JP2008224543A - Measuring method of saliva buffer performance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring saliva buffer performance quickly, accurately and simply, even in the case of a subject having a small secretion amount of saliva. <P>SOLUTION: Saliva is filtered with a filter medium carrying a saliva buffer performance inspection reagent, to thereby separate the saliva into a component held on the filter medium and a filtrate, and the number of caries related bacteria is measured by utilizing the component on the filter medium, and the saliva buffer performance is measured by a colored state of the filtrate, to thereby measure the saliva buffer performance quickly and accurately, even in the case of the subject having a small secretion amount of saliva. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for diagnosing the risk of caries development by measuring the buffer capacity of saliva. More specifically, the present invention relates to a method for measuring saliva buffering ability with a filtrate produced by filtering saliva with a filter medium carrying a saliva buffering ability test reagent.

  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, periodontal disease-related bacteria and opportunistic bacteria are strongly involved in the occurrence of caries, periodontal disease, and aspiration pneumonia. In recent years, it has been clarified that microorganisms present in the oral cavity may be involved in the development of diseases that have not been related to infectious diseases, such as heart disease, gastric ulcer, and cancer. Microbiological examinations are important not only in the dental field but also in the medical field.

The measurement of microorganisms in saliva has been carried out 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. (Non-Patent Documents 1 and 2, Patent Document 1). In general, the results of immunoassays can be quickly determined because the time required for the antigen-antibody reaction is short.

  Saliva is important in protecting against infections caused by microorganisms present in the oral cavity. Saliva protects a living body from microorganisms by (1) cleaning action, (2) buffering action, (3) antibacterial action, etc. (Non-patent Document 3). The cleaning action is an action of washing away microorganisms from the oral cavity and generally increases in proportion to the amount of saliva secretion. The buffering action is to keep the oral pH constant around neutrality, and is exerted by bicarbonate, phosphate, protein, etc. in saliva to prevent the onset of disease by maintaining oral homeostasis. To do. The antibacterial action is said to be exerted by peroxidase, lactoferrin, antibodies, etc. contained in saliva. Among these, it has been found that the buffering action is particularly important for disease prevention.

  For the above reasons, it is important to measure the saliva buffering action (hereinafter sometimes referred to as saliva buffering ability) in addition to the number of microorganisms in order to determine the risk of developing diseases caused by oral microorganisms. .

  For example, even if the number of microorganisms in saliva is large, the risk of disease development differs between people with high saliva buffering capacity and those with low saliva buffering capacity, and even when the saliva buffering capacity is low, The risk of developing the disease is different. Therefore, in order to accurately determine the risk of disease occurrence and effectively prevent it, it is important to measure the total number of microorganisms in the oral cavity and the saliva buffering capacity at the same time and make a comprehensive determination.

  It is known that the microorganism concentration in saliva and the saliva buffering capacity fluctuate within the day. Therefore, the situation in the oral cavity cannot be determined accurately even if the number of microorganisms and the saliva buffer capacity are measured and compared using saliva with different collection times. For these reasons, when measuring the number of microorganisms and the saliva buffer capacity, it can be said that it is desirable to simultaneously measure using the same sample.

  Generally, when measuring the number of microorganisms in the oral cavity by a culture method, about 0.01 to 0.1 mL of saliva is required, but when carrying out an immunoassay, a larger amount of saliva (0.3 About 0.5 mL) is necessary (for example, Non-Patent Document 3). The reason is as follows. In an immunological measurement method, an antibody against an antigen present on the surface of a microorganism is usually used. Since microorganisms having pathogenicity in saliva are often covered with biofilms such as dental plaque, antibodies cannot be contacted as they are and antigen-antibody reaction does not occur. In order to cause an antigen-antibody reaction, it is necessary to perform some kind of pretreatment to remove the biofilm to expose the antigen, or to extract the antigen from the cells. However, even if pretreatment is performed, the biofilm cannot be completely removed, all antigens cannot be extracted, or some antigens are destroyed by pretreatment, etc. Since not all antigens can be measured, a large amount of saliva is required as compared with the culture method.

  Here, the saliva is carried out by chewing a chewing object such as paraffin pellets and gums for a certain period of time and collecting the secreted saliva. It is possible to determine the amount of salivary secretion that is one of the important indicators for comprehensive determination of the risk of occurrence.

  The saliva buffering ability can be determined by adding a certain amount of acid to the saliva collected in this manner and measuring the pH. The method of visually confirming the color state of the pH indicator and comparing it with a standard colorimetric table prepared in advance at each pH value can be measured without the need for an instrument. It can be said that this is one of the preferred embodiments. For example, Yamada et al. Have proposed a method in which a pH indicator and an acidic buffer are held in an absorbent carrier, saliva is poured on the carrier, and the color tone is visually confirmed, a so-called test paper method (Patent Document 2). . This method can measure the saliva buffering capacity with a very small amount of saliva of about 0.005 to 0.03 mL, but the viscosity can be measured only after saliva soaks into the carrier and is mixed with the reagent. In saliva, since the penetration of saliva into the carrier tends to be non-uniform, there is a drawback that the pH indicator on the carrier becomes mottled and the determination of the saliva buffering capacity may be difficult.

