JPS60142925A - Preparation of antibody of human t-cell leukemia virus - Google Patents

Abstract

PURPOSE:To obtain the titled antibody, by administering an immune antigen comprising a complex of peptide related to human T-cell leukemia virus and a carrier to a mammal, collecting a formed antibody. CONSTITUTION:An immune antigen comprising a complex of peptide related to human T-cell leukemia virus (separated from adult T-cell leukemia) selected from the group consisting of peptides shown by the formula I , II, III and IVand a carrier is administered to a mammal, a formed antibody is collected, to prepare stably a large amount of an antibody of human T-cell leukemia virus. The specific antibody can be used for purification of protein related to virus by bonding it to a carrier for affinity chromatography, using the carrier for the chromatography, it is useful for diagnosing infection of human T-cell leukemia virus, also useful for diagnosis, research, etc. of adult T-cell leukemia such as human T-cell leukemia, T-cell lymphosarcoma, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to human leukemia virus (Adljlt T-c
ell L eukemia V 1rus ;ΔTL
V or Human T-cell 1-eukesia
Production of a new antibody specific for virus: HTLV) 3!
! ! Regarding the law.

In Suimei IS, when amino acids, peptides, protective groups, active groups, etc. are indicated by abbreviations, IUPACI
1LJB regulations or common symbols in the field concerned, examples of which are listed below. Furthermore, if there are possible optical isomers for amino acids, etc., only one isomer is shown unless otherwise specified.

Ser; Serine leu; r = + Isine Thr: Threonine Sn; Asparagine G10: Glutamine Gl
u; glutamic acid ■S: lysine pro; proline Tyr: tyrosine Trpnitributophan)-1i
s; histidine A sp; aspartate GIV nigericine lle; isoleucine Ala: alanine P
he: Phenylalanine Aro; Arginine Tos; p-1-luenesulfonyl group BOC: ff1
Tertiary butoxycarbonyl body ONP: p-nitrophenoxy group 1321: benzyl group QBzl; benzyloxy group C12-871: 2,6-dichlorobenzyl group Cl-
7:2-Chlorbenzyloxycarbonyl human leukemia virus is isolated from adult T-cell leukemia (ATL),
This is a virus that is attracting attention for its association with the disease. The present inventors, Yoshihichi and Kanno, used genetic engineering techniques to clone (atoning) the provirus gene integrated into the DNA of a host cell, and determined its entire base sequence. Based on this, we established the basis for the diagnosis, treatment, and prevention of the disease and the virus infection. The determined nucleotide sequence encoding the envelope protein precursor of the above virus gene is known (Proc, Natl.
Acad, Sci. , USA. 80 (1 983
), Bioche II listry, 11 362
1), the outer shell protein precursor consists of 488 flil amino acids.

Based on the above basic information and as a result of further research, the present inventors discovered a specific peptide that can be a hapten of a protein related to human leukemia virus (outer protein).
ATLV
or has specific reactivity with HTLV and related peptides, such as ΔTLV or HTLVgll-linked protein, etc.
We have succeeded in obtaining a new antibody that can be used for production and diagnosis of ΔT'LV or HTLV infection, and have now completed the present invention.

That is, the present invention provides a peptide represented by the formula (1), the formula H-Pro-8sn-Ar(1-Δsn
- G Iy - G ly - G ly-Tyr-
OH (2) Peptide represented by the formula % Formula % (3) Peptide represented by the formula H-Gly-Leu-ΔSp-L eu - LeU
- P he - T rDGlu Qln aly
, GIy Leu Tyr OH (4) An immunogenic antigen consisting of a complex of a human leukemia virus-related peptide selected from the group consisting of a carrier and a carrier is administered to a mammal, and the produced antibodies are collected. The present invention relates to a method for producing a characteristic human leukemia virus antibody.

According to the present invention, the above formulas (1) to 1 can be easily synthesized by simple operations using easily available commercially available amino acids.
Based on the use of an immunizing antigen prepared using the peptide represented by (4) as a hapten, antibodies that exhibit specific reactivity to virus-related proteins can be obtained easily, easily, and stably. . The specific antibody can be used for the purification of the virus III-linked protein by, for example, binding it to a carrier for affinity chromatography and using it in the chromatography, and can also be used to purify the virus-related protein in various immunoassay methods. It can be used as an antibody to diagnose human leukemia virus infection, and even
It is useful for diagnosis, research, etc. of mature T cell leukemia/lymphoma such as adult T cell leukemia and homotypic TIII cell lymphoma, and related diseases.

The peptides represented by the above formulas (1) to (4) can be synthesized using conventional peptide synthesis methods, specifically “The Peptide (The Peptide)”.
Peptides) J Volume 1 (1966) (S
By Chroder and Luhke, Δca
de1c press.

New York, LJSA) or “Peptide Synthesis”
[Azumaya et al., Maruzen Co., Ltd. (1975)], for example, the azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method (
p-nido-O phenyl ester method, N-hydroxysuccinimide ester method, cyanomethyl ester method, etc.), method using Woodward reagent K, carbodiimidazole method, redox method, DCC/7 active (HONB
, t-10Bt.

It can be manufactured by the HO8u) method or the like. In the above method, both solid phase synthesis method and liquid phase synthesis method can be applied.

