JPH0160036B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel N-terminal peptide of β-type interferon. 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. Arg: Arginine Asn: Asparagine Ser: Serine Gln: Glutamine Met: Methionine Leu: Leucine Phe: Phenylalanine Tyr: Tyrosine Gly: Glycine Z: Carbobenzoxy group Su: Succinimide group Tos: p-Toluenesulfonyl group Boc: Tertiary butoxycarbonyl group Interferon is an antiviral (glyco) protein produced when living cells are infected with a virus, and its use to prevent or treat viral diseases is attracting attention. Human interferon is α-type interferon (Leucocyte Interferon, Lympho blastoid
To date, it has been elucidated that it is a mixture of interferon, β-type interferon (Fibroblast interferon), and gamma-type interferon (Immune interferon). However, a technique for purifying such human interferon into a single type of glycoprotein has not yet been developed. The ultimate objective of the present invention is to isolate and purify β-type interferon from human interferon using an antibody that specifically reacts with β-type interferon. Although many antibodies that react with human β-type interferon have been known, none of these antibodies has specific reactivity with human β-type interferon. The inventors
We have been conducting intensive research on methods to select human β-type interferon with high sensitivity and obtain antibodies that exhibit specific reactivity against human β-type interferon. As a result, the general formula R-Leu-Gln-Arg-Ser-Ser-OH (1) [wherein R is a hydrogen atom, H-Phe- group, H-Leu
―Gly―Phe― group, H―Tyr―Asn―Leu―Leu―
Gly―Phe― group or H―Met―Ser―Tyr―Asn―
Indicates Leu-Leu-Gly-Phe- group. ], successfully synthesized an antigen using the peptide as a hapten, and subsequently succeeded in creating an antibody with high specificity for the target human β-type interferon from the antigen, The present invention has now been completed. By using the peptide represented by the above general formula (1) of the present invention as a hapten, it is allowed to react with a carrier in the presence of a hapten-carrier binding reagent to produce a human β-type interferon antigen consisting of a peptide-carrier complex. obtain. Furthermore, a human β-interferon antibody having specific reactivity to human β-interferon can be produced from this human β-interferon antigen. Furthermore, this human β-type interferon antibody can be used to purify human β-type interferon by incorporating it into an affinity chromatography carrier. The peptide of general formula (1) used in the present invention is a new compound, and such a peptide can be produced by the following method. The peptide of general formula (1) can be produced by conventional peptide synthesis methods. Either a solid phase synthesis method or a liquid phase synthesis method may be used, but the liquid phase synthesis method is often advantageous. Such means of peptide synthesis are described, for example, in “The Peptides”, Volume 1 (1966), Schröder
and Luhke, Academic Press, New York,
USA or "Peptide Synthesis", written by Izumiya et al., Maruzen Co., Ltd. (1975), such as the azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, etc. ―
Nitrophenyl ester method, N-hydroxysuccinimide ester method, cyanomethyl ester method, etc., methods using Woodward's reagent K, carbodiimidazole method, redox method, DCC/additive (e.g. HONB, HOBt, HOSu) method, etc. can be given. The peptide of general formula (1) can be easily synthesized using general polypeptide synthesis methods, such as the so-called stepwise method in which amino acids are sequentially condensed one by one to the terminal amino acid, or the method in which the amino acids are divided into several fragments and coupled together. Can be manufactured. 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. If the resulting condensate has a protecting group, it can be produced by removing the protecting group using a conventional method. It is often desirable to protect arginine in the reaction process for producing the peptide of general formula (1), and in the final step, the protecting group is removed from the protected peptide in which at least one of the constituent amino acid residues of the peptide is protected. In many cases, it can be produced by eliminating all of them. Protection of functional groups that should not participate in the reaction of raw materials, protective groups, removal of the protective groups, activation of functional groups that participate in the reaction, etc. can also be appropriately selected from known methods or methods. Examples of the protecting group for the amino group of the raw material include carbobenzoxy, tert-butyloxycarbonyl,
tert-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, o-nitrophenylsulfenyl, diphenylphosphine Examples include wild boar oil. Examples of protective groups for carboxyl groups include alkyl esters (e.g. methyl,
ethyl, propyl, butyl, tert-butyl, etc.), benzyl ester group, p-nitrobenzyl ester group, p-methoxybenzyl ester group, p-chlorobenzyl ester group, benzhydryl ester group, carbobenzoxyhydrazide group , tert-butyloxycarbonyl hydrazide group, trityl hydrazide group, and the like. As a protecting group for the guanidino group of arginine,
Examples include nitro group, tosyl group, p-methoxybenzenesulfonyl group, carbobenzoxy, isobornyloxycarbonyl, adamantyloxycarbonyl, and the like. The guanidino group may also be protected in the form of an acid (eg, benzenesulfonic acid, toluenesulfonic acid, hydrochloric acid, sulfuric acid, etc.) salt. Methionine may be protected in the form of sulfoxide. Examples of raw materials with activated carboxyl groups include the corresponding acid chloride,
Acid anhydrides or mixed acid anhydrides, azides, active esters (methyl alcohol, ethyl alcohol, benzyl alcohol, pentachlorophenol, p-
Examples include esters with nitrophenol, N-hydroxysuccinimide, N-hydroxybenztriazole, N-hydroxy-5-norbornene-2,3-dicarboximide, etc. The peptide bond-forming reaction may be carried out in the presence of a condensing agent (eg, dicyclohexylcarbodiimide, a carbodiimide reagent such as carbodiimidazole, tetraethylpyrophosphite). More specifically, the peptide represented by the above general formula (1) is produced according to the method shown below. That is, among the peptides of general formula (1), peptides in which R represents a hydrogen atom are produced according to the following (), and R is H-
A peptide showing a Phe- group is produced according to the following (), a peptide in which R shows a H-Leu-Gly-Phe- group is produced according to the following (), and a peptide in which R is H-Tyr.
