JPS59122446A - Peptide relating to gamma-interferon - Google Patents

Abstract

NEW MATERIAL:The peptide of formula I and II (R is H or H-Tyr group). USE:Antigen for the antibody for purifying human gamma-type interferon. PROCESS:For example, for the preparation of the objective substance by a solid- phase synthesis, a C-terminated amino acid is bonded to an inert carrier (e.g. chloromethylated resin, benzohydryl resin, etc.) through the carboxyl group, the amino-protecting group are removed, the amino-protected amino acids are condensed by the condensation of the reactive amino group and the reactive carboxyl group according to the amino acid sequence of formula I and II, step by step, and the peptide is separated from the insoluble carrier after the completion of the synthesis of the whole sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel human γ-interferon related peptides.

In this specification, when amino acids, peptides, protecting groups, active groups, etc. are indicated by abbreviations, IUPAC,
The IUB regulations or symbols commonly used in the field shall be followed, examples of which are listed below. In addition, if an optical isomer is possible for an amino acid, etc., the isomer is indicated unless otherwise specified.

S sr; serine 1-eu; leucine Asn
; Asparagine Ala: Alanine Gin: Glutamine Glu: Glutamic acid Aro: Arginine L
ys: Lysine Tyr; Tyrosine phe
: Phenylalanine Met: Methionine ASI):
Aspartic acid GIV nigericin ONP: p-nitrophenoxy group Tos; p-) luenesulfonyl group Boc; tertiary butoxycarbonyl group Bzl; benzyl group 0Bzl; benzyloxy group CIQ 821: 2.6 ')') 0) Libe ', /
d) LiMC1-Z: 2-chlorobenzyloxycarbonyl interferon is an antiviral glycoprotein or protein that is produced when living cells are infected with a virus, and its use can be used to prevent or treat viral diseases. It is believed that this is possible and has been attracting attention in recent years. The currently elucidated human interferon is β-type interferon (Flbro blast Interferon).
on), β-type interferon (Leucocyte
s Interferon, Lyldh.

bla8t01d +nterreron) and γ-type interferon (I n+une 1nterfe
ron). However, a technique for purifying these interferons into a single glycoprotein or protein has not yet been established.

The present inventors believe that human γ-interferon can be purified by antigen-antibody reaction by using antibodies that specifically react with human γ-interferon, and based on this idea, human γ-interferon can be purified with high sensitivity. We have been conducting intensive research to obtain antibodies that exhibit specific reactivity against the selected interferon. In the process, they succeeded in synthesizing a peptide having the amino acid sequence of a specific site of human γ-type interferon and using this as a hapten to synthesize an antigen. We found that it was possible to obtain antibodies for The present invention was completed based on this new knowledge.

That is, the present invention is based on the general formula % (1) [wherein R represents a hydrogen atom or an H-Tyr group]. ] and the general formula % formula % (2) [wherein R is the same as above. ] Human γ- selected from the group consisting of peptides represented by
Concerning interferon-related peptides.

The peptides represented by the above general formulas (1) and (2) of the present invention can be easily synthesized by simple operations using easily available commercially available amino acids. Moreover, based on the fact that each of these peptides has a specific amino acid sequence, by using them as haptens,
Certain antigens with a defined 1m site can be created in large I. From the antigen thus obtained, antibodies with high specificity for human γ-type 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 to produce human γ-type interferon II, for example, by binding it to a carrier for affinity chromatography and utilizing it in the chromatography.

The peptides represented by general formulas (1) and (2) of the present invention can be synthesized using conventional peptide synthesis methods, specifically [The Peptides J Volume 1 (196
(6 years) (5 Chroder and Luhke, Academlc press, New York
K, LISA) or “Peptide Synthesis J [written by Azumaya et al.
Maruzen Co., Ltd. (1975). N-hydroxysuccinimide ester method, cyanomethyl ester method, etc.), method using Woodward reagent K, carbo-5-diimidazole method, redox method, DCC/additive (HONB, HOBt, HO8Ij') method, etc. can. In the above method, both the in-phase synthesis method and the 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. manufactured by the method.

