NZ702764B2 - Method for detecting specific substance in milk - Google Patents
Method for detecting specific substance in milk Download PDFInfo
- Publication number
- NZ702764B2 NZ702764B2 NZ702764A NZ70276412A NZ702764B2 NZ 702764 B2 NZ702764 B2 NZ 702764B2 NZ 702764 A NZ702764 A NZ 702764A NZ 70276412 A NZ70276412 A NZ 70276412A NZ 702764 B2 NZ702764 B2 NZ 702764B2
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- NZ
- New Zealand
- Prior art keywords
- milk
- specific substance
- antibody
- immunochromatographic
- substance
- Prior art date
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Abstract
immunochromatographic method for detecting specific substances in milk, wherein the method comprises: (1) a step of making the milk contact a test piece, having a first portion in which a first antibody tagged for the specific substance or the labeled specific substance is stored, a second portion that is connected downstream of the first portion and to which a second antibody for the specific substance is fixed, and a third portion that is connected upstream of the first portion or the second portion and that has an aperture capable of removing milk fat globules in the milk, said milk contacting the test piece at the third portion or further upstream of the same; and (2) a step of having the milk flow to the second portion or further downstream of the same and providing a detectable signal by the tag at the second portion or further downstream of the same. n that is connected downstream of the first portion and to which a second antibody for the specific substance is fixed, and a third portion that is connected upstream of the first portion or the second portion and that has an aperture capable of removing milk fat globules in the milk, said milk contacting the test piece at the third portion or further upstream of the same; and (2) a step of having the milk flow to the second portion or further downstream of the same and providing a detectable signal by the tag at the second portion or further downstream of the same.
Description
Specification
Title of the Invention: Method for detecting specific nce in milk
Technical Field
The present invention relates to an immunochromatographic method and
immunochromatographic device for detecting a substance in milk by using an antigenantibody
reaction.
Background Art
Milk of livestock animals, of which typical examples are cow, sheep, and goat,
is not sterile, and may be inated with certain microorganisms due to diseases or
environment. In particular, it is known that animals with a disease caused by infection
of a microorganism often discharge a lot of the rganisms into milk. Typical
diseases of livestock animals caused by infection of a microorganism include mastitis.
Mastitis is inflammation of the fer system or milk gland tissue, and it is
caused largely by invasion, ecesis, and proliferation of a microorganism in the udder.
Although many kinds of animals contract mastitis, it is said that, especially concerning
cow's mastitis in dairy cows, 15 to 40% of the whole dairy cows contract mastitis, and
thus it is one of the extremely important diseases for dairy s. If a dairy cow
contracts mastitis, not only the milk synthesis function is ted to result in reduction
of lactation amount, or even stop of lactation as the case may be, but also us
economical losses such as cost of medical treatment and penalty concerning milk price
are imposed on dairy farmers. Furthermore, it also increases the labor of dairy farmers,
since, for example, g of teats suffering from mastitis must be separately
performed for preventing ion.
Mastitis is caused by infection of various microorganisms, but antibiotics that
ts efficacy against mastitis may differ depending on type of causative
microorganism, and certain types of microorganisms have different characteristics
concerning, for example, transmission to other teats or individuals, or post—infectious
ng thereof differs. Therefore, it is extremely important to quickly and
iently identify the causative microorganism existing in milk.
As methods for identifying a causative microorganism of an infectious disease,
there are known ation-based identification method, ased identification
method, and antigen-antibody reaction—based identification method. Although the
current mainstream of the method for identifying a ive microorganism of is
of livestock animals is the cultivation-based identification method, the ion thereof
is cated, and in on, it has a problem of requiring several days for obtaining
results. There has also been reported an identification method based on detection of a
specific gene by a gene amplification method (PCR method) atent document 1).
Although results can be obtained in about one day by this method, it still has a problem
of requiring special instruments and maneuvers. In recent years, there has been
developed an apparatus for detecting Staphylococcus aureus, which is one of the
causative microorganisms of mastitis, or Escherichia coli by e plasmon resonance
(SPR) on the basis of an antigen-antibody reaction atent document 2). For this
apparatus, an antibody against an antigen specific to each bacterium is prepared, and
this antibody is fixed on an SPR chip and used. Although this method enables quick
detection of a specific causative microorganism, it requires a special apparatus, and
therefore it is difficult to perform measurement by this method on practical dairy spots
etc.
Simple measurement devices which enable quick and convenient measurement
in the home or clinic for biosamples such as blood and urine utilizing
immunochromatography (immunochromatographic device) have widely been used (for
example, refer to Patent document 1). The method performed in such devices uses a
test strip sing test paper containing a first antibody specific to a target substance
of measurement (antigen) and labeled with coloring particles such as gold colloid, and a
porous membrane on which a second antibody for capturing the target nce of
measurement is immobilized, which test paper and porous membrane are linked
together. If a test sample containing a target substance of measurement is dropped
onto the strip, the target substance of measurement binds with the antibody labeled with
coloring particles and/or the antibody immobilized on the porous membrane, resulting a
visually distinguishable line or the like on the membrane. Therefore, by confirming
presence or absence of such a line or the like, presence or absence of the substance to be
ed can be ed.
Although Patent document 1 teaches that the aforementioned method can be
applied to, besides blood (whole blood), plasma, serum, urine, saliva, sputum, sweat,
and so forth, it does not teach application of the entioned method to milk of
livestock animals, and does not teach nor suggest the problems encountered at the time
of such application at all. r, although s for applying an
immunochromatographic method to biosamples such as blood (whole blood) as a test
sample are also sed in Patent documents 2 to 4, these patent documents do not
teach nor suggest application of immunochromatographic method to milk of livestock
animals, either.
For a simple measurement device that enables quick and convenient
measurement for milk of livestock animals as a test sample by using an
chromatographic method at practical dairy spots, Patent document 5 proposes a
method of using the immunochromatographic method for milk as a test sample for the
purpose of inspecting causative microorganisms of mastitis of livestock animals. This
patent document teaches that milk fat globules and casein contained in milk inhibit
detection by the immunochromatographic , and it is preferred that they are
removed in advance before the test. Although this patent document discloses a method
of removing milk fat globules and casein by leaving milk standing, separating a cream
layer, and performing a treatment with a surfactant, it does not teach l of milk fat
globules with a filter.
Further, although Patent documents 2 to 4 teach techniques of removing
contaminants with a physical filter or a membrane showing chemical affinity for use in
applying immunochromatographic method, the s described in these nces
are methods of applying the immunochromatographic method to biosamples such as
whole blood as described above, and they are not methods of applying it to milk of
livestock animals. Patent documents 1 to 4 do not teach nor suggest the problems
encountered at the time of applying the immunochromatographic method to milk of livestock
animals, and do not teach any means for solving the problems at all.
