JPS582749A - Adsorbent for affinity chromatography - Google Patents

Abstract

PURPOSE:To obtain an adsorbent easily carrying out the selective adsorption of human beta type interferon and desorbs easily under a mild condition, by fixing an antibody obtained by using an antigen composed of peptid expressed by a prescribed general formula on an insoluble carrier. CONSTITUTION:A human beta type interferon antibody composed of a peptid- carrier complex is prepared by reacting to an antibody under the existence of haptencarrier coupling reagent by using peptid expresed by general formula as hapten. Next, this antigen is given to a mammal an an antibody having the singularity of the human beta type interferon which is produced in a living body, is sampled. Further, an insoluble supporting material is treated with cyanobromide and obtained activated material is coupled with the antibody under a mild condition. Then, fixation of the antibody is carried out and an adsorbent is obtained. This adsorbent is preserved in a physiological saline solution.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an adsorbent for affinity chromatography.

In this specification, when amino acids, peptides, protective groups, active groups, etc. are expressed by abbreviations, IUPAC, IU
The regulations in Section B or the symbols commonly used 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.

Ary poison arf-nine Axn: Asparagus Str:t.

Gln-Diltamine Net', Metisaijishi Ltu: 0 IshishiP: Phenylalanyl T1. :Tyrosine Glf :Glycine Z :Carbovecilyl group S□ :Succinic acid group Tas :Toluecysulfonyl group B, :Tertiary butylene dicarbonyl group It is an antiviral glycoprotein produced when infected, and its use to prevent or treat viral diseases is attracting attention. Therefore, human interferon is α-type interferon;
n IIL-her blastaid In
terferon).

β-type I, Turf Ion (Fibrtrblast)
Interferon) and γ-type Ishitafue □ y
Until now, it has been elucidated that it is a mixture of (Immune Interferon).

However, the technology to purify such human interferon into a single type of glycoprotein has not yet been developed.

The object of the present invention is to isolate and purify β-type isterferon from human interferon.

That is, the present invention is based on the general formula % [wherein R1gl hydrogen atom, H-Pkt- group, H-Lzu
-Human β-type interferon antigen consisting of a complex of the N-terminal peptide of β-type interferon and a carrier.
The present invention relates to an adsorbent for affinity chromatotraffy, characterized in that it is made by immobilizing a β-type antibody obtained by administering to a mammal on an insoluble support.

By using the adsorbent for affinitake 0 magnetography of the present invention, it is possible to isolate and purify the β-type interferon from human ishita-fue 0. In other words, it is possible to selectively adsorb only the β-type interferon, and also to prevent the desorption of human β-type interferon from the adsorbent for affinitake O adsorption to which human β-type interferon has been adsorbed. It can be carried out with simple operations under mild conditions, and therefore does not deactivate the human β-type enzyme activity and can produce highly purified human β-type protein.
type interferon can be obtained quantitatively.

The Affinitake 0 adsorbent for magnetography of the present invention is manufactured by the method shown below. That is, the above general formula (1)
By using the peptide represented by as paraten, human β-type interferon antigen consisting of a peptide carrier complex can be produced by reacting with a carrier in the presence of a Yazutesy carrier-binding reagent, and then this β-type Ishitafue 0
It is possible to produce a β-type interferon antibody that has specific reactivity to human β-interferon from human β-interferon antigens, and to use this β-type interferon antibody as an adsorbent for affinity chromatography. The adsorbent of the present invention is produced by containing the above.

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 include, for example, “TA/Pt tl
Ties”, Volume 1 (+966).

5thr”tittr and Luhkt, Ac
academic Press, NztuYard,
U, S, A, or "Peptide Synthesis" is described in Azumaya et al., Maruzen Co., Ltd. (1975), and includes, for example, the azide method, the lylide method, the acid anhydride method, the mixed acid anhydride method,
(DCC method, active ester method) For example, 1. Method using Wood's 9T reagent K, carpodiimidalyl method, fermentation reduction method, D
CC/Additive (e.g. HONB, HOBt, HO
5st) method etc.〇The peptide of general formula (1) can be synthesized by a general polypeptide synthesis method, for example, by dividing it into several fragments using the so-called Stellarwise method, in which amino acids are sequentially condensed one by one to the terminal amino acid. It can be easily manufactured by following a method such as cup linking. More specifically, the above-mentioned peptide is divided into two parts at any position of the bond.+1 A raw material having a reactive carboxyl group corresponding to one of the two fragments and a reactive amino group corresponding to the other fragment. When the raw materials are condensed using a conventional method for peptide synthesis and the resulting condensate has a protecting group, it can be produced by removing the protective 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, at least one of the constituent amino acid residues of the peptide is protected from a protected peptide. In many cases, it can be produced by eliminating all groups.

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.

As the protecting group for the amino group of the raw material, for example, carboben, ni+shi, tire-butyl-dicarbonyl-tart
-Ami41 dicarbonyl 1 isobornyl dicarbonyl, t-methoxybenzyl dicarbonyl-%2-
Examples include 0-besidyloxycarbonyl-1 atamacyl-oxycarbonyl-1-fluoro-7-ethyl-phthalyl-formyl-10-di-O-phenylsulfenyl-1-diphenylphosphinothioyl. Examples of protective groups for carboxyl groups include alkylene esters (eg, ester groups such as methyl, ethyl butyl, tart-butyl, etc.), besidyl ester groups, and benzyl ester groups Sl'. -methox dibenzyl ester group 171-riO rubesidyl ester group -besiz dibenzyl ester group, carbobenly + dibenzyl ester group, tIrt-
Examples thereof include a butyl-o-decacarbonyl-dracito group, a trityl-dracito group, and the like.

Examples of protective groups for the taanidino group of arufine include a nido group, a tosyl group, and a methoxyl group: Jt! Examples thereof include a disulfonyl group, a carbobenyl group, an isobornyl dicarbonyl group, an atamantyl dicarbonyl group, and the like. Further, the quanidino group may be protected in the form of an acid salt (eg, benzenesulfone Itsukushima tornidisulfonic acid, hydrochloric acid, sulfuric acid, etc.).

Methionine may be protected in the form of sulfo+side. Raw materials with activated carbo+syl groups include, for example, the corresponding acid 0ride, acid anhydride or mixed acid anhydride, azide, active ester (methyl alcohol 1 ethyl alcohol, benzyl alcohol 1 pentac 0 0 phenol 1 Enol, y-eFa + Shisakushini!do1N-shido01 dibenztriaryl-%N-
Examples include esters of 0 + 5-norbornene-2,3-dicarboxylic acid, etc. The peptide bond-forming reaction may be carried out in the presence of a condensing agent (eg, tricarboxylic reagents such as dihydrylcarbodiimide, carposiimidalyl, tetraethyl phosphite).

More specifically, the peptide represented by the above general formula (1) is produced according to the method shown below. That is, general formula (1)
Among the peptides, peptides in which R represents a hydrogen atom are produced according to the following (1), and peptides in which R represents H-Pkz-d are produced according to the following (II), and R is H-Lz
u -G / y -P A z- group is produced according to the following (2), a - group is produced according to the following (transition), and R is H-Me l-5ty-Tyr- A
A peptide exhibiting an xn-Liu-Lzu-Gly-Phz- group is produced according to the following (7).

(1) A-Lg u-Gl n -B(2) (4) H-Lzu-Gl n-Ary-5zr-5et -0
N(1) CH-Lzu-
Gin-Ary-5er-5zr-ON(ll)C H-Phz-Ltu-Gln-Ary-5tr-5zr
-ONH-Leu-Gly -Phz -Leu-Gl
n-Ar(1-5tr-5tr-OHA-Tyr-A
'xn-Lzu-Lzu-Gly-Pkt-Ltu-
Gln -C H-Tyr -Asn-Leu -Lay -Gly
-Pkz -Ltu-Gln-Ary-5ty -5p
y -0H(7)H-T y r-As n-Lx u
-Lx u-Gly-PA t-1,. -Gln-A
rg-5et-5et-ORPhz-Ltu-C;In
-Arg-5et-5tr-ORH-Net-5zr-
Tyr-Azn-Lzu-Ltu-Gly-Pkt-L
xu-Gln-Arg-5et-5tr-OR (above (
In 1) to (V), A represents a protecting group for an amino group, B represents a hydroxyl group or an active group for a carboxyl group SC represents a protective group for an alkyl quanidino group, respectively 0) It peptide (
The reaction between 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 the peptide condensation reaction, such as anhydrous or water-containing dimethylformamide. Examples include dimethyl sulfonate, dilysine, dilysine, 1 form, 1 tetramethane, 1 tetrahydrofuran, 1 N-methyl acetate, 1 N-methyl phosphoric acid triamide, and mixed solvents thereof. .peptide(
The proportion 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 equivalent amount to 5 times the amount of the former, preferably an equivalent amount to 1.
It is best to use 5 times the amount. The reaction temperature is appropriately selected from the range known to be usable for peptide bond forming reactions, usually from -40°C to about 60°C, preferably about -20°C.
to about 40°C, as appropriate. The reaction time is generally about several minutes to 30 hours.

