JP2007139556A - New analysis method and kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analysis method and a kit for collecting an analysis sample from an analysis object or an analysis individual by using an analysis sample collecting apparatus capable of recovering an analysis material in the sample selectively with a high recovery rate, and analyzing the analysis material in the acquired analysis sample with high specificity and excellent high sensitivity. <P>SOLUTION: In the swab-shaped analysis sample collecting apparatus having a head part and a shaft part for recovering the analysis material in the analysis sample selectively with a recovery rate of 50% or higher, a means for collecting liquid by utilizing a capillary phenomenon is arranged on the head part. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention provides an analytical sample collection device capable of selectively recovering an analyte in an analytical sample at a high recovery rate, and a specific qualitative and analytical method for an analyte in a sample using the sample collection device. The present invention relates to an analysis method and apparatus for quantitative determination, and a kit including the sampling instrument.

  In a medical field, it is desired to provide an apparatus and a method that can easily and quickly measure an analyte in an analyte sample. For example, an immunochromatographic analysis method is rapidly spreading due to its simplicity. Immunochromatographic analysis methods are roughly classified into a lateral flow type and a flow-through type according to their structures. In any case, a sample to be analyzed is collected from a subject, the sample is mixed and reacted with a reagent, the mixture is developed on a solid phase part such as a membrane, and reacted with a reagent provided in advance on the solid phase part. This is an analytical method for qualitatively and quantitatively analyzing analytes by looking at the reaction results.

JP-T-2002-508193 discloses a method for detecting influenza virus infection in which a sample is obtained by wiping or aspirating an individual's nasal passage or pharynx with a cotton swab.
Special Table 2002-508193 Publication

  An object of the present invention is obtained by collecting an analyte from an analyte or a subject using an analyte sampling device that can selectively collect an analyte in the sample at a high recovery rate. An analysis method and a kit for analyzing an analyte in an analysis sample with high specificity and excellent sensitivity.

  Conventionally, cotton swabs, platinum ears, spoon tools, and the like have been used as sample collection means for collecting samples from an object to be analyzed or a subject. In particular, when a human body is a subject and a nasal wipe, nasal aspirate, or throat swab is used as a sample to be analyzed, a cotton swab has been mainly used as a sample collecting means. Until now, improvement of specificity and high sensitivity of measurement methods and apparatuses have been studied, but analysis methods and kits including examination of sample collection means for analysis have not been sufficiently studied.

  As a result of diligent research, the present inventors have collected an analyte using an analyte sample collection device that can selectively collect an analyte that collects an analyte in an analyte at a high recovery rate. As a result, it was found that analysis with high specificity and high sensitivity can be performed by using this for analysis, and the present invention has been completed.

Aspects of the present invention are as follows.
[1] A swab-shaped sample-collecting device for selective collection of a substance to be analyzed in a sample to be analyzed with a recovery rate of 50% or more.
[2] The sample-collecting device to be analyzed according to [1], having a head and a shaft, and means for collecting a liquid using capillary action is arranged on the head.
[3] The shape or size of the axial vertical cross section of the head changes depending on the position in the axial direction, and is formed to become thicker than the shaft toward the tip, utilizing the head shape and capillary action [1] or [2] the sample collecting device to be analyzed, wherein the external shape of the means for collecting the liquid is similar.
[4] The sample-collecting device to be analyzed according to any one of [1] to [3], wherein the means for collecting the liquid using the capillary phenomenon is made of a fibrous material and has a uniform thickness of 0.5 mm to 5 mm.
[5] In a means for collecting a liquid using capillary action, fibers made of natural products or artificial products are arranged in a brush shape, arranged in a vertical direction toward the axial center of the swab shaft portion, and a fineness of 1.0 to The analysis sample collecting instrument according to any one of [1] to [4], which is 4.0 dtex.
[6] Analyze the analyte characterized by collecting the analyte using the analyte sampling instrument according to any one of [1] to [5] and measuring the analyte in the analyte how to.
[7] A means for collecting a liquid using the capillary phenomenon of the sample-collecting device for analysis according to any one of [1] to [5] is brought into contact with the sample to be analyzed containing the substance to be analyzed. The method for analyzing an analyte according to [6], wherein the analyte is selectively released from the means, and the released analyte is detected.
[8] Further, the analyte is selectively released from the means for collecting the liquid using capillary action, and is filtered with an analyte sample filtering instrument to detect the analyte. [6] Or [7] a method for analyzing an analyte.
[9] A kit for detecting a substance to be analyzed in a sample to be analyzed, including the sample collecting instrument for analysis according to any one of [1] to [5].
[10] The kit for detecting a substance to be analyzed in the sample to be analyzed according to [9], further including a sample filtering device to be analyzed.

  An analyte to be measured from an analyte sample in which various substances are mixed by using an analyte sample collecting device capable of selectively recovering the analyte in the analyte sample of the present invention at a high recovery rate Can be selectively recovered at a high recovery rate, and a highly sensitive analysis with superior specificity compared to conventional methods and devices can be performed, making it easier, faster, more accurate and easier to judge than conventional methods Inspection can be performed.

