U.S. patent application number 10/460754 was filed with the patent office on 2004-02-26 for prostaglandin d synthase-specific monoclonal antibody.
This patent application is currently assigned to Maruha Corporation. Invention is credited to Nishikawa, Masazumi, Oda, Hiroshi, Saji, Fumitaka, Sato, Nobuyuki, Seiki, Kosuke, Urade, Yoshihiro.
Application Number | 20040038314 10/460754 |
Document ID | / |
Family ID | 27463568 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040038314 |
Kind Code |
A1 |
Oda, Hiroshi ; et
al. |
February 26, 2004 |
Prostaglandin D synthase-specific monoclonal antibody
Abstract
The invention relates to a monoclonal antibody specifically
recognizing lipocalin-type prostaglandin D synthase (L-PGDS), a
hybridoma producing said monoclonal antibody, methods for detection
of L-PGDS or diseases by said monoclonal antibody, and a kit for
detection of L-PGDS by said monoclonal antibody. According to the
invention, there is provided a monoclonal antibody specific to
L-PGDS.
Inventors: |
Oda, Hiroshi; (Ibaraki,
JP) ; Sato, Nobuyuki; (Ibaraki, JP) ;
Nishikawa, Masazumi; (Ibaraki, JP) ; Seiki,
Kosuke; (Ibaraki, JP) ; Urade, Yoshihiro;
(Kyoto, JP) ; Saji, Fumitaka; (Hyogo, JP) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
45 ROCKEFELLER PLAZA, SUITE 2800
NEW YORK
NY
10111
US
|
Assignee: |
Maruha Corporation
Osaka Bioscience Institute, a Japanese corporation
|
Family ID: |
27463568 |
Appl. No.: |
10/460754 |
Filed: |
June 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10460754 |
Jun 12, 2003 |
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09066357 |
Apr 28, 1998 |
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6605705 |
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09066357 |
Apr 28, 1998 |
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PCT/JP96/03190 |
Oct 31, 1996 |
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Current U.S.
Class: |
435/7.2 ;
435/338; 435/7.5; 530/388.26 |
Current CPC
Class: |
Y10S 530/809 20130101;
C07K 16/40 20130101 |
Class at
Publication: |
435/7.2 ;
530/388.26; 435/338; 435/7.5 |
International
Class: |
G01N 033/53; G01N
033/567; C12N 005/06; C07K 016/40 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 31, 1995 |
JP |
283859/1995 |
Mar 14, 1996 |
JP |
57841/1996 |
Jun 10, 1996 |
JP |
147689/1996 |
Aug 22, 1996 |
JP |
221530/1996 |
Claims
1. A monoclonal antibody specifically recognizing lipocalin-type
prostaglandin D synthase (L-PGDS).
2. The monoclonal antibody according to claim 1, wherein the
subclass of said monoclonal antibody is immunoglobulin G1 or
G2.
3. A hybridoma obtained by cell fusion between antibody-producing
cells from an animal immunized with L-PGDS and myeloma cells and
producing a monoclonal antibody of claim 1 or 2.
4. A method for detection of L-PGDS, wherein L-PGDS is detected by
a monoclonal antibody of claim 1 or 2.
5. A method for detection of a disease, wherein L-PGDS is detected
by a monoclonal antibody of claim 1 or 2.
6. The method according to claim 5, wherein the disease is
oligospermia.
7. A kit for detection of L-PGDS, which is selected from the group
consisting of: a reagent containing the monoclonal antibody of
claim 1 or 2 labeled with an enzyme, and a substrate solution; a
reagent containing the monoclonal antibody of claim 1 or 2, said
monoclonal antibody being labeled with an enzyme, or an
enzyme-labeled polyclonal antibody recognizing human L-PGDS, and a
substrate solution; a reagent containing the monoclonal antibody of
claim 1 or 2 which is biotinylated, enzyme-labeled avidin or
streptoavidin, and a substrate solution; and a reagent containing
the monoclonal antibody of claim 1 or 2, said monoclonal antibody
being biotinylated, or a biotinylated polyclonal antibody
recognizing human L-PGDS, enzyme-labeled avidin or streptoavidin,
and a substrate solution.
8. A method for detection of a disease, wherein the disease is
detected using the kit for detection of L-PGDS described in claim
7.
9. The method according to claim 8, wherein the disease is
oligospermia.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a monoclonal antibody
specific to human L-PGDS present predominantly in cerebrospinal
fluid (CSF), a hybridoma producing said monoclonal antibody,
methods for detection of L-PGDS or diseases by said monoclonal
antibody, and a kit for detection of L-PGDS by said monoclonal
antibody.
BACKGROUND OF THE INVENTION
[0002] Prostaglandin D is a biologically active substance
synthesized in animal tissues from arachidonic acid released from a
biomembrane upon stimulation, and it is produced from prostaglandin
H (a common precursor of prostaglandin families produced by
cyclo-oxygenase) by prostaglandin D synthase (PGDS).
[0003] The presence of two types of PGDS, glutathione-independent
L-PGDS and glutathione-dependent spleen type PGDS, is known
(Shimizu et, al., J. Biol. Chem., 25A, 5222-5228 (1979); Urade et
al., J. Biol. Chem., 260, 12410-12415 (1985); Christ-Hazelhof and
Nugteren, Biochim. Biophys. Acta, 572, 43-51 (1979); Urade et al.,
J. Biol. Chem., 262, 3820-3825 (1987). The former is known to be
predominantly located in the central nervous system such as brain,
epididymis, spinal cord, retina, and inner ear (Urade et al., J.
Biol. Chem., 26, 12410-12415 (1985); Ueno et al., J. Neurochem.,
45, 483-489 (1985); Urade et al., J. Biol. Chem., 2, 3820-3825
(1987); Goh et al., Biochim. Biophys. Acta, 921, 302-311 (1987);
and Tachibana et al., Proc. Natl. Acad. Sci. USA, 84, 7677-7680
(1987)), and the latter is known to be distributed broadly in
almost all peripheral organs including spleen, bone marrow,
digestive organs, thymus, and skin (Ujihara et al., Arch. Biochem.
Biophys., 260, 521-531 (1988); and Ujihara et al., J. Invest.,
Dermatol., 90, 448-451 (1988)).
[0004] On the other hand, a protein called .beta.-trace was
observed to be present specifically in human CSF, but its
physiological function remained unrevealed (Causen, J. Proc. Soc.
Exp. Biol. Med., 107, 170-172 (1961)).
[0005] A certain correlation between .beta.-trace known as a
protein specific to CSF and severe brain disorders or certain
diseases (multiple sclerosis, brain tumors, Meckel's syndrome and
paraproteinemia) was noted from the observation that .beta.-trace
levels depend on such disorders (Ericsson et al., Neurology, 19,
606-610 (1969); Olsson et al., J. Neurol. Neurosurg. Psychiat. 37,
302-311 (1974); Link, J. Neurol. Sci., 16, 103-114 (1972); Whistsed
and Penny, Clinica Chimica Acta, 50, 111-118 (1974); and Chemke et
al., Clinical Genetics, 11, 285-289 (1977)). However, the exact
correlation between .beta.-trace and such disorders could not be
determined because the physiological function of .beta.-trace still
remained unrevealed and because there was no tool available for
determining the exact amount (concentration) of .beta.-trace.
[0006] Recently, the nucleotide sequence of cDNA coding for L-PGDS
was reported (Nagata et al., Proc. Natl. Acad. Sci. USA, 88,
4020-4024 (1991)), and production of L-PGDS by genetic
recombination became feasible. The L-PGDS thus produced was
examined and its amino acid sequence was estimated and compared
with an N-terminal partial amino acid sequence of human
.beta.-trace in searching for their homology (Kuruvilla et al.,
Brain Research, 56, 337-340 (1991), Zahn et al., Neuroscience
Letters, 154, 93-95 (1993)) or with the amino acid sequence of
purified human .beta.-trace (Hoffmann et al., J. Neurochem., 61(2),
451-456 (1993)), and further immunological examination was made
using polyclonal antibodies (Watanabe et al., Biochem. Biophys.
Res. Communication, 203, 1110-1116 (1994)). These studies revealed
that .beta.-trace was identical with L-PGDS.
