[CANCER RESEARCH 52. 6153-6157, November 15. 1992]
Mouse Monoclonal Antibodies Which Recognize a Human (/?l-4)Galactosyltransferase
Associated with Tumor in Body Fluids
Morito Uemura,1 Takashi Sakaguchi, Takao Uejima, Shiro Nozawa, and Hisashi Narimatsu
Development Center A'o. 3, Konica Corporation, .\'o. I Sakura-Machi, Hino-Shi, Tokyo 191 [M. L'., T. S., T. L'.J; Department of Obstetrics and (jynecology, Keio
University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160 [S. NJ; and Institute of Life Science, Soka University, 1-236 Tangi-cho,
Hachioji-Shi, Tokyo 192 [H. N.¡.Japan
ABSTRACT
Mouse monoclonal antibodies against human (/31-4)galactosyltransferase (GalT) puntini from human ovarian tumor effusion fluids
were prepared and characterized. GalT purified from normal human
plasma showed a single diffused band in nondenaturing polyacrylamide
gel electrophoresis, but GalT purified from human ovarian tumor effu
sion fluids showed several oligomeric bands and a monomi-rii band in
nondenaturing polyacrylamide gel electrophoresis. These oligomeric
bands were dissociated into monomer by urea treatment and polymer
ized by a 2-mercaptoethanol treatment. Nine monoclonal antibodies
(MAb) were prepared by immunization of purified GalT from human
ovarian tumor effusion fluids and classified into three groups. Type I
M Alis (MAb8611, MAb8913, and MAb8919) reacted only to the GalT
monomer. Type II MAbs (MAb4880, MAb8507, and MAb8628) re
acted to both the GalT monomer and the GalT polymer. Type III MAbs
(MAb7907, MAb8513, and MAb8677) reacted only to the GalT poly
mer. These MAbs except MAb7907 could recover GalT enzyme activity
from effusion fluids by immunoprecipitation. A fraction passed through
MAb8513 attillili, chromatography still showed reactivity to MAb8919,
demonstrating that an epitope of MAb8513 resides on a minor part of
GalT. A sandwich immunoassay (MAb8513-MAb8628HRP) was de
veloped, and serum samples from ovarian cancer patients and benign
ovarian patients were tested. The levels of sandwich immunoassay of
serum samples from cancer were elevated significantly compared to
those from benign and did not necessarily correlate to total GalT en
zyme activity in serum samples. These results suggested that MAb8513
(Type III) might recognize a unique GalT associated with tumor (GAT).
INTRODUCTION
GalT2 catalyzes the transfer of galactose from UDP-galactose to glycoproteins with jY-acetylglucosamine as acceptor res
idues. GalT is widespread in mammalian tissues, present as
both membrane-bound and soluble forms (1). The membranebound form of GalT is considered as a suitable marker enzyme
for golgi membranes (2). The membrane-bound form on the cell
surface proposed by Shur (3) may have certain functions in
cell-cell interactions (4,5). The soluble forms of GalT have been
detected in various body fluids including milk, serum, and both
pleural and ascitic effusions (6). Measurement of GalT enzyme
activity in serum was reported as a useful prognostic marker in
various malignant tumors including ovarian cancers (7, 8), but
clinical application of the measurement of GalT to a cancer
marker has been restricted because of false positives in benign
diseases such as inflammation, regenerative processes, and he
patic diseases (9, 10). While tumor-associated GalT isoenzymes
Received 6/8/92; accepted 9/11/92.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in accord
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed.
2 The abbreviations used are: GalT, (tf1-4)galactosyltransferase; MAb, mono
clonal antibody: PBS. phosphate-buffered saline; BSA. bovine serum albumin;
GAT, galactosyltransferase associated with tumor; ELISA, enzyme-linked nimm
nosorbent assay; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro
phoresis; 2-ME, 2-mercaptoethanol: IFE, isoelectric focusing electrophoresis;
OVA, ovalbumin: «-LA,n-lactalbumin; HRP. horseradish peroxidase; cDNA,
complementary DNA.
such as GT-II have been reported (11-17), these reports reveal
some confusion concerning the identification, quantification,
and physical properties of tumor-associated GalT isoforms
(18-21). To clear these problems, immunological approaches
have been taken in preparations and characterizations of mon
oclonal antibodies to serum GalTs (22, 23). Although we re
ported characterization of GT-II using MAb3872 (24-26),
analysis of a molecule recognized by MAb3872 has not been
done yet. In this paper, we clarified that GT-II showing slow
migrating bands in nondenaturing PAGE consists of an isoform
of GalT that tends to aggregate and named it as GAT. Then, we
present preparation and characterization of several mouse antihuman GalT MAbs, one of which was specifically reacting to
GAT.
