JPH0124799B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to monoclonal antibodies, specifically human adult T-cell leukemia-associated antigen (ATLA, Adult T-
The present invention relates to a monoclonal antibody having a molecular weight of 28,000 and having reactivity with ATLA. Two typical adult T-cell leukemia (ATL)-related cell lines are known today.
One of them was reported in 1980 by Miyoshi et al.
The MT-1 strain was derived from peripheral blood leukemia cells of ATL patients [Gann 71, 155-156 (1980)], and the other one was the same strain that was also reported in 1981 by Miyoshi et al.
The MT-2 strain [Gann 72, 978-981 (1981)] was established by mixed culture of peripheral blood leukemia cells of ATL patients and normal cord blood leukocytes. Above MT-
Both strains 1 and MT-2 are reactive with almost all ATL patient serum, and therefore
It was confirmed that the patient had ATLA [Proc.
Natl.Acad.Sci., USA, Vol.78, No.10, 6476−
6480 (1981) and Nature, 294, 770−771
(1981)]. Cells that have ATLA (ATLA-positive cells)
also has type C virus particles and that the virus is a retrovirus [Proc. Natl.
Acad.Sci., USA, Vol.79, 2031−2035 (1982)]
were demonstrated, and the virus was named ATL virus (ATLV). In 1982, Yamamoto and Hinuma labeled these cells in advance with ³⁵ S-methionine, subjected the cell extract to specific immunoprecipitation with ATL patient serum, and then precipitated the precipitate with sodium dodecyl sulfate (SDS). -Polyacrylamide gel electrophoresis (SDS
As a result of fractionation based on molecular weight (-PAGE) and search using autoradiography, the existence of several specific proteins was proved, and the above-mentioned
It was demonstrated that ATLA is an ATLV-related antigen [Symposium supported by a cancer research grant from the Ministry of Health and Welfare; Adult T-cell leukemia and lymphoma (ATL) and its pathogenesis;
Life Science Center Publishing, pp137-138,
Published January 5, 1982]. It is already known to date that various cancers are caused by viruses in many animal species. These include bovine lymphoma, feline leukemia, and avian sarcoma. Even in humans, a small number of cancers, such as Burkitt's lymphoma, which causes jaw tumors in African children, are thought to be caused by viruses. Elucidation of the mechanism of carcinogenesis by oncogenic viruses is rapidly being elucidated at the animal level based on genetic engineering methods, but the important point in this case is the elucidation of the effects of proteins and glycoproteins as gene products. It is necessary to cross over. The most powerful method is to identify and quantify the substances using specific antibodies against the respective gene products. On the other hand, this is also related to the fact that ATL carries a retrovirus as its causative factor.As with the case of hepatitis B virus, ATLV is expected to be infected during blood circulation from a virus carrier, and a follow-up investigation is already possible. Post-transfer transmission from virus carriers to non-infected individuals has been reported in many cases. Screening virus carriers from blood circulation solutions may be the most effective preventive method to prevent the spread of this disease, but this requires specific, sensitive, and rapid measurement of ATLV as an antigen. Therefore, specific antibodies are required for antigen quantification, and also for the preparation of antigens used for antibody measurement. The purpose of the present invention is to provide a monoclonal antibody (ATLA antibody) specific for the above ATLA (i.e., ATLV-related antigen), and that the antibody is effective against ATLV-immunized immune cells and myeloma cells (myeloma cells). It was discovered and completed by the discovery that it can be produced by a hybridomer established by a cell fusion method with a cell. Specifically, the present invention provides monoclonal antibody GIN-14 and monoclonal antibody GIN-14, which are produced by a fusion cell of a mammalian immune cell immunized with ATLV and a mammalian myeloma cell, and are characterized by having specific reactivity with ATLA. Regarding 2. As detailed below, the monoclonal antibody of the present invention can be obtained by culturing selected hybridomas created by cell fusion of specific immune cells and myeloma cells, and by immunofluorescence. Monoclonal antibody GIN-14 has ATLA specificity in that it specifically binds to ATLA-positive cells identified in patient serum and stains only their cytoplasm.
ATLA (p28) in the fraction with a molecular weight of 28,000 was precipitated, and monoclonal antibody GIN-2 was precipitated in the fraction with a molecular weight of 28,000 (p20) and 19,000 (p19).