The peptide showing -Asn-Leu-Leu-Gly-Phe- group is produced according to the following (), and R is H-
Met―Ser―Tyr―Asn―Len―Len―Gly―Phe―
The peptide showing the group is produced according to the following (). [In the above () to (), A represents a protecting group for an amino group, B represents an active group for a hydroxyl group or a carboxyl group, and C represents a protective group for a guanidino group of arginine. ] The reaction between peptide (2) and peptide (3) can be carried out in the presence of a solvent. The solvent can be appropriately selected from those known to be usable in peptide condensation reactions, such as anhydrous or water-containing dimethylformamide, dimethyl sulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, ethyl acetate, and N-methyl. Examples include pyrrolidone, hexamethylphosphoric acid triamide, and a mixed solvent thereof. The ratio of peptide (3) and peptide (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 appropriately selected from the range known to be usable for peptide bond formation reactions, and is usually about -40°C.
to about 60°C, preferably from about -20 to about 40°C. The reaction time is generally about several minutes to 30 hours. Reaction between peptide (5) and amino acid (6), peptide (8)
Reaction between and peptide (9), peptide (11) and peptide (12)
The reaction between peptide (14) and peptide (15) can both be carried out in the same manner as the reaction between peptide (2) and peptide (3). After completion of the above condensation reaction, A of the obtained peptide (4)
The protecting groups of and C can be removed by conventional methods. Such a method includes, for example, a reduction method using metallic sodium in liquid ammonia.
Metallic sodium is used in such an amount that the reaction solution turns permanent blue for about 30 seconds to 10 minutes. This reduction is usually carried out at about -40 to -70°C. Removal of protecting groups A and C from peptide (7), peptide (10), peptide (13) or peptide (16) is
This is carried out in the same manner as the method for removing protecting groups A and C from peptide (4). Peptide (3) used in () above is produced, for example, by the method shown below. [In the above, R ¹ represents a lower alkyl group.
A, B and C are the same as above. ] The reaction between amino acid (17) and amino acid (18) can be carried out in the same manner as the reaction between peptide (2) and peptide (3). The protecting group A of peptide (19) can be removed by a conventional method. Such methods include, for example, reductive methods (e.g. hydrogenation using catalysts such as palladium, palladium black, etc., reduction with metallic sodium in liquid ammonia), acidolysis (e.g. trifluoroacetic acid,
Examples include acidolysis with strong acids such as hydrogen fluoride, methanesulfonic acid, and hydrobromic acid. For example, in the case of hydrogenation using a catalyst, it can be carried out at a hydrogen pressure of 1 atmosphere and at a temperature of 0 to 40°C. The amount of catalyst used is usually about 100 mg to 1 g, and generally 1 to 1 g.
The reaction completes in about 48 hours. In the case of acidolysis, it is usually carried out without a solvent 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.
Further, for the reduction with metallic sodium in liquid ammonia, the conditions already described above can be employed. The reaction between peptide (20) and amino acid (21) can be carried out in the same manner as the reaction between peptide (2) and peptide (3). Hydrolyze peptide (22) to produce peptide (23)
The reaction for obtaining is carried out, for example, in the presence of a mineral acid such as hydrochloric acid or sulfuric acid or an alkali such as sodium hydroxide or potassium hydroxide in a solvent such as water, methanol, ethanol or dioxane. The reaction is usually carried out at 20 to 50°C for about 30 minutes to 3 hours. Examples of methods for removing the protecting group A from the peptide (23) include hydrogenation using a catalyst such as palladium or palladium black, and acidolysis. As conditions for these methods, any of the conditions already mentioned above can be adopted. Peptide (5) used in () above is produced by removing protecting group A from peptide (4). Similarly, peptide (8) used in the above () is produced by removing the protecting group A from the peptide (7), and peptide (11) used in the above () is produced from the peptide (10) by removing the protecting group A.
The peptide (14) produced by removing peptide (14) and used in () above is the peptide (13)
It is produced by removing the protecting group A from .
Examples of methods for removing the protecting group A from peptide (4), peptide (7), peptide (10) and peptide (13) include hydrogenation using a catalyst such as palladium or palladium black, acidolysis and the like. As conditions for these methods, any of the conditions already mentioned above can be adopted. Peptide (12) used in the above () is produced, for example, by the method shown below. A―Asn―B (24) H―Leu―OR ¹ (25) ―――――――→ A―Asn―Leu―OR ¹ (26) ――――→ H―Asn―Leu―OR ¹ ( 27) ^| 12) [In the above, R ¹ , A, B and C are the same as above. ] The reaction between amino acid (24) and amino acid (25) and the reaction between peptide (27) and amino acid (28) can both be carried out in the same manner as the reaction between peptide (2) and peptide (3). . Peptide (26)
To remove protecting group A from the peptide (19)
Any method for removing the protecting group A from can be applied. Further, the reaction of hydrolyzing peptide (29) to obtain peptide (30) can be carried out under the same conditions as the reaction of hydrolyzing peptide (22) to obtain peptide (23). Preferred methods for obtaining peptide (12) from peptide (30) include, for example, the mixed acid anhydride method and the azide method. 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),
Examples include organic bases such as 1,4-diazabicyclo[2,2,2]octane (DABCO), and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, and sodium hydrogen carbonate. Any solvent commonly used in the mixed acid anhydride method can be used, and specifically, halogenated hydrocarbons such as methylene chloride, chloroform, and dichloroethane,
Aromatic hydrocarbons such as benzene, toluene, xylene, diethyl ether, tetrahydrofuran,
Ethers such as dimethoxyethane, methyl acetate,
Examples include esters such as ethyl acetate, 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, and the reaction time is generally 5 minutes to 10 hours, preferably 5 minutes to 2 hours. In the azidation method, first, an activated carboxyl group, for example, a carboxyl group activated with an alcohol such as methyl alcohol, ethyl alcohol, or benzyl alcohol, is reacted with hydrazine hydrate in a suitable solvent. Examples of the solvent used include dioxane, dimethylformamide, dimethyl sulfoxide, and a mixed solvent thereof. The amount of hydrazine hydrate to be used is usually 5 to 20 times, preferably 5 to 10 times, by molar amount relative to the activated carboxyl group. The reaction is usually carried out at -10°C or lower, preferably at -20 to -10°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 nitrous acid compound in a suitable solvent in the presence of an acid. Hydrochloric acid is usually used as the acid. Examples of the solvent include dioxane, dimethylformamide, dimethyl sulfoxide, and a mixed solvent thereof.
