DESCRIPTION
Title of the Invention: Immunoassay of Cofilin 1 Protein
Technical Field
[0001]
The present invention relates to an immunoassay of cofilin 1
protein , an anti-cofilin 1 monoclonal antibody, a method for
determining whether or not gastrointestinal cancer is developing, and
a kit for cofilin 1 protein quantification.
Background Art
[0002]
Cofilin 1 (cofilin, non-muscle isoform 18 kDa phosphoprotein)
protein is a cytoskeleton binding protein belonging to the
ADF/COFILIN family and is one of the most highly conserved proteins
among mammals. The amino acid sequence of the cofilin 1 protein
has been determined for humans, mice, rats, chimpanzees, cattle, dogs,
and the like, and is known to show 98% or higher identity among these
species. It has been revealed that the cofilin 1 protein is involved in
various biological phenomena including regulation of cell morphology
and motility (Non-patent Literature 1), cytokinesis (Non-patent
Literature 2), endocytosis (Non-patent Literature 3), and the like.
Furthermore, the protein is also expressed at high levels in cancer
tissues and cancer cells from lung cancer (Non-patent Literature 4),
cancer of pancreas (Non-patent Literature 5), breast cancer
(Non-patent Literature 6), ovarian cancer (Non-patent Literature 7) or
hepatic cancer (Non-patent Literature 8). Involvement of the cofilin

1 protein in the progression of cancer has also been reported
(Non-patent Literature 9).
Prior Art Literature
Non-patent Literature
[0003]
Non-patent Literature 1: Bugyi B. et al., 2010, Annual Review of
Biophysics, Vol. 39, p.449-470
Non-patent Literature 2: Kaji N. et al., 2008, Journal of Biological
Chemistry, Vol. 283, p. 4983-4992
Non-patent Literature 3: Okreglak V. et al., 2007, The Journal of Cell
Biology, Vol. 178, p. 1251-1264
Non-patent Literature 4: Keshamouni VG. et al., 2006, Journal of
Proteome Research, Vol. 5, p.1143-1154
Non-patent Literature 5: Sinha P. et al., 1999, Electrophoresis, Vol.
20, p. 2952-2960
Non-patent Literature 6: Zhang Y. et al., 2010, The Journal of
International Medical Research, Vol. 38, p.1042-1048
Non-patent Literature 7: Martoglio A. et al., 2000, Molecular
Medicine, Vol. 6, p.750-765
Non-patent Literature 8: Ding S. et al., 2004, Proteomics, Vol. 4,
p.982-994
Non-patent Literature 9: Yamaguchi H. et al. 2007, Biochemica et
Biophysica Acta, Vol. 1773, p. 642-652
Summary of the Invention
Problem to be Solved by the Invention
[0004]
The present inventors have found in recent years that the blood

levels of the cofilin 1 protein are elevated in early gastric cancer
patients, and thus have developed a method for detecting gastric
cancer, by which gastric cancer can be detected at an early stage using
the cofilin 1 protein, or a mutant and/or a fragment of the cofilin 1
protein as a diagnostic marker useful for early detection of gastric
cancer. If the quantification of the cofilin 1 protein becomes
possible, this can be expected to lead to early detection and effective
clinical treatment for gastrointestinal cancer including gastric cancer.
[0005]
However, existing methods for detecting the cofilin 1 protein
are limited to non-quantitative immunoassays with low sensitivity
using such as the Western blot method. Development of a new
immunoassay for clinical application remains as an unsolved problem.
[0006]
Hence, an object of the present invention is to provide an
immunoassay for quantitatively detecting cofilin 1 protein with high
sensitivity.
Means for Solving the Problem
[0007]
As a result of intensive studies to achieve the above object, the
present inventors have found that the cofilin 1 protein can be
quantitatively detected with high sensitivity by combining 2 or more
types of monoclonal antibody, which separately specifically recognize
epitopes that are present on different specific peptide regions of the
amino acid sequence constituting the cofilin 1 protein, and then by
immunologically mesuring cofilin 1 and/or a fragment thereof. The
present invention has been completed based on the above findings and

provides the following (1) to (21).
[0008]
(1) An anti-cofilin 1 monoclonal antibody or a fragment thereof,
which specifically recognizes an epitope that is present in any one of
the following peptide regions:
[0009]
a) a peptide region comprising at least the amino acid sequence shown
in SEQ ID NO: 5 in the amino acid sequence shown in SEQ ID NO: 1;
b) a peptide region having a deletion, a substitution, or an addition of
1 or several amino acids with respect to the amino acid sequence of
the peptide region of a) above; and
c) a peptide region having 90% or more identity with the amino acid
sequence of the peptide region of a) above.
(2) The anti-cofilin 1 monoclonal antibody or a fragment thereof
according to (1), wherein in the light chain, CDR1 comprises the
sequence shown in SEQ ID NO: 8, CDR2 comprises the sequence
shown in SEQ ID NO: 9, and CDR3 comprises the sequence shown in
SEQ ID NO: 10, and in the heavy chain, CDR1 comprises the sequence
shown in SEQ ID NO: 11, CDR2 comprises the sequence shown in SEQ
ID NO: 12, and CDR3 comprises the sequence shown in SEQ ID NO:
13.
[0010]
(3) The anti-cofilin 1 monoclonal antibody or a fragment thereof
according to (1), wherein in the light chain, CDR1 comprises the
sequence shown in SEQ ID NO: 14, CDR2 comprises the sequence
shown in SEQ ID NO: 15, and CDR3 comprises the sequence shown in
SEQ ID NO: 16, and in the heavy chain, CDR1 comprises the sequence

shown in SEQ ID NO: 17, CDR2 comprises the sequence shown in SEQ
ID NO: 18, and CDR3 comprises the sequence shown in SEQ ID NO:
19.
[0011]
(4) An anti-cofilin 1 monoclonal antibody or a fragment thereof,
which specifically recognizes an epitope that is present in any one of
the following peptide regions:
[0012]
a) a peptide region comprising at least the amino acid sequence
shown in SEQ ID NO: 6 in the amino acid sequence shown in SEQ ID
NO: 1.
b) a peptide region having a deletion, a substitution, or an addition
of 1 or several amino acids with respect to the amino acid sequence of
the peptide region of a) above; and
c) a peptide region having 90% or more homology with the amino
acid sequence of the peptide region of a) above.
(5) The anti-cofilin 1 monoclonal antibody or a fragment thereof
according to (4), wherein in the light chain, CDR1 comprises the
sequence shown in SEQ ID NO: 20, CDR2 comprises the sequence
shown in SEQ ID NO: 21, and CDR3 comprises the sequence shown in
SEQ ID NO: 22, and in the heavy chain, CDR1 comprises the sequence
shown in SEQ ID NO: 23, CDR2 comprises the sequence shown in SEQ
ID NO: 24, and CDR3 comprises the sequence shown in SEQ ID NO:
25.
[0013]
(6) An anti-cofilin 1 monoclonal antibody or a fragment thereof,
which specifically recognizes an epitope that is present in any one of

the following peptide regions:
[0014]
a) a peptide region comprising at least the amino acid sequence
shown in SEQ ID NO: 7 in the amino acid sequence shown in SEQ ID
NO: 2;
b) a peptide region having a deletion, a substitution, or an addition
of 1 or several amino acids with respect to the amino acid sequence of
the peptide region of a) above; and
c) a peptide region having 90% or more identity with the amino acid
sequence of the peptide region of a) above.
(7) The anti-cofilin 1 monoclonal antibody or a fragment thereof
according to (6), wherein in the light chain, CDR1 comprises the
sequence shown in SEQ ID NO: 26, CDR2 comprises the sequence
shown in SEQ ID NO: 27, and CDR3 comprises the sequence shown in
SEQ ID NO: 28, and in the heavy chain, CDR1 comprises the sequence
shown in SEQ ID NO: 29, CDR2 comprises the sequence shown in SEQ
ID NO: 30, and CDR3 comprises the sequence shown in SEQ ID NO:
31.
[0015]
(8) An immunoassay of cofilin 1 protein , comprising measuring
cofilin 1 and/or a fragment thereof in a sample using 2 or more types
of anti-cofilin 1 monoclonal antibody and/or fragments thereof that
specifically recognize different epitopes on the amino acid sequence
of the cofilin 1 protein.
[0016]
(9) The immunoassay according to (8), wherein the above different
epitopes are present in the peptide regions consisting of the amino

acid sequences shown in SEQ ID NO: 1 and/or 2.
[0017]
(10) The immunoassay according to (9), wherein the above different
epitopes are present in a peptide region comprising at least the amino
acid sequence shown in SEQ ID NO: 3 in the peptide region consisting
of the amino acid sequence shown in SEQ ID NO: 1, and/or a peptide
region comprising at least the amino acid sequence shown in SEQ ID
NO: 4 in the peptide region consisting of the amino acid sequence
shown in SEQ ID NO: 2.
[0018]
(11) The immunoassay according to (9), wherein the above different
epitopes are present in the peptide regions comprising at least the
amino acid sequences shown in SEQ ID NO: 5 and/or 6 in the peptide
region consisting of the amino acid sequence shown in SEQ ID NO: 1,
and/or a peptide region comprising at least the amino acid sequence
shown in SEQ ID NO: 7 in the peptide region consisting of the amino
acid sequence shown in SEQ ID NO: 2.
[0019]
(12) The immunoassay according to (8), wherein the above different
epitopes are separately present in two peptide regions selected from
the peptide regions consisting of the amino acid sequences shown in
SEQ ID NOS: 5 to 7.
[0020]
(13) The immunoassay according to (9), wherein the above different
epitopes are present in a peptide region consisting of 6 or more and 21
or less continuous amino acids in the peptide regions consisting of the
amino acid sequences shown in SEQ ID NO: 1 and/or 2.

[0021]
(14) The immunoassay according to (8), wherein the above 2 or more
types of anti-cofilin 1 monoclonal antibody and/or fragments thereof
are selected from the anti-cofilin 1 monoclonal antibodies or
fragments thereof of (2), (3), (5), and (7).
[0022]
(15) The immunoassay according to (14), wherein the two types of
anti-cofilin 1 monoclonal antibody and/or fragment thereof constitute
any one of the following combinations of anti-cofilin 1 monoclonal
antibodies and/or fragments thereof:
[0023]
a) a combination of the anti-cofilin 1 monoclonal antibodies and/or
the fragments thereof separately described in (2) and (5);
b) a combination of the anti-cofilin 1 monoclonal antibodies and/or
fragments thereof separately described in (2) and (7);
c) a combination of the anti-cofilin 1 monoclonal antibodies and/or
fragments thereof separately described in (3) and (5); and
d) a combination of the anti-cofilin 1 monoclonal antibodies and/or
fragments thereof separately described in (5) and (7).

(16) The immunoassay according to any one of (8) to (15), wherein the
sample is blood, urine, cell supernatant, cell extract, tissue extract,
gastric juice, saliva, lymph fluid, lacrimal fluid, or seminal fluid.
[0024]
(17) A method for determining whether or not gastrointestinal cancer
is developing, comprising the steps of:
measuring the amounts of the cofilin 1 protein and/or a fragment
thereof in a sample derived from a subject and a healthy subject using

the immunoassay of any one of (8) to (16);
comparing the amounts of the cofilin 1 protein and/or a fragment
thereof measured in the above measurement step, and then determining
that the subject is affected by gastrointestinal cancer if the amount of
the cofilin 1 protein and/or a fragment thereof in the subject is
statistically significantly higher than that of the healthy subject.
[0025]
(18) The determination method according to (17), wherein the
gastrointestinal cancer is early gastrointestinal cancer.
[0026]
(19) The determination method according to (17) or (18), wherein the
gastrointestinal cancer is gastric cancer.
[0027]
(20) A kit for cofilin 1 protein quantification, containing 2 or more
types of anti-cofilin 1 monoclonal antibody and/or fragments thereof,
which specifically recognize different epitopes of the cofilin 1
protein.
[0028]
(21) The kit according to (20), containing 2 or more types of the
anti-cofilin 1 monoclonal antibody or the fragments thereof of any one
of (l)to (7).
[0029]
This description includes the disclosure of the description and
drawings of Japanese Patent Application No. 2010-274879, which is
base of the priority of the present application.
Effects of the Invention
[0030]

According to the immunoassay of the present invention, the
cofilin 1 protein in a sample can be quantitatively detected with high
sensitivity compared with conventional methods. Furthermore,
immunoassay can be conveniently completed with simple experimental
procedures.
[0031]
Moreover, according to the method of the present invention for
determining whether or not gastrointestinal cancer is developing,
whether or not a subject is affected by gastrointestinal cancer can be
determined at an early stage by measuring the amount of cofilin 1
contained in a sample such as blood of a subject.
Brief Description of the Drawings
[0032]
Fig. 1 shows the primary structures of deletion mutants used for
epitope analysis, and each peptide region and the sequence
identification number of the amino acid sequence thereof.
Fig. 2 shows the results of Western blotting performed for a
sample of a cell line expressing a human cofilin 1 protein. " + "
indicates the result for HEK293 cells in which pCMV-Mychuman
cofilin 1 was introduced, and "-" indicates the result for HEK293
cells in which only pCMV-Myc vector was introduced.
Figs. 3A-3E show the detection sensitivity for the cofilin 1
protein, as detected by sandwich ELISA using two different types of
anti-cofilin 1 monoclonal antibody.
Fig. 4 shows the results of evaluating the detection sensitivity
by sandwich ELISA for measurement of the cofilin 1 protein. Fig. 4B
is a graph showing an expanded view of the circular broken-line

portion in Fig. 4A. In Fig. 4B, the value indicated by an arrow is the
minimum detection limit of the ELISA method.
Fig. 5 is a graph showing the results of detecting the cofilin 1
protein in blood plasma of gastric cancer patients and healthy subjects
by sandwich ELISA.
Fig. 6 is a graph showing the results of detecting the cofilin 1
protein in sera of gastric cancer patients and healthy subjects by
sandwich ELISA.
Embodiments for Carrying Out the Invention
[0033]
1. Anti-cofilin 1 monoclonal antibody and fragment thereof
A 1st embodiment of the present invention relates to an
anti-cofilin 1 monoclonal antibody and a fragment thereof.
[0034]
1-1. Anti-cofilin 1 monoclonal antibody
"Cofilin 1" includes the human cofilin 1 protein consisting of
the amino acid sequence under GenBank accession NP005498.1 or a
natural mutant thereof, or mammalian orthologs or natural mutants
thereof showing 95% or more homology with the human cofilin 1
protein, such as a rat cofilin 1 protein consisting of the amino acid
sequence under GenBank accession NP_058843, a mouse cofilin 1
protein consisting of the amino acid sequence under GenBank
accession NP_031713.1, a chimpanzee cofilin 1 protein consisting of
the amino acid sequence under GenBank accession NP_001170183.1, a
cattle cofilin 1 protein consisting of the amino acid sequence under
GenBank accession NP_001015655, or a dog cofilin 1 protein
consisting of the amino acid sequence under GenBank accession