Usually, the peptide of the present invention is synthesized according to the general polypeptide synthesis method described above, for example, by the so-called stepwise method in which amino acids are sequentially condensed one by one to the terminal amino acid, or by dividing into several 7-ragments and coupling them. manufactured by the method. More specifically, when employing, for example, a solid phase synthesis method, the C-terminal amino acid is bound to an insoluble carrier via its carboxyl group. The insoluble carrier is not particularly limited as long as it has a good bonding property with the reactive carboxyl group, and examples thereof include halogenomethyl resins such as chloromethyl resin and bromomethyl resin, hydroxymethyl resin, phenol resin, and tart-alkyloxycarbonyl hydrazide. Chemical resin etc. can be used.

Next, after removing the amino protecting group, amino group-protected amino acids are sequentially bonded by a condensation reaction with the reactive amino group and the reactive carboxyl group according to the amino acid sequences represented by formulas (1) to (4). After synthesizing the entire sequence, the peptide is removed from the insoluble carrier.

In the above, ΔrO1TyrSGlu, Gln, T
hr.

It is preferred that the side chain functional groups of the lys, ASD, and Ser amino acids are protected by a conventional protecting group, and the protecting group is removed after the reaction is completed. Furthermore, the functional groups involved in the reaction are usually activated. Each of these reaction methods is well known, and the reagents used therein are also appropriately selected from known methods.

Examples of protecting groups for amino groups include benzyloxycarbonyl, Boa, tert-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, C+-Z, adamantyloxycarbonyl del, phthalyl, formyl, 0-nitro Examples include phenylsulfenyl and diphenylphosphinothioyl groups.

Maintenance of Δrg! ! ! ! Examples of the group include Tos, nitro, benzyloxycarbonyl, amyloxycarbonyl group, and the like.

Preservation of hydroxyl groups in Scr and Tl+r [E] includes, for example, Bzl, tart-butyl, acetyl, and tetrahydropyranyl groups.

As a protecting group for the hydroxyl group of Tyr, for example, Bzl, CI
2-Bzl, benzyloxycarbonyl, acedel, T
Examples include os group.

For example, benzyloxycarbonyl, CI Z. Cl 2 Bzl,
Examples include 3oc and 1゛OS groups.

As the protecting group for the carboxyl group of Glu and Δsp,
For example, benzyl alcohol, methanol, ethanol,
Esterification with tert-butanol etc. is carried out.

Examples of activated carboxyl groups include the corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, activated esters (pentaf OD phenol, p-
Nitrophenol, N-hydroxysuccinimide, N
-Hydroxybenz1-lyazole, N-hydroxy-
Examples include esters with 5-norbornene-2,3-dicarboximide, etc. The peptide bond forming reaction may be carried out in the presence of a condensing agent such as a carbodiimide reagent such as dicyclohexylcarbodiimide or carbodiimidazole, or tetraethylpyrophosphine.

Hereinafter, the method for producing the above-mentioned specific peptide used in the present invention will be specifically explained using the peptide of the above formula (1) as an example and a reaction scheme.

[Reaction scheme-1] A-Lys-OH (a) ↓ 8-L VS- R Blind (mouth) ↓ H-Lys-R' (c) ↓ A-T hr - OH (2) 8-Th
r-Lys-R' (E) △-Tyr-3er-1-eu-Tyr-Leu-Pl
ea-pr.

-Hls-Trp-T11r-LVS-R' (to) ↓ H-Tyr-5er-L eu-Tyr-1eu-Pl
ea-Pr.

-Hls-”rrp-Tl+r-Lys-OH(5)(
In the formula, A represents an amino group! ! ! fM and R1 indicate an insoluble carrier. ) In the above, preferred examples of R1 include 3oc1 benzyloxycarbonyl group and p-methoxybenzyloxycarbonyl group, and preferred examples of R1 include chloromethylated polystyrene.

Furthermore, in each reaction, when the amino acid used has a side chain functional group that does not participate in the reaction, it is protected by the above-mentioned protecting group as usual, and this is removed simultaneously with the removal of the insoluble carrier R1.

In the above method, the reaction between the amino acid (a) and the insoluble carrier R1 is carried out by utilizing the reactive carboxyl group of the amino acid (a) and bonding it to R1 according to a conventional method. For example, when chloromethylated polystyrene is used, the reaction is carried out in a suitable solvent in the presence of a basic compound such as triethylamine, potassium 1XjQrj-butoxide, fatsium carbonate, cesium hydroxide, and the like.

As a solvent, for example, dimethylformamide (DMF)
, dimethyl sulfoxide (DMSO), pyridine, chloroborum, di(non, di(J)), tetrahydrofuran, N-methylpyrrolidone, trimethylphosphoric acid triamide, etc., or a mixed solvent thereof. The reaction is usually completed at about 0 to 85°C, preferably about 25 to 80°C, for about several minutes to 24 hours.

The ratio of amino acid and insoluble carrier to be used is usually such that the former is used in excess of 1 equivalent of the latter, generally 1 to 3 times.

The elimination reaction of the thus obtained immobilized amino acid of general formula (b) to the protecting group is carried out by a conventional method. Such methods include, for example, hydrogenation using catalysts such as palladium, palladium black, etc., reductive methods such as reduction with metallic sodium in liquid ammonia, trifluoroacetic acid, hydrochloric acid, hydrogen fluoride, methanesulfonic acid, bromide. Examples include acidolysis using strong acids such as hydrogen acid. Hydrogenation using the above catalyst can be carried out, for example, at a hydrogen pressure of 1 atmosphere and at a temperature of 0 to 40°C. Usually 1001H to 1g of catalyst is used in the tank.
The process is generally completed in about 1 to 48 hours. In addition, the above acidolysis is carried out without a solvent, usually at 0.
~about 30℃, preferably about 0~20℃ for about 15 minutes~
It takes about one hour to complete. The amount of acid used in the tank should normally be about 5 to 10 times the amount of the raw material compound. When only the protecting group Δ is removed in the acidolysis, it is preferable to use trifluoroacetic acid or hydrochloric acid as the acid. Furthermore, the reduction with metal sodium thorium in liquid ammonia is carried out using positive metal stroium such that the reaction solution is colored permanent blue for about 30 seconds to 10 minutes.
It can be carried out usually at about -40°C to -70°C.

