KR19990008649A - Mouse Monoclonal Antibodies to Surface Antigen Pre-S 1 of Non-Hepatitis Virus, Hybridoma Cell Line Producing the Same, and Method for Preparing the Same - Google Patents

Abstract

The present invention relates to a monoclonal antibody that binds to surface antigen pre-S1 (pre-S1) of hepatitis B virus (HBV), a hybridoma cell line producing the same, and a method for producing the same. Specifically, the present invention relates to a mouse monoclonal antibody that specifically recognizes an epitope comprising amino acids 43-49 of HBV surface antigen pre-S1, which can neutralize several subtypes and mutant HBV to prevent HBV infection. It can be widely used to prevent and treat.

Description

Mouse monoclonal antibody against surface antigen pre-S1 of hepatitis virus, a hybridoma cell line producing the same, and a method of preparing the same.

The present invention relates to a monoclonal antibody that binds to the surface antigen pre-S1 (pre-S1) of hepatitis B virus (hereinafter abbreviated as HBV), a hybridoma cell line producing the same, and a method for producing the same. .

More specifically, the present invention relates to a mouse monoclonal antibody that specifically recognizes an epitope comprising amino acids 43-49 of HBV surface antigen pre-S1, which can neutralize several subtypes and variant HBVs. It can be widely used to prevent and treat infections.

Hepatitis B virus (HBV) is a pathogen that invades humans and causes chronic and acute hepatitis and, if worse, causes cirrhosis and liver cancer, an estimated 300 million people worldwide (Tiollais Buendia, Sci). Am., 264: 48, 1991).

HBV infections include HB immunoglobulin (HBIG) to prevent HBV infection, especially in newborns born to HBV-positive mothers, health care workers exposed to HBV, or patients undergoing liver transplantation due to HBV-related liver disease. (Beasley, et al., Lancet, 2: 1099, 1983; Todo, et al., Hepatol., 13: 619, 1991). However, since HBIG, which is currently used, is extracted from plasma, it has a low specificity and is exposed to a contaminant, which requires a continuous supply of human blood for mass production. This disadvantage can be overcome by developing and using monoclonal antibodies against the surface antigens of HBV.

The coating of HBV consists of three proteins, specifically, a major protein containing S antigen, a middle protein containing S antigen and a pre-S2 antigen, and a S antigen, pre-S2 antigen and a pre- It consists of a large protein comprising the S1 antigen (Neurath, AR and Kent, SBH, Adv. Vir. Res., 34: 65-142, 1988). All of these surface antigen proteins induce antibodies that neutralize and neutralize HBV, in particular antibodies induced by the free-S site are associated with virus clearance and recovery from acute HBV infection and with an immune response to S antigen ( Non-responsiveness) (Iwarson, S. et al., J. Med. Virol. 16: 89-96, 1985; Itoh, Y. et al., Proc. Natl. Acad. Sci. USA, 84: 9174-9178, 1986; Neurath, AR et al., Vaccine, 7: 234-236, 1989; Budkowska, A. et al., J. Med. Virol., 20: 111-125, 1986; Milich, DR et al., Proc. Natl. Acad. Sci. USA, 82: 8168-8172, 1985; Neurath, AR et al., J. Med. Virol. 17: 119-125, 1985; Milich, DR et al. , J. Immunol., 137: 315-322, 1986). In particular, amino acid positions 21-47 of the pre-S1 protein bind to the HBV receptor of human hepatocytes and play a decisive role in infecting the hepatocytes, so monoclonal antibodies specific for these sites may play an important role in virus neutralization. Neurath, et al., Cell, 46: 429, 1986; Pontisso, et al., Virol., 173: 533, 1989; Neurath, et al., Vaccine, 7: 234, 1989).

Accordingly, the present inventors obtained a pre-S1 peptide using GST-pre-S1 fusion protein combined with glutathione-S-transferase, and combined keyhole limpet hemocyanin (hereinafter abbreviated as KLH) to mice. The hybridoma cell line was prepared by injection, and the monoclonal antibody binding to pre-S1 was separated therefrom to confirm that the epitope containing amino acid positions 43-49 was recognized.

It is an object of the present invention to provide a mouse monoclonal antibody that binds to surface antigen pre-S1 of hepatitis B virus (HBV), a hybridoma cell line producing the same, and a method for producing the same.

