U.S. patent application number 10/723748 was filed with the patent office on 2005-02-24 for monoclonal antibody recognizing phosphatidylinositol-3,4-diphosphate.
This patent application is currently assigned to Yasuhisa Fukui and Medical & Biological Laboratories, Co., Ltd.. Invention is credited to Fukui, Yasuhisa, Nagata, Satoshi, Saito, Naoaki, Shirai, Ryuichi.
Application Number | 20050043514 10/723748 |
Document ID | / |
Family ID | 17204454 |
Filed Date | 2005-02-24 |
United States Patent
Application |
20050043514 |
Kind Code |
A1 |
Fukui, Yasuhisa ; et
al. |
February 24, 2005 |
Monoclonal antibody recognizing
phosphatidylinositol-3,4-diphosphate
Abstract
A novel monoclonal antibody that specifically recognizes
phosphatidylinositol-3,4-biphosphate (PI-3,4-P2) but does not
cross-react with structurally similar phospholipid antigens is
advantageous for PI-3,4-P2-specific immunoassay. The gene in the
variable regions of the monoclonal antibody has been identified,
which enables producing recombinant antibodies.
Inventors: |
Fukui, Yasuhisa; (Tokyo,
JP) ; Nagata, Satoshi; (Rockville, MD) ;
Shirai, Ryuichi; (Ikoma-shi, JP) ; Saito, Naoaki;
(Kobe-shi, JP) |
Correspondence
Address: |
Peter F. Corless
EDWARDS & ANGELL, LLP
P.O. Box 55874
Boston
MA
02205
US
|
Assignee: |
Yasuhisa Fukui and Medical &
Biological Laboratories, Co., Ltd.
|
Family ID: |
17204454 |
Appl. No.: |
10/723748 |
Filed: |
November 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10723748 |
Nov 25, 2003 |
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09518737 |
Mar 3, 2000 |
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6709833 |
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Current U.S.
Class: |
530/387.1 |
Current CPC
Class: |
Y10S 435/975 20130101;
C07K 16/18 20130101; G01N 33/5308 20130101 |
Class at
Publication: |
530/387.1 |
International
Class: |
C07K 016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 3, 1999 |
JP |
11/250209 |
Claims
What is claimed is:
1. An antibody specifically recognizing
phosphatidylinositol-3,4-biphospha- te.
2. The antibody of claim 1, wherein the antibody is a monoclonal
antibody.
3. The antibody of claim 2, which recognizes an antigenic
determinant comprising an inositol group and a glycerol backbone in
phosphatidylinositol-3,4-biphosphate.
4. The antibody of claims 1, which is substantially
non-cross-reactive, with at least one compound selected from the
group consisting of phosphatidylinositol-4,15-bisphospate,
phosphatidylinositol-3,4,5-triphos- phate
phosphatidylinositol-1,4,5-triphosphate, and
phisphatidylinositol-1,- 3,4,5-tetraphosphate.
5. A hybridoma producing the antibody of claim 2.
6. The hybridoma of claim 5, which has the properties of the
deposit identified by the accession No. FERM-BP-6849.
7. A method of producing the antibody of claim 2, the method
comprising culturing the hybridoma of claim 5.
8. A variable region of immunoglobulin heavy chain specifically
binding to phosphatidylinositol-3,4-biphosphate, comprising an
amino acid sequence set forth in SEQ ID NO: 2 or an amino acid
sequence of SEQ ID NO: 2 in which one or more amino acid residues
are substituted, deleted or added.
9. A variable region of immunoglobulin light chain specifically
binding to phosphatidylinositol-3,4-biphosphate, comprising an
amino acid sequence set forth in SEQ ID NO: 4 or an amino acid
sequence of SEQ ID NO: 4 in which one or more amino acid residues
are substituted, deleted or added.
10. CDR1 in immunoglobulin heavy chains specifically binding to
phosphatidylinositol-3,4-biphosphate, comprising an amino acid
sequence set forth in SEQ ID NO: 5 or an amino acid sequence of SEQ
ID NO: 5 in which one or more amino acid residues are substituted,
deleted or added.
11. CDR2 in immunoglobulin heavy chains specifically binding to
phosphatidylinositol-3,4-biphosphate, comprising an amino acid
sequence set forth in SEQ ID. NO: 6 or an amino acid sequence of
SEQ ID NO: 6 in which one or more amino acid residues are
substituted, deleted or added.
12. CDR3 in immunoglobulin heavy chains specifically binding to
phosphatidylinositol-3,4-biphosphate, comprising an amino acid
sequence set forth in SEQ ID NO: 7 or an amino acid sequence of SEQ
ID NO: 7 in which one or more amino acid residues are substituted,
deleted or added.
13. CDR1 in immunoglobulin light chains specifically binding to
phosphatidylinositol-3,4-biphosphate, comprising an amino acid
sequence set forth in SEQ ID NO: 8 or an amino acid sequence of SEQ
ID NO: 8 in which one or more amino acid residues are substituted,
deleted or added.
14. CDR2 in immunoglobulin light chains specifically binding to
phosphatidylinositol-3,4-biphosphate comprising an amino acid
sequence set forth in SEQ ID NO: 9 or an amino acid sequence of SEQ
ID NO: 9 in which one or more amino acid residues are substituted,
deleted or added.
15. CDR3 immunoglobulin light chains specifically binding to
phosphatidylinositol-3,4-biphosphate, comprising an amino acid
sequence set forth in SEQ ID NO: 10 or an amino acid sequence of
SEQ ID NO: 10 in which one or more amino acid residues are
substituted, deleted or added.
16. An immunogen composition for use in producing an antibody
specifically recognizing phosphatidylinositol-3,4-biphosphate,
comprising a mixture of a dead Salmonella cell as an adjuvant and
phosphatidylinositol-3,4-biphos- phate.
17. A method for producing an antibody specifically recognizing
phosphatidylinositol-3,4-biphosphate, the method comprising using
an immunogen composition of claim 16 for immunization.
18. An immunoassay method which comprises the steps of reacting the
antibody specifically recognizing
phosphatidylinositol-3,4-biphosphate or a variable region thereof
with phosphatidylinositol-3,4-biphosphate present in a sample, and
detecting the binding based on an immunological reaction between
the antibody or a variable region thereof and the biphosphate.
19. An immunoassay method of claim 18, which comprises observing
the degree to which the immunological reaction between the antibody
or a variable region thereof and an antigenic determinant
recognized thereby is inhibited by
phosphatidylinositol-3,4-biphosphate present in a sample.
20. A kit for immunoassay for phosphatidylinositol-3,4-biphosphate
comprising the antibody of specifically recognizing
phosphatidylinositol-3,4-biphosphate or a variable region thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a monoclonal antibody to
phosphatidylinositol-3,4-diphosphate and a method for immunoassay
using the monoclonal antibody.
BACKGROUND OF THE INVENTION
[0002] Formerly phospholipids in living organisms was merely
appreciated as constituents of cell membranes. Recently, however,
inositol phospholipid, a member of phospholipids, has been found to
play an important role in the intracellular signal transduction
system. In particular, metabolism of phosphatidylinositol (PI), a
phospholipid present in biomembranes, has been extensively
investigated because abnormality of this system is known to induce
aberrant cell proliferation and causes cancers.
