JP5586842B2 - Simple membrane assay method and kit using sample filtration filter - Google Patents

Description

  The present invention relates to a detection method, a detection apparatus, and a detection kit for specifically qualitatively or quantitatively measuring an analyte in a specimen sample.

  Recently, a simple test that uses antigen-antibody reaction or enzyme reaction to detect or quantify various measurement items such as infection with pathogens such as viruses and bacteria, presence of pregnancy, blood glucose level in a few minutes to tens of minutes. Reagents or kits have been developed. Pathogen component proteins, human ciliary gonadotropin (hCG), blood glucose, and the like are targets for detection or quantification. Many of the simple test reagents do not require special equipment and are easy to operate and inexpensive. For example, simple test reagents for pregnancy diagnosis are sold as OTC at general pharmacies. Unlike other test reagents, simple test reagents for measuring infection with pathogens are widely used in general hospitals and clinics in addition to large hospitals and medical test centers. These facilities are often the first medical institutions visited by patients, and if specimens collected from patients are found to be infected on the spot, symptoms can be treated as soon as possible. The importance of test reagents in medicine is increasing.

  Currently, immunoassay methods, that is, immunoaggregation methods and immunomembrane assay methods, particularly assay methods using membranes such as nitrocellulose and membranes, such as filters, are generally known as simple test methods. It is roughly classified into the formula membrane assay method. The former allows the solution containing the detection object to pass through the film coated with the detection substance for the detection object in the vertical direction, and the latter allows the solution to be developed in the horizontal direction. In either case, a membrane solid phase substance that specifically binds to the detected substance, a detected substance, and a complex of a labeled substance that specifically binds to the detected substance is formed on the film to detect or quantify the label. This is common in that detection or quantification of the detected substance is possible, but recently it is simpler and can be detected in a shorter period of time. Therefore, the lateral flow membrane assay method (also referred to as immunochromatography) is used. ) Has become mainstream.

  However, in a simple test method based on a membrane assay using such a membrane or filter, in the analysis of a sample actually collected from a patient, it is determined that the detected object is positive even though it is not present in the sample. So-called false positives may occur. If a false positive reaction occurs when measuring the infection of a pathogen, it will give incorrect information about the disease, so it will not only delay the cause identification but also take inappropriate measures, making the medical condition more serious, etc. It can have serious consequences. Therefore, suppressing false positives is an extremely important issue in view of the main purpose of the simple test method.

  The cause of false positives is not completely known. However, it is thought that the contamination contained in the specimen is one of the causes. In a simple membrane assay method using an assay device equipped with a membrane to which a capture substance is bound to capture the detection target, a sample such as a cotton swab from the pharynx or nasal cavity of the patient where the detection target is expected to exist is detected. Use a sampling device to collect the wiping solution and float it in a buffer solution, or collect a part from secretions and excreta such as nasal discharge and stool containing the object to be detected, and float in the buffer solution for membrane assay. In this case, in addition to the detection target, cells, secretions, excrement components, and the like that have fallen from the sample collection site may be mixed in the sample. Since such contaminants include various biological components including viscous substances such as proteoglucan and glycolipid, when the specimen sample is added as it is onto a membrane such as a membrane, a part of the above components is present. It is thought that it adheres on or in the membrane. In particular, a membrane having a pore size or retained particle size of about 0.5 to 15 μm is often used, but if a component having a size comparable to this pore size or retained particle size is added, the pores in the membrane are blocked. It is conceivable to inhibit the movement of components in the solution. It is considered that a non-specific reaction occurs due to such a phenomenon, and so-called false positive occurs in which it is determined that the object to be detected is positive even though the detection target does not exist in the sample.

  In addition, depending on the content and properties of the above contaminants, the pores in the membrane may be clogged to a considerable extent or completely, making it extremely difficult or impossible for the specimen to pass through the membrane. As a result, the reaction rate may become extremely slow or measurement may become impossible, and preventing this is a major problem.

  In order to solve the above problems, it is effective to filter the specimen sample in advance and add it onto the membrane (see Patent Documents 1 and 2). Since the false positive causative substances in the specimen sample are removed by filtration, it is possible to prevent these substances from adhering to the membrane of the assay device or causing them to cause false positives. However, when filtering with a filter, the substance in the sample may be clogged in the filter, making it impossible to add the assay device onto the membrane. One means for solving this problem is to devise the configuration of the sample filtration filter. By using a sample filtration filter obtained by stacking several small filters from a filter having a large pore diameter or retained particle diameter, it is possible to avoid clogging of the specimen sample to some extent during filtration. However, a new problem arises here. First, by stacking a number of filters, the thickness of the filter increases, and when a commonly used cellulose filter paper or the like is used, the sample sample is absorbed, so the amount of the sample sample is reduced. In particular, to increase sensitivity, it is necessary to reduce the buffer solution as much as possible and increase the concentration of the analyte in the sample. However, if the amount of absorption by the filter is large, the buffer solution must be increased accordingly. Sensitivity will decrease. In addition, if the number of filters is increased, the possibility that the object to be detected is more adsorbed on the filter increases. As means for avoiding these problems, it is conceivable to increase the filtration area. However, since the amount of the buffer solution is limited, it cannot be increased without limitation, and a great effect cannot be expected. From the above, achieving the filtration of the specimen sample without sacrificing the sensitivity has been an important issue for the construction of the measurement system of the kit.

JP 2000-88851 A JP 2004-28875 A

  An object of the present invention is to provide a specimen sample filtration method capable of preventing false positives and clogging while maintaining sensitivity in a simple specimen inspection method using a membrane assay method, and highly accurate detection or quantification of an object to be detected. And a kit for use in such a method.

  That is, an object of the present invention is to use a filter including a rigid filter in a simple membrane assay method of a detection object in a specimen sample using an assay device including a membrane to which a capture reagent for capturing the detection object is bound. This is solved by a method characterized in that a sample sample is filtered using a configured filter and then dropped on a membrane to detect the presence of an object to be detected in the sample sample.

Moreover, the subject of this invention is the simple membrane assay kit for test | inspecting presence of the to-be-detected object in a sample sample containing the following;
(1) An assay sample including a filter tube for a specimen sample including a filtration filter constituted by a filter including a strong rigidity filter, and (2) a membrane bound with a capture substance for capturing an object to be detected in the specimen sample. apparatus,
It is solved by.

  A preferred embodiment of the present invention is the above method or kit, wherein the rigid filter is a sintered filter.

  A particularly preferred embodiment of the present invention is the above method or kit, characterized in that the material of the sintered filter is selected from the group consisting of polypropylene, polyethylene, polystyrene, polymethyl methacrylate, alumina, zirconia and silicon.

  In another preferred embodiment of the present invention, the filtration filter is formed by stacking two or more filters, and one of the coarse filters has a pore diameter or a retained particle diameter of not less than the pore diameter of the membrane and not more than 200 μm. One sheet for filtration is the above-mentioned method or kit in which the pore diameter or the retained particle diameter is not more than the pore diameter of the membrane. The rigid filter can be used for both fine filtration and coarse filtration, but the pore size or retained particle size of at least one of the rigid filters is not less than the pore size of the membrane, and preferably not more than 200 μm.

  Moreover, the other preferable embodiment of this invention is the said method using a cotton swab as a specimen collection instrument, or the said kit containing a cotton swab.

  Also, a particularly preferred embodiment of the present invention is the above method, wherein the swab is a brush swab, or the kit comprising a brush swab.

  Moreover, the preferable embodiment of this invention is the said method or kit whose specimen sample is a biological sample.

  Further, in a particularly preferred embodiment of the present invention, the specimen is a pharyngeal wipe, nasal wipe, nasal aspirate, nasal wash, alveolar wash, stool suspension or rectal wipe of a human or other animal. Method or kit.

  In addition, a particularly preferred embodiment of the present invention is the above method or kit, wherein the detected substance in the specimen is a microorganism causing respiratory infection or a microorganism causing diarrhea.

  Further, in a particularly preferred embodiment of the present invention, the detected substance in the sample is selected from the group consisting of influenza virus, RS virus, adenovirus, group A streptococcus, pneumonia mycoplasma, norovirus, rotavirus, sapovirus and diarrhea adenovirus. The above-described method or kit, which is a pathogenic microorganism or a substance derived from the microorganism or an antibody against them.

  Further, a particularly preferred embodiment of the present invention is the above method or kit relating to a flow-through type or lateral flow type simple membrane assay method.

  When detecting or quantifying a substance to be detected in a sample using the method or apparatus of the present invention, the sample is a sample containing a large amount of impurities such as a pharyngeal wiping solution, a nasal wiping solution, a nasal aspirate, and an alveolar lavage solution. In addition, impurities can be removed without reducing the amount of the sample by using the sample collection device and / or the filtration filter. As a result, accurate results can be obtained without being affected by impurities.

Hereinafter, the present invention will be described in detail.
(Simple membrane assay method and assay device)
The method of the present invention is a simple membrane assay method for an object to be detected in a specimen sample using an assay device including a membrane to which a capture substance for capturing the object to be detected is bound, and includes a filter including a rigid filter A specimen sample prepared from a specimen containing a detection object using a filtration filter constituted by the above is filtered and dropped on the membrane, and the presence or absence of the detection object in the specimen sample is detected or quantified. It is a method to do.

  In this specification, the “simple membrane assay method” refers to the presence or absence of an analyte in a specimen sample using an assay device including a membrane on which a capture substance that specifically binds to the analyte is immobilized. This is a simple inspection method in a short time. Typically, a detection target is reacted with the capture substance and the labeling substance to form a sandwich-like complex having a structure of supplementary substance-detection target-labeling substance on the membrane, and the labeling substance is detected. This is a method for detecting the presence of this complex. Examples of the reaction between the detected substance and the capture substance and the labeling substance include antigen-antibody reaction, reaction between other receptor and receptor, specific binding reaction between biotin and avidin, reaction between DNAs having complementary sequences, and the like. It is done. In addition, the simple inspection method of the present invention is not limited to the types of the detection target, the membrane, and the labeling substance as long as they are used in such a method.