More specifically, for example, when employing an in-phase synthesis method, the C-terminal amino acid is bound to an 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 includes, for example, hegenomethyl resins such as chloromethyl resin and polymethyl resin, benzhydrylamine resin, hydroxymethyl resin, and phenol resin. , tert-alkyloxycarbonyl hydrazide resin, etc. can be used.

Then, after removing the amino protecting group, general formulas (1) and (
According to the amino acid sequence represented by 2), 6-amino group-protected amino acids are sequentially bonded by a condensation reaction with their reactive amino groups and reactive carboxyl groups, and the synthesis is performed step by step. After synthesizing the entire sequence, the peptide is synthesized. It is produced by removing it from an insoluble carrier.

In the above, it is preferable to protect the side chain functional groups of the amino acids tyrosine, glutamic acid, arginine, lysine, aspartic acid, and serine. Layered. 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-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl, adamantyloxycarbonylloacetyl, phthalyl, formyl, 0 -nitrophenylsulfenyl, diphenylphosphinothioyl groups, and the like.

As a protecting group for a side chain functional group, as a protecting group for a carboxyl group of aspartic acid and glutamic acid, for example, BZ
I,D-methoxybenzyl, p-nitrobenzyl, trityl, methyl, ethyl, tert-butyl groups, and the like.

Examples of protecting groups for the hydroxyl group of serine include Bzl and te.
Examples include rt-butyl, acetyl, and tetrahydropyranyl groups.

Examples of protective groups for the 7-mino group of arginine include nitro, Tos, and benzyloxycarbonyl groups.

Examples of the protecting group for the amino group of lysine include benzyloxycarbonyl group.

Examples of protecting groups for the hydroxyl group of tyrosine include BZI, C
I, -Bzl, benzyloxycarbonyl, acetyl,
Examples include Tos group.

Examples of activated carboxyl groups include the corresponding acid chlorides, 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.). 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' (o) ↓ H-Phe-R' (A) ↓ A - A sp- O H (2) -〇- A -ASD-Phe-R' (E)H - p h
e - A sn - 3 sr - A sn-1
ys-1 ys-Lys-A rlJ-A 8
11- A Sll- P he- R' (
f)↓ A-Tyr-OH (g) A -
Tyr - P he - A sn - S sr
-A sn-1 ys-1 ya-Lys-Ara-
AsD-Asp-Phe-R' (chi)↓ @ - Tyr - p he - A sn - S
er-A sn-1 ys-1 ys-Lys-
Ara-Asfl-Asp-Phe-OH (1
a) [In the formula, A represents a protecting group for an amino group and R1 represents an insoluble carrier. ) In the above, A is preferably 3oc.

Preferred examples of R1 include benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, and chloromethylated polystyrene.

In addition, in each reaction, if the amino acid used has a side chain functional group that does not participate in the reaction, as usual,
protected by the above-mentioned protecting group, which is an insoluble carrier R1
It is released at the same time as the release of .

In the above method, the reaction between the amino acid (a) and the insoluble carrier R1 is carried out by combining the amino acid (a) with R1 using the reactive carboxyl group of the amino acid (a) according to a conventional method. For example, when chloromethylated polystyrene is used, the reaction is carried out in a suitable solvent such as triethylamine, potassium tert-butoxide, cesium carbonate, cesium hydroxide, etc. It is carried out in the presence of a basic compound.

Examples of the solvent include dimethylformamide, dimethylsulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, N-methylpyrrolidone, hexamethylphosphoric triamide, and a mixed solvent thereof. The above reaction is usually 0
The process is completed in a few minutes to 24 hours at a temperature of ~85°C, preferably about 25~80°C. The ratio of amino acid and insoluble carrier used is usually an excess amount of the former to 1 equivalent of the latter, generally 1 to 3
It is best to use double equivalents.