The reaction between the resulting solid-phase amino acid of general formula (c) and amino Wi(d) (or its carboxyl group activated) is carried out in the presence of a solvent. Various known substances commonly used in synthetic reactions, such as anhydrous dimethylformamide, dimethyl sulfoxide, pyridine, chloroborm, dioxane,
dichloromethane, tetrahydrofuran, ethyl acetate, N
Examples include -methylpyrrolidone, hexamethylphosphoric triamide, and a mixed solvent thereof. The reaction may be carried out using reagents used in ordinary peptide bond forming reactions, such as N,N-dicyclohexylcarbodiimide (DCC), N-ethyl-N'-dimethylaminocarbodiimide, 1-ethyl-3 -diisopropylaminocarbodiimide, 1-cyclohexyl-3-
This can be carried out in the presence of a dehydration condensation agent such as carbodiimides such as (2-morpholinyl-4-ethyl)carbodiimide. There is no particular limitation on the ratio of the amino acid (c) to the amino acid (d), but usually the former is used at an equimolar to 10 times the molar ratio, preferably an equimolar to 5 times the molar ratio of the former. It is better to do so.

There is no particular limitation on the amount of dehydration condensation agent used, and usually amino acids (
It is preferably used in about the same molar amount as (d). The reaction temperature is within the usual range used for peptide bond forming reactions, generally about -40°C to about 60°C, preferably about -20°C.
The reaction temperature is appropriately selected from the range of .degree. C. to about 40" C.The reaction time is generally about several minutes to about 30 hours.

Thus, the peptide of the general formula (E) represented by
Hls-01(, Δ-Pro-Of-1, A-Phe-
OH1Δ-L eu -OH1Δ-Tyr-01-1,
Δ-Leu-OH1Δ-3er-OH1Δ-Tyr-
This is carried out by successively condensing each amino acid or side chain functional group of OH with a protected one or an activated carboxy group, and thus a peptide represented by the general formula (H) can be derived. . These synthesis reactions and elimination reactions to form one group are carried out in the same manner as described above.

The obtained peptide (H) is similarly induced into a peptide represented by formula (1) by removing the protecting group Δ1I1211t, removing the protecting group from the side chain functional group of the amino acid, and removing the insoluble carrier R1. Ru. Here, the elimination reaction of the protective group of the side chain functional group and the insoluble carrier R1 can be carried out in the same manner as the elimination reaction of the protective group Δ, and in this case, it is preferable to use hydrogen fluoride or hydrobromic acid as the acid. Note that each amino acid used in the above method may be a known commercially available product.

The peptide of 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.

Moreover, each peptide represented by general formulas (2) to (4) is also produced according to the above method.

The peptide thus obtained is 1251°131
Radioactive substances such as ■, peroxidase (POX), chymotrypsinogen, procarboxypeptidase, glyceraldehyde 3-phosphate dehydrogenase, amylase, phosphorylase, D-Nase, P-Nasc
By introducing various enzyme reagents such as , β-galac-1 hesidase, glucose-6-phosphide dehydrogenase, ornithine decarboxylase, etc., the radioimmunoassay (RIA) method or enzyme immunoassay ( It can be used as a labeled antigen for use in the EIA) method. Introduction of the above-mentioned radioactive substance can be carried out by a conventional method. For example, radioactive iodine can be prepared by oxidative iodination using phosphoramine T (W).

M, ) lunter and F, C, Gree
nwood; Nature.

194. 495 (1962), Biochem J
.

(See Wang, 144, (1963)) carried out by a hat,
The enzyme reagent can be introduced using conventional coupling methods such as the method of Erlanger (B, F, Erlanger) et al.
Δacta, Endocrino1. 3upD1. ．． 1
68.

206 (1972)) and force [1-le (M, l-1
゜Karol et al.'s method (proc, Na mouth, △
cad, 3 ci,.

USA, 57. 713 (1967)).

Hereinafter, a method for producing an immunizing antigen using the above-mentioned peptide as a hubten will be described in detail.

The above-mentioned antigen is produced by using any of the peptides (1) to (4) as habuten, and reacting this with a suitable carrier in the presence of a habuten-carrier binding reagent. As the carrier bound to Habten in the above, a wide range of high-molecular natural or synthetic proteins commonly used in the preparation of antigens can be used. Examples of such carriers include animal serum albumins such as horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, and sheep serum albumin; horse serum globulin, bovine serum globulin, horse blood antiglobulin, and human serum albumin. Animal serum globulin such as U purine, sheep serum globulin [1 purines: animal thyroglobulin such as equine thyroglobulin, bovine thyroglobulin, rabbit thyroglobulin, human thyroglobulin, ovine thyroglobulin; equine hemoglobulin, cattle Animal hemoglobulins such as hemoglobulin, Urigi hemopropurin, human hemoglobulin, and ovine hemoglobulin; Animal hemocyanins such as keyhole linbet hemocyanin (KLH); proteins extracted from roundworms (Ascaris extract,
JP-A-56-16414, J, Immun, 1
1 1. 260-268 (1973), J.J.
mmun,, 122. 302 ~・308 (197
9), J. Immun., 98. 893-900 (
1967) and Δm, J, Pl+ysio1. .

