JP2002340902A - Method for measuring microorganisms or microbial components and measuring kit using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply and accurately measuring microorganisms or microbial components regardless of the variation/diversity of an object to be measured without being affected by impurities in a sample. SOLUTION: In the method for measuring microorganisms or microbial components in a specimen, the specimen is reacted with protein specifically bonded to polysaccharides constituting envelopes or cell walls of microorganisms to detect the polysaccharides bonded to protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring a microorganism or a microorganism component using a protein that specifically binds to a polysaccharide constituting the outer membrane or cell wall of the microorganism, and a measurement kit used for the method.

[0002]

2. Description of the Related Art In the fields of medicine and food, it is essential to confirm the presence of microorganisms and to avoid contamination in order to ensure safety for humans and the like. Is required. Therefore, microbial tests are used for diagnosis at medical sites, safety tests for pharmaceuticals, etc.
It is performed in a wide range of fields, such as inspection of water and food at manufacturing sites.

On the other hand, not only living microorganisms,
It is known that some polysaccharides constituting microorganisms have various biological activities themselves, and their relation to diseases has been discussed (Lisa, Vol. 6, No. 9, pp 854-858, 1
999). Therefore, it is also important to avoid contamination of such substances.

For example, lipopolysaccharide (hereinafter referred to as “LP”), which is one of the polysaccharides constituting the outer membrane present on the surface of Gram-negative bacteria,
S)) is composed of a lipid called lipid A and a polysaccharide covalently bonded to the lipid A, which promotes the reaction of the coagulation and fibrinolysis system as an endotoxin, and has an effect on the immune system such as adjuvant action and mitogen activity. Platelet / leukopenia,
Impact on the circulatory system such as shock, Shwartzm
an) It is known to exhibit various biological activities such as reaction and heat generation.

[0005] Further, β-1,3-glucan (hereinafter referred to as “βG”), which is one of the polysaccharides constituting the cell wall of fungi such as yeasts, molds, and mushrooms, has a variety of structures. Induction of various cytokines, activation of complement, antitumor activity, action as allergen, LPS
It is known to exhibit various biological activities such as enhancement of the physiological action of.

[0006] Furthermore, peptidoglycan (hereinafter referred to as "P") is one of the polysaccharides constituting the cell wall of most prokaryotes.
G ") is a polymer of a glycopeptide containing N-acetylmuramic acid or N-glucolylamuramic acid and a D-amino acid, which, like βG and LPS, is also a macrophage, T lymphocyte and B lymphocyte. Have various effects on immune response cells. Specifically, destruction of platelets, promotion of proliferation of fibroblasts, promotion of bone resorption, activation of complement, enhancement or suppression of humoral immune response, enhancement of cellular immunity, stimulation of cell endothelial system, transient It is known to have effects such as leukopenia and subsequent increase in, increase in the action of interferon-inducing factors, enhancement of natural resistance, induction of fever, and enhancement of sensitivity to LPS.

[0007] Therefore, it is strongly required in manufacturing and medical fields to accurately detect the presence of such microorganisms and components of these microorganisms and to prevent their contamination, and some of them are obliged.

[0008] At present, as a method for measuring microorganisms or substances derived from microorganisms, there is known a method for measuring by using an antibody against these. However, this method enables highly sensitive measurement of microorganisms and microbial components used as an immunogen, but on the other hand, because antibodies strictly identify antigens, it is necessary to use a method other than the microorganism used as the immunogen. Even if a microorganism or a microorganism used as an immunogen has a mutation in the antigen, it is highly likely that the antigen cannot be detected.

[0009] Culture methods, PCR methods and the like are also used for detecting microorganisms. Among them, in the culture method, detection of microorganisms takes time, and depending on the selection of the medium and conditions, the growth of the target microorganism does not occur, so that detection may not be possible.
In addition, since the PCR method can only amplify a specific gene sequence, there is a problem that only a known specific microorganism can be measured. Further, most of these methods are methods for measuring living microorganisms themselves, and cannot be used for detecting microorganism components.

[0010] For these reasons, at present, the presence of microorganisms is detected by measuring LPS, βG or PG, which are polysaccharides constituting the outer membrane and cell wall of the microorganisms.

