Hybridomas and monoclonal antibodies which specifically bind human basic fibroblast growth factor

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.

The present invention 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 referred to as bFGF, in 
the present specification) is a basic polypeptide hormone which is 
secreted mainly from the pituitary gland and which has a molecular weight 
of about 17,000. It was first isolated as a factor showing potent growth 
promoting action on fibroblasts 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.: National 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 quantitative determination 
of bFGF has not been established to date. For these reasons, much remains 
unknown of basic information 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 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 of bFGF is achieved by adding bFGF to 
3T3 cells which have been cultured at reduced serum concentration to 
attenuate their DNA synthesizing potency, and counting back bFGF 
concentration from the degree of recovery of DNA synthesizing potency. 
However, this method is faulty in that the procedure is delicate and 
determination errors are great, due to the use of cells, and in addition 
much time is needed to obtain results. It is therefore desired that a 
simple and accurate means of bFGF determination will be developed for the 
abovementioned purpose. 
Taking note of the above-mentioned circumstances, the present inventors 
made various investigations to find any practical means of bFGF 
determination, and prepared a monoclonal antibody which combines 
specifically 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 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 cloned hybridoma comprising a splenic cell from a mammal immunized 
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; 
(4) a method for producing a monoclonal antibody which combines 
specifically with bFGF, which 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 defined 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 (Japanese Patent Application No. 241053/1986 
which corresponds to European Patent Publication No. 237,966; European 
Molecular Biology Organization (EMBO) Journal, 5, 2523 (1986)). 
______________________________________ 
Polypeptides which includs the amino acid sequence: 
______________________________________ 
Phe--Phe--Leu--Arg--Ile--His--Pro--Asp--Gly--Arg--Val-- 
Asp--Gly--Val--Arg--Glu--Lys--Ser--Asp--Pro 
(I) 
______________________________________ 
are preferred. 
More preferably, the polypeptides are represented by the 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--Lys--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--Ser 
(II) 
______________________________________ 
wherein X represents Thr or Ser; Y represents Ser when X is Thr or Y 
represents Pro when X is Ser. 
For obtaining human bFGF (also briefly referred to as hbFGF), an expression 
vector which contains a DNA segment having a base sequence encoding, for 
example, the above-mentioned hbFGF protein polypeptide, can be produced, 
for example, by: 
(a) Isolating an RNA encoding hbFGF; 
(b) Synthesizing from said RNA a single-stranded complementary DNA (cDNA) 
and then a double-stranded DNA; 
(c) Inserting said complementary DNA into a plasmoid; 
(d) Transforming a host with the resultant recombinant plasmid; 
(e) Cultivating the transformant thus obtained, then isolating the plasmid 
which contains the desired DNA from the transformant by an appropriate 
method, for example, 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. 
RNAs 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 (ATCC No. CCL-75) and IMR90 (ATCC No. 
CCL-186). Said cell lines WI38 and IMR90 are listed in the Catalogue of 
Cell Lines & Hybridomas, 5th edition, 1985, published by the American Type 
Culture Collection. 
By inserting the expression vector thus obtained into an appropriate host 
(e.g., Escherichia coli, Bacillus 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 acid includes deletion of at least one 
bFGF-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. 
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 
homology with bFGF and which exhibit activities similar to those of bFGF. 
As 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 bFGF: 
Met-Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser the 14 residues in the amino 
terminal of human bFGF: 
Met-Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe-Pro the 41 residues in 
the amino terminal of human bFGF: 
##STR1## 
or the 61 residues in the carboxyl terminal of human bFGF: 
##STR2## 
As for the number of bFGF-constituent amino acids that may be substituted 
by other amino acids before substitution in mutein is lost, it may be any 
number, as long as any characteristic of bFGF is not lost. 
As examples of constituent amino acids before substitution, mention may be 
made of cysteine and other amino acids but cysteine is preferable. 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. 
As specific examples of such neutral amino acids, mention may be made of 
glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine, 
histidine, tryptophan, serine, threonine and methionine. Serine and 
threonine are preferable. 
When the constituent amino acid before substitution is other than cysteine, 
the substituted amino acids are selected from amino acids which are 
different from the amino acid before 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 arginine, examples 
of the substituted amino acids include glutamine, threonine, leucine, 
phenylalanine and aspartic acid; glutamine is preferable. 
When the constituent amino acid before substitution is glycine, examples of 
the substituted amino acids include threonine, leucine, phenylalanine, 
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, examples of 
the substituted amino acids include serine, leucine, proline, glycine, 
lysine, and aspartic acid; serine is preferable. 
The constituent amino acid before substitution are preferably aspartic 
acid, arginine, glycine, serine and valine. 
The substituted amino acid is preferably asparagine, glutamine, arginine, 
threonine, methionine, serine, and leucine. 
The embodiment on the substitution in the mutein 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. 31-50. Mutagenesis 
which is directed to oligonucleotide is described in Smith, M. and Gillam, 
S., 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 DNA 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 elongated by DNA polymerase to allow it to form a 
mutational heteroduplex, 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 
(Difco Laboratories). 
When a carrier protein complex is used, the coupling ratio of carrier 
protein to bFGF is about 5 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 preferably 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 
xnonoclonal 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 
intravenously (in particular, subcutaneously). In addition, immunizing 
interval, immunizing dose, etc. are also highly variable, allowing various 
methods to be carried out; the method in which immunization is conducted 
about 2 to 6 times at intervals of 2 weeks, and splenic cells taken out 
about 1 to 5 times, preferably about 2 to 4 days after the final 
immunization are used, for example, is commonly used. It is desirable that 
an immunizing dose of more than about 0.1 .mu.g, preferably about 10 to 
300 .mu.g for each mouse, calculated on the peptide amount basis, be used 
in each injection. It is also desirable that a fusion experiment 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 
(HGPRT.sup.-) or thymidine kinase deficiency (TK.sup.-). As the lymphoid 
cell line, myeloma cell is preferred, and the myeloma cell there is 
mentioned myeloma P3-X63-Ag. 8UI (Ichimori et al.: Journal of 
Immunological Methods, 80, 55 (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 Iscore medium 
and Ham F-12 medium in a 1:1 ratio (hereinafter referred Co as IH medium), 
and a fusing agent such as Sendal virus or polyethylene glycol (PEG) is 
used. Of course, dimethyl sulfoxide (DMSO) and/or other fusion promoters 
can also be added. The following are normally used: a degree of 
polymerization 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 achieved by about 4 to 10 minutes of PEG 6000 treatment at 
an about 35 to 55% concentration. The fused cells can be selectively grown 
using the hypoxanthine-aminopterin-thymidine medium (HAT 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 assayed for the production of antibody to an immunized peptide by 
a method such as the radioimmunoassay (RIA) method or enzyme immunoassay 
(EIA) method. These methods are also widely modifiable. As an example of 
preferred method of assay, a method using EIA is described below. To a 
carrier such as cellulose beads the rabbit anti-mouse immunoglobulin 
antibody, for 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.degree. to 40.degree. C.; this definition also applied 
hereinafter) for the specified time. After the reaction product is well 
washed, a peptide labeled with enzyme (prepared by coupling of an enzyme 
and a peptide in accordance with a routine method, followed by 
purification) is added, and reaction is carried out at constant 
temperature for the specified time. After the reaction product is well 
washed, an enzyme substrate is added, and reaction is carried out at 
constant temperature for the specified time, whereafter the resulting 
chromogenic substance can be assayed by absorptiometry or fluorometry. 
It is desirable that the cells, which showed proliferation in the selective 
medium 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 antibody 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, G. 
E. et al.; Journal of American Medical Association, 199, 549 (1967)). 
Specifically, 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.times.10.sup.4 to 1.times.10.sup.7, 
preferably about 5.times.10.sup.5 to 2.times.10.sup.6 hybridoma cells are 
intraperitoneally inoculated to a mouse of BALB/c or similar strain, 
previously inoculated with mineral oil etc., and about 7 to 20 days later, 
preferably 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 DEAE-cellulose column 
chromatography, whereby the desired monoclonal antibody can easily be 
isolated as a pure immunoglobulin. 
A monoclonal antibody which combines specifically with bFGF is thus 
obtained. 
The monoclonal antibody of the present invention combines specifically with 
the immunogen peptide bFGF. The monoclonal antibody of the present 
invention may also combine with a bFGF other than the immunogen peptide. 
