U.S. patent application number 12/567944 was filed with the patent office on 2010-01-21 for method for examining colorectal cancer and colorectal adenoma.
This patent application is currently assigned to Japan Science and Technology Agency. Invention is credited to Hideaki Hosokawa, Mineko Izawa, Reiji Kannagi, Takashi Muramatsu, Kenji Uchimura.
Application Number | 20100015632 12/567944 |
Document ID | / |
Family ID | 34213585 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100015632 |
Kind Code |
A1 |
Kannagi; Reiji ; et
al. |
January 21, 2010 |
METHOD FOR EXAMINING COLORECTAL CANCER AND COLORECTAL ADENOMA
Abstract
The present invention provides a method for examining colorectal
cancer and colorectal adenoma, which enables to detect colorectal
cancer patients and patients at high risk of colorectal cancer at a
high probability and is useful for diagnosis of colorectal cancer
and colorectal adenoma, and provides the examination reagents
thereof. There are significant differences in the distribution of
GlcNAc-6-sulfotransferase isozymes, sulfation enzymes of sugar
residues, among non-cancer colorectal tissues, colorectal cancer
tissues and colorectal adenoma tissues. Furthermore, colorectal
cancers and adenomas are detected specifically by assaying a
definite range of GlcNAc-6-sulfated sugar residues in tissues from
patients or feces samples. MECA-79 antibody (Pharmingen, catalog
No. 09961D, Distributor: Becton Dickinson), reacting with
GlcNAc-6-sulfated sugar residues, which are produced specifically
by the enzyme present in colorectal cancer and colorectal adenoma
tissues could be used for the examination of colorectal cancers and
colorectal adenomas.
Inventors: |
Kannagi; Reiji; (Nagoya-shi,
JP) ; Izawa; Mineko; (Seto-shi, JP) ;
Muramatsu; Takashi; (Nagoya-shi, JP) ; Uchimura;
Kenji; (San Francisco, CA) ; Hosokawa; Hideaki;
(Osaka, JP) |
Correspondence
Address: |
JENKINS, WILSON, TAYLOR & HUNT, P. A.
Suite 1200 UNIVERSITY TOWER, 3100 TOWER BLVD.,
DURHAM
NC
27707
US
|
Assignee: |
Japan Science and Technology
Agency
Saitama
JP
|
Family ID: |
34213585 |
Appl. No.: |
12/567944 |
Filed: |
September 28, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10568544 |
Sep 22, 2006 |
7601348 |
|
|
PCT/JP04/09805 |
Jul 9, 2004 |
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12567944 |
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Current U.S.
Class: |
435/6.11 ;
435/7.23 |
Current CPC
Class: |
G01N 2400/02 20130101;
G01N 2333/91097 20130101; G01N 33/57419 20130101 |
Class at
Publication: |
435/6 ;
435/7.23 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/574 20060101 G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 20, 2003 |
JP |
2003-296216 |
Claims
1. An examination reagent for colorectal cancer and colorectal
adenoma comprising, as a major component, an antibody (including
MECA-79 antibody) reacting specifically with an antigen carrying
sugar residues, which is present in cells expressing HEC-GlcNAc6ST
gene and is absent or almost absent in cells expressing GlcNAc6ST-1
or GlcNAc6ST gene.
2. An examination reagent for colorectal cancer and colorectal
adenoma comprising, as a major component, an antibody (including
MECA-79 antibody) reacting specifically with an antigen carrying
sugar residues, which is present in cells transduced with
HEC-GlcNAc6ST gene and is absent or almost absent in cells
transduced with GlcNAc6ST-1 or GlcNAc6ST gene.
3. An examination reagent for colorectal cancer and colorectal
adenoma comprising, as a major component, an antibody (including
MECA-79 antibody) reacting specifically with an antigen carrying
sugar residues, which are present in tissues, body fluid or feces
of patients with colorectal cancer and colorectal adenoma and
expressed by the following general formula: R1-Gal .beta.1-3/4
(SO.sub.3-6) GlcNAc .beta.1-R2 where, R1 represents sugar residues
added by other enzymes and is not limited in structure. Gal .beta.
represents .beta. galactose, GlcNAc .beta. represents .beta.
