JP5124211B2 - Treatment method for intraoral samples in immunochromatography - Google Patents

Description

  The present invention relates to the processing of a sample for immunochromatographic examination, and more particularly to a pretreatment method for a specimen derived from an oral cavity for examination or diagnosis of periodontal disease.

  Periodontal disease is a general term for diseases in the periodontal tissue, which is a tissue for supporting the teeth, and is a multifactorial disease caused by various risks. Among them, the periodontal disease is a necessary condition. It is infection of the periodontal pocket site of pathogenic bacteria. This periodontal disease begins with inflammation of the gingiva, collapses the periodontal tissue, and eventually leads to tooth loss. As described above, periodontal disease has a serious effect on teeth, but it progresses slowly and there is almost no pain, so it worsens without subjective symptoms. Thus, it is considered that examining what kind of bacteria are present in the periodontal pocket is important for early detection of periodontal disease and for examining the risk of periodontal disease.

  It is known that there are many periodontopathic bacteria, but as shown in Non-patent document 1, it is infected with three strains of Porphyromonas gingivalis, Bacteroides forsysus and Treponema denticola called “Red Complex” It is said that the risk of suffering from periodontal disease is the highest. At present, as a means for detecting bacteria, an inspection method using real-time PCR and a method for detecting and inspecting enzymes secreted by the three bacterial species are put into practical use. At the laboratory level, bacteria may be examined using an enzyme immunoassay or radioimmunoassay utilizing an antigen-antibody reaction.

  However, in the above inspection method, a specific dedicated device is necessary, or it is necessary to perform a plurality of complicated inspection processes with specialized knowledge. For this reason, the time until the inspection is performed and the result is obtained. However, it was not possible to carry out a simple inspection from the viewpoint of cost and labor.

  On the other hand, the immunochromatography method, which is one of the technologies utilizing antigen-antibody reaction, is performed by dropping the bacteria contained in the collected specimen as an antigen onto a dedicated strip coated with an antibody that recognizes the antigen. It is a method that can know the presence and amount of antigen. This exclusive strip mainly uses a porous membrane such as nitrocellulose, and the antigen is detected by binding to the antibody by moving in the porous membrane by capillary action. Therefore, only the specimen is dropped as an operation, and the time until the sample containing the antigen and antibody moves and reacts is about 30 minutes. Therefore, if this immunochromatography method is used, the test can be performed. It can be performed simply.

  However, in the examination or diagnosis of periodontal disease, specimens collected from the oral cavity, especially saliva and mouthwash discharge liquid, are very mucous and therefore move through the porous membrane as they are by capillary action. Is very difficult. Therefore, it is necessary to process these specimens in advance to facilitate movement by capillary action. In the treatment of these specimens, degradation due to chemical substances is often seen, but if the degradation is excessive, the target antigen or antibody is also degraded, making it impossible to detect the specimen. . In addition, if the chemical substance causes denaturation of molecules or the like, accurate detection of the specimen is impaired. Therefore, it is necessary to process these specimens using appropriate substances.

  For example, in Patent Document 1, saliva is treated with alkali (NaOH) in a test for measuring or identifying caries-causing bacteria (mutans streptococci) by immunochromatography, and then acid treatment (tartaric acid / citric acid). A method of decomposing mucin and glucan in saliva by neutralizing and then treating with a nonionic surfactant or amphoteric surfactant is disclosed. However, in this processing method, a plurality of solution materials are required for the sample processing, and accordingly, a plurality of processing steps are performed. Cost concerns are also needed.

On the other hand, Patent Document 2 discloses that a sample (nasal discharge, sputum, throat swab, etc.) collected from a living body for influenza testing is used as a nonionic surfactant (polyoxyethylene) when testing influenza by immunochromatography. And a method of treating with a pretreatment liquid containing 0.3 M alkali metal ions (sodium ions). However, these techniques are not disclosed at all for the treatment of saliva and mouthwash discharge liquid.
JP 2002-357599 A JP-A-2005-24323 Socransky SS, et al., “Microbial complexes subgingival plaque”, Journal of Clinical Periodontology, Vol. 25, pp. 134-144 (19-98). )

  As described above, a method for processing saliva in the examination of caries-causing bacteria has been known, but it is not as simple as requiring a plurality of materials and processing actions. On the other hand, there was a method for processing a biological specimen in influenza testing, but it was not saliva or mouthwash discharge liquid, nor did it specify the examination or diagnosis of periodontal disease.

