U.S. patent application number 09/297603 was filed with the patent office on 2001-12-13 for fluorescence polarization method.
Invention is credited to MIYAZAKI, JINSEI, NAKAYAMA, HIROSHI.
Application Number | 20010051331 09/297603 |
Document ID | / |
Family ID | 17062994 |
Filed Date | 2001-12-13 |
United States Patent
Application |
20010051331 |
Kind Code |
A1 |
NAKAYAMA, HIROSHI ; et
al. |
December 13, 2001 |
FLUORESCENCE POLARIZATION METHOD
Abstract
A fluorescence polarization method for analyzing an assay-object
in a sample is provided. The fluorescence polarization method
includes the steps of: (a) providing a fluorescent-labeled protein
in which a protein is covalently bound to a fluorochrome(s),
wherein the protein is capable of specifically binding to the
assay-object; (b) allowing the fluorescent-labeled protein to bind
to the assay-object; and (c) measuring a change in the degree of
fluorescence polarization which has taken place in the
fluorescent-labeled protein by its binding to the assay-object.
Inventors: |
NAKAYAMA, HIROSHI; (OSAKA,
JP) ; MIYAZAKI, JINSEI; (OSAKA, JP) |
Correspondence
Address: |
IOTA PI LAW GROUP
350 CAMBRIDGE AVENUE SUITE 250
P O BOX 60850
PALO ALTO
CA
94306-0850
US
|
Family ID: |
17062994 |
Appl. No.: |
09/297603 |
Filed: |
July 23, 1999 |
PCT Filed: |
September 4, 1998 |
PCT NO: |
PCT/JP98/03988 |
Current U.S.
Class: |
435/5 |
Current CPC
Class: |
G01N 33/542
20130101 |
Class at
Publication: |
435/5 |
International
Class: |
C12Q 001/70; C12P
013/14 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 5, 1997 |
JP |
9-240672 |
Claims
1. A fluorescence polarization method for analyzing an assay-object
in a sample, the method comprising the steps of: (a) providing a
fluorescent-labeled protein in which a protein is covalently bound
to a fluorochrome(s), wherein the protein is an antibody, a
receptor or an inhibitor, which is capable of specifically binding
to the assay-object; (b) allowing the fluorescent-labeled protein
to bind to the assay-object; and (c) measuring a change in the
degree of fluorescence polarization which has taken place in the
fluorescent-labeled protein by its binding to the assay-object.
2. A fluorescence polarization method according to claim 1, wherein
the antibody is a polyclonal antibody, a monoclonal antibody, a
chimeric antibody, a Fab antibody or a (Fab)2 antibody.
3. A fluorescence polarization method according to claim 1, wherein
the assay-object is a biological substance, a microorganism, a
virus, a pharmaceutical, an environmental pollutant or an abused
drug.
4. A fluorescence polarization method according to claim 3, wherein
the biological substance is a peptide, a protein, a lipid, a
saccharide or a nucleic acid.
5. A fluorescence polarization method according to claim 4, wherein
the protein has a molecular weight of 500,000 or more.
6. A fluorescence polarization method according to claim 4, wherein
the protein is an antibody, a hormone, an inflammation marker, a
coagulation factor, an apolipoprotein, a high density lipoprotein
(HDL), a low density lipoprotein (LDL), a glycosylated albumin, a
glycosylated hemoglobin, a hemoglobin, a cancer marker or an
enzyme.
7. A fluorescence polarization method according to claim 6, wherein
the hormone is chorionic gonadotropin, thyroid-stimulating hormone,
progesterone, follicular forming hormone, parathyroid-stimulating
hormone, adrenocorticotropic hormone, or insulin.
8. A fluorescence polarization method according to claim 6, wherein
the inflammation marker is C-reactive protein (CRP), al-antitrypsin
(.alpha.1-AT), .alpha.1-antichymotrypsin (.alpha.1-X),
.alpha.1-acid glycoprotein (.alpha.1-AG), haptoglobin (Hp),
ceruloplasmin (Cp), the 9th component of complement (C9), the 4th
component of complement (C4), the 3rd component of complement (C3),
complement factor B (B), fibrinogen (Fbg), serum amyloid A (SAA),
C1 inhibitor (C1I), a sialoglycoprotein, an acid-soluble protein
(ASP) or an immunosuppressive acidic protein (IAP).
9. A fluorescence polarization method according to claim 1, wherein
the fluorochrome has a functional group which can bind to a
primary, secondary or tertiary amino group, a carboxyl group, a
thiol group, a phenyl group, a phenol group or a hydroxyl
group.
10. A fluorescence polarization method according to claim 1,
wherein a lifetime of fluorescence of the fluorochrome is in the
range of 10 nanoseconds to 200 nanoseconds.
11. A fluorescence polarization method according to claim 1,
wherein the fluorochrome has a skeletal structure of rhodamine,
pyrene, dialkylaminonaphthalene or cyanin.
12. A reagent for use in a fluorescence polarization method for
analyzing an assay-object in a sample, the reagent comprising a
fluorescent-labeled protein in which a protein is covalently bound
to a fluorochrome(s), wherein the protein is an antibody, a
receptor or an inhibitor, which is capable of specifically binding
to the assay-object.