[0007] Prostaglandin D occurring abundantly in the central nervous
system functions, in one physiological action, as a neuromodulator
of several central actions including sleep promotion. Prostaglandin
D synthase is considered as a key enzyme for sleep-wake activities
(Hayashi, FASEB J., 5, 2575-2581 (1991)), and it is believed that
at least a part of the L-PGDS secreted from competent cells is
accumulated in CSF (Watanabe et al., Biochem. Biophys. Res.
Communication, 203, 1110-1116 (1994)).
[0008] Accordingly, the analysis of L-PGDS distribution etc. in the
central nervous system is useful for detection of diseases in the
central nervous system, and it is expected that L-PGDS levels in
CSF or humor can also be used as an indicator in early diagnosis
and prognostic observations for other diseases caused by
abnormalities in the central nervous system. It is further expected
that L-PGDS (or .beta.-trace) can be used for examination of a
reproduction ability, diagnosis of fetal growth, etc. because this
enzyme is distributed in such humors derived from genital organs,
as semen, oviduct fluid and amniotic fluid, as well. For such
applications, there is demand for antibodies specifically
recognizing L-PGDS.
[0009] Nevertheless, such antibodies have still not been
established with high specificity to meet such demand.
DISCLOSURE OF THE INVENTION
[0010] The object of the present invention is to provide a
monoclonal antibody specifically recognizing L-PGDS, a hybridoma
producing said antibody, methods for detection of L-PGDS or
diseases by said monoclonal antibody, and a kit for detection of
L-PGDS by said monoclonal antibody.
[0011] As a result of their eager researches, the present inventors
have successfully obtained a monoclonal antibody specifically
recognizing L-PGDS from a hybridoma prepared by cell fusion between
myeloma cells and antibody-producing cells from an animal immunized
with L-PGDS which is a major protein in human CSF, and they thereby
arrived at the present invention.
[0012] That is, the present invention relates to a monoclonal
antibody specifically recognizing L-PGDS. The subclass of such
monoclonal antibody includes immunoglobulin G1 or G2.
[0013] Further, the present invention relates to a hybridoma
producing said monoclonal antibody by cell fusion between myeloma
cells and antibody-producing cells from an animal immunized with
L-PGDS
[0014] Further, the present invention relates to methods for
detection of L-PGDS or diseases by said monoclonal antibody. An
example of such diseases is oligospermia.
[0015] Further, the present invention relates to a kit for
detection of L-PGDS, which is selected from a reagent containing
said monoclonal antibody labeled with an enzyme and a substrate
solution, and a reagent containing said monoclonal antibody
obtained by biotination, enzyme-labeled avidin, and a
substrate.
[0016] Further, the present invention relates to a method for
detection of diseases by said kit for detection of L-PGDS. An
example of such diseases is oligospermia.
[0017] Hereinafter, the present invention is described in
detail.
[0018] 1. Production of the Monoclonal Antibody
[0019] Production of the present monoclonal antibody against L-PGDS
consists of the steps of:
[0020] (1) Preparation of an antigen;
[0021] (2) Immunization and preparation of antibody-producing
cells;
[0022] (3) Establishment of an antibody-titration system;
[0023] (4) Cell fusion;
[0024] (5) Selecting and cloning hybridomas; and
[0025] (6) Isolation of the monoclonal antibody.
[0026] Hereinafter, each step is described.
[0027] (1) Preparation of an Antigen
[0028] L-PGDS can be produced in large amounts in a usual manner by
E. coli and CHO cells etc. with its known cDNA (Nagata et al.,
Proc. Natl. Acad. Sci. USA, 88, 4020-4024 (1991)). In this
production of L-PGDS, a recombinant DNA containing the cDNA for
L-PGDS is constructed and transformed into a microorganism, which
is then cultured to produce the enzyme. The L-PGDS thus produced
can be purified from the culture by conventional means.
[0029] Then, an immunogen is prepared by dissolving the resulting
L-PGDS in a buffer and then adding an adjuvant to it. Examples of
such adjuvants are Freund complete adjuvant, Freund incomplete
adjuvant, BCG, Hunter's Titermax (CytRx Corporation), key hole
limpet hemocyanin-containing oil, etc., and any of them can be
mixed.
[0030] (2) Immunization and Preparation of Antibody-producing
Cells
[0031] The immunogen thus obtained is administrated as antigen into
mammals such as horse, monkey, dog, pig, cow, goat, sheep, rabbit,
guinea pig, hamster and mouse, or birds such as pigeon and chicken.
In particular, mouse, rat, guinea pig, rabbit and goat are
preferably used. Any of the known immunization methods may be
employed preferably using i.v., s.c., or i.p. administration.
Immunization intervals are not particularly limited, and the
immunogen is given 2 to 10 times, preferably 2 to 5 times,
preferably at intervals of several days to several weeks, more
preferably 1 to 3 weeks.
[0032] 1 to 10 days preferably 2 to 5 days after the final
immunization, antibody-producing cells are prepared from the
animal. Examples of such antibody-producing cells are spleen cells,
lymph node cells, thymocytes and peripheral blood cells, and
generally spleen cells are used conventionally. In the case of
mouse, 0.01 .mu.g to 1,000 .mu.g, preferably 1 to 300 .mu.g antigen
is given per animal in one administration.
[0033] (3) Establishment of an Antibody-titration System
[0034] It is necessary to establish a system of measuring the
antibody titer in serum from the immunized animal or in a culture
supernatant from the antibody-producing cells, so that the immune
response level of the immunized animal can be confirmed and the
desired hybridoma can be selected from the fusion cells. For
example, the antibody can be detected conventionally using known
methods such as enzyme immunoassay (EIA), radioimmunoassay (RIA),
enzyme-linked immunosorbent assay (ELISA) and fluorescence
immunoassay (FIA). Although the present method for detecting the
antibody is not limited to the above methods, ELISA is conveniently
used for easy operations. Usually, L-PGDS is put to each well of a
96-well plastic microtiter plate and left at room temperature to
immobilize the enzyme onto the well. Then, the unbound sites of the
L-PGDS as antigen are blocked with calf serum albumin, fetal bovine
serum, skim milk, or gelatin. Then, an antiserum diluted with
phosphate buffered saline (referred to hereinafter as PBS) or a
culture supernatant of the hybridoma is added to each well.
Subsequently, commercial secondary antibody labeled with an enzyme
or fluorescent compound or with biotin is added to each well,
followed by adding a coloration substrate. The coloration occurring
can be determined in a photometer or fluorometer to quantify the
antibody against L-PGDS.
[0035] (4) Cell Fusion
[0036] The myeloma cells to be subjected to cell fusion with the
antibody-producing cells are those derived from animal species such
as mouse, rat, and human, easily available to those skilled in the
art. The cell line used is preferably drug-resistant, which upon
fusion with the antibody-producing cells, is rendered survivable in
a selection medium such as HAT medium. A cell line resistant to
8-azaguanine is generally used. Because such cell line lacks
hypoxanthine-guanine phosphoribosyltransferase (HGPRT-), it can not
grow in hypoxanthine-aminopterine-thymidine (HAT) medium. The cell
line used is preferably cells not secreting immunoglobulins.
[0037] Examples of such myeloma cells are mouse myeloma cell lines
such as P3X63Ag8 (ATCC TIB-9; Nature, 256, 495-497 (1978)),
P3X63Ag8U.1 (P3U1) (ATCC CRL-1580; Current Topics in Microbiology
and Immunology, al, 1-7 (1978), P3X63Ag8.653 (ATCC TIB-18; European
J. Immunology, 6, 511-519 (1976)), and P2/NSI/1-Ag4-1 (ATCC
CRL-1581; Nature, 276, 269-270 (1978)); rat myeloma cell lines such
as for example 210. RCY.Agl.2.3 (Y3-Agl.2.3) (ATCC CRL-1631;
Nature, 277, 131-133 (1979)); human myeloma cell lines such as
U-266-AR1 (Proc. Natl. Acad. Sci. USA, 77, 5429 (1980)), GM 1500
(Nature, 288, 488 (1980)), and KR-4 (Proc. Natl. Acad. Sci. USA,
79, 6651 (1982)).
