U.S. patent application number 11/773393 was filed with the patent office on 2007-11-22 for reagent for determining laminin 5 antigen in biological sample and assay method.
Invention is credited to Masahiko Katayama, Noriko Sanzen, Kiyotoshi Sekiguchi.
Application Number | 20070269835 11/773393 |
Document ID | / |
Family ID | 27347338 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070269835 |
Kind Code |
A1 |
Katayama; Masahiko ; et
al. |
November 22, 2007 |
REAGENT FOR DETERMINING LAMININ 5 ANTIGEN IN BIOLOGICAL SAMPLE AND
ASSAY METHOD
Abstract
A method of determining a laminin 5 antigen in a biological
sample, comprising the steps of bringing an antibody reactive to a
laminin 5 .gamma.2 chain N-terminal fragment into contact with the
biological sample; measuring a reaction of the antibody; and
determining an amount of the laminin 5 antigen based on a
measurement result of the reaction, as well as, a method of
detecting a laminin 5-producing tumor cell, a method of examining
acute respiratory distress syndrome and a method of evaluating
malignancy of a malignant tumor based on the assay method.
Inventors: |
Katayama; Masahiko;
(Tukubashi, JP) ; Sanzen; Noriko; (Aichi-gun,
JP) ; Sekiguchi; Kiyotoshi; (Osaka, JP) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Family ID: |
27347338 |
Appl. No.: |
11/773393 |
Filed: |
July 3, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10487321 |
Feb 17, 2004 |
7256001 |
|
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PCT/JP02/08347 |
Aug 19, 2002 |
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11773393 |
Jul 3, 2007 |
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Current U.S.
Class: |
435/7.2 |
Current CPC
Class: |
C07K 16/18 20130101;
G01N 33/6893 20130101; G01N 2800/38 20130101; G01N 33/57492
20130101; G01N 2333/7055 20130101 |
Class at
Publication: |
435/007.2 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 17, 2001 |
JP |
2001-247685 |
Feb 7, 2002 |
JP |
2002-31181 |
Apr 9, 2002 |
JP |
2002-106468 |
Claims
1. A method of determining .beta.4 integrin expression, comprising
steps of: bringing an antibody reactive to a laminin 5 into contact
with a cell culture supernatant; measuring a reaction of the
antibody; and determining an expression amount of a .beta.4
integrin in the cell based on the measurement result of the
reaction.
2. The method of claim 1, wherein the antibody is a monoclonal
antibody produced from a cell deposited with an accession number
selected from the group consisting of: FERM BP-8133, FERM BP-8134,
FERM BP-8135, FERM BP-8136, FERM BP-8137, FERM BP-8140, and
combinations thereof.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method and a reagent for
determining a laminin 5 antigen in a biological sample.
BACKGROUND ART
[0002] A basal lamina is constituted by an extracellular matrix
component which is mainly a collagen, and exists universally in an
organism. One of macromolecular proteins constituting the basal
lamina is laminin (hereinafter, often abbreviated as LN). The LN is
classified into more than ten kinds based on the structure thereof,
which are different in functions and localized tissues, and is
classified by adding a number at the end, e.g., LN1 or LN2. Every
LN is constituted by a complex formed from three polypeptide chains
each having a different amino acid sequence, and gives a cross-like
molecular form in an electron microscope observation. The three
polypeptide chains are respectively called .alpha. chain, .beta.
chain, and .gamma. chain, and each has several molecular species
(.alpha.1 to .alpha.5, .beta.1 to .beta.3, and .gamma.1 and
.gamma.2).
[0003] In the basal lamia between epithelial cells and a connective
tissue backing the epithelial cells, there exists a cell adhesion
structure peculiar to the epithelial cells. As an extracellular
matrix-constituting protein which exists mainly in the above
structure, laminin 5 is known (hereinafter, often abbreviated as
LN5). Physiological characteristics of LN5 is that it is the only
LN in the above LN group, produced solely from epithelial cells and
that it has an activity to promote adhesion to the basal lamia and
motility of epithelial cells. As for the epithelial cell, it is
known phenomena that it adheres strongly to LN5 and the basal lamia
containing LN5 via a specific receptor called integrin on a cell
membrane of the epithelial cell itself, and that it migrates
aggressively.
[0004] The LN5 is constituted by a complex formed from one .alpha.3
chain, one .beta.3 chain, and one .gamma.2 chain. The .gamma.2
chain, in particular, is considered to be specific to LN5, and is
not included in the other LN molecular species [Dev. Dyn., 218,
213-234 (2000)]. In addition, it is found that LN5 is partially
degraded by a proteolytic enzyme (protease) when secreted from the
epithelial cells, and it is indicated that, especially by shedding
an N-terminal part of the .gamma.2 chain, the remaining molecular
entity of LN5 from which the N-terminal part was released has an
increased activity for promoting cell movement [J. Cell Biol., 148,
615-624 (2000)].
[0005] That is, the amount of LN5 .gamma.2 chain fragments released
from the epithelial cells, etc. reflects an LN5 production amount
in the cells, and it considered to be a potential index for
measuring an epithelial cell motility-promoting activity of LN5 in
an epithelial tissue.
[0006] Recently, a lot of study results reported that an expression
of the LN5 increased in a malignant tumor tissue from epithelial
cells. There are many reports, in particular, that the LN5
expression level correlates well with an invasiveness of a
malignant tumor, and its application as a pathological marker for
the purpose of in vitro diagnosis of cancer is considered to be
possible [J. Natl. Cancer Inst., 91, 1882-1887 (1999) and Cancer,
85, 2315-2321 (1999)].
[0007] However, in the above studies, there is mainly employed a
method in that a pathologic tissue is removed from a body of a
patient through an operation to prepare a section, and then an
expression site of a target protein is immunstained with an
antibody. Such method lacks quantitativeness and versatility in
terms of practical application to in vitro diagnosis in medical
places, and also has a problem of imposing physical strains on a
patient, for example, a surgical operation and a tissue biopsy.
Therefore, an establishment of a simple and quick method of
determining a level of an LN5 antigen in a biological sample, such
as blood, which can be sampled relatively safely is desired.
[0008] Recently, there has been reported an enzyme-linked
immunosorbent assay (ELISA) using two kinds of monoclonal
antibodies to LN5 (a monoclonal antibody BM165 against an .alpha.3
chain and a monoclonal antibody 6F12 against a .beta.3 chain) [J.
Immunol. Meth., 224, 161-169 (1999)]. However, this report shows no
example that an LN5 antigen in a biological sample is detected by
ELISA.
DISCLOSURE OF THE INVENTION
[0009] An object of the present invention is to provide a method of
determining an LN5 antigen in a small amount of a biological sample
accurately and simply, using an antibody that specifically binds to
LN5.
