U.S. patent application number 11/394463 was filed with the patent office on 2006-08-03 for reagent for an immunoassay.
This patent application is currently assigned to Wako Pure Chemical Industries, Ltd.. Invention is credited to Hiromichi Adachi, Minoru Fujita, Kyoichi Sumida.
Application Number | 20060172351 11/394463 |
Document ID | / |
Family ID | 34373384 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060172351 |
Kind Code |
A1 |
Sumida; Kyoichi ; et
al. |
August 3, 2006 |
Reagent for an immunoassay
Abstract
The present invention relates to (1) A reagent for an
immunoassay of a target substance existing in a free form and a
bound form in a specimen, comprising a latex 1 which is immobilized
with a monoclonal antibody 1 for the target substance, and a latex
2 which has a different mean particle size from the latex 1 and is
immobilized with a monoclonal antibody 2 having a different
recognition site for the target substance from the antibody 1, (2)
An immunoassay method comprising reacting the target substance with
the reagent of (1) and determining an amount of the substance based
on the result of an agglutination reaction among the target
substance, the latex1 and the latex2, and (3) A reagent kit
comprising a reagent of (1) and a reagent containing an
agglutination accelerator for an antigen-antibody reaction.
Inventors: |
Sumida; Kyoichi;
(Amagasaki-shi, JP) ; Fujita; Minoru;
(Amagasaki-shi, JP) ; Adachi; Hiromichi;
(Amagasaki-shi, JP) |
Correspondence
Address: |
HAMRE, SCHUMANN, MUELLER & LARSON, P.C.
P.O. BOX 2902-0902
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Wako Pure Chemical Industries,
Ltd.
Chuo-Ku
JP
|
Family ID: |
34373384 |
Appl. No.: |
11/394463 |
Filed: |
March 30, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10867912 |
Jun 15, 2004 |
|
|
|
11394463 |
Mar 30, 2006 |
|
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Current U.S.
Class: |
435/7.23 |
Current CPC
Class: |
G01N 33/54313
20130101 |
Class at
Publication: |
435/007.23 |
International
Class: |
G01N 33/574 20060101
G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 30, 2003 |
JP |
2003-340028 |
Claims
1-11. (canceled)
12. An immunoassay method for a target substance existing in a free
form and a bound form in a specimen, comprising reacting the target
substance with a first latex particle [1] which is immobilized with
a first monoclonal antibody [1] for the target substance, and a
second latex particle [2] which has a different mean particle size
from the first latex particle [1] and is immobilized with a second
monoclonal antibody [2] having a different recognition site for the
target substance from the first monoclonal antibody [1], and
determining an amount of the substance based on the result of an
agglutination reaction among the target substance, the first latex
particle [1] and the second latex particle [2].
13. The method according to claim 12, wherein the result of the
agglutination reaction is a change in turbidity.
14. The method according to claim 13, wherein the target substance
is prostate-specific antigen.
15. A method according to claim 14, wherein the first latex
particle [1] is one having the mean particle size of 0.05-0.18
.mu.m and the second latex particle [2] is one having a mean
particle size of 0.18-0.5 .mu.m.
16. The method according to claim 12, wherein the reaction of the
target substance with the first latex particle [1] and the second
latex particle [2] is performed in the presence of a polymer having
a side chain of the group represented by the general formula [1]:
##STR6## (wherein each of R.sup.1-R.sup.3 is an alkyl group
optionally having a hydrogen atom or a hydroxyl group
independently, and R.sup.4 is an alkylene group)
17. The method according to claim 16, wherein the polymer having a
side chain of the group represented by the general formula [1] is a
polymer having a monomer unit derived from the monomer represented
by the general formula [2]: ##STR7## (wherein R.sup.5 is an
alkylene group optionally having a substituent and an oxygen atom
in the chain, R.sup.6 is a hydrogen atom or a methyl group, X is an
oxygen atom or a --NH-- group, and R.sup.1-R.sup.4 are the same as
the above).
Description
[0001] This application is a division of application Ser. No.
10/867,912, filed Jun. 15, 2004, entitled A REAGENT FOR AN
IMMUNOASSAY.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field
[0003] The present invention relates to a reagent for a high
sensitive immunoassay method, an immunoassay method using said
reagent and a reagent kit comprising said reagent.
[0004] 2. Related Art
[0005] It has been known that some biological substances exist both
in a free form and a bound form. A representative example thereof
is the prostate-specific antigen (PSA) which has been known to
exist in a bound form of PSA and .alpha..sub.1-antichymotrypsin
(ACT) in blood, as well as in a free form of PSA itself. Since such
substances exhibit a change in a ratio of the free form and the
bound form depending on an amount of substance bound with the free
form as well as in vivo circumstances, both forms should be assayed
in the assay of such substances. However, there is a problem that
in an immunoassay of such substances by applying antigen-antibody
reaction, a high precise assay is difficult due to different
binding rate of the antibody to the free form and the bound form.
