JP3781768B2 - Microbial extraction method and filtration apparatus used in the method - Google Patents

Description

  The present invention relates to a method for concentrating Streptococcus mutans and / or Streptococcus sobrinus in an extraction liquid containing saliva, dental plaque, or a mixture thereof by filtration, and extracting a sugar chain antigen possessed by the microorganism, and Filtration apparatus therefor, and a highly sensitive measurement method for Streptococcus mutans and / or Streptococcus sobrinus that determines the amount of sugar chain antigen in the sugar chain antigen extract prepared as described above by an immunological measurement method .

  Microbiological testing is an important test item in medical testing, food testing, and environmental measurement.

  A wide variety of microorganisms exist in the oral cavity, and it is known that microorganisms that cause infections may exist among these microorganisms. For example, it is known that caries-related bacteria, specifically, Streptococcus mutans and Streptococcus sobrinus are strongly involved in the occurrence of caries.

  The measurement of microorganisms has been performed by a conventional culture method, and there is a problem that it takes a long time until the result is known. However, an immunological measurement method has been developed, and the test time has been greatly shortened. The immunological measurement method is a method of detecting a microorganism with high sensitivity using an antibody against a specific antigen possessed by the microorganism.

  Detection sensitivity can be further enhanced by measuring an antigen extract prepared by extracting an antigen from a microorganism by an immunological measurement method. The reasons for this are considered to be 1) the antigen is solubilized and becomes a low molecule, so that the antigen-antibody reaction is likely to proceed, and 2) the epitope is exposed.

  In particular, when an antigen is measured by immunochromatography, in which a solution containing the antigen is developed on a development membrane and the antigen is detected by an antigen-antibody reaction, the extractable antigen has a better developability than the insoluble antigen. High sensitivity measurement can be expected by extracting.

  Various methods such as surfactant treatment, enzyme treatment, and heat treatment have been put to practical use as methods for extracting microbial antigens. Nitrous acid extraction methods (Takei Takei, Osaka University Medical Journal. 35: 93-109, 1990.). This method is particularly effective for extraction of sugar chain antigens of microorganisms belonging to the genus Streptococcus such as group A streptococci and oral streptococci.

  This nitrite extraction method is a method in which an aqueous solution of nitrous acid is reacted with a sugar chain, and the sugar chain is cleaved and released from the microbial cells. A general nitrite extraction method is a method in which nitrite is added to an extraction liquid containing microorganisms. Mixing an aqueous solution (usually sodium nitrite) and an aqueous acid solution (acetic acid, hydrochloric acid, nitric acid, etc.) (hereinafter, these solutions are also collectively referred to as “nitrite extraction reagents”) to produce nitrous acid, This is carried out by reacting the microorganism with the nitrous acid for a sufficient time. Since the above reaction solution is strongly acidic, a base [2-amino-2-hydroxymethyl-1,3-propanediol (hereinafter also abbreviated as “Tris”), sodium hydroxide, sodium hydrogen carbonate, etc.] is added after the reaction. It is general to analyze after adding and neutralizing the reaction solution.

  However, when extracting sugar chain antigens of the caries-related bacteria of Streptococcus mutans and Streptococcus sobrinus from the extraction liquid containing saliva, plaque, or a mixture thereof, these are included in the extraction liquid. Since the concentration of microorganisms is dilute, a glycan antigen extract was prepared by adding a nitrous acid extraction reagent directly to the extraction solution. It was difficult to extract sugar chain antigens.

  Therefore, Streptococcus mutans and Streptococcus sobrinus sugar chain antigens can be easily extracted from the extracted liquid containing saliva, plaque, or a mixture thereof at a concentration that can be measured by an immunoassay. It was desired to develop a method.

  The inventors of the present invention have continually studied to solve the above problems. As a result, when extracting the sugar chain antigen possessed by the Streptococcus mutans or Streptococcus sobrinus from the extraction liquid containing saliva, plaque, or a mixture thereof, concentration by filtration using a filter medium having a specific pore size The present invention has been completed by finding that the above-mentioned problems can be solved by combining nitrous acid extraction method with nitrous acid extraction method.

  That is, the present invention filters Streptococcus mutans and / or Streptococcus mutans retained on the filter medium by filtering an extraction liquid containing saliva, plaque, or a mixture thereof with a filter medium having a pore diameter of 0.8 to 2 μm. A method for extracting a microbial antigen characterized by extracting a sugar chain antigen possessed by the microorganism by allowing a nitrous acid aqueous solution to act on Sobrinus.

  Further, the present invention is a method for measuring the amount of sugar chain antigen contained in the sugar chain antigen extract extracted by the above method by an immunological measurement method, and based on the measurement result, Streptococcus There is also provided a method for measuring a microorganism characterized by determining the amount of mutans and / or Streptococcus sobrinus.

  Furthermore, the present invention is to filter the liquid to be extracted comprising saliva, plaque, or a mixture thereof with a filter medium, and to act the nitrite aqueous solution on Streptococcus mutans and / or Streptococcus sobrinus held on the filter medium A filtration device used for carrying out a method for extracting a sugar chain antigen possessed by the microorganism, wherein a filtration material having a pore diameter of 0.8 to 2 μm is used as the filtration material. A filtration device for microbial antigen extraction is also provided.

  By the method for extracting a microbial antigen of the present invention, Streptococcus mutans or Streptococcus sobrinus present in an extract containing saliva or plaque can be easily and rapidly concentrated to easily prepare a sugar chain antigen extract. it can. By analyzing this sugar chain antigen extract with an immunological measurement method, the concentration of the caries-related bacteria in the extract can be easily determined with high sensitivity even though the concentration is low. Is possible.

