CA1296275C - Monoclonal antibody, hybridoma, their production and use thereof - Google Patents

Abstract

MONOCLONAL ANTIBODY,HYBRIDOMA, THEIR PRODUCTIONAND
AND USE THEREOF

Abstract of the Disclosure A monoclonal antibody is produced from a cloned hybridoma, and the monoclonal antibody combines specifically with basic fibroblast growth factor (bFGF). Therefore, the monoclonal antibody can be advantageously used for assay reagents on bFGF or for purification of bFGF.

Description

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The subject matter of this application is closely rela-ted to copending Canadian Application Nos. 531,955, 559,540 and 559,536~
The present inven-tion relates to monoclonal antibodies which combine specifically with basic fibroblast growth factor, hybridomas, their production and use thereof.
The basic fibroblast growth factor (also briefly refer-red to as bFGF, in the present specification) is a basic polypep-tide hormone which is secreted mainly from the pituitary gland and which has a molecular weight of about 17,000. It was first isola-ted as a fac-tor showing potent growth promoting action on fibro-blasts such as BALB/C3T3 cells [D. Gospodarowicz: Nature, 249,123 (1974)]. Later, however, it was revealed that it exhibits growth promoting action on almost all mesoderm-derived cells [D.
Gospodarowicz et al.: ~ational Cancer Institute Monograph, 48, 109 (1978)]. The neovascularizing activity of bFGF, among others, conjointly with its cell growth promoting activity, suggests the possibility of its use as a therapeutic agent for lesions and burns and as a preventive/therapeutic agent for thrombosis, arteriosclerosis and the like.
The quantity of naturally occurring human bFGF is very small, and attempts to obtain this factor from human tissues have encountered serious difficulties arising from various restrictions and limitations. In addition, any method that is easily usable for quantitatlve determination of bFGF has not been established to date. For these reasons, much remains unknown of basic informa-tion which is essential for developing bFGF as a drug, such as the properties of bFGF.
Therefore, the development of bFGF as a drug will be facilitated if further basic information about bFGF is known, for example, the distribution of bFGF in vivo and the manner of its production.
In addition, to accurately determine the quantity of bFGF is important in purifying this protein from gene ~'

2~
- la - 24205-772 recombinants. Moreover, it is very important to -trace blood FGF
concentration in animals which have had bFGF administered thereto, but blood bFGF cannot be determined by the conventional method using 3T3 cells, due to the mingling of serum in the sample.
Usually, the determination o~ bFGF is achieved by adding bFGF to 3T3 cells which have been cultured at reduced serum concentration to a-ttenuate their DNA synthesizing potency, and counting back bFGF

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concentratioll from the degree of recovery of DN~ synthesizing potency.
However, this method is faulty in that the yrocedure is clelicate and deterlninatio~l errors are great, due to the use Or cells, and in addition much time is needed to obtain results. It is thererore desired that a simple and accurate means of bFGF cleterminatioll will be developed ~or the above-mentioned purpose.
Taking note of the above-mentioned circumstances, the present inventors macle various investigations to find any practical means of bFGF
determination, and prepared a monoclonal antibody which combines specifïcally with bFGF and which enables the determination thereof`. The present inventors conducted further researches based on this achievement, and as a result have now developed the present invention The present invention provides:
(1) a monoclonal antibody which combines specifically with basic fibroblast growth factor (bFGF), the monoclonal antibody having the characteristics:
(a~ it has a molecular weight of` about 1~LO to 1~0 kilodaltons, (b) it does not cross-react with acidic fibroblast growth ractor, and (c) it belongs to the irnmulloglobulill class IgM or IgG;
(2) a cloned hybridoma comprising a splenic cell from a mammal immullized with bFGF and a homogenic or heterogenic lymphoid cell;

(3) a method for producing a cloned hybridoma comprising a splenic cell from a mammal immunized with bFGF and a homogenic or heterogenic lymphoid cell, which comprises subjecting said splenic cell and said lymphoid cell to cell fusion followed by cloning;
(~) a methocl for producing a monoclonal antibody which combines specifically with bFGF, whieh comprises growing a cloned hybridoma comprising a splenic cell from a mammal immunized with said factor and a homogenic or heterogenic lymphoid cell in liquid medium or mammalian abdomen to allow the hybridoma to produce and accumulate the monoclonal antibody;
(5) a method for purifying bFGF, which comprises treating a material containing crude bFGF with the use of the monoclonal antibody derlned in said item (1); and (6) a method for detecting or measuring bFGF, which comprises using, as antibody, the monoclonal antibody defined in said item (1).
As the bFGF for immunizing mammals, any bFGF can be included, as long as it is a bFGF of a warm-blooded mammal. Its mutein can also be used, so in the present specification "basic fibroblast growth factor (bFGF)" may include its mutein unless otherwise specified.
As representative examples of such mammalian bFGF, mention may be made of bovine bFGF [Proceedings of the National Academy of Sciences, USA, 82, 6507 (1985)] and human bFGF European Patent Publication No. 237,966, European Molecular Biology Organization (EMBO) Journal 5,2523 (1986)].
Polypeptides which includes the amino acid sequence:
Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro are preferred.
~ore preferably, the polypeptides are represented by the 20 formula:
Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe-Pro-Pro-Gly-His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe~Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-Cys-Ala-Asn-Arg-Tyr-Leu-Ala-Met-L,ys-Glu-Asp-Gly-Arg-Leu-Leu-Ala-Ser-Lys-Cys-Val-Thr-Asp-Glu-Cys-Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-X-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Gly-Y-Lys-Thr-Gly~Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-5er :(II) wherein X represents Thr or Ser: Y represents Ser when X is Thr or Y represents Pro when X is Ser.

- 3a - 24205-772 For obtaining human bFGF (also briefly referred to as hbFGF3, an expression vector which contains a DNA segment having a base sequence encoding, for example, the abovementioned hbFGF
protein polypeptide, can be produced, for example, by:

:

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(a) Isolating an RNA encoding hbFGF;
(b)Synthesizing from said RNA a single-strandetl complementary DNA (cDNA) and then a double-stranded DNA;
(c) Inserting said complementary DNA into a plasmid;
(d) Transforming a host with the resultant recombinant plasmid;
(e)Cultivating the transformant thus obtained, then isolating the plasmid which contains the clesired DNA from the transformant by an appropriate method, for exarmple, the colony hybridization method using a DNA probe;
(f~ Cleaving off the desired cloned DNA from said plasmid; and (g) Inserting said cloned DNA into a vehicle at a site downstream from a promoter.
E~NAs encoding hbFGF can be obtained from a wide variety of hbFGF-producing cells such as human pituitary-derived cells or human fibroblasts.
Such human fibroblasts include WI38 (~TCC No. CCL-75) and IMR90 (~TCC
No. CCL-186). Said cell lines WI38 and IMR90 are listed in the Catalogue of Cell Lines & Hybridomas, ~th edition, 1986, published by the American Type ~ulture Collection.
By inserting the expression vector thus obtained into an appropriate host (e.g., Escher~ch~a coli, B~cillus subitlis, yeasts, animal cells), and cultivating the obtained transformant in a medium, human bFGF can be produced.
The muteins in the present invention essentially have the amino acid sequence of the original peptide or protein; but variations include an addition of amino acid(s), deletion of constituent amino acid(s) and substitution of constituent amino acid(s) by other amino acid(s).
Such addition of amino acid includes addition of at least one amino acid. Such deletion of constituent amino acicl includes deletion of at least onebFGF-constituent amino acid. Such substitution of constituent amino acid by other amino acids includes substitution of at least one bFGF-constituent amino acid by other amino acid.

~2~ 5 The at least one amino acid in the mutein which has at least one amino acid added to bFGF excludes methionine deriving from initiation codon used for peptide expression and signal peptide.
The number of added amino acids is at least 1, but it may be any one, as long as bFGF characteristics are not lost. Preferable amino acids should include some or all of the amino acid sequences of proteins which have homol-ogy with bFGF and which exhibit activities similar to those of bFGF.
~ s for the number of deleted bFGF-constituent amino acids in the present mutein which lacks at least one bFGF-constituent amino acid, it may be any one, as long as any characteristic of bFGF is not lost.
Examples of such deleted constituent amino acids include:
the deletion of amino acids from amino terminal or carboxyl terminal; the deletion of the 10 residues in the amino terminal of human bIi GF:
Met-Pro-Ala-Leu-Pro-Glu-~sp-Gly-Gly~er the 14 residues in the amino terminal of human bFGF:
~ et-Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly~Ser-Gly-Ala-Phe-Pro the 41 residues in the amino terminal of human bFGF:

Met-Pro~Ala-Leu- .... -Val or the 61 residues in the carboxyl terminal of`hurman bFGF:

Lys-Cys- ... -Lys-Ser As for the number of bFGF-constituent amino acids that may be substituted by other amino acids before substitutioll in mutein is lost, it may be any number, as long as any characteristic of bFGF is not lost.
~ s examples of constituent amino acids berore substitution, mention may be made of cysteine and other amino acids but cysteine is pre~erable. As the constituent amino acid other than cysteine which may be substituted for, examples include but are not limited to aspartic acid, arginine, glycine, serine, valine and so forth.
When the constituent amino acid before substitution is cysteine, the substituted amino acids are preferably, for example, neutral amino acids. l~s specific examples of such neutral amino acids~ mention may be made of glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine, .

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histidine, tryptophan, serine, threonine and methionine. Serine and threonille are preferable.
When the constituent amino acid before subsl;itution is other than cysteine, the substituted amino acids are selected f`rom amino acids which are different from the amino acid be~ore substitution in a property such as hydrophilicity, hydrophobicity or electric charge.
When the constituent amino acid before substitution is aspartic acid, examples of the substituted amino acids include asparagine, threonine, valine, phenylalanine and arginine; asparagine and arginine are preferable.
When the constituent amino acid before substitution is ar~inine, examples of the substituted amino acids include glutamine, threonine, leucine, phenylalanirle and aspartic acid; glutamine is preferable.
When the constituent amino acid be~ore substitution is glycine, examples Or the substituted amino acids include threonine, leucine, phenylalanille, serine, glutamic acid, and arginine; threonine is preferable.
When the constituent amino acid before substitution is serine, examples of the substituted amino acids include methionine, alanine, leucine, cysteine, glutamine, arginine and aspartic acid; methionine is preferable.
When the constituent amino acid before substitution is valine, ea~amples of the substituted amino acids include serine, leucine, proline, glycine, lysine, and aspartic acid; serine is preferable.
The constituent amino acid before substitution are pre~erably aspartic acid, arginine, gl~cine, serine and valine.
The substituted amino acid is preferably asparagine, glutamine, arginine, threonine, methionine, serine, and leucine.
The embodiment on the substitution in the rnutein wherein there is a substitution of serine for cysteine (i.e. cysteine is replaced by serine) is most preferred.
In said substitution, there may be at least 2 substitutions and two or three substitutions are preferred.
The muteins in the present invention include combination of 2 or 3 of the above-mentioned additions, deletions and substitutions.
For producing said muteins, site-directed mutagenesis is carried out.
This technique is well-known, and is described in Lather, R. F. and Lecoq, J.
P., Genetic Engineering, Academic Press (1983), pp. 31-~0. Mutagenesis which is directed to oligonucleotide is described in Smith, M. and Gillam, S., 3L~9~;27S

Genetic Engineering: Principles and Methods, Prenam Press (1981), vol. 3, pp. 1-32 For producing a structural gene encoding said mutein, for example:
(a) a single-stranded DN~ consisting of a single strand of a structural gene of bFGF is hybridized with a mutant oligonecleotide primer (the above-mentioned primer is complementary to a region including the cysteine codon to be replaced by this single strand or, as the case may be, an antisense triplet which forms a pair with this codon, except that discrepancies with codons for amino acid coding other than the relevant codon or, as the case -may be, with antisense triplets are permitted.), (b) the primer is elongate~ by DNA polymerase to allow it to form a mutational hetèroduplex, and (c) this mutational heteroduplex is replicated.
The phage DNA carrying the mutated gene is then isolated and inserted into a plasmid.
The plasmid thus obtained is used to transform an appropriate host (as mentioned above), and the resulting transformant is cultured in a medium, whereby mutein can be produced.
In immunizing said bFGF, the bFGF may be prepared in a complex form with a carrier protein before use.
Such carrier proteins include, for example, Freund's complete adjuvant tDifco Laboratories).
When a carrier protein complex is used, the coupling ratio of carrier protein to bFGF is about G to 30 times (carrier/bFGF, ratio by weight). It is preferable that the ratio be about 15 to 20 times.
For coupling between hapten and carrier, various condensing agents can be used, but glutaraldehyde, carbodiimide, etc. are pre~erably used.
In immunizing mammals by means of bFGF or a protein complex, laboratory animals such as sheep, goats, rabbits, guinea pigs, rats and mice may be used, and rats and mice, especially mice, are preferred for obtaining monoclonal antibodies. As to the method of immunization, immunization is possible via any route such as subcutaneous, intraperitoneal, intravenous, intramuscular or intracutaneous injection, but it is preferable that the immunogen be injected mainly subcutaneously, intraperitoneally or ~29~7S

intravellously (in particular, subcutaneously~. In addition, imlnunizing interval, immunizing dose, etc. are also highly variable, allowing various methocls to be carried out; the methocl in which immuni~ation is conducted about 2 to 6 times at intervals of 2 weeks, and splenic cells taken out about 1 to ~; times, preferably about 2 to 4 days after the final immunization are used,for example, is commonly used. It is desirable that an iimnunizing dose of more than about 0.1 ~lg, preferably about 10 to 300 ~g for each mouse, calculated on the peptide amount basis, be used in each injection. It is also desirable that a fusion experirnent using a splenic cell be conducted after certification of increase in blood antibody titer by local blood sampling prior to excision of the spleen.
In the above-mentioned cell fusion of a splenic cell with a lymphoid cell, an excised mouse splenic cell, for example, is fused with an appropriate homogenic or heterogenic (preferably homogenic) lymphoid cell line having a marker such as hypoxanthine-guanine phosphoribosyltransferase deficiency (HGPl~T-) or thymidine kinase deficiency (TK-). ~s the lymphoid cell line, myeloma cell is preferred, and the myeloma cell there is mentioned myeloma P3-~63-Ag 8UI (Ichimori et al.: Journal of Immunological Methods, 80, 65 (1985)~. This fusion can be executed via e.g. the method developed by Kohler and Milstein ~Nature, 256, 495 (1975)]. For example, myeloma cells and splenic cells, in an about 1:5 ratio, are suspended in a medium prepared by mixing together Iskov medium and Eam F-12 medium in a 1:1 ratio (hereinafter referred to as III medium), and a fusing agent such as Sendai virus or polyethylene glycol (PEC~) is used. Of course, climethyl sulfoxide (DMSO) and/or other ~usion promoters can also be added. The following are normally used: a degree of polymeri~ation for the PEG of about 1000 to 6000, a treating time of about 0.5 to 30 minutes and a PEG concentration of about 10 to 80%. Efficient fusion can be achieYed by about 4 to 10 minutes of PEG
6000 treatl~ent at an about 35 to 55% concentration. The fusecl cells can be selectively grown using the hypoxanthine-aminopterin-thymidine medium [~IAT medium; Nature, 256,495 (1975)] or the like.
The culture supernatant of grown cells can be subjected to screening for the production of the desired antibody, and screening for antibody titer can be conducted as follows: In this case, the culture supernatant can first be assayedfor the production of antibody to an immunized peptide by a method such as the radioimrnunoassay (RIA) method or en~me immunoassay (EI~) method.

