JPH04169195A - Anti-ed-b monoclonal antibody - Google Patents

Abstract

NEW MATERIAL:The subject antibody recognizing the ED-B amino acid sequence of formula. USE:Immunoassay of carcinomatous fibronectin. PREPARATION:The antibody can be produced by ordinary process using an immunogen consisting of a fused protein of the 91 amino acids of the ED-B region of formula and protein A.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to anti-ED-B monoclonal antibodies, more specifically fibronectin (F
N) relates to novel monoclonal antibodies against the above-mentioned FN, especially of the type contained in cancer tissues.

Conventional technology FN was first reported as a plasma protein by Morrison et al. in 1948 [Morriso et al.
n, P, R, et al, J, Am, Che
m, Soc,.

70, 3103 (194B)], a group of multifunctional glycoproteins that are widely distributed in various tissues and body fluids, and are involved in a variety of biological phenomena such as cell migration, differentiation, proliferation, and canceration as cell adhesion factors. [Kiyotoshi Sekiguchi, Cell Engineering, 4 (6), 485-497 (19
85)].

Conventionally, there are two molecular species of FN: FN synthesized in the liver and present in the blood is called plasma FN (pFN), and FN present on the surface of cultured cells and in the culture medium is cellular FN (pFN). cFN), but the molecular diversity of these FNs is due to variable splicing of gene early transcripts (cFN).
It has been clarified that this occurs due to (alternative splicing). Regions that undergo such variable splicing include ED-A, ED-B, and cs
It is thought that there are three regions called ``molecular molecule'', and a large number of molecular species are generated depending on the combination of expression of the secondary regions.

On the other hand, the type of FN contained in cancer tissue (hereinafter [cancerous FN
J) is an FN with abnormally high expression of the ED-B region, and is known as an FN with an ED-B region consisting of 91 amino acids.
rdi, et al,, The EMBOJour
nal, 61. (8), 2337-23
42 (1987) Ko.

Under such current circumstances, in order to advance research on the cancerous FN at the molecular level, and to enable specific measurement (detection) and purification of its molecular species, and by extension, to enable diagnosis of cancer, there is a need. It is demanded in this industry.

An object of the present invention is to provide means that meet the above requirements. That is, the present invention specifically recognizes ED-B,
Therefore, it is desirable to provide a monoclonal antibody with reactive specificity for cancerous FN, to identify a peptide related to ED-B, which can in particular serve as an immunogen for the production of said monoclonal antibody and as a tracer for the determination of cancerous FN. peptides, and further utilize the same to provide the desired cancerous FN.
Another object of the present invention is to provide a technique for measuring ED-B not only in a conventional solid phase system but also in a liquid phase system.

Means for Solving the Problems According to the present invention, there is provided an anti-ED-B monoclonal antibody characterized by recognizing the amino acid sequence of ED-B represented by the following formula (1).

Formula (1): % Formula % Further, according to the present invention, a peptide consisting of a fusion protein of 91 amino acids of the ED-B region represented by the amino acid sequence of the above formula (1) and protein A is provided.

In the above and following specification, when amino acids, peptides, protecting groups, active groups, etc. are indicated by abbreviations, they shall comply with the I UPAC regulations or common symbols in the field. The same applies to the representation of nucleic acids in base sequences.

The above-mentioned specific anti-ED-B monoclonal antibody provided by the present invention is an antibody that specifically recognizes EI)-B, and has reaction specificity to ED-B or FN having the region, that is, cancerous FN. It is characterized by having.

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 antibody of the present invention is useful for immunological purification of the above-mentioned ED-B or cancerous FN by, for example, affinity chromatography.

In addition, the above-mentioned specific peptide (E
D-B protein A fusion peptide) is the anti-ED of the present invention.
-B It is useful as an immunogen for producing antibody, and can also be effectively used as a tracer (label) in the above measurement method.

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 according to the boat fishing method using a fusion protein of 91 amino acids of the ED-B region represented by the above formula (1) and protein A as an immunogen.
Hanfland, P., Chem, Phys.

Lipids, 15.105 (1975): H
anfland, P., Chem.

Phys, Lipids, 10.201 (197
6): Koscielak.

J., Eur, J., Biochem.
Schiff, 214 (1978).

The above-mentioned ED-B region is known and its gene has also been determined.

More specifically, in the above method, for example, a fusion cell (hybridoma) of a mammalian plasma cell (immune cell) immunized with the above immunogen and a mammalian plasmacytoma cell is created, and from this, FN ED- This is carried out by selecting a clone that produces the desired antibody (monoclonal antibody) that recognizes the B region and culturing the clone.

The antibody of the present invention may be a crude antibody solution obtained by the above method, that is, the culture supernatant of an antibody-producing hybridoma or mouse ascites as it is. It may be purified by affinity chromatography or the like.

In the production of the antibody of the present invention, the fusion protein of the 91 amino acids of the ED-B region of FN and protein A used as an immunogen is particularly suitable as long as it has at least the amino acid sequence represented by the above formula (1). There are no limitations, and examples include cancerous FN prepared from cancer tissue, cancerous FN produced according to genetic recombination technology, the ED-B region of these cancerous FNs or fragments thereof, and synthetic peptides having the above-mentioned specific amino acid sequence. A fusion protein of any of the above and protein A may be used. Among these, particularly preferred are those obtained by using the 91 amino acids of the ED-B region of the present invention as a hapten.

The above fusion protein of EI)-B region 91 amino acids and protein A can be produced by genetic engineering techniques, more preferably using an established cell line of cancerous FN having the ED-B region of FN. .

The details are as follows.

That is, first, a cultured and established cell line that produces cancerous FN, such as normal diploid fibroblast Wr-38 typically isolated from human fetal lung tissue, is transformed (carcinomatized) with tumor virus SV40. Wr-38VA1, an established cell line obtained by
From 3 cells, the guanidine thiocyanate method [Chirg
Win, J. M. et al.

Biochemis, try, 18.5294-52
99 (1979)], this R
From NA to poly(A”) on oligo dT cellulose column
) RNA was screened, followed by the method of Kawasaki and Wong (
Kawasaki and Wang, PCR Tech
nology, H.A. Erlich, ed.

5tockton Press, New York,
p89-98 (1989)] l: Therefore, the polymerase chain reaccidine method (hereinafter referred to as rP
5aiki, R.

