JPH02487A - Monoclonal antibody, hybridoma, its production and use thereof - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to monoclonal antibodies that specifically bind to basic fibroblast growth factor, hybridomas, methods for producing them, and uses thereof.

Prior Art Basic Fibroblast Growth Factor (herein referred to as bFG)
It is sometimes abbreviated as F. ) is a basic polypeptide pormon with a molecular weight of 17,000 mainly secreted from the pituitary gland, and was initially isolated as a factor that exhibits a strong proliferation-promoting effect on linear blast cells such as BALB/c3T3 cells [D
, Gospodarovicz;
ture) 249:123 (1974)].

However, it was subsequently found that it exhibits a proliferation-promoting effect on almost all cells derived from mesoderm [D].

Gospodarowicz et al.; National Cancer Institute Monograph (Nation
alCancer In5 position Mono
graph) 48; I 09 (1978)]. Among them, bFGF's angiogenic effect, combined with its cell proliferation effect, shows its potential as a therapeutic agent for injuries and burns, and as a prophylactic agent for thrombosis, arteriosclerosis, and the like.

Problems to be Solved by the Invention Naturally occurring human bFGP is extremely scarce, and attempts to obtain it from human tissue have been extremely difficult due to various restrictions. Furthermore, until now, no easy-to-use method for quantifying bFGF has been established, and for these reasons, there are many unknowns regarding basic knowledge such as the properties of bFGF, which is essential for developing bFGF as a drug. many.

Therefore, many basic knowledge regarding bFGP, such as bF
If we can know the distribution of GI';' in the living body and its production mode, it will be easier to develop bFGF as a pharmaceutical product.

Furthermore, accurately knowing the amount of bPGF is also important when purifying this protein from a genetically recombinant plant. Furthermore, it is very important to track the blood FGFe levels of animals administered with bFGF, but this cannot be measured using the conventional method using 3T3 cells because of the contamination of the sample with serum. Normally, bFGF is measured by culturing at a lower serum concentration and
Add bFGF to 3T3 cells with reduced NA synthesis, and calculate backwards based on how much DNA synthesis ability is recovered.

However, since this method uses cells, the operations are delicate, measurement errors are large, and it takes a long time to obtain results. Therefore, it is desired to develop a simple and accurate means for measuring FGF for the above purpose.

Means for Solving the Problems In view of the above-mentioned circumstances, the present inventors have conducted various studies to find a practical means for measuring bFGF, and have developed a monoclonal antibody that specifically binds to bFGF, which makes the measurement possible. As a result of further research based on this, the present invention was completed.

The present invention provides (1) a monoclonal antibody that has the following properties and specifically binds to bFGF.

(a) Molecular weight; approximately 140-160 kilodaltons (b) Does not cross-react with acidic fibroblast growth factors.

(c) The immunoglobulin class belongs to IgM or IgG.

(2) A cloned hybridoma consisting of spleen cells of a mammal immunized with bFGF and homologous or heterologous lymphoid cells.

(3) A clone consisting of spleen cells and lymphoid cells characterized by cell fusion of spleen cells of a mammal immunized with bFGF and homologous or heterologous lymphoid cells, and cloning. A method for producing hybridomas.

(4) Cloned hybridomas consisting of spleen cells of a mammal immunized with bFGF and homologous or heterologous lymphoid cells are grown in a liquid medium or intraperitoneally of the mammal to produce monoclonal antibodies. , accumulate,
A method for producing a monoclonal antibody that specifically binds to the factor, which comprises collecting the antibody.

(5) A method for purifying bFGF, which is characterized by using the monoclonal antibody according to item (1) above, and (6) a method for detecting and quantifying bFGF, which is characterized by using the monoclonal antibody according to item (1) above. It is.

The bFGF for immunizing mammals may be any bFGF' of warm-blooded mammals. Alternatively, the mutein may be used. Therefore, herein, "basic fibroblast growth factor (bFGF)"
may also include the mutin unless otherwise specified.

Representative examples of the mammalian bFGF include, for example, bovine bFGP [procedure in the
National Academy of Sciences (Pro
c, Natl, Acad, Scf,) U
SA, Vol. 82, p. 6507 (1985)], Human bF G F [Japanese Patent Application No. 61-241053 (European Patent Application Publication No. 237,966), European Molecular Biology. Organization Journal (European)
Molecular Biology Organiz
ation (EM [30) Journal) No. 5
Vol., p. 2523 (1986)].

More specifically, Phe -Phe -Leu -Arg -I le
-His -Pro -A sp -G ly -A
rg -V al -Asp -G Iy -V a
lArg-Glu-Lys-Ser-Asp-Pro
A polypeptide containing the amino acid sequence shown in (1) is preferred.

Furthermore, the general formula % formula % ([) [wherein, X represents Thr or Ser, and Xh<Thr
When , Y indicates Ser, and when X is Ser, Y indicates Pro. ] is preferred.

To obtain human bFGF (hereinafter sometimes abbreviated as hbFGF), for example, the above-mentioned hbF' CF
D having a base sequence encoding a protein polypeptide
Expression vectors containing NA are, for example, (a) hb
Isolate the RNA encoding FGP; (b) separate the RNA;
Synthesize single-stranded complementary DNA (cDNA) and then double-stranded DNA from A, (c) integrate the complementary DNA into a plasmid, (d) transform a host with the obtained recombinant plasmid, and (e) After culturing the obtained transformant, a plasmid containing the desired DNA is isolated from the transformant by an appropriate method, such as colony hybridization using a DNA probe, and (to) a plasmid containing the desired DNA is isolated from the plasmid. cloned DNA
(g) ligating the cloned DNA downstream of bromocous in a vehicle.

RNA encoding hbF G F can be used for various hbFG
It can be obtained from F-producing cells, such as human pituitary-derived cells or human fibroblasts. Examples of the human fibrinoblasts include WI38 (ATCC number CCL-75) and IMR9Q (ATCC number CCL-186). The above cells W138 and 1MR90 were obtained from The American Type Culture Collection (Th
e A mericanTypeCulture
Catalog in Cell Lying and Hybridomas (co11ection)
Ogue of Ce1l L 1nes &
Hybridmas) 5th edition
n, l 985.

The expression vector thus obtained was transferred to an appropriate host (
For example, human bFGF' can be produced by incorporating it into E. coli, Bacillus subtilis, yeast, animal cells) and culturing the obtained transformant in a medium.

The mutin in the present invention is originally a mutated amino acid sequence of an original peptide or protein,
Therefore, such mutations include addition of amino acids, deletion of constituent amino acids, and substitution with other amino acids.

Examples of the addition of amino acids include those in which at least 61 amino acids are added.

The deletion of the constituent amino acids includes at least one bF
' Examples include those lacking GP-constituent amino acids.

At least 61 b
Examples include those in which a PGF-constituting amino acid is substituted with another amino acid.

If at least bFGF! The at least one amino acid in the mutin to which the amino acid I is added does not include methionine, which is based on the initiation codon used to express the peptide, or a signal peptide.

The number of amino acids added is at least one, but any number may be used as long as the characteristics of bFGF are not lost. More preferably, it includes part or all of the amino acid sequence of a protein that is homologous to bFGF and exhibits similar activity.

The number of deficient constitutive amino acids in a mutin in which at least one bFGF' constitutive amino acid of bFGF is deficient may be any number as long as it does not lose the characteristic of bF'GF.

Examples of the deficient constituent amino acids include human bFG
Amino-terminal lO residue of F: Met-Pro-A I
a Leu-Pro-G! u-Asp Gly-Gly-Ser 14 amino-terminal residues of human bFGF: Met-Pr
o-A Ia-Leu-Pro-G lu-Asp-
G ly - G Iy - S er - G ly -
A Ia - P he - Pro.

41 residues on the amino terminal side of human bFGF; and 6I residues on the carboxyl terminal side of bFGF.

At least I in mutin in which at least Ifll of bFGF constituent amino acids are substituted with another amino acid (as the number of bF G F' constituent amino acids in [! Any number is fine.

Examples of substituted amino acids constituting moe include amino acids other than stein and cysteine.

Cystine is particularly preferred. In addition to cysteine, the constituent amino acids to be substituted include aspartic acid, arginine, and glinone. Examples include valine.

When the substituted moe constituent amino acid is cysteine, the substituted amino acid is preferably, for example, a neutral amino acid. Specific examples of the neutral amino acid include glycine, valine, alanine, loinine, isoleucine, and thyronone. Phenylalanine, histidine, tryptophan, serine.

Examples include threonine and methionine. Particularly preferred are serine and threonine.

When the constituent amino acid to be substituted is other than cysteine, the other substituted amino acid may be, for example, in terms of hydrophilicity, hydrophobicity, or Choose one that has properties different from amino acids. Specifically, when the amino acid to be substituted is aspartic acid, the amino acids after substitution are asparagine threonine, valine, phenylalanine,
Examples include arginine, and asparagine and arginine are particularly preferred.

If the amino acid before substitution is arginine, the amino acid after substitution is glutamine.

Examples include threonine, leucine, phenylalanine, and aspartic acid, with glutamine being particularly preferred.

When the constituent amino acid to be substituted is glycine, examples of the substituted amino acid include threonine, leucine, phenylalanine, serine 9 glutamic acid, and arginine, with threonine being particularly preferred.

If the constituent amino acid before substitution is serine,
Examples of the amino acid after substitution include methionine, alanine, leucine, cysteine, glutamine, arginine, and aspartic acid, with methionine being particularly preferred.

When the constituent amino acid to be replaced is valine,
Examples of the amino acid after substitution include serine, loinone, proline, glycine, lysine, and aspartic acid, with serine being particularly preferred.

As the original constituent amino acids of H to be substituted, aspartic acid, arginine, glycine, serine, and valine are preferred.

Preferred amino acids after substitution are asparagine, glutamine, arginine, threonine, methionine, serine, and leucine.

The most preferred substituted mutin is one in which the constituent amino acid cysteine is replaced with serine.

In the above substitutions, two or more substitutions may be performed simultaneously. In particular, it is preferred that two or three constituent amino acids are substituted.

The mutin of the present invention may be a combination of two or three of the above additions, deletions, and substitutions.

To produce the mutin, site-directed mutagenesis technology is used.
esis) will be adopted. The technology is well known and has been developed by Lather, R.F. and Lecoq, J.P.
), Genetic Engineering (Geneii
C Engineering), Academic Press (1983), pp. 31-50. Oligonucleotide-directed mutagenesis has been described by M. Smith, M., and Gilam.
lam, S.), Genetic Engineering: Principles and Methods, Plenum Press (1981), 3 volumes.
1-32.

In order to produce the structural gene encoding the mutin, for example, (a) a single chain D consisting of a single chain of the bFGF structural gene;
Hybridize the NA with the mutant oligonucleotide primer (the primer is complementary to the region containing the codon for cysteine to be replaced with this single strand, or optionally the antisense triplet that pairs with this codon). (b), (b)
a) extending the primer by a DNA polymerase to form a mutant heteroduplex; and (c) replicating the mutant heteroduplex.

Next, a phage DN carrying the mutated gene
A is isolated and incorporated into a plasmid.

A mutin can be produced by transforming a suitable host (same as above) with the plasmid thus obtained and culturing the obtained transformant in a medium.

When immunizing bF with F, bF'GF' may be made into a complex with a carrier protein and then used for immunization.

Examples of the carrier protein include Freund's complete adjuvant (manufactured by Difco).

When using a carrier protein complex, the coupling ratio between the carrier protein and bFGF' is approximately 5 to 30 times (
carrier/bF G F :weight ratio).

Desirably, about 15 to 20 times is used.

Further, various condensing agents can be used for coupling the hapten and the carrier, and glutaraldehyde, carbodiimide, etc. are conveniently used.

When immunizing with bFGF or protein complexes, the mammals to be immunized are experimental animals such as sheep, goats, rabbits, guinea pigs, rats, and mice.In order to obtain monoclonal antibodies, rats and mice are used. Preferably, mice are particularly preferred. For example, when immunizing mice, immunization can be done by any route such as subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal, but mainly subcutaneous, intraperitoneal, or intravenous (especially subcutaneous) injection is possible. It is preferable to do so.

In addition, the immunization interval, immunization m, etc. are highly variable, and various methods are possible, but for example, immunization about 2 to 6 times at 2-week intervals,
A method using spleen cells extracted about 1 to 5 times, preferably about 2 to 4 days after the final immunization, is often used. For immunization, it is desirable to use about 0.1 μg or more of the peptide per mouse, preferably about IO μg to 300 μg per mouse. Furthermore, it is desirable to perform a partial blood sampling before removing the spleen, confirm an increase in the antibody titer in the blood, and then perform a fusion experiment using spleen cells.

