JPS61124377A - Novel hybridoma, preparation and use thereof - Google Patents

Abstract

PURPOSE:To obtain a hybridoma capable of producing high-purity antibody in high efficiency, by fusing a lymphocyte hybridoma of human and heteroanimal with a human lymphocyte capable of producing a specific antibody, selecting the desired hybridoma from the fused cell, and cloning the hybridoma. CONSTITUTION:Activated human lymphocyte (e.g. peripheral blood lymphocyte) and a lymphoidocyte strain of a heteroanimal (e.g. mouse) are fused together by treating in a medium containing 10-80wt% polyethylene glycol having a polymerization degree of 1,000-6,000 for 0.5-30min. The fused cell is cultured in a selective medium added with 8-azaguanine, etc., to obtain a hybridoma cell (heterohybridoma) introduced with a marker. Separately, a human lymphocyte infected with hepatitis B virus and positive to the anti-hepatitis B virus surface antigen antibody is separated in vitro, stimulated with an antigen, and is fused with the above cell. The obtained fused cell is proliferated in a selective medium. The antibody value of the supernatant liquid is determined, and the cell positive to the test is selected.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel hybridoma, its production method and uses.

Conventional technology The method for producing monoclonal antibodies using hybridomas, which was developed by Koehler and Milstein and has become popular in recent years, is capable of producing antibodies that exhibit monospecificity for each antigenic determinant, and that has become popular in recent years. It has many advantages, such as being able to freely obtain large quantities of homogeneous specimens with good reproducibility. In this sense, the method of obtaining monoclonal antibodies (hereinafter sometimes abbreviated as MoAb) using hybridomas has been highly evaluated for its effectiveness in many fields. In addition, its use has been developed not only for the detection of antigens, but also for the purification of trace components and its application to diagnostic agents.Furthermore, its application to preventive and therapeutic drugs is being considered.
Ru.

Among these uses, mouse MoAb can be used in cases other than direct administration to humans such as prophylactic and therapeutic drugs, but in the case of prophylactic and therapeutic drugs, mouse MoAb is a foreign protein to humans. It is not appropriate to use mouse MoAbs. This is because when mouse proteins are administered, antibodies are induced due to the mouse proteins entering the human body, which not only reduces the effect but also poses the risk of allergic reactions caused by foreign proteins. be.

In general, research on MoAbs lags far behind that on mice. The reason is h) MoAb producing strain t[i
! This is because the method to create them is more difficult than that of mice, and there are few success stories.

) Representative methods for producing MoAb-producing strains include methods using human-human hybridomas, methods using human-mouse heterohybridomas, and methods using Epstein-Bar vir (UA; hereinafter E).
There is a method using established cells transformed with BV (abbreviated as BV). Although many research results using these methods have been reported recently, it has been pointed out that each method has drawbacks in practical terms. Human chromosomes tend to fall off, and the ability to produce human antibodies is unstable. Furthermore, it is difficult to clone the antibody-producing cells obtained from EBv transformants. For this reason, it is said that when the rate of increase of cells that do not produce the target antibody is rapid, the stability of antibody production decreases.

Although there are still few reports regarding these experimental systems, the recently reported method of fusing the Maunu human heterohybridoma as a parent strain with antibody-producing cells to obtain a hybridoma that produces the desired human antibody overcomes these drawbacks. This is a useful method for obtaining a human MoAb-producing strain that has improved [Proceedings of the National Academy of Sciences, 80°7308-7312 (1983)].

Hepatitis B virus (HBv) is said to be the last remaining serious infectious disease, and it has a potential of 3.1 million people worldwide, mainly in Asia, and 2.7% of the total population in Japan. It is said that there is a patient. Many of these potential patients are so-called asymptomatic hepatitis B dog surface antigen (HBaAg) carriers, but there is a close relationship between persistent HBV infection and chronic hepatitis, liver cirrhosis, and even liver cancer. It is said that there is
The problem of how to prevent and treat HBV infection is an extremely important problem in preventive and therapeutic medicine.