MATERIALS
AND METHODS
Materials. Reagents were obtained from the following sources:
a-LA, goat anti-human IgG affinity column, UDP-galactose, horserad
ish peroxidase, trichloroacetic acid, and OVA from Sigma Chemical
Co., St. Louis, MO; UDP-pHjgalactose from New England Nuclear,
Boston, MA; Affi-Gel 10 from Bio-Rad Laboratories, Richmond, CA;
peroxidase-conjugated goat anti-mouse (IgG+IgM) from Tago Immunodiagnostics, Burlingame, CA; anti-mouse immunoglobulin typing kit
from Miles Laboratories, Elkhart, IN; Immunopiate MaxiSorp from
Nunc Inter Med Co., Roskilde, Denmark; MPL+TDM Emulsion from
Ribi Immunochem Research Inc., Hamilton, MT; Immobilen PVDF
membrane from Millipore Co., Bedford, MA; Immunostain Kit from
Konica Co., Tokyo, Japan: GF-2000 from Pierce Chemical Co., Rockford, IL.
Human Fluids. Ascitic fluids were drawn from peritoneal cavity of
patients with malignant ovarian tumors in Keio University School of
Medicine, Tokyo, Japan. Serum samples were obtained from the Na
tional Cancer Institute Tumor Serum Bank maintained by the Mayo
Clinic, Rochester, MN. All samples were stored frozen at -40'C and
were thawed at 4°Cprior to use.
Immunization and Cell Fusion. GalT was purified from ascitic fluids
of cancer patients by a-LA and anti-human IgG affinity chromatographies as described previously (24). BALB/c mice were immunized i.p.
with 0.1 ml of 20 Mgof purified GalT/mouse in 0.1 ml of MPL+TDM
Emulsion (27). Three weeks after, each mouse received a i.p. booster of
0.1 ml of GalT (20 ^g) in 0.1 ml of MPL+TDM Emulsion. Two weeks
after the booster, the mice received 50 ^g GalT in 0.1 ml of PBS i.v.
Three days following the booster, the mice were sacrificed, the spleens
were removed, and cell fusions were performed as described previously
(28). Briefly, X63-Ag8.653 mouse myeloma cells (5 x IO7) were fused
with spleen cells (3 x IO8) in 1 ml of 50% polyethylene glycol, and the
fusion mixture was distributed into three 96-well microtiter plates.
Hybridomas were selected in hypoxanthine-aminopterin-thymidine
medium and screened for antibody production by ELISA. The class and
subclass of each MAb were determined using the commercially avail
able kit.
Production of MAbs and Preparation of MAb-HRP Conjugates.
MAbs were prepared by injecting hybridoma cells (5 x 10*) into the
peritoneal cavities of BALB/c mice and subsequently ascitic fluids were
collected. Each MAb was purified by fast protein liquid chromatogra
phy (Pharmacia LKB) equipped with a Mono-Q column for IgG or
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ANTIBODIES
TO GAT IN BODY FLUIDS
Table 1 Properties of anti-GalT MAbs
Superóse 6 for IgM after 50% ammonium sulfate precipitation. MAbIsotyping
and
inhibition
assays were determined by ELISA as described in
HRP conjugates were prepared as described previously (29).
Inhibition Assay. Microtiter plates were coated overnight at 4°C "Materials and Methods." Inhibition activities of each MAb against MAb4880-
with 0.1 ml of purified GalT (1 ng/m\ in PBS). The plates were washed
three times with PBS, blocked with 0.2 ml of 1% BSA/PBS for l h at
37°C,and then incubated for l h at 37°Cwith suspension containing
both O.I ml of MAb-HRP conjugate in 1% BSA/PBS and 0.1 ml of each
MAb supernatant. After three washes with PBS, the remaining HRP
was measured by developing 0.1 ml of 0.04% (w/v) o-phenylenediamine
in a buffer (pH 6.0) containing 100 mM citric acid, 200 miviNa2HPO4
and 0.01% H2O2. After a 15-min incubation, the reaction was stopped
by adding 0.025 ml of 9 M H2SO4. The absorbance of each well was
assessed in an automatic ELISA plate spectrophotometer (Bio-Rad) at
492 nm.
