Method for pretreatment of specimen for immunochromatographic test

Abstract

The present invention addresses the problem of providing a method which, in immunochromatography that uses saliva and that is for a minute amount of antigen or antibody such as for SARS-CoV-2, makes it possible to detect the immunochromatographic antigen or antibody with good sensitivity even when saliva is used.　The present invention relates to a method for pretreatment of a specimen for an immunochromatographic test, said method being characterized in that saliva is subjected to (1) a step for removing a viscous component in the saliva by passing the saliva through a porous body that can support the viscous component, (2) a step for passing the liquid which has passed through the porous body through a filter having a pore diameter of 0.1-10 μm, and (3) a step for concentrating an antigen or antibody in the liquid that has passed through the filter.

Description

Specimen pretreatment method for immunochromatographic testing

[0001] The present invention relates to a method for pretreating a specimen for immunochromatographic testing of saliva, a pretreatment kit for said sample, and an immunochromatographic testing method.

[0002] COVID-19, the novel coronavirus disease caused by COVID-19, was first identified near Wuhan, China, in 2019 and has since become a global pandemic. Currently, IgG antibodies, IgM antibodies, and neutralizing antibodies, which are types of immunoglobulins present in the blood that specifically react to the nucleocapsid protein and spike protein (hereinafter referred to as "S antigen") of SARS-CoV-2, have been identified. Furthermore, antiviral drugs, neutralizing antibody drugs, immunomodulatory drugs, and other drug therapies for COVID-19 have been developed. Meanwhile, vaccines for preventing COVID-19 have been developed, and the effectiveness of vaccination has been confirmed. However, there is currently no non-invasive method for easily checking antibody fluctuations before and after vaccination or determining the timing of the next vaccination, and there is a need for practical application of such methods.

[0003] Regarding SARS-CoV-2 antibodies, measuring devices based on the immunoassay method chemiluminescence immunoassay (CLIA) and kits capable of measuring antibodies in whole blood, serum, or plasma using immunochromatography have been developed, and performance evaluation tests are being conducted (Non-Patent Document 1). Immunochromatography is simple and rapid, allowing diagnostic results to be determined in approximately 10 to 20 minutes by simply dropping the sample. However, while blood sample collection is considered relatively easy, it is problematic in that it is invasive for the subject. Therefore, there is a need to establish a method for measuring antibodies using non-invasively collected biological samples such as saliva and nasal secretions. In addition, collection of saliva and nasal secretions can be performed even in situations where the availability of collection devices is limited or strict hygiene practices may not be adhered to.

[0004] Problems with conventional immunoassays using non-invasively collected highly viscous biological samples such as saliva and nasal secretions include inaccurate results due to nonspecific reactions and false positives. While some currently available saliva collection devices are capable of removing mucus from saliva (see Patent Document 1), they require centrifugation and are therefore inconvenient. Additionally, some devices use a filter to remove viscous components (see Patent Document 2), but these devices fail to achieve sufficient detection sensitivity when the target substance is an antibody present in extremely small amounts in saliva.

[0005] Furthermore, methods for eliminating non-specific reactions have been disclosed, such as a method using a pretreatment solution containing a nonionic surfactant and alkali metal ions (Patent Document 3) and a method using a pretreatment solution containing a water-soluble ammonium polymer (Patent Document 4). However, it is necessary to check in advance whether these methods will affect the test, and it is also anticipated that the pretreatment solution will dilute the target antibody titer (the amount of antibody against the antigen).

[0006] Furthermore, there is a correlation between the neutralizing activity of SARS-CoV-2 antibodies against the S antigen in saliva and serum (Non-Patent Document 2), but it has been reported that the antibody titer in saliva is more than 1,000 times lower than that in blood (Non-Patent Document 3), and accurate measurement cannot be performed using conventional immunochromatography methods that use saliva as a sample.

[0007] U.S. Patent No. 4,774,962, Japanese Patent No. 4,131,966, Japanese Patent Application Laid-Open No. 2005-24323, Japanese Patent No. 5,334,742

[0008] Report on the simultaneous performance evaluation test of antibody test kits for the novel coronavirus (July 14, 2021, National Institute of Health Sciences) Microbiol Spectr. 2021 Oct 31; 9(2): e0069321 Pathog Immun. 2021 Jun 7; 6(1): 116-134

[0009] Therefore, an object of the present invention is to provide a method for immunochromatography of minute amounts of antigens or antibodies, such as SARS-CoV-2, using saliva, which can detect immunochromatographic antigens or antibodies with high sensitivity even when saliva is used.