It was confirmed that ATLA was precipitated. Therefore, the antibody of the present invention can be used as a probe for ATLV- or ATLA-bearing human lymphoid cells in vivo or in vitro, and can also be used for analysis of ATLV-specific antigens, as well as for infection, diagnosis, treatment, prevention, etc. of ATL or ATLV. It's useful. The method for producing the monoclonal antibody (ATLA antibody) of the present invention will be described in detail below. The ATLA antibody of the present invention is obtained by fusing the immune cells of a mammal immunized with ATLV with myeloma cells to create a hybridoma, from which a monoclonal antibody having the above characteristics is isolated. In the above method of the present invention, one of the parent cells used for cell fusion is an immune cell that serves as an immunogen.
It is prepared by immunizing a mammal with ATLV using a conventional method. Here, ATLV as an immunizing antigen is not particularly limited, and for example, in addition to the above-mentioned MT-1 and MT-2 cells,
YAM, TER, ASO, OKI, KEN, SAT,
Cells isolated by a conventional method from a culture solution of known ATLA-positive cells such as OKU [Science, 217, 737-739 (1982)] cells can be used. the above
ATLV is separated by a conventional centrifugation method or the like, and may be further purified by a density gradient ultracentrifugation method or the like. The mammal to be immunized with ATLV is not particularly limited, but is preferably selected in consideration of compatibility with the myeloma cells used for cell fusion, and mice, rats, etc. are generally used. The immunization method is also carried out by a general method, for example, ATLV is diluted to an appropriate concentration with a normal buffer, etc., made into a suspension with Freund's auxiliary solution, etc., and administered to the animal by subcutaneous injection etc. .
A booster immunization was performed 3 to 5 weeks after the initial immunization, and the total dose was 2 to 3 x ¹⁰⁵ cells/ml of cultured ATLA.
ATLV obtained from approximately 600ml of positive cell culture fluid
It is preferable to adjust the amount to approximately 1/mouse.
As the immune cells, it is preferable to use spleen cells extracted about 3 days after the final immunization. In addition, the mammalian myeloma cells (myeloma cells) as the other parent cells to be fused with the immune cells obtained as described above may be selected from various known cell lines, such as p3 (p3/×63-Ag8 ) [Nature, 256, 495
-497 (1975)], p3-U1 [Current Topics in
Microbiology and Immunology, 81; 1-7
(1978)], NS-1 [Eur.J.Immunol., 6; 511-
519 (1976)], MPC-11 [Cell, 8; 405-415
(1976)], SP2/0 [Nature, 276; 269-270
(1978)]. FO [J. Immunol. Meth., 35; 1-21
(1980)], ×63.6.55.3. [J.Immunol., 123; 1548−
1550 (1979)], S194 [J.Exp.Med., 148; 313-323
(1978)] and R210 in rats [Nature,
277; 131-133 (1979)] etc. are used. The fusion reaction between the above immune cells and bone marrow tumor cells is
Basically, known methods such as the method of Oi and Herzenberg [Selected Methods in Cellular Immunology,
p351-371, WHFreeman & Co., USA Publishing (1980)], etc. More specifically, the fusion reaction is carried out in a conventional nutrient medium, for example in the presence of a fusion promoter. As the fusion promoter, commonly used polyethylene glycol (PEG), Sendai virus (HVJ), etc. are used, and if desired, an adjuvant such as dimethyl sulfoxide may be added to increase the fusion efficiency. The ratio of immune cells to myeloma cells to be used is the same as in normal methods; for example, about 1 to 10 times as many immune cells as myeloma cells may be used. As the medium for the above fusion, for example, RPMI-1640, which is used for the proliferation of the above myeloma cell line, may be used.
Medium, MEN medium, and any other medium commonly used for this type of cell culture can be used, and it is usually best to omit serum supplements such as fetal calf serum (FCS). For fusion, predetermined amounts of the above immune cells and myeloma cells are mixed well in the above medium, and a PEG solution preheated to about 37°C, for example, with an average molecular weight of 1000 to 6000, is added.