Among them, for measurement of LPS and βG, a Limulus reagent utilizing a horseshoe crab blood cell extract is used. The measurement method using this Limulus reagent
Although it is a simple and highly sensitive measurement method, it uses the blood coagulation cascade mechanism, which is one of the biological defense reactions in horseshoe crab blood cell extracts, so it can use proteases, protease inhibitors, protein denaturants, chelating agents, Ions, strong acids
There are many factors that interfere with the measurement, such as strong alkalis, and there is a problem that these must be removed or prepared by diluting the sample.

Further, as for the measurement of PG and βG, a method using silkworm body fluid has been reported (FEMS Immunology an).
d Medical Microbiology, 15, 129-134, 1996.). This method utilizes the fact that the protease cascade present in silkworm body fluid is activated by PG and βG, and quantifies PG and βG by measuring the melanin finally generated by the activated cascade. Things. Although products utilizing this principle are already on the market, this reagent is easily affected by the salt concentration in the sample, and in many cases, both PG and βG are present in the sample. In such a case, since the same reaction is performed for both samples, only a combined measurement value can be obtained, that is, there is a problem that only PG cannot be measured.

Regarding the above method, a method has been reported in which, in order to specifically measure PG, a component that reacts with βG is removed from the silkworm body fluid and only a component that specifically reacts with PG is used. Japanese Patent Publication No. Hei 7-114707). However, in this method, PG or βG from the external environment is liable to be mixed during the operation, the activity of the enzyme constituting the reaction system in the silkworm body fluid is easily reduced, and the system which reacts with βG during affinity chromatography is easily generated. However, there is a drawback that there is a high possibility that the particles are activated. Other methods include a method of removing a system that reacts with βG using a βG-recognition protein antibody or the like, a method of removing βG from a sample (Japanese Patent Laid-Open No. 11-196895), and a method of removing poly (1 → 3) A method using a saccharide compound such as glucoside that inhibits a reaction system to βG (JP-A-11-255805)
However, it is difficult to completely prevent contamination of PG or βG from the external environment during the preparation operation. That is, PG and βG in reagent preparation
Even a slight contamination of the compound may activate the reaction system, resulting in a decrease in the reactivity to PG, and it is highly likely that it is difficult to obtain reproducibility of the measured value because the degree is not constant. .

[0014]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and is not affected by contaminants in a sample. It is an object of the present invention to provide a method for simply and accurately measuring a microorganism component.

[0015]

Means for Solving the Problems The present inventors first studied a method for detecting a microorganism, and as a result, incorporated a protein that specifically binds to a polysaccharide constituting an outer membrane or a cell wall of the microorganism into a measurement system. As a result, they have found that the presence of microorganisms can be detected more accurately than in the conventional method. Further, they found that this method can detect not only living microorganisms but also the presence of microbial components, and have completed the present invention.

That is, the present invention is characterized in that a sample reacts with a protein that specifically binds to a polysaccharide constituting the outer membrane or cell wall of a microorganism, and the polysaccharide bound to the protein is detected. It is intended to provide a method for measuring a microorganism or a microorganism component in a sample.

The present invention also relates to a method in which a solid phase on which a protein that specifically binds to a polysaccharide constituting the outer membrane or cell wall of a microorganism is immobilized, and a sample are reacted with the solid phase. Detecting a polysaccharide constituting the outer membrane or cell wall of the microorganism with a protein that specifically binds to the polysaccharide constituting the outer membrane or cell wall of the microorganism labeled with a labeling substance; A method for measuring a microorganism component is provided.

The present invention further provides the following components (a) and (b): (a) a solid phase on which a protein that specifically binds to a polysaccharide constituting the outer membrane or cell wall of a microorganism is immobilized; An object of the present invention is to provide a kit for measuring a microorganism or a microorganism component containing a protein that specifically binds to a polysaccharide constituting an outer membrane or a cell wall of a microorganism labeled with a labeling substance.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a protein which specifically binds to a polysaccharide constituting the outer membrane or cell wall of a microorganism (hereinafter referred to as "membrane polysaccharide") to a microorganism or a specimen from which a microbial component is to be detected. (Hereafter, "specific binding protein"
) To detect a complex formed by binding of the specific binding protein and the membrane polysaccharide.

