U.S. patent application number 10/697886 was filed with the patent office on 2004-12-16 for monoclonal antibody recognizing c-terminus of hbnp.
This patent application is currently assigned to Shinogi Seiyaku Kabushiki Kaisha. Invention is credited to Igano, Ken'ichi, Inouye, Ken, Kono, Masao, Tsuji, Tetsuo, Yamauchi, Akira.
Application Number | 20040253655 10/697886 |
Document ID | / |
Family ID | 18193724 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253655 |
Kind Code |
A1 |
Tsuji, Tetsuo ; et
al. |
December 16, 2004 |
Monoclonal antibody recognizing C-terminus of hBNP
Abstract
A hybridoma producing a monoclonal antibody recognizing the
C-terminus of human brain natriuretic peptide (hBNP) was cultivated
in a medium or the abdominal cavity of a mouse to recover the
monoclonal antibody from the medium or ascites accumulated in the
abdominal cavity. An immunoassay for hBNP was established using the
monoclonal antibody. The immunoassay for hBNP of the invention is
so sensitive that the minimum detection limit is 1 pg/ml and can
therefore determine the hBNP level in blood plasma directly,
without the extraction of hBNP from blood plasma. It is useful for
diagnosing diseases such as hypertension and the like, and states
of the heart, kidney, and the like by using the increase/decrease
of the hBNP level as an index.
Inventors: |
Tsuji, Tetsuo; (Nara,
JP) ; Inouye, Ken; (Hyogo, JP) ; Yamauchi,
Akira; (Osaka, JP) ; Kono, Masao; (Osaka,
JP) ; Igano, Ken'ichi; (Nara, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Shinogi Seiyaku Kabushiki
Kaisha
|
Family ID: |
18193724 |
Appl. No.: |
10/697886 |
Filed: |
October 31, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10697886 |
Oct 31, 2003 |
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09942709 |
Aug 31, 2001 |
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6677124 |
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09942709 |
Aug 31, 2001 |
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08749031 |
Nov 14, 1996 |
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09942709 |
Aug 31, 2001 |
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08236013 |
May 2, 1994 |
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09942709 |
Aug 31, 2001 |
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07976457 |
Nov 13, 1992 |
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Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
C07K 16/26 20130101 |
Class at
Publication: |
435/007.92 |
International
Class: |
G01N 033/53; G01N
033/537; G01N 033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 14, 1991 |
JP |
326961/1991 |
Claims
1. An immunoassay for human brain natriuretic peptide, hBNP,
comprising the steps of: (a) contacting a solution suspected of
containing hBNP with an enzyme-conjugated or radioisotope-labeled
Fab' fragment of an antibody which is reactive with a first,
N-terminal region of hBNP and an antibody reactive with a second,
C-terminal region of hBNP having an amino acid sequence
lys-val-leu-arg-arg-his (SEQ ID NO:2), to produce complexes of said
enzyme-conjugated or radioisotope-labeled Fab' fragment, said hBNP,
and said antibody reactive with a second region of hBNP; (b)
contacting said complexes of step (a) with an immobilized antibody
reactive with the Fc fragment of said antibody reactive with a
second region of hBNP to produce further complexes of said
enzyme-conjugated or radioisotope-labeled Fab' fragment, said hBNP,
said antibody reactive with a second region of hBNP, and said
immobilized antibody; (c) recovering and washing said further
complexes of step (b); (d) when an enzyme-conjugated antibody is
employed in step (a), contacting said further complexes of step (c)
with a substrate of said enzyme in an appropriate reaction buffer
and incubating so as to allow formation of the enzymatic reaction
end product; (e) determining the amount of said end product formed
in step (d) when an enzyme-conjugated antibody is employed in step
(a), or determining the amount of radioactivity bound to said
further complexes of step (c) when a radioisotope-labeled antibody
is employed in step (a); and (f) relating the amount of said end
product formed in step (e) or the amount of radioactivity bound to
said further complexes of step (e) to the amount of said hBNP via
the use of a standard curve for hBNP.
2. The immunoassay of claim 1, wherein said Fab' fragment of an
antibody which is reactive with a first region of hBNP recognizes
the intramolecular disulfide bridged loop structure of hBNP.
3. The immunoassay of claim 2, wherein said first antibody is
produced by hybridoma KY-hBNP-II, FERM BP-2863.
4. The immunoassay of claim 1, herein said second antibody is
produced by hybridoma BC203, FERM BP-3515.
5. The immunoassay of claim 4, wherein said first antibody
recognizes the intramolecular disulfide bridged loop of hBNP.
6. An immunoassay for human brain natriuretic peptide, hBNP,
comprising the steps of: (a) contacting a solution suspected of
containing hBNP with an enzyme-conjugated or radioisotope-labeled
Fab' fragment of an antibody which is reactive with a first,
N-terminal region of hBNP and an antibody that specifically binds
to a second, C-terminal region of hBNP, having an amino acid
sequence lys-val-leu-arg-arg-his (SEQ ID NO:2), which binding site
includes the C-terminal his residue, to produce complexes of said
enzyme-conjugated or radioisotope-labeled Fab' fragment, said hBNP,
and said antibody reactive with a second region of hBNP; (b)
contacting said complexes of step (a) with an immobilized antibody
reactive with the Fc fragment of said antibody reactive with a
second region of hBNP to produce further complexes of said
enzyme-conjugated or radioisotope-labeled Fab' fragment, said hBNP,
said antibody reactive with a second region of hBNP, and said
immobilized antibody; (c) recovering and washing said further
complexes of step (b); (d) when an enzyme-conjugated antibody is
employed in step (a), contacting said further complexes of step (c)
with a substrate of said enzyme in an appropriate reaction buffer
and incubating so as to allow formation of the enzymatic reaction
end product; (e) determining the amount of said end product formed
in step (d) when an enzyme-conjugated antibody is employed in step
(a), or determining the amount of radioactivity bound to said
further complexes of step (c) when a radioisotope-labeled antibody
is employed in step (a); and (f) relating the amount of said end
product formed in step (e) or the amount of radioactivity bound to
said further complexes of step (e) to the amount of said hBNP by a
standard curve for hBNP.
