JP2003215126A - Method for extracting microbe antigen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a simple method for extracting a saccharide chain antigen by concentrating the microbes having the saccharied chain antigen in a liquid to be extracted and to determine the quantity of the microbes in the liquid to be extracted by using the obtained saccharide chain extracted liquid. <P>SOLUTION: The saccharide chain antigen is extracted by filtering the liquid to be extracted which may contain the microbes having the saccharide chain antigen by a filter perforation, preferably by a filter perforation in which the pore size is 0.8-2 μm and reacting the microbes held on the filter perforation with nitrous acid solution. The quantity of the saccharide chain antigen is determined by immunoassay method using the antibody united with the saccharide chain antigen. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for concentrating a microorganism in a liquid to be extracted by filtration to extract a sugar chain antigen of the microorganism, a filtering device therefor, and a sugar chain prepared as described above. The present invention relates to a highly sensitive assay method for microorganisms, which determines the amount of sugar chain antigen in an antigen extract by an immunological assay method.

[0002]

2. Description of the Related Art Microbial inspection is an important inspection item in medical inspection, food inspection, or environmental measurement.

It is known that a wide variety of microorganisms are present in the oral cavity, and among these microorganisms, there may be microorganisms that cause infectious diseases. For example, it is known that caries and periodontal disease are strongly associated with caries and periodontal disease-related bacteria. In recent years, it has become clear that there are cases where microorganisms present in the oral cavity are involved in the development of diseases that were previously thought to be unrelated to infectious diseases, such as heart disease, gastric ulcer, and cancer. Microbial tests are becoming important not only in the dental field but also in the medical field.

The measurement of microorganisms has been conventionally carried out by a culture method, and there was a problem that it took a long time until the result was clarified, but an immunological measurement method was developed and the examination time was greatly shortened. The immunological measurement method is a method of detecting the microorganism with high sensitivity by using an antibody against a specific antigen possessed by the microorganism.

The detection sensitivity can be further enhanced by measuring the antigen extract prepared by extracting the antigen from the microorganism by an immunological measuring method. The reason is 1)
It is considered that the antigen is solubilized and becomes a low molecular weight compound, so that the antigen-antibody reaction is likely to proceed, 2) the epitope is exposed, and the like.

In particular, when an antigen-containing solution is spread on the spreading membrane and the antigen is measured by an immunochromatographic method in which the antigen is detected by an antigen-antibody reaction, the extracted antigen has a better spreading property than the insoluble antigen. Highly sensitive measurement can be expected by extracting the antigen from the microorganism.

As a method for extracting a microbial antigen, various methods such as surfactant treatment, enzyme treatment, and heat treatment have been put into practical use. As a method for extracting a sugar chain antigen, a nitrite extraction method (Takei Takei. Osaka Univ. Medical Journal 35: 93-109, 199.
0. )It has been known. The method is particularly effective for the extraction of sugar chain antigens of microorganisms belonging to the genus Streptococcus such as group A streptococci and oral streptococci.

This nitrous acid extraction method is a method in which an aqueous nitrite solution is reacted with sugar chains and the sugar chains are cleaved to be released from the cells. A general nitrite extraction method is a liquid to be extracted containing microorganisms. Nitrite by mixing an aqueous nitrite solution (in most cases sodium nitrite) and an aqueous acid solution (acetic acid, hydrochloric acid, nitric acid, etc.) (hereinafter, these solutions are also collectively referred to as "nitrous acid extraction reagents"). It is carried out by allowing the microorganisms to react with the nitrous acid for a sufficient time. Since the above reaction solution is strongly acidic, the base [2-amino-2-
Hydroxymethyl-1,3-propanediol (hereinafter,
[Tris]), sodium hydroxide, sodium hydrogen carbonate, etc.] is added to neutralize the reaction solution, and then the analysis is generally performed.

[0009]

However, when extracting the sugar chain antigen of the microorganism belonging to the genus Streptococcus from the liquid to be extracted containing saliva, dental plaque, or a mixture thereof, etc., the liquid to be extracted is Since the concentration of microorganisms contained is low, it was sufficient to add a nitrous acid extraction reagent directly to the liquid to be extracted to prepare a sugar chain antigen extract. It was difficult to extract the sugar chain antigen at the concentration.

Therefore, even when the content of the microorganism having the sugar chain antigen contained in the liquid to be extracted is low, the sugar chain antigen can be easily extracted at a concentration at which the measurement by the immunological measurement method is sufficiently possible. It was desired to develop a method.

[0011]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to solve the above problems. As a result, when extracting the sugar chain antigen from the liquid to be extracted that may contain a microorganism having a sugar chain antigen, by combining the concentration by filtration and the nitrite extraction method, the above problems The inventors have found that they can be solved and completed the present invention.

That is, according to the present invention, a liquid to be extracted which may contain a microorganism having a sugar chain antigen is filtered with a filter medium, and the microorganism retained on the filter medium is treated with an aqueous nitrite solution. A method for extracting a microbial antigen, which comprises extracting the sugar chain antigen.

In the present invention, the amount of sugar chain antigen contained in the sugar chain antigen extract extracted by the above method is measured by an immunological measuring method, and the sugar chain antigen contained in the liquid to be extracted is measured based on the measurement result. Also provided is a method for measuring a microorganism, which comprises determining the amount of the microorganism having a sugar chain antigen.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, microorganisms include bacteria, genus Rickettsia, genus Chlamydia, genus Mycoplasma (M).
yplasma) and unicellular eukaryotes.

The liquid to be extracted used in the present invention is not particularly limited as long as it may contain a microorganism having a sugar chain antigen, but generally, the content of the microorganism having the sugar chain antigen is tested. It is intended to do. Such a liquid to be extracted is usually in a state in which a microorganism having a sugar chain antigen to be measured and other insoluble matter are mixed.

Examples of the liquid to be extracted are culture liquid of microorganisms, suspension of microorganisms, food and its suspension, body fluids such as pharyngeal swab, saliva, plasma, serum and urine, or suspension of dental plaque. Liquid etc. are mentioned. In the case of extracting a sugar chain antigen from a microorganism having a sugar chain antigen present in the oral cavity, the liquid to be extracted generally contains saliva collected from the oral cavity, dental plaque, or a mixture thereof. .