The monoclonal antibody of the present invention is a monoclonal antibody 
to the immunogen peptide bFGF or its mutein. As the present monoclonal 
antibody reacts with only bFGF (and its mutein), the present monoclonal 
antibody combines specifically 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 specifically with bFGF, the monoclonal antibody of the 
present invention is very useful as a reagent for bFGF assay. It also 
facilitates bFGF assay in living organs and tissues, so it is very useful 
also in obtaining basic information about bFGF (e.g., distribution in 
vivo). In the detection procedure on the bFGF in living organs and 
tissues, the measuring by EIA method or fluorescent antibody technique are 
generally employed. In order to measure the amount in the living organs 
and tissues, it is always employed Western blotting method on protein. In 
this method, a crude extract or partial purified sample of the extract is 
subjected to electrophoresis with acylamide gel, transferring to membrane 
filter, and then detection with HRP-anti bFGF antibody. 
In addition, it is thought that 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 
proliferation-promoting bFGF is neutralized, and the antibody is expected 
to exhibit cancer cell proliferation inhibition, that is, to act as an 
anticancer substance. In addition, the antibody can be used to determine 
the bFGF in bFGF-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 affinity column can be prepared to use the antibody 
as a reagent for bFGF purification. 
As the EIA method and RIA method for detecting or measuring bFGF, there are 
mentioned the following procedure. 
For example, a purified antibody is fixed on 96 wells plastic plate (e.g. 
Immunoplate, Nunc, Denmark) at about 0.1 to 10 .mu.g/well, glass beads, 
plastic beads. The fixation is carried out at about 4.degree. C. for 
overnight, or at room temperature for about 0.4 to 4 hours, in case of 
plastic. In case of glass beads, the fixation is carried out in accordance 
with the method described in Proc. Natl. Acad. Sci. USA, 80, 
3513.times.3516 (1983). Thus obtained plate or beads to which the antibody 
has been fixed is subjected to adsorption reaction with the antigen bFGF 
(or its mutein). The adsorption reaction is generally carried out at room 
temperature for about 0.2 to 2 hours, preferably about 4.degree. C. 
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 RIA. As the enzyme 
to label an antibody, there are exemplified by horse radish peroxidase 
(HRP), alkaline phosphatase. As the examples of radioisotope labeling, 
there are mentioned .sup.125 I. 
In case of EIA, a substrate such as 2,2'-adino-di(3-ethylbenzothiazoline 
sulfonate (6)) is employed for coloring when HRP is employed as a labeling 
enzyme. 
In case of RIA, 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. 
As the EIA method, there are mentioned sandwich EIA method, competitive EIA 
method, indirect EIA method. In the sandwich method, two antibodies are 
bound by mediating the antigen, bFGF. In the competitive EIA method, an 
antibody is fixed to a carrier, antigen bFGF which has been combined with 
a labeling enzyme or radioisotope, and a sample, so as to react 
competitively, and then measuring the amount of labeled ant/gem 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 fixed) 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 purification, efficient purification can be 
achieved by, for example, the method in which the purified relevant 
antibody, after 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 containing bFGF such as culture supernatant or disrupted 
bacterial cells is adsorbed to the antibody affinity column, and the 
column is washed, whereafter bFGF is eluted with a chaotropic reagent such 
as KSCN (potassium thiocyanate) 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, purified 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 efficient adsorption 
specifically of bFGF after coupling, and enables the appropriate elution 
of bFGF after the adsorption; as the 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-10 is used as appropriate in the 
manner as described below. The reaction between Affi-Gel- 10 and 
antibodies is carried out in a buffer such as a solution of about 0.001 to 
1M, preferably about 0.1M bicarbonate. As to reaction conditions, the 
reaction can be carried out at about 0.degree. to 20.degree. C. for about 
10 to 24 hours at any pH level, but about 4.degree. C., about 4 hours, and 
pH about 3 to 10 is preferably used for reaction conditions. Since more 
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 Affi-Gel-10 and antibodies to be mixed 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 final concentration of about 0.05 to 0.1M of a compound 
having a primary amino group such as ethanolamine hydrochloric acid or 
glycine is added and reaction is carried out at about 4.degree. 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 4.degree. C. 
overnight. 
In purification using the above-mentioned antibody column, for example a 
sample containing bFGF is dissolved in a buffer such as a phosphate buffer 
or a Tris hydrochloric acid and is adsorbed to the antibody column. 
Thereafter, the column is washed with the same buffer, and bFGF is then 
eluted. As eluents, there can be used slightly acidic solutions such as 
acetic acid solutions, solutions containing polyethylene glycol, solutions 
containing peptide 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 buffer 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 95% (w/w). 
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 added stabilizers such as sorbitol, mannitol, 
dextrose, maltose and glycerol. 
The hbFGF thus obtained possesses growth promoting activity of fibroblast 
cells and endothelial cells and angiogenic activity, and its toxicity is 
low; therefore, the hbFGF can be used 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 hbFGF 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 glucose 
and/or other adjuvant or adjuvants. Tablets, capsules and other 
pharmaceutical compositions can also be prepared in accordance with a 
conventional method. When prepared for 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, for example, to 
the above warm-blooded mammals in an appropriate amount selected from the 
range of from about 1 ng to 100 .mu.g/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 .mu.g, more preferably 0.1 to 10 .mu.g per liter 
of medium. The mutein of 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 t/me of use, for example, 
antiserum, is saved. Furthermore, bFGF purification 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 IU-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 specified, in the L form. 
DNA: Deoxyribonucleic acid 
cDNA: Complementary deoxyribonucleic acid 
A: Adenine 
T: Thymine 
G: Guanine 
C: Cytosine 
RNA: Ribonucleic acid 
dATP: Deoxyadenosine triphosphate 
dTTP: Deoxythymidine triphosphate 
dGTP: Deoxyguanosine triphosphate 
dCTP: Deoxycytidine triphosphate 
ATP: Adenosine triphosphate 
Tdr: Thymidine 
EDTA: Ethylenediaminetetraacetic acid 
SDS: 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: Arginine 
His: Histidine 
Phe: Phenylalanine 
Tyr: Tyrosine 
Trp: Tryptophan 
Pro: Proline 
Ash: 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 for Y in the 
abovementioned amino acid sequence (II), said Met is numbered as the 
first.

EXAMPLES 
The present invention 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 coli x1776 as the host as produced by 
inserting cDNA synthesized from human foreskin derived primary culture 
cell mRNA into the pCD vector (Okayama et al.: Molecular and Cellular 
Biology, 3, 280 (1983)) was provided by Dr. Okayarea at the National 
Institute of Child Health and Human Development, Bethesda, USA. The 
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 Escherichia coli DH1 was infected with this DNA. A 
cDNA library comprising about 2.times.10.sup.6 clones was thus produced 
with Escherichia coli DH1 as the host. 
The above cDNA library with Escherichia coli DH1 used therein was plated on 
10 pieces of nitrocellulose filter (Millipore's HATF filter) in an amount 
of about 5.times.10.sup.4 clones per filter. Using these filters as master 
filters, 20 replica filters were prepared in 10 pairs corresponding to the 
master filters. Escherichia coli cells on these replica filters were lysed 
with a 0.5N NaOH solution and plasmid DNAs exposed and denatured were 
immobilized on the filters (Grunstein, M. & Hogness, D. S.: Proc. Natl. 
Acad. Sci. USA, 72, 3961 (1975)). 
Based on the amino acid sequence of bovine basic fibroblast 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 covering 
amino acid Nos. 13-20 (Pro-Pro-Gly-His-Phe-Lys-Asp-Pro) and amino acid 
Nos. 89-96 (Thr-Asp-Glu-Cys-Phe-Phe-Phe-Glu), respectively were chemically 
synthesized. (In some codons, the third letter was selected arbitrarily. 
Thus, the base sequences synthesized were 5' GG.sup.A /.sub.G TC.sup.T 
/.sub.C TT .sup.A /.sub.G AA.sup.A /G TGGCCAGGAGG and 5' TC.sup.A /G 
AA.sup.A /.sub.G AA.sup.A /.sub.G AA.sup.A /.sub.G CA.sup.T /.sub.C 
TCGTCGGT, with each underlined base being the one selected.) These 
oligonucleotides were labeled with .sup.32 p at the 5' end by treating 
said oligonucleotides in 50 .mu.l of reaction mixture [0.1 .mu.g of 
oligonucleotide, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl.sub.2, 10 mM 
mercaptoethanol, 50 .mu.Ci .lambda.-.sup.32 P ATP (&gt;5,000 Ci/mmole), 3 
units of T4 polynucleotide kinase (Takara Shuzo, Japan) at 37.degree. C. 
for 1 hour. 