The specific binding protein used in the present invention refers to a protein capable of recognizing a polysaccharide constituting an outer membrane or a cell wall of a microorganism and forming a stable conjugate with the polysaccharide. The specific binding protein includes a peptidoglycan recognition protein (hereinafter referred to as “PGRP”) that specifically binds to PG constituting the cell wall of most prokaryotes.
LPS-binding protein that specifically binds to LPS constituting the outer membrane of Gram-negative bacteria (Eur. J. Biochem. V).
ol. 1248, pp. 217-224, 1997.), a βG recognition protein that specifically binds to βG constituting the cell wall of fungi (The
Journal of Biological Chemistry Vol.263, No24, pp.
12056-12062, 1988.).

The above-mentioned specific binding proteins are all known ones.
Purification from silkworm body fluid (The Journal of Biological
Chemistry Vol.271, No.23, pp.13854-13860, 199
6.), a method of producing using a recombinant (JP-A-10-179171)
No.) have been reported, but are not particularly limited thereto, and any method may be used as long as it can produce a binding protein.

Among the above-mentioned methods for producing a specific binding protein, a particularly preferable method is a method of infecting silkworms with a recombinant baculovirus to express the recombinant specific binding protein, and collecting and purifying a body fluid.
In this case, it becomes possible to obtain a required amount of specific binding protein from a relatively small amount of material. As a method for purifying the specific binding protein obtained as described above, for example, a method using an affinity column (The Journal of B
iological Chemistry Vol.271, No.23, pp.13854-1386
0, 1996.) can be used, but the method is not particularly limited as long as the method can purify the binding protein.

In the measurement method of the present invention, a particularly preferable example is a method in which a specific binding protein is immobilized on a solid phase surface, and a membrane polysaccharide in a sample is bound to the immobilized protein. (Hereinafter, referred to as "sandwich assay"). This sandwich measurement method is a method widely used in a measurement method using an antibody. In this case, the antibody to be immobilized and the detection antibody usually need to recognize different epitopes. However, in the method of the present invention, since the membrane polysaccharide to be measured has a polymer-like repeating structure or a large number of specific structures, one specific binding protein that recognizes this specific unit is used. The sandwich measurement method can be performed using only the specific binding protein.

The carrier used for immobilization is not particularly limited as long as it can immobilize a sufficient amount of a specific binding protein capable of binding to a membrane polysaccharide, and a plate made of a substance usually used as a carrier can be used. Those having a shape such as a membrane, a tube, a bead, and a gel can be used. The immobilization method is not particularly limited as long as it is a method capable of immobilizing a sufficient amount of a specific binding protein capable of binding to a membrane polysaccharide, and is directly immobilized on a carrier, a method using a spacer, an antibody Any method can be used for bonding.

However, the solid phase material capable of maximizing the properties of the specific binding protein is preferably one having a functional group for covalent bonding on the surface of the carrier, and particularly when PGRP is used as the specific binding protein, Is preferably a group having a carboxyl group.
It is particularly preferable to use a plate carbo for LISA (manufactured by Sumitomo Bakelite) or the like. Also, as a method for bonding to the solid phase, more preferable results can be obtained by using a method using carbodiimide as a condensing agent.

In order to form a complex by binding a membrane polysaccharide to the specific binding protein immobilized on the solid phase, impurities are non-specifically adsorbed to the solid phase or the binding protein before the complex is formed. In order to prevent this, it is preferable to perform a blocking treatment on the solid phase in advance. The blocking substance contained in the solution used for the blocking treatment at this stage (hereinafter referred to as “first blocking agent”) is to prevent non-specific adsorption of contaminants as much as possible and to bind the binding protein to the microorganism component. What has the property which does not inhibit is used. Examples of the block in the first blocking agent include serum proteins, milk component proteins such as casein and skim milk, and gelatin. The blocking agent differs depending on the type of the binding protein used in the measurement and the type of the microbial component to be measured, and a blocking body must be selected accordingly. Specifically, when peptidoglycan is measured using PGRP as a binding protein, a serum albumin such as bovine serum albumin is preferred as a block contained in the first blocking agent.

There is no particular limitation on the method of forming a complex by binding the membrane polysaccharide to the solid phase on which the specific binding protein is immobilized, thus obtaining, for example, a plate having several wells. A method of adding a sample liquid to each well of the solid phase, or a method of adding a granular solid phase to the sample liquid, and the like can be given. Alternatively, a swab or the like having a location to be measured wiped may be used as a sample, and the sample may be copied onto a solid phase, or the sample may be suspended in an appropriate solution for use. After that, it is preferable to remove unbound contaminants by washing using an appropriate washing solution. By this process, it becomes possible to specifically bind only the microorganism component to be measured to the solid phase.

