JPH0276598A - Anti ed-b antibody - Google Patents

Abstract

PURPOSE:To obtain an antibody having specific reactivity to carcinomatous FN by administering peptide, etc., having amino acid sequence expressed by a specific formula to a mammal as an immunogen and producing a desired antibody in vivo. CONSTITUTION:A peptide or protein having at least amino acid sequence expressed by formula I, formula II or formula III (A is alanine, D is aspartic acid, N is asparagine, I is isoleucine, L is leucine, G is glycine, P is proline, S is serine, E is glutamic acid, Q is glutamine, T is threonine, F is phenylalanine, V is valine, C is cysteine, Y is tyrosine) is administered to a mammal as an immunogen to produce the antibody capable of specifically recognizing ED-B and having specific reactivity to cancerous FN in vivo.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to fibronetatin (fibr'onectin).
n :FN), especially the above FN of the type contained in cancer tissue
Concerning novel antibodies against.

Conventional technology "N is a group of multifunctional glycoproteins that are widely distributed in various tissues and body fluids, and as a cell adhesion factor, it is involved in a variety of biological phenomena such as cell migration, differentiation, proliferation, and canceration. It is known that (Kiyotoshi Sekiguchi, Cell Engineering, Vol. 4)
．． No, 6. D485-497 (1985))
.

Furthermore, conventionally, the above-mentioned FN includes those present in the extracellular matrix (cell type FN: CFN) and FN in plasma (
It was known that there are two molecular species, plasma type FN: pFN), but the molecular diversity of FN is due to variable splicing of gene early transcripts (alternat
raw zirco was revealed by ive splicing). There are three regions that undergo such variable splicing, called ED-A, ED-B, and ll1cs, and it is thought that a large number of molecular species are generated by the combination of expression of the secondary regions.

On the other hand, the type of FN contained in cancer tissue (hereinafter referred to as “cancerous FN”)
(abbreviated as J) is F with abnormally high expression of ED-B.
N and F having ED-8 consisting of 91 amino acids
(Luciano Zardi, known as N.
et at,, The EMBO Journal, V
ol.

6. No, 8. p2337-2342 (1987))
.

Problems to be Solved by the Invention In this state, in order to advance research on the cancerous FN at the molecular level, it is possible to measure (detect) or purify the cancerous FN specific to its molecular species, and to diagnose cancer. There is a need in the industry for a means to make this possible.

The object of the present invention is to provide means that meet the above requirements. That is, the present invention provides an antibody that specifically recognizes the FD-B and therefore has specific reactivity with cancerous FN, and provides a peptide related to the ED-B, particularly for the production of the antibody. The purpose of the present invention is to provide a specific peptide that can serve as a tracer for measuring hapten and cancerous FN, and to provide a technique for measuring desired cancerous FN or ED-8 using the same. .

Means for Solving the Problems According to the present invention, there is provided an anti-FD-B antibody characterized by having an antigenic determinant in the amino acid sequence of the following formula (1), (2), or (3). .

(1):EGIPIFEDFVDSSVGY(2):Y
TV Ding GLEPGIDYDIS (3):NGGESAP
According to the present invention, the above formula (1), (
An FD-8 peptide represented by the amino acid sequence of 2) or (3) is provided.

In the present specification above and below, when amino acids, peptides, protecting groups, active groups, etc. are indicated by abbreviations, they shall comply with the IUPAC regulations or common symbols in the field. Examples of these are listed below. In addition, when an amino acid or the like may have optical isomers, one isomer is shown unless otherwise specified.

A; Alanine D; Asparaquinic acid N; Asparaquine I; Isoleucine L; Leucine G; Glycine P; Proline S; Serine [; Glutamine M Q; Glutamine; Threonine F; Phenylalanine ■; Valine C; Cystine Y; Tyrosine'ros; p-toluenesulfonyl group BOC; tertiary butoxycarbonyl group BZI: penzyl group) IBZI; p-methyl-xybenzyl group 0Bzl
; Benzyloxy group Cl2Bzl; 2,6-dichlorobenzyl group CI
-Z: 2-thalolbenzyloxycarbonyl group Br-Z
;2-Bromobenzyloxycarmonyl The above-mentioned specific anti-FD-B antibody provided by the basic invention is ED-B
An antibody that recognizes an antigenic site specific to FD-8
or FN having said region, i.e., having specific reactivity to cancerous "N", said CFN, 1m) FN of other molecular species such as FN.
It is distinguished from N by not exhibiting cross-reactivity.

Therefore, the antibody of the present invention can be used as a specific antibody in immunoassay for ED-B or cancerous FN,
This makes it possible to establish highly sensitive, highly accurate, and simple measuring methods. Furthermore, if the above measurement method can be established, it will be possible to provide cancer screening and diagnostic techniques, and it will also be extremely useful for basic research such as research and elucidation of carcinogenic mechanisms.