Binding Assay. Purified GalT was incubated with 10 mM 2-ME at
37°Cfor l h and then applied to fast protein liquid chromatography
HRP or MAb7907-HRP were shown at measurement of A492 nm.
withType
Reaction
IType
8913
89194880
IgG2a
IgGlIgGl
>2.000
>2.0000.309
>2.000
>2.000>2.000
8507
86287907
IgGl
IgGlIgM
0.235
0.059>2.000
>2.000
>2.0000.192
IIType
IIIMAb8611
>2.000
8513
IgM
0.013
>2.000MAb7907-HRP>2.000
IgGlMAb4880-HRP>2.000
8677IsotypeIgGl
0.112
equipped with Superóse 12. Fractions were pooled separately according
to whether they eluted earlier or later than the standard M, ~ 200,000
protein. Each pooled fraction was biotinated as described in the previ
ous report (28). Microtiter plates were coated with 0.1 ml of purified
MAb (10 /jg/ml in PBS) and blocked with 1% BSA/PBS as described
above. The plates were incubated with 0.1 ml of biotinated GalT diluted
with 1% BSA/PBS for l h at 37°C.After three washes with PBS, 0.1 ml
of a 1:2000 dilution of avidin-HRP with 1% BSA/PBS was added and
incubated for 30 min at room temperature. HRP activity remaining on
the wells was measured as described above.
Sandwich Assay. Microtiter plates were coated with 0.1 ml of
MAb8513 or MAb8919 (10 Mg/ml in PBS) and blocked with 1% BSA/
PBS. Afterwards, the plates were incubated with a 0.05-ml serum sam
ples and 0.1 ml buffer (pH 6.5) containing 20 min Na2HPO4 and l M
NaCl, for 2 h at 37°C.After three washes with PBS, the plates received
0.1 ml of MAb8628-HRP in 1% BSA/PBS and were incubated for l h
at room temperature and HRP activity was measured.
Assay of GalT Activity. GalT activity was determined by using UDP[•'Hjgalactoseand OVA as substrates, as described previously (24).
GalT activity bound to MAb was assayed by immunoadsorption using
GF-2000-immobilized MAb, as described previously (24).
Electrophoresis. Nondenaturing polyacrylamide electrophoresis (8%;
10x10 cm) was carried out as described previously (15). After elec
trophoresis, a sensitive silver-staining procedure was used to visualize
proteins. SDS-PAGE (12.5%) and (pi 4.0-6.5) IFE were performed by
using the PHAST system (Pharmacia, Piscataway, N.J.). Western blot
ting of SDS-PAGE and IFE onto PVDF membranes were performed as
described in the procedure handbook of PHAST system. After blotting,
PVDF membranes were treated overnight with 3% BSA/PBS, and then
incubated with MAb8628-HRP (2 Mg/ml in 1% BSA/PBS) for 2 h at
37°C.After three washes, the membranes were visualized by Konica
Immunostain HRP (Konica), as described in manufacturer's guide.
MAb7907, MAb8513, and MAb8677 inhibited MAb7907HRP conjugates; MAb4880, MAb8507, and MAb8628 inhib
ited MAb4880-HRP conjugates; MAb8611, MAb8913, and
MAb8919 inhibited neither MAb7907-HRP nor MAb4880HRP conjugates. Therefore, nine MAbs were classified: type I,
MAb8611, MAb8913, and MAb8919; type II, MAb4880,
MAb8607, and MAb8628; type III, MAb7907, MAb8513, and
MAb8677.
Electrophoretic Analysis of Purified GalTs. GalT was puri
fied by using two step affinity Chromatograph ¡esof a-LA from
ascitic fluids. The purified GalT was revealed on SDS-PAGE,
followed by silver staining (Fig. IA). The duplicated lanes were
transferred onto PVDF membranes and then immunostained
with MAb8628 (Fig. IB). A single broad band appeared at a
molecular weight of 50,000 (Fig. 1B, Lane 1). Lane 2 of Fig. l B
showing minor bands at Mr 100,000 and the origin of the gel
suggested that GalT might aggregate and form homopolymers
under heat treatment with 2-ME. IFE of the purified GalT from
ascitic fluid showed multiple bands between pH 4.5 and 6.0
(Fig. 2A, Lane 1) and still the same pattern shifted to a higher
pH under treatment with neuraminidase (Fig. 2A, Lane 2). The
immunostained bands with MAb8628 showed a similar pattern
corresponding to the protein bands (Fig. 2B). There was no
apparently specific band seen in the stained bands among nine
monoclonal antibodies (data not shown). An additional exper
iment using MAb3872, which had been reported previously
(24) to react to the GT-II molecule, was done to determine
whether MAb3872 detects the same molecule as the other nine
MAbs. As shown in Fig. 2C, the bands stained with MAb3872
Affinity Chromatography. Purified MAb8513 (20 mg) was immobi
lized on 2 ml of Affi-Gel 10 as described in manufacturer's guides. The
were distinct from those with the others. This result suggested
that MAb8628 and MAb3872 recognize different molecules.