  Therefore, the problem to be solved by the present invention is that the above-mentioned oral-derived specimen necessary for conducting a more accurate and simple examination when examining or diagnosing periodontal disease using an oral-derived specimen by immunochromatography. It is to provide a processing method.

  In order to solve the above-mentioned problems, the present inventors have made extensive studies on the treatment of the oral cavity-derived specimen. As a result, the oral cavity-derived specimen has an anionic surfactant, preferably sodium dodecyl sulfate or sodium deoxycholate. The present inventors have found that periodontal diseases can be inspected or diagnosed accurately and simply by subjecting a specimen to a sample treatment.

  That is, the present invention provides a sample processing method for a sample derived from the oral cavity, characterized in that an anionic surfactant is used for a sample derived from an oral cavity for periodontal disease testing or diagnosis by immunochromatography. This is a method for processing a specimen.

  In the specimen treatment method, the anionic surfactant is preferably dodecyl sulfate or deoxycholate.

  As described in detail above, in the case of examining or diagnosing periodontal disease by immunochromatography, by treating a sample derived from the oral cavity with sodium dodecyl sulfate or sodium deoxycholate, an immunochromatographic strip is obtained. Periodontal diseases can be examined or diagnosed accurately and simply without causing clogging.

  In the present invention, the sample to be measured is not particularly limited as long as it is derived from the oral cavity, and preferred examples include saliva, mouthwash discharge, gingival crevicular fluid, or plaque.

  A method for collecting these specimens is not particularly limited, and a known method can be adopted. For example, saliva can be collected by stimulating saliva using chewing gum base or paraffin wax, unstimulated saliva at rest, swabs or water-absorbing paper. Stimulated saliva that can be processed in a short time and facilitates sample processing is most desirable. Further, the mouthwash discharge liquid may contain water, physiological saline, a buffer solution, or the like and then move the mouth well to perform rinsing, or it may be discharged immediately after being contained in the mouth. Examples of plaques include those obtained by scraping with a scaler or toothbrush and suspending in water, physiological saline, buffer solution, or the like, water washed after brushing, physiological saline, buffer solution, or the like.

  The anionic surfactant used in the sample treatment method of the present invention is not particularly limited, but alkyl sulfate ester salts (such as sodium dodecyl sulfate), bile salts (such as sodium deoxycholate), and sodium lauryl sulfosuccinate. , Sodium α-olefin sulfonate, sodium polyoxyethylene lauryl ether sulfate, sodium polyoxyethylene lauryl ether phosphate, sodium lauryl benzene sulfonate and the like. In the present invention, alkyl sulfate ester salts and bile salts are particularly preferable, and sodium dodecyl sulfate or sodium deoxycholate is most suitable.

  Said anionic surfactant can be added so that it may become a density | concentration of 0.001-0.1%, when using it for the process of a test substance.

  In addition to the above-mentioned anionic surfactant, the sample processing method of the present invention can be used by including components that can be generally used by those skilled in the art. Specifically, components that constitute a buffer solution that can be maintained at an optimum pH for the reaction, other organic acids, inorganic acids, metal salts, and the like can be included. Examples of the buffer include phosphoric acid, boric acid, Tris buffer and the like. The pH is not particularly limited, but is preferably neutral (pH 5.0 to 9.0). Specific examples of the organic acid include tartaric acid, citric acid, oxalic acid, malonic acid, succinic acid, acetic acid, formic acid and the like, and inorganic acids include sulfuric acid, hydrochloric acid, nitric acid, nitrous acid and the like. Examples of the metal salt include sodium salt, potassium salt, magnesium salt, lithium salt and the like.

  The sample treatment method may be a normal method. When a liquid sample is treated, a liquid containing the anionic surfactant is added to the sample, and mixed by inversion mixing, shaking, stirring, or the like. The liquid and the sample may be mixed. In addition, when saliva is collected using a swab or water-absorbing paper, the water-absorbing body is immersed in a liquid containing the anionic surfactant and mixed, and then the liquid and the specimen are squeezed out of the water-absorbing body. Can be processed. In addition, the above anionic surfactant is directly applied directly to a part upstream of the antigen detection site on the immunochromatography, such as a sample pad or a part upstream of the antigen detection site of the nitrocellulose membrane. By impregnating with the liquid etc. which contain, it is also possible to process simultaneously with the movement of the specimen on immunochromatography. The processing time is not particularly limited, but is preferably within 2 hours after the processing, and it is possible to perform an inspection immediately after the processing. If reaction temperature is room temperature, it will suffice if it reacts at 5 to 35 degreeC.

