JPH05244990A - Rat il-1 antibody and method for measuring rat il-1 - Google Patents

Abstract

PURPOSE:To obtain the subject antibody, prepared by using rat interleukin-1 s(IL-1s) as an immunogen, having specific reactivity toward rat IL-1alpha or rat IL-1beta and useful for high-sensitivity immunoassay, etc., for the rat IL-Is. CONSTITUTION:The objective anti-rat IL-1 monoclonal antibody having specific reactivity toward rat interleukin-1 (IL-1)alpha or rat interleukin-1 (IL-1) beta is obtained by administering the rat IL-1alpha, or rat IL-1beta as an immunogen together with a complete Freund's adjuvant liquid into the abdominal cavity of a Ba1b/c mouse, immunizing the mouse, collecting a cell of the spleen after the final immunization, fusing the collected cell to a cell of a murine myeloma, culturing the fused cell in an HAT culture medium, providing a hybridoma, cloning the hybridoma according to a limiting dilution method, affording a monoclone and then culturing the monocloned hybridoma. This antibody has specific reactivity toward the rat IL-1alpha or rat IL-1beta.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a monoclonal antibody against rat interleukin-1 (IL-1), its use in an enzyme immunoassay (particularly a highly sensitive ELISA assay), and rat IL-by a three-step sandwich method. 1
The present invention relates to an immunoassay method for α or rat IL-1β.

[0002]

2. Description of the Related Art IL-1 was introduced by Gerry in 1972.
y) et al. reported it as a mouse thymocyte growth promoting factor present in human monocyte culture supernatant [Gery, I., et al.,
J. Exp. Med., 139 , 128 (1972)]. Then, mouse, human and rabbit IL-1s were successively purified, their cDNAs were cloned, and their structures were elucidated. According to et al., IL-1 is found in monocytes, macrophages, keratinocytes, NK cells, T cells,
Produced by many cells such as B cells, vascular endothelial cells, neutrophils, etc., it acts on many cells and shows a wide variety of biological activities, such as immunity, inflammation, hematopoiesis, endocrine, cranial nerve, and homeostasis Was reported to be involved in the reaction in [Oppenheim, JJ, et al., Immunol. Today, 7 , 45 (19
86)]. Also, IL-1 is classified into two types, α type (pI = 5) and β type (pI = 7-8), depending on the difference in their isoelectric points, but they bind to the same receptor. (Matsushi
ma, K., et al., J. Immunmol., 136, 4496 (1986)], it was reported that almost no difference in biological activity was observed in many reactions. Type [Conlon, PJ, et al., J. Immunol., 1
39 , 98 (1987)].

The amino acid sequences of IL-1α and IL-1β show considerable homology, with human IL-1α and β being 26% and mouse IL-1α and β being 22%, respectively. 62 between human IL-1α and mouse IL-1α
%, And 64% homology between human IL-1α and rabbit IL-1α, and especially the homology between the same types is remarkable.

As described above, the physiological function of IL-1 showing a wide variety of biological activities on many cells is gradually being elucidated, but the role of IL-1 is still unsolved. Many problems remain.

[0005]

As one means for solving the above problems, the study of IL-1 in an animal model is considered, and the rat IL-1 gene as a pathological model and recombinant rat by its use are considered. A rat IL-1 assay system, especially rat I, for establishing the IL-1 production technology and the kinetics and role of rat IL-1 in the pathological model
Development of a measurement system excellent in sensitivity, specificity, accuracy, reproducibility, etc. for L-1, and further anti-rat I used in the measurement system
There is a demand in the art for the development of L-1 monoclonal antibody and the like. However, there are currently no reports on such assay systems and antibodies.

Therefore, an object of the present invention is to provide a rat IL-1 assay system which has been demanded in the art and an anti-rat IL-1 monoclonal antibody utilized therein.

[0007] As a result of intensive studies conducted by the present inventors, the rat IL-1 gene, particularly the rat IL-1α gene, which was previously established by the present inventors, and recombinant rat IL-1α by its use. Manufacturing technology [Japanese Patent Laid-Open No. 1-1682
No. 86] and rat IL-1β gene [JP-A-64]
-71493], and a rat IL-1 assay system that meets the above-mentioned object, and an anti-rat I used for the assay system
We have succeeded in providing the L-1 monoclonal antibody, and completed the present invention here.

[0008]

That is, according to the present invention, it is formed by fusing mammalian immune cells immunized with rat IL-1α or rat IL-1β as an immunizing antigen and mammalian bone marrow cells. Anti-rat IL-1 monoclonal antibody having specific reactivity with rat IL-1α or rat IL-1β produced by the hybridoma, particularly hybridoma KOCO1801
No. 702) and KOCO1901 (Ministry of Industrial Science, Microbiology Research Institute No. 127)
No. 03) is provided.

Further, according to the present invention, in the enzyme immunoassay method, the above-mentioned monoclonal antibody or anti-rat IL-1 polyclonal antibody is immobilized on the first antibody, and the above-mentioned monoclonal antibody or anti-rat IL-1 polyclonal antibody. Rat I is prepared by a three-step sandwich method in which a labeled antibody is reacted with the second antibody using a second antibody.
A method for measuring rat IL-1 for measuring L-1 is provided.

In the following specification, amino acids,
IUPAC and IUPAC-I are used when abbreviations are used for peptides, nucleotide sequences, nucleic acids, restriction enzymes, etc.
The UB nomenclature and regulations and "Guidelines for preparing specifications including base sequences or amino acid sequences" (November 1990, Patent Office Coordination Division Examination Standards Office) shall be followed. When amino acids and the like may have optical isomers, the L-form is shown unless otherwise specified.

By using the monoclonal antibody of the present invention, rat IL-1α and rat IL having biological activity are obtained.
It is possible to provide a new immunoassay method capable of easily measuring with high sensitivity and high accuracy by distinguishing each -1β. The immunoassay can follow, for example, a sandwich method as a conventional enzyme immunoassay, and by using the antibody of the present invention as a specific antibody, rat IL-1α or rat IL-1β can be quantified. , Associated with rat I
Research and elucidation of the physiological function of L-1 can be performed. Moreover, according to the present invention, rat IL-1α and rat IL-1
It is possible to provide a monoclonal antibody specific to each of β, and by using the monoclonal antibody, for example, rat IL-1α and rat IL-1 can be obtained by affinity chromatography or the like.
Specific purification of β can be performed.

The method for producing the antibody of the present invention and the immunoassay method using the thus obtained antibody of the present invention will be described in detail below.

The antibody of the present invention can be produced by using rat IL-1α or rat IL-1β as an immunogen. More specifically, the antibody of the present invention is, for example, a fused cell (hybridoma, hybridoma) of a mammalian plasma cell (immune cell) immunized with the above-mentioned immunogen and a mammalian plasmacytoma cell.
A) can be prepared, a clone producing an antibody (monoclonal antibody) recognizing a desired rat IL-1α or rat IL-1β can be selected therefrom, and the clone can be produced and collected by culturing the clone.

In the above method, rat IL-1α or rat IL-1β can be used as the immunizing antigen. Further, it is also possible to use a rat IL-1α or rat IL-1β as a hapten, which is obtained by binding it to a carrier with a binding reagent according to a general technique for producing an immunogen. The rat IL-1α or rat IL-1β used as an immunogen or a hapten thereof is not particularly limited, and it is a culture containing rat IL-1α or rat IL-1β which is conventionally known in vitro and is induced. The supernatant, purified product thereof, rat IL-1α or rat IL-1β produced according to gene recombination technology, and a synthetic peptide equivalent having a partial amino acid sequence thereof may be used. Among them, particularly preferable examples include recombinant (recombinant) IL-1α or IL-1β obtained by a gene recombination technique, or a similar drug having a partial structure thereof. Specific examples of such recombinant rat IL-1α include, for example, JP-A-1-168.
No. 286, and recombinant rat IL
As -1β, for example, a gene obtained by using the gene described in JP-A-64-71493 and the gene recombination technique described in the above-mentioned publication can be used.

Basically, the production of the monoclonal antibody of the present invention using the above-mentioned immunogen can be carried out by a conventional method [for example, Hanfland, P., Chem. Phys. Lipids, 15 , 105 (197).
5): Hanfland, P., Chem.Phys.Lipids, 10 , 201 (1976)
: Koscielak, J., Eur. J. Biochem., 37 , 214 (1978) etc.].

