U.S. patent application number 14/465336 was filed with the patent office on 2015-02-19 for method and kit for isolating target cell.
The applicant listed for this patent is SAMSUNG ELECTRONICS CO., LTD.. Invention is credited to Jeong-gun LEE, Jong-myeon PARK.
Application Number | 20150050669 14/465336 |
Document ID | / |
Family ID | 44953275 |
Filed Date | 2015-02-19 |
United States Patent
Application |
20150050669 |
Kind Code |
A1 |
PARK; Jong-myeon ; et
al. |
February 19, 2015 |
METHOD AND KIT FOR ISOLATING TARGET CELL
Abstract
A particle comprising at least one polymer having negative or
positive charges and at least one antibody bound to the polymer,
wherein the antibody specifically binds to a surface marker of at
least one target cell, and kit comprising same.
Inventors: |
PARK; Jong-myeon; (Incheon,
KR) ; LEE; Jeong-gun; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SAMSUNG ELECTRONICS CO., LTD. |
Suwon-si |
|
KR |
|
|
Family ID: |
44953275 |
Appl. No.: |
14/465336 |
Filed: |
August 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13582931 |
Nov 13, 2012 |
8846322 |
|
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PCT/KR2011/001561 |
Mar 7, 2011 |
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14465336 |
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Current U.S.
Class: |
435/7.23 ;
521/32 |
Current CPC
Class: |
G01N 33/543 20130101;
C07K 17/08 20130101; G01N 33/57492 20130101; C07K 16/30 20130101;
G01N 33/574 20130101; G01N 33/54313 20130101; G01N 33/56966
20130101; G01N 33/54353 20130101; Y10T 428/2982 20150115; G01N
33/57484 20130101 |
Class at
Publication: |
435/7.23 ;
521/32 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C07K 16/30 20060101 C07K016/30; C07K 17/08 20060101
C07K017/08 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 5, 2010 |
KR |
10-2010-0020079 |
Mar 3, 2011 |
KR |
10-2011-0019095 |
Claims
1. A particle comprising at least one polymer having negative or
positive charges and at least one antibody bound to the polymer,
wherein the antibody specifically binds to a surface marker of at
least one target cell.
2. The particle of claim 1, wherein the at least one polymer has
negative charges, and the particle is bound to at least one second
particle comprising at least one polymer having positive charges,
or at least one second particle comprising at least one polymer
having positive charges and at least one antibody bound to the
polymer of the second particle.
3. The particle of claim 1, wherein the at least one polymer has
positive charges, and the particle is bound to at least one second
particle comprising at least one polymer having negative charges,
or at least one second particle comprising at least one polymer
having negative charges and at least one antibody bound to the
polymer of the second particle.
4. The particle of claim 1, wherein the charges are negative, and
the polymer is polystyrenesulfonate, polyacrylic acid,
polymethacrylic acid, polyalcohol, polyphosphate, polymaleic acid,
hyaluronic acid, or a combination thereof.
5. The particle of claim 1, wherein the charges are positive, and
the polymer is polyaniline, polypyrrol, polyethyleneimine,
polylysine, chitosan, or a combination thereof.
6. The particle of claim 1, wherein the particle further comprises
a protein which links the polymer to the antibody, and the protein
is protein G, protein L, protein A, protein LA, protein AG, or a
combination thereof.
7. The particle of claim 1, wherein the particle is a polystyrene
particle, latex particle, metal particle, glass particle, magnetic
particle, or a combination thereof.
8. The particle of claim I, wherein the target cell is a
circulating tumor cell, cancer stem cell, immunocyte, fetal stem
cell, fetal cell, cancer cell, or tumor cell.
9. The particle of claim 1, wherein the particle has a diameter of
about 100 nm to about 5 .mu.m.
10. A kit for isolating a target cell from a biological sample, the
kit comprising: a) a particle of claim 1 having positive charges
and a particle of claim 1 having negative charges; b) a particle of
claim 1 having negative charges and a particle comprising at least
one polymer having positive charges; or c) a particle of claim 1
having positive charges and a particle comprising at least one
polymer having negative charges.
11. The kit of claim 10, wherein the kit further comprises a filter
having pores, optionally having a diameter of about 1 .mu.m to
about 100 .mu.m, about 3 .mu.m to about 50 .mu.m, or about 8 .mu.m
to about 30 .mu.m.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to methods and kits for
isolating a target cell by using particles comprising polymers
having positive charge and negative charge and at least one
antibody bound to polymers.
BACKGROUND ART
[0002] The majority of deaths associated with malignant tumor are
due to the metastasis of the original tumor cells to tissues and
organs distant from the initial tumor. Accordingly, early diagnosis
of metastasis is a critical factor for the survival of a cancer
patient, and early diagnosis of tumor and monitoring of tumor
growth are considered as very important factors for successful
treatment of a cancer patient. The diagnosis of cancer usually uses
diagnosis techniques by histopathology. The histopathological
diagnosis technique is a method of using a tissue sample from a
living subject to diagnose a cancer. Such a histopathological
approach allows a tumor cell to be directly observed. However, it
may be incorrect whether there is a tumor from a tissue site
selected in order to obtain a sample from a living subject, and
only data about a particular site obtained from the living subject
are provided and thus it is difficult to know whether tumor has
metastasized to another site. For this reason, the applicability in
diagnosing and monitoring tumors may be limited.
[0003] It is known that circulating tumor cells (CTCs) are found
from a patient before the tumor is originally detected.
Accordingly, CTCs may play an important role in early diagnosis and
prognosis of cancers. In addition, because cancer usually
metastasizes through blood, CTC may be a marker for determining
whether cancer has metastasized. Even after cancer cells have been
removed by surgery, CTCs may be still exist and cancer may reoccur.
However, very small amounts of these CTCs are found in blood and
the cells are themselves weak, and thus it is very difficult to
detect them and grasp the number of the cells. Accordingly, there
still remains a need for a diagnosis method that is highly
sensitive to detect CTCs, cancer cells, or cancer stem cells in a
patient's body.
[0004] CTC separation methods by using magnetic nanoparticles are
described in the related art. However, the method according to the
related art is disadvantageous because the processes are very
complicated, for example, the method of separating serum from
blood, and using the affinity of biotin and streptavidin even in a
magnetic separation process have a risk of losing of CTCs in the
separation steps.