FIG. 1 shows the expression of GST-preS1 fusion protein in E. coli and electrophoresis of 12.5% SDS-polyacrylamide gel electrophoresis to determine the epitope of the monoclonal antibody KR127 of the present invention (a), Westing blot (b) Show the results,

Lane 1: GST; Lane M: Standard Protein

Lane 2: GST-preS1 (1-11); Lane 3: GST-preS1 (1-20);

Lane 4: GST-preS1 (1-28); Lane 5: GST-preS1 (1-35);

Lane 6: GST-preS1 (1-42); Lane 7: GST-preS1 (1-49);

Lane 8: GST-preS1 (1-56)

2 is a graph showing the antigen binding affinity of the monoclonal antibody KR127 of the present invention,

Figure 3 is a monoclonal antibody KR127 of the present invention confirmed the ability to bind to hepatitis B virus (HBV) by immunoprecipitation,

NC: negative control

Figure 4 shows the expression of several subtypes of GST-preS1 (1-56) fusion protein in Escherichia coli to confirm the ability of the monoclonal antibody KR127 of the present invention to bind to several subtypes and variants of HBV. Gel electrophoresis (a) Western blot (b) shows the results.

Lane 1: GST; Lane M: standard protein;

Lane 2: pre-S1 (1-56) of the adr subtype;

Lane 3: pre-S1 (1-56) of the adw subtype;

Lane 4: pre-S1 (1-56) of the ayw subtype;

Lane 5: pre-S1 (1-56) of the adr subtype with the 25 th amino acid substituted with leucine

In order to achieve the above object, the present invention provides a hybridoma cell line by binding KLH to HBV surface antigen pre-S1 to immunize mice, and from this, an epitope comprising amino acids 43-49 of pre-S1 It provides a mouse monoclonal antibody that recognizes.

Hereinafter, the present invention will be described in detail.

In order to obtain a monoclonal antibody that recognizes the surface antigen pre-S1 site of hepatitis B virus (HBV), a peptide consisting of 56 amino terminal amino acids of pre-S1 is first prepared.

The present inventors have combined a gene encoding the 56 amino terminal amino acids of HBV surface antigen pre-S1 with a glutathione-S-transferase (GST) gene to form a fusion protein that is water-soluble in E. coli. Mass expression of S1 protein has been performed (Korean Patent Application No. 95-9865).

Pre-S1 peptide obtained from the fusion protein obtained by the above method was combined with keyhole limpet hemocyanin (KLH), and then its antigenicity was greater than that of the pre-S1 peptide alone. Used to Next, the splenocytes and myeloma cells of the mouse immunized with the antigen are fused by the conventional method, and a clone showing specificity for the pre-S1 peptide is selected by an indirect ELISA method. Of these, hybridoma cell lines producing antibodies with isotype IgG are selected and cells which stably secrete antibodies are passaged continuously. As a result, a hybridoma cell line producing the monoclonal antibody KR127 (Accession No .: KCTC 0289 BP) of the present invention having specificity for the epitope of pre-S1 was selected.

In order to determine the exact epitope of the pre-S1 region to which the monoclonal antibody KR127 produced in the hybridoma cell line reacts, the present invention uses an expression plasmid containing the pre-S1 gene to obtain the 3'- of the pre-S1 gene. Various plasmid vectors are prepared, with some of the ends cleaved.

Specifically, the pre-S1 gene present in the plasmid vector pGSTpreS1-56 was cut from the 3′-end to 1-11, 1-20, 1-28, 1-42 and 1-49 of the pre-S1 region. Plasmid vectors containing genes encoding amino acids 1 and 56, etc., are prepared by polymerase chain reaction (PCR), and are used to produce pre-S1 peptides of various sizes.

As a result of Western blot of the peptides expressed above by SDS-polyacrylamide gel electrophoresis, it was confirmed that the monoclonal antibody KR127 of the present invention recognized an epitope containing amino acids 43-49 of the pre-S1 region. (See FIG. 1 )

The present invention uses the monoclonal antibody KR127 to investigate antibody binding ability and antigen binding affinity by indirect ELISA method (see FIG. 2 ), and analyze HBV particle immune sedimentation by Southern blot (see FIG. 3 ).