Infection with HBV is said to occur through blood, and there are horizontal transmissions such as blood transfusions and artificial dialysis in patients, contamination through needles and scalpels in medical workers, and infection of infants born to HBsAg carriers. Vertical transmission is possible. Anti-HBaA is a preventive method for these infections.
There are two types of immunotherapy: passive immunotherapy, which involves administering g-antibodies, and active immunotherapy, which involves administering vaccines. With recent rapid advances in genetic manipulation methods and peptide synthesis methods, expectations for active immunotherapy are greatly increasing. However, active immunotherapy alone is difficult to apply to immunocompromised individuals with decreased antibody production ability.
In addition, passive immunization is necessary in cases where early effects are expected, such as in cases of mother-to-child infection or medical contamination, and complete prevention requires countermeasures from both active and passive immunization. is necessary.

Most of the γ-globulin preparations currently on the market as materials for passive immunization contain anti-F! Although it contains BsAg antibodies, its titer is low (we cannot expect much effect on preventing HBV infection.On the other hand, human immunoglobulin preparations with high titre anti-HBaAg antibodies (HBBO3 has not been tested in many clinical trials). Although its efficacy and safety are widely recognized through @, BIG acquisition has significant limitations in terms of materials as it uses human blood with high antibody titer.

From this point of view, anti-HBsAg human MoAb-producing strains can be used to freely produce large amounts of homogeneous anti-HBsAg human MoAb.
The method for producing anti-HBa is highly useful, but so far no anti-HBa
Regarding cell lines that produce Ag human MoAb, there have been no reports of cell lines producing this antibody using hybridoma techniques. The reason for this is thought to be the drawbacks pointed out in the human hybridoma or mouse-human hybridoma experimental systems as mentioned above.

The only report on anti-HBaAg human MoAb is the production method using IBv transformants (Japanese Patent Application Laid-Open No. 55-31
However, as mentioned above, cloning of cells is difficult, so a time-consuming method of seeding a relatively small number of cells and repeating passages is used. Strictly speaking, it lacks credibility as a single antibody.

Problems to be Solved by the Invention The present inventors have intensively compared and studied highly practical methods for producing human anti-HBaAg monoclonal μ antibodies, which have important implications for the prevention and treatment of HBV infection. We succeeded in producing for the first time an anti-HBaAg human MoAb-producing hybridoma, which had not been obtained so far.

The present inventors produced a hybridoma producing this antibody, and further used this hybridoma to
b was produced, its properties were clarified, and the present invention was completed.

Means for Solving the Problems The present invention provides: (1) an anti-HBaAgMoA comprising a hybridoma of a human lymphoid cell and a lymphoid cell line of a xenogeneic animal, and a human lymphoid cell that produces an anti-llBsAg antibody;
A cloned Hyzuridoma producing bi.

(2) and) Cell fusion of a hybridoma of a lymphoid cell and a lymphoid cell line of a xenogeneic animal with a human lymphoid cell that produces an anti-HBaAg antibody, and producing an anti-HBsAg MoAb from the resulting fused cell. A method for producing a cloned hybridoma, which comprises selecting a hybridoma and cloning the hybridoma, and (3) anti-HBsAg M using the above hybridoma.

The present invention provides a method for producing MoAb, which is characterized by producing and accumulating Ab, and collecting the residue.

Regarding the above hybridoma of a human lymphoid cell and a lymphoid cell line of a xenogeneic animal, examples of the human lymphoid cell include normal lymphocytes obtained from healthy individuals and human lymphoid cell lines. Normal lymphocytes may be from culm, lymph nodes, peripheral blood, etc., but peripheral blood lymphocytes (PBL) are advantageously used from the viewpoint of easy availability. Although these lymphoid cells may be used as they are, it is preferable to activate them in advance with a bozoclonal mitogen (such as Hawkelyid mitogen).

Cell lines derived from lymphocytes of mammals other than humans (e.g., experimental animals such as mice and rats) are examples of lymphocyte-like cell lines from xenogeneic animals. , cell fusion efficiency.

Mouse myeloma cell line with excellent proliferative properties, and Dwaf
Cells with markers such as the f-hyboxanthin-guanine-phospholiposyltranuphue-deficient strain (HGPRT'' strain) are advantageously used.

