JPH06169792A - Monoclonal antibody against non-structural region ns3 of non-a, non-b type hepatitis virus, hybridoma producing the same, and measurement and purification of the virus antigen with the monoclonal antibody - Google Patents

Abstract

PURPOSE:To obtain the subject antibody capable of detecting and determining a viral antigen in high sensitivity, accuracy and specificity and of being effectively used for the purification of the viral antigen. CONSTITUTION:This monoclonal antibody specifically binding to an epitope on the polypeptide antigen in the non-structural region NS3 of a non-A, non-B type hepatitis virus, a hybridoma producing the monoclonal antibody, and the method for measuring and purifying the viral antibody with the monoclonal antibody.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a monoclonal antibody against a non-A non-B hepatitis virus-related antigen, and more specifically to a monoclonal antibody against a non-A non-B hepatitis virus nonstructural region NS-3. The present invention also relates to hybridomas producing such monoclonal antibodies. The present invention further relates to a method for immunologically measuring and purifying non-A non-B hepatitis virus using the monoclonal antibody.

[0002]

2. Description of the Related Art Non-A non-B hepatitis virus is a virus different from hepatitis A virus and hepatitis B virus, and it is known that a part of chronic hepatitis shifts to cirrhosis and liver cancer due to infection with this virus. However, there is great interest in its biochemical and physiological functions.

Regarding the diagnosis of non-A non-B hepatitis, in the early stage, hepatitis A, hepatitis B and D, as well as known viruses that cause liver damage (for example, cytomegalovirus and Epstein-Barr virus). The mainstream of this is the so-called exclusion diagnosis method, in which a non-A non-B hepatitis is diagnosed when the above does not apply to the diagnosis of hepatitis. After that, as a method to improve the diagnostic accuracy,
Development of a method for detecting an antibody using a protein obtained by genetically expressing a non-hepatitis B virus gene as an antigen and a method for detecting the presence of the virus gene by a gene amplification method (PCR) or a DNA probe method Was done.

The identity of the non-A non-B hepatitis virus is still unknown because the amount of viral particles in the patient's body, and therefore the amount of viral antigen, is extremely small and mutation is likely to occur. Technology has become an effective means of diagnosing non-A non-B hepatitis.

Under these circumstances, virus detection using a polyclonal or monoclonal antibody against the virus has been attracting attention. For example, International Application Publication No. WO92 / 07001 (ABBOTT LAB
Oratories), Chir is a specific example of an antibody capable of selectively binding to a non-A non-B hepatitis virus antigen.
A monoclonal antibody against the on C-100 protein (NS4 region; European Patent Application Publication No. 0318216) is disclosed. In addition, International Application Publication No. WO92 / 08738 by the same applicant describes non-A non-B hepatitis virus C-100 protein (NS4 region), 3
Monoclonal antibodies that specifically bind to 3C protein (NS3 region) or CORE protein have been described.

[0006]

As described above, non-A
It is known that the non-hepatitis B virus has very few virus particles per se in the body of an infected patient and also causes a high degree of mutation generally observed in RNA virus species. Therefore, the amount of antibody against the virus-related antigen contained in the blood of the patient is expected to be small, and the need for higher diagnostic accuracy has been desired.

Under such circumstances, if a specific antibody against a non-A non-B hepatitis virus-related antigen was developed, especially a monoclonal antibody, due to its high degree of specificity, analysis for the presence of the virus in blood was carried out. It is expected that the reliability will be further improved.

[0008] Further, in general, the monoclonal antibody can be applied to the purification technique of the peptide expressed by the genetic engineering technique, and even in the field of non-A non-B hepatitis virus, the expressed peptide can be purified. Development of applicable monoclonal antibodies has been desired.

[0009]

The present invention provides a monoclonal antibody that specifically binds to an epitope on a non-A non-B hepatitis virus nonstructural region NS3 polypeptide antigen having the amino acid sequence set forth in SEQ ID NO: 1. To do.