NP_533231.1.
[0035]
Here, the term "natural mutant" refers to a mutant existing in
nature, such as a mutant having a deletion, a substitution, or an
addition of one or several amino acids with respect to the above amino
acid sequences, and a mutant having 90% or more, 92% or more or
94% or more, preferably 95% or more, more preferably about 97% or
more, and further preferably about 99% or more identity with the
above amino acid sequences. The term "sequence identity" is the
percentage (%) of the number of identical amino acid residues in an
amino acid sequence with respect to the total number of amino acid
residues (including the number of gaps) of the other amino acid
sequence, when gaps are introduced or not introduced so as to
maximize the agreement between the two amino acid sequences
aligned. The term "several" refers to integers between 2 and 10,
such as integers of 2 to 7, 2 to 5, 2 to 4, and 2 to 3. Specific
examples of a natural mutant include mutants based on polymorphism
such as SNP (single nucleotide polymorphism) and splice mutants.
The above substitution is preferably conservative amino acid
substitution. If it is conservative amino acid substitution, the
relevant mutant can have a structure or properties substantially
equivalent to the cofilin 1 protein having any one of the above amino
acid sequences. The conservative amino acids include nonpolar
amino acids (glycine, alanine, phenylalanine, valine, leucine,
isoleucine, methionine, proline, and tryptophan) and polar amino acids
(amino acids other than nonpolar amino acids), charged amino acids
(acidic amino acids (aspartic acids and glutamic acid) and basic amino

acids (arginine, histidine, and lysine)) and uncharged amino acids
(amino acids other than charged amino acids), aromatic amino acids
(phenylalanine, tryptophan, and tyrosine), branched amino acids
(leucine, isoleucine, and valine), and aliphatic amino acids (glycine,
alanine, leucine, isoleucine, and valine).
[0036]
The term "monoclonal antibody" as used herein refers to a
single immunoglobulin, or a polypeptide comprising a framework
region (hereinafter, referred to as "FR") thereof and a
complementarity determining region (hereinafter, referred to as
"CDR"), which is capable of specifically recognizing and binding to a
specific antigen. Therefore, the term "anti-cofilin 1 monoclonal
antibody" of the present invention refers to a polypeptide capable of
specifically recognizing and binding to the cofilin 1 protein. The
expression "specifically recognizing and binding to" means that a
polypeptide has no or extremely weak cross-reactivity and neither
recognizes nor binds or almost never recognizes and binds to antigens
other than a target antigen.
[0037]
A typical immunoglobulin molecule is composed as a tetramer in
which two sets of polypeptide chains referred to as a heavy chain and
a light chain are interconnected via disulfide bonds. The heavy chain
consists of the N terminal heavy chain variable region (H chain V
region, hereinafter, referred to as "VH") and the C terminal heavy
chain constant region (H chain C region, hereinafter, referred to as
"CH"). The light chain consists of the N terminal light chain
variable region (L chain V region, hereinafter, referred to as "VL")

and the C terminal light chain constant region (L chain C region,
hereinafter, referred to as "CL"). VH and VL are particularly
important in that VH and VL are involved in antibody binding
specificity. VH and VL each consists of about 110 amino acid
residues and contains three CDRs (CDR1, CDR2, CDR3) directly
involved in binding specificity with an antigen and four FRs (FR1,
FR2, FR3, FR4) that function as a framework structure of the variable
region. CDRs are known to form a three-dimensional structure with
an antigen molecule, so as to determine antibody specificity (E. A.
Kabat et al, 1991, Sequences of proteins of immunological interest,
Vol. 1, eds. 5, NIH publication). Whereas the amino acid sequences
of constant regions are almost the same among intraspecies antibodies,
the amino acid sequences of CDRs have high variability among
antibodies, and thus CDRs are also referred to as hyper variable
regions. In a variable region, the above CDRs and FRs are placed in
order of, from the amino acid terminus to the carboxy terminal
direction, FR1, CDR1, FR2, CDR2, FR3, CDR3, and then FR4.
Within an immunoglobulin molecule, VL and VH face each other to
form a dimer, so as to form an antigen binding site. Examples of
known immunoglobulin classes include IgG, IgM, IgA, IgE, and IgD.
The antibody of the present invention may be of any class. A
preferable class is IgG.
[0038]
The anti-cofilin 1 monoclonal antibody of the present invention
is characterized by specifically recognizing an epitope that is present
in: (1) a peptide region comprising at least the amino acid sequence
shown in SEQ ID NO: 5 or 6 in the amino acid sequence shown in SEQ

ID NO: 1; (2) a peptide region comprising at least the amino acid
sequence shown in SEQ ID NO: 7 in the amino acid sequence shown in
SEQ ID NO: 2; (3) a peptide region having a deletion, a substitution,
or an addition of 1 or several amino acids with respect to the amino
acid sequence of the peptide region of (1) or (2) above; or (4) a
peptide region having 90% or more identity with the amino acid
sequence of the peptide region of (1) or (2) above.
[0039]
The amino acid sequence shown in SEQ ID NO: 1 is the amino
acid sequence of the C terminal peptide region (C-terminal 5 amino
acids in M region and C1-C3 regions in Fig. 1) of the human cofilin 1
protein, corresponding to positions 122 to 166 when the initiator
methionine is located at position 1. The amino acid sequence shown
in SEQ ID NO: 5 is the amino acid sequence of the C1 region in Fig.
1, corresponding to positions 158 to 166 of the human cofilin 1
protein. The amino acid sequence shown in SEQ ID NO: 6 is the
amino acid sequence of the C3 region in Fig. 1, corresponding to
positions 127 to 146 of the human cofilin 1 protein. In the peptide
region consisting of the amino acid sequence shown in SEQ ID NO: 5
or 6, an epitope effective for preparing an anti-cofilin 1 antibody with
high-sensitivity is present.
[0040]
Specific examples of the anti-cofilin 1 monoclonal antibody
recognizing an epitope that is present in the C1 region shown in SEQ
ID NO: 5 include antibody clones represented by antibody clone names
1E2 and 2C4 in Table 1 in Example 3 described later. The 1E2 clone
is characterized in that, in the light chain, CDR1 consists of the

sequence shown in SEQ ID NO: 8, CDR2 consists of the sequence
shown in SEQ ID NO: 9, and CDR3 consists of the sequence shown in
SEQ ID NO: 10, and in the heavy chain, CDR1 consists of the
sequence shown in SEQ ID NO: 11, CDR2 consists of the sequence
shown in SEQ ID NO: 12, and CDR3 consists of the sequence shown in
SEQ ID NO: 13. Moreover, the 2C4 clone is characterized in that in
the light chain, CDR1 consists of the sequence shown in SEQ ID NO:
14, CDR2 consists of the sequence shown in SEQ ID NO: 15, and
CDR3 consists of the sequence shown in SEQ ID NO: 16, and in the
heavy chain, CDR1 consists of the sequence shown in SEQ ID NO: 17,
CDR2 consists of the sequence shown in SEQ ID NO: 18, and CDR3
consists of the sequence shown in SEQ ID NO: 19.
[0041]
Specific examples of the anti-cofilin 1 monoclonal antibody
recognizing an epitope that is present in the C3 region shown in SEQ
ID NO: 6 include an antibody clone represented by antibody clone
name of 4E12 in Table 1. The 4E12 clone is characterized in that, in
the light chain, CDR1 consists of the sequence shown in SEQ ID NO:
20, CDR2 consists of the sequence shown in SEQ ID NO: 21, and
CDR3 consists of the sequence shown in SEQ ID NO: 22, and in the
heavy chain, CDR1 consists of the sequence shown in SEQ ID NO: 23,
CDR2 consists of the sequence shown in SEQ ID NO: 24, and CDR3
consists of the sequence shown in SEQ ID NO: 25. In addition, the
C3 region is composed of only 9 amino acids, and thus the C3 region
likely constitutes an epitope itself. Furthermore, the amino acid
sequence shown in SEQ ID NO: 2 is the amino acid sequence of the N
terminal peptide region (N1 and N2 regions and N terminal 5 amino

acids in M region in Fig. 1) of the human cofilin 1 protein,
corresponding to positions 1 to 54 when initiator methionine is
located at position 1. The amino acid sequence shown in SEQ ID
NO: 7 is the amino acid sequence of the N2 region in Fig. 1,
corresponding to a sequence of positions 29 to 49 of the human cofilin
1 protein. In the peptide region consisting of the amino acid
sequence shown in SEQ ID NO: 7, an epitope effective for preparing
an anti-human cofilin 1 antibody with high sensitivity is present.
[0042]
Specific examples of the anti-cofilin 1 monoclonal antibody
recognizing an epitope in the N2 region shown in SEQ ID NO: 7
include an antibody clone represented by antibody clone name of 4F12
in Table 1. The 4F12 clone is characterized in that, in the light
chain, CDR1 consists of the sequence shown in SEQ ID NO: 26, CDR2
consists of the sequence shown in SEQ ID NO: 27, and CDR3 consists
of the sequence shown in SEQ ID NO: 28, and in the heavy chain,
CDR1 consists of the sequence shown in SEQ ID NO: 29, CDR2
consists of the sequence shown in SEQ ID NO: 30, and CDR3 consists
of the sequence shown in SEQ ID NO: 31.
[0043]
Antibodies useful in the present invention may be derived from
all animal sources including birds and mammals. Examples thereof
include mice, rats, guinea pigs, rabbits, goats, donkeys, sheep,
camels, horses, chickens and humans. In general, antibodies of mice,
rats, or rabbits are preferably used.
[0044]
Moreover, the "antibody" in the present invention may be a

multispecific antibody. The term "multispecific antibody" refers to a
multivalent antibody; that is, an antibody having multiple antigen
binding sites within one molecule, in which each antigen binding site
binds to a different epitope. In the case of a bispecific antibody such
as IgG having two antigen binding sites, each antigen binding site
binds to a different epitope. In the present invention, a multispecific
antibody has antigen binding sites, so as to be able to bind to different
epitopes that are present in the cofilin 1 protein. These antibodies can
be obtained by artificially altering IgG or the like by a known method
using a DNA recombination technique.
[0045]
1-2. Anti-cofilin 1 monoclonal antibody fragment
The term "a fragment thereof" in "monoclonal antibody or a
fragment thereof" as used herein refers to a partial fragment of the
above monoclonal antibody, and specifically refers to a polypeptide
chain or a complex thereof having activity substantially equivalent to
the antigen-specific binding activity of the antibody. For example, it
may involve an antibody portion containing at least one antigen
binding site described above; that is, a polypeptide chain or a complex
thereof having at least a set of VL and VH. Specific examples
thereof include many sufficiently characterized antibody fragments or
the like obtained by cleaving an immunoglobulin with various
peptidases. More specific examples thereof include Fab, F(ab')2, and
Fab'. Fab is a fragment that is generated by cleaving an IgG
molecule with papain at a site closer to the N-terminal side than to the
hinge disulfide bonds, which is composed of a polypeptide consisting
of VH and CHI adjacent to VH among 3 domains (CH1, CH2, CH3)

constituting CH and a light chain. F(ab')2 is a Fab' dimer that is
generated by cleaving an IgG molecule with pepsin at a site closer to
the C-terminal side than to the hinge disulfide bonds. Fab' has an H
chain that is somewhat longer than that of Fab since Fab' contains the
hinge region, but Fab' has a structure substantially equivalent to that
of Fab (Fundamental Immunology, Paul ed., 3d ed., 1993.). Fab' can
be obtained by reducing F(ab')2 under mild conditions, so as to cleave
the hinge disulfide bonds. All of these antibody fragments contain
antigen binding sites and are capable of specifically binding to
antigens (specifically, in the present invention, the cofilin 1 protein
or a fragment thereof).
[0046]
The monoclonal antibody fragment of the present invention can
be chemically synthesized or synthesized using a recombinant DNA
technique. Examples thereof are antibody fragments that are newly
synthesized using the recombinant DNA technique. Specific
examples of the monoclonal antibody fragment include, but are not
limited to, a monomeric polypeptide molecule prepared by artificially
linking one or more VLs and one or more VHs of the monoclonal
antibody of the present invention via a linker peptide or the like
having an appropriate length and an appropriate sequence, and a
multimeric polypeptide thereof. Examples of such a polypeptide
include single-stranded Fv (scFv: single chain Fragment of variable
region)(see Pierce catalog and Handbook, 1994-1995, Pierce Chemical
co., Rockford, IL), and synthetic antibodies such as a diabody, a
triabody, or a tetrabody. In an immunoglobulin molecule, VL and VH
are generally located on separate polypeptide chains (L chain and H

chain, respectively). Single-stranded Fv is a synthetic antibody
fragment prepared by ligating these variable regions with a flexible
linker having a sufficient length, so as to have a structure in which
one polypeptide chain contains VL and VH. Within single-stranded
Fv, both variable regions can self-assemble to form one functional
antigen binding site. Single-stranded Fv can be obtained by
incorporating recombinant DNA encoding the Fv into a phage
genome using a known technique for expression. A diabody is a
molecule having a structure based on the dimeric structure of
single-stranded Fv (Holliger et al, 1993, Proc. Natl. Acad. Sci U.S.A.,
90: 6444-6448). For example, when the length of the above linker is
shorter than about 12 amino acid residues, two variable sites within
the single-stranded Fv are unable to self-assemble. However, through
the formation of a diabody and specifically interaction between the
two single-stranded Fvs, VL of one of the Fv chains becomes possible
to self-assemble with VH of the other Fv chain, so that two functional
antigen binding sites can be formed (Marvin et al, 2005, Acta
Pharmacol. Sin., 26: 649-658). Furthermore, a cysteine residue is
added to the C terminus of single-stranded Fy, making it possible to
form a disulfide bond of the two Fy chains, and thus a stable diabody
can also be formed (Alafsen et al, 2004, Prot. Engr. Des. Sel., 17:
21-27). As described above, a diabody is a divalent antibody
fragment. The two antigen binding sites thereof are not required to
bind to the same epitope and they may have bispecificity by which
they recognize and specifically bind to different epitopes. A triabody
and a tetrabody have, similarly to a diabody, a trimeric structure and a
terameric structure, respectively, on the basis of the single-stranded

Fv structure. Such a triabody and a tetrabody are trivalent and
tetravalent antibody fragments, respectively, or may be multispecific
antibodies. Moreover, the antibody fragment of the present invention
may be an antibody fragment that is identified using a phage display
library (e.g., see McCafferty et al., 1990, Nature, Vol. 348, 522-554)
and have antigen binding capacity. In addition to the literature, see
Kuby, J., Immunology, 3rd Ed., 1998, W. H. Freeman & Co., New York,
for example.
[0047]
1-3. Other characteristics of anti-cofilin 1 monoclonal antibody and
fragment thereof
The antibody or a fragment thereof of the present invention can
be modified. The modification as used herein includes any of
functional modification (e.g., glycosylation) required for the antibody
or a fragment thereof of the present invention to have activity of
specifically binding to the cofilin 1 protein, and labeling required for
detection of the antibody or a fragment thereof of the present
invention. Examples of the aforementioned labels for the antibodies
include fluorescent dyes (FITC, rhodamine, Texas Red, Cy3, and Cy5),
fluorescent proteins (e.g., PE, APC, and GFP), enzymes (e.g.,
horseradish peroxidase, alkaline phosphatase, and glucose oxidase),
and biotin or (strept)avidin. Furthermore, glycosylation of the
antibody of the present invention may be altered to adjust the
antibody affinity for a target antigen. Such modification can be
achieved by, for example, changing one or more glycosylation sites
within the antibody sequence. More specifically, one or more amino
acid substitutions are introduced into an amino acid sequence

constituting one or more glycosylation sites within FR, so as to
remove the glycosylation sites, so that the loss of glycosylation at the
sites can be realized, for example. Such deglycosylation is effective
for increasing antibody affinity for an antigen (U.S. Patent Nos.
5714350 and 6350861).
[0048]
A monoclonal antibody or a fragment thereof to be used in the
present invention is preferably verified in advance for cross-reactivity
with other antigens (proteins or fragments thereof) before use in order
to confirm the specificity to the cofilin 1 protein or a fragment
thereof. Regarding the antibody or a fragment thereof of the present
invention, examples of an antigen, for which cross-reactivity should
be confirmed, include, proteins belonging to the ADF/COFILIN family
and particularly a cofilin 2 protein structurally analogous to the
cofilin 1 protein. The cross-reactivity of an antibody or a fragment
thereof to be used in the present invention is more preferably
confirmed in advance for other proteins sharing a common partial
structure with the cofilin 1 protein, other than the above proteins.
For confirmation of cross-reactivity, an ELISA method using the
cofilin 1 protein as an antigen can be employed. By coexisting with
another antigen protein to be confirmed for cross-reactivity at a
reaction site of an antibody to be tested for reaction specificity; that
is, a site for reaction of an anti-cofilin 1 monoclonal antibody and a
fragment thereof with the cofilin 1 protein, cross-reactivity can be
confirmed based on observation of the competitive state of the two.
Screening can be rapidly performed by such a method for confirmation
of cross-reactivity using the principle of competitive inhibition, since

the preparation of reaction systems is not required for all antigens.
[0049]
1-4. Method for preparing anti-cofilin 1 monoclonal antibody and
hybridoma
The anti-cofilin 1 monoclonal antibody of the present invention
or a hybridoma producing the antibody can be prepared by the method
described below. Examples of the method are not limited to this
method and the anti-cofilin 1 monoclonal antibody and such a
hybridoma can also be prepared by all other methods known in the art.
[0050]
1-4-1. Method for preparing anti-cofilin 1 monoclonal antibody
Methods for preparing an anti-cofilin 1 monoclonal antibody
specifically binding to an epitope that is present in a peptide region
consisting of any one of the amino acid sequences shown in SEQ ID
NOS: 1 to 7 from among amino acid sequences constituting the cofilin
1 protein are: a method that involves preparing a monoclonal antibody
using the full-length cofilin 1 protein as an immunogen, and then
screening for an antibody that specifically binds to a peptide region
consisting of any one of the amino acid sequences shown in SEQ ID
NOS: 1 to 7; and a method for preparing a monoclonal antibody using
in advance a peptide that consists of a cofilin 1 protein fragment
shown in SEQ ID NOS: 1 to 7, as an immunogen.
[0051]
(1) Preparation of cofilin 1 protein
The cofilin 1 protein to be used as an immunogen (antigen) can
be prepared by the following method, for example.
[0052]