7. The immunoassay of claim 4, wherein said first antibody is
produce by hybridoma KY-hBNP, FERM BP-2863.
8. An immunoassay for human brain natriuretic peptide, hBNP,
comprising the steps of: (a) contacting a solution suspected of
containing hBNP with a Fab' fragment of an antibody which is
reactive with a first, N-terminal region of hBNP and an antibody
that specifically binds to a second, C-terminal region of hBNP,
having an amino acid sequence lys-val-leu-arg-arg-his (SEQ ID
NO:2), which binding site includes the C-terminal his residue, to
produce complexes of said Fab' fragment, said hBNP, and said
antibody reactive with a second region of hBNP; (b) contacting said
complexes of step (a) with an immobilized antibody reactive with
the Fc fragment of said antibody reactive with a second region of
hBNP to produce further complexes of said Fab' fragment, said hBNP,
said antibody reactive with a second region of hBNP, and said
immobilized antibody; (c) recovering and washing said further
complexes of step (b); (d) determining the amount of said further
complexes and (e) relating the amount of said further complexes
determined in step (d) to the amount of said hBNP by a standard
curve for hBNP.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a monoclonal antibody recognizing
the C-terminus of hBNP, a hybridoma producing the monoclonal
antibody, a method of producing the monoclonal antibody comprising
cultivating the hybridoma in a medium or an abdominal cavity of a
mouse and recovering said monoclonal antibody from said medium or
ascites in said abdominal cavity, and an immunoassay for hBNP with
use of said monoclonal antibody.
[0003] 2. Related Art
[0004] Brain natriuretic peptide (BNP) in the porcine brain was
first reported by Matsuo et al, Nature 332, 78-81 (1988). There
exist porcine (p) BNP-26 of 26 amino acid residues and pBNP-32 of
32 residues. These has peripheral and central actions similar to
those of atrium natriuretic peptide (ANP) and play an important
role in the homeostasis of body fluid and the control of blood
pressure together with ANP. BNP was suggested to be produced in and
secreted from the heart in human (Biochem. Biophys. Res. Commun.
159, 1427-1434 (1989)), and BNP in the human heart has recently be
isolated and characterized (FEBS Lett. 259, 341-345 (1990)). Human
BNP (hBNP) comprises 32 amino acid residues identical with the
sequence 77-108 of hBNP precursor.
SUMMARY
[0005] As mentioned above, because BNP plays an important role in
the homeostasis of body fluid and the control of blood pressure,
the determination of hBNP in the blood by an immunoassay etc. seems
useful for diagnosing diseases such as hypertension and the like
and states of heart, kidney and the like with taking an
increase/decrease of hBNP level as an index. However, an average
level of hBNP in the blood of normal adults is 0.9.+-.0.07 fmol/ml
(3.12.+-.0.24 pg/ml) (J. Clin. Invest. 87, 1402-1412 (1991)) and
such a low level has made it impossible to directly assay hBNP in
the blood plasma without an extraction.
[0006] The inventors of this invention made an effort to solve the
problem as mentioned above and succeeded in preparing a monoclonal
antibody recognizing the C-terminus of hBNP and thereby
establishing a specific sandwich radioimmunoassay for hBNP.
[0007] The immunoassay for hBNP provided by this invention is so
sensitive that its minimum detection limit is 1 pg/ml and can
therefore determine an hBNP level in plasma directly, that is,
without extracting hBNP from plasma. It is useful for diagnosing
diseases such as hypertension and the like and states of a heart, a
kidney and the like with taking an increase/decrease of hBNP level
as an index.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows a standard curve of the sandwich
radioimmunoassay of this invention.
[0009] FIG. 2 shows a standard curve of the competitive
radioimmunoassay using the monoclonal antibody of this
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0010] This invention provides a monoclonal antibody recognizing
the C-terminus of hBNP and a hybridoma producing the same. The
monoclonal antibody of this invention reacts with hBNP but not with
hBNP having an additional Tyr residue at the C-terminus. It is
unlikely that the monoclonal antibody reacts with hBNP having any
additional amino acid residue except His at the C-terminus.
Therefore, it is concluded that the monoclonal antibody of this
invention mainly recognizes His residue at the C-terminus of hBNP,
especially, a carboxyl group of the His residue.
[0011] The monoclonal antibody of this invention may be produced by
a hybridoma BC203. This hybridoma BC203 has been deposited in the
Fermentation Research Institute at 1-3, Higashi 1-chome,
Tsukuba-shi, Ibaraki-ken, as Mouse Hybridoma BC203 with an
accession number FERM BP-3515 under the Budapest Treaty since Aug.
20, 1991.
[0012] The hybridoma of this invention may be produced as follows.
A peptide having a sequence around the C-terminus of hBNP,
preferably hBNP(27-32), is prepared and conjugated to bovine serum
albumin, bovine thyroglobulin or the like to increase its
antigenicity. Thus obtained conjugate is emulsified with an
appropriate adjuvant such as Freund's complete adjuvant to be used
for the immunization of mice.