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These methods are also widely modifiable. ~s an example Or preferred method of assay, a metllod using EI~ is clescribed below. To a carrier such as cellulose beads the rabbit anti-mouse immunoglobulin antibody, ~or example, is beforehand coupled in accordance with a routine method, and the culture supernatant to be assayed and mouse serum are added thereto, and reaction is carried out at constant temperature (which means about 4 to 40C; this defïnition also applied hereinafter) for the specifled time. ~fter the reaction procluct is well washed, a peptide labeled with enzyme (prepared by coupling OI an enzyme and a peptide in accordance with a routine method, followed by purifïcation) is added, and reaction is carried out at constant temperature for the specifïed time. After the reaction product is well washed, an enzyme substrate is added, and reaction is carried out at constant temperature for the specîfied time, whereafter the resulting chromogenic substance can be assayed by ab~orptiometry or fluorometl y.
It is desirable that the cells, which showed proliferation in the selective meclium and secreted antibodies which combined with peptide used for the immunization, were subjected to cloning by limiting dilution analysis etc.
The supernatant of the cloned cells is subjected to screening in the same manner as above to increase cells in the cells high in antibody titer, whereby monoclonal alltibo~y producing hybridoma clones showing reactivity to the immunized peptide are obtained.
The hybridoma thus cloned is grown in liquid medium, for example, a medium prepared by adding about 0.1 to 40% bovine serum to RPMI-1640 [Moore, Gl. E. et al.; Journal of American Medical Association, 199, 549 (19~7)]. Speci~lcally, said monoclonal antibody can be obtained from the medium cultured for about 2 to 10 days, preferably about 3 to 5 days. The antibody can also be obtained from ascites fluids of mice which are intraperitoneally inoculated the hybridoma. For this purpose, in the case of mice, for example, about 1 X 10~ to 1 X 107, preferably about 5 X 105 to ~ X 106 hybridoma cells are intraperitoneally inoculated to a mouse of B~LB/c or similar strain, previously inoculated with mineral oil etc., and about 7 to 20 days later, prefera~ly about 10 to 14 days later ascites fluid is collected. The antibody produced and accumulated in the ascites is subjected to, for example, ammonium sulfate fractionation and DE~E-cellulose column chromatography, whereby the desired monoclonal antibody can easily be isolated as a pure immunoglobulin.

~9Ç;275 ~ monoclonal antibody which combines specifically with bFGF is thus obtained.
The monoclonal antibody of`the present invention combines speci~lcally with the immunogen peptide bFGF. The rnonoclonal antibody o~ the present invention may also combine with a bFGF other than the immunogen peptide.
The monoclonal antibody of the present inveIltion is a monoclonal antibody to the immunogen peptide bFGF or its mutein. ~s the present monoclonal antibody reacts with only bFGF (and its mutein), the present monoclonal antibody combines specifïcally with bFGF.
As shown in Example 3 below, when human bFGF is used as an immunogen, a monoclonal antibody belonging to the immunoglobulin class IgM is obtained in some cases.
Since combining speci~lcally with bFGF, the monoclonal antibody of the present invention is very useful as a reagent for bFGF assay. It also facilitates bFG~ assay in living organs and tissues, so it is very useful also in obtaining basic information about bFGF (e.g., distribution in uivo). In the detection procedure on the bFGF in living organs and tissues, the measuring by EI~ method or fluorescenl; antibody technique are generally employed. In order to measure the amount in the living organs and tissues, it is always employed Wesl;ern blotting method on protein. In this method, a crude extract or partial purif-led sample of the extract is subjected to electrophoresis with acylamide gel, transferring to membrane filter, and then detection with HRP antî b:FGF antibody.
In addition, it is thought l;hat some cancer cells produce bFGF by themselves to continue their proliferation on the basis of the bFGF. When anti-bFGF antibody is allowed to act on such cancer, the prolif-eration-promoting bFGF is neutralized, and the antibody is expected to exhibit cancer cell prolifer~tion inhibition, that is, to act as an anticancer substance. In addition, the antibody can be use~ to determine the bFGF in bFG~F-producing cancer, so it can also be applied to cancer diagnostic reagents. Moreover, based on the avidity of the said antibody to bFGF, an antibody afrlnity column can be prepared to use the antibody as a reagent for bFGF purification.
~ s the EIA method and RLA method for detecting or measuring bFGF, there are mentioned the follwing procedure.
For example, a purified antibody is -f~lxed on 96 wells plastic plate (e.g.
Immunoplate, Nunc, Denmark) at about 0.1 to 10 ~g/well, glass beads, plastic ~L;29~;275 beads. The fïxation is carried out at about a,~oC for overnight, or at room temperature for about 0.4 to a~ hours, in case of plastic. In case of glass beads, the rlxation is carried out in accordance with the method described in Proc.
Natl. Acad. ~ci. US~, 80, 3513-3616 (1983). ~`hus obtained plate or beads to which the a~tibody has been ~lxecl is subjected to adsorption reaction with the antigen bF~F (or its mutein). The adsorption reaction is generally carried out at room temperature for about 0.2 to 2 hours, preferably about 4C
overnight. After the antigen-antibody reaction, adsorption reaction is carried out by adding an antibody which has been labeled with an enzyme in case of EIA or labeled with a radioisotope in case of RI~. As the enzyme to label an antibody, there are exemplif~led by horse radish peroxidase (HRP), alkaline phosphatase. As the examples of radioisotope labeling, there are mentioned 125I.
In case of EIl~, a substrate such as 2,2'-adino-di [3-ethylbenzothiazoline sulfonate (6)] is employed for coloring when HRP is employed as a labeling en~yIlle.
In case of RI~, the radioactivity is measured by scintillation counter.
The measuring the bFGF is carried out by comparing the absorbancy or radioactivity with those of the known amount of bFGF.
l~s the EI~ method, there are mentioned sandwich EIA method, competitive EI~ method, indirect EI~ method. In the sandwich method, two alltibodies are bound by mediating the antigell, bFGF. In the competitive EIA method, an antibody is fixed to a carrier, antigell bFGF which has been combined with a 'la'beling enzyme or radioisol~ope, and a sample, so as to reactcompetitively, and then measuring the amount of labeled antigen. In this competitive EIA method, the reaction conditions, and measuring the amount of bound antibody to antigen are the same as the sandwich EIA method. As the indirect EIA method, a sample and an antibody (which is not ~lxed) are reacted with each other, the unbound antibody is measured by a plate to which the antigen is fixed and an anti-mouse antibody. In this indirect EIA
method, the reaction conditions and measuring method are those as mentioned above.
For the purpose of bFGF purif~lcation~ efficient purification can be achieved by, for example, the method in which the purified relevant antibody, af`ter coupling with an appropriate carrier such as the activated agarose bead in accordance with a routine method, is packed in a column, the crude sample ~29~2~5 containing bFGF such as culture supernatant or disrupted bacterial cells is aclsorbed to the antibody aîrinity column, alld the column is washed, whereaf`l;er bFG-F is eluted with a chaotropic reagent such as KSCN
(potassium l;hiocyanate) or under such slightly acidic conditions that bFGF is never inactivated.
The preparation of antibody column is carried out by coupling the monoclonal antibody of the present invention, puri~led from, for example, ascites fluid inoculated with hybridoma, with an appropriate carrier, by the method as follows:
Any carrier can be used, as long as it ensures the efrlcient adsorption specif~lcally of bFGF after coupling, and enables the appropriate elution of bFGF after tlle adsorption; as She carrier there are mentioned polymer of agarose, cellulose or acrylamide, and as the carrier, for example, polyacrylamide gel beads activated so that primary amino group in protein is easily combined with, such as Affi-Gel~iO is usecl as appropriate in the manner as described below. The reaction between Af~l-Gei~10 and antibodies is carried out in a buffer such as a solution of about 0.001 to lM, preferably about O.lM bicarbonate. As to reaction conditions, the reaction can be carried out at about 0 to 20C for about 10 to 24 hours at any pE level, but about 4C, about 4 hours, and pH about 3 to 10 is preferably used ~or reaction conditions.
Since m~re antibodies are adsorbed as the amount of antibodies per 1 m of Affi-Gel-10 increases, as long as the amount is less than about 50 mg;
therefore, any quantitative ratio between ~rfï-Gel-10 and antibodies to be mia~ed together can be chosen within this range, but about 10 to 30 mg of antibodies is used as appropriate, considering binding efficiency and purifying efficiency in affinity column chromatography. These antibody-carrier conjugates can be used for an antibody colum by packing in an appropriate column after blocking the remaining unreacted active groups by a method such as the method in which the conjugates, after being well washed with the buffer used for the reaction, are kept standing several days, or the method in which a f~lnal concentration of about 0.05 to 0.1 M of a compound haying a primary amino group such as ethanolamine hy~lrochloric acid or glycine is added and reaction is carried out at about ~C for about 1 to 4 hours, or about 1 to 5% of protein such as bovine serum albumin (BSA) is added and reaction is carried out at 4C overnight.

Irac~e rnar k ~;~9~ 75 In purirication using the above-rnentioned antibody column, for example a sample containing bF~F is dissolved in a buffer such as a phosphate bu~fer or a Tris hydrochloric acid and is adsorbed to the antibody columll. Thereafter, the colunm is washed with the same buffer, and bFC~F is then eluted. ~s eluents, there can be used slightly acidic solutions such as acetic acid solutions, solutions containing polyethylene glycol, solutions containing pepticle which is more likely to combine with antibodies than the sample, high concentration salt solutions, and solutions prepared by combining these, and those which do not considerably accelerate the decomposition of bFGF are preferred.
The column effluent is neutralized with a buf~er by a routine method.
Where necessary, a purifying procedure using the above antibody column can be again carried out.
In this way, substantially pure bFGF mutein protein can be obtained.
The substantially pure bFGF mutated protein according to this invention includes products whose bFGF mutated protein content is not less than 90%
(w/w) and, more preferably, products whose bFGF mutein content is not less than 9~o (wlw).
The bFGF solution thus obtained are subjected to dialysis, and, if necessary, can be made into a powder by lyophilization. In the lyophilization, there may be adcled stabilizers such as sorbitol, mannitol, dextrose, maltose and glycerol.
The hbFGF thus obtained possesses growlh promoting activity of fibroblast cells and endothelial cells and angiogenic activity, and its toxicityis low; therefore, the hbFGF can be Ised as a cure promoter for burns, wounds, postoperative tissues, etc., or as a therapeutic agent for thrombosis, arteriosclerosis, etc. which is based on its neovascularizing effect.
Furthermore, it can be used as a reagent for promoting cell cultivation.
For its pharmaceutical use, the hbFGI? can be safely administered to warm-blooded mammals (e.g. humans, mice, rats, hamsters, rabbits, dogs, cats) parenterally or orally either per se in a powder form or in the form of pharmaceutical compositions (e.g., injection, tablet, capsule, solution, ointment) made up together with pharmacologically acceptable carriers, excipients and/or diluents.
Injectable preparations can be produced by a conventional method using, for example, physiological saline or an aqueous solution containing - 1'1-~29~ 75 glucose and/or other adjuvant or adjuvanls. Tablets, capsules ancl other pharmaceutical compositions can also be prepared in accordance with a conventional method. When prepared ror use as a pharmaceutical, care should be taken that aseptic conditions are used and that the resultant product is sterile, low in pyrogens and endotoxins.
When used for the above pharmaceutical purposes, the hbFGF is administered, ~or example, to the above warm-blooded mammals in an appropriate amount selected ~rom the range of ~rom about 1 ng to 100 llg/kg body weight a day according to the route of administration, symptoms, etc.
When used as a reagent for promoting cell cultivation, the hbFGF is added to the medium preferably in an amount of 0.01 to 10 ~g, more preferably 0.1 to lO~g per liter of medium.
The mutein o~hbFGF can also be used as with the above hbFGF.
Thus, since combining specifically with bFGF, the monoclonal antibodies of the present invention can be advantageously used for bFGF
assay reagents and for purifying bFGF.
In addition, of the monoclonal antibodies of the present invention, those which are high in antibody valency are advantageous in that, when they are used as bFGF assay reagents, the amount of other reagents prepared at the time of use, for example, antiserum, is saved. Furthermore, bFGF
purif~lcation to higher degree can be achieved by the use thereof.
In the specification and drawings of the present invention, the abbreviations used for bases, amino acids and so on are those recommended by the IUPAC-IUB Commission on Biochemical Nomenclature or those conventionally used in the art. Examples thereof are given below. Amino acids for which optical isomerism is possible are, unless otherwise specifled, intheLform.
DNA : Deoxyribonucleic acid cDNA : Complementary deoxyribonucleic acid A : Adenine T : ~hymine G : Guanine C : Cytosine RNA : Ribonucleic acid d 7~ j dATP : Deoxyadenosine triphosphate dlY~P : Deoxythymidine triphosphate dGTP : Deoxyguanosine triphosphate dCTP : Deoxycytidine triphosphate ATP : Adenosine triphosphate Tdr : Thymidine EDT~: Ethylenediaminetetraacetic acid SlDS : Sodium dodecyl sulfate Gly : Glycine Ala : Alanine Val : Valine Leu : Leucine Ile : Isoleucine Ser : Serine Thr : Threonine Cys : Cysteine Met : Methionine Glu : Glutamic acid Asp : Aspartic acid Lys : Lysine Arg , ~rginille :EIis : Histidine Phe : Phenylalanine Tyr : I'yrosine Trp : Tryptophan Pro : Proline Asn : Asparagine Gln : Glutamine In Reference Examples mentioned below, the human bFGF constituent amino acids shall be numbered according to the rule, in which Met added to the N-terminal of the peptide having Thr for X and Ser l`or Y in the above-mentioned amino acid sequence (II), said Met is numbered as the ~lrst.