[0038] The antibody-producing cells are obtained from spleen cells,
lymph node cells, thymocytes, and peripheral blood cells. Briefly,
the antibody-producing cells are prepared as follows: Tissues such
as spleen, lymph node or thymus are excised or blood are collected
from the immunized animals. These tissues are disrupted and then
suspended in a buffer such as PBS or in a medium such as DMEM, RPMI
1640 and E-RDF. This cell suspension is filtered through e.g. a
#200-250 stainless mesh and then centrifuged to give the desired
antibody-producing cells.
[0039] Then, the antibody-producing cells are subjected to cell
fusion with myeloma cells.
[0040] Before cell fusion, myeloma cells suitable for antibody
production are selected. 10.sup.6 to 10.sup.8 cells/ml
antibody-producing cells are mixed with 10.sup.6 to 10.sup.8
cells/ml myeloma cells at a ratio of from 1:1 to 1:10 in an animal
cell growth medium, e.g. Eagle's minimal essential medium (MEM),
Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium, or
E-RDF medium. To enhance cell fusion, these cells are mixed at a
ratio of e.g. 1:6 and then incubated for 1 to 15 minutes in the
presence of a fusion enhancer in e.g. RPMI-1640 medium containing
dimethylsulfoxide at a temperature of 30 to 37.degree. C. This
fusion enhancer may be polyethylene glycol with an average
molecular weight of 1,000 to 6,000, polyvinyl alcohol or Sendai
virus. Alternatively, the antibody-producing cells can be subjected
to cell fusion with myeloma cells by electric stimulation (e.g.
electroporation) in a commercial cell fusion apparatus.
[0041] (5) Selection and Cloning of Hybridomas
[0042] After cell fusion, the cells are screened for desired
hybridomas. The selective growth of the fusion cells in a selection
medium may be used for screening as follows: The cell suspension is
diluted 5- to 10-fold with e.g. E-RDF medium containing 15% fetal
bovine serum and then put to each well of a microtiter plate at
about 10.sup.2 to 10.sup.6 cells/well, followed by addition of a
selection medium (e.g. HAT medium) to each well. Thereafter, the
cells are incubated while the selection medium is exchanged with
fresh one at suitable intervals.
[0043] Where myeloma cells are from an 8-azaguanine-resistant
strain and HAT medium is used as selection medium, the myeloma
cells and antibody-producing cells, if they fail to fuse, will die
during in vitro culture in about 7 days and antibody-producing
cells in 10 days. Hence, hybridomas can be obtained from those
cells beginning to grow after the 10th day of culture.
[0044] The hybridomas are screened for the desired ones by
examining their supernatants on the presence of the antibodies
against L-PGDS. This screening step can be carried out by any of
the conventional methods. For example, a supernatant (first
antibody) from hybridomas grown in each well is put to a well with
L-PGDS immobilized on it, then a labeled secondary antibody is
added to the well and incubated, and the binding ability of the
secondary antibody is then examined in enzyme immunoassays (EIA,
ELISA), RIA, etc.
[0045] In more detail, the screening of hybridomas is carried out
as follows: Natural or recombinant L-PGDS, which was used as
immunogen, is immobilized as antigen onto a 96-well microtiter
plate. A culture supernatant expected to contain the monoclonal
antibody is added to each well and reacted with the immobilized
antigen. Then, the antigen-bound monoclonal antibody, if any, is
reacted with another antibody (enzyme-labeled anti-immunoglobulin
antibody). Alternatively, said immobilized monoclonal antibody is
reacted with a biotinylated anti-immunoglobulin antibody and then
with enzyme-labeled avidin. Finally, each well is colored by adding
an enzyme substrate solution. The hybridomas whose culture
supernatants are colored in the wells having the immobilized
natural or recombinant L-PGDS are those producing antibodies having
the ability to bind to the L-PGDS.
[0046] These hybridomas can be cloned in conventional methods
including limiting dilution, soft agar cloning, fibrin gel cloning
and fluorescence excitation cell sorting to give the desired
monoclonal antibody-producing hybridoma.
[0047] (6) Isolation of the Monoclonal Antibody
[0048] From the resulting hybridoma, the monoclonal antibody can be
isolated using conventional methods such as cell culture method,
ascites transudate method, etc.
[0049] In the cell culture method, the hybridoma is cultured for 2
to 14 days in a medium such as RPMI-1640, MEM, or E-RDF containing
10 to 20% calf serum or in a serum-free medium under conventional
culture conditions, e.g. 37.degree. C., 5% CO.sub.2. The antibody
can be obtained from the culture.
[0050] In the ascites transudate method, a mineral oil such as
pristane (2,6,10,14-tetramethylpentadecane) is administrated by
i.p. to the same mammal species as the mammal from which the
myeloma cells were derived. Then, the hybridoma, 1.times.10.sup.7
to 1.times.10.sup.9 cells, preferably 5.times.10.sup.7 to
1.times.10.sup.8 cells, are administrated by i.p. to the animal,
and a large amount of hybridoma cells are grown in the animal.
After 1 to 4 weeks, preferably 2 to 3 weeks, ascites fluid or serum
is collected from the animal.
[0051] If it is necessary to purify the antibody from the ascites
fluid or serum, it can be purified by conventional methods such as
salting-out with ammonium sulfate, ion-exchange chromatography on
anion exchanger e.g. DEAE cellulose, affinity chromatography on
Protein A Sepharose, and gel filtration, and these may be used
singly or in combination.
[0052] 2. Method of Detecting L-PGDS by the Monoclonal Antibody of
the Present Invention
[0053] The method of detecting L-PGDS according to the present
invention can be carried out using said monoclonal antibody, as
follows:
[0054] A 96-well microtiter plate is coated with a diluted sample
such as CSF, serum etc. and then blocked with e.g. 0.2% gelatin in
PBS. Then, the monoclonal antibody of the present invention,
labeled with an enzyme, is added to each well and then incubated;
alternatively, the monoclonal antibody labeled with biotin is added
to each well, then the plate is washed, enzyme-labeled avidin or
streptoavidin is added to each well, and the plate is further
incubated. Then, the plate is washed and a coloration substrate
such as ABTS (2,2'-azino-di-(3-ethyl-benzothiazoline- -6-sulfonic
acid)) is added to each well. L-PGDS can be determined by examining
this coloration in the colorimetric method.
[0055] In another embodiment of the present invention, a 96-well
microtiter plate is coated with the diluted monoclonal antibody of
the present invention and then blocked with e.g. 0.2% gelatin in
PBS. Then, a diluted sample such as CSF, serum etc. is added to
each well and the plate is incubated. After washing the plate,
another enzyme-labeled monoclonal or polyclonal antibody solution
is added to each well and the plate is incubated; alternatively,
the monoclonal antibody or polyclonal antibody labeled with biotin
is added to each well, then the plate is washed, enzyme-labeled
avidin or streptoavidin is added to each well, and the plate is
further incubated. Then, the plate is washed and a coloration
substrate such as ABTS (2,2'-azino-di-(3-ethyl-benzothiazoline-
-6-sulfonic acid)) is added to each well. The L-PGDS can be
determined by examining this coloration in the calorimetric method.
In this manner, it is possible to detect and quantify L-PGDS.
[0056] 3. A Reagent for Measurement of L-PGDS by the Monoclonal
Antibody of the Present Invention
[0057] The monoclonal antibody of the present invention is useful
as a reagent for measurement of L-PGDS because it specifically
binds to L-PGDS. Furthermore, the present monoclonal antibody is
useful as a reagent in determining the presence and distribution of
not only L-PGDS as antigen but also other similar antigens having
the same epitope as that of L-PGDS, as well as fragments of L-PGDS
in biological samples such as organs, tissues, cells and humor.
Hence, the antibody of the present invention is useful as a reagent
for such measurement and diagnosis. The detection or measurement of
L-PGDS in organs, tissues, cells and humor can be effected using
quantitative or qualitative means such as EIA, ELISA, RIA, FIA,
Western blot technique and immunohistochemistry, etc.
[0058] 4. A Kit for Detection of L-PGDS
[0059] The kit of the present invention, if an enzyme is used as a
label for detection, contains the following ingredients:
[0060] (1) monoclonal antibody labeled with an enzyme; and
[0061] (2) substrate.