Next, in order to detect a membrane polysaccharide bound to the specific binding protein immobilized on the solid phase, a binding protein labeled with a labeling substance (hereinafter referred to as “labeled binding protein”) is added, and the reaction is carried out. Let it. The labeling substance used for the labeling binding protein is not particularly limited as long as it can be used for detecting a membrane polysaccharide without inhibiting the binding function of the binding protein, and various substances are used. By selecting a labeling substance that can be detected with high sensitivity even in a trace amount, highly sensitive measurement can be performed. Examples of such labeling substances include alkaline phosphatase, peroxidase,
enzymes such as β-galactosidase, radioisotopes, GF
Fluorescent substances such as P and fluorescein; and luminescent agents such as luciferase. A plurality of the labeling substances may be combined. The binding method of the labeling substance to the binding protein may be a known binding method to the protein. Of course, instead of a method of directly binding a labeling substance, an indirect binding method such as binding of biotin or the like to a specific binding protein and binding of a labeling substance bound to avidin or the like may be used.

When the labeled binding protein is added to the solid phase, a blocking agent that simultaneously inhibits non-specific adsorption (hereinafter, referred to as a blocking agent) to prevent the labeled binding protein from adsorbing to the solid phase or the immobilized binding protein. "Referred to as a" second blocking agent ").
Highly sensitive measurement becomes possible. Examples of the block body contained in such a second blocking agent include serum proteins,
Milk component proteins such as casein and skim milk, and gelatin. The second blocking agent differs depending on the type of the membrane polysaccharide to be measured and the type of the binding protein used in the measurement, and a blocking body must be selected accordingly. Specifically, when PG is measured using PGRP as the binding protein, it is preferable to use a milk component protein such as casein or skim milk as a block contained in the second blocking agent.

Further, between each step, it is preferable to wash with a phosphate buffer solution containing a surfactant such as Tween-20 (manufactured by Wako Pure Chemical Industries) in order to eliminate impurities and the like.

Finally, the amount of the membrane-bound polysaccharide bound to the solid phase is determined by measuring the amount of the label-bound protein.
Detect the presence of microorganisms or microbial components. In the measurement method of the present invention, the amount of the membrane polysaccharide is measured by measuring a labeling substance of a label-bound protein, and a measuring device used for the measurement is a spectrophotometer, a microplate reader, a liquid scintillation counter, a fluorescence spectrometer. It can be appropriately selected and used according to the labeling substance such as a photometer and a luminometer. Further, when a fluorescent substance or a luminescent substance is used as a labeling substance, it is possible to confirm the properties of the microorganism or the microorganism component under a microscope.

In order to easily carry out the measuring method of the present invention described above, a kit for measuring a microorganism component can be used. As an example of this detection kit, a kit comprising the following combinations can be mentioned.

Components (a) and (b), (a) a solid phase on which a protein that specifically binds to a polysaccharide constituting the outer membrane or cell wall of the microorganism is immobilized, and (b) a microorganism labeled with a labeling substance. A kit for measuring a microorganism or a microorganism component containing a protein that specifically binds to a polysaccharide constituting the outer membrane or cell wall of the present invention.

The kit further comprises the first and second
Or a phosphate buffer solution containing an appropriate surfactant can be added.

Specific examples of particularly preferred assay kits for carrying out the method of the present invention include the following.

(I) a solid phase in which a specific binding protein is bound to a carboxyl group on the surface of a carrier using carbodiimide; (ii) a specific binding protein labeled with a fluorescent substance or a luminescent substance; (iii) a first block containing serum albumin (Iv) Second blocking agent containing milk component protein (v) Phosphate buffer containing surfactant

[0037]

The measuring method of the present invention detects the polysaccharides constituting the outer membrane or cell wall of a microorganism, and detects the presence of the microorganism or the components of the microorganism therefrom. That is,
LPS, β are commonly used in the outer membrane or cell wall of microorganisms.
This is based on the finding that polysaccharides having a polymeric repetition or a large number of specific structures, such as G, PG and teichoic acid, exist in the structure.

