U.S. patent application number 14/008441 was filed with the patent office on 2014-03-20 for psa assay and reagent therefor.
This patent application is currently assigned to SEKISUI MEDICAL CO., LTD.. The applicant listed for this patent is Shinichiro Kitahara, Yasushi Nakamura, Shinya Nakayama, Tomo Shimizu, Yuki Takahashi. Invention is credited to Shinichiro Kitahara, Yasushi Nakamura, Shinya Nakayama, Tomo Shimizu, Yuki Takahashi.
Application Number | 20140080143 14/008441 |
Document ID | / |
Family ID | 46931192 |
Filed Date | 2014-03-20 |
United States Patent
Application |
20140080143 |
Kind Code |
A1 |
Takahashi; Yuki ; et
al. |
March 20, 2014 |
PSA ASSAY AND REAGENT THEREFOR
Abstract
Provided is a method for the simple and highly accurate assay of
PSA by a one-step reaction that does not use carriers having
different average grain sizes. Also provided is a reagent therefor.
The PSA assay method comprises sensitizing insoluble carriers
having the same average grain size within a range of greater than
0.20 .mu.m but 0.40 .mu.m or less using two types of anti-PSA
monoclonal antibodies having different epitopes that are anti-PSA
monoclonal antibodies that will react with both free PSA and
PSA-ACT, which is a complex of free PSA and
.alpha.1-antichymotrypsin, and bringing the carriers into contact
with a sample in the presence of an aggregation promoter.
Inventors: |
Takahashi; Yuki;
(Ryugasaki-shi, JP) ; Shimizu; Tomo;
(Ryugasaki-shi, JP) ; Nakamura; Yasushi;
(Ryugasaki-shi, JP) ; Nakayama; Shinya;
(Ryugasaki-shi, JP) ; Kitahara; Shinichiro;
(Ryugasaki-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Takahashi; Yuki
Shimizu; Tomo
Nakamura; Yasushi
Nakayama; Shinya
Kitahara; Shinichiro |
Ryugasaki-shi
Ryugasaki-shi
Ryugasaki-shi
Ryugasaki-shi
Ryugasaki-shi |
|
JP
JP
JP
JP
JP |
|
|
Assignee: |
SEKISUI MEDICAL CO., LTD.
Tokyo
JP
|
Family ID: |
46931192 |
Appl. No.: |
14/008441 |
Filed: |
March 28, 2012 |
PCT Filed: |
March 28, 2012 |
PCT NO: |
PCT/JP2012/058056 |
371 Date: |
December 4, 2013 |
Current U.S.
Class: |
435/7.4 |
Current CPC
Class: |
G01N 33/573 20130101;
G01N 33/543 20130101; G01N 33/57434 20130101; G01N 2333/96433
20130101; G01N 33/577 20130101 |
Class at
Publication: |
435/7.4 |
International
Class: |
G01N 33/573 20060101
G01N033/573 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 2011 |
JP |
2011-069161 |
Claims
1. A PSA assay comprising using insoluble carriers immobilizing
thereon two kinds of anti-PSA monoclonal antibodies, and bringing
the insoluble carriers into contact with a sample in the presence
of an agglutination accelerator, wherein the two kinds of anti-PSA
monoclonal antibodies can react with both free PSA and complex of
free PSA and .alpha.1-antichymotrypsin (PSA-ACT) and recognize
different epitopes, and the insoluble carriers have an identical
average particle size that is more than 0.20 .mu.m and equal to or
less than 0.40 .mu.m.
2. The PSA assay according to claim 1, wherein the agglutination
accelerator is one or more agglutination accelerators selected from
polyethylene glycol, a polysaccharide, polyvinylpyrrolidone,
polyvinyl chloride, a poly-.gamma.-glutamate, and
poly(2-methacryloyloxyethylphosphorylcholine).
3. The PSA assay according to claim 1, wherein the insoluble
carriers are of one or more type selected from particles comprising
materials derived from latex particles of synthetic polymers,
silica, alumina, carbon blacks, metal compounds, metals, ceramics,
and/or magnetic substances.
4. The PSA assay according to claim 1, wherein the concentration of
the agglutination accelerator is adjusted so that an equimolar
response to free PSA and complex of free PSA and
.alpha.1-antichymotrypsin (PSA-ACT) is obtained.
5. The PSA assay according to claim 2, wherein the polysaccharide
is one or more polysaccharides selected from dextran, pullulan, and
alkylated polysaccharides including methyl cellulose and ethyl
cellulose.
6. The PSA assay according to claim 3, wherein the synthetic
polymer is one or more synthetic polymers selected from
polystyrene, a styrene-sulfonic acid copolymer, a
styrene-methacrylic acid copolymer, an
acrylonitrile-butadiene-styrene copolymer, a vinyl
chloride-acrylate copolymer, and a vinyl acetate-acrylate
copolymer.
7. The PSA assay according to any one of claims 1 to 6, wherein the
two kinds of anti-PSA monoclonal antibodies have a ratio (fKd/cKd)
of a dissociation constant (fKd) for free PSA to a dissociation
constant (cKd) for complex of free PSA and
.alpha.1-antichymotrypsin (PSA-ACT) of more than 0.1 and equal to
or less than 2.0, and have a dissociation constant (fKd) for free
PSA of 10 nM or less.
8. A PSA assay reagent comprising at least 1) and 2); 1)
antibody-immobilized carriers prepared by using insoluble carriers
immobilizing thereon two kinds of anti-PSA monoclonal antibodies,
wherein the two kinds of anti-PSA monoclonal antibodies can react
with both free PSA and complex of free PSA and
.alpha.1-antichymotrypsin (PSA-ACT) and recognize different
epitopes, and the insoluble carriers have an identical average
particle size that is more than 0.20 .mu.m and equal to or less
than 0.40 .mu.m 2) one or more agglutination accelerators selected
from polyethylene glycol, a polysaccharide, polyvinylpyrrolidone,
polyvinyl chloride, a poly-.gamma.-glutamate, and
poly(2-methacryloyloxyethylphosphorylcholine).
9. The PSA assay according to claim 1, wherein the two kinds of
anti-PSA monoclonal antibodies have a ratio (fKd/cKd) of a
dissociation constant (fKd) for free PSA to a dissociation constant
(cKd) for complex of free PSA and .alpha.1-antichymotrypsin
(PSA-ACT) of more than 0.1 and equal to or less than 2.0 and have a
dissociation constant (fKd) for free PSA of 10 nM or less, the
agglutination accelerator is one or more agglutination accelerators
selected from polyethylene glycol, a polysaccharide,
polyvinylpyrrolidone, polyvinyl chloride, a poly-.gamma.-glutamate,
and poly(2-methacryloyloxyethylphosphorylcholine), and the amount
of the agglutination accelerator is adjusted so that an equimolar
response to free PSA and complex of free PSA and
.alpha.1-antichymotrypsin (PSA-ACT) is obtained.
