JPWO2013073558A1 - Liquid sample inspection method, sample extraction nozzle, sample extraction container, and liquid sample inspection kit - Google Patents

Abstract

The present invention provides a liquid sample inspection tool having a chromatographic medium to which a substance that captures an object to be detected is bound after the liquid sample prepared from the sample is filtered by a filtration filter. In the liquid sample inspection method for detection or quantification, the filtration filter includes a glass fiber filter having a pore size of 2.5 μm or more and a resin filter made of an organic polymer foamable resin material. About. According to the present invention, an object to be detected in a specimen can be accurately detected by an immunoassay method without causing poor development.

Description

  The present invention relates to a liquid sample inspection method using an immunoassay method, a sample extraction nozzle, a sample extraction container, and a liquid sample inspection kit. This application claims priority on November 14, 2011 based on Japanese Patent Application No. 2011-249130 for which it applied to Japan, and uses the content here.

2. Description of the Related Art Conventionally, simple test reagents or kits have been provided that can perform various tests such as infection of pathogens such as bacteria and viruses, presence of pregnancy, and measurement of blood glucose levels in a short time of several minutes to tens of minutes. As a test method using these test kits, an immunoassay method is widely used because it is simpler and can be detected in a shorter time.
However, when testing by immunoassay, in the analysis of the sample actually collected from the patient, the detected object may be determined to be positive even though it is not present in the sample (so-called false positive). May be determined to be negative even though it is present in the sample (so-called false negative). Such misjudgment delays the identification of the cause and may impede early treatment and early treatment. Therefore, a filtration filter that suppresses misjudgment and an assay kit having the filtration filter have been developed.

  The misjudgment is that non-specific reactions occur as a result of contaminants contained in the sample added on the chromatographic medium of the assay device blocking the pores in the chromatographic medium and inhibiting the movement of components in the solution. It is thought to occur by what happens. Therefore, it can be expected that erroneous determination can be suppressed by filtering in advance a liquid sample prepared by diluting the specimen with an appropriate buffer. In addition, when the sample dropped on the chromatographic medium is not sufficiently developed, the chromatographic medium is broadly stained, and it may be difficult to determine whether it is positive or negative. Therefore, by diluting the specimen with an appropriate buffer and filtering the obtained liquid sample, contaminants contained in the specimen can be removed in advance to reduce such development failure.

In the filtration of a liquid sample, in order to prevent clogging and to prevent a decrease in detection sensitivity of an object to be detected due to an increase in buffer solution, in Patent Document 1, the occurrence of clogging is particularly caused by deformation of filter holes or pores. Focusing on the fact that the filter is based on the above, a strong rigidity filter is used for the filtration tube.
In Patent Document 1, a liquid sample obtained by extracting a specimen into a specimen extract is placed in a filtration tube, filtered through a filtration filter attached to the tip, and filtered when the filtrate is dropped onto a chromatographic medium in an immunoassay device. As the filter, a high-rigidity filter having rigidity that is difficult to be crushed in the thickness direction of the filter or difficult to deform such as a curve is adopted. More specifically, even when the pressure applied to the filter by liquid or air when filtering a liquid sample exceeds 0.2 MPa, the amount of change in thickness before filtration versus filtration is not more than 5%, preferably Is not more than 2%, and has rigidity that is difficult to deform such as bending, a filter that does not have a radius of curvature of the filter of 2 cm or less during filtration even when the same pressure as described above is applied. .

JP 2009-36781 A

  In the method described in Patent Document 1, there is a description about the reduction of false positives, but about improvement when it is difficult to judge positive or negative as a result of chromatographic media being stained broadly due to poor chromatographic development. There is no description at all.

  Therefore, the present invention provides a sample extraction nozzle, a sample extraction container, a liquid sample test kit including the sample extraction nozzle, and a liquid sample test kit that can be easily and cost-effectively prevented from developing defects and having good operability even during testing. A liquid sample inspection method using a sample extraction nozzle, a sample extraction container, or a liquid sample inspection kit is provided.