13. A fluorescence polarization method for analyzing a bacteria or
a virus in a sample, the method comprising the steps of: (a)
providing a fluorescent-labeled antibody in which an antibody is
covalently bound to a fluorochrome, wherein the antibody is capable
of specifically binding to the bacteria or the virus; (b) allowing
the fluorescent-labeled antibody to bind to the bacteria or the
virus; and (c) measuring a change in the degree of fluorescence
polarization which has taken place in the fluorescent-labeled
antibody by its binding to the bacteria or the virus.
14. A fluorescence polarization method according to claim 13,
wherein the antibody is a polyclonal antibody, a monoclonal
antibody, a chimeric antibody, a Fab antibody or a (Fab)2
antibody.
15. A fluorescence polarization method according to claim 13,
wherein the bacteria is selected from the group consisting of
Rhodospirillaceae, Chromatiaceae, Chlorobiaceae, Myxococcaceae,
Archangiaceae, Cystobacteraceae, Polyangiaceae, Cytophagaceae,
Beggiatoaceae, Simonsiellaceae, Leucotrichaceae, Achromatiaceae,
Pelonemataceae, Spirochaetaceae, Spirillaceae, Pseudomonadaceae,
Azotobacteraceae, Rhizobiaceae, Methylomonadaceae,
Halobacteriaceae, Enterobacteriaceae, Vibrionaceae, Bacteroidaceae,
Neisseriaceae, Veillonellaceae, Organisms oxidizing ammonia or
nitrite, Organisms metabolizing sulfer and sulfer compounds,
Organisms depositing iron and/or manganese oxides, Siderocapsaceae,
Methanobacteriaceae, Aerobic and/or facultatively anaerobic
Micrococcaceae, Streptococcaceae, Anaerobic Peptococcaceae,
Bacillaceae, Lactobacillaceae, Coryneform group of bacteria,
Propionibacteriaceae, Actinomycetaceae, Mycobacteriaceae,
Frankiaceae, Actinoplanaceae, Dermatophilaceae, Nocardiaceae,
Streptomycetaceae, Micromonosporaceae, Rickettsiaceae,
Bartonellaceae, Anaplasmataceae, Chlamydiaceae, Mycoplasmataceae
and Acholeplasmataceae.
16. A fluorescence polarization method according to claim 13,
wherein the virus is selected from the group consisting of
Enterovirus, Cardiovirus, Rhinovirus, Aphthovirus, Calicivirus,
Orbivirus, Reovirus, Rotavirus, Abibirnavirus, Piscibirnavirus,
Entomobirnavirus, Alphavirus, Rubivirus, Pestivirus, Flavivirus,
Influenzavirus, Pneumovirus, Paramyxovirus, Morbillivirus,
Vesiculovirus, Lyssavirus, Coronavirus, Bunyavirus, Arenavirus,
Human immunodeficiency virus, Hepatitis A virus, Hepatitis B virus
and Hepatitis C virus.
17. A fluorescence polarization method according to claim 13,
wherein the fluorochrome has a functional group which can bind to a
primary, secondary or tertiary amino group, a carboxyl group, a
thiol group, a phenyl group, a phenol group or a hydroxyl
group.
18. A fluorescence polarization method according to claim 13,
wherein a lifetime of fluorescence of the fluorochrome is in the
range of 10 nanoseconds to 200 nanoseconds.
19. A fluorescence polarization method according to claim 13,
wherein the fluorochrome has a skeletal structure of rhodamine,
pyrene, dialkylaminonaphthalene or cyanin.
20. A reagent according to claim 12, wherein the assay-object is a
bacteria or a virus, and the specifically-binding protein is an
antibody.
Description
TECHNICAL FIELD
[0001] The present invention relates to a fluorescence polarization
method for analyzing an assay-object in a sample. In particular,
the present invention relates to a fluorescence polarization method
useful in analyzing a bacteria or a virus in a sample. The present
invention is useful in the fields of art of medical diagnosis,
environmental assay, and food control relevant to food poisoning
and infectious diseases.
BACKGROUND ART
[0002] The fluorescence polarization method is known in the art as
a method for assaying a substance in a sample. The method is based
on the principle that when a fluorescent-labeled compound is
excited by linearly polarized light, the fluorescence emitted from
the compound has a degree of polarization which is in proportion to
the molecular weight thereof.
[0003] As a fluorescence polarization method which has been
developed, there is a fluorescence polarization immunossay based on
an antigen-antibody reaction.
[0004] For example, U.S. Pat. No. 4,902,630 discloses an assay
method (FIG. 8 is a concept diagram showing the assay principle) in
which a body fluid (particularly, a blood) containing CRP (5), an
assay-object, is added to a mixed solution which contains: a
"tracer" (6) obtained by binding fluorescein, a fluorochrome, to
C-reactive protein (CRP); and an antibody (4) which specifically
binds to CRP. CRP (5) in the sample is assayed based on competition
between the tracer (6) and CRP (5) for the antibody (4) in the
mixed solution. Since this assay system is based on a competitive
reaction, it requires a two-step binding reaction, thereby
complicating the assay operation. Moreover, fluorescein used in the
assay has a short lifetime of the fluorescence, and thus it is
difficult to apply it in an assay for a high-molecular-weight
substance.