In order to produce a hybridoma (heterohybridoma) of the above-mentioned human lymphoid cells and a lymphoid cell line of a heterologous animal, these two cells are treated with a fusion agent such as Sendai virus or polyethylene glyco-A/(PEG). Using),
Fusion can be achieved by methods such as electrical stimulation. An example in which PEG is used will be given, but of course the method is not limited to this method. The degree of polymerization of PEG is usually 1ooo-6
000, processing time 0.5-30 minutes, concentration 10-80
% etc., but as an example of preferable conditions, PgG
Efficient fusion can be achieved by treating at 6000t-35-55% for 4 to 10 minutes. Although it is possible to combine each ear as a medium for selective growth of the fused cells in the first stage, one example that is advantageously used is H.
When fusing a GPRT mouse myeloma cell line with human PBL, the fused cells can be selectively enriched using, for example, hypoxanthine, amitubzoline, thymidine medium (HAT medium), azaserine, hypoxanthine medium (AH medium), etc. can.

The second stage of cell fusion is a process in which the above-mentioned heterohybridoma and human lymphoid cells producing anti-HBsAg antibodies are fused. As an advantageous example of a parent heterohybridoma, a case where a heterohybridoma consisting of a fusion of an HGPRT-mouse myeloma cell line and human PBL is used will be described below. The above-mentioned heterohybridoma has HGPRT, but it is possible to insert a marker such as - with HGPR again. For this purpose, the purpose can be achieved by, for example, adding 8-azaguanine (8AZG) or the like to the medium and increasing its concentration sequentially. The heterohybridoma into which a marker has been introduced in this way can be used as a parent strain for the second stage of cell fusion. Before actually fusing with the target anti-RBaAg antibody-producing cells, multiple car-introduced heterohybridomas are combined with PBL to improve fusion efficiency, hybridoma proliferation, ease of cloning, and human MoAb production ability. It is very effective to check the stability and select the optimal parent heterohybridoma strain. In this selection, it is preferable to select a heterohybridoma that does not secrete antibodies.

On the other hand, anti-HBaAg to be fused with heterohybridoma
The human lymphoid cells that produce antibodies may be normal human lymphocytes, or established cell lines such as EBV) transformants may also be used. Anti-f, which is thought to have been infected with IIIBY as an antigen in the past, is separated from human lymphocytes.
Human lymphocytes positive for iBaAg antibodies were taken out in vitro and stimulated with an antigen (1!BeAg), voriklona μ, lymphocyte mitogen (Hawklyde mitogen, Staphylococcus aculeus quowane, etc.), and then transformed into heterohybridoma. By fusing,
The desired hybridoma can be obtained more efficiently. Furthermore, a method of stimulating in vitro-extracted lymphocytes as described above and further infecting them with IBM can also be applied as a method for temporarily immortalizing cells. Transformation by EBv becomes more efficient when EBY is added externally, but it is also known that transformation may occur spontaneously by simply culturing in vitro even without the addition of EBY. However, cloning of the EBV transformant itself is difficult, and therefore antibody production is often unstable, so by fusing this DBv transformant with a heterohybridoma,
The obtained hybridoma can be cloned,
It is also advantageous because antibody production is stabilized. This cell fusion can be performed in exactly the same manner as the first stage cell fusion. In this case, selective growth of rehybridoma can be obtained by using HAT medium or AH medium as a selective medium;
When using cell lines such as EBV) transformants as human cells, the purpose can be achieved by further adding ouabain to the above selective medium.

The anti-■BsAg antibody titer is measured for the supernatant of each hybridoma grown in the selective medium in the second stage of cell fusion, and positive cells are selected. Although various modified methods are possible for measuring anti-HBa g antibodies, it can be easily measured, for example, by enzyme immunoassay (EIA method) using a commercially available Ausub IrA kit or the like.

Anti-HBsA that showed growth on selective medium and was determined by EIA method
Sea urchin cells that have been found to produce g antibodies can be cyclocloned by limiting dilution method or the like. The cloned cells are screened in the same manner and the number of sea urchin μ cells with a high antibody titer is increased to obtain the desired hybridoma clone.

HBsAgK has four subtypes (Adr, adw
.

It is known that MoAb of any subtype can be produced according to the present invention, but which type of MoAb obtained depends on the antigen of each subtype. This can be easily determined by a blocking test using HBaAg.