The cell fusion of the above-mentioned spleen cells and lymphoid cells can be carried out by, for example, using isolated mouse spleen cells with hypoxandine-guanine-phosphoribosyltransferase-deficient (HGPRT) spleen cells.
suitable allogeneic or xenogeneic (preferably allogeneic) markers such as ``'') or thymidine kinase deficiency (TK-).
fused with a lymphoid cell line. As the lymphoid cell line, myeloma is preferable, and an example thereof is myeloma P3-X63-Ag・8UI (Ichimori et al. Journal, Saibu Immunological Methods 8055).
1985)). For example, the method of Keller and Milstein et al. [Nature 256:
495 (1975)]. For example, myeloma cells and callus cells are approximately 1
For example, a medium in which Iskot medium and Ham's F-12 medium are mixed at a ratio of 1=5 (hereinafter referred to as IH medium).
Sendai virus, a fusion agent such as polyethylene glycol (PEG) is used. Of course, it is possible to add dimethyl sulfoxide (DMSO) and other fusion promoters. The degree of polymerization of PEG is usually about 10
00-6000. The time is about 0.5 to 30 minutes, the concentration is about 1
0% to 80%, etc., but as an example of preferable conditions, PEG 6000 is used at about 35 to 55% and about 4 to I
By treating with O, efficient fusion can be achieved. The fused cells are hyboxanthine-aminopterin-
Thymidine medium [HAT medium; Nature, 256, 49
5 (1975)], etc., selective propagation can be performed.

The culture supernatant of the proliferated cells can be screened to determine whether the desired antibody is produced or not, and the antibody titer can be screened as follows. That is, in this case, as a first step, the presence or absence of antibody production against the immunized peptide is determined by radioimmunoassay (RIA) or enzyme immunoassay (E).
This can be investigated using methods such as the IA) method, but various modifications of these methods are also possible. As an example of a preferred measurement method, one method using EIA will be described. For example, a rabbit anti-mouse immunoglobulin antibody is coupled to a carrier such as cellulose beads according to a conventional method, and the culture supernatant to be measured or mouse serum is added thereto, and the mixture is incubated at a constant temperature (approximately 4 to 40°C) for a certain period of time. (The same applies below). After this, the reaction product is thoroughly washed, and an enzyme-labeled peptide (purified after coupling the enzyme and peptide according to a conventional method) is added, and the reaction is allowed to proceed for a certain period of time at a constant temperature. After thoroughly washing the reaction product, an enzyme substrate is added and allowed to react for a certain period of time at a constant temperature. Thereafter, the colored product produced can be measured by absorbance or fluorescence.

It is desirable to clone cells in wells that have proliferated in the selective medium and have antibody activity against the peptide used for immunization using a limiting dilution method or the like. By similarly screening the supernatant of the cloned cells and increasing the number of cells in wells with high antibody titers, monoclonal antibody-producing hybridoma clones that exhibit reactivity with the immunized peptide can be obtained.

Hybridomas thus cloned are grown in liquid culture. Specifically, for example, a liquid medium such as RPM T-1640 [Moore.

G, E, et al, Journal of the American Medical Association (J, Am,
Med.

^5soc,) 199. 549 (1967)] with about 0.1 to 40% bovine serum added to about 2 to IO.
The monoclonal antibody can be obtained from the culture solution by culturing for days, preferably about 3 to 5 days. Alternatively, antibodies can be obtained by intraperitoneally inoculating the cells into a mammal, proliferating the cells, and collecting ascites fluid.

For this purpose, for example, in the case of mice, about 1 x l
O' to IxlO', preferably about 5XI05 to 2X
Inoculate 10' hybridomas intraperitoneally, approximately 7 to 2
Ascites fluid is collected after 0 days, preferably about 10-14 days.

Antibodies produced and accumulated in ascites can be detected by, for example, ammonium sulfate fraction, DEAE
- Monoclonal antibodies can be easily isolated as pure immunoglobulins by cellulose column chromatography or the like.

In this way, a monoclonal antibody that specifically binds to bFGF is obtained.

The monoclonal antibody of the present invention specifically binds to bF'GF' of the immunogenic peptide.

The monoclonal antibody of the present invention may bind to bF(,P) different from the immunogenic peptide used during production.

The monoclonal antibody of the present invention is a monoclonal antibody directed against the immunogenic peptide bFGF or its mutin.

The monoclonal antibody of the present invention is a monoclonal antibody that specifically binds to bF GF, which has the property that the antibody binds only to bl:'GF' (and its mutin).

As shown in Example 3 below, when human bFGF is used as an antigen, a monoclonal antibody belonging to the rgM immunoglobulin class may be obtained.

Since the monoclonal antibody of the present invention specifically binds to bFGF, it is extremely useful as a reagent for measuring bFGF. Furthermore, facilitating the measurement of bF'GF in living organs and tissues is extremely useful from the perspective of obtaining basic knowledge regarding bFGF (eg, biodistribution). Detection of bFGF in nine tissues of living organs is usually carried out by enzyme immunoassay (ERA method) or the like, or by fluorescent antibody method or radioimmunoassay (RIA method).

In addition, the protein Western blotting method is effective in determining the size of bFGF present in each tissue of these organs. This method involves performing acrylamide electrophoresis on a crude extract derived from one organ tissue or a partially purified sample thereof, and then
Transfer to a membrane filter and detect with RP-conjugated anti-bFGF antibody.

Further, it is thought that some cancer cells themselves produce bFGF and continue to proliferate due to the bFGF. When an anti-bF'GF' antibody is applied to such cancers, bF'GF, which promotes proliferation, is neutralized, and it is expected to inhibit the proliferation of cancer cells, that is, act as an anticancer substance. In addition, in cancers that produce bFGF, antibodies can be used to
GF can be quantified, and it can also be applied as a diagnostic test for cancer. Furthermore, by utilizing the binding ability between the antibody and bFGF, an antibody affinity column can be prepared and used as a reagent for bF'GF purification.

EIA used to detect and quantify bI'GF'
For the RIA method or RIA method, for example, purified antibodies are
．． 1 to 10 μg/well, immobilized on a carrier such as a 96-well plastic plate (eg, Nunc, Immunoplate, Denmark), glass beads, plastic beads, or the like. Fixation is carried out in the case of plastics by reacting at about 4° C. overnight or at room temperature for about 0.5 to 4 hours. For glass, e.g. Proc, Natl, Acad
, Sci, USA, Vol. 80.

3513-3516 (1983). In addition, various commercially available plates for immobilizing antibodies (Nunc Co., Ltd., etc., as mentioned above) can also be used.

A solution containing the antigen bFGF (or its mutin) is added to the plate or beads on which the antibody has been immobilized as described above, and an adsorption reaction is performed. The adsorption reaction may be carried out at room temperature for about 0.2 to 2 hours, preferably at about 4°C - overnight.

After the antigen-antibody binding reaction, an enzyme-labeled (in the case of EIA) or radiolabeled (in the case of RrA) antibody is added to perform an adsorption reaction. Horseradish peroxidase (1-(RP), alkaline phosphatase, etc.) are often used as enzymes, and 12aI is often used as a radiolabel.For enzyme-labeled enzymes, reaction substrates such as ■2,2 in the case of IRP are often used. 'Azinodi-[3-ethylbenzothiazolinsul71nate (6)) (2,2'
-Adino-di[3-ethylbenzoth
iazoline 5ulronate (6)), etc., and measure the absorbance. For radiolabeled samples, unbound radioactivity is measured using a scintillation counter. The absorbance or radioactivity of the sample can be quantified by comparing it to a value for a known amount of bFGF.

Sandwich ET sandwiching the antigen between the above two types of antibodies
In the case of method A, the competitive EIA method and the indirect ElΔ method, which are known per se, are carried out. In the competitive EIA method, an antibody is immobilized on a carrier, and an enzyme or radiolabeled antigen bFGF and a sample are added, reacted, and quantified. The reaction conditions and the measurement of the amount of label are carried out in the same manner as described above. Indirect ErA
As a method, a sample is reacted with an unfixed antibody, and the unadsorbed antibody is quantified using an antigen-fixed plate and an anti-mouse labeled antibody. The reaction conditions and the measurement of the amount of label are carried out in the same manner as described above.

In order to purify bFGF, the purified antibody is coupled to a suitable carrier such as activated agarose gel beads according to a conventional method, and then filled in a column and cultured with culture supernatant or crushed bacterial cells. A sample containing rllbFGF is applied to an antibody affinity column, adsorbed, washed, and then treated with a chaotropic reagent such as KSCN (potassium thionoanate), or in weakly acidic conditions that do not deactivate bFGP. Efficient purification can be achieved by elution methods and the like.

An antibody column can be produced by the following method, for example, by coupling the monoclonal antibody of the present invention purified from ascites inoculated with a hybridoma to an appropriate carrier.

The carrier used may be any carrier as long as bFGF can be specifically and efficiently adsorbed after coupling and then appropriately eluted, such as agarose, cellulose or acrylamide polymers. An example of this is polyacrylamide gel beads activated to facilitate binding of primary amines of proteins.
For example, Affigel-10 (manufactured by Bio-Rad) is conveniently used in the manner described below. Affigel-
The reaction between 10 and the antibody is about 0.001 to IM.

The reaction is preferably carried out in a buffer such as about 0.1M bicarbonate. The reaction conditions are about 0° to 20°C, about IO minutes to 24 hours, various pH values are possible, but preferably,
Conditions of about 4° C., about 4 hours, and pH about 3 to IO are used. The ratio of Affi-Gel-10 and antibody to be mixed can be adjusted within this range, up to approximately 50 mg of antibody per Affi-Gel I, as the larger the amount of antibody, the more antibody will be attached, but it may be adjusted to suit binding efficiency and affinity column chromatography. Approximately 10 to 30 mg of antibody is conveniently used considering the purification efficiency in graphics. After washing the antibody-carrier conjugate thus prepared thoroughly with the buffer used in the reaction, it is left to stand for several days, or it is preferably treated with a primary amine such as ethanolamine or glycine hydrochloride at a final concentration of about 005-010M. The remaining compounds are added and allowed to react at about 4° C. for about 1 to 4 hours. Alternatively, use as an antibody column by blocking remaining unreacted active groups by incubating 1-5% live blood TN albumin (BSA) or other protein at 4°C, and then packing it into an appropriate column. You can do it.

When purifying with the antibody column described above, for example, b
Dissolve the PGF protein-containing bounty in a near-neutral buffer, such as phosphate buffer or Tris/HCl buffer, and adsorb it onto an antibody column. Next, after washing the column with the same buffer, bFGF is eluted. Examples of eluents include weakly acidic solutions such as acetic acid solutions, solutions containing polyethylene glycol,
A solution containing a peptide that binds more easily to the antibody than the sample.

A highly concentrated salt solution or a combination thereof can be used, and preferably one that does not promote the decomposition of bFGF too much.

The column eluate is neutralized with a buffer solution in a conventional manner.

If necessary, the purification operation using the antibody column described above can be performed again.

In this way, substantially pure FGF is obtained that is substantially free of pyrodiene and endotoxin. The substantially pure FGF of the present invention includes one having a protein content of 90% (tv/v) or more of bFGF, more preferably 95% (w/w) of hbFGF.

The bFGF protein solution obtained here is subjected to dialysis, and if necessary, it can be made into a powder by freeze-drying.

Stabilizers such as sorbitol, mannitol, dextrose, maltose, and glycerol can be added during freeze-drying.

HBL"GF obtained in this way has the effect of promoting the proliferation of nematoblasts, preventing the proliferation of endothelial cells, and angiogenesis, and has low toxicity, so it can be used in burns, wounds, and after surgery. It can be used as a healing accelerator for tissues, etc., or as a therapeutic agent for thrombosis, arteriosclerosis, etc. due to angiogenic action.It can also be used as a reagent for promoting cell culture.

To use the hbFGF as a medicine, it can be used as a powder as it is, or in a pharmaceutical composition (e.g., one tablet for injection, a capsule, a liquid, an ointment) together with other pharmacologically acceptable carriers, excipients, and diluents. It can be safely administered parenterally or orally to warm-blooded mammals (eg, humans, mice, rats, hamsters, rabbits, dogs, cats).

Injection preparations are carried out in a conventional manner using, for example, physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules may also be prepared according to conventional methods.

When the hbFGF is used as the above-mentioned medicine, for example, about 1 ng to 100 μg/kg of 1B1 is administered to the above-mentioned warm-blooded mammals, taking into consideration the route of administration, symptoms, etc.
An appropriate dose is selected and administered.

Furthermore, when the hbF G F is used as a reagent for promoting cell culture, the medium Il! Approximately 0.01-
It is preferable to add it to the medium in an amount of 10 μg, more preferably about 0.1 to 10 μg.

In addition, the mutin of hbFGF is also the above-mentioned hbF GF
It can be used in the same way.

In the specification and drawings of the present invention, when bases, amino acids, etc. are indicated by abbreviations, IUPAC-IUB Co
n++++1sion on BiocheIIl
The abbreviations are based on the abbreviations based on the ical Nomenclature or the abbreviations commonly used in the field, examples of which are given below. In addition, when an amino acid can have optical isomers, unless otherwise specified, it shall indicate the unity.

