JPH0160480B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel human gamma-interferon-related peptides. In this specification, when amino acids, peptides, protecting groups, active groups, etc. are indicated by abbreviations, they shall comply with the regulations of IUPAC and IUB or the common symbols in the field, and examples thereof are listed below.
Furthermore, when an amino acid or the like may have optical isomers, the L-isomer is indicated unless otherwise specified. Ser; serine Leu; leucine Asn; asparagine Ala; alanine Gln; glutamine Glu; glutamic acid Arg; arginine Lys; lysine Tyr; tyrosine Phe; phenylalanine Met; methionine Asp; aspartic acid Gly; glycine ONP; p-nitrophenoxy group Tos; p-toluenesulfonyl group Boc; tertiary butoxycarbonyl group Bzl; benzyl group OBzl; benzyloxy group Cl ₂ -Bzl; 2,6-dichlorobenzyl group Cl-Z; 2-chlorobenzyloxycarbonyl group Interferon is It is an antiviral glycoprotein or protein that is produced when living cells are infected with a virus, and its use has been attracting attention in recent years as it is believed that it is possible to prevent or treat viral diseases. The currently elucidated human interferons are β-type interferon (Fibro blast interferon), α-type interferon (Leucocyte interferon, Lympho blastoid interferon).
It is classified into γ-type interferon (interferon) and γ-type interferon (immune interferon). However, a technique for purifying these interferons into a single glycoprotein or protein has not yet been established. The present inventors believed that human γ-interferon could be purified by antigen-antibody reaction by using antibodies that specifically react with human γ-interferon, and based on this idea, human γ-interferon could be purified with high sensitivity. γ
We have selected a type of interferon and have been conducting intensive research to obtain antibodies that exhibit specific reactivity to the interferon. In the process, they synthesized a peptide with the amino acid sequence of a specific site of human γ-interferon, succeeded in synthesizing an antigen using this as a hapten, and obtained specific reactivity from the antigen to human γ-interferon. It has been found that it is possible to obtain a desired antibody having the following properties. The present invention was completed based on this new knowledge. That is, the present invention has the general formula R-Phe-Asn-Ser-Asn-Lys-Lys-Lys-
Arg-Asp-Asp-Phe-OH (1) [In the formula, R represents a hydrogen atom or a H-Tyr group. ] This relates to a human γ-interferon-related peptide selected from the group consisting of peptides represented by the following. The peptide represented by the above general formula (1) of the present invention can be easily synthesized by simple operations using easily available commercially available amino acids. Furthermore, since each peptide has a specific amino acid sequence, by using it as a hapten, a certain antigen with a clear recognition site can be produced in large quantities.
From the antigen thus obtained, antibodies with high specificity for human γ-interferon can be obtained in large quantities and always stably, compared to when natural γ-interferon is used as the antigen. The antibody can be used for purification of human γ-interferon by binding it to, for example, an affinity chromatography carrier and utilizing the chromatography. The peptide represented by the general formula (1) of the present invention can be synthesized using a conventional peptide synthesis method, specifically, according to "The Peptides", Volume 1 (1966) [Schroder
and Luhke, Academic press, New York,
USA] or "Peptide Synthesis" [Izumiya 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-nitrophenyl ester method, N-hydroxysuccinimide ester method, cyanomethyl ester method, etc.), method using Woodward's reagent K,
Carbodiimidazole method, redox method, DCC/
It can be manufactured by additive methods (HONB, HOBt, HOSu), etc. 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 prepared in accordance with the general polypeptide synthesis method described above, for example, by sequentially adding 1 amino acid to the terminal amino acid.
