JPS5835157A - Lymphoblastoid interferon having peptide at terminal n and its derivative - Google Patents

Abstract

NEW MATERIAL:The human lymphoblastoid interferon having peptide at terminal N of formulaI(R is H or group of formula II-IV) and its derivative. EXAMPLE:H-Thr-His-Ser-Lue-Gly-Asn-Arg-Arg-Ala-Leu-Ile-Leu-Leu-Ala-Gln- OH USE:For the preparatiopn of an antibody having specificity to human lymphoblastoid interferon and useful for the purification of the interferon by affinity chromatography. PROCESS:The peptide of formulaIcan be synthesized by the conventional peptide synthesis (e.g. azide process, chloride process, PCC process, etc.), e.g. the stepwise process comprising the stepwise condensation of respective amino acids to the terminal amino acid, or the coupling of several fragments constituting the peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention has no novel trim hob 5 stoid interface 0
This invention relates to N-terminal peptides and derivatives thereof.

In this specification, when amino acids, peptides, membrane-retaining groups, active groups, etc. are expressed by abbreviations, IUPAC, IU
The regulations in Section B or the symbols commonly used in this field shall be followed, examples of which are listed below. Furthermore, when an amino acid or the like can have different optical isomers, only one isomer is shown unless otherwise specified.

L-Iris: 0 Isin 11g : Iso 0 Ishishi ALa: Alanyl (d*: Kurta Σshi Thr: ) Leonine His: Shisutijishi Say: tlisiaty: Talysin As: Asparagus f-Arg: Al-Koniji Asp: Asuba 54 silicic acid pro: Z: carbovecilyl group Su: succinic acid group Top: p-toluenesulfonyl group 1ine: tertiary dicarbonyl group It is an antiviral glycoprotein or protein that is produced when infected with viruses, and it has been attracting attention in recent years as it is believed that it can be used to prevent or treat viral diseases. There are currently 0 elucidated human interfaces (Leucocyts).
es Intarfaravh, Lymp 5 o Haz
te*d circuit tarfgran), β-type isitafue O'xi (FihroblaztIrLtgrfar
aF&) and r-type interface 0ci (1wmu@gIn
tarfartnh), but no technology has yet been developed to purify these tarfarts into a single glycoprotein or protein.

The present invention relates to a new antibody for the purpose of providing a technique for isolating and purifying human α-type interferovirus, particularly limboplastoid interferovirus, an antigen for producing the antibody, and a method suitable for producing the antigen. The purpose of this research is to provide tools and techniques for acquiring them.

As a result of intensive research for the above-mentioned purpose, the present inventors have discovered a specific N-terminal peptide of a human trimester newly synthesized by the present inventors and its 0Il.
When a conductor is used, a novel plastoid interfacial antigen can be produced, and when the antigen is used, a new antibody having specificity for the plastoid interfacial antigen can be produced. By using the antibody in Affinity Take 5 fee,
We have found that it is possible to purify the target human 41Wiisitafue0. The present invention has been completed based on the above-mentioned new findings.According to the present invention, it is possible to produce plastoid isiterphein by a simple operation from synthetic beetide, which can be easily produced in large quantities. Antibodies that are useful for purification and exhibit specific reactivity can be produced industrially and advantageously, thus establishing a technology for purifying the present plastoid ester membrane.

The new synthetic peptide according to the present invention has the following general formula: 1)
It is expressed as

R-L-μm7Z--Law-L-Iris-AL@-(Aru〇H+1) [In the formula, R is a hydrogen atom, H-Thr-His
-5ay-Lgu -GLy-Ash-Arc -Ar
g-Arc group, H-5ay-lap -Lag -pr
o-GL@-Thr-1ha-5at-Zgm-GLy
-Azn -Arg -Arg -Arc group, or H-
Tyr-5ay-Asp-Lau-Pro -01%-
Thr-His-5#r-Law-Gly-Arm
-Arg-Arg -Arc group. ] It is a peptide corresponding to the N-terminal peptide chain or a derivative thereof. These methods are commonly used for peptide synthesis, specifically, "ThgPaptidaz".
) J Volume 1 (1966) (5chr''”jdtr
add f, uhh-author, AcarLgmic Pr
ays, May York.

U, S, A,”J or “Peptide Synthesis” [written by Azumaya et al.

Maruzen Co., Ltd. (1975)], for example, the azide method, the acid anhydride method, the acid anhydride method,
Mixed acid anhydride method, DCC method, active ester method (P-nide 0
Phenyl ester method, N1ZFO + succinic acid! ester method, cyanomethyl ester method, etc.), method using Woodward reagent X, carboxy:! Taril method, redox method, DCC/additive (HONB, HOB
t, H2Sx) method, etc. In the above method, both solid phase synthesis method and liquid phase synthesis method can be applied, but liquid phase synthesis method is preferable.

Normally, the compound of general formula (1) is synthesized according to the general polypeptide synthesis method described above, for example, by the so-called step quiz method in which amino acids are sequentially condensed one by one to the terminal amino acid, or by dividing into several compounds. It is manufactured by a method of chromatography and calapuri sifting. In detail, the above-mentioned Peptide Co., Ltd. has two types of 5-fold fragments that are bisected at any position of the bond.
A raw material having a reactive carboxyl group corresponding to one of tamento and a raw material having a reactive amino group corresponding to frucmesito on the other side are condensed by a conventional method for peptide synthesis,
When the resulting condensate has a protecting group, it can be produced by removing the protecting group by conventional means. Furthermore, the general formula (
When using aspartic acid in the reaction step for producing the peptide in 1), it is often desirable to protect it, and in the final step, at least one of the amino acid residues constituting betutide is usually protected. Remove all protecting groups from the protected peptide.

In addition, in the synthesis reaction step of bezutide of the above general formula (1),
Functional groups that should not participate in the reaction are protected by conventional protective groups, and after the reaction is completed, the film-retaining group is removed. Furthermore, the functional groups involved in the reaction are usually activated. These reaction methods are known, and the reagents used therein can be appropriately selected from known methods.

Examples of protecting groups for amino groups include carboxylic acid, tart-butylic dicarbonyl, tart-amylic dicarbonyl, isobornyl dicarbonyl, P-meth+cybecidyl dicarbonyl, 2-dicarbonyl, and dicarbonyl, atamantyl dicarbonyl, triple oatyl, phthalyl, formyl,
Examples include O-nitrophenylsulfenyl, diphenylsulfinothioyl, and the like. Examples of protective groups for carboxyl groups include alkyl esters (e.g. ester groups such as methyl, ethyl, divinyl, butyl, tart-butyl, etc.), benzyl esters, P-nitrobenzyl esters, P-meth+cybesidyl esters. , P-tri-O-benzyl ester, benzyl-benzyl ester, carbobenly+dihydrazide, tart-dimethyl-dicarbonyl hydrazide, trityl hydrazide, and the like.

Examples of the quanidino group-protecting group of alf-nidi include nido0, tosyl, P-meth+cybecysulfonyl, carbobecyly+cy, isopornylo-dicarbonyl, atamasitylo-dicarbonyl, and the like. Further, the quanidino group may be maintained in the form of a salt of an appropriate acid such as becisesulfosic acid, tornidisulfonic acid, hydrochloric acid, and sulfuric acid.

The hydroxyl group of threonide, for example, can be preserved by etherification if it is esterified, but it is not necessarily necessary to preserve it as a film. Groups suitable for this esterification include, for example, lower alkanoyl such as acetyl, aroyl such as benlyyl, groups derived from sulfuric acid such as penlyyl dicarbonyl, and ethyl dicarbonyl. Examples of groups suitable for etherification include benzyl, tetrahydrohyranyl, tart-butyl, and the like.

Activated carboxyl groups include, for example, the corresponding acid 05 ide, acid anhydride or mixed acid anhydride, azide, active ester (methyl alcohol, ethyl alcohol, benzyl alcohol, Besitak 00 phenol, P
- ditrophenol, N-human 0 + cisakusiciimide,
Examples include N-dibenztriaryl, N-dibenztriaryl, ester with diimide in carboxylic acid, and the like. The peptide bond forming reaction is carried out using a condensing agent such as dicarbodiimide,
Karuboshii! In some cases, the reaction can be carried out in the presence of a carbodiimide reagent such as Taryl or Tetraella 110 Sphite.

Depending on the type of group represented by R, the peptide represented by the above general formula (1) can be expressed as (I) as shown below.
It is manufactured according to the method of ~(5).

(I) When R represents a hydrogen atom A-, Itα-B (2) ↓ H-GZs-OH1a) A-ALa-Gin-OH (4) ↓ H-Ala-Gln-OR (5) ↓ A- , Lgu-B(6) A-Law-Ala-Gin-OH(71H-La
w-ALa-GZs-OH11) ↓ A-Lm Juku-B(
6) A-Law-Law-Ala-Gin-OH (8) ↓ H-LaI&-Lgu-ALa-Gin-OH (101
↓ A-ILa-B (11) A-ILa-L-μmL-Juku-ALa-GL-ON
(lkon↓ H-ILa-Law-Law-ALa-Gin-OH(
1:i↓ A-L-μ-B(6) A-Lgu-ILg-Law-Law-ALa-GZs
-OH(14↓ H-Lau-ILg-Law-Law-ALa-(da
-OH (11(u) R is E-Thr-Hip-5a
y-Law-GLy-AyF&-Arg-Arg-AL
When indicating a group, A-Arg-B (l groan↓ H-ALa-B η A-Arg-ALa-B Ql H-Arg-Ala-B Fist 4 C ↓ A-Asn-B @ ↓ A-Asn -Arc -Arc -ALa-B (2
4Lag-ALa-GLn-OH@ ↓ H-Azn-Arg-Arg-Ale-Law-IZa
-Lgu-Lau-ALa-GLn-OH@A-Thr-Hip-5ay-Lash-(dy-As
h -Arc--OH@↓ -QHfl14 However, 10 is based on the following process.