Furthermore, the above-mentioned antibody of the present invention can be used to obtain the above-mentioned ED-B or cancerous [N
useful for immunological purification of

In addition, the above-mentioned specific peptide (E
D-8 peptide) consists of an antigenic site specific to ED-B, and is therefore useful as a hapten for the production of the above-mentioned anti-FD-B antibody. It can also be effectively used as a body).

The method for producing the antibody of the present invention will be described in detail below.

The antibody of the present invention can be produced by a conventional method using a peptide or protein having at least the amino acid sequence represented by formulas (1), (2), and (3) above as an immunogen. For example, a method for producing desired antibodies in vivo by administering (immunizing) the above immunogen to a mammal, plasma cells (immune cells) of a mammal immunized with the above immunogen, and plasma cells of a mammal. It can be obtained by, for example, creating a hybridoma with tumor cells and selecting and culturing a clone that produces the desired antibody (monoclonal antibody).

In producing the antibodies of the present invention, the above-mentioned peptides or proteins used as immunogens are of the formulas (1), (2) and (
There is no particular limitation as long as it has at least the amino acid sequence represented by 3), and for example, cancerous FN prepared from cancer tissue, cancerous FN produced according to gene transfer technology, and FD- of these cancerous FNs. Any of the B region or 7 fragments thereof, a synthetic peptide having the above-mentioned specific amino acid sequence, etc. may be used. Among these, particularly preferred are those obtained by using the ED-8 peptide of the present invention as a hapten.

In the above method, there are no particular restrictions on the mammal to be immunized, but when producing the antibody of the present invention using a hybridoma, consideration should be given to compatibility with the plasmacytoma cells used for cell fusion. It is preferable to select
Generally, mice, rats, etc. are advantageously used.

In the above method for producing antibodies, immunization can be carried out by a conventional method, for example, by administering an immunogen to a mammal by intravenous, intradermal, subcutaneous, or intraperitoneal injection. More specifically, the immunogen is administered to the test animal several times every 2 to 14 days, if desired in combination with a conventional adjuvant 1, and the total dose is about 10 to 100 μq for mice, for example, at home. For rabbits, this can be done by adjusting the amount to about 0.2 to 2.0 mg. The antibody is collected by collecting blood from the immunized animal one to two weeks after the final administration, centrifuging the blood, and separating the serum. Furthermore, as the immune cells used in the production of the monoclonal antibody, it is preferable to use spleen cells extracted about 3 days after the final administration.

As the mammalian plasmacytoma cell as the other parent cell to be fused with the above-mentioned immune cell, there are various known mammalian plasmacytoma cells,
For example, p3 (p3/x63-Ag8) (Nature
, 256, 495-497 (1975)), p3-
U] (Current topic in Micro
obiology and Immunology
.

8'l, 1-7 (1978)), NS-1 (Eur.

J, I+nmuno1. ．． 6.511-519 (
1976) ), MPC-11 (Cell, 8.4
05-415 (1976)), 5P210 (Natsu
re, 276°269-270 (1978)), F
O(J.

Immunol, Methl, 35.1-21 (
1980)), X63.6.5.3. (J, I
mmunol,, 123°154.8-1550 (1
979)), 3194 [J, EXI), Med
,, 148. 313-323 (1978)), etc.
Rats [Puru R210 [Nature, 277.1]
31-133 (1979)) can be used.

The above-mentioned fusion reaction between immune cells and plasmacytoma cells can be carried out using known methods, such as the method of Milstein et al. (Method in Enzymology).
Vol. 73, I)l)3 (1981)).

More specifically, the above fusion reaction is carried out in a conventional medium in the presence of a conventional fusion promoter, such as polyethylene glycol (PEG), Sendai virus (HV, J), etc., and the medium also contains the fusion agent. Auxiliary agents such as dimethyl sulfoxide may be added as necessary to increase efficiency. The ratio of immune cells to plasmacytoma cells used is the same as in conventional methods; for example, about 1 to 10 times the amount of immune cells to plasmacytoma cells can be used. As a medium for the fusion reaction, various media commonly used for the proliferation of the above-mentioned plasmacytoma cells, such as RPMI-1640, can be used.
Examples include medium, MEM medium, and other media commonly used for this type of cell culture, and the secondary medium is usually fetal bovine serum (F
It is best to use serum replacement fluids such as e2). For fusion, a predetermined amount of the above immune cells and plasmacytoma cells are mixed well in the above medium, and a PEG solution preheated to about 37°C, for example, one with an average molecular weight of about 1000 to 6000, is added to a normal medium. This is done by adding and mixing at a concentration of 30 to 60 W/V%. Thereafter, desired hybridomas are formed by repeating the steps of sequentially adding an appropriate medium, centrifuging, and removing the supernatant.