K., et al., 5science, 230.
1350-1354 (1985)) using ED-B
Synthesize cDNA encoding the region.

That is, after synthesizing single-stranded cDNA with reverse transcriptase using a random hexamer as a primer, 5'-CA
GAGCTCCTGCACTTTTGA-3' upstream primer, 3'-TGTGACTGTGTTGTTTT
Sac I encoding the ED-B region on FN cDNA was obtained by PCR using GCC-5° as a downstream primer.
- Pvu II region can be amplified. The two primers used here are not particularly limited to the above nucleotide sequences, and the target Sac I or Pvu II
Any item that includes the site may be used. After cutting the double-stranded c DNA obtained above with Sac I and Pvu II, a plasmid containing F N c DNA was obtained.
'5 [K, Sekiguchi et al.
.

Biochemistry, 25.4936-494
1 (1986) FN cDN excised from Ko, 6
．． (7) Together with the Pve II-Acc I fragment, plasmid pGEM4 [Sac
I-Acc 1 site, FN(7)ED-B
cDNA clone (pG
EMBl) can be obtained.

Next, the cDNA encoding the region containing ED-B was recovered from the above pGEMB1 as an Eco RI-Pst I fragment, and this was inserted into the protein A gene fusion vector p
RrT2T [Eco RI manufactured by Pharmacia Co., Ltd.]
Insert into the Pst I site and insert the desired protein A and ED.
An expression vector pPAB1 for the fusion protein with -B is obtained.

Transformation of a host with the above expression vector can be carried out using, for example, Escherichia coli N4830 [obtained from Pharmacia] having the λCr857 temperature-sensitive repressor as a host cell using the calcium phosphate method [D].

Hanahan, D. M., Glover, ed.
,, DNA cloning.

vol, 1. p109-135. IRL Pres.
After culturing the thus obtained transformant in LB medium, the method of Hanahan and Meselsono [f(anaha
n, D., and Meselson, M., G.
en.

By cloning, the desired protein A.
ED-B fusion protein positive clones can be obtained.

Production of the target fusion protein is carried out after isolating the above positive clone.
This can be carried out by culturing and applying heat induction, and the resulting protein can be released and recovered from the bacterial cells by ultrasonic disruption, and can be purified by chromatography using an immunoglobulin solubilization column. A purified desired immunogen is thus obtained.

In the above method, the ED-B gene is divided into a Sac I-Pvu II fragment encoding the ED-B region and a sono-downstream (r) Pvu II-Acc I fragment, which are cloned into the pGEM4 vector. However, there is no particular need for this; for example, from the beginning Sac I - Acc
The I fragment can also be amplified by PCR and used.

Furthermore, the above gene was synthesized by the phosphite triester method [N
It is also possible to carry out total synthesis by chemically synthesizing nucleic acids according to conventional methods such as those described in J. Ature, 310.105 (1984).

In the production of the monoclonal antibody of the present invention, the mammal to be immunized with the immunogen, that is, the fusion protein of protein A and ED-B region, is not particularly limited, but the plasmacytoma cells used for cell fusion and It is preferable that the animal be selected in consideration of the compatibility of the animal, and mice, rats, etc. are generally advantageously used.

Immunization is carried out by a general method, for example, by intravenously administering the above-mentioned immunogen or an immunizing antigen bound to a carrier (heterologous protein with high antigenicity) using an appropriate binding reagent as described below, to the feeder animal.
It can be administered by intracranial, subcutaneous, or intraperitoneal injection.

In the production of the above-mentioned immunizing antigen, a wide range of high-molecular natural or synthetic proteins commonly used in the production of antigens can be used as carriers.

Examples of the carrier include animal serum albumins such as horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, and sheep serum albumin; horse serum globulin, bovine serum globulin, rabbit serum globulin, human serum globulin, and sheep serum. Animal serum globulin such as globulin; animal thyroglobulin such as equine thyroglobulin, bovine thyroglobulin, rabbit thyroglobulin, human thyroglobulin, ovine thyroglobulin; equine hemoglobin, bovine hemoglobin, rabbit hemoglobin, human hemoglobin, ovine hemoglobin Animal hemoglobins such as keyhole limpet hemocyanin (KLH); Proteins extracted from roundworms (Ascaris extract, JP-A-56-16414, J
, Immun, 260-268 (1973)
J. Immun., 122.302-308 (197
9), J. Immun., 98.893-900 (
1967) and Am.

J, Physiol, 575-578
(1960) or further purified versions of these); examples include polylysine, polyglutamic acid, lysine-glutamic acid copolymers, and copolymers containing lysine or ornithine.

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; for example, N, which crosslinks thiol groups.

N'-〇-phenylene dimaleimide, N, N'-
simaleimide compounds such as m-phenylene dimaleimide;
Cross-linking amino groups and thiol groups, such as metamaleimidobenzoyl-N-hydroxysuccinimide ester, 4-(maleimidomethyl)-cyclohexane-1-carboxyl-N'-hydroxysuccinimide ester, N-succinimidyl-3-(2 -Maleimidocarboxyl-N-hydroxysuccinimide esters such as -pyridyldicyclo)propionate (SPDP); reagents used in normal peptide bond-forming reactions that form amide bonds between amino groups and carboxyl groups, such as N,
N-dicyclohexylcarbodiimide (DCC), N
-ethyl-N'-dimethylaminocarbodiimide, 1-
Ethyl-3-diisopropylaminocarbodiimide, ■
Examples include dehydration condensation agents such as carbodiimides such as -cyclohexyl-3-(2-morpholinyl-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 production reaction of the immunizing antigen using the above-mentioned hapten, carrier protein, hapten-carrier binding reagent, spacer, etc. can be carried out by a conventional method, and is generally performed using an aqueous solution or p) (5 to 10
The reaction is carried out in a normal buffer solution having a pH of about 6 to 9, preferably at about 0 to 40°C, preferably around room 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. Through the above process, 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.

The antigen obtained after the reaction is processed according to conventional methods, such as dialysis,
It can be easily isolated and purified by gel filtration, fractional precipitation, etc.