  Generally, saliva collected from subjects with low saliva secretion is known to have high viscosity. In such cases, it is often difficult to measure the saliva buffer capacity by the test paper method for the above reasons. However, in such a test paper method, it is known that saliva for measurement is subjected to filtration treatment to remove viscous substances such as insoluble matter and mucin in the saliva (Patent Document 3). However, this conventional method requires a two-step operation in which saliva is filtered to prepare a filtrate, and then the filtrate is brought into contact with a saliva buffer capacity test reagent.

  On the other hand, a method in which saliva is added to a test tube containing a dry powder of a reagent (lactic acid, pH indicator) in advance, the lid is shaken to dissolve the reagent, and the color tone of the pH indicator is visually determined, a so-called determination tube A method has also been proposed (Non-Patent Document 4). In this method, since it is not necessary to soak the reagent in the carrier, saliva buffering ability can be measured relatively easily even with high-viscosity saliva, but about 0.5 to 2 mL of saliva is necessary for the measurement. is there.

  When the test subject is an elderly person or an infant, there are cases where only 0.5 mL or less of saliva can be collected for reasons such as a small amount of saliva secretion or a difficulty in spitting saliva. In this case, the amount of saliva secretion can be measured even if the amount of secretion is small, but there has been a problem that the number of microorganisms in saliva and the saliva buffering capacity cannot be measured simultaneously and quickly.

Edited by The Biochemical Society of Japan, Laboratory for Neonatal Chemistry 12 Molecular Immunology III Antigens / Antibodies / Captures, Tokyo Chemical Dojin, 1992 From Healthcare Dental Clinic Excellent preventive dentistry 17 + α, Dental Diamond Co., Ltd., 2006 Saliva, by Jorma Tenobu, Japan Finnish Caries Prevention Society, 1999 Yoshihide Okazaki et al., Journal of Pediatric Dentistry, 38: 615-621, 2000 JP 2003-215126 A Japanese Patent No. 1574981 JP 2002-323493 A

  The present invention has been made in view of the above circumstances, and provides a method for easily and rapidly measuring saliva buffering capacity in addition to the number of microorganisms in saliva, even for saliva collected from a subject having a small amount of saliva secretion. The purpose is to do.

  The present inventor has intensively studied to solve the above problems. As a result, the saliva is filtered by the filter medium carrying the saliva buffer capacity test reagent, so that the microorganisms in the saliva are retained on the filter medium, and at the same time, the saliva and the saliva buffer capacity test reagent are efficiently contacted. It was found that the saliva buffer capacity can be easily measured by measuring the coloration of the filtrate produced as a result of filtration. Further studies have been made and the present invention has been completed.

  That is, the present invention is a method for measuring the saliva buffer capacity using a saliva filtrate produced by filtering saliva with a filter medium and measuring the amount of microorganisms in the saliva from components retained on the filter medium. The saliva buffering capacity is measured by filtering the saliva with a filter medium carrying a test reagent containing a pH indicator and an acid, and measuring the coloration state of the filtrate.

  Another aspect of the present invention is a filtration device comprising a filter medium carrying a saliva buffer capacity test reagent, which can be used for the measurement of the saliva buffer capacity.

  According to the measurement method of the present invention, the risk of disease occurrence can be evaluated easily and quickly with high accuracy by filtering saliva and measuring the microorganisms retained on the filter medium and the buffer capacity of the filtrate. . Saliva collected from the subject is about 0.3 mL to 20 mL when collected by a conventional method (collecting saliva chewed by a subject such as a chewing gum for 5 minutes), and this collected sample is used in the present invention. Apply to the method. Since the method of the present invention can be used to measure both the number of microorganisms and the saliva buffering capacity, the method of the present invention sufficiently performs both measurements even when the amount of saliva is small, for example, 0.3 mL to 0.5 mL. Possible and preferred.

  In the method for measuring saliva buffering capacity of the present invention, the saliva is filtered with a filter medium carrying a saliva buffering capacity test reagent to separate the insoluble matter containing microorganisms and the filtrate, and the amount of microorganisms in the saliva using the former insoluble matter While measuring the salivary buffer capacity using the latter filtrate.

  In the present invention, both resting saliva and stimulated saliva can be used, but it is preferable to use stimulated saliva for reasons such as reproducibility and ease of sample collection. Stimulated saliva can be collected, for example, by spitting saliva while chewing a chewing object such as gum or paraffin pellets for a certain period of time.