This is done by adding about 30% to 60% W/V% to a normal medium and mixing. Thereafter, desired fused cells (hybridomas) are formed by repeating the steps of sequentially adding an appropriate medium, centrifuging, and removing the supernatant. The resulting desired hybridoma is isolated by culturing it in a conventional selection medium, such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT medium may be carried out for a sufficient period of time, usually from several days to several weeks, to kill cells other than the target hybridoma (unfused cells, etc.). The hybridoma thus obtained is searched for a strain producing the antibody of interest and single cloned according to the usual limiting dilution method. The above-mentioned search for the desired hybridoma can be carried out, for example, by the method proposed by Hinuma et al.
Indirect immunofluorescence method of ATLA antibody [Proc. Natl. Acad.
Sci.USA., Vol. 78, No. 10, pp. 6476-6480 (1981)] and enzyme-linked immunoassay (EIA), neutralization reaction method,
It is carried out by various methods commonly used for antibody detection, such as precipitation reaction method, complement fixation reaction method, agglutination reaction method, Ouchterlony method, RIA method ["Hybridoma method and monoclonal antibody" R Co., Ltd.]
Published by &D Planning, pp30-53, March 1982
5th of the month]. The thus obtained hybridoma producing the monoclonal antibody of the present invention can be subcultured in a normal medium and can be easily stored for a long period of time in liquid nitrogen. Representative examples of this hybridoma include GIN-2 and
An example is GIN-14. These are held available for distribution by the inventors. The ATLA antibody of the present invention can be produced from the specific hybridoma obtained as described above by culturing the hybridoma according to a conventional method and separating the desired antibody from the culture supernatant, or by adapting the hybridoma to this method. A method is adopted in which the antibody is administered to a sexually active mammal, allowed to grow, and the desired antibody is isolated from the ascites. The former method is good for obtaining high purity products, and the latter method is excellent for mass production. Thus, according to the present invention, a monoclonal antibody having reactivity with ATLA, particularly p28, can be obtained as shown in the test examples below. Next, reference examples, working examples, and test examples will be given and explained. The cultured cell lines used in Reference Examples, Examples, and Test Examples are all known and are as shown below. (1) ATLA-positive cell line MT-1 [Gann71, 155-156 (1980)] and
MT-2 cell line [Gann72, 978-981 (1981)]
was mainly used. In addition, the following several types
ATLA positive cell line [Science, 217, p737-739
(1982)] was used. i.e., healthy adult peripheral blood lymphocytes (PBL) and lethally irradiated MTs.
-2 was established by mixed culture with
YAM, TER and ASO strains, IL in vitro
KEN established by culturing peripheral blood lymphocytes of ATL patients under continuous supply of -2;
OKI and SAT lines, as well as spontaneous cell lines from ATL patients, whose growth is IL-2 independent
Each used OKI stock. (2) ATLA-negative cell line The following cell line was used as a control. T cell line; Molt-4 [Biochem.Biophys.Res.Commun.,
51, 529-535 (1973)] CCRF-CEM [Cancer, 18, 522-529 (1965)] RPMI 8402 [J. Natl. Cancer Inst., 55, 11-
14 (1975)] TALL-1 [Nature (London), 267843-844
(1977)] HPB-ALL [Int.J.Cancer21, 166-170
(1978)] HPB-MLT [same as above] B cell line; TL-1 [Jpn.J.Exp.Med., 50, 423-434
(1980)] BJA-B [Biomedicine, 22, 276-284
(1975)] Daudi [Cancer Res., 23, 1300-1310 (1968)] P3HR-1 [J. Virol., 1, 1045-1051 (1967)] Raji [J. Clin. Pathol., 18, 261- 273 (1965)] LCL-TAN [J.Immunol., 128, 1241-1245
(1982)] LCL-KIT [same as above] non-T, non-B cell line; HPB-NULL [obtained from Professor Shigeru Morikawa, Shimane Medical University] HL-60 [Proc. Natl. Acad. Sci., 75, 2458-
2462 (1978)] K-562 [J.Natl.Cancer.Inst., 50, 535-538
(1973)] In addition, long-term cultured Cytotoxic T cells,
YT-13 and TC-SUG#14 [J.Immunol., 128,
1241-1245 (1982) and 128, 1749-1752
(1982)] and fresh PBL were also used as ATLA-negative cells. (3) Each of the above cells was incubated in RPMI-1640 medium for 10 to 20 days.
