JPH09211000A - Sensing method for yellow staphylococcus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To sense yellow staphylococcus simply, quickly, and accurately by using fowl immune globulin as an antibody. SOLUTION: The immunity is measured, for example by the sandwich ELISA method, and examples of the antibody are the primary and/or secondary antibody. Example of the fowl immune globulin (IgY) is yellow staphylococcus, concretely a one which reacts with Protein A peculiarly. Even though the specimen consists of a substance coupling non-immunologically with an antibody originating from mammalia or a mixture thereof with bacteria, etc., to produce such substance, the yellow staphylococcus can be sensed peculiarly and with a good sensitivity by using IgY.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting Staphylococcus aureus.

[0002]

2. Description of the Related Art Staphylococcus aureu
s) is a kind of pathogenic bacteria that causes various diseases in humans and animals. When Staphylococcus aureus contaminates food and grows there, extracellular toxin (enterotoxin) is produced. 2 when you eat food containing enterotoxin
Symptoms of acute gastroenteritis appear after ~ 6 hours, followed by vomiting,
Causes abdominal pain and diarrhea. In severe cases, slight fever accompanies marked intoxication symptoms such as decreased blood pressure, chest distress, cloudy consciousness, and decreased pulse rate, and may require emergency hospitalization. MRSA (mesitillin-resistant Staphylococcus aureus), which is a type of Staphylococcus aureus, is a causative bacterium of nosocomial infectious diseases, which has become a major social problem.

[0003] Since Staphylococcus aureus is closely related to humans in terms of food hygiene and medicine, it is required to establish a simple, quick and highly accurate method for detecting S. aureus. Until now, the detection method of Staphylococcus aureus has mainly been a culture method using a selective separation medium. In the culturing method, first, preculture is performed for 48 hours using a mannitol salt medium or the like. Next, pure culture is carried out for 24 hours to identify the bacteria, and further the identification work of the bacteria is carried out using rabbit plasma and PS latex (Food Hygiene Inspection Guidelines, Microorganisms Edition). In the cultivation method, the preservability of the selective separation medium and the complexity of preparing the medium are problems, and the cultivation takes much time.

As a detection method other than the culture method, DNA for a ribosomal RNA gene specific to Staphylococcus aureus is used.
A method using a probe (Japanese Patent Laid-Open No. 5-49477) is known, but this method is complicated as a daily inspection method.
There is a problem in that it is not simple in that NA extraction operation is required and that the cost is higher than that in the culture method.

As a detection method other than the above, an enzyme immunoassay method (Enzyme-linke) using an antibody against a thermonuclease or enterotoxin produced by Staphylococcus aureus
d immunosorbent assay, ELISA method [Archiv fur Lebensmittelhygiene.35,97 (1984), JP-A-6-88824], polystyrene latex sensitized with human fibrinogen and immunoglobulin G (hereinafter referred to as "IgG") A method utilizing the fact that particles specifically react with protein A produced by Staphylococcus aureus to cause an agglutination reaction (Japanese Patent Publication No. 2-502942) is known.

In the above-mentioned agglutination reaction method or ELISA method, mammal-derived IgG (eg, mouse, rabbit, etc.) is used as the antibody. However, the method using mammalian-derived IgG has a problem that the IgG binds to protein A produced by S. aureus non-immunologically. Protein A is a protein produced by the majority of S. aureus (85-95%) and occupies 5% of cell wall components. Protein A non-immunologically binds to the Fc fragment of IgG of mammals (eg, human, mouse, rabbit, etc.).

The term "non-immunological binding" means binding of an antigen to a region other than the V region which is the antigen binding site of an antibody. In addition, IgG derived from mammals is
It also binds to protein G produced by ptococci in a non-immunological manner (Neochemistry Laboratory, Molecular Immunology III). Therefore, in the method using IgG derived from mammal,
When the bacterial cells of Group G Streptococci or protein G were contaminated, there was a problem that the antibody showed cross-reactivity with these contaminants and specific detection of S. aureus was difficult.

[0008]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a simple, rapid and highly accurate method for detecting Staphylococcus aureus.

[0009]

Means for Solving the Problems As a result of intensive research based on the above problems, the present inventor has found that by using chicken immunoglobulin as an antibody in a method for detecting Staphylococcus aureus, yellow The inventors have found that cocci can be detected, and completed the present invention. That is,
The present invention is a method for detecting Staphylococcus aureus characterized by using chicken immunoglobulin as an antibody.