199. 575-578 (1960) or further produced by vJ): polylysine,
Examples include polyglutamic acid, lysine-glutamic acid copolymers, and copolymers containing lysine or ornithine.

As the hubten-carrier binding reagent, a wide variety of those commonly used in the preparation of antigens can be used.

Specifically, diazonium compounds such as bisdiazotized benzidine (BDB), bisdiazotized-3゜3'-dianisidine (BDD), etc., which cross-link diL]sine, histidine, and 1-rib1-fan. Crosslinking between amino groups, such as aliphatic dialdehydes such as glyoxal, malondialdehyde, glucuraldehyde, succinaldehyde, and azibaldehyde; Crosslinking between thiol groups;
For example, N, N'-o-phenylene dimaleimide, N,
Simaleimide compounds such as N'-m-phenylene dimaleimide; those that crosslink amino groups and thiol groups, such as metamaleimidobenzoyl-N-hydroxysuccinimide ester, 4-(maleimidomedyl)-cycUhex'tin-1-carboxyl -N'-Maleimide carboxyl such as hydroxysuccinimide ester -N-
Hydroxysuccinimide esters; reagents used in the usual peptide bond-forming reaction to form an amide bond between an amino group and a carboxyl group, such as N,N-dicyclo-4-zylcarbodiimide, N-di-N'-dimethylaminocarbodiimide, 1- Ethyl-3-cyclohexyl 1-bilaminocarbodiimide, 1-cyclohexyl-3
Examples include 1B2 water condensing agents such as carbodiimides such as -(2-morpholinyl-4-ethyl)carbodiimide. In addition, as the above-mentioned Habten-carrier binding reagent,
It is also possible to use a combination of a diazonium arylcarboxylic acid such as p-diazonium phenylacetic acid and a conventional peptide bond forming reaction reagent, such as the above dehydration condensation agent.

The production reaction of the above-mentioned immune antigen can be carried out, for example, in an aqueous solution or at pH
7 to 10 in normal N salmon solution, preferably 1) 88 to 9 loose! ! i? [l! The process is carried out at a temperature of 0 to 40°C, preferably around air temperature. The reaction is usually completed in about 1 to 24 hours, preferably 2 to 5 hours. Typical buffer solutions used in the above may include the following.

0.2N helium hydroxide - 0.2M boric acid - 0.2
M potassium chloride spinning solution, 0.2M charcoal n'2sb 0, 2M boric acid
〇, 2 MJHI: Power IJ Ram Iy! WJ'a, 0
．． 05M Putrium Tetraborate-0.2M Boric Acid-0,0
5M1i]~Lium Pi buffer solution, 0.1M potassium dihydrogen phosphate-0.05M sodium tetraate buffer solution In L13 above, the proportions of Habten, Habten-carrier binding reagent and carrier can be determined as appropriate. Usually, the carrier is about 1 to 6 times heavier than Habten, preferably 1 to 5 times heavier.
t4m, and Habten-carrier binding reagent 1 to 10 times.
It is best to use about double the mole.

Through the above reaction, a desired immunizing antigen consisting of a bebudid-carrier complex in which a carrier and habten are bound is obtained through the mediation of a habten-carrier binding reagent.

After completion of the reaction, the antigen can be easily isolated and purified according to conventional methods, such as dialysis, gel filtration, and fractional precipitation.

The immunizing antigen thus obtained usually has an average of 5 to 60 moles of peptide bound to 1 mole of protein, and all of these make it possible to subsequently produce antibodies with high specificity for the antigen.

The antibody of the present invention can be produced using the antigen by administering the antigen to a mammal, producing the desired antibody in vivo, and collecting the antibody.

There are no particular restrictions on the mammal used for antibody production, but it is usually preferable to use a horse or a guinea pig.For antibody production, a predetermined amount of the antigen obtained as described above is added to physiological saline. dilute to an appropriate concentration with
Adjuvant) may be mixed to prepare an F3'FJ solution, which may be administered to a mammal. For example, inject the above suspension into one's legs (0.1 to 5 m of antigen).
(no+/times) and thereafter administered every 2 weeks for 2 to 10 months, preferably 4 to 6 months, for immunization. The antibody is collected by collecting blood from the immunized animal 1 to 2 weeks after the final administration of the suspension, centrifuging the blood, and separating the serum. According to the above method, an antibody having excellent specificity for the immunizing antigen used can be obtained, and this antibody can be used in the R1A method, ElA method, etc. to determine human leukemia virus-related proteins.

Below, in order to explain the present invention in more detail, formulas (1) to (
An example of the production of the peptide represented by 4), an example of the production of an immunizing antigen from the peptide, and an example of the production of the antibody of the present invention from the antigen will be given, but the present invention is not limited to these.

Incidentally, the Rf value in each production example was measured using a thin layer Chroma 1 to Graphee on silica gel using the following mixed solvent.

Rf'・n-butanol-vinegar! Iti-water (4:1:5
) Rf2...n-butanol-pyridine-acetic acid-water (
30:20:6:24) <Production of peptide> Production example 1 ■ Potassium tert-butoxide 471 ml per suspension f
7) Boc-Ly in DMSO solution 36.7111111
Dissolve 7.92J of s(CI-Z)-OH and add chloromethylated polystyrene resin (Protein Research Foundation Foundation, 2% divinylbenzene, mesh 200-40o) 1
Add 4.46 CI and react at 80°C for 30 minutes.