The present invention relates to the following.
[1] After the liquid sample with the sample prepared is filtered by a filtration filter, the detected object in the liquid sample is detected on a liquid sample inspection tool having a chromatographic medium combined with a substance that captures the detected object. Alternatively, in the liquid sample inspection method for quantification, the filtration filter includes a glass fiber filter having a pore diameter of 2.5 μm or more and a resin filter made of an organic polymer foamable resin material.
[2] A method for detecting or quantifying the object to be detected in the liquid sample specifically binds a solution containing the object to be detected and a labeling substance that specifically binds to the object to be detected to the object to be detected. The liquid sample inspection method according to [1], wherein the chromatographic medium coated with the capture reagent is passed by a vertical method or developed in a horizontal direction.
[3] The liquid sample inspection method according to [1] or [2], wherein the chromatographic medium has an average pore size of 0.5 to 50 μm, preferably 10 to 30 μm.
[4] The liquid sample inspection method according to any one of [1] to [3], wherein the resin filter has a porosity of 70% or more.
[5] The liquid sample inspection method according to any one of [1] to [4], wherein the resin filter is formed using polyvinyl alcohol.
[6] The liquid sample inspection method according to [1] to [5], wherein the detected object is an influenza virus, adenovirus, pneumonia mycoplasma or norovirus, or a protein derived from these pathogenic microorganisms or an antibody against them.
[7] In a sample extraction nozzle for filtering a liquid sample containing a sample through a filter and dropping the sample on a liquid sample inspection tool, the filter includes a glass fiber filter having a pore size of 2.5 μm or more, and foaming of an organic polymer Sample extraction nozzle provided with a resin filter made of a conductive resin material.
[8] The specimen extraction nozzle is
A combined portion that is liquid-tightly contained in the open end of the sample extraction container body;
A flange engaged with an edge of the opening end,
A nozzle part that is made to flow while reducing the amount of the liquid sample so that the liquid sample can be suitably dropped, and
The specimen extraction nozzle according to [7], wherein the filtration filter is provided in the joint portion.
[9] The specimen extraction nozzle according to [7] or [8], wherein the resin filter has a porosity of 70% or more.
[10] The sample extraction nozzle according to any one of [7] to [9], wherein polyvinyl alcohol is used as the organic polymer.
[11] A sample extraction container comprising the sample extraction nozzle according to any one of [7] to [10] and a container main body to which the sample extraction nozzle is attached.
[12] The container body has a bottomed cylindrical shape in which one end side of the container body is open and the other end is closed, and the opening end portion is configured to be covered with a lid. Specimen extraction container.
[13] The specimen extraction container according to [12], wherein a male screw portion is formed on an outer peripheral surface of the opening end portion, and a flange portion projecting outward is provided in the vicinity of a terminal end side of the male screw portion. .
[14] The lid includes a circular top plate portion and a side wall portion that rises from an outer edge of the top plate portion, and a female screw that can be screwed into the male screw portion of the container body on the inner wall of the side wall portion. The specimen extraction container according to [12] or [13], wherein a portion is formed.
[15] A liquid sample test comprising a liquid sample test tool having a chromatographic medium combined with a substance that captures the object to be detected, and the specimen extraction container according to any one of [11] to [14]. kit.

  According to the present invention, there is an effect that an object to be detected in a specimen can be accurately detected by an immunoassay method without causing poor development. In addition, there is also an effect that the inspection tool can be manufactured easily and at low cost even during inspection.

It is sectional drawing which showed the liquid sample test | inspection tool of the liquid sample test kit shown as one Embodiment of this invention, Comprising: The same figure (a)-(c) showed each process by which a liquid sample is test | inspected. It is. It is the side view which showed the container main body of the liquid sample test kit shown as one Embodiment of this invention, and its cover in the cross sectional view. It is a side view of the sample extraction nozzle of the liquid sample test kit shown as one embodiment of the present invention. It is a bottom view of the sample extraction nozzle of the liquid sample test kit shown as one embodiment of the present invention. It is the perspective view which showed typically the filtration filter of the liquid sample test | inspection kit shown as one Embodiment of this invention. It is the figure which showed the dyeing | staining result on the membrane after dripping and developing a liquid sample to the liquid sample test | inspection tool using the extraction nozzle of Example 1 and Comparative Examples 1-5. It is the figure which showed an example of the division | segmentation method of a membrane in the case of determining whether development is unsatisfactory using a RGB ratio.

Hereinafter, a liquid sample inspection method and a liquid sample inspection kit according to an embodiment of the present invention will be described in detail.
The method of the present invention is a so-called immunoassay method in which an object to be detected in a liquid sample is detected using a liquid sample inspection tool (so-called test strip) having a chromatographic medium to which a capture substance for capturing the object to be detected is bound. A liquid sample inspection method for detecting or quantifying an object to be detected in the liquid sample on the liquid sample inspection tool after the liquid sample prepared for the specimen is filtered by a filtration filter. It is a method characterized by filtering through a filter comprising a glass fiber filter having a pore diameter of 5 μm or more and a resin filter made of an organic polymer foamable resin material.

  This immunoassay method generally uses a measurement principle collectively referred to as a so-called sandwich method, and specifically binds to a detection target, a capture substance that specifically binds to the detection target, and a detection target. In this method, three types of complexes of the labeling substance are formed on the chromatographic medium, the labeling substance is detected, and the complex, that is, the detected substance is detected or quantified. Examples of the reaction between the detection target, the capture substance, and the labeling substance include an antigen-antibody reaction and a binding reaction between a ligand and a receptor.

  The liquid sample inspection kit of the present invention using the above method includes a liquid sample inspection tool 1 having a chromatographic medium 5 to which a substance Q for capturing an object to be detected shown in FIG. 1, and FIGS. And a sample extraction container 2 equipped with a sample extraction nozzle 9 for filtering the liquid sample and dropping it onto the liquid sample inspection instrument 1 shown in FIG. 4 having a pore diameter of 2.5 μm or more, and a resin filter 3a made of an organic polymer foamable resin material (hereinafter sometimes referred to as “foamable resin filter”) 3b. It is configured with.

