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

Abstract

The application discloses a monoclonal 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. 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 an existing antibody 3A3, the stability and the 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 a monoclonal antibody against hepatitis B virus e antigen, its preparation method, and its uses. Background Technology

[0002] Hepatitis B (HBV) is an infectious disease caused by hepatitis B virus (HBV) infection, characterized primarily by liver inflammation and liver function impairment. It maintains a high incidence and disease burden globally. Hepatitis B e antigen (HBeAg) is a soluble non-structural protein encoded by the pre-C and C regions of the HBV gene. It is mainly found in HBsAg-positive serum, appearing slightly later than HBsAg during the acute infection phase. It is an important serological marker of active viral replication and high infectivity. The presence of HBeAg usually indicates a high infection, low response phase, and serological conversion (disappearance of HBeAg and appearance of anti-HBe) is often associated with a decrease in viral replication levels and clinical remission.

[0003] Currently, hepatitis B serological testing mainly uses the "two and a half pairs of hepatitis B markers" combination, including HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc. HBeAg detection methods mainly include enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECL), and time-resolved fluorescence immunoassay (TRFIA). However, current HBeAg testing reagents still have a significant problem of false negatives in practical applications. The main reason is the high variability of the HBV genome, especially mutations at key sites in the pre-C region and C region. Although some mutations are silent mutations, some mutations can lead to a decrease in HBeAg expression or changes in antigenic epitopes, making existing detection antibodies unable to effectively recognize them, thus resulting in false negative HBeAg test results. This type of false negative seriously affects the accurate clinical assessment of patients' infection status, disease progression, and treatment efficacy.

[0004] It is worth noting that such false negatives are not due to the lack of HBeAg expression, but rather to the insufficient ability of the antibody used in the detection system to recognize mutant strains. Therefore, developing an anti-HBeAg monoclonal antibody that can broadly recognize both wild-type and mutant HBeAg, possesses high sensitivity, high specificity, and a low false negative rate, and applying it to downstream diagnostic reagent production has significant clinical application value and market prospects. 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 in the amino acid sequence shown in SEQ ID NO:13. In some embodiments, the CDR shown is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some embodiments, the CDR shown is defined according to the Kabat numbering system.

[0010] In some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:7. In some embodiments, the CDRs shown are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some embodiments, the CDRs shown are defined according to the Kabat numbering system.

[0011] In some embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in SEQ ID NO:4, 5, and 6, or have at least 95% identity with them. In some specific embodiments, LCDR1, LCDR2, and LCDR3 each comprise amino acid sequences as shown in SEQ ID NO:4, 5, and 6. In some specific embodiments, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO:4, 5, and 6, respectively.

[0012] In some embodiments, HCDR1, HCDR2, and HCDR3 respectively comprise amino acid sequences as shown in SEQ ID NO:1, 2, and 3, or have at least 95% identity with them. In some specific embodiments, HCDR1, HCDR2, and HCDR3 respectively comprise amino acid sequences as shown in SEQ ID NO:1, 2, and 3. In some specific embodiments, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are 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, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:13 or having at least 95% identity with it. In some specific embodiments, the light 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:13.

[0015] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7 or having at least 95% identity with it. In some specific embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7. In some specific embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7.

[0016] In some specific embodiments, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7.

[0017] 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.

[0018] 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.

[0019] In some embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:17 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:17. In some specific embodiments, the amino acid sequence of the light chain is as shown in SEQ ID NO:17.

[0020] In some embodiments, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:11 or having at least 95% identity with it. In some specific embodiments, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:11. In some specific embodiments, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:11.

[0021] The present invention also provides a biomaterial.

[0022] 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.

[0023] In some embodiments, the gene sequence encoding the heavy chain variable region of the nucleic acid molecule is shown in SEQ ID NO: 8; and the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 14.

[0024] 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: 12; and the gene sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO: 18.

[0025] The present invention also provides a method for preparing antibodies or antigen-binding fragments thereof as described in any of the above claims.

[0026] 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.

[0027] The present invention also provides a composition against human hepatitis B e antigen.

[0028] In some embodiments, the composition comprises the antibody or antigen-binding fragment thereof described in any of the preceding embodiments.

[0029] 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.

[0030] 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.

[0031] 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

[0032] Figure 1 This shows the chromatographic pattern of the target protein. The peak of the target protein is highlighted in red.

