A HBV S protein-specific monoclonal antibody HBV-S-4H7 and its use in preparing a detection kit

Abstract

The present invention relates to a HBV S protein-specific monoclonal antibody, HBV-S-4H7, and its use in the preparation of a detection kit. Based on the amino acid sequences of different HBV S proteins, multiple sequence alignment, and online epitope screening software, the present invention ultimately selects a preferred epitope peptide and immunizes mice to prepare a broad-spectrum monoclonal antibody. The antibody has good binding properties. Based on the role of the S protein in the virus, neutralization experiments confirm that the monoclonal antibody prepared by the present invention also has good virus neutralization effect, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the biological field, and more specifically to an HBV S protein-specific monoclonal antibody HBV-S-4H7 and an application thereof in preparing a detection kit. Background Art

[0002] Viral hepatitis is an infectious disease characterized by liver disease and caused by various hepatitis viruses. Clinically, it presents with loss of appetite, nausea, upper abdominal discomfort, pain in the liver area, and fatigue. Some patients may experience jaundice, fever, and hepatomegaly with impaired liver function. In some cases, the condition may become chronic, even developing cirrhosis, and a few may develop liver cancer. The etiological classification of viral hepatitis currently recognizes five types of hepatitis viruses: HAV, HBV, HCV, HDV, and HEV. With the exception of hepatitis B virus, which is a DNA virus, all others are RNA viruses.

[0003] Currently, testing for viral hepatitis primarily focuses on testing for hepatitis virus markers. In patients with hepatitis A and acute hepatitis, a positive serum anti-HAV IgG test indicates recent HAV infection, while a positive anti-HAV IgG test indicates previous infection and pre-existing immunity. Regarding hepatitis B, HBsAg and anti-HBs testing: HBsAg positivity indicates current HBV infection, while anti-HBs, a protective antibody, indicates pre-existing immunity. Chronic HBsAg carriers are diagnosed based on the absence of clinical symptoms and signs, normal liver function, and persistent HBsAg positivity for at least six months. HBeAg and anti-HBe testing: HBeAg positivity indicates active HBV replication and high infectivity. A shift from HBeAg positivity to anti-HBe positivity indicates disease remission and decreased infectivity. HBcAg and anti-HBc testing: HBcAg positivity directly reflects the presence of intact HBV particles, indicating active HBV replication and is rarely used clinically due to the complexity of the testing method. Anti-HBc is a hallmark of HBV infection, and a positive anti-HBc IgM test indicates early-stage infection and viral replication. In chronic mild hepatitis B and HBsAg carriers, positive HBsAg, HBeAg, and anti-HBc tests indicate high infectiousness and are difficult to detect. In hepatitis C, because the amount of antigen in the blood is too low to be detected, only antibodies can be tested. Anti-HCV is a marker of HCV infection, not a protective antibody. Using nested reverse transcription PCR, a positive serum HCV RNA test indicates active viral replication and infectiousness. In hepatitis D, HDV is a defective virus that relies on HBsAg for replication, which can manifest as simultaneous HDV and HBV infection. HDAg only appears in the blood for a few days, followed by the development of IgM anti-HDV. In chronic HDV infection, anti-HDV IgG levels remain elevated. Detection of HDV RNA in serum is a more direct and specific diagnostic method. Anti-HEV IgM antibodies are detected in the serum of patients with acute hepatitis E. IgG antibody titers are very low in convalescent serum, and anti-HEV IgG persists in serum for less than a year. Therefore, both anti-HEV IgM and anti-HEV IgG can be used as indicators of recent HEV infection. For hepatitis G, RT-PCR can detect HGV RNA and is an effective method for early diagnosis and monitoring of viremia. However, anti-HGV IgM and IgG antibodies are not yet mature, resulting in low detection rates and discrepancies between RT-PCR results.