A-L-μ-B (61 ↓ H-GLy-B @ A-Law-GLy-B @4 ↓ x-Law-cLy-B C(l) ↓ A-5ar-B (2) A-5ar- Lgu-GLy-B -↓ H-5ar-Law-GLy-B @4)↓ A-
Thy-His-B @IA-Thy-His-5a
y-Law-GLy-B - US GIT) R is H-5
et-Asp-Law-Pro-Gin-Thr-
Hlp -Say -Lau-C;ly -Ash-A
rg -Ag-ALa group A-Pro-B@η ↓ H-GA-B- A-Pro -Gja-B k4 ↓ H-Pro - to An-B 141 ・↓ A-Lau-B i@ ) A-Lau-Pro -GLn-B -↓ H-Lag-Pro-Gin-B IdJ↓ A-A
sp-B@3 A-Asp-Law-Pro -Gjs-B M↓ Tomi A-Asp-Lgu-Pro -Gin -B (45
↓ M-Asp-Lgu-Pro -GLn-B i41↓
A-Ear-E@A-5ay-Asp-Law-Pro-Gin-B
@ηH-Thr-Hip-5ay-Lag-GLy
-Azn↓ -Arg -Arc -ALa-Law
-ILm-Lath -Lau-ALa-GZa-OH
@1 -ILa-L-μmL-μ-Am-GL-OHM↓ avtR is H-Tyr -Say-Asp-Law-P
ro-GLn-Thr-Hip-5ar-Law-G
In the case of Ly-Ass-Arg-Arg -ALa group A-5ay-Asp-Law-Pro -Gin-B
@η↓ H-5ay -Asp-Lgu=-Pro -GLn-
B i4↓ A-Tyr-B 〇A-Tyr-5ay-Asp-Law-Pro -GL
n-B Lau-Thr-His -Sar -Law
-GLy -Ash↓ -Arg-Arg -ALa
-Lgu-ILg-Lgu -jvu-ALa-GLn
-OHel A-Tyr-5ay-Asp-Law-Pry-Gln
-Thr--Lsu-11g-Law-Law-ALa
-GLn-OHI3↓ -Law-ILa -Law-Law-ALa-GmuOI - [In the above methods (I) to GV), A is a protecting group for an amino group, and B is an active group for a hydroxyl group or a carboxyl group. , C represents a guanidino group-protecting group of an alkyl atom, and D represents a protective group for an asb-4'J acid, respectively. ]In the above, A
Preferably, the active group of the carboxyl group shown in B is a dicarbonyl group in Bat%Z%P-meth+cybecidyl, etc., and the active group of the carboxyl group shown in B is preferably a dicarbonyl group in the N-human 0+sysuccinimide group, an isobutyl group, etc. Examples of mixed acid anhydride residues, azide, etc. are preferable as C, and preferable examples are nido 0, tosyl, etc., and preferable examples of D are besidyl oxy groups.

In the above method (I), amino acid (2) and amino acid (3)
) can be carried out in the presence of a solvent.

As a solvent, various solvents known to be usable in the betutide condensation reaction may be used, such as anhydrous, water-containing methylformanide, dimethylsulfonate, pyridyl, and methylformanide.
Solvents such as 0-form, diol-methane, dichloromethane, tetrahydrobrassi, ethyl acetate, N-methyl bi-hydro-hydride, hexamethyl phosphoric triamide, or a mixture thereof may be used. The ratio of 1 ml of amino acid to amino acid (2) is not particularly limited and can be appropriately selected within a wide range, but usually the latter is used in an equivalent amount to 5 times the amount of the former, preferably 1 ml to 5 times the amount of the latter, preferably 1 ml of the former. It is best to use 5 times the amount. The reaction temperature is within a range known to be usable for peptide bond formation reactions, usually about -40 to about 60°C, preferably about -2
The temperature is appropriately selected from the range of 0 to about 40°C. The reaction time is generally from several minutes to about 30 hours.

Method (I) Bezutide (i) and amino acids (6) in K
reaction between peptide (1) and amino acid (6),
The reaction between bezutide (to) and the amino acid (rr) and the reaction between the bezutide ring and the amino acid (6) are the same as the above amino acid (11).
It can be carried out in the same manner as the reaction between and amino acid (3). In addition, in method (If), the reaction between amino acids and amino acids, the reaction between bezutido and amino acid, the reaction between bezutide and amino acids (foundation), and the reaction between bezutide and peptides (If9) The reaction, the reaction between Bezutide and peptide, the reaction between amino acid (6) and amino acid 4, the reaction between Bezutide and amino acid, and the reaction between Peptide and Bezutide are also carried out in the same manner. Furthermore, in method GII), the reaction between an amino acid (goods) and an amino acid, the reaction between peptide and an amino acid (6), the reaction between a peptide and an amino acid, and the reaction between a peptide and an amino acid (good) can be carried out. Reactions and reactions between Bezutide and Bezutide 4-dos, and method QV) The reaction between peptides and amino acids in K and the reaction between Bezutide and Bezutide are also as described above. You can do it in the same way.

Peptide +41, (71, (
9), hit, (14, exposed, -1 (to), (goods), -1-
The elimination reaction of the retaining group A of 1-1, -1←η, - and - is carried out in a conventional manner. The method is as follows:
For example, reductive methods (e.g. hydrogenation using vanadium, vandium kurogo catalysts, reduction with metallic sodium in liquid ammonia), acidolysis (e.g. trifluoroacetic acid, hydrogen fluoride, metadisulfonic acid, hydrobromic acid) Acidolysis with strong acids such as

Hydrogenation using the above catalyst can be carried out using, for example, hydrogen pressure type atmospheric pressure, 0
It is possible to produce seeds that are carried out at ~40°C. The amount of catalyst to be used is usually about 100 V to IF, and the reaction line generally ends in about 1 to 48 hours. The above acidolysis is carried out without a solvent, usually at about θ to 30°C, preferably from 0 to 20°C, and the reaction is generally completed in about 15 minutes to 1 hour. The amount of acid to be used is usually about 5 to 10 times the amount of the raw material compound. In addition, in the reduction with metallic sodium in liquid asimonia, metallic sodium is used in such an amount that the reaction solution is colored permanaged blue for about 30 seconds to 10 minutes. This reduction is carried out by applying electricity to -40 to -70℃.
It is held in Wado.

Furthermore, the protecting group (C) of bezutide (2) and the protecting group (1) of bezutide can be similarly deprotected by the reductive method described above.

Amino acids used in the above methods (I) to (5)
(3), 01), ring, (goods), (goods), (b), - and (goods) may be known commercially available products, and peptides (i14,
(Taiwan), -1-1 (Treasury), (47), and - can be used as known commercial products or those obtained by the noodle expansion synthetic acid anhydride method, the azidation method, or the like. The mixed acid anhydride method is carried out using a ruhalocarboxylic acid in alcohol in the presence of a basic compound in a suitable solvent. Examples of the alkoxylic acid used include methyl formate, methyl formate, ethyl formate, ethyl formate, and isobutyl formate. Examples of the basic compounds include triethyl amici, trimethyl amici, pyridine, dimethyl anilis, N-methyl ester, 1,5-diazapisic O(4,3,01 nonene-5CDBN), 1-5
'-, :;Azapisik o (5,4,03 undet sea 5CDBU), 1,4-diazapisik 0 (2,2,2)
Organic bases such as Octasi (DABCO), potassium carbonate,
Examples include inorganic bases such as sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate. The solvents that can be used include various solvents commonly used in the mixed acid anhydride method, specific examples of which include halogenated hydrocarbons such as Ka methylene chloride, Ri00 form, and Dig00 ethane, and aromatic compounds such as benzene, tornidi, and medium silane. Hydrogen, diethyl ether, tetrahydrobrush, ethers such as dimethic acid, esters such as methyl acetate, ethyl acetate, N, N-dimethyl, dimethyl sulfate, he + samethyl phosphoric acid triamide, etc. Examples include 0 tonic polar solvents. The reaction is -
The reaction is carried out at a temperature of 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, in the azidation method, first activated carboxyl group,
For example, this reaction is carried out by reacting a carboxyl group activated with an alcohol such as methyl alcohol, ethyl alcohol, or besidyl alcohol with a hydrate in a suitable solvent.