N-acetylglucosamine, Gal .beta.1-3/4 represents binding of 1
position of Gal .beta. and 3 position and/or 4 position of GlcNAc
.beta., (SO.sub.3-6) represents addition of a sulfate group to 6
position of GlcNAc .beta., R2 represents -3GalNAc .alpha., -3Gal
.beta. or -2Man.alpha. and binds to 1 position of GlcNAc
.beta..
4. An examination reagent for colorectal cancer and colorectal
adenoma comprising MECA-79 antibody as a major component.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional patent application of U.S.
patent application Ser. No. 10/568,544, filed Sep. 22, 2006, which
is a national stage application of International Application No.
PCT/JP2004/009805, filed Jul. 9, 2004, and which claims benefit of
Japanese Patent Application No. 2003-296216 filed Aug. 20, 2003,
the disclosures of each of which are incorporated herein In their
entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a method for examining
human colorectal cancers and colorectal adenomas, and to antibodies
and examination reagents thereof.
[0004] 2. Prior Art
[0005] Since the number of colorectal cancer patients is increasing
year by year, a method for early detection is necessary. Although,
immunological fecal occult blood test and various tumor markers are
used at present for the examination of colorectal cancers, these
methods do not have satisfactory positive rates. Namely, the
positive rate of immunological fecal occult blood test used for the
examination of colorectal cancers is 50-60%. As to the tumor
markers of colorectal cancers, carcino-embryonic antigen (CEA),
CA19-9, STX, which are used for examining the therapeutic effect
and for monitoring recurrence, are not satisfactory as tumor
markers for early detection of colorectal cancers.
[0006] The colon is divided into right-half and left-half drawing a
line at the flexure coli and colorectal cancers in right-half
result frequently in pseudo-positive by fecal occult blood test
using an anti-hemoglobin antibody, which is pervasively used for
the detection of colorectal cancers. Therefore, development of a
test method contributing to increased early diagnosis rate
particularly of the right-half colorectal cancers is expecting. As
to the laboratory diagnosis of colorectal adenomas, which is
regarded as the birthplace of colorectal cancers, suitable methods
of detecting are not available and people rely inevitably on
endoscopy at the present time. Simple method of diagnosing
colorectal adenomas will be beneficial to classify patients into
groups necessary and unnecessary for endoscopy, and contribute to
early detection of colorectal cancers.
[0007] Although the sulfation of sugar residues is active in a
normal large bowel, it is known to be remarkably reduced in
colorectal cancers. Namely, both 3'-sulfation of galactose and
6-sulfation of N-acetylglucosamine (hereinafter referred to as
[GlcNAc]), which are abundant in colorectum, are reduced (reference
1). A number of GlcNAc-6-sulfotransferase isozymes have been known
in colorectal cancer tissues and in non-cancer colorectal tissues
of patients, and I-GlcNAc6ST is significantly decreased in course
of carcinogenesis, which leads to the reduced sulfation of sugar
residues in colorectal cancer (reference 2). While, GlcNAc6ST-1,
one of the isozymes in a normal colorectum, does not show
significant changes in the level in course of carcinogenesis.
Furthermore, HEC-GlcNAc6ST, another isozyme, increases
significantly in colorectal cancer (reference 3).
[0008] HEC-GlcNAc6ST, which increases in colorectal cancers,
synthesizes 6-sulfated GlcNAc and carries out sulfation of GlcNAc
in various sugar residues. Therefore, there are a huge variety of
the structures of intra-cellularly synthesized sugar residues and
their antigenicity. Since GlcNAc6ST-1 and I-GlcNAc6ST also
synthesize 6-sulfatedGlcNAc, only the fact that 6-sulfatedGlcNAc is
synthesized from HEC-GlcNAc6ST cannot be used as a specific method
for diagnosis of colorectal cancer. However, it is known that the
substrate selectivity of GlcNAc6ST-1 and I-GlcNAc6ST is more
specific than that of HEC-GlcNAc6ST (reference 3, 4). Therefore,
certain 6-sulfated sugar residues might be produced by
HEC-GlcNAc6ST, but not by GlcNAc6ST nor by I-GlcNAc6ST. However, an
actual system for diagnosis of colorectal cancers has not been
established.
