U.S. patent application number 11/165348 was filed with the patent office on 2005-10-27 for human lung adenocarcinoma-related monoclonal antibody and antigen and immunaossay method which uses the same.
This patent application is currently assigned to Fujirebio Inc.. Invention is credited to Tsuji, Kouichi, Uwano, Takashi, Yoshida, Yoshihiro.
Application Number | 20050238652 11/165348 |
Document ID | / |
Family ID | 26392856 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050238652 |
Kind Code |
A1 |
Tsuji, Kouichi ; et
al. |
October 27, 2005 |
Human lung adenocarcinoma-related monoclonal antibody and antigen
and immunaossay method which uses the same
Abstract
The monoclonal antibody produced by a hybridoma cell strain
TRD-L1, -L2 or -L3 which is obtained by the fusion between mouse
myeloma cells and spleen cells of a mouse immunized with a human
lung adenocarcinoma cell secretion component reacts in a specific
fashion with a glycoprotein antigen that has a molecular weight of
200 kD or more (SDS-PAGE) and is present in human lung
adenocarcinoma cells. It can be used efficiently in cancer
diagnosis.
Inventors: |
Tsuji, Kouichi;
(Ashigarakami-gun, JP) ; Yoshida, Yoshihiro;
(Ashigarakami-gun, JP) ; Uwano, Takashi;
(Ashigarakami-gun, JP) |
Correspondence
Address: |
BUCHANAN INGERSOLL PC
(INCLUDING BURNS, DOANE, SWECKER & MATHIS)
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
Fujirebio Inc.
Tokyo
JP
|
Family ID: |
26392856 |
Appl. No.: |
11/165348 |
Filed: |
June 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11165348 |
Jun 24, 2005 |
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09350899 |
Jul 12, 1999 |
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6919435 |
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09350899 |
Jul 12, 1999 |
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08913315 |
Sep 12, 1997 |
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6015680 |
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08913315 |
Sep 12, 1997 |
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PCT/JP96/00613 |
Mar 13, 1996 |
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Current U.S.
Class: |
424/184.1 |
Current CPC
Class: |
C07K 16/3023 20130101;
G01N 33/57423 20130101; A61P 35/00 20180101; Y10S 530/828 20130101;
C07K 14/435 20130101 |
Class at
Publication: |
424/184.1 |
International
Class: |
G01N 033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 13, 1995 |
JP |
7-52260 |
Apr 20, 1995 |
JP |
7-94071 |
Claims
1-9. (canceled)
10. A monoclonal antibody or antigen binding fragment binding to a
glycoprotein antigen which is produced from a human pulmonary
adenocarcinoma cell and has a molecular weight of 200 kD or more
determined by SDS-PAGE, the glycoprotein antigen reacting with PNA
lectin and not reacting with GNA lectin, SNA lectin and DSA
lectin.
11. The monoclonal antibody or antigen binding fragment according
to claim 1, wherein a sugar chain terminal structure of the
glycoprotein antigen has Gal (1-3) GalNAc structure and does not
have Mannose, NeuAc (2-6) Gal and Gal (1-4) GlcNAc structures.
12. The monoclonal antibody or antigen binding fragment according
to claim 1, wherein the antibody or fragment is of IgM isotype.
13. The monoclonal antibody or antigen binding fragment according
to claim 1, wherein the antibody or fragment is a fragment selected
from the group of a Fab, Fab(ab).sub.2 and a Fv fragment.
14. The monoclonal antibody or antigen binding fragment according
to claim 1, wherein the antibody or fragment directly or indirectly
is attached to a detectable label.
15. An immunoassay method for diagnosis of human pulmonary
adenocarcinoma, the method comprising: contacting a sample of a
subject with a monoclonal antibody or antigen binding fragment
binding to a glycoprotein antigen which is produced from the human
pulmonary adenocarcinoma cell and has a molecular weight of 200 kD
or more determined by SDS-PAGE, the glycoprotein antigen reacting
with PNA lectin and not reacting with GNA lectin, SNA lectin and
DSA lectin; and measuring binding amount obtained on the basis of
the contacting between the antibody or fragment and the
glycoprotein antigen in the sample.
16. The immunoassay method according to claim 6, wherein a sugar
chain terminal structure of the glycoprotein antigen has Gal (1-3)
GalNAc structure and does not have Mannose, NeuAc (2-6) Gal and Gal
(1-4) GlcNAc structures.
17. The immunoassay method according to claim 6, wherein the sample
is human serum or human tissue.
18. The immunoassay method according to claim 6, wherein the
antibody or fragment directly or indirectly is attached to a
detectable label.
19. The immunoassay method according to claim 6, wherein the
immunoassay method is a sandwich enzyme assay method.
20. The immunoassay method according to claim 9, wherein the label
is an enzyme.
Description
TECHNICAL FIELD
[0001] Human lung carcinoma is classified into four principal
histological types. That is, lung adenocarcinoma, squamous cell
carcinoma, small cell carcinoma and large cell carcinoma. This
invention relates to a monoclonal antibody having. reactivity with
human lung adenocarcinoma, to a glycoprotein antigen recognizable
by this antibody and to a detection method and a therapeutic drug
of human lung adenocarcinoma in which the monoclonal antibody is
used. Also, this invention can be used in the diagnosis and
treatment of human lung adenocarcinoma and is applicable to the
field of diagnostic drugs and therapeutic drugs of lung
adenocarcinoma.
BACKGROUND ART
[0002] As a detection method of lung adenocarcinoma, a method has
been known in which human lung adenocarcinoma is detected by
measuring it using antibodies for carcinoembryonic antigen (CEA)
and sialyl SSEA-1 (SLX) known as tumor markers. However, in the
diagnostic method which detects these tumor markers, not only the
positive ratio on lung adenocarcinoma is 40 to 50% but also they
are detected in other tissue type of lung carcinoma, benign lung
diseases and other organ cancers including colon cancer, stomach
cancer and breast cancer. On the other hand, Maimonis P. et al.
(Cancer Res., 50, 6738(1990)) have reported a monoclonal antibody
DF-L1 obtained using a lung adenocarcinoma tissue as the immunogen.
However, the antigen which is detected using this antibody is
present in sera of patients of not only lung adenocarcinoma but
also squamous cell carcinoma, breast cancer and the like.
