An antibody against hepatitis b virus e antigen and its preparation method and use

Abstract

The application discloses an antibody against hepatitis B virus e antigen and a preparation method and application thereof, and relates to the field of hepatitis B antigen detection. Specifically, the antibody provided by the application has HCDR1-3 as shown in SEQ ID NO:1, 2 and 3 respectively and LCDR1-3 as shown in SEQ ID NO:4, 5 and 6 respectively; or has HCDR1-3 as shown in SEQ ID NO:7, 8 and 9 respectively and LCDR1-3 as shown in SEQ ID NO:10, 11 and 12 respectively. The provided antibody can specifically recognize human hepatitis B e antigen, the positive coincidence rate, the negative coincidence rate and the total coincidence rate of a detection reagent constructed using the antibody and the Abbott control reagent are all 100%; compared with existing antibodies, the stability and repeatability of detection are significantly improved, and the early detection rate of the reagent kit has important value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to antibodies against hepatitis B virus e antigen, their preparation methods, and uses. Background Technology

[0002] Hepatitis B virus (HBV) infection is one of the leading causes of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. Hepatitis B e antigen (HBeAg), as an important serological marker of active viral replication, directly impacts antiviral treatment decisions and efficacy assessments in patients with chronic hepatitis B.

[0003] Currently, clinical detection of HBeAg mainly relies on immunological methods, such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay. These methods typically use monoclonal antibodies targeting specific epitopes of HBeAg as the core capture or detection reagent. However, HBV exhibits high genetic variability, particularly mutations in the pre-C region, which are prevalent in clinical isolates. Pre-C mutations can alter the molecular structure of HBeAg, preventing existing monoclonal antibodies from binding effectively and resulting in false-negative results. This missed detection can lead to misjudgment of a patient's viral replication status in clinical practice, delaying antiviral treatment and even increasing the risk of disease progression.

[0004] Existing research indicates that the mutation frequency in the pre-C region varies significantly among HBV isolates of different genotypes (e.g., B and C) and from different geographical origins, making it difficult for monoclonal antibodies that recognize a single epitope to achieve broad-spectrum coverage. Therefore, developing monoclonal antibodies capable of recognizing multiple pre-C region mutant HBeAg with high affinity and high sensitivity to improve the clinical accuracy of HBeAg detection reagents has significant clinical application value and market potential. Summary of the Invention

[0005] To improve upon this invention, a monoclonal antibody against hepatitis B e antigen was designed and screened. The antibody was then validated against existing positive reagent kits and other highly efficient detection antibodies, fully demonstrating its detection advantages. Specifically, the technical solution includes the following:

[0006] On the one hand, this application provides a binding molecule against hepatitis B e antigen.

[0007] In some embodiments, the binding molecule is an antibody against hepatitis B e antigen or an antigen-binding fragment thereof.

[0008] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region.

[0009] In some embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:14; in some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:13. In some specific embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:14; and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:13.

[0010] In some embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:26; in some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:25; and in some embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO:26; and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO:25. In some specific implementations, the CDR shown is defined according to the numbering system of Kabat, IMGT, Chothia, AbM, or Contact.

[0011] In some specific implementations, the CDR shown is defined according to Kabat's numbering system.

[0012] In some embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:4, 5, and 6, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:1, 2, and 3, respectively. In some specific embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in SEQ ID NO:4, 5, and 6, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in SEQ ID NO:1, 2, and 3, respectively.

[0013] In some specific embodiments, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:4, 5, and 6, respectively, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:1, 2, and 3, respectively.

[0014] In some embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:10, 11, and 12, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in or having at least 95% identity with SEQ ID NO:7, 8, and 9, respectively. In some specific embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in SEQ ID NO:10, 11, and 12, respectively, and HCDR1, HCDR2, and HCDR3 each comprise amino acid sequences as shown in SEQ ID NO:7, 8, and 9, respectively.

[0015] In some specific embodiments, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:10, 11, and 12, respectively, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:7, 8, and 9, respectively.

[0016] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:14, and the heavy chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:13; in some specific embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:14, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:13; in some specific embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:13.