[0005] Japanese Patent Application No. 62-38363 discloses an
immunoassay apparatus for an assay of an antigen or antibody
utilizing an immunoreaction. It is taught that a
fluorescent-labeled antibody can be used in the assay. However, the
application describes no specific embodiment of the assay. As an
assay-object, it only accounts for a substance, such as a
therapeutic drug existing in the blood (e.g., digoxin), whose
molecular weight is clearly lower than that of a coexisting
protein.
[0006] Thus, there has been a demand for development of a
fluorescence polarization method with a simple assay reaction
system and an easy assay operation, and particularly a method which
is suitable for an assay of a high-molecular-weight substance.
DISCLOSURE OF THE INVENTION
[0007] An object of the present invention is to provide a
fluorescence polarization method for analyzing an assay-object
contained in a sample by measuring a change in the degree of the
fluorescence polarization thereof, particularly a method which is
suitable for an assay of a high-molecular-weight substance. Another
object of the present invention is to provide a reagent for
analyzing an assay-object contained in a sample utilizing a
fluorescence polarization method.
[0008] The present invention relates to a fluorescence polarization
method for analyzing an assay-object in a sample, the method
including the steps of: (a) providing a fluorescent-labeled protein
in which a protein is covalently bound to a fluorochrome(s),
wherein the protein is an antibody, a receptor or an inhibitor,
which is capable of specifically binding to the assay-object; (b)
allowing the fluorescent-labeled protein to bind to the
assay-object; and (c) measuring a change in the degree of
fluorescence polarization which has taken place in the
fluorescent-labeled protein by its binding to the assay-object.
[0009] In the above-described method, the antibody capable of
specifically binding to the assay-object may be a polyclonal
antibody, a monoclonal antibody, a chimeric antibody, a Fab
antibody or a (Fab)2 antibody.
[0010] In the above-described method, the assay-object may be a
biological substance; a microorganism including a bacteria; a
virus; a drug; an environmental pollutant or an abused drug. The
biological substance may be a peptide, a protein, a lipid, a
saccharide or a nucleic acid. The protein as a biological substance
may have a molecular weight of 500,000 or more.
[0011] The protein as a biological substance may be an antibody, a
hormone, an inflammation marker, a coagulation factor, an
apolipoprotein, a high density lipoprotein (HDL), a low density
lipoprotein (LDL), a glycosylated albumin, a glycosylated
hemoglobin, a hemoglobin, a cancer marker or an enzyme.
[0012] The hormone may be chorionic gonadotropin,
thyroid-stimulating hormone, progesterone, follicular forming
hormone, parathyroid-stimulating hormone, adrenocorticotropic
hormone, or insulin.
[0013] The inflammation marker may be C-reactive protein (CRP),
.alpha.1-antitrypsin (.alpha.1-AT), .alpha.1-antichymotrypsin
(.alpha.1-X), .alpha.1-acid glycoprotein (.alpha.1-AG), haptoglobin
(Hp), ceruloplasmin (Cp), the 9th component of complement (C9), the
4th component of complement (C4), the 3rd component of complement
(C3), complement factor B (B), fibrinogen (Fbg), serum amyloid A
(SAA), C1 inhibitor (C1I), a sialoglycoprotein (i.e., a
glycoprotein to which a sialic acid is bound), an acid-soluble
protein (ASP) or an immunosuppressive acidic protein (IAP).
[0014] The present invention also relates to a reagent for use in a
fluorescence polarization method for analyzing an assay-object in a
sample, the reagent including a fluorescent-labeled protein in
which a protein is covalently bound to a fluorochrome, wherein the
protein is an antibody, a receptor or an inhibitor, which is
capable of specifically binding to the assay-object.
[0015] The present invention further relates to a fluorescence
polarization method for analyzing a bacteria or a virus in a
sample, the method including the steps of: (a) providing a
fluorescent-labeled antibody in which an antibody is covalently
bound to a fluorochrome, wherein the antibody is capable of
specifically binding to the bacteria or the virus; (b) allowing the
fluorescent-labeled antibody to bind to the bacteria or the virus;
and (c) measuring a change in the degree of fluorescence
polarization which has taken place in the fluorescent-labeled
antibody by its binding to the bacteria or the virus.
[0016] In the above-described method, the antibody may be a
polyclonal antibody, a monoclonal antibody, a chimeric antibody, a
Fab antibody or a (Fab)2 antibody.
[0017] In the above-described method, the bacteria may be selected
from the group consisting of Rhodospirillaceae, Chromatiaceae,
Chlorobiaceae, Myxococcaceae, Archangiaceae, Cystobacteraceae,
Polyangiaceae, Cytophagaceae, Beggiatoaceae, Simonsiellaceae,
Leucotrichaceae, Achromatiaceae, Pelonemataceae, Spirochaetaceae,
Spirillaceae, Pseudomonadaceae, Azotobacteraceae, Rhizobiaceae,
Methylomonadaceae, Halobacteriaceae, Enterobacteriaceae,
Vibrionaceae, Bacteroidaceae, Neisseriaceae, Veillonellaceae,
Organisms oxidizing ammonia or nitrite, Organisms metabolizing
sulfer and sulfer compounds, Organisms depositing iron and/or
manganese oxides, Siderocapsaceae, Methanobacteriaceae, Aerobic
and/or facultatively anaerobic Micrococcaceae, Streptococcaceae,
Anaerobic Peptococcaceae, Bacillaceae, Lactobacillaceae, Corynef
orm group of bacteria, Propionibacteriaceae, Actinomycetaceae,
Mycobacteriaceae, Frankiaceae, Actinoplanaceae, Dermatophilaceae,
Nocardiaceae, Streptomycetaceae, Micromonosporaceae,
Rickettsiaceae, Bartonellaceae, Anaplasmataceae, Chlamydiaceae,
Mycoplasmataceae and Acholeplasmataceae.