Description
TECHNICAL FIELD
[0001] The invention relates to an antigen assay and a reagent that
make it possible to measure a free antigen, and a complex of a free
antigen and a coexisting substance in a sample, by immune
agglutination so that an equimolar response is obtained. In
particular, the invention relates to an assay of prostate-specific
antigen, and a reagent therefor.
BACKGROUND ART
[0002] Prostate cancer is a malignant disease that is observed in
men. A large number of patients suffer from prostate cancer in the
United States and Europe. In recent years, the number of patients
who suffer from prostate cancer has rapidly increased in Japan.
Since prostate cancer grows slowly, and may effectively be treated
by radiotherapy or anti-androgenic therapy, it is important to find
prostate cancer in an early stage.
[0003] Prostate-specific antigen (hereinafter may be referred to as
"PSA") is a glycoprotein (serine protease) that is secreted from
prostate epithelial cells, and has a molecular weight of 33,000 to
34,000 Da. Since a person who suffers a prostate disease shows an
increase in PSA level in blood as compared with a healthy person,
PSA is useful for early detection of a prostatic disease
(particularly prostate cancer). PSA is classified into complex-type
PSA that binds to a protease inhibitor in blood, and free PSA
(hereinafter may be referred to as "fPSA"). Most of the PSA in
blood is complex-type PSA, and present as complex of PSA and
.alpha.1-antichymotrypsin (hereinafter may be referred to as
"PSA-ACT"), complex of PSA and .alpha.2-macroglobulin, or the like.
fPSA and PSA-ACT can be measured by an immunoassay.
[0004] An assay based on agglutination (immunoagglutination assay)
that utilizes a latex or the like is used as the immunoassay.
However, since an anti-PSA monoclonal antibody has different
reactivity with fPSA and PSA-ACT, it may be difficult to accurately
measure the total PSA level.
[0005] In order to solve the above problem, PTL 1 proposes an
immunoagglutination assay reagent and an assay that utilizes the
same, wherein the immunoagglutination assay reagent including (1) a
first particle suspension that includes first insoluble carrier
particles immobilizing thereon a first monoclonal antibody that can
bind to a free measurement target substance and a complex of the
free measurement target substance and the corresponding binding
molecule, (2) a second particle suspension that includes second
insoluble carrier particles immobilizing thereon a second
monoclonal antibody that can bind to the free measurement target
substance and a complex of the free measurement target substance
and the corresponding binding molecule, and does not compete with
the first monoclonal antibody, and (3) a third particle suspension
that includes third insoluble carrier particles immobilizing
thereon a third monoclonal antibody that does not recognize the
free measurement target substance, but recognizes a complex of the
free measurement target substance and the corresponding binding
molecule.
[0006] PTL 2 proposes an assay reagent and an assay that utilizes
the same, wherein the assay reagent adjusting reactivity with a
free antigen and a complex-type antigen by using carriers having a
smaller particle size among two or more types of carriers that
differ in particle size and immobilizing thereon at least one
monoclonal antibody among three monoclonal antibodies that have
reactivity with a free antigen and a complex-type antigen and
differ in recognition site, and using carriers having a larger
particle size among the two or more types of carriers and
immobilizing thereon the remaining monoclonal antibodies.
[0007] PTL 3 proposes a two-step reaction immunoassay that includes
(1) reacting a sample that includes a free measurement target
substance and a complex-type measurement target substance with a
latex 1 on which a monoclonal antibody 1 to the measurement target
substance is immobilized to obtain a reaction product 1, and (2)
reacting the reaction product 1 with a latex 2 on which a
monoclonal antibody 2 that differs in recognition site from the
monoclonal antibody 1 is immobilized to obtain a reaction product
2.
[0008] PTL 4 proposes a prostate-specific antigen immunoassay
reagent and an assay that utilizes the same, wherein the
immunoassay reagent including a latex 1 on which a monoclonal
antibody 1 that has reactivity with free PSA and PSA complex is
immobilized, and a latex 2 on which a monoclonal antibody 2 that
has reactivity with free PSA and PSA complex is immobilized, the
monoclonal antibody 2 differing in recognition site from the
monoclonal antibody 1, and the latex 2 differing in average
particle size from the latex 1.
CITATION LIST
Patent Literature
[0009] PTL 1: JP-A-2001-108681 [0010] PTL 2: WO2006/068206 [0011]
PTL 3: JP-A-2007-163319 [0012] PTL 4: Japanese Patent No.
4241301
SUMMARY OF INVENTION
Technical Problem
[0013] However, when using the method disclosed in PTL 1, a
decrease in relative measured value is observed as the mixing ratio
of PSA-ACT increases (see the measurement results when a
polystyrene latex having an average particle size of 0.78 .mu.m and
immobilizing an anti-PSA monoclonal antibody thereon, and fPSA and
PSA-ACT are mixed in a different ratio). Specifically, when only
PSA-ACT is used, the relative measured value to free PSA is 81.43%
and an equimolar response is not obtained. The method disclosed in
PTL 2 has a problem in that it is necessary to combine latexes that
differ in particle size, and the method disclosed in PTL 3 has a
problem in that it is necessary to employ a two-step reaction in
which the second monoclonal antibody is reacted after reacting the
first monoclonal antibody. The method disclosed in PTL 4 also has a
problem in that it is necessary to combine latexes that differ in
particle size. PTL 4 discloses a comparative example in which
latexes having an identical average particle size (0.22 .mu.m) are
used. However, an agglutination accelerator is not used, and an
equimolar response is not obtained.
[0014] In view of the above situation, development of a method that
can more easily and accurately measure the total PSA level (i.e.,
the sum of fPSA and PSA-ACT that can be measured by an immunoassay)
has been desired.
[0015] Specifically, an object of the invention is to provide an
assay that easily and accurately measures PSA by a one-step
reaction without using carriers that differ in average particle
size, and a reagent used therefor. Note that the expression
"measurement of PSA" or "assay of PSA" used herein refers to
measurement or assay of the total PSA level unless otherwise
specified.
Solution to Problem
[0016] Several aspects of the invention that achieve the above
object include the following.