More specifically, the immunization is carried out by diluting the immunogen to an appropriate concentration with a phosphate buffer solution containing physiological saline (PBS), physiological saline, etc., and using it together with a usual adjuvant if desired. The test animals were administered several times every 2 to 14 days, and the total dose was
For example, about 10 to 100 μg for mice, and about 0.0 μg for rabbits. 2-2. This can be done by making it about 0 ■. As the adjuvant, pertussis vaccine, complete Freund's adjuvant, alum, etc. can be used.

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.

As the immune cells used in the production of the monoclonal antibody, it is preferable to use lung 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/X63-Ag3 (X6 3) [N
ature, 256. 495-497 (19
75') Ko, p 3/X63 Ag8.
Ul (P3U1) [CurrentTopics
in Microbiology and Immunochemistry
nology, 81°1-7 (197B)],
P3/NS I-1-Agl-1-A 3-1)
[Eur, J. Immunol, 6.511
-519 (1976)], S p 210-A g
14 (S p 210) [Nature, 276,
269-270 (1978) ], F O [J.

Immunol, Meth, 35.1-21 (
1980) and 210 in rats. RCY3.
Ag1.2.3゜(Y3) [Nature, 27
7, 131 (1979), etc. 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. [Methocl fn Enzymology,
73.3 (1981) et al. More specifically, the above fusion reaction is carried out in a normal medium in the presence of a normal fusion promoter, such as polyethylene glycol (PEG), Sendai virus (TVJ), etc. An auxiliary agent such as dimethyl sulfoxide may be added as necessary to increase the Further, a method using electrical processing (electrofusion) or the like may be appropriately employed. The ratio of immune cells to plasmacytoma cells used is the same as in conventional methods; for example, the ratio of immune cells to plasmacytoma cells is about 1 to 10 times that of plasmacytoma cells.

As a medium for the fusion reaction, various media commonly used for the proliferation of the above-mentioned plasmacytoma cells, such as RPMI-164, can be used.
Examples include 0 medium, MEM medium, and other media commonly used for this type of cell culture.
It is best to remove serum replacement fluids such as FCS).

For cell fusion, predetermined amounts of the above immune cells and plasmacytoma cells are thoroughly mixed in the above medium, and then pre-warmed to about 37°C.
This is carried out by adding an EG solution, for example, one having an average molecular weight of about 1,000 to 6,000, to a normal medium at a concentration of about 30 to 5 Qw/v% and mixing. 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 HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT 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.

Search for target antibody-producing strains can be performed, for example, by the ELTSA method [Engvall, E., Meth, Enzym et al.
ol,, 70.419-439 (1980)],
Plaque method, spot method, agglutination reaction method, Octagney
Various methods commonly used for detecting antibodies, such as (Ouchterlony) method and radioimmunoassay (RrA) method [``Hybridoma method and monoclonal antibodies'', published by R&D Planning Co., Ltd., pp. 30-53, 1982 March 5], and the above-mentioned immunizing antigen can be used for this search.

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.

The monoclonal antibody of the present invention can be collected from the above-mentioned hybridoma by culturing the hybridoma according to a conventional method and obtaining the culture supernatant, or by administering the hybridoma to a mammal compatible with the hybridoma and allowing the hybridoma to proliferate. The method used is to obtain the information as follows. 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-B monoclonal antibody of the present invention can be produced.

More specifically, the antibody of the present invention can be used to easily and specifically purify the EI)-B region of FN by conventional purification methods such as immunoprecipitation and affinity chromatography. be able to. Further, by using the antibody of the present invention, cancerous FN in a body fluid or the like can be specifically measured by immunoreaction in the sample. Such methods include conventional immunological means such as a conventional competitive method, radioimmunoassay (RIA) using the Sand-Deutsch method, enzyme-linked immunosorbent assay (ELISA), and agglutination method, and the operations of each method include , procedures, etc. can follow conventional methods.

More specifically, for example, when carrying out a competitive method, cancerous FN in a sample to be measured and a certain amount of inactivated FN ED-B are combined with the present invention labeled with a labeling agent. Competitive reaction with a certain amount of antibody, then ED-B of insolubilized FN
and the labeled antibody and the unbound labeled antibody are separated,
By measuring the labeling activity of either of them, cancerous FN in the sample can be quantified. Furthermore, when carrying out the Sand-Deutsch method, the substance to be measured (specimen) and the insolubilized antibody of the present invention are reacted to form an ED-B insolubilized antibody complex of FN, and a certain amount of labeled antibody is added to this complex. Cancerous FN in a specimen can be quantified in the same manner as described above by reacting and then measuring the labeling activity or unbound labeling activity of the conjugate of the complex and the labeled antibody formed.

In the various assay methods described above, body fluids such as blood, urine, cell tissue fluid, etc. can be used as the specimen, and among these, blood, particularly serum or plasma, is preferred.

Preparation of the antibody of the present invention and labeled antibody labeled with a labeling agent is as follows:
This can be carried out according to a conventional method using an appropriate labeling agent. Common labeling agents include I, 1311, radioactive substances such as tritium, glucoamylase, peroxidase (POX), chymotrypsinogen, procarboxypeptidase, glyceraldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, Examples include various enzyme reagents such as alkaline phosphatase, D-Nase, P-Nase, β-galactosidase, glucose-6-phosphinidobuhydrogenase, and ornithine decarboxylase. As a labeling method, for example, in the case of radioactive iodine, an oxidative iodination method using chloramine T [W, M, Hunter and F, C.

Greenwood; Nature, 194.4
95 (1962); Biochem, Jl, 8
[A
cta, Endocrinol, 5upp1. ,1
68.206 (1972)] and 7'70-ru (M,
[Proc, Natl.
, Acad, Sci, , U, S, A, , 57.
713 (1967)].

Furthermore, the ED-
B, for example, one that is physically or chemically immobilized on a plate, can be produced by chemically or physically bonding the antibody of the present invention or ED-B to a suitable insoluble carrier. The carriers used include cellulose powder, Sephadex, Sepharose, polystyrene, filter paper, carboxymethyl cellulose, ion exchange resin, dextran, plastic film, plastic tube, nylon, glass beads, silk, polyamine-ethyl vinyl ether.
Examples include maleic acid copolymers, amino acid copolymers, and ethylene-maleic acid copolymers. The above-mentioned insolubilization can be carried out by covalent bonding methods such as diazo method, peptide method, alkylation method, carrier bonding method using a crosslinking reagent (geltal aldehyde, hexamethylene isocyanate, etc. are used as the crosslinking reagent),
This can be carried out by various chemical reaction means such as a carrier binding method using a UgI reaction, an ion binding method using a carrier such as an ion exchange resin, a physical adsorption method using a porous glass such as glass beads as a carrier, and the like.