The cofilin 1 protein may be of a natural, a recombinant, or a
synthetic cofilin 1 protein synthesized by, e.g., peptide synthesis,
chemically synthesizing the whole or a part of the amino acid
sequence. A natural cofilin 1 protein can be recovered from a sample
such as a body fluid (e.g., blood or urine) or a culture supernatant of
cultured cells by known protein separation and purification techniques
such as gel chromatography, ion exchange chromatography, or affinity
chromatography. A recombinant cofilin 1 protein can be expressed in
microorganisms, insect cells, or animal cells into which DNA
encoding the protein has been introduced and then recovered from the
cells using known protein separation and purification techniques. A
synthetic cofilin 1 protein can be synthesized by a technique known in
the art (e.g., a solid phase peptide synthesis method) using, for
example, published information in amino acid sequence of cofilin 1
protein. To the thus synthesized cofilin 1 protein, a carrier protein
such as KLH (keyhole lympet hemocyanin), OVA (ovalbumin), or BSA
(bovine serum albumin) may be ligated.
[0053]
When the cofilin 1 protein fragments shown in SEQ ID NOS: 1
to 7 are used as immunogens, any one of a natural cofilin 1 protein
fragment, a recombinant cofilin 1 protein fragment, or a synthetic
cofilin 1 protein fragment can also be used herein. As such a cofilin
1 protein fragment, an oligo peptide or a polypeptide comprising, in
the sequences shown in SEQ ID NOS: 1 to 7, continuous 6 or more, 7
or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13
or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more,
19 or more, 20 or more, 21 or more, 22 or more, or 23 or more amino

acid residues can be used as an antigen.
[0054]
When a natural cofilin 1 protein fragment is used as an
immunogen, for example, the purified cofilin 1 protein is treated with
appropriate protease such as trypsin, the peaks are fractionated with a
reverse phase column, the amino acid sequence of a peptide contained
in each peak was determined with a mass spectrometer, and then a
peak corresponding to a partial sequence shown in SEQ ID NOS: 1 to
7 or a part thereof can be used as an immunogen.
[0055]
When partial amino acid sequences of a recombinant cofilin 1
protein are used as immunogens, among the above DNA sequences
encoding the cofilin 1 protein, the partial amino acid sequences shown
in SEQ ID NOS: 1 to 7 or partial DNA sequences encoding portions
thereof are inserted to expression vectors in a manner similar to that
for preparation of the full-length cofilin 1 protein, the vectors are
introduced into various cells, and thus recombinant cofilin 1 proteins
comprising the partial amino acid sequences shown in SEQ ID NOS: 1
to 7 or portions thereof can be obtained.
[0056]
Preparation of recombinant cofilin 1 protein fragments
(hereinafter, referred to as "cofilin 1 protein fragments") shown in
SEQ ID NOS: 1 to 7 is as described in detail below.
[0057]
(a) Preparation of polynucleotide encoding recombinant cofilin 1
protein fragment
As a vector to be used for expression of a cofilin 1 protein

fragment, a phage or a plasmid capable of autonomously replicating in
a host microorganism can be used. Examples of such a plasmid
include Escherichia coli-derived plasmids (e.g., pET30a, pGEX6p,
pUC118, pUC119, pUC18, and pUC19), Bacillus subtilis-derived
plasmids (e.g., pUB 110 and pTP5) and yeast-derived plasmids (e.g.,
YEp13, YEp24, and YCp50). Examples of a phage include λ phages
(e.g., λ gt11 and λ ZAP). Furthermore, animal viruses such as a
vaccinia virus and insect virus vectors such as baculovirus can also be
used herein.
[0058]
A method that is employed for insertion of a polynucleotide
encoding a cofilin 1 protein fragment into the above vector involves,
for example, cleaving the purified polynucleotide with an appropriate
restriction enzyme, and then ligating the resultant into a vector
cleaved with an appropriate restriction enzyme, with the use of DNA
ligase or the like.
[0059]
(b) Introduction of cofilin 1 protein fragment expression vector into
host
The obtained cofilin 1 protein fragment expression vector is
introduced into a host capable of expressing the expression vector,
and then a cofilin 1 protein fragment-expressing transformant is
obtained. A host to be used herein is not particularly limited, as long
as it is a host appropriate for a vector to be used herein and can
express the cofilin 1 protein. For example, bacteria (e.g.,
Escherichia coli and Bacillus subtilis), yeast, insect cells, animal
cells (COS cells and CHO cells (Journal of immunology, 1998, Vol.

160, 3393-3402)), and the like are preferably used. A method for
introducing the above vector into bacteria is not particularly limited,
as long as it is a known method for introducing the vector into
bacteria. Examples thereof include a heat shock method, a method
using calcium ion, and an electroporation method. All of these
techniques are known in the art and described in various literatures.
For example, see Sambrook, J. et. A1., 1989, Molecular Cloning: A
Laboratory Manual Second Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York. Also, for transformation of animal
cells, a lipofectin method (PNAS, 1989, Vol. 86, 6077), (PNAS, 1987,
Vol. 84, 7413), an electroporation method, a calcium phosphate
method (Virology, 1973, Vol. 52, 456-467), a DEAE-Dextran method
and the like are preferably employed herein.
[0060]
When a bacterium is used as a host, a cofilin 1 protein fragment
expression vector is preferably autonomously replicable within the
bacterium, and at the same time is preferably composed of a promoter
sequence, a ribosome binding sequence, a DNA sequence encoding a
cofilin 1 protein fragment, and a transcription termination sequence.
Also, a gene encoding a promoter-controlling regulatory factor may
also be contained. Any promoter can be used herein as long as it is
capable of functioning in a host such as Escherichia coli.
[0061]
When eukaryotic cells such as yeast, animal cells, or insect cells
are used as host cells, a cofilin 1 protein fragment-expressing
transformant can be obtained according to techniques known in the art
similarly. A cofilin 1 protein fragment expression vector to be used

in eukaryotic cells contains, in addition to a promoter sequence and a
DNA sequence encoding a cofilin 1 protein fragment, a cis element,
such as an enhancer, splicing signals (e.g., donor site, acceptor site,
and branch point), a poly A addition signal, a selection marker
sequence, a ribosome binding sequence (SD sequence), and the like
that are ligated as necessary.
[0062]
(c) Culture of transformant and expression of recombinant cofilin 1
protein fragment
Subsequently, the above prepared transformant is cultured. A
method for culturing transformant in a medium is according to a
general method that is employed for culturing a host. For example,
when a bacterium is used as a host, a medium contains a carbon
source, a nitrogen source, inorganic salts, and the like that are
assimilable by bacteria and enables bacterial growth and
proliferation. The examples of such a medium are not particularly
limited. Either natural medium or synthetic medium can be used
herein. A more specific example thereof is, but is not limited to, an
LB medium. Furthermore, for selective culture of a transformant,
antibiotics such as ampicillin or tetracycline may be added into a
medium if necessary. Culture is generally performed under aerobic
conditions such as aeration-agitation culture at 37°C for 6 to 24
hours. During the culture period, the pH is preferably maintained at
around neutral pH. pH adjustment is performed using inorganic or
organic acid, an alkaline solution, or the like. When a transformant
is an animal cell such as a CHO cell, host cells may be seeded into
DMEM (Gibco) at 1 × 105 cells/mL and then cultured in a 5% CO2

incubator at 37°C. Antibiotics such as ampicillin or tetracycline may
also be added to a medium during culture, if necessary.
[0063]
When the above cofilin 1 protein fragment expression vector is a
protein expression-inducing vector containing a protein expression
control system (e.g., when a host is a bacterium, examples of the
system are a repressor gene and an operator, etc.), the above
transformant should be subjected to predetermined treatment so as to
induce the expression of the cofilin 1 protein fragment. Methods for
inducing expression differ depending on protein expression control
systems contained in vectors, and thus induction treatment appropriate
for the relevant system is performed. For example, a protein
expression control system that is most generally employed for a
protein expression-inducing vector using a bacterium as a host
comprises a lac repressor gene and a lac operator. This system can
induce expression via IPTG (isopropyl-1-tio-P-D-galactoside)
treatment. For expression of the cofilin 1 protein of interest in a
transformant having a cofilin 1 protein expression vector that contains
the system, an appropriate amount of IPTG (e.g., a final concentration
of 1 mM) may be added into the medium.
[0064]
(d) Extraction and/or recovery of recombinant cofilin 1 protein
fragment
After culture, when a cofilin 1 protein fragment is produced
within microorganisms or cells, microorganisms or cells are collected
and then disrupted, so that the protein can be extracted. Also, when
a cofilin 1 protein fragment is produced outside the microorganisms or

cells, the culture solution may be directly used, or microorganisms or
cells are removed by centrifugation or the like and then a supernatant
may be used. Subsequently, general protein purification methods
such as ammonium sulfate precipitation, gel chromatography, ion
exchange chromatography, and affinity chromatography are
independently used or used in an appropriate combination thereof, so
that the cofilin 1 protein can be isolated and purified from the culture
product. Obtainment of a cofilin 1 protein fragment may be
confirmed by SDS-polyacrylamide gel electrophoresis or the like.
[0065]
(2) Preparation of anti-cofilin 1 protein fragment antibody-producing
cells
A recombinant cofilin 1 protein fragment obtained in (1) above
as an immunogen is dissolved in a buffer, so as to prepare an
immunogen solution. At this time, for effective immunization, an
adjuvant may be added if necessary. Examples of an adjuvant include
commercially available Freund's complete adjuvant (FCA) and
Freund's incomplete adjuvant (FIA), which may be used independently
or mixed and then used.
[0066]
Next, the above-prepared immunogen solution is administered to
a mammal such as a rat, a mouse (e.g., BALB/c (inbred line mouse)),
or a rabbit for immunization. Examples of a method for
administration of an immunogen include, but are not limited to,
subcutaneous injection using FIA or FCA, intraperitoneal injection
using FIA, and intravenous injection using 0.15 mol/L sodium
chloride. A single dose of an immunogen is appropriately determined

depending on animal types to be immunized, route of administration,
and the like, ranging from about 50 µg to 200 µg per animal.
Furthermore, immunization intervals are not particularly limited such
that after primary immunization, a booster is performed 2 to 6 times
and preferably 3 to 4 times at intervals of several days to several
weeks and at intervals of preferably 1 to 4 weeks. After primary
immunization, antibody titer in the serum of an immunized animal is
measured by ELISA (Enzyme-Linked Immunosorbent Assay) or the
like. When the antibody titer reaches a plateau, an immunogen is
intravenously or intraperitoneally injected for final immunization.
Two to 5 days and preferably 3 days after the final immunization,
antibody-producing cells are collected.
[0067]
1-4-2. Method for preparing hybridoma producing anti-cofilin 1
partial sequence monoclonal antibody
(1) Recovery of antibody-producing cells from immunized animal
and cell fusion
Cell fusion of antibody-producing cells obtained from an
immunized animal to myeloma cells is performed, so that a hybridoma
producing a monoclonal antibody that specifically recognizes a partial
sequence of the cofilin 1 protein can be prepared. Examples of
antibody-producing cells include spleen cells, lymph node cells, and
peripheral blood cells. Preferable examples thereof are spleen cells
or local lymph node cells. As myeloma cells to be fused to
antibody-producing cells, established cells derived from a mouse or
the like, which are generally available, can be used. A preferable
cell line to be used herein has drug selectivity and has a property such

that it cannot survive in a HAT selective medium (containing
hypoxanthine, aminopterin, and thymine) in an unfused state, but can
grow only in a state of being fused to antibody-producing cells.
Also, preferable established cells are derived from an animal of the
same species or lines of an immunized animal. Specific examples of
myeloma cells include BALB/c mouse-derived
hypoxanthine•guanine.phosphoribosyl.transferase (HGPRT)-deficient
cell lines such as the P3X62-Ag.8 cell line (ATCCTIB9), the
P3X63-Ag. 8. U1 cell line (JCRB9085), the P3/NSI/l-Ag4-l cell line
(JCRB0009), the P3X63Ag8.653 cell line (JCRB0028) and the
Sp2/0-Agl4 cell line (JCRB0029).
[0068]
For cell fusion of the above myeloma cells to
antibody-producing cells, in an animal cell culture medium such as
serum-free DMEM or an RPMI1640 medium, antibody-producing cells
and myeloma cells are mixed at a ratio ranging from about 1:1 to 20:1,
and then a fusion reaction is conducted in the presence of a cell fusion
accelerator. As a cell fusion accelerator, polyethylene glycol or the
like having an average molecular weight of 1,500 Da-4,000 Da can be
used at a concentration of about 10%-80%. Moreover, if necessary,
an adjunct such as dimethyl sulfoxide can be used in combination in
order to increase fusion efficiency. Furthermore, antibody-producing
cells and myeloma cells can be fused to each other using a
commercially available cell fusion apparatus that uses electrical
stimulation (e.g., electroporation) (Nature, 1977, Vol. 266, 550-552).
[0069]
(2) Selection of hybridoma of interest

A method for selecting hybridomas producing an anti-cofilin 1
fragment monoclonal antibody of interest from cells after cell fusion
treatment involves appropriately diluting a cell suspension with fetal
calf serum-containing RPMI1640 medium or the like, seeding cells
onto a 96-well microtiter plate at about 2xl06 cells/well, adding a
selective medium to each well, and then culturing while appropriately
exchanging selective media. The temperature for culture ranges from
20°C to 40°C and is preferably about 37°C. When myeloma cells are
of an HGPRT-deficient cell line or a thymidine kinase (TK)-deficient
cell line, only hybridomas of antibody-producing cells and myeloma
cells can be selectively grown and proliferated with the use of a
selective medium (HAT medium) containing
hypoxanthine-aminopterin-thymidine. Thus, cells that grow around
about 10 days after the initiation of culture in the selective medium
can be selected as hybridomas of interest.
[0070]
Hybridomas that are selected with a HAT medium are subjected
to screening using as an indicator the binding activities to a natural or
recombinant cofilin 1 protein or peptide regions consisting of the
amino acid sequences shown in SEQ ID NOS: 1 to 7. Subsequently,
hybridomas producing an antibody that has binding activity to the
cofilin 1 protein are tested for cross-reactivity. Specifically, the
binding activity to another ADF/COFFILIN family or the like is verified, and
hybridomas with acceptable cross-reactivity are selected. The term
"acceptable cross-reactivity" refers to the ignorable degree of
cross-reactivity in terms of application of an antibody of interest.
For example, in the case of a monoclonal antibody to be used for

immunoassay, if signal intensity resulting from a cross-reaction in the
final measurement system can be suppressed to a level between the
background level and less than 1% of signal intensity resulting from a
specific reaction, it can be concluded that no cross-reaction virtually
takes place.
[0071]
For confirmation of reactivity to the cofilin 1 protein or
cross-reactivity to another ADF/COFILIN family, for example, an
ELISA method can be used. In the ELISA method, a microplate is
prepared by immobilizing the cofilin 1 protein or a fragment thereof
as an antigen, and then a sample prepared by appropriately diluting a
culture supernatant of the above hybridoma is added to the microplate
for reaction. After sufficient reaction, wells are washed, and then a
labeled secondary antibody against the immunoglobulin is added for
further reaction. Wells are washed again, and then the label of the
secondary antibody bound to wells is finally measured, so that the
binding activity of the antibody (existing in the culture supernatant)
to the antigen can be quantitatively found.
[0072]
Hybridomas can also be selected using recombinant DNA
techniques. First, mRNA is extracted from a hybridoma group
obtained according to the above method. A known method, for
example, described in Example 1 may be employed for mRNA
extraction. Next, cDNA of the above mRNA is obtained using oligo
dT primers or random primers. PCR is carried out using the cDNA as a
template and a primer set containing the nucleotide sequence of a
signal sequence located upstream of a gene encoding a variable region

and the nucleotide sequence on the constant region side. The thus
obtained amplification product is inserted to an appropriate cloning
vector for cloning, and thus a variable region gene library of an
antibody produced by the hybridomas can be obtained. In a more
specific example, PCR is carried out using, but not limited to, Mouse
Ig Primer (Novagen), the amplification product (mouse
immunoglobulin variable region cDNA) is inserted to an Eco R I site of
ZERO BLUNT PCR TOPO Vector (Invitrogen) for cloning, and then
the thus obtained vector group can be used as a gene library encoding
variable region amino acid sequences. Subsequently, a probe is
designed based on the amino acid sequence of a variable region or
each CDR disclosed above in the present invention, positive clones are
screened for from the above library, and thus hybridomas producing
the antibody of the present invention can be selected.
[0073]
(3) Antibody production using hybridoma
Hybridomas in the present invention can be prepared in the form
of ascites using mice, so that they can be used for antibody
production. Specifically, hybridomas are intraperitoneally inoculated
to mice, from which cells used for fusion partners upon hybridoma
preparation are derived or nude mice, ascites are appropriately
collected, and thus an ascites solution containing the antibody can be
recovered. More specifically, hybridomas prepared with SP/0 cells as
fusion partners are inoculated intraperitoneally to BALB/c mice 10
days after pristine inoculation, and thus an ascites solution containing
the antibody can be recovered.
[0074]

