JPH0160037B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel human limboblastoid interferon N-terminal peptide. In this specification, when amino acids, peptides, protective groups, active groups, etc. are indicated by abbreviations
The IUPAC, IUB regulations or common symbols in the field shall be followed, examples of which are listed below.
Furthermore, when an amino acid or the like can have optical isomers, the L-isomer is indicated unless otherwise specified. Leu: Leucine Ile: Isoleucine Ala: Alanine Gln: Glutamine Thr: Threonine His: Histidine Ser: Serine Gly: Glycine Asn: Asparagine Arg: Arginine Asp: Aspartic acid Pro: Proline Z: Carbobenzoxy group Su: Succinimide group Tos : P-toluenesulfonyl group Boc: Tertiary butoxycarbonyl group Interferon is an antiviral glycoprotein or protein that is produced when living cells are infected with a virus, and its use can prevent viral diseases. It has been attracting attention in recent years as it is thought to be possible to prevent or treat. The currently elucidated human interferons are α-type interferon (Leucocyte Interferon, Lympho blastoid
Interferon (interferon), β-type interferon (Fibroblast interferon), and gamma-type interferon (Immune interferon), but the technology to purify these interferons into single glycoproteins or proteins has not yet been developed. do not have. The present invention provides a new antibody for the purpose of isolating and purifying human α-type interferon, particularly limboblastoid interferon, an antigen for producing the antibody, a hapten suitable for producing the antigen, and a method for producing these. The purpose is to provide technology for acquiring As a result of intensive research for the above-mentioned purpose, the present inventors have discovered that when using the specific N-unterminated peptide of human limboblastoid interferon newly synthesized by the present inventors, a novel human limboblastoid interferon can be obtained. An interferon antigen can be produced, and by using this antigen, a new antibody having specificity for human limboblastoid interferon can be produced, and by using this antibody in alignment chromatography, Target human α
We have discovered that it is possible to purify type interferon. The present invention has been completed based on the above-mentioned new findings.According to the present invention, a specific protein useful for the purification of human limphoblastoid interferon by a simple operation from a synthetic peptide that can be easily produced in large quantities has been developed. Antibodies exhibiting reactivity can be produced industrially and advantageously, thus establishing a technology for purifying human limphoblastoid interferon. The new synthetic peptide according to the present invention is represented by the following general formula (1). R-Leu-Ile-Leu-Leu-Ala-Gln-OH [In the formula, R is a hydrogen atom or H-Thr-His-Ser
-Leu-Gly-Asn-Arg-Arg-Ala group. ] The two new synthetic peptides represented by the above general formula (1) are peptides corresponding to the N-terminal peptide chain of human lymphoblastoid interferon. These are methods commonly used for peptide synthesis,
Specifically, “The Peptides”
Volume 1 (1966) [Schro¨der and Luhke,
Academic Press, New York, US/A.] or "Peptide Synthesis" [Izumiya et al., Maruzen Co., Ltd. (1975)]. Anhydride method, DCC method, active ester method (p-nitrophenyl ester method, N-hydroxysuccinimide ester method, cyanomethyl ester method, etc.),
It can be produced by a method using Woodward's reagent K, a carbodiimidazole method, a redox method, a DCC/additive (HONB, HOBt, HOSu) method, etc. In the above method, both solid phase synthesis method and liquid phase synthesis method can be applied. The peptide of general formula (1) is usually synthesized 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 coupling into several fragments. It is manufactured by a method of More specifically, the above-mentioned peptide consists of a raw material having a reactive carboxyl group corresponding to one of two types of fragments that are divided into two at an arbitrary position of the bond, and a raw material having a reactive amino group corresponding to the other fragment. is condensed by conventional means of peptide synthesis, and when the resulting condensate has a protecting group,
It can be produced by removing the protecting group in a conventional manner. Furthermore, when aspartic acid is used in the reaction step to produce the peptide of general formula (1), it is often desirable to protect it, and in the final step, at least one of the constituent amino acid residues of the peptide is usually protected. Remove all protecting groups from the protected peptide. In addition, in the synthesis reaction step of the peptide of general formula (1) above, functional groups that should not participate in the reaction are protected with a conventional protecting group, and the protecting group is removed after the reaction is completed. 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 carbobenzoxy, tert-butyloxycarbonyl, tert-
Amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, o-nitrophenylsulfenyl, diphenylphosphithioyl, etc. can be mentioned. Examples of protective groups for carboxyl groups include alkyl esters (eg, ester groups such as methyl, ethyl, propyl, butyl, and tert-butyl), benzyl esters, p-nitrobenzyl esters, p-methoxybenzyl esters, and p-methoxybenzyl esters.
-chlorobenzyl ester, benzhydryl ester, carbobenzoxy hydrazide, tert-butyloxycarbonyl hydrazide, trityl hydrazide and the like. Examples of the guanidino group-protecting group for arginine include nitro, tosyl, p-methoxybenzenesulfonyl, carbobenzoxy, isobornyloxycarbonyl, adamantyloxycarbonyl, and the like. The guanidino group may also be protected in the form of a salt of a suitable acid such as benzenesulfonic acid, toluenesulfonic acid, hydrochloric acid, sulfuric acid and the like. The hydroxyl group of threonine can be, but need not be, protected, for example by esterification or etherification. Examples of groups suitable for this esterification include lower alkanoyl such as acetyl, aroyl such as benzoyl, and groups derived from carbonic acid such as benzoyloxycarbonyl and ethyloxycarbonyl. Examples of groups suitable for etherification include benzyl, tetrahydropyranyl, and tert-butyl. Examples of activated carboxyl groups include:
For example, the corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, active esters (methyl alcohol, ethyl alcohol, benzyl alcohol,
Pentachlorophenol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxybenztriazole, N-hydroxy-5-
esters with 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 tetraethylpyrophosphite. The peptide represented by the above general formula (1) is produced more specifically according to the methods () to () shown below, depending on the type of group represented by R. () When R represents a hydrogen atom, A-Ala-B(2) ↓ H-Gln-OH(3) A-Ala-Gln-OH(4) ↓ H-Ala-Gln-OH(5) ↓ A- Leu-B(6) A-Leu-Ala-Glu-OH(7) ↓ H-Leu-Ala-Gln-OH(8) ↓ A-Leu-B(6) A-Leu-Leu-Ala-Gln- OH(9) ↓ H-Leu-Leu-Ala-Gln-OH(10) ↓ A-Ile-B(11) A-Ile-Leu-Leu-Ala-Gln-OH(12) ↓ H-Ile-Leu -Leu-Ala-Gln-OH(13) ↓ A-Leu-B(6) A-Leu-Ile-Leu-Leu-Ala-Gln-OH(14) ↓ H-Leu-Ile-Leu-Leu-Ala -Gln-OH(15) () R is H-Thr-His-Ser-Leu-Gly-
When indicating an Asn-Arg-Arg-Ala group [Table] [Table] [In the above methods () and (), A is a protecting group for an amino group, B is an active group for a hydroxyl group or carboxyl group, and C is a guanidino group for arginine. The protecting groups are shown respectively. ] In the above, A is preferably Boc, Z,
p-methoxybenzyloxycarbonyl group, etc.
