Vol. 175, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 291-297
February 28, 1991
ESTABLISHMENT
Yuzo
OF
MONOCLONAL
ANTIBODIES
ACIDIC
FIBROBLAST
GROWTH
Ichimori,
Yumiko Kinoshita,
Masaharu
Chemistry
Research Research
Senot,
Laboratories,
Tatsuya Koichl
Division,
Ltd.,
Osaka
HUMAN
FACTOR
Watanabe!,
Kondo
tBiotechnology
and Development Industries
Received January
and
AGAINST
Research
Laboratories,
Takeda Chemical
532, Japan
£i, 1991
SUMMARY: Four kinds of hybridomas secreting monoclonal antibodies (MAbs) against human acidic fibrobtast growth factor (haFGF) were established using recombinant haFGF as an immunogen. The recognition sites of four Mhbs designated AFl-52, 81, ll4 and 1el0 for the haFGF molecule were examined by binding studies with synthetic polypeptides and with amino-terminal truncated forms of haFGF. These experiments suggested that AFI-52, II4, and lCI0 Mhbs recognize epitopes within the ]-5, 44-132 and 6-43 amino acid sequences, respectively. However, the epitope recognized by the AF|-81 MAb could not be determined. The sandwich EIA method constructed w i t h t h e s e MAbs w a s s e n s i t i v e to 1.9 pg/well of haFGF and had no cross-reactivity w i t h h u m a n b a s i c FGF, b o v i n e aFGF o r t h e h s t - ] gene product. Q 1991 ~oad~mioP~s~, z~o
Fibroblast a wide
fibroblasts, angiogenic values:
endothelial activity
(bFCF)
A
in vivo
(1).
form with
(}).
number
like
of
FGFs
proteins,
proteins
share
findings
have
course
cultured FGF
(4),
30-90
% amino
revealed
that
cells.
bFGF
(aFGF)
from
FGF-5
by distinct
malignant
function
FGF-6
homology
behave
tissues
new oneogenes
(6),
sequence
that FGFs might
they possess
They
two forms
with
including also
have
by their
, and a basic one with
are encoded
Recently,
(9),
into
activity
in vitro
genes,
pl
a pl they
(2).
derived
acid
glial
ave separated
and
(3).
int-2
and
5.0-7.0
homology
physiological
reported
myoblasts FGFs
cells
to have mitogenie cells
aFGF
mRNAs
hst-]
are known
and mesoderm-derived
a pl of
Although
and
of normal
been
(FGFs)
cells,
55 % amino acid sequence
synthesize
has
factors
of neuroeetoderm-
an acidic
of 9.6 share
growth
variety
(7) were
with
as growth
but also
transforming
were
which
aFGF
factors
isolated.
under
to
FGFThese
and bFGF.
These
not only
in the
in carcinogenesis. activity
found
encode
Indeed,
it
some conditions
(8,9). The important MAbs
quantification for
against
and a useful
the human
of
diagnosis bFGF
detection
have method
FCFs of
from
human
malignant
already
been
for human
diseases. established
aFGF
291
t~ssues
(haFGF)
and
fluids
EIA
methods
(10,11).
is and
therefore specific
However,
a MAb
have not been established.
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 175, No. 1, 1991
In against
this
report,
haFgF and
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
we d e s c r i b e the p r e p a r a t i o n and c h a r a c t e r i z a t i o n of MAbs the
c o n s t r u c t i o n of
a
s e n s i t i v e sandwich
EIA method f o r
measurement of i t using these MAbs.
MATERIALS AND METHODS Materials: BALB/cmice were purchased from Shizuoka Animal Center (Hamamatsu, Japan). Human bFGF (hbFGF) and h s t - ] mutein (amino-terminal region 1-27 aa truncated form of h s t - ] ) used in the experiments are described elsewhere (I2,13). Synthetic p o l y p e p t i d e s , haFGF[I-9] (amino-terminal peptide of haFGF,]9 aa) and haFgF[133-140] (carboxyl-termina] peptide of haFGF, ]33-140 aa), were provided by Dr. Wakimasu (Tsukuba Research I n s t i t u t e of Takeda Chemical I n d . , Tsukuba, Japan). Bovine aFGF (baFgF) was obtained from R&D Systems, Inc. (Minneapolis, MN). Preparation of haFgF and i t s truncated forms: haFGFwas p u r i f i e d to apparent homogeneity f r o m recombinant E . c o l i (14). E . c o l i expression systems for Mutein I (amino-terminal l-5 aa truncated form of haFGF) and MuteinH (aminoterminal ]-43 aa t r u n c a t e d form of haFGF) w e r e c o n s t r u c t e d based on the expression system for haFgF (]4). Mutein[ was p u r i f i e d by heparin a f f i n i t y HPLC from the b a c t e r i a l e x t r a c t ( ] 4 ) . MuteinH was s o [ u b i l i z e d from i n c l u s i o n bodies with b M urea and then p u r i f i e d by O-Sepharose column chromatography in the presence of 3 M urea.