Furthermore, hybridomas in the present invention can be used
for antibody production by culturing the hybridomas using appropriate
medium. Specifically, a culture supernatant containing the antibody
can be obtained by seeding hybridomas into hybridoma SFM medium
(Gibco) at 1x105 cells/mL, culturing cells at 37°C in a 5% CO2
incubator until all hybridomas had died. The example thereof is not
limited thereto.
[0075]
(4) Method for preparing recombinant anti-cofilin 1 protein fragment
monoclonal antibody
The antibody or a fragment thereof of the present invention can
also be obtained by recombinant DNA procedures using the cDNA
sequence encoding the amino acid sequence of a monoclonal antibody
that specifically recognizes a cofilin 1 fragment disclosed in the
present invention.
[0076]
For example, with the use of a DNA sequence encoding the
amino acid sequence that encodes a variable region of an antibody
derived from the anti-cofilin 1 protein fragment monoclonal
antibody-producing hybridoma obtained by the techniques in
"1-4-2(2)" above, the nucleotide sequences of VH and VL are ligated
to the nucleotide sequences encoding arbitrary CL and CH,
respectively, and then each polynucleotide is incorporated into an
appropriate expression vector. After the vector is introduced into
host cells, the resultant can be expressed as a complete
immunoglobulin molecule. Moreover, with the use of a CDR grafted
antibody technique, the amino acid sequence of the CDR sequence,

from among the amino acid sequences encoding variable regions
obtained by the techniques in "1-4-2(2)" above, is incorporated into
each CDR of an arbitrary immunoglobulin to result in a variable
region. A polynucleotide encoding the variable region is
incorporated into an appropriate expression vector, the vector is
introduced into host cells, and then the resultant may be expressed as
a complete immunoglobulin molecule. At this time, a heavy chain
and a light chain may be expressed in the same host cell and then a
dimer consisting of the heavy chain and the light chain can be
produced. Specifically, for example, cells are co-transformed with a
light chain expression vector and a heavy chain expression vector, and
then the antibody according to the present invention can also be
obtained from the transformed cells. Alternatively, a polynucleotide
encoding the above amino acid sequence can be directly incorporated
into an appropriate expression vector, the vector is introduced into
host cells, and then the resultants can also be expressed as fragments
of the immunoglobulin molecule. Alternatively, as described above,
polynucleotides encoding VL and VH or a light chain and a heavy
chain, respectively, comprising the above amino acid sequences are
linked with an appropriate linker, and then the resultant is
incorporated into a phage. Thus, the resultant can be expressed as
single-stranded Fv or a synthetic antibody fragment such as a diabody.
Moreover, with the use of recently developed genetic engineering
techniques, genes encoding a heavy chain and a light chain are
artificially shuffled using a phage display antibody technique
(Brinkmann et al, 1995, J Immunol Methods, 182, 41-50, International
Patent Publication W097/13844 and WO90/02809) by which a

recombinant antibody is expressed on phage surfaces, the thus
diversified single-stranded Fv antibodies are expressed as phage
fusion proteins, and thus specific antibodies can also be obtained.
[0077]
Preparation of a polynucleotide encoding a recombinant
anti-cofilin 1 protein fragment monoclonal antibody or a fragment
thereof, construction of a vector into which the polynucleotide has
been incorporated, and a method for introducing the vector into a host
may be carried out using a recombinant DNA technique known in the
art as described in 1-4-1., "Method for preparing anti-cofilin 1
monoclonal antibody" above. The recombinant anti-cofilin 1 protein
antibody or a fragment thereof of interest can be obtained from a
culture solution of transformed cells or from within the cells.
[0078]
As immunoglobulin expression vectors, for example, plasmids,
phagemids, cosmids, viral vectors (e.g., SV40 virus-based vector, EB
virus based vector, and BPV based vector), and the like can be used,
but examples thereof are not limited thereto. For example, a BCMGS
Neo vector that is one type of BPV-based vector is a desirable vector
since a foreign gene is efficiently expressed by transformation of
COS7 cells or the like with the BCMGS Neo vector (Hajime
Karasuyama "Cattle Papillomavirus Vector,", Ed., Masami Muramatsu
and Hiroto Okayama, Experimental Medicine Separate Volume:
Genetic Engineering Handbook, 1991, YODOSHA, 297-299).
[0079]
The above vector may contain, in addition to a polynucleotide
encoding an antibody or a fragment thereof, regulatory elements

required for expression of such an antibody or a fragment thereof
(e.g., a promoter, an enhancer, a terminator, a polyadenylation site,
and a splicing site), or a selection marker, as necessary.
[0080]
As hosts for transformation, in addition to hosts described in
1-4-1., "Method for preparing anti-cofilin 1 monoclonal antibody"
above, SP2/0 (mouse myeloma) cells (European Journal of Cancer
Research Preview(l 996) Vol. 5, 512-519; Cancer Research (1990)
Vol.50, 1495-1502) are preferably used.
[0081]
Host cells in the present invention containing a vector
expressing an antibody or a fragment thereof are cultured according to
a conventional method and thus the antibody can be produced in the
culture supernatant or within host cells. Specifically, when CHO
cells are used as host cells, host cells are seeded into DMEM (Gibco)
at 1x105 cells/mL, cultured in a 5% CO2 incubator at 37°C, and thus
the culture supernatant containing the antibody can be obtained.
When Escherichia coli cells are used as host cells, E. coli cells are
seeded and cultured in general medium such as LB medium to be used
for culturing Escherichia coli, so as to induce protein expression, and
thus the antibody can be produced in a culture supernatant or within
host cells.
[0082]
In addition, when an antibody or a fragment thereof as an
expression product contains a constant region, the product can be
purified and/or recovered from a culture supernatant or a cell
disruption solution using a protein A column, a protein G column, an

anti-immunoglobulin antibody affinity column, or the like.
Meanwhile, when an expression product is expressed in a form
containing no constant region, and thus is composed of only a variable
region, the above purification method cannot be applied thereto.
Hence, another appropriate purification method is applied. For
example, an expression product is expressed to have a structure in
which a tag sequence advantageous for purification, such as a
histidine tag, has been fused to the C terminus, so that purification is
possible by affinity chromatography using a ligand corresponding
thereto. If an expression product is not a fusion protein with a tag, it
can be purified according to a conventional method for protein
purification such as ammonium sulfate precipitation, ion exchange
chromatography, reverse phase chromatography, gel filtration
chromatography, or hydroxy apatite chromatography.
[0083]
1-4-3. Confirmation of epitope on cofilin 1 protein to be recognized
by the obtained anti-cofilin 1 monoclonal antibody
A sequence on the cofilin 1 protein to be specifically recognized
by the obtained anti-cofilin 1 monoclonal antibody; that is, an epitope
sequence can be determined by preparing various deletion mutant
genes using PCR and the like based on the gene of the protein, and
then analyzing the binding of the monoclonal antibody to mutant
cofilin 1 proteins obtained from the mutant genes. Specifically, this
is carried out by the following method. First, fragments with various
lengths are prepared by deleting 10 to 400 nucleotides from the 5'
terminal side, or the 3' terminal side, or both ends (the 5' terminus
and the 3' terminus) of the cofilin 1 gene. These fragments are

inserted into vectors and various expression vectors are constructed.
Such a method for preparing gene fragments with various deletion
mutations is described in "Seikagaku Jikken Koza 2 (Biochemical
Experimental Lecture Series 2), Vol. 1, Idenshi Kenkyu-ho (Method
for Genetic Research) II, pp. 289-305, Ed., The Japanese Biochemical
Society." First, various deletion mutant proteins and the full-length
cofilin 1 protein are prepared by the above method from host cells
into which expression vectors of different deletion mutants and the
expression vector of the full-length cofilin 1 protein have been
introduced. Subsequently, the binding of the anti-cofilin 1
monoclonal antibody to various deletion mutants and the full-length
protein is evaluated by an ELISA method using these proteins as
antigens. When the monoclonal antibody loses the binding in a
mutant prepared by introducing a deletion of a specific amino acid
sequence into the full-length sequence, it can be concluded that at
least a part of the region (from among the epitope sequence of the
monoclonal antibody) is contained in the amino acid sequence having
the relevant deletion. Moreover, the number of epitope sequences
can further be narrowed down by evaluating the reactivity of the
monoclonal antibody to different 2 types of and preferably 3 types of
deletion mutant.
[0084]
An epitope sequence on the cofilin 1 protein to be recognized by
the obtained anti-cofilin 1 monoclonal antibody can also be confirmed
by the following method.
[0085]
First, the cofilin 1 protein subjected to reductive alkylation is

reacted with an anti-cofilin 1 monoclonal antibody to form an
antigen-antibody complex, followed by proteolysis using appropriate
protease such as trypsin. Even subjected to proteolysis, antibodies
are not easily digested with trypsin. Hence, an antigen-antibody
complex can be recovered using ProteinG sepharose or the like. At
this time, as a result of digestion by proteolysis with protease, antigen
portions other than those protected by binding to antibodies are
digested. The recovered antigen-antibody complex is analyzed by
LC-MS, and thus portions protected by binding to antibodies; that is,
an epitope on the cofilin 1 protein to be recognized by the antibody
can be identified.
[0086]
Furthermore, an epitope sequence on the cofilin 1 protein to be
recognized by the anti-cofilin 1 monoclonal antibody can be confirmed
by, for example, a competitive method using a synthetic peptide.
First, synthetic peptides each consisting of 6 to 21 amino acids of the
amino acid sequence constituting the cofilin 1 protein are prepared by
a solid phase synthesis method or the like. In the above experiment
for confirming the binding to the cofilin 1 protein using the ELISA
method, the synthetic peptides are caused to act when the anti-cofilin
1 monoclonal antibody is reacted with the immobilized cofilin 1
protein. When the inhibition of the binding of the anti-cofilin 1
monoclonal antibody is confirmed, it can be concluded that the amino
acid sequences of the synthetic peptides are epitope sequences to be
recognized by the anti-cofilin 1 monoclonal antibody.
[0087]
2. Immunoassay of Cofilin 1 protein

A 2nd embodiment of the present invention relates to an
immunoassay of cofilin 1 protein . The immunoassay of the present
invention is characterized by measuring cofilin 1 and/or a fragment
thereof in a sample using 2 or more types of anti-cofilin 1 monoclonal
antibody and/or fragments thereof specifically recognizing different
epitopes on the amino acid sequence of the cofilin 1 protein. With
the use of the monoclonal antibody or a fragment thereof,
measurement excellent in quantification of and detection sensitivity
for the cofilin 1 protein can be realized.
[0088]
The term "sample" as used in the assay of the present invention
refers to various samples that can contain the cofilin 1 protein and/or
a fragment thereof. Examples thereof include cultured cells, a cell
homogenate, culture supernatants, and mammalian samples, which
contain DNA encoding the cofilin 1 protein or a fragment thereof.
The term "mammalian sample" refers to biological samples derived
from all mammals, such as tissues collected from mammals (e.g.,
tissue collected postoperatively), and body fluids such as blood,
lymph fluid, urine, spinal fluid, saliva, and seminal fluid. Such a
mammalian sample is preferably blood or urine. Examples of blood
as used in the present invention include serum, blood plasma, and
interstitial fluid. Also, types of mammal are not particularly limited
and are preferably primates and more preferably humans.
[0089]
Specific examples of a peptide region in which an epitope is
present include peptide regions consisting of the amino acid sequences
shown in SEQ ID NOS: 1 and 2. Of the peptide region consisting of

the amino acid sequence shown in SEQ ID NO: 1, a peptide region
comprising at least the amino acid sequence shown in SEQ ID NO: 3 is
more preferable. Of the peptide region consisting of the amino acid
sequence shown in SEQ ID NO: 2, a peptide region comprising at least
the amino acid sequence shown in SEQ ID NO: 4 is more preferable.
Furthermore, of the peptide region consisting of the amino acid
sequence shown in SEQ ID NO: 1, a peptide region comprising at least
the amino acid sequences shown in SEQ ID NO: 5 and 6 is further
more preferable. Of the peptide region consisting of the amino acid
sequence shown in SEQ ID NO: 2, a peptide region comprising at least
the amino acid sequence shown in SEQ ID NO: 7 is further more
preferable. The peptide regions consisting of the amino acid
sequences shown in SEQ ID NOS: 5, 6, and 7 are even more preferable
as peptide regions in which epitopes are present. This is because
epitopes effective for preparation of high-sensitivity antibodies are
present in these peptide regions as described above.
[0090]
Epitopes in the present invention are not particularly limited, as
long as they are present on the cofilin 1 protein or a fragment thereof.
For example, different epitopes may be present at positions away from
each other on the cofilin 1 protein or present at positions close to
each other. Furthermore, when each of different epitopes is present
in any one of the peptide regions consisting of the amino acid
sequences shown in SEQ ID NOS: 1 to 7 above, different epitopes may
be present separately on different peptide regions, or present on a
single peptide region if the peptide region has a sufficient length of
amino acids, so that it can contain multiple epitopes. When epitopes

are present on different peptide regions, combinations of peptide
regions are not particularly limited. For example, epitopes may be
separately present in the peptide regions consisting of the amino acid
sequences shown in SEQ ID NOS: 1 and 2, respectively.
Alternatively, epitopes may be present separately in the peptide
regions consisting of the amino acid sequences shown in SEQ ID NOS:
5 and 6 on the peptide region consisting of the amino acid sequence
shown in SEQ ID NO: 1. Alternatively, epitopes may be separately
present in the peptide regions consisting of the amino acid sequences
shown in SEQ ID NOS: 5 and 6, SEQ ID NOS: 5 and 7, or SEQ ID
NOS: 6 and 7, respectively.
[0091]
For 2 or more types of anti-cofilin 1 monoclonal antibody and/or
fragments thereof to be used in this embodiment, the anti-cofilin 1
monoclonal antibody and/or a fragment thereof described in the above
embodiment 1 can be used. Examples thereof include a combination
of 2 or more types of anti-cofilin 1 monoclonal antibody and/or
fragments thereof selected from antibody clone groups listed in Table
1 below, preferably an antibody clone group containing at least 1E2,
2C4, 2D12, 2F5, 3F11, 3G2, 4F12, 4E12, and 4G10, and more
preferably an antibody clone group containing at least 1E2, 2C4,
4F12, and 4E12, or, combinations of at least 4F12 and 1E2, 4F12 and
4E12, 1E2 and 3F11, 1E2 and 4E12, 2C4 and 4E12, 3G2 and 4E12,
4G10 and 4E12, 2D12 and 1E2, 2D12 and 2C4, and 4E12 and 2F5.
[0092]
Examples of a combination of more preferable 2 or more types
of anti-cofilin 1 monoclonal antibody and/or fragments thereof to be

used in this embodiment include combinations selected from the group
consisting of (1) a combination of an anti-cofilin 1 monoclonal
antibody or a fragment thereof, in which, at least, in the light chain,
CDR1 comprises the sequence shown in SEQ ID NO: 8, CDR2
comprises the sequence shown in SEQ ID NO: 9, and CDR3 comprises
the sequence shown in SEQ ID NO: 10, and in the heavy chain, CDR1
comprises the sequence shown in SEQ ID NO: 11, CDR2 comprises the
sequence shown in SEQ ID NO: 12, and CDR3 comprises the sequence
shown in SEQ ID NO: 13, (2) a combination of an anti-cofilin 1
monoclonal antibody or a fragment thereof, in which in the light
chain, CDR1 comprises the sequence shown in SEQ ID NO: 14, CDR2
comprises the sequence shown in SEQ ID NO: 15, and CDR3
comprises the sequence shown in SEQ ID NO: 16, and in the heavy
chain, CDR1 comprises the sequence shown in SEQ ID NO: 17, CDR2
comprises the sequence shown in SEQ ID NO: 18, and CDR3
comprises the sequence shown in SEQ ID NO: 19, (3) a combination of
an anti-cofilin 1 monoclonal antibody or a fragment thereof, in which
in the light chain, CDR1 comprises the sequence shown in SEQ ID
NO: 20, CDR2 comprises the sequence shown in SEQ ID NO: 21, and
CDR3 comprises the sequence shown in SEQ ID NO: 22, and in the
heavy chain, CDR1 comprises the sequence shown in SEQ ID NO: 23,
CDR2 comprises the sequence shown in SEQ ID NO: 24, and CDR3
comprises the sequence shown in SEQ ID NO: 25, and (4) a
combination of an anti-cofilin 1 monoclonal antibody or a fragment
thereof, in which in the light chain, CDR1 comprises the sequence
shown in SEQ ID NO: 26, CDR2 comprises the sequence shown in SEQ
ID NO: 27, and CDR3 comprises the sequence shown in SEQ ID NO:

28, and in the heavy chain, CDR1 comprises the sequence shown in
SEQ ID NO: 29, CDR2 comprises the sequence shown in SEQ ID NO:
30, and CDR3 comprises the sequence shown in SEQ ID NO: 31.
[0093]
Examples of a combination of further preferable 2 or more types
of anti-cofilin 1 monoclonal antibody and/or fragments thereof to be
used in this embodiment include (1) a combination of an antibody or a
fragment thereof, in which, at least, in the light chain, CDR1
consisting of the sequence shown in SEQ ID NO: 8, CDR2 consisting
of the sequence shown in SEQ ID NO: 9, and CDR3 consisting of the
sequence shown in SEQ ID NO: 10, and in the heavy chain, CDR1
consisting of the sequence shown in SEQ ID NO: 11, CDR2 consisting
of the sequence shown in SEQ ID NO: 12, and CDR3 consisting of the
sequence shown in SEQ ID NO: 13, with an antibody or a fragment
thereof, in which, in the light chain, CDR1 consisting of the sequence
shown in SEQ ID NO: 26, CDR2 consisting of the sequence shown in
SEQ ID NO: 27, and CDR3 consisting of the sequence shown in SEQ
ID NO: 28, and in the heavy chain, CDR1 consisting of the sequence
shown in SEQ ID NO: 29, CDR2 consisting of the sequence shown in
SEQ ID NO: 30, and CDR3 consisting of the sequence shown in SEQ
ID NO: 31, (2) a combination of an antibody or a fragment thereof, in
which, at least, in the light chain, CDR1 consisting of the sequence
shown in SEQ ID NO: 8, CDR2 consisting of the sequence shown in
SEQ ID NO: 9, and CDR3 consisting of the sequence shown in SEQ ID
NO: 10, and in the heavy chain, CDR1 consisting of the sequence
shown in SEQ ID NO: 11, CDR2 consisting of the sequence shown in
SEQ ID NO: 12, and CDR3 consisting of the sequence shown in SEQ

ID NO: 13, with an antibody or a fragment thereof, in which, in the
light chain, CDR1 consisting of the sequence shown in SEQ ID NO:
20, CDR2 consisting of the sequence shown in SEQ ID NO: 21, and
CDR3 consisting of the sequence shown in SEQ ID NO: 22, and in the
heavy chain, CDR1 consisting of the sequence shown in SEQ ID NO:
23, CDR2 consisting of the sequence shown in SEQ ID NO: 24, and
CDR3 consisting of the sequence shown in SEQ ID NO: 25, (3) a
combination of an antibody or a fragment thereof, in which, at least,
in the light chain, CDR1 consisting of the sequence shown in SEQ ID
NO: 14, CDR2 consisting of the sequence shown in SEQ ID NO: 15,
and CDR3 consisting of the sequence shown in SEQ ID NO: 16, and in
the heavy chain, CDR1 consisting of the sequence shown in SEQ ID
NO: 17, CDR2 consisting of the sequence shown in SEQ ID NO: 18,
and CDR3 consisting of the sequence shown in SEQ ID NO: 19, with
an antibody or a fragment thereof, in which, in the light chain, CDR1
consisting of the sequence shown in SEQ ID NO: 20, CDR2 consisting
of the sequence shown in SEQ ID NO: 21, and CDR3 consisting of the
sequence shown in SEQ ID NO: 22, and in the heavy chain, CDR1
consisting of the sequence shown in SEQ ID NO: 23, CDR2 consisting
of the sequence shown in SEQ ID NO: 24, and CDR3 consisting of the
sequence shown in SEQ ID NO: 25, and (4) a combination of an
antibody or a fragment thereof, in which, at least, in the light chain,
CDR1 consisting of the sequence shown in SEQ ID NO: 20, CDR2
consisting of the sequence shown in SEQ ID NO: 21, and CDR3
consisting of the sequence shown in SEQ ID NO: 22, and in the heavy
chain, CDR1 consisting of the sequence shown in SEQ ID NO: 23,
CDR2 consisting of the sequence shown in SEQ ID NO: 24, and CDR3

consisting of the sequence shown in SEQ ID NO: 25, with an antibody
or a fragment thereof, in which, in the light chain, CDR1 consisting of
the sequence shown in SEQ ID NO: 26, CDR2 consisting of the
sequence shown in SEQ ID NO: 27, and CDR3 consisting of the
sequence shown in SEQ ID NO: 28, and in the heavy chain, CDR1
consisting of the sequence shown in SEQ ID NO: 29, CDR2 consisting
of the sequence shown in SEQ ID NO: 30, and CDR3 consisting of the
sequence shown in SEQ ID NO: 31.
[0094]
The amino acid sequences shown in SEQ ID NOS: 1 to 7 that are
partial sequences of the amino acid sequence constituting the cofilin 1
protein show 100% homology in humans, mice, rats, cattle, dogs and
chimpanzees, since the amino acid sequences correspond to regions
highly conserved among mammals. The use of antibodies recognizing
these sequences makes it possible to construct a versatile cofilin 1
protein measurement system accommodating samples from various
mammalian species.
[0095]
The immunoassay of the present invention can make use of a
known immunoassay using 2 or more types of antibody and/or
fragments thereof. For example, the immunoassay can be carried out
by an immunoassay using a labeled antibody, such as an ELISA
method, an EIA method, a fluorescence immunoassay, a
radioimmunoassay, or a luminescence immunoassay, a surface plasmon
resonance method (SPR method), or a quarts crystal microbalance
measurement method (QCM method). Moreover, the immunoassay of
the present invention can be achieved using a visually method or by an

optical method by mesuring the transmitted light or the scattered
light, resulting from the generation of immune complex agglutinates
as a result of immunonephelometry, latex agglutination reaction, latex
turbidimetry, hemagglutination reaction, particle agglutination
reaction, or the like.
[0096]
The ELISA (Enzyme-Linked Immunosorbent Assay) method is
also referred to as "enzyme immunosorbent analysis method" and is a
method for quantifying a target antigen by detecting the target antigen
contained in a trace amount in a sample based on color optical density
or fluorescence intensity resulting from an antigen-antibody reaction
using an enzyme-labeled antibody or antigen and the action of the
enzyme. Specifically, the antibody or a fragment thereof of the
present invention or the cofilin 1 protein or a fragment thereof is
immobilized on a solid-phase support, and then an immunological
reaction between the antibody or the like and the cofilin 1 protein or
the like is enzymatically detected. Methods such as a direct
method, an indirect method, and a sandwich method are known. In
the present invention, particularly the sandwich method is preferably
applied. The sandwich method involves binding a 1st antibody
(immobilized antibody) immobilized on a solid-phase support to an
antigen, adding a 2nd antibody (labeled antibody/primary antibody)
recognizing an epitope differing from that of the 1st antibody so as to
bind it to the antigen, and then detecting the label when the 2nd
antibody is a labeled antibody, or detecting using a tertiary antibody
(secondary antibody) when the 2nd antibody is a primary antibody.
For detailed information on measurement methods for the ELISA

method, see known methods (Ed., Japanese Society of Laboratory
Medicine "Clinical Pathology, Extra Edition, Vol. 53, Immunoassay
for Clinical Examination -Techniques and Applications-," The Clinical
Pathology Press, 1983, Ed., Eiji Ishikawa, "Enzyme Immunoassay," 3rd
edition, Igaku-Shoin Ltd., 1987, Ed., Tsunehiro Kitagawa et al.,
"Protein Nucleic Acid Enzyme, Separate Volume No. 31, Enzyme
Immunoassay," KYORITSU SHUPPAN CO., LTD, 1987, Ed., Minoru
Irie "Radioimmunoassay," Kodansha Scientific Ltd., 1974, Ed.,
Minoru Irie "Radioimmunoassay 2," Kodansha Scientific Ltd., 1979).
[0097]
As the above solid-phase support, an insoluble carrier (support)
in the form of beads, microplates, test tubes, sticks, test species, or
the like can be used, which is made of material such as polystyrene,
polycarbonate, polyvinyl toluene, polypropylene, polyethylene,
polyvinyl chloride, nylon, polymethacrylate, latex, gelatin, agarose,
cellulose, sepharose, glass, metal, ceramics, or magnetic material.
The antibody or a fragment thereof of the present invention or the
cofilin 1 protein or a fragment thereof can be immobilized onto a
solid-phase support by binding it to a support by a known method such
as a physical adsorption method, a chemical binding method, or a
method using the methods in combination.
[0098]
As the above labeling substances, in the case of the ELISA
method, for example, peroxidase (POD), alkaline phosphatase,
β-galactosidase, urease, catalase, glucose oxidase, lactate
dehydrogenase, amylase, a biotin-avidin complex, or the like; in the
case of fluorescence immunoassay, fluorescein isothiocyanate,

tetramethylrhodamine isothiocyanate, substituted rhodamine
isothiocyanate, dichlorotriazine isothiocyanate, Alexa480,
AlexaFluor488, or the like; and in the case of radioimmunoassay,
tritium (3H), iodine 125(125I), iodine 131(I31I), or the like; can be
used, but are not limited thereto. Furthermore, in the case of
luminescence immunoassay, an NADH-FMNH -luciferase system, a
luminol-hydrogen peroxide-POD system, an acridinium ester system, a
dioxetane compound system, or the like can be used. As a method for
binding a labeling substance to an antibody, in the case of the ELISA
method, a known method such as a glutaraldehyde method, a
maleimide method, a pyridyl disulfide method, or a periodic acid
method can be used; and in the case of radioimmunoassay, a known
method such as a chloramine T method or Bolton-Hunter method can
be used.
[0099]
A case, in which the immunoassay of the present invention is
carried out by a sandwich ELISA method using anti-cofilin 1 protein
monoclonal antibodies specifically and differently recognizing
epitopes that are present in the peptide regions consisting of the
amino acid sequences shown in SEQ ID NOS: 5 and 6, is specifically
described as an example. However, the embodiments of the present
invention are not limited thereto.
[0100]
First, a monoclonal antibody specifically recognizing an epitope
that is present in the peptide region consisting of the amino acid
sequence shown in SEQ ID NO: 5 is immobilized on an insoluble
support. Antibodies to be immobilized (immobilized antibodies) may

be of one type or of several types. Next, a sample containing the
cofilin 1 protein is caused to act on the surface of an immobilized
antibody, and then an antigen-antibody complex consisting of the
immobilized antibody and the cofilin 1 protein is formed on the
surface of the solid-phase support. Subsequently, the support is
sufficiently washed with a wash solution, so as to remove substances
unbound to the immobilized antibodies. Subsequently, labeling is
performed using a monoclonal antibody specifically recognizing an
epitope that is present in the peptide region consisting of the amino
acid sequence shown in SEQ ID NO: 6 to obtain a labeled antibody.
The labeled antibody is caused to act on the support to which the
complex of the immobilized antibody and the cofilin 1 protein has
been bound. The immobilized antibody and the labeled antibody
recognize different epitopes of the cofilin 1 protein, respectively, so
that on the solid-phase support, a ternary complex consisting of
immobilized antibody/cofilin 1 protein/labeled antibody is formed.
After unbound labeled antibodies are sufficiently washed off with a
wash solution, the cofilin 1 protein existing in the sample can be
detected and quantified through detection of the label of the labeled
antibody of the ternary complex. The labeled antibodies may be of 1
type or several types. Preferably 2 or more types of labeled antibody
are used and 3 types of labeled antibody are more preferably used.
Also, when animal species from which an immobilized antibody is
derived differs from the animal species from which a labeled antibody
is derived, an unlabeled antibody is caused as a primary antibody
without labeling to act on an immobilized antibody/cofilin 1 protein
complex, and then detection can also be carried out using a labeled

secondary antibody that recognizes the primary antibody. In
addition, an antibody used for immobilization and an antibody used
for labeling can also be used vice versa.
[0101]
Furthermore, a sample containing a labeled antibody and the
cofilin 1 protein is mixed in advance to cause them to form an
antigen-antibody complex, and then the complex can be caused to act
on an immobilized antibody. When an antibody to be immobilized is
biotin-labeled in advance, a sample containing a biotinylated and
immobilized antibody and the cofilin 1 protein is mixed with an
antibody labeled with a label other than biotin to form an
antigen-antibody complex. When the complex is caused to act on a
support onto which avidin has been immobilized, the antigen-antibody
complex can be detected using labeling other than biotinylation.
[0102]
Furthermore, in the immunoassay of the present invention,
immunochromatographic test strips can also be used. An
immunochromatographic test strip is composed of, for example, a
sample reservoir made of a material that can easily absorbs a sample,
a reagent part containing a diagnostic agent of the present invention, a
development part where the reaction product of the sample and the
diagnostic agent is developed, a labeling part where the reaction
product that has been developed is colored, and a display part where
the colored reaction product is developed. Commercially available
pregnancy diagnostic agents have forms similar thereto. The
principle of the immunoassay is as described below. First, a sample
is added to the sample reservoir, the sample reservoir absorbs the

sample to cause it to reach the reagent part. Subsequently, an
antigen-antibody reaction of the cofilin 1 protein in the sample and
the above anti-cofilin 1 partial sequence monoclonal antibody or a
fragment thereof takes place at the reagent part, and then the reaction
complex migrates in the development part to reach the labeling part.
At the labeling part, the above reaction complex reacts with the
labeled secondary antibody. When the reaction product from the
reaction with the labeled secondary antibody is developed to reach the
display part, color development is observed. The above test strip for
immunochromatography has extremely low invasiveness, providing no
distress and no risk caused by the use of reagents to users. Hence, the
test strip can be used for monitoring at home, making it possible to
review the results and perform treatment (e.g., surgical resection) at
the medical institution level. In such a manner, the results can be led
to prevent metastasis and relapse. In addition, the test strip can be
currently produced inexpensively in large quantities by a production
method as described in JP Patent Publication (Kokai) No. H10-54830
A (1998), for example.
[0103]
Immunoassay using immunochromatographic test strips is as
specifically described below with reference to an example wherein a
monoclonal antibody (referred to as "1st antibody") specifically
recognizing an epitope that is present in the peptide region consisting
of the amino acid sequence shown in SEQ ID NO: 5 and a monoclonal
antibody (referred to as "2nd antibody") specifically recognizing an
epitope that is present in the peptide region consisting of the amino
acid sequence shown in SEQ ID NO: 6 are used. First, a sample

containing the cofilin 1 protein is brought into contact with a sample
reservoir, and then the sample reservoir absorbs the sample to cause it
to reach a reagent part. Subsequently, at the reagent part, an
antigen-antibody reaction takes place between the cofilin 1 protein in
the sample and the 1st antibody, and then the thus formed
antigen-antibody complex migrates through a development part so as
to reach a labeling part. At the labeling part, a reaction takes place
between the above antigen-antibody complex and the labeled 2nd
antibody, the reaction product resulting from the reaction with the
labeled 2nd antibody is developed to reach a display part, and thus
color development is observed.
[0104]
In the assay of the present invention, a surface plasmon
resonance method (SPR method) can also be used. The surface
plasmon resonance phenomenon refers to a phenomenon in which
reflected light intensity is significantly attenuated when a thin metal
film is irradiated with a laser beam at a specific angle of incidence
(resonance angle). Adsorbates on the surface of the thin metal film
can be measured with high sensitivity using an SPR sensor using the
principle of the SPR phenomenon. Therefore, an antibody and/or a
target antigen is immobilized in advance on the surface of the thin
metal film, a sample is caused to pass over the surface of the thin
metal film, and thus differences in measured (before and after the
passage of the sample) values of adsorbates on the surface of the thin
metal film resulting from an antigen-antibody reaction can be
detected. A substitution method, an indirect competitive method, and
the like are known and any of these methods may be used herein.