  An assay device including a membrane (also referred to as a membrane assay device) as used in the present specification is a device including a membrane on which a capture substance that specifically binds to an object to be detected is immobilized. As such an assay apparatus, it is preferable to use a flow-through membrane assay method or a lateral flow membrane assay method because it is simple and rapid. In the flow-through membrane assay method, a solution containing an object to be detected is passed in a direction perpendicular to a membrane coated with a capture reagent or a detection substance that specifically binds to the object to be detected. By forming a complex of a capture substance that specifically binds to the target substance, an object to be detected, and a label substance that specifically binds to the object to be detected, and detecting or quantifying the label, Perform quantification. The lateral flow membrane assay method differs from the flow-through membrane assay method in that the same membrane is used to spread the solution containing the analyte on the membrane in the horizontal direction, but the detection principle of the analyte is the same. It is. Of the two methods, the lateral flow membrane assay is more preferable from the viewpoint of simplicity and short measurement time.

  A specific example of an assay device using a lateral flow membrane assay is a device as shown in FIGS. 1 and 2, for example.

  1 is a plan view of the apparatus, and FIG. 2 is a cut end surface taken along the line I-I ′ of FIG. 1. In FIGS. 1 and 2, a is a sample dropping pad installed to drop the prepared specimen sample. b is a labeling substance drying pad on which the labeling substance is dried and held. c is a sample absorption pad for absorbing the dropped solution. d is a membrane to which a capture substance that specifically binds to the detection target is bound, and e, f, and g are positions on the d where the capture substance that specifically binds to the detection target is bound in a line. Show. Further, a substance capable of binding the labeling substance to a position h downstream of the position where the capture substance is bound, for example, a substance having reactivity equivalent to the detection target or an antibody binding to the labeling substance may be solid-phased. Good. Thus, it can be confirmed for each test that the labeling substance flows from the labeling substance drying pad on the membrane to which the trapping substance is bound. If the filter is clogged during filtration, the sample sample is absorbed by the filter and a sufficient amount of sample sample cannot be filtered, or the membrane of the assay device is clogged, the h position is Color development is not recognized. i is a plastic backing sheet for fixing members and increasing strength. j is a transparent plastic laminate covering a part of the sample dropping pad, the labeling substance drying pad, the sample absorbing pad, and the membrane.

  Examples of the membrane in the membrane assay apparatus include porous materials such as paper and nitrocellulose, or membranes made of a carrier such as a fibrous matrix, silica used for thin layer chromatography, fine granular cellulose, nylon 6,6, and the like. be able to. Preferred porous materials for conducting a lateral flow membrane assay with high sensitivity and specificity include microporous cellulose esters, for example, cellulose esters with aliphatic carboxylic acids such as alkane carboxylic acids, particularly preferably nitro Examples include microporous materials made from cellulose. Moreover, the mixture of the said cellulose ester and nitrocellulose can also be used suitably. The average pore diameter of the membrane is often 0.5 to 15 μm.

Below, an example of a more specific procedure is shown about the method of this invention, and this invention is demonstrated.
(1) A sample sample collected directly from a pharynx, nasal cavity, rectum or the like of a patient suspected of being infected with a virus or bacteria using a sample collection device such as a cotton swab is suspended in a sample suspension as described later. Alternatively, a specimen sample collected from a subject to be analyzed such as a nasal aspirate, urine, or feces collected from a patient by using a specimen collection device such as a cotton swab is suspended in a specimen suspension as described later.
(2) The suspended liquid is filtered in a specimen sample filtration tube equipped with a filtration filter.

(3) Add this filtrate by dropping or the like to a membrane bound with a capture reagent that specifically binds to the analyte in the assay device equipped with the membrane and captures the analyte. Capture on membrane. At this time, by dropping the filtrate onto the sample dropping pad, the detection object flows through the membrane by capillary action.

(4) A labeling substance, which is a detection reagent that specifically binds to the object to be detected, is added onto the membrane by dropping or the like to form a capture reagent / object to be detected / labeled substance complex. At this time, the labeling substance may be incorporated in the assay device in advance. In this case, by adding the suspension liquid containing the specimen sample to the assay apparatus, when the suspension liquid flows on the assay apparatus and reaches the pad portion containing the detection reagent, the detection reagent dissolves in the suspension liquid, and the analyte At the same time, it reaches the membrane and forms a complex with the capture reagent of the membrane.

(5) The presence or absence of an object to be detected in the specimen sample is measured by detecting the presence of the complex with the labeling substance in the complex. Prior to detecting the presence of the complex, the reaction may be stopped using a reaction stop solution, if necessary.

(Sample collection device)
As an instrument for collecting the specimen, a cotton swab, platinum ear, dropper, spoon tool or the like can be used. In particular, when a human body is a subject and a nasal swab, nasal aspirate, throat swab, alveolar lavage, stool suspension or rectal wiping fluid is used as a specimen, a cotton swab is mainly used as a specimen collection device.

  As shown in FIG. 3, a conventionally used cotton swab is formed by a cotton ball in which a sample collection portion k1 is wound around a head m at one end of a shaft portion l. Considering the purpose of the original sample collection device, which collects the sample and then releases it from the swab for use in the test, this conventional swab requires more cotton than necessary, and the sample enters the deep part of the cotton and is held there. In the step of releasing the specimen, there is a problem that the object to be detected in the specimen is not sufficiently released.

  Recently, a new type of cotton swab in which fibers made of natural products or artificial products are arranged in a brush shape and liquid is collected using capillary action is commercially available and is called a brush-like swab. The brush-like swab can be particularly preferably used in the present invention for the following reasons. The brush-like swab can be produced by, for example, a method called flocking, in which short cut fibers (floc) are attached to the handle of a swab to which an adhesive is applied by static electricity. Also, with this type of swab, it is possible to collect a solid or viscous substance that is a dissolved or mixed material in a liquid. In this case, not only the capillary phenomenon but also a solid or viscous substance is collected in the sample collection site of the instrument in the present invention. It is collected by using the binding by adsorption. Since the surface area is greatly increased as compared with a conventional cotton swab, the amount of sample collected is large, and more objects to be detected in the sample can be adsorbed. In addition, when suspended in suspended liquid, unlike conventional cotton swabs, suspended liquid can easily enter between fibers, so that the detected object is easily released and the detected object is efficiently collected in the suspended liquid. can do.

  FIG. 4 shows an example of a brush-like swab. A specimen collection portion k2 in which fibers are arranged in a brush shape on the surface of the head m at one end of the shaft portion l is provided. There are no particular limitations on the material of the shaft portion l and the head portion m, but a substance that is inferior in liquid absorptivity than the specimen collection portion k2 and has appropriate hardness and appropriate flexibility is suitable. A molded product of wood, plastic, urethane resin, paper, metal, or the like is preferable, and a molded product of a mixture of these materials may be used. For the purpose of adjusting the hardness, flexibility, liquid absorbency, etc., it may be treated with chemicals as necessary. The shaft portion l and the head portion m may be made of different materials or may be made of the same material. Moreover, it is good also as separate molding and it does not matter even as integral molding. There is no particular limitation on the material of the fibers constituting the specimen collection site k2, but for example, a material in which fine fibers are combined, woven, knitted, or formed as an aggregate can be used, for example, cloth, woven fabric, non-woven fabric, Cotton-like and sponge-like items can be used. Natural materials and artificial materials can be used, and natural products such as cotton and silk, processed products of natural products such as absorbent cotton, chemical processed products of natural products, and artificial products such as nylon fibers can be used. Any material can be used in combination or in layers. It is preferable that the fine fibers are arranged in a brush shape and are arranged in a substantially vertical direction toward the axial center of the shaft portion. The fiber portion arranged in a brush shape can absorb and hold the liquid by capillary action. The thickness of the brush-like fiber portion can be appropriately selected, but is about 0.5 to 5 mm, preferably about 1 to 2 mm. The brush-like fiber part that collects liquid by utilizing capillary action, the shaft part and the head part are usually the target shape with respect to the central axis of the shaft part, and the vertical cross section with respect to the axial direction is circular around the axis. The shape may be changed as necessary, or a brush-like fiber portion that collects liquid using capillary action may be selectively disposed. The fibers arranged in a brush shape are preferably arranged in a substantially vertical direction toward the axis center of the shaft portion. The thickness and arrangement density of the fibers can be appropriately selected according to the purpose and material, but the fineness is preferably 1.0 to 4.0 dtex.

(Filtration filter)
In the method of the present invention, a specimen collected from a patient is suspended in a specimen suspension and used as a specimen sample, and then filtered using a filtration filter to prevent clogging or false positives of the membrane in the assay device. . In particular, when a brush-like swab is used as a specimen collection device, it is possible to collect more detection objects than when using a normal swab, but at the same time cells, secretions and excretion that have fallen from the specimen collection site. Since more components are collected, membrane clogging and false positives are more likely to occur. Therefore, the importance of the filtration process becomes higher. In order to prevent clogging and false positives of the membrane and clogging of the filtration filter itself, the filtration filter may be combined not only with one type but also with several different materials, those with different pore diameters or retained particle diameters. In the invention, it is necessary to include at least one type of rigid filter.

The rigid filter mentioned here refers to a filter that is not easily deformed by an external force.
Place the specimen sample suspended in the specimen suspension in the filtration tube, filter through a filtration filter including a rigid filter attached to the tip, and filter the filtrate when dropping the filtrate onto the membrane in the membrane assay device. Receives the pressure of liquid or air such as the specimen sample in the tube. At this time, the filter may be crushed or deformed in the thickness direction, but these phenomena block the flow path of the filtrate inside the filter and cause the filter itself to be clogged. The rigid filter used in the present invention is a method in which a specimen sample suspended in a specimen suspension is placed in a filtration tube and filtered through a filtration filter including a rigid filter attached to the tip, and the filtrate is passed through a membrane in a membrane assay device. It refers to a filter having rigidity that is difficult to be crushed in the thickness direction of the filter when it is dropped onto the filter or to be deformed such as curved. In other words, it refers to a filter having rigidity that makes it difficult to block the flow path of the filtrate inside the filter even by the pressure of the filtrate.