[0033] Figure 2 This represents an SDS-PAGE electrophoresis image. Channel M corresponds to the protein marker; channel 1 corresponds to the 10F5 antibody. Detailed Implementation

[0034] The following description of specific embodiments further illustrates this application, but it 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.

[0035] 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.

[0036] 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.

[0037] 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 ).

[0038] 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.

[0039] 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 of 100,000 or higher were selected for fusion. Three days before fusion, mice were immunized intraperitoneally at a dose of 100 μg / mouse without adjuvant.

[0040] 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).

[0041] 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.

[0042] 2) Preparation of spleen cells BALB / c mice that have completed animal immunization were enucleated to collect blood, and the serum was separated by centrifugation to serve as a positive control serum for antibody detection.

[0043] 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 r / min for 10 minutes. The supernatant was discarded, 10 ml of culture medium was added, and a small amount was taken and diluted 10 times for counting.

[0044] 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 and fused using PEG. The mixtures were 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, 13 positive hybridoma cell lines were finally identified.

[0045] 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.

[0046] 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.

[0047] Table 1. Results of OD value measurement ; 2.3 Production and purification of monoclonal antibodies a) 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.

[0048] b) Ascites purification 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 hours. After filtration through a 0.22 μm filter, the antibody was aliquoted and stored at -20°C for subsequent detection and validation.

[0049] 2.4 Characterization of Monoclonal Antibodies Antibody concentration determination: The absorbance values ​​(A280) and (A260) of the monoclonal antibody at 280 nm and 260 nm were determined by ultraviolet spectrophotometry. The protein content of HBe-10F5 was found to be 5 mg / mL. The protein content was calculated using the following formula: Protein content (mg / mL) = (A280 × dilution factor) / 1.35.

[0050] Antibody molecular weight determination: Monoclonal antibodies were measured using SDS-PAGE, such as... Figure 2 As shown, the heavy chain of the monoclonal antibody HBe-10F5 is approximately 46 kDa, and the light chain is approximately 25 kDa.

[0051] ELISA titer determination: The purified ascites fluid monoclonal antibody was determined by indirect ELISA, with a negative control of irrelevant antibodies. The results are shown in Table 2. The results showed that the purified titer was greater than 1:100,000, and the negative background was clean.

[0052] Table 2. Results of indirect ELISA assay ; Example 3: Application of 10F5 antibody in chemiluminescent reagent 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 magnetic microparticle concentration was 10 mg / mL, the reaction temperature was 37℃, and the reaction time was 30 min. The reacted magnetic microparticles were then conjugated with 3C3 monoclonal antibody (see CN 119661697 B). The ratio of magnetic microparticles to antibody was 10 μg of 3C3 antibody per milligram of magnetic microparticles. The conjugation process was carried out at 25℃ for 5 h. After conjugation, the magnetic microparticles were washed three times with washing buffer, and then added to a phosphate buffer solution containing 2% glycine, 1% BSA, 0.05% Tween 20, 0.05% ProClin 300, and pH 7.4 to achieve a magnetic microparticle concentration of 10 mg / mL. The solution was incubated at 25℃ for 2 h and then stored at 2-8℃ for later use.

[0053] 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.

[0054] (2) Take 1 mg of 3A3 and 10F5 antibodies respectively, adjust the concentration to 1 mg / mL, add 0.034 mg of SMCC respectively, and react at 37℃ for 1 h.

[0055] (3) Divide the alkaline phosphatase that has completed the reaction into two tubes, add 3A3 and 10F5 antibodies that have been treated with SMCC to the tubes respectively, 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 to the tubes respectively, let stand for 1 hour, add 1 mL of glycerol, and store at -20°C for later use.

[0056] 3.3 Reagent Performance Testing Magnetic microparticles coated with 3C3 antibody were diluted 20-fold with phosphate buffer containing 0.15M sodium chloride. Alkaline phosphatase-labeled 3A3 and 10F5 antibodies were diluted 1000-fold with phosphate buffer containing 0.15M sodium chloride before testing. The test samples were 1068 clinical samples tested using the Abbott Laboratories Hepatitis B virus e antigen assay kit (chemiluminescent microparticle immunoassay).

[0057] 3.3.1 Potency and Precision Testing Magnetic microparticles coated with 3C3 antibody were diluted 20-fold with phosphate buffer containing 0.15M sodium chloride; alkaline phosphatase-labeled 3A3 and 10F5 antibodies were diluted 1000-fold with phosphate buffer containing 0.15M sodium chloride; the detection signals of samples at different concentrations were detected by chemiluminescent microparticle immunoassay.