[0010] The inventors of the present invention focused on the
degradation of an LN5.gamma.2 chain in various examinations of a
method of determining an LN5 antigen in a biological sample. Thus,
the inventors of the present invention prepared monoclonal
antibodies recognizing respective chains of LN5, and
immunologically analyzed an LN5 antigen in culture supernatants of
epithelial tumor cells. As a result, it was found that many
epithelial tumor cells secreted LN5 with its .gamma.2 chain shed
into culture supernatants thereof. Furthermore, it was confirmed
that the prepared monoclonal antibody against the .gamma.2 chain
was reactive to a .gamma.2 chain fragment released due to the
protease degradation of LN5.
[0011] As a result of further analyses, a plurality of kinds of
monoclonal antibodies reactive to the released .gamma.2 chain
fragment were prepared, to prepare a sandwich-type immunological
assay reagent using two different monoclonal antibodies reactive to
the .gamma.2 chain fragment. It was also found that an LN5 antigen
in a blood serum and a blood plasma could be determined efficiently
by using the assay reagent using two different monoclonal
antibodies reactive to the LN5 .gamma.2 chain fragment.
[0012] As explained above, the .gamma.2 chain is a constituent
specific to LN5, and not included in the other LN molecular
species. That is, the assay reagent using the antibody against the
.gamma.2 chain fragment allows to determine any molecule of a
.gamma.2 chain fragment, a nonfragmented .gamma.2 chain, a .beta.3
chain-.gamma.2 chain complex, or an .alpha.3 chain-.beta.3
chain-.gamma.2 chain complex in a biological sample. It is
therefore considered that the reagent determines most of LN5
antigens and thus may provide a useful index which reflects the
LN5-producing amount in an epithelial tissue for grasping a
pathological condition.
[0013] It was also experimentally examined what kind of in vivo
phenomena an increase of an LN5 antigen in a biological sample
reflects. Eleven kinds of human pancreatic tumor cell lines were
cultured in a liquid medium in the presence of a 10% bovine fetal
serum for a given period, and LN5 concentrations in the culture
supernatants and expression levels of various integrins which are
adhesion molecular receptors expressed were compared. Six kinds of
LN5-producing lines obviously had a tendency to express higher
expression level of .beta.4 integrin in comparison to five kinds of
LN5-nonproducing lines. That is, it was experimentally proved that
the in vivo LN5 antigen-producing amount may be an index for the
.beta.4 integrin expression level in epithelial cells in an in vivo
epithelial tissue.
[0014] Further, a human tumor producing LN5 was transplanted to a
nude mouse and it was thus found that a growth of the tumor and the
LN5 concentration in a blood serum correlated each other, thereby
elucidating that determining LN5 in a blood serum allows monitoring
of the growth of an LN5-producing tumor.
[0015] In addition, it was hypothesized that an LN5 metabolism in
an organism increases in pulmonary inflammatory disease, based on
the fact that much LN5 is included especially in a pulmonary
epithelial tissue among organs in an organism. Under the
hypothesis, the LN5 antigen concentration in blood plasma specimens
sampled from patients suffering from acute respiratory distress
syndrome (hereinafter, abbreviated as ARDS) and in blood plasma
specimens sampled from healthy subjects was determined in
accordance with the above method. It was found that the
concentration in blood was obviously higher for a group of the
patients suffering from acute respiratory distress syndrome, in
comparison with the group of healthy subjects, thereby discovering
that the method was extremely useful for clinical diagnosis of the
disease.
[0016] The present invention was completed based on the above
findings. That is, the present invention provides the
following.
[0017] 1. A method of determining a laminin 5 antigen in a
biological sample, comprising the steps of:
[0018] bringing an antibody reactive to a laminin 5 .gamma.2 chain
N-terminal fragment into contact with the biological sample;
[0019] measuring a reaction of the antibody; and
[0020] determining an amount of the laminin 5 antigen based on a
measurement result of the reaction.
[0021] 2. A method according to item 1, wherein the antibody is a
monoclonal antibody produced from a cell deposited with an
accession number of FERM BP-8136, FERM BP-8133, or FERM
BP-8134.
[0022] 3. A reagent for determining a laminin 5 antigen in a
biological sample, comprising an antibody reactive to a laminin 5
.gamma.2 chain N-terminal fragment.
[0023] 4. A reagent for determining a laminin 5 antigen according
to item 3, wherein the antibody is a monoclonal antibody produced
from a cell deposited with an accession number of FERM BP-8136,
FERM BP-8133, or FERM BP-8134.
[0024] 5. A monoclonal antibody produced from a cell deposited with
an accession number of FERM BP-8136, FERM BP-8133, or FERM
BP-8134.
[0025] 6. A method of detecting a laminin 5-producing tumor cell,
comprising the steps of:
[0026] determining a laminin 5 antigen in a biological sample by
the method as defined in item 1 or 2; and
[0027] detecting a laminin 5-producing tumor cell based on an assay
result of the laminin 5 antigen.
[0028] 7. A detection reagent for detecting a laminin 5-producing
tumor cell, comprising the reagent for determining as defined in
item 3 or 4.
[0029] 8. A method of examining acute respiratory distress
syndrome, comprising the steps of:
[0030] determining a laminin 5 antigen in a biological sample by
the method as defined in item 1 or 2; and
[0031] examining acute respiratory distress syndrome based on an
assay result of the laminin 5 antigen.
[0032] 9. An examination reagent for examining acute respiratory
distress syndrome, comprising the reagent for determining as
defined in item 3 or 4.
[0033] 10. A method of evaluating malignancy of a malignant tumor,
comprising the steps of:
[0034] determining a laminin 5 antigen in a biological sample by
the method as defined in item 1 or 2; and
[0035] evaluating malignancy of a malignant tumor based on an assay
result of the laminin 5 antigen.
[0036] 11. An examination reagent for evaluating malignancy of a
malignant tumor, comprising the reagent for determining as
described in item 3 or 4.
[0037] 12. A method of determining .beta.4 integrin expression,
comprising the steps of:
[0038] bringing an antibody reactive to a laminin 5 into contact
with a cell culture supernatant;
[0039] measuring a reaction of the antibody; and
[0040] determining an expression level of a .beta.4 integrin in the
cell based on the measurement result of the reaction.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is a graph showing reactivity of each of monoclonal
antibodies (19, 57, 2B10, and 8C2) to LN5 from culture mediums of
MNK45 cells and A-172 cells. The term "antiserum" refers to a
reaction of anti-human LN5 mouse polyclonal antibody. The term
"PBS" refers to a reaction without addition of an antibody. The
horizontal axis of the graph indicates an absorbance value in
ELISA.
[0042] FIG. 2 shows a result of an immunoprecipitation experiment
of the LN5 antigen in an HT-1080 cell culture medium using each
antibody. Numerical values in left indicate migration positions of
a molecular weight marker (kDa). The term "supernatant" refers to a
migration image of total proteins. The term "gel only" refers to a
migration image of proteins from gel used in the
immunoprecipitation. The term "pAb LN-5" refers to a migration
image of proteins imminoprecipitated using a polyclonal antibody to
human LN5. The term "pAb .gamma.2" refers to a migration image of
the proteins immunoprecipitated using a polyclonal antibody to
human LN5 .gamma.2 chain. The term "mAb .beta.3" refers to a
migration image of the proteins immunoprecipitated using a
monoclonal antibody to a human LN5 .beta.3 chain.