In order to solve such the problem, JP-A-09-234068 proposes an
assay reagent for equimolar reaction with both free form and bound
form, and an assay method using the same are proposed. Since this
method is, however, the assay method by ELISA, it can be possible
to assay only in the specific equipment and is impossible to apply
in the common autoanalyzer. Although the latex turbidimetry is
known as a high sensitive immunoassay method by applying the common
autoanalyzer, there is also a problem that the equimolar assay of
both free form and bound form is difficult in such a method, even
if the latex sensitized (immobilized) with the antibody which
exhibits equimolar reaction in ELISA is used. Owing to these
problems, a development of a method is demanded, which can assay
the substance existing in the free form and the bound form in the
organism and can be applied in the common autoanalyzer.
[0006] In JP-A-10-123137, an immunoassay method is disclosed, in
which at least two types of different monoclonal antibodies for a
specific antigen are carried by an insoluble carrier such as latex
and the insoluble carrier having different mean particle diameter
is used. However, in this method, the free form and the bound form
in the specimen are not assayed.
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0007] An object of the present invention is to provide a reagent
for immunoassay using two or more types of antibodies which are
immobilized in two or more types of latex having different mean
particle sizes and for equimolar assay of a substance existing both
in the free form and the bound form in the organism with high
sensitivity and high precision, an assay method using said reagent,
and a reagent kit containing said reagent. The present invention
provides the following means for solving the problems. Means for
Solving the Problems
[0008] (1) A reagent for an immunoassay of a target substance
existing in a free form and a bound form in a specimen, comprising
a latex 1 which is immobilized with a monoclonal antibody 1 for the
target substance, and a latex 2 which has a different mean particle
size from the latex 1 and is immobilized with a monoclonal antibody
2 having a different recognition site for the target substance from
the antibody 1.
[0009] (2) An immunoassay method for a target substance existing in
a free form and a bound form in a specimen, comprising reacting the
target substance with a latex 1 which is immobilized with a
monoclonal antibody 1 for the target substance, and a latex 2 which
has a different mean particle size from the latex 1 and is
immobilized with a monoclonal antibody 2 having a different
recognition site for the target substance from the antibody 1, and
determining an amount of the substance based on the result of an
agglutination reaction among the target substance, the latex1 and
the latex2.
[0010] (3) A reagent kit for an immunoassay of a target substance
existing in a free form and a bound form in a specimen, comprising
a reagent for an immunoassay containing a latex 1 which is
immobilized with a monoclonal antibody 1 for the target substance,
and a latex 2 which has a different mean particle size from the
latex 1 and is immobilized with a monoclonal antibody 2 having a
different recognition site for the target substance from the
antibody 1, and a reagent containing an agglutination accelerator
for an antigen-antibody reaction.
EFFECT OF THE INVENTION
[0011] The present invention relates to the highly sensitive and
highly precise reagent for immunoassay of the target substance
existing in the free form and the bound form in the specimen, the
assay method using said reagent, and the reagent kit containing
said reagent. By using the assay method of the present invention,
both target substances in the free form and the bound form can be
precisely assayed in an equimolar.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows the calibration curve prepared from the optical
density of the free PSA and the complex PSA obtained in Example 2
and each concentration thereof, i.e. a comparison with the
reactivity between the forms of PSA and the latex immobilizing the
antibody.
DESCRIPTION OF CODES
[0013] A mark -.circle-solid.- shows assay result of Example 2
using the free form PSA, and a mark -.largecircle.- shows assay
result of Example 2 using the complex form PSA.
BEST MODE FOR CARRYING OUT THE INVENTION
[0014] A target substance in the present invention is not
particularly limited, so long as it has the free form and the bound
form, but the substance having both forms in the specimen to be
assayed are preferable. The free form designated herein means, for
example, the substance existing alone in the specimen such as the
biological specimen. The bound form designated herein means, for
example, a complex which is bound with the free form hereinabove
and the substance having an affinity to the target substance (the
affinity substance). Specifically, the substance having both forms
includes prostate-specific antigen (PSA), protein C, elastase,
cathepsin G, thrombin, C.sub.1-esterase, plasmin and tissue-type
plasminogen activator, and among them, a preferable example thereof
is PSA. The affinity substance in the bound form is not
particularly limited, so long as it is one having an affinity to
the target substance and an ability to bind therewith.
Specifically, .alpha..sub.1-antichymotrypsin or protein C inhibitor
for the case when the target substance is PSA; protein C inhibitor
for the case when the target substance is protein C;
.alpha..sub.1-protease inhibitor for the case when the target
substance is elastase; .alpha..sub.1-antichymotrypsin for the case
when the target substance is cathepsin G; anti-thrombin III for the
case when the target substance is thrombin; C.sub.1 inhibitor for
the case when the target substance is C.sub.1-esterase;
.alpha..sub.2-antiplasmin for the case when the target substance is
plasmin; and plasminogen activator inhibitor 1 can be included for
the case when the target substance is tissue-type plasminogen
activator, respectively.
[0015] A monoclonal antibody for the target substance in the
present invention is not particularly limited so long as it has
reactivity with the target substance. The origin thereof is not
particularly limited. Further, the monoclonal antibody of the
commercially available product or the monoclonal antibody produced
by the known method using cell fusion technique or gene
recombination technology [Eur. J. Immunol. 6, 511 (1976)] and
having properties described in the above can be used.
