JP2008128797A - Method of measuring saliva buffer capacity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of measuring saliva buffer capacity even when a subject has a small saliva secretion quantity. <P>SOLUTION: Saliva is divided into a component held on a filter material and a filtered liquid by the filter material with diameter of 0.8-2 μm; the number of bacteria causing erosion is measured using the component on the filter material, and the filtered liquid is brought into contact with an absorbent carrier carrying a test reagent containing a pH indicator; the pH is measured, and the saliva buffer capacity is measured. Thus, even when the subject has a small saliva secretion quantity, the saliva buffer capacity can be measured rapidly and accurately. <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 the saliva buffering ability with a filtrate produced by filtering saliva with a filter medium in order to measure the number of caries-causing bacteria.

  In recent years, attempts have been made to determine the risk of caries development in the human oral cavity and prevent caries. The determination of caries occurrence risk is determined by measuring factors involved in caries occurrence (caries risk factor) and comprehensively judging these results. Caries risk factors such as the number of caries-causing bacteria such as mutans streptococci and lactobacillus in saliva, saliva properties such as salivary secretion and saliva buffering capacity, plaque adhesion, number of meals and fluoride usage Each item, such as a custom, is examined (for example, refer nonpatent literature 1).

  The following methods have been developed as methods for examining the above items. The number of mutans streptococci and the number of Lactobacillus are measured by a culture method using a selective medium. ing. The saliva secretion amount is measured by collecting saliva secreted in a certain time in a measuring cup or the like. The saliva buffering ability is measured by a method of examining the pH after mixing a certain amount of acid and a certain amount of saliva, and examining the color change of the pH indicator. Lifestyles such as the amount of plaque adhering, the number of meals, and the use status of fluoride are carried out by dentist inspection and patient interview.

  At some dental clinics, all of the above-mentioned inspection items are examined and scored, and attempts have been made to prevent caries by reducing the total of caries risk factors in the patient's oral cavity below a certain level. (See, for example, Non-Patent Document 1).

  Among the above caries risk factors, it is said that in particular the number of mutans streptococci, salivary secretion, and saliva buffering capacity are closely related to caries risk. Carbohydrates in food and sugar, which is a degradation product, are rapidly metabolized by mutans streptococci present in plaque to produce acid, resulting in a decrease in pH in the plaque and the surface of the teeth. Calcium ions and phosphate ions are eluted in plaque and saliva (decalcification). However, as mentioned above, even if teeth decalcify due to the action of mutans streptococci, if the pH in the plaque returns to near neutrality due to the cleaning and buffering action of saliva, Calcium ions and phosphate ions are deposited again on the tooth surface (remineralization). Caries are a phenomenon in which the balance between demineralization and remineralization is lost and demineralization proceeds under the surface of the enamel. For the above reasons, it is important to measure the saliva secretion amount and saliva buffering capacity in addition to the number of mutans streptococci in order to determine the risk of caries development.

  For example, even if the number of mutans streptococci in saliva is large, the risk of caries development is different between people with low saliva secretion and saliva buffer capacity and those with low saliva buffer capacity, and even when saliva secretion capacity or saliva buffer capacity is low, The risk of caries development differs between people with a high number of mutans streptococci in the oral cavity and those with a low number. Therefore, in order to determine the risk of the occurrence of caries, it is important to make a comprehensive determination by simultaneously measuring the number of mutans streptococci in the oral cavity, the saliva secretion amount, and the saliva buffering capacity.

  It is known that the number of mutans streptococci in the oral cavity, salivary secretion, and saliva buffering capacity vary within the day. Since the measurement results of caries risk factors vary depending on the time at which saliva is collected, using the saliva with different collection times, the number of mutans streptococci and the saliva buffering capacity can be measured and compared, so that the situation in the oral cavity can be accurately determined. Cannot judge. For these reasons, when measuring the number of caries-causing bacteria, the amount of saliva secreted, and the saliva buffering capacity, it can be said that it is desirable to simultaneously measure using the same sample.