[0043] FIG. 3 shows a result of an immunoprecipitation experiment
of the LN5 antigen in an A-431 cell culture medium using each
antibody. Numerical values in left indicate migration positions of
a molecular weight marker (kDa). The term "supernatant" refers to a
migration image of total proteins. The term "gel only" refers to a
migration image of proteins from gel used in the
immunoprecipitation. The term "pAb LN-5" refers to a migration
image of proteins imminoprecipitated using a polyclonal antibody to
human LN5. The term "pAb .gamma.2" refers to a migration image of
the proteins immunoprecipitated using a polyclonal antibody to
human LN5 .gamma.2 chain. The term "mAb 33" refers to a migration
image of the proteins immunoprecipitated using a monoclonal
antibody to a human LN5 .beta.3 chain.
[0044] FIG. 4 shows a measurement result of LN5 concentration in
culture supernatants of eleven kinds of human pancreatic tumor cell
lines and MKN45 human gastric tumor cell line. The term
".alpha.3.gamma.2" refers to values measured by ELISA using a
combination of an 8C3 (anti-.gamma.2 monoclonal
antibody)-immobilized plate and an enzyme-labeled 2B10
(anti-.alpha.3 chain monoclonal antibody). The term
".beta.3.gamma.2" refers to values measured by ELISA using a
combination of an 8C3 (anti-.gamma.2 monoclonal
antibody)-immobilized plate and an enzyme-labeled 57 (anti-.beta.3
chain monoclonal antibody). The term ".alpha.3.beta.3" refers to
values measured by ELISA using a combination of a 57 (anti-.beta.3
monoclonal antibody)-immobilized plate and an enzyme-labeled 2B10
(anti-.alpha.3 chain monoclonal antibody).
[0045] FIG. 5 shows a result of comparing an expression level of
each integrin molecule from eleven kinds of the human pancreatic
tumor cell lines. There are shown the mean value and standard
deviation of each integrin expression level of the LN5-producing
lines (KP-2, KP-3, AsPC-1, HPAC, SUIT-2, and BxPC-3) and the mean
value and standard deviation of each integrin expression level of
the LN5-nonproducing lines (KP-1, KP-4, MIApaca-II, PSN-1, and
PANC-1).
[0046] FIG. 6 shows a result of a Western blot experiment of the
LN5 antigen in a BxPC-3 cell culture medium using each monoclonal
antibody. Numerical values in right refer to migration positions of
a molecular weight marker (kDa). The term "8C2" refers to a
reactive antigen by the 8C2 monoclonal antibody; the term "2B10"
refers to a reactive antigen by the 2B10 monoclonal antibody; the
term "18-4" refers to a reactive antigen by the KP2-LN5-8C2E 18-4
monoclonal antibody; the term "12-1" refers to a reactive antigen
by the KP2-LN5-8C2E 12-1 monoclonal antigen; and the term "control"
refers to a reactive antigen by a control antibody. The three kinds
of 8C2, KP2-LN5-8C2E 12-1, and KP2-LN5-8C2E 18-4 are obviously
reactive to a fragment having a molecular weight of about
50,000.
[0047] FIG. 7 shows a calibration curve in an
electrochemiluminescence immunoassay using the KP2-LN5-8C2E 18-4
monoclonal antibody-immobilized beads and a ruthenium-labeled
KP2-LN5-8C2E 12-1 monoclonal antibody.
[0048] FIG. 8 shows changes in the weight of a primary tumor
(.circle-solid.) and in the concentration of an LN5 antigen in a
blood serum (.largecircle.) of a nude mouse transplanted with the
HPAC cell, at 2, 3, 4, and 5 weeks after the transplantation.
[0049] FIG. 9 shows a graph showing changes in the weight of a
primary tumor (.circle-solid.) and in the concentration of an LN5
antigen in a blood serum (.largecircle.) of a nude mouse
transplanted with the MIApaca-II cell, at 2, 3, 4, and 5 weeks
after the transplantation.
[0050] FIG. 10 shows changes in the weight of a primary tumor
(.circle-solid.) and in the concentration of an LN5 antigen in a
blood serum (.largecircle.) of a nude mouse transplanted with the
KP-1 cell, at 2, 3, 4, and 5 weeks after the transplantation.
[0051] FIG. 11 is shows distribution, a mean value, and a standard
deviation of an LN antigen concentration in a blood plasma for 27
ARDS patients and 15 healthy persons. The ARDS patients were
classified into 19 survived cases (mild cases) and 8 non-survived
cases (serious cases).
[0052] FIG. 12 shows distribution of the LN5 antigen concentration
in a blood serum for 31 patients suffering from benign digestive
disease, 9 patients suffering from intraductal papillary-mucinous
tumor, and 155 patients suffering from malignant digestive
disease.
BEST MODE FOR CARRYING OUT THE INVENTION
[0053] An LN5 .gamma.2 chain N-terminal fragment in the present
description refers to an N-terminal side fragment among the
fragments generated by degradation of a .gamma.2 chain. The
N-terminal fragment is usually a fragment which may be released
into a biological sample, i.e., a fragment which may exist in a
biological sample, which is a liquid sample, or in a liquid
fraction prepared from a biological sample. Examples of such an
N-terminal fragment include a fragment of a molecular weight of
about 50,000, containing the domains IV and V of the .gamma.2
chain.
[0054] An antibody reactive to an LN5 .gamma.2 chain N-terminal
fragment in the present description refers to an antibody that is
immunochemically bound to an LN5 .gamma.2 chain N-terminal
fragment, i.e., an antibody showing an antigen-antibody
reaction.
[0055] A biological sample in the present description is a body
fluid such as a blood serum, a blood plasma, cerebrospinal fluid,
ascites, urine, tears, sweat, or saliva, excrement, tissue extract,
etc. There is no particular limitation provided that the biological
sample is normally sampled at a medical institution, etc., but a
blood specimen from a subject, such as a blood serum or a blood
plasma is particularly preferable.
[0056] An LN5 antigen in the present description is an antigen with
which an antibody reactive to an LN5 .gamma.2 chain N-terminal
fragment reacts immunochemically, and includes an LN5 .gamma.2
chain and an N-terminal fragment thereof and a complex including a
.gamma.2 chain.
[0057] The step of bringing a biological sample into contact with
an antibody reactive to an LN5 .gamma.2 chain N-terminal fragment,
the step of measuring the reaction of the antibody, and the step of
determining a level of the LN5 antigen based on a measurement for
the reaction, in the method of determining an LN5 antigen in a
biological sample of the present invention, may be similar to those
in a method of determining an antigen using a general
immunochemical method, except using an antibody reactive to an LN5
.gamma.2 chain N-terminal fragment as the antibody.