Examples of nitrite compounds include sodium nitrite, isoamyl nitrite, nitrosyl chloride, etc., and the nitrite compound is usually used in an amount of 1 to 2 times the amount of the hydrazine derivative, preferably 1 to 1.5 times the amount by mole of the hydrazine derivative. It is better to use twice the molar amount. 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. A labeled peptide, which is a raw material for producing a labeled antigen used in the RIA method, is produced by introducing radioactive iodine such as I ¹²⁵ or I ¹³¹ into the peptide produced above. The radioactive iodine can be introduced by conventional iodination methods, such as oxidative iodination 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. In the method of the present invention, a peptide represented by the above general formula (1) is used as a hapten. As the carrier bound to the hapten, a wide range of high-molecular natural or synthetic proteins commonly used in the preparation of antigens can be used, such as horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, and sheep serum. Animal serum albumins such as albumin, horse serum globulin,
Animal serum globulins such as bovine serum globulin, rabbit serum globulin, human serum globulin, and ovine serum globulin; animal thyroglobulins such as equine thyroglobulin, bovine thyroglobulin, rabbit thyroglobulin, human thyroglobulin, and ovine thyroglobulin; horse hemoglobin, bovine hemoglobin,
Animal hemoglobulins such as rabbit hemoglobulin, human hemoglobulin, and sheep hemoglobulin;
Animal hemocyanins, proteins extracted from roundworms (Ascaris extract, see patent application No. 1983-92781), polylysine, polyglutamic acid, lysine.
Examples include glutamic acid copolymers, copolymers containing lysine or ornithine, and the like. The Ascaris extract will be detailed below. Ascaris extract is derived from 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 such as water, physiological saline, 50% methanol or ethanol aqueous solution, and near-neutral buffer solutions can be used, 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 Ascaris porcine 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 obtained in this way 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 Ascaris extract used in the present invention is described, for example, in 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. A wide variety of hapten-carrier binding reagents that are commonly used in the preparation of antigens can be used, and examples include gluoxal, malondialdehyde, glutaraldehyde, and sulfate, which cross-link amino groups. Aliphatic dialdehydes such as cynaldehyde and adipaldehyde, crosslinking between thiol groups, such as N,N'-o-phenylene dimaleimide, N,N'-m-phenylene dimaleimide, etc. dimaleimide compounds that crosslink amino groups and thiol groups, such as 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)carbodiimide, and 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 antigen of the present invention is produced by reacting the above hapten with a carrier in the presence of a hapten-carrier binding reagent. The above reaction can be carried out in aqueous solution or at pH 7.
-10 in a normal buffer solution, preferably at a pH of 8 to 9, at 0 to 40°C, preferably around room temperature,
The reaction is 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; 2 to 6 times the weight, preferably 3 to 5 times the weight, and the hapten-carrier binding reagent 5
It is best to use ~10 times the molar amount. Through the above reaction, an antigen of human β-type interferon, which is composed of the peptide-carrier complex of the present invention 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. The antigen of the present invention obtained in this way usually has an average of 5 to 20 moles of peptide bound to 1 mole of protein, and in both cases, it is possible to continue producing antibodies with high reproducibility and high specificity for human β-type interferon. However, those in which the binding molar ratio of the peptide to the above-mentioned protein is 1:8 to 15 are particularly preferable because they have even higher specificity and can produce antibodies with high titer and high sensitivity. In producing antibodies using the antigen obtained above, a conventional method can be employed in which the antigen is administered to a mammal and the antibodies produced in the body are collected.
There are no particular restrictions on the mammal used for antibody production, but it is usually desirable to use rabbits and 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 (0.5 to 5 mg/dose as antigen amount)
and then every 2 weeks for 2 to 10 months, preferably 4 to 6
It is sufficient to administer the drug for several months to achieve 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, according to the method of the present invention, based on the specificity of the antigen used, it is possible to produce antibodies that are always stable, have excellent specificity for human β-type interferon, and have high titers and high sensitivity. It has the advantage of being able to be obtained with good reproducibility. As mentioned above, the antibody thus obtained has particularly excellent specificity for human β-type interferon, and can be used to quantify human β-type interferon with high precision by the RIA method, which is desired in this field. It is a useful thing that makes it possible. In addition, the antibody is
By labeling this with an enzyme or a fluorescent substance, it can be used in enzyme immunoassay (EIA), florescence immunoassay (FIA), etc. Furthermore, the antibody can be made into an insolubilized antibody by reacting with a known insolubilizing substance. Reference examples and examples will be given below to explain the present invention in more detail, but the present invention is not limited thereto. The Rf value in the reference example was measured by layer chromatography on silica gel using the following mixed solvent. Rf〓...1-butanol-acetic acid-water (4:1:
5) Rf =...1-butanol-pyridine-acetic acid-water (15:10:3:12) <Peptide synthesis> Reference example 1 Production of Z-Ser-Ser-OMe Z-Ser-NHNH ₂ 5.06g to 50ml of dimethylformamide and 6.66 ml of 6N-HCl/dioxane, and after cooling to -15°C, add 2.68 ml of isoamyl nitrite and stir for 5 minutes. then 5.60
ml by adding triethylamine to neutralize. This solution is added to 30 ml of dimethylformamide solution containing 3.11 g of H-Ser.OMe.HCl and 2.80 ml of triethylamine, and stirred at 4° C. for 20 hours. Dimethylformamide was distilled off, and the resulting residue was extracted with ethyl acetate, washed with water, and then dried over Glauber's salt. After distilling off ethyl acetate, ether was added to crystallize, reprecipitation was performed with ethyl acetate-ether, and Z-Ser-Ser
- Obtain 3.75g of OMe. Rf value: 0.64, Rf value: 0.77 Elemental analysis value (as C ₁₅ H ₂₀ N ₂ O ₇ ) C H N Calculated value (%) 52.94 5.92 8.23 Actual value (%) 52.67 5.89 8.35 Reference example 2 Z-Arg Production of (Tos)-Ser-Ser-OMe 2.5 g of Z-Ser-Ser-OMe was dissolved in 50 ml of methanol and 7.34 ml of 1N-HCl, and in the presence of 500 mg of palladium black at 20°C and 1 atm hydrogen pressure. Perform catalytic reduction to obtain H-Ser-Ser-OMe.HCl. Rf value: 0.08 Add 3.39 g of Z-Arg(Tos)-OH to 40 ml of tetrahydrofuran and 0.75 ml of N-methylmorpholine, and after cooling to -15°C, add 0.97 ml of isobutyl chloroformate and stir vigorously for 30 seconds. . H-Ser-Ser-OMe・HCl obtained above,
20ml dimethylformamide and triethylamine
Add 1.03 ml and stir for 1 minute, then stir at 0°C for 5 minutes, at 40°C for 2 minutes, and at room temperature for 15 minutes. After distilling off the tetrahydrofuran and dimethylformamide, the mixture is extracted with ethyl acetate, washed three times with 1N citric acid, then five times with water, and dried over Glauber's salt.