Reaction between peptide (5) and amino acid (6), peptide (
8) Reaction between peptide Ql) and bedutide (9), reaction between peptide Ql) and peptide (2), and peptide Q4 and bedutide Qf9
The reaction with peptide (2) and peptide (3) can be carried out in the same manner as the reaction between peptide (2) and peptide (3).゛After the completion of the above condensation reaction, the protecting groups of A and C of the resulting peptide (4) can be removed by a conventional method. Such a method includes, for example, a reduction method using metallic sodium in liquid ascinia.

Metallic sodium causes the reaction solution to turn a permanent blue color.
It is used in such an amount that it remains colored for about 0 seconds to 10 minutes. This reduction is usually carried out at about -40 to -7 et.

Peptide (7), Peptide QO1 Peptide (To) and G Removal of protecting groups A and C from peptide α6 & Same method as removing protecting groups A and C from peptide (4) It is carried out as follows.

Pezuchido (shouma analogy Gf) used in (1) above
It is manufactured by the method shown below.

H-5zr-OR” (To) A-5ay-B A-5ay-5tr
-OR"Qη a-h-5
tr-5tr -OR'' (E) A-Ary-5Ir-5lr -OR 翰A-Arq-5zr-5zr-OH ・翰(3) [In the above, Ri represents a lower alkyl group.

JXB and C are the same as above. ] Reaction 1 between amino acid α and amino acid (1) can be carried out in the same manner as the reaction between peptide (2) and peptide (3).

The protecting group A of the peptide chain can be removed using a conventional method. Such a method includes, for example, a reductive method (for example, radium,
Hydrogenation using a catalyst such as radium black, reduction with metal sodium in liquid ammonia) S acidolysis (e.g. trifluoroacetic acid, hydrogen fluoride, methanesulfosic acid,
Acidolysis with strong alcohol such as hydrobromic acid), etc. For example, in the case of hydrogenation using a catalyst, hydrogen pressure 1
It can be carried out at atmospheric pressure and 0 to 40°C. The amount of catalyst used is usually about 100111 to 1f.1 Generally 1 to 4
The reaction is completed in about 8 hours. In addition, in the case of acidolysis, it is usually about 0 to 30°C, preferably 0 to 20°C without a solvent.
The reaction is carried out at °C and generally completes in about 15 minutes to 1 hour. The amount of acid used is usually 5% based on the raw material compound.
It is best to use about 10 times the amount. Furthermore, the conditions already described above can be used for the reduction with metallic sodium in liquid asimonia.

The reaction between Pezunato and the amino acid (c) is the reaction of the above peptide (
The reaction can be carried out in the same manner as the reaction between 2) and peptide (3).

For example, the reaction of hydrolyzing peptide ■ to obtain peptide (sol) is carried out in the presence of a mineral acid such as hydrochloric acid or sulfuric acid, an alkali such as sodium hydroxide or potassium hydroxide, water, methanol 1 ethanol, or a solvent such as diosan. It is done inside. 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 peptides 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.

The peptide (5) used in (II) above is produced by removing the protecting group Δ from the peptide (4). Similarly, the peptide (8) used in the above (2) is produced by removing the protecting group A from the peptide (7), and the peptide a1 used in the above (transformation) is protected from the peptide α1. Peptide a4, which is produced by removing group A and used in (9) above, is produced by removing protecting group A from a peptide. Examples of methods for removing the protecting group A from peptide (4), peptide (7), peptide α and peptide (2) include hydrogenation using a catalyst such as palladium 1 palladium black, acidolysis, and the like. As conditions for these methods, any of the conditions already mentioned above can be adopted.

The peptide (b) used in the above (conversion) is produced, for example, by the method shown below.
n-Lx u-OR engineering ■
(e)---m-tH-A x n -Lz u
-07? Kokuso↓ A-Tyr-Asn-Lxu-OH (2) [In the above, R", B and C are the same as above.]?
・The reaction between amino acid and amino acid (amino acid) and the reaction between peptide erection and amino acid (2) are both caused by the above-mentioned peptide (2).
It can be carried out in the same manner as the reaction between and peptide (3). To remove the protecting group A from the peptide (E), any of the methods for removing the protecting group A from the peptide Q (I) described above can be applied.In addition, the reaction for hydrolyzing the peptide chain to obtain the peptide Z can be carried out using the above-mentioned peptide Q (I). can be carried out under the same conditions as the reaction to obtain peptides by hydrolyzing .

Preferred methods for obtaining peptides from peptides include, for example, the mixed acid anhydride method and the azide method.

The mixed acid anhydride method is carried out using an alkoxycarboxylic acid in the presence of a basic compound in a suitable solvent. The alkoxylic acid used is, for example, 00 methyl formate 1 methyl heptaformate 00 ethyl formate 1 oV
, ethyl formate, oo isobutyl formate, and the like.

Examples of basic compounds include triethylamine, trimethylaminopyridine, 1. ; Methylanilisi 1N-
Methylmorpholysyl, 1,5-diazapico (413
t o ) none: I-5 (DBN), 1,5-diazajisifu o (514-O) undetcy 5 (DBU),
1.4-Diazazischiff O(2,2,2)octasi(DA
Examples include organic bases such as charcoal-potassium, sodium carbonate, potassium bicarbonate, and sodium monocarbohydrate. The solvent used can be any of the solvents commonly used in the mixed acid anhydride method, and specifically, 1 methyl chloride, 1 0 form, 1 sidium, 1 sulfuric acid hydrocarbon such as ethane, 1 benzene, toluene, Aromatic hydrocarbons (diethyl ether, etc.) 1) Ethers (metobushi), etc. (1) Methyl acetate
Examples include esters such as ethyl acetate, 1N, N-dimethylformamide, dimethylsulfonate, and non-polymerizable polar solvents such as methylphosphoric acid triazide. The reaction is carried out at -20 DEG to 100 DEG C., preferably -20 DEG to 50 DEG 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 carboxylic group activated with an alcohol such as methyl alcohol, ethyl alcohol, benzyl alcohol, etc., is dissolved in a suitable solvent. All you have to do is react. Examples of the solvent that can be used include dioxane-1-dimethylformamide, dimethylsulfo+cyto, a mixed solvent thereof, and the like. The amount of cydrazine hydrate to be used is usually 5 to 20 times, preferably 5 to 10 times, the amount of activated h-rubodecyl group. The reaction is usually carried out at a temperature of ζ50°C or less, preferably from -20 to \50''C.

In this way, a compound in which the carboxyl group of the terminal amino acid is substituted with didrazine (a didrazine derivative can be produced. It is produced by reacting the dorazine derivative obtained above with a nitrous acid compound in a solvent. Hydrochloric acid is usually used as the acid. Examples of the solvent include dioxane, dimethylformamide, dimethylsulfo+cyto, or a mixture thereof. Solvents, etc. Examples of nitrite compounds include sodium nitrite,
Isoamyl nitrite, nitrite chloride, etc. can be mentioned, and such nitrite compounds are usually used in a molar amount equivalent to 2 times, preferably 1 to 1.5 times the amount of the dorazine derivative.
It is best to use the same amount.

The reaction is usually carried out at -20 to 0°C, preferably -20 to -1
The reaction is carried out at 0°C and generally completes in about 5 to 10 minutes.

The peptide of general formula (1) produced as described above can be separated from the reaction mixture by means such as extraction, distribution, etc.
It is isolated and purified by h-lambda 0 madography and the like.

The labeled peptide, which is the raw material for producing the labeled antigen used in the RIA method, is produced by introducing radioactive iodine (1) into the peptide produced above.The introduction of the radioactive iodine is generally carried out by Conventional iodination methods, such as oxidative iodination using phosphorus (F, M, Hunte
r and F, C0Grztnasoad; Na
turI, 194. P495 (1962), Bio
ckzm, 10g9. See P 114 (1963)]
This is done by For example, the above iodination method is carried out in a suitable solvent such as 0.2M phosphate buffer (PH-7,4) in the presence of phosphoric acid at room temperature for about 10 to 30 seconds. It will be done. For example, when introducing one radioactive iodine into tyrosine, the ratio of the peptide, radioactive iodine, and phosphor T to be used is, for example, when one radioactive iodine is introduced into tyrosine, it is The radioactive iodine is about 13 F, and the radioactive iodine is about 10 to 10.
It is best to use about 0 nanotons of radioactive iodine, and when introducing two radioactive iodines into tyrosine, it is recommended to use about 2 milliliters of radioactive iodine per nanogram of tyrosine molecules contained in the peptide. It is preferable to use about 10 to 100 nano-T. The radioactive iodine-labeled peptide thus produced is isolated and purified by conventional separation means such as extraction, partitioning, column chromatography, dialysis, and the like.