[0003] Thus, the monoclonal antibodies so far developed can hardly
be regarded as diagnostic means specific for lung adenocarcinoma
and are nor sufficient for screening, definite diagnosis and
monitoring of metastasis, recidivation. In addition, lung cancers
have different properties in terms of cell proliferation,
development and the like, due to different tissue type of each lung
cancer. Because of this, the corresponding therapeutic method
varies depending on each tissue type. In consequence, it is
extremely important to find an antigen and an antibody peculiar to
lung adenocarcinoma for the diagnosis of lung adenocarcinoma, and
furthermore, for the treatment of lung adenocarcinoma.
DISCLOSURE OF THE INVENTION
[0004] Taking the aforementioned actual circumstances into
consideration, the inventors of the present invention have
conducted intensive studies on lung adenocarcinoma cells and found
as the result a high molecular weight glycoprotein which is
peculiarly present in the culture broth of an established lung
adenocarcinoma cell line Calu-3 (ATCC HTB-55) and in sera of lung
adenocarcinoma patients but is hardly detectable in normal human
sera, from glycoproteins which are present in the culture broth of
the established lung adenocarcinoma cell line Calu-3 (ATCC HTB-55)
and can perform cross reaction with the monoclonal antibody DF-L1
prepared by Maimonis P. et al. (Cancer Res., 50, 6738 (1990)).
Also, hybridomas TRD-L1, TRD-L2 and TRD-L3 were established using
this glycoprotein as the immunogen, and antibodies produced by
these cells were obtained. Thereafter, the present invention has
been accomplished by further finding that, when said monoclonal
antibodies are used, a lung adenocarcinoma-originated antigen can
be isolated and said antigen can be measured accurately.
[0005] The present invention is as follows.
[0006] (1) A monoclonal antibody which binds to a glycoprotein
antigen that has a molecular weight of 200 kD or more (SDS-PAGE),
exists in human lung adenocarcinoma cells and is secreted by human
lung adenocarcinoma.
[0007] (2) The monoclonal antibody according to the above item (1)
wherein said glycoprotein antigen is an antigen which further
reacts with MAA lectin and PNA lectin but does not react with GNA
lectin, SNA lectin and DSA lectin.
[0008] (3) The monoclonal antibody according to the above item (1)
or (2) wherein its isotype is IgM.
[0009] (4) The monoclonal antibody according to any one of the
above items (1) to (3) wherein it is produced by a hybridoma cell
strain TRD-L1 (original designation FERM P-14878 assigned by
National Institute of Bioscience and Human Technology, Agency of
Industrial Science and Technology, Higashi 1-1-3, Tsukuba-shi,
Ibaraki, Japan, which has been transferred to the authority on Feb.
8, 1996, under Budapest Treaty (designation FERM BP-5383)), TRD-L2
(designation FERM P-14879 assigned by National Institute of
Bioscience and Human Technology, Agency of Industrial Science and
Technology) or TRD-L3 (designation FERM P-14880 assigned by
National Institute. of Bioscience and Human Technology, Agency of
Industrial Science and Technology) obtained by the fusion between
mouse myeloma cells and spleen cells of a mouse immunized with a
human lung adenocarcinoma cell secretion component.
[0010] (5) The monoclonal antibody according to any one of the
above items (1) to (4) wherein the antibody is an Fab, F(ab).sub.2
or Fv fragment.
[0011] (6) The monoclonal antibody according to any one of the
above items (1) to (5) wherein it is linked with a marker which can
generate a detectable signal.
[0012] (7) The monoclonal antibody according to the above item (6)
wherein said marker is an enzyme, a radioactive material, a
fluorescent material or a metal colloid illuminant.
[0013] (8) A glycoprotein antigen that has a molecular weight of
200 kD or more (SDS-PAGE), exists in human lung adenocarcinoma
cells and is secreted by human adenocarcinoma.
[0014] (9) The glycoprotein antigen according to the above item (8)
wherein it further reacts with MAA lectin and PNA lectin but does
not react with GNA lectin, SNA lectin and DSA lectin.
[0015] (10) An iunmunoassay method for cancer diagnosis use which
comprises using the monoclonal antibody of any one of the
aforementioned items (1) to (7).
[0016] (11) The immunoassay method for cancer diagnosis use
according to the above item (10) wherein the glycoprotein antigen
of the aforementioned item (8) or (9) is measured using the
monoclonal antibody of any one of the aforementioned items (1) to
(7).
[0017] (12) An immunoassay kit for use in internal cancer diagnosis
to specify cancers and their metastatic regions, which uses the
monoclonal antibody of any one of the aforementioned items (1) to
(7).
[0018] (13) The glycoprotein antigen of the aforementioned item (8)
or (9) which is a cancer vaccine.
[0019] (14) A monoclonal antibody in which a drug for cancer
treatment use is linked to the monoclonal antibody of any one of
the aforementioned items (1) to (7).
[0020] (15) A method for treating lung adenocarcinoma, which
comprises administering a cancer vaccine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows photographs of electrophoresis as schematic
illustration, in which FIG. 1(A) shows a result of PAS staining and
FIG. 1(B) shows a result of immunoblotting.
[0022] FIG. 2 shows results of the measurement of an antigen in
sera of normal volunteers and patients of lung adenocarcinoma,
benign lung diseases and other cancers, detected at an absorbance
of 492 nm in Inventive Example 6.
[0023] FIG. 3 shows results of the absorbance measurement in which
antigens recognizable by DF-L1 or TDR-L1 and prepared from cancer
cells and normal volunteer sera are measured, wherein (A) is a case
of the use of TDR-L1 and (B) is a case of the use of DF-L1.
[0024] FIG. 4 shows specificity of measuring systems in which the
TRD-L1 antibody is used, wherein FIG. 4(A) shows specificity in
lung diseases, and FIG. 4(B) in lung cancers and FIG. 4(C) in other
cancers.
[0025] FIG. 5 to FIG. 9 shows results of the measurement of CEA, CA
19-9, CA 125, CA 50 and CA 15-3, obtained in Inventive Example
8.
OUTLINE OF THE INVENTION
[0026] The following describes the present invention in detail.
First, mouse spleen cells prepared using a glycoprotein as the
immunogen which is present in the culture broth of the established
lung adenocarcinoma cell line Calu-3 (ATCC HTB-55) and can perform
cross reaction with the monoclonal antibody DF-L1 are subjected to
cell fusion with mouse myeloma cells, thereby preparing hybridomas,
subsequently selecting a hybridoma capable of producing a
monoclonal antibody having low reactivity with a protein
recognizable by DF-L1 which is present in normal human serum and
having high reactivity with a protein that exists in the culture
broth of Calu-3 (ATCC HTB-55). This hybridoma is further cultured
in a medium or administered into the abdominal cavity of mice to
effect formation of ascitic fluid. The present invention provides a
monoclonal antibody obtained from the culture broth or ascitic
fluid and an antigen which can be recognized by this antibody. It
also provides a method for the measurement of said antigen making
use of said monoclonal antibody.