In the method, the mammal to be immunized with the immunizing antigen is not particularly limited, but in consideration of compatibility with plasmacytoma cells used for cell fusion, it is generally mouse, rat or the like. Is 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, for example, in the case of a mouse,
The immune antigen is diluted to a suitable concentration with physiological saline-containing phosphate buffer (PBS), physiological saline, etc., optionally combined with an ordinary adjuvant, and administered to test animals several times every 2 to 14 days, It is preferable that the total dose is about 100 to 500 μg / mouse. As the adjuvant, pertussis vaccine, complete Freund's adjuvant, alum, etc. can be preferably used. Moreover, as an immunogen,
It is preferable to use splenocytes extracted about 3 days after the final administration.

The mammalian plasmacytoma cell as the other parent cell to be fused with the above-mentioned immune cell is various known cells such as p3 / × 63-Ag8 (X63).
[Nature, 256, 495-497 (1975)], p3 / X63-A
g8. U1 (P3U1) [Current Topics in Microbio
logy and Imunology, 81 , 1-7 (1978)], P3 / NSI
-1-Ag4-1 (NS-1) [Eur. J. Immunol., 6,
511-519 (1976)], Sp2 / 0-Ag14 (Sp2 /
0) [Nature, 276, 269-270 (1978)], FO [J. Imm
unol. Meth., 35 , 1-21 (1980)], and 210 in rats. RCY3. Ag 1.2.3. (Y3) [Natur
e, 277, 131-133 (1979)] and the like can be used.

The fusion reaction between the above-mentioned immune cells and plasmacytoma cells can be carried out by a known method, for example, Milstein.
The method can be carried out according to the method [Method in Enzymology, 73 , 3 (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. Further, a method using electric treatment (electric fusion) can be appropriately adopted. The ratio of immune cells to plasmacytoma cells used is the same as in the conventional method. For example, the ratio of immune cells to plasmacytoma cells is about 1
It is usual to use about 10 times. Examples of the medium at the time of the fusion reaction include various types usually used for the growth of the above-mentioned plasmacytoma cells, for example, RPMI-1640 medium, MEM medium, and other types commonly used for this kind of cell culture. It is advisable to remove serum supplement such as fetal calf serum (FCS) from the isoculture medium. Fusion is a predetermined amount of the immune cells and plasmacytoma cells, well mixed in the medium,
A PEG solution preheated to about 37 ° C., for example, one having an average molecular weight of about 1000 to 6000 is added to a normal medium at about 30.
It is carried out by adding and mixing at a concentration of -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.

Isolation of the desired hybridoma 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 for killing cells (unfused cells etc.) other than the target hybridoma, usually several days to several weeks. The hybridoma thus obtained is subjected to the search for the desired antibody and the monocloning by the 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 (198
0)], plaque method, spot method, agglutination reaction method, Ouchterlony method, radioimmunoassay (RI
A) various methods generally used for detecting antibodies ["Hybridoma method and monoclonal antibody", published by R & D Planning Co., Ltd., pp. 30-53, 1982 3
May 5]], and the above immunogen can be used for this search.

The thus obtained hybridoma producing the desired monoclonal antibody recognizing rat IL-1α or rat IL-1β can be subcultured in a usual medium and stored in liquid nitrogen for a long period of time. You can

The monoclonal antibody of the present invention can be collected from the above hybridoma by culturing the hybridoma according to a conventional method to obtain a culture supernatant thereof, or by administering the hybridoma to a mammal compatible with the hybridoma and growing the hybridoma. The method of obtaining ascites is adopted. The former method is
It is suitable for obtaining high-purity antibody, and the latter method is suitable for mass production of antibody.

The antibody-producing hybridoma culture supernatant and mouse ascites obtained according to the above method can be used as they are as a crude antibody solution. In addition, they can be subjected to an ammonium sulfate fractionation, salting out, gel filtration according to a conventional method. Method, ion exchange chromatography, affinity chromatography such as protein A column chromatography, and the like to obtain a purified antibody.

The monoclonal antibody of the present invention is used in a conventional purification means such as immunoprecipitation method or affinity chromatography to prepare rat IL-1.
Each of α and rat IL-1β can be conveniently and specifically purified.

By using the antibody of the present invention in an immunoassay, each of rat IL-1α and rat IL-1β in a sample can be specifically detected and measured by an immune reaction. As the immunoassay method, an ordinary competitive method, RIA method by sandwich method, E
The LISA method, the agglutination method, etc. can be exemplified, and the operations, procedures, etc. of these methods can follow conventional methods.

In particular, the present invention provides a highly sensitive immunoassay method by the three-step sandwich method using the monoclonal antibody of the present invention. This method is typically carried out as follows, for example. That is, the antibody of the present invention immobilized on a suitable carrier such as a 96-well plate is used as the first antibody, and this is combined with rat IL-1α or rat IL-1α.
-1β standard solution and a measurement substance (rat IL-1α such as an experimental sample or a sample containing rat IL-1β) are allowed to react overnight at room temperature [first step], and then as a second antibody Anti-rat IL-1α rabbit serum (rabbit anti-rat IL-1α polyclonal antibody) or anti-rat IL-1β rabbit serum (rabbit anti-rat IL-1β polyclonal antibody) was added to the above plate, and at room temperature for about 2 hours. By reacting the second antibody with the reaction product of the first step (reaction product of the antibody of the present invention and the substance to be measured) [second step], further, an enzyme-labeled anti-rabbit IgG antibody, etc. A certain amount of the labeled antibody is reacted with the reaction product of the second step (reaction complex of the antibody of the present invention, the substance to be measured and the second antibody) for about 2 hours at room temperature [third step],
Next, after separating and removing the unbound labeled antibody from the conjugate of the reaction complex and the labeled antibody obtained in the above third step, a coloring solution is added to cause the coloring reaction to stop the coloring reaction with 2N sulfuric acid. It is carried out by measuring the absorbance of the coloring reaction solution. Thus rat IL-1α in the sample
Alternatively, rat IL-1β can be quantified.

In the above three-step sandwich method, rabbit or sheep rabbit anti-rat IL is used as the first antibody.
-1α polyclonal antibody or rabbit anti-rat IL-1β
It is also possible to use a polyclonal antibody or sheep anti-rat IL-1α polyclonal antibody or sheep anti-rat IL-1β polyclonal antibody, and use the antibody of the present invention as the second antibody. Furthermore, as a first antibody, rabbit anti-rat IL-
1α polyclonal antibody or rabbit anti-rat IL-1β polyclonal antibody is used, and sheep anti-rat IL-1α polyclonal antibody or sheep anti-rat IL-1 is used as the second antibody.
It is also possible to use a β-polyclonal antibody (a reaction in which the first antibody is a rabbit polyclonal antibody and the second antibody is a sheep polyclonal antibody), and vice versa (the first antibody is a sheep polyclonal antibody and the second antibody is a rabbit polyclonal antibody). It is also possible to carry out a reaction which is a polyclonal antibody.

In the above, the anti-rat IL-1α monoclonal antibody, the anti-rat IL-1β monoclonal antibody, the rabbit or sheep anti-rat IL-1α polyclonal antibody and the rabbit or sheep anti-rat IL-1β polyclonal antibody were immobilized ( The insolubilization) can be carried out by physically or chemically binding the above antibody to the insoluble carrier according to a conventional method. Examples of the carrier for insolubilization include polystyrene, Sephadex, ion exchange resin,
Plastic tubes, amino copolymers, etc. can be used,
The insolubilization is a chemical reaction such as a diazo method as a covalent bond method, a peptide method, an alkylation method, a carrier binding method by a crosslinking reagent, a carrier binding method by a Ugi reaction, or an ionic bonding method using a carrier such as an ion exchange resin, It can be performed by a physical adsorption method using porous glass such as glass beads as a carrier.

The above-mentioned polyclonal antibody is not particularly limited as long as it recognizes rat IL-1α or rat IL-1β, that is, has the ability to bind to rat IL-1α or rat IL-1β. An antiserum produced in vivo by administering the antibody of the present invention or the immune antigen disclosed in the production of the antibody to a mammal can be used, and this can be collected by a conventional method.

The labeled antibody used for labeling in the third step may be a known antibody, for example, obtained by immunizing an already commercially available animal such as mouse, rat, guinea pig, rabbit, sheep, goat, horse or cow. Antiserum
Anti-immunoglobulin antibody labeled with an enzyme such as peroxidase (POD), alkaline phosphatase, β-D-galactosidase, and acid phosphatase, that is, PO
D-labeled goat anti-rabbit IgG antibody, POD-labeled goat anti-mouse IgG antibody and the like can be used. This enzyme labeling can be performed according to a conventional method.