[0018] In the above-described method, the virus may be selected
from the group consisting of Enterovirus, Cardiovirus, Rhinovirus,
Aphthovirus, Calicivirus, Orbivirus, Reovirus, Rotavirus,
Abibirnavirus, Piscibirnavirus, Entomobirnavirus, Alphavirus,
Rubivirus, Pestivirus, Flavivirus, Influenzavirus, Pneumovirus,
Paramyxovirus, Morbillivirus, Vesiculovirus, Lyssavirus,
Coronavirus, Bunyavirus, Arenavirus, Human immunodeficiency virus,
Hepatitis A virus, Hepatitis B virus and Hepatitis C virus.
[0019] In the fluorescence polarization method of the present
invention, fluorochrome may have a functional group which can bind
to a primary, secondary or tertiary amino group, a carboxyl group,
a thiol group, a phenyl group, a phenol group or a hydroxyl group.
The lifetime of the fluorescence of the fluorochrome may be in the
range of 10 nanoseconds to 200 nanoseconds. The fluorochrome may
have a skeletal structure of rhodamine, pyrene,
dialkylaminonaphthalene or cyanin.
[0020] The present invention also relates to a reagent for use in a
fluorescence polarization method for analyzing a bacteria or a
virus in a sample, the reagent including a fluorescent-labeled
antibody in which an antibody is covalently bound to a
fluorochrome, wherein the antibody is capable of specifically
binding to the assay-object.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a concept diagram showing the principle of the
present invention in the case where a fluorescent-labeled antibody
is used.
[0022] FIG. 2 is a diagram showing a synthesis scheme of a
succinimidyl-1-pyrenebutanoic acid.
[0023] FIG. 3 is a concept diagram showing the labeling of an
antibody with fluorochromes.
[0024] FIG. 4 is a graph showing measurement results of the
fluorescence polarization method of the present invention for
C-reactive protein (CRP).
[0025] FIG. 5 is a graph showing measurement results of the
fluorescence polarization method of the present invention for a
high density lipoprotein (HDL).
[0026] FIG. 6 is a graph showing measurement results of the
fluorescence polarization method of the present invention for a low
density lipoprotein (LDL).
[0027] FIG. 7 is a graph showing measurement results of the
fluorescence polarization method of the present invention for E.
coli 0157.
[0028] FIG. 8 is a concept diagram showing the assay principle of a
conventional fluorescence polarization method.
BEST MODE FOR CARRYING OUT THE INVENTION
[0029] In an assay system of the method of the present invention, a
protein is labeled with a fluorescent substance, wherein the
protein is an antibody, a receptor or an inhibitor, which is
capable of specifically binding to the assay-object. By mixing the
fluorescent-labeled protein with the assay-object, the presence of
the assay-object can be confirmed. FIG. 1 shows the principle of
the present invention in the case where a fluorescent-labeled
antibody is used. By mixing a fluorescent-labeled antibody, which
is obtained by labeling an antibody (1) which is capable of
specifically binding to the assay-object with a fluorochrome (2),
with the assay-object (3), the presence of the assay-object (3) is
confirmed. Since the reaction required for the assay is a
single-step reaction, the reaction system can be simplified,
thereby simplifying the assay operation as well as shortening the
assay time.
[0030] In the assay system of the present invention, a change in
molecular weight concomitant with the binding of the
fluorescent-labeled protein to the assay-object is measured as a
change over time in the molecular orientation. Thus, by selecting
the fluorochrome in consideration of the change in the molecular
weight before and after the binding, it is possible to assay
various assay-objects of high molecular weight (about 500,000 or
more), e.g., an assay-object having a size which is equal to or
greater than a virus (about 20 nanometers (nm) or more as a
particle). The same type of fluorochrome can be used for various
assay-objects with different molecular weights so long as the
fluorochrome has a lifetime of fluorescence corresponding to the
change in the molecular weight before and after the binding.
[0031] The present invention will be described below in further
detail.
[0032] With the fluorescence polarization method of the present
invention, it is possible to analyze (i.e., quantify, detect or
identify) an assay-object in a sample based on the above-described
principle of the fluorescence polarization method.
[0033] By covalently binding the protein, which is classified
either as an antibody, a receptor or an inhibitor, and which is
capable of specifically binding to the assay-object (hereinafter,
referred to as the "specifically-binding protein"), with a
fluorochrome, a fluorescent-labeled protein useful in the method of
the present invention is provided.