  FIG. 1 is a cross-sectional view of a liquid sample inspection tool 1 as viewed from a direction orthogonal to the longitudinal direction. FIGS. 1A to 1C are diagrams showing each step of inspecting an object P to be detected by the liquid sample inspection tool 1. Is schematically shown. As shown in FIG. 1A, the liquid sample inspection tool 1 includes a sample pad 4 for dropping a liquid sample S, which is a prepared specimen, in order from one end side on a substrate formed on a strip. A chromatographic medium 5 to which a capture substance Q such as an antibody that specifically binds to the detected substance P is bound, and an absorption pad 6 are arranged, and a conjugate pad 7 interposed between the sample pad 4 and the chromatographic medium 5 is further provided. It is prepared for. Here, the liquid sample inspection tool 1 includes a back side piece and a front side piece having a determination display window through which the inspection result can be visually recognized. The sample pad 4 and the chromatographic medium 5 are attached to a rod-like container assembled by fitting them together. The absorbent pad 6 and the conjugate pad 7 can be accommodated and used. Moreover, the back side piece and the front side piece may be integrally formed. As the container, a known material such as a resin material such as a plastic or an elastomer can be used, and can be appropriately selected depending on the purpose, such as transparency, opacity, moldability, and strength.

As an immunoassay method, a flow-through method and an immunochromatography method (lateral flow method) are known. In the flow-through method, a solution containing the detection target P and a labeling substance that specifically binds to the detection target are perpendicular to the chromatographic medium 5 coated with a capture reagent that specifically binds to the detection target P. The detection substance is detected or quantified by forming a complex of the capture substance Q, the detection object P, and the labeling substance that pass through in the direction and specifically binding to the detection object P on the film, and detecting or quantifying the label. Perform detection or quantification. The immunochromatography method uses the same chromatographic medium 5 and the principle of detecting the detection object P is the same as that of the flow-through method. However, the liquid sample S containing the detection object P is horizontally oriented with respect to the chromatography medium 5. It differs from the flow-through method in that it is deployed. Of the two methods, the immunochromatography method is more preferable from the viewpoint of simplicity and speed.
In the immunochromatography method, a labeling substance, which is a detection reagent that specifically binds to the detected substance P, is added onto the chromatographic medium 5 by dropping or the like to form a complex of the capture reagent, the detected object and the labeled substance. And a method in which a conjugate pad 7 containing a labeled compound bonded to a capture substance Q such as an antibody that specifically binds to the detection object P is incorporated in the liquid sample inspection tool 1 in advance.
As such a liquid sample inspection tool 1, it is preferable to use a method generally known as an immunochromatography method from the viewpoint of eliminating development defects.

In the liquid sample inspection according to the present specification, whether or not the development of the used liquid sample in the detection region of the membrane (chromatography medium) is defective can be confirmed by the following visual method.
(Visual judgment method)
After the liquid sample containing the object to be detected is developed in the membrane of the chromatographic medium, it is visually confirmed whether or not the target sample on the membrane is developed to the region where the target band is detected (detection region).

  When visually confirming whether or not the development is poor, it is determined that the development is good when the target band is detected and the other regions on the membrane are hardly stained. On the other hand, if the target band on the membrane is darkly stained up to the area where the target band is detected, and if the target band detection area is not expanded, the entire membrane is stained and the target band is detected. If the vicinity of the area where the band is detected is dark and broadly stained, it is determined that the development is poor.

  Whether or not the development determined by visual observation is defective can be confirmed by, for example, quantifying using the following RGB ratio.

(Judgment method using RGB ratio)
Of the regions on the membrane, for one or more regions that affect the visibility of the band, the color of each region is extracted, the RGB value is analyzed, and the RGB ratio is calculated.
A case where it is confirmed whether or not the development is defective by calculating the RGB ratio will be specifically described with reference to FIG.
First, as shown in FIG. 6, the membrane 26 is divided into one or more regions including bands (three regions 7 to 9 in FIG. 6) and other regions.
In addition, as the membrane dividing method, at least one location including a band is set, and the number of divisions can be appropriately set according to the size of the detection region of the filter membrane.
Next, the color of each area | region is extracted about the area | region (3 areas of 7-9 in FIG. 6) which influences the visibility of a band, and an RGB value is analyzed.
The RGB value of each region may be measured using a commercially available RGB measuring device or software. For example, a device such as a color analyzer RGB-1002 (manufactured by Sato Corporation) or general-purpose paint software (Microsoft Paint) (Microsoft), Photoshop (Adobe), Corel Painter (Corel, etc.) can be used. When the RGB measuring apparatus is not used, the image of the determination portion can be taken into a PC and the RGB value can be calculated using image analysis software.
The RGB ratio is obtained from the RGB value of each region measured above.
The RGB ratio can be obtained by R value− (average value of G value and B value).
The lower the RGB ratio is, the closer the color is to white, so it is considered that the development is better. Further, the higher the RGB ratio is, the closer the color is to red, so the development is considered to be poor. In the liquid sample inspection method according to the present invention, when the RGB ratio was 15 or less, it was judged that the development was good, and when it was larger than 15, the development was judged as poor.

  Examples of the chromatographic medium 5 include porous materials such as paper, glass fiber filter paper, and nitrocellulose. Particularly preferred raw materials include nitrocellulose. The average pore diameter of the chromatographic medium 5 is often 0.5 to 50 μm, preferably 10 to 30 μm.

The sample extraction container 2 includes a container main body 10 that stores a liquid sample S obtained by extracting a sample from the sample extract W shown in FIG. 2, and a sample extraction nozzle 9 shown in FIGS. It is equipped with.
As shown in FIG. 2, the container body 10 is formed in a bottomed cylindrical shape that is open at one end side and closed at the other end, and the lid 11 can be covered with the opening end portion 10 a.