Among microorganisms having a sugar chain antigen contained in saliva and dental plaque, examples of microorganisms that cause infectious diseases include caries-related bacteria, periodontal disease-related bacteria, upper respiratory tract-related bacteria, and opportunistic bacteria. Etc. When a concrete example is displayed for each of them, the caries-related bacteria are Streptococcus mutans (Streptococcus
utans), Streptococcus sobrinus (St)
Bacteria belonging to mutans streptococci such as reptococcus sobrinus, bacteria belonging to the genus Lactobacillus, and bacteria belonging to the genus Actinomyces are Porphyromonas gingivalis (Porphyromion) as periodontal disease-related bacteria.
lis), Actinobacillus actinomycetem comitance (Actinobatillus actinom)
ycetemcomitans), Bacteroides forsyth
us), Campylobacter rectus
bacterium rectus), Eikenella corrodens, Treponema denticola
cola), Prevotella Intermedia (Prev
ottera intermedia), Prevotella
Nigresense (Prevotella nigres
cens), Fusobacterium nucleatane (Fus
Obacterium nucleatum) is a group A streptococcus (Group)
A Streptococcus, Mycoplasma pneumo
niae), Chlamydia pneumoniae (Chlami)
dia pneumoniae) and the like are, as opportunistic infection bacteria, Candida sp. (Candida sp.), Staphylococcus aureus (Staphylococcus aure)
us), Pseudomonas aerug
inosa), Streptococcus pneumoniae
pneumoniae), Haemophilus influenzae (Haemophilus influenzae)
e), Klebsiella pneumoniae (Klebsiell)
a pneumoniae), Serratia marcescens, and the like.

In the extraction method of the present invention, the liquid to be extracted as described above is first filtered using a filter medium to retain the microorganisms present in the liquid to be extracted on the filter medium. By this operation, the microorganism having the sugar chain antigen in the liquid to be extracted is well concentrated on the filter medium. In the present invention, retention of microorganisms on the filter medium means not only those retained on the surface of the filter medium but also those trapped in the pores thereof.

[0019] Conventionally, in a microorganism inspection by an immunological measuring method, when the liquid to be extracted contains a large amount of insoluble matter when concentrating the microorganisms in the liquid to be extracted, there is a risk of clogging and the like. The centrifuge method is mainly adopted rather than the method. However, the centrifugation method requires an expensive centrifuge, and first, the liquid to be extracted must be centrifuged to precipitate the microorganisms, and then the supernatant must be discarded. It was complicated and took time to concentrate. Therefore, it is extremely significant to concentrate a microorganism having a sugar chain antigen by a filtration method capable of performing an operation in a short time using a simple filtration device and utilize this for the inspection of the microorganism.

In the present invention, as the filter medium, a film-shaped or layered filter medium made of a known material can be used without limitation as long as it is stable even after the extraction with nitrous acid. Examples of such a filter material include a screen filter such as a membrane filter and a depth filter such as glass fiber filter paper and filter paper. When filtering a sample liquid containing microorganisms, the material of the membrane filter usually used is cellulose acetate, polyvinylidene fluoride (hereinafter abbreviated as "PVDF"), nylon, etc., but these materials are Since it is slightly unstable under strongly acidic conditions during nitrous acid extraction, it is preferable to use a depth filter made of the glass fiber or filter paper or a screen filter made of a material having sufficient resistance to acid.

In the present invention, the pore size of the filter medium is 90% of that of the microorganism containing the sugar chain antigen to be measured contained in the liquid to be extracted.
It is preferable that the pore diameter is at least 100%.

Here, the size of the microorganism is, for example, Streptococcus mutans or Streptococcus.
Sobrinus should be 0.5-0.9 μm,
In most cases, it is in the range of 0.3 to 1.0 μm. Therefore, in the step of preparing an analyte liquid (sugar chain antigen extract) in a microbiological test, when the extract is filtered, the filter medium should have a pore size sufficiently smaller than the size of the microorganism, specifically, It is common practice to use filter media having a pore size of 0.22 to 0.45 μm.

Also in the present invention, if the liquid to be extracted is filtered using the filter medium having the above-mentioned small pore size, the microorganism having the sugar chain antigen can be satisfactorily captured. Then, the filtration rate of the liquid becomes slower, and when other insoluble substances are mixed in the liquid to be extracted, clogging easily occurs. Then, when clogging occurs in the filter medium in this manner, the extraction efficiency is also increased in the step following the above step, in which the aqueous solution of nitrite is allowed to act on the microorganism having the sugar chain antigen retained on the filter medium. It decreases a little.

From such a background, in the present invention, the filter medium is 0.8 to 2 μm, and more preferably 0.8 to 1.6 μm.
It is preferable to use one having a pore size of m. The pore size is usually slightly larger than the size of the microorganism having the sugar chain antigen retained on the filter medium, but in the case of the microorganism having the sugar chain antigen, the filter medium having such a large pore size is used. However, it can be captured with almost no flow down. Therefore, the filtration speed is greatly improved,
Furthermore, clogging due to other insoluble materials is extremely unlikely to occur,
It becomes possible to more efficiently extract the sugar chain antigen using the aqueous nitrite solution.

In the present invention, the pore size of the filter medium means a value at which the filter medium captures 98% or more of particles larger than the pore size. When the filter material is a screen filter such as a membrane filter, the pore size displayed in the commercial filter media catalog corresponds to the pore size of the present invention, and when the filter media is a depth filter such as glass fiber filter paper or filter paper, the commercial filter media is used. The retained particle size and the particle retention capacity displayed in the catalog correspond to the pore size of the present invention.

The microorganisms retained on the filter medium are preferably washed by flowing a cleaning liquid through the filter device. When the amount of the sugar chain antigen extracted by the method of the present invention is measured by an immunological measuring method, the occurrence of false positives due to a nonspecific reaction can be reduced by performing the above washing.

The filtration method is characterized in that the concentrated microorganisms can be washed easily and in a short time. For example, in the extraction method of the present invention, when the washing operation of the microorganisms retained on the filter medium in this way is added, the washing can be completed in a short time by a simple operation of adding the washing liquid to the filtering device and flowing it down. . On the other hand, when washing the microorganisms by the centrifugation method, the washing operation of the concentrated microorganisms is performed by centrifuging the concentrated liquid and discarding the supernatant, and then suspending the obtained precipitate in the washing liquid. It must be carried out by a complicated method in which the solution becomes turbid and is further centrifuged to discard the supernatant, which usually requires 2 to 5 times the operation time of the filtration method.