  In particular, a sugar chain antigen is extracted from Streptococcus mutans and / or Streptococcus sobrinus in the above-mentioned extract, and the sugar chain antigen extract is measured by an immunochromatography method, a latex agglutination method, or a flow-through immunoassay method. For example, it becomes possible to measure the above-mentioned caries-related bacteria quickly and easily at a dental clinic or at home, and a new caries diagnosis system can be constructed.

  The liquid to be extracted used in the present invention comprises saliva collected from the oral cavity, plaque, or a mixture thereof. Such a liquid to be extracted may contain caries-related bacteria such as Streptococcus mutans and Streptococcus sobrinus, which are microorganisms to be measured by the present invention. When microorganisms having the sugar chain antigen to be measured are contained, these microorganisms are mixed with other insoluble substances.

  In the extraction method of the present invention, the liquid to be extracted as described above is first filtered using a filter medium having a pore size of 0.8 to 2 μm, and Streptococcus mutans and / or Streptococcus sobrinus present in the liquid to be extracted is removed. Hold on filter media. By this operation, the caries-related bacteria in the extraction liquid are well concentrated on the filter medium. In the present invention, the retention of these microorganisms on the filter medium includes not only those retained on the surface of the filter medium but also those trapped in the pores.

  Conventionally, in the microbiological examination etc. by immunological measurement method, when the extraction liquid contains a lot of insoluble matter when concentrating the microorganisms in the extraction liquid, it is more than the filtration method which is dangerous such as clogging. Centrifugation is mainly used. However, the centrifugation method requires an expensive centrifuge, and must first be performed by centrifuging the liquid to be extracted to precipitate microorganisms and then discarding the supernatant. There is a problem that it is complicated and takes time to concentrate. Therefore, it is extremely significant to concentrate microorganisms having sugar chain antigens by using a filtration method that can be performed in a short time using a simple filtration apparatus, and to use them for testing such microorganisms.

  In the present invention, as the filter medium, any membrane or layered filter medium made of a known material can be used without limitation as long as it is stable even after nitrous acid extraction. Examples of such a filter medium include a screen filter such as a membrane filter, a depth filter such as glass fiber filter paper and filter paper, and the like. When filtering a sample liquid containing microorganisms, the material of a membrane filter that is usually used is cellulose acetate, polyvinylidene fluoride (hereinafter abbreviated as “PVDF”), nylon, etc., but these materials are: Since it is somewhat unstable under strongly acidic conditions at the time of 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.

  Here, the size of Streptococcus mutans and Streptococcus sobrinus is in the range of 0.5 to 0.9 μm. Therefore, in the preparation process of the specimen liquid in the examination of these microorganisms, when filtering the extraction liquid containing these microorganisms, as a filter medium, the pore size sufficiently smaller than the size of the microorganism, specifically, It is usual to use filter media having a pore size of 0.22 to 0.45 μm.

  Even in the present invention, if the liquid to be extracted is filtered using a filter medium having the above-mentioned small pore diameter, Streptococcus mutans and Streptococcus sobrinus can be satisfactorily captured. When the liquid filtration rate is slow and other insoluble substances are mixed in the liquid to be extracted, clogging is likely to occur. And when clogging occurs in the filter medium in this way, the extraction efficiency is slightly lowered also in the process of applying a nitrous acid aqueous solution to the microorganisms held on the filter medium following the above process.

  From such a background, in the present invention, a filter medium having a pore diameter of 0.8 to 2 μm, more preferably 0.8 to 1.6 μm is used. The pore diameter is usually slightly larger than the size of the Streptococcus mutans or Streptococcus sobrinus retained on the filter medium, but in the case of these caries-related bacteria, the filter medium has a large pore diameter to this extent. Can be captured with almost no flow. Therefore, the filtration rate is greatly improved, and clogging with other insoluble matters is hardly caused, and the sugar chain antigen can be extracted more efficiently using the aqueous nitrite solution.

  In the present invention, the pore diameter of the filter medium refers to a value at which the filter medium captures 98% or more of particles larger than the pore diameter. When the filter medium 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 The reserved particle size and particle retention capacity displayed in the catalog correspond to the pore size of the present invention.

  The Streptococcus mutans and / or Streptococcus sobrinus held on the filter medium is preferably washed by flowing a washing liquid through the filtration device. When the amount of sugar chain antigen extracted by the method of the present invention is measured by an immunological measurement method, the occurrence of false positives due to non-specific reactions can be reduced by performing the above washing.

  According to the filtration method, these concentrated microorganisms can be easily and quickly washed. For example, in the extraction method of the present invention, when a washing operation of Streptococcus mutans and / or Streptococcus sobrinus held on the filter medium in this way is added, washing is performed by a simple operation of adding a washing liquid to the filtration device and causing it to flow down. Can be completed in a short time. In contrast, when these microorganisms are washed by centrifugation, the concentrated microorganisms are washed by centrifuging the liquid to be concentrated and discarding the supernatant, and then using the resulting precipitate as a washing liquid. It must be carried out by a cumbersome method of suspending, further centrifuging, and discarding the supernatant, and usually requires 2 to 5 times the operation time of the above filtration method.

  Furthermore, if washing is performed in the filtration method in this way, the washing efficiency can be increased compared to washing by the centrifugal method, and as shown in the examples described later, the measurement target concentrated by the centrifugal method can be obtained. Sugar chain antigens extracted from microorganisms also have a great effect that accurate measurement is possible even when the immunological measurement method is performed with a low-specificity antibody that can detect a nonspecific reaction. .