The thus-labeled oligonucleotides of the above two kinds were individually 
hybridized as probes with the replica filters. The hybridization reaction 
was conducted in 10 ml of a 100 .mu.g/ml denatured salmon sperm DNA 
solution containing 10 .mu.Ci of probe in 5.times.SSPE (180 mM NaCl, 10 mM 
NaH.sub.2 PO.sub.4, 1 mM EDTA (pH 7.4)) and 5.times.Denhardt's with 0.1% 
SDS at 35.degree. C. for 16 hours. After reaction, the filters were washed 
with a 0.1% SDS solution in 5.times.SSC (0.15M NaCl, 0.015M sodium 
citrate) three times each at room temperature for 30 minutes and then two 
times each at 45.degree. C. for 30 minutes (T. Maniatis et al.: "Molecular 
Cloning", Cold Spring Harbor 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 DH1/pTB627 (IFO 14494, FERM 
BP-1280)) capable of reacting with the two kinds of probe was obtained 
from among 5.times.10.sup.5 colonies. 
(2) The plasmid DNA (pTB627) was extracted from the strain obtained above 
in (1) (Escherichia coli K12 DH1/pTB627 (IFO 14494, FERM BP-1280)) by the 
alkaline extraction method (Nucleic Acids 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 Example 2 
(Expression of hbFGF-encoding gene in Escherichia 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 AvaI 
and BalI, whereby a 0.44 kb DNA fragment containing the hbFGF-encoding 
region was obtained. A BglII linker, pCAGATCTG, was ligated with this DNA 
fragment at its Bali cleavage site (blunt end) by T4 DNA ligase, and a 
0.44 Kb AvaI-BglII DNA fragment was isolated. T4 DNA ligase was allowed to 
act on this 0.44 Kb AvaI-BglII fragment to thereby cause ligation between 
the BglII cleavage sites. Then, DNA polymerase (Klenow fragment) reaction 
was carried out in the presence of dXTPs to render the AvaI cleavage sites 
blunt. This DNA fragment was ligated with phosphorylated synthetic 
oligonucleotides, .sup.5' AATTCTATGCCAGCATTGC.sup.3' and .sup.5' 
GCAATGCTGGCATAG.sup.3', in the presence of T4 DNA ligase. An about 0.46 kb 
DNA fragment was then prepared by cleavage with EcoRI-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 DNA 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-BglII 
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 EcoRI-BgIII DNA fragment containing the hbFGF-encoding gene region 
by T4 DNA ligase reaction, whereby an hbFGF expression plasmid, pTB669, 
was constructed. 
This plasmid pTB669 was used to transform Escherichia coli DH1 to give a 
transformant carrying the plasmid pTB669, namely Escherichia coli 
DH1/pTB669. 
pTB669 was also used in the same manner to transform the Escherichia coli 
strains K12 MM294 and C600 to give Escherichia coli K12 MM294/pTB669 (IFO 
14532, FERM BP-1281) and E. coli C600/pTB669, respectively. 
Reference Example 3 
(Purification 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 .mu.g/ml 
tetracycline. When the Klett value reached about 200, 
3-.beta.-indolylacrylic acid was added to 25 .mu.g/ml, and the cultivation 
was continued for 4 more hours. Thereafter, cells were harvested and 
suspended in one twentieth volume of 10% sucrose solution in 20 mM 
Tris-HCl, pH 7.6. To this suspension were added phenylmethylsulfonyl 
fluoride (PMSF) to 1 mM (final concentration), EDTA to 10 mM, NaCl to 
0.1M, spermidine hydrochloride to 10 mM and lysozyme to 100 .mu.g/ml. 
After allowing to stand at 0.degree. C. 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. 
A 25-ml portion of this extract (as prepared from 500 ml of culture broth) 
was passed through a DEAE-cellulose (DE52, Whatman, England) column 
(diameter 2.times.10 cm) equilibrated with 0.2M NaCl solution in 20 mM 
Tris-HCl, pH 7.6 to thereby remove nucleic acid components in the extract. 
The effluent from the column and the column washings resultant from 
washing with 0.2M NaCl solution in 20 mM Tris-HCl, pH 7.6 were collected 
and combined (DEAE effluent fraction 44 ml). 
A 14-ml portion of this fraction was applied to a high performance liquid 
chromatograph (Gilson, France) equipped with a heparin column Shodex 
AF-pak HR-894 (8 mm ID.times.5 cm, Showa Denko, Japan). The column was 
washed with 20 mM Tris-HCl, pH 7.6. Thereafter, elution was performed on a 
linear gradient of 0.5-2M NaCl in 20 mM Tris-HCl buffer, pH 7.6, (eluent 
volume 60 ml, flow rate 1.0 ml/min). 
The hbFGF eluted by this procedure showed a single band in 
SDS-polyacrylamide gel electrophoresis, and it was thus found to be 
sufficiently purified and suitable for use as an antigen. The assay of 
hbFGF was conducted using the following conditions. 
A Nunc 96-well microtiter plate (fiat bottomed) was sown with mouse 
BALB/c3T3 cells (2.times.10.sup.3 cells per well) with DMEM medium 
containing 5% calf serum (0.2 ml per well) and the cells were cultured. 
Next day, the medium 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 DME medium containing 0.5% BSA 
were added in an amount of 10 .mu.l per well. After 20 hours of continued 
cultivation, 2 .mu.l of .sup.3 H-Tdr (5 Ci/mmol, 0.5 mCi/ml RCC Amersham) 
was added to each well. Six hours later, cells in each well were scraped 
off by treatment with phosphate buffer (PBS) containing 0.2% trypsin and 
0.02% EDTA and collected on a glass filter using a Titertek cell 
harvester, and the quantity of .sup.3 H-Tdr taken up by the cells was 
measured using a scintillation counter. 
Reference Example 4 
(Production of recombinant DNA having mutein-encoding base sequence) 
(1) Cloning of human bFGF gene M13 vector 
The plasmid pTB669 obtained in Reference Example 2 was digested with the 
restriction enzymes EcoRI and BamHI. Phage vector M13mp8 (J. Messing: 
Methods in Enzymology, 101, 20-78 (1983)) replicative form (RF) DNA was 
digested with the restriction enzymes EcoRI and BamHI. The DNA fragment 
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 Research, 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 dideoxynucleotide synthetic 
chain termination method (J. Messing et al.: Nucleic Acids Research, 9, 
309 (1981)), whereby it was confirmed that human bFGF DNA was accurately 
inserted. 
From this M13PO clone was purified 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: 
##STR3## 
(the primer for converting Cys 26 to Ser (the recognition sequence for 
restriction enzyme Rsa I disappears)) was treated with 9 units of T4 
kinase at 37.degree. C. for 1 hour in 50 .mu.l of a solution containing 
0.1 mM adenosine triphosphate (ATP), 50 mM hydroxymethylaminomethane 
hydrochloride (Tris-HCl), pH 8.0, 10 mM MgCl.sub.2 and 5 mM dithiothreitol 
(DTT). This kinase-treated primer (12 picomoles) was heated at 67.degree. 
C. for 5 minutes and then at 42.degree. C. for 25 minutes in 50 .mu.l of a 
mixture containing 50 mM NACl, 1.0 mM Tris-HCl, pH 8.0, 10 mM MgCl.sub.2 
and 10 mM .beta.-mercaptoethanol, whereby the primer was hybridized to 5 
.mu.g of single-stranded (ss)M13-PO DNA. After annealing, the mixture was 
cooled on ice, and was added to 50 .mu.l of a reaction mixture containing 
0.5 mM dideoxynucleotide triphosphate (dNTP), 80 mM Tris-HCl, pH 7.4, 8 mM 
MgCl.sub.2, 100 mM NaCl, 9 units of DNA polymerase I Klenow fragment, 0.5 
mM ATP and 2 units of T4 DNA ligase, and reaction was carried out at 
37.degree. C. for 3 hours and 25.degree. C. for 2 hours. The reaction was 
terminated by adding 2 .mu.l 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-purified 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) Si te-directed mutagenesis 
The procedure of the above term (2) was repeated, but the synthetic 
oligonucleotide primer used was 
##STR4## 
which converts cysteine 70 to serine (A recognition sequence for 
restriction enzyme HaeII is produced). 