[0017] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:26, and the heavy chain variable region comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:25. In some specific embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:26, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:25. In some specific embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:26, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:25.

[0018] In some embodiments, the antibody is a murine antibody, a chimeric antibody, a recombinant antibody, or a humanized antibody; the antigen-binding fragment is Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, or dAb.

[0019] In some embodiments, the antibody or its antigen-binding fragment further includes an immunoglobulin Fc region. In some specific embodiments, the Fc is selected from IgG1, IgG2, IgG3, or IgG4.

[0020] In some embodiments, the light chain comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:18, and the heavy chain comprises an amino acid sequence as shown in or having at least 95% identity with SEQ ID NO:17. In some specific embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:18, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:17. In some specific embodiments, the amino acid sequence of the light chain is as shown in SEQ ID NO:18, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:17.

[0021] In some embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:30 or having at least 95% identity with it, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:29 or having at least 95% identity with it. In some specific embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:30, and the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:29. In some specific embodiments, the amino acid sequence of the light chain is as shown in SEQ ID NO:30, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO:29.

[0022] The present invention also provides a biomaterial.

[0023] In some embodiments, the biomaterial includes any one of the following: (1) A nucleic acid molecule, wherein the nucleic acid molecule encodes an antibody or an antigen-binding fragment thereof as described in any of the places above; (2) Expression vectors / vector groups containing any of the nucleic acid molecules described above; (3) A host cell containing any of the nucleic acid molecules or expression vectors / vector groups described above.

[0024] In some embodiments, the gene sequence encoding the heavy chain variable region of the nucleic acid molecule is shown in SEQ ID NO: 19; and the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 20.

[0025] In some embodiments, the gene sequence encoding the heavy chain of the nucleic acid molecule, which encodes the antibody or its antigen-binding fragment, is shown in SEQ ID NO: 21; and the gene sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO: 22.

[0026] In some embodiments, the gene sequence encoding the heavy chain variable region of the nucleic acid molecule is shown in SEQ ID NO: 31; and the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 32.

[0027] In some embodiments, the gene sequence encoding the heavy chain of the nucleic acid molecule, which encodes the antibody or its antigen-binding fragment, is shown in SEQ ID NO: 35; and the gene sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO: 36.

[0028] The present invention also provides a method for preparing antibodies or antigen-binding fragments thereof as described in any of the above claims.

[0029] In some embodiments, the method includes culturing host cells as described in any of the preceding embodiments. In some specific embodiments, the cultured host cells express an antibody or an antigen-binding fragment thereof. In some specific embodiments, the method further includes isolating the antibody or the antigen-binding fragment thereof. In some specific embodiments, the method further includes purifying the antibody or the antigen-binding fragment thereof.

[0030] The present invention also provides a composition against human hepatitis B e antigen.

[0031] In some embodiments, the composition comprises the antibody or antigen-binding fragment thereof described in any of the preceding embodiments.

[0032] The present invention also provides the use of an antibody or antigen-binding fragment thereof as described in any of the preceding claims, or a composition as described in any of the preceding claims.

[0033] In some implementations, the application includes: products for preparing anti-human hepatitis B e antigen, or for detecting human hepatitis B e antigen not for the purpose of disease diagnosis and treatment; said products are test kits, test strips, or test chips.

[0034] In some specific implementations, the detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunochromatographic kit, an immunoturbidimetric detection kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit, or a radioimmunoassay kit; the test strip is a colloidal gold test strip or an ELISA test strip. Attached Figure Description

[0035] Figure 1 This shows the chromatographic pattern of the target protein. The peak of the target protein is highlighted in red.

[0036] Figure 2 This represents an SDS-PAGE electrophoresis image. Channel M corresponds to the protein marker; channel 1 in A corresponds to the 5F5 antibody, and channel 1 in B corresponds to the 14D6 antibody. Detailed Implementation

[0037] The following description of specific embodiments further illustrates this application, but this is not intended to limit the scope of this disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from its basic ideas and scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products; the methods described, unless otherwise specified, are all conventional techniques in the art.