  Examples of microorganisms that cause infections contained in saliva according to the present invention include caries-related bacteria, periodontal disease-related bacteria, upper respiratory tract infection-causing bacteria, opportunistic infection bacteria, and the like. When specific examples are displayed for each, the caries-related bacteria include Streptococcus mutans, Streptococcus sobrinus, and other bacteria belonging to mutans streptococci, Lactobacillus, Bacteria belonging to the genus Actinomyces include porphyromonas gingivalis, actinomycetemcomitomitus Bacterium, and actinomycetem bacterium bacterium. ), Treponema denticola, Prevotella intermedia, Prevotella nigrescens, etc., and the upper respiratory tract-causing bacteria, A group of Streptococcus (Gr. (Micoplasma pneumoniae), Chlamydia pneumoniae, etc., and Candida sp., Staphylococcus aureus, and Pseudomonas (Pseudomonas) are examples of opportunistic infections.

  Microorganism can be measured quickly and accurately by, for example, extracting an antigen from the microorganism and measuring the extracted antigen by an immunoassay. For example, microorganisms are concentrated on a filter medium by filtering saliva, and antigens are extracted, thereby enabling highly sensitive immunological measurement. As a method for extracting an antigen, a known method can be used without any limitation. As a suitable example, a nitrite extraction method can be shown. The nitrous acid extraction method can be performed by a known method (for example, see Patent Document 1).

  Hereinafter, the nitrous acid extraction method will be described. The nitrite extraction method is a method in which a nitrite aqueous solution and an acid aqueous solution are dropped onto a filter medium to form nitrous acid, and a sugar chain antigen is extracted from the resulting nitrous acid aqueous solution. Examples of the aqueous nitrite solution include 0.5 to 8M sodium nitrite and potassium nitrite, and examples of the aqueous acid solution include 0.5 to 4M acetic acid, nitric acid, hydrochloric acid, and citric acid. For example, 0.005 to 0.05 mL of the nitrite aqueous solution and the acid aqueous solution as described above are added to the filter medium and allowed to stand at 15 to 50 ° C. for 1 to 10 minutes to cause the reaction on the filter medium.

  What is necessary is just to collect | recover the extract after reaction by pressurizing by the method similar to filtration of saliva. Since the reaction solution is strongly acidic, for example, an aqueous base solution such as 1 to 3 M sodium hydroxide, 0.5 to 2 M Tris, 0.5 to 1 M sodium bicarbonate is added to adjust the pH of the extract to 7.0 to 8.5. It is preferable to adjust to.

  The base aqueous solution may be added after the antigen extract is recovered from the filter medium, or the extract may be recovered after the extract is added to the filter medium before the extract is recovered. From the viewpoint, it is more preferable to add to the filter medium before recovery. Alternatively, an aqueous nitrite solution and an aqueous acid solution are mixed in advance to prepare an aqueous nitrous acid solution (for example, a mixture of 0.2 to 4M sodium nitrite and 0.5 to 4M acetic acid). Nitrous acid extraction can also be carried out by filtering the filtration membrane. In this case, 0.2 to 0.5 mL of a nitrous acid aqueous solution prepared by mixing a sodium nitrite aqueous solution and an acetic acid aqueous solution was added, filtered, and left at 5 to 50 ° C. for 1 to 10 minutes to extract nitrous acid. The neutralization after the reaction is preferably performed by adding 0.5 to 1.0 M Tris to a 100 to 800 μL filter membrane and collecting the sugar chain antigen extract by filtration under pressure.

  As the filter medium used in the method for measuring the saliva buffering capacity of the present invention, a membrane or layered filter medium that is stable in the above extraction operation can be used without limitation. Examples of such a filter medium include a screen filter such as a membrane filter, and a depth filter such as a filter paper and a glass fiber filter paper. From the purpose of stably carrying a certain amount of a saliva buffer capacity test reagent as described later. A depth filter can be used more suitably.

  In the case of a screen filter, the pore size of the filter medium is a numerical value calculated from the pressure when air is pushed out through the pores of a membrane filter wetted with a liquid described in Japanese Industrial Standard (JIS K3832) (bubble point method) In the case of a depth filter, it is a numerical value obtained from the leaked particle size when barium sulfate or the like described in Japanese Industrial Standard (JIS P 3801) is naturally filtered. In general, when the filter medium is a screen filter such as a membrane filter, the pore diameter displayed in the commercially available filter medium catalog corresponds to the pore diameter of the filter medium obtained by the above method, and the filter medium is like glass fiber filter paper or filter paper. In the case of a depth filter, the retained particle diameter and the particle retention capacity displayed in the commercially available filter media catalog correspond to the pore diameter determined by the above method.