Examples of solvents that can be used include Dionaka San, Dimethyl Honlumua! For example, solvents such as dimethyl chloride, dimethyl chloride, or a mixed solvent thereof can be used. The amount of Shidorajishi hydrate used is usually 5 to 20% based on the activated carboxyl group.
The amount should be 7 times the molar amount, preferably 5 to 10 times the molar amount. The reaction is usually carried out at a temperature of 50°C or lower, preferably -20 to 30°C.
CK is performed. In this way, it is possible to produce a compound in which the carboxyl group of the terminal amino acid is substituted with didrazine (hydro-5-disi derivative). A compound in which the carboxyl group of the terminal amino acid is substituted with azide can be produced by reacting the above-obtained doradish visiting conductor with a nitrous acid compound in a suitable solvent in the presence of an acid. Hydrochloric acid is usually used as the acid. Examples of solvents that can be used include dioxins, dimethylformamide, dimethylformamide, and mixed solvents thereof. Examples of nona-nitrite compounds include sodium nitrite, isoamyl nitrite, and chloride 0
Sil, etc. can be used. Such a nitrous acid compound is generally used in an amount of 1 to 2 times the molar amount, preferably 7 molar to 1.5 times the molar amount of the hydrochloride derivative. The reaction is usually carried out at -20 to 0°C, preferably -20 to -10°C, and is generally completed in about 5 to 10 minutes.

The peptide of general formula (1) produced as described above is isolated and purified from the reaction mixture by means of separation such as extraction, partitioning, column flow, etc.

In this way, the N-terminal peptide of the synthetic heptide, ie, tricyphodlastoid isiterphae0, represented by the general formula (0) and its derivatives are obtained.

The synthetic betutide obtained in this way has 1125,
z If you introduce radioactive iodine such as L34L, it will be better.
It can be used as a labeled peptide, which is a raw material for producing a labeled antigen used in radioimmune assay (RIA method). The above radioactive iodine can be introduced by the usual iodination method, for example, the oxidative iodination method using Olamin T [W,
M, Hμnear d-F, C, Greenwo
od; Nature, 194, 95 to P'
C1962), B no. och-ko, J, 89. P114
(196'S)]. For example, the iodination method described above is carried out in a suitable solvent such as 0.2M phosphoric acid buffer (pH = 7.4) in the presence of phosphoric acid buffer at room temperature for about 30 seconds. . The proportions of bezutide, radioactive iodine, and tiro-53 molecules used are, for example, when one radioactive iodine is introduced into tio-tide, 1 nanoL of tyro-53 molecules contained in pezurad.
About 1 mm of radioactive 3-D is applied to the
It is best to use about 10 to 100 nanotol of Lamin T.
Also, when introducing two radioactive iodines into tyrosine,
Radioactive iodine is added to 2.0 μm per 1 nano℃ of 90-synthene molecules contained in Bezurad. :Su+1-ri1 degree, ri0u! ShiT
It is preferable to use about 10 to 100 nanomoles. The thus-produced radioactive iodine-labeled ebetide can be extracted, distributed, and columnarized using conventional separation methods.
It is isolated and purified by dialysis.

The peptides obtained in this way can be stored fresh if necessary by freeze-drying.

Furthermore, the synthetic peptide represented by the above general formula (superscript) can be used as a material for producing Shitrim Honzu5 Stoid Ishita Ifue O Anti-M.

The method for producing the above-mentioned trimmed protein interfering antigen will be described in detail below.

The present plastoid interferon antigen in human trim includes at least one of the synthetic betatides represented by the above general formula (1).
It is produced by converting the seeds into Yazutesi and reacting them with a carrier in the presence of a Yazutesi carrier-binding reagent.

In the above method, the carrier to be bound to Yaduteshi is:
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 Alz! Animal serum albumin, such as bovine serum albumin, rabbit f serum albumin, human serum albumin, and azalea serum albumin! species, horse serum globulin, bovine serum globulin, rabbit upper serum globulin, human serum globulin,
Serum of animals such as sheep serum, etc., horse serum, cow chicken, rabbit, human chicken, shitsujichi, etc. Chi0ri0zurin of animals such as, 7ri0bishi to horses, ℃ri0purin to cows, rabbit hemog Opurin, 7riOpuril to humans, and sheep! : Animal hemolymph such as Ri0purine, animal hemocyanin, protein extracted from roundworms (Ascaris extract, see JP-A-56-16414), boririjisi, borikurtanisic acid, Examples include a lysine-curtamic acid copolymer, a copolymer containing lysine-curtamic acid, and lysine-curtamic acid copolymer.

The Ascaris extract will be detailed below.

The Ascaris extraction method is based on the Ascaris extraction method.
It is extracted from the ground product of Uuwn) according to the usual protein extraction method. Extraction solvents such as water, physiological saline, 50
Various known protein extraction solvents such as timethanol, an aqueous ethanol solution, and a near-neutral buffer solution can be used, and physiological saline is particularly preferably used. The above-mentioned extract is specifically produced by, for example, the following method. That is, after removing the contents of the pig incubator and washing it with physiological saline, it is preferably shredded to facilitate extraction, and the shredded material is added to a protein extraction solvent such as physiological saline. Extract while stirring at ℃. This extraction is usually carried out at low temperatures, preferably from about 2° to 10°C.

The extract thus obtained is then centrifuged, the supernatant is collected, and after dialysis, the dialysate is freeze-dried, or the dialysate is centrifuged again, the supernatant is collected, suspended matter is removed, and then frozen. By drying, the desired Ascaris extract is produced. This can be further carried out using conventional protein purification methods, such as dialysis, gel filtration, adsorption, etc., as necessary.
After purification, it can be used in the present invention.

Further, the above-mentioned Ascaris extract is, for example, J, 1wna
- μle,.

11L260-26B (1973), J, 1 set of connections.

122, '302-30B (1979), 1.11 Deception, 98°893-900 (1967) and Am,
J, Phyziol.

199.575-578 (1960) or further purified versions thereof.

A wide range of reagents commonly used for preparing antigens can be used as the reagent for binding to the carrier.Specifically, those that cross-link amino groups, such as Tario+Sall and MA0:J dialdehyde, can be used. , tartar aldahyde, succisialdehyde, aliphatic dialdehydes of the adiboaldase type, crosslinking thiol groups with thiol groups, such as N,N'-e-phenylesidimale! Do, N,
Shimmering imide compounds such as N'-11-phenylene dimaleimide, which cross-link amino groups and thiol groups, such as metamaleimide besylyyl-N-hydro+cissuccinimide ester, Imayachi (maley:domethyl)-Schiff 0
Hechu San-1-Carbo Naka Shiru N'-Citro + Sisukushii! Maleimidocarboxyl-N-hydroester, etc. ester compounds, reagents used in the usual ethyl bond-forming reaction for forming an amide bond between an amino group and a sil group in a carboxyl group, such as N,N-dicyclo
Hesi-diylcarbodiimide, N-ethyl-N'-dimethylaminocarbodiimide, 1-ethyl-3-diisodiylaminocarbodiimide, l-Schif0+silu3-(
2-1 Dehydration condensation agents such as carbodiimides such as linyl-4-ethyl)carbodiimide, sialinium arylcarboxylic acids such as enyl acetic acid, and ordinary benzitide bond-forming reaction reagents, such as the above dehydration condensation agents. A combination can also be used.

The reaction for producing the antigen of the present invention may be carried out in an aqueous solution or at pH 7.
The reaction is carried out at 0 to 40° C., preferably around room temperature, in a conventional buffer solution having a pH of 8 to 9, preferably a buffer solution having a pH of 8 to 9. 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-0,2I
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 2 to 6 times the weight of the carrier, preferably 3 to 5 times the weight, and 5 to 10 times the weight of the hapten-carrier binding reagent. Through the above reaction, a human plastid interferon antigen consisting of a peptide-carrier complex in which a carrier and a hapten are bound through the mediation of a hapten-carrier binding reagent is obtained.

The antigen obtained after the reaction is completed according to the conventional method, for example, dialysis method,
It can be easily isolated and purified by gel V filtration method, fractional precipitation method, etc. 9. The antigen can be preserved by conventional lyophilization methods.

Thus, a tricyphoplastoid interferon represented by the above general formula (1) consisting of a complex of at least 1 m of the N-terminal compound of tricyphoplastoid interferon and its derivative and a carrier: J5-pheon Obtain the antigen. The antigen usually contains an average of 5 to 20 betutides per mole of protein.
Both of these antibodies are capable of producing antibodies with high specificity against trimophoblastoid interferon with good reproducibility. In particular, the binding ratio of the peptide to the above protein is 1;8 to 15.
It is preferable that antibodies with higher specificity, higher titer, and higher sensitivity can be produced.

Preparation of antibodies using the antigen obtained above is carried out as follows. That is, it is carried out by administering the above-mentioned antigen to a mammal and collecting antibodies produced in the body.

There are no particular limitations on the mammal used for antibody production, but it is usually desirable to use a rabbit cut. For antibody production, a predetermined amount of the antigen obtained above is diluted with physiological saline to an appropriate concentration, and a supplementary solution of Frauside (Coll11pLatt FrauyL'zA K1
1a1%t) to prepare a suspension, which may be administered to mammals. For example, rabbits may be immunized by subcutaneously injecting the above suspension (antigen amount: 0.5 to 511 v/dose) and then administering it every two weeks for 2 to 10 months, preferably 4 to 6 months. Antibodies are collected after the final administration of the suspension, when antibodies are produced in large quantities, usually between 1 and 2 of the final administration.
After a week, blood is collected from the immunized animals, centrifuged, and serum is separated and collected. In particular, according to the method of the present invention, based on the specificity of the antigen used, it is possible to obtain antibodies with extremely high specificity, high titer, and high sensitivity for human plastidoid spores. There are advantages.

