Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
MEASURING METHOD USING WHOLE BLOOD SAMPLE
TECHNICAL FIELD
The present invention relates to a method for measuring a
substance to be examined in a whole blood sample using said
whole blood sample as it is. More particularly, the present
invention relates to a method for measuring a substance to be
examined in a whole blood sample using said whole blood sample
as it is, without preparing a serum or plasma sample from a
blood collected from a patient or the like.
BACKGROUND ART
An immunoassay has been widely used when measuring a
substance to be examined (analyte) in a sample, particularly
the analyte present at a trace amount in a biological sample.
Most of such trace analytes are generally contained in the
sample only in an amount of a .g/ml unit or less. For
example, there is a detectability limitation in an
immunodiffusion or laser nephelometer method wherein a complex
produced from an antigen-antibody reaction is directly
measured. Therefore, such trace analytes in the biological
sample may be measured more accurately by a method wherein one
of the antigen or antibody is labeled with a suitable
substance and a signal originating therefrom is detected,
namely, a label-immunoassay, or the like. The label-
immunoassay may be carried out in many manners. For example,
there may be mentioned a forward sandwich assay or a delayed
one-step sandwich assay wherein a sample is brought into
contact with an insoluble carrier covered with a first
immunological partner of an analyte; after a washing treatment
is optionally carried out, the resulting first immunological
partner-analyte-complex carried on the insoluble carrier is
brought into contact with a labeled second immunological
partner; the resulting complex of the insoluble carrier-first
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immunological partner-analyte-labeled second immunological
partner, and a portion not containing said complex are
separated; and a signal originating from the label contained
in one of the complex or the portion without said complex is
detected: a one-step sandwich assay wherein a sample is
brought into contact with an insoluble carrier covered with a
first immunological partner of an analyte, and at the same
time with a labeled second immunological partner; the
resulting complex of the insoluble carrier-first immunological
partner-analyte-labeled second immunological partner, and a
portion not containing said complex are separated; and a
signal originating from the label contained in one of the
complex or the portion without said complex is detected: a
reverse sandwich assay wherein a sample is brought into
contact with a labeled first immunological partner of an
analyte; the resulting labeled first immunological partner-
analyte-complex is brought into contact with a second
immunological partner carried on an insoluble carrier; the
resulting complex of the labeled first immunological partner-
analyte-second immunological partner-insoluble carrier, and a
portion not containing said complex are separated; and a
signal originating from the label contained in one of the
complex or the portion without said complex is detected: an
immunoinhibition method wherein a sample is brought into
contact with a labeled first immunological partner (preferably
a labeled monoclonal antibody in case of an antibody) of an
analyte, and then with a substance which shows a function same
as that of the analyte and is carried on an insoluble carrier,
or an immunological partner to the analyte, said partner being
carried on an insoluble carrier; the resulting immunological
partner-substance showing the function same as that of the
analyte-insoluble carrier complex, and a portion not
containing said complex are separated; and a signal
originating from the label contained in one of the complex or
the portion without said complex is detected: or a competitive
method wherein a sample is competitively brought into contact
with an insoluble carrier covered with an immunological
partner to an analyte, and a substance which shows a function
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same as that of the analyte; the resulting insoluble carrier-
immunological partner-substance showing the function same as
that of the analyte complex, and a portion not containing said
complex are separated; and a signal originating from the label
contained in one of the complex or the portion without said
complex is detected.
Further, the label-immunoassay may be classified, in view
of the label used, under, for example, an EIA wherein an
enzyme is used as a label, an immunoagglutination method
wherein erythrocytes or latex particles are used as a carrier,
and the resulting aggregates are visually observed, RIA
wherein an isotope is used as a label and the like. In these
methods, the amount of the complex produced is increased with
the contacting time of the sample with the immunological
partner to the analyte. Therefore, the contacting of the
sample with the immunological partner is lengthily continued
to reach the equilibrium state, namely until the amount of the
complex produced is not changed.
The RIA method requires particular equipment and has a
problem of radioactive wastes, and thus, has gradually been
replaced with the EIA method or the like. Further, when a
blood sample is used in all the above-mentioned conventional
methods, the collected whole blood sample was not used as they
are, but a serum or plasma sample was prepared from the
collected whole blood, and then an assay was carried out.
When a whole blood sample containing insoluble components such
as hemocytes is used in general methods other than the above
methods, the insoluble components would possibly interfere
with the value measured. For example, when the lights as
detecting signals originating from the labels are measured in
a visible light range in a homogeneous assay wherein B/F
separation is not carried out, a value with a positive error
may be obtained. In agglutination, an accurate determination
cannot be carried out, because substances, such as hemocytes,
which produce turbidity independently of the desired
agglutination exist. on the other hand, the B/F separation
assay may be conducted by reacting a whole blood sample with
an antibody immobilized on an insoluble carrier, washing out
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the whole blood, and then reacting with a labeled antibody or
the like. However, the result of the above assay is different
from that of an assay using a serum sample, because there
exist a lot of hemocyte components in the whole blood.
