 in Example 3 of the present invention in identifying HBc of different HBV genotypes.

[0042] Figure 3 This is a schematic diagram showing the results of using the monoclonal antibody cAbA1 in Example 4 of the present invention to identify HBc in HBV-infected cells by Western blot method.

[0043] Figure 4 This is a schematic diagram showing the results of using the monoclonal antibody cAbA1 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 monoclonal antibody cAbA1 in flow cytometry to detect HBc in HBV-infected cells in Example 6 of the present invention.

[0045] Figure 6 This is a schematic diagram of the results of using the monoclonal antibody cAbA1 in the immunospot assay to detect HBc in HBV-infected cells in Example 7 of the present invention.

[0046] Figure 7 This is a schematic diagram showing the results of using the monoclonal antibody cAbA1 in Example 8 of the present invention to detect intracellular HBc in HBV-infected liver tissue by immunohistochemistry. Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify the source are all commercially available raw materials. The equipment used in each step of the following embodiments are all conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0048] It should be noted that, unless otherwise specified, the embodiments and features described in 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 this is not intended to limit the scope of the invention.

[0049] Example 1: Antibody screening, expression, and purification

[0050] This study was approved by the Ethics Committee of Shenzhen Third People's Hospital (Approval No.: 2021-030). Written informed consent was provided by all participants, which was used 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 preserved in liquid nitrogen, and liver tissue was embedded in paraffin blocks.

[0051] This example uses one PBMC sample. The monoclonal antibody screening and purification steps are as follows:

[0052] (1) Monoclonal antibody screening

[0053] Resuscitation of cryopreserved 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 viable cells. Assume a cell count of 1 × 10⁻⁶ cells. 6Peripheral blood mononuclear cells were aliquoted into tubes and stained at 4°C for 30 minutes with 50 μL of staining buffer (PBS + 2% fetal bovine serum) containing Live / Dead dye (L34964, Invitrogen). After washing twice with PBS, peripheral blood mononuclear cells were resuspended in 50 μL of staining buffer with 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) and stained at 4°C for 30 minutes. After washing twice with PBS, peripheral blood mononuclear cells were resuspended in 50 μL staining buffer with DyLight 550 (84531, Invitrogen) and DyLight 650 (84536, Invitrogen) labeled genotype C HBc (P2301, Shenzhen Innosys Biotechnology Co., Ltd.) 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. Following the method 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.), RT-PCR and nested PCR were performed to amplify the variable regions of the heavy and light chains, respectively. After sequencing of the PCR amplification products by Sangon Biotech (Shanghai) Co., Ltd., 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, ultimately preparing a large number of antibody heavy and light chain plasmids.

[0055] (2) Antibody expression and purification

[0056] The monoclonal antibody in this embodiment was obtained by expression and purification in 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 cells 6 293F cells per mL were cultured for 2 hours in a shaker at 37°C with 8% CO2.

[0058] 2. Prepare transfection plasmid A: Take 200 μg each of antibody heavy chain plasmid and antibody light chain plasmid, and dilute them together in 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 stand for 5 minutes;

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

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

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

[0063] 7. Antibody purification: Open the cap of the ProteinA 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 0.1M glycine-hydrochloric acid solution (pH=3.0); place the eluent in a 30KD ultrafiltration concentration tube, centrifuge at 3500g at 4℃ until the liquid in the concentration tube is less than 1mL; discard the waste liquid in the collection tube and fill the concentration tube with PBS solution, centrifuge under the same conditions until the liquid in the concentration tube is less than 1mL, and determine the antibody concentration.

[0064] Example 2: Enzyme-linked immunosorbent assay (ELISA) to verify the binding ability of monoclonal antibody cAbA1

