JP2017181131A - Method for removing protease in saliva - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique for measuring the concentration of a measuring target material with high sensitivity by reducing the influence of protease derived from saliva.SOLUTION: The present invention relates to a method for removing protease from saliva by filtering the saliva. The method preferably uses a filter with hole diameters of 0.2 μm to 0.45 μm for filtering protease from saliva, and the filter is desirably a syringe filter. The present invention also relates to a method for measuring a measuring target material in the saliva including processes of: removing protease from saliva; and measuring a measuring target material in the saliva.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for removing proteases in saliva.

It is useful from a medical point of view to clarify the concentration of a specific substance in saliva. For example, if endotoxin is present in a certain amount or more in saliva, it can be seen that periodontal diseases such as gingivitis are likely to be caused. Therefore, whether endotoxin is measured or not can be determined.
A protease reaction may be used to measure the concentration of these substances. For example, as a method for measuring endotoxin, a method based on the Limulus test is generally known. In the Limulus test, endotoxin and Limulus reagent are reacted to obtain a plurality of types of substances having protease activity, and the protease activity of the reaction solution is detected. As a detection method, gelation measurement or colorimetric method is generally used. For example, in Patent Document 1, the concentration of endotoxin is measured by detection using a fluorescent reaction or the like.
However, when measuring the concentration of a substance in saliva, there is a problem that the detection reaction proceeds due to the saliva-derived protease remaining in the sample, and the substance to be measured cannot be detected accurately.
Therefore, attempts have been made to add a protease inhibitor to saliva to inhibit saliva-derived protease reaction, or to denature saliva-derived protease by performing denaturation treatment such as pH change or heating (Patent Document 2). .
However, addition of a protease inhibitor or denaturation treatment also reduces the protease activity of the reaction solution derived from the substance to be measured. Therefore, a method capable of measuring the concentration of the measurement target substance with high sensitivity by reducing the influence of protease derived from saliva has been demanded.

Japanese Patent Laying-Open No. 2015-141088 Japanese Patent Publication No. 3-500369

  An object of the present invention is to provide a method capable of measuring the concentration of a substance to be measured with high sensitivity by reducing the influence of protease derived from saliva.

In order to solve the above problems, the present invention employs the following configuration.
[1] A method for removing protease in saliva, wherein the protease is removed by filtering saliva.
[2] The method for removing saliva protease according to [1], wherein the saliva is filtered using a filter having a pore size of 0.2 μm to 0.45 μm.
[3] The method for removing protease in saliva according to [2], wherein the filter is a syringe filter.
[4] A removal step of removing protease in saliva by the method for removing protease in saliva according to any one of [1] to [3], and a measurement step of measuring a substance to be measured in saliva. Method for measuring target substance in saliva.
[5] The method for measuring a substance in saliva according to [4], wherein the substance to be measured is endotoxin.
[6] The saliva target substance measurement method according to [4] or [5], wherein the measurement step measures a concentration of the measurement target substance.
[7] The method for measuring a target substance in saliva according to any one of [4] to [6], wherein the measurement step is a Limulus test.

  According to the method for removing protease in saliva of the present invention, the concentration of a measurement target substance can be measured with high sensitivity by reducing the influence of protease derived from saliva.

It is a schematic diagram which shows one aspect | mode of the removal method of the protease in saliva of this application.

The method for removing protease in saliva according to the present invention is a method for removing protease by filtering saliva.
Proteases are present in saliva. When an attempt is made to measure a compound present in saliva, the presence of a protease in saliva may affect the measurement result and prevent high-sensitivity measurement of the measurement object.
As a result of diligent research, the present inventors filtered saliva as a measurement target, and even when the measurement substance is a compound having a molecular weight equivalent to or higher than that of protease, the measurement target substance remains, The inventors have found a technique for removing protease in saliva and have completed the present invention.