The resulting desired hybridomas are isolated by culturing them in a conventional selection medium, such as HA Ding medium (a medium containing hypoxanthine, aminopterin, and thymidine). The culture in the HA medium may be carried out for a sufficient period of time, usually from several days to several weeks, to kill cells other than the target hybridoma (unfused cells, etc.). The hybridoma thus obtained is subjected to the search for the desired antibody and single cloning by the usual limiting dilution method.

Searching for target antibody-producing strains can be performed, for example, by ELISA method (Eng.
vall, E, ) Ieth, Enzymol,
70.419-439 (1980)), plaque method,
spot method, agglutination reaction method,
Various methods commonly used for detecting antibodies, such as the rlony) method and the radioimmunoassay (RIA) method [“Hybridoma method and monoclonal antibodies”, R& Co., Ltd.
Published by D Planning, pp. 30-53, March 5, 1981.
This search can be carried out according to the above-mentioned FD-B of the present invention.
The use of peptides is preferred.

The thus obtained hybridoma producing the desired monoclonal antibody of the present invention can be subcultured in a conventional medium and can be stored for a long period of time in liquid nitrogen.

Collection of the antibody of the present invention from the above hybridoma 1. The hybridoma is cultured according to a conventional method and obtained as a culture supernatant, or the hybridoma is administered to a compatible mammal and grown, and obtained as ascites. method etc. will be adopted. The former method is suitable for obtaining highly purified antibodies, and the latter method is suitable for mass production of antibodies.

Furthermore, the antibody obtained as described above can be further purified by conventional means such as salting out, gel filtration, and affinity chromatography.

In this way, the anti-ED-8 antibody of the present invention can be produced.

Hereinafter, the ED-8 peptide of the present invention and its use will be described in detail.

The ED-B peptide of the present invention can be easily produced by simple operations using readily available commercially available amino acids, for example, according to conventional chemical synthesis methods. The chemical synthesis method is, more specifically, a normal peptide synthesis method, i in vivo G,: har f
The peptides J Volume 1 (1
966) (Schr6der and Luhk
e-author, Hachicademic press, New Yo
rk, tJsA) or "Peptide Synthesis" [by Azumaya et al., Maruzen Co., Ltd. (1975)], for example, the azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCCL activity. Ester method (p-nitrophenyl ester method, N-hydroxysuccinimide ester method, cyan methyl ester method, etc.), method using Woodward reagent K, carbodiimidazole method, redox method, DCC/additive (HONB).

This can be carried out by the HO2C, HO3U) method, etc. In the above method, both the in-phase synthesis method and the liquid-phase synthesis method can be applied. Usually, the peptide of the present invention is synthesized in accordance with the general polypeptide synthesis method described above, for example, by sequentially adding 1 amino acid to the terminal amino acid.
It is produced by a so-called stepwise method in which amino acids are condensed one by one, or by a method in which several fragments are coupled together.

More specifically, for example, when employing the same phase synthesis method, a C-terminal amino acid (with a protected amino group) is first bonded to an insoluble carrier via its carboxyl group. The insoluble carrier is not particularly limited as long as it has binding properties with reactive carboxyl groups, such as chloromethyl resin,
Halogenomethyl resins such as bromomethyl resins, hydroxymethyl resins, phenol resins, tert-alkyloxycarbonylhydracitated resins, etc. can be used. Next, after removing the amine protecting group, amino group-protected amino acids are sequentially bonded according to the amino acid sequence of the target peptide through a condensation reaction with the reactive amino group and the reactive carboxyl group, and the entire sequence is synthesized step by step. After synthesis, the peptide of the present invention can be obtained by removing the peptide from the insoluble carrier.

Each amino acid having a side chain functional group in the above various methods,
For example, it is preferable to protect the side chain functional groups of Y, E, T, C, S, etc., using a conventional protecting group, and after the reaction is completed, the protecting group is removed. Furthermore, the functional groups involved in the reaction are usually activated. Each of these reaction methods is known, and the reagents used therein are also appropriately selected from known methods.

Examples of protecting groups for amino groups include benzyloxycarbonyl, BOC, tert-amyloxycarbonyl,
Examples include isobornyloxycarbonyl, ρ-methoxybenzyloxycarunthyloxysulfonyl, trifluoroacetyl, phthalyl, formyl, 0-nitrophenylsulfenyl, diphenyl small sulfinothioyl group, and the like.

Examples of carboxyl groups include alkyl esters (methyl, ethyl, propyl, butyl, tert-
alkyl esters such as butyl), BZI esters, ρ
Examples include groups that can form nitrobenzyl ester, MBZI ester, p-chlorobenzyl ester, benzhydryl ester tert to butyloxycar small nyl hydrazide, trityl hydrazide, and the like.