The peptides thus obtained can be stored by lyophilization, if necessary.

In the method of the present invention, the above general formula (1
) is used.

As the carrier bound to octatene, a wide range of high-molecular natural or synthetic proteins commonly used in the preparation of antigens can be used, such as horse serum aldimine, bovine serum albumin, rabbit f serum alpine, and human serum alpine. Animal serum albumins such as serum albumin, horse serum albumin, bovine serum albumin, rabbit serum albumin,
Animal serum such as human serum 0 purine, sheep serum 1) ozurin, etc. , Shitsujichi 0ri 0 pudding and other animals to 1 horse r Niji 0jishi, cow to 7 ji 0zu rin 1 rabbit f to V: ri O pudding, human to 7ri 0 pudding ) Proteins extracted from roundworms (Ascaris extract, patent application 1972-
92781), polylysine 1 polyglutamic acid 1
Examples include a copolymer containing Rigi-Sicurtaethyl-9, a copolymer containing Rigi-Si or ornithine, and the like.
The B. Ascaris extract will be described in detail below.

Ascaris extract is an extract of Ascaris spp.
It is extracted from the pulverized product of um) according to the usual protein extraction method. Examples of the extraction solvent include water, physiological saline, and
Various known protein extraction solvents can be used, such as % methanol or ethanol aqueous solution, buffer solutions around neutrality, etc., and it is particularly preferable to use physiological saline.

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 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. In this way, a desired Ascaris extract is produced. This protein can be used in the present invention after being further purified by conventional protein purification methods such as dialysis, gel filtration, adsorption, and chromatography, if necessary.

Further, the Ascaris extract used in the present invention is, for example, J.
Immuno, III, 260-268 (1973),
J. Immun.

122.302-308 (1979), J. Immun.
,, 98°893~900 (+967) and Am, J
,Pkyxial,.

199.575-578 (, +960) or further purified versions thereof.As the carrier-binding reagent, those commonly used in the preparation of antigens are widely used. Specifically, aliphatic dialdehydes such as thaliosulfate, malondialdehyde, coulteraldehyde, succicialdehyde, adipaldehyde, and thiol groups can be used to crosslink amino groups. For example, N, "-'-fe:17:J,7?L
'IE) % 7vI"-"-': A cimaleimide compound of phenylecidimaleimide, which cross-links the p-3sono thiol group, for example, metamaleimide besiliyl N-human 0 bovine cissuccinimide ester 54
-(Maletritomethyl)-Schiff0hesan-1-carbo+sil-y-sido0 Maleimidocarboxyl-N-eFo+cissuccinimide ester compound 1 Trino group and carboxysil group Reagents used in the usual peptide bond forming reaction to form an amide bond between
N-ethyl-N'-dimethylaenocarbodiimide, l
-ethyl-3-diiso-pyraenocarbodiide,
l-schiff0 to bovine syl-3-(2-v: luforinyl-4-
Examples include dehydration condensation agents such as carbosiimides such as ethyl)carbosiimide, but further examples include dehydration condensation agents such as carbosiimides such as -mono-dialinium phenylacetic acid and ordinary betutide bond-forming reaction reagents, such as the above-mentioned dehydration condensation agents. It is also possible to use a combination with a condensing agent.

The antigen of the present invention is produced by combining the above-mentioned compound and carrier in a buffer solution of Steps 8 to 9 at 0 to 40°C, preferably at 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 aqueous solution - 0.2M Sodium chloride - 0.2M
Potassium chloride buffer, 0.2M carbonate @J')L-0, 2M*')m-0,
2M potassium chloride buffer, 0.05M sodium tetraboron-0.2M boron 1? -0
, 05M sodium dihydrogen buffer, 0.1M potassium dihydrogen phosphorus-0.05M sodium borate buffer In the above, the proportions of the hapten, the 8-carrier binding reagent and the carrier can be determined as appropriate; On the other hand, it is preferable that the carrier is 2 to 6 times the weight, preferably 3 to 5 times the weight, and the carrier binding reagent is 5 to 10 times the weight. Through the above reaction, an antigen of human β-type Ishita protein comprising 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 according to 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 thus obtained is usually a protein 1t.
The peptide has an average of 5 to 20 v-nyl bonds to the protein, and all of them continue to be reproducible and enable the production of highly specific antibodies against cytoβ-type interferon. A binding ratio of peptide to peptide of 1:8 to 15 is preferable because it allows production of antibodies with higher specificity and 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 vivo are collected.

There are no particular restrictions on the mammal used for antibody production, but it is usually desirable to use rabbits or rats. For antibody production, dilute a predetermined amount of the antigen obtained above with physiological saline to an appropriate concentration, and add auxiliary solutions (
Complete Frzund'zAdjuvant
) to prepare a suspension and administer it to a mammal. For example, rabbits may be immunized by subcutaneously injecting the above suspension (antigen amount: 0.5 to 511 qI/dose), followed by administration every two weeks for 2 to 10 months, preferably 4 to 6 months. 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 always has excellent specificity against the existing β-type isiter enzyme, and has high titer and high It has the advantage of being able to obtain sensitive antibodies with good reproducibility.

When immobilizing the human β-type isiterfue obtained above on an insoluble support, any of the conventional methods for immobilizing biological substances can be used. It is preferred to use This diazibromide-activated polysaccharide is a method in which an insoluble support is treated with diazibromide, and the resulting activated product is combined with a biological material under mild conditions to immobilize the biological material. . When treating an insoluble support with diazibromide, for example, using a basic compound such as sodium hydroxide or sodium hydrogen carbonate, the insoluble support is kept at l'H11 to 12 and heated at room temperature in a solvent such as water or acetonitrile for about 1 It is sufficient to process for about 12 minutes. The amount of diazibromide to be used relative to the insoluble support is usually approximately equal.

Insoluble supports have low nonspecific adsorption to biological substances in general, high porosity, functional groups that can immobilize biological substances under mild conditions, and chemically and physically Any conventionally known insoluble support can be used as long as it is sufficiently stable, such as athinoethylcellulose,
Carboxyl dimethyl cell 0-su, 7-dimethyl cell 0-
cell 0-su yarn support such as s, door-anilino cel 0-su,
Examples include cross-linked fiber-based supports such as Sephadex and CM-Sephadex (manufactured by Pharmacia), and hemlock fiber supports such as T Edge 0-Screen B1 Sepha 0-F 48.

When the thus obtained diazidibromide-activated support is coupled with a human β-type interfacial antibody, for example, the diazibromide-activated support is added at a concentration of 30 to 80% of the human β-interfacial antibody. Using twice the weight, use a suitable solvent, such as 0.1't sodium hydrogen reconstituted water 0.
5 Contains 1-nyl sodium chloride, 13H8.4> In an aqueous solution, usually about 0 to 40°C, preferably about 1 to 8°C
The reaction may be carried out for 0 to 20 hours. In this way, the adsorbent for affinity chromatography of the present invention is manufactured. The adsorbent may be stored in a suitable solvent, such as physiological saline.

In addition, the human β-type isiterfeon antibody obtained above is
As mentioned above, it has particularly excellent specificity for β-type interferon in livestock, and enables the quantitative determination of β-interface in livestock using the EIA method, which is desired in this field, with high precision. It is useful. Further, the antibody can be used in enzyme immunoassay (EIA) method, fluorescence immunoassay (FIG) 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.

, ``-'' Below, reference examples and examples are given 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 using a thin layer of silica gel using the following mixed solvent.

Rf...1-butanol-acetic acid-water (4:1:5
)■ Rf...1-butanol-hyridine-g[-water(
15:10:3:12) <Peptide synthesis> Reference example! Zukyou Z-5tr-5tr -OMz f) Manufacturing Z-! yr-
NHNH25,Q 6f with 50ml dimethylformamide and 6.66+wl 6N-HCI/,
; Dissolve in O+san, cool to -15°C, add isoamylsuitolite 2.68s+j, and stir for 5 minutes.

Then 5.60 g/triethylamine is added to neutralize. This solution was mixed with H-5zr-OMz-HCI3,11
Add to a 30-proof dimethylformamide solution containing f and 2.80 g+/ of triethylamine, and stir at 4°C for 20 hours. Dimethylformamide was distilled off, the resulting residue was extracted with ethyl acetate, washed with water, and dried with sodium sulfate. -5zr-5zr-
Obtain Oug 3.75 f.