[0058] In the assay method of the present invention, an LN5
.gamma.2 chain N-terminal fragment which is degraded to be secreted
in a body fluid and thereby can be determined in a biological
sample, is preferably determined by means of an immunochemical
method using an antibody, reactive to the LN5 .gamma.2 chain
N-terminal fragment preferably a monoclonal antibody, more
preferably a monoclonal antibody produced from a cell deposited
with an accession number of FERM BP-8136, FERM BP-8133, or FERM
BP-8134.
[0059] Examples of the immunochemical method include, but not
particularly limited to, a latex agglutination method, a
competition method, and a sandwich method. In the competition
method and the sandwich method, an LN5 antigen or an antibody is
labeled with a labeling substance such as a radioactive label, an
enzyme label, an electrochemiluminescence (ECL) label, or a
fluorescence label to be detected by a method suitable to the
label. Preferably, the sandwich method using an antibody labeled
with an enzyme label or an electrochemiluminescence label, more
preferably, the sandwich method using an antibody labeled with an
electrochemiluminescence label is suitable.
[0060] An assay reagent of the present invention, which is an assay
reagent used in the assay method of the present invention, includes
at least an antibody reactive to the LN5 .gamma.2 chain N-terminal
fragment, preferably a monoclonal antibody, more preferably a
monoclonal antibody produced from a cell deposited with an
accession number of FERM BP-8136, FERM BP-8133, or FERM BP-8134,
and may optionally include a standard antigen of LN5 and a
detection reagent for a label depending on the kind of the label,
e.g., an enzyme substrate in case of the enzyme label. The assay
reagent may also include an appropriate buffer. An antibody may be
bonded to a carrier or a label acceptable for use in an assay
reagent to form an immobilized antibody or a labeled antibody. The
assay reagent of the present invention may be manufactured by
selectively using a technique generally used for manufacturing a
reagent containing an antibody. In the case that the assay reagent
is composed of a plurality of constituents, the reagent may be in a
kit.
[0061] The antibody reactive to the LN5 .gamma.2 chain N-terminal
fragment is not particularly limited provided that the antibody is
the one having an ability to bind to the LN5 .gamma.2 chain
N-terminal fragment, such as a polyclonal antibody derived from an
antiserum obtained by immunizing a purified LN5 .gamma.2 chain
fragment with an experimental animal. However, a mouse monoclonal
antibody is the best because of its excellent reaction specificity
and its inexpensive manufacturing cost.
[0062] The method of preparing a monoclonal antibody will be
explained below. A human LN5 may be purified in accordance with a
method already reported (Int. J. Cancer, 76, 63-72 (1998)). The
purified LN5 or the purified LN5 .gamma.2 chain fragment is
dissolved in a saline as an immunological antigen to be
administered to an experimental animal with an appropriate
adjuvant. Examples of the experimental animal include one available
from a breeder, and especially a Balb/C mouse may be frequently
used, but not limited thereto. The administration of the antigen is
repeated several times at intervals of several weeks. At 3 days
after the last administration, the spleen is extracted from the
immunized animal. The extracted spleen is dispersed into single
cells, which are subjected to cell fusion with pre-cultured mouse
myeloma cells in the presence of a polyethylene glycol reagent.
After the cell fusion, selective culture using a drug is conducted
to select only a hybridoma cell producing a monoclonal antibody
reactive to the LN5 .gamma.2 chain N-terminal fragment. After that,
several times of cloning operations of an antibody-producing cell
bring about a complete monoclonal antibody-producing cell. The
specificity of the monoclonal antibody can be identified, for
example, by a Western blot analysis, etc. of the purified antigen
used for immunization which is separated by electrophoresis
[Antibodies: a Laboratory Manual, by Ed Harlow & D. Lane, Cold
Spring Harbar Laboratory Press (1988)].
[0063] The antibody produced from the cell deposited to
International Patent Organism Depositary of National Institute of
Advanced Industrial Science and Technology with the accession
number of FERM BP-8136, FERM BP-8133, or FERM BP-8134 may be used
as the monoclonal antibody.
[0064] The ECL method will be explained below. While various
measurement methods are known for the ECL measurement of an LN5
antigen in a biological sample, as one having remarkable simplicity
and high quantitativeness, the sandwich method simultaneously using
a first antibody-immobilized magnetic bead and a second antibody
labeled with a ruthenium complex is preferable. In this case, the
first antibody and the second antibody desirably bind to different
antigenic determinants respectively while the antibodies
specifically react to the LN5 .gamma.2 chain fragment.
[0065] The standard antigen may be prepared by purifying a human
tumor cell line, e.g., a culture supernatant of a human tumor cell
line and then by determining its protein concentration by
absorbance analysis, etc., and may be used by being diluted with a
diluent to an appropriate concentration on use.
[0066] An example of the assay procedure will be explained below,
but the present invention is not limited thereto.
[0067] An LN5 .gamma.2 chain fragment antibody (the first antibody)
is immobilized on a commercially available magnetic bead. The
immobilization may be via a covalent bond or a noncovalent bond.
Then, for inhibiting nonspecific binding of other molecules with
the magnetic bead, a blocking protein such as milk casein is
adsorbed to the beads.
[0068] A standard LN5 antigen solution, the concentration of which
is known in advance, or a subjective biological sample is then
added thereto and the whole is stirred for a given time. After the
LN5 antigen in the sample is adsorbed to the surface of an
antibody-bound particle, the particle is washed. Next, another
anti-LN5 .gamma.2 chain fragment antibody (the second antibody)
labeled with a chemiluminescent complex such as ruthenium is added
in an appropriate concentration. The sample is stirred for a given
time to form a complex of the first antibody, the LN5 antigen, and
the second antibody on the magnetic bead.
[0069] Then, after the bead has been washed, the bead is placed
between a pair of electrodes in a special device and is charged
with electricity to make the complex as a marker emit light for
measuring the emission intensity. At this point, an emission level
corresponding to the level of the ruthenium marker is obtained.
Comparison of the emission level of the subjective biological
sample with the emission level of the standard sample using a
calibration curve, etc. allows one to know the LN5 antigen level in
the subjective biological sample accurately.
[0070] The method of detecting an LN5-producing tumor cell, the
method of examining acute respiratory distress syndrome, and the
method of determining the malignancy of a malignant tumor of the
present invention are characterized by determining an LN5 antigen
in a biological sample, to conduct detection, examination, and
determination based on the assay result.
[0071] The assay of the LN5 antigen in a biological sample is
conducted in accordance with the assay method of the present
invention. Using the assay result for the LN5 antigen as an index
allows to detect an LN5-producing tumor cell, to examine acute
respiratory distress syndrome, and to determine the malignancy of a
malignant tumor. For example, if the quantitative level of the LN5
antigen is higher than the normal value, it may be recognized that
an LN5-producing tumor cell is detected, that acute respiratory
distress syndrome is detected, and that the malignancy of a
malignant tumor is determined to be high.