Prior art references
Patent nts
Patent document 1: Japanese Patent Unexamined Publication (KOKAI) No. 1-244370 (EP
03223605)
Patent nt 2: Japanese Patent Unexamined Publication (KOHYO) No. 2003-512624 (WO
2001/29558)
Patent document 3: Japanese Patent Unexamined Publication (KOHYO) No. 11-505327
(US 6197598)
Patent document 4: Japanese Patent Unexamined Publication (KOKAI) No. 2002-214236
Patent document 5: International Patent ation WO02/075310
Non-patent documents
Non-patent document 1: J. Dairy. Sci., 84:74-83
Non-patent document 2: JRA Advanced Livestock Management System Utilization Report (Heisei
18 to 20 fiscal years), pp.58-65
Summary of the Invention
Object to be Achieved by the Invention
An object to be achieved by the present invention is to provide an
immunochromatographic method and immunochromatographic device for ing a substance in
milk of a ock animal using an antigen-antibody reaction.
Means for Achieving the Object
The inventor of the present invention examined identification of causative
microorganisms of is on the basis of an antigen-antibody reaction using an
immunochromatographic device. However, when the or of the t invention examined
whether a microorganism could actually be detected by the immunochromatographic method using
milk as a sample, it was found that the antigen-
antibody on did not advance on a test strip of the immunochromatographic device.
The inventor of the present ion investigated the cause of the no smooth advance
of antigen—antibody reaction on the test strip, and as a result, considered that the cause
of the above phenomenon might be clogging of the test strip linked with a porous
membrane in the immunochromatographic device with a lot of milk fat globules
contained in milk, resulting in insufficient flow of a developing solution.
Immunochromatographic devices usually use a test strip having a pore diameter of
several tens to several hundreds nm for transportation by the developing solution, and in
order to obtain appropriate rate of the antigen—antibody reaction. Milk immediately
after g contains a lot of milk fat globules having a diameter of around 1 to ten and
several micrometers (although the particle diameter of milk fat globules in marketed
milk or the like is made to be 1 pm or r by a homogenization ent of the
milk fat globules in fresh milk, milk not subjected to the homogenization treatment
contains milk fat es having a wide range of le sizes), and it was considered
that the particle size distribution of the milk fat globules was extremely larger than that
of erythrocytes, and therefore they causes clogging of the membrane to make the
measurement difficult.
The inventor of the present invention conducted various researches in order to
achieve the aforementioned object, as a result, found that ement of a specific
substance contained in milk by the immunochromatographic method was enabled by
trapping a part of milk fat globules contained in the milk by a sizing treatment on the
upstream side of the test strip used in the immunochromatographic method, and
accomplished the present invention.
Thus, the present invention is as follows.
[001 5]
An immunochromatographic method for detecting a specific substance in milk,
which comprises:
(1) the step of contacting the milk with a test strip having a first part ing a labeled
first antibody directed to the specific substance or retaining the c nce that is
labeled, a second part disposed downstream from the first part, on which a second
antibody directed to the c substance is immobilized, and a third part disposed
upstream from the first part or the second part and having voids enabling removal of
milk fat globules contained in the milk, at the third part or a further am part, and
(2) the step of flowing the milk up to the second part or a further downstream part to
obtain a detectable signal of the label at the second part or a r downstream part.
[001 6]
The method according to [1], wherein the immunochromatographic method is a
lateral flow type method.
The method according to [l] or [2], wherein a labeled first antibody directed to the
specific substance is retained at the first part.
The method ing to any one of [l] to [3], wherein the specific substance is a
component of a ium, or a substance secreted by a bacterium.
The method according to any one of [l] to [4], wherein at least one or both of the
first antibody and the second antibody are monoclonal antibodies.
[001 7]
The method according to [5], wherein the first antibody and the second antibody are
monoclonal antibodies.
[001 8]
The method according to any one of [l] to [6], wherein retention particle size of the
voids of the third part is 1 to 3.5 pm.
The method according to any one of [l] to [7], wherein the third part is constituted
by two or more kinds of members having voids that can remove milk fat globules of
different particle sizes.
The method according to [8], wherein the third part is constituted by a first member
disposed downstream and a second member disposed upstream, and retention particle
size of the second member is larger than retention le size of the first member.
The method according to [9], wherein the ion particle size of the first member
is 1.0 to 2.0 pm, and the retention particle size of the second member is 3.0 to 3.5 pm.
[001 9]
[l l] The method according to any one of [l] to [10], which comprises the step of
flowing milk subjected to a treatment with a lytic enzyme up to the second part or a
further downstream part.
The method according to [l 1], wherein the lytic enzyme is an autolysin.
The method ing to [l 1], wherein a staphylococcus contained in the milk is
detected by using lysostaphin as the lytic enzyme.
A method for diagnosing whether a causative microorganism of mastitis of a
livestock animal is a staphylococcus or not, which comprises:
the step of obtaining milk containing the causative rganism from the livestock
animal ing from mastitis, the step of mixing a lytic enzyme in the milk so that a
c substance existing in cells of the causative microorganism is released out of the
cells, and the step of determining presence or absence of the specific nce or
measuring existing amount of the specific substance by an logical method
using an antibody directed to the specific substance as an antigen.
The method according to [14], wherein whether the causative microorganism is a
staphylococcus or not is diagnosed by using lysostaphin as the lytic enzyme.
An immunochromatographic device for detecting a specific substance contained in
milk, which comprises a test strip having a first part retaining a labeled first antibody
directed to the specific substance or retaining the specific substance that is labeled, a
second part disposed downstream from the first part, on which a second antibody
directed to the specific substance is lized, and a third part disposed upstream
from the first part or the second part and having voids enabling removal of milk fat
globules contained in the milk.
The immunochromatographic device according to [16], wherein the first part or the
second part retains a lytic enzyme or a surfactant.
A ion kit consisting of an additive solution containing a lytic enzyme or a
surfactant, and the immunochromatographic device according to [16].
Effect of the Invention
According to the present invention, presence or absence of a specific substance
in milk can be quickly and conveniently detected on site. In particular, when it is
desired to diagnose cow's mastitis, if a visually recognizable label is used, the detection
can be carried out in dairy farms without using any apparatus etc., and a treatment for
removing the cream layer is also unnecessary. ore, the causative microorganism
can be quickly identified before further ation of the condition of the disease, and
right ent policies such as selection of riate otics, and measure for
preventing expansion of the infection can be determined at an early stage.
Brief Description of the Drawings
[002 1 ]
[Fig. 1] Fig. 1 shows a schematic sectional view of the test strip of the
immunochromatographic device used in Example 1, which comprises a labeled
antibody—impregnated member 1 (first part), a membrane carrier 2 for chromatographic
development (second part), a part 3 for capturing, a member 4 for sample addition, a
member 5 for absorption, a substrate 6, and a member 7 for removal of fat globules
(third part).
[Fig. 2] Fig. 2 shows a schematic sectional view of another example of the test strip of
the immunochromatographic device.
[Fig. 3] Fig. 3 shows a schematic sectional view of r r example of the test
strip of the immunochromatographic device.
[Fig. 4] Fig. 4 shows detection ivity-improving effect of use of an autolysin.
[Fig. 5] Fig. 5 shows the results of the ement of Staphylococcus aureus added to
cow's milk based on ELISA using achromopeptidase and lysostaphin.
[Fig. 6] Fig. 6 shows a schematic sectional view of the test strip of the
immunochromatographic device used in Example 6, which comprises a labeled
antibody-impregnated member 1 (first part), a membrane carrier 2 for chromatographic
development (second part), a part 3 for capturing, a member 4 for sample addition, a
member 6 for absorption, and a substrate 6.