  The container body 10 is formed of a synthetic resin. A male screw portion 12 is formed on the outer peripheral surface of the opening end portion 10a, and the container main body 10 is formed outwardly in the vicinity of the terminal end side (the container main body 10 bottom side). It has a flange portion 13 projecting over the surface. In consideration of the operation of dropping the specimen extract W from the specimen extraction container 2 to the liquid sample testing instrument 1 during the examination, it is preferable that the container body 10 includes an elastically deformable material. . The elastically deformable material is a material having elasticity and flexibility, and any material having chemical resistance and moldability is preferable, and a material not accompanied by odor is more preferable. Specifically, a thermoplastic resin, a silicone resin, a thermoplastic elastomer, etc. are illustrated. These resins may be combined. In the case of a thermoplastic resin, for example, in the case of an olefin resin, it is known that moldability is improved by combining with a thermoplastic resin elastomer and a hydrogenated styrene thermoplastic resin elastomer. Examples of the thermoplastic resin include polyethylene such as high density polyethylene (HDPE), low density polyethylene (LDPE), and metallocene-catalyzed linear low density polyethylene (LLDPE), polypropylene, polyvinyl chloride, polystyrene, EVA, and the like. These may be a polymer blend or polymer alloy in which two or more types are combined.

  The lid 11 has a circular top plate portion 11a and a side wall portion 11b that rises from the outer edge of the top plate portion 11a, and a female screw that can be screwed into the male screw portion 13 of the container body 10 on the inner wall of the side wall portion 11b. A portion 14 is formed, and the lid 11 is detachable from the container body 10.

  As shown in FIG. 3A, the sample extraction nozzle 9 has its base end side 9a fitted into the open end 10a of the container body 10, and the liquid sample S containing the sample is dropped from the nozzle tip 9b side. Is. The sample extraction nozzle 9 includes a fitting portion 15 that is liquid-tightly fitted in the opening end portion 10a of the container body 10, a flange 16 that is engaged with the edge of the opening end portion 10a, and an amount of the liquid sample S. The nozzle part 17 is made to flow while being squeezed so that the liquid sample S can be suitably dropped, and the filter 3 is provided in the fitting part 15.

The inner diameter 17a of the nozzle portion 17 is formed to be slightly smaller than the inner diameter 15a of the fitting portion 15 and has a narrow step portion 19 between the fitting portion 15 and the filtration filter 3 is fitted. The peripheral edge 18 of the filtration filter 3 can be locked to the stepped portion 19 when arranged in the portion 15.
As shown in FIGS. 3A and 3B, the inner hole of the nozzle portion 17 is formed in a substantially circular cross section, and the first tapered portion 20 and the first tapered portion 20 that are gradually reduced in diameter from the step portion 19 toward the tip. And the second taper portion 21 formed so that the inner wall gradually expands toward the tip of the nozzle portion 17 on the tip side of the first taper portion 20 and the wall surface of the first taper portion 20 and the second taper portion 21, The ridges 22A and 22B respectively formed from the step portion 19 toward the tip of the nozzle portion 17 are annularly arranged at equal intervals.

As shown in FIG. 4, the outer shape of the filtration filter 3 is formed in a disk shape by laminating a glass fiber filter 3a having a pore diameter of at least 2.5 μm or more and a foamable resin filter 3b. It is slightly larger than the inner diameter of the fitting portion 15 of the specimen extraction nozzle 9 shown in FIG. 3A. Further, in the filtration filter 3, the foamable resin filter 3b is disposed on the distal end side, the proximal end side, or both of the specimen extraction nozzle 9 rather than the glass fiber filter 3a, and protects the glass fiber filter 3a and is liquid. The sample is filtered.
Further, the sample such as the nasal swab taken in the sample extract W in the container body 10 contains components such as cells and secretions detached from the sample collection site such as the nasal cavity. Are formed with a large number of pores 25 having a predetermined pore diameter, and an intended object P to be detected can be prevented so as to prevent erroneous determination in the chromatographic medium 5 of the liquid sample inspection tool 1 shown in FIG. It can be suitably filtered.

The filtration filter 3 may be produced by combining three or more types of filters.
When combining several filtration filters 3 with different pore diameters or retention particle diameters, a filter with a large pore diameter or retention particle diameter is on the upstream side (container body 10 side), and a small filter is on the downstream side (nozzle part 17 side). In order to avoid clogging of the liquid sample S at the time of filtration, it is preferable to arrange the two filters so as to overlap each other. For example, when two filters are combined, it is preferable to use a filter having a large pore size or retained particle size as the first-stage filter and a filter having a small pore diameter as the second-stage filter.

  The minimum pore size or the retained particle size of the filter having the minimum pore size or the retained particle size among the filters constituting the filtration filter 3 is preferably equal to or smaller than the pore size of the chromatographic medium 5. That is, for example, when the pore diameter of the chromatographic medium 5 to be used is 10 μm or more, it is preferable that at least one of the filtration filters 3 has a pore diameter of the pore 25 of 10 μm or less.

  The filtration filter 3 can be used for both fine filtration and coarse filtration. However, the pore diameter or the retained particle diameter of at least one kind of filtration filter 3 is equal to or smaller than the pore diameter of the chromatographic medium 5 and is 10 μm or less. preferable.