(1) A PSA assay comprising using insoluble carriers immobilizing
thereon two kinds of anti-PSA monoclonal antibodies, and bringing
the insoluble carriers into contact with a sample in the presence
of an agglutination accelerator, wherein the two kinds of anti-PSA
monoclonal antibodies can react with both free PSA and complex of
free PSA and .alpha.1-antichymotrypsin (PSA-ACT) and recognize
different epitopes, and the insoluble carriers have an identical
average particle size that is more than 0.20 .mu.m and equal to or
less than 0.40 .mu.m. (2) The PSA assay according to (1), wherein
the agglutination accelerator is one or more agglutination
accelerators selected from polyethylene glycol, a polysaccharide,
polyvinylpyrrolidone, polyvinyl chloride, a poly-.gamma.-glutamate,
and poly(2-methacryloyloxyethylphosphorylcholine). (3) The PSA
assay according to (1), wherein the insoluble carriers are of one
or more type selected from particles comprising materials derived
from latex particles of synthetic polymers, silica, alumina, carbon
blacks, metal compounds, metals, ceramics, and/or magnetic
substances. (4) The PSA assay according to (1), wherein the
concentration of the agglutination accelerator is adjusted so that
an equimolar response to free PSA and complex of free PSA and
.alpha.1-antichymotrypsin (PSA-ACT) is obtained. (5) The PSA assay
according to (2), wherein the polysaccharide is one or more
polysaccharides selected from dextran, pullulan, and alkylated
polysaccharides including methyl cellulose and ethyl cellulose. (6)
The PSA assay according to (3), wherein the synthetic polymer is
one or more synthetic polymers selected from polystyrene, a
styrene-sulfonic acid copolymer, a styrene-methacrylic acid
copolymer, an acrylonitrile-butadiene-styrene copolymer, a vinyl
chloride-acrylate copolymer, and a vinyl acetate-acrylate
copolymer. (7) The PSA assay according to any one of (1) to (6),
wherein the two kinds of anti-PSA monoclonal antibodies have a
ratio (fKd/cKd) of a dissociation constant (fKd) for free PSA to a
dissociation constant (cKd) for complex of free PSA and
.alpha.1-antichymotrypsin (PSA-ACT) of more than 0.1 and equal to
or less than 2.0, and have a dissociation constant (fKd) for free
PSA of 10 nM or less. (8) A PSA assay reagent comprising at least
1) and 2): 1) antibody-immobilized carriers prepared by using
insoluble carriers immobilizing thereon two kinds of anti-PSA
monoclonal antibodies, wherein the two kinds of anti-PSA monoclonal
antibodies can react with both free PSA and complex of free PSA and
.alpha.1-antichymotrypsin (PSA-ACT) and recognize different
epitopes, and the insoluble carriers have an identical average
particle size that is more than 0.20 .mu.m and equal to or less
than 0.40 .mu.m; and/or 2) one or more agglutination accelerators
selected from polyethylene glycol, a polysaccharide,
polyvinylpyrrolidone, polyvinyl chloride, a poly-.gamma.-glutamate,
and poly(2-methacryloyloxyethylphosphorylcholine). (9) The PSA
assay according to (1), wherein the two kinds of anti-PSA
monoclonal antibodies have a ratio (fKd/cKd) of a dissociation
constant (fKd) for free PSA to a dissociation constant (cKd) for
complex of free PSA and .alpha.1-antichymotrypsin (PSA-ACT) of more
than 0.1 and equal to or less than 2.0 and have a dissociation
constant (fKd) for free PSA of 10 nM or less, the agglutination
accelerator is one or more agglutination accelerators selected from
polyethylene glycol, a polysaccharide, polyvinylpyrrolidone,
polyvinyl chloride, a poly-.gamma.-glutamate, and
poly(2-methacryloyloxyethylphosphorylcholine), and the amount of
the agglutination accelerator is adjusted so that an equimolar
response to free PSA and complex of free PSA and
.alpha.1-antichymotrypsin (PSA-ACT) is obtained.
Advantageous Effects of Invention
[0017] The assay according to the aspect of the invention can
easily and accurately measure PSA by a one-step reaction without
using carriers that differ in average particle size. Therefore, it
is possible to implement high-accuracy measurement using a
general-purpose automatic analyzer without requiring a special
device.
DESCRIPTION OF EMBODIMENTS
(Measurement Target Substance)
[0018] The embodiments of the invention are targeted at
prostate-specific antigen (PSA) (i.e., measurement target
substance). PSA in plasma that can be measured by immunoassay
include free PSA (fPSA) and complex of free PSA (fPSA) and
.alpha.1-antichymotrypsin (PSA-ACT).
[0019] The target sample is not particularly limited as long as the
sample includes PSA, but is preferably blood, serum, plasma, or the
like.
(Anti-PSA Monoclonal Antibody)
[0020] At least two kinds of anti-PSA monoclonal antibodies that
can react with both fPSA and PSA-ACT and recognize different
epitopes are used as monoclonal antibodies to PSA. The difference
in epitope between the two kinds of anti-PSA monoclonal antibodies
can be determined by confirming whether normal sandwich immunoassay
using PSA (antigen) and these antibodies is possible or not.
(Selection of Antibodies)
[0021] The antibodies are selected as described below.
[0022] It is desirable to select an antibody that has high
reactivity (i.e., high titer) with PSA-ACT and fPSA by ELISA or the
like. It is desirable to select carriers that ensure sufficient
measurement sensitivity. The carriers immobilize thereon two kinds
of antibodies arbitrarily selected from the antibodies selected to
have a high titer, and a combination of antibodies are selected so
that the reactivity with PSA-ACT and fPSA shows small difference
and sufficient measurement sensitivity is obtained.
[0023] It is preferable that the anti-PSA monoclonal antibodies
have a ratio (fKd/cKd) of the dissociation constant (fKd) for free
PSA to the dissociation constant (cKd) for complex of free PSA and
.alpha.1-antichymotrypsin (PSA-ACT) of more than 0.1, and more
preferably more than 0.2. The upper limit of the ratio (fKd/cKd) is
preferably 2.0 or less, and more preferably 1.5 or less. The
dissociation constant (fKd) for free PSA is preferably 10 nM or
less, and more preferably 6 nM or less.
[0024] The monoclonal antibodies may include, for example, an Fab
fragment obtained by partial hydrolysis by papain or the like, an
F(ab').sub.2 fragment obtained by partial hydrolysis by pepsin or
the like, and/or an Fab' fragment obtained by reduction of an
F(ab').sub.2 fragment.
(Insoluble Carriers)
[0025] The insoluble carriers immobilizing antibodies thereon are
not particularly limited, but are preferably selected from
particles comprising materials derived from latex particles of
synthetic polymers, silica, alumina, carbon blacks, metal
compounds, metals, ceramics, and/or magnetic substances. The
synthetic polymer is preferably one or more synthetic polymers
selected from polystyrene, a styrene-sulfonic acid copolymer, a
styrene-methacrylic acid copolymer, an
acrylonitrile-butadiene-styrene copolymer, a vinyl
chloride-acrylate copolymer, and a vinyl acetate-acrylate
copolymer.
[0026] It is particularly preferable to use particles of a
polystyrene latex obtained by soap-free emulsion polymerization.
The polystyrene latex may be produced, for example, by the method
disclosed in WO2003/005031. Specifically, a reaction vessel
containing water as a solvent is charged with specific amounts of a
styrene monomer, an initiator (e.g., potassium persulfate), and a
polymerization stabilizer (e.g., sodium styrene sulfonate). After
optionally adding emulsifier (e.g., sodium lauryl sulfate), the
mixture is heated with stirring in a nitrogen atmosphere to effect
polymerization.