[0013] For the immunization the emulsion is intraperitoneally,
intravenously or subcutaneously inoculated several times into mice
at intervals of a few weeks. The spleen is excised 3-5 days after
the last immunization to be used as antibody-producing cells. Thus
obtained antibody-producing cells are fused with myeloma cells
having an appropriate marker to prepare hybridomas.
[0014] A medium to be used for the preparation of hybridomas
includes Eagle's MEM, Dulbecco's modified medium, RPMI-1640 and the
like conventional. Myeloma cells are fused with spleen cells
(myeloma cells:spleen cells=about 1:5). As to a fusing agent 50% of
polyethyleneglycol (PEG) may preferably be chosen for higher yield
of fusion cells. Fusion cells are selected according to the HAT
method. Thus obtained hybridomas are screened by a competitive
radioimmunoassay or the like using their cultivation supernatants
to get hybridomas producing desired immunoglobulin. The obtained
hybridomas were subcloned in order to make hybridomas monoclonal.
In brief, they are sown in a 96-well plate at a concentration of
less than one cell per well and clones grown are again screened.
This subcloning is repeated to yield monoclonal hybridomas.
[0015] This invention further provides a method of producing the
above-mentioned monoclonal antibody, which method comprises
cultivating the above-mentioned hybridoma in a medium or in an
abdominal cavity of a mouse and recovering said monoclonal antibody
from said medium or from ascites accumulated in said abdominal
cavity.
[0016] In brief, the above-mentioned hybridoma is cultivated in an
incubator (in vitro) or in an animal (in vivo). For the in vitro
system a medium may be selected from the conventional ones, as
mentioned above, fortified with fetal calf serum (FCS), in which
medium the hybridoma is cultivated for 3-5 days to recover the
monoclonal antibody from the supernatant of the medium. For the in
vivo system the hybridoma is inoculated into an abdominal cavity of
a mammal and after 1-3 weeks ascites accumulated in the abdominal
cavity is recovered, from which the monoclonal antibody is
recovered. The in vivo system is preferable to the in vitro system
because the monoclonal antibody can efficiently be produced in the
in vivo system much more than in the in vitro system.
[0017] Monoclonal antibody BC203 of this invention recognizes hBNP
at around the C-terminus. As described in the Example, BC203 does
not bind to [Tyr.sup.33]-hBNP, that is, hBNP which has additional
Tyr residue at the C-terminus that is prepared by linking Tyr
residue to the C-terminal residue His.sup.32 of hBNP. This
indicates that BC203 recognizes the C-terminal residue His.sup.32
of hBNP.
[0018] This invention provides an immunoassay for hBNP
characterized in that hBNP is sandwiched between the
above-mentioned monoclonal antibody A and an antibody B which
recognizes hBNP at the site different from that recognized by said
antibody A. A preferable embodiment of said antibody B is a
monoclonal antibody, especially., recognizing the ring structure of
hBNP, for example, monoclonal antibody KY-hBNP-II (Japanese Patent
Application No. 2-99623). The hybridoma KY-hBNP-II producing this
antibody has been deposited in the Fermentation Research Institute
at 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken as Mouse
Hybridoma KY-hBNP-II with an accession No. FERM BP-2863 under the
Budapest Treaty since Apr. 11, 1990.
[0019] An immunoassay of this invention is briefly explained below
with taking a sandwich radioimmunoassay as an example.
[0020] (1) Preparation of Antibody Labeled with Isotope
[0021] An IgG fraction of either of the above-mentioned antibodies
A and B is digested with pepsin to give F(ab').sub.2 fragment,
which is reduced with 2-mercaptoethanol to yield Fab' fragment. A
method of preparing Fab' from IgG is detailed in J. Immunoassay 4,
209-327 (1983), which is also applicable to this invention. Fab'
fragment thus obtained is labeled with an isotope according to the
conventional methods such as Chloramine T method, a method using
maleimide-monoiodo(.sup.125I)tyramine and the like. The labeled
antibody is purified by gel filtration, column chromatography or
the like.
[0022] (2) Preparation of Second Antibody Immobilized
[0023] According to the manner described in Japanese KOKAI
62-132172, an anti-mouse IgG Fc fragment antibody is immobilized on
a commercially available carrier for an antigen-antibody reaction,
such as beads, a ball, a tube or a plate made of glass or
synthesized resin, which is usually used for the conventional
immunoassays.
[0024] (3) Assay of hBNP
[0025] A standard solution or a sample of hBNP is allowed to react
with the labeled antibody A or B (Fab') of the above step (1) and
the other antibody B or A in a tube. The immobilized anti-mouse Fc
of the above step (2) is added thereto to be allowed to react.
After centrifugation, the supernatant is removed for washing.
Radioactivity of the labeled antibody precipitated is
determined.
[0026] Further, the antibody A or B different from the labeled
antibody can be immobilized on a carrier and used as an immobilized
antibody. In this case, hBNP is allowed to react with the labeled
antibody A or B and the immobilized antibody B or A, respectively.
After centrifugation the supernatant is removed for washing and
radioactivity of the labeled antibody precipitated is
determined.
[0027] The method of this invention includes not only
radioimmunoassay but also enzyme immunoassay (EIA). An
enzyme-conjugated antibody for EIA is prepared as follows.
[0028] An IgG fraction of either the above-mentioned antibody A or
B is digested with pepsin to give F(ab').sub.2 fragment, which is
reduced with 2-mercaptoethylamine to yield Fab' fragment. A method
of preparing Fab' from IgG is detailed in J. Immunoassay 4, 209-327
(1983), which is also applicable to this invention.