  Measurement of caries-causing bacteria in saliva has been carried out by the conventional culture method, but there was a drawback that it took a relatively long time of 2 days or more until the results were revealed, but an immunological measurement method was developed. Inspection time has been greatly reduced. 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 2 and 3). When measuring caries-causing bacteria by the culture method, about 0.01 to 0.1 mL of saliva is required, but when performing an immunoassay, a larger amount of saliva (about 0.3 to 0.5 mL) than the culture method ) Saliva is necessary (for example, Non-Patent Document 3). The reason is as follows. In an immunoassay, an antibody against an antigen present on the surface of a cariogenic bacteria is usually used. Since the caries-causing bacteria in saliva are covered with dental plaque, the antibody cannot be contacted as it is, and the antigen-antibody reaction does not occur. In order to cause an antigen-antibody reaction, it is necessary to perform some pretreatment to remove plaque and expose the antigen, or to extract the antigen from the cells. However, even if pretreatment is performed, dental plaque cannot be completely removed, all antigens cannot be extracted, and some antigens are destroyed by pretreatment. Since not all antigens can be measured, a large amount of saliva is required as compared with the culture method.

  Saliva buffering ability can be measured by adding a certain amount of acid to saliva and measuring the pH. The method of visually confirming the color of the pH indicator and comparing it with a standard colorimetric table prepared in advance at each pH value is particularly preferable from the viewpoint of simplicity and speed, because it can be measured without requiring an instrument. It can be said that this is one of the embodiments. For example, Yamada et al. Have proposed a method of holding a pH indicator and an acidic buffer in an absorbent carrier, adding saliva to the carrier and visually checking the color tone, a so-called test paper method (Patent Document 1). . 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 uneven, there is a drawback that the color of the pH indicator becomes mottled and 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 used for measurement is subjected to filtration treatment to remove impurities and mucous substances such as mucin in the saliva (Patent Document 2). However, the filter medium specifically used in this conventional example is a glass fiber filter AP15 (manufactured by Millipore), and its pore diameter is a very small one having a catalog value of 0.2 to 0.6 μm. Thus, when the pore diameter is small as described above, the filter medium is likely to be clogged by the insoluble matter in the saliva, and only a small amount of saliva can be filtered. When measuring only the saliva buffering capacity, it can be used without any problem, but when measuring the number of mutans streptococci from the same saliva, the amount of mutans streptococci cannot be filtered because the amount of saliva required for the measurement cannot be filtered. There is a problem that it cannot be measured accurately. Furthermore, Okazaki et al. Add saliva to a test tube containing a dry powder of a reagent (lactic acid, pH indicator) in advance, and cover and shake to dissolve the reagent, and visually determine the color tone of the pH indicator. The so-called determination tube method is 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.

  If the test subject is an elderly person or an infant, the saliva secretion amount may be low, or the saliva may not be discharged well. There was a problem that both could not be measured.

Clinical Carriology, Takashi Kumagai, Bio-Dental Publishing Co., Ltd. 1996 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 Yoshihide Okazaki et al., Journal of Pediatric Dentistry, 38: 615-621, 2000 Japanese Patent No. 1574981 JP 2002-323493 A

  The present invention has been made in view of the above circumstances, measuring caries-causing bacteria and saliva buffering capacity, and determining the caries development risk from both measured values, up to saliva buffering capacity with a small amount of saliva, It is an object of the present invention to provide a method capable of measuring quickly and accurately.

  The present inventors have intensively studied to solve the above problems. As a result, by filtering the saliva with a filter medium having a pore diameter of 0.8 to 2 μm, the saliva buffering ability can be accurately measured even with the filtrate from which insoluble matter containing caries-causing bacteria has been removed. The present inventors have found that the viscosity of the saliva is reduced and the uniformity of the penetration of the saliva filtrate into the absorbent carrier is improved even in the test paper method, so that the pH can be easily determined. And further examination was advanced and it came to complete this invention.

  That is, the present invention uses the saliva filtrate produced when the saliva is filtered through a filter medium having a pore size of 0.8 to 2 μm and the number of caries-causing bacteria is measured from the components retained on the filter medium. Is a method for measuring saliva buffering capacity, characterized in that

  According to the measurement method of the present invention, the caries risk can be efficiently evaluated by filtering saliva and measuring the number of caries-causing bacteria and the buffer capacity of the filtrate retained on the filter medium. 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 it can be used for the measurement of both the number of caries-causing bacteria and the saliva buffering capacity, the method of the present invention can measure both sufficiently even when the amount of saliva is small, for example, 0.3 mL to 0.5 mL. Is feasible and suitable.