[0009] On the other hand, the monoclonal antibody (MECA-79
antibody, reference 5), commercially available as an antibody
against an immunological homing receptor of lymphocytes, is known
to react with chemically synthesized GlcNAc6-sulfated sugar
residues (reference 7). Moreover, the antigens recognizable by the
antibody (MECA-79) are reported to emerge on the cell surface, when
a mouse gene encoding HEC-GlcNAc6ST enzyme is transduced into CHO
cells (hamster ovary cells) (reference 7). However, it has not been
known whether the antigens are recognizable by MECA-79 antibody
emerged on human cancer cells. [0010] reference 1: Izawa, M. et
al., Cancer Res., 60: 1410-1416, 2000. [0011] reference 2: Abstract
of the 22nd Research Meeting of Japan Molecular Tumor Maker pp
42-43, 2002. [0012] reference 3: Seko, A. et al., Glycobiology,
10:919-929, 2000 [0013] reference 4: Seko, A. et al., Glycobiology,
12:379-388, 2002 [0014] reference 5: Streeter, P. R. et al., J.
Cell Biol. 107: 1853-1862, 1988. [0015] reference 6: Bruehl, R. E.
et al., J. Biol. Chem. 275: 32642-32648, 2000 [0016] reference 7:
Yeh, J. C. et al., Cell 105: 957-969, 2001.
Problems to be Solved by the Invention
[0017] The present invention provides a method for examining
colorectal cancer and colorectal adenoma, which enables to detect
colorectal cancer patients and patients at high risk of colorectal
cancer at a high probability and is useful for diagnosis of
colorectal cancer and adenoma, and provides the examination
reagents thereof.
Means to Solve the Problems
[0018] The present inventors discovered that there are significant
differences in the distribution of GlcNAc-6-sulfotransferase
isozymes, sulfation enzymes of sugar residues, between non-cancer
colorectal tissues and colorectal cancer tissues or colorectal
adenoma tissues, as a result of investigations. Then the inventors
found that colorectal cancers and adenomas could be detected
specifically by assaying 6-sulfated sugar residues, which are
synthesized only by HEC-GlcNAc6ST, but not by GlcNAc6ST-1 nor by
I-GlcNAc6ST, in tissues of patients and in fecal samples.
Previously, many antibodies such as AG223 (Biochem. (Tokyo),
124:670-678,1998), G152, G72, AG97, AG107, AG273, G2706, G27011,
G27039 (all above, J. Biol. Chem., 273:11225-11233,1998) are known
as those reacting with GlcNAc-6-sulfated sugar residues. Meanwhile,
MECA-79 antibody (Pharmingen, catalog No. 09961D, distributor:
Becton Dickinson), which is commercially available as immunological
homing receptor of lymphocytes, is known to react in some way with
GlcNAc-6-sulfated sugar residues (reference 6).
[0019] The inventors made a search for an antibody, which have
little reactivity to such cells as normal colorectal epithelial
cells expressing GlcNAc-6-sulfated sugar residues, but have strong
reactivity to cells with such GlcNAc-6-sulfated sugar residues as
expressed in cancer cells, by screenings these antibodies. Then the
obtained antibodies were assayed against samples from patients and
were shown to be highly positive to colorectal cancer cells. The
above results lead to completion of the present invention.
[0020] In other words, the present invention is a method for
examining colorectal cancer and colorectal adenoma comprising
assaying the reactivity of an antibody to tissues, body fluid or
feces of patients, or extracts thereof, wherein said antibodies
react with such antigen that is present in cells expressing
HEC-GlcNAc6ST gene encoding GlcNAc-6-sulfotransferase and that is
absent or almost absent in cells expressing GlcNAc6ST-1 or
I-GlcNAc6ST gene. The antigen may be present in cells transduced
with HEC-GlcNAc6ST gene and is absent or almost absent in cells
transduced with GlcNAc6ST-1 gene or I-GlcNAc6ST gene.