It is produced by a so-called stepwise method in which amino acids are condensed one by one, or by a method in which they are divided into several fragments and coupled together. More specifically, for example, when solid phase synthesis is employed, C
The terminal amino acid is attached to the insoluble carrier via its carboxyl group. The insoluble carrier is not particularly limited as long as it has a bonding property with a reactive carboxyl group, and examples thereof include halogenomethyl resins such as chloromethyl resin and bromomethyl resin, benzhydrylamine resin, hydroxymethyl resin, phenol resin, and tert- Alkyloxycarbonyl hydrazide resins and the like can be used. Next, after removing the amino protecting group, the amino group-protected amino acids are sequentially bonded by condensation reaction with the reactive amino group and the reactive carboxyl group according to the amino acid sequence represented by the general formula (1), and the synthesis is performed step by step. , is produced by synthesizing the entire sequence and then removing the peptide from the insoluble carrier. In the above, each of the amino acids tyrosine, glutamic acid, arginine, lysine, aspartic acid, and serine preferably has its side chain functional group protected.This is protected by a conventional protecting group, and after the reaction is completed, the protecting group is removed. Will be detached. Furthermore, the functional groups involved in the reaction are usually activated. Each of these reaction methods is well known, and the reagents used therein are also appropriately selected from known methods. Examples of protecting groups for amino groups include benzyloxycarbonyl, tert-butyloxycarbonyl, tert-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl, adamantyloxycarbonyl, Examples include trifluoroacetyl, phthalyl, formyl, o-nitrophenylsulfenyl, diphenylphosphinothioyl groups, and the like. Examples of protecting groups for side chain functional groups include Bzl, p-methoxybenzyl, p-nitrobenzyl, trityl, methyl, ethyl, and tert as protecting groups for carboxyl groups of aspartic acid and glutamic acid.
-butyl group, etc. As a protecting group for the hydroxyl group of serine, for example,
Examples include Bzl, tert-butyl, acetyl, and tetrahydropyranyl groups. Examples of the protecting group for the amino group of arginine include nitro, Tos, and benzyloxycarbonyl groups. Examples of the protecting group for the amino group of lysine include benzyloxycarbonyl, Cl--Z, Boc, and Toc groups. As a protecting group for the hydroxyl group of tyrosine, for example,
Examples include Bzl, Cl ₂ -Bzl, benzyloxycarbonyl, acetyl, and Tos group. Examples of activated carboxyl groups include:
For example, the corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, active esters (pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenztriazole, N-hydroxy-5-norbornene-
esters with 2,3-dicarboximide, etc.). The peptide bond forming reaction may be carried out in the presence of a condensing agent such as a carbodiimide reagent such as dicyclohexylcarbodiimide or carbodiimidazole, or tetraethylpyrophosphine. Hereinafter, an example of the production of the peptide of the present invention will be specifically explained using a reaction scheme. (Reaction scheme 1) A-Phe-OH (a) ↓ A-Phe-R ¹ (b) ↓ H-Phe-R ¹ (c) ↓A-Asp-OH (d) A-Asp-Phe-R ¹ (H) ↓ ↓ ↓ H―Phe―Asn―Ser―Asn―Lys―Lys―Lys―
Arg―Asp―Asp―Phe―R ¹ (F) ↓A―Tyr―OH (G) A―Tyr―Phe―Asn―Ser―Asn―Lys―Lys
―Lys―Arg―Asp―Asp―Phe―R ¹ (chi) ↓ H―Tyr―Phe―Asn―Ser―Asn―Lys―Lys
-Lys-Arg-Asp-Asp-Phe-OH (1a) [In the formula, A represents a protecting group for an amino group and R ¹ represents an insoluble carrier. ] In the above, preferable A is
Preferred examples of R ¹ include Boc, benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, and chloromethylated polystyrene. Furthermore, in each reaction, when the amino acid used has a side chain functional group that does not participate in the reaction, it is protected by the above-mentioned protecting group as usual, and this is removed at the same time as the insoluble carrier R ¹ is removed. In the above method, the amino acid (a) and the insoluble carrier
The reaction with R ¹ is carried out by utilizing the reactive carboxyl group of the amino acid (a) and bonding it to R ¹ according to a conventional method. For example, when using chloromethylated polystyrene, the reaction is carried out in a suitable solvent, such as triethylamine, potassium tert.
-Carried out in the presence of basic compounds such as butoxide, cesium carbonate, cesium hydroxide, etc. Examples of solvents include dimethylformamide, dimethylsulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, N-
Examples include methylpyrrolidone, hexamethylphosphoric acid triamide, and a mixed solvent thereof. The above reaction is usually carried out at 0 to 85°C, preferably at 25°C.
The process will be completed in a few minutes to 24 hours at a temperature of ~80℃. The ratio of the amino acid and the insoluble carrier to be used is usually such that the former is used in an excess amount, generally 1 to 3 times equivalent, to 1 equivalent of the latter. The elimination reaction of the protecting group A of the immobilized amino acid of the general formula (b) thus obtained is carried out by a conventional method.
