JPS60246322A - Antibody to human interleukin-2 - Google Patents

Abstract

PURPOSE:An antibody capable of specifically recognizing partial structure on the terminal side of amino acid sequence of human interleukin-2 (IL-2) and making the purification and measurement (detection) of IL-2 possible. CONSTITUTION:An antibody obtained by using a complex of a peptide having at least 6 amino acid sequences in human interleukin-2 (IL-2), preferably a human IL-2 related peptide of formula I (R is H or H-Tyr-; X is peptide having 1-3 amino acids from the terminal N side of the peptide chain of -Thr-Lys-Lys-) with a carrier as an immunological antigen, cultivating fused cells formed from plasma cells of an animal immunized with the above-mentioned antigen or collecting an antibody formed by administration of the above-mentioned antigen to a mammal, and having reactivity toward decapeptide expressed by formula II.

Description

DETAILED DESCRIPTION OF THE INVENTION Contrary to 1-1 The present invention relates to novel antibodies against human interleukin-2.

Background technology Interleukin-2 (hereinafter abbreviated as rlL-2J) is
Conventional Tl1lf! Growth factor (TCGF), as it was also called, is a soluble protein that regulates lymphocyte reactivity and promotes long-term culture of lymphocytes in vitro (Swith, K, A, T- cell gr
low factor, lII*uno1. Rev
, vol. 151.337 (1980)). The IL-2 is typically produced by stimulating lymphocytes of various mammals with mitogens such as phytohemagglutinin (P)-IA), funcanavalin A (CONA), and Hawkelyde mitogen (PKW). Utilizing its biological activity, it can be used as a reagent to promote the proliferation of various Tll cells in vitro and in vitro, and to treat various immunodeficiency diseases and malignant neoplastic diseases caused by immunodeficiency Tll cells. It is being used in research on abnormal immune responses, such as, and in clinical clinics, such as.

The object of the present invention is to provide an antibody that enables the above-mentioned IL-2 semen-1 measurement (detection), particularly an antibody that has specific reactivity to human IL-2, and a method for producing the antibody. Another object of the present invention is to provide a specific IL-29il peptide that can be used as a hapten for the purpose of the present invention, and a technique for producing the above-mentioned IL-2 antibody using the same.

According to the present invention, there is provided an antibody that can specifically recognize the N-terminal partial structure of the amino acid sequence of human 1m-2. ! fl
1 provides an antibody against human IL-2 characterized by having reactivity with a decapeptide represented by the formula %.

The antibody of the present invention has the following amino acid sequence of human IL-2:
A peptide having a sequence of at least about 6 amino acids from the N-terminal side, preferably having the general formula % formula % (1) [wherein R represents a hydrogen atom or H-1r-, and X is -
Thr-LyS-1ys- indicates a peptide or amino acid residue having 1 to 3 amino acids counted from the N-terminus in the peptide chain. ] A complex of a human i[-2-related peptide represented by It is produced by fusing animal plasma cells with mammalian plasmacytoma cells, cloning the fused cells that produce antibodies against human IL-2, and collecting antibodies from the clones.

In this specification, when amino acids, peptides, protective groups, active groups, etc. are indicated by abbreviations, ILIPAC
, IUB regulations or common symbols in the field, examples of which are listed below. In addition, if there are optical isomers for amino acids, etc., unless otherwise specified, 1
The body shall be shown.

Tyr: Tyrosine Ala; Alanine Pro Nibroline Thr: Threonine 3er; Serine Ly
s: lysine 2; benzyloxycarbonyl group 3 oc; tertiary butoxycarbonyl group 0NII:
p-nitrophenyloxy group Bzl; benzyl group 0Bzl; benzyloxy group O8u: N-hydroxysuccinimide group OMe; methoxy group Tos; p-toluenesulfonyl basic Peptide represented by the above general formula 1) for production of the invention antibody uses easily available commercially available amino acids,
It can be easily synthesized with simple operations. In addition, the peptide can be used as a hapten to create an immunizing antigen, and when the antigen is used, antibodies having specific reactivity against IL-2 can be easily obtained in large quantities and stably. can. The antibody of the present invention thus obtained has the characteristic of being able to specifically recognize the amino acid sequence portion of the N-terminus (Ala) side of human IL-2, and can be used, for example, by binding it to a carrier for affinity chromatography. ,
By using this chromatography, etc., human
It can be used for the purification of IL-2, and as a specific antibody in various immunoassay methods for IL-2, thereby enabling clinical diagnosis and research of abnormal immune responses.

The chemical synthesis method for the peptide of the present invention will be described in detail below.

The peptide of the present invention can be synthesized using a conventional peptide synthesis method, as described in "The Peptides J Volume 1 (1966) (3 Chroder and L.
uhke@, Academic press, New
York, LJSA) or "peptide synthesis" according to the methods described in Azumaya et al., Maruzen Co., Ltd. (1975), for example, the azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, Active ester method (p
-Nitrophenylene ester method, N-hydroxysuccinimide ester method, cyanomethyl ester method, etc.), method using Woodward reagent K, carbodiimidazole method, redox method, DCC/additive (HONB, H
It can be manufactured by the OBt 1 HO 8 u ) method. In the above method, both solid phase synthesis method and liquid phase synthesis method can be applied. Usually, the peptide of the present invention is coupled according to the general polypeptide synthesis method described above, for example, by the so-called stepwise method in which amino acids are sequentially condensed one by one to the terminal amino acid, or by dividing into several 7-ragments and coupling them. Reported by method.

In any of the above methods, each amino acid having a side chain functional group, such as Tyr, Thr, Ser, etc., preferably has its side chain functional group protected. Afterwards the protecting group is removed. Furthermore, the functional groups involved in the reaction are usually activated. These reaction methods are well known, and the reagents used therein can be appropriately selected from known methods.

Examples of protecting groups for amino groups include benzyloxycarbonyl, Boc, tctrt-amyloxycarbonyl, isobornyloxycarbonyl, ρ-methoxybenzyloxycarbonyl, 2-lyloropenzyloxycarbonyl, adamantyloxycarbonyl, Examples include trifluoroacetyl, phthalyl, formyl, 0-nitrophenylsulfenyl, diphenylphosphinothioyl groups, and the like.

Examples of carboxyl protecting groups include alkyl esters (e.g. methyl, ethyl, propyl, butyl, tert
-alkyl esters such as butyl), [3zl ester, p-nitrobenzyl ester, p-methoxybenzyl ester, p-chlorobenzyl ester, benzhydryl ester, carbobenzoxyhydrazide, tert-
Examples include groups that can form butyloxycarbonyl hydrazide, trityl hydrazide, and the like.

The hydroxyl groups of Thr and Ser can be, but do not necessarily need to be protected, for example by esterification or etherification. Groups suitable for this esterification include, for example, lower alkanoyl of an acetyl group, aroyl such as benzoyl, and groups derived from carbonic acid such as benzoyloxycarbonyl and ethyloxycarbonyl. Examples of groups suitable for etherification include benzyl, tetrahydropyranyl, and tert-butyl groups.