  The pore diameter of the filter medium may be appropriately selected in consideration of the size of the microorganism to be measured and the filtration efficiency of saliva. Since microorganisms are generally 0.3 to 1.0 μm in size, it is usual to filter using a filter medium having a pore diameter of less than 1.0 μm and a membrane filter of 0.22 μm or 0.45 μm as the filter medium. In many cases, microorganisms in the oral cavity are covered with a biofilm such as plaque, so that microorganisms can be captured on the filter medium even when a filter medium having a pore size larger than the size of a general microorganism is used. In addition, high-viscosity polymers such as mucin and insolubles are present in saliva, so if a filter medium with a small pore size is used, the filtration pressure during filtration increases due to clogging of the filter medium, The saliva filtration speed and the amount of saliva that can be filtered are reduced. For example, when measuring caries-related bacteria, as described in Patent Document 1, the cell size of mutans streptococci is in the range of 0.5 to 0.9 μm, but 0.8 to 2 μm. By using a filter medium having a pore size, it is possible to efficiently carry out both the capture of bacterial cells on the filter medium and the filtration of saliva.

  In the present invention, the amount of microorganisms in saliva is usually measured by quantifying the antigen extract prepared as described above by an immunological measurement method. 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 thereof can be used 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 a microorganism sugar chain antigen (hereinafter also simply referred to as an antibody) is immobilized on latex beads. After preparing a measurement reagent composed of sensitized antibody-sensitized particles, the measurement reagent and a sugar chain antigen extract are mixed, and the degree of aggregation of the sensitized particles after the antigen-antibody reaction is visually or optically measured. It can be measured by detecting.

[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 an 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 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 colloid or various particles as labels are characterized by simple operation.

  In the present invention, as described above, saliva is filtered with a filter medium, and when the amount of microorganisms in saliva is measured from components (insoluble matter) retained on the filter medium, the saliva filtrate generated by the filtration is used. The buffer capacity of the saliva is also measured. As a result, not only the number of microorganisms contained in saliva, but also the buffering action of the saliva can be known at the same time, and the overall risk of the occurrence of diseases caused by microorganisms in the oral cavity can be determined with high accuracy. It becomes possible to judge by.

  Thus, the greatest feature of the present invention is that the saliva buffer capacity measurement using the saliva filtrate is efficiently performed using a filter medium carrying a saliva buffer capacity test reagent containing a pH indicator and an acid. There is. That is, the filtrate filtered using the filter medium carrying the saliva buffer capacity test reagent exhibits the color tone of the pH indicator according to the buffer capacity due to the action of the saliva buffer capacity test reagent. Thus, the strength of the buffer capacity can be measured easily and quickly. In particular, according to this method, since the color state of the pH indicator can be observed as a uniform color tone of the filtrate, when the saliva is infiltrated on the carrier soaked with the saliva buffering capacity test reagent, the color state is observed. Such a variation in color tone is unlikely to occur (particularly when the viscosity of saliva to be measured is high) and can be measured with high accuracy.

  Even if the buffer capacity of the filtrate obtained by filtering saliva in this way is measured, the measurement result is substantially the same as that obtained when the saliva is directly subjected to measurement.

The reagent for testing saliva buffering ability used in the present invention contains a pH indicator and an acid. In the present invention, the pH indicator refers to a reagent that changes color by adding / eliminating protons by acid-base reaction, and any known one can be used without any limitation. As specific examples of such pH indicators, new experimental chemistry course 9 Analytical Chemistry II (published by Maruzen Co., Ltd. The Chemical Society of Japan, edited by the Chemical Society of Japan), page 181 (neutralization titration indicator), indicators (chlorophenol red, bromo) Cresol purple, chlorophenol red, etc.). Moreover, as an acid, a well-known thing can be used without being specifically limited. For example, tartaric acid, citric acid, malic acid, lactic acid, sulfosalicylic acid, oxalic acid, hydrochloric acid and the like can be used (for example, see Patent Document 2).
Since the pH distribution range after mixing with the saliva filtrate varies depending on the type and amount of acid used, a pH indicator having a color change range suitable for each pH range is appropriately selected (see, for example, Patent Document 2). Preferred combinations of these acids and pH indicators include tartaric acid, citric acid, malic acid, lactic acid, oxalic acid and chlorophenol red, bromocresol purple, oxalic acid and bromocresol green, sulfosalicylic acid and congo red. Examples thereof include a combination and a combination of hydrochloric acid and bromocresol green. These acids and pH indicators may be used singly or a plurality of them may be used in appropriate mixture.

In the saliva buffer capacity test reagent of the present invention, the amount of the pH indicator and the acid used per test is such that when the saliva is filtered through a filter medium carrying the saliva test reagent and the filtrate is prepared, The concentration is adjusted to 0.01 to 0.5 N in the case of acid and 10 to 1000 ppm in the case of pH indicator. Although affected by the amount of saliva to be filtered, in order for the saliva buffer capacity test reagent to be contained in the filtrate at such a concentration, the loading amount of the pH indicator and the acid is usually the former per 1 cm ^{2 of the} filter medium. In the range of 0.0001 mg to 50 mg, more preferably in the range of 0.001 to 15 mg, and the latter in the range of 0.0001 mg to 5 mg, more preferably in the range of 0.001 mg to 0.5 mg. It is.