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, hydrochloric acid, hydrogen fluoride, 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 atmosphere and at a temperature of 0 to 40°C. The amount of catalyst used is usually 100 mg to 1
The reaction is generally completed in about 1 to 48 hours. The acidolysis is carried out without a solvent, usually at about 0 to 30°C, preferably about 0 to 20°C, for about 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 protecting group A is removed in the acidolysis, it is preferable to use trifluoroacetic acid or hydrochloric acid as the acid. Furthermore, the above reduction with sodium gold oxide in liquid ammonia is usually carried out at about -40°C to -70°C using an amount of metallic sodium such that the reaction solution turns a permanent blue color for about 30 seconds to 10 minutes. obtain. The reaction between the resulting immobilized amino acid of general formula (c) and amino acid (d) (or its carboxyl group activated) is carried out in the presence of a solvent. Examples of the solvent include various known solvents commonly used in peptide condensation reactions, such as anhydrous dimethylformamide, dimethyl sulfoxide (DMSO),
Pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, ethyl acetate, N-
Examples include methylpyrrolidone, hexamethylphosphoric acid triamide, and a mixed solvent thereof. In addition, the reaction may be carried out using reagents used in ordinary peptide bond forming reactions, such as N, N,
N-dicyclohexylcarbodiimide (DCC),
In the presence of a dehydration condensing agent such as carbodiimides such as N-ethyl-N'-dimethylaminocarbodiimide, 1-ethyl-3-diisopropylaminocarbodiimide, and 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide. can be done. The ratio of the amino acid (c) and the amino acid (d) to be used is not particularly limited, but the latter is usually used in an equimolar amount to 10 times the molar amount of the former, preferably an equimolar amount to 5 times the molar amount of the latter. It is better. There is no particular limitation on the amount of the dehydration condensing agent used, and it is usually used in an equivalent molar amount to the amino acid (d). The reaction temperature is appropriately selected from the usual range used for peptide bond-forming reactions, generally from about -40°C to about 60°C, preferably from about -20°C to about 40°C. The reaction time is generally about several minutes to 30 hours. The thus obtained peptide of the general formula (E), after removing the protecting group A in the same manner as above, has the following amino acid sequence represented by the general formula (1): A-Asp-OH, A-
Arg―OH, A―Lys―OH, A―Lys―OH, A
-Lys-OH, A-Asn-OH, A-Ser-OH, A
-Asn-OH, A-Phe-OH, A-Tyr-OH amino acids or activated versions of their carboxyl groups are carried out by sequential condensation reaction, and thus represented by the general formula (H). It can be derivatized into peptides. The condensation reaction and the elimination reaction of the protecting group A are carried out in the same manner as described above. In addition, the obtained peptide (H) can be obtained by similarly removing the protecting group A, the protecting group of the side chain functional group of the amino acid, and the insoluble carrier R ¹ in the general formula (1).
is induced into peptide (1a) representing an H-Tyr group. Here, the elimination reaction of the protecting group of the side chain functional group of the amino acid and the insoluble carrier R ¹ can be carried out in the same manner as the elimination reaction of the protecting group A, and in this case, hydrogen fluoride or hydrobromic acid may be used as the acid. is preferred. In addition, the peptide of the present invention in which R represents a hydrogen atom can be obtained by removing the protecting group of the side chain functional group of the amino acid and the insoluble carrier R ¹ from the peptide of the general formula (f) obtained in the above reaction in the same manner as above. It can be manufactured by separating it. Note that each amino acid used in the above method may be a known commercially available product. The peptide of the present invention having general formula (1) produced as described above is isolated and purified from the reaction mixture by conventional peptide separation means such as extraction, partitioning, column chromatography, etc. The peptide of the present invention thus obtained is
Radioactive substances such as ¹²⁵ and ¹³¹ , peroxidase (POX), chymotrypsinogen, procarboxypeptidase, glyceraldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, D
- By introducing various enzyme reagents such as Nase, P-Nase, β-galactosidase, glucose-6-phosphate dehydrogenase, ornithine decarboxylase, radioimmunoassay (RIA) method or enzyme immunoassay It can be used as a labeled antigen for use in the EIA method. Introduction of the above-mentioned radioactive substance can be carried out by a conventional method. For example, radioactive iodine can be prepared by oxidative iodination using chloramine T [WM
Hunter and F.C. Greenwood; Nature, 194 ,
495 (1962), Biochem J. 89 , 144, (1963)]
etc. Specifically, the reaction is carried out in a suitable solvent such as 0.2M phosphate buffer (PH=7.4) in the presence of chloramine T at around room temperature for about 10 to 30 seconds. The ratio of peptide, radioactive iodine, and chloramine T used is, for example, when introducing one radioactive iodine per tyrosine, approximately 1 millicury of radioactive iodine and 10 ml of chloramine T per 1 nanomole of tyrosine molecules contained in the peptide.