[0003] It was commonly believed that inositol phospholipids are
synthesized as follows: the 4-position of PI is phosphorylated by
the action of phosphatidylinositol-4-kinase (PI4K) to generate
phosphatidylinositol-4-monophosphate the 5-position thereof is then
phosphorylated by the action of
phosphatidylinositol-4-monophosphate-5-ki- nase to generate
phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2), which is
degraded into inositol triphosphate (IP3) and diacylglycerol (DG)
by extracellular stimulation. However, Cantley et al. (Rameh, L. E.
and Cantley, L. C., J. Biol. Chem. Vol. 274, 8347-8350, 1999)
discovered phosphatidylinositol-3-kinase (PI3K) that mediates
phosphorylation at the 3-position of the inositol: ring. They also
demonstrated the products of this enzyme reaction,
phosphatidylinositol-3-monophosphate (PI-3-P),
phosphatidylinositol-3,4-bisphosphate, and
phosphatidylinositol-3,4,5-tri- phosphate (PI-3,4,5-P3).
Phosphatidylinositol-3,4-bisphosphate is also generated by
dephosphorylation of the 5-position of PI-3,4,5-P3. Herein,
phosphatidylinositol-3,4-bisphosphate is abbreviated as PI-3,4-P2
in principle. The structure of PI-3,4-P2 is schematically shown
below. 1
[0004] PI3K is known to be involved in the signal transduction
system of insulin. It is becoming clear that PI-3,4,5-P3 and
PI-3,4-P2, which are produced by the action of PI3K, an enzyme
activated by a stimulus such as insulin also activate kinases such
as phosphoinositidin kinase-1 (PKD-1) and Akt/PKB to generate a
survival signal that suppresses the cell death (apoptosis) (Coffer,
P. J. et al., Biochem. J., Vol. 335, 1-13, 1998). This means that
PI-3,4'-P2 suppresses apoptosis by activating Akt/PKB and thus
takes part in survival of cells.
[0005] Based on this knowledge, specific detection of these
phospholipids in the cells and clarification of the dynamics
thereof have been demanded to shed light on not only mechanisms of
intracellular signal transduction and apoptosis but also
pathogenesis of cancers and other diseases. However, no method of
detecting and measuring PI-3,4-P2 exclusive of other
phosphatidylinositol-polyphosphates is known.
[0006] Antibodies that specifically recognize PI-3,4-P2 are useful
for purification and immunoassay of PI-3,4-P2, and serve as
inhibitors of PI-3,4-P2. However, in general; poor antigenicity of
phospholipids make it difficult to obtain antibodies against them.
Furthermore, PI-3,4-P2 is difficult to be obtained in large
quantities. These problems have prevented the development of an
immunoassay technique for PI-3,4-P2. Immunoassays are so excellent
analytical methods as to achieve a high sensitivity and accuracy by
a simple manipulation. For further investigation of signal
transduction, an immunological assay method for PI-3,4-P2 has been
strongly desired.
SUMMARY OF THE INVENTION
[0007] An objective of the present invention is to provide an
antibody specifically recognizing PI-3,4-P2 and an immunological
assay method using the antibody. More specifically, the present
invention seeks to provide a novel antibody specifically
recognizing PI-3,4-P2 and a simple method for determining PI-3,4-P2
with high sensitivity, like enzyme immunoassay, without requiring
any special facilities.
[0008] Producing an anti-PI-3,4-P2 antibody is a problem because it
is difficult to obtain a large quantity of antigens and the poor
antigenicity of phospholipids used as antigens makes it difficult
to produce an antibody of high titer. The inventors have solved the
former problem by chemical synthesis of PI-3,4-P2 that enables it
to be produced in large quantities. The inventors have also
overcome the problem of poor antigenicity by enhancing the
antigenicity using an immunogen obtained through adsorption of
PI-3,4-P2 to dead Salmonella cells. In this way, the inventors have
succeeded in producing a novel monoclonal antibody that binds
specifically to PI-3,4-P2. Using the antibody, an immunological
assay specific to PI-3,4-P2 in the living organism can be
performed-successfully. Furthermore, the inventors have isolated a
gene encoding the amino acid sequence that constitutes variable
regions of the antibody and have determined the nucleotide
sequence, which will enable producing recombinant antibodies. The
inventors have also found that topological PI-3,4-P2 distribution
in cells can be identified and inhibitors specific to the function
of PI-3,4-P2 can be developed, using the antibody of the present
invention.
[0009] Specifically, the present invention relates to the following
antibody, monoclonal antibody, variable regions thereof, hybridoma
producing the antibody, and an immunological assay method using the
antibody.
[0010] (1) An antibody specifically recognizing
phosphatidylinositol-3,4-b- iphosphate.
[0011] (2) The antibody of (1), wherein the antibody is a
monoclonal antibody.
[0012] (3) The antibody of (2), which recognizes an antigenic
determinant comprising an inositol group and a glycerol backbone in
phosphatidylinositol-3,4-biphosphate.
[0013] (4) The antibody of (1), which is substantially
non-cross-reactive with at least one compound selected from the
group consisting of phosphatidylinositol-4,5-bisphospate,
phosphatidylinbsitol-3,4,5-triphosp- hate
phosphatidylinositol-1,4,5-triphosphate, and
phisphatidylinositol-1,3- ,4,5-tetraphosphate.
[0014] (5) A hybridoma producing the antibody of (2).
[0015] (6) The hybridoma of (5), which has the properties of the
deposit identified by the accession No. FERM-BP-6849.
[0016] (7) A method of producing the antibody of (2), the method
comprising culturing the hybridoma of (5)
[0017] (8) A variable region of immunoglobulin heavy chain
specifically binding to phosphatidylinositol-3,4-biphosphate,
comprising an amino acid sequence set forth in SEQ ID NO: 2 or an
amino acid sequence of SEQ ID NO: 2 in which one or more amino acid
residues are substituted, deleted or added.
[0018] (9) A variable region of immunoglobulin light chain
specifically binding to phosphatidylinositol-3,4-biphosphate,
comprising an amino acid sequence set forth in SEQ ID NO: 4 or an
amino acid sequence of SEQ ID NO: 4 in which one or more amino acid
residues are substituted, deleted or added.
[0019] (10) CDR1 in immunoglobulin heavy chains specifically
binding to phosphatidylinositol-3,4-biphosphate, comprising an
amino acid sequence set forth in SEQ ID NO: 5 or an amino acid
sequence of SEQ. ID NO: 5 in which one or more amino acid residues
are substituted, deleted or added.
[0020] (11) CDR2 in immunoglobulin heavy chains specifically
binding to phosphatidylinositol-3,4-biphosphate, comprising an
amino acid sequence set forth in SEQ ID NO: 6 or an amino acid
sequence of SEQ ID NO: 6 in which one or more amino acid-residues
are substituted, deleted or added.
[0021] (12) CDR3 in immunoglobulin heavy chains specifically
binding to phosphatidylinositol-3,4-biphosphate, comprising an
amino acid sequence set forth in SEQ ID NO: 7 or an amino acid
sequence of SEQ ID NO: 7 in which one or more amino acid residues
are substituted, deleted or added.