[0016] In the monoclonal antibody for the target substance in the
present invention, Fab fragment obtained by partial degradation
using papain; F(ab').sub.2 fragment obtained by partial degradation
using pepsin; and Fab' fragment obtained by reduction of
F(ab').sub.2 fragment, namely, antibody fragment can all be
included. Such fragments are preferably used, because the
non-specific reaction can be avoided in the assay of the target
substance.
[0017] Two or more types of the monoclonal antibody for the target
substance in the present invention have the different antibody
recognition site in the target substance. In the case when two
types of the monoclonal antibody are used, the one having the
acidic isoelectric point (pI) and the other having the neutral
isoelectric point are preferably used. Specific examples of the
acidic pI are 3-5, preferably 4-5, and the neutral pI are 6-8,
preferably 7-8. Further, in the case when three or more types of
the antibody are used, the antibody having pI according to the
above may be used.
[0018] A latex used in the present invention is not particularly
limited so long as it is commonly used in this field. Preferable
examples thereof are styrene-based latex such as polystyrene latex,
and acrylic acid-based latex. Among these latex particles, the
polystyrene latex particles obtained by emulsion polymerization
without using emulsifying agent are most preferable, because they
can adsorb protein or peptide smoothly due to strong hydrophobic
surface thereof and have properties to be stably dispersed in a
solution without using any emulsifying agent due to repulsive force
of negative charges on the surface. Various modified latex (e.g.
carboxylic acid modified latex, in which carboxyl group is
introduced into the above polystyrene) and magnetic latex (a latex
including magnetic particles) can also be used if necessary.
[0019] The latex used in the present invention may be commercially
available latex, and the latex particle having small mean particle
size, namely, one having large surface area per unit weight is
preferable due to sensitizing the antibody on the surface
effectively. Specifically, two types of particles having different
mean particle sizes selected from particles generally having
0.05-2.4 .mu.m, preferably 0.05-1.0 .mu.m, more preferably
0.05-0.28 .mu.m, can preferably be used in combination. In a
preferable combination of particle sizes, one is selected from the
range of particle size of generally 0.05-0.3, preferably 0.05-0.18
.mu.m, and the other is selected from the range of particle size of
generally 0.18-0.5, preferably 0.18-0.28 .mu.m. Equimolar assay of
the target substance existing both in the free form and the bound
form can be performed by using the latex particles having such
different mean particle sizes in combination. In addition,
difference between the particle sizes of two types of the latex
should preferably be at least 0.05 nm.
[0020] A reagent for an immunoassay of the present invention
comprises a latex which is immobilized with a monoclonal antibody
for the target substance, and another latex which has a different
mean particle size from the above latex and is immobilized with a
monoclonal antibody having a different recognition site from the
antibody in the target substance. Specifically, for example, in the
case when the target substance is PSA, the reagent comprising the
latex immobilized with PSA monoclonal antibody having, for example,
a mean particle size of 0.05-0.18 .mu.m, and the latex immobilized
with PSA antibody having the recognition site different from the
above PSA antibody having, for example, a mean particle size of
0.18-0.28 .mu.m can be included. Using the latexes having such
particle sizes, equimolar assay of the free form and the bound form
can be performed. When two types of latexes are mixed, a ratio of
volumes is preferably 1:10-10:1, more preferably 1:10-2:1, in the
ratio of latexes having larger and smaller mean particle sizes. In
the case of using three or more types, two types thereof are set to
be the above ratio, and other type is set to a volume depending on
the above.
[0021] A solvent used for preparing the reagent may be any solvent
so long as it does not have a property to inhibit binding the
monoclonal antibody with the target substance, and include, for
example, buffer solution having buffering action around the neutral
range at pH 5.0-10.0, preferably at pH 6.5-8.5, such as phosphate
buffer, Tris buffer, Good's buffer, glycine buffer and borate
buffer. Concentration of buffering agent in the buffer is generally
selected from a range of 10-500 mM, preferably 10-300 mM. Further,
the solution may contain, for example, sugars, salts such as NaCl,
surface active agents and antiseptics, so long as the amount
thereof does not inhibit binding the monoclonal antibody with the
target substance.
[0022] A method for immobilizing the monoclonal antibody with the
latex can be performed according to the known method. For example,
the monoclonal antibody and the latex are suspended in the buffer,
reacted at 20-30.degree. C. for 2-3 hours, and the mixture is
treated with centrifugation, blocking, and the like, which are
commonly performed as post-treatment in this field, to obtain the
immobilized monoclonal antibody with latex.