This technique is known in the art. See Kazuhiro Nagata and Hiroshi
Handa, Experimental Methods for Realtime Analysis of Biomaterial
Interaction, Springer-Verlag Tokyo, Tokyo, 2000, for example.
[0105]
Moreover, a quartz crystal microbalance measurement method
(QCM method) can also be used for the assay of the present invention.
This method uses a phenomenon such that when a substance is
adsorbed to the surface of an electrode installed in a quartz resonator,
the resonance frequency of the quartz resonator decreases in
accordance with the mass. A QCM sensor using the method is a mass
measuring sensor capable of quantitatively capturing very small
quantities of an adsorbate based on changes in water resonance
frequency. This technique is known in the art. For example, see J.
Christopher Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo, G. M.
Whitesides, 2005, Self-Assembled Monolayers of a Form of
Nanotechnology, Chemical Review, 105: 1103-1169; Toyosaka
Moriizumi, Takamichi Nakamoto, 1997, Sensor Engineering, Shokodo
Co., Ltd.
[0106]
3. Method for determining whether or not gastrointestinal cancer is
developing
The 3rd embodiment of the present invention relates to a method
for determining whether or not gastrointestinal cancer is developing.
The determination method of the embodiment is characterized by
measuring the amount of the cofilin 1 protein and/or a fragment
thereof in a sample as a marker for detecting gastrointestinal cancer,
and then determining the presence or the absence of gastrointestinal

cancer in a subject based on the ratio of the amount of the cofilin 1
protein and/or a fragment thereof in the subject to the amount of the
same in a healthy subject. The method of the embodiment comprises
a measurement step (1) and a determination step (2). Each step is as
specifically described below.
[0107]
(1) Measurement step
The term "measurement step" refers to a step for measuring the
amount of the cofilin 1 protein in a sample from a subject and the
same in a sample from a healthy subject using the assay described in
the 2nd embodiment above.
[0108]
The term "subject" refers to an individual subject to be
subjected to the determination method of the present invention.
Subject types are not particularly limited, as long as the subjects are
mammals. Preferably, a subject is a human (hereinafter, when a
subject is a human, the subject is referred to as "subject" in the
Description).
[0109]
The term "healthy subject" refers to an individual not affected
by at least gastrointestinal cancer, and preferably refers to a healthy
individual subject. Such a healthy subject is required to be of the
same biological species as that of a subject. For example, in the case
of a subject, a healthy subject must also be a human (in the
Description, in this case, hereinafter referred to as "healthy subject").
The physical conditions of a healthy subject are preferably the same
as or analogous to those of a subject. Physical conditions involve,

for example, in the case of a human, race, gender, age, height, body
weight, and the like.
[0110]
Samples to be used in the embodiment are mammalian samples
since the samples are derived from subjects or healthy subjects.
Specifically, examples of such samples include all biological samples
derived from mammals, such as tissue collected from a mammal (e.g.,
samples collected postoperatively), and body fluids (e.g., blood,
lymph fluid, urine, spinal fluid, saliva, and seminal fluid). A sample
to be used herein is preferably blood, and is particularly preferably
serum or blood plasma. Since measured values must be compared,
the types of samples from a subject and a healthy subject must be the
same in principle. For example, when a sample from a subject is
serum, a sample from a healthy subject is also desired to be serum in
principle.
[0111]
A method for measuring the cofilin 1 protein in a sample may be
carried out according to the assay of the 2nd embodiment, but the
explanation for the detailed information thereof is omitted.
[0112]
(2) Determination step
The term "determination step" refers to a step that involves
comparing the amount of the cofilin 1 protein and/or a fragment
thereof in a sample from a subject with the amount of the same in a
sample from a healthy subject as measured by the above measurement
step, and then determining that the subject is affected by
gastrointestinal cancer when the amount of the cofilin 1 protein and/or

a fragment of the subject is significantly statistically higher than that
of the healthy subject. The term "gastrointestinal cancer" refers to a
primary malignant tumor that is developed in a digestive system
organ. Examples of gastrointestinal cancer include esophageal
cancer, gastric cancer, duodenal cancer, small intestine carcinoma
(including jejunal cancer and ileum cancer), large-bowel cancer
(including cancer of cecum, cancer of the colon, and rectal cancer),
and cancer of pancreas. Gastrointestinal cancer that is particularly
preferable as an object to be determined by the embodiment is gastric
cancer. However, an example thereof is not limited to gastrointestinal
cancer, cancer to be determined herein may also be an epithelial tumor
such as breast cancer, liver cancer, and lung cancer, or skin cancer
such as malignant melanomas.
[0113]
In this step, the amount of the cofilin 1 protein and/or a
fragment thereof obtained by the above measurement step from a
sample of a subject is compared herein with the amount of the same
from a sample of a healthy subject. The amounts to be compared may
be relative amounts such as concentrations, or absolute quantities. In
the case of absolute quantity, the amounts of samples subjected to
measurement should be adjusted in advance to be equivalent to each
other between a subject and a healthy subject, or should be converted
so that the amounts are equivalent to each other based on the ratio of
the amount of the sample from the subject to the amount of the sample
from the healthy subject (which are subjected to measurement). In
this step, based on the result such that the amount of the cofilin 1
protein and/or a fragment thereof in a sample from a subject is

statistically significantly higher or less than that from a healthy
subject, a subject exhibiting the amount of the cofilin 1 protein and/or
a fragment thereof significantly higher than the other is classified into
a group of subjects affected by gastrointestinal cancer; or a subject
exhibiting no significant difference is classified into a group not
affected by gastrointestinal cancer. A subject classified into such a
group of subjects affected by gastrointestinal cancer is determined to
have gastrointestinal cancer or determined to highly likely have
gastrointestinal cancer. On the other hand, a subject classified into a
group of subjects not affected by gastrointestinal cancer is determined
to unlikely have gastrointestinal cancer, although the possibility of
having gastrointestinal cancer cannot be completely eliminated.
[0114]
Here, the term "statistically significantly" means that, for
example, the significance level of the obtained value is less than 5%,
1%, or 0.1%. Therefore, "statistically significantly high(er)" means
that when a quantitative difference in the amount of the cofilin 1
protein and/or a fragment thereof between a subject and a healthy
subject is statistically processed, there is a significant difference
between the two and the amount of the protein in the subject is higher
than that in the healthy subject. In general, cases, in which the
amount of the cofilin 1 protein in a blood sample of a subject is 2-fold
or more, preferably 3-fold or more, more preferably 4-fold or more,
and most preferably 5-fold or more than that of a healthy subject, fall
under the above category. If a quantitative difference is 3-fold or
more, it can be said that the reliability is high, and the quantitative
difference is statistically significantly high. As statistical methods

for statistical processing, known statistical methods, by which the
presence or the absence of significance can be determined, may be
appropriately used, and are not particularly limited. For example, a
student's t-test method or multiple comparison procedures can be
employed herein.
[0115]
The amount of the cofilin 1 protein in a blood sample of a
healthy subject can be measured every time when the amount of the
cofilin 1 protein in a blood sample from a subject is measured, or the
amount of the cofilin 1 protein measured in advance can also be used.
In particular, the amounts of the cofilin 1 protein of healthy subjects
with various physical conditions are measured in advance and then the
values are input to a computer to form a database. Through the entry
of physical conditions of subjects into the computer, the amount of the
cofilin 1 protein of a healthy subject who has physical conditions
optimum for comparison with the relevant subject can be used
immediately and conveniently.
[0116]
Gastrointestinal cancer stages to be subjected to the present
invention are not particularly limited, ranging from early
gastrointestinal cancer to terminal gastrointestinal cancer. In
particular, the determination method of the present invention has high
sensitivity for the antibody and/or a fragment thereof of the
embodiment 1, so that it is excellent in that even gastrointestinal
cancer at an early stage, the detection of which has been difficult with
conventional methods, can be detected. The term "(early)
gastrointestinal cancer at an early stage" refers to gastrointestinal

cancer characterized in that it is limited to a spot (intramucosal)
where tumor is developed and does not invade the surrounding tissue
or the infiltration range is limited locally even if there is the invasion
of surrounding tissue. Early detection of gastrointestinal cancer
significantly improves the 5-year survival rate, so that the present
invention capable of determining the disease at an early stage is
practically highly beneficial.
[0117]
As described above, according to the method of the present
invention for determining whether or not gastrointestinal cancer is
developing, a marker for detection of gastrointestinal cancer in a
blood sample is immunologically assayed using an antibody, so as to
be able to conveniently and rapidly determine at an early stage if a
subject is affected by gastrointestinal cancer.
The determination method of the present invention makes it
possible to determine if a subject is affected by gastrointestinal
cancer even at an early stage using a blood plasma sample, a serum
sample or the like from the subject.
[0118]
4. Kit for cofilin 1 protein quantification using the assay of the
present invention
The 4th embodiment of the present invention relates to a kit for
cofilin 1 protein quantification. The kit of this embodiment is
characterized by comprising, as essential components, 2 or more types
of anti-cofilin 1 monoclonal antibody and/or fragments thereof
specifically recognizing different epitopes on the amino acid sequence
constituting the cofilin 1 protein. Examples of the above antibodies

and/or fragments thereof contained in this kit include the antibodies
and/or fragments thereof described in the embodiment 1 above.
Examples of a combination of 2 or more types of antibody and/or
fragments thereof contained in the kit include combinations of the
antibodies and/or fragments thereof described in the embodiment 2
above. In addition, the kit may further contain a labeled secondary
antibody, a substrate required for detection of a label, a positive
control or a negative control, a buffer (to be used for dilution or
washing of a sample) and/or instructions, and the like, as necessary.
[0119]
According to the present invention, the cofilin 1 protein that is
contained in an appropriate sample such as blood can be easily and
conveniently measured by the immunoassay as described in the 1st
embodiment above. Furthermore, based on the result and the method
of the 3rd embodiment above, the convenient and rapid determination
of the presence or the absence of gastrointestinal cancer in a subject
from which a sample is obtained becomes possible.
Examples
[0120]
The present invention is described more specifically based on
the Examples, but the present invention is not limited by these
examples.
[0121]
Preparation of recombinant cofilin 1 protein by
Escherichia coli
(Preparation of human cofilin 1 gene)

For preparation of a recombinant human cofilin 1 protein to be
used as an immunogen for an antibody, human cofilin 1 mRNA was
prepared from HEK293 cells of a human fetal kidney cell line.
mRNA was specifically prepared using a Qia shredder and an RNeasy
mini kit (Qiagen) in accordance with the protocols included therewith.
[0122]
Next, cDNA was synthesized using reverse transcriptase
Superscriptll (Invitrogen) and the aforementioned obtained total
mRNA as a template, and then a human cDNA library was constructed.
A reverse transcription reaction was conducted in accordance with the
protocols included with the enzyme.
[0123]
Subsequently, PCR was carried out using the thus obtained
human cDNA library as a template and a primer set consisting of the
nucleotide sequences shown in SEQ ID NOS: 32 and 35. The
nucleotide sequence shown in SEQ ID NO: 32 contains a portion of the
5' terminal region of the human cofilin 1 gene and a BamH I
recognition sequence upstream of such portion. The nucleotide
sequence shown in SEQ ID NO: 35 contains a portion of the 3'
terminal region of the human cofilin 1 gene and an EcoR I recognition
sequence downstream of the portion. A PCR solution was prepared
using KOD (Toyobo Co., Ltd.) as DNA polymerase, so that it
contained 10 ng of a cDNA library and 10 pmol of each primer in
accordance with the protocols included with KOD. Reaction
conditions are as follows. PCR was carried out by repeating 35 times
a cycle consisting of, after 5 minutes of heating at a temperature of
94°C and keeping the temperature at 94°C for 15 seconds, 55°C for 30

seconds, and 68°C for 30 seconds, followed by keeping the final
temperature at 68°C for 4 minutes. The thus amplified DNA fragment
was purified using Wizard SV Gel and PCR Clean-up System
(Promega). A PCR product with a full length of about 500 bp was
obtained by the reaction.
[0124]
The obtained DNA fragment was cleaved with BamH I and
EcoR I, and then a ligation reaction was conducted to incorporate the
resultant into open circular pET30a (Novagen) that had been subjected
to cleavage with BamH. I and EcoR I, and treatment with BAP.
Ligation High (Toyobo Co., Ltd.) was used as DNA ligase, and then
the reaction was conducted in accordance with the protocols included
therewith. Subsequently, competent cells were transformed using a
solution after the ligation reaction. Competent cells were
specifically prepared using a DH5oc Escherichia coli strain (Takara
Bio Inc.) in accordance with the protocols included therewith.
Transformed cells were applied onto an LB plate containing antibiotic
kanamycin (100 µg/mL), and then cultured at 37°C overnight. The
thus obtained transformant was cultured in an LB liquid medium
containing 100 µg/mL kanamycin at 37°C overnight, and thus
pET30a_cofilin 1 of interest was obtained by Mini-Prep.
[0125]
Subsequently, PCR was carried out using the obtained
pET30a_cofilin 1 gene (10 ng) as a template and a primer set
consisting of the nucleotide sequences shown in SEQ ID NOS: 39 and
40. The nucleotide sequence shown in SEQ ID NO: 39 contained a
portion of the 5' terminal region of the human cofilin 1 gene and an

EcoR I recognition sequence upstream of the portion. The nucleotide
sequence shown in SEQ ID NO: 40 contained a portion of the 3'
terminal region of the human cofilin 1 gene and a Bgl II recognition
sequence downstream of the portion. A PCR solution was prepared
using KOD (Toyobo Co., Ltd.) as DNA polymerase in accordance with
the protocols included with KOD, so that it contained 10 ng of the
cDNA library and 10 pmol of each primer. Reaction conditions are as
follows. PCR was carried out by repeating 35 times a cycle consisting
of, after 5 minutes of heating at a temperature of 94°C and keeping
the temperature at 94°C for 15 seconds, 55°C for 30 seconds, and
68°C for 30 seconds, followed by keeping the final temperature at
68°C for 4 minutes. The thus amplified DNA fragment was purified
using Wizard SV Gel and PCR Clean-up System (Promega). A PCR
product with a full length of about 500 bp was obtained by the
reaction.
[0126]
The obtained DNA fragment was cleaved with EcoR I and Bgl II,
and then a ligation reaction was conducted to incorporate the resultant
into open circular pCMV-Myc (Clontech) that had been subjected to
cleavage with EcoR I and BamH I, and treatment with BAP. Ligation
High (Toyobo Co., Ltd.) was used as DNA ligase, and then the
reaction was conducted in accordance with the protocols included
therewith. Subsequently, competent cells were transformed using a
solution after the ligation reaction. Competent cells were
specifically prepared using a DH5α Escherichia coli strain (Takara
Bio Inc.) in accordance with the protocols included therewith.
Transformed cells were applied onto an LB plate containing antibiotic

kanamycin (50 µg/mL), and then cultured at 37°C overnight. The
thus obtained transformant was cultured in an LB liquid medium
containing 100 µg/mL ampicillin at 37°C overnight, and thus
pCMV-Myc_human cofilin 1 of interest was obtained by Mini-Prep.
[0127]
(Preparation of recombinant human cofilin 1 protein)
For preparation of a recombinant human cofilin 1 protein,
Rosetta-Gami 2 Escherichia coli strain (Novagen) was transformed
with pET30a_cofilin 1. The obtained transformant was pre-cultured
in 10 mL of an LB medium containing kanamycin and chloramphenicol
at 37°C overnight. Next, the pre-cultured product was seeded in 1 L
of the same medium and then cultured at 37°C for 5 hours. IPTG
with a final concentration of 0.25 mM was added, cells were cultured
at 32°C for 12 hours, to induce the expression of the recombinant
human cofilin 1 protein of interest, and then centrifugation was
performed to collect microorganisms.
[0128]
The obtained microorganisms were washed with PBS, and then
an insoluble fraction was prepared as a precipitate using B-PER
(PIERCE). The procedure was specifically performed in accordance
with the protocols included therewith. Next, the insoluble fraction
was solubilized with 6 M urea, and then a histidine-tagged human
cofilin 1 protein was adsorbed to TALON Metal Affinity Resin
(CLONETECH). The protein-adsorbed resin was washed with 6 M
urea containing 10 mM imidazole, and then eluted using a 6 M urea
solution containing 1 M imidazole.
[0129]

