A SIMPLE METHOD FOR COLLECTING A SAMPLE OF A MUCOSAL FLUID ADHERED TO THE MUCOSAL SURFACE OF THE NASAL CAVITY AND A TEST KIT FOR DETECTING AN ANALYTE IN THE SAMPLE

Abstract

This invention provides a method for analyzing an analyte in a sample in a rapid and simple manner with high accuracy with the use of a sample of a mucosal fluid adhered to the mucosal surface of the nasal cavity collected using a nasal spray device in a less invasive and simple manner. The method comprises detecting an infectious disease by applying a washing liquid to the nasal cavity of a subject suspected of an infectious disease to wash the mucosal surface of the nasal cavity and using, as a sample, the collected wash liquid containing the mucosal fluid adhered to the mucosal surface of the nasal cavity.

Claims

1. A method for detecting an infectious disease comprising applying a washing liquid to the nasal cavity of a subject suspected of an infectious disease to wash the mucosal surface of the nasal cavity and using, as a sample, the collected washing liquid containing the mucosal fluid adhered to the mucosal surface of the nasal cavity.

2. The method according to claim 1, wherein the amount of the washing liquid applied to the nasal cavity is 200 to 800 μl.

3. The method according to claim 1, wherein the method for applying a washing liquid to the nasal cavity is spraying.

4. The method according to claim 1, wherein the infectious disease is an infectious disease caused by influenza viruses.

5. The method according to claim 1, which is a method of immunological detection.

6. The method according to claim 1, which is a method of gene amplification.

7. A test kit used for detecting an infectious disease by applying a washing liquid to the nasal cavity of a subject suspected of an infectious disease to wash the mucosal surface of the nasal cavity and using, as a sample, the collected washing liquid containing the mucosal fluid adhered to the mucosal surface of the nasal cavity, which comprises a test reagent for detecting an infectious disease and a nasal spray device containing a washing liquid for washing the mucosal membrane of the nasal cavity.

8. The test kit according to claim 7, wherein the nasal spray device is a single-use nasal spray device and the device contains a single dose of 200 to 800 μl of a wash liquid to be applied to the nasal cavity.

9. The test kit according to claim 7, wherein the method for applying a washing liquid to the nasal cavity is spraying.

10. The test kit according to claim 7, wherein the infectious disease is an infectious disease caused by influenza viruses.

11. The test kit according to claim 7, which is a kit for immunological testing.

12. The test kit according to claim 7, which is a kit for gene amplification testing.

13. A method for collecting a sample that contains a washing liquid containing a mucosal fluid adhered to the mucosal surface of the nasal cavity comprising applying a washing liquid to the nasal cavity of a subject suspected of an infectious disease to wash the mucosal surface of the nasal cavity.

14. The method according to claim 13, wherein the amount of the washing liquid applied to the nasal cavity is 200 to 800 μl.

15. The method according to claim 13, wherein the method for applying a washing liquid to the nasal cavity is spraying.

16. The method according to claim 13, wherein the infectious disease is an infectious disease caused by influenza viruses.

Description

EXAMPLES

[0058] Hereafter, the present invention is described in greater detail with reference to the examples, although the present invention is not limited to the examples.

Example 1

1. Preparation of Nasal Spray Device

[0059] A 20-ml nasal spray container (Kinshi Seisakusho) was filled with 10 ml of physiological saline (Otsuka Pharmaceutical Co., Ltd.) to prepare a nasal spray device. 2. Collection of easily-collectible samples of mucosal fluids adhered to the mucosal surface of the nasal cavity

[0060] Washing liquids in amounts of 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, and 800 μl were sprayed to the nasal cavity of each of 3 healthy adult subjects with the use of the nasal spray device, the liquids discharged to the entrance of the nose were absorbed to Ex Swabs 003T and collected, and the collected liquids were designated as easily-collectible samples of mucosal fluids adhered to the mucosal surface of the nasal cavity.

3. Functional Test at the Time of Collection

[0061] When collecting the easily-collectible samples of mucosal fluids adhered to the mucosal surface of the nasal cavity in 2. above, the 3 healthy adult subjects were subjected to evaluation in terms of ease of collection, a feeling of suffocation, and difficulty in fluid flow to the throat at each spray dose.

4. Comparison and Examination

[0062] On the basis of the results shown in Table 1, the optimal spray dose was considered to be 300 μl to 500 μl, although the optimal spray dose would vary among individuals. Because healthy adult subjects were employed, different results may be obtained in the case of child subjects. In addition, an optimal dose may vary among races. Thus, the optimal dose is not limited to the dose indicated above.