  In the present invention, the method for supporting the saliva buffering capacity test reagent having the above-described configuration on the filter medium to be used may drop a certain amount of reagent on the filter medium, or put the filter medium in a container containing a certain amount of reagent. You may implement by making the reagent in a container inhale. Saliva may be filtered while the filter medium is wet with the test reagent, but it is preferable to dry the filter medium for ease of operation. The filter medium soaked with the test reagent may be dried by a known method. For example, it may be carried out by keeping the temperature at normal pressure (30 to 80 ° C.) or by reducing the pressure with a vacuum pump or the like.

  In addition to these acids and pH indicators, substances that do not change the pH can be added to the test reagent. For example, when preparing a filter medium supporting a saliva buffer capacity in a dry state, polyethylene glycol, polyvinyl pyrrolidone, a surfactant (polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid) is used for the purpose of improving the resolubility of the reagent. A wetting agent such as an ester) may be included in the test reagent component so that the concentration in the reagent is 0.05 to 5%.

  Specific production examples of the filter medium carrying the saliva buffering capacity test reagent are shown below. When filtering 0.3 mL of saliva using a filter medium carrying a saliva buffer capacity test reagent in a dry state, 0.1-5 N tartaric acid, 100-10000 ppm chlorophenol red, 0.05-5% A reagent for testing saliva buffering ability in which polyethylene glycol is dissolved in ethanol is added to the glass fiber filter paper in an amount that makes the above-mentioned suitable loading amount, and the filter medium may be dried by incubating at 30 to 80 ° C. for 10 to 60 minutes. .

  In the present invention, saliva filtration using the filter medium can be performed according to a general filtration method. For example, it can be carried out by using a syringe filter equipped with a filter medium carrying a saliva buffer capacity test reagent and pressurizing with a syringe. Alternatively, for example, it is possible to perform filtration by using a filtration device having a structure capable of attaching and detaching the lid as shown in FIG. 1 and injecting saliva into the filtration device and then closing and pressurizing the lid.

  In the present invention, the saliva buffering capacity is measured by filtering saliva using a filter medium carrying a saliva buffering capacity test reagent as described above, and measuring the pH of the resulting filtrate according to its color state. The The measurement of the colored state of the filtrate is usually carried out by visually confirming the color tone of the filtrate. The color tone at each pH is examined in advance, a colorimetric table is created, the pH is determined by comparing with the colorimetric table, and the saliva buffering capacity is determined. For example, when a test reagent is prepared from tartaric acid and chlorophenol red, the mixed solution of the test drug and saliva has a color tone as shown in the following table depending on pH. The higher the saliva buffering capacity is, the higher the pH of the mixed solution is, so that it can be determined as shown in Table 1 according to the color tone.

  As described above, the number of microorganisms in the oral cavity, the saliva secretion amount, and the saliva buffering capacity are efficiently measured. According to the method for measuring the saliva buffering capacity of the present invention, both the number of microorganisms and the saliva buffering capacity have been difficult to measure at the same time, even a subject with a small amount of saliva secretion can be measured easily at the same time. Is possible.

  The filter device of the present invention is not particularly limited in material, shape, size, etc., as long as the filter medium carrying the saliva buffering capacity test reagent is contained in its constituent elements. From the viewpoint of performing pressure filtration, the filtration device body is made of a flexible material (for example, plastic such as polypropylene), and the filter medium stably carries a certain amount of saliva filtration and saliva buffer capacity test reagent. A material such as filter paper or glass fiber is preferable. As a specific example of such a filtering device, a commercially available disposable syringe fixed with a glass fiber filter paper carrying a saliva buffer capacity test reagent, or a syringe filter equipped with a glass fiber filter paper carrying a saliva buffer capacity test reagent Alternatively, the filtration device having the structure shown in FIG. 1 can be displayed.

  Hereinafter, a specific example of the filtration device will be described with reference to FIG. Note that the shape of the filtration device shown in FIG. 1 is merely an example of the filtration device of the present invention, and the filtration device of the present invention is not limited to the shape shown in these drawings.

  Filter material is set on the bottom surface of the filter device body, and after injecting saliva, the lid is closed and pressure is applied to perform filtration, but the filter material may be held on the bottom surface with an adhesive or not particularly bonded. Further, it may be held on the bottom surface only by the frictional force.

  The filtration device preferably has a structure that can be sealed so that when the lid is closed and pressurized, the lid is held on the main body and the pressure does not escape to the outside. For this purpose, the lid can be held not only by frictional force as shown in FIG. 1 but also by screw screws or other sealing methods. It is preferable in terms of operability that the pressurization is performed by placing the syringe in the syringe insertion port of the lid and pressurizing.

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

Production Example 1 [Preparation of purified polyclonal antibody against Streptococcus mutans]
(1) [Preparation of cell sample suspension]
Brain Heart Infusion (hereinafter sometimes abbreviated as “BHI”) (DIFCO) (3.7 g) was dissolved in 100 mL of pure water and then autoclaved to prepare a BHI liquid medium. After culturing Ingbritt (Streptococcus mutans, serotype c) in 2 ml of BHI liquid medium under anaerobic conditions (N ₂ : H ₂ : CO ₂ = 80: 10: 10) at 37 ° C. for 5 hours, the culture solution Was centrifuged at 4000 g for 5 minutes, the medium components in the supernatant were removed, and the cell pellets were collected.