Next, from the obtained elution fraction, protein refolding was
carried out. First, the fraction was dialyzed overnight against a PBS
solution supplemented with 6 M urea, PBS was gradually added to the
dialysis solution until the final concentration of urea in the dialysis
solution reached 1 M for dilution. Finally, the resultant was dialyzed
against a newly prepared PBS solution overnight. The obtained
refolding solution was subjected to acrylamide gel electrophoresis.
Purification of a histidine tagged human cofilin 1 protein having a
molecular weight of about 16,000 Da was confirmed by Coomassie
brilliant blue staining.
[0130]
Preparation and selection of mouse monoclonal antibody
against human cofilin 1 protein
(Production of anti-human cofilin 1 antibody-producing mouse)
50 µL of a 1 mg/mL human cofilin 1 protein solution obtained in
Example 1 was mixed with 50 µL of a Sigma adjuvant system (Sigma).
The total amount of the solution was intraperitoneally administered to
a 6-week-old BALB/c mouse. After 2 weeks and 4 weeks, similarly
prepared human cofilin 1 protein solution was administered in the
same volume. Subsequently, 100 µL of blood was collected via mouse
tail vein. The sample was left to stand overnight and then
centrifuged at 5000 × g for 5 minutes, and thus the supernatant was
collected as serum.
[0131]
100 µL of the 0.1 µg/mL human cofilin 1 protein solution was
added to each well of a flat bottom 96-well plate (Nunc), followed by
overnight immobilization. After the protein solution in each well

was discarded, 400 µL of a blocking solution diluted 4-fold (PBS-T
containing 1% BSA) was poured thereinto and then the plate was left
to stand for 1 hour at room temperature. Subsequently, the plate was
washed with PBS-T so as to prepare a plate to which the human cofilin
1 protein had been immobilized. A serum sample obtained above was
diluted 1000-fold, 100 µL of the solution was added to each well of
the plate to which the human cofilin 1 protein had been immobilized,
and then the plate was left to stand at room temperature for 1 hour.
Subsequently, solutions within wells were discarded, the plate was
washed with PBS-T, 100 µL of an HRP-labeled anti-mouse IgG
solution (GE Healthcare) was added, and then the plate was further
left to stand at room temperature for 1 hour. Solutions within wells
were discarded, the plate was washed with PBS-T, and then 100 µL of
a TMB solution was added for 15 minutes of reaction. Color
development resulting from the reaction was confirmed with
absorbance at 450 nm. It was determined that an antibody against the
human cofilin 1 protein was produced in mice from which blood
samples with colors developed had been collected.
[0132]
(Preparation of anti-human cofilin 1 monoclonal antibody)
A human cofilin 1 protein solution prepared in a manner similar
to the above was intraperitoneally administered to a mouse for which
antibody production against the human cofilin 1 protein had been
confirmed. 3 days later, splenectomy was carried out. The excised
spleen was punctured with a syringe, and then an RPMI1640 medium
(GIBCO) was injected to extrude spleen cells, so that a spleen cell
solution was obtained. The obtained spleen cell solution was

centrifuged at 1200 rpm for 7 minutes, to remove a supernatant, and
then the resultant was washed with an RPMI1640 medium. The
resultant was suspended again in an RPMI1640 medium, the number of
cells was counted, and thus a SP2/0 myeloma cell solution containing
SP2/0 myeloma cells in number 1/10 the number of spleen cells was
prepared. Both cell solutions were mixed, and the mixture was
centrifuged at 2200 rpm for 10 minutes, following which the
supernatant was discarded. Cells were loosened by tapping, 1 mL
of a solution prepared by mixing PEG (ROCHE) with HBSS (GIBCO)
at a ratio of 5:1 was added to the cells, and then the solution was
stirred. In the following procedures, solutions or media used herein
were all maintained at 37°C and then used, unless particularly
specified.
[0133]
An RPMI1640 medium (9 mL) was gradually added to the cell
solution supplemented with PEG and HBSS for 5 minutes. The
solution was slowly mixed, centrifuged at 2200 rpm for 10 minutes,
and thus the supernatant was removed. The obtained precipitated
cells were suspended in an RPMI1640 medium supplemented with
15% FCS and HAT (ROCHE). The suspension was added to a 96-well
cell culture plate (Greiner Bio-One) at 200 µL per well, followed by 1
week of culture at 37°C under 5% CO2.
[0134]
Colonies that had grown under conditions in which HAT had
been added thereto were determined to be colonies of hybridomas
resulting from the fusion of spleen cells to myeloma cells. The
supernatant of each well in which the colonies had grown was diluted

5-fold, and then 100 µL of the solution was added to each well of the
plate to which the human cofilin 1 protein had been immobilized, and
then whether or not antibody production was taking place was
determined by a method similar to the above. Wells for which antibody
production had been confirmed were designated as being positive.
Colonies in positive wells were suspended in an RPMI medium
containing 15% FCS and HT (Invitrogen), and then cloning of positive
clones was carried out by limiting dilution. 39 types of hybridoma
obtained as a result of cloning were conditioned to an SFM medium,
and then antibody production was carried out. Hybridomas were
seeded into a 100% SFM medium (60 mL) at 1x105 cells/mL and then
cultured for 10 days until all cells had died. Then the culture
solution was centrifuged at 3000 rpm for 15 minutes to remove the
cells. Antibodies contained in the obtained culture supernatants were
purified using ProSep-vA Ultra chromatography (Millipore).
[0135]
Epitope mapping of cofilin 1 protein antibody
(Preparation of deletion mutant)
The thus obtained 39 types of purified antibody were subjected
to epitope sequencing by the following techniques. First, 5 types of
human cofilin 1 protein deletion mutant (mutant A to mutant E) in
Example 1 and the full-length human cofilin 1 protein as GST fusion
proteins were prepared (Fig. 1).
[0136]
PCR was carried out for mutant A using pET30a_cofilin 1 gene
(10 ng) as a template and a primer set consisting of the nucleotide
sequences shown in SEQ ID NOS: 32 and 37, for mutant B using the

same template and a primer set consisting of the nucleotide sequences
shown in SEQ ID NOS: 33 and 35, for mutant C using the same
template and a primer set consisting of the nucleotide sequences
shown in SEQ ID NOS: 32 and 38, for mutant D using the same
template and a primer set consisting of the nucleotide sequences
shown in SEQ ID NOS: 32 and 36, for mutant E using the same
template and a primer set consisting of the nucleotide sequences
shown in SEQ ID NOS: 34 and 38, and for the full-length human
cofilin 1 using the same template and a primer set consisting of the
nucleotide sequences shown in SEQ ID NOS: 32 and 35, so that each
gene was obtained. The nucleotide sequences shown in SEQ ID NOS:
32, 33, and 34 contained a portion of the 5' terminal region of the
human cofilin 1 gene and a BamH I recognition sequence upstream of
the portion. The nucleotide sequences shown in SEQ ID NOS: 35 to
38 contained a portion of the 3' terminal region of the human cofilin 1
gene and an EcoR I recognition sequence downstream of the portion.
[0137]
The obtained 6 types of PCR product were each cleaved with
BamH I and EcoR I, and then a ligation reaction was conducted to
incorporate the resultant into open circular pGEX6P-l (GE
Healthcare) that had been subjected to cleavage with BamE I and
EcoR I, and treatment with BAP. Ligation High (Toyobo Co., Ltd.)
was used as DNA ligase, and then the reaction was conducted in
accordance with the protocols included therewith. Subsequently,
competent cells were transformed using a solution obtained after the
ligation reaction. Competent cells were specifically prepared using a
DH5α Escherichia coli strain (Takara Bio Inc.) in accordance with the

protocols included therewith. Transformed cells were applied onto
an LB plate containing antibiotic ampicillin (100 p,g/mL), and then
cultured at 37°C overnight. The thus obtained transformant was
cultured in an LB liquid medium containing 100 µg/mL ampicillin at
37°C overnight, and thus 6 types of expression vector constructed by
introducing the full-length human cofilin 1 protein of interest and the
5 types of deletion mutant gene were obtained by Mini-Prep.
[0138]
The above 6 types of expression vector were each transformed
into Rosetta-Gami2 Escherichia coli strain (Novagen). With
procedures described below, recombinant GST fusion proteins of the
full-length human cofilin 1 and the 5 types of deletion mutant were
prepared. First, the transformant was pre-cultured overnight at 37°C
in 10 mL of an LB medium containing ampicillin and chloramphenicol.
Next, the pre-cultured cells were seeded in 1 L of the same medium
and then cultured at 37°C for 5 hours. IPTG having a final
concentration of 0.5 mM was added and then the cells were cultured at
37°C for 12 hours, so as to induce the expression of the recombinant
human cofilin 1 protein of interest. Microorganisms were then
collected by centrifugation.
[0139]
The obtained microorganisms were washed with PBS and then a
soluble fraction was obtained using B-PER (PIERCE). The procedure
was specifically carried out in accordance with the protocols included
therewith. Next, the recombinant GST fusion protein in the soluble
fraction was adsorbed to glutathione sepharose 4B (GE Healthcare).
The resin to which the protein had been adsorbed was washed with

TBS (150 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA, pH8.0), and then
eluted with TBS containing 10 mM reduced glutathione. The
obtained GST fusion protein was adjusted to 1 mg/mL and then the
solution was used for the following experiment.
[0140]
(Evaluation of reactivity of monoclonal antibody to human cofilin 1
deletion mutant)
A solution (1 µg/mL) of each recombinant GST fusion protein of
the full-length human cofilin 1 and 5 types of deletion mutant was
prepared. The solution (100 µL each) was poured into each well of a
flat bottom 96-well plate (Nunc), followed by 3 hours of
immobilization. The GST fusion protein solution within each well
was discarded, 400 µL of a blocking solution (PBS-T containing 1%
BSA) was poured into each well, and then the plate was left to stand
for 1 hour at 4°C. Subsequently, the solution was discarded, the
plate was washed with PBS-T, and thus a plate to which an antigen
protein had been immobilized was prepared. Also, a blank plate was
also prepared by performing only a blocking reaction without
performing immobilization of the GST fusion protein. Next, each of
the 39 types of anti-human cofilin 1 monoclonal antibody diluted with
PBS-T containing 1% BSA to have a final concentration of 1 µg/mL
was poured into the blank plate and the plate to which the GST fusion
protein had been immobilized, followed by 1 hour of reaction at room
temperature. The solution within each well was discarded. The
plate was washed with PBS-T, and then 100 µL of an HRP-labeled
anti-mouse IgG antibody solution (GE Healthcare) was added for 1
hour of reaction at room temperature. Subsequently, after washing

with PBS-T, 100 µL of the TMB solution was added for 1 minute of
color reaction. The reaction was stopped by adding 100 µL of a 2 N
sulfuric acid solution. Color development was confirmed by
measuring absorbance at 450 nm. When the absorbance was higher
by 0.5 or more than that of a blank well, " reactivity" was confirmed,
suggesting the binding of the immobilized recombinant GST fusion
protein to the monoclonal antibody added. The binding of each of
the 39 types of anti-human cofilin 1 monoclonal antibody to the GST
fusion protein is as shown in Table 1. In Table 1, a result in which
"reactivity" was confirmed is indicated with "o" and a result in which
"non-reactivity" was confirmed is indicated with "x."
[0141]
(Identification of epitope sequence)
Epitope sequences were determined using as indices differences
in reactivity of the 39 types of anti-human cofilin 1 monoclonal
antibody to the full-length human cofilin 1 and the 5 types of deletion
mutant. For example, the 4E12 antibody reacted to only the
full-length human cofilin 1 protein and mutant B but did not react to
other deletion mutants. Therefore, at least a partial sequence of the
epitope sequence of the 4E12 antibody is present in the full-length
human cofilin 1 protein and mutant B, but is contained in the C3
region shown in SEQ ID NO: 4 corresponding to a deleted region in
other deletion mutants (Fig. 1).
[0142]
Similar analysis was conducted for other clones. As a result,
the epitopes to be recognized by the 39 types of anti-human cofilin 1
monoclonal antibody were classified by their locations; that is, each

epitope was present in any one of the peptide regions (N1, N2, M, C1,
C2, and C3) consisting of partial sequences of the human cofilin 1
protein (Table 1).
Table 1

[0143]
Construction of ELISA for measurement of human cofilin
1 protein
(Human cofilin 1 protein expression in HEK293 cells)
HEK293 cells (2xl06 cells) were cultured overnight in DMEM
(GIBCO) containing 10% FCS using cell culture dishes (Corning).
pCMV-Myc_human cofilin 1 or pCMV-Myc was introduced into the
cells using lipofectamine 2000 (Invitrogen) in accordance with the
protocols included therewith. After 2 days of culture, the medium
was removed. After washing with 10 mL of PBS, 1.0 mL of a 1%
NP40 buffer (1% NP40, 150 mM NaCl, 5 mM EDTA, 100 mM Tris-Cl,
and pH 8.0) was added, and then cells were left to stand for 30
minutes at 4°C. Cells were suspended in 1% NP40 buffer, and then
the solution was transferred to 1-mL centrifugal tubes. After
centrifugation at 15,000 × g, supernatants were collected. Each
sample (5 µL) was denatured with an LDS sample buffer (Invitrogen)
and then subjected to electrophoresis using NuPage 4-12% Bis-Tris
Gel (Invitrogen). The proteins were transferred to a PVDF
membrane. The resultant underwent reaction with a rabbit polyclonal
antibody (Proteintech Group), and then with a peroxidase-labeled
secondary antibody. Proteins that have immunologically reacted were
exposed to X-ray films using Western Lightning Plus-ECL (Perkin
Elmer) for visualization. As a result, the band representing the
human cofilin 1 protein was detected for only cells into which
pCMV-Myc_human cofilin 1 had been introduced (Fig. 2).
[0144]
(Selection of antibody combination)

For selection of a combination of 2 types of antibody for
sandwich ELISA, by which high-sensitivity detection is possible, 1521
combinations (39x39 combinations) obtained from the 39 types of
antibody and the 39 types of biotin-labeled antibody were compared
for superiority and inferiority in detection sensitivity in sandwich
ELISA. ELISA was carried out using as a positive control sample, a
cell extract into which pCMV-Myc_cofilin 1 had been introduced and
using as a negative control, a cell extract into which pCMV-Myc had
been introduced. Superiority and inferiority in detection sensitivity
were determined using as an index a value found by subtracting the
measured value of the negative control from the measured value of the
positive control.
[0145]
First, a solution (3 µg/mL) of each of the 39 types of purified
antibody was prepared. The solution (100 µL each) was added to
each well of a flat bottom 96-well plate (Nune), followed by 5 hours
of immobilization. The purified antibody solution within each well
was discarded. A blocking solution (400 µL) (PBS-T containing 1%
BSA) was poured thereinto and then the plate was left to stand
overnight at 4°C. Subsequently, the solution was discarded and then
the plate was washed with PBS-T, so that a plate to which the purified
antibody had been immobilized was prepared. Next, a protein extract
of cells into which pCMV-Myc_human cofilin 1 had been introduced
or a protein extract of cells into which pCMV-Myc had been
introduced was diluted 10-fold with 1% NP40 buffer. The solution
(100 µL) was added to 39 wells per type of immobilized antibody,
followed by 1 hour of reaction at room temperature. Subsequently,

the solution within each well was discarded, and then the plate was
washed with PBS-T. The thus obtained 39 types of biotinylated
antibody were diluted with 1% NP40 buffer containing 1% BSA to 0.5
µg/mL. Each diluted solution was added to wells wherein the extract
of the pCMV-Myc_human cofilin 1-introduced cells had been reacted
and to wells wherein the extract of the pCMV-Myc-introduced cells
had been reacted, followed by 1 hour of reaction at room temperature.
The solution within each well was discarded and then the plate was
washed with PBS-T, followed by 1 hour of reaction with 100 uX of an
avidin-HRP solution (R&D) at room temperature. Furthermore, the
avidin-HRP solution was discarded, the plate was washed with PBS-T,
and then 100 µL of a TMB solution was added for 5 minutes of
reaction. The reaction was stopped by adding 100 µL of a 2N
sulfuric acid solution. Color development was confirmed by
measuring absorbance at 450 nm. For all combinations of the 39
types of immobilized antibody and the 39 types of biotin-labeled
antibody, the human cofilin 1 protein concentrations of a positive
control and a negative control were measured. Antibody combinations
with high sensitivity were determined on the basis of the value found
by subtracting the value of a negative control from the value of a
positive control. As a result, it was revealed that the human cofilin 1
protein can be detected with particularly high sensitivity with the use
of 2 types of anti-cofilin 1 monoclonal antibody specifically
recognizing any one of C1 region, C3 region, and N2 region (Fig. 3).
[0146]
Analysis of amino acid sequence of anti-cofilin 1
monoclonal antibody

(Determination of monoclonal antibody light chain and heavy chain
cDNA sequences and the amino acid sequences using hybridomas)
For the 4 types of antibody required for the selected 5 types of
antibody combination, the cDNA sequences and the amino acid
sequences of light chain and heavy chain were determined. First, a
hybridoma producing each antibody was cultured using an RPMI1640
medium supplemented with 15% FCS at 37°C under 5% CO2 to 1x106
cells/mL. Subsequently, the culture solution was centrifuged at 1200
rpm for 5 minutes, thereby collecting cells. From the thus collected
hybridomas, mRNA was prepared. mRNA preparation was
specifically carried out using a Qia shredder and an RNeazy mini kit
(Qiagen) in accordance with the protocols included therewith. Next,
cDNA was synthesized using reverse transcriptase SuperscriptII
(Invitrogen), the obtained Total mRNA as a template, and oligo dT
primers, so that a cDNA library was constructed.
[0147]
PCR was carried out using the cDNA library obtained for each
hybridoma as a template and Mouse Ig Primers (Novagen). The
amplification product (mouse immunoglobulin variable region cDNA)
was subjected to ligation using a ZERO BLUNT TOPO PCR cloning
Kit (Invitrogen). Next, competent cells were transformed with each
ligation reaction solution. Competent cells were specifically
transformed using DH5a (Takara Bio Inc.) in accordance with the
protocols included therewith. Transformed cells were applied onto
an LB plate containing 50 µg/mL kanamycin and then cultured at 37°C
overnight. Four clones each of the transformant derived from each
amplification product were seeded in LB liquid medium containing 50