[0038] Example 1: Preparation of Immunogen 1.1 Sample Pretreatment Positive serum from hepatitis B patients was collected, centrifuged at 10,000 rpm and 4°C for 30 min, and filtered at 0.45 μm.

[0039] 1.2 Ammonium sulfate precipitation Collect the pretreated serum, add 15% solid ammonium sulfate according to the mass-volume ratio, stir thoroughly to dissolve, stir at 4°C for 15 h, centrifuge at 10000 r at 4°C for 30 min, collect the precipitate, redissolve it in Tris solution at pH 8.5, filter at 0.45 μm to prepare for the next step.

[0040] 1.3 Gel Filtration Chromatography Equilibrate the Chromdex 75 PG column (16mm x 600mm) thoroughly with mobile phase (20mM Tris, pH 8.5), load 3ml of the pretreated protein, and maintain a flow rate of 0.2ml / min. Collect 1.5ml portions per tube. The target protein is highlighted in red. Collect peak 4. Its chromatogram (e.g., [image not provided]) is shown below. Figure 1 ).

[0041] 1.4 Density gradient centrifugation Prepare sucrose density gradient solutions with mass fractions of 20%, 30%, 40%, 50%, and 60% using 20 mM PBS. Filter each solution through a 0.22 μm filter and then spread them evenly in ultracentrifuge tubes according to density from highest to lowest. Spread 1 ml of the prepared protein concentrate on top of the density gradient sucrose solution and centrifuge at 30,000 r / min for 4 h at 20 °C. After centrifugation, aspirate the protein bands and dialyze them against PBS at 4 °C; collect the protein and store it at -20 °C.

[0042] Example 2: Preparation of monoclonal antibody against human hepatitis B e antigen 2.1 Animal Immunization Five 6-8 week old female Balb / c mice were immunized. The purified hepatitis B e antigen was mixed with Freund's complete adjuvant at a dose of 50 μg / mouse, completely emulsified, and administered in 200 μL per mouse per immunization. The mice were immunized for the first time at multiple sites on the back, groin, and axilla. Two weeks later, a second immunization was administered at the same dose and route, but with an incomplete adjuvant. Fifteen days later, a third immunization was administered at the same dose, with Freund's incomplete adjuvant added, via intraperitoneal injection. One week later, blood was collected from the tails of all immunized mice, and serum antibody titers were measured by ELISA. Mice with serum titers exceeding 100,000 were selected for fusion cell preparation. Three days before fusion, mice were immunized intraperitoneally at a dose of 100 μg / mouse without adjuvant.

[0043] 2.2 Construction of B cells (1) Culture and preparation of myeloma cell lines This embodiment uses the SP2 / 0 myeloma cell line, which exhibits excellent growth and fusion efficiency, with a doubling time of 10-12 hours. Myeloma cells in the logarithmic growth phase, with optimal cell morphology and viability, are selected for fusion. Myeloma cells should undergo adaptive culture before fusion to allow them to grow to their optimal state (i.e., the logarithmic growth phase).

[0044] On the day of fusion, gently blow myeloma cells off the bottle wall with a bent dropper and collect them in a 50ml centrifuge tube or fusion tube. Centrifuge at 1000r / min for 5-10min. Discard the supernatant, add 30ml of culture medium to the precipitate, centrifuge and wash once, centrifuge for 5-10min. Discard the supernatant, mix the precipitate with 20ml of culture medium and set aside.

[0045] (2) Preparation of spleen cells BALB / c mice that have already undergone animal immunization were enucleated to collect blood, and the serum was separated using a centrifuge to serve as a positive control serum for antibody detection.

[0046] Mice were euthanized by cervical dislocation and immersed in 75% alcohol for 5 minutes. The spleen was aseptically removed in a laminar flow hood and placed in a petri dish containing 10 ml of culture medium. The spleen was gently washed and the surrounding connective tissue was removed. The spleen was punctured with a sterile syringe until it turned white. The spleen cells in the petri dish were collected into a 50 ml centrifuge tube and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, 10 ml of culture medium was added, and a small amount of 10-fold dilution was taken for counting.