In addition, the present invention, in order to confirm that the monoclonal antibody KR127 binds to various HBV subtypes and variants, HBV pre-S1 variants of various subtypes are prepared and Western blot is performed using the same (see FIG. 4 ). As a result, the monoclonal antibody of the present invention shows excellent immune activity against HBV such as adw and ayw subtypes including adr subtype

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the examples.

Example 1 Preparation of a Peptide Consisting of 56 Free-S1 Amino Terminals

E. coli BL21 (DE3) containing the plasmid vector pGSTpreS1-56 (KCTC 8645P) was incubated in a 1 liter LB medium with an absorbance (OD) of 0.5, incubated at 37 ° C for 3 hours with 0.1 mM IPTG, and then 4000 Centrifugation at xg for 10 minutes and the precipitate was recovered and dissolved in 20 ml of Tris buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 0.02% NaN ₃ ). The suspension was sonicated to disrupt cells and centrifuged at 12000 xg for 10 minutes to obtain supernatant.

In order to purify the pre-S1 protein from the fusion protein expressed above, the supernatant of the crushed cells was first applied to a glutathione-agarose column equilibrated with Tris buffer (Sigma, USA), and the 5 mM reduced glutathione solution. Flowing to recover the bound fusion protein. Next, 50 μg of the fusion protein was dissolved in 50 μl of a digestion buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 2.5 mM CaCl ₂ ), and 0.2 μg of thrombin was added and reacted at room temperature for 2 hours. The pre-S1 peptide that did not bind to the column by application to an agarose column was isolated from the fusion protein.

Example 2 Preparation of KLH-preS1 Protein

0.55 mg (0.09 μM) of the pre-S1 peptide obtained in Example 1 was dissolved in 50 mM phosphate buffer (pH 8.6), and 0.025 mg (0.18 μM) 2-iminothiolane was added for 3 hours at room temperature. Reacted for a while. The reaction solution was applied to a Sephadex G-25 column to remove byproducts, and then 1 mg of maleimide activated KLH (maleimide activated KLH; keyhole limpet hemocyanin, manufactured by Pierce) was added and reacted at room temperature for 2.5 hours. Dialysis was performed for 2 days with Tris-HCl buffer (pH 7.4). The dialysate was loaded onto a Sephacryl S-200 column and pre-S1 bound with KLH in Tris-HCl buffer (50 mM Tris-HCl, pH7.4, 1 mM EDTA, 0.2 mM PMSF, 0.5 M NaCl, NaN ₃ 0.02%). Protein (KLH-preS1) was eluted.

The eluate obtained above was quantified using the BCA kit (Pierce Co., Ltd.), a protein quantification kit, and the final protein concentration was 0.175 mg / ml, from which a total of 1.7 ml of antigen was obtained.

Example 3 Antigenicity of KLH-preS1 Protein

1 μg of the KLH-preS1 protein prepared in Example 2, 1 μg of KLH, and 100 ng of the pre-S1 peptide prepared in Example 1 were each immobilized in an Eliza plate well using a coating buffer. Thus, monoclonal antibody 1B3 (Kim, et al., Korean J. Immunol., 18: 367, 1996), which recognizes an epitope at amino acid positions 1-20 of the pre-S1 protein, was 1.5-100 ng per well. It was added and reacted for 2 hours at 37 ℃. The plates were washed with phosphate buffer added 0.05% Tween 20, reacted with anti-mouse IgG-HRP (manufactured by Sigma), and then a substrate solution containing OPD (o-phenylenediamine) and hydrogen peroxide was added at 492 nm. Absorbance was measured. The results are shown in Table 1.

1B3 (ng) Immunologist 0 1.56 3.13 6.25 12.5 25 50 100 KLH 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 KLH-preS1 0.05 0.2 0.29 0.58 0.81 1.18 1.6 2.2 preS1 0.05 0.09 0.13 0.19 0.32 0.55 0.85 1.27

Fixation of KLH alone did not increase the absorbance, whereas pre-S1 protein and KLH-pre-S1 protein was confirmed that the absorbance increases as the concentration increases, KLH-pre-S1 protein acts as an immunogen. Particularly, in the case of binding KLH, the reactivity is increased, and thus, the present invention uses an experiment to bind KLH.