That is, as a method for specifically detecting various proteins, a method using an antibody is known and widely used, but this method requires the presence of an antigen prior to the production of the antibody. Therefore, there is a problem in detecting unknown microorganisms. Further, when detection is performed by a sandwich method using an antibody, there is a problem that an antibody that recognizes two different epitopes is required for one detected substance (for example, a microorganism).

On the other hand, in the method of the present invention, the presence of microorganisms and the like is detected by focusing on the membrane constituents of the microorganisms and detecting them using a protein that recognizes the rough structure of the polysaccharide and specifically binds thereto. A wide variety of microorganisms that cannot be dealt with by antibodies because they are to be detected can be detected. Further, since the structure of the membrane component has a polymer-like repeating structure as described above, one type of specific binding protein can be used for immobilization and labeling.

[0040]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the invention thereto.

Example 1 Measurement of PG: (1) Preparation of PGRP plate Purified PGRP (The Journal of Biological Chemistry,
Vol.271, No.23, pp13854-13860, 1996.)
/ Ml of a phosphate buffer solution (pH 5.8) containing water-soluble carbodiimide (manufactured by Dojindo Laboratories) at a concentration of 50
It was diluted to give μg / ml. This diluted solution is used for ELI
Dispense 100 μl into each well of SA plate carbo (manufactured by Sumitomo Bakelite), and shake slowly at 4 ° C.
PGRP was allowed to bind to the plate by incubating overnight. The next day, the reaction solution was discarded and 0.05% Tween was added.
Phosphate buffer solution 30 containing n-20 (manufactured by Wako Pure Chemical Industries)
The plate was washed three times with 0 μl (hereinafter referred to as “washing solution”). Then 1% bovine serum albumin (SIGM)
A) was dispensed in 300 μl portions each containing a phosphate buffer solution.
Plate blocking was performed. After incubation at 30 ° C. for 10 minutes, the supernatant was discarded to prepare a PGRP plate. This plate was used for the following measurements.

(2) Measurement of PG PG (manufactured by Wako Pure Chemical Industries) was diluted with a phosphate buffer solution containing 1% bovine serum albumin to prepare a two-fold dilution series having a maximum concentration of 50 ng / ml ( 50, 25, 12.
5, 6.25, 3.125 ng / ml). 100 μl of the PG diluent was added to each well of the PGRP plate,
PGRP and PG by shaking gently at
Was combined.

After washing with a washing solution three times, PG labeled with biotin was used using an ECL protein biotinylation system (manufactured by Amersham Pharmacia Biotech).
RP was diluted with a phosphate buffer solution containing 0.5% casein (manufactured by Merck) to a concentration of 10 μg / ml, and 100 μl was dispensed into each well. Next, the mixture was incubated at 30 ° C. for 1 hour with gentle shaking, and then washed three times with a washing solution. Then, 100 μl of peroxidase-labeled streptavidin (manufactured by Amersham Pharmacia Biotech) diluted 1000-fold with a phosphate buffer solution containing 0.5% casein was added, and the mixture was incubated at 25 ° C. for 1 hour to bind streptavidin to biotin. .

The well solution was discarded, and the well was washed four times with a washing solution, and 100 μl of TMB reagent (manufactured by KPL) was added.
The color was developed by incubating with. 1 hour later
The reaction was stopped by adding 100 μl of M phosphoric acid, and the absorbance at 450 nm was measured using a microplate reader.

(3) Results A graph was prepared with the PG concentration on the horizontal axis and the absorbance on the vertical axis. As shown in FIG. 1, the PG concentration and the absorbance showed a clear correlation.

Example 2 Selection of Blocks after Immobilization of PGRP: (1) Preparation of PGRP Plate Purified PGRP (The Journal of Biological Chemistry,
Vol.271, No.23, pp13854-13860, 1996.)
g / ml water-soluble carbodiimide (manufactured by Dojindo Laboratories)
In a phosphate buffer solution (pH 5.8) containing
It was diluted to be 00 μg / ml. This diluted solution is
100 μl was dispensed into each well of a LISA plate carbo (Sumitomo Bakelite) and incubated at 37 ° C. for 2 hours to bind PGRP to the plate. The reaction solution was discarded and 0.05% Tween-20 was added.
300 μl of phosphate buffer solution containing (manufactured by Wako Pure Chemical Industries)
(Hereinafter, referred to as “washing solution”), the plate was washed three times. Then, as the block body contained in the first blocking agent, 1
Phosphate buffer solution containing 30% bovine serum albumin (Sigma) or 5% skim milk (Snow Brand Milk)
The plate was blocked by dispensing 0 μl each.
After incubating at 37 ° C for 5 minutes, the supernatant was discarded and PGRP
A plate was made. This plate was used for the following measurements.