[0029] The Fab' fragment thus obtained is conjugated with an
enzyme. An enzyme to be conjugated includes alkaline phosphatase,
.beta.-D-galactosidase, peroxidase, glucose oxidase and so on.
Horse radish peroxidase is preferably applied to the present
method. A bridging agent to be used for the conjugation includes
N,N'-o-phenylene-dimaleimid- e, 4-(N-maleimidomethyl)cyclohexanoic
acid N-succinimide ester, 6-maleimidohexanoic acid N-succinimide
ester, 3-(2-pyridylthio)-propionic acid N-succinimide ester,
4,4'-dithiopyridine and other well known bridging agents. These
bridging agents can be used to conjugate an antibody with an enzyme
according to the conventional methods suitable for the properties
of the agents.
[0030] The present immunoassay is highly sensitive with the minimum
detection limit being 1 pg/ml and extremely specific for hBNP,
thereby enabling the direct assay of hBNP in blood plasma without
an extraction.
[0031] The present invention is detailed in the following
Example.
[0032] Abbreviation in the Example; Boc: t-butyloxycarbonyl, Tos:
tosyl, PAM: 4-(oxymethyl)-phenylacetamidomethyl, Cl-Z:
2-chloro-benzyloxycarbony- l, Br-Z: 2-bromo-benzyloxycarbonyl, Bom:
benzyloxymethyl, Bzl: benzyl, cHex: cyclohexyl, maleimide-monoiodo
[.sup.125I]tyramine:
N-[2-4[-hydroxy-3([.sup.125I]iode)phenyl]ethyl]-4-(maleimidomethyl)-1-cyc-
lohexanecarboxamide.
EXAMPLE
I. Preparation of Hybridoma Producing Anti-hBNP Monoclonal Antibody
and Production of Antibody
[0033] (1) Preparation of Conjugate for Immunization Synthesis of
hBNP fragment (27-32)
[0034] Boc-Lys(Cl-Z)-Val-Leu-Arg(Tos)-His(Tos)-PAM-Resin, wherein
protected amino acids were purchased from Applied Biosystems, was
synthesized from Boc-His(Tos)-PAM-Resin (Applied Biosystems)(0.25
mmol) by an amino acid synthesizer such as SHIONOGI SRL-02
according to the usual solid phase method. This was deprotected
with HF/anisole. The crude peptide thus obtained was subjected to
reversed phase chromatography (column: Wako Pure Chemical RQ-2,
24.times.360 mm) with linear gradient of 0.50% CH.sub.3CN/0.1%
CF.sub.3COOH for purification to give 59 mg of the desired
peptide.
[0035] The obtained preparation gave a single peak, retention time:
6.04 min, on analytical HPLC using a column of Nucleosil 5C.sub.18
(4.6.times.150 mm, Shinwa Chemical Industries Ltd.), under the
condition of solvent: 5% CH.sub.3CN/0.1% CF.sub.3COOH, flow rate:
1.8 ml/min and detection: 220 nm.
[0036] The peptide was hydrolyzed with 6M HCl at 110.degree. C. for
24 hr by HITACHI automatic amino acid analyzer Type 835 and the
result of amino acid analysis of the hydrolyzate is described
below.
[0037] Val 0.95(1); Leu 1.00(1); Lys 0.90(1); His 0.95(1); Arg
2.00(2)
[0038] Preparation of Conjugate
[0039] An aqueous solution of bovine thyroglobulin (40 mg/2.0 ml)
was mixed with an aqueous solution of hBNP fragment (27-32) (16
mg/1.0 ml). To the mixture was added an aqueous solution of
1-ethyl-3(3-dimethylamino- propyl)carbodiimide hydrochloride (46
mg/0.6 ml) and stirred at 0.degree. C. for 2 hr. The reaction
mixture was dialyzed against distilled water at 4.degree. C. for 2
days and the dialysate was lyophilized. A portion of the dialysate
with or without being gel-filtrated was subjected to amino acid
analysis to calculate the conjugation ratio, which was 29 molecules
of hBNP fragment (27-32) per bovine thyroglobulin molecule.
[0040] (2) Immunization
[0041] A suspension of the obtained hBNP fragment (27-32)--bovine
thyroglobulin conjugate in physiological saline (conjugate
concentration: 4 mg/ml) was emulsified with an equal volume of
Freund's complete adjuvant. The emulsion was subcutaneously
injected 5 times to BALB/c mice (female, 6 weeks old) at intervals
of 3 weeks at a dose of 0.1 ml. The amount injected to one mouse in
one injection was 200 .mu.g as the conjugate and 5.6 .mu.g as the
hapten hBNP fragment (27-32). Three weeks after the last
immunization, 400 .mu.g of hBNP fragment (27-32)--bovine
thyroglobulin conjugate (11.2 .mu.g of the hapten) was suspended in
physiological saline and 0.1 ml of the suspension was
intraperitoneally injected as a booster to each of the mice once a
day for 2 consecutive days.
[0042] (3) Cell Fusion
[0043] The spleens of the mice were excised 3 days after the
booster and the spleen cells were put in 0.17 M ammonium chloride
solution with ice-cooling for 5 min to destroy erythrocytes. The
remaining cells were suspended in RPMI 1640 medium to prepare a
splenic lymphocyte preparation to be used for cell fusion. Splenic
lymphocytes (1.88.times.10.sup.8) thus obtained were mixed with
8-azaguanine resistant myeloma cells (X63.653, 3.76.times.10.sup.7)
suspended in RPMI 1640 medium. After centrifugation the supernatant
was removed, and to the cell precipitate was added 0.8 ml of 50%
polyethyleneglycol (m.w. 4,000, Merck) in RPMI 1640 medium with
agitation by a pipet in 1 min and further agitated for 1.5 min.