[0034] The specifically-binding protein has a desired binding
property with respect to the assay-object. The specifically-binding
protein is a protein, which is classified as any of antibodies,
receptors or inhibitors, so long as it has a functional group which
allows for the binding to the fluorochrome. An antibody is
particularly preferred for its broad spectrum of applications. The
antibody type includes a polyclonal antibody, a monoclonal
antibody, a chimeric antibody, a Fab antibody and a (Fab)2
antibody. Any type of antibody can be applied to the method of the
present invention. A receptor can be used in the case where the
assay-object acts as a ligand for the receptor. For example, an
inhibitor can be used in the case where the assay-object is an
enzyme.
[0035] For the fluorochrome, those having a functional group which
can be covalently bound to a functional group of the
specifically-binding protein (typically, a primary, secondary or
tertiary amino group, a carboxyl group, a thiol group, a phenyl
group, a phenol group or a hydroxyl group) are utilized.
Especially, in the case where a protein such as an antibody is used
as the specifically-binding protein, in terms of the binding
efficiency, a fluorochrome having an activated functional group
(e.g., a halogenated sulphonyl group, a succinimidized carboxyl
group, or an isothiocyanated primary amino group) is desired.
[0036] The number of fluorochrome molecules bound to one molecule
of the labeled object (i.e., the specifically-binding protein) can
be varied arbitrarily. It is preferred in order to increase the
detection sensitivity to bind 2 or more fluorochromes. However,
when more fluorochromes than necessary are bound, it may adversely
affect a property of the specifically-binding protein. For example,
it may reduce the affinity, solubility, or the like, of the
antibody. Therefore, the above-described binding number is
preferably 10 or less and, more preferably, one.
[0037] When selecting the skeletal structure of the fluorochrome to
be used, the excitation wavelength, the fluorescence wavelength,
the Stokes shift and the lifetime of the fluorescence are
important. Preferably, either or both of the excitation wavelength
and/or the fluorescence wavelength exist in the visible light
wavelength range (i.e., 300 nm to 700 nm). Preferably, the
difference in wavelength between the excitation wavelength and the
fluorescence wavelength (i.e., the Stokes shift) is at least 20 nm
or more. The lifetime of the fluorescence (the fluorescence
relaxation time) of the fluorochrome is typically selected from the
range of about 10 nanoseconds to about 1,000 nanoseconds and,
preferably, selected from the range of about 10 nanoseconds to
about 200 nanoseconds. A fluorochrome having an excessively long
fluorescence lifetime which exceeds about 1,000 nanoseconds is not
preferred since the degree of polarization can hardly be maintained
because of the long lifetime no matter how big the assay-object is.
In selecting the lifetime of the fluorescence, the change in
molecular weight of the fluorescent-labeled protein through the
binding to the assay-object is taken into consideration. This is
because the degree of polarization of fluorescence emitted from the
fluorescent-labeled protein bound to the assay-object is in a
proportional relationship with the size of the molecule.
[0038] Specifically, when the change in the molecular weight is
about 5,000 to about 50,000 (i.e., when the molecular weight of the
assay-object is several thousands to several ten thousands), a
fluorochrome having a lifetime of the fluorescence of about 1 to
about 15 nanoseconds is preferred. Examples of such a fluorochrome
include cyanin and rhodamine. When the change in the molecular
weight is about 50,000 to about 500,000 (i.e., when the molecular
weight of the assay-object is about several ten thousands to
several hundred thousands), a fluorochrome having a lifetime of the
fluorescence of about 10 nanoseconds to about 150 nanoseconds is
preferred. Examples of such a fluorochrome include
dialkylaminonaphthalenes and pyrene derivatives. When the change in
the molecular weight is about 500,000 to about 5,000,000 (i.e.,
when the molecular weight of the assay-object is about several
hundred thousands to several millions), a fluorochrome having a
lifetime of the fluorescence of about 100 nanoseconds to about
1,000 nanoseconds is preferred. Examples of such a fluorochrome
include pyrene derivatives and metal complexes.
[0039] From the above-described points of view, preferred examples
of fluorochrome include fluorochromes having a skeletal structure
of rhodamine, pyrene, a dialkylaminonaphthalene, cyanin, or the
like. A particularly preferred fluorochrome may be a fluorochrome
having a skeletal structure of a dialkylaminonaphthalene or
pyrene.
[0040] The reaction for forming the covalent bond between the
specifically-binding protein and the fluorochrome can be carried
out according to conditions well known to those skilled in the art.
When the specifically-binding protein has a primary, secondary or
tertiary amino group, a carboxyl group, a thiol group, a phenyl
group, a phenol group or a hydroxyl group, the covalent bond can be
formed by reacting the specifically-binding protein and the
fluorochrome having an activated functional group normally at room
temperature for several hours. After the completion of the
reaction, the unreacted fluorochrome can be easily removed by an
ordinary method (e.g., gel filtration or dialysis). The
specifically-binding protein and the fluorochrome can be bound
directly or can be bound indirectly via a bifunctional linker
molecule, or the like.
[0041] Using the above-described fluorescent-labeled protein, it is
possible to analyze the assay-object in a sample as follows.
