Note: Descriptions are shown in the official language in which they were submitted.
2180830
SPECIFICATIONS
ANTI-ANNEXIN-V MONOCLONAL ANTIBODIES. AND PREPARATION AND USE
THEREOF
Technical Field
The present invention relates to anti-annexin-V monoclonal
antibodies having binding specificities to antigenic determinant
sites on annexin-V, a protein present in blood, plasma and/or
1o serum of humans and mammalian animals, and, more particularly, to
such anti-annexin-V monoclonal antibodies suitable for use in the
immunological determination of the concentration of human
annexin-V in the blood, plasma and/or serum, being present
particulary in the human cardiac muscles as a marker of
myocardial infarction and angina pectoris.
The present invention further relates to hybridoma cell
lines producing said anti-annexin-V monoclonal antibodies and,
more particularly, to such hybridoma cell lines producing the
monoclonal antibodies for use in the detection and quantitative
2o analysis of human cardiac-muscle annexin-V as a marker of
myocardial infarction and angina pectoris.
The present invention also relates to a method for the
detection of annexin-V in a sample and, more particularly, to a
method for the detection or quantitative analysis of human
cardiac-muscle annexin-V in the blood and/or serum as a marker of
myocardial infarction and angina pectoris. The present invention
further relates to human cardiac muscle annexin-V in the plasma
and/or serum, monoclonal antibodies for use in the detection and
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quantitative analysis thereof and hybridoma producing such
monoclonal antibodies.
In addition, the present invention relates to the
development and utilization of anti-annexin-V antibodies for the
measurement of annexin-V of humans and mammals and the hybridoma
cell lines producing such antibodies and, more particularly, to
the development and utilization of the anti-human-annexin-V
antibodies for use in quick diagnosis of diseases causing sudden
cellular necrosis such as myocardial infarction and angina
1o pectoris causing an ischemic disorder, through the concentration
measurement of annexin-V, a protein in the cells or blood.
Background Art
Annexin is a calcium-binding protein being present in
tissues and cells of humans and various animals, particularly
in their cytoplasmic solubles. This protein is composed of
families defined by the amino acid sequences and, at present,
annexin I through XII are known. The protein binds to
phospholipids and actin depending upon the calcium
concentration, and is known to have anti-inflammatory and
2o anticoagulant functions.
In patients suffering from tissue or cellular necrosis, due
to myocardial infarction, for example, various substances
contained in the necrosed cardiac-muscle cells leak into blood.
The current diagnosis of acute myocardial infarction is conducted
by detecting such substances in the blood and the substances are
generally called myocardial-infarction markers.
Such myocardial-infarction markers to be measured in
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biochemical tests for use in the diagnosis of acute myocardial
infarction include LD-1, AST, creatin kinase (CK), creatin kinase
MB-fraction (CK-MB), myoglobin, lactic dehydrogenase (LDH),
myosin light-chain I, and troponin-T (TnT). While the results or
the analytical values of these myocardial-infarction markers may
be employed independently or in combination in the diagnosis of
myocardial-infarction, the prevailing method is to employ them in
combination.
In the case of an ischemic disease such as angina pectoris,
arrhythmia is observed arrhythmia within several hours after
the onset of the attack, and may lead to arrhythmia death.
Quick diagnosis and treatment at the early stage are therefore
needed in myocardial infarction and angina pectoris.
However, regarding the above-mentioned various types of
myocardial-infarction markers (all are substances leaking from
the cardiac muscles into the blood) to be examined in biochemical
tests for the diagnosis of acute myocardial-infarction, it has
been reported that such myocardial-infarction markers reach
their respective peak concentrations in the blood 7 to 78 hours
after the attack of myocardial infarction. In addition, the time
in which the myocardial infarction markers reach the maximal or
peak concentration in the blood may vary considerably from one
patient to another. Thus, these markers for the diagnosis of
myocardial infarction are considered to be problematic since they
make it difficult to establish a quick diagnosis of myocardial
infarction and hence they do not contribute to quick selection of
therapeutic measures for the patients.
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The employment of the results obtained by measuring two or
more markers of the aforesaid myocardial-infarction markers for
more reliable diagnosis of myocardial infarction is also
problematic and far from meeting the need for early-stage
therapy of myocardial infarction patients because it takes a
long time for the respective myocardial-infarction markers to
reach the maximal (peak) concentrations in the blood and such
time varies depending upon the patient examined.
In the case of angina pectoris, marker substances to be
1o examined in biochemical tests for diagnozing the disease have not
yet been identified. Thus, biochemical diagnosis of angina
pectoris is generally considered to be difficult and a need is
felt for the establishment of diagnosis for angina pectoris
through biochemical tests.
The present invention is directed to overcoming the problems
in the conventional biochemical tests so as to enable quick
diagnosis of myocardial infarction and angina pectoris.
Disclosure of the Invention
It has been found that, among annexin-proteins present in
human cardiac muscles, human annexin-V, occurring in the solubles
of the cardiac-muscle cells and having a molecular weight of
35,000, i.e. smaller than that of creatin kinase (81,000),
generally leaks from the necrosed cardiac muscles of myocardial
infarction or angina-pectoris patients in the early stage of the
diseases. Thus the present applicant has already disclosed a
method for determining the concentration of human annexin-V in
human blood, plasma and/or serum and a method for diagnosing
myocardial infarction and/or angina pectoris, using dog cardiac-
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muscle annexin-V polyclonal antibodies which specifically cross-
react with such human annexin-V (Japanese Laid-Open Pat.
Application No.72147/1995).
Thus, human annexin-V is a novel marker of myocardial
infarction whose concentration (in the blood, plasma and serum)
reaches the peak within one to several hours after the attack of
myocardial infarction.
The present inventors therefore made extensive studies to
provide antibodies capable of specifically recognizing human
annexin-V and suitable for use in a method for the assay of human
annexin-V in a sensitive, simple and quick manner and found that
hybridoma cells, obtained from anti-human-annexin-V-producing
lymphocytes and myeloma cells using the conventional cell fusion
technique, produce stable anti-human-annexin-V monoclonal
antibodies meeting the above-mentioned object for the completion
of the present invention.
The present invention relates to an improvement on the
invention of the aforesaid application and is directed to the
provision of diagnostic agents and diagnostic methods, by which
2o highly reliable diagnosis of myocardial infarction and/or angina
pectoris can be made in a short time after the attack of
myocardial infarction or angina pectoris, as well as the
provisions of detection substances for detecting and
quantitatively analyzing the myocardial-infarction marker or
angina pectoris marker and the preparation and use thereof.
Thus, the present invention is directed to an anti-annexin-V
monoclonal antibody characterized by having a binding specificity
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to antigenic determinant site on annexin-V as antigenic protein
and belonging to immunoglobulin G class, to a hybridoma cell line
characterized by being capable of producing an anti-annexin-V
monoclonal antibody having a binding specificity to antigenic
determinant site on annexin-V as antigenic protein and belonging
to immunoglobulin G class, to a diagnostic agent for myocardial
infarction and angina pectoris characterized by comprising an
anti-annexin-V monoclonal antibody having a binding specificity
to antigenic determinant site on annexin-V as antigenic protein
and belonging to immunoglobulin G class, and also to a diagnostic
agent for myocardial infarction and angina pectoris characterized
by comprising a reagent containing a first anti-annexin-V
monoclonal antibody having a binding specificity to antigenic
determinant site on annexin-V as antigenic protein and belonging
to immunoglobulin G class and a reagent containing a second anti-
annexin-V monoclonal or polyclonal antibody having a binding
specificity to antigenic determinant site on annexin-V as
antigenic protein and belonging to immunoglobulin G class, with
the second antibody being labeled.
In addition, the present invention is directed to a method
for diagnosing myocardial infarction and angina pectoris
characterized by comprising an antigen-antibody reaction of
annexin-V in a sample with an anti-annexin-V monoclonal antibody
to form an annexin-V antigen/anti-annexin-V monoclonal antibody
complex and assaying the formed annexin-V antigen/anti-annexin-V
monoclonal antibody complex, to a method for diagnosing
myocardial infarction and angina pectoris characterized by
comprising causing an antigen-antibody reaction of annexin-V in a
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sample with an anti-annexin-V monoclonal antibody to form an
annexin-V antigen/anti-annexin-V monoclonal antibody complex,
allowing the antigenic site of annexin-V of the formed annexin-V
antigen/anti-annexin-V monoclonal antibody complex to bound with
a labeled anti-annexin-V polyclonal or monoclonal antibody so as
to form a labeled form of said annexin-V antigen/anti-annexin-V
monoclonal antibody complex bound with the polyclonal or
monoclonal antibody, and quantitatively analyzing the labeled
form of the complex, to a method for analyzing human cardiac
1o muscle or related annexin-V in a sample characterized by
comprising causing an antigen-antibody reaction of human annexin-
V in the sample with an anti-annexin-V monoclonal antibody to
form an annexin-V antigen/anti-annexin-V monoclonal antibody
complex and quantitatively analyzing the formed annexin-V
antigen/anti-annexin-V monoclonal antibody complex, and also to a
method for analyzing annexin-V in a sample characterized by
comprising causing an antigen-antibody reaction of human annexin-
V in the sample with a first anti-annexin-V monoclonal antibody
having a specificity to antigenic determinant site on annexin-V
antigenic protein to form an annexin-V antigen/anti-annexin-V
monoclonal antibody complex, allowing the antigenic site of human
annexin-V of the formed annexin-V antigen/anti-annexin-V
monoclonal antibody complex to be bound with an anti-annexin-V
polyclonal or second monoclonal labeled antibody so as to form a
labeled form of said annexin-V antigen/anti-annexin-V monoclonal
antibody complex bound with the polyclonal or the second
monoclonal antibody, and quantitatively analyzing the labeled
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form of the complex.
The present invention is further directed to a method for
preparing anti-annexin-V monoclonal antibodies which comprises
fusing lymphocytes induced from mice immunized with annexin-V
with myeloma cells to form hybridoma cells, and growing the
formed hybridoma cells to produce anti-annexin-V monoclonal
antibodies having binding specificities to antigenic determinant
sites on annexin-V antigenic protein.
Human annexin-V to be used as the antigen in the present
invention is a protein which may have a molecular weight of 32 to
35 kilodalton and may be one occurring in the human heart.
The human heart to be used in the present invention is one
extracted from a human cadaver and maintaining its activities.
The antigen protein - human annexin-V protein occurring in
human heart - is found in the soluble fractions of the heart and
purified by removing the connective tissues and lipids from the
cardiac tissue of a human cadaver. The hybridoma cell lines to
be used in the present invention for producing anti-human-
annexin-V monoclonal antibodies are formed by means of the cell
2o fusion of lymphocytic cells with myeloma cells, in which the
lymphocytic cells are spleen (a lymphoid organ) including spleen
cells and white pulp of spleen or plasmablast cells prepared from
lymph nodes (e.g. lymphoid nodule) and being in the process of
the differentiation to plasma cells, from a mammalian animal such
as a mouse or the like which was immunized with human annexin-V
as the antigen extracted and purified from human cardiac tissue.
The myeloma cell strain to be used in the present invention
is generally a hypoxanthine-guanine-phosphoribosyltransferase
8
CA 02180830 2004-12-23
(HGPRT)-deficient one or thymidine-kinase(TK)-deficient
one. The HGPRT-deficient strains suitable for use include 8-
azaguanine(8-AG)-resistant strain and 6-thioguanine(6-TG)-
resistant strain while,the TK-deficient strains suitable for
use include bromodeoxyuridine(BUdR)-resistant strain.
The cell fusion is conducted using a cell fusion
accelerator, in which polyethylene glycol (PEG) is primarily used
for the cell fusion in the present invention.
The cell fusion accelerators usable in the present invention
1o include, by substance name or trade name, PEG600, PEG400,
PEG1000, PEG3000, PEG6000, GlucamTM E-10, GlucamTM E-20,
GlucamTM P-10, polyeth lene
y glycol methyl ester (PEG
methyl ester) (molecular weight: 350, 2000 and 5000), PEG 1000,
polyglycol P15-200, pluronic F38 polyol Prilled,
Decaglycerol, Camul* 101 Butyrate, Tetronic~304 polyol,
triglycerol, polyvinylpyrroridone (molecular weight: 1000) and
glycerin.
The anti-human-annexin-V monoclonal antibodies in the
present invention can be produced by fusing lymphocytic
2o plasmablast cells extracted from the lymphoid organs of mice or
other animals immunized against annexin-V as the antigen with
myeloma cells to form hybridomas and growing the hybridomas with,
for example, a HAT culture medium, in which the hybridomas for
the selective proliferation are, for example, anti-human-annexin-
V-monoclonal-antibody-producing hybridoma cell lines HCA-627 and
HDA-907 deposited at the National Institute of Bioscience and
v Human-Technology, the Agency of Industrial Science and Technology
trademark
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of Japan, an International Depositary Authority for the deposit
of microorganism, under the numbers FERM BP-5284 and FERM BP-
5286, respectively.
The selection of the hybridoma cell lines can be made
through a HAT-selection, an ouabain-selection or the like.-
The anti-human-annexin-V monoclonal antibodies in the
present invention can be obtained by culturing the above-
mentioned hybridoma clones by a conventional culture process such
as the high density culture process, the spinner-flask culture
1o process or the like and purifying the antibodies from the culture
supernatant by means of affinity chromatography using a carrier
bound with protein-A or a carrier bound with anti-mouse-
immunoglobulin.
In the present invention, the anti-human-annexin-V
15 monoclonal antibodies can be produced by injecting the hybridoma
cell lines obtained through the above-mentioned culture into the
abdominal cavities of prestene-pretreated immunocompromised mice,
salting out, with ammonium sulfate or sodium sulfate, the ascitic
fluid induced by the hybridoma cell lines, and purifying the
2o fluid by means of ion-exchange chromatography using DEAF
cellulose to obtain the immunoglobulin (IgG) fractions.
A hybridoma cell line as termed with respect to the
present invention means not only a hybridoma cell just obtained
by cell-fusion but also hybridoma cells at any passages
25 subsequent to the primary culture.
The screening for the resultant hybridoma cell lines can be
conducted by detecting the anti-human-annexin-V monoclonal
antibodies secreted by the hybridomas in the culture by such
2180830
methods as immunoassay, radioimmunoassay and ELISA.
In the present invention, the process of screening for the
hybridoma cell lines has been found to be able to obtain
various anti-human-annexin-V monoclonal antibodies having
different specific reactivities with human-annexin-V antigen.
By a method similar to that for producing anti-human-
annexin-V monoclonal antibodies there can be obtained anti-
annexin-V monoclonal antibodies having specific reactivities
with the antigenic determinant sites of the annexin-V antigens of
l0 dogs and other mammalian animals, by immunizing the mammalian
animals with the annexin-V antigens derived from dogs or the
like, cell-fusing the lymphocytic cells with myeloma cells to
produce the hybridoma cell lines.
The anti-annexin-V monoclonal antibodies in the present
invention are defined to include such anti-annexin-V monoclonal
antibodies from mammalian animals as well as anti-human-annexin-V
monoclonal antibodies.
Thus, cloning is carried out for the hybridoma cell lines
which have been screened for the ability to secrete anti
human-annexin-V monoclonal antibodies.
The cloning of the hybridoma cell lines in the present
invention can be made by such techniques as limiting dilution,
soft agar method, fibrin gel method, cell sorter method and the
like.
The hybridoma cell lines with an ability to produce anti-
human-annexin-V monoclonal antibodies thus obtained are subjected
to a large-scale fermentation to produce anti-human-annexin-V
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monoclonal antibodies.
The anti-human-annexin-V monoclonal antibodies of the
present invention have specific reactivities with the antigenic
determinant sites of human annexin-V, a protein antigen. They
are antibodies featuring an antigen-antibody reaction with the
antigenic protein (human annexin-V) and will thus bind to human
annexin-V present in human blood, plasma and/or serum, thereby
enabling highly specific and sensitive detection and quantitative
analysis of human annexin-V in human blood, plasma and/or serum
1o through such techniques as immunoassay, radioimmunoassay, ELISA
and the like.
The anti-human-annexin-V monoclonal antibodies of the
present invention exhibit specific reactivities with the
antigenic determinant sites of human annexin-V, a protein
15, occurring in the human heart, thereby enabling highly specific
and sensitive detection and/or quantification of human annexin-V
in the blood, plasma and/or serum, through such techniques as
immunoassay, radioimmunoassay, ELISA and the like, as they bind
to the antigenic protein, human annexin-V, present in the human
20 blood, plasma and/or serum.