  The rigid filter is not easily deformed, so clogging is unlikely to occur. Further, since it can be laminated or processed to a thickness of about several millimeters to several centimeters, the fact that the effective filtration area can be increased is also a factor that prevents clogging. In the present invention, it is necessary to include a strong filter that does not cause such deformation.

  For example, even if the pressure applied to the filter by liquid or air when the specimen sample is filtered exceeds 0.2 [MPa], the thickness at the time of pre-filtration vs. filtration The amount of change in thickness does not exceed 5%, preferably 2% or more, and is rigid enough to prevent deformation such as bending. This means that the radius of curvature is not less than 2cm.

  As the strong filter, various metal fibers are wound, metal fiber nonwoven fabrics are used, plastics are foamed, metal mesh is laminated in layers, metal, plastics, ceramic powder, etc. And sintered filters in which metal fibers are bonded with heat or pressure, and any of them can be used in the present invention.

  The sintered filter is a filter that is manufactured by directly bonding (sintering) powder particles such as metal, ceramic, plastic, and metal fibers by heat and pressure. Among strong filters, sintered filters are 1) inexpensive, 2) pore size can be controlled by particle size, 3) suitable for mass production, and 4) low affinity with biological materials. Therefore, it does not specifically bind an object to be detected, 5) hardly absorbs moisture in the liquid, and 6) it can be produced with various pore sizes depending on the size of the particle, so that it contains a biological substance. Preferred for sample filtration.

  Materials for the sintered filter include polypropylene, polyethylene (low density polyethylene, high density polyethylene, ultra high molecular weight polyethylene), polystyrene, polymethyl methacrylate resin, alumina, zirconia, silicon, silicon tetrafluoroethylene polymer, stainless steel Although steel etc. can be mentioned, the thing using resin, such as a polypropylene, polyethylene, a polystyrene, a polymethylmethacrylate, is preferable from the surface of workability.

  The sample filtration filter may be produced by combining two or more types of strong rigidity filters. Moreover, you may combine with filters other than a rigid filter and a rigid filter.

  Although the rigid filter can be used for both fine filtration and coarse filtration, the pore diameter or retained particle diameter of at least one kind of rigid filter is not less than the pore diameter of the membrane, and preferably not more than 200 μm.

  For example, a polypropylene rigid filter has a pore size of 50 to 200 μm, a low density polyethylene rigid filter has a pore size of 30 to 70 μm, a high density polyethylene rigid filter has a pore size of 10 to 50 μm, and an ultrahigh molecular weight polyethylene rigid filter The pore size of the polystyrene rigid filter is 100 to 200 μm, and the pore size of the tetrafluoroethylene polymer strong filter is 30 to 100 μm. A plurality of strong rigidity filters having different materials and / or hole diameters may be used in combination.

  For example, a membrane filter can be used as a filter for microfiltration. The material is cellulose, glass fiber, silica fiber, nitrocellulose, cellulose ester, a mixture of nitrocellulose and cellulose ester, polyethersulfone, polysulfone, tetrafluoride. A suitable one can be appropriately selected from among ethylene resin, vinylidene fluoride resin, polycarbonate, polypropylene, polyamide, nylon 6,6, polyester, cotton, stainless steel fiber, etc. It is necessary to make the amount of the sample suspension absorbed less.

  It is preferable that the minimum pore diameter or the retained particle diameter of the filter having the smallest pore diameter or the retained particle diameter among the filters constituting the filtration filter is not more than the pore diameter of the membrane. That is, for example, when the pore diameter of the membrane to be used is 10 μm or more, it is preferable that at least one of the filtration filters has a pore diameter of 10 μm or less.

  For example, when two filters are used in combination with another type of filter other than the strong rigidity filter and the strong rigidity filter (hereinafter sometimes referred to as a non-rigid rigidity filter), the filter is a filtration nozzle for a filtration tube described later. (In this case, the first-stage filter is the first-stage filter that contacts the specimen sample first, and the specimen sample passes through the first-stage filter and enters the assay device.) As a combination of the first stage filter and the second stage filter, there are a combination of a strong filter and a strong filter, and a combination of a strong filter and a non-rigid filter. A filter close to the nozzle tip is sometimes referred to as a downstream filter, and a filter that comes into contact with a specimen sample earlier is sometimes referred to as an upstream filter. When combining the filters, it is preferable to combine filters having different pore diameters, and at least one of the two types of filters preferably has a pore diameter smaller than that of the membrane filter. Since all of the strong filters trap contaminants not only on the filter surface but also inside, a certain thickness is required. The required thickness naturally depends on the amount of contaminants and the pore size and size of the filter, but it is generally rigid when 100 to 2000 μl of a sample sample required for a simple membrane assay is filtered with a sample filtration filter with a diameter of about 5 to 20 mm. The thickness of the filter or the non-rigid filter is not limited, but is preferably 0.5 mm to 7 mm, more preferably 1 mm to 5 mm. The total thickness of the sample filtration filter is preferably about 2 mm to 10 mm.

  When combining several filters with different pore diameters or retained particle diameters, it is necessary to place a filter with a large pore diameter or retained particle diameter upstream and a small filter downstream so that both filters overlap. This is preferable in order to avoid clogging of the specimen sample at the time. For example, when two filters are combined, it is preferable to use a filter having a large pore diameter or retained particle diameter as the first-stage filter and a filter having a small pore diameter as the second-stage filter.

  The size of the filter used depends on the size of the specimen suspension tube used in the assay, but is, for example, circular and has a diameter of 0.5 to 1.5 cm.

(Filtration tube for specimen sample)
In the simple membrane assay method or kit of the present invention, the filtration filter is preferably used by being attached to the tip of a specimen sample filtration tube. That is, a method in which a specimen sample suspended in a specimen suspension is put in a filtration tube, filtered through a filtration filter attached to the tip, and the filtrate is dropped onto a membrane in a membrane assay apparatus is simple and preferable. The schematic diagram of one embodiment of this filtration tube is shown in FIG.5 and FIG.6. For example, as shown in FIG. 5 and FIG. 6, the filtration tube has a shape including a tip portion (filtration nozzle) n and a main body portion p, and the filtration filters o1 to o3 are provided inside the tip portion as shown in FIG. It is provided. In the filtration nozzle of FIG. 5, the lower part of the drawing is the bottom surface of the nozzle, and the filtration filters o1, o2, and o3 are a third-stage filter, a second-stage filter, and a first-stage filter, respectively. A prepared specimen sample in which a specimen is suspended in a suspended liquid is placed in a main body as shown in FIG. 6, and a tip portion equipped with a filtration filter is attached. The specimen sample is filtered through a filtration filter, and the filtrate is dropped onto the membrane assay device. When the main body is made of a flexible material such as polyethylene or polyethylene terephthalate (PET), the sample can be easily removed by attaching a filtration filter and applying pressure by hand with the sample in the sample. Since it can filter, it is preferable.

(Detected object)
Examples of detected substances in the present invention include influenza virus, RS virus, adenovirus, group A streptococcus, pneumonia mycoplasma, pathogenic microorganisms such as norovirus, rotavirus, sapovirus, diarrhea adenovirus, and the like Substances such as proteins derived from pathogenic microorganisms or antibodies to them, peptide hormones, steroids, bioactive amines, vitamins, prostaglandins, tetracycline and other antibiotics, toxins produced by bacteria, various tumor markers, pesticides And nucleotides complementary to a nucleic acid component derived from a pathogenic microorganism. When the above pathogenic microorganisms or substances such as proteins derived from the pathogenic microorganisms or antibodies against them are used as non-detected substances, in particular, influenza virus, RS (Respiratory Syncytial) virus, adenovirus, group A streptococcus, pneumonia mycoplasma, rotavirus In addition, it is extremely useful for diagnosis of pathogens such as Norovirus that are very popular and need to be identified in a very short time.

  Samples to be analyzed by the test method of the present invention are pharyngeal wiping fluid, nasal wiping fluid, nasal aspirate, alveolar lavage fluid, stool suspension, plasma, serum, urine, saliva, amniotic fluid, spinal fluid, pus, organ Extracts, biological samples such as various tissue extracts, food extracts, culture supernatants, clean water, sewage, lake water, river water, seawater, soil extracts, and sludge extracts can be used, but are not limited thereto. Among them, it is particularly useful when a biological sample is used as a specimen, and is extremely useful when a pharyngeal wiping liquid, nasal wiping liquid, nasal aspirate, nasal washing liquid, alveolar washing liquid, rectal wiping liquid or stool suspension is used as a specimen. Useful. The biological sample may be derived from a human or a non-human animal.

(Capture substance)
A capture substance for capturing a detection target is a substance that forms a complex by binding to the detection target by a specific reaction such as an antigen-antibody reaction. Therefore, it is natural that the capture substance to be used differs depending on the object to be detected. However, in general, when the object to be detected is bacteria, viruses, hormones, other clinical markers, etc., they specifically react to bind to them. Polyclonal antibodies, monoclonal antibodies and the like. In addition, virus antigens, virus hollow particles, recombinant E. coli expressed protein, recombinant yeast expressed protein and the like can be mentioned. As a method for binding such a capturing substance to the above-described membrane surface, physical adsorption may be used, or chemical binding may be used. The immobilization can be performed by a known method for immobilizing a protein on a solid phase such as a nitrocellulose membrane. The membrane to which the capture substance is bound is prepared, for example, by adsorbing a solution obtained by diluting the capture substance in a buffer solution or the like and then drying the membrane. What is necessary is just to solidify a supplementary substance in the shape of a line, for example.

(Sample suspension)
In the present invention, the specimen suspension is a solution for suspending or suspending a specimen that may contain an object to be detected collected from a patient or the environment. A sample sample is added to the assay device by dropping or the like and used for the assay.

  The collected specimen contains various substances in addition to the object to be detected. For example, when collecting specimens from humans for pathogen infection measurements, they are often collected from the nasal cavity, pharynx, or rectum with a cotton swab, etc., but in this specimen, in addition to patient-derived tissue fragments and secretions, It may contain the pathogen tissue that is detected. Some of these exist as aggregates in the sample suspension, and some exist in a dissolved state in the sample. If the specimen sample is added to the assay device in a state containing these, a false positive may occur due to a non-specific reaction. In order to remove aggregates, a method of filtering through a membrane for filtration before adding to the assay device is effective. However, in the case of lysate, it cannot be removed by filtration. It is necessary to devise the composition.