[0042] The sample containing the assay-object and the
fluorescent-labeled protein are mixed with each other in a solution
so as to measure the degree of fluorescence polarization of the
fluorescent-labeled protein in the mixed solution. If necessary,
the degree of fluorescence polarization of the fluorescent-labeled
protein in the absence of the assay-object is also measured. Any
polarization measurement apparatus can be used for measuring the
degree of fluorescence polarization. The measurement is performed
at a mild temperature (about 10 degree centigrade (.degree. C.) to
about 40.degree. C.) and, preferably, at a constant
temperature.
[0043] The measurement of the degree of fluorescence polarization
can be performed by measuring the degree after a predetermined time
from the mixing of the assay-object and the fluorescent-labeled
protein, or by measuring a change in the degree of fluorescence
polarization for a unit of time. By taking a measurement at the
time when the binding between the assay-object and the
fluorescent-labeled protein has been completely finished, more
reproducible measurement values are obtained. By measuring the
change in the degree of fluorescence polarization for a unit of
time while the binding reaction between the assay-object and the
fluorescent-labeled protein is in progress, on the other hand, a
quicker measurement is possible. For the purpose of identifying the
assay-object, a measurement value of a degree of fluorescence
polarization for a known standard sample is compared with the
measurement value for the unknown assay-object in the sample. For
the purpose of quantifying the assay-object contained in the
sample, a standard curve is provided through a measurement of the
degree of fluorescence polarization using a solution containing a
known concentration of the assay-object so as to compare it with
the measurement value for the sample.
[0044] With the fluorescence polarization method of the present
invention, it is possible to perform a quick measurement for a
protein having a high molecular weight, particularly about
1,000,000 or more, and a bacteria or a virus, which has been
difficult in the past. Therefore, the method of the present
invention is preferred in assaying an object, particularly, those
having a high molecular weight. The fluorescence polarization
method of the present invention is particularly useful in that it
is possible to identify the type of bacteria or virus.
[0045] The sample intended to be used with the method of the
present invention is a material including an assay-object desired
to be analyzed in any fields of art including medical diagnosis,
environmental assay, and food control. Exemplary samples for
medical diagnosis include body fluids including a blood, a lymph,
and a tissue fluid. Exemplary samples for environmental assay
include materials collected from soil, a river, the air, or the
like. Exemplary samples for food control include an extract from
ground meat, and an extract from a chopping board. The sample can
be in any form so long as it can be used with the method of the
present invention.
[0046] The assay-objects of the method of the present invention
include, though are not limited to, a biological substance, a
microorganism, a virus, a pharmaceutical, an environmental
pollutant and an abused drug. Preferably, the assay-object is a
high molecular weight substance having a molecular weight of about
20,000 to 30,000 or more, more preferably, about 100,000 to 200,000
or more, even more preferably, about 500,000 or more and, most
preferably, about a million or more. Alternatively, the
assay-object is preferably a substance having a size of about 2 nm
or more, more preferably, about 10 nm or more and, most preferably,
about 20 nm or more.
[0047] The biological substance refers to any organic or inorganic
substance existing in the body of a human or other mammal. Typical
examples of the biological substance include a peptide, a protein,
a lipid, a saccharide and a nucleic acid. Herein, the peptide as a
biological substance refers to those having a molecular weight of
less than about 1,000. The protein as a biological substance refers
those having a molecular weight of about 1,000 or more. Examples of
the protein include an antibody, a hormone, an inflammation marker,
a coagulation factor, an apolipoprotein, a high density lipoprotein
(HDL), a low density lipoprotein (LDL), a glycosylated albumin, a
glycosylated hemoglobin, a hemoglobin, a cancer marker and an
enzyme. Examples of the cancer marker include, though not limited
to, .alpha.-fetoprotein (AFP), CEA, CA19-9, ferritin, an
immunosuppressive acidic protein (IAP), .beta..sub.2-microglobulin
(BMG), TPA, polyamine, a polyamine fraction, basic fetoprotein
(BFP), SCC antigen, neural specific enolase (NSE),
sialylLe-iantigen (SLX), CYFRA21-1, CA15-3, BGA225, estrogen
receptor (ER), progesterone receptor (PgR), 5'-nucleotide
phosphodiesterase isozyme-V (5'-NPD-V), vitamin K deficiency
protein II (PIVKA-II), CA19-9, elastase I, CA50, SPan-1, DUPAN-2,
KMO I, NCC-ST-439, CA125, CA130, CA72-4, sialyl Tn antigen (STN),
SP.sub.4, fee-HOG-.beta., PAP, PA, .gamma.-seminoprotein
(.gamma.-Sm), and the like.
[0048] The microorganism includes a bacteria, a fungus and a
protozoan. As the assay-object, a bacteria may be important,
Enterobacteriaceae and Vibrionaceae may be more important, and
salmonella and enterohemorrhagic Escherichia coli belonging to
Enterobacteriaceae and Vibrio parahaemolyticus belonging to
Vibrionaceae may be particularly important. The virus includes a
bacterial virus, a plant virus and an animal virus. As the
assay-object, Human immunodeficiency virus (AIDS virus), Hepatitis
A virus, Hepatitis B virus and Hepatitis C virus may be
particularly important. The pharmaceutical includes any agent used
for treating or diagnosing a human or other mammals. The
environmental pollutant includes any substance causing
environmental pollution which can be detected from soil, a river,
the air, or the like. The abused drug refers to a drug, intake of
which by a human is restricted by law or regulation, and which has
been used in violation of the restriction.