Subsequently, the precipitate was suspended in 5 mL of phosphate physiological saline buffer (pH 7.4) (hereinafter sometimes abbreviated as PBS), and the same centrifugation operation was performed three times to wash the precipitate. Thereafter, the obtained bacterial cell precipitate was suspended in PBS, adjusted to A ₆₀₀ = 1.0, and used as an Ingbritt cell sample suspension. In addition, after ultrasonically treating the bacterial cell sample suspension, it is appropriately diluted and then added to a BHI medium plate, and the number of colonies produced is counted and multiplied by the dilution factor of the bacterial cell sample suspension. The bacterial cell concentration of the sample suspension was determined to be about 1 × 10 ⁹ cells / mL.

(2) [Preparation of antiserum against Streptococcus mutans]
Immunization was performed as follows. That is, in the first week, 0.5 mL of Ingbritt cell sample 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 bacterial cell sample suspension was injected into the auricle intravenously to the rabbit 5 times for 5 consecutive days. In the third week, 2.0 mL of the bacterial cell sample 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.

(3) [Purification of polyclonal antibody against Streptococcus mutans]
Ingbritt was cultured at 37 ° C. for 12 hours under anaerobic conditions in 1 L of autoclaved BHI liquid medium. 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. Next, the precipitate was suspended in 100 mL of PBS and subjected to the same centrifugation three times to wash the precipitate.

The Ingbritt cells were washed, suspended in 0.1 M Tris-HCl buffer (pH 8.0), and adjusted to A ₆₀₀ = 15. Pronase (Wako Pure Chemical Industries, Ltd.) was added to this so that it might become 5 mg / mL, and it heat-retained at 37 degreeC for 1 hour. After completion of the reaction, the mixture was centrifuged to collect the cell precipitate. Subsequently, the precipitate was suspended in 20 mL of PBS and subjected to the same centrifugation three times to wash the precipitate. Subsequently, it was washed 3 times with 20 mL of 0.1 M glycine hydrochloride buffer (pH 2.0), and further washed 3 times with 20 mL of PBS to prepare a protease-treated cell suspension (A ₆₀₀ = 12.5).

  Next, the protease-treated cell suspension and 0.5 mL of the antiserum prepared in (2) were mixed and reacted at 4 ° C. for 60 minutes. The mixed solution was centrifuged at 4000 g for 5 minutes to recover the cells. The cells were suspended in 10 mL of PBS and washed by performing the same centrifugation operation three times.

  Subsequently, the cells are suspended in 0.5 mL of 0.1 M glycine hydrochloride buffer (pH 2.0), the adsorbed antibody is eluted, the supernatant is recovered by centrifugation, and 1 M Tris-HCl (pH 9.0) is collected. Was added to adjust the pH to 7.4. The same elution operation was performed 4 times, and the protein amount of each fraction was measured by absorbance at 280 nm.

Next, the eluate was added to a column packed with 1 mL of Protein A-Sepharose (Amersham Pharmacia Biotech) previously equilibrated with PBS, washed with 5 mL, and then 5 mL of 0.1 M glycine-HCl buffer (pH 3.0). 1M Tris-hydrochloric acid (pH 9.0) was immediately added to adjust to pH 7.4. The eluted fraction of IgG was confirmed by measuring _A280 . The IgG fraction was collected and dialyzed against 0.01 M phosphate buffer (4 ° C., 3 days).

As described above, about 1 mg of a polyclonal antibody obtained by purifying antiserum (0.5 mL) with protease-treated cells was obtained.
Production Example 2 [Production of immunochromatographic device]
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.

Next, 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 while 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 while stirring (final concentration of skim milk 1%), and left at room temperature for 30 minutes. The reaction solution was then centrifuged at 10 ° C. and 10,000 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 sometimes referred to as “gold colloid-labeled antibody”) was stored at 4 ° C.

  1 mg / mL of the antibody and anti-rabbit IgG prepared in Production Example 1 on a detection line and a control determination line on a development membrane composed of a nitrocellulose membrane (MILLIPORE, Hi-Flow Plus Membrane, HF180, 25 mm × 6 mm), respectively 1 μL of (H + L) polyclonal antibody (ICN Pharmaceuticals) was 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.

Further, the conjugate pad (MILLIPORE, 7.5 mm × 6 mm) was removed after being shaken in a 0.5% PVA-0.5% sucrose aqueous solution for 1 minute, and dried in a desiccator for 60 minutes while sucking with a vacuum pump. did. 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 (MILLIPORE, 17 mm × 6 mm) was removed after being shaken in a 1% Tween 20-PBS aqueous solution for 1 minute, and dried in a desiccator for 60 minutes while sucking with a vacuum pump. The absorbent pad (MILLIPORE, 20 mm × 6 mm) was used untreated.