TABLE-US-00001 TABLE 1 3 Healthy subjects Spray dose (μl) Evaluation points in functional test A B C 200 Ease of collection Fine Poor Fine Feeling of suffocation Very good Very good Very good Difficulty in fluid flow to throat Very good Very good Very good 300 Ease of collection Very good Good Good Feeling of suffocation Very good Good Very good Difficulty in fluid flow to throat Very good Very good Very good 400 Ease of collection Very good Very good Very good Feeling of suffocation Very good Fine Very good Difficulty in fluid flow to throat Very good Fine Very good 500 Ease of collection Very good Very good Very good Feeling of suffocation Very good Poor Very good Difficulty in fluid flow to throat Good Poor Good 600 Ease of collection Very good Very good Good Feeling of suffocation Good Poor Very good Difficulty in fluid flow to throat Fine Poor Fine 700 Ease of collection Good Very good Fine Feeling of suffocation Good Poor Very good Difficulty in fluid flow to throat Fine Poor Poor 800 Ease of collection Fine Very good Fine Feeling of suffocation Good Poor Very good Difficulty in fluid flow to throat Fine Poor Poor

Example 2

1. SDS-PAGE Analysis of Easily-Collectible Samples of Mucosal Fluids Adhered to the Mucosal Surface of the Nasal Cavity

[0063] The easily-collectible samples of mucosal fluids adhered to the mucosal surface of the nasal cavity collected in Example 1 2. were suspended in 200 μl of physiological saline. The samples were fractionated, reagents were added thereto to adjust the final concentration to 62.5 mM Tris-HCl (pH 6.5), 10(w/v)% glycerol, 2.3(w/v)% SDS, and 0.05(w/v)% BPB (dye), the resultant was subjected to thermal denaturation at 95° C. for 5 minutes, and SDS-PAGE was performed in accordance with a conventional technique.

2. Analysis of Total Protein Level Using Densitometry Analyzer

[0064] With the use of a densitometry analyzer (BioRad), the CBB-stained gel obtained in SDS-PAGE performed in 1. above was analyzed, the band density in each lane was assayed, and the total density level was designated as the density score.

3. Comparison and Examination

[0065] Table 2 shows the results of measurement of the density score. While the results of evaluation varied among individuals, as shown in Table 2, the protein levels in the easily-collectible samples of mucosal fluids adhered to the mucosal surface of the nasal cavity at a spray dose of 400 to 700 μl were found to be high. Because healthy adult subjects were employed, different results may be obtained in the case of patients with symptoms or child subjects. In addition, an optimal dose may vary among races. Thus, the optimal dose is not limited to the dose indicated above.

TABLE-US-00002 TABLE 2 Density Score 3 Healthy subjects Spray dose (μl) A B C 200 106.4 104.6 107.5 300 110.5 105.9 119.0 400 109.5 110.1 152.4 500 119.2 106.1 113.4 600 129.5 102.0 100.0 700 100.0 103.8 127.8 800 106.8 100.0 101.1

Example 3

1. SDS-PAGE Analysis of Nasal Aspirate Samples and Easily-Collectible Samples of Mucosal Fluids Adhered to the Mucosal Surface of the Nasal Cavity

[0066] Samples were collected from 10 nasal aspirate samples using Mentip P1503 (J.C.B. Industry Limited) and suspended in 200 μl of physiological saline to prepare SDS-PAGE samples. Also, the easily-collectible samples of mucosal fluids adhered to the mucosal surface of the nasal cavity collected from 3 healthy subjects by spraying 400 μl of the liquidss in 2. of Example 1 were mixed to prepare SDS-PAGE samples. The samples were fractionated, reagents were added thereto to adjust the final concentration to 62.5 mM Tris-HCl (pH 6.5), 10(w/v)% glycerol, 2.3(w/v)% SDS, and 0.05(w/v)% BPB (dye), the resultants were subjected to thermal denaturation at 95° C. for 5 minutes, and SDS-PAGE was performed in accordance with a conventional technique.

2. Analysis of Total Protein Level Using Densitometry Analyzer

[0067] With the use of a densitometry analyzer (BioRad), the CBB-stained gel obtained in SDS-PAGE performed in 1. above was analyzed, the band density in each lane was assayed, and the total density level (the density score) was determined.

3. Comparison and Examination

[0068] Table 3 shows the results of measurement of the density scores. While the results varied among samples, as shown in Table 3, no significant differences were observed in the amount of the collected proteins between the samples of the nasal aspirates and the easily-collectible samples of mucosal fluids adhered to the mucosal surface of the nasal cavity when the samples were mixed. It was thus considered that influenza viruses could be tested with the use of the easily-collectible samples of mucosal fluids adhered to the mucosal surface of the nasal cavity according to the present invention.