It is best to use about 100 nanomoles of radioactive iodine, and when introducing two radioactive iodines per tyrosine, use about 2 millicuries of radioactive iodine and 10 to 100 nanomoles of chloramine T per 1 nanomol of tyrosine molecules contained in the peptide. It is best to use it in moderation.
The radioactive iodine-labeled peptide thus produced is subjected to conventional separation means such as extraction,
It is isolated and purified by partitioning, column chromatography, dialysis, etc. The peptides thus obtained can be stored by lyophilization, if necessary. The enzyme reagent can be introduced using conventional coupling methods such as the method of BFE Erlanger et al. [Acta. Endocrinol. Suppl., 168 , 206 (1972)] and the method of MH Karol et al. [Proc. Natl.
Acad. Sci., USA., 57 , 713 (1967)]. That is, peptides and enzymes are mixed at pH 4 in the presence of an oxidizing agent such as NaIO ₄ .
-6 buffer, e.g. 1mM acetate buffer (PH4.4)
in a room temperature for 2 to 5 hours, and then
This is done by reduction with NaBH ₄ or the like.
The enzyme is used in an amount of about 1 to 3 times the molar amount per mole of peptide. It is preferable that the oxidizing agent is used in an amount of about 100 to 300 times the molar amount of the peptide, and the reducing agent is used in an amount of about 1 to 2 times the molar amount of the oxidizing agent. The enzyme-labeled peptide thus produced can be isolated, purified, and stored in the same manner as the radioactive iodine-labeled peptide. Hereinafter, a method for producing an antigen using the peptide of the present invention as a hapten will be described in detail. The above antigen is produced by using the peptide of the present invention as a hapten and reacting it with a suitable carrier in the presence of a hapten-carrier binding reagent. As the carrier bound to the hapten in the above, a wide range of high-molecular natural or synthetic proteins commonly used in the preparation of antigens can be used. Examples of the carrier include horse serum albumin, bovine serum albumin,
Animal serum albumins such as rabbit serum albumin, human serum albumin, and sheep serum albumin;
Animal serum globulins such as horse serum globulin, bovine serum globulin, rabbit serum globulin, human serum globulin, and sheep serum globulin; Thyroglobulins; horse hemoglobulin, bovine hemoglobulin, rabbit hemoglobulin,
Animal hemoglobulins such as human hemoglobulin and sheep hemoglobulin; animal hemocyanins such as keyhole limpet hemocyanin (KLH); proteins extracted from roundworms (Ascaris extract, Japanese Patent Application Laid-open No. 16414/1983, J .Immun.，
111, 260-268 (1973), J.Immun., 122 , 302-
308 (1979), J.Immun., 98 , 893-900 (1967) and Am.J.Physol., 199 , 575-578 (1960) or further purified versions thereof); polylysine, Examples include polyglutamic acid, lysine-glutamic acid copolymers, copolymers containing lysine or ornithine, and the like. As the hapten-carrier binding reagent, a wide variety of those commonly used in the production of antigens can be used. Specifically, amino groups are cross-linked, such as glyoxal, bisdiazotized benzidine (BDB), malondialdehyde,
Aliphatic dialdehydes such as glutaraldehyde, succinaldehyde, adipaldehyde; crosslinking between thiol groups, such as N,N'-o-phenylene dimaleimide, N,
Dimaleimide compounds such as N'-m-phenylene dimaleimide; cross-linking amino groups and thiol groups, such as metamaleimidobenzoyl-N-
Maleimidocarboxyl-N-hydroxysuccinimide esters such as hydroxysuccinimide ester and 4-(maleimidomethyl)-cyclohexane-1-carboxyl-N'-hydroxysuccinimide ester; normal peptide bond formation by amide bonding between an amino group and a carboxyl group Reagents used in the reaction, such as N,N'-dicyclohexylcarbodiimide, N-ethyl-
N'-dimethylaminocarbodiimide, 1-ethyl-3-diisopropylaminocarbodiimide,
1-Cyclohexyl-3-(2-morpholinyl-
Examples include dehydration condensation agents such as carbodiimides such as 4-ethyl)carbodiimide. Furthermore, as the hapten-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
In a normal buffer solution of PH 7-10, preferably PH 8-9
The reaction is carried out in a buffer solution at 0 to 40°C, preferably around room temperature. The reaction is usually completed in about 1 to 24 hours, preferably 3 to 5 hours. Typical buffer solutions used in the above may include the following. 0.2N sodium hydroxide - 0.2M boric acid - 0.2M potassium chloride buffer, 0.2M sodium carbonate - 0.2M boric acid - 0.2M potassium chloride buffer, 0.05M sodium tetraborate - 0.2M boric acid -
0.05M sodium chloride buffer, 0.1M potassium dihydrogen phosphate-0.05M sodium tetraborate buffer In the above, the proportions of hapten, hapten-carrier binding reagent, and carrier can be determined as appropriate;
Usually, the carrier is about 2 to 6 times the weight of the hapten.