DNA: Deoxyribonucleic acid cDNA: Complementary deoxyribonucleic acid NA ATP TTP G T P CTP TP dr DTA DS 1y la al eu ie er Thr: Adenine: Thymine: Guanine: Cytosine: Ribonucleic acid: Deoxyadenosine triphosphate: Deoxythymidine triphosphate Acid: Deokinganosine triphosphate/deoxycytidine triphosphate: Adenosine triphosphate: Thymidine: Ethylenediaminetetraacetic acid nidodecyl sulfate sodium Glycine: Alanine: Valine: Leucine; Isoleucine: Serine: Threonine Cys Nijistine Met: Methionine Glu: Glutamic acid Asp + Aspartic acid Lys : Lysine Arg : Arginine His : Histidine Phe : Phenylalanine Tyr : Tyrosine Trp Nitributophane Pro Nibroline Asn + Asparagine Gln : Glutamine In the reference examples described below, the numbers of the constituent amino acids of human bFGF are as described in Moe's In the amino acid sequence ([r), X is Th
In the amino acid sequence in which Met is added to the N-terminus of a peptide where r and Y h < S er, the Met is counted as the first.

Mouse HbF99 cells, mouse HbF'+61 cells and mouse HbF I obtained in Example 2 (3) described below
Each of the 65 cells has been deposited with the Research Institute for Fermentation (rFo) since January 28, 1986 under the following accession numbers.

Mouse I (bF99 cells: IFO50122 Mouse HbFI 61 cells: IPO50123 Mouse I (bF
I65 cells: IPO50124 (mouse hybridoma obtained in Example 4 below) (bFI 2.H
bP52. HbP78 and HbF98 are respectively
It has been deposited with IF'O since August 17, 1988 under the following accession number.

Mouse HbF I2 cells: IFO50142 Mouse H
bF52 cells: [PO50143 mouse HbF 78 cells: IFO50144 mouse) rbF 98 cells: IP
o 50145 Among the transformants produced in the Reference Examples and Examples shown below, those with accession numbers are certified by the Institute for Fermentation (IFO) and the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry. (FR■). Please check their accession numbers and mills as follows:
Shown in the table. In addition, in Table 1, if a FERM P number and a FERM BP number are listed together, the deposit was originally deposited domestically and a deposit number indicated by the PERMP number was attached, and the deposit was changed to a deposit based on the Budapest Treaty. It is assigned an accession number indicated by the F'ERM BP number and is stored at the same research institute (r'RI).

(Left below) Kanuratane: The present invention will be explained in more detail with reference to the following examples.
The present invention is not limited to these.

Reference Example + (Construction of a plasmid containing the gene encoding hbFGP) (1) Single # of cDNA-containing plasmid: cDNA synthesized from human foreskin-derived primary cultured cell mRNA was used as a CD vector [Okayama et al., Molecular &amp;・Cellular Biology (Molecular a)
nd Ce1lular Biology), 3,
280 (1983)] using E. coli x1776 as a host.
ional In5titude of Chi
ld Healthand) - 1 time man D
development, Bethesda.

Received a grant from Dr. Okayama of U.S.A. This cDN
Alkaline method from A library (Birlbojm.

H, C, & Doly, J.
Research (Nucleic Ac1ds Re5e
arch), 1.

1513 (1979)] to extract plasmid DNA,
This DNA was used to infect Escherichia coli I) 1-E 1, and approximately 2
A cDNA library consisting of ×10 clones was created using E. coli Dr(I) as a host.

The cDNAΔ library using E. coli DHI was filtered through a nitrocellulose filter (Millipore).

1 - Approximately 5 X I O' on (A TF filter)
Clone/Sow 10 sheets to form a filter,
A total of 20 replica filters, each consisting of two master filters, were made. 0.5N of E. coli on this replica filter
Dissolve and expose denatured plasmid DNA in NaOH solution
was fixed on the filter [Grunstein, M.
& Ilogness, D. S., Proc.

Natl, Acad, Sci, USA 72,
3961 (1975)].

On the other hand, [P
roc, Natl, Acad, Sci, USA
82゜6507 (1985)] Amino acid No. based on the amino acid sequence of bovine basic fibroblast growth factor.

13-20 (Pro-Pro-Gly-His-Phe
-Lys-Asp-Pro) and amino acid No. 8
9-96 (Thr-Asp-GIu-Cys
-Phe -PhePhe-Glu), base sequences corresponding to these amino acid sequences (some codons are 3
Fix the th character arbitrarily. 5'GGA/each
TC'r/cTTA/GAA/GTG to CCNGG 80G, and 5 to TC/GA//GAA〆 A A/AAA/GCAT/TCGTCGGT.

GC--Underlined bases indicate fixed bases. ) was chemically synthesized. This oligonucleotide was treated with a reaction solution of 50 μQ using T4 polynucleotide kinase (manufactured by Takara Shuzo) [0.1 Mg of oligonucleotide.

50IIIM Tris-)ICI pHB, 0.
I OmMgCIt, I OmM Mercaptoethanol, 50p Ci 7-”F ATP (>
5000 Ci/mmole).

3 units T4 nucleotide kinase]
℃ for 1 hour, and the 5' end of the oligonucleotide was
Labeled with 1p.

Two types of oligonucleotides labeled by the above method were used as probes and were separately associated with a replica filter on which DNA was immobilized. The association reaction was performed using 5X SSPE [I 80mM NaCl, 10mM
Na1l, PO, ImM EDTA (pi-17,
4)], 5 x Denhardt's, 0;
I%SDS.

3 in 100 μg/- of denatured salmon sperm DNA solution 10-
The reaction was carried out at 5°C for 16 hours, and after the reaction, the filter was
, I 5M NaCl, 0.015MS odiun
citrate] O,I% in SDS solution at room temperature.
Washed 3 times for 0 minutes at 45°C, and 2 times for 30 minutes at 45°C [T
, Maniatis et al., “Molecular Ct.
oning”Co1d Spring 1larb
or Laboratory, P.

309 (February 1982).

Take a radioautogram from the washed filter, and superimpose the radioautograms of two replica filters for one set of bacterial strains that react to both of the two types of probes4.
I searched for it by working. By this method, 5 X I
fffg [E scherichiacolt] reacts to two types of probes from Oclonies.
K12 DHI/pTB627 (IFO+4494
．． FERM BP-1280)] was obtained.

(2), the bacterial strain obtained in (1) above [E 5cherich
ja coliK12 DI port/pTB627 (
IFO14494, FERM BP-1280) was used to extract plasmid DNA (pTB627) using the alkaline method.
Nucleic Assists Research (Nucleic＾
cids Research), I, 1513
(1979)).

(3) The base sequence of cDNA encoding hbF'GF was synthesized using the dideoxynucleotide synthetic chain termination method [J.

Messing et al., Nucleic Acids Research), 9.
．． 309 (1981)] and the amino acid sequence deduced from that sequence is shown in FIG.

Reference Example 2 (Expression of hbFGF-encoding gene in E. coli) Construction of hbFGF expression plasmid pTf3669: Plasmid pTB627 containing hbFGP cDNA obtained in Moeki Reference Example 1 (2) was digested with restriction enzymes Aval and B.
A 0.44 Kb DNA fragment containing the region encoding hbF GF was obtained by cutting with a1l. An 8glll linker-pCAGATCTG was ligated to the Ba1l cleavage site (blunt end) of this DNA fragment using T, DNA ligase to separate a 0.44 Kb Aval-BglII DNA fragment. This 0.44Kb Ava113gll
l React the DNA fragment with T and DNA ligase and
After linking the glII cleavage sites, a DNA polymerase (Klenow fragment) reaction was performed in the presence of dXTP to blunt the Aval cleavage site. This DN
The synthetic oligonucleotide after the phosphorylation reaction is added to the A fragment.
"AA'l'TCTATGCCAGCATTGC" and "GCAATGC1'GGCATAG" were ligated with 74 DNA ligase and cut with EcoRI-Bgl It to prepare an approximately 0.46 Kb DNA fragment.Meanwhile,
Plasmid ptrp781 with trp promoter
(Kurokava, T. et al. Nucleic Δcids Res.
, )I I , 30773085(+983))D
NA was cut with Pstl and blunted by T, DNA polymerase reaction.

The Econ
By cutting with r-r3gl II, an approximately 3.2 Kb DNA fragment containing the trp promoter tetracycline resistance gene and the plasmid replication initiation site was isolated.

0.46 containing the gene region encoding hbFGF
Kb EcoRI-Bgl [DNA fragment and this 3.
The 2Kb DNA fragments were ligated by T4 DNA ligase reaction to create hbFGF expression plasmid pTB669.
was built.

By transforming Escherichia coli DHI using this plasmid pT8669, a strain E5cherichia col 1DHI/ containing plasmid pTB669 was obtained.
pTB669 was obtained.

Furthermore, in the same manner, using pTB669, E. coli K
12 MM294. Alternatively, transform C600 to Escherichia coli K Ig
MM294/1) TB669 (IFO14532°FE
nM BP-1281), E. coli C6
00/pTI3669 were obtained, respectively.

Reference Example 3 (Human basic fibroblast growth factor (hbFG)
F) Purification) Escherlchia col obtained in Reference Example 2
i K IgMM294/I)T[3669(IFO1
453,2°FERM BP-1281) with 1% glucose.

M9 medium containing 0.4% casamino acids, 8 μg/ln1 tetracycline [T. maniatis (T.

Maniatis et al., Molecular Cloning
Laboratory-v Manual, (Maniatis, T.
, et alMolecular Cloning
(1982), A Laboratory Manu
al, Col1d Spring l1arborL
aboratory, Co1d Spring
Irabor NY.

USA)], and when the K 1ef value reached approximately 200, 3β indolyl acrylic acid was added at a concentration of 25 μg/d, and the cells were further cultured for 4 hours. After culturing, the bacterial cells were collected and treated with 20mM Tris・HCI at I/20m,
It was suspended in a pH 7.6, I 0% sucrose solution.

Add phenylmethylsulfonyl fluoride (
PMSF) Ia+M, EDTA 10mM, Na
C1 at 0, 1 M, spermidine hydrochloride at OmM
, lysozyme was added at a concentration of 100 μg/− (all final concentrations), and after standing at 0° C. for 45 minutes, ultrasonication was applied for 30 seconds. This solution was centrifuged for 30 minutes at 1800 Orpm (serval centrifuge, 5S34o-tar) to obtain a supernatant, which was used as a bacterial cell extract.

20ml of this extract (prepared from 500d of culture solution)
M Tris-HCI pH7,6,0,2MNa
DEAE cellulose (DE52.
Whatman Co.) column, (diameter 2XIOc+n),
Nucleic acid components in the extract were removed. The flow-through from the column and 20 mM Tris-HCI, pH 7,
The column washes with 6,0,2M NaCl solution were combined and collected (DEAE pass-through fraction 44d).

This fraction 14- was applied to a heparin column 5hodex A
F-pak HR-894 (8mIIIDx5c
The mixture was applied to a high performance liquid chromatography apparatus (Gilson Co., Ltd.) equipped with a high-performance liquid chromatography system (M, manufactured by Showa Denko). The column was coated with 20mM Tris.
-I-rcl pH 7,6 solution 5 then 20mM Tr
is-HCI pH7, 6,0,5M NaCl solution followed by 20ltlM Tris-HCI pH
7,6, Linear gradient elution from 0.5M to 2M NaCl in buffer
elution, 60! , flow rate 1.0-/ml).

The hbF GP eluted by this operation was 5DS-
It showed a single band on polyacrylamide gel electrophoresis and was found to be well purified and suitable for use as an antigen. The test for hbFGF was conducted under the following conditions.

D containing mouse BALB/c3T3 cells with 5% calf serum
Tank 96 six microtiter plates with MEM medium (
2 x 10'' seeds per hole (flat bottom) were seeded in 0.2 medium and cultured the next day.

The medium was replaced with DMEM medium containing 0.5% calf serum.

After culturing for 3 days, the bacterial cell extract was serially diluted 5 times with DME medium containing 0.5% BSA, and the IQ was determined per well.
μ was added, cultured, and after 20 hours, 'H-Tdr (5C
i/a+mol, 0.5mC1/藏 RCCAmers
ham) was added to each of the 6 wells. Zero cells after 6 hours
．． The cells were peeled off by treatment with phosphate buffer (PI3S) containing 2% trypsin and 2% EDTA, and the cells were collected on a glass filter using a titer tick cell harvester to determine the amount of 3H-thymidine incorporated into the cells. was measured using a scintillation counter.