Further, the results cannot be adjusted by multiplying a
constant coefficient, because each individual has a different
amount of hemocytes. Therefore, the result obtained from a
whole blood sample should be adjusted by measuring a
hematocrit value of the blood examined so as to obtain a
diagnostic criterion from the whole blood sample. Taking into
account such troublesome procedure, a serum or plasma sample
prepared from a whole blood sample was used even in an assay
wherein B/F separation was carried out.
Many attempts to avoid the influence of the insoluble
components have been made. For example, Japanese Examined
Patent Publication (Kokoku) No. 02-51150 discloses a method to
avoid the influence of the insoluble components by adding a
hemolyzing agent to whole blood before agglutination.
Japanese Unexamined Patent Publication (Kokai) No. 01-237454
discloses a method to enhance detectability by enzymatic
digestion or exposure to a mild acid to remove interfering
substances and expose binding sites in a whole blood sample.
Japanese Unexamined Patent Publication (Kokai) No. 01-165964
discloses a method to reduce the influence of the insoluble
components by treating a whole blood with neuraminidase to
liberate latent tumor-associated antigens. Further, as an
attempt to conduct measurement without removing insoluble
components, a method for detecting hepatitis virus by a dried
filter blood is disclosed in Japanese Unexamined Patent
Publication (Kokai) No. 64-63868. In this method, a filter
paper stained with blood is air-dried, the dried filter paper
is cut up to a piece with a predetermined surface area, and
the piece, a buffer to extract the blood components contained
the piece, and beads coated with antibodies are reacted for
about 20 hours. However, the above method requires the
pretreatment procedures of the air-drying of the blood on the
filter paper and cutting of the dried paper. The above method
does not use a whole blood sample as it is, but uses the blood
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components extracted from the filter paper.
In addition to the above-mentioned methods using
immunoreaction in liquid, an analyte may be detected using a
whole blood sample, by a method using a dry immunoassay
element called dry chemistry [Japanese Unexamined Patent
Publication (Kokai) No. 01-112159], a method using so-called
biosensor [Japanese Unexamined Patent Publication (Kokai) No.
04-502671], or a method using immunochromatography [Japanese
Unexamined Patent Publication (Kokai) No. 06-947181.
In the above-mentioned conventional methods wherein
immunoreaction is carried out in liquid, an additional
procedure (pretreatment) is required before the
immunoreaction, and thus the assay procedure becomes
troublesome. In many cases, the above methods are not
suitable for treating many samples for many examination items.
The method using a dry immunoassay element and a whole blood
sample is not satisfied with accuracy. The methods using a
biosensor or immunochromatography requires particular
equipment.
Accordingly, the object of the present invention is to
provide an assaying means capable of rapidly measuring an
analyte in a whole blood sample, using a whole blood as a
sample without pretreatment of the blood. More particularly,
the object is to provide an assaying means capable of
obtaining a result for an analyte from a whole blood sample,
identical to that from a conventional sample, such as serum or
plasma, even when a problem associated with the whole blood
sample, such as troublesome variation of existing ratio of
insoluble components and soluble components, is encountered,
to thereby enable a rapid examination without pretreatment of
the collected whole blood sample, and extremely save labor in
a general examination.
DISCLOSURE OF INVENTION
According to the present invention, a concentration of a
substance to be examined (analyte) in a whole blood sample can
be determined using a simple and rapid assay, particularly an
immunoassay, as in a method wherein a serum or plasma sample
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is used. Thus, the procedure of separating serum or plasma
becomes unnecessary. Further a whole blood sample is brought
into contact with an insoluble carrier coated with
specifically binding partners (particularly immunological
partners) for a short period of time in the present invention,
and thus, a result can be obtained rapidly.
The above object can be achieved by the present invention
relating to a method for measuring a substance to be examined
(analyte), characterized in that a sufficient amount of a
whole blood sample is brought into contact with an insoluble
carrier coated with a first partner specifically bindable to
said substance to be examined (analyte) for a short period of
time; a complex of said substance to be examined (analyte) and
said first partner is brought into contact with a second
partner which is labeled and capable of specifically binding
to said first partner, said complex being carried on said
insoluble carrier; a resulting complex of said insoluble
carrier-said first partner-said substance to be examined
(analyte)-said labeled second partner, and a portion without
said resulting complex are separated from eath other; and a
signal originating from said label contained in one of said
separated portions is detected. The first partner may be same
as or different from the second partner. The first and second
partners may appropriately be selected on the basis of the
analyte.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 is a graph illustrating correlation between a
serum sample and a whole blood sample, in the case of HBs
antigen assay.
Figure 2 is a graph illustrating correlation between a
serum sample and a whole blood sample, in the case of HBs
antibody assay.
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be described in detail
hereinafter.