  The method of the present invention is carried out using an analytical sample collection instrument that can selectively recover an analyte in the analytical sample of the present invention at a high recovery rate. Moreover, the apparatus and kit of the present invention include an analytical sample collection instrument that can selectively recover an analyte in the analyzed sample of the present invention at a high recovery rate.

  The analysis method used in the method of the present invention and the reaction form to be observed are not particularly limited. For example, an analysis method using an immunochemical reaction such as an antigen-antibody reaction, an analysis method using a chemical reaction, a method using a gene, or any other method may be used. Analytical methods using immunochemical reactions include, for example, SRID (Single radial immunodiffusion) method classified as sedimentation reaction, immunotrophic method or immunoturbidimetric method, direct agglutination reaction classified as agglutination reaction, indirect agglutination reaction or There are reverse passive agglutination reactions, RIA (Radioimmunoassay) method classified as labeling method, EIA (Enzyme immunoassay) method or fluorescence / luminescence immunization method, methods classified into complement binding reaction, etc., but any method can be used . Analytical methods using chemical reactions include, for example, a method in which an enzymatic decomposition reaction is performed to analyze the decomposition products and products, hydrolysis, dehydration reaction, addition polymerization, condensation polymerization (condensation polymerization), oxidation reaction, There is a method of analyzing a decomposition product or a product by causing a chemical reaction such as a reduction reaction or a neutralization reaction, but any form may be used.

  The analysis method used in the method of the present invention is not particularly limited. For example, observation with a microscope, observation with a sorting medium, slide latex reagent for observation of agglutination reaction, fluorescent antibody method reagent, enzyme immunoassay reagent, metabolite detection reagent, immunochromatography reagent, various methods for detecting genes and nucleic acids Etc., but any method is acceptable. These analysis methods may be performed by a technique, by an apparatus or a kit applying the above analysis method, or by a machine such as an immunological automatic analyzer, biochemical automatic analyzer or PCR apparatus. .

  In the method of the present invention, an analyte sample is collected using an analyte sample collecting device that selectively collects an analyte at a high recovery rate, and the analyte sample is transferred from the analyte sample collecting means to the test application means. The test application means is not particularly limited. For example, there are a culture medium, a plate, a microplate, a preparation, a test tube, a tube, a tube for centrifugation, a chromatostrip, etc., but any test application means may be used. The movement of the sample to be analyzed is not particularly limited, and may be carried out by smearing, dissolving or suspending in a suitable liquid, or smearing the dissolved liquid or suspension, and any method may be used. . Further, the sample-collecting device to be analyzed or a part of the sample-collecting device can be directly used for analysis, or the sample-collecting device to be analyzed and the test application means can be integrated.

  There are no particular limitations on the analyte to be used in the method of the present invention, and in addition to soil, air, river water, seawater, culture media, medical instruments, animals such as walls, floors, doorknobs, elevator buttons, escalator belts, and humans An environmental article or the like (hereinafter referred to as an environmental material) to which a substance emitted from the product adheres can be an object to be analyzed.

  There is no particular limitation on the subject used in the method of the present invention. Animals, humans, microorganisms, and all living organisms can be subjects.

  There is no particular limitation on the analyte to be used in the method of the present invention. For example, any substance such as protozoa, fungus, bacteria, mycoplasma, rickettsia, chlamydia, and virus can be the analyte. Any substance such as a toxin, enzyme, polypeptide, protein, sugar chain, glycoprotein, lipid, DNA, RNA or the like can be an analyte. The whole substance or a part of the substance can be the analyte. In addition, antibodies using them as antigens can also be analytes.

  There is no particular limitation on the sample to be analyzed used in the method of the present invention, but the method of the present invention is an analyte sample collection device that can selectively recover the analyte in the sample to be analyzed at a high recovery rate. In this case, it is necessary to apply the sampling device. Specifically, substances in soil, air, water, culture medium, and medical substances and deposits on environmental articles themselves can be analyzed samples, but if necessary, dissolve or suspend them in an appropriate liquid. The solution can be used as a sample to be analyzed by performing a treatment such as the above. Blood, urine, sputum, saliva, vomit, feces, and other substances in the body of humans and human bodies, substances that have been released into the body, and substances that have been removed from the body themselves can also be samples to be analyzed. The solution can be used as a sample to be analyzed by performing a treatment such as dissolving or suspending in a liquid.

  Here, a sample to be analyzed is collected from the subject or the object to be analyzed, and the substance to be analyzed is contained in the sample to be analyzed. The analyte can be selectively recovered from the sample to be analyzed by using an analyte sampling device that can selectively recover the analyte in the sample of the present invention at a high recovery rate.

  When an analyte sample is collected using an analyte sample collection device that can selectively recover an analyte in the analyte sample of the present invention used in the method of the present invention at a high recovery rate, In particular, it means that the analyte can be selectively recovered from a large number of substances that are mixed together. In the present invention, such a state is also referred to as an irreversible state of adsorption / dissociation of the analyte.

  In the present invention, “selectively recovering the analyte in the sample to be analyzed at a high recovery rate” may be “dissociating the analyte in the sample to be analyzed at a high dissociation rate”. In other words, an analyte sample collection device capable of selectively recovering an analyte in a sample of the present invention at a high recovery rate is an irreversible analyte that dissociates the analyte in the analyte at a high dissociation rate. Analytical sample collection device, the analysis sample collected in the step of collecting from the analyte or individual to be analyzed, the next step, for example, a process of dissolving or suspending the analysis sample in a liquid or In the process of providing and mixing with the reagent used for detection of the analyte, the amount of the analyte to be retained in the irreversible analyte sampling instrument used for collection is small, and the amount released in the solution or reagent is increased. Refers to analytical sample collection means.