The polypeptide shown in SEQ ID NO: 1 has an amino acid sequence determined by the present applicant as a part of the nonstructural NS3 region of the viral genome (Japanese Patent Application No. 3-1
89268), which is clearly different from the HCV33C protein disclosed in WO92 / 08738 mentioned above. Therefore, the monoclonal antibody of the present invention is novel.

The monoclonal antibody of the present invention includes all antibodies capable of specifically binding to any epitope on the polypeptide antigen having the amino acid sequence shown in SEQ ID NO: 1. Specific examples of such monoclonal antibodies according to embodiments of the present invention include monoclonal antibodies 1H10, 2F4, 3F9, 1G6 and 3D3. The isotype of these antibodies was IgG1. In addition, the antibody of the present invention contains the NS3 antibody in serum and N
It is possible to inhibit the reaction of S3 antigen.

The monoclonal antibody of the present invention is a plasmacytoma of a mammal, which is obtained by immunizing a mammalian plasma cell (immune cell) immunized with a polypeptide having all or part of the amino acid sequence shown in SEQ ID NO: 1 as an immunizing antigen. A hybridoma can be produced by fusing with cells, and a clone that produces an antibody that specifically recognizes only the above-mentioned antigen can be selected and obtained from this clone.

Here, the mammal to be immunized is not particularly limited, but it is preferable to select it in consideration of compatibility with the plasmacytoma cell used for cell fusion, and generally mouse, rat and the like are advantageous. used. In addition, immunization is performed by a general method, for example, by administering an immunizing antigen intraperitoneally, subcutaneously or intravenously to a mammal. More specifically, the polypeptide is added to physiological phosphate buffer (P
BS) or physiological saline or the like to an appropriate concentration, and this is administered to the animal several times, if necessary, in combination with a usual adjuvant. As the immune cells, it is preferable to use splenocytes that have been extracted about 3 days after the final administration of the polypeptide of the non-A non-B hepatitis virus non-structural region (NS-3). As a mammalian plasmacytoma (cell) as the other parent cell fused with an immune cell, various cell lines already known, for example, P3 (P
3 / X63Ag8) [Nature 256,495-
497 (1975)], P3-U1 [Current
Topics in Microbiology and
Immunology 81, 1-7 (198)
7)], NS-1 [Eur. J. Immunol. 6,
511-519 (1976)], SP2 / 0 [Nat
ure 276, 269-270 (1980)], F
O [J. Immunol. Meth. 35, 1-21
(1980)], X63-6, 5, 3, [J. Immu
nol. 123, 1548-1550 (197)
9)], S194 [J. EXP. Med. 148, 3
13-323 (1978)], and also R210 [Nature 277, 131-133 in rats.
(1979)] and the like are used.

The fusion reaction between the immune cells and plasmacytoma is basically carried out by a known method, for example, Milstein.
Et al. [Methods Enzymol. 73,
3-46 (1981)] and the like.

Specifically, the fusion reaction is carried out in a usual nutrient medium in the presence of a fusion promoter, for example. Commonly used fusion promoters such as polyethylene glycol (PEG) and Sendai virus (HV)
J) or the like is used, and an auxiliary agent such as dimethyl sulfoxide (DMSO) can be added and used to further enhance the fusion efficiency. The mixing ratio of the immune cells and the plasmacytoma cells is the same as the usual method, and for example, the immune cells may be used about 1 to 10 times the plasmacytoma cells.

The medium used for the fusion is, for example, RPMI-164 used to grow the plasmacytoma cell line.
Various ordinary mediums used for culturing cells of this type, such as 0 medium or MEM medium, can be used, and it is usually preferable to remove serum supplement such as fetal calf serum (FCS). In the fusion, a predetermined amount of the immune cells and plasmacytoma cells are mixed well in the medium, and a PEG solution preheated to about 37 ° C., for example, one having an average molecular weight of about 1000 to 6000 is added to the normal medium in about It is carried out by adding and mixing at a concentration of 30 to 60% (w / v). After that, a desired hybridoma is formed by repeating the operation of sequentially adding an appropriate medium, centrifuging, and removing the supernatant.

The isolation of hybridomas is carried out by culturing in a normal selection medium, for example, a medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Culturing in the HAT medium may be carried out for a time sufficient to kill cells other than the target hybridoma (unfused cells etc.), usually for several days to several weeks.