Example 4 Preparation of Mouse Monoclonal Antibody KR127

20 μg of the KLH-pre-S1 protein prepared in Example 2 was mixed with the same volume of an antigenic enhancer (Hunter's Titer Max adjuvant, manufactured by Sigma), which was 17.5 intraperitoneally and subcutaneously of 10-week-old female Balb / c mice, respectively. μg and 8.7 μg were injected, and the procedure was repeated four times at monthly intervals. After the fourth intraperitoneal injection, mice with high antibody titers were selected for cell fusion.

To collect feeder cells, one day before cell fusion, 8 ml of a 0.34 M sugar solution was added to the peritoneum of healthy mice, which was then sucked up again to collect cells in the abdominal cavity, followed by centrifugation and HAT medium (GIBCO Corporation Product) into aliquots in 96-well plates to 10,000 cells per well and incubated in 37 ° C., CO ₂ incubator. In addition, SP2 / 0-Ag14 myeloma cell line to be fused with splenocytes from two weeks ago was also cultured in a medium made by mixing IMEM (GIBCO Co.) and 10% fetal bovine serum.

The spleens were removed from the mice selected above, washed well with DMEM (GIBCO Co., Ltd.), crushed well in a Petri dish with a glass rod, and the cell suspension was left in a 15 ml tube, and the supernatant was transferred to a new tube. In addition, SP2 / 0-Ag14 myeloma cell line was also harvested by centrifugation and suspended in 10 ml DMEM to measure the number of cells with the spleen cells above. Ten million SP2 / 0-Ag14 myeloma cells and 100 million splenocytes were transferred to a 50 ml tube, mixed, centrifuged at 200 xg for 5 minutes to remove supernatant, and left in a beaker filled with water at 37 ° C for 2 minutes. In addition, 1 ml of PEG solution (manufactured by GIBCO) was added for 1 minute while gently shaking the tube by gently tapping the tube and soaking in 37 ° C water. Then centrifuge at 100 xg for 2 minutes, slowly add 5 ml of DMEM solution over 3 minutes, add 5 ml of DMEM solution slowly over 2 minutes, centrifuge at 200 xg to recover the cells, and place in 30 ml of HAT medium. Carefully suspended. This was allowed to stand at 37 ° C. in a CO ₂ incubator for 30 minutes, and then 100 μl per well was dispensed into 96-well plates with feeder cells pre-incubated, and after 4 days, 70 μl HAT medium was added and grown for 2 weeks or longer. It was.

Indirect Eliza method was used to select clones showing specificity for pre-S1 protein. 100 μl of hybridoma culture solution was added to a plate coated with 1 μg of GST-preS1-56 fusion protein per well, and reacted at 37 ° C. for 1 hour, followed by anti-mouse IgG conjugated with HRP (horseradish peroxidase manufactured by Sigma). It was further reacted with 1/1000 dilution of IgM for 1 hour. The plate was washed with a phosphate buffer solution containing 0.05% Tween 20, and a substrate solution containing OPD (manufactured by Sigma) and H ₂ O ₂ was added, and the absorbance was measured at 492 nm.

Hybridoma cell lines showing specificity for pre-S1 were selected and their isotypes were determined using an isotyping kit (BM, Germany) by the sandwich Eliza method, of which IgG-type antibodies were produced. Hybridoma cell lines were selected. The results are shown in Table 2.

Clone Isotype KR1 IgG2a, κ KR2 IgG1, κ KR3 IgG1, κ KR4 IgG1, κ KR5 IgG, κ KR6 IgG2a, κ

Among them, the monoclonal antibody that reliably secretes the antibody KR1 and sub-cloned the cells in a dilution method containing 0.2 cells per well to maintain stability and specificity for the pre-S1 epitope. KR127 was selected. In the present invention, only monoclonal antibody KR127 was selected and its properties were investigated by various processes.

The monoclonal antibody KR127 of the present invention was deposited with the accession number KCTC 0289BP dated December 19, 1996 to the Genetic Bank of Korea Institute of Science and Technology.

Example 5 Epitope Determination of Monoclonal Antibody KR127

The pre-S1 gene in the plasmid vector pGSTpreS1-56 of Example 1 was partially cut from the 3′-end to 1-11, 1-20, 1-28, 1-35, 1- of the pre-S1 site. Seven plasmid vectors were prepared by PCR to contain genes encoding amino acids 42, 1-49 and 1-56.

In this case, oligonucleotides having the following sequences were used as primers in the 5'-end.