Examples of the active group of the carboxyl group shown in B are preferably active esters such as N-hydroxysuccinimide, mixed acid anhydride residues such as isobutyloxycarbonyl, azide, etc., and examples of C are preferably nitro, tosyl, etc. can. In the above method (), amino acid (2) and amino acid
The reaction with (3) can be carried out in the presence of a solvent. As the solvent, various solvents known to be usable in the peptide condensation reaction, such as anhydrous or hydrous dimethylformamide, dimethyl sulfoxide, pyridine, chloroform, dioxane,
Dichloromethane, tetrahydrofuran, ethyl acetate, N-methylpyrrolidone, hexamethylphosphoric triamide, or a mixed solvent thereof may be used. The ratio of amino acid (3) and amino acid (2) to be used is not particularly limited and can be appropriately selected within a wide range, but usually the latter is used in an equal amount to 5 times the amount of the former, preferably an equal amount to It is best to use 1.5 times the amount.
The reaction temperature is within a range known to be usable for peptide bond forming reactions, usually about -40 to about 60°C;
Preferably, the temperature is appropriately selected from the range of about -20 to about 40°C. The reaction time is generally about several minutes to 30 hours. Reaction of peptide (5) and amino acid (6), reaction of peptide (8) and amino acid (6), reaction of peptide (10) and amino acid (11), and reaction of peptide (13) and amino acid in method () The reaction with (6) can be carried out in the same manner as the reaction between amino acid (2) and amino acid (3) above. Also in method (), the reaction between amino acid (16) and amino acid (17), the reaction between peptide (19) and amino acid (16), the reaction between peptide (21) and amino acid (22), and the reaction between peptide (24) and peptide (15)
reaction between peptide (26) and peptide (36), reaction between amino acid (6) and amino acid (29), reaction between peptide (31) and amino acid (32), and reaction between peptide (34) and peptide ( The reaction with 35) can be carried out in the same manner. Peptides (4), (7), obtained by each of the above reactions
(9), (12), (14), (18), (20), (25), (27)
, (30)
The removal reaction of the protecting group A of (33) is carried out by a conventional method. Such methods include, for example, reductive methods (e.g. hydrogenation using catalysts such as palladium or palladium black, reduction with metallic sodium in liquid ammonia), acidolysis (e.g. trifluoroacetic acid, hydrogen fluoride, methanesulfonic acid, hydrogen bromide, etc.). Acidolysis with strong acids such as acids), etc. Hydrogenation using the above catalyst can be carried out, for example, at a hydrogen pressure of 1
It can be carried out at atmospheric pressure and 0 to 40°C. The amount of catalyst used is usually about 100 mg to 1 g, and generally 1 g.
The reaction completes in about 48 hours. The above acidolysis is carried out without a solvent, usually at about 0 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 ammonia, metallic sodium is used in such an amount that the reaction solution is colored permanently blue for about 30 seconds to 10 minutes. This reduction is usually −40 to −70
It is carried out at around ℃. Furthermore, the protecting group (C) of peptide 23 can be similarly deprotected by the reductive method described above. Amino acids 2, 6, 11, 16, 22 and 32 used in the above methods () and () may be known commercially available products, and peptides 18, 23, 35 and 36 may be known commercially available products or mixed acid anhydrides. Those obtained by the method, azide method, etc. can be used. The mixed acid anhydride method is carried out using an alkylhalocarboxylic acid in the presence of a basic compound in a suitable solvent. Examples of the alkylhalocarboxylic acids used include methyl chloroformate, methyl bromoformate, ethyl chloroformate, ethyl bromoformate, isobutyl chloroformate, and the like. Examples of basic compounds include triethylamine, trimethylamine, pyridine, dimethylaniline, N-methylmorpholine, 1,5-diazabicyclo[4,3,
0] nonene-5 (DBN), 1,5-diazabicyclo[5,4,0] undecene-5 (DBU), 1,
Examples include organic bases such as 4-diazabicyclo[2,2,2]octane (DABCO), and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate. The solvents used include various solvents commonly used in the mixed acid anhydride method, specifically halogenated hydrocarbons such as methylene chloride, chloroform, and dichloroethane, aromatic hydrocarbons such as benzene, toluene, and xylene, diethyl ether, Examples include ethers such as tetrahydrofuran and dimethoxyethane, esters such as methyl acetate and ethyl acetate, and aprotic polar solvents such as N,N-dimethylformamide, dimethyl sulfoxide, and hexamethylphosphoric triamide. The reaction is carried out at -20 to 100°C, preferably -20 to 50°C.
The reaction time is generally 5 minutes to 10 hours, preferably 5 minutes to 2 hours. The azidation method is carried out by first reacting an activated carboxyl group with an alcohol such as methyl alcohol, ethyl alcohol, or benzyl alcohol with hydrazine hydrate in an appropriate solvent. It will be done. Examples of solvents used include dioxane,
Examples include dimethylformamide, dimethylsulfoxide, and a mixed solvent thereof. The amount of hydrazine hydrate to be used is usually 5 to 20 times, preferably 5 to 10 times, the amount of activated carboxyl groups. The reaction is usually
It is carried out at a temperature of 50°C or lower, preferably -20 to 30°C. In this way, a compound (hydrazine derivative) in which the carboxyl group of the terminal amino acid is substituted with hydrazine can be produced. A compound in which the carboxyl group of the terminal amino acid is substituted with azide is produced by reacting the hydrazine derivative obtained above with a nitrite compound in a suitable solvent in the presence of an acid. Hydrochloric acid is usually used as the acid. As the solvent, for example, dioxane, dimethylformamide, dimethyl sulfoxide, or a mixed solvent thereof can be used. Further, as the nitrite compound, for example, sodium nitrite, isoamyl nitrite, nitrosyl chloride, etc. can be used. Such a nitrite compound is generally used in an amount of equimolar to 2 times the molar amount, preferably equimolar to 1.5 times the molar amount of the hydrazine 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 peptide separation means such as extraction, partitioning, column chromatography, etc. In this way, a synthetic peptide represented by the general formula (1), ie, the N-terminal peptide of human lymphoblastoid interferon, is obtained. The synthetic peptide thus obtained is
By introducing radioactive iodine such as I ¹²⁵ or I ¹³¹ , it can be used as a labeled peptide, which is a raw material for producing labeled antigens used in radioim assay (RIA method). The above radioactive iodine can be introduced by a conventional iodination method, for example, an oxidative iodination method using chloramine T [WMHunter and FC
Greenwood; Nature, 194 , P495 (1962),
Biochem. J. 89 , P114 (1963)]. For example, the iodination method described above is carried out in a suitable solvent such as 0.2M phosphate buffer (PH=7.4) in the presence of chloramine T at around room temperature for about 10 to 30 seconds. The ratio of the peptide, radioactive iodine and chloramine T used is, for example, when one radioactive iodine is introduced into tyrosine,
It is best to use about 1 millicurie of radioactive iodine and about 10 to 100 nanomoles of chloramine T per 1 nanomole of tyrosine molecules contained in the peptide. Also, when two radioactive iodines are introduced into tyrosine, Tyrosine molecule included 1
It is preferable to use about 2 millicuries of radioactive iodine and about 10 to 100 nanomoles of chloramine T per nanomole. The radioactive iodine-labeled peptide thus produced is isolated and purified by conventional separation means such as extraction, partitioning, column chromatography, dialysis, etc. The peptides thus obtained can be stored by lyophilization, if necessary. Furthermore, the synthetic peptide represented by the above general formula (1) can be used as a hapten for producing human limphoblastoid interferon antigen. The method for producing the human limboblastoid interferon antigen described above will be described in detail below. The human limboblastoid interferon antigen uses at least one kind of synthetic peptide represented by the above general formula (1) as a hapten.