In addition, the fact that the obtained cells are truly hybridomas means that
It is clear from the fact that it grows in HAT medium, but it can be determined more clearly by further chromosomal analysis.

Using the hybridoma of the present invention described above, anti-1iBaA
Anti-HBaAgMoAb can be produced by producing and accumulating gMoAb and collecting it.

The production and accumulation of the MoAb is performed by incubating the hybridoma of the present invention.

Incubation is carried out in a liquid medium or intraperitoneally in the animal (usually in the peritoneal cavity of a mate mouse Tomoura mammal), and the hybridoma of the present invention is preferably cultured in a liquid medium.

As a medium, for example, a composition for animal cell culture [mixed medium such as Iskot medium and Ha412 medium (K/H medium)
, RI'M11640 medium, etc.) by subjecting fetal bovine serum, fetal bovine serum that does not contain gamma p-purine, or mammalian serum to a purification process that includes an inactivation process of mixed microorganisms, salting out, and desalting process. Examples include compositions for culturing animal cells derived from mammalian serum [Japanese Patent Application No. 59-521 (filed on January 7, 1980)].

Cultivation is usually carried out for 5 to 10 days, preferably at 30'-38°C.
Perform at 7°C.

Furthermore, anti-HBsAg M was produced and accumulated.

Abs can be purified by conventional protein purification means, if desired.
i can be purified.

For example, the culture supernatant is removed by centrifugation, salted out (usually using 30-60% ammonium sulfate), the precipitate is separated by centrifugation, and after dialysis, it is precipitated by centrifugation, etc. Remove the liquid and use the supernatant to collect the supernatant.
Purified MoAb can be produced by subjecting it to a cellulose cuff (e.g., cellulose cuff, etc.) and eluting it.

The monoclonal μ antibody produced according to the present invention has the following properties.

(1) Specifically binds to HBsAg (2) Four subtypes of HBsAg (adw, ad
r.

&7W, ayr) 'Ia' binds to # subtype (3) SDS-polyacrylamide/l/2 that completely matches the molecular weight of standard immunoglobulin chain and light chain in electrophoresis Only the book band is shown.

Anti-F! produced according to the present invention! BsAg monoclonal
The antibody is homogeneous and has a high titer, and is a human immunoglypurin preparation (HB Engineering G) with known anti-EIBaAg antibody activity [New England Journal Opmedicun, 291
．． 1378 (1974)), it can be used for the diagnosis, prevention, and treatment of hepatitis B cirrhosis. Regarding the amount (dose) to be used, the antibody titer corresponding to the known EIB Engineering G may be used.

Effects and Examples The present invention will be explained in more detail below using Reference Examples and Examples, but the present invention is not limited thereto.

The (mouse/human)-human hybridoma HBI[-43,1 disclosed in the Examples has been deposited with the Institute for Fermentation Research (IF'O) under deposit number F'O-C;:"QQo7. .

Reference Example 1 Method for Measuring Human Antibodies The method for measuring human antibodies was the enzyme-linked immunosorbent assay (Intech 8A method). That is, goat anti-human rgGt or engineered gMt-15μf/m
0,0 containing 0.01M NaCl so that l
The mixture was suspended in 1M 9-phosphate buffer (polio), and 100 μj was dispensed into each sea urchin (96-well μmicrobe V-), followed by reaction at 4° C. for 24 hours. After the reaction, 100μJ of phosphate buffer containing 2% bovine serum albumin (BSA) was dispensed in order to block the excess binding sites in the wells, and treated at 4°C for 24 hours to prepare plate V-) using ILI3AK. .

Pour 100μ of the culture supernatant onto the plate thus prepared! The mixture was added and reacted at 24°C for 3 hours. After the reaction, wash well with physiological saline, and add horseradishbeμ okindase (HR
100 μl of goat anti-human XgG or engineered gM antibody labeled with P) was added to each sea urchin/L'K and reacted at room temperature for 3 hours. After the reaction, each sea urchin /L/ was thoroughly washed with physiological saline, and 100μm of an enzyme substrate solution prepared by adding 22q of osophenylene diamine and 10μj of H2O2 to 10ml of 0.1M citric acid solution was added to each sea urchin. In addition to y, the enzymatic reaction was carried out at room temperature for 15 minutes and the reaction was stopped with 1N sulfuric acid.