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Brief Description of the Drawings Figure 1 shows the human bFGF-encoding base sequence as deter--mined in Reference Example 1 and tlle amino acid sequellce deducible from said base sequence.
Figure 2 shows the polyacrylamide gel electrophoresis patterns on immunoprecipitation as obtained in Example 5.
Figure 3 shows the antibody valencies as obtained in Example 6.
Figure 4 shows the results of competitive inhibition experiment to MoAb 12 by the various peptides as obtained in Example 7.
Figure 5 shows the results of competitive inhibition experiment to MoAb 52 by the various peptides as obtained in Example 7.
Figure 6 shows the results of competitive inhibition experiment to Mo~b 78 by the various peptides as obtained in Example 7.
Figure 7 shows the results of competitive inhibition experiment to Mol~b 98 by the various peptides, as obtained in E~ample 7.
Figure 8 shows the results of competitive inhibition experiment on bFGF mutein of`Mo~b 52 as obtained in Example 7.
Figure 9 shows the results of competitive inhibition experiment on bFGF mutein of MoAb as obtained in Example 7.
Figure 10 shows the pattern of polyacrylamide gel electrophoresis as obtained in Example 9.
Figure 11 shows the quantification of bFGF by EIA using MoAb 52 and HRP-MoAb 78.
Figure 12 shows the quantification of bFGF by EIA using MoAb 98 and HRP~MoAb 78.
Figure 13 shows the detection of bFGF by Western blotting method using monoclonal antibody 78 as obtained in Example 12.
The mouse HbF99 cell, mouse HbF161 cell and rnouse HbF165 cell obtained in Example 2 (3) to be described later have been deposited at Institute for Fermentation, Osaka (IFO), Japan since January 28, 1987 respectively under the following accession numbers:
Mouse HbF99 cell : IFS:) 50122 Mouse HbF161 cell: IFO 50123 Mouse HbF165 cell: IFO 50124 ,......................................... .

- ~7-~L29~iZ75 The mouse hybridomas HbF12, EbF52, HbF78, and EIbF98 obtained in Example 4 to be described later have respectively been deposited at the IFO
since August 17,1987 under the following accession numbers:
Mouse HbF12 cell : IFO 50142 Mouse HbF52 cell : IFO 50143 Mouse HbF78 cell : IFO ~0144 Mouse HbF98 cell : IFO 60145 The following transformants which were produced in the Reference Exàmples mentioned below were deposited at IFO and at the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (FRI), Japan under the accession numbers on the deposit dates shown in Table 1 (The deposit dates are indicated in parentheses.). As to the deposit number of FRI, FERM BP number denotes the number of deposit under the Budapest Treaty; and in case both FERM P
number and FERM BP number are described, it shows that the deposit under the number of FERM P has been converted to the deposit under Budapest Treaty and the transformants have been stored at FRI under the number of FERM BP.

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P __ i Examples The presenl; invenl;ion will now be illustrated in more detail by means of the following working examples, but the present invention is never limited thereby.
Reference Example 1 (Construction of a plasmid containing an hbFGF-encoding gene) (1) Isolation of cDNA-containing plasmid:
A cDNA library with Escherichia col~ x1776 as the host as produced by inserting cDNA synthesized from human foreskin derived primary culture cell m~NA into the pCD vector [Okayama et al.: Molecular and Cellular Biology, 3, 280 (1983)] was provided by Dr. Okayama at the National Institute of Child Eealth and Human Development, Bethesda, USA. l'he plasmid DNA was extracted from this cDNA library by the alkaline extraction method [Birnboim, H. C. and Doly, J.: Nucleic Acids Research, 1, 1513 (1979)] and Escher~chia coli DH1 was infected with this DNA. A cDNA
library comprising about 2 X 106 clones was thus produced with Escherichia col~ DII1 as the host.
The above cDNA library with l~sc~eric11ia coli DEI1 used therein was plated on 10 pieces o~ nitrocellulose filter (Millipore's HATF filter) in an amount of about 5 X 104 clones per fïlter. Using these filters as master ~llters, 20 replica ~llters were prepared in 10 pairs corresponding to the master f~llters. Escherichia coli cells on these replica fllters were lysed with a 0.5 N NaOH solution and plasmid DNAs exposed and denatured were immobilized on the ~llters ~Grunstein, M. &~ ~Iogness, D. S.: Proc. Natl. Acad.
Sci. USA, 72, 3961(1975)].
Based on the amiIlo acid sequence of bovine basic ~lbroblast growth factor as reported by F. Esch et al. [Proc. Natl. Acad. Sci. USA, 82, 6507 ~1985)], base sequences corresponding to two amino acid sequences cosTering amino acid Nos. 13-20 (Pro-Pro-Gly-Eis-Phe-Lys-Asp-Pro) and amino acid Nos. 89-96 (Thr-Asp-Gllu-~ys-Phe-Phe-Phe-Glu), respectively were chemically synthesized. (In some codons, the third letter was selected arbitrarily. Thus, the base sequences synthesized were 5' GGA/G TCrl~/C TT
AiG AAA/~ TGGCCAGGAGG and ~' TCA/G AAAIG AAAIG AAAIG CAT/C
TCGTCGGT, with each underlined base being the one selected.) These oligonucleotides were labeled with 32p at the 5' end by treating said 129~i275 oligonucleotides in 50 ~e of reaction mixture [0.111g of oligonucleotide, ~OmM
Tris-HCl, pE 8.0, 10 mM MgC12, 10 mM mercaptoethanol, 50 llCi ~ 32p ATP
(>5,000 Ci/mmole), 3 units of T4 polynucleotide kinase (Takara Shuzo, Japan)] at 37~C for 1 hour.
The thus-labeled oligon-lcleotides of the above two kinds were individually hybridized as probes with the replica filters. The hybridization reaction was conducted in 10m~ of a lOO~g/me denatured salmon sperm DNA
solution containing 1011Ci of probe in ~; X SSPE [180 mM NaCl, 10 mM
NaH2PO~, 1 mM EDTA (p~I 7.4)] and 5 X Denhardt's with 0.1% SDS at 35C
for 16 hours. After reaction, the rllters were washed with a 0.1% SDS solution in 5 X SSC [0.15 M NaCl, 0.015 M sodium citrate] three times each at room temperature for 3Q minutes ancl then two times each at 45C for 30 minutes [T. Maniatis et al.: "Molecular Cloning", Cold Spring ~Iarbor Laboratory, p.
309 (1982)].
Radioautograms were taken for the washed filters. A bacterial strain capable of reacting with the both kinds of probe was searched for by superposing the radioautograms for each pair of replica filters. In this manner, a strain [Escherichia coli K12 l)E1/pTB627 (IFO 14494, FERM BP-1280)] capable of reacting with the two kincls of probe was obtained from among 5 X 106 colonies.
t2) The plasmid DNl~ (pTB627) was extracted from the strain obtained above in (1) ~Escherichia coli K12 DH1/pl'B627 (IFO 14494, FE~M BP-1280~]
by the alkaline extraction method [Nucleic l~cids Research, 1, 1513 (1979)]
and purified.
(3) Then, the base sequence of the cDNA portion encoding hbFGF was determined by the dideoxynucleotide synthetic chain termination method [J.
Messing et al.: Nucleic Acids Research, 9, 309 (1981)]. The amino acid sequence deduced from said base sequence is shown in Fig.1.
~ Reference Exa~le 2 (Expression of hbFGF-ensoding gene in Escher ichia coli) Construction of hbFGF expression plasmid pTB669:
The plasmid pTB627 obtained in Reference Example 1 (2) mentioned above and containing the hbFGF cDNA was cleaved with the restriction enzymes AuaI and BalI, whereby a 0.44 kb DNA fragment containing the hbFGF-encoding region was obtained. A BglII linker, pC~GATCTG, was ~L2~3~;2~75i ligatecl with this DNA rragment at its BalI cleavage site (blunt end) by T4 DNA ligase, and a 0.44 Kb AuaI-BglII DNA fi~aglnent was isolated~ T4 DNA
ligase was allowed to act on this 0.44 Kb AuaI-BglII fragment to thereby cause ligatioll between the Bglll cleavage sites. 'L'hen, DNA polymerase (Klenow fragment) reaction was carried out in the presence of dX'l'Ps to render the AuaI cleavage sites blunt. This DNA rragment was ligated with phosphorylated synthetic oligonucleoticles, 5A~'IYrCTATGCCAGCA'I'rrGC3 and !j GCAATGCTGGCA'I'AG3, in the presence of T4 DNA ligase. An about 0.46 kb DNA fragment was then prepared by cleavage with ~coRI-BglII.
Separately, the trp promoter-containing plasmid ptrp781 [Kurokawa, T. et al.: Nucleic Acids Research, 11, 3077-3085 (1983~] was cleaved with PstI and rendered blunt-ended by T4 DN~ polymerase reaction. The BglII linker pCAGATCTG was joined to the above cleavage product at the blunt ends thereof by T4 DNA ligase reaction; then the ligation product was cleaved with EcoRI-BgtlJI and an about 3.2 kb DNA fragment containing the trp promoter, the tetracycline resistance gene and the plasmid replication origin was isolated. This 3.2 kb DNA fragment was ligated with the above-mentioned 0.46 kb Eco~I-Bglll DNA fragment containing the hbFGF-encoding gene region by T~ DNA ligase reaction, whereby an hbFGF expression plasmid, pTB669, was constructed.
This plasmid pTB669 was used to transform l~scherichia coli DH1 to give a transrormallt carrying the plasmid pTB669, naInely Escherichia coli DEI1/pT13669.
pTB669 was also used in the same manner to transform the Escherichia coli strains K12 MM294 and C600 to give 1~scheric11ia coli K12 MM294/pTB6G9 (IFO 14~32, FERM BP-1281) and E. coli C600/pTB669, respectively.
Reference Example 3 (Purif~lcation of human basic fibroblast growth factor (hbFGF)) Escherichia coli K12 MM294/pTB669 (IFO 14532, FERM BP-1281) as obtained in Reference Example 2 was cultivated in M9 medium [Maniatis, T.
et al.: Molecular Cloning (1982), A Laboratory Manual, Cold Spring Harbor Laboratory, USA] containing 1% glucose, 0.4% casamino acid and 8 ~ug/m~
tetracycline. When the Klettvalue reached about 200,3-~3-indolylacrylic acid was added to 25 ~ug/m~, and the cultivation was continued for 4 more hours.
Thereafter, cells were harvested and suspended in one twentieth volume of ~9~75 lU% sucrose solution in 20 mM'l'ris-EICl, plI7.6. 'l'o tllis susl)ension were aclded phellyllllelhylsulfonyl fluoride (PMSF) lo 1 mM (rlnal concentral;ion), EDTl~ ~o 10m~, NaCl to 0.1M, spermidille hydlochlolide to 10 mM and lysozyme to 100 ~g/me. A~ter allowing to stand at 0C Çor 45 minutes, the whole mixture was sonicated for 30 seconds. The sonication product was centrifuged at 18,000 rpm (Sorvall centrifuge, SS 34 rotor, US~) ~or 30 minutes to give a supernatant, which was used as the cell extract.
~ 25-me portion of this extract (as prepared from 500 me of culture broth) was passed through a DE:~E-cellulose (DIE52, Whalman, England) columu ~diameler 2 X 10 cm) equilibrated wil;h 0.2 M NaCl solution in 20 mM
Tris-HCl, pH 7.ff to thereby remove nucleic acid components in the extract.
The efrluent from the column and the column wasllings resultant from washing with 0.2 M NaCl solution in 20 lnM Tris-EICl, pI-I 7.6 were collected and combined (DE1~13 effluent fraction 44 m~).
A 14-me portion of this fraction was applied to a high performance liquid chrolnatograph (Gilson, France) equipped with a heparin column Shodex~F-pak EIR-894 (8 mln ID X 5 cm, Showa Denko, Japan). 'l'he colu}nn was washed with 20 mM Tris-IICl, pH 7.G. Thereafter, elution was performed on a lineal gradient of 0.5-2 M NaCl in 20 lnM Tris-lICl buffer, pH 7.6, (eluent volume 60 m~, flow rate 1.0 nle/lnin).
The hbFGF eluted by this procedure showed a single band in SDS-polyacrylalnide gel electrophoresis, and it was thus ~ound to be sufficiently purified and suil;able for use as an antigen. The assay of llbFGF was conducted USillg l;he following conditions.
~ Nunc 9G-well microtiter plate (flat bottomed) was sown with mouse B~LB/c3T3 cells (2 X 103 cells per well) with DMEM medium containing 5%
calf serum (0.2 me per well) and the cells were cultured. Next day, the lnedium was replaced with DMEM medium containing 0.5% calf serum.
After 3 days of cultivation, dilutions of the cell extract as prepared by serial 5-fold dilution with DM13 medium containirlg 0.5% BSA were added in an amount of 10 lle per well. ~fter 20 hours of continued cultivation, 2 1l~ of 3H-Tdr (5 Ci/mmoe, 0.5 mCi/me ~CC ~mersllam~ was added to each well. Six hours later, cells in each well were scraped of~ by treatment with pllosphate bufrer (PBS) containing 0.2% trypsin and 0.02% EDTA and collected on a glass fllter Usillg a Titertel~cell harvester, and the quanl ity of 3M-Tdr takenup by the cells was measured Usillg a scintillation counter.