While human annexin-V concentration in the blood of a normal
adult is found, for example, to be 5.6 ng/ml at the highest, it
has been found by the application of the present invention that
the human annexin-V concentration with a patient suffering
25 myocardial infarction may be, for example, as high as 90 ng/ml
even in the stage when the CK value indicated is normal and also
that the human annexin-V concentration of a patient suffering
angina pectoris may be as high as 29 ng/ml even in the stage when
12
CA 02180830 2006-08-14
the CK value indicated is normal. The present invention
therefore enables a diagnosis of the diseases at earlier
stages than possible with the conventional diagnosis
based on the CK value.
According to one aspect of the present invention,
there is provided an anti-annexin-V monoclonal antibody
produced by a hybridoma cell line which is selected from
the group consisting of the hybridoma cell lines
deposited as FERM BP-5284 and FERM BP-5286 at the
International Depository Authority for the deposit of
microorganisms, said anti-annexin-V monoclonal antibody
being cross-reactive with annexin-V from the heart cells
of one or more mammalian species.
According to another aspect of the present
invention, there is provided a hybridoma cell line
selected from the group consisting of the hybridoma cell
lines deposited as FERM BP-5284 and FERM BP-5286 at the
International Depository Authority for the deposit of
microorganisms, said hybridoma cell line having the
ability to produce an anti-annexin-V monoclonal antibody
which has a binding specificity to an antigenic
determinant on annexin-V antigenic protein and which is
capable of cross-reacting with annexin-V from the heart
of one or more mammalian species.
According to still another aspect of the present
invention, there is provided a diagnostic agent for
myocardial infarction and angina pectoris which
comprises:
a first reagent containing a first anti-annexin-V
monoclonal antibody being produced by a hybridoma cell
line selected from the group consisting of the hybridoma
cell lines deposited as FERM BP-5284 and FERM BP-5286 at
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CA 02180830 2006-08-14
the International Depository Authority for the deposit
of microorganisms;
a second reagent containing a second anti-annexin-V
monoclonal antibody being produced by the other
hybridoma cell line selected from said group, or
polyclonal antibody, wherein the antibody of the second
reagent is labelled;
wherein the antibodies of both reagents are cross-
reactive with annexin-V from human heart cells and the
annexin-V antigen is annexin-V from heart cells of one
or more mammalian species.
According to yet another aspect of the present
invention, there is provided a method for analyzing
annexin-V in human cardiac muscle or in a sample which
comprises causing an antigen-antibody reaction of human
annexin-V in the human cardiac muscle or the sample with
an anti-annexin-V monoclonal antibody produced by a
hybridoma cell line selected from the group consisting
of the hybridoma cell lines deposited as FERM BP-5284
and FERM BP-5286 at the International Depository
Authority for the deposit of microorganisms, to form an
annexin-V antigen-anti-annexin-V monoclonal antibody
complex and quantitatively analyzing the formed annexin
V antigen-anti-annexin-V monoclonal antibody complex.
According to a further aspect of the present
invention, there is provided a method for analyzing
annexin-V in a sample which comprises the steps of:
causing an antigen-antibody reaction of human
annexin-V in the sample with a first anti-annexin-V
monoclonal antibody produced by a hybridoma cell line
selected from the group consisting of the hybridoma cell
lines deposited as FERM BP-5284 and FERM BP-5286 at the
13a
CA 02180830 2006-08-14
International Depository Authority for the deposit of
microorganisms to form an annexin-V antigen-anti-
annexin-V monoclonal antibody complex,
allowing the antigenic site of human annexin-V of
the formed annexin-V antigenic-anti-annexin-V monoclonal
antibody complex to be bound with a labelled second
anti-annexin-V monoclonal antibody or anti-annexin-V
polyclonal antibody, so as to form a labelled from of
said annexin-V antigen-anti-annexin-V monoclonal
polyclonal antibody complex bound with labelled anti-
annexin-V polyclonal antibody or labelled the second
anti-annexin-V monoclonal antibody;
quantitatively analyzing the labelled form of the
complex and;
said labelled second anti-annexin monoclonal
antibody is produced by labelling second anti-annexin-V
monoclonal antibody being different from the first anti-
annexin-V monoclonal antibody and produced by a
hybridoma cell line which is selected from the group
consisting of the hybridoma cell line deposited as FERM
BP-5284 and FERM BP-5286 at the International Depository
Authority for the deposit of microorganisms.
Brief Description of the Drawings
Fig. 1 illustrates a calibration curve for use in
ELISA system for a working example, in which the anti-
human-annexin-V monoclonal antibodies of the present
invention are employed to determine the concentration of
8M-urea-treated human annexin-V. The ELISA system is
made up of a combination of a HRPO-labelled anti-human-
annexin-V monoclonal antibody produced by HCA-155H, an
anti-human-annexin-V monoclonal antibody-producing
hybridoma cell line clone, and having a specific
13b
CA 02180830 2006-08-14
activity with human annexin-V, and another anti-human-
annexin-V monoclonal antibody as the solid phase,
produced by HCA-290, another anti-human-annexin-V
monoclonal antibody-producing hybridoma cell line clone,
and having a specific activity with human annexin-V.
In the Figure, the ordinate indicates difference
absorbance obtained by subtracting an absorbance
measurement at the secondary wavelength 690nm from an
absorbance measurement at the primary wavelength 492nm
and the abscissa indicates the concentration of human
annexin-V, in which each ~ mark represents the average
difference absorbance and the length of the arrows
extending upward or downward from each ~ mark represents
the average ~2SD respectively.
Fig. 2 illustrates a calibration curve for use in
ELISA system for another working example, in which the
a r ~ ; ~" ,..,-, .,
13c
2180830
annexin-V monoclonal antibodies of the present invention are
employed to determine the concentration of 8M-urea-treated human
annexin-V. The ELISA system is made up of a combination of a
HRPO-labeled anti-human-annexin-V monoclonal antibody produced by
HCA-155H, an anti-human-annexin-V monoclonal antibody-producing
hybridoma cell line clone, and having a specific activity with
human annexin-V, and another anti-human-annexin-V monoclonal
antibody as the solid phase, produced by HCA-57HDR, another anti-
human-annexin-V monoclonal antibody-producing hybridoma cell line
1o clone, and having a specific activity with human annexin-V.
In the Figure, the ordinate indicates difference absorbance
obtained by subtracting an absorbance measurement at the
secondary wavelength 690nm from an absorbance measurement at the
primary wavelength 492nm and the abscissa indicates the
concentration of human annexin-V, in which each ~ mark
represents the average difference absorbance and the length of
the arrows extending upward or downward from each ~ mark
represents the average ~2SD respectively.
Fig. 3 illustrates a calibration curve for use in ELISA
2o system for a further working example different from the examples
as shown by Figs. 1 and 2, in which the anti-human-annexin-V
monoclonal antibodies of the present invention are employed to
determine the concentration of 8M-urea-treated human annexin-V.
The ELISA system is made up of a combination of a HRPO-labeled
anti-human-annexin-V monoclonal antibody produced by HCA-155H, an
anti-human-annexin-V monoclonal antibody-producing hybridoma cell
line clone, and having a specific activity with human annexin-V,
and another anti-human-annexin-V monoclonal antibody as the solid
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phase, produced by HCA-293HDR, another anti-human-annexin-V
monoclonal antibody-producing hybridoma cell line clone, and
having a specific activity with human annexin-V.
In the Figure, the ordinate indicates difference absorbance
obtained by subtracting an absorbance measurement at the
secondary wavelength 690nm from an absorbance measurement at the
primary wavelength 492nm and the abscissa indicates the
concentration of human annexin-V, in which each 0 mark
represents the average difference absorbance and the length
the arrows extending upward or downward from each 0 mark
represents the average ~2SD respectively.
Fig. 4 illustrates a calibration curve for use in ELISA
system for a still further working example different from the
examples as shown by Figs. 1 through 3, in which the anti-human-
15' annexin-V monoclonal antibodies of the present invention are
employed to determine the concentration of 8M-urea-treated human
annexin-V. The ELISA system is made up of a combination of a
HRPO-labeled anti-human-annexin-V monoclonal antibody produced by
HCA-155H, an anti-human-annexin-V monoclonal antibody-producing
hybridoma cell line clone, and having a specific activity with
human annexin-V, and another anti-human-annexin-V monoclonal
antibody as the solid phase, produced by HCA-350HDR, another
anti-human-annexin-V monoclonal antibody-producing hybridoma cell
line clone, and having a specific activity with human annexin-V.
In the Figure, the ordinate indicates difference absorbance
obtained by subtracting an absorbance measurement at the
secondary wavelength 690nm from an absorbance measurement at the
2180830
primary wavelength 492nm and the abscissa indicates the
concentration of human annexin-V, in which each ~ mark
represents the average difference absorbance and the length of
the arrows extending upward or downward from each ~ mark
represents the average ~2SD respectively.
Fig. 5 illustrates a calibration curve for use in ELISA
system for a working example different from the examples as
shown by Figs. 1 through 4, in which the anti-human-annexin-V
monoclonal antibodies of the present invention are employed to
determine the concentration of 8M-urea-treated human annexin-V.
The ELISA system is made up of a combination of a HRPO-labeled
anti-human-annexin-V monoclonal antibody produced by HCA-660HD,
an anti-human-annexin-V monoclonal antibody-producing hybridoma
cell line clone, and having a specific activity with human
annexin-V, and another anti-human-annexin-V monoclonal antibody
as the solid phase, produced by HCA-290HDR, another anti-human-
annexin-V monoclonal antibody-producing hybridoma cell line
clone, and having a specific activity with human annexin-V.
In the Figure, the ordinate indicates difference absorbance
obtained by subtracting an absorbance measurement at the
secondary wavelength 690nm from an absorbance measurement at the
primary wavelength 492nm and the abscissa indicates the
concentration of human annexin-V, in which each 0 mark
represents the average difference absorbance and the length of
the arrows extending upward or downward from each 0 mark
represents the average ~2SD respectively.
Fig. 6 illustrates a calibration curve for use in ELISA
system for a further working example different from the examples
16
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as shown by Figs. 1 through 5, in which the anti-human-annexin-V
monoclonal antibodies of the present invention are employed to
determine the concentration of 8M-urea-treated human annexin-V.
The ELISA system is made up of a combination of a HRPO-labeled
anti-human-annexin-V monoclonal antibody produced by HCA-660HD,
an anti-human-annexin-V monoclonal antibody-producing hybridoma
cell line clone, and having a specific activity with human
annexin-V, and another anti-human-annexin-V monoclonal antibody
as the solid phase, produced by HCA-57HDR, another anti-human-
l0 annexin-V monoclonal antibody-producing hybridoma cell line
clone, and having a specific activity with human annexin-V.
In the Figure, the ordinate indicates difference absorbance
obtained by subtracting an absorbance measurement at the
secondary wavelength 690nm from an absorbance measurement at the
primary wavelength 492nm and the abscissa indicates the
concentration of human annexin-V, in which each ~ mark
represents the average difference absorbance and the length of
the arrows extending upward or downward from each ~ mark
represents the average ~2SD respectively.
Fig. 7 illustrates a calibration curve for use in ELISA
system for a still further working example different from the
examples as shown by Figs. 1 through 6, in which the anti-human-
annexin-V monoclonal antibodies of the present invention are
employed to determine the concentration of 8M-urea-treated human
annexin-V. The ELISA system is made up of a combination of a
HRPO-labeled anti-human-annexin-V monoclonal antibody produced by
HCA-660HD, an anti-human-annexin-V monoclonal antibody-producing
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hybridoma cell line clone, and having a specific activity with
human annexin-V, and another anti-human-annexin-V monoclonal
antibody as the solid phase, produced by HCA-293HDR, another
anti-human-annexin-V monoclonal antibody-producing hybridoma cell
line clone, and having a specific activity with human anneXin-V.
In the Figure, the ordinate indicates difference absorbance
obtained by subtracting an absorbance measurement at the
secondary wavelength 690nm from an absorbance measurement at the
primary wavelength 492nm and the abscissa indicates the
l0 concentration of human annexin-V, in which each 0 mark
represents the average difference absorbance and the length of
the arrows extending upward or downward from each 0 mark
represents the average ~2SD respectively.
Fig. 8 illustrates a calibration curve for use in ELISA
15 system for another working example different from the examples as
shown by Figs. 1 through 7, in which the anti-human-annexin-V
monoclonal antibodies of the present invention are employed to
determine the concentration of 8M-urea-treated human annexin-V.
The ELISA system is made up of a combination of a HRPO-labeled
2o anti-human-annexin-V monoclonal antibody produced by HCA-660HD,
an anti-human-annexin-V monoclonal antibody-producing hybridoma
cell line clone, and having a specific activity with human
annexin-V, and another anti-human-annexin-V monoclonal antibody
as the solid phase, produced by HCA-350HDR, another anti-human-
25 annexin-V monoclonal antibody-producing hybridoma cell line
clone, and having a specific activity with human annexin-V.
In the Figure, the ordinate indicates difference absorbance
obtained by subtracting an absorbance measurement at the
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secondary wavelength 690nm from an absorbance measurement at the
primary wavelength 492nm and the abscissa indicates the
concentration of human annexin-V, in which each 0 mark
represents the average difference absorbance with the arrows
extending upward or downward from each 0 mark represents
the average ~2SD respectively.
Fig. 9 illustrates a calibration curve for use in ELISA
system for a working example, in which the anti-human-annexin-V
monoclonal antibodies of the present invention are employed to
determine the concentration of native human annexin-V. The ELISA
system is made up of a combination of a HRPO-labeled anti-dog-
32KP polyclonal antibody, and an anti-human-annexin-V monoclonal
antibody as the solid phase, produced by HCA-627, an anti-human-
annexin-V monoclonal antibody-producing hybridoma cell line
clone, deposited under the number FERM BP-5284 at the
International Depositary Authority for the deposit of
microorganisms and having a specific activity with native human
annexin-V.
In the Figure, the ordinate indicates difference absorbance
obtained by subtracting an absorbance measurement at the
secondary wavelength 690nm from an absorbance measurement at the
primary wavelength 492nm and the abscissa indicates the
concentration of human annexin-V, in which each 0 mark
represents the average difference absorbance and the length of
the arrows extending upward or downward from each 0 mark
represents the average ~2SD respectively.
Fig.lO is a microscopic photograph, under 200 diameter
19
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magnification, of human cardiac muscle tissue subjected to a
specific staining by indirect enzyme antibody technique in which
anti-human-annexin-V monoclonal antibody HCA-627 was employed as
the primary antibody and HRPO-labeled-anti-mouse-immunoglobulin
antibody was employed as the secondary antibody. The 'figure
demonstrates that human annexin-V was stained in the form o,f
uneven aggregates in the cytoplasm within the human heart of a
patient with sudden death.
Fig. 11 is a microscopic photograph, under a 200 diameter
magnification, of human cardiac muscle tissue for comparison with
the example as shown by Fig. 10. The figure demonstrates that
without the use of the anti-human-annexin-V monoclonal antibody,
it is impossible to stain human annexin-V and therefore to locate
the distribution thereof.
Fig. 12 illustrates a calibration curve for use in ELISA
system for a working example, in which the anti-human-annexin-V
monoclonal antibodies of the present invention are employed to
determine the concentration of native human annexin-V. The ELISA
system is made up of a combination of a HRPO-labeled anti-
2o annexin-V monoclonal antibody, produced by HDA-907, an anti-
annexin-V monoclonal antibody-producing hybridoma cell line
clone, deposited under the number FERM BP-5286 at the
International Depositary Authority for the deposit of
microorganisms and having a specific activity with native human
annexin-V, and another anti-human annexin-V monoclonal antibody
as the solid-phase, produced by HCA-627, an anti-human-
annexin-V monoclonal antibody-producing hybridoma cell line
clone and having a specific activity with native human
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annexin-V.
In the Figure, the ordinate indicates absorbance obtained
by subtracting an absorbance measurement at the secondary
wavelength 690nm from an absorbance measurement at the primary
wavelength 492nm and the abscissa indicates the
concentration of human annexin-V, in which each ~ mark
represents the average difference absorbance measured four
times and the length of the arrows extending upward or
downward from each 0 mark represents the average ~2SD
1o respectively.
Fig. 13 shows the results of dilution tests performed on
plasma samples by an ELISA system for assay of native human
annexin-V concentration using the anti-annexin-V monoclonal
antibodies of the present invention. The ELISA system was
constructed using a combination of HRPO-labeled antihuman-
annexin-V monoclonal antibody produced by anti-annexin-V
monoclonal antibody-producing hybridoma cell line clone HDA-
907 (deposited under the number FERM BP-5286 at the
International Depositary Authority for deposit of
microorganism) and having a specific reactivity with
annexin-V and anti-human-annexin-V monoclonal antibody as
solid phase produced by anti-annexin-V monoclonal antibody-
producing hybridoma cell line clone HCA-627 (deposited under
the number FERM BP-5284 at the International Depositary
Authority for deposit of microorganism) and having a specific
reactivity with native human annexin-V.