(4) Site-directed mutagenesis 
The procedure of the above term (2) was repeated, but the synthetic 
oligonucleotide primer used was 
##STR5## 
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 
##STR6## 
which converts cysteine 93 to serine (A recognition sequence for 
restriction enzyme HinfI 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 cooled to 4.degree. C., and 
the plaques from each plate were transferred to two round nitrocellulose 
filters by superposing a dry filter on the agar plate for 5 minutes in the 
case of the first filter, or by superposing a dry filter for 15 minutes in 
the case of the second filter. Then, the filters were placed on a thick 
filter paper and immersed in a solution containing 0.2N NaOH, 1.5M NaCl 
and 0.2% Triton X-100, and then on a filter paper in a solution containing 
0.5M Tris-HCl, pH 7.5 and 1.5M NaCl for 5 more minutes, to thereby 
neutralize the filters. The filters were washed on a filter by immersing 
them twice in 2.times.SSC (standard sodium citrate). The filters were then 
allowed to dry in air, after which they were dried at 80.degree. C. in a 
vacuum oven for 2 hours. The duplicated filters were subjected to 
prehybridization at 55.degree. C. for 4 hours in 10 ml/filter DNA 
hybridization buffer (5.times.SSC), pit .times.Denhardt's solution 
(polyvinylpyrrolidone, Ficoll, and bovine serum albumin, 1.times.=0.02% 
for each)/0.1% sodium dodecylsulfate (SDS)/50 mM sodium phosphate buffer, 
pH 7.0/100 .mu.g/ml denatured salmon sperm DNA. The oligonucleotide primer 
was hybridized to 10.sup.5 cpm/ml at 42.degree. C. for 24 hours. The 
filters were washed in washing buffers containing 0.1% SDS and decreasing 
amounts of SSC at 50.degree. C. for 30 minutes for each wash. That is, the 
filters were washed first with the buffer containing 2.times.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 filters were washed until no detectable 
radioactivity remained on the control filters containing unmutated M13-PO 
plaques. The minimum SSC concentration used was 0.1.times.SSC. The filters 
were air-dried and exposed to film at -70.degree. C. 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 of the kinase-treated 
oligonucleotide probe. None of the control plaques hybridized to the 
probe, while 3 to 10 of the mutated M13-PO plaques hybridized to 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 AGC 
(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 had been 
converted to Ser, M13-P4. 
Reference Example 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 Example 4 was 
cleaved with the restriction enzymes EcoRI and PstI to thereby obtain an 
about 0.5 kb DNA fragment including the human bFGF mutein-encoding region. 
Separately, the trp promoter-containing plasmid ptrp781 (Kurokawa, T. et 
al.: Nucleic Acids Res., 11, 3077-3085 (1983)) was cleaved with 
EcoRI-PstI, 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 plasmid, pTB739, was constructed. 
This plasmid pTB739 was used to transform Escherichia coli DH1 to give a 
transformant carrying the plasmid pTB739 containing the mutein-encoding 
gene, namely Escherichia coli DH1/pTB739 (IFO 14575, FERM BP-1641). 
(2) Preparation of cell extract 
The above transformant was cultivated in M9 medium containing 1% glucose, 
0.4% casamino acid and 8 .mu.g/ml tetracycline. When the Klett value was 
about 200, 3-.beta.-indolylacrylic acid was added to 25 .mu.g/ml, and the 
cultivation was continued for 4 more hours. Thereafter, cells were 
harvested and suspended in one twentieth volume of 10% sucrose solution in 
20 mM Tris-HCl, pH 7.6. To this suspension were added phenylmethylsulfonyl 
fluoride (PMSF) to 1 mM (final concentration), EDTA to 10 mM, NaCl to 
0.1M, spermidine hydrochloride to 10 mM and lysozyme to 100 .mu.g/ml. 
After allowing to stand at 0.degree. C. 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) Human bFGF activity of cell extract 
A Nunc 96-well microtiter plate (fiat bottomed) was sown with mouse 
BALB/c3T3 cells (2.times.10.sup.3 cells per well) with DMEM medium 
containing 5% calf serum (0.2 ml per well) and the cells were cultured. 
Next day, the medium 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 DME medium containing 0.5% BSA 
were added in an amount of 10 .mu.l per well. After 20 hours of continued 
cultivation, 2 .mu.l of .sup.3 H-Tdr (5 Ci/mmol, 0.5 mCi/ml RCC Amersham) 
was added to each well. Six hours later, cells in each well were scraped 
off by treatment with phosphate buffer (PBS) containing 0.2% trypsin and 
0.02% EDTA and collected on a glass filter using a Titertek cell 
harvester, and the quantity of .sup.3 H-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 
(Expression 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 EcoRI and PstI 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 3.2 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 abovementioned 0.5 kb 
EcoRI-PstI DNA fragment containing a gene region encoding a human bFGF 
mutein were ligated together by T4 DNA ligase reaction to construct the 
plasmid pTB742 for the expression of a human bFGF mutein. 
Using this plasmid pTB742, Escherichia coli DH1 was transformed, whereby 
the strain Escherichia coli DH1/pTB742 (IFO ].4584, FERM BP-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) Human bFGF activity of the bacterial cell extract: 
A determination was made of the human bFGF activity on the bacterial cell 
extract obtained in (2) above, by the method described in Reference 
Example 5 (3). 
The bacterial cell extract of E. coli DH1/pTB742 thereby tested exhibited 
FGF activity. 
The mutein CS2, in which Cys at the 70-position of human bFGF had been 
replaced by Set, was thus obtained. 
Reference Example 7 
(Expression in Escherichia coli of gene encoding human bFGF mutein) 
(1) Construction of the plasmid pTB743 for human bFGF mutein expression: 
The M13-P3 replicative form (RF) Obtained in Reference Example 4 above was 
cleaved using the restriction enzymes EcoRI and PstI 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 3.2 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 abovementioned 0.5 kb 
EcoRI-PstI DNA fragment containing a gene region encoding a human bFGF 
mutein were ligated together by T4 DNA ligase reaction to construct the 
plasmid pTB743 for the expression of human bFGF mutein. 
Using this plasmid pTB743, Escherichia coli DH1 was transformed, whereby 
the strain Escherichia coli DHI/pTB743 (IFO 14585, FERM BP-1643) was 
obtained, which harbors the plasmid pTB743 containing the mutein-encoding 
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) 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 
Example 5 (3). 
The bacterial cell extract of E. coli DH1/pTB743 thereby tested exhibited 
FGF activity. 
The mutein CS3, in which Cys at the 88-position of human bFGF had been 
replaced by Ser, was thus obtained. 
Reference Example 8 
(Expression in Escherichia coli of gene which encodes human bFGF mutein) 
(1) Construction of the plasmid pTB744 for human bFGF mutein expression: 
The M13-P4 replicative form (RF) obtained in Reference Example 4 above was 
cleaved using the restriction enzymes EcoRI and PstI 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 EcogI-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 abovementioned 0.5 kb 
EcogI-PstI DNA fragment containing a gene region encoding a human bFGF 
mutein were ligated together by T4 DNA ligase reaction to construct the 
plasmid pTB744 for the expression of a human bFGF mutein. 
Using this plasmid pTB744, Escherichia coli DH1 was transformed, whereby 
the strain Escherichia coli DH1/pTB744 (IFO 14586, FERM BP-1644) was 
obtained, which harbors the plasmid pTB744 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) 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 
Example 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 Set 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 4.degree. C., and the 
plaque from each plate was transferred to 2 round nitrocellulose filters 
by keeping a dry filter placed 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 5 minutes on thick filter papers 
immersed in 0.2N NaOH, 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 0.5M Tris-HCl (pH 7.5) and 1.5M NaCl. The filters were 
twice washed on filters immersed in 2.times.SSC (standard sodium citrate) 
in the same manner, and were allowed to dry, and this was followed by 
drying at 80.degree. C. for 2 hours in a vacuum oven. The overlapped 
filters were subjected to prehybridization at 55.degree. C. for 4 hours 
with 10 ml/filter of a DNA hybridization buffer solution (5.times.SSC) 
having a pH-value of 7.0 containing 4.times.Denhardt's solution 
(polyvinylpyrrolidone, Ficoll and bovine serum albumin, 1.times.=0.02%), 
0.1% sodium dodecyl sulfate (SDS), 50 mM sodium phosphate-buffered 
solution having a pH-value of 7.0 and 100 .mu.g/ml denatured salmon sperm 
DNA. Hybridization was carried out at 42.degree. C. for 24 hours with 
10.sup.5 cpm/ml of an oligonucleotide primer. The filters were each washed 
in a buffer solution for washing containing 0.1% SDS and a reduced amount 
of SSC at 50.degree. C. for 30 minutes. The filters were then first washed 
with a buffer solution containing 2.times.SSC; the control filters, 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.times.SSC. The 
filters were allowed to dry in air, and radioautographs were taken by 2 to 
3 days of exposure at -70.degree. C. 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-P 2 plaques 
hybridized to the probe. 
One of the mutated M13-P2 plaques was taken, and was inoculated to a JM105 
culture medium. From the resulting supernatant an ssDNA was prepared, and 
from the bacterial cell pellets a double-stranded (ds) DNA was prepared. 
Analyses were made of the base sequences using appropriate oligonucleotide 
primers and ssDNAs. 
As a result, it was respectively confirmed that the TGC (Cys-26) codon had 
been changed to a TCT (Ser) codon; the TGT (Cys-88) codon, to a TCT (Set) 
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 Ser-encoding codons was named M13-P12; the phage in which the 
codons Cys-70 and-88 had become Seroencoding codons, M13-P23; and the 
phage in which the codons Cys-70 and-93 had become Ser-encoding codons, 
M13-P24. 
