U.S. patent application number 09/120192 was filed with the patent office on 2001-07-26 for immunoassay method and immunoassay kit.
Invention is credited to MORI, KENJIRO, OKADA, KEISAKU, SENDA, SHUJI.
Application Number | 20010009765 09/120192 |
Document ID | / |
Family ID | 16634826 |
Filed Date | 2001-07-26 |
United States Patent
Application |
20010009765 |
Kind Code |
A1 |
OKADA, KEISAKU ; et
al. |
July 26, 2001 |
IMMUNOASSAY METHOD AND IMMUNOASSAY KIT
Abstract
An immunoassay method comprising bringing an immobilized phase
comprising, at different positions on a water-absorbable base
material, at least two first immunity substances capable of
specifically binding with at least two kinds of assay target
substances selected from the group consisting of
verotoxin-producing Escherichia coli, verotoxin and human
hemoglobin contained in a test sample, into contact with a test
sample and a liquid containing labeled immunity substances each
comprising a second immunity substance that is labeled with colored
particles and capable of binding with said assay target substance,
thereby to form an assay target substance-labeled immunity
substance complex and to bind said complex with respective first
immunity substances at the immobilized phase. The immunoassay
method, the immunoassay device and the immunoassay kit of the
present invention enable easy and simultaneous analysis of O157
(VTEC), VT and Hb in a test sample, by adsorption of the assay
target substances on an immobilized phase and evaluation of the
developed color.
Inventors: |
OKADA, KEISAKU; (OSAKA,
JP) ; MORI, KENJIRO; (OSAKA, JP) ; SENDA,
SHUJI; (OSAKA, JP) |
Correspondence
Address: |
McGUIREWOODS LLP
1750 TYSONS BOULEVARD
SUITE 1800
McLEAN
VA
22102
US
|
Family ID: |
16634826 |
Appl. No.: |
09/120192 |
Filed: |
July 22, 1998 |
Current U.S.
Class: |
435/7.32 |
Current CPC
Class: |
Y10S 436/81 20130101;
G01N 33/558 20130101 |
Class at
Publication: |
435/7.32 |
International
Class: |
G01N 033/554 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 7, 1997 |
JP |
213177/1997 |
Jan 27, 1997 |
JP |
ZZZZZZZZZZZZZZ |
Jul 19, 1995 |
JP |
ZZZZZZZZZZZZZZ |
Nov 16, 1995 |
JP |
ZZZZZZZZZZZZZZ |
Claims
What is claimed is:
1. An immunoassay method comprising bringing an immobilized phase
comprising, at different positions on a water-absorbable base
material, at least two first immunity substances capable of
specifically binding with at least two kinds of assay target
substances selected from the group consisting of
verotoxin-producing Escherichia coli, verotoxin and human
hemoglobin contained in a test sample, into contact with a test
sample and a liquid containing labeled immunity substances each
comprising a second immunity substance that is labeled with colored
particles and capable of binding with said assay target substance,
thereby to form an assay target substance-labeled immunity
substance complex and to bind said complex with respective first
immunity substances at the immobilized phase.
2. The immunoassay method of claim 1, wherein the contact is made
by flowing a mixture of the test sample and the liquid, so that it
is absorbed from one end of the water-absorbable base material,
thereby to bind said complex with the first immunity substance.
3. The immunoassay method of claim 1, wherein the contact is made
by flowing the test sample, so that it is absorbed from one end of
the water-absorbable base material, thereby to bind the assay
target substance with the first immunity substance, and then
flowing the liquid to allow absorption thereof by the base
material, thereby to bind said labeled immunity substance with the
assay target substance.
4. The immunoassay method of claim 1, wherein the contact is made
by having the test sample absorbed halfway up to the immobilized
phase, allowing the liquid to be absorbed from one end of the
water-absorbable base material, thereby to form a complex of said
labeled immunity substance and the assay target substance, and
binding said complex with the first immunity substance at the
immobilized phase.
5. The immunoassay method of claim 1, wherein the contact is made
by forming a label phase at a position halfway up to the
immobilized phase, the label phase comprising the labeled immunity
substance in such a manner that the labeled immunity substance can
be released from the base material upon contact with water,
allowing the test sample to be absorbed from one end of the
water-absorbable base material, thereby to form a complex of said
labeled immunity substance and the assay target substance, and
binding said complex with the first immunity substance at the
immobilized phase.
6. An immunoassay device comprising an immobilized phase comprising
plural first immunity substances each capable of specifically
binding with an assay target substance immobilized on a
water-absorbable base material, and a label phase comprising a
labeled immunity substance comprising a second immunity substance
that is labeled with colored particles and capable of binding with
said assay target substance, in such a manner that the labeled
immunity substance can be released from the base material upon
contact with water, said immobilized phase comprising at least two
first immunity substances capable of specifically binding with at
least two kinds of assay target substances selected from the group
consisting of verotoxin-producing Escherichia coli, verotoxin and
human hemoglobin contained in a test sample, said first immunity
substances being immobilized on different positions on the base
material.
7. An immunoassay kit comprising an immobilized phase comprising,
on a water-absorbable base material, plural immobilized first
immunity substances each capable of specifically binding with an
assay target substance, and a liquid containing labeled immunity
substances each comprising a second immunity substance that is
labeled with colored particles and capable of binding with said
assay target substance, said assay target substance being at least
two kinds of assay target substances selected from the group
consisting of verotoxin-producing Escherichia coli, verotoxin and
human hemoglobin.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to an immunoassay method, an
immunoassay device and an immunoassay kit, permitting easy, quick
and highly accurate detection of at least two kinds of test
substances from verotoxin-producing Escherichia coli, verotoxin and
human hemoglobin in a test sample simultaneously on the same base
material.
BACKGROUND OF THE INVENTION
[0002] The O157, which is a verotoxin-producing Escherichia coli
posing serious problems in recent years, enters the body with
foodstuff, which is the main infectious source, and causes the
onset of disease after about 4 to 9 days of incubation period.