The elimination reaction of the protecting group A of the homomorphized 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. Usage of catalyst is usually 100m! 11~
The amount is preferably about 1 g, and the reaction process generally takes about 1 to 48 hours to complete. In addition, the above acidolysis is carried out in the absence of a solvent, usually at a temperature of about 0 to 30°C, preferably about 0 to 20°C, at a temperature of about 15°C.
The process takes about a minute to an hour. The amount of acid to be 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,
Preference is given to using trifluoroacetic acid or hydrochloric acid as acid. Furthermore, the above reduction with metallic sodium in liquid ammonia turns the reaction solution into permanent blue for 30 seconds or more.
This can be carried out usually at about -40°C to -70°C using an amount of metallic sodium that remains colored for about 10 minutes.

Then, the reaction between the homomorphized amino acid of the general formula (c) and the 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-methylpyrrolidone, and hexamethylphosphorus. Examples include acid triamides and mixed solvents thereof. In addition, the reaction may be performed using reagents used in ordinary peptide bond forming reactions, such as N,N-dicyclohexylcarbodiimide (DCC), N-ethyl-
N'-dimethylaminocarbodiimide, 1-ethyl-3
The reaction can be carried out in the presence of a dehydration condensation agent such as carbodiimides such as -diisopropylaminocarbodiimide 13-3-(2-morpholinyl-4-ethyl)carbodiimide. The ratio of amino acid (c) and amino acid (ii) 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. , l12 There is no particular limitation on the amount of the water condensing agent used, and it is usually for the amino acid (2),
Preferably, about an equimolar amount is used. The reaction temperature is within the usual range used for peptide bond-forming reactions, generally about -
The temperature is appropriately selected from the range of 40°C to about 60°C, preferably about -20°C to about 40°C. Reaction time is generally several minutes to 30
It is said to be about an hour.

The peptide of the general formula (e) thus obtained follows the amino acid sequence represented by the pAIL general formula (1) of the protecting group A, as described above, and has the following amino acid sequence: A-A SD-0 1-(, A
-AI"CI-OH%A-LVS-OH1A-LVS-
OH,A-Lys-OH%A-Asn-OH,A-Se
r-OH1A-Asn-OH, A-Phe-01-1
.

This is carried out by sequential condensation reaction with each amino acid of A-Tyr-OH or its activated carboxyl group, and thus it is possible to derive the peptide represented by the general formula (H). can. 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, removing the protecting group of the side chain functional group of the amino acid, and removing the insoluble carrier R1, so that R in the general formula (1) is H- Tyr
derived from peptide (1a) representing the group. Here, the elimination reaction of the protecting group of the side chain functional group of the amino acid and the insoluble carrier R+ can be carried out in the same manner as the elimination reaction of the protecting group A, and in this case, it is preferable to use hydrogen fluoride or hydrobromic acid as the acid. preferable.

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 R1 from the peptide of the general formula (I) obtained in the above reaction in the same manner as above. It can be manufactured by Note that each amino acid used in the above method may be a known commercially available product.

The peptide of the present invention of 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.

Furthermore, the peptide of the present invention represented by general formula (2) also includes:
It can be manufactured in the same manner as above.

The peptide of the present invention obtained in this way has +25
), 131 1 and other radioactive substances and peroxidase (P
OX), chymotrypsinogen, procarboxypeptidase, glyceraldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, D-Nase, P
-Nase, β-galactosidase, glucose-6
By introducing various enzyme reagents such as -7 osophate dehydrogenase and ornithine decarboxylase, it can be used as a labeled antigen for use in radioimmunoassay (RIA) or enzyme immunoassay (EIA). 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 (W, M, H
unter and F, C, Greenwoo
d: Nature, 194. 495 (1962)
, Blochei+J, 89. 144, (196
3) etc.). Specifically, in a suitable solvent such as 0.2M phosphate buffer (pH-7,4) in the presence of chloramine T at around room temperature,
It takes about seconds. The ratio of peptide, radioactive iodine, and chloramine T to be used is, for example, when introducing one radioactive iodine per tyrosine, approximately 1 milliKy of radioactive iodine and chloramine T per 1 nanomole of tyrosine molecules contained in the peptide. It is best to use about 10 to 100 nmoles of radioactive iodine, and when introducing 2 radioactive iodines per tyrosine, use about 2 milliquiries of radioactive iodine and 10 to 100 ml of chloramine T per 1 nmole of tyrosine molecules contained in the peptide. It is preferable to use about 100 nanomoles. The radioactive iodine-labeled peptide thus produced is isolated and purified by conventional separation means such as extraction, partitioning, column chromatography, dialysis, and the like. The peptide thus obtained can be lyophilized and stored if necessary.

