Antibody against hepatitis B surface antigen and its application and detection kit

Abstract

The present invention relates to the field of antibody drug technology, particularly to antibodies against hepatitis B surface antigen (HBsAg), their applications, and detection kits. Disclosed are a monoclonal antibody and its sequence, which binds to an epitope located within the highly hydrophilic domains 117-134 of the HBsAg. The antibodies of the present invention can be used for medical treatment and diagnosis, for example, in the treatment and / or diagnosis of liver diseases caused by the HBV, such as acute and chronic hepatitis, cirrhosis, and primary liver cancer, and have promising clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody drugs, and in particular to antibodies against hepatitis B surface antigens, applications thereof, and a detection kit. Background Art

[0002] Hepatitis B virus (HBV) is a major cause of serious liver disease, including acute and chronic hepatitis, cirrhosis, and primary liver cancer. People with chronic HBV infection can transmit the infection for many years, but safe and effective vaccines are available to prevent HBV infection. According to a conference statement from the National Institutes of Health (NIH), the natural history of chronic HBV infection is a dynamic process divided into three phases: immune tolerance, immune activation (IA), and the inactive carrier state.

[0003] Hepatitis B virus (HBV) is a member of the hepadnaviridae family of viruses and can cause acute or chronic liver disease. The virion consists of an outer lipid envelope and a core composed of icosahedral proteins called a nucleocapsid. The nucleocapsid encapsulates viral DNA and DNA polymerase, which possesses reverse transcriptase activity. The outer membrane proteins include an intercalating protein involved in viral attachment and entry. The virus is one of the smallest enveloped animal viruses, with virions measuring 42 nanometers in diameter. They exist in polymorphic forms, including large spherical particles, small spherical particles, and tubular particles. These particles are non-infectious and form the surface of the virion, which is composed of lipids and proteins. This is the surface antigen (HBsAg), which is produced during the viral life cycle. Mutations in the primary hydrophilic region of the HBV surface antigen can alter the protein's antigenicity and affect the secretion and formation of virions. They can also interfere with the detection of surface antigens in commercial kits, leading to vaccine ineffectiveness and the failure of hepatitis B immune globulin (HBIG) therapy. Therefore, it is necessary to obtain a new antibody that can recognize multiple hepatitis B surface antigens to better facilitate the treatment of hepatitis B disease and the development of detection kits. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an anti-HBsAg antibody and its application and detection kit.

[0005] The present invention provides a monoclonal antibody against hepatitis B surface antigen, wherein the amino acid sequence of the CDR1 region of the heavy chain is GFSLSDYW, the amino acid sequence of the CDR2 region is IRNKPYNYAT, and the amino acid sequence of the CDR3 region is TGGFDF; or a sequence having at least 80% sequence homology with the above sequences;

[0006] The amino acid sequence of the CDR1 region of the light chain is QSVNTD, the amino acid sequence of the CDR2 region is SAS, and the amino acid sequence of the CDR3 region is QHHYLPPYT, or a sequence having at least 80% sequence homology with the above sequences.

[0007] In the monoclonal antibody against hepatitis B surface antigen of the present invention,

[0008] The four FR regions of its heavy chain have the amino acid sequences shown in SEQ ID NOs: 7, 8, 9 and 10, respectively, or sequences having at least 80% sequence homology thereto;

[0009] The four FR regions of the light chain have the amino acid sequences shown in SEQ ID NOs: 11, 12, 13 and 14, respectively, or sequences having at least 80% sequence homology thereto.

[0010] In some embodiments, the monoclonal antibodies against hepatitis B surface antigen provided by the present invention are

[0011] The amino acid sequences of the three CDR regions of its heavy chain are GFSLSDYW, IRNKPYNYAT, and TGGFDF;

[0012] The amino acid sequences of the three CDR regions of its light chain are QSVNTD, SAS, and QHHYLPPYT;

[0013] The amino acid sequences of the four FR regions of its heavy chain are shown in SEQ ID NOs: 7, 8, 9 and 10, respectively;

[0014] The amino acid sequences of the four FR regions of its light chain are shown in SEQ ID NOs: 11, 12, 13 and 14, respectively.