In the assay method of the present invention, examples of experimental samples used as specimens include rat serum or plasma blood, cell tissue fluid, lymph fluid, thymus fluid, ascites fluid, amniotic fluid, gastric fluid, urine, pancreatic fluid, bone marrow fluid, Any of various body fluids such as saliva may be used, but blood, particularly serum or plasma, is preferred.

As the solvent used in the above-mentioned measuring system, various ordinary ones which do not adversely influence the reaction can be used, and examples thereof include a citrate buffer solution, a phosphate buffer solution, a Tris hydrochloric acid buffer solution, and an acetic acid buffer solution. It is preferable to use a buffer solution having a pH of about 5.0 to 9.0. In the present invention, the solvent contains about 0.1 to 30 w / v% of serum (which does not contain rat IL-1α or rat IL-1β to be measured) and / or about 0.1%. ~ 2M NaCl
It is preferable to include since it is more suitable for the purpose of the above assay method.

The immunoreaction conditions in the immunoassay are not particularly limited and may be the same as those in the usual measuring method of this kind. Generally about 45 ° C. or less, preferably about 4-40.
The immunoreaction may be performed under a temperature condition of about C for about 1 to 80 hours.

In the immunoassay method of the present invention, the separation of the solid-liquid phase (the conjugate of the reaction complex and the labeled antibody in the 3-step sandwich-the unbound labeled antibody) after completion of the immune reaction is performed, for example, by centrifugation. It can be carried out by a usual method such as separation, filtration, decantation and washing.

The enzyme labeling activity of each substance thus separated can be measured according to various known methods depending on the type of enzyme used. The coloring solution used at that time is an ordinary one, for example, o-phenylenediamine (OPD) when peroxidase is used.
Etc. can be used, and the color reaction can be stopped by a conventional method, for example, by adding an appropriate enzyme activity inhibitor such as 1 to 4 N sulfuric acid to the reaction solution.

Thus, according to the method of the present invention, a trace amount of rat IL-1α or rat I such as an experimental sample of rat is obtained.
Using a sample containing L-1β as a sample, rat IL-1α or rat IL-1β in the sample can be quantified with high accuracy and high sensitivity by a simple operation.

A particularly convenient method for carrying out the above immunoassay method of the present invention is to use the above-mentioned first antibody, labeled antibody and second antibody as reagents.
This is a method using a measurement kit containing an antibody. In the rat IL-1α or rat IL-1β antibody reagent in the kit, for example, a stabilizer and / or a preservative such as glycerol or bovine serum protein can be added and present. This antibody reagent is preferably freeze-dried, and the kit may contain a water-soluble or water-miscible solvent. Further, the antibody reagent may contain a buffer solution for keeping the reconstituted reagent system at a constant pH and a preservative and / or stabilizer for preventing deterioration of the sample. Although the buffer solution is not an essential component of the kit reagent, it has a pH of about 5.0-9.
It is preferable to use one set to 0. The reconstitution agent is
It preferably contains water, but part or all of the water may be replaced with a solvent miscible with water. Examples of the solvent miscible with water include well-known solvents such as glycerin, alcohols and glycol ethers.

[0038]

According to the present invention, an antibody specific to rat IL-1α or rat IL-1β, particularly rat IL-1
α monoclonal antibody and rat IL-1β monochrome antibody
Null antibodies are provided. The use of the monoclonal antibody has extremely high measurement sensitivity and excellent specificity, and therefore the rat IL in a sample containing rat IL-1α or rat IL-1β at an extremely low concentration, such as an experimental rat sample. An assay method capable of accurately measuring -1α or rat IL-1β is provided.

[0039]

The present invention will be described in more detail below with reference to reference examples and examples, but the present invention is not limited to these examples.

[0040]

Reference Example 1 Production of rat IL-1α Production of rat IL-1α can be performed by a known general gene recombination technique. The technique is disclosed, for example, in Japanese Patent Laid-Open No. 1-1.
It is described in Japanese Patent No. 68286, and an example according to this is shown below.

Measurement of Cell Proliferation Inhibitory Activity (GIF Activity) A 96-well microplate (manufactured by Corning) was charged with 0.1 ml of the test solution diluted to various concentrations, and then 2 × human melanoma cells A375 were placed in each well. 10 ⁴ pieces / m
Eagles MEM containing 10% FCS contained at a concentration of 1
The suspension (0.1 ml) is added, and the cells are cultured in a carbon dioxide incubator (manufactured by Nafco) for 4 days. After completion of the culture, 0.05 ml of 0.05% neutral red (manufactured by Wako Pure Chemical Industries, Ltd.) is added to each well, and the mixture is cultured at 37 ° C for 2 hours. After removing the supernatant, 0.3 ml of phosphate buffered saline (PBS) is gently added to each well to wash the well. After removing the washing solution, 0.1 ml of a monosodium phosphate-ethanol equivalent mixture was added to each well, and the mixture was shaken with a micromixer for several minutes to measure the amount of dye incorporated into cells for 96 well-microtitration plate. Using a photometer (Titer Check Multiscan, manufactured by Flow Laboratories), the absorbance is measured at 540 mμ to determine the growth inhibitory activity. Take the reciprocal of the dilution ratio of the test group showing 50% inhibition of cell proliferation in the control group (control group), that is, the test group showing the measured absorbance value of 1/2 of the measured absorbance value of the control group,
This is defined as a GIF activity unit. Therefore, for example, this GI
When the F activity is 10 units, this test solution has an activity of inhibiting cell growth by 50% even when diluted 10-fold.

Measurement of lymphocyte activating factor (LAF activity) The measurement of lymphocyte activating factor is performed by the method of Oppenheim et al. [JJ Oppenheim, et al., J. Immunol., 116 , 1466 (197).
6)] was carried out as follows.

That is, thymocytes collected from 4- to 5-week-old male Balb / c mice were treated with RPMI containing 10% FCS.
After preparing 1 × 10 ⁷ cells / ml in -1640 medium, 1/100 volume of 200 μg / ml phytohemagglutinin (PHA, manufactured by Burroughs-Wellcome) was added to give a final concentration of 2 μg / ml. To be prepared.
100 μl of this cell suspension is added to each well of a 96-well plate to which 100 μl of sample solution has been added in advance, and the cells are incubated at 37 ° C. under 5% CO ₂ . 48 hours later, [
³ H] -thymidine was added at 0.5 μCi / well, and further 2
After culturing for 4 hours, the amount of [ ³ H] -thymidine incorporated into the cells is measured by a liquid scintillation counter. The LAF activity is defined as 1 unit at a value of 50% of the maximum uptake amount by using recombinant human IL-1β.

Construction of plasmid ptrp-RT-IL-1α containing rat IL-1α cDNA and expression in E. coli Rat IL isolated from cDNA library prepared from mRNA obtained from rat peritoneal macrophages
-1α cDNA plasmid pcD-RT-IL-1α
(3) is cleaved with restriction enzymes AccI and HincII to give a 870 bp DN containing rat IL-1α cDNA.
Isolate the A fragment by agarose gel electrophoresis,
Purified. This was further cleaved with a restriction enzyme HaeIII to isolate a 530 bp HaeIII-AccI DNA fragment by agarose gel electrophoresis and purified, and then blunt-ended with DNA polymerase I Klenow fragment.

A vector pTM having a tryptophan promoter cleaved with the restriction enzyme HindIII
1 [Nippon text man, metabolism, 23 , 289 (1985)] E. coli DNA
Polymerase I Klenow Fragment (Takara Shuzo)
After blunting with blunt end, this was isolated and purified as described above, and the blunt-ended DNA fragment was ligated using a DNA ligation kit (Takara Shuzo), and then Escherichia coli HB101 competent cell was prepared. Transformation was performed to obtain a transformant having the plasmid pTM1-RT-IL-1α.

From the obtained transformants, the alkaline lysis method [T. Maniatis, EFFritsch and J. Sambrook, Molecula] was used.
r Cloning A Laboratory Manual, 368, Cold Spring Ha
borLaboratory, 1982], the above-mentioned plasmid was taken out, cleaved with restriction enzymes EcoRI and BamHI, and then 1.2-containing the tryptophan promoter and rat IL-1α cDNA by agarose gel electrophoresis.
The kb DNA fragment was isolated and purified.