In the Figure, the ordinate shows human annexin-V
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concentration measured on the original solution and the series of
diluted solution of each sample and the abscissa represents
1/dilution rate for each sample.
The Best Mode for Carrying out the Invention
While the present invention will be further illustrated by
the examples given below, the invention is not to be limited by
the following examples and description.
In the following examples the symbol M represents molarity
and it indicates, for a solution of mixture, the mol
per liter of the solution.
EXAMPLE 1: Hybridoma Preparation
(1) Purification of human annexin-V
The heart was excised from a human adult cadaver. After
clearing the blood, the right ventricle was excised from the
heart, followed by the removal of the connective tissue and the
lipids therefrom. These operations were carried out in ice or
at a 4 °C temperature.
The heart was added with a buffer solution of the following
composition in a ratio of ten times as much of the heart weight:
250mM, sucrose 0.5mM ethylene glycol bis(2-aminoethyl ether)
tetraacetate (EGTA), 1mM phenylmethane sulfonyl fluoride
(PMSF) and lOmM tris (tris(hydroxymethyl) aminomethane)HC1,
pH7.4.
The heart was then homogenized by a homogenizer and the
homogenate was centrifuged at 3000xg for 15 minutes. The
separated supernatant is added with 1M CaCl2 solution to give a
final concentration of 2mM for mixing, followed by centrifugation
at 28,OOOxg for an hour. To the residue was added 2m1 of lOmM
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EGTA for suspending the residue. The residue suspension was
centrifuged at 28,OOOxg for 1 hour.
The supernatant was subjected to gel filtration with
Sephacryl S-300 (trade name) column available from Pharmacia Co.
for elution with pH 7.4 buffer solution (H) containing O.1M NaCl
and 30mM Tris-HCl. The fraction containing protein having a
molecular weight of 35 KDa was recovered and passed through an
ion-exchange column (Biogelagarose: trade name) with an eluent of
mM Tris HC1 solution (pH 7.4) containing NaCl in the
to concentration range of 0 to 0.3M for purification by means of
NaCl-concentration-gradient elution.
The human annexin-V thus purified was divided into two
portions, one of which was freeze-dried and then dissolved by
O.1M sodium phosphate buffer (pH 7.b) containing 8M urea, The
other part was freeze-dried and dissolved by O.1M sodium
phosphate buffer (pH 7.6). The two parts were both preserved at
4°C. These parts of purified human annexin-V were measured for
purity with sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) to quantify the protein
concentration.
Identification was also made of the purified annexin-V
protein: Peptide fractions were prepared by adding
lysylendopeptidase and keeping the mixture at 37°C for 15 hours
so as to allow the human annexin-V protein to react with
lysylendopeptidase. The peptides thus obtained were analyzed
for amino acid sequence on a Shimadzu~PPSQ-10 protein sequences
to determine the amino acid sequences of the two peptide
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fractions, based on Edman method (cf. Edman P. "A method for the
determination of the amino acid sequence in peptide", Arch.
Biochem, Biophys., 1949, Vo1.22, page 475).
As the N-terminal amino acid sequences of the two peptides
from the purified protein are Glu-Tyr-Gly-Ser-Ser-Leu-Glu for the
first and Gly-Thr-Asp-Glu-Glu-Lys-Phe-Ile-Thr-Ile-Phe-Gly-Thr for
the second, the protein was identified as annexin-V.
(2) Mice
BALB/c inbred-strain female mice 5 to 8 weeks of age were
to maintained on a diet of standard pellets feeding water ad libitum
in an animal-breeding chamber kept at the temperature of 23+1°C
and the humidity of 70~.
(3) Immunization
The human annexin-V 100 micrograms/0.5m1 as prepared in (1)
was mixed with an equal amount of Freund's complete adjuvant to
be emulsified. The human annexin-V in emulsion was administered
as the antigen into the abdominal cavities of four female mice 5
weeks of age in a dose of 15-40 micrograms of the purified
annexin-V per mouse. The administration was made every two weeks
for two months for the immunization of the mice. The mice were
determined for the antibody titer to select mice having a high
titer, which were administered, three weeks after, with 50
micrograms of the purified human annexin-V as a booster
intravenously through the mice tails for final immunization.
(4) Cell fusion
Three days after final immunization, the spleens were
excised from BALB/c mice and the spleen cells were suspended in
EMEM culture medium to prepare a suspension of spleen cells. The
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spleen cells were then washed four times with EMEM medium
(available from Nissui Co.) and determined fox the number of
cells. The result was that 7x108 living spleen cells were
obtained.
Cell fusion was conducted using P3-X63-Ag8 653 culture
cells, hereinafter referred to as X63 cells, as parent cell
strain, which are 2-amino-6-oxy-8-azapurine (8-azaguanine)-
resistant and derived from BALB/c mouse myeloma. X63 cells in
the logarithmic growth phase were employed to subculture in
RPMI-1640 medium (at a concentration of 20 micrograms/ml and with
8-azaguanine) (available from GIHCO Co.), containing 10~ of
immobilized fetal calf serum (hereinafter referred to as FCS)
available from Intergen Co. Further cultivation was conducted,
from three days prior to the cell fusion, in RPMI-1640 medium
containing 10$ of FCS but not containing 8-azaguanine, in which
the cells in the logarithmic growth phase were also employed.
Following washing with RPMI-1640 medium three times, X63 cells
were determined for the number of cells, with the result that the
number of living X63 cells was 7x10.
Polyethylene glycol-4000 available from Sigma Co. was
dissolved in RPMI-1640 medium to give a concentration of 50~
(w/v) for use in the cell fusion.
The spleen cells and X63 cells were mixed in a ratio of the
spleen cells . X63 cells = 10:1. The mixture was centrifuged at
1500 rpm for five minutes, the supernatant was removed and the
cells were well disintegrated to be used in the cell fusion. The
cell fusion was carried out, using polyethylene glycol prepared
CA 02180830 2004-12-23
in the aforesaid manner and kept at 37°C, in accordance with the
method described in "Kdhller and Milstein . Nature, Vo1.256,
pages 495-497 (1975)" and "European Journal of Immunology, Vol.6,
pages 511-519 (1976)".
The cell lines following the cell fusion for the hylaridoma
formation were suspended in HAT selection medium containing RPMI-
1640 added with FCS of a 10$ concentration, 1x10-4M hypoxanthine,
4x10-7M aminopterine and 1.6x10-5M thymidine, to give a spleen
cell concentration of 2.0x106 per ml. Each 50 microliters
1o aliquot of the cell suspension was distributed in 96 wells, and
incubated an incubator kept at 37°C temperature and 95~ humidity
under a 8% C02 atmosphere. One drop of the HAT medium was added
to each well on the first day and the second day from the start
of the incubation, followed by the addition of two drops of the
15 same medium on the seventh and ninth day from the start for
further incubation. Then incubation was carried out in a HAT-
free culture medium. Approx. ten days to two weeks after the
start, screening was made for hybridoma cell clones producing
anti-human-annexin-V monoclonal antibodies capable of
2o specifically reacting with human annexin-V treated with urea of
8M concentration (8M-urea-treated human annexin-V) and/or with
human annexin-V not treated with 8M urea (native human annexin-
V). The screening was done by ELISA using a human annexin-V
absorption test and microplates in which human annexin-V as the
25 antigenic protein is adsorbed onto a solid phase.
All the clones of established hybridoma cell lines
producing anti-human-annexin-V monoclonal antibodies as well as
anti-dog-32KP polyclonal antibody are sensitized for solid
26
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phase. In addition, the anti-human-annexin-V monoclonal
antibodies produced from such clones as well as anti-dog-32KP
. polyclonal antibody are labeled with biotin. Thus, a variety
of ELISA methods were employed: the solid-phase-coupled clones
and anti-dog-32KP polyclonal antibody are used in combination
with the biotin-labeled anti-human-annexin-V monoclonal
antibodies and also anti-dog-32KP polyclonal antibody, in order
to detect clones) producing anti-human-annexin-V monoclonal
antibodies which will specifically react with 8M-urea-
treated human annexin-V. native human annexin-V and native
human annexin-V in the human blood, serum or plasma not treated
with 8M urea.
(5) Screening
As cell clones appeared ten days after the start of
incubation, an absorption test with human annexin-V as the
antigen was carried out by ELISA for the supernatants of
hybridoma cell lines.
It was found by the present inventors that the antigenic
activity of human annexin-V treated with 8M urea solution is
significantly higher than that of native human annexin-V, with
anti-32KP polyclonal antibodies described later. Thus, the
human annexin-V absorption test was carried out using the two
types of antigens: human annexin-V treated with 8M urea
(hereinafter referred to as (8M-urea-treated human annexin-V)
and human annexin-V with no such urea-treatment (hereinafter
referred to as native human annexin-V).
Thus, a first set is constituted of 50 microliters per well
27
CA 02180830 2004-12-23
of 8M-urea-treated human annexin-V solution (the 8M-urea-treated
human annexin-V concentration being 200ng/ml) and 50 microliters
per well of the culture supernatant of hybridoma cell line
distributed each U-bottomed well of a microtiterplate,
while a second set is constituted of 50 microliters per
well of native human annexin-V (the native human annexin-V
concentration being 200ng/ml) and 50 microliters per well of
the supernatant of hybridoma cell line distributed in each U-
bottomed well of another microtiterplate. Each well was added
l0 with 50 microliters of 20~ suspension of Sepharose*4B combined
with anti-mouse-immunoglobulin antibody and allowed to stand
for 10 minutes following stirring for one hour. With each set,
when it was observed that the anti-mouse-immunoglobulin antibody-
combined Sepharose*4B completely sedimented on the well bottom,
15 the respective supernatants 25 microliters were employed to
measure remaining human annexin-V concentration therein by
means of the human annexin-V ELISA method.
In this measurement, if any anti-human-annexin-V
monoclonal antibodies against human annexin-V was present
in the culture supernatant of hybridoma cell line, reactions
will occur between human annexin-V and the anti-human-
annexin-V monoclonal antibodies, followed by a reaction with
anti-mouse-immunoglobulin monoclonal antibody-combined
Sepharose* 4B, resulting in the formation of antigen-antibody
25 complexes which are to sediment. This will lead to a
decrease in the human annexin-V content remaining in the
supernatant and it is therefore possible to identify the
presence of any anti-human-annexin-V monoclonal antibodies
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through the measurement of the human annexin-V
concentration in the supernatant.
After the screening was completed in the above-mentioned
manner for anti-human-annexin-V monoclonal antibodies capable of
reacting with human annexin-V as the antigenic protein, a test
was conducted to study the difference in epitopes with which the
anti-human-annexin-V monoclonal antibodies will react.
Thus, 8M-urea-treated human annexin-V and native annexin-V
as the antigenic solutions, each having been prepared to have a
1o concentration of 1 microgram/ml, were absorbed onto a
microtiterplate in an amount of 50 microliters per well, followed
by washing three times with phosphate buffer solution
(hereinafter referred to as PBS) containing 0.05$ Tween~20, a
Tween~ surfactant, and blocking with PBS containing 1$ BSA to
prepare plates with solid phase-coupled human annexin-V antigens.
A solid phase-coupled human annexin-V antigen in the plates
thus obtained was allowed to react, for one hour at room
temperature, with a culture supernatant of the hybridoma cell
lines producing monoclonal antibodies capable of reacting with
2o human annexin-V screened by the human annexin-V absorption test.
The resulting antigen-antibody complexes were subjected to three
times of washing with the washing solution and allowed to react,
for thirty minutes at room temperature, with anti-mouse-
immunoglobulin antibodies (from goats) labeled with horseradish
peroxidase (hereinafter referred to as HRPO). Following the
reaction, the resultant antigen-antibody complexes were subjected
to washing four times with the washing solution and allowed to
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react, at room temperature for five minutes, with OPD substrate
solution containing O.1M phosphate-citrate buffer solution,
. 2mg/ml o-phenylenediamine and 4mM H202, and then the
reaction was terminated with 2N H2S04 and absorbance
measurement was performed by the dual wavelength method at' 492nm
as the primary wavelength and at 690nm as the secondary
wavelength on an ELISA plate reader.
At the primary wavelength 492nm absorbance is measured with
the substrate undergone the reaction due to the bound labeled
antibodies, while at the secondary wavelength the absorbance
reflects cracks or stains of the microtiterplates. Accordingly,
the subtraction of the absorbance at the secondary wavelength
from that at the primary wavelength makes it possible to
determine the absorbance reflecting the amount of the enzyme on
the labeled antibodies which have bound to the solid phase-
coupled antigen in proportion to the amount of the antigen.
(6) Studies on reactivities with solid phases sensitized
with annexin-V antigens from various organs and animals
Studies were made of the anti-human-annexin-V monoclonal
2o antibodies thus obtained for the species-dependent specificities
with annexin-V derived from various human organs and animals,
using the solid phases sensitized in the above-mentioned manner
for human annexin-V.
With each annexin purified from the various human organs and
animals, a solution was prepared to give a concentration of 1
microgram/ml with O.1M phosphate buffer at pH 7.5. 50
microliters of each antigen solution was distributed in a 96-well
microtiterplate for the adsorption overnight at 4°C, followed by
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washing twice with the washing solution and blocking with the
blocking solution. The solid phases sensitized with the various
annexin-V antigens were allowed to react with the anti-human-
annexin-V monoclonal antibodies obtained by the example herein.
The reaction products were assayed by the ELISA method in the
same manner for the screening, for determining the reactivities
of the anti-human-annexin-V monoclonal antibodies as obtained by
the present invention with the respective annexin-V antigens.
(7) Studies on reaction specificities by Western blotting
Homogenized specimens of the human cardiac muscle, the human
liver, the human kidney, the human lungs, the beagle's cardiac
muscle and the rat's cardiac muscle were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
using 10 to 20$ polyacrylamide gel to fractionate the protein
fractions. The protein fractions are then transferred into
nitrocellulose membranes, followed by a blocking with PBS
containing 3~ skim milk and 1$ BSA. Then the protein fractions
fixed onto the membranes were allowed to react with the
respective monoclonal antibodies to examine reactive protein
fractions derived from the various organs and animals.
(8) Establishment of hybridoma cell lines producing
monoclonal antibodies
Based on the above-mentioned studies, eighty-four strains
were screened which are hybridoma cell line clones capable of
reacting with 8M-urea-treated human annexin-V, through the
absorption test using human cardiac muscle annexin-V. From such
strains twenty-one clone strains were strictly selected depending
31
2 ~ soa3o
upon the difference in reactivities with type of annexin-V
derived from the various organs and animals. Thus, the twenty-
one strains were classified into the following five groups
depending upon the reactivities:
(i) Clones which exhibit a high reactivity in the 8M-urea-
treated human cardiac muscle annexin-V absorption test, but
do not exhibit reactivities with any of the antigen-
sensitized solid phases: five strains (clone HCA-212, clone
HCA-290, clone HCA-656, clone HCA-713 and clone HCA-805).
(ii) Clones which exhibit a high reactivity in the 8M-urea-
treated human cardiac muscle annexin-V absorption test and also
exhibit a high reactivity with the human-annexin-V-sensitized
solid phase, but exhibit almost no or a low reactivity with the
beagle dog- and rat-derived-annexin-V-sensitized solid phases:
eight strains (clone HCA-69H, clone HCA-155H, clone HCA-231H,
clone HCA-671H, clone HCA-770H, clone HCA-784H, clone HCA-803H
and clone HCA-838H).
(iii) Clones which exhibit high reactivities in the 8M-urea
treated human cardiac muscle annexin-V absorption test, as well
as with all of the solid phases sensitized with annexin-V
antigens derived from the various human organs, the beagle's and
the rat's cardiac muscles: four strains (clone HCA-57HDR, clone
HCA-293HDR, clone HCA-350HDR and clone HCA-805HDR).
(iv) Clones which exhibit high reactivities in the 8M-urea
treated human cardiac muscle annexin-V absorption test and also
with the solid phases sensitized with annexin-V antigens from the
human cardiac muscle, the human kidney or the human liver, but
exhibit only low reactivities with the solid phases sensitized
32
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with annexin-V antigens from the beagle's and the rat's cardiac
muscles: three strains (clone HCA-507HD, clone HCA-660HD and
clone HCA-646HD).
