JPH03244394A - Monoclonal antibody, hybridoma, its production and use thereof - Google Patents

Abstract

PURPOSE:To produce a monoclonal antibody by carrying out cell fusion of a mammalian splenic cell immunized with an acidic fibroblastic cell growth factor (aFGF) to a lymphoid cell and proliferating the resultant hybridoma. CONSTITUTION:A mammalian splenic cell immunized with an aFGF protein is subjected to cell fusion to a homologous or heterologous lymphoid cell and cloning is then performed to clone the fused cells and synthesize a hybridoma, which is subsequently proliferated in a liquid culture medium or in the abdominal cavity of a mammal to produce and accumulate a monoclonal antibody. The resultant monoclonal antibody is then recovered to afford a monoclonal antibody against the aFGF protein. The aforementioned monoclonal antibody has 140000-160000 molecular weight and will not carry out cross reaction with a basic fibroblastic cell growth factor.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to acidic fibroblast growth factor (herein referred to as
It is sometimes abbreviated as aFGF. ) Monoclonal antibodies against proteins, hybridomas, their production methods, and their uses.

Prior Art aF G F is an acidic polypeptide hormone with a molecular weight of 16,000 that is mainly localized in the brain and retina.
It exhibits a proliferation-promoting effect on almost all cells derived from fibroblasts and mesoderm, such as LB/c3T3 cells. [D
, Gospodarowicz et al; Endocrin
e Reviews 8: 95 (1987)] In addition, this factor exhibits an angiogenic effect in vivo, and in combination with the above-mentioned cell proliferation effect, it can be used as a therapeutic agent for injuries and burns, and as a prophylactic agent for thrombosis, arteriosclerosis, etc. Show possibilities.

Problems to be Solved by the Invention Human aFGF exists in nature in extremely small amounts, and attempts to obtain it from human tissues have been extremely difficult due to various restrictions. Furthermore, up until now, no method has been established that can be easily used to quantify aFGF, and for these reasons, there are still some unknown points regarding the basic knowledge regarding the properties of aFGF, which is essential for developing aFGF as a drug. Or very many.

Therefore, if a lot of basic information regarding aF GF is known, such as the distribution of aF GF in the living body and its production mode, the development of aF GF as a pharmaceutical will become easier.

Furthermore, accurately knowing the amount of aFGF is also important when purifying this protein from a genetically recombinant plant. Furthermore, it is very important to track the level of FGFJ in the blood of animals administered with aFGF, or it cannot be measured using the conventional method using 3T3 small capsules due to the contamination of the sample. Normally, aFGF measurement: aFGF was applied to 3T3 cells that were cultured with reduced blue concentration and reduced DNA synthesis.
is added, and it is calculated backwards depending on the amount of DNl\synthetic ability and how much power is recovered.

However, this method has the drawbacks of using cells, requiring delicate operations, large measurement errors, and requiring a long time to obtain results. Therefore, it is desired to develop a simple and accurate means for measuring aFGF for the above purpose.

Means for Solving the Problems In view of the above-mentioned circumstances, the present inventors conducted various studies to find a practical means of measuring aFGF protein, and as a result, created a monoclonal antibody against aFGF protein that would enable the measurement. As a result of further research based on this, the present invention was completed.

The present invention provides (1) a monoclonal antibody against acidic fibroblast growth factor (aFGF) protein having the following properties: (a) Molecular weight: about 140,000 to 160,000;
(b) Does not cross-react with basic fibroblast growth factor (bFGF).

(C) The immunoglobulin class belongs to IgG; (2)
, a cloned hybridoma consisting of spleen cells of a mammal immunized with acidic fibroblast growth factor (aFGF) protein and homologous or xenogeneic lymphoid cells; (3) acidic fibroblast growth factor; (aF G F ) The method for producing a hybridoma according to (1) above, which comprises cloning spleen cells of a mammal immunized with protein and homologous or heterologous lymphoid cells; (4);
Propagating the hybridoma described in (1) above in a liquid medium or intraperitoneally of a mammal to produce a monoclonal antibody,
The above (1) characterized by accumulating and collecting the accumulated
) Method for producing the monoclonal antibody described in (5), a method for detecting and quantifying aFGF protein characterized by using the monoclonal antibody described in (1) above, and (6), the monoclonal antibody described in (1) above. This is a method for purifying aFGF protein, which is characterized by using.

The aFGF protein to be used to immunize mammals may be any a-FGF protein derived from hematopoietic mammals, or its mutein.

Representative examples of the mammalian aFGF include, for example, bovine aFGF [G, Gimenez-Gall
ego et al.

5science 230: 1385 (1985)]
and [F.

Esch et al., Biochem, Biophy
s, Res, Commun.

133:544 (1985) L Human aF G F
CGGimenez-Gallego et al., Bi
ochem, Biophys, Res.

Common, Sat. 38: 611 (1986)]. a As FGF, the above-mentioned document Bioc
hem.

Biophys, Res, Commun, Sat38
: 611 (1985) is preferred.

To obtain human aF G F, for example, the above-mentioned aFG
An expression vector containing a DNA having a base sequence encoding the F protein polypeptide may be prepared and introduced into a suitable host cell to produce aFGF. As an expression vector, for example, (a) an RNA encoding aF GF is isolated, and (b) a single-stranded complementary D is isolated from the RNA.
Synthesizing NA (cDNA) and then double-stranded DNA, (c) incorporating the complementary DNA into a plasmid, (d) transforming a host with the obtained recombinant plasmid, and (e) obtaining the obtained transformant. After culturing, 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) the desired cloned DNA is isolated from the plasmid.
It can be produced by excising A and (g) ligating the cloned DNA downstream of a promoter in a vehicle.

RNA encoding aFGF can be obtained from various aFGF-producing tissues, such as human brain tissue or human retinal tissue.

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

The mutin in the present invention is originally a mutated amino acid sequence of an original peptide or protein,
It has aFGF action. 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 addition of at least one amino acid.

The deletion of the constituent amino acids includes at least one aF
Examples include those in which GF constituent amino acids are deleted.

The deletion type mutin mentioned above includes one consisting of a polypeptide chain of 90 to 133 consecutive amino acid sequences of aFGF, and one consisting of 120 to 133 consecutive polypeptide chains of aFGF amino acid sequence.
Furthermore, those consisting of 12.5 to 131 consecutive polypeptide chains of the amino acid sequence of aFGF + 131 to 133 consecutive polypeptide chains of the amino acid sequence of aFGF There are several things that can be mentioned.

The deletion type mutin of the present invention is preferably one in which an amino acid is deleted from the N-terminus.

This deleted mutin may have up to three amino acids deleted from the C-terminus of the basic mature aFGF polypeptide.

Specific examples of deletion-type mutins of the present invention include, for example,
human aFGF with the N-terminal 8 amino acids deleted, human aFGF with the N-terminal 9 amino acids deleted, human aFGF with the N-terminal 11 amino acids deleted, human) aFGF with the N-terminal 12 amino acids deleted, human aFGF with the N-terminal 20 amino acids deleted, human aFGF with the N-terminal 43 amino acids deleted, etc. Can be mentioned.

The above-mentioned deletion-type mutins include those in which one amino acid is deleted from the N-terminus of human aFGF, those in which five amino acids are deleted from the N-terminus, and those in which one amino acid is deleted from the N-terminus of human aFGF.
Bovine aFG, which is missing the last 6 amino acids
' Consisting of the 1st to 15th amino acids from the N-terminus of F, the 7th to 41st amino acids from the N-terminus of bovine aF G
The amino acid may consist of the 1st to 41st amino acids from the N-terminus of human aFGF, or the 7th to 41st amino acids from the N-terminus of human aFGF.

Furthermore, the aFGF mutin used in the present invention is the 16th position of mature bovine aFGF.

One or more cysteine residues at positions 47 and 83 may be substituted with other amino acids and/or methionine may be added to the N-terminus.

Furthermore, the aFGF mutin used in the present invention may be a human and/or bovine aFGF mutin having 139, 140 or 154 amino acid residues.

The above mutin having 154 amino acids has the following amino acid sequence: A 1a-G 1u-G 1y-G lu-l 1e-
Thr-Thr-Phe-Thr-A 1a-LeuT
Examples include those in which the C-terminus of hr-Glu-Lys is added to the tip of Phe at the N-terminus of aFGF (see EP-319,052).
.