gel was subsequently washed as the following manner. After a washing
buffer (pH 7.3) containing 20 miviNa2HPO4 and 1 MNaCl, an eluting
GalT was purified from normal human plasma in the same
buffer (pH 7.3) containing 20 mivi Na2HPO4 and 3 M KSCN, and
manner. The purified GalTs from ascitic fluids of cancer pa
followed with PBS, 5 ml of human tumor ascitic fluids were applied to
tients or normal human plasma were run in 8% nondenaturing
the gel packed into a 1- x 2.5-cm column. The column was washed with
PAGE (Fig. 3). A plot of the log molecular weight versus rela
20 ml of the washing buffer and eluted with 10 ml of the eluting buffer.
tive migration (assuming a A/r 48,000 monomer and each ob
Each 1-ml fraction was collected and assayed by the sandwich assays as
served band as a multimer of the monomer) yielded a straight
described above.
line (correlation coefficient, 0.98) (24). The molecular weight of
each
band was estimated by this linear semilog plot. The GalT
RESULTS
from normal plasma showed a single broad band (GT-I) at A/r
Preparation and Classification of Monoclonal Antibodies.
50,000, but the GalT from ascitic fluids of cancer patients
showed slow migrating bands, mainly as a Mr 100,000 dimer, in
BALB/c mice were immunized with purified GalT from cancer
ascitic fluids in MPL+TDM Emulsion. Cell fusions were per
addition to the same band as seen in normal plasma. These slow
formed five times and resulted in obtaining nine positive hybri- migrating bands moved toward the origin of the gel under 10
HIM2-ME treatment, but toward the Rr value corresponding to
domas against purified GalT which were screened by ELISA.
The nine monoclonal antibodies were cloned successfully, and GT-I under treatment with 4 M urea. There was no change seen
in the GalT from normal plasma under treatment with either
their classes and subtypes were determined (Table 1). Inhi
bitions among MAbs against GalT were tested (Table 1). 2-ME or urea.
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MOUSE MONOCLONAL
ANTIBODIES
TO GAT IN BODY FLUIDS
Each MAb immobilized on GF-2000-activated trisacrylamide gel was incubated with ascitic fluids, and the enzyme
activity of GalT on the gel was assayed. The enzyme activities
could be recovered from the gels immobilized with each MAb
except MAb7907 (Fig. 5). Ascitic fluids from cancer patients
were applied to the affinity column immobilized with
MAb8513 on agarose. Both the passed through and bound
B
«-Origin^
1
«-92.5K-*
4-66.
^Origin
2K-»
«-45K-*
4-lOOKd
4-31K-*
-Dye
Front-
Fig. 1. SDS-PAGE (12.5%) of purified GalT from ascilic fluids. A, silver
staining; B, Western blotting by MAb8628. Lane 1, 1 ng of GalT treated without
2-ME; Lane 2, 1 ng of GalT treated with 2-ME. Arrows, molecular weight protein
standards: phosphorylase (M, 92,500), BSA (M, 66,200), ovalbumin (M, 45,000),
and carbonic anhydrase (M, 31,000). K, thousands.
B
Dye Front
pH
Fig. 3. Nondenaturing 8% polyacrylamide electrophoresis of purified GalT
from normal plasma and ascitic fluids. Lanes I, 2, and 3, 1 ng of GalT from
normal plasma; Lanes 4, 5, and 6. I »jg
of GalT from ascitic fluids; Lanes I and
4, not treated; Lanes 2 and 5, treated with 10 mM 2-ME; Lanes 3 and 6. treated
with 4 M urea. AW,molecular weight in thousands.