The plastoid interferon antibody obtained in this way has particularly excellent specificity for tricypholastoid interferon, and can be used for plastoid interferon production using the RfI method, which is desired in this field. This is a useful product that enables the quantification of Idoishiterfue 0 with high precision. The antibody can also be used in the enzyme immunoassay (xzA) method and the fluorescence immunoassay (F) method by labeling it with an enzyme or a fluorescent substance.
IA) Can be used for law, etc. 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 producing the present invention, an antigen using the same, and an antibody from the antigen will be given below, but the present invention is not limited thereto.

The Rf value in each production example was measured using a thin layer on silica gel using the following mixed solvent.

R7K...l-butanol-acetic acid-water (4:1:5)
RfI...1-Butanol-pyridic acid vinegar 112-water (5:10:3:12) Synthesis of peptide> Production example 1 1. Production of Atα-GL-OH Z-ALa
-05u To a 4-8Of tetrahydrofu5silo solution, a 40-solution of H-Gl*-OK 2.199 in water and 2.1 OIlj of triethylua and trichloride were added, and the mixture was stirred at room temperature for 20 hours. The water was distilled off and the residue was extracted with butanol. The extract was washed with 2% acetic acid, and the tanol was distilled off. The precipitated material was collected in a furnace and reprecipitated with methanol-ethyl acetate to give ;lney At--(A
@-OH3-87t is obtained.

Rf, 0.41 Rf, 0.56 Elemental analysis value (as C□6H2□N306) Calculated value (H) C54,69R6,02#11.96 Actual value (H) C54,50#6.31 11. 622 (→,
Production of H-Mα-GA Rou〇H Z-ALa-GL Route-
OH3,5Of was dissolved in water 5o- and methanol 30- and catalytically reduced using palladium as a catalyst to obtain H-ALa.
-GZa -ON is obtained.

7? f, 0.04 2(A), 2-Law-ALa-Gin-OHO production Z -Lgu-O5tb 3.97 t, above (
') Te obtained H-ALa-Gl-OH and triethylanisi 1.39mt dimethyl fluorine! Dissolve at 50 mK and stir at room temperature for 20 hours. Dimethylformamide was distilled off, and the remaining liquid was extracted with ethyl acetate. The extract solution is 1N
- Wash 3 times with quesic acid and 5 times with water. Ethyl acetate was distilled off, ether was added to the residue, the precipitate was removed, dried, and reprecipitated with methanol-ethyl acetate.
-Law-Ale-(dn-OK 2. Obtain l5f.

Rf, 0.62 Elemental analysis value (as C22H32407) Calculated value CTo
) C56,88H6,94N 12.06 Actual value (
h) C56,41H6,80N 12.183(a
), H-Law -ALa-GZa-OH (Q production Z-Lgu-ALa-GLn -OK 2. l Of
Add 20 ml of acetic acid solution containing K 25-hydrogen bromide and leave at room temperature for 1 hour. Add dry ether to the reaction solution and
-LaM-ALa-Gin-OK is obtained.

Rf”: 0.10 3(h), H-Law-Law-ALa-Gin
-OHO production Z-4gs-O5u 1-96 f,)
ReI Chi'Ata! Shi0.63d and H-L-Magazine-AL
Dissolve in a-01%-oHt-dimethylformamide 5 (14) and stir at room temperature for 20 hours.

5 Methyl lumamide is distilled off, Otori M quesiic acid is added to the residue, the precipitated crystals are taken off, and the crystals are washed with water until the P solution becomes neutral and dried. Washed with methanol-ethyl acetate to give Z-LaK-LaM-ALa-GLa-0H1,
Get 63f.

ff7”: 0.58 Rf, 0.64 Elemental analysis value (as C28II, 3N508) Calculated value (
*) C5B, 22 H7, 50ff12.12 actual value (%) C57, 85M, 7.90 #11.
964(a), H-Lau-Lau-Ala-G
in-OHO production Z-Lag-L-Iris-ALa-GZs
-OH1,50f K 25 acetic acid solution containing hydrogen thibromide 2
Add 0 Mt and stir at room temperature for 1 hour. Dry ether was added to the reaction solution, the precipitated solid was collected by F, and H-Law
-Law -ALa -GLn -OH is obtained.

17.0.19 4(h), Z-ILm-Lgl&-Law-ALa
-Production of GLa-OH Z-11a -05u 1-4
1 f, H-Lem-Lgvb-ALa obtained above
-Ghb-OH and triethylamine 0.36- are dissolved in dimethylformamide 50- and stirred at room temperature for 20 hours. Dimethylformamide was distilled off, and the residue was
Add IN citric acid, collect the precipitated crystals and wash with hot methanol to give Z-Ha-Lau-Lam-ALa-
GLa-OH1,l7f is obtained.

H7,0,61 Rf, 0.11 Elemental analysis value (as C3, y, N6o) Calculated value (chi'
) C59, Violet 1 ff7.87 N 12.1
6 Actual side value C1) C59, 23H7, 80N 12.
025(a), H-IZg -LgI&-Lau-
ALa-GLn-OHO production Z-11g -Lau-L
Add 25-thiobromide-containing acetic acid 15- to aw-Ala-GZn-OH1,1Of and stir at room temperature for 1 hour. Dry ether was added to the reaction solution, and the precipitated solid was collected with F and H
Obtain -ILm-Lgu-ALa -GL-U.

Rf”, 0.25 5(A), Z-Law-ILm-Law-Law-
Preparation of Ala-Gln-OH Z-Law-O5μ0.69F, H-H obtained above
a-La@L-LaW-ALa-Gtn-OH and triethylamine 0,221+7 in dimethylformamide 5
Qm and stirred at room temperature for 20 hours. Dimethylformamide is distilled off, succinic acid is added to the residue, and the precipitated substance is collected in F, washed with water and dried until the F solution becomes neutral. Wash with hot methanol and remove Z-1g gourd-ILg
-Labuty-Law-ALa -Gin -OK 1.1
We get 0'f.

Rf, 0.58 Rf, 0.71 Elemental analysis value (as C4°H65N70□) Calculated value (
h) C59,75H8,15N12.19 actual measurement value (
It) C59,6048,02ff11.926,
H-Lau-Ha -Law-Law-ALa -
(drb-OHOH Z-Law -ILg-Law-
Lgu-ALa-GLn-OHO,5Of was dissolved in methanol 50- and 10 m acetic acid 10- and catalytically reduced using palladium as a catalyst to obtain H-Lgu-Ha-Law -
118-Ala-Gin-OH is obtained. This will be referred to as "peptide A" hereinafter.

, Rf": 0.23 Rf": 0.61 Elemental analysis value (as C32N, N708/2H20)
Calculated value (9g) C54,45H8,99N 13.
89 actual measurement value (chi) 054.3Of8.81 N
13.98 Production Example 2 1. Boc-ALa-NHNH1OH-made Boc-A
La-OH4,99t%ME2-NH-Z 4.36
and 5.44 f of dicyclohydrylcarbodiimide were dissolved in 150 wjK of dofuran and stirred at 4°C for 20 hours. The precipitated solid was removed by distillation, the F solution was distilled off, ether was added, the precipitate was collected in a furnace, and reprecipitated from ether and petroleum ether to give fjoc-ALa-NHNHl 7
．． 03 Obtain f.

Rf, 0.79 Rf, 0.81 Elemental analysis value (as C□6H23N305) Calculated value (chi) C56,96H6,87N 12.45 Actual value (
*) C56,81H6,49N 12.342(a
), Production of H-ALa-NHNHl Boc-ALa
-NHNH23,0Of trifluoroacetic acid 10s17
After being left at room temperature for 15 minutes, trifluoroacetic acid was distilled off and dried to obtain H-AL a -NHNHl.

J? 7.0.51 2 (phrase, Bat-Arj(NO2)-AZa-NH
Production of NHZ Ihe-Arc(NO2)-ON 2.8
4f is dissolved in a solution of Nato 5 human O fladi 4011j and N-methyl 1 holin 0.914, and after cooling to -15°C, 1.17 m of isobutylchloroformate is added and stirred vigorously for 30 seconds. To this H-AL @-NHNHl
(D, ; Methyl luma:do 201u solution and triethylamine 1.24-solution are added and stirred for 1 minute.
℃ for 5 minutes, then 40℃ for 2 minutes, then warmed at room temperature for 15 minutes.
Stir for a minute. After distilling off tetrahydrofuran and dimethylformamide, the mixture is extracted with ethyl acetate. Inject the extract
After washing with citric acid, followed by saturated sodium bicarbonate, and then saturated brine, ethyl acetate was distilled off, and reprecipitated with ethyl acetate-ether. Bac-Arl(NO2) -ALa
-NHNH23-70t is obtained.

Rf”: 0.68 Rf, 0.79 Elemental analysis value (as C22H34N808) Calculated value (%
) C49,06H6,36N20.80 actual measurement value (chi) C49,40H6,72 #20.433 ((E)
, Production of H-Arc(NO2)-ALa-NHNH2 Boc-Arg(NO2)-AltL-NHNJIZ3
．． 67t was dissolved in 15m of trifluoroacetic acid, left at room temperature for 15 minutes, crystallized by adding dry ether, and the crystals were collected by F to form H-Ag (NO2) -ALa-NHNHz.
get.