  As the microorganism cleaning solution, for example, a buffer solution, an acid aqueous solution, an alkaline aqueous solution, a surfactant aqueous solution or the like can be used.

  The extraction operation of the liquid to be extracted can be performed according to a general filtration method. In addition to gravity, filtration may be performed under pressure or under reduced pressure. Preferably, it is preferable to use a syringe filter in which a filter medium is fixed, inject the liquid to be extracted, and then pressurize with a syringe. Further, for example, it is possible to filter by fixing the filter medium in the syringe, dispensing the liquid to be extracted into the syringe with the plunger removed, and inserting the plunger into the syringe and pressurizing it.

  Further, for example, as shown in FIG. 1, a main body container (3) having a cylindrical shape or the like in which the upper surface of the container is open, the drainage port (1) protrudes from the bottom wall, and the filter medium (2) is fixed inside. ) And a filtration device (6) comprising a lid portion (5) protruding from the syringe insertion port (4) on the top plate portion, which can be fitted to the upper surface of the main body container (3). This is also a preferred embodiment. That is, according to the filtration device (6), the liquid to be extracted is poured into the container from the upper surface opening of the container body, and then the lid (5) is fitted to the upper surface of the container body. Attach the tip of the pressurizing syringe to the syringe insertion port (4), press the plunger to pressurize the inside of the container, and discharge the filtrate from the drainage port (1). And filtered under pressure. Further, according to the filtration device (6), when the washing operation after the filtration is performed, and further, after that, when the Streptococcus mutans and / or Streptococcus sobrinus held on the filter medium is allowed to act on the aqueous nitrite solution In addition, the operation can be efficiently performed by the same procedure.

  Here, in the filtration apparatus 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 bonded.

  When the filter medium (2) is directly fixed to the bottom of the main body container, when filtering the liquid to be extracted (for example, when the viscosity is high or contains a large amount of insoluble matter), the filtration speed is increased. If the pressure is increased more than necessary, a part of the filter medium (2) is pushed out into the drainage port (1), and the filtration rate may be significantly reduced. 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, entry of the filter medium (2) into the drainage port (1) is more preferable. As the support filter, for example, those used for the purpose of preventing the outflow of the chromatography carrier from the column at the bottom of a general open column for liquid chromatography, specifically, a glass filter, a polypropylene filter, a 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 (5) to the upper surface of the main body container (3) may be performed not only by a frictional force but also by a screw screw or other sealing method.

  Next, in the method for extracting microbial antigens of the present invention, a nitrite aqueous solution is allowed to act on Streptococcus mutans and / or Streptococcus sobrinus held on a filter medium by the above-described method, whereby sugars possessed by these microorganisms are obtained. Extract the chain antigen. As a method of allowing a nitrous acid aqueous solution to act on a microorganism, a known method known as a nitrous acid extraction method can be used without limitation. A nitrous acid aqueous solution can be generally prepared by mixing a nitrite aqueous solution and an acid aqueous solution. From the viewpoint of extraction efficiency and accuracy in subsequent immunological measurement methods, 0.2-8M nitrite and 0.3-4M acid were used for the liquid volume of the mixture. It is desirable to use a nitrous acid aqueous solution obtained by mixing each solution in such an amount.

The amount of the nitrous acid aqueous solution used is 5 to 400 μl, preferably 20 to 300 μl per 1 cm ² of the area of the upper surface of the filter medium. The temperature at which the aqueous nitrous acid solution acts is preferably 15 to 50 ° C., and the working time is preferably 1 to 10 minutes.

A specific method for causing a nitrite aqueous solution to act on Streptococcus mutans and / or Streptococcus sobrinus is performed, for example, by supplying a nitrite aqueous solution and an acid aqueous solution onto a filter medium and generating nitrous acid on the filter medium. be able to. As the nitrite aqueous solution, for example, 0.2-8M sodium nitrite, potassium nitrite, etc., and as the acid aqueous solution, for example, 0.5-4M acetic acid, nitric acid, hydrochloric acid, citric acid, etc. 1 cm ² per the may be added adopted from the range of 5~200μl so that the mixing ratio of each quantity.

  In the conventional nitrous acid extraction method, during extraction of sugar chain antigens, that is, by coexistence of microorganisms that extract sugar chain antigens, a nitrite aqueous solution and an acid aqueous solution are reacted to form a nitrous acid aqueous solution, thereby producing microorganisms. In the present invention, a nitrous acid aqueous solution is prepared in advance by such a reaction, and this is applied by filtering it onto the filter medium holding the microorganisms. Is more preferable. Streptococcus mutans and Streptococcus sobrinus retained on the filter medium are usually mixed with other insoluble materials, and some of them penetrate into the pores. In order to act, it is more effective to prepare a uniform solution in advance as described above and then pour it into the filter medium. This further improves the extraction efficiency of the glycan antigen and shortens the extraction time. become.

  When preparing a nitrous acid aqueous solution in advance, a nitrite aqueous solution (sodium nitrite, potassium nitrite, etc.) and an acid aqueous solution (acetic acid, nitric acid, hydrochloric acid, citric acid, etc.) are mixed as appropriate in the above ratio. Then, a nitrous acid aqueous solution may be prepared. From the viewpoint of the stability of nitrous acid, it is preferable not to leave a long period of time until it is used after blending, but if it is stored at 2 to 25 ° C., even if it is stored for about 4 hours, there is almost no problem in practical use. Does not occur.

