JP2007046959A - Kit for immuno-chromatographic measurement - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To quickly and sufficiently mix an analyte contained in a specimen with a labeled substance coupled immunologically, by mixing simply the specimen with an extraction solvent, to be subjected quickly to immuno-chromatographic development. <P>SOLUTION: This kit for immuno-chromatographic measurement has: at least an extracting container for extracting the analyte from the specimen by mixing simply the specimen with a development solvent; and a film carrier for chromatography-developing the extracted analyte together with the development solvent. The film carrier is provided at least with a capture portion formed by solidifying the first substance coupled immunologically with the analyte, the extracting container contains the second substance coupled immunologically with the analyte, in a condition allowing the immunological coupling with the analyte, and the second substance is labelled with the labeling substance. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  In the present invention, a sample collected from a patient's nose, throat, conjunctiva, etc. can be easily mixed with a developing solvent, and this can be immediately chromatographed on a membrane carrier for immunochromatographic measurement to measure the analyte. The present invention relates to an immunochromatography measurement kit.

  Test samples for immunochromatography were preliminarily placed in a container such as a test tube with biological samples such as tears, eyelids, sputum, saliva, stool, gauze, cotton swabs, etc. It is immersed in an extraction solvent to disperse or dissolve the substance to be analyzed and prepared as an extract. The measurement is performed by sucking up this extract with a pipette or the like, and then injecting the extract into a sample injection portion of a test strip for immunochromatography measurement so as to develop the test strip.

  In the conventional test strip for immunochromatography, when an analyte is an antigen, a membrane carrier having a capture site formed by immobilizing the first antibody against the antigen, and a second antibody against the antigen And an impregnated member impregnated with a label, and the impregnated member is disposed adjacent to the sample injection portion. Therefore, when the extract is injected into the sample injection part, the antigen contained in the extract penetrates into the impregnated member to form a conjugate with the labeled second antibody, and the conjugate further becomes a membrane carrier. The substance to be analyzed is detected by being chromatographed and captured at the capture site.

JP 2003-344406 A JP 2004-271341 A

  However, in the configuration of the conventional test strip for immunochromatography measurement as described above, the extract immediately passes through the impregnated member, moves to the membrane carrier and is chromatographed, and the antigen contained in the extract in the impregnated member The time for contact with the second antibody was not always sufficient.

  Therefore, the present invention simply mixes a sample collected from a patient with an extraction solvent, quickly and sufficiently mixes the analyte to be contained in the sample and the labeling substance immunologically bound thereto, and quickly An object of the present invention is to provide an immunochromatography measurement kit that can be used for immunochromatographic development.

  According to the present invention, an extraction container for extracting a specimen to be analyzed from a specimen by mixing the specimen with a developing solvent, a membrane carrier for chromatographically developing the extracted substance to be analyzed together with the developing solvent, An immunochromatography measurement kit comprising at least a capture site formed by immobilizing a first substance that immunologically binds to the substance to be analyzed. The container comprises a second substance that immunologically binds to the analyte, in a state capable of immunologically binding to the analyte, and the second substance is labeled with a labeling substance. An immunochromatography measurement kit is provided.

  Further, in order to chromatograph on a membrane carrier having at least a capture site formed by immobilizing a first substance that immunologically binds to a substance to be analyzed, the specimen is mixed with a developing solvent from the specimen. An extraction container for extracting an analysis target substance, wherein the extraction container is capable of immunologically binding a second substance that immunologically binds to the analysis target substance with the analysis target substance. An extraction container for immunochromatography measurement is provided, wherein the second substance is labeled with a labeling substance.

  Further, according to the present invention, there is provided a membrane carrier used for chromatographic development of an immunologically bound product of the analyte to be extracted and the second substance extracted from the extraction container, There is provided a membrane carrier for immunochromatography measurement, comprising at least a capture site formed by immobilizing a first substance that immunologically binds to the substance.