[Fig. 7] Fig. 7 shows the results of detection of Staphylococcus aureus contained in milk
by the immunochromatographic method using lysostaphin as a lytic enzyme.
Modes for Carrying out the ion
Hereafter, the t invention will be explained in more detail.
The immunochromatographic device of the present invention is a device for
detecting a specific nce contained in milk by the immunochromatographic
method, which comprises a test strip having a first part retaining a labeled first antibody
ed to the specific substance or the specific substance that is labeled, a second part
disposed downstream from the first part, on which a second antibody directed to the
c substance is immobilized, and a third part disposed upstream from the first part
or the second part and having voids enabling removal of milk fat globules in the milk.
Specific es of the structure of the test strip include those of the test strips of
which tic sectional views are shown in Figs. 1, 2, and 3. In Fig. 1, the member
4 for sample addition and the member 7 for removal of fat globules (third part) are
integrally disposed upstream from the labeled dy-impregnated member (first part)
1. In Fig. 2, the member 7 for removal of fat globules (third part) is disposed
downstream from the labeled antibody-impregnated member (first part) 1, and upstream
from the membrane carrier 2 for chromatographic development (second part). In Fig.
3, the member 7 for removal of fat globules (third part) is disposed downstream from
the member 4 for sample addition, and upstream from the d antibody—impregnated
member (first part) 1.
The immunochromatographic device can be produced in a known manner by
using marketed materials.
The material used for the first part is not particularly d, so long as a
material ng immunochromatography is chosen, but preferred examples e a
fiber matrix of cellulose derivative etc., filter paper, glass fiber, cloth, cotton, and so
forth.
The material used for the second part is not particularly limited, so long as a
material enabling immunochromatography is chosen, but preferred examples include
cellulose nitrate, mixed cellulose nitrate ester, nylidene fluoride, nylon, and so
forth.
The material used for the third part preferably has voids that enable removal of
milk fat globules contained in milk and having a diameter of about 1 to ten and several
micrometers. The third part must be disposed upstream from the aforementioned
second part consisting of a porous membrane having a pore diameter of several tens to
several hundreds nm, and preferably disposed upstream from the aforementioned first
part, i.e., at a on at which a sample solution first contacts with and passes through
the test strip.
The voids of the third part may have a size that enables removal of milk fat
globules, and ion particle size is preferably 0.1 to 10 um, more preferably 1 to 3.5
um. The material is not particularly limited, so long as a material having voids
showing a retention particle size within the aforementioned range is chosen, but
preferred es include a matrix of fibers such as cellulose derivatives, filter paper,
glass fiber, cloth, cotton, and so forth. The ion particle size means such a particle
size of milk fat globules that milk fat globules having a particle size not smaller than the
retention particle size cannot pass through the voids and retained by third part, and
substantially corresponds to average pore size of the voids of the third part, and 50% or
more, preferably 60% or more, more preferably 70% or more, still more preferably 80%
or more, particularly preferably 90% or more, most preferably 98% or more, of milk fat
globules having a particle size not r than the ion particle size cannot pass
through the voids and retained by the third part. Ratio of milk fat globules to be
ed can be measured by a method well known to those d in the art. For
example, it is described that the retention particle size of GF/B provided by GE
Healthcare Bioscience is 1.0 urn in the catalogue thereof (particle retention), and such a
particle size as mentioned above can be confirmed by a method well known to those
skilled in the art.
The aforementioned third part may consist of a single kind of material having a
specific retention particle size, or may consist of a laminate comprising materials having
different retention particle sizes and integrally adhered so that the retention le size
becomes smaller stepwise, in order to increase milk fat globule separation efficiency.
Such a third part as mentioned above constituted by two or more kinds of members that
can remove milk fat globules of different particle sizes constitutes a preferred
ment of the present ion, and in a more preferred ment of the
present invention, the third part is constituted with a first member disposed downstream
and a second member disposed upstream, and the retention particle size of the second
member is larger than the retention particle size of the first member. When the third
part is constituted with such two kinds of members, it is preferred that the retention
particle size of the first member disposed downstream is 1.0 to 2.0 um, and the retention
particle size of the second member disposed am is 3.0 to 3.5 um. In order to
detect a specific substance from milk containing milk fat globules of high concentration
and wide particle size distribution, preferably such milk undiluted after milking, with
high sensitivity, it is preferred that the third part is constituted with a combination of a
member having a small retention particle size and a member having a large retention
particle size.
The aforementioned first part retains a labeled first antibody directed to a
specific substance, or a labeled specific substance. If the first part retains a labeled
first antibody directed to a c substance, the specific substance can be detected by
the sandwich assay method. Further, if the first part retains a labeled specific
nce, the specific substance can be detected by the competition method. Since
the sandwich assay method shows high detection sensitivity and gives a line indicating
detection of dy as a positive result, it is more preferred for the present invention,
and therefore the first part preferably retains a labeled first dy directed to a
specific substance.
When the first part is made to retain a labeled first antibody directed to a
specific substance, two kinds of antibodies, the first dy directed to the c
substance, and a second dy directed to the specific nce. In order to enable
detection of the specific substance by the sandwich assay method, the entioned
first antibody and second antibody are antibodies that can simultaneously bind to the
specific substance, and it is preferred that the epitope of the specific substance to be
recognized by the aforementioned first antibody is different from the e of the
specific substance to be recognized by the aforementioned second antibody.
[003 1]
In the present invention, in order to obtain a detectable signal, the first antibody
or the specific substance retained by the first part is labeled. Examples of the label
used for the present invention include a coloring particle, enzyme, radioisotope, and so
forth, and it is preferable to use a coloring particle that can be visually detected without
any l equipment. Examples of the coloring particle include metal microparticles
such as those of gold and platinum, nonmetallic particles, latex les, and so forth,
but it is not limited to these. The coloring particle may have any size so long as the
ng particle have such a size that it can be transported downstream through the
inside of the voids of the test strip, but it preferably has a size of 1 nm to 10 um, more
preferably 5 nm to 1 pm, still more preferably 10 to 100 nm, in diameter.
[003 2]
The specific substance measured by the present invention may be any
substance so long as it is a substance that can be measured by the
immunochromatographic method, but it is preferably a ent of a ium or a
substance that is secreted by a bacterium. The specific substance is more preferably
the L7/L12 ribosomal protein of a bacterium. High detection sensitivity can be
obtained for the L7/Ll2 mal protein, since it exists in cells in a large copy number.
Further, as shown in the examples mentioned later, an antibody with which a specific
bacterium as a cause of mastitis can be distinguished from other bacteria at a species or
genus level can be actually obtained by a known . Type of the ium is not
particularly d, and it may be a gram—positive bacterium, or a gram—negative
ium. Examples include, for example, gram-positive bacteria such as
staphylococci (bacteria belonging to the genus Staphylococcus), preferably
Staphylococcus aureus, and so forth, Escherichia coli, bacteria belonging to the genus
Klebsiella, and so forth, but it is not limited to these.