Here, the immunoassay method includes a sandwich ELISA method, and the antibodies include primary and / or secondary antibodies. Further, as chicken immunoglobulin, Staphylococcus aureus, specifically protein A
Those that react specifically with Furthermore, the present invention is a reagent for detecting Staphylococcus aureus that contains chicken immunoglobulin that specifically reacts with Staphylococcus aureus.

Hereinafter, the present invention will be described in detail. The present invention is characterized by using chicken immunoglobulin (hereinafter also referred to as “IgY”) as an antibody in the method for detecting Staphylococcus aureus. IgY is different from antibodies obtained from mammals, and protein A produced by Staphylococcus aureus and protein G produced by Group G Streptococci
It is known that it does not bind non-immunologically.

[0012] Therefore, by using IgY, even Staphylococcus aureus is specific even in a sample in which a substance that non-immunologically binds to a mammal-derived antibody and a microorganism that produces it are mixed. It is possible to detect with high sensitivity. As IgY of the present invention, either a polyclonal antibody or a monoclonal antibody can be used.

The production of polyclonal IgY can be carried out according to a conventional method. For example, the antigen is suspended in Freund's adjuvant, or the antigen is directly administered to chickens for immunization and boosting. After the final booster immunization, blood is collected after a certain period of time, and serum is obtained according to a conventional method. Ammonium sulfate or the like is added to the obtained serum for salting out to obtain a protein fraction. The protein fraction is applied to a carrier-bound protein G column and the polyclonal antibody is adsorbed on the column. The adsorbed fraction is eluted according to a conventional method to obtain a polyclonal antibody.

As a method for efficiently preparing polyclonal IgY, a method for purifying a polyclonal antibody from chicken eggs produced by an antigen-administered chicken can be used ([J. Vet. Med. B33, 609 (1986)]). The specific preparation method is described in Example 4. Note that the purification of the polyclonal antibody from chicken eggs can also be performed using a commercially available purification kit (Pharmacia).

The production of monoclonal IgY can also be carried out according to a conventional method. For example, a method of purifying a monoclonal antibody from a fused cell (hybridoma) of a chicken B lymphoblast-derived thymidine kinase-deficient strain and a chicken splenocyte immunized can be used (JP-A-2-186980).

As a method for detecting Staphylococcus aureus of the present invention, a known immunological measurement method can be used as long as it utilizes an antigen-antibody reaction. Examples thereof include agglutination reaction method, enzyme immunoassay method (EIA method), radioimmunoassay method (RIA method), and fluorescence immunoassay method (FIA method). Among them, EIA method, especially sandwich ELIS
Method A (Enzyme-linked immunosorbent assay) is a preferable method because of its extremely high sensitivity. Side switch EL
The detection method of the present invention using the ISA method is shown below.

In the sandwich ELISA method, first, an antibody (primary antibody) is immobilized on a solid carrier. The immobilized primary antibody is called a solid-phased antibody. Then, after the antigen in the sample is bound to the immobilized antibody, the unbound antigen is removed. Then, an antibody (secondary antibody) labeled with an appropriate enzyme is added to bind to the antigen on the immobilized antibody. Alternatively, the secondary antibody is not labeled and an antibody (3
There is also a method of labeling a secondary antibody). Finally, the activity of the labeling substance bound to the immobilized antibody is measured to determine the presence or absence of the antigen and its amount.

When the sandwich ELISA method is used in the present invention, only one of the primary antibody and the secondary antibody may be IgY, or both may be IgY. When IgY is used as the primary antibody, the secondary antibody may be IgY or an antibody derived from a non-chicken animal (eg, mammalian IgG such as rabbit, mouse and goat). When a non-chicken animal-derived antibody is used as the primary antibody, IgY is used as the secondary antibody. As the antibody derived from a non-chicken animal, either a polyclonal antibody or a monoclonal antibody can be used.

Preparation of a polyclonal antibody derived from a non-chicken animal can be carried out by a conventional method. For example, a polyclonal antibody is obtained from the serum of an animal (for example, rabbit, mouse, goat, etc.) immunized with the antigen by the same method as that for obtaining the polyclonal antibody IgY. The specific method is described in Example 1.