As for the substitution with other amino acids, at least one a
Examples include those in which the F GF constituent amino acid is substituted with another amino acid.

The at least one amino acid in a mutin in which a small amount (both one amino acid and one amino acid is added to aF, G, and F) does not include methionine, which is based on the start 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 added as long as the characteristics of aFGF are not lost.More preferably, it is homologous to aFGF.
This includes part of the amino acid sequence or complete amino acid sequence of a protein that exhibits similar activity.

The number of deficient constitutive amino acids in a mutin in which at least one aFGF constitutive amino acid of aFGF is deficient may be any number as long as the characteristics of aFGF are not lost.

At least one aFGF constituent amino acid of aFGF is substituted with another amino acid. Any number is fine as long as you don't lose any.

Examples of the constituent amino acids before substitution include those other than thusteine and cysteine.

Cystine is particularly preferred. Constituent amino acids other than cysteine before substitution include aspartic acid, arginine, glycine, and valine.

If the constituent amino acid before substitution is cysteine, the substituted amino acid may be, for example, a neutral amino acid.
) acids are preferred. Specific examples of the neutral amino acids include glycine, valine, alanine, leucine, inleucine, tyrosine, phenylalanine, histidine,
tryptophan, serine.

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

When the constituent amino acid before substitution is other than cysteine, the substituted amino acid may be, for example, in terms of hydrophilicity, hydrophobicity, or charge of the amino acid.
Choose an amino acid that has properties different from those of the amino acid before being substituted. Specifically, it is replaced. If the previous amino acid is aspartic acid, the substituted amino acid is asparagine.

Examples include threonine, valine, phenylalanine, and arginine, with asparagine and arginine being particularly preferred.

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

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

If the constituent amino acid before substitution is glycine, the amino acids after substitution are threonine, leucine, phenylalanine, and serine.

Examples include glutamic acid and alkynine, 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, alkinine, and aspartic acid, with methionine being particularly preferred.

If the constituent amino acid before substitution is valine,
Examples of the amino acid after substitution include senone, leucine, proline, glycine, lysine, and aspartic acid, with serine being particularly preferred.

The original constituent amino acids before substitution are preferably aspartic acid, arginine, glycine, serine, and valine.

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

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.

The aF GF mutin used in the present invention has one or more cysteine residues at positions 16, 83, and 1.17 of mature human aF GF substituted with other amino acid residues. And/or methionine may be added to the N-terminus.

To produce the mutin, site-directed mutagenesis technology is used.
nesir) will be adopted. The technique is well known and has been described by Lather, R.F. and J.P. Recoq, J.P.
), Genetic Engineering (Gene
tic Engineering), 2nd Century Press, 1983, pp. 31-50. Oligonucleotide-directed mutagenesis is described in Smith, M. and Gillam, S., Genete Inc. Engineering: Principles and Methods, Plenum Press (L98.
1) Volume 3, pages 1-32.

In order to produce a structural gene encoding the mutin, for example, (a) one consisting of a single chain of the aF GF structural gene
The double-stranded DNA is hybridized with the mutant oligonucleotide primer (for the region containing the codon for cysteine that cannot be replaced by this single strand, or, optionally, an antisense triplet that pairs with this codon). The above-mentioned primers are complementary, except for the mismatch of the codon with other amino acid encoding codons or, in some cases, with antisense triplets.)
, (b) Extend the brimer with DNA polymerase to form a mutant heteroduple.
(c) replicating this mutant heterodimer.

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

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

In the present invention, the mutin lacking the constituent amino acids of aFGF as an antigen is preferably a mutin consisting of 110 or more of the amino acid sequence of aFGF.

When immunizing with the aFGF protein, aFGF may be used as a complex with a carrier protein for immunization.

Examples of the carrier protein include bovine serum albumin, bovine thyroglobulin, and hemocyanin.

When using a carrier protein complex, the coupling ratio between the carrier protein and aFGF protein is about 0.1 to
It is used at a ratio of 30 times (carrier/aFGF protein/ff1ffi). Desirably, about 0.5 to 5 times is used.

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

When immunizing with aFGF protein or protein complex, the mammals to be immunized include sheep, goats, rabbits, guinea pigs,
Although experimental animals such as rats and mice can be used, rats and mice are preferred for obtaining monoclonal antibodies.

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 dose, etc. are highly variable, and various methods are possible.
A method using spleen cells extracted after 5 days, preferably about 2 to 4 days, is often used.

The immunization dose is approximately 0.1 peptide per mouse.
It is desirable to use µg or more, preferably about 10 µg to 300 µg. Furthermore, before removing the spleen, it is desirable to perform a partial blood sampling, confirm an increase in antibody titer in the blood, and then perform a fusion experiment using spleen cells.

The above cell fusion between spleen cells and lymphoid cells can be carried out by, for example, using isolated mouse spleen cells with hypoxanthine-guanine-phosphoribosyltransferase deficient (HGPRT)
-) or appropriate allogeneic or xenogeneic (preferably allogeneic) myeloma bearing markers such as thymidine kinase deficiency (TK-) [e.g., P3-X63-Ag・8Ul (Ichimori
Other Journal of Immunological Methods 80
55 (1985)) and a lymphoid cell line. For example, the method of Keller and Milstein et al. [Nature 256: 49
5 (1975) 3. For example, myeloma cells and lung cells may be mixed in a ratio of about 1=5, for example, Iscot medium and Ham's F-12 medium.
: Sendai virus, polyethylene glycol (P
A fusing agent such as EG) is used. Of course, it is also possible to add dimethyl sulfoxide (DMSO) and other fusion promoters.

The degree of polymerization of PEG is usually about 1000-60'OO.

As an example of preferred conditions, the time is about 0.5 to 30 minutes, the concentration is about 10% to 80%, etc.
By treating 000 at a concentration of about 35 to 55% for about 4 to 10 minutes, efficient fusion can be achieved. The fused cells are
Hypoxanthine-aminopterin-thymidine medium [HA
T medium: Nature λ5, 495 (1975) 3, etc. can be used to selectively proliferate.

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 to this and 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 caprijig of the enzyme and peptide according to a conventional method) is added and reacted 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 at a constant temperature for a certain period of time, after which the colored product produced can be measured by absorbance, fluorescence, or the like.

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 RP MI 1640 [Moore.

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

As5oc, ) 1 degree 9. 549 (1967)l
Approximately 2 to 1
By culturing for 0 days, preferably about 3 to 5 days,
The monoclonal antibody can be obtained from the culture solution.

Alternatively, antibodies can be obtained by intraperitoneally inoculating the cells into a mammal, allowing the cells to proliferate, and collecting ascites fluid.

For this purpose, for example, in the case of mice, approximately 1×1
0' to lXl0', preferably about 5X 10' 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 against the aF G )" protein is obtained.

The monoclonal antibody of the present invention specifically binds to the aFGF protein of the immunogenic peptide.

Note that the monoclonal antibody of the present invention may bind to aFGF protein different from the immunogen peptide used during production.

Since the monoclonal antibody of the present invention binds to aFGF protein, it is extremely useful as a reagent for measuring aFGF protein. Furthermore, basic knowledge regarding aFGF (
For example, it is extremely useful for obtaining biodistribution). Detection of aFGF in nine tissues of living organs is usually performed by enzyme immunoassay (EIA) or the like, or by fluorescent antibody method or radioimmunoassay (RIA). In addition, a protein Western blotting method is effective in determining the size of aFGF present in these six organs and tissues. This method involves performing acrylamide electrophoresis on a crude extract derived from one organ tissue or a partially purified sample thereof, and then transferring it to a membrane filter.
Detection is performed using a P-linked anti-aF GF protein antibody. In addition, some cancer cells themselves produce aFGF, and the aFGF
It is possible that the proliferation may continue due to F.

When anti-aFGF protein antibodies act on such cancers, aFGF, which promotes proliferation, is neutralized, inhibiting the proliferation of cancer cells,
In other words, it is expected to act as an anti-cancer substance. Also aF
In cancers that produce GF, aFGF can be quantified using antibodies, and can also be applied to cancer diagnostic tests. Furthermore, by utilizing the binding ability between the antibody and aFGF, an antibody affinity column can be prepared and used as a reagent for purifying aFGF.

The antibody molecule used to detect and quantify aFGF may be IgG or a fragment thereof.