  The membrane carrier of the present invention is not particularly limited as long as the first substance can be immobilized and the analysis target substance can be chromatographed. Usually, the developing solvent is capillarized by capillary action. A mesh-like or porous membrane member that can be infiltrated is used. Specific examples of such membrane carriers include nitrocellulose membrane filters, other cellulose membranes, nylon membranes, glass fiber membranes, etc. Of these, nitrocellulose membrane filters are preferably used. The shape and dimensions of the membrane carrier can be appropriately selected depending on the application, but are usually provided in the form of strips, that is, strips. The commercially available product is provided as a strip for immunochromatography measurement housed in an elongated container with a sample addition member overlapped and connected to one end of the membrane carrier strip and an absorption member overlapped and connected to the other end. .

In the present invention, when the substance to be analyzed is an antigen, both the first substance and the second substance are antibodies against the antigen. In this case, the first substance and the second substance must bind to different sites of the antigen. The antibody used as the first substance and the second substance may be a monoclonal antibody or a polyclonal antibody, but from the viewpoint of reaction specificity, both the first and second substances are monoclonal antibodies. Preferably there is.
In the present invention, when the substance to be analyzed is an antibody, an antigen to which the antibody specifically binds is used as the first substance, and an antibody against the antibody is used as the second substance. The antibody used as the second substance may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody from the viewpoint of reaction specificity.

  The first substance is fixed to the membrane carrier by a known means to form a capture site for the substance to be analyzed. The shape of the capture site is not particularly limited, and can be, for example, a line shape that crosses the width direction of the strip-shaped film carrier or a spot shape.

  The second substance may be contained in the extraction container in a state labeled with an appropriate labeling substance and immunologically bindable with the analyte. The labeled second substance may be easily dissolved in the developing solvent by contact with the developing solvent in the extraction container at the time of use. For example, the labeled second substance is applied to the inner surface of the container. Alternatively, it may be included in a container in a state of being impregnated with a suitable impregnating member. According to a preferred embodiment, the impregnated member impregnated with the labeled second substance is placed in the discharge path of the nozzle of the extraction container and analyzed when the contents of the container are discharged through the nozzle. The target substance and the impregnated member are configured to contact each other.

  The labeling substance used for labeling the second substance may be any substance that can label the substance, and examples thereof include a color labeling substance, an enzyme labeling substance, and a radiation labeling substance. Among these, it is preferable to use a color labeling substance from the viewpoint that the color change at the capturing site can be determined quickly and easily by observing with the naked eye. Examples of the color labeling substance include metal colloids such as gold colloid and platinum colloid, synthetic latex such as polystyrene latex colored with respective pigments such as red and blue, and latex such as natural rubber latex. Of these, metal colloids such as gold colloid are particularly preferred.

  The impregnated member is a member that holds the labeled second substance in an unfixed state, infiltrates the developing solvent, and allows the immunologically bound substance of the analyte and the second substance to pass through with the developing solvent. If it is, there will be no restriction | limiting in particular, For example, the glass fiber nonwoven fabric which consists of felt, a glass filter, glass fiber, etc., cellulose cloth (filter paper, nitrocellulose membrane, etc.), porous plastic cloth, such as polyethylene and polypropylene, etc. are used. it can. The labeled second substance can be easily held on the impregnation member by, for example, impregnating the suspension into the impregnation member and drying the suspension.

  The developing solvent used in the present invention is not particularly defined as long as it can extract a substance to be analyzed by dissolving or dispersing a sample, and examples thereof include a buffer solution having a pH of 4 to 10. Can be selected from physiological saline, phosphate buffer, Tris buffer, HEPES (2-hydroxypiperazine-N′-2-ethanesulfonic acid) buffer, etc., but physiological saline, phosphate buffer and Tris buffer Liquid is preferred. In order to enhance the performance as a developing solvent, the buffer solution is an additive as described in JP-A-2003-344406, that is, a polymer containing an ethylenically unsaturated monomer containing a phosphorylcholine group as a constituent unit. An auxiliary agent such as coalescence may be contained.

  Examples of specimens that can be used in the present invention include biological samples such as urine, stool, saliva, nasal discharge, sputum, blood, serum, and the like, and these are simply dispersed or dissolved in a developing solvent in the extraction container of the present invention. A test sample in which the substance to be analyzed is uniformly dispersed is obtained. For example, a test sample can be prepared by simply immersing or shaking a gauze or cotton swab from the affected area such as the pharynx in a developing solvent.