The hybridoma thus obtained is subjected to a conventional limiting dilution method to search for a target antibody-producing strain and to perform monocloning.

The production strain can be searched by, for example, ELISA.
Method, agglutination method, octalony method, plaque method, spot method, radioimmunoassay method, and the like, according to various methods generally used for antibody detection.

The hybridoma producing the desired monoclonal antibody thus obtained can be subcultured in an ordinary medium and can be stored in liquid nitrogen for a long period of time.

The monoclonal antibody of the present invention can be collected from the hybridoma by culturing the hybridoma according to a conventional method, and then proliferating the hybridoma as a culture supernatant or by administering the hybridoma to a mammal having compatibility with the hybridoma. Is used. Furthermore, the antibody obtained as described above can be purified by a conventional purification means such as a salting-out method, a gel filtration method, or affinity chromatography.

Accordingly, the present invention also provides a hybridoma producing a monoclonal antibody which specifically binds to an epitope on the non-A non-B hepatitis virus nonstructural region NS3 polypeptide antigen having the amino acid sequence shown in SEQ ID NO: 1. provide.

Specific examples of hybridomas include hybridomas HC7-1H10, HC7-2F4 and HC7-3.
F9, HC7-1G6 and HC7-3D3 can be mentioned, and monoclonal antibodies 1H10 and 2F, respectively.
It produces 4, 3F9, 1G6 and 3D3. By using the method shown in Test Example 1 for these hybridomas,
It is a monoclonal antibody that specifically recognizes a part of the amino acid sequence shown in SEQ ID NO: 1.

[0024]

[Table 1] Hybridoma recognition site HC7-1H10 ⁹⁴ Met ………… ²²¹ Met HC7-2F4 ²²¹ Met …… ²⁵³ Glu HC7-3F9 ⁹⁴ Met ………… ²⁵³ Glu HC7-1G6 ⁹⁴ Met ………… ²⁵³ Glu HC7-3D3 ⁴⁹ Ser ………… ²²¹ Met Especially hybridomas HC7-1H10, HC7-2F
4, HC7-3F9 and HC7-3D3 are 1992 1
It was deposited at the Institute of Microbial Science and Technology on February 1, and the deposit numbers are Micromachine Research Institute No. 13316 and No. 13 respectively.
Nos. 313, 13315, and 13314 are assigned.

The present invention also provides a method for immunologically measuring a non-A non-B hepatitis virus-related antigen using the above monoclonal antibody.

Specifically, this method involves contacting a specimen (eg, blood) suspected of containing the antigen with one or more monoclonal antibodies as defined above to form an antigen-antibody complex. And then detecting or quantifying the complex. Detection or quantification is carried out by conventional means, eg
Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EI)
A), the agglutination method and the like, and both the solid phase method and the homogeneous method can be used. By the method of the present invention, high sensitivity,
A polypeptide derived from the non-A non-B hepatitis virus non-structural region NS3 can be easily measured with high accuracy and high specificity.

The present invention further provides a method for purifying non-A non-B hepatitis virus-related antigens using a monoclonal antibody as defined above.

Specifically, this method comprises binding the monoclonal antibody to a natural or synthetic polymer carrier to form an affinity column, and a liquid containing a non-A non-B hepatitis virus-related antigen in the column ( For example, it includes passing through the blood or an extract of cultured bacterial cells) to bind the antigen to the antibody, and then eluting the target antigen from the column.
Here, agarose gel, sepharose and the like can be mentioned as suitable carriers, and acidic glycine- can be used as an eluent.
A hydrochloric acid buffer solution, a PBS containing a surfactant and the like can be used. For the preparation of the antibody column, it is convenient to use a conventional CNBr-activated agarose gel.

By the above method, the non-A non-B hepatitis virus non-structural region NS3 polypeptide antigen can be easily and specifically purified.

[0030]

The present invention will be more specifically described with reference to the following non-limiting examples.