Reference 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 Reference Example 5 (1) to construct 
the plasmid pTB762 for human bFGF mutein expression. 
Using this plasmid pTB762, Escherichia coli MM294 was transformed, whereby 
the strain Escherichia coli MM294/pTB762 (IFO 14613, FERM BP-1645) was 
obtained, which harbors the plasmid pTB762 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) 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 
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 obtained. 
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 EcoRI and BamHI. Phage vector M13mp8 (J. Messing, 
Methods in Enzymology, 101, 20.about.78 (1983)) replicative form (RF) DNA 
was mixed with a human bFGF DNA fragment derived from pTB669, previously 
digested with EcoRI and BamHI. The mixture was then subjected to ligation 
using T4 DNA ligase. The resulting ligated DNA was transformed into 
infectable 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 DNA 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-specific mutagenesis: 
In the presence of 0.1 mM adenosine triphosphate (ATP), 50 mM 
hydroxymethylaminomethane hydrochloride (Tris-HCl) having a pH-value of 
8.0, 10 mM MgCl.sub.2, 5 mM dithiothreitol (DTT) and 9 units of T4 kinase, 
in a total amount of 50 .mu.l, 40 picomoles of the synthetic 
oligonucleotide: 
EQU 5'-CGGGCATGAATTCGCCGCT-3' 
(primer for producing in the base sequence a recognition site for the 
restriction enzyme EcoRI, and subsequently changing Pro-14 to Met) was 
treated with T4 kinase at 37.degree. C. for 1 hour. This kinase-treated 
primer (12 picomoles) was heated at 67.degree. C. for 5 minutes, and at 
42.degree. C. for 25 minutes, in 50 .mu.l of a mixture containing 50 mM 
NaCl, 1.0 mM Tris-HCl having a pH-value of 8.0, 10 mM MgCl.sub.2 and 10 mM 
.beta.-mercaptoethanol, whereby it was hybridized to 5 .mu.g of the 
single-stranded (ss) M13-PO DNA. The annealed mixture was then cooled on 
ice, and was added to 50 .mu.l of a reaction mixture containing 0.5 mM 
deoxynucleotide triphosphate (dNTP), 80 mM Tris-HCl having a pH-value of 
7.4, 8 mM MgCl.sub.2, 100 mM NaCl, 9 units of DNA polymerase I Klenow 
fragment, 0.5 mM ATP and 2 units of T4 DNA ligase, and reaction was 
carried out at 37.degree. C. for 3 hours, and at 25.degree. C. for 2 
hours, whereafter the reaction was stopped by adding 2 .mu.l 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-purified RF-type DNA 
was transformed into infectable JM 105 cells; the resulting transformant 
cells were spread over an agar plate, and were cultured overnight to 
obtain phage plaques. 
(3) Site-directed mutagenesis: 
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 NcoI, 
and concurrently changes Gly-9 to Thr and Ser-10 to Met, respectively: 
(4) Site-directed mutagenesis: 
The procedure of the above term (2) was repeated but the synthetic 
oligonucleotide primer used was: 5'-TAACACCTAAGAAGCCAG-3' which produces 
in the base sequence a recognition site for the restriction enzyme All II, 
and concurrently changes the Lys-87-encoding codon to a termination codon. 
(5) Site-directed mutagenesis: 
The procedure of the above term (2) was repeated but the synthetic 
oligonucleotide primer used was: 5'-CCGGACTCCGTTAACTCGG-3' which produces 
in the base sequence a recognition site for the restriction enzyme HpaI, 
and concurrently changes Asp-42 to Ash. 
(6) Site-directed mutagenesis: 
The procedure of the above term (2) was repeated but the synthetic 
oligonucleotide primer used was: 5'-CTTCTCCTGGACTCCGTCAAC-3' which deletes 
the recognition site for the restriction enzyme HpaII in the base 
sequence, and concurrently changes Arg-45 to Gln. 
(7) Screening and identification of plaques which were mutagenic: 
Plates containing mutated M13-PO plaques (above term (1)) and 2 plates 
containing unmutated M13-PO phage plaques were cooled to 4.degree. C., and 
the plaques from each plate were transferred to 2 round nitrocellulose 
filters by keeping a dry filter placed 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 5 minutes on thick filter 
papers immersed in 0.2N NaOH, 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 0.5M Tris-HCl having a pH-value of 7.5 and 1.5M NaCl. 
The filters were twice washed on filters immersed in 2.times.SSC (Standard 
Sodium Citrate) in the same manner, and were allowed to dry, and this was 
followed by drying at 80.degree. C. for 2 hours in a vacuum oven. The 
overlapped filters were subjected to prehybridization at 55.degree. C. for 
4 hours with 10 ml/filter of a DNA hybridization buffer solution 
(5.times.SSC) having a pH-value of 7.0 containing 4.times.Denhardt's 
solution (polyvinylpyrrolidone, Ficoll and bovine serum albumin, 
1.times.32 0.02%), 0.1% sodium dodecyl sulfate (SDS), 50 mM sodium 
phosphate-buffered solution having a pH-value of 7.0 and 100 .mu.g/ml 
denatured salmon sperm DNA. Hybridization was carried out at 42.degree. C. 
for 24 hours with 10.sup.5 cpm/ml of an oligonucleotide primer. Each 
filter was washed at 50.degree. C. for 30 minutes in a buffer solution for 
washing containing 0.1% SDS and a reduced amount of SSC. The filters were 
then first washed with a buffer solution containing 2.times.SSC; the 
control filters, 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.times.SSC. The 
filters were allowed to dry in air, and autoradiographs were taken by 2 to 
3 days of exposure at -70.degree. C. Screening was carried out of 10,000 
mutated M13-PO plaques and 100 unmutated control plaques by means of an 
oligonucleotide probe treated with 32P-.gamma.-ATP. None of the control 
plaques hybridized to the probe, while 3 to 10 of the mutated M13-PO 
plaques hybridized to the probe. 
One of the mutated M13-PO plaques was taken, and was inoculated to a JM105 
culture medium. From the resulting supernatant an ssDNA was prepared, and 
from the bacterial cell pellets a double-stranded (ds) DNA was prepared. 
Analyses were made of the base sequences using appropriate oligonucleotide 
primers and ssDNAs. 
As a result, it was respectively confirmed that the GGC (Gly-9) codon had 
been changed to an ACC (Thr) codon and the AGC (Ser-10) codon had been 
changed to an ATG (Met) codon; the CCG (Pro-14) codon, to an ATG (Met) 
codon; the AAA (Lys-87) codon, to a TAA (termination) codon; the GAC 
(Asp-42) codon, to an AAC (Ash-42) codon; and the CGG (Arg-45) codon, to a 
CAG (GIn-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 had become a Met-encoding codon 
was named M13-PN 10; 
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 codon had become an Asn-encoding codon, 
M13-PDN42; and, 
the phage in which the Arg-45 codon had become a GIn-encoding codon, 
M13-PRQ45. 
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 (RF) obtained in Example 11 above was treated 
in the manner described in Reference Example 5 (1) to construct the 
plasmid pTB795 for human bFGF mutein. 
Using this plasmid pTB795, Escherichia coli MM294 was transformed, whereby 
the strain Escherichia coli MM294/pTB795 (IFO 14700, FERM BP-1660) was 
obtained, which contains the plasmid pTB795 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) 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 
Example 5 (3). 
The bacterial cell extract from E. coli MM294/pTB795 thereby tested 
exhibited FGF 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 had 
been deleted, was thus obtained. 
Reference Example 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 MM294/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-mentioned 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 87position to Ser 
at the 147-position had been deleted, and the supernatant was then used 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 5 (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, FERM BP-1662) was 
obtained, which harbors the plasmid pTB797 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) 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 
Example 5 (3). 
The bacterial cell extract from E. coli MM294/pTB797 thereby tested 
exhibited FGF activity. The mutein DN42, in which Asp at the 42-position 
of human bFGF had been replaced by Ash, was thus obtained. 
Reference Example 15 
(Expression in Escherichia coli of gene which encodes human bFGF mutein) 
(1) Construction of the plasmid pTB855 for human bFGF mutein expression: 
The DNA of the plasmid pTB669 which was obtained in the above mentioned 
Reference Example 2 was cleaved with a restriction enzyme HincII, and it 
was ligated with EcoRI linker p(5' CATGAATTCATG 3') under T4 DNA ligase 
reaction. Thus obtained DNA was further cleaved with a restriction enzymes 
EcoR I and PstI to recover a DNA 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 EcoR I-PstI, to obtain the plasmid pTB855 for human 
bFGF mutein expression was constructed. 
Using this plasmid 855, Escherichia 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 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/pTB855 thereby tested 
exhibited FGF activity. The mutein N41, in which the amino acid sequence 
of from Pro at the 2-position to Val at the 41-position of human bFGF had 
been deleted, was thus obtained. 