[0047] (3) Cell fusion Three days prior to fusion, positive mice were boosted with immunization. Mouse spleen cells and myeloma cells (sp2 / 0) were mixed at a ratio of 10:1, fused with PEG, and then cultured in HAT selective medium. Ten days later, the hybridoma cell supernatant was screened using ELISA. The selected positive hybridoma cells were cloned using limiting dilution. After five rounds of screening, multiple positive hybridoma cell lines were finally identified.

[0048] (4) Screening of hybridoma cells (ELISA method) Hepatitis B e antigen (HBeAg) at 10 μg / mL, 50 μL / well, was coated onto a 96-well plate and incubated overnight at 4°C. The liquid in the wells was discarded the following morning, and the plates were washed three times with washing buffer, patted dry, and 100 μL of blocking buffer was added to each well for blocking at 37°C for 2 h. The plates were then washed twice and patted dry. 100 μL of culture supernatant from 10 hybridoma cell lines to be tested was added to each well, along with positive, negative, and blank control standards. The plates were incubated at 37°C for 0.5 h, washed four times, and patted dry. Horseradish enzyme-labeled goat anti-mouse IgG (1:10000), 100 μL, was added to each well of the ELISA plate and incubated at 37°C for 30 min. The plates were washed four times and patted dry.

[0049] Finally, 100 μL of TMB substrate chromogenic solution was added to each well, and the reaction was terminated with 50 μL of 2 mol / L dilute hydrochloric acid per well after 15 min of development. As shown in Table 1, the OD values ​​of each well were measured at dual wavelengths of 450 nm and 630 nm. The blank well value was below 0.02, and the negative well value was below 0.1, showing a clear distinction between positive and negative. This indicates that hybridoma cells anti-human hepatitis B e antigen secrete antibodies that specifically recognize human hepatitis B e antigen. The titer in mouse ocular blood was measured using the same method, reaching 1:100,000, which is suitable for cell fusion.

[0050] Table 1. Results of OD value measurement ; 2.3 Production and purification of monoclonal antibodies (1) Preparation of ascites Two hybridoma cell lines with relatively strong hybridoma cell lines were selected for ascites production. Balb / c mice weighing approximately 25 grams were selected, and each mouse was injected intraperitoneally with 500 μL of liquid paraffin. Two weeks later, hybridoma cells containing anti-human hepatitis B e antigen were injected intraperitoneally at a dose of 10. 6 Each mouse was observed for 10 days. Once the abdomen was significantly distended, the ascites fluid was collected, centrifuged at 10,000 rpm for 10 minutes, the supernatant was collected, impurities were removed, and the fluid was stored at -20°C.

[0051] (2) Purification of ascites The ascites fluid was removed from the -20°C freezer, thawed, and centrifuged at 10,000 rpm. The supernatant was filtered through a 0.22 μm filter. Affinity purification was performed according to the instructions for the protein-A column from Tiandi Renhe. The purified antibody was collected, dialyzed against 20 mM pH 7.4 PBS, and collected after 72 h. After filtration through a 0.22 μm filter, the antibody was aliquoted and stored at -20°C for subsequent detection and validation.

[0052] 2.4 Characterization of Monoclonal Antibodies According to routine gene sequencing, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the 5F5 antibody are shown in SEQ ID NO:1-3, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:4-6, as detailed in Table 2 below.

[0053] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the 14D6 antibody are shown in SEQ ID NO:7-9, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NO:10-12. See Table 2 below for details.

[0054] Table 2. Distribution of antibody CDR sequences (Kabat number) ; Antibody concentration determination: The absorbance values ​​(A280) and (A260) of the monoclonal antibodies at 280 nm and 260 nm were determined by ultraviolet spectrophotometry. The resulting protein contents were HBe-5F5 antibody (5 mg / mL) and HBe-14D6 antibody (3 mg / mL). The protein content was calculated using the following formula: Protein content (mg / mL) = (A280 × dilution factor) / 1.35.

[0055] Antibody molecular weight determination: Monoclonal antibodies were measured using SDS-PAGE, such as... Figure 2 As shown, the heavy chain of the monoclonal antibody is approximately 46 kDa, and the light chain is approximately 25 kDa.