[0015] In some specific embodiments, in the monoclonal antibody against hepatitis B surface antigen of the present invention,

[0016] The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 15;

[0017] The light chain variable region thereof has the amino acid sequence shown in SEQ ID NO:5.

[0018] The monoclonal antibody provided by the present invention further comprises a constant region, wherein the constant region of the heavy chain is any one of mouse IgG1; and the constant region of the light chain is of κ type.

[0019] The advantages of the antibody of the present invention are high sensitivity, strong specificity, fast recognition speed and strong binding ability.

[0020] The present invention also provides a nucleic acid encoding the monoclonal antibody against hepatitis B surface antigen.

[0021] The present invention provides an expression vector comprising a nucleic acid encoding the monoclonal antibody. In some embodiments, the backbone vector in the expression vector is pCMV3.

[0022] The present invention provides host cells transformed or transfected with the expression vector. In some embodiments, the host comprises HEK 293T cells and CHO cells.

[0023] The present invention also provides a method for preparing the monoclonal antibody against hepatitis B surface antigen, which comprises: culturing the host cell and inducing the expression of the monoclonal antibody.

[0024] The present invention also provides a chemically labeled or biologically labeled monoclonal antibody against hepatitis B surface antigen.

[0025] The chemical label is an isotope, an immunotoxin, and / or a chemical drug; the biological label is biotin, avidin, or an enzyme label. The enzyme label is preferably horseradish peroxidase or alkaline phosphatase. The immunotoxin is preferably aflatoxin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe lectin, modeccin, PAP, herbivorin, gelonin, or luffa toxin.

[0026] The present invention provides a conjugate prepared by coupling the monoclonal antibody or conjugate with a solid medium or a semisolid medium. The medium in the conjugate of the present invention is selected from magnetic microparticles, magnetic beads or latex.

[0027] The present invention also provides the use of the cloned antibody, the binder and / or the conjugate in preparing a product for detecting hepatitis B virus surface antigen.

[0028] The present invention provides a kit comprising the monoclonal antibody, the chemically labeled or biolabeled monoclonal antibody, and / or a conjugate obtained by coupling the monoclonal antibody with a solid medium or a semisolid medium. In some embodiments, the kit is a magnetic microparticle chemiluminescent hepatitis B kit.

[0029] The kit can be used to detect HBsAg or its mutant strains.

[0030] The present invention also provides a method for detecting hepatitis B virus surface antigen.

[0031] The method comprises using the kit to detect hepatitis B virus surface antigen.

[0032] The detection methods described herein include both diagnostic and non-diagnostic testing. Non-diagnostic testing can be used in scientific research or experimental simulations. Diagnostic testing includes clinical testing for hepatitis B virus surface antigen activity in patients.

[0033] The present invention provides use of the monoclonal antibody, the nucleic acid, the expression vector, the host cell, the monoclonal antibody and / or the conjugate in preparing drugs for preventing and treating hepatitis B.

[0034] The present invention also provides a medicine comprising the monoclonal antibody, the nucleic acid, the expression vector, the host cell, the monoclonal antibody and / or the conjugate.

[0035] The medicine provided by the present invention also includes a substance prepared from the monoclonal antibody or a fragment thereof.

[0036] The present invention also provides a method for treating hepatitis B disease, which comprises using the drug to treat the hepatitis B disease.

[0037] The present invention provides antibodies against hepatitis B surface antigen (HBsAg), their applications, and a detection kit. Disclosed are a monoclonal antibody and its sequence, which binds to an epitope located within the highly hydrophilic domains 117-134 of the HBsAg. The antibodies provided herein can be used for medical treatment and diagnosis, for example, in the treatment and / or diagnosis of HBV-induced liver diseases such as acute and chronic hepatitis, cirrhosis, and primary liver cancer. The detection kit provided herein has excellent specificity and high sensitivity, and has promising clinical application prospects. DETAILED DESCRIPTION

[0038] The present invention provides antibodies against hepatitis B surface antigen and their applications and detection kits. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0039] SEQ ID NO: 1: GFSLSDYW

[0040] SEQ ID NO: 2: IRNKPYNYAT

[0041] SEQ ID NO: 3: TGGFDF

[0042] SEQ ID NO: 4: QSVNTD

[0043] SEQ ID NO:6: QHHYLPPYT

[0044] The four FR regions of the heavy chain have the amino acid sequences shown in SEQ ID NOs: 7, 8, 9 and 10, respectively.