[0005] Accordingly, there still remains a need for a method for
efficiently separating tumor cells from a biological sample and an
apparatus associated with that.
DISCLOSURE OF INVENTION
Technical Problem
[0006] Provided are particles comprising at least one polymer
having negative charges and at least one antibody bound to the
polymer, wherein the antibody specifically binds to a surface
marker of at least one target cell.
[0007] Provided are particles comprising at least one polymer
having positive charges and at least one antibody bound to the
polymer, wherein the antibody specifically binds to a surface
marker of at least one target cell.
[0008] Provided are methods and kits for isolating a target cell
from a biological sample.
[0009] Additional aspects will be set forth in part in the
description which follows and, in part, will be apparent from the
description, or may be learned by practice of the presented
embodiments.
Solution to Problem
[0010] According to an aspect of the present invention, particles
comprising at least one polymer having negative charges and at
least one antibody bound to the polymer, wherein the antibody
specifically binds to a surface marker of at least one target cell,
are provided.
[0011] The term "particle" used herein refers to a particle that
can be changed electrostatic properties according to the pH of the
ambient environment. According to an exemplary embodiment, the
particle may be a charge reversible bead.
[0012] The term "polymer" used herein refers to a class of
macromolecules composed of repeating monomers. The polymer may
include homopolymers, heteropolymers, or copoloymers. The polymer
may include linear polymers or branched polymers.
[0013] The polymer serves to change electrostatic properties of the
particle according to the pH of the ambient environment. The
polymer having negative charges may be selected from the group
consisting of polystyrenesulfonate, polyacrylic acid,
polymethacrylic acid, polyalcohol, polyphosphate, polymaleic acid,
hyaluronic acid, and any combinations thereof, but it is not
limited thereto. For example, when a particle comprising polymaleic
acid is present in a solution having a pH value higher than a pKa
value of the polymaleic acid, the particle may be negative. On the
other hand, when the particle comprising polymaleic acid is present
in a solution having a pH value lower than a pKa value of the
polymaleic acid, the particle may be neutral.
[0014] According to an exemplary embodiment, the polymer may have a
molecular weight of about 1,000 to about 100,000 or of about 3,000
to about 50,000. The polymer may be linked to the particle to
increase the charge density. Also, the polymer may provide
functional groups to bind at least one antibody, which at least one
antibody specifically binds to a surface marker of a target
cell.
[0015] According to another aspect of the present invention,
particles comprising at least one polymer having positive charges
and at least one antibody bound to the polymer, wherein the
antibody specifically binds to a surface marker of at least one
target cell, are provided.
[0016] The polymer serves to change electrostatic properties of the
particle according to the pH of the ambient environment. As
described above, the polymer having positive charges may be
selected from the group of, for example, polyaniline, polypyrrol,
polyethyleneimine, polylysine, chitosan, and any combinations
thereof, but it is not limited thereto. For example, when a
particle comprising polyethyleneimine is present in a solution
having a pH value lower than a pKa value of the polyethyleneimine,
the particle may be positive. On the other hand, when the particle
comprising polyethyleneimine is present in a solution having a pH
value higher than a pKa value of the polyethyleneimine, the
particle may be neutral.
[0017] According to an exemplary embodiment, the polymer may have a
molecular weight of about 100 to about 50,000 and of about 400 to
about 25,000. The polymer may be linked to the particle to increase
the charge density. Also, the polymer may provide functional groups
to bind at least one antibody, which at least one antibody
specifically binds to a surface marker of a target cell.
[0018] At least one antibody specifically binds to a surface marker
of at least one target cell.
[0019] The term "target cell" used herein refers to a cell having a
surface marker on a cell surface. The target cell may be selected
from the group of, for example, circulating tumor cell, cancer stem
cell, immunocyte, fetal stem cell, fetal cell, cancer cell, tumor
cell, and any combinations thereof, but it is not limited
thereto.
[0020] The term "surface marker" used herein refers to any
substance which exists on the surface of the target cell and may
differentiate the target cell from other cells in a biological
sample. The surface marker may be selected from the group
consisting of protein, polysaccharide, lipid, nucleic acid, and any
combinations thereof, but it is not limited thereto. According to
an exemplary embodiment, the surface marker may be a protein which
is specifically expressed in a target cell and displayed on a cell
membrane. For example, the protein may be selected from the group
consisting of estrogen receptor, progesterone receptor,
synaptophysin, mucin 1 (MUC 1), Bcl-2, MIB1/Ki67, cyclin D1, cyclin
E, p27, topoisomerase Ha, cyclooxygenase 2, ERK1/ERK2, phosphor-S6
ribosomal protein, CK5, CK8, CK17, vimentin, epithelial cell
adhesion molecule (EpCAM), c-Met, cytokeratines, Her2, EGFR, p53,
p63, E-cadherin, fragile histidine triad, protein tyrosine
phosphatase, .beta.-catenin, p16, c-kit, endothelin-1, endothelin
receptor-.alpha., endothelin receptor-.beta., chemokine (CXC motif)
receptor 4, breast cancer resistance protein, ABCA3, MGMT, and any
combinations thereof, but it is not limited thereto.