[0056] ELISA titer determination: The purified ascites monoclonal antibody was determined by indirect ELISA, as shown in Table 3. The results showed that the purified titer was greater than 1:100000.

[0057] Table 3. Results of indirect ELISA assay ; Experimental Example 3: Application of 5F5 and 14D6 antibodies in chemiluminescent reagents for hepatitis B virus e antigen 3.1 Preparation of monoclonal antibodies coated with magnetic microparticles: Magnetic microparticles with a particle size of 1.5-3 μm, along with EDC and NHS, were added to a 50 mM MES solution at pH 5.0 at a mass ratio of 5:1:2. The concentration of magnetic microparticles was 10 mg / mL, the reaction temperature was 37°C, and the reaction time was 30 min. The reacted magnetic microparticles were then conjugated with 3C3 (patent number: CN 119661697 B) and 5F5 monoclonal antibodies, respectively. The ratio of magnetic microparticles to antibodies was 10 μg of 3C3 or 5F5 antibody per milligram of magnetic microparticles. The conjugation process was carried out at 25°C for 5 h. After coupling, the magnetic microparticles were washed three times with washing solution, and then phosphate buffer containing 2% glycine, 1% BSA, 0.05% Tween 20, 0.05% ProClin 300, and pH=7.4 was added to make the magnetic microparticle concentration 10 mg / mL. After standing at 25°C for 2 hours, the mixture was stored at 2-8°C for later use.

[0058] 3.2 Preparation of alkaline phosphatase-labeled monoclonal antibodies (1) Take 2.0 mg AP and dilute it to 10 mg / mL with phosphate buffer containing 0.15 M sodium chloride, and add 0.08 mg TR to it. React at 37°C for 1 h.

[0059] (2) Take 1 mg of 3A3 (patent number: CN 119661697 B) and 14D6 antibody respectively, adjust the concentration to 1 mg / mL, add 0.034 mg of SMCC respectively, and react at 37°C for 1 h.

[0060] (3) Divide the alkaline phosphatase that has completed the reaction into two tubes, add 3A3 and 14D6 antibodies that have been treated with SMCC, mix them evenly at a mass ratio of 1:1, and react at 25°C for 1 hour. After the reaction is completed, add 0.1 mL of phosphate buffer containing 0.15 M sodium chloride, 2% cysteine, and pH=7.4, let stand for 1 hour, add 1 mL of glycerol, and store at -20°C for later use.

[0061] 3.3 Reagent Performance Testing Magnetic microparticles coated with 3C3 and 5F5 antibodies were diluted 20-fold with phosphate buffer containing 0.15M sodium chloride, and alkaline phosphatase-labeled 3A3 and 14D6 antibodies were diluted 1000-fold with phosphate buffer containing 0.15M sodium chloride before testing. The commercially available positive test samples were 1068 clinical samples tested using Abbott Laboratories' Hepatitis B e Antigen Assay Kit and control reagents (3C3-3A3).

[0062] 3.3.1 Stability Test Experimental objective: The stability of the test reagent (5F5-14D6) and the control reagent (3C3-3A3) described in this invention under different storage conditions was investigated to verify its applicability.

[0063] Experimental methods: Magnetic microparticles coated with 3C3 and 5F5 antibodies were diluted 20-fold with phosphate buffer containing 0.15M sodium chloride, mixed thoroughly, and divided into two portions. One portion was labeled for storage at 4℃, and the other at 37℃. Alkaline phosphatase-labeled 3A3 and 14D6 antibodies were diluted 1000-fold with phosphate buffer containing 0.15M sodium chloride, mixed thoroughly, and divided into two portions. One portion was labeled for storage at 4℃, and the other at 37℃. After 7 days, the samples were taken out simultaneously. The test reagent (5F5-14D6) stored at 4℃ and the control reagent (3C3-3A3) stored at 4℃ and the control reagent stored at 37℃ were compared. The detection signals of samples at different concentrations were detected by chemiluminescent microparticle immunoassay.