(Particle Size of Insoluble Carriers)
[0027] The average particle size of the insoluble carriers is more
than 0.20 .mu.m and equal to or less than 0.40 .mu.m, preferably
more than 0.22 .mu.m and equal to or less than 0.40 .mu.m, more
preferably equal to or more than 0.23 and equal to or less than
0.40 .mu.m, and still more preferably equal to or more than 0.23
and equal to or less than 0.34 .mu.m. If the average particle size
of the insoluble carriers is 0.20 .mu.m or less, or exceeds 0.40
.mu.m, it may be difficult to obtain an equimolar response. Note
that the term "average particle size (.mu.m)" used herein in
connection with the insoluble carriers refers to a value that is
obtained by image analysis using a transmission electron microscope
(see the examples) and rounded off to two decimal places. Note that
the average particle size can be calculated to four decimal places
by image analysis.
[0028] In case that the average particle size is more than 0.20
.mu.m and equal to or less than 0.40 .mu.m, it is considered that
two types of particles (e.g. prepared in different lots) have an
identical average particle size when the average particle sizes of
the two types of particles satisfy the relationship
"M-N.ltoreq.P+Q" when the average particle size (.+-.SD) of
particles A is M.+-.N, the average particle size (.+-.SD) of
particles B is P.+-.Q, M>P, and N and Q are 0.02 .mu.m or
less.
[0029] For example, when the average particle size of particles A
is 0.22.+-.0.02 .mu.m and the average particle size of particles B
is 0.21.+-.0.02 .mu.m, M-N is 0.20 and P+Q is 0.23 (i.e., the above
relationship is satisfied). Therefore, it is considered that the
particles A and the particles B have an identical average particle
size.
[0030] When the average particle size of particles A is
0.40.+-.0.02 .mu.m and the average particle size of particles B is
0.37.+-.0.02 .mu.m, M-N is 0.38 and P+Q is 0.39 (i.e., the above
relationship is satisfied). Therefore, it is considered that the
particles A and the particles B have an identical average particle
size.
[0031] A mixture of particles wherein the particles are considered
to have an identical average particle size as mentioned above and
are mixed in an arbitrary ratio is within the scope of the
invention.
[0032] According to the embodiments of the invention, two or more
carriers have an identical average size, but a combination of
carriers made from different materials is not excluded. A
combination of carriers having different average particle sizes is
also not excluded, as long as the carrier therefrom has an average
particle size within the above range and is used in the main
reaction to obtain an equimolar response.
(Immobilization of Antibodies)
[0033] The insoluble carriers may immobilize the antibodies thereon
by a generally-used physical adsorption method, and a chemical
binding method, an immunobinding method, or the like is used as
well. The carriers which have respectively immobilize thereon two
kinds of monoclonal antibodies that recognize different epitopes
are normally mixed in an appropriate ratio (see the examples), but
the two kinds of monoclonal antibodies may be mixed beforehand in
an appropriate ratio to be immobilized on the carriers.
(Agglutination Accelerator)
[0034] The agglutination accelerator is not particularly limited as
long as the agglutination accelerator promotes agglutination of the
insoluble carriers that immobilize the antibodies thereon via an
antigen-antibody reaction. Examples of the agglutination
accelerator include polyethylene glycol, a polysaccharide,
polyvinylpyrrolidone, polyvinyl chloride, a poly-.gamma.-glutamate,
poly(2-methacryloyloxyethylphosphorylcholine) (hereinafter may be
referred to as "MPC polymer"), and the like. The polysaccharide is
preferably one or more polysaccharides selected from dextran,
pullulan, and alkylated polysaccharides including methyl cellulose
and ethyl cellulose. The poly-.gamma.-glutamate may be an alkali
metal salt (e.g., sodium, potassium, or lithium salt), an
alkaline-earth metal salt (e.g., magnesium, calcium, or barium
salt), or an ammonium salt.
[0035] Among these, polyethylene glycol and polyvinylpyrrolidone
are preferable, and polyethylene glycol is more preferable. Note
that a plurality of agglutination accelerators may be used in
combination.
[0036] Products that differ in molecular weight are commercially
available as the agglutination accelerator, and may be
appropriately selected taking account of water-solubility and the
like. The number average molecular weight of polyethylene glycol is
preferably 3,000 to 1,000,000, for example, more preferably 5,000
to 100,000, and particularly preferably 5,000 to 50,000. When the
number average molecular weight of polyethylene glycol is 3,000 to
1,000,000, excellent measurement sensitivity can be achieved.
Polyvinylpyrrolidone may have a weight average molecular weight of
25,000 to 1,200,000, for example, and preferably 40,000 to 360,000.
The poly-.gamma.-glutamate may have a weight average molecular
weight of 200,000 to 6,000,000, for example. The MPC polymer may
have a molecular weight of 5,000 to 5,000,000, for example, and
preferably 500,000 to 2,000,000.
[0037] It is preferable to appropriately set the concentration of
the agglutination accelerator so that an equimolar response is
obtained taking account of the average particle size of the
carriers and the type of the agglutination accelerator. For
example, the concentration of the agglutination accelerator is
preferably adjusted so that the final concentration of the
agglutination accelerator is 0.1 to 5 wt %, more preferably 0.2 to
2 wt %, and particularly preferably 0.2 to 0.6 wt %, when the
agglutination accelerator is brought into contact with the sample.
When the final concentration of the agglutination accelerator is
0.1 to 5 wt %, excellent measurement sensitivity can be
achieved.
(Additive)
[0038] A saccharide (e.g., glucose and sucrose), an inorganic salt
(e.g., sodium chloride), a surfactant (e.g., polyoxyethylene
sorbitan monostearate), a preservative (e.g., sodium azide), and/or
a non-specific reaction inhibitor (e.g., IgG antibody derived from
a normal animal) may be added to the reagent as long as the
reaction between the anti-PSA monoclonal antibody and PSA is not
hindered.
[0039] The content of the saccharide in the reagent is preferably
about 0.1 to about 10 wt %, the content of the inorganic salt in
the reagent is preferably about 0.01 to about 5 wt %, the content
of the surfactant in the reagent is preferably about 0.02 to about
5 wt %, the content of the preservative in the reagent is
preferably about 0.001 to about 0.1 wt %, and the content of the
non-specific reaction inhibitor in the reagent is preferably about
0.001 to about 5 wt %.
(Buffer)
[0040] The antigen-antibody reaction according to the embodiments
of the invention is effected in a buffer. The type, the
concentration, and the pH of the buffer are not particularly
limited as long as the antigen-antibody reaction occurs. Example of
the buffer includes a phosphate buffer, a Tris-HCl buffer, a
carbonate buffer, a glycine buffer, a Good's buffer, and the like.
The concentration of the buffer is about 3 to about 500 mM,
preferably 5 to 100 mM, and more preferably 5 to 50 mM. It is
preferable that the buffer have a pH in a neutral to basic region
(normally 6.5 to 9.5).