On the other hand, the plasmid pUC119 (manufactured by Takara Shuzo) was cleaved with restriction enzymes EcoRI and BamHI,
Between the EcoRI-BamHI cleavage site, the above 1.2
The kb DNA fragment was ligated using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.), and E. coli M
V1184 strain to obtain the desired plasmid ptr
A transformant having p-RT-IL-1α was obtained.

Single-stranded DNA phage was prepared using the obtained transformant and helper phage M13K07 (manufactured by Takara Shuzo), and extracted with phenol to recover single-stranded DNA ptrp-RT-IL-1α. did.

All the above operations were in accordance with the Takara Shuzo manual.

Excess bases were removed and ATG was inserted by the in vitro mutagenesis method using the single-stranded DNA obtained above as a template and the synthetic oligonucleotides shown below as primers. The primer was synthesized using an automatic synthesizer (manufactured by Applied Biosystems) and has the following base sequence.

5'-GGGTATCGATAATGTCAGCACCT-3 'The above-mentioned in vitro mutagenesis is an oligonucleotide-directed in vitro mutagenesis system (Oligonucleotide-dire) manufactured by Amersham.
cted in vitro mutagenesis system) according to the manual of the company. Transformation of the finally synthesized double-stranded DNA was performed using Escherichia coli JM109 strain. Plasmid DNA was prepared from the obtained transformant by the alkaline lysis method, its restriction enzyme map was prepared, the DNA base sequence was confirmed, and the target mutant plasmid ptrp-RT-IL-1α was identified.

The above outline is as shown in FIG.

Rat IL-1αc confirmed above
The nucleotide sequence of the DNA region and the amino acid sequence of rat IL-1α encoded by the nucleotide sequence are shown in FIGS. 2 and 3.

Transformed strain JM109 / p obtained above
trp-RT-IL-1α, ampicillin 50 μg /
LB containing 20 ml of L-tryptophan and L-tryptophan
Medium [1% tryptophan (manufactured by Difco), 0.5%
Yeast extract (manufactured by Difco) and 1% NaCl] 20 m
Incubation was carried out at 37 ° C. in 1 overnight with shaking.

10 ml of this culture solution was added to M9 minimal medium containing 1% casamino acid [0.6% Na ₂ HPO ₄ , 0.3% K].
H ₂ PO ₄ , 0.05% NaCl, 0.1% NH ₄ C
1, 2 mM MgSO ₄ , 0.2% glucose and 0.
The cells were inoculated into 500 ml of 1 mM CaCO _{2 and} cultured at 37 ° C. for 8 hours with shaking. After harvesting the cells,
It was suspended in a ₂ HPO ₄ , left to stand at low temperature (4 ° C.) overnight, and then dialyzed for 2 days against 10 mM Tris-HCl buffer (pH 8.0).

The dialysate thus obtained was centrifuged (10
(000 rpm, 30 minutes) to separate the supernatant from the precipitate.

Measurement of LAF activity and GIF activity Regarding the culture supernatant obtained above, the LAF activity was measured, and as a result, an activity of about 2 × 10 ⁶ units was recognized in terms of E. coli extract in 1 ml of culture solution. Was given. The GIF activity of the supernatant was measured and found to be about 5.6 × 10 ^6.
It was a unit.

Purification of Target Polypeptide The culture supernatant obtained above was purified by the following purification procedure.

That is, JM109 / pt obtained above
3 liters of rp-RT-IL-1α was similarly cultured, and the obtained cells were suspended in 25 ml of 1M Na ₂ HPO ₄ ,
It was left overnight at 4 ° C. Then, it was dialyzed for 2 days against 10 mM Tris / HCl buffer (pH 8.0). The dialysate obtained was centrifuged to separate the supernatant and the precipitate. The thus-obtained supernatant was subjected to ion exchange chromatography (SP-HPLC) using a GTi Ultrochrome Hyperformance Liquid Chromatography System (manufactured by LKB) under the following conditions.

Column: TSK gel SP-5PW (7.5
Eluent: A = 50 mM sodium acetate (pH 5.5) B = 0.5 M NaCl-containing 50 mM sodium acetate (pH 5.5) Flow rate: 1 ml / min Fraction volume: 1 ml / min / tube Fraction No. by SP-HPLC 20-5
0 was collected, concentrated by ultrafiltration using a YM-5 membrane filter, and the concentrate was subjected to high performance gel chromatography (GF-HPLC) under the following conditions.

Column: TSK gel G2000SW (2
Eluent: PBS ^(-) Flow rate: 3 ml / min Fraction volume: 12 ml / 4 min / tube Fraction No. by GF-HPLC described above. As 16, the target purified rat IL-1α was obtained. This was used as an immunogen for producing the rat IL-1α monoclonal antibody of the present invention.

The molecular weight of the purified rat IL-1α obtained above was calculated according to sodium dodecyl sulfate (SDS) -polyacrylamide gel (PAGE) electrophoresis.
The method is the method of Laemuli [Laemuli, UK, Nature, 227,
680-685 (1970)] with some modifications. Here, the polyacrylamide gel used was 3.9% for concentration and 15% for separation. The staining was performed using Coomassie Brilliant Bull (CBB).

As a result of the above SDS-PAGE, the molecular weight of purified rat IL-1α was confirmed to be about 17.3 kd.
The molecular weight of IL-1α calculated from the amino acid composition is 17.8 kd. In addition, the purified rat IL-1α
The GIF activity of E. coli was about 1 × 1 as measured by the above method.
It was 0 ⁷ U / mg protein.

[0064]

[Reference Example 2] Production of rat IL-1β Regarding the rat IL-1β gene, it has already been disclosed in JP-A-64-
It is disclosed in Japanese Patent No. 71493. The rat IL-1β deduced from the sequence of this gene consists of 268 amino acid residues. From the study of human IL-1β, I
It has been clarified that L-1β is synthesized as a precursor in the cell, is processed, and is then released outside the cell as mature IL-1β. Human IL-1β
It has been previously reported that the mature amino-terminus of E. coli is alanine at position 117 of the amino acid sequence deduced from human IL-1β cDNA [CJ March et al., Natur.
e, 315, 641-647 (1985); Y.Hirai et al., Gaun Monogr
aph on Cancer Research, 34 , 155-166 (1988)]. In addition, regarding rabbit IL-1β, rabbit IL-1βcDN
It has been reported that the 117th alanine in the amino acid sequence deduced from A is the amino terminal amino acid of mature IL-1β [S. Mori et al., Biochem. Biophys. Re.
v. Communi., 150, 1237-1243 (1988)]. On the other hand, regarding mouse IL-β, the amino terminus of mature IL-1β is unknown, but from the comparison with the amino acid sequence of human IL-1β, the 118th valine is assumed to be the mature amino terminus, It has been reported that an expression product in which 152 amino acid residues after the 118th position are expressed in E. coli has IL-1 activity [JJ Huang et al., J. Immunol., 140, 3].
838-3843 (1988)].

As for rat IL-1β, the amino-terminal amino acid of its mature form is unknown as described above. Therefore, the present inventors have determined the amino acid sequences of human, rabbit, and mouse IL-1β and rat IL-1β. Comparing with the amino acid sequence, valine at the 117th position shows mature IL-
It was presumed to be the amino terminal of 1β, and based on this, 152 amino acid residues after the 117th position of rat IL-1β were expressed in Escherichia coli as follows.

Rat IL-1β expression plasmid pt
Preparation of rpIL-2X / ratIL-1β Rat IL-1β cDNA shown in Example of Japanese Patent Laid-Open No. 64-71493 (rat IL-1β gene)
ΛRIL-5 DNA having the following is cleaved with restriction enzyme EcoRI, rat IL-1β cDNA of about 1.35 kb is separated by agarose gel electrophoresis, and agarose gel is prepared using GENECLEAN (manufactured by BIO101) which is a kit for DNA purification. More isolated and purified. The following D
The NA fragment was isolated and purified in the same manner. on the other hand,
Plasmid pcDE [T. Nishida et al., J. Biochem., 1
05, 351-357 (1989)] was cleaved with the restriction enzyme EcoRI, and the resulting rat IL-1β cDNA and T4 DNA were cleaved.
Ligation with ligase, pcDEratIL-1β
Got

The plasmid pcDEratIL-β was cleaved with the restriction enzyme EcoRI to give rat I of about 1.35 kb.
L-1β cDNA was obtained. This rat IL-1βcDN
A is partially cleaved with the restriction enzyme PvuII to give a carboxyl-terminal 147 amino acid residue-containing region of about 0.
A 9 kb PvuII-EcoRI DNA fragment was obtained.