[Measurement target and target substance]
The present invention uses saliva as a measurement target. As the sample, saliva collected from the oral cavity of the subject or one obtained by diluting the saliva 10 to 100,000 times can be used. In terms of ease of filtration, it is preferable to use a sample obtained by diluting saliva 500 to 1000 times. The saliva is diluted using a diluted solution that does not affect the measurement target and protease. The dilution liquid is preferably purified water or water for injection.
The substance to be measured is a substance that exists or may exist in saliva. For example, endotoxin, β glucan and the like can be mentioned. The present invention is particularly suitable when the protease in saliva interferes with the measurement of the substance to be measured. That is, the substance to be measured is preferably a substance whose measurement is hindered by the presence of protease. Examples of the substance whose measurement is hindered by the presence of protease include a substance that utilizes a protease reaction for measurement of an object to be measured, such as endotoxin.

  "Endotoxin (endotoxin)" is one of the components constituting the outer membrane of Gram-negative bacteria, and the main body of its activity is LPS (lipopolysaccharide: lipopolysaccharide). Endotoxin exists in a state where it is bound to cells in the living body or released by lysis.

  Endotoxin is known to be an important factor related to periodontal disease. If endotoxin is present in saliva in a certain amount or more, it is easy to cause periodontal diseases such as gingivitis, so it is possible to determine whether or not to perform medical treatment by clarifying the concentration of endotoxin it can.

[Protease]
Protease is a protein that acts as an enzyme for proteolytic reaction. Examples of the protease include endopeptidase, aminopeptidase, carboxypeptidase and the like. The protease removed in the removal method of the present invention is a protease contained in saliva, and such a protease is trypsin-like produced by P. gingivalis bacteria which are bacteria in the oral cavity. Protein is considered. The protease removed in the saliva target substance measuring method of the present invention includes a protease that hinders the measurement of the target substance in saliva.

[Saliva filtration]
It does not specifically limit as the method of filtration which filters saliva, It can carry out by the method used normally. Examples of the filtration method include vacuum filtration, pressure filtration, and centrifugal filtration using a filter. Since the operation is easy, it is preferable to perform pressure filtration using a syringe filter.
A syringe filter is a filtering device used by being attached to a syringe.
One embodiment of a pressure filtration method using a syringe filter is shown in FIG. In the syringe filter 1, a housing 1a and a filter 1b that can be attached to a syringe are integrated. The syringe 2 includes a cylindrical portion tip 2a, a cylindrical portion 2b, and a plunger 2c. Pressure filtration using the syringe filter 1 is performed as follows.
The saliva sample S is filled in the cylindrical part 2 b of the syringe 2, and the syringe filter 1 is attached to the cylindrical part tip 2 a of the syringe 2. By pushing the plunger 2 c of the syringe 2, the internal pressure in the cylindrical portion 2 b of the syringe 2 is increased, the saliva sample S is passed through the syringe filter 1, and the filtered saliva sample S is received in the container 3.
As a filter used for filtration, those having a pore diameter of 0.2 μm or more and 0.45 μm or less are preferable, and those having a pore diameter of 0.2 μm or more and 0.30 μm or less are more preferable. If the pore diameter is less than or equal to the preferred upper limit value, the protease in saliva can be sufficiently removed. The lower limit of the pore size of the filter used for filtration can be determined by the size of the substance to be measured. For example, when the substance to be measured is endotoxin, the pore diameter is preferably 0.2 μm or more.
The material of the filter used for filtration is preferably PTFE, and more preferably hydrophilic PTFE.
PTFE represents tetrafluoroethylene.
Since the protease in saliva is removed by the above method, highly sensitive measurement can be performed on the measurement target substance in saliva.