[0062] The kit of the present invention, if modified with the
sandwich ELISA method, contains the following ingredients:
[0063] (1) monoclonal antibody;
[0064] (2) monoclonal or polyclonal antibody labeled with an
enzyme; and
[0065] (3) substrate.
[0066] The kit of the present invention, if modified with the
biotin-avidin method, contains the following ingredients:
[0067] (1) biotinated monoclonal antibody;
[0068] (2) enzyme-labeled avidin or streptoavidin; and
[0069] (3) substrate.
[0070] The kit of the present invention, if modified with the
sandwich ELISA and biotin-avidin methods, contains the following
ingredients:
[0071] (1) monoclonal antibody;
[0072] (2) biotinated monoclonal or polyclonal antibody;
[0073] (3) enzyme-labeled avidin or streptoavidin; and
[0074] (4) substrate.
[0075] In the ingredients, the "monoclonal antibody" means the
monoclonal antibody of the present invention. The "polyclonal
antibody" means an antibody contained in serum from an animal
immunized with L-PGDS, and it can be prepared in the following
manner.
[0076] (1) Preparation of the Antigen
[0077] L-PGDS can be produced in large amounts in a usual manner by
E. coli and CHO cells etc. with its known cDNA (Nagata et al.,
Proc. Natl. Acad. Sci. USA, 88, 4020-4024 (1991)). For production
of L-PGDS, a recombinant DNA containing the cDNA for L-PGDS is
constructed and transformed into a microorganism, and the
transformant is cultured to produce the enzyme. The resulting
L-PGDS can be purified from the culture by conventional means.
[0078] L-PGDS thus obtained is dissolved in a buffer, and an
immunogen is prepared by adding an adjuvant to it. Examples of
adjuvants are Freund complete adjuvant, Freund incomplete adjuvant,
BCG, Hunter's Titermax (CytRx Corporation), key hole limpet
hemocyanin-containing oil, etc., and any of them can be mixed.
[0079] (2) Immunization and Preparation of Blood
[0080] The immunogen thus obtained is administrated into mammals
such as horse, monkey, dog, pig, cow, goat, sheep, rabbit, guinea
pig, hamster and mouse, or birds such as pigeon and chicken, among
which mouse, rat, guinea pig, rabbit and goat are preferably used.
Any of the known immunization methods can be employed preferably
via i.v., s.c., or i.p. administration. Immunization intervals are
not particularly limited, and this administration is carried out 2
to 10 times, preferably 2 to 5 times, at intervals of preferably
several days to several weeks, more preferably 1 to 3 weeks.
[0081] Antibody titer in blood from the immunized animal is
determined according to the above method in (3) "1. Production of
the monoclonal antibody". Blood samples found to have high antibody
titer are left at room temperature or 4.degree. C. and centrifuged
to give serum containing the polyclonal antibody.
[0082] If it is necessary to purify the polyclonal antibody from
the serum, it can be purified by conventional methods such as
salting-out with ammonium sulfate, ion-exchange chromatography on
anion exchanger e.g. DEAE cellulose, affinity chromatography on
Protein A Sepharose, and gel filtration separating molecules
depending on molecular weight and structure, and these may be used
in singly or in combination.
[0083] According to the present invention, various diseases can be
detected by use of the present kit for detection of L-PGDS. For
example, oligospermia can be diagnosed readily and rapidly by the
present kit using the monoclonal antibody.
[0084] The monoclonal antibody of the present invention can also be
used to purify L-PGDS. That is, the monoclonal antibody of the
present invention is coupled in a usual manner to carriers such as
agarose, cellulose, acrylamide gel, commercially available
self-made affinity carriers and then washed. L-PGDS can be purified
easily with high yield by elution from the column with a suitable
solvent or buffer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0085] FIG. 1 is a photograph showing a profile in SDS-PAGE.
[0086] FIG. 2 is a photograph showing a profile in SDS-PAGE and
Western blotting of CSF and purified L-PGDS.
[0087] FIG. 3 is a photograph showing a profile in Western blotting
of purified L-PGDS treated with N-glycanase.
[0088] FIG. 4 shows the result of an epitope mapping of each
monoclonal antibody.
[0089] FIG. 5 is a calibration curve for L-PGDS (by monoclonal
antibody 1B7).
[0090] FIG. 6 is a calibration curve for L-PGDS (by monoclonal
antibody 6F5).
[0091] FIG. 7 is a calibration curve for L-PGDS (by sandwich ELISA
method using monoclonal antibody 1B7 as primary antibody and
biotinylated polyclonal antibody as secondary antibody).
[0092] FIG. 8 is a calibration curve for L-PGDS (by sandwich ELISA
method using monoclonal antibody 6F5 as primary antibody and
biotinylated polyclonal antibody as secondary antibody).
[0093] FIG. 9 is a calibration curve for L-PGDS (by sandwich ELISA
method using monoclonal antibody 1B7 as primary antibody and
biotinylated polyclonal antibody (A), biotinylated 7B5 (B) and
biotinylated 10A3 (C) as secondary antibodies).
[0094] FIG. 10 is a calibration curve for L-PGDS (by sandwich ELISA
method using monoclonal antibody 10A3 as primary antibody and
biotinylated polyclonal antibody (A) and biotinylated 1B7 (B) as
secondary antibodies).
[0095] FIG. 11 is a calibration curve for L-PGDS (by sandwich ELISA
method using monoclonal antibody 7F5 as primary antibody and
biotinylated polyclonal antibody (A) and biotinylated 1B7 (B) as
secondary antibodies).
[0096] FIG. 12 is a drawing showing a calibration curve prepared
using L-PGDS and calibration curves for L-PGDS which were prepared
using several samples.
[0097] FIG. 13 is a photograph showing a profile in Western
blotting.
[0098] FIG. 14 shows the result of measurement of L-PGDS.
[0099] FIG. 15 is a photograph showing a profile in SDS-PAGE of
L-PGDS purified by monoclonal antibody.
BEST MODE FOR CARRYING OUT THE INVENTION
[0100] Hereinafter, the present invention is described in more
detail by reference to Examples. However, the present invention is
not limited to the Examples.
Example 1
Production of the Monoclonal Antibody
[0101] (1) Preparation of the Antigen
[0102] L-PGDS was prepared as antigen by genetic engineering.
[0103] A GST gene fusion system (Pharmacia) was used for expression
of the antigen in E. coli and purification. The following procedure
was carried out for fusion of L-PGDS with GST protein.
[0104] A 185 bp product was obtained by amplifying a region of cDNA
coding for N-terminal region of L-PGDS by polymerase chain reaction
(PCR) in the following manner.
[0105] The primer nucleotide sequences used are:
[0106] Ec23ALA: Sequence ID NO:1.
[0107] 78NMUTA: Sequence ID NO:2.
[0108] PCR was carried out using Taq DNA polymerase (Takara Shuzo
Co., Ltd.), restriction enzymes EcoRI and XhoI, and T4 DNA ligase
(Takara Shuzo) where 1 cycle reaction (94.degree. C. for 5 seconds,
45.degree. C. for 3 seconds, and at 72.degree. C. for 5 seconds)
was repeated 28 times.
[0109] With these primers, the partial nucleotide sequence between
the 152 to 327 positions (from guanine (G) to cytosine (C),
corresponding to the partial amino acid sequence between the
N-terminal (alanine) and the 81 position (serine) on the amino acid
sequence of the mature protein excluding its signal sequence) was
amplified and an EcoRI site was introduced into the 5'-terminal.
Because this PCR product had an XhoI site at the 238 position, the
product was subcloned by digestion with restriction enzymes EcoRI
and XhoI. A recombinant DNA was obtained by replacement, by the
subcloned product, of the corresponding N-terminal region of the
cDNA for native L-PGDS (Nagata et al., Proc. Natl. Acad. Sci. USA,
88, 4020-4024 (1991)). This recombinant DNA was inserted into an
EcoRI site of vector pGEX-2T (Pharmacia) which was selected for GST
fusion protein.
[0110] Alternatively, a 521 bp product was obtained by amplifying
that region of cDNA coding for the whole mature protein of L-PGDS
by PCR as described below.