  Moreover, in the present invention, saliva filtered with a filter medium has both insoluble matter content and viscosity decreased, so that saliva buffering ability can be easily measured as compared to saliva before filtration. Among subjects, it is not unusual for the amount of stimulated saliva collected while chewing a gum or the like to chew a subject for 5 minutes, so there is a risk of caries development of such subjects. When evaluating, the said effect of this invention is exhibited notably.

  This effect can be exhibited more remarkably when the saliva buffer capacity is measured by the test paper method. That is, the viscosity of saliva is reduced by the filtration, and the operability of measurement is improved. In particular, when the viscosity of saliva is high, the filtrate whose viscosity has been reduced by the filtration greatly improves the permeability to the test paper, and the color of the pH indicator can be made uniform.

  In the method for measuring saliva buffering capacity of the present invention, saliva is filtered to separate into insoluble matter containing caries-causing bacteria and filtrate, and the saliva buffer capacity is measured using the 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.

It is preferable that the saliva used for the method of the present invention has a high viscosity because the improvement effect is remarkably exhibited in the measurement of the saliva buffering ability. Generally, the viscosity of saliva and the time required for filtration are in a proportional relationship. When filtering saliva in accordance with an embodiment of the present invention to be described later, specifically, a filtration device having the structure of FIG. 1 prepared by setting a glass fiber filter paper (for example, GA-100 manufactured by Advantech Toyo Co., Ltd.) having a pore diameter of 1 μm. After adding 0.3 mL of saliva to the (filtration surface area 0.95 cm ² ) and removing the plunger of the 2.5 mL syringe (eg, Terumo), the plunger is attached to the syringe outer cylinder. When filtration is performed by pressurizing by inserting and holding down, the time required for filtration of the total amount of added saliva is 20 seconds or more (the amount of saliva secretion is relatively large) In the case of saliva with low viscosity, the time required for filtration is about several seconds to 10 seconds), which can be said to be saliva with high viscosity. In the present invention, good measurement can be performed even when the viscosity of saliva collected from a subject to be filtered is 20 seconds or longer, more preferably 25 to 60 seconds, as described above.

  In the present invention, caries-causing bacteria refer to microorganisms present in the oral cavity that are deeply involved in the occurrence or development of caries. Specific examples include mutans streptococci and lactobacilli, but Streptococcus mutans and Streptococcus sobrinus, which are said to be particularly highly related to the occurrence of dental caries. It is particularly preferable to measure.

  The caries-causing bacteria can be measured quickly and accurately, for example, by extracting an antigen from the caries-causing bacteria and measuring the extracted antigen by an immunoassay. For example, caries-causing bacteria are concentrated on a filter medium by filtering saliva, and antigens are extracted, thereby enabling highly sensitive immunological measurement. As the extraction method, a known method can be used without any limitation. As a suitable example, a nitrous acid extraction method can be shown. The nitrous acid extraction method can be carried out by a known method (for example, the method described in JP-A-2003-215126). 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, a basic aqueous solution such as 1 to 3 M sodium hydroxide, 0.5 to 2 M Tris, 0.5 to 1 M sodium hydrogen carbonate or the like is added to adjust the pH of the extract to 7.0 to 8. It is preferable to adjust to 5. 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 nitrite solution (0.2-4M sodium nitrite, 0.5-4M acetic acid), and the filtration membrane is filtered with the aqueous nitrite solution. Nitrous acid extraction can also be performed. 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 performed, and 0.5 to 1.0 M Tris is added to a 100 to 800 μL filter membrane, and the sugar chain antigen extract is recovered by pressure filtration.

  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 filter paper and glass fiber filter paper.

  In the case of a screen filter, the pore diameter of the filter medium in the present invention 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) ( In the case of a depth filter, it is a numerical value obtained from a leaked particle diameter 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 hole diameter displayed in the commercially available filter medium catalog corresponds to the hole diameter of the present invention obtained by the above method, and the filter medium is made of glass fiber filter paper or filter paper. In the case of such 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 of the present invention.