[0065] One antibody was selected from the antibodies obtained in Example 1 for monoclonal antibody expression and related functional verification, and named cAbA1. The amino acid sequences of CDR1-H, CDR2-H, and CDR3-H of the heavy chain variable region VH of cAbA1 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. 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 cAbA1 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively. CDR2-L is “GVS”. 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. The nucleic acid sequence encoding CDR2-L is “GGTGTCTCC”. The amino acid sequence of the cAbA1 heavy chain variable region VH 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 cAbA1 light chain variable region VL 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 monoclonal antibody cAbA1 to the HBV core protein HBc was detected by ELISA. 2 μg / mL, 100 μL / well of genotype C HBc (P2301, Shenzhen Innosys Biotechnology Co., Ltd.) was coated into 96-well microplates and incubated overnight at 4°C. The plates were washed 5 times with 0.05% Tween-20 PBS solution (PBST). 200 μL / well of blocking buffer (5% skim milk + 2% BSA, prepared in PBS) was prepared and blocked at room temperature for 1 hour. After washing 3 times, the cAbA1 antibody was diluted to a maximum concentration of 10 μg / mL with blocking buffer and then serially diluted 5-fold (7 dilutions total). 100 μL of each diluted antibody was added to each well of the plate washed 3 times with PBST and incubated at 37°C for 1 hour. Wash the plate 5 times with PBST. Dilute HRP-goat anti-human IgG (ZB-2304, Zhongshan Jinqiao) 1:5000 in blocking buffer, 100 μL / well, and incubate at 37°C for 1 hour. Wash the plate 5 times with PBST. Mix colorimetric solutions A and B (E661007, Sangon Biotech) 1:1, 100 μL / well, and incubate at room temperature in the dark for 20 minutes. Finally, add 50 μL of 2 mol / L H2SO4 to stop the reaction. Measure the optical density at 450 nm (OD) using a Varioskan LUX multimodal microplate reader (Thermo Scientific).

[0067] The binding activity of the monoclonal antibody cAbA1 to the HBV core protein HBc was analyzed by ELISA. The results are as follows: Figure 1 As shown, the monoclonal antibody cAbA1 can specifically bind to HBc of HBV genotype C, exhibiting strong binding ability and a half-maximal effective concentration (EC50). 50 It was 2.73 ng / mL.

[0068] Example 3: Flow cytometry verification of the broad-spectrum recognition capability of monoclonal antibody cAbA1

[0069] This embodiment uses flow cytometry to verify that the monoclonal antibody cAbA1 recognizes intracellularly expressed HBc 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, with cells transfected with the empty Vector vector serving as a control. After 48 hours, cells were digested with 0.5% trypsin and the process was terminated by adding complete DMEM medium containing 10% FBS. Cells were gently pipetted and observed under a microscope until more than 90% of the cells were in a single-cell state. The single-cell suspension was transferred to a 15mL centrifuge tube and centrifuged at 400g for 5 minutes. The supernatant was discarded, and 3mL of complete DMEM was added for culture. 20μL of the cell suspension was taken, and 20μL of AO / PI dye (RE010212, Countstar) was added. The viable cell count was determined to be 5×10⁶ cells / year. 5 Cells / tubes. Cells were fixed and permeabilized using a cell fixation / permeabilization kit (554714, BDBiosciences), centrifuged at 400g for 5 minutes, and the supernatant was discarded. Then, 5 μg / mL cAbA1 was added to each flow cytometry tube, and the cells were 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 Data were acquired using an A3 flow cytometer (BD Biososciences), and analyzed using FlowJo software V10.9 (BD Biososciences). Experimental results are as follows: Figure 2 As shown.

[0071] The results showed that cAbA1 antibody can clearly detect intracellularly expressed HBc and can widely identify HBc from different HBV genotypes A, B, C, D, E, F, G, H, I and J.

[0072] Example 4: Verification of the function of monoclonal antibody cAbA1 using Western blot experiment

[0073] This embodiment uses the monoclonal antibody cAbA1 in a Western blot method to identify HBc in HBV-infected cells. The specific experimental steps are as follows:

[0074] This experiment was conducted in a certified biosafety level 2 laboratory. 1.5 × 10⁻⁶ 6HepG2-NTCP cells were seeded 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 a concentration of 1800 copies / cell was added to DMEM medium containing 4% PEG8000 (89510, Sigma), 2.5% DMSO, and 5% FBS, mixed well, and then added to the HepG2-NTCP cells. Twenty hours after HBV infection, the cells were diluted three times with PBS and cultured in DMEM medium containing 2.5% DMSO and 16% FBS. The medium was changed every two days. On day 7, the HepG2-NTCP cells were washed twice with PBS, and 300 μL of 1.5× Protein Loading Buffer (DL101-02, TransGold) was added to collect the cells. Cell samples treated at 100°C for 10 minutes were subjected to SDS-PAGE electrophoresis and then transferred to PVDF membranes, blocked with 5% skim milk at room temperature for 1 hour. After washing twice with TBST, 0.5 μg / mL cAbA1 was added and incubated overnight at 4°C. After washing five times with TBST, HRP-goat anti-human IgG (ZB-2304, Zhongshan Jinqiao) diluted 1:5000 was added and incubated at room temperature for 1 hour. After washing five times with TBST, the membrane was developed for color, and the experimental results are as follows. Figure 3 As shown.