It is produced by reacting with a carrier in the presence of a carrier-binding reagent. As the carrier bound to the hapten in the above method, 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 animal serum albumins such as horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, and sheep serum albumin, horse serum globulin, bovine serum globulin, rabbit serum globulin, human serum globulin,
Animal serum globulin such as sheep serum globulin, horse thyroglobulin, bovine thyroglobulin, rabbit thyroglobulin, human thyroglobulin, animal thyroglobulin such as sheep thyroglobulin, horse hemoglobin, bovine hemoglobulin, rabbit hemoglobulin, human Hemoglobulin, animal hemoglobulins such as sheep hemoglobulin, animal hemocyanins, proteins extracted from roundworms (Ascaris extract, see JP-A No. 16414/1983), polylysine, polyglutamic acid, lysine-glutamic acid copolymer Copolymers, copolymers containing lysine or ornithine, etc. can be mentioned. The Ascaris extract is detailed below. The Ascaris extraction method uses Ascaris
It is extracted from the pulverized product of (suum) according to the usual protein extraction method. As the extraction solvent, various known protein extraction solvents can be used, such as water, physiological saline, 50% methanol or ethanol aqueous solution, and near-neutral buffer solutions, with physiological saline being particularly preferred.
More specifically, the above extract is produced, for example, as follows. That is, after removing the contents of the porcine roundworm 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 and homogenized. while extracting. This extraction is usually carried out at low temperatures, preferably at about 2 DEG to 10 DEG 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, and the suspended matter is removed, followed by freeze-drying. Thereby, the desired Ascaris extract is produced. This can be used in the present invention after further purification by conventional protein purification methods such as dialysis, gel filtration, adsorption, chromatography, etc., if necessary. Further, the above-mentioned Ascaris extract may be used, for example.
J.Immun., 111 , 260-268 (1973), J.Immun.,
122, 302-308 (1979), J.Immun., 98 , 893-900
(1967) and Am.J.Physiol., 199 , 575-578
(1960) or further purified versions thereof. As the hapten-carrier binding reagent, a wide range of reagents that are commonly used in the preparation of antigens can be used. Aliphatic dialdehydes such as cynaldehyde and adipaldehyde, crosslinking between thiol groups, such as N,N'-o-phenylene dimaleimide, N,N'-m-phenylene dimaleimide, etc. The dimaleimide compound crosslinks the amino group and the thiol group. For example, metamaleimidobenzoyl-N-hydroxysuccinimide ester, 4
-(maleimidomethyl)-cyclohexane-1-
Maleimide carboxyl-N-hydroxysuccinimide ester compounds such as carboxyl-N'-hydroxysuccinimide ester, reagents used in the usual peptide bond-forming reaction to form an amide bond between an amino group and a carboxyl group, such as N,N-dicyclohexylcarbodiimide, N ―
Ethyl-N'-dimethylaminocarbodiimide,
Examples include dehydration condensation agents such as carbodiimides such as 1-ethyl-3-diisopropylaminocarbodiimide and 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbosiimide, and further examples include P-diazoniumphenylacetic acid. It is also possible to use a combination of diazonium arylcarboxylic acids such as and ordinary peptide bond-forming reaction reagents, such as the dehydration condensation agent described above. The reaction for producing the antigen of the present invention is carried out, for example, in an aqueous solution or a normal buffer solution with a pH of 7 to 10, preferably with a pH of 8 to 9.
The reaction is carried out in a buffer solution at 0 to 40°C, preferably around room temperature. The reaction is usually completed in about 1 to 24 hours, preferably 3 to 5 hours. Typical buffer solutions used in the above may include the following. 0.2N sodium hydroxide - 0.2M boric acid - 0.2M potassium chloride buffer, 0.2M sodium carbonate - 0.2M boric acid - 0.2M potassium chloride buffer, 0.05M sodium tetraborate - 0.2M boric acid -
0.05M sodium chloride buffer, 0.1M potassium dihydrogen phosphate-0.05M sodium tetraborate buffer In the above, the proportions of hapten, hapten-carrier binding reagent, and carrier can be determined as appropriate;
Generally, it is preferable to use the carrier in an amount of 2 to 6 times the weight of the hapten, preferably 3 to 5 times the weight, and the hapten-carrier binding reagent to be used in an amount of about 5 to 10 times the weight. Through the above reaction, a human limphoblastoid interferon antigen consisting of a peptide-carrier complex in which a carrier and a hapten are bound is obtained through the mediation of a hapten-carrier binding reagent. The antigen obtained after completion of the reaction can be easily isolated and purified by conventional methods such as dialysis, gel filtration, and fractional precipitation. Moreover, the antigen can be preserved by conventional freeze-drying methods. In this way, a human lymphoblastoid interferon antigen consisting of a complex of at least one N-terminal peptide of human lymphoblastoid interferon represented by the general formula (1) and a carrier is obtained. The antigen usually has an average of 5 to 20 moles of peptide bound to 1 mole of protein, and all of these antigens allow for the production of antibodies with high reproducibility and high specificity against human limphoblastoid interferon. be. In particular, those with a binding molar ratio of peptide to protein of 1:8 to 15,
This method is preferable because it allows production of antibodies with higher specificity, higher titer, and higher sensitivity. 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 living body. There are no particular restrictions on the mammal used for antibody production, but it is usually desirable to use rabbits or guinea pigs. For antibody production, dilute the prescribed amount of the antigen obtained above with physiological saline to an appropriate concentration, and add Freund's auxiliary solution (Complete
Freund's Adjuvant) to prepare a suspension, which may then be administered to a mammal. For example, intradermally inject the above suspension into rabbits (the amount of antigen is 0.5~
5 mg/dose) and then administered every 2 weeks for 2 to 10 months, preferably 4 to 6 months, for immunization. Antibodies are collected by collecting blood from the immunized animal at a time when large amounts of antibodies are produced after the final administration of the suspension, usually 1 to 2 weeks after the final administration, and centrifuging the blood, followed by separating and collecting the serum. It is done by doing.