5'-CGAGGATCCATGGGAGGTTGGTCTTCC-3 '

In the case of 3'-end, oligonucleotides each having the following sequence were used.

Up to the 11th, 5'-CGTGAATTCGCCTTGTCGAGGTTTGGA-3 ',

Until the 20th, 5'-GCTGAATTCATTGGGAACAGAAAGATT-3 ',

Until the 28th, 5'-GCTGAATTCGTGATCGGGAAAGAATCC-3 ',

Up to the 35th, 5'-GCTGAATTCTCCGAACGCAGGGTCCAA-3 ',

Until the 42nd, 5'-GCTGAATTCATCTGGATTGTTGGAGTT-3 ',

Up to the 49th, 5'-GCTGAATTCCTTGTTGGGGTTGAAGTC-3 ',

Up to 56th 5'-CCGAATTCATTTCCGTCTGGCCAGTG-3 '

The plasmid vector thus produced was transformed into E. coli DH5α by CaCl ₂ method and then cultured in LB medium.

The expression of the fusion protein was induced by the method of Example 1 from each E. coli, subjected to 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (see FIG. 1A ), and then westernized using the monoclonal antibody KR127. Blot (see FIG. 1B ).

As shown in FIG. 1 , the monoclonal antibody KR127 does not bind to GST-preS1 (1-20) protein to which the peptides of amino acids 1-20 of pre-S1 are fused (lanes 1-2) and pre-S1. From the GST-preS1 (1-28) fused to the peptides of amino acids 1-28 of (Lane 3-8), the epitope of the monoclonal antibody KR127 is located amino acid positions 21-28 of pre-S1 It was found to include.

Example 6 Investigation of antigen binding capacity of monoclonal antibody KR127

Monoclonal antibody KR127 was incubated for one week in PFMHII (Gibco Co., Ltd.) medium, isolated and purified by Protein G-affinity chromatography, and then 1 μg of GST-pre-S1 fusion protein was purified by Elisa plate. Antigen binding ability was measured by indirect ELISA method using anti-mouse IgG-HRP by binding to and reacting the following monoclonal antibody KR127 at various concentrations. The results are shown in Table 3.

KR359 (ng) 0 0.25 0.5 One 2 4 6 8 10 12 15 20 40 80 100 200 A492 0.11 0.18 0.23 0.36 0.67 1.02 1.31 1.58 1.63 1.79 2.02 2.37 3.12 3.29 3.30 3.52

As shown in Table 3, monoclonal antibody KR127 binds to 4 ng of GST-preS1 protein and exhibits an absorbance of 1.02 at 492 nm.

Example 7 Investigation of Antigen Binding Affinity of Monoclonal Antibody KR127

4 ng of monoclonal antibody KR127 was combined with various concentrations of pre-S1 competing antigens as follows, and then the mixture was added to wells coated with 1 μg pre-S1 antigen and affinity was measured by performing an indirect ELISA method ( FIG. 2 ). As shown in FIG. 2 , the affinity for the pre-S1 peptide of the monoclonal antibody KR127 was about 1.7 × 10 ⁸ / M.

Example 8 HBV Particle Immunoprecipitation Study of Monoclonal Antibody KR127

30 μg of the plasmid vector pHBV5.2 (Ryu, et al., J. Med. Virol., 52: 226-233, 1997) with HBV genomic DNA of the adr subtype previously prepared to prepare HBV was electrophoresed. 3x10 ⁷ HepG2 cells were infected by electroporation and incubated for 15 days in H medium. PEG 6000 was added to the culture solution to a final concentration of 6%, and reacted at 4 ° C. for 12 hours to precipitate HBV, followed by centrifugation at 10000 × g for 30 minutes. Virus particles were suspended in 1/50 volume of phosphate buffer with 25% fetal calf serum and stored at minus 70 ° C.

10 μg of the monoclonal antibody KR127 was bound to protein A-agarose (protein A-agarose, Pharmacia), mixed with the virus particles, and reacted at room temperature overnight. After washing 5 times with phosphate buffer, 10 μg of tRNA was added to the immunocomplex and the viral DNA was extracted by the Hert method (Hirt, J. Mol. Biol., 26: 365, 1967). Viral DNA was analyzed by Southern blot with a probe of radiolabeled α-P ³² HBV genomic DNA. As a result, by observing the HBV genomic DNA band of the circular (Relaxed Circular) form (see Fig. 3 ), it was confirmed that the monoclonal antibody KR127 binds to the HBV particles and immunoprecipitates HBV.