% heat-inactivated fetal calf serum (FCS),
The cells were cultured in a mixed medium containing 100 IU/ml penicillin, 100 μg/ml streptomycin, and 4 mM L-glutamine. IL-2-dependent cells are produced using the supernatant of cell culture obtained by stimulating human splenocytes with PHA and allogeneic cells as IL-2 [Microbiology and
Immunology, 25, 1077-1086 (1981)]
(V/V) was added to the above mixed medium and cultured. Each cell was maintained at a concentration of 2-3 x ¹⁰⁵ cells/ml every 3-4 days. Reference Example 1 MT-2 cells were cultured for 5 days at a concentration of 3 x 10 ⁵ cells/ml of the above mixed medium, and then incubated at 4°C and 1500 rpm for 20
Centrifuge for minutes. The supernatant was further heated at 4℃ and 3000rpm.
Centrifugation was performed for 15 minutes to obtain 4 supernatants. This was done using a Beckman JA-20 rotor (Beckman).
Centrifuge at 20,000 rpm for 1 hour at ℃. The resulting precipitate was added to 10 ml of TEN buffer (1mM EDTA, 100mM
20mM Tris-HCl buffer with NaCl, PH = 7.5)
Suspended in
min/mm ² ) for 20 minutes, and then ultracentrifuged again in the same manner as above. 6 ml of the obtained sediment
Crude ATLV was obtained by suspending it in TEN buffer. the
1.5 ml was stored frozen at -20°C for initial immunization. The remainder was further partially purified by sucrose density gradient centrifugation [Proc. Natl. Acad. Sci., Vol. 79, 2031-
2035 (1982)]. That is, after ultracentrifugation in the same manner as above, the precipitate was suspended in 1.5 ml of TEN buffer, and this was
The mixture was layered on 35 ml of ~60% sucrose density gradient and ultracentrifuged at 27,000 rpm for 18 hours at 4°C using a Beckman SW27 rotor (Beckman). Density 1.15
Collect a band of ~1.16 viral particles and TEN
After diluting with a buffer solution, the mixture was further centrifuged at 45,000 rpm for 1 hour at 4°C using a Beckman SW50.1 rotor (Beckman). The precipitate was suspended in 1 ml of TEN buffer and stored at -20°C (ATLV standard). Reference example 2 Immunization and cell fusion For five male BALB/c mice aged 6 weeks,
0.3 ml of the above crude ATLV (approx.
(equivalent to ATLV obtained from 200 ml) was suspended in the same amount of Freund's complete adjuvant and then subcutaneously immunized. After 30 days, 0.13ml of the above ATLV standard
(obtained from approximately 400 ml of MT-2 cell culture medium)
(equivalent to ATLV) was injected intraperitoneally into the previously immunized mice without mixing with Ajiyu Band. Cell fusion was performed using a method described by Oy and Herzenberg et al. in 1980, with some modifications. That is, on the third day after the booster injection, the spleen was removed and its spleen cells were 3.2×10 ⁸ and 3.2×10 ⁷ .
P3／×63−Ag8 cells [Selected Methods in
cellular Immunology, p351−371, WH
Freeman & Co., USA Publishing (1980)]
After washing with RPMI-1640 medium, mix this,
Centrifugation was performed at 1000 rpm for 10 minutes. The cell pellet obtained by removing the supernatant was added to RPMI containing 42.5% polyethylene glycol-1000 (PEG-100) and 15% dimethyl sulfoxide (DMSO) heated to 37°C.
Add 1 ml of 1640 medium and shake for 1 minute.
1 ml of RPMI-1640 medium was added and shaken for 1 minute. Further, 1 ml of RPMI-1640 medium was added and shaken for 1 minute, and a total of 7 ml of the same medium was added at 0.5 ml/min. and similarly shaken. this
Centrifuge at 1500 rpm for 5 minutes, remove the supernatant, and reduce to 15%
10 ml of RPMI-1640 medium containing FCS was gently added to obtain a cell suspension. The suspension was placed in a 96-well flat tissue culture plate (manufactured by NUNC).