Rf, 0.20 3(b), Btsc-Arg(NO2)-Arg(
NO2) -ALa-NHNH2 (D production Bee-Arg(NO2) -OH2,l 7 f is tetrafluorinated and N-methyl ℃ ruphori.
After cooling to -15°C, add 900 isobutyl lumates and 0.8911j of lumate and stir vigorously for 30 seconds.

To this, H -ATg (N02) obtained in the above (-)
-AL a -NHNHz dimethylformamide 3
Add 9 m and triethylamine 0.95 m solution and add 1
Stir for a minute. After warming at 0°C for 5 minutes, then at 40°C for 2 minutes, stir at room temperature for 15 minutes. The tetrahydrofuran and dimethylformamide are distilled off, and the residue is extracted with ethyl acetate. The extract was washed successively with IN-quesiic acid and a saturated aqueous sodium hydrogen carbonate solution, and then ethyl acetate was distilled off. Bat-A was reprecipitated with ethyl acetate-ether.
ry<No2)-Ar! ICN02)-ALa-NH
NH13,7Of is obtained.

RfX: 0.58 Rf, 0.75 Elemental analysis value (as C28H45N□301□) Calculated value (%) C45,46#6.13 N24.61 Real 11JI (%) C45,13H5,71N24.
514(a), H-Arg(NO2)-Arg(N
o2) -Ala-NHNH2 production Boc -Arc (NO2) -Arg (NO2)
-AlaaNHNHz 3 , OO2 is dissolved in 20 m of trifluoroacetic acid, left at room temperature for 15 minutes, and then crystallized by adding dry ether.

The crystals are taken out in a furnace and H-Arg (NO2)-Arg (
702) -ALa -NHNHz is obtained.

Rf”: 0.11 4(j+), BaC-, 4Jl&-Arc(NO2
) -Arg(No2)-ALa-NHNHz productionH-Arg(No2)-Arc(NO2)-Ata-
NHNHz tDMF 50ml/additionally, triethylamine 0.56d and 13oc-As-0NH5
2.17F and left at room temperature for 20 hours. Dimethylformamide is distilled off and the residue is extracted with butanol. After washing the extract with dithiacetic acid, ether was added to crystallize it, and the crystals were collected in a furnace and reprecipitated with methanol-acetic acid to give lJmc -Azn -Arc (NO2) -Arc.
(No. 2)-ALa-NHNH22,64t is obtained.

Rf”: 0.40 Rf: 0.72 Elemental analysis value (C, □H5□N□50□3) Calculated value (%) C45,01N6.02 N24.60 Actual value (-) C44,80N5.85 N24.1
24(C), BoC-Azn-Ar! I-Arg-
Preparation of ALa-NHNH2 Boc-Ash-Ar! 1c
NO2)-AryCN02)-Ma-NHNHz2
．． 5Or was dissolved in a mixture of methanol 30- and 30-thiacetic acid and catalytically reduced using palladium as a catalyst to obtain Boc-A.
ss-Arl-Arc-ALa-NHNH22,2
Get 0t.

Rf, 0.06 Rf, 0.40 Elemental analysis value (02447N, 307・2CH, (:H
-1120) Calculation m (ls) C43,80N7.48 N2
3.71 actual value (*) ('43.51 N7.6
2 N23,455, Z-Ll-Gty-oC2H5
) Manufactured N-methyl t-phosphoric acid 1.86- was dissolved in 500 m of tetrahydrofuric acid, and Z-L-Tsuru-0H4,8
Add 5f. Cool to -15℃ and add isobutyl 900
Add Lumate 2.41i1 and stir vigorously for 30 seconds. A solution of 2.54 f of H-GLy-QC2H5.HCl in 40 WIl of dimethylformamide and 2.56 liters of triethyl anil are added to this, and the mixture is stirred for 1 minute. After warming at 0°C for 5 minutes, then at 40°C for 2 minutes, stir at room temperature for 15 minutes. Nato 5 hydrobrush and dimethylformamide are distilled off, 1M citric acid is added to the residue, and the precipitated crystals are collected in a furnace. The F solution was washed with water until it became neutral, and the precipitated crystals were removed from F, dried, and reprecipitated with ethyl acetate-ether to form Z-L.
ea-GLy-QC2H54,68f is obtained.

Rf, 0.80 Rf, 0.77 Elemental analysis value (as C□8H26N205) Calculated value (chi) C61,7Of7.48 ff7.99 Actual value (%) C61,51N7.32 N7.806 (
α), H-L-μm (dybeni 2I5 production Z-
L#u-GLy-()C:2H53,12F
) H-Law 50d and ■N hydrochloric acid 8.90a (H-Law) and catalytically reduced using palladium as a catalyst.
- (Obtain 2H5 in the dye.

Rf'': 0.41 6(h), Production of Z-5-r-L-μ-GLy-OC2H5 Z-5ar-NHNH22,48f was dimethytolumanid 20+j and 6N hydrochloric acid/;
After dissolving dK and cooling to -15°C, isoanyl nitrite 1.
Add 31- and stir for 5 minutes. Then, 14.11 ml of triethylanil was added to neutralize. The above (obtained by →) H−L g I & −GL y −0CaH=, ・I
HCl)! JIfJl)'! Add the above reaction solution to 1.24 mo dimethyl fluoride solution and add 4
Stir at ℃ for 20 hours.

After distilling off dimethylformamide, the residue was extracted with ethyl acetate, and the extract was washed successively with IN-citric acid and saturated brine, and then dried over magnesium sulfate. After distilling off ethyl acetate, it was reprecipitated with ethyl acetate to give Z-5ay-Law-
'2.64 f is obtained for GLy-QC2.

ff/": 0.78 Rf": 0.85 Elemental analysis value (as c2□H3□N30.) Total g value C 俤) C57,65ff7.14 N9.60 Actual value (*
) C57,60H6,88H9,637(a),
H-5at-Law-GLy-QC2, 0 manufacturing Z-
5et-Law-GLy-QC2jr52.50 t was dissolved in 10-acetic acid IQil and methanol 5°0-,
By catalytic reduction using palladium as a catalyst, H-5at-L
Get 2jrs on aw-GLybe・J? /”:0.31 7 (A), Z-Thr-His-5ay-Law
-GLy-QC2H, production Z-Thr-Hip-NH
NH22,54ft,; Methylformamide 20-
and 6N-hydrochloric acid/diosane 4,19- dissolved in -1
After cooling to 5°C, 0.8411j of isoamyl nitrite is added and stirred for 5 minutes. Then triethylamine 3.511
Add 1111 to neutralize. H-5 obtained in (α) above
ay-Law-(dy-QC2H, and 0.79 m of triethylamine in a dimethylformamide 20-solution,
Add the above reaction solution and stir at 4°C for 20 hours. After distilling off dimethylformamide, the residue was extracted with ditanol,
Wash the extract with water. The solvent was distilled off and recrystallized from methanol-ethyl acetate to give Z-Thr-His-5ay-L.
aw-GLy-QC2H54-31f is obtained.

Rf, 0.35 Rf, 0.71 Elemental analysis value (as C3□, N70.・H20) Calculated value C'l) C64, OOH8,14#16.85
Actual value (chi) C64,48#8.1ON16.54
f3 (a), Z-Tkr-Hit-5ay-La
Production of w-GLy-NHNH2 2-Tkr-His-5
at-Law-GLy-(X:'2jr54-3 Of
Dissolve in methanol 20Ilj, and add Shidora! Add 3.18 ml of Shi.L hydrate and leave at room temperature for 20 hours.

Ether is added to the reaction solution, and the precipitated crystals are collected and dried. Wash with hot methanol and remove Z-Thr-1ha-5.
ay-Law-GLy-NHNH22-55t is obtained.

Rf, 0.17 Rf, 0.57 Elemental analysis value (as C2943N909) Calculated value (%)
C52,64H6,55N19.05 fruit 11jl[
(*) C52,55H6,44#19.09Bib
), H-Ass-Arg-Arc-AL@-Lg+
a-ILa -L-Juku-Lau-ALa-GL*-OH
Manufacture of Bee-Ass-Ar! 1-Arc-ALa-NHN
H20-78f 't Dimethyl Hon Lumuaed 8- and 6
Dissolved in N-hydrochloric acid/diosane 1.031L/-15
After cooling to <0>C, 0.16 - of isoamyl nitrite is added and stirred for 5 minutes.

Then, 0.87% of triethylamine is added to neutralize.

Bepuyodo A i.e. H-Lal&-ILa -Lgu-La
w -ALg-GZs-〇H,) Rechilua! ::J0
．． The above reaction solution was added to a mixture of 87m, dimethylformamide 2011/and hesamethyllisic acid triamide 10-, stirred at 4°C for 24 hours, and then EaC-As
h-Arl-Ag-ΔLa-NHNH20-39t was converted to azide, and Kuzono was added thereto, followed by stirring for 48 hours. Dimethylformamide is distilled off and the residue is extracted with ditanol. The extract is washed with water and the butanol is distilled off. Add ether to the residue to crystallize it, and collect the precipitated crystals. Wash with water and dry with phosphorus pentoxide. Obtained Boa-Az
f&-Arc-Arg-ALa-Law-11a-La
w-Law-ALa-Gin -OH was dissolved in triple-O-acetic acid 3- and allowed to stand at room temperature for 15 minutes. Dry ether was added to precipitate the precipitate. 50-acetic acid) to obtain H-
ALa-Artl-Arc-ALa-Law-ILa-
Obtain Law-LaIl&-ALa-GLn-OH120v.