  The specimen suspension usually contains a salt and a buffer for maintaining a constant pH. As the buffer solution, a buffer solution usually used in immunological tests can be used, and examples include Tris buffer solution, phosphate buffer solution, Good's buffer solution and the like. Furthermore, a surfactant can be contained in the specimen suspension for the purpose of reducing nonspecific reactions within a range that does not inhibit specific agglutination reactions. As surfactants, Triton X-100 (trade name): polyethylene glycol mono-р-isooctyl phenyl ether, Tween 20: polyoxyethylene sorbitan monolaurate, Tween 80: polyoxyethylene sorbitan monooleate, Nonidet P -40: nonidet P-40, ZWITTERGENT 3-14: n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, CHAPS: 3-[(3-colamidopropyl) dimethylammonio ] Propanesulfonic acid, sodium dodecyl sulfate (SDS) or the like, or a mixture of two or more of these can be used.

  In addition, the sample suspension may contain proteins such as bovine serum albumin, immunoglobulin and casein, serum from rabbits and mice, and the like for the purpose of reducing non-specific reactions.

  In addition, for example, when the detection target has a cell wall like bacteria and the binding site of the labeling substance or capture substance to the detection target is inside the bacteria, the specimen is suspended in the specimen suspension. Only the binding sites are not easily exposed to the outside, and detection / quantification may not be possible. In this case, pretreatment such as addition of alkali such as NaOH, neutralization, or heat treatment may be necessary.

  Although the above-mentioned device can reduce the occurrence of false positives, it cannot be completely prevented.

(Detection reagent)
In the present specification, the detection reagent is capable of specifically binding to an object to be detected and forming a complex with the object to be detected. The labeling substance means a detection reagent labeled so that it can be detected by some means after forming a complex with an object to be detected. For example, when the object to be detected is an antigenic substance such as a virus, an antibody against the virus and labeled with an enzyme or the like can be used. When labeled with an enzyme in this way, the complex can be detected by adding a substrate of the enzyme that produces a substance detectable by a colorimetric method or a fluorescence method by a reaction catalyzed by the enzyme. It can be carried out. Examples of the detection reagent before labeling are the same as those described for the capture reagent. Examples of the label include an enzyme, a fluorescent label, a magnetic label, a radioisotope, a gold colloid, and a colored latex. The detection reagent may be added to the assay device after the sample sample is added to the assay device, or may be incorporated in the assay device in advance. When the detection reagent is incorporated into the assay device, for example, a pad impregnated with the detection reagent and dried may be incorporated between the sample dropping pad and the membrane containing the capture substance. In this case, by adding the suspension liquid containing the specimen sample to the assay apparatus, when the suspension liquid flows on the assay apparatus and reaches the pad portion containing the detection reagent, the detection reagent dissolves in the suspension liquid, and the analyte At the same time, it reaches the membrane and forms a complex with the capture reagent of the membrane. For the pad portion containing the detection reagent, for example, a nonwoven fabric made of cellulose, glass fiber, or the like is used.

  When an enzyme label is used, examples of the enzyme used include alkaline phosphatase, peroxidase, and glucose-6-phosphate dehydrogenase.

  When an enzyme label is used as a label for a labeling substance, a substrate that is usually a substrate for the enzyme and generates a substance detectable by a colorimetric method or a fluorescence method by a reaction catalyzed by the enzyme is added. . Specific examples include 5-bromo-4-chloro-3-indolyl phosphate (BCIP) / nitrotetrazolium blue (NBT), tetramethylbenzidine (TMB), and glucose-6-phosphate NAD +.

(Reaction stop solution)
The kit of the present invention may further contain a reaction stop solution for stopping the reaction between the enzyme and the substrate, for example, if necessary. Examples of such a reaction stop solution include citric acid and sulfuric acid.

  The device of the present invention may include an absorbent pad portion. The absorption pad portion is provided at one end of the enzyme immunoassay device of the present invention, and promotes the flow of the liquid on the membrane by absorbing the liquid developing the device. The absorbent pad portion is made of a water-absorbing material so that a large amount of liquid can be absorbed. For example, a nonwoven fabric made of cellulose, glass fiber, or the like is used. The size is not particularly limited, but is usually about 3 mm to 15 mm × 10 mm to 40 mm and a thickness of about 0.5 mm to 3 mm.

(Simple membrane assay kit)
The simple membrane assay kit of the present invention is a kit used in the above-described simple membrane assay method of the present invention. The simple membrane assay kit of the present invention comprises at least the following (i) and (ii).
(I) An assay device comprising a filtration filter composed of a filter including a rigid filter, and (ii) a membrane bound with a capture substance for capturing an object to be detected.

  The filtration filter is preferably attached to the specimen sample filtration tube described above.

  The kit may contain a sample collection device such as a cotton swab, if necessary. As the sample collection device, a brush-like swab is preferable.

  The kit may further contain a sample suspension, a cleaning solution composition, and / or a labeling substance, if necessary. As the cleaning composition, physiological saline, Tris buffer, phosphate buffer, Good's buffer, etc. can be used, and sodium chloride, surfactant, bovine serum albumin, sodium azide, etc. are added as appropriate. Also good. In addition, when the label of the labeling substance is an enzyme label, it can contain an enzyme substrate, a reaction stop solution, and the like described later.

  Further, if necessary, a negative control solution consisting only of a buffer for examining the activity of the kit, and a positive control consisting of a buffer containing an object to be detected such as an antigenic substance may be included.

Hereinafter, the present invention will be described in detail based on examples. The present invention is not limited to the following examples.
[Example 1] Detection of influenza virus and RS virus in nasal aspirate by lateral flow immunochromatographic assay 1
1. Production of monoclonal antibody (1) Production of anti-influenza A virus NP (Nucleoprotein; nucleoprotein) monoclonal antibody (mouse) Immunized with purified influenza A virus antigen, and spleen was removed from BALB / c mice maintained for a certain period of time. The cells were fused with mouse myeloma cells (P3 × 63) by the method of Keller et al. (Kohler et al., Nature, vol. 256, p495-497 (1975)).

  The obtained fused cells (hybridomas) are maintained in a 37 ° C. incubator, and purification of the cells (monocloning) is performed while confirming the antibody activity of the supernatant by ELISA using an influenza A virus NP antigen solid phase plate. went.

  The obtained two cell lines were each intraperitoneally administered to pristane-treated BALB / c mice, and about 2 weeks later, antibody-containing ascites was collected. IgG was purified from the obtained ascites by affinity purification using Protein A column chromatography (Amersham) to obtain two types of purified anti-influenza A virus NP monoclonal antibodies.

(2) Anti-influenza B virus NP monoclonal antibody (mouse)
The spleen was excised from BALB / c mice that had been immunized with purified influenza B virus antigen and maintained for a certain period of time, and the mouse myeloma was prepared by the method of Keller et al., Nature, vol. 256, p495-497 (1975). Fused with cells (P3x63). The obtained fused cells (hybridomas) are maintained in an incubator at 37 ° C., and cell purification is performed while confirming the antibody activity of the supernatant by ELISA using an influenza B virus NP antigen solid phase plate (monoclonal clone). ). The obtained two cell lines were each intraperitoneally administered to pristane-treated BALB / c mice, and about 2 weeks later, antibody-containing ascites was collected. IgG was purified from the obtained ascites by Protein A column chromatography (Amersham) to obtain two types of purified anti-influenza B virus NP monoclonal antibodies.

(3) RS virus FP monoclonal antibody (mouse)
A spleen was excised from a BALB / c mouse that had been immunized with a polypeptide obtained by synthesizing a part of the amino acid sequence of FP (Fusion Protein) of human RS virus, and the method of Keller et al., Nature, vol, 256, p495-497 (1975)) and fused with mouse myeloma cells (P3 × 63).

  The obtained fused cells (hybridoma) were maintained in a 37 ° C. incubator, and the cells were purified (monocloned) while confirming the antibody activity of the supernatant by ELISA using an RS virus FP antigen solid phase plate. .

  The obtained two cell lines were each intraperitoneally administered to pristane-treated BALB / c mice, and about 2 weeks later, antibody-containing ascites was collected. IgG was purified from the obtained ascites by affinity purification using Protein A column chromatography (Amersham) to obtain two types of purified anti-RS virus FP monoclonal antibodies.

2. Preparation of labeled anti-influenza virus antibody (labeling substance) (1) Preparation of latex particle-labeled anti-influenza A virus antibody One type of anti-influenza A virus NP monoclonal antibody is 50 mM MES (2-Morpholinoethanesulfonic acid, monohydrate) After dialysis with a buffer solution (pH 6.0), 10 mL of a solution diluted with the same buffer solution was prepared so that OD _{280 nm} = 0.5. Next, mixed with 10 (W / V)% red polystyrene latex particles (particle size 0.45μm, surface functional group is carboxyl group, functional group density 65Å ² / COOH group; Magsphere) so that the liquid volume ratio is 40: 1. And reacted. Next, 1 (W / V)% EDAC (N- (3-Dimethlaminopropyl) -N′-ethylcarbodiimide hydrochloride; Sigma) was added to a final concentration of 0.1%, followed by reaction for 2 hours. After washing, it floats in 20mL of the final suspension (5mM Tris, 0.04 (W / V)% BSA (bovine serum albumin), 0.4M trehalose, 0.2 (V / V)% TritonX-100). The latex particles were dispersed.

(2) Preparation of latex particle-labeled anti-influenza B virus antibody One type of anti-influenza B virus NP monoclonal antibody was prepared in a 50 mM MES (2-Morpholinoethanesulfonic acid, monohydrate) buffer solution (pH 6.0). After dialysis, 10 mL of a solution diluted with the same buffer was prepared so that OD _280nm = 0.5. Then 10 (W / V)% blue polystyrene latex particles (particle size 0.45 [mu] m, the surface functional group is a carboxyl group, a functional group density 65 Å ² / COOH group; Magsphere Co.) and liquid volume ratio of 40: mixed at a 1 And reacted. Next, 1 (W / V)% EDAC (N- (3-Dimethlaminopropyl) -N′-ethylcarbodiimide hydrochloride; Sigma) was added to a final concentration of 0.1%, followed by reaction for 2 hours. After washing, the sample was 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 an ultrasonic dispersion device (Olympus) The latex particles were dispersed.