Tyr's water! ! Examples of protecting groups include Bzl, C
l 2-BZI, benzyloxycarbonyl, acetyl, TO3 group, and the like.

Examples of the protecting group for the amino group of Lys include benzyloxycarbonyl group, CI Z, C12-Bzl, 30
Examples include C, 70S groups, and the like.

Examples of activated carboxyl groups include the corresponding lorides, acid anhydrides or mixed acid anhydrides, azides, active esters (pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenz esters with triazole, N-hydroxy-5-norbornene-2,3-dicarboximide, etc.).

In the above method, the condensation reaction (peptide bond formation reaction) between the reactive amino group and the reactive carboxyl group can be carried out in the presence of a solvent. As solvents, various solvents known to be usable for peptide bond formation may be used, such as anhydrous or hydrous dimethylformamide, dimethyl sulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, ethyl acetate, N-methylpyrrolidone, hexane. Methyl phosphoric acid triamide or a mixed solvent thereof may be used. There is no particular limitation on the ratio of the raw material compounds used, but it is usually an equimolar amount to 5 times the molar amount of one to the other, preferably an equimolar amount to 1.5 times the molar amount of the other. The reaction temperature is within the usual range used for peptide bond formation reactions, generally from about -40°C to about 60°C.
, preferably from the range of about -20°C to about 40°C. The reaction time is generally from several minutes to about 30:1Ii1.

When employing the mixed acid anhydride method, in the presence of a basic compound in a suitable solvent, an alkyl halocarbon, such as methyl chloroformate, methyl bromoIate, methyl chloroformate, ethyl bromoIIate, chloro This is carried out using isobutyl formate or the like. Examples of basic compounds include triethylamine, trimethylamine, pyridine, dimethylaniline, N-methylmorpholyl, 1,5-diazabicyclo(4,3,0)nonene-5 (DBN), 1,5-diazabicyclo(5,4,0 ) Undesen-5 (DBtJ),
1,4-Diazabicyclo(2,2゜2)octane (DA
Organic bases such as BCO) and invisible bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate can be used.

In addition, WJts includes various solvents commonly used in the mixed acid anhydride method, specifically halogenated hydrocarbons such as methylene chloride, chloroform, and dichloroethane, aromatic hydrocarbons such as benzene, toluene, and xylene, diethyl ether,
Ethers such as tetrahydrofuran and dimethoxyethane, esters such as methyl acetate and ethyl acetate, aprotic polar solvents such as N,N-dimethylformamide, dimethyl sulfoxide, and hexamethylphosphoric triamide, etc. can be used. The reaction is usually carried out at -20 to 100°C, preferably -20 to 50°C, and the reaction time is generally 5 minutes to 10 hours, preferably 5 minutes to 2 hours.

In addition, when adopting the azidation method, first, activated carboxyl groups, for example, activated carboxyl groups with alcohol such as methyl alcohol, ethyl alcohol, benzyl alcohol, etc., are reacted with hydrazine hydrate in a suitable solvent. This is done by As the solvent, for example, dioxane, dithylformamide, dimethylformamide, or a mixed solvent thereof can be used. The amount of hydrazine hydrate to be used is usually 5 to 20 times, preferably 5 to 10 times, by molar amount relative to the activated carboxyl group. The reaction is usually carried out at 50°C or lower, preferably at -20°C.
Performed at ~30°C. Compounds in which the carboxyl group moiety is substituted with hydrazine (hydrazine derivatives)
can be manufactured.

A compound in which the carboxyl group moiety is substituted with azide is produced by reacting the hydrazine derivative obtained above with a nitrite compound in a suitable solvent in the presence of an acid. Hydrochloric acid is usually used as the acid. As the solvent, for example, dioxane, dimethylformamide, dimethyl sulfoxide, or a mixed solvent thereof can be used. Further, as the nitrite compound, for example, sodium nitrite, isoamyl nitrite, nitrosyl chloride, etc. can be used. Such a reduced nitrate compound is generally used in an amount of equimolar to 2 times the molar amount of the hydrazine derivative, preferably in an amount of equimolar to 1.5 times the molar amount. The reaction is usually carried out at -20 to 0°C, preferably at -20°C.
The reaction is carried out at -10°C and generally completes in about 5 to 10 minutes.

The peptide bond forming reaction can also be carried out in the presence of a condensing agent such as dicyclohexylcarbodiimide (DCC), a carbodiimide reagent such as carbodiimidazole, or tetraethylpyrophosphine.

In each of the above reaction steps and in the final step, when it is necessary to remove the protecting group, this is carried out according to a conventional elimination reaction. Examples of such methods include hydrogenation using a catalyst such as palladium or palladium black, reductive methods such as reduction with metallic sodium in liquid ammonia, trifluoroacetic acid, hydrogen fluoride on the side of chloride, methanesulfonic acid, and hydrobromic acid. Examples include acidolysis using strong acids such as

Hydrogenation using the above catalyst can be carried out, for example, at a hydrogen pressure of 1 atm, 0
It can be carried out at ~40°C. The amount of catalyst used is usually 1
It is best to use 1 to 1 g of 100I, and generally 1 to 48
The time interval ends. In addition, the acidolysis mentioned above is
Without solvent, usually about 0 to 30°C, preferably about 0 to 20°C
The process takes approximately 15 minutes to 1 hour. The amount of acid used is usually about 5 to 10 times the amount of the raw material compound. When only the 7-mino group is removed in the acidolysis, trifluoroacetic acid or hydrochloric acid is preferably used as the acid. Further, the above reduction with metallic sodium in liquid ammonia can be carried out using the highest metallic sodium such that the reaction solution is colored permanent blue for about 30 seconds to 10 minutes, usually at about -40°C to -70°C. .

The peptide of general formula (1) produced as described above can be separated from the reaction mixture by a means such as extraction, distribution, etc.
It is isolated and purified by column chromatography or the like.

In this way, the IL-2-related peptide of the present invention represented by general formula (1) is obtained.

The peptide represented by the above general formula (1) of the present invention can be used as a hubten of an immunizing antigen for obtaining an antibody having specific reactivity with human ll-2. In addition, in various immunoassay methods for human IL-2 using the above-mentioned antibodies, such as radioimmunoassay (RIA) method or enzyme immunoassay (EIA) method, the peptide of the present invention can be used with radioactive substances such as 1251, 131 1, etc. , peroxidase (POX), chymotrypsinogen,
BuO carboxypeptidase, glyceraldehyde-3
-Phosphate dehydrogenase, amylase, phosphorylase, D
-Nase, P-Nase, β-galactosidase, glucose-6-7 osphaate dehydrogenase, ornithine decarboxylase, and other enzyme reagents can be introduced and used as labeled antigens. The above-mentioned radioactive substance can be introduced by a conventional method, for example, the oxidative iodination method using chloramine T (W, M, Hunter and
F, C, Greenwood; Nature, 19
4. 495 (1962), BiochemJ, 8
144, (1963)) etc.,
The enzyme reagent can be introduced using conventional coupling methods, such as the method of Erlanger et al.
Acta, Endocrinol, suppl
,, 168.