(eg, F(ab')*, Fab' or Fab). Among these, the antibody molecule to which the labeling agent is directly bound is preferably Fab'.

The monoclonal antibody of the present invention can be used as a reagent in immunochemical measurement of aFGF.

In the immunochemical assay, when two or more types of antibodies are used at the same time, particularly when three types of antibodies are used at the same time, results with good detection sensitivity can be obtained.

By immunochemical measurement of aFGF protein, it is possible to measure the amount of aFGF protein in living tissues and body fluids, and thereby, as mentioned above (for example, angiogenic factors in various tissues and body fluids) can be measured. Measuring it is thought to be useful for cancer diagnosis.

As the antibody retained on the carrier, an anti-aFGF protein antibody is used.

The labeling agent-conjugated antibody used in the assay method of the present invention is one in which the labeling agent is directly bound to an anti-aFGF protein antibody that has a different antigen-determining site from that of the antibody held on the carrier.

Anti-aFG used in the immunochemical assay method of the present invention
Any F protein antibody may be used as long as it has the ability to bind to aF GF protein.

Examples of the carrier in the antibody supported on a carrier used in the method for measuring aFGF protein include gel particles (e.g., agarose gel [e.g., Sepharose 4B,
Sepharose 6B (manufactured by Pharmacia Fine Chemicals (Sweden)), dextran gel [e.g., Sephadex G-75, Sephadex C-too, Sephadex G-200 (manufactured by Pharmacia Fine Chemicals (Sweden)]), polyacrylamide Gel [e.g., Biogel P-30, Biogel P-60, Biogel P-100 (manufactured by Bio-Rad Laboratories (USA))], cellulose particles [e.g., Avicel (manufactured by Fukkasei)]
, ion-exchanged cellulose (e.g., diethylaminoethyl cellulose, carboxymethyl cellulose)], physical adsorbent [e.g., glass (e.g., glass bulbs, glass rods, aminoalkyl glass spheres, aminoalkyl glass rods),
Silicon pieces, styrene resins (e.g., polystyrene spheres, polystyrene particles), immunoassay plates (e.g., manufactured by Nunc (Denmark)), ion exchange resins (e.g., weakly acidic cation exchange resins [e.g., Amberlite IRC- 5
0 (manufactured by Rohm & Haas (USA)), Theocarp 226 (manufactured by Palmchit (West Germany))], weakly basic anion exchange resins (e.g., Amberlite IR-4B,
Examples include DOWEX 3 (manufactured by Dow Chemical Company (USA)).

In order to retain the antibody on a carrier, a known conventional method can be applied, but for example, "Metabolism", Vol. 8 (1971),
Examples include the bromcyan method and the geltaraldehyde method described on page 696.

Alternatively, as a simpler method, it may be physically adsorbed onto the surface of the carrier.

Examples of the labeling agent in an antibody bound to a labeling agent include a radioactive isotope, an enzyme, a fluorescent substance, a luminescent substance, etc., and it is preferable to use an enzyme.

As an enzyme, one that is stable and has a high specific activity is preferable.
peroxidase, alkaline phosphatase, β-D-
Galactosidase, glucose oxidase, etc. can be used, but peroxidase is preferred. As peroxidase, those of various origins can be used, examples of which include peroxidase obtained from horseradish, pina cible, fig, sesame, fava bean, corn, etc. In particular, peroxidase obtained from horseradish Horseradish peroxidase extracted from
(HRP) is preferred.

In order to utilize the thiol group of Fab' as an antibody molecule in binding peroxidase and antibody, it is convenient to use a peroxidase into which a maleimide group has been introduced in advance.

A method for introducing a maleimide group into peroxidase is to introduce the maleimide group via an amino group of peroxidase. For that purpose, N-succinimidyl-maleimido-carboxylate derivatives can be used, preferably N-(γ-maleimidobutyloxy)succinimide (sometimes abbreviated as GMBS). Therefore, a certain group may be present between the maleimide group and the peroxidase.

・To react GMBS with peroxidase, both are heated at about 10 to 50°C in a buffer solution with a po of about 6 to 8.
The reaction is carried out by reacting at a temperature of about 10 minutes to 24 hours. As the buffer solution, for example, pH 7.0
Examples include 0.01 phosphate buffer. The maleimidated peroxidase thus obtained can be purified, for example, by gel chroma-dolphin. Examples of carriers used in gel chromatography include Sephadex G-25, [
Manufactured by Pharmacia Fine Chemicals (Sweden)]
, Biogel P-2 [manufactured by Bio-Rad Laboratories (USA)], and the like.

The reaction between maleimidating peroxidase and antibody molecules is
Both were mixed in a buffer solution at a temperature of about 0°C to 40°C for about 1 hour.
This can be carried out by reacting for 48 hours to 48 hours. Examples of the buffer include O.1M phosphate buffer containing 5mM ethylenediaminetetraacetic acid sodium salt at pH 6.0. The bell-okindase-labeled antibody thus obtained can be purified, for example, by gel chromatography. Examples of carriers used when carrying out the Kel chromatography include Sephatex G-25 [manufactured by Farman Fine Chemical Co., Ltd. (Sueten)], Biokel P-2 [
Hioshi.

One example is Konato manufactured by TO Laboratories (USA).

Furthermore, a thiol group may be introduced into peroxidase and reacted with a maleimidated antibody molecule.

Direct binding of an enzyme other than peroxidase to an antibody can be carried out in the same manner as for peroxidase, and the per se known glutaraldehyde method, periodic acid method, water-soluble carbodiimide method, etc. can be used.

Test samples in the measurement system of the present invention include urine, serum,
Examples include body fluids such as plasma and cerebrospinal fluid, extracts of animal cells and bacterial cells, and culture supernatants thereof.

As an example of the measurement method of the present invention, a case in which the labeling agent is peroxidase will be specifically explained below, but it is not limited to peroxidase.

First, ■: The aF to be measured is added to the antibody retained on the carrier.
After performing an antigen-antibody reaction by adding the GF-containing analyte, the peroxidase obtained above and anti-aF
A conjugate with a GF protein antibody is added and reacted.

Samples to be tested in this measurement system include body fluids such as urine, serum, plasma, and cerebrospinal fluid, extracts of animal cells or bacterial cells, or culture supernatants thereof.

(2) Add a peroxidase substrate to the reaction product obtained in step 20, and measure the absorbance or fluorescence intensity of the resulting substance to determine the enzyme activity of the reaction product.

(2) The above procedures (1) to (2) are performed in advance on a known amount of a standard solution of aFGF, and the relationship between aFGF and absorbance or fluorescence intensity is prepared as a standard curve.

(2) The absorbance or fluorescence intensity obtained for the analyte (test sample) containing an unknown amount of aF GF is applied to a standard curve, and the amount of aF GF in the analyte is measured by C.

The monoclonal antibody of the present invention can be used to detect aFGF protein.
When used for quantitative determination, high detection sensitivity can be obtained. Therefore, by this detection method, for example, trace amounts of aFGF produced by cancer cells can be detected.

The aFGF protein can be purified using an antibody obtained using a complex of a monoclonal antibody against the aFGF protein and a protein for xeria as an immunogen. This includes:
This can be done by performing affinity column chromatography using the antibody.

The affinity column chromatography can be performed, for example, by coupling the antibody to a suitable carrier, filling a column with the antibody, passing a solution containing the aFGF protein through the column to adsorb it, and then eluting it.

Examples of the carrier include carriers similar to those described above. In particular, gel particles and various synthetic resins are conveniently used. For example, CNBr-acti
vated 5epharose 4 B (manufactured by Pharmacia Fine Chemicals), Affigel-1O,
Affigel 15 (manufactured by Bioland Laboratories)
Examples include.

In order to couple antibodies to carriers, known conventional methods can be applied, such as those described in "Metabolism", Vol. 8 (1971).
Examples include the Bromsian method and the geltaraldehyde method, which are described in 1999, p. 696. Further, a method using water-soluble carbodiimide, an active ester method, etc. can also be used, but as a simpler method, it may be physically adsorbed onto the surface of the carrier.

In order to perform purification using the antibody column thus obtained, aF GF protein in a near-neutral buffer is adsorbed onto the antibody column filled with a carrier bound to the antibody. Next, after washing the column with the same buffer, the specifically adsorbed aFGF protein is eluted. Elution of specifically absorbed antibodies is carried out using, for example, a low pH or high pH buffer, or a buffer containing a high concentration of salt.