(v) A clone which exhibit high reactivities in the 8M-urea-
treated human cardiac muscle annexin-V absorption test as well as
in the native human cardiac muscle annexin-V absorption test: one
strain (clone HCA-627).
Thus, there were obtained monoclonal antibodies having
different reactivities as classified into the five groups
1o mentioned in the above.
The reaction specificities of these monoclonal antibodies
are shown in Table 1 below.
20
33
Image
Image
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CA 02180830 2004-12-23
(9) Cloning
The hybridoma cell lines producing monoclonal antibodies as
classified into the above-mentioned five groups were subjected to
cloning by the limiting dilution to obtain a single clone. In
performing the cloning, thymus cells prepared from BALB/c mice
and suspended in HAT medium was added as the feeder cell at a
rate of 1x106 per well.
(10) Identification of mouse immunoglobulin subclass
Identification of mouse immunoglobulin subclass was made
with the monoclonal antibodies produced by the hybridoma cell
lines which have been prepared as having a single clone by means
of the above-mentioned cloning. The culture supernatants of the
respective hybridoma cell lines were assayed for the mouse
immunoglobulin subclass identification using a MONOAb~typing kit
(available from Zymed Co.)
The result was that seven clones have IgG1 type, eleven
clones IgG2a type and three clones IgG2b type, with regard to the
H chain. Regarding the L chain, all the clones were found to
have k chain (Table 1).
The respective clones classified into the five groups are
shown in Table 1.
EXAMPLE 2: Production of Monoclonal Antibodies
(1) Harvest of ascitic fluid
In order to obtain a higher concentration of anti-human-
cardiac-muscle-annexin-V monoclonal antibodies, the various
hybridoma cells are grown and the resulting respective clones
were inoculated, following washing three times with EMEM culture
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37
2 ~ goa3o
solution, into the ascitic cavities of HALB/c mice which have
been administered with 2,6,10,14-tetramethyl pentadecane
(pristane) in advance into their ascitic cavities. The ascitic
fluids were harvested seven to fourteen days after the
inoculation of the hybridoma cell lines into the ascitic
cavities. The ascitic fluids thus obtained were applied to
centrifuge to remove the cellular and residual fractions. The
supernatant fractions were pooled for keeping at 4°C.
(2) Purification for the anti-human cardiac-muscle-annexin-V
1o monoclonal antibodies from the ascitic fluids
The respective anti-human-cardiac-muscle-annexin-V
monoclonal antibodies were purified by a combination of salting-
out and ion-exchange chromatography to recover the purified IgG
fractions.
Thus, 20m1 of each clone was added with an equal amount of
phosphate-buffered saline (PBS), followed by the addition of
anhydrous sodium sulfate to give a final concentration of
20%(w/v) with stirring at room temperature. The mixture was
stirred for an additional one hour.
20~ Centrifugation was then performed at 12,000 rpm with a high-
speed centrifuge (available form Hitachi. Co.) and the resulting
residue fraction was dissolved in approx. 10 ml of phosphate-
buffered saline. The solution was dialyzed three times against
mM sodium phosphate buffer solution (pH 7.0) as the outer
solution. On completing the dialysis, from the dialyzed solution
the IgG fraction of the anti-human-cardiac-muscle-annexin-V
monoclonal antibody was purified by ion-exchange chromatography
38
CA 02180830 2004-12-23
using a DEAE (diethylaminoethyl cellulose) column DE-52
(available from Whatman Co.) measuring 1.5 cm in inner diameter
and 8 cm in length equilibrated with 20 mM sodium phosphate
buffer solution (pH 7.0), from which the purified IIgG fraction
was obtained as the pass-through fraction or as the eluent
fraction a~ a NaCl concentration in the range of 30 mM
gradationally up to 50 mM formed with 20 mM sodium phosphate
buffer solution (pH 7.0) containing 30 mM to 50 mM NaCl. Each
of the purified clones was determined for the purity of IgG
fraction, by high performance liquid chromatography using a
TSK gel G3000SW column (available from Tosoh. Co.) and sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
using PhastSystem* available from Pharmacia Co. The purity
was found to be 95$ or higher.
EXAMPLE 3: ELISA Assay System for Human Cardiac Muscle
Annexin-V
(1) Preparation of anti-human-cardiac-muscle-annexin-V
monoclonal antibodies for HRPO-labeling
The IgG fractions of clones HCA-155 and HCA-660HD were
purified in the manner as described in Example 2 to study the
application to ELISA system for human cardiac muscle annexin-V
assay.
(2) Preparation of HRPO-labeled anti-human-cardiac-muscle-
annexin-V monoclonal antibodies
(A) Preparation of anti-human-cardiac-muscle-annexin-V
monoclonal antibodies F(ab')2
For labeling the IgG fractions of the anti-human-cardiac-
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muscle-annexin-V monoclonal antibodies, each 10 mg of the anti-
human-cardiac-muscle-annexin-V monoclonal antibodies was
subjected to centrifugal filtration with a centrifugal
concentrator (Centricon 10 available from Amicon Co.) to give a
final volume of 1 ml. The concentrated antibody was dialyzed
against 0.1 M sodium acetate buffer (pH 4.0) containing 0.2 M
NaCl as the solvent.
The IgG solution resulting from the dialysis was added with
a solution of pepsin (available from Sigma Co.) dissolved in 1 M
1o sodium acetate buffer (pH 4.0) containing 0.2 M NaCl, so that the
pepsin is 4~ of the IgG content, followed by reaction at 37°C for
6 to 16 hours. On completing the reaction, the resultant was
applied to molecular-sieve chromatography using a Sephadex* G-150
column (available from Pharmacia Co.) for gel filtration,
15 which has been equilibrated with 0.1 M sodium borate buffer (pH
8.0), to obtain the F(ab')2 fragments of the respective
anti-human-cardiac-muscle-annexin-V monoclonal antibodies.
(H) Preparation of anti-human-cardiac-muscle-annexin-V
monoclonal Fab'-SH
2o Each of the anti-human-cardiac-muscle-annexin-V monoclonal
antibodies F(ab')2 as prepared in the aforesaid (A) was subjected
to centrifugation for further concentration by a Centricon*
centrifugal concentrator to prepare the concentrated F(ab')2
fractions of the respective anti-human-cardiac-muscle-annexin-V
25 monoclonal antibodies.
The concentrated fraction was added with 0.1 ml of 100 mM 2-
mercaptoethylamine hydrochloride (available from Kishida
trademark 40
CA 02180830 2004-12-23
Chemicals Co.) solution for reaction at 37°C for 9b minutes. On
completing the reaction, the resultant was applied to a Sephadex*
G-25 column for equilibrium gel filtration (available from
Pharmacia Co.) measuring 1.6 cm in diameter and 20 cm in length
which has been equilibrated with 0.1 M sodium phosphate buffer
(pH 6.0) containing 1 mM EDTA, for fractional purification of the
Fab'-SH fraction, followed by centrifugal concentration by a
Centricon~l0 centrifugal concentrator, to give a final volume of
1 ml. Thus, the concentrated Fab'-SH fractions of clones HCA-
155H and HCA-660HD anti-human-cardiac-muscle-annexin-V monoclonal
antibodies were prepared.
(C) Preparation of HRPO maleimide
Ten mg (as a protein content) of HRPO (available from
Boehlinger Co.) was dissolved in 1 ml of 0.1 M sodium
phosphate buffer (pH 6.0). The resulting solution was added
with 100 microliters solution of N-hydroxysuccinimide ester
(available from Zeeben Chemicals Co.) dissolved in
dimethylformamide (DMF) (available from Kishida Chemicals
Co.) to give a final concentration of 25 mg/ml, followed
2o by reaction at 30°C for 60 minutes to prepare maleimide ester
of HRPO. On completing the reaction, the solution was
centrifuged for five minutes at 3000 rpm. The supernatant was
applied to a SephadeX G-25 column for equilbrium gel filtration
(available from Pharmacia Co.) measuring 1.6 cm in diameter
and 20 cm in length, which has been equilibrated with 0.1 M
sodium phosphate buffer (pH 6.0), in order to purify HRPO
maleimide. The purified fraction of HRPO maleimide was then
trademark
41
CA 02180830 2004-12-23
subjected to centrifugal concentration by a Centricon 10
centrifugal concentrator to prepare the concentrated fraction of
HRPO maleimide.
(D) Preparation of HRPO-labeled anti-human-cardiac-muscle-
annexin-V monoclonal Fab' antibodies '
The concentrated Fab'-SH fraction of each anti-human-
cardiac-muscle-annexin-V monoclonal antibody and the concentrated
fraction of HRPO maleimide were mixed together at a molar ratio
of 1:1, for reaction at 4°C for 15 to 24 hours. On completing
the reaction, 2-mercaptoethylamine was added to give a
concentration of 2 mM in the reaction solution, followed by
reaction at 37°C for 20 minutes in order to block unaltered HRPO
maleimide. The resultant was then subjected to molecular-sieve
gelchromatography using a Ultragel~ACA44 column (available from
lg Pharmacia Co.) equilibrated with 20 mM sodium phosphate - sodium
citrate buffer containing 0.15 M NaCl and 2.5mM EDTA (pH 5.6) and
measuring 1.6 cm in diameter and 65 cm in length, for removing
unaltered Fab'-SH of the anti-human-cardiac-muscle-annexin-V
monoclonal antibodies and unaltered HRPO maleimide and purifying
the HRPO-labeled anti-human-cardiac-muscle-annexin-V monoclonal
Fab' antibodies (hereinafter referred to as HRPO-labeled anti-
human-cardiac-muscle-annexin-V monoclonal antibodies).
(3) Preparation of anti-dog-32KP polyclonal antibodies IgG
Anti-dog-32KP-annexin-V polyclonal antiserum was obtained by
immunizing rabbits with dog-derived purified 32KP annexin-V
antigen. To three ml of the antiserum was added an equal amount
of phosphate-buffered saline (PBS), followed by the addition of
anhydrous sodium sulfate to give a final concentration of 20%
* trademark
42
2180830
with stirring and an additional stirring for one hour at room
temperature. The resultant was then centrifuged at 12,000 rpm
for 10 minutes, and the residue obtained was dissolved in approx.
3 ml of saline. The solution was then subjected to dialysis
against 20 mM sodium phosphate (pH 7.0) as the solvent. On
completing the dialysis, the resulting solution was applied to
ion-exchange chromatography using a DEAF cellulose DE-52 column
(available from Whatman Co.) equilibrated with 20 mM sodium
phosphate buffer (pH 7.0) and measuring 1.5 cm in diameter and 6
cm in length, to purify the IgG fraction of anti-dog-32KP-
annexin-V polyclonal antibody. From 3 ml of the anti-dog-32KP=
annexin-V polyclonal antibody-containing antiserum there was
obtained 13 mg of the purified IgG fraction.
(4) Preparation of HRPO-labeled anti-dog-32KP-annexin-V
15, polyclonal antibody
(A) Preparation of anti-dog-32KP-annexin-V polyclonal
antibody F(ab')2
For labeling the purified IgG fraction of the anti-dog-32KP
annexin-V polyclonal antibody, 12 mg of aforesaid antibody
was applied to a Centricon 10 centrifugal concentrator
(available from Amicon Co.) to give a concentrated volume of 1
ml, followed by dialysis against 0.1 M sodium acetate
buffer (pH 4.5) containing 0.2 M NaCl.
The antibody solution resulting from the dialysis was added
with a solution of pepsin (available from Sigma Co.) dissolved in
0.1 M sodium acetate (pH 4.5) containing 0.2 M NaCl to give a
concentration of 4% based on the IgG content, followed by
43
CA 02180830 2004-12-23
reaction at 37°C for 16 hours. On completing the reaction, the
resultant was applied to molecular-sieve chromatography using a
Sephadex*G-150 column for equilibrium gel filtration (available
from Pharmacia Co.) equilibrated with 0.1 M sodium borate buffer
(pH 8.0) and measuring 1.6 cm in diameter and 65 cm in length, to
fractionate and purify the F(ab')2 fraction of the antibody.
The resulting fraction was subjected to dialysis against sodium
phosphate buffer (pH 6.0) containing 1 mM EDTA as the solvent. On
completing dialysis, the dialyzed solution was applied to
1o Centrifugal concentration by a Centricori 10 centrifugal
concentrator to give a final volume of 1 ml. The solution
resulting from the dialysis was used to prepare the labeled
antibody as the anti-dog-32KP-annexin-V F(ab')2 polyclonal
antibody. Thus, from 12 mg of the IgG fraction of the anti-
15 dog-32KP polyclonal antibody obtained in the above-mentioned
manner, there was prepared approx. 7 mg of the F(ab')2 fraction.
(B) Preparation of anti-dog-32KP-annexin-V polyclonal Fab'-
SH
To 7 mg/ml solution of anti-dog-32KP polyclonal antibody
F(ab')2 fraction as prepared in above (A) was added 0.1 ml of 100
mM 2-mercaptoethylamine chloride (available from Kishida
Chemicals Co.) for reaction at 37°C for 90 minutes. On
completing the reaction, the resultant was applied to a Sephadex*
G-25 column (available fro Pharmacia Co.) for equilibrium gel
25 filtration measuring 1.6 cm in diameter and 20 cm in length
equilibrated with 0.1 M sodium phosphate buffer (pH 6.0)
containing 1 mM EDTA, to purify the Fab'-SH fraction. The
fraction was then subjected to centrifugal~concentration by a
* trademark
44
2180830
Centricon 10 centrifugal concentrator to a volume of 1 ml. Thus,
from 7 mg of the F(ab')2, there was obtained 6.1 mg of anti-dog-
32KP annexin-V polyclonal antibodies Fab'-SH.
(C) Preparation of HRPO maleimide
Ten mg (as a protein content) of HRPO (available from
Boehlinger C0.) was dissolved in 1 ml of 0.1 M sodium
phosphate buffer (pH 6.0). The resulting solution was added
with 100 microliters solution of N-hydroxysuccinimide ester
(available from Zeeben Chemicals Co.) dissolved in
to dimethylformamide (DMF) (available from Kishida Chemicals
Co.) to give a final concentration of 25 mg/ml, followed by
reaction at 30°C for 60 minutes to prepare maleimide ester of
HRPO. On completing the reaction, the solution was centrifuged
for five minutes at 3000 rpm. The supernatant was applied to a
15 Sephadex G-25 column for equilibrium gel filtration (available
from Pharmacia Co.) measuring 1.6 cm in diameter and 20 cm in
length, which has been equilibrated with 0.1 M sodium phosphate
buffer (pH 6.0), in order to purify HRPO maleimide. The
purified fraction of HRPO maleimide was then subjected to
20 (centrifugal) concentration by a Centricon 10 centrifugal
concentrator to prepare the concentrated fraction of HRPO
maleimide.
(D) Preparation of HRPO-labeled anti-dog-32KP-annexin-V
polyclonal antibody Fab'
25 The Fab'-SH fraction of anti-dog-32KP-annexin-V polyclonal
antibody thus obtained was mixed with the HRPO maleimide
fraction at a molar ratio of 1:1, for reaction at 4°C for 15 to
CA 02180830 2004-12-23
24 hours. Then the resultant was added with 2-mercaptoethylamine
hydrochloride to give a concentration of 2 mM in the reaction
solution, followed by reaction at 37°C for 20 minutes to block
unaltered HRPO maleimide. The resultant was then subjected to
gelchromatography using a Ultrogel ACA44 column (available from
Pharmacia Co.) equilibrated with 20 mM sodium phosphate-sodium
citrate buffer (pH 5.6) containing 0.15 M NaCl and 2.5 mM EDTA
and measuring 1.6 cm in diameter and 65 cm in length, for
removing unaltered anti-dog-32KP-annexin-V polyclonal antibody
Fab'-SH fraction and unaltered HRPO maleimide and purifying HRPO-
labeled anti-dog-32KP-annexin-V polyclonal antibody
(hereinafter referred to as HRPO-labelled antibodies).
(E) HRPO activity determination
In determining the HRPO enzyme activity of the HRPO-labeled
antibodies, 2.98 ml of 0.1 M sodium phosphate buffer (pH 7.0)
containing 0.2$ phenol, 0.5 mM hydrogen peroxide and 0.15 mg/ml
4-amino antipyrine were added with 20.microliters of the HRPO-
labeled antibodies to give a total volume of 3.0 ml, followed by
reaction at 37°C for five minutes, and measurement of absorbance
at 500nm by means of the Rate assay. HRPO activity was
determined by measuring the difference in absorbance (0 Abs)
per one minute.