[0027] More illustratively, it provides a monoclonal antibody which
is produced by a hybridoma TRD-L1, TRD-L2 or TRD-L3, belongs to the
class IgM and reacts with lung adenocarcinoma cells, said
monoclonal antibody being able to recognize a glycoprotein having a
molecular weight of 200 kD or more. The following describes a
production method of the monoclonal antibody of the present
invention in detail.
[0028] (1) Preparation of Immunogen
[0029] An established lung adenocarcinoma cell line, such as Calu-3
(ATCC HTB-55) for example, is cultured in RPMI 1640 medium or MEM
medium and then the culture supernatant fluid is recovered. After
removing insoluble matter from the thus recovered culture
supernatant fluid by centrifugation or using a filter, this is
applied to a monoclonal antibody DF-L1-linked affinity
chromatography column to effect adsorption of the antigen in the
culture supernatant fluid. After subsequent washing with PBS, the
elution is effected using 3 M KSCN. After the elution, dialysis and
concentration are carried out and then the lung adenocarcinoma
established cell-originated antigen comprised of a glycoprotein
fraction of 200 kD or more in molecular weight is recovered using a
gel chromatography column and used as the immunogen.
[0030] (2) Immunization of Animals and Preparation of Hybridoma
Cells
[0031] Mice of 3 to 10 weeks of age, preferably 6 weeks of age, are
immunized using the just described antigen derived from the lung
adenocarcinoma established cell line as the immunogen to prepare
hybridoma cells from spleen lymph node and peripheral blood. With
regard to the immunization method, the immunogen (1 to 100
.mu.g/animal) is administered, together with an appropriate
adjuvant such as Freundis complete adjuvant, Freundis incomplete
adjuvant or pertussis vaccine, subcutaneous or into the abdominal
cavity of the animals. Thereafter, the immunogen is administered 2
to 5 times at intervals of 1 to 2 weeks. In subjecting to cell
fusion, the immunogen is administered to the immunized mice (1 to
100 .mu.g/animal) 3 to 4 days prior to the fusion treatment and
then spleen tissues are excised to prepare spleen cells.
[0032] (3) Myeloma Cells
[0033] An established cell line is used as the myeloma cells. For
example, though not particularly limited, 8-azaguanine-resistant
mouse (BALB/c) myeloma cell strains P3-X63Ag8-U1, SP2/O--Ag4,
PC-X63-Ag8 and the like are desirable.
[0034] (4) Cell Fusion
[0035] Cell fusion is effected by mixing myeloma cells and antibody
producer cells generally in RPMI medium, MEM medium or the like
medium. They are mixed in such a ratio that the number of cells
becomes antibody producer cells:myeloma cells=5-10:1 and
centrifuged and then the thus precipitated cells are thoroughly
loosened and subjected to fusion using polyethylene glycol while
stirring or using an electrofusion apparatus. The polyethylene
glycol to be used may preferably have a molecular weight of 1,000
to 6,000. Selection of fused cells is carried out using HAT
medium.
[0036] (5) Selection of Hybridoma
[0037] Selection of hybridoma is carried out and checking culture
medium of hybridomas by the following enzyme immunoassay or the
like means. Since this method is merely an example of the checking
method, the present invention is not restricted by this method, and
various alterations and modifications can be made.
[0038] Enzyme Immunoassay
[0039] The aforementioned Calu-3 cancer cell-derived antigen
(protein, 0.5 to 5 .mu.g/ml) is dispensed in 50 to 200 .mu.l
portions into wells of a 96 F aminoplate, a normal human
serum-derived antigen (protein, 0.5 to 5 .mu.g/ml) is distributed
in 50 to 200 .mu.l/well portions in the same manner, and their
respective bonding is effected using glutaraldehyde in the usual
way. After removing the supernatant fluid by suction, each of the
plates is washed with PBS-Tween 20 and block (blocking) of the
bound residues is carried out by adding 300 .mu.l of 1% BSA-PBS.
After removing the supernatant fluid by suction, the plate is
washed with PBS-Tween 20. A hybridoma culture supernatant fluid is
dispensed in 40 to 100 .mu.l portions into wells of the plate and
allowed to undergo the reaction overnight at 4.degree. C. or for 1
hour at 37.degree. C. After removing the supernatant fluid by
aspiration, the plate is washed with PBS-Tween 20. After the
washing, an anti-mouse immunoglobulin antibody-peroxidase conjugate
is dispensed in 100 .mu.l portions into the wells to carry out 1
hour of reaction at 37.degree. C. After the reaction, the plate is
again washed, dispensed with 0.1 M citrate buffer (pH 4.2)
containing O-phenylenediamine dihydrochloride or
tetramethylbenzidine and hydrogen peroxide in 100 .mu.l/well
portions and then allowed to stand for 30 minutes at room
temperature. After stopping the reaction by dispensing 2 N sulfuric
acid in 50 .mu.l/well portions, OD at 492 nm or OD at 450 nm is
measured. In this case, a hybridoma having higher reactivity with
the cancer cell strain-derived antigen than that with the normal
human serum-derived antigen is selected.
[0040] A hybridoma which produces the monoclonal antibody of the
present invention is obtained by carrying out cloning of the thus
obtained antibody producing hybridoma by limiting dilution analysis
or the like means. In order to produce the monoclonal antibody of
the present invention in a large amount making use of the thus
obtained hybridoma, as a first step, the cells are cultured in a
large scale or, alternatively, pristane or the like mineral oil is
administered into the abdominal cavity of mice, subsequently
carrying out intraperitoneal administration of said hybridoma, and
then the ascitic fluid is collected several days thereafter. Next,
the monoclonal antibody is separated and purified from the thus
obtained culture broth or ascitic fluid for example in accordance
with the usual antibody separation purification techniques. The
thus obtained monoclonal antibody of the present invention reacts
with the human lung adenocarcinoma-derived glycoprotein but hardly
with the normal human-derived glycoprotein.
[0041] The monoclonal antibody of the present invention produced by
the hybridoma TRD-L1, TRD-L2 or TRD-L3 obtained in this manner can
be classified as IgM isotype, but the antibody is not necessarily
in its intact form after its production by the cells and may be in
the form of Fab, F(ab)2, Fv or the like useful fragment. Such a
useful fragment can be obtained from its original antibody by its
peptidase hydrolysis using papain or pepsin. The term useful
fragment means that it can bind to the binding site of the antigen
of the same origin in competition with the original antibody.