  Furthermore, according to the present invention, the sample containing the anionic surfactant is one of the constituent members, and the oral-derived sample is processed by the processing method, and the immunochromatography for periodontal disease test or diagnosis is characterized. An inspection kit can also be provided.

  In order to understand the present invention, examples will be shown and described below, but the present invention is not limited to these examples.

Test Example 1 Effect of Anionic Surfactant on Saliva Specimen This test example was conducted for the purpose of confirming the effect of an anionic surfactant when saliva was used as a specimen.

  0.9 mL of 50 mM phosphate buffer was added to 0.1 mL of a commercially available gold colloid solution (BBI) having a colloidal particle shape of 40 nm to adjust the pH to 9.0. On the other hand, the monoclonal antibody specifically recognizing Porphyromonas gingivalis (hereinafter referred to as “Pg”) according to the present invention is referred to as “Monoclonal antibody experiment manual” (cf. published by Koji Toyama, Tamie Ando, Kodansha) Based on the establishment of hybridomas by cell fusion as shown in the above, the anti-Pg monoclonal antibody prepared by a conventional method was prepared to 60 μg / mL using distilled water. 100 μL of the anti-Pg monoclonal antibody solution was added to the gold colloid suspension, and the mixture was allowed to stand for 10 minutes after stirring. Next, 55 μL of 1% PEG 20000 and 110 μL of 10% BSA were added and centrifuged at 10,000 rpm at 10 ° C. for 15 minutes, and the supernatant was removed. Next, it is resuspended in 500 μL of 20 mM TBS buffer (hereinafter referred to as “gold colloid storage buffer”) containing 1% BSA, 0.1% PEG 20000, and 0.1% sodium azide, and again at 10 ° C. and 10000 rpm for 15 minutes. Centrifuged. After removing the supernatant again, 100 μL of colloidal gold storage buffer was added and suspended to obtain a colloidal gold labeled antibody.

  As shown in FIG. 1, a detection unit (test line) 4 and an antigen-antibody reaction confirmation unit (control line) on a development membrane 3 composed of a nitrocellulose membrane (Millipore, Hi-Flow Plus membrane, HF120, 25 mm × 5 mm). ) 5 mg / mL anti-Pg monoclonal antibody and 0.25 mg / mL commercially available anti-mouse IgG polyclonal anti (SIGMA) were applied in a straight line and incubated at 50 ° C. for 30 minutes. The antibody-immobilized membrane was impregnated with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5% BSA for 30 minutes, and then 20 mM Tris containing 0.5% sucrose and 0.05% sodium cholate. -After impregnation in HCl buffer (pH 7.4) for 60 minutes, the antibody-immobilized membrane was taken out and allowed to stand overnight at room temperature and dried.

  25 μL of a mixture of 10 μL of colloidal gold-labeled antibody mixed with 10 μL of distilled water and 20 μL of 20 mM TBS buffer containing 5% sucrose and 0.05% PEG 20000 is applied to glass fiber (Millipore, 8 mm × 5 mm) overnight. The conjugate pad 2 was obtained by vacuum drying.

  The prepared antibody-immobilized membrane 3 and conjugate pad 2, the sample pad 1 (Millipore, 18 mm × 5 mm), and the absorption pad 6 (Millipore, 20 mm × 5 mm) are supported on a support 7 (Millipore, 60 mm × 5 mm). As shown in FIG. 1, the films were bonded together to form an immunochromatographic strip.

  To a solution (pH 7.4) containing 10 mM Tris-HCl buffer and 0.9% NaCl, sodium dodecyl sulfate was added to a concentration of 0.05%, and this was used as a sample treatment solution (Example 1). Furthermore, it replaced with the said sodium dodecyl sulfate and used what added the sodium deoxycholate (Example 2), Triton X-100, polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene hardening castor as others What added oil (HCO-60) was used (Comparative Examples 1-3). Moreover, the thing which did not add the said sodium dodecyl sulfate was also used (comparative example 4).

  A healthy person chewed the gum base for 5 minutes, and the secreted stimulated saliva was collected. The stimulated saliva is confirmed by the real-time PCR method that the number of Pg bacteria is below the detection limit of the measuring instrument (Applied Biosystems 7500 Fast), and then the Pg suspension with a known concentration of 1/1000 in the stimulated saliva is used. The solution was added to 107 cells / mL as a positive sample, and the stimulated saliva was used as a negative sample. 20 μL of each sample was added to 180 μL of the sample treatment solution and mixed by inversion to prepare a measurement sample.