[0023] A reagent kit for immunoassay of the present invention is
not particularly limited, so long as the kit comprises the above
described reagent for the immunoassay of the present invention and
the reagent containing agglutination accelerator for
antigen-antibody reaction. The agglutination accelerator for the
antigen-antibody reaction may be any substance having accelerating
action for the agglutination reaction of the antigen-antibody
reaction. Specifically, the accelerator includes, for example, a
polymer having a side chain of the group represented by the
following general formula [1]: ##STR1## (wherein each of
R.sup.1-R.sup.3 is an alkyl group optionally having a hydrogen atom
or a hydroxyl group independently, and R.sup.4 is an alkylene
group). More specifically, the accelerator includes a polymer
having a monomer unit derived from the monomer represented by the
following general formula [2]: ##STR2## (wherein R.sup.5 is an
alkylene group optionally having a substituent and an oxygen atom
in the chain, R.sup.6 is a hydrogen atom or a methyl group, X is an
oxygen atom or a --NH-- group, and R.sup.1-R.sup.4 are the same as
the above).
[0024] In the above general formula [1] or [2], the alkyl group of
R.sup.1-R.sup.3 optionally having a hydroxyl group may be any of
straight, branched and cyclic chains, and is a group generally
having 1-6 carbon atoms, preferably 1-4 carbon atoms, more
preferably 1-2 carbon atoms, and most preferably 1 carbon atom.
Specifically, the alkyl group includes, for example, methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,
n-pentyl, isopentyl, sec-pentyl, tert-pentyl, n-hexyl, isohexyl,
sec-hexyl, tert-hexyl, cyclopropyl, cyclohexyl and cyclopentyl
groups. Among them, methyl and ethyl groups are preferable, and
methyl group is more preferable.
[0025] The alkyl group having a hydroxyl group includes an alkyl
group, in which 1-2 hydrogen atoms, preferably 1 hydrogen atom, is
substituted by a hydroxyl group. Specifically, the alkyl group
having a hydroxyl group includes, for example, hydroxymethyl,
hydroxyethyl, hydroxy-n-propyl, hydroxy-isopropyl, hydroxy-n-butyl,
hydroxy-isobutyl, hydroxy-sec-butyl, hydroxy-tert-butyl,
hydroxy-n-pentyl, hydroxy-isopentyl, hydroxy-sec-pentyl,
hydroxy-tert-pentyl, hydroxy-n-hexyl, hydroxy-isohexyl,
hydroxy-sec-hexyl, hydroxy-tert-hexyl, hydroxy-cyclopropyl,
hydroxy-cyclohexyl and hydroxy-cyclopentyl groups. Among them,
hydroxymethyl and hydroxyethyl groups are preferable.
[0026] The alkylene group represented by R.sup.4 includes, for
example, an alkylene group having 1-6 carbon atoms, preferably 2-3
carbon atoms, and may be any of straight, branched and cyclic
chains. Specifically, the alkylene group includes, for example,
methylene, ethylene, propylene, trimethylene, butylene,
1-ethylehtylene, 2-methyltrimethylene, 2-ethyltrimethylene,
hexylene, cyclopropylene, cyclobutylene, cyclopentylene and
cyclohexylene groups. Among them, preferable examples are ethylene,
propylene and trimethylene groups.
[0027] In the alkylene group represented by R.sup.5 optionally
having a substituent and an oxygen atom in the chain in the general
formula [2], examples of the alkylene group without an oxygen atom
include, for example, the alkylene group having 1-10 carbon atoms,
preferably 1-6 carbon atoms, more preferably 2-6 carbon atoms,
which may be any of straight, branched and cyclic chains.
Specifically, the alkylene group includes, for example, methylene,
ethylene, propylene, trimethylene, butylene, 1-ethylehtylene,
2-methyltrimethylene, 2-ethyltrimethylene, hexylene,
cyclopropylene, cyclobutylene, cyclopentylene and cyclohexylene
groups. Examples of the substituent include an alkoxyl group having
1-6 carbon atoms, preferably 1-3 carbon atoms (which may be any of
straight, branched and cyclic chains). More specifically, the
substituent includes, for example, methoxy, ethoxy, n-propoxy,
isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy,
n-pentyloxy, isopentyloxy, sec-pentyloxy, tert-pentyloxy,
n-hexyloxy, isohexyloxy, sec-hexyloxy, tert-hexyloxy, cyclopropoxy,
cyclohexyloxy and cyclopentyloxy groups. Further, the substituent
includes a halogen atom such as fluorine atom, chlorine atom,
bromine atom and iodine atom. Among them, preferable examples are
ethylene, propylene, trimethylene and butylene groups. In the case
of the substituent having oxygen atoms in the chain, the number of
oxygen atom are 1-5, preferably 1-3, and more specifically, the
substitutent includes a group of
--(C.sub.2H.sub.4O)--C.sub.2H.sub.4--, wherein n is an integer of
1-5. In the alkylene group represented by R.sup.5 optionally having
a substituent and an oxygen atom in the chain hereinabove, ethylene
and propylene groups are preferable, and ethylene group is more
preferable.
[0028] A polymer having a constitutional unit based on the monomer
having the group represented by the general formula [1] hereinabove
as a side chain, may be a commercially available product, or a
product synthesized by the methods described in JP-A-10-45794,
JP-A-2000-239696 or the like.