The resin was thoroughly washed with DMSO, ethanol, 50% acetic acid, water, ethanol, and methylene chloride in that order, and dried under reduced pressure to give 3 oc -1-ys (CI-Z) of 16.0 (]).
- Obtain resin.

After hydrolyzing a portion, amino acid analysis was performed and the result was 0.272 mmol of amino acid/g resin.

■ Boa-Lys (CI-Z) obtained in ■ above
- After washing resin 3, OO three times with 30 mQ of chloroform,
509G 1-Riful A Add to 30 m2 of a chloroform solution of acetic acid (1-FA) and react at room temperature for 20 minutes.

(Cleanse the H fat once with 30rj2 of form, 5 times with 30 times of JB methylene chloride, 3 times with 30m2 of a 10% 1-ethylamine methylene chloride solution, then 30m2 of methylene chloride.
H-Lys(CI-7)-resin is obtained by washing each time with Q for 6 times.

25 mg of a solution of 0.63 g of Boc-TI+r(Bzl)-OH dissolved in methylene chloride was added to the above H-Lys(C
I-Z)-resin, and then 5 ml of a solution of 0.42 J of Dcc dissolved in methylene chloride was added and reacted at room temperature for 2 hours. After washing the resin 6 times with 30 mG of methylene chloride, 0.63 (] of Boc-Tbr(Bzl) -OH
and] 031 g of 1-hydroxybenzotriazole to 25 mQ of methylene chloride, then 0.42 g of DCC
A solution prepared by dissolving R in methylene chloride is added and the reaction is carried out again in the same manner (two-wheel coupling method). The resin was thoroughly washed with methylene chloride and Boc-'T'hr (Bzl)
-Lys(CI-Z)-resin is obtained.

■ In the same way as above ■, Boc-Tbr (Bzl)
-Lys(CI-Z)- Resin is converted into DI2Boc, and then the following amino acids, side chain functional group protected amino acids, or carboxyl group activated amino acids are sequentially subjected to condensation and Boc removal reaction.

Boc-Tr++-OH0,62g Boc-His-OH0,84a Boc-1')ro-OH,0,44QBoc-Phc
-OH0,540 Boa-Leu-Of-LH200,510Boc--
1-yr(cl p Bzl) -0HO,96°Boa -Leu-OH-H200,51'JBoc-
8er(Bzl)-OH0,57(IBoc--ryr
(Cl 2 Bzl) OH0,96(1 Thus HTyr (CI 2 Bzl) -8er(
Bzl)-1cu-Tyr(cl2-3zl)-1
eu-Phe-Pro-H1s-Trp-Thr (
5.2 g of Bzl)-Lys(CI-Z)-resin are obtained. Of this, 1.30o is anisole 1.5mQ, 1
．． Dissolved in 2-ethanedithiol 0.751110 and hydrogen fluoride 154 and heated at -20°C for 30 minutes, then at 0°C for 30 minutes.
After incubation for 0 minutes, excess hydrogen fluoride was distilled off under reduced pressure, and the residue was extracted with 10% acetic acid and washed with ether. The aqueous layer was freeze-dried, then purified by gel filtration using Sephadex G-25 (Pharmacia, eluent 50% acetic acid), and further separated twice using Sephadex LH-20 (Pharmacia, 1111MHCQ). and the target peptide (H-Tyr-8er-Lau-
Tyr-Leu-Phe-Pro-His-Trp-T
hr-Lys-OH) to obtain 581. This peptide is hereinafter referred to as "peptide A."

Rfvi: R (1 = 0.03 Rf 2 = 0.58 Amino acid analysis (n: (analyzed with Hitachi model 835) Analysis value Thr (1) 0.96 Ser (1) 0.94 Leu (2) 2. 03 Tyr(2) 2.11 Phe(1) 1.02 Trp(1) 0.93 Lys(1) 1.02 )-1is(1) 1.01 Pro(1) 0.97 The above amino acid analysis values is the result measured after hydrolysis with 6N-hydrochloric acid, and the same is true for each side below.Therefore, Asn and Gl are present in the peptide obtained on each side.
If n is present, they are quantified as Δsp and Glu, respectively.

Production Example 2 3oc-T I'(C
I2 Z)-Resin (0,294s+mo110 mfl
i) 3 g was sequentially condensed with each of the following amino acids or derivatives thereof in the same manner as in Production Example 1. II! IB oc reaction.

Boc-Gly-OH0,39°Boa-Gly-OH0,39°Boa-Gly-OH0,39°Boa-Asn (1°os)-OH0,78aBoc
-Δrl-ros)-OH0,95aBoc-Asn(
1-os)-0HO, 78QBoc-Pro-OH0,
489 Thus, @ -pro-Asn(Tos)-Δrg
(Tos)-Asn('ros)-Gly-Gly-a
ly-1-yr(C12-Z)-1 resin 3.14 g. 1.0g of this was mixed with 110mG of fluoride water and 1 piece of anisole, heated to -20°C for 30 minutes, then heated to 0°C for 30 minutes.
The bales were coated with ink for 0 minutes, excess hydrogen fluoride was distilled off under reduced pressure, extracted with °UIO% acetic acid, washed with ether, and freeze-dried.

Next, gel filtration was performed using Sephadex G-25 (Pharmacia, eluent 1M acetic acid), and further HPLC (10%
Acetoni-1-lyl 10.05% trifluoroacetic acid, flow rate 1.0
/min, 0DS120T, '1.6X250+I1m,
The target peptide (
H-Pro-Asn-Δ"a-Δ5n-Gly Gly
36 mg of Gly-7yr OH) are obtained. This peptide is hereinafter referred to as "betide B."