[0075] Western blot results showed that, compared with uninfected HBV cells, HBV-infected cells had the same β-actin expression level in the internal reference band, and the total number of cells was similar; however, the HBc-specific band was only expressed in infected cells and could be identified by the monoclonal antibody cAbA1. This indicates that the monoclonal antibody cAbA1 can specifically bind to intracellular HBc after HBV infection.

[0076] Example 5: Verification of the function of monoclonal antibody cAbA1 using immunofluorescence assay (IFA)

[0077] This embodiment uses the monoclonal antibody cAbA1 for IFA detection to identify HBc in HBV-infected cells. The specific experimental steps are as follows:

[0078] The HBV infection procedure was the same as 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 one wash with PBS, the cells were permeabilized for 30 minutes with PBS solution containing 0.2% Triton X-100, and then QuickBlock was added. TMImmunostaining blocking buffer (P0260, Beyotime) was used, and incubated at room temperature for 1 hour. After washing twice with PBS, 10 μg / mL cAbA1 antibody was added and incubated overnight at 4°C. After washing 5 times with PBS, Alexa Fluor was added at a 1:1000 ratio. TM 488-Goat anti-Human IgG (H+L) Secondary Antibody (A11013, Thermo Fisher) was incubated at room temperature for 1 hour. After washing three times with PBS, DAPI staining solution (C1002, Beyotime) was added and incubated for 10 minutes. After washing twice with PBS, images were taken using a fluorescence microscope. The experimental results are shown below. Figure 4 As shown.

[0079] The results showed strong HBc fluorescence intensity under the field of view, indicating that the monoclonal antibody cAbA1 can clearly recognize HBc in the cytoplasm and nucleus of HBV-infected cells.

[0080] Example 6: Verification of the ability of monoclonal antibody cAbA1 to recognize HBV-infected cells using flow cytometry

[0081] In this embodiment, the monoclonal antibody cAbA1 was applied to flow cytometry to verify its recognition of HBc in HBV-infected cells. The specific experimental steps are as follows:

[0082] The HBV infection procedure in this experiment was the same as in Example 4. Seven days after infection, the cells were washed twice with PBS, and then digested at 37°C for 5 minutes with 0.5% trypsin. Afterwards, DMEM medium with 10% FBS was added to terminate the digestion, and the cells were gently pipetted until more than 90% of the cells were in a single-cell state. The single-cell suspension was transferred to a 15mL centrifuge tube and centrifuged at 300g for 5 minutes. The supernatant was discarded, and 3mL of complete DMEM medium was added. 20μL of the cell suspension was mixed with an equal volume of AO / PI dye, and viable cells were counted at 5×10⁻⁶. 5 Cells / tubes were dispensed. Subsequent staining and data collection procedures were the same as in Example 3, and the experimental results are as follows. Figure 5 As shown.

[0083] The results showed that the monoclonal antibody cAbA1 could specifically recognize HBc in HBV-infected cells, clearly distinguishing between HBV-infected and non-infected cells.

[0084] Example 7: Verification of HBV infection detection by monoclonal antibody cAbA1 using live virus immunospot assay

[0085] In this embodiment, the monoclonal antibody cAbA1 is used in an immunospot assay to detect HBc in HBV-infected cells. The specific experimental steps are as follows:

[0086] This experiment was conducted in a certified biosafety level 2 laboratory. 0.43 × 10⁻⁶ 5 HepG2-NTCP cells were seeded in 96-well plates coated with type I rat tail collagen (354236, Corning) and cultured for more than 18 hours. HBV virus was added to DMEM medium containing 4% PEG8000 (89510, Sigma), 2.5% DMSO, and 5% FBS at concentrations of 1800, 600, 300, and 0 gene copies / cell, respectively, and then added to the HepG2-NTCP cells. Twenty hours after HBV infection, the cells were diluted three times with PBS and cultured in DMEM medium containing 2.5% DMSO and 16% FBS. The medium was changed every two days. On day 7, HepG2-NTCP cells were washed twice with PBS and fixed with 4% paraformaldehyde solution at room temperature for 30 minutes. After one PBS wash, the cells were permeabilized with PBS solution containing 0.2% Triton X-100 for 30 minutes. Then, QuickBlock™ immunostaining blocking solution (P0260, Beyotime) was added, and the cells were incubated at room temperature for 1 hour. After washing twice with PBS, 10 μg / mL cAbA1 antibody was added and incubated overnight at 4°C. After washing five times with PBS, HRP-goat anti-human IgG (ZB-2304, Zhongshan Jinqiao) diluted 1:5000 was added and incubated at room temperature for 1 hour. After washing five times with PBS, KPL TrueBlue peroxidase substrate (Seracare Life Sciences) was used for color development. Cell imaging was performed using a Cytation 7 (BioTek) multi-well microplate reader to capture images and count the number of HBV-infected cells. Experimental results are as follows. Figure 6 As shown.

[0087] The results showed that cAbA1 could clearly identify HBV-infected cells and the number of spots decreased as the viral load of HBV decreased, indicating that cAbA1 can be used in immunospot assays to detect the HBV core protein HBc and is dose-dependent.

[0088] Example 8: Verification of the ability of monoclonal antibody cAbA1 to recognize HBV-infected liver tissue using immunohistochemistry.

[0089] In this embodiment, the monoclonal antibody cAbA1 was applied to IHC detection to verify its recognition of 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 fixed in formalin, embedded in paraffin, sectioned, dewaxed in xylene, and hydrated with ethanol of different concentrations. After rinsing with tap water for 1 minute, 100 μL of endogenous peroxidase inhibitor (Kit-0014, Maixin Biotechnology Development Co., Ltd.) was added to the tissues and incubated at room temperature for 10 minutes. After washing three times with PBS, 100 μL of antibody dilution buffer (ABD-0030, Maixin Biotechnology Development Co., Ltd.) was added, and the tissues were blocked at room temperature for 1 hour. After washing three times with PBS, 100 μL of 20 μg / mL HRP-conjugated cAbA1 antibody (cAbA1-HRP) was added, and the tissues were incubated at room temperature for 2 hours. After washing five times with PBS, DAB reagent (Kit-0014, Maixin Biotechnology Development Co., Ltd.) was used for staining at room temperature for 5-10 minutes. After rinsing with tap water, hematoxylin staining was performed for 20 seconds, and the PBS returned to blue after rinsing with tap water. After dehydration, clearing, and sealing, images were taken using a Motic VM1000 (Motic Electric Group Co., Ltd.). The experimental results are as follows: Figure 7 As shown.

[0091] The results showed that cAbA1 could clearly recognize HBc in HBV-infected hepatocytes in Donor 1 and Donor 2 liver tissues. The distribution of HBc differed between the two types of hepatocytes; it was present in both the cytoplasm and nucleus of Donor 1 hepatocytes, while it was mainly found in the cytoplasm of Donor 2 hepatocytes. This indicates that cAbA1 can be used for clinical immunohistochemical detection of the core protein HBc in the liver tissues of HBV patients.

[0092] As can be seen from the above embodiments, the monoclonal antibody cAbA1 obtained by screening in this 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 merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody cAbA1 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, GVS and SEQ ID NO.5, respectively.

2. The monoclonal antibody cAbA1 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region VH of the monoclonal antibody is 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 shown in SEQ ID NO.12 or an amino acid sequence having at least 80% identity thereto.

3. A biomaterial related to the monoclonal antibody cAbA1 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 cAbA1 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 GGTGTCTCC; 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 cAbA1 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 cAbA1 is transfected into a host cell; the transfected host cell is cultured and the supernatant is collected and purified to obtain the monoclonal antibody cAbA1.

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 cAbA1 according to any one of claims 1 to 2, the biomaterial according to any one of claims 3 to 5, or the monoclonal antibody cAbA1 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 cAbA1 according to any one of claims 1 to 2, or the monoclonal antibody cAbA1 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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