(3) Preparation of latex particle-labeled anti-RS virus antibody One type of anti-RS virus FP monoclonal antibody was dialyzed with 50 mM MES buffer solution (pH 6.0) and diluted with the same buffer solution so that _OD280nm = 0.5. 10 mL of the solution was prepared. Then 10 (W / V)% green polystyrene latex particles (particle size 0.49 .mu.m, the surface functional group is a carboxyl group, a functional group density 69 Å ² / COOH group; Merck) and liquid volume ratio of 40: mixed at a 1 And reacted. Next, 1 (W / V)% EDAC (N- (3-Dimethlaminopropyl) -N′-ethylcarbodiimide hydrochloride; Sigma) was added to a final concentration of 0.1%, followed by reaction for 2 hours. After washing, the sample was 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 an ultrasonic dispersion device (Olympus) The latex particles were dispersed.

3. 2. Drying of latex particle labeled antibody (1) Mixing of latex particle labeled antibody The latex concentration of the latex particle-labeled anti-type A influenza virus antibody, latex particle-labeled anti-type B influenza virus antibody and latex particle-labeled anti-RS virus antibody prepared in (1), (2) and (3) is 0.3 (W / V)%, so that 2. (1) After dilution with the final suspension described above, the mixture was stirred at 150 rpm for 5 minutes at room temperature and mixed in equal amounts.

(2) Production of latex particle-labeled antibody dry pad The latex particle-labeled antibody prepared in (1) was 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). After spraying, warm air of 50 ° C. was blown for 1 minute to dry, thereby preparing a latex particle-labeled antibody drying pad.

4). Preparation of antibody for membrane solid phase (1) Preparation of anti-influenza A virus antibody for solid phase The purified anti-influenza A virus NP monoclonal antibody prepared in (1), which was not used for labeling, was dialyzed against a solid phase solution (10 mM Tris-HCl (pH 7.5)), and 0.22 μm filtered after dialysis. The solution was diluted with a solid phase solution so that OD _{280 nm} = 4.0, and an anti-influenza A virus antibody for solid phase was prepared.

(2) Preparation of anti-influenza B virus antibody for solid phase The purified anti-influenza B virus NP monoclonal antibody prepared in (2), which was not used for labeling, was dialyzed against a solid phase solution (10 mM Tris-HCl (pH 7.5)), and 0.22 μm filtered after dialysis. The solution was diluted with a solid phase solution so that OD _280nm = 3.0, and an anti-type B influenza virus antibody for solid phase was prepared.

(3) Preparation of anti-RS virus antibody for solid phase The purified anti-RS virus FP monoclonal antibody prepared in (3), which was not used for labeling, was dialyzed against a solid phase solution (10 mM Tris-HCl (pH 7.5), 1 (W / V)% trehalose). After dialysis, 0.22 μm filtration was performed, and dilution with a solid phase solution was performed so that OD _{280 nm} = 3.0 to prepare an anti-RS virus antibody for solid phase.

5. Production of Lateral Flow Membrane Assay Device for Detection of Influenza and RS Virus A lateral flow membrane assay device for detection of influenza and RS virus was used having the same configuration as that shown in FIGS.

The membrane used was a nitrocellulose membrane (pore diameter 12 μm; manufactured by Whatman) sheet (white) having a width of 3 cm and a length of 10 cm (d in FIGS. 1 and 2). A positive pressure spraying device with a coating amount of 1 μL / cm of anti-influenza A virus antibody for solid phase at a position e of 6 mm away from one end of the long axis side (this end is the upstream end and the opposite side is the downstream end) (BioJet; BioDot) was applied in a line, and a positive pressure spraying device (BioJet; BioDot) was applied at a coating amount of 1 μL / cm for anti-influenza B virus antibody for solid phase at a position f 10 mm away. The solid-phase anti-RS virus antibody was applied linearly at a coating amount of 1 μL / cm at a position of 14 mm away using a positive pressure spray device (BioJet; BioDot). The anti-mouse IgG antibody was diluted to OD _280nm = 1.0 at a position h of 22 mm away, and applied in a linear manner using a positive pressure spray device (BioJet; BioDot) at a coating amount of 1 μL / cm. After the application, it was dried by blowing warm air of 45 ° C. for 10 minutes.

  Next, in order to fix the member and increase the strength, a plastic backing sheet (BioDot) is bonded to the opposite side (this surface is the lower surface) of the membrane antibody application surface (this surface is the upper surface). (I in FIG. 2).

  Next, the latex particle labeled antibody drying pad (b in FIGS. 1 and 2) prepared in 3 (2) above is cut into a width of 15 mm and a length of 10 cm so that the upstream end of the membrane overlaps with the upper surface of the membrane by 2 mm. Place and paste a cellulose filter paper (Wattman) 23 mm wide x 10 cm long on the top of the latex particle-labeled antibody drying pad so as to overlap 13 mm. A).

  Next, cellulose filter paper (Wattman Co., Ltd.) having a width of 30 mm and a length of 10 cm is attached to the upper surface of the membrane so as to overlap the downstream end of the membrane by 5 mm, and a sample absorption pad (FIG. 1 and FIG. 2 c) and did.

  Next, 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 dropping pad (j in FIG. 2).

  Finally, the membrane assay apparatus shown in FIGS. 1 and 2 was produced by cutting along the long axis direction by 5 mm.

6). Preparation of sample filtration filter Prepare a nozzle for filtration as shown in Fig. 5n, punch the member shown in Table 1 into a circle (diameter 0.7cm), and load it on the bottom of the nozzle, as shown in Table 2 A sample filtration filter was prepared. When two or more members were stacked as a filtration filter, they were counted as the first, second, and third steps from the bottom side.

7). Detection of influenza virus and RS virus (1) Sample collection and sample preparation Nasal aspirate was collected from patients clinically suspected of influenza virus or RS virus infection. First, a part thereof was suspended in 2 mL of virus isolation medium, and using this suspension, it was confirmed by RT-PCR method whether influenza virus gene or RS virus gene was present in the specimen. The RT-PCR method for detecting influenza virus genes was performed by the method of Shimizu (Journal of Infectious Diseases, Vol. 71, No. 6, p522-526). The RT-PCR method for detecting RS virus gene was performed by the method of Stockton et al. (Journal of Clinical Microbiology, Vol. 36, p2990-2995, 1998). As a result, 10 samples of nasal aspirate fluid from which influenza A virus gene was detected, 10 samples of nasal aspirate fluid from which influenza B virus gene was detected, 10 samples of nasal aspirate fluid from which RS virus gene was detected, and Ten specimens of nasal aspirate from which no gene was detected were selected and used in subsequent tests.

  A conventional cotton swab (with a water absorption of 90 μL of sample collection part; microRheologics, Inc.) and a cotton swab wound around the shaft to form a cotton ball on the selected nasal aspirate fluid. A sample suspension (100mL; Nippon Cotton Swab Co., Ltd.) was soaked for 10 seconds and then pulled up and dispensed into a tube as shown in Fig. 6 (20 mM MES buffer (pH 6.0), 1 (W / V)% TritonX-100, 5 (W / V)% arginine hydrochloride, 1.0 (W / V)% bovine serum albumin) were floated in 0.3 mL, and specimen samples were prepared. A total of six specimen samples were prepared using three brush-like swabs and three conventional cotton swabs per specimen of nasal aspirate.

(2) Detection by membrane assay method 6. At the tip of six tubes containing specimen samples prepared from one specimen of nasal aspirate. Two each of the various types of sample filtration filters prepared in the above were attached. Table 3 shows combinations of the types of cotton swabs used for specimen collection and the types of sample filtration filters.

The collected filtrate is passed through the nozzle for filtration and collected in a tube, and then the above 5. The sample dripping pad side of the lateral flow membrane assay device for detecting influenza and RS virus prepared in 1 was immersed in the solution. 10 minutes later, the assay device is observed, and the case where color development is observed at the position h (control line) in FIG. 1 or FIG. 2 is effective, and the color development of the colored latex used for the labeling (position e) In case of red), positive for influenza A virus, positive coloration of colored latex at position f (in this case, blue), positive for influenza B virus, position at g When color development of colored latex (green in this case) was observed, it was judged as positive for RS virus, and negative when no color development was observed at any position. Also, the case where no color development was observed at the position h was regarded as invalid.
The results of the test are shown in Table 3.

Explanation of results Positive type A influenza: When tested by a membrane assay using a sample prepared from a sample from which the type A influenza virus gene was detected by RT-PCR, only the type A influenza virus was determined to be positive. In case of influenza B positive: when tested by membrane assay using a sample prepared from a sample from which the influenza B virus gene was detected by RT-PCR, only influenza B virus was determined to be positive Case
RS positive: When a sample sample prepared from a sample in which the RS virus gene was detected by RT-PCR was tested by the membrane assay method, only RS virus was determined to be negative. When a sample prepared from a sample in which no gene was detected was tested as negative when tested by a membrane assay method Non-specific: A gene was detected from the sample by RT-PCR when tested If the virus that was not detected was determined to be positive by the membrane assay (in addition to the virus for which the gene was detected by RT-PCR, the virus for which the gene was not detected was also determined to be positive by the membrane assay. Including the case where
Invalid: When color development is not recognized at the position h in the membrane assay method, invalid or false due to clogging by filtering the specimen sample prepared from the nasal aspirate with a sample filtration filter containing a sintered filter in a simple membrane assay It was found that measurement with high sensitivity and specificity is possible by preventing positiveness and combining with a brush-like swab.

[Example 2] Detection of influenza virus and RS virus in nasal aspirate by lateral flow immunochromatographic assay 2
1. Production of Lateral Flow Membrane Assay Device for Detection of Influenza and RS Virus The same one used in Example 1 was used.