[0021] The antigen may be represented by the formula below:
R1-Gal .beta.1-3/4(SO.sub.3-6) GlcNAc .beta.1-R2
where, R1 represents sugar residues added by other enzymes and is
not limited in structure. Gal .beta. represents .beta. galactose,
GlcNAc .beta. represents .beta. N-acetylglucosamine, Gal
.beta.1-3/4 represents binding of 1 position of Gal .beta. and 3
position and/or 4 position of GlcNAc .beta., (SO.sub.3-6)
represents addition of a sulfate group to 6 position of GlcNAc
.beta., R2 represents -3GalNAc .alpha., -3Gal .beta. or
-2Man.alpha. and binds to 1 position of GlcNAc .beta.. As the
antibody, MECA-79 antibody (Phamingen, catalog No. 09961D, shown in
FIGS. 6 and 7) is preferably used.
[0022] Also, the present invention is a method for examining
colorectal cancer and colorectal adenoma, which comprises
reactivity of MECA-79 antibody or its equivalent with tissues, body
fluid, feces or extracts thereof of examination subjects.
[0023] Furthermore, the present invention is any of said methods
comprising the reaction of a labeled probe to said antibody and
qualitative or quantitative assay of the label. The preferable
method for examination comprises fixing the antigens present in
tissues, body fluid or feces or extracts thereof of patients to a
membrane, reacting with the antibody, reacting with a labeled probe
and detecting the label. It is preferable to insert washing
procedures appropriately between the above processes. Said probe
includes anti-human-IgG, antibody, protein G, protein A, and
protein L. These probes are usually labeled. Said labels include a
radioactive isotope (125I) and enzymes (peroxidase, alkaline
phosphatase). An antibody with enzyme may involve observation of a
change (i.e. color change) by the reaction between the enzyme and
the substrate.
[0024] Moreover, the present invention is an antibody reacting
specifically with an antigen carrying sugar residues, which are
present in cells expressing HEC-GlcNAc6ST gene encoding
GlcNAc-6-sulfotransferase and are absent or almost absent in cells
expressing GlcNAc6ST-1 or GlcNAc6ST gene (excluding MECA-79
antibody). Still furthermore, the present invention is an antibody
reacting specifically with an antigen carrying sugar residues,
which are present in cells transduced with HEC-GlcNAc6ST gene
encoding GlcNAc-6-sulfotransferase and are absent or almost absent
in cells transduced with GlcNAc6ST-1 or GlcNAc6ST gene (excluding
MECA-79 antibody).
[0025] Still moreover, the present invention is an antibody
(excluding MECA-79 antibody) reacting specifically with an antigen
carrying sugar residues, which are present in tissues, body fluid
or feces of patients with colorectal cancers and colorectal
adenomas and expressed by the following general formula:
R1-Gal .beta.1-3/4 (SO.sub.3-6) GlcNAc .beta.1-R2
(in the formula, each note is the same to the above).
[0026] Also, the present invention is an examination reagent of
colorectal cancer and colorectal adenoma comprising any of these
antibodies, including MECA-79 antibody, as a main component.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows the results of flowcytometric analysis using
MECA-79 antibody on colorectal cancer cells (COLO201 cells) and on
normal colorectal epithelial cells (SW480 cells treated with
Tricostatine A). a shows the enzyme specificity of colorectal
cancer cells (COLO201 cells) and normal colorectal epithelial cells
(SW480 cells). b shows the results of flowcytometric analysis on
the reactivity of anti-6-sulfated sugar residues antibody with the
above 2 kinds of cells. The ordinate shows the cell frequency (the
number of cells) and the abscissa axis shows the fluorescence
(Arbitrary Unit).
[0028] FIG. 2 shows the result of the flowcytometric analysis on
the reactivity of MECA-79 antibody with cells transduced with
HEC-GlcNAc6ST gene, GlcNAc6ST-1 gene or I-GlcNAc6ST gene. The
ordinate shows the cell frequency (the number of cells) and the
abscissa axis shows the fluorescence (Arbitrary Unit).
[0029] FIG. 3 shows the photographs of tissues stained with MECA-79
antibody of a colorectal cancer patient. Ca shows a colorectal
cancer tissue and N shows non-cancer colorectum tissue.
[0030] FIG. 4 shows the photographs of tissues of colorectal cancer
and normal colorectum of a patient stained with MECA-79 antibody.