[Measurement of substances to be measured]
The method for measuring a substance in saliva according to the present invention includes a removing step for removing protease in saliva by the method for removing a protease in saliva described above, and a measuring step for measuring a substance to be measured in saliva.
Examples of the measurement include measurement for examining the concentration, structure, and activity of the substance to be measured in saliva.
If the measurement is usually performed by a measurement method that is hindered by the presence of a protease, the effect of improving the measurement sensitivity by performing the removal step appears significantly.
For example, when the substance to be measured is endotoxin, the concentration can be measured by a Limulus test. The Limulus test is a measurement method for measuring the concentration of endotoxin by reacting an endotoxin with a Limulus reagent to obtain a plurality of types of substances having protease activity and detecting the protease activity of the reaction solution. The detection is performed using, for example, an optical measurement using a gelation test, a colorimetric method, a fluorescence reaction, a biochemiluminescence reaction, or the like. The reaction between endotoxin and Limulus reagent specifically includes the following three-step reactions (1) to (3).
(1) A step in which endotoxin binds tightly to factor C and converts factor C into an active form.
(2) Next, the active factor C converts the factor B into an active form.
(3) Next, the active factor B generates a clotting enzyme from a procoagulase.
Since the active factor C, active factor B, and coagulase produced at each stage have sequence-specific protease activity, the endotoxin concentration can be determined by detecting the protease activity.

Protease activity can be detected, for example, as follows.
A luminescent synthetic substrate formed by binding a luminescent substrate to a peptide is added to a measurement sample for detecting protease activity and reacted. After the reaction, a reagent containing a luminescent enzyme and other compounds necessary for the luminescent reaction is added, and the amount of luminescence is measured.
When the endotoxin concentration is measured using a Limulus reagent, a luminescent synthetic substrate formed by binding a luminescent substrate to a peptide is an active factor C produced by the activation of factor C, as well as active factor C It has a structure in which the bond between the luminescent substrate and the peptide is cleaved by the action of any one of activated factor B and coagulase produced by the action. Therefore, when the luminescent synthetic substrate is added to the measurement sample, the synthetic substrate is cleaved and the luminescent substrate is released.
As the luminescent substrate, aminoluciferin can be preferably used. As the peptide that binds to the luminescent substrate, the amide bond with aminoluciferin at the C-terminus of the peptide is any of active factor C, active factor B, and coagulation enzyme. Or any amino acid sequence that is cleaved by one protease activity.
The luminescent enzyme is an enzyme that generates light by catalyzing the bioluminescence of the luminescent substrate released from the luminescent synthetic substrate. When the luminescent substrate is aminoluciferin, the luminescent enzyme is luciferase, and other compounds required for the luminescent reaction are ATP and divalent metal ions.
When a reagent containing a luminescent enzyme and other compounds necessary for the luminescent reaction is added, the measurement sample emits light due to the reaction between the released luminescent substrate and the luminescent enzyme.
The concentration of the substance to be measured can be calculated from the light emission amount of the measurement sample.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by the following description.
(filtration)
The sample was filtered as follows.
A sample to be filtered was packed in a syringe, and a syringe filter was attached to the syringe for pressure filtration.
(Limulus reaction)
To 200 μL of the sample, 30 μL of Limulus reagent (manufactured by Wako Pure Chemical Industries, Ltd., horseshoe crab blood cell extract ES-II lyophilized product) was added and reacted at 37 ° C. for 5 minutes.
(Measurement of luminescence)
The measurement of luminescence was performed as follows.
For 200 μL of sample, 5 μg of synthetic substrate (Toa DKK Corporation) in which aminoluciferin is bound to the peptide, 10 μg of luciferase (Toago DKK Corporation), 8.8 μg of ATP (Wako Pure Chemical Industries, Ltd.), A lyophilized reagent containing 7.5 μg of divalent metal ions (magnesium acetate: manufactured by Kanto Chemical Co., Inc.) was added, and the amount of luminescence was measured 10 times every 0.1 second after 44 seconds after the addition. The total of the 10 measurements obtained was taken as the amount of luminescence. The amount of luminescence was measured several times for the sample, and the average value was taken as the average amount of luminescence of the sample.
(Blank measurement)
In the absence of protease, the influence of a synthetic substrate in which aminoluciferin was bound to a peptide on the amount of luminescence was examined as follows.
Using pure water as a sample, the light emission amount was measured twice by the above light emission amount measurement method, and the average light emission amount was calculated. As a result, the average light emission amount was 174.
In the following description, a value that does not exclude the blank is used as the average light emission amount.