The present invention comprises using a whole blood sample
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taken from a patient or the like by any processes, such as
blood collection, or a synthetic sample containing an analyte
or an analogue thereof, and immunologically measuring the
analyte in said sample. The term "whole blood sample" used
herein means a sample which has not been treated to remove
insoluble components from blood taken from a patient or the
like, and thus contains insoluble components. Therefore, a
serum sample or a plasma sample is not involved in the whole
blood sample. The whole blood sample involves a sample
prepared by adding, to a collected blood, an anticoagulant,
such as EDTA, sodium citrate, heparin, sodium fluoride, or
sodium oxalate, and/or a blood preservative, such as CPD
comprising sodium citrate, citric acid, glucose and sodium
dihydrogenphosphate, or the like. Further, a sample prepared
by diluting a blood collected from a patient or the like with,
for example, a buffer having an anticoagulant function is also
involved in the above whole blood sample. A sample prepared
by treating a blood collected from a patient or the like as in
the above manner, and allowing to stand for a long period of
time, for example, for a durable term of a blood preservative,
i.e., for 21 days or more at 4 to 6 C, is also involved in
the above whole blood sample. The "synthetic sample
containing an analyte or an analogue thereof" is a sample for
use of quantitative determination of the present invention, or
for control of accuracy of the present invention. The
synthetic sample may contain or need not contain natural or
synthetic insoluble components, such as hemocytes, latex beads
or the like. Preferably, the synthetic sample does not
contain insoluble components in view of workability. Further,
the synthetic sample need not contain an analyte or an
analogue thereof, if it contains a substance which shows
correlation with the analyte.
The analyte in the present invention is not limited, so
long as it is generally contained in blood, particularly in a
trace amount. There may be mentioned, for example, proteins,
polysaccharides, lipids, haptens, nucleic acids, or a complex
or fragment thereof. More particularly, the analyte is, for
example, an infectious disease-related marker, such as an HBs
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antigen, HBs antibody, HIV-1 antibody, HIV-2 antibody, HTLV-I
antibody or treponema antibody, a tumor-associated antigen,
such as AFP, CRP or CEA, a coagulation-fibrinogenolysis-
related marker, such as plasminogen, antithrombin-III, D-dimer
or thrombin-antithrombin-III, a hormone, a cytosine, an
enzyme, or a drug, such as antiepileptic or digoxin. Further,
the analyte is also DNA or RNA having a specific sequence, or
a polynucleotide fragment thereof.
The partner specifically binding to the analyte.is a
partner which immunologically binds specifically to the
analyte, i.e., an immunological partner, for example, an
immunological substance, such as an antigen or antibody, to
proteins, polysaccharides, lipids, nucleic acids, or a complex
or fragment thereof. For example, the partner is an antigen
or antibody to the infectious disease-related marker, or an
antibody to the tumor-associated antigen, more particularly an
HBs antibody when the analyte is an HBs antigen, an HIV-1
antigen when the analyte is an HIV-1 antibody. Further, when
the analyte is a nucleic acid, the partner may be not only the
antibody thereto, but also the complementary nucleic acid,
such as complementary DNA or RNA, or a fragment thereof
prepared by treating with a restriction enzyme, or a synthetic
polynucleotide thereof, having at least a part of, preferably
all of, the sequence complementary to that of the nucleic acid
to be examined. The antibody as the immunological partner may
be a monoclonal antibody or polyclonal antibody, or a fragment
prepared by treating with an enzyme. An immunoglobulin which
is not treated or treated with an enzyme, such as IgG or
F(ab')2 prepared by pepsin digestion, may be also used. As
the antigen, a product obtained from a virus lysate by a
common process, such as density gradient centrifugation, or a
peptide or recombinant protein corresponding to a part of the
antigen may be also used. The specifically binding partner
without a label is immobilized to an insoluble carrier and
used as the first specifically binding partner, and the
specifically binding partner with a label is used as the
second specifically binding partner. The present invention
wherein immunological partners are used as the specifically
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binding partners will be described hereinafter. It is to be
understood that the following description may also be applied
to the present invention wherein the complementary nucleic
acids are used as the specifically binding partners.
When the present invention is carried out, an insoluble
carrier is coated with the first immunological partner to the
analyte, for example, by a conventionally known process, such
as the physical adsorption or covalent bond as described in
"Koso-Meneki-Sokutei-Ho (Enzyme Immunoassay)". More
particularly, the process comprises bringing the insoluble
carrier into contact with a solution containing the
immunological partner at room temperature for several hours,
washing with an appropriate buffer, and if necessary, air-
drying. After the carrier is coated with the immunological
partner, if necessary, the carrier may be blocked with, for
example, bovine serum albumin or skimmed milk, or excess
functional groups may be blocked with, for example, lysine, to
prevent components other than the analyte from adsorbing to
the insoluble carrier. The resulting insoluble carrier coated
with the immunological partner may be stored as it is, or as a
swelled form in a suitable buffer, such as a phosphate buffer.
In the present invention, any known insoluble carrier,
such as a tube, plate, membrane, film or bead, may be used.
The carrier may be made of natural or synthetic material, such
as cellulose, polyamide, polystyrene or polyethylene, or a
combination thereof, or a material prepared by adding an
inorganic material such as iron thereto to obtain a suitable
strength. The surface of the insoluble carrier is preferably
treated by a known process, for example, suitable anion groups
are introduced to the surface, to prevent substances other
than the analyte or the labeled immunological partner from
nonspecifically adsorbing to the surface. Further, suitable
functional groups, such as a carboxyl or amino group, are
preferably introduced to the surface of the insoluble carrier
by a known process so that the insoluble carrier may easily be
coated with the immunological partner to the analyte. The
bead as the immunological partner may be porous or nonporous,
but nonporous one with good washability being preferable. The
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diameter of the bead is preferably 0.1 .m to 7 mm. When the
bead is washed by filtration using a filter or suction using a
nozzle, the bead having a diameter greater than those of
insoluble components in a whole blood sample, for example, 10
.m or more, is more preferably used. The bead containing a
magnetic material, such as ferrite, may be washed by
collecting the beads by a magnet, without a filter, and thus
the diameter of such bead may be 10 m or less, preferably 0.1
to 5 m.