(5) Preparation of anti-human-cardiac-muscle-annexin-V
monoclonal antibodies solid phase
As monoclonal antibodies for use in the solid phase in an
ELISA system for the assay of human cardiac muscle annexin-V
clones HCA-290, HCA-627, HCA-57HDR, HCA-293HDR and HCA-350HDR
trademark
46
CA 02180830 2004-12-23
were selected and the IgG fractions of the respective clones were
purified from the ascitic fluids in the same manner
described in Example 3.
The IgG fraction of each of the anti-human annexin-V
monoclonal antibodies was adjusted to have a concentration-of 30
micrograms/ml with 0.1 M sodium phosphate buffer (pH 7.5)
containing 0.1$ sodium azide, and distributed in a
microtiterplate for ELISA (available from Nunc Co.) at a rate of
100 microliters per well, for sensitization at 4°C overnight.
to Then each well of the microtiterplate was washed three times with
phosphate-buffered saline (PBS) containing 0.05 Tween~ 20
surfactant, and then added with 300 microliters of PBS containing
1~ BSA (as the blocking solution), followed by a further blocking
operation at 4°C overnight, to prepare_antibody-plates sensitized
with anti-human-cardiac-muscle-annexin-V monoclonal antibodies
(hereinafter referred to as anti-human-annexin-V monoclonal
antibody plates).
(6) Studies on ELISA assay system using anti-human-annexin-
V monoclonal antibodies
2o The anti-human-annexin-V monoclonal antibody plates prepared
in the above-mentioned manner were added, after the blocking
solution discarded, with 10 mM sodium phosphate buffer (pH 7.0)
containing 1$ BSA, 0.15 M NaCl and 5 mM EDTA at a rate of 100
microliters per well. Standard antigen solutions were prepared,
with 8M-urea-treated human annexin-V antigen (obtained by the
treatment with 8M urea), and native annexin-V (obtained by no
treatment with 8M urea), to give concentrations of 1.5625 ng/ml,
trademark
47
2180830
3.125 ng/ml, 6.25 ng/ml, 12.5 ng/ml, 25 ng/ml, 50 ng/ml and 100
ng/ml. The respective standard antigen solutions were added to
the well at a rate of 20 microliters per well, followed by
stirring and reaction for one hour at room temperature. On
completing the reaction, each well was washed three times with
the washing solution. To each of the washed wells were added the
HRPO-labeled anti-human-annexin-V monoclonal antibodies Fab'
(from clones HCA-155H and HCA-660HD) at a rate of 100 microliters
per well, for reaction at room temperature for thirty minutes.
1o Following the reaction, each well was washed six times with
washing solution and then added with 100 microliters of OPD
substrate solution containing 2 mg/ml o-phenylenediamine and 4 mM
H202, for reaction for thirty minutes. The reaction was
terminated by adding 2N H2S04 solution at a rate of 100
microliters per well. Absorbance was determined on an ELISA
plate reader by the dual wavelength method at 492nm as the
primary and at 690nm as the secondary.
The absorbance herein intended is obtained by subtracting an
absorbance measurement at the secondary wavelength 690nm from
that at the primary wavelength 492nm.
The results of the absorbance measured are given in Table
2 below, with respect to 8M-urea-treated-human annexin-V
standard antigen solutions.
48
2180830
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53
2180830
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54
2180830
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55
2180830
H ~y tv
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56
2180830
Figures 1 thorough 8 illustrate calibration curves for
human annexin-V, in which the results of measurement as given in
Tables 2-1 through 2-8 are plotted in diagrams with the absiccia
being the concentration of human annexin-V antigen and the
ordinate being the difference in the absorbance measured between
at 492nm and at 690nm.
The calibration curves are excellent ones as the absorbance
increases with increasing concentration of human annexin-V
substantially linearly as high as a concentration of 100 ng/ml.
1o The use of these calibration curves enables reliable assay of
human annexin-V concentration in samples to be examined (plasma
and serum). It is also possible to determine the concentration
of human annexin-V with good reproducibility even as low as
a concentration of 1 ng/ml, as can be seen Figure 1.
The calibration curves as shown by Figures 1 through 8 are
of reliable use for the determination of the concentration of
human annexin-V.
With the native human annexin-V without 8M-urea-treatment as
the standard antigen, the absorbance detected was approx. 5$ to
10% of that with 8M-urea-treated human annexin-V, demonstrating
that the system has a low reactivity to the native human annexin-
V antigen.
EXAMPLE 4: Assay of Human Annexin-V Using Anti-human-
annexin-V Monoclonal Antibody HCA-627 and
HRPO-labeled-anti-dog-32KP-annexin-V Polyclonal
Antibody
57
2180830
Assay of human annexin-V concentration was conducted by
ELISA using anti-human-annexin-V monoclonal antibody HCA-627,
which has been found to exhibit a high reactivity with native
annexin-V without 8M-urea treatment, in combination with anti-
dog-32KP-annexin-V polyclonal antibody.
Thus, 100 microliters of anti-human-annexin-V monoclonal
antibody HCA-627, secreted from hybridoma cell line HCA-627
strain capable of producing an anti-human-annexin-V monoclonal
antibody, was distributed in wells at a rate of 100 microliters
to per well, to prepare solid-phase well coupled with anti-human-
annexin-V monoclonal antibody in the manner for preparing the
solid-phase as described in Example 4 (5).
To the solid phase wells was added lOmM sodium phosphate
buffer (pH 7.0) containing 1~ BSA, 0.15M NaCl and 5mM EDTA at a
15 rate 100 microliters per well.
Following the distribution of the reaction buffer, each well
was added with a standard solution. As the standard solution in
this Example were used standard solutions of 8M-urea treated
human annexin-V antigen and standard solutions of native human
2o annexin-V antigen. Thus, one set of the solid phase wells were
added with the standard solution of 8M-urea-treated human
annexin-V whereas the other set of the solid phase wells were
added with the standard solutions of native annexin-V antigen.
Following the distribution of the standard solutions, an antigen-
25 antibody reaction was allowed to take place in each well with
stirring for one hour. After the antigen-antibody reaction,
each well was washed four times with the washing solution and
then added with 100 microliters of the HRPO-labeled-anti-dog-
58
2180830
annexin-V polyclonal Fab' antibody (100mU per ml of the
reaction buffer) followed by an antigen-antibody reaction for
thirty minutes with stirring. Then, each well was washed
eight times with the washing solution and added with 100
microliters of OPD substrate solution for the color production
reaction at room temperature for thirty minutes.
With the lapse of the time for the color production, each
well was added with the reaction-terminating solution ( 2N H2S04
solution) to terminate the reaction, followed by absorbance
1o measurements at the primary wavelength 492nm and secondary
wavelength 690nm on an ELISA plate reader to assay the samples
and the standard solutions for the concentration of human
annexin-V.
The absorbance was obtained by subtracting an absorbance
15 measured at the secondary wavelength 690nm from that at the
primary wavelength 492nm. Thus, a calibration curve was prepared
by plotting the absorbance data against the human annexin-V
concentration with reference to the antigen protein standard
solution. Based on such calibration curve, a sample can be
20 calibrated for the concentration of the antigenic protein, human
annexin-V.
In the Table 3 below, there are given absorbance data on the
standard antigen of native annexin-V obtained by ELISA using
anti-human annexin-V monoclonal antibody HCA-627 and the HRPO-
25 labeled-anti-dog-32KP polyclonal antibody.
59
2180830
1 :sCa O Q~u~u1~ d~t'
(0V] N M l~N N V~N I~
-ri S-.1N O O O ~ N d'00Ln
X ~ t
N ~ O O O O O O O '-
C ~2
I N
C ~ O~a0N ~ I~I~l~O
(d (O ~ M l0N ~ N N CO
E L-I O O O ~ N d'COd'
O
O O O O O O O
1
Cn -ri
H -+.)
a
W ~ ~ N I~1D01l~N O V~01
O LT ~ M L(1.-.-.-N QO
~, '(~ 01 (0Gl O O O ~'N d'00M
f0 v N O O O O O O O ~-
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N
'O
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i-~.~ N
wi 01
+.~ d' b O
(~" ~Q,'1..1-r1 .-r-r-N N QD~ Lf1
> .~ t0+~ O O O O O O O Wit'
I II 't7f0~1O O O O O O O O
-I ~ -,.-I~
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N O ~ U1C.a
U
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Pr .~ O1OpM N aDM 10N
-rl ~ Ul ~ M l0N ~ M N -
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x ~ . . . . . . .
z N ~ w v o 0 0 0 0 0 0 -
M U1
'I-iI f0 U
O b~ .~ 1JS...I
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0 0 0 0 0 0 0 0
O N
E
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o rx o
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o >~ +~~
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U ~ !~ N ~ O ~ M l~N Ll1O O
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v
0 0
U
c0
E1
2180830
Figure 9 illustrates a calibration curve prepared from the
results of the assay as given in Table 3. Thus, the reliable
calibration curve was obtained for the concentration of the
native human annexin-V antigen in the ELISA system using the
anti-human-annexin-V monoclonal antibody HCA-627 and the HRPO-
labeled anti-dog-32KP polyclonal antibody.
EXAMPLE 5: Detection and Quantitative Analysis of Human
Annexin-V in Samples from Patients with
Myocardial Infarction, by ELISA Using Anti-
human-annexin-V Monoclonal Antibody HCA-627 and
HRPO-labeled-anti-dog-32KP Polyclonal Fab'.
The combination of anti-human-annexin-V monoclonal antibody
HCA-627 and HRPO-labeled-anti-dog-32KP polyclonal Fab' prepared
in the above-mentioned manner, which has been found to have an
excellent sensitivity in assaying the native human annexin-V, was
examined for its applicability to the assay of human annexin-V
concentration in the clinical samples from patients with
myocardial infarction.
Thus, the solid phase composed of anti-human-annexin-V
monoclonal antibody was added, after disharging the solution,
with 10 mM sodium phosphate (pH 7.0) containing 1~ HSA, 0.15M
NaCl and 5mM EDTA at a rate of 100 microliters per well. Antigen
standard solutions of native human annexin-V (1.5625ng/ml,
3.125ng/ml, 6.25ng/ml, 12.5ng/ml, 25ng/ml, 50ng/ml and 100ng/ml)
and human plasma samples are added each at a rate of 20
microliters per well, followed by stirring and an antigen-
antibody reaction for one hour at room temperature. With the
61
2180830
lapse of the time for the antigen-antibody reaction, each well
was washed three times with the washing solution. Then, to the
wells was added HRPO-labeled-anti-dog-32KP polyclonal Fab'
antibody having been prepared to have an appropriate
concentration (100mU per ml of the reaction buffer) at a rate of
100 microliters per well, followed by an antigen-antibody
reaction for thirty minutes at room temperature. With the lapse
of the time for the antigen-antibody reaction, each well was
washed eight times with the washing solution.
1o Following the washing, each well was added with 100
microliters of OPD substrate solution containing O.lm phosphate-
citrate buffer, 2mg/ml o-phenylenediamine and 4mM H202, for color
production reaction for thirty minutes at room temperature.
With the lapse of the time for the color production
reaction, each well was added with 2N H2S04 as reaction
terminating solution to terminate the color production reaction.
The color produced in each well was assayed at the primary
wavelength 492nm and at the secondary wavelength 690nm. The
assay was conducted by measuring absorbance on an ELISA plate
reader at the primary wavelength 492nm and at the secondary
wavelength 690nm. The absorbance for a sample to be examined or
for a standard solution was obtained by subtracting an absorbance
measurement at the secondary wavelength 492nm from that at the
primary wavelength 690nm, to determine the concentration of human
cardiac-muscle annexin-V in the sample or the standard solution.
The measurements for the human plasma samples were obtained
by reading the human annexin-V concentration in the respective
62
CA 02180830 2004-12-23
samples with reference to the calibration curve which has been
prepared based on the absorbance measurements of the standard
solutions of varying human annexin-V concentration.
EXAMPLE 6: Application of the Enzyme-Antibody Method of the
Invention to the Assay of Blood Samples
(1) Diagnosis of myocardial infarction
A 75-year-old man with a pain in the chest at 6:00 a.m. was
hospitalized at 8:30 a.m. of the same day on suspicion of acute
~ myocardial infarction because of abnormality on the
electrocardiogram and the pain in the chest. At 9:00 a.m. of the
day (i.e. three hours from the the attack) while CK (creatine
phosphate enzyme) value indicated 108U/L in the normal range,
. annexin-V was found to be an increased value of 90.4ng/ml. With
the lapse of three days from attack, annexin-V decreased down to
l3.lng/ml. The abnormality on the electrocardiogram in this case
indicated the finding of acute myocardial infarction and the
assay of human annexin-V concentration in the plasma sample was
found to be of use in a quick diagnosis of myocardial infarction.
2o It was later established that 70$ of patients are diagnostic
of myocardial infarction, in cases where the annexin-v
concentrations in the plasma of the patients were determined to
be 50ng/ml or more as the annexin-V concentration in the blood of
the patient with myocardial infarction is relatively high.
(2) Diagnosis of angina pectoris
An 58-year-old man, who complained of two times of pains in
the chest attacked at 2:30 p.m. and 6:30 p.m. and continued over
63
2180830
to 15 minutes, was hospitalized at 7:30 of the same day on
suspicion of angina pectoris because of abnormality in the
electrocardiogram and the relatively short pain in the chest.
While the CK value was a normal value of 83U/L, the annexin-V
concentration was an increased value of 29.9ng/ml. The annexin-V
value decreased down to 29.9ng/ml at 12:00 and l5.lng/ml at 15:00
of the same day, demonstrating the restoration to normal. The
case was assessed to be suspicions of angina pectoris because of
the clinical symptom and the abnormality on the
1o electrocardiodiagram was then diagnostic of angina pectoris, in
which it was found that the concentration of human annexin-V in
the plasma also increases in a case of angina pectoris.
The concentration of human annexin-V in the blood of a
patient with angina pectoris is much higher than the normal
value, although it is relatively low as compared with in the case
of myocardial infarction. It was later established that 70~ of
the patients were diagnostic of angina pectoris, in cases where
the human annexin-V concentrations in the plasma of the patients
were determined to be 20 to 50ng/ml.
(3) Annexin-V concentration in the blood from normal adults
Annexin-V concentration in the blood from normal male adults
24 to 76 years of age were determined to be 0.9 to 11.3ng/ml,
whereas those from normal female adults 24 to 76 years of age
were 0.8 to 10.6ng/ml, with the average being 6.2ng/ml, the
maximum 11.3ng/ml and the minimum 0.8ng/ml. However, there were
observed no age-dependent variations.
64
2180830
EXAMPLE 7: Application of the Enzyme Antibody Method to
Cardiac Muscle Tissue Specific Staining
(1) The anti-human-annexin-V monoclonal antibodies obtained
in Example 3 was applicable not only to an ELISA method but to
staining specifically localized or distributed areas of human
annexin-V in the cardiac muscle tissue by means of an indirect
enzyme antibody method.
Furthermore, the use of the HRPO-labeled anti-human-
annexin-V antibodies prepared in Example 4 makes it possible to
l0 make a short-time specific staining of the cardiac muscle tissue
by means of a direct enzyme antibody method.
The following is given by taking the indirect enzyme
antibody method as an example: paraffin-treated or frozen slices
of the cardiac muscle tissue were fixed on a slide glass and
allowed, following the paraffin-removal or blocking, to react
with the anti-human annexin-V monoclonal antibodies having been
prepared to have an appropriate concentration (IgG concentration:
500 to 1000 micrograms/ml) as the primary antibody at room
temperature over an hour in a humid box. The resultant was then
washed with phosplate-buffered saline (PBS) three times for five
minutes each, followed by a reaction with HRPO-labeled anti-
mouse-immunoglobulin antibodies as the secondary antibody for
thirty minutes in a humid box. The resultant was washed three
times for five minutes each, followed by addition of substrate
solution of 4-aminoantipyrine type or diaminobenzidine for
reaction over thirty minutes. On completing reaction, the slide-
glass was washed with PHS and then sealed with a sealant such as
CA 02180830 2004-12-23
glycerol through a covering glass, for a microscopic observation
of the stained areas.
The results are given in Figures 10 and 11, in which the
distribution of localized human annexin-V in the cardiac muscle
tissue was stained with brown color and developed in -black,
demonstrating that the invention is applicable to a tissue-
staining for-cardiac muscle by means of an enzyme antibody method
as well as to an ELISA.
EXAMPLE 8: Hybridoma Preparation
(1) Mice
Inbred-strain BALB/c female mice 5 to 8 weeks of age were
maintained on a diet of the standard pellets, feeding water ad
libitum in an animal-breeding chamber (23+1°C, humidity 70$).