  The substance to be analyzed is not particularly limited as long as it can be detected by immunochromatography. For example, it is contained in tuberculosis bacteria contained in sputum, pathogenic bacteria such as O-157 contained in stool, and nasal wipes. Influenza virus, viruses such as adenovirus contained in throat swabs, antigens such as C-reactive protein contained in blood, biopolymers such as various antibodies, and environmental hormone-like substances present in the environment Trace substances are included.

  According to the immunochromatography measurement kit of the present invention, a developing solvent is put in an extraction container, and a biological sample or a swab that wipes the affected area is put in the extracting container to dissolve or disperse the specimen in the developing solvent. Inside the extraction container, the second substance labeled with the labeling substance and the analyte can be immunologically combined. Thus, by injecting this immunologically bound substance into the membrane carrier from the extraction container together with the developing solvent and chromatographing, the immunologically bound substance is captured by the first substance at the capturing site of the membrane carrier. Therefore, by measuring the accumulation degree of the labeling substance, it is possible to measure the detection target substance, semi-quantitative measurement, and the like. In particular, when measuring a plurality of analytes at the same time, the extraction container may contain a plurality of second substances, but each of the analytes is good with each corresponding second substance. Therefore, even when chromatographically developed on a membrane carrier, measurement can be performed with good sensitivity.

  Hereinafter, although the present invention will be described based on the illustrated specific examples, the present invention is not limited to the specific examples.

  FIG. 1 shows the configuration of a specific example of an extraction container according to the present invention, where numeral 1 indicates a container body, numeral 2 indicates a nozzle, and numeral 3 indicates a nozzle lid. The main body 1 has a generally tubular shape, one end of which is an open end 11 and the other end is a closed end 12. As shown in the drawing, the main body 1 has a side wall portion 14 adjacent to the open end 11 having a larger diameter than the side wall portion 13 adjacent to the closed end 12 of the main body 1, and generally toward the closed end 12. It is tapered. The side wall portion 14 adjacent to the open end 11 has a circular cross section, and the side wall portion 13 adjacent to the closed end 12 is formed to have a flat shape such as an elliptical cross section. May be easily deformed, and the contents may be easily discharged from the extraction container. The main body 1 is preferably transparent or translucent so that the inside can be seen through from the outside.

  As is apparent from FIG. 1, a threaded portion 15 is provided on the side wall near the opening end 11 of the main body 1 so that a nozzle 2 or a lid 2 'described later can be detachably attached thereto.

  The inner dimension of the main body 1 is preferably formed to a dimension that can accommodate the entire cotton swab used for wiping the biological sample and the affected part. In this case, the analysis target substance in a state where the swab is accommodated in the container is used. Extraction can be performed, and after use, the container can be discarded in a state in which a cotton swab is accommodated. The main body 1 can be generally produced by molding by a known molding method such as injection molding or blow molding using a plastic material resistant to the developing solvent to be used. Examples of the plastic material include polyethylene, polypropylene, and the like, but it is preferable to use polyethylene.

  The side wall portion 13 adjacent to the closed end 12 of the main body 1 is preferably formed more flexibly than the side wall portion adjacent to the open end 11 of the main body 1. Specifically, the side wall portion 13 can be pushed and deformed by hand. However, it is preferable that the side wall portion 13 has flexibility enough to return to the original shape when the hand is released. For this purpose, for example, an annular housing 141 projecting in a direction perpendicular to the longitudinal axis of the main body 1 is integrally formed with the main body 1 at a position of the side wall portion 14 located slightly closer to the closed end 12 from the threaded portion 15. Can be provided. In this case, the side wall portion 14 is reinforced by the casing 141, whereas the side wall portion 13 adjacent to the closed end 12 is not reinforced, so that the side wall portion 14 becomes more flexible than the side wall portion 14. Alternatively, the thickness of the side wall portion 13 adjacent to the closed end 12 may be made thinner than the thickness of the side wall portion 14 located on the opening end 11 side. By comprising in this way, when dripping an extract from the nozzle 2, a user can press the side wall part 13 easily with a finger so that an extract can be quickly discharged from the nozzle 2. It is convenient.