Bloody feces is a symptom seen from the early stages of the
infection and, in some cases, hemolytic anemia, renal failure and
thrombocytopenia follow due to the action of verotoxin produced by
O157. The disease may ultimately progress to cause hemolytic uremic
syndrome (HUS).
[0003] The operation to detect verotoxin-producing Escherichia coli
in foodstuff and patients is extremely complicated and requires
many days before results are obtained. However, an immunological
assay method has recently enabled a comparatively easy detection of
the causative source.
[0004] A specific detection method includes a method (trademark
EHEC-TEC ELISA TEST SYSTEM, manufactured by Organon Teknika Corp.)
for detecting O157 antigen, comprising culturing a food using mTSB
(Tripticase Soy Broth Modified) medium and applying an ELISA method
(enzyme-linked immunosorbent assay method). For the detection of
verotoxin production by Escherichia coli separated from food, a
method (trademark, Verotox-F "SEIKEN", manufactured by Denka Seiken
Co., LTD.) includes culture thereof in a CA-YE medium and detection
of verotoxin 1 and verotoxin 2 by latex agglutination test using
supernatant as a test sample.
[0005] A method for detecting O157 in a test sample from a patient
includes use of a latex agglutination test (trademark Escherichia
coli O157detection kit "UNI", manufactured by UNIPATH LTD.).
[0006] The above-mentioned methods detect O157 and verotoxin as a
single test item, which means that they cannot be detected
concurrently. In addition, these methods require enrichment before
detection. Thus, they are time-consuming and require complicated
manipulation.
[0007] On the other hand, an immunity chromatography method has
recently been drawing attention as a method permitting quick and
easy immunoassay. In this method, the following steps are taken.
That is, an immobilized phase on which an immunity substance
capable of binding with an assay target substance in a test sample
is immobilized on a water absorbable base material, and a label
phase comprising a labeled immunity substance capable of binding
with said assay target substance, in such a manner that the labeled
immunity substance can be released from said base material upon
contact with water, are set at specific intervals to give a test
strip, and a test sample is absorbed from one end on the label
phase side of the strip. Then, the labeled immunity substance is
released from the label phase, bound with the assay target
substance to form a labeled immunity substance--assay target
substance complex, and this complex is bound with an immobilized
immunity substance at the aforementioned immobilized phase. By
assaying the labeled immunity substance bound at the immobilized
phase, the assay target substance in the test sample can be
assayed.
[0008] The label to be used to give the labeled immunity substance
is exemplified by colloidal metallic particle, enzyme, fluorescent
material, phosphorescent material, coloring material and water
dispersible polymer particles bound with or containing enzyme,
fluorescent material, phosphorescent material, coloring material
and the like. In particular, a labeled immunity substance wherein
water dispersible polymer particles colored with a
fluorophosphorescent material, coloring material (e.g., dye and
pigment) and the like are bound with an immunity substance by
physical adsorption, and a labeled immunity substance obtained by
binding gold colloidal particle with an immunity substance are
widely used due to high determination sensitivity and easiness.
[0009] In the present invention, an immunity chromatography method
is used to detect verotoxin-producing Escherichia coli which is
represented by O157 raising great concerns these days, verotoxin
and human hemoglobin associated with intestinal hemorrhage, and an
immunoassay method is provided that permits easy, quick, highly
accurate and simultaneous detection of these assay target
substances.
SUMMARY OF THE INVENTION
[0010] According to the present invention, it has been found that,
in a conventional immunity chromatography method, the use of a
labeled immunity substance wherein a second immunity substance
capable of binding with said assay target substances has been
labeled with colored particles and an immobilized phase comprising
at least two first immunity substances capable of specifically
binding with at least two kinds of assay target substances selected
from verotoxin-producing Escherichia coli, verotoxin and human
hemoglobin in a test sample, the first immunity substances being
immobilized on different positions on a water-absorbable base
material, enables simultaneous detection of plural assay target
substances in a test sample.
[0011] Thus, the present invention provides the following.
[0012] (1) An immunoassay method comprising bringing an immobilized
phase comprising, at different positions on a water-absorbable base
material, at least two first immunity substances capable of
specifically binding with at least two kinds of assay target
substances selected from the group consisting of
verotoxin-producing Escherichia coli, verotoxin and human
hemoglobin contained in a test sample, into contact with a test
sample and a liquid containing labeled immunity substances each
comprising a second immunity substance that is labeled with colored
particles and capable of binding with said assay target substance,
thereby to form an assay target substance-labeled immunity
substance complex and to bind said complex with respective first
immunity substances at the immobilized phase.
[0013] (2) The immunoassay method of above (1), wherein the contact
is made by flowing a mixture of the test sample and the liquid, so
that it is absorbed from one end of the water-absorbable base
material, thereby to bind said complex with the first immunity
substance.
[0014] (3) The immunoassay method of above (1), wherein the contact
is made by flowing the test sample, so that it is absorbed from one
end of the water-absorbable base material, thereby to bind the
assay target substance with the first immunity substance, and then
flowing the liquid to allow absorption thereof by the base
material, thereby to bind said labeled immunity substance with the
assay target substance.
[0015] (4) The immunoassay method of above (1), wherein the contact
is made by having the test sample absorbed halfway up to the
immobilized phase, allowing the liquid to be absorbed from one end
of the water-absorbable base material, thereby to form a complex of
said labeled immunity substance and the assay target substance, and
binding said complex with the first immunity substance at the
immobilized phase.
[0016] (5) The immunoassay method of above (1), wherein the contact
is made by forming a label phase at a position halfway up to the
immobilized phase, the label phase comprising the labeled immunity
substance in such a manner that the labeled immunity substance can
be released from the base material upon contact with water,
allowing the test sample to be absorbed from one end of the
water-absorbable base material, thereby to form a complex of said
labeled immunity substance and the assay target substance, and
binding said complex with the first immunity substance at the
immobilized phase.