[0004] Of course, with the continuous advancements in clinical medicine, immunology, and molecular biology in recent years, numerous new, highly sensitive detection methods, such as chemiluminescence, time-resolved fluorescence, and surface-enhanced Raman spectroscopy, have begun to be applied in hepatitis testing. Hepatitis B virus (HBV), the pathogen that causes hepatitis B (HBV), belongs to the Hepadnaviridae family. Among HBV carriers, 50% to 75% develop chronic hepatitis B with active viral replication. The estimated five-year incidence of progression from chronic hepatitis B to cirrhosis is 2%-20%, from compensated cirrhosis to decompensated liver disease is 20%-23%, and from compensated cirrhosis to liver cancer is 6%-15%. Chronic hepatitis B is the primary risk factor for progression to cirrhosis, liver failure, and hepatocellular carcinoma. HBV is highly contagious; inoculation of 0.00004 mL of virus-containing blood is sufficient to infect a person.

[0005] Radioimmunoassay (RIA) is also a commonly used method for HBV detection. The most commonly used RIA is the solid-phase radioimmunoassay (SPRIA). This method uses radioactive isotopes labeled with known antigens or antibodies, allowing them to bind to the corresponding antibodies or antigens to be tested. A counter measures the Cpm value of the isotope, thereby estimating the amount of antigen or antibody in the sample. This method has high sensitivity, but requires strict experimental conditions, making it difficult to promote at the grassroots level.

[0006] Enzyme immunoassay (EIA) is a commonly used method for detecting HBV, with the enzyme-linked immunosorbent assay (ELISA) being the most common. Polystyrene adsorption pores are typically used as carriers to adsorb specific antigens or antibodies. These antibodies then bind to the corresponding antibodies or antigens in the sample being tested. Enzyme-catalyzed color development occurs, and the result can be determined visually based on the depth of the color (qualitative). A colorimeter (enzyme reader) can also be used for precise determination (quantitative). This method is currently being widely used in various institutions. However, the hepatitis B virus frequently mutates, which greatly complicates its diagnosis and treatment. Therefore, an accurate and rapid laboratory test for detecting the hepatitis B virus is urgently needed. Summary of the Invention

[0007] The present invention provides a monoclonal antibody capable of broad-spectrum detection of HBV and a kit or test strip using the same.

[0008] In one aspect, the present invention provides a broad-spectrum monoclonal antibody HBV-S-4H7 for detecting HBV.

[0009] In the present application, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0010] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 1, and its amino acid sequence is

[0011]

[0012] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and its amino acid sequence is

[0013]

[0014] Also provided are antibodies derived from antibodies or antibody binding fragments as described herein. Typically, as used herein, derivatives provided herein are substantially similar to antibodies or antibody binding fragments as described herein. For example, they can include one or more conservative substitutions in their amino acid sequences, or can include chemical modifications. Derivatives and modified peptides / polypeptides / proteins are all considered to be "structurally similar," meaning that they retain the structure (e.g., secondary, tertiary or quaternary structure) of the parent molecule and are expected to interact with the antigen in the same manner as the parent molecule.

[0015] A class of synthetically derived antibodies or antigen-binding portions can be produced by conservatively mutating residues on the parent molecule to produce peptides, polypeptides or proteins that retain the same activity as the parent molecule. Representative conservative substitutions are known in the art and are also summarized here.

[0016] In general, conservative substitutions can be made at any position as long as the desired activity is retained. So-called conservative exchanges can be made, in which the replaced amino acid has similar properties to the original amino acid, such as Asp for Glu, Asn for Gln, Val for Lie, Leu for Lie, and Ser for Thr.

[0017] For example, amino acids with similar properties can be aliphatic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine); hydroxy or sulfur / selenium-containing amino acids (e.g., serine, cysteine, selenocysteine, threonine, methionine); cyclic amino acids (e.g., proline); aromatic amino acids (e.g., phenylalanine, tyrosine, tryptophan); basic amino acids (e.g., histidine, lysine, arginine); or acidic and its amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine). Deletion refers to the replacement of an amino acid by a direct bond. The position of the deletion includes the end of the polypeptide and the connection between the various protein domains. Insertion is the introduction of an amino acid into the polypeptide chain, with the direct bond formally replaced by one or more amino acids. The amino acid sequence can be adjusted with the aid of computer simulation programs known in the art, which can produce monoclonal antibodies with, for example, improved activity or altered regulation.