Rf values: Rf + =0.01 Rf 2 =0. 26 amino acid analysis v1: (analyzed with Hitachi 835 model) Analysis value Pro (1) 0.98 Δ5p (2) 1.89 Δrll (1) 1.04 Gly (3) 3.10 Tyr (1) 0.99 Production Example 3 Boo-ArO<TOli) in the same manner as in Production Example 1
-Produce 0.075m101/Q resin of resin, Part 5
0, the following amino acids or derivatives thereof were sequentially subjected to double coupling reaction and 1L'2BOc conversion reaction in the same manner as in (1) and (2) of Preparation Example 1.

Boc-△5nONP 0.33゜Boa-GlnONP 0.34゜Boc-Ala-OH0,18a Boa-Ala-OH0,I Bg 13oc-Tyr(Cl 9-Z)-OH0,450 Thus f-1-TVr( Cl2-Z) Ala-Δ
1a-Qln-,a,sn-Δro(Tos)-resin 5
．． Multiply 190 by 1. Of this, 1.7317 was hydrogen fluoride2
0m1? and anisole 2- and heated at -20°C for 30
After incubating at 0°C for 30 minutes and removing excess hydrogen fluoride under reduced pressure, the mixture was extracted with a 10% aqueous acetic acid solution, washed with ether, and freeze-dried.

Next, gel filtration was performed using Sephadex G-10 (Pharmacia, eluent: 10% acetic acid), Sephadex LH-
20 (Pharmacia, eluent 1111M hydrochloric acid), further HPLC (7.5% acetonitrile 10.05% trifluoroacetic acid, flow rate 1.011If?/min, Cafum 0DS12
The target peptide (H-Tyr-Al
a-A Ia-G ln-ASrl-8rg-0H)
291 (l) is obtained.Hereinafter, this peptide will be referred to as "peptide C
”.

Rf value: Rfl=0.01 Rf2=0.26 Amino acid analysis value: (analyzed with Hitachi model 835) Analysis value Tyr(1) 0.98 Δ1a(2) 2.07 Glu(1) 1.04 Δ5o( 1) 0.92 Arg(1) 1.00 Production Example 4 3o(i-TVr(CI 2
-Bzl)-resin 0,29 mmol/a tree fil
e.Production t, 3 g thereof was subjected to double coupling reaction and deboa deoxidation reaction in sequence with the following amino acids or their MFs forms in the same manner as in (1) and (2) of Production Example 1.

Boc-L cu-OH-H200,55QBoc-G
IV-OH0,39Q Boc GIV OHO,39g Boc-GlnONP O,81゜Boc-Glu(OBzl)-OHO,74aBoa-
Trp-OH0,67a Boa-Pha-OHO,59Q Boa-1,eu-OH” H2O0,550Boc
-I-eu-oH” H200,55QBoc-Δ 5
IT(OBzl) -o ト1 0.71+113oc
-Loll-OH-H2O0,55QBoc-Gly-
OHO, 39a ka<LTH-GIV-LOu-Δ5D(OBzl)-L
eu-Leu-Pbe-Trp-Glu(OBzl)
-Glnaly Gly Leu, Tyr(Cl 2-
Z)-Resin 4.31+7 is obtained.

1g of it is hydrogen fluoride 10111Q, anisole 111Q
and 0.5 mQ of 1,2-ethanedithiol, -
After incubation at 20°C for 30 minutes and then at 0°C for 30 minutes, excess hydrogen fluoride is distilled off under reduced pressure, extracted with a 50% acetic acid aqueous solution, washed with ether, and then freeze-dried. Next, gel filtration was performed using Sephadex LH-20 (Pharmacia, 1mfvHjjIff ammonium hydrogen), and further Hr'LC (29% acetonitrile 10.IM
Ammonium fit acid, D H-8,0, flow rate 1,0μ
Q/min,) J Ram 0DS-120T, 4.6gmx25
The target peptide (H-Gly-Leu-8sp-L
eu -L eu-P he -Trll-G lu
-G ln-G +y-G IV -L eu -T
43mo of Vr-OH) is obtained. Hereinafter, this will be referred to as "peptide D".

Rf value: Rf l = 0.02 Rf 2 = (L 69 Amino acid analysis W: (Analyzed with Hitachi model 835) Analysis value TVr (1) 1.05 Leu (4) 4.09 GIV (3) 2.88 Glu(2) 2.04 Tr+1(1) 0.97 Pl+c(1) 1.Q5 Δ5p(1) 0.91 <Production of immune antigen> Production example 1 ■ 2111g to the peptide obtained in peptide production example 1
(1,392 mmol) and Ascaris extract protein 4m
g, fish distilled water3. Oml and add Dishiku L to this solution]
Hexylcarbodiimide (DCC) 0.571110
Add and stir at room temperature for 2 minutes. Then the reaction mixture was
The immune antigen 5 was dialyzed against distilled water at 4°C for several days, lyophilized, and
．． Obtain 8 mg. Hereinafter, this antigen will be referred to as [antigen Ia J].

Antigen Ia has an average of 0.164 μnof of peptide 8 bound to Ascaris 1+ng.