Ca shows cancer tissues and N shows non-cancer colorectum tissues
in a to c. N shows a non-adenoamotous colorectum tissue and A shows
adenomatous cells in d. The black part (brown in color) shows the
presence of antigens recognizable by the antibody and the gray part
(faint blue in color) shows control staining with methylene
blue.
[0031] FIG. 5 shows the reactivity of MECA-79 antibody with fecal
extracts of patients. Each row from above shows colorectal cancer 8
cases, colorectal adenoma 8 cases, benign 8 cases and healthy
control 8 cases, respectively.
[0032] FIG. 6 shows a catalog of MECA-79 antibody (Pharmingen,
catalog No. 09961D).
[0033] FIG. 7 shows a catalog of MECA-79 antibody (Pharmingen,
catalog No. 09961D).
DETAILED DESCRIPTION OF THE INVENTION
[0034] The structure of 6-sulfated sugar residues, which are little
synthesized by GlcNAc6ST-1 or by I-GlcNAc6ST, but are synthesized
only by HEC-GlcNAc6ST is expressed as the following general
formula:
R1-Gal .beta.1-3/4 (SO.sub.3-6) GlcNAc .beta.1-R2.
[0035] GlcNAc.beta., which is the substrate of
GlcNAc6-sulfotransferase in a body, is carried by various sugar
residue carrier. R2 shows the carrier.
[0036] HEC-GlcNAc6ST has been known to transfer sulfate residues to
all kinds of GlcNAc.beta.-R2 previously tested according to both
our research and other people's research (references 4 and 7). In
contrast, GlcNAc6ST-1 and I-GlcNAc6ST transfer sulfate residues
only to such GlcNAc.beta.-R2 as accompanied with a specific form of
R2. The case, which HEC-GlcNAc6ST but not GlcNAc6ST-1 nor
I-GlcNAc6ST can transfer sulfate residues, is known as a case when
R2 is -3GalNAc.alpha. (the structure after sulfation is
SO.sub.3.sup.--6GlcNAc.beta.1-3GalNAc.alpha.), a case when R2 is
-3Gal.beta. (the structure after sulfation is
SO.sub.3.sup.--6GlcNAc.beta.1-3Gal.beta.) and a case when R2 is
-2Man.alpha. (the structure after sulfation is
SO.sub.3.sup.--6GlcNAc.beta.1-2Man.alpha.) (J. Biol. Chem., 277:
3979-3984, 2002 and Glycobiology, 12: 379-388, 2002). In the
examination method of the present invention, a specific antibodies
to any of the three cases and antibodies cross reacting to all
three sugar residues may be usable.
[0037] GlcNAc-6-sulfotransferase adds sulfate group to distal
GlcNAc of sugar residues and synthesizes 6-sulfated GlcNAc (i.e.
SO.sub.3.sup.--6GlcNAc) intra-cellularly. However, after synthesis
of distal 6-sulfated GlcNAc of sugar residues, the modified sugar
residues are further added sugar residues (R1) by other enzyme
groups intra-cellularly, and a large variety of the structure and
antigenicity of the sugar residues are finally synthesized and
produced from cells. Generally the structure added to 6-sulfated
GlcNAc is Gal.beta.1-4 and Gal.beta.1-3 (referred to as
Gal.beta.1-3/4). Moreover, it is known that NeuAc.alpha.2-3/6,
SO.sub.3.sup.--3/6, and Fuc .alpha.1-2/3/4 are added to the
6-sulfated GlcNAc. The R1 part is added after the synthesis of
6-sulfated GlcNAc by GlcNAc-6-sulfotransferase. Therefore, R1 part
is not related to the substrate specificity of such
GlcNAc-6-sulfotransferases as HEC-GlcNAc6ST, GlcNAc6ST-1 and
I-GlcNAc6ST.
[0038] The antigens with the above sugar residues are present in
cancer tissues obtained from colorectal cancer patients by biopsy
or by surgical operation, and present in such samples as serum,
ascites and feces containing the antigens. Also, the antigen may be
easily extracted from these samples using phosphate buffered
saline. Also, the 5 antibody against this sugar residue antigen
could be obtained by known arts producing antibodies (e.g. Methods
in Enzymology, 312: 160-179, 2000; Methods in Molecular Biology,
199: 203-218, 2002 et al.).