[0029] A constitutional unit based on the monomer represented by
the general formula [2] hereinabove includes a constitutional unit
having the above described R.sup.1-R.sup.6, and specifically
includes a constitutional unit based on the monomer represented by
the following general formula [5]: ##STR3##
[0030] In the case when the polymer having the constitutional unit
based on the monomer represented by the general formula [2]
hereinabove is a copolymer, examples of the monomer unit other than
the constitutional unit based on the monomer represented by the
general formula [2] hereinabove can be the monomer unit derived
from the monomer selected from a group consisting of acrylic acid
or acrylates thereof, methacrylic acid or methacrylates thereof,
acrylamide or N-substited acrylamides, methacrylamide or
N-substited methacrylamides, and styrene or derivatives thereof.
Two or more types of these monomer units may be contained in the
copolymer. A ratio of the constitutional unit based on the monomer
represented by the general formula [2] hereinabove is generally 20%
and more to under 100%, preferably 30 to 95%, more preferably 30 to
90%.
[0031] In the monomer unit other than the constitutional unit based
on the monomer represented by the general formula [2] hereinabove,
the acrylate includes alkyl acrylate and aralkyl acrylate; and the
methacrylate includes alkyl methacrylate and aralkyl methacrylate.
The N-substited acrylamide includes N-alkyl acrylamide and
N-aralkyl acrylamide. The N-substited methacrylamide includes
N-alkyl methacrylamide and N-aralkyl methacrylamide. The styrene
derivatives include .alpha.-methyl styrene and styrene or
.alpha.-methyl styrene having a substituent.
[0032] An alkyl group in the above described alkyl acrylate, alkyl
methacrylate, N-alkyl acrylamide and N-alkyl methacrylamide may be
any of straight, branched and cyclic chains, and carbon atoms
thereof are generally 1-6, and more preferably 1-4. Specifically,
the alkyl group includes, for example, methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,
isopentyl, sec-pentyl, tert-pentyl, n-hexyl, isohexyl, sec-hexyl,
tert-hexyl, cyclopropyl, cyclohexyl and cyclopentyl groups. The
alkyl group may optionally have a substituent, which includes
hydroxyl group, lower-alkoxyl group having 1-3 carbon atoms and
trialkylammonio group (An alkyl group thereof includes, for
example, an alkyl group having 1-3 carbon atoms such as methyl,
ethyl, propyl and isopropyl groups. When the substitutent is
trialkylammonio group, since the substituent is positively charged,
a counter anion is generally bound. Such counter anion includes a
halide ion such as fluoride ion, chloride ion, bromide ion and
iodide ion). The alkyl group having a substituent includes, for
example, a group represented by the following formula:
--(CH.sub.2CH.sub.2O).sub.m--R.sup.7 (wherein R.sup.7 is an alkyl
group having 1-3 carbon atoms and m is 1-100).
[0033] An aralkyl group in the above described aralkyl acrylate,
aralkyl methacrylate, N-aralkyl acrylamide and N-aralkyl
methacrylamide includes the one having 1-10 carbon atoms.
Specifically, the aralkyl group includes, for example, benzyl,
phenylethyl, phenylpropyl and phenylbutyl groups.
[0034] A substituent for styrene or .alpha.-methyl styrene
includes, for example, an alkyl group which generally has straight,
branched or cyclic chain having 1-6 carbon atoms, preferably 1-4
carbon atoms (specifically, for example, methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,
isopentyl, sec-pentyl, tert-pentyl, n-hexyl, isohexyl, sec-hexyl,
tert-hexyl, cyclopropyl, cyclohexyl and cyclopentyl groups); for
example, an aralkyl group which generally has straight, branched or
cyclic chain having 1-6 carbon atoms, preferably 1-4 carbon atoms
(specifically, for example, methoxy, ethoxy, n-propoxy, isopropoxy,
n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy,
isopentyloxy, sec-pentyloxy, tert-pentyloxy, n-hexyloxy,
isohexyloxy, sec-hexyloxy, tert-hexyloxy, cyclopropoxy,
cyclohexyloxy and cyclopentyloxy groups); a halogen atom such as
fluorine atom, chlorine atom, bromine atom and iodine atom,
carboxyl group, hydroxy group and amino group.