The reaction (immune reaction) in the above assay method can be carried out at a temperature of usually 45° C. or lower, preferably 4 to 40° C., for several hours to 24 hours.

Thus, by using the antibody of the present invention, cancerous FN or ED-B-bearing FN in a specimen can be measured easily and with high precision.

Setting up a purification system and measurement system using such an antibody of the present invention, and modification or application thereof are obvious to those skilled in the art.

Effects of the Invention According to the present invention, FN anti-ED-B monoclonal antibody and FN ED-B monoclonal antibody as an immunogen for its production.
Protein A fusion proteins are provided. Utilization of the antibodies of the present invention provides research on cancerous FN and methods for diagnosing and treating cancer.

EXAMPLES Examples will be given below to explain the present invention in more detail, but the present invention is not limited thereto.

Example I Production of ED-B/Protein A fusion protein ■ E of FN
Preparation of Sac I-Pvu II fragment containing the D-B region a) Culture of cells WI-38VA13 cells were used in this example.

These cells are a cell line obtained by transforming normal diploid fibroblasts WI-38 isolated from human fetal lung tissue with tumor virus SV40, and were developed by Girardi (A, J.
Girardi) et al. [Ann, Med, Exp, Biol, F.
enn,.

Growth, 242-254 (1966), A.
It has been deposited with the TCC as ATCCCCL 75.1.

The above WI38VA13 cells were transformed into fresh knee (R,1
, Freshney) “Culture of Animal Cells”
) j (r) written (Anan RLis, Inc.
, New York, 1983).

Approximately 10 cells of WI38VA13 cells suspended by trypsin treatment were plated on 10 15-culture dishes (Falcon tissue culture dishes #3025), and DME medium (Dulbetzco's modified Eagle medium, Gibco Co., Ltd.) containing 10% FC3 (fetal bovine serum) was plated. 37 in the presence of 5% CO2 using
After culturing at 50° C. for 5 days, the cells were detached from the culture dish using a rubber policeman, and about 1 g of WI38VA13 cells were collected by centrifugation (500×g, 5 minutes).

b) Preparation of cDNA library Approximately 1 g of cells obtained in a) above was mixed with 1511 GTC homogenate buffer [5,3M guanidinium thiocyanate, 0.02M N-sodium lauryl sarcosyl, 0
．． The mixture was added to a Botter type homogenizer containing 03M trisodium citrate, 0.8% β-mercaptoethanol, and 0.7% Antifoam 289 (defoaming agent, manufactured by Sigma).

After 10 reciprocations, the contents were transferred to a beaker and 2011
Attach a 22G needle to the syringe and forcefully pass it through three times to shear.

Place about 41 A' of 5.7 M cesium chloride and OoIM EDTA in a centrifuge tube, and add about 8 A' of the above homogenate on top. After layering n, 20°C, 3.200 Orpm
Total RNA was collected by centrifugation for 0 hours.

Dilute total RNA to a concentration of 5 μ/11 or less and incubate at 65°C for 7
After incubation for minutes, it was rapidly cooled in water for 2 minutes. Equal volume of 2x oligo dT binding buffer [1,0 M NaC
A', 20 m m Lis-HCl.

pH 7.5] and 1/100 volume of 20% SDS were added and mixed well. Then oligo dT binding buffer [0,
5M NaCA7, 10mM Tris-HCl, pH 7
, 5, 0, 1% SDS] was added to an oligo dT cellulose column (manufactured by Bio-Rad) equilibrated with 1% SDS. The unadsorbed fraction was reacted again at 65°C for 7 minutes, rapidly cooled in water for 2 minutes, and then added to the column again. Wash the column with 10 volumes of oligo dT binding buffer and add another 10 volumes of oligo dT binding buffer.
T wash solution [0,1M NaCA', 10mM)
Washing was performed using HC/, pH 7, 5, 0, 1% SDS. A'' RNA bound to oligo-dT cellulose was purified by oligo-dT eluate [10 mM Tris-HCl, pH 7,
5.0.05% SDS]. 1 in the eluate
/25 volumes of 5M NaCl and 2.5 volumes of ethanol were added, mixed well and left at -20°C overnight. This was then centrifuged at 1200 rpm for 15 minutes to precipitate poly A'' RNA, which was suspended again in 70% ethanol and centrifuged in the same manner. The precipitate was dried and then dissolved in an appropriate amount of water.

From the poly A'' RNA obtained by the above method, the method of Kawasaki and Wang [Kawasaki and Wang,
PCR Technology, H, A, Erlic
h, ed,, 5tockton Press.

New York, p89-98 (1989)], the region encoding the ED-B region of FN cDNA was amplified by the polymerase chain-reaxidin method.

C) Synthesis of Primers The following two oligodeoxynucleotide primers were prepared.

Upstream primer (Sac I site): 5'-CAGA
GCTCCTGCACTTTTGA-3° upstream primer (Pvu II site): 3'-TGTGACTGT
GTTGTTTGCC-5' Preparation of the above primer is as follows:
Synthesis was performed using an automatic DNA synthesizer (manufactured by Applied Biosystems, model 380A) from β-cyanoethylphosphoramidite derivatives of four types of bases by a solid phase method.

Deprotection of the synthesized oligonucleotide and release from the solid support were performed by heating at 55°C for 10 hours in concentrated ammonia water. The synthetic oligonucleotides thus prepared were purified by HPLC to finally obtain about 50 μg of target oligonucleotides as upstream and downstream primers, respectively.

The purified oligonucleotide obtained was diluted with TE buffer [10 m
M) Lis-HC/, pH 7, 4, 1mM EDTA] and stored at -20°C.

d) -Synthesis of end-strand cDNA Place 10μ in a 0.5xl tube (manufactured by Eppendorf).
l of 2x reaction buffer [40mM Tris-HCl, pH
8, 4, 100mM KCI.