[0021] The definition of the term "specifically binding" used
herein is the same as the definition of the term typically known to
those skilled in the art, and the term refers to an immunological
response through specific interaction of an antigen and an
antibody. An antibody specifically binding to a surface marker of
the target cell may be construed as including a complete antibody
as well as antigen binding fragments of the antibody molecule. A
naturally occurring complete antibody, or immunoglobulin, includes
four polypeptides: two full-length light chains and two full-length
heavy chains, in which each light chain is linked to a heavy chain
by disulfide bonds. Each heavy chain has a constant region and a
variable region. Similarly, each light chain has a constant region
and a variable region. There are five heavy chain classes
(isotypes): gamma (.gamma.), mu (.mu.), alpha (.alpha.), delta
(.delta.), or epsilon (.epsilon.), and additionally several
subclasses gamma 1 (.gamma.1), gamma 2(.gamma.2), gamma
3(.gamma.3), gamma 4(.gamma.4), alpha 1(.alpha.1), and alpha
2(.alpha.2). The light chain constant region can be either kappa
(.kappa.) or lambda (.lamda.) type. The term "antigen binding
fragment" used herein refers to fragments of an intact
immunoglobulin, and any part of a polypeptide including antigen
binding regions having the ability to specifically bind to the
antigen. For example, the antigen binding fragment may be a
F(ab').sub.2 fragment, a Fab' fragment, a Fab fragment, a Fv
fragment, or a scFv fragment, but is not limited thereto. A Fab
fragment has one antigen binding site and contains the variable
regions of a light chain and a heavy chain, the constant region of
the light chain, and the first constant region CH1 of the heavy
chain. A Fab' fragment is different from the Fab fragment in that
the Fab' fragment additionally includes the hinge region of the
heavy chain, including at least one cysteine residue at the
C-terminal of the heavy chain CH1 region. The F(ab).sub.2fragment
is produced whereby cysteine residues of the Fab' fragment are
joined by a disulfide bond at the hinge region. A Fv fragment is
the minimal antibody fragment having only heavy chain variable
regions and light chain variable regions, and a recombinant
technique for producing the Fv fragment is well known in the art.
Two-chain Fv fragments may have a structure in which heavy chain
variable regions are linked to light chain variable regions by a
non-covalent bond. Single-chain Fv fragments generally may have a
dimer structure as in the two-chain Fv fragments in which heavy
chain variable regions are covalently bound to light chain variable
regions via a peptide linker or heavy and light chain variable
regions are directly linked to each other at the C-terminal
thereof. The antigen binding fragment may be obtained using a
protease (for example, a whole antibody is digested with papain to
obtain Fab fragments, and is digested with pepsin to obtain
F(ab').sub.2 fragments), and may be prepared by a genetic
recombinant technique.
[0022] The antibody may be a monoclonal antibody, a bispecific
antibody, a non-human antibody, a human antibody, a humanized
antibody, a chimeric antibody, single chain Fvs (scFV) fragments, a
single chain antibody, Fab fragments, F(ab') fragments,
disulfide-bond Fvs (sdFV) fragments, an anti-idiotype (anti-Id)
antibody, and epitope-binding fragments of these antibodies, but is
not limited thereto. In an antibody specifically binding to a
surface marker of the target cell, a constant region of the
antibody may be bound to a polymer linked to the particle such that
an antigen binding site may be exposed.
[0023] According to an exemplary embodiment, the particle may
further include a protein. According to an exemplary embodiment,
the protein may link the polymer to the antibody. The protein may
be selected from the group consisting of protein G, protein L,
protein A, protein LA, protein AG, and any combinations thereof.
The protein is a microorganism-derived protein which binds to a
heavy-chain constant region of immunoglobulin. The protein
conventionally used for purification of antibodies. By linking the
protein to the polymer and antibody, the directionality may be
given such that an antigen binding site of the antibody may be
directed toward a surface marker of a target cell.
[0024] According to an exemplary embodiment, the particle may be
selected from the group consisting of polystyrene particle, latex
particle, metal particle, glass particle, magnetic particle, and
any combinations thereof, but it is not limited thereto. In
addition, according to an exemplary embodiment, for example, the
particle may have a diameter of about 10 nm to about 10 .mu.m,
about 100 nm to about 5.mu., or about 1 .mu.m to about 3 .mu.m. The
particle can increase the size of a target cell by binding to the
surface marker of the marget cell. For example, because a cancer
cell in blood is about 14 um to about 24 um in size. A white blood
cell ranging from about 10 um to about 20 um in size. Thus, it is
difficult to selectively isolate the cancer cell from the blood.
According to an exemplary embodiment, because the particle links to
at least one polymer having negative charges or positive charges,
and to which at least one antibody specifically binding to a
surface marker of at least one target cell is bound, the particle
is positioned around the target cell by binding to the surface
marker of the target cell. Thus, the binding of the particle may
increase the size of the particle to allow the target cell to be
selectively isolated from the blood.
[0025] According to another aspect of the present invention, a
method for isolating a target cell from a biological sample
includes: a) contacting a particle comprising at least one polymer
having negative charges and at least one antibody bound to the
polymer, wherein the antibody specifically binds to a surface
marker of at least one target cell, with a biological sample in a
solution; b) adding a particle comprising at least one polymer
having positive charges or a particle comprising at least one
polymer having positive charges and at least one antibody bound to
the polymer, wherein the antibody specifically binds to a surface
marker of at least one target cell, into the mixed solution of step
a); and c) adjusting a pH value of the mixed solution of step
b).
[0026] The method for isolating a target cell will be described in
detail with each of the following steps:
[0027] The method may include: a) contacting a particle comprising
at least one polymer having negative charges and at least one
antibody bound to the polymer, wherein the antibody specifically
binds to a surface marker of at least one target cell, with a
biological sample in a solution.
[0028] According to an exemplary embodiment, the biological sample
may be any biological sample in which the target cell may be
present. For example, the sample may be selected from the group
consisting of a biopsy sample, a tissue sample, a cell suspension
including a separated cell suspended in a liquid medium, a cell
culture, and any combinations thereof. In addition, the biological
sample may be an animal body fluid. The body fluid may be selected
from the group consisting of blood, bone marrow fluid, lymph fluid,
saliva, lachrymal fluid, urine, mucous fluid, amniotic fluid, and
any combinations thereof, but it is not limited thereto.
[0029] For example, in order to isolate a circulating tumor cell,
blood may be used as the biological sample.
[0030] The contacting may be performed by adding a particle
comprising at least one polymer having negative charges and at
least one antibody bound to the polymer, wherein the antibody
specifically binds to a surface marker of at least one target cell,
into a solution including the biological sample. According to an
exemplary embodiment, the contacting may be performed in a solution
having a pH value equal to or higher than a pKa value of the
polymer having negative charges. Then, the particle may be neutral
in the solution. The solution serves to provide an environment in
which a biological sample and the particle may be stably reacted,
and any buffer well known in the art may be used as the solution.
The solution may be phosphate buffered saline (PBS) or phosphate
buffered saline Tween (PBST), but it is not limited thereto.