The aforementioned antibody can be prepared by the method described in
International Patent Publication WOOD/06603. When the bacterial ribosomal protein
L7/L12 is the n, the antibody can be ed by using a full length protein or a
partial peptide of the bacterial ribosomal protein L7/L12 as an antigen, but it is
preferably prepared by using a full length n as an antigen. An antiserum
containing an antibody (polyclonal antibody) that recognizes the L7/L12 ribosomal
n can be obtained by inoculating such a partial peptide or full length protein as
mentioned above as it is or crosslinked with a carrier protein to an animal, together with
an adjuvant as ed, and collecting the serum of the animal. Further, the antibody
can also be purified from the antiserum, and used. Examples of the animal used for
the inoculation include sheep, horse, goat, , mouse, rat, and so forth, and sheep,
rabbit, and so forth are especially preferred for preparing polyclonal antibodies.
Further, it is more preferable to use, as the antibody, a monoclonal antibody obtained by
a known method in which a hybridoma cell is prepared, and in such a case, mouse is
preferred as the animal. As such a monoclonal antibody, if a monoclonal antibody that
reacts with the ribosomal protein L7/L12 of a specific bacterium that causes mastitis,
but does not react with the ribosomal protein L7/L12 of a ium that causes mastitis
other than the above specific bacterium is retrieved by screening, it can be utilized for
diagnosing whether an animal suffers from infection by the bacterium or not.
An antibody that recognizes a substance other than the mal protein
L7/L12 as an antigen may also be used, so long as the antibody is a monoclonal
antibody that reacts with a component of a specific bacterium that causes mastitis or a
substance secreted by such a bacterium, but does not react with a ent of a
ium that causes mastitis other than the foregoing bacterium or a substance
secreted by such a bacterium.
r, as the monoclonal antibody, it is preferable to use a monoclonal
antibody of which antigen-antibody reaction is not inhibited with any contaminants
other than the specific substance contained in milk. For example, milk contains a large
amount of proteins such as casein, and they may inhibit the reaction of the specific
nce and the monoclonal antibody. As the monoclonal antibody directed to the
specific substance prepared in a conventional manner, for example, a monoclonal
antibody of which antigen-antibody reaction is not ted by casein or the like, or a
monoclonal antibody of which antigen-antibody on is hardly ed by casein or
the like may be preferably chosen and used. Such a monoclonal antibody can be easily
prepared by preparing monoclonal antibodies that specifically react with an antigen in a
usual manner, and then selecting a monoclonal dy of which n-antibody
reaction is not substantially ted by a contaminant such as casein by examining
whether the antigen—antibody reaction is inhibited or not in the presence of the
inant.
In the present invention, the test strip described above may be used as it is as an
immunochromatographic device, or the test strip may be stored in a case to constitute an
immunochromatographic device. In the former case, if a large volume of milk is used
as a sample, the immunochromatographic device is preferably used by directly
immersing one end of the test strip into the sample contained in a container. In the
latter case, if the volume of milk as a sample is small, the immunochromatographic
device is preferably used by measuring a predetermined volume of the sample with a
pipette or the like, and dropping the sample to the test strip. In the latter case, the case
may have any shape so long as the test strip can be stored. The case may be formed
with any material, and preferred examples include polypropylene, polycarbonate, and so
forth.
[003 7]
The chromatographic device of the present invention can also be
provided as a kit comprising a container such as microtube, and an additive on, for
example, an additive solution containing a lytic enzyme or surfactant for lysing the
bacterium to elute the ribosomal n L7/Ll2 into the solution.
The immunochromatographic method of the present invention is an
immunochromatographic method for detecting a specific substance contained in milk,
which comprises:
(1) the step of contacting the milk with a test strip having a first part ing a labeled
first dy directed to the specific substance or retaining the specific substance that is
labeled, a second part disposed downstream from the first part, on which a second
antibody directed to the specific substance is immobilized, and a third part disposed
am from the first part and the second part and having voids enabling removal of
milk fat es contained in the milk, at the third part or a further upstream part, and
(2) the step of flowing the milk up to the second part or a further ream part to
obtain a detectable signal of the label at the second part or a further downstream part.
[003 9]
Milk is contacted with the aforementioned third part or a member for sample
addition locating at a further upstream position, as it is, or as a mixed solution
containing the additive solution. When the aforementioned c substance is a
substance existing in cells of a bacterium, the bacterium can be lysed by directly adding
a surfactant ingredient to the milk, or the aforementioned additive solution, or making
the surfactant ingredient to be retained in a part locating upstream from the
entioned second part, preferably a part locating upstream from the
aforementioned first part (as for the cell lysis treatment with a surfactant, refer to
Japanese Patent Unexamined Publication ) No. 61—111464). These techniques
may be appropriately ed. As another component of the additive solution, an
appropriate buffer (for example, MOPSO etc.) can be used, although the component is
not particularly limited so long as a component that does not inhibit the antigen-
antibody reaction is chosen.
As another embodiment, by directly adding a lytic enzyme to milk, or adding it
to the ve solution, or immobilizing a lytic enzyme upstream from the
aforementioned second part, preferably upstream from the aforementioned first part,
milk subjected to the treatment with the lytic , in which the ic substance
contained in the cells is exposed out of the cells, can be flown up to the second part or a
further downstream part, and thus the c substance existing in the cells of the
bacterium can be detected at high sensitivity. These techniques may be appropriately
combined. In a preferred embodiment, the lytic enzyme can be directly added to milk,
or it can be contained in the aforementioned additive solution, and in a particularly
preferred embodiment, the aforementioned additive solution containing a lytic enzyme
can be added to milk.
Type of the lytic enzyme is not particularly limited, and two or more kinds of
arbitrary lytic enzymes may also be used in combination. Since infections of
ichia coli, bacteria belonging to the genus ella, and Staphylococcus aureus
as etiologic bacteria of mastitis ntly occur, it is also preferable to use one or more
kinds of lytic enzymes that can t a bacteriolytic action against these
microorganisms in combination. For example, one or two or more kinds of lytic
enzymes selected from lysozyme, lysostaphin, pepsin, glucosidase, galactosidase,
achromopeptidase, B-N—acetylglucosaminidase, and so forth can be used. For e,
there has been proposed a method of using lysozyme and a cell membrane lytic agent as
the lytic enzyme (Japanese Patent ined Publication (KOKAI) No. 63—167799).
As the lytic , many of autolysins exhibit high lytic effect, and such
autolysins are preferred (refer to Fig. 4). Autolysin refers to an enzyme that is
produced by a bacterium itself, and lyses the bacterium itself. Although details of the
reason why such autolysis occurs remain unknown, it is considered that, when a
bacterium extends and divides, it plays a role of lysing and cleaving the bacterial cell.
As bacterial autolysins, there are known lysostaphin, acetylglucosaminidase,
achromopeptidase, and so forth.
When the ce of Staphylococcus aureus is especially suspected as an
etiologic bacterium of mastitis, it may be preferable to use lysostaphin that specifically
exhibits a bacteriolytic action against Staphylococcus aureus independently or in
combination with another lytic enzyme, since lysis rate for Staphylococcus aureus
exhibited by tants is low. Japanese Patent Unexamined Publication (KOKAI)
No. 11-28099 discloses a method of lysing Staphylococcus aureus with lysostaphin, and
those skilled in the art can easily obtain lysostaphin as a lytic enzyme. The entire
disclosures of the aforementioned patent nt are incorporated into the disclosures
of the present specification by reference.