[0064] 3.4 Experimental Results As shown in Table 4, the positive concordance rate, negative concordance rate, and overall concordance rate of the reagent described in this invention are all 100% with Abbott's control reagent. All indicators show that the results of the "test reagent" and the "Abbott reagent" are completely consistent with each other, with no difference whatsoever. Kappa=1, indicating perfect consistency. This demonstrates that the detection reagent prepared by the antibody of this invention has fully met the requirements of commercial grade.

[0065] Table 4. Chemiluminescent microparticle immunoassay results of test reagents and Abbott reagents ; As shown in Table 5, the antibody pair 5F5-14D6 provided by this invention, compared with the existing antibody pair 3C3-3A3, achieved a 100% concordance rate for negative and positive results, as well as a 100% overall concordance rate in the tested samples. After being stored at 37°C for 7 days, the detection signal attenuation rate of the test reagent (5F5-14D6) was 5%; in contrast, the control reagent (3C3-3A3) showed an 8% signal attenuation rate on the same day, indicating that the test reagent (5F5-14D6) has superior anti-degradation ability. The core advantage of this antibody pair 5F5-14D6 lies in its better reagent stability, thus improving diagnostic accuracy.

[0066] Table 5. Chemiluminescent microparticle immunoassay results for test reagents and control reagents ; Example 4: Application of 5F5 and 14D6 antibodies in hepatitis B virus e antigen latex chromatography test strips 4.1 Preparation of coating membrane 5F5 and goat anti-mouse IgG were diluted to 1 mg / mL with 10 mM phosphate buffer (pH 7.4), and then streaked onto the T and C lines of a nitrocellulose membrane at a volume of 1 μL / cm. The membranes were then dried in a 37°C oven for 20 hours before use.

[0067] 4.2 Preparation of Latex Pads 100 μL of 400 nm latex microspheres (4%) were added to 5 mL of MES buffer, followed by 100 μL of 20 mg / mL EDC solution and 500 μL of 20 mg / mL NHS solution. The mixture was stirred at room temperature for 1 h to activate the microspheres. After centrifugation at 10,000 rpm for 10 min to remove the supernatant, 5 mL of MES buffer was added. The mixture was then sonicated in a water bath for 10-20 s. 400 μg of 14D6 antibody (purchased from Wuhan Aokebotai Biotechnology Co., Ltd., catalog number A0911) was added to the solution. The mixture was stirred at room temperature for 2 h to react. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 6 min to remove the supernatant. 4 mL of microsphere storage solution was added. The mixture was sonicated in an ice bath for 7 min and stirred at room temperature for 2 h. The mixture was then incubated at 37 °C overnight. The storage concentration of the microspheres was 1 mg / mL (0.1%).

[0068] The 14D6-labeled microspheres at a storage concentration of 1 mg / mL were diluted to 0.5 mg / mL with microsphere preservation solution and sprayed onto the treated conjugate pads using an XYZ three-dimensional gold spraying spectrometer at a spray rate of 2 μL / cm. The conjugate pads were then dried in a 37°C oven for 20 hours. The dried conjugate pads were then cut to 300 mm × 8 mm dimensions for later use.

[0069] 4.3 Reagent Performance Testing The prepared 5F5 coated membrane was combined with a 14D6 latex pad, and after attaching the sample pad, it was cut into 3mm test strips. The strips were then tested with the National Reference Material for Rapid Diagnostic Reagent of Hepatitis B Virus e Antigen purchased from the China National Institutes for Food and Drug Control and 509 clinical samples tested with Shanghai Kehua HBeAg reagent collected from hospitals.

[0070] The results of testing the national reference material for the rapid diagnostic reagent for hepatitis B virus e antigen are as follows: (1) Negative compliance rate: The compliance rate (- / -) of 15 negative national reference samples N1 to N15 is 15 / 15, which meets the requirements; (2) Positive compliance rate: The positive compliance rate (+ / +) of 10 positive national reference samples P1 to P10 is 10 / 10, which is higher than the standard requirement of 9 / 10; (3) Precision: The precision reference sample was repeatedly tested 10 times, and all results were positive with uniform color development, which meets the requirements; (4) Minimum detection limit: The minimum detection limit of the reference standard is 0.1 IU / mL, which is far higher than the standard requirement of 5 IU / mL.