Each component of the immunochromatography strip prepared as shown in FIG. 2 was placed on a plastic support, and the immunochromatography strip as shown in FIG. 3 was assembled.
Example 1 [Measurement of saliva buffering capacity by a filtration device equipped with a filter medium carrying a saliva buffering capacity test reagent]
(1) Collection of saliva and measurement of the number of Streptococcus mutans bacteria by culture method Five subjects (A, B, C, D, E) were bitten with paraffin pellets for 5 minutes, and saliva was collected. Table 2 shows the amount of saliva secretion.

The collected saliva was appropriately diluted and 0.05 mL was added onto the MSB solid medium, and cultured at 37 ° C. under anaerobic conditions for 48 hours. The number of colonies generated on the MSB solid medium was counted, and the concentration of mutans streptococci was calculated as number / mL from the dilution rate of saliva. The identification of Streptococcus mutans on MSB medium is based on the morphological classification of colonies, and for morphologically indistinguishable colonies, the serotype-specific antibodies of mutans streptococci after pure culture of the colonies Streptococcus mutans was identified by an immunological measurement method using a biochemical method and a biochemical method such as a sugar fermentation test.
(2) Measurement of saliva buffering capacity Tartrate, chlorophenol red and polyethylene glycol (average molecular weight 3000) are dissolved in ethanol to prepare a saliva buffer capacity test reagent containing 0.45N tartaric acid, 3000 ppm chlorophenol red and 1% polyethylene glycol. did. Glass fiber filter paper “GA-100” (manufactured by Advantech Toyo Co., Ltd.) with a pore size (value obtained from the leaked particle size when barium sulfate or the like described in Japanese Industrial Standard (JIS P 3801) is naturally filtered) is circular. cut (area 0.95 cm ² of filtering surface), the saliva buffer capacity test reagents 0.03mL was dropped into the filter media to dry the filter media GA-100 by incubation for 30 minutes at 50 ° C.. This filter medium GA-100 was set in an SC cartridge (Marem Co., Ltd.) to produce a filtration device having the structure of FIG. 0.3 mL each of saliva collected from the above five subjects was added to the filtration device, the lid was closed, the sample was pressurized with a syringe and filtered, and the saliva buffer capacity was determined according to Table 1 from the color of the filtrate. The saliva was able to filter the entire 0.3 mL. Judgment was carried out by three judges (a, i, c).

  Moreover, the determination by the saliva buffering capacity measuring method by the conventional method was implemented by the oral tester buffer (Tokuyama Dental) which is a determination tube method reagent. According to the instruction manual of the kit, 0.5 mL of saliva was added to the determination tube and mixed well with the reagent in the determination tube to determine the buffer capacity. Yellow was judged as low buffer capacity, orange as medium buffer capacity, and red purple as high buffer capacity. Judgment was performed by three judges. The results are shown in Table 2.

The saliva buffering ability of the conventional method determined using saliva and the saliva buffering ability determined by the method of the present invention are the same results, and it was revealed that the saliva buffering ability can be accurately determined by the method of the present invention.
Reference Example 1 [Measurement of the number of Streptococcus mutans bacteria in saliva using components retained on the filter medium in Example 1]
(1) Extraction of sugar chain antigen of Streptococcus mutans Into the filtration device obtained by filtering saliva in Example 1 (2), 0.5 mL of 0.1 M NaOH solution was added to the filtration device, followed by filtration. The microorganisms on the filter membrane and the filter membrane were washed by adding 5 mL of PBS to the filter and filtering. A 1M sodium nitrite solution and a 2M acetic acid solution were mixed at a ratio of 1: 1 to prepare an aqueous nitrous acid solution, and 0.3 mL of the aqueous nitrous acid solution was added to a filter and filtered. After leaving at room temperature for 2 minutes, 0.09 mL of 1M Tris (pH unadjusted) containing 0.05% Tween 20 was added, and the sugar chain antigen extract was recovered by pressurizing the filtration device with a syringe.
(4) Measurement of sugar chain antigen extract using immunochromatography device 0.1 mL of sugar chain antigen extract was added to the sample pad of the immunochromatography device manufactured in Production Example 2, and the presence or absence of a spot 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 3.

  As shown in Table 3, a spot color intensity correlated with the concentration of Streptococcus mutans obtained by the culture method was obtained.

From these results, it was found that the amount of saliva secretion, the saliva buffering capacity and the number of Streptococcus mutans bacteria can be accurately and rapidly measured by the method of the present invention.
Example 2 [Measurement of Salivary Buffering Capacity of Subjects with Low Salivary Secretion]
Saliva was collected from subjects F, G, and H with low saliva secretion by the same method as in Example 1 (1). The number of Streptococcus mutans bacteria was measured by a culture method.