The resulting immunological partner-coated insoluble
carrier is then brought into contact with a whole blood sample
to form a complex of the analyte and the immunological partner
on the insoluble carrier. In this step of the present
invention, it is preferable that a lot of liquid substances,
such as a buffer for diluting the sample, are not contained.
An assay system wherein an immunoreaction is carried out
for a long period of time is generally called an end-point
assay, and is based on the completion of the immunoreaction of
all the analyte in the sample added to the assay system. When
a whole blood having a hematocrit value of 30 % is used as a
sample in this assay, the amount of the analyte is 70 % of
that contained in the same amount of the serum, and thus the
result would be found to be 70 % of that obtained from the
corresponding assay using the same amount of the serum sample.
If a whole blood having a hematocrit value of 50 % is used as
a sample in this assay, the result would be found to be 50 %
of that obtained from the corresponding assay using the same
amount of the serum sample. Therefore, an adjustment by a
hematocrit value must be made so as to secure
interchangeability of the result with that obtained from the
assay using a serum sample.
On the contrary, the present invention is characterized in
that a whole blood sample is brought into contact with the
immunological partner-coated insoluble carrier for a short
period of time from an initial stage of the reaction, and the
reaction is ceased or the measuring is carried out before the
end-point of the reaction. Thus, all the analytes in the
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sample are not reacted with the immunological partners on the
insoluble carriers, but the analytes are reacted with the
partners within a limited time in an amount dependent on and
proportional to the concentration of the analytes. In this
case, the concentration of the analyte in a whole blood is
same as that in a serum. Therefore, the hemocytes present in
the sample do not interfere the results. The result obtained
from a whole blood sample is unexpectedly excellently
corresponding to that obtained from a serum sample.
The concrete contacting time varies with a kind or
concentration of the analyte used, a kind of the immunological
partners used, a surface area of the insoluble carrier, or the
like. The reaction time range within which the result from a
whole blood sample is identical to that from a serum or plasma
sample obtained from said whole blood may be easily determined
by a simple pilot test.
The whole blood sample may be brought into contact with
beads used as the insoluble carrier in such an amount that the
beads are freely movable therein. The amount of the whole
blood sample brought into contact with the insoluble carrier
coated with the immunological partner may be determined by the
following test.
For example, when the analyte is an antigen and the
immunological partner thereof is an antibody, polystyrene
beads (diameter = 100 m) are coated with a predetermined
amount of the antibodies, and an assay is carried out with
changing the amount of the whole blood sample and the reaction
time for a predetermined constant amount of beads. Then, the
conditions to obtain the results identical to those from a
serum sample may be found.
For example, when an HBs antigen is used as the analyte,
1.7 g and 0.12 g of anti-HBs rabbit-specific antibodies
F(ab')2 are used as the first and second immunological
partners, respectively, and 5 mg of polystyrene beads
(diameter = 100 m; surface area = 2.5 cm2) are used as the
insoluble carrier, the contacting time may be 24 hours or
less, preferably 30 seconds to 30 minutes, more preferably 1
to 10 minutes.
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A complex of the analyte in the whole blood sample and the
first immunological partner on the insoluble carrier is formed
by said contact, and then, washed with a phosphate buffer or
the like, if necessary. It is not necessary, but preferable
to perform the washing. This is because that if a buffer
containing the labeled second immunological partner is used
upon adding the second partner in the next step, the whole
blood sample is diluted with the buffer, and the amount of the
analyte is substantially changed. On the contrary to the
former step, however, it is possible to minimize the influence
of other liquid components in which the second immunological
partner may be contained. For example, the influence may be
reduced by adding the labeled second immunological partner
after the analyte in the sample and the immunological partner
on the insoluble carrier nearly reach a state of equilibrium,
or adding the buffer containing the labeled second
immunological partner in an amount smaller than that of the
whole blood sample. The influence may be further reduced by
combining the above two methods. It is not necessary to
perform the washing procedure, when the labeled second
immunological partner is a powder material, or powdery
partners are encapsulated. If the washing is performed, the
buffer containing the labeled second immunological partner
used upon adding the second partner in the next step does not
affect that the result of the whole blood sample is made
identical to the result of the serum or plasma sample obtained
from said whole blood sample.
In the next step, the second immunological partner labeled
with a suitable labeling material is brought into contact with
the complex. The contacting time of the labeled second
immunological partner with the complex of the immunological
partner-coated insoluble carrier and the analyte is not
particularly limited, but may be several hours or less as in a
conventional method. In view of a rapid assay, the contacting
time is 1 hour or less, preferably 30 minutes or less, more
preferably 10 minutes or less. In this step, a complex of the
immunological partner on the insoluble carrier/the analyte/the
labeled second immunological partner is formed, and washed
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with a suitable buffer to remove unbound portion of the
labeled second immunological partner.