[0022] (13) CDR1 in immunoglobulin light chains specifically
binding to phosphatidylinositol-3,4-biphosphate, comprising an
amino acid sequence set forth in SEQ ID NO: 8 or an amino acid
sequence of SEQ ID NO: 8 in which one or more amino acid residues
are substituted, deleted or added.
[0023] (14) CDR2 in immunoglobulin light chains specifically
binding to, phosphatidylinositol-3,4-biphosphate, comprising an
amino acid sequence set forth in SEQ ID NO: 9 or an amino acid
sequence of SEQ ID NO: 9 in which one or more amino acid residues
are substituted, deleted or added.
[0024] (15) CDR3 in immunoglobulin light chains specifically
binding to phosphatidylinositol-3.4-biphosphate, comprising an
amino acid sequence set forth in SEQ ID NO: 10 or an amino acid
sequence of SEQ. ID NO: 10 in which one or more amino acid residues
are substituted, deleted or added.
[0025] (16) An immunogen composition for use in producing an
antibody specifically recognizing
phosphatidylinositol-3,4-biphosphate, comprising a mixture of a
dead Salmonella cell as an adjuvant and
phosphatidylinositol-3,4-biphosphate.
[0026] (17) A method for producing an antibody specifically
recognizing phosphatidylinositol-3,4-biphosphate, the method
comprising using an immunogen composition of (16) for
immunization.
[0027] (18) An immunoassay method which comprises the steps of
reacting the antibody specifically recognizing
phosphatidylinositol-3,4-biphosphat- e or a variable region thereof
with phosphatidylinositol-3,4-biphosphate present in a sample, and
detecting the binding based on an immunological reaction between
the antibody or a variable region thereof and the biphosphate.
[0028] (19) An immunoassay method of (18), which comprises
observing the degree to which the immunological reaction between
the antibody or a variable region thereof and an antigenic
determinant recognized thereby is inhibited by
phosphatidylinositol-3,4-biphosphate present in a sample.
[0029] (20) A kit for immunoassay for
phosphatidylinositol-3,4-biphosphate comprising the antibody
specifically recognizing phosphatidylinositol-3,4- -biphosphate or
a variable region thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is a graph showing the results of indirect
enzyme-linked immunosorbent assay (hereinafter referred to as
ELISA) performed to verify the cross-reactivity of the antibody 8C2
with various inositol polyphosphates. The ordinate designates the
binding (%) of the antibody 8C2, and the abscissa designates the
concentration (ng/mL) of competitive compounds.
[0031] FIG. 2 is a graph showing the results of liposome lysis
assay performed to verify the binding activity of the antibody 8C2
to various phospholipids with similar structures. The ordinate
designates the lysis (%) of liposomes, and the abscissa designates
the dilution(10 .sup.-n) of the antibody.
[0032] FIG. 3 is a graph showing the results of liposome lysis
assay performed to verify the binding-activity of the antibody 8C2
to various compounds with similar structures. The ordinate
designates the lysis (%) of liposomes, and the abscissa designates
the dilution(10.sup.-n) of the antibody.
[0033] FIG. 4 is a graph showing the reactivity of the antibody 8C2
to inositol compounds with similar structures using the competitive
reaction in the liposome lysis assay. The ordinate designates the
lysis (%) of liposomes, and the abscissa designates the
concentrations (M) of the competitive compounds.
[0034] FIG. 5 is a graph showing the reactivity of the antibody 8C2
to PI-3,4-P2 with various length side chains using the competitive
reaction in the liposome lysis assay. The ordinate designates the
lysis (%) of liposomes, and the abscissa designates the
concentrations (M) of the competitive compounds.
[0035] FIG. 6 schematically shows a putative hypervariable region
(complementarity determining region (CDR)) in the variable region
in each of the light chains and the heavy chains of the monoclonal
antibody 8C2.
[0036] FIG. 7 is photographs showing the results of immunostaining
of PI-3,4-P2 induced by the H.sub.2O.sub.2 treatment. The upper six
photographs represent the case with no addition of waltmannine, and
the lower six photographs the case with addition of
waltmannine.
[0037] FIG. 8 is photographs showing the specificities of 8C2
determined by the competitive reaction with PI-3,4-P2 analogs. A
and B represents the case with no competitive compound, C and D the
case with 50 .mu.M phosphatidylcholine, E and F the case with 50
.mu.M PI-3,4-P2, and G and H the case with 50 .mu.M PI-4,5-P2.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The antibody of the present invention specifically
recognizes PI-3,4-P2 but is not substantially cross-reactive with
PI-4,5-P2. Throughout the specification, the term "specific to
PI-3,4-P2" means that the antibody can recognize the phosphorylated
state of PI-3,4-P2 and immunologically distinguish PI-3,4-P2 from
other phosphorylated compounds. The antibody of the present
invention can be produced by the following immunological
procedures. PI-3,4-P2 is suspended together with the killed
Salmonella cells, and the resulting suspension is used as an
immunogen. In more detail, lipopolysaccharides are removed from the
dead Salmonella cells (Galanos C., Eur. J. Biochem., 24, 116-122,
1971). PI-3,14-P2 is then coated onto the dead cells to prepare a
suspension for use as an immunogen (Umeda M., J. Immun., 137,
3264-3269, 1986). PI-3,4-P2 may be purified from cells or
chemically synthesized. PI-3,4-P2 can be produced in accordance
with the methods for synthesizing known compounds with similar
structures to PI-3,4-P2(Thum O., Chen J., Prestwich G. D.,
Tetrahedron Lett. 37, 9017-9020, 1996; Shirai R. et al. ibid. 39,
9485-9488, 1998, Shirai R. et al., ibid. 40, 1693-1696, 1999;
Sawada T. et al., Chem. Pharm. Bull. 45,1521-1523, 1997). An animal
suitable for immunization is immunized with PI-3,4-P2. When the
antibody titer increased, the blood sample is collected. The
antibody may be polyclonal or monoclonal. The monoclonal antibody,
is more advantageous in that an antibody with a higher specificity
can be selected. Furthermore, when the monoclonal antibody is
established, it enables cloning cDNA coding for the amino acid
sequences that constructs the variable regions of the monoclonal
antibody having a desired binding activity, as will be described
later.
[0039] The monoclonal antibody can be produced by cloning
antibody-producing cells. In general, antibody-producing cells
taken out from the immunized animal are subjected to cell fusion
with an appropriate fusion partner. The resulting hybridomas are
then screened in terms of the activity of the produced antibodies
(Gulfre G., Nature, 266, 550-552, 1977). When mice are employed as
the animal for immunization, mouse-derived myeloma cells such as
P3-X63-Ag.653 are adequate as the fusion partner. The hybridomas
subjected to HAT selection are screened first in terms of the
binding activity to PI-3,4-P2. The hybridomas producing the
antibodies that have the binding activity to PI-3,4-P2 are then
subjected to across-reactivity test. In this test, the binding
activity to other-phospholipid antigens is examined to screen
hybridomas having an acceptable cross-reactivity. Acceptable
cross-reactivity means a cross-reactivity that can be disregarded
for the desired use of the antibody. When the monoclonal antibody
is employed for an immunological assay, it has no substantial
cross-reactivity if a signal provided by the cross-reactivity is
reduced to the level of the background in the final assay
system.