[0018] Further, the antibody can comprise an immunoglobulin heavy chain variable region having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or 100% sequence identity to SEQ ID NO:2, and an immunoglobulin light chain variable region having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or 100% sequence identity to SEQ ID NO:1.

[0019] The present invention also discloses the use of the above HBV antibody in preparing a kit for detecting HBV in a sample by ELISA.

[0020] The present invention also discloses the use of the above HBV antibody in detecting the HBV content in a sample by double antibody sandwich ELISA.

[0021] Preferably, the application procedure is: coating with HBV antibodies, washing the plates, blocking with gelatin, washing the plates, adding the test sample for incubation, washing the plates, adding biotin-labeled HBV antibodies for incubation, washing the plates, adding HRP-labeled avidin for incubation, washing the plates, adding TMB for color development, and reading with a microplate reader after adding the stop solution.

[0022] The present invention also discloses an HBV detection kit, which is characterized by comprising the above-mentioned HBV antibody.

[0023] Furthermore, in order to label monoclonal antibodies, for example, various pigments and colloids can be used as pigments for binding markers such as enzymes to monoclonal antibodies, for example, FITC (Fluorescein5-isothiocyanate), rhodamine, and fluorescent labeling pigments such as fluorescein can be used. They are highly versatile and easy to obtain. FITC is preferably FITC, which can be purchased from Sigma (strain) as a colloid. For example, as an enzyme that can use gold colloid, for example, peroxidase, alkaline phosphatase, luciferase, β-galactosidase, etc. can be used to bind to the above-mentioned monoclonal antibodies, and a known method can be used.

[0024] Furthermore, the present invention provides an HBV colloidal gold test strip, wherein the test strip includes a colloidal gold-labeled antibody, and the colloidal gold-labeled monoclonal antibody is prepared by coupling colloidal gold with the monoclonal antibody of the present invention;

[0025] The preparation of the colloidal gold comprises the following steps: heating chloroauric acid, adding trisodium citrate, and continuing to heat until the solution changes from light yellow to bluish black and finally to bright red, continuing to heat after the color stabilizes, and then cooling to obtain a colloidal gold solution;

[0026] The preparation of the colloidal gold-labeled monoclonal antibody comprises the following steps: adjusting the colloidal gold solution to a weak alkaline state, adding the monoclonal antibody of the present invention, then sequentially adding PEG and BSA to continue the reaction to obtain a colloidal gold-labeled monoclonal antibody precipitate, and resuspending the solution in PBS to obtain the colloidal gold-labeled monoclonal antibody;

[0027] The components of the test strip include a base plate, a sample pad, a nitrocellulose membrane, and a water absorbent pad, which are connected in sequence; and a detection line and a quality control line are provided on the nitrocellulose membrane.

[0028] The test strip is assembled by overlapping the sample pad, nitrocellulose membrane and water absorbent pad on the bottom plate in sequence, and the sample pad and water absorbent pad are respectively pressed 1 to 2 mm above the nitrocellulose membrane.

[0029] Furthermore, the monoclonal antibody of the present invention can also be used to inhibit the proliferation of HBV and to prepare corresponding therapeutic drugs.