5mM MgCl2.0.2mg/zl! Nuclease-free bovine serum albumin, 2mM dATP, 2m
M dGTP, 2mM dCTP, 2mM TTP,
2 units 7. I RNasin (manufactured by o ln), 100 pnllo1 random hexamer (manufactured by Pharmacia)] and approximately 1μ which had been heat treated at 90°C for 5 minutes.
After mixing with 9 μl of a solution containing g of RNA, add 1 μl of mouse Moloney leukemia virus-derived reverse transcriptase (approximately 2
00 units) at room temperature for 10 minutes, then at 42°C for 30 minutes.
By incubating for a minute, -terminal strand cDNA was synthesized.

The reaction solution was heated at 95° C. for 10 minutes to stop the reaction.

e) Amplification of Sac I-Pvu II fragment d above
) - 20 ul of a double-stranded cDNA solution containing 5 Q pmol each of upstream and downstream primers in 1x PcR reaction buffer [20 mM) Lis-HCl, pH 8, 4, 50 mM
KCI, 2.5mM MgCl2.0.1■/11
Nuclease-free bovine serum albuminco and 5 units of T
aq polymerase (manufactured by PerkinElmer/Siegis,
After adding 1 μl of mineral oil and layering with 100 μl of mineral oil, the operation of heating at 95°C for 1.5 minutes, then at 50°C for 3 minutes, and further at 72°C for 3 minutes was repeated 35 times to obtain the desired ED- Sac I-Pvu I encoding region B
Amplify the I ([)NA fragment. After the reaction is completed, 10
of Sac T and incubated at 37° C. for 2 hours to amplify Sac I-Pvu11 cDNA.
The 5' Sac I site of the A fragment was exposed.

Regarding the above reaction solution, in the presence of ethidium bromide,
By performing electrophoresis using a 1.5% agarose gel using the Hae III-degraded DNA fragment of φX174 DNA as a molecular weight marker, the Sac I Pvu II fragment was amplified to the desired size of 385 base pairs. I confirmed that there is.

f) Purification of Sac I-Pvu II fragment e above
) Sac I − separated on agarose gel according to
The Pvu II fragment was prepared using the method of Dretzen et al.
zen, G, M,, et al,, Anal,
Biochem.

112, 295-298 (1981)] DEAE cellulose membrane (manufactured by S&S, NA45)
After adsorption onto the top, the adsorbed DNA fragments were dissolved in elution buffer [50mM) Lis-HCA', pH 8.0, IMN.
aCl, 10 mM EDTA] from the DEAE cellulose membrane, followed by cold ethanol precipitation to obtain the desired Sac I-Pvu II fragment (approximately 10
00n was collected.

■ )' N (Di'J APvu II -Ace
Preparation of I fragment a) Human fibronectin cDNA clone isolated by Sekiguchi et al. I) L F 5 [
Sekiguchi.

K., et al., Biochemistry,
25. p4936-4941 (1986)] 2
0 μg was added to 50 μl of reaction buffer [10 mM Tris-H
Cl! , pH 7, 5, 7mM MgC12, 60mM
NaCl, 7mM 2-mercaptoethanol, 0.01
% bovine serum albumin and add 20 units of Pv to this.
u II and Acc 1 (both manufactured by Takara Shuzo Co., Ltd.) were added and reacted at 37°C for 2 hours. After the reaction, electrophoresis was performed using a 1% agarose gel to determine the desired Pvu.
The II-Acc I fragment (226 base pairs) was separated, and then the desired DNA fragment (approximately 500 ng) was recovered using a DEAE cellulose membrane as described in (1)-f) above.

■ p of Sac I-Acc I fragment of FN cDNA
Cloning into GEM4 5 μg of pGEM4 (manufactured by Promega) was added to 20 μl of reaction buffer [10 mM Tris-HC/X pH 7, 5.7 mM
MgC/2.60mM NaCA', 7mM 2-mercaptoethanol, 0.01% bovine serum albumin] and 10 units of 5acI (Takara Shuzo) and 1
0 units of AccI (manufactured by Takara Shuzo Co., Ltd.) was added and incubated at 37°C for 2 hours to cleave the polylinker region of pGEM4 at the Sac I and Acc 1 sites. The reaction product was treated with phenol and then cleaved by ethanol precipitation. Collect plasmid DNA and add 48 μl of this
reaction buffer [50mM) Lis-HCl! , pH 9,0
, 0.1mM ZuC12, 1mM MgCl2.
The solution was dissolved in 1mM Spermida, and 20 units of fresh small intestine alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.) was added thereto, and the mixture was heated at 37°C for 15 minutes and then at 56°C for 15 minutes to dephosphorylate the 5' end. I did it.

2.5 μ of 10% SDS! After the addition, the enzyme was inactivated by heating at 68°C for 15 minutes, followed by phenol extraction and ethanol precipitation to recover plasmid DNA with its 5' end dephosphorylated.

Next, 20 ng of the above plasmid DNA and the above ■ and ■
20 ng of each cDNA fragment obtained in 2
4μl of ligation buffer [66mM Tris-HC
1, pH 7,6, 5mM MgC/2.5mM dithiothreidoyl, 1mM ATP], and 74DN was dissolved in this.
Add 300 units of A ligase (manufactured by Takara Shuzo Co., Ltd.) and incubate at 16°C for 16 hours.
A cDNA fragment from the Sac 1 site to the Acc I site encoding the ED-B region of FN was inserted into the I-Acc I site.

Next, 1 μl of this reaction solution was taken out, mixed with 100 μl of E. coli HBIOI competent cells (manufactured by Takara Shuzo Co., Ltd.), and incubated under water cooling for 30 minutes, then at 42°C for 90 minutes.
After incubation for 0 seconds, the plasmid DNA was introduced into E. coli. To this was added 1 xi of LB medium [1% Bacto-tryptone, 0.5% yeast extract, 1% salt] and cultured with shaking at 37°C for 1 hour, then aliquoted 100 μl and added 50 μg of ampicillin/ LB including xi
Agar plate [1.5% Bacto agar, 1% Bacto tryptone, 0.5% yeast extract, 1% table salt,
Plasmid DNA was incubated at 37°C for 14 hours.
Approximately 200 E. coli colonies were obtained. 12 pieces were randomly collected from these and 50μg/
After culturing in LB medium containing xi ampicillin, Birnbimand Doly [Birnbimand Doly]
c) Modified method [Molecular Cloning, A
Laboratory Manual, T, Man
Iatis et al., ed.

p368-369 (19B2)], plasmid DNA was recovered from each colony. Eco RI and Ps
A plasmid clone (pGEMBl) with the expected insertion sequence of approximately 600 base pairs was generated by double digestion with tI.
were selected.