The hydroxyl groups of S and D can be protected, for example, by esterification or [j, etherification, but do not necessarily need to be protected. Groups suitable for this esterification include lower alkanoyl groups such as acetyl, aroyl groups such as benzoyl, and groups derived from carbonic acid such as benzoyloxycarbonyl and ethyloxycarbonyl. Examples of groups suitable for etherification include benzyl, tetrahydropyranyl, and tert-butyl groups.

As a protecting group for the hydroxyl group of Y, for example, BZI, C12B
Examples include zl, Br-Z, benzyloxycarbonyl, acetyl, TOS group, and the like.

As the protecting group for the thiol group of C, )IBz l, BZ
I, p-methylbenzyl group and the like.

The carboxyl group of E is protected by esterification with benzyl alcohol, methanol, ethanol, tert-butanol, etc.

Examples of activated carboxyl groups include the corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, active esters (pentachlorophenol, p-21
~lophenol, N-e droxysuccinimide, N-
Hydroxybenztriazole, N-hydroxy-5-
esters with norbornene-2,3-dicarfoximite, etc.).

In the above method, the condensation reaction (peptide bond formation reaction) between the reactive amino group and the reactive carboxyl group can be carried out in the presence of a solvent. Various solvents known to be usable for peptide bond formation, such as anhydrous or aqueous dimethylformamide (DMF), dimethyl sulfoxide (DMSO), pyridine, chloroform, dioxane, dichloromethane, and tetrahydrofuran (THF), can be used as the solvent.
, ethyl acetate, N-methylpyrrolidone, hexamethylphosphoric acid triamide (HMPA), or a mixed solvent thereof may be used. The ratio of the same raw material compounds to be used is not particularly limited, but usually an equimolar amount to 5 times the molar amount of one to the other,
Preferably, the amount is equimolar to 1.5 times the molar amount.

The reaction temperature is appropriately selected from the usual range used for peptide bond forming reactions, generally from about -40°C to about 60'C, preferably from about -20'C to about 40'C. The reaction time is generally about several minutes to 30 hours.

The mixed acid anhydride method is carried out using an alkylhalocarboxylic acid such as methyl chloroformate, methyl bromoformate, ethyl chloroformate, edyl bromoate, isobutyl chloroformate, etc. in a suitable solvent in the presence of a basic compound. . Examples of basic compounds include triethylamine, trimethylamine, pyridine, dimethylaniline, N-methylmorpholine, 1,5-diazabicycloC4,3,○]nonene-5 (DBN>, 1,5-diazadicyclo(5,4, .

O] Arous bases such as Lourensen-5 (DBU), 1,4-diazabicyclo[2,2,2)t-furan (DABCO), and inorganic bases such as potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc. can be used. As the solvent, various solvents commonly used in the mixed acid anhydride method, specifically halogenated hydrocarbons such as methylene chloride, chloroform, and dichloroethane, aromatic hydrocarbons such as benzene, toluene, and xylene, diethyl ether, THF, Ethers such as dimethoxyethane, methyl acetate, ethyl acetate, etc. (D esters, aprotic polar solvents such as DMF, DMSOSHMPA, etc.) can be used.

The reaction is usually carried out at -20 to 100°C, preferably -20 to 50°C.
The reaction time is generally 5 minutes to 10 hours, preferably 5 minutes to 2 hours.

In addition, in the azidation method, first the activated carboxyl group,
For example, this is carried out by reacting a carboxyl group activated with an alcohol such as methyl alcohol, ethyl alcohol, or benzyl alcohol with hydrazine hydrate in a suitable solvent. Examples of the solvent include dioxane and DMF.

DMSO or a mixed solvent thereof can be used.

The amount of hydrazine hydrate to be used is usually 5 to 20 times the molar amount, preferably 5 to 1 times the amount of activated carboxyl group.
It is preferable to use 0 times the molar amount. The reaction is usually below 50'C.
It is preferably carried out at -20 to 30'C. Thus, a compound in which the carboxyl group moiety is substituted with hydrazine (
hydrazine derivatives).

A compound in which the carboxyl group moiety is substituted with azide is produced by reacting the hydrazine derivative obtained above with a nitrite compound in a suitable solvent in the presence of an acid. The acid is usually hydrochloric acid, and the solvent is dioxane, D
MF, DMSO or a mixed solvent thereof, and nitrite compounds such as sodium nitrite, isoamyl nitrite,
Nitrosyl chloride and the like can be used respectively. Such a nitrite compound is generally used in an equimolar to 2-fold molar amount, preferably in an equimolar to 1.5-fold molar amount, relative to the hydrazine derivative. The reaction is usually -20-0'C1 preferably -20
The reaction is carried out at ~-10'C and generally completes in about 5 to 10 minutes.

The peptide bond forming reaction can also be carried out in the presence of a condensing agent such as dicyclohexylcarbodiimide (DCC), a carbodiimide reagent such as carbodiimidazole, and the like.