The solvent was distilled off, reprecipitation was performed with methanol-ethyl acetate, and ether, and Z-Arg(Tos)-Ser-
Obtain 3.65 g of Ser-OMe. Rf value: 0.51, Rf value: 0.73 Elemental analysis value (as C ₂₈ H ₃₈ N ₆ O ₁₀ S) C H N Calculated value (%) 51.68 5.88 12.91 Actual value (%) 51.62 5.82 12.73 Reference example 3 Z- Production of Arg(Tos)-Ser-Ser-OH Dissolve 3.5g of Z-Arg(Tos)-Ser-Ser-OMe in 50ml of methanol and 10ml of water, and prepare 1N-
Add 8.06 ml of NaOH, stir at room temperature for 45 minutes, then neutralize by adding 7.24 ml of 1N-HCl, and after distilling off methanol, extract with n-butanol. After washing with water
- Butanol and water were distilled off, ether was added to the residue to crystallize it, reprecipitation was performed from methanol-ethyl acetate, and Z-Arg(Tos)-Ser-Ser-
Obtain 2.60g of OH. Rf value: 0.28, Rf value: 0.57 Elemental analysis value (as C ₂₇ H ₃₆ N ₆ O ₁₀ S・1/2H ₂ O) C H N Calculated value (%) 50.22 5.77 13.02 Actual value (%) 50.24 5.62 13.20 Reference Example 4 Production of H-Arg(Tos)-Ser-Ser-OH Dissolve 1.35g of Z-Arg(Tos)-Ser-Ser-OH in 50ml of methanol and 10ml of 10% acetic acid to obtain 500mg.
catalytic reduction in the presence of Pd at 20°C and normal pressure
Get Arg(Tos)-Ser-Ser-OH. Rf value: 0.034 Reference example 5 Production of Z-Asn-Leu-OEt 5.32 g of Z-Asn-OH was added to 60 g of tetrahydrofuran
Using 40 ml of dimethylformamide solution containing 2.64 ml of isobutyl chloroformate, 3.91 g of H-Leu-OEt・HCl and 2.80 ml of triethylamine dissolved in 10 ml of dimethylformamide and 2.04 ml of N-methylmorpholine, the procedure of Reference Example 2 was carried out. Carry out the reaction according to the method. After distilling off the solvent, a 1 molar aqueous citric acid solution was added, the precipitate was collected, washed with water, dried under reduced pressure,
Recrystallize from ethyl acetate to Z-Asn-Leu-OEt
Obtain 6.1g. Rf value: 0.71, Rf value: 0.80 Elemental analysis value (as C ₂₀ H ₂₉ N ₃ O ₆ ) C H N Calculated value (%) 58.95 7.17 10.31 Actual value (%) 58.99 7.23 10.26 Reference example 6 Z-Tyr -Production of Asn-Leu-OEt 3.01g of Z-Asn-Leu-OEt and 50ml of methanol
and dissolved in 7.4ml of 1N-HCl, palladium black 500
Catalytic reduction was carried out at room temperature and normal pressure in the presence of H
Obtain -Asn-Leu-OEt・HCl. Rf〓 value: 0.26 H-Asn-Leu-OEt・HCl obtained above was mixed with 50 ml of dimethylformamide and triethylamine.
Dissolve in 1.03 ml, then add 3.35 g of Z-Tyr-ONHS and leave at room temperature for 20 hours. After distilling off dimethylformamide, add a 1M aqueous citric acid solution, collect the precipitate, wash with water, and reprecipitate with methanol-ethyl acetate to obtain 3.58 g of Z-Tyr-Asn-Leu-OEt.
get. Rf value: 0.73, Rf value: 0.82 Elemental analysis value (as C ₂₉ H ₃₈ N ₄ O ₈ ) C H N Calculated value (%) 61.04 6.71 9.82 Actual value (%) 60.84 6.70 9.39 Reference example 7 Z-Tyr -Production of Asn-Leu-NHNH ₂ Z-Tyr-Asn-Leu-OEt2.78g in methanol
Dissolve in 30 ml of hydrazine hydrate, add 1.21 ml of hydrazine hydrate, let stand overnight, collect crystals, wash with a small amount of methanol, and obtain Z-Try-Asn-Leu-NHNH ₂
Obtain 3.10g. Rf value: 0.56, Rf value: 0.75 Elemental analysis value (as C ₂₇ H ₃₆ N ₆ O ₇ ) C H N Calculated value (%) 58.26 6.52 15.10 Actual value (%) 57.91 6.56 15.12 Example 1 (a) Boc―Leu―Gln―Arg(Tos)―Ser―Ser―
Production of OH 0.84 g of Boc-Leu-Gln-OH was dissolved in 30 ml of tetrahydrofuran and 0.24 ml of N-methylmorpholine, and 0.31 ml of isobutyl chloroformate and H-Arg(Tos)-Ser-Ser obtained in Reference Example 4 were dissolved. -OH
The procedure is carried out according to the method of Reference Example 2. After distilling off tetrahydrofuran and dimethylformamide, the residue is extracted with butanol. After washing the butanol layer with 2% acetic acid, the butanol was distilled off and reprecipitated with methanol-ethyl acetate.