[0040] A desired antibody of the present invention is not
substantially cross-reactive with the compounds shown below, which
are structurally similar to PI-3,4-P2. A particularly preferable
antibody of the present invention, as described in Examples below,
distinguishes PI-3,4-P2 from any of these similar compounds:
[0041] phosphatidylinositol-4,5-bisphosphate;
[0042] phosphatidylinositol-3,4,5-triphosphate; and
[0043] phosphatidylinositol-1,3,4,5-tetraphosphate.
[0044] ELISA or liposome lysis assay is useful for verifying the
reactivity of the antibody to PI-3,4-P2 or the cross-reactivity
with other phospholipid antigens. In ELISA, microtiter plates
coated with an antigen whose reactivity is to be observed are
prepared. A sample solution obtained by suitably diluting the
hybridoma supernatants is then added to the wells of the microtiter
plates to initiate a reaction. After a thorough reaction, the wells
are washed, and a second antibody to immunoglobulin is then added
for further reaction The second antibody finally bound to each well
is assayed. Thus, the binding activity of the antibody present in
the culture supernatant to the antigen can be quantitatively
determined. ELISA has been demonstrated for an antibody using a
phospholipid antigen (Umeda M., J. Immun., 136, 2562-2567,
1986).
[0045] The liposome lysis assay utilizes the phenomenon that when
an antibody reacts with an antigen-coated liposome, the liposome
lyses by the action of the complement. Since the action of the
complement is utilized, the technique is called
complement-dependent liposome lysis assay. Liposomes comprise
phospholipid antigens to be examined in addition to dicetyl
phosphate (hereinafter abbreviated as DCP), dimyristoyl
phosphatidylcholine (hereinafter abbreviated as DMPC), and
cholesterol. These lipid components are dissolved in an appropriate
organic solvent. The solution is then dried to prepare a lipid
film. When the film is added to an aqueous solvent and the mixture
is agitated vigorously liposomes of multilamellar structure are
formed. In the liposomes thus prepared, phospholipid antigens are
taken up as membrane-constructing components. For this reason, an
antigenic structure close to the antigen present in actual cell
membranes is presented. It is thus appropriate for screening the
monoclonal antibody. For easy screening, a fluorescent dye can be
enclosed in the liposomes as a lysis marker. Typical examples of
the fluorescent dye include 4-methylumbelliferyl phosphate and
calcein. When the antibody binds to this liposome-constructing
phospholipid antigen in the presence of its complement the liposome
is broken down to release the fluorescent dye in the liposome. This
phenomenon is observed as increased fluorescence intensity in the
liquid phase. Complement-dependent liposome lysis assay using
phospholipid antigens such as PI-4,5-P2 is known (Molec. Immun.,
26, 1025-1031, 1988). The phospholipids used to verify the
cross-reactivity are phospholipid antigens having a similar
structure. The cross-reactivity should be verified with analogous
substances having a similar partial structure. Specific examples of
such compounds are given below, and their structural
characteristics will be shown in examples below.
[0046] PC phosphatidylcholine
[0047] PS phosphatidylserine
[0048] PA phosphatidic acid
[0049] PI phosphatidylinositol
[0050] PE phosphatidylethanolamine
[0051] PI-4,5-P2 phosphatidylinositol-4,5-bisphospate
[0052] IP3 1,4,5-inositol triphosphate
[0053] IP4 1,3,4,5-inositol tetraphosphate
[0054] IP6 1,2,3,4,5,6-inositol hexaphosphate
[0055] In both ELISA and liposome lysis assay, the cross-reactivity
of the antibody with other phospholipid antigens can be verified by
the reaction-system using PI-3,4-P2 as an antigen. That is to the
reaction system containing PI-3,4-P2 and the antibody to be
examined for its specificity are added other antigens to be
examined for the cross-reactivity with the antibody and the
competitive reaction is then observed to confirm the
cross-reactivity. This technique for verifying the cross-reactivity
by means of the competitive inhibition is useful for rapid
screening since it is unnecessary to prepare the reaction system
for all antigens.
[0056] The procedures described above can yield an antibody of the
present invention that has binding activity to PI-3,4-P2 and can
immunologically distinguish PI-3,4-P2 from structurally similar
antigens such as PI-4,5-P2.
[0057] The monoclonal antibody of the present invention is produced
by, for example, the hybridoma 8C2-FNL, which has been deposited
under accession No. FERM BP-6849. The monoclonal antibody of the
present invention can be obtained from a hybridoma by culturing the
hybridoma cells under appropriate conditions and collecting the
antibodies produced by the cells. If the hybridoma is a
homohybridoma, it can be inoculated into syngeneic animals
intraperitoneally and cultured in vivo. In this case, monoclonal
antibodies are collected as ascitic fluid. In the case of
heterohybridomas, such cells can be cultured in vivo using a nude
mouse as a host.
[0058] In vitro culture, as well as in vivo culture, is commonly;
performed in an appropriate culture environment. An essential
medium such as RPMI1640 or DMEM is typically used as a culture
medium for hybridomas. Additives such as animal sera may be added
to the medium to maintain high antibody productivity. Monoclonal
antibodies can be recovered from culture supernatant of in vitro
culture of hybridoma. Culture supernatant can be harvested by
either separating it from cells after culturing or successively
collecting it during culturing when a hollow fiber culture system
is used.
[0059] Monoclonal antibodies collected as ascitic fluid or culture
supernatant are fractionated by salting out with saturated ammonium
sulfate to isolate the immunoglobulin fraction, and then subjected
to purification processes such as gel filtration and ion-exchange
chromatography to obtain the monoclonal antibody of the present
invention. If the monoclonal antibodies are IgGs, other
purification methods such as affinity chromatography using protein
A or G column are also effective.
[0060] The present invention further provides the amino acid
sequences constituting the variable regions of the novel antibody
having a desired binding activity to PI-3,4-P2 and the nucleotide
sequences encoding the same. More specifically, the present
invention provides the, immunoglobulin variable regions containing:
the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO:
4. The present invention further provides cDNA encoding the
immunoglobulin variable regions containing the nucleotide sequences
set forth in SEQ ID NO: 1 and SEQ ID NO: 3. SEQ. ID NO: 1 and SEQ
ID NO: 2 are deduced from the heavy chain and, SEQ ID NO: 3 and SEQ
ID NO: 4 from the light chain, in the immunoglobulin molecule.
These amino acid sequences or cDNA nucleotide sequences are not
necessarily identical but may vary so long as the specific binding
activity to PI-3,4-P2 is maintained. As will be later described,
particularly the site corresponding to CDR is highly variable. In
the CDR region, even amino acids may vary some occasions.
[0061] In general, each immunoglobulin molecule consists of heavy
chains having a larger molecular weight and light chains having a
smaller molecular weight. The heavy and light chains each carries a
region called "a variable region" in about 110 amino acid residues
at the N-terminus, which are different between the molecules.
Variable regions of a heavy chain and a light chain are designated
VH and VL, respectively. The antigen-binding site is formed by
forming a dimer through electrostatic interaction between the heavy
chain variable region VH and the light chain variable region VL.