206 (1972)) and Force Roll (M, H.

Karol et al.'s method (Proc, Natl, Acad
, Sc1. .

USA, 57. 713 (1967)).

The labeled peptide thus produced is produced into a single mts by conventional separation means such as extraction, partitioning, column chromatography, dialysis, etc. The labeled peptide thus obtained can be lyophilized and stored if necessary.

Hereinafter, a method for producing an IL-2 antigen (immunization antigen) using the peptide of the present invention represented by the above general formula (1) as a hubten will be described in detail.

The peptide of the present invention is used as an immunizing antigen by reacting with a suitable carrier in the presence of a hubten-carrier binding reagent. As the carrier, eight molecules of natural or synthetic proteins commonly used in the preparation of antigens can be widely used. Examples of the carrier include primary serum albumins such as horse serum albumin, bovine IIh serum albumin, rabbit serum albumin, human serum albumin, and sheep serum albumin; horse serum globulin, bovine serum globulin, rabbit serum globulin, Self-extinguishing animal purines such as human serum globulin and sheep serum globulin: horse thyroglobulin, bovine thyroglobulin,
Animal thyroglobulins such as rabbit thyroglobulin, human thyroglobulin, and ovine thyroglobulin; Animal hemoglobulins such as equine hemoglobulin, bovine hemoglobulin, rabbit hemoglobulin, human hemoglobulin, and ovine hemoglobulin; keyhole linbet Hemocyanin (KL
Proteins extracted from hemocyanin roundworms of animals such as I-1 (Ascaris extract, JP-A-56-1641)
Publication No. 4, J, [mmun,, 111. 260-26
8 (1973), J. Iuun, 122.

302-308 (1979), J, riimu
n.

98. 893-900 (1967) and AIl, J.
.

Physiol, 199. 575-578 (1
Polylysine, polyglutamic acid, lysine-glutamic acid copolymers, copolymers containing lysine or ornithine, and the like can be mentioned.

As the hubten-carrier binding reagent, a wide variety of those commonly used in the preparation of antigens can be used.

Specifically, tyrosine, histidine, and tryptophan are cross-linked, for example, bisdiazotized benzidine (
Diazonium compounds such as BDB), bisdiazotized-3゜3'-dianisidine (BDD); Aliphatic dialdehydes such as aldehydes:
Cross-linking between thiol groups, for example N
, N'-o-phenylene dimaleimide, N , N'-
11-Phenyleimide compounds such as phenylene dimaleimide; crosslinking amino groups and thiol groups, such as metamaleimidobenzoyl-N-hydroxysuccinimide ester, 4-(maleimidomethyl)-cyclohexane-1-carboxyl-N'-hydroxysuccinimide Maleimidocarboxyl-N-hydroxysuccinimide esters such as esters; reagents used in the usual peptide bond-forming reaction to form an amide bond between an amino group and a carboxyl group, such as N,N-dicyclohexylcarbodiimide, N-ethyl-N'-dimethyl Aminocarbodiimide-3-diisopropylaminocarbodiimide, 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)
Examples include dehydration condensation agents such as carbodiimides such as carbodiimide. Further, as the hubten-carrier binding reagent, a combination of a diazonium arylcarboxylic acid such as p-diazonium phenylacetic acid and a conventional peptide bond-forming reaction reagent, such as the dehydration condensation agent described above, can also be used.

The above antigen production reaction can be carried out, for example, in an aqueous solution or at pH 7 to
The reaction is usually carried out in a normal buffer solution at 0 to 40°C, preferably around room temperature, for about 1 to 24 hours. Through the above process, a desired antigen consisting of a peptide-carrier complex in which a carrier and habten are bound is obtained through the mediation of a habten-carrier binding reagent. The antigen obtained from the reaction bale can be easily purified by conventional methods such as dialysis, gel filtration, and fractional precipitation.
Ill purification is possible.

Human IL-
The production of antibodies having reactivity with 2 is carried out, for example, by administering the above-mentioned antigen to a mammal, causing the desired antibody to be produced in vivo, and collecting the antibody. The present invention provides such IL-
The present invention also provides a method for producing antibodies against 2. There are no particular restrictions on the mammal used for antibody production, but it is usually preferable to use rabbits, goats, guinea pigs, and the like. For antibody production, a predetermined amount of antigen is diluted with physiological saline to an appropriate concentration, and 70% Indian auxiliary solution (Comp
let's rend 's Adjuvant
) to prepare a suspension, which may be administered to a mammal for immunization. The immunization can be carried out, for example, by intracavernally injecting the above suspension into rabbits (antigen amount: 0.5 to 5 g/dose).
Thereafter, the drug is administered every 2 weeks for 2 to 10 months, preferably 4 to 6 months. Antibodies are collected by collecting blood from the immunized animal at the time when antibodies are produced multiple times after Iin administration of the suspension, usually 1 to 2 weeks after the final administration, and centrifuging the blood to separate the serum. This is done by sampling. According to the above, based on the specificity of the antigen used, it is possible to obtain an antibody with excellent specificity for human IL-2, high titer, and high sensitivity.

The present invention also provides a mammal immunized with the above-mentioned immunizing antigen.
li cells (immune cells), mammalian plasma cells 111B
Fuse with the cyst to create a pibridoma, and from this IL
The present invention also provides a method for selecting a clone that produces an antibody against IL-2 and obtaining an antibody (monoclonal antibody) against IL-2 from the clone.

The mammal to be immunized above is not particularly limited, but is preferably selected in consideration of compatibility with the plasmacytoma iv used for cell fusion, and mice, rats, etc. are generally used. The immunization method can be based on the method described above, and it is preferable to use 1/8 spleen Wi extracted about 3 days after the final immunization as the immune cells.

In addition, as the mammalian plasmacytoma cell as the other parent cell to be fused with the above immune cell, various known cell lines such as I) 3 (+1 3/X63-AU
8) (Nature, 256, 495-497 (
1975)), D3-Ul (CurrentT
opics in Microbiology an
dIsiunolooy, 81:1-7 (19
78) ), NS-1 (Eur. J. Immuno
l. , 6: 51 1-519 (1976)), M.
PC-11 (Cell.

8; 405-415 (1976)), SP210 (
Nature, 276; 269-27ON978))
, FO (J. Iawunol. Meth.

35; 1-21 (1980)), x63.6.

5, 3. (J. Immunol., 1 23 +
1 548-1550 (1979)), 8194
L.J. Exp.

Med. , 148:313-323 (1978))
W, R 2 1 0 in rats (Nature
, 277:131-133 (1979)) and the like are used.