As the low pH buffer solution, for example, a 0.0.
17M glycine-hydrochloric acid buffer, pH 1.8, 0.1
M Secondary sodium citrate-hydrochloric acid buffer and the like.

Examples of the high pH buffer include aqueous ammonia at pH 11 and 0.2M sodium borate buffer at pH 11.7.

As the buffer containing the high concentration of salt, for example, 6M
Examples include guanidine hydrochloric acid solution and 7M urea solution.

The above elution may be performed by a batch method or by a method using a column.

The antibody eluate is purified, for example, by dialysis. For example, when eluting with a low pH buffer, for example, 0.1M sodium carbonate buffer (pH 10.5), and when eluating with a high pt-t buffer, for example, 0.1M glycine-hydrochloric acid buffer (pH 3). , 0) and then, for example, 0.1%
0.02M phosphate saline buffer (pH 8,
0). Alternatively, the antibody solution eluted with a buffer containing a high concentration of salt can be directly dialyzed against the above-mentioned phosphate saline buffer and stored. Alternatively, the eluate or dialysate may be lyophilized and stored as a lyophilized specimen.

The aFGF protein purified in this way has an extremely high unit content and can be used, for example, as a wound healing promoter, and also has a neuronal proliferation effect, making it effective for the treatment of various neurological disorders. Available.

In order to use the aFGF protein as a medicament for the above-mentioned treatment, it can be used as a powder as it is, or in a pharmaceutical composition (e.g.
Injection 1 tablet, capsule, liquid.

ointments), mixed-breed animals (e.g., humans, mice, rats).

It can be safely administered parenterally or orally to hamsters, rabbits, dogs, cats).

The above-mentioned formulation as a pharmaceutical composition is carried out according to a conventional method.

When using aFGF protein as the above-mentioned medicine,
For example, the drug is administered to the above-mentioned warm-blooded animals by selecting an appropriate daily dose from about Long to 10 μg/kg in consideration of the route of administration, symptoms, etc.

Furthermore, the aFGF protein purified in this manner can be used as a test for promoting cell culture. In this case, the aFGF protein is preferably added to the medium in an amount of about 0.1 to 10 μg per medium 112.

In the specification and drawings of the present invention, when bases, amino acids, etc. are indicated by abbreviations, IUPAC-IUB
Collmision on Biochem
It is based on the abbreviations according to the ical culture or the abbreviations commonly used in the field, and examples thereof are shown below. Furthermore, if an amino acid can have optical isomers, unless otherwise specified, the L-isomer is assumed to be indicated.

NA DNA NA ATP TTP GTP CTP T.P. dr D.T.A. DS G,Gly :deoxyribonucleic acid : Complementary deoxyliponucleic acid :Adenine dithymine :guanine :Cytosine :ribonucleic acid : Deoxyadenone triphosphate :Deoxythymidine triphosphate : Deoxyguanosine triphosphate : Deoxycytidine triphosphate :Adenosine triphosphate : Thymidine :Ethylenediaminetetraacetic acid sodium nidodecyl sulfate :glycine A.Ala V, Vat L.Leu 1, l1e S, Ser T, Thr C, Cys M, Met E, Glu D.Asp. K, Lys R, Arg H, His F, Phe Y, Tyr W,Trp P, Pr.

N, Asn Q, Gin lz: Alanine: Valine: Leucine: Isoleucine: Serine: Threonine: Cysteine: Methionine Glutamic acid: Aspartic acid: Lysine: Arginine: Histidine: Phenylalanine; Tyrosine Tryptophan proline: Asparagine: Glutamine: 2-chloro Penzyloxycarbonyl BrZ: 2-bromobenzyloxycarbonyl Bzl: Benzyl Boa: t-butoxycarbonyl The hybridomas produced in the examples described below were obtained from the Fermentation Research Institute (IFO), Ministry of International Trade and Industry. The mortar and deposit number deposited with the Institute of Microbial Technology (FRI) are shown in Table 1 below.The FERM BP number of FRl indicates the deposit number based on the Budapest Treaty.

(Margin below) Combinant human basic FGF (hereinafter sometimes abbreviated as rhbFGF) used in the examples described below is obtained from the European Patent Application Publication (hereinafter also abbreviated as EP publication). ) Manufactured by the method described in Publication No. 237,966. That is, the rhbF G F
is a transformant of Escherichia coli (Escherichia coli).
hia coli) K12 MM294/pTB669
(I Fo 14532, FERM BP-12
81) by the method described in Examples 1.3 and 6 or 8 of EP Publication No. 237,966,
It is purified.

Transformant E described above, coli K 12 MM2
94/pTB669 has been deposited with IFO and FRI. The entrustment numbers and dies are shown in Table 2 below. Regarding the deposit with FRI, it was initially deposited domestically and a deposit number indicated by the FERMP number was attached, but the deposit was changed to deposit based on the Budapest Treaty and was transferred to FE.
It is assigned an accession number indicated by the RM BP number and is stored in Ft.

Transformant Escherichia coli MM2 produced in the reference example described below
94(DE3)/pLysS, pTB975. E,co
li MM294(DE3)/pLysS, pTB
1069 and E. coli MM294 (DE3)
/pLysS, pTB1070 is IFO and FRI
It has been deposited in Their accession numbers and dies are shown in Table 3 below.

(The following is a blank space) Examples The present invention will be explained in more detail with reference examples and examples below, but the present invention is not limited by these in any way.

Reference Example 1 (Preparation of recombinant human aFGF) Human aF
G F, Biotechnology 5, 96
0 (1987); Journal or Bio
Logical Chemistry 2631647
1 (198g), and IC5U 5hort
Report volume 8. Advances
in Gene Technology: Pro
tein Engineering and P
production.

Proceedings of the 1988 M
iami Bio, /TechnologyWinte
r Symposium, IRL Press, p
It was manufactured by the following method with reference to the method described in AGE 110.

(a) Construction of expression plasmid The chemically synthesized human haFGF cDNA (Fig. 1) was
U C18 [Methods in Enzymolo
gy, 10120-78 (1983)).
Transformant Ecoli K12 MM294/pTB917 obtained by introducing the into Escherichia coli K12MM294
has been deposited with IFO as IFO15093. )
Cut with BspMl and make large fragment
This site was made blunt-ended by the reaction described above, and then digested with BamHI to prepare a 0.45 Kb DNA fragment. The vector DNA contained pET3c (5tudier, F, W
et al. J, 1AolBiol ↓89: 113-1
30 (1986)), pE and T3c were cut with Ndel, large fragment was made into blunt ends, and Ncol linker-5'- was added with T4 DNA ligase.
CCΔTGG-3' was conjugated. This plasmid is N
cut the site with DNA polymerase lar
BamHI after smoothing by ge fragment
Cut it with , remove the SIO array, and add the preceding 0, 45 there.
The Kb blunt B spM rBamHI fragment was integrated using T4 DNA ligase to create pT B97.
5 was obtained (Figure 2).

(b) Expression of human aFGF cDNA in E. coli Next, R of T7 phage was injected into E. coli MM294 strain.
λ phage DE incorporating the NA polymerase gene
3 (Studier, F, W, et al. J, Mo1.B
iol, 189: 113-130 (1986)
) was lysogenized, and the plasmid pLys 5 (Studier,
F, W, et al. J, Mo1. Biol, 189:1
13-130 (1986)) was introduced into E. coli MM2.
94(DE3)/pLys S strain was created. pTB975 was introduced into this E. coli strain, and E. coli ~4M2'9
4 (D E 3 )/p1. yss, pTB975 (
IFO14936, FER~I BP-2599). 35μg/d Anpinlin, 10μg of this bacteria
Culture at 37°C in a medium containing chloramphenicol, and when the turbidity reached Klett I 70, add isopropyl β-D thiogalactone (IPTG) to a final concentration of O, V mM. In addition, the culture was continued for an additional 3 hours. Bacterial cells were collected by centrifugation, washed with water-cooled PBS, and then recollected and stored at -20°C until use.