Further, by performing the washing in the filtration method as described above, the washing efficiency can be improved as compared with the washing by the centrifugation method, and as shown in Examples described later, the sugar concentrated by the centrifugation method is used. With sugar chain antigens extracted from microorganisms having chain antigens, accurate measurement is possible even when an immunological measurement method is carried out with an antibody having low specificity such that a nonspecific reaction is detected. The effect is also obtained.

As the washing liquid for microorganisms, for example, a liquid such as a buffer solution, an acid aqueous solution, an alkaline aqueous solution, and a surfactant aqueous solution can be used.

The extraction operation of the liquid to be extracted can be carried out according to a general filtration method. In addition to gravity, filtration may be performed under pressure or under reduced pressure. It is preferable to use a syringe filter in which a filter medium is fixed, to inject the liquid to be extracted, and then pressurize using the syringe. Alternatively, for example, the filter material may be fixed in the syringe, the liquid to be extracted may be dispensed into the syringe with the plunger removed, and the plunger may be inserted into the syringe and pressurized to perform filtration.

Further, for example, as shown in FIG. 1, the upper surface of the container is opened, the drainage port (1) is projected on the bottom wall, and the filter medium is provided inside.
Main body container (3) consisting of a fixed cylindrical shape (2)
And a filtration device comprising a lid part (5) that can be fitted to the upper surface of the main body container (3) and has a syringe insertion port (4) protruding from the top plate part.
It is also a preferable embodiment to carry out filtration using (6). That is,
According to this filtering device (6), the liquid to be extracted is injected into the container from the opening on the upper surface of the container body, and then the lid portion (5) is fitted on the upper surface of the container body, and then the syringe. Outlet (4)
By attaching the tip of the pressurizing syringe to the container and pressing the plunger to pressurize the inside of the container and discharge the filtrate from the drainage port (1), the liquid to be extracted can be rapidly pressure-filtered by a simple operation. . Further, according to such a filtration device (6), even after the washing operation after the filtration, and further, when the aqueous solution of nitrite is allowed to act on the microorganisms retained on the filter medium, the same procedure is performed efficiently. Can be operated.

Here, in the filtration device having the above structure, the main body container (3) is preferably made of a flexible material (for example, plastic such as polypropylene). With respect to the main body container (3), the filter medium (2) may be held on the bottom surface by an adhesive, or may be held on the bottom surface only by a frictional force without being particularly adhered.

When the filter medium (2) is directly fixed to the bottom surface of the main body container, the liquid to be extracted having a slow filtration rate (for example, a high viscosity,
When filtering a large amount of insoluble matter, etc.), if the pressure is increased more than necessary to increase the filtration rate, the filter medium (2)
Part of the water is extruded into the drainage port (1), and the filtration rate may be significantly reduced, so caution is required. In such a case, if a support filter, a mesh sheet or the like is placed between the filter medium (2) and the bottom surface, it is possible to prevent the filter medium (2) from entering the drainage port (1), which is more preferable. The support filter, for example, those used for the purpose of preventing outflow from the column of the chromatography carrier in the open column bottom for general liquid chromatography, specifically, glass filter, polypropylene filter, polyester filter, A polyamide filter or the like can be used. As the mesh sheet, for example, a commercially available mesh sheet made of the same material as the support filter can be used.

Further, the fitting of the lid portion (5) to the upper surface of the main body container (3) may be performed not only by frictional force but also by screw screw or other sealing method.

Next, in the method for extracting a microbial antigen of the present invention, when the nitrous acid aqueous solution is allowed to act on the microorganism retained on the filter medium by the above-mentioned method, the microorganism has a sugar chain antigen, The sugar chain antigen is extracted. A known method known as a nitrous acid extraction method can be used without limitation as a method of causing the nitrous acid aqueous solution to act on the microorganism. The nitrite aqueous solution can be generally prepared by mixing the nitrite aqueous solution and the acid aqueous solution. From the viewpoint of extraction efficiency and accuracy in the subsequent immunological assay, it was determined that 0.2 to 8 M nitrite and 0.3 to 4 M acid were used with respect to the volume of the mixed solution. It is desirable to use an aqueous nitrous acid solution obtained by mixing the respective solutions in such amounts.

The amount of the aqueous nitrous acid solution used is 5 to 400 μl, preferably 20 to 30 per 1 cm ² of the upper surface of the filter medium.
0 μl is preferred. The temperature at which the nitrous acid aqueous solution acts is
The temperature is preferably 15 to 50 ° C., and the working time is preferably 1 to 10 minutes.

A specific method of causing the aqueous nitrite solution to act on the microorganisms can be carried out, for example, by supplying an aqueous nitrite solution and an aqueous acid solution onto a filter medium to generate nitrous acid on the filter medium. As an aqueous nitrite solution, for example,
0.2-8M sodium nitrite, potassium nitrite, etc. as an acid aqueous solution, for example, 0.5-4M acetic acid, nitric acid, hydrochloric acid, citric acid, etc.
5 to 20 so that the mixing ratio of each of the above amounts per m ²
It may be added by selecting from the range of 0 μl.

In the conventional nitrite extraction method, an aqueous nitrite solution is produced by reacting an aqueous nitrite solution with an aqueous acid solution when the sugar chain antigen is extracted, that is, in the presence of a microorganism that extracts the sugar chain antigen. Although it is a general method to act on the microorganisms in the present invention, in the present invention, an aqueous nitrite solution is prepared in advance by such a reaction, and it is applied by pouring it onto the filter medium on which the microorganisms are retained by the filtration. More preferably. The microorganisms retained on the filter medium are usually
Some of them are mixed with other insoluble substances and even penetrate into the inside of the pores.To make them uniformly act on the nitrous acid aqueous solution, first prepare a uniform solution as described above. It is more effective to pour it into the filter medium,
As a result, the extraction efficiency of the sugar chain antigen is further improved and the extraction time can be shortened.

When the nitrite aqueous solution is prepared in advance, the nitrite aqueous solution (sodium nitrite, potassium nitrite, etc.) and the acid aqueous solution (acetic acid, nitric acid, hydrochloric acid, citric acid, etc.) are adjusted to the above ratio. It may be prepared by mixing appropriately with the above to prepare an aqueous nitrite solution. From the viewpoint of the stability of nitrous acid, it is preferable not to leave it for a long period of time from the preparation to the use, but if it is stored at 2 to 25 ° C., even if it is stored for about 4 hours, there is practically no problem. Does not happen.