The above-mentioned fusion reaction between the immune cells and the plasma cell Ill cells is basically carried out using known methods such as Milestein (Mllstein).
Method in Enzyio et al.
logy, vol, 73. DD3 (198
1) ), 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 plasmacytoma cells used is the same as in normal methods; for example, about 1 to 10 times as many immune cells as plasmacytoma cells may be used. The medium for the fusion is RPMI-1640, such as that used for the growth of the plasmacytoma cell line.
Medium, MEM medium, and various other usual media used for this type of cell culture can be used, usually tallow-engorged Wi (F
It is best to use self-extinguishing fluids such as e2). Fusion is carried out by thoroughly mixing a predetermined amount of the immune cells and plasmacytoma cells in a supernatant medium, and adding a PEGI solution pre-warmed to about 37°C, for example, one with an average molecular weight of about 6,000, to a normal medium. This is done by adding and mixing at a concentration of about 30 to 60 W/V%. By repeating the steps of sequentially adding an appropriate medium, centrifuging, and removing the supernatant, the desired fusion II hybridoma is formed.

The resulting desired hybridomas are isolated by culturing them 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 search for the producing strain can be performed, for example, by the ELISA method (Enova
l, E, , Meth, Enzyiol, ,
7 Q.

419-439 (1980)), plaque method, spot method, agglutination reaction method, OCT method, RIA method, etc. [``Hybridoma method and monoclonal antibodies''] , published by R&D Planning Co., Ltd., pp30-53,
March 5, 1982]. More specifically, for example, a plate coated with purified IL-2 or the peptide represented by the general formula (1) of the present invention is used, and this is reacted with the culture supernatant of a test hybridoma to obtain the purified IL-2.
An example of a method is to confirm the presence of an antibody that binds to 2 or a peptide by reacting with enzyme-labeled anti-mouse immunoglobulin (if a mouse is used for fusion) according to a conventional method, and to select a desired antibody-producing strain. Ru.

The thus obtained hybridoma producing a monoclonal antibody against IL-2 can be subcultured in a conventional medium and can be easily stored for a long period of time in liquid nitrogen.

Representative examples of this hybridoma are shown in Examples below, and these are maintained by the present inventors in a distributable state.

11 from specific hybridomas obtained as above.
-2 antibody can be collected by culturing the hybridoma according to a conventional method and separating the desired antibody from the culture supernatant, or by administering the hybridoma to a compatible mammal, allowing it to grow, and collecting the desired antibody from its ascites. Methods such as separating antibodies are adopted. 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, as shown in the test examples below, IL-
A monoclonal antibody having reactivity with 2 is obtained.

The present invention also provides the human IL-2 antibody obtained as described above, which has the characteristic of specifically recognizing the amino acid sequence site on the N-terminal side of human IL-2. Therefore, this antibody can be used, for example, in the RIA method to detect human lm.
-2 constant R can be achieved with high accuracy. Furthermore, the antibody can be used in enzyme immunoassay (EIA) 1, fluorescence immunoassay (FIA), etc. by labeling it with an enzyme or a fluorescent substance.

Furthermore, the antibody can be made into an insolubilized antibody by reacting with a known insolubilizing substance.

In order to explain the present invention in more detail, examples of the production of the peptide of general formula (1), an antigen using the same, and an antibody from the antigen will be given below, but the present invention is not limited thereto.

In addition, in each production example, each amino acid shows the L form. Further, Rflia was measured by thin layer chromatography on silica gel using the following mixed solvent.

Rf +-CHC93-mer9/-le (15:1)Rf
2...CHCQ 3-methanol (9:1) Rf3.
・・CHCQ3-methanol-acetic acid (9: 1: 0.
5) Rrt...n-butanol-vinegar-pyridine-water (4
: 1: 1: 2) Rf5...n-butanol-acetic acid-pyridine-water (1
5: 3:10:12) Production of peptide> 1 Preparation example 1 1) Z(OMe)-Lys(Z)-Lys(Z)-Q
Production of 3zl H-Lys(Z)-0Bzl・Tos-OH (7.6g
) was dissolved in a mixture of dimethylformamide (DMF) (5IIg>) and tetrahydrofuran (THF) (50m) and triethylamine (Et s N) <1.9+ under water cooling.
+a) and add Z (OMe)-Lys to this solution.
(Z)-OH(6,Oo), butanol (Bt 0H)
(1,8a) and dicyclohexylcarbodiimide (
DCC) (7,6Q') are added sequentially and stirred at room temperature for 15 hours. The reaction solution was treated with tP, PI was concentrated under reduced pressure,
The pickles were extracted with ethyl acetate, and the ethyl acetate layer was washed sequentially with 10% citric acid, 5% sodium bicarbonate (NaHCOs), and saturated saline (NaC9) water, and then dried over anhydrous sodium chloride (Na2304). , concentrate under reduced pressure. The residue is recrystallized from ethyl acetate and petroleum ether to obtain the target peptide.

Yield 8.2J (yield 76.6%) a+p, 75-78°C, Rf 1=0.75, [α)1
2--4.0° (c-1,0,DMF)l) 2) Boa-Thr(Bzl)-Lys(Z)-LY
Preparation of s(Z)-0Bzl Z (OMe)-Lys(Z)-Lys'(Z)-O
Bzl (8,00) in the presence of anisole (51Q),
Treat with TFA (20m+> for 40 minutes at 0°C. Diethyl ether (Et2o>-petroleum ether (1:1)
) and dry the resulting oil under reduced pressure. This is T
Dissolved in HF (20 Wtl), ET s N (1,
4m? ) to this water-cooled solution, Boc-Thr (
Bzl)-0H mixed acid anhydride (Bo
Add c-Thr(Bzl)-OH(3,Hl) and isobutyl chloroformate (adjusted from 1,3m>),
Stir for an hour. The solvent was distilled off under reduced pressure, the residue was extracted with ethyl acetate, and the washing procedure was then carried out according to 1).
The desired peptide is obtained by recrystallization from ethyl acetate and petroleum ether.

Yield 6.20 (67,1%) IRll, 150-152°C, Rf +-0,79,3
) Preparation of Boc-8er (Bzl)-8er-OBzl H-3er-OBzl-Tos-OH (13,1a
) was dissolved in THF (200 m), and Ets was added under water cooling.
Neutralize with N (4.7 ml) and add Boc- to this solution.
3er(Bzl)-OH(10,OQ), Bt 0
f-1 (4.6 g) and DCC (7,0Q) were added sequentially and stirred at room temperature for 18 hours. The reaction solution is filtered, and the filtrate is concentrated under reduced pressure. The residue is purified according to 1) above to obtain the target peptide.

Yield 12. Oo (75,0%) Garden p, 80-83 ℃, Rf 2-0. 82
, (a), -+1.6° (c-1,9, MeOH)4
) Production of Boc-8er (Bzl) -3er-NHNH2 Boa-8er (Bzl) -8er-OBzl (4
,7g) in MeOH (30m12) and diluted to 80%
Add hydrazine hydrate (5-) and incubate at room temperature for 24 hours!
! ! do. The precipitated crystals are collected, washed with water, and dried to obtain the desired peptide.