Reference Example 4 (Production of recombinant DNA having a nucleotide sequence encoding mutin) (1) Cloning of M13 vector of human bFGF gene: Plasmid pT and B669 obtained in Reference Example 2 were digested with restriction enzymes ECOR [and BamHI]. It was digested with Phage vector MI3mp8 (Clay Messing (JMes)
sing), Methods in Engineering 101
．． 20-78 (1983)) replicative (RF) DNA was digested with restriction enzymes EcoRI and Ball1ltl, and pTB that had been previously digested with EcoRI and BamHI was prepared.
669-derived human IGF' DNA fragment.

The mixture is then ligated with T4 DNA ligase, and the ligated DNA
A was transformed into Escherichia coli-JMI05 strain capable of infection, and the cells were plated on a plate using xgat as an indicator scale [Clay Metzing et al., Nucleic Acids Research (Nucleic Ac1ds R)]
) (1981) Vol. 9, pp. 309-321], the plaques containing the recombinant phage (white plaques) were picked up, and the nucleotide sequence of the recombined portion was determined using the dideoxynucleotide synthetic chain termination method (J. Messing et al., Nucleotid Technologies, Inc.). Research (Nucleic A
c1ds Res, )9, 309 (+981))
It was confirmed that the human bFGF DNA was correctly inserted as determined by .

Single-stranded phage DNA from this M13PO clone
A was purified and used as a template for site-directed mutagenesis using synthetic oligonucleotides.

(2) Site-directed mutagenesis 0, 1 mM adenosine triphosphate (ATP), 50 mM
Hydroxymethylaminomethane hydrochloride (Tris-HC1
) pH 8,0, I OmM MgCIt, 5mM dithiothreitol (DTT) and in the presence of 9 units of T4 kinase, synthesize oligonucleotides 5et OCT <3' (brimer (restriction enzyme) to change cys26 to Ser) in 50 μρ. (Rsal recognition sequence disappears.) 40 picomole was treated with T4 kinase at 37°C for 1 hour.50
iM NaCl, l, On+M tris summer fcI,
pH 8,0% 10mM MgCIt and 10mM
Mixture containing β-mercaptoethanol 50μσ
5 μg of single-stranded (ss) M l 3-PO DNA by heating the kinased brimer (12 pmol) at 67° C. for 5 min and 42° C. for 25 min.
were allowed to hybridize. The annealed mixture was then cooled over water and treated with 0,5 mM dideoxynucleotide triphosphates (dNTPs), 80IIIM Tris-MCI, p
H7,4, 8mM MgCIt, 1001M Na
C1, 9 units of DNA polymerase IKlenow fragment;
0,5 mM ATP and 2 units of T4 DNA licase were added to 50 μa of the reaction mixture and incubated at 37°C.
3 hours at 25°C and 2 hours at 25°C, 0.2mM E
DTA2uQ was added to stop the reaction. It was used to transform JM 105 cells capable of infection, the bacteria were grown overnight, and 5sDNA was isolated from the culture supernatant. This 5sDNA was used as a template for the second cycle of brimer extension, and the gel-purified RF-type DNA was transformed into JMI O5 cells capable of infection, plated on agar plates, and cultured overnight, resulting in phage plaques. Obtained.

(3) Site-directed mutagenesis: Repeat the procedure in (2) above, but change the synthetic oligonucleotide primer used from one that encodes cysteine 70 to one that encodes serine, and change Hae ■ C 3. ′. (A recognition sequence for the restriction enzyme Haell is generated.) (4) Site-directed mutagenesis: Repeat the procedure in (2) above, except that the synthetic oligonucleotide primer used encodes cysteine 88. It encodes serine from /MuT AGT <3'. (A recognition sequence for the restriction enzyme Alul is generated.) (5) Specific site-directed mutagenesis: Repeat the procedure in (2) above, except that the synthetic oligonucleotide primer used encodes cysteine 93. It is assumed that 1nfl TCC<3' is used to encrypt serine. (A recognition sequence for restriction enzyme 1-1ir+4I is generated) (6) Sieving and identification of mutagenic thickened plaques: Plates containing mutated M13-PO plaques (see item (1) above) and unmutated M13-PO
The two plates containing phage plaques were cooled to 4°C, and the plaques from each plate were layered onto two nitrocellulose round filters, in the case of the first filter, the dried filters were layered onto an agar plate for 5 min. For the second filter, the transfer was carried out for 15 minutes. Next, 0.2N Na
Oh.

1.5M NaCl and 0.2% Triton X-100
Place the filters on thick filter paper soaked in water for 5 minutes, then add 0.5M Tris-HC1, pH 7, 5, and 1.5M
Neutralization was achieved by layering on filter paper soaked in NaCl for an additional 5 minutes.

The filters were washed twice in a similar manner on filters soaked in 2X SSC (Xianxian Citrate), dried, and dried in a vacuum drying oven at 80° C. for 2 hours. Add duplicate filters to toy DNA hybridization solution (
5x s S C), I) H7,0,4X Denhard's solution (polyvinylpyrrolidone.

Ficoll and bovine serum albumin, 1× = 0.02% each
), O, 1% sodium dodecyl sulfate (S D S
).

50mM sodium phosphate buffer, pH 7, 0 and 100
Prehybridization was performed with μg/− of denatured salmon sperm DNA for 4 hours at 55°C. Oligonucleotide primers were hybridized to 105 cpm/- for 24 hours at 42°C. Filters were washed at 50° C. for 30 minutes each in wash buffer containing 0.1% SDS and decreasing amounts of SSC. Filters were first washed with buffer containing 2X SSC and control filters containing non-mutated M13-PO plaques were tested for the presence of radioactivity using a Geiger counter. 5sceti was reduced stepwise and the filters were washed until no detectable radioactivity remained on control filters with unmutated to 113-PO plaques. The minimum concentration of SSC used is 0. lX
It was 5SC. Filters were air dried and autoradiographed at -70°C for 2-3 days. 10,000 mutated M13-PO plaques and 100 unmutated control plaques were kinased and screened with the Jniolibonucleotide probe 1. In the control plaques there was no hybridization with the probe, whereas in the mutated M13PO plaques 3 to IO
individuals formed hybrids with the probe.

Taking the mutant M13-PO plaque IIIJ,
It was inoculated into JMI05 culture medium. 5sDNA was prepared from the supernatant, and 2 m(ds) DNAs were prepared from the bacterial pellet. Appropriate oligonucleotide primers and 5sDN
The base sequence was analyzed using A.

As a result, the TGCCCYS26) codon is changed to TCT(Se
r) Converted to a codon, TGT (cys70) codon converted to AGC (Set) codon, TG
The T (cys88) codon has been converted to a TCT (Set) codon, and the TGT (cys93) codon has been converted to a TCT (Set) codon.
It was confirmed that each codon had been converted to a (Set) codon.

Among the mutated Ml3-PO phages, the codon Cys-
26 is set as Ml3-PI.

Ml 3- with codon CYS-70 set
P2. : A set of 1 ton Cys-88 is M
l 3-P3. The one in which the codon Cys-93 was set was designated as M13-P4.

Reference Example 5 Expression of a gene encoding human bFGF mutin in E. coli: (1) Plasmid pT for human bFGF mutin expression
Construction of B739 The replicative form (RF') of Ml3-PI obtained in Reference Example 4 was treated with restriction enzymes EcoRr and Pst.
An approximately 0.5 Kb DNA fragment containing the human bFGF mutin-encoding region was obtained.

On the other hand, plasmid ptrp with trp promoter
781 (Kurokawa, T. et al. Nuclic Acids Research)
ds Res, )-U Engineering, 3077-3085
(+983)) DNA to Econ l -Pstr
3.2 containing the trp promoter, tetracycline resistance gene and plasmid replication initiation site.
Kb DNA fragment was isolated. The 0.5 Kb E containing the gene region encoding the mutin of human bFGF
coRI-PstI DNA fragment and this 3.2K
b DNA fragments were ligated by T4 DNA ligase reaction to create plasmid pTB7 for human bFGF mutin expression.
39 was constructed.

Transform Escherichia coli DHI using this plasmid pTB739.E5ch strain containing plasmid pTB739 containing the gene encoding mutin.
erichia col i DHI/pT
B739 (IFO14575, FERM BP-16
41) was obtained.

(2) Preparation of bacterial cell extract The transformant obtained in (1) above was mixed with 1% glucose,
The cells were cultured in M9 medium containing 0.4% casamino acids and 8 μg/n1 of tetracycline, and when KIett15 reached approximately 200, 3β indolyl acrylic acid was added to 25 μg/rn1 and cultured for an additional 4 hours. After culturing, the bacterial cells were collected and treated with 20mM Tris-
Added to HCl, pH 7, 6, 10% sucrose solution. To this suspension, phenylmethylsulfonyl fluoride (PMSF) was added at ImM, EDTA was added at I OmM,
NaCl 0.1M, spermidine hydrochloride 10mM,
Lysozyme was added at a concentration of 100 μg/d (all final concentrations), and after being left at 0° C. for 45 minutes, ultrasonication was applied for 30 seconds. This solution was spun at 18,000 rpm (Serval centrifuge).

The mixture was centrifuged for 30 minutes to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract D containing 5% calf serum of mouse BΔLB/c3T3 cells
Tank 96-well microtiter plate with MEM medium (
2XIO' cells per hole (flat bottom) were seeded in a 0.2 medium and cultured on the next day.

The medium was replaced with DMEM medium containing 0.5% calf serum.

After culturing for 3 days, the bacterial cell extract was serially diluted 5 times with DME medium containing 0.5% BSA, and 10 microorganisms were added per well.
uρ was added, cultured, and 20 hours later, 3H-thymidine (
5C4/mmol, 0.5mC1/KuraRCCA mer
2 μQ of sham) was added to each person. After 6 hours, the cells were peeled off using a phosphate buffer solution (PBS) containing 0.2% trypsin and 0.2% EDTA, and collected on a glass filter using a titer tick cell harvester. The amount of 3H-thymidine incorporated was measured using a scintillation counter.

As a result, the bacterial cell extract of E. coli DHI/pTB739 showed FGF activity.

In this way, Cys at position 26 of human bFGF is 5e
rf:substituted mutin C8I was obtained.

Reference Example 6 (Expression of gene encoding human bFGF mutin in E. coli) (1) Plasmid p for human bFGF mutin expression
Construction of TB742 The replicative form (RF) of M2S-P2 obtained in Reference Example 4 was treated with restriction enzymes EcoRI and Pstl.
An approximately 0.5 Kb DNA fragment containing the human bFGF mutin-encoding region was obtained.

On the other hand, plasmid ptrp with trp promoter
781 DNA was cut with EcoR[-PstI and t
Approximately 3.2 Kb DNA containing rp bromocouple, tetracycline resistance gene and plasmid replication initiation site
The fragments were separated. The 0.5Kb EcoRI-Pstl containing the gene region encoding the mutin of human bFGF
DNA fragment and this 3.2Kb DNA fragment
4 DNA ligase reaction to construct plasmid 1) TB7-12 for human bFGF mutin expression.

By transforming E. coli DI11 using this plasmid pTB742, a strain E5 containing plasmid pTI3742 containing the gene encoding mutin was obtained.
cherichia coli DHI/p
TB742 (IFO14584, FERM BP16
42) was obtained.

(2) Preparation of bacterial cell extract The above transformant was cultured in the same manner as in Reference Example 5 (2) to obtain superl+W, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Regarding the bacterial cell extract obtained in (2) above, Reference Example 5 (3)
) Human bFGF activity was measured in the same manner.

As a result, the bacterial cell extract of B. coli DHI/pTB742 showed FGF activity.

In this way, Cys at position 70 of human bFGF becomes Se
A mutin CS2 substituted with t was obtained.

Reference Example 7 (Expression in Escherichia coli of gene encoding human bFGF mutin) (1) Construction of plasmid pTB743 for expressing human br'CF mutin Replicative form of M+ 3-P3 obtained in Reference Example 4 above (RF) with restriction enzymes EcoRI and Pst
An approximately 0.5 Kb DNA fragment containing the human bFGF' mutin-encoding region was obtained by cleaving with r.

On the other hand, plasmid ptr with trp promoter
p781 DNA was cut with EcoRI-PstI to obtain approximately 3.2 Kb DNA containing the trp promoter, tetracycline resistance gene and plasmid replication initiation site.
Fragment A was isolated. The 0.5Kb EcoRI-Ps containing the gene region encoding the mutin of human bFGF
tl DNA fragment and this 3.2Kb DNA fragment
4 DNA ligase reaction to construct a plasmid pTB743 for expressing human bFGF mutin.

Plasmid containing the gene encoding mutin by transforming E. coli DHI using this plasmid pTB743 1) Esch strain containing TB743
erichia coli D Hl /pTB7
43 (IFO14585, FERM BP-1643
) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Regarding the bacterial cell extract obtained in (2) above, Reference Example 5 (3)
) Human bFGF activity was measured in the same manner.

As a result, E, colt DHl/pTB 743
The bacterial cell extract showed FGF activity.