■ Recovery of Eco RI - Pst I fragment from pGEMBl a) Isolation of plasmid DNA The E. coli strain containing the plasmid clone pGEMB1 obtained in (■) above was cultured using LB medium 5002/ containing 50 μg/yl of ampicillin. The cells were cultured at ℃ for 12 hours. Thereafter, the bacterial cells were collected by centrifugation at 5000Xg for 10 minutes, and subjected to alkaline lysis method [Molecular Cl
oning, A Laboratory Manua
l, T.

Maniatis et al, ed, p90
-91 (1982)] plasmid DNA was isolated from D as follows.

That is, the bacterial cells were suspended in buffer I [50mM glucose, 25mM lyso-HCl, pH 8.0, 10mM EDTA] containing 8 yl of lysozyme 5μ/yl, and after being left at room temperature for 5 minutes, 16 zl of lysozyme was added to this.
A 0.2N NaOH/1% SDS solution was added thereto, mixed quickly, and lysed under water cooling for 10 minutes. Next 1211
An ice-cooled 5M potassium acetate solution (pH 4,8) was added and mixed, and the mixture was further left to stand under water cooling for 10 minutes.

After the above, centrifuge at 2000 rpm for 20 minutes at 4°C, transfer 16 Il of the supernatant 32zA' to two Corex glass centrifuge tubes, add 1011 isopropanol to each tube, and leave for 15 minutes at room temperature. , 12000X
g, centrifuged at 15°C for 30 minutes, and plasmid DNA was recovered as a precipitate.

After air-drying this precipitate, 8 xi of TE buffer [10 m
M) Lis-HCII, pH 8,0,0,1m M E
DT A ], add 8 g of cesium chloride and 0.4xl of 1/xi ethidium bromide solution, mix well, and centrifuge at 200 rpm for 5 minutes at room temperature to remove insoluble matter. Ta. The supernatant was transferred to a 12PA seal tube (manufactured by Hitachi King), and the upper part of the tube was filled with mineral oil, and then incubated at 55,000 rpm for 16 hours.
Centrifugation at °C allowed for the formation of plasmid DNA bands. Next, the plasmid DNA was collected using a syringe needle, and the desired pGEMB1 plasmid D was recovered by ethanol precipitation.
NA (approximately 200 μg) was obtained.

b) Collection of Eco RI to Pst I fragment a) above
5 μg of pGEMB1 plasmid DNA obtained in 2
5 μl of Eco RI-pst I reaction buffer [1
0mM) Lis-HCl! , pH 7, 5, 10mM
gCA'2.50mM NaCA', 1mM dithiothreitol], 10 units of Eco RI and 10
unit of Pst I and incubated for 2 hours at 37°C to convert the plasmid DNA into Eco R1 sites and ps
Cut at the tI site. The desired Eco RI-
The Pst I fragment was separated and the desired DNA fragment (approximately 30
01g) was collected.

■ Insertion of Eco RI-Pst I fragment into pRIT2T a) Production of plasmid vector 2 μg of protein A gene fusion vector pRIT2T (manufactured by Pharmacia) was dissolved in 20 μl of Eco old-Pst I reaction buffer, and Eco R1 and Pst 10 units of each of I and I were added and incubated at 37° C. for 2 hours to cleave the plasmid DNA at the Eco RI-Pst I site. Plasmid DNA cleaved by ethanol precipitation after phenol extraction of the reaction product
was recovered, and its 5' end was dephosphorylated using live small intestine alkaline phosphatase according to the method described in (1) above, and after phenol extraction was performed again, 1 μg of the desired plasmid vector was obtained by ethanol precipitation.

b) Eco RI-Pst into plasmid vector
Insertion of the pRIT2T plasmid 201g, which was cleaved with Eco RI and Pst I according to a) above and its 5' end was dephosphorylated, and the (7) ECORI-Pst I fragment derived from pGEMB1 prepared in ① above. 20''g was dissolved in 24 μl of the ligation buffer described in ① above,
Add 300 units of T4 DNA ligase to this and make 16
Eco RI-P from pGEMB1 was incubated at ℃ for 16 hours to incubate the polylinker region of pRI72T.
The st I fragment was inserted.

C) Preparation of transformants Using 1 μl of the reaction solution obtained in b) above, transform Escherichia coli E, coli HBI old strain according to the method described in ① above, and place about 50 μl on a 9 cm LB agar plate. Obtained several colonies. From these, 12 colonies were randomly collected separately and LB containing 1.57/ml of ampicillin was collected.
After culturing in medium, Birnboin and Dolly [Birnboin]
bim and Doly] (y) Plasmid DNA was collected from each colony by a modified method. The thus obtained plasmid DNA (approximately 1 μg) was treated with Eco RI
- After dissolving in Pst I reaction buffer 1-Ott, 1, 5 units each of Eco RI and Pst I were added and incubated at 37°C for 2 hours. Analysis by electrophoresis identified a clone (pPABl) in which a 623 base pair Eco RI-Pst fragment was produced.

d) Culture the E. coli strain containing plasmid pPAB1 identified in C) above using LB medium containing 500 zl of ampicillin, and use the alkaline lysis method shown in a) of ① above to incubate approximately 300 ml of the desired plasmid DNA pPAB1.
μg was obtained.

■ Introduction of plasmid pPAB1 into Escherichia coli N4830 The pPAB1 plasmid DNA obtained in step (■) above was introduced into E. coli N4830 by Mandel and Hjga (Mandel and Hjga).
y) +) Calcium diacid method [J, Mo1. Bj
ol., Suzu, 15'i (1970)] was introduced into Escherichia coli N4830 (obtained from Pharmacia) as follows.

That is, 37% of E. coli N4830 in 100z/LB medium
The cells were cultured with shaking at ℃, and the culture was stopped when the bacterial cell density reached approximately 5 x 107/xll, and the cells were rapidly cooled in a water bath. 400
After collecting bacteria by centrifugation at 0xg for 5 minutes at 4°C, the pellet was
Suspend in 0 zl of ice-cold 50mM calcium chloride-10mM Tris-HCl (pH 8,0) and incubate for 1 hour in a water bath.
After standing still for 5 minutes, it was centrifuged at 4000Xg for 5 minutes at 4°C.