(2) Inmmunization
Dog-annexin-V extracted and purified from dog cardiac
muscles (cf. Japanese Laid-open patent application No.72147/
1995) and the human annexin-V purified from human cardiac muscles
as described in Example 1 were each dissolved in O.1M sodium
phosphate buffer (pH 7.6) and dialyzed against the same buffer.
Following the dialysis, the respective annexin-V antigens was
prepared so as to give solutions of a concentration of lmg/ml,
which were distributed into EppPndorf tubesat a rate of 50
microliters per tube, followed by freeze-drying at -400°C to
keep the purified native dog- and human-cardiac muscle-
derived annexin-V antigens for immunization.
Dog annexin-V 0.2m1 (200 micrograms) as prepared in the
above-mentioned manner was added with 0.3m1 of physiological
66
CA 02180830 2004-12-23
saline to give a volume of 0.5mI. The antigen solution thus
prepared was mixed with 0.5m1 of Freund's complete adjuvant,
followed by thorough emulsification. The dog annexin-V in
emulsion as the antigen was administered into the abdominal
cavities of four female mice of 5 week-of age in a dose of
the purified dog annexin-V 40 micrograms per mouse for the
intial immunization.
Dog annexin-V antigen 0.2m1 (200 micrograms) as prepared in
the aforesaid manner was taken in a test tube, to which was added
l0 0.3m1 of physiological saline to give a volume of 0.5m1. The
solution thus prepared was added with 0.5m1 of RIBI Adjuvant
System (RAS) MPL+TDM Emulsion* R-700 (available from RIBI. Immuno
Chem. Research Inc., Hamilton, MT., USA), in which lml of
plysiological saline was added per vial for making the solution.
The resultant mixture was vigorously stirred in a vortex mixer
for approx. three minutes for emulsification. With this dog
annexin-V antigen in emulsion the mice were immunized as booster
immunizations two weeks, four weeks and six weeks after the
intial immunization in a dose of 50 maicrograms per mouse. A
2o further booster immunization was carried out twenty-three weeks
after the initial immunization in a dose of the dog annexin-V
antigen 30 micrograms per mouse, with the antigen having been
prepared in the same manner as mentioned above using RIBI
Adjuvant System (RAS) MPL+TDM Emulsion, R-700. Following the
booster immunization the blood was collected from the respective
mice at intervals to prepare sera, which were assayed by the
aforesaid absorption test for antibody titers against native dog
trademark
67
2180830
cardiac muscle- and human-annexin-V to examine the production of
specific antibodies. Based on the result of the assay for the
antibody titer, mice were selected having a high titer. The
selected mice were administered, twenty-six weeks after the
initial immunization, with the native annexin-V (having been
prepared by dissolving in lml of physiological saline to give a
concentration of 50 micrograms/ml) intravenously through the
mice tails as a booster. At this point the mouse sera were again
assayed for the antibody titer to select mice having a higher
titer. The selected mice were administered, one week after the
preceding booster immunization, with lml of the native human
annexin-V (having been prepared with physiological saline to give
a concentration of 100 micrograms/ml) slowly and intravenously
through the tails of the mice for final immunization.
(3) Preparation of spleen cells
Three days after the final immunization, the spleen were
excised from the BALB/c mice in the same manner as described in
Example 1. The excised spleen cells were suspended in EMEM
culture medium (available from Nissui Co.) to prepare a
2o suspension of spleen cells. The spleen cells were washed four
times with EMEM culture medium and determined for the number of
cells. The resultant spleen cells are 4.7x108.
(4) Preparation of parent strain
Cell fusion was conducted using P3-X63-Ag8.653 culture cells
(hereinafter referred to as X63 cells) as parent cell strain,
which are 2-amino-6-oxy-8-azapurine (8-Azaguanine) -resistant and
derived from BALB/c mouse myeloma.
X63 cells in the logarithmic growth phase were employed to
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subculture in RPMI-1640 medium (in a concentration of 20
micrograms/ml and with 8-azaguanine) (available from GIBCO Co.)
containing 10% of immobilized fatal calf serum (hereinafter
referred to as FCS). Further cultivation was carried out, from
three days in advance to the cell fusion, in RPMI-1640 culture
medium containing 10% FCS but not containing 8-azaguanine to
maintain the growth ability in the logarithmic phase. The X 63
cells were washed three times with RPMI-1640 medium and
determined for the number of cells. The number of living X63
cells was 1.5x108.
(5) Cell fusion
Polyethylene glycol-400 available from Sigma Co. was
dissolved in RPMI-1640 culture medium to give a concentration of
50% (W/V), followed by heating at 37°C for use.
The spleen cells and X63 cells were mixed together in a
ratio of the spleen cells: the X63 cells=10:1. The mixture was
centrifuged at 1500 rpm for five minutes, the supernatant was
removed and the pelletized cells were thoroughly disintegrated
for use in cell fusion. The cell fusion was conducted, using
polyethylene glycol prepared in the aforesaid manner and kept at
37°C, in accordance with the method described in "Kohler and
Milstein: Nature, Vo1.256, pages 495-497 (1975)" and "European
Journal of Immunology, Vol.6, pages 511-519 (1976)".
The cell line following the cell fusion were suspended in
HAT selection medium containing RPMI-1640 added with FCS of a 10%
concentration, 1x10-4 hypoxanthine, 4x10-7 aminopterin and
1.6x10-5M thymidine, to give a spleen-cell concentration of
69
CA 02180830 2004-12-23
2.0x106 per ml. The cell suspension was distributed in a 96-well
microtitreplate at a rate of 50 microliters per well, and
incubated in an incubator kept at a 37°C temperature and a 95$
humidity under a 8% COZ atomsphere. One drop of the HAT medium
was added to each well on the first day and the second day from
the start of the incubation, followed by the addition of two
drops of the same medium seven and nine days after start, for
further incubation. Then, incubation was carried out in a HAT-
free culture medium. Approx. ten days to two weeks after the
1o start of incubation, screening was made, as the first
screening, for clones producing anti-annexin-V monoclonal
antibodies having a common reactivity to native human annexin-
V and native dog annexin-V, in which the screening was
done by, an annexin-V absorption test using the native human
15 annexin-V and dog annexin-V antigens.
The selected clones were then subjected to the
second screening by means of a capture method for monoclonal
antibodies, through which were selected hybridoma cell line
clones producing monoclonal antibodies suitable for use in a
O sandwich ELISA system for a sensitive assay of annexin-V in
combination with monoclonal antibodies HCA-627. The
monoclonal antibodies HCA-627 are produced by hybridoma
cell line HCA-627 strain which has been deposited under the
number BP-5284 at the National Institute of Bioscience and
25 Human-Technology, the Agency of Industrial Aeience and Technology
of Japan.
(6) Screening
(i) First Screening
CA 02180830 2004-12-23
As cell clones appeared ten days after the start of
incubation, the absorption test with native dog- and human-
annexin-V as the antigens was carried out by ELISA for the
supernatants of the hybridoma cell line cultures.
It was found by the present inventors that an ELISA system
employing a combination the monoclonal antibodies HCA-627 bound
to the cells as the solid phase and the HRPO-labeled anti-dog-
32KP polyclonal antibodies can be applied to the assay of native
human annexin-V and dog annexin-V antigens. The system was thus
1o utilized to screen monoclonal antibodies which will efficiently
react with the native annexin-V.
Thus, the first set was constituted of the supernatant
of hybridoma cell line clone culture obtained by the subject
cell fusion and the native human annexin-V antigen solution
(100ng/ml) with each being distributed in U-bottomed
wells of a microtitreplate at a rate of 50 microliters per
well, while the second set was constituted of the
supernatant of the same hybridoma cell line clone culture and
the native dog annexin-V antigen solution (100ng/ml) with
2o each being distributed in a plurality of U-bottomed wells of
another microtitreplate at a rate of 50 microliters per well.
Each set was added with 50 microliters of 20$ suspension of
Sepharose~4B combined with anti-mouse-immunoglobulin antibody and
allowed to stand for 10 minutes following stirring for the one
hour. With each set, when it was observed that the anti-mouse
immunoglobulin antibody-combined sepharose~ 4B completely
sedimented on the well-bottoms, the respective supernatants 50
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71
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microtilers were assayed for human annexin-V or dog annexin-V
content remaining therein by means of the aforesaid annexin-V
ELISA method.
(ii) Selection of monoclonal antibodies by. the first screening
In this assay, if any anti-cardiac-muscle-anriexin-V
monoclonal antibodies against native dog- and human-annexin-V
remain in the culture supernatant of a hybridoma cell line, a
reaction will occur between the native dog- or human-annexin-V
and the anti-human-annexin-V monoclonal antibodies, followed by a
reaction with anti-mouse-immunoglobulin monoclonal antibody-
combined Sepharose~ 4B, resulting in the formation of antigen-
antibody complexes which are to sediment. This will lead to a
decrease in the annexin-V content remaining in the supernatant
and it is therefore possible to identify the occurrence of any
anti-annexin-V monoclonal antibodies.
(iii) Second screening
If there were successfully selected anti-annexin-V
. monoclonal antibodies having a reactivity with native human- and
dog-annexin-V antigens, screening was then made, through the
0 following capture ELISA, for hybridoma cell line clones
producing any monoclonal antibodies suitable for establishing a
sandwich ELISA system for sensitive assay of annexin-V, in
combination with the monoclonal antibodies HCA-627 produced by
hybridoma cell line HCA-627 strain which has been deposited as an
International Deposit under the number FERM BP-5284.
(iv) Capture ELISA method
A solution was prepared of the IgG fraction of polyclonal
antibody having a specificity to the Fc portion of goat-derived
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anti-mouse immunoglobulin (available from Nordic Co.), with O.1M
sodium phosphate solution (pH 7.5) containing 0.1% sodium azide
to give a concentration of 50 microliters/ml. The solution was
distributed in a 96-well titerplate (available from Nunk Co,) at
a rate of 50 microliters per well for adsorption at 4°C
overnight.
The resultant was then washed three times with washing
phosphate buffer (hereinafter referred to washing solution)
containing 0.05% Tween-20, followed by blocking with PHS
1o containing 1% BSA to prepare anti-mouse-immunoglobulin G-Fc
polyclonal antibody-bound wells as the solid phase.
(v) Based on the results of the absorption test with native
human- and dog-annexin-V, the culture supernatants of hybridoma
cell lines producing monoclonal antibodies with a high reactivity
15 with the native human- and dog annexin-V was added into the wells
bound with the anti-mouse-immunoglobulin G-Fc polyclonal
antibody as the solid phase at a rate 50 microliters per well,
following the removal of the blocking solution from each well.
Upon the completion of reaction for one hour at room temperature,
2o each well was washed three times with the washing solution. With
a phosphate buffer of pH 7.0 containing 1% HSA and. 5mM EDTA
(hereinafter reaction buffer solution), a solution of native
human annexin-V was prepared to give a concentration of 100ng/ml.
To each well was added 50 microliters of the solution of native
25 human annexin-V, for a further reaction over one hour, followed
by washing three times with the washing solution. HRPO-labeled
HCA-627-Fab' antibody were conditioned with the reaction buffer
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73
2180830
solution to have an appropriate concentration, and then added to
the respective wells for reaction over thirty minutes. The
dilution of the HRPO-labeled antibodies was done in such manner
that the mouse immunoglobulin was lOmicrograms/ml, in order to
block the adsorption of the HRPO-labeled antibodies onto the
well bound with the anti-mouse -immunoglobulin G-Fc polyclonal
antibodies as the solid phate. On completing the reaction, each
well was washed three times with the washing solution, followed
by reaction with OPD substrate solution (containing O.1M
1o phosphate-citrate buffer added with 2mg/ml o-phenylenediamine and
4mM H202) for five minutes at room temperature. The reaction was
terminated with 2N H2S04, to determine absorbance at the primary
wavelength 492nm and at the secondary wavelength 690nm on an
ELISA plate reader. Measurements at the primary wavelength 492nm
i5 are attributed to the substrate chromogen produced by the bound
labeled-antibody, whereas the measurements at the secondary
wavelength reflect flaws or stains on the microtiterplate.
Hy subtracting an absorbance measurement at the secondary
wavelength from an absorbance measurement at the primary
2o wavelength, it is possible to determine the true absorbance in
response to the amount of chromogen changed due to the enzyme on
the labeled-antibodies which have been bound to antigen in
proportion to the amount of the antigen bound to the solid-phase.
This second screening enables obtainment of an annexin-V
25 monoclonal antibody having reactivities with the native human
and dog-annexin-V antigens and being capable of recognizing an
antigen determinant site different from another site on the
indentical annexin-V molecule to be recognized by anti-annexin-V
74
2180830
monoclonal antibody HCA-627, thereby providing an optimal
combination with the anti-annexin-V monoclonal antibody HCA-627
for a sandwich ELISA system. Thus, a hybridoma cell line can be
obtained which produces the monoclonal antibody to provide the
optimal combination with the anti-human-annexin-V monoclonal
antibody HCA-627 for the sandwich ELISA system.
(7) Studies on reactivities with the solid-phases, sensitized
with annexin-V antigens derived from various animals
Studies were made on animal-dependent reactivities of the
various anti-annexin-V monoclonal antibodies identified by the
aforesaid screening, by allowing them to react with
microtiterplate solid phases composed of annexin-V antigens
extracted and purified from various animals.
(8) Preparation of microtiterplates with solid phase-coupled
native annexin-V antigens
Native annexin-V antigens extracted and purified from the
human heart, the dog heart, the rat heart and the bovine lung
were each adjusted with O.1M phosphate buffer at pH 7.5 to give
an antigen solution of 1 microgram/ml. Each antigen solution was
distributed in a 96-well microtiterplate (available from Nunk
Co.) at a rate of 50 microliters per well, followed by adsorption
at 4°C overnight, washing three times with the washing solution
and then blocking at 4°C overnight with the blocking solution.
Thus, the solid phase wells sensitized with various annexin-V
antigens were prepared for use.
Studies were made of the anti-annexin-V monoclonal
antibodies produced by the hybridoma cell lines obtained by the
218J830
above described screening, with respect to their reactivities
with the various annexin-V antigens bound to the plates as solid
phase. Thus, reaction was performed, at room temperature for one
hour, of the respective culture supernatants of hybridoma cell
line clones producing the various anti-annexin-V monoclonal
antibodies (the original solutions) or their purified IgG
fractions (prepared to give a final concentration of 1
microgram/ml) with the respective solid phase wells sensitized
with the native annexin-V antigens derived from the various
l0 animals, followed by washing three times with the washing
solution. A further reaction was carried out with HRPO-labeled
anti-mouse-immunoglobulin antibodies (derived from goats) at room
temperature for thirty minutes, followed by washing four times
with washing solution and reaction with OPD substrate solution
(containing 0.1M phosphate-citrate 4mM H202) at room
temperature for thirty minutes. Then the reaction was
terminated with 2N H2S04 solution for absorbance assay at the
primary wavelength 492nm and the secondary wavelength 690nm
on an ELISA plate reader. Measurements at the primary
2o wavelength 492nm are attributed to the substrate chromogen
produced by the bound labeled-antibody, whereas the
measurements at the secondary wavelength reflects the
detection of flaws or stains on the microtiterplate.
By subtracting an absorbance measurement at the secondary
wavelength from an absorbance measurement at the primary
wavelength, it is possible to determine the true absorbance in
response to the amount of chromogen changed due to the enzyme on
the labeled-antibodies which have been bound to antigen in
76
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proportion to the amount of the antigen bound to the solid-phase.
(9) Cloning
Based on the absorption test using the native human- and
dog-annexin-V and through the above described capture ELISA
system, the selection was made of hybridoma cell line clones
which produce anti-annexin-V monoclonal antibodies to provide a
prospective sensitive sandwich ELISA system for assaying native
annexin-V in combination with the anti-annexin-V monoclonal
antibody produced by hybridoma cell line HCA-627 strain
(deposited under the number FERM PB-5284 at the National
Institute of Bioscience and Human-Technology,the Agency of
Industrial Science and Technology, Japan). The hybridoma cell
line clones were subjected to a cloning operation by means of
limiting dilution to obtain a single clone. In performing the
cloning, thymus cells prepared from BALB/c mice and suspended in
HAT medium was added as feeder cells at 1x106 per well.
(10) Identification of mouse immunoglobulin subclass
Identification of mouse immunoglobulin subclass was made
with respect to the monoclonal antibodies produced by the
2o hybridoma cell lines which have been prepared as having a single
clone by means of the above-mentioned cloning. The culture
supernatants of the respective hybridoma cell lines were assayed
for the mouse immunoglobulin subclass identification using
MONOAb typing kit (available from Zymed Co.).