The variable region consists of three complementarity determining
regions (CDRs) and four frameworks. The CDR forms a complementary
steric structure with the antigen molecule and determines the
specificity of the antibody. The three CDRs inserted between the
four framework regions (FRs) are present like a mosaic in the
variable region (E. A. Kobat et al., Sequences of proteins: of
immunological interest, vol. I, 5th edition, NIH Publication,
1991). The amino acid sequences of FRs are well conserved, but
those of CDR are highly variable and may thus be called
hypervariable regions. Among the amino acid sequences of the
antibody specifically recognizing PI-3,4-P2 a CDR that determines
the binding activity to antigens has been clarified in the present
invention. Thus, the present invention further provides the CDR
shown below (numbering the N-terminal amino acid residue as 1 in
SEQ ID NO: 1 or 3), in which the number in parentheses corresponds
to the SEQ ID number.
1 CDR Heavy chain Light chain CDR1 26-32(5) 24-39(8) CDR2 52-57(6)
55-61(9) CDR3 99-109(7) 94-102(10)
[0062] The cDNAs bearing the nucleotide sequences coding, the
variable regions in immunoglobulin molecules can be cloned from
hybridomas that produce the monoclonal antibody to PI-3,4-P2. For
example, the hybridoma of the present invention, deposited under
the accession No. FERM BP-6849, is preferable as a starting
material the cloning. More specifically, PCR is performed using the
signal sequence of the gene in the variable regions and the
nucleotide sequence in the constant regions. The amplified product
is introduced into an appropriate cloning vector for further
amplification to produce a library of variable genes. Since the
site corresponding to CDR will be a sequence specific to the
variable regions of the present invention, positive clones are
screened from the library of the variable regions using the site as
a probe. The resulting cDNA is inserted into a phage by linking the
light and heavy chain variable region genes through an appropriate
linker. Thus, the cDNA may be expressed as a single-stranded
antibody (so-called scFV). Alternatively, immunoglobulins cDNA are
inserted into a known vector for expression to utilize the same for
producing immunoglobulins. Examples of the vector for expressing
immunoglobulins without limit include a SV40 virus-based vector and
a BPV (papilloma virus)-based vector. For example, BCMGS Neo
vector, one of the BPV vectors (Hajime Karasuyama, Bovine Papilloma
Virus Vector from the extra issue of JIKKEN IGAKU (Experimental
Medicine): IDENSHI KOGAKU (Genetic Engineering) Handbook, edited by
Masami Muramatsu & Hiroto Okayama. Yodosha Publishing Co., pp.
297-299,1991), is desirable since the vector transfect to COS7
cells to express a foreign gene efficiently.
[0063] Alternatively, the specificity of the antibody of the
present invention may also be artificially reconstructed by
incorporating the CDR into the framework of an optional
immunoglobulin molecule. Such a technique is called the CDR
grafting antibody technique, (P. T. Jones et al., Nature, 321, 522,
1986) and has already been established for humanizing mouse
immunoglobulins. The CDRs of the present invention include not only
those completely identical but also variants so long as the
specificity to PI-3,4-P2 is maintained. That is, the CDR amino acid
sequences in which one or more amino acid residues are modified may
also be used as the CDR sequence. The modified amino acid residues
in the amino acid sequences of the CDR variant are preferably 30%
or less, more preferably 20% or less, most preferably 10% or less,
within the entire CDR. Any FR can be used as the FR into which the
CDRs are to be-incorporated. The CDRs of the present invention are
originally derived from mouse immunoglobulins. However, the CDRs
may be inserted into FRs of not only mouse immunoglobulins but also
immunoglobulins of other species. The cDNAs encoding the variable
regions thus constructed may be expressed by incorporating the same
into the vectors described above.
[0064] In introducing, mutations into CDRs, the above-described
phage vector maybe employed. The phage vector can express the
antibody activity rapidly and hence can rapidly: screen mutants.
Moreover, the phage vector expresses the antibody molecule on the
surface of host bacteria in an amount sufficient for screening. A
mutation introduced into the CDRs remarkably increases the antibody
binding activity. Thus, a single-stranded antibody having an
improved binding activity can be produced using the CDRs of the
present invention.
[0065] The variable regions and CDR-incorporated variable regions
provided by the present invention may be expressed in their
original forms. Alternatively, these variable regions may be
expressed as complete immunoglobulin molecules by linking to a gene
encoding the constant regions
[0066] To express variable regions by the cDNA incorporated vector
or by the vector bearing the insert obtained by linking CDR alone
to a certain FR, a dimer of the heavy and light chains can be
produced by expressing heavy chain variable regions and light chain
variable regions in, the same host cell. This can be done by
co-transformation of a host cell with a light chain expression
vector and a humanized heavy chain expression vector. The antibody
of the present invention can be produced from the transformant.
Preferred examples of the host for the transformation include:
Chinese hamster ovary (CHO) cells (A. Wright & S. L. Morrison,
J. Immunol., 160., 3393-3402, 1998) and SP2/0 cells (mouse myeloma)
(K. Motmans et al., Eur. J. Cancer Prev., S5, 512-5.19, 1996; R. P.
Junghans et al., Cancer Res., 50, 1495-1502.1990). The
transformation can be performed by the lipofectin method (R. W.
Malone et al., Proc. Natl. Acad. Sci. USA, 86, 6077, 1989, P. L.
Felgner et al., Proc. Natl. Acad. Sci. USA, 84, 7413, 1987), the
electroporation method, the calcium phosphate method (F. L. Graham
& A. J van der Eb, Virology, 52, 456-467, 1973), or the
DEAE-Dextran method.
[0067] When the expressed variable region is accompanied by the
constant region, the expression product may be purified through a
protein A column, a protein G column, an anti-immunoglobulin
antibody affinity column, etc. to recover the product as a purified
protein. When only the variable region is expressed, these
techniques for purification do not apply. In that case other
suitable purification methods should be selected. For example, if
the variable region is expressed as the product fused to a protein
such as a histidine tag at the C-terminus, then the expression
product is purified by affinity chromatography using the
corresponding ligand
[0068] According to the present invention, PI-3,4-P2 can be
immunologically assayed using the thus produced monoclonal antibody
or variable regions thereof. Immunological assay of PI-3,4-P2 was
impossible by conventional methods since an antibody itself
specific to PI-3,4-P2 was not available. The antibody of the
present invention provides excellent specificity to PI-3,4-P2 and
hence provides an ideal immunological method for assaying
PI-3,4-P2.
[0069] According to the present invention, PI-3,4-P2 can also be
assayed by observing: the degree of PI-3,4-P2 inhibition, utilizing
the phenomenon that the binding between PI-3,4-P2 and the antibody
of the present invention (including the variable regions) is
inhibited by PI-3,4-P2 originating from a sample to be analyzed.