Reference Example 16 
(Expression in Escherichia coli of gene which encodes human bFGF mutein) 
(1) Construction of the plasmid pTB856 for 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 BamHI so 
as to obtain BamHI recognition site in the bFGF gene. The site was further 
cleaved with Escherichia coli DNA polymerase I in the presence of dATP, 
dCTP, dGTP, dTTP 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 cleaved site under T4 DNA 
ligase reaction, the plasmid pTB856 for human bFGF mutein expression was 
constructed. 
Using this plasmid pTB856, Escherichia coli MM294 was transformed, whereby 
the strain Escherichia coli MM294/pTB856 which contains the plasmid pTB856 
having the mutein-encoding gene. 
(2) Preparation of bacterial cell extract: 
The above-mentioned transformant was cultured by the method as in Reference 
Example 5 (2) to give a supernatant which contains the mutein C129, in 
which the amino acid sequence of from Lys at the 130-position to Ser at 
the 147-position had been deleted, was thus obtained, and then the 
supernatant which was then used as a bacterial cell extract. 
Reference Example 17 
(Production of H-Leu-Pro-Met-Ser-Ala-Lys-Ser-OH) 
Boc-Ser(Bzl)-resin (696 rag, 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 
to cause condensation reaction: 
Boc-Lys(Z)-OH, Boc-Ala-OH, Boc-Ser(Bzl)-OH, Boc-Met-OH, Boc-Pro-OH, 
Boc-Leu-OH 
Bzl: benzyl 
Boc: t-butoxycarbonyl 
Z: benzyloxycarbonyl 
By said procedure, 1.08 g of 
Boc-Leu-Pro-Met-Ser(Bzl)-Ala-Lys(Z)-Ser(Bzl)-resin was produced. 400 mg of 
the peptide-resin was incubated in 5.0 ml of hydrogen fluoride containing 
0.5 ml of anisole and 0.5 ml of dimethylsulfide at 0.degree. C. for 60 
minutes to recover the peptide from resin. The excess amount of hydrogen 
fluoride was removed by distillation under reduced pressure to give a 
residue. The residue was washed with d/ethyl ether, and extracted with 30 
ml of water, and lyophilized. The lyophylizate was dissolved in 5 ml of 
water, and the solution was subjected to ion-exchange employing Amberlite 
IRA-400 (acetate form) resin (column 2.times.5 cm, elution solvent: 
water). The eluate was concentrated under reduced pressure, and subjected 
to gel filtration with Sephadex LH-20 (Pharmacia, column 2.5.times.125 cm, 
elution solvent: 1N acetic 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) [.alpha.].sup.25.sub.D -8.1 (c=0.11, 1N acetic acid) Amino 
acid analysis: Ser. 2.08, Pro 1.06, Ala 1.00, Met 0.98, Leu 1.03, Lys0.99. 
Example 1 
(Immunization) 
BALB/c mice (female, 4-week old) had the antigen human bFGF (as obtained in 
Reference Example 3) in solution in 0.4 ml of Freund's complete adjuvant 
(Difco Laboratories, USA) injected intraperitoneally. Three weeks later, 
10 .mu.g of the antigen hbFGF in solution in 0.4 ml of Freund's incomplete 
adjuvant was intraperitoneally administered. 3 weeks later, the same 
additional immunization was carried out, and two weeks later, 10 .mu.g of 
human bFGF in saline was intraperitoneally inoculated. 
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 Iscove's medium and Ham Of12 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 
containing 10% fetal bovine serum under an atmosphere of 5% carbon dioxide 
and 95% air. 
Cell fusion was conducted in accordance with the method established by 
Kohler and Milstein (Kohler, G. and Milstein, C.: Nature, 256,495 (1975)). 
2.9.times.10.sup.7 cells of the above myeloma cell line and 
1.5.times.10.sup.8 immunized lymphocytes obtained by the above-mentioned 
method were mixed together and centrifuged, and 45% polyethylene glycol 
6000 (hereinafter referred to as PEG 6000) in 0.3 ml of IH medium was 
dropwise added. The PEG 6000 solution was preheated to 37.degree. C., and 
was gradually added. Five minutes later, the 37.degree. C.-preheated IH 
medium was added at a rate of 0.5 ml per minute to make 10 me. The 
solution was then centrifuged at room temperature at 600 rpm for 15 
minutes, and the supernatant was removed. This cell precipitate was 
suspended in 200 ml of IH medium containing 20% calf serum, and this 
suspension was transferred to a 24-well microplate (Linbro) in an amount 
of 2 ml per well. One day later, IH medium (containing 20% calf serum) 
supplemented with HAT (1.times.10.sup.-4 M hipoxanthine, 4.times.10.sup.-7 
M aminopterin, 1.6.times.10.sup.-5 M thymidine) (hereinafter referred to 
as HAT medium) was added to the microplate in an amount of 1 ml per well, 
and, a further three day, one half amount of the medium was replaced with 
HAT 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 
.mu.l 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, the 
horse radish peroxidase (HRP)-labeled anti-mouse IgG goat antibody (Miles) 
was added as the secondary antibody, and incubated at room temperature for 
2 hours. The secondary antibody was removed, and, after thoroughly washing 
the wells, coloring reaction was carried out in the presence of added 
reaction substrate (ErA method). By this method potent valency was 
observed in 3 wells. 
(3) 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 HbF99 cell (IFO 50122), the mouse HbF161 
cell (IFO 50123) and the mouse HbF165 cell (IFO 50124) were obtained. 
The results of the determination of antibody titers in supernatants of 
these cell lines are shown in Table 2. 
TABLE 2 
______________________________________ 
Culture Supernatant bFGF- 
HbF HbF HbF Parent Line 
Immunized Mouse 
Dilution 
99 161 165 Myeloma Cell 
Serum 
______________________________________ 
X 64 1.93 1.08 0.66 0.02 -- 
X 128 1.72 0.63 0.51 0.02 -- 
X 3200 -- -- -- -- 1.93 
X 6400 -- -- -- -- 1.25 
______________________________________ 
Note: 
The numerical figures in the above Table 1 represent absorbances at 492 n 
wavelength; blank (--) means that determination was not made. 
The cloned cells were stored in IH medium containing 20% calf serum and 
having dimethylsulfoxide (DMSO) added thereto to 10% under liquid nitrogen 
atmosphere. 
Example 3 
(Immunoglobulin class of monoclonal antibodies) 
The mouse antibodies obtained in Example 2 were reacted with various 
immunoglobulin standards by means of the Mouse-Typer subisotyping kit 
(Bio-Rad). The results are presented in Table 3. 
TABLE 3 
______________________________________ 
Immuno- Monoclonal 
globulin Antibodies According to the Invention 
Standard MoAb99 MoAb161 MoAb165 
______________________________________ 
IgG 1 - - - 
IgG 2a - - - 
IgG 2b - - - 
IgG 3 - - - 
IgM + + + 
IgA - - - 
______________________________________ 
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), HbF45, HbF47, HbF52 (IFO 50143), HbF78 
(IFO 50144) and HbF98 (IFO 50145) were obtained. 2.times.10.sup.6 cells 
each hybridoma were intraperitoneally inoculated to mice to which 0.5 ml 
of mineral oil were preinjected. 
After 10 days, 2 to 4 ml of ascites per mouse were collected, and 
monoclonal antibodies MoAb12, MoAb45, MoAb47, MoAb52, MoAb78 and MoAb98 
were obtained, from said hybridomas, respectively, in accordance with the 
method described in Example 2 (4). 
By the method described in Example 3', determinations were made of the 
immunoglobulin class of said monoclonal antibodies, whereby the following 
results shown in Table 4 were obtained. 
TABLE 4 
______________________________________ 
Immunoglobulin 
Monoclonal Antibody Class 
______________________________________ 
MoAb12 IgG 1 
MoAb45 IgG 1 
MoAb47 IgM 
MoAb52 IgG 2b 
MoAb78 IgG 2b 
MoAb98 IgG 1 
______________________________________ 
Example 5 
(1) Preparation of radiolabeled hbFGF 
Using the transformant Escherichia coli MM294/pTB669 (IFO 14532, FERM 
BP-1281) described in Reference Example 2, hbFGF radiolabeled with .sup.35 
S was obtained in the following manner. 