[0072] The assay reagent of the present invention may be used in
applications including detection of an LN5-producing tumor cell,
examination of acute respiratory distress syndrome, and
determination of the malignancy of a malignant tumor.
[0073] The present invention also provides a method of determining
the .beta.4 integrin expression of a cell. The method includes the
steps of: bringing a cell culture supernatant into contact with an
antibody reactive to an LN5; measuring the reaction of the
antibody; and determining an expression level of a .beta.4 integrin
in the cell based on the measurement for the reaction.
[0074] The step of bringing a cell culture supernatant into contact
with an antibody reactive to an LN5 and the step of measuring the
reaction of the antibody may be similar to those of the method of
determining the antigen using a general immunochemical method,
except using an antibody reactive to LN5. The immunochemical method
may be similar to that explained above for the assay method of the
present invention.
[0075] The antibody reactive to LN5 is preferably a monoclonal
antibody. Examples of such a monoclonal antibody include a
monoclonal antibody produced from a cell deposited with an
accession number of FERM BP-8133, FERM BP-8134, FERM BP-8135, FERM
BP-8136, FERM BP-8137, or FERM BP-8140.
[0076] The measurement of the reaction of the antibody reactive to
LN5 reflects the LN5-producing level in a culture supernatant, and
the .beta.4 integrin expression level is determined by using the
measurement as an index.
EXAMPLES
[0077] The present invention will be described in reference with
detailed examples hereinafter, but is not limited thereto.
Example 1
Preparation of Monoclonal Antibody Against LN5
[0078] The human LN5 was purified in accordance with a method
already reported [Int. J. Cancer, 76, 63-72 (1998)]. Specifically,
a human breast tumor cell line MKN45 (obtained from Japan Cancer
Research Resources Bank) was cultured in a liquid medium containing
a 2.5% bovine fetal serum and 100 ng/mL phorbol 12-myristate
13-acetate (manufactured by from Wako Pure Chemical Industries,
Ltd.). Then, a culture supernatant was sampled, followed by
removing cell components by centrifugation. After that, a protease
inhibitor was added to the supernatant. The culture supernatant was
applied to a column packed with a gel to which an anti-LN5 .gamma.2
chain polyclonal antibody was immobilized, followed by sufficiently
washing with a phosphate buffer saline (PBS). The LN5 bound to the
column was eluted using a 0.1 M glycine-hydrochloride buffer (pH
3.0). The LN5 eluted from the column was promptly neutralized and
was then sufficiently dialyzed against PBS in a dialysis tube. The
LN5 preparation thus purified was analyzed by polyacrylamide
electrophoresis to identify the presence of an .alpha.3 chain, a
.beta.3 chain, and a .gamma.2 chain.
[0079] The purified LN5 was intraperitoneally administered to
Balb/C female mice (6 weeks of age) in an amount of 0.015 mg per
mouse with Complete Freund's Adjuvant (manufactured by Sigma
Aldrich Corporation). After 3 weeks, the same amount of LN5 was
intraperitoneally administered with Incomplete Freund's Adjuvant
(manufactured by Sigma Aldrich Corporation).
[0080] After an additional 3 weeks, only purified LN5 was
administered to the mice in an amount of 0.03 mg per mouse. After 3
days from the last immunization, spleens were removed from the
mice. The operations after that were conducted in a clean bench.
The removed spleens were dispersed using a mesh, and were mixed
with pre-cultured Sp2/0-Ag14 mouse myeloma cells, followed by cell
fusion in the presence of 50% polyethylene glycol 1500
(manufactured by Boehringer Mannheim). The fused hybridoma cells
were separated into several 96-well microculture plates and were
cultured in an RPMI 1640 liquid medium containing a 10% bovine
fetal serum and an HAT reagent (manufactured by ICN Biomedicals
Inc.) for 1 to 2 weeks. During this period, only a hybridoma cell
stably producing a monoclonal antibody survived while the nonfused
myeloma cells and mouse spleen cells were dead.
[0081] The cell producing a monoclonal antibody against LN5 was
selected by ELISA using an antigen-immobilized plate. Specifically,
a culture supernatant was sampled at the time when a colony of a
hybridoma cell had sufficiently grown, and was added into a 96-well
microplate (manufactured by Nalge Nunc International) to which an
immunogen adsorbed in an immobilized phase, to be allowed to react
with the monoclonal antibody in the supernatant. A peroxidase
conjugate of the second antibody reactive to a mouse IgG
(manufactured by American Qualex, Inc.) was then added to the
supernatant in an appropriate concentration. The plate was washed
after a given period of time, and then a solution of
o-phenylenediamine enzyme substrate color developer (manufactured
by Wako Pure Chemical Industries, Ltd.) and hydrogen peroxide was
added. Four lines of the target monoclonal antibody-producing
hybridoma were selected based on the presence or absence of color
development. The selected 4 lines were cloned several times and
were named 19, 57, 8C2, and 2B10. The large scale manufacture of
each monoclonal antibody was carried out according to an ordinary
method by intraperitoneally inoculating the hybridoma to a mouse,
and then by purifying the antibody from ascites of the mouse by
affinity chromatography using protein G fixed Sepharose gel
(manufactured by Pharmacia).
[0082] The obtained monoclonal antibody was examined for
specificity using LN5 produced from a human breast carcinoma cell
line MKN45, a human glioblastoma cell line A-172, a human
leiomyosarcoma cell line HT-1080, and a human epidermal carcinoma
cell line A-431. The A-172 cell was obtained from American Type
Culture Collection, and the HT-1080 cell and the A-431 cell were
obtained from Japan Cancer Research Resources Bank.
[0083] Each cell was cultured in a tissue culture flask using an
RPMI 1640 medium containing a 10% bovine fetal serum, and a culture
supernatant was sampled. LN5 was purified from the culture
supernatant by the above-mentioned method. The cell line A-172 is
known to secret a .gamma.2 chain singly, and the purified LN5
mainly contains the .gamma.2 chain. On the other hand, the LN5 from
the culture supernatant of the MKN45 cell contains each of the
.alpha.3 chain, the .crclbar.3 chain, and .gamma.2 chain in
approximately the same amount. The reactivity of the 4 monoclonal
antibodies to the two kinds of LN5s was compared by ELISA in
accordance with the above-described method. As a result, because
only 8C2 strongly reacted with the two, the 8C2 monoclonal antibody
was found to recognize the LN5 .gamma.2 chain (FIG. 1).
[0084] Furthermore, when culturing each of the cell lines HT-1080
and A-431, secreted proteins were labeled with an express .sup.35S
protein labeling mix reagent (manufactured by NEN Life Science).
The culture supernatant was subjected to immunoprecipitation of
antigen proteins using the 4 monoclonal antibodies. Specifically,
to a given amount of the .sup.35S-labeled cell culture supernatant,
an antibody was added and further a gel to which an anti-mouse IgG
antibody was immobilized was added. After stirring gently for a
while, the gel was separated by low-speed centrifugation and was
heated as it was in an SDS-containing sample solution, followed by
separating the antigen protein adsorbing to the gel by
electrophoresis.