[Removal of protease in saliva]
In order to confirm that the protease in saliva was removed by filtration, Sample 1 was prepared as follows and the amount of luminescence before and after filtration was measured.
(Sample 1)
The subject after chewing with water for injection (Otsuka Pharmaceutical Factory Co., Ltd., Otsuka distilled water) chewed the experimental gum, and collected about 2 mL of saliva that came out. The collected saliva was diluted 1000 times with water for injection to obtain Sample 1.
Half of the diluent was sample 1 (no filtration), and the remaining half of the diluent was filtered by the above method to give sample 1 (with filtration). In the filtration, an Advantech filter, 13HP020AN, a hydrophilic PTFE membrane, and a pore diameter of 0.2 μm were used as a syringe filter.
For Sample 1 (without filtration) and Sample 1 (with filtration), the light emission amount was measured twice without performing the Limulus reaction, and the average light emission amount was calculated respectively. The results are shown in Table 1.
The average luminescence amount of Sample 1 (without filtration) was 803, whereas the average luminescence amount of Sample 1 (with filtration) was 152, which was similar to the blank. This indicates that by performing filtration using a filter, the protease in saliva could be reduced to the same level as the blank. That is, by performing filtration using a filter, almost 100% of the protease in saliva could be removed.

[Endotoxin remaining confirmation]
In order to confirm that endotoxin remains in the sample even after filtering the sample that is saliva, Sample 2 was prepared as described below, and the amount of luminescence before and after filtration was measured.
(Sample 2)
A standard sample derived from P. gingivalis (InvivoGen (USA), LPS-PG) is dissolved in water for injection (Otsuka Pharmaceutical Factory, Otsuka distilled water) to give a 1 mg / mL LPS solution. Was prepared as a standard solution. The standard solution was diluted 100,000 times to obtain Sample 2.
Half of the diluent was sample 2 (no filtration), and the remaining half of the diluent was filtered by the above method to give sample 2 (with filtration). In the filtration, an Advantech filter, 13HP020AN, a hydrophilic PTFE membrane, and a pore diameter of 0.2 μm were used as a syringe filter.
Sample 2 (without filtration) and sample 2 (with filtration) were subjected to Limulus reaction, and then the light emission amount was measured four times, and the average light emission amount was calculated for each. The results are shown in Table 1.

  The average luminescence amount of Sample 2 (with filtration) was 83% of the average luminescence amount of Sample 2 (without filtration). This result shows that endotoxin remains sufficiently in the sample even after filtration.

[Confirmation of contribution of saliva components in protease removal]
In order to show that the removal of protease by filtration contributes to the presence of protease in saliva, sample 3 was prepared and the amount of luminescence was measured as follows to calculate the average amount of luminescence.
(Samples 3 and 4)
Trypsin (manufactured by SIGMA-ALDRICH, Trypsin solution porcine pancreas) as a protease was dissolved in pure water to give a final concentration of 0.25 μg / mL, and this was used as sample 3.
Sample 4 was prepared by dissolving trypsin (manufactured by SIGMA-ALDRICH, Trypsin solution porcine pancreas) as a protease in a 0.1% calcium phosphate suspension so as to have a final concentration of 0.25 μg / mL.
Samples 3 and 4 were filtered by the above method to obtain Sample 3 (with filtration) and Sample 4 (with filtration). In the filtration, an Advantech filter, 13HP020AN, a hydrophilic PTFE membrane, and a pore diameter of 0.2 μm were used as a syringe filter.
For sample 3 (with filtration) and sample 4 (with filtration), the light emission amount was measured four times without performing the Limulus reaction, and the average light emission amount was calculated. The results are shown in Table 1.