It is preferable to use about 3 to 5 times the weight and about 5 to 10 times the mole of the hapten-carrier binding reagent.
Through the above reaction, a human γ-type interferon antigen consisting of a peptide-carrier complex in which a carrier and a hapten are bound is obtained through the mediation of a hapten-carrier binding reagent. The antigen obtained after completion of the reaction can be easily isolated and purified by conventional methods such as dialysis, gel filtration, and fractional precipitation. The antigen thus obtained usually has an average of 5 to 20 moles of peptide bound to 1 mole of protein, and any of these makes it possible to subsequently produce antibodies with high specificity for the antigen. . Production of antibodies using the antigen is carried out by administering the antigen to a mammal, causing the desired antibody to be produced in vivo, and collecting the antibody. There are no particular restrictions on the mammal used for antibody production, but rabbits and guinea pigs are usually preferably used. For antibody production, dilute the prescribed amount of the antigen obtained above with physiological saline to an appropriate concentration, and add Freund's auxiliary solution (Complete
Freund's Ajuvant) to prepare a suspension;
This may be administered to a mammal. For example, intradermally inject the above suspension into rabbits (the amount of antigen is 0.5
~5 mg/dose) and then administered every two weeks for 2 to 10 months, preferably 4 to 6 months, for immunization. Antibodies were collected from 1 to 1 of the final administration of the above suspension.
After two weeks, blood is collected from the immunized animal, centrifuged, and serum is separated. According to the above, an antibody with excellent specificity for the antigen used can be obtained, and this is RIA
It can be used to quantify human γ-type interferon using methods such as EIA method and EIA method. Below, in order to explain the present invention in more detail, general formula
Examples of production of the peptide of the present invention represented by (1) and production of antigens and antibodies from the peptides obtained thereby will be given, but the present invention is not limited thereto. The Rf value in each production example was measured by thin layer chromatography on silica gel using the following mixed solvent. Rf ¹ ...n-butanol-acetic acid-water (4:1:
5) Rf ² ...n-butanol-acetic acid-pyridine-water (15:3:10:12) <Production of peptide> Production example 1 12.4 milliequivalents of potassium tert-butoxide
Dissolve 3.65 g of Boc-Phe-OH in 35 ml of DMSO solution and add chloromethylated polystyrene resin (1.32
Add 5 g of milliequivalent/g resin (Protein Research Foundation) and react at 80°C for 30 minutes. The resin was thoroughly washed with DMSO, 50% acetic acid/chloroform, and methylene chloride in this order, and dried under reduced pressure.
6.04 g of Boc-Phe-resin is obtained. As a result of amino acid analysis after partially hydrolyzing the amino acid
It was 0.17 mmol/g resin. After washing 5.88 g of the Boc-Phe-resin obtained above three times with 30 ml of chloroform, it was added to 30 ml of a chloroform solution of 50% trifluoroacetic acid (TFA).
Incubate for 20 minutes at room temperature. Chloroform the resin
Once with 30 ml, 5 times with 30 ml of methylene chloride, and 3 times with 30 ml of 10% triethylamine in methylene chloride.