Example 1) Non-A non-B hepatitis virus non-structural region (NS3) poly
In the preparation of the recombinant peptide for producing the peptide, the method described in Japanese Patent Application No. 3-189268 of the present applicant was used. That is, the vector pBR322 was treated with the restriction enzyme HaeII to pAI153, and the tryptophan operon promoter, E. Non-A non-B type by synthesizing cDNA by a random primer method from a gene encoding a protein (anthranilic acid synthase) fragment derived from E. coli and viral mRNA extracted from serum of Japanese chronic hepatitis patients and cloning with λgt II phage. Sequentially incorporating the target cDNA encoding the non-structural protein of hepatitis virus,
An expression vector was constructed, and the expression vector was used to transform the E. coli host sensitized with calcium chloride. coli K
It was introduced into C600 strain derived from 12 strains to obtain a recombinant. Cultivate the recombinant, crush the cells with an ultrasonic cell crusher,
The extracted protein was purified by the column chromatography method.

2) Production of hybridoma Each non-A non-B hepatitis virus non-structural region NS3 peptide obtained by the above method was dissolved in physiological saline to obtain 1.0 mg /
ml, mix with an equal volume of Freund's complete adjuvant,
Suspended. The obtained suspension containing 0.01 to 0.05 mg of the non-A non-B hepatitis virus nonstructural region NS3 peptide was intraperitoneally administered to 4 to 6 week-old BALB / c mice. After about 8 weeks, immunized animals were given the same concentration of non-A as above.
A portion of the non-hepatitis B virus nonstructural region NS3 peptide containing 0.005-0.03 mg of physiological saline was administered into the tail vein, and three days after the administration, the spleen was aseptically removed from the immunized animal.

Next, the spleen was disentangled into individual cells using a mesh and washed 3 times with RPMI-1640 medium. 8
-A mouse myeloma cell line P3X in the logarithmic growth phase, which was cultured in the presence of azaguanine for several days and completely eliminated the revertants.
63Ag8 [Nature 256, 495-497]
(1975)] was washed in the same manner as above, and then 1.8 × 1
0 ⁷ cells and 1.0 × 10 ^{8 of the} spleen cells were placed in a 50 ml centrifuge tube and mixed. After centrifugation at 200 xg for 5 minutes, the supernatant was removed and PEG4000 (Merck) kept at 37 ° C was used.
The cells were fused by adding 1 ml of RPMI-1640 medium containing 50%.

The fused cells, after removing PEG by centrifugation, were subjected to hypoxanthine, aminopterin and thymidine (hereinafter abbreviated as HAT) using a 96-well plate.
The cells were cultured in RPMI-1640 medium containing 1 to 2 weeks to grow only hybridomas. Thereafter, the cells were grown in a medium containing no HAT, and after about 2 weeks, a clone producing the desired antibody was searched by the ELISA method shown in Test Example 1, and a hybridoma producing the monoclonal antibody of the present invention having a desired reaction specificity. Got

The obtained hybridoma was designated as HC7-1H1.
0, HC7-2F4, HC7-3F9, HC7-1G6
And HC7-3D3, of which HC7-1G6
4 types of hybridomas except the above were deposited at the Institute of Microbial Science and Technology on December 1, 1992. The deposit numbers are, in order, the Micromachine Research Institute, No. 13316, No. 13313, and No. 1
3315 and 13314.

Rabbit anti-mouse Ig each isotype antibody (Z
by the double immunodiffusion method using Ymed), the monoclonal antibody (1H1) produced by these hybridomas.
The isotype of 0, 1G6, 2F4, 3F9 and 3D3) was found to be IgG1.

Test Example 1. Antibody reaction by ELISA
The non-A non-B hepatitis virus non-structural region NS3 polypeptide used as an immunity antigen was treated with phosphate buffer pH 7.4 (P
It was dissolved in BS) and the concentration was adjusted to 1 μg / ml.
Dispense 50 μl into each well of a well microplate,
Adsorb overnight at 4 ° C. After adsorption, 0.05% Twee
Wash with n-20-containing PBS (-) (T-PBS) three times, and add 3% gelatin-containing PBS (-) to 1 well of 200 μm.
It was dispensed by 1 and treated at room temperature for 1 hour.