[0010] Therefore, the present inventors conducted extensive research to easily and efficiently extract high concentrations of target antigens or target antibodies from saliva. As a result, they discovered that by combining three steps of (1) passing the saliva through a porous body capable of supporting the viscous components in the saliva to remove the viscous components, (2) passing the liquid that has passed through the porous body through a filter with a pore size of 0.1 μm to 10 μm, and (3) concentrating the antigen or antibody in the liquid that has passed through the filter, only the viscous components can be removed in step (1), the target antigen or target antibody can be quickly filtered in step (2), and the target antigen or target antibody concentration in the sample can be sufficiently increased by step (3), dramatically increasing the detection sensitivity by immunochromatography, and thus completed the present invention.

[0011] That is, the present invention provides the following inventions [1] to

[16] . [1] A pretreatment method for a specimen for immunochromatography, comprising the steps of (1) passing saliva through a porous body capable of retaining viscous components in the saliva to remove the viscous components, (2) passing the liquid that has passed through the porous body through a filter with a pore size of 0.1 μm to 10 μm, and (3) concentrating antigens or antibodies in the liquid that has passed through the filter. [2] The pretreatment method according to [1], wherein the porous body has pores that allow low-viscosity components in the saliva to pass but not mucin when a pressure of 1000 kPa or less is applied. [3] The pretreatment method according to [1] or [2], wherein the porous body has pores with a pore size of 10 μm to 3000 μm. [4] The pretreatment method according to any of [1] to [3], wherein the porous body is disposed at the tip of a saliva collection tool. [5] The pretreatment method according to any of [1] to [4], wherein the filter is a one- to four-layer filter. [6] The pretreatment method according to any one of [1] to [5], wherein the antigen or antibody concentration step is antigen or antibody affinity chromatography. [7] The pretreatment method according to any one of [1] to [6], wherein the target antigen or target antibody is SARS-CoV-2 or a SARS-CoV-2 antibody. [8] An immunochromatographic test method for antigens or antibodies in saliva, comprising subjecting a specimen obtained by the pretreatment step according to any one of [1] to [7] to an immunochromatographic antigen or antibody test kit. [9] A pretreatment kit for specimens for immunochromatographic testing, comprising: (a) a porous body capable of retaining viscous components in saliva; (b) a filter with a pore diameter of 0.1 μm to 10 μm; and (c) an antigen or antibody concentration device.

[10] The pretreatment kit according to [9], wherein the porous body has pores that allow low-viscosity components in saliva to pass but not mucin when subjected to a pressure of 1,000 kPa or less.

[11] The pretreatment kit according to [9] or

[10] , wherein the porous body has pores with a diameter of 10 μm to 3000 μm.

[12] The pretreatment kit according to any one of [9] to

[11] , wherein the porous body is disposed at the tip of a saliva collection tool.

[13] The pretreatment kit according to any one of [9] to

[12] , wherein the filter is a 1- to 4-layer filter.

[14] A pretreatment kit according to any one of [9] to

[13] , wherein the antigen or antibody concentrating device is an antigen or antibody affinity chromatography device.

[15] A pretreatment kit according to any one of [9] to

[14] , wherein the target antigen or target antibody is SARS-CoV-2 or a SARS-CoV-2 antibody.

[16] A kit for testing for immunochromatographic antigens or antibodies in saliva, comprising the pretreatment kit according to any one of [9] to

[15] , and an immunochromatographic antigen or antibody test kit.

[0012] According to the present invention, only viscous components can be removed in step (1), the target antigen or target antibody can be quickly filtered in step (2), and the concentration of the target antigen or target antibody in the sample is sufficiently increased in step (3), dramatically increasing the detection sensitivity by immunochromatography. Therefore, the present invention is particularly useful for immunochromatographic testing of SARS-CoV-2 or SARS-CoV-2 antibodies in saliva.