(2) Measurement of PG PG (manufactured by Wako Pure Chemical Industries) was diluted with a phosphate buffer solution containing 1% bovine serum albumin or 5% skim milk,
A two-fold dilution series having a maximum concentration of 20 μg / ml was prepared (20, 10, 5, 2.5, 1.25 μg / m 2).
l). 100 μl of the PG dilution corresponding to each well of the blocked PGRP plate prepared above
PGRP for 1 hour at 37 ° C.
And PG were bound.

After washing three times with a washing solution, PG labeled with biotin was used using an ECL protein biotinylation system (manufactured by Amersham Pharmacia Biotech).
Prepare RP, dilute it with phosphate buffer containing 5% skim milk to a concentration of 10 μg / ml,
100 μl was dispensed into each well. Then at 37 ° C for 1
After incubation for an hour, washing was performed three times with a washing solution. Then, 100 μl of peroxidase-labeled streptavidin (manufactured by Amersham Pharmacia) diluted 160-fold with a phosphate buffer solution containing 0.5% casein was added, and the mixture was incubated at 37 ° C. for 1 hour to bind the streptavidin to biotin.

The solution was discarded from the well, and the well was washed four times with a washing solution, and 100 μl of ABTS reagent (manufactured by KPL) was added.
Color was developed by incubation at 5 ° C. After 30 minutes, the reaction was stopped by adding 100 μl of a stop solution, and the absorbance at 405 nm was measured using a microplate reader.

(3) Results A graph was prepared by plotting the PG concentration on the horizontal axis and the absorbance on the vertical axis. As shown in FIG. 2, when the bovine serum albumin was used as a block, a measurement curve of PG could be drawn. On the other hand, those using skim milk as a block
Since no was detected, a measurement curve could not be drawn.

Example 3 Selection of a Block at the Time of Binding of Labeled PGRP: (1) Preparation of PGRP Plate Purified PGRP (The Journal of Biological Chemistry,
Vol.271, No.23, pp13854-13860, 1996.)
/ Ml of a phosphate buffer solution (pH 5.8) containing water-soluble carbodiimide (manufactured by Dojindo Laboratories) at a concentration of 50
It was diluted to give μg / ml. This diluted solution is used for ELI
100 μl was dispensed into each well of an SA plate carbo (Sumitomo Bakelite) and incubated at 37 ° C. for 2 hours to bind PGRP to the plate. The reaction solution was discarded, and washing was performed three times with 300 μl of a phosphate buffer solution containing 0.05% Tween-20 (manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as “washing solution”). Next, 300 μl of a phosphate buffer solution containing 1% bovine serum albumin (manufactured by SIGMA) was dispensed to block the plate. After incubation at 37 ° C for 10 minutes, the supernatant was discarded and PG
An RP plate was prepared. This plate was used for the following measurements.

(2) Measurement of PG PG (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with a phosphate buffer solution containing 1% bovine serum albumin, and two 2-fold dilution series having a maximum concentration of 10 μg / ml were prepared. (10, 5, 2.
5, 1.25 μg / ml). 100 μl of the PG diluent is added to each well of the PGRP plate and incubated at 37 ° C. for 2 hours 3
PGRP and PG were bound by incubating for 0 minutes.

After washing with a washing solution three times, PG labeled with biotin was used using an ECL protein biotinylation system (manufactured by Amersham Pharmacia Biotech).
RP was diluted with a phosphate buffer solution containing 1% bovine serum albumin or 2% skim milk (Snow Brand Milk Products) to a concentration of 10 μg / ml, and 100 μl was dispensed into each well of each dilution series. . After incubation at 37 ° C. for 1 hour, washing was performed three times with a washing solution. Then 0.5%
100 μl of peroxidase-labeled streptavidin (manufactured by Amersham Pharmacia Biotech) diluted 500-fold with a phosphate buffer solution containing casein was added, and 37
Strepavidin was bound to biotin by incubation at 1 ° C for 1 hour.