[0035] A monomer unit other than the constitutional unit based on
the monomer represented by the general formula [2] hereinabove
includes, for example, a monomer unit derived from methacrylic
acid, methyl methacrylate, ethyl methacrylate, propyl methacrylate,
butyl methacrylate, dodecyl methacrylate, octadecyl methacrylate,
2-ethylhexyl methacrylate, lauryl methacrylate, stearyl
methacrylate, 2-trimethylammonioethyl methacrylate, benzyl
methacrylate, phenylethyl methacrylate, acrylic acid, methyl
acrylate, ethyl acrylate, butyl acrylate, 2-ethylhexyl acrylate,
lauryl acrylate, stearyl acrylate, 2-trimethylammonioethyl
acrylate, benzyl acrylate, phenylethyl acrylate, acrylamide,
N-methyl acrylamide, N-ethyl acrylamide, N-butyl acrylamide,
N-2-ethylhexyl acrylamide, N-lauryl acrylamide, N-stearyl
acrylamide, N-2-trimethylammonioethyl acrylamide, N-benzyl
acrylamide, N-phenylethyl acrylamide, methacrylamide, N-methyl
methacrylamide, N-ethyl methacrylamide, N-butyl methacrylamide,
N-2-ethylhexyl methacrylamide, N-lauryl methacrylamide, N-stearyl
methacrylamide, N-2-trimethylammonioethyl methacrylamide, N-benzyl
methacrylamide, N-phenylethyl methacrylamide, styrene,
carboxystyrene, hydroxystyrene, aminostyrene, methylstyrene,
ethylstyrene, methoxystyrene, ethoxystyrene, chlorostyrene,
bromostyrene, .alpha.-methylstyrene, .alpha.-methyl-carboxystyrene,
.alpha.-methyl-hydroxystyrene, .alpha.-methyl-aminostyrene,
.alpha.-methyl-methylstyrene, .alpha.-methyl-ethylstyrene,
.alpha.-methyl-methoxystyrene, .alpha.-methyl-ethoxystyrene,
.alpha.-methyl-chlorostyrene, .alpha.-methyl-bromostyrene,
N,N,N-triethylammoniumethyl methacrylate bromide, N,N,
N-trimethylammoniumethyl methacrylate chloride,
N,N-diethyl-N-propylammoniumethyl methacrylatebromide,
N,N,N-trimethylammonium-2-hydroxypropyl methacrylate chloride (QM),
and N,N,N-trimethylammoniummethyl styrene bromide. Further, a
monomer unit represented by the following general formula [4] can
also be exemplified: ##STR4## (wherein m is 1-100).
[0036] Among the above exemplified monomer units, the monomer unit
derived from methacrylic acid, stearyl methacrylate, benzyl
methacrylate, butyl methacrylate, dodecyl methacrylate, octadecyl
methacrylate and N,N, N-trimethylammonium-2-hydroxypropyl
methacrylate chloride (QM), and the monomer unit represented by the
general formula [4] are preferable.
[0037] In the agglutination accelerator hereinabove, a polymer
comprising the monomer unit represented by the following general
formula [5]; ##STR5## a copolymer comprising the monomer unit
represented by the general formula [5] and butyl methacrylate; a
copolymer comprising the monomer unit represented by the general
formula [5] and the monomer unit represented by the general formula
[4] hereinabove; a copolymer comprising the monomer unit
represented by the general formula [5] and octadecyl methacrylate;
a copolymer comprising the monomer unit represented by the general
formula [5] and octadodecyl methacrylate; and a copolymer
comprising the monomer unit represented by the general formula [5]
and benzyl methacrylate are preferable. Among them, the copolymer
comprising the monomer unit represented by the general formula [5]
and benzyl methacrylate is more preferable.
[0038] Concentration of the agglutination accelerator hereinabove
in the reaction is generally 0.1-20 w/v %, preferably 0.1-10 w/v %,
more preferably 0.1-5 w/v %. Two or more types of the above
agglutination accelerator may be used, and in such a case, a
concentration range is preferably set as described above.
[0039] Production process of the monomer and the polymer used as
the agglutination accelerator hereinabove can be performed
according to the description in JP-A-2002-365296.
[0040] Guanidine, guanidine salts or derivatives thereof as a
nonspecific reaction suppressor may be added to the reagent
containing agglutination accelerator for antigen-antibody reaction.
The guanidine salts include, for example, guanidine hydrochloride,
guanidine carbonate, guanidine thiocyanate, guanidine sulfate,
guanidine nitrate and guanidine phosphate. The guanidine
derivatives include, for example, arginine, guanidinobenzoic acid,
guanidinoglutaric acid, guanidinosuccinic acid and guanidinoacetic
acid. Two or more additives may be added. Amount of the additive is
not particularly limited, so long as the objective effect can be
obtained. Preferable amount of the additive is generally set to the
concentration of 10-700 mM in the reagent, and 7.5-525 mM during
the reaction.
[0041] The solvent used for preparation of the reagent containing
agglutination accelerator for antigen-antibody reaction in the
present invention is same as used in the reagent for assay of the
present invention.
[0042] Immunoassay method of the present invention comprises
reacting a specimen (for example, serum, plasma, urine, spinal
fluid, extract solution of human tissue or human cells, etc.)
containing the target substance existing in the free form and the
bound form in the organism or in the specimen, with a reagent
containing the latex 1 immobilized with the monoclonal antibody 1
for the target substance and the latex 2 having a mean particle
size different from the latex 1 and being immobilized with the
monoclonal antibody 2 having a different recognition site for the
target substance from the antibody 1, and determining an amount of
the substance based on the result of the agglutination reaction
among the target substance, the latex1 and the latex2.
Specifically, for example, the assay method for PSA used as the
target substance is performed as follows. A specimen containing PSA
and a reagent containing a monoclonal antibody to PSA immobilizing
(carrying or sensitizing) latex having, for example, a mean
particle size of 150 nm and a monoclonal antibody to PSA, which has
a different recognition site from the above monoclonal antibody,
immobilizing (sensitizing) a latex having, for example, a mean
particle size of 280 nm are reacted; an extent of the thus
generated agglutination is measured by photometry; and a
concentration is determined from the previously prepared
calibration curve of the standard substance. Wavelength for
measurement of optical density is generally at 340-1000 nm,
preferably at 500-900 nm. Measurement of the extent of
agglutination is not limited to the photometry and can be selected
from any known methods, such as the nephelometry and the counting
immunoassay.