The binding rate between this peptide and Ascaris was determined by further testing the obtained antigen with Refadex G-50 (eluate: physiological saline, detection: OI) at 280 nm, flow rate: 3 mQ/hour, fractional m: 1 mQ each. ), the presence of unreacted Ascaris and peptide was not observed. Therefore, the fraction of peptide bound to Ascaris and other products (peptide 2-pendant) were determined by gel filtration.
The fraction of peptide 2m is separated and labeled t+q
Create a calibration curve of iri and find the ω of the dimer,
This value was subtracted from the peptide cloth used as the starting raw material, and all of the values were assumed to be bound to Ascaris. The same applies to the following example of IFI synthesis of antigen.

■ 3mg of peptide A and 6m of Ascaris extract protein
g, O, IM phosphorus rA112 gastric juice (pH=7.2
) 3.0 Peng, and 0.46 mg (4, 176 μmol) of 0.1% geltaraldehyde was added to this solution, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was then dialyzed for 3 days at 4°C against distilled water, wire dried, and the immunizing antigen was 5.7 m
get g. Hereinafter, this antigen will be referred to as "antigen Ib and I. Antigen Ib has an average of 0.149 μmol of peptide 8 bound to Ascaris l1nill.

Production Example 2 5Il1g (5Il1g of peptide B obtained in Peptide Production Example 2)
, 995 mmol) and Ascaris extract protein 10 mg
was added to a 0.16M borate buffer (I]H=9.0) containing 0.13M sodium chloride, and B was added to this solution.
DB solution 3.35111 (I) was added and stirred at 4°C for 2 hours. The above BDBrFJ solution was prepared by adding 83.25 m0 of benzidine to a mixed solvent of 0.2N acid-resistant 2011Q and 3 m12 of dimedylbol amide (DMF), and cooling with water. Then, a solution of 1 to 87.03 mg of sodium nitrite to 87.03 mg of nitrous acid in 2 tablespoons of distilled water was gradually added, and the mixture was adjusted by stirring for 30 minutes.Then, the reaction mixture was heated to 4°C with distilled water for 3 days. Dialysis and freeze-drying yield 13.7 mg of the immunizing antigen.Hereinafter, this antigen will be referred to as "antigen ■".Antigen ■ contains an average of 0.269 μmol of peptide B per 1n+g of Ascaris.
It is a combination.

Production Example 3 ■ 8.7 mg of the immunizing antigen is obtained in the same manner as in Antigen Production Example 1 (2) using peptide C, which was used in Peptide Production Example 3, in place of peptide Δ. Hereinafter, this antigen will be referred to as “antigen 1”.
It's called 11aJ. Antigen 11[a has an average of 0.373 μmol of peptide C bound to 1 mole of Ascaris.

(2) Using peptide C obtained in Peptide Production Example 3 instead of Peptide B, immunogen 8.61 is obtained in the same manner as in Antigen Production Example 2 and li1. Below, we will refer to this antigen as 1 antigen 11
It's called 1bJ. Antigen mb has an average of 0.313 μmol of peptide C bound to 1 mg of Ascaris.

Production Example 4 ■ Using peptide D obtained in Peptide Production Example 4 instead of Peptide A, 8.6 mg of an immunizing antigen is obtained in the same manner as in Step 2 of Antigen Production Example 1 above. Hereinafter, this antigen will be referred to as [antigen I
It's called VaJ. Antigen IVa has an average of 0.269 μmol of peptide D bound to Ascaris 111 (+).

(2) Using Peptide D obtained in Peptide Production Example 4 instead of Peptide B, 8.5 mg of an immunizing antigen is obtained in the same manner as in Antigen Production Example 2 above. Hereinafter, this antigen will be referred to as [antigen IVI
It's called IJ. The antigen ``vb'' has an average of 0.224 μmol of peptide C bound to 1 mg of Ascaris.

Production of Antibodies> Production Example 1 Before and after dissolving 500 μg each of the antigens A and Ib obtained in Production Example 1 of Immunizing Antigen in 1.5% physiological saline, 1.5 mQ of Freund's auxiliary solution was added to the solution. The prepared suspension was applied to several New-7ealand eel
white rabbits, 2. 5-3.0k
o) subcutaneously administered 500 μg/body of antigen exposure for - times according to the procedure shown in the immunization schedule below,
Then, administer the same amount as the first dose three times every month thereafter. Seven days after the final administration, whole blood is collected from the test animal and centrifuged to obtain antiserum (antibody).

Antibody IIJ was collected from each rabbit administered with antigen Ia.
Antibody Δ” and “Antibody B”. Antibodies obtained from rabbits administered with antigen Ib were used as "antibody C" for each rabbit.
, "Antibody D" and "Antibody E".

<Immunization schedule> Period (weeks) Antigen inoculation 0 1st cohabitant species 2 2nd vaccination 4 3rd cohabitant species 6 4th cohabitant species 8 5th cohabitant species 10 6th cohabitant species Production example 2 Production example of immunizing antigen 2 -4 obtained 1 [antigen ■, ■a, 1 [1
immunization using IVa, IVa, and IVb, respectively, according to the same immunization schedule as in Antibody Production Example 1, whole blood was collected after the fifth cohabitant of the antigen, and the target antibody was obtained in the same manner.

Each antibody obtained is labeled with the antibody number shown in Table 1 below for each immunization antigen and rabbit used.

Table 1 - Production of labeled peptide> (1) Peptide 8 obtained in Peptide Production Example 1 is labeled using chloramine T as follows.