[0017] (6) An immunoassay device comprising an immobilized phase
comprising plural first immunity substances each capable of
specifically binding with an assay target substance immobilized on
a water-absorbable base material, and a label phase comprising a
labeled immunity substance comprising a second immunity substance
that is labeled with colored particles and capable of binding with
said assay target substance, in such a manner that the labeled
immunity substance can be released from the base material upon
contact with water, said immobilized phase comprising at least two
first immunity substances capable of specifically binding with at
least two kinds of assay target substances selected from the group
consisting of verotoxin-producing Escherichia coli, verotoxin and
human hemoglobin contained in a test sample, said first immunity
substances being immobilized on different positions on the base
material.
[0018] (7) An immunoassay kit comprising an immobilized phase
comprising, on a water-absorbable base material, plural immobilized
first immunity substances each capable of specifically binding with
an assay target substance, and a liquid containing labeled immunity
substances each comprising a second immunity substance that is
labeled with colored particles and capable of binding with said
assay target substance, said assay target substance being at least
two kinds of assay target substances selected from the group
consisting of verotoxin-producing Escherichia coli, verotoxin and
human hemoglobin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a plane view schematically showing one embodiment
of the immunoassay strip of the present invention prepared in
Example 1. In the Figure, 1 is a water-absorbable base material, 2
is an immobilized phase and 3 is a polyester nonwoven fabric.
[0020] FIG. 2 is a cross sectional view along the line X-X' of the
immunoassay strip of FIG. 1. In the Figure, 1 is a water-absorbable
base material, 2 is an immobilized phase, 3 is a polyester nonwoven
fabric and 4 is a polyester film.
[0021] FIG. 3 is a plane view schematically showing one embodiment
of the immunoassay strip of the present invention prepared in
Example 4. In the Figure, 1 is a water-absorbable base material, 2
is an immobilized phase, 3 is a polyester nonwoven fabric and 5 is
a label phase.
[0022] FIG. 4 is a cross sectional view along the line X-X' of the
immunoassay strip of FIG. 3. In the Figure, 1 is a water-absorbable
base material, 2 is an immobilized phase, 3 is a polyester nonwoven
fabric, 4 is a polyester film, and 5 is a label phase.
DETAILED DESCRIPTION OF THE INVENTION
[0023] In the present invention, the first immunity substance and
the second immunity substance are antibodies that specifically bind
with verotoxin-producing Escherichia coli (hereinafter to be
referred to as VPEC), verotoxin (hereinafter to be referred to as
VT) or human hemoglobin thereinafter to be referred to as Hb) as an
assay target substance. The first immunity substance and the second
immunity substance bind with the same assay target substance.
According to the present invention wherein simultaneous assay of
two or more assay target substances can be achieved, plural first
immunity substances and the second immunity substances can be used.
They specifically bind with at least two kinds of assay target
substances from among the above-mentioned assay target substances.
The immunity substance may be known and may be the one used in
sandwich assay method depending on the assay target substance to be
assayed. The first immunity substance to be immobilized to form an
immobilized phase and the second immunity substance to be used as a
labeled immunity substance may be the same, or two kinds of
antibodies recognizing different epitopes may be used, though
subject to variation depending on the kind of antibody to be used
and assay targets. As the immunity substance, a monoclonal antibody
and a polyclonal antibody can be used. When one of the immunity
substances is a monoclonal antibody, the other immunity substance
is one that recognizes different epitope from the epitope said
monoclonal antibody recognizes.
[0024] In the present invention, moreover, VTEC to be detected may
be of various serum groups, which are exemplified by VTECs of serum
groups of O157, O26, O111, O18, O114, O115, O128, O145 and the
like. Inclusive of H antigen in the protein moiety of flagella,
they are designated O157:H7, O157:H-, O26:H11, O26:H-, O111:H-,
O18:H-, O114:H-, O115:H19, O128:H2, O145:H- and the like. Of these,
the serum groups of VTEC that are effectively used in the present
invention are O157, O26 and O111, which are specifically O157:H7,
O157:H-, O26:H111, O26:H-, O111:H-and the like. VT in case of human
includes VT-1 and VT-2 having different physicochemical properties
and immunological properties. In the present invention, these two
kinds of verotoxins can be detected simultaneously.
[0025] The water-absorbable base material to be used in the present
invention, is capable of absorbing a test sample containing an
assay target substance, such as a solution extracted from foodstuff
and culture supernatant, feces suspension (dissolved) and the like.
Alternatively, a base material capable of absorbing diluted
solutions of the above-mentioned with a buffer, a liquid containing
a labeled immunity substance and the like can be used. These are
free of limitation as long as they are capable of absorbing the
above-mentioned test samples. In the present invention, a
water-absorbable base material is used to secure time for
sufficient reaction between the assay target substance in a test
sample and the labeled immunity substance or first immunity
substance on the immobilized phase. When the water-absorbable base
material shows inferior water absorption, the time necessary for an
assay target substance to reach an immobilized phase, or when a
label phase is set, to reach said label phase, becomes longer and a
quick assay is not attainable.
[0026] On the other hand, when the water-absorbable base material
shows too high a water absorption, the time necessary for
sufficient reaction of an assay target substance in a test sample
with a labeled immunity substance or a first immunity substance on
the immobilized phase cannot be secured, thus making an accurate
assay unattainable. Preferable examples of the base material
include nonwoven fabric, filter paper, glass fiber fabric, glass
filter, nitrocellulose filter, porous material and the like. These
base materials have suitable water absorption speed and allow easy,
visual confirmation when colored particles bind to develop
color.
[0027] In view of the above, the water absorption of the
water-absorbable base material in the present invention is
preferably demonstrated by about 0.5-5 cm of the length of the part
of water absorption upon immersing one end of a 5 mm wide
rectangular water-absorbable base material in water and leaving the
strip for one minute.