[0030] Beneficial effects

[0031] The present invention is based on the amino acid sequences of S proteins of different HBVs, multiple sequence alignment, and online software screening of antigen epitopes, and ultimately selects better antigen epitope peptides. Mice are immunized to prepare broad-spectrum monoclonal antibodies. The antibodies have good binding properties. Based on the role of the S protein in the virus, neutralization experiments confirm that the monoclonal antibodies prepared by the present invention also have good virus neutralization effects, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Alignment results of broad-spectrum peptide sites

[0033] Figure 2 Monoclonal antibody Western-blot test results

[0034] Figure 3 Monoclonal antibody subtype identification results

[0035] Figure 4 Monoclonal antibody specificity identification results

[0036] Figure 5 Monoclonal antibody neutralization experiment results DETAILED DESCRIPTION

[0037] The present invention may be more readily understood through the following description of certain embodiments of the invention and the detailed description of the Examples included therein. Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, as such embodiments are necessarily diverse. It should also be understood that the terminology used in this specification is intended only to illustrate specific embodiments and is not intended to be limiting, as the scope of the present invention is defined solely by the appended claims.

[0038] Example 1 Screening of HBV S protein epitope peptides

[0039] Based on the amino acid sequences of different HBV S proteins, multiple sequence alignment, and antigen epitope online software screening, the best antigen epitope peptide was finally selected as cpgyrwmclrrfiiflfilllclifllvlld (named HBV-S broad-spectrum peptide). Although the a determinant cluster cttpaqgnsmf of the S protein has the ability to exist in any serum subtype, Figure 1 As can be seen, the sequence is not completely conserved among different HBV species. Therefore, the broad-spectrum peptides screened in this application have a broader spectrum of properties. Shanghai Bioengineering was commissioned to synthesize the peptides for use as immunogens. The antigenic peptides were conjugated to KLH for later use.

[0040] Example 2 Preparation of HBV S protein monoclonal antibodies

[0041] Five SPF-grade, 6-week-old female Balb / c mice were selected and housed for two weeks to acclimate them to the environment. The antigen polypeptide HBV-S broad-spectrum peptide-KLH coupling complex prepared in Example 1 was diluted with physiological saline to prepare a 1 mg / ml solution. For the first immunization, 200 μg of the immunogen was emulsified with Freund's complete adjuvant at a ratio of 1:1 and injected subcutaneously at multiple points. Two weeks later, 200 μg of the immunogen was emulsified with Freund's incomplete adjuvant at a ratio of 1:1 and injected subcutaneously at multiple points. Two weeks later, 150 μg of the immunogen was diluted with physiological saline and injected intraperitoneally for booster immunization. Two weeks later, serum was collected for titer detection.

[0042] Determination of serum antibody titer of immune mice: dissolve the immunogenic polypeptide HBV-S broad-spectrum peptide in the coating solution and dilute it to 20 μg / ml, add 100 μl to each well, and let it stand at 4°C overnight; soak and wash 3 times, 3 minutes each time; add 200 μl of 2% BSA blocking solution to each well and incubate at 37°C for 2 hours; soak and wash 3 times, 3 minutes each time; add the serum of each mouse diluted in a gradient of 1:2500, 1:5000, 1:10000, 1:15000, 1:17500, 1:20000, 1:30000, and 1:40000, respectively. Clear, incubate 3 wells at 37°C for 1 hour; soak and wash 3 times, 3 minutes each time; add enzyme-labeled rabbit anti-mouse secondary antibody, diluted 1:5000, incubate at 37°C for 1 hour; soak and wash 3 times, 3 minutes each time; add a colorimetric solution mixed with OPD and hydrogen peroxide, and stop the reaction after 10 minutes; measure the OD value at a wavelength of 490nm. The serum titers of the five mice are 1:15000, 1:17500, 1:17500, 1:20000, and 1:15000, respectively. The serum titer of mouse No. 4 is the highest, so mouse No. 4 is selected for cell fusion.