  As shown in FIG. 1, the nozzle 2 can be detachably attached to the opening end 11 of the main body 1. In the illustrated example, the nozzle 2 includes a cylindrical portion 21, one end of which includes a flange portion 22, and the other end of which has a smaller diameter and a tapered nozzle portion 23 that communicates with the cylindrical portion 21. It protrudes. On the inner peripheral surface of the cylindrical portion 21, a female screw portion 24 that is screwed with the threaded portion 15 of the main body 1 is formed. The outer peripheral surface of the cylindrical portion 21 is knurled so that the nozzle 2 can be easily attached and detached. It is preferable that the flange portion 22 is in close contact with the casing 141 of the main body 1 when attached to the main body 1. Further, it is possible to improve the adhesion with the opening end 11 of the main body 1 by providing a packing or a sealing material inside the cylindrical portion 21. The outer peripheral surface of the nozzle portion 23 is provided with a male threaded threaded portion 25 in a region adjacent to the cylindrical portion 21. A lid 3 for closing the nozzle portion 23 can be detachably attached to the nozzle 2. In the illustrated example, the lid body 3 has a substantially truncated cone shape with one end closed and the other end opened, and has an internal thread portion 31 that can be screwed into the threaded portion 25 of the nozzle 2 on its inner peripheral surface. . The outer peripheral surface of the lid 3 is provided with a ridge extending in the axial direction thereof so that the user can easily attach and detach the lid 3.

  In the illustrated example, a filter 4 ′ and a disc-shaped impregnation member 4 impregnated with a labeled second substance are disposed inside the nozzle 2. Specifically, a filter 4 ′ having a diameter substantially the same as the inner diameter of the cylindrical portion 21 is disposed in contact with a step portion 26 that forms a boundary between the cylindrical portion 21 and the nozzle portion 23. An impregnation member 4 having the same shape as the filter 4 ′ is disposed on the filter 4 ′. The filter 4 ′ and the impregnating member 4 are normally prevented from falling off the nozzle 2 by the female threaded portion 24 projecting inward from the inner surface of the cylindrical portion 21, but the filter 4 ′ can be connected to the stepped portion 26 as necessary. The impregnated member 4 may be fixed to the filter 4 'by bonding with an adhesive or the like. Thus, since the impregnation member 4 is disposed in the discharge path 27 of the nozzle, when the contents of the container are discharged from the nozzle 2, the impregnation member 4 is discharged via the impregnation member 4, and An immunological binding with the second substance is ensured. The filter 4 ′ is not particularly limited as long as it allows a conjugate of the substance to be analyzed and the labeled second substance to pass through. For example, the same material as the impregnation member 4 can be used.

  Each of the nozzle 2 and the lid 3 can be formed by molding a plastic material using a known molding method such as injection molding or blow molding, like the main body 1. Examples of the plastic material include polyethylene and polypropylene, but it is preferable to use a material harder than the main body 1 so as not to be easily deformed, and it is particularly preferable to use polypropylene.

  In the illustrated example, the nozzle 2 and the main body 1 are coupled to each other by screwing. However, as long as the nozzle 2 and the main body 1 can be detachably attached, various other methods such as a snap-fitting method can be used. Can be adopted. Similarly, as long as the nozzle 2 and the lid 3 can be detachably attached, various other methods such as a snap-fitting type can be adopted.

  In addition, the extraction container of the present invention may be provided in a state in which a predetermined amount of developing solvent is accommodated in advance and closed with a lid 2 ′ as shown in FIG. The lid 2 ′ may be anything that can be screwed into the threaded portion 15, and more preferably has excellent sealing performance. In this case, the nozzle 2 can be attached as an accessory of the extraction container. Further, a sample collection tool such as a cotton swab may be sterilized and attached as an accessory to the extraction container of the present invention.

  Next, a specific example of the membrane carrier of the present invention will be described with reference to FIG. In the illustrated example, the membrane carrier 6 is combined with a sample addition member 7 and an absorption member 8 to form a so-called immunochromatographic measurement test strip, and can simultaneously measure two analytes. ing.