In particular, the method of the present invention has the advantage that, based on the specificity of the antigen used, it is possible to obtain antibodies with extremely high specificity for human limphoblastoid interferon, and with high titer and high sensitivity. be. The human lymphoblastoid interferon antibody thus obtained has particularly excellent specificity for human lymphoblastoid interferon as described above, and is suitable for quantifying human lymphoblastoid interferon by the RIA method, which is desired in this field. This is a useful tool that enables high accuracy. Further, the antibody can be used in enzyme immunoassay (EIA) method, florescence immunoassay (FIA) method, etc. by labeling it with an enzyme or a fluorescent substance. Furthermore, the antibody can be made into an insolubilized antibody by reacting with a known insolubilizing substance. In order to explain the present invention in more detail, examples of the production of the peptide of 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 R value in each production example was measured by thin layer chromatography on silica gel using the following mixed solvent. R =...1-butanol-acetic acid-water (4:1:
5) R =...1-butanol-pyridine-acetic acid-water (15:10:3:12) <Synthesis of peptide> Production example 1 1 Production of Z-Ala-Gln-OH Z-Ala-OSu4.80g of tetrahydrofuran 60
A solution of 2.19 g of H-Gln-OH in 40 ml of water and 2.10 ml of triethylamine are added to the ml solution, and the mixture is stirred at room temperature for 20 hours. Tetrahydrofuran and water are distilled off, and the residue is extracted with n-butanol. The extract is washed with 2% acetic acid and the butanol is distilled off. The precipitated material was collected, reprecipitated with methanol-ethyl acetate, and Z
-Ala-Gln-OH3.87g is obtained. R〓:0.41 R〓:0.56 Elemental analysis value (as C ₁₆ H ₂₁ N ₃ O ₆ ) Calculated value (%) C54.69 H6.02 N11.96 Actual value (%) C54.50 H6.31 N11.62 2(a) Production of H-Ala-Gln-OH 3.50 g of Z-Ala-Gln-OH is dissolved in 50 ml of water and 30 ml of methanol, and subjected to catalytic reduction using palladium as a catalyst to obtain H-Ala-Gln-OH. R〓: 0.04 2(b) Production of Z-Leu-Ala-Gln-OH Z-Leu-OSu3.97g, H-Ala obtained in above (a)
-Gln-OH and 1.39 ml of triethylamine are dissolved in 50 ml of dimethylformamide and stirred at room temperature for 20 hours. Dimethylformamide is distilled off and the residue is extracted with ethyl acetate. Wash the extract three times with 1N citric acid and five times with water. Ethyl acetate is distilled off, ether is added to the residue, the precipitate is dried, and reprecipitated from methanol-ethyl acetate to obtain 2.15 g of Z-Leu-Ala-Gln-OH. R〓: 0.49 R〓: 0.62 Elemental analysis value (as C ₂₂ H ₃₂ N ₄ O ₇ ) Calculated value (%) C56.88 H6.94 N12.06 Actual value (%) C56.41 H6.80 N12.18 3(a) Production of H-Leu-Aln-OH Add 20 ml of 25% hydrogen bromide-containing acetic acid solution to 2.10 g of Z-Leu-Ala-Gln-OH and leave at room temperature for 1 hour. Add dry ether to the reaction solution to form H-Leu-
Obtain Ala-Gln-OH. R〓: 0.10 3(b) Production of H-Leu-Leu-Ala-Gln-OH Z-Leu-OSu 1.96g, triethylamine 0.63
ml and H-Leu-Ala-Glu-OH are dissolved in 50 ml of dimethylformamide and stirred at room temperature for 20 hours.
Dimethylformamide is distilled off, 1M citric acid is added to the residue, and the precipitated crystals are collected, washed with water until the liquid becomes neutral, and dried. methanol-
Wash with ethyl acetate, Z-Leu-Leu-Ala-
Obtain 1.63 g of Gla-OH. R: 0.58 R: 0.64 Elemental analysis value (as C ₂₈ H ₄₃ N ₅ O ₈ ) Calculated value (%) C58.22 H7.50 N12.12 Actual value (%) C57.85 H7.90 N11.96 4(a) Production of H-Leu-Leu-Ala-Gln-OH 25% in 1.50g of Z-Leu-Leu-Ala-Gln-OH
Add 20 ml of hydrogen bromide-containing acetic acid solution and stir at room temperature for 1 hour. Add dry ether to the reaction solution, remove the precipitated solid, and prepare H-Leu-Leu-Ala-Gln.
- Get OH. R〓: 0.19 4(b) Production of Z-Ile-Leu-Leu-Ala-Gln-OH Z-Ile-OSu1.41g, H-Leu obtained above
-Leu-Ala-Gln-OH and triethylamine
Dissolve 0.36 ml in 50 ml of dimethylformamide and stir at room temperature for 20 hours. Dimethylformamide was distilled off, 1N citric acid was added to the residue, the precipitated crystals were collected, washed with hot methanol, and Z-Ile
-Leu-Ieu-Ala-Gln-OH1.17g is obtained. R〓:0.61 R〓:0.71 Elemental analysis value (as C ₃₄ H ₅₄ N ₆ O ₉ ) Calculated value (%) C59.11 H7.87 N12.16 Actual value (%) C59.23 H7.80 N12.02 5(a) Production of H-Ile-Leu-Leu-Ala-Gln-OH To 1.10 g of Z-Ile-Leu-Leu-Ala-Gln-OH
Add 15 ml of acetic acid containing 25% hydrogen bromide and stir at room temperature for 1 hour. Add dry ether to the reaction solution, remove the precipitated solid and prepare H-Ile-Leu-Leu-Ala-
Obtain Gln-OH. R〓:0.25 5(b) Z―Leu―Ile―Leu―Leu―Ala―Gln―
Production of OH Z-Leu-OSu0.69g, H-Ile obtained above
-Leu-Leu-Ala-Gln-OH and 0.22 ml of triethylamine are dissolved in 50 ml of dimethylformamide and stirred at room temperature for 20 hours. Dimethylformamide is distilled off, 1N succinic acid is added to the residue, the precipitated substance is collected, washed with water until the liquid becomes neutral, and dried. Wash with hot methanol and remove from Z-Leu-Ile.
