JPH06339394A - Monoclonal antibody against human interleukin-1alpha - Google Patents

Abstract

PURPOSE:To obtain a method for immune certification using an antibody specific to human IL-1alpha, e.g. capable of simply measuring human IL-1alpha in high sensitivity and in high precision while distinguishing the human IL-la from human IL-1beta CONSTITUTION:A monoclonal antibody against human interleukin-1alpha produced by an antibody forming cell deposited as indication of KOCO301 (FERM BP-1,554) in Fermentation Research Institute.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to interleukin-1.
The present invention relates to an antibody against (IL-1), specifically, a novel antibody against human IL-1 which enables immunological purification and measurement of IL-1.

[0002]

2. Description of the Related Art Human IL-1 is known to be produced not only by macrophages and monocytes but also by many cells and exhibit various molecular forms and biological activities. Currently, the human IL-1 has two different isoelectric points, namely, IL-1.
α and IL-1β are known, and their primary structures have been clarified, respectively [Nature, 315 , p64].
1 (1985); Exp. Med., 164 , p237 (198
6) etc.].

[0003] The above-mentioned IL-1 has been variously studied for its application as a medicine, and for example, for the study of various immunodeficiency diseases and abnormal immune responses and for clinical diagnosis such as
In particular, its measurement in clinical samples is of interest.

At present, however, a bioassay (biological assay method) is known as a technique for measuring the IL-1. In this method, IL-1 is measured as the active amount of a test sample. . However, this method is inferior in operability and accuracy, and it is necessary to always consider the presence of a component that interferes with the measured value. Moreover, in the above method, IL-1α
And since IL-1β shows the same activity, there was a big drawback that it was not possible to measure them separately.

[0005]

An object of the present invention is to provide an antibody against the above human IL-1α.

[0006] Another object of the present invention is to provide an antibody against human IL-1 which can be used in a new immunological measurement method capable of distinguishing human IL-1α from human IL-1β.

Further, it is an object of the present invention to provide an antibody against human IL-1 which can be used for suppressing (neutralizing) the action of IL-1 in various diseases accompanied by abnormal production of IL-1. .

Another object of the present invention is to provide a technique for producing the above antibody.

[0009]

According to the present invention, human I
Monoclonal antibody having specific reactivity with L-1α, especially KOCO301
(Ministry of Industrial Technology, Article 1554, FERM BP-155
4) There is provided a monoclonal antibody against human interleukin-1α produced by the antibody-producing cells deposited under the designation of, and a method for producing the same.

By using the antibody of the present invention, human IL-1
Provided is a new immunoassay method (immunoassay method) that can distinguish α from human IL-1β by high sensitivity, high accuracy, and simple measurement.

Further, since the antibody of the present invention is specific to human IL-1α, its use also provides a means for specifically purifying them by a technique such as affinity chromatography.

Further, the antibody of the present invention includes an antibody of a type having a neutralizing activity on the biological activity of human IL-1, and such an antibody can be used for various diseases associated with abnormal production of IL-1, such as chronic diseases. In chronic inflammatory diseases such as rheumatoid arthritis, thyroiditis, hepatitis, nephritis, arteriosclerosis, vasculitis such as Kawasaki disease, generalized intravascular coagulation syndrome, blood cancer, etc., enhanced IL-1 based on its abnormal production A drug which is useful for suppressing or neutralizing the biological activity of Escherichia coli, and is extremely valuable in the treatment of such various diseases is provided.

The antibody of the present invention can be produced using human IL-1α as an immunogen. More specifically, for example, a fused cell (hybridoma) of a mammalian plasma cell (immune cell) immunized with the above immunogen and a mammalian plasmacytoma cell is prepared, and human IL-1α is recognized therefrom. It can be produced by selecting a clone producing a desired antibody and culturing the clone.

The human IL-1α as an immunogen used in the above method is not particularly limited, and a culture supernatant containing human IL-1α derived in vitro, a purified preparation thereof, and a gene set, which are already known in vitro, are used. Any of human IL-1α produced according to the replacement technique and synthetic peptides having an amino acid sequence of a part thereof may be used.

The mammal immunized with the immunizing antigen in the above method is not particularly limited, but is preferably selected in consideration of compatibility with the plasmacytoma cells used for cell fusion, and in general, Mice, rats, etc. are advantageously used.

Immunization can be carried out by a general method, for example, by administering the above-mentioned immunizing antigen to a mammal by intravenous, intradermal, subcutaneous or intraperitoneal injection. More specifically,
It is preferable to administer the immunizing antigen to the test animal several times every 2 to 14 days, optionally in combination with an ordinary adjuvant, so that the total dose will be about 100 to 500 μg / mouse. As the immunizing antigen, it is preferable to use spleen cells extracted about 3 days after the final administration.