In each of the above reaction steps and in the final step, when it is necessary to remove the protecting group, this is carried out according to a conventional elimination reaction. Examples of such methods include hydrogenation using a catalyst such as palladium or palladium black, reductive methods such as reduction with metallic sodium in liquid ammonia, trifluoroacetic acid, hydrochloric acid, hydrogen fluoride, methanesulfonic acid, and hydrobromic acid. Examples include acidolysis using strong acids such as

Hydrogenation using the above catalyst can be carried out, for example, at a hydrogen pressure of 1 atm, 0
It can be carried out at -40°C. The amount of catalyst used is usually 100m
The amount is preferably about 1 g, and the reaction is generally completed in about 1 to 48 hours. The acidolysis is carried out without a solvent, usually at about 0 to 30'C, preferably about 0 to 20'C, for about 15 minutes to 1 hour. The amount of acid to be used is usually about 5 to 8 O (about 5 to 8) based on the raw material compound. When removing only the protecting group of the amino group in the acidolysis, use trifluoroacetic acid or hydrochloric acid as the acid. Further, the above reduction with metallic sodium in liquid ammonia is carried out using an amount of metal sodium to lithium such that the reaction solution is colored permanent blue for about 30 seconds to 10 minutes, and is usually heated at -40'C. ~-70'
It can be done at level C.

The peptide of the present invention produced as described above is isolated and purified from the reaction mixture by peptide separation means such as extraction, partitioning, reversed phase high performance liquid chromatography, etc.

The thus obtained peptide of the present invention is the anti-ED-
It is useful for the production of immunogens that can be used for the production of 8 antibodies.

Such an immunogen can be obtained by binding the above-mentioned peptide of the present invention as a hapten to a conventional carrier protein, either directly or via an appropriate spacer, and this binding reaction can be carried out using a conventional hapten-carrier binding reagent. It can be carried out by common methods.

In the above, as the spacer, any of various compounds having at least two or more functional groups for the above-mentioned binding in one molecule can be used, and this includes a part of the hapten-carrier binding reagent. Suitable spacers include:
Mention may be made of the usual amino acids such as cysteine, lysine, glycine, in particular cis9. A secondary spacer can be optionally attached to the N-terminus and/or C-terminus of the peptide of the present invention, and therefore, the present invention also provides a peptide that further has such an amino acid.

The reaction between the amino acid as the spacer and the hapten can be carried out in the same manner as in the production of the peptide of the present invention described above.

In addition, in the above method for producing an immunogen, as the carrier protein used, a wide range of high-molecular natural or synthetic proteins commonly used in the production of antigens can be used. Examples of the carrier include horse serum albumin, bovine serum albumin,
Animal serum albumins such as rabbit serum albumin, human serum albumin, and sheep serum albumin; animal serum globulins such as horse serum globulin, bovine serum globulin, rabbit serum globulin, human serum globulin, and sheep serum globulin; equine thyroglobulin; Animal thyroglobulins such as raw thyroglobulin, rabbit thyroglobulin, human thyroglobulin, and ovine thyroglobulin; animal hemoglobins such as equine hemoglobin, bovine hemoglobin, rabbit hemoglobin, human hemoglobin, and ovine hemoglobin; keyhole limpet hemocyanin ( Animal hemocyanins such as KLH); proteins extracted from roundworms (Ascar
Squirrel extract, JP 56-16414@Publication, J, Im
mtJn6, 111, 260-268 (1973),
J.Immun., 122.302-308 (1
979), J. Immun., 98.893-900
(1967) and Am, J. Physiol, 1
99.575-578 (1960) or further purified products thereof>; examples include polylysine, polyglutamic acid, lysine-glutamic acid copolymers, copolymers containing lysine or ornithine, and the like.

As the hapten-carrier binding reagent, a wide variety of those commonly used for preparing antigens can be used.

Specifically, tyrosine, histidine, and tryptophan are cross-linked, for example, bisdiazotized benzidine (
Diazonium compounds such as BDB), bisdiazotized-3゜3'-dianisidine (BDD); Aliphatic dialdehydes such as aldehydes; crosslinking diol groups and thiol groups, for example N.

N'-〇-phenylene dimaleimide, N, N'-m
-Cimalimide compounds such as phenylene dimaleimide; those that crosslink amino groups and thiol groups, such as metamaleimidobenzoyl-N-didroxysuccinimide ester, 4-(maleimidomethyl)-cyclohexane-
Maleimidocarboxyl-N-hydroxysuccinimide esters such as 1-carboxyl-N'-hydroxysuccinimide ester and N-succinimidyl-3-(2-pyridyldicyclo)propione-1-(SPDP); amino group and carboxyl group A reagent used in a normal peptide bond forming reaction to form an amide bond with, for example, N,
N-dicyclohexylcal diimide (DCC>, N
-ethyl-N'-dimethylaminocarbodiimide, 1-
Ethyl-3-diisopropylaminocarposiimide, 1
Examples include dehydration condensation agents such as carbodiimides such as -cyclohexyl-3-(2-molpolinyl-4-ethyl)carbodiimide. As the hapten-carrier binding reagent, a combination of a diazonium arylcarboxylic acid such as p-diazonium phenylacetic acid and a conventional peptide bond forming reaction reagent, such as the dehydration condensation agent described above, can also be used.