[0043] Mice were sacrificed and spleen cells were prepared. The spleen cells were fused with myeloma cells according to conventional fusion methods in the art, and cultured and screened using HAT and HT culture media. Positive clones were screened using the indirect ELISA method. When the ratio of the absorbance value of the well containing the clone to the absorbance value of the negative control well in two tests was greater than 2.1, the well was determined to be a positive well. According to statistics, there were a total of 23 clones with good positive results. The positive clone cells were subcloned 4 times until all the cloned cells in the 96-well cell culture plate were positive. Three monoclonal positive hybridoma cells were obtained, namely HBV-S-2F6, HBV-S-4G4 and HBV-S-4H7. Among them, HBV-S-2F6 and HBV-S-4H7 had the strongest positive reactions and were used subsequently.

[0044] The in vivo induction method was used. The animals used were 10-week-old female purebred BALB / c mice. Each mouse was injected with 500 μL of liquid paraffin 7 days in advance. When the number of the two hybridoma cells HBV-S-2F6 and HBV-S-4H7 reached 3×10 6 The cells were injected into the peritoneal cavity of mice at 37 ℃ and 1 ℃, and the ascites was collected after the abdomen of the mice swelled. The cells were centrifuged at 2500 rpm for 20 min, and the clear ascites was taken for titer determination: 50 μL of the ascites collected from each hybridoma cell line was taken out and serially diluted at a ratio of 1:100-1:51200. The ascites titer was determined by indirect ELISA. The results are shown in Table 1.

[0045] Table 1 Ascites titer of monoclonal antibodies

[0046] Monoclonal antibody type potency HBV-S-2F6 1：25600 HBV-S-4H7 1：51200

[0047] The ascites was centrifuged at low temperature and filtered through a microporous membrane to remove large clots and fat droplets. Cell debris and small particulate matter were removed by high-speed centrifugation at 10,000 g for 15 minutes (4°C). With stirring, 5.0 ml of saturated ammonium sulfate solution was added dropwise. Slow stirring was continued for 30 minutes. Centrifugation was performed at 10,000 rpm for 15 minutes. The supernatant was discarded, and the precipitate was suspended in 1 / 3 saturated ammonium sulfate, stirred for 30 minutes, and centrifuged using the same method. The previous step was repeated twice. The precipitate was dissolved in 1.5 ml of PBS (0.01 mol / L PH 7.2) to obtain the purified two antibodies. The antibody concentration was adjusted to 2 mg / mL and stored at 4°C.

[0048] Example 3 Western-blot identification of HBV-S-4H7 monoclonal antibody

[0049] Protein sample preparation: Place HBV-S broad-spectrum peptide in an EP tube at a ratio of 5:4:1 peptide:SDS:DTT in a centrifuge tube. Boil the sample in boiling water for 10 minutes and then cool on ice for 5 minutes. Use the protein sample directly for Western blotting.

[0050] The Western blot method steps are as follows:

[0051] (1) Prepare SDS-PAGE, with a 15% separating gel at the bottom and a 5% stacking gel at the top;

[0052] (2) Sample loading: Load the peptide;

[0053] (3) Electrophoresis: Run the stacking gel at 90V for about 30 minutes, and the separation gel at 120V until it reaches the bottom.

[0054] (4) Transfer: The order of stacking from top to bottom during transfer is: filter paper - protein gel - NC membrane - filter paper. Transfer conditions: 56 mA, about 40 min. After the transfer is completed, place the membrane in 20 mL of 5% skim milk blocking solution and block at 4°C overnight.

[0055] (5) Antibody incubation:

[0056] 1) Pour off the blocking solution and wash the membrane with PBST for 5 times, 5 minutes each time. Then dilute the prepared monoclonal antibody at a 1:1000 dilution in PBST and incubate at 4°C for 8 hours.

[0057] 2) Recover the primary antibody and wash with PBST five times, 5 minutes each time;

[0058] 3) Add goat anti-mouse IgG-HRP secondary antibody, dilute 1:5000 in PBST, and incubate at 37°C on a shaker for 2 h;

[0059] 4) Recover the secondary antibody and wash with PBST five times, 5 minutes each time;

[0060] 5) Store the prepared ECL exposure solution away from light. Place the membrane in an exposure box and allow it to react with the exposure solution for 30 seconds for imaging.