As the other parental cell, a mammalian plasmacytoma cell to be fused with the above-mentioned immune cell, various known cells such as p3 (p3 / x63-Ag) can be used.
8) [Nature, 256 , 495-497 (1975)],
p3-U1 [Current Topics in Microbiology and
Immunology, 81 , 1-7 (1978)], NS-1 [E
ur.J. Immunol., 6 , 511-519 (1976)], M
PC-11 [Cell, 8 , 405-415 (1976)],
SP2 / 0 [Nature, 276 , 269-270 (197
8)], FO [J. Immunol. Meth., 35 , 1-21
(1980)], × 63.6.5.3. [J. Immunol., 1
23 , 1548-1550 (1979)] S194 [J. E
xp. Med., 148 , 313-323 (1978)] and R210 in the rat [Nature, 277 , 131-13].
3 (1979)] and the like.

The fusion reaction between the above immune cells and plasmacytoma cells can be carried out by a known method, for example, the method of Milstein et al. [Metho
d in Enzymology, Vol. 73, pp3 (1981)] and the like. More specifically, the above fusion reaction is carried out in an ordinary medium in the presence of an ordinary fusion promoter such as polyethylene glycol (PEG), Sendai virus (HVJ), etc. An auxiliary agent such as dimethyl sulfoxide may be added as necessary to increase the amount. The ratio of immune cells to plasmacytoma cells used is the same as in the usual method. For example, it is common to use about 1 to 10 times more immune cells than plasmacytoma cells. As the medium for the fusion reaction, various media commonly used for the growth of plasmacytoma cells, such as RP
Examples include MI-1640 medium, MEM medium, and other mediums commonly used for this kind of cell culture. Usually, it is preferable to remove serum supplement such as fetal calf serum (FCS) from the medium. In the fusion, a predetermined amount of the above immune cells and plasmacytoma cells are mixed well in the above 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 ordinary medium. 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 the desired hybridomas obtained is
It is carried out by culturing in an ordinary selection medium, for example, HAT medium (medium containing hypoxanthine, aminopterin and thymidine). 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 several days to several weeks. The hybridoma thus obtained is subjected to a search for a desired antibody and a monocloning by a usual limiting dilution method.

The target antibody producing strain can be searched by, for example, ELIS.
Method A [Engvall, E., Meth. Enzymol., 70 , 419-
439 (1980)], plaque method, spot method, agglutination reaction method, Ouchterlony method, radioimmunoassay (RIA) method, and other various methods commonly used for antibody detection [“hybridoma method and monoclonal method”]. Antibodies, "R & D Planning, Inc., 30-53
Page, March 5, 1982],
The immunogen described above can be used for this search.

The thus-obtained hybridoma producing the desired monoclonal antibody that recognizes human IL-1α can be subcultured in an ordinary medium and can be stored in liquid nitrogen for a long period of time.

The desired antibody can be collected from the hybridoma by a method of culturing the hybridoma according to a conventional method to obtain a culture supernatant thereof, or by administering the hybridoma to a mammal having compatibility with the hybridoma and proliferating it. And the like are adopted. The former method is suitable for obtaining highly pure antibody, and the latter method is suitable for mass production of antibody.

The antibody obtained as described above can be further purified by ordinary means such as salting out, gel filtration, affinity chromatography and the like.

The thus obtained monoclonal antibody of the present invention has specific reactivity with human IL-1α.

The antibody of the present invention also includes a type of antibody having a neutralizing activity against the biological activity of human IL-1. Such an antibody is suitable for specifically measuring human IL-1 having biological activity. In addition, antibodies of such a type having neutralizing activity, particularly antibodies of the type recognizing a site involved in binding to an IL-1 receptor on an IL-1 molecule, are various types of antibodies with abnormal production of IL-1 described above. It is suitable for application to diseases.

Further, the antibody of the present invention also includes an antibody of the type which recognizes different sites of human IL-1 molecule, has no steric hindrance to each other, and can simultaneously bind to human IL-1 molecule. Therefore, such an antibody is extremely useful for use in an immunoassay such as a sandwich method.

In addition, the antibody of the present invention includes an antibody of a type which is particularly excellent in reactivity in a liquid phase system or a solid phase system. They are highly preferred for application in liquid phase and solid phase immunoassays.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, a monoclonal antibody specific for human IL-1α is provided, and by adopting the antibody of the present invention, the measurement sensitivity is extremely high and the specificity is excellent.
Therefore, very low concentrations of human I, eg in clinical samples, etc.
There is provided a measurement method by an immunoassay method capable of accurately measuring the human IL-1α in a sample containing L-1α.