[0085] The cell line HT-1080 is known to express no .alpha.3 chain
and to secrete a complex of a .beta.3 chain and a .gamma.2 chain.
On the other hand, the cell line A-431 is known to express all the
chains. As a result of the electrophoresis analysis for the
immunoprecipitate, both of the 19 and the 57 strongly reacted with
the antigen (.beta.3 chain), which migrated to a position of a
molecular weight of around 145 kDa, from an HT-1080 supematant.
While, the 8C2 strongly reacted with the antigen (.gamma.2 chain),
which migrated to a position of a molecular weight of around 155
kDa, from the HT-1080 supernatant (FIG. 2). The 2B10 reactive
antigen was not detected in the HT-1080 supernatant. Any of the 4
monoclonal antibodies precipitated a 3-chain complex of the
.alpha.3 chain, the .beta.3 chain, and the .gamma.2 chain from the
A-431 cell culture supernatant (FIG. 3).
[0086] The above experimental results elucidated that the 19 and
the 57 reacted with the .beta.3 chain, that the 2B10 reacted with
the .alpha.3 chain, and that the 8C2 reacted with the .gamma.2
chain. Because both of the 19 and the 57 reacted with the .beta.3
chain similarly, only the 57 was used in the examination below.
Example 2
Determining the Ln5 Level in Human Tumor Cell Culture Supernatant
Using Monoclonal Antibody
[0087] Eleven human pancreatic tumor cell lines (KP-1, KP-2, KP-3,
KP-4, AsPC-1, HPAC, SUIT-2, MIApaca-II, PSN-1, BxPC-3, PANC-1
:MIApaca-II (CRL-1420), PANC-1 (CRL-1469), AsPC-1 (CRL-1682), and
HPAC (CRL-2119) were obtained from ATCC. The KP-1 to KP-4, the
SUIT-2, and the BxPC-3 were gratefully given from Dr. Akihiro
Funakoshi of National Kyushu Cancer Center. The PSN-1 was
gratefully given from Dr. Kazuto Nishio of National Cancer Center)
were cultured in an RPMI-1640 medium supplemented with a 10% bovine
fetal serum in a culture flask, to sample the culture supernatant
thereof at a stage of sufficient growth of the lines. The culture
supernatant was aseptically sealed and stored at 4.degree. C. until
use.
[0088] Among the 3 monoclonal antibodies (57, 2B10, and 8C2) which
were prepared and the specificity of which was identified in
Example 1, the 57 and 2B10 monoclonal antibodies were labeled with
horseradish peroxidase (manufactured by Boehringer Mannheim) by a
periodate method. Each of the labeled monoclonal antibodies was
sufficiently dialyzed against PBS, was filter-sterilized using a
membrane filter (manufactured by Nihon Millipore), and was then
stored at 4.degree. C. The purified monoclonal antibody of 57 or
8C2 was diluted with PBS to a concentration of 0.01 mg/mL and was
added in an amount of 0.2 mL to each well of a MAXISOAP 96-well
microplate (manufactured by Nalge Nunc International K.K.) to
immobilize each antibody. Those plates were sealed so as not to be
dried and stood at 4.degree. C. for 12 hours or more. Then, the
antibody solution in the plate was discarded, and a 1% skimmed
milk-containing PBS solution was added into each well in an amount
of 0.2 mL, followed by standing for 1 hour to block the plate.
[0089] A human LN5 standard sample (derived from the MKN45 cell)
was diluted with a 1% skim milk-containing PBS solution to prepare
solutions having concentrations of 1000, 500, 250, 125, 62.5,
31.25, and 15.625 ng/mL. The blocking solution was discarded from
the antibody-immobilized plate in which the blocking was completed.
Then, each of the human LN5 level standard samples and already
sampled 11 human pancreatic tumor cell culture supernatants was
added into a well in an amount of 0.1 mL. After standing at room
temperature for 2 hours, the solution was discarded and the wells
were washed with PBS sufficiently. The peroxidase-conjugate of the
monoclonal antibody 57 or 2B10 described above was appropriately
diluted with a 1% skim milk-containing PBS to add into each well in
an amount of 0.1 mL. After standing at room temperature for 2
hours, the solution was discarded, and the wells were sufficiently
washed with PBS. An ABTS enzyme substrate solution (manufactured by
Roche Diagnostics K.K.) was added to each well in an amount of 0.1
mL and was stood at room temperature for 10 minutes with stirring
sometimes. A 2 mM aqueous solution of sodium azide was additionally
added to each well in an amount of 0.1 mL, followed by sufficiently
mixing to terminate the reaction. The absorbance at 492 nm of each
well was measured using the T-max microplate reader (manufactured
by Molecular Devices Corporation) and a calibration curve was made
based on the absorbance of the standard sample, to calculate the
LN5 level in each culture supernatant.
[0090] As a result of the measurement, the LN5 production in a part
of the human pancreatic tumor cell lines was confirmed, and the LN5
measured value by ELISA using the 8C2 monoclonal antibody as an
immobilized antibody was obviously lower than the LN5 measured
value by ELISA not using the 8C2 monoclonal antibody (FIG. 4).
However, in the culture supernatant of the cell line MKN45, such
tendency as value-decreasing was not detected. The MKN45-derived
LN5 is known not to fragment at the .gamma.2 chain thereof, so that
the 8C2 was considered to have reaction specificity to a fragment
site of the .gamma.2 chain to be released.
[0091] Also, the 11 kinds of human pancreas cancer culture cells
were collected from the flasks with a trypsin treatment
simultaneously with the sampling of the culture supernatant. After
the cell number had been counted, the cells were added into
microtubes with the same cell number respectively. To the
respective tubes, the monoclonal antibodies against the human
.alpha.1-, .alpha.2-, .alpha.3-, .alpha.5-, .alpha.6-, .beta.1-,
.beta.3-, and .beta.4 integrins (all manufactured by CHEMICON
International, Inc.) were added, and the tubes were stood for 30
minutes with ice-cooling, followed by washing the cells with PBS.
To the tubes, the FITC-labeled second antibody were further added
and the tubes were stood again for 30 minutes with ice-cooling.
Finally, after washing with PBS, a 1% paraformaldehyde fixing
solution was added to fix the cell surface antigen. Each fixed cell
suspension was passed through the FACScan cell analyzer
(manufactured by Nippon Becton Dickinson Company Ltd.) to measure
the fluorescence intensity mean value of the cell surface, thereby
determining each integrin expression level in the surface of a cell
membrane. The fluorescence intensity mean value was represented as
a ratio to the background mean value (Relative Mean
Fluorescence).
[0092] Among the 11 human pancreatic tumor cell lines, the integrin
expression of the 6 LN5-producing lines and the 5 LN5-nonproducing
lines were compared (FIG. 5). While the expression of .beta.1
integrin, etc. in the LN5-producing lines was approximately in
equal to that in the LN5-nonproducing lines, the expression of
.beta.4 integrin in the LN5-producing lines obviously increased.