[0061] See the results Figure 2 It can be found that HBV-S-4H7 reacts with the S protein polypeptide, and an obvious specific band appears on the transfer membrane, indicating that the HBV-S-4H7 monoclonal antibody specifically reacts with the S protein and the band is single.

[0062] Example 4 HBV-S-4H7 Monoclonal Antibody Subtype Identification

[0063] Add the prepared HBV-S-4H7 monoclonal antibody to the sample wells of the strips, 50 μL per well; without incubation, add 1× goat anti-mouse IgA+IgM+IgG-HRP to the sample wells, 50 μL per well. Mix gently on a mixer, or tap the sides of the plate rack gently with your hands for 1 minute. Cover with a sealing film and incubate at room temperature for 1 hour. Wash 3 times with 1×PBST and pat dry. Add the freshly prepared color development solution to the wells, 100 μL per well. Color development method: A solution: B solution = 1:100. Color development at room temperature in the dark for 15 minutes. Add stop solution to each well, 100 μL per well. Interpretation of results. The results are read using an enzyme reader at OD450. The well with the darkest color or the highest OD value corresponds to the corresponding subtype. The results are as follows: Figure 3 shown.

[0064] from Figure 3 The results show that HBV-S-4H7 monoclonal antibody is of IgG2a subtype, Kappa chain.

[0065] Example 5 Affinity identification of HBV-S-4H7 monoclonal antibody

[0066] Using the AMC sensor, the purified HBV-S-4H7 antibody was diluted to 10 μg / mL with PBST, and the antigen polypeptide S-broad-spectrum peptide was gradiently diluted with PBST: 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.3 μg / mL, 15.6 μg / mL, 7.80 μg / mL, and 3.9 μg / mL.

[0067] Run time: 65 s equilibration in buffer, 340 s antibody immobilization in antibody solution, 250 s incubation in buffer, 500 s binding in antigen solution, 1600 s dissociation in buffer, sensor regeneration with 10 mM pH 1.69 GLY solution and buffer, and data output. KD represents the equilibrium dissociation constant, or affinity. Results are shown in Table 1 below.

[0068] Table 1 Affinity test results of HBV-S-4H7 antibodies

[0069] Antibody name Dissociation constant HBV-S-4H7 antibody 6.32E-10

[0070] It can be seen from the data in Table 1 that the HBV-S-4H7 antibody has a good affinity for the antigen.

[0071] Example 6 Sequencing and Identification of Variable Regions of HBV-S-4H7 Monoclonal Antibody

[0072] The heavy chain variable region and light chain variable region sequences of HBV-S-4H7 monoclonal antibody were obtained by sequencing using the degenerate primer amplification method. Specifically, HBV-S-4H7 hybridoma monoclonal cells were collected, and total cellular RNA was extracted using a kit. RNA integrity was detected by 1% agarose gel electrophoresis, and RNA concentration was determined by a nucleic acid quantitative analyzer. Using an RNA reverse transcription kit, 1 μg of RNA was reverse transcribed into cDNA. Using 5 μl of cDNA as a template, the PCR reaction system and PCR program were configured according to conventional light and heavy chain primer amplification primers and reaction systems in the art, wherein the annealing temperature was adjusted using a gradient cooling method. Then, a second round of PCR was performed directly using 1 μl of the first-round PCR product as a template. The PCR reaction system and program were the same as above. All second-round PCR products were loaded onto a 1% agarose gel for electrophoresis, and specific target bands of appropriate sizes were cut out and recovered from the gel. The cloning vector was ligated and the competent cells were transformed. Monoclonal colonies were picked, and after colony activation, sequencing was commissioned to Shanghai Biotech. After sequencing, the VL and VH amino acid sequences of the HBV-S-4H7 antibody were obtained, as shown in SEQ ID NOs: 1 and 2, respectively.