[0111] The primer nucleotide sequences used are:
[0112] forward primer: Sequence ID NO:3.
[0113] reverse primer: Sequence ID NO:4.
[0114] PCR was carried out using Taq DNA polymerase (Takara Shuzo)
where 1 cycle reaction (94.degree. C. for 5 seconds, 45.degree. C.
for 3 seconds, and at 72.degree. C. for 5 seconds) was repeated 28
times.
[0115] With these primers, the region.between the 152 to 656
positions (from guanine (G) to adenine (A), corresponding to the
region between the N-terminal (alanine) to the C-terminal
(glutamine) of the amino acid sequence of the mature protein
excluding its signal sequence) was amplified, and a BamHI site was
introduced at the 5'-terminal and an EcoRI site at the 3'-terminal.
The amplified DNA was inserted into EcoRI/BamHI sites of vector
pGEX-2T (Pharmacia) for GST fusion protein.
[0116] The resulting expression vector for GST-L-PGDS fusion
protein was transformed in a usual manner into E. coli DH5.alpha.
or JM109. The fusion protein produced by the transformant was
recovered by selective absorption onto affinity chromatography
beads (Pharmacia) and subsequent elution with thrombin according to
manufacture's instructions. In this manner, about 2 mg L-PGDS was
obtained from 100 ml culture of the transformant.
[0117] Proteins produced by 2 independent clones (E. coli
DH5.alpha./pGDS2 and E. coli DH5.alpha./pGDS7) were analyzed by
SDS-PAGE on 10-20% gradient gel. The results are shown in FIG.
1.
[0118] In FIG. 1, lane 1 shows bands of molecular-weight markers;
lanes 2 to 5, from one of the above clones; lanes 6 to 9, from the
other clone; lanes 2 and 6, homogenates of the respective clones;
lanes 3 and 7, fractions not absorbed onto the affinity column;
lanes 4 and 8, fractions eluted with thrombin; and lanes 5 and 9,
fractions eluted with a buffer containing reduced glutathione. In
lanes 2 and 6, a band corresponding to a molecular weight of about
45 kDa is the fusion protein, and this band is scarcely observed in
the GST-unbound fractions (lanes 3 and 7). The fractions eluted
with glutathione (lanes 5 and 9) showed the fusion protein band and
GST band (molecular weight of about 25 kDa), which were not eluted
with thrombin.
[0119] According to these results in SDS-PAGE, L-PGDS was produced
in E. coli in the form of an about 45 kDa fusion protein with GST,
and the protein with a molecular weight of about 20 kDa, eluted
with thrombin (lanes 4 and 8), is L-PGDS itself because this
molecular weight corresponds to the molecular weight of 20 kDa
deduced from the nucleotide sequence for L-PGDS. (2) Preparation of
the antibody-producing cells A 0.5 ml solution containing 500,
.mu.g L-PGDS obtained in (1) was mixed with 0.5 ml Freund complete
adjuvant and emulsified for 3 to 5 minutes. As antigen, 100 .mu.l
of the emulsion was administrated by s.c. into the tail rump of a
BALB/c mouse. 3 weeks after the first immunization, the same volume
of another antigen emulsion in Freund incomplete adjuvant was
administrated by i.p. to the mouse for boosting. 3 weeks after the
second immunization, 100 .mu.g antigen (100 .mu.g antigen/200 .mu.l
PBS) was administrated to each mouse via its tail vein. 3 days
after the final immunization, the spleen was excised from the
immunized mouse and disrupted in E-RDF medium to give a cell
suspension.
[0120] (3) Cell Fusion
[0121] The suspended 1.times.10.sup.8 spleen cells were subject to
cell fusion with 1.times.10.sup.7 mouse myeloma cells P3-X63-Ag8-U1
(P3-U1) or P3-X-63-Ag8.653 in 50% (W/V) PEG (molecular weight
1,500, Boehringer Mannheim) according to the method of Oi and
Herzenberg (Selected Methods in Cellular Immunology, 351-371, W. H.
Freeman & Co., USA press, 1980).
[0122] (4) Selection of the Hybridoma
[0123] According to the above-mentioned method of Oi et al., the
desired hybridoma was selected in HAT medium, i.e. E-RDF medium
containing 1.36 mg/dl hypoxanthine, 19.1 .mu.g/dl aminopterin, 387
.mu.g/dl thymidine, 10% fetal bovine serum and 5% Origen HCF
(IGEN).
[0124] (5) Selection of the Monoclonal Antibodies
[0125] The antibody-positive cells in 15 wells were cloned by
repeating limiting dilution at least twice. The resulting 6 clones
were cultured to give strains producing a significant amount of the
monoclonal antibody specific to L-PGDS, as follows:
[0126] 1.times.10.sup.7 hybridoma cells were cultured at 37.degree.
C. for 4 days in 5% Co.sub.2 in the 225 cm.sup.2 flask containing
50 ml E-RDF medium with 10% fetal calf serum. Among the resulting 6
cell lines, 5 lines were selected. The results are shown in Table
1.
1TABLE 1 number of number of cell number of antibody- number of
antibody Antigen total wells growth wells positive wells
established wells hPGDS 960 778 15 6 hPGDS: human lipocalin-type
prostaglandin D synthase.
[0127] In Table 1, "number of cell growth wells" means the number
of wells where hybridomas could grow in selective culture in HAT
medium; "number of antibody-positive wells", the number of wells
where antibody production was detected by ELISA using the antigen
prepared in Example 1 (1); and "number of antibody established
wells", the number of wells where hybridomas producing the specific
antibody were established by cloning. The 5 selected cell lines
were designated 1B7, 6F5, 7F5, 9A6 and 10A3, respectively. The
antibodies produced by these cell lines were given the same
designations as above. The cell lines 1B7, 6F5, 7F5, 9A6 and 10A3
have been deposited as FERM BP-5709 (original deposit date:
September 21, 1995), FERM BP-5710 (original deposit date: September
21, 1995), FERM BP-5711 (original deposit date: June 6, 1996), FERM
BP-5712 (original deposit date: June 6, 1996) and FERM BP-5713
(original deposit date: Jun. 6, 1996), respectively, with National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology (Higashi 1-1-3, Tsukuba City, Ibaragi Pref.,
Japan).
[0128] (6) Production of the Monoclonal Antibodies
[0129] Each of the cell lines 1B7, 6F5, 7F5, 9A6 and 10A3, selected
above in (5), was administrated by i.p. to mice. Remaining
resulting cell strain, 6B9 was also administrated by i.p. to mice.
Briefly, 1 ml pristane was administrated by i.p. to each mouse. 2
weeks thereafter, 1 X10.sup.8 hybridoma cells were inoculated
intraperitoneally to the mouse, and 2 weeks thereafter, the
transuded ascites was collected from the mouse.
[0130] The collected ascites was applied to Protein A affinity
column chromatography according to a conventional method.
[0131] As a result, 3 to 10 mg/ml antibodies against L-PGDS were
obtained.
[0132] Typing of the antibodies of 1B7, 6F5 with Isotyping Kit
(RPN29, AMERSHAM) indicated that all of them belong to IgGl
subclass and possess .lambda. light chain.
EXAMPLE 2
Properties of the Monoclonal Antibodies
[0133] (1) Specificity Examination by ELISA
[0134] A 96-well microtiter plate was coated with various types of
L-PGDS and blocked with 0.2% gelatin in PBS. After blocking, each
of the 6 antibodies obtained in Example 1 (5) was examined for
specificity in ELISA after adding 50 .mu.l antibody solution to
each well. The results are shown in Table 2.
2TABLE 2 antigen 1B7 6B9 6F5 7F5 9A6 10A3 E. coli antigen
.smallcircle. .smallcircle. .smallcircle. .smallcircle.
.smallcircle. .smallcircle. E. coli lysate x x x x x x CHO antigen
.smallcircle. .smallcircle. .smallcircle. .smallcircle. x
.smallcircle. CSF antigen .smallcircle. .smallcircle. .smallcircle.
x x x .smallcircle.: Reactivity was observed. x: No reactivity was
observed. The antigens in the table are as follows: E. coli
antigen, expressed in E. coli and purified by affinity
chromatography; E. coli lysate, carrying a vector not containing
L-PGDS cDNA (negative control); CHO antigen, expressed in CHO cells
and purified by a conventional method; and CSF antigen, purified
from CSF by a conventional method.