  Microorganisms and other insoluble matter retained on the filter medium penetrate not only into the surface of the filter medium but also into the pores, so the supplied nitrous acid solution generally does not flow under gravity. . Therefore, until Streptococcus mutans or Streptococcus sobrinus held inside the pores, the nitrous acid aqueous solution is not sufficiently effective, so in the present invention, the supplied nitrous acid aqueous solution is pressurized and flowed down at the beginning of supply. Is preferred. As a result, although the excess amount of the nitrous acid aqueous solution is discharged, the necessary amount is uniformly distributed to every corner of the pores of the filter medium. The extraction efficiency is further improved.

  Here, the flow of the nitrous acid aqueous solution under pressure is preferably finished within at least 2 minutes after the liquid is poured into the filtration device. If it is allowed to flow down after this time has elapsed, the extraction of the sugar chain antigen will proceed to a considerable extent, so that the excess amount of the discharged liquid will be accompanied by the released sugar chain antigen so that the effective amount disappears. Become. In the method of producing a nitrite aqueous solution by reacting a nitrite aqueous solution and an acid aqueous solution at the time of sugar chain antigen extraction, both liquids were supplied to the filter medium, and then the mixed liquid was allowed to flow down in such a short time. Then, since there exists a possibility that the uniform nitrous acid aqueous solution may not be produced | generated, the method of preparing nitrous acid aqueous solution prior to supply to a filter medium is advantageous also from this viewpoint.

  In the present invention, the sugar chain antigen extract is recovered by allowing the nitrous acid aqueous solution to act on Streptococcus mutans and / or Streptococcus sobrinus held on the filter medium by the above nitrous acid extraction method, and then flowing down the liquid components. You can do that. At that time, as described above, when the excess amount of the nitrous acid aqueous solution is pressurized and discharged at the beginning of the action, the amount of the nitrous acid aqueous solution contained in the filter medium is small, and the released sugar chain antigen is Since it is insufficient for sufficient recovery, it is preferable to add an appropriate amount of recovery liquid. This recovered solution may be water or the like, but is a basic aqueous solution in which the pH of the recovered sugar chain antigen extract is 7.0 to 8.5 in order to neutralize strong acidity due to the remaining acid. Is preferred.

  As such a basic aqueous solution, it is preferable to use 1 to 3 M sodium hydroxide aqueous solution, 0.5 to 2 M tris aqueous solution, 0.5 to 1 M sodium hydrogen carbonate aqueous solution, or the like. The remaining acid can be neutralized by collecting the sugar chain antigen extract and then mixing the basic aqueous solution with a predetermined concentration. From the viewpoint, as described 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 sample liquid, and the amount of the sugar chain antigen contained is quantified by an immunological measurement method, and the Streptococcus Determine the amount of mutans and / or Streptococcus sobrinus. Hereinafter, a method for measuring this microorganism will be described.

  In the present invention, the immunological measurement method uses a monoclonal antibody or a polyclonal antibody that binds to a sugar chain antigen possessed by Streptococcus mutans or Streptococcus sobrinus present in the sugar chain antigen extract. The globulin class in the antibody is not particularly limited, and all currently known globulin classes can be used. Furthermore, not only normal antibody molecules but also partial degradation products (Fab, Fab ′, Fab′2, etc.) of the antibody, and active fragments of the antibody (antigen recognition site of the antibody) can be used.

  Such antibodies are known, and specific examples of antibodies against Streptococcus mutans include Zbl. Bakt. Hyg. (1987) Polyclonal antibodies described in A265, pages 330-339 and antibodies against Streptococcus sobrinus include the polyclonal antibodies and monoclonal antibodies described in Osaka University Medical Journal (1990) 35: 93-109. Any antibody that can bind to a sugar chain antigen of Streptococcus mutans or Streptococcus sobrinus can be used without limitation, and polyclonal antibodies and monoclonal antibodies prepared by known methods can be used.

  For example, a polyclonal antibody can be obtained by immunizing an immunized animal with Streptococcus mutans cells or 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 an antibody having cross-reactivity with other microorganisms.

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

  Hereinafter, these immunological measurement methods will be described.

[Immunoagglutination]
This method is a method for 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. Semi-quantitative methods include latex agglutination and microtiter methods, and quantitative measurement methods include latex quantification and the like.

  For example, when the amount of antigen in a sugar chain antigen extract is immunologically measured using the latex agglutination method, an antibody that binds to the sugar chain antigen of the microorganism (hereinafter simply referred to as “antibody”) on latex beads. The measurement reagent and the sugar chain antigen extract are mixed, and the degree of aggregation of the sensitized particles after the antigen-antibody reaction is visually or optically mixed. It can be measured by detecting by a measuring method or the like.

[Optical immunoassay method]
This method is a method for detecting a change in turbidity of an aggregate resulting from an antigen-antibody reaction when an antigen-antibody reaction is carried out by contacting an antibody and a sugar chain antigen extract, or a thin antibody on which an antibody is immobilized. A method in which a sugar chain antigen extract is brought into contact with a layer (hereinafter also referred to as an “antibody layer”), and a change in the refractive index of the antibody layer resulting from the antigen-antibody reaction is detected as a change in transmitted light, surface plasmon wave, etc. It is a method for optically detecting the presence or absence of an antigen-antibody reaction.