[0013]

[0023] Figures 1A and 1B show the results of steps (1) and (2) of the present invention. Left: Saliva filtered through one filter; Center: Saliva filtered through two filters; Right (reference): Saliva filtered through one filter and mixed for 3 minutes with 10% hexadimethrine bromide at a 1:1 ratio (saliva diluted 2-fold with a final concentration of 5% hexadimethrine bromide). Figures 1A and 1B show the results of steps (1), (2), and (3) of the present invention. Top: filtrate (non-concentrated sample); Bottom: eluate (concentrated sample). Figure 1B shows a syringe-type saliva neutralizing antibody concentration and collection device. The left side shows the filter 1, saliva neutralizing antibody concentration and collection device 2, and saliva filtrate collection device 3 separated, while the right side shows them inserted. Figure 1C shows a plunger-type saliva collection device 14. It has a pressing portion 15, a syringe rod 16, a gasket 17, and a saliva absorber 18. 1 is a diagram showing a state in which a plunger-type saliva collection device 14 is inserted into a syringe-type saliva neutralizing antibody concentrated collection device, and saliva filtration is performed.

[0033] FIG. 1 is a diagram showing a saliva neutralizing antibody concentrated collection device 2 and an insertable dropper portion 20 containing an eluate, and a saliva antibody immunochromatographic test kit 21 that can be inserted into the saliva neutralizing antibody concentrated collection device 2.

[0034] FIG. 1 is a diagram showing a state in which the dropper portion 20 containing the eluate is inserted into the saliva neutralizing antibody concentrated collection device 2, and the saliva neutralizing antibody concentrated collection device 2 is inserted into the saliva antibody immunochromatographic test kit 21.

[0035] FIG. 1 is a longitudinal cross-sectional view of a saliva filtrate collection device 26.

[0036] FIG. 1 is a diagram showing a state in which a plunger-type saliva collection device 14 is inserted into a filter 1, saliva filtration is performed, and the saliva filtrate is collected by the saliva filtrate collection device 26.

[0037] FIG. 1 is a longitudinal cross-sectional view of a saliva neutralizing antibody concentrated collection device 30.

[0038] FIG. 1 is a cross-sectional view showing a saliva neutralizing antibody concentrated collection device 30 inserted into a saliva filtrate collection device 26 containing saliva filtrate. 14 is a longitudinal cross-sectional view of the eluate container 38. FIG. 15 is a diagram showing the saliva neutralizing antibody concentrated collection device 30 being removed from the saliva filtrate collection device 26 and inserted into the eluate container 38. FIG. 16 is a diagram showing the saliva neutralizing antibody concentrated collection device 30 inserted into the eluate container 38. FIG. 17 is a diagram showing an immunochromatographic test paper 45 inserted into the neutralizing antibody concentrate 42 obtained in FIG. 14.

[0014] One aspect of the present invention is a method for pretreating a specimen for immunochromatographic testing, comprising the steps of: (1) passing saliva through a porous body capable of retaining viscous components in the saliva to remove the viscous components; (2) passing the liquid that has passed through the porous body through a filter having a pore diameter of 0.1 μm to 10 μm; and (3) concentrating antigens or antibodies in the liquid that has passed through the filter.

[0015] The pretreatment method of the present invention targets saliva. Saliva includes secretions secreted into the oral cavity from various salivary glands, such as the parotid gland, submandibular gland, and sublingual gland, as well as gingival crevicular fluid. Saliva can be collected by the subject or by another person. Because gingival crevicular fluid contains a large amount of antibodies, it is preferable to collect saliva after massaging the gums. Specifically, with the subject's mouth closed, the subject presses his or her fingers against the upper and lower gums from above the skin, slides the fingers back and forth, and stimulates the oral cavity with the tongue to promote saliva secretion before collecting saliva. While saliva can be collected directly from the oral cavity by the subject, it is preferable to collect it using a saliva collection tool. As the saliva collection tool, a dropper or a saliva collection tool having a porous tip capable of retaining viscous components in saliva is preferred, and a saliva collection tool having a porous tip capable of retaining viscous components in saliva is even more preferred.

[0016] The detection target of the test method of the present invention is an antigen or antibody in saliva. Examples of antigens or antibodies include various viruses or antiviral antibodies, bacteria or antibacterial antibodies, fungi or antifungal antibodies, and exogenous antigens and autoantigens in immune reactions or their antibodies. Examples of viruses include influenza virus, SARS-CoV-2, herpes simplex virus, rubella virus, varicella virus, mumps virus, and cytomegalovirus. Examples of bacteria include Mycoplasma salivarius, Streptococcus mutans, and Treponema pallidum. Examples of fungi include the genus Candida and the genus Geotrichum. The detection target of the test method of the present invention is preferably SARS-CoV-2 or an anti-SARS-CoV-2 antibody, and more preferably an anti-SARS-CoV-2 antibody.