(c) Purification of human aF G
mM Tris-HCl2 (pH 7,4), L O
mM EDTA, Q6~lNaC1, 10% N~3
Sugar, 0.25mM PMS F? Then, egg white lysozyme was added at a concentration of 0.5 mg/d. After leaving in water for 1 hour, incubate at 37°C for 5 minutes, perform ultrasonic treatment (20 seconds, twice) under water cooling, and centrifuge (S
ORVALL.

18 Krpm, 30 min, 4°C) to obtain a supernatant. This supernatant was diluted with ice-cold 20mM Tris for 200d.
-HCρ (pH 7,4), mixed with 1 mM EDTA, 20mM Tris-HCl2 (pH 7,4), 1
It was applied to a heparin Sepharose column (diameter 2.5×4 Cin) equilibrated with mM EDTAo, 2M NaC12. The column was washed with 150 m of 20 mM Tris-HCC' (
pH 7,4), 1mM EDTA, 0.5M Na
After washing with C12, 20mM Tris-HCl2 (1
)H7,4), LmM EDTA.

Proteins were eluted with 1.5M NaCf2. The eluate was fractionated every 6 days, the OD was monitored, and the second peak fractions (numbers 8 to 11, total 24 days) were collected (Figure 3).

For this fraction 22d, an equal amount of 20mM Tris-HC
(! (pH 7,4), 1mM EDTA, 2M (NH
-) Mix tS04 and add 20mM Tris-H.
Cl2 (pH 7,4), 1mM EDTA, IM (N
H4) tso.

Phenyl Sepharose column (diameter 2.5X) equilibrated with
8c+a) (flow rate 0, 53112/win
).

After washing the column with 20 m of the same buffer, IM to OM
ammonium sulfate linear concentration gradient (flow rate 0, 5 y
tl/sin, gradient time 200 minutes), eluted fractions 40-55 were collected (Figure 4), and purified human aF
GF.

(d) Reversed-phase C HPLC A 1.2 mg/m solution of purified human aFGF was mixed with 0.25 d of 0.1% trifluoroacetic acid (TFA) and subjected to reverse-phase C HPLC.
4 column (VYDAC) and O11% TFA.
The elution pattern was examined by applying a linear concentration gradient from 0% to 90% acetonitrile. Flow rate 17/nin,,
The gradient time was 60 minutes (Figure 5).

(e) The activity of bioactive human aFGF was determined by the method of Sasada et al. (Sasada et al., Mo1. Ce1l Biol, 8:5
88 594 (198 g)), mouse BALB/
The induction of DNA synthesis in c3T3 cells was measured using [3H]thymidine incorporation as an indicator. When adding the sample, add heparin (SIGMA Gr.
ade I) solutions were mixed.

Reference Example 2 (Production of partial peptide of aFGF) (1)
H-Phe-Asn-Leu-Pro-Pro-G
ly-Asn-Tyr-Lys-OH (human aF G
Boc-
Lys (CQZ) phenylacetamide (PAM) resin 0
, 5 mmole, the following amino acids were sequentially subjected to condensation and de-Boc reaction.

Boa-Tyr(BrZ)-OH Boc-Asn-OH Boa-Gly-OH Boc-Pro-OH Boa-Pro-OH Boc-Leu-OH Boc-Asn-OH Boa-Phe-OH Thus, Boc-Phe -Asn-Leu-Pro-
Pro-Gly-Asn-Tyr(BrZ)-Lys(
1.13 g of C12Z)-PAM resin was obtained.

This was mixed with 1.34d of anisole and 1.34d of dimethyl sulfide.
, 34- in hydrogen fluoride 13d at 0°C for 160 minutes, the hydrogen fluoride was distilled off under reduced pressure, the residue was washed with ethyl ether, and the target product was extracted with IN acetic acid 501t1,
This was ion-exchanged using an Amberlite IRA-400 (acetic acid type) column (2Z5 cm), and the eluate was freeze-dried. This was dissolved in 30% acetic acid 10j112, and Sephadex G-50 (manufactured by Pharmacia; column: 5X
The product was purified by gel filtration using 110 cm (elution H: 30% acetic acid) to obtain 267 mg of a partially purified product. Set this to 0
．． Dissolve in 20% IN acetic acid, CM-52 (Whatman Co., Ltd.; column 2.2 x 18 cm; elution method: gradient elution with 0.01 M ammonium acetate aqueous solution (pH 4,5) - 0.15 M ammonium acetate aqueous solution (pH 6,5). ) to obtain the desired product.

Yield 213 mg Rf O,50 (n-but/-ru:hyricin:acetic acid:
Water = 5:5:1:4) Amino acid analysis value: Asp 1.98; Gly
1.00; LeuO,99: TyrO,9
7; Phe 1.00: Lys O, 98
;Pr.

2.1O (2) H-Leu-Pro-Leu-Pro-Va
l-3er-Ser-Asp-OH (human (bovine)
Production of aF G F (133-140)) Boc-Asp (OBz12)-PAM resin 0 using an Applied Biosystems Model 430A fully automatic peptide synthesizer.
, 5 mmole, the following amino acids were sequentially subjected to condensation and de-Boc reaction.

Boc-3et(Bz12)-0H Boc-3et(Bz(2)-0H Boa-Val-OH Boc-Pro-OH Boc-Leu-OR Boc-Pro=OR Boc-Leu-OR Thus, Boc-Leu -Pro-Leu-Pro-
Vat-3et(Bze)Set(Bzff)-As
p(OBz12)-PAM resin 1. Obtained lOg.

Of this, 500mg of anisole 0.6d and dimethyl sulfide 0.6j! Hydrogen fluoride containing I26. After treatment with O'C in Od for 60 minutes, hydrogen fluoride was distilled off under reduced pressure,
After washing the residue with ethyl ether, the target product was extracted with 40ml of IN-acetic acid, and
(acetic acid type) column (2×5 cm), and the eluate was lyophilized. This was dissolved in 5 m of IN acetic acid, and Sephadex LH-20 (manufactured by Farmana Co., Ltd.; column:
The desired product was purified by gel filtration using 2.5×1 25 cm; eluent: IN acetic acid).

Yield: 120 mg Rr O, 43 (ethyl acetate:buta/l:acetic acid water
:1:l:1) Amino acid analysis value: ^sp 1.00; Ser 1.
95; Pr.

2.06; Val O, 98; Leu
2.01 Reference Example 3 (Amine terminal deleted recombinant human)
Preparation of aFGF) (a) Construction of expression plasmid The chemically synthesized cDNA of human aFGF (Fig. 1) was
UCl 8 [Methods in Enzymol
After cutting the plasmid pTB917, which had been integrated into SmaI (Fig. 6) or PvuIl (Fig. 7), into T.
Ncol linker-5'-CCA by DNA ligase
TGG-3' was conjugated. These plasmids were converted into Nc
A DNA fragment of 0.41 kb5+10.3 kb was prepared by cutting with Ol and BamHI. Vector DN
A contains pE with the T7 phage φ10 promoter.
T 8 C[5tudier, F, W, (Br
ookhaven National Labs U.

Received a donation from S.A. ] was used. Cut pET8c with Ncol and BamHI and insert the 0°4
A 1 kb or 0.3 kb DNA fragment was integrated using T4 DNA ligase to obtain pTB1069 (Figure 6) or pTB1070 (Figure 7), respectively.

(b) Expression of amino-terminal deleted human aFGF cDNA in Escherichia coli λ phage DE3 [5tud
ier, F, W, et al + J, Mo1
．． Biol: 189 +11:3-130(
1986)] was lysogenized, and a plasmid pL ysS (
Studier, F., et al.
J, Mo1. Biol.

189: 113-130 (1986)),
Escherichia coli MM 2
94 (D E 3 )/pLysS strain was created. By introducing pT81069 or pTB1070 into this E. coli strain, E. coli MM294 (DE 3)/pLysS,
pTB 1069 (I FO14937, FERM
BP-2600) or Escherichia coli ~1M294 (DE3
)/I) LysS, pTB1070 (IFO14938
, FERM BP2601). These bacteria were treated with 35 μg/d ampicillin and 10 μg/d, respectively.
Cultured at 37°C in a medium containing g/d chloramphenicol.
When the turbidity reached Klett 120, isopropyl βD-thiogalactone was added to a final concentration of 0.5 mM, and the culture was continued for an additional 2 hours.

Bacterial cells were collected by centrifugation, washed with water-cooled phosphate bath, fardoserine (PBS), collected, and stored at -20°C until use.