[Labeled immunoassay]
The method comprises contacting an antigen with a measurement reagent containing a labeled antibody obtained by binding various labeled substances such as radioactive substances, enzymes, various dyes, colloids, and various particles to an antibody, and a sugar chain antigen extract. After performing the antibody reaction, the amount of the labeling substance bound to the antigen in the sugar chain antigen extract, ie, the radioactivity, enzyme activity, fluorescence intensity, coloring, etc. derived from the labeling substance is measured to extract the sugar chain antigen. This is a method for detecting and quantifying an antigen in a liquid.

  In this method, for example, an antigen-antibody reaction is performed by contacting a measurement reagent comprising an insoluble carrier (particle, membrane, immunoplate, etc.) on which an antibody is immobilized with a sugar chain antigen extract, and then the antibody is labeled with a labeling substance. After further antigen-antibody reaction by contacting with another measurement reagent containing the labeled antibody, the amount of the labeled substance is measured, or the sugar chain antigen extract and the sugar chain antigen labeled with the labeled substance are mixed After the antigen-antibody reaction by contacting with a measurement reagent consisting of an insoluble carrier with immobilized antibody, the antigen in the sugar chain antigen extract is detected and quantified by measuring the amount of the labeled substance bound to the antibody. can do.

  Labeling substances include radioactive iodine, radioactive carbon, etc. as radioactive substances, peroxidase, alkaline phosphatase, galactosidase, etc. as enzymes, various dyes, fluorescent dyes such as fluorescein isothiocyanate, tetramethylrhodamine, etc., colloidal gold colloid Carbon colloids and the like, and various latex particles such as colored latex particles can be used. When performing enzyme labeling, methods such as direct labeling through covalent bonds such as thiol group and maleimide group, amino group and aldehyde group, or labeling via a biotin-avidin complex can be used.

  In addition, alkaline phosphatase and peroxidase are used as labeling enzymes, and in the case of the former enzyme, a chemiluminescent substance such as a dioxetane derivative, and in the case of the latter enzyme, a chemiluminescent substance such as a luminol derivative is used as the enzyme substrate. When used as, 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 employed, and can be applied to known non-competitive methods, competitive methods, sandwich methods, and the like. In addition to the antibody, a secondary antibody labeled with each of the labeling substances described above, a substance capable of binding to an antibody such as protein A, and the like can also be used for detection and quantification of a sugar chain antigen.

  In the labeled immunoassay method, a conventionally used method can be used without particular limitation depending on the label to be used. Among them, a radioimmunoassay method using a radioactive substance as a label, an enzyme immunoassay method using an enzyme as a label, Fluorescent immunoassay methods that use dyes, especially fluorescent dyes as labels, and chemiluminescent immunoassay methods that use chemiluminescent substances as labels for enzymes as labels are highly quantitative, so when performing highly accurate quantitative measurements It is preferable to adopt these measurement methods. In addition, the flow-through immunoassay method, immunochromatography method, and latex agglutination method using colloids or various particles as labels are characterized by simple operation.

  Among these methods, the immunochromatography method is a simple and quick method that can be carried out at 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) that can be suitably used in the immunoassay method of the present invention. Like the conventional strip, the strip (7) is a member made of a porous support according to the purpose, that is, a sample pad (8) for temporarily absorbing and holding a sugar chain antigen extract, Conjugate pad (9) for temporarily holding labeled antibody, development membrane (10) on which detection antibody is immobilized, and sugar chain antigen extract developed from sample pad (8) The absorbent pads (11) are joined in this order.

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

  Each dimension (length) shown in FIG. 2 and FIG. 3 is merely an indication of the size, and the size of the strip used in the immunochromatography method of the present invention is shown in these drawings. It is not limited to the value that can be obtained.

  The material used for the porous support constituting 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, as the sample pad (8), it is preferable to use filter paper, blotting paper or the like, and as the conjugate pad (9), it is preferable to use glass fiber cloth, polypropylene nonwoven fabric, glass filter or the like. Further, it is preferable to use nitrocellulose or nitrocellulose containing cellulose acetate as the developing membrane (10), and it is preferable to use filter paper, blotting paper, or the like as the absorbent pad (11).

  The method for immobilizing the detection antibody and the control antibody on the development membrane (10) is not particularly limited, and any conventionally known physical adsorption method or covalent bond method can be used without any limitation, and it can be mixed with other proteins. It can also be fixed. In addition, in order to suppress non-specific adsorption of components of the sugar chain antigen extract to the development membrane, or to improve the wettability of the sugar chain antigen extract to the development membrane, the development membrane after antibody immobilization can be obtained by a known method. Blocking treatment can also be performed with proteins, lipids, polymer compounds and the like. The protein or the like used for this blocking treatment is not particularly limited, but BSA, skim milk, etc. that are used for the purpose of suppressing non-specific reactions in a general immunological measurement method are suitable. In addition, in order to adjust the water absorption of the development membrane so that the sugar chain antigen extract uniformly develops the development membrane, the surface of the development membrane can be coated or impregnated with a hydrophilic polymer or a surfactant. .

  For the conjugate pad (9), when the sugar chain antigen extract was added, the water-soluble heavy weight was so extracted that the complex of the sugar chain antigen extract and the labeled antibody was easily detached from this part. After blocking with a saccharide such as coalescence or saccharose, add a labeled antibody and dry it, or mix the labeled antibody with a water-soluble polymer or saccharide such as saccharose before applying to the conjugate pad and drying. Is preferred.

  Examples of the water-soluble polymer include polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, cellulose ether (such as methyl cellulose, ethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, oxyethyl cellulose, and cyanethyl cellulose), gelatin, and the like.