[0068] Next, the synthetic oligodeoxynucleotide 5 _'OH
-CTAGAACGGAGGACTCATTGATGGTTCCCATTAGACAG- _OH 3'and 5 '
_OH -CTGTCTAATGGGAACCATCAATGAGTCCTCCGTT- _OH 3 ', T4
The PvuII-EcoR obtained above was obtained by phosphorylating its 5'end using a polynucleotide kinase.
After ligation to the I DNA fragment using T4 DNA ligase,
Cleaved with restriction enzymes XbaI and SalI to give about 0.75
A kb XbaI-SalI DNA fragment was obtained. on the other hand,
Human IL-2 expression plasmid ptrpIL-2D8Δ
(See JP-A-63-12958), the restriction enzyme X
After digestion with baI and SalI, an about 4.2 kb XbaI-SalI DNA fragment containing the tryptophan promoter and the amino-terminal 59 amino acid residues of IL-2 was obtained. This DNA fragment was treated with alkaline phosphatase derived from calf intestine to dephosphorylate its 5'end, and then the XbaI-SalI DNA obtained above was obtained.
The fragment was ligated with T4 DNA ligase to produce E. coli H
B101 strain and transformed with the desired plasmid ptrp
Escherichia coli HB10 having IL-2X / ratIL-1β
Got 1.

Further, this plasmid was used as Escherichia coli SG210.
58 strains [MR Maurizi et al., J. Bacteriol., 164, 112
4-1135 (1985)] and transformed into E. coli SG21058.
/PtrpIL-2X.ratIL-1β was obtained.

The above outline is shown in FIG.

PtrpIL-2X.r thus obtained
As shown in FIG. 5, atIL-1β, tryptophan.
It encodes two types of polypeptides in the transcription unit from the promoter, one of which is generated by the translation initiation codon, the amino terminal 60 amino acid residue of human IL-2, and the linker portion by the synthetic oligodeoxynucleotide. It consists of 4 amino acid residues. The other consists of the translation initiation codon and 152 amino acid residues of rat IL-1β. Such a two-cistron
In the expression system, the second cistron (2nd
The rat IL-1β gene, which is a cistron, is translated by the ribosome binding to the downstream of the first cistron (the second SD sequence present in the sequence of the linker portion in this case). ..

E. coli SG21058 / ptrpIL
-2X · ratIL-1β culture E. coli SG21058 / ptrpIL-2X · ratI
LB medium containing 50 μg / ml ampicillin of L-1β [T. Maniatis et al., Molecular Cloning, A Laborat
ory Manual, pp.440, Cold Spring Harbor Laboratory
(1992)], after overnight culture at 37 ° C., the culture solution was 5 m
1 with 1% casamino acid, 5 μg / ml thiamine hydrochloride, 2
M9 medium supplemented with 0 μg / ml L-cysteine and 50 μg / ml ampicillin (however, the glucose concentration was 0.1%).
4%) [T. Maniatis et al., Molecular Clonin
g, A Laboratory Manual, pp.440, Cold Spring Harbor
Laboratory (1982)] 500ml (2l flask)
And cultured at 37 ° C. for 8 hours. It should be noted that the culture was carried out in a total of 3 l using 6 2 l flasks. Purification of ratIL-1β RatIL-1β was purified as follows using E. coli (SG21058) culture solution (3 l) containing the ptrpIL-2X · ratIL-1β plasmid as a starting material.

That is, after collecting the bacterial cells by centrifugation,
The cells were suspended in 1M Na ₂ HPO ₄ (50 ml) and left in a cold room overnight. Subsequently, the suspension was dialyzed against 5 mM Tris.HCl (pH 8.0) (15 l) for 2 days in a cold room (external solution was exchanged 3 times). Next, the supernatant was obtained by centrifugation (16000 g), and this was used for purification.

First, purification by SP-HPLC was carried out under the following conditions.

Column: TSK gel SP-5PW (7.5
Eluent: A = 20 mM phosphate buffer (pH 6.0) B = 0.5 M NaCl-containing 20 mM phosphate buffer (p)
H8.0) Flow rate: 1 ml / min Model using HPLC: GTi Ultrochrome High Performance Liquid Chromatography System (LKB
The target product-containing fractions are collected, concentrated by ultrafiltration (using YM-5 membrane), and then subjected to the next gel filtration HP.
It was subjected to LC purification. The GIF activity after SP-HPLC was about 5 × 10 ³ units / mg protein.

The conditions for gel filtration HPLC are as follows.

Column: TSK gel G2000SW × L
(7.5 x 300 mm, manufactured by Tosoh Corporation) Eluent: PBS ^(-) Flow rate: 1 ml / min HPLC model: GTi Ultrochrome High Performance Liquid Chromatography System (LKB)
Target product-containing fraction (GIF activity is about 1.6 x 10
⁴ units / mg protein) were collected and purified by SP-HPLC in the same manner as described above using this product. The GIF activity of the obtained purified product is about 1 × 10 ⁵ units /
It was mg protein.

[0078]

Example 1 Production of Monoclonal Antibody of the Present Invention Production of Rat IL-1α Monoclonal Antibody Rat IL-1α obtained in Reference Example 1 was used as an immunogen (hereinafter abbreviated as “rRT-IL-1α”), The following method was performed.

That is, 20 μg of the above rRT-IL-1α was mixed with an equal amount of Freund's complete adjuvant solution and emulsified,
The obtained suspension was used as a male Balb / c mouse (8 weeks old).
The mice were immunized by intraperitoneal administration with 40 μl / mouse. Two
The suspension was boosted 3 times every other week by the same route for immunization. Two to three weeks later, 20 μg of the same suspension was intravenously administered as the final immunization. All the following operations were performed aseptically and the reagents were kept at 37 ° C.

Two to three days after the final immunization, the spleen was excised from one mouse, and splenocytes were excised from the excised spleen.
It was crushed in ml of RPMI-1640 medium (Nissui Pharmaceutical Co., Ltd.). Next, the cells are centrifuged (1500 rpm, 5 minutes), the cell pellet is scraped off, and the red blood cells present in the cells in the pellet are washed with 0.83% ammonium chloride solution.
Dissolve by treatment for ~ 2 minutes. The cells obtained above were collected as sensitized lymphocyte cells and warmed to 37 ° C.
The plate was washed 3 times with I-1640 medium.

Next, mouse myeloma cells [ATCC CR
L1597, P3U1, Current Topics in Microbiog
y., 81 , 1-7 (1978)] with 15% FCS-containing RPMI-
1640 medium was subcultured in a medium containing 100 μM of 8-azaguanine, and this was used as myeloma cells and washed in the same manner as the above lymphocyte cells.

The sensitized cells and the myeloma cells were mixed in a 50 ml tube so that the cell number ratio was 10: 1, centrifuged (1500 rpm, 5 minutes), and the supernatant was collected with a Pasteur pipette. It was completely removed and the pellet was loosened. These operations were performed in a water tank kept at 37 ° C. Then 45% polyethylene glycol-4000
(Bering, Mannheim, Yamanouchi, "PE
G) (2 g) is slowly added dropwise with stirring for 30 seconds, then left for 30 seconds, then 5 ml of FCS-free RPMI-1640 medium is slowly added over about 2 minutes, and left for 1 minute. After adding 5 ml of the culture solution and leaving it for 3 minutes, centrifugation (1500 rpm,
5 minutes), the supernatant was removed with a Pasteur pipette, and the resulting pellet was added to 50 ml of RPMI-containing 10% FCS.
The cell suspension was suspended in 1640 medium at 1 × 10 ⁵ cells / ml.

Next, this suspension was dispensed into four 24-well plates (manufactured by Corning Incorporated) at 1 ml / well and 37
The cells were cultured in an incubator at 5 ° C, 5% CO ₂ and 100% humidity. 24 hours later, 1 ml / well 10% FC
S-added hypoxanthine 1 × 10 ⁻⁴ M, aminopterin 4
RPM containing x 10 ^-7 M and thymidine 1.6 x 10 ^-5 M
I-1640 medium (hereinafter abbreviated as "HAT medium") was added. After that, on the second and third days of the supernatant, HAT medium was added to half of the medium to change the liquid. After that, the liquid change was performed every 2 to 3 days. The supernatant was aspirated in the same manner on the 6th day, and hypoxanthine 1 × 10 ⁻⁴ M and thymidine 1.6 ×
The medium was replaced with RPMI-1640 medium containing 10 ⁻⁵ M (hereinafter abbreviated as “HT medium”). After that, RPMI-1640
Growth was maintained in the medium.