The naturally occurring type lysostaphin is a zinc protease produced by
Staphylocuccus simulans, and it is known that it hydrolyzes glycylglycine bonds in the
glycopeptide chains of the cell wall peptidoglycans of Staphylococcus aureus or
congeneric bacterium thereof to lyse the bacterium. In this cation, the term
lysostaphin also means, besides the naturally occurring type lysostaphin, a mutant
lysostaphin having the amino acid sequence of the naturally ing type lysostaphin,
but ing a mutation such as addition, deletion, and/or substitution of one or more
amino acid residues in such a degree that the aforementioned hydrolysis activity is not
lost. Further, the term lysostaphin also means a modified type lysostaphin consisting
of the naturally occurring type lysostaphin or a mutant lysostaphin bound with another
compound, for example, saccharide, polyethylene glycol, or the like. These
lysostaphins can be obtained by the cultivation—based method or genetic ering
technique disclosed in Japanese Patent ined Publication (KOKAI) No. 11-
28099, or by purchasing a commercial product. When a lysostaphin is used as the lytic
enzyme, for example, an antigen consisting of the ribosomal L7/L12 protein of
Staphylococcus aureus can be detected by the chromatographic method, or such
an antigen as mentioned above can also be detected with a ation of an
immunochromatographic method and another immunological assay method, or another
immunological assay method d of the immunochromatographic method.
es of the immunological assay method other than the immunochromatographic
method include, for example, the ination reaction method, enzyme—linked
immunosorbent assay ), radioimmunoassay (RIA), fluorescent immunoassay
(FIA), and so forth, but it is not limited to these.
The antigen—antibody reaction can be detected by the sandwich assay method
using the "labeled first antibody directed to the specific substance" retained by the first
part, and the "second dy ed to the specific nce" immobilized by the
second part. Alternatively, the antigen-antibody reaction may be detected by the
competitive assay method using the labeled specific substance retained by first part and
the antibody directed to the specific substance immobilized on the second part.
However, in the t invention, the sandwich assay method that provides high
detection sensitivity and gives a line indicating detection of antibody as a ve result
is preferred.
The immunochromatographic methods are roughly classified into those of the
lateral flow type using transfer of a liquid by arity in a test strip disposed in the
transverse direction, and those of the flow through type in which a liquid is passed
through a vertically disposed test strip from the top to the bottom thereof mainly by
gravity. Although both the lateral flow type and flow through type methods may be
used in the present invention, the lateral flow type methods are more preferred.
The present invention will be further explained with reference to the following
examples. However, the present invention is not limited by these examples.
Examples
Example 1: Confirmation of liquid flow of milk sample in immunochromatographic
method
(1) Preparation of immunochromatographic device
The immunochromatographic device shown in Fig. l as a schematic sectional
view was ed as follows.
(a) Preparation of ribosomal protein L7/L12 antibody
As the antibody to be labeled with gold colloid, lococcus aureus
ribosomal protein L7/L12 monoclonal dy was used. ing to the method
described in International Patent ation W000/06603, Example 5, the
Staphylococcus aureus L7/L12 ribosomal protein was obtained, and the monoclonal
antibody was prepared by using this protein. As the monoclonal antibody, a
combination of two kinds of monoclonal antibodies (SA-l and SA—2) that can
simultaneously bind to different sites of the aforementioned L7/L12 ribosomal protein.
It was confirmed that the combination of the onal antibodies SA—l and
SA-2 react with the ribosomal protein L7/L12 of Staphylococcus aureus, but does not
react with the ribosomal protein L7/L12 of bacteria that cause is other than
Staphylococcus aureus, and they are antibodies of which reactions are not inhibited by
components of milk, as follows.
The monoclonal antibody SA—l (10 ug/ml) and PBS (100 ul) were poured into
each well of a 96—well ELISA plate (Maxsorp ELISA Plate, Nunc), and adsorption of
the antibody was allowed overnight at 4°C. After the supernatant was removed, a 1%
bovine serum albumin solution (in PBS, 200 pl) was added, and the reaction was
allowed at room temperature for 1 hour to attain blocking. After the supernatant was
removed, each well was washed several times with a washing solution (0.02% Tween 20,
PBS), each of the various bacteria shown in Table 1 (about 1 X 108 ml) diluted 10
times with 0.5% Triton X-100, PBS (100 pl) was added to the well, and the reaction was
allowed at room temperature for 1 hour. Further, after the atant was removed,
the monoclonal antibody SA—2 labeled with dase and diluted with 0.02% Tween
, PBS to a final concentration of 1 ug/ml (100 pl) was added, and the reaction was
allowed at room temperature for 1 hour. After the supernatant was d, each well
was further washed several times with the g solution, then a TMB solution (KPL,
100 u/l) was added to each well, and the reaction was allowed at room temperature for
minutes. Then, 1 mol/l hydrochloric acid (100 pl) was added to terminate the
reaction, absorbance was measured at 450 nm, and on the basis of the difference of the
absorbance from the signal of the ve control (solution not containing bacterium),
reactivity was evaluated. The results are shown in Table l. The positive s (+)
mean that the difference of the absorbance ed after ELISA and the absorbance of
the negative control was at least 0.5 or larger, and the negative results (—) mean that the
difference of the absorbance obtained after ELISA and the absorbance of the negative
control was not larger than 0.1.
[Table 1]
Enterrococcusfaecalis -—-
ichia coli -—
hilus influenzae -—
Haemophilus parahaemolilicus-—
hilus parainfluenzac -—
Klebsiella pneumoniae -—
Legionella pneumophila -—
lla catarrharis -—
Mycopzasmapneumm _
Neisseria gonorrhoeae -—
Neisseria lactamica -—
Propionibacteriumacens -—
Proteus mirabilis -—
The monoclonal antibody SA-l (10 ug/ml) and PBS (100 pl) were poured into
each well of a l ELISA plate (Maxsorp ELISA Plate, Nunc), and adsorption of
the antibody was allowed overnight at 4°C. After the supernatant was removed, a 1%
bovine serum n solution (in PBS, 200 pl) was added, and the reaction was
allowed at room temperature for 1 hour to attain blocking. After the supernatant was
removed, each well was washed several times with a washing solution (0.02% Tween 20,
PBS), Staphylococcus aureus (about 1 X 108 cells/ml) d 10 times with 0.5% Triton
X-100, PBS (20 ul) and milk (commercial cow's milk to drink, 80 ul) were added to the
well, and the reaction was allowed at room temperature for 1 hour. Further, after the
supernatant was removed, the monoclonal antibody SA—2 labeled with peroxidase and
diluted with 0.02% Tween 20, PBS to a final concentration of l ug/ml (100 pl) was
added, and the reaction was allowed at room temperature for 1 hour. After the
supernatant was removed, each well was further washed several times with the washing
solution, then the TMB solution (KPL, 100 u/l) was added, and the reaction was
allowed at room temperature for 10 minutes. Then, 1 mol/l hydrochloric acid (100 pl)
was added to terminate the reaction, then absorbance was measured at 450 nm, and on
the basis of the difference of the absorbance from the signal of the positive l
(solution not ning milk), reactivity was evaluated to confirm that the reactivity
was not reduced by milk.