6
I
OD:492ni
20
15
p H 4
0-»
1.0
Fig. 2. Isoelectric focusing electrophoresis (pi 4.0-6.5) of purified GalT from
ascitic fluids. A, silver staining; B. Western blotting with MAb8628; C, Western
blotting with MAb3872; Lane I, 1 *igof GalT; Lane 2, 1 ng of GalT treated with
neuraminidase.
05
Immunological Analysis of CalTs Using Three Types of
MAbs. The purified GalT from cancer patients was treated
with 2-ME and then fractionated to high molecular weight
GalT and low molecular weight GalT by gel filtration chromatography as described in "Materials and Methods." While
MAb8628 (type II) reacted to both high and low molecular
weight GalT, the others, M Ab8919 (type I) and MAb8513 (type
III), had selective binding against a low molecular weight GalT
and a high molecular weight GalT, respectively (Fig. 4).
—¿-0—
-0
0 1:T
1:300
I :IOO
]H*bB919
I:T
1:300
I : 100
[TypeD|HAD86?8
1:T I :300
I : 100
[Typel]NADI513
Fig. 4. Binding assay for MAbs to high and low molecular weight GalTs. The
procedure is described in "Materials and Methods." Type I, MAb8919; type II,
MAb8628; and type III, MAb8513. were coated on microtiter plates. The biotinated GalT of the high molecular weight fraction (O) or the low molecular weight
fraction (•)was incubated in a 3-fold serial dilution.
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MOUSE MONOCLONAL
ANTIBODIES
DISCUSSION
X 10 3cpm
0.8 0.7 .=•°6 I
°5"
° 0.4 0.3 0.2 -
n
O.I 0.0
4880
7907 8507
8513
8611
8628
8677
8913
8919
Fig. 5. Enzyme assay of GalT precipitated by each MAb from ascitic fluids.
Each MAb immobilized on GF-2000 gel was incubated with ascitic fluids.
After »ashes,the GalT enzyme activity on the gel was measured as described in
"Materials and Methods."
On»
TO GAT IN BODY FLUIDS
I'jss IhfiHiph
There was no difference in molecular weight and isoelectric
points between GalTs purified from tumor effusion fluids and
normal plasma. However, there were slower migrating bands
seen in nondenaturing PAGE of GalT purified from tumor
effusion fluids corresponding to GT-II as was reported (15).
Moreover, when the GalT was heat treated with 2-ME, the
GalT purified from tumor effusion fluids proceeded to aggre
gate itself and revealed the strong band at the origin of nonde
naturing PAGE. When the GalT was treated with urea, the
GT-II bands disappeared and shifted to the position corre
sponding to the band of GT-I in normal plasma. Meanwhile,
only the GT-I was seen in the nondenaturing PAGE of the GalT
purified from normal plasma; there was no change under treat
ment with 2-ME or urea. These results suggested that a human
GAT having characteristics to aggregate itself easily might exist
in tumor effusion fluids, so that its partially aggregated confor
mation in human body fluids could be detected mainly as a
dimer form on nondenaturing PAGE as the GT-II.
MAbs were successfully prepared to GalT from cancer pa
tients and classified into three types. One of them, type III, was
characterized and proved to react to GAT. From the results of
binding assays to GAT, it could not be defined whether the
epitope on GAT to type III MAbs might be on conformational
sites of the aggregated GAT or on an intact molecule of GAT.
In either case, it was suggested that a structural difference of
GAT related to such aggregation could form a unique epitope to
type III MAbs. According to the elusion profile of MAb8513immobilized affinity chromatography, the GAT assayed by
MAb8513-MAb8628HRP
was considered to be a minor por
tion of GalT assayed by MAb8919-MAb8628HRP.
Mean
while, when the concentrations of GalT and GAT in the pa
tients' sera with ovarian cancer and benign ovarian cysts were
Washing
Fig. 6. Elusion profile of MAh85l3 affinity chromatography. The ascitic fluids
were applied to the column and eluted as described in "Materials and Methods."
Each fraction was measured by the sandwich immunoassay. MAb85l3MAD8628HRP (•)or MAb8919-MAb8628HRP (O).
fractions to this column were assayed by both MAb8513MAb8628HRP and MAb8919-MAb8628HRP
sandwich immunoassays. While no protein from the passed through frac
tions was detected by MAb8513-MAb8628HRP,
a reasonable
amount of protein was recovered from the eluted fractions.
Meanwhile, a higher amount of protein was detected by
MAb8919-MAb8628HRP
from the passed through fractions
than from the eluted fraction (Fig. 6).