[0031] According to an exemplary embodiment, the method may further
include, before the contacting, pre-treating the biological sample
to isolate cells from the biological sample. The cell refers to any
cells in the biological sample including the target cell. For
example, the pre-treatment may be performed by reducing or removing
other materials except for the cells from the sample. The
pre-treatment may be selected from the group consisting of
centrifugation, filtration, chromatography such as affinity
chromatography, and any combinations thereof. For example, when the
biological sample is blood, plasma or protein may be removed
through the pre-treatment.
[0032] In addition, the method may further include, after the
contacting, washing the particles unbound to the target cell to
remove them.
[0033] The washing may be achieved by performing at least one
selected from the group consisting of flowing a washing solution,
centrifugation, filtration, chromatography, and any combinations
thereof to remove or reduce materials other than particles bound to
the target cell. The washing solution may be selected from the
group consisting of water, buffer (e.g., PBS), physiological
saline, and any combinations thereof, but it is not limited
thereto.
[0034] Subsequently, the method may include: b) adding a particle
comprising at least one polymer having positive charges or a
particle comprising at least one polymer having positive charges
and at least one antibody bound to the polymer, wherein the
antibody specifically binds to a surface marker of at least one
target cell, into the mixed solution of step a).
[0035] In the step a), a particle comprising at least one polymer
having negative charges and at least one antibody bound to the
polymer, wherein the antibody specifically binds to a surface
marker of at least one target cell, is present in a state in which
the particle is specifically bound to the target cell in the
biological sample. Accordingly, when the particle comprising at
least one polymer having positive charges or a particle comprising
at least one polymer having positive charges and at least one
antibody bound to the polymer, wherein the antibody specifically
binds to a surface marker of at least one target cell, is added
into the solution of stap a), the particle may be suspended in the
mixed solution. In addition, because a pH value of the mixed
solution of step a) may be equal to or lower than a pKa value of
the polymer having negative charges, a particle comprising at least
one polymer having positive charges or a particle comprising at
least one polymer having positive charges and at least one antibody
bound to the polymer, wherein the antibody specifically binds to a
surface marker of at least one target cell, may be positive.
[0036] Finally, the method may include: c) adjusting a pH value of
the mixed solution of step b).
[0037] According to an exemplary embodiment, the adjusting may be
performed by adjusting a pH value of the mixed solution of step b)
to have a pH value higher than a pKa value of the polymer having
negative charges and lower than a pKa value of the polymer having
positive charges. For example, when the polymer having negative
charges is polymaleic acid and the polymer having positive charges
is polyethyleneimine, a pH value in the mixed solution of step b)
may be adjusted to higher than about 4 and lower than about 10,
specifically about 6 or higher and about 7 or lower.
[0038] The pH value may be adjusted by using any acid or base known
in the art, which may increase or decrease the pH value. By
adjusting the pH value such that it has a value higher than a pKa
value of the polymer having negative charges and lower than a pKa
value of the polymer having positive charges in the step, a
particle comprising at least one polymer having positive charges
and at least one antibody bound to the polymer, wherein the
antibody specifically binds to a surface marker of at least one
target cell, may be negative. Accordingly, an electrostatic
attraction forms between the particles having negative charges and
the particles having positive charges, and thus aggregation may be
occur between particles. Therefore, the overall size of the target
cell may be increased, thereby the target cell can be
differentiated other cells in a biological sample.
[0039] According to an exemplary embodiment, the method further
comprises: d) isolating the target cell from the mixed solution of
step c). According to an exemplary embodiment, the isolating may be
achieved by performing one or more selected from the group
consisting of centrifugation, filtration, chromatography, and any
combinations thereof. For example, a filter having pores with a
size smaaler than that of the increase-sized target cell may be
used to isolate the target cell, to allow other cells in a
biological sample to pass and prevent the target cell whose overall
size has been increased by electrostatic aggregation from
passing.
[0040] According to an exemplary embodiment, the method may
comprise: e-1) adjusting a pH value of the mixed solution of step
d) such that the pH value has a value lower than a pKa value of the
polymer having negative charges. According to an exemplary
embodiment, the method further comprises: f) detecting the target
cell.
[0041] The step e-1) is a pre-step for detecting the isolated
target cell and a process of removing electrostatically aggregated
particles unbound to the surface marker of the target cell from the
cell whose overall size has been increased. This is step e-1) to
adjust the pH value of the mixed solution of step d) in order to
remove electrostatic attraction between the particles. That is, by
decreasing a pH of the mixed solution of step d), an aggregated
particle comprising a polymer having positive charges is
dissociated. For example, when the polymer having negative charges
is polymaleic acid, and the polymer having positive charges is
polyethyleneimine, a pH value of the solution of step e-1) may be
adjusted to about 1 or higher and about 5 or lower, or about 2 or
higher and about 4 or lower. Subsequently, the target cell in which
electrostatically aggregated particle has been removed as above may
be detected by electrical or optical methods well known in the art.
For example, the target cell may be detected by fluorescent
material bound to the particle. In addition, the target cell
isolated for detection may be cultured according to cultivation
methods well known in the art to be appropriately used for
experimental purposes.
[0042] According to another aspect of the present invention, a
method for isolating a target cell from a biological sample, the
method comprising: a) contacting a particle comprising at least one
polymer having positive charges and at least one antibody bound to
the polymer, wherein the antibody specifically binds to a surface
marker of at least one target cell, with the biological sample in a
solution; b) adding a particle comprising at least one polymer
having negative charges or a particle comprising at least one
polymer having negative charges and at least one antibody bound to
the polymer, wherein the antibody specifically binds to a surface
marker of at least one target cell, into the mixed solution of step
a); and c) adjusting a pH value of the mixed solution of step
b).
[0043] Because the method for isolating a target cell described
above, each step will be described by omitting what are common
between the two methods in order to avoid the excessive complexity
of the specification:
[0044] The method may include: a) contacting a particle comprising
at least one polymer having positive charges and at least one
antibody bound to the polymer, wherein the antibody specifically
binds to a surface marker of at least one target cell, with the
biological sample in a solution.
[0045] According to an exemplary embodiment, the contacting may be
performed in a solution having a pH value equal to or smaller than
a pKa value of the polymer having positive charges. Then, the
particle may be neutral in the solution.