[0049] The present invention also provides a reagent including a
fluorescent-labeled protein which is suitable for use in the
above-described method. The fluorescent-labeled protein may be
provided in various forms such as a dry form, a solution form
wherein the protein is dissolved in a buffer solution, or the
like.
[0050] The following examples of the invention are intended to
illustrate, but not to limit, the present invention.
EXAMPLES
[0051] Hereinbelow, the results of measurements for high molecular
weight assay-objects: C-reactive protein (CRP; molecular weight:
120,000); ahigh density lipoprotein (HDL; molecular weight: about
400,000): a low density lipoprotein (LDL; molecular weight
3,000,000); and enterohemorrhagic E. coli 0157 (E. coli 0157; size:
2 .mu.m) according to the present invention will be described. An
anti-CRP polyclonal antibody, an anti-HDL polyclonal antibody, an
anti-LDL polyclonal antibody, and anti-E. coli 0157 polyclonal
antibody, each labeled with a pyrene derivative, were used in the
measurements. The pyrene derivative is a fluorochrome having a
fluorescence lifetime of about 50 nanoseconds to about 500
nanoseconds which correspond to a change in molecular weight from
about 100,000 to about 5,000,000. F-4000 manufactured by Hitachi
Ltd. was used as an apparatus for measuring the degree of
fluorescence polarization.
Example 1
[0052] Synthesis of Succinimidyl-1-pyrenebutanoic Acid (SPB)
[0053] SPB was prepared according to the synthesis scheme shown in
FIG. 2. In 20 ml of dimethylsulfoxide (DMF), 2.25 g of
1-pyrenebutanoic acid (7.8 mmol: obtained from Molecular Probes,
Inc.) and 1.2 g of N-hydroxy succinimide (10.4 mmol: obtained from
Wako Pure Chemical Industries, Ltd.) were dissolved. After the
mixed solution was cooled to 0.degree. C., 2.69 g of
1,3-dichlorohexylcarbodiimide (13.1 mmol: DCC) was added thereto.
Then, the mixture was reacted at 0.degree. C. for 24 hours while
stirring. After the reacted solution was filtered with a 0.45 .mu.m
filter, the filtrate was collected and dried using an evaporator.
Then, the filtrate was recrystallized from 95% ethanol, thereby
obtaining 1.88 g of succinimidyl-1-pyrenebutanoic acid (yield:
70%).
[0054] The obtained SPB was analyzed by an IR spectrometer and NMR.
The infrared absorption of the carboxyl group (1692 cm.sup.-1)
disappeared, and the infrared absorption of the imide group (1783
cm.sup.-1, 1785 cm.sup.-1 and 1817 cm.sup.-1) appeared. Also from
the results of NMR for .sup.1H and .sup.13C, the product was
identified as SPB.
Example 2
[0055] Preparation of Pyrene-labeled Polyclonal Antibody
[0056] A pyrene-labeled polyclonal antibody was prepared as
described below, using an anti-CRP polyclonal antibody (obtained
from Bio Reactive), an anti-HDL polyclonal antibody (obtained from
Cosmo Bio Co., Ltd.), an anti-LDL polyclonal antibody (obtained
from Cosmo Bio Co., Ltd.), and an anti-E. coli 0157 polyclonal
antibody (obtained from Funakoshi) together with SPB synthesized in
Example 1 above.
[0057] A respective solution (1000 .mu.l) containing 2.0 mg/ml of
each of the polyclonal antibodies in a phosphate-buffered saline
(PBS), pH 7.4, was mixed with a solution (20 .mu.l) containing 1.29
mg/ml of SPB (5-fold amount of antibody) dissolved in
dimethylsulfoxide (DMSO). These mixed solutions were reacted at
room temperature for 4 hours while stirring. The reacted solutions
were each subjected to Sephadex G-25 gel filtration column
(Pharmacia) (size: 10.times.60 mm, flow rate: about 2 ml/min).
Unreacted SPB was removed, and fractions containing the
pyrene-labeled polyclonal antibody were collected.
[0058] The collected fractions were used to evaluate the labeling
amount and the fluorescence property of the prepared pyrene-labeled
polyclonal antibody.
[0059] The labeling amount was measured using an
ultraviolet/visible spectrometer (manufactured by Shimadzu Corp.,
UV-1600PC), confirming labeling of: 1.1 pyrenes per one molecule of
the anti-CRP polyclonalantibody; 0.9 pyrene per one molecule of the
anti-HDL polyclonal antibody; 0.5 pyrene per one molecule of the
anti-LDL polyclonal antibody; and 1.2 pyrenes per one molecule of
the anti-E. coli 0157 polyclonal antibody. FIG. 3 shows an image of
pyrene binding to an antibody.
[0060] The fluorescence property was measured using a fluorescence
spectrometer (manufactured by Shimadzu Corp., RF-5300PC), finding
that the fluorescence property of pyrene bound to each of the
labeled antibodies was such that the excitation wavelength was 330
nm and the resulting fluorescence wavelengths were 373 nm and 397
nm. Since the fluorescence intensity was greater at 397 nm, as the
measurement conditions to be used with the fluorescence
polarization method, it was determined to utilize the excitation
wavelength of 330 nm and the fluorescence wavelength of 397 nm.