(Agglutination Signal Measurement Method)
[0041] The agglutination signal may be measured using an arbitrary
method used to measure agglutination. For example, the
agglutination signal may be measured by measuring the absorbance,
the particle count, the particle size, scattered light, or the
like.
[0042] For example, PSA is measured as described below.
[0043] Specifically, agglutination occurs when at least two kinds
of antibodies that can react with both fPSA and PSA-ACT are
immobilized on carriers (e.g., latex) and thereafter react with a
sample that includes fPSA and/or PSA-ACT. The total PSA level in
the sample can be determined by measuring the degree of
agglutination, and comparing the measured degree of agglutination
with the degree of agglutination when using a standard solution
having a known PSA level. For example, the degree of agglutination
is preferably detected as a change in absorbance by utilizing a
general-purpose biochemical automatic analyzer. In this case, it is
preferable to use a change in absorbance at a wavelength of 500 to
900 nm for determination of the degree of agglutination.
(Equimolar Response)
[0044] Since an equimolar response to fPSA and PSA-ACT can be
obtained by utilizing the assay according to the embodiments of the
invention, PSA can thereby be quantitatively determined with high
accuracy.
[0045] The expression "equimolar response to fPSA and PSA-ACT" used
herein means that the ratio of a signal obtained by measuring a
sample that includes only fPSA to a signal obtained by measuring a
sample that includes only PSA-ACT (equal mol with fPSA) is
approximately 1:1.
[0046] For example, the ratio (PSA-ACT/fPSA) (hereinafter may be
referred to as "c/f ratio") of agglutination signals per equal mol
of fPSA and PSA-ACT is calculated, and it is considered that an
equimolar response is obtained when the c/f ratio is 85 to 115%
(most preferably 90 to 110%).
(Method for Producing Reagent)
[0047] The PSA assay reagent according to the embodiments of the
invention is produced as described below.
[0048] Specifically, the PSA assay reagent may be produced by
providing carriers having immobilized thereon two kinds of
antibodies that can react with both PSA-ACT and fPSA and differ in
recognition site, setting the mixing ratio of the carriers having
immobilized thereon two kinds of antibodies and have an identical
average particle size (more than 0.20 .mu.m and equal to or less
than 0.40 .mu.m) within a specific range, and adjusting the
concentration of the agglutination accelerator so that an equimolar
response to PSA-ACT and fPSA is obtained. The mixing ratio is 1:10
to 10:1, preferably 1:5 to 5:1, and more preferably 1:2 to 2:1.
[0049] The invention is further described below by way of examples.
Note that the invention is not limited to the following
examples.
EXAMPLES
(Preparation of Monoclonal Antibodies)
1. Immunization
(1) Immunogen
[0050] Human semen-derived purified PSA (SCIPAC Ltd., Code No.
P117-7, degree of purification: 96%) was used as an immunogen. The
purified PSA was used after dialysis using 20 mM PBS (pH: 7.2).
(2) Immunization Method
[0051] The above PSA solution and a complete Freund's adjuvant
(CFA) (GIBCO) were mixed and emulsified in a ratio of 1:1, and
administered subcutaneously to the back of 6-week-old female Balb/C
mice in an amount of 25 .mu.g PSA/mouse. Additional immunization
was performed three times at intervals of 2 weeks, and the PSA
solution (25 .mu.g PSA/mouse) was administered intraperitoneally 3
days before cell fusion.
2. Cell Fusion
[0052] The spleen was removed from each mouse immunized with PSA to
collect spleen cells. The spleen cells and mouse myeloma cells
SP2/O--Ag14 were mixed in a ratio of 6:1, and fused in the presence
of 50% polyethylene glycol 1540 (Wako Pure Chemical Industries,
Ltd.). The fused cells were suspended in an HAT medium so that the
number of spleen cells was 2.5.times.10.sup.6, and dispensed onto a
96-well culture plate (CORNING Inc.) in an amount of 0.2 ml/well.
The fused cells were cultured at 37.degree. C. for 2 weeks in a 5%
CO.sub.2 incubator.
3. Screening
[0053] The culture supernatant in each well of the culture plate
into which the fused cells were dispensed was subjected to ELISA
(see below) to select wells that reacted with both fPSA and
PSA-ACT.
(1) Material: Antigen
[0054] 1) PSA: SCIPAC, Code No. P117-7 2) PSA-ACT: SCIPAC, Code No.
P192-3
(2) Method
[0055] 1) An ELISA plate (Nunc) was coated (50 .mu.l/well) with a
goat anti-mouse IgG (Fc) antibody (Jackson Inc.) (5 .mu.g/ml), and
allowed to stand at 4.degree. C. overnight. 2) After washing the
ELISA plate with a washing solution (0.05% Tween 20-PBS) three
times (400 .mu.l/well), a blocking reagent (0.05% Tween 20-PBS) was
dispensed into each well in an amount of 200 .mu.l/well, and the
ELISA plate was allowed to stand at room temperature for 1 hour. 3)
After removing the blocking reagent, the culture supernatant in
each well of the culture plate into which the fused cells were
dispensed, was dispensed into each well of the ELISA plate in an
amount of 50 .mu.l/well, and the ELISA plate was allowed to stand
at room temperature for 1 hour. 4) After washing the ELISA plate
with a washing solution (0.05% Tween 20-PBS) three times, fPSA or
PSA-ACT diluted with 0.05% Tween 20-PBS to 5 ng/ml was dispensed
into each well in an amount of 50 .mu.l/well, and the ELISA plate
was allowed to stand at room temperature for 1 hour. 5) After
washing the ELISA plate with a washing solution three times, an
HRP-rabbit anti-human PSA antibody (.times.500) was dispensed into
each well in an amount of 50 .mu.l/well, and the ELISA plate was
allowed to stand at room temperature for 1 hour. Note that the
HRP-rabbit anti-human PSA antibody was prepared by a periodic acid
method using a rabbit anti-human PSA antibody (DAKO) and peroxidase
(Toyobo Co., Ltd.). 6) After washing the ELISA plate three times,
an OPD color reagent was dispensed into each well in an amount of
50 .mu.l/well, and the ELISA plate was allowed to stand at room
temperature for 10 minutes. 7) A stop solution (1.5 N sulfuric
acid) was dispensed into each well in an amount of 50 .mu.l/well to
terminate the reaction, and the absorbance at a wavelength of 492
nm was measured using a plate leader.
4. Cloning
[0056] The cell lines in the wells that reacted with both fPSA and
PSA-ACT during the above screening were cloned by a limiting
dilution method to establish hybridomas. 28 types of established
hybridomas were thus obtained.
5. Preparation of Monoclonal Antibodies
[0057] The hybridomas (0.5.times.10.sup.6 cells) obtained by
cloning were administered intraperitoneally to 8-week-old female
Balb/C mice to which 0.5 ml of pristane was administered
intraperitoneally 2 weeks ago. Abdominal fluid was collected when 2
weeks had elapsed, and an IgG fraction was purified using a protein
A column (Amersham plc). Purified fractions of the 28 types of
monoclonal antibodies were thus obtained.