(11) Establishment of hybridoma cell lines producing anti-
annexin-V monoclonal antibodies
As a result of the first screening for the subject cell
77
2180830
fusion, 200 strains were obtained which were positive in the
absorption test using the native human- and dog-annexin-V
antigen. In addition, the second screening through the above
described capture ELISA were selected 8 clones, which can
establish the sandwich ELISA system in combination with anti-
annexin-V monoclonal antibody produced by hybridoma cell line
HCA-627 strain deposited as an International Deposit under the
number FERM BP-5284. Cloning through the limiting solution was
conducted with regard to said eight elones to establish the
1o respective hybridoma cell clones. Table 4 below gives reaction
specificities of the anti-annexin-V monoclonal antibodies
obtained by the subject cell fusion, No.2.
In table 4 there are given, for comparison, the reactivities
of clones HCA-627, HCA-155H, HCA-507HD and HCA-293HDR obtained in
15~ the previous cell fusion of Example 1.
Different from the clones established in Example 1 (HCA-
115H, HCA-507HD and HCA-293HRD), all the anti-annexin-V
monoclonal antibodies produced by hybridoma cell line clones
established in the subject Example demonstrate to have a high
20 reactivity with the native human- and dog-annexin-V (cf. the
absorption tests in Table 4).
It is also shown from the screening by the capture ELISA
system that the clones are able to provide an efficient and
sensitive sandwich ELISA system in combination with the anti-
25 annexin-V monoclonal antibody produced by hybridoma cell line
clone HCA-627 strain (deposited as an International Deposit under
the number FERM BP-5284) (cf. the Capture Method in Table 4).
In addition, from the studies of the reaction system in
78
2180830
which annexin-V antigens from humans and the various animals are
bound as solid phase it was found that the hybridoma cell line
clones established in the subject Example have a reactivity as
monoclonal antibody totally different from some of the clones
established in Example 1, HCA-115H, HCA-507HD and HCA-293HDR, as
they do not exhibit any reactivity with the antigens of bound
annexin-V as the solid phase derived from such animals (cf. Table
4), although they exhibit a strong reactivity with native
annexin-V in the absorption test, i.e. with the antigens in
1p liquid phase.
As can be seen from the above, the eight strains as screened
by the capture ELISA system, anti-annexin-V monoclonal antibody-
producing clones, are all capable of providing a sandwich ELISA
system in combination with the monoclonal antibody clone HCA-627
15 (Table 4) and recognizing an epitope totally different from one
to be recognized by the clone HCA-627.
As given in Table 4, the immunoglobulin subtypes of the
anti-annexin-V monoclonal antibodies produced by the eight
strains of hybridoma cell line clones established in the subject
2o Example are all IgG type, with respect to the H chain. Regarding
the L chain all the clones have k-chain (cf. Table4).
79
2180830
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2180830
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81
CA 02180830 2004-12-23
EXAMPLE 9: Production of Monoclonal Antibodies
(1) Harvesting of ascitic fluids
In order to obtain anti-annexin-V monoclonal antibodies of a
higher concentration the various hybridoma cell lines are grown
and the resulting respective clones were innoculated, following
washing three times with EMEM culture solution, into the ascitic
cavities of BALH/c mice which have been administered with 2, 6,
10, 14-tetramethyl pentadecane (pristane) in advance into their
ascitic cavities. The ascitic fluids were harvested seven to
fourteen days after the innoculation of the hybridoma cell lines
into the ascitic casities. The ascitic fluids thus obtained were
applied to a centrifuge to remove the cellular and residual
fractions. The supernatant fractions were pooled for keeping at
4°C with the addition of sodium azide as antiseptic.
(2) Purification of monoclonal antibodies from ascitic fluids
The IgG fractions of the monoclonal antibodies were obtained
by applying the ascitic fluids obtained from the hybridoma cell
lines to affinitychromatograpy using ConSep~LC100 device and a
protein-A column (available from Perceptive Co.). The purified
2o IgG fractions of the respective clones were determined for purity
by HPLC using a TSK*gel G3000SW column (available from Tosoh Co.)
and SDS-PAGE using PhastSystem~ (available from Pharmacia Co.).
The result was that all the monoclonal antibodies from the
hybridoma cell lines have a purity of 98$ or more.
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CA 02180830 2004-12-23
EXAMPLE 10 . Studies on ELISA System for Assay of Human
Annexin-V
(1) Preparation of HRPO-labeled anti-annexin-V monoclonal
antibodies
Studies were made on the applicability of the monoclonal
antibodies as estatleshed in the above to an ELISA system for
assay of native human annexin-V.
Thus, as representatives there were selected anti-annexin-V
monoclonal antibodies HDA-676, HDA-907, HDA-1606 and HDA-1937,
which had been produced by the anti-annexin-V monoclonal
antibody-producing hybridoma cell line clones. The selected
monoclonal antibodies were applied to affinity purification using
a ConSepLC 100 device and a protein-A column (available from
Perceptive Co.) to purify the IgG fractions.
(2) Preparation of Horseradish peroxidase (HRPO) labeled
Monoclonal antibodies
(A) Preparation of anti-human-annexin-V monoclonal antibodies
F(ab')2
For labeling the IgG fractions of the anti-annexin-X
monoclonal antibodies, the purified IgG fractions of the
respective anti-annexin-V monoclonal antibodies HDA-676, HDA-907
(deposited as an International under the number FERM BP-5286 at
the National Institute of Bioscience and Human-Technology, the
Agency of Industrial Science and Technology of Japan), HDA-1606
and HDA-1937 were each concentrated by a Centricon 10 centifugal
concentrator (available from Amicon Co.) to give a concentration
of lml from lOmg. The resultant was then dialyzed against O.1M
~ trademark
83
2180830
sodium acetate buffer (pH 3.7) containing 0.2M NaCl as solvent.
Each dialyzed IgG solution was added with pepsin in an amount of
40 of IgG for reaction at 37°C over 2 to 4 hours. On completing
the reaction, the resultant was applied to molecular-sieve
chromatography using a Sephadex G-150 column (available from
Pharmacia Co.) equilibrated with O.1M borate buffer (pH 8.0) to
obtain the F(ab')2 fragment. With every clone, lOmg of the IgG
fraction provided approx. 5mg of F(ab')2 fraction with a high
yield. There was almost no difference in yield among the clones.
to (B) Preparation of anti-annexin-V monoclonal antibodies Fab'-SH
Each of the anti-annexin-V monoclonal antibodies F(ab')2
from HDA-676, HDA-907, HDA-1606 and HDA-1937, as prepared in the
above method (A), was subjected centrifugal concentration by a
Centricon 10 centrifugal concentrator to give lml of each anti-
annexin-V monoclonal antibody F(ab')2 fraction.
To each concentrated fraction lml was added with O.lml of
100mM 2-mercaptoethylamine hydrochloride solution (available from
Kishida Chemicals Co.) for reaction at 370°C for 90 minutes. On
completing the reaction, the resultant was purified using a
2o Sephadex G-25 column for equilibrium gel filtration measuring
l.6cm in diameter and 20cm in length (available from Pharmacia
Co.) and equilibrated with O.1M sodium phosphate buffer (pH 6.0)
containing 1mM EDTA, to fractionate the Fab'-SH fraction.
Following the fractionation, the resultant purified fraction was
pooled for centifugal concentration with a Centricon 10
centifugal concentrator to give a volume of lml. Thus, the
concentrated anti-annexin-V monoclonal antibodies Fab'-SH
fractions were obtained from the respective clones . HDA-676,
84
CA 02180830 2004-12-23
HDA-907, HDA-1606 and HDA-1937.
(C) Preparation of HRPO maleimide
20mg (as protein content) of HRPO (available from
Boehringer Co.) was dissolved in lml of O.1M sodium phosphate
buffer (pH 6.0). The resulting solution was added with I00
microliters of solution of N-hydroxysuccinimi ester
(available from Zeeben Chemicals Co.) dissolved in dimethyl
formanide (DMF ) ( available from Kishida Chemicals Co.) to
give a final concentration of 25mg/1, followed by reaction at
30°C for 60 minutes to prepare maleimide ester of HRPO. On
completing the reaction, the solution was centrifuged for
five minutes at 3000 rpm. The supernatant was applied to
a Sephadex G-25 column for equilibrated gel filtration
(available from Pharmacia Co.) measuring l.6cm in diameter
and 20cm in length, which has been equilibrated with O.1M
sodium phosphate buffer (pH 6.0), in order to purify HRPO
maleimide. The purified fraction of HRPO maleimide was
then subjected to centrifugal concentration by a Centricon 10
centrifugal concentrator to prepare the concentrated fraction of
HRPO maleimide.
(D) Preparation of HRPO-labeled anti-annexin-V monoclonal
antibodies Fab'
The concentrated Fab'-SH fraction of the anti-annexin-V
monoclonal antibody was mixed with the concentrated fraction of
HRPO maleimide at a molar ratio of 1 . 1, for reaction at 4°C for
15 to 24 hours. On completing the reaction, 2-mercaptoethylamine
was added to give a final concentration of 2mM in the reaction
2180830
solution, followed by reaction at 37°C for 20 minutes in order to
block unaltered HRPO maleimide. The resultant was then subjected
to molecular-sieve gelchromatography using a Ultragel ACA44
column (available from Pharmacia Co.) equilibrated with 20mM
sodium phosphate-sodium citrate buffer (pH 5.6) containing 0.15M
NaCl and 2.5M EDTA and measuring l.6cm in diameter and 65cm
in length, for removing unaltered Fab'-SH of the auti-
annexin-V monoclonal autibodies and unaltered HRPO maleimide and
purifying the respective, HRPO-labeled anti-annexin-V
1o monoclonal antibodies Fab' (hereinafter referred to as HRPO-
labeled anti-annexin-V monoclonal antibodies).
(E) Activity determination of HRPO-labeled antibodies
In determining HRPO enzyme activity of the HRPO-labeled
antibodies, 2.98m1 of O.1M sodium phosphate buffer (pH 7.0)
containing 0.2% phenol, 0.5mM hydrogen peroxide and 0.15mg/ml 4-
amino antipyrine was added with 20microliters of the HRPO-
labeled antibody to give a total volume of 3.Oml, followed by
reaction at 37°C for five minutes, and measurement of absorbance
at 500nm by means of the Rate assay. HRPO activity was
determined by measuring the difference in absorbance (0 Abs)
for one minute.
(6) Preparation of anti-annexin-V monoclonal antibody solid
phase coupled wells.
In order to obtain monoclonal antibody for use as solid
phase in an ELISA system for assay of human annexin-V, monoclonal
antibody HDA-627, produced by hybridoma cell line HCA-627 strain
which has been deposited under the number FERM BP-5284 at the
International Depositary Authority for the deposit of
86
2180830
microorganism, was applied to affinity purification using
ConSepLC-100 device and a protein A column (available from
Perceptive Co.) to purify the IgG fraction from the ascitic
fluid.
The IgG fraction of anti-annexin-V monoclonal antibody HCA-
627 was adjusted to have a concentration of 30micrograms/ml with
O.1M sodium phosphate buffer (pH 7.5) containing 0.1% sodium
azide, and distributed in a microtitreplate for ELISA (available
from Nunk Co.) at a rate of 100 microliters per well, for
sensitization at 4°C overnight. Then each well of the
microtitreplate was washed three times with phosphate-buffered
saline (PBS) containing 0.050 Tween 20 surfactant, and then added
with 300 microliters of PHS containing 1% BSA (as the blocking
solution), followed by a further blocking operation at 4°C
overnight, to prepare antibody-plates sensitired with anti-human-
annexin-V monoclonal autibodies (hereinafter referred to as auti-
human-annexin-V monoclonal antibody plates).
(7) Studies on ELISA assay system using anti-annexin-V
monoclonal antibodies
The anti-annexin-V monoclonal antibody plates as prepared
above were added, following discarding the blocking solution,
with phosphate buffer of pH 7,0 (reaction buffer) containing 1~
HSA and 5mM EDTA at a rate of 100 microliters per well. Standard
antigen solutions were prepared so as to give native annexin-V
standard antigen concentrations of 1.563ng/ml, 3.125ng/ml
6.25ng/ml, 12.5ng/ml, 25ng/ml, 50ng/ml and 100ng/ml. The
respective standard antigen solutions were added to the wells at
87
2180830
a rate of 20 microliters per well, followed by stirring and
reaction at room temperature for one hour. On completing the
reaction, each well was washed three times with the washing
solution. Then, to the wells were added the HRPO-labeled anti-
s annexin-V monoclonal antibodies Fab' (clones . HDA-676, HDA-907,
HDA-1606 and HDA-1937 ) at a rate of 100 microliters per well,
. for reaction at room temperature for thirty minutes. Following
the reaction, each well was washed six times with the washing
solution and then added with 100 microliters of OPD substrate
1o solution containing O.1M phosphate-citrate buffer added with
2mg/ml o-phenylenediamine and 4mM H202, for reaction for thirty
minutes. The reaction was terminated by adding 2N H2S04 solution
at a rate of 100 microliters per well. Absorbance was determined'
on an ELISA plate reader at the primary wavelength 492nm and at
15 the secondary wavelength 690nm.
The absorbance herein intended is obtained by subtracting an
absorbance measurement at the secondary wanelength 690nm from
that at the primary wanelength 492nm.
The results of absorbance measurements with respect to the
20 representative native annexin-V standard antigen solutions were
given in Table 5.
88
Z I $oa3o
I
X '~1.-I 00~ O ~ ODl0L(1M
> 00N d1M tf1crM l0
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89
2180830
Fig. 12 illustrates a calibration curve for human annexin-V
obtained by plotting the measurements on the human annexin-V
antigen solutions with standard antigen solution absorbance as
ordinate and human annexin-V antigen concentration as abscissa.
The calibration curve as shown in Fig. 12 is excellent one,
as it demonstrates that the absorbance rises with increasing
concentration of human annexin-V, substantially linearly from a
low concentration up to as high as 100ng/ml.
The calibration curve also indicates that the subject
1o example enjoys a wider dynamic range and hence provides a more
sensitive system, as compared with the calibration curve for
native human annexin-V antigen in which there was employed the
combination of the monoclonal antibody as solid phase produced by
hybridoma cell line clone HCA-627 strain deposited under the
number FERM BP-5284 as an International Deposit with HRPO-
labeled anti-32KP polyclonal Fab' antibody.
It should be noted, however, that the combination of the
anti-annexin-V monoclonal antibody HCA-627 as solid phase coupled
to wells and the HRPO-labeled anti-dog-32KP annexin-V polyclonal
Fab' antibody is not problematic at all in sensitivity for assay
of human annexin-V.
The use of the calibration curve as obtained in the subject
example (Fig. 12) enabled us to assay annexin-V concentration in
the blood, plasma and serum in a highly reproducible and reliable
manner from a low concentration to a high concentration, in which
there was employed a combination of the monoclonal antibody
produced by hybridoma cell line HCA-627 strain deposited as an
International Deposit under the number FERM BP-5284 with the
2180830
HRPO-labeled monoclonal antibody produced by hybridoma cell line
HDA-907 strain also deposited as an International Deposit under
the number FERM BP-5286.
The calibration curve of Fig. 12 was obtained by employing
an extremely low concentration of HRPO-labeled anti-annexin-V
monoclonal antibody HDA-907-Fab' which may have a HRPO activity
of 3 to 4mU/ml. It is thus possible to provide a more sensitive
system.
As can be seen from Table 5 and Fig. l2 on the calibration
curve obtained with the standard antigen, the CV values are
within 3.6% for four times of measurements of the standard
antigen concentration in the range of from a human annexin-V
concentration as low as lng/ml up to as high as 100ng/ml,
demonstrating the system enables a highly reproducible assay of
annexin-V concentration. From the foregoing, the subject ELISA
system using the aforesaid monoclonal antibodies for assay of
annexin-V will be fully satisfactory in use as an ELISA system to
determine native annexin-V concentration in the human blood.
As shown in Figs. 1 through 8, in a case where native human
annexin-V antigen without 8M-urea-treatment was used as standard
antigen in an ELISA system using the combination of the
monoclonal antibodies, the absorbance was very small, for
example, as in Example 1, only about 5o to about 10$ of 8M-urea
treated antigen. Hy contrast, the ELISA system using the
monoclonal antibodies as obtained in the subject example has made
a remarkable improvement in reactivity with native human annexin-
V antigen.