One inhibition assay that realizes such an assay principle utilizes
immobilized PI-3,4-P2. In more detail, PI-3,4--P2 is adsorbed onto
a container like a microtiter plate. A sample solution is added to
the plate. PI-3,4-P2 can be physically adsorbed onto the container
wall after it is dissolved in an appropriate carrier such as
phosphatidylcholine. The antibody of the present invention is then
added, causing a competitive reaction between PI-31-4-P2 adsorbed
on the container and PI-3,4-P2 in the sample solution with the
antibody of the present, invention. The antibody bound (or unbound)
to the solid phase can be readily assayed by labeling the antibody
with an appropriate marker. The quantity of PI-3,4-P2 present in
the sample solution can then be determined by comparison with the
results obtained from a standard solution. The antibody can be
labeled with a marker such as an enzyme, a fluorescence or a
luminiferous substance. PI-3,4-P2 can be assayed in a biological
sample solution such as a tissue, a cultured cell, or a body fluid
like blood or serum. The foregoing competitive reaction may also be
performed by immobilizing the antibody of the present invention
onto the wall of a container. In this case, the labeled PI-3,4-P2
is reacted with the antibody concurrently with a
PI-3,4-P2-containing sample.
[0070] The sample for the immunological assay may be either liquid
or solid. For example, a tissue specimen is immunologically stained
to observe the presence or absence of PI-3,4-P2 or localization of
PI-3,4-P2. In a preferred embodiment, the antibody of the present
invention recognizes the epitope formed by the inositol group and
the glycerol backbone of PI-3,4-P2. Since the epitope is assumed to
be exposed on the surface of the cell membrane, the antibody is
useful for staining a tissue specimen. In this case, localization
of several phospholipids may be observed in the same sample by
using the antibody in combination with another antibody
specifically recognizing a phospholipid, e.g., PI-4,5-P2. A known
double-staining technique involves staining the same sample using
different antibodies each labeled with fluorescent dyes having
different wavelengths.
[0071] The present invention further provides a kit for use in the
immunoassay described above. More specifically, the kit of the
present invention comprises the antibody of the present invention,
a substrate required for detecting the label, positive control,
negative control, and a buffered solution used for diluting and
washing a sample.
[0072] The present invention provides a monoclonal antibody that
specifically binds to PI-3,4-P2. The present invention further
provides an immunological assay method using the antibody. The
experimental results revealed that the antibody recognizes, as the
epitope not only the: inositol group but also the glycerol backbone
of PI-3,4-P2. Thus, the antibody of the present invention can
distinguish PI-3,4-P2 from other inositol compounds.
[0073] The present invention further provides the gene encoding
the, variable regions of the antibody and hence enables producing
recombinant antibodies. Since the antibody of the present invention
is highly specific to PI-3,4-P2, the location of PI-3,4-P2 in cells
can be identified. Alternatively, signal transduction by PI-3,4-P2
to the downstream can be inhibited using the specificity of the
antibody to PI-3,4-P2 to investigate any affect caused. The present
invention facilitates conducting studies that could not be
conducted by conventional assay methods.
[0074] The present invention will be described below in more detail
with reference to examples However, this invention is not to be
construed to be limited to those examples.
EXAMPLE 1
Producing Anti-PI-3-4-P2 Monoclonal Antibody
[0075] To produce anti-PI-3,4-P2 antibody, synthesized PI-3,4-P2
was coated onto dead Salmonella cells as an adjuvant. The coated
cells were then used as an immunogen. Namely, Salmonella minnesota
was cultured overnight and collect the cells. The cells were
centrifuged and washed twice with distilled water and once with
diethyl ether, then dried in vacuo. The cells were then dispersed
in a 1% aqueous acetic acid solution. The dispersion was heated at
100.degree. C. for 2 hours to remove liposaccharide-linked
oligosaccharides (Galanos C., Eur. J. Biochem., 24, 116-122, 1971).
The thus treated cells were washed and coated with 4 .mu.g of
PI-3,4-P2 per 50 .mu.g cells. The resulting suspension was used as
the immunogen (for single-use) (Umeda M., J. Immun., 137,
3264-3269, 1986). PI-3,4-P2 was chemically synthesized by the known
method (Thum O., Chen J., Prestwich G. D., Tetrahedron Lett. 37,
9017-9020, 1996; Shirai R. et al. ibid. 39, 9485-9488, 1998, Shirai
R. et al., ibid. 40, 1693-1696, 1999; Sawada T. et al., Chem.
Pharm. Bull. 45, 1521-1523, 1997). The immunogen was injected into
Balb/c mice via the tail veins a few times every other week. In the
mice with an increased antibody titer, the spleen cells were fused
with myeloma cells P3-X63-Ag.653 to produce hybridomas.
[0076] Following the HAT selection, hybridoma supernatants were
screened for the antibodies in terms of the binding activity to
PI-3,4-P2. The binding activity of the antibodies was screened by
liposome lysis assay. At the same time, the antibodies that
produced positive clones were tested for the cross-reactivity by
indirect ELISA.
[0077] In the liposome lysis assay, PI-3, 4-P2 (<1%),
phosphatidylcholine (40%), cholesterol (40%) and dicetyl phosphate
(10%) were dissolved in chloroform and distilled to dryness under
reduced pressure to prepare a lipid film. After a highly
concentrated aqueous solution of calcein (fluorescent dye) was
added to the film, the mixture was vigorously stirred to prepare
calcein marker-enclosed multilamellar liposomes. The culture
supernatant was-added to the liposomes together with the complement
so that the antigen-antibody binding occurred to activate the
complement and break the membrane. When the antibody bound to
PI-3,4-P2 is present in the culture supernatant, the highly
concentrated calcein in the liposomes is released so that the
concentration of calcein is reduced, causing fluorescence to be
emitted. By measuring the fluorescent intensity, the activity of
the antibody with respect to the antigen was determined.
[0078] Indirect ELISA was performed as follows. First, a solution
of 100 ng/mL of PI-3,4-P2 in 5 .mu.g/mL of phosphatidylcholine
(carrier) was added in microtiter plates. It was allowed to stand
overnight at room temperature for coating, then dried. The wells
were then incubated with blocking buffer (1% bovine serum albumin
(BSA), 10 mM Hepes-buffered saline. (HBS, pH 7.6)) for 30 minutes
at room temperature. The blocked antigen-coated plates were washed,
sealed and stored in a refrigerator until use.
[0079] After 100 .mu.L each of PIP, PI-4,5-P2, IP3, IP4 and IP6
(serially diluted to 10.sup.0 to 10.sup.4 ng/mL with 0.5% BSA/HBS)
and 100 .mu.L of hybridoma supernatants were added to the wells of
the antigen-sensitized plates, incubation was carried out at room
temperature for 2 hours. The reaction solution was removed. After
washing with HBS, a second antibody (alkaline phosphatase-labeled
anti-mouse IgG3 or anti-mouse IgM; diluted to 1/2000 with 0.5%
BSA/HBS) was added to the system followed by incubation at room
temperature for 2 hours. After completion of the reaction, the
unreacted antibodies were removed. The wells were again washed with
HBS, and p-nitrophenyl phosphate (PNPP) was added to the wells to
measure the activity of the alkaline phosphatase remaining in the
wells. When the antibody contained in the culture supernatants was
specific to PI-3,4-P2, the immune reaction: between the antibody
and PI-3,4-P2 proceeded without competitive inhibition by the
various co-existing compounds. Thus, the higher alkaline
phosphatase activity is retained in the solid phase. In contrast,
when the antibody was not reactive with PI-3,4-P2 or was
cross-reactive with the various co-existing antigens., the number
of antibodies that reacted with PI-3,4-P2 on the solid phase was
reduced due to competitive inhibition. As a result, the alkaline
phosphatase activity retained on the solid phase became lower. The
results of the culture supernatant of hybridoma 8C2-FNL obtained
by. ELISA are shown in FIG. 1. Since 8C2-FNL produces the antibody
that reacts only with PI-3,4-P2, a concentration-dependent decrease
in binding percent (taking: the absorbance obtained with the
culture supernatant alone as 100%) is observed only for
PI-3,4-P2.