The enzyme reagent can be introduced using conventional coupling methods such as the method of Erlanger (B, F, Erlanger) et al.
Acta, Endocrlnol, 5upp1. ．． 1
68. 206 (1972)) and force rolls (M, H
, Karol) et al.'s method (Proe, Natl, A
cad, 3cl. LISA.

57. 713 (1967)). In other words, peptides and enzymes are
In the presence of an oxidizing agent such as al0L, in a pH 4 to 6 buffer such as 1 M acetate buffer (DH4,4), the reaction is carried out at around room temperature for 2 to 5 hours, and then reduced with Na BN2, etc. It is done. The enzyme is used in an amount of about 1 to 3 times the molar amount per mole of peptide. The oxidizing agent is preferably used in an amount of about 100 to 300 times the mole of the peptide, and the reducing agent is preferably used in an amount of about 1 to 2 times the mole of the oxidizing agent. The enzyme-labeled peptide thus produced can be isolated and stored in the same manner as the radioiodine-labeled peptide described above.

18- 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 uses the peptide of the present invention as a hapten, and the hapten-carrier bond l! It is produced by reacting with a suitable carrier in the presence of an IT drug. As the carrier bound to the hapten in the above, a wide range of high-molecular natural or synthetic proteins commonly used for preparing antigens can be used. Examples of the carrier include animal serum albumins such as horse serum albumin, bovine serum albumin, 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; equine thyroglobulin, bovine thyroglobulin, rabbit thyroglobulin, human thyroglobulin,
Animal thyroglobulins such as ovine thyroglobulin; animal hemoglobulins such as horse hemoglobulin, bovine hemoglobulin, rabbit hemoglobulin, human hemoglobulin, and ovine hemoglobulin; animal hemocyanins such as keyhole linbet hemocyanin (KLH) Protein extracted from roundworms (Ascaris extract, Japanese Patent Publication No. 1983
-16414 publication, J, Iu+un.

111.260-26B (1973), J.

Igvun,, 1 22. 302 to 308 (
1979), J, l1iun, 98. 893～
900 (1967) and A■, J, Physi
of, Joshishi, 575-578 (1960) or further purified versions of these); polylysine, polyglutamic acid, lysine-glutamic acid copolymer,
Examples include copolymers containing lysine or ornithine.

As the hapten-carrier binding reagent, a wide variety of those commonly used for preparing antigens can be used.

Specifically, amino groups are cross-linked, such as glyoxal, bisdiazotized benzidine (
Aliphatic dialdehydes such as BDB), malondialdehyde, geltaraldehyde, succinaldehyde, and azibaldehyde: Crosslinking between thiol groups, such as N, N'-o-phenylene dimaleimide, N .