Since the microorganisms and other insoluble substances retained on the filter medium have penetrated not only to the surface of the filter medium but also to the inside of the pores, the supplied aqueous nitrite solution hardly flows down under gravity. Target. Therefore, the nitrous acid aqueous solution is hard to act sufficiently up to the microorganisms retained inside the pores, and therefore, in the present invention, it is preferable to pressurize the supplied nitrite aqueous solution to flow it down at the beginning of supply. As a result, although the excess amount of the nitrous acid aqueous solution is discharged, the necessary amount is evenly distributed to all the pores of the filter medium.
By setting the action time of nitrite to the above-mentioned time, the extraction efficiency of the sugar chain antigen is further improved.

Here, it is preferable that the flow of the nitrous acid aqueous solution under pressure is completed within at least 2 minutes after the liquid is poured into the filtration device. If it is allowed to flow down after a lapse of more time, the extraction of the sugar chain antigen will proceed to a considerable extent,
The released sugar chain antigen accompanies the excess amount of liquid that is discharged, and the effective amount disappears. In the method of producing an aqueous nitrite solution by reacting an aqueous nitrite solution with an aqueous acid solution at the time of extracting a sugar chain antigen, both solutions were supplied to a filter medium, and then the mixture was flown under pressure for such a short time. Then, since there is a possibility that a uniform nitrite aqueous solution is not generated, from this viewpoint, the method of preparing the nitrite aqueous solution prior to the supply to the filter medium is advantageous.

In the present invention, the sugar chain antigen extract may be obtained by allowing the microorganisms retained on the filter medium to react with the aqueous nitrite solution by the above-mentioned nitrite extraction method, and then allowing the liquid component to flow down and be recovered. At that time, as described above, when the excess amount of the nitrite aqueous solution was pressurized and discharged at the beginning of the action, the amount of the nitrite aqueous solution contained in the filter medium was small, and the liberated sugar chain antigen was removed. Since it is insufficient for sufficient recovery, it is preferable to additionally flow an appropriate amount of recovery liquid. The recovered liquid may be water or the like, but the pH of the recovered sugar chain antigen extract is 7. Since it neutralizes the strong acidity due to the residual acid.
It is preferable that the basic aqueous solution be 0 to 8.5.

Such a basic aqueous solution is 1 to 3
It is preferable to use an M sodium hydroxide aqueous solution, a 0.5 to 2 M tris aqueous solution, a 0.5 to 1 M sodium hydrogen carbonate aqueous solution, or the like. The residual acid can be neutralized by collecting the sugar chain antigen extract and then mixing the basic aqueous solution with a predetermined concentration, but the sensitivity of the immunoassay can be improved. From a perspective, as mentioned above,
It is more preferable to carry out the recovery liquid as a basic aqueous solution.

In the present invention, the sugar chain antigen extract extracted by the above method is used as a test liquid, and the amount of the sugar chain antigen contained in the extract is quantified by an immunological measurement method. Determine the microbial load of. The method for measuring this microorganism will be described below.

In the immunological assay method of the present invention, a monoclonal antibody or a polyclonal antibody that binds to the sugar chain antigen present in the sugar chain antigen extract is used. The globulin class in the antibody is not particularly limited, and all globulin classes currently known can be used. Furthermore, not only ordinary antibody molecules but also partial degradation products (Fab, F
ab ', Fab'2, etc.) and active fragments of the antibody (antigen recognition site of antibody) can also be used.

For example, when the amount of the sugar chain antigen in the sugar chain antigen extract of Streptococcus mutans or Streptococcus sobrinus is measured and the amount of Streptococcus mutans or Streptococcus sobrinus in the liquid to be extracted is determined, , An antibody that reacts with the sugar chain antigen of these bacteria is used.

Such an antibody is known, and as a specific example, an antibody against Streptococcus mutans is Zbl. Bakt. Hyg. (1987) A
As the polyclonal antibody described in Vol. 265, pages 330-339, and the antibody against Streptococcus sobrinus, Vol. 35, 93-10, Osaka University Medical Journal (1990).
Examples of the polyclonal antibody and the monoclonal antibody described on page 9 are not particularly limited as long as they are antibodies that can bind to the sugar chain antigens of Streptococcus mutans and Streptococcus sobrinus, and polyclonal antibodies and monoclonal antibodies produced by known methods. Can be used.

For example, a polyclonal antibody can be obtained by immunizing an immunized animal with Streptococcus mutans cells, Streptococcus sobrinus cells, or a sugar chain antigen extract obtained from these cells. The obtained polyclonal antibody is preferably used with higher specificity by purification treatment such as removal of antibodies having cross-reactivity with other microorganisms.

As a specific immunological measurement method, conventionally known methods such as an immunoaggregation method, an optical immunoassay method, a labeled immunoassay method, and a combination of these methods can be employed without limitation.

The immunological measurement methods will be described below.

[Immune agglutination method] This method is a method of detecting and quantifying an antigen in a sugar chain antigen extract using an agglutination reaction of an insoluble carrier based on an antigen-antibody reaction. The semi-quantitative method includes a latex agglutination method, a microtiter method and the like, and the quantitative measurement method includes a latex quantitative method.

For example, when the amount of antigen in the sugar chain antigen extract is immunologically measured by using the latex agglutination method,
After preparing a measurement reagent consisting of antibody-sensitized particles in which an antibody that binds to a sugar chain antigen of a microorganism (hereinafter also simply referred to as “antibody”) is immobilized on latex beads, the measurement reagent and a sugar chain antigen extract are mixed. The degree of agglutination of the sensitized particles after the antigen-antibody reaction can be measured by visual detection or by an optical measurement method or the like.

[Optical immunoassay method] This method detects a change in the turbidity of aggregates resulting from the antigen-antibody reaction when the antibody and the sugar chain antigen extract are contacted to carry out the antigen-antibody reaction. The method or a thin layer on which an antibody is immobilized (hereinafter, also referred to as “antibody layer”) is brought into contact with a sugar chain antigen extract, and changes in the refractive index of the antibody layer resulting from the antigen-antibody reaction are reflected by transmitted light or surface plasmon waves It is a method of optically detecting the presence or absence of an antigen-antibody reaction, such as a method of detecting the change as

[Labeled immunoassay method] The method is a measuring reagent containing a labeled antibody obtained by binding various labeling substances such as radioactive substances, enzymes, various dyes, colloids and various particles to an antibody, and a sugar chain. After carrying out an antigen-antibody reaction by contacting with the antigen extract, the amount of the labeling substance bound to the antigen in the sugar chain antigen extract, that is, radioactivity derived from the labeling substance, enzyme activity,
It is a method of detecting and quantifying an antigen in a sugar chain antigen extract by measuring fluorescence intensity, coloration and the like.