Thereafter, 2 ml of RPMI 1640 medium was added in 2 min and then 2
ml in 1 min with agitation. Further, 18 ml of RPMI 1640 medium was
added dropwise with gentle agitation. After centrifugation the
supernatant was removed and the precipitated cells were suspended
in 100 ml of HAT medium (20% FCS--RPMI 1640 medium with
1.times.10.sup.-4 M hypoxanthine, 4.times.10.sup.-7 M aminopterin
and 1.6.times.10.sup.-5 M thymidine), 0.1 ml of which was put in
each well of seven 96-well cell culture plates (Costar
Corporation). In each of the wells 5.times.10.sup.4 cells of mouse
spleen cells in 0.1 ml of HAT medium had previously been put as a
feeder. Thereafter, the cells were incubated with exchanging a half
amount of HAT medium into fresh one at intervals of a few days. The
growth of hybridomas was observed about 10 days later. The number
of wells where hybridomas grew was 670 (99.7%) in total.
[0044] (4) Selection of Hybridoma
[0045] Supernatants of wells in which hybridomas grew were assayed
for the production of anti-hBNP antibody according to the
competitive radioimmunoassay. In brief, 50 .mu.l of a supernatant
was mixed in a tube with 150 .mu.l of Assay buffer (described later
in item V) and 100 .mu.l of .sup.125I-labeled hBNP (described in
item V) diluted with Assay buffer by about 15,000 cpm/tube and was
incubated at 4.degree. C. for at least 16 hr. Then, 100 .mu.l of
2.5% bovine gamma-globulin and 500 .mu.l of 25% polyethyleneglycol
6000 (Yoneyama Yakuhin Kogyo Co., Ltd.) were added thereto,
agitated and centrifuged at 3,000 rpm for 20 min. The supernatant
was removed and radioactivity in the precipitate was determined by
a gamma counter (Aloka ARC-600). Radioactivity of at least 5,000
cpm (about 5 times as much as nonspecific binding) was taken as
positive to select 10 wells in total.
[0046] (5) Cloning
[0047] Hybridomas selected in item (4) were cloned by the limiting
dilution method. They were incubated in 96-well cell culture plates
at a concentration of 1 cell/200 .mu.l/well. The supernatants of
wells in which hybridomas grew were subjected to the
radioimmunoassay of item (4). Hybridomas of antibody-positive wells
were expanded. A series of these steps was repeated a few times to
establish antibody-producing cell line BC203.
[0048] (6) Preparation of Ascites
[0049] The hybridoma thus established was transplanted into an
abdominal cavity to prepare ascites of high antibody-concentration.
Briefly, about 1.times.10.sup.7 hybridomas suspended in RPMI 1640
were injected intraperitoneally to a mouse (male BALB/c) to which
0.5 ml of pristane had previously been injected intraperitoneally.
Ascites was recovered at an appropriate timing during 1-3 weeks
after the injection. After removing cells in the ascites by
centrifugation, the obtained supernatant to which 0.1% sodium azide
was added was kept frozen. Thus, the ascites BC203A containing
monoclonal antibody BC203 produced by the hybridoma BC203 was
obtained.
II. Determination of Class/Subclass of Monoclonal Antibody
[0050] Class/subclass of the monoclonal antibody produced by the
hybridoma was determined by the immunodiffusion method using
antibodies specific to each class/subclass of mouse immunoglobulins
(Serotec, Mouse monoclonal antibody-typing kit). As a result, a
precipitation line was observed between ascites BC203A and
anti-IgG.sub.1 antibody, indicating that the antibody BC203 belongs
to IgG.sub.1.
III. Determination of Affinity (Binding Constant) of Monoclonal
Antibody
[0051] A standard curve for hBNP was made by the method described
in item V. According to the Scatchard's method radioactivity of
precipitate/radioactivity of supernatant was plotted in vertical
axis and a molar concentration of antibody bound to antigen in
horizontal axis on each concentration of hBNP. The binding constant
of the antibody was determined to be 1.3.times.10.sup.9 M.sup.-1
from the inclination of thus obtained line.
IV. Specificity of Monoclonal Antibody
[0052] (1) Synthesis of Tyr.sup.33-.sup.125I-Labeled hBNP
[0053] Purified Tyr.sup.33-hBNP (7.0 mg) was prepared from
Boc-Tyr(Br-Z)-PAM-Resin (Applied Biosystems, 0.25 mmol Tyr)
according to the synthesis of Tyr.sup.0-hBNP-32 in the Referential
Example using an amino acid synthesizer Applied Biosystems 430A,
and was labeled in the same manner as in the Referential Example to
give the desired compound.
[0054] The obtained preparation gave a single peak (Retention time:
5.98 min) under the condition of column: YMC A-302 S-5 120A ODS,
4.6.times.150 mm (YMC Co., Ltd.); Solvent: 19.0% CH.sub.3CN/0.1%
CF.sub.3COOH; Flow Rate: 1.0 ml/min; and Detection: 220 nm. A
portion of the obtained preparation was hydrolyzed with 6 M HCl at
110.degree. C. for 20 h in the presence of phenol and subjected to
an amino acid analysis to give the following result.