  Next, as in Example 1 (2), the saliva buffer capacity of the subject was measured by the method for measuring saliva buffer capacity of the present invention.

  The measurement by the conventional method was carried out by the test paper method because the current test subject has a small amount of saliva secretion, and thus the saliva buffering ability cannot be measured by the determination tube method as in Example 1. In general, saliva collected from a subject with a small amount of saliva secretion often has a high viscosity, and if it is used directly, it may not penetrate uniformly and it may be difficult to measure the saliva buffer capacity by the test paper method. For this reason, as described in Patent Document 3, an attempt was made to lower the viscosity of saliva by filtering saliva with a filter medium that does not carry a saliva buffering capacity test reagent. Except for using the filter medium GA-100 that does not carry a saliva buffering capacity test reagent, the same filtration apparatus as shown in FIG. 1 was prepared, and 0.1 mL of saliva was filtered. The saliva buffer capacity was measured with the filtrate produced at this time. Saliva buffering ability was measured by Dentobuff Strip (Oral Care), which is a test paper method reagent, using the filtrate. According to the instruction manual, a drop of the filtrate was dropped on the strip and the coloration after 5 minutes was observed. Judgment was made with yellow as low buffer capacity, green as medium buffer capacity, and blue as high buffer capacity. Judgment was carried out by three judges (a, i, c). Further, saliva was dropped directly on the strip without filtering saliva, and saliva buffering ability was measured. The results are shown in Table 4.

When the amount of saliva secretion was small, the saliva buffering ability could not be determined accurately because the viscosity of saliva was high and did not uniformly penetrate the test paper. However, the determination could be easily made by reducing the viscosity by filtration. In the saliva buffer capacity measuring method of the present invention, the saliva and the saliva buffer capacity test reagent are efficiently mixed simply by filtering the saliva with a filter medium, and the saliva buffer capacity can be determined more easily and quickly than the conventional method. It was.
Reference Example 2 [Measurement of the number of Streptococcus mutans bacteria in saliva using components retained on the filter medium in Example 2]
After carrying out Example 2, the number of Streptococcus mutans bacteria was measured by the same method as in Reference Example 1 (1) (2), using a filtration device that filtered saliva according to the method for measuring saliva buffering ability. Table 5 shows the amount of saliva secretion, the number of Streptococcus mutans measured by the culture method, and the results of immunochromatography.

  From these results, it can be seen that the saliva secretion capacity, saliva buffer capacity and the number of Streptococcus mutans bacteria can be measured accurately and rapidly even in the case of a subject with low saliva secretion volume by the method for measuring saliva buffer capacity of the present invention. It was. Saliva collected from patients with low saliva secretion often has a high viscosity, and when measuring the saliva buffering capacity using the test paper method, it may be difficult to determine because saliva is difficult to penetrate into the test paper. It has been known. In this result as well, when saliva was directly measured by the test paper method, the judgment could vary. In order to avoid this, a method of reducing the viscosity of saliva by using a filtrate obtained by filtering saliva and measuring the saliva buffer capacity using the filtrate has been known. It was complicated because it was necessary to perform two operations for measuring the performance. According to the saliva buffering capacity measuring method of the present invention, saliva buffering capacity can be measured simply by filtering saliva with a filter medium, and saliva buffering capacity can be measured easily and quickly.

  Furthermore, if microorganisms are held on the filter medium using the filtration device of the present invention, the number of microorganisms can be measured quickly and easily, and the risk of developing a disease of a subject can be determined more accurately. The method for measuring the saliva buffering capacity of the present invention is particularly effective in the case of a subject having a low saliva secretion amount, which has heretofore been difficult to measure both the number of microorganisms and the saliva buffering capacity.

This figure is a schematic view of a filtration apparatus used in the antigen extraction method of the present invention. This figure is a schematic view of each member of the strip used in the immunochromatography method of the present invention. This figure is a side view of a strip used in the immunochromatography method of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Drainage port 2 ... The filter medium which carry | supported the saliva buffering capacity test reagent 3 ... Container main body 4 ... Syringe insertion port 5 ... Lid part 6 ... Filtration apparatus 7 ... Strip 8 ... Sample pad 9 ... Conjugate pad 10 ... Development membrane 11 ... Absorption pad 12 ... Detection line 13 ... Control judgment line

Claims (3)

  A method of measuring the buffer capacity of saliva using a saliva filtrate produced by filtering saliva through a filter medium and measuring the amount of microorganisms in the saliva from components retained on the filter medium, the pH indicator and A method for measuring saliva buffering capacity, which is carried out by filtering saliva with a filter medium carrying an acid-containing saliva buffering capacity test reagent and measuring the coloration state of the filtrate.   2. The method for measuring saliva buffering capacity according to claim 1, wherein the microorganism in the saliva for measuring the amount of microorganisms is a caries-associated bacterium.   A filtration device comprising a filter medium carrying a saliva buffer capacity test reagent containing a pH indicator and an acid.

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