[0039] The GlcNAc-6-sulfated sugar residues detected by the present
invention are positive not only in colorectal cancers but also in
colorectal adenomas, which are regarded as the birthplace of
colorectal cancers. Therefore, using the sugar residues as a target
of screening test, a group of patients with colorectal adenomas,
for whom endoscopic examination or follow-up is required, could be
detected. Compared with the occult blood test with anti-hemoglobin
antibody, which is currently used for screening of colorectal
cancers, the present method has higher yield of detection of
colorectal adenomas. Also, 6-sulfated sugar residues are originally
abundant in right-half of colorectum, if a colorectum is separated
in right-half and left-half, therefore the above method of
diagnosis is particularly useful for the diagnosis of colorectal
cancers and colorectal adenomas generated in right-half colorectum.
Since colorectal cancers in right-half are not frequently positive
by fecal occult blood test using anti-hemoglobin antibody,
concomitant use of the present method may contribute to increased
yield of positive diagnosis in right-half colorectal cancers.
[0040] The following examples are provided to illustrate the
present invention, but are not intended to limit the scope
thereof.
Example 1
[0041] Gene expression of GlcNAc-6-sulfotransferase isozymes was
examined by RT-PCR on human-derived colorectal cancer cells and on
normal colorectal epithelial cells. In the RT-PCR analysis, PCR
primers for detection of the expression of HEC-GlcNAc6ST gene
(Genebank, AF131235) are synthetic oligonucleotides of SEQ ID NO. 1
for upper strand side and those of SEQ ID NO. 2 for lower strand
side (Tm=59.degree. C.), those for GlcNAc6ST-1 gene (Genebank,
AB011451) are synthetic oligonucleotides of SEQ ID NO. 3 for upper
strand side and those of SEQ ID NO. 4 for lower strand side
(Tm=62.degree. C.) and those for I-GlcNAc6ST gene (Genebank,
AF176838) are synthetic oligonucleotides of SEQ ID NO. 5 for upper
strand side and those of SEQ ID NO. 6 for lower strand side
(Tm=60.degree. C.). The results are shown in FIG. 1a. COLO201 cells
are typical cells showing colorectal cancer pattern, which
expresses strongly HEC-GlcNAc6ST gene and little GlcNAc6ST-1 and
I-GlcNAc6ST genes. TSA-SW480 cells treated SW480 cells with
tricostatinA are typical cells showing normal epithelial pattern,
which little expresses HEC-GlcNAc6ST gene, but significantly
expresses GlcNAc6ST and I-GlcNAc6ST genes.
[0042] Then, a number of anti6-sulfated sugar residues antibody
were screened based on the reactivity to the above two kinds of
cells. In other words, antibodies, which react well with COLO201
cells and not react with TSA-SW480 cells, were searched. The
screening of the reactivity between cells and antibodies was
performed by flowcytometric analysis by use of FACScan (Becton
Dickinson) stained cells with indirect fluorescent antibody method
(the first antibody 1.0 .mu.g/ml, 4.degree. C., 30 min, the second
antibody rabbit anti-rat IgM antibody (Zymed Laboratories),
4.degree. C., 30 min). A typical result of the analysis is shown in
FIG. 1b. MECA-79 antibody (Pharmingen, catalog No. 09961D,
Distributor: Becton Dickinson) showed strong reactivity with
COLO201 cells, but showed little reactivity with TSA-SW480 cells.
The above result showed that MECA-79 antibody is a preferable
antibody for diagnosis of colorectal cancers. While, G72 antibody
used as a control (J. Biol. Chem., 273: 11225-11233, 1998) reacted
significantly with both COLO201 cells and TSA-SW480 cells and is
not appropriate for diagnosis of colorectal cancers.
Example 2
[0043] In this example, cells transduced with HEC-GlcNAc6ST gene,
GlcNAc6ST-1 gene or I-GlcNAc6ST gene were prepared. Then
flowcytometric analysis for these cells was performed using MECA-79
antibody.
[0044] For the preparation of cells transduced with HEC-GlcNAc6ST
gene, the gene (Genebank, AF131235) inserted into pCDNA3.1 vector
was used. For those with GlcNAc6ST-1 gene, the gene (Genebank,
AB011451) inserted into pIRES1hygro vector was used. For those with
I-GlcNAc6ST gene, the gene (Genebank, AF176838) inserted into
pCDNA3.1 vector was used. The flowcytometric analysis was performed
as in Example 1.