[0017] The collected saliva is subjected to (1) a step (step (1)) in which viscous components in saliva are removed by passing the viscous components through a porous body capable of retaining the viscous components. This step (1) is important because saliva contains viscous components, such as mucin, which cannot be filtered using a conventional filter. A porous body capable of retaining the viscous components in saliva is preferably one having pores that allow low-viscosity components in saliva to pass but not mucin when a pressure of 1000 kPa or less is applied. Here, the pressure refers to the pressure applied when the porous body in a syringe is pressed from above. Preferably, a porous body has pores that allow low-viscosity components (liquids containing antigens or antibodies) in saliva to pass but not mucin when a pressure of 500 kPa or less is applied using a plunger or the like. Specifically, a porous body having pores with a pore diameter of 10 μm to 3000 μm is preferred, and a porous body having pores with a pore diameter of 10 μm to 1500 μm is more preferred. Examples of such porous bodies include chemical fibers, cotton, sponges, and filter paper. Examples of sponges include polyurethane-based, polyvinyl alcohol-based, fluorine-based, and polypropylene-polyethylene polymer sponges. This porous body is preferably disposed in a saliva collection tool (see FIG. 4). The saliva collection tool shown in FIG. 4 is an example of a plunger-type saliva collection tool that not only has a porous body but also applies pressure. The operation of step (1) preferably involves collecting saliva using the saliva collection tool (see FIG. 4) that has a porous body at its tip that can retain viscous components in saliva, and then applying pressure with a plunger or the like to pass the saliva through a porous body that can retain viscous components in saliva, thereby removing the viscous components (see FIG. 5).

[0018] Next, a step (step (2)) is carried out in which the liquid that has passed through the porous body is passed through a filter with a pore diameter of 0.1 μm to 10 μm. In this step, a liquid containing antigens, antibodies, and low-molecular-weight components is separated from the liquid that has passed through the porous body. Examples of filters that can be used include filters used for filtering ordinary solutions and suspensions, such as cellulose acetate filters, polypropylene filters, nylon filters, and polytetrafluoroethylene filters. This filter may consist of a single layer, or two to four layers of filters with different pore diameters. Step (2) is also preferably carried out by applying pressure using a plunger or the like, as shown in Figure 5.

[0019] Next, a step (step (3)) of concentrating the antigen or antibody in the filter-passing liquid is carried out. Concentrating the antigen or antibody in the filter-passing liquid improves the detection sensitivity of the immunochromatography method. A concentrating device such as an antigen purification kit or an antibody purification kit is used for this concentration step. Specifically, antigen or antibody affinity chromatography is used. In the case of antibodies, Sepharose using a protein that binds to the antibody, such as Protein L Sepharose or Protein G Sepharose, is used. In the case of antigens, Sepharose in which Protein L or Protein G bound to the antibody is cross-linked with other bonds is used. When affinity chromatography is used, the antigen or antibody is bound to the concentrating device, and the antigen or antibody bound to the device is eluted and dripped into the immunochromatography test kit (see Figures 5 to 7).

[0020] Another aspect of the present invention is a pretreatment kit for immunochromatographic test specimens, comprising (a) a porous body capable of retaining viscous components in saliva, (b) a filter with a pore size of 0.1 μm to 10 μm, and (c) an antigen or antibody concentrator. The pretreatment is preferably carried out using this pretreatment kit. The (a) porous body capable of retaining viscous components in saliva, (b) a filter with a pore size of 0.1 μm to 10 μm, and (c) an antigen or antibody concentrator can be the same as those described in steps (1), (2), and (3) above.