The immunogen production reaction using the above-mentioned hapten, carrier protein, hapten-carrier binding reagent, spacer, etc. can be carried out according to a conventional method, and is generally carried out in an aqueous solution or a conventional buffer solution with a pl of about 5 to 10, preferably - 28=1) The reaction is carried out in a buffer solution of about H6 to 9, at a temperature of O to 40'C1, preferably near the lowest temperature. The reaction is usually completed in about 2 to 5 hours.

In the above, the ratio of the hapten, the hapten-carrier binding reagent, and the carrier to be used can be determined as appropriate, but it is usually about 0.5 to 5 times the weight of the carrier to the hapten, preferably 1 to 2 times the weight of the carrier.
It is preferable to use about twice the weight and about 1 to 30 times the mole of the hapten-carrier binding reagent. As described above, a desired immunizing antigen consisting of a hapten-carrier complex in which a carrier and a hapten are bound via a spacer or directly can be obtained.

After the completion of the reaction, the antigen is removed using conventional methods, such as dialysis,
It can be easily isolated and purified by gel filtration, fractional precipitation, etc.

Furthermore, the ED-8 peptide of the present invention has 1251.
Radioactive substances such as 131 guns, peroxidase (POX)
, chymotrypsinogen, procalphochydipeptidase, glyceraldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, D-Nase, P-Na
se, β-galactosidase, glucose-6-phA
By introducing various enzyme reagents such as sulfate dehydrogenase and ornithine decal small xylase, it can be used as a tracer for radioimmunoassay (RIA) or enzyme immunoassay (FIA). 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 chloramine (W, M, HIJnte).
rand F, C, GreenWOOd; 1atu
re, 194.

495 (1962), Biochem, J., 8
9. 'I 44° (1963)], and the introduction of the enzyme reagent is carried out using conventional coupling methods, such as the method of Erlanger (B, F, Erlanger) et al.
Acta.

Endocrinol, 5upp1. , 168.206
(1972) ) and force roll (14, 11, ar
ol) et al.'s method (Proc.

Natl, Acad, Sci., USA, 57. 7
13 (1967)) and the like.

In the above, among the peptides of the present invention, a peptide that does not contain tyrosine in its amino acid sequence can optionally have tyrosine attached to its N-terminus and/or C-terminus, for example, to facilitate the above-mentioned iodination. The present invention also provides the above BiD-B peptide to which such tyrosine is bound.

EXAMPLES Below, in order to explain the present invention in more detail, examples of producing the peptide of the present invention, producing an immunogen from the peptide, and producing an antibody from the antigen will be given, but the present invention is not limited thereto. .

<Production of peptide> Production example 1 Production of the peptide of the present invention represented by formula (1) Peptide synthesis was performed using a peptide synthesizer model 430A manufactured by Applied Biosystems.

Using BO Kano Kushistein PAM resin (1% divinylbenzene cross-linked polystyrene, 0.559 mmol/Q) as a starting material resin, the following 3oc-amino acid derivatives were introduced sequentially from the C-terminal side, and the activator vessel shown in Table 1 below was prepared. A peptide synthesis reaction was carried out according to a program consisting of a concentrator vessel, a concentrator vessel, and a reaction vessel.The condensation reaction was carried out by the symmetric acid anhydride method.

Boc-L-E (OBzl) Boc-L-G Boc-L-I・1/2H20 13oc-L-P 3oc-L-F Boc-L-D (OBzl) 3oc-L-V [3oc-L- 3 Boc-LY (Br-Z) The peptide chain was sequentially extended according to the above program. After the final condensation reaction, the protected peptide resin was treated with HF for 1 hour with O'C in the presence of anisole and ethyl methyl sulfide to deprotect the group and remove the resin, and then washed alternately with ethyl acetate and chloroform. , dried, extracted with 2M acetic acid, and lyophilized to obtain the target peptide.

Yield: Protected peptide resin 1.7g Crude peptide approximately 400mg Next, the crude peptide was purified by HPLC under the following conditions.

Column: YMC pack D-ODS-5 (2,1x25
cm) Eluent: 0.1% TFA 10.075% TFA.

99.9% CH3CN 70/30 → 40/60 (1 hour) Detection: 206 nm Flow rate: 5 + nQ/min to obtain 250 mQ of purified peptide (hereinafter referred to as "Peptide B-1") (yield 63%, (from crude peptide)
.

The physical properties of the obtained peptide B-1 were as follows.