-Leu-Leu-Ala-Gln-OH1.10g is obtained. R〓:0.58 R〓:0.71 Elemental analysis value (as C ₄₀ H ₆₅ N ₇ O ₁₀ ) Calculated value (%) C59.75 H8.15 N12.19 Actual value (%) C59.60 H8.02 N11.92 6 H-Leu-Ile-Leu-Leu-Ala-Gln-OH
Production of Z-Leu-Ile-Leu-Leu-Ala-Gln-
Dissolve 0.50 g of OH in 50 ml of methanol and 10 ml of 10% acetic acid, and perform catalytic reduction using palladium as a catalyst.
We get H-Leu-Ile-Leu-Leu-Ala-Gln-OH. This will be referred to as "peptide A" hereinafter. R〓: 0.23 R〓: 0.61 Elemental analysis value (as C ₃₂ H ₅₉ N ₇ O ₈・2H ₂ O) Calculated value (%) C54.45 H8.99 N13.89 Actual value (%) C54.30 H8. 81 N13.98 Production example 2 1 Production of Boc-Ala-NHNHZ Boc-Ala-OH4.99g, _NH2- NH-Z4.36g
and 5.44 g of dicyclohexylcarbodiimide were dissolved in 150 ml of tetrahydrofuran and stirred at 4°C for 20 hours. Remove the precipitated solid, evaporate the liquid, add ether to collect the precipitate, reprecipitate from ether and petroleum ether, and obtain Boc-Ala-
Obtain NHNHZ7.03g. R〓: 0.79 R〓: 0.81 Elemental analysis value (as C ₁₆ H ₂₃ N ₃ O ₅ ) Calculated value (%) C56.96 H6.87 N12.45 Actual value (%) C56.81 H6.49 N12.34 2(a) Production of H-Ala-NHNHZ 3.00 g of Boc-Ala-NHNHZ is dissolved in 10 ml of trifluoroacetic acid, and after being left at room temperature for 15 minutes, the trifluoroacetic acid is distilled off and dried to obtain H-Ala-NHNHZ. R〓: 0.51 2(b) Production of Boc-Arg(NO ₂ )-Ala-NHNHZ Dissolve 2.84 g of Boc-Arg(NO ₂ )-OH in 40 ml of tetrahydrofuran and 0.91 ml of N-methylmorpholine solution and heat to -15℃. After cooling, add 1.17 ml of isobutyl chloroformate and stir vigorously for 30 seconds. This is dimethylformamide of H-Ala-NHNHZ.
Add 20 ml solution and 1.24 ml triethylamine solution and 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. After distilling off tetrahydrofuran and dimethylformamide, the mixture is extracted with ethyl acetate. The extract was washed with 1N citric acid, then saturated sodium bicarbonate, and then saturated saline, and the ethyl acetate was distilled off.
Reprecipitate with ether to form Boc―Arg(NO ₂ )―Ala―
Obtain NHNHZ3.70g. R〓:0.68 R〓〓:0.79 Elemental analysis value (as C ₂₂ H ₃₄ N ₈ O ₈ ) Actual value (%) C49.06 H6.36 N20.80 Actual value (%) C49.40 H6.72 N20. 43 3(a) Production of H-Arg(NO ₂ )-Ala-NHNHZ Dissolve 3.67 g of Boc-Arg(NO ₂ )-Ala-NHNHZ in 15 ml of trifluoroacetic acid, leave it at room temperature for 15 minutes, and then add dry ether. The mixture is added and crystallized, and the crystals are taken to obtain H-Arg(NO ₂ )-Ala-NHNHZ. R〓:0.20 3(b) Boc―Arg(NO ₂ )―Arg(NO ₂ )―Ala―
Production of NHNHZ 2.17 g of Boc-Arg(NO ₂ )-OH is dissolved in 50 ml of tetrahydrofuran and 0.69 ml of N-methylmorpholine, and after cooling to -15°C, 0.89 ml of isobutyl chloroformate is added and stirred vigorously for 30 seconds. Add to this the H-Arg(NO ₂ )-Ala- obtained in (a) above.
Add a solution of NHNHZ in 30 ml of dimethylformamide and 0.95 ml of triethylamine and stir for 1 minute. Warm at 0°C for 5 minutes, then at 40°C for 2 minutes, then stir at room temperature for 15 minutes. Tetrahydrofuran and dimethylformamide are distilled off, and the residue is extracted with ethyl acetate. The extract is washed successively with 1N citric acid and a saturated aqueous sodium bicarbonate solution, and then ethyl acetate is distilled off. Boc-Arg(NO ₂ )-Arg(NO ₂ ) by reprecipitation with ethyl acetate-ether
-Ala-NHNHZ3.70g is obtained. R〓:0.58 R〓:0.75 Elemental analysis value (as C ₂₈ H ₄₅ N ₁₃ O ₁₁ ) Actual value (%) C45.46 H6.13 N24.61 Actual value (%) C45.13 H5.71 N24.51 4(a) H―Arg(NO ₂ )―Arg(NO ₂ )―Ala―
Production of NHNHZ Boc―Arg(NO ₂ )―Arg(NO ₂ )―Ala―
Dissolve 3.00 g of NHNHZ in 20 ml of trifluoroacetic acid, let stand at room temperature for 15 minutes, and then add dry ether to crystallize. Take the crystal and H-Arg(NO ₂ )
-Arg(NO ₂ )-Ala-NHNHZ is obtained. R〓：0.11 4(b) Boc－Asn－Arg(NO ₂ )－Arg(NO ₂ )－
Production of Ala―NHNHZ H―Arg(NO ₂ )―Arg(NO ₂ )―Ala―
Dissolve NHNHZ in 50 ml of DMF, add 0.56 ml of triethylamine and 2.17 g of Boc-Asn-ONHS, and leave at room temperature for 20 hours. Dimethylformamide is distilled off and the residue is extracted with butanol. After washing the extract with 2% acetic acid, add ether to crystallize it, collect the crystals, and reprecipitate from methanol-acetic acid to form Boc-Asn-Arg(NO ₂ )-Arg(NO ₂ ).
-Ala-NHNHZ2.64g is obtained. R〓:0.40 R〓:0.72 Elemental analysis value (as C ₃₂ H ₅₁ N ₁₅ O ₁₃ ) Calculated value (%) C45.01 H6.02 N24.60 Actual value (%) C44.80 H5.85 N24.12 4(c) Production of Boc-Asn-Arg-Arg-Ala-NHNH ₂ Boc-Asn-Arg(NO ₂ )-Arg(NO ₂ )-Ala
-Dissolve 2.50 g of NHNHZ in a mixture of 30 ml of methanol and 30% acetic acid, and perform catalytic reduction using palladium as a catalyst to obtain 2.20 g of Boc-Asn-Arg-Arg-NHNH ₂ . R〓:0.06 R〓:0.40 Elemental analysis value (C ₂₄ H ₄₇ N ₁₃ O ₇・2CH ₃ CO ₂ H・H ₂ O
) Calculated value (%) C43.80 H7.48 N23.71 Actual value (%) C43.51 H7.62 N23.45 5 Production of Z-Leu-Gly-OC ₂ H ₅ 1.86ml of N-methylmorpholine Dissolve in 60ml of tetrahydrofuran and add 4.85g of Z-Leu-OH.