In this way, Cys at position 88 of human bFGF becomes Se
Mutin CS3 substituted with r was obtained.

Reference Example 8 (Expression of gene encoding human bFGP mutin in E. coli) (1) Plasmid p for human bFGF mutin expression
Construction of TB744 The replicative form (RP) of M13-P4 obtained in Reference Example 4 was treated with the restriction enzymes Ecofl [and Pst
[] to obtain an approximately 0.5 Kb DNA fragment containing the human bFGF mutin-encoding region.

On the other hand, plasmid ptrp with trp promoter
781 DNA was cut with EcoRI-PstI to obtain approximately 3.2 Kb DNA containing the trp promoter 7 tetracycline resistance gene and plasmid replication initiation site.
The fragments were separated. The 0.5KbEcon I-Ps containing the gene region encoding the mutin of human bFGP
The tl' DNA fragment and this 3.2 Kb DNA fragment were ligated by T4 DNA ligase reaction to construct a plasmid pTB744 for expressing human bFGF mutin.

By transforming Escherichia coli DHI using this plasmid pTB744, strain E5ch containing plasmid pTB744 containing the gene encoding mutin was obtained.
erichia col i D H1/ p'r
B744 (IFO14586, FERM BP-16
44) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Regarding the bacterial cell extract obtained in (2) above, Reference Example 5 (3)
) Human bFGF' activity was measured in the same manner as in (2009).

As a result, the bacterial cell extract of E. coli DHI/pTB744 showed PGF activity.

In this way, Cys at position 93 of human bFGF becomes Se
Mutin C94 substituted with t was obtained.

Reference Example 9 (Screening and identification of mutagenic thickened plaques) Plates containing mutated M+3-P2 phage plaques obtained in Reference Example 4 and unmutated M+3 obtained in Reference Example 4 - Cool the two plates containing P2 phage plaques to 4°C and place the plaques from each plate onto two nitrocellulose round filters.
The dried filters were transferred onto an agar plate in a 5 minute overlay for the first filter and 15 minutes for the second filter. Next, 0.2N NaOH, 1.5M Na
Place the filters on thick filter paper soaked in C1 and 0.2% Triton X-100 for 5 minutes, then soak in 0.5M Tris-100.
Neutralization was achieved by overlapping for an additional 5 minutes onto filter paper soaked with HCI, pl-17, 5, and 1.5M NaCl. The filters were washed twice in a similar manner on filters soaked in 2xsscceA (subcitrate), dried and dried in a vacuum drying oven at 80°C for 2 hours.

Duplicate filters were prepared with 10 portions per filter of DNA hybridization buffer (5xSSC), pH 7.0, Ix Denhard's solution (polyvinylpyrrolidone, Ficoll and bovine serum albumin, lx = 0.02% each).

0.1% sodium dodecyl sulfate (SDS), 50mM
Sodium phosphate buffer, pH 7.0 and 100μg/
Speciation was performed at 55° C. for 4 hours with denatured salmon sperm DNA. Oligonucleotide primers were then hybridized for 24 hours at 42° C. using 10′ cpm. Filters were washed at 50° C. for 30 minutes each in wash buffer containing 0.1% SDS and decreasing amounts of SSC. Filters were first washed with buffer containing 2X SSC and control filters containing non-mutated M13-P2 plaques were tested for the presence of radioactivity using a Geiger counter. Filters were washed by decreasing the 5SCJ5 degrees in steps until no detectable radioactivity remained on control filters with unmutated M+3-P2 plaques. The minimum concentration of SSC used is 0. Ix
It was SSC. Filters were air dried and autoradiographed at -70°C for 2-3 days. 10,000 mutated M13-P2 plaques and 100 non-mutated control plaques were screened with a kinased oligonucleotide probe.

In control plaques there was no hybridization with the probe, whereas in mutated Ml3-P2 plaques 3~
IO hybridized with the probe.

One of the mutant M13-P2 plaques was picked up and JM
I05 culture medium was inoculated. Make 5sDNA from the supernatant and make 2 DNA from the bacterial pellet 8! (ds) Made DNA. Appropriate oligonucleotide primers and 5sDNA
The base sequence was analyzed using

As a result, the TGC (cys26) codon becomes TCT
(Ser) codon, TGT (cys8
8) The codon has been converted to a TCT (Set) codon, and the TGT (cys93) codon has been converted to a TCT (Ser) codon.
) was confirmed to have been converted into a codon.

Among the mutated Ml3-P2 furnos, the codon Cys-
26 and -70 are set as Ml 3-P
I 2. Ml3-P23.codon Cys-70 and -88 become Ser. When the codons Cys-70 and -93 become Set, they are respectively referred to as M13-P24.

Reference Example 10 (Expression in Escherichia coli of gene encoding human bFGF mutin) (1) Construction of plasmid pTB762 for expressing human bF'GF mutin Replicative form (RF) of Ml3-P23 obtained in Reference Example 9 above was treated in the same manner as in Reference Example 5(I) to obtain human bFGF mutin expression plasmid pTI37.
62 was constructed.

Using this plasmid pTB762, E. coli MM294
Strain Esc containing plasmid pTB762 containing the gene encoding mutin
herichia coli MM 294/I)
TE101 (IFO14613, PERM BP-1
645) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of V4 body extract (2) above
Human bFGF activity was measured for the bacterial cell extract obtained in the same manner as in Reference Example 5(3).

As a result, E. coli MM294/I)TB
The cell extract of No. 762 showed FGF activity.

In this way, mutin CS23 in which Cys at positions 70 and 88 of human bFGF were substituted with Set was obtained.

Reference Example II (Production of recombinant DNA having a nucleotide sequence encoding mutin) (1) Cloning of M13 vector of human bFGF gene: Plasmid pTB669 obtained in Reference Example 2 was digested with restriction enzymes EcoRI and BamH. Ta. Phage vector MI3mp8 (Jay Messing U.

Messing), Methods in Engineering 101.20-78 (+983)) Replication Type (RF) D
NA was digested with restriction enzymes EcoRr and BamHI,
pT previously digested with EC0RI and BamHI
8669-derived human bFGF DNA fragment. The mixture is then ligated with T4 DNA ligase, and the ligation D
NA was transformed into Escherichia coli-JMI05 strain capable of infecting cells, and the cells were plated on a plate using Xgal as an indicator balance [J. Messing et al., Nucleic Ac1ds Research.
Res, ) (1981) Vol. 9, pp. 309-321],
The plaque containing the recombinant phage (white plaque) was picked up and the base sequence of the recombined portion was extracted using the dideoxynucleotide synthetic chain termination method (J. Messing et al., Nucleic Acids Research).
Ac1ds Res, )9. 309 (1981)
) to confirm that the human bFGF DNA was correctly inserted.

Single-stranded phage DNA was purified from this MI3-PO clone and used as a template for site-directed mutagenesis using synthetic oligonucleotides.

(2) Site-directed mutagenesis 0, ImM adenosic acid phosphate (ATP), 50mM
M hydroxymethylaminomethane hydrochloride (Tris-HC
I) Synthetic oligonucleotide 5'-CGGGCATGAATTCGCCGCT3' [recognition site for restriction enzyme EcoRI in base sequence 40 picomole of the primer for generating Pro14 and changing Pro14 to Met was treated with T4 kinase at 37°C for 1 hour. 50mM NaCl, 1.0mM Tris-
HCl, I) + (8,0, IOmMMgCI, and l0
Mixture 5 containing mM β-mercaptoethanol
This kinased brimer (12
single-stranded (ss) Ml 3-PODNA by heating picomoles) at 67 °C for 5 min and 42 °C for 25 min.
5 μg were hybridized. The annealed mixture was then cooled over water and treated with 0.5mM each deoxynucleotide triphosphate (dNTP), 80mM Tris-I-(cI, p
l-17,4,8mM MgCl, 100mM
Add to 50 μι reaction mixture containing NaCl, 9 units of DNA polymerase I Klenov fragment, 0.5 mM ATP and 2 units of T4 DNA licase and incubate at 37°C.
3 hours at 25°C and 2 hours at 25°C, 0.2mM E
The reaction was stopped by adding 2 μQ of DTA. It was used to transform JM 105 cells capable of infection, the bacteria were grown overnight, and 5sDNA was isolated from the culture supernatant.

Using this ssDNA as a template in the second cycle of brimer extension, gel-purified RF-type DNA was transformed into infection-competent JMI05 cells, plated on agar plates, and incubated overnight to form phage plaques. was gotten.

(3) Site-directed mutagenesis: Repeat the procedure in (2) above, except that the synthetic oligonucleotide primer used generates a recognition site for the restriction enzyme Ncol in the base sequence, and at the same time generates a recognition site for the restriction enzyme Ncol. Thr
In this case, 5etlO is changed to Met, which is 5'-CGCCCATGGTGCCCATCCTC-3'.

(4) Site-directed mutagenesis: Repeat the procedure in (2) above, except that the synthetic oligonucleotide primer used contains restriction enzyme A in the base sequence.
It creates a recognition site for r1n and at the same time changes the codon encoding Lys87 to a stop codon, which is designated as 5'-TAACACCTTAAGAAGCCAG-3'.

(5) Site-directed mutagenesis: Repeat the procedure in (2) above, except that the synthetic oligonucleotide primer used has restriction enzyme 1 in the base sequence.
Generate a recognition site for 4paf and simultaneously convert Asp42 into As
n is changed to 5'-CCGGACTCCGTTAACTCGG3'.

(6) Site-directed mutagenesis: Repeat the procedure in (2) above, except that the synthetic oligonucleotide primer used is different from the restriction enzyme H in the base sequence.
The recognition site of parI is deleted, and at the same time Arg! ! 5 is changed to Gln, 5'-CTTCTCCTGGA
CTCCGTCAAC-3'.

(7) Screening and identification of mutagenic thickened plaques: Plates containing mutated Ml3-PO plaques ((+) section above) and unmutated Ml3-PO
The two plates containing phage plaques were cooled to 4°C, and the plaques from each plate were layered onto two nitrocellulose round filters, in the case of the first filter, the dried filters were layered onto an agar plate for 5 min. For the second filter, the transfer was carried out for 15 minutes. Next, 0.2N Na
OH1,5M NaCl and 0.2% Triton X-1
Place the filters on thick filter paper soaked in 00 for 5 minutes,
The filters were then neutralized by layering for an additional 5 minutes onto filter papers soaked with 0.5 M) Lith-1-ICI, p) (7, 5, and 1.5 M NaCl. Filters were coated in a similar manner with 2x SSC (
The filters were washed twice on a filter soaked in standard citrate salt, dried, and dried in a vacuum drying oven at 80° C. for 2 hours. Duplicate filters were diluted with 10 ml of DNA hybridization buffer (5X SSC) per filter, II) H7.0,
4x Denhard solution (polyvinylpyrrolidone.

Ficoll and bovine serum albumin, ■×=0.02% each
), 0.1% sodium dodecyl sulfate (SDS).

50mM sodium phosphate buffer, I) 87.0 and 10
0 μg/- of denatured salmon sperm DNA at 55°C.
time, pre-hybridization. Oligonucleotide primer 10′ cpm N at 42 °C for 24 h. Species were formed. Filters were washed at 50° C. for 30 minutes each in wash buffer containing 0.1% SDS and decreasing amounts of SSC. Filters were first washed with buffer containing 2xSSC and control filters containing unmutated M13-PO plaques were tested for the presence of radioactivity using a Geiger counter. The filters were washed in steps of 5 scA until no detectable radioactivity remained on the control filter containing unmutated M+3-PO plaques. The minimum concentration of SSC used is 0. lX5S
It was C. Air dry the filter at -70°C for 2~
After 3 days of exposure, an autoradiograph was taken. 10,000 mutated M13PO plaques and 100 non-mutated control plaques were screened with a 32P-γ-ATP kinased oligonucleotide probe. There were no control plaques that hybridized with the probe, whereas 3 to 10 mutated M13-PO plaques hybridized with the probe.

One of the mutant M13-PO plaques was picked up and J
M105 culture medium was inoculated. 5sDNA from supernatant
Make two bottles from bacterial pellets! 1 (ds) DNA was created. Appropriate oligonucleotide primer and 5s
The base sequence was analyzed using DNA.

As a result, the AGC (Serl O) codon becomes ATG (M
et) converted to a codon, CCG (Pro+4)
The codon was converted to an A T G (M eL) codon.

AAA (LYS87) codon was converted to TAA (stop) codon, GAC (Asp42) codon was converted to AA
Conversion to C (Asn42) codon, CGG (
It was confirmed that the Arg45) codon was converted to the CAG (Gin45) codon.

Among the mutated Ml 3-PO phages, codon 5er
The one where lO becomes Met is Ml3-PNIO, and the one where Kodo7PraI4 becomes Met is Ml3-
PH10 and Ml3- with codon Lys87 turned into a stop codon.
PC86 and Ml3-PD with codon Asp42 changed to Asn
N42 and Ml3-PR with codon Arg45 changed to Gin
We decided to refer to them as Q45.