The H-Phe-resin was obtained by washing twice with 30 ml of methylene chloride, and then six times with 30 ml of methylene chloride. Add the above H-Phe-resin to 25 ml of a solution of 0.81 g of Boc-Asp(OBzl)-OH dissolved in methylene chloride, then add 5 ml of a solution of 0.51 g of DCC dissolved in methylene chloride, and react at room temperature for 2 hours. let After washing the resin 6 times with 30ml of methylene chloride,
Add 0.81 g of Boc-Asp(OBzl)-OH and 0.34 g of 1-hydroxybenzotriazole (HOBt) to 25 ml of methylene chloride solution, then add DCC.
Add 5 ml of a solution of 0.51 g dissolved in methylene chloride and react in the same manner again (double coupling method). Wash the resin thoroughly with methylene chloride.
Obtain Boc-Asp(OBzl)-Phe-resin. In the same way as above, Boc―Asp(OBzl)―
The Phe-resin is de-Boc-reacted, and then the following amino acids are sequentially subjected to condensation and de-Boc reaction. Boc-Asp(OBzl)-OH 0.81g Boc-Arg(Tos)-OH 1.11g Boc-Lys(Cl-Z)-OH 1.04g repeated 3 times Boc-Asn-ONP 0.88g and HOBt 0.34g Boc-Ser (Bzl)-OH 0.74g Boc-Asn-ONP 0.88g and HOBt 0.34g Boc-Phe-OH 0.66g Thus H-Phe-Asn-Ser (Bzl)-Asn-
Lys(Cl―Z)―Lys(Cl―Z)―Lys(Cl―Z)―
Arg(Tos)-Asp(OBzl)-Asp(OBzl)-Phe
- Obtain 7.79 g of resin. Dissolve 3.75 g of this in 4 ml of anisole and 40 ml of hydrogen fluoride, and
After reacting for 30 minutes at 0°C, hydrogen fluoride is distilled off, and the residue is dried, extracted with 10% acetic acid, washed with ether, and freeze-dried. Next, gel filtration was performed using Cephadex G-10 (Pharmacia, eluent: 10% acetic acid), and then purification was performed using LH-20 (Pharmacia, eluent: 1/1000N-HCl) to obtain H-Phe-Asn-Ser-. Asn―Lys―Lys―Lys
- Obtain 350 mg of Arg-Asp-Asp-Phe-OH.
This peptide is hereinafter referred to as "peptide A." Rf value: Rf ¹ = 0.01 Rf ² = 0.35 Elemental analysis value: (as C ₈₁ H ₉₃ N ₁₉ O ₁₈・8H ₂ O・5CH ₃ COOH) C(%) H(%) N(%) Theoretical value 46.73 7.13 14.58 Analysis value 46.48 7.40 14.46 Production example 2 H-Phe-Asn-Ser (Bzl) obtained in Production Example 1 above
―Asn―Lys(Cl―Z)―Lys(Cl―Z)―Lys(Cl
―Z)―Arg(Tos)―Asp(OBzl)―Asp(OBzl)
―Phe―resin 4.04g and Boc―Tyr(Cl ₂ ―Bzl)―
1.06 g of OH was reacted in the same manner as in Preparation Example 1-, and then deprotected and resin-removed reactions were performed in the same manner as in 1-, purified in the same manner, and H-Tyr-
Phe―Asn―Ser―Asn―Lys―Lys―Lys―Arg―
258 mg of Asp-Asp-Phe-OH [hereinafter referred to as "peptide B"] is obtained. Rf value: Rf ¹ = 0.01 Rf ² = 0.36 Elemental analysis value: (as C ₇₀ H ₁₀₂ N ₂₀ O ₂₀・10H ₂ O・5CH ₃ COOH) C(%) H(%) N(%) Theoretical value 47.47 7.07 13.84 Analysis value 47.20 7.30 13.92 <Production of antigen> Production example 1 5.55mg of peptide B obtained in peptide production example 2
and 10.23 mg of bovine serum albumin (BSA)
0.16M borate buffer (0.13M NaCl, PH=
9.0) Dissolve in 2ml. In this solution, 1.64mg of BDB
Add 3.0 ml of the same buffer and stir at 4°C for 5 hours. The reaction mixture is then dialyzed against 1 part of water, changing the water 5 times during the dialysis. Thereafter, the solution containing the peptide-protein complex is freeze-dried to obtain 17.27 mg of human γ-interferon antigen. Hereinafter, this antigen will be referred to as an "antigen." The antigen is an average of 8 moles of peptide B bound to 1 mole of BSA. Furthermore, this peptide B
The binding rate with BSA was determined by adding the obtained antigen to Sephadex G-50 (eluent: physiological saline, detection:
OD 280nm, flow rate: 3ml/hour, fractional amount: 1
Since the presence of unreacted BSA and peptide B was not observed when the gel filtration was carried out with 2 ml of peptide B, the fraction of peptide B bound to BSA and other products (2 ml of peptide body)
A standard concentration curve of the peptide dimer is created, the amount of the dimer is determined, and the amount subtracted from the amount of peptide B used as the starting material is calculated as BSA. This was determined assuming that they are connected. The same applies to the following antigen production examples. Production Example 4 The target antigen is obtained in the same manner as in Preparation Example 1 of the antigen, except that peptide A is used instead of peptide B. <Production of antibody> Production example 1 After dissolving 30 μg of the antigen obtained in antigen production example 1 in 1.5 ml of physiological saline, add Freund's auxiliary solution to this.