  As a labeling substance for preparing the labeled antibody, a labeling substance that can be visually confirmed such as gold colloid, carbon colloid, and colored latex can be used, and a radioactive substance and a fluorescent substance can also be used. Alternatively, an enzyme can be used as a labeling substance, and a substrate can 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 can be performed by a method generally employed conventionally.

  In the immunochromatography method, a 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 the sugar chain is left at room temperature for 1 to 30 minutes. The labeled antibody is expanded to the detection line (12) together with the antigen extract. Based on the principle of the sandwich method, the glycan antigen of the microorganism bound to the labeled antibody in the developing solution is detected on the detection line (12). By detecting the labeled antibody bound to the captured sugar chain antigen and measuring the presence or absence of the sugar chain antigen possessed by Streptococcus mutans or Streptococcus sobrinus in the sugar chain antigen extract Can do. At this time, the labeled antibody is normally developed together with the sugar chain antigen extract. This is because the antibody that reacts with the labeled antibody is immobilized on the control judgment line (13) downstream of the detection line (12). This can be confirmed by detecting the labeled antibody at the position.

  The position, order, shape, number, etc. of the detection line (12) and the control determination line (13) on the development membrane (10) shown in FIG. 2 are merely examples, and are not particularly limited.

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

  In the measurement by the immunochromatography method, for example, the amount of the antigen in the sugar chain antigen extract is quantified quickly and easily by performing the following procedure, and based on this value, the measurement in the extracted solution is performed. The microbial content of the subject can be determined.

  That is, first, a sugar chain antigen extract (for example, 100 to 200 μl) is collected, added to the sample pad of the strip, allowed to stand for a predetermined time (for example, 1 to 30 minutes), and then the strip detection line (12 ) And the control determination line (13), whether or not the sugar chain antigen is present can be determined by determining whether or not it is colored. Since the antigen concentration in the sugar chain antigen extract and the concentration of the microorganism to be measured in the extract are in a proportional relationship, the amount of the microorganism in the extract is determined based on the amount of antigen.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to a following example.

Production Example 1 [Preparation and purification of polyclonal antibody against Streptococcus mutans]
(1) [Preparation of antiserum against Streptococcus mutans]
Brain Heart Infusion (abbreviated as “BHI”; DIFCO) 3.7 g dissolved in 100 ml of ultrapure water, autoclaved to prepare a BHI liquid medium, and Ingbritt (Streptococcus mutans) at 37 ° C., 18 Incubate for an hour under anaerobic conditions. The culture solution was centrifuged at 4000 g for 5 minutes to remove the culture medium component of the supernatant and collect the bacterial cell precipitate. Subsequently, the precipitate was suspended in 100 ml of phosphate physiological saline buffer (pH 7.4) (hereinafter also abbreviated as “PBS”) and subjected to the same centrifugation three times to wash the precipitate. .

The cell pellet was suspended in PBS and adjusted to A ₆₀₀ = 1.0 to prepare a cell suspension of Ingbritt. In addition, after ultrasonically treating the bacterial cell suspension, it is appropriately diluted and then added onto a BHI medium plate, and the resulting colony count is counted and multiplied by the dilution factor of the bacterial cell suspension. When the bacterial cell concentration of the liquid was determined, it was about 1 × 10 ⁹ cells / ml.

  Immunization was performed as follows.

  In the first week, 0.5 ml of the bacterial cell suspension was injected into the auricular vein to the rabbit 5 times for 5 consecutive days. In the second week, 1.0 ml of the cell suspension was injected into the ear vein of the rabbit 5 times for 5 consecutive days. In the third week, 2.0 ml of the bacterial cell suspension was injected into the ear vein of the rabbit 5 times for 5 consecutive days. The fourth week was immunized as in the third week. After confirming the increase in titer by the degree of agglutination reaction of the bacterial cells using a slide glass, blood was collected according to a standard method one week after the final immunization to obtain an antiserum against Streptococcus mutans.

(2) [Purification of polyclonal antibody against Streptococcus mutans]
Challis (Streptococcus gordonii) was cultured in a BHI medium in the same manner as in (1) above, and then washed with PBS to prepare 30 ml of a cell suspension containing 2 × 10 ¹² cells / ml. Next, the cell suspension and 0.5 ml of antiserum were mixed and reacted at 4 ° C. for 60 minutes. The mixture was centrifuged at 4000 g for 5 minutes, and the supernatant was collected and filtered through a 0.22 μm filter.

Next, the supernatant sample was added to a column packed with 1 ml of Protein A-Sepharose (Amersham Pharmacia Biotech) previously equilibrated with PBS, the column was washed with 5 ml of PBS, and then 5 ml of 0.1 M glycine-HCl buffer. The mixture was eluted with a liquid (pH 3.0), and immediately 1M Tris-hydrochloric acid (pH 9.0) was added to adjust to pH 7.4. The eluted fraction of IgG was confirmed by measuring _A280 . About 5 mg of IgG was recovered from 0.5 ml of antiserum.

Example 1 Comparative Example 1 [Filtration of Extracted Liquid by Filtration Device]
The test subject chewed the paraffin pellets for 5 minutes, and the secreted saliva was collected as an extraction liquid.

  A glass fiber filter paper or filter paper was set on an SC cartridge (manufactured by Maruemu Co., Ltd.) to produce a filter device having the structure of FIG. The glass fiber filter paper and filter paper used in Table 1 were used. Experiment No. Nos. 3, 4, 5 and 6 are filter papers having a pore diameter in the range of 0.8 to 2.0 μm and are examples of the present invention. Nos. 1, 2, 7, 8, and 9 are comparative examples for the present invention in which the pore diameter of the filter paper is outside the above range.