The monoclonal antibody can also be prepared by a conventional method. For example, spleen cells of a non-chicken animal immunized with an antigen are fused with myeloma cells to obtain a hybridoma, which is inoculated into the abdominal cavity of an animal such as BALB / c mouse, and ascites is collected from the animal. After the obtained ascites fluid is clarified by centrifugation or the like, it is applied to a column on which protein G is immobilized to adsorb the antibody. The monoclonal antibody can be purified by eluting the adsorbed antibody according to a conventional method. The specific method is described in Example 3. In addition to this, the hybridoma is cultured in a conventional culture medium,
It is also possible to use a method for purifying a monoclonal antibody secreted in the culture medium.

The antigen with which the antibody of the present invention specifically reacts may be any of the biological components of Staphylococcus aureus, such as protein (protein A, thermonuclease, enterotoxin, etc.), nucleic acid, polysaccharide,
Lipids and other cell surface components can be used. When the antibody of the present invention is obtained by immunizing an animal having antibody-producing ability, the antigen to be administered is not only the purified product of the above-mentioned biological components, but also the whole bacterial cell (eg, heat-treated bacterial cell, formalin-treated bacterial cell, etc.). May be

In order to detect and quantify Staphylococcus aureus with high accuracy, it is preferable to select a biological component specific to S. aureus as an antigen.
In particular, protein A is the majority of Staphylococcus aureus (85-95
%) Produced by the cell wall protein and is suitable as an antigen.

In the sandwich ELISA method, first, a primary antibody is immobilized on a solid support to prepare a solid-phased antibody. As the solid carrier, for example, various microtiter plates, microtiter tubes, magnetic particles and the like can be used. If the primary antibody is diluted with an appropriate buffer solution (eg, carbonate buffer solution, phosphate buffer solution, borate buffer solution, etc.) and then contacted with a solid carrier, and incubated at 4 to 37 ° C. for 30 minutes or more, 1 The secondary antibody is immobilized on a solid support.

Next, the solid carrier is treated with a surfactant [Tween 20
(Manufactured by Bio-Rad) and the like] (for example,
Unadsorbed antibody is removed by washing several times with phosphate buffer, Tris buffer, borate buffer, etc.).

Next, in order to block the free binding groups on the surface of the solid support, the solid support is brought into contact with the blocking buffer with the blocking buffer. As a blocking buffer, bovine serum albumin (BSA), ovalbumin
A buffer solution containing (OVA), skim milk, etc. (eg, phosphate buffer solution, Tris buffer solution, borate buffer solution, etc.) can be used. The blocking treatment is preferably performed at 4 to 37 ° C for 30 minutes or more.

Next, the solid support is washed with a buffer containing a surfactant to remove the blocking buffer. This washing can be performed in the same manner as the washing after immobilization of the above-mentioned monoclonal antibody or polyclonal antibody. Thus, the immobilized antibody is prepared.

Next, the immobilized antibody is contacted with a sample suspected of containing Staphylococcus aureus. As a result, Staphylococcus aureus in the sample specifically binds to the immobilized antibody. This contact is preferably performed at 4 to 37 ° C for 30 minutes or more. When diluting the sample, phosphate buffer, Tris buffer, borate buffer, physiological saline, etc. can be used. Next, the immobilized antibody is washed as described above.

Next, the immobilized antibody is brought into contact with a buffer solution containing a secondary antibody (eg, phosphate buffer solution, Tris buffer solution, borate buffer solution, etc.). This contact is preferably performed at 4 to 37 ° C for 30 minutes or more. As a result, the secondary antibody
It specifically binds to Staphylococcus aureus immobilized on the immobilized antibody. Next, washing is performed as described above to remove unbound secondary antibody.

In order to detect the secondary antibody immobilized on the immobilized antibody, the secondary antibody is labeled with an appropriate enzyme. As the enzyme, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, luciferase, acetate kinase and the like can be used.

When peroxidase is used as the enzyme, hydrogen peroxide is used as the substrate and 2, as the coloring reagent.
2'-azinodi- (3-ethylbenzthiazoline-6-
Sulfonic acid) (ABTS), 3,3 ′, 5,5′-tetramethylbenzidine and the like are used. The specific method is described in Example 6.