[0080] By this screening, four clones that produced an antibody
reactive only with PI-3,4-P2 were established. Designations of
these clones are summarized in the table below. The relative
reactivity of the clone to PI-3,4-P2 was defined as the inverse of
the concentration (ml/.mu.g) required for lysis of 40% of the
liposomes. The reactivity of the antibody produced by the 8C2-FNL
(Hereinafter, 8C2 denotes a monoclonal antibody unless otherwise
noted.), which showed the highest reactivity to PI-3,4-P2, was
further investigated.
2 Relative reactivity to Clone Class PI-3, 4-P2 8C2 IgG3 417 12D10
IgG3 12 3E10 IgG3 26 3C7 IgG2b 83
EXAMPLE 2
Specificity of Anti-PI-3,4-P2 Monoclonal Antibody 8C2 (IgG3)
[0081] Among the antibodies obtained in Example 1, 8C2 reacted only
with PI-3,4-P2 and showed the highest specificity to PI-3,4-P2. The
reactivity of this antibody was further analyzed by liposome lysis
assay. Prior to the assay, pristane-treated mice were
intraperitoneally inoculated with hybridoma 8C2-FNL. Immunoglobulin
was purified from the ascetic fluid by ammonium sulfate
fractionation and used as monoclonal antibody 8C2. The following
lipid antigens were used for the liposome lysis assay.
[0082] phosphatidylcholine (PC)
[0083] phosphatidylserine (PS)
[0084] phosphatidic acid (PA)
[0085] phosphatidylinositol (PI)
[0086] phosphatidylethanolamine (PE)
[0087] Liposomes prepared for the assay consisted of 50%
cholesterol and 40% phosphatidylcholine as the main constituents
and 10% of various different lipids as the remaining constituents.
The liposomes different in composition were prepared in the same
manner. The binding activity of 8C2 was assayed by varying dilution
of the antibody within 10.sup.-1 to 10.sup.-7 based on the
liposomes. Lysis of the liposomes was observed to be
concentration-dependent only in the liposomes containing PI-3,4-P2.
In contrast, no lysis was observed in the liposomes containing
other membrane-constructing phospholipids such as PC, PS, PA, PI,
or PE, even when using the dilution with the highest antibody
concentration (10.sup.-1) prepared for the assay. The results are
shown in FIG. 2. In FIG. 2, liposome lysis (%) refers to a
percentage when the fluorescent intensity is made 100% when all of
the liposomes reacted were lysed. The results reveal that 8C2
reacted only with PI-3,4-P2 in the phospholipids. To obtain more
detailed information on an epitope recognized by 8C2, the
cross-reactivity of phosphatidylinositol with various
phosphorylated antigen derivatives was examined.
[0088] Liposomes prepared for the assay consisted of 50%
cholesterol, 40% phosphatidylcholine, and 9% dicetyl phosphate as
the main constituents and 1% of the following phosphorylated
derivatives as the remaining constituents.
[0089] PI
[0090] PI4P (phosphatidylinositol phosphate)
[0091] PI-3,4-P2
[0092] PI-4 5-P2
[0093] N-PIP3 (natural PIP3, purchased from Alexis)
[0094] S-PIP3 (chemically synthesized PIP3)
[0095] PA (phosphatidic acid)
[0096] PC
[0097] PE
[0098] PS
[0099] cardiolipin
[0100] The assay was performed by varying the antibody dilution
from 10.sup.-1 to 10.sup.-6. As in the previous experiment, lysis
of the liposomes was observed to be concentration-dependent in the
liposomes containing PI-3,4-P2. However, no reaction was observed
in the liposomes containing any other phospholipids, even when the
dilution with the highest antibody concentration prepared for the
assay was used (FIG. 3). The antibody exhibited only 1% or less
cross-reactivity to PI-4,5-P2, which is structurally very similar
to PI-3,4-P2, compared with the reactivity to PI-3,4-P2. The
foregoing results reveal that the phosphate group at the 4-position
of the inositol group plays the important role for antigen
recognition of 8C2', and that the phosphate group at the 3-position
also participates in the epitope configuration.
EXAMPLE 3
Epitope of Anti-PI-3,4-P2 Monoclonal Antibody 8C2 (IgG3)
[0101] To identify the recognition site of the 8C2 antibody, the
above liposome lysis assay was performed using as competitors
inositol polyphosphates (30 nM, 100 nM, 300 nM, 1 .mu.M, 3 .mu.M,
and 10 .mu.M) having a similar configuration to the inositol group
of PI-3,4-P2. The inositol polyphosphates used are given below.
[0102] inositol-1,4,5-triphosphate (IP3)
[0103] inositol-1,3,4,5-tetraphosphate (IP4)
[0104] inositol-1,2,3,4,5,6-hexaphosphate(IP6):
[0105] When free. PI-3,4-P2 was added as the competitor, absorbance
was reduced in a concentration-dependent manner by competition with
the fixed PI-3,4-P2. Other inositol polyphosphates, such as IP3,
IP4 and IP6 were not affected as shown in FIG. 4.
[0106] These results suggest that the glycerol backbone is involved
in the recognition, site of the antibody. Since the absorbance
reduction depended on the concentration of PI-3,4-P2 added, the
antibody of the present invention makes the immunoassay possible,
based on the competitive reaction of PI-3,4-P2. Furthermore, the
antibody of the present invention is not affected by various other
compounds having similar configurations. Thus, the present
invention can provide a simple assay system which is excellent in
specificity to PI-3,4-P2.
[0107] To determine whether the side chains of PI-3,4-P2 constitute
a recognition site for the antibody, phosphatidylserine (PS) and
PI-3,4-P2 with side chains of different length were examined for
their reactivity by liposome lysis assay. The experiments were
conducted using the compounds of the formulae shown below in which
the lengths of the side chains are different: 2
[0108] As a result, the degree of liposome lysis of PS remained
constant in a concentration-independent manner, and the competition
with PS was not observed. In contrast, the degree of liposome lysis
was reduced as the concentration of C16PI-314-P2 or C4PI-3,4-P2 was
increased (FIG. 5). This result revealed that the antibody of the
present invention recognizes the inositol ring portion independent
of the difference of the length of the side chains.
[0109] The hybridoma 8C2-FNL that produces the antibody of the
present invention has been deposited under accession No. FERM
BP-6849 at National Institute of Bioscience and Human-Technology
Agency of Industrial Science and Technology., of 1-3. Higashi.
1-chome, Tsukuba-shi, Ibaraki, 305-8566 Japan since Aug. 18, 1999
(date of original deposition).