However, the above examples of the monoclonal antibody of the
present invention are unique isotype antibodies which bind to
specific determining sites on respective antigens and are derived
from mice, though not particularly limited thereto. In consequence,
the monoclonal antibody of the present invention which is derived
from mouse, human or the like mammals or other origin or any
combination thereof can be used, provided that the antibody has a
function to bind to the specified specific determining site on the
specified antigen in the same manner. Also, even when isotype of
the antibody is IgG, IgA, IgE or the like other class, it can be
used in the same manner.
[0042] (6) Preparation of Antigen
[0043] The human lung adenocarcinoma-derived antigen to be obtained
by the present invention can be produced for example by making use
of the monoclonal antibody of the present invention which is
obtained in the aforementioned manner. That is, it can be produced
from sera of lung adenocarcinoma patients or cells of a lung
adenocarcinoma established cell line. For. example, Calu-3 (ATCC
HTB-55) is cultured in RPMI 1640 medium or MEM medium and the
culture supernatant fluid is recovered, or serum of a lung
adenocarcinoma patient is obtained in the usual way. Insoluble
matter is removed from the thus recovered culture supernatant fluid
or serum of a lung adenocarcinoma patient by centrifugation or
using a filter, and the resulting sample is applied and adsorbed to
an affinity chromatography column whose packing is linked by the
monoclonal antibody of the present invention. After washing with an
appropriate buffer (PBS for example), elution is carried out with 3
M KSCN. After the elution, dialysis and concentration are carried
out and then fractions of 200 kD or more in molecular weight are
recovered using a gel filtration chromatography column. The antigen
obtained in this manner has a molecular weight of about 200 kD or
more (SDS-PAGE) and is a sugar chain-containing glycoprotein,
because it is stained by PAS staining. The human lung
adenocarcinoma-derived antigen obtained in this manner can be used
in the treatment of human lung adenocarcinoma as an attenuated
vaccine or an inactivated vaccine.
[0044] (7) Detection of Antigen
[0045] In order to measure the antigen of the present invention
using an antibody specific for the antigen of the present
invention, it can be carried out in accordance with a known
immunoassay method, for example to measure the presence of a
malignant state in the lung tissue. The term malignant state means
the presence of atypical tumor cells and the like simultaneously
including a cancer. The presence of a tumor in a sample is
confirmed by allowing the sample to react with the monoclonal
antibody of the present invention and then detecting an immune
complex linked to the sample. An example of the present invention
is a method for the detection of tumor cells in excised tissues. A
tumor section obtained by excision is fixed on a slideglass in the
usual way. The thus obtained tumor section is allowed to react with
the monoclonal antibody of the present invention. With regard to
the reaction conditions, the reaction is carried out for example in
an appropriate container such as a Petri dish. After removing
non-specifically bound antibodies by washing, subsequent reaction
is carried out with a second antibody which can react with said
antibody and is labeled with a detectable marker. The marker is a
substance which can generate a detectable signal, such as a
radioactive element, a fluorescent material, an enzyme or the like.
Since binding of the antibody reflects the presence of a malignant
material, the presence of the malignant material can be confirmed
by detecting the detectable signal. In addition, binding of said
monoclonal antibody to a sample can also be measured by using the
monoclonal antibody to which a radioactive material, an enzyme or
the like marker capable of generating a detectable signal is linked
by covalent bonding. Covalent bonding of a marker to the antibody
or antibody fragment can be effected by a usually used method. In
this connection, this method is not limited to excised tissues, and
it can be used in the detection of cancer cells in sputum, alveoli
wash solution and the like.
[0046] When an antibody specific for the antigen of the present
invention is used, the antigen of the present invention in human
secretions (blood, urine, sputum and the like) can be measured.
This measurement can be carried out in accordance with a known
immunoassay method. Examples of the antibody specific for the
antigen of the present invention include both polyclonal and
monoclonal antibodies which can be used alone or a combination
thereof. Examples of the immunoassay include enzyme immunoassay,
radioimmunoassay, fluoroimmunoassay, chemiluminescence/biolu-
minescence immunoassay, latex agglutination immunoassay and the
like. As the enzyme immunoassay, optional known modifications can
be used. For example, the antigen can be measured directly or
competitively making use of a homogenous phase enzyme immunoassay,
a solid phase method, a heterogeneous enzyme immunoassay, a
sandwich enzyme immunoassay and the like. Of these methods,
sandwich enzyme immunoassay is particularly desirable. In this
method, a monoclonal antibody linked to a solid carrier is allowed
to react with a test solution and then, after washing, a solution
of an enzyme-labeled monoclonal or polyclonal antibody which has
reactivity with the antigen is dispensed. After washing, amount of
the antigen in the sample is measured based on the enzyme-substrate
reaction. In this connection, the monoclonal antibody to be used in
the latter reaction is desirably an antibody which can bind to a
site which is different from the specific binding site recognizable
by the monoclonal antibody linked to the solid carrier, except for
a case in which the antigen has a plurality of sites to which the
solid phase monoclonal antibody can be linked. Examples of the
carrier to be used include a microplate, a test tube, beads or fine
particles made of polystyreine, polyethylene or polyvinyl chloride,
a test tube, beads or a filter paper made of glass, or a sheet of
dextran, cellulose acetate or cellulose, as well as similar
materials thereof. Also, examples of the desirable enzyme to be
used in the enzyme immunoassay of the present invention include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase
and the like. Examples of other assay methods of the present
invention include radioimmunoassay in which a radioactive marker is
used, fluoroimmunoassay in which a fluorescent marker is used,
chemiluminescence/bioluminescence immunoassay in which a
luminescent marker is used and latex agglutination immunoassay in
which a latex marker is used.
[0047] In carrying out the present invention, it is desirable to
prepare in advance an assay kit which comprises a combination of
the monoclonal antibody of the present invention, the antigen of
the present invention and other accessories. For example, in the
case of sandwich enzyme immunoassay, it contains a monoclonal
antibody linked to an appropriate carrier, a freeze-dried
preparation or a solution of an enzyme-labeled monoclonal antibody
which can bind to the same antigen together with the monoclonal
antibody or of a polyclonal antibody labeled with the enzyme in the
same manner, a standard solution of purified antigen, a buffer
solution, a washing solution, pipettes, a reaction container and
the like. In this connection, the polyclonal antibody to be used in
the assay of the present invention can be collected easily from an
animal immunized against the antigen of the present invention in
the usual way. For example, a rabbit is immunized several times
using the antigen of the present invention, and when the antibody
titer against said antigen reaches maximum, blood is collected to
separate and purify the antibody fraction in the usual way.