  The average luminescence amount of Sample 4 (with filtration) was 0.34% of the average luminescence amount of Sample 3 (with filtration). This indicates that the protease cannot be removed simply by filtering the aqueous protease solution with a filter, and the protease is removed when filtration is performed in the presence of calcium phosphate. This is presumably because the apparent molecular weight of the protease increased due to the binding of calcium phosphate to the protease. It was suggested that the amount of luminescence decreased by filtration in the presence of the saliva of Sample 1 was due to the calcium phosphate in the saliva being associated with the protease.

[Endotoxin concentration in saliva]
The concentration of endotoxin in the saliva was calculated as follows by filtering the saliva to remove the protease.
(Sample 5)
The subject after chewing with water for injection (Otsuka Pharmaceutical Factory Co., Ltd., Otsuka distilled water) chewed the experimental gum, and collected about 2 mL of saliva that came out. The collected saliva was diluted 1000 times with water for injection to obtain sample 5.
Half of the diluent was sample 5 (no filtration), and the remaining half of the diluent was filtered by the above method to give sample 5 (with filtration). In the filtration, an Advantech filter, 13HP020AN, a hydrophilic PTFE membrane, and a pore diameter of 0.2 μm were used as a syringe filter.
For sample 5 (without filtration) and sample 5 (with filtration), the luminescence was measured four times after the Limulus reaction, and the average luminescence was calculated respectively. The results are shown in Table 1.
Since Sample 5 (without filtration) has a large light emission amount, the light emission amount was measured by diluting 10 times, and the obtained value was multiplied by 10 and used to calculate the average light emission amount.
The ratio (%) of the average luminescence amount of Sample 5 (with filtration) to Sample 5 (without filtration) was 12%. This suggests that the protease in the sample has been sufficiently removed.
(Create a calibration curve)
A standard sample derived from P. gingivalis (InvivoGen (USA), LPS-PG) was dissolved in water for injection (Otsuka Pharmaceutical Co., Ltd., Otsuka distilled water), and Standard Endotoxin (STD ) Was prepared so as to have a concentration of 0, 1, 3, 5, 10 ng / mL and filtered. In the filtration, an Advantech filter, 13HP020AN, a hydrophilic PTFE membrane, and a pore diameter of 0.2 μm were used as a syringe filter.
After performing the Limulus reaction, the amount of luminescence was measured four times, and the average amount of luminescence was calculated for each. The results are shown in Table 2. A calibration curve was prepared for the average luminescence and STD concentration. A calibration curve represented by the following formula (1) was obtained.
Light emission amount = STD concentration (ng / mL) × 13631 + 5416.3 (1)

(Calculation of salivary endotoxin concentration)
The endotoxin concentration in saliva (diluted 1000 times) was calculated to be 4.2 ng / mL from the amount of luminescence of the above formula (1) and sample 5 (with filtration). That is, the concentration of endotoxin in saliva was 4.2 μg / mL.

  According to the method for removing protease in saliva of the present invention, the concentration of a measurement target substance can be measured with high sensitivity by reducing the influence of protease derived from saliva. Therefore, the present invention is extremely important in industry.

DESCRIPTION OF SYMBOLS 1 Syringe filter 1a Housing 1b Filter 2 Syringe 2a Pipe | tube part front-end | tip 2b Pipe | tube part 2c Plunger 3 Container S Saliva sample

Claims (7)

  A method for removing protease in saliva, wherein the protease is removed by filtering saliva.   The method for removing protease in saliva according to claim 1, wherein the saliva is filtered using a filter having a pore size of 0.2 μm to 0.45 μm.   The method for removing protease in saliva according to claim 2, wherein the filter is a syringe filter.   A saliva target substance measurement comprising: a removal step of removing protease in saliva by the method for removing protease in saliva according to any one of claims 1 to 3; and a measurement step of measuring a measurement target substance in saliva. Method.   The method for measuring a substance in saliva according to claim 4, wherein the substance to be measured is endotoxin.   The saliva target substance measurement method according to claim 4 or 5, wherein the measurement step measures the concentration of the substance to be measured.   The method for measuring a target substance in saliva according to any one of claims 4 to 6, wherein the measurement step is a Limulus test.

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