In the present invention, any conventionally known
labeling materials may be used. For example, an enzyme, such
as peroxidase or alkaline phosphatase, a bioluminescent
substance, such as luciferin-luciferase, a chemiluminescent
substance, such as luminol, acridine derivative or adamantane
derivative, fluorescent substance, such as fluorescein
isothiocyanate, a metal, such as gold colloid, a radioactive
material, such as 32P, or the like may be used as a label.
The label may be sensitized with an enzyme cycling or the
like. It is preferable to use the chemiluminescent substance,
such as luminol, acridine derivative or adamantane derivative.
The second immunological partner may be labeled with the
labeling material by a known process. For example, the
labeling material may be bound to the second immunological
partner by a physical-binding method wherein the material and
the partner are directly admixed, a glutaraldehyde method
wherein the material and the partner are bound via a suitable
linker, a periodate method, a maleimide method, a pyridine-
disulfide method, a method wherein the material and the
partner are bound via an avidin and biotin, or the like. The
resulting labeled material may be stored in a suitable
solution, or preferably phosphate buffer. The necessary
amount of the solution may be added to the complex, after
diluted if necessary. In this stage, a conventionally known
blocking agent, such as bovine serum albumin or skimmed milk,
a preservative, such as sodium azide, or a signal-amplifying
agent, such as p-iode-phenol may be added.
The complex of the immunological partner on the insoluble
carrier/the analyte/the labeled second immunological partner
which is formed in the former step is washed with a suitable
buffer, to separate a portion containing the complex (washed
residue) and the other portion not containing the complex
(washings). Then, the signal originating from the label in
one of the separated portions, preferably the portion
containing the complex (washed residue) may be detected to
qualitatively or quantitatively determine the analyte in the
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sample.
When an enzyme is used as the label, a substrate and
chromogene or the like may be added. After the reaction is
performed in a predetermined term, a color strength may be
spectroscopically measured. When a chemiluminescent
substance, such as an acridine derivative, is used as the
label, hydrogen peroxide and an alkaline solution by which the
acridine derivative emits may be added, at the same time or
separately. After the reaction is performed in a
predetermined term, an intensity of chemiluminescence may be
measured by a photomultiplier. In this case, a signal-
amplifying agent, such as hydrochloric acid, may be added if
necessary, before the addition of hydrogen peroxide and the
alkaline solution, or together with hydrogen peroxide. The
analyte in the whole blood sample may be measured in a short
period of time by measuring the resulting signal originating
from the label.
According to the present invention, as explained above,
the analyte in the whole blood sample may be quickly measured,
using the whole blood without pretreatment, as a sample.
Further, the present invention can shorten the contacting time
of the sample and the immunological partner. Furthermore, a
surprisingly advantageous effect that the analyte result from
the whole blood sample shows a correlation with the analyte
result from the conventional sample, such as a serum or plasma
sample, can be obtained by selecting preferable conditions of
the contacting time, the amount of the sample, the amount of
the insoluble carrier (the surface area of the carrier or the
amount of the immunological partner coated), and so on.
Because the pretreatment of the collected blood which is
required in conventional methods may be omitted, and the
contacting time may be shortened as above, thus, the present
invention can achieve a rapid examination and an extreme
labor-saving in a general examination. The present invention
may be applied not only to the above forward sandwich assay or
delayed one-step sandwich assay, but also to all the general
label-immunoassays, such as a one-step sandwich assay, reverse
sandwich assay, or competitive assay. The sample which may be
.CA 02172840 2004-03-04
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used in the present invention is not limited only to the whole
blood sample, but the other biological components, such as
serum, plasma, urine, or the like may be measured.
EXAMPLE
The present invention now will be further illustrated by,
but is by no means limited to, the following examples.
Examole 1: Preparation of beads coated with anti-HBs
antibodies
The antiserum obtained from a rabbit immunized with HBs
antigens was purified as follows. The antiserum was treated
with an HBs antigen-immobilized,*Sepharose 4B column, and the
unbound serum components were thoroughly washed with 20 mM
phosphate buffer, pH 7.0 (hereinafter referred to as PBS)
containing 0.15 M NaCl. Then, the specific antibodies were
eluted by PBS containing 3M sodium thiocyanate, and the elute
was dialyzed to 50 mM acetate buffer (pH 4.5). The specific
antibodies were digested with 2 % by weight (based on the
antibodies weight) of pepsin at 37 C for 16 hours. 1 M tris
solution was added and pH was adjusted to pH 8 to cease the
reaction. Thereafter, F(ab')2 was fractionated through
*Sephacryl S-200 column (equilibrated with PBS). The resulting
anti-HBs specific antibody F(ab')2 fraction was diluted with
PBS to 100 g/ml. Polystyrene beads (diameter = 100 m) were
added thereto in an amount of 0.35 g/1 ml of the antibody
solution, and admixed therewith by a rotator at 37 C for 4
hours to obtain coated beads. The beads was washed with PBS
three times, and stored in the form of 10 % suspension in PBS.