Analysis by HPLC: 5ynchropackRP 8 (4, 1x 250
HPLC (eluent: 0.1% FA10
．． 075%TFA-99.9%CH30N=10010
→40/60.35 minutes, detection 206nm, flow rate 2.0
mQ/min) results are shown in FIG.

In the figure, the vertical axis is the absorbance at 206 nm (OD> (shown as a solid line) and the gradient of CH3ON (O → 60%).
(not shown by a broken line), and the horizontal axis shows elution time (minutes).

Amino acid analysis values: Amino acid analysis results analyzed using Hitachi L-8500 are shown in Table 2.

Table 2 The above table shows the value of 1.00 mol. In addition, the numerical value in parentheses of the analytical value indicates an integer ratio.

The above amino acid analysis values are the results measured after hydrolysis with 6N-hydrochloric acid (110°C, 124 hours).

Production Example 2 In the same manner as Production Example 1, the peptide of the present invention represented by the formula (2) (hereinafter referred to as "peptide B-2") was produced.

The HPLC analysis pattern is shown in FIG. 2 in the same manner as FIG. 1.

The amino acid analysis values are shown in Table 3 below.

Amino acid analysis value: (Analyzed using Hitachi Model 1-8500) The table above (the value of yield is expressed as 2 moles), and the values in parentheses (y) are the same as above.

<Production of antigen (immunogen)> Production example 1 Peptide synthesis Production example 1
It was dissolved in 0.1M sodium phosphate buffer (pH 7.5
> 1.0 m2, and
1 mQ of the above phosphoric acid buffer containing LH was added and mixed.Next, 1 mQ of 20mM glutaraldehyde solution was slowly added dropwise to this, and the mixture was stirred at the lowest temperature for 30 minutes to react. .

The resulting reaction product was dialyzed against phosphate buffered saline (PBS) at 4°C for 2 days to remove the desired antigen (antigen-
1> was obtained.

Production Example 2 Peptide Synthesis Using peptide B-2 obtained in Production Example 2, a desired antigen (antigen-2) was obtained in the same manner as in the above Production Example.

<Production of antibodies of the present invention> Production example 1 Production of polyclonal antibodies Antigens obtained in antigen production examples (1 of antigen-1 and antigen-2:
After diluting >0.5 mQ of 1 mixture with 1.0 mQ of PBS, 1.5 m12 of adjuvant (complete
Freund's adjuvant) was mixed and emulsified, and 1.011Q of this was placed on the backs of two rabbits for 30 minutes.
It was administered intradermally at ~4011N sites. Further booster immunizations were performed in the same manner three times at two-week intervals. Thus, it was confirmed by immunoplotting that both test animals produced the desired antibodies.

One week after the final immunization, whole blood was collected from the test animals, left overnight at 4°C, and then centrifuged to obtain the desired antibody (antiserum) of the present invention.

Production Example 2 Monoclonal antibody production Antigens obtained in the antigen production example (1 of antigen-1 and antigen-2:
After diluting 0.05 mQ of 1 mixture with 0.5 mQ of PBS, 0.5 m2 of adjuvant (complete mixture) was diluted with 0.5 mQ of PBS.
Freund's adjuvant) was mixed and emulsified, and this was subcutaneously administered in 5 to 10 locations on the backs of 5 BALB/c mice (8 weeks old) at 0.2 m each. Booster immunization was performed four times in the same manner. Three days after the final administration, spleen cells were removed from the spleen of each mouse, and the red blood cells present in the cells were incubated with 0.83% ammonium chloride solution at 4°C. The cells obtained above were treated as sensitized lymphocytes for 1 to 2 minutes to melt and remove them.
Washed several times with RPMI-1640 medium.

On the other hand, HGPRT-deficient BALB/c-derived P3U1 cells were placed in RPMI-1640 medium containing 15% FC8.
The cells were subcultured in a medium containing 100 μM of 8-azaguanine, and used as myeloma cells.

5 x 107 myeloma cells were mixed with 5 x 108 sensitized cells prepared above, and the resulting cell mixture was mixed with 5 x 108 sensitized cells.
After centrifugation at 00Xg, 35% polyethylene glycol]-
Cell fusion was performed by adding 4 mQ of Lu 1500 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.).

Hybridomas that grow in HA medium are transferred to BALB
/c mouse thymocytes were used as feeder cells to perform the limiting dilution method (1'1ethOd in Enzymol
oqy, 73.3 (1981)).

The search for target antibody-producing clones by ELISA was carried out using as an indicator the reactivity with cell type FN purified from the culture supernatant of WI-38VA13, a human normal fibroblast cell WI-38 exacerbated with tumor virus SV40. The above cloning was carried out four times while confirming that there was no reactivity with type FN, and the desired monoclonal antibody was obtained.

One of the obtained hybridomas producing the desired monoclonal antibody is designated as 10AI-CF525J. This item was sent to the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Microbial Industry Research Institute (Kikoken).
It has been deposited under the designation AL-CF525j, and its deposit number is [Kikoken Bacteria No. 10052 (FFRM P
-10052)".