Rf value = 0.6 Now, Rf value: 0.7? Elemental analysis value (as CL5H2°N207) CHN Calculated value (%”) 52,94 5.92 8.2
3 Actual measurement value (phantom 52.67 5,89 8.35 Reference example 2 Production of Z-Arq (Tag)-5zr-5tr-OMz Z-5tr-5tr -0ut 2.5 f for 50 m
H-5z r-5z r was dissolved in 1 part of methanol and 7.34 g of IN-MCI and subjected to catalytic reduction at 20°C and 1 atm of hydrogen pressure in the presence of 500 m of palladium black.
ORf value to obtain -0Mg? 0.08 Z', -Arg(Tos) -OH3,399 to 4
0m1 of tetrahydrofuran and 0.75m+/N-
In addition to methyl hexafluoride, after cooling to -15°C, 0.97-
Add isoptiluc 00 formate and stir vigorously for 30 seconds.

The above Tefl Rahle H-5py-5ir-Ohh, HC
IS=; Add 2ml of methylformamide and 3ml of triethylamine LO, stir for 1 minute, and then add 2ml of methylformamide and 3ml of triethylamine.
Stir for 5 minutes at 'C, 2 minutes at 40°C, and 15 minutes at room temperature. After distilling off tetrahydrofuran and dimethylformamide, the extract was extracted with ethyl acetate, washed three times with IN-I silicic acid, then five times with water, and dried over sodium sulfate. The solvent was distilled off and reprecipitation was performed with methanol-ethyl acetate and ether to give Z-Ary(Tax)-5et-5zr-O
Obtain ug 3,659.

Rf value: 0.5 I, Rf" value: 0.73 Elemental analysis value ('251H36N6'□. As S) CH'
# Calculated value (%) 51.68 5.88 J2.9J
Actual value (%) 51.62 5.82 12.73 Reference example 3 Z-Arg(Tax)-5tr-5ty-OHC7
) Manufacturing Z-Arg (Tax) -5er-5tr-Ou
g 3,5 f m) 50 ml and water IC) a
Add lN-NaOH8,06+w/, stir at room temperature for 45 minutes, then lN-MCI7
．． Add 24 liters of water for neutralization, and after distilling off methanol, extract with n-butanol. After washing with water, n-butanol and water were distilled off, ether was added to the residue to crystallize it, reprecipitation was performed from methanol-ethyl acetate, and Z-Ar g (TOx
)-5t r-5t r -OR2,6Qf is obtained.

Rf value: 0.2B, Rf It value: 0.57 Elemental analysis value (C2 wa H36N60 engineering. As %H20) CHN
Calculated value (%) 50,22 5.77 13.02★
Measured values (phantom 50.2 current 5.62 13.20 Reference example 4 Production of H-Ary(Tax)-5zr-5er-OH P
H-Arg(T
17s)-8Ir-5ty-OR is obtained.

Rf value: 0.034 Reference example 5 2-Arg-Ltu-OEI (7) Production Z-Axn
-ON 5.32ftttet5sitO") run 60m
+Z% dimethylformua! Dissolved in 10 liters of cod and 2.0 + 11 It of N-methyl cellulose,
Formate 2.64111 S//-Lx u-OE
/ -EC13,91F and triethylamine 2.80
The reaction is carried out according to the procedure of Reference Example 2 using a dimethylformamide solution of 4C)tl containing g/. After distilling off the solvent, 1 molar aqueous citric acid solution was added, and the precipitate was collected in a furnace, washed with water,
Dry under reduced pressure and recrystallize from ethyl acetate to obtain Z-Azn-
Law-OEt 6. Get l l.

Rf value: 0.71, R1 value: o, s O elemental analysis value (
(as C2♂29N306) CHN Calculated value (%) 58.95 7.17 10.31 Actual value (%) 58,99 7.23 10.26 Reference example 6 Production of Z-Tyr-Asn-Lzu-OEt Z- Azn
-Ltu-OE13. Q l f mul)-7115
The mixture was dissolved in 7.4 ml of Ogo and I N -MCI, and subjected to catalytic reduction at room temperature and normal pressure in the presence of Palladium Black 5004 to obtain H-Ax n-Lgu-OEI -HCl.

Rf value: 0.26 Above “C’ Obtain h 6 H-Ax n-Lt b-O
El-ECJ ft =, ,! Chilformamide 5o
Dissolve in Mt and triethylamine 1.03*/, then add 2-Tyr-ONNS 3.35 f,
Leave at room temperature for 20 hours. Precipitate with dimethylpolyte and Z-T
yr-Azn-Ltu-OEt 3.58 f'lt
Profit; S.

R/value: 0.73, R/value: 0.82 Elemental analysis value (as C29H351N401!l) HN Calculated value (%> 61.04 6.71 9.82 Actual value (%) 60,84 6,70 9.39 Reference Example 7 Z-Tyr-Azn-Ltu-NHNH2 production Z-T
yr-Axn-Ltu-OEt 2,73 fj9)
- Wash with L alcohol and Z-Tyr-Ax n-Lt
u-NHNH23,10f is obtained.

Bt I value: 0.56, Rf]1 value: 0.75 Elemental analysis value - (as C27H36N607) CHN Calculated value (%) 58,26 6.52 15.10 Actual value (%) 57,91 6.56 15.12 Example 1 (a) Boc-Ltu-Gin-Ary (Tax)
-5ty-5ir -Production of -011
．． Dissolved in 24 ml of isobutylchloride formate 0.
The procedure is carried out in accordance with the method of Reference Example 2 using No. 31 I. After washing with tetrachloric acid, butanol was distilled off and reprecipitated with methanol-ethyl acetate to give Bat-Ltu-Gin-Arg (
Tax) -5ir-5zr -OH1,54j'.

Rf value: 0.18, Rf value: 0.57 Elemental analysis value (C35H57N90., S-H2O tri-
c) CHN Calculated value (%) 48.77 6.90 14.62 Actual value (%) 48.76 6.61 14.78 (b
) H-Ltu-Gin-Ary-5zr-5tr-
OH production Bo c-Lz u-Gl n-Arq (
To z )-5t r-5t r-0H1,50f
Tori'7 JL of 10 * / 000 acetic acid was allowed to stand for 15 minutes at room temperature to remove Bat, and then crystallized by adding dry ether to H-Lzu-Gin-Ary (Tax) LSzr.
-5tr-OR is obtained. Rf value: 0.04 H-Ltu-Gl n'-Arg (Tax) obtained above, -5
Dissolve zr-5zr-ON in 25 ml of liquid ammonia and add metal storium barrel. When the reaction solution turns blue, 1 g of dry am7ium chloride is added, and then the ammonia is distilled off. The residue was dissolved in 50% acetic acid, Sephadex G-10 (2,2X851, eluent 50% acetic acid) and then LH-20 (2,2X80α, eluent 0.
001 N-HCl) and purified to obtain H-Lzu-Gl n-Arf-5ir-5zr-O.
Get H.

Rf value: 0.0+, Rf value: 0.34 (cv)
: -42,5°CCO,250,0,001N-MC
I) Elemental analysis value (C23H4, N909.4 N20
．． , CH, COOH) CHN Calculated value (%) Now 1.60 7.68 17.46 Actual value (%) 41.20 7.93 17.91 Example 2 (a) Z-Pkt-Ltu-Gln- Arg(Ta
x) -5et-5tr -ON Production Example 1) "t' Obtained H-Lzu-Gl n-Ar
y(Ttyz)-5ty-5tr-ON with 30% of dimethylformamide and 0.24% of triethylamine.
g/, then Z-Pkv-0NH5Q,
Add 77 f and leave it at room temperature for 20 hours. After distilling off dimethylformamide*, extract the residue with butanol and dilute with 2%
Wash with acetic acid. The butanol layer was concentrated, ether was added to crystallize, the obtained crystals were separated, and the crystals were washed with hot ethanol to give Z-Pke-Lzu-Gin-Arg.
(Toe)-5'Jr-5zr-ON 1.40 g
get.

Rf value: 0.22, Rf value: 0.59 Elemental analysis value (C47H64N engineering. O engineering 4S-N20) CHN Calculated value (%) 54,12 6.28 13.43 Actual value (%) 54,42 6.38 13.84 (b
) H-Pkz-Ltu-Ginl-Ary-5tr
-Production of 5zr-ORZ-Phz-Leu-Gln-Arg(Tax)-5z
Z-removal of r-5zr-ON20IIf was carried out in the same manner as in Example 1(b) using a piece of sodium chloride in a liquid acidic solution.
De-Tera, tfay"7x G-19, (2,2
X85cIII, eluent 10% acetic acid), and further LH-2
0 (2,2X 80nls' eluate 0.00
Purified by gel filtration with 1 #-HCO to obtain II-Pk.
t-Lest-Gin-Ary-5et, -5zr -
OHL' Get 2 capes.