Add. Cool to -15°C, add 2.41 ml of isobutyl chloroformate, and stir vigorously for 30 seconds. To this, add 40 ml of dimethylformamide solution of 2.54 g of H-Gly-OC ₂ H ₅ HCl and triethylamine.
Add 2.56ml and stir for 1 minute. 5 minutes at 0℃
It is then warmed to 40°C for 2 minutes and then stirred at room temperature for 15 minutes. Tetrahydrofuran and dimethylformamide are distilled off, 1M citric acid is added to the residue, and the precipitated crystals are collected. The solution was washed with water until it became neutral, the precipitated crystals were dried, and reprecipitated with ethyl acetate-ether to form Z-Leu-Gly-OC ₂ H ₅ 4.68
get g. R〓:0.80 R〓:0.77 Elemental analysis value (as C ₁₈ H ₂₆ N ₂ O ₅ ) Calculated value (%) C61.70 H7.48 N7.99 Actual value (%) C61.51 H7.32 N7.80 6(a) Production of H-Leu-Gly-OC ₂ H ₅ Add 3.12 g of Z-Leu-Gly-OC ₂ H ₅ to 50 g of methanol.
ml and 8.90 ml of 1N hydrochloric acid and catalytically reduced it using palladium as a catalyst to form H-Leu-Gly-OC ₂ H ₅
get. R: 0.41 6(b) Production of Z-Ser-Leu-Gly-OC ₂ H ₅ Dissolve 2.48 g of Z-Ser-NHNH ₂ in 20 ml of dimethylformamide and 4.89 ml of 6N hydrochloric acid/dioxane, and cool to -15°C. Afterwards, add 1.31 ml of isoamyl nitrite and stir for 5 minutes. Then triethylamine
Add 4.11ml to neutralize. H- obtained in (a) above
Leu―Gly―OC ₂ H ₅・1HCl and triethylamine
Add the above reaction solution to 1.24 ml of a 10 ml solution of dimethylformamide, and stir at 4°C for 20 hours. After distilling off dimethylformamide, the residue is extracted with ethyl acetate, and the extract is washed successively with 1N citric acid and saturated brine, and then dried over magnesium sulfate. After distilling off ethyl acetate, it is reprecipitated from ethyl acetate to give Z
-Ser-Leu-Gly-OC ₂ H ₅ H ₅ 2.64g is obtained. R〓:0.78 R〓:0.85 Elemental analysis value (as C ₂₁ H ₃₁ N ₃ O ₇ ) Calculated value (%) C57.65 H7.14 N9.60 Actual value (%) C57.60 H6.88 N9.63 7(a) Production of H-Ser-Leu-Gly-OC ₂ H ₅ Add 2.50 g of Z-Ser-Leu-Gly-OC ₂ H ₅ to 10% acetic acid.
H-Ser-Leu-Gly-
_{Obtain OC2H5} . R〓:0.31 7(b) Z―Thr―His―Ser―Leu―Gly―OC ₂ H ₅
Production of Z-Thr-His-NHNH ₂ 2.54g and dimethylformamide 20ml and 6N-hydrochloric acid/dioxane 4.19g
ml and after cooling to -15℃, isoamyl nitrite
Add 0.84ml and stir for 5 minutes. Next, add 3.51 ml of triethylamine to neutralize. The above reaction solution is added to a 20 ml solution of H-Ser-Leu-Gly-OC ₈ E obtained in above (a) and 0.79 ml of triethylamine in dimethylformamide, and the mixture is stirred at 4°C for 20 hours. After distilling off dimethylformamide, the residue is extracted with butanol, and the extract is washed with water. The solvent was distilled off and recrystallized from methanol-ethyl acetate to give Z-Hhr-
4.31 g of His-GSer-Leu-Gly⊂-OC ₂ H ₅ is obtained. R〓: 0.35 R〓: 0.71 Elemental analysis value (as C ₃₁ H ₄₅ N ₇ O ₃・H ₂ O) Calculated value (%) C64.00 H8.14 N16.85 Actual value (%) C64.48 H8. 10 N16.54 8(a) Z―Thr―His―Ser―Leu―Gly―
Production of NHNH ₂ Z―Hhr―His―Ser―Leu―Gly―OC ₂ H ₅ 4.30
Dissolve g in 20 ml of methanol, add 3.18 ml of hydrazine monohydrate, and leave at room temperature for 20 hours. Add ether to the reaction solution, collect the precipitated crystals, and dry. Wash with hot methanol and Z-Hhr-His.
-Ser-Leu-Gly-NHNH ₂ 2.55g is obtained. R〓: 0.17 R〓: 0.57 Elemental analysis value (as C ₂₉ H ₄₃ N ₉ O ₉ ) Calculated value (%) C52.64 H6.55 N19.05 Actual value (%) C52.55 H6.44 N19.09 8(b) H―Asn―Arg―Arg―Ala―Leu―Ile―
Production of Leu-Leu-Ala-Gln-OH Boc-Asn-Arg-Arg-Ala-NHNH ₂ 0.78g
was dissolved in 8 ml of dimethylformamide and 1.03 ml of 6N hydrochloric acid/dioxane, and after cooling to -15°C, 0.16 ml of isoamyl nitrite was added and stirred for 5 minutes. Next, add 0.87 ml of triethylamine to neutralize. Peptide A or H-Leu-Ile-Leu-Leu-Ala-
Add the above reaction solution to a mixture of Gln-OH, 0.87 ml of triethylamine, 20 ml of dimethylformamide, and 10 ml of hexamethylphosphoric acid triamide, and add the above reaction solution at 4°C.
Stir for 24 hours, then add Boc-Asn-Arg-Arg
-Ala-NHNH ₂ 0.39 converted to azide is added and stirred for 48 hours. Dimethylformamide is distilled off and the residue is extracted with butanol. The extract is washed with water and the butanol is distilled off. Ether is added to the residue to crystallize it, and the precipitated crystals are collected. Wash with water and dry with phosphorus pentoxide. Obtained Boc-
Asn―Arg―Arg―Ala―Leu―Ile―Leu―Ala―
Dissolve Gln-OH in 3 ml of trifluoroacetic acid,
After being left at room temperature for a minute, dry ether was added to precipitate the precipitate, which was then dried and then
25 (eluent 50% acetic acid) to obtain H-Asn-
Arg―Arg―Ala―Leu―Ile―Leu―Leu―Ala―
Obtain 120mg of Gln-OH. R〓:0.01 R〓:0.35 Elemental analysis value (C ₅₁ H ₉₄ N ₁₈ O ₁₃・2CH ₃ COOH・
5H ₂ O) Calculated value (%) C47.95 H8.19 N18.30 Actual value (%) C47.66 H8.41 N18.62 [α] ²⁵ _D : -185.44 (C = 0.57, 1M acetic acid) 9 H-Hhr-His-Ser-Leu-Gly-Asn-
Arg―Arg―Ala―Leu―Ile―Leu―Leu―Ala
-Production of Gln-OH Z-Thr-His-Ser-Leu-Gly-NHNH ₂ 125
mg to 10 ml of dimethylformamide and 6N-hydrochloric acid/
Dissolve in 0.125 ml of dioxane, cool to -15°C, add 0.025 ml of isoamyl nitrite, and stir for 5 minutes. Next, add 0.105 ml of triethylamine to neutralize.