In the present invention and the specification of the present application, the “pore diameter” means the largest pore among the pores 25 of the filter, and can be measured by, for example, a bubble point method. Further, the “retained particle diameter” means the maximum value of the diameter of the particles passing through the filter, which is leaked when, for example, barium sulfate defined in JIS 3801 and JIS Z 8901 is naturally filtered. By calculating the particle retention rate from the absorbance of the solution before and after filtration, by obtaining the maximum value of the particle size or by naturally filtering latex particles with a fixed particle size diluted with ultra-pure water (Milli-Q Water) Can be sought. The retained particle diameter substantially corresponds to the pore diameter of the filtration filter.
The porosity can also be referred to as a porosity, and is measured by a fully automatic pore size distribution measuring device (mercury porosimeter).

  Each thickness of the glass fiber filter 3a and the foamable resin filter 3b may be 0.05 mm to 35 mm, preferably 0.1 mm to 10 mm, and more preferably 1 mm to 5 mm. When the filtration filter 3 is produced by combining two or more types of flexible filters, the thickness of the entire filter is preferably 0.1 to 50 mm, more preferably 0.5 to 30 mm, and even more preferably 1 to 10 mm. The thicknesses of the glass fiber filter 3a and the foamable resin filter 3b are not limited to those having the ratio shown in FIG.

  The filtration filter 3 used in the present invention includes a glass fiber filter 3a and a foamable resin filter 3b. When the filtration filter 3 is formed of the glass fiber filter 3a and the foamable resin filter 3b, the glass fiber filter 3a constituting the filtration filter 3 may be a single sheet, of the same type or different types. Two or more glass fiber filters 3a may be combined. Similarly, the foamable resin filter 3b constituting the filtration filter 3 may be one, or two or more of the same or different types of foamable resin filters 3b may be combined.

  The glass fiber filter 3a is obtained by stacking glass fibers so that the pore diameter or the retained particle diameter is 2.5 μm or more, preferably 2.5 to 3.5 μm. Since the glass fiber filter 3a used in the present invention has a relatively large pore diameter or retained particle diameter, it has flexibility. Although there is no restriction | limiting in particular in the thickness of the glass fiber filter 3a with which the filtration filter 3 is provided, It is preferable that it is 200 micrometers-1500 micrometers from the point of filterability and a softness | flexibility.

  The porosity of the glass fiber filter 3a used in the present invention is preferably in the range of 40% to 90%. When the porosity of the glass fiber filter 3a is 40% or more, clogging is hardly caused by foreign matters in the liquid sample, and when the specimen is a biological sample, the biological sample is adsorbed. It becomes difficult to clog, and even when continuous measurement is performed, an increase in the filtration pressure of the filter due to clogging can be suppressed. Further, when the porosity of the glass fiber filter 3a is 90% or less, excessive diffusion of the liquid sample in the filter can be suppressed, and more accurate measurement can be performed. In addition, since the filter is provided with an appropriate strength, it is possible to suppress damage due to the pressure of the liquid mobile phase during handling or measurement of the filter.

  The foamable resin filter 3b has a function of protecting the glass fiber filter 3a and preventing it from being damaged by filtration pressure. For this reason, the foamable resin filter 3b does not need to cover the entire surface of the glass fiber filter 3a, and may be laminated so as to cover only a part of the glass fiber filter 3a, such as an O-ring shape. In addition, since the foamable resin filter 3b can function also as a filtration filter, it is preferable to form it so that the whole surface of the filter 3a made from glass fiber may be covered.

  The foamable resin filter 3b preferably has a relatively high flexibility. Therefore, the porosity of the foamable resin filter 3b is preferably 70% or more, more preferably 70 to 98%, still more preferably 80% to 98%, still more preferably 85% to 98%, and 85% to 95. % Is particularly preferred. Moreover, 50-800 micrometers is preferable, as for the hole diameter of the foamable resin filter 3b used by this invention, or a retention particle diameter, 50-300 micrometers is more preferable, and 50-100 micrometers is more preferable.

The foamable resin filter 3b is preferably made of a foam of one or more organic polymers selected from the group consisting of polyamide-based polymers, polyolefin-based polymers, and polyvinyl-based polymers. Specifically, polyamide, aramid, polycarbonate, polyacetal, polyester, polyphenylene ether, polyphenylene sulfide, polyether ether ketone, polyallyl ether ketone, polyamide imide, polyimide, polyether imide, polysulfone, polyether sulfone, polyethylene, polypropylene, Polymethylpentene, polystyrene, polyvinyl chloride, polymethyl methacrylate, cellulose acetate, polymethylpentene, polybutene, polycarbonate, polyarylate, polyethylene oxide, polyparamethylstyrene, polyallylamine, polyvinyl alcohol, polyvinyl acetal, polyvinyl ether, polyvinyl Butyral, polyvinyl formal, polybutylene terephthalate, polyester Terephthalate, acrylic resin, methacrylic resin, xylene resin, amino resin, guanamine resin, melamine resin, urea resin, unsaturated polyester resin, vinyl ester resin, diallyl phthalate resin, epoxy resin, polyurethane, silicon resin, polyimide, alkyd resin, Examples include one obtained by molding one or a mixture of two or more selected from the group consisting of furan resin, phenol resin, fluororesin, poly-p-hydroxybenzoic acid, and maleic acid resin into a foam.
In the present invention, it is preferable to use a filter made of a foamable resin composed of a polyvinyl foamable resin, particularly a polyvinyl alcohol such as polyvinyl alcohol (PVA), or a polyvinyl acetal.