Rf value: 0.01, Rf” value: 0.35 (a)
Knee 37.36C00,249,0,001N-H(
4) Elemental analysis value (C32H52N engineering. O engineering. 7 N20.
As CH, C00N) HN Calculated value (%) 44.24 7.64 15.17 Actual value (%) 44.27 ? , 10 15.60 Example 3 (a) Z-Lzu-Gly-Pkz-Lzu-Gi
Production of n-Ary('ryz)-5zr-5zr-OH Z-Lz u -G l -y -NHNH2Q,
39 f with 5 ml of dimethylformamide and 6 N
-HC1/'': 0-15 g/s of triethyl nitride dissolved in 0.58 d of triethyl nitride.
49wt1 and H-Pke-Ltu-Gln-Ary
(Tax)-5tr-8tr -OHl, Q
3 was added in an equivalent amount and the reaction was carried out at 4°C for 20 hours. After extraction with butanol and washing with 2% acetic acid, butanol and acetic acid are distilled off, and ether is added to crystallize.

Separate the crystals, wash them with hot methanol, and add Z-L.
zu-Gly-Phz-Lzu-Gl n-Arg(T
ax )-5z, r-5zr -OHO, 65f are obtained.

R/value i0.19, Rf value: 0.60 Elemental analysis value (C55H781'1.2', S, N20
) CHN Calculated value (%) 54.44 6.64 13.85 Actual value (%) 54.74 6.6j13.98 (b)
H-Ltu-Gly-Pkz-Lzu-Gin-A
Production of ry-5ir-5zr-OH Z-Ltu-Gly -Ph t-Ltu-Gl
n-Ar q (Toz)-5t r-5tr
-OH'l Q 1151 was de-Zed and de-Tosized in the same manner as in Example 1(b), and tfadex (1; -1
0′″=(2,2X85cIII, eluent 10% acetic acid)
, further LH-20C2, 2 x 80CIIl, eluent 0
．． Purified by gel filtration with 0OIA'-HCl)
Ltu-Gly-Pht-Ltu-Gln-Ary-5
OR to obtain zr-5zr-OHl lq Rf value: 0.01, Rf value: 0.40 [mountain] 20 knee 27.6°CCO, 228, 0,001N-MCI)
Elemental analysis value (C4゜H66N engineering, O engineering, ・6 H20,
CHN Calculated value (%) 46.92 7.68 15.63 Actual value (%) 46.89 7.25 15.51 Example 4 (a) //-Tyr-Azn-Ltu-Ltu-G
ly-Pkt-Lzaz-Gl n-Ary(Tax
)-5tr-5et-OR production Z-Lzu-Gl
y-Phz-Leu-Gln-Arg(Tax)
-5zr-5zr -OH600q)9)-JLI5
H-Lzu-Gly-Pkz-Ltu-Gln-A was dissolved in 10 ml of 0ml/10% acetic acid and subjected to catalytic reduction at room temperature and normal pressure in the presence of 50011g of palladium.
ry(Tax)-5zr-5Ir-OH is obtained.
Rf value: O, Ig Z-Tyr-Axn-Lz (, u-NHNH242Qq
l to dimethylformamide 5IId and 6N ++ H
CI/Geo+Dimension 7" Dissolved in 0.37m, Isooatrinitrite 0,, IOg/and) !J Engineering f
Jl/7 E:/0. ．． Use 31 ml,
An azide compound was produced according to the method of Reference Example 1, and this solution was converted into H-Ltu-Gly-Pkz-Lzu-Gl n-
Ary(Tas)-5zr-5tr-OH+Samechi11. It is added to a mixed solution of phosphoric acid triazide IOg/ and dimethylformamide IoII/ and stirred 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.
ry(Tos)-5t, r-5zr-OH ometa)
- JL, 30, / and 10% acetic acid 10 + /, after catalytic reduction in the presence of palladium at room temperature and normal pressure, Fadex G-25 (3XI203, eluent 50%
H-Tyr-Asn
-Lzu-Lzu-Gly -Fire-Lzu-G
ln-Arg(Tos)-5ir-5et-OH6
Obtain 50 capes.

Rf value: 0.05, Rf value: 0.53 Elemental analysis value (as C66H98N♂60 engineering, S・2H20) CHN Calculated value (%) 53,28 6.91 15.06 Actual value (%") 53, 10 6.43 14.84 (
b) H-Tyr-Asn-Lzu-Gly-Phi
-Ltu-Gin-Arq, -5tr-5zr-OH (
D Production H-Tyr-As n-Lzu- obtained in above (a)
Lzu-Gl y-Pht-Ltu-Gl n-Arq
(Tos)-5t r-5zr -OR
Tos was removed in the same manner as <b>, and tFadex G-10 (2,2X85cIII, eluent 10% acetic acid)
, Sarani LH-20 (2,2X80CM%0.001
Purified by gel filtration with N-MCI > H-Tyr-
Asn-Lzu-C1y-Phi-Lz, u-Gln-
Ary-5tr-5tr-OH9q is obtained.

RyI value: 0.0+S R1” value: 0.42 [
Out) Knee 24.3°CCO,078,O,0OI
N-11CI) Elemental analysis value (C59H92N□.O engineering,, 15 R20, CH3CO0H) CHN Calculated value (%) 45.01? ,80 13.77
Actual value (%) 45.21 7.31 14.22 Example 5 (a) Z-Mzl-5tr-Tyr-Asn-Lt
u-Ltu-Gly-Phg-Ltu-Gl n-A
Preparation of rq(Tax)-5tr-5zr-OH Z-M, 1-5t'r-NHNHl 59 q/ was dissolved in dimethyJL, formamide 5 ml and 6N, HC4/dio+san 0.21 M/ An azide compound was produced according to the method of Reference Example 1 using 0.055 ml of trinitrite and 0.17 g of triethylamine, and then this solution was converted into H-Tyr-As n-Lx u-L.
x u-Gly-Pkt-Lx u-Gln-Ar
g(Tax)-5ty-5zr-OH3Q Q Ilg
, 5 ml of dimethylformamide, and 5 liters of dimethylphosphoric acid triamide, and heated at 4°C for 2 hours.
Stir for 4 hours. Furthermore, Z-Net-5et-NHNH
Add 2.31811v and stir at 4°C for 72 hours.

Purification was carried out in the same manner as in Example 4(a), and Z-Hz
t -5t r-Tyr-Azn-Lzu-Ltu-G
ly-Pkz-Lzu-Gln-Ary(Tos
)-5tr-5zr-ON is obtained.

(h) H-Met-5zr-Tyr-Asn-Lz
u-Lzu-Gly -Pkz-Lx u-Gl n-
Arg-5t r-5t r -ON (7) Production Z-Hz 1-5t r-Tyr- obtained in above (a)
Ax n-Lx u=Lz u-Gly-Pit-L
x u-Gl n-Arg, (T x )-5tr
-5zr-OR was de-Zed and de-taxed in the same manner as in Example 1 (b), and tfadex G-25
C3X12 (111, eluent 50% acetic acid), plus LH
・-20 (2X85c!II, eluent 0.001N-H
H,-Me t-5tr-Ty
r-Axn-Lzu-Lzu-Gly-Pht, -L
zu-Gln-Ary-5Ir-5tr-OHI I
Get 9q.

Rf value: 0.01, Rf value: 0.45 (d,)20
: -15,7°(CQ, 25 now, 0.001N-
MCI) Elemental analysis value (C67H engineering. 6N engineering 8'2°S,
5 R20, CH3CO0H) CHN Calculated value (%) 49.75 7.26 15.13 Actual value (%) 49.80 6.92 14.91 Amino acid analysis value All: 0.95, Str: 3.15, Gl n
: 1.03, Met: 0.92, Gly: 1.05
, Lzu: 3.08, TFr: 1.00, Phi
:Q, 98, Art: 0.96 Preparation of Antigen> Example 6 The peptide (hereinafter abbreviated as "Pettide A") obtained from the above Example I Cb) loIIF and bovine serum Alzumishi (hereinafter " (abbreviated as “BSA”) 50gIg
Ni buffer (0, Iv nil, /'H7,0) 2ml
Laugh. 0.1 t of tartar aldide solution (0.11 g) was added to this solution, and the mixture was stirred at room temperature for 5 hours. The reaction mixture is then dialyzed for 48 hours at 4° C. against water 14. Change the water 5 times during dialysis. Thereafter, the solution containing the protein complex was lyophilized to obtain β-type interferon antigen (hereinafter abbreviated as "antigen I") 45III.
Example 7: The peptide obtained in Example 2(b) above (hereinafter abbreviated as "Peptide B") and B5A25q/ were added to ammonium acetate buffer (0.1 t, #7.0). Add 0.11 g of 0.1 molar tartaraldehyde solution to this solution, stir at room temperature for 5 hours, and freeze-dry the solution containing the protein complex.
:J antigen (hereinafter abbreviated as "antigen") 23W is obtained.