Reference Example 12 (Expression in Escherichia coli of gene encoding human bF'GF mutin) (1) Construction of plasmid pTI3795 for expressing human IIF'GF' mutin Replicative form of Ml3-PH10 obtained in Reference Example 11 above (RF) was treated in the same manner as in Reference Example 5 (1), and a human bFGF mutin expression plasmid pTB was prepared.
795 was constructed. By transforming E. Ila MM294 using this plasmid pTB795, the plasmid pTB795 containing the mutin-encoding gene was transformed.
Strains including TB795 Escherichia co
ljMM294/pTB795 (IFO14700, F
ERM BP1660) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bPGF activity of bacterial cell extract Regarding the bacterial cell extract obtained in (2) above, Reference Example 5 (3)
) Human bFGF activity was measured in the same manner. As a result, the bacterial cell extract of E. coli MM294/pTB795 showed FGF activity. In this way, mutin NI4 lacking human bFGF Pro2 to Pro14 was obtained.

Reference Example 13 (Expression of gene encoding human bFGF mutin in E. coli) (1) Plasmid pT for human bFGF mutin expression
Construction of B796 The replicative form (RF) of MI3-PC86 obtained in Moeki Reference Example 11 was treated in the same manner as in Reference Example 5 (I) to create a plasmid pTB for expressing human bFGF mutin.
796 was constructed. By transforming E. coli MM294 using this plasmid pTB796, a plasmid pTB containing the gene encoding mutin was transformed.
Escherichia coli strains containing 796
MM294/pTB796 (IFO14701, FER
MBP1661) was obtained.

(2) Preparation of bacterial cell extract The above transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was obtained from a strain containing mutin C86 lacking amino acids after Lys87 of human bFGF. A body extract was obtained.

Reference Example 14 (Expression of gene encoding human bFGP mutin in E. coli) (1) Plasmid p for human bFGP mutin expression
Construction of TB797 The replicative form (RF') of M13-PDN42 obtained in Reference Example 11 of Moeki was treated in the same manner as in Reference Example 5 (1) to create a plasmid p for expressing human bFGP mutin.
TB797 was constructed. By transforming E. coli MM294 using this plasmid pTB797, a plasmid pTB797 containing the gene encoding mutin was transformed.
Strains including TB797 Escherichia co
liMM294/pTB797 (IFO14702, F
ERM BP-1662) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Regarding the bacterial cell extract obtained in (2) above, Reference Example 5 (3)
) The human l-bF GF activity was measured in the same manner as in (2009).

As a result, E, colt MM294/pTB
The bacterial cell extract of 797 showed FGF activity. In this way, mutin DN42 in which Asp42 of human bFGF was replaced with Asn was obtained.

Reference Example +5 (Expression of gene encoding human bFGP mutin in E. coli) (1) Plasmid pTB for human bFGF mutin expression
Construction of 855 DNA of plasmid pTB669 obtained in Reference Example 2 above
A was cut with the restriction enzyme Hinall, and EcoR linker-1) (5'-CATGAATTCATG-3') was cut with T
4 DNA ligase reaction. Furthermore, it was cut with restriction enzymes EcoRI and Pstl to give approximately 0.35 kb.
DNA fragments were isolated. This DNA fragment and a DNA fragment of about 3 to 2 kb obtained by cleaving plasmid ptrp781 with EcoRIPstl (Reference Example 5 (1)) were ligated by T4 DNA ligase reaction to construct plasmid pTB855 for expressing human bFGF mutin.

Using this plasmid pTB855, E. coli MM294
strain E containing plasmid pTB855 containing the gene encoding mutin,
E.coli MM294/I'1TB855 was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Regarding the bacterial cell extract obtained in (2) above, Reference Example 5 (3)
) Human bF GF activity was measured in the same manner as in . As a result, E. coIi MM294/pTB855
The bacterial cell extract showed FGP activity.

In this way, human bFGF P'02 to Va14
Mutin N41 lacking up to 1 was obtained.

Reference Example 16 (Expression in Escherichia coli of gene encoding human bPGF mutin) (1) Construction of plasmid pTB856 for expressing human bFGF' mutin DN of plasmid pTB669 obtained in Reference Example 2 above
Partially cleave A with restriction enzyme Ba1m1-II to obtain bF
Only the B, 1mHI recognition site within the GP gene was cleaved. The cleavage sites are dA T P , dCT P , dG
TP.

The ends were made blunt using E. coli DNA polymerase f in the presence of dTTP, and Nher linker-p (5'CTAGCT
AGCTAG-3') was ligated by T4 DNA ligase reaction. Treated with restriction enzyme Nhel and further T4
The cleavage site was joined by a DNA ligase reaction, and human b
A plasmid pTB856 for expressing FGF' mutin was constructed.

Using this plasmid pTB856, E. coli MM294
strain E containing plasmid pTB856 containing the gene encoding mutin,
E. coli MM294/pTB856 was obtained.

(2) Preparation of bacterial cell extract The above transformant was cultured in the same manner as in Reference Example 5 (2) to obtain the upper 1R, which contains mutin Cl29 lacking amino acids after Lys130 of human IGF'. A bacterial cell extract was obtained.

Reference Example 17 H-Leu-Pro-Met-Set-Ala-Lys
-5er-OH (bFGF[I 40-146]) ProductionBoc-Ser(Bz()-kiRF169
From 6 mg (0.72 mmo (7 g resin)), the following amino acids were sequentially condensed and debo
c reaction.

day oc-Lys(Z)-OH, Boc-Ala-OH,
Boc-Ser(Bz12)-OH, Boc-Met-
OH, Boc -Pro-OHBoc-Leu-OH F3zQ: Benzyl Boc: t-Butoxycarbonyl Z: Benzyloxycarbonyl thus Boc-Leu-Pro-Met-Ser(B
zρ)-Ala-Lys(Z)-5er(Bz12)
- 1.08 g of resin was obtained. Of this, 400 mg was incubated at 0° C. for 60 minutes in hydrogen fluoride containing 0.5 ml of anisole and 0.5 ml of trimethyl sulfide for 60 minutes, and then taken out from the resin. Excess hydrogen fluoride was distilled off under reduced pressure, and the residue was washed with E[,0, extracted with 30 d of water, and lyophilized. The crude product obtained was dissolved in water 5- and Amberlite R.
RA-400 (acetic acid type) resin (column 2 x 5 c+
a, eluent: water), the eluate was concentrated under reduced pressure, and purified by gel filtration using Sephadex LH-20 (Pharmacia Co., Ltd., column 2.5 x + 25 cm, eluent: 1N acetic acid). Obtained the object. Collection r;i
I I 8 mg (78,8%) Rf O, 22 (ethyl acetate: acetic acid: butanol: water = 1: I: I 2■
) [α] D 8pi (c=0. ratio IN acetic acid) Amino acid analysis value: Ser 2.08. Pro 1.06
．． Ala 1.00, Met 11.9g, Le
u 1.03. Lys O,99 Example 1 (Immunization) For BALB/C mice (female 4-week withdrawal), 10 gg of the antigen human bFGF (obtained in Reference Example 3) dissolved in 0.41 RIl of complete Freund's adjuvant (manufactured by Difco) was used. ) was injected intraperitoneally. Three weeks later, IOμ dissolved in Freund's incomplete adjuvant Q,4,d
The antigen hbFGF of g was re-administered intraperitoneally. Further 3 weeks later, a similar booster immunization was performed, and 2 weeks later, 10 gg of human bFGF dissolved in physiological saline was intraperitoneally inoculated.

Example 2 (1) Cell Fusion Four days after the final administration of antigen, the spleen was removed from the immunized mouse shown in Example 1 to obtain cells used for cell fusion. These cells were suspended in a medium (hereinafter abbreviated as IH medium) in which Iskot's medium and Ham's F-12 medium were mixed at a ratio of III.

Mouse myeloma cells P3-X63-Ag・801 are
5% in RPMr1640 medium containing 10% fetal bovine serum.
Subculture was carried out under conditions of carbon dioxide gas and 95% air.

Cell fusion is performed by the method established by Köhler and Milstein et al. [Kohler, G., and Milstein.

C1: Nature +- (Nature) 256. 49
5 (B75)]. Myeloma cell 2 above
．． 9 x I07 cells and 1.5XIo' immunized lymphocytes obtained by the method described above were mixed, centrifuged, and 0.3
45% polyethylene glycol 6000 (hereinafter referred to as PEG6000) dissolved in the IH medium of d was added dropwise. PEG
The 6000 solution was preheated to 37°C and slowly added dropwise. After 5 minutes, IH medium prewarmed to 37°C was added to the IH medium for 1 minute.
After adding 5 d each to obtain IO-, the mixture was centrifuged at 600 rpm for 15 minutes at room temperature, and the supernatant was removed. This cell precipitate was suspended in 200 ml of ■H medium containing 20% calf serum, and plated in groups of 2 in 24-well microplates (Linpro). One day later, HAT (hyboxanthin I x I O-'M, aminopterin 4XIO''?M, thymidine I, 6X
10-5M) containing IH medium (containing 20% calf serum)
(hereinafter referred to as HAT medium) was added to each well in an amount of 1 d, and 1/2 of the medium was replaced with HAT medium every 3 days. Cells grown in this manner are hybrid cells.

(2) Search for antibody-producing cells In advance, 100% of the hybrid cell culture supernatant was placed in a 96-well polystyrene microtiter plate on which human bFGF was immobilized.
μQ was added and incubated at room temperature for 2 hours. After removing the culture supernatant and washing, a horseradish peroxidase (HRP')-labeled anti-mouse TgG goat antibody (Miles) was added as a secondary antibody, and the mixture was incubated at room temperature for 2 hours. 2
After removing the next antibody and thoroughly washing the wells, a reaction substrate was added to perform a color reaction (EIA method). Strong binding valencies were observed in three wells using this method.

(3) Cloning of hybrid cells The cells in these wells were placed in a 96-well microtiter plate in which IO'flJ/well of mouse thymocytes were seeded as vegetative cells in advance at 0.5 cells per well. Then, cloning was performed. the result,
Three clones, mouse HbF99 cells (IFO501
22), mouse HbFI61 cells (IFO50123)
, mouse HbF165 cells (IFO50124) were obtained.

Table 2 shows the results of measuring the antibody titer in these cell supernatants.

Table 2 In the table, the results show absorption at a wavelength of 492 nm.

− means not measured.

The cloned cells were incubated in IH containing 20% calf serum.
Dimethyl sulfoxide (DMS) was added to the medium to a concentration of 10%.
O) was added and stored in liquid nitrogen.

Example 3 (Immunoglobulin class of monoclonal antibody) Mouse antibody subclass detection kit obtained in Example 2 (
(Bio-Rad) and reacted with various standard immunoglobulins. The results are shown in Table 3.

Table 3 In the table, + indicates a positive reaction. indicates a negative reaction.

From Table 3, MoAb99, MoAbl 61 and M
The immunoglobulin class of oAb165 is IgM.
It can be seen that it belongs to

Example 4 Mouse BA L immunized by the method described in Example I
The spleen was collected from B/c and infected with no.
l"12 (IF'0 50142), HbF45.Hb
F47. HbF52 (rFo 50143), HbF
78 (■F0 50144) and I-(bF' 98
(I Fo 50145) was obtained.

Hybridoma HbF I 2. HbF45. HbF4
7, LIbF 52 , HbF 78.1-1bF
98, 2xlO'lIl cells each were inoculated into mice that had previously been intraperitoneally administered with 0.5 ml of mineral oil. After 10 days, 2 to 4 days of ascites was collected from each animal, and monoclonal antibodies MoAbl2, MoAb45, MoAb were collected from each hybridoma.
47MoAb52. MoAb78. MoAb98 was obtained.

The immunoglobulin classes of these monoclonal antibodies were determined according to the method described in Example 3, and the results were determined as shown in Table 4 below.

Table 4 Example 5 (1) Preparation of radiolabeled hbFGF E. coli MM294/pTB669 (described in Reference Example 2)
I F'0 14532. FERM BP-1281
), hbFGF radiolabeled with 35S was obtained by the following method.

The above E. coli MM294/pTB 669 was cultured in the medium described in Reference Example 3 until the Klett value reached 200. This culture solution was added to M9 (M et-) medium.
I put it in. M9 (Met-) medium is a medium obtained by adding amino acids with the following composition to M9 medium containing 1% glucose and 8βg/d tetracycline.

L-Chironn 83.9 L-valine 94.0 Amino acid composition L-alanine L-arginine hydrochloride L-asparagine hydrate L-aspartic acid L-cysteine disodium salt -Glutamic acid L-glutamine L-glycino L-histidine hydrochloride l hydrate Shi-isoleucine L-Loin Lysine hydrochloride L-phenylalanine L-proline L-serine L-threonine 25.0 mg/Q 28.4 30.0 82.8 75.0 584.0 30.0 42.0 tos,.