(c) Purification of aFGF lacking 5 amino terminal residues 75
d E. coli MM294 (DE3) pLys collected from culture.
S, pTB1069 (IFO14937°FERM
IQ, d(7) water-cooled 10mM
Tris-HCC (pH 7,4), 10mMEDT
A, 0.2M NaCl, 10% sucrose, 0.25mM
It was suspended in phenylmethylsulfonyl fluoride (PMSF), and egg white lysozyme was added at a concentration of 0.5 mg/filter. After being left in water for 1 hour, incubated at 37°C for 5 minutes, sonicated under water cooling, and centrifuged (SO
RVALL, 18K rpm, 30 minutes.

4°C) to obtain a supernatant. This supernatant was diluted with 20 mM T
Heparin H equilibrated with risHCl2 (pH 7,4)
It was applied to a PLC column (0.8 cm in diameter x 5 cm).

Coat the column with 20mM Tris-HCl2(+)H7,4
), after washing with 0.6 M NaCQ, 2 M N from OM
aCQ linear concentration gradient (flow rate 1 m/min, gradient time 1
time) and fractionated every ld. Eluted fraction 28
-32 were collected (Figure 8). By the above operations, the N-terminal 5 residue-deleted aFGF having the amino acid sequence shown in FIG.
4.2 mg of mutin (hereinafter sometimes referred to as rN5-deficient aFGFJ) was obtained.

(d) Purification of human aFGF with amino terminal 43 residues deleted 12! E. coli MM294 (DE3) collected from M culture
pLyss, pTB1070 (IFO14938, FE
RM, BP-2601) cells were cooled with water at 1042
mM Tris-HCl2 (pH 7,4), l Om
MEDTA, 0.2M NaCQ, 10% sucrose, 0.
It was suspended in 25mM PMSF, and egg white lysozyme was added at a concentration of 0.5 g/IIt1. After leaving it in ice for 1 hour, it was kept warm at 37°C for 5 minutes, and subjected to ultrasonication while cooling with water.
Centrifuged. The precipitate was suspended in 2 M NaCQ and centrifuged again, and the resulting precipitate was added to 15j112 of 6 M urea.

20mM Tris-HCC (pH 7,4), 10mM
It was suspended in dithiothreitol (DTT) and kept warm on water for 3 hours with occasional stirring. After centrifuging this solution, the supernatant was mixed with 3M urea, 20mM Tris-HC.
It was applied to a Q-Sepharose column (diameter 2.5 cm x 8 cm) equilibrated with Q (pH 7,4). After washing the column with the buffer used for equilibration, the OM to IM NaC
A Q linear concentration gradient was applied over 160 minutes at a flow rate of 0.6 m/min and fractionated every 2.5 d (Figure 9).

The eluted fractions 14-19 were collected and treated with 20mM Tri of 2Q.
s-HC (2 (pH 7,4), 5i+M after overnight dialysis against DTT, 3Q 20mM Tris-HCl
2 (pH 7,4), and dialyzed against 1 mM DTT for 3 hours. By the above operations, the N-terminal 43 residue-deleted aFGF mutin (hereinafter referred to as [
It is also called N43-deficient aFGFJ. ) 3.2m
I got g.

Example 1 (1) Immunity.

BALB/c mice (♀8 weeks old) were given 0.0% physiological saline.
100 μg of the antigen human aFGF (obtained in Reference Example 1) dissolved in 3d and the same amount of complete Freund's adjuvant (Difco) were mixed and injected subcutaneously. 3
A week later, a mixture of the same amount of antigen and 0.3 d of Freund's incomplete adjuvant was re-administered subcutaneously. Further 3 weeks later, a similar booster immunization was performed, and 2 weeks later, 100 μg of human aFGF dissolved in physiological saline was intravenously inoculated.

(2) Cell fusion: Three days after the final administration of the antigen, the spleen was removed from the immunized mouse obtained in (1) above to obtain cells used for cell fusion. These cells were suspended in a medium (hereinafter abbreviated as ``H medium'') in which Iskot's medium and Ham's F12 medium were mixed at a ratio of 1:1.

Mouse myeloma cells P3-X63-Ag-8Ul are 1
5 in RPMI 1640 medium containing 0% 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.

C,; Fuichi+-(Nature) 25-β-,4
95 (1975). 3.2 x 107 myeloma cells and 1.6 x 10" immunized lymphocytes obtained by the above method were mixed, centrifuged, and 0.3
45% polyethylene glycol 6000 (hereinafter referred to as PE06000) dissolved in No. 7 IH medium was added dropwise.

The PEG6000 solution was preheated to 37°C and slowly added dropwise. After 8 minutes, add 0.53112 per minute to IH medium prewarmed to 37°C to make 10d, then add 600% at room temperature.
The mixture was centrifuged for 15 minutes and the supernatant was removed. This cell precipitate is
Suspended in IH medium 200d containing 0% calf serum,
100 μg each was plated in 960 wells of a well microplate (Nunc). One day later, HAT (hyboxanthin 1 x 10-'M, aminopterin 4 x 10-7
IH medium (containing 20% calf serum) (hereinafter referred to as HAT medium) containing M, thymidine 1.6 x 10-'M).

) was added to each well in an amount of 100 μσ, and 1/2 the volume of the medium was replaced with HAT medium every 3 days. Cells grown in this manner are hybrid cells.

(3) Search for antibody-producing cells: Purified by the method described in Reference Example 1 or combined aFG
F to 10dg/m. O1M carbonate buffer (p
H8,5), and 100 μQ of the solution was placed in each well of a 96-well immunoplate (manufactured by Nunc) and incubated at 4° C. overnight to bind aFGF to the solid phase. 0.15M
0.01M phosphate buffer containing NaCc (pH 7,
After washing with 1%
200A1i2 of 0 and oIM phosphate buffer containing bovine serum albumin (BSA) were injected into each well and stored in a cool place until use. To the 96-well immunoplate bound with human aFGF obtained as described above, 100 μQ each of hybrid cell culture Tsuchiura was added and incubated at room temperature for 2 hours. After removing the culture supernatant and washing, a horseradish peroxidase (HRP)-labeled anti-mouse IgG Yaki antibody (Kabbell) was added as a secondary antibody, and the mixture was incubated at room temperature for 2 hours. After removing the secondary antibody and rinsing the wells thoroughly, add Veloki/Tase substrate solution. Night (002%H,
02 and 0.15% 07 p containing L/N diamine
Add 100 μQ of sodium citrate buffer (H5.5), react at 25°C for 10 minutes, and add 100 μg of 2N-sulfuric acid.
After stopping the enzyme reaction by adding
law). By this method, the presence of bound antibodies in 12 wells was observed.

(4) Cloning of hybrid cells: The cells in these wells were placed in a 96-well microplate in which 5XIO5 cells/well of mouse thymocytes were seeded as feeder cells in advance at 0.5 cells per well. Sowing and cloning. As a result, one representative clone cell was obtained from each well, and a total of 12 clone cells, namely AFi52 (
IFO50204, FERM BP-2607).

API-81 (IFO50205, FERMBP-26
81), API-113, AFL114 (IFO502
06, FERM BP2608), HaFIHll (
IFO50198) HaF IF5(IFO5020
0), HaF IC10 (IFO50197, FERM
BP-2605), HaF2F9 (IFO502
01), HaF2E'6 (IFO50202, FERM
BP2606), HaF2B7 (I FO5019
9).

HaFIAlo (IFO50203) and HaFIF
9 (IFO50196) was obtained.