After stopping the reaction, titer Tetsukuma Mutiscan ()-sha)
The amount of colored pigment was measured at wavelength 492JLO+ using
The amount of antibody was quantified by comparison with the following.

゛Reference Example 2 Production of mouse-human heterohybridoma and production of 8-azaguanine-resistant strain (1) Production of mouse-human heterohybridoma Ficoll-Hypaque from human peripheral blood of healthy individuals
Lymphocytes (PBL) were separated by specific gravity centrifugation using PBL, and suspended at 2×10/d in Ko-H medium (Ko-H-10F') containing 10% non-mobilized beef tallow serum. . This lymphocyte suspension was dispensed into a tissue culture flask (Corning Inc. AR-1507 Funuco), and when the concentration reached 32μ9/d, Borkfeet mit-diene (PWM
) and cultured for 3 days at 37°C in a carbon dioxide gas bubbler.

After culture, lymphocytes and HGPRT-deficient mouse myeloma cells P3-X63-Ag8. Ul (P2H4) [Current Topics in Microbiology and Immunology, 81.1-7 (1978)], 2:1
Cell fusion was carried out by mixing the cells at a cell ratio of 100% and treating with 45% PEG6000 (manufactured by Kotzholite Co., Ltd.) for 7 minutes.

After fusion, engineer pancreatic cancer cells into 1'I-10F' and 2X10'
The cells were suspended at a temperature of 1 d/d, and then seeded at 1 d each in a Linpro 24-well mackerel, and cultured at 37° C. in a carbon dioxide atmosphere. After 1124 hours of culture, HAT (hyboxanthin: 1 x 10'M, aminodederine: 4 to 10
M, thymidine 16X10-5M) containing X・Fl-1
Add 1 m of 03' (HAT medium).IIIA
T selection culture was started. Additionally, 3.
5. After 8 odd days of 7 days, discard 1 d of the old solution and add 1 ml of new HAT medium, 9.1! AT selection culture was continued. Hybridoma growth is 10 minutes after cell fusion.
It was observed on the 14th.

(2) Obtaining an 8-azaguanine-resistant strain The mouse-human heterohybridoma obtained in (1) with excellent proliferation ability and no antibody production was obtained by Ii
In order to make the ATg-sensitive strain, 8-azaguanine (8-AZG
) Culture was performed in supplemented medium.

That is, RP containing 8-AZ G') at a concentration of 1 μM
Culture was started at V knee 10r, and the concentration of 8-AZG was 1 μM.
The culture was continued by increasing the concentration by 2 times.

HA strains that became resistant to 100 μM 8-AZG
Tg susceptibility was examined and six susceptible strains (HM1 to HM6) were obtained.

Example 1 (+3 Comparison of fusion between each m mouse and the toheterohybridoma strain Tohuman PBL Cell fusion efficiency of each of the six HAT-sensitive mouse-human heterohybridoma strains obtained in Reference Example 2'zP3U1
In order to compare with that of
Cell fusion was performed with PBL that had been cultured in the presence of PBL for 5 to 7 days. Table 1 shows the results.
When No. 4 was used as the parent strain, although the fusion efficiency was high, no hybridoma capable of stably producing antibodies was obtained. On the other hand, when a mouse-human heterohybridoma was used as the parent strain, the fusion efficiency was higher than that of P2H4, especially in HM3 to HM5, and the antibody production of the obtained hybridoma was also stable.

(Toma) Production of anti-HB8Ag antibody-producing hybridoma Mouse-human heterohybridoma HM-5 obtained in Example 1 (1) and anti-HBsAg antibody-positive human PBL''k
32tt 9jml of lymphocytes obtained by treatment with PWM for 2 days were mixed at a ratio of 1:2, and 45% PK060
00 (manufactured by Kotsuholite) and 37°C.