  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, and the filtrate was recovered. The liquid to be extracted and the filtrate were appropriately diluted, and 100 μl was added onto a solid medium of Mits Salivarius bacitracin (hereinafter also abbreviated as “MSB”) and cultured at 37 ° C. under anaerobic conditions for 48 hours.

  As an example of filtration with a membrane filter, filtration of the liquid to be extracted with Millex HV (PVDF membrane with a pore diameter of 0.45 μm, diameter of 13 mm) was also performed. The number of colonies generated on the MSB medium was counted, and the retention rate [(the number of bacteria in the extraction liquid−the number of bacteria in the filtrate) / the number of bacteria in the extraction liquid × 100] was calculated. The results are shown in Table 1.

  The bacteria that grow on the MSB medium are mainly Streptococcus mutans and Streptococcus sobrinus, so that the cells have a size of 0.5 to 0.9 μm. When a filter medium having a pore size smaller than the cells was used (Experiment No. 1, 2, 9), clogging occurred at an early stage, and the amount of liquid that could be filtered was less than 0.5 ml. In particular, when a membrane filter was used, only 0.1 ml could be filtered.

  The trapping rate by the filter medium is 90% or more up to a pore diameter of 0.8 to 2.0 μm, particularly 95% or more in the case of 0.8 to 1.6 μm. It was confirmed that Streptococcus mutans and Streptococcus sobrinus were trapped on the filter medium. In addition, the amount of analyte liquid that can be filtered with a filter medium of 0.8 to 2.0 μm was 1.0 ml.

Example 2 [Extraction of sugar chain antigen from extraction liquid containing saliva and plaque and measurement by immunochromatography]
(1) [Collection of extraction liquid and measurement of bacterial count by culture method]
According to the method of Example 1, specimens containing saliva and plaque were obtained from 18 different subjects. Each specimen was appropriately diluted, and 100 μl was added onto MSB solid medium and BHI solid medium, and cultured at 37 ° C. under anaerobic conditions for 24-48 hours. The number of colonies generated on the MSB and BHI solid medium was counted, and the concentration of mutans streptococci was calculated as number / ml from the dilution rate of the extraction solution.

  The identification of Streptococcus mutans on MSB and BHI media is based on the morphological classification of colonies and, for morphologically indistinguishable colonies, serotype-specific mutans streptococci after pure culture of the colonies. Streptococcus mutans was identified by an immunological assay method using various antibodies 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 through a filtration device in which GA-100 (pore size: 1.0 μm) was set. Next, 0.5 ml of 0.1 M NaOH solution was added to the filtration device and filtered, and further 0.5 ml of PBS was added to the filtration device and filtered to wash the microorganisms on the filter material and the filter material. Add 20 μl of 2M acetic acid solution and 20 μl of 1M sodium nitrite aqueous solution to the filter medium, let stand for 2 minutes or 10 minutes at room temperature, and then add 90 μl of 1M Tris containing 0.05% Tween20 (pH not prepared) Then, the sugar chain antigen extract (100 to 140 μl) was recovered by pressurizing the filtration device with a syringe.

(3) [Measurement of sugar chain antigen extract by immunochromatographic strip]
88 μl of 100 mM K ₂ CO ₃ was added to 10 ml of a commercially available gold colloid solution (EY Laboratory) having a colloid particle size of 40 nm to adjust the pH to 9.0, followed by 0.22 μm filter treatment. The absorbance at 520 nm of the colloidal gold solution was measured and found to be A ₅₂₀ = 1.0.

Subsequently, 64 μl of the 2 mM borate buffer solution (pH 9.0) of the antibody prepared in Production Example 1 adjusted to 1 mg / ml was added to the gold colloid solution with stirring, and left at room temperature for 5 minutes. Next, 1.1 ml of 10% skim milk-2 mM borate buffer (pH 9.0) was added with stirring (final concentration of skim milk 1%) and left at room temperature for 30 minutes. Next, the reaction solution was centrifuged at 10 ° C., 10000 g for 30 minutes, and after removing the supernatant, 2 ml of 2 mM PBS (pH 7.4) was added to resuspend the lower gold colloid fraction. When the absorbance at 520 nm of the resuspended fraction was measured, A ₅₂₀ = 4.9. The obtained gold colloid fraction (hereinafter also abbreviated as “gold colloid-labeled antibody”) was stored at 4 ° C.

  Production example of 1 mg / ml on the detection line (12) and the control judgment line (13) on the development membrane (10) composed of a nitrocellulose membrane (MILLIPORE, Hi-Flow Plus Membrane, HF180, 25 mm × 6 mm) The antibody prepared in 1 and 1 μl of anti-rabbit IgG (H + L) polyclonal antibody (ICN Pharmaceuticals) were spotted and dried in an incubator at 37 ° C. for 60 minutes to immobilize the antibody. The antibody-immobilized membrane was shaken in a 1% skim milk-0.1% Triton X100 aqueous solution at room temperature for 5 minutes. Next, the membrane was taken out in a 10 mM phosphate buffer solution (pH 7.4) at room temperature for 10 minutes and then taken out, and dried in a desiccator for 60 minutes while sucking with a vacuum pump.