Example 9 Investigation of the Avidity of Monoclonal Antibody KR127 against HBV Multiple Subtypes

In order to investigate the binding capacity of HBV of various subtypes of the monoclonal antibody KR127 of the present invention, the amino acid sequences of various variants of HBV pre-S1 listed in the GenBank library were examined and listed as follows.

D23680 (adr) 20 NPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWP 53

X14193 (adr) 20 ------------------------------- R-- 53

A32621 (adw) 20 ------------------------------- Q-- 53

M74498 (adw) 20 ---------------------------- I ----- 53

A32626 20 ------------- V -------------- I ----- 53

X01587 65 ------------------- H -------------- 98

S81946 (adr) 20 ----- L ---------------------------- 53

A32624 20 ---------------------------- V--D-- 53

U55223 (ayw) 7 TS --------------- R --------------- T-- 42

A32622 (ayw) 7 TS --------------- R--TA ----------- T-- 42

To investigate the antigen binding capacity of HBV to other subtypes or variants of the monoclonal antibody KR127, the first of each of the pre-S1 variants of the adr, adw, ayw, and 25th amino acids phenylalanine of the adr subtype was substituted with leucine. DNAs containing the first amino acid to the 56 th amino acid were synthesized by PCR. The nucleotide sequence of the primer used at this time and the plasmid DNA used as a template are as shown in Table 4.

Template DNA 5 'primer 3 'primer adr subtype pHBV315 5'-CGAGGATCCATGGGAGGTTGGTCTTCC-3 ' 5'-CCGAATTCATTTCCGTCTGGCCAGTG-3 ' adw subtype pAM6 5'-CGAGGATCCATGGGAGGTTGGTCTTCC-3 ' 5'-CCGAATTCGTTGGCTGCTGGCCAGTG-3 ' ayw subtype pREP8-preS1 5'-CGGAATTCATATGGGGACGAATCGGGCC-3 ' 5'-CCGAATTCGTTGGCGTCTGGCCAGGT-3 ' adr (F → L) subtype pHBV315 5'-CGAGGATCCATGGGAGGTTGGTCTTCC-3'5'-AATCCTCTGGGATTCCTTCCCGATCACCAG-3 '(mutant primer) 5'-CCGAATTCATTTCCGTCTGGCCAGTG-3'5'-CTGGTGATCGGGAAGGAATCCCAGAGGATT-3 '(mutant primer)

The DNA for each of the pre-S1 variants was inserted and replaced with pre-S1 DNA contained in the cloning position of the plasmid vector pGSTpreS1-56 of Example 1, transformed into Escherichia coli, and then the fusion protein was prepared in the same manner as in Example 1. Induced production. Cell extracts of this recombinant Escherichia coli were each analyzed by 12.5% SDS-polyacrylamide gel electrophoresis (see FIG. 4A ), then transferred to nitocellulose membrane and Western blotted with monoclonal antibody KR127 (see FIG. 4B ).

As a result , as shown in FIG. 4 , the monoclonal antibody KR127 binds well to the pre-S1 antigen of other subtype and variant viruses. Therefore, it was confirmed that the monoclonal antibody KR127 (KCTC 0289BP) of the present invention exhibited excellent immune activity against not only adr subtypes but also overall HBV such as adw and ayw.

As described above, the monoclonal antibodies of the present invention can be widely used for the prevention and treatment of various diseases caused by various subtype and variant HBV infections as well as neutralizing viruses by recognizing specific amino acid sites of the pre-S1 peptide. .

Claims (4)

Monoclonal antibody KR127 (Accession Number: KCTC 0289BP), which recognizes an epitope comprising amino acids 43-49 of the surface antigen pre-S1 (pre-S1) of hepatitis B virus (HBV). A hybridoma cell line producing the monoclonal antibody of claim 1. Immunization of mice by binding KLH to HBV surface antigen pre-S1, and then fusion of the spleen cells of the mice with myeloma cells from which the hybridoma cell line producing the monoclonal antibody of claim 1 is selected. Method of making cell line. Use of the monoclonal antibody of claim 1 for the prevention and treatment of HBV infection, including adr, adw, ayr and ayw subtypes.