[0045] SEQ ID NO:7:ETKLDESGGGLVQPGRPMKLSCVAS

[0046] SEQ ID NO:8:MNWVRQSPEKGLEWVAQ

[0047] SEQ ID NO:9:YYLDSVKGRFTISRDDSKSSVYLQMNNLRTEDTGLYYC

[0048] SEQ ID NO: 10: WGQGTALTVSS

[0049] The four FR regions of the light chain have the amino acid sequences shown in SEQ ID NOs: 11, 12, 13 and 14, respectively.

[0050] SEQ ID NO:11:ETKLDESGGGLVQPGRPMKLSCVAS

[0051] SEQ ID NO:12:MNWVRQSPEKGLEWVAQ

[0052] SEQ ID NO:13:YYLDSVKGRFTISRDDSKSSVYLQMNNLRTEDTGLYYC

[0053] SEQ ID NO: 14: WGQGTALTVSS

[0054] Heavy chain variable region (SEQ ID NO: 15):

[0055] ETKLDESGGGLVQPGRPMKLSCVASGFSLSDYWMNWVRQSPEKGLEWVAQIRNKPYNYATYYLDSVKGRFTISRDDSKSSVYLQMNNLRTEDTGLYYCTGGFDFWGQGTALTVSS

[0056] Light chain variable region (SEQ ID NO: 5):

[0057] DIQMIQSHKSMSTSIGDRVTITCKASQSVNTDVAWHQQKPGQSPKLLIYSASYRFTGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCQHHYLPPYTFGGGTKLEIK

[0058] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0059] Example 1 Preparation of anti-HBsAg monoclonal antibodies

[0060] 1. Mouse immunization

[0061] HBsAg protein, fully emulsified with Freund's complete adjuvant, was intraperitoneally immunized into 5-week-old female Balb / c mice at a primary dose of 100 μg / mouse. Second and third immunizations were administered 21 and 42 days after the first immunization, respectively, at a dose of 50 μg / mouse. Approximately 10 days after the third immunization, tail blood was collected, and serum titers were determined indirectly using a 96-well plate coated with HBsAg.

[0062] 2. Hybridoma Cell Preparation

[0063] Select indirect method to detect serum titer greater than 10 4 Mice were given an intrasplenic booster immunization at a dose of 100 μg / mouse. Three days after the booster immunization, the spleens of the mice were harvested and fused with mouse myeloma cells NS1 at a ratio of 10:1. The fused cells were cultured in DMEM medium containing HAT (Gibco).

[0064] About 6 to 7 days after fusion, the specific antibody content in the cell culture supernatant was detected indirectly using a 96-well plate coated with HBsAg. Positive wells with an OD value of not less than 0.5 were selected and subcloned three times using the limiting dilution method. Finally, hybridoma cell lines that could stably secrete anti-HBsAg were obtained.

[0065] 3. Purification of anti-HBsAg monoclonal antibodies

[0066] The obtained mouse hybridoma cells capable of stably secreting anti-HBsAg are injected into the peritoneal cavity of mice, and the ascites is collected and purified by SPA to obtain anti-HBsAg monoclonal antibodies with a purity of more than 90%.

[0067] 4. Anti-HBsAg monoclonal antibody titer detection

[0068] HBsAg (ad) and HBsAg (ay) proteins were diluted to 1 μg / ml and 4 μg / ml, respectively, in 0.05 mmol / L CB buffer, pH 9.6. 50 μl was added to each well of a 96-well ELISA plate (Corning) and coated overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with 1% Casein (100 μl / well) at 37°C for 2 hours. Purified monoclonal antibodies (5 mg / ml) were serially diluted starting from 10 μg / ml in 0.05 mmol / L CB buffer, pH 9.6. The diluted antibodies were added to the HBsAg-coated ELISA plate. 50 μl / well of 0.05 mmol / L CB buffer, pH 9.6, was added to the negative control wells. After incubation at 37°C for 30 minutes, wash the plate five times with PBST, pat dry, add 100 μl / well of 1:4000 diluted HRP-goat anti-mouse IgG (purchased from SIGMA), and incubate at 37°C for 30 minutes. Wash the plate five times with PBST, pat dry, add 100 μl / well of standard enzyme immunoassay substrate, and incubate at room temperature in the dark for 10 minutes. Terminate the reaction with 50 μl of 0.1 mol / L sulfuric acid, and measure absorbance at 450 nm. The test results are shown in Table 1 below:

[0069] Table 1: Anti-HBsAg antibody titer test results

[0070]

[0071] As shown in Table 1, the OD value of the negative control was lower than 0.2, the titer of the anti-HBsAg monoclonal antibody recognizing ad subtype HBsAg reached above 0.0097 μg / ml, and the titer of recognizing ay subtype HBsAg reached above 0.019 μg / ml, showing a relatively high titer.

[0072] 5. Identification of anti-HBsAg monoclonal antibody subtypes

[0073] HBsAg (ad) protein was diluted to 1 μg / ml and coated overnight on a 96-well ELISA plate, which was then blocked with 1% Casein. Purified anti-HBsAg monoclonal antibody (5 mg / ml) was diluted 1:1000 in 0.05 mmol / L CB buffer, pH 9.6. The subtype of the purified anti-HBsAg antibody was indirectly identified using a mouse monoclonal antibody subtype identification reagent. The results are shown in Table 2.

[0074] Table 2: Anti-HBsAg antibody subtype identification results

[0075]

[0076] As shown in Table 2, the subtype of anti-HBsAg monoclonal antibodies is IgG1.

[0077] 6. Detection of the ability of anti-HBsAg monoclonal antibodies to recognize mutant strains (antibodies screened in the above examples)

[0078] Anti-HBsAg monoclonal antibody was coated on magnetic beads and used in combination with anti-HBs sheep polyclonal enzyme-labeled antibody to detect recombinant HBsAg mutant strains. The test results are shown in Table 3:

[0079] Table 3

[0080]

[0081]

[0082]

[0083] As can be seen from Table 3, the anti-HBsAg antibodies had weak ability to recognize the T123N and C124R mutants, but could recognize the other mutants.

[0084] The luminescence values of anti-HBsAg antibodies diluted to different concentrations for detecting different subtype mutants were measured to prepare a magnetic particle chemiluminescence hepatitis B test kit. The results are shown in Table 4. It can be seen that the detection limit of the present invention can be as low as 0.98 ng / mL (the luminescence value is higher than S1 in Table 5), with good specificity and high sensitivity.

[0085] Table 4

[0086]

[0087] 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 monoclonal antibody against hepatitis B surface antigen, characterized in that: The amino acid sequence of the CDR1 region of its heavy chain is GFSLSDYW, the amino acid sequence of the CDR2 region is IRNKPYNYAT, and the amino acid sequence of the CDR3 region is TGGFDF; The amino acid sequence of the CDR1 region of its light chain is QSVNTD, the amino acid sequence of the CDR2 region is SAS, and the amino acid sequence of the CDR3 region is QHHYLPPYT.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 15; The amino acid sequence of its light chain variable region is shown in SEQ ID NO:

16.

3. A nucleic acid encoding the monoclonal antibody according to claim 1 or 2.

4. An expression vector, characterized in that The invention also comprises a nucleic acid encoding the monoclonal antibody according to claim 1 or 2.

5. A host cell transformed or transfected with the expression vector according to claim 4.

6. The monoclonal antibody according to claim 1 or 2 which is chemically labeled or biolabeled.

7. A conjugate prepared by coupling the monoclonal antibody according to claim 1 or 2 or the monoclonal antibody according to claim 6 with a solid medium or a semisolid medium.

8. Use of the cloned antibody according to claim 1 or 2, the nucleic acid according to claim 3, the expression vector according to claim 4, the host cell according to claim 5, the monoclonal antibody according to claim 6 and / or the conjugate according to claim 7 in the preparation of a product for detecting hepatitis B virus surface antigen.

9. A kit for detecting hepatitis B virus surface antigen, characterized in that: It comprises the monoclonal antibody according to claim 1 or 2, the monoclonal antibody according to claim 6 and / or the conjugate according to claim 7.

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