Exam8le 2: Preparation of acridinium ester-labeled anti-HBs
antibodies
The anti-HBs antibody F(ab')2 fraction prepared in Example
1 was diluted with 0.1 M phosphate buffer (pH 8.0) to 0.25
mg/ml. To the antibody liquid (1 ml), a solution (50 l) of
0.125 mg/ml 10-methyl-9-{4-[2-(succinimidyloxycarbonyl)-
ethyl]phenyloxycarbonyl}-acridinium fluorosulfonate in
dimethylformamide was added. The reaction was performed under
shaking at room temperature for 30 minutes. Thereafter, 0.5
ml of 0.2 M glycine buffer (pH 8.0) as a blocking agent was
*Trade-mark
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added, and the reaction was performed under shaking for
further 1 hour. The reaction mixture was treated through PD-
column (Pharmacia) equilibrated with physiological salt
solution to remove low-molecular luminescent substances.
Examole 3: Measurement of HBs antigen concentrations in whole
blood _ sample
From the HBs antibodies-coated beads containing suspension
prepared in Example 1, an aliquot containing 5 mg of the beads
was taken by a pipette to a reaction vessel. 100 l of a
whole blood sample with EDTA was added thereto and the
reaction was performed at 37 C for 3 minutes under shaking.
The reaction mixture was washed with a washing solution (10 mM
phosphate buffer, pH 7.0, containing 0.15 M NaCl and 0.1 %
Tween 20) four times. After adding thereto 100 l of a liquid
prepared by diluting the luminescent substance-labeled HBs
antibody prepared in Example 2 with the above washing solution
to 1/100 and 200 l of a dilution solution (PBS containing
0.05 % *Tween 20), the reaction was performed at 37 C for 5
minutes under shaking. Then, the reaction mixture was washed
with the washing solution four times. Emission was caused by
adding 100 l of 0.1 N-HC1 aqueous solution and then 300 .l of
0.1 M sodium hydroxide solution containing 20 mM hydrogen
peroxide. The chemiluminescent intensity was measured by a
photomultiplier.
As a control test, the above procedure was repeated except
that the serum sample prepared from the above whole blood
sample was used, and the chemiluminescent intensity was
obtained. The correlation between the serum sample and the
whole blood sample was shown in Fig. 1. The coefficient of
correlation for 287 samples was 0.9958, and the regression
line was y = 0.9863 x + 114 (x = serum; y = whole blood). The
chemiluminescent intensities of both cases were well
correlated.
Example 4: Chanaes in result of HBsantiaen concentration for
standing time of whole blood samnles
The whole blood samples were taken from the HBs-negative
and HBS-positive patients by a blood collecting tube with
adding EDTA (Terumo) and placed into test tubes in an amount
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of 5 ml. After allowing to stand for 15 minutes, 30 minutes,
or 60 minutes, the procedure described in Example 3 was
repeated, and a chemiluminescent intensity originating from
the HBs antigen. The whole blood samples were stirred only
shortly after collected, but allowed to stand at room
temperature. The results are shown in Table 1.
Table 1
chemiluminescent intensities (counts)
after blood collection (minutes)
0 min 15 min 30 min 60 min
later later later later
HBs-negative sample 2,185 2,159 2,226 2,148
HBs-positive sample 46,666 46,270 46,136 47,253
The precipitation of the hemocytes was visually observed
while time elapsed after the whole samples were placed into
the test tubes. However, substantial differences due to the
standing time were not observed. When the immunoassay
procedure was carried out, 100 l of aliquot was taken from
the sample in the test tube, at the position of 3 mm under the
liquid level thereof.
Example 5: Preparation of HBs antigen-coated beads
HBs antigen-positive human plasma was fractionated by
density gradient centrifugation (density of cesium chloride =
1.04 to 1.20; 48000 rpm; 210 minutes) to obtain virus
particles. The virus particles were solubilized with Tween 80
to obtain the HBs antigen. Electrophoresis of the solubilized
HBs antigen revealed that it mainly contained HBs antigen
proteins having molecular weights of 24,000 and 27,000. The
HBs antigen was diluted with 0.1 M glycine-NaOH buffer to 40
g/ml. To 1 ml of the dilution, 0.35 g of styrene beads
(diameter = 100 .m) was added, and the whole was stirred at 37
C for 16 hours to coat the beads with the antigens. The
coated beads were washed with PBS five times, and stored as 10
% suspension in PBS.
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Examgle 6: Preparation of acridinium ester-labeled HBs
antigens
The HBs antigen prepared in Example 5 was diluted with 0.1
M phosphate buffer (pH 8.0) to 0.25 mg/ml. To the antigen
liquid (1 ml), a solution (50 l) of 0.125 mg/ml 10-methyl-9-
{4-[2-(succinimidyloxycarbonyl)-ethyl]phenyloxycarbonyl}-
acridinium fluorosulfonate in dimethylformamide was added.
The reaction was performed under shaking at room temperature
for 30 minutes. Thereafter, 0.5 ml of 0.2 M glycine buffer
(pH 8.0) as a blocking agent was added, and the reaction was
performed under shaking for further 1 hour. The reaction
mixture was dialyzed to physiological salt solution three
times to prepare the luminescent substance-labeled HBs
antigen.
Examnle 7: Measurement of HBs antibody concentrations in whole
blood sample
From the HBs antigens-coated beads containing suspension
prepared in Example 5, an aliquot containing 5 mg of the beads
was taken by a pipette to a reaction vessel. 100 l of a
whole blood sample with EDTA was added thereto and the
reaction was performed at 37 C for 5 minutes under shaking.