Simaleimide compounds such as N'-m-phenylene dimaleimide; crosslinking amino groups and thiol groups, such as metamaleimidobenzoyl-N-hydroxysuccinimide ester, 4-(maleimidomethyl)-cyclohexane-1-carboxyl-N' -Maleimidocarboxyl-N-hydroxysuccinimide esters such as hydroxysuccinimide 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-disyc0hexylcarbodiimide, N- Ethyl-N'-dimethylaminocarbodiimide, 1-ethyl-3-diisopropylaminocarbodiimide, 1-cyclohexyl-3-(2-
Examples include dehydration condensation agents such as carbodiimides such as morpholinyl-4-ethyl)carbodiimide. 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 using, for example, an aqueous solution or EIH7
~10, preferably at pH 8 to 9, 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 the hapten, the hapten-carrier binding reagent, and the carrier can be determined as appropriate; It is preferable to use the carrier in an amount of about 2 to 6 times the weight, preferably about 3 to 5 times the weight, and the hapten-carrier 22-body binding reagent in an amount of about 5 to 10 times the weight.

Through the above reaction, a human gamma interferon antigen consisting of a peptide-carrier complex in which a carrier and habten are bound is obtained through the mediation of a hapten-carrier binding reagent.

After the completion of the reaction, the antigen is removed using conventional methods, such as dialysis,
It can be easily isolated and purified by gel filtration, fractional precipitation, etc.

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 limitations on the mammal used for antibody production, but it is usually preferable to use rabbits and guinea pigs. For antibody production, a predetermined amount of the antigen obtained above is diluted with physiological saline to an appropriate concentration, and a 70% Indian supplementary solution (Co-pleteFreund's A
juvant) to prepare a suspension, which may be administered to a mammal. For example, the above suspension is injected subcutaneously into rabbits (one time of 0.5 to 5 ma as the amount of antigen),
Thereafter, the vaccine may be administered every two weeks for 2 to 10 months, preferably 4 to 6 months, for immunization. The antibody is collected by collecting blood from the immunized animal 1 to 2 weeks after the final administration of the suspension, centrifuging the blood, and separating the serum. According to the above method, an antibody having excellent specificity for the antigen used can be obtained, and this can be achieved by RIA method, EI method, etc.
It can be used in method A etc. to quantify human γ-type interferon.

In order to explain the present invention in more detail, examples of production of the peptides of the present invention represented by general formulas (1) and (2) and production examples of antigens and antibodies from the peptides obtained thereby will be given below. It is not limited to.

In addition, Rfi! in each manufacturing example! was measured by thin layer chromatography on silica gel using the following mixed solvent.

Rf+/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 ■ Potassium tert-butoxide 12.4 meq/DMSO solution 3 Solution 3, 65a was dissolved, and chloromethylated polystyrene resin (1.32 meq/ g resin, Protein Research Foundation Foundation) 5Ω was added and reacted 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 to obtain Boc-P.
He-resin 6.040 is obtained.

Amino acid analysis after hydrolysis of a portion revealed that the amino acid content was 0.17 mmol/g resin.

■Boc-Phe-resin obtained in the above ■5.8
After washing 8CI three times with chloroform 301Q, 50%
Trifluoroacetic acid (TFA) in chloroform solution 30cm
Add to Q and allow to react at room temperature for 20 minutes. The resin was washed once with chloroform 30112, 5 times with methylene chloride 3025-1 (i), 3 times with 10% triethylamine in methylene chloride 301Q, then 6 times with methylene chloride 301Q.
Wash each time to obtain H-phe=resin.

Boa-Asp(OBzl)-0H(7)0.81(1
'Fr Dissolved in methylene chloride 25mQ of the above H
-Phe- resin is added, then 5 Q of a solution of 0.51 (+) of DCC dissolved in methylene chloride is added, and the reaction is allowed to proceed for 2 hours at room temperature. After washing the resin 6 times with 30 Q of methylene chloride, Boc- 0.81g of Asp(OBzl)-OH
and 1-hydroxybenzotriazole (HOBt)
A solution of 0.34a in methylene chloride is added to 25Q, followed by a solution of 0.51(I) in methylene chloride of DCC.
Add IQ and react in the same way again (coupling method). Wash the resin thoroughly with methylene chloride and transfer to Boa-AS.
p(OBzl)-Phe-resin is obtained.

■ In the same way as above ■, B oc- A sp (
The BOC-resin is removed from the O B zl )-Phe-resin, and then the following amino acids are sequentially subjected to condensation and dislocation OC reactions.