In this method, for example, a measuring reagent consisting of an insoluble carrier (particles, membrane, immunoplate, etc.) on which an antibody is immobilized is brought into contact with a sugar chain antigen extract to carry out an antigen-antibody reaction, and then the antibody is labeled. By contacting another measurement reagent containing a labeled antibody labeled with a substance and further performing an antigen-antibody reaction, the amount of the labeled substance is measured, or the sugar chain antigen extract and the sugar chain antigen labeled with the labeled substance The antigen in the sugar chain antigen extract is measured by measuring the amount of the labeling substance bound to the antibody by mixing the antigen and the antigen-antibody reaction by contacting with a measurement reagent consisting of an insoluble carrier on which the antibody is immobilized. It can be detected and quantified.

As the labeling substance, radioactive iodine, radioactive carbon and the like as radioactive substances, and peroxidase, alkaline phosphatase, galactosidase and the like as enzymes,
Fluorescent dyes such as fluorescein isothiocyanate and tetramethylrhodamine can be used as various dyes, gold colloid and carbon colloid can be used as colloids, and colored latex particles can be used as various particles. When carrying out enzyme labeling, methods such as direct labeling with a covalent bond such as a thiol group and a maleimide group or an amino group and an aldehyde group, or labeling via a biotin-avidin complex can be used.

Alkaline phosphatase and peroxidase are used as the labeling enzymes, and in the case of the former enzyme, a chemiluminescent substance such as a dioxetane derivative is used, and in the case of the latter enzyme, a chemiluminescent substance such as a luminol derivative is used. When used as a substrate for an enzyme, the luminescence of the substrate can also be detected.

The operations, procedures and the like in these various labeled immunoassay methods are not particularly different from those generally adopted, and can be based on known non-competitive method, competitive method, sandwich method and the like. Further, a substance capable of binding to an antibody such as a secondary antibody or protein A labeled with each of the above-mentioned labeling substances can be used together with the antibody to detect and quantify the sugar chain antigen.

In the labeled immunoassay, conventionally used methods can be used without particular limitation depending on the label used. Among them, a radioimmunoassay using a radioactive substance as a label and an enzyme immunoassay using an enzyme as a label. Measuring method,
Fluorescent immunoassay methods that use dyes, especially fluorescent dyes as labels, chemiluminescent immunoassay methods that use chemiluminescent substances as enzyme substrates as labels, etc. have high quantitative properties, and thus are suitable for highly accurate quantitative measurements. It is preferable to adopt these measuring methods. Further, the flow-through immunoassay method, the immunochromatography method, and the latex agglutination method, which use colloids or various particles as a label, are characterized by easy operation.

Among these methods, the immunochromatographic method is a simple and rapid method which can be carried out in a dental clinic or at home. The method will be described in detail below with reference to the drawings.

2 and 3 show the structure of a typical strip (7) which can be preferably used in the immunological assay method of the present invention. Like the conventional strip, the strip (7) is a sample pad (8) for temporarily absorbing and retaining each member composed of a porous support according to the purpose, that is, a sugar chain antigen extract. Conjugate pad (9) for temporarily holding labeled antibody, development membrane (10) on which detection antibody is immobilized, and sample pad
The absorbent pad (11) for absorbing the sugar chain antigen extract developed from (8) has a structure joined in this order.

On the conjugate pad (9) of the strip (7), an antibody (hereinafter, referred to as “labeled antibody”) for labeling the antigen in the sugar chain antigen extract is retained by being applied and dried. There is. As the labeled antibody, for example, an antibody labeled with a labeling substance such as gold colloid is used. In addition, the detection line (12) on the development membrane (10)
A detection antibody for capturing the antigen in the sugar chain antigen extract is immobilized on the.

The dimensions (lengths) shown in FIGS. 2 and 3 are merely indications of the size, and the sizes of the strips used in the immunochromatography method of the present invention are as follows. It is not limited to the values shown in the figure.

The material used for the porous support which constitutes each member is not particularly limited, and is appropriately selected from a hygroscopic material, a porous material, a fibrous material and the like according to the purpose of each member.
For example, it is preferable to use filter paper, blotter paper or the like as the sample pad (8), and it is preferable to use glass fiber cloth, polypropylene non-woven fabric, glass filter or the like as the conjugate pad (9). Further, it is preferable to use nitrocellulose, nitrocellulose containing cellulose acetate or the like as the spreading membrane (10), and filter paper, blotter paper or the like as the absorbent pad (11).

The method of immobilizing the detection antibody and the control antibody on the developing membrane (10) is not particularly limited, and any conventionally known physical adsorption method or covalent binding method can be used without any limitation, and other proteins can be used. It is also possible to immobilize it by mixing with etc. Further, in order to suppress the non-specific adsorption of the sugar chain antigen extract solution component to the development membrane, or to improve the wettability of the sugar chain antigen extract solution to the development membrane, the development membrane after antibody immobilization is performed by a known method. It is also possible to carry out a blocking treatment with a protein, a lipid, a polymer compound or the like. The protein or the like used in this blocking treatment is not particularly limited, but BSA, skim milk, etc. which are used for the purpose of suppressing non-specific reaction in a general immunological assay method are preferable. Further, in order to adjust the water absorption of the spreading membrane so that the sugar chain antigen extract spreads uniformly on the spreading membrane, it is possible to coat or impregnate the surface of the spreading membrane with a hydrophilic polymer or a surfactant. .

Regarding the conjugate pad (9), when the sugar chain antigen extract was added, the complex of the sugar chain antigen extract and the labeled antibody was easily desorbed from this part. Pre-blocking with water-soluble polymer or saccharides such as saccharose, followed by addition of labeled antibody and drying, or mixing of labeled antibody with water-soluble polymer or saccharide such as sucrose before application to conjugate pad And dried.

As the above water-soluble polymer, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, cellulose ether (methyl cellulose, ethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, oxyethyl cellulose, cyanethyl cellulose, etc.), gelatin and the like can be used.

As the labeling substance for preparing the labeled antibody, a labeled substance such as gold colloid, carbon colloid, colored latex and the like which can be visually confirmed can be used, and a radioactive substance and a fluorescent substance can also be used. Alternatively, an enzyme may be used as a labeling substance, and a substrate may be added after the antigen-antibody reaction to detect a compound produced by the enzyme reaction. The labeling of the antibody of the present invention is not particularly limited, and it can be carried out by a method generally adopted in the past.