Then, 50 l of a liquid prepared by diluting the luminescent
substance-labeled HBs antigen prepared in Example 6 with the
above washing solution to 1/100, and the reaction was
performed for 5 minutes under shaking. The reaction mixture
was washed with the washing solution four times. Emission was
caused by adding 100 .l of 0.1 N-HC1 aqueous solution and then
300 l of 0.1 N sodium hydroxide solution containing 20 mM
hydrogen peroxide. The chemiluminescent intensity was
measured by a photomultiplier.
As a control test, the above procedure was repeated except
that the serum sample prepared from the above whole blood
sample was used, and the chemiluminescent intensity was
obtained. The correlation between the serum sample and the
whole blood sample was shown in Fig. 2. The coefficient of
correlation for 286 samples was 0.9927, and the regression
line was y = 1.017 x + 207 (x = serum; y = whole blood). The
chemiluminescent intensities of both cases were well
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correlated.
Example 8: Preparation of anti-HBs antibody-coated maanetic
beads
Magnetic beads (tosylation-activated *Dynabeads; particle
size = 0.5 m; 2 ml) were collected by a magnet (MPC) to
remove the supernatant. The beads were washed by adding 4 ml
of 50 mM borate buffer (pH 9.5), and then collected by MPC.
The supernatant was removed with suction, and 1 ml of the
above buffer was added to the beads to obtain the suspension.
The anti-HBS-specific antibody F(ab')2 fraction prepared
in Example 1 was dialyzed to 50 mM borate buffer (pH 9.5). To
1 ml of the antibody liquid prepared by adjusting the
concentration to 0.5 mg/ml, the bead suspension was added
dropwise, and admixed with gently stirring in an incubator at
37 C for 24 hours. After the beads were collected by the
MPC, the supernatant was removed with suction. To the beads,
4 ml of bovine serum albumin (BSA) liquid prepared by
adjusting the concentration to 1 mg/ml with 20 mM phosphate
buffer (pH 7.0) containing 0.15 M NaCl was added, and the
whole was stirred at 4 C overnight to block the excess
functional groups. After washed with PBS four times as above,
the beads were stored in 2 ml of PBS containing 0.05 % sodium
azide and 1 mg/ml BSA.
Examiple 9: Relation between reaction time and HBs antiaen
concentrations
From the HBs antibodies-coated beads containing suspension
prepared in Example 1, an aliquot containing 5 mg of the beads
was taken by a pipette to a reaction vessel. 100 l of a
whole blood sample with EDTA (HBs antigen = 25 U/ml) was added
thereto and the reaction was performed at 37 C for 1 minute,
3 minutes, 5 minutes, 15 minutes, 30 minutes, 1 hour, 3 hours,
7 hours, 24 hours, or 48 hours under shaking. The reaction
mixture was washed with a washing solution (10 mM phosphate
buffer, pH 7.0, containing 0.15 M NaCl and 0.1 % Tween 20)
four times. After adding thereto 100 l of a liquid prepared
by diluting the luminescent substance-labeled HBs antibody
prepared in Example 2 with the above washing solution to 1/100
and 200 l of a dilution solution (PBS containing 0.05 % Tween
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20), the reaction was performed at 37 C for 5 minutes under
shaking. Then, the reaction mixture was washed with the
washing solution four times. Emission was caused by adding
100 l of 0.1 N HC1 aqueous solution and then 300 l of 0.1 M
sodium hydroxide solution containing 20 mM hydrogen peroxide.
The chemiluminescent intensity was measured by a
photomultiplier.
As a control test, the above procedure was repeated except
that the serum sample prepared from the above whole blood.
The results are shown in Table 2. It is apparent from Table 2
that the measurements (chemiluminescent intensities) are
almost the same within 1 to 30 minutes of the contacting time
of the sample and the antibody-beads, between the EDTA-added
whole blood sample and the serum sample separated therefrom.
Table 2
Time Whole blood Serum
1 minute 6114 5850
3 minutes 16026 16164
minutes 25060 24940
minutes 52368 53176
30 minutes 79953 87234
1 hour 92035 121869
3 hours 108694 145286
7 hours 113945 169346
24 hours 116523 172345
48 hours 118692 176692
Examnle 10: Relation between IHBs antigen concentrations and
hematocrit values
A blood sample was taken from an HBs-positive patient by a
blood collecting tube with adding EDTA (Terumo) and the
hematocrit value thereof was adjusted to 70 %. Then, the
blood samples having hematocrit values of 0 %, 10%, 20 %, 30
%, 40 %, 50 %, 60 %, and 70 % were prepared by diluting the
whole blood sample with the plasma taken from the same
patient. Then, the procedure described in Example 3 was
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repeated, and a chemiluminescent intensity originating from
the HBs antigen was measured. The results are shown in Table
3. In this Example, blood samples having hematocrit values of
more than 70 % were not used, because such samples were
difficult to be collected.