[0028] For adjusting the water absorption of a base material, the
surface of the base material may be covered with a hydrophilic
polymer or surfactant, or immersed therein. Moreover, the
water-absorbable base material may be made from a single material,
or a continuous base material obtained by connecting heterogeneous
materials with an optional adhesive means may be used.
[0029] In the present invention, the shape of the water-absorbable
base material is not particularly limited as long as an assay
target substance can be developed. For example, a rectangular sheet
(strip) or a rod-shaped base material may be preferably used.
[0030] In the present invention, by the immobilized phase is meant
the area where a first immunity substance capable of binding with
an assay target substance is immobilized on a water-absorbable base
material. The method for immobilizing the first immunity substance
(preparation method of immobilized phase) on a water-absorbable
base material is not particularly limited. A conventionally known
physical method and a covalent bond method are suitable. In
particular, the covalent bond method is preferable, wherein the
immunity substance is hardly released from the base material. When
the water-absorbable base material does not have a functional group
for the above-mentioned covalent bond method, a base material is
prepared using a polymer having a suitable functional group and
attached to the water-absorbable base material to the extent the
water absorption property is not impaired. Alternatively, a
solution containing a first immunity substance and a hydrophilic
polymer is applied to a water-absorbable base material and immersed
into a coagulant solvent to coagulate the above-mentioned
hydrophilic polymer, whereby an immobilized phase can be prepared.
Examples of the hydrophilic polymer include hydroxypropylmethyl
cellulose, poly(vinyl alcohol), hydroxyethyl cellulose and the
like. Examples of the coagulant solvent include acetone, ethanol,
methanol, ether and the like.
[0031] The amount of the first immunity substance to be immobilized
varies depending on the kind and property of the immunity substance
to be used. In general terms, an antibody to VTEC is applied in an
amount of 0.001-0.5 mg/cm.sup.2, an antibody to VT is applied in an
amount of 0.01-1 mg/cm.sup.2, an antibody to Hb is applied in an
amount of 0.01-1 mg/cm.sup.2.
[0032] The immobilized phase in the present invention comprises at
least two first immunity substances immobilized on the
water-absorbable base material at different positions. In order to
trap the second immunity substances labeled with colored particles
(labeled immunity substances) that migrated by liquid absorption to
be mentioned later, at the immobilized phase, and to allow color
development of each assay target substance, the first immunity
substances are preferably immobilized on the water-absorbable base
material at an interval of not less than 1 mm, preferably 5 mm, so
that the developed colors will not mingle with the adjacent
color.
[0033] The labeled immunity substance in the present invention
comprises colored particles that label a second immunity substance
capable of binding with an assay target substance. The colored
particles may be any as long as they can be perceived visually.
They are exemplified by colloidal particles of metals such as gold,
silver, copper and the like, pigments represented by Sudan blue,
Sudan red IV, Sudan III, Oilorange, Quinizarin green and the like,
colored latex wherein latex has been colored with dye, and the
like. From the aspect of visual observation, gold colloid and
colored latex in blue and red, green or orange are preferably used,
with particular preference given to colored latex made from water
dispersible polymer particles colored in blue or red, in view of
water dispersibility, dispersion stability and easy adjustment of
detection sensitivity of assay target substance.
[0034] The size of the aforementioned colored particles is set to
fall in the range of 0.01-3 .mu.m, preferably 0.05-0.5 .mu.m, in
view of storage stability and easiness of preparation. When the
particle size is too small, the degree of coloring per particle is
also small, such that visual observation becomes difficult due to
poor color development upon binding with the immobilized phase.
When the size is too large, a slight agglomeration of colored
particles may cause clogging in the water-absorbable base material,
which in turn may result in degraded water absorption and
non-specific color development.
[0035] The colored particles may be bound with the second immunity
substance by a method conventionally known, such as a covalent bond
method, physical adsorption method, ionic bond method and the like.
In view of the absence of release of immunity substance after
binding and superior stability, a covalent bond method is
preferably employed. For the detection of plural assay target
substances in a test sample, the corresponding plural immunity
substances are bound with different kinds of colored particles in
the present invention. The colored particles used for this purpose
may have the same color or different color.
[0036] In the present invention, the labeled immunity substance is
dispersed in a buffer etc. to give a liquid, which may be absorbed
from one end of the water-absorbable base material having an
immobilized phase. Alternatively, it may be contained in the
water-absorbable base material, so that a labeled immunity
substance can be released from the water-absorbable base material
upon contact with water, when the labeled immunity substance
contacts with the test sample. In the present invention, the label
phase refers to an area where the labeled immunity substance is
comprised in such a manner that enables release thereof from the
base material upon contact with water.
[0037] The buffer in which the labeled immunity substance is
dispersed has a pH and salt concentration that do not inhibit
antigen-antibody reaction. The labeled immunity substance
concentration during detection is 0.005-5%, preferably 0.01-0.5%.
When the concentration is too low, the particles bound with the
immobilized phase decreases in number, thus resulting in poor color
development. When it is too high, not only an economic problem but
also an obscure color development of the immobilized phase occur,
that is caused by excess colored particles remaining in the area
other than the immobilized phase. Hereinafter the liquid containing
a labeled immunity substance is also referred to as a labeled
immunity substance liquid.
[0038] The labeled immunity substance is contained in the
water-absorbable base material (preparation of a label phase) by,
for example, applying a solution containing a labeled immunity
substance to a water-absorbable base material and drying same under
suitable conditions. The solution may be dried by lyophilization. A
different method includes dispersing a labeled immunity substance
in a water soluble polymer or a saccharose solution, applying this
dispersion liquid to a water-absorbable base material and drying.
This method is advantageous for the preparation of the immunoassay
strip of the present invention. When brought into contact with a
test sample, a water soluble polymer or saccharose is easily
solubilized in water and the labeled immunity substance is quickly
released from the base material and reacts with the assay target
substance. By adjusting the concentration of the water soluble
polymer or saccharose, a solution having a suitable viscosity can
be obtained, so that a labeled immunity substance can be contained
in a specific area of the water-absorbable base material and the
labeled immunity substance is free of coagulation or deformation
upon drying. After drying, moreover, the labeled immunity substance
is hardly released from the water-absorbable base material.