[0021] The immunochromatographic test specimen pretreatment kit of the present invention will be described with reference to the drawings. Figure 4 shows a plunger-type saliva collection device 14. It includes a pressing portion 15, a syringe rod 16, a gasket 17, and a saliva absorber 18. Figure 3 shows a syringe-type saliva neutralizing antibody concentration and collection device. The left side of Figure 3 shows the filter 1, saliva neutralizing antibody concentration and collection device 2, and saliva filtrate collection device 3 separated, while the right side shows them inserted together. Filter 1 has an upper opening 4 and a lower opening 7, and includes filtration filters 5 and 6. The filtration filters may be single-layered or multi-layered. It is preferable that the filter hole diameter of the lower filter in filtration filters 5 and 6 is smaller. Saliva neutralizing antibody concentration and collection device 2 has an upper opening 8 and a lower opening 10, and includes a neutralizing antibody concentration column 9. Saliva filtrate collection device 3 has an upper opening 11 and a lower closing portion 13, and preferably includes an air hole 12. Figure 5 shows the state in which a plunger-type saliva collection device 14 is inserted into a syringe-type saliva neutralizing antibody concentrated collection device 1 and saliva is filtered. Reference numeral 19 denotes the saliva filtrate. Figure 6 shows a dropper portion 20 containing an eluate that can be inserted into the saliva neutralizing antibody concentrated collection device 2, and a saliva antibody immunochromatographic test kit 21 that can be inserted into the saliva neutralizing antibody concentrated collection device 2. Reference numeral 22 denotes the dropper bulb containing the eluate, 23 denotes the lower opening from which the eluate comes out, 24 denotes a sample addition portion for neutralizing antibodies eluted from the saliva neutralizing antibody concentrated collection device 2, and 25 denotes the saliva antibody immunochromatographic test kit. The lower opening 23 is configured to be inserted into the upper opening 8 of the saliva neutralizing antibody concentrated collection device 2, and the lower opening 10 of the saliva neutralizing antibody concentrated collection device 2 is configured to be inserted into the sample addition portion 24. 7 is a diagram showing a configuration in which the eluate-containing dropper part 20 is inserted into the saliva neutralizing antibody concentrated collection device 2, and the saliva neutralizing antibody concentrated collection device 2 is inserted into the saliva antibody immunochromatography test kit 21. By pouring the eluate in the dropper part 20 into the saliva neutralizing antibody concentrated collection device 2, the neutralizing antibodies in the saliva neutralizing antibody concentrated collection device 2 are eluted and the eluate is added to the sample addition part 24.

[0022] 8 to 15 show devices for concentrating and testing saliva neutralizing antibodies in a form different from that shown in FIGS. 3 to 7 . FIG. 8 is a longitudinal cross-sectional view of a saliva filtrate collector 26. It has an upper opening 27 and a lower closing portion 28. FIG. 9 shows a state in which a plunger-type saliva collection device 14 is inserted into the filter 1, saliva is filtered, and the saliva filtrate is collected by the saliva filtrate collector 26. Reference numeral 29 denotes the saliva filtrate. FIG. 10 is a longitudinal cross-sectional view of a saliva neutralizing antibody concentrated collector 30. The saliva neutralizing antibody concentrated collector 30 has an upper opening 31 and a lower opening 36 in an empty tube 33. The upper opening 31 may have a pressing portion 32, and the lower opening 36 has a neutralizing antibody concentration column 34 and a gasket 35. FIG. 11 shows a cross-sectional view of the saliva neutralizing antibody concentrated collector 30 inserted into the saliva filtrate collector 26 containing saliva filtrate. Reference numeral 37 denotes the saliva filtrate after passing through the saliva neutralizing antibody concentration collector 30. FIG. 12 is a longitudinal cross-sectional view of the eluate container 38. It has an upper opening 39 and a lower closing opening 41, and reference numeral 40 denotes the eluate. FIG. 13 is a diagram showing the saliva neutralizing antibody concentration collector 30 being removed from the saliva filtrate collector 26 and inserted into the eluate container 38. FIG. 14 is a diagram showing the saliva neutralizing antibody concentration collector 30 inserted into the eluate container 38. Reference numeral 42 denotes the neutralizing antibody concentrate eluted from the neutralizing antibody concentration column 34 of the saliva neutralizing antibody concentration collector 30. The obtained neutralizing antibody concentrate 42 may be directly added as a sample to the opening 44 of a saliva antibody immunochromatography test kit 43. FIG. 15 is a diagram showing an immunochromatography test paper 45 inserted into the neutralizing antibody concentrate 42 obtained in FIG. 14.

[0023] Another aspect of the present invention is an immunochromatographic test method for detecting antigens or antibodies in saliva, characterized in that the sample obtained by the pretreatment step is subjected to an immunochromatographic antigen or antibody test kit. Here, we briefly explain an immunochromatographic test kit using colored latex particles to detect target antibodies. A typical immunochromatographic test kit has a sample application section, a target antigen-labeled colored particle holding section, and an antibody detection section to which the target antigen is immobilized. First, the sample obtained by the pretreatment step is dropped into the sample application section, and the sample is allowed to develop toward the target antigen-labeled colored particle holding section. If the target antibody is present in the sample, the target antibody and the target antigen-labeled colored latex particles react, and the complex is captured in the antibody detection section to which the target antigen is immobilized, resulting in the appearance of a colored band. The color tone of the band, etc., allows the amount of target antibody contained in the sample to be roughly determined.