* Trademark ~.~9~X75 Reference Example 4 (Production of recombinant DNA having mutein-encoding base sequence) (1) Cloning of hulnan bFGF gene M13 vector The plasmid pTB669 obtained in Reference 13xample 2 was digested with lhe restriction enzymes EcoRI and BamHI. Yhage vector M13mp8 [J. Messing: Methods in Enzymology, 101, 20-78 (1983)] replicative form (RF) DNA was digestecl with the restriction enzymes EcoRI and BclmHI. The DNA
fragrnent thus obtained was mixed with the human bFGF DNA fragment derived from the pTB669 which was previously digested with EcoRI and BamHI. The mixture was then ligated together by T4 DNA ligase. The ligated DNA was transformed into infectable cells of Escherichia coli JM105 strain, and the cells were sown on a plate containing Xgal as the indicator species [J. Messing et al.: Nucleic Acids E~esearch, 9, 309-321 (1981)]. The plaque containing the recombinant phage (white plaque) was picked up, and the base sequence of the recombinated segment was determined by the dicleoxynucleotide synthetic chain ter~nination method [J. Messing et al.:
Nucleic ~cids ~esearch, 9, 309 (1981)], whereby it was conf~lrmed that human bFGF DNA was accurately inserted.
From this M13PO clone was purifiecl single-stranded phage DNA, which was used as a template for site-directed mutagenesis using synthetic oligonucleotide.
(2) Site-specific mutagenesis Forty picomoles of the synthetic oligonucleotide:
S er S ' ~ C G T T C T T G C T G T A G A G C C
( R s a ~ ) G CT <.3' [the primer for con~lerting Cys 26 to Ser (the recognition sequence for restriction enzyme Rsa I disappears)] was treated with 9 units of T4 kinase at 37C for 1 hour in 50 11~ of a solution containing 0.1 mM adenosine triphosphate (AIlP), 50 mM hydro~ymethylaminomethane hydrochloride (Tris-HCl), pH 8.0, 10 mM MgCl2 and 5 mM dithiothreitol (DTT). This kinase-treated primer (12 picomoles) was heated at 67C for 5 minutes and then at 42C for 25 minutes in ~0 ~ of a mixture containing 50 mM NaCl, 1.0 ':

~ . ~

., .

~L~99~275 mM 'rris-HCl, pH 8.0, 10 mM MgC12 and 10 mM l3-mercaptoethanol, whereby the primer was hybridized to 5 1l~ of single-stranded (ss)M13-PO DN~. After annealing, the mixture was cooled on ice, and was added to {jO ye Or a reaction mixture containing 0.5 mM dideoxynucleotide triphosphate (dNTP), 80 mM
Tris-~ICl, pII 7.4, 8 mM MgC12, 100 mM NaCl, 9 units of DNA polymerase I
Klenow fra~nent, 0.5 mM ATP and 2 units of T4 DNA ligase, and reaction was carried out at 37C for 3 hours and 25C for 2 hours. The reaction was terminated by adding 2~ of 0.2 mM EDTA. The reaction product was used to transform infectable JM 105 cells, and the cells were grown overnight.
Thereafter, ssDNA was isolated from the medium's supernatant. Using this ssDNA as the template for the second cycle of primer elongation, gel-purirled RF DNA was transformed into infectable JM 105 cells. The cells were sown over an agar plate and cultivated overnight, whereby a phage plaque was obtained.
(3) Site-directed mutagenesis The procedure of the above term (2) was repeated, but the synthetic oligonucleoti~leprimerused was S er 5'>AACGATTAGCGCTCACTC
II ae 11 C ~3' which converts cysteine 70 to serine (~ recognition sequence for restriction enzyme HaeJI is produced).

(4) Site-directed mutagenesis The procedure of the above term (2) was repeated, but the synthetic oligonucleotide primer used was ~ .
S er

5'>GTAACAGACTTAGAAGCT
. Alul AGT < 3 which converts cysteine 88 to serine (A recognition sequence for restriction enzyme AluI is produced).
(5) Site-directed mutagenesis The procedure of the above term (2) was repeated, but the oligonucleotide primer used was 1~9~i275 s er 5 ' > T C G A A C A A G A ~ A G=C l` C

T c c < 3' which converts cysteine 93 to serine (A recognition sequence for restriction enzyme IIinf I is produced).
(6~ Screening and identification of mutated plaques Plates containing mutated M13-PO plaques [above term (1)] and two plates containing unmutated M13-PO phage plaques were coolecl to 4C, and the plaques from each plate were transferred to two round nitrocellulose fïlters by superposing a dry filter on the agar plate for 5 minutes in the case of the first f~llter, or by superposing a clry ~llter for 15 minutes in the case of the seconcl rllter. Then, the filters were placed on a thick filter paper and j~ immersed in a solution containing 0.2 N NaOH, 1.5 M NaCl arld 0.2% Triton X-100, and then on a ~llter paper in a solution containing 0.5 M Tris-HCl, pEI 7.~ and 1.5 M NaCl for 5 more minutes, to thereby neutralize the filters.
The filters were washed on a filter by immersing them twice in 2 X SSC
(standard sodium citrate). The filters were then allowed l;o dry in air, after which they were dried at 80C in a vacuum oven for 2 hours. The duplicated fïlters were subjected to prehybridization at 55C for 4 hours in 10 m~ llter I)NA hybridization buffer (5 X SSC), pH 7.0/4 X Denhardt's solution (polyvinylpyrrolidolle, Ficoll, and bovine serum albumin, 1 X = 0.02% for each)/0.1% sodium dodecylsulfate (SDS)/50 mM sodium phosphate buffer, pE 7.0/100 ~g/me denatured salmon sperm DNA. The oligonucleotide primer was hybridized to 105 cpm/m~ at 42C for 24 hours. The f`ilters were washed in washing buffers containing 0.1% SDS and decreasing amounts of SSC at 50~C
for 30 minutes ~or each wash. That is, the filters were washed first with the buffer containing 2 X SSC, and the control filters containing unmutated M13-PO plaques were examined for radioactivity by means of a Geiger counter. While reducing SSC concentration step by step, the ~llters were washed until no detectable radioactivity remained on the control filters containing unmutated M13-PO plaques. ~he minimum SSC concentration used was 0.1 X SSC. The filters were air-dried and e~posed to ~llm at--70C
for 2 to 3 days to thereby take radioautograms. A total of 10,000 mutated M13-PO plaques and 100 unmutated control plaques were screened by means ~ rC~ ~fna r ~

~;29~7S

of the kinase-l;reated oligonucleotide probe. None of the control plaques hybridized to the probe, while 3 to 10 Or the mutated M13-PO plaques hybridized ts~ the probe.
One of the mutated M13-O plaques was picked up and inoculated to JM105 medium. From the supernatant was prepared ssDNA, and from the cell pellet was prepared double-stranded (ds) DNA. Using appropriate oligonucleotide primers and ssDNAs, the base sequences were analyzed.
As a result, it was respectively confirmed that TGC (Cys26) codon had been converted to TCT (Ser) codon, TGT (Cys70) codon had been converted to ~GC (Ser) codon, TGT (Cys88) codon had been converted to TCT (Ser) codon, and TGT (Cys93) codon had been converted to TCT (Ser) codon.
Of the mutated M13-PO phages, the phage in which the codon Cys-26 had been converted to Ser was designated M13-P1; the phage in which the codon Cys-70 had been Ser, M13-P2; the phage in which the codon Cys-88 had been converted to Ser, M13-P3; and the phage in which the codon Cys-93 hacl been converted to Ser, M13-P4.
Reference l3xample 5 (Expression of human bFGF mutein-encoding gene in Escherichia coli) (1) Construction of human bFGF mutein expression plasmid pTB739) The M13-P1 replicative form (RF) obtained above in Reference Exarnple 4 was cleaved with the restriction enzymes EcoRI and PstI to thereby obtain an about 0.5 kb DN~ fragment including the human bFGF
mutein-encoding region.
Separately, the trp promoter-containing plasmid ptrp781 [Kurokawa, T. et al.: Nucleic Acids Res., 113 3077-3085 (1983)] was cleaved with EcoRI-Ps~I, and an about 3.2 kb DNA fragment containing the trp promoter, the tetracycline resistance gene and the plasmid replication origin was isolated.
This 3.2 kb DNA fragment was ligated with the above-mentioned 0.5 kb .EcoRI-PstI DNA fragment containing the human bFGF mutein-encoding gene region by T4 DNA ligase reaction, whereby a human bFGF mutein expression plasmisl, pTB739, was constructed.
This plasmid pTB739 was used to transrorm Escherichia coli DE1 to give a transformant carrying the plasmid pTB739 containing the mutein-encoding gene, namely Escherichia coli DH1/pTB739 (IFO 14575, l?ERM BP-~41).

;27S

(2) Preparation of cell extract The above transrormant was cultivated in M9 medium containing 1%
glucose, 0.4% casamino acid and 8 ~g/m~ tetracycline. When the Klett value was about 200, 3-~-indolylacrylic acid was added to 25 llg/me, and the cultivation was co;ntinued for 4 more hours. Thereafter, cells were harvested and suspended in one twentieth volume of 10% sucrose solution in 20 mM
Tris-:EICl, p:EI7.6. To this suspension were added phenylmethylsulfonyl fluoride (PMSF) to 1 mM (final concentration), EDTA to 10 mM, NaCl to 0.1 M, spermidine hydrochloride to 10 mM and lysozyme to 100 llg/me. After allowing to stand at 0C for 45 minutes, the whole mixture was sonicated for 30 seconds. The sonication product was centrifuged at 18,000 rpm (Sorvall centrifuge, SS 34 rotor, USA) for 30 minutes to give a supernatant, which was used as the cell extract.
(3) Euman bFGF activity o~cell extract ~ Nunc 96-well microtiter plate (flat bottomed) was sown with mouse BALB/c3T3 cells (2 X 103 cells per well) with DME~ medium containing ~%
calf serum (0.2 m~ per well) and the cells were cultured. Next day, the medium was replaced with DMEM medium containing 0.5% calf serum.
l~fter 3 days of cultivation, dilutions of the cell extract as prepared by serial 5-fold dilution with DME meclium containing 0.5% BSA were added in an amount of 10 lle per well. ~fter 20 hours of continued cultivation, 2 ~ of 3H-Tdr (~ Ci/mmoe, 0.~ mCi/me RCC ~mersham) was added to each well. Six hours later, cells in each well were scraped of~ by t~eatment with phosphate buffer (PBS) containing 0.2% trypsi~ and 0.02% EDTA and collected on a glass filter using a Titertek cell harvester, and the quantity of 3H-Tdr taken up by the cells was measured using a scintillation counter.
The cell extract of E. coli DH1/pTB739 thereby tested showed FGF
activity.
The mutein CS1 in which the 26-position Cys of human bFGF had been replaced by Ser was thus obtained.
Reference Example 6 (:E xpression in Escherichia coli of gene encoding human bFGF mutein) (1) Construction of the plasmid pTB742 for human bFGF mutein expression:
The M13-P2 replicative form (RF) obtained in Reference Example 4 above was cleaved using the restriction enzymes EcoR I and Pst I to obtain an ~L2~3~i275 about Q.5 kb DN~ fragment containing a region whic:h encodes a human bFGF mutein.
Separately, a plasmid ptrp781 DNA containing a trp promoter was cleaved using Eco~ PstI to separate an about 3.2 kb DNA fragment containing a trp promoter, a tetracycline resistance gene and a plasmid replication initiation site. This 3.2kb DNA fragment and the above-mentioned 0.5kb EcoRI-PstI I)NA fragment containirlg a gene region encoding a human bFGF mutein were ligated together by T4 DNA ligase reaction to construct the plasmid pTB742 ror the expression of a human bFGF
mutein.
Using this plasmid pTB742, Escherichia coli DH1 was transformed, whereby the strain Esc11erichi~ coli DE1/pTB742 (IFO 14584, FERM 13P-1642) was obtained, which harbors the plasmid pTB742 containing the mutein-encoding gene.
(2) Preparation of bacterial cell extract:
The above-mentioned transformant was cultured by the method described in Reference Example 5 (2) to give a supernatant, which was then used as a bacterial cell extract.
(3) Euman bFGF activity of the bacterial cell extract:
A cletermination was ma~e of the human bFGF activity on the bacterial cell extract obtained in (2) above, by the method described in Reference Exarmple 5 (3).
The bacterial cell extract of E. col~ Dl-I1/pTB742 thereby tested exhibited FGF activity.
The mutein CS2, in which Cys at the 70-position of human bFGF had been replaced by Ser, was thus obtained.
Reference Example 7 (Expression in Escherichia coli of gene encoding human bFGF mutein) (1) Construction of the plasmid pTB743 t`or human bFGF mutein expression:
The M13-P3 replicative form (RF) obtained in Reference Example 4 above was cleaved using the restriction enzymes EcoR I and Pst I to obtain an about 0.5 kb DNA fragment containing a region which encodes a human bFGF mutein.
Separately, a plasmid ptrp781 DNA containing a trp promoter was cleaved using EcoRI-PstI to separate an about 3.2kb DNA fragment containing a trp promoter, a tetracycline resistance gene and a plasmid ~L2~9Çi2~75 replication initiation site. This 3.2kb DNA rragment and the above-mentioned 0.5kb EcoRI-PstI DNA -rragment containing a gene region encoding a human bFGF mutein were ligated together by '1l4 DNA ligase reaction to construct the plasmid pTB743 for the expression of human bFGF
mutein.
Using this plasmid pTB7439 Escherichia coli DII1 was trans~ormed, whereby the strain Escherichia coli DH1/p'rl3743 (IFO 14585, FElRM
BP-1643) was obtained, which harbors the plasmid pTB743 containing the mutein-encodillg gene.
(2) Preparation of bacterial cell extract:
The above-mentioned transformant was cultured in the manner described in Reference Example 5 (2) to give a supernatant, which was then used as a bacterial cell extract.
(3) Euman bFGF activity of the bacterial cell extract:
A determination was made of the human bFGF activity o~ the bacterial celi extract obtained in ~2) above, by the rnethod described in Reference Example 5 (3).
The bacterial cell extract o~ E. coli DIIl/pTB743 thereby tested exhibited F~F activity.
The mutein CS3, in which Cys at the 88-position of hulllan bFGF had been replaced by Ser, was thus obtained.
Reference Example 8 (Expressioll ill Escherichia coli of gene which encodes human bFGF mutein) (1) Construction of the plasmid pTB744 for human bFGF rnutein expression:
The 1!![13-P4 replicative form (RF) obtained in Reference Example 4 above was cleaved using the restriction enzymes EcoR I and Pst I to obtain an about 0.5 kb DNA fragment containing a region which encodes a human bFGF mutein.
Separately, a plasmid ptrp781 I)NA containing a trp promoter was cleaved using EcoRI-PstI to separate an about 3.2 kb DNA fragment containing a trp promoter, a tetracycline resistance gene and a plasmid replication initiation site. This 3.2 kb DNA fragment and the above-mentioned 0.6kb EcoRI-PstI DNA fragment containing a gene region encoding a human bFGF mutein were ligated together by T4 DNA ligase reaction to construct the plasrnid pTB744 for the expression of a human bFGF
mutein.