The cells were dispensed at 1×10 ⁶ cells/well. HAT medium (containing 10 ^-4 M hypoxanthine, 4 x 10 ^-7 M aminopterin, 1.6 x 10 ^-5 M thymidine and 15% FCS)
Selection in RPMI-1640 medium was started 24 hours later, and cells were grown by replacing half the medium in each well after 3, 5, 8, 12, and 14 days, and then twice a week. I let it happen. Two to three weeks after the cell fusion, the culture supernatant of the proliferating cells was subjected to ATLA antibody screening.
ATLA antibodies were measured by indirect immunofluorescence as described by Hinuma et al. in 1981. That is,
The hybridoma culture supernatant was air-dried and then incubated with an acetone-fixed smear of MT-1 or MT-2 cells. After incubation at 37°C for 30 minutes, the reaction was washed twice with 0.05M phosphate buffer (PBS) containing physiological concentrations of saline, and then anti-mouse IgG rabbit F (ab ') The FITC-labeled reagent ₂ was incubated with a 1:30 diluted solution at 37°C for 30 minutes. After washing with PBS again, the cell smear is
The cells were treated with PBS containing 50% glycerol and immunofluorescence was measured. Measurement of the immunofluorescence of acetone-fixed MT-1 cells was very useful in distinguishing ATLA antibodies from non-specific antibodies in hybridoma culture supernatants. This is because only 1-5% of MT-1 cells
cells are ATLA-positive cells, and they are also mostly multinucleated giant cells. Thus, the criterion for hybridoma screening for ATLA antibodies in the initial culture is that they produce antibodies that primarily bind to ATLA-positive cells and do not react with other ATLA-negative cells. It was hot. Hybridoma cell proliferation was observed in all 384 wells. Of these, 33 wells
The patient was positive for ATLA antibody. Select 10 of these ATLA antibody-positive cultures, expand the culture, and repeat 1 week after initial screening.
I tested for ATLA antibodies. A culture solution with a high titer of ATLA antibody and a weak non-specific reaction was cloned by limiting dilution method to give 0.5 hybridomas/well, and the selected clones were cloned again in the same manner. Monoclonal strains of clone No. GIN-2 and GIN-14 were thus obtained. The cell line is identified by the monoclonal antibodies it produces, and these have the characteristics shown in the test examples below. The above GIN-2 and GIN-14 are stably stored in liquid air. Example 1 Each of GIN-2 and GIN-14 obtained in Reference Example 2 above was treated with 10% FCS, 100 IU/ml penicillin, and 100μ
g/ml streptomycin and 4mM L-glutamine in RPMI-1640 medium at 2 x ¹⁰⁶ cells/ml in a 5% carbon dioxide incubator.
Culture was carried out at 37°C for 48 hours. The culture solution was centrifuged (3000 rpm, 10 minutes) to obtain about 1000 ml of each culture supernatant containing the monoclonal antibody of the present invention. Below, culture supernatants derived from hybridomas GIN-2 and GIN-14 were used as "GIN-2 antibody" and "GIN-2 antibody," respectively.
14 Antibody”. Test example (1) IgG subclass Ochterlonie test GIN-2 antibody and GIN obtained in Example 1 above
Each of the -14 antibodies was concentrated approximately 10-fold by the addition of 50% saturated ammonium sulfate solution and placed in the center of an Ouchterlonie plate in PBS containing 1.5% agar. Subclass-specific rabbit serum for mouse IgG ₁ , IgG ₂ a, IgG ₂ b, and IgG ₃ purchased from Miles Lab. was placed in the surrounding wells, and after 1 to 2 days, the subclass of the monoclonal antibody was detected. was determined by immunoprecipitation lines appearing within the agar gel. Results The GIN-2 antibody contained IgG ₁ and IgG ₃ , while the GIN-14 antibody contained only IgG ₁ . Since the P3/×63-Ag8 myeloma cells used for cell fusion constantly secrete IgG ₁ ,
Has specific binding activity to ATLA
GIN-2 and GIN-14 antibodies are IgG ₃
and IgG ₁ subclass. (2) Analysis based on immunofluorescence method Various lymphoid cells and fresh human lymphocytes
The reactivity with GIN-2 and GIN-14 antibodies and its specificity were searched according to the indirect immunofluorescence method described above. In addition, indirect immunofluorescence is a method reported by Klein et al. in 1966 [Proc. Natl.