The well solution was discarded, and the well was washed four times with a washing solution, and 100 μl of ABTS reagent (manufactured by KPL) was added.
Color was developed by incubating at ° C. After 30 minutes the reaction was stopped by adding 100 μl of stop solution,
The absorbance at 405 nm was measured using a microplate reader.

(3) Results A graph was prepared with the PG concentration on the horizontal axis and the absorbance on the vertical axis. As shown in FIG. 3, the measurement curve of peptidoglycan was drawn in the case of using skim milk as a block, as shown in FIG. In the case of using bovine serum albumin as a block, the measurement curve could not be drawn because the absorbance was 2.0 or more in all cases including the PG amount of 0.

Example 4 Measurement of βG: (1) Preparation of PGRP Plate Purified PGRP (The Journal of Biological Chemistry,
Vol.271, No.23, pp13854-13860, 1996.)
/ Ml of a phosphate buffer solution (pH 5.8) containing a water-soluble carbodiimide (manufactured by Dojindo Laboratories) at a concentration of 50 μg /
It was diluted to a volume of ml. This diluted solution is added to each well of an ELISA plate carbo (Sumitomo Bakelite).
PGRP was bound to the plate by dispensing 00 μl each and incubating overnight at 4 ° C. with gentle shaking. The next day, the reaction solution was discarded, and 0.05% Tween-
300μ phosphate buffer solution containing 20 (manufactured by Wako Pure Chemical Industries)
1 (hereinafter referred to as “washing solution”) three times. Next, 300 μl of a phosphate buffer solution containing 1% bovine serum albumin (manufactured by SIGMA) was dispensed to block the plate. After incubation at 30 ° C for 10 minutes,
The supernatant was discarded to prepare a PGRP plate. This plate was used for the following measurements.

(2) Measurement of βG As βG, carboxymethyl curdlan (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with a phosphate buffer solution containing 1% bovine serum albumin to give a 2-fold dilution with a maximum concentration of 50 μg / ml. Series were created (50, 25, 12.5, 6.25, 3.12
5 μg / ml). As a control, PG (manufactured by Wako Pure Chemical Industries)
Was similarly diluted to prepare a two-fold dilution series having a maximum concentration of 50 ng / ml (50, 25, 12.5, 6.25,
3.125 ng / ml). 100 μl of βG or PG diluent is added to each well of the PGRP plate at 30 ° C.
PGRP and βG or PG by binding slowly for 3 hours.

After washing with a washing solution three times, PG labeled with biotin was used using an ECL protein biotinylation system (manufactured by Amersham Pharmacia Biotech).
RP was diluted with a phosphate buffer solution containing 0.5% casein (manufactured by Merck) to a concentration of 10 μg / ml, and 100 μl was dispensed into each well. Next, the mixture was incubated at 30 ° C. for 1 hour with gentle shaking, and then washed three times with a washing solution. Then, a peroxidase-labeled streptavidin (Amersham Pharmacia) diluted 1000-fold with a phosphate buffer solution containing 0.5% casein.
Biotech) (100 μl) was added, and incubated at 25 ° C. for 1 hour to bind streptavidin to biotin.

The solution was discarded from the well, and the well was washed four times with a washing solution, and 100 μl of TMB reagent (manufactured by KPL) was added.
Color was developed by incubating at ° C. One hour later, the reaction was stopped by adding 100 μl of 1M phosphoric acid, and the absorbance at 450 nm was measured using a microplate reader.

(3) Results A graph was prepared with the PG and βG concentrations on the horizontal axis and the absorbance on the vertical axis. As shown in FIG. 4, although a measurement curve could be drawn for PG, βG was not detected. The measurement curve could not be drawn.

[0061]

The present invention relates to a method for detecting a microorganism or a microbial component by measuring a membrane polysaccharide using a specific binding protein, wherein the microorganism is unknown or mutated. Regardless of whether they are present, they can detect the presence of microorganisms and their components widely. In addition, by performing the method of the present invention while changing the membrane polysaccharide to be measured, it is possible to detect not only the presence of microorganisms but also the classification of microorganisms. .

Therefore, the present invention is useful for applications such as diagnosis at medical sites, safety tests for pharmaceuticals, etc., and inspection of water and food at manufacturing sites.