  The recovery rate of the analyte is the next step, for example, a process of dissolving or suspending in a liquid, with respect to the total amount of the analyte collected in the step of selectively collecting from the analyte or subject, or It can be represented by the ratio of the total amount of the analyte released in the process of using the reagent. The analyte is selectively recovered from the sample to be analyzed with a high recovery rate, that is, the analyte in the sample to be analyzed is recovered with a high recovery rate. When the sample to be analyzed is collected, and the sample to be analyzed retained in the sampling device is subjected to a treatment such as dissolving or suspending in a liquid as described above, or when it is subjected to a reagent used for detection and mixed. When the ratio of the amount contained in the analyte to the amount dissolved or suspended in the liquid or mixed with the reagent used for detection is compared between the various substances contained in the analyte This means that the ratio of analytes is high. In this case, it is desirable that the ratio of the analyte is higher than that of the other substance, but if the other substance does not affect the detection of the analyte, it may be equal to or lower than the substance. Specifically, an analyte sample containing an analyte is collected in a process of collecting an analyte in the analyte sample using an analyte sample collection device that can selectively collect the analyte in a high recovery rate. In the step of, for example, the step of dissolving or suspending in a solution or the step of releasing the substance held in the instrument in the step of applying to the reagent used for detection of the analyte and mixing, etc. Since the amount of the substance that remains in the collection device of the present invention by adsorption or the like is large, if the amount used for the test is small, it cannot be said that the substance is recovered at a high recovery rate. When the amount remaining in the collection device is small and the amount used for the test is large, it is said to be collected at a high recovery rate.

  There are no particular limitations on the material, structure, and form of the analyte sampling device that can selectively recover the analyte in the sample of the present invention at a high recovery rate, but a material that collects liquid using capillary action Those having a structure and form are desirable. As a material for collecting a liquid by utilizing capillary action and its structure, for example, a material in which fine fibers are combined, woven, knitted or formed as an aggregate can be used. 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 of these materials can be used in combination or in layers. There is no particular limitation on the structure and form of an analyte sample collection device that can selectively collect an analyte in the sample of the present invention at a high recovery rate, and the above-mentioned material that collects liquid using capillary action It may be used as it is without any special processing, or a material for collecting a liquid by using a capillary phenomenon may be formed into an appropriate shape or size, or a liquid by using a capillary phenomenon. The material to be collected may be formed into a cotton swab attached to the tip of a rod-shaped object made of paper, resin or the like with an appropriate shape or size. Collecting liquid using capillary phenomenon Analytical sample collecting instrument capable of selectively recovering the analyte in the sample to be analyzed of the present invention at a high recovery rate mainly utilizes the capillary phenomenon. Although it is possible to collect solids and viscous substances that are dissolved or mixed in a liquid, in this case, not only capillary phenomenon but also solids and viscous substances are absorbed by the sample collection site of the instrument according to the present invention. Collect using combining.

  The material, structure, and form of an analyte sample collection instrument that can selectively recover an analyte in an analyte sample of the present invention at a high recovery rate are the object to be analyzed, the subject, the analyte sample, and the analyte. It can select suitably according to a substance. In order to efficiently collect analytes and use them for testing, they can be selected as appropriate.

  The analytical sample collection device capable of selectively recovering the analyte in the analytical sample of the present invention at a high recovery rate is not an analytical sample collection device that collects the entire collected analytical sample at a high recovery rate. This is a sample-collecting device for collecting an analyte to be analyzed in a sample to be analyzed with a high recovery rate. For example, in a test to determine the presence or abundance of viral antigens in a human pharynx, nasal passage or nasal cavity to confirm influenza infection, wipe the pharynx, nasal passage or nasal cavity with a conventional cotton swab It is an analyte present in the cotton part of a cotton swab in the process of subjecting the sample to be analyzed to a test, such as collecting or absorbing the substance obtained by aspirating the substance in the nasal cavity. In many cases, the antigen remains in the cotton parts, which is not preferable for achieving high specificity and high sensitivity analysis. However, according to the method of collecting an analyte using an analyte sample collecting device that can selectively collect an analyte in the analyte of the present invention at a high recovery rate, and using this for analysis, High specificity and high sensitivity analysis and testing can be performed.

  In addition, in tests that measure the presence or amount of analytes such as bacteria, their products, and viruses that are present and adhered to soil, air, river water, seawater, culture media, medical instruments and environmental materials, In the process of collecting the analyte with a cotton swab and subjecting the sample to be analyzed to the test, the antigen that is the analyte present in the cotton in the conventional cotton swab often remains in the cotton. It was not preferable to achieve high specificity and high sensitivity analysis. However, according to the method of collecting an analyte using an analyte sample collecting device that can selectively collect an analyte in the analyte of the present invention at a high recovery rate, and using this for analysis, High specificity and high sensitivity analysis and testing can be performed.