That is, 5μ0 of the above peptide in a 0.5M phosphate ms solution (
D H7,5> 10uQ to Na, (” 5(1(ca
rrier free N, E, N, ) Add 20 μQ of 1 milli-Koley's 0.5 molar phosphate buffer, then 20 μQ of chloramine T, 0.5 molar phosphate j!
! Add 20 μe of the solution. Stir for 25 seconds at room temperature to dissolve sodium metabiite (Na 2520s)
The reaction is terminated by adding 20 μQ of 0 μg of 0.5 M Phosphorus R salt buffer. Next, 10 μQ of a 10% cold butrium iodide aqueous solution was added to the reaction mixture, and the reaction mixture was diluted with the empty group of Sephadex G-25 (1,0 to 500 L elution).
, 0.2M ammonium acetate W1 containing i% BSA and 0.01% Na N3! Solution J, E) l-15,5) is used to remove peptide 8 labeled with 125■ in ff1FjL.

The radioactivity of the labeled peptide was 255 μC1/μg.

■ Peptide'! Peptide B obtained in R3fi examples 2-4
~D + by the same method as in the above labeled peptide production example.
251 to obtain labeled peptide-BD.

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

That is, the antibody was diluted with physiological saline at 10.1o2 and 10', respectively.
, 10 μM, 105......Diluted to 100μQ each, 1251tFIF! Peptide (1q labeled peptide above diluted to approximately 9500 cpm) 0.111 and 0.05 molar phosphate! 1 box liquid (pH=7.4) (0,2596B
SA, 10mM EDTA and 0.02% NaN) was added and incubated at 4°C for 24 hours. °C, 3
0 minutes, 3000 rpm) caused no reaction (15 times did not occur) +251c3! The binding rate (%) of the antibody to the 1251-labeled peptide at each dilution concentration is measured. Antibody 1 on the vertical axis
The binding rate with the 25 I-labeled peptide (96) and the dilution factor of the antibody are plotted on the horizontal axis, and the binding rate is plotted at each S degree. Determine the dilution ratio of the antibody at which binding is 50%, that is, the titer of the antibody. Table 2 below shows the results obtained for antibodies A to 0 obtained in the above antibody production examples.

Table 2 Antibody No. 5096 Binding rate Antibody 8 2800 〃 B 20000 n'C5000 〃 D 9000 11 E 37000 11 F 20000 〃 G 25000 I! H12000 〃 I 12700 1no J2750 〃 K 20000! r 1 260000, M 60000 〃 N 3700'' 0 1750000 As test samples for ATLA specificity test of antibodies, various concentrations of peptide Δ and the following ΔTL8 (ATLA-associated anNgen
) Use +J pull.

ATLA sample: MT-2 (Nature, 296.p770-771
(1981))'s culture! Add 30 mQ of physiological saline to 5 x 10' R111 cells, homogenize, centrifuge for 1 hour (105,000 x (+)), collect the supernatant, and reduce the protein amount by
The protein amount of 1g/4 (,, is measured by the color method using Tonein-TPJ, a total protein quantitative reagent made by Ogaki Assay Institute) is adjusted to v4 (hereinafter referred to as I'MT-TPJ).
2).

and 0.25% BSA and 5 mmol E as standard diluents.
0.05 Morlin containing DTA and 0.0296 NaN3! l1jl! Buffer (pl-17,4) is used.

In each test tube, add 0.211G of standard diluent and sample o.
, 1 mG to the antibody obtained in the antibody production example or B (final,
5000 times dilution for antibody, 25000 times for antibody B
@diluted) 0.1- and 125 corresponding to each of the above antibodies
■Labeled peptide (labeled peptide A obtained above diluted to about 110,000 cp) 0.1+
1 and incubated at 4°C for 72 hours,
Normal Porcinc 5
Add 1all of eru11>, then add 11W0.5- of activated carbon coated with dextran,
The antibody and +25 II
The conjugate (B) with the R-labeled peptide is separated from the unreacted (unbound) +25'l-labeled peptide (F), its radiation is counted, and the amount of (B) at the concentration and dilution rate of each test sample is determined. Earn your own rate (8%). The results obtained are shown in FIGS. 1 and 2.

Figure 1 shows the results when the antibody was used, and Figure 2 shows the results when the antibody was used. In each figure, the II axis is the binding percentage.
(B%/BoX100: However, BO is the test sample sr!I
(8%) when 0 is O), and the horizontal axis is the sample concentration (concentration to peptide and protein 1r!1 of MT-2 supernatant).
) is shown. Furthermore, in FIGS. 1 and 2, the curve tlA (a) represents peptide A, and the curve (b) represents MT-21 serum. From each figure, the antibodies that can be improved by the present invention are △
It can be seen that it has T LΔ reactivity.

In the above, a ΔTLΔ specificity test was conducted in the same manner using each of Antibodies C to O in place of Antibody A or B (labeled peptides were also replaced with those corresponding to each antibody), and each antibody was found to be similar. It was confirmed that it has ATLA reactivity of /j0

[Brief explanation of drawings]

FIGS. 1 and 2 are curves showing the affinity of the peptide Δ and ΔTLΔ samples with the antibody obtained according to the present invention, respectively. (that's all) /

Claims (1)

[Claims] A peptide represented by the formula H-Pro-Δsn-A rg-ΔSn-G +y-
Peptide represented by GIV-Gly-Tyr-OH, peptide represented by formula % and formula 1-Gly-Leu-Asp-Leu-Leu
-Phc -'rrp Glu-Gln-Gly-G
The method is characterized in that an immunogen consisting of a complex of a human leukemia virus aS peptide selected from the group consisting of peptides represented by lyLcu-■yrOH and a carrier is administered to a breast milk object, and the produced antibodies are collected. A method for producing human leukemia virus antibodies.