26- Boc-Asp(OBzl)-OH0,81gBoa-
Aro(Tos)-OH1, 11ΩBoa-Lys(C
I-Z) -OH1,04Q repeated 3 times Boc-Asn-ONP 0.880 and H
OBt 0.34ΩBoa-8e
r(Bzl)-OH0,74QBoa-Asn-ONP
O, asp and HOBt
0.340Boc-Phe-OH0,66G Thus H-Phe-Asn-8ar(Bzl) -A
sn-Lys(CI-Z)-Lys(CI-Z)-Ly
s(CI-Z)-Aro(Tos)-Asp(OBzl
)-A8D(OBzl)-Phe-resin 7.790 is obtained.

Of this, 3.750 were divided into 4 anisole Q and 4 hydrogen fluoride.
Dissolve in 0-Q and incubate at -20℃ for 30 minutes, then at 0℃ for 30 minutes.
After reacting for 1 minute, hydrogen fluoride was distilled off, and the residue was dried.
Extract with 0% acetic acid, wash with ether, and freeze-dry. Then Sephadex G-10 (Pharmacia,
Eluent: 10% acetic acid) followed by LH-20
(Pharmacia, eluent, 1/100ON-HCI!
) l-phe-Asn-8er-A
sn-1ys-L VS-L VS-A ri A 8
0- A S+1- P ho-OH17) 350 ■g
get. This peptide is hereinafter referred to as "peptide A."

Rf value: Rf l-0,01Rf 2-0.35 Elemental analysis value: (Os + He a N+ e O+ a ・8H2
As 0.5CHsCOOH) C (%) H (%) N (%) Theoretical value 46.73 7.13 14.58 Analysis If
46.48 7.40 14.46 Production Example 2 H-Phe-Asn-8et (Bz
l)-Asn-Lys(CI-Z)-Lys(CI-Z
)-Lys(CI-Z)-Aro(Tos)-As
p(OBzl) -Atp(OBzl) -Phe-Resin 4.04a with Boa-Tyr(C12Bzl) O
H1,06(l) was reacted in the same manner as in Preparation Example 1-■, followed by deprotection and resin removal reaction in the same manner as in 1-■, and purified in the same manner to obtain H-Tyr-P he
-A sn -S er -A sn -L VB -
L vS-L VS -Ara-ASI)-ADD-
Phe-OH (hereinafter referred to as "peptide B") 258 Peng 0
get.

Rf value: Rfl-0,01Rf2-0.36 Elemental analysis value: (CyoH+o2N2o02o ・10Ha0・5CH
aCOOH) C (%) H (%) N (%) Theoretical IF 47,47 7.07 13.84 Analysis value 47.20 7.30 13.92 Production example 3 Benzhydrylamine resin (0.56 meq. The following amino acids were sequentially subjected to condensation and de-Boc reaction in the same manner as in Production Examples 1-1 and 1-2 using 1.789/g resin (Protein Research Foundation Foundation).

29- Boa-Glu-OB, zl 0. 840
Boa-8or(Bzl)-OH0,74QBoa-A
la-OH0,470 Boa-Aro(Tos)-OH'1.07a twice B
oa-Gly-OH0,43゜Boa-Gln-ONP 0.92゜Bo
c -P he-0)1 0.66゜Boa
-Leu-OH”HQOO,62gBoC-Met-O
H0,620 Boa-Gln-ONP 0.92゜Boa
-8er(Bzl)-OH0,74a Thus, H-8
er(Bzl) -Gln-Met-L eu-Phe
-Gln-Gly-Aro(Toa) -Ar.