This indicates that the production level of LN5 may be an index for
.beta.4 integrin expression.
Example 3
Preparation of Monoclonal Antibody to Degraded Fragment of .gamma.2
Chain
[0093] (1) Purification of Degraded Fragment of .gamma.2 Chain
[0094] A purified 8C2 monoclonal antibody was cross-linked to a
bromcyan-activated Sepharose (manufactured by Pharmacia) in
accordance with an instruction manual to prepare an
antibody-immobilized gel. A human pancreatic tumor cell line KP-2
or BxPC-3 was cultured in a large amount in an RPMI-1640 liquid
medium containing a 10% bovine fetal serum in a tissue culture
flask. After sufficient cultivation, 1 L of the culture supernatant
thereof was sampled and centrifuged to remove cell components. The
culture supernatant was diluted with PBS and was then applied to a
column packed with the 8C2 monoclonal antibody-immbolized gel.
After the column had been sufficiently washed with PBS, the LN5
antigen bound to the column was eluted with 8 M urea-containing
PBS. The eluted fraction was dialyzed against PBS in a dialysis
tube to remove urea. After a sufficient dialysis, the fraction was
concentrated using a Centricon centrifugal concentrator
(manufactured by Amicon), thereby preparing a purified human LN5
antigen.
[0095] The purified product was separated by 4 to 20% gradient-SDS
polyacrylamide electrophoresis (manufactured by Daiichi Pure
Chemicals Co., Ltd.). The gel was protein-stained using a quick CBB
stain solution (manufactured by Wako Pure Chemical Industries,
Ltd.) to analyze the molecular weight of the LN5 antigen.
Additionally, the LN5 antigen was electrically transferred from the
electrophoresed gel to an Immobilon-P membrane (manufactured by
Nihon Millipore) and was immuno-stained with the 8C2 monoclonal
antibody (FIG. 6). The degraded product to which the 8C2 monoclonal
antibody reacted had an estimated molecular weight of around
50,000, and was considered to be a fragment derived from an
N-terminal (N-terminal fragment) of a .gamma.2 chain, which was
suggested to be released by a proteolytic cleavage in a previous
report (Science, 277, 225-228 (1997)). The purified human LN5
.gamma.2 chain N-terminal fragment obtained here was separated in
tubes without preservatives and was stored under freezing until
use.
[0096] (2) Preparation of Monoclonal Antibody to Decgraded Fragment
of .gamma.2 Chain
[0097] A novel monoclonal antibody was prepared using the
freeze-stored purified .gamma.2 chain N-terminal fragment derived
from a KP-2 cell as an immunogen. The preparation of the monoclonal
antibody was conducted in accordance with the method described in
Example 1. Newly obtained were two kinds of monoclonal antibodies
KP2-LN5-8C2E 12-1 (hereinafter, abbreviated as 12-1) and
KP2-LN5-8C2E 18-4 (hereinafter, abbreviated as 18-4). It was
confirmed by a Western blot analysis that both the monoclonal
antibodies were reactive to the .gamma.2 chain fragment having a
molecular weight of around 50,000 similarly to 8C2 (FIG. 6).
[0098] Each monoclonal antibody was manufactured in a large amount
by obtaining a culture supernatant of a hybridoma cell using a
Hybridoma-SFM serum-free medium (manufactured by GIBCO Industries,
Inc.) and conducting affinity purification using a protein
A-immobilized gel. It was confirmed by a Mouse-Typer Isotyping
Panel reagent (manufactured by Bio-Rad Laboratories, Ltd.) that the
obtained monoclonal antibodies were each of an IgG1 type.
[0099] The obtained monoclonal antibody-producing cells (12-1,
18-4, 2B10, 8C2, 19, and 57) were deposited to International Patent
Organism Depositary of National Institute of Advanced Industrial
Science and Technology (address: AIST Tsukuba Central 6, 1-1-1
Higashi, Tsukuba, Ibaraki, Japan, 305-8566) with the accession
numbers of FRPM P-18465, FRPM P-18466, FRPM P-18467, FRPM P-18468,
FRPM P-18469, and FRPM P-18470, respectively, on Aug. 16, 2001, and
were converted to international accession under the Budapest Treaty
on Aug. 1, 2002 (Aug. 5, 2002 for 57), being given the accession
numbers of FERM BP-8133, FERM BP-8134, FERM BP-8135, FERM BP-8136,
FERM BP-8137, and FERM BP-8140, respectively.
[0100] (3) Assay of LN5 Degraded Products in Biological Sample
[0101] The two kinds of LN5 .gamma.2 chain monoclonal antibodies
obtained in (2) (12-1 and 18-4) were prepared as purified IgGs.
[0102] The 18-4 monoclonal antibody purified product was diluted
with PBS to a final concentration of 0.2 mg/mL. A suspension (0.25
mL) of Dynabeads M-450 epoxyl (manufactured by Dynal Biotech) was
added to 1 mL of the antibody solution, and sealed in a
polypropylene container followed by gently stirring at room
temperature for 4 hours. The mixture was then stood at 4.degree. C.
for around 12 hours for stabilization of the bonds. Then, 2 mL of a
PBS solution containing 1% skim milk, 0.1% sodium azide, and 2 mM
EDTA (hereinafter, abbreviated as SM/PBS) was added to the
antibody-bound beads, for the purpose of blocking the surplus
binding sites in the bead surfaces. The mixture was stood as it was
for around 12 hours to stabilize the blocking. Then, the beads were
washed twice with PBS and was diluted with the SM/PBS solution in
20-fold and stored in a liquid state at 4.degree. C. until use.
[0103] A solution of 12-1 antibody purified product with a final
concentration of 6 mg/mL was prepared. About 2 mg of ruthenium
(manufactured by IGEN International, Inc.) was added to 1 mL of the
antibody solution and stood at room temperature for 2 hours. Then,
a mixture (2 mL) of 0.2 M glycine/PBS (pH 7.8) was added to block
the surplus reactive sites. The ruthenium-labeled antibody solution
was applied to ultrogel AcA44 gel chromatography and eluted with
PBS containing 0.1% sodium azide to separate the labeled antibody
which had been first eluted. The ruthenium labeled 12-1 monoclonal
antibody thus prepared was stored at 4.degree. C. until use as it
was.
[0104] A chicken serum (manufactured by JRH Biosciences, Inc.) and
the SM/PBS were mixed in equal amounts and used as a diluent for
LN5 standard products and a diluent for labeled antibodies
(hereinafter, abbreviated as Diluent). The MKN45 cell-derived
purified LN5 obtained in Example 1 above was used as the standard
products and diluted with the Diluent in LN5 concentrations of
1000, 500, 250, 125, 62.5, 31.25, and 15.625 ng/mL to prepare
standard product solutions. Also, the each ruthenium-labeled
antibody was diluted with the Diluent in 100-fold. Measurement of a
biological sample was conducted using an automatic clinical
analyzer (Picolumi 8220: manufactured by Sanko Junyaku Co, Ltd.).