2. Preparation of sample filtration filter Prepare a nozzle for filtration as shown in Fig. 5n, punch out the members shown in Table 4 in a circular shape (0.7cm in diameter), load them on the bottom of the nozzle, and configure as shown in Table 5 A sample filtration filter was prepared. When two or more members were stacked as a filtration filter, they were counted as the first, second, and third steps from the bottom side. Of the two types of filters included in the sample filtration filter, a filter with a small pore size is called a fine filtration filter, and a filter with a large pore size is called a coarse filtration filter.

3. Detection of influenza virus and RS virus (1) Collection of specimen and preparation of specimen sample From the nasal aspirate used in Example 1, nasal aspiration in which influenza A virus gene was detected by the method described in Example 1 10 samples of liquid, 7 samples of nasal aspirate fluid in which influenza B virus gene was detected, 10 samples of nasal aspirate fluid in which RS virus gene was detected, and 10 samples of nasal aspirate fluid in which no gene was detected Selected and used for subsequent tests.

  A sample-like suspension (20 mM MES buffer (pH 6.0), 1) which was pulled up after immersing a brush-like swab whose head is made of nylon fiber in nasal aspirate for 10 seconds and then dispensed into a tube as shown in FIG. (W / V)% TritonX-100, 5 (W / V)% arginine hydrochloride, 1.0 (W / V)% bovine serum albumin) were suspended in 0.3 mL, and a specimen sample was prepared. Six specimen samples were prepared for one specimen of nasal aspirate.

(2) Detection by membrane assay method The above-mentioned 2. is attached to the tip of six tubes containing specimen samples prepared from one specimen of nasal aspirate. Each of the various sample filtration filters A, D, E, F, G, and H prepared in Step 1 was attached, and the test was performed in the same manner as in Example 1.

  The results of the test are shown in Table 6. The explanation of the results is the same as that described in Table 3 of Example 1.

  Even if any sample filtration filter of DH was used, the effect was seen compared with the case where the sample filtration filter was not used, but the pore size of the filter for microfiltration was larger than the pore size of the membrane, or for coarse filtration. When a filter with a filter pore size of 200 μm or more was used, a sample determined to be non-specific or invalid was generated.

[Example 3] Detection of influenza virus and RS virus in nasal wipes by lateral flow immunochromatographic assay Production of Lateral Flow Membrane Assay Device for Detection of Influenza and RS Virus The same one used in Example 1 was used.
2. Production of Sample Filtration Filter A, C and D among the sample filtration filters produced in Examples 1 and 2 were used.

3. Detection of influenza virus and RS virus (1) Sample collection and sample preparation Brushed swabs made of nylon fibers from patients clinically suspected of influenza virus or RS virus infection (water absorption in sample collection part) 90 μL; microRheologics) was used to collect 3 nasal wipes and 1 pharyngeal wipe. First of all, the pharyngeal wiping solution was suspended in 2 mL of virus isolation medium, and this suspension was used to perform 7. of Example 1. (1) The presence of influenza virus gene or RS virus gene in the specimen was confirmed by the RT-PCR method described. As a result, 5 patients with influenza A virus gene were detected from the collected throat swabs, 5 patients with influenza B virus gene were detected, and 5 patients with RS virus gene detected. And 5 patients who did not detect any gene, and the sample suspension (20 mM MES buffer (pH 6.0)) in which the nasal wipes collected from the patients were dispensed into a tube as shown in FIG. 1 (W / V)% TritonX-100, 5 (W / V)% arginine hydrochloride, 1.0 (W / V)% bovine serum albumin) were suspended in 0.3 mL, and specimen samples were prepared.

(2) Detection by membrane assay 2. At the tip of three tubes containing specimen samples prepared from nasal wipes collected from one patient. Each of the sample filtration filters A, C, and D prepared in 1 was attached, and the test was performed in the same manner as in Example 1.

  The test results are shown in Table 7. The explanation of the results is the same as that described in Table 3 of Example 1.

  It was found that the sample sample prepared from the nasal wiping solution in the simple membrane assay was filtered with a sample filtration filter including a sintered filter to prevent invalidity and false positives due to clogging.

[Example 4] Detection of influenza virus and RS virus in throat swabs by lateral flow immunochromatographic assay Production of Lateral Flow Membrane Assay Device for Detection of Influenza and RS Virus The same one used in Example 1 was used.
2. Production of Sample Filtration Filter A, C and D among the sample filtration filters produced in Examples 1 and 2 were used.

3. Detection of influenza virus and RS virus (1) Sample collection and sample preparation Brushed swabs made of nylon fibers from patients clinically suspected of influenza virus or RS virus infection (water absorption in sample collection part) 90 μL; microRheologics) was used to collect 3 pharyngeal wipes and 1 nasal wipe. First of all, the nasal wiping solution was suspended in 2 mL of virus isolation medium, and this suspension was used in Example 7. (1) The presence of influenza virus gene or RS virus gene in the specimen was confirmed by the RT-PCR method described. As a result, 5 patients with influenza A virus gene were detected from the collected throat swabs, 5 patients with influenza B virus gene were detected, and 5 patients with RS virus gene detected. And 5 patients who did not detect any gene, and the sample suspension (20 mM MES buffer (pH 6.0)) in which the nasal wipes collected from the patients were dispensed into a tube as shown in FIG. 1 (W / V)% TritonX-100, 5 (W / V)% arginine hydrochloride, 1.0 (W / V)% bovine serum albumin) were suspended in 0.3 mL, and specimen samples were prepared.

(2) Detection by membrane assay 2. At the tip of three tubes containing specimen samples prepared from a throat swab collected from one patient. Each of the sample filtration filters A, C, and D prepared in 1 was attached, and the test was performed in the same manner as in Example 1.
The test results are shown in Table 8. The explanation of the results is the same as that described in Table 3 of Example 1.

簡易メンブレンアッセイにおいて咽頭拭い液から調製した検体試料を、焼結フィルターを含む試料ろ過フィルターでろ過することにより、目詰まりによる無効や偽陽性を防ぐことが判明した。

It was found that the sample sample prepared from the pharyngeal wiping solution in the simple membrane assay was filtered with a sample filtration filter including a sintered filter to prevent invalidity and false positives due to clogging.

[Example 5] Detection of adenovirus in throat swab by lateral flow immunochromatographic assay Production of monoclonal antibody (1) Adenovirus hexon protein monoclonal antibody (mouse)
A spleen was excised from a BALB / c mouse that had been immunized with a polypeptide obtained by synthesizing a part of the amino acid sequence of human adenovirus hexon protein and maintained for a certain period of time (Kohler et al., Nature, vol, 256 , p495-497 (1975)) and fused with mouse myeloma cells (P3 × 63).

  Maintain the resulting fused cells (hybridomas) in a 37 ° C incubator and purify the cells (monocloning) while confirming the antibody activity of the supernatant by ELISA using an adenovirus hexon protein antigen solid phase plate. went.

  The obtained two cell lines were each intraperitoneally administered to pristane-treated BALB / c mice, and about 2 weeks later, antibody-containing ascites was collected. IgG was purified from the obtained ascites by affinity purification using Protein A column chromatography (Amersham) to obtain two types of purified anti-adenovirus hexon protein monoclonal antibodies.

2. Preparation of labeled anti-adenovirus hexon protein antibody (labeling substance) (1) Preparation of latex particle-labeled anti-adenovirus hexon protein antibody One type of anti-adenovirus hexon protein antibody is 50 mM MES (2-Morpholinoethanesulfonic acid, monohydrate) Dojindo Chemical Co., Ltd.) After dialysis with a buffer solution (pH 6.0), 10 mL of a solution diluted with the same buffer solution was prepared so that _OD280nm = 0.5. Then 10 (W / V)% blue polystyrene latex particles (particle size 0.45 [mu] m, the surface functional group is a carboxyl group, a functional group density 65 Å ² / COOH group; Magsphere Co.) and liquid volume ratio of 40: mixed at a 1 And reacted. Next, 1 (W / V)% EDAC (N- (3-Dimethlaminopropyl) -N′-ethylcarbodiimide hydrochloride; Sigma) was added to a final concentration of 0.1%, followed by reaction for 2 hours. After washing, the suspension is 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). (Olympus) to disperse latex particles.

3. 1. Drying of latex particle labeled antibody (1) Preparation of latex particle labeled antibody dry pad The latex particle-labeled antibody prepared in (1) was sprayed on the whole surface of a cellulose nonwoven fabric having a width of 15 mm wound in a reel at a coating amount of 8 μL / cm using a positive pressure spraying device (BioJet; BioDot). After spraying, warm air of 50 ° C. was blown for 1 minute to dry, thereby preparing a latex particle-labeled antibody drying pad.

4). Preparation of antibody for membrane solid phase (1) Preparation of anti-adenovirus hexon protein antibody for solid phase The purified anti-adenovirus hexon protein antibody prepared in (1), which was not used for labeling, was dialyzed against a solid phase solution (10 mM Tris-HCl (pH 7.5)), 0.22 μm filtered after dialysis, A solid phase anti-adenovirus hexon protein antibody was prepared by diluting with a solid phase solution so that _OD280nm = 4.0.

5. Production of Lateral Flow Membrane Assay Device for Detection of Adenovirus A lateral flow membrane assay device for detection of adenovirus was used having the same configuration as that shown in FIGS.

The membrane used was a nitrocellulose membrane (pore diameter 12 μm; manufactured by Whatman) sheet (white) having a width of 3 cm and a length of 10 cm (d in FIGS. 1 and 2). Positive-pressure spraying of anti-adenovirus hexon protein antibody for solid phase at a coating amount of 1 μL / cm 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) Using a device (BioJet; BioDot), the anti-mouse IgG antibody was diluted to OD _280nm = 1.0 at a position h of 22 mm away, and a positive pressure spray device (BioJet; BioDot) was applied linearly. Nothing was applied to the positions of f and g. After the application, it was dried by blowing warm air of 45 ° C. for 10 minutes.