(Tos) -A Ia-8er (Bzl) -Gl
u (0Bzl)-resin 3.230 is obtained. 1 of these
．． 5g of hydrogen fluoride was mixed with 15IQ of hydrogen fluoride, 1 of anisole, 5IQ and 0.5mQ of ethanediol and heated at -20°C for 30 minutes.
After stirring for 30 minutes at 0°C, hydrogen fluoride was distilled off.
Dry the residue. Extract with 10% acetic acid, wash with ether, and freeze-dry. Then, it was purified with H-3er-Q In-Met
-L eu -P he-G In -G +y -
A NJ -A rQ -A Ia -S er -G
235 UQ of In-OH is obtained. Hereinafter, this will be referred to as "peptide C".

Rf value: Rfl-0,01Rf2-0.38 Elemental analysis value: (Cs a H9v N210t a S・4H20・
(as 2CH3C0OH) C (%) H (%) N (%) Theoretical value 46.52 7.12 18.37 Analytical value
46.43 7.38 18.51 Production Example 4 H-8er(Bzl)-Gln-Me obtained in Production Example 3
t-Leu-Phe-Gln-Gly-Ara(Tos
) -Ar.

(Tos) -Ala-8et(Bzl) -Glu(
OBzl)-0.8(l k:Boc-Tyr(
0.260 of C12Bzl)-OH was added to the above Production Example 1-■
The reaction was carried out in the same manner as in 1-0, and then the deprotection group and resin removal reaction were carried out in the same manner as in 1-0, and the same purification was performed to obtain H-T.
yr -S er -G In -M at -L
eu -P he -G In -G IV-A rQ
-A rQ-A Ia-Ser-G In-OH 9
Obtain 5-g. Hereinafter, this will be referred to as peptide DJ.

Rr value: Rfl-0,01Rf2-0.39 Elemental analysis value: (CayH+oaN2202oS・5H20°2CHs
As C0OH) C (%) H (%) N (%) Theory li1 47,86 7.01 17.29 Analysis (148,107,2517,03 <Production of antigen> Production example 1 Obtained in peptide production example 2 5.55 mo of peptide B and 10.23 mg of bovine serum albumin (BSA)!0.
16M *W1 Salt a** (0,13M Na
C1, pH-9, 0>2i+ Dissolve in Q. To this solution, add 3.0 I of the same buffer of BDB 1.641111.
Add Q and stir at 4°C for 5 hours. The reaction mixture is then dialyzed against 1Q of water, changing the water 5 times during the dialysis. Thereafter, the solution containing the peptide-protein complex was freeze-dried to obtain human γ-type interferon antigen 17.271 (+. This antigen will be referred to as "antigen ■" hereinafter.

Antigen ■ has an average of 8 moles of peptide B bound to moles of B5Al. In addition, the binding rate of this peptide 8 and BSA is as follows:
(Eluent: physiological saline, detection: oo 280nl, flow rate: 3m + 1/hour, fraction volume: 11Q each) When subjected to gel tF, the presence of unreacted BSA and peptide B was not observed. The fraction of peptide B bound to BSA and the fraction of other products (21 bodies of peptide B) were separated by the gel filtration, and a calibration curve of the standard concentration of the peptide dimer was created. This value was calculated by subtracting this amount from the amount of peptide B used as a starting material, assuming that the entire amount was bound to BSA. The same applies to the following antigen production examples.

33- Production Example 2 5Q and BS of Peptide C obtained in Peptide Production Example 3
Dissolve 25 g of A in 4 Q of water. Dicyclohexylcarposiimide (DCG) 2001 (] was added to this solution and stirred at room temperature for 5 hours.Then, the reaction mixture was mixed with 2Q of water.
Dialyze at 4° C. for 48 hours. During dialysis 5
Replace the water. Thereafter, the solution containing the peptide-protein complex is freeze-dried to obtain the human γ-type interferon antigen. Hereinafter, this antigen will be referred to as "antigen ■." Antigen ■ is B5A
On average, 12 moles of peptide C were bound to 1 mole.

Production Example 3 Peptide C 410 and BS obtained in Peptide Production Example 3
Dissolve 20+o of A in 2Q of ammonium acetate l1ii solution (0.1 mol, pH-7.0).