  The recovery rate of the analyte is 50% or more, preferably 60% or more when using an analyte sample collecting device that can selectively recover the analyte in the analyte of the present invention at a high recovery rate. More preferably, it is 70% or more, particularly preferably 80% or more, and most preferably 90% or more.

  Specific examples of an analyte sample collecting instrument capable of selectively recovering an analyte in an analyte sample of the present invention at a high recovery rate, a method of using the extractor instrument, and an apparatus or kit including the extractor instrument will be described below. explain.

  FIG. 1 shows an example of an analyte sample collection device that can selectively recover an analyte in an analyte sample of the present invention at a high recovery rate. A shaft portion 2 and a head portion 3 are provided, and a means 1 for collecting liquid on the surface of the head portion 3 using a capillary phenomenon is provided. The material of the shaft portion 2 and the head portion 3 is not particularly limited, but a substance having a moderate hardness and a suitable flexibility is suitable as it is inferior in liquid absorbability to the liquid collecting means 1 utilizing the capillary phenomenon. A molded product of wood, plastic, urethane resin, paper, or metal 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 part 2 and the head part 3 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 means 1 for collecting the liquid by utilizing the capillary phenomenon, 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, cotton-like and sponge-like materials 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 of these materials can be used in combination or in layers. Although the thickness of the arrangement of the means 1 for collecting liquid utilizing capillary action can be selected as appropriate, it is preferably about 0.5 to 5 mm. The means 1 for collecting liquid utilizing capillary action, the shaft portion 2 and the head portion 3 are usually of a target shape with respect to the central axis of the shaft portion, and the vertical cross section with respect to the axial direction is circular around the axis. The shape may be changed as necessary, or the means 1 for collecting a liquid using capillary action may be selectively disposed.

  As shown in FIG. 3, a conventional cotton swab wraps cotton around a shaft portion whose vertical cross-sectional shape with respect to the axial direction is substantially the same in any portion in the axial direction to form a cotton ball. This conventional cotton swab requires more cotton than necessary and covers the deep part of the cotton in consideration of the purpose of the original sample-collecting device, in which the sample to be analyzed is collected and the sample to be analyzed is released from the swab and used for the test. There is a problem that the analyte in the sample to be analyzed is not sufficiently released in the step of entering the analyte and holding it there and releasing the next analyte. By using an analyte sample collecting device that can selectively recover an analyte in an analyte sample of the present invention at a high recovery rate, the analyte in the analyte sample can be selectively recovered at a high recovery rate. Therefore, an analysis method for analyzing with high specificity and excellent sensitivity is achieved.

  A cotton swab-shaped instrument is an example of an analyte sample collection instrument that can selectively recover an analyte in the analyte sample of the present invention at a high recovery rate. The swab-like device is formed such that the shape or size of the axial vertical cross section of the head 3 changes at a position in the axial direction and becomes thicker than the shaft 2 toward the tip. Specifically, the head 3 is molded in advance into a shape that is substantially similar to the shape of the cotton ball portion, which is a portion for collecting the analyte of the final collection instrument, and a capillary phenomenon is formed on the head 3. It is formed by fixing by winding, spraying, adhering and / or welding the means 1 for collecting liquid using In such an analyte sample collecting device, since there is no thick part in the means 1 for collecting liquid using the capillary phenomenon, there is a possibility that the substance to be analyzed stays inside the means 1 for collecting liquid using the capillary phenomenon. Analyze the analyte in the next step, for example, in the process of dissolving or suspending the sample in the liquid, or in the step of mixing with the reagent used for detection of the analyte. The amount remaining in the sampling means is small.

  A liquid for performing treatment such as dissolution or suspension can be recovered at a high recovery rate by sufficiently immersing the liquid into the deep part of the means 1 for collecting the liquid using capillary action. . However, when the portion where the liquid is collected using the capillary phenomenon is thick, it is not possible to sufficiently infiltrate the deep part of the means 1 for collecting the liquid using the capillary phenomenon, and the analyte is dissolved or dissociated. In particular, when the substance is contained in a liquid for dissolving or dissociating, the action does not sufficiently function, and the substance to be analyzed is large and remains in the sample collecting means.

  The analyte sample collecting device capable of selectively recovering the analyte in the sample of the present invention at a high recovery rate does not include a thick portion in the means 1 for collecting a liquid utilizing capillary action and is substantially uniform. The liquid can be soaked in the thickness so as to be dissolved or suspended to the deep part of the means 1 for collecting the liquid using the capillary phenomenon, and the analyte can be recovered at a high recovery rate. be able to. As a result, it is possible to provide an analysis method and apparatus for analyzing with high specificity and excellent sensitivity. The thickness of the means 1 for collecting the liquid utilizing the capillary phenomenon can be appropriately selected according to the purpose and material, and is preferably about 0.5 to 5 mm, more preferably about 1 to 2 mm.

  FIG. 2 shows another example of the sample-collecting instrument to be analyzed that can selectively recover the analyte in the sample of the present invention at a high recovery rate. A shaft portion 2 and a head portion 3 are provided, and a means 1 for collecting liquid on the surface of the head portion 3 using a capillary phenomenon is provided. The materials and structures of the shaft portion 2 and the head portion 3 are the same as described in FIG.