Immunization: Eight-week-old female BALB/c mice were immunized 3 times at 3week i n t e r v a l s . T h e y were immunized f i r s t subcutaneously ( s . c . ) with I00 ~g of recombinant haFgF e m u l s i f i e d in Freund's complete adjuvant and the 2nd and 3rd time s . c . with the s a m e dose of recombinant haFgF e m u l s i f i e d in Freund's incomplete adjuvant. Following the t h i r d immunization, they were intravenously infused with ]00 ~g of recombinant haFgF in s a l i n e 3 days p r i o r to s a c r i f i c e . Fusion procedure: Three days a f t e r the f i n a l immunization, the spleen c e l l s from immunized mice were mixed with 8 - a z a g u a n i n e - r e s i s t a n t murine myeloma P3Xb3-Ag.8U] (P3U]) c e l l s at a r a t i o of 5:] and fused by a m o d i f i c a t i o n of the procedure d e s c r i b e d previously (15). The c u l t u r e supernatants from the w e l l s , in which the growth of hybrid c e l l s was observed, w e r e assayed for presence of anti-haFgF antibody by an enzyme linked immunosorbent assay (ELISA) method. ELISA for a n t i b o d i e s to h a F G F : P u r i f i e d recombinant haFGF was d i s s o l v e d in 0.0l M sodium bicarbonate b u f f e r (pH 8.5) at a c o n c e n t r a t i o n of I0 ug/ml, and ]00 ~l of the s o l u t i o n was added to each well of a 96-well m i c r o t i t e r p l a t e . The p l a t e was incubated overnight at 4 ° C , and the f l u i d removed. Residual p r o t e i n binding s i t e s in the wells were blocked by adding Buffer A (phosphate buffered s a l i n e (PBS, pH 7.2) containing 25 % Block Ace (Snow Brand Milk products Co., Japan)) followed by incubation overnight at 4°C. One hundred m i c r o l i t e r s of each hybridoma c u l t u r e supernatant was added to each of the haFGF-coated wells and incubated for 2 hr at 2 5 ° C . After washing the p l a t e 5 times with PBS, 100 ~l of a 5000-fold d i l u t e d s o l u t i o n of h o r s e r a d i s h peroxidase (HRP) conjugated goat anti-mouse IgG (Organon Teknika Corp., West Chester, PA) was added to each well. Following a 2 hr incubation at 25°C, the p l a t e was washed 5 times with PBS and ]00 ~l of p e r o x i d a s e - s u b s t r a t e (22 mg of o -phenylenediamine and 10 ~l of hydrogen peroxide in I0 ml of 0.] M c i t r a t e b u f f e r , pH 5.5) was added. A f t e r 30 min. of r e a c t i o n at 25°C, ]00 ~l of 4 N s u l f u r i c acid was added to the w e l l s , and the absorbance of each well was measured at 4q2 nm using a microplate reader (MTP-32, CORONA Co. L t d . , JAPAN ).
Neutralizing activity of antibody t o haFGF: Mitogenic activity o f haFGF was monitored using BALB/c 3T3 clone A31-1-1 c e l l s as previously d e s c r i b e d (8) and the e f f e c t s of hybridoma supernatant a g a i n s t t h i s a c t i v i t y w e r e i n v e s t i g a t e d . B r i e f l y , 2×103 A3I-I-I c e l l s were suspended in ]60 pl of Dulbecco's modified E a g l e ' s medium supplemented with 0.5 % c a l f serum, then 40 ul of a 5 - f o l d s e r i a l l y d i l u t e d hybridoma supernatant and I0 ~I of a mitogen s o l u t i o n containing 20 pg of haFgF and 10 ~g of heparin were added. After the c e l l s were 292
Vol. 175, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
incubated for 18 hr at 37°0, they were pulse-labeled with [3H]-thymidine (37kBq/well) for 5 hr, and subjected to radioactivity determination.
Antibody
production
and
purification:
Each
hybridoma
was
injected
intraperitoneally (i.p.) at 2X10 ~ cells into BALB/c female mice that had received 0.5 ml of mineral oil i.p.. Ascitic fluids were collected 7-10 days after the injections. MAbs were purified by ammonium sulfate precipitation followed by hydroxyapatite HPLC column chromatography. Purified AFI-52 MAb was conjugated with HRP described previously (10). Sandwich EIA for haFGF: Each purified MAb was dissolved in 0.01 M sodium bicarbonate buffer (pH 8.5) at a concentration of 10 ~g/ml. One hundred microliters of each solution or a mixture of the three were added to each well of a q6-well microtiter plate. One hundred microliters of various concentrations of haFGF diluted with Buffer A was added to each well and incubated overnight at 4°C. The plate was then processed for measurement of AFI-52-HRP conjugate binding activity (added from a 200-fold dilution) as described above under "ELISA for antibodies to haFGF". For serum study, the sandwich EIA method was modified as follows. Sixty microliters of the sera or "0" serum containing the standard haFGF and 180 ~i of Buffer B (Buffer A containing 0.3b M NaCI and 13 ~g/ml unrelated mouse IgG) were mixed. The "0" serum was pooled-sera which passed through the AF1-114-coupled Sepharose 4B. One hundred microliters of this mixture were added to each well and incubated overnight at 4°C, then the detection of immuno-reactive haFGF was measured.