Example 8 Peptide (hereinafter abbreviated as "peptide C") 4q and B5A15q obtained in Example 3(b) above were added to acetate buffer solution (0.1 t//7.0) 2 Dissolve the leakage. Add 0.11 gt of 0.17 l tartaraldehyde solution to this solution and stir at room temperature for 5 hours. The reaction mixture is then dialyzed for 48 hours at 4° C. against 2 portions of water. Water was replaced 5 times during dialysis. Thereafter, the solution containing the peptide-protein complex is freeze-dried to obtain human β-type interferon antigen (hereinafter abbreviated as "antigen tablet") 17 cape.

Example 9 The peptide obtained in Example 4(b) above (hereinafter abbreviated as "Peptide D") and B5A12q/ were added to acetanium acetate buffer (0,1'El, pHT, 0). 211j
dissolve in Add 0.1 mol of tartar to this solution.

Add 0.11 - of aldehyde solution and stir at room temperature for 5 hours. The reaction mixture is then dialyzed for 48 hours at 4 DEG C. against 1 g of water. Change the water 5 times during dialysis. Thereafter, the solution containing the peptide-protein complex was freeze-dried to obtain 14 d of human β-type isitaphen antigen (hereinafter abbreviated as "antigen 2").

Example 10 811jg of the peptide obtained in Example 5(b) above (hereinafter abbreviated as "petutide E") and 12q/B5A were added to an ammonium acetate buffer (0.1 L Tamedo H7,O)2
Dissolve in ml. To this solution was added 0.11 g of 0.17 rutal aldehyde solution, and the mixture was stirred at room temperature for 5 hours. The reaction mixture is then dialyzed for 48 hours at 11°C in water. Change the water 5 times during dialysis. Thereafter, the solution containing the beta-protein complex is freeze-dried to obtain human β-type interferon antigen (hereinafter abbreviated as "antigen V") 31 cape.

Example 11 Peptide E8■ and B5A25q) are dissolved in 4s+/2 of water. Add silica diimide to this solution + silcaredimide (
DCC) and stirred at room temperature for 5 hours.

The reaction mixture is then dialyzed against 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 human β-type interferon antigen (hereinafter abbreviated as "antigen 2") 29 cape.

Production of antibodies> Example 12 250 μf of antigen I obtained in Example 6 was added to 1.5 m+1
After dissolving in physiological saline, add 1.5 m of Furuiside auxiliary solution.
The suspension prepared by adding 1.1 liters of
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 β-type isitafe antibody of the present invention (hereinafter abbreviated as "antibody!").

Example 13 Antigen II 25 μf obtained in Example 7 was heated for 1.5 s/
After dissolving in saline solution, add 1.5 s of fluoroside auxiliary solution.
The suspension prepared by adding / to 4 rabbits (2, 5 to 3,
(0 kg) subcutaneously, and the same amount is administered 6 times every 2 weeks. Thereafter, the same amount as the first dose was administered three times every month. Seven days after the final administration, blood was collected from the test animals and centrifuged to collect antiserum.
Type Ishitafue Oshi antibody (hereinafter abbreviated as “antibody ■”)
get.

Example 14 Antigen III obtained in Example 8 25 μf for 1.5 s
/ After dissolving in physiological saline, add 1.5 ml of Supplementary Solution of Furuiside.
The suspension prepared by adding s/
．． 0#), and the same amount is administered 6 times every 12 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 β-type isiterfeon antibody of the present invention (hereinafter abbreviated as "antibody ■").

Example 15 After dissolving 25 μf of the antigen N obtained in Example 9 in 1.5 s/ of physiological saline, an auxiliary solution of 70 indica (1.5 s/) was added.
The suspension prepared by adding
#), and the same amount is administered 6 times every 2 weeks. Thereafter, the same amount as the initial dose is administered three times every month. Blood was collected from the test animals 7 days after the final administration,
The antiserum is collected by centrifugation to obtain the human β-type isiterfeon antibody of the present invention (hereinafter abbreviated as "antibody ■").

Example 16 Example 10r116t14 antigen V 251tfltt
After dissolving in physiological saline at 1.5 s/mL, a suspension prepared by adding 1.5 s/mL of 70% Indian auxiliary solution was added to S4 rabbits (2
, 5-3.0 kg), and the same amount is administered six times every two weeks. Thereafter, the same amount as the first dose was administered three times every month. Seven days after the final administration, blood was collected from the test animals.1. The antiserum was collected by centrifugation, and the antiserum was collected, and the antiserum was collected, and the antiserum was collected, and the antiserum was collected.
) is obtained.

Example 17 Using antigen 1 obtained in Example 11 and treating in the same manner as in Example 12, a high titer antibody (hereinafter abbreviated as [antibody Y]) having specificity for human β-type interferon was produced. ) is obtained.

Example 18 Production of 0-labeled peptide (1) //-Met-5tr-Tyr-Asn-L
zu-Ltu-Gly -Phe-Law-Gl n-
Arg-5tr-5zr-OR is labeled using a method using RomiT.

That is, add 5 μf of the above heptide to 20 μl of 0.2 t of lysinate buffer (door H7, 4) [125 Summer] No (M
EN) l Mike 0 + Add Ury's 0.25 luricate buffer, then Ola E y 73.5 */mlo
Add 20 μl of O,2'e L lysinate buffer. Sodium metabisulfite (#a2 s2 o5
Terminate the reaction by adding 50 μl of 10 M lysinate buffer. The reaction mixture was diluted with DEAE
) - Seven Fadex A-25 columns (1, OX3 (1
m) (eluent 0.1% BSA and 0.01% #
0.17 lutris-salt buffer containing a7''3 (H8,6). The first fraction was purified by ion exchange using 0.0-molecular fluoride to obtain the above peptide labeled with I.

(2) H-Met-5zr-Tyr-Azn-Lz
u-Ltsi-Gly-Pkt-Lzu-Gl n-
Arg-5t r-5t r-ON is labeled by a method using rayic acid T.

That is, (1) JVa (MEN) 1 was added to 20 μl of 0.27 μl phosphate buffer (///7.4) containing 5 μf of the above peptide.
? Add 0.21 ml of Iku Ogyu 11J- lurin salt reduction buffer, then add 20 µl of 0.27 lurin phosphate buffer of 6 Lamisi T3.5W/G. Leave it for 20 seconds at room temperature.

2,,4sf/l of cod in 0.2M phosphate buffer, metabisulfite tri,l')&,(NL!S,,05)(D
50 pl 0.2 M! J:/Terminate the reaction by adding acid salt buffer. 5p-p reaction mixture
The fourth fraction was applied to a Fadex C-25 column (1, OX3 (1 m) (eluent: 50 ml lurine salt buffer)), which was the fraction of the above heptide labeled with 125 I.

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

That is, each antibody was dissolved in physiological saline at 10% 102.10
', 10. 10...Diluted by 2 times (initial)
100゛μ4 of each of them! Labeled peptide (the labeled peptide obtained above was diluted to about 10,000 ppm) O.I ml and 0.1 heptaphosphate buffer (pH-7.4) (0.1%, BSA , Q, 1
0.2 g/containing 5 mol sodium chloride and 0.01% NaN3 was added and incubated at 4°C for 24 hours. and centrifugation method (4°C, 15 minutes, 30 minutes)
00 rplpl) to separate from the unreacted (unbound) I-labeled peptide, count the radiation, and measure the binding rate (illusion) of the antibody to the I-labeled peptide at each dilution concentration. Take the binding rate (%) with the labeled peptide and the dilution factor (initial concentration) of the antibody on the horizontal axis, and calculate the binding rate at each concentration.When the binding rate is 50%,
The dilution ratio of each antibody, that is, the titer of the antibody, is determined as follows.

Antibody titer Antibody V 52,000 Antibody Vl 100,000 or higher )-, the 1st to 13th betutide chains of cytoβ-type interferon and cytoto-β-type interferon (manufactured by Hayashibara Research Institute, L-dohablazlaid IntIrferes)
, Let, No. 800928).

Also, 0.1% BSA as a standard diluent.

0.1 molar sodium phosphate buffer containing 0.15 eL NaC6 and 0.01% NaN5 (door #7.4)
use.