[0039] Examples of the water soluble polymer to be used in the
above-mentioned way include poly(vinylpyrrolidone), poly(vinyl
alcohol), poly(ethylene glycol), cellulose ether (e.g., methyl
cellulose, ethyl cellulose, carboxymethyl cellulose, carboxyethyl
cellulose, oxyethyl cellulose, cyan ethyl cellulose and the like),
gelatin and the like.
[0040] In the present invention, the distance between the
above-mentioned immobilized phase and the part where absorption of
the test sample and/or the liquid containing labeled immunity
substance is initiated (hereinafter to be referred to as liquid
absorption part), or when an immunoassay strip having a label phase
is used, the distance between the immobilized phase and the label
phase, is 1-6 cm, preferably about 3-4 cm. When the distance is too
great, the assay target substance may not reach the immobilized
phase, or color development becomes too intense, or assay takes too
long. Conversely, when the distance is too short, the color
development at the immobilized phase becomes inconsistent or too
low.
[0041] The amount of the labeled immunity substance to be applied
in the label phase is free of particular limitation as long as it
is sufficient to achieve the concentration of the labeled immunity
substance during the detection. The amount is about 0.005-5
mg/cm.sup.2 in the weight of a latex solid.
[0042] The liquid absorption part is not particularly limited as
long as it does not prevent transfer of the test sample or a liquid
containing a labeled immunity substance toward the water-absorbable
base material. It may be the same base material, or a laminate of
nonwoven fabric or woven fabric attached to said water absorbable
base material.
[0043] In the present invention, moreover, an immobilized phase
where the first immunity substance is immobilized, and a
water-absorbable base material having a liquid absorption part is
also referred to as an immunoassay device of the present invention.
The shape of the immunoassay device of the present invention is
free of limitation and may be, for example, a sheet, strip or rod .
This immunoassay device may contain an immobilized phase, a liquid
absorption part and a label phase.
[0044] The inventive immunoassay method includes the following
methods. In a first method, a test sample to be subjected to an
assay and a labeled immunity substance liquid are mixed. At this
time, plural assay target substances (VTEC, VT, Hb) in the test
sample are each bound with the labeled immunity substance to
respectively form a labeled immunity substance--assay target
substance complex [labeled immunity substance (colored
particle--second immunity substance)--assay target substance].
Then, a mixture of the test sample and the labeled immunity
substance liquid is absorbed from one end of the water-absorbable
base material having an immobilized phase. The complex formed in
the mixture moves through the water-absorbable base material along
with the movement of the liquid to reach the immobilized phase. At
the immobilized phase, the complex is bound with the first immunity
substance immobilized on the immobilized phase to newly form a
labeled immunocomplex comprising [labeled immunity substance
(colored particle--second immunity substance)--assay target
substance--first immunity substance], whereby the complex is
immobilized and bound on the immobilized phase.
[0045] In a second method, a test sample alone is absorbed from one
end of the water-absorbable base material having an immobilized
phase. Each assay target substance in the test sample is bound with
the first immunity substance on the immobilized phase (assay target
substance--first immunity substance). According to this method, the
test sample is liquid, and when solid, it is dissolved in a
suitable buffer etc. and subjected to a treatment such as
suspending, so that it can be absorbed by the water-absorbable base
material. Then, the labeled immunity substance liquid is absorbed,
whereby each labeled immunity substance forms a complex with the
assay target substance bound with the immobilized phase to give a
labeled immunocomplex comprising [labeled immunity substance
(colored particle--second immunity substance)--assay target
substance--first immunity substance], whereby the complex is
immobilized and bound on the immobilized phase.
[0046] According to a third method, a labeled immunity substance
liquid is absorbed from one end of the water-absorbable base
material having an immobilized phase. A liquid or solid test sample
is absorbed or applied at halfway up to the immobilized phase. The
plural assay target substances in the test sample respectively form
a complex with the labeled immunity substance. Thus formed complex
moves through the water-absorbable base material along with the
movement of the liquid to reach the immobilized phase. At the
immobilized phase, the complex binds with the first immunity
substance bound with the immobilized phase to newly form a labeled
immunocomplex comprising [labeled immunity substance (colored
particle--second immunity substance)--assay target substance--first
immunity substance], whereby the complex is immobilized and bound
on the immobilized phase.
[0047] When the inventive immunoassay strip on which a label phase
has been previously formed is to be used, the following method is
employed. First, a test sample to be subjected to an assay is
absorbed from one end of the water-absorbable base material having
an immobilized phase. In this method, also, the test sample is
liquid, and when solid, it is dissolved in a suitable buffer etc.
and subjected to a treatment such as suspending, so that it can be
absorbed by the water-absorbable base material. The test sample
moves through the water-absorbable base material to reach the label
phase and liberates the labeled immunity substance from the base
material. The plural assay target substances (VTEC, VT, Hb)
contained in the test sample bind with respective labeled immunity
substances to each form a complex of the labeled immunity substance
and the assay target substance [labeled immunity substance (colored
particle--second immunity substance)--assay target substance].
Then, the thus formed complex moves through the water-absorbable
base material along with the movement of the liquid to reach the
immobilized phase. At the immobilized phase, the complex binds with
the first immunity substance bound with the immobilized phase to
newly form a labeled immunocomplex comprising [labeled immunity
substance (colored particle--second immunity substance)--assay
target substance--first immunity substance], whereby the complex is
immobilized and bound on the immobilized phase.
[0048] The colored particles constituting the labeled immunity
substance gather at one part due to immobilization, where they
collectively produce clear color development that enables visual
confirmation of the existence of the assay target substance.