[0135] The results in Table 2 suggested that at least 3 antibodies
1B7, 6B9 and 6F5 specifically recognize L-PGDS itself.
[0136] (2) Specificity Examination by Western Blotting
[0137] The above antigens were electrophoresed by SDS-PAGE on 16%
isocratic gel and then subjected to Western blot analysis using 5
kinds of antibody. The results are shown in Table 3.
3 TABLE 3 antigen 1B7 6B9 6F5 9A6 10A3 E. coli antigen
.smallcircle. .smallcircle. .smallcircle. .smallcircle.
.smallcircle. .smallcircle. E. coli lysate x x x x x CHO antigen
.smallcircle. .smallcircle. .smallcircle. x O .smallcircle.: A
signal was observed. x: No signal was observed.
[0138] A signal, appearing in an immunoblot profile obtained in
coloration after Western blotting, was detected at a position
corresponding to the molecular weight (about 20 to 30 kDa) of
prostaglandin D synthase. Hence, the antigens showing such signals
are considered specific to prostaglandin D synthase.
[0139] FIG. 2 shows the results of Western blot analysis using CSF
antigen and CSF itself.
[0140] Each lane A shows CSF where 2 .mu.l was applied. Each lane B
shows L-PGDS (CSF antigen) purified from CSF where 50 ng was
applied. Silver staining indicated a large number of proteins in
CSF, and the purified L-PGDS appears as a broad band with a
molecular weight of about 27 kD.
[0141] Each monoclonal antibody was used in Western blotting. The
results revealed that all monoclonal antibodies (1B7, 6B9, 6F5,
7F5, 9A6 and 10A3) are reactive exclusively to the single protein
(lane A) corresponding to the purified L-PGDS (lane B), indicating
that these antibodies specifically recognize L-PGDS without
reacting to other contaminants in CSF.
[0142] In addition, the purified L-PGDS was treated with
N-glycanase and then subjected to Western blot analysis. The
results are shown in FIG. 3. Each lane A shows the sample treated
with glycanase, and each lane B shows the sample not treated with
glycanase. As shown in FIG. 3, there is no significant difference
in band density before and after treatment with glycanase in the
case of 1B7, 6B9, 6F5, 7F5, and 10A3, while in the case of 9A6, the
intensity of the band was significantly increased after treatment
with glycanase, suggesting that 9A6 recognizes a glycosylation site
or therearound.
[0143] Then, the isotype of each monoclonal antibody was determined
using a mouse monoclonal antibody typing kit (RPN29, Amersham). The
results are shown in Table 4. Further, their Kd values toward the
E. coli antigen and CSF antigen were determined by the method of
Friguet et al. (J. Immunol. Methods, 77, 305-319 (1985)). The
results are shown in Table 4.
4TABLE 4 monoclonal immunoglobulin dissociation constant (nM)
antibody subclass E. coli antigen CSF antigen 1B7
IgG.sub.1(.lambda.) 5.4 3.9 6B9 IgG.sub.1(.kappa.) >1000
>1000 6F5 IgG.sub.1(.lambda.) 13.2 10.3 7F5 IgG.sub.1(.kappa.)
0.65 4.1 9A6 IgG.sub.2a(.kappa.) 7.42 >1000 10A3
IgG.sub.1(.kappa.) 0.53 3.9
[0144] 1B7, 6F5, 7F5 and 10A3 showed high affinities for the E.
coli antigen and CSF antigen, indicating that these monoclonal
antibodies recognize the surface of native L-PGDS molecule. 9A6
showed a high affinity for the E. coli antigen, but it showed least
reactivity to the CSF antigen. The results, along with these
results in FIG. 3, suggested that 9A6 recognizes a glycosylation or
therearound. 6B9 showed reactivity in Western blotting as shown in
FIG. 2, but showed no reactivity to the E. coli antigen or the CSF
antigen, suggesting its recognition of the denatured molecule of
L-PGDS.
[0145] Then, deletion mutants of L-PGDS were prepared by truncation
of its partial N-terminal amino acid sequence step by step and used
as antigens for epitope mapping of their monoclonal antibodies. The
PCR primers used in this experiment are shown below and the results
are shown in FIG. 4. The antigens used in this experiment were
prepared in essentially the same way as in Example 1(1) except that
the L-PGDS product was recovered in the form of a fusion protein
with GST by boiling in 1% SDS, not by treatment with thrombin.
[0146] forward primer A (amino acids 1 to 7): SEQ ID NO:3.
[0147] forward primer B (amino acids 7 to 12): SEQ ID NO:5.
[0148] forward primer C (amino acids 13 to 18): SEQ ID NO:6.
[0149] forward primer D (amino acids 30 to 35): SEQ ID NO:7.
[0150] forward primer E (amino acids 52 to 57): SEQ ID NO:8.
[0151] forward primer F (amino acids 68 to 73): SEQ ID NO:9.
[0152] forward primer G (amino acids 85 to 90): SEQ ID NO:10.
[0153] forward primer H (amino acids 99 to 105): SEQ ID NO:11.
[0154] forward primer I (amino acids 118 to 123): SEQ ID NO:12.
[0155] forward primer J (amino acids 134 to 139): SEQ ID NO:13.
[0156] forward primer K (amino acids 152 to 158): SEQ ID NO:14.
[0157] reverse primer (amino acids 163 to 168): SEQ ID NO:4.
[0158] FIG. 4 suggested that 7F5 and 10A3 recognize a sequence in
Ala.sup.1-Val.sup.6; 9A6, Gln.sup.13-Asn.sup.29; 6F5,
Tyr.sup.85-Val.sup.98; and 1B7 and 6B9, Gly.sup.118-Pro.sup.133
[0159] (3) Preparation of Calibration Curves
[0160] The above E. coli antigen was used for preparation of
calibration curves for L-PGDS. The monoclonal antibodies used were
1B7 and 6F5. A 96-well microtiter plate was coated with diluted E.
coli antigen and blocked with 0.2% gelatin in PBS. After blocking,
the biotinylated monoclonal antibody 1B7 or 6F5 was added to each
well and incubated. Then, the plate was washed, and a
streptoavidin-horseradish peroxidase conjugate was added to each
well and incubated. The plate was washed, and a coloration
substrate ABTS (2,2'-azino-di-(3-ethyl-benzothiazoline-6-sul- fonic
acid) was added to each well. L-PGDS was determined by measuring
this coloration by the calorimetric method. The results are shown
in FIGS. 5 and 6 for 1B7 and 6F5, respectively.
[0161] Then, the sandwich ELISA method was used for calibration
curves for L-PGDS. A 96-well microtiter plate was coated with
diluted monoclonal antibody 1B7 or 6F5 and blocked with 0.2%
gelatin in PBS. After blocking, the diluted E. coli antigen was
added to each well and incubated. Then, the plate was washed, and
the biotinylated polyclonal antibody was added to each well and
incubated. Then, the plate was washed, and a
streptoavidin-horseradish peroxidase conjugate was added to each
well and incubated. The plate was washed, and a coloration
substrate ABTS (2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulfonic
acid) was added to each well. L-PGDS was determined by measuring
this coloration by the calorimetric method. The results are shown
in FIGS. 7 and 8 for 1B7 and 6F5, respectively. As can be seen from
FIGS. 5 to 8, about 10-fold higher sensitivity was attained using
the sandwich ELISA method.
[0162] In another embodiment of the present invention, the sandwich
ELISA method and the biotinylated polyclonal or monoclonal antibody
were used for preparation of calibration curves. A 96-well
microtiter plate was coated with diluted monoclonal antibody 1B7,
10A3 or 7F5 as primary antibody and blocked with 0.2% gelatin in
PBS. The diluted E. coli antigen was then added to each well and
incubated. Then, the plate was washed, and the biotinylated
polyclonal antibody or the biotinylated monoclonal antibody 1B7,
7F5 or 10A3 was added as secondary antibody to each well and
incubated. The plate was washed, and a streptoavidin-horseradish
peroxidase conjugate was added to each well and incubated. The
plate was washed, and a coloration substrate ABTS
(2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) was added
to each well. L-PGDS was determined by measuring this coloration by
the calorimetric method.