In each test tube, add standard diluent 0-2 mes test sample 0-I ml% antibody VO obtained in Example 16,
After adding 0.1 ml of lml (labeled peptide obtained above and diluting the labeled peptide obtained above to approximately 17 rpm) and incubating at 4°C for 48 hours, Normal sheep serum (ntyrmal)
Add 0.1 ml of 5heep slrum),
Then a suspension of activated carbon coated with De+Strashi 0.5
ml, leave at 4°C for 30 minutes, then incubate at 4°C for 30 minutes.
Centrifugation was performed for 30 minutes at 00 rpm to separate the conjugate of the antibody 25 and the l-labeled peptide and the unreacted (non-binding) l-labeled peptide, and the radiation was counted, and the amount of the antibody used was determined. The binding rate (Bo
) as 100%', the concentration of each test sample is 125'
□ Determine the percentage of the conjugate ■ between the antibody and the labeled peptide at each concentration and dilution rate. The results obtained are shown in FIG. The vertical axis in Figure 1 is the binding percentage (B/B).

X100), and the horizontal axis shows the concentration of the test sample (human β-type interferon 1 to 13th peptide chains, human β-type interferon and human d-type interferon). In addition, in the figure, the song S (a) represents the 1st to 13th peptide chains of the β-type interferon 0, the curve O represents the peptide chain of the β-type interferon 0, and the curve 09 represents the peptide chains of the η-type interferon 0. 0n is shown respectively. From FIG. 1, antibody V shows clearly distinct curves in its reactivity to human d-type interferon and to β-type interferon. .7×10
It can be seen that it is a highly specific antibody with no cross-over up to the unit/tan.

Further, in the same manner as above, antibody (2) is also tested for specificity of human β-type protein. The results obtained are shown in FIG.

FIG. 2 is a graph showing the specificity of antibody ■.

In Fig. 2, the vertical axis represents the bond 96CB/BO x 100), and the horizontal axis represents the test sample (human β-type
3rd peptide chain, human β-type interferon 0 and human α
In addition, the curve () shows the concentration of human β-type Isitafe 0, the 1st to 13th bezutide chains of human β-type Isitafe 0, and the curve (H) shows the concentration of human β-type I, Taaffe 0. (f) indicates human α-type interferon 0, respectively.

Also, add 2 g of the standard diluent Q mentioned above to each test tube.
1 Example 2.3 and now obtained Bezutide 0.1 ml, antibody V or 1 dMO, Im and 1251 labeled peptide 0.1
Add g+/ and react under the same conditions as above. Antibody V obtained from peptide chains Nos. 1 to 13 of β-type Isitaphe 0 or 1-1
Table 1 shows the binding form of each peptide, assuming that the binding reactivity with peptide No. 3 is 100%.

Table 1 Sensitivity measurement In each test tube, add 0.21 dl of standard diluent, 0.1 g of peptide chains 1 to 13 of Nido β-type interferon, and the antibody obtained in Example 16 or the antibody obtained in Example 17. The obtained antibodies MO, Ig/ and ■125-labeled peptide (the labeled peptide obtained above was diluted to about 10,000 tpm) O, I + wl were added and incubated at 4°C for 48 hours. Add 0.1 m of normal sheep serum, then add 0.5 wl of activated carbon suspension coated with Di+Stran.
was added, left at 4°C for 30 minutes, then heated at 4°C at 130°C.
Centrifugation was performed for 30 minutes at 00 rpm to separate the conjugate of the antibody and the 1125-labeled peptide and the unreacted (unbound) l-labeled peptide, and the radiation was counted to determine the power of the antibody used. The binding rate (Bo ) corresponding to the titer is 100%, and the antibody and 1125 label at each concentration and dilution rate of each test sample are
Determine the percentage of bonding with the peptide. The results obtained are shown in FIG. The vertical axis in Figure 3 is the bonding percentage (-B/Box
lQO), and the horizontal axis is human β-type
The concentration of the 3rd peptide chain is shown. Curve (G) in the original drawing
The curve (A) is the one using the antibody ■, and the curve (A) is the one using the antibody ■.

As is clear from Figure 3, the minimum detection sensitivity is 13 for antibody ■.
hicogram/m s antibody ■ is 5 hicogram/we.

Production of adsorbent> Example 19 (a) Antibody V obtained in Example 16 0.74ml
was dissolved in 2.26 ml of distilled water, then 3 g of a saturated aqueous acinanium sulfate solution was added, and the mixture was left at 4°C for 3 hours. Next, add 3 ml of distilled water and leave at 4℃ for 3 hours.
t-le. Centrifugation at 4℃-t' for 15 minutes (3500rpH
1) Dissolve the precipitate in distilled water and add 8 d of antibody aqueous solution.
Obtain ゞ゛ (0D2190=1,157)
. Next, it was dialyzed three times against distilled water and further dialyzed against 0.1M sodium bicarbonate (pH 8,4) containing 0.5M sodium chloride to obtain 7 liters of an aqueous antibody solution (OD280=0.923).
.

(b) 1 g of dry diazibromide-activated Cephas 4E
After washing with 200ml of 3M hydrochloric acid aqueous solution, 30ml of 0.1M sodium bicarbonate containing 0.5M salt and 7ml of the dialyzed antibody aqueous solution obtained in (d) above are added, and incubated at room temperature for 2 hours. .

Filtrate through a class filter, dissolve the resulting gel in 40 ml of 1M hexaethanolamine CL (H8, 36), and incubate at room temperature for 2 hours. O, 1M 7t date buffer CI'H4,O> containing 0.5M NaCl, followed by four washes with 0.1M Borate buffer containing 0.5M NaCl. Suspend in physiological saline to obtain a β-type A, T-F0 sheet, T-F4B adsorbent.

In addition, 35gt of 0D28 furnace liquid was filtered through a glass filter.
0 is 0.004, and most of the antibodies are
It can be seen that it is fixed to B.

Example 20 (a) Antibody 1 obtained in Example 1-2 0.741
was dissolved in 2.26 g/distilled water, then 3 ml of saturated ammonium sulfate aqueous solution was added, and the mixture was left to stand at °C for 3 hours. Next, add 3 ml of distilled water and leave at 4°C for 3 hours. Centrifuge at 4°C for 15 minutes (C3500rl'W1) and dissolve the precipitate in distilled water to obtain an aqueous antibody solution 8d (
0D28o=1.154). Next, it was dialyzed three times against distilled water, and further dialyzed against 0.1 Np sodium hydrogen oxide containing 0.5 M sodium chloride (Toh H8,4) to prepare 7 ml of the antibody aqueous solution.
(OD2&, = 0.921).

(b) Dry diazibromide activated Cepha D-su 4B If
After washing with 200 ml of 1 mm HCl aqueous solution, 0.
Contains 5M salt 0. Add 30 ml of IM sodium bicarbonate and 7 ml of the dialyzed aqueous antibody solution obtained in (a) above, and stir at room temperature for 2 hours.

Filter with a class filter and the resulting gel is 1Mv:)
Engineering9) -1vAE:/-CL (fiH8,36)40
ml and incubated at room temperature for 2 hours. 06
0, 1 M acetate buffer CI containing 5 M NaCl)
Circulating washing with H4, O>, then 0.5M salt containing 0.5M salt.
Wash 4 times with 1 M borate buffer. Suspend in physiological saline to obtain human β-type isitase 4
Obtain B sucking and 1 body.

In addition, 0D28 of 35ml of furnace liquid filtered with a glass filter
1O is Sail 004, and most of the antibodies are Sephas 0-4B
It can be seen that it is fixed.

Example 21 (2) Antibody II O,74 obtained in Example 13
m/ is dissolved in 2.26 g/distilled water, then 3 ml of a saturated aqueous sodium sulfate solution is added, and the mixture is left at 4°C for 3 hours. Next, add 3 ml of distilled water and leave at 4°C for 3 hours. Centrifuge at 4°C for 15 minutes (3500 rpm) and dissolve the precipitate in distilled water to obtain 8 ml of antibody aqueous solution (0D280.=1.155). Then, dilute with distilled water for 30 minutes.
The antibody aqueous solution was dialyzed twice and further dialyzed against 0.1M sodium hydrogen suboxide containing 0.5M sodium chloride (door #8.4).
(OD280=0.921).

Cb) After washing dry diazibromide-activated Cephas 4BIf↑9 with 161 M hydrochloric acid aqueous solution 20011, 0.5
Add 30 ml of 0.1 M sodium bicarbonate containing M sodium chloride and the dialyzed aqueous antibody solution 71j obtained in (2) above, and stir at room temperature for 2 hours.

Filter with a glass filter, and the resulting gel is 1Mv:)
Engineering 9) LEE:/-CL(/'#8.36')40
ml and incubate for 2 hours at room temperature. 0.
Circulate wash with 0.1 M'' Pt tate buffer (7#4.0) containing 5M saline, then wash 4 times with 0.I# borate buffer containing 0.5M saline in saline. By suspending it, a human β-type isiter 4B adsorbent is obtained.