(b) Gold colloid-labeled antibody-impregnated member
A gold colloid solution (particle size 60 nm, 0.9 mL, BB International) was
mixed with 0.1 M ium phosphate, pH 7.5, the monoclonal antibody SA-2 to be
labeled with gold colloid (100 ug/mL) was added to the mixture, and the mixture was
left standing at room temperature for 5 minutes so that the dy bound to the gold
colloid particle surfaces. Then, a 10% aqueous solution of bovine serum albumin
(BSA) was added at a final concentration of 1% in the gold colloid on, and the
remaining surfaces of the gold colloid particles were blocked with BSA to prepare a
solution of the monoclonal antibody SA-2 labeled with gold colloid (henceforth referred
to as "gold colloid-labeled antibody"). This solution was centrifuged (at 15000 x rpm
for 5 minutes) to precipitate the gold colloid-labeled antibody, and the supernatant was
removed to obtain the gold colloid-labeled antibody. This gold d-labeled
antibody was suspended in 20 mM Tris-hydrochloric acid buffer (pH 8.2) containing
0.25% BSA, 2.5% sucrose, and 35 mM NaCl to obtain a gold colloid-labeled antibody
solution. A glass fiber pad of a strip-like shape (10 mm x 300 mm) was impregnated
with the gold colloid-labeled antibody solution (2 mL), and dried at room temperature
under reduced re to obtain a gold colloid-labeled antibody-impregnated member 1
(first part).
(0) Part for capturing complex of antigen and gold colloid-labeled antibody
A ellulose membrane having a width of 25 mm and a length of 300 mm
was prepared as a membrane carrier 2 for chromatographic pment with
chromatography medium (second part).
A on containing the monoclonal antibody SA—l (1.5 mg/mL) was applied
in the shape of a line in a volume of l uL/cm to the membrane r 2 for
chromatographic development at a position of 10 mm from the end on the side of the
chromatography development starting point, and dried at 50°C for 30 minutes, and then
the membrane carrier was immersed in a 0.5% sucrose solution for 30 minutes, and
dried overnight at room temperature to obtain a part 3 for capturing the complex of the
Staphylococcus aureus ribosomal n L7/Ll2 antigen and the gold colloid-labeled
(d) Preparation of immunochromatographic device
In addition to the aforementioned labeled antibody-impregnated member 1 and
membrane r 2 for chromatographic development, cotton cloth serving as the
member 4 for sample addition and the member 7 for removal of fat globules (third part),
and filter paper as the member 5 for absorption were prepared. After these members
were adhered on the substrate 6 (thickness 254 um, made of polystyrene, having
adhesive for ng members), the substrate was cut in a width of 5 mm to prepare the
immunochromatographic device of which sectional view is shown in Fig. 1. As the
member 4 for sample addition, the members shown in Table 2 were used.
[Table 2]
Member for sample addition (manufacturer) Retention particle Material Thickness
size (um) (um)
GF/B (GE Healthcare Bioscience) Glass fiber 980
GF/AVA (GE Healthcare Bioscience) Glass fiber 299
MFl (GE care Bioscience) Glass fiber 357
VFl (GE Healthcare Bioscience) Glass fiber 701
VF2 (GE Healthcare Bioscience) Glass fiber 764
GF/DVA (GE care Bioscience) Glass fiber 776
NE107 (Asahi Kasei Fibers) Several 10s to Cellulose 410
several 100$
UR601 (Asahi Kasei Fibers) Several 10s to Cellulose 520
several 1005
(2) Test
ement for cow's milk using the immunochromatographic device was
performed as follows. A fresh milk sample (100 pl) from which Staphylococcus
aureus was detected was put into a microtube, an additive solution (150 pl, 1% Tween
, 0.05 M NaCl, 0.1 M MOPSO as final concentrations, pH 7.5) was added to the
sample and mixed. The immunochromatographic device obtained in (I) mentioned
above was immersed into the above mixed solution from the member 4 for sample
addition, chromatographic development was carried out by the l flow method, and
it was examined whether the developing solution would flow to the member 5 for
absorption.
The results are shown in Table 3. The symbol + tes that the developing
solution flowed to the member 5 for absorption, and the symbol — indicates that the
developing solution stopped on the way to the member 5 for absorption. Only when a
member showing a retention particle size not larger than 3.5 um was used, the solution
flowed to the member 5 for absorption. In the other cases, the liquid flow stopped in
the middle of the membrane carrier 2 for chromatographic development.
[Table 3]
Member for sample addition (manufacturer)
GF/B (GE Healthcare Bioscience)
GF/AVA (GE Healthcare Bioscience)
MFl (GE Healthcare ence)
VFl (GE care Bioscience)
VF2 (GE Healthcare Bioscience)
GF/DVA (GE Healthcare Bioscience)
NE107 (Asahi Kasei )
UR601 (Asahi Kasei Fibers) -
Example 2: Detection of Staphylococcus aureus in milk by immunochromatographic
method
(1) Preparation of immunochromatographic device
According to the method described in e 1, the immunochromatographic
device shown in Fig. l was ed. As the member serving as the member 4 for
sample addition and the member 7 for removal of fat globules (third part), GF/DVA
mentioned in Table 2 was used.
(2) Test
Measurement for cow's milk using the immunochromatographic device was
performed as follows. Each of fresh milk samples 1 to 6 (100 pl) was put into a
microtube, an additive solution (150 pl, 1% Tween 20, 0.05 M NaCl, 0.1 M MOPSO as
final concentrations, pH 7.5) was added to the sample and mixed. The aforementioned
immunochromatographic device was immersed into the above mixed solution from the
member 4 for sample addition, the device was left standing at room temperature for 15
minutes to carry out chromatographic development by the lateral flow method, and then
whether the complex of the mal protein L7/L12 n and the gold colloid—
labeled dy was captured by the aforementioned part 3 for capturing or not was
visually ined on the basis of the presence or absence of a reddish purple line that
became more or less conspicuous in tion to the capture amount.
Separately, in order to confirm the ce or absence of Staphylococcus
aureus in the milk samples, detection was also med by the cultivation-based
method. Each milk sample (100 pl) was inoculated to Trypticase Soy Agar II with 5%
Sheep Blood (Becton Dickinson Japan) or the Staphylococcus aureus ion medium,
X-SA Agar Medium (Nissui), then incubation was performed at 370C for 24 hours, and
the colonies appeared were confirmed.
The results of the visual determination and the results of the cultivation—based
method are shown in Table 4. In the results of the cultivation—based method, the
symbol + indicates that a colony of Staphylococcus aureus was observed, and the
symbol - indicates that colony of Staphylococcus aureus was not observed. In the
results obtained by determination using the kit, the symbol + indicates that a reddish
purple line could be visually observed, and the symbol — indicates that such a line could
not be visually observed.
[Table 4]
I Sample No. I s obtained by cultivation—based method I Results obtained by
(detected bacterium) determination with kit
1 _ _
2 _ _
3 _ _
(Streptococcus)
4 _ _
(Streptococcus)
+ +
(Staphylococcus aureus)
6 + +
(Staphylococcus aureus)
For the two milk samples for which no bacteria were detected, any line did not
appear. Also for the two samples for which ococcus was detected, any line did
not appear. On the other hand, for the samples for which Staphylococcus aureus was
detected, lines appeared, and thus Staphylococcus aureus could be detected.