In the immunochromatographic method, the sugar chain antigen extract is added to the sample pad (8) located in the vicinity of the conjugate pad (9) of the strip (7) and left at room temperature for 1 to 30 minutes. The labeled antibody is developed along with the sugar chain antigen extract to the detection line (12), and based on the principle of the sandwich method, the sugar chain antigen of the microorganism bound to the labeled antibody in the developing solution is detected by the detection antibody detection line ( 12) The presence or absence of the sugar chain antigen of the microorganism in the sugar chain antigen extract or the amount thereof can be measured by capturing on the above and detecting the labeled antibody bound to the sugar chain antigen of the captured microorganism.
At this time, the labeled antibody is normally developed together with the sugar chain antigen extract, that the antibody that reacts with the labeled antibody is immobilized in the control determination line (13) downstream of the detection line (12). It can be confirmed by detecting the labeled antibody at the position.

The development membrane (10) shown in FIG.
Upper detection line (12) and control decision line (13)
The position, the order, the shape, the number, etc. of are only examples and are not particularly limited.

The immunochromatography method using a strip coated with the labeled antibody in advance and dried has been described above. The labeled antibody is mixed with the sugar chain antigen extract in advance to carry out the antigen-antibody reaction, and then the It can also be measured by a method of adding the mixed solution to one end of the porous support on which the detection antibody is immobilized.

In the measurement by the immunochromatography method, the amount of the antigen in the sugar chain antigen extract is quantified quickly and simply by performing the measurement in the following procedure,
Based on this value, the amount of microorganisms in the liquid to be extracted can be determined.

That is, first, a sugar chain antigen extract (eg 10
0-200 μl) was collected, added to the sample pad of the strip, and allowed to stand for a predetermined time (for example, 1 to 30 minutes), and then the detection line (12) and the control determination line (13) of the strip were colored. Whether or not the sugar chain antigen is present or the amount thereof can be known by judging the presence or absence or the state thereof. Since the concentration of the antigen in the sugar chain antigen extract and the concentration of the microorganism in the liquid to be extracted are in a proportional relationship, the amount of the microorganism in the liquid to be extracted is determined based on the amount of the antigen.

[0074]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to the following examples.

Production Example 1 [Preparation and purification of polyclonal antibody against Streptococcus mutans] (1) [Preparation of antiserum against Streptococcus mutans] Brain heart infusion, also abbreviated as "BHI";
3.7 g of DIFCO) was dissolved in 100 ml of ultrapure water and then autoclaved to prepare a BHI liquid medium,
Ingbritt (Streptococcus mutans) was cultured under anaerobic conditions at 37 ° C. for 18 hours. The culture solution was centrifuged at 4000 g for 5 minutes to remove the culture medium components of the supernatant and collect the bacterial cell precipitate. Then 100 ml of the precipitate
Phosphate saline buffer solution (pH 7.4) (hereinafter referred to as "PB
S), and the same centrifugation was repeated 3 times to wash the precipitate.

The cell pellet was suspended in PBS and A ₆₀₀ = 1.
The suspension was adjusted to 0 to prepare a suspension of Ingbritt cells. The microbial cell suspension is sonicated and then appropriately diluted and then added to a BHI medium plate, the number of colonies generated is counted, and the cell suspension is multiplied by the dilution factor of the microbial cell suspension. When the cell concentration of the liquid was determined, it was about 1 × 10 ⁹ cells / ml
Met.

Immunization was performed as follows.

During the first week, 0.5 ml of the bacterial cell suspension was intravenously injected into the rabbit auricularly 5 times for 5 consecutive days. During the second week, 1.0 ml of the bacterial cell suspension was intravenously injected into the rabbit 5 times for 5 consecutive days. In the 3rd week, add 2.0 ml of the bacterial cell suspension to 5
The rabbits were intravenously injected with auricular vein 5 times on consecutive days. The fourth week was immunized in the same manner as the third week. After confirming the increase in titer based on the degree of agglutination of bacterial cells using a slide glass, one week after the final immunization, blood was collected according to a standard method to obtain antiserum against Streptococcus mutans.

(2) [Purification of Polyclonal Antibody Against Streptococcus mutans] In BHI medium, the same method as in (1) above was carried out.
After culturing is (Streptococcus gordonii), it was washed with PBS to prepare 30 ml of a cell suspension containing 2 × 10 ¹² cells / ml. Then, the bacterial cell suspension was mixed with 0.5 ml of antiserum and reacted at 4 ° C. for 60 minutes. Mix 4
After centrifugation at 000g for 5 minutes, the supernatant is collected and 0.22 μm
It was filtered through a filter.

Next, 1 was equilibrated with PBS in advance.
The supernatant sample was added to a column packed with ml of protein A-Sepharose (Amersham Pharmacia Biotech), and the column was washed with 5 ml of PBS, followed by addition of 5 ml of 0.
Elute with 1 M glycine-hydrochloric acid buffer (pH 3.0),
Immediately add 1 M Tris-hydrochloric acid (pH 9.0) to adjust the pH.
Adjusted to 7.4. The elution fraction of IgG was confirmed by measuring A ₂₈₀ . About 0.5 ml of antiserum
mg IgG was recovered.

Example 1 [Filtration of Extracted Liquid by Filter] Paraffin pellets were bitten by a subject for 5 minutes, and secreted saliva was collected as an extracted liquid.

A glass fiber filter paper or a filter paper was set in an SC cartridge (Maruem Co., Ltd.) to prepare a filter device having the structure shown in FIG. The glass fiber filter paper and the filter paper shown in Table 1 were used.

1 ml of the liquid to be extracted was added to the filtration device, the lid was closed and pressure filtration was performed with a syringe to collect the filtrate. Appropriately dilute the liquid to be extracted and the filtrate, and add 100 μl of Mitis
Salivarius bacitracin (hereinafter, also abbreviated as “MSB”) was added onto a solid medium and cultured at 37 ° C. under anaerobic conditions for 48 hours.

As an example of filtration with a membrane filter, Millex HV (PVDF membrane having a pore size of 0.45 μm,
The liquid to be extracted was also filtered with a diameter of 13 mm). MS
The number of colonies generated on the B medium was counted, and the retention rate [(the number of bacteria in the liquid to be extracted-the number of bacteria in the filtrate) / the number of bacteria in the liquid to be extracted x 1
00] was calculated. The results are shown in Table 1.