Inasmuch as the immobilized phase comprises first immunity
substances corresponding to plural assay target substances at
different positions in the present invention, at least two kinds of
assay target substances from VTEC, VT and Hb can be detected for
their presence.
[0049] Therefore, the present invention enables simultaneous
detection of VTEC and VT present in food or Hb co-existent with
VTEC and/or VT present in feces.
[0050] The immunoassay method of the present invention is
advantageously embodied by the inventive immunoassay kit or
immunoassay device. The water-absorbable base material comprising
an immobilized phase on which a first immunity substance capable of
binding with an assay target substance has been immobilized, and a
liquid containing a labeled immunity substance bound with a second
immunity substance that is labeled with colored particles and
capable of binding with said assay target substance, or a label
phase comprising said labeled immunity substance, that constitute
the kit, are the same as those mentioned above.
[0051] When an assay is done using the inventive immunoassay
method, immunoassay device or assay kit, a test sample in a liquid
of 1-500 .mu.l, an assay target substance of VTEC of
10.sup.2-10.sup.9 cfu, VT of 0.01-100,000 ng and Hb of 0.5-500,000
ng, can be preferably assayed, though subject to change depending
on the kind and size of water-absorbable base material to be used
and properties of immunity substance to be used.
[0052] The present invention is described in more detail by way of
examples, to which the present invention is not limited.
[0053] Example 1
[0054] Detection of assay target substance (1)
[0055] 1) Preparation of labeled immunity substance liquid
[0056] To a dispersion (3 ml) containing blue-colored carboxylated
polystyrene latex particles (solid concentration 5 wt % average
particle size 0.1 .mu.m, 0.01M-borate buffer, pH 8) were added
water soluble carbodiimide (1 ml, 1 mg/ml, 0.01M-borate buffer, pH
8) and anti-Escherichia coli O157:H7 antibody (1 ml, goat IgG
(manufactured by Kirkegaard & Perry Laboratories Inc.), 1
mg/ml, 0.01M-borate buffer, pH 8), and the mixture was reacted at
10.degree. C. for 3 hr. The reaction mixture was washed by
centrifugation using 0.01 M borate buffer (pH 8) as a washing
solution to give blue-colored latex particle-labeled
anti-Escherichia coli O157:H7 antibody (solid concentration 2 wt
%).
[0057] In the same manner as above, anti-Verotoxin 1 antibody
(mouse IgG, 1 mg/ml) and anti-verotoxin 2 antibody (mouse IgG, 1
mg/ml) were independently bound with a separate dispersion of
green-colored carboxylated polystyrene latex particles (average
particle size 0.1 .mu.m).
[0058] In the same manner as above, anti-human hemoglobin antibody
(rabbit IgG (manufactured by Nippon Bio-Test Laboratories Inc.), 5
mg/ml) was bound with red-colored carboxylated polystyrene latex
particles (average particle size 0.1 .mu.m).
[0059] Then, each latex particle-labeled antibody was suspended in
0.01 M-borate buffer (pH 8, each solid concentration 2 wt %).
[0060] 2) Preparation of immobilized phase
[0061] On a nitrocellulose membrane (pore size 8 .mu.m, 6
mm.times.60 mm, corresponding to 1 in Figures), at 30 mm from one
end (2-4 in Figures), anti-Escherichia coli O157:H7 antibody (goat
IgG, 1 mg/ml, 0.1 M phosphate buffer, pH 7.4) was applied, at 25 mm
(2-3 in Figures), anti-verotoxin 1 antibody (rabbit IgG, 2 mg/ml)
was applied, at 20 mm (2-2 in Figures), anti-verotoxin 2 antibody
(rabbit IgG, 2 mg/ml) was applied, and at 15 mm (2-1 in Figures),
anti-human hemoglobin antibody (rabbit IgG, 1 mg/ml) was applied in
an amount of 1.5 .mu.l each for draw a line with a dispenser.
[0062] This membrane was immersed in an aqueous solution of bovine
serum albumin (1 wt %) and polyoxyethylene (10) octylphenylether
(Wako Pure Chemical, 0.1 wt %) for 10 minutes and dried at
40.degree. C. for 2 hr.
[0063] Then, a polyester film (90 .mu.m thick, corresponding to 4
in Figures) was adhered to the back side of this membrane (opposite
side from the surface on which antibody was applied) with a spray
glue.
[0064] At 0-8 mm from the opposite end from the application of
antibody, a polyester nonwoven fabric (6 mm.times.8 mm, thickness
2.5 mm, corresponding to 3 in Figures) was adhered to give the
inventive immunoassay strip.
[0065] 3) Assay
[0066] A test sample was prepared, which comprised 0.1 M phosphate
buffer (containing NaCl (0.9 wt %), pH 7.4) and Escherichia coli
O157:H7, verotoxin type 1, verotoxin type 2 and human hemoglobin
dispersed therein at concentrations shown in Tables 1-3.
[0067] This test sample was mixed with the suspensions of colored
latex labeled antibody (labeled immunity substance) prepared in 1)
above to a solid concentration of 0.02 wt % each. After mixing and
stirring, the mixed solution (60 .mu.l) was dropwise added to the
test strip of polyester nonwoven fabric prepared in 2) above, and
the presence or absence of color development in 20 minutes was
visually observed.
[0068] Tables 1-3 show the assay results when each assay target
substance was used alone or in combination in the test sample.
Escherichia coli O157:H7 used did not produce verotoxin, so that
assay results of a mixed test sample would not be influenced. In
each Table, the evaluation criteria were as follows.