As described above, use of the immunochromatographic device of the present
ion made it possible to develop a milk sample containing milk fat globules
without clogging in membrane, and perform accurate measurement.
Example 3: Measurement with milk at high tration by chromatographic
method
The immunochromatographic device shown in Fig. 1 was ed by the
method described in Example 1, and liquid flow was ed with changing ratio of
milk in samples. As the member for sample addition, those mentioned in Table 3 were
used. A fresh milk sample and an additive solution were mixed at various ratios
(additive solution contained 1% Tween 20, 0.05 M NaCl, 0.1 M MOPSO as final
trations, pH 7.5, total liquid volume was 250 pl), the immunochromatographic
device was immersed into the mixture from the member for sample addition,
chromatographic development was carried out by the lateral flow method, and whether
the total volume of the mixture was developed or not was examined.
The results are shown in Table 5. The symbol + indicates that the developing
solution flowed to the member 5 for absorption, and the symbol — indicates that the
ping on stopped on the way to the member 5 for tion. When the
ratio of milk was 50%, the total volume of the mixture flowed to the member 5 for
absorption with the members for sample addition other than VFl. When the ratio of
milk was 70%, for all of the members for sample addition, the flow of the mixture
stopped in the middle of the membrane carrier 2 for chromatographic development, or
even if the mixture d the member 5 for tion, flow of the mixture stopped,
and thus correct measurement could not be performed, as a result.
[Table 5]
Member for sample addition Result (ratio of milk, %)
(manufacturer)
GF/AVA (GE Healthcare Bioscience)
MFl (GE Healthcare Bioscience)
VFl (GE care Bioscience)
VF2 (GE Healthcare Bioscience)
GF/DVA (GE Healthcare Bioscience)
Then, two of the members for sample addition were d, and used to
perform the same test as mentioned above. Two of the members for sample addition
were stacked as shown in Fig. 3. As the members for sample addition, GF/AVA and
GF/DVA were used. GF/DVA was disposed on the upstream side (member 4 for
sample addition), and GF/AVA was disposed on the downstream side (member 7 for
removal of fat globules), and the test was performed with various lengths of the
members. The milk ratio was 80%, and the volume of the sample was 250 pl. The
results are shown in Table 6 (as for "Length" and "Overlap" mentioned in the table, refer
to Fig. 3). The symbol + te that the developing solution flowed to the member 5
for absorption. Even when the milk rate was 80%, the liquid flow did not stop, and
correct measurement could be performed. Furthermore, even when the lengths of two
of the members for sample addition were changed, correct measurement could be
performed.
[Table 6]
Length (mm) Overlap
GF/DVA (mm)
-----+++
w ---+
w«41 II
21 11 _ o
As bed above, by using two kinds of the members for sample addition
having different retention particle sizes in combination, milk fat globules could be
removed, the milk samples could be developed without clogging in the membrane, and
thus highly sensitive and correct ement was enabled, even when milk was used
at high concentration.
Example 4: Effect of lytic enzyme in detection of Staphylococcus aureus by enzyme-
linked immunosorbent assay (ELISA)
(a) Preparation of monoclonal antibody against ribosomal protein L7/L12
The monoclonal antibodies SA—l and SA—2 mentioned in Example 1 were used.
(b) Confirmation of effect of lytic enzyme in ELISA
The effect of lytic enzyme was confirmed as follows.
lococcus aureus was treated with each of the tant and lytic
enzymes shown in Table 7 at 37°C for 20 minutes in 10 mM Tris-HCl (pH 8.0), and
then used for ELISA, in which absorbance was measured in the same manner as that of
Example 1, (a). Staphylococcus aureus was adjusted to a final concentration of 2.5 x
105 (cells/ml) with physiological saline. As the negative control, phosphate ed
saline (PBS) was used. For the positive control, a nonionic surfactant, Triton X—100,
was used. The final concentration was 1%, and the ent was performed overnight
at room temperature.
[Table 7]
. Concentration
Surfactant or lytlc enzyme Type Manufacturer
(final concentration)
PBS Buffer Wako Pure Chemical Industries -
Triton X-100 Surfactant SIGMA 1.0%
Wako Pure Chemical Industries 5 (pg/ml)
Achromopeptidase Wako Pure Chemical Industries 5 (pg/ml)
Lysostaphin Wako Pure al Industries 5 (pg/ml)
The results are shown in Fig. 4.
Compared with PBS used as the negative control, higher ance was
obtained with the surfactant and all of the lytic enzymes. Compared with the
surfactant Triton X-100, which is a commonly used lysing agent, achromopeptidase and
lysostaphin, which are autolysins, gave twice higher absorbance vales.
Example 5: Effect of lytic enzyme in case of using milk
By using achromopeptidase (Wako Pure Chemical ries) and lysostaphin
(Wako Pure Chemical Industries), which showed high lysing effect in Example 4,
Staphylococcus aureus added to cow's milk was measured by ELISA. As the cow's
milk, commercial cow's milk to drink was used at a ratio of O to 80% (remainder was 10
mM Tris-HCl (pH 8.0)), and Staphylococcus aureus was adjusted to a final
concentration of l x 105 /ml) with physiological saline. Concentrations of
achromopeptidase and lysostaphin were adjusted by using 10 mM Tris-HCl (pH 8.0),
and they were mixed with Staphylococcus aureus samples at a final concentration of 5
ug/ml. The measurement by ELISA was performed in the same manner as that of
Example 4.
The results are shown in Fig. 5. When achromopeptidase was used, the
absorbance decreased with increase of the ratio of milk. On the other hand, when
lysostaphin was used, regardless of the ratio of milk, a fixed absorbance was observed.
Thus, the results indicate that achromopeptidase cannot t ent effect in the
presence of milk of high concentration.
Example 6: Detection of Staphylococcus aureus in milk by immunochromatographic
method
(1) Preparation of immunochromatographic device
An immunochromatographic device was prepared as follows.
(a) Gold colloid—labeled antibody-impregnated member
The gold d-labeled antibody-impregnated member described in Example
1 was used.
(b) Part for capturing complex of n and gold colloid—labeled antibody
This part was ed in the same manner as that of Example 1.
(c) Preparation of immunochromatographic device
In addition to the aforementioned labeled antibody-impregnated member 1 and
membrane carrier 2 for chromatographic development, GF/DVA (filter member
consisting of glass fibers having a thickness of 776 um, and a retention particle size of
3.5 mm, GE care Bioscience) as a member serving as both the member 4 for
sample addition and the member 7 for removal of fat globules (third part), and filter
paper as the member 5 for absorption were prepared. After these members were
d on the substrate 6 (thickness 254 um, made of polystyrene, having adhesive for
adhering members), the substrate was cut in a width of 5 mm to prepare the
immunochromatographic device of which sectional view is shown in Fig. 6.
[008 1 ]
(2) Test
Measurement for cow's milk using the immunochromatographic device was
performed as follows.