[0163] The results are shown in FIGS. 9 to 11.
[0164] FIG. 9 shows the calibration curves using monoclonal
antibody 1B7 as primary antibody, where curve A makes use of the
biotinylated polyclonal antibody as secondary antibody; curve B,
biotinylated 7F5 as secondary antibody; and curve C, biotinylated
10A3 as secondary antibody.
[0165] FIG. 10 shows the resulting calibration curves using
monoclonal antibody 10A3 as primary antibody, where curve A makes
use of the biotinylated polyclonal antibody as secondary antibody;
and curve B, biotinylated 1B7 as secondary antibody.
[0166] FIG. 11 shows the calibration curves using monoclonal
antibody 7F5 as primary antibody, where curve A makes use of the
biotinylated polyclonal antibody as secondary antibody; and curve
B, biotinylated 1B7 as secondary antibody.
[0167] As can be seen from FIGS. 9 to 11, high sensitivity can be
attained using 10A3 or 7F5 as primary antibody and the biotinylated
polyclonal antibody, or biotinylated monoclonal antibody 1B7, as
secondary antibody.
EXAMPLE 3
Detection of L-PGDS Derived from Various Human Tissues
[0168] (1) Detection of L-PGDS in CSF
[0169] A 96-well microtiter plate was coated with diluted
monoclonal antibody 1B7 or 6F5 and then blocked with 0.2% gelatin
in PBS. Diluted human CSF was then added to each well and
incubated. Separately, the polyclonal antibody prepared using a
rabbit as an immunized animal was purified by affinity column
chromatography on Protein A and then biotinylated with
NHS-LC-Biotinylation Kit (PIERCE).
[0170] Subsequently, the above microtiter plate was washed, and the
biotinylated polyclonal antibody was added to each well and then
incubated. Subsequently, a streptoavidin-horseradish peroxidase
conjugate was added to each well and incubated. The plate was
washed, and a coloration substrate ABTS
(2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulfo- nic acid) was
added to each well. L-PGDS was determined by measuring this
coloration by the calorimetric method. The results are shown in
Table 5.
5 TABLE 5 Sample No. 1 2 3 4 5 L-PGDS (.mu.g/ml) 23 31 20 10 18
[0171] (2) Detection of L-PGDS in Blood
[0172] L-PGDS in human serum obtained by centrifugation of human
blood was determined by the same method as in (1) above except that
diluted serum was used. The results are shown in Table 6.
6 TABLE 6 Sample No. 1 2 3 4 5 L-PGDS (.mu.g/ml) 0.45 0.29 0.21
0.41 0.26
[0173] (3) Detection of L-PGDS in Amniotic Fluid
[0174] L-PGDS in human amniotic fluid was determined by the same
method as (1) above except that diluted amniotic fluid was used.
The results are shown in Table 7.
7 TABLE 7 Sample No. 1 2 3 4 5 L-PGDS (.mu.g/ml) 5.5 1.2 1.3 2.9
1.8
[0175] (4) Detection of L-PGDS in Semen Supernatant
[0176] L-PGDS in human seminal plasma was determined by the same
method in (1) above except that diluted seminal plasma was used.
The results are shown in Table 8.
8 TABLE 8 Sample No. 1 2 3 4 5 L-PGDS (.mu.g/ml) 4.1 12 23 10
8.3
[0177] (5) Detection of L-PGDS in Follicular Fluid
[0178] L-PGDS in human follicular fluid was determined by the same
method in (1) above except that diluted follicular fluid was used.
The results are shown in Table 9.
9 TABLE 9 Sample No. 1 2 3 4 5 L-PGDS (.mu.g/ml) 0.21 0.11 0.11
0.15 0.13
[0179] (6) Detection of L-PGDS in Urine
[0180] L-PGDS in human urine was determined by the same method
in
[0181] (1) above except that diluted urine was used. The results
are shown in Table 10.
10 TABLE 10 Sample No. 1 2 3 4 5 L-PGDS (.mu.g/ml) 1.07 0.65 2.45
1.62 0.32
[0182] (7) Detection of L-PGDS in Various Humors
[0183] The sandwich ELISA system (7F5 monoclonal antibody.times.
biotinylated 1B7 monoclonal antibody) established in Example 2(3)
was used for quantifying L-PGDS in human CSF, serum and urine. The
results (means (.+-.SE)) were compared with those of previous
literatures. In this assay, TMBLUE (Intergen-CDP) was used as
colouring substrate in place of ABTS.
[0184] The measurement means are shown in Table 11.A, and those of
the literatures are shown in Table 11.B. L-PGDS levels in amniotic
fluid and seminal plasma were also determined in the same manner,
and the results (means (.+-.SE)) are shown in Table 11.A, as
well.
11 TABLE 11.A sample number of samples means (.+-.SE) (.mu.g/ml)
CSF 38 12.07 .+-. 1.26 serum 12 0.27 .+-. 0.01 urine 10 1.56 .+-.
0.30* amniotic fluid 52 2.55 .+-. 0.22 seminal plasma 32 13.01 .+-.
1.72 *Excretion amount (mg) per day
[0185]
12TABLE 11.B number means sample of samples (.+-.SD) (.mu.g/ml)
literature CSF 12 40 Pepe,A.J. and Hochwald, G.M. (1967)
Proc.Soc.Exp.Biol. 126, 630-633 CSF 59 26(.+-.6) Link,H. and
Olsson,J.E. (1972) Acta Neurol.Scadinav. 48, 57-68 CSF 35
27(.+-.1.5) Olsson,J.E., Link,H., and Muller,R. (1976) J.Neurol.
Sci. 27,233-245 CSF 192 33(.+-.11) Felgenhauer,K., Schdlich, H.J.,
and Nekic,M. (1987) Kiln.Wochenschr. 65, 764-768 serum 25
3.9(.+-.0.16) Olsson,J.E., Link,H., and Nosslin,B. (1973)
J.Neurochem. 21, 1153-1159 urine 15 3.6.about.53.9* Whitsed,H. and
Penny,R. (1974) Clin.Chim.Acta 50,119-128 *Excretion amount (mg)
per day (not mean).
[0186] As shown in the tables, the present means are lower than
those of the literatures, particularly with respect to serum
considered to have contaminants in abundance. This difference may
result from the fact that L-PGDS might be overestimated in the
methods of the literatures because of their low specificity, and
high specificity in the present assay system is clarified.
[0187] A typical calibration curve prepared in these measurements
is shown in FIG. 12.
[0188] Curve A is a calibration curve where L-PGDS purified from
CSF or the recombinant L-PGDS prepared in Example 1(1) was used as
the standard substance. Curves B, C, D, E, and F are calibration
curves prepared using CSF, seminal plasma, urine, amniotic fluid,
and serum, respectively. As can be seen from FIG. 12, the sample
calibration curves are nearly parallel with the calibration curve
A, so L-PGDS can be specifically determined in the present assay
system without any influence of contaminants in a sample.
[0189] Then, an additive test in the present assay system was
carried out. Human CSF, serum, urine, and seminal plasma were
diluted respectively to adjust their L-PGDS contents to about 20
ng/ml. Purified L-PGDS (CSF antigen) from CSF was added in an
amount of 15 ng/ml to each sample. The results indicated that good
recovery (98.5 to 104.6%) was obtained in every sample as shown in
Table 12.
13 TABLE 12 dilution degree L-PGDS (ng/ml) recovery sample (-fold)
initial conc. addition recovery (%) CSF 1 350 20.69 15.00 35.65
99.7 CSF 2 600 21.17 15.00 36.31 100.9 serum 1 15 20.71 15.00 36.21
103.3 serum 2 15 13.67 15.00 33.96 101.9 urine 1 40 20.86 15.00
36.55 104.6 urine 2 40 18.56 15.00 33.34 98.5 seminal 500 16.82
15.00 31.70 99.2 plasma 1 seminal 1000 19.51 15.00 34.93 102.8
plasma 2
EXAMPLE 4
[0190] L-PGDS in 5 samples (seminal plasma) was detected by Western
blotting using the monoclonal antibody 1B7. The results are shown
in FIG. 13. As shown in FIG. 13, L-PGDS was detected specifically,
indicating that this monoclonal antibody is reactive exclusively to
L-PGDS in the presence of other contaminants.