[0045] The results are shown in FIG. 2. MECA-79 antibody reacted
strongly with those cells transduced with HEC-GlcNAc6ST, but
reacted with slightly those cells transduced with GlcNAc6ST gene or
I-GlcNAc6ST gene.
Example 3
[0046] Colorectal cancer tissues derived from patients (31 cases)
were stained with immunohistological staining using MECA-79
antibody. For immunohistological staining, frozen sections with 10
.mu.m thick were used, 1.0 .mu.g/ml MECA-79 antibody was used as
the first antibody, and a reagent kit (Vectastain) of Vecta Co.
containing anti-rat IgM antibody was used as the second antibody
according to the protocol of the company.
[0047] The results are shown in FIG. 3. The antibody does not react
with non-cancer colorectal mucosa (N) (0 case/31 ca31 cases, 32%).
Positive reaction rate is higher for cancers in right-half
colorectum (60%) and lower for those in left-half colon (19%).
[0048] A typical stained photographs are shown in FIG. 4.
[0049] FIG. 4a shows the stained photograph of a colorectal cancer
tissue (ca) and a colorectal non-cancer tissue (N) of a patient.
The colorectal cancer tissue is strongly stained but the non-cancer
tissue is little stained.
[0050] FIG. 4b shows the photograph of the same tissue stained
using AG107 antibody.
[0051] Since AG107 antibody reacts with general GlcNAc-6-sulfated
sugar residues, the non-cancer tissue (N) was stained well much
more than the cancer tissue (Ca) in contrast to 4a and
GlcNAc-6-sulfated sugar residues only could not be used for the
specific detection of cancer tissues. Namely, use of such antibody
as MECA-79, which detects the specific GlcNAc-6-sulfated sugar
residues abundant in colorectal cancers and colorectal adenomas,
could be applied for the specific detection.
[0052] FIG. 4c shows the example of the expression of the sugar
residues in a colorectal cancer tissue derived from right-half
colorectum. That the cancer tissue is strongly stained shows strong
expression of the sugar residues.
[0053] FIG. 4d shows the expression in an adenomatous polyposis
coli. N shows non-adenomatous colorectal tissue and A shows
adenomatous cells. The adenomatous part of A is well stained by
MECA-79 antibody. GlcNAc-6-sulfated sugar residues, which could be
detected by MECA-79 antibody, are expressed abundantly in
non-cancer adenomatous polyposis as well as in cancer tissues.
Adenomatous polyposis coli, which is benign by itself but is
regarded as the birthplace of colorectal cancer, could be detected
credibly.
Example 4
[0054] In this example, an enzyme-linked immunosorbent assay, which
is a simple qualitative test and uses MECA-79 antibody for the
reaction with fecal extracts of colorectal cancer patient,
confirmed the emergence of GlcNAc-6-sulfated sugar residues in
feces of patients. Sometimes sugar residue antigens are decomposed
by enzymes secreted from fecal bacterium and could not be detected
in feces. Therefore, the confirmation is important for the
practicability of the present invention. 0.1 g human feces was
dispersed in 1 ml fecal extraction buffer (10 mM PBS, 1% BSA, pH
7.5), centrifuged at 8,000 g for 15 min at 4.degree. C., and the
supernatant was recovered. To 40 .mu.l supernatant, 120 .mu.l
extraction buffer was added and the sample was prepared. The sample
was blotted to a PVDF membrane (Immobilon, Milipor, Lot K2JN2659B)
by suction and the membrane was reacted with MECA-79 antibody,
rabbit anti-rat IgM antibody, POD labeled goat anti-rabbit IgG
antibody, and avidin-biotin complex solution, sequentially, after
blocked for nonspecific reactions, and stained in NTB solution.
[0055] The results are shown in FIG. 5.
[0056] Positive results are 4 cases in 8 cases of colorectal
cancers (50%) and 4 cases in 8 cases of colorectal adenomas (50%).
For benign disorders cases, 1 case gives a slight positive result
and almost all cases of normal healthy subjects are negative.
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