[0038] PBS (-) containing 3% gelatin was removed, and 50 µl each of hybridoma culture supernatant, crude or purified monoclonal antibody, etc. were added to the wells and reacted at room temperature for 1 hour. After the reaction, wash 3 times with T-PBS,
2% polyvinylpyrrolidone, 0.05% Tween-2
50 μl of enzyme-labeled anti-mouse IgG + M antibody (Jackson) diluted 5,000-fold with 0-containing PBS (−) was added to each well, and the mixture was reacted at room temperature for 1 hour. Unreacted antibody was removed by washing with T-PBS three times, and 50 μl of ortho-phenylenediamine solution (Wako Pure Chemical Industries, Ltd.) was added to each well to react, and after 10 to 30 minutes at room temperature, 20
The reaction was stopped with a% sulfuric acid solution, and the absorbance (A492) at a wavelength of 492 nm was measured to assay the antibody reaction.

The results are shown in FIG. Monoclonal antibody 1
H10, 1G6, 2F4 and 3F9 have antibody concentrations of about 0.
Absorbance of 2.5 to 2.6 (max) at 2 μg / ml (4
92 nm) and monoclonal antibody 3D3 gave an absorbance of about 2.0 (max) at the same concentration.

Test Example 2. Western blotting method
Demonstration of reaction specificity of antibody by non-A non-B hepatitis B virus non-structural region NS3 polypeptide crude product was subjected to electrophoresis using sodium dodecyl sulfate (SDS-PAGE method), and then the gel was subjected to polyvinylidene difluoride. It was brought into close contact with a (PVDF) film, and the gel side was used as a cathode and the PVDF film side was used as an anode for transfer, and the protein electrophoresed on the gel was transferred to the PVDF film.

The transferred PVDF membrane was immersed in 4% Block Ace (Snow Brand Milk Products Co., Ltd.), 2% BSA-containing 0.1M phosphate buffer (pH 7.4) at 4 ° C. overnight for blocking, and 0.05 After washing with Tris-HCl buffer (pH 7.0) containing% Tween-20, the monoclonal antibody prepared from the hybridoma obtained as the above 2) as the primary antibody was reacted at room temperature for 1 hour.

After the reaction, the membrane was washed thoroughly with T-TBS and washed with 2
% Polyvinylpyrrolidone, 0.05% Tween-20
An enzyme-labeled anti-mouse IgG + M antibody (Jackson) diluted 5000 times with PBS (-) was used as a secondary antibody,
The reaction was carried out at room temperature for 1 hour. Then, PVDF membrane is TT
It was washed with BS and developed with a 0.1% 4-chloro-1-naphthol solution and a 0.2% aqueous hydrogen peroxide solution.

A non-A non-B hepatitis patient serum was used as a positive control, and an anti-human IgG antibody was used in place of the anti-mouse IgG antibody as a negative control.

The results are shown in FIG. Monoclonal antibody 1
It was found that all of G6, 1H10, 2F4, 3D3 and 3F9 formed an antigen-antibody complex only with a band near the size of 30 kDa, similarly to the positive control. The negative control did not develop any color.

Test Example 3. Purification proof human non-A, non-B hepatitis virus nonstructural region NS3 peptide inhibition by antibody sensitized plates monoclonal antibody 0.1μ
g / ml, 1 μg / ml, 5 μg / ml, 10 μg / m
100 μl of each of 1 concentration was added to each well, reacted for 1 hour at room temperature, washed 3 times with T-PBS, and measured according to the Imcheck HCVAb kit (International Reagents Co., Ltd.) measuring method. At this time, the monoclonal antibodies used were 2F4, 3D3, and 2F4 / 3D3 mixture. In addition, as antiserum, non-A non-B hepatitis patient serum (N
o. 27, 28, 29, 30 and 31) were used.

The results are shown in FIG. Regardless of which antibody was used, the antigen-antibody complex level (A492 /
It is clear that the present monoclonal antibody binds to the NS3 polypeptide antigen, that is, inhibits the binding of the antiserum, because of the decrease of 600). It can be seen that the inhibitory effect is even more pronounced when using a mixture of antibodies.