105.0 146.0 66.0 42.0 95.0 Culture in M9 (M et-) medium until the Klett value reaches 200, and add 25μ of 3β indole acrylic acid.
g/d, and culture was continued for an additional 2 hours.

After this, l- was fractionated and 10 μCi of 35S-Met (
Specific activity > I 000 Ci/mmol) was administered at 30
It was cultured separately. After culturing, the bacteria were collected and a bacterial cell extract was obtained according to the method described in Reference Example 3.

In addition, the vector plasmid ptrp7 described in Reference Example 2
The same treatment was performed on E. coli MM294 having 8 I to obtain a labeled bacterial cell extract.

(2) Immunoprecipitated protein A (manufactured by BRL) was prepared according to the manufacturer's instructions and a 10% solution was used. An unlabeled E. coli extract was obtained from E. coli MM 294 /ptrp781 by the method described in Reference Example 3.

1 d of ascites obtained in Example 4 was added to 100 μl of unlabeled E. coli extract.
After mixing with a and leaving for 1 hour at 4°C, labeled bacterial cell extract (MM
294 /I) trp781 or MM29.1/
pTB669) was added at IO'cpm and incubated at 4°C overnight.

Add 100 μg of unlabeled E. coli extract to 100 μQ of Protein A I 0% solution, mix, leave at 4°C overnight, centrifuge, and add 100 μQ of NETBN solution [150 mM NaCl,
5mM EDTA, 50mM TrisCl(1)H
7,5), O, 1% 13SA, 0.05% NP40 (Non-idet P -40)] again! 8
I was so angry. A mixture of the above-treated labeled extract and ascites was added and mixed, and the mixture was incubated at 4°C overnight. This was centrifuged, and the precipitate was suspended in 500 μe of NETBN solution. After repeating this operation 5 times to remove unadsorbed labeling substances, the sample was finally BBed into a 50 μσ electrophoresis sample buffer and subjected to polyacrylamide gel electrophoresis. Laemmli, U, K.

[Nature, 227. 680 (197
0)] was followed. After electrophoresis, the gel was immersed in 50% trichloroacetic acid (TCA) for 1 hour, washed 4 times for 30 minutes each with distilled water to remove TCA, and then soaked in dimethyl sulfoquine (DMSO).
Soaked for 1 hour. This was followed by immersion in DMSO containing 10% 2.5 diphenyloxazole (DPO) for 1 hour. After washing the gel three times with distilled water for 30 minutes each, the gel was dried. A radioautogram was then taken to observe the immunoprecipitation pattern.

This is shown in FIG. From FIG. 2, monoclonal antibodies MoAbl 2 , MoAb52, MoAb78
.

MoAb98 was found to bind to hbFGF in the bacterial cell extract.

Example 6 Among the monoclonal antibodies obtained in Example 4, Example 5
4 antibodies that showed immunoprecipitation in monoclonal antibody MoAbl 2. Antibody binding titers were measured for MoAb52°MoAb78 and MoAb98 by limiting dilution method.

That is, the monoclonal antibody Mo obtained in Example 4
Abl 2. MoAb52. MoAb78 or M
The ascites fluid containing oAb98 was diluted with IH medium containing 10% fetal bovine serum, and the EI shown in Example 2 (2) was prepared.
Antibody 1 in the diluted solution was measured by Method A. The results are shown in FIG. In Figure 3, -〇---- is M
The results for oAb12 are Mo
The results for Ab52 are shown, +- shows the results for MoAb78, and -1--1- shows the results for MoAb98.

From FIG. 3, it can be seen that ascites containing these antibodies has a limiting dilution value of lXIO' or more, and therefore these antibodies have a very high antibody binding titer.

Example 7 (Determination of Antigen Recognition Sites) The antigen recognition sites of the four antibodies whose antibody binding values were measured in Example 6 were examined by competitive binding inhibition experiments.

As a competitor, hbFGF synthetic peptide Pepl obtained in Reference Example 3: Pro-Ala-Leu-Pro
-Glu-Asp-Gly-Gly-Ser-Tyr (
Tyr added to N-terminal amino acids 2-10) [Regulatory P
eptides), 10.309-317 (1985)
), Pep2: Leu-Pro-Met-Ser-A
la-Lys-Set (corresponding to amino acids +42-147) (obtained in Reference Example 17) 5 and deletion type bFG
F Mutins, Mutin N14 (obtained in Reference Example!2) and Mutin N41 (obtained in Reference Example 15) were used.

Synthetic peptides! Prepared to a concentration of 00 μg/d, 1O
IH medium containing % FC8 [Iscot and Ham F-1
2 mixed medium (1:1)). Mutin NI
4 and N41 are Reference Example 12(2) or 15(2)
The extract obtained by the method shown in 10%F
It was used after being diluted with rH medium containing C9. Antibody amount is A41
Regarding the ascites fluid obtained in Example 4, MoAbI 2. MoAb52. MoA
For b98, 5XI05 times, for MoAb78, 5
Diluted 10' times. As a diluent, 10% FC8
An IH medium containing . The diluted competitor was added to the diluted antibody solution, stirred, and then kept at 37°C for 30 minutes. The amount of unbound antibody contained in this solution was determined by the ETA shown in Example 2 (2).
It was measured by the method.

Figure 4 shows the results when synthetic peptides were used.

It is shown in FIGS. 5, 6 and 7. Figure 4 shows the results for monoclonal antibody MoAb12, Figure 5 shows the results for monoclonal antibody MoAb52, and Figure 6 shows the results for monoclonal antibody MoAb52.
The figure shows the results for monoclonal antibody MoA bl8, and FIG. 7 shows the results for monoclonal antibody MoAb98. In addition, in these figures, -・- is h
bFGF, ---0----One is Pepl, ---
--■---- shows the results using Pep2 as a competitor. In the figure, the vertical axis indicates the light absorption (based on wavelength 4150I11) of the coloring agent.

From FIG. 4 and FIG. 6, monoclonal antibody MoAh
12 and MoAb78 were competitively inhibited by Pep I. This revealed that monoclonal antibodies MoAb+2 and MoAb78 recognize the N-terminal amino acids 2 to 10 of hbFGF.

From FIGS. 5 and 7, MoAb52. It was found that MoAb98 was not inhibited by these synthetic peptides.

In order to determine the recognition site of these antibodies, Reference Example 12
A competitive inhibition experiment using Mutin NI4 obtained in Example 15 and N41 obtained in Example 15 was conducted in the same manner as above, and the results for MoAb52 are shown in Figure 8, and the results for MoAb52 are shown in Figure 9.
98 results are shown. In addition, in these figures, -1
- is hbFGF, ~---- is Mutin N14,
-110--- indicates the results using Mutin N41 as a competitor. In the figure, the vertical axis indicates the absorption of the coloring agent (at a wavelength of 415 nm). The horizontal axis is reference example 3
The total protein content of the E. coli crude extract obtained by the method described above is shown.

From FIG. 8 and FIG. 9, monoclonal antibody MoAb
52. MoAb98 was competitively inhibited by mutin N+4 but not by mutin N41.

This revealed that monoclonal antibodies MoAb52 and MoAb98 recognize amino acids 15 to 41 of hbFGF.

Next, bovine acidic F GF (baF G
F) (R&D 5ystea+s Inc.
A competitive inhibition experiment was conducted in the same manner as described above using hbFGF obtained in Reference Example 3 and the hbFGF obtained in Reference Example 3, and the results shown in Table 5 below were obtained. The numerical value represents the amount of unbound antibody obtained by ET using the method described in Example 2 (2).
The absorbance due to color development of A is shown.

Based on the above, the recognition sites of the four types of monoclonal antibodies are summarized as shown in Table 6 below.

Table 6 From this, monoclonal antibody M o A bl
2, MoAb 529MoAb 78 and MoA
b98 was found not to cross-react with bovine acidic FGF.

In the table, "+" indicates that there was competitive inhibition, and "-" indicates that there was no competitive inhibition.

Example 8 Purification of antibodies from ascites Monoclonal antibody M by the method of Example 4.

Abl 2. MoAb52. MoAb78 MoAb
98 was inoculated into 10 mice each, and ascites fluid 20 to 3
I got 0d. The ascites fluid was centrifuged at 200 Orpm (Hitachi refrigerated centrifuge) to remove cells, and then centrifuged in a Subinco SW2
The mixture was centrifuged at 4°C for 2 hours in an S rotor (Beckman, USA) to remove insoluble proteins, fats, etc. For the supernatant,
Ammonium sulfate was added to achieve 40% saturation, and the mixture was gently stirred in water for 1 hour. Precipitate with Zabal 5S34
I5000r at 4°C using 0-tar (DuPont, USA)
After centrifugation at pm and collection, buffer y-I C20mM
Tris-HCI (pr(7,9), 4 Qn+M
The protein was dissolved in NaCl) at a protein concentration of Iθ to 15 mg/h4, and dialyzed against Buffer 1 at 4°C overnight. This solution was mixed with DE equilibrated with buffer l.
It was passed through AE-cellulose DE52 (Whatman, USA) and adsorbed. Elution with buffer 1 and 0.4M NaC
An 8 degree gradient was performed with buffer 1 containing l. The immunoglobulin fraction was collected and precipitated by addition of 40% saturated ammonium sulfate, and the precipitate was precipitated with buffer 2 (0,
Protein concentration is IO~20mg in 1M NaHC03)
/! The mixture was combed and dialyzed against Buffer 2 at 4°C for two nights. Dialysate was changed overnight.

Furthermore, it was further purified using a hydroxyapatite column (HC 8-column). At this time, lOn+M sodium phosphate buffer (pH 6,8) was used as the starting buffer, and 500 mM sodium phosphate buffer y- (pH 6,8) was used as the elution buffer. Elution was performed by a linear gradient from starting buffer to elution buffer. Fractions containing antibodies were stored at 4°C.

Example 9 (Purification of hbFGF mutin using antibody column) Affigel 10 (Bio-Rad, USA) 5- was placed on a sintered filter, washed with 10 times the volume of water-cooled isopropanol under suction, and then washed with 10 times the volume of water-cooled isopropanol. Wash with water-cooled distilled water,
Transferred to reactor. 15mg monoclonal antibody MoA
5 to 15 times of b78 (dissolved in Buffer 2 of Example 8 or phosphate buffer) was added and reacted at 4°C. Monoethanolamine (pH 8.0) was added to the reaction solution to a concentration of 0.01M, and the mixture was allowed to react at room temperature for 1 hour.
Unreacted reactive groups were inactivated. After this, apply the gel to Example 8.
The gel was washed with 10 times the volume of Buffer 2.

After that, the 2d gel was packed into the column and the initial buffer (2d) was packed into the column.
0mM Tris-HCI (pH 7,6), 1mM E
DTA, 0.15M NaCl, 0.05% NP40)
Equilibrated with.

On the other hand, the transformant Escherichia coli D containing mutin C34 obtained in Reference Example 8(2)
The extract of the HI/pTB744 culture solution was diluted 3 times with the initial buffer, and the diluted solution was applied to the above column at a flow rate of 20°C. #
It was allowed to flow at 1/h and adsorbed. After adsorption, initial buffer 2
The column was washed with 0-, followed by hot buffer y-(
20mM Tris-HCI(+)H7,6).

ImM EDTA, LM NaCl, 0,05% NP4
0) 20In1 elution buffer A (0.2M acetate buffer (pH 4,5), 0.2M NaCI) 20utl
, elution buffer y-B (0,2M acetic acid (PH2,5), Q
, 2M NaCl). The flow rate was 20 d/h and the entire process was carried out at 4°C.

Both fractions were subjected to 17.25% acrylamide electrophoresis (Laemili, Nature (N.
ature) 277.680 (1970)). Staining was carried out using the silver staining method. The results are shown in FIG. In addition, the 10th
In the figure, MK is the molecular weight marker, A is the crude extract, B is the flow-through fraction, C is the elution fraction with high salt concentration buffer, D is the elution fraction with elution buffer A, and E is the fraction eluted with elution buffer A.
2 shows the results of the elution fraction with elution buffer B, respectively. For D and E fractions, immediately after elution from the column, IMTris-HCI (pH 9,5) was added to I) H
I gave it a 7.5. The FGF activity of each sample was measured by the method described in Reference Example 5 (3). The results are shown in Table 7. Note that in Table 7, A to E are the same as those in FIG. 1O described above.

Table 7: 0.69' FGF activity using 3H-thymidine incorporation as an index,
Bovine bFGF used as a trademark product (to Takara Sake Brewery,
) Expressed in equivalent weight.

From the above, it was found that the antibody column using monoclonal antibody MoAb78 efficiently adsorbed bFGF mutin CS = 1, and by the above procedure, bFGF mutin CS = 1 was adsorbed efficiently.
Mutin CS4 was successfully purified.