[0048] (8) Internal Diagnosis
[0049] The antibody of the present invention can be applied to a
method in which primary and metastatic layers of lung
adenocarcinoma in a patient are transformed into images, in
accordance with a known technique on malignant melanoma (Larson S.
M. et al., J. Clin. Invest., 72, 2101 (1983)). The antibody or a
fragment thereof is labeled with a radioactive material and
administered to a patient by intravenous injection, and then the
affected parts of the patient are transformed into images making
use of PET method or the like. Examples of the material for
radioactive labeling include 99-Tc, 131-I and 125-I.
[0050] In addition, the monoclonal antibody of the present
invention may be used as a diagnosis kit for the practice of the
aforementioned methods. An example of such a case is a combination
of the monoclonal antibody and a marker-linked material which can
detect the antigen, or the monoclonal antibody labeled with a
detectable marker. Said diagnosis kit contains a buffer solution
necessary for the detection method and a protein stabilizing agent
such as a polysaccharide, and an active substance for reducing
background interference in the assay, a control reagent, a test
apparatus and the like may also be used as occasion demands.
[0051] (9) Therapeutic Method
[0052] The monoclonal antibody of the present invention can also be
used in the treatment of lung adenocarcinoma, for example by
pharmaceutically combining it with a variety of toxins having
toxicity to cancer cells, an anticancer drug or a radioactive
material in accordance with a known method. In addition, a method
in which a patient is immunized against the antigen of the present
invention in its purified form, a fragment of said antigen or a
modified form of said antigen can be exemplified as a method for
providing immunological antitumor activity.
EXAMPLES
[0053] The following describes the present invention further in
detail with reference to examples.
Inventive Example 1
[0054] Preparation of Monoclonal Antibody
[0055] An established lung adenocarcinoma cell line, Calu-3 (ATCC
HTB-55), was cultured in RPMI 1640 medium containing 5% FBS and
then the culture supernatant fluid was recovered. The thus
recovered culture supernatant fluid was centrifuged and the
insoluble matter was removed using a 0.45 .mu.m filter, and then
the thus prepared sample was applied to an affinity chromatography
column whose packing has been linked by a monoclonal antibody
DF-L1, thereby effecting adsorption of the antigen. After
subsequent washing with 10 mM PBS (phosphate buffer, pH 7.4), the
elution was effected using 3 MKSCN. After the elution, the eluate
was dialyzed and concentrated and then fractionated by a gel
chromatography using Sephacryl S-300 (manufactured by Pharmacia) to
recover and concentrate a fraction of 200 kD or more in molecular
weight. The thus obtained glycoprotein was used as the immunogen.
This was made into an emulsion with Freundis complete adjuvant and
Freundis incomplete adjuvant, and 1 .mu.g of it was subcutaneously
administered to a BALB/c mouse of 6 weeks of age. The immunization
step was repeated three times at intervals of 2 weeks, and the
final immunization was effected by intravenous injection of the
same amount of the aforementioned glycoprotein. Three days
thereafter, the spleen was aseptically excised to prepare
2.5.times.10.sup.-7 of spleen cells. After hemolysis of
contaminated erythrocytes with Geyis solution, the cells were
washed twice with RPMI 1640 medium.
[0056] Antibody non-producing SP2/0-Ag4 mouse myeloma cells were
used in the subsequent cell fusion. The SP2/0-Ag4 cells were
cultured and maintained in RPMI 1640 medium containing 10% fetal
calf serum (FCS, manufactured by Flow) contained in a culture flask
(manufactured by Sumitomo Bakelite), and the cells at logarithmic
growth phase were used in the cell fusion. The myeloma cells were
recovered, adjusted to a density of 5.times.10.sup.-6 cells and
then washed twice with RPMI 1640 medium. The washed spleen cells
and myeloma cells were thoroughly mixed in a fusion buffer and
subjected to 5 minutes of centrifugation at 1,000 rpm to remove the
culture solution. The resulting pellet was again suspended in the
fusion buffer. The cell fusion was carried out by an electrofusion
method using a cell fusion apparatus (SSH-2, manufactured by
Shimadzu Corp.). After the cell fusion, the cells were recovered in
a container which contained 30 ml of RPMI medium supplemented with
10% FCS and then cultured for 30 minutes under conditions of
37.degree. C. and 5% CO.sub.2/95% air. After 5 minutes of
centrifugation at 1,000 rpm, the cells were re-suspended in RPMI
1640 medium containing 10% FCS and 10% HCF (manufactured by ORIGEN)
and dispensed in 0.1 ml portions into wells of a 96 F plate
(manufactured by Costar). This was cultured under conditions of
37.degree. C. and 5% CO.sub.2/95% air, and a HAT selection was
carried out on the next day by adding 0.1 ml of HAT medium (RPMI
1640 medium containing 10% FCS, hypoxanthine, aminopterin and
thymidine). After the fusion, half the volume of the medium was
exchanged with HAT medium on the fourth day, and the culture
supernatant fluids were collected on the tenth day to measure their
antibody activity using an enzyme immunoassay method.
[0057] The immunoassay was carried out in the following manner.
That is, the cancer cell-derived antigen and the same amount of a
normal human serum-derived antigen (antigen containing 1 .mu.g/ml
of protein) were dispensed in 100 .mu.l portions into wells of a 96
F aminoplate, and their bonding was effected using glutaraldehyde
in the usual way. After removing the supernatant fluid by suction,
the plate was washed with PBS-Tween 20, and block (blocking) of the
bound residues was carried out by adding 300 .mu.l of 1% BSA-PBS.
After removing the supernatant fluid by suction, the plate was
washed with PBS-Tween 20. A hybridoma culture supernatant fluid was
dispensed in 50 .mu.l portions into wells of the plate and allowed
to undergo 1 hour of reaction at 37.degree. C. After removing the
supernatant fluid by suction, this was again washed with PBS-Tween
20. After the washing, an anti-mouse immunoglobulin
antibody-peroxidase conjugate was dispensed in 100 .mu.l portions
into the wells to carry out 1 hour of reaction at 37.degree. C.