Obtain 1.54 g of Arg(Tos)-Ser-Ser-OH. Rf value: 0.18, Rf value: 0.57 Elemental analysis value (as C ₃₅ H ₅₇ N ₉ O ₁₃ S・H ₂ O) C H N Calculated value (%) 48.77 6.90 14.62 Actual value (%) 48.76 6.61 14.78 ( b) Production of H-Leu-Gln-Arg-Ser-Ser-OH Boc-Leu-Gln-Arg (Tos)-Ser-Ser-
1.50 g of OH was left in 10 ml of trifluoroacetic acid for 15 minutes at room temperature to remove Boc, and crystallized by adding dry ether to form H-Leu-Gln-Arg(Tos)-Ser-
Get Ser-OH. Rf〓 value: 0.04 H-Leu-Gln-Arg(Tos) obtained above-
Dissolve Ser―Ser―OH in 25ml of liquid ammonia,
Add a small piece of metallic sodium. When the reaction solution turns blue, 1 g of dry ammonium chloride is added, and then the ammonia is distilled off. Dissolve the residue in 50% acetic acid and add Sephadex G-10 (2.2 x 85 cm,
Eluent: 50% acetic acid) and gel filtration using LH-20 (2.2 x 80 cm, eluent: 0.001N-HCl) to obtain H-Leu-Gln-Arg-Ser-Ser-OH. Rf value: 0.01, Rf value: 0.34 [α] ²⁰ _D : −42.5° (C0.230, 0.001N—HCl) Elemental analysis value (C ₂₃ H ₄₃ N ₉ O ₉・4H ₂ O・
CH ₃ COOH) C H N Calculated value (%) 41.60 7.68 17.46 Actual value (%) 41.20 7.93 17.91 Example 2 (a) Z―Phe―Leu―Gln―Arg(Tos)―Ser―
Production of Ser-OH H-Leu-Gln-Arg obtained in Example 1(b)
(Tos)-Ser-Ser-OH was dissolved in 30 ml of dimethylformamide and 0.24 ml of triethylamine, then 0.77 g of Z-Phe-ONHS was added and the mixture was heated for 20 min at room temperature.
Leave it for a while. After distilling off dimethylformamide, the residue was extracted with butanol and washed with 2% acetic acid. The butanol layer was concentrated, ether was added to crystallize, the obtained crystals were collected, and the crystals were washed with hot ethanol to obtain Z-Phe-Leu-Gln-Arg.
Obtain 1.40g of (Tos)-Ser-Ser-OH. Rf value: 0.22, Rf value: 0.59 Elemental analysis value (as C ₄₇ H ₆₄ N ₁₀ O ₁₄ S・H ₂ O) C H N Calculated value (%) 54.12 6.28 13.43 Actual value (%) 54.42 6.38 13.84 ( b) H-Phe-Leu-Gln-Arg-Ser-Ser-OH
Production of Z―Phe―Leu―Gln―Arg(Tos)―Ser―Ser
- 20 mg of OH was de-Zed and de-Tosized using metallic sodium pieces in liquid ammonia in the same manner as in Example 1(b).
% acetic acid), and further LH-20 (2.2 x 80 cm, eluent
Purified by gel filtration with 0.001N HCl)
Obtain 12 mg of Phe-Leu-Gln-Arg-Ser-Ser-OH. Rf value: 0.01, Rf value: 0.35 [α] ²⁰ _D : −37.3° (C0.249, 0.001N—HCl) Elemental analysis value (C ₃₂ H ₅₂ N ₁₀ O ₁₀・7H ₂ O・
CH ₃ COOH) C H N Calculated value (%) 44.24 7.64 15.17 Actual value (%) 44.27 7.10 15.60 Example 3 (a) Z-Leu-Gly-Phe-Leu-Gln-Arg
Production of (Tos)-Ser-Ser-OH Z-Leu-Gly-NHNH ₂ 0.39g in dimethylformamide 5ml and 6N-HCl/dioxane 0.58g
ml, isoamylnitrite 0.15ml, triethylamine 0.49ml and H-Phe-Leu-Gln-
A reaction was carried out in the same manner as in Reference Example 1 using a solution of 1.00 g of Arg(Tos)-Ser-Ser-OH in 10 ml of dimethylformamide, and an equivalent amount of Z-Leu-Gly N ₃ was added and the reaction was continued at 4°C for 20 hours. Let's do it. After extraction with butanol and washing with 2% acetic acid, butanol and acetic acid are distilled off, and ether is added to crystallize. Take the crystals, wash them with hot methanol, and
Leu-Gly-Phe-Leu-Gln-Arg(Tos)-Ser
-Ser-OH 0.65g is obtained. Rf value: 0.19, Rf value: 0.60 Elemental analysis value (as C ₅₅ H ₇₈ N ₁₂ O ₁₆ S・H ₂ O) C H N Calculated value (%) 54.44 6.64 13.85 Actual value (%) 54.74 6.66 13.98 ( b) H-Leu-Gly-Phe-Leu-Gln-Arg-Ser
- Production of Ser-OH Z-Leu-Gly-Phe-Leu-Gln-Arg (Tos)
20 mg of -Ser-Ser-OH was de-Zed and de-Tosized in the same manner as in Example 1(b), and converted into Cephadex G-10.