[0043] As described hereinabove, according to the immunoassay of
the present invention, both target substances in the free form and
the bound form can be assayed precisely in equimolar reaction. The
equimolar reaction of the free form and the bound form in the
present invention means the reaction binding the monoclonal
antibody immobilized with latex to both forms in a ratio of
approximately 1:1. Specifically, for example, the equimolar
reaction in PSA can be defined as follows. The purified free form
of PSA and the ACT-PSA bound form obtained from Stanford University
are diluted stepwisely and each solution is assayed. The
theoretical values calculated by the designated value and the
actual measured values are plotted on the X-axis and the Y-axis
respectively, and a slope of the dilution straight line is obtained
by the regression equation, and a ratio of both slopes (the slope
of the free form PSA/the slope of the ACT-PSA bound form) is
expressed as a deviation .gamma.. When .gamma. ranges is
0.9.ltoreq..gamma.<1.1, the reaction is regarded as the
completely equimolar reaction. When .gamma. ranges is
0.8.ltoreq..gamma.<0.9 and 1.1.ltoreq..gamma.<1.2, the
reaction is regarded as the nearly equimolar reaction (Symposium on
65th Meeting of the Japanese Urological Association).
[0044] The present invention will be further explained by using
Examples, but the present invention is not limited by these
Examples.
EXAMPLE
Example 1
Assay of Prostate-Specific Antigen (PSA) by Latex
Immunonephelometry
(1) Preparation of Anti-Human PSA Antibody Sensitized (Immobilized)
Latex Test Solution
[0045] With 0.5 ml of 50 mM borate buffer (pH 7.1) containing 0.3
mg of anti-human PSA mouse monoclonal antibody (clone No. PSA10)
(Wako Pure Chemical Industries, Ltd.), 0.5 ml of 50 mM borate
buffer (pH 7.1) suspended with polystyrene latex (mean particle
size 0.22 .mu.m or 0.28 .mu.m, Sekisui Chemical Co. Ltd.) to adjust
the concentration at 2% (W/V) was mixed, and the mixture was
reacted at 25.degree. C. for 2 hours. Thereafter, the latex
isolated from the mixture by centrifugation was washed with 50 mM
borate buffer (pH 7.1). The latex was suspended in 50 mM borate
buffer (pH 7.3) containing 0.5% (W/V) of BSA to adjust the
concentration of the latex at 1% (W/V), to obtain the anti-human
PSA antibody sensitized latex test solution [1].
[0046] In the similar way hereinabove, 0.5 ml of 50 mM borate
buffer (pH 7.1) containing 0.7 mg of anti-human PSA mouse
monoclonal antibody (clone No. PSA14) (Wako Pure Chemical
Industries, Ltd.) and 0.5 ml of 2% (W/V) polystyrene latex (mean
particle size 0.12 .mu.m or 0.15 .mu.m, Sekisui Chemical Co. Ltd.)
were mixed to prepare the anti-human PSA antibody sensitized latex
test solution [2].
(2) Specimens
[0047] The standard sample of free form PSA and the standard sample
of complex PSA (Stanford University) were diluted with 10 mM
phosphate buffer (containing 0.85% NaCl, pH7.0) containing 1% BSA
to prepare 25 ng/ml solution. The phosphate buffer is used as a
blank reagent.
(3) Reagents
I. First Test Solution
[0048] HEPES-NaOH buffer (pH 7.0) containing 0.1% BSA and 1% NaCl
was prepared as the first test solution.
II. Second Test Solution
[0049] Each of the anti-human PSA antibody sensitized latex test
solutions [1] and [2] prepared in the above (1) was adjusted to
0.1% (W/V) of latex, and the both solutions were mixed in equal
amount to prepare the second test solution.
(4) Assay Method
[0050] Assay of PSA concentration was performed by using
autoanalyzer (JOEL BM-8) under the following condition.
Specimen: 5 .mu.l
First test solution: 90 .mu.l
Second test solution: 30 .mu.l
Assay method: 2 point end method (34-65)
Main wavelength: 571 nm
(5) Results
[0051] Optical densities (turbidities) obtained are shown in Table
1. The values in the table are indicated by 10,000-fold values of
the obtained optical densities subtracted with the blank value. The
deviation .gamma. is a value dividing the slope of the free form
PSA (measured value/theoretical value) by the slope of the complex
PSA (measured value/theoretical value). When this value becomes the
closer to 1.0, the more likely the equimolar binding of the
antibody sensitized latex with the free form and the complex form
occurs.