  In the example shown in the figure, the membrane carrier 6 is composed of a strip-shaped nitrocellulose membrane filter having a width of 5 mm and a length of 36 mm, and is attached to the middle of the adhesive sheet 5 having the same width. On the membrane carrier 6, the chromatographic start point side, that is, the left side of FIG. 2 (hereinafter referred to as “upstream side”. The opposite side, that is, the chromatographic development direction side in FIG. 2 is hereinafter referred to as “downstream side”. 2), two kinds of first substances are fixed at positions 7.5 mm and 10.5 mm from the end, respectively, and a capture site 61 (61a and 61b) for the substance to be analyzed is formed. When detecting each of influenza virus types A and B simultaneously, for example, an antibody that reacts with influenza virus type A but does not react with type B is immobilized at capture site 61a, and reacts with influenza virus type B at capture site 61b. However, antibodies that do not react with type A can be immobilized. Further, a so-called control line 62 is provided at a position 15 mm from the upstream end of the membrane carrier 6. This control line is used to confirm that the reaction has been performed regardless of the presence or absence of the analyte, and is usually a substance that immunologically specifically binds to the second substance (analyte) Can be formed on the membrane carrier 6. For example, when a mouse antibody is used as the second substance, an antibody against the mouse antibody can be used.

  In the present invention, not limited to the example shown in the figure, when detecting a single analyte, the capture site 61 may be single, and for semi-quantifying a single analyte, In order to detect three or more substances to be analyzed simultaneously, three or more capture sites 61 may be provided. When detecting influenza virus types A and B simultaneously as described above, the labeled second substance may consist of only antibodies that cross-react with influenza virus types A and B, or An antibody that reacts with influenza virus type A but does not react with type B may consist of an antibody that reacts with influenza virus type B but not with type A. In the latter case, each antibody has a different color. Labeling with the labeling substance to be presented is advantageous because the capture site 61a and the capture site 61b exhibit different colors and are therefore easily distinguishable.

  On the upstream side of the membrane carrier 6, a sample addition member 7 having the same width as the membrane carrier 6 is disposed in a state where the downstream end thereof is overlapped and connected to the upstream end of the membrane carrier 6, The remaining part of the sample addition member 7 is stuck and fixed to the adhesive sheet 5. The sample addition member 7 can be the same as that used for a known immunochromatographic measurement test strip. For example, a porous synthetic resin such as porous polyethylene or porous polypropylene can be used. In addition to sheets or films, paper made of cellulose such as filter paper and cotton cloth, woven cloth or non-woven cloth can be used.

  On the downstream side of the membrane carrier 6, an absorbing member 8 having the same width as that of the membrane carrier 6 is arranged in a state where the upstream side portion is overlapped and connected to the downstream side portion of the membrane carrier 6, The remaining part of the absorbing member 8 is stuck and fixed to the adhesive sheet 5. The absorbent member 8 may be the same as that used for known immunochromatographic measurement test strips, and may be made of any material that can quickly absorb and retain liquid, such as cotton cloth, filter paper In addition, a porous plastic nonwoven fabric made of polyethylene, polypropylene, or the like can be used, and filter paper is particularly suitable.

  Further, in the case of a commercial product, the immunochromatographic measurement test strip of FIG. 2 is usually provided by being housed in a plastic case 9 as shown in FIG. In the case 9, a sample injection hole 93 and a determination hole 94 are opened above the sample addition member 7 and above a region extending from the capture site 61 to the control line 62, respectively.

  In the example of FIG. 3, the case 9 includes a lid body 91 and a main body 92 as shown in FIG. The test strip shown in FIG. 2 is placed in the main body 92, and the sample injection hole 93 and the determination hole 94 are formed in the lid 91. In addition, six air holes 95 are formed in the lid 91 above the absorbing member 8 in order to promote the absorption.

Hereinafter, a method of using the illustrated specific example will be described.
First, in a state where the nozzle 2 of the container shown in FIG. 1 is removed from the main body 1, a required amount of developing solvent is put into the main body 1 from the open end 11 of the main body 1. Thereafter, after cleaning the biological sample and the affected part with the cotton swab 16, the cotton swab 16 is inserted into the main body 1 from the open end 11 of the main body 1, and the nozzle 2 is attached to the main body 1 with the nozzle part 23 closed with the lid 3. . Then, by shaking the sealed container by hand, the specimen adhering to the cotton swab 16 is dissolved or dispersed in the developing solvent.