When a rigid filter is used as the filtration filter 3 as in the immunoassay kit described in Patent Document 1, the filtration filter 3 is fitted when fitted in the fitting portion 15 formed in the sample extraction nozzle 9. It does not elastically deform according to the shape in the joint portion 15. Therefore, in order to reliably filter the liquid sample, the size of the rigid filter and the size in the fitting portion 15 must be matched with high precision so that the strong filter is closely fitted in the fitting portion 15. However, in the above-described high-rigidity filter, a physical dimensional error is likely to occur between the fitting portion 15 and the high-rigidity filter, so that the thickness of the strong-rigid filter needs to be increased. There was a problem that it took time.
Further, even if a strong rigidity filter is manufactured by substantially eliminating the physical dimensional error between the fitting portion 15 and the strong rigidity filter, the strong rigidity filter has a strong rigidity in the fitting portion 15 because of its hardness. There is a problem that it is difficult to insert and arrange the filter, and it is difficult to manufacture the specimen extraction nozzle 9 appropriately.
On the other hand, even during the test operation, when pre-processing the sample solution, the pressure is applied too much and the operability may be reduced, or the filter may come off. There were also issues to be addressed.

On the other hand, in the present invention, the filtration filter 3 in which the glass fiber filter 3a and the foamable resin filter 3b are laminated has flexibility, so that the fitting portion 15 has a structure as shown in FIG. 3A. 4 is elastically deformed (contracted) in accordance with the inner diameter of the fitting portion 15 when being inserted and arranged, and is elastically restored in a state where the flat plate surface 3t of the filtration filter 3 shown in FIG. Thus, the peripheral surface 23 of the filtration filter 3 is fitted to the inner wall surface 24 of the fitting portion 15 while being urged.
That is, since the filtration filter 3 of the sample extraction container 2 has flexibility, it can be placed in the sample extraction container 2 while being fitted. Therefore, it is possible to easily arrange the filtration filter 3 in the fitting portion 15 without the process of matching the dimensions of the filtration filter 3 and the dimensions in the fitting portion 15 with high precision, and the specimen extraction container 2 or liquid The manufacturing cost of the sample inspection kit can be reduced.
Further, since a physical dimensional error hardly occurs between the fitting portion 15 and the filtration filter 3, the liquid sample S can be surely passed through the filtration filter 3. Therefore, it is possible to reliably place the filter 3 while making the filter 3 as thin as possible, and the liquid sample S can be reliably filtered in a short time.

  Examples of the detection object P include influenza viruses, RS viruses, adenoviruses, pathogenic microorganisms such as diarrhea, pathogenic microorganisms such as pneumonia mycoplasma, rotavirus and norovirus, or substances such as proteins derived from the pathogenic microorganisms, or antibodies thereto. Examples include physiologically active substances, toxins produced by bacteria, and various tumor markers. When using the above-mentioned pathogenic microorganisms or substances such as proteins derived from the pathogenic microorganisms or antibodies against them, there is a possibility of exhibiting acute symptoms such as influenza virus, adenovirus, pneumonia mycoplasma and norovirus. It is extremely useful for diagnosis of pathogens that need to be identified in a short time.

Specimens include nasal wipes, nasal aspirates, nasal discharge, throat wipes, saliva, sputum mucosal secretions, stool suspension, urine, blood components, organ extracts, various tissue extracts, or Examples include a diluted solution obtained by diluting these with an appropriate buffer.
It is also possible to use a non-biological sample as a specimen. When a biological sample is used as a specimen, the present invention is extremely useful when a nasal wiping liquid, nasal suction liquid, nasal irrigation liquid, pharyngeal wiping liquid, or stool suspension is used as a specimen.

  The capture substance Q for capturing the detection target P is a substance that binds to the detection target P by a specific reaction to form a complex. Therefore, generally, when the detected substance P is a bacterium, a hormone, a virus or the like and a substance derived therefrom, or other biomarkers, a polyclonal antibody or a monoclonal antibody that specifically binds to these can be used. As a method for binding such a capturing substance Q to the above-mentioned chromatographic medium surface, a known method for immobilizing a protein on a nitrocellulose membrane or the like can be used. The chromatographic medium 5 to which the capture substance Q is bound is prepared, for example, by adsorbing a solution obtained by diluting the capture substance Q in a buffer solution or the like and then drying the solution. The trapping substance Q may be fixed in a spot shape or a line shape, for example.

  The labeling substance is a substance that specifically binds to an object to be detected and forms a complex so that it can be detected by some means. Generally, the same ones as described for the capture reagent are labeled with an enzyme, a noble metal colloid or the like. For example, when the detection object P is an antigenic substance such as a virus, an antibody against the virus, which is labeled with a noble metal colloid or the like, can be used. Specific examples of the labeling substance include enzymes, fluorescent labels, radioisotopes, noble metal colloids, colored latex and the like. The labeling substance may be added to the liquid sample tester 1 after adding the liquid sample to the liquid sample tester 1 or may be incorporated in the liquid sample tester 1 in advance. When incorporated in the liquid sample inspection tool 1 in advance, the labeling substance is incorporated in a state of being included in the pad in a form called a conjugate pad 7. For the pad containing the labeling substance, for example, a nonwoven fabric made of cellulose, glass fiber or the like is used.