The obtained precipitate was resuspended in 7 ill of a water-cooled solution of 50 mM calcium chloride-10 mM Tris-HCl (pH 8,0), and allowed to stand still in water cooling. Add 10 μl of the pPAB1 plasmid solution (plasmid DNA
(containing 100 g of A) was added, left to stand for 30 minutes in a water bath, heated for 2 minutes in a warm vortex at 42°C, and then
LB medium was added and incubated at 37°C for 1 hour. 100 μl of the E. coli suspension obtained in this manner was plated on an LB agar medium containing ampicillin with the above composition,
Transformed E. coli colonies were generated on agar plates by incubation at 37°C for 14 hours.

■ Isolation of protein A-ED-B fusion protein The transformant obtained in step (1) above (E. coli N4830 transformed with plasmid pPABl) was incubated with 500 zl of LB medium for 30 minutes.
After culturing with shaking at 0°C for 14 hours, 500 zl of LB medium preheated to 54°C was added, and cultured with shaking in a water bath at 42°C for 90 minutes to express protein A-ED-B fusion protein. was induced.

Thereafter, the cells were collected by centrifugation at 5000Xg for 15 minutes at 4°C, and then added to ice-cold Tris-buffered saline [50
mM) Lis-HCl, pH 7,6, 150mM Na
C/] by suspending the mixture at 100 y/cm and subjecting it to ultrasonic crushing in a water bath (using a Bransono Sonifier-250, repeating the treatment for 3 minutes three times at an output setting of 7). The proteins in the bacterial cells were released. Approximately L of this crushed liquid
Centrifuge ool/(16000Xg, 20 minutes, 4℃)
After collecting the supernatant fractional 9511 and diluting it by adding 300 zl of Tris-buffered saline, approximately 1011 Ig
G-Sepharose 6 First Flow (manufactured by Pharmacia) was attached to the collar 11 filled with protein
The D-B fusion protein was adsorbed onto the column. 1 column
Tris-buffered saline of 007/, then 5m of 2011
After washing with M ammonium acetate solution (pH 5,0), the adsorbed proteins were eluted with 0.5M acetic acid solution. The protein A-ED-B fusion protein thus obtained was dialyzed against Tris-buffered saline for two days and nights.
The desired antigenic mass was obtained.

Example 2 Preparation of hybridoma After diluting 0.05 μl of the purified ED-B-Protein A fusion protein obtained in Example 1 with 0.5xl of PBS, the same amount of Freund's complete adjuvant (complete
E. Freuncl's adjuvant) was mixed and emulsified, and 0.2 zl of this was subcutaneously administered to male Balb/c7 mice (8 weeks old). Thereafter, the mice were immunized in the same manner four times with additional administration every two weeks, and the lungs were removed 3 days after the final immunization.

Take out the tile cells from the excised lung, and incubate the red blood cells present in the cells with 0.83% ammonium chloride solution at 4°C for 1 to 2 hours.
It was treated for 1 minute to melt and remove. The cells obtained above were collected as sensitized lymphocytes, and RPMI-
Washed three times with 1640 medium.

Next, mouse myeloma cells [P3U1, Current to
pics 1n Microbiology and
Immunology, 73°p3 (1981)
etc.], R containing 15% FC8 (fetal bovine serum)
The cells were subcultured in a PMI-1640 medium to which 100 μM of 8-azaguanine was added, and the cells were used as myeloma cells and washed.

The above myeloma cells and myeloma cells were mixed in a 501/2 tube at a cell number ratio of 10:1, the resulting cell mixture was centrifuged at 500Xg for 5 minutes, and the supernatant was completely removed with a Pasteur pipette. did. These operations were performed in a water tank kept at 37°C.

Next, 35% polyethylene glycol 1500 (manufactured by Wako Tsumugi Co., Ltd., hereinafter abbreviated as PEG) 411 was added, stirred slowly for 1 to 2 minutes, left for 1 minute, and then heated to 37°C.
Fe2-free RPMI-1640 medium 2 kept warm at
Add xl slowly for about 1 minute, leave it for 1 minute,
Add the same wave 4yl and leave it for 2 minutes, then add the same wave 4xl
was added and left for 4 minutes. Next, 1 was kept warm at 37°C.
Streptomycin sulfate at 5% FC8.0.05 titer/l, Penicillin G potassium at 60,000 U/l, 54■
RPMI-1640 (hereinafter referred to as complete RPMT-16) containing /l gentamicin and lxipyruvate.
After adding 811 (referred to as 40 medium) for 2 to 3 minutes,
Centrifugation was performed at 500×g for 5 minutes. Aspirate the supernatant and
The cells were suspended in complete RPMI-1640 medium kept at 37°C at a density of 106 tile cells/xi. Next, 0.1xl of this suspension was dispensed into a 96-well plate (manufactured by Coaster) at 37°C, 5% CO2, 1
The cells were cultured for 24 hours in an incubator at 00% humidity.

Then, in each well, add 1 × 10 M1 aminopterin, 4 × 10 M1 aminopterin, and 1.6 × 10 thymidine to each well.
0.1 zl of complete RPMI-1640 medium supplemented with 10% FC8 containing M (hereinafter referred to as HAT medium) was added. Thereafter, the supernatant was diluted to 0.0% on the 2nd and 3rd day, respectively.
127 volumes were aspirated, and 0.1x fresh HAT medium was added to replace the solution. Thereafter, the above liquid exchange was performed every 2 to 3 days. On the 6th day, the supernatant was similarly aspirated and replaced with complete RPMT-1640 medium (hereinafter referred to as HT medium) containing 1 x 10 M of hypoxanthine and 1.6 x 10 M of thymidine. Thereafter, growth was maintained in complete RPMI-1640 medium.

Colonies became visible to the naked eye in 10 to 14 days after cell fusion by the above procedure. When the cells occupied 1/4 of the bottom area of the 96-well plate, enzyme immunoassay (EL) using human placenta-derived FN retaining ED-B as an antigen was performed.
The culture supernatant was tested using the ISA method, and positive wells were immediately subjected to the limiting dilution method (Method in Enz.
Hybridoma cloning was carried out using the following method.