TABLE-US-00003 TABLE 3 Samples Density Score Easily-collectible sample of mucosal fluid 153.5 adhered to mucosal surface of nasal cavity Nasal aspirate 1 144.0 Nasal aspirate 2 115.9 Nasal aspirate 3 201.0 Nasal aspirate 4 140.8 Nasal aspirate 5 96.5 Nasal aspirate 6 118.0 Nasal aspirate 7 120.2 Nasal aspirate 8 102.0 Nasal aspirate 9 187.5 Nasal aspirate 10 176.2 Mixture of nasal aspirates 1 to 10 161.0

Example 4

1. Preparation of Anti-Influenza a Virus Antibodies

[0069] BALB/c mice were immunized with inactivated influenza A viruses and raised for a given period of time. The spleens were removed from the mice and fused to mouse myeloma cells (P3X63) in accordance with the method of Kohler et al. (Kohler et al., Nature, vol. 256, pp. 495-497, 1975). The resulting fused cells (hybridomas) were maintained in an incubator at 37° C., and cells were purified (monoclonalized) while observing the antibody activity of the supernatant by ELISA using a plate comprising an antigen extracted from Helicobacter pylori immobilized thereon. The 2 resulting cell lines were administered intraperitoneally to the pristane-treated BALB/c mice, and the antibody-containing ascites fluids were collected approximately 2 weeks later.

[0070] IgG was purified from the obtained ascites by affinity chromatography using a protein A column to obtain 2 types of purified anti-influenza A virus antibodies.

2. Preparation of Anti-Influenza B Virus Antibodies

[0071] BALB/c mice were immunized with inactivated influenza B viruses and raised for a given period of time. The spleens were removed from the mice and fused to mouse myeloma cells (P3X63) in accordance with the method of Kohler et al. (Kohler et al., Nature, vol. 256, pp. 495-497, 1975). The resulting fused cells (hybridomas) were maintained in an incubator at 37° C., and cells were purified (monoclonalized) while observing the antibody activity of the supernatant by ELISA using a plate comprising an antigen extracted from Helicobacter pylori immobilized thereon. The 2 resulting cell lines were administered intraperitoneally to the pristane-treated BALB/c mice, and the antibody-containing ascites fluids were collected approximately 2 weeks later.

[0072] IgG was purified from the obtained ascites by affinity chromatography using a protein A column to obtain 2 types of purified anti-influenza B virus antibodies.

3. Preparation of Labeled Anti-Influenza a Virus Antibodies

[0073] One of the anti-influenza A virus antibodies was dialyzed against a 50 mM MES (2-morpholinoethanesulfonic acid, monohydrate, Dojindo Laboratories) buffer (pH 6.0) solution, and 10 ml of a solution was prepared by diluting the dialyzed antibody solution with the same buffer to adjust O.D. 280 nm to 0.5. Subsequently, the resultant was mixed with 10(w/v)% blue polystyrene latex particles (particle diameter: 0.45 μm; surface functional group: carboxyl; functional group density: 65 Å2/COOH group; Magsphere) to result in the liquid volume ratio of 40:1, and the reaction was allowed to proceed. Subsequently, 1(w/v)% EDAC (N-(3-dimethlaminopropyl)-N′-ethylcarbodiimide hydrochloride (Sigma)) was added to the final concentration of 0.1%, and the reaction was then allowed to proceed for 2 hours. The reaction product was washed, suspended in 20 ml of the final suspension (5 mM Tris, 0.04(w/v)% BSA (bovine serum albumin), 0.4 M trehalose, 0.2(v/v)% TritonX-100), and applied to an ultrasonic dispersion apparatus (Olympus) to disperse latex particles.

4. Preparation of Labeled Anti-Influenza B Virus Antibodies

[0074] One of the anti-influenza B virus antibodies was dialyzed against a 50 mM MES (2-morpholinoethanesulfonic acid, monohydrate, Dojindo Laboratories) buffer (pH 6.0) solution, and 10 ml of a solution was prepared by diluting the dialyzed antibody solution with the same buffer to adjust O.D. 280 nm to 0.5. Subsequently, the resultant was mixed with 10(w/v)% blue polystyrene latex particles (particle diameter: 0.45 μm; surface functional group: carboxyl; functional group density: 65 Å2/COOH group; Magsphere) to result in the liquid volume ratio of 40:1, and the reaction was allowed to proceed. Subsequently, 1(w/v)% EDAC (N-(3-dimethlaminopropyl)-N′-ethylcarbodiimide hydrochloride (Sigma)) was added to the final concentration of 0.1%, and the reaction was then allowed to proceed for 2 hours. The reaction product was washed, suspended in 20 ml of the final suspension (5 mM Tris, 0.04(w/v)% BSA (bovine serum albumin), 0.4 M trehalose, 0.2(v/v)% TritonX-100), and applied to an ultrasonic dispersion apparatus (Olympus) to disperse latex particles.