[0024] Another aspect of the present invention is a kit for testing for immunochromatographic antigens or antibodies in saliva, comprising (a) a porous body capable of retaining viscous components in saliva, (b) a filter with a pore size of 0.1 μm to 10 μm, (c) an antigen or antibody concentrator, and (d) an immunochromatographic antigen or antibody test kit. The (a) porous body capable of retaining viscous components in saliva, (b) a filter with a pore size of 0.1 μm to 10 μm, and (c) an antigen or antibody concentrator may be those described in steps (1), (2), and (3) above. Furthermore, the (d) immunochromatographic antigen or antibody test kit may be the same as the conventional immunochromatographic test kit.

[0025] The immunochromatographic test specimen pretreatment method and immunochromatographic test method for antigens or antibodies in saliva of the present invention are particularly useful for anti-SARS-CoV-2 antibody testing methods using saliva, for which there has not previously been a simple and highly sensitive detection method.

[0026] Next, the present invention will be further explained with reference to examples, but the present invention is not limited to these examples and various applications are possible within the scope of the technical idea of ​​the present invention.

[0027] The abbreviations used in the following examples have the following meanings: Syringe: Terumo disposable syringe 5 mL Filter: Advantec syringe filter (0.80 μm) Nanosep: Nanosep centrifugal filtration device (0.45 μm) Saliva absorber (porous material): swab removed from Salivette (saliva collection kit, Sarstedt Co., Ltd.) Antibody purification kit: spin column type IgG antibody purification kit (Cosmo Bio) APK-10G Antibody measurement kit: Kohjin Bio KBM COVID-19 IgG / IgM (whole blood, plasma, serum)

[0028] Comparative Example 1 With the subject's mouth closed, the subject's gums were massaged for two minutes by pressing a finger against the skin of the upper and lower gums and sliding the finger from side to side. At the same time, the tongue was used to stimulate the gums and other parts of the oral cavity to promote saliva secretion, and 3 mL of saliva was collected into a paper cup. 2 mL of the collected saliva was placed in a syringe equipped with a filter, and pressure was applied with the plunger. The test results showed that the pressure applied to the plunger did not allow the saliva to pass through the filter.

[0029] Example 1: As in Comparative Example 1, 3 mL of saliva was collected in a paper cup and absorbed into a softened swab (saliva absorber). The saliva-absorbed swab was placed in a syringe connected to one or two filters, and filtered by applying pressure with the plunger. The filtrate was collected, and 500 μL of the filtrate was added to a Nanosep filter. The sample was centrifuged at 4000 rpm for 5, 10, and 30 seconds in a small tabletop centrifuge. Viscosity was evaluated using the amount of permeation through the Nanosep filter as an index. The results are shown in Figure 1. The test results showed that saliva filtered twice exhibited higher permeability than saliva filtered once.

[0030] Example 2: Saliva was absorbed into a swab (saliva absorber) in the same manner as in Example 1. The saliva-absorbed swab was placed in a syringe connected to two filters, and filtration was performed by applying pressure with the plunger. The filtrate was collected, and 500 μL of the filtrate was passed through a column in an antibody purification kit and eluted with 50 μL of the buffer solution provided with the kit (10-fold concentrated: theoretical value). 20 μL of the eluate (concentrated sample) was added dropwise to a blood antibody measurement kit and compared with 20 μL of the filtrate (non-concentrated sample). The results are shown in Figure 2. As a result of the test, IgG antibodies were not detected in the non-concentrated sample, but IgG antibodies were detected in the concentrated sample.

[0031] Example 3: After performing a gingival massage similar to that in Comparative Example 1, a plunger-type saliva collection device (Figure 4) with a soft saliva absorber attached to its tip was placed in the oral cavity to collect saliva. The saliva collection device, which had absorbed the saliva, was inserted into a syringe-type filtration and concentration device incorporating a filtration filter and an antibody concentration column, and pressure was applied to the plunger to filter the saliva (Figure 5), while the antibodies in the filtrate were concentrated in the antibody concentration column. The antibody concentration column was removed from the filtration and concentration device and attached to the immunochromatography plate. A tube containing an eluent (e.g., citrate buffer solution) was then attached to the top of the antibody concentration column (Figure 7). Pressure was then applied around the tube to elute the antibodies, and several drops of the eluent were dripped into the sample addition section of the immunochromatography test kit.