[0055] Asp 1.05(1); Ser 5.46(6); Glu 1.04(1); Gly 5.21(5); Val
1.97(2);
[0056] Met 1.94(2); Ile 0.97(1); Leu 2.00(2); Tyr 1.01(1); Phe
1.00(1);
[0057] Lys 3.01(3); His 0.95(1); Arg 3.88(4); Pro 1.05(1)
[0058] (2) Specificity of Antibody
[0059] Tyr.sup.33-.sup.125I-labeled hBNP, wherein an additional Tyr
residue was introduced into the C-terminal His residue of hBNP, did
not bind to the monoclonal antibody of this invention in the
radioimmunoassay of item I-(4), indicating that the monoclonal
antibody of this invention specifically binds to hBNP at the
C-terminal His residue.
V. Sandwich Radioimmunoassay for hBNP Using Monoclonal Antibody
(Direct Method)
[0060] Sandwich radioimmunoassay for hBNP was established using the
monoclonal antibody BC203 described in item I and .sup.125I-labeled
Fab' of anti-hBNP(1-32) antibody (hereinafter referred to as
KY-hBNP-II) produced by hybridoma KY-hBNP-II (Japanese Patent
Application No. 2-99623).
[0061] (1) Synthesis of .sup.125I-labeled KY-hBNP-II Fab'
[0062] Purification of Ascites: Ascites (3 ml) from hybridoma
KY-hBNP-II was purified by Protein G sepharose 4FF, MAb TrapTM G
(Pharmacia). Ascites was 2-fold diluted with Binding Buffer
(attached to Protein G sepharose. 4FF, MAb TrapTM G Kit) and loaded
on Protein G sepharose 4FF column (3 ml) previously equilibrated
with Binding Buffer. Impurities other than IgG was removed by
washing with 30 ml of Binding Buffer. After fractionation with 15
ml of Eluting Buffer (attached to Protein G sepharose 4FF, MAb
TrapTM G Kit) to give 1 ml-fractions, IgG fractions were collected
according to absorbance of at least 0.2 at 280 nm and concentrated
by Centricon-100.TM. (Amicon). The buffer was exchanged into 0.1 M
citrate buffer (pH 4.1) to give 18 mg of IgG fraction.
[0063] Preparation of F(ab').sub.2: To an IgG solution of
KY-hBNP-II (4.57 mg/2.19 ml) as obtained above was added pepsin
(porcine gastric mucosa, Sigma) by 2.5% (W/V) and then sodium
chloride by 0.1 M. After 1 hr incubation at 37.degree. C. the
mixture was gel-filtrated with Sephadex G-100 column (1.5.times.58
cm)(Bio-Rad) equilibrated with 0.1 M sodium borate buffer (pH 8.0)
to give F(ab').sub.2. Indigested IgG contained therein was removed
by Affigel Protein A MAPS-II (Bio-Rad) for purification. In brief,
fractions not adsorbed on Affigel Protein A were collected and
concentrated by Centricon-30.TM. (Amicon). The buffer was exchanged
into 0.1 M sodium phosphate buffer (pH 6.0) containing 5 mM EDTA to
give 1.4 mg of F(ab').sub.2 fraction.
[0064] Preparation of Fab': To an F(ab').sub.2 solution of
KY-hBNP-II (0.4 mg/70 .mu.l) was added 0.1 M 2-mercaptoethylamine
hydrochloride (in 0.1 M sodium phosphate buffer (pH 6.0) containing
5 mM EDTA) to give a final concentration of 10 mM. After 90 min
incubation at 37.degree. C. the mixture was filtrated with TSK gel
2000XL (0.78.times.30 cm, TOSOH CORPORATION) equilibrated with 0.1
M sodium phosphate buffer (pH 6.0) containing 5 mM EDTA to give
Fab' preparation. The Fab' preparation was concentrated to 60 .mu.l
with Centricon-30.TM. (Amicon) to prepare Fab' fraction (0.2 mg/60
.mu.l).
[0065] Preparation of Maleimide-monoiodo(.sup.125I)tyramine:
Maleimidotyramine (15 .mu.l, 1 mg/ml DMSO) was put in a glass tube,
to which 60 .mu.l of 0.2 M sodium phosphate buffer (pH7.0) and 208
MBq (5.6 mCi) of Na.sup.125I were added. After addition of 10 .mu.l
of 0.5% chloramine T in 0.2 M sodium phosphate buffer (pH 7.0), the
mixture was vigorously agitated for 30 sec and then subjected to a
reversed phase HPLC using Nucleosil 10C18 column (0.46.times.30 cm)
equilibrated with a reversed phase HPLC developing solvent (0.1%
TFA:CH.sub.3CN:CH.sub.3OH=5:- 3:2) to collect
Maleimide-monoiodo(.sup.125I)tyramine fractions. The fractions were
evaporated to dryness under nitrogen atmosphere at 60.degree. C.
and then dissolved in 50 .mu.l of 0.1 M sodium phosphate buffer (pH
6.0) containing 2% dimethyl sulfoxide (DMSO) and 5 mM EDTA.
[0066] .sup.125I-Labeling of KY-hBNP-II Fab': KY-hBNP-II Fab' (70
.mu.g) was put in a glass tube, to which 130 MBq of
maleimide-monoiodo(.sup.125I- )tyramine was added, followed by 90
min incubation at room temperature. To the mixture was added 0.1 M
sodium phosphate buffer (pH 6.0) containing 1% S-carboxymethyl BSA,
0.02% N-ethylmaleimide and 5 mM EDTA. The resulting mixture was
gel-filtrated with PD-10 (Sephadex G10, Bio-Rad) column
equilibrated with 0.1 M sodium phosphate buffer (pH 6.0) containing
5 mM EDTA, and then with Superose 12 column (Pharmacia,
1.0.times.30 cm) equilibrated with 0.1 M sodium phosphate buffer
(pH 6.0) containing 5 mM EDTA for purification of labeled antibody.