Yield: 14.0g (85.1%) -0,147~149.5°C, Rf3~0.
27, [α)13−+ 5,9° (c−1,7
, DMF) 5) Boa-8ar (Bzl)-8er-
Thr(Bzl)-Lys(Z)-Lys(Z)
-0BzlノfJ Boc-Thr (Bzl) -
Lys(Z)-Lys(Z)-OBzl(6,
Oo) in the presence of anisole (3IQ) with TFA
(15 ml) at 0° C. for 1 hour. Excess TFA
is distilled off under reduced pressure, and the resulting oil is washed with hexane and dried under reduced pressure.

Dissolve this in DMF (51111) and Et3N (0
, 9G), and added 3oc-8et (B
zl) 5er-N3(Boc-3er(Bzl)Se
r-NHNH2 (prepared from 2,6 g)) in DMF solution (
5-) and stirred at 4°C for 48 hours. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in 10% citric acid, 5% NaHCO
After sequentially washing at S and +20, dry. Ethyl acetate-M
The target peptide is obtained by reprecipitation from eOH and petroleum ether.

Yield 4.9g <63.6%) mp, 132-134°C, Rf I -0,74, (
α), -+3.8° (c-1,L DMF)6) B
oc-8er(Bzl)-8er(Bzl)-8er-
Thr(Bzl) -Lys(Z) -Lys(Z
) -Production of Q3zl Protected pentapeptide ester (4,5
g) in the presence of anisole (3WIJ), TFA (
10) at 0°C for 1 hour. The precipitate formed by adding ether is collected and dried under reduced pressure. This powder is D
Dissolved in MF (2011G), Et 3 N (0,
512), and added Boc-8er (Bzl) to this solution.
) -0Su (1,5g) and Et a N (0,5
+i2) and stirred at room temperature for 24 hours. After distilling off the solvent under reduced pressure and washing the remaining residue according to 5), CHCQ
The target peptide is obtained by reprecipitation from a and petroleum ether.

Yield 114.0!1 <76.9%) Ill, 167-169. 5°C, Rf + ~0.7
2, [α), -+7.4° (c-1, L DMF)
7) Boc-Thr (Bzl) -8ep (Bzl)
-8er(Bzl)-3er-Thr(Bzl)-Ly
Production of S(Z 1-Lys(Z)-0BZI) Protected hexapeptide ester (4, Oa
) is treated with TFA according to 6). The obtained powder was dissolved in DMF (10Cl), and Et 3 N
(0,81111> and Boc-Thr(Bzl)-0S
Add u (1,20) and stir at room temperature for 48 hours. The solvent is distilled off under reduced pressure, and the residue is washed according to 5), followed by reprecipitation from acetic ether-MeOH and ether to obtain the target peptide.

Yield 13.1 (81.1%) 1.188-190°C, Rf + ~0.76, (α)
12, +3, 2° (c-1,9, DMF)6
Amino acid analysis value of N-H09 hydrolyzate: Thr2.1
3.5er3.07, LyS2.0O (lj's recovery rate, 80.0%).

8) Production of Boc-Ala-pro-Q)l Et 3
80% aqueous dioxane (1
00m), dissolve Pro (5,39>) and add Bo to this.
Add a-Ala-ONp (16.0 g) and stir at room temperature for 4 hours.
Stir for 8 hours. The reaction solution was concentrated under reduced pressure to reduce the residual amount to 5%.
After dissolving in Na HCO3 and washing with ethyl acetate, the aqueous layer is acidified with citric acid and the resulting oil is extracted with ethyl acetate. The ethyl acetate layer is washed with water, dried, and concentrated under reduced pressure, and the residue is recrystallized from ethyl acetate and petroleum ether to obtain the desired veptidide.

Yield 10. OQ (74,1%) Ill, 125-127℃, Rf a-0,46, [
α), -97,4°C (C=1.6, MeOH) 9) Boc-Ala-Pro-Thr (Bzl)-8
er(Bzl) -8er-Thr(Bzl) -Ly
Production of s(Z)-Lus(Z)-0Bzl 6) The protected heptapeptide ester (3,0111) obtained in 7) above
Treat with TEA according to .

The obtained powder was dissolved in DMF (10g1), neutralized with Et3N (0,311!+) under water cooling, and Boc-Ala-Pro-OH (0,6a) was added to this water-cooled solution.
, Bt OH (0,3!It) and DCC (0,5o
) and stirred at room temperature for 48 hours. The reaction solution was concentrated under reduced pressure, and the residue was washed according to 5).
Recrystallize twice from OH to obtain the target peptide.

Yield 2.2o (66.1%) mp, 180-183℃, Rf 2-0.73, [α
)D--8,6° (c-1,6,DMF)6NHCQ
Amino acid analysis value of hydrolyzate: Thr2.10.5e
r3.02, Proo, 82, AIao, 84, Lys
2.0O1 (Lys recovery rate 287.0%).

10) Z-Tyr-Ala-Pro-Thr (Bzl
)-8er(Bzl)-8er(8zl)-8ep
-Thr(Bzl)-Lys(Z)-Lys(Z)-
Production of OBzl The protected nonapeptide ester (2, Oa
) is treated with TFA according to 6).

The obtained powder was dissolved in DMF (!M) and Ej 3
Neutralize with N (0,15all) and add Z to this ice-cold solution.
-Tyr-N3 (Z-Tyr-NHNH2(0,5o
)] DMF solution (5-) and Ej 3N (
0.2 m) and stirred at 4°C for 24 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (elution: CHC93MeOH-40:
1) Obtain the target peptide.

Yield 1. Oo (43.3%) 111.173-175°C, Rf 2 -0. 42
, (α door--9.7℃ (c-1,0, DMF) 6Nl
Amino acid analysis value of -1cQ hydrolyzate = Thr2.14
．． 5er2.99, Prol, 09, Alal, 00.
Tyre, 94, LVSL, 90 (Ala recovery rate, 8
5.5%).

11) H-Tyr-Ala-Pro-Thr-8er
-8er -S er -T hr -L ys-L
Production of ys-OH The protected decapeptide ester (200+ao) obtained in 10) above was mixed with metacresol (0.1mf).
2) in the presence of 1M trifluoromethanesulfonic acid (TFMSA)-thioanisole/TFA (3-),
Treat at 0°C for 2 hours. Ether was added, the resulting precipitate was collected, and H1! After dissolving in O(30112), Amberlite I RA-4
00 (acetate type) for 30 minutes at room temperature, and the solvent was distilled off under reduced pressure. The residue was dissolved in a small amount of 0.2N acetic acid, and Sephadex G-15 (2,
Qx80C1m) column (eluent: 0,2N acetic acid) to purify @-7yr-AIa-Pro-Thr.
-S er -S er -S er-T hr -L
ys-Lys-OH is obtained. This will be referred to as "peptide B" hereinafter.