Example 3
[0061] Measurement of CRP with Pyrene-labeled Anti-CRP Polyclonal
Antibody
[0062] A solution (700 .mu.l) containing the pyrene-labeled
anti-CRP polyclonal antibody at 400 .mu.g/ml was placed into a
cuvette (5.times.5 mm) so as to measure the degree of fluorescence
polarization. The measurement conditions for the pyrene-labeled
anti-CRP polyclonal antibody were as follows: a measurement
temperature of 35.degree. C., an excitation wavelength of 330 nm, a
fluorescence wavelength of 397 nm and a G factor of 0.942.
[0063] A CRP solution having a CRP concentration of 0 to 50 mg/dl
(obtained from O.E.M. Concepts, Inc.) was prepared. The
above-described antibody solution (700 .mu.l) and the CRP solution
(30 .mu.l) were mixed with each other, and stirred at 35.degree. C.
for 0.5 minute, after which the degree of fluorescence polarization
was measured under the above-described conditions for 0.5 minute,
thereby measuring the change in the degree of fluorescence
polarization. As a result, it was found that up to a 30 mg/dl
concentration could be measured for CRP. The results are shown in
Figure 4.
Example 4
[0064] Measurement of HDL with Pyrene-labeled Anti-HDL Polyclonal
Antibody
[0065] A solution (700 .mu.l) containing the pyrene-labeled
anti-HDL polyclonal antibody at 400 .mu.g/ml was placed into a
cuvette (5.times.5 mm) so as to measure the degree of fluorescence
polarization. The measurement conditions for the pyrene-labeled
anti-HDL polyclonal antibody were as follows: a measurement
temperature of 35.degree. C., an excitation wavelength of 330 nm, a
fluorescence wavelength of 397 nm and a G factor of 0.943.
[0066] An HDL solution having an HDL concentration of 0 to 500
mg/dl (obtained from Cosmo Bio Co., Ltd.) was prepared. The
above-described antibody solution (700 .mu.l) and the HDL solution
(5 .mu.l) were mixed with each other, and stirred at 35.degree. C
for 0.5 minute, after which the degree of fluorescence polarization
was measured under the above-described conditions for 0.5 minute,
thereby measuring the change in the degree of fluorescence
polarization. As a result, it was found that up to a 500 mg/dl
concentration could be measured for HDL. The results are shown in
FIG. 5.
Example 5
[0067] Measurement of LDL with Pyrene-labeled Anti-LDL Polyclonal
Antibody
[0068] A solution (700 .mu.l) containing the pyrene-labeled
anti-LDL polyclonal antibody at 400 .mu.g/ml was placed into a
cuvette (5.times.5 mm) so as to measure the degree of fluorescence
polarization. The measurement conditions for the pyrene-labeled
anti-LDL polyclonal antibody were as follows: a measurement
temperature of 35.degree. C., an excitation wavelength of 330 nm, a
fluorescence wavelength of 397 nm and a G factor of 0.943.
[0069] An LDL solution having an LDL concentration of 0 to 1650
mg/dl (obtained from Cosmo Bio Co., Ltd.) was prepared. The
above-described antibody solution (700 .mu.l) and the LDL solution
(5 .mu.l) were mixed with each other, and stirred at 35.degree. C.
for 0.5 minute, after which the degree of fluorescence polarization
was measured under the above-described conditions for 0.5 minute,
thereby measuring the change in the degree of fluorescence
polarization. As a result, it was found that up to a 1000 mg/dl
concentration could be measured for LDL. The results are shown in
FIG. 6.
Example 6
[0070] Measurement of LDL with Pyrene-labeled Anti-E. coli 0157
Polyclonal Antibody
[0071] A solution (700 .mu.l) containing the pyrene-labeled anti-E.
coli 0157 polyclonal antibody at 400 .mu.g/ml was placed into a
cuvette (5.times.5 mm) so as to measure the degree of fluorescence
polarization. The measurement conditions for the pyrene-labeled
anti-E. coli 0157 polyclonal antibody were as follows: a
measurement temperature of 35.degree. C., an excitation wavelength
of 330 nm, a fluorescence wavelength of 397 nm and a G factor of
0.942.
[0072] An E. coli 0157 (obtained from Funakoshi) solution was added
to the above-described antibody solution (700 .mu.l) so that the
final concentration is 0 to 1.2.times.10.sup.9 cells/ml. The mixed
solution was stirred at 35.degree. C. for 0.5 minute, after which
the degree of fluorescence polarization was measured under the
above-described conditions for 0.5 minute, thereby measuring the
change in the degree of fluorescence polarization. As a result, it
was found that up to a 1.0.times.10.sup.8 cells/ml concentration
could be measured for E. coli 0157. The results are shown in FIG.
7.
[0073] Industrial Applicability
[0074] The method of the present invention provides a fluorescence
polarization method with a simple assay reaction system and an easy
assay operation, and particularly a method which is suitable for an
assay of a high-molecular-weight substance.
* * * * *