Post-fusion EI where cell fusion was performed by treatment for 7 minutes
M-5 cells were suspended in NiH-10)' at 2XI05/d, and placed in a Linpro 24-well multi-day tube at 1XI05/d.
The seeds were sown at 37° C. and cultured in a carbon dioxide gas atmosphere. After 24 hours of culture, 1j) HAT selective culture was started by adding 111 liters of HAT medium. Furthermore, after 8 odd days of 3, 5, and 7 days from the day when it was first added, add 1 wl of the old solution.
The medium was discarded, 1 d of fresh HAT medium was added, and selective culture was continued. 10 to 14 days after cell fusion, hybridoma growth was observed in 56 sea urchins out of 80 seeded sea urchins, and 3
After foreshadowing, anti-HBaAg antibodies in the culture supernatant were measured using the enzyme immunoassay (EIA) method using a commercially available "Ausub IIA" kit (Dynabot). That is, polystyrene balls coated with HBsAg4 were used. Add 200μ! of each hybridoma culture supernatant to
After reacting at 0° C. for 2 hours, it was thoroughly washed with physiological saline. After washing, add TIRP-labeled HBsAg and
The reaction was carried out at 0°C for 2 hours. After the incubation, the polystyrene po μ was thoroughly washed with physiological saline, 200 μ of the substrate solution included in the kit was added, and the enzyme was diluted for 15 minutes at room temperature.
The reaction was stopped with 1N sulfuric acid. After stopping, the amount of dye was quantified using multiscan at a wavelength of 492 nm. As a result, anti-HBsA was found in the culture supernatant of 15 sea urchin μ in 56 ways.
The presence of g antibodies was observed.

(II) Cloning of anti-HBaAg antibody-producing hybridoma 2 sea urchins/l/(HBI[[-zs, Hv
Each hybridoma (N-43) was cloned by the limiting dilution method. In other words, the number of hybridomas was 2/d by floating on the H-10F', 96
0.0 mm per sea urchin in a hole microplate (manufactured by Nunc).
1 wl was dispensed. When dispensing, add breast WaIm cells from BALB/C mice as feeder cells to the sea urchin at 5 x 10 cells. In this way, after approximately 2 weeks, when cell proliferation is observed, the supernatant is collected and the presence or absence of antibodies is determined using the "Ausub E Engineering A" kit described in Example 1 (1). I investigated it using the E-E construction law. As a result, antibody activity was observed in 21 out of 37 clones for IIIB-25 and in 19 out of 37 clones for EBBN-43. Among the above 40 clones, Toshihiko (mouse/human)-human hybridoma HBI[-43
, 1 is fast proliferating and anti-HBI! Production of IAg antibodies was also high.

Example 2 Production of monoclonal p-sy antibody (1) Monoclonal antibody productivity of hybridoma (mouse/human)-human hybridoma obtained in Example 1 (1) ×10
The cells were suspended at a concentration of 4 cells/Mt, and 1 gl each was seeded in a Linpro 24-well multi-dish. After seeding, the culture supernatant was collected day after day, and the number of viable cells and antibody amount were determined. The number of living cells was determined by staining with trivan blue, and the antibody was quantified by the method described in Reference Example 1.

As a result, it was found that the proliferation doubling time of the hybridoma was 29 hours, the antibody was of the TgG class, and the production amount was λ8 μg/wl.

(It) HBs mug recognized by monoclonal antibody
MoAb HEM-43゜1-containing supernatant obtained in Subtype Example 2 (1) was used at 2500! unpurified antigen preparations of each subtype of HBsAg (adrlam, adw
10 types, ayr 1 type, ta, yv 5 types; - all HBa
Ag (derived from human blood) and 25 μm of each were mixed and reacted at 37° C. for 60 minutes (pre-incubation). After the reaction, each reaction solution and purified ad
3m HB a Ag k of r, adw and ayw
:f-) 1. Rinse sheep red blood R (SRBC) at room temperature for 6 hours.
It reacted for 0 minutes (passive hemagglutination reaction).

As a result of the above, IH3sAg (32
H
M o A b to BaAg'(:!-) sheep red blood cells
Binding of HBI[-43, ■ was suppressed, and inhibition of hemagglutination was observed. This indicates that the above MoAb recognizes α-buttermint as a common antigen. That is, H
oAbHBI[-43,1 represents the four subtypes of llBaAg (adw, adr.

ayw, ayr).