The standard product, a blood serum, and a blood plasma were each
collected by 0.2 mL and added into dedicated reaction tubes, which
were mounted to a dedicated reaction tube rack. The prepared
antibody-bound beads and the labeled antibody were also contained
in dedicated vessels, which were mounted to the automatic clinical
analyzer. The process of automatic measurement was as follows. At
first, 0.025 mL of the antibody-bound beads were added to the
reaction tube and stirred intermittently for 9 minutes for carrying
out the reaction with the first antibody. After removing the
solution from the reaction tube by suction, washing with the
washing solution was carried out twice. After washing, 0.2 mL of
the ruthenium-labeled antibody solution was added and stirred
intermittently for 9 minutes for carrying out the reaction with the
second antibody. After removing the solution from the reaction tube
by suction, washing with the washing solution was carried out
twice. After the washing, 0.3 ML of emission electrolytic solution
was added to measure the emission level.
[0105] A calibration curve was constructed based on the results for
the standard antigens measured simultaneously to calculate the
antigen level. The emission count number only for the Diluent was
set as a blank value. Satisfactory measurements were possible in an
antigen concentration range of 1000 to 15.625 ng/mL (FIG. 7).
Example 4
Ln5 Antigen in Blood Serum from Nude Mouse Transplanted with Human
Tumor
[0106] Orthotopic transplantation of human pancreatic tumor cell
into nude mouse
[0107] Three human pancreatic tumor cell lines (HPAC, MIApaca-II,
and KP-1) were each cultured in an RPMI-1640 medium supplemented
with a 10% bovine fetal serum in a culture flask, and only cells
were collected by a trypsin-EDTA solution treatment at a
sufficiently grown stage. The cells were well washed and were
orthotopically transplanted into pancreases of 8 week-aged nude
mice (manufactured by Charles River Japan Inc.) under anesthesia.
The mice transplanted with cells were bred in a clean room. At 2,
3, 4, and 5 weeks after the transplantation, three mice were
randomly selected to sample blood from their hearts under ether
anesthesia. The sampled whole blood was stood at 4.degree. C.
overnight and was then subjected to centrifugation to separate only
sera. The separated sera were stored under freezing until
measurement. Furthermore, the mice were dissected after the blood
sampling to extract portions of primary pancreatic tumor to be
weighed.
[0108] (2) Measurement of LN5 Degraded Products in Mouse Serum
[0109] The LN5 antigens in the mice sera sampled in the previous
item were measured in accordance with the detection method
described in (3) in Example 3. The measurements revealed that the
LN5 antigen derived from the transplanted cell was obviously
released in the blood of the nude mouse transplanted with the HPAC
cell. Those measurements also confirmed that the concentration of
the LN5 antigens in blood increased in correlation with the
enlargement of the primary tumor (FIG. 8).
[0110] Further, the enlargement of the primary tumor was recognized
in the nude mouse transplanted with the MIApaca-II cell having no
LN5 antigen-production ability, while the increase in the
concentration of the LN5 antigen in blood was not detected at all
(FIG. 9).
[0111] Further, the experimental results in Example 2 have
confirmed that the KP-1 cell has no ability to secrete the .alpha.3
chain and the .beta.3 chain, and thus the cell has been considered
to produce no LN5 antigen. However, in this examination, an
increase in the LN5 antigen concentration correlating to the
enlargement of the primary tumor was detected in the blood of the
KP-1-transplanted nude mouse (FIG. 10). Therefore, it was shown
that the KP-1 cell is a cell having a characteristic of producing a
.beta.2 chain monomer of LN5, and it was thus shown that the LN5
antigen assay in accordance with the present assay method is useful
for detecting a tumor of a .gamma.2 chain monomer
producing-type.
[0112] The above results have shown that the present assay method
allows one to monitor the production of various kinds of LN5
antigens in a primary tumor.
Example 5
LN5 Antigen concentration in blood of Patients Suffering from ARDS
(Acute Respiratory Distress Syndrome)
[0113] From the fact that a lung epithelial tissue is especially
rich in LN5 among internal organs, the inventors hypothesized that
the internal LN5 metabolism in a lung inflammatory disease
increases. Thus, the LN5 antigen concentrations in blood plasma
specimens sampled from healthy subjects and ARDS patients were
determined in accordance with the assay method described in (3) in
Example 3.
[0114] FIG. 11 shows the LN5 antigen concentrations in a blood
plasma of 15 healthy persons, 19 survived ARDS patients, and 8
non-survived ARDS patients. The LN5 antigen concentrations in a
blood plasma were 60.8 ng/mL, 158 ng/mL, and 250.9 ng/mL,
respectively, showing that the concentrations in blood obviously
increased in the group of ARDS patients. This finding confirmed
that the LN5 antigen would be a novel marker in blood for diagnosis
of ARDS and determination of the disease severity thereof.
Example 6
LN5 Antigen Concentrations in Blood of Patients Suffering from
Various Digestive Organ Cancers
[0115] The inventors hypothesized that LN5 is highly produced from
epithelial malignant tumor cells. Thus, LN5 antigen concentrations
in serum specimens sampled from 31 patients suffering from benign
digestive diseases (8 diabetes patients and 25 chronic pancreatitis
patients), 9 patients suffering from intraductal papillary-mucinous
tumors, and 155 patients suffering from malignant digestive
diseases (36 pancreatic carcinoma patients, 18 pancreatic carcinoma
patients (with liver metastasis), 10 gastric cancer patients, 16
gallbladder and bile duct cancer patients, and 75 hepatic cancer
patients) were determined in accordance with the assay method
described in (3) in Example 3.
[0116] FIG. 12 shows the LN5 antigen concentrations in blood sera
of 8 diabetes patients, 25 chronic pancreatitis patients, 9
patients suffering from intraductal papillary-mucinous tumor
diseases, 36 pancreatic carcinoma patients, 18 pancreatic carcinoma
patients (with liver metastasis), 10 gastric cancer patients, 16
gallbladder and bile duct cancer patients, and 75 hepatic cancer
patients. Mean values of the LN5 antigen concentrations in blood
sera were 54.2 ng/mL, 50.6 ng/mL, 35.0 ng/mL, 68.3 ng/mL, 165.9
ng/mL, 111.0 ng/mL, 136.0 ng/mL, and 121.9 ng/mL, respectively,
showing that the concentrations in blood obviously increased in the
group of the patients suffering from various digestive organ
cancers. Additionally, there was detected a tendency of increasing
the concentration in patients with liver metastasis. The detection
confirmed that the LN5 antigen would be a novel marker in blood for
determination of the malignancy of digestive organ cancers.
INDUSTRIAL APPLICABILITY
[0117] The present invention provides the reagent and the method of
determining an LN5 antigen in a biological sample. Thus, it becomes
possible to detect a tumor cell producing an LN5, to examine acute
respiratory distress syndrome, and to determine the malignancy of a
malignant tumor.
* * * * *