Colonies became visible to the naked eye 7 to 10 days after the fusion, and the supernatant was obtained in Reference Example 1 from the time when the cells occupied 1/4 of the bottom area of the 24-well plate. Enzyme-linked immunosorbent assay using rRT-IL-1α as an antigen (E
LISA method) and positive wells were immediately cloned by the limiting dilution method [Methods in Enzymology, 73 , 3 (1981)].

That is, a 96-well microplate for ELISA (manufactured by Nunc) was used to immunize with the immunogen r used for immunization.
An antigen solution prepared by diluting RT-IL-1α in PBS to 10 μg / ml was added to the immunoplate at 100 μm.
Each 1 / well was dispensed and left standing at 4 ° C. for 2 hours for coating. Then, after removing the antigen solution, 0.1
% BSA and PBS were added at 400 μl / well each, and the mixture was allowed to stand at room temperature for 1 hour, 0.1% BSA and PBS were removed, and the supernatant was added at 100 μl / well. After overnight incubation at room temperature, 0.05% Tween (Tween
) -20 and PBS were washed 3 times. Then 10
0 μl / well HPRO (horseradish peroxidase) labeled rabbit anti-mouse IgG antibody (manufactured by Bio-Rad)
Was added and incubated at room temperature for 2 hours, then 0.05
Washed 5 times with% Tween-20 and PBS. Finally,
100 μl / well of ortho-phenylenediamine (OP
D) Substrate solution [4 mg OPD, 4 μl 30% hydrogen peroxide solution, 10 ml citrate buffer solution (pH 5.0)] was added,
After sufficient color development, 50 μl of 2N sulfuric acid solution was added to the well to stop the reaction.

Coloring of the substrate was carried out by measuring the absorbance at 490 nm using an Elisa Analyzer (manufactured by Titer Tech).

The hybridoma in the well which became positive in the above ELISA was immediately cloned by the limiting dilution method. That is, Balb / c mouse splenic gland cells were used as feeder cells, diluted with 10% FCS-supplemented RPMI-1640 medium, and added to a 96-well plate at 2 × 10 ⁶ cells / 100 μm.
It was prepared and dispensed so as to have 1 / well. ELI above
The cells in the wells colored with SA were diluted with RPMI-1640 medium supplemented with 10% FCS so as to be 3 cells / ml, and
100 μl / well was dispensed into a 6-well plate.
When the clones grew to a certain extent, screening was carried out again in the same manner as described above, single clones were subjected to ascitesification or cryopreservation, and those that were not were screened again as described above.

The above cloning was repeated 5 times to obtain a hybridoma producing the monoclonal antibody of the present invention having a desired reaction specificity. This is "KOCO1801"
I named it.

The clone KOCO1801 was cloned into RPM.
I-1640 medium was cultured under 5% CO ₂ conditions at 37 ° C. for 96 hours in a 50 ml flask. The culture solution was centrifuged at 3000 rpm for 10 minutes to obtain a culture supernatant containing the target monoclonal antibody. The obtained clone was selected.

The above-mentioned monoclonal antibody-producing clone KOCO1801 of the present invention has been deposited at the Institute of Microbial Technology, Ministry of International Trade and Industry, Institute of Microbial Science and Technology (MICO) under the designation "KOCO1801", and the deposit number is "MICO The first
2702 (FERM P-12702) ".

Preparation of ascites The hybridoma obtained above was subjected to 5% C with the above culture medium.
The cells were cultured under O ₂ conditions at 37 ° C. for 48 hours in a 50 ml flask, the culture solution was centrifuged at 1500 rpm for 5 minutes, and the obtained pellet was suspended in 2.5 ml of PBS. Next, two to three days in advance, pristane (2, 6, 1
0.5 ml of the above cell suspension was intraperitoneally administered to 5 Balb / c mice that had been inoculated with 0,14-tetramethylpentadecane (Aldrich).
After 10 to 14 days, the accumulated ascites of only the first one was aseptically collected to obtain ascites containing the antibody of the present invention. This ascites fluid was centrifuged at 1500 rpm for 5 minutes, and the pellet was
DMSO (dimethyl sulfoxide, Dojindo) and 10
The cells were suspended in RPMI-1640 medium supplemented with% FCS and stored frozen. The rest of the supernatant was combined with 4 ascites fluids for subsequent purification.

Purification of Monoclonal Antibody of the Present Invention Purification of the antibody of the present invention from the ascites fluid obtained above was performed using an antibody purification kit (MAPS-II kit manufactured by Bio-Rad) as follows.

That is, protein A was equilibrated in advance with about 20 ml of a binding buffer solution (utilizing the reagent attached to the kit dissolved in water), and a double amount of the binding buffer solution was added to the ascites and mixed, and the mixture was mixed at 4 ° C After standing for 2 hours, the mixture was centrifuged at 3000 rpm for 30 minutes. The resulting supernatant was filtered through a 45 μm membrane filter (Millex HA, Millipore).
The filtrate was applied to a protein A column and washed with about 30 ml of binding buffer. Next, IgG was eluted with 10 ml of an elution buffer (using the reagent attached to the kit dissolved in water), and 1 ml of 1 M Tris-hydrochloric acid buffer was added for neutralization.
Further, it was dialyzed against PBS at 4 ° C. overnight.

Using the obtained dialysate, absorption at 280 nm was measured to determine the antibody concentration. As a result, the purified antibody concentration was 0.16 mg / ml.

[0095]

Example 2 Characterization of rat IL-1α monoclonal antibody Subclass of antibody rRT-IL-1α antigen was diluted with PBS to 10 μl / m
Then, 100 μl / well was dispensed to the immunoplate, and the plate was left standing at 4 ° C. for 2 hours for coating. Next, after removing the antigen solution, 0.1% BSA and PBS were added at 400 μl / well, and the mixture was allowed to stand at room temperature for 1 hour, then 0.1% BSA and PBS were removed, and the supernatant was removed to 100%.
μl / well was added. After overnight incubation at room temperature, wells were washed 3 times with 0.05% Tween-20 and PBS.

Next, anti-mouse IgG ₁ , IgG _2a and Ig
G _2b , IgG ₃ and IgM polyclonal antibodies (rabbit) (all manufactured by Bionetics) were diluted 1000-fold with 1% BSA and PBS, added 100 μl / well each, and incubated at room temperature for 2 hours, and then diluted with 0. 0
Wells were washed 5 times with 5% Tween-20 and PBS. Finally, add 100 μl / well of substrate solution and add about 5
After color reaction for 10 minutes to sufficiently develop color, 100 μl / well of 2N sulfuric acid solution was added to each well to stop the reaction.

As a result of the above reaction, the rat IL-1α of the present invention was obtained.
The subtype of the monoclonal antibody was IgG _2a .

Antibody Production Level The culture supernatant obtained in Example 1 was centrifuged and the supernatant was adjusted to 10%.
FCS-added RPMI-1640 medium at 37 ° C., 5% C
The cells were cultured in vitro under O ₂ conditions for 10 days. KO in culture supernatant when hybridoma reaches maximum cell density
The amount of IgG _{2a of} CO1801 was about 10 μg / ml.

SDS-PAGE An equal volume of sample buffer (10%) to the purified rat IL-1α monoclonal antibody of the present invention obtained in Example 1
Glycerol, 5% 2-mercaptoethanol, 2.5
% SDS, 30 mM Tris-HCl buffer (pH 6.8))
The mixture of and was mixed with the Laemmli method [Laemmli, UK, Na
ture, 227, 680-685 (1970)] and analyzed by SDS-PAGE using a 15% polyacrylamide gel. The SDS used in the SDS-PAGE was manufactured by Wako Pure Chemical Industries, Ltd., and the molecular weight markers used were phosphorylase B (molecular weight: 94000), albumin (molecular weight: 67,000), and ovalbumin (molecular weight: 43).
000), carbonic anhydrase (molecular weight: 30
000), trypsin inhibitor (molecular weight: 2010
0) and α-lactalbumin (molecular weight: 14400)
And are all manufactured by Pharmacia.

The results of the above SDS-PAGE are shown in FIG.

In the figure, lane 1 shows the molecular weight marker, and lane 2 shows the rat IL-1α monoclonal antibody (AN of the present invention).
OC1801) and lane 3 show molecular weight markers.