Comparative Example 1
Assay of PSA Using Latexes Having Equal Mean Particle Size
[0052] Latexes were prepared by the same method as in Example 1,
and assay was performed by the same way as in Example 1, except
that 0.5 ml of 50 mM borate buffer (pH 7.1) containing 0.6 mg each
of two types of anti-human PSA mouse monoclonal antibody (clone
Nos. PSA 10 and PSA 14) (Wako Pure Chemical Industries, Ltd.) and
0.5 ml of 50 mM borate buffer (pH 7.1) suspended with polystyrene
latex (mean particle size 0.22 .mu.m, Sekisui Chemical Co. Ltd.) to
adjust the concentration at 2% (W/V) were mixed and reacted at
25.degree. C. for 2 hours. The obtained results and deviation
.gamma. are shown in Table 1 together with the results of Example
1. TABLE-US-00001 TABLE 1 Comparative Example 1 Example 1 Particle
size of 0.28 .mu.m 0.22 .mu.m 0.22 .mu.m antibody sensitized latex
[1] Particle size of 0.12 .mu.m 0.15 .mu.m 0.15 .mu.m 0.22 .mu.m
antibody sensitized latex [2] Concentration of PSA (ng/ml) Reagent
blank -72 -44 -30 -69 Sensitivity (O.D. .times. 10,000) Free PSA 25
ng/ml 602 984 484 1,136 Complex PSA 25 ng/ml 629 898 426 825
Deviation .gamma. 0.96 1.10 1.14 1.38
[0053] As obvious from Table 1, in the method combining with the
latex having different mean particle size, the deviation shows
approximately 1.0, indicating possible equimolar reaction with the
free PSA and the complex PSA. Contrary to that, in the method
combining with the latex having identical mean particle size, the
deviation shows 1.38, indicating stronger reaction with the free
PSA as compared with the complex PSA.
Example 2
(1) Standard Specimens
[0054] The standard sample of free form PSA and the standard sample
of complex PSA (Stanford University) were diluted with 10 mM
phosphate buffer (containing 0.85% NaCl, pH7.0) containing 1% BSA
to prepare 0, 5, 10, 25 and 50 ng/ml solutions, respectively.
(2) Reagents
I. First Test Solution
[0055] The first test solution prepared in Example 1 was used.
II. Second Test Solution
[0056] In the antibody sensitized latexes prepared in Example 1,
the antibody sensitized latex [1] was prepared using the latex
having a mean particle size of 0.28 .mu.m, and the antibody
sensitized latex [2] was prepared using the latex having a mean
particle size of 0.15 .mu.m, and the solutions were mixed with the
same way as in Example 1.
(3) Assay Method
[0057] Assay of PSA concentration was performed by using
autoanalyzer (Toshiba Corp. TBA-120FR) under the following
condition.
Specimen: 12 .mu.l
First test solution: 120 .mu.l
Second test solution: 40 .mu.l
Assay method: 2 point end method (20-33)
Main wavelength: 572 nm
(4) Results
[0058] The optical densities (turbidities) obtained and the
deviation .gamma. are shown in Table 2. The values in the table are
indicated by 10,000-fold values of the measured optical densities
subtracted with the blank value. The measured values in the table
are values calculated by using the calibration curve prepared from
the standard substance of free form PSA. A relationship between the
measured optical densities of the free form PSA and the complex
form PSA and each concentration (a graph) is shown in FIG. 1.
Comparative Example 2
Assay of PSA Using Latexes Having Equal Mean Particle Size
[0059] Assay was performed by the same way as in Example 2, except
that two types of the antibody sensitized latex (mean particle size
0.22 .mu.m) prepared in Comparative Example 1 were used. The
obtained results and the deviation .gamma. are shown in Table 2
together with the results of Example 2. TABLE-US-00002 TABLE 2
Example 2 Comparative Example 2 Particle size of 0.28 .mu.m 0.22
.mu.m antibody sensitized latex [1] Particle size of 0.15 .mu.m
0.22 .mu.m antibody sensitized latex [2] Concentration of PSA
(ng/ml) Reagent blank 9 -23 Optical Measured Optical Measured
Density value(ng/ml) Density value(ng/ml) Free PSA 5 298 5.0 300
5.0 Complex PSA 5 291 4.9 239 4.0 Free/Complex (%) 102% 125% Free
PSA 10 761 10.0 707 10.0 Complex PSA 10 759 10.0 567 8.3
Free/Complex (%) 100% 120% Free PSA 25 2221 25.0 2293 25.0 Complex
PSA 25 2335 27.1 1751 19.9 Free/Complex (%) 92% 126% Free PSA 50
3580 50.0 4592 50.0 Complex PSA 50 3755 53.2 4013 41.2 Free/Complex
(%) 94% 121% Deviation .gamma. 0.93 1.22
[0060] In Table 2, in the method of combining use of the latexes
having different mean particle sizes, since almost equimolar
reaction is indicated in any concentration of PSA and the deviation
0.93 shows a value near to 1.0, it can be understood that the
equimolar reaction system is substantially carried out. Further, in
the case of using the latex having equal mean particle size, the
reactivity of the free PSA is higher than the complex PSA in all
concentrations, and the deviation exhibits 1.22, indicating no
equimolar reaction.
[0061] This application is based on Japanese Patent Application
serial No. JP2003-340028 filed in Japan Patent Office on Sep. 30,
2003, the contents of which are hereby incorporated by
reference.
* * * * *