µg/mL kanamycin and then cultured at 37°C overnight. As a result of
preparing a vector DNA solution from each culture solution by
Mini-Prep, 4 types each of vector solution (into which DNA encoding
the monoclonal antibody had been incorporated) were obtained per
amplification product.
[0148]
For the thus obtained vector solutions, DNA sequence analysis
was conducted for regions encoding the monoclonal antibodies using
an M13 primer. Analysis was conducted using a 3130 λ 1 genetic
analyzer (Applied Biosystems). Clones having no stop codon in
insertion regions were determined to have the DNA sequence encoding
the monoclonal antibody of interest. The light chain and heavy chain
DNA sequences of the above 4 antibodies (1E2, 2C4, 4E12, 4F12)
were determined. For the thus determined DNA sequences, the amino
acid sequences encoding the DNA sequences were determined based on
the frequency of codon usage of Escherichia coli. Thus, the
sequences shown in SEQ ID NOS: 8 to 31 could be obtained.
[0149]
The amino acid sequences shown in SEQ ID NOS: 8 to 31 were
obtained as the amino acid sequences encoding 1E2. More
specifically, SEQ ID NOS: 8, 9, and 10 encode 1E2 light chain CDR1,
CDR2, and CDR3, respectively, and the amino acid sequences shown
in SEQ ID NOS: 11, 12, and 13 encode 1E2 heavy chain CDR1, CDR2,
and CDR3, respectively.
[0150]
The amino acid sequences shown in SEQ ID NOS: 14 to 19
were obtained as the amino acid sequences encoding 2C4. More

specifically, SEQ ID NOS: 14 to 16 encode 2C4 light chain CDR1 to
3, respectively. Also the amino acid sequences shown in SEQ ID
NOS: 17 to 19 encode 2C4 heavy chain CDR1 to 3, respectively.
[0151]
The amino acid sequences shown in SEQ ID NOS: 20 to 25 were
obtained as the amino acid sequences encoding 4E12. More
specifically, SEQ ID NOS: 20 to 22 encode 4E12 light chain CDR1 to
3, respectively. The amino acid sequences shown in SEQ ID NOS: 23
to 25 encode 4E12 heavy chain CDR1 to 3, respectively.
[0152]
The amino acid sequences shown in SEQ ID NOS: 26 to 31 were
obtained as the amino acid sequences encoding 4F12. More
specifically, SEQ ID NOS: 26 to 28 encode 4F12 light chain CDR1 to
3, respectively. The amino acid sequences shown in SEQ ID NOS: 29
to 31 encode 4F12 heavy chain CDR1 to 3, respectively.
[0153]
Detection of human cofilin 1 protein by sandwich ELISA
method using monoclonal antibody 1E2 and monoclonal antibody 4E12
It was thus revealed in Example 3 that the human cofilin 1
protein can be detected with the highest sensitivity by the sandwich
ELISA method using: from among the amino acid sequence shown in
SEQ ID NO: 1, the 1E2 monoclonal antibody specifically recognizing
at least an epitope that is present in the peptide region consisting of
the amino acid sequence shown in SEQ ID NO: 5; and from among the
amino acid sequence shown in SEQ ID NO: 1, a biotin-labeled 4E12
monoclonal antibody specifically recognizing at least an epitope that
is present in a peptide region consisting of the amino acid sequence

shown in SEQ ID NO: 6. Hence, a His-human cofilin 1 protein was
serially diluted with TBS buffer containing 1% BSA and 1% NP40
from 1000 ng/mL to 10 pg/mL. Detection sensitivity of the sandwich
ELISA was calculated by measurement using the diluted solutions as
samples. As a result, the minimum detection limit (the minimum
concentration at which the mean value + 2SD does not overlap with
the mean value of subject - 2SD) of the ELISA method using a
combination of 1E2 and 4E12 was 10 pg/mL (Fig. 4 (indicated with an
arrow).
[0154]
Detection of gastric cancer by measurement of human
cofilin 1 protein in blood plasma
The human cofilin 1 protein in blood plasma samples from
gastric cancer patients and healthy subjects were detected by the
sandwich ELISA method using biotin-labeled 1E2 and biotin-labeled
4E12. Blood was collected from 51 gastric cancer patients (stage I:
24 patients, stage II: 6 patients, stage III: 11 patients, stage IV: 10
patients) and 20 control healthy subjects using a Benedict II blood
vacuuming tube (Terumo Corporation), thereby obtaining blood plasma
samples. Each blood plasma sample was diluted 10-fold with TBS
buffer containing 1% BSA and 1% NP40, and then 100 JLIL thereof was
used as a sample.
[0155]
First, a 3 µg/mL solution of 1E2 anti-human cofilin 1 antibody
was prepared, and then 100 µL each of the solution was added to each
well of a flat bottom 96-well plate (Nunc), followed by 5 hours of
immobilization. The purified antibody solution within each well was

discarded, 400 µL of a blocking solution (PBS-T containing 1% BSA)
was poured thereinto, and then the plate was left to stand overnight at
4°C. The blocking solution was removed, the plate was washed once
with 400 uX of PBS-T, and then 100 uL of a blood plasma sample was
added, followed by 1 hour of reaction at room temperature. The
solution within each well was discarded, the plate was washed with
PBS-T, and then biotin-labeled 4E12 diluted with TBS buffer
containing 1% NP40 to 0.5 µg/mL was added at 100 µL per well,
followed by 1 hour of reaction at room temperature. Subsequently,
the solution within each well was discarded, the plate was washed
with PBS-T, and then 100 µL of an avidin-HRP solution (R&D)
underwent reaction for 1 hour at room temperature. Thereafter, the
avidin-HRP solution was discarded, the plate was washed with PBS-T,
and then 100 µL of the TMB solution was added for 5 minutes of
reaction. The reaction was stopped by adding 100 µL of a 2N
sulfuric acid solution. Color development was confirmed by
measuring absorbance at 450 nm. As a result, the human cofilin 1
protein was detected at statistically significantly higher levels in the
blood plasma samples from early and advanced gastric cancer patients
than blood plasma samples from control healthy subjects (Fig. 5).
[0156]
Detection of gastric cancer by measurement of human
cofilin 1 protein in serum
The human cofilin 1 in sera of gastric cancer patients and
healthy subjects were detected by the sandwich ELISA method using
biotin-labeled 1E2 and biotin-labeled 4E12. Blood was collected
from 15 gastric cancer patients (stage I: 7 patients; stage II: 1 patient;

stage III: 4 patients; stage IV: 3 patients) and 5 control healthy
subjects using a Benedict II blood vacuuming tube (Terumo
Corporation), so as to obtain sera. Each serum sample was diluted
10-fold with TBS buffer containing 1% BSA and 1% NP40, and then
100 fiL of each solution was used as a sample.
[0157]
First, a 3 µg/mL solution of 1E2 anti-human cofilin 1 antibody
was prepared. 100 µL each of the solution was added to each well
of a flat bottom 96-well plate (Nunc), followed by 5 hours of
immobilization. The purified antibody solution within each well was
discarded, 400 µL of a blocking solution (PBS-T containing 1% BSA)
was poured thereinto, and then the plate was left to stand overnight at
4°C. The blocking solution was removed, the plate was washed once
with 400 µL of PBS-T, and then 100 µL of a serum sample was added,
followed by 1 hour of reaction at room temperature. The solution
within each well was discarded, the plate was washed with PBS-T, and
then biotin-labeled 4E12 diluted with TBS buffer containing 1% NP40
to 0.5 µg/mL was added at 100 µL per well, followed by 1 hour of
reaction at room temperature. Subsequently, the solution within each
well was discarded, the plate was washed with PBS-T, and then 100 µL
of an avidin-HRP solution (R&D) underwent reaction for 1 hour at
room temperature. Thereafter, the avidin-HRP solution was
discarded, the plate was washed with PBS-T, and 100 µL of the TMB
solution was added for 5 minutes of reaction. The reaction was
stopped by adding 100 µL of a 2N sulfuric acid solution. Color
development was confirmed by measuring absorbance at 450 nm. As
a result, the human cofilin 1 protein was detected at statistically

significantly higher levels in serum samples from early and advanced
gastric cancer patients than serum samples from control healthy
subjects (Fig. 6).
Industrial Applicability
[0158]
The present invention enables to quantitatively detect with high
sensitivity the cofilin 1 protein that is present in biological samples
derived from various mammalian cells. Furthermore, according to
the method of the present invention, early gastrointestinal cancer can
be detected with high sensitivity using patients' blood. Hence, the
method of the present invention is also useful as a method for
screening for an early cancer patient using a noninvasive testing
technique. Moreover, the method of the present invention is also
useful as an assay system to be used in basic research for the purpose
of elucidating a cytoskeleton regulatory mechanism in which the
cofilin 1 protein is involved.
[0159]
All publications, patents, and patent applications cited herein
are incorporated herein by reference in their entirety.

WE CLAIMS
1. An anti-cofilin 1 monoclonal antibody or a fragment thereof, which
specifically recognizes an epitope that is present in any one of the
following peptide regions:
a) a peptide region comprising at least the amino acid sequence shown
in SEQ ID NO: 5 in the amino acid sequence shown in SEQ ID NO: 1;
b) a peptide region having a deletion, a substitution, or an addition of
1 or several amino acids with respect to the amino acid sequence of
the peptide region of a); and
c) a peptide region having 90% or more identity with the amino acid
sequence of the peptide region of a);
2. The anti-cofilin 1 monoclonal antibody or a fragment thereof
according to claim 1, wherein
in the light chain, CDR1 comprises the sequence shown in SEQ ID
NO: 8, CDR2 comprises the sequence shown in SEQ ID NO: 9, and
CDR3 comprises the sequence shown in SEQ ID NO: 10, and
in the heavy chain, CDR1 comprises the sequence shown in SEQ ID
NO: 11, CDR2 comprises the sequence shown in SEQ ID NO: 12, and
CDR3 comprises the sequence shown in SEQ ID NO: 13.
3. The anti-cofilin 1 monoclonal antibody or a fragment thereof
according to claim 1, wherein
in the light chain, CDR1 comprises the sequence shown in SEQ ID
NO: 14, CDR2 comprises the sequence shown in SEQ ID NO: 15, and

CDR3 comprises the sequence shown in SEQ ID NO: 16, and
in the heavy chain, CDR1 comprises the sequence shown in SEQ ID
NO: 17, CDR2 comprises the sequence shown in SEQ ID NO: 18, and
CDR3 comprises the sequence shown in SEQ ID NO: 19.
4. An anti-cofilin 1 monoclonal antibody or a fragment thereof, which
specifically recognizes an epitope that is present in any one of the
following peptide regions:
a) a peptide region comprising at least the amino acid sequence
shown in SEQ ID NO: 6 in the amino acid sequence shown in SEQ ID
NO: 1.
b) a peptide region having a deletion, a substitution, or an addition
of 1 or several amino acids with respect to the amino acid sequence of
the peptide region of a); and
c) a peptide region having 90% or more homology with the amino
acid sequence of the peptide region of a).
5. The anti-cofilin 1 monoclonal antibody or a fragment thereof
according to claim 4, wherein in the light chain, CDR1 comprises the
sequence shown in SEQ ID NO: 20, CDR2 comprises the sequence
shown in SEQ ID NO: 21, and CDR3 comprises the sequence shown in
SEQ ID NO: 22, and in the heavy chain, CDR1 comprises the sequence
shown in SEQ ID NO: 23, CDR2 comprises the sequence shown in SEQ
ID NO: 24, and CDR3 comprises the sequence shown in SEQ ID NO:
25.
6. An anti-cofilin 1 monoclonal antibody or a fragment thereof, which

specifically recognizes an epitope that is present in any one of the
following peptide regions:
a) a peptide region comprising at least the amino acid sequence
shown in SEQ ID NO: 7 in the amino acid sequence shown in SEQ ID
NO: 2;
b) a peptide region having a deletion, a substitution, or an addition
of 1 or several amino acids with respect to the amino acid sequence of
the peptide region of a); and
c) a peptide region having 90% or more identity with the amino acid
sequence of the peptide region of a).
7. The anti-cofilin 1 monoclonal antibody or a fragment thereof
according to claim 6, wherein
in the light chain, CDR1 comprises the sequence shown in SEQ ID
NO: 26, CDR2 comprises the sequence shown in SEQ ID NO: 27, and
CDR3 comprises the sequence shown in SEQ ID NO: 28, and
in the heavy chain, CDR1 comprises the sequence shown in SEQ ID
NO: 29, CDR2 comprises the sequence shown in SEQ ID NO: 30, and
CDR3 comprises the sequence shown in SEQ ID NO: 31.
8. An immunoassay of cofilin 1 protein, comprising measuring cofilin
1 and/or a fragment thereof in a sample using 2 or more types of
anti-cofilin 1 monoclonal antibody and/or fragments thereof that
specifically recognize different epitopes on the amino acid sequence
of the cofilin 1 protein.
9. The immunoassay according to claim 8, wherein the different

epitopes are present in the peptide regions consisting of the amino
acid sequences shown in SEQ ID NO: 1 and/or 2.
10. The immunoassay according to claim 9, wherein the different
epitopes are present in a peptide region comprising at least the amino
acid sequence shown in SEQ ID NO: 3 in the peptide region consisting
of the amino acid sequence shown in SEQ ID NO: 1, and/or a peptide
region comprising at least the amino acid sequence shown in SEQ ID
NO: 4 in the peptide region consisting of the amino acid sequence
shown in SEQ ID NO: 2.
11. The immunoassay according to claim 9, wherein the different
epitopes are present in the peptide regions comprising at least the
amino acid sequences shown in SEQ ID NO: 5 and/or 6 in the peptide
region consisting of the amino acid sequence shown in SEQ ID NO: 1,
and/or a peptide region comprising at least the amino acid sequence
shown in SEQ ID NO: 7 in the peptide region consisting of the amino
acid sequence shown in SEQ ID NO: 2.
12. The immunoassay according to claim 8, wherein the different
epitopes are separately present in two peptide regions selected from
the peptide regions consisting of the amino acid sequences shown in
SEQ ID NOS: 5 to 7.
13. The immunoassay according to claim 9, wherein the different
epitopes are present in a peptide region consisting of 6 or more and 21
or less continuous amino acids in the peptide regions consisting of the

amino acid sequences shown in SEQ ID NO: 1 and/or 2.
14. The immunoassay according to claim 8, wherein the above 2 or
more types of anti-cofilin 1 monoclonal antibody and/or fragments
thereof are selected from the anti-cofilin 1 monoclonal antibodies or
fragments thereof of claims 2, 3, 5, and 7.
15. The immunoassay according to claim 14, wherein the 2 types of
anti-cofilin 1 monoclonal antibody and/or fragments thereof constitute
any one of the following combinations of anti-cofilin 1 monoclonal
antibodies and/or fragments thereof:

a) a combination of the anti-cofilin 1 monoclonal antibodies and/or
the fragments thereof separately described in claims 2 and 5;
b) a combination of the anti-cofilin 1 monoclonal antibodies and/or
fragments thereof separately described in claims 2 and 7;
c) a combination of the anti-cofilin 1 monoclonal antibodies and/or
fragments thereof separately described in claims 3 and 5; and
d) a combination of the anti-cofilin 1 monoclonal antibodies and/or
fragments thereof separately described in claims 5 and 7.

16. The immunoassay according to any one of claims 8 to 15, wherein
the sample is blood, urine, cell supernatant, cell extract, tissue
extract, gastric juice, saliva, lymph fluid, lacrimal fluid, or seminal
fluid.
17. A method for determining whether or not gastrointestinal cancer is
developing, comprising the steps of:

measuring the amounts of the cofilin 1 protein and/or a fragment
thereof in a sample derived from a subject and a healthy subject using
the immunoassay of any one of claims 8 to 16;
comparing the amounts of the cofilin 1 protein and/or a fragment
thereof measured in the above measurement step, and then determining
that the subject is affected by gastrointestinal cancer if the amount of
the cofilin 1 protein and/or a fragment thereof in the subject is
statistically significantly higher than that of the healthy subject.
18. The determination method according to claim 17, wherein the
gastrointestinal cancer is early gastrointestinal cancer.
19. The determination method according to claim 17 or 18, wherein
the gastrointestinal cancer is gastric cancer.
20. A kit for cofilin 1 protein quantification, containing 2 or more
types of anti-cofilin 1 monoclonal antibody and/or fragments thereof,
which specifically recognize different epitopes of the cofilin 1
protein.
21. The kit according to claim 20, containing 2 or more types of the
anti-cofilin 1 monoclonal antibody or the fragments thereof of any one
of claims 1 to 7.