Furthermore, 0D28o of the furnace liquid 351 filtered with a glass filter
It can be seen that most of the antibodies are immobilized on Sephas 004B.

Example 22 (a) Dissolve 0.74 sl of the antibody tablet obtained in Example 14 in 2.26 l of distilled water, then add 3 ml of a saturated aqueous solution of sodium sulfate, and leave at 4°C for 3 hours. . Next, add 3 ml of distilled water and leave at 4°C for 3 hours.

Centrifuge at 4°C for 15 minutes (35001 × 0) and dissolve the precipitate in distilled water to obtain 8 ml of antibody aqueous solution (0D2).
80=I, i56). Next, dialysis was performed three times against distilled water, and further 0.5M salt-containing 0.5M sodium chloride solution was used. Dialyze with IMp sodium hydrogen pate (-H8,4) to obtain antibody aqueous solution 71j (
OD2&)=0.922).

(b) Dry diazibromide activated Cephas 4B
After washing II with 200 ml of 113 M hydrochloric acid aqueous solution,
．． Contains 5M salt 0. IM sodium bicarbonate 30ml
Then, 7 ml of the dialyzed antibody aqueous solution obtained in (a) above is added, and incubated at room temperature for 2 hours.

Filter through a glass filter, dissolve the resulting gel in 40 ml of 1M ethanolic acid CL (pH 8,36), and incubate at room temperature for 2 hours. 0,1 M acetate buffer CI'H4,O containing 0.5 M NaCl)
and then four times with O,+U borate buffer containing 0.5M NaCl. It is suspended in physiological saline to obtain a β-type isiterfue 0-sheet t-face 4B adsorbent.

In addition, filter LfcF liquid 3511/0D with a glass filter.
280 is 0.004, and it can be seen that most of the antibody is immobilized on tFase D-4B.

Example 23 (a) Dissolve 0.74 ml of the antibody W obtained in Example 15 in 2.26 g+/ of distilled water, then add 3 ml of saturated aci7ium sulfate aqueous solution in □, and leave it at 4°C for 3 hours. . Next, add 3 ml of distilled water and leave at 4°C for 3 hours. Centrifuge for 15 minutes at 4°C (C3C550Orl)
Then, the precipitate was dissolved in distilled water to obtain 8 ml of an aqueous antibody solution (on280=1.!53). Next, add 3 with distilled water.
The antibody aqueous solution was dialyzed twice and further dialyzed against 0.1 M sodium bicarbonate containing 0.5 M sodium chloride (pH 8.4).
ml (OD28o=0.920).

(b) Dry diazibromide activated Cephas 4B If
After washing with 13M hydrochloric acid aqueous solution 200g, 0.5M
Add 30 ml of 0.1 M sodium bicarbonate containing sodium chloride and 7 d of the dialyzed antibody aqueous solution obtained in (a) above, and incubate for 2 hours at room temperature.

Filter through a class filter, dissolve the resulting gel in 40 ml of 1M heptanoethanolic acid CL (#8.36), and incubate for 2 hours at room temperature. o, 5Mf
Sail IM acetate buffer containing k salt <1'H4,o
> and then four times with 0.1 M borate buffer containing 0.5 M saline. The suspension is suspended in physiological saline to obtain a β-type isiterphae0cephae0-su4B adsorbent.

In addition, 0D28 of 35ml of furnace liquid filtered with a glass filter
0 is 0.004, and the antibody is mostly Sepharose 4B.
It can be seen that it is fixed.

Example 24 (a) Example! Antibody obtained in 7 ■0.74vtl
was dissolved in 2.26 g/distilled water, then 3 ml of a saturated acinanium sulfate aqueous solution was added, and the mixture was left at 4°C for 3 hours. Next, 3 fIt of distilled water was added and the mixture was left at 4°C for 3 hours. Centrifuge for 15 minutes at 4°C (C3500rfitPI) and dissolve the precipitate in distilled water to obtain 8 ml of an aqueous antibody solution (OD28o = 1.158). Next, it was dialyzed three times against distilled water, and further dialyzed against 0.5 Mf & salt containing 0.1 # sodium hydrogen oxide (-H8,4>) to give 7 ml of the antibody aqueous solution.
(0D280=0.924).

(b) Dry cyanogen bromide activated Cephas 4B
After adding IF and washing with 200 ml of 1mM hydrochloric acid aqueous solution,
．． Contains 5M salt 0. IM sodium bicarbonate 30ml
Then, add 7 t of the dialyzed aqueous antibody solution obtained in (a) above, and incubate for 2 hours at room temperature.

Filter the resulting gel with a class filter at 1'v:)
eta) ItiPE:1-CL (///8.36
) Dissolve in 4-Os+l and incubate for 2 hours at room temperature. Circulating wash with 0.1 N acetate buffer (1)//4.0) containing 0.5 M NaCl, followed by 0.5 M NaCl.
Wash 4 times with 0.1 M borate buffer containing M NaCl.

It is suspended in physiological saline to obtain a β-type ferrocycephae 4B adsorbent.

In addition, 0D2 of 35yxl of furnace liquid filtered with a glass filter.
80 is 0.004, and most of the antibodies are
It can be seen that it is fixed to B.

Example 25 Cell 0-su (Toyo 0!/A2) was cut into 0.5 c IR squares and weighed to about 1 f. This cellulose is diazibromide
11.0~11.5
Add sodium hydroxide solution to keep it at room temperature for 6~
Perform 8 minute activation. After activation, remove the reaction solution with a glass filter and cool.
．． Washed several times with 5M sodium bicarbonate buffer (pH 8,4), suspended in the same buffer after washing, and prepared in Example 19 (d
) Antibody aqueous solution (□Z)280=0 obtained in the same manner as
．． 918) and treated in the same manner as in Example +9(b) to obtain a human β-type interferase adsorbent. Furthermore, the furnace liquid filtered with a glass filter is 0D28oO1.
004, indicating that most of the antibody was immobilized on the cells.

Example 26 Suspending cross-linked de+strane (Sephadex G-25) IF in water to increase the water velocity of cross-linked de+tran
Add 0 μl at once and stir vigorously for 1 to 2 minutes, then remove the reaction solution on a glass filter. a, washed several times with 0.1 M sodium bicarbonate buffer containing 5X* salt (door H8, 4), and suspended in the same buffer after washing, Example 19
Antibody aqueous solution obtained in the same manner as in (a) (0D28o=
0.921 ) and treated in the same manner as in Example +9(b) to obtain a cross-linked human β-type isitase adsorbent.

In addition, 0D28o of the furnace liquid filtered with 4 glass filters is 0.
．． 004, and it can be seen that most of the antibody is immobilized on the bridge.

Example 27 β-type Ishitafue 0 (Tokyo Metropolitan Institute of Clinical Medicine, 0D280=11.8) 0.511 (Protein 5
．． 91F) in physiological saline, and then this solution was used to prepare the β-type isiterfue obtained in Example 19(b).
It was packed into an affinity chromatography using Fast 4B adsorbent, and 0.15 M salt containing 0.1
Elute with M lysinate buffer (door #7.4).

0.5M* when OD = 0.0002
It is purified by elution with a salt-containing 800.5M acetate buffer (H2, 5), and β-type isiterfue is separated from the adsorbent.

Before affinity chromatography, the converted OD value (value when protein is 1III when OD28° = 1, the same applies hereinafter) of human β-type Ishitafue was 5.9, and human β-type Ishitafue was 0. In the immune response, 1
3240 units/llt, 1.5 x 10 units/
In contrast, the converted OD value of Shito β-type Ishitafue after affinity take 0 is 0.
．． 059, and +2050 f/me, 1.37
It shows an activity of unit/q.

Therefore, the specific activity before affinity chromatography is 2.54 X I 051. Knit/IIg Decha, while Affinity Chroma “After Totaraf・° is °2
, 3'2ξ×107 units/IIg 91.3
Indicates the value equivalent to double.

The above results show that by using the adsorbent of the present invention, it is possible to purify human β-type isiterphein 91 times or more in one step, and the recovery rate is almost quantitative.

Furthermore, when affinity chromatography was performed in the same manner as above using the adsorbents of the present invention obtained in Examples 20 to 26, they showed good separation and purification ability as above.

[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 1 to 13 of β-type interference 0 and binding % for antibodies of the present invention. (The above) Agent: Patent attorney Eiji Saegusa +l

Claims (1)

[Claims] ■ General formula [wherein R is a hydrogen atom, H-Pht- group, //-Ltu-
Gly-Pht- group, H-Tyr-Asn-Leu-L
tu-Gly -Pkt- group or H-Met-5tr-
Tyr-Axn-Lzu -Lzu-Gly-Phe-
Indicates the group. ] A human β-interferon antigen consisting of a complex of the N-terminal peptide of human β-interferon represented by Affinity characterized by being fixed