Using this plasmid pTB744, Escheric1~ia coli DH1 was transformed, whereby the strain Escherichia col~ DIIl/pTB744 (IFO 14586, FERM
BP-1644) was obtained, which harbors the plasmid pTB744 containing the mutein-erlcoding gene.
(2) Preparation of bacterial cell extract:
The above-mentioned transformant was cultured by the method described in Referellce Example 5 (2) to give a supernatant, which was then used as a bacterial cell extract.
(3) EIuman bFGF activity of the bacterial cell extract:
A determination was made of the human bFGF activity of the bacterial celi extract obtained in (2) above, by the method described in Reference E~ample 5 (3).
The bacterial cell extract from E. coli DH1/pTB744 thereby tested exhibited FGF activity.
The mutein CS4, in which Cys at the 93-position in human bFGF had been replaced by Ser was thus obtained.
Reference Example 9 (Screening and identification of plaques which were made mutagenic) Plates containing mutated M13-P2 phage plaques obtained in Reference Example 4 and two plates containing unmutated M13-P2 phage plaques obtained in Reference Example 4 were cooled to 4C, and the plaque from each plate was transferred to 2 round nitrocellulose fïlters by keeping a dry filter placecl on the agar plate for 5 minutes in the case of the 1st filter, and for 15 minutes in the case of the 2nd filter. The filters were then kept placed for ~ minutes on thick filter papers immersed in 0.2N NaOE, 1.5M
NaCl and 0.2% Triton X-100, after which they were neutralized by keeping them placed for 5 more minutes on filter papers immersed in 0.6M Tris-:EICl (pH 7.5) and 1.5M NaCl. The filters were twice washed on filters i-mmersed in 2 X SSC (standard sodium citrate) in the same manner, and were allowed to dry, and this was followecl by drying at 80C for 2 hours in a vacuum oven.
I'he overlapped filters were subjected to prehybridization at 55C for 4 hours with 10 m~/fïlter of a DNA hybridization buffer solution (5 X SSC) having a pH-value of 7.0 containing 4 X Denhardt's solution (polyvinylpyrrolidone, Ficoll and bovine serum albumin, 1 X = 0.02%), 0.1% sodium dodecyl sulfate (SDS), 60mM sodium phosphate-buffered solution having a pH-value of 7.0 and 100 ~g/m~ denatured salmon sperm DNA. Hybridization was carried out 7~;

at 42C for 2~ hours with 105 cpm/m~ of all oligonucleotide primer. The filters were each washed in a bu~fer solution for washing containing 0.1% SDS and a reduced amount of SSC at 50C for 30 minutes. The 11ters were then iirst washed with a buffer solution containing 2 X SSC; the control rllters, which contained unmutated M13-P2 plaques, were examined for radioactivity using a Geiger counter. While stepwise reducing SSC concentration, the control filters were washed until no detectable radioactivity remained on the filters.
The minimum of the used SSC concentrations was 0.1 X SSC. The ~llters were allowed to dry in air, and radioautographs were taken by 2 to 3 days of exposure at--70C. Screening was carried out of 10~000 mutated M13-P2 plaques and 100 unmutated control plaques using a kinase-treated oligonucleotide probe. None of the control plaques hybridized to the probe, while 3 to 10 of the mutated M13-P2 plaques hybridized to the probe.
One of the mutated M13-P2 plaques was taken, ancl was inoculated to a JM105 culture medium. From the resulting supernatant an ssDNA was prepared, and Çrom the bacterial cell pellets a double-stranded (ds) DNA was prepared. ~nalyses were made of` the base sequences using appropriate oligonucleotide primers and ssDN~s.
~ s a result, it was respectively confilrrned that the 'l'GC (Cys-26) codonhad been changed to a TCT (Ser) codon; the TGT (Cys-88) codon, to a TCT
(Ser) codon; and the TGT (Cys-93) codon, to a TCT (Ser) codon.
Of the mutated M13-P2 phages, the phage in which the codons Cys-26 and -70 had become Serencoding codons was named M13-P12; the phage in which the codons Cys-70 and -88 had become Ser-encoding codons, M13-P23;
and the phage in which the codons Cys-70 and -93 had become Ser-encoding codons, ~I13-P24.
Ref~rence Example 10 (Expression in Escherichia coli of gene encoding human bFGF mutein) (1) Construction of the plasmid pTB762 for human bFGF mutein expression:
The M13-P23 replicative form (RF) obtained in Reference Example 9 above was treated in the manner described in Re~erence Example 5 (1~ to construct the plasmid pTB762 for human bFGF mutein expression.
Using this plasmid pTB762, Escherichia coli MM294 was transÇormed, whereby the strain Escherichia coli MM294/pTB762 (IFO 14613, F:13RM
BP-1645) was obtained, which harbors the plasmid pTB762 containing the mutein-encuding gene.

, ; .

~29$~75 (2~ Preparation of bacterial cell extract:
The above-mentioned transformant was cultured by the method des-cribed in Re~erence Example 5 (2) to give a supernatallt, which was then used as a bacterial cell extract.
(3) EIuman bFGF activity Or ~he bacterial cell extract:
A determination was made of the human bFGF activity of the bacterial cell extract obtained in (2) above, by the method described in Reference Example 5 (3).
The bacterial cell extract from E. coli MM294/pTB762 thereby tested exhibited FGF activity.
The mutein CS23, in which Cys at the 70-position and at the 88-position had been replaced by Ser, was thus obtainecl.
Reference Example 11 (Production of recombinant DNAs having mutein-encoding base sequence) (1) Cloning of M13 vector for human bFGF gene:
The plasmid pTB669 obtained in Reference example 2 was digested with the restriction enzymes EcoR I and BamH I. Phage vector M13mp8 [J.
Messing, Methods in ~nzymology, 101, 20 ~ 78 (1983)] replicative form (RF) DNA was mi~ed with a human bFGF DNA rragment derived from pTB669, previously digested with EcoR I and BamII I. The mixtul e was then subjected to ligation USillg T4 DNA ligase. The resulting ligated DNA was transformed into in~ectable cells of the strain Escherichia coli JM105; the transformant cells were spread over a plate whose indicator species was Xgal [J. Messing et al., Nucleic Acids Research, 9, 309-321 (1981)]; the plaque containing the recombinant phage ~white plaque) was picked up; the base sequence of the recombinated region was determined by the dideoxynucleotide synthesis chain termination method [J. Messing et al., Nucleic Acids Research, 9, 309 (1981)], whereby it was confirmed that the human bFGF DN~ had been accurately inserted.
From this M13-PO clone was purified a single-stranded phage DNA, which was used as a template for site-directed mutagenesis using a synthetic oligonucleotide.
(2) Site-specificmutagenesis:
In the presence of 0.1mM adenosine triphosphate (ATP), 50 mM
hydroxymethylaminomethane hydrochloride (Tris-HCl) having a pH-value of 8.0, 10 mM MgCl2, 5 mM dithiothreitol (D1`T) and 9 units of T4 kinase, in a ~ 6275 total amoullt or 50 ~ e, 40 picomoles of the synthetic oligonucleotide:
5'-CGGGC~TGAA'l"rCGCCGC'I'-3' [primer for producing in the base sequence a recognition site for the restriction enzyme EcoRI, and subsequelltly changing Pro-14 to Met] was treated with T4 kinase at 37C for 1 hour. This kinase-treated primer (12 picomoles) was heated at 67C for 5 minutes, and at 42C for 25 minutes, in 50 ~e of a mixture containing 50 mM NaCl, 1.0 mM Tris-EICl having a pH-value o~ 8.0, 10 mM MgCl2 and 10 mM ~3-mercaptoethanol, whereby it was hybridized to 5 llg of the single-strandecl (ss) ~13-PO DNA. The annealed mixture was then cooled on ice, and was addecl to 50 1l~ of a reaction mixture containing 0.5 mM deoxynucleotide triphosphate (dNTP), 80mM Tris-HCl having a pEI-value of 7.4, 8 mM MgCl2, 100 mM NaCl, 9 units of DNA
polymerase I Klenow rragment, 0.5 mM ATP and 2 units of T4 DNA ligase, and reaction was carried ouS at 37C ror 3 hours, and at 25C for 2 hours, whereafter the reaction was stopped by adding 2 ~ of 0.2 mM EDTA. The reaction product was used to transform infectable JM 105 cells; the transformant cells were allowed to grow overnight, whereafter an ssDNA was isolated from the culture medium supernatant. Using this ssDNA as a template for the 2nd cycle of primer elongation, gel-purirled RF-type DNA
was trans~ormed into infectable JM 105 cells; the resulting transformant cells were spreacl over an agar plate, and were cultured overnight to obtain phage plaques.
(3) Site-directedmutagenesis:
The procedure of the above term (2) was repeated but the synthetic oligonucleotide primer used was: 5'-CGCCCATGGTGCCATCCTC-3' which produces in the base sequence a recognition site for the restriction enzyme Nco I, and concurrently changes Gly-9 to Thr and Ser-10 to Met, respectively.
(4) Site-directedmutagenesis:
The procedure of the above term (2) was repeated but the synthetic oligonucleotide primer used was: 5'-TAAC~CC'rrrAAGAAGCCAG-3' which produces in the base sequence a recognition site fior the restriction enzyme Afl II, and concurrently changes the Lys-87-encoding codon to a termination codon.
(5) Site-directedmutagenesis:
The procedure of the above term (2~ was repeated but thesynthetic oligonucleotide primer used was: 5'-CCGGl~CTCCGTT~ C'l`CGC.-3' which S
produces in l;he base sequence a recognition site for the restriction enzyme Hpa I, and concurrently changes Asp-42 to Asn.

(6) Site-clirected mutagenesis:
The procedure of the above term (2) was repeated but the syntlletic oligonucleotide primer used was: ~'-CTTCTCC'l'GACTCCG'l'CAAC-3' which deletes the recognition site for the restriction enzyme IIpa II in the base sequence, and concurrently changes Ar~-45 to Gl n.

(7) Screening and identification of plaques which were mutagenic:
Plates containing mutated M13-PO plaques [above term (1)] and 2 plates containing unmutated M~3-PO phage plaques were cooled to 4"C, and the plaques from each plate were transferred to 2 round nitrocellulose ~llters by keeping a dry fïlter placed on the agar plate fior 5 minutes in the case of the 1st filter, and f`or 15 minutes in the case of the 2nd filter. The filters were then kept placed for 5 minutes Oll thick fllter papers immersed in 0.2N NaOlI, 1.5M NaCl ancl 0.2% Triton X-100, after which they were neutralized by keeping them placed for 5 more minutes Oll ~ilter papers immersed in 0.~M
Tris-HCl having a pH-value of 7.~ and 1.5M NaCl. The filters were twice washed on filters immerse~l in 2 X SSC (Standard Sodium Citrate) in the same manner, a~d were allowed to dry, and this was followecl by drying at 80C for 2 hours in a vacuum oven. The overlapped filters were subjected to prehybridization at 5~C for 4 hours with 10 m~/filter of a DNA hybridization b~ er solution (5 X SSC) having a p~ value of 7.0 containing 4 X Denhardt's solution (polyvinylpyrrolidone, Ficoll and bovine serum albumin, 1 X ~ 0 02%), 0.1% sodium dodecyl sulfate (SDS), ~0 mM sodium phosphate-buffered solution having a pEI-value of 7.0 and 100 ~ug/m~ denatured salmon sperm DNA. :EIybridization was carried out at 42C for 24 hours with 105 cpm/m~ of an oligonucleotide primer. Each filter was washed at 50C for 30 minutes in a buffer solution for washing containing ().1% SDS and a reduced amount of SSC. The fillters were then first washed with a buffer solution containing 2 X SSC; the control f-~lters, which contained unmutated M13-PO plaques, were examined for radioactivity using a Geiger counter.
While stepwise reducing SSC concentration, the control filters, which contained unmutated M13-PO plaques, were washed until no detectable radioactivity remained on the filters. The minimum of the used SSC
concentrations was 0.1 X SSC. The filters were allowed to dry in air, and autoradiographs were taken by 2 to 3 days of exposure at--7()C. Screening ':

~;~9Çi275 was carried out of 10,000 mutated M13-PO plaques and 100 unmutated control plaques by means of an oligonucleotide probe treated with 32P-r-ATP. None of the cont;rol plaques hybridized to the probe, while 3 to 10 of the mutated M13-PO plaques hybridized to the probe.
One of l;he -mutated M13-PO plaques was taken, and was inoculated to a JM106 culture medium. From the resulting supernatant an ssI)NA was prepared, and from the bacterial cell pellets a double-stranded (cls) DNA was prepared. Analyses were made of the base seq uences using appropriate oligo-nucleol;ide primers and ssDNAs.
As a result, it was respectively con-rlrmed that the G~C (Gly-9) codon had been changed to an ACC (Thr) codon and the ~GC (Ser-10) codon had been changed to an ATG (Met) codon; the CCG (~ro-14) codon, to an ArrG
(Met) codon; the A~ (.Lys-87) codon, to a TA~ (termination) codon; the GA~
(Asp-42) codon, to an AAC (Asn-42) codon; and the CGG (Ar~-46) codon, to a CAG (Gln-45) codon.
Of the mutated M13-PO phages, the phage in which Gly-9 codon had become a Thr-encoding codon and the Ser-10 codon hacl become a Met-encoding codon was narned M13-PN10;
the phage in which the Pro-14 codon had become a Met-encoding codon, M13-PN14;
the phage in which the Lys-87 codon had become a termination codon, M13-PC86;
the phage in which the Asp-42 coclon had become an Asn-encodillg codon, M13-PDN42; ancl, the phage in which the Arg-45 codon had become a Gln-encoding codon, Reference Example 12 (Expression in Escherichia coli of gene which encodes human bFGF mutein) (1) Construction of the plasmid pTB795 for human bFGF mutein expression:
The M13-PN14 replicative form (l~F) obtained in Example 11 above was treated in the manner described in Reference Example 5 (1) to construct the plasmid pTB79~ for human bFGF mutein.
Using this plasmid pTB795, Escherichia coli MM294 was transf~rmed, whereby the strain Escherichia coli MM294/pTB7g5 (IFO 14700, FERM BP-1660) was obtained, which contains the plasmid pTB795 containing the mutein-encoding gene.