  The pore size of the filter medium needs to be a pore size through which caries-causing bacteria cannot pass. Streptococcus mutans and Streptococcus sobrinus, which are suitable measurement targets of the present invention, have a cell size of 0.5 to 0.9 μm. Therefore, in order to retain these bacteria on the filter medium, a filter medium having a pore size sufficiently smaller than the size of the cells, specifically, a pore size of 0.22 μm to 0.45 μm is usually used. Is the law. Also in the present invention, if saliva is filtered using the filter medium having the small pore diameter, Streptococcus mutans and Streptococcus sobrinus can be supplemented well. In addition, since saliva usually contains other insoluble matter, clogging is likely to occur. When the filter medium is clogged in this way, the extraction efficiency is slightly lowered in the antigen extraction process from the bacteria 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 supplemented with almost no flow. Therefore, the filtration rate is also greatly improved. Furthermore, when saliva is filtered using a filtration membrane of the same area, it can be seen that about 10 times the amount of saliva can be filtered with a filtration membrane having a pore diameter of 0.8-2 μm compared to a filtration membrane having a pore diameter of 0.45 μm. In addition, clogging due to other insoluble materials is extremely unlikely to occur.

  The fact that clogging is less likely to occur is important for highly sensitive measurement of caries-causing bacteria. For example, when antigen is extracted by the nitrous acid extraction method described above, the amount of reagent added at the time of nitrous acid extraction increases in proportion to the area of the filtration surface of the filter medium, and the amount of sugar chain antigen extract recovered from the filter medium increases. To increase. Since the amount of liquid that can be used in immunoassay is limited (generally 0.15 mL or less), even if a large amount of saliva can be filtered, the area of the filtration surface is large and the amount of sugar chain antigen extract When the number of cells increases, only a part of them can be analyzed by immunoassay, so that highly sensitive measurement becomes impossible. In order to realize highly sensitive measurement, the relationship between the amount of saliva to be filtered and the area of the filtration surface of the filter medium needs to be in a suitable range. As described above, the amount of saliva that can be filtered per area of the filter medium is proportional to the pore diameter of the filter medium (the amount of saliva that can be filtered decreases when the pore diameter is small), so a filter medium with an appropriate pore diameter, for example, 0.8 to 2 μm. Filter media must be used.

  For example, saliva can be filtered by a filtering device having a structure as shown in FIG. The filtration device body and the lid are preferably made of a flexible material, for example, a plastic such as polypropylene. 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.

  In the present invention, the number of caries-causing bacteria 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, the saliva buffer capacity is measured using a filtrate prepared by filtering saliva with a filter medium. Even if the buffer capacity of the filtrate obtained by filtering saliva is measured, the measurement result is substantially the same as when the saliva is directly subjected to measurement.

  The saliva buffering capacity is measured by bringing the saliva filtrate prepared as described above into contact with a test reagent and determining the pH visually or the like. The buffer capacity of the filtrate may be measured by adding a certain amount of acid to the saliva and dropping the pH indicator directly onto the saliva to confirm the pH in the colored state. Since the viscosity of this filtrate is lowered, it is preferable to carry out the method by measuring the pH by bringing the filtrate into contact with an absorbent carrier carrying a test reagent containing a pH indicator. That is, the methods represented by these test paper methods are particularly difficult to absorb into the carrier and cause variation in color development when, for example, high-viscosity saliva is provided, but when the above filtrate is used, The problem is alleviated and accurate judgment can be made.

  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.).

  Here, the test reagent containing a pH indicator is usually composed of an acid such as lactic acid, hydrochloric acid, citric acid, and tartaric acid in addition to the pH indicator. If necessary, wetting agents such as polyethylene glycol, polyvinyl pyrrolidone, surfactants (polyoxyethylene alkyl ether type, polyoxyethylene sorbitan fatty acid ester type, etc.) to promote the penetration of saliva into test paper Can also be included in the test reagent component. The contact method between the absorbent carrier such as paper carrying these test reagents and the filtrate can be carried out by a method suitable for the reagent form. For example, when the test reagent is a strip in which acid, pH indicator, etc. are held in a strip-shaped absorbent carrier in a dry state, the amount of saliva (about 0.005 to 0.03 mL) that the determination unit is sufficiently moistened The filtrate can be added dropwise. In addition, the buffer capacity of the filtrate may be measured by putting the filtrate into a container and inserting the determination part of the strip into the filtrate in the container.