Antibody 10AL-C of the present invention produced by the above hybridoma
The subclass of F525j was determined by the Ophthalony method.

As a result, the subclass of the above antibody was c+G2a.

<Reactivity of various antibodies of the present invention to N> ■ The reactivity of the antibodies of the present invention (antiserum) obtained in the above antibody production example 1 to various FNs was investigated as follows.

As a FN standard (1) Plasma type FN purified from human plasma
(P), (2) cell type FN purified from the culture supernatant of normal human fibroblast cell WI-38 (nC), (3) same W
Cell W made from I-38 that became malignant with tumor virus SV40
Cell type FN purified from the culture supernatant of I-38VA13
(tc) and (4) Fetal tissue type F purified from human placenta
Four types of N (f) were used.

These FNs were analyzed in 5D using a 6% polyacrylamide gel.
S-gel electrophoresis was performed, and the separated proteins were further transferred onto a nitrocellulose membrane by electroblotting.

First of all, this is a 77 strike green (Fast Green).
) was used for protein staining and photographs were taken.

Separately, the nitrocellulose membrane was reacted with the antibody of the present invention diluted 1000 times with PBS containing 2% bovine serum albumin (BSΔ) for 2 hours at room temperature, and then 0.1% B
Washed three times with PBS containing SA, then washed with 2
The reaction was carried out for 30 minutes in PBS containing %BSΔ. Set this to 0
．． After washing three times with PBS containing 1% BSA, it was air-dried and placed in close contact with X-ray film to obtain an autoradiogram.

The above results are shown in FIG. The left panel of Figure 3 is
This shows the results after the above fast green staining,
The right panel of FIG. 3 shows the results of the autoradiogram described above. Furthermore, each lane in the figure indicates each of the FN specimens described above.

From the figure or panel, it can be seen that approximately equal amounts of each FN were transferred to the nitrocellulose membrane. Furthermore, from the right panel of the figure, the antibody of the present invention does not react with plasma type "N(p)", cell type FN (nc; and ↑C) and fetal tissue type FN (
It is clear that it reacts only with f). Furthermore, the antibody of the present invention binds more strongly to FN produced by virus-induced cells (↑C) than to FN produced by normal fibroblasts (nc>) and fetal tissue type FN (f). This indicates that the use of the antibody of the present invention can reduce cancerous FN.
It is supported that FNs can be distinguished from other FNs.

■ In addition, various F
The reactivity to N was investigated as follows.

That is, FN purified from human plasma and WI-38VA1
ELI was performed using a 96-well plastic plate in which cell type FN purified from the culture supernatant of 3.
The reactivity of the antibodies of the present invention to each of these FNs was investigated by the SA method. The antibody was used by diluting the culture supernatant of rOAL-CF525J with RPMI-1640 medium containing 15% FC3.

The above results are shown in FIG.

In the figure, the vertical axis shows the OD at 492 nm, and the horizontal axis shows the dilution factor of the antibody. (1) is the result for cell type FN, and (2) is the result for plasma type FN.

From the figure, it is clear that the antibody of the present invention does not react with plasma type FN, but reacts with cell type FN (tc) in a dose-dependent manner.

Next, in the same manner as in the above ①, 4 to 15% of the 4 types of FN were added.
% gradient polyacrylamide gel and transferred to a nitrocellulose membrane by electroblotting.
The reactivity with 25 was investigated. The antibody of the present invention was used by diluting the culture supernatant 50 times with PBS containing 2% BSA.

Other conditions were the same as those described in (2) above.

The results of the above test are the same as those shown in Figure 3, and from this, the antibody OAL of the present invention, -CF525, has the same reactivity as the polyclonal antibody of the present invention obtained from domestic rabbits shown in (2) above. It became clear that there was.

[Brief explanation of the drawing]

Figure 1 shows the peptide ED obtained in Production Example 1 of the peptide of the present invention.
A graph showing the results of HPLC analysis of Bi, Figure 2 is the HPLC of peptide EDB-2 obtained in Production Example 2 of the peptide of the present invention.
A graph showing the results of C analysis, FIG. 3 shows the reactivity of the antibody obtained in Production Example 1 of the antibody of the present invention to various FN
A photograph in place of a drawing showing the results of GE-8DS gel electrophoresis and autoradiogram, and FIG. 4 shows the results of examining the reactivity of the antibody of the present invention obtained in antibody production example 2 with various FNs. This is a graph showing. (that's all)

Claims (1)

[Scope of Claims] 1. An anti-ED-B antibody characterized by having an antigenic determinant in the amino acid sequence of the following formula (1), (2), or (3). (1):EGIPIFEDFVDSSVGY(2):Y
TVTGLEPGIDYDIS (3):NGGESAPTTLTQQT