[0058] Potency determination: The test reagent (3C3-10F5) and the control reagent (3C3-3A3) were tested on samples of different concentrations under the same conditions, and their luminescence values ​​(RLU) were recorded.

[0059] Precision determination: The test reagent (3C3-10F5) and the control reagent (3C3-3A3) were used to repeatedly test the positive control at low, medium and high concentration levels (n≥20), and the coefficient of variation (CV%) of the measured concentration was calculated to evaluate the precision.

[0060] 3.4 Experimental Results The comparison test results between the antibody to be tested and the Abbott reagent kit are shown in Table 3 below. The positive concordance rate, negative concordance rate and total concordance rate of the reagent described in this invention and the Abbott control reagent are all 100%. All indicators show that the results of the "test reagent" and the "Abbott reagent" are completely consistent with each other, with no difference. Kappa=1, which is perfect consistency.

[0061] Table 3. Antibody performance testing ; The comparison results between the test reagent and the control reagent are shown in Table 4 below. The antibody 10F5 provided by this invention, compared with the existing antibody 3A3, showed a 100% concordance rate for negative and positive results and a 100% overall concordance rate in the tested samples. In the repeatability test, the test reagent performed better. Data showed that the detection precision (coefficient of variation, CV) of the test reagent was 5%, which was significantly better than the precision of the control reagent (CV was 8%). It could also significantly improve the signal value of the detection system. Specifically, under the same detection sensitivity, the test reagent showed a stronger signal response capability, and its potency was about 30% higher than that of the control reagent. This series of performance improvements significantly improved the stability and repeatability of the detection, which is of great value for the early detection rate of the kit.

[0062] Table 4. Antibody performance testing ; Example 4: Application of 10F5 antibody in hepatitis B virus e antigen latex chromatography test strips 4.1 Preparation of coating membrane 3C3 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.

[0063] 4.2 Preparation of Latex Pads Add 100 μL of 400 nm latex microspheres (4%) to 5 mL of MES buffer, then add 100 μL of 20 mg / mL EDC solution, followed by 500 μL of 20 mg / mL NHS solution. Activate at room temperature with stirring for 1 h, centrifuge at 10000 rpm for 10 min to remove the supernatant, add 5 mL of MES buffer, sonicate in a water bath for 10-20 s, and quickly add to 400 μg of 10F5 antibody solution. Stir at room temperature for 2 h, centrifuge at 10000 rpm for 6 min after the reaction is complete, discard the supernatant, add 4 mL of microsphere storage solution, sonicate on ice for 7 min, stir at room temperature for 2 h, and incubate overnight at 37 °C. The microsphere storage concentration is 1 mg / mL (0.1%).

[0064] The 10F5 microspheres, with a storage concentration of 1 mg / mL, were diluted to 0.5 mg / mL with microsphere preservation solution. The solution was then sprayed onto the treated conjugate pads using an XYZ three-dimensional gold spraying spectrometer at a spray rate of 2 μL / cm, and dried in a 37°C oven for 20 hours. The dried conjugate pads were then cut to 300 mm × 8 mm dimensions and labeled as 10F5 latex pads for later use.

[0065] 4.3 Reagent Performance Testing The prepared 3C3 coated membrane was combined with a 10F5 latex pad, and after attaching the sample pad, it was cut into 3mm test strips. The test strips were tested using the national reference material for rapid diagnostic reagents for 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.

[0066] 4.4 Experimental Results 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.

[0067] The results of the clinical sample comparison test are shown in Table 5 below. The test strip made with 3C3 coated with 10F5 label has a 100% consistency rate with the control reagent, indicating that the consistency is good and the specificity and sensitivity of the antibody are both 100%.