(2.2 x 85cm, eluent 10% acetic acid), and LH-20
(2.2 x 80 cm, eluent 0.001N-HCl) to purify H-Leu-Gly-Phe-Leu-Gln-
Obtain 11 mg of Arg―Ser―Ser―OH. Rf value: 0.01, Rf value: 0.40 [α] ²⁰ _D : −27.6° (C0.228, 0.001N—HCl) Elemental analysis value (C ₄₀ H ₆₆ N ₁₂ O ₁₂・6H ₂ O・
CH ₃ COOH) C H H Calculated value (%) 46.92 7.68 15.63 Actual value (%) 46.89 7.25 15.51 Example 4 (a) H-Tyr-Asn-Leu-Leu-Gly-Phe-
Production of Leu－Gln－Arg(Tos)－Ser－Ser－OH Z－Leu－Gly－Phe－Leu－Gln－Arg(Tos)
-Ser-Ser-OH 600mg with methanol 50ml and 10
H-Leu-Gly-
Phe―Leu―Gln―Arg(Tos)―Ser―Ser―OH
get. Rf〓 value: 0.18 Dissolve 420 mg of Z-Tyr-Asn-Leu-NHNH ₂ in 5 ml of dimethylformamide and 0.37 ml of 6N-HCl/dioxane, and add isoamyl nitrite.
Using 0.10 ml and 0.31 ml of triethylamine, an azide compound was produced according to the method of Reference Example 1, and this solution was converted into H-Leu-Gly-Phe-Leu-Gln-Arg.
Add to a mixed solution of (Tos)-Ser-Ser-OH of 10 ml of hexamethylphosphoric acid triamide and 10 ml of dimethylformamide, and stir at 4°C for 20 hours. Dimethylformamide was distilled off, the residue was extracted with butanol, and after washing with 2% acetic acid, the butanol was distilled off.
Add ether to the residue and crystallize, then obtain Z-Tyr-Asn-Leu-Leu-Gly-Phe-Leu
-Gln-Arg(Tos)-Ser-Ser-OH was dissolved in 30 ml of methanol and 10 ml of 10% acetic acid, and after catalytic reduction in the presence of palladium at room temperature and normal pressure, Sephadex G-25 (3 x 120 cm, elution H-Tyr-Asn-Leu-Leu-Gly-
Phe―Leu―Gln―Arg(Tos)―Ser―Ser―OH
Get 650mg. Rf value: 0.05, Rf value: 0.53 Elemental analysis value (as C ₆₆ H ₉₈ N ₁₆ O ₁₉ S・2H ₂ O) C H N Calculated value (%) 53.28 6.91 15.06 Actual value (%) 53.10 6.43 14.84 ( b) H-Tyr-Asn-Leu-Gly-Phe-Leu-
Production of Gln-Arg-Ser-Ser-OH H-Tyr-Asn-Leu-Leu obtained in (a) above
―Gly―Phe―Leu―Gln―Arg(Tos)―Ser―
Ser-OH was removed from Tos in the same manner as in Example 1(b), and Sephadex G-10 (2.2 x 85 cm, eluent 10
% acetic acid), and further LH-20 (2.2 x 80cm, 0.001N-
H-Tyr-Asn was purified by gel filtration with HCl).
―Leu―Gly―Phe―Leu―Gln―Arg―Ser―Ser
- Obtain 9 mg of OH. Rf value: 0.01, Rf value: 0.42 [α] ²⁰ _D : −24.3° (C0.078, 0.001N—HCl) Elemental analysis value (C ₅₉ H ₉₂ N ₁₀ O ₁₇・15H ₂ O・
CH ₃ COOH) C H N Calculated value (%) 45.01 7.80 13.77 Actual value (%) 45.21 7.31 14.22 Example 5 (a) Z―Met―Ser―Tyr―Asn―Leu―Leu―
Gly―Phe―Leu―Gln―Arg(Tos)―Ser―
Production of Ser-OH 159 mg of Z-Met-Ser-NHNH ₂ was added to 5 ml of dimethylformamide and 0.21 6N-HCl/dioxane.
ml, the azide compound was produced according to the method of Reference Example 1 using 0.055 ml of isoamyl nitrite and 0.17 ml of triethylamine, and then this solution was dissolved in H-Tyr-Asn-Leu-Leu-Gly-Phe-Leu.
Add to a mixed solution of 300 mg of -Gln-Arg(Tos)-Ser-Ser-OH, 5 ml of dimethylformamide and 5 ml of hexamethylphosphoric acid triamide, and stir at 4°C for 24 hours. Furthermore, Z-Met-Ser-NHNH ₂
Add 318 mg and stir at 4°C for 72 hours. Example 4
Purification is carried out in the same manner as in a, and Z-Met-Ser-
Tyr―Asn―Leu―Leu―Gly―Phe―Leu―Gln―
Get Arg(Tos)-Ser-Ser-OH. (b) H-Met-Ser-Tyr-Asn-Leu-Leu-
Gly―Phe―Leu―Gln―Arg―Ser―Ser―OH
Production of Z-Met-Ser-Tyr obtained in (a) above
―Asn―Leu―Leu―Gly―Phe―Leu―Gln―
Arg(Tos)-Ser-Ser-OH was removed from Z and Tos in the same manner as in Example 1(b), and Sephadex G-25 (3 x 120 cm, eluent: 50% acetic acid) was used.
Furthermore, LH-20 (2 x 85cm, eluent 0.001N-
HCl), H-Met-Ser-Tyr-Asn
―Leu―Leu―Gly―Phe―Leu―Gln―Arg―
Obtain 119mg of Ser―Ser―OH. Rf value: 0.01, Rf value: 0.45 [α] ²⁰ _D : −15.7° (C0.254, 0.001N−HCl) Elemental analysis value (C ₆₇ H ₁₀₆ N ₁₈ O ₂₀ S・5H ₂ O・
CH ₃ COOH) C H N Calculated value (%) 49.75 7.26 15.13 Actual value (%) 49.80 6.92 14.91 Amino acid analysis value Asp: 0.95, Ser: 3.15, Gln: 1.03, Met: 0.92, Gly: 1.05, Leu: 3.08 , Tyr: 1.00, Phe: 0.98, Arg: 0.96 <Production of antigen> Example 6 10 mg of the peptide obtained in Example 1(b) above (hereinafter abbreviated as "Peptide A") and bovine serum albumin (hereinafter "BSA") ”) was 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 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 is freeze-dried to obtain 45 mg of human β-type interferon antigen (hereinafter abbreviated as "antigen"). Example 7 5 mg of the peptide obtained in Example 2(b) above (hereinafter abbreviated as "peptide B") and 20 mg of 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 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 is freeze-dried to obtain 23 mg of human β-type interferon antigen (hereinafter abbreviated as "antigen"). Example 8 4 mg of the peptide obtained in Example 3(b) above (hereinafter abbreviated as "Peptide C") and 15 mg of BSA were dissolved in 2 ml of ammonium acetate buffer (0.1 mol, PH7.0). Add 0.11 ml of 0.1 molar glutaraldehyde solution to this solution and stir at room temperature for 5 hours.