After washing, 0.1 M citrate buffer (pH 4.2) containing
O-phenylenediamine dihydrochloride or tetramethylbenzidine and
hydrogen peroxide was dispensed in 100 .mu.l/well portions and then
allowed to stand for 30 minutes at room temperature. After stopping
the reaction by dispensing 2 N sulfuric acid in 50 .mu.l/well
portions, OD at 492 nm was measured (OD 450 nm in the case of
TMBZ). In this case, a hybridoma having higher reactivity with the
cancer cell strain-derived antigen than that with the normal human
serum-derived antigen was selected.
[0058] A hybridoma capable of producing the monoclonal antibody of
the present invention was obtained by carrying out cloning of the
thus obtained antibody producing hybridoma by limiting dilution
analysis or the like means. In order to produce the monoclonal
antibody of the present invention in a large amount making use of
the thus obtained hybridoma, the cells were cultured in a large
scale. Next, the monoclonal antibody of the present invention was
separated and purified from the thus obtained culture broth in
accordance with the usual antibody separation purification
techniques. The thus obtained monoclonal antibody of the present
invention reacts with human lung adenocarcinoma but hardly with
normal human. When its isotype was confirmed using an isotype kit
(manufactured by Bio-Rad), said antibody was classified as IgM
isotype.
Inventive Example 2
[0059] Purification of Antigen
[0060] The established lung adenocarcinoma cell line, Calu-3 (ATCC
HTB-55), was cultured in RPMI 1640 medium containing 5% FBS and
then the culture supernatant fluid was recovered. The thus
recovered culture supernatant fluid was centrifuged, the insoluble
matter was removed using a 0.45 .mu.m filter, the thus prepared
sample was applied to an affinity chromatography column whose
packing has been linked by the monoclonal antibody TRD-L1, TRD-L2
or TRD-L3 in the usual way, thereby effecting adsorption of the
antigen, and then the column was washed with 10 mM PBS (phosphate
buffer, pH 7.4). After the washing, the elution was effected using
3 M KSCN. After the elution, the eluate was dialyzed and
concentrated and then fractionated by a gel chromatography using
Sephacryl S-300 (manufactured by Pharmacia) to recover and
concentrate a fraction which reacted with the antibody.
Inventive Example 3
[0061] Biochemical Properties of Antigen
[0062] Each of the antigens reactive with TRD-L1, TRD-L2 and TRD-L3
was analyzed by SDS-polyacrylamide gel electrophoresis, Western
immunoblotting analysis, lectin analysis and PAS staining. Purified
antigen was separated on 4% gel by the Laemmli method in the usual
way. A portion of the gel was used in PAS staining. The results
thus obtained are shown in FIG. 1(A). In the lectin analysis, an
antigen purified in the same manner from normal human serum using
DF-L1 was also analyzed in the same manner.
[0063] In this connection, Western immunoblotting was carried out
in the following manner. That is, separated protein was transferred
on a PVDF membrane (manufactured by Bio-Rad) in the usual way.
After the transfer, blocking with 1% BSA-PBS was carried out at
room temperature for 60 minutes. After the blocking, the monoclonal
antibody was allowed to undergo overnight reaction at 4.degree. C.
After washing with PBS-Tween 20, alkaline phosphatase-labeled
rabbit anti-mouse immunoglobulin was allowed to undergo 1 hour of
reaction at room temperature. After the reaction, the washing was
repeated and color development was effected by adding BCIP/NBT
solution. The results thus obtained are shown in FIG. 1(B). The
band was found in the region of 200 kD or more.
[0064] Also, the lectin analysis was carried out by effecting
blocking of the sample after its transfer on PVDF membrane in the
same manner as the case of Western blotting, allowing
digoxigenin-labeled lectin (manufactured by Boehringer-Mannheim) to
undergo 2 hours of reaction at room temperature, carrying out
washing, allowing alkaline phosphatase-labeled digoxigenin antibody
(manufactured by Boehringer-Mannheim) to undergo 1 hour of reaction
at room temperature, carrying out washing and then effecting color
development by adding BCIP/NBT solution.
[0065] As shown in FIG. 1, it can be said that the antigen has a
molecular weight of 200 kD or more based on the results of
SDS-polyacrylamide gel electrophoresis and Western immunoblotting
and is a glycoprotein because it is strongly stained by PAS
staining.
[0066] Also, based on the lectinanalysis, this is aglycoprotein
which reacts strongly with MAA lectin and PNA lectin but does not
react with GNA lectin, SNA lectin and DSA lectin. The results are
shown in Table 1. Based on its reactivity with lectin, it seems
that the sugar chain terminal structure of this antigen has
NeuAc(2-3)Gal and Gal(1-3)GalNAc structures but does not have
mannose, NeuAc(2-6)Gal, NeuAc(2-6)GalNAc and Gal(1-4)GlcNAc
structures.
1TABLE 1 Reactivity of TRD-L1 reactive antigen with lectin Lectin
Reactivity GNA - SNA - MAA + PNA + DSA - -: negative +:
positive
[0067] On the other hand, the DF-L1 reactive antigen is a
glycoprotein which reacts strongly with MAA lectin, SNA lectin and
DSA lectin and weakly with PNA lectin but does not react with GNA
lectin, and it seems that the sugar chain terminal structure of the
DF-L1 reactive antigen has NeuAc(2-3)Gal, Gal(1-3)GalNAc,
NeuAc(2-6)Gal, NeuAc(2-6)GalNAc and Gal(1-4)GlcNAc structures.
Inventive Example 4
[0068] Immunohistochemical Assay
[0069] Frozen blocks of lung adenocarcinoma, squamous cell
carcinoma, small cell carcinoma and normal tissue were sliced to a
thickness of 9 microns using a cryostat to prepare slide glass
non-fixed frozen sections. Each of the frozen sections was
subjected to 10 minutes of reaction with 0.5% hydrogen peroxide to
effect blocking of endogenous peroxidase. This was washed with 20
mM phosphate buffer containing 0.154 M NaCl (PBS) and then
subjected to 10 minutes of reaction with 10% rabbit serum in PBS.
After washing again with PBS, a monoclonal antibody was added
thereto to carry out 60 minutes of reaction. This was again washed
with PBS and allowed to undergo reaction with peroxidase-labeled
rabbit anti-mouse immunoglobulin (manufactured by Cappel) which has
been diluted 200 times with PBS. After 60 minutes of the reaction
and subsequent washing with PBS, a color developing kit
(manufactured by Funakoshi Pharmaceutical) containing
diaminobenzidine and hydrogen peroxide was added as the substrate.
and 15 minutes of reaction was carried out. This was washed again,
subjected to nuclear staining with a hematoxylin solution,
dehydrated and then embedded. In this connection, all of the above
reactions were carried out at room temperature. The thus treated
sections were observed under a microscope at 100.times.
magnification, and a section in which 10% or more of cells were
stained brown was judged positive. The results are shown in Table
2. As the result, the monoclonal antibody of the present invention
showed highly positive reaction against lung adenocarcinoma.