[0029]

EXAMPLES Examples will be given below to explain the present invention in more detail, but the present invention is not limited to these.

Example 1 Production of antibody against human IL-1α Human IL-1α [Nature, 315 , p641 (198)
5)] was intraperitoneally administered to BALB / C mice together with complete Freund's adjuvant. Immunization was performed by boosting the same amount twice with incomplete adjuvant every 3 to 4 weeks. Three to four weeks later, 30 μg of a human IL-1β saline solution was intravenously administered as the final immunization. Three to four days after the final immunization, cell fusion was performed according to a conventional method [see Method in Enzymology, 73 , pp3 (1981), etc.].
That is, the cell fusion is performed by immunizing the splenocytes and myeloma cells [P3U1, Current Topics in Microbiology and I].
mmunology, 81 , 1-7 (1978)] at a ratio of 10: 1 and polyethylene glycol (PEG-4000).

After selecting the hybridomas in HAT medium,
The supernatant was applied to the human IL-1β-coated 96-well microplate and a peroxidase-labeled goat anti-mouse globulin antibody [E. Wai. [Manufactured by EY Lab.], And tested by an enzyme immunoassay method to detect a target antibody-producing strain against human IL-1β.

Cloning is repeated by the limiting dilution method to obtain a desired antibody-producing clone for human IL-1α, from which the antibody AN for human IL-1α of the present invention AN
OC301 was obtained. The characteristics of this product were determined according to the following methods.

Antibody Subclass Mouse Antibody Subclass Detection Kit (Bio-Rad (B
io-Rad)).

Antibody production level It was shown by the amount of IgG (μg / ml) in the culture supernatant when the hybridoma reached the maximum cell density.

The titer was determined according to the following.

RIA: Iodogen method [B. B. R. C.,
80,849-857 (1978)], 100 μl of human IL-1α labeled with ¹²⁵ I (about 10,000 cp)
m), 100 μl of a diluted culture supernatant of hybridoma, 0.01% NaN ₃ , 5 mM EDTA and 0.1% B
Reaction by mixing 100 μl of PBS containing SA (4 ° C)
50 μl of normal goat serum as a carrier protein
1 and then 600 μl of 20% PEG in PBS was added and mixed, and the mixture was allowed to stand in an ice bath for 20 minutes and then centrifuged to measure the radioactivity of the precipitate with a γ-counter. The dilution ratio of the culture supernatant in which ¹²⁵ I-labeled IL-1α comes to 15% of the sediment is R
The IA titer was used.

EIA: 20 μ in 96-well microplate
Dispense human IL-1α adjusted to g / ml (100 μl
/ Well) and left for 1 hour at room temperature. After washing with PBS, block with BSA to prevent nonspecific adsorption, and
It was washed with S-Tween 20. Diluted hybrid
Aliquot the culture supernatant of horse mackerel (100 μl / well) at 37 ℃
The mixture was reacted for 2 hours, washed, and further peroxidase-labeled anti-mouse immunoglobulin (X500, manufactured by Capel Co.).
0) was added at 100 μl / well and reacted in the same manner.
After washing, the bound enzyme activity is colorimetrically analyzed and OD ₄₉₂ =
The EIA titer was determined by using the dilution ratio of the culture supernatant of 1.0.

Cross-reactivity [0038] Western blotting [Western [Western] was used using test proteins (both human) produced by gene recombination technology.
blotting; Proc. Natl. Acad. Sci., USA, 76 , 4
350 (1979); Anal. Biochem., 112 , 195.
(1981)].

Coupling constant (kd) Scatchard plot analysis (Scatchard p
lot analysys).

Neutralizing activity LAF activity [J. Immunol., 116 , 1466 (197)
6)].

The results of obtaining the above-mentioned characteristics are as follows.

Subclass: IgG _2a antibody production level: 75 μg / ml Titer: RIA × 3200, EIA × 2000 Cross-reactivity: No cross-reactivity with IL-2, IL-1β, GM-CSF and TNF.

Binding constant (kd): 3.6 × 10 ^-8 M / L Neutralizing activity: Yes.

The hybridomas (antibody ANOC301-producing hybridomas) obtained in the above-mentioned examples were KOCO3 tested by the Institute of Microbiology (Ministry of Industrial Science), Ministry of International Trade and Industry.
No. 01, the Micro Engineering Research Institute No. 1554 (FERM BP-155
Deposited as 4).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location // C12N 15/06 (C12P 21/08 C12R 1:91)

Claims (1)

[Claims] 1. The Ministry of International Trade and Industry, Institute of Industrial Technology, Microbial Industry Research Institute
OCO301 (Microtech Research Institute No. 1554, FERM B
P-1554) A monoclonal antibody against human interleukin-1α produced by the antibody-producing cell deposited under the designation.