The reaction mixture 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 is freeze-dried to obtain 17 mg of human β-type interferon antigen (hereinafter abbreviated as "antigen"). Example 9 4 mg of the peptide obtained in Example 4(b) above (hereinafter abbreviated as "Peptide D") and 12 mg of 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 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 is freeze-dried to obtain 14 mg of human β-type interferon antigen (hereinafter abbreviated as "antigen"). Example 10 8 mg of the peptide obtained in Example 5(b) above (hereinafter abbreviated as "Peptide E") and 25 mg of 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 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 is freeze-dried to obtain 31 mg of human β-type interferon antigen (hereinafter abbreviated as "antigen"). Example 11 8 mg of peptide E and 25 mg of BSA are dissolved in 4 ml of water. Add dicyclohexyl carbodiimide (DCC) to this solution and stir at room temperature for 5 hours. The reaction mixture is then dialyzed against 2 portions of water at 4° C. for 48 hours. Change the water 5 times during dialysis. The solution containing the peptide-protein complex was then lyophilized to produce human β-β.
29 mg of type interferon antigen (hereinafter abbreviated as "antigen") was obtained. <Production of antibody> Example 12 Dissolve 250 μg of the antigen obtained in Example 6 in 1.5 ml of physiological saline, then add 1.5 ml of Freund's auxiliary solution.
The suspension prepared by adding
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 is collected from the test animal and centrifuged to collect antiserum to obtain the human β-type interferon antibody (hereinafter abbreviated as "antibody") of the present invention. Example 13 After dissolving 250 μg of the antigen obtained in Example 7 in 1.5 ml of physiological saline, 1.5 ml of Freund's auxiliary solution was added.
The suspension prepared by adding
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 is collected from the test animal and centrifuged to collect antiserum to obtain the human β-type interferon antibody (hereinafter abbreviated as "antibody") of the present invention. Example 14 After dissolving 25 μg of the antigen obtained in Example 8 in 1.5 ml of physiological saline, 1.5 ml of Freund's auxiliary solution was added.
The suspension prepared by adding
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 is collected from the test animal and centrifuged to collect antiserum to obtain the human β-type interferon antibody (hereinafter abbreviated as "antibody") of the present invention. Example 15 After dissolving 25 μg of the antigen obtained in Example 9 in 1.5 ml of physiological saline, 1.5 ml of Freund's auxiliary solution was added.
The suspension prepared by adding
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 is collected from the test animal and centrifuged to collect antiserum to obtain the human β-type interferon antibody (hereinafter abbreviated as "antibody") of the present invention. Example 16 After dissolving 25 μg of the antigen obtained in Example 10 in 1.5 ml of physiological saline, 1.5 ml of Freund's auxiliary solution was added.
The suspension prepared by adding
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 is collected from the test animal and centrifuged to collect antiserum to obtain the human β-type interferon antibody (hereinafter abbreviated as "antibody") of the present invention. Example 17 Using the antigen obtained in Example 11, the above Example
When treated in the same manner as in 12, a high titer antibody (hereinafter abbreviated as "antibody") having specificity for human β-type interferon is obtained. Reference example Γ Production of labeled peptide (1) H-Met-Ser-Tyr-Asn-Leu-Leu-
Gly―Phe―Leu―Gln―Aug―Ser―Ser―OH
is labeled by a method using chloramine T. That is, to 20 μl of 0.2 molar phosphate buffer (PH 7.4) containing 5 μg of the above peptide, 0.2 molar phosphate buffer containing 1 microcury of [ ¹²⁵ ]Na (MEN) was added, and then 3.5 mg/ml of chloramine T was added. Add 20 µl of 0.2 molar phosphate buffer. Leave the reaction for 20 seconds at room temperature and terminate the reaction by adding 50 μl of 2.4 mg/ml sodium metadisulfate in 0.2 M phosphate buffer. The reaction mixture was converted into DEAE-Sephadex A.
-25 column (1.0 x 30cm) (eluate
0.1M Tris-HCl buffer containing 0.1% BSA and 0.01% _NaN3 , PH8.6). The first fraction was purified by ion exchange chromatography to ¹²⁵
The above peptide labeled with is obtained. (2) H-Met-Ser-Tyr-Asn-Leu-Leu-
Gly―Phe―Leu―Gln―Arg―Ser―Ser―OH
is labeled by a method using chloramine T. That is, to 20 μl of 0.2 molar phosphate buffer (PH 7.4) containing 5 μg of the above peptide, 0.2 molar phosphate buffer containing 1 microcury of [ ¹²⁵ ]Na (MEN) was added, and then 3.5 mg/ml of chloramine T was added. Add 20 µl of 0.2 molar phosphate buffer. Leave at room temperature for 20 seconds and terminate the reaction by adding 50 μl of 2.4 mg/ml sodium metadisulfate in 0.2 M phosphate buffer. The reaction mixture is applied to a column (1.0 x 30 cm) of SP-Sephadex C-25 (eluent 50 mmol phosphate buffer). The fourth fraction is the fraction of the above peptide labeled with ¹²⁵ . Measurement of Γ titer Measure the titer of the antibody obtained above as follows. That is, the antibody was dissolved in physiological saline at 10, 10 ² ,
10 ³ , 10 ⁴ , 10 ⁵ ... dilute (initial) twice,
To 100 μl of each of these, add 0.1 ml of ^125- labeled peptide (the labeled peptide obtained above diluted to about 10,000 cpm) and 0.1 molar phosphate buffer (PH = 7.4) [0.1% BSA, 0.15 molar]. Add 0.2 ml of sodium chloride and 0.01% _NaN3 and incubate at 4°C.