2 TABLE 2 Lung squamous Lung small Lung cell cell Lung normal
adenocarcinoma carcinoma carcinoma tissue TRD- 5/5 0/5 0/3 0/2 L1
TRD- 5/5 0/5 0/3 0/2 L2 TRD- 5/5 0/5 0/3 0/2 L3 DF-L1 5/5 5/5 0/3
1/2
Inventive Example 5
[0070] Enzyme Immunoassay
[0071] Each of normal human serum, serum of lung adenocarcinoma
patient and a culture supernatant fluid of the lung adenocarcinoma
established cell line Calu-3 (ATCC HTB-55) was centrifuged and the
insoluble matter was removed using a 0.45 .mu.m filter, and then
the thus prepared sample was applied to an affinity chromatography
column whose packing has been linked by the monoclonal antibody
DF-L1, thereby effecting adsorption of the antigen, subsequently
washing with 10 mM PBS (phosphate buffer, pH 7.4). After washing,
the elution was effected using 3 M KSCN. After the elution, the
eluate was dialyzed and concentrated to obtain DF-L1-linked
antigen. The same amount of each of the thus obtained antigen
(antigen containing 1 .mu.g/ml of protein) was dispensed in 100
.mu.l portions into wells of a 96 F aminoplate (manufactured by
Sumitomo Bakelite), and their bonding was effected using
glutaraldehyde in the usual way. After removing the supernatant
fluid by suction, the plate was washed with PBS-Tween 20, and block
(blocking) of the bound residues was carried out by adding 300
.mu.l of 1% BSA-PBS. After removing the supernatant fluid again by
suction, the plate was washed with PBS-Tween 20. A monoclonal
antibody solution (1 .mu.g/ml) was dispensed in 100 .mu.l portions
into wells of the plate and allowed to undergo overnight reaction
at 4.degree. C.
[0072] After removing the supernatant fluid by suction, this was
washed with PBS-Tween 20. After the washing, an anti-mouse
immunoglobulin antibody-peroxidase conjugate was dispensed in 100
.mu.l portions into the wells to carry out 2 hours of reaction at
room temperature. After washing, 0.1 M citrate buffer (pH 4.2)
containing O-phenylenediamine dihydrochloride and hydrogen peroxide
was dispensed in 100 .mu.l/well portions and then allowed to stand
for 30 minutes at room temperature. After dispensing 2 N sulfuric
acid in 50 .mu.l/well portions, OD at 492 nm was finally measured
to confirm the presence of antigen. The results are shown in Table
3. The antigen of the present invention was secreted in the serum
of lung adenocarcinoma patient and the culture supernatant fluid of
lung adenocarcinoma established cell line, and the monoclonal
antibody of the present invention reacted with this antigen most
strongly.
3 TABLE 3 Absorbance at 492 nm Lung adenocarcinoma Normal human
Calu-3 patient serum TRD-L1 0.480 0.478 0.048 TRD-L2 0.351 0.422
0.021 TRD-L3 1.034 1.271 0.094 DF-L1 1.333 1.233 1.235
Inventive Example 6
[0073] Using 10 serum samples of normal volunteers, 5 serum samples
of lung adenocarcinoma patients, 5 serum samples of benign lung
disease patients and 5 serum samples of other cancer patients, the
amount of antigen in the serum of each patient was measured by
sandwich enzyme immunoassay. That is, a monoclonal antibody
solution (5 .mu.g/ml, 0.05 M carbonate buffer (pH 9.6)) was
dispensed in 100 .mu.l portions into wells of a 96 F immunoplate
(manufactured by Nunc) and allowed to undergo overnight reaction at
4.degree. C. After removing the solution by aspiration, PBS
containing 1% BSA was dispensed to carry out blocking. After
washing with PBS-Tween 20, each of the serum samples which has been
diluted 20 times with PBS solution containing 5% BSA was dispensed
in 100 .mu.l portions into the wells to carry out 1 hour of
reaction at room temperature. After washing the plate with
PBS-Tween 20, peroxidase-labeled DF-L1 antibody was dispensed in
100 .mu.l portions into the wells. After 1 hour of reaction at room
temperature and subsequent washing with PBS-Tween 20, 0.1 M citrate
buffer (pH 4.2) containing O-phenylenediamine dihydrochloride and
hydrogen peroxide was dispensed in 100 .mu.l/well portions and
allowed to stand for 30 minutes at room temperature, and then 2 N
sulfuric acid was dispensed in 50 .mu.l/well portions. Finally
measuring OD at 492 nm, the presence of antigen was confirmed. The
results are shown in FIG. 2. Since the antigen of the present
invention is secreted in sera of lung adenocarcinoma patients most
frequently, it can be said that measurement of said antigen making
use of the monoclonal antibody of the present invention is useful
for the diagnosis of lung adenocarcinoma.
Inventive Example 7
[0074] Comparison of Antibody Characteristics
[0075] Using a DF-L1 monoclonal antibody-linked affinity
chromatography, antigen was purified from a culture supernatant
fluid of the lung adenocarcinoma cell line Calu-3 in the same
manner as described in Inventive Example 2. Concentration of the
purified antigen was measured by the method of Maimonis P. et al.
(Cancer Res., 51, 3838, 1991). In the same manner, antigen was
purified from pooled normal human sera, and the amount of antigen
in the normal human purified antigen solution was measured by the
same sandwich enzyme immunoassay. After the measurement, the amount
of each of these antigens was adjusted to 2.9 to 1,000 units/ml by
diluting it with 10 mM PBS, and the following measurement was
carried out by the method of Maimons P. et al. in which DF-L1
antibody was used and the method of Inventive Example 6 in which
TRD-L1 antibody was used.
[0076] That is, each of the antibody solutions (5 .mu.g/ml, 0.5 M
carbonate buffer (pH 9.6)) of DF-L1 antibody and TRD-L1 antibody
was dispensed in 100 .mu.l portions into wells of two 96 F
immunoplates (manufactured by Nunc) and allowed to undergo
overnight reaction at 4.degree. C. After removing the solution by
suction, PBS containing 1% BSA was dispensed to carry out blocking.
After washing with PBS-Tween 20 solution, each of the diluted
cell-derived and normal human-derived antigen solutions was
dispensed to carry out 1 hour of reaction at room temperature.