(Preliminary Consideration of Combination of Monoclonal
Antibodies)
[0058] Each of the 28 types of monoclonal antibodies is immobilized
on a latex, and agglutination of the latex was observed as
described below. Two kinds of antibodies (#91 antibody and #51
antibody) that showed a large signal, and combinations thereof with
the latex were selected.
1. Selection of Antibodies
[0059] (1) A 1% latex (0.3 .mu.m) solution diluted with 20 mM
Tris-HCl (pH: 8.5) and each monoclonal antibody solution (0.5 Abs)
were mixed in a ratio of 1:1 (v/v), and the mixture was stirred at
4.degree. C. for 2 hours. (2) After the addition of 20 mM Tris-HCl
(pH: 8.5) including 0.4% BSA (2 vol), the mixture was stirred at
4.degree. C. for 1 hour. (3) The supernatant liquid was removed by
centrifugation, and suspended in 5 mM MOPS (pH: 7.0) so that the
absorbance at 600 nm was 2 Abs. (4) Two kinds of MoAb-Lx
(antibody-immobilized latexes) were selected, and mixed in a ratio
of 1:1 (v/v) to obtain a reagent 2 (the reagent 2 was prepared
using all of the combinations). (5) 26 ng/ml of fPSA and PSA-ACT
were measured using a reagent 1 (30 mM HEPES buffer (pH: 7.0)
including 0.1% BSA, 0.5 M NaCl, 0.3% polyvinylpyrrolidone K-90 (PVP
K-90)) and the reagent 2 utilizing a Hitachi 7170 automatic
analyzer. (6) Two groups of antibodies that were considered to
recognize different epitopes were obtained from the combinations of
monoclonal antibodies that showed agglutination. i) #63217, #63251,
and #63279 ii) #63214 and #63291
[0060] Agglutination was observed using the combinations of the
antibodies of groups i) and ii).
[0061] The hybridomas that produce the antibodies #63251, #63279,
and #63291 are deposited at International Patent Organism
Depositary (IPOD), National Institute of Advanced Industrial
Science and Technology as accession numbers FERM BP-11453, FERM
BP-11454, and FERM BP-11455. The monoclonal antibodies obtained
from these hybridomas may be referred to as "#51 antibody", "#79
antibody", and "#91 antibody", which respectively corresponds to
the first- and second-final numbers of the identification
numbers.
(Production of Latexes that Differ in Particle Size) 1) 0.2 .mu.m
or less
[0062] A glass reaction vessel (2 l) equipped with a stirrer, a
reflux condenser, a thermometer, a nitrogen inlet tube, a heating
oil bath, and the like was charged with 1200 g of water, 200 g of a
styrene monomer, 1.2 g of potassium persulfate, and 0.2 g of sodium
styrene sulfonate, and the atmosphere inside the reaction vessel
was sufficiently replaced with nitrogen with stirring (about 200
rpm). After polymerizing the monomer at 70.degree. C. for about 18
hours, the reaction solution was filtered through filter paper
(ADVANTEC No. 2) to obtain latex particles. The average particle
size (.+-.SD) of the latex particles was determined by
photographing the latex particles using a transmission electron
microscope, randomly selecting three fields of view, subjecting 100
or more latex particles within each field of view to image analysis
to determine the average particle size (.+-.SD) of the latex
particles for each field of view, and averaging the average
particle size (.+-.SD) of the latex particles for each field of
view. The average particle size thus determined was 0.19 .mu.m
(.+-.0.01 .mu.m).
2-1) 0.23 .mu.m
[0063] Latex particles were obtained in the same manner as in
section 1), except that 1200 g of water, 200 g of a styrene
monomer, 2.4 g of potassium persulfate, and 0.1 g of sodium styrene
sulfonate were used. The average particle size of the latex
particles thus obtained was 0.23 .mu.m (.+-.0.01 .mu.m).
2-2) 0.25 .mu.m
[0064] Latex particles were obtained in the same manner as in
section 1), except that 1200 g of water, 200 g of a styrene
monomer, 0.9 g of potassium persulfate, and 0.2 g of sodium styrene
sulfonate were used. The average particle size of the latex
particles thus obtained was 0.25 .mu.m (.+-.0.01 .mu.m).
2-3) 0.29 .mu.m
[0065] Latex particles were obtained in the same manner as in
section 1), except that 1200 g of water, 200 g of a styrene
monomer, 1.3 g of potassium persulfate, and 0.1 g of sodium styrene
sulfonate were used. The average particle size of the latex
particles thus obtained was 0.29 .mu.m (.+-.0.01 .mu.m).
2-4) 0.34 .mu.m
[0066] Latex particles were obtained in the same manner as in
section 1), except that 1200 g of water, 200 g of a styrene
monomer, 1.3 g of potassium persulfate, and 0.08 g of sodium
styrene sulfonate were used. The average particle size of the latex
particles thus obtained was 0.34 .mu.m (.+-.0.01 .mu.m).
2-5) 0.40 .mu.m
[0067] Latex particles were obtained in the same manner as in
section 1), except that 1200 g of water, 200 g of a styrene
monomer, 1.3 g of potassium persulfate, and 0.03 g of sodium
styrene sulfonate were used. The average particle size of the latex
particles thus obtained was 0.40 .mu.m (.+-.0.01 .mu.m).
3) More than 0.4 .mu.m
[0068] Latex particles were obtained in the same manner as in
section 1), except that 1200 g of water, 200 g of a styrene
monomer, 1.2 g of potassium persulfate, and 0.01 g of sodium
styrene sulfonate were used. The average particle size of the latex
particles thus obtained was 0.42 .mu.m (.+-.0.01 .mu.m).
(Preparation of Antibody-Immobilized Latex)
[0069] Two kinds of monoclonal antibodies (#91 and #51) obtained as
described above are immobilized on carriers using the following
materials and method.
1. Material
(1) Anti-PSA Monoclonal Antibody
[0070] #63291 (#91) #63251 (#51) (These monoclonal antibodies were
dissolved in PBS.)
(2) Latex
[0071] Polystyrene latexes (average particle size: 0.19 to 42
.mu.m) obtained as described above
2. Preparation of Antibody-Immobilized Latex Liquid
[0072] (1) Preparation of #91 antibody-latex complex
(#91Lx)-containing liquid 1) The latex and the #91 antibody were
respectively diluted with a 20 mM glycine buffer (pH: 9) to prepare
a 1% latex liquid and a #91 antibody liquid (0.4 mg/ml). The latex
liquid and the #91 antibody liquid were mixed (1:1, v/v), and the
mixture was stirred for about 1 hour. 2) A blocking reagent (10%
BSA) was added to the mixture (0.1:2, v/v), and the mixture was
stirred for about 1 hour. 3) The supernatant liquid was removed by
centrifugation, suspended in a 5 mM MOPS buffer (pH: 7.0), and
diluted so that the absorbance at a wavelength of 600 nm was 3
Abs/ml to obtain a #91 antibody-immobilized latex (#91Lx) liquid.