Acad. Sci., 55, 1628-1635 (1966)]. The results are shown in Table 1, Reference Figure 1 and Reference Figure 2 below.

【table】

[Table] From Table 1 above, GIN-2 and GIN-14 antibodies react only with ATLA-positive cell lines that were previously shown to contain ATLA using ATLA antibody-positive human serum using indirect immunofluorescence. did. The percentage of fluorescence-positive cells of ATLA-positive cells stained with both monoclonal antibodies was very close to that stained by anti-ATLA human serum. T cell lines, B cell lines, non-T, non-B cell lines, normal IL-2 dependent Cytotoxic T cell lines, normal peripheral blood lymphocytes, fresh peripheral blood lymphocytes isolated from ATLA patients, etc.ATLA
No reactivity was shown with negative cell lines. Reference Figure 1 shows the stained image of cells when the GIN-2 antibody of the present invention is used in the indirect immunofluorescence method using MT-2 cells.
Similarly, shows an image of cells stained using GIN-14 antibody. From both figures, these monoclonal antibodies are
Only the cytoplasm of fixed cells of the ATLA-positive cell line was stained, but the membranes of live cells were not stained. In addition, GIN-2 and GIN-14 based on indirect immunofluorescence
The properties of the immunofluorescence labeled by the antibodies were different. In other words, as shown in Reference Figure 1, GIN-2 antibody stains a part of the cytoplasm, whereas
The GIN-14 antibody stained the entire cytoplasm as shown in Reference Figure 2. These discoveries are GIN-2
This suggests that the GIN-14 and GIN-14 monoclonal antibodies recognize different antigens. (3) Analysis based on immunoprecipitation method Target polypeptides of GIN-2 and GIN-14 antibodies were searched for by immunoprecipitation method and SDS-PAGE analysis. MT-2 cells were substituted with ^35S -methionine and lysed with NP40, and purified ATLV preparations collected from the resulting cell extract and culture supernatant were solubilized with NP40. I thought about it. i.e., a radioactive specific activity of 800-1000 Ci/m in a medium lacking methionine.
MT-2 was cultured in 10 ml of cell culture medium supplemented with mole L-[ ^35S ]methionine at a concentration of 25 μCi/ml.
Cells were incubated at 37°C at a concentration of 5 x ¹⁰⁶ cells per ml.
Incubation was carried out for 10 hours. Cells were subsequently washed thoroughly three times with PBS followed by 0.14M NaCl, 3mM _MgCl2 , 1mM dithiothreitol, 2mM phenylmethylsulfonyl fluoride (PMSF), 10mM Tris.
1 ml of ice-cold low salt concentration extraction buffer containing 0.5% NP40 (Hani Kagaku Co., Ltd.) was added to -HCl, pH 8.0, and dissolved. NP-40 soluble cytoplasmic extract, stirred with a vortex mixer, then placed in ice water.
After standing for 20 minutes, the mixture was centrifuged at 12,000g at 4°C for 10 minutes to obtain a supernatant (cell extract). The ATLV specimen obtained in the same manner as in Reference Example 1 above was
After centrifugal washing three times with PBS, the protein was dissolved in the ice-cold extraction buffer described above to a concentration of approximately 0.1 mg protein/ml. 1 mCi of ¹²⁵ I-Na salt (N.
To this, 20μ of 50mg/ml Crosilane T in 0.2M-PBS (PH=7.2) was added, and after reacting for 15 seconds at room temperature, 100mg/ml of DMSO in 0.2M-PBS was added.
(PH=7.2) 50μ was added to stop the reaction. After the reaction was completed, 100 μ of 1 mg/ml ¹²⁵ I-Na salt in PBS was added, and further 200 μ of 1% BSA in PBS was added. This mixed solution was applied to a Cephadex G-10 (Pharmacia) column (1φ x 20cm) and eluted with PBS.
A radioactive fraction of the excluded volume (void volume fraction) was obtained ( ¹²⁵ I-labeled ATLV preparation). After dispensing the above cell extract or ¹²⁵ I-labeled ATLV preparation into several portions, incubation was carried out at 4°C for 2 hours with 200μ of the hybridoma culture supernatant, and then 1.5μ of anti-mouse 1gG rabbit serum was added. Incubation was continued for an additional 2 hours. Furthermore, 200μ of Staphylococcus aureus suspension (Pharmacia) (10% W/V) was added, and after incubation at 4°C for 2 hours, the bacteria were removed.