H-Asn-Arg-Arg-Ala-Leu-Ile-Leu-
Leu-Ala-Gln-OH110mg and triethylamine
The above reaction solution was added to a solution of 0.013 ml of dimethylformamide and 6 ml of hexamethylphosphoric acid triamide, and the mixture was stirred at 4°C for 24 hours. Further Z-
Add 125 mg of azidated Hhr-His-Ser-Leu-Gly-NHNH ₂ and stir for 48 hours. Dimethylformamide is distilled off and the residue is extracted with butanol. Wash the extract with water. butanol is distilled off,
Reprecipitate with methanol-ethyl acetate. Obtained Z―Hhr―His―Ser―Len―Gly―Asn―Arg―
Arg―Ala―Leu―Ile―Leu―Leu―Ala―Gln―
OH is dissolved in 50 ml of methanol and 10 ml of 10% acetic acid and catalytically reduced using palladium as a catalyst. After removing the catalyst, methanol was distilled off, and the resulting residue was purified with Sephadex G-25 (eluent: 50% acetic acid) to obtain H-Hhr-His-Ser-Leu-Gly-
Asn―Arg―Arg―Ala―Leu―Ile―Leu―Ala―
Obtain 125 mg of Gln-OH. Hereinafter, this will be referred to as "peptide B". R〓:0.01 R〓:0.38 Elemental analysis value (C ₇₂ H ₁₂₅ N ₂₅ O ₂₀・2CH ₃ COOH・
4H ₂ O) Calculated value (%) C49.21 H7.77 N18.87 Actual value (%) C49.60 H7.92 N18.54 [α] ²⁵ _D : -66.76 (C = 0.42, 1M acetic acid) Manufacture Example 3 1(a) H―Ser―Asp―Leu―PPro―Gln―
Manufacturing of NHNHBoc Z―Ser―Asp―Leu―Pro―Gln―
Dissolve 1.03g of NNHHBoc in 50ml of methanol,
Catalytic reduction using palladium as a catalyst to produce H-Ser
-Asp-Leu-Pro-Gln-NHNHBoc is obtained. R〓:0.06 1(b) Z―Hyr―Ser―Asp―Leu―Pro―Gln―
Production of NHNHBoc Z-Tyr-ONHS 0.79g was added to H obtained in (a) above.
-Ser-Asp-Leu-Pro-Gln-NHNHBoc in 20 ml of dimethylformamide solution and left at room temperature for 20 hours. After distilling off the dimethylformamide, the residue is extracted with ethyl acetate. The extract is washed successively with 1N citric acid and saturated brine, and ethyl acetate is distilled off. Reprecipitate with methanol-ethyl acetate to form Z-Tyr-Ser-Asp-Leu-Pro-Gln-
Obtain 588 mg of NNHHBoc. R〓:0.51 R〓:0.69 Elemental analysis value (as C ₅₂ H ₆₉ N ₉ O ₁₅ ) Calculated value (%) C58.91 H6.56 N11.89 Actual value (%) C58.53 H6.22 N12.28 2 H-Tyr-Ser-Asp-Leu-Pro-Gln-
Thr―His―Ser―Leu―Gly―Asn―Arg―Arg
―Ala―Leu―Ile―Leu―Leu―Ala―Gln―
Manufacturing of OH Z―Tyr―Ser―Asp―Leu―Pro―Gln―
Dissolve 44 mg of NNHHBoc in 3 ml of dimethylformamide and 0.0225 ml of 6N hydrochloric acid/dioxane, -15
After cooling to ℃, add 0.0060 ml of isoamyl nitrite,
Stir for a minute. Then triethylamine 0.0252ml
Add to neutralize. Peptide B or H-Thr-His-Ser-Leu-
Gly―Asn―Arg―Arg―Ala―Leu―Ile―Leu―
Leu-Ala-Gln-OH, 25 mg dimethylformamide 5 ml and hexamethyl phosphoric triamide 2 ml
Add the above reaction solution to the solution and stir at 4°C for 20 hours. Furthermore, Z-Tyr-Ser-Asp-Leu-
Add azidated Pro-Gln-NHNHBoc (44 mg) and 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. Obtained Z-Tyr-Ser-Asp-Leu-Pro-
Gln―Thr―His―Ser―Leu―Gly―Asn―Arg―
Arg―Ala―Leu―Ile―Leu―Leu―Ala―Gln―
Dissolve OH in 30 ml of methanol and perform catalytic reduction using palladium as a catalyst. The catalyst was removed, methanol was distilled off, and the resulting residue was purified with Sephadex G-25 (eluent: 50% acetic acid), followed by LH-20 (eluent: 1/1000N hydrochloric acid), and purified with H-Tyr-Ser- Asp―Leu―Pro―Gln―
Thr―His―Ser―Leu―Gly―Asn―Arg―Ala―
Obtain 18 mg of Leu-Ile-Leu-Leu-Ala-Gln-OH. Hereinafter, this will be referred to as "peptide C". R〓:0.01 R〓:0.38 Elemental analysis value (C ₁₀₄ H ₁₆₉ N ₃₂ O ₃₁・2CH ₃ COOH・
5H ₂ O) Calculated value (%) C50.40 H7.32 N17.41 Actual value (%) C50.72 H7.67 N17.03 [α] ²⁵ _D : -77.88 (C = 0.22, 1M acetic acid) < Production of antigen> Production example 1 Peptide synthesis Peptide A 5 obtained in Production example 1
mg and 15 mg of bovine serum albumin (hereinafter abbreviated as "BSA") are dissolved in 2 ml of ammonium acetate buffer (0.1 mol, PH 7.0). Add 0.11 ml of 0.1 molar glutaraldehyde solution to this solution and stir at room temperature for 5 hours. The reaction mixture was then stirred for 48 h, 4
Dialyze against 1 ml of water at °C. Change the water 5 times during dialysis. Thereafter, the solution containing the peptide-protein complex was lyophilized to produce 18 mg of human limphoblastoid interferon antigen (hereinafter referred to as "antigen").
get. This antigen has peptide A for 1 mole of BSA.
is combined with an average of 12 moles. Production example 2 Peptide synthesis 5 mg of peptide B obtained in production example 2
and 20 mg of BSA in ammonium acetate buffer (0.1
Mol, PH7.0) Dissolve to 2 ml. Add 0.11 ml of 0.1 molar glutaraldehyde solution to this solution and stir at room temperature for 5 hours. The reaction mixture is then dialyzed against 1 part water at 4°C for 48 hours. Change the water 5 times during dialysis. Thereafter, the solution containing the peptide-protein complex was lyophilized to produce 23 mg of human limphoblastoid interferon antigen (hereinafter referred to as "antigen").
get. The obtained antigen has an average of 9 moles of peptide B bound to 1 mole of BSA. <Production of antibody> Production example 1 100 μg of the antigen obtained in antigen production example 1 was added to 1.5 ml.