[0046] According to an exemplary embodiment, the method may further
include, before the contacting, pre-treating the biological sample
to isolate cells from the biological sample. In addition, the
method may further include, after the contacting, washing the
particles unbound to the target cell.
[0047] Subsequently, the method include: b) adding a particle
comprising at least one polymer having negative charges or a
particle comprising at least one polymer having negative charges
and at least one antibody bound to the polymer, wherein the
antibody specifically binds to a surface marker of at least one
target cell, into the mixed solution of step a).
[0048] In the step a), a particle comprising at least one polymer
having positive charges and at least one antibody bound to the
polymer, wherein the antibody specifically binds to a surface
marker of at least one target cell is specifically bound to the
target cell in the biological sample. When the particle comprising
at least one polymer having negative charges or a particle
comprising at least one polymer having negative charges and at
least one antibody bound to the polymer, wherein the antibody
specifically binds to a surface marker of at least one target cell,
is added into the mixed solution of step a), the particle may be
suspended in the mixed solution. In addition, because a pH value of
the mixed solution of step a) may be equal to or higher than a pKa
value of the polymer having positive charges, a particle comprising
a polymer having negative charges or a particle comprising a
polymer having negative charges and at least one antibody bound to
the polymer, wherein the antibody specifically binds to a surface
marker of at least one target cell, may be negative.
[0049] Finally, the method may include: c) adjusting a pH value of
the mixed solution of step b).
[0050] According to an exemplary embodiment, the pH value of the
mixed solution of step b) is adjusted to a value higher than a pKa
value of the polymer having negative charges and lower than a pKa
value of the polymer having positive charges. For example, when the
polymer having positive charges is polyethyleneimine and the
polymer having negative charges is polymaleic acid, a pH value in
the mixed solution of step b) may be adjusted to higher than about
4 and lower than about 10, specifically about 6 or higher and about
7 or lower.
[0051] The pH value may be adjusted by using any acid or base known
in the art, which may increase or decrease the pH value. By
adjusting the pH value such that it has a value higher than a pKa
value of the polymer having negative charges and lower than a pKa
value of the polymer having positive charges in the step, a
particle comprising at least one polymer having positive charges
and at least one antibody bound to the polymer, wherein the
antibody specifically binds to a surface marker of at least one
target cell, may be positive, while a particle comprising the
polymer having negative charges, the particle including an antibody
specifically binding to an surface marker of at least one target
cell bound to the polymer, may be negative. Accordingly, an
electrostatic attraction forms between the particles having
negative charges and the particles having positive charges, and
thus aggregation may occur between particles. Therefore, the
overall size of the target cell may be increased, thereby the
target cell can be differentiated other cells in a biological
sample.
[0052] According to an exemplary embodiment, the method may further
comprise: d) isolating the target cell from the mixed solution of
step c). According to an exemplary embodiment, the isolating may be
achieved by performing one or more selected from the group
consisting of centrifugation, filtration, chromatography, and any
combinations thereof.
[0053] According to an exemplary embodiment, the method may
comprise: e-2) adjusting a pH value of the mixed solution of step
d) such that the pH value has a value higher than a pKa value of
the polymer having positive charges. According to an exemplary
embodiment, the method further comprises: f) detecting the target
cell. The step e-2) is a pre-step for detecting the isolated target
cell and a process of removing electrostatically aggregated
particles unbound to the surface marker of the target cell from the
cell whose overall size has been increased. This is step e-2) to
adjust the pH value of the mixed solution of step d) in order to
remove electrostatic attraction between the particles. That is, by
increasing a pH of the mixed solution of step d), an aggregated
particle comprising a polymer having negative charges is
dissociated. For example, when the polymer having positive charges
is polyethyleneimine and the polymer having negative charges is
polymaleic acid, a pH value of the solution of step e-2) may be
adjusted to about 9 or higher and about 14 or lower, specifically
about 10 or higher and about 12 or lower. Subsequently, the target
cell in which electrostatically aggregated particle has been
removed as above may be detected by electrical or optical methods
well known in the art. In addition, the target cell isolated for
detection may be cultured according to cultivation methods well
known in the art to be appropriately used for experimental
purposes.
[0054] According to another aspect of the present invention, a kit
for isolating a target cell from a biological sample, the kit
comprising: a) a particle comprising at least one polymer having
negative charges and at least one antibody bound to the polymer,
wherein the antibody specifically binds to a surface marker of at
least one target cell and a particle comprising at least one
polymer having positive charges and at least one antibody bound to
the polymer, wherein the antibody specifically binds to a surface
marker of at least one target cell; b) the particle comprising at
least one polymer having negative charges and at least one antibody
bound to the polymer, wherein the antibody specifically binds to a
surface marker of at least one target cell and a particle
comprising at least one polymer having positive charges; or c) the
particle comprising at least one polymer having positive charges
and at least one antibody bound to the polymer, wherein the
antibody specifically binds to a surface marker of at least one
target cell and a particle comprising at least one polymer having
negative charges.
[0055] According to an exemplary embodiment, the kit may further
comprise a filter having pores.
[0056] The kit may be manufactured by varying the antibody
according to the type of a target cell to be isolated and by
varying a pore diameter according to the size of a target cell.
According to an exemplary embodiment, the kit may be manufactured
to have a pore diameter of, for example, about 1 .mu.m to about 100
.mu.m, about 3 .mu.m to about 50 .mu.m, or about 8 .mu.m to about
30 .mu.m.
[0057] One or more embodiments of the present invention will be
described in further detail with reference to the following
examples. These examples are for illustrative purposes only and are
not intended to limit the scope of the one or more embodiments of
the present invention.