The filtrate was put in glass tubes each containing 10 .mu.l of 1%
S-carboxymethyl BSA solution to give a fraction of purified
.sup.125I-KY-hBNP-II Fab' (about 26-40 MBq, i.e., 0.7-1.1 mCi).
[0067] (2) Preparation of Immobilized Second Antibody
[0068] Anti-mouse IgG Fc fragment rabbit serum (Rockland) was
immobilized on Immunobeads matrix (Bio-Rad) according to the method
described in Japanese Kokai No. 62-132172.
[0069] (3) Reagents
[0070] Assay buffer: 0.1 M phosphate buffer (pH 7.0) containing
0.372 g disodium ethylenediaminetetraacetate, 0.063 g cystine
dihydrochloride, 0.1 g sodium azide, 10.sup.6 KIU aprotinin and 1 g
bovine serum albumin each per liter.
[0071] Diluted ascites: BC203A 1000-fold diluted with Assay
Buffer.
[0072] hBNP standard: 1, 4, 10, 40, 100, 400 and 1000 pg of hBNP in
1 ml of Assay buffer.
[0073] (4) Method
[0074] In Shionogi tube (trade name) were put 100 .mu.l of hBNP
standard, 100 .mu.l of .sup.125I-labeled KY-hBNP-II Fab' and 100
.mu.l of Diluted ascites (BC203A). After at least 16 h incubation
at 4.degree. C., a suspension (0.1 ml) of anti-mouse IgG Fc
fragment rabbit serum immobilized on Immunobeads (2 mg/ml) was
added and incubated at 4.degree. C. for 4 h. After centrifugation
at 3,000 rpm for 10 min, the supernatant was removed and the
precipitate was washed with 1 ml of Washing solution (0.01M
phosphate buffer, pH 7.0, containing 0.1% Tween-80 and 0.9% sodium
chloride). After 10 min centrifugation at 3,000 rpm, the
supernatant was removed and radioactivity of the precipitated
.sup.125I-labeled antibody was determined by a gamma counter (Aloka
ARC-600).
[0075] (5) Standard Curve
[0076] A typical standard curve of the sandwich radioimmunoassay is
shown in FIG. 1. It showed good linearity in the assay range from 1
pg/ml to 1,000 pg/ml. The minimum detection limit was 1 pg/ml. In
FIG. 1,--.largecircle.--(Ascites) shows a standard curve obtained
by the above-mentioned method and--.DELTA.--(BC2035) shows a
standard curve obtained by the same method as mentioned above
except the use of a monoclonal antibody from a supernatant of in
vitro culture of hybridoma BC203 in place of Diluted ascites
(BC203A).
[0077] (6) Plasma hBNP Levels in Healthy Subjects
[0078] Plasma hBNP levels in healthy subjects were determined by
the immunoassay (direct method) of this invention and ranged from 1
pg/ml to 15 pg/ml as shown in Table 1, which was enough within the
range detectable by the immunoassay of this invention.
1 TABLE 1 Plasma No. hBNP level (pg/ml) 1 10.9 2 3.7 3 5.3 4 6.1 5
8.0 6 4.8 7 1 8 2.2 9 2.7 10 13.9 11 12.8 12 14.7 13 2.0 14 5.1 15
5.2
[0079] (7) Comparison Between Direct and Extraction Methods
[0080] A comparison was made between the direct method of this
invention which directly assays BNP in plasma and the extraction
method which assays BNP in extract from plasma indirectly. For the
extraction method, BNP was extracted from plasma with Sep-pak
C.sub.18 cartridge (trade name, Waters) and thereafter assayed by
the same method as the above-mentioned sandwich radioimmunoassay.
As shown in Table 2, values determined by the direct method and the
extraction method showed good correlativity (coefficient of
correlation: r=0.98). The direct method of this invention showed
good enough sensitivity and reliability even without extraction of
BNP and is a very simple method in comparison with the extraction
method.
2 TABLE 2 hBNP level (pg/ml) Plasma No. Extraction Method Direct
Method 1 57.6 71.9 2 175 196 3 548 650 4 17.0 24.9 5 1,040 1,117 6
1,191 1,120 7 4.8 4.2 8 344 400 9 1,273 969 10 769 833 11 14.5 12.8
12 2.3 2.0 13 5.1 5.2
REFERENTIAL EXAMPLE
Radioimmunoassay Using Monoclonal Antibody
[0081] Competitive radioimmunoassay was carried out with use of the
monoclonal antibody of this invention.
[0082] (1) Synthesis of .sup.125I-labeled hBNP Synthesis of
Tyr.sup.0-hBNP-32
[0083] Because hBNP has no Tyr residue to be labeled,
Tyr.sup.0-hBNP, i.e., hBNP having additional Tyr residue at the
N-terminus, was synthesized. Namely,
Boc-Tyr(Br-Z)-Ser(Bzl)-Pro-Lsy(Cl-Z)-Met-Val-Gln-Gly-
-Ser(Bzl)-Gly-Cys(4-CH.sub.3OBzl)-Phe-Gly-Arg(Tos)-Lys(Cl-Z)-Met-Asp(OcHex-
)-Arg-Ile-Ser(Bzl)-Ser(Bzl)-Ser(Bzl)-Ser(Bzl)-Gly-Leu-Gly-Cys(4-CH.sub.3OB-
zl)-Lys(Cl-Z)-Val-Leu-Arg(Tos)-Arg(Tos)-His(Bom)-PAM-resin was
synthesized from 0.45 mmol of Boc-His(Bom)-PAM-resin (NOVA Biochem
AG) according to the usual solid phase method using an amino acid
synthesizer, Applied Biosystems 430A. A half amount of the obtained
peptide was deprotected with HF/p-crezole/dimethylsulfide and
diluted with distilled water. After adjusting pH to 9 with aqueous
ammonium, the mixture was agitated at room temperature for 24 h to
introduce S-S bridge. Thus obtained crude peptide was subjected to
reversed phase chromatography (column: YMC S-50 120A ODS AM-type,
30.times.200 mm) with a linear gradient of 0-40% CH.sub.3CN/0.1%
CF.sub.3COOH, then to HPLC fractionation (column: .mu.-bondasphere
15C18 300A, 30.times.300 mm (Waters)) with a linear gradient of
12-22% CH.sub.3CN/0.1% CF.sub.3COOH, and further purified by means
of a column of YMC 342-5 S-5 120A ODS (20.times.150 mm) with a
solvent of 19.5% CH.sub.3CN/0.1% CF.sub.3COOH to give 3.9 mg of the
desired peptide.