~,9~ 75 (2) Preparation of bacterial cell extract:
The above-rnentioned transformant was cultured by the method described in Reference Example ~ (2) to give a supernatant, which was then used as a bacterial cell extract.
(3) Human bFGF activity of the bacterial cell extract:
A determination was made of the human bFGF activity of the bacterial cell extract obtained in (2) above, by the method described in Reference :Ex~nple 5 (3).
The bacterial cell extract from E. coli MM294/pTB795 thereby tested exhibited E`GF activity. The mutein N14, in which the amino acid sequence of from Pro at the 2-position to Pro at the 14-position of human bFGF hacl been deleted, was thus obtained.
Reference Exa nple 13 (Expression in Escherichia coli of gene which encodes human bFGF mutein) (1) Construction of the plasmid pTB796 for human bFGF mutein expression:
The M13-PC86 replicative form (RF) obtained in Reference Example 11 above was treated in the manner described in Reference Example 5 (1) to construct the plasmid pTB796 for human bFGF mutein.
Using this plasmid pTB796, Escherichia coli MM294 was transformed, whereby the strain Escherichia coli MM294c/pTB796 (IFO 14701, FERM BP-1661) was obtained, which contains the plasmid pTB796 containing the mutein-encoding gene.
(2) Preparation of bacterial cell extract:
The above-mentionecl transformant was cultured by the method described in Reference Example 5 (2) to give a supernatant which contains the mutein C86, in which the amino acid sequence of from Lys at the 87-position to Ser at the 147-pos;tion hacl been deleted, and the supernatant was then usecl as a bacterial cell extract.
Reference Example 14 (Expression in Escherichia coli of gene which encodes human bFGF mutein) (1) Construction of the plasmid pTB797 for human bFGF mutein expression:
The M13-PDN42 replicative form (RF) obtained in Reference Example 11 above was treated in the manner described in Reference Example 6 (1) to construct the plasmid pTB797 for human bFGF mutein.
Using this plasmid pTB797, Escherichia coli MM294 was transformed, whereby the strain Escherichia coli MM294/pTB797 (IFO 14702, F~,RM BP-1662) was obtained, which harbors the plasmi~ pTB797 containing the mutein-encodin g gene.
(2) Preparation o~bacterial cell extract:
The above-mentioned transformant was cultured by the method described in Rererence 13xample 5 (2) to give a supernatant, which was then used as a bacterial cell extract.
(3) Human bFGF activity of the bacterial cell extract:
A determination was made of the human bFGF activity of the bacterial cell extract obtained in (2) above, by the methl)d described in Reference Example 5 (3).
'I'he bacterial cell extract from E. coli MM294/pT:13797 thereby tested exhibited FGF activity. The mutein DN42, in which Asp at the 42-position of human bFGF had been replaced by Asn, was thus obtained.
Reference Example 16 (Expression in Eschericllia coli of gene which encodes human bFGF mutein ) (1) Construction of the plasmid pTB865 for human bFGF mutein expression:
The DNA of the plasmid pTB669 which was obtained in the above mentioned Ref`erence Example 2 was cleaved with a restriction enzyme HincII, and it was ligated with Eco~I linker p(5' C~TGA~TTCATG 3') under T4 DNA ligase reaction. Thus obtained DNA was further cleaved with a restriction enzymes EcoRI and PstI to recover a DN~ fragment of about 0.35 kb. This DNA fragment was ligated with the about 3.2 kb DNA
fragment obtained in Reference Example 5 (1), the fragment being obtained by cleaving the plasmid ptrp781 with EcoRI-PstI, to obtain the plasmid pTB855 for human bFGF mutein expression was constructed.
Using this plasmid 855, Esc1~erichia, coli MM294 was transformed, whereby the strain :Escherichia coli MM294/pTB855, which contains the plasmid pTB855 having the mutein -encoding gene.
(2) Preparation of bacterial cell extract:
The above-mentioned transformant was cultured by the method described in Reference Example 5 (2) to give a supernatant, which was then used as a bacterial cell extract.
(3~ Human bFGF activity of the bacteriaI cell extract:
A determination was made of the human bFGF activity of the bacterial cell extract obtained in (2) above, by the method described in Reference Example 5 (3).

~L~9~275 The bacterial cell extract from E. coli MM294/p'rB85{; thereby tested exhibited FGF activity. The mutein N41, in which the amino acid sequence of f`rom Pro at the 2-position to Val at the 41-position o~ human bFGF had been deleted, was thus obtained.
Ref`erence Example 16 (Æxpression in Escher ichia coli of gene which encodes human bFGF mutein ) (1) Construction of the plasmid pTB856 ~or human bFGF mutein expression:
The DNA of the plasmid pTB669 which was obtained in the above mentioned Reference Example 2 was partly cleaved with a restriction enzyme BamEII so as to obtain BamHI recognition site in the bl?GF gene. The site was further cleaved with Escherichicl coli DNA polymerase I in the presence of dA L'P, clCTP, dGTP, clTTP to give blunt end. This DNA is ligated with NheI
linker p(5' CTAGCTAGCTAG 3'~ under T4 DNA ligase reaction. After treating with the restriction enzyme NheI and ligating the clea~Ted site under T4 DNA ligase reaction, the plasmid pTB856 for human bFGF mutein expression was constructed.
Using this plasmicl pTB856, Escherichia coli MM294 was transformed, whereby the sl;rain Escherichia coli MM294/pTB856 which contains the plasmid pTB856 having the mutein-encoding gene.
(2) Preparation of bacterial cell e2ctract:
The above-mentioned transformant was cultured by the method as in Reference Example ~ (2) to give a supernatant which contains the mutein C129, in which the amino acid sequence of ~rom Lys at the 130-position to Ser at the 147-position hacl been deleted, was thus obtained, and then the supernatant which was then used as a hacterial cell extract.
Reference Example 17 (Production of H-Le-u-Pro-Met-Ser-Ala-Lys-Ser-OH) Boc-Ser(Bzl)-resin (696 mg, 0.72 m mol/g resin~ was applied to automatic peptide synthesizer Type 430A (Applied Biosystems, U.S.A.), and the following amino acids were applied to the synthesizer in that order so as tocause condensation reaction:
Boc-Lys(Z)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-O~I, Boc-Met-OH, Boc-Pro-OH, Boc-Leu-OH
Bzl: benzyl Boc: t-butoxycarbonyl Z: benzyloxycarbonyl - ~o -~L~9~27S
By said procedure, 1.08 g of Boc-Leu-Pro-Met-Ser(Bzl)-~la-l,ys(Z)-Ser(Bzl)-resin was produced. 400 rng of the pepticle-resin was incubatecl in 5.0ml of hydrogen fluoride colltaining 0.5 ml of anisole ancl 0.5 ml of climethylsulrlde at 0C for 60 millutes to recover the pepli~le from r esin. Theexcess amount Or hydrogen fluoride was removed by distillation under reduced pressure to give a residue. The residue was washed with diethyl ether, and extracted with 30 ml of water, and lyophilized. The lyophylizate vvas dissolved in 5 ml of water, and the solution was subjected to ion-exchange p~ employing ~mberlite3~RA-400 (acetate rorm) resin (column 2 x 5 cm, elution solvent: water). '11he eluate was concen,~rated under reduced pressure, and X subjected to gel filtration with Sephadex~;E-20 (Pharmacia, column 2.5 x 125 cm, elution solvent: lN acel,ic acid) to obtain the peptide H-Leu-Pro-Met-Ser-Ala-Lys-Ser-OH .
Yield: 118 mg (78.8%) Rf value: 0.22 ~ethyl acetate: acetic acid: butanol: water--1:1:1:1) [alD -8.1 (c--0.11, lN acetic acid) Amino acid analysis: Ser. 2.08, Pro 1.06, Ala 1.00, Met 0.98, Leu 1.03, Lys 0.99.
xample :l (Immunization) 13ALB/c mice (female, 4-week old) had the antigen human bFGF (as obtained in Reference Example 3) in solution in 0.4 me of Freund's complete adjuvant (DiÇco Laboratories, USl~) injected intraperitoneally. Three weeks later, 10 ~ug o~ the antigen hbFGF in solution in 0.4 m~ of Freund's incomplete adjuvant was intraperitoneally admillistered. 3 weeks later, the same additional immunization was carried out, and two weeks later, 10 llg of human bFGF in saline was intraperitoneally inoculatecl.
Example 2 (1) Cell fusion From the immunized mice mentioned in Example 1, the spleen was excised 4 days after final antigen challenge to thereby obtain cells to be used for cell fusion. These cells were suspended in a medium prepared by mixing together Isokov medium and Ham F-12 medium in a ratio of 1:1 (hereinafter referred to as IH medium).
The mouse myeloma cell P3-X63-Ag 8UI was subcultured in RPMI
1640 medium containinK 10% fetal bo~Tine serum under an atmosphere of 5%
;~

I fc~de~?ark ~9~2~5 carbon dioxide ancl 95% air.
Cell fusion was conducted in accordance with the rnethod established by Kohler an~ Mils~ein [Kohler, G~. and ~ilstein, C.: Nature, 256, 495 (1975)].
2.~ x 107 cells of the above myeloma cell line and 1.5 x 108 immunized lymphocytes obtained by the above-mentioned method were mixed together and centrifuged, and 4~% polyethylene glycol 6000 (hereinafter referred to as PEG 6000) in 0.3 m~ of EI medium was dropwise added. The PEG 6000 solutioll was preheated to 37C, and was gradually added. Five minutes later, the 37~C-preheated IE medium was added at a rate of 0.5 me per minute to make 10 m~. The solution was then centri~uged at room temperature at 600 rpm for 15 minutes, and the supernatant was removed. This cell precipitate was suspended in 200 me of IH meclium containing 20% calf serum, and this suspension was transferred to a 24-well microplate (Linbro) in an amount of 2 m~ per well. One day later, IH medium (containing 20% calf serum) supplemented with HAT (1 X 10-1 ~ hipo~anthine, 4 X 10-7 M aminopterin, 1.6 X 10-~ M thymidine) (hereinafter referred to as HAT medium) was added to the microplate in an amount of 1 m~' per well, and, a ~urther three day, one half amount of the medium was replaced with I~lAT medium.
The cells thus grown are hybrid cells.
(2) Search for antibody-producing cells Previously, the hybridoma conditioned medium was added in an amount of 100 ~ue per well to a 96-well polystyrene microtiter plate which had had human bFGF immobilized thereto, and incubation was carried out at room temperature for 2 hours. The medium was removed, and, after washing, khe horse radish peroxiclase (~IRP)-labeled anti-mouse IgG goat antibody (Miles) was added as the secondary antibody, and incubated at room temperature for ~ hours. The secondary antibody was removed, and, after thoroughly washing the wells, coloring reaction was carried out in the presence of added reaction substrate (EIA method). By this method potent valency was observed in 3 wells.
(33 Cloning of hybrid cells Cells in each of these three cells were sown to 0.5 cell per well to a 96-well microtiter plate which had had 104 cells/well mouse thymocytes as vegetative cells sown thereon, and cloning was carried out. As a result, three clones, namely the mouse Hb~'99 cell (IFO 60122), the mouse HbF161 cell (IFO 50123) and the mouse IlbF165 cell (IFO 50124) were obtained.
The results o~ the determination o~ antibody titers in supernatants of these cell lines are shown in Table 2.
Table 2 Culture Supernatant bFGF-. .
Dllutlon Hbl~ HbF ~IbF Parent Line Immunized Mouse 99 161 165Myeloma Cell Serum _ X 6~ 1.93 1.08 0.66 0.02 X 128 1.72 0.63 0.51 0.02 X3200 _ _ _ _ 1.93 X6~00 ~ ~ - - 1.25 Note: The numerical rlgures in the above Table 1 represent absorbances at 492 nm wavelength; blank (--) means that determination was not made.
The cloned cells wexe stored in IH medium containing 20% calf serum and having dimethylsulfoxide tDMSO) added thereto to 10% under liquid nitrogen atmosphere.
Example 3 (Immunoglobulin class of monoclonal antibodies) The mouse antibodiés obtained in Exalllple 2 were reacted with various immunoglobulin standards by means of the ~ouse-Typer subisotyping kit (Bio-Rad). The results are presented in Table 3.

~29~275 Table 3 Immuno- Monoclonal Antibodies According to the Invention ~ lub li ~ MoAb99 MoAb161 MoAbl65 IgG 2a _ _ IgG 2b _ _ IgM t +

Note: ~ indicates positive for the reaction, and--indicates negative for the reaction.
From Table 2 it is obvious that HbF99, HbF161, and HbF165, all belong to the immunoglobulin class IgM.

Example 4 Spleens were collected from BALB/c mice immunized by the method described in Example 1, and, by the methods described in Example 2 (1), (2) and (3), the hybridomas HbF12 (IFO 50142), EbF45, HbF47, HbF52 (IFO
50143), HbF78 (IFO 50144) and HbF98 (IFO 50145) were obtained. 2 x 106 cells eaeh hybridomà were intraperitoneally inoculated to mice to which 0.
m~ of mineral oil were prei~ected.
After 10 days, 2 to 4m~ of ascites per mouse were collected, and monoclonal antibodies MoAbl2, MoAb45, MoAb47, MoAb52, MoAb78 and MoAb98 were obtained, frorn said hybridomas, respectively, in accordance with the method described in Example 2 (4).
By the method described in Ea~ample 3, deterrninations were made of the immunoglobulill class of said monoclonal antibodies, whereby the following results shown in Table 4 were obtained.