After washing with PBS-Tween 20 solution, peroxidase-labeled DF-L1
antibody was dispensed in 100 .mu.l portions into the wells. After
1 hour of reaction at room temperature and subsequent washing with
PBS-Tween 20 solution, 0.1 M citrate buffer (pH 4.2) containing
O-phenylenediamine dihydrochloride and hydrogen peroxide was
dispensed in 100 .mu.l/well portions and allowed to stand for 30
minutes at room temperature, and then 2 N sulfuric acid was
dispensed in 50 .mu.l/well portions. Finally measuring OD at 492
nm, the presence of antigen was confirmed. The amount of antigen in
serum sample is shown in FIG. 3 as the strength of color
development, namely as the absorbance at a wave length of 492 nm.
In this case, the amount of antigen shown in the drawing was
measured by the method reported by Maimonis et al.
[0077] (Results)
[0078] In FIG. 3, the reactivity of cell-derived and normal
human-derived antigen solutions obtained in Inventive Example 7 by
respective assay methods was shown as the strength of color
development. That is, in comparison with the assay method in which
DF-L1 antibody is solely used, the assay method of the present
invention in which TRD-L1 antibody is used recognizes the antigen
specific to lung adenocarcinoma cells, so that measurement of said
antigen making use of this antibody is useful for the diagnosis of
lung adenocarcinoma, and this information can be used broadly in
the diagnosis of cancers including metastatic cancers.
Inventive Example 8
[0079] Detailed Evaluation by Samples from Patients
[0080] In order to confirm clinical usefulness of the TRD-L1-aided
assay method, the amount of antigen in serum of each patient was
measured by the same method of Inventive Example 6, using 46 serum
samples of normal volunteers (Normal), 425 serum samples of lung
adenocarcinoma patients (Lung Cancer), 240 serum samples of benign
lung disease patients (Benign), 27 lung adenocarcinoma serum
samples (Adeno), 9 squamous cell carcinoma serum samples (Sque), 5
small cell carcinoma serum samples (Small), 2 large cell carcinoma
serum samples (Large), 45 serum samples of stomach cancer patients
(Stomach), 44 serum samples of breast cancer patients (Breast), 21
serum samples of colon cancer patients (Colon) and 5 serum samples
of other cancer patients (Misc). The amount of antigen was
calculated by deciding previously measured samples of patients as
optional values and using the values as standards.
[0081] That is, a TRD-L1 monoclonal antibody solution (5 .mu.g/ml,
0.5 M carbonate buffer (pH 9.6)) was dispensed in 100 .mu.l
portions into wells of a 96 F immunoplate (manufactured by Nunc),
subsequently carrying out blocking. After washing with PBS-Tween 20
solution, each of the serum samples which has been diluted 6 times
with PBS solution containing 5% BSA and four standard solutions
having known concentrations (0, 20, 100, 200 units/ml) was
dispensed in 120 .mu.l portions into the wells to carry out 1 hour
of reaction at room temperature. After washing with PBS-Tween 20
solution, peroxidase-labeled DF-L1 antibody was dispensed in 100
.mu.l portions into the wells. After 1 hour of reaction at room
temperature and subsequent washing with PBS-Tween 20 solution, 0.1
M citrate buffer (pH 4.2) containing O-phenylenediamine
dihydrochloride and hydrogen peroxide was dispensed in 100
.mu.l/well portions and allowed to stand for 30 minutes at room
temperature, and then 2 N sulfuric acid was dispensed in 50
.mu.l/well portions. The amount of antigen in serum samples of
patients was determined by measuring the strength of color
development, namely absorbance at a wave length of 492 nm, and
comparing the value with a calibration curve prepared from the
concentration of standards and the absorbance.
[0082] When the serum concentration exceeded 200 units/ml, the
measurement was carried out after further dilution.
[0083] (Results)
[0084] FIG. 4 shows the amount of antigen in sera of normal
volunteers and patients of lung adenocarcinoma, benign lung
diseases and other cancers, obtained in Inventive Example 8. Since
said antigen of the present invention is secreted in sera of lung
adenocarcinoma patients most frequently, measurement of said
antigen making use of said monoclonal antibody is useful for the
diagnosis of lung adenocarcinoma.
Inventive Example 9
[0085] Reactivity with Other Tumor Antigens
[0086] Reactivity of the antibody and assay method of the present
invention with known tumor antigens CEA, CA19-9, CA125, CA50 and
CA15-3, glycoprotein antigens similar to the inventive antigen, was
measured by the same method of Inventive Example 6. Each antigen
was diluted with 10 mM PBS, and the amount of antigen was measured
as the absorbance at a wave length of 492 nm.
[0087] That is, a TRD-L1 monoclonal antibody solution (5 .mu.g/ml,
0.5 M carbonate buffer (pH 9.6)) was dispensed in 100 .mu.l
portions into wells of a 96 F immunoplate (manufactured by Nunc)
and allowed to undergo overnight reaction at 4.degree. C. After
removing the solution by suction, PBS containing 1% BSA was
dispensed to carry out blocking. After washing with PBS-Tween 20
solution, each of the antigen solutions was dispensed in 100 .mu.l
portions into the wells to carry out 1 hour of reaction at room
temperature. After washing with PBS-Tween 20 solution,
peroxidase-labeled DF-L1 antibody was dispensed in 100 .mu.l
portions into the wells. After 1 hour of reaction at room
temperature and subsequent washing with PBS-Tween 20 solution, 0.1
M citrate buffer (pH 4.2) containing O-phenylenediamine
dihydrochloride and hydrogen peroxide was dispensed in 100
.mu.l/well portions and allowed to stand for 30 minutes at room
temperature, and then 2 N sulfuric acid was dispensed in 50
.mu.l/well portions. Finally measuring OD at 492 nm, the presence
of antigen was confirmed.
[0088] (Results)
[0089] FIG. 5 to FIG. 9 are graphs showing results of the
measurement of TRD-L1 antibody linked to CEA, CA19-9, CA125, CA50
or CA15-3, obtained in Inventive Example 9, which are compared with
the results of TRD-L1 antigen. That is, said antigen and antibody
of the present invention are different from other glycoprotein
tumor antigens.
INDUSTRIAL APPLICABILITY
[0090] Novel monoclonal antibody and antigen can be obtained by the
present invention, and an immunoassay method which uses said
monoclonal antibody and antigen renders possible definite diagnosis
and prognostic judgment of lung adenocarcinoma by detecting lung
adenocarcinoma cells in tissues and measuring the amount of antigen
in human body fluids such as blood.
* * * * *