Yield 98.7ao (82.3%) Rf t -0,11, Rf 5-0.506NHC1
;! Amino acid analysis value of hydrolyzate = Thr2.04.5
er2.89, Prol, 04, Alal, 00. Ty
re, 96, Lysl, 93° Production example 2 1) Boa-8er (Bzl) -3er-OBz
Production of H-8er-OBZI-Tos・OH (13,
1+1) in THF (200 m2), Fj
After neutralizing with 3N (4,711Q), Boc-8er
(Bzl) -OH(10,OQ), Bt0H(
4,69) and DCC (7, Oa) were added sequentially and stirred at room temperature for 24 hours. The reaction solution was filtered, the furnace solution was concentrated under reduced pressure, the residue was extracted with ethyl acetate, and 10% citric acid,
Wash sequentially with 5% Na HCOs and saturated NaC9 water. After drying, ethyl acetate is distilled off under reduced pressure, and the residue is reconstituted from ethyl acetate-petroleum ether to obtain the target peptide.

Yield 12. OQ (75,0%) If), 78-83°C, Rf 2-0.82, ((Z is -+1.6" (C-1,9, Me 0H) 2) Bo
a-8ep(8zl)-8er(Bzl)-8er-O
Production of B zl Boc-8er (Bzl) -8er-OBzl (4
,7g) in the presence of anisole (31111), TF
Treat with A(9,4IQ) for 60 minutes in a water bath.

TFA is distilled off under reduced pressure and dried under reduced pressure. This oil was dissolved in DMF (519) and Et 3N (1,
39WIJ) and added Boc-8er (B
zl)-OH mixed acid anhydride (Boc-5er(Bzl
) -OH (3,09) and isobutyl chloroformate (1,
310) and 411i1! under cooling. Stir. The reaction solution was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, washed and dried according to 1), the solvent was distilled off under reduced pressure, and the residue was recrystallized from ethyl acetate-petroleum ether to obtain the target peptide. obtain.

Yield 5.4a (83.1% ip, 92-101°C, Rf 2-0.78(cr)
a -7,3° (c-1,4, Me 0H)3) B
oc-Thr(Bzl)-8er(Bzl)-8er(
Bzl) -8ep-Production of OBzl8oc-8er(
8zln-8er(Bzl)-8er-OBzl(4,
0g)+72')-ruf) in the presence of TFA (81fl
) for 40 minutes. Then, the process was carried out according to 2), the obtained oil was dissolved in DMF (6-), and Et 3
Neutralize with N (0.9 m) and add 8 oc-Thr to this solution.
(8zl>-0H mixed acid anhydride (Boc-Thr(B
zl) prepared from -OH (1,9+1) and isobutyl chloroformate (0,8I112)) and stirred for 4 hours under cooling. Thereafter, the target product is isolated according to 2) and recrystallized from ethyl acetate-petroleum ether to obtain the target peptide.

Yield 2.5o (48.1%) ■p, 132-136℃, Rf 2-0.84, ((Z
)D-+4. Oo(Q-1,7,MeOH)4) B
Production of oa-Ala-Pro-OH H-Pro-OH (
5,30) to 80% including EC3N(13,90>
Dissolved in aqueous dioxane (100m+), Boc-A
Add Ia-ON p (16, Of; J) and stir at room temperature for 48 hours. The reaction solution was concentrated under reduced pressure, and the residue was
% NaHCOs, washed with ethyl acetate, the aqueous layer was acidified with citric acid, the resulting oil was extracted with ethyl acetate, the ethyl acetate layer was washed with saturated NaOQ water, dried, and the solvent was evaporated under reduced pressure. The residue is recrystallized from ethyl acetate-petroleum ether to obtain the target peptide.

Yield 10. O(J (74,1%) mp, 124-127 °C, Rf s -0,46, (
α) −−97,4° (c-1,6, MeOH) 5) Production of Z-Tyr-Ala-Pro-OH Boa
-Ala-Pro-OH (4,0Q) is treated with TFA (8m)'t' for 40 minutes in the presence of anisole.

According to WI2), the obtained oil was mixed with DMF (10 m
) k, dissolved and neutralized with EtaN (2,0119), and the water-cooled solution was dissolved into azide (Z-Tyr-NHNH2 (4,8
o)) in DMF solution (191Q),
Stir at 4°C for 48 hours. The reaction solution is concentrated under reduced pressure, the residue is extracted with ethyl acetate, and the ethyl acetate layer is washed successively with 10% citric acid and saturated NaCQ water, and then dried. Ethyl acetate was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: CHCQ 3 Me
OH+20-90+15:5) to obtain the target peptide.

Yield 12.50 (36.2%), amorphous powder, R13-
0,45 [α)22--50.2° (c-2,3, MeO)1
) 6) Z-Tyr-Ala-Pro-Thr (Bzl)
-3er(Bzl) -8er(Bzl) -8er
-Production of OBzl Boa-Thr (Bzl) -3er (Bzl) -8
er(8zl) -8ar-08zl (1,79>) is treated with TFA (5m) in the presence of anisole for 60 min. Et20-petroleum ether (1:11) is added and the resulting precipitate is washed with petroleum ether. , dry. Dissolve this in DMF (511Q) and prepare Et, N (0,2
Z-Tyr-Ala-
Pro-OH(1,OQ), BtOH(0,29o
) and DCC (0.43 g) were added, and after stirring at room temperature for 48 hours, the reaction solution was filtered, and the furnace solution was concentrated under reduced pressure to remove the remaining residue.
Wash sequentially with 0% citric acid, 5% Na HCO3 and saturated NaCQ water and dry. This is purified using silica gel column chromatography to obtain the target peptide.

Yield 11.3g (54,4%), u, 120-125℃, [2-0,41, [α)22=-17,3° (c-1,5, DMF)7
) l-1-Tyr-Ala-Pro-Thr-8er
-8er-8er-Of (preparation of 6 above), the protected hepigpeptide (500-g) was added to M
e OH-DMF-acetic acid (151112-51112-
The mixture was dissolved in a mixed solution of 2-1 and catalytically reduced using palladium black as a catalyst at room temperature for 28 hours. After removing the catalyst, the furnace solution was concentrated under reduced pressure, and the residue was washed with ether and dried.

3% vinegar! (5112), gelatinized with Sephadex (3Hhadex) G-10 and H-T
Vr-Ala-Pr.

-Thr-Ser-Ser-Ser-OH is obtained. Hereinafter, this will be referred to as "peptide A."

Yield 1260u (87.3%) Rf, -0.15, Rf 5-0.29, (α)''--
70,0' (c-1,4,0,2N acetic acid) <Production of antigen> Production example 1 Peptide 1 Preparation 510 of peptide A obtained in Example 2 was added with Ascaris extract protein solution (ASC, A2s).

-1,35) 5- was added, and to this solution was added 1°55 mL of 0.1% glutaraldehyde solution (GA) dissolved in O.1M phosphate buffer <rtH-7,2), and at room temperature Stir for 2 hours. Then cool the reaction mixture! (4℃)
The desired immune antigen 1 was dialyzed against physiological saline for 3 days within the
0m(+ is obtained.Hereinafter, this antigen will be referred to as rlL-2<A>-G
I'll call you AJ.

Production Example 2 An immunizing antigen is obtained in the same manner as in Production Example 1 above, using peptide B obtained in Peptide Production Example 1 instead of peptide A. This is called rlL-2(B)-GAJ.