After dissolving in physiological saline, add Freund's auxiliary solution to
The suspension prepared by adding 1.5 ml was added to three rabbits (2.5
~3.0Kg), 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 human limboblastoid interferon antibody (hereinafter referred to as "antibody") of the present invention. Production Example 2 After dissolving 20 μg of the antigen obtained in Antigen Production Example 2 in 1.5 ml of physiological saline, add Freund's auxiliary solution.
The suspension prepared by adding 1.5 ml to 7 rabbits (2.5
~3.0Kg), 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 human limboblastoid interferon antibody (hereinafter referred to as "antibody") of the present invention. Production of Γ-labeled peptide H-Tyr-Ser-Asp-Leu-Pro-Gln-Hhr
-His-Ser-Leu-Gly-Asn-Arg-Arg-Ala
-Leu-Ile-Leu-Leu-Ala-Gln-OH, ie, peptide C, is labeled using chloramine T as follows. That is, 5 μg of the above peptide was added to 20 μl of 0.5 molar phosphate buffer (PH7.0) with Na[ ₁₂₅ ] (carrier free NE
N.) Add 1 millicurie of 0.5 molar phosphate buffer, then add 20 μl of 0.5 molar phosphate buffer of 70 mg chloramine T/ml. The reaction is terminated by stirring at room temperature for 30 seconds and adding 50 μl of 60 mg/ml sodium metabisulfite (Na ₂ S ₂ O ₅ ) in 0.5 M phosphate buffer. Next, 100 μl of a cold 1% aqueous sodium iodide solution was added to the reaction mixture, and the reaction mixture was transferred to a Sephadex G-25 column (1.0 x 30 cm).
(Eluent: 0.05 M phosphate buffer containing 0.25% BSA, 10 mM EDTA and _0.02 % NaN,
PH7.4). The 13th and 14th fractions are the above peptide labeled with ¹²⁵ . Γ Measurement of titer Measure the antibody and titer obtained above as follows. That is, each antibody was diluted with physiological saline for 10 min,
10 ² , 10 ³ , 10 ⁴ , 10 ⁵ …diluted (initial)
Then, add 0.1 ml of ^125- labeled peptide (the labeled peptide obtained above was diluted to approximately 9500 cpm) and 0.05 M phosphate buffer (PH = 7.4) [0.25% BSA, 10 mM] to 100 μl of each of these.
Add 0.2 ml of EDTA and 0.02% _NaN3 ,
Incubate for 24 hours at °C to remove the generated antibodies and
The conjugate with ^125- labeled antigen was separated by dextran-activated charcoal method and centrifugation method (4°C, 30 minutes, 3000 rpm).
The final reaction (not binding) is separated from the ^125- labeled peptide, the radiation is counted, and the binding rate (%) of the antibody to ^{the 125-} labeled peptide at each diluted concentration is measured. The binding rate (%) of the antibody to the ^125- labeled peptide is plotted on the vertical axis, and the dilution factor (initial concentration) of the antibody is plotted on the horizontal axis, and the binding rate is plotted at each concentration. Determine the dilution ratio of the pore at which the binding rate is 50%, that is, the titer of the antibody. The results are shown in Table 1 below. [Table] Specificity test of human rimhoblastoid interferon of Γ antibody Test samples were human β-type interferon (manufactured by Tokyo Metropolitan Clinical Research Institute, specific activity 3×) at various concentrations.
10 ⁶ U/mg protein) and human α-type interferon (manufactured by Hayashibara Research Institute, Limphoblastoid Interferon Lot. No. 800928 and Cantel). Also 0.25% BSA, 5mM EDTA and 0.02% _NaN3 as labeling diluents.
Use 0.05 molar phosphate buffer (PH7.4) containing Add 0.2 ml of standard diluent and sample to each test tube.
Add 0.1 ml of the antibody obtained in Antibody Production Examples 1 and 2 (potency 200,000) and 0.1 ml of the ^125- labeled peptide (the labeled peptide obtained above diluted to about 2800 cpm). , 72 at 4℃
After incubating for an hour, add 0.1 ml of normal porcine serum, then add 0.5 ml of dextran-coated activated charcoal suspension, leave for 30 minutes at 4°C, then incubate at 4°C, 3000 rpm.
Centrifuge for 30 minutes under the conditions of
Conjugate and terminal reaction with ^125- labeled peptide (no binding) Separate the ^125- labeled peptide, count its radiation, and set the binding rate (Bo) corresponding to the titer of the antibody used as 100% for each test sample. Determine the percentage of the conjugate (B) between the antibody and the ^125- labeled peptide by applying the concentration and dilution rate. From the results obtained, both the antibodies derived from the peptides A and B of the present invention are clearly distinguished in their reactivity to human α-type interferon and reactivity to human β-type interferon, and are highly reactive to human α-type interferon. It was found that this antibody is a highly specific antibody that shows strong specific reactivity against human β-type interferon, but does not cross-react with human β-type interferon up to 3.0×10 ⁶ units/ml. In addition, the binding rate of the antibody and peptide obtained in the above antigen production examples 1 and 2 and BSA is as follows:
When each antigen obtained was further gel-filtered with Sephadex G-50 (eluent: physiological saline, detection: OD280nm, flow rate: 3ml/hour, fractional volume: 1ml each), the presence of unreacted BSA and peptides was eliminated. Since this was not observed, the fraction of the peptide bound to BSA and the fraction of other products (peptide dimers) were separated by the gel filtration,
Create a calibration curve of standard concentration of peptide dimer,
Determine the amount of the above dimer and subtract it from the amount of peptide used as the starting material.
This was determined assuming that it is bound to BSA.

Claims (1)

[Claims] 1 General formula R-Leu-Ile-Leu-Leu-Ala-Gln-OH [In the formula, R is a hydrogen atom or H-Thr-His-Ser
-Leu-Gly-Asn-Arg-Arg-Alg group] An N-terminal peptide of human limphoblastoid interferon.