[0073] Example 7 Specificity Identification of HBV-S-4H7 Monoclonal Antibody

[0074] The ELISA plate coated with immunogen S-broad spectrum peptide, S epitope peptide 2 (cttpaqgtsmypsccctkpseg), recombinant hepatitis B vaccine (Saccharomyces cerevisiae) (Shenzhen Kangtai Biological Products Co., Ltd.), G9P[8] type inactivated rotavirus (purchased from Peking Union Medical College), BSA, Enterococcus faecium lysate, and Escherichia coli lysate was added with 1:3000 diluted antibodies. At the same time, negative control wells were set up. A450nm was measured by ELISA. The absorbance value of the sample well (S) / the absorbance value of the negative control well (N) ≥ 2.1 was judged as positive. The results are as follows Figure 4 shown.

[0075] from Figure 4From the specific identification results of the ELISA system, it can be seen that the P / N value of HBV-S-4H7 monoclonal antibody against S-broad-spectrum peptide and recombinant hepatitis B vaccine is significantly greater than 2.1, reaching (16.18±0.34) and (14.59±0.43) respectively. The test results for other substances are negative, indicating that the system can specifically detect HBV S protein, and also that HBV-S-4H7 monoclonal antibody has a good ability to distinguish hepatitis B virus from other substances.

[0076] Example 8 Detection of the Virus Neutralization Ability of HBV-S-4H7 Monoclonal Antibody

[0077] Since the S protein is the main pathogenic protein of the virus, monoclonal antibodies that inhibit the S protein theoretically have the ability to neutralize the virus. The Shanghai Pasteur Institute of the Chinese Academy of Sciences was commissioned to conduct virus neutralization testing. Specifically, HepG2-NTCP cells were seeded into 24-well plates at 2×105 cells / well and allowed to adhere. The cells were divided into experimental and control groups. The experimental group received 1.0×10 ayw serotype virus. 7 Cells were incubated with serially diluted HBV-S-4H7 antibodies (1, 0.5, 0.1, and 0.01 mg / ml) at an MOI of 500. A control group (MOCK group) was incubated with virus and PBS. Cells in each group were mixed in DMEM (10% FBS, 2.5% DMSO, 4% PEG 8000) and infected overnight. The cells were washed five times with PBS, and the medium was changed every two days. HBeAg levels in the culture medium were measured on day 5. Detection was performed using the Hepatitis B virus e antigen (HBeAg) detection kit (chemiluminescence method) (Shanghai Kehua Bioengineering Co., Ltd., National Medical Device Registration No. 20143402150).

[0078] like Figure 5 As shown, compared with the control group, HBeAg levels in cells co-incubated with HBV-S-4H7 antibody in the experimental group decreased significantly. In particular, at a concentration of 1 mg / mL, the OD450 was only (0.16±0.02), compared to (1.78±0.07) in the control group (significant difference, P<0.01). This indicates that HBV-S-4H7 antibody has a neutralizing effect on HBV. As the incubation antibody concentration decreases, the amount of HBeAg increases, indicating that the antibody's ability to neutralize the virus decreases, suggesting that the neutralization effect of HBV-S-4H7 antibody on HBV virus is dose-dependent.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A broad-spectrum monoclonal antibody HBV-S-4H7 for detecting HBV, characterized in that The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 1, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

2.

2. Use of the monoclonal antibody according to claim 1 in the preparation of a reagent for detecting HBV S protein.

3. Use of the monoclonal antibody according to claim 1 in preparing a kit for detecting HBV.

4. Use of the monoclonal antibody according to claim 1 in preparing a test strip for detecting HBV.

5. The use according to claim 2 or 3, characterized in that The monoclonal antibody is labeled with a detectable enzyme.

6. The use according to claim 4, characterized in that The monoclonal antibody is labeled with colloidal gold.

7. Use of the HBV monoclonal antibody HBV-S-4H7 according to claim 1 in the preparation of a drug for inhibiting HBV proliferation.

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