[0085]

[Table 1]

Bacteria that grow in MSB medium are mainly Streptococcus mutans and Streptococcus.
Since it is sobrinus, the size of the bacterial cell is 0.5 to 0.9 μm. When a filter medium having a pore size smaller than that of the bacterial cells was used (Experiment No. 1, 2, 9), clogging occurred early and the amount of liquid that could be filtered was less than 0.5 ml. In particular,
When the membrane filter was used, only 0.1 ml could be filtered.

The capture rate by the filter medium is 0.8 to 2.0 for the pore size.
90% or more up to μm, particularly 95 at 0.8 to 1.6 μm
% Or more, and it was confirmed that Streptococcus mutans and Streptococcus sobrinus in the oral cavity were captured on the filter medium within this range even if the pore size was larger than the size of the bacterium itself. In addition, the amount of the test liquid that can be filtered was 1.0 ml with a filter medium of 0.8 to 2.0 μm.

Example 2 [Extraction of Sugar Chain Antigen from Extracted Liquid Containing Saliva and Plaque and Measurement by Immunochromatography] (1) [Collection of Extracted Liquid and Measurement of Bacterial Count by Culture Method] Different Samples containing saliva and plaque were obtained from 18 subjects according to the method of Example 1, respectively. Each sample was appropriately diluted, 100 μl was added to the MSB solid medium and the BHI solid medium, and cultured at 37 ° C. under anaerobic conditions for 24 to 48 hours. The number of colonies generated on MSB and BHI solid medium was counted, and the concentration of mutans streptococci was calculated as the number / ml from the dilution ratio of the liquid to be extracted, and shown in Table 2.

Discrimination of Streptococcus mutans on MSB and BHI media is based on morphological classification of colonies, and for colonies that are morphologically indistinguishable, sera of mutans streptococci after pure culture of the colonies. Streptococcus mutans was identified by an immunological measurement method using a type-specific antibody and a biochemical method such as a sugar fermentation test.

(2) [Preparation of sugar chain antigen extract] 0.5 ml of the liquid to be extracted collected in (1) was filtered by a filter equipped with GA-100. Then 0.5m
Microorganisms on the filter medium and the filter medium were washed by adding 1 l of 0.1 M NaOH solution to the filter device and filtering, and further adding 0.5 ml of PBS to the filter device and filtering. To the filter medium, 20 μl of 2M acetic acid solution and 20 μl of 1M sodium nitrite aqueous solution were added, followed by 2 minutes at room temperature, or 1
After leaving for 0 minutes, 90 μl of 0.05% Tween
1 M Tris containing 20 (pH unprepared) was added, and the sugar chain antigen extract (10
0-140 μl) was collected.

(3) [Measurement of sugar chain antigen extract by immunochromatography strip] A commercially available gold colloid solution having a colloidal particle size of 40 nm (EY
Laboratory) 10 ml with 100 mM K ₂ CO
88 μl of ₃ was added to adjust the pH to 9.0, and then 0.22
μm filtered. 520nm of gold colloid solution
When the absorbance of was measured, it was _A520 = 1.0.

Next, Production Example 1 adjusted to 1 mg / ml
2 mM borate buffer solution (pH 9.0) of the antibody prepared in
64 μl was added to the above gold colloid solution with stirring, and the mixture was allowed to stand at room temperature for 5 minutes. Then, 1.1% of 10% skim milk-2 mM borate buffer (pH 9.0) was added with stirring (final skim milk concentration 1%), and the mixture was allowed to stand at room temperature 3
It was left for 0 minutes. Then, the reaction solution is heated at 10 ° C., 10,000
Centrifuge for 30 minutes and remove the supernatant, then add 2 ml of 2 mM
PBS (pH 7.4) was added to resuspend the lower layer gold colloid fraction. When the absorbance at 520 nm of the resuspended fraction was measured, it was A ₅₂₀ = 4.9. The obtained gold colloid fraction (hereinafter, also abbreviated as "gold colloid labeled antibody") was stored at 4 ° C.

Nitrocellulose Membrane (MILLI
PORE, Hi-Flow Plus Membra
ne, HF180, 25 mm × 6 mm) on a detection membrane (12) and a control judgment line (13) on a development membrane (10), respectively, containing 1 mg / ml of the antibody prepared in Production Example 1 and anti-rabbit IgG (H + L). ) Polyclonal antibody (ICN Pharmaceuticals) 1μ
1 was spotted and dried in an incubator at 37 ° C. for 60 minutes to immobilize the antibody. 1 for the antibody-immobilized membrane
% Skim milk-0.1% Triton X100 aqueous solution was shaken at room temperature for 5 minutes. Then, the membrane was placed in a 10 mM phosphate buffer (pH 7.4) at room temperature for 10 minutes.
After shaking for a minute, the product was taken out and dried in a desiccator for 60 minutes while sucking with a vacuum pump.

In addition, the conjugate pad (9) (MIL
LIPORE, 7.5 mm x 6 mm) 0.5% PV
After shaking for 1 minute in an A-0.5% sucrose aqueous solution, it was taken out and dried in a desiccator for 60 minutes while sucking with a vacuum pump. A ₅₂₀ = 1.
25 μl of the gold colloid-labeled antibody adjusted to 0 was added, and dried in a desiccator for 60 minutes while sucking with a vacuum pump. Furthermore, the sample pad (8) (MILLIPORE
Company, 17 mm x 6 mm) 1% Tween20-PBS
It was shaken in the aqueous solution for 1 minute, taken out, and dried in a desiccator for 60 minutes while sucking with a vacuum pump. The absorbent pad (11) (MILLIPORE, 20 mm x 6 m
m) was used untreated.

The components of the immunochromatographic strip as shown in FIG. 2 thus prepared were placed on a plastic support, and the immunochromatographic strip as shown in FIG. 3 was assembled.

Immunochromatography Method 100 μl of the sugar chain antigen extract was added onto the sample pad (8) of the strip, and after 10 minutes, the presence or absence of spots was determined. The judgment is
The degree of the gold colloid captured on the detection antibody spot was visually identified in four levels (++++: strong positive, ++: positive, +: weak positive, −: negative). The results are shown in Table 2.

[0097]

[Table 2]

As shown in Table 2, the spot color intensity correlated with the concentration of Streptococcus mutans obtained by the culture method was obtained. The spot strength was stronger when the action time of the nitrite aqueous solution was 10 minutes than 2 minutes. According to the measuring method of the present invention, the amount of antigen in the sugar chain antigen extract, in other words, the amount of Streptococcus mutans in the liquid to be extracted could be detected in a concentration-dependent manner.