[0032] 1: Filter 2: Saliva neutralizing antibody concentrated collection device 3: Saliva filtrate collection device 4: Upper opening of filter 5: Filtration filter 6: Filtration filter 7: Lower opening of filter 8: Upper opening of saliva neutralizing antibody concentrated collection device 9: Neutralizing antibody concentration column 10: Lower opening of saliva neutralizing antibody concentrated collection device 11: Upper opening of saliva filtrate collection section 12: Air hole 13: Lower opening of saliva filtrate collection section 14: Plunger-type saliva collection device 15: Pressing section 16: Syringe rod 17: Gasket 18: Saliva absorber 19: Saliva filtrate 20: Dropper section containing eluate 21: Saliva antibody immunochromatography test kit 22: Dropper bulb containing eluate 23: Lower opening from which eluate comes out 24: Neutralizing antibody sample addition section 25: Saliva antibody immunochromatography test kit 26: Saliva filtrate collector 27: Upper opening of saliva filtrate collector 28: Lower opening of saliva filtrate collector 29: Saliva filtrate 30: Saliva neutralizing antibody concentrated collector 31: Upper opening of saliva neutralizing antibody concentrated collector 32: Pressing part 33: Empty tube 34: Neutralizing antibody concentrated column 35: Gasket 36: Lower opening of saliva neutralizing antibody concentrated collector 37: Saliva filtrate 38: Elution solution container 39: Upper opening 40: Elution solution 41: Lower opening 42: Neutralizing antibody concentrated solution 43: Saliva antibody immunochromatography test kit 44: Opening of kit 45: Immunochromatography test paper

Claims

1. A method for pretreatment of a sample for immunochromatography, characterized by subjecting saliva to the following steps: (1) passing it through a porous material capable of supporting viscous components in the saliva to remove the viscous components; (2) passing the liquid that has passed through the porous material through a filter with a pore diameter of 0.1 μm to 10 μm; and (3) concentrating the antigen or antibody in the liquid that has passed through the filter.

2. The pretreatment method according to claim 1, wherein the porous body is a porous body having voids that allow low-viscosity components in saliva to pass through but not mucin when pressed with a pressure of 1000 kPa or less.

3. The pretreatment method according to claim 1, wherein the porous body is a porous body having pores with a diameter of 10 μm to 3000 μm.

4. The pretreatment method according to claim 1, wherein the porous body is disposed at the tip of the saliva collection device.

5. The pretreatment method according to claim 1, wherein the filter is a filter with 1 to 4 layers.

6. The pretreatment method according to claim 1, wherein the antigen or antibody concentration step is affinity chromatography of the antigen or antibody.

7. The pretreatment method according to claim 1, wherein the target antigen or target antibody is SARS-CoV-2 or SARS-CoV-2 antibody.

8. An immunochromatographic test method for antigens or antibodies in saliva, characterized by applying a sample obtained by a pretreatment step according to any one of claims 1 to 7 to an immunochromatographic antigen or antibody test kit.

9. A sample pretreatment kit for immunochromatographic testing, characterized by comprising: (a) a porous body capable of supporting viscous components in saliva; (b) a filter with a pore diameter of 0.1 μm to 10 μm; and (c) an antigen or antibody concentration device.

10. The pretreatment kit according to claim 9, wherein the porous body has voids that allow low-viscosity components in saliva to pass through but not mucin when pressed with a pressure of 1000 kPa or less.

11. The pretreatment kit according to claim 9, wherein the porous body is a porous body having pores with a diameter of 10 μm to 3000 μm.

12. The pretreatment kit according to claim 9, wherein the porous body is disposed at the tip of the saliva collection device.

13. The pretreatment kit according to claim 9, wherein the filter is a filter with 1 to 4 layers.

14. The pretreatment kit according to claim 9, wherein the antigen or antibody concentration device is an antigen or antibody affinity chromatography device.

15. The pretreatment kit according to claim 9, wherein the target antigen or target antibody is SARS-CoV-2 or SARS-CoV-2 antibody.

16. A kit for testing immunochromatographic antigens or antibodies in saliva, comprising a pretreatment kit according to any one of claims 9 to 15, and an immunochromatographic antigen or antibody test kit.