  There is no particular limitation on the material of the means 1 for collecting the liquid using the capillary phenomenon, but it is formed by, for example, thin fibers being fixed to the head 3. The fixing method is not limited, but it can be formed by spraying, bonding and / or welding. 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 of these materials can be used in combination. 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 thickness of the fine fibers and the arrangement density can be appropriately selected according to the purpose and material, but the fineness is preferably 1.0 to 4.0 dtex, and the thickness of the fibers can be appropriately selected, but 0.5 to About 5 mm is preferable, and about 1 to 2 mm is more preferable.

  The means 1 for collecting liquid utilizing capillary action, the shaft 2 and the head 3 are usually shapes with respect to the central axis of the swab, and the vertical cross section with respect to the axial direction is circular around the axis, but is necessary Depending on the shape, the shape may be changed, or the means 1 for collecting the liquid using the capillary phenomenon may be selectively disposed.

  In addition, the analysis method of the present invention can use an analyte sample filtering instrument in addition to an analyte sample collecting instrument that can selectively recover the analyte in the analyte sample of the present invention at a high recovery rate. . The analyte sample filtering instrument is dropped from the means 1 for collecting liquid using the capillary phenomenon of the analyte sample collecting instrument that can selectively recover the analyte in the analyte sample of the present invention at a high recovery rate. By capturing the fibrous substance, unnecessary nonspecific reaction can be suppressed. The analysis sample filtering device may be applied and used in any way, but for example, a process for performing a process such as dissolving or suspending the collected analysis sample containing the analyzed substance in a liquid, for example, Is preferably applied after. In the next step, for example, the step of dissolving or suspending the sample to be analyzed containing the collected analyte in the liquid, the sample to be analyzed may be rubbed or rubbed. There is a possibility that the fibrous substance of the means 1 for collecting the liquid using the capillary phenomenon of the sample collecting instrument to be dropped off, and this substance may cause a non-specific reaction. A non-specific reaction can be suppressed by capturing the fibrous substance using an analysis sample filtering instrument.

  The present invention also includes an analysis apparatus and an analysis kit including an analysis sample filtering instrument in addition to an analysis sample collecting instrument capable of selectively recovering an analyte in the analysis sample of the present invention at a high recovery rate. To do. The analysis device and the analysis kit include a measurement device such as an immunochromatography analysis device, a reagent, and the like.

  By collecting the sample to be analyzed using an analytical sample collection device that can selectively collect the analyte in the sample to be analyzed at a high recovery rate, and subjecting it to analysis and testing, high specificity and It is possible to analyze with excellent sensitivity. There are no particular limitations on the analysis and testing, but examples include analysis and testing for simple and rapid measurement of substances in test samples at medical sites. For example, qualitative and quantitative analysis methods using immunochromatographic analysis methods. Applicable. Substance obtained by wiping with a sample collection device that selectively collects the analyte in the pharynx, nasal passage, or nasal cavity of the patient with a high recovery rate, or collecting the analyte in the nasal cavity Highly specific by applying a sample to be analyzed to a pre-prepared immunochromatographic analyzer, for example, by absorbing the sample in a sample collection device that selectively collects the analyte at a high recovery rate Highly sensitive analysis and testing can be performed.

The present invention will be specifically described below based on examples of the present invention. The present invention is not limited to the following examples.
[Example 1] Detection of influenza virus using a flow-through detection apparatus using an analyte sample collection device capable of selectively recovering an analyte in an analyte sample of the present invention at a high recovery rate (1 ) Preparation of antibody-bound gold colloid
10 mL of colloidal gold solution (OD ₅₂₀ = 1.0) was taken and the pH was adjusted to 7.0 with 100 mM potassium carbonate. The anti-influenza A virus monoclonal antibody purified by affinity using a protein A column was dialyzed and centrifuged with a 2 mM boric acid solution, and then prepared with a 2 mM boric acid solution to a concentration of 100 μg / mL. An amount of the prepared anti-influenza A virus monoclonal antibody at a final concentration of 4 μg / mL was added to the gold colloid with sufficient stirring. After 5 minutes, 1 mL of 10% BSA was added, and gently stirred with a rotator for 10 minutes. The whole amount was transferred to a centrifuge tube and centrifuged at 14000 rpm for 30 minutes at 4 ° C. After centrifugation, the supernatant is aspirated and discarded, and the resulting anti-influenza A virus monoclonal antibody-conjugated gold colloid is added to 1 mL of a solution containing a final concentration of 20 mM Tris-HCl buffer (pH 8.0), 1% BSA, 150 mM sodium chloride. Floated.

10 mL of colloidal gold solution (OD ₅₂₀ = 1.0) was taken and the pH was adjusted to 7.0 with 100 mM potassium carbonate. The anti-influenza B virus monoclonal antibody affinity-purified with a protein A column was dialyzed and centrifuged with a 2 mM boric acid solution, and then prepared with a 2 mM boric acid solution to a concentration of 100 μg / mL. An amount of 4 μg / mL final concentration of the prepared anti-influenza B virus monoclonal antibody was added to the colloidal gold with sufficient stirring. After 5 minutes, 1 mL of 10% BSA was added, and gently stirred with a rotator for 10 minutes. The whole amount was transferred to a centrifuge tube and centrifuged at 14000 rpm for 30 minutes at 4 ° C. After centrifugation, the supernatant is aspirated and discarded, and the resulting anti-influenza B virus monoclonal antibody-conjugated gold colloid is added to 1 mL of a solution containing a final concentration of 20 mM Tris-HCl buffer (pH 8.0), 1% BSA, 150 mM sodium chloride. Floated.