A milk sample (100 pl) containing a known amount of Staphylococcus aureus
was put into a microtube, an ve solution (150 pl, 1% Triton X-100, 5 ug/ml of
lysostaphin, 0.1 M MOPSO as final concentrations, pH 7.5) was added to the sample
and mixed. As the milk, commercial product to drink was used. For comparison, the
test was also performed with the additive solution not ning lysostaphin (1% Triton
X—100, 0.1 M MOPSO as final concentrations, pH 7.5). The aforementioned
immunochromatographic device was immersed into the above mixed solution from the
member 4 for sample addition, the device was left standing at room temperature for 30
minutes to carry out chromatographic development by the lateral flow method, and then
whether the complex of the ribosomal protein L7/Ll2 antigen and the gold colloid-
labeled antibody was captured by the aforementioned part 3 for capturing or not was
determined by quantifying a reddish purple line, which became more or less
conspicuous in tion to the capture amount, using an apparatus, FASTKIT
Immunochromatographic Reader n 20-A (Becton Dickinson Japan).
The results are shown in Fig. 7. When the additive solution containing
aphin was used, it was found that the curve shifted to the low concentration side
from the on of the curve obtained with the additive solution containing Triton X-
100 d of lysostaphin, as a result, and Staphylococcus aureus can be detected with
about 10 times higher sensitivity.
Example 7: ement for milk sample by immunochromatographic method
An immunochromatographic device was prepared in the same manner as that
of Example 6.
A fresh milk sample (100 pl) from which Staphylococcus aureus was detected
was put into a microtube, an additive solution (150 pl) was added to the sample, and
mixed at room temperature. As the additive solution, two kinds of the additive
solutions described in Example 6 were used. The measurement was performed in the
same manner as that of Example 6, and then a reddish purple line appeared was visually
evaluated ive +, negative —).
On the other hand, in order to confirm the number of Staphylococcus aureus in
the milk samples, quantification was performed by the cultivation-based method. Each
milk sample (100 ul) was inoculated to Trypticase Soy Agar II with 5% Sheep Blood
(Becton Dickinson Japan) or the Staphylococcus aureus selection medium, X-SA Agar
Medium (Nissui), then incubation was performed at 37°C for 24 hours, and the colonies
appeared were d.
The number of Staphylococcus aureus counted by the cultivation-based method
and the determination results obtained by the immunochromatographic method are
shown in Table 8. When lysostaphin was not added, 1 x 104 (cfu/ml) or fewer
Staphylococcus aureus could not be detected, but when aphin was added, it
became possible to detect up to 5 X 103 of Staphylococcus .
[Table 8]
Sample Number of Results of measurement with kit
No. Staphylococcus aureus With Without
(cfu/ml) lysostaphin lysostaphin
Industrial Applicability
The present invention can be used for diagnosis of infectious diseases of
livestock animals.
Claims (23)
- [Claim 1] An immunochromatographic method for detecting a specific substance contained in milk, which comprises: (1) the step of ting the milk with a test strip having a first part retaining a labeled first antibody directed to the specific substance or retaining the specific substance that is labeled, a second part disposed downstream from the first part, on which a second dy directed to the specific substance is lized, and a third part disposed am from the first part or the second part and having voids enabling removal of milk fat globules contained in the milk, at the third part or a further upstream part, and (2) the step of flowing the milk up to the second part or a further downstream part to obtain a detectable signal of the label at the second part or a further downstream part, wherein the method is performed to diagnose cow's mastitis.
- [Claim 2] The method according to claim 1, wherein the immunochromatographic method is a lateral flow type method.
- [Claim 3] The method according to claim 1 or 2, wherein a labeled first antibody ed to the specific substance is retained at the first part.
- [Claim 4] The method according to any one of claims 1 to 3, wherein the specific substance is a component of a bacterium, or a substance secreted by a ium.
- [Claim 5] The method according to any one of claims 1 to 4, wherein at least one or both of the first antibody and the second antibody are monoclonal antibodies.
- [Claim 6] The method according to claim 5, wherein the first antibody and the second antibody are monoclonal antibodies.
- [Claim 7] The method according to any one of claims 1 to 6, wherein the voids of the third part have a retention particle size of 1 to 3.5 µm.
- [Claim 8] The method according to any one of claims 1 to 7, wherein the third part is constituted by two or more kinds of members having voids that can remove milk fat globules of ent particle sizes.
- [Claim 9] The method according to claim 8, wherein the third part is constituted by a first member disposed downstream and a second member disposed upstream, and retention particle size of the second member is larger than retention particle size of the first member.
- [Claim 10] The method according to claim 9, wherein the ion particle size of the first member is 1.0 to 2.0 µm, and the retention le size of the second member is 3.0 to 3.5 µm.
- [Claim 11] The method according to any one of claims 1 to 10, which further comprises the step of lysing a bacterium in which the specific substance exists to elute the specific substance into the milk, before the step of (1).
- [Claim 12] The method according to claim 11, wherein the lysing is performed with a lytic .
- [Claim 13] The method according to claim 12, wherein the lytic enzyme is an autolysin.
- [Claim 14] The method ing to claim 12, wherein a lococcus contained in the milk is detected by using lysostaphin as the lytic enzyme.
- [Claim 15] A method for diagnosing whether a ive microorganism of mastitis of a livestock animal is a staphylococcus or not, which comprises: the step of obtaining milk containing the causative microorganism from the livestock animal suffering from mastitis, the step of mixing a lytic enzyme in the milk so that a specific substance existing in cells of the causative microorganism is released out of the cells, and the step of determining presence or absence of the specific substance or measuring existing amount of the specific substance by an immunological method using an antibody directed to the specific substance as an antigen.
- [Claim 16] The method according to claim 15, n r the causative microorganism is a staphylococcus or not is diagnosed by using lysostaphin as the lytic enzyme.
- [Claim 17] An immunochromatographic device for detecting a specific substance contained in milk, which comprises a test strip having a first part retaining a labeled first dy directed to the specific substance or the ic substance that is labeled, a second part ed downstream from the first part, on which a second antibody directed to the specific substance is immobilized, and a third part disposed upstream from the first part or the second part and having voids enabling removal of milk fat globules contained in the milk, wherein the specific substance is a ent of a etiologic bacterium of cow’s mastitis, or a substance secreted by a etiologic bacterium of cow’s mastitis.
- [Claim 18] The immunochromatographic device according to claim 17, wherein the first part or the second part retains a lytic enzyme or a surfactant.
- [Claim 19] A ion kit consisting of an additive on containing a lytic enzyme or a surfactant, and the immunochromatographic device according to claim 18.
- [Claim 20] An immunochromatographic method according to claim 1, substantially as herein described with reference to any one of the examples and/or figures.
- [Claim 21] A method according to claim 15, ntially as herein described with reference to any one of the examples and/or figures.
- [Claim 22] An chromatographic device according to claim 17, substantially as herein described with reference to any one of the examples and/or figures.
- [Claim 23] A detection kit according to claim 19, substantially as herein described with reference to any one of the examples and/or figures.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/JP2012/065150 WO2013186885A1 (en) | 2012-06-13 | 2012-06-13 | Method for detecting specific substances in milk |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ702764A NZ702764A (en) | 2016-02-26 |
NZ702764B2 true NZ702764B2 (en) | 2016-05-27 |
Family
ID=
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