  The measurement of pH is carried out by visually confirming the color tone after contacting the test reagent with the saliva 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, the higher the pH of the mixed solution. Therefore, it can be determined as shown in Table 1 according to the color tone.

  As described above, the number of caries-causing bacteria and the saliva buffering capacity are efficiently measured. Evaluation of the caries occurrence risk in consideration of the number of caries-causing bacteria and saliva buffer capacity measured in the present invention is performed as follows. That is, it is known that caries does not develop due to a single cause, but occurs when a plurality of factors (caries risk factors) that promote caries are in a state suitable for caries. Therefore, if as many caries risk factors as possible are measured and the risk of caries occurrence is comprehensively determined from these results, the accuracy of the determination will be improved. Known caries risk factors include the number of germs causing caries in saliva, saliva properties such as saliva secretion and saliva buffering capacity, the amount of plaque adhering, the number of meals and the use of fluoride, etc. By simultaneously measuring and comprehensively determining the caries risk factor, it is possible to determine the caries risk of the subject much more accurately than judging only from the measurement results of the individual caries risk factors. Among these caries risk factors, the amount of plaque adhering, the number of meals and the use of fluoride can be ascertained by conventional dental examinations (inspection, interview, etc.). The amount of secretion and the ability to buffer saliva cannot be measured by examinations conventionally performed in dentistry. It is necessary to collect the stimulated saliva and measure the amount of saliva secretion, and then, for example, to measure the number of caries-causing bacteria and the saliva buffer capacity according to the method of the present invention. According to the method for measuring the salivary buffering capacity of the present invention, both of the subjects having a low saliva secretion amount, which has been difficult to measure simultaneously the number of caries-causing bacteria and the saliva buffering capacity, have been measured at the same time. Is possible.

  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.
Reference Example 1 [Measurement of saliva buffering capacity by saliva and filtrate]
Five subjects (A, B, C, D, E) chewed paraffin pellets for 5 minutes and collected saliva. Table 2 shows the amount of saliva secretion.

On the other hand, a glass fiber filter paper “GA-” having a pore size (value obtained from the leaked particle size when natural filtration of barium sulfate described in Japanese Industrial Standard (JIS P 3801)) is 1.0 μm is applied to an SC cartridge (Maruem). 100 ”(manufactured by Advantech Toyo Co., Ltd.) was set (filtration surface area 0.95 cm ² ) to produce a filtration device having the structure of FIG. 0.7 mL each of saliva collected from the above five subjects was added to the filtration device, and the lid was closed and pressurized with a syringe for filtration.

  The buffer capacity of saliva and filtrate was measured with an oral tester buffer (Tokuyama Dental), which is a determination tube method reagent. According to the instruction manual of the kit, saliva or filtrate was added to a 0.5 mL determination tube and mixed well with the reagent in the determination tube to determine the buffer capacity. Judgment was carried out by three judges (A, B, and U), and yellow was judged to have a low buffer capacity, orange to a medium buffer capacity, and reddish purple to be a high buffer capacity. The results are shown in Table 2.

  The buffering capacity of saliva and filtrate measured by the judgment tube method which is not easily affected by the viscosity of saliva was the same result. From this, it was clarified that the buffer capacity can be measured in the same manner as saliva even when the filtrate is used.

Example 1
(1) Collection of saliva and measurement of the number of Streptococcus mutans bacteria by culture method Paraffin pellets were added to subject A of Reference Example 1 and the other four subjects (F, G, H, I, J). Chewing for minutes and collecting saliva. Table 3 shows the saliva secretion amount of each subject.

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) Filtration of saliva and preparation of sugar chain antigen extract 0.3 mL of saliva A, F, G, H, I, and J was filtered with GA-100 in the same manner as in Reference Example 1, and the filtrate was collected. A total amount of 0.3 mL of saliva could be filtered. Table 3 shows the time required for filtering saliva.