[Brief description of the drawings]

FIG. 1 is a drawing showing the relationship between PG concentration and absorbance (λ = 450 nm).

FIG. 2 is a drawing showing the relationship between PG concentration and absorbance (λ = 405 nm).

FIG. 3 is a drawing showing the relationship between PG concentration and absorbance (λ = 405 nm).

FIG. 4: PG concentration and βG concentration and absorbance (λ = 45
0 nm). that's all

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/543 541 G01N 33/543 541B 541Z 33/547 33/547

Claims (16)

[Claims] 1. A method for reacting a sample with a protein that specifically binds to a polysaccharide constituting an outer membrane or a cell wall of a microorganism, and detecting the polysaccharide bound to the protein. A method for measuring microorganisms or microbial components. 2. A sample and a solid phase on which a protein that specifically binds to a polysaccharide constituting the outer membrane or cell wall of a microorganism is allowed to react with a sample, and then the outer membrane of the microorganism bound to the protein on the solid phase Or the polysaccharides that make up the cell wall,
A method for measuring a microorganism or a microbial component in a specimen, wherein the detection is performed with a protein that specifically binds to a polysaccharide constituting an outer membrane or a cell wall of the microorganism labeled with a labeling substance. 3. A protein that specifically binds to a polysaccharide constituting the outer membrane or cell wall of a microorganism immobilized on a solid phase, and a polysaccharide constituting the outer membrane or cell wall of the microorganism labeled with a labeling substance. 3. The method for measuring a microorganism or a microorganism component according to claim 1, wherein the protein that specifically binds is the same protein. 4. Polysaccharides constituting the outer membrane or cell wall of the microorganism are lipopolysaccharides constituting the outer membrane present on the surface of Gram-negative bacteria, and β-1 constituting the cell wall of fungi such as yeasts, molds and mushrooms. 4. The method for measuring microorganisms or microbial components according to claim 1, which is a peptidoglycan constituting a cell wall of most prokaryotes, or 3-glucan. 5. The polysaccharide constituting the outer membrane or cell wall of the microorganism is a peptidoglycan, and the protein specifically binding to the polysaccharide constituting the outer membrane or cell wall of the microorganism is a peptidoglycan recognition protein. The method according to any one of claims 1 to 4, wherein is a prokaryotic microorganism or a component thereof. 6. A reaction between a specimen and a protein that specifically binds to a polysaccharide constituting an outer membrane or a cell wall of a microorganism, wherein at least one compound selected from serum proteins, milk component proteins, and gelatin is blocked. The method for measuring a microorganism or a microbial component according to any one of claims 1 to 5, wherein the method is added as a component. 7. The method according to claim 6, wherein the block is serum albumin. 8. A conjugate of a polysaccharide constituting an outer membrane or a cell wall of a microorganism and a protein which specifically binds to the polysaccharide is a protein which specifically binds to a microorganism component labeled with a labeling substance. The microorganism or the microorganism according to any one of claims 2 to 7, wherein in the labeling reaction, at least one selected from serum protein, milk component protein, and gelatin is added as a block. Method for measuring microbial components. 9. The method according to claim 8, wherein the block is a milk component protein. 10. The carrier according to claim 2 or 3, wherein the carrier used for the solid phase has a carboxyl group on its surface.
The method for measuring a microorganism or a microorganism component according to the above item. 11. The method according to claim 2, wherein the immobilization is performed using carbodiimide. 12. The following components (a) and (b): (a) a solid phase on which a protein that specifically binds to a polysaccharide constituting the outer membrane or cell wall of a microorganism is immobilized; and (b) a labeling substance. A kit for measuring a microorganism or a microorganism component containing a protein that specifically binds to a polysaccharide constituting the outer membrane or cell wall of a labeled microorganism. 13. The kit according to claim 12, wherein the labeling substance of the component (b) is an enzyme, a radioactive substance, a fluorescent substance or a luminescent substance. 14. The kit according to claim 12, further comprising serum albumin as the first blocking agent and milk component protein as the second blocking agent. 15. The kit for measuring a microorganism or a microorganism component according to claim 12, wherein the carrier used for the solid phase has a carboxyl group on its surface. 16. The kit according to claim 12, wherein the immobilization is performed using carbodiimide.