Clinical Evaluation of Patients' Sera. Serum samples of
patients with malignant ovarian tumors (N = 19) and be
nign ovarian cysts (N = 18) were assayed by MAb8513MAb8628HRP sandwich immunoassay and total GalT enzyme
activity assay (Fig. 7). The values of serum samples with ma
lignant ovarian tumors in the sandwich immunoassay were el
evated significantly (P < 0.05) compared to those of serum
samples with benign ovarian cysts. In the case of setting a cutoff
value at AW2 mm = 0.45, 12 of 19 (63%) sera with malignant
ovarian tumors and 3 of 18 (17%) sera with benign ovarian cysts
were positive in the MAb8513-MAb8628HRP
sandwich im
munoassay. Meanwhile, there was no significant difference in
the values of samples between malignant ovarian tumors and
benign ovarian cysts in the total GalT enzyme activity assay.
measured, the levels of GAT in the sera with cancer were ele
vated significantly compared to the levels of sera without can
cer. Eight samples of 20 cancer patients were negative in this
assay. It is not known that GAT production might be dependent
on cancer progress or histológica! types. As described by other
X|0'3cpm
OD/492nm
>2.0
1.5
0.5
Cancer
Benign
MAb8M3-MAh8628HRP
Cancer
Benign
Tolal GalT Aclivily
Fig. 7. Clinical evaluation of serum samples with malignant ovarian tumors
(N = 19) and ovarian benign cysts (N = 18). A 0.1-ml sample of each serum was
measured by MAb85l3-MAb8628HRP
sandwich immunoassay. At the same
time. lOfil of each serum were measured by total GalT enzyme activity assay. The
cutoff values indicated by the horizontal dotted line were determined by mean +
2 SD in normal sera.
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MOUSE MONOCLONAL
ANTIBODIES
reports, there was no significant difference in the levels of total
GalT in patients' sera between ovarian cancer and benign
ovarian cysts (9, 10). These results suggest that MAb8513MAb8628HRP sandwich assay to quantify GAT concentration
in serum might be useful for serological cancer diagnosis.
All MAbs in this report could not block GalT enzyme activity
and more or less immunoprecipitated enzyme activity in case of
using OVA as an acceptor. It was unknown why MAb7907
showed a much less recovery of enzyme activity, although
MAb7907 indicated the same characteristics as MAb8513 in
the binding assay and Western blotting.
We previously reported about MAb3872 that was thought to
be specific to GT-II (24). Although both MAb3872 and the
other MAbs in this report have been prepared by immunization
of purified GalT by «-LAaffinity chromatography, the correc
tion on the antigen recognized by MAb3872 should be taken
here. We concluded that MAb3872 reacted to an unrelated
protein binding to GalT, which was copurified with «-LAaf
finity chromatography. The Western blotting pattern of IFE in
Fig. 2 suggests that MAb8628 and MAb3872 might recognize
different molecules than each other. In addition, by determina
tion of partial amino acid sequences on the proteins recovered
by affinity chromatography using MAb3872 or MAb8628, the
former had no shared sequences with those deduced from the
GalT cDNA, but the latter had amino acid sequences identical
with theirs (data not shown). The complex of the GalTs and the
binding protein might be detected as the slower migrating bands
in nondenaturing PAGE as GT-II bands as well. MAb3872
could not detect any band in Western blotting transferred from
SDS-PAGE, and the same characteristic MAb was reported by
the others (30). This binding protein might form the complex
with GAT, not whole GalT, as well as the self-aggregation of
GAT, but it is unknown whether the binding protein would be
specific protein to GAT. Therefore, the GT-II represents just a
phenomenon seen as slower migrating bands in nondenaturing
PAGE as a result of the self-aggregation of GAT or the com
plex of GAT with the binding protein. The analysis of GAT on
a molecular basis must be needed to clarify the physical prop
erty of GAT as a cancer-related protein. Thus it is being studied
further by cDNA cloning what the differences among enzymes
recovered by the three types of MAbs are and whether those
enzymes are similar to products encoded by cDNAs which had
been already cloned. In the following paper (31 ), we will present
the results of cDNA cloning of GAT using these MAbs.
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TO GAT IN BODY FLUIDS
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1992 American Association for Cancer Research.
Mouse Monoclonal Antibodies Which Recognize a Human (β1−
4)Galactosyl-transferase Associated with Tumor in Body Fluids
Morito Uemura, Takashi Sakaguchi, Takao Uejima, et al.
Cancer Res 1992;52:6153-6157.
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