From the sum of the molecular weights of the heavy chain and the light chain by SDS-PAGE, the molecular weight of the antibody was 160 kd.

[0103]

[Reference Example 3] Production of anti-rat IL-1α polyclonal antibody 1 mg / rRT-IL-1α obtained in Reference Example 1
ml was dissolved in a PBS solution, and an equal amount of Freund's complete adjuvant solution was added thereto to prepare a suspension. Add this suspension to several rabbits (New-Zealand White Rabbit, weight 3.
Immunization was carried out by subcutaneously administering a single dose of 20 to 100 μg / rabbit (0 to 3.5 kg) as a rRT-IL-1α dose every 2 weeks. After immunization by administering 6 times, blood was collected from each rabbit to obtain antiserum.

From the above antisera, the method of Ishikawa et al. [J. Immuno
Ammonium sulfate fractionation and diethylaminoethyl-cellulose column fractionation according to assay, 4 , 209 (1983)].
gG was collected and purified by affinity chromatography using purified product rRT-IL-1α to obtain the desired rabbit anti-rat IL-1α polyclonal antibody.

In the same manner as above, rRT was performed on sheep (Suffolk breed, weight 50-60 kg) instead of rabbits.
-One dose of 100 μg / sheep as IL-1α amount is 2
Immunization was carried out by subcutaneously administering 7 times every other week, and desired sheep anti-rat IL-1α polyclonal antibody was obtained from each sheep.

Titer of anti-rat IL-1α polyclonal antibody Dilutions of each antibody obtained above and in various dilution ratios (diluted with 0.1% BSA and PBS) 100 μ
In 1 l, 10,000 cpm / 100 μl of ¹²⁵ I-labeled rat IL-1α (standard rat IL-1α labeled with Na ¹²⁵ I by the iodogen method) (0.1% BSA and PBS).
, Then 50 μl of 2% bovine γ-globulin and 0.
250% of 1% BSA and PBS were added, stirred and reacted overnight at room temperature. 25% PEG-6000 in the reaction product
PBS solution (500 μl) was added, stirred and left at room temperature for 1 hour. After further centrifugation at 3000 rpm for 15 minutes,
The supernatant was discarded and the radioactivity of the precipitate was measured with a γ-counter.

By the above, ¹²⁵ I-labeled rat IL-1α
The dilution ratio of the antiserum capable of binding to 50% of was determined and used as the titer.

As a result, the titer of rabbit anti-rat IL-1α polyclonal antibody was 256000, and the titer of sheep anti-rat IL-1α polyclonal antibody was 12.
It was 8000.

[0109]

Example 3 Production of Rat IL-1β Monoclonal Antibody of the Present Invention Production of Rat IL-1β Monoclonal Antibody The rat IL-1β obtained in Reference Example 2 was used as an immunogen (hereinafter abbreviated as “rRT-IL-1β”). Use as
A rat IL-1β monoclonal antibody was produced in the same manner as in Example 1.

That is, lymphocyte cells sensitized with rRT-IL-1β were prepared and fused with mouse myeloma cells, and the fused cells were cultured by an ELISA method using rRT-IL-1β as an immunogen. Cloning was performed by a limiting dilution method from wells tested and positive.

The above cloning was performed 5 times to obtain a hybridoma producing the monoclonal antibody of the present invention having a desired reaction specificity. This is "KOCO1901"
I named it.

The above clone KOCO1901 was cultured in the same manner as in Example 2 and then centrifuged to obtain a culture supernatant containing the desired monoclonal antibody, and a clone was selected.

The above-mentioned monoclonal antibody-producing clone KOCO1901 of the present invention has been deposited at the Institute for Microbial Technology, Ministry of International Trade and Industry (MICRO) with the designation "KOCO1901", and the deposit number is "MICO The first
2703 (FERM P-12703) ".

Preparation of ascites Ascites containing the antibody of the present invention was obtained from the hybridoma obtained above by treating in the same manner as in Example 1. The ascites was centrifuged at 1500 rpm for 5 minutes, and the pellet was added with RPMI-164 containing 10% DMSO and 10% FCS.
It was suspended in 0 medium and stored frozen. The rest of the supernatant was combined with 4 ascites fluids for subsequent purification.

Purification of the Monoclonal Antibody of the Present Invention Purification of the antibody of the present invention from the ascites fluid obtained above was carried out according to Example 1
The same procedure as in No. 2 was performed, and the dialysate obtained was used to
The antibody concentration was determined by measuring the absorption at 0 nm.

As a result, the purified antibody concentration was 1.53 mg /
It was ml.

[0117]

Example 4 Characteristics of rat IL-1β monoclonal antibody rRT-IL-1β obtained in Reference Example 2 as an immunogen
The following test results were obtained using.

Subclass of Antibody The subclass of the rat IL-1β monoclonal antibody of the present invention determined in the same manner as in Example 2 was IgG ₁ .

Antibody Production Level I of KOCO1901 in the culture supernatant when the hybridoma determined in the same manner as in Example 2 reached the maximum cell density.
The amount of gG ₁ was about 12 μg / ml.

SDS-PAGE The results of SDS-PAGE obtained in the same manner as in Example 2 are also shown in FIG.

Lanes 1 and 2 in the figure are rat IL-1β monoclonal antibodies of the present invention (ANOC1901).
Lane 3 shows the molecular weight marker.

From the sum of the molecular weights of the heavy chain and the light chain obtained by SDS-PAGE, the molecular weight of the antibody was 150 kd.

[0123]

[Reference Example 4] Production of anti-rat IL-1β polyclonal antibody 1 mg / rRT-IL-1β obtained in Reference Example 2
ml was dissolved in a PBS solution, and an equal amount of Freund's complete adjuvant solution was added thereto to prepare a suspension. Add this suspension to several rabbits (New-Zealand White Rabbit, weight 3.
Immunization was carried out by subcutaneously administering a single dose of 20 to 100 μg / rabbit (0 to 3.5 kg) as a rRT-IL-1β amount every 2 weeks. After immunization by administering 6 times, blood was collected from each rabbit to obtain antiserum.

From the above antisera, the method of Ishikawa et al. [J. Immuno
Ammonium sulfate fractionation and diethylaminoethyl-cellulose column fractionation according to assay, 4 , 209 (1983)].
gG was collected and purified by affinity chromatography using purified product rRT-IL-1β to obtain the desired rabbit anti-rat IL-1β polyclonal antibody.

In the same manner as above, rRT was performed on sheep (Suffolk breed, weight 50-60 kg) instead of rabbits.
-One dose of 100 μg / sheep as IL-1β amount is 2
Immunization was carried out by subcutaneous administration seven times every week, and desired sheep anti-rat IL-1β polyclonal antibody was obtained from each sheep.

Titer of anti-rat IL-1β polyclonal antibody Dilutions of each antibody obtained above and in various dilution ratios (diluted with 0.1% BSA and PBS) 100 μ
In 1 l, ¹²⁵ c-labeled rat IL-1β (standard rat IL-1β labeled with Na ¹²⁵ I by the iodogen method) was 10,000 cpm / 100 μl (0.1% BSA and PBS).
, Then 50 μl of 2% bovine γ-globulin and 0.
250% of 1% BSA and PBS were added, stirred and reacted overnight at room temperature. 25% PEG-6000 in the reaction product
PBS solution (500 μl) was added, stirred and left at room temperature for 1 hour. After further centrifugation at 3000 rpm for 15 minutes,
The supernatant was discarded and the radioactivity of the precipitate was measured with a γ-counter.

Based on the above, ¹²⁵ I-labeled rat IL-1α
The dilution ratio of the antiserum capable of binding to 50% of was determined and used as the titer.

As a result, the titer of rabbit anti-rat IL-1β polyclonal antibody was 16000, and the titer of sheep anti-rat IL-1β polyclonal antibody was 640.
It was 00.

[0129]

Example 5 Preparation of Standard Curve by ELISA Method Preparation of Standard Curve of Rat IL-1α In this example, the sheep anti-rat IL-1α polyclonal antibody obtained in Reference Example 3 was immobilized as the first antibody, and After reacting each standard solution in which the purified preparation rRT-IL-1α was dissolved, and further reacting the rabbit anti-rat IL-1α polyclonal antibody obtained in Reference Example 3 as the second antibody, HR was used.
React with P-labeled goat anti-rabbit IgG antibody and use o as a substrate
Solid phase ELIS based on the 3-step sandwich method of the present invention using phenylenediamine (OPD, Sigma)
This is an example of system A and was carried out as follows.