Next, 0.5 mL of 0.1 M NaOH solution was added to the filtration device and filtered, and then 0.5 mL of PBS was added to the filtration device and filtered to wash the microorganisms on the filtration membrane and the filtration membrane. . 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.
(3) 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 4.

As shown in Table 4, a spot color intensity correlated with the concentration of Streptococcus mutans obtained by the culture method was obtained.
(4) Measurement of saliva buffer capacity by filtrate Using the filtrate collected in (2), saliva buffer capacity was measured by Dentobuff Strip (Oral Care) which is a test paper method reagent. According to the instruction manual of the kit, a drop of the test paper filtrate was dropped 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). The results are shown in Table 5.

When the filtrate was used, the viscosity of saliva decreased, so that the filtrate uniformly penetrated the test paper. The same results were obtained with three judges, and the judgment was easy.
Comparative Example 1 [Measurement of the number of Streptococcus mutans bacteria when a filter medium having a small pore size is used]
Saliva was filtered by the same method as in Example 1 except that glass fiber filter paper “GA-55” (manufactured by Advantech Toyo Co., Ltd.) having a pore size of 0.6 μm was used as the filter medium. Subsequently, the number of bacteria was measured with an immunochromatographic device in the same manner as in Example 1. The results are shown in Table 6. As saliva, A, F, G, H, and I used in Example 1 were used. J could not be filtered because the remaining amount was small.

When GA-55 was used, only 0.15 mL of saliva could be filtered, so the spot color development was weaker than in the case of GA-100, and it was not positive unless the number of bacteria was 5 × 10 ⁵ cells / mL or more. When GA-55 was used, the sensitivity was insufficient.

Comparative Example 2 [Measurement of saliva buffering ability using saliva directly]
Saliva buffer capacity was measured by the same method as in Example 1 (4) using saliva without filtering. The results are shown in Table 7. In this case, in saliva from subjects G, H, I, and J, which is saliva that is highly viscous and takes a long time to filter, the saliva does not uniformly penetrate the test paper. It was. As a result, the results differ depending on the judge.

Example 2, Comparative Example 3
Saliva was collected from subject G, which is saliva with high viscosity, by the same method as in Example 1 (1) (saliva volume 2 mL). Using this saliva, filtration of 0.3 mL of saliva was carried out in the same manner as in Example 1 (2), except that the glass fiber filter paper shown in Table 8 was used as a filtration device, and that having the structure of FIG. 1 using filter paper was used. The sugar chain antigen was extracted, and the number of Streptococcus mutans bacteria was measured by immunochromatography using the sugar chain antigen extract in the same manner as in Example 1 (3). Experiment No. in which the pore diameter of the filter medium is within the scope of the present invention. 1-4, the results were the same as in Example 1. However, in Experiment No. 1, the pore size of the filter medium exceeded the scope of the present invention. In No. 5, spots were not detected because the mutans bacteria were not sufficiently retained on the filter medium.

  Next, the buffer capacity of each filtrate was measured in the same manner as in Example 1 (4), and the results are shown in Table 9. In Experiments N0.1 to 4 in which the pore diameter of the filter medium was within the scope of the present invention, all of the three judges showed the same result and could easily be judged. On the other hand, Experiment No. in which the pore diameter of the filter media exceeds the range of the present invention. In the case of 5, the viscosity of the saliva was not sufficiently reduced, so the color of the test paper became slightly mottled, and the results of the three judges varied.

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 ... 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)

  The saliva is filtered through a filter medium having a pore diameter of 0.8 to 2 μm, and the buffer capacity of the saliva is measured using the filtrate of saliva generated when the number of caries-causing bacteria is measured from the components retained on the filter medium. And measuring method of saliva buffer capacity.   The method for measuring the saliva buffer capacity according to claim 1, wherein the filtrate buffer capacity is measured by bringing the filtrate into contact with an absorbent carrier carrying a test reagent containing a pH indicator and measuring the pH.   The method for measuring the saliva buffering capacity according to claim 1 or 2, wherein the amount of saliva collected from the subject for filtration is 0.3 to 0.5 mL.

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