[0071] 4.4 Experimental Results The results of the clinical sample comparison test are shown in Table 6 below. The results show that the test strip made with 5F5 coated with 14D6 label has a 100% concordance rate with the control reagent, indicating that the consistency is good and the specificity and sensitivity of the antibody are both 100%.

[0072] Table 6. Latex chromatography results ; The relevant sequences of this invention are as follows: > 5F5 antibody heavy chain variable region EVQLQESGAGLVPSSQSLSITCTVSGFSLISFGVHWVRQCPGKGLEWLGVIWTGGSTDYNAAFMSRLSISKDNSKSQVFFKMNSLQTDDTAIYYCARGNGYYFDYWGQGTTLTVSS SEQ ID NO:13 > 5F5 antibody light chain variable region DIVMTQSPSSLAVSTGEKVTMSCKSSQSLLNSNTRKNNLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLWTFGGGTKLEIK SEQ ID NO:14 > 5F5 antibody heavy chain constant region AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO:15 > 5F5 antibody light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 16 > 5F5 antibody heavy chain EVQLQESGAGLVPSSQSLSITCTVSGFSLISFGVHWVRQCPGKGLEWLGVIWTGGSTDYNAAFMSRLSISKDNSKSQVFFKMNSLQTDDTAIYYCARGNGYYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 17 > 5F5 antibody light chain DIVMTQSPSSLAVSTGEKVTMSCKSSQSLLNSNTRKNNLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 18 > 5F5 antibody heavy chain variable region gene sequence gaggtccagctgcaagagtcaggagctggcctagtgccgtcctcacagagcctgtccatcacctgcacagtctctggtttctcattaattagttttggtgtacactgggttcgccagtgtccaggaaagggtctggagtggctgggagtgatatggactggtggtagcacagactataatgcagctttcatgtccagactgagcatcagcaaggacaactccaaaagccaagttttctttaaaatgaatagtctacaaactgatgacacagccatttactactgtgccagagggaacggctactactttgactactggggccaaggcaccactctcacagtctcctca SEQ ID NO: 19 > 5F5 antibody light chain variable region gene sequence Gacattgtgatgacacagtctccatcctccctggctgtgtcaacaggagagaaggtcactatgagctgtaaatccagtcagagtctgctcaacagtaatacccgaaagaacaacttggcttggtaccagcagaaaccaggacagtctcctaaactcctgatctactgggcgtccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccatcagcagtgtgcaggctgaagacctggcagtttattactgcaagcaatcttataatctgtggacgttcggtggaggcaccaagctggaaatcaaa SEQ ID NO: 20 > 5F5 antibody heavy chain constant region gene sequence Gccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagcagctcagtgactgtcccctccagcacctggcccagcgagaccgtcacctgcaacgttgcccacccggccagcagcaccaaggtggacaagaaaattgtgcccagggattgtggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaaccccgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaa SEQ ID NO:21 > 5F5 antibody light chain constant region gene sequence CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGA ACAGTTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT SEQ ID NO:22 > 5F5 antibody heavy chain gene sequence SEQ ID NO: 23 > 5F5 antibody light chain gene sequence GACATTGTGATGACACAGTCTCCATCCTCCCTGGCTGTGTCAACAGGAGAGAAGGTCACTATGAGCTGTAAATCCAGTCAGAGTCTGCTCAACAGTAATACCCGAAAGAACAACTTGGCTTGGTACCAGCAGAAACCAGGACAGTCTCCTAAACTCCTGATCTACTGGGCGTCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT SEQ ID NO: 24 > 14D6 antibody heavy chain variable region QVQLQESGPELVKPGTSMKISCKASGYSFTAYTMNWVKQSHGKNLEWIGLINPYSGDSSFNHMFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGTTIITTWFAYWGQGTLVTVSA SEQ ID NO: 25 > 14D6 antibody light chain variable region DVVMTQTPLPPPVSLGDQASISCRSSQSLVHRSENTFLHWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTVFTLKISRVEAEDLGVYFCSQSTHVPFTFGAGTKLELK SEQ ID NO:26 > 14D6 antibody heavy chain constant region AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO:27 > 14D6 antibody light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:28 > 14D6 antibody heavy chain QVQLQESGPELVKPGTSMKISCKASGYSFTAYTMNWVKQSHGKNLEWIGLINPYSGDSSFNHMFEGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGTTIITTWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 29 > 14D6 antibody light chain DVVMTQTPLPPPVSLGDQASISCRSSQSLVHRSENTFLHWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTVFTLKISRVEAEDLGVYFCSQSTHVPFTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 30 > 14D6 antibody heavy chain variable region gene sequence