It was centrifuged at 12000g for 2 minutes. This precipitate was mixed with 20mM Tris-HCl, PH7.6, 0.5M
Wash twice with 30% sucrose in wash buffer containing NaCl, 1mM EDTA, 0.5% NP40 and 1% desoxycholate, followed by 10mM NaCl.
The cells were washed twice with 10mM Tris-HCl, pH 7.6. The antigen-antibody-bacteria complex was transferred to 70μ of electrophoresis buffer (0.0625M Tris-HCl, PH6.8, 2% SDS,
4% mercaptoethanol, 10% Glyalol,
0.05% Bromophenol Blue) and heated in boiling water for 3 minutes. 30μ of the above sample was subjected to analytical SDS-polyacrylamide gel electrophoresis. Electrophoresis in SDS-polyacrylamide slab gels was carried out under a constant voltage of 100 V in a 10% resolving gel with a 5% concentrating gel as reported by Laemmli in 1970 [Nature,
227, 680-685 (1970)]. After electrophoresis, gel 1974 Bonner
and Laslcey et al. [Europian J. of Biochemistry., 46, 83-88]
(1974)] To determine the molecular weight, a molecular weight determination kit manufactured by Pharmacia was used. This kit contains phosphorylase B (molecular weight 94000), bovine serum albumin (M.
W. = approx. 67,000), ovalbumin (MW = approx.
43000), carbonic anhydrase (MW approx. 30000), Fava metrypsin inhibitor (MW
= approx. 20100), and α-lactone albumin (M.
W. = approx. 14400). Immunoprecipitation using ATLA-positive or -negative human serum was performed in accordance with the method previously reported by Yamamoto and Hinuma in 1982 [Symposium supported by the Ministry of Health and Welfare Cancer Research Grant; Adult T-cell leukemia, lymphoma ( ATL) and its pathogenesis, Life Science Center Publishing, p137-138, published January 5, 1981]. The results using the MT-2 cell extract are shown in FIG. 2. In the figure, the vertical axis represents the molecular weight, and the abbreviations for each lane are as follows. (−) HS; ATLA antibody-negative human serum ×63; culture supernatant of P3/×63-Ag8 (+) HS; ATLA antibody-positive human serum GIN-1; GIN-14 antibody GIN-2; GIN-2 antibody. As is clear from Figure 1, GIN-2 and
It was found that each GIN-14 monoclonal antibody recognized a different antigen or antigenic determinant of ATLA. GIN-2 of MT-2 cell extract
The band specifically precipitated by the antibody is
Among p28 and p20, p19 slightly precipitated, whereas in GIN-14, only p28 precipitated. Some faint bands of precipitated radioactive material were also observed in this immunoprecipitation analysis. These are p36 and p23, which also produce more sedimentation lines when treated with GIN-2 or GIN-14 antibodies than after treatment with human ATLA antibody-negative serum or myeloma supernatant. Indicated. Although not shown in the data, ¹²⁵ I− labeling
No specific sedimentation line from the ATLV preparation could be observed with either leukemia 2 or GIN-14 monoclonal antibodies.

[Brief explanation of drawings]

Figure 1 shows the SDS-SDS of antigenic substances specifically precipitated by the antibody of the present invention from MT-2 cell extract.
It is a drawing showing the PAGE analysis results.

Claims (1)

[Scope of Claims] 1. A human adult T-cell leukemia-related antigen produced by a fusion cell of a mammalian immune cell immunized with a human adult T-cell leukemia virus and a mammalian myeloma cell, and mainly having a molecular weight of 28,000. A monoclonal antibody characterized by having specific reactivity with. 2 Human adult T with molecular weight fractions of 20,000 and 19,000
2. The monoclonal antibody according to claim 1, which has specific reactivity with cellular leukemia-related antigens. 3 Human adult T with molecular weight fractions of 20,000 and 19,000
The monoclonal antibody according to claim 1, which has no specific reactivity with cellular leukemia-related antigens.