, - ~4 -~;29~75 Table 4 Monoclonal Antibody Immunoglobulin Class MoAbl2 IgG 1 Mo~b45 IgG 1 MoAb47 IgM
MoAb52 IgG 2b MoAb78 IgG 2b MoAb98 _ _ IgG 1 Example 5 (1) Preparation of radiolabeled hbF~F
Using the transformant Escherichia coli MM294/pTB669 (IFO 14532, FE~M J3P-1281) described in E~eference :Example 2, hbFGF radiolabeled with 35S was obtained in the fullowing mamler.
The above Escherichia coli MM294/pTB669 was cultivated in the medium described in Reference Exarnple 3 until the Klett value was 200.
This culture broth in the one fi:~th amount was poured into M9 (Met~) medium. The M9 (Met~) medium was prepared by supplementing the M9 medium containing 1% glucose, 8 ~lg/m~ tetracycline, and the amino acid shown below:
Amino acid composition L-alanine 25.0 mg/~
L-arginine hydrochloride 84.0 mg/~
L-asparaginemonohydrate 28.4 mg/~
L-aspartic acid 30.0 mg/~
L-cysteine disodium salt 82.8 mg/~
L-glutamic acid 75.0 mg/~
L-glutamine ~84.0 mg/~
L-glycine 30.0 mg/~
L -histidine hydrochloride monohydrate 42.0 mg/~
L-isoleucine 105.0 mg/~
L-leucine 105.0 mg/~
L-lysine hydrochloride 146.0 mg/~
L-phenylalanine 66.0 mg/e L-prGliIle 40.0 mg/~

3~96Z75 L-serine 42.0 m~/~
L-threonine 95.0 mgle L-tyrosine 83.9 mg/~
L-valine 94.0 mgle Cultivation was carried out in the M9 (Met~) medium until the Klett value was 200, and 3-~3-indoleacrylic acid was aclded to 25 ~g/me, and the cultivation was continued ~or 2 more hours. Thereafter, a l-m~ portion of the culture broth was collected, and 10 ~uCi of 35S-Met (specirlc activity > 1000 Ci/mmoO was added, and cultivation was carried out for 30 minutes. After cultivation, cells were harvested, and a cell extract was obtained in accordance with the method described in Reference Example 3.
The Escherichia coli MM294 carrying the vector plasmid ptrp781 and described in Reference Example 2 was subjected to the same procedure to thereby obtain a labeled cell extract.
(2~ Immunoprecipitation A 10% solution of Protein ~ (BRL) was prepared in accordance with the instruction manual thereof. An unlabeled Es~cherichia coli cell extract was obtained by treating 13scherichia coli MM294/ptrp781 by the method of Reference E~ample 3.
One milliliter of the ascites fluid obtained in Example 4 was mixed with 100 }1~ of the unlabeled Escherich~a coli cell extract, and the mixture, after being allowed to stand at 4C for 1 hour, had 10~ cpm of the labeled cell extract (MM294/ptrp781 or MM294/pTB669) added thereto, and was allowed to stand at 4C overnight.
To 100 ,u~ of the 10% solution of Protein A, was added 100 ~e of the unlabeled Escherichia coli cell extract, and the mixture, after being allowed to stand at 4C overnigllt, was centrifuged and again suspended in 100 ~u~ of NETBN solution [150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl (pH 7.5), 0.1%
BSA, 0.05% Non-idet (NP)-40]. To this suspension was added the above-treated mixture of labeled cell extract of ascites, and the mixture was allowed to stand at 4C overnight. This mixture was then centrifuged, and the resulting precipitate was s-uspended in ~00 ~ of NETBN solution. This procedure was repeated ~ times to thereby remove unadsorbed labeled substance, and the pellets were resuspended with 50 m~ of electrophoresis sample buf~er. The polyacrylamidegel electrophoresis was performed in 9~7~

accordance with the method of Laemmli, U. K. [Nature, 227, 680 (1970)].
Af ter migration, the gel was immersed in 50% trichloroacetic acid (rl'CA) for 1hour and was washed four tirnes with distilled water ror 3û minutes Çor each wash to thereby remove the TCA, a~ter which the gel was immersed in dimethylsulroxide (DMSO) for 1 hour. Therearter, the gel was immersed in DMSO containing 10% 2, 5-diphenyloxazole (DPO) for 1 hour. After washing three times with distilled water for 30 minutes f`or each wash, the gel was dried. The dried gel was radioautographed, and the immunoprecipitation pattern was examined. The radioautograms are shown in Fig. 2. From Fig. 2, the monoclonal antibodies MoAbl2, MoAb52, MoAb78 and MoAb98 were found to combine with hbFGF in cell extract.
Example 6 The antibody valencies of the 4 lines which showed immunoprecipita-tion in Example 6, selected from the monoclonal antibodies described in Example 4, namely the monoclonal antibodies MoAb 12, Mo~b ~2, MoAb 78 and MoAb 98 were determined ~y the limiting dilution method.
That is, each of the ascites fluids of monoclonal antibody MoAb 12, MoAb 52, MoAb 78 or Mo~b 98 obtained in Example 4 was diluted with IH
medium containing 10% fetal bovine serum, and the quantity of antibodies in the dilution was determiIled by the EIA method mentioned in Example 2 (2).
The results are shown in Fig. 3. In Fig 3, - - - - O - - - - indicates the results ~or MoAb 12, ~ - - - - indicates the results Eor MoAb ~2, 11~1 indicates the results for MoAb 78, and - - - - V - - - - indicates the results for MoAb98.
Fig. 3 shows that the ascites containing the above antibody show a limiting dilution rate of more than 1 X 106, that is, these 4 antibodies are very high in antibody valency.
Exam~le 7 (Determination of recognition site to the antigen) Recognition sites of the four antibodies, which show high antibody valencies obtained in Example 6, to the antigen were determined by competitive analysis.
As competitors, hbFGF obtained in Reference Example 3, synthetic peptide Pep 1: Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Thr (which is ob-tained by adding Tyr to the polypeptide of N-terminal amino acids Nos. 2 to 10, Regulatory Peptides, 10, 309-317(1985~), synthetic peptide Pep 2:
!

9~2~S

Leu-Pro-Met-Ser-Ala-Lys-Ser (which corresponds l;o the amino acids Nos.142 to 147 obtained in Reference Example 17), bFGF mutein N14 ( obtained in Reference Example 12), and bFGF mutein N41 (obtained in Reference Example 15).
The synthetic peptides were adjusted to the concentration of 100 }Ig/m~
and diluted with IH medium (Iscov and Eam F12 mixed medium at a ratio with 1:1) containing 10% FCS. The extract containing the mutein N14 or N41 obtained in I~eference Example 12 (2) or 15 (2) was diluted with IEI medium containing 10% ~CS. The antibodies, obtained in Example 4, were diluted to their binding titers showing between 0.7 and 1.0 with absorbance at 416 nm.
So, dilution ~actor about MoAb12, MoAb62 and MoAb98 was 5 x 105, and about MoAb98 was 5 x 104. In the above dilution, EI medium containing 10%
FCS was used as a d.iluent. To the diluted antibody solution were added the diluted competitor. After stirring the mixture, the diluent was warmed at 37C for 30 minutes. The amount of the unbound antibody was measured by EIA method shown in Example 2 (2). The results in case of using synthetic pepticle are shown in Figures 4 to 7. Fig. 4 shows the results on the monoclollal antibody Mo~b 12, Fig. 5 shows the results on the monoclonal antibody MoAb 52, Fig. 6 shows the results on the monoclonal antibody MoAb 78, and Fig. 7 shows the results on the monoclonal antibody MoAb 98.
In these figures,--~51 denotes the results of hbFGF, ~ O - - - -denotes the results of Pep 1, - - - - -1~11 - - - - - denotes the results of Pep 2, these being used as competitor, and the vertical axis denotes the absorption at 415 mr~.
As shown in the Figures 4 and 6, the monoclonal antibodies MoAb 12 and MoAb 78 are competitively inhibited to combine with bFGF and Pep 1.
This indicates that the monoclonal antibodies MoAb 12 and MoAb 78 recognize the N-terminal amino acids of Nos. 2 to 10.
As shown in the Fig. ~ and Fig. 7, the monoclonal antibodies MoAb 62, Mo~b 98 are not competitively inhibited to Pep 1 and Pep 2.
In order to determine the recognition site of the monoclonal antibodies, the competition analyses were conducted using the mutein N14 (obtained in Reference E~ample (2~ or N41 (obtained in Reference Example 16) by the method described in the above, and the results are shown in Figures 8 and 9.
In these figures,--~--denotes the results of hbFGF, - - - - V - - - -denotes the results of the mutein N14, - - - - O - - - - denotes the results of the ~'9Ç~275 mutein N41, and the vertical axis denotes the absorption at 415 mm, and the horizontal axis shows the total protein in the E. coli extract obtained in Reference Example 3.
As shown in Fig. 8 and Fig. 9, the monoclonal alltibodies MoAb 52 and MoAb 98 are competitively inhibited to combine with mutein N14, but not with mutein N41. This indicates that the monoclonal antibodies MoAb 52 and MoAb 98 recognize the amino acids of Nos.15 to 41.
The competitive analysis was carried out on bovine acidic FGF (baFGF) [purchased from R & D Systems Inc., USA] and human bFGF obtained in ReÇerence Example 3 by the method described in the above, and the results are shown in Table 5. The results are indicated by optical density of the substrate by EIA method.

Table 5 .
baFGF hbFGF
10 llg/m~ 80 ng/m~ 10 }lg/m~ 80 ng/me MoAbl2 1.049 0.975 0.396 1.077 MoAb52 0.997 0.923 0.230 0.722 Mo~b78 0.978 0.962 0.062 0.523 MoAb98 0.948 0.921 0.095 1.356 As sbown in Table 5, the monoclonal antibodies MoAb 12, MoAb 52, MoAb 78 and Mo~b 98 do not cross-react with bovine bFGF.
All of the above results are shown in the following Table 6.

~_2q~275 Table 6 . . _ _ _ Monoclonal ~ntibody HbF

_ .
hbFGF
baFGF _ _ _ _ N~ _ ~ -- ~
N41 _ _ _ _ Pepl + _ ~ _ Pep2 _ _ _ _ II1 the Table 6, + denotes that they are competitive, and - denotes that they are not cornpetitive.
xamPle 8 (Purification of the monoclonal antibody f`rom ascites) The monoclonal antibody Mo~b 12, MoAb 52, MoAb 78 or MoAb 98 was inoculated to 10 mice, and 20 to 30 m~ of ascites were~collected. I'he ascites ,~ were subjected to centrifugation at 2,000 rpm (Hitachi refrigrated centrifuge) <~ to remove cells, and further subjected to centrifugatiorl with Spinco~W 28 roter (Beckman, USA) at 4C for 2 hours to remove the insoluble proteins and fats. To the supernatant thus obtained there is added ammonium sulfate so as to 40% saturation, and the mixture was stirred gently in ice bath for 1 hour.
k The precip;tate was subjected to centrifugation with Sorval SS34 roter tDupont, USA) at 4C, 15,000rpm. The pellet was dissolved in Buffer 1 [20mM Tris HC1 (pH 7.9), 40mM NaCl] so that the protein concentration is 10 to 15 mg/m~. Thus obtained solution is dialyzed for the Buffer 1 at 4C
overnight. Thus~btained solution is passed through the column of DEAE-cellulose (DE-52, Whatman, USA) to adsorb. The elution was carried out employing the linear gradient from Buffer 1 to 0.4M NaCl.
The immunoglobulin fractions were recovered. To the fraction were added 40% saturated ammonium sulfate so as to emerge precipitate. The precipitate was dissolved in Bu~fer 2 (0.1M NaHCO3) so as to the protein concentration being 10 to 20 mg/m~, and the solution was subjected to dialysis to Buffer 2 at 4C for two overnights. The Buffer 2 were replaced every day.
.~
r~ac1-e~mc~ r k ~9~2'75 ~ urthermore, the dialyzate is subjectecl l;o hy~roxy apatite column (I-ICl~ column). As the initiation bufrer, lOmM sodium phosphate buffer (pE 6.8) was used~ and as the elution bufrer, 500m~ soclium phosphate buffer (pEI6.8) was used. I'he elution was carried out by linear gradient elution from the initial buffer to the elution buffer. 1~hus obtained eluate containing the antibody was preserved at 4C.
Example 9 (Purifïcation of hbFGF by antibody column) 5m~ of Affi-Gel-10 (Bio-Rad, USA) was put on sintered filter, was washed with ten volumes of ice-coled isopropanol and with ten times of ice-cooled distilled water. Thus obtained gel is transfered to a reaction vessel. Tothe gel was mixed with 15 mg of monoclonal antibody MoAb 78 in the volume of 5 to 15 m~ (dissolved in Buffer 2 or phosphate buf~er, in E~ample 8) to reactat 4C overnight.
Monoetllanol amine (pH 8.0) was added to the reaction mi~ture with the concentration of 0.01M, and leave at room temperature for one hour to inactivate the unreacted site. The gel was washed with 10 times (volume) of Buffer 2 (Example 8). 2 m-e of the gel was pacl{ed into a column, and the colu~ml was equilibrated with the initiation buffer [20mM Tris-ECl (pH 7.6), 1mM EDTA, 0.1~M NaCl, 0.05% NY-40].
On the other hand, the extracts of a transformant Escherichia coli DE1/pTB744 (the extracts containing mutein CS4), is diluted three times by the addition of the initial buf~er. The diluent was applied to said column at a flow rate of 20 me/hour to adsorb the mutein CS4 to the antibody. After the adsorption, the column was washed with 20 me of the initial bufrer, and then the elution was carried out by using 20 me of high salt burfer [20mM Tris ~ICl (pH 7.6), lmM EDTA, lM NaCl, 0.0~% NP-40], 20 me of an elution buffer A
~0.2M acetate buf~er (pH 4.5), 0.2M NaCl], 20 m~ of` an elution buffer B [0.2M
acetic acid (pH2.6), 0.2M NaCl] in that order, wherein the flow rate is 20 ~;n~/hour and the temperature is 4C. Thus obtained fractions were subjected to electrophoresis in accordance with the method described in Laemmli, Nature 277, 6flO (1970) employing 17.2~% acrylamide. Proteins were detected by silver staining.
The results are shown in Figure 10. In Figure 10, MK denotes the result of molecular marker, A denotes that of the crude extract, B denotes that of flow through fractions, C denotes that of the eluent of the high salt buffer, D denotes that of the elution bufrer A, and E denotes that of the elution buffer B. Just after the elution, the pH of D fraction and E ~raction was adJusted to pH 7.5 by adding lM Tris-HCl (pl-I 9.~). The FGF activity on the ~ractions were measured in accordance with the method of Reference Example 5 (3). The results are shown in Table 7. In Table 7~ A to E denote the same as above.
Table 7 . ._ . . ._ _ _ _ I
Protein (A) FGF Activity* ~B)B/~
(llg) (,~ug) . . ._ .~
A. 63000 96 0.0015 B 63000 19 0.0003 C 28 0.003 0.0011 D 17 0.049 0.0030 l _ E 33 23 0.69 Note: *: The FGF activity is shown in equivalent amount of the bovine pituitary derived bFGF (perchared ~rom Takara Shuzo, Japan) on the basis of incorporation of 3II-thymidine.
:l~xample 10 (Measurement of hbFGF mutein by EIA methocl using monoclonal alltibody) (1) The antibody MoAb 78 obtained in 13xample 4 was subjected to purification from ascites fluid in accordance with the method of Example 8.
Thus obtained antibody was concentrated to more than 2 mg/m~, and subjected to dialysis in 0.2M sodium phosphate buffer (pEI 7.0~. To thus obtained 1.4m~ solution of monoclonal antibody MoAb 78 (concentration 2.8 mg/m~), 50 ~u~ of S-acetylmercaptosuccinic anhydride (Alclrich Co., U.S.A) dissolved in N, N'-dimethylformamide was added so as to reach the concentration of 11.5 mg/m~. The air in the reaction vessel is replaced by nitrogen gas. The vessel was sealed, and subjected to stirring so as to cause the reaction of introducing SH group. The unreacted S-acetylmercaptosuccinic acid anhydride was inactivated by the treatment for 10 minutes with 130 11~ of 0.2M Tris HCl (pH 7.0), 13 1l~ of 0.2M EDTA and 130 lue of 2M hydroxyamine (pH 7.0). The MoAb 78 was recovered by gel fil-tration using a column packed with Sephadex G-25 (cliameter 1 cm X 80 cm, Pharmacia, Sweden) (flow rate: 20 m~/hour).
(2) 10 mg of` horse radish peroxidase (HRP, 13ehringer Manheim, Grade I, West Germany) was dissolved in 1.4 m~ of 0.1M phosphate buffer (pH 6.8).
On the other hand, 14 mg of N-hydroxysuccinimide ester of N-(4-corboxy cyclohexyl methyl) maleimide was dissolved in 335 lle of DMF, and 100 ~ of thus obtained solution was added to the ERP solution above mentioned. The air in the reaction vessel was replaced by nitrogen gas, and the vessel was sealed. After 1 hour reaction at room temperature, proteirls of the portion of HRP introduced with maleimide group were recovered by gel filtratioxl using a colomn packed with Sephadex G-25 as in the above (f).
(3) 6 m~ of the portion of the monoclonal antibody MoAb 78 introduced with SH group obtained in the above (1) and 2 m~ o~ the portion of ERP introduced with maleimide group obtained in the above (2) were mixed, and the mixture was concentrated to 1 m~ using collodion bag (Sartorius, West Germany) ~ under reduced pressure at 4C i~or 20 hours. After the reaction, the unreacted k HRP introduced with SEI group was removed with the use of~Ultrogei~cA44 (LKB Co., diameter 1 cm X 80 cm, Sweden) colutrm (flow rate: 10 m~/hour).
In the eluates, the fraction containing 2.4 I~ P/antibody has the most high HRP number per antibody molecule. The product thus obtained was employed in the EIA in the following it0m (4).
(a~) The monoclonal alltibody Mo~b 52 was purifïed by the manner described in the above (1). The monoclonal antibody MoAb ~2 was diluted with PBS so as to be 10 ~ug/m~ or 20 llg/m~, and the diluent was poured into Immunoplate (Nunc, Denmark) so as to be 100 ~I~/well. The plate was kept standing at 4C
overnight to adsorb the monoclonal antibody MoAb 52 to the plate. After removing the antibody which is not adsorbed, the plate was washed with PBS
thrice, PBS containing 0.01% merthiolate and 1% bovine serum albumin (BSA) was added to the plate at 200 ll~/well, and the plate was kept standing at 4C overnight.
(5) The cell extract containing bFGF mutein C86 obtained in Reference Example 3 was diluted with PBS containing 0.1% BS~. From the plate obtained in the above (4), BS~ solution was removed, the plate was washed with PBS four times, and the diluted bFGF mutein C86 was added to the plate so as to be 100 ~e/well to adsorb to the plate at 4C overnight. The unreacted mutein C86 was rernoved, and the plate was washed with PBS four times.