(2) Drying of colloidal gold Using the positive pressure spraying device (BioJet, BioJet), the amount of anti-type A and anti-type B influenza monoclonal antibody-conjugated gold colloid prepared in the previous section was 8.0OD ₅₂₀ , 10μL / cm And at a speed, each was sprayed onto a 10 mm x 300 mm polystyrene nonwoven fabric. Subsequently, it was dried under reduced pressure in a vacuum apparatus for 1 hour to obtain dry gold colloid pads (A type and B type). At the time of use, it cut | judged and used by 10 mm x 7 mm space | interval.

(3) Production of flow-through detection device A flow-through detection device, which is one form of immunochromatographic analysis, was used as the detection device. The apparatus used for the flow-through detection method includes at least a sheet-like carrier to which a receptor for an analyte is bound. The method using this apparatus is as follows. The sample liquid containing the analyte is brought into contact with the colored particles to which the first receptor for the analyte is bound, and then passed through the sheet-like carrier in the apparatus, whereby the colored particles are formed on the surface of the sheet-like carrier. A / first receptor / analyte / second receptor complex is formed and detected. A schematic diagram of this apparatus is shown in FIGS. 4 is a plan view of the apparatus, and FIG. 5 is an AA cut end view of FIG. 4 and 5, reference numeral 5 denotes a concentrator having an opening for dropping the prepared sample (specimen solution) and having a hole 6 for allowing the sample to pass through the bottom surface. The left hole is A hole, and the right hole is B hole. 7 is a sheet-like carrier to which a receptor for the analyte is bound, and 8 is a member that absorbs liquid. A nitrocellulose sheet was used as the sheet carrier.

  In this example, the colored particles are colloidal gold, the first receptor is an anti-influenza monoclonal antibody bound to the gold colloid, the analyte is an influenza virus antigen, and the second receptor is an anti-influenza monoclonal antibody bound to a nitrocellulose sheet. To do.

  Immobilization on a nitrocellulose sheet was carried out as follows using an anti-influenza virus monoclonal antibody. An anti-influenza A virus monoclonal antibody and an anti-influenza B virus monoclonal antibody purified by affinity purification using a protein A column were prepared. The buffer in which the antibody was suspended was replaced with 0.1 mM trehalose-added 10 mM citrate buffer (pH 4.0) using a Sephadex G-25 gel filtration column. Dilute with 10 mM citrate buffer (pH 4.0) with 0.1% trehalose so that the absorbance at 280 nm is 1.0, and detect the anti-influenza A virus monoclonal antibody at 10 μL / device (device). Drip onto the nitrocellulose in the hole, drop the anti-influenza B virus monoclonal antibody onto the nitrocellulose in the B hole of the detection device so as to be 10 μL / device (device), then let stand at 45 ° C. for 40 minutes and dry . After the device is dried, the detection device is constructed by leaning one dry gold colloid pad (A type) made and cut in (2) into A hole and one dry gold colloid pad (B type) into B hole. Was completed.

(4) Samples Samples were obtained from 4 patients diagnosed with influenza.
(5) Detection method A sample which is considered to contain influenza virus is collected from the nasal cavity by the irreversible sample collecting means of the present invention having the structure shown in FIG. The product was suspended in 1.5 mL. 1 w / v% BSA, 20 mM MES buffer (pH 6.0), 50 mM NaCl, 4 w / v% Triton X-100 (trade name), 2 w / v% arginine.

  The solution was filtered through a filter (pore size: 0.22 μm), and 500 μL each was added dropwise so that the dry gold colloid pad was immersed in the A hole and B hole of the detection device (device). After all the liquid has been absorbed by the membrane member, remove the concentrator, and if the membrane member of the part that adsorbs the anti-influenza virus monoclonal antibody of A-hole is colored in the color of gold colloid (for example, red to reddish brown) , It is confirmed that influenza A virus is present in the sample (positive for type A). If there is no change in color tone and the color of the membrane member remains, influenza A virus is not present in the sample. In addition, if the part of the membrane member adsorbing the anti-influenza virus monoclonal antibody in the B hole is colored in the color of colloidal gold (for example, red to reddish brown), the influenza B virus is present in the sample Confirmed (type B positive). If there is no change in color tone and the color of the membrane member remains, influenza B virus is not present in the sample. If neither A hole nor B hole is colored, it means that neither A nor B influenza viruses exist (negative).

[Comparative Example 1] Detection of influenza virus with a flow-through detection device using a conventional sample collecting instrument
(1) Preparation of colloidal gold antibody The same procedure as in Example 1 was performed.
(2) Drying of colloidal gold antibody The same procedure as in Example 1 was performed.
(3) Immobilization of antibody on diagnostic membrane filter and production of detection device The same procedure as in Example 1 was performed.
(4) Sample The same sample as in Example 1 was used.
(5) Detection method A sample was collected with a cotton swab from a nasal aspirate obtained from a sample suspected of containing influenza virus using a suction catheter, and suspended in 1.5 mL of the following sample suspension preparation medium composition. 1 w / v% BSA, 20 mM MES buffer (pH 6.0), 50 mM NaCl, 4 w / v% Triton X-100 (trade name), 2 w / v% arginine. A commercially available cotton swab (manufactured by Nippon Cotton Swab Co., Ltd.) was used as a conventional sample collecting instrument.