R7,0,0I Rf, 0.35 Elemental analysis value (c5□H,4N□80□3・2 CH,C
As 0OH・5H20) Calculated value (chi) C47,95H8,19#18.30
Actual measurement value C'lk) C47, 66H8, 41NI8.
62 [α) 25 knees 185.44 (C = 0.57.1M
acetic acid) “9, H-Thr-H*z-5ay-Law
-GLy-Azn-Arg-Art-ALa-Law
-ILm-Lg pot-LatL-ALa-Gin-OH production Z-Thr-His-5at-Lets-Gly-NH
Dissolve NH2125"II in dimethylformamide l〇- and 6N-hydrochloric acid/;O+san 0.125i1, -
After cooling to 15°C, add 0.025% of isoamyl nitrite.
Stir for a minute. Then, 0.105 m of triethyl amyl was added to neutralize. H-Azn-Ar51-Arg-ALa
-Law-7J#-Lgu-Leg-ALg-Gts-
The above reaction solution was added to a solution of OH110"f and triethyl amyl 0,013- in dimethylformamide 1Qil and hedecathyl phosphoric acid triamide 6-, and the solution was heated at 4°C for 24 hours.
Stir for an hour. SaraK Z-Thr-His-5at-
Add the azide of Law-GLy-NHNH21251at and stir for 48 hours. Dimethylformamide is distilled off and the residue is extracted with butanol. Wash the extract with water. Distilling off butanol -Law -11g -Lgu
-Lets-ALa-Gin-OH in methanol 5Qa
/ and 10-acetic acid IQd and catalytically reduced using palladium as a catalyst. The catalyst was removed by F, and methanol was then distilled off, and the resulting residue was purified with Sephadex G-25C eluent (50-acetic acid) to obtain H-Thr-His-5ay.
-Law-GLy-Ash-Arg -Arg-ALa
-Lgu-ILa-Lgu-Law-ALa-Gin-
Obtain OH125Q. Hereinafter, this will be referred to as "Bezuchido B".

Rf, 0.01 R7,0,38 Elemental analysis value (C72H127N25020°2 CH3
CO0H.

As 4H20) Calculated value (chi) C49, 21111, 77N18.8
7 Actual value (%) C49, 60H7, 92N18.5
4[α), -66,76CC=0.42% 1M acetic acid) Production Example 3 1(g), Production of H-GLm-NHNHBoc Z-
rGLn-NHNHBoe 7-00 t is dissolved in methanol 59m and catalytically reduced using palladium as a catalyst to obtain H-GL s -NHNHBo c.

Rf, 0.37 1(A), 4%-NHNI& in Z-pro-. Manufacture of Z-pry-OH4-41f to 5) 5)
0d and N-methyl heptaphosphorus 1. Dissolve in g□+7,
After cooling to -15℃,
34 Add N7 and stir vigorously for 30 seconds. To this, H -GZ n -NHNHBo obtained in the above (α)
cf) Add methyl formamide 30-solution and stir for 1 minute.

After warming at 0°C for 5 minutes, then at 40°C for 2 minutes, stir at room temperature for 15 minutes. The tetrahydrofluoride and dimethylformamide are distilled off and the residue is extracted with ethyl acetate containing a small amount of butanol. The extract was washed successively with 1N citric acid, a saturated aqueous sodium bicarbonate solution, and saturated brine, and then with hot ethyl acetate to obtain Z-pro-Gin-NHN.
HBc5.6? get f.

Rf, 0.60 Rf, O, '16 Elemental analysis value (as '23H33'5'7) Calculated value (%
) C56, 20H6, 77N13.25 actual measurement value ('
II) C55,97H6,68N14.152((
L), Production of H-pro-Gin-NHNH13oc 5.50 t of Z -pro-Gin-NHNHBc was dissolved in 501 methanol and catalytically reduced using palladium as a catalyst to produce , Z/-prO-GLn-NHNHBc.
get.

Rf”: 0.09 2(A), Z-Law-pre-GJs-NHNE
Preparation of Bet 77-pr obtained from Z-Lag-0NH54-05t in the above (α).

-OL+%-NHNElk,, dimethylformamide 100- and triethyl a! In addition to the 1.56 resistant solution,
Leave at room temperature for 20 hours. After distilling off the dimethylformamide, the residue is extracted with ethyl acetate. The extract is washed successively with IN-citric acid and saturated saline. Z-Lag-pr was obtained by reprecipitation with ethyl acetate-ether.

-GL s -NHNHBt-c 3.72 f is obtained.

Rf, 0.68 A1.0.80 Elemental analysis value (as '29II, 4N6'8) Calculated value (
h) C57,60H7,33N 13.90 [IJ
Value C%＞C57,21H7,08N13.583(
a), H-Law-prO-GZs-NHNHBc
Production of 'Z-Law-pry-Gin-NHNMk3.
5Of was dissolved in methanol 5Qm and catalytically reduced using palladium as a catalyst to obtain H-Law-pre -(Js-
Obtain NHNHBc.

Rf, 0.14 3(b), Z-Asp(OCH2-C6H5) -
Production of Law-pry -GZs -NHNHBo c Z-Asp(OCH2-C6H5) -OH2,27f
Tetra, dissolved in dorofuran 30- and N-methyl 0.65°C, and after cooling to -15°C, isobutyl 90-
Add 0.8411 d of Lumate and stir vigorously for 30 seconds. This was followed by 9H-LgK-pr obtained in (g) above.

-Gl s -NHNEB-c dimethylformamide 20- and triethylamine 0.81 dil solution was added,
Stir for 1 minute. After warming for 5 minutes at 0°C and 2 minutes at 40°C, stir at room temperature for 15 minutes. The organic matter and dimethylformamide were distilled off, and the residual liquid was extracted with ethyl acetate. The extract is washed successively with IN-citric acid, a saturated aqueous sodium bicarbonate solution, and saturated brine, and the extract is distilled off. Reprecipitation with ethyl acetate-ether-petroleum ether to give Z-Asp(OCH2-C6H5)-Lgv
&-pro-GLs-NHNHBoc 4. QQ
get f.

Rf”: 0.6B Rf”:0.7? Elemental analysis value (as C4°H55N70.) Calculated value (
S) C59,32H6,84ff12.11 actual measurement value (IG) ('58.88 H6,65A'11.
724(a), H-Asp-Leu-PrO-Gj
Preparation of s-NHNHBoc Z-Asp(OCH2-C6
M, 3> -Law-Pro-Gin -NHNEBe
t2.0Of methanol 501 and I〇-acetic acid! 0d
and catalytically reduced H- using palladium black as a catalyst.
Asp-Law-pro-GLm-NHNHlk is obtained.

#7.0.08 4(A), Z-5ay-Asp-Law-Pro
-Production of GLn-NHNHBocZ-5ay-NHNH20,75f t-dimethylformamide 15- and 6N-hydrochloric acid/, ;O + Sashimi 1.48
After cooling to -15°C, add 0.39 il of isoamyl nitrite and stir for 5 minutes. Then, 1.24111j of triethylamine was added to neutralize. H -Asp -Lg u -Pre -Gi obtained in the above (α)
n -NHNHBdDjijchi L book rumuaed lQs+j
The above reaction solution was added to the triethyl amyl 0.34-solution and stirred at 4°C for 20 hours. Dimethylformamide is distilled off and the residue is extracted with butanol. The extract is washed with water and the butanol is distilled off. Recrystallized from methanol-ethyl acetate to give Z-5ay-Asp-Lag-pro.
-GLn-NHNHBocl, 52f is obtained.

Rf, 0.45 Rf, 0.67 5, H-5ay-Asp-Law-pro-GL
n-Tkr-Mix-5at-Lgm-GLn-As
h-Arg-Arc-ALa-Lmu-ILa-”L
Production of mu-Law-ALa-GLs-OH Z-5ay
-,4JFF-Law-prO-GZs-NHNHBo
c 116 W was removed by TFA2111, and the precipitate obtained by adding anhydrous ether was dried on a furnace plate, and dimethyl chloride was added. Do5i1j and 6N-hydrochloric acid/Gio medium sashi 0.0
? 2iE/, and after cooling to -15°C, 0.019 - of isoamyl nitrite was added and stirred for 5 minutes. Then, 0.081 m of triethylamine was added to neutralize.

Bezutide B or H-Thr-His-5at-Law-
GLy -Azn-Ass-Arc-ALa-Lau-
11a-Lgu-Law-Alt-GLn-OH80
Add the above reaction solution to the dimethyl phosphoric acid triamide 5111 and hexamethyl phosphoric acid triamide 6-solution of "f", and heat at 4°C.
Stir for 20 hours. Furthermore, Z-5ay-, 4JFF
-Law-Pro-GZs-NHNH%tAzide 1164 is added and stirred for 28 hours. Dimethylformamide is distilled off and the residue is extracted with ditanol.