Add 0.1 molar geltaraldehyde solution to this solution.
11112 and stirred at room temperature for 5 hours. The reaction mixture is then dialyzed for 48 hours at 4° C. against 1Q of water. Thereafter, the solution containing the peptide-protein complex was lyophilized to obtain 22% of the human gamma interferon antigen.
1 (l) is obtained.Hereinafter, this antigen will be referred to as "antigen ■".

The obtained antigen (2) has an average of 9 moles of peptide C bound to 1 mole of B5A.

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. In addition, in Preparation Example 2 of the antigen, peptide D was substituted for peptide C.
The target antigen is obtained in the same manner using .

Production of Antibody> Production Example 1 After dissolving 30 μg of the antigen obtained in Antigen Production Example 1 in 1.5 μg of physiological saline, add 1.5 μg of Freund's auxiliary solution to this.
The suspension prepared by adding IQ was added to seven rabbits (New Zealand white rabbits).
bblts) (2.5-3.0 kO) is administered 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 antisera, thereby obtaining human γ-type interferon antibodies (hereinafter referred to as "antibodies 1 to 2").

Production Example 2 Human γ-type interferon antibodies (antibodies ① to XVI) were obtained in the same manner as in Production Example 1 above, except that 60 μg of antigen ① was used for each of 10 rabbits.

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 in 0.5 molar phosphate II solution.
1 (DH7,5) 10μQ with Na (”51) (carr
ier free N, E, N, ) Add 20 μQ of 0.5 molar phosphate mti solution of 1 millicurie, then add 20 μQ of 0.5 molar phosphate buffer of 60 μQ of chloramine T. Stir for 25 seconds at room temperature.
0Ig of sodium metabisulfite (~82SQ Owl
The reaction is terminated by adding 20 μQ of 0.5 M phosphorus*mm buffer solution. Next, 100 μQ of a 1% cold aqueous sodium iodide solution was added to the reaction mixture, and the reaction mixture was subjected to high performance liquid chromatography [10 to 40% ethanol grad
lentlo, 1M Tris HcQ1 buffer (1) H8,O
) 1,0g+Q/i+in, reversed phase chromatography] to obtain 125I S-labeled peptide B. The radioactivity of the labeled peptide was 1128 μC1/μg.

Measurement of 0 titer The titer of the antibody obtained above is measured as follows.

10,102,103 of each antibody in physiological saline.
．． 104.105......Diluted (initial) twice, and added 125 labeled peptide (approximately 9500Q of the labeled peptide obtained above) to 100μQ of each of these.
D-) 0.1111 and 0.
05 molar phosphate buffer (pH-7,4) (0,25
%BSA, 10i+ MED-A and 0.02%Na
Add 0.2IQ (containing Ns) and incubate at 4°C for 24 hours to form a conjugate of antibody and 1125-labeled antigen.
Dextran - unreacted (does not bind) by charcoal method and centrifugation method (4°C, 30 minutes, 3000 ml)
The antibody is separated from the 11125-labeled peptide, its radiation is counted, and the binding rate (%) of the antibody to the 1125-labeled peptide at each dilution concentration is measured. Antibody 112 on the vertical axis
The binding rate (%) with the 5-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. The dilution ratio of the antibody at which the binding rate is 50%, that is, the titer of the antibody is determined. The results obtained for antibodies I-XVI are shown in Table 1 below.

1st surface force Antibody titer I 3280 rX 3000I
<1000 x 18400m 2000
XI 44000TV 19200 XII 1
80000V 80000 XI[[168003
8- Vl 52000 XrV 20
0000Vl <1000XV
30800■ Death
XVI 12800XVI 78
00 All antibodies obtained according to the present invention react with high specificity to human γ-type interferon.

(that's all) 39- =321-

Claims (1)

[Claims] ■ General formula % formula % [In the formula, R represents a hydrogen atom or an H-Tyr group. ] and the general formula % formula % [wherein R is the same as above. ] Human γ- selected from the group consisting of peptides represented by
Interferon-related peptides.