e ~nG~

:~96Z~5 The monoclo~al antibody conjugated with EII~P (I-IRP-MoAb 78) obtained in the above (3) was diluted with PBS containing 0.1% BSA to 1/300, and the diluent was added to the plate so as to be 1ûO ll~/well. The reaction was carried out for 4 hours at room temperature. ~fter removing the antibody, the plate was washed with PBS for 6 times, substrate for oxidase (Bio. Rad Co.
U.S.A) was added to the plate so as to be 100 lle/well. Quantiflcation was accomplished by absorbance measurements at 415 nm, and it was conf~lrmed that a small amount of the mutein C86 was produced.
~6) In Figure 11, the detection curve ;s shown in case that the amount of monoclonal antibody MoAb 52 which is fixed to the plate is 1 llg/well (- - - O - --), and 2 ~g/well ( - ~--). The horizontal axis shows the concentration of bFGF added, and the vertical axis shows the absorbance at 415 nm of the solution caused by HRP-MoAb78.
From the Figure 11, it is taught that the concentration of 0.5 ng/m~ of bFGF can be detected, when the monoclonal antibody MoAb 52 is adsorbed to the plate in an amount of 2 ~Ig/well.
(7) The monoclonal antibody ~oAb 98 was adsorbed to the plate in an amount of 2~g/well, according to the method of the above (4), and the measurement of absorbance at 415 nm was carried out in accordance with the method of the above (5). The results are shown in Figure 12. The horizontal and vertical axes show the same as those of Figure 11. From Figure 12, it is taught that at least 0.5 ng/m~ of bFGF can he detected by using the monoclonal antibody MoAb 98.
Example 11 (The measurement of hbFGF mutein by EIA method employing monoclonal antibody) The cell extract containing human bFGF mutein C86 obtained in Reference Example 13 was treated with the manner of Example 9 to measure the expression amount of the mutein. The results indicate that the mutein C86 is expressed in the cell in a slight amount.
_xample 12 (The measurement of hbFGF mutein by EIA method employing monoclonal antibody) The cell extract containing human bFGF mutein C129 obtained in Reference Example 14 was treated with the manner of Example 9 to measure - ~4-~5 the expression amount of the mutein. The results indicate that the mutein C129 is expressed in the cell in a slight amount.
Example 13 (Detection of hbFGF by the method of Western blotting) hbFGF obtained in Reference Example 3 was subjected to electro-phoresis employing 17.2~% acrylamide gel ~Laemmli, Nature, 277, 680-685 ~1970)], and it was trans~erred [Journal of I3iochemical and Biophysical Methods, 10, 203-209 (1984)] on the membrane of nitrocellulose by using Sartoblot~Sartorius, West Germany). This membrane was washed with TBS
(20mM Tris-HCl (pH 7.5), O.~M NaCl] ~or 5 minutes twice, and kept standing in TBS containing 4% BSA at roo-m temperature for one hour to block the unreacted material on the membrane. Thus obtained membrane was washed Y with T:E~S containing 0.05% Twee~20 ('lVrBS) for ~i minutes twice.
The monoclonal antibody MoAb 12 or MoAb 78 was diluted with TTBS
containing 1% gelatin so as to 1/3000. To thus obtained dilution said nitrocellulose membrane was inserted, and reaction was carried out overnight. After the reaction, the reaction liquid was removed, and the membrane was washed with I"rBS for 5 minutes twice.
The secondary antibody, i.e., anti mouse IgG goat serum (Bio Rad, USA) labeled with HRP, was diluted to 1/3000 by TTBS containing 1%
gelatin.
To the membralle obtained above was added the diluted secondary antibody, and reaction was carried out at room temperature for one hour. The membrane thus obtained was washed with ~r:3s for 5 minutes thrice, and then washed with TBS for 5 minutes twice. After that, to the membrane was added 0.05% 4-chloro-1-naphtol as substrate and 0.01~% hydrogen peroxide, and the reaction was carried out for 1~ minutes.
In Figure 13, the results of Western blotting in case the monoclonal antibody MoAb 78 was used as primary antibody. The lane 1 shows the results of 1 llg of bFGF, the lane 2 shows the results of 300 llg of bFGF, the lane 3 shows the results of 100 ~g of bFGF, wherein bFGF was electrophoresised and transferred. M shows a marker, and the numerals in vertical axis show molecular weight. bFGF was detected in the same sensitivity when the monoclonal antibody MoAb 1~ instead of MoAb 78.

'~ ~
~r~C~e- rna,rk

Claims (29)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A monoclonal antibody which combines specifically with basic fibro-blast growth factor (bFGF), the antibody having the characteristics:
(a) it has a molecular weight of about 140 to 160 kilodaltons, (b) it does not cross-react with acidic fibroblast growth factor, and (c) it belongs to the immunoglobulin class IgM or IgG.

2. A monoclonal antibody as claimed in Claim 1, wherein the bFGF is a polypeptide which includes the amino acid sequence:
.

3. A cloned hybridoma comprising a splenic cell from a mammal immunized with basic fibroblast growth factor (bFGF) and a homogenic or heterogenic lymphoid cell fused with the splanic cell.

4. A hybridoma as claimed in Claim 3, wherein the mammal is a mouse.

5. A hybridoma as claimed in Claim 3, wherein the lymphoid cell is a myeloma cell.

6. A hybridoma as claimed in Claim 3, wherein the bFGF is a polypeptide which includes the amino acid sequence:
.

7. A method for producing a monoclonal antibody which combines specifically with basic fibroblast growth factor (bFGF) and has the characteristics:
(a) it has molecular weight of about 140 to 160 kilodaltons, (b) it does not cross-react with acidic fibroblast growth factor, and (c) it belongs to the immunoglobulin class IgM or IgG, which comprises growing a cloned hybridoma comprising a splenic cell from a mammal immunized with said bFGF and a homogenic or heterogenic lymphoid cell in liquid medium or mammalian abdomen to allow the hybridoma to produce and accumulate the monoclonal antibody.

8. A method as claimed in Claim 7, wherein the mammal is a mouse.

9. A method as claimed in Claim 7, wherein the lymphoid cell is a myeloma cell.

10. A method as claimed in Claim 7, wherein the bFGF is a polypeptide which includes the amino acid sequence:
.

11. A method for producing a cloned hybridoma comprising a splenic cell from mammal immunized with basic fibroblast growth factor (bFGF) and a homogenic or heterogenic lymphoid cell, which comprises subjecting said splenic cell and said lymphoid cell to cell fusion followed by cloning.

12. A method as claimed in Claim 11, wherein the mammal is a mouse.

13. A method as claimed in Claim 11, wherein the lymphoid cell is a myeloma cell.

14. A method as claimed in Claim 11, wherein the bFGF is a polypeptide which includes the amino acid sequence:
.

15. A method for purifying basic fibroblast growth factor (bFGF), which comprises treating a material containing crude bFGF with the use of a monoclonal antibody which combines specifically with bFGF and has the characteristics:
(a) it has molecular weight of about 140 to 160 kilodaltons, (b) it does not cross-react with acidic fibroblast growth factor, and (c) it belongs to the immunoglobulin class IgM or IgG.

16. A method as claimed in Claim 15, wherein the bFGF is a polypeptide which includes the amino acid sequence:
.

17. A method for detecting or measuring basic fibroblast growth factor (bFGF), which comprises using, as antibody, a monoclonal antibody which combines specifically with bFGF and has the characteristics:
(a) it has molecular weight of about 140 to 160 kilodaltons, (b) it does not cross-react with acidic fibroblast growth factor, and (c) it belongs to the immunoglobulin class IgM or IgG.

18. A method as claimed in Claim 17, wherein the bFGF is a polypeptide which includes the amino acid sequence:
.

19. A substantially pure monoclonal antibody as claimed in claim 1, wherein the bFGF is a polypeptide having the following amino acid sequence:
(II) wherein X represents Thr or Ser; Y represents Ser when X is Thr or Y represents Pro when X is Ser.

20. A monoclonal antibody as claimed in claim 19, wherein the bFGF is human bFGF having the principal amino acid sequence as shown in claim 19 in which Y is Ser and X is Thr.

21. A monoclonal antibody as claimed in claim 20, wherein 9 amino acid residues Pro-Ala-Leu.......Gly-Gly-Ser from the amino terminal are lacking from the sequence.

22. A monoclonal antibody as claimed in claim 20, wherein 13 amino acid residues Pro-Ala-Leu........Ala-Phe-Pro from the amino terminal are lacking from the sequence.

23. A monoclonal antibody as claimed in claim 20, wherein 40 amino acid residues Pro-Ala-Leu..........Gly-Arg-Val from the amino terminal are lacking from the sequence.

24. A monoclonal antibody as claimed in claim 20, wherein 61 amino acid residues Lys-Lys-Val........Ala-Lys-Ser from the carboxyl terminal are lacking from the sequence.

25. A monoclonal antibody as claimed in claim 20, wherein in the amino acid sequence at least one cysteine is replaced by a neutral amino acid selected from the group consisting of gly-cine, valine, alanine, leucine, isoleucine, tyrosine, phenyl-alanine, histidine, tryptophan, serine, threonine and methionine.

26. A monoclonal antibody as claimed in claim 25, wherein the neutral amino acid is serine.

27. A monoclonal antibody as claimed in claim 20, wherein in the amino acid sequence, aspartic acid is replaced by aspara-gine or arginine; arginine is replaced by glutamine; glycine is replaced by threonine; serine is replaced by methionine; or valine is replaced by serine.

28. A method as claimed in claim 16, wherein an affinity column prepared from the purified monoclonal antibody is used.

29. A method as claimed in claim 18, which comprises:

contacting bFGF with plate or beads to which the purified monoclonal antibody is fixed, thereby adsorbing bFGF, adding the purified monoclonal antibody which is labeled with a color-forming enzyme or a radio isotope, thereby adsorbing the antibody to the adsorbed bFGF, and then measuring the amount of the enzyme or the radio isotope adsorbed by adding the substrate of the enzyme thereby forming a color or by counting the radio activity.