5. Preparation of Latex Particle-Labeled Antibody Drying Pad

[0075] The latex particle-labeled anti-influenza A and B virus antibodies obtained in 3. and 4. above were mixed and sprayed on the entire surface of a cellulose nonwoven fabric having a width of 15 mm wound in a reel shape at a coating amount of 8 μl/cm using a positive pressure spray device (BioJet; BioDot). Thereafter, warm air at 50° C. was blown for 1 minute to dry, thereby preparing a latex particle-labeled antibody drying pad.

6. Preparation of Antibody for Solid-Phase Membrane

[0076] The purified anti-influenza A virus antibody prepared in 1. above, which was not used for labeling, was dialyzed against a solid solution (10 mM Tris-HCl (pH 8.0)), followed by filtration with a 0.22 μm filter. The solution was diluted with a solid solution to adjust OD 280 nm to 3.0, and an anti-influenza A virus antibody for solid phase was prepared.

[0077] The purified anti-influenza B virus antibody prepared in 2. above, which was not used for labeling, was dialyzed against a solid solution (10 mM Tris-HCl (pH 8.0)), followed by filtration with a 0.22 μm filter. The solution was diluted with a solid solution to adjust OD 280 nm to 3.0, and an anti-influenza B virus antibody for solid phase was prepared.

7. Preparation of Lateral-Flow Membrane Assay Device for Detection of Influenza Viruses

[0078] A nitrocellulose membrane (pore diameter: 12 μm; Whatman) sheet (white) having a width of 3 cm and a length of 10 cm was used. With the use of a positive pressure spray apparatus (BioJet; BioDot), the anti-influenza A virus antibody for solid phase was applied linearly at a position 6 mm away from one end of the long axis (this end is the upstream end and the opposite side is the downstream end) at a coating amount of 1 μl/cm, the anti-influenza B virus antibody for solid phase was applied linearly at a position 8 mm away from one end of the long axis at a coating amount of 1 μl/cm, and the anti-mouse IgG antibody diluted to OD 280 nm of 1.0 was applied linearly at a position 13 mm away from one end of the long axis at a coating amount of 1 μl/cm. Thereafter, warm air at 45° C. was brown for 30 minutes to dry the membrane.

[0079] In order to fix the member and increase the strength, subsequently, a plastic backing sheet (BioDot) was bonded to the opposite side (this surface is the lower surface) of the membrane surface on which the antibody has been applied (this surface is the upper surface).

[0080] Subsequently, the latex particle-labeled antibody drying pad prepared in 5. above was cut into a width of 15 mm and a length of 10 cm, so that the upstream end of the membrane would overlap with the upper surface of the membrane by 2 mm, and a cellulose filter paper (Wattman) having a width of 23 mm and a length of 10 cm was applied on top of the latex particle-labeled antibody drying pad to overlap by 13 mm. Thus, a sample application pad was prepared.

[0081] Subsequently, a cellulose filter paper (Wattman) having a width of 30 mm and a length of 10 cm was applied on top of the membrane to overlap with the downstream end of the membrane by 5 mm. Thus, a sample absorption pad was prepared.

[0082] Subsequently, the entire upper surface was covered with a transparent plastic laminate (Adhesive Research) except for a width of 5 mm at the upstream end of the sample application pad.

[0083] In the end, the resultant was cut along the long axis by 5 mm to prepare a membrane assay apparatus.

8. Detection of Influenza Viruses

[0084] Nasopharyngeal swab and nasal swab specimens were collected using an influenza virus antigen test kit, QuickNave™-Flu 2 (Denka Company Limited), for diagnosis of influenza virus infection. Five patients diagnosed positive(+) for the influenza A virus, five patients diagnosed positive(+) for the influenza B virus, and five patients diagnosed negative(−) for the influenza viruses on the basis of the overall finding were designated as the subjects. To the nasal cavity of each subject, 400 μl of physiological saline was sprayed with the use of the nasal spray device prepared in 1 of Example 1, the liquids discharged to the entrance of the nose were absorbed to Ex Swabs 003T (Denka Company Limited) and collected, and the collected liquids were designated as easily-collectible samples of mucosal fluids adhered to the mucosal surface of the nasal cavity. The tips of the cotton swabs used for collecting the samples were soaked in 0.2 ml of a sample suspension buffer (a phosphate buffer (pH 7.4) containing Tween 20 (0.05(w/v)%) and bovine serum albumin (0.1(w/v)%)), and substances adhered to the tips were squeezed out into the sample suspension buffer to prepare a sample solution.