Example 10 (Quantification of hbP GF mutin using monoclonal antibody by EIA method) (1) Monoclonal antibody 78 obtained in Example 4
was purified from ascites by the method of Example 8.

The antibody thus obtained was 2mF! , /1n1 or more, and then dialyzed against 0.2M sodium phosphate buffer (pH 7.0).

2, 8 mg/min monoclonal antibody MOΔb78
NN'- so that Il is 5 mg/d for 1.4 d.
S- dissolved in dimethylformamide (DMF)
Acetylmercaptosuccinic anhydride (A 1
drich, USA) was added at 50 μρ. Replace the air in the reactor with nitrogen gas, seal it, and leave it at room temperature for 1 hour. The SH group was introduced. Unreacted S-acetylmercaptosuccinic anhydride was added to 130 μQ of 0.2 MTr.
is-HCf! (pH 47,0), I 3μ (!0.
2MEDTA, 2M hydroxyamine (p
i-17,0) was added and treated at room temperature for 10 minutes to inactivate.

Monoclonal antibody MoAb78 was prepared using Sephadex G.
-25 (diameter 1 cn+x80 cm, Pharmacia, Sweden) was used for fractionation using a gel filtration column (flow rate 20 m/h).

(2) Horseradish peroxidase (hereinafter f (RP Base Germannheim, West Germany, Grade 1)
10 mg in 1.4-O,IM phosphate buffer y-(p
1-16,8). 14 mg of N-hydroxysuccinimide ester of N-(4-carboxocyclohexylmethyl)maleimide was dissolved in 335 μQ of DMF, and 100 μQ of this was added to the HRP solution. The air in the reactor was replaced with nitrogen, the reactor was tightly stoppered, and the reactor was stirred at room temperature for 1 hour. After that, H
The P fraction was collected.

(3) Antibody MoA with SH group introduced in (1) above
b78 fraction 6- and Hr(P fraction 2d) into which a maleimide group was introduced in (2) above were mixed and placed in a collodion bag (
The mixture was concentrated to Id under reduced pressure using a sieve (manufactured by Sartorius, West Germany) and reacted at 4° C. for 20 hours. After the reaction, the HRP-introduced antibody was applied to Bovine Trogel AcA44 (LKB, Sweden, diameter 1 cm x 80 cm) and separated (flow rate 10 d/h). Among the elution peak fractions, the two fractions with the highest number of HRP per antibody molecule were 2.4 HRP/
It was an antibody.

This was used for the next EIA (4).

(4) MoAb52 was purified by the same method as in (1) above. 10 μg/- or 20 μg/- of this MoAb 52
It was diluted with PBS to give a concentration of μg/Ml, injected at 100 μa/well into an immunoplate (Nunc, Denmark), and allowed to adsorb at 4° C. overnight. After removing unadsorbed antibodies, wash 3 times with PBS,
μ% merthiolate, 1% bovine serum albumin (BSA)
200 μQ/well of PBS containing PBS was added and incubated at 4° C. overnight.

(5) Human bFGF obtained in Reference Example 3 in 0.1% BS
It was diluted with PBS containing A. Remove the BSA solution from the plate prepared in (4) above, wash it 4 times with PBS,
Add 100 μa/well of diluted human bFGF and incubate at 4°C.
- Adsorption was performed at night. After removing unreacted human bFGF, P
Wash 4 times with BS, and add the HRP-binding antibody (HRPMoAb78) prepared in (3) above to PB containing 0.1% BSA.
The mixture was selected with S at 1/300, added at 100 μQ/well, and allowed to react at room temperature for 4 hours. After removing the antibody, it was washed 6 times with PBS, and peroxidase substrate (BioRad, USA) was added once.
00 μQ/well was added for color development and colorimetric determination.

(6) First! The figure shows worms immobilized on a plate with monoclonal antibody M o Ab 52 at 1 μg/well (--
10-----) and 2 μg/Ueno--).

The horizontal axis is the added bFGFa degree, and the vertical axis is the HRP-M.

It represents the absorbance developed by Ab78. From this figure, it was found that when MoAb52 was adsorbed to the plate at 2 μg/well, bFGF at a concentration of 0.5 ng/− could be detected.

(7) Apply MoAb98 to the plate using the method described in (4) above.
μg/well was adsorbed, and bFGF was colorimetrically quantified by the method described in (5) above. FIG. 12 shows the results.

The horizontal and vertical axes represent the same things as in FIG. 11 of (6) above. From this figure, it can be seen that MoA 698 is less concentrated in the same Ep as MoAb52 than in bF at a concentration of 5 ng/-.
It was found that GF can be detected.

Example II (Quantification of hbFGF mutin using monoclonal antibody by ETA method) The extract containing human bFGF mutin 086 obtained in Example 13 was treated in the same manner as in Example 9 to determine the expression level of the mutin. investigated. As a result, it was found that 086 was expressed in E. coli, albeit in a trace amount.

Example +2 (Quantification of hbFGF mutin using monoclonal antibody by EIA method) The extract containing human bFGF mutin C129 obtained in Reference Example +4 was treated and examined in the same manner as in Example 9. As a result, it was found that mutin Cl29 was produced in small amounts within E. coli.

Example 13 (hb by Western blotting
Detection of FGP) hbFGP obtained by the method described in Reference N3 was 1725%
Acrylamide gel electrophoresis (L aemmli, U
.

K. (1970) Nature 277.680-6
85) and then transferred onto a nitrocellulose membrane using Zardoblot (Sartorius, West Germany).
Journal of Biochemical and Biophysical Methods
chemical andB 1ophysical
Methods), 10 203-209 (+
984)). This membrane was soaked in TBS (20mM Tris-
The membrane was washed twice with HCI (pH 7,5), 0.5M NaCI) for 5 minutes each, and left in TBS containing 4% BSA at room temperature for 1 hour to block unreacted areas on the membrane.

The cells were washed twice with TBS containing 0.05% Tween 20 (TTBS) for 5 minutes each. Monoclonal antibody MoAb12. obtained in Example 4. Alternatively, the above nitrocellulose membrane was placed in a solution in which MoAb78 was diluted 3000 times with TTBS containing 1% gelatin, and the reaction was allowed to occur overnight at room temperature. Remove the reaction solution, wash the membrane twice for 5 minutes each with TTBS, and then add secondary antibody diluted 3000 times with TT [3S] containing 1% gelatin, HRPti-recognizing mouse IgG goat serum (Bio-Rad, USA). The mixture was added, and the reaction was carried out at room temperature for 1 hour.

Wash the membrane three times for 5 minutes each with TTBS, then wash the membrane for an additional 5 minutes with TBS.
Wash twice for 2 minutes, then add 0.05% as a coloring agent 4
-Chloro-1-naphthol and 0.015% hydrogen peroxide were added, and the reaction was carried out for 15 minutes.

FIG. 13 shows the results of Western blotting using monoclonal antibody MoAb78 as the primary antibody. Lane 1 contained 1 μg of hbFGF, Lane 2 received 300 μg of hbFGF.
ng of hbFGF was electrophoresed and 1100 n of hbFGF was electrophoresed in 3 and transferred. M represents a marker, and the number on the left is its molecular weight.

Note that MoAb I 2 was used instead of MoAb78 above.
It was also possible to detect bFGF with the same response when using .

Effects of the Invention Since the monoclonal antibody of the present invention specifically binds to bFGF, it can be usefully used as a reagent for measuring bFGF and for purifying bFGF.

Furthermore, among the monoclonal antibodies of the present invention, those with a high antibody binding value have the advantage that when used as a bpcpIiJ++ regular reagent, the amount of other reagents such as antiserum used at the same time can be reduced. Furthermore, there is an advantage that bFGF' can be purified to a higher degree.

[Brief explanation of the drawing]

FIG. 1 shows the nucleotide sequence encoding bFGF obtained in Reference Example I and the amino acid sequence deduced from it. FIG. 2 shows the immunoprecipitation pattern obtained in Example 5 and analyzed by polyacrylamide gel electrophoresis. FIG. 3 shows the antibody binding titers obtained in Example 6. Figure 4 shows the MoA of various peptides obtained in Example 7.
The results of a competitive binding inhibition experiment for b12 are shown. Figure 5 shows the MoA of various peptides obtained in Example 7.
The results of a competitive binding inhibition experiment for b52 are shown. Figure 6 shows the MoA of various peptides obtained in Example 7.
The results of a competitive binding inhibition experiment for b78 are shown. Figure 7 shows the MoA of various peptides obtained in Example 7.
The results of competitive binding inhibition experiments for b9B are shown. FIG. 8 shows the results of a competitive inhibition experiment of bFGF' mutin against MoAb52 obtained in Example 7. FIG. 9 shows the M of bFGP mutin obtained in Example 7.
Shows the results of competitive inhibition experiments against oAb98. FIG. 1O shows the electrophoretic pattern obtained in Example 9. FIG. 11 shows MQAb52,H obtained in Example 10.
The results of quantifying bFGF by ErA using RP-MoAb78 are shown. FIG. 12 shows MoAb98 and H obtained in Example 10.
The results of quantitative determination of bFGF using RP-MoAb7B are shown. FIG. 13 shows the results of Western blotting using the monoclonal antibody MoAb78 obtained in Example 12. 0.1 0.51.0 5.010 50100 October l force (pg/ml) -L futures (2g7ml) 0.1 0.51.0 5.010 50 warehouses (P
g/ml) Bald Man (Pg/ml) Magic Rib (mg/ml) 0.0050. Ol 0.050.1 妓δ咄0.51.0 (mg/ml) 5.010 bFGF '→sa/6 i- (ng/ml) ・, &- bFGF ;i, t (ng/ml)

Claims (20)

[Claims] (1), has the following properties, basic fibroblast growth factor (
Monoclonal antibody that specifically binds to bFGF): (a) Molecular weight: approximately 140-160 kilodaltons (b) Does not cross-react with acidic fibroblast growth factor. (c) The immunoglobulin class belongs to IgM or IgG. (2) The monoclonal antibody according to claim 1, wherein bFGF is a polypeptide comprising the amino acid sequence: [There is a gene sequence]. (3) A cloned hybridoma consisting of spleen cells of a mammal immunized with basic fibroblast growth factor (bFGF) and homologous or heterologous lymphoid cells. (4) The hybridoma according to claim 3, wherein the mammal is a mouse. (5) The hybridoma according to claim 3, wherein the lymphoid cells are myeloma. (6) The hybridoma according to claim 3, wherein bFGF is a polypeptide comprising the following amino acid sequence: [There is a gene sequence]. (7) The spleen cells of a mammal immunized with basic fibroblast growth factor (bFGF) are fused with homologous or heterologous lymphoid cells and cloned. A method for producing a cloned hybridoma comprising lymphoid cells. (8) The production method according to claim 7, wherein the mammal is a mouse. (9) The method according to claim 7, wherein the lymphoid cells are myeloma. (10), bFGF has an amino acid sequence: [There is a gene sequence] The production method according to claim 7, which is a polypeptide comprising. (11), cloned hybridomas consisting of spleen cells of a mammal immunized with basic fibroblast growth factor (bFGF) and homologous or xenogeneic lymphoid cells were grown in liquid culture or intraperitoneally in a mammal. A method for producing a monoclonal antibody that specifically binds to the factor and has the following properties, which is characterized by multiplying the monoclonal antibody, producing and accumulating the monoclonal antibody, and collecting the monoclonal antibody: (a) Molecular weight: about 140 ~160 kilodaltons (b) Does not cross-react with acidic fibroblast growth factor. (c) The immunoglobulin class belongs to IgM or IgG. (12) The production method according to claim 11, wherein the mammal is a mouse. (13) Claim 1, wherein the lymphoid cells are myeloma.
1. The manufacturing method described in 1. (14), bFGF has an amino acid sequence: [There is a gene sequence] The production method according to claim 11, which is a polypeptide comprising. (15), a monoclonal antibody that has the following properties and specifically binds to basic fibroblast growth factor (bFGF): (a) Molecular weight: approximately 140 to 160 kilodaltons (b) Acidic fibroblast growth factor Does not cross-react with (c) The immunoglobulin class belongs to IgM or IgG. A method for purifying bFGF, characterized by using. (16), bFGF has an amino acid sequence: [There is a gene sequence] [There is a gene sequence] The purification method according to claim 15, which is a polypeptide comprising. (17) The purification method according to claim 15, wherein affinity chromatography is performed. (18), a monoclonal antibody that has the following properties and specifically binds to basic fibroblast growth factor (bFGF): (a) Molecular weight: approximately 140 to 160 kilodaltons (b) Acidic fibroblast growth factor Does not cross-react with (c) The immunoglobulin class belongs to IgM or IgG. A method for detecting and quantifying bFGF, characterized by using. (19), bFGF has an amino acid sequence: [There is a gene sequence] The detection method according to claim 18, which is a polypeptide comprising. (20) The detection and quantification method according to claim 18, wherein enzyme immunoassay is performed.