  Then, only the lid 3 is removed, and as shown in FIG. 3A, the sample 2 in the extraction container is injected into the sample injection hole 93 with the nozzle 2 facing downward. At that time, the extract passes through the impregnating member 4 disposed in the discharge path 27 of the nozzle 3 and is discharged from the nozzle 3. At this time, the analysis target substance of the extract and the second substance come into contact with each other, Both immunological conjugates are formed. Since the used swab and the extract are both contained in the container, there is little risk of contamination of the surroundings even when the specimen is infectious, and the nozzle 2 is sealed with the lid 3. The container can be disposed of in a state and is easy to handle.

  Thus, the extract added to the sample addition member 7 from the sample injection hole 93 is chromatographed from the upstream end of the membrane carrier 7 toward the downstream side, and the conjugate of the substance to be analyzed and the second substance is captured. The second substance that has accumulated in the region 61 and did not bind to the substance to be analyzed accumulates in the control line 62. When the labeling substance of the second substance is a colored labeling substance such as a colloidal gold, the capture site 61 is colored when the substance to be analyzed is present. Therefore, by measuring the degree of coloration, the substance to be analyzed is measured. Can be detected or semi-quantified. Since the control line is always colored when the second substance is chromatographed regardless of the presence or absence of the substance to be analyzed, it can be confirmed whether the chromatographic development has been performed normally.

  FIG. 4 shows another specific example of the extraction container of the present invention. 4, the same elements as those in FIG. 1 are denoted by the same reference numerals. In FIG. 4, the container body 1 has a substantially cylindrical shape, one end of which is an open end 11 and the other end is a closed end 12. On the inner peripheral surface in the vicinity of the opening end 11 of the container main body 1, an annular convex portion 15 ′ that engages with a nozzle 2 described later is formed.

  The nozzle 2 in FIG. 4 includes a slightly tapered cylindrical portion 21 that is inserted into and fitted into the open end 11 of the container body 1, and a nozzle portion 23 that projects from one end of the cylindrical portion 21 and communicates therewith. Yes. The nozzle portion 23 has a smaller diameter than the cylindrical portion 21, and a flange portion 22 having a larger diameter than the cylindrical portion 21 protrudes from the outer surface of the connecting portion between the nozzle portion 23 and the cylindrical portion 21. A concave portion 24 ′ that engages with the convex portion 15 ′ of the main body 1 is formed on the outer peripheral surface near the flange portion 22 of the cylindrical portion 21.

  In the nozzle 2 of FIG. 4, the impregnating member 4 having a diameter substantially the same as the inner diameter of the cylindrical portion 21 is disposed in contact with a step portion 26 that forms a boundary between the cylindrical portion 21 and the nozzle portion 23. ing. Then, a small cylindrical member 29 having an outer diameter substantially the same as the inner diameter of the cylindrical portion 21 is inserted and fitted into the cylindrical portion 21, so that the impregnated member is interposed between the small cylindrical member 29 and the step portion 26. By sandwiching 4, the impregnation member 4 is prevented from falling off the nozzle 2. Thus, since the impregnation member 4 is disposed in the discharge path 27 of the nozzle, when the contents of the container are discharged from the nozzle 2, the impregnation member 4 is discharged via the impregnation member 4, and An immunological binding with the second substance is ensured.

Hereinafter, a method of using the specific example of FIG. 4 will be described.
First, the nozzle 2 of the container shown in FIG. 4 is removed from the main body 1 to be in the state shown in FIG. 4B, and a required amount of developing solvent is put into the main body 1 from the open end 11 of the main body 1. Thereafter, after the biological sample and the affected part are wiped with a cotton swab, the cotton swab is inserted into the main body 1 from the open end 11 of the main body 1, the cylindrical portion 21 of the nozzle 2 is inserted into the open end 11 of the main body 1, and the former recess 24 ' Is brought into a state shown in FIG. 4A by engaging the latter convex portion 15 'with a snap. Then, by shaking the container by hand, the specimen attached to the cotton of the cotton swab 16 is dissolved or dispersed in the developing solvent.