  Next, in order to fix the member and increase the strength, a plastic backing sheet (BioDot) is bonded to the opposite side (this surface is the lower surface) of the membrane antibody application surface (this surface is the upper surface). (I in FIG. 2).

  Next, the latex particle labeled antibody drying pad (b in FIGS. 1 and 2) prepared in 3 (2) above is cut into a width of 15 mm and a length of 10 cm so that the upstream end of the membrane overlaps with the upper surface of the membrane by 2 mm. Place and paste a cellulose filter paper (Wattman) 23 mm wide x 10 cm long on the top of the latex particle-labeled antibody drying pad so as to overlap 13 mm. A).

  Next, cellulose filter paper (Wattman Co., Ltd.) having a width of 30 mm and a length of 10 cm is attached to the upper surface of the membrane so as to overlap the downstream end of the membrane by 5 mm, and a sample absorption pad (FIG. 1 and FIG. 2 c) and did.

  Next, 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 dropping pad (j in FIG. 2).

  Finally, the membrane assay apparatus shown in FIGS. 1 and 2 was produced by cutting along the long axis direction by 5 mm.

6). Production of Sample Filtration Filter A, C and D among the sample filtration filters produced in Examples 1 and 2 were used.

7). Detection of adenovirus (1) Collection of specimen and preparation of specimen A brush-like swab with a head of nylon fiber from a patient suspected of clinically adenovirus infection (water absorption of specimen collection part: 90μL; microRheologics) 4 pharyngeal wipes were collected. First, one of them is suspended in 2 mL of virus isolation medium, and this suspension is used to the method of Fujimoto et al. (Single-tube multiplex PCR for rapid and sensitive diagnosis of subgenus B and other subgenera adenovirus in clinical samples. Microbiology and Immunology. 44. 821-826, 2000), the presence of adenovirus gene in the specimen was confirmed by PCR. Based on the results, 5 patients with adenovirus gene detected and 5 patients with no adenovirus gene detected were selected from the collected pharyngeal wipes, and the pharyngeal wipes collected from these patients are shown in FIG. Sample suspension (20 mM MES buffer (pH 6.0), 1 (W / V)% TritonX-100, 5 (W / V)% arginine hydrochloride, 1.0 (W / V) )% Bovine serum albumin) each suspended in 0.3 mL to prepare a specimen sample.

(2) Detection by membrane assay 2. At the tip of three tubes containing specimen samples prepared from a throat swab collected from one patient. Each of the sample filtration filters A, C, and D prepared in 1 was attached, and the test was performed in the same manner as in Example 1.

  The test results are shown in Table 9. Ten minutes later, the assay apparatus is observed, and the case where blue color development is recognized at the position (control line) h in FIG. 1 or FIG. 2 is effective, and when blue color development is recognized at the position e, adeno is observed. The virus was judged positive. Also, the case where no color development was observed at the position h was regarded as invalid.

Description of results Adenovirus positive: Negative when adenovirus is positive when tested by membrane assay using a sample prepared from a sample in which adenovirus gene was detected by PCR: Adenovirus by PCR When it is determined to be negative when tested by a membrane assay using a sample prepared from a sample in which no viral gene was detected Non-specific: Adenoviral gene was detected from the sample by PCR when tested When it is determined to be adenovirus-positive when tested in a membrane assay using a sample prepared from a sample that did not exist Invalid: When no color development is observed at position h in the membrane assay

  It was found that the sample sample prepared from the pharyngeal wiping solution in the simple membrane assay was filtered with a sample filtration filter including a sintered filter to prevent invalidity and false positives due to clogging.

[Example 6] Detection of norovirus in rectal wipes by lateral flow immunochromatographic assay Production of monoclonal antibody (1) Anti-Norovirus capsid protein monoclonal antibody (mouse)
Viral hollow particles (VLPs) were prepared by protein expression by recombinant baculovirus introduced with a gene encoding capsid protein of Norovirus. The spleen was removed from BALB / c mice that had been immunized and maintained for a certain period of time, and mouse myeloma cells (P3 × 63) were obtained by the method of Keller et al. (Kohler et al., Nature, vol. 256, p495-497 (1975)). )

  The obtained fused cells (hybridoma) were maintained in a 37 ° C. incubator, and the cells were purified (monocloned) while confirming the antibody activity of the supernatant by ELISA using a VLPs solid phase plate.

  The obtained two cell lines were each intraperitoneally administered to pristane-treated BALB / c mice, and about 2 weeks later, antibody-containing ascites was collected. IgG was purified from the obtained ascites by affinity purification using Protein A column chromatography (Amersham) to obtain two purified anti-norovirus capsid protein monoclonal antibodies.

2. Preparation of labeled anti-norovirus capsid protein antibody (labeling substance) (1) Preparation of latex particle-labeled anti-norovirus capsid protein antibody One type of anti-norovirus capsid protein antibody was buffered with 50 mM MES (2-Morpholinoethanesulfonic acid, monohydrate). After dialysis with a solution (pH 6.0), 10 mL of a solution diluted with the same buffer was prepared so that OD _{280 nm} = 0.5. Then 10 (W / V)% blue polystyrene latex particles (particle size 0.45 [mu] m, the surface functional group is a carboxyl group, a functional group density 65 Å ² / COOH group; Magsphere Co.) and liquid volume ratio of 40: mixed at a 1 And reacted. Next, 1 (W / V)% EDAC (N- (3-Dimethlaminopropyl) -N′-ethylcarbodiimide hydrochloride; Sigma) was added to a final concentration of 0.1%, followed by reaction for 2 hours. After washing, the suspension is 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). (Olympus) to disperse latex particles.

3. 1. Drying of latex particle labeled antibody (1) Preparation of latex particle labeled antibody dry pad The latex particle-labeled antibody prepared in (1) was sprayed on the whole surface of a cellulose nonwoven fabric having a width of 15 mm wound in a reel at a coating amount of 8 μL / cm using a positive pressure spraying device (BioJet; BioDot). After spraying, warm air of 50 ° C. was blown for 1 minute to dry, thereby preparing a latex particle-labeled antibody drying pad.

4). Preparation of antibody for membrane solid phase (1) Preparation of anti-norovirus capsid protein antibody for solid phase The purified anti-Norovirus capsid protein antibody prepared in (1), which was not used for labeling, was dialyzed against a solid phase solution (10 mM Tris-HCl (pH 8.0)), filtered through 0.22 μm, and OD _{280 nm} A solid phase anti-Norovirus capsid protein antibody was prepared by diluting with a solid phase solution so that = 3.0.

5. Production of Lateral Flow Membrane Assay Device for Detection of Norovirus A lateral flow membrane assay device for detection of norovirus was used having the same configuration as that shown in FIGS.

The membrane used was a nitrocellulose membrane (pore diameter 12 μm; manufactured by Whatman) sheet (white) having a width of 3 cm and a length of 10 cm (d in FIGS. 1 and 2). A positive pressure spraying device with an application amount of 1 μL / cm of the anti-norovirus capsid protein antibody for solid phase at a position e of 6 mm away from one end of the long axis side (this end is the upstream end and the opposite side is the downstream end) BioJet; BioDot) was applied linearly, anti-mouse IgG antibody was diluted to OD _280nm = 1.0 at a position h of 22 mm away, and a positive pressure spray device (BioJet; BioDot) was applied at a coating amount of 1 μL / cm. ) Was applied linearly. Nothing was applied to the positions of f and g. After the application, it was dried by blowing warm air of 45 ° C. for 10 minutes.

  Next, in order to fix the member and increase the strength, a plastic backing sheet (BioDot) is bonded to the opposite side (this surface is the lower surface) of the membrane antibody application surface (this surface is the upper surface). (I in FIG. 2).

  Next, the latex particle labeled antibody drying pad (b in FIGS. 1 and 2) prepared in 3 (2) above is cut into a width of 15 mm and a length of 10 cm so that the upstream end of the membrane overlaps with the upper surface of the membrane by 2 mm. Place and paste a cellulose filter paper (Wattman) 23 mm wide x 10 cm long on the top of the latex particle-labeled antibody drying pad so as to overlap 13 mm. A).

  Next, cellulose filter paper (Wattman Co., Ltd.) having a width of 30 mm and a length of 10 cm is attached to the upper surface of the membrane so as to overlap the downstream end of the membrane by 5 mm, and a sample absorption pad (FIG. 1 and FIG. 2 c) and did.

  Next, 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 dropping pad (j in FIG. 2).

  Finally, the membrane assay apparatus shown in FIGS. 1 and 2 was produced by cutting along the long axis direction by 5 mm.

6). Production of Sample Filtration Filter A, C and D among the sample filtration filters produced in Examples 1 and 2 were used.

7). Detection of norovirus (1) Collection of specimen and preparation of specimen using clinically suspected norovirus infection patients with a brush-like swab (90 μL of sample collection part; microRheologics) Four rectal wipes were collected. First, one of them is suspended in 2 mL of virus isolation medium, and this suspension is used to monitor the RT-PCR method (No. 1105001, food safety supervision dated November 5, 2003, Food Safety Department, Ministry of Health, Labor and Welfare, Food Safety Department) According to the section manager's notice), it was confirmed whether the norovirus gene was present in the sample. From the results, 5 patients with norovirus gene detected and 5 patients with norovirus gene detected were selected from the collected rectal wipes, and the rectal wipes collected from these patients were as shown in FIG. Sample suspension (20 mM MES buffer (pH 6.0), 1 (W / V)% TritonX-100, 5 (W / V)% Arginine hydrochloride, 1.0 (W / V)% Bovine serum albumin) was suspended in 0.3 mL, and specimen samples were prepared.

(2) Detection by membrane assay 2. At the tip of three tubes containing specimen samples prepared from rectal wipes collected from one patient. Each of the sample filtration filters A, C, and D prepared in 1 was attached, and the test was performed in the same manner as in Example 1.

  Table 10 shows the results of the test. Ten minutes later, the assay apparatus is observed, and when blue color is observed at the position h (control line) in FIG. 1 or FIG. 2, it is effective, and when blue color is observed at the position e, norovirus Positive. Also, the case where no color development was observed at the position h was regarded as invalid.