Test Example 4. Non-A non-B hepatitis by antibody
Quantification of Rus nonstructural region NS3 polypeptide 1 μg of purified monoclonal antibody on microplate
50 μl / ml concentration was added to each well, reacted at room temperature for 1 hour or at 4 ° C. overnight, washed 3 times with T-PBS, and 1% BSA-containing PBS (−) was added to 200 μl per well.
Dispense each by 1 and treat at room temperature for 1 hour.

Non-A except PBS (-) containing 1% BSA
50 μl of a solution containing the non-hepatitis B virus non-structural region NS3 polypeptide was added to each well and reacted at room temperature for 1 hour. After the reaction, the plate was washed 3 times with T-PBS, and 50 μl of 1 well of a plate in which biotin (PIERE) was bound to a monoclonal antibody having a different recognition site from the monoclonal antibody sensitized to the plate was added and reacted at room temperature for 1 hour. rear,
Wash 3 times with T-PBS, 2% polyvinylpyrrolidone,
200 with PBS (-) containing 0.05% Tween-20
Peroxidase-labeled avidin (Vec) diluted 0-fold
50 μl each was added to each well and reacted at room temperature for 1 hour. After the reaction, the plate was washed 3 times with T-PBS, and orthophenylenediamine solution (Wako Pure Chemical Industries) was added to 50 wells per well.
The non-A non-B hepatitis B virus non-structural region NS3 polypeptide was measured by adding μl and reacting at room temperature for 10 to 30 minutes, stopping the reaction with a 20% sulfuric acid solution, and measuring the absorbance (A492) at a wavelength of 492 nm. .

The results are shown in FIG. From FIG. 4, the monoclonal antibody is non-A non-B hepatitis virus non-structural region NS3.
It can be seen that the antigen-antibody reaction with the polypeptide depends on the concentration. This indicates that the antibody can be used to quantify the NS3 polypeptide.

3) Large-scale preparation of monoclonal antibody The large-scale preparation of the monoclonal antibody from the hybridoma obtained in the above 2) was carried out by the following method.

(I) Method by cell culture The clone obtained in 2) above was cultured in RPMI-1640 medium in a 5% carbon dioxide gas incubator at 37 ° C. for 48 hours.
After culturing for a period of time and centrifuging the culture broth at 1500 × g for 10 minutes, a culture supernatant containing the desired monoclonal antibody is obtained.

(Ii) Method using mouse ascites From the clone obtained in 2) above, cells 3 × 10 ⁵ to 1 were obtained.
* 10 ⁶ pieces are preliminarily used for pristane (Aldrich
Balb that had been inoculated with Chemicals)
/ C strain mouse was intraperitoneally administered. After 10 to 14 days, the accumulated ascites was collected to obtain ascites containing the antibody of the present invention.

4) Non-A non-B type by monoclonal antibody
Purification of hepatitis virus-related antigen The present antibody was added to a carbonate buffer (pH 8.5) at 5 to 10 μg /
Add the dissolved ml to CNBr-activated agarose gel (swell volume 1 ml) and react at 4 ° C. with stirring overnight. The antibody that did not bind to the gel was removed by centrifugation and 0.2
An equal amount of M glycine buffer (pH 8.5) is mixed with the gel solution, and the mixture is reacted at room temperature for 2 hours with stirring. After the reaction, the plate is washed with a carbonate buffer (pH 8.5) and then with a 0.2 M glycine buffer (pH 8.5). The same operation is repeated 2-3 times.

A solution of the protein extracted from the transformant culture prepared according to the method of 1) above was added to the antibody-binding column thus prepared, and 0.5% Tween-8 was added.
The peak eluted with 0-containing PBS (-) is immediately neutralized with 1M Tris-HCl buffer (pH 8.0).

As a result, the non-A non-B hepatitis virus nonstructural region NS3 polypeptide antigen having a purity of 90% or more was purified.