The extract was washed with water, the methanol was distilled off, and petroleum ether was added to the residue to crystallize it.
Reprecipitate with ethyl acetate.

The obtained Z-5ay-Asp-Law-pro-GL
n-Tkr-Hip-5ay-Law-GLy-As
n-Arg-Arg-ALa-Law-ILm-La
w-Law-ALa-GLn-OH in methanol 30+
j and 10-acetic acid in 30wLl, and a contact amount of palladium was added to give 58q of H-54D Asp-Law-Pro-
Gin-Thr-Hip-5ay-Law-GLy-
Ass-Arg-Arg-Ala-Lgu-7Z#
-Law-Lgu-ALa-GZs-OH is obtained. Hereinafter, this will be referred to as "Bezuchido C".

Rf”: 0.01 J?f”: 0.3? yc 'a analysisfli (C9,,,H□63N3□
029-2 CM, as C0OH-7H20) Calculated value (%) C48,54 #7.61 #17.
72 actual measurement value (chi) ('48.14 Ml, 50
#17.4B[α]':: -85,70<c=o,
23.1M acetic acid) Production Example 4 1(a), H-5ay-Asp-Lag-pre-
Production of GLn-NHNHBIC Z-5ay-Asp-Law-pry-GLn-NHN
HBacl, 03f was dissolved in methanol 50+j and catalytically reduced using palladium as a catalyst to obtain H-5et-
Asp-Law-pro-Gin-NHNHBoc is obtained.

Rf”: 0.06 1(A), Z-Tyr-, Sgr-Asp-Law
Production of -pry -Gin-NHNHBac Z-Tyr-ONH5O, 79t was converted into H-5at -Asp -Lat&-pry -GL obtained in the above (α)
rb-NHNHB-c dimethylformamide 20iu
Add to the solution and leave at room temperature for 20 hours. After distilling off the dimethyl fluoride, the residue is extracted with ethyl acetate. The extract is washed successively with IN-queshiic acid and saturated brine, and ethyl acetate is distilled off. Reprecipitate with methanol-ethyl acetate to give Z-Tyr-5ay-Asp-Law-pre-G
in -NHNHBac 588"f is obtained.

Rf”: 0.51 Rf”, 0.69 Elemental analysis value (as 052H69N90 engineering) Calculated value (
%) C5B, 91 N6.56 N11.89
Actual measurement value (chi) 05B, 53 N6.22 #1
2.282, H-Tyr-5ay-Asp-Law
-pro-GLn-Thr-HisSay-Law-G
Ly-Azn-Arg-Arg-Alt-Lg! &-I
Production of La -Lag-Lgu-ALa-GLx-OH Z-Tyr-5ay-, 4JF-Lag-Pro-GL
n-NHNHB=c44 cape dimethylformamide 3w
Dissolved in J and 6N-hydrochloric acid 751 silicate 0.0225114 and cooled to -15°C. Isoamyl nitrite 0.0060
ml and stir for 5 minutes. Then, 0.0252 m of triethylamine is added to neutralize.

Peptide B or H-Thr-Hit-5ay-Law-
GLy -Azn-Ag-Arg-Ala-LaML-
Ha-Lel&-Law-ALa-Gln-OH,
25'Q O,; Methylformamide 51Li! ! The above reaction solution was added to the 2-solution of methyllysic acid triamide and stirred at 4° C. for 20 hours. Furthermore, Z-T
yr-5ay-Asp-Law-pro-Gin
-Add NHNHBac44 Ki to azide%
Stir for 24 hours. The solvent is distilled off, the residue is extracted with butanol-water, ether is added, and the precipitated crystals are collected.

The obtained Z -Tyr -Ear -Asp -LaK
- and @-Gtn -Thr-Haz-5ay-Lgu
-GLy-Ass-Ary -Arg-ALa -La
K-dissolved and catalytically reduced using palladium as a catalyst.

The residue obtained by removing the catalytic F and distilling off the methanol was treated with Sephadex G-25C eluate (5o thiacetic acid), followed by LH-
20 (eluent 4°.N hydrochloric acid) and purified with H-Tyr.
-5ty-Asp-Law-, pro-GZn-T
hr -Hiz-5ay-Law-GLy-Asvb-
Arg-Artl-ALa-Law-IZ#-Lgu
-La&&-ALa -Gtn-OH18N is obtained. Hereinafter, this will be referred to as "peptide D".

Rf”: 0.Q I Rf”, 0.38 Elemental analysis value (C□., 4H, 69N3203□・2C
As H3CO0H-5H20) Calculated value (%) C50,40H7,32N 17.4
1 Actual value C%) C50,72N7.67 N17
．． 0325.

[α), -77,88 (C-0,22,1M acetic acid) <Production of antigen> Production example l Synthesis of peptide. 15q of AJ) was added to acymonium acetate buffer (0.17L, pH 7).
,0) comb to 211. To this solution was added 0.1 liter of curtaraldehyde solution. Add l l m and 5 ml at room temperature.
Stir for an hour. The reaction mixture is then dialyzed for 48 hours at 4° C. against 14 ml of water. Change the water 5 times during dialysis. Thereafter, the solution containing the peptide-protein complex is lyophilized to obtain Trimpholastoid Isiter Fe 0 antigen (hereinafter referred to as "antigen I") 1811F.

This antigen I has peptide A bound to 41 t of BS at an average of 12 t.

Production Example 2 Betutide synthesis 5■ and B of Betutide B obtained in Production Example 2
20IIf of SA was dissolved in acetate a:J heptanium buffer (0,1
Jlz%P#7.0) dissolve to 2mJ. O in the Koya solution,
Add 0.11 d of diluteal aldide solution and stir at room temperature for 5 hours. The reaction mixture is then dialyzed for 48 hours at 4° C. against 1 t of water. The water was changed 5 times during dialysis.

Thereafter, the solution containing the peptide-protein complex is freeze-dried to obtain human limhoplastoid interfae antigen 23q (hereinafter referred to as "antigen ■").

The obtained antigen was isolated from peptide B against ESA 17.
are bonded at an average of 9°C.

Production Example 3 Bezutide C (2) obtained in Peptide Synthesis Production Example 3 4.5
Add 25 ml of cape and BSA to acetic acid buffer (
Dissolve to 0.1E, pH 7.0>2111. To this solution, add 1.0-
and stirred at room temperature for 5 hours. Then the reaction mixture was
Dialyze against water IL for 8 hours at 4°C. Change the water 5 times during dialysis. Thereafter, the solution containing the peptide-protein complex is freeze-dried to obtain the human plastidoid antigen 27Iv (hereinafter referred to as "antigen 2").

The obtained antigen (2) has an average of 10 tons of betutide C bound to B5Al7.

Production Example 4 5xay of 9 peptide D and 25 Iv of BSA obtained in Bezutide Synthesis Production Example were mixed with acetate slow extraction solution (0.1V).
pH 7,0) 2-. Add 0.1 to this solution.
Add 11m of Enil's cultaraldehyde solution Q,
Stir at room temperature for 5 hours. The reaction mixture was then heated for 48 hours.
Dialyze against water 11 at 4°C. Change the water 5 times during dialysis. Thereafter, the solution containing the peptide-protein complex was freeze-dried to obtain 28"lF of trimmed plastoid interferon antigen (hereinafter referred to as "antigen 2").

The obtained antigen ■ was peptide D against BS and 411LK.
has an average of 97 bonds.

Production Example 5 Bezutide Synthesis 4.5 capes of Bezutide C obtained in Production Example 3 and B5A25W9 are dissolved in 4 liters of water. Add to this solution 0 to 100% carbon dioxide (CDC) 20011f.
and stirred at room temperature for 5 hours. The reaction mixture was then diluted with 2 ml of water.
Dialyze at 4° C. for 48 hours. Change the water 5 times during dialysis. Thereafter, the solution containing the Bezutide protein complex was lyophilized to produce 0% human rimhoplastoid protein complex.
:, I antigen (hereinafter referred to as "antigen V") 27.511F is obtained.

The obtained antigen V was
has an average of 12V nyl bonds.

Preparation Example 6 Peptide Synthesis 4.5 ml of Peptide D obtained in Preparation Example 4 and B5A25q are dissolved in water. This solution contains 0
Heshi Silcar Body! Add 200 kg of DCC and stir at room temperature for 5 hours. The reaction mixture was then diluted with 2 parts of water at 4°C.
Dialysis takes 48 hours. Change the water 5 times during dialysis. Thereafter, the solution containing the beta-protein complex was lyophilized to obtain trimmed plastoid protein antigen 29q (hereinafter referred to as "antigen 2").

The resulting nine antigens (1) have nine moles of bezutide D bound to B5A25q, L on average.

<Production of antibody> Production example 1 100 μf of antigen I obtained in antigen production example 1 was transferred to 1.5 m
After dissolving in physiological saline, add 1.5 ml of Furuiside auxiliary solution.
A suspension was prepared by adding - to three rabbits (2,5-3.
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 animal and centrifuged to collect antiserum to obtain the human plastoid interferon antibody of the present invention (hereinafter referred to as "antibody I").