Description of results Norovirus positive: When norovirus is positive when tested by membrane assay using a sample prepared from a sample with norovirus gene detected by RT-PCR: Negative: RT-PCR When it is judged negative when tested by membrane assay using a sample prepared from a sample in which norovirus gene was not detected Non-specific: When tested, RT-PCR detects norovirus gene from sample When norovirus is determined to be positive when tested in a membrane assay using a specimen prepared from a specimen that has not been tested, invalid: When no color is observed at position h in the membrane assay

  It was found that the sample sample prepared from the rectal wiping solution in a simple membrane assay was filtered with a sample filtration filter including a sintered filter to prevent invalidity and false positives due to clogging.

[Example 7] Detection of norovirus in feces by lateral flow immunochromatographic assay Preparation of Lateral Flow Membrane Assay Device for Norovirus Detection The same one used in Example 6 was used.
2. Production of Sample Filtration Filter A, C and D among the sample filtration filters produced in Examples 1 and 2 were used.

3. Detection of norovirus (1) Collection of specimen and preparation of specimen A brush-like swab made of nylon fiber from the stool of a patient suspected of having a clinically norovirus infection (water absorption of the specimen collection part: 90μL; microRheologics) Using this, 4 specimens were collected. First, one of them is suspended in 2 mL of virus isolation medium, and this suspension is used to monitor the RT-PCR method (No. 1105001, food safety supervision dated November 5, 2003, Food Safety Department, Ministry of Health, Labor and Welfare, Food Safety Department) According to the section manager's notice), it was confirmed whether the norovirus gene was present in the sample. Based on the results, five patients with norovirus gene detected from the collected feces and five patients with no norovirus gene detected were selected, and the feces collected from the patients were placed in a tube as shown in FIG. Dispensed specimen suspension (20 mM MES buffer (pH 6.0), 1 (W / V)% TritonX-100, 5 (W / V)% arginine hydrochloride, 1.0 (W / V)% bovine serum albumin) Sample samples were prepared by floating in 0.3 mL.

(2) Detection by membrane assay 2. At the tip of three tubes containing specimen samples prepared from stool collected from one patient. Each of the sample filtration filters A, C, and D prepared in 1 was attached, and the test was performed in the same manner as in Example 1.

  The test results are shown in Table 11. Ten minutes later, the assay apparatus is observed, and when blue color is observed at the position h (control line) in FIG. 1 or FIG. 2, it is effective, and when blue color is observed at the position e, norovirus Positive. Also, the case where no color development was observed at the position h was regarded as invalid.

Explanation of results Same as Example 6.
It was found that the sample sample prepared from stool in a simple membrane assay was filtered with a sample filtration filter including a sintered filter to prevent invalidity and false positives due to clogging.

  The method and apparatus of the present invention can be used for quantification or detection of a specific substance in a subject, and can be used for detection and diagnosis of a disease by measuring a substance related to the disease.

It is a top view of the lateral flow type membrane assay apparatus which is one embodiment of this invention. FIG. 2 is an end view taken along the line I-I ′ of FIG. 1. It is a figure which shows an example of the conventional cotton swab. It is a figure which shows an example of a brush-shaped cotton swab. It is a structural diagram showing an example of a sample filtration filter. It is a structural diagram showing an example of a sample tube.

Explanation of symbols

a: Sample dropping pad b: Latex particle-labeled antibody drying pad (labeling substance drying pad)
c: Sample absorption pad d: Nitrocellulose membranes e to g: A position where a capture substance that specifically binds to an object to be detected is bound in a line on d. h: A substance that can bind a labeling substance is d. Position i connected in line above: backing sheet j: transparent plastic laminate k1: sample collection part k2 made of cotton balls wound with cotton k: sample collection part l with fibers arranged in a brush shape l: shaft part m: Head n: Filtration nozzles o1 to o3: Filtration filter p: Sample suspension tube

Claims (17)

In a simple membrane assay method comprising detecting or quantifying the presence of an analyte in a sample on an assay device comprising a membrane to which a capture reagent for capturing the analyte in the sample is bound, human or other Collect a specimen selected from the group consisting of animal pharyngeal wipe, nasal wipe, nasal aspirate, nasal wash, alveolar wash, stool suspension, and rectal wipe, and float the collected sample in the sample suspension A sample is prepared, and the prepared sample is subjected to pressure filtration using a filtration filter including a rigid filter, wherein the rigid filter has a thickness of 0.5 mm to 7 mm and a pore diameter of 10 to 200 μm. Even if the pressure applied to the filter by liquid or air when the specimen sample is filtered exceeds 0.2 [MPa], the amount of change in thickness before filtration vs. filtration does not exceed 5%, or filtration Hour filter Not curvature radius and 2cm or less, a strong rigid filter having a hard rigid block the flow path of the filter inside the filtrate simple membrane assay method.   Even if the pressure applied to the filter by liquid or air when the rigid filter filters the specimen sample exceeds 0.2 [MPa], the amount of change in thickness before filtration vs. filtration does not exceed 2%, or The simple membrane assay method according to claim 1, wherein the filter is a highly rigid filter that does not have a radius of curvature of 2 cm or less during filtration.   It is characterized by pressure-filtering a sample prepared from a specimen by applying pressure to the inside of the filtration tube made of a flexible material and capable of filtering the sample prepared from the specimen. The simple membrane assay method according to claim 1 or 2.   The simple membrane assay method according to any one of claims 1 to 3, wherein the rigid filter is a sintered filter.   The simple membrane assay method according to claim 4, wherein the material of the sintered filter is selected from the group consisting of polypropylene, low density polyethylene, high density polyethylene, ultrahigh molecular weight polyethylene, polystyrene, tetrafluoroethylene polymer, and polymethyl methacrylate. .   When the sintered filter material is a polypropylene rigid filter, the pore size of the filter is 50-200 μm. When the low-density polyethylene rigid filter is 30-70 μm, the pore size of the filter is 30-70 μm. The pore size of the filter is 10 to 50 μm, the pore size of the filter is 10 to 20 μm in the case of ultra-high molecular weight polyethylene rigid filter, the pore size of the filter is 100 to 200 μm in the case of polystyrene rigid filter, and is made of tetrafluoroethylene polymer The simple membrane assay method according to claim 5, wherein the pore size of the filter in the case of a highly rigid filter is 30 to 100 μm.   The simple membrane assay method according to any one of claims 1 to 6, wherein the detected substance in the sample is a microorganism causing respiratory infection, a substance derived from the microorganism, or an antibody against them.   8. The simple substance according to claim 7, wherein the detected substance in the sample is a pathogenic microorganism selected from the group consisting of influenza virus, RS virus, adenovirus, group A streptococci and mycoplasma pneumonia, a substance derived from the microorganism, or an antibody against them. Membrane assay method.   The simple membrane assay method according to any one of claims 1 to 6, wherein the detected substance in the specimen is a microorganism causing diarrhea, a substance derived from the microorganism, or an antibody against them.   The simple membrane assay method according to claim 9, wherein the detected substance in the sample is a pathogenic microorganism selected from the group consisting of norovirus, rotavirus, sapovirus and diarrhea adenovirus, a substance derived from the microorganism, or an antibody thereto. .   The simple membrane assay method according to any one of claims 1 to 10, which is a flow-through type or lateral flow type membrane assay method.   The filtration filter is configured by stacking a plurality of filters having different pore diameters, and the pore diameter of the filter having the minimum pore diameter is equal to or smaller than the pore diameter of the membrane of the assay device including the membrane, and the pore diameter of the filter having the maximum pore diameter is the membrane. The simple membrane assay method according to any one of claims 1 to 11, wherein the simple membrane assay method is at least 200 µm.   13. The simplified membrane assay method according to claim 12, wherein the sample is first passed through a filter having a maximum pore size and then passed through a filter having a smaller pore size. Objects to be detected in a specimen selected from the group consisting of a pharyngeal wipe, nasal wipe, nasal aspirate, nasal wash, alveolar wash, stool suspension, and rectal wipe from humans or other animals , including: Simple membrane assay kit to test for the presence of
(1) The thickness is 0.5mm to 7mm, the pore size is 10 to 200μm, and even if the pressure applied to the filter by liquid or air when the specimen sample is filtered exceeds 0.2 [MPa], it is filtered before filtration. Includes a rigid filter with a rigidity that does not block the flow path of the filtrate inside the filter , and the amount of change in thickness at the time does not exceed 5% or the radius of curvature of the filter during filtration does not become 2 cm or less An assay device comprising a pressure filtration tube for a specimen sample provided with a filtration filter constituted by a filter, and (2) a membrane bound with a capture substance for capturing an object to be detected in the specimen.   Even if the pressure applied to the filter by liquid or air when filtering the sample exceeds 0.2 [MPa], the amount of change in the thickness before filtration vs. filtration does not exceed 2%, or the filter during filtration The simple membrane assay kit according to claim 14, comprising a pressure filtration tube for a specimen sample provided with a filtration filter comprising a filter including a strong rigidity filter whose curvature radius is not 2 cm or less. The simple membrane assay kit according to claim 14 or 15, further comprising at least one of the following (3) to (6):
(3) Specimen suspension,
(4) Cleaning liquid composition,
(5) Labeling substance, and (6) Control solution.   The filtration filter is configured by stacking a plurality of filters having different pore diameters, and the pore diameter of the filter having the minimum pore diameter is equal to or smaller than the pore diameter of the membrane of the assay device including the membrane, and the pore diameter of the filter having the maximum pore diameter is the membrane. The pore size of the filter is 50 to 200 μm when the material of the sintered filter is a polypropylene rigid filter, and the rigid filter is a low-density polyethylene strong filter. The pore size of the filter in the case of 30 to 70 μm, the pore size of the filter in the case of the high-density polyethylene strong filter is 10 to 50 μm, the pore size of the filter in the case of the ultra high molecular weight polyethylene strong filter is 10 to 20 μm, made of polystyrene In the case of a strong rigidity filter, the pore size of the filter is 100 to 200 μm, Filter having a pore size in the case of the ethylene polymer manufactured by strong rigid filter is 30 to 100 [mu] m, simple membrane assay kit according to any one of claims 14 to 16.

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