Incubate for 24 hours and generate antibodies and ¹²⁵
The conjugate with the labeled antigen was separated from the unreacted (unbound) ¹²⁵ labeled peptide by the dextran-activated charcoal method and the centrifugation method (4°C, 15 minutes, 3000 rpm), the radiation was counted, and the antibody at each dilution concentration was separated. Binding rate (%) with ^125- labeled peptide
Measure. 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. The dilution ratio of the antibody at which the binding rate is 50%, that is, the titer of the antibody, is determined as follows. Antibody titer: Antibody 52,000 Antibody: 100,000 or more
10 ⁶ U/mg protein), peptide chains 1 to 13 of human β-type interferon, and human α-type interferon (Lympho blastoid, manufactured by Hayashibara Institute)
Interferon, Lot. No. 800928). Also standard diluents include 0.1% BSA, 0.15M NaCl and
Use 0.1 molar sodium phosphate buffer (PH7.4) containing 0.01% _NaN3 . Add 0.2 ml of standard diluent and sample to each test tube.
0.1ml, antibody V0.1ml obtained in Example 16 (titer
52,000) and ^125- labeled peptide (diluted to approximately 10,000 cpm of the labeled peptide obtained above) and incubated at 4°C for 48 hours.
Add 0.1 ml of dextran-coated activated charcoal suspension, leave to stand at 4°C for 30 minutes, and then centrifuge at 4°C and 3000 rpm for 30 minutes to remove antibodies and ¹²⁵ The conjugate with the labeled peptide and the unreacted (unbound) I ¹²⁵ labeled peptide are separated, the radiation is counted, and the binding rate (Bo) corresponding to the titer of the antibody used is set as 100%, and each test sample is antibody at a concentration and dilution of
Determine the percentage of the conjugate (B) with the ^125- labeled peptide. The results obtained are shown in FIG. In Figure 1, the vertical axis shows the binding % (B/Bo x 100), and the horizontal axis shows the test samples (human β-type interferon 1st to 13th peptide chains, human β-type interferon, and human α-type interferon). ). In addition, in the figure, curve A indicates the 1st to 13th lines of human β-type interferon.
Curve B shows human β-type interferon, and curve C shows human α-type interferon. From FIG. 1, the antibody shows clearly distinct curves in reactivity to human α-type interferon and reactivity to ^human β-type interferon. It can be seen that this is a highly specific antibody with no cross-over up to ml. Also, in the same way as above, human β
Perform type interferon specificity test. The results obtained are shown in FIG. FIG. 2 is a graph showing the specificity of antibodies.
In Figure 2, the vertical axis shows the binding % (B/Bo x 100), and the horizontal axis shows the test sample (human β-type interferon No. 1 to 13).
Curve 2 shows the concentration of peptide chains 1 to 13 of human β-type interferon, and curve E shows the concentration of human β-type interferon. , curves B and B respectively represent human α-type interferon. Specificity tests will also be performed on antibodies and. That is, 0.2 ml of the above-mentioned standard diluent, 0.1 ml of the above-mentioned test sample, 0.1 ml of the antibody, and 0.1 ml of the ^125- labeled peptide are placed in each test tube and reacted under the same conditions as above. Binding of each antibody when the binding reactivity between the antibody obtained from the peptide chains of human β-type interferon Nos. 1 to 13 or the peptides of human β-type interferon Nos. 1 to 13 is taken as 100%. The percentages are shown in Table 1.

[Table] In addition, when the specific reactivity of the antibodies against human β-type interferon was investigated, each antibody showed high specificity for human β-type interferon. Γ Sensitivity measurement In each test tube, add 0.2 ml of standard diluent, 0.1 ml of human β-type interferon peptide chains 1 to 13,
Add 0.1 ml of the antibody obtained in Example 16 or the antibody obtained in Example 17 and ^{0.1 ml} of the 125-labeled peptide (the labeled peptide obtained above diluted to about 10,000 cpm), and incubate at 4°C for 48 hours. After incubating for an hour, add 0.1 ml of normal sheep serum, then add 0.5 ml of dextran-coated activated charcoal suspension, leave for 30 minutes at 4°C, then 4°C,
Centrifugation was performed for 30 minutes at 3000 rpm to separate the conjugate of the antibody and ^125- labeled peptide and the unreacted (unbound) ^125- labeled peptide, and the radiation was counted and determined to be equivalent to the titer of the antibody used. Assuming that the binding rate (Bo) is 100%, determine the percentage of conjugate B between the antibody and the ^125- labeled peptide at the concentration and dilution rate of each test sample. The results obtained are shown in FIG. The vertical axis in Figure 3 is the bond percentage (B/Bo
×100), and the horizontal axis indicates the concentration of human β-type interferon peptide chains 1 to 13. In the figure, the curved line G is the one using an antibody, and the curve H is the one using the antibody. As is clear from Figure 3, the minimum detection sensitivity is 13 picograms/ml for antibodies and 5 picograms/ml for antibodies.
ml.

[Brief explanation of drawings]

FIGS. 1 and 2 are graphs showing the specificity of the antibodies of the present invention. FIG. 3 is a graph showing the relationship between the concentration of peptide chains of human β-type interferon Nos. 1 to 13 and binding % for the antibody of the present invention.

Claims (1)

[Claims] 1 General formula R-Leu-Gln-Arg-Ser-Ser-OH [In the formula, R is a hydrogen atom, H-Phe- group, H-Leu
-Gly-Phe- group or H-Tyr-Asn-Leu-Leu
-Gly-Phe- indicates group. ] N-terminal peptide of human β-type interferon represented by