Sheep anti-rat IL-obtained in Reference Example 3
1α polyclonal antibody was diluted with PBS to 10 μg / ml of protein A purified product, and 100 μ was added to the immunoplate.
1 / well and dispensed at 4 ° C. for 2 hours, then 0.
Wash solution containing 10% Tween-20 (10 mM PB
S, pH 7.2) to wash away the antibody solution, then 0.2.
400 μl / well of 1% BSA and 0.14 M NaCl (pH 7.2) was dispensed, left standing at room temperature for 1 hour to block, and then the above washing solution 250 to 300 μl / well.
The wells were washed 3 times to remove 0.1% BSA and PBS to prepare a solid phase plate.

Then, add 150 to each well of the plate.
μl of reaction buffer [1M NaCl, 20% FCS,
25 mM PBS containing 1% BSA, 10 mM EDTA, 0.05% thimerosal and 0.05% CHAPS (3-[(3-chloramidopropyl) dimethylamino] -1-propanesulfonate, manufactured by Katayama), pH 6.
5] is added, and further 1.5 pg / ml-10000 pg / ml
100 μl / well of rRT-IL-1α antigen standard solution diluted to a predetermined concentration of 100 ml were overlaid and incubated at room temperature overnight for reaction. Furthermore, plate 250 ~
300 μl / well wash (0.05% Tween-2
0.1% BSA and PB after washing 3 times with 0 and PBS)
S, 5000 times, 10000 times, 20000 times and 40
100 μl / well of the rabbit anti-IL-1α polyclonal antibody solution diluted 000 times each was added, and the mixture was incubated at room temperature for 2 hours.

After further washing three times with the above washing solution, HRP
A labeled anti-rabbit IgG antibody solution (manufactured by Bio-Rad) was diluted 5000-fold with 0.1% BSA and PBS to give 10
0 μl / well was added and incubated at room temperature for 2 hours for reaction. Then, wash the plate with the same washing solution.
Washed twice, and finally 100 μl / well of substrate solution (o
-Add phenylenediamine (OPD) and 0.015% hydrogen peroxide solution, and react at room temperature for about 5-10 minutes.
The reaction was stopped by adding 100 μl / well of N-sulfuric acid solution.

The absorbance of the solution in each well at 492 nm was measured, a standard curve was prepared, and the amount of rat IL-1α in each sample was determined from this.

The above results are shown in FIG.

In the figure, the vertical axis represents the absorbance (OD) at 492 nm and the horizontal axis represents the rat IL-1α concentration.

From the figure, the EL of rat IL-1α of the present invention is shown.
It is clear that the ISA system can reproducibly draw a good dose-response curve between 20 pg / ml and 3000 pg / ml.

Preparation of standard curve for rat IL-1β In this example, the sheep anti-rat IL-1β polyclonal antibody obtained in Reference Example 4 was immobilized as a first antibody, and purified standard rRT-IL-1β was added thereto. After each dissolved standard solution was reacted and the anti-rat IL-1β monoclonal antibody of the present invention obtained in Example 3 was reacted as a second antibody, HR was used.
React with P-labeled goat anti-rabbit IgG antibody and use o as a substrate
Solid phase ELIS based on the 3-step sandwich method of the present invention using phenylenediamine (OPD, Sigma)
This is an example of system A and was carried out as follows.

Sheep anti-rat IL-obtained in Reference Example 4
The 1β polyclonal antibody was diluted with PBS to 10 μg / ml of protein A purified product, and 100 μ was added to the immunoplate.
1 / well and dispensed at 4 ° C. for 2 hours, then 0.
Wash solution containing 10% Tween-20 (10 mM PB
S, pH 7.2) to wash away the antibody solution, then 0.2.
400 μl / well of 1% BSA and 0.14 M NaCl (pH 7.2) was dispensed, left standing at room temperature for 1 hour to block, and then the above washing solution 250 to 300 μl / well.
The wells were washed 3 times to remove 0.1% BSA and PBS to prepare a solid phase plate.

Then, add 150 to each well of the plate.
μl of reaction buffer [1M NaCl, 20% FCS,
25 mM PBS containing 1% BSA, 10 mM EDTA, 0.05% thimerosal and 0.05% CHAPS (3-[(3-chloramidopropyl) dimethylamino] -1-propanesulfonate, manufactured by Katayama), pH 6.
5] is added, and 0.15 ng / ml to 333 ng / m is further added.
100 μl / well of rRT-IL-1β antigen standard solution diluted to a predetermined concentration of 1 were overlaid and incubated at room temperature overnight for reaction. In addition, plate 250-3
00 μl / well wash (0.05% Tween-20
And PBS) three times, then 0.1% BSA and PBS
100 μl / well of the anti-rat IL-1β monoclonal antibody solution of the present invention diluted to a predetermined concentration with was added and incubated at room temperature for 2 hours.

After further washing three times with the above washing solution, HRP
A labeled anti-rabbit IgG antibody solution (manufactured by Bio-Rad) was diluted 5000-fold with 0.1% BSA and PBS to give 10
0 μl / well was added and incubated at room temperature for 2 hours for reaction. Then, wash the plate with the same washing solution.
Washed twice, and finally 100 μl / well of substrate solution (o
-Add phenylenediamine (OPD) and 0.015% hydrogen peroxide solution, and react at room temperature for about 5-10 minutes.
The reaction was stopped by adding 100 μl / well of N-sulfuric acid solution.

The absorbance of the solution in each well at 492 nm was measured, a standard curve was prepared, and the rat IL-1β amount in each sample was determined from this.

The above results are shown in FIG.

In the figure, the vertical axis shows the absorbance (OD) at 492 nm, and the horizontal axis shows the rat IL-1β concentration.

From the figure, the EL of rat IL-1β of the present invention is shown.
It is clear that the ISA system can reproducibly draw good dose response curves between 2 ng / ml and 300 ng / ml.

[Brief description of drawings]

FIG. 1 shows a schematic diagram of construction of a rat IL-1α expression plasmid according to Reference Example 1.

FIG. 2 shows the nucleotide sequence of rat IL-1α cDNA region and the corresponding amino acid sequence.

FIG. 3 shows the nucleotide sequence of rat IL-1α cDNA region and the corresponding amino acid sequence.

FIG. 4 shows a schematic diagram of construction of a rat IL-1β expression plasmid according to Reference Example 2.

FIG. 5 is a restriction map of a plasmid containing a gene encoding rat IL-1β.

FIG. 6 shows the nucleotide sequence of rat IL-1β cDNA region and the corresponding amino acid sequence.

FIG. 7 shows the nucleotide sequence of rat IL-1β cDNA region and the corresponding amino acid sequence.

FIG. 8: SDS-PAGE of anti-rat IL-1α monoclonal antibody of the present invention and anti-rat IL-1β monoclonal antibody of the present invention determined according to Example 2 and Example 4.
The analysis results are shown.

FIG. 9 shows an ELISA system standard curve in measuring rat IL-1α according to the 3-step sandwich method shown in Example 5-.

FIG. 10 shows an ELISA system standard curve in measuring rat IL-1β according to the three-step sandwich method shown in Example 5-.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // C12N 5/20 15/06 (C12P 21/08 C12R 1:91) (72) Motoichi Anichi 35-6 Yashita, Sasakino, Matsushige-cho, Itano-gun, Tokushima Prefecture (72) Inventor Shoichi Adachi 3493-9 Ishihara-cho, Takasaki-shi, Gunma Prefecture

Claims (4)

[Claims] 1. A rat IL-1α or a rat IL-1 produced by a hybridoma formed by fusing mammalian immune cells immunized with rat IL-1α or rat IL-1β as an immunizing antigen and mammalian bone marrow cells. -1β
Anti-rat IL- characterized by having specific reactivity to
1 monoclonal antibody. 2. The antibody according to claim 1, wherein the hybridoma is KOCO1801 (Microtechnology Research Institute No. 12702). 3. The antibody according to claim 1, wherein the hybridoma is KOCO1901 (Microtechnology Research Institute, No. 12703). 4. An enzyme-linked immunosorbent assay, wherein the monoclonal antibody or anti-rat IL-1 polyclonal antibody of claim 1 is immobilized on a first antibody, and the monoclonal antibody or anti-rat IL-1 of claim 1 is immobilized. A method for measuring rat IL-1 which comprises measuring a rat IL-1 by a three-step sandwich method in which a labeled antibody and the above second antibody are reacted with a second antibody which is a polyclonal antibody.