  The sample extract W is a solution for extracting and suspending a detection object from a sample that may contain the detection object collected from a patient or the environment. An extract obtained by extracting or suspending the sample with the sample extract W is added as a liquid sample to the liquid sample test tool 1 by dropping or the like, and provided to the liquid sample test tool 1.

  A sample collected from a patient or the environment may contain a substance that affects the salt composition or pH of the sample extract W. When the sample extract W whose properties have changed is used, there is a possibility that the detection of the detection object may be adversely affected. Therefore, the sample extract W usually has a buffer for maintaining a constant salt and pH. included. As the buffer solution, a buffer solution or the like usually used in an immunological test can be used, and examples include a Tris buffer solution, a phosphate buffer solution, and a Good buffer solution. Furthermore, the sample extract W can contain a surfactant for the purpose of preventing non-specific reactions within a range that does not inhibit specific agglutination reactions. The surfactant is not particularly limited, and a known surfactant can be used. Specific examples include Briji 35, dodecyl-β-D-maltoside, octyl-β-D-glucoside, Triton X-100, CHAPS, Zwittergent 3-12, sodium dodecyl sulfate, Tween 20, Tween 80, Nonidet P40 and the like. These may be used in combination of two or more.

  For the purpose of preventing non-specific reactions, the sample extract W can also contain proteins such as bovine serum albumin, immunoglobulin and casein, amino acids such as glycine, and serum such as rabbits and mice. is there.

Next, the usage method and action of the liquid sample test kit will be described using a virus diagnostic kit for diagnosing influenza virus infection as an example.
When examining the presence or absence of influenza virus using a liquid sample test kit, first, for example, a cotton swab is used as a specimen collecting device (not shown), and the swab is inserted into the nasal cavity and the mucous membrane is rubbed several times. Collect the epidermis.

  In accordance with the collection of the mucosal epidermis, the lid 11 shown in FIG. 2 is removed from the container body 10, and the cotton swab from which the sample is collected is immersed in the sample extract W in the container body 10. Then, the cotton swab is pinched or moved up and down several times from the outside of the container body 10 to sufficiently infiltrate the cotton swab into the specimen extract W, and then the cotton swab is pulled out from the container body 10 so that the specimen extract W is squeezed.

After that, as shown in FIG. 3A, the fitting portion 15 of the sample extraction nozzle 9 is fitted into the opening end portion 10a of the container main body 10 to obtain a sample extraction container 2 in which a liquid sample can be dropped.
Then, when the intermediate portion (body portion) 10b of the container main body 10 shown in FIG. 2 is held with the nozzle tip 9b side of the sample extraction container 2 facing downward, the liquid sample flows from the container main body 10 toward the sample extraction nozzle 9. And passes through the filtration filter 3 installed in the fitting portion 15.

  In this way, the liquid sample S is filtered by the filtration filter 3 and moves to the tip of the nozzle portion 17 of the specimen extraction nozzle 9. Therefore, the liquid sample S is further grasped by strongly gripping the body portion 10b of the container body 10. It can be dripped on the sample pad 4 of the liquid sample inspection tool 1 shown to 1 (a).

  As shown in FIG. 1A, when the liquid sample S is added to the sample pad 4, the liquid sample S penetrates into the conjugate pad 7 and penetrates into the pad 7 as shown in FIG. While reacting with the supported specific antibody, it permeates the chromatographic medium 5 as shown in FIG. When an antigen that is the detection target P exists in the liquid sample S, an antigen-antibody reaction occurs between the antigen and the antibody fixed to the chromatographic medium 5, and a colored label appears on the chromatographic medium 5. Therefore, the inspection result can be known by visually recognizing this colored marker in the determination region of the chromatographic medium 5. Further, the excess liquid sample S that has permeated the chromatographic medium 5 is absorbed by the absorption pad 6. At this time, since the absorption pad 6 is colored, it is possible to confirm the excess or deficiency of the liquid sample by visually checking the degree of coloring in the liquid amount confirmation region.

  As described above, according to the sample extraction container 2 and the liquid sample test kit of the present invention, since the filtration filter 3 has flexibility, the filtration filter 3 is placed in the fitting portion 15 of the sample extraction nozzle 9. It can be placed in a fit. Therefore, it is possible to easily dispose the filtration filter 3 in the fitting portion 15 without the step of matching the diameter size of the filtration filter 3 and the inner diameter size of the fitting portion 15 with high accuracy. The effect that the manufacturing cost of the sample inspection kit can be suppressed is obtained.

  Further, the filtration filter 3 can be arranged in a state of being urged against the inner wall of the fitting portion 15, and a physical dimensional error is unlikely to occur between the fitting portion 15 of the sample extraction nozzle 9 and the filtration filter 3. Therefore, the liquid sample S can be reliably filtered by the filtration filter 3. Therefore, the effect that the liquid sample S can be reliably filtered in a short time while the filter 3 is made as thin as possible is obtained.

  Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. However, the present invention is not limited by the following description.