[0084] Thus obtained preparation of the desired peptide gave a
single peak (retention time: 9.10 min) on analytical HPLC (column:
Cosmosil 5C.sub.18, 4.6.times.150 mm (NACALAI TESQUE, INC.);
solvent: 20% CH.sub.3CN/0.1% CF.sub.3COOH; flow rate: 1.0 ml/min;
detection: 220 nm).
[0085] The peptide was hydrolyzed with 6 M HCl at 110.degree. C.
for 20 h in the presence of phenol and subjected to amino acid
analysis to give the following result.
[0086] Asp 0.99(1); Ser 4.98(6): Glu 0.97(1); Gly 4.75(5); Val
1.90(2); Met 1.82(2); Ile 0.95(1); Leu 2.02(2); Tyr 0.85(1); Phe
0.93(1); Lys 2.79(3); His 1.00(1); Arg 3.78(4); Pro 0.89(1).
[0087] (Theoretical values in parentheses and an uncorrected value
for Ser)
[0088] Labeling by Na.sup.125I
[0089] Tyr.sup.0-hBNP (10 .mu.g) was mixed with 0.05 ml of 0.5 M
phosphate buffer (pH 7.5) and 18.8 MBq. of Na.sup.125I (Amersham).
To the mixture was added 0.01 ml of 0.2% chloramine T, followed by
30 sec agitation. The mixture was mixed with 1% sodium
metabisulfite, to which 0.01 ml of 10% potassium iodide was added.
The resulting mixture was subjected to HPLC (Column: YMC-A-302 S-5
12A ODS, 4.6.times.150 mm) with a gradient elution by
CH.sub.3CN/CF.sub.3COOH to give 4.2 Mbeq. of the labeled peptide as
a monoiodo form at a retention time of 19 min.
[0090] (2) Reagents
[0091] Assay buffer: 0.1 M phosphate buffer (pH 7.0) containing
0.372 g disodium ethylenediaminetetraacetate, 0.063 g cystine
dihydrochloride, 0.1 g sodium azide, 10.sup.6 KIU aprotinin and 1 g
bovine serum albumin each per liter.
[0092] Diluted ascites: BC203A 42,000-fold diluted with Assay
Buffer
[0093] hBNP standard: 3-fold serial dilutions (0.046-99.9 ng/ml) of
commercially available hBNP (Peptide Institute, Inc.) with Assay
buffer.
[0094] 2.5% bovine gammaglobulin: bovine gammaglobulin dissolved in
Assay buffer.
[0095] 25% polyethyleneglycol: polyethyleneglycol 6000 (Yoneyama
Yakuhin Kogyo Co., Ltd.) dissolved in Assay buffer without bovine
serum albumin.
[0096] (3) Method
[0097] To Shionogi tube containing 100 .mu.l of hBNP standard or a
sample, 100 .mu.l of .sup.125I-labeled hBNP and 100 .mu.l of
Diluted Ascites were added, mixed and incubated at 4.degree. C.
overnight. Then, 100 .mu.l of 2.3% bovine gammaglobulin cooled to
4.degree. C. and 500 .mu.l of 25% polyethyleneglycol were added and
agitated immediately thereafter. After a centrifugation at
4.degree. C. at 3,000 rpm for 20 min, the supernatant was removed
and radioactivity in the precipitate was determined by a gamma
counter (Aloka ARC-600).
[0098] (4) Standard Curve
[0099] A standard curve of this radioimmunoassay was shown in FIG.
2. Sensitivities as 90% and 50% inhibitory concentrations were 1.3
ng/ml and 11.8 ng/ml, respectively. In FIG.
2,--.largecircle.--(Ascites) indicates the standard curve obtained
by the above-mentioned method and--.circle-solid.--(27-32 No. 1-F)
indicates that obtained by the same as the above-mentioned method
except the use of the final antiserum of the mouse immunized by
hBNP(27-32) in place of Diluted ascites.
Sequence CWU 1
1
3 1 33 PRT Artificial Sequence synthetic amino acid according to
the usual solid phase method 1 Tyr Ser Pro Lys Met Val Gln Gly Ser
Gly Cys Phe Gly Arg Lys Met 1 5 10 15 Asp Arg Ile Ser Ser Ser Ser
Gly Leu Gly Cys Lys Val Leu Arg Arg 20 25 30 His 2 6 PRT Homo
sapiens 2 Lys Val Leu Arg Arg His 1 5 3 5 PRT Artificial Sequence
synthetic amino acid according to the usual solid phase method 3
Lys Val Leu Arg His 1 5
* * * * *