In addition, conjugate pad (9) (MILLIPORE, 7.5 mm × 6 mm) was taken out after shaking for 1 minute in 0.5% PVA-0.5% aqueous sucrose solution, and desiccator for 60 minutes while sucking with a vacuum pump. Dried in. 25 μl of gold colloid-labeled antibody adjusted to A ₅₂₀ = 1.0 was added to the conjugate pad and dried in a desiccator for 60 minutes while sucking with a vacuum pump. Further, the sample pad (8) (MILLIPORE, 17 mm × 6 mm) was shaken in a 1% Tween 20-PBS aqueous solution for 1 minute and then taken out and dried in a desiccator for 60 minutes while being sucked with a vacuum pump. The absorbent pad (11) (MILLIPORE, 20 mm × 6 mm) was used untreated.

  Each component of the immunochromatographic strip prepared as shown in FIG. 2 was placed on a plastic support, and the immunochromatographic strip as shown in FIG. 3 was assembled.

  On the sample pad (8) of the immunochromatography method strip, 100 μl of the sugar chain antigen extract was added, and the presence or absence of spots was determined after 10 minutes. In the determination, the degree of colloidal gold captured on the detection antibody spot was visually identified in four stages (++: strong positive, ++: positive, +: weak positive, −: negative). The results are shown in Table 2.

  As shown in Table 2, a spot color intensity correlated with the concentration of Streptococcus mutans obtained by the culture method was obtained. As for the action time of the nitrous acid aqueous solution, the spot intensity was stronger at 10 minutes than at 2 minutes. The amount of antigen in the sugar chain antigen extract, in other words, the amount of Streptococcus mutans in the extract, could be detected in a concentration-dependent manner by the measurement method of the present invention.

Comparative Example 2 [Extraction of Sugar Chain Antigen from Extracted Liquid Containing Saliva and Plaque by Centrifugation and Measurement by Immunochromatography]
(1) [Preparation of sugar chain antigen extract]
The extraction liquid (0.5 ml) containing saliva and plaque collected in Example 1 is centrifuged at 10,000 rpm for 5 minutes, the precipitate is suspended in 0.5 ml of 0.1 M NaOH, and the supernatant is obtained by centrifugation. Removed. Subsequently, the precipitate was suspended in 0.5 ml of PBS, and the supernatant was removed by centrifugation.

  After the same operation with PBS was performed again, 20 μl of 2M acetic acid solution and 20 μl of 1M sodium nitrite aqueous solution were added to the precipitate and mixed well, and then allowed to stand at room temperature for 10 minutes. 90 μl of 1M Tris (pH not prepared) containing 0.05% Tween 20 was added to prepare a sugar chain antigen extract.

(2) [Measurement of sugar chain antigen extract by immunochromatographic strip]
The sugar chain antigen extract prepared in (1) above was measured by the same method as in Example 2 (3). The results are shown in Table 3.

  As shown in Table 3, some of the samples in which Streptococcus mutans were not detected by the culture method were positive (Samples 14, 15, and 17). This is probably because the non-specific reaction-causing substance contained in the extraction liquid could not be sufficiently removed by washing with a centrifugal separation method, so that it became positive due to the non-specific reaction.

Example 3 [Nitrous acid extraction with a previously prepared aqueous nitrite solution]
After the liquid to be extracted was filtered by the same method as in Example 2 (1), the microorganisms and the filter medium on the filter medium were washed with 0.5 ml of 0.1 M NaOH solution. A 1M sodium nitrite solution and a 2M acetic acid solution are mixed at a ratio of 1: 1 to prepare an aqueous nitrous acid solution, 0.2 ml of the aqueous nitrous acid solution is added to the filter, and the excess amount is quickly lowered by pressurizing with a syringe. It was. After leaving at room temperature for 2 minutes, 90 μl of 1M Tris (pH unadjusted) aqueous solution containing 0.05% Tween20 is added, and the sugar chain antigen extract (100 to 140 μl) is recovered by pressurizing the filtration device with a syringe. did. The sugar chain antigen extract was measured by the same method as in Example 2 (3). The results are shown in Table 4.

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

This figure is a schematic view of a filtration apparatus used in the method for extracting a microbial antigen of the present invention. This figure is the schematic of each member of the strip used when the measuring method of microorganisms of this invention is implemented by the immunochromatography method. This figure is a side view of the strip of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Drainage port 2 ... Filter medium 3 ... Container main body 4 ... Syringe insertion port 5 ... Cover part 6 ... Filtration apparatus 7 ... Strip 8 ... Sample pad 9 ... Conjugate pad 10 ... Deployment membrane 11 ... Absorption pad 12 ... Detection line 13 ... Control judgment line

Claims (3)

A liquid to be extracted comprising saliva, plaque, or a mixture thereof is filtered through a filter medium having a pore size of 0.8 to 2 μm, and a nitrous acid aqueous solution is applied to Streptococcus mutans and / or Streptococcus sobrinus held on the filter medium. A method for extracting a microbial antigen, which comprises extracting a sugar chain antigen possessed by the microorganism by acting. The amount of the sugar chain antigen contained in the sugar chain antigen extract extracted by the method according to claim 1 is measured by an immunological measurement method, and Streptococcus mutans contained in the extraction liquid based on the measurement result. And / or determining the amount of Streptococcus sobrinus. By filtering a liquid to be extracted containing saliva, dental plaque, or a mixture thereof with a filter medium, and reacting Streptococcus mutans and / or Streptococcus sobrinus held on the filter medium with an aqueous nitrite solution, the microorganism Filtration device used for carrying out the method for extracting a sugar chain antigen possessed by a microorganism, wherein a filtration material having a pore diameter of 0.8 to 2 μm is used as the filtration material. .

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