RESULTS
Hybridoma s e c r e t i n g a high
antibody
seeded the
antibodies
titer
into 960 wells
growth
to
against
haFGF
in
haFGF:
their
of microculture
of hybridomas
Spleen
sera
were
plates.
was observed
cells
fused
from mice
with
One to 2 weeks
in 390 wells.
P3UI
showing
ceils
and
after the fusion,
Culture
supernatants
were
harvested from each well and assayed for the presence of haFGF antibody using an ELISA method.
Hybridomas
from 4 wells
were
cloned
by limiting dilution
from
BALB/c
mice
their monoclonal studies.
The
as
feeder
ceils.
antibodies
The
subclass
of
that of AFI-II4 and ]CI0 MAbs was 71~
haFGF
were
of
forms
of
114
MAb
by
(haFGF[I-9]
haFGF
(Mutein I
but not
bound
haFGF[133-140] These
results
within
the
recognition
recognition
recognized
polypeptides
haFGF[]-9]
MAb
to
MAbs,
that AFI-52,
44-132
and
6-43
haFGF.
activity
Next, of
haFGF
we to
neutralizing activity
determine
and
BALB/c
selected
MAbs
was
72b~,
1-43
aa
these
(data not shown).
293
to
The AF1-52
and
and
MAbs
can
However,
to ]).
The
it bound only to truncated forms
neutralize of
to
epitopes
respectively.
because
all
not
(Table
ICl0 MAb recognize
sequences,
bound
The AFI-
but
but not to M u t e i n H
the
truncated
MAb
form)
of
synthetic
form).
or to amino-terminal
these
cells.
MAbs
truncated
and and acid
regions
amino-terminal
I-5 aa truncated
to M u t e i n /
amino
which
of
examined.
AF]-I]4
whether 3T3
were
to
MAb could not be determined
examined
to haFGF
1C10) were used for all
and AFI-8]
binding
(amino-terminal
1CI0 MAb bound
site of AFI-8]
To
haFGF[]33-140])
M u t e i n H (amino-terminal
and the
titer
(5×I0 s cells/ml)
.
haFGF but not to the synthetic polypeptides of
AFI-52
site:
four
clones
AF]-114 and
and M u t e i n H ) was
to M u t e i n l
indicate I-5,
the and
of thymocytes
representative
(AF]-52, AF]-81,
immunoglobulin
Determination
that showed high antibody
in the presence
these
the mitogenie MAbs
had
no
Vol. 175, No. 1, 1991
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
1.
Binding
ability
o f MAb t o v a r i o u s Binding
MAb
haFGF
AFl-52
+~'
AF1-81
+
AF]-II4
+
1010
+
antigens
ability"
Pepl-9
Pep133-140
+
-'
-
-
-
-
+
-
+
-
MuteinI
Mutein
+
One hundred microliters of hybridoma supernatant was added to each well coated with antigen: (Pepl-9: amino-terminal peptide of haFGF, ]-9 aa ; P e p 1 3 3 - 1 4 0 : carboxyl-terminal peptide of haFGF, 133-140 aa ; Hutein I : amino-terminal ]-5 aa truncated form of haFGF; H u t e i n M : aminoterminal 1-43 aa truncated form of haFGF.). The binding of MAb to the well was examined with goat anti-mouse IgG antibody labeled with HRP. h Bound. Not bound.
Construction
of
quantified
by a
them and
(AF]-52 ]C10)
EIA
sandwich
HAb)
or
plates.
from
coated
wells
].5 p g / w e l l
was
the
microtiter
was
method
haFGF:
To
examine
whether
EIA,
four
kinds
of HAbs
were
labeled
with
HRP.
Each
the o t h e r
mixture As with
for
of
shown
all in
three
Fig. I~
the m i x t u r e .
was
the
The
of
used
most
purified
to
coat
limit
could
be
and used.
One of
(AF]-8],
AF]-]]4
HAbs
sensitive
detection
haFGF
wells
result
using
of was
this
96-well obtained
EIA
method
of haFGF.
1.O 1.0 04 04 CrJ
v
v ¢D
~_ JQ o c9