91
2180830
It was found that the HRPO-labeled Fab' antibodies of the
anti-annexin-V monoclonal antibody clones (HDA-676, HDA-907, HDA-
1606 and HDA-1937) having a reactivity with human- and dog-
annexin-V and selected by the screening operation in the subject
example all exhibit a high reactivity with human annexin-V and
therefore provide a good combination with HCA-627 as shown in
Example 1 in obtaining an excellent calibration curve for human
annexin-V antigen concentration.
(8) Possibility of Detection and Quantitative Analysis of
Human Annexin-V in Samples by Sandwich ELISA in which
Anti-Human-Annexin-V Monoclonal Antibody (HCA-627)
and Anti-Native-Human- and Dog-Annexin-V Monoclonal
Antibody Clone (HDA-907) are used
From among possible combinations of the monoclonal
antibodies as prepared above for use in sensitive assay of native
human annexin-V, as a representative was selected the combination
of anti-annexin-V monoclonal antibody HCA-627 as solid phase
coupled to wells and HRPO-labeled anti-annexin-V monoclonal
antibody HDA-907-Fab to study its application to assay of
clinical samples by ELISA.
Thus, into the anti-human-annexin-V monoclonal antibody HCA
. 627-coupled plates was added, after discarding the blocking
solution, reaction buffer solution (sodium phosphate buffer at pH
7.0 containing 1% BSA and 5mM EDTA)at a rate of 100 microliters
per well. An antigen standard solution of native human cardiac
mustle-derived annexin-V (Ong/ml, 1.5625ng/ml, 3.125ng/ml,
6.25ng/ml, 12.5ng/ml, 25ng/ml, 50ng/ml or 100ng/ml) and a human
plasma sample are added each at a rate of 20 microtiters per
92
2180830
well, followed by stirring and reaction for one hour at
room temperature. Each well was washed four times with the
washing solution. Then there was added the HRPO-labeled anti-
annexin-V monoclonal antibody HDA-907-Fab' having been prepared
to have a concentration of 4mU/ml as HRPO enzyme activity, at a
rate of 100 microliters per well for reaction for thirty
minutes at room temperature, followed by washing four times
with the washing solution. After the washing the wells
were added with OPD substrate solution (containing O.1M
phosphate-citrate buffer added with 2mg/ml o-phenylenediamine
and 4mM H202) at a rate of 100 microliters per well, followed
by absorbance measurements at the primary wanelength 492nm
and at the secondary wanelength 690nm on an ELISA plate
reader.
The human plasma samples were determined for native human
annexin-V concentration by reading the calibration curve which
was prepared by plotting the absorbance against varying
concentration of human cardiac muscle-derived annexin-V as
standard antigen. The data for the calibration curve are given
in Table 5 and Fig. l2. As mentioned previously, the absorbance
increased with increasing concentration of the human annexin-V
standard antigen.
(9) Sample dilution tests.
Dilution tests were performed on a plasma sample prepared by
adding purified annexin-V antigen to normal plasma(Sample 1), a
plasma sample with a low value of annexin-V concentration (Sample
2) and a plasma sample with a high value of annexin-V
93
2180830
concentration (Sample 3). Each sample was subjected to a series
of successive doubling dilution. Each series of samples,
including the original sample solution, were assayed for annexin-
V concentration by the ELISA system for annexin-V assay using the
combination of the monoclonal antibody clone HCA-627 as solid
phase coupled to well and the HRPO-labeled monoclonal antibody
clone HDA-907 -Fab'.
The results are given in Table 6, Tables 6-1 through 6-3 and
Fig. l3.
15
25
94
2180830
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95
2180830
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96
2180830
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97
2180830
With Sample 1, a sample prepared by adding annexin-V to
normal plasma, the annexin-V concentration in blood varies with
the dilution rate, forming a straight dilution line directing
toward the origin. With both Sample 2 and Sample 3 (a sample
having a high annexin-V concentration), the annexin-V measurement
varies in a close relationship with the dilution rate, also
forming a substantially straight dilution line directing toward
the origin.
Thus, as it was found that the annexin-V measurement is
closely related with the dilution rate, forming a straight
dilution line directing toward the origin for all samples with or
without annexin-V addition, the subject system can provide an
assay system for quantitating the annexin-V concentration in
samples without interference by possible components in the
plasma.
In addition, for the purpose of confirming the reliability
of the subject system for assay of clinical samples, recovery
tests were conducted by adding human annexin-V as purified
annexin-V antigen to plasma and serum samples.
(10) Human annexin-V recovery tests in ELISA system for
assay of human annexin-V
With respect to the ELISA system as studied above in which
there are used anti-annexin-V monoclonal antibody HCA-627 as
solid phase coupled to wells and HRPO-labeled anti-annexin-V
monoclonal antibody HDA-907-Fab', recovery tests for human
annexin-V were conducted to examine whether purified annexin-V
antigens as added to plasma or serum are recovered without any
influence on the measurements of annexin-V by components present
98
2180830
in the plasma or serum.
(11) Procedure for recovery test
Purified human annexin-V antigen solutions were prepared to
have a concentration of 40.8ng/ml, 14.6ng/ml and 4.7ng/ml. To
each of three human plasma samples and two human serum samples
was added one of the purified human annexin-V antigens having the
three different(high, middle and low) concentrations in a
proportion of 9 volumes of sample to 1 volume of antigen to
observe annexin-V concentration in each sample. The human
annexin-V antigen solution was replaced by buffer solution which
was also added to each sample in the same proportion as in the
case of human annexin-V antigen, so as to observe human annexin-V
concentration in each sample per se. Human annexin-V antigen
concentration as added was determined through assay of a sample-
like solution prepared by adding human annexin-V antigen to
reaction buffer solution instead of the plasma or serum sample
in the same proportion as in such sample.
The concentration of human annexin-V recovered was obtained
by subtracting measured human annexin-V concentration of a sample
2o per se from measured human annexin-V concentration as added, as
given by the following equation(1):
The concentration of annexin-V antigen recovered
(ng/ml) - Measured human annexin-V concentration of a sample
added with the antigen of the respective concentration
(ng/ml) - Measured human annexin-V concentration of the
per se (ng/ml) ..... (1)
The recovery rate at the concentration of annexin-V
99
2180830
recovered was obtained by the following equation (2):
Recovery Rate (%)_ (Concentration of human annexin-V
recovered when added with the antigen of the respective
concentration/Human annexin-V concentration as added)x100
..... (2)
The results of the recovery tests are summarized in Table 7.
15
25
100
2180830
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101
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The result of the recovery test was excellent in the plasma
samples with from a low value (1.27ng/ml) up to a high value
(42.Omg/ml) of annexin-V concentration in sample per se, as the
antigen recovery rate was between 96% and 1090 in the
concentration range of human annexin-V antigen added (from
4.7ng/ml to 40.8ng/ml).
The recovery rate was also excellent with respect to the
serum samples, as it was between 91% and 106% in the
concentration range of human annexin-V antigen added, i.e. from a
low concentration (4.7ng/ml) up to a high concentration
(40.8ng/ml).
As can be seen from the foregoing, the subject ELISA system
for assay of annexin-V has a good prospect of application to the
assay of clinical samples because it has enabled reliable
15~ quantitative analysis without being influenced by components of
the plasma and serum.
EXAMPLE 11: Application of the Present Invention to Analysis
of Blood Sample by Enzyme Antibody Method
(1) Pretreatment of collected blood
Intvavenuously collected whole blood was placed without
delay into a 1.8m1 collecting tube containing 0.2m1 of 3.8%
sodium citrate, followed by mild stirring and centrifugation at
2500 rpm for 10 minute to isolate plasma. The isolated plasma
was immediately or, following after frozen for keeping until use
for keeping, supplied for use in the assay of annexin-V
concentration.
In the case when EDTA salt is used in place of sodium
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citrate, 3 to 5 mM EDTA 2 potassium or EDTA 2 sodium may be
placed in the blood collecting tube to isolate plasma following
centrifugation.
(2) Diagnosis of myocardial infarction
Analysis of blood collected three hours after the onset from
a 72-year-old lady and having no symptom of hemolysis indicated
CK (creatine phosphate enzyme) value of 50U/L, a normal value,
and annexin-V concentration in plasma of 16.4ng/ml, an increased
value. The case was later affirmatively diagnostic of myocardial
infection because of elevation of CK value up to 1108U/L and a
typical abnormality on the electrocardiogram. Annexin-V
concentration in plasma returned to a normal value three days
after the onset of the disease.
This case indicates that the assay of annexin-V
concentration in plasma in the first sampling of blood with no
hemolysis is of use in diagnosis of myocardial infarction.
Annexin-V concentration in plasma of a patient with myocardial
infection is relatively high in an early stage of the disease
(within 6 hours after the onset of attack). From the analysis of
blood samples without homolysis from patients complaining of
pains in the chest, it was later established that 70% of the
cases, where annexin-V concentration in blood was determined to
show lOng/ml or more, is diagnostic of myocardial infarction.
(3) Diagnosis of angina pectoris
Blood with no hemolysis from a 55-year-old man who
complained of a pain in the chest three hours after a first pain,
was collected three and a half hours after the onset and analyzed
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for annexin-V concentration in plasma, with the result that the
concentration was 5.8ng/ml. Analysis was also conducted on blood
samples from the same patient thereafter for CK values in the
blood with the result that the values were 140U/L or less, all a
normal value. Coronary angiography taken later showed a high
degree of stenosis on the right coronary artery. Thus, based
on this fact with reference to the clinical symtoms, the case
was affirmatively diagnozed as angina pectoris.
This case demonstrates that the analysis of blood with no
l0 hemolysis to determine annexin-V concentration in plasma is of
use in the diagnosis of angina pectoris. While annexin-V
concentration in plasma of a patient with annexin-V angina
pectoris is relatively low as compared with a case of myocardial
infarction, approx. 700 of cases, where CK value of a patient
15 complaining of pains in the chest was not diagnostic of angina
pectoris but the annexin-V concentration in plasma showed 5ng/ml
or more, were diagnozed as angina pectoris.
(4) Annexin-V concentration in normal adults
Annexin-V concentration in plasma from blood with no
2o hemolysis of normal males 22 to 75 years of age is in the range
of 0.6 to l.9ng/ml, whereas that of normal females 24 to78 years
of age is in the range of 0.5 to l.7ng/ml, with the average being
l.Ong/ml, the maximum l.9ng/ml and the minimum 0.5ng/ml. There
was observed neither sex-dependent nor age-dependent variations.
25 Hemolysis will bring a higher value of annexin-V
concentration measurement in plasma because annexin-V leaks into
the plasma from the decomposed leukocytes, platelets and
erythrocytes. In treating blood sample following the collection,
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care should therefore be taken not to cause hemolysis to occur,
for example, through use of a fine collecting needle or avoidance
of vigorous shaking of blood samples. Slight hemolysis will give
no substantial influence on the assay of annexin-V concentration
in blood, enabling such assay. Thus, the assay of
annexin-V concentration in plasma is of use in the diagnosis of
myocardial and angina pectoris.
(5) Comparison with example 7
In Example 7 the numerical values of analysis, including the
normal values, are somewhat higher than those in Example 9. This
is because the analysis in Example 7 includes measurement of
annexin-V concentration due to hemolysis which have occurred
during the treatment of the blood samples.
Example 7 therefore suggests that, as long as blood samples
are treated in the same manner, assay of annexin-V concentration
in plasma is of use in the diagnosis of myocardial infarction and
angina pectoris even if hemolysis occurs.
(6) As can be seen from the description of the invention, anti
human-annexin-V monoclonal antibody HCA-627 and anti-human
annexin-V monoclonal antibody HDA-907 obtained by the present
invention are monoclonal antibodies which are capable of
specifically recognizing an annexin-V antigenic molecule through
different antigenic determinants on an identical annexin-V
molecule.
The sandwich ELISA system using the two monoclonal
antibodies enables the assay of annexin-V antigen in a highly
sensitive manner, providing excellent quantitative analysis as
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can be seen from the satisfactory results of the dilution tests
and the recovery tests.
In addition, the two monoclonal antibodies are capable of
cross-reacting with both human-annexin-V antigen and dog annexin-
V antigen. The two cross-reacting monoclonal antibodies are
considered to recognize antigenic determinants common to the
different types of annexin-V antigen molecules. Thus, in
preparing standard antigen for assay annexin-V, such monoclonal
antibodies make it possible to provide less expensive dog-derived
annexin-V without causing the ethical issue with respect to
extraction and purification of human-derived annexin-V.
Industrial Applicability
The present invention is directed to the production of novel
anti-human-annexin-V monoclonal antibodies by culturing hybridoma
cell lines HCA-627 strain (deposited on November 6, 1995 as an
International Deposit under the number FERM BP-5284 at the
National Institute of Bioscience and Human-Technology, the Agency
of Industrial Science and Technology of Japan, at Higashi
1-1-3, Tukuba-City, Ibaraki-Pref., Japan, an International
Depositary Authority for the deposit of
microorganisms) and HDA-907 strain (deposited on November 7, 1995
as an International Deposit under the number FERM HP-5286 at the
National Institute of Bioscience and Human-Technology, the Agency
of Industrial Science and Technology of Japan, at Higashi 1-
1-3, Tukuba-City, Ibaraki-Pref., Japan, an International
Depositary Authority for the deposit of
microorganisms), in which the hybridoma cells were prepared by
extracting lymphocytic plasmablast cells from lymphoid organs of
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mammalian animals such as mice immunized with human annexin-V, an
antigenic protein from human heart, and fusing lymphocytic
plasmablast cells with myeloma cells.
The anti-human-annexin-V monoclonal antibodies obtained in
the present invention are ones capable of recognizing
specifically human annexin-V as the antigenic protein.
In addition to the anti-human-annexin-V monoclonal
antibodies there can be also obtained, according to the present
invention, anti-annexin-V monoclonal antibodies which will cross-
react with annexin-V as antigenic protein derived from various
animals (e. g. beagles and rats).
These cross-reacting anti-annexin-V monoclonal antibodies
are considered to have an ability to recognize antigenic
determinant sites occurring commonly on the annexin-V protein
molecules. Such sites having antigenic determinants common to
the annexin-V protein molecules are important ones for
maintaining the molecular structure of annexin-V reserved among
different animal species.
The use of the anti-human-annexin-V monoclonal antibodies of
the present invention makes it possible to diagnose patients with
ischemic diseases such as myocardial infarction and angina
pectoris by means of ELISA, for example, through the measurement
of human annexin-V concentration in the blood. Thus, although
conventionally thought impossible, myocardial infarction and
angina pectoris can be diagnosed quickly and reliably even at the
early stages of the diseases, and the reliable diagnosis can be
conducted free of influence by rheumatoid factor.
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Another exemplarly advantage of the present invention is
that it can be applied in autopsy or post mortem to stain the
cardiac muscle tissue using the anti-human-annexin-V monoclonal
antibodies so as to detect the distribution of human annexin-V
through the cardiac muscle tissue.
In a further aspect of the present invention, there can be
obtained anti-human-annexin-V monoclonal antibodies which are
different in reactivity and are capable of recognizing different
antigenic determinants on a human annexin-V molecule. By
l0 selecting, from a group of such anti-human-annexin-V monoclonal
antibodies, two different types of anti-human-annexin-V
monoclonal antibodies different from each other in antigenic
determinant for recognition and combining them, it is possible
to determine human annexin-V with a high sensitivity and
15~ reproducibility by means of ELISA.
Thus, by the combination of the anti-human-annexin-V
monoclonal antibodies having different specificities to different
antigenic determinants, preparation is now possible of highly
reliable reagents for the assay of myocardial infarction and
20 angina pectoris.
The anti-human-annexin-V monoclonal antibodies of the
present invention are also suitable for use in tissue-staining,
in addition to their use in the ELISA system for the assay of
human annexin-V. Through the tissue-staining analysis, it is
25 possible to identify the areas in the myocardial tissue of human
heart where human anexin localizes or distributes, and of the
areas where human annexin-V passes from the myocardial tissue due
to ischemia, thereby greatly contributing to molecular
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physiological and other studies. It is also possible to recover
annexin-V molecules of a high purity from the tissue extracts
through the purification by means of affinity chromatography
using the anti-human-annexin-V monoclonal antibodies or the anti-
s annexin-V monoclonal antibodies. Further, identification of
annexin-V molecules can be made by means of Western blotting.
As can be seen from the above, the anti-human-annexin-V
monoclonal antibodies and the anti-annexin-V monoclonal
antibodies obtained from various animals according to the present
invention can be used in molecular and physiological studies on
annexin-V occurring in humans and various animals, thereby making
a great contribution in the wide range of areas including both
basic medicine and clinical medicine.
20
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