Exam8le 11: Chanaes of HBs antiaen concentrations by diluting
samples
From the HBs antibodies-coated beads containing suspension
prepared in Example 1, an aliquot containing 5 mg of the beads
was taken by a pipette to a reaction vessel. Further, 100 .l
of each of the whole blood samples prepared by diluting to
have the above hematocrit values as in Example 10, and 100 .l
of the washing solution used in Example 3 were added thereto.
Then, the procedure described in Example 3 was repeated, and a
chemiluminescent intensity originating from the HBs antigen
was measured. The results are shown in Table 3.
Example 12: Changes of HBs antigen concentrations without
primary washing (1)
From the HBs antibodies-coated beads containing suspension
prepared in Example 1, an aliquot containing 5 mg of the beads
was taken by a pipette to a reaction vessel. Further, 100 .l
of each of the whole blood samples prepared by diluting to
have the above hematocrit values as in Example 10 was added
thereto, and the reaction was performed at 37 C for 5 minutes
under shaking. After adding to the reaction mixture 100 l of
a liquid prepared by diluting the luminescent substance-
labeled HBs antibody prepared in Example 2 with the washing
solution used in Example 3 to 1/100 and 100 l of a dilution
solution (PBS containing 0.05 % Tween 20), the reaction was
performed at 37 C for 5 minutes under shaking. Then, the
procedure described in Example 3 was repeated, and a
chemiluminescent intensity originating from the HBs antigen
was measured. The results are shown in Table 3.
Example 13: Changes of HBs antigen concentrations without
primary washing (2)
From the HBs antibodies-coated beads containing suspension
prepared in Example 1, an aliquot containing 5 mg of the beads
was taken by a pipette to a reaction vessel. Further, 100 l
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of each of the whole blood samples prepared by diluting to
have the above hematocrit values as in Example 12 was added
thereto, and the reaction was performed at 37 C for 5 minutes
under shaking. After adding to the reaction mixture 10 l of
a liquid prepared by diluting the luminescent substance-
labeled HBs antibody prepared in Example 2 with the washing
solution used in Example 3 to 1/27.5, the reaction was
performed at 37 C for 5 minutes under shaking. Then, the
procedure described in Example 3 was repeated, and a
chemiluminescent intensity originating from the HBs antigen
was measured. The results are shown in Table 3.
Table 3
Hematocrit
value Examole 10 Example 11 Examnle 12 Examnle 13
0% 17764 17566 16486 18182
10% 17506 16376 16284 18286
20% 17682 15250 15752 17978
80% 17340 14122 15426 18000
40% 17982 12874 14876 17620
50% 17860 11700 14720 17782
60% 17886 10258 14450 17894
70% 17368 7974 13118 17460
(Figures are emission counts)
From the results of Examples 10 to 13, it was confirmed
that hematocrit value does not produce a difference of or does
not affect the emission, if other liquid components are absent
upon contacting the whole blood sample containing the HBs
antigens and the beads coated with HBs antibodies (Example
10); and that the changes in the measurements due to the
hematocrit values are very small when the HBs antibodies
labeled with acridinium esters were added in a 1/10 th amount
of the sample, after contacting the whole blood sample
containing the HBs antigens and the beads coated with HBs
antibodies, but not carrying out the washing procedure
(Example 13). The reason is assumed that the formation of the
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23 -
complex of the HBs antigen in the whole blood sample and the
HBs antibodies-coated bead proceeds before the HBs antibodies
labeled with acridinium esters were added, and the influence
by the addition is very small in comparison with that to the
measurements by the change of the sample calculated from
hematocrit value.
Example 14: Measurement of HBs antigen concentrations in whole
blood sam8le using magnetic beads
The suspension (50 l) of the HBs antibodies-coated
magnetic beads prepared in Example 8 was placed in a reaction
vessel, and collected by the MPC to remove liquid components
with suction. After adding 100 l of the whole blood sample
with added EDTA, the whole was reacted at 37 C for 3 minutes
under shaking. The reaction mixture was washed with the
washing solution described in Example 3 four times. After
adding thereto 100 l of a liquid prepared by diluting the
luminescent substance-labeled HBs antibody prepared in Example
2 with the above washing solution to 1/100 and 200 l of the
dilution solution described in Example 3, the reaction was
performed at 37 C for 5 minutes under shaking. Then, the
reaction mixture was washed with the washing solution four
times. Emission was caused by adding 100 l of 0.1 N HC1
aqueous solution and then 300 .l of 0.1 M sodium hydroxide
solution containing 20 mM hydrogen peroxide. The
chemiluminescent intensity was measured by a photomultiplier.
As a control test, the above procedure was repeated except
that the serum sample prepared from the above whole blood, and
the chemiluminescent intensity was measured. The coefficient
of correlation for 50 samples was 0.9892, and the regression
line was y = 0.9739 x + 189 (x = serum; y = whole blood). The
chemiluminescent intensities of both cases were well
correlated.
INDUSTRIAL APPLICABILITY
According to the present invention, the whole blood can be
used as a sample without pretreatment, and the analyte in the
whole blood sample can be quickly measured. Therefore, the
present invention enables a rapid examination without the
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pretreatment of the collected blood which is required in
conventional methods, and achieves an extreme labor-saving in
a general examination.
As above, the present invention was explained with
reference to particular embodiments, but modifications and
improvements obvious to those skilled in the art are included
in the scope of the present invention.