(2) Preparation of #51 antibody-latex complex (#51Lx)-containing
liquid 1) The latex and the #51 antibody were respectively diluted
with a 20 mM Tris buffer (pH: 8) to prepare a 1% latex liquid and a
#51 antibody liquid (0.4 mg/ml). The latex liquid and the #51
antibody liquid were mixed (1:1, v/v), and the mixture was stirred
for about 1 hour. 2) A blocking reagent (10% BSA) was added to the
mixture (0.1:2, v/v), and the mixture was stirred for about 1 hour.
3) The supernatant liquid was removed by centrifugation, suspended
in a 5 mM MOPS buffer (pH: 7.0), and diluted so that the absorbance
at a wavelength of 600 nm was 3 Abs/ml to obtain a #51
antibody-immobilized latex (#51Lx) liquid.
Test Example 1
(Determination of Change in c/f Ratio Due to Change in Average
Particle Size of Latex)
[0073] fPSA and PSA-ACT (concentration: 5 ng/ml) were used as
measurement samples, and the reactivity with fPSA and PSA-ACT was
determined using the following PSA measurement reagents and
measurement conditions. The ratio "PSA-ACT/fPSA" (c/f ratio) of the
reactivity with PSA-ACT to the reactivity with fPSA was determined.
The results are shown in Table 1.
(1) PSA Measurement Reagent
First Reagent (Buffer)
[0074] 30 mM HEPES buffer (pH: 7.0) including 0.5 M KCl, 0.1% BSA
(Proliant), and 0.3% PVP K-90
Second Reagent (Antibody-Immobilized Latex Liquid)
[0075] 5 mM MOPS buffer (pH: 7.0) including mouse anti-PSA
monoclonal antibody-immobilized latex particles (mixing ratio of
two kinds of antibody-immobilized particle, #91Lx-containing
liquid: #51Lx-containing liquid=1:1.
[0076] Each combination of the antibody-containing liquids used in
this example is prepared by respectively using latex particles
having an identical average particle size (0.19 to 0.42 .mu.m) and
immobilizing thereon the #91 antibody or the #51 antibody.
(2) Measurement Conditions
[0077] Measurement system: Hitachi 7170 automatic analyzer
(H-7170)
[0078] Measurement parameters:
[0079] Analysis method: 2-point end method (measurement points: 19
to 34)
[0080] Amount of liquid (.mu.l): 14.4/90/90
[0081] Measurement wavelength (nm): 570 (main)/800 (sub)
[0082] Calibration: spline
[0083] Calibration was performed using a Nanopia (registered
trademark) PSA calibrator (PSA concentration: 0, 4.2, 10, and 29
ng/ml, Sekisui Medical Co., Ltd.).
(3) Measurement Sample
[0084] The measurement samples were prepared by dissolving fPSA and
PSA-ACT (Fitzgerald) in PBS (pH: 7.4) including 1% BSA and 0.1%
NaN.sub.3.
TABLE-US-00001 TABLE 1 Test Test Test Test Test Test Test Example
Example Example Example Example Example Example 1-1 1-2 1-3 1-4 1-5
1-6 1-7 Average particle 0.42 0.40 0.34 0.29 0.25 0.23 0.19 size
(.mu.m) c/f ratio (%) 70.6 86.0 95.1 95.0 88.0 90.0 79.0
[0085] According to the results shown in Table 1, it was confirmed
that the c/f ratio was 85% or more (close to 100%) and an equimolar
response was obtained when the average particle size was more than
0.20 .mu.m and equal to or less than 0.40 .mu.m.
[0086] The c/f ratio was less than 80% and an equimolar response
was not obtained when the average particle size was 0.20 .mu.m or
less, or exceeded 0.40 .mu.m.
Test Example 2
(Adjustment of c/f Ratio by Addition of Agglutination
Accelerator)
[0087] fPSA and PSA-ACT (concentration: 35 ng/ml) were used as
measurement samples. The reactivity with fPSA and PSA-ACT was
determined using the following PSA measurement reagents under the
same measurement conditions as those employed in Test Example 1,
and the ratio "PSA-ACT/fPSA" (c/f ratio) of the reactivity with
PSA-ACT to the reactivity with fPSA was determined. The results are
shown in Tables 2 to 4.
(1) PSA Measurement Reagent
First Reagent (Buffer)
[0088] 30 mM HEPES buffer (pH: 7.0) including 0.5 M KCl, 0.1% BSA
(Proliant), and 0 to 0.8% agglutination accelerator (PEG
(polyethylene glycol) (the numerical value is the number average
molecular weight), PVP (polyvinylpyrrolidone) (molecular weight of
PVP K-30: 40,000, molecular weight of PVP K-90: 360,000, Wako Pure
Chemical Industries, Ltd.), MPC polymer (molecular weight:
1,000,000), Pullulan PI-20 (molecular weight: about 200,000,
Hayashibara Co., Ltd.), or PGA-Na (poly-.gamma.-sodium glutamate)
(the numerical value is the number average molecular weight) (see
Tables 2 to 4))
[0089] (The agglutination accelerator was not added in the
comparative example.)
Second Reagent (Antibody-Immobilized Latex Liquid)
[0090] 5 mM MOPS buffer (pH: 7.0) including mouse anti-PSA
monoclonal antibody-immobilized latex particles (mixing ratio of
two kinds of antibody-immobilized particle, #91Lx-containing
liquid: #51Lx-containing liquid=1:1).
[0091] The antibody-containing liquid used in this example is
prepared by using latex having an identical average particle size
(0.29 .mu.m) and immobilizing thereon the #91 antibody and the #51
antibody.
TABLE-US-00002 TABLE 2 Test Test Test Test Test Test Comparative
Example Example Example Example Example Example Example 2-1 2-2 2-3
2-4 2-5 2-6 Agglutination Not added PEG6000 PEG20000 accelerator --
0.2% 0.3% 0.6% 0.2% 0.3% 0.6% Amount c/f ratio (%) 82.3 84.4 85.4
88.6 85.5 87.4 93.7
TABLE-US-00003 TABLE 3 Test Test Test Test Test Test Test Example
Example Example Example Example Example Example 2-7 2-8 2-9 2-10
2-11 2-12 2-13 Agglutination PVP K-30 PVP K-90 MPC polymer
accelerator 0.2% 0.3% 0.6% 0.2% 0.3% 0.6% 0.8% Amount c/f ratio (%)
83.0 84.7 87.7 88.3 92.7 96.6 99.7
TABLE-US-00004 TABLE 4 Test Test Test Test Test Test Example
Example Example Example Example Example 2-14 2-15 2-16 2-17 2-18
2-19 Agglutination Pullulan 200,000 1,500,000 4,000,000 accelerator
to 500,000 to 2,500,000 to 6,000,000 Amount PGA-Na PGA-Na PGA-Na
0.2% 0.3% 0.6% 0.20% 0.25% 0.30% c/f ratio (%) 88.1 89.5 93.7 93.9
94.9 97.0
[0092] According to the results shown in Tables 2 to 4, it was
confirmed that the c/f ratio approached from about 82% to 100% due
to the addition of the agglutination accelerator and an increase in
the amount of the agglutination accelerator, and an equimolar
response (c/f ratio: 85% or more) was obtained (showing an effect
increasing the c/f ratio to about 100%) by a one-step reaction by
appropriately adjusting the amount of the agglutination
accelerator. Note that the term "one-step reaction" refers to
adding and reacting two kinds of anti-PSA monoclonal antibodies at
one time.