[0069] +:line-like color development found on immobilized phase
[0070] -:line-like color development not found on immobilized
phase
1TABLE 1 Test sample O157:H7 (cfu/ml) 10.sup.5 10.sup.5 10.sup.5
10.sup.5 0 10.sup.5 Verotoxin 1 (ng/ml) 5 5 5 0 5 5 Verotoxin 2
(ng/ml) 5 5 0 5 5 0 Human hemoglobin (ng/ml) 100 0 100 100 100 0
Immobilized Anti- O157:H 7 antibody + + + + - + phase
Anti-Verotoxin 1 antibody + + + - + + Anti-Verotoxin 2 antibody + +
- + + - Anti-Human hemoglobin + - + + + - antibody
[0071]
2TABLE 2 Test sample O157:H7 (cfu/ml) 10.sup.5 10.sup.5 0 0 0
Verotoxin 1 (ng/ml) 0 0 5 5 0 Verotoxin 2 (ng/ml) 5 0 5 0 5 Human
hemoglobin 0 100 0 100 100 (ng/ml) Immobilized Anti-O157:H7 + + - -
- phase antibody Anti-Verotoxin 1 - - + + - antibody Anti-Verotoxin
2 + - + - + antibody Anti-Human - + - + + hemoglobin antibody
[0072]
3TABLE 3 Test sample O157:H7 (cfu/ml) 10.sup.5 0 0 0 0 Verotoxin 1
(ng/ml) 0 5 0 0 0 Verotoxin 2 (ng/ml) 0 0 5 0 0 Human hemoglobin 0
0 0 100 0 ng/ml Immobilized Anti-O157:H7 + - - - - phase antibody
Anti-Verotoxin 1 - + - - - antibody Anti-Verotoxin 2 - - + - -
antibody Anti-Human - - - + - hemoglobin antibody
[0073] Example 2
[0074] Detection of assay target substance (2)
[0075] In the same manner as in Example 1, the test samples (60
.mu.l) prepared to have the concentrations shown in Tables 4-6 were
dropwise added to the polyester nonwoven fabric part of the test
strip prepared in Example 1, 2), and developed to the immobilized
phase. Then, 0.1 M phosphate buffer (containing NaCl (0.9 wt %), pH
7.4) was added to dilute the solid component of each labeled
immunity substance to a concentration of 0.02 wt %. The obtained
labeled immunity substance liquid (60 .mu.l) was dropwise added to
the polyester nonwoven fabric part of the above-mentioned test
strips and the presence or absence of the color development at the
immobilized phase was visually observed 20 minutes later.
[0076] Tables 4-6 show the assay results when each assay target
substance was used alone or in combination in the test sample.
4TABLE 4 Test sample O157:H7 (cfu/ml) 10.sup.5 10.sup.5 10.sup.5
10.sup.5 0 10.sup.5 Verotoxin 1 (ng/ml) 5 5 5 0 5 5 Verotoxin 2
(ng/ml) 5 5 0 5 5 0 Human hemoglobin (ng/ml) 100 0 100 100 100 0
Immobilized Anti- O157:H 7 antibody + + + + - + phase
Anti-Verotoxin 1 antibody + + + - + + Anti-Verotoxin 2 antibody + +
- + + - Anti-Human hemoglobin + - + + + - antibody
[0077]
5TABLE 5 Test sample O157:H7 (cfu/ml) 10.sup.5 10.sup.5 0 0 0
Verotoxin 1 (ng/ml) 0 0 5 5 0 Verotoxin 2 (ng/ml) 5 0 5 0 5 Human
hemoglobin 0 100 0 100 100 ng/ml Immobilized Anti-O157:H7 + + - - -
phase antibody Anti-Verotoxin 1 - - + + - antibody Anti-Verotoxin 2
+ - + - + antibody Anti-Human - + - + + hemoglobin antibody
[0078]
6TABLE 6 Test sample O157:H7 (cfu/ml) 10.sup.5 0 0 0 0 Verotoxin 1
(ng/ml) 0 5 0 0 0 Verotoxin 2 (ng/ml) 0 0 5 0 0 Human hemoglobin 0
0 0 100 0 ng/ml Immobilized Anti-O157:H7 + - - - - phase antibody
Anti-Verotoxin 1 - + - - - antibody Anti-Verotoxin 2 - - + - -
antibody Anti-Human - - - + - hemoglobin antibody
[0079] Example 3
[0080] Detection of assay target substance (3)
[0081] In the same manner as in Example 1, the test samples (2
.mu.l) prepared to have the concentrations shown in Tables 7-9 were
absorbed in the surface side of the immunoassay strip prepared in
Example 1, 2) at 12-20 mm from the opposite end from the part where
the antibody was applied (immobilized phase). Then, 0.1 M phosphate
buffer (containing NaCl (0.9 wt %), pH 7.4) was added to dilute the
solid component of each labeled immunity substance to a
concentration of 0.02 wt %. The labeled immunity substance liquid
(60 .mu.l) obtained upon mixture was dropwise added to the
polyester nonwoven fabric part of the above-mentioned test strips
and the presence or absence of the color development at the
immobilized phase was visually observed 20 minutes later.
[0082] Tables 7-9 show the assay results when each assay target
substance was used alone or in combination in the test sample.