The suspension prepared by adding 1.5 ml was added to each of 7 rabbits (New-Zealand white rabbits) (2.5
~3.0Kg) subcutaneously, and the same amount is administered 6 times every 2 weeks. Thereafter, the same amount as the first dose was administered three times every month. Seven days after the final administration, blood is collected from the test animals and centrifuged to collect antiserum, thereby obtaining human γ-type interferon antibodies (hereinafter referred to as "antibodies"). Production Example 2 In Production Example 1 above, human γ-interferon antibodies (antibodies to
) are obtained respectively. <Production of labeled peptide> Peptide B obtained in Peptide Production Example 2 is labeled using chloramine T as follows. That is, 5 μg of the above peptide was added to 10 μg of 0.5 M phosphate buffer (PH7.5) with Na[ ¹²⁵ ] (carrier free N.
EN) Add 20 μ of 0.5 molar phosphate buffer of 1 millicurie, then 0.5 μ of 60 μ of chloramine T.
Add 20μ of molar phosphate buffer. The reaction is terminated by stirring at room temperature for 25 seconds and adding 20 μg of 150 μg of sodium metabisulfite (Na ₂ S ₂ O ₅ ) in 0.5 M phosphate buffer. Then add 1% to the reaction solution.
Add 100μ of a cold aqueous sodium iodide solution and analyze the reaction mixture by high performance liquid chromatography [10-40%
Purify with ethanol gradient/0.1M Tris Hcl buffer (PH8.0) 1.0ml/min, reversed phase chromatography to obtain ¹²⁵ -labeled peptide B. The radioactivity of the labeled peptide was 1128 μCi/μg. Measurement of Γ titer Measure the titer of the antibody obtained above as follows. That is, the antibody was dissolved in physiological saline at 10, 10 ² ,
10 ³ , 10 ⁴ , 10 ⁵ ... dilute (initial) twice,
To 100 μl of each of these, add 0.1 ml of ^125- labeled peptide (the labeled peptide obtained above was diluted to approximately 9500 cpm) and 0.05 molar phosphate buffer (PH = 7.4) [0.25% BSA, 10 m
Add 0.2 ml of MEDTA and 0.02% _NaN3 ,
Incubate at 4°C for 24 hours to combine the generated antibodies and
The conjugate with ^{the 125-} labeled antigen was separated by the dextran-activated charcoal method and centrifugation (4°C, 30 minutes,
3000 rpm) to separate from unreacted (unbound) ^125- labeled peptide, count the radiation, and measure the binding rate (%) of the antibody to ^125- labeled peptide at each dilution concentration. Antibody on the vertical axis
The binding rate (%) with the ^125- labeled peptide and the dilution factor (initial concentration) of the antibody are plotted on the horizontal axis, and the binding rate is plotted at each concentration. Combine rate is 50
% of the dilution of the antibody, that is, the titer of the antibody. The results obtained for antibodies ~ are shown in Table 1 below. [Table] [Table] All antibodies obtained according to the present invention are human γ
It responds with high specificity to type interferon.

Claims (1)

[Claims] 1 General formula R-Phe-Asn-Ser-Asn-Lys-Lys-Lys-
Arg-Asp-Asp-Phe-OH [In the formula, R represents a hydrogen atom or a H-Thr group. ] A human γ-interferon-related peptide represented by