The above Escherichia coli MM294/pTB669 was cultivated in the medium 
described in Reference Example 3 until the Klett value was 200. This 
culture broth in the one fifth amount was poured into M9 (Met.sup.-) 
medium. The M9 (Met.sup.-) medium was prepared by supplementing the M9 
medium containing 1% glucose, 8 .mu.g/ml tetracycline, and the amino acid 
shown below: 
______________________________________ 
Amino acid composition 
______________________________________ 
L-alanine 25.0 mg/l 
L-arginine hydrochloride 
84.0 mg/l 
L-asparagine monohydrate 
28.4 mg/l 
L-aspartic acid 30.0 mg/l 
L-cysteine disodium salt 
82.8 mg/l 
L-glutamic acid 75.0 mg/l 
L-glutamine 584.0 mg/l 
L-glycine 30.0 mg/l 
L-histidine hydrochloride monohydrate 
42.0 mg/l 
L-isoleucine 105.0 mg/l 
L-leucine 105.0 mg/l 
L-lysine hydrochloride 146.0 mg/l 
L-phenylalanine 66.0 mg/l 
L-proline 40.0 mg/l 
L-serine 42.0 mg/l 
L-threonine 95.0 mg/l 
L-tyrosine 83.9 mg/l 
L-valine 94.0 mg/l 
______________________________________ 
Cultivation was carried out in the M9 (Met.sup.-) medium until the Klett 
value was 200, and 3-.beta.-indoleacrylic acid was added to 25 .mu.g/ml, 
and the cultivation was continued for 2 more hours. Thereafter, a 1-ml 
portion of the culture broth was collected, and 10 .mu.Ci of .sup.35 S-Met 
(specific activity &gt;1000 Ci/mmol) 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 A (BRL) was prepared in accordance with the 
instruction manual thereof. An unlabeled Escherichia coli cell extract was 
obtained by treating Escherichia coli MM294/ptrp781 by the method of 
Reference Example 3. 
One milliliter of the ascites fluid obtained in Example 4 was mixed with 
100 .mu.l of the unlabeled Escherichia coli cell extract, and the mixture, 
after being allowed to stand at 4.degree. C. for 1 hour, had 10.sup.6 cpm 
of the labeled cell extract (MM294/ptrp781 or MM294/pTB669) added thereto, 
and was allowed to stand at 4.degree. C. overnight. 
To 100 .mu.l of the 10% solution of Protein A, was added 100 .mu.l of the 
unlabeled Escherichia coli cell extract, and the mixture, after being 
allowed to stand at 4.degree. C. overnight, was centrifuged and again 
suspended in 100 .mu.l 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 4.degree. C. overnight. This 
mixture was then centrifuged, and the resulting precipitate was suspended 
in 500 .mu.l of NETBN solution. This procedure was repeated 5 times to 
thereby remove unadsorbed labeled substance, and the pellets were 
resuspended with 50 ml of electrophoresis sample buffer. The 
polyacrylamidegel electrophoresis was performed in accordance with the 
method of Laemmli, U. K. (Nature, 227, 680 (1970)). After migration, the 
gel was immersed in 50% trichloroacetic acid (TCA) for 1 hour and was 
washed four times with distilled water for 30 minutes for each wash to 
thereby remove the TCA, after which the gel was immersed in 
dimethylsulfoxide (DMSO) for 1 hour. Thereafter, 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 for 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 MoAb12, MoAb52, MoAb78 and MoAb98 were found to 
combine with hbFGF in cell extract. 
Example 6 
The antibody valencies of the 4 lines which showed immunoprecipitation in 
Example 5, selected from the monoclonal antibodies described in Example 4, 
namely the monoclonal antibodies MoAb 12, MoAb 52, MoAb 78 and MeAb 98 
were determined by the limiting dilution method. 
That is, each of the ascites fluids of monoclonal antibody MoAb 12, MoAb 
52, MoAb 78 or MoAb 98 obtained in Example 4 was diluted with IH medium 
containing 10% fetal bovine serum, and the quantity of antibodies in the 
dilution was determined by the EIA method mentioned in Example 2 (2). The 
results are shown in FIG. 3. In FIG. 3, - - - - .largecircle. - - - - 
indicates the results for MoAb 12,-- - -- ---.quadrature.-- - -- - 
--indicates the results for MoAb 52,-- -- indicates the results for MoAb 
78, and - - - - .gradient. - - - - indicates the results for MoAb98. 
FIG. 3 shows that the ascites containing the above antibody show a limiting 
dilution rate of more than 1.times.10.sup.6, that is, these 4 antibodies 
are very high in antibody valency. 
Example 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 obtained 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: 
Leu-Pro-Met-Ser-Ala-Lys-Ser (which corresponds to 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 .mu.g/ml 
and diluted with IH medium (Iscove and Ham F12 mixed medium at a ratio 
with 1:1)containing 10% FCS. The extract containing the mutein N14or N41 
obtained in Reference Example 12 (2) or 15 (2) was diluted with IH medium 
containing 10% FCS. The antibodies, obtained in Example 4, were diluted to 
their binding titers showing between 0.7 and 1.0 with absorbance at 415 
nm. So, dilution factor about MoAb12, MoAb52 and MoAb98 was 
5.times.10.sup.5, and about MoAb98 was 5.times.10.sup.4. In the above 
dilution, IH medium containing 10% FCS was used as a diluent. To the 
diluted antibody solution were added the diluted competitor. After 
stirring the mixture, the diluent was warmed at 37.degree. C. 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 peptide 
are shown in FIGS. 4 to 7. FIG. 4 shows the results on the monoclonal 
antibody MoAb 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, -- --denotes the results of hbFGF, - - - - .largecircle. 
- - - - denotes the results of Pep 1, -- - -- - --denotes the results of 
Pep 2, these being used as competitor, and the vertical axis denotes the 
absorption at 415 mm. 
As shown in the FIGS. 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. 5 and FIG. 7, the monoclonal antibodies MoAb 52, MoAb 
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 Example (2) or N41 (obtained in Reference Example 15) by the 
method described in the above, and the results are shown in FIGS. 8 and 9. 
In these figures, -- --denotes the results of hbFGF, - - - - V - - - - 
denotes the results of the mutein N14, - - - - .largecircle. - - - - 
denotes the results of the 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 antibodies 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 
Reference 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 .mu.g/ml 80 ng/ml 10 .mu.g/ml 
80 ng/ml 
______________________________________ 
MoAb12 1.049 0.975 0.396 1.077 
MoAb52 0.997 0.923 0.230 0.722 
MoAb78 0.978 0.962 0.062 0.523 
MoAb98 0.948 0.921 0.095 1.356 
______________________________________ 
As shown in Table 5, the monoclonal antibodies MoAb 12, MoAb 52, MoAb 78 
and MoAb 98 do not cross-react with bovine aFGF. 
All of the above results are shown in the following Table 6. 
TABLE 6 
______________________________________ 
Monoclonal Antibody HbF 
12 52 78 98 
______________________________________ 
hbFGF + + + + 
baFGF - - - - 
N14 - + - + 
N41 - - - - 
Pep1 + - + - 
Pep2 - - - - 
______________________________________ 
In the Table 6, + denotes that they are competitive, and - denotes that 
they are not competitive. 
Example 8 
(Purification of the monoclonal antibody from ascites) 
The monoclonal antibody MoAb 12, MoAb 52, MoAb 78 or MoAb 98 was inoculated 
to 10 mice, and 20 to 30 ml of ascites were collected. The ascites were 
subjected to centrifugation at 2,000 rpm (Hitachi refrigrated centrifuge) 
to remove cells, and further subjected to centrifugation with Spinco SW 28 
roter (Beckman, USA) at 4.degree. C. 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. The precipitate was subjected to 
centrifugation with Sorval SS34 roter (Dupont, USA) at 4.degree. C., 
15,000 rpm. The pellet was dissolved in Buffer 1 (20 mM Tris. HCl (pH 
7.9), 40 mM NaCl) so that the protein concentration is 10 to 15 mg/ml. 
Thus obtained solution is dialyzed for the Buffer I at 4.degree. C. 
overnight. Thus obtained 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 Buffer 2 (0.1M NaHCO.sub.3) so as to the protein 
concentration being 10 to 20 mg/ml, and the solution was subjected to 
dialysis to Buffer 2 at 4.degree. C. for two overnights. The Buffer 2 were 
replaced every day. 
Furthermore, the dialyzate is subjected to hydroxy apatite column (HCA 
column). As the initiation buffer, 10 mM sodium phosphate buffer (pH 6.8) 
was used, and as the elution buffer, 500 mM sodium phosphate buffer 
(pH6.8) was used. The elution was carried out by linear gradient elution 
from the initial buffer to the elution buffer. Thus obtained eluate 
containing the antibody was preserved at 4.degree. C. 
Example 9 
(Purification of hbFGF by antibody column) 
5 ml of Affi-Gel-10 (Bio-Rad, USA) was put on sintered filter, was washed 
with ten volumes of ice-cooled isopropanol and with ten times of 
ice-cooled distilled water. Thus obtained gel is transferred to a reaction 
vessel. To the gel was mixed with 15 mg of monoclonal antibody MoAb 78 in 
the volume of 5 to 15 ml (dissolved in Buffer 2 or phosphate buffer, in 
Example 8) to react at 4.degree. C. overnight. 