[0085] The sample application pad of the lateral-flow membrane assay apparatus for influenza virus detection prepared in 7. was soaked in the sample solution. The assay apparatus was inspected 10 minutes later. The case in which color development was observed at the position (control line) where the anti-mouse IgG antibody had been applied was regarded as valid. When color development was observed at the position where the anti-influenza A virus antibody for solid-phase had been applied, the subject was evaluated positive(+) for the influenza A virus. When color development was observed at the position where the anti-influenza B virus antibody for solid-phase had been applied, the subject was evaluated positive(+) for the influenza B virus. When color development was not observed in any position, the subject was evaluated negative(−). The case in which no color development was observed on the control line was regarded as invalid.

9. Comparison and Examination

[0086] Table 4 shows the results of assays. The results shown in Table 4 demonstrate that all the test results attained with the samples according to the present invention were consistent on the basis of comparison of the results of the test performed by collecting nasopharyngeal swab and nasal swab specimens using QuickNave™-Flu 2 and the overall findings.

TABLE-US-00004 TABLE 4 Lateral-flow membrane assay apparatus QuickNavi-Flu2 Easily-collectible sample nasopharyngeal of mucosal fluid Sample swab and nasal adhered to mucosal Overall No. swab specimen surface of nasal cavity finding No. 1 — — — No. 2 A+ A+ A+ No. 3 — — — No. 4 B+ B+ B+ No. 5 A+ A+ A+ No. 6 B+ B+ B+ No. 7 A+ A+ A+ No. 8 A+ A+ A+ No. 9 — — — No. 10 B+ B+ B+ No. 11 — — — No. 12 B+ B+ B+ No. 13 A+ A+ A+ No. 14 B+ B+ B+ No. 15 — — —

Example 5

1. Sample Collection for Comparison of Influenza Virus Levels

[0087] Nasopharyngeal swab and nasal swab specimens were collected using an influenza virus antigen test kit, QuickNave™-Flu 2 (Denka Company Limited), for diagnosis of influenza virus infection. To the nasal cavity of a subject diagnosed positive(+) for the influenza A virus on the basis of the overall finding, 400 μl of physiological saline was sprayed with the use of the nasal spray device prepared in 1. of Example 1, the liquid discharged to the entrance of the nose was absorbed to Ex Swabs 003T (Denka Company Limited) and collected as an easily-collectible sample of a mucosal fluid adhered to the mucosal surface of the nasal cavity, and the collected sample was suspended in 200 μl of physiological saline to prepare a qPCR sample. The residual liquid of the nasopharyngeal swab and nasal swab specimens obtained from the same subject with the use of QuickNave™-Flu 2 was used as a control sample.

2. Real-Time PCR Analysis

[0088] With the use of the QIAamp Viral RNA Mini Kit (a kit for nucleic acid extraction, QIAGEN), nucleic acids were extracted from the 2 samples obtained in 1. above and analyzed using the PCR apparatus (Applied Biosystems QuantStudio™ 3, Thermo Fisher Scientific) with the addition of a given PCR sample.

3. Comparison and Examination

[0089] Table 5 shows the results of assays. The results shown in Table 5 demonstrate that the virus level in the easily-collectible sample of a mucosal fluid adhered to the mucosal surface of the nasal cavity was equivalent to 1.39×10.sup.6 copies/ml and the virus level in the control sample of nasopharyngeal swab and nasal swab specimens was equivalent to 1.31×10.sup.5 copies/ml. The virus level in the easily-collectible sample of a mucosal fluid adhered to the mucosal surface of the nasal cavity was higher than the virus level in the nasopharyngeal swab and nasal swab specimens according to a conventional technique. This indicates that the easily-collectible sample of a mucosal fluid adhered to the mucosal surface of the nasal cavity enables more stable virus collection compared with the nasopharyngeal swab and nasal swab specimens and that such easily-collectible sample can be used for clinical testing.

TABLE-US-00005 TABLE 5 Real time PCR analysis Easily-collectible sample of mucosal QuickNavi-Flu2 fluid adhered to nasopharyngeal swab and mucosal surface Sample No. nasal swab specimen of nasal cavity No. 16 1.31 × 10.sup.5 1.39 × 10.sup.6

INDUSTRIAL APPLICABILITY

[0090] The present invention can be used for detection of respiratory infectious diseases.

[0091] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.