BRIEF DESCRIPTION OF DRAWINGS
[0058] These and/or other aspects will become apparent and more
readily appreciated from the following description of the
embodiments, taken in conjunction with the accompanying drawings of
which:
[0059] FIGS. 1 and 2 are views schematically illustrating a method
for isolating a target cell according to an exemplary embodiment
;
[0060] FIG. 3 is a schematic view of a particle having negative
charges, the particle having an antibody, according to an exemplary
embodiment;
[0061] FIG. 4 is a set of fluorescent microscopic photos
illustrating results of aggregation and dissociation between
CRBs(CRB1-1 or CRB1-2 and CRB2-1, CRB2-2 or CRB2-3) according to an
exemplary embodiment ;
[0062] FIG. 5 is a set of fluorescent microscopic photos
illustrating results of binding between CRB1s (CRB1-1, CRB1-1-EpC,
CRB1-1-GEpC, CRB1-2, CRB1-2-EpC or CRB1-2-GEpC) and MCF-7 cells,
according to an exemplary embodiment;
[0063] FIG. 6 is a fluorescent microscopic photo illustrating a
result of binding between CRB1-1-GEpC and white blood cells
according to an exemplary embodiment;
[0064] FIG. 7 is a set of fluorescent microscopic photos
illustrating results of binding between CRB1-1GEpC and MCF-7 cells
and aggregation and dissociation between CRB1-1-GEpC, which are
bound to MCF-7 cells, and CRB2-1 according to an exemplary
embodiment;
[0065] FIG. 8 is a graph illustrating results of separating MCF-7
to which CRB1-1GEpC is bound by using a fine filter, according to
an exemplary embodiment;
[0066] FIG. 9 and FIG. 10 are fluorescent microscopic photos and a
graph illustrating a result of binding between CRB1-1-GEpC and
MCF-7 cell included in whole blood which is not pre-treated,
according to an exemplary embodiment;
[0067] FIG. 11, FIG. 12 and FIG. 13 are graphs of normal
distribution illustrating sizes of MCF-7 cells according to
aggregation reactions between CRB1-1-GEpC, which are bound to MCF-7
cell and CRB2-1;
[0068] FIG. 14 is a graph of normal distribution illustrating sizes
of MCF-7 cells to which
[0069] CRB1-1-GEpC is bound according to the size of particles
used; and
[0070] FIG. 15 is a schematic view of a kit for isolating a target
cell according to an exemplary embodiment.
MODE FOR THE INVENTION
[0071] Reference will now be made in detail to embodiments,
examples of which are illustrated in the accompanying drawings,
wherein like reference numerals refer to the like elements
throughout. In this regard, the present embodiments may have
different forms and should not be construed as being limited to the
descriptions set forth herein. Accordingly, the embodiments are
merely described below, by referring to the figures, to explain
aspects of the present description.
[0072] FIGS. 1 and 2 are schematic views of a method for isolating
a target cell according to an exemplary embodiment, and show a
method for isolating circulating tumor cells from blood.
[0073] Hemocyte cells such as white blood cell and red blood cell
are present together in a blood sample including circulating tumor
cells, and the size of the circulating tumor cells is similar to
that of white blood cell in the blood sample and thus the
circulating tumor cells may not be isolated by size. Accordingly,
the overall size of the circulating tumor cells may be increased by
the method and thus the circulating tumor cells may be isolated
from other hemocyte cells by filtration.
[0074] In order to increase the overall size of the circulating
tumor cells, the method utilizes a principle of aggregating
particles on the surface of the cell. In order to aggregate
particles on the cell surface, a polymer, whose electrostatic
properties may be changed according to the pH value of the ambient
environment, is linked to the particles, and a marker (for example,
EpCAM and/or C-Met) shown on the surface of the circulating tumor
cells is used for the particles bound to the polymer in order to
specifically aggregate the circulating cancer cells only.
[0075] Referring to FIG. 1 to describe the method, a blood
including circulating tumor cells is first obtained, the sample is
suspended in a buffer with a pH of about 3 to about 4, and a
particle, whose surface has an acrylic acid-maleic acid copolymer
or a styrene sulfonic acid-maleic acid copolymer and to which at
least one antibody of EpCAM and/or C-Met is bound, is added into
the buffer to contact the circulating tumor cells with the
particle. At the time, the antibody of EpCAM and/or C-Met bound to
the particle binds specifically to EpCAM and/or C-Met in
circulating tumor cells and thus the particle to which the antibody
is bound is positioned around the circulating tumor cell through
the antibody. In addition, the acrylic acid-maleic acid copolymer
or the styrene sulfonic acid-maleic acid copolymer, linked to the
particle, has a pKa value of about 4, and thus the particle is
neutral in the buffer.
[0076] Subsequently, when a particle having polyethyleneimine is
added into the buffer and a pH of the buffer is increased to about
6 to about 7, an imine group on the particle having
polyethyleneimine shows positive charges while a carboxylic group
or a sulfonic group on the particle having the acrylic acid-maleic
acid copolymer or the styrene sulfonic acid-maleic acid copolymer
shows negative charges. As shown in FIG. 1, a particle having
polyethyleneimine, the particle dissociated around a particle
specifically bound to EpCAM and/or C-Met in the existing
circulating tumor cells, may be aggregated by electrostatic
attraction. The overall size of the circulating tumor cells is
increased by the aggregated particles, and as a result, the cells
have sizes much bigger than those of other hemocyte cells in a
blood sample. The circulating tumor cells with their sizes
increased by this method may be isolated through filtration. When a
pH of the buffer is reduced to about 4 as in the initial step prior
to the isolating or the pH reduction is performed according to the
purpose so as to remove the aggregated particles after the
isolating, the particle having the polyethyleneimine becomes
neutral as shown in FIG. 1. As a result, the particle may be
dissociated. The Examples show experimental results according to
the principle.
EXAMPLE 1
Manufacture of a Particle Having Charges (Charge Reversible Bead,
CRB)
[0077] A particle having negative charges in a neutral pH and a
particle having positive charges in a neutral pH were each
manufactured in the following manner.