caggtccagctgcaggagtctggacctgagctggtgaagcctggaacttcaatgaagatatcctgcaaggcttctggttactcattcactgcctacaccatgaactgggtgaagcagagccatggaaagaaccttgagtggattggacttattaatccttacagtggtgattctagctttaatcatatgttcgagggcaaggccacattaactgtggacaagtcatccagcacagcctacatggagctcctcagtctgacatctgaagactctgcagtctattactgtgcaagatcggggactactattattacgacctggtttgcttactggggccaagggactctggtcactgtctctgca SEQ ID NO: 31 > 14D6 antibody light chain variable region gene sequence gatgttgtgatgacccaaactccactccccccgcctgtcagtcttggagatcaggcctccatctcttgcagatctagtcagagccttgtacacagaagtgaaaatacctttttacattggtacctgcagaagccaggccagtctccaaagctcctgatctacagagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagttttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatgttcctttcacgttcggtgctgggaccaagctggagctgaaa SEQ ID NO: 32 > 14D6 antibody heavy chain constant region gene sequence gccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagcagctcagtgactgtcccctccagcacctggcccagcgagaccgtcacctgcaacgttgcccacccggccagcagcaccaaggtggacaagaaaattgtgcccagggattgtggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaaccccgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaa SEQ ID NO:33 > 14D6 Antibody Light Chain Constant Region Gene Sequence cgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctga acagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt SEQ ID NO:34 > 14D6 Antibody Heavy Chain Gene Sequence SEQ ID NO:35 > 14D6 Antibody Light Chain Gene Sequence gatgttgtgatgacccaaactccactccccccgcctgtcagtcttggagatcaggcctccatctcttgcagatctagtcagagccttgtacacagaagtgaaaatacctttttacattggtacctgcagaagccaggccagtctccaaagctcctgatctacag agtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagttttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatgttcctttcacgttcggtgctgggaccaagctgg agctgaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtc ctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt SEQ ID NO:36 The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. An antibody or antigen-binding fragment thereof against hepatitis B e antigen, characterized in that, The antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region as shown below: The light chain variable region includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:14, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:

13. The CDR is defined according to the Kabat numbering system.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:4, 5, and 6, respectively, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:1, 2, and 3, respectively.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO:14, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

13.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a murine antibody, a chimeric antibody, or a humanized antibody; the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, scFv, or dsFv.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, It also includes the immunoglobulin Fc region, wherein the Fc is selected from IgG1, IgG2, IgG3 or IgG4.

6. A biomaterial, characterized in that, The biomaterial includes any one of the following: (1) A nucleic acid molecule, wherein the nucleic acid molecule encodes an antibody or an antigen-binding fragment thereof as described in any one of claims 1-5; (2) An expression vector / vector group containing the nucleic acid molecules described in (1); (3) A host cell containing the nucleic acid molecule described in (1) or the expression vector / vector group described in (2).

7. A method for preparing the antibody or antigen-binding fragment thereof as described in any one of claims 1-5, characterized in that, The method includes culturing the host cells as described in claim 6 to express an antibody or an antigen-binding fragment thereof; and includes isolating or purifying the antibody or the antigen-binding fragment thereof.

8. A composition against human hepatitis B e antigen, characterized in that, Includes the antibody or antigen-binding fragment thereof as described in any one of claims 1-5.

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