[0068] Table 5. Comparison Results of Clinical Samples Tested ; The relevant sequences of this invention are as follows: > 10F5 antibody HCDR1 amino acid sequence (Kabat numbering rules) DYHMY SEQ ID NO:1 > 10F5 antibody HCDR2 amino acid sequence (Kabat numbering rules) YISIGGGSIAYSDTVEG SEQ ID NO:2 > 10F5 antibody HCDR3 amino acid sequence (Kabat numbering rules) HDYYGDYAMDY SEQ ID NO:3 > 10F5 antibody LCDR1 amino acid sequence (Kabat numbering rules) RASQEISGYLT SEQ ID NO:4 > 10F5 antibody LCDR2 amino acid sequence (Kabat numbering rules) VASTLDS SEQ ID NO:5 > 10F5 antibody LCDR3 amino acid sequence (Kabat numbering rules) LQYASSPYT SEQ ID NO:6 > 10F5 antibody heavy chain variable region LVQLQQSGGGLVRPGGSLKLSCGTSGFTFSDYHMYWFRQTPEKRLEWVAYISIGGGSIAYSDTVEGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARHDYYGDYAMDYWGQGTSVTVSS SEQ ID NO:7 > 10F5 antibody heavy chain variable region gene sequence ttggtccagctgcagcagtctgggggaggcttagtgcggcctggagggtccctgaaactctcctgtggaacctctggattcactttcagtgactatcacatgtattggtttcgccagactccagagaagaggctggaatgggtcgcatacataagtattggtggtggtagtattgcttattcagacactgtagagggccgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagccgtctgaagtctgaggacacagccatgtattactgtgcaagacacgattactacggtgactatgctatggactactggggtcaaggaacctcagtcaccgtctcctca SEQ ID NO: 8 > 10F5 antibody heavy chain constant region AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 9 > 10F5 antibody heavy chain constant region gene sequence gccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagcagctcagtgactgtcccctccagcacctggcccagcgagaccgtcacctgcaacgttgcccacccggccagcagcaccaaggtggacaagaaaattgtgcccagggattgtggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaaccccgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaa SEQ ID NO:10 > 10F5 antibody heavy chain LVQLQQSGGGLVRPPGSLKLSCGTSGFTFSDYHMYWFRQTPEKRLEWVAYISIGGGSIAYSDTVEGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARHDYYGDYAMDYWGQGTSVTVSSAKTTPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO:11 > 10F5 Antibody SEQ ID NO:12 > 10F5 antibody light chain variable region DIVLTQSPSSLSASLGERVSLTCRASQEISGYLTWLQQKPDGTIKRLIYVASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASSPYTFGGGTKLEIK SEQ ID NO:13 > 10F5 antibody light chain variable region gene sequence gacattgtgctgacccagtctccatcctccttatctgcctctctgggagaaagagtcagtctcacttgtcgggcaagtcaggaaattagtggttacttaacctggcttcagcagaaaccagatggaactattaaacgcctgatctacgtcgcatccactt tagattctggtgtccctaaaaggttcagtggcagtaggtctgggtcagattattctctcaccatcagcagccttgagtctgaagattttgcagactattactgtctacaatatgctagttctccgtacacgttcggaggggggaccaagctggaaataaaa SEQ ID NO:14 > 10F5 antibody light chain constant region RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:15 > 10F5 antibody light chain constant region gene sequence CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT SEQ ID NO: 16 > 10F5 antibody light chain DIVLTQSPSSLSASLGERVSLTCRASQEISGYLTWLQQKPDGTIKRLIYVASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASSPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 17 > 10F5 antibody light chain gene sequence gacattgtgctgacccagtctccatcctccttatctgcctctctgggagaaagagtcagtctcacttgtcgggcaagtcaggaaattagtggttacttaacctggcttcagcagaaaccagatggaactattaaacgcctgatctacgtcgcatccactt tagattctggtgtccctaaaaggttcagtggcagtaggtctgggtcagattattctctcaccatcagcagccttgagtctgaagattttgcagactattactgtctacaatatgctagttctccgtacacgttcggaggggggaccaagctggaaataaaa cgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctga acagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt SEQ ID NO:18 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 against hepatitis B e antigen or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes LCDR1, LCDR2, and LCDR3, and the heavy chain variable region includes HCDR1, HCDR2, and HCDR3; 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.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

7.

3. 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.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, It also includes the immunoglobulin Fc region, wherein the Fc is selected from IgG1, IgG2, IgG3 or IgG4.

5. 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 1-4; (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).

6. A method for preparing the antibody or antigen-binding fragment thereof as described in any one of claims 1-4, characterized in that, The method includes culturing the host cells as described in claim 5 to express an antibody or an antigen-binding fragment thereof; and optionally includes isolating or purifying the antibody or the antigen-binding fragment thereof.

7. 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 1-4.

8. The use of an antibody or antigen-binding fragment thereof as described in any one of claims 1-4, or the composition as described in claim 7, characterized in that, Products used to prepare 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.

9. The application according to claim 8, characterized in that, 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.

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