[0078] First, polystyrene beads (Polysciences, Inc) with a diameter
of about 1 .mu.m to about 3 .mu.m were prepared, and then a
particle having negative charges in a neutral pH was manufactured
by treating the polystyrene beads with N-hydroxysuccinimide
(NHS)/1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
(EDC) and linking an acrylic acid-maleic acid copolymer or a
styrene sulfonic acid-maleic acid copolymer to the polystyrene
beads. A polystyrene particle having the acrylic acid-maleic acid
copolymer was referred to as CRB 1-1, and a polystyrene particle
having the styrene sulfornic acid-maleic acid copolymer was
referred to as CRB1-2. In addition, a particle having positive
charges in a neutral pH was manufactured by treating the
polystyrene beads with NHS/EDC and linking a linear
polyethyleneimine polymer, a branched polyethyleneimine polymer, or
chitosan to the polystyrene beads. A polystyrene particle having
the linear polyethyleneimine polymer was referred to as CRB2-1, a
polystyrene particle having the branched polyethylene polymer was
referred to as CRB2-2, and a polystyrene particle having chitosan
was referred to as CRB2-3. The CRB2-1, CRB2-2, and CRB2-3 were
manufactured by linking Texas Red for giving a red fluorescence to
them.
EXAMPLE 2
Manufacture of a Particle Having Negative Charges, the Particle to
Which an Antibody is Bound
[0079] As a cell used as a target cell to be isolated according to
the principle was a breast cancer cell line MCF-7 (Korea Cell Line
Bank), an antibody (Human EpCAM/TROP1 Fluorescein MAb (Clone
158206), FAB9601F, R&S system) specifically binding to EpCAM as
a surface marker of a cancer cell existing therein was selected.
Subsequently, the CRB1-1 or CRB1-2 manufactured in Example 1 was
put into a PBS solution including 5% BSA, an antibody (0.65 mg/ml)
specifically binding to EpCAM is added into the resulting solution,
and the mixture was gently stirred at room temperature for about 2
hours. As a result, CRB 1-1 (referred to as CRB1-1-EpC) or CRB1-2
(referred to as CRB1-2-EpC) having an antibody specifically binding
to EpCAM was manufactured. In addition, in order to enhance the
directionality during binding of the antibody, CRB 1-1 or CRB 1-2
was put into a PBS solution including 5% BSA, protein G (0.65
mg/ml) was added to the resulting solution for reaction for about 2
hours, and then an antibody (0.65 mg/ml) specifically binding to
EpCAM was added into the reaction mixture while being gently
stirred for about 2 hours. As a result, an antibody specifically
binding to EpCAM and CRB1-1 (referred to as CRB1-1-GEpC) or CRB1-2
(referred to as CRB1-2-GEpC), to which protein G is bound, was
manufactured. FIG. 3 shows a schematic view of the particle having
an antibody.
EXAMPLE 3
Identification of Aggregation and Dissociation Between CRBs
[0080] When the CRBs manufacture in Examples 1 and 2 were mixed,
experiments were performed to see whether the CRBs may be
aggregated and dissociated according to the pH of the ambient
environment. Each of 30 .mu.l of CRB2-1, CRB2-1, and CRB2-3 was
mixed with each of 30 .mu.l of CRB 1-1 in a test tube including a
PBS solution, a NaOH solution was added portionwise into the
resulting solution, the pH was titrated to about 7.4, and the
mixture was allowed to stand for about 1 hour. For CRB 1-2, the
aggregation and dissociation was performed in the same manner as
above. In the process, NaOH was added for reaction for about 1
hour, HCl was added for reaction for about 1 hour, and then a
fluorescent microscope (Olympus IX-81) it was used to identify
whether the particles had been aggregated and dissociated. It was
identified by an observation of the intensity of fluorescence of
Texas Red through the fluorescent microscope that the two different
kinds of particles had been aggregated and that the two different
kinds of particles had been dissociated when the pH of the ambient
environment was decreased to about 4 (FIG. 4).
EXAMPLE 4
Identification of Binding Between CRB1 and Cancer Cell
[0081] Each of 30 .mu.l of the CRB1-EpC, CRB1-2-EpC, CRB1-1-GEpC,
or CRB1-2-GEpC manufactured in Example 2 was added into breast
cancer cell MCF-7 (1.times.10.sup.5 cells) in DMEM medium and
allowed to stand for about 1 hour. It was identified by the
fluorescence intensity of fluorescein using a fluorescent
microscope (Olympus IX-81) whether MCF-7 had been bound to
CRB1-1-EpC, CRB1-2-EpC, CRB1-1-GEpC, or CRB1-2-GEpC (FIG. 5). As a
result, it was confirmed that the added particles had bound to
cancer cell to increase the overall size of the cancer cell than
the original size of the cancer cell. On the contrary, the same
experiment as in the Example was performed as a control experiment
to see whether white blood cell isolated from a blood sample had
been bound to CRB-1, and it was identified that the binding as
above had not occurred in white blood cell (FIG. 6).
EXAMPLE 5
Identification of Aggregation and Dissociation Between Cancer Cell
to Which CRB1 is Bound and CRB2
[0082] 30 .mu.l of the CRB1-1-GEpC manufactured in Example 2 and
breast cancer cell MCF-7 (1.times.10.sup.5 cells)stained with
Hoechst 33342 were mixed in a test tube including a PBS solution,
the resulting solution was allowed to stand for about 1 hour, and
then it was identified by a fluorescent microscope (Olympus IX-81)
whether MCF-7 had been bound to CRB1-1-GEpC. As a result, it was
confirmed that CRB1-1-GEpC had been bound to MCF-7 (FIG. 7).
Subsequently, 30 .mu.l of the CRB2-1 manufactured in Example 1 was
added into MCF-7 to which CRB1-1-GEpC was bound, a NaOH solution
was added portionwise into the resulting solution, the pH was
titrated to about 7.4, the mixture was allowed to stand for about 1
hour, and then an observation was made by using a fluorescent
microscope (Olympus IX-81) (FIG. 7). As a result, it was identified
that the added CRB2-1 was aggregated in MCF-7 to which CRB1-1-GEpC
was bound to increase the overall size than the original size of
MCF-7. Subsequently, a HCl solution was added portionwise into the
solution, the pH was titrated to about 3, and the mixture was
allowed to stand for about 1 hour. As a result, when the pH of the
solution was lowered to about 3, it was identified that the
aggregated CRB2-2 had been dissociated from the MCF-7 to which
CRB1-1-GEpC had been bound (FIG. 7).