Test Example 3
(Determination of Reactivity Using Different Antibody
Combinations)
[0093] The c/f ratio was determined using a combination of the
monoclonal antibodies #79 and #91, #14 and #51, #91 and #17, or #14
and #17 instead of the combination of the monoclonal antibodies #51
and #91, and using the following measurement reagent including a
0.6% MPC polymer as the agglutination accelerator. The remaining
conditions were the same as those employed in Test Example 1. fPSA
and PSA-ACT (concentration: 35 ng/ml) were used as samples.
(1) PSA Measurement Reagent
First Reagent (Buffer)
[0094] 30 mM HEPES buffer (pH: 7.0) including 0.5 M KCl, 0.1% BSA
(Proliant), and 0.6% MPC polymer
[0095] The results are shown in Table 5.
TABLE-US-00005 TABLE 5 Test Test Test Test Example Example Example
Example 3-1 3-2 3-3 3-4 Combination of #79 antibody #14 antibody
#91 antibody #14 antibody antibodies #91 antibody #51 antibody #17
antibody #17 antibody c/f ratio (%) 96.4 89.0 91.2 101.9
[0096] According to the results shown in Table 5, it was confirmed
that an equimolar response was obtained (c/f ratio: 89.0 to 101.9%)
by using any of the above combinations.
(Kd Value of Monoclonal Antibodies Used in Test Examples)
[0097] Table 6 shows the Kd values of the monoclonal antibodies
used in the test examples for fPSA and PSA-ACT. As shown in Table
6, it was confirmed that it is preferable that the anti-PSA
monoclonal antibodies have a ratio (fKd/cKd) of the dissociation
constant (fKd) for free PSA to the dissociation constant (cKd) for
complex of free PSA and .alpha.1-antichymotrypsin (PSA-ACT) of more
than 0.1 and equal to or less than 2.0, and have a dissociation
constant (fKd) for free PSA of 10 nM or less.
TABLE-US-00006 TABLE 6 Kd value (nM) fKd cKd fKd/cKd #14 antibody
5.58 23.61 0.24 #17 antibody 1.72 4.29 0.40 #51 antibody 0.82 1.39
0.59 #79 antibody 0.72 0.57 1.26 #91 antibody 0.1 0.25 0.40
[0098] The c/f ratio was 52.4%, and an equimolar response was not
obtained when using the #16 antibody (fKd=1.33 nM, cKd=23.18 nM,
fKd/cKd=0.057) instead of the #91 antibody of the Test Example
3-3.
[0099] The Kd value in Table 6 was determined under the following
experimental conditions.
1. Biacore (registered trademark) system and dedicated reagent (GE
Healthcare (former Biacore), (i) to (viii) indicate the product
name and the catalog No. from Biacore (currently available from GE
Healthcare)) (i) Biacore (registered trademark) T100: JJ-1037-02
(Biacore)
(ii) Series S Sensor Chip CM5: BR-1005-30 (Biacore)
[0100] (iii) Amine Coupling Kit: BR-1000-50 (Biacore)
(iv) Acetate 5.0: BR-1003-51 (Biacore)
(v) .alpha.-Mouse Immunoglobulins: BR-1005-14 (Biacore)
(vi) Glycine 1.5: BR-1003-54 (Biacore)
[0101] (vii) Glycine 2.0: BR-1003-55 (Biacore) (viii)
HBS-EP+10.times.(running buffer): BR-1006-69 (Biacore) (adjusted to
pH 8.5 with NaOH, and 10-fold diluted with purified water prior to
use) 2. Test method (i) The sensor chip CM5 on which .alpha.-Mouse
Immunogloblins is immobilized is placed in the Biacore system. (ii)
The anti-PSA antibodies are respectively diluted to 1.0 .mu.g/ml
with the running buffer (HBS-EP), and added for 60 seconds at a
flow rate of 30 .mu.l/min. (iii) The PSA antigen RSA or PSA-ACT) is
diluted to 5.0 .mu.g/ml with the running buffer (HBS-EP), and added
for 120 seconds at a flow rate of 30 .mu.l/min. (iv) A free running
operation is performed using the running buffer (HBS-EP) to effect
dissociation for 120 seconds (flow rate: 30 .mu.l/min). (v) The
sensor chip is regenerated using a regenerant (Glycine 1.75
prepared by mixing Glycine 1.5 and Glycine 2.0 in a ratio of
1:1).
INDUSTRIAL APPLICABILITY
[0102] The assay and the reagent according to the embodiments of
the invention can easily and accurately measure PSA using a
general-purpose automatic analyzer, and are useful for early
detection of prostatic diseases (particularly prostate cancer).
[Reference to the Deposited Microorganism]
[0103] (1) FERM BP-11453 (Hybridoma #63251 producing the
#51-antibody)
[0104] i) Name and address of depository institution at which the
biological materials were deposited.
[0105] International Patent Organism Depositary, National Institute
of Advanced Industrial Science and Technology Tsukuba Central 6,
1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
[0106] ii) Date of biological material deposit in the depository
institution in i).
[0107] Feb. 19, 2010
[0108] iii) Accession number for the deposition assigned by the
depository institution in i).
[0109] FERM BP-11453
(2) FERM BP-11454 (Hybridoma #63279 producing the #79-antibody)
[0110] i) Name and address of depository institution at which the
biological materials were deposited.
[0111] International Patent Organism Depositary, National Institute
of Advanced Industrial Science and Technology Tsukuba Central 6,
1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
[0112] ii) Date of biological material deposit in the depository
institution in i).
[0113] Feb. 19, 2010
[0114] iii) Accession number for the deposition assigned by the
depository institution in i).
[0115] FERM BP-11454
(3) FERM BP-11455 (Hybridoma #63291 producing the #91-antibody)
[0116] i) Name and address of depository institution at which the
biological materials were deposited.
[0117] International Patent Organism Depositary, National Institute
of Advanced Industrial Science and Technology Tsukuba Central 6,
1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
[0118] ii) Date of biological material deposit in the depository
institution in i).
[0119] Feb. 19, 2010
[0120] iii) Accession number for the deposition assigned by the
depository institution in i).
[0121] FERM BP-11455
* * * * *