In the sandwich ELISA method, 2
The fixed amount of the secondary antibody reflects the number of fixed Staphylococcus aureus, that is, the number of Staphylococcus aureus in the sample. Therefore, by measuring the fixed amount of the secondary antibody,
The amount of Staphylococcus aureus in a sample can be predicted.

As an alternative to the sandwich ELISA method,
There is also a method of enzymatically labeling an antibody against the secondary antibody (tertiary antibody) without enzymatically labeling the secondary antibody. As a third antibody,
An antibody of an animal different from the animal used for preparing the secondary antibody, which is an antibody against immunoglobulin of the animal species used for preparing the secondary antibody, or a fragment thereof is used. For example, when the secondary antibody is IgY, goat IgG that specifically reacts with IgY may be used as the tertiary antibody. For the labeling of the tertiary antibody, the same method as that for labeling the secondary antibody can be used.

[0033]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to these examples. In this example, Staphylococcus aureus was detected by the sandwich ELISA method. Rabbit immunoglobulin (polyclonal antibody) was used as the primary antibody.
IgY (polyclonal antibody) was used as the next antibody. The secondary antibody was labeled with peroxidase.

Example 1 Preparation of Primary Antibody (Rabbit-Derived Polyclonal Antibody) Staphylococcus aureus 494 (9-16)
Strain; donated by National Institute of Preventive Health, Department of Food Hygiene and Microbiology]
The overnight culture solution was heat-treated at 100 ° C. for 15 minutes and then collected by centrifugation. Suspend the cells in physiological saline, 10 mg
/ ml concentration. Freund's complete and incomplete adjuvants were added to the cell suspension to emulsify. The emulsion was intraperitoneally injected into rabbits three times every three weeks for immunization and booster immunization. Blood was collected 6 days after the final immunization to obtain serum according to a standard method. Ammonium sulfate was added to the obtained serum so as to be 33% saturated, and the salted-out product was collected by centrifugation.
After dissolving the salted out product in physiological phosphate buffer (PBS),
The lysate was dialyzed against PBS to remove ammonium sulfate. The lysate after dialysis is treated with High Trap Protein G
The sample was applied to a column (Pharmacia) and the immunoglobulin was adsorbed to the column. After washing the column with neutral PBS, the immunoglobulin adsorbed on the column was eluted with a glycine hydrochloride buffer (pH 2.7) to obtain a polyclonal antibody.

Example 2 Preparation of Secondary Antibody A chicken-derived anti-protein A antibody (polyclonal antibody) was used as a secondary antibody. The secondary antibody preparation is Lsch
It was performed according to the method of J. Vet. Med. B33, 609 (1986)!

In a protein A (Pharmacia) solution,
Freund's complete adjuvant was added and emulsified. The emulsion was injected into chickens (white leghorn) every 10 days for a total of 4 injections for immunization and booster immunization. Eggs were collected 5 days after the final immunization to separate the yolk. 4 times as much yolk as 0.
After diluting with 0.01 M phosphate buffer (pH 7.6) containing 1 M NaCl, polyethylene glycol (Mw.8000) was added so as to be 3.5%. This was centrifuged at 14,000 xg for 20 minutes. Polyethylene glycol (Mw.8000) was added to the centrifugation supernatant so that the final concentration was 12%. The precipitated fraction was applied to a column packed with DEAE-cellulose, and a fraction eluted with a 250 mM potassium phosphate (pH 8.0) solution was obtained. Ammonium sulfate was added to the elution fraction so as to be 50% saturated, and the salted out product was collected by centrifugation. The salted out product was dissolved in physiological saline and dialyzed against PBS to obtain a secondary antibody fraction.

Example 3 Enzyme Labeling of Secondary Antibody The secondary antibody was labeled using peroxidase as a labeling enzyme by the method of Nakane et al. (J. Histochem. Cytochem. 22,
1974). First, peroxidase is oxidized with periodate and then bound to a secondary antibody, and then N
It was stabilized by reducing with aBH ₄ . The reaction solution was dialyzed against PBS to remove the precipitate, and then purified by gel filtration to obtain a labeled secondary antibody.

Example 4 Detection of Staphylococcus aureus by ELISA (1) In each well of a 96-well microplate (Sumitomo Bakelite Co., Ltd.), a primary antibody solution [10 μg / ml primary antibody,
100% of 0.1% carbonate buffer (pH 9.0)] was injected and incubated at room temperature for 2 hours. After removing the primary antibody solution from the wells, 200 μl of a washing solution (phosphate buffer solution containing Tween 20) was added to each well, and the washing solution was removed after 3 minutes. The well was washed by repeating this washing operation three more times.