EXAMPLE 4
Identification of Hypervariable Regions (CDR) of Anti-PI-3,4-P2
Monoclonal Antibody
[0110] cDNA encoding the variable regions of the monoclonal
antibody 8C2 of the present invention that specifically recognizes
PI-3,4-P2 was cloned. RNA extracted from the hybridomas was
subjected to RT-PCR for amplification of cDNA encoding the variable
regions, using signal peptide and constant region sequences as
primers. First the hybridoma 8C2-FNL was incubated in DMEM/10% FCS
to prepare poly A.sup.+ RNA. Single-stranded cDNA was synthesized
from 5 .mu.g of the poly A.sup.+ RNA. PCR was performed for 30
cycles, one cycle consisting of 94.degree. C. for minute,
55.degree. C. for 2 minutes and 72.degree. C. for 2 minutes.
Restriction enzyme recognition sites corresponding to the cloning
sites of pBluescript, which is a cloning vector, are provided at
the 5' end of the primer used.
[0111] Bands of about 400 bp and about 300 bp were isolated from
the amplification products obtained using the heavy chain and light
chain, respectively, by agarose gel electrophoresis and
independently inserted into the cloning vector pBluescript. After
the cloning, the vector was collected. The nucleotide sequence of
the insert was confirmed by the dideoxy method using the vector
primer and [.alpha.-.sup.32P] dATP (F. Sanger, Science, 214,
1205-1210, 1981).
[0112] The amino acid sequence the thus obtained gene encodes was
deduced, and hypervariable regions CDR were identified by the
Chothia Numbering Scheme
(http://www.biochem.ucl.ac.uk/-martin/abs/GeneralInfo.html#kabatnu-
m, Al-Lazikani et al., J. Molec. Biol., 273, 927-948, 1997). The
results are shown below.
3 CDR Heavy chain Light chain CDR1 26-32(5) 24-39(8) CDR2 52-57(6)
55-61(9) CDR3 99-109(7) 94-102(10)
EXAMPLE 5
Immunostaining of PI-3,4-P2 Induced by H.sub.2O.sub.2 Treatment
[0113] 293 cells (Japanese Collection of Research Bioresources No.
JCRB9068) were cultured on cover slips, to which wortmannin, an
inhibitor of PI3 kinase, was added or not added, followed by
treatment with 10 mM H.sub.2O.sub.2 for 0, 3, or 10 minutes. The
cells were fixed in PBS containing 10% formalin at room temperature
for 10 minutes, and then reacted in 0.1% Tween 20 at room
temperature for 10 minutes to render the cells permeable. The cells
were incubated in Dulbecco's medium supplemented with 10 calf serum
for 10 min or longer at room temperature for blocking, then reacted
with the supernatant from the culture of 8C2-FNL hybridoma as it
was in a humidified chamber at room temperature for 2 hours or
more, thereby completing the primary reaction; (culture medium:
Iscove's modified Dulbecco's medium supplemented with 0.10% fetal
bovine serum). Subsequently, FITC-labeled anti-mouse-IgG3.
(Southern Biotechnology, 1100-02) was diluted to 1/100: with the
blocking buffer and allowed to react with the above reaction
mixture for 2 hours or longer at room temperature to complete the
secondary reaction. After the reaction, the cells were mounted in
90% glycerol for observation.
[0114] As a result, staining for PI-3,4-P2 was observed three and
ten minutes after the H.sub.2O.sub.2 treatment when wortmannin was
not added prior to the induction of PI-3,4-P2 production by
H.sub.2O.sub.2 treatment, and the staining intensity increased with
time. In contrast, no staining of the cells was observed after the
H.sub.2O.sub.2 treatment when wortmannin was added, confirming that
the antibody of the present invention is reactive with PI-3,4-P2
(FIG. 7).
[0115] To examine the specificity of 8C2, phosphatidylcholine (PC),
PI-3,4-P2, or PI-4,5-P2 was added to the culture medium of 293
cells and their effects on the immunoreaction were determined. As a
result, PC and PI-4,5-P2 did not compete with PI-3,4-P2, and
fluorescence produced by PI-3,4-P2 staining in the cells was
observed. In contrast, fluorescence was not observed in the cells
to which PI-3,4-P2 was added because the antibody was reacted with
PI-3,4-P2 added (FIG. 8). These results confirmed that the antibody
of the present invention is specific to PI-3,4-P2.
Sequence CWU 1
1
10 1 372 DNA Mus musculus CDS (1)..(372) 1 gag gtg caa ctg gtg gag
tct ggg gga gac tta gtg aaa cct gga ggg 48 Glu Val Gln Leu Val Glu
Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 tcc gtg aaa ctc
tcc tgt gca gcc tct gga ttc act ttc agt agc tat 96 Ser Val Lys Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 ggc atg
tct tgg gct cgc cag act cca gac aag agg ctg gag tgg gtc 144 Gly Met
Ser Trp Ala Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45
gca agc att agt agt ggt ggt agt tac acc tac tat cca gac agt gtg 192
Ala Ser Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50
55 60 aag ggg cga ttc acc atc tcc aga gac aat gcc aag aac acc ctg
tac 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr 65 70 75 80 ctg caa atg agc agt ctg aag tct gag gac aca gcc atg
tac tac tgt 288 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95 gca aga cag agg ggc tat gtt aac ttc ggg att
gct tac tgg ggc caa 336 Ala Arg Gln Arg Gly Tyr Val Asn Phe Gly Ile
Ala Tyr Trp Gly Gln 100 105 110 ggg act ctg gtc act gtc tct gca gct
aca aca aca 372 Gly Thr Leu Val Thr Val Ser Ala Ala Thr Thr Thr 115
120 2 124 PRT Mus musculus 2 Glu Val Gln Leu Val Glu Ser Gly Gly
Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Val Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Ala
Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile
Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr
Cys 85 90 95 Ala Arg Gln Arg Gly Tyr Val Asn Phe Gly Ile Ala Tyr
Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala Ala Thr Thr
Thr 115 120 3 336 DNA Mus musculus CDS (1)..(336) 3 gat gtt gtg atg
acc caa act cca ctc tcc ctg cct gtc agt ctt gga 48 Asp Val Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 gat cag
gcc tcc atc tct tgc aga tct agt cag agc ctt gta cac agt 96 Asp Gln
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30
aat gga aac acc tat tta cat tgg tac ctc cag aaa cca ggc cag tct 144
Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc
cca 192 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60 gac agg ttc agt ggc agt gga aca agg aca gat ttc aca
ctc aag atc 240 Asp Arg Phe Ser Gly Ser Gly Thr Arg Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga gtt tat
ttc tgc tct caa agt 288 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr
Phe Cys Ser Gln Ser 85 90 95 aca cat gtt ccg tac acg ttc gga ggg
ggg acc aag ctg gaa ata aaa 336 Thr His Val Pro Tyr Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105 110 4 112 PRT Mus musculus 4
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5
10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His
Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Thr Arg
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 5 7 PRT Mus
musculus 5 Gly Phe Thr Phe Ser Ser Tyr 1 5 6 6 PRT Mus musculus 6
Ser Ser Gly Gly Ser Tyr 1 5 7 11 PRT Mus musculus 7 Gln Arg Gly Tyr
Val Asn Phe Gly Ile Ala Tyr 1 5 10 8 16 PRT Mus musculus 8 Arg Ser
Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 9
7 PRT Mus musculus 9 Lys Val Ser Asn Arg Phe Ser 1 5 10 9 PRT Mus
musculus 10 Ser Gln Ser Thr His Val Pro Tyr Thr 1 5
* * * * *
References