Comparative Example 1 [Extraction of Sugar Chain Antigen from Extracted Liquid Containing Saliva and Plaque by Centrifugation Method and Measurement by Immunochromatography Method] (1) [Preparation of Sugar Chain Antigen Extract Solution] Example 1 Extracted liquid containing saliva and plaque collected in (0.5
(ml) was centrifuged at 10,000 rpm for 5 minutes, the precipitate was suspended in 0.5 ml of 0.1 M NaOH, and the supernatant was removed by centrifugation. The precipitate is then added to 0.5 ml of PBS
It was suspended in and the supernatant was removed by centrifugation.

After performing the same operation with PBS again, 20 μl of 2M acetic acid solution and 20 μl of 1M were added to the precipitate.
An aqueous sodium nitrite solution was added and mixed well, and the mixture was left at room temperature for 10 minutes. 90 μl of 0.05% Tween
A sugar chain antigen extract was prepared by adding 1 M Tris containing n20 (pH not yet adjusted).

(2) [Measurement of sugar chain antigen extract using immunochromatographic strip] The sugar chain antigen extract prepared in (1) above was used in Example 2 (3).
The measurement was performed in the same manner as in. The results are shown in Table 3.

[0102]

[Table 3]

As shown in Table 3, some of the samples in which Streptococcus mutans were not detected by the culture method became positive (samples 14, 15, 17). This is probably because the non-specific reaction-causing substance contained in the liquid to be extracted could not be sufficiently removed by washing by the centrifugation method, and thus became positive due to the non-specific reaction.

Example 3 [Extraction of nitrous acid with a previously prepared nitrous acid aqueous solution] In the same manner as in Example 2 (1), the liquid to be extracted was filtered, and then 0.5 ml of 0.1 M NaOH solution was applied to the filter medium. The microorganisms and the filter medium were washed. A 1M sodium nitrite solution and a 2M acetic acid solution were mixed at a ratio of 1: 1 to prepare an aqueous nitrite solution, and 0.2 ml of the aqueous nitrite solution was added to a filtration device.
Pressurized with a syringe to allow the excess amount to flow down immediately. After standing at room temperature for 2 minutes, 90 μl of 0.05% Tween
A 1 M tris (pH unprepared) aqueous solution containing n20 was added, and the sugar chain antigen extract (100 to 140 μl) was collected by pressurizing the filtration device with a syringe. Example 2 (3)
The sugar chain antigen extract was measured by the same method as described above. The results are shown in Table 4.

[0105]

[Table 4]

Even if the reaction time of nitrous acid extraction was shortened to 2 minutes, the same result as that of the reaction in Example 1 for 10 minutes was obtained.

[0107]

By the method for extracting a microbial antigen of the present invention,
Microorganisms having sugar chain antigens present in the liquid to be extracted can be simply and quickly concentrated to easily prepare a sugar chain antigen extract. By analyzing this sugar chain antigen extract with an immunological measurement method, even when the concentration of the above-mentioned microorganisms in the liquid to be extracted is low, the value can be easily obtained with high sensitivity.

In particular, a sugar chain antigen is extracted from Streptococcus mutans or Streptococcus sobrinus in an extract containing saliva or dental plaque, and the sugar chain antigen extract is subjected to immunochromatography, latex agglutination, flow-through immunization. According to the measuring method of the present invention, which is measured by the measuring method, in a dental clinic or at home, quickly and easily Streptococcus mutans or Streptococcus
It is possible to measure sobrinus and build a new caries diagnosis system.

[Brief description of drawings]

FIG. 1 is a schematic view of a filtration device used in the method for extracting a microbial antigen of the present invention.

FIG. 2 is a schematic view of each member of a strip used when the method for measuring a microorganism of the present invention is carried out by an immunochromatography method.

FIG. 3 is a side view of the strip of FIG.

[Explanation of symbols]

1. Drainage port 2 ... Filter material 3 ... Container body 4 ... Syringe insertion port 5 ... Lid 6 ... Filtration device 7 ... strip 8: Sample pad 9: Conjugate pad 10 ... Deployment membrane 11 ... Absorption pad 12 ... Detection line 13 ... Control judgment line

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/569 G01N 33/569 F // (C12M 1/12 C12R 1:46 C12R 1:46) (C12M 1/34 C12R 1:46) (C12N 1/20 C12R 1:46) (72) Inventor Naohiro Hanyu 1-38-9 Taito, Taito-ku, Tokyo F-term in Tokuyama Dental Co., Ltd. (reference) 2G052 AA29 AA33 AB17 AD32 EA03 FC02 FC11 FC15 GA30 JA04 JA07 4B029 AA09 BB02 CC01 HA06 4B065 AA34X BC31 BD18 BD32 CA46

Claims (8)

[Claims] 1. The above sugar chain antigen by filtering a liquid to be extracted, which may contain a microorganism having a sugar chain antigen, with a filter medium, and allowing the microorganism retained on the filter medium to act on an aqueous nitrite solution. A method for extracting a microbial antigen, which comprises: 2. The method for extracting a microbial antigen according to claim 1, wherein the nitrite aqueous solution is allowed to act on the microorganisms retained on the filter medium by pouring the nitrite aqueous solution prepared in advance into the filter medium. . 3. The method for extracting a microbial antigen according to claim 1 or 2, wherein the liquid to be extracted comprises saliva, dental plaque, or a mixture thereof. 4. The method for extracting a microbial antigen according to claim 1, wherein a filter medium having a pore size of 0.8 to 2 μm is used. 5. The microorganism having a sugar chain antigen is Streptococcus mutans or Streptococcus.
The method for extracting a microbial antigen according to any one of claims 1 to 3, which is sobrinus. 6. The sugar chain antigen is obtained by filtering a liquid to be extracted, which may contain a microorganism having a sugar chain antigen, with a filter material, and allowing the microorganisms retained on the filter material to act on an aqueous nitrite solution. A filter device used for carrying out the method for extracting a microbial antigen, comprising a filter medium having a pore size of 0.8 to 2 μm as a filter medium. 7. The amount of sugar chain antigen contained in the sugar chain antigen extract extracted by the method according to claim 1, is measured by an immunological measuring method, and the amount of sugar chain antigen is measured based on the measurement result. A method for measuring a microorganism, which comprises determining the amount of a microorganism having a sugar chain antigen contained in an extract. 8. The method for measuring a microorganism according to claim 7, wherein the immunological measurement method is an immunochromatography method.