[0056]

INDUSTRIAL APPLICABILITY The monoclonal antibody of the present invention can detect and quantify a polypeptide antigen derived from the non-A non-hepatitis B virus non-structural region NS3 in a sample with high sensitivity, high accuracy and high specificity. However, it has the advantage that it can be used for effective purification of the polypeptide antigens of various origins.

[0057]

[Sequence list]

SEQ ID NO: 1 Sequence length: 253 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys ^{1 5 10 15} Ser Thr Lys Val Pro Ala Ala Tyr Ala Ser Gln Gly Tyr Lys Val Leu ^{20 25 30} Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met ^{35 40 45} Ser Lys Ala His Gly Thr Asp Pro Asn Ile Arg Thr Gly Val Arg Thr ^{50 55 60} Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu ^{65 70 75 80} Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Met Cys Asp ^{85 90 95} Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val ^{100 105 110} Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr ^{115 120 125} Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu ^{130 135 140} Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys Gly Ile ^{145 150 155 160} Pro Ile Glu Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser ^{165 170 175} Lys Lys Lys Cys Asp Glu Leu Al a Ala Lys Leu Ser Gly Leu Gly Ile ^{180 185 190} Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr ^{195 200 205} Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr ^{210 215 220} Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln ^{225 230 235 240} Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu ^{245 250}

[Brief description of drawings]

FIG. 1 shows the monoclonal antibody 1H10, 1G.
The antibody titer of 6, 2F4, 3F9, and 3D3 is shown by the result of measuring by ELISA method using non-A non-B hepatitis virus non-structural region NS3 polypeptide as an immunizing antigen.

FIG. 2 shows the monoclonal antibodies 1H10, 1G.
FIG. 3 is a diagram demonstrating the reaction specificity of 6, 2F4, 3F9 and 3D3 with the non-A non-B hepatitis virus non-structural region NS3 peptide by Western blotting.

FIG. 3 shows that monoclonal antibodies 2F4, 3D3
And 2F4 / 3D3 mixture demonstrate inhibition of binding of non-A non-B hepatitis virus nonstructural region NS3 antigen polypeptide to non-A non-B hepatitis patient serum.

FIG. 4 This figure shows monoclonal antibodies 2F4, 3D3
FIG. 3 demonstrates the quantification of non-A non-B hepatitis virus nonstructural region NS3 polypeptide by.

Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location G01N 33/577 B 9015-2J // C12N 15/06 (C12P 21/08 C12R 1:91) (72) Invention Takayuki Yamamoto 1-2-1 Muroya, Nishi-ku, Kobe-shi, Hyogo International R & D Center, Inc. (72) Inventor Hiroyuki Mori 1-2-1 Muroya, Nishi-ku, Kobe-shi, Hyogo International R & D Center (72) ) Inventor Yosuke Ota 1-2-2 Muroya, Nishi-ku, Kobe-shi, Hyogo Prefecture Kokusai Reagents Co., Ltd. R & D Center

Claims (6)

[Claims] 1. A monoclonal antibody that specifically binds to an epitope on a non-A non-B hepatitis virus nonstructural region NS3 polypeptide antigen having the amino acid sequence set forth in SEQ ID NO: 1. 2. A monoclonal antibody HC7-1H10,
The monoclonal antibody according to claim 1, which is secreted by a hybridoma selected from the group consisting of HC7-2F4, HC7-3F9, HC7-1G6 and HC7-3D3. 3. A hybridoma producing the monoclonal antibody according to claim 1 or 2. 4. The hybridoma is HC7-1H10, H
C7-2F4, HC7-3F9, HC7-1G6 or H
The hybridoma according to claim 3, which is C7-3D3. 5. A method for immunologically measuring a non-A non-B hepatitis virus-related antigen, comprising a specimen expected to contain the antigen and one or more monoclonal antibodies according to claim 1 or 2. Are contacted with each other to form an antigen-antibody complex, and then the complex is detected or quantified. 6. A monoclonal antibody according to claim 1 or 2 is bound to a natural or synthetic polymer carrier to form an affinity column, and a liquid containing a non-A non-B hepatitis virus-related antigen is contained in the column. A method of purifying a non-A non-B hepatitis virus-related antigen, which comprises passing through to bind the antigen to the antibody and then eluting the antigen from the column.