Next, 200 μl of blocking solution (phosphate buffer containing skim milk) was added to each well and incubated at 37 ° C. for 1 hour to block non-specific binding sites in the wells. After removing the blocking solution from the wells, 3
Washed twice.

(2) As a sample solution, Staphylococcus aureus [St
aphylococcus aureus 494 (9-16) strain] for 24 hours and a diluted solution with physiological saline were prepared. 100 sample solution
μl was added to each well and incubated at 37 ° C. for 1 hour. After removing the sample solution from the wells, the wells were washed three times.

(3) The labeled secondary antibody was diluted with the above blocking solution to a concentration of 1 μg / ml, 100 μl was added to each well, and the mixture was incubated at 37 ° C. for 1 hour. After removing the labeled secondary antibody solution from the wells, the wells were washed three times with the above washing solution.

(4) To each well, add 100 μl of the substrate solution (0.003%, hydrogen peroxide, 0.04%, ABTS) and incubate at 37 ° C. for 1 hour.
After incubating for 5 minutes to cause an enzymatic reaction, 100 μl of a stop solution (1% sodium lauryl sulfate) was added to stop the reaction. The microplate was mounted on a plate reader (Labo Systems) and the absorbance at 405 nm was measured. The concentration of S. aureus in the sample solution
The relationship with the absorbance at nm is shown in FIG.

As is clear from FIG. 1, in the assay method of the present invention, the concentration of a 24-hour culture of Staphylococcus aureus
A volume dependence was confirmed with the absorbance at nm.
Therefore, it can be seen that Staphylococcus aureus can be detected easily, quickly and highly accurately by the method of the present invention.

Example 5 Using the same method as in Example 4, the concentration of various staphylococci including Staphylococcus aureus, and
The relationship with the absorbance at 05 nm was investigated. The strain used was Staphylococcus aureus (Staphylococcus aureus 494 strain),
Staphylococcus (Staphylococcus capitis T13 strain) and Staphylococcus epidermidis T6 strain (All strains are donated by National Institute of Preventive Health and Food Sanitation Microorganisms Division).

FIG. 2 shows the results. As is apparent from FIG. 2, in the assay of the present invention, the concentration of 24-hour cultures of a strain of staphylococcus different from S. aureus did not show a dose-dependent relationship between the absorbance at 405 nm. That is, it was confirmed that the assay method of the present invention does not show cross-reactivity with staphylococci other than S. aureus.

Example 6 The same method as in Example 4 was used to examine the specificity of bacteria different from staphylococci. The strain used was Escherichia coli W
P2 strain, Salmonella typhimurium TA100
Strain) (All strains are distributed from the National Institute of Health Sciences, Department of Mutation and Genetics). The results are shown in FIG. As is clear from FIG. 3, it was confirmed that the assay method of the present invention does not show cross-reactivity with bacteria of a different type from Staphylococcus aureus.

[0047]

The present invention provides a simple, rapid and highly accurate method for detecting Staphylococcus aureus.

[Brief description of drawings]

FIG. 1 is a diagram showing the relationship between the concentration of Staphylococcus aureus and the absorbance.

FIG. 2 is a diagram showing the relationship between the concentration of various staphylococci and the absorbance.

FIG. 3 is a diagram showing the relationship between the concentration of bacteria other than various staphylococci and the absorbance.

Claims (6)

[Claims] 1. A method for detecting Staphylococcus aureus, which comprises using chicken immunoglobulin as an antibody. 2. The method for detecting Staphylococcus aureus according to claim 1, wherein the detection method is a sandwich ELISA method. 3. The method for detecting Staphylococcus aureus according to claim 1 or 2, wherein the antibody is a primary and / or secondary antibody. 4. The method for detecting S. aureus according to claim 1, wherein the chicken immunoglobulin specifically reacts with S. aureus. 5. The chicken immunoglobulin is protein A.
The method for detecting Staphylococcus aureus according to any one of claims 1 to 3, which specifically reacts with. 6. A reagent for detecting Staphylococcus aureus comprising a chicken immunoglobulin which specifically reacts with Staphylococcus aureus.