(5) Production of antibodies: Hybridoma clone API-52, AFL8 obtained by cloning in (4) above], AFL
113. AFL 114. HaFIFII, Ha
FIF6. HaFIClo, HaF2F9. )(aF2
E6. HaF2B7. HaF lAl0 and HaFI
F9 was injected intraperitoneally into BALB/c mice, which had previously been intraperitoneally administered with 05d of mineral oil, to induce ascites formation by injecting 1×106 mice per mouse into ascitic fluid. After administering the hybridoma into the peritoneal cavity,
After o days, ascitic fluid was collected. Each ascites obtained approx.
Obakara, Staelin et al.
Monoclonal antibodies were purified according to the method of 981)E. First, to remove fibrin-like material from ascites, 10
．． After centrifuging at 000 rpm for 15 minutes, phosphate buffer-saline solution (PBS: 8.1mM-Na, HPO, 5rn
M KHtPO, 27mM KCQ, 137mM
The sample was diluted with NaC (!, pl(7,2) to a concentration exhibiting an ultraviolet absorption (Atso) of 12 to 14 at 280 nm. After dilution, a 47% saturated ammonium sulfate solution was added to the sample.
salting out for 60 minutes with stirring at 4°C, and then centrifuged (10,000 rpm, 15°C).
) to obtain a precipitate. The precipitate was diluted with 50mM Na.
Contains CQ 20mM)! The product was dissolved in JS buffer solution (pH 7, 9) and dialyzed against 2Q of the same solution. 2 hours later,
The dialysate was replaced with the same new 2σ dialysate, and dialysis was continued for an additional 15 hours. After dialysis, centrifugation was performed at 10,000 rpm for 15 minutes to remove the precipitate, and the supernatant was transferred to A2. . The value of 20~
The concentration was adjusted to 30. This sample was applied to a 20 d DEAE cellulose column (Whatman DE, ) equilibrated with a sufficient amount of Tris buffer containing 50 mM NaCQ, and then washed with Tris buffer containing 50 mM NaCQ (after washing, 500 mM 500 mM
Fractionation was performed using a concentration gradient salt solution of the same buffer containing NaC (!) at a flow rate of 1.5 ml/111 min, the flow-through fraction was concentrated, and each purified monoclonal antibody A
F 1-52. AF l-81゜API-113,A
FL 114. HaFIHll, HaFIF5. H
aF IC10, HaF2F9゜HaF2E6. HaF
2B7. HaFIAlo and HaFIF9 were obtained.

To confirm the purity of antibodies, the method of Laemli et al. [Nature,
5DS according to 227.680-685 (19yo))
- using polyacrylamide gel electrophoresis. That is, the fraction that passed through the DEAE cellulose column after salting out with ammonium sulfate was reduced with 2-mercaptoethanol, and the fraction was reduced to 2.5 at 180 ports using Kel with an acrylamide concentration of 10%.
Time migration was performed. As a result, all monoclonal antibodies had H chains before and after molecule ff152 and L chains before and after 28.
Two bands of strands were observed.

(6) Measurement of antibody subclass: Antibody subclass was measured by the following method. That is, reference example 1
100 μQ of each hybridoma culture supernatant obtained in the above or cloned into a 96-well immunoplate coated with combinant aFGF was added and incubated at room temperature for 2 hours. After removing the culture supernatant and washing, remove rabbit anti-mouse IgG1゜IgG2a, IgG2b.
, IgG 3 , K chain, and λ chain antibodies (Kabbell) were added at 100 μσ and incubated at room temperature for 2 hours. HRP after removing each antibody and washing
A labeled goat anti-rabbit IgG antibody (Cabbell) was added and incubated at room temperature for 2 hours. After removing the labeled antibody and washing thoroughly, an enzyme reaction was performed by the method described in (3) above, and the absorbance was measured. As a result, monoclonal antibody AP
It was found that two types, I-52 and AF 1-81, were of the IgG2bK type, and the other 10 types of monoclonal antibodies were all of the IgGIK type subclass.

(7) Examination of antibody recognition site: Regarding which site of aFGF the antibody recognizes,
We investigated using the following method.

rhaFGF obtained in Reference Example 1, partial peptides 1 to 9 of human aFGF obtained in Reference Example 2 (human aFGF (1-9)), and human aFGF 1
Partial peptide 33-140 (human aFGF (133-
140)), combined human basic fibroblast growth factor (rhbFGF) obtained by the method described in EP Publication No. 237.966, and N5-deficient aFGF and N43-deficient aFGF obtained in Reference Example 3. FGF was diluted with O, 0, 1M carbonate buffer (pH 8,5) to a concentration of 10 μg/ll12, and 100 μQ of the diluted solution was placed in each well of a 96-well immunoplate at 4°C overnight to incubate each antigen on the solid phase. were combined. After washing with 0.01M phosphate buffer (pH 7.0) containing 0.15M NaCl2, 0.01M phosphate buffer (pH 7.0) containing 1% bovine serum albumin (BSA) was used.
200 μC of 01M phosphate buffer was injected into each well and stored in a cool place until use. The culture supernatants of the 12 types of cloned cells obtained in (4) above were dispensed into 100 μσ portions onto the plates prepared as above, and ERA was performed using the same method as described in (3) above. , the results shown in Table 9 were obtained.

From the results shown in Table 9, monoclonal antibody API-
52 is the 1-5 amino acid sequence of aFGF, monoclonal antibody API-113゜IHIl, IF
5. Six species of ICl0.2F9,287 are 6-
43 amino acid sequence, monoclonal antibody AP
I-114, 2E6゜A10.1F9 are aFG
It was thought that the antigenic determinant present within the 44-132 amino acid sequence of F. Monoclonal antibody API-81 is a whole mole of aFGF.
cule, but does not recognize N5-deficient aFGF, nor does it recognize a peptide consisting of the 1-9 amino acid sequence of aFGF, so the recognition site could not be specified.

(Left below) Example 2 (1) Enzyme-labeled and purified antibody API-52 antibody (4.2 mg/1.41II2
) was added with S-acetylmercaptosuccinic anhydride at a concentration of 0.4 mg/m, and reacted at room temperature for 1 hour to introduce an SH group. Tris-HCl buffer (pH 7)
, 0), EDTA, and hydroxylamine to be 15mM, 1.5mM, and 15mM, respectively, to inactivate unreacted components, and then separated by Sephadex G-25 column chromatography to remove SH groups. The introduced antibody fraction was obtained.

On the other hand, horseradinosuberoxidase (HRP)
0.1M sodium phosphate solution containing 10n+g (pH
7,0) N to 1.4d so that it becomes 1.5n+g/d
-(γ-maleimidobutyryloxy)succinimide was added and the mixture was reacted at room temperature for 1 hour to introduce a maleimide group. Maleimidated HRP is Sephadex G-2
Separation was performed using 5-column chromatography. Mix the separated maleimidized HRP and the antibody into which SH groups have been introduced, and
Coupling reactions were performed overnight at °C. Ultrogel Δc after reaction
Separation was performed by t\44 column chromatography to obtain HRP-labeled AF L-52 antibody.

(2) Preparation of antibody-bound solid phase Purified AAFI-sl, API-114, and ICl0 antibodies were each diluted to 10 ug/d with 0.01 M carbonate buffer (pH 8, 5), and each alone or three types of 100 μQ of the antibody mixture was placed in each well of a 96-well immunoplate and incubated at 4°C overnight to bind the antibody to the solid phase. After washing with 0,01M phosphate buffer (pH 7°0) containing 0,15M NaCQ, 200ug of OOI Ni phosphate buffer containing 1% BSA was injected into the wells and kept in a cool place until use. Saved to.

(3) 100 μQ of the aFGF solution prepared to the concentration of the standard EIA method,
It was placed in a well bound to the antibody and incubated overnight at 4°C. a After removing the FGF solution, add 100 μl of HRP-labeled API-52 antibody diluted 200 times per well.
e and incubated at room temperature for 2 hours. After removing the labeled antibody, an HRP base solution was prepared, and the following enzymatic reaction was performed in the same manner as described in Example 1, and the absorbance at 492 nm was measured. As a result, three types of monoclonal antibodies (AF
When a mixture of L-81, AFL-114, and ICl0) is used as a solid phase, the sensitivity is better than when each antibody is used alone, and the detection limit for aFGF is extremely high at 1.5 pg/well. Detection was possible at low concentrations (Figure 10). In Figure 10, △ indicates monoclonal antibody API-
The results of 81 were obtained using monoclonal antibody API-114.
○ indicates the result of monoclonal antibody IC10,
* indicates the results for a mixture of three types of monoclonal antibodies, respectively.

a The detection sensitivity of FGF was best when three types of monoclonal antibodies were used (although the sensitivity was lower in all cases except one of them (see Figure 11).

That is, in FIG. 11, Mu is monoclonal antibody A.
The result is for the FL-81, AF1-114 mixture, △ is the result for the monoclonal antibody 1cIO, AFl-81 mixture, and ○ is the result for the monoclonal antibody ICl0. The results for the API-114 mixture are shown, and the * represents the results for the mixture of three monoclonal antibodies.