  The solution was filtered through a filter (pore size: 0.22 μm), and 500 μL each was added dropwise so that the dry gold colloid pad was immersed in the A hole and B hole of the detection device (device). After all the liquid has been absorbed by the membrane member, remove the concentrator, and if the membrane member of the part that adsorbs the anti-influenza virus monoclonal antibody of A-hole is colored in the color of gold colloid (for example, red to reddish brown) , It is confirmed that influenza A virus is present in the sample (positive for type A). If there is no change in color tone and the color of the membrane member remains, influenza A virus is not present in the sample. In addition, if the part of the membrane member adsorbing the anti-influenza virus monoclonal antibody in the B hole is colored in the color of colloidal gold (for example, red to reddish brown), the influenza B virus is present in the sample Confirmed (type B positive). If there is no change in color tone and the color of the membrane member remains, influenza B virus is not present in the sample. If neither A hole nor B hole is colored, it means that neither A nor B influenza viruses exist (negative).

  The results of Example 1 and Comparative Example 1 are shown in Table 1. As shown in Table 1, in Comparative Example 1, one of the four positive specimens became negative, but in Example 1 using the composition of the present invention, all were type A positive. In the confirmation test by PCR method, it was confirmed that all were A type positive.

[Example 2] Influenza virus detection sensitivity measurement using a flow-through detection apparatus using an analyte sample collection device capable of selectively recovering an analyte in an analyte sample of the present invention at a high recovery rate
(1) Preparation of colloidal gold antibody The same procedure as in Example 1 was performed.
(2) Drying of colloidal gold antibody The same procedure as in Example 1 was performed.
(3) Immobilization of antibody on diagnostic membrane filter and production of detection device The same procedure as in Example 1 was performed.
(4) Preparation of Analyzing Sample Collecting Device With respect to the analyzing sample collecting device shown in FIG. 2 used in the method of the present invention, two types for nasal and pharynx were prepared according to the sample collection site, and the water absorption was measured. The water absorption was measured according to the following procedure.
1) Weigh the sample before immersing it in the sample suspension.
2) Weigh in sample suspension (specific gravity: 0.995) for 20 seconds and then weigh
3) Find the difference and use the amount of water absorbed per cotton swab.
4) The average value in terms of volume was taken as the amount of water absorbed by the sample collection device to be analyzed.
Table 2 shows the results of water absorption measured by the above procedure.

(5) Samples Samples were influenza virus virus Beijing strain (A / Beijing / 262/95) and influenza B virus Shandong strain (B / Shandong / 7/97), MDCK (a cultured cell derived from canine kidney epithelial cells). Madin-Darbycanine kidney) A culture solution grown in cells diluted with a virus isolation medium was used.

(6) Detection method When a 200 to 6400-fold dilution of virus dilution is prepared and the amount of water absorption determined above for each sample collection device to be analyzed is added directly to the specimen suspension (1.5 mL) ( Control) and to what extent each sample-collecting device was allowed to be water-absorbed and then floated in the suspended liquid were confirmed to be positive for each magnification.

  As the suspension, the following sample suspension preparation medium composition was used. 1 w / v% BSA, 20 mM MES buffer (pH 6.0), 50 mM NaCl, 4 w / v% Triton X-100 (trade name), 2 w / v% arginine.

  After filtering 1.5 mL of the virus dilution and suspension mixture above with a filter (pore size 0.22 μm), add 500 μL each so that the dried gold colloid pad is immersed in the A hole and B hole of the detection device (device). did. After all the liquid has been absorbed by the membrane member, remove the concentrator, and if the membrane member where the anti-influenza virus monoclonal antibody is adsorbed is colored in the color of gold colloid (for example, red to reddish brown) Is confirmed to have influenza A virus (positive for type A). If there is no change in color tone and the color of the membrane member remains, the presence of influenza virus in the sample cannot be confirmed. In addition, if the part of the membrane member adsorbing the anti-influenza virus monoclonal antibody in the B hole is colored in the color of colloidal gold (for example, red to reddish brown), the influenza B virus is present in the sample Confirmed (type B positive). If there is no change in color tone and the color of the membrane member remains, influenza B virus is not present in the sample. If neither A hole nor B hole is colored, it means that neither A nor B influenza viruses exist (negative).

[Comparative Example 2] Measurement of influenza virus detection sensitivity using a flow-through detection device using a conventional sample collection device
(1) Preparation of colloidal gold antibody The same procedure as in Example 1 was performed.
(2) Drying of colloidal gold antibody The same procedure as in Example 1 was performed.
(3) Immobilization of antibody on diagnostic membrane filter and production of detection device The same procedure as in Example 1 was performed.
(4) Preparation of the sample collection device to be analyzed The swab shown in Fig. 3 used in the conventional method (Hospital swab made by Japan Cotton Swab Co., Nose: Paper shaft swab 150mm-Single cotton 3.0mm type, Pharynx: Paper shaft swab 150mm -One piece of cotton 4.5mm type) was prepared for nasal and pharynx depending on the sample collection site, and the water absorption was measured. The water absorption was measured in the same manner as in Example 2.
Table 3 shows the results of the measured water absorption.