100 μL of a positive sample or negative sample as a measurement sample was dropped on the immunochromatography strip, and the movement of the measurement sample was confirmed for 30 minutes. After dropping of each measurement sample, the movement of the colloidal gold labeled antibody (red purple) impregnated in the conjugate pad is visually confirmed, and clogging occurs for those in which no movement of the colloidal gold labeled antibody was observed. It was supposed to be.

  After the clogging was confirmed as described above, the detection of the specimen in the immunochromatographic strip was examined using the specimen treatment liquid in which the clogging did not occur. Specifically, in the same manner as described above, 100 μL of a positive sample or negative sample as a measurement sample was dropped, and Pg detection was examined for 30 minutes. After dropping the sample for measurement, the Pg detection part (test line) and the antigen-antibody reaction confirmation part (control line) were visually determined, and the reaction was confirmed as “+”, and the reaction could not be confirmed. The case was defined as “−”.

The results are shown in Table 2. When sodium dodecyl sulfate was used as the sample treatment solution and when sodium deoxycholate was used as the sample treatment solution, the positive sample was determined as “+”, and the negative sample was “−”. It was determined. On the other hand, for the other sample treatment solutions, all negative samples were determined as “+”, and Pg could not be accurately determined. In addition, all the control lines were determined as “+”, and it was confirmed that the antigen-antibody reaction was performed.

Test Example 2 Effect of Anionic Surfactant on Mouthwash Discharge Sample The purpose of this test example was to confirm the effect of the surfactant when using the mouthwash discharge solution as a sample.

  According to Test Example 1, an immunochromatographic strip was prepared.

  To a solution (pH 7.4) containing 10 mM Tris-HCl buffer and 0.9% NaCl, sodium dodecyl sulfate was added to 0.05%, and this was used as a sample treatment solution (Example 3). Furthermore, it replaced with the said sodium dodecyl sulfate, and used what added the sodium deoxycholate (Example 4), Triton X-100, polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene hardening castor as others What added oil (HCO-60) was used (Comparative Examples 5-7). Moreover, the thing which did not add the said sodium dodecyl sulfate was also used (comparative example 8).

  A healthy person was rinsed with 15 mL of sterile distilled water for 10 seconds, and the discharged material was collected as a mouthwash discharge. The mouthwash discharge liquid is confirmed to have a Pg bacterial count below the detection limit of the measuring instrument (Applied Biosystems 7500 Fast) by real-time PCR, and then the concentration of 1/1000 of the mouthwash discharge liquid is known. A Pg suspension was added to prepare 107 samples / mL, and a positive sample was used. 20 μL of the sample treatment solution was added to 180 μL of each sample, and mixed by inversion to prepare a measurement sample.

In the same manner as in Test Example 1, 100 μL of a positive sample or negative sample as a measurement sample was dropped on the immunochromatography strip, and the movement of the measurement sample was confirmed for 30 minutes. After dropping of each measurement sample, the movement of the colloidal gold labeled antibody (red purple) impregnated in the conjugate pad is visually confirmed, and clogging occurs for those in which no movement of the colloidal gold labeled antibody was observed. It was supposed to be.

The results are as shown in Table 4, and in the same manner as in Test Example 1, when sodium dodecyl sulfate was used as the sample treatment solution and when sodium deoxycholate was used as the sample treatment solution, the positive sample was determined as “+”. Negative specimens were determined as “−”. On the other hand, for the other sample treatment solutions, all negative samples were determined as “+”, and Pg could not be accurately determined. In addition, all the control lines were determined as “+”, and it was confirmed that the antigen-antibody reaction was performed.

It is a figure which shows the outline of the strip for immunochromatography.

Explanation of symbols

1 Sample Pad 2 Conjugate Pad 3 Nitrocellulose Membrane 4 Test Line 5 Control Line 6 Absorption Pad 7 Support Mount

Claims (2)

For periodontal disease examination or diagnosis using immunochromatographic strip , dodecyl sulfate or deoxycholate is added to saliva or mouthwash discharge solution and used as a sample for measurement without filtration. A characteristic inspection method . Use saliva or mouthwash as a measurement sample without performing filtration for periodontal disease testing or diagnosis using immunochromatography strips pre-added with dodecyl sulfate or deoxycholate Inspection method characterized by

Images