(1) Production of monoclonal antibodies (mouse/human) - human hybridoma! IBM-4
3,1t, fetal bovine serum 3Mg from which the globulin fraction has been removed
The cells were suspended in a medium containing Z1, insulin 2 μf/ld, transferrin 2 μ9/ml + ethanolamine 2 μy, and selenite Z5×10 −8 M and cultured at 37° C. for 4 hours. Culture supernatant 10j with
Add ammonium sulfate to a concentration of 47%,
Salting out was carried out for 60 minutes with stirring at 4C, followed by centrifugation (10 minutes).
ρOO rotation for 15 minutes) 1- to obtain a precipitate. The precipitate was suspended in a 20mM) Lith buffer solution (pH 7,9) containing 5QmM NaCl, and dialyzed against the same solution 2j. After 2 hours, the dialysate was replaced with 21 new identical dialysates, and dialysis was continued for an additional 15 hours. After dialysis, the mixture was centrifuged seven times at 10,000 rpm for 15 minutes to remove the precipitate, and the supernatant was adjusted to have an A28o value of 20 to 3o. This sample was diluted with a sufficient amount of 50mM-NaCl-containing Tris buffer solution.
Apply to tA/loin kahum (Whatman Dg52),
After thorough washing with a Tris buffer solution containing 50 mM NaCl, fractionation was performed using a concentration gradient salt solution of the same buffer containing 50 mM to 500 mM Hack at a flow rate of L5 gt/min to concentrate the decoys. Monoclonal antibody HB
IIr-43,1 was obtained. To confirm the purity of the antibody, the method of Laemli et al.
5DS-Polyacrylamide spike/according to 1970))
l/electrophoresis was used. That is, ammonium sulfate analysis and DEAE
The amount passed through the cellulose column was reduced with 2-mercaptoethanol-μ and 10% SDS was added.

The electrophoresis was carried out using 180 bottles for 15 hours. As a result, two bands were observed: a ■ chain at a molecular weight of about 52 kiloda tons and an L chain at a molecular weight of about 28 kilodatons.

Qv) Monoclonal antibody subclass HBM-4
3.1-e-Noclonal antibodies were prepared using the antibody purified in Example 2(i) and sheep anti-human egg G1. Engineering g
G2. Engineering gG3. The results were investigated using agar precipitation reaction (Immunological Methods Gemu Difagemin Techniques Puwotsu Well Oxford (1964) with Meiss Publishing Co., Ltd.).The results showed that the reaction between monoclonal antibodies and sheep anti-human) IgG1 One band was observed between the monoclonal antibodies and no bands were observed between the monoclonal antibodies and other anti-human antibodies.Therefore, it was determined that the monoclonal antibodies belonged to IgG1 sapcufus (Table 2). .

Table 2 Antigen Antibody Presence of sedimentation line Monoclonal antibody of the present invention Technique gG1 +Ip
G2-〃
G3-〃G
4-Effects of the Invention The cloned hybridoma producing the anti-hepatitis B virus surface antigen monoclonal antibody of the present invention has been completely cloned, and is used to produce the highly purified monoclonal antibody. It can be manufactured efficiently.

Claims (3)

[Claims] (1) An anti-hepatitis B virus surface antigen monoclonal antibody consisting of a hybridoma of a human lymphoid cell and a lymphoid cell line of a xenogeneic animal, and a human lymphoid cell that produces an anti-hepatitis B virus surface antigen antibody. A cloned hybridoma that produces. (2) Cell fusion of a hybridoma of human lymphoid cells and a lymphoid cell line of a xenogeneic animal with human lymphoid cells that produce anti-hepatitis B virus surface antigen antibodies, and use the resulting fused cells as anti-hepatitis B 1. A method for producing a cloned hybridoma, which comprises selecting a hybridoma that produces a hepatitis virus surface antigen monoclonal antibody and cloning the hybridoma. (3) An anti-hepatitis B virus surface antigen monoclonal antibody consisting of a hybridoma of a human lymphoid cell and a lymphoid cell line of a xenogeneic animal, and a human lymphoid cell that produces an anti-hepatitis B virus surface antigen antibody. producing and accumulating anti-hepatitis B virus surface antigen monoclonal antibodies using the cloned hybridomas produced;
A method for producing the monoclonal antibody, which comprises collecting the monoclonal antibody.