Production example 3 ASC to peptide A3l1+l! Add 0.M and add 0.3M to this solution containing 0.13M sodium chloride <Na CQ >.
Add 16M sodium chloride solution (pH-9,0>5IIIg) and add sodium diazobenzidine (BDB) 3.9311
Add 11 and stir at 4°C for 2 hours. The reaction mixture is then dialyzed against saline for 3 days in low room temperature to obtain the desired immune anti-13!10+ag. The post-reaction mixture is dialyzed against physiological saline in a cold room for 3 days to obtain 10Q of the desired immunizing antigen. This is r I L-2(A>-B
It's called DBJ.

Production Example 4 An immunizing antigen is obtained in the same manner as in Production Example 3 above, using peptide B instead of peptide A. This is r I L-
It is called 2(B)-BDBJ.

Production example 5 Peptide A5Img and bovine serum albumin (BSA) 5
mg of ammonium acetate buffer (0.1 mol, D H7
, 0) 2I1giC solution Kashi, Z (Add 0.114 of 0.1 mol glutaraldehyde solution (GA) to D solution I,
Stir at room temperature for 5 hours. The reaction mixture was then heated for 48 hours.
Dialyze against 1Q water at 4°C. Change the water 5 times during dialysis. In this way, 10 σ of the desired immunizing antigen is obtained.

<Production of antibodies> Production example 1 Antigen Kiri 100μQ of each antigen obtained in Examples 1 to 5
/m in physiological saline, add Freund's complete adjuvant solution (Difco) 1.5-m to each antigen solution to create a suspension, and add several rabbits (N e
w-Z ealand whiterabbits)
(3.0 to 3.5 ko), repeated subcutaneous administration every 2 weeks at an antigen inoculation dose of 100 μg/rabbit, and after 6 or more sensitizations, blood was collected from the test animal, centrifuged, and antiserum was extracted. Collect and obtain the desired antibodies. The obtained antibodies are labeled with the antibody number shown in Table 1 for each rabbit.

<Measurement of antibody titer> Antibody titer was measured by the enzyme immunoassay method described below.

Peptide 81m! A plate for enzyme immunoreaction (Nunc
Ltd., Denmark). 10,102.103.10', 105.1 of each antibody in physiological saline
Diluted 0.8 times, put 100 μQ of each into a peptide B binding plate, and reacted at 37° C. for 1 hour. Peroxidase-labeled goat anti-rabbit globulin antibodies (E, Y, Lab, U, S, A, >1
Add 00μQ and react at 37°C for 1 hour. Next, after removing unbound substances by washing, orthophenylenediamine [1'l (2,5■ LIT?), masiquilobane buffer: citric acid 7g, phosphorus 1!2 sodium salt 23.89o
and 3% hydrogen peroxide solution 5- solution made up to 19 with distilled water) 1
After 30 minutes, 100 μQ of 2N sulfuric acid W was added to stop the reaction, and the absorbance at a wavelength of 492 rv was measured using a microplate photometer. The absorbance (binding rate of antibody) is plotted on the vertical axis and the dilution factor of the antibody is plotted on the horizontal axis, and the absorbance is plotted at each dilution factor. The dilution ratio of the antibody at which the absorbance was 50% was determined and determined as the titer of each antibody.

Table 1 shows the results obtained for each antibody obtained in the above antibody production example.

Table 1: Antibody IL-2 specificity test> (1) Inhibition of binding rate by synthetic fragment of 11-2 Add a divine concentration of peptide B as an IL-2 fragment to 100 μQ of antibody J diluted 50,000 times.
The reaction was carried out at ℃ for 1 hour. Pour 0.1 mG of the reaction solution into an enzyme immunoplate to which peptide B has been bound in advance and incubate at 37°C for 1 hour.
Allow time to react. Thereafter, an enzyme immunoreaction was performed using the same method as described above for measuring the antibody titer.
The absorbance at 11 was measured.

FIG. 1 shows the results of the binding inhibition reaction by peptide B at various concentrations. In Figure 1, the vertical axis is the absorbance (OD490n
m), and the horizontal axis shows the concentration of TL-27 fragment (peptide B). It was found from the cough diagram that the binding of antibody J to the plate was inhibited in proportion to the increase in the concentration of peptide B.

(2) Binding to IL-2 Human IL-2 in itw4 is produced in Escherichia coli using a tryptophan promoter, using a conventional method (Smith, K.A.).

Iveunol, Rev, vol 51.33
7 (1980)) by ammonium sulfate precipitation, ion-exchange resin Sepharose, gel filtration, and high-resolution liquid chromatography, or using combinant interleukin-2 (rlL, -2).

Human IL-2 recombinant DNA was expressed in E. coli using the following method. That is, human tonsil Metsenger RNA
Plasmid pH1G5-3 containing IL-2c DNA cloned from (Maeda, S, N
1shino, N, 5hiiada.

Hirano, T., Qnoue et al.
B10Chell.

B 1ophys, Res, Coo,, vol.
115.

Restriction enzyme 1- from 1040-1047 (1983))
IL-2c DN by decomposition of 1 indl[[ and pvu
A DNA fragment containing A was isolated by agarose gel electrophoresis, this DNA fragment was further cut with restriction enzymes Ho+Ai, and the r)NAF+ fragment encoding Il--2 was isolated by agarose gel electrophoresis f71. 'IIP! I had it.

After reacting this DNA fragment with 74 DNA polymerase, synthetic oligonucleotide (5') CGATAATGGC
A (3') and (5'> TGCCATTAT (3
) (only the latter oligonucleotide had its 5' end phosphorylated in advance) were ligated using T4 DNA ligase. A DNA fragment encoding IL-2 bound to this synthetic oligonucleotide and restriction enzyme CQ
a Plasmid D ATl 53 cut with I and T4
It was ligated with DNA ligase and transformed into Dai 11188101. oATi 11-2-2/HB101 containing the desired plasmid was obtained from the transformed bacterium. This II
Plasmid pA from ATm IL-2-2/HB101
"rllL2-2 was prepared, and after cutting this plasmid with the restriction enzyme cQa i, the DNA encoding I[-2
Fragments were isolated by agarose gel electrophoresis. On the other hand, the trp promoter and S hlne D aloarn
Fill vector plasmid IITM1 containing o sequence
It was cut with limiter C; Qa I, ligated with a DNA fragment encoding I) ATIIL-2 using T4ONA ligase, and transformed into Escherichia coli HB101. ptrp IL-208/H8 containing the desired plasmid from the transformed bacterium
I got 101. This Nrpg+ IL-2D8/1-I
After culturing BloI overnight at 37°C in LB medium containing 25 μg/− of ampicillin, 0.5% casamino acids and 25 μQ
/WIJ Inoculate M9 medium containing ampicillin and grow at 37°C.
After culturing for 9 hours at
-100, the lysate was centrifuged at 100,000 x g, and the supernatant was collected.

r IL-2 with 0.05M1 charcoal! ! Buffer w1 (pH 9,
6) and statically digested at room temperature for 15 hours to bind to an enzyme immunoplate (LlnC). Each antibody was diluted 30.100 times and 1000 times with physiological saline, and 100 μQ of each was placed in each well of an enzyme immunoplate bound to r 1m-2, and reacted at 37° C. for 1 hour.