Monoethanol amine (pH 8.0) was added to the reaction mixture 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 ml of the gel was packed into a column, and the 
column was equilibrated with the initiation buffer (20 mM Tris.HCl (pH 
7.6), 1 mM EDTA, 0.15M NaCl, 0.05% NP-40). 
On the other hand, the extracts of a transformant Escherichia coli 
DH1/pTB744 (the extracts containing mutein CS4), is diluted three times by 
the addition of the initial buffer. 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 ml of the initial 
buffer, and then the elution was carried out by using 20 ml of high salt 
buffer (20 mM Tris. HCl (pH 7.6), 1 mM EDTA, 1M NACl, 0.05% NP-40), 20 ml 
of an elution buffer A (0.2M acetate buffer (pH 4.5), 0.2M NaCl), 20 ml of 
an elution buffer B (0.2M acetic acid (pH 2.5), 0.2M NaCl) in that order, 
wherein the flow rate is 20 ml/hour and the temperature is 4.degree. C. 
Thus obtained fractions were subjected to electrophoresis in accordance 
with the method described in Laemmli, Nature 277, 680 (1970) employing 
17.25% acrylamide. Proteins were detected by silver staining. 
The results are shown in FIG. 10. In FIG. 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 buffer A, and E denotes that of the 
elution buffer B. Just after the elution, the pH of D fraction and E 
fraction was adjusted to pH 7.5 by adding 1M Tris. HCl (pH 9.5). The FGF 
activity on the fractions were measured in accordance with the method of 
Reference Example (3). The results are shown in Table 7. In Table 7, A to 
E denote the same as above. 
TABLE 7 
______________________________________ 
Protein (A) FGF Activity* (B) 
(.mu.g) (.mu.g) B/A 
______________________________________ 
A 63000 96 0.0015 
B 63000 19 0.0003 
C 28 0.003 0.0011 
D 17 0.049 0.0030 
E 33 23 0.69 
______________________________________ 
Note: 
*The FGF activity is shown in equivalent amount of the bovine pituitary 
derived bFGF purchased from Takara Shuzo, Japan) on the basis of 
incorporation of .sup.3 Hthymidine. 
Example 10 
(Measurement of hbFGF mutein by EIA method using monoclonal antibody) 
(1) The antibody MoAb 78 obtained in Example 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/ml, and 
subjected to dialysis in 0.2M sodium phosphate buffer (pH 7.0). To thus 
obtained 1.4 ml solution of monoclonal antibody MoAb 78 (concentration 2.8 
mg/ml), 50 .mu.l of S-acetylmercaptosuccinic anhydride (Aldrich Co., 
U.S.A) dissolved in N, N'-dimethylformamide was added so as to reach the 
concentration of 11.5 mg/ml. 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 minutes with 130 .mu.l of 0.2M Tris.HCl (pH 7.0), 13 .mu.l of 0.2M 
EDTA and 130 .mu.l of 2M hydroxyamine (pH 7.0). The MoAb 78 was recovered 
by gel filtration using a column packed with Sephadex G-25 (diameter 1 
cm.times.80 cm, Pharmacia, Sweden) (flow rate: 20 ml/hour). 
(2) 10 mg of horse radish peroxidase (HRP, Behringer Manheim, Grade I, West 
Germany) was dissolved in 1.4 ml 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 .mu.l of DMF, and 100 .mu.l of thus 
obtained solution was added to the HRP 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, proteins of the portion 
of HRP introduced with maleimide group were recovered by gel filtration 
using a colomn packed with Sephadex G-25 as in the above (f). 
(3) 6 ml of the portion of the monoclonal antibody MoAb 78 introduced with 
SH group obtained in the above (1) and 2 ml of the portion of HRP 
introduced with maleimide group obtained in the above (2) were mixed, and 
the mixture was concentrated to 1 ml using collodion bag (Sartorius, West 
Germany) under reduced pressure at 4.degree. C. for 20 hours. After the 
reaction, the unreacted HRP introduced with SH group was removed with the 
use of Ultrogel AcA44 (LKB Co., diameter 1 cm.times.80 cm, Sweden) column 
(flow rate: 10 ml/hour). In the eluates, the fraction containing 2.4 
HRP/antibody has the most high HRP number per antibody molecule. The 
product thus obtained was employed in the EIA in the following item (4). 
(4) The monoclonal antibody MoAb 52 was purified by the manner described in 
the above (1). The monoclonal antibody MoAb 52 was diluted with PBS so as 
to be 10 .mu.g/ml or 20 .mu.g/ml, and the diluent was poured into 
Immunoplate (Nunc, Denmark) so as to be 100 .mu.l/well. The plate was kept 
standing at 4.degree. C. 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 pe/well, and the 
plate was kept standing at 4.degree. C. overnight. 
(5) The cell extract containing bFGF mutein C86 obtained in Reference 
Example 3 was diluted with PBS containing 0.1% BSA. From the plate 
obtained in the above (4), BSA 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 .mu.l/well to adsorb to the plate at 4.degree. C. 
overnight. The unreacted mutein C86 was removed, and the plate was washed 
with PBS four times. The monoclonal antibody conjugated with HRP (HRP-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 100 .mu.l/well. 
The reaction was carried out for 4 hours at room temperature. After 
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 .mu.l/well. Quantification was accomplished by absorbance 
measurements at 415 nm, and it was confirmed that a small amount of the 
mutein C86 was produced. 
(6) In FIG. 11, the detection curve is shown in case that the amount of 
monoclonal antibody MoAb 52 which is fixed to the plate is 1 .mu.g/well (- 
- - .largecircle. - - -), and 2 .mu.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 FIG. 11, it is taught that the concentration of 0.5 ng/ml of bFGF 
can be detected, when the monoclonal antibody MoAb 52 is adsorbed to the 
plate in an amount of 2 .mu.g/well. 
(7) The monoclonal antibody MoAb 98 was adsorbed to the plate in an amount 
of 2 .mu.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 FIG. 12. The horizontal 
and vertical axes show the same as those of FIG. 11. From FIG. 12, it is 
taught that at least 0.5 ng/ml of bFGF can be 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. 
Example 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 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 electrophoresis 
employing 17.25% acrylamide gel (Laemmli, Nature, 277, 680-685 (1970)), 
and it was transferred (Journal of Biochemical and Biophysical Methods, 
10, 203-209 (1984)) on the membrane of nitrocellulose by using Sartoblot 
(Sartorius, West Germany). This membrane was washed with TBS (20 mM 
Tris.HCl (pH 7.5), 0.5M NaCl) for 5 minutes twice, and kept standing in 
TBS containing 4% BSA at room temperature for one hour to block the 
unreacted material on the membrane. Thus obtained membrane was washed with 
TBS containing 0.05% Tween 20 (TTBS) for 5 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 TTBS 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 membrane 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 TTBS 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 0.015% hydrogen peroxide, and 
the reaction was carried out for 15 minutes. 
In FIG. 13, the results of Western blotting in case the monoclonal antibody 
MoAb 78 was used as primary antibody. The lane I shows the results of 1 
.mu.g of bFGF, the lane 2 shows the results of 300 .mu.g of bFGF, the lane 
3 shows the results of 100 .mu.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 12 instead of MoAb 78. 
The following references, which are referred to for their disclosures at 
various points in this application, are incorporated herein by reference. 
Nature, 249, 123 (1974) 
National Cencer Institute Monograph, 48, 109(1978) 
Proceedings of the National Academy of Sciences, USA, 82, 6507 (1985) 
European Patent Publication No. 237, 966 
European Molecular Biology Organization (EMBO) Journal, 5, 2523(1986) 
Genetic Enginnering, Academic Press (1983), pp. 31-50 
Genetic Enginnering: Principles and Methods, Prenum Press (1981), vol. 3, 
pp. 1-32 
Journal of Immunological Methods, 80, 55(1985) 
Nature, 256, 495(1975) 
Journal of American Medical Association, 199, 549(1967) 
Proc. Natl. Acad. Sci. USA, 80, 3513-3516(1983) 
Molecular and Cellular Biology, 3, 280(1983) 
Nucleic Acids Research 1, 1513(1979) 
Proc. Natl. Acad. Sci. USA, 72, 3961(1975) 
Nucleic Acids Research, 9, 309(1981) 
Nucleic Acids Research, 11, 3077-3085(1983) 
Molecular Cloning (1982), A Laboratory Manual, Cold Spring Harbor 
Laboratory, USA. 
Methods in Enzymology, 101, 20-78(1983) 
Nature, 227, 680(1970) 
Regulatory Peptides, 10, 309-317 (1985) 
Journal of Biochemical and Biophysical Methods, 10, 203-209(1984)