7TABLE 7 Test sample O157:H7 (cfu/ml) 10.sup.6 10.sup.6 10.sup.6
10.sup.6 0 10.sup.6 Verotoxin 1 (ng/ml) 150 150 150 0 150 150
Verotoxin 2 (ng/ml) 150 150 0 150 150 0 Human hemoglobin (ng/ml) 3
0 3 3 3 0 Immobilized Anti- O157:H 7 antibody + + + + - + phase
Anti-Verotoxin 1 antibody + + + - + + Anti-Verotoxin 2 antibody + +
- + + - Anti-Human hemoglobin + - + + + - antibody
[0083]
8TABLE 8 Test sample O157:H7 (cfu/ml) 10.sup.6 10.sup.6 0 0 0
Verotoxin 1 (ng/ml) 0 0 150 150 0 Verotoxin 2 (ng/ml) 150 0 150 0
150 Human hemoglobin 0 3 0 3 3 ng/ml Immobilized Anti-O157:H7 + + -
- - phase antibody Anti-Verotoxin 1 - - + + - antibody
Anti-Verotoxin 2 + - + - + antibody Anti-Human - + - + + hemoglobin
antibody
[0084]
9TABLE 9 Test sample O157:H7 (cfu/ml) 10.sup.6 10.sup.6 0 0 0
Verotoxin 1 (ng/ml) 0 150 0 0 0 Verotoxin 2 (ng/ml) 0 0 150 0 0
Human hemoglobin 0 0 0 3 0 ng/ml Immobilized Anti-O157:H7 + - - - -
phase antibody Anti-Verotoxin 1 - + - - - antibody Anti-Verotoxin 2
- - + - - antibody Anti-Human - - - + - hemoglobin antibody
[0085] Example 4
[0086] Detection of assay target substance (4)
[0087] 1) Preparation of immunoassay strip
[0088] The blue-colored latex particle-labeled anti-Escherichia
coli O157:H7 antibody dispersion (1 ml, solid concentration 2 wt %)
prepared in Example 1, 1), green-colored latex particle-labeled
anti-verotoxin 1 antibody dispersion (1 ml, solid concentration 2
wt %), green-colored latex particle-labeled anti-verotoxin 2
antibody dispersion (1 ml, solid concentration 2 wt %), red-colored
latex particle-labeled anti-human hemoglobin antibody dispersion (1
ml, solid concentration 2 wt %) and aqueous solution of saccharose
(6 ml, 20 wt %) were mixed and a rayon nonwoven fabric (6
mm.times.8 mm) was impregnated with this mixture (10 .mu.l) and
dried at 40.degree. C. for 2 hr.
[0089] This nonwoven fabric containing colored latex
particle-labeled antibodies was adhered to the front surface of the
membrane having an immobilized phase prepared in Example 1, 2)
wherein a polyester film (90 .mu.m thick) was adhered to the back
(the opposite side from the antibody application side) as in
Example 1, at 12-20 mm from the opposite end from the immobilized
phase. In addition, polyester nonwoven fabric (6 mm.times.8 mm,
thickness 2.5 mm, corresponding to 3 in Figures) was adhered at 0-8
mm to give the immunoassay strip of the present invention.
[0090] 2) Assay
[0091] Escherichia coli O157:H7, verotoxin type 1, verotoxin type 2
and human hemoglobin were dispersed in 0.1 M phosphate buffer
(containing NaCl (0.9 wt %), pH 7.4) to the concentrations shown in
Tables 10-14 to give test samples.
[0092] The obtained test sample (60 .mu.l) was dropwise added to
the polyester nonwoven fabric part of the above-mentioned test
strips and the presence or absence of the color development at the
immobilized phase was visually observed 20 minutes later.
[0093] Tables 10-13 show the assay results when each assay target
substance was used alone or in combination in the test sample.
Table 14 shows the assay results when each test sample was mixed
and subjected to the assay. Escherichia coli O157:H7 used did not
produce verotoxin, so that assay results of a mixed test sample
would not be influenced. In each Table, the evaluation criteria
were as in Example 1.
10TABLE 10 Test sample of E. coli O157:H7 alone E. coli O157:H7
concentration (cells, cfu/ml) Immobilized phase 10.sup.3 10.sup.4
10.sup.5 10.sup.6 10.sup.7 Anti-E. coli O157:H7 - - + + + antibody
immobilized phase Anti-verotoxin 1 antibody - - - - - immobilized
phase Anti-verotoxin 2 antibody - - - - - immobilized phase
Anti-human hemoglobin - - - - - antibody immobilized phase
[0094]
11TABLE 11 Test sample of Verotoxin 1 alone Verotoxin 1
concentration (ng/ml) Immobilized phase 0.5 1.0 5.0 10 50 Anti-E.
Coli O157:H7 antibody - - - - - immobilized phase Anti-verotoxin 1
antibody - + + + + immobilized phase Anti-verotoxin 2 antibody - -
- - - immobilized phase Anti-human hemoglobin - - - - - antibody
immobilized phase
[0095]
12TABLE 12 Test sample of Verotoxin 2 alone Verotoxin 2
concentration (ng/ml) Immobilized phase 0.5 1.0 5.0 10 50 Anti-E.
Coli O157:H7 antibody - - - - - immobilized phase Anti-verotoxin 1
antibody - - - - - immobilized phase Anti-verotoxin 2 antibody - -
+ + + immobilized phase Anti-human hemoglobin - - - - - antibody
immobilized phase
[0096]
13TABLE 13 Test sample of human hemoglobin alone human hemoglobin
concentration (ng/ml) Immobilized phase 10 50 100 500 1000 Anti-E.
coli O157:H7 - - - - - antibody immobilized phase Anti-verotoxin 1
antibody - - - - - immobilized phase Anti-verotoxin 2 antibody - -
- - - immobilized phase Anti-human hemoglobin - + + + + antibody
immobilized phase
[0097]
14TABLE 14 Test sample mixture of assay target substance Test
sample O157:H7 (cfu/ml) 10.sup.5 10.sup.5 10.sup.5 10.sup.5
10.sup.5 0 Verotoxin 1 (ng/ml) 5 5 0 5 0 5 Verotoxin 2 (ng/ml) 5 5
0 0 5 5 Human hemoglobin (ng/ml) 100 0 100 100 100 100 Immobilized
Anti- O157:H7 antibody + + + + + - phase Anti-Verotoxin 1 antibody
+ + - + - + Anti-Verotoxin 2 antibody + + - - + + Anti-Human
hemoglobin + - + + + + antibody
[0098] As is evident from the above results, the immunoassay
method, the immunoassay device and the immunoassay kit of the
present invention enable easy and simultaneous analysis of O157
(VIEC), VT and Hb in a test sample, and enable detection with
precision at the same level as individual analysis. Inasmuch as the
analysis depends on color development, visual observation gives
qualitative or semi-qualitative analysis. Using an optical device,
quantitative analysis becomes possible.
[0099] This application is based on application No. 213177/1997
filed in Japan, the content of which is incorporated hereinto by
reference.
* * * * *