[0191] Then, the sandwich ELISA system (monoclonal antibody
1B7.times. biotinated polyclonal antibody) established in Example 2
was used to quantify L-PGDS in 1 ml seminal plasma from each of
healthy normal persons (40 cases) and patients with oligospermia
(10 cases). The number of spermatozoa in 1 ml semen was determined
with a Makler counting chamber. The results are shown in Table
13.
14 number of spermatozoa L-PGDS(.mu.g/ml) sample no.
(.times.10.sup.4 spermatozoa/ml) subject by ELISA 1 0 oligospermia
patient 2.6 2 100 oligospermia patient 0.8 3 100 oligospermia
patient 1.0 4 800 oligospermia patient 3.8 5 800 oligospermia
patient 2.2 6 1000 oligospermia patient 0.9 7 1800 oligospermia
patient 1.0 8 2000 oligospermia patient 5.0 9 2000 oligospermia
patient 4.8 10 2000 oligospermia patient 2.6 11 2500 healthy person
4.3 12 3000 healthy person 7.5 13 3000 healthy person 23.6 14 3000
healthy person 30.0 15 3000 healthy person 9.5 16 3000 healthy
person 2.5 17 3000 healthy person 4.4 18 4000 healthy person 6.0 19
4000 healthy person 30.0 20 4200 healthy person 8.0 21 5000 healthy
person 1.9 22 6000 healthy person 42.0 23 6000 healthy person 1.5
24 6000 healthy person 3.8 25 6500 healthy person 2.0 26 7000
healthy person 3.2 27 7000 healthy person 8.2 28 8000 healthy
person 9.6 29 8000 healthy person 14.0 30 8500 healthy person 6.0
31 8800 healthy person 3.0 32 9000 healthy person 8.0 33 9000
healthy person 7.1 34 10000 healthy person 10.1 35 10000 healthy
person 3.0 36 10000 healthy person 7.5 37 10000 healthy person 6.0
38 11000 healthy person 0.3 39 12000 healthy person 17.0 40 12000
healthy person 2.6 41 12000 healthy person 7.0 42 12000 healthy
person 1.1 43 13000 healthy person 10.5 44 13900 healthy person
30.0 45 15000 healthy person 16.0 46 15000 healthy person 5.1 47
15000 healthy person 3.5 48 16000 healthy person 13.0 49 17400
healthy person 15.0 50 18000 healthy person 6.2
[0192] The results indicated that L-PGDS levels are 9.75.+-.1.486
.mu.g/ml (healthy persons) and 2.470.+-.0.509 .mu.g/ml
(oligospermia patients), and the difference therebetween is
statistically significant at P.ltoreq.0.0008 level (Mann-Whitney
method) or P<0.0192 level (Fisher method), in FIG. 14. Because
many samples can be dealt with in a short time using the kit of the
present invention, it is useful for diagnosis of oligospermia.
Example 5
[0193] One-step purification of L-PGDS from CSF was attempted by
coupling each of monoclonal antibodies 1B7, 6F5, 9A6, 7F5 and 10A3
to carriers by use of an Affi-Gel Hz immunoaffinity kit
(Bio-Rad).
[0194] First, 10 ml gel with each monoclonal antibody coupled
thereto was equilibrated with PBS. A CSF solution, prepared by
diluting 10 ml of CSF at least 3-fold with PBS, was applied to the
gel. The gel was washed with 20 ml PBS containing 2 M NaCl, then 30
ml PBS containing 0.1% Triton X-100, and finally with 50 ml PBS,
and the protein was eluted with 0.1 M sodium citrate (pH 3.0).
Using any kind of antibody, L-PGDS can be efficiently adsorbed onto
the gel and eluted with 0.1 M sodium citrate (pH 3.0), and the
resulting preparation having enzymatic activity was almost
homologous in SDS-PAGE. The yield was about 80% and the product was
purified as high as 37-fold relative to the original CSF.
[0195] One Example of such purification is shown in FIG. 15.
[0196] Industrial Applicability
[0197] According to the present invention, there is provided the
monoclonal antibody specific to L-PGDS.
[0198] The analysis of the distribution of L-PGDS in the central
nervous system is useful for detection of diseases in the central
nervous system, and it is expected that L-PGDS levels in CSF or
humor can also be used as an indicator in early diagnosis and
prognostic observations for other diseases caused by abnormalities
in the central nervous system. It is further expected that L-PGDS
(.beta.trace) can be used for examination of a reproduction
ability, diagnosis of fetal growth, etc. because this enzyme is
distributed in such humors derived from genital organs, as semen,
oviduct fluid and amniotic fluid, as well. Further, it was recently
revealed in our study that the gene for L-PGDS is expressed in the
heart too and therefore the distribution and levels of L-PGDS in
blood and other humors can also be used for diagnosis of diseases
in the circulatory organs.
[0199] Accordingly, the monoclonal antibody provided according to
the present invention is useful as a reagent in studying the
expression, tissue distribution, physiological action etc. of
L-PGDS, and as a reagent for pathological diagnosis of various
diseases in the central nervous system as well as in the genital
and circulatory organs.
Sequence CWU 1
1
15 1 22 DNA Artificial Sequence primer 1 gggaattcat gcacccgagg cc
22 2 20 DNA Artificial Sequence primer 2 gaggtcaggg cgaagccacc 20 3
29 DNA Artificial Sequence primer 3 ccggatccgc acccgaggcc caggtctcc
29 4 30 DNA Artificial Sequence primer 4 atgaattcac tattgttccg
tcatgcactt 30 5 26 DNA Artificial Sequence primer 5 ccggatcctc
cgtgcagccc aacttc 26 6 26 DNA Artificial Sequence primer 6
ccggatccca gccggacaag ttcctg 26 7 26 DNA Artificial Sequence primer
7 ccggatcctc gagctggctc caggag 26 8 26 DNA Artificial Sequence
primer 8 ccggatccga tggtggcttc aacctg 26 9 26 DNA Artificial
Sequence primer 9 ccggatccga gacccgaacc atgctg 26 10 25 DNA
Artificial Sequence primer 10 ccggatccta ccggagtccc cactg 25 11 27
DNA Artificial Sequence primer 11 ccggatccgt ggagactgac tacgacc 27
12 26 DNA Artificial Sequence primer 12 ccggatccgg cgaggacttc
cgcatg 26 13 26 DNA Artificial Sequence primer 13 ccggatccag
ggctgagtta aaggag 26 14 29 DNA Artificial Sequence primer 14
ccggatccga ggattccatt gtcttcctg 29 15 168 PRT Homo sapiens 15 Ala
Pro Glu Ala Gln Val Ser Val Gln Pro Asn Phe Gln Pro Asp Lys 1 5 10
15 Phe Leu Gly Arg Trp Phe Ser Ala Gly Leu Ala Ser Asn Ser Ser Trp
20 25 30 Leu Gln Glu Lys Lys Ala Ala Leu Ser Met Cys Lys Ser Val
Val Ala 35 40 45 Pro Ala Ala Asp Gly Gly Phe Asn Leu Thr Ser Thr
Phe Leu Arg Lys 50 55 60 Asn Gln Cys Glu Thr Arg Thr Met Leu Leu
Gln Pro Gly Asp Ser Leu 65 70 75 80 Gly Ser Tyr Ser Tyr Arg Ser Pro
His Trp Gly Ser Thr Tyr Ser Val 85 90 95 Ser Val Val Glu Thr Asp
Tyr Asp His Tyr Ala Leu Leu Tyr Ser Gln 100 105 110 Gly Ser Lys Gly
Pro Gly Glu Asp Phe Arg Met Ala Thr Leu Tyr Ser 115 120 125 Arg Thr
Gln Thr Pro Arg Ala Glu Leu Lys Glu Lys Phe Thr Ala Phe 130 135 140
Cys Lys Ala Gln Gly Phe Thr Glu Asp Ser Ile Val Phe Leu Pro Gln 145
150 155 160 Thr Asp Lys Cys Met Thr Glu Gln 165
* * * * *