[Example 1, Comparative Examples 1 to 5]
(Test method)
Extraction nozzles of Example 1 and Comparative Examples 1 to 5 were prepared using the filter types and pore size filters shown in Table 1 as filtration filters. In addition, about the extraction nozzle of Example 1, in order of a glass fiber filter and a PVA filter from the dripping port side of a nozzle, and about the extraction nozzle of the comparative example 5, a glass fiber filter and a sintered filter from the dripping port side of a nozzle Each filter was laminated in the order of.
As a liquid sample, a pseudo nasal swab prepared by adding a biologically-derived viscous substance to an amount equivalent to 40% to a sample extract attached to a commercially available influenza test kit (Check FluA / B, manufactured by Alfresa Pharma) Was used.
First, a liquid sample was placed in a sample extraction container prepared by setting each extraction nozzle in a sample extraction container main body (manufactured by LLDPE), and filtration was performed by applying pressure by pinching the middle of the container main body. 80 μL of the obtained filtrate was added to the test plate dropping part (sample pad) of the kit and developed, and the presence or absence of development failure in the determination region of the membrane (chromatography medium) was visually confirmed. The results are shown in Table 1 and FIG. In the column of “Development” in Table 1, A indicates that the expansion occurred satisfactorily and the target band was detected. B indicates that the target band was detected, but other areas on the membrane were also broadened due to poor development. “C” means that the membrane was broadly stained due to poor development, and the target band could not be detected.

As a result, as shown in FIG. 5, when the extraction nozzle of Example 1 was used, the target band was detected, and other regions on the membrane were hardly stained, and the dropped liquid sample was found in the membrane. It was confirmed that it was developed well. On the other hand, in the case of using Comparative Examples 1 and 2 in which the glass fiber filter was not used, the area just before the area where the target band on the membrane was detected was stained deeply, and the development was poor, and the liquid The sample was not expanded to the target band detection region. In addition, when Comparative Examples 3 and 4 including only a glass fiber filter were used, the target band was detected, but the other areas were also widely stained and the background was high, and the development was also good. There wasn't. Furthermore, when using the extraction nozzle of Comparative Example 5 that combines a glass fiber filter and a sintered filter, the liquid sample was developed on the membrane longer than in Comparative Examples 1 and 2, but the entire membrane was The target band could not be confirmed because it was stained, and particularly in the vicinity of the region where the target band was detected, it was stained deeply and broadly.
In addition, with respect to the degree of development on the membrane, three regions that affect the visibility of the band among the regions 1 to 9 shown in FIG. 6 with respect to Example 1 and Comparative Examples 3 and 4 in which the target band was detected. For (7-9), the color of each region was extracted, the RGB value was analyzed, and the RGB ratio was calculated. The RGB ratio was obtained from the following formula 1. The results are shown in Table 2.
For the measurement of RGB values, an image captured on a PC was calculated using Microsoft Paint (manufactured by Microsoft Corporation), and the RGB ratio was calculated from the following equation.
RGB ratio = R value− (average of G value and B value) (1)
As a result, Comparative Examples 3 and 4 have a higher RGB ratio than Example 1, so that it was confirmed numerically that the development was poor.

  The liquid sample inspection method of the present invention can detect an object in a specimen with high accuracy without causing poor development by immunoassay. For example, infection with pathogens such as bacteria and viruses, pregnancy It can be suitably used for the presence / absence, blood glucose level measurement and the like.

DESCRIPTION OF SYMBOLS 1 Liquid sample inspection tool 2 Sample extraction container 3 Filtration filter 3a Glass fiber filter 3b Resin filter (foamable resin filter)
5 Chromatographic medium 9 Specimen extraction nozzle P Detected object Q Capture substance S Liquid sample W Specimen extract

Claims (8)

After the liquid sample with the sample prepared is filtered by a filtration filter, the detected object in the liquid sample is detected or quantified on a liquid sample inspection tool having a chromatographic medium bound with a substance that captures the detected object. In the liquid sample inspection method,
The said filtration filter is a liquid sample inspection method provided with the filter made from glass fiber which has a hole diameter of 2.5 micrometers or more, and the resin filter which consists of a foaming resin material of an organic polymer. The liquid sample inspection method according to claim 1,
A liquid sample inspection method, wherein the porosity of the resin filter is 70% or more. In the liquid sample inspection method according to claim 1 or 2,
The resin filter is a liquid sample inspection method formed using polyvinyl alcohol. In a sample extraction nozzle that filters a liquid sample containing a sample with a filtration filter and drops it on a liquid sample inspection tool,
The filter is a specimen extraction nozzle provided with a glass fiber filter having a pore diameter of 2.5 μm or more and a resin filter made of an organic polymer foamable resin material. In the specimen extraction nozzle according to claim 4,
The specimen extraction nozzle, wherein the porosity of the resin filter is 70% or more. In the specimen extraction nozzle according to claim 4 or 5,
A specimen extraction nozzle using polyvinyl alcohol as the organic polymer.   A sample extraction container comprising: the sample extraction nozzle according to any one of claims 4 to 6; and a container body to which the sample extraction nozzle is attached.   A liquid sample test kit comprising: a liquid sample test tool having a chromatographic medium combined with a substance that captures an object to be detected; and the sample extraction container according to claim 7.