(4) Examination of specificity of sandwich EIA In order to examine whether the sandwich EIA set up is specific for human aFGF, rhbFG prepared at various concentrations was used.
Sandwich EIA was performed using F as an antigen. That is, 100 μQ of the above antigen preparation solution was added to the well to which the three types of antibody mixture had been bound and incubated overnight at 4°C.

After removing the antigen, HRP-labeled AFI diluted 200 times
52 antibody was added at 100 uQ per well, and the presence or absence of reactivity was examined in the same manner as above. As a result, rhb
Even at a high concentration of FGF of 800 ng/Ml, it was not detected by this sandwich EIA, indicating that the system is actually a system that specifically recognizes aFGF (Figure 12). 12th
In the figure, * indicates the results for aFGF, and ○ indicates the results for bFGF.

(5) Effect of heparin 4 monoclonal antibodies (API-52, AFl-8
We investigated whether heparin affects the aFGF binding of 1, API-114, ICl0). That is,
When each antibody was added to the aFGF-binding well prepared by the method described in Example 1, heparin was allowed to coexist to examine changes in antibody binding. The results are shown in Table 10.

Monoclonal antibody heparin (μg/, m) A Fl -520,9610,8340,887A
F 1-81 0.278 0.278
0.224AF 1-114 1.4
98 1.478 1.398 As is clear from Table 10, heparin 10mg/4,
It was found that even at a concentration of 100 mg/rm, the binding of none of the antibodies to aFGF was inhibited, and these antibodies did not recognize the heparin binding site of aFGF. Next, due to the presence of heparin, sandwich EIA
We also investigated whether it would be affected. That is,
Heparin was added to aFGF solutions prepared at various concentrations.
ug/m or 100 μs/d, and subjected to Santoi, Chi's EIA. As a result, the recognition of aFGF by this EIA was hardly suppressed even in the presence of heparin b (the detection sensitivity was 2 pg/m). well and high sensitivity was maintained (Fig. 13). In Fig. 13, ◯ indicates the results without the addition of heparin, ○ indicates the results with the addition of heparin at 100 μs/m,
Δ indicates the results of adding 10 μg/d of heparin.

Example 3 (1) Preparation of antibody-binding resin CNBr-activated 5ephar
ose 4 B lg on a glass filter.
Repeat washing and swelling with mM hydrochloric acid ('200d).

After washing the gel with coupling buffer (0.1M carbonate buffer (pH 8,0) containing 0.5M NaCQ), the AF 1-81°HaF obtained in Example 1 (5)
LC10, AF l l 14 each containing 2111 g of each monoclonal antibody, 2 pulls 1.
Add the gel to the fur, make the total volume 10ml with coupling buffer, and incubate at 4°C for 20 hours.

Take the gel on a glass filter and transfer it to 0゜2M.
Transfer to Gly NaOH (pH 8,0) and stir at room temperature for 2 hours.

Take the gel and add coupling buffer and 0°5M
Wash with 0.1M acetate buffer containing NaCl2,
．． Store at 4°C in 02M phosphate buffer (pH 7,0).

(2) Purification of human aFGF Apply the gel 0.5d prepared in (1) to a column (inner diameter 0°8c).
m') and thoroughly washed with buffer A (0.05M Heves buffer (pH 7.5) containing 0.15M NaCl2).

E. coli MM294 (DE 3) obtained in Reference Example 3(b)
)/pLysS, pT B 975 was cultured and the resulting bacterial cells were cultured in 1 m of water-cooled 10 mM Tris-HC (
1(p)17.4), 10mM EDTA, 0.2M
NaCQ.

Suspend in 10% sucrose and 0.25 mM phenylmethylsulfonyl fluoride (PMSF), and add egg white lysozyme to a concentration of 0.5+ng/m. After standing in water for 1 hour, incubate at 37°C for 5 minutes, perform ultrasonic treatment on water cooling, and centrifuge (SORVA L Ll 8
Krpm, 30 min at 4°C) to obtain the supernatant, of which 5
0 μQ is applied to the above column.

After thoroughly washing the column with buffer A, add buffer B (02M
aFGF is eluted with green-hydrochloric acid buffer (pH 2,0). The eluate is medium +13 on IM Tris. The biological activity of aFGF in the eluate was determined using Reference Example 1 (
When examined using the method of Sasama et al. described in e), the reference example + (d
) It shows almost the same specific activity as aFGF obtained in .

Since the monoclonal antibody of the present invention specifically binds to aFGF and has a high binding ability, it can be advantageously used as a reagent for determining aFGF.

[Brief explanation of drawings]

Figure 1 shows the aFGF cDNA used in Reference Example 1.
This shows the placement of the winnings. Figure 2 shows plasmid pTB97 obtained in Reference Example 1.
5 is shown. FIG. 3 shows the elution pattern obtained in Reference Example 1. FIG. 4 shows the elution pattern obtained in Reference Example 1. FIG. 5 shows the elution pattern obtained in Reference Example 1. FIG. 6 shows the N-terminal 5 residue deleted aF obtained in Reference Example 3.
A construction diagram of Hector pTB1069 for GF expression is shown. Figure 7 shows the N-terminal 43 residue deletion type a obtained in Reference Example 3.
A construction diagram of Hector pTB1070 for FGF expression is shown. Figure 8 shows N-terminal 5 residue deleted aFG obtained in Reference Example 3.
Fig. 9 shows the elution pattern of PLC column (heparin used for F purification).
The elution pattern from the Q Sepharose column used for FGF purification is shown. Figure 10 shows the sandwich E obtained in Example 2 (3).
The results of detection sensitivity of aFGF by IA are shown. FIG. 11 shows the results of detection sensitivity of aFGF by Santoy and Chi EIA obtained in Example 5 (3). FIG. 12 shows the specificity results of Santoy and 1,000 EIA obtained in Example 2 (4). FIG. 13 shows the san[・i,
We present the results of the effect of heparin on the EIA. Figure 14 shows the N-terminal 5 residue deletion "1" obtained in Example 3.
The amino acid sequence of h a F GF mutin is shown. Note that N1 at the N-terminus represents methionine due to the translation initiation futon. FIG. 15 shows the amino acid sequence of the N-terminal 43 residue-deleted haFGF mutiino obtained in Reference Example 3. Note that M at the N-terminus replaces methionine due to the translation start codon.

Claims (18)

[Claims] (1) Acidic fibroblast growth factor (a
Monoclonal antibody against FGF) protein: (a) Molecular weight: approximately 140,000-160,000 (b) Does not cross-react with basic fibroblast growth factor (bFGF). (c) The immunoglobulin class belongs to IgG. (2) A cloned hybridoma consisting of spleen cells of a mammal immunized with acidic fibroblast growth factor (aFGF) protein and homologous or heterologous lymphoid cells. (3) The hybridoma according to claim 2, wherein the mammal is a mouse. (4) The hybridoma according to claim 2, wherein the lymphoid cells are myeloma. (5) Claim 2, wherein the aFGF protein is human aFGF.
Hybridoma described. (6) Cell fusion of spleen cells of a mammal immunized with acidic fibroblast growth factor (aFGF) protein and homologous or heterologous lymphoid cells and cloning. Hybridoma production method. (7) The production method according to claim 6, wherein the mammal is a mouse. (8) The production method according to claim 6, wherein the lymphoid cells are myeloma. (9), Claim 6, wherein the aFGF protein is human aFGF.
Manufacturing method described. (10) Production of the monoclonal antibody according to claim 1, which comprises growing the hybridoma according to claim 2 in a liquid medium or intraperitoneally of a mammal, producing and accumulating the monoclonal antibody, and collecting the monoclonal antibody. Law. (11) The production method according to claim 10, wherein the mammal is a mouse. (12) Claim 1, wherein the lymphoid cells are myeloma.
The manufacturing method described in 0. (13) The production method according to claim 10, wherein the aFGF protein is human aFGF. (14) A method for detecting and quantifying aFGF protein, which comprises using the monoclonal antibody according to claim 1. (15) The detection/quantification method according to claim 14, wherein the aFGF protein is human aFGF. (16) The detection/quantification method according to claim 14, wherein enzyme immunoassay is performed. (17) A method for purifying aFGF protein, which comprises using the monoclonal antibody according to claim 1. (18), the purification method according to claim 17, wherein the aFGF protein is human aFGF.