(5) Sample The same sample as in Example 2 was used.
(6) Detection method When a 200-fold to 6400-fold dilution of the virus dilution is prepared and the amount of water absorption determined above for each cotton swab is added directly to the sample suspension (1.5 mL) (control), It was confirmed to what extent each case could be determined as positive when the cotton swab was absorbed in water and then suspended in the suspension.

  As the suspension, the following sample suspension preparation medium composition was used. 1 w / v% BSA, 20 mM MES buffer (pH 6.0), 50 mM NaCl, 4 w / v% Triton X-100 (trade name), 2 w / v% arginine.

  After filtering 1.5 mL of the mixture of the seed solution and suspension above with a filter (pore size 0.22 μm), 500 μL each was added dropwise so that the dry gold colloid pad was immersed in the A hole and B hole of the detection device (device). . After all the liquid has been absorbed by the membrane member, remove the concentrator, and if the membrane member of the part that adsorbs the anti-influenza virus monoclonal antibody of A-hole is colored in the color of gold colloid (for example, red to reddish brown) , It is confirmed that the influenza virus is present in the sample (type A positive). If there is no change in color tone and the color of the membrane member remains, the presence of influenza virus in the sample cannot be confirmed. In addition, if the part of the membrane member adsorbing the anti-influenza virus monoclonal antibody in the B hole is colored in the color of colloidal gold (for example, red to reddish brown), the influenza B virus is present in the sample Confirmed (type B positive). If there is no change in color tone and the color of the membrane member remains, influenza B virus is not present in the sample. If neither A hole nor B hole is colored, it means that neither A nor B influenza viruses exist (negative).

  The results of Example 2 and Comparative Example 2 are shown in Table 2, Table 3, Table 4, and Table 5. As shown in Table 2 and Table 3, although the water absorption of Example 2 is less than that of Comparative Example 2 for both nasal and pharyngeal areas, as shown in Table 4 and Table 5, the Examples 2 was twice as sensitive as Comparative Example 2, and it was confirmed that the detection was possible without inferior sensitivity compared to the case where the virus dilution of the control test was directly added to the sample suspension.

  The method of the present invention is an excellent method capable of analyzing an analyte in an analyte sample with high specificity and excellent sensitivity.

It is the figure which showed an example of the analysis material collection instrument of the cotton swab which selectively collect | recovers the to-be-analyzed substance of this invention with a high recovery rate. It is the figure which showed an example of the analysis material collection instrument of the cotton swab which selectively collect | recovers the to-be-analyzed substance of this invention with a high recovery rate. It is a figure which shows the conventional cotton swab. It is a top view of an example of the analyzer used with the method of the present invention. It is an AA cutting | disconnection end elevation of FIG.

Explanation of symbols

1: Means for collecting liquid utilizing capillary action 2: Shaft portion 3: Head portion 4: Device 5: Concentrator 6: Hole 7: Sheet-like carrier 8: Liquid absorbing member

Claims (10)

  A swab-shaped sample-collecting device that selectively collects a substance to be analyzed in a sample to be analyzed at a recovery rate of 50% or more.   2. The sample-collecting device to be analyzed according to claim 1, wherein said sample-collecting device has a head and a shaft, and means for collecting liquid using capillary action is disposed on the head.   The shape or size of the axial vertical cross section of the head changes depending on the position in the axial direction, and it is formed so as to be thicker than the shaft toward the tip. The sample collecting instrument to be analyzed according to claim 1 or 2, wherein the outer shape of the means for collecting is similar.   4. A sample collecting instrument to be analyzed according to claim 1, wherein the means for collecting the liquid using the capillary phenomenon is made of a fibrous material and has a uniform thickness of 0.5 mm to 5 mm.   In the means of collecting liquid using capillary action, fibers made of natural or artificial materials are arranged in a brush shape, arranged vertically toward the axis center of the swab shaft, and the fineness is 1.0 to 4.0 dtex. The analysis sample collecting instrument according to any one of claims 1 to 4.   An analyte is collected by collecting the analyte using the analyte sampling instrument according to any one of claims 1 to 5 and measuring the analyte in the analyte. Method.   A means for collecting a liquid using the capillary phenomenon of the sample-collecting device for analysis according to any one of claims 1 to 5 is brought into contact with the sample to be analyzed containing an analyte, and the sample to be analyzed is placed in the means. 7. The method for analyzing an analyte according to claim 6, wherein the analyte is selectively released from the means, and the released analyte is detected.   Further, the substance to be analyzed selectively discharged from the means for collecting the liquid using capillary action is filtered by a sample filtering device to detect the substance to be analyzed. A method for analyzing the analyte.   A kit for detecting a substance to be analyzed in a sample to be analyzed, comprising the sample collecting instrument to be analyzed according to any one of claims 1 to 5.   Furthermore, the kit for detecting the to-be-analyzed substance in the to-be-analyzed sample containing the to-be-analyzed sample filtration instrument.

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