After washing and removing unreacted substances, peroxidase-labeled goat anti-rabbit globulin antibodies (E, Y) were used.

Add 100 uQ of ab, U, S, A, ) and react at 37°C for 1 hour. Next, add 100 µe of an ortho-phenylenediamine solution from which unreacted antibodies have been removed by washing, and add
After reacting for a minute, 2 regulations @l! The reaction was stopped by adding 100 μQ, and the wavelength was measured at 4921 with a microplate photometer.
The absorbance of 1 was measured.

The vertical axis represents absorbance (antibody binding rate), and the horizontal axis represents each antibody (antibody N).
o, A-L) were taken and the absorbance of each dilution factor was plotted. Antibody A-L1. The results obtained regarding: are shown in Figure 2. From FIG. 2, it was found that each antibody produced by immunization with peptides A and B binds to rlL-2 produced by genetic recombination technology.

<Production of monoclonal antibody> (1) Sensitization of mice with immunization antigen Mix 111111 of the immunization antigen IL-2(B)-GA (100 μg/-) with an equal volume of 70 India complete adjuvant to prepare a suspension. BAL8/C mice were sensitized intraperitoneally at a dose of 50 μg/mouse at a time, then sensitized twice at 3-week intervals using the same method, and the final dose was administered by IL.
-2 (B) GA physiological saline 0.11 flll/1112
is administered intraperitoneally (50 μΩ/mouse).

(2) Preparation of sensitized lymphocytes for cell fusion Pile cell suspension mu was prepared from the mouse spleen 48 days after the final immunization. Red blood cells present in the tile cells were thawed and removed by treatment with 0.83% ammonium chloride solution at 4°C for 10 minutes.

The cells obtained in this manner were used as sensitized lymphocytes and washed several times with RPMI-1640 medium.

(3) Preparation of mini 0-ma gun for fusion HGPRT
Missing 11BALB/C derived P3-X63-/l 8-U1
Myeloma (G.

Qalfre & C, MHstein, Metho
d 1nEnzyIoloay, vol 73.
p. 3 (1981)), 100% of 8-azaguanine was added to RPMI-1640 medium containing 10% tallow serum.
Subcultured in medium supplemented with μM.

(4) Generation of fused cells (hybridoma) lX10'
P3-X63-At+8-U1 myeloma cells were
It was mixed with 1×10 8 cannonballs prepared from L-2(B)-GA-sensitized BALB/C mice. For cell fusion, the above cell mixture was centrifuged at 500Xll, and then cell fusion was performed using 35% polyethylene glycol-2000 (Wako Pure Chemical Industries, Ltd.). The basic fusion operation is Milestein (M
Method CG of i 1stein ) et al.

Ga1fre & C, Milsteln, Meth
odd in Enzymology, vol. 7
3. p3 (1981)).

Hybridomas growing in HAT medium are transferred to BALB.
/C thymocytes were used as feeder cells and cloned by limiting dilution method.

Table 2 below shows the anti-IL-2 antibody activity (absorbance at 0D49211 measured according to the antibody titer measurement method described above) of each clone and its culture supernatant.

Table 2 According to Table 2, clone No. 26-3.26-32.2
6-45.26-46.26-62.26-73 was found to produce antibodies that bind to peptide B.

In addition, Table 3 below shows the <IL-2 specificity test of antibodies>
The binding rate with rlL-2 measured in the same manner as (2) is shown.

Table 3 From Table 3, clone No. 26-3.26-32.26
-45.26-46.26-62.26-73 was found to bind to rlL-2 produced by genetic recombination.

Clone N, which showed the strongest binding strength by immunodiffusion method,
The antibody produced by O,26-3 was found to belong to the [IG2a subclass.

Illumination of IL-2 using a monoclonal antibody binding column
Clone No. 2 producing antibodies against IL-2
6-3 by intraperitoneal transplantation of BALB/c mice or by high-density continuous culture! ! Equipment (Shibata Vario Glass■)
Antibodies were prepared in large quantities by growing flllI in culture using a cell culture method using (G).

Ga1fre & C, Milsteln: Meth
ods InE nzy*ology, vol 7
3, p3 (1981)).

Immunoglobulin was purified by ammonium sulfate salting out method and DEAE ion exchange chromatography [Medical Chemistry Experimental Method Course 4, Immunochemistry, Nakayama Nikuten, p. 75-90].

Next, immunoglobulin was bound to bromide-activated Cephas 4B (Pharmacia Japan) (Cuatre
cases, P, &C, 8, Anrinser+
:Methods in Enzymology vo
l 22゜p345. (1971)) produced an IL-2 monoclonal antibody-binding column.

The column (gel volume 14i0) was soaked in a phosphate buffered salt solution of 501111.
After washing with 5aline: PBS, pH 7,4>, a crude 11-2 solution 1- produced in Escherichia coli by a genetic recombination method was applied to the column, reacted at room temperature for 1 hour, and then I L
-2 to the antibody. The column was washed with PBS50- to remove substances that did not bind to the antibody, and then the IL-2 bound to the antibody was washed with 0.1 M glycine-HCl buffer (p
H3) was eluted. The eluted protein solution was immediately neutralized with unoxidized sodium, dialyzed against PBS, and then concentrated.

The purity of IL-2 purified by adsorption chromatography using an antibody column was determined by 5DS-PAG F
(Sodium dodecyl 5ulfate
-Polyacrylag+ide Get elec
trophoresis). (L
aeuli U, K, , Nature.

227.680 (1970)).

Phosphorylase B (94K), bovine albumin (68K)
), ovalbumin (43K), charcoal 11115! Hydrogen enzyme (30K), trypsin inhibitor (20,1
K) and alpha-lactalbumin (14,4K>) were prepared using standard proteins for molecular weight measurement (Sigma and Pharmacia).
(manufactured by Fine Chemicals). After completion of electrophoresis, proteins were stained with 0.1% Coomassie Brilliant Blue solution. Figure 3 shows the migration pattern.

IL-2 eluted from the antibody column has a molecular weight of approximately 16K (
Shows a single band of 16,000) with high purity.
It turned out that it was.

[Brief explanation of drawings]

FIG. 1 is a graph showing the binding property between the antibody of the present invention and synthetic IL-2 peptide, and FIG. 2 is a graph showing the reactivity between the antibody of the present invention and I[-2 produced by genetic recombination method. FIG. 3 is a graph showing the electrophoretic pattern of 11-2 produced by the genetic recombination method and subjected to IIIFj using the monoclonal antibody-binding column of the present invention. (That's all) Figure 6 STD old-2-16

Claims (1)

[Scope of Claims] An antibody against human interleukin-2 characterized by having reactivity with a decapeptide represented by 0 formula % formula %.