Production Example 2 After dissolving 20μt of the antigen ■ obtained in Antigen Production Example 2 in 1.5d of physiological saline, add 70% of India's auxiliary solution to 1.511% of the solution.
The suspension prepared by adding 1 to 7 rabbits (2, 5 to 3.
Administer subcutaneously to 0 Kg') and administer the same amount 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 was collected from the test animals and centrifuged to collect antiserum.
).

Production Example 3 After dissolving 20 µf of the antigen (1) obtained in Antigen Production Example 3 in 1.5-mL physiological saline, a suspension prepared by adding 1.5-mL Supplementary Solution of Furuiside was prepared using 7 chickens. Rabbit (2.5~3.0
Kg), and the same amount is administered 6 times every 2 weeks. After that, the same amount as the initial dose was administered three times every month. Seven days after the final administration, blood is collected from the test animal and centrifuged to collect antiserum to obtain the antiserum antibody of the present invention (hereinafter referred to as "antibody ■").

Production Example 4 100 μt of antigen ■ obtained in Antigen Production Example 3 was placed in a 1.5 m
After dissolving in physiological saline, add 1.5% of Freund's auxiliary solution.
The suspension prepared by adding ml of
Administer subcutaneously on >3.04 days and administer the same amount six times every two 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 animal and centrifuged to collect the antiserum to obtain the plastidoid antibody of the present invention (hereinafter referred to as "Antibody ■").

Production Example 5 After dissolving the antigen (1) obtained in Antigen Production Example 4 in 20jft1.5- of physiological saline, add 1.511% of Freund's auxiliary solution.
The suspension prepared by adding / was added to 7 rabbits (2.5-5.
011) K 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 animal and centrifuged to collect antiserum to obtain the plastidoid antibody of the present invention (hereinafter referred to as "antibody V").

Production example 6 Antigen production example 10θμft-1,5- of the antigen just obtained
After dissolving in physiological saline, add 70% Indian auxiliary solution, 5%
The suspension prepared by adding - was added to three rabbits (2,5-3.
0 kg), 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 was collected from the test animals and centrifuged to collect antiserum.
).

Production Example 7 Using 20 µf of Antigen Production Example (2) and anti-w obtained in Example 1, a trimhoplastoid isitaph antibody (hereinafter referred to as "antibody 1") was obtained in the same manner as in Production Example 3 above.

Production Example 8 In Antigen Production Example 5, using 100 µl of II antigen matrix, perform the same procedure as in Production Example 4 above to prepare a trimmed plastotoy:
J9-Fxo antibody (hereinafter referred to as "antibody antibody") is obtained.

Production Example 9 Using 20 µf of the antigen 1 obtained in Antigen Production Example 6, the same procedure as in Production Example 3 above is used to obtain a trimmed 5-stoid interferon antibody (hereinafter referred to as "antibody group").

ata Example 10 Using the antigen wotooμl obtained in Antigen Production Example 6, immunize three rabbits in the same manner as in Production Example 4 above, then collect blood from the test animals, separate one heart, and collect antiserum. Then, the human plastoid starter antibody of the present invention (hereinafter referred to as "antibody X") is obtained.

Production of 0-labeled peptide H"Tyr-5ay-Asp-Law-pre = G,
n-Tru-Hip-5ar-Law-GLy-A
jts-Arg-Arg-Ala-Law-ILg
-L#[mu]mL#[mu]mAt[alpha]-OL~H, that is, peptide D, is labeled as follows by a method using 0 lamici.

That is, 5 μt of the above-mentioned Bezutide and 20 μt of 0.5 t of lysinate buffer (pff 7.0) KN5Ic1251] (c
arrier fret N, E, N, ) Add 1 microt of 0.57 lysinate buffer of Bovine Yury, then add 2oμt of 0.5'e lysinate buffer of 053 T70 x/- . 60q/y by stirring for 30 seconds at room temperature
d of sodium metabisulfite (Kg2S205).
The reaction is terminated by adding 50 μt of 5M lysinate buffer. Next, 100 μt of a cold sodium iodide aqueous solution of 1-1 was added to the reaction mixture, and the reaction mixture was poured into
25O column (1, OX 90cts) (eluent: 0.25% BSA% 10*M EDTA and 0.25% BSA% 10*M EDTA).
0.05'E Nyl phosphate slow release solution containing 021G Naps, pH 7.4). The 13th and 14th fractions are the above peptides labeled with 125.

Measurement of 0 titer Measure the titer of the antibody obtained above as follows:

10,102,103 of each antibody in physiological saline.
゜10, 10... Dilute (initial) twice and dilute each of these to 100. 1125-labeled peptide (labeled betutide obtained above was diluted to about 95,000 pm) O, 1 m and 0.05'e luricate buffer (PH = 7.4) (0,25 mt) %B
SA%10whMEDTA and C)-0211NaN5
0.21117 (containing 100% of the total number of antibodies) was added and incubated at 4°C for 24 hours.
minutes, 3000 rllllL)
L4b) Separate from 1125-labeled Bezutide, remove its radiation, and measure the binding rate (-) of the antibody to I-labeled Bezutide at each dilution concentration. Antibody 112 on the vertical axis
The binding rate with the 5-labeled peptide (91) and the dilution factor (initial concentration) of the antibody are plotted on the horizontal axis, and the binding rate is calculated at each concentration. The dilution factor of the antibody at which the binding rate is 501, that is, the titer of the antibody is determined. The results are shown in Table 1 below.

Table 1: Antibody and Plastoid Interferase Specificity Test Samples of various concentrations of seven β-interferases (
Manufactured by Tokyo Metropolitan Clinical Research Institute, specific activity 5 x 106 U/MIj
of 9 peptide C, i.e., the 9 peptide C obtained in Vep fFO & Kai Production Example 3, and the Vepti F chain of this peptide interferon and the 9 peptide α-type interferon [manufactured by Taishu Institute,
Rim Hon Plastoid Isita Hue O:, ILot, No.
, 800928 and Cantyl (manufactured by Cantor)]. Also, as a standard diluent, 0-25*ESA, 5
Use 0.05v:luricate buffer (pf?-4) containing rnM EDTA and 0.02 S ONaps.

1 standard diluent 0.2 d, test sample 0.1 in each test tube
M, Antibody production example 0.1-(titer of 20) of the antibody 1 obtained just now
10,000) and I labeled betutide (labeled betutide obtained above diluted to about 2800 epm) 0
．． 1- and incubated at 4°C for 72 hours,
Normal porcine serum
erm) was added to the mixture, then coated with jishi-strashi, added with 0.51 of a suspension of activated carbon, allowed to stand at 4°C for 30 minutes, and then centrifuged at 4°C for 30 minutes at 3000 rpws. Separate the conjugate of the antibody and 1125-labeled peptide and the unreacted (not bound) 1125-labeled peptide, remove the radiation, and obtain the binding rate (BO) corresponding to the titer of the antibody used. Assuming 100*, calculate the percentage of the conjugate (B) between the antibody and I-labeled beuttide at the concentration and dilution rate of each test sample.
The vertical axis in Figure 1 is the bond* (B/Be x 1
00), and the horizontal axis shows the concentration of the test sample (the peptide chain of Bezutide C, i.e., human lim plastoid obtained in Peptide Synthesis and Production Example 3, human β-type Ishita-Fue 0 and human α-type Ishita-Fue 0). . In addition, in the figure, curve (a) represents the peptide chain of peptide C, i.e., the trim-type protein interferon; Curve (c) is human α
Figure 1 shows the type interferotype 1 (manufactured by Taisei Research Institute), the front curve (d), the β-interferotype 0, and the β-interferotype 0, respectively.Antibody V is 7 to α-type interferotype: JK. The reactivity and the reactivity to human β-type interferon 0 are clearly differentiated curves, and this clearly indicates that β-type interferon 0 is different from 5. It can be seen that this is a highly specific antibody with no cross-over up to OX lO'l nits/d.

In addition, the binding rate of peptides and BSA in antigens 1 to V obtained in the above antigen production examples 1 to 6 can be determined by comparing the obtained antigens with IIct Fadex G-50 , 0D280m, flow rate 23-/hour, fractional amount: 1d) to remove unreacted BSA.
In many cases, the presence of VeptiF is not recognized.The gel filtration is used to separate the VeptiFO fraction bound to BSI from the residue of other products (VeptiF dimer), and to remove the VeptiF dimer. Create a calibration curve for the standard concentration of
The amount of the dimer is determined, and this value is subtracted from the amount of the peptide used as the starting material, assuming that all 5siyc bonds are present.

[Brief explanation of the drawing]

The third imIl is obtained according to the present invention and shows the specificity of the trimmed plastoid antibody. (or more) 15″.

Claims (1)

[Claims] ■ General formula % formula % [In the formula, ia hydrogen atom, H-Tkr-His-3ay-L
aw-Gjy-Az-Arg-Arg-Al@ group, H
-5ay -Asp-Law-Pro -Glrh -
Thr-Hip-5ay-Law-GLy-Ash
-Arc -Arg-Arg group, or H-TFy-5a
y-Asp-Law-Pro-GJa-Thr-H
is-5et-Las-GLy-Ash-Ary71-
Arg-At reef base is shown. ]. Trimmed plastoid isiterfue 0s N-terminal betutide and its derivatives, characterized in that it is represented by "°".