That is, 10% FC8-added RPMI-1640 prepared to contain 1 x 108 Ba1b/c mouse thymocytes.
Using medium 2011, add 6 hybridomas to 3 cells/well, 1 cell/well, and 0.3 cells/well.
Cloning was performed by distributing 0.2 zl onto well plates to establish the desired hybridoma.

The above cloning was carried out using human normal fibroblast W-38 cell lines WT-38VA, which were aggravated with tumor virus SV40.
Cancerous FN and placenta-derived FN purified from the culture supernatant of No. 13
Using the reactivity with FN as an indicator, the above cloning was performed four times while confirming that there was no reactivity with plasma type FN,
Four hybridoma strains producing the monoclonal antibody of the present invention having the desired reaction specificity were obtained.

rOAL-TFN-01j to rOAL-, respectively.
It was named TFN-04J.

Clones 0AL-TFN-01 to 0AL obtained above
- TFN-04 in complete RPMI-1640 medium,
The cells were cultured at 37° C. for 96 hours under 5% CO2 conditions.

The culture solution was centrifuged at 300 Orpm for 10 minutes to obtain a culture supernatant containing the monoclonal antibody of interest.

One of the obtained clones (the antibody-producing hybridoma of the present invention 0AL-TFN-01) was selected.

The monoclonal antibody-producing cells have been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under the designation rOAL-TFN-01J, and the deposit number is
No. 0 (FERM P-11540) J.

1 x 106 pieces of the above clone 0AL-TFN-01,
The mixture was administered intraperitoneally to Ba1b/c mice that had been previously inoculated with pristane (manufactured by Aldrich). After 10 to 14 days, the accumulated ascites was collected to obtain ascites containing the antibody of the present invention.

The antibodies in the ascites were purified using gel chromatography (using Sephacryl-3-300) and anion exchange chromatography (using Q-Sepharose),
Purified antibody 0AL-TFN-01 was obtained.

The properties of the monoclonal antibody of the present invention obtained above are shown below as Example 3.

Example 3 Properties of the antibody of the present invention (1) Antibody subclass The subclass of the antibody of the present invention was determined using a mouse monoclonal antibody subclass identification kit (manufactured by Bio-Rad).

As a result, the subclass of the above antibody was IgM.

■ Antibody production level The culture supernatant obtained in Example 2 was centrifuged, and the supernatant was
%FC8 supplemented RPMI-1640 medium at 37°C, 5
The cells were cultured in vitro for 10 days under conditions of % CO2.

The amount of 1 gM of 0AL-TFN-01 in the culture supernatant when the hybridoma reaches the maximum cell density is approximately 5 μg/
It was xi.

■ A 96-well polystyrene microplate (NUNC) coated (4°C, 24 hours) with 2 μg/well of purified FN derived from placenta (extracted with urea after homogenizing the placenta) retaining antibody titer EI)-B. ), 1% BS
A Dulbets cholic acid buffer (pH 7.2, hereinafter referred to as D'P)
After blocking with BS (abbreviated as BS) for 24 hours at 4°C, 50 μl of the culture supernatant containing the antibody of the present invention obtained in Example 2 was added to each well of the plate, and reacted at room temperature for 3 hours. After washing three times with washing buffer (D' PBS + 0.05% Tween 20), F
The antibody bound to the ED-B/Protein A fusion protein of N. was measured.

As a result, sufficient color development was observed when the culture supernatant was diluted 1XloI times.

■ Standard curve by ELISA method 25 μg of the monoclonal antibody of the present invention in D'PBS
/zl, 100 μl of this was added to each well of a 96-well microplate, fixed overnight at 4°C, and then washed with D'PBS (containing 0.05% Tween 20). Each well was then injected with D'PBS, 0
．． 300 μl each of 0.5% thimerosal and 0 and 5% bovine serum albumin (BSA) were added, and blocking was performed overnight at 4°C. After blocking, D' PBS
(containing 0.05% Tween 20) and inject each well with 0.01M phosphate buffer [0.1% NP-40 (containing NO.
NIDENT P-40, manufactured by Sigma), 0.05% thimerosal, 10% FC3, pH 5.5] 100μ
I put l. Furthermore, in each well, FN (pFN) purified from human plasma diluted to various concentrations and human normal fibroblast Wr-38 cells Wr-38 infected with tumor virus were added.
Cancerous FN (cF) purified from the culture supernatant of -38VA13.
After adding 20 μl of each of N) and incubating at room temperature for 2.5 hours, 0.05% Tween 20
The cells were washed 6 times with D'PBS containing D'PBS.

Furthermore, a biotinylate label (Bio
(tinylated) anti-FN monoclonal antibody [0AL-pF 11.5, established using Sigma's pFN as an immunogen, Clinical Pathology, vol. 35 supplement, 1
987, pH9; The 18th Congre
ss of the International A
s5organization of MedicalLa
laboratory technologists, A.
bstructs, p225 (198B), etc.]
(100μl/well of X100O fold diluted solution), D
'PBS (100μ//well), 0.1% CH
APS (3-[(3-floramidopropyl)dimethylammonio]-1-propanesulfonate), 0.1%
BSA, 0.05% thimerosal solution; A buffer 1-0
After adding 0 μl, the mixture was incubated for 2.5 hours and washed six times with D'PBS washing buffer containing 0.05% Tween 20.

Next, 100 μl/well of avidin-peroxidase complex (manufactured by Bio-Rad) dissolved in A buffer was added, followed by incubation for 1 hour. After washing the plate with washing buffer, 0-phenylenediamine solution (O
PD solution) at 100μ per well! Add 10 at room temperature
After reacting for a minute, 100 μl of 2N sulfuric acid was added to stop the reaction, and the absorbance at 4921 m was measured.

The above results are shown in Figure 1.

In the figure, the vertical axis shows the absorbance (OD) at 492 nm, and the horizontal axis shows the concentration of FN. (1) is the result of cancerous FN, (2
) is the result of plasma type FN.

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

[Brief explanation of drawings]

FIG. 1 is a graph showing the results of examining the reactivity of the antibodies of the present invention to various FNs. (that's all)

Claims (1)

[Scope of Claims] [1] An anti-ED-B monoclonal antibody characterized by recognizing the amino acid sequence of ED-B represented by the following formula (1). Formula (1): [There is a gene sequence. [2] The anti-ED-B monoclonal antibody according to claim [1], which is obtained using a fusion protein of 91 amino acids of the ED-B region and protein A as an immunogen.