[0083] The CRB2-1 had been dissociated, and then only the MCF-7 to
which CRB1-1-GEpC had been bound was isolated by using a fine
filter with a pore size of about 7 .mu.m to about 40 M. A solution
including MCF-7 to which CRB 1-1 had been bound was flowed into the
fine filter at a rate of about 10 ul/min, and an operation was
performed such that about 100 cells per ml might be included in the
solution. As shown in FIG. 8, an experiment was performed 5 times,
and it was confirmed that the average recovery ratio of MCF-7 to
which CRB1-1-GEpC had been bound was about 91% and the isolation of
MCF-7 to which CRB1-1-GEpC had been bound was excellent when a fine
filter with a pore size of about 20 .mu.m to about 25 .mu.m was
used. On the contrary, the isolated cell was inoculated into a cell
culture plate including a DMEM medium and the content was cultured
under conditions of 37.degree. C. and 5% CO.sub.2for about 24
hours. As a result, it was identified that the cell had been grown
in the same way as in the MCF-7 cell as a control group.
EXAMPLE 6
Test of Binding Between Cancer Cell Included in Blood Without a
Pre-Treatment and CRB1
[0084] 30 .mu.l of the CRB1-1-GEpC manufactured in Example 2 was
added into 1 mg of blood including 50 cells of MCF-7, the resulting
solution was allowed to stand for about 1 hour, and it was observed
by a fluorescent microscope (Olympus IX-81) in Bright Field mode
whether CRB1-1-GEpC had been bound to MCF-7 (FIG. 9). In order to
calculate a binding ratio of CRB1-1-GEpC to MCF-7, the amount of
the CRB1-1-GEpC bound to the cell was converted into an area. A PBS
solution including 50 cells of MCF-7 was used as a comparison
group. As shown in FIG. 9 and FIG. 10, even when a blood sample
including MCF-7 was not subjected to a pretreatment, it was
identified that CRB1-1-GEpC has been bound to MCF-7 cell.
EXAMPLE 7
Size comparison test between a cancer cell to which CRB1 is bound,
aggregated by CRB2, and a cancer cell to which CRB1 is bound
according to the size of particle
[0085] The CRB1-1-GEpC manufactured in Example 2 was used to allow
the CRB1-1-GEpC to bind to MCF-7 cell in the same manner as in
Example 5, and then the sizes of MCF-7 cells to which CRB1-1-GEpC
had been bound were observed by a fluorescent microscope (Olympus
IX-81) in Bright Field mode (n=100). White blood cell was used as a
comparison experiment instead of MCF-7 cell while MCF-7 to which
CRB1-1-GEpC had not been bound was used as a control group (FIG.
11). As shown in FIG. 12, the size of MCF-7 to which CRB1-1-GEpC
had been bound was about 20 .mu.m, and it was identified that the
size had been increased by about 4 .mu.m to about 10 .mu.m, as
compared to the size of MCF7 to which CRB1-1-GEpC had not been
bound. In addition, CRB1-1-GEpC was bound to the MCF-7 cell, and
then an aggregation reaction was performed by adding CRB2-1 into
the mixture. As shown in FIG. 13, it was identified that the size
had been increased by about 8 .mu.m to about 20 .mu.m, as compared
to the size of MCF-7 to which CRB1-1-GEpC had not been bound. The
size of monocyte in white blood cell was known to be about 14 .mu.m
to about 20 .mu.m. Accordingly, when CRB1-1-GEpC bound to MCF-7
cell and CRB2-1 was aggregated to the mixture, the difference in
sizes of other white blood cells may be increased to isolate the
MCF-7 cell in blood. In addition, even when CRB2-1 was not
aggregated, the difference in sizes of other white blood cells may
be generated by increasing the size of polystyrene bead in the
CRB1-1-GEpC. As shown in FIG. 14, when the size of polystyrene bead
with which CRB1-1-GEpC was made was increased from about 1 .mu.m to
about 3 .mu.m, it was identified that the size of MCF-7 cell to
which CRB1-1-GEpC was bound had been increased by about 2
.mu.m.
EXAMPLE 8
A Target Cell Isolation Method Using a Kit for Isolating a Target
Cell
[0086] FIG. 15 is a schematic view of a kit for isolating a target
cell according to an exemplary embodiment. Referring to FIG. 12, an
isolation and detection process of the circulating tumor cells will
be described with reference to examples.
[0087] First, a blood sample is suspended in a buffer with a pH of
about 3 to about 4, and the suspension is injected into an upper
opening 130 of a filter column 100. In order to prevent a sample
from spilling, a lower opening 140 of the filter column 100 may be
sealed with a cap 150. Because CRB1-1-EpC, CRB1-2-EpC, CRB1-1-GEpC,
or CRB1-2-GEpC is included in the filter column 100, the particle
may be contacted with circulating tumor cell in the blood sample.
Subsequently, CRB2-1, CRB2-2, or CRB2-3 is added into the upper
opening 130 of the filter column, followed by pH adjustment with a
buffer included in the filter column 100 to about 6 to about 7. At
the time, the pH may be adjusted by adding an acid solution into
the filter column 100. Subsequently, as described above, an
aggregation reaction occurs in the filter column 100, and the sizes
of circulating tumor cells have been increased by the aggregated
particles to prevent the cells from passing through pores 120 in a
filter 110. Accordingly, the cells remains in the filter column 100
while other hemocyte cells and polymers pass through the filter 110
to be drained into the lower opening 140 of the filter column 100.
Subsequently, in order to detect the circulating tumor cells, a
base solution may be added into the filter column 100 and CRB2-1,
CRB2-2, or CRB2-3 may be isolated and removed from the filter
column 100. A circulating tumor cell from which the CRB2-1, CRB2-2,
or CRB2-3 has been removed may be isolated in a state in which
CRB1-1-EpC, CRB1-2-EpC, CRB1-1-GEpC, or CRB1-2-GEpC is bound to the
cell. The isolated cell may be cultured in the same way as in
methods known in the art. After the culture, trypsin may be treated
to separately isolate only circulating tumor cells.
[0088] A target cell in a biological sample may be efficiently
isolated and detected by using a method and kit for isolating the
target cell from the biological sample according to an exemplary
embodiment.
[0089] It should be understood that the exemplary embodiments
described therein should be considered in a descriptive sense only
and not for purposes of limitation. Descriptions of features or
aspects within each embodiment should typically be considered as
available for other similar features or aspects in other
embodiments.
* * * * *