  Then, as shown in FIG. 3A, the nozzle 2 is directed downward, and the sample extraction liquid in the extraction container is injected into the sample injection hole 93. At that time, the extract passes through the impregnating member 4 disposed in the discharge path 27 of the nozzle 2 and is discharged from the nozzle 2. At this time, the analysis target substance of the extract and the second substance come into contact with each other, Both immunological conjugates are formed. As in the case of the extraction container of FIG. 1, since the used swab and the extract are both contained in the container, there is a risk of contaminating the surroundings even if the specimen is infectious. The container can be discarded with the discharge path 27 of the nozzle 2 closed by an appropriate means, and handling is easy. Then, the extract added to the sample addition member 7 through the sample injection hole 93 is chromatographed in the same manner as in FIG. 3, and immunochromatography measurement can be performed.

  FIG. 5 shows still another specific example of the extraction container of the present invention. 5, the same elements as those in FIG. 4 are denoted by the same reference numerals. In the extraction container of FIG. 5, a filter 4 ′ having the same shape as the impregnation member 4 is arranged instead of the impregnation member 4 of FIG. 4, and the cylindrical impregnation member 4 is filled in the nozzle portion 23 of the nozzle 2. Except for this, it has the same configuration as the extraction container of FIG. Thus, the extraction container in FIG. 5 can be used in the same manner as in FIG. 4. Specifically, the extraction liquid in the container is impregnated with the filter 4 ′ disposed in the discharge path 27 of the nozzle 2. It passes through the member 4 and is discharged from the nozzle 2. At this time, the substance to be analyzed in the extract and the second substance come into contact with each other to form an immunological conjugate of both, which can be subjected to immunochromatographic measurement. it can. In the case of FIG. 5, it is preferable to use a glass fiber nonwoven fabric made of glass filter or glass fiber as the impregnating member 4.

  In order to detect influenza virus types A and B simultaneously, a test strip for immunochromatography measurement shown in FIG. 2 was prepared. A membrane filter made of nitrocellulose was used as the membrane carrier 6, cotton cloth was used as the sample addition member 7, and filter paper was used as the absorption member. Here, as a first substance constituting the capture site 61a, a monoclonal antibody that is reactive to influenza virus type A nucleoprotein but not reactive to influenza virus type B is used, and the first material constituting the capture site 61b. A monoclonal antibody that is reactive to influenza virus type B nucleoprotein but not influenza virus type A was used.

  An extraction container shown in FIG. 4 was prepared as an extraction container used with the immunochromatographic measurement test strip. Here, as a second substance to be impregnated into the impregnating member 4, a monoclonal antibody having cross-reactivity with both influenza virus A type nucleoprotein and B type nucleoprotein is prepared, and this is labeled with colloidal gold by a conventional method. used. As the impregnating member 4, a felt having a square thickness of about 4 mm cut into about 7.5 mm square was used. In addition, as an equivalent product of the impregnated member 4, a glass fiber nonwoven fabric having a thickness of about 0.5 mm cut into a disk shape can be used.

  A commercially available influenza virus type A nucleoprotein was diluted 1600 times, 800 times, and 400 times in a developing solvent (phosphate buffer solution) to prepare a specimen dilution solution. Only a developing solvent was prepared as a blank. Then, a predetermined amount of each sample diluent is injected into the container body 1 of the extraction container, a nozzle 2 is attached as shown in FIG. 4A, and about 100 μl of the sample diluent is applied from the nozzle 2 to the test strip. The sample was dropped onto the sample addition member 7, and after 15 minutes, the coloration at the capture site 61a (A line) and the capture site 61b (B line) was evaluated with the naked eye. The results are shown in Table 1. Evaluation was performed according to the following criteria.

++: Strong coloration was observed.
+: Ordinary coloration was observed.
±: Very little coloration was observed.
−: Coloration was not recognized.

  As a control, a commercially available influenza virus antigen detection reagent “Capilia Flu A + B” (trade name) was used, and 100 μl of the sample dilution was dropped, and the same evaluation as described above was performed. The antibody used in this commercially available reagent is the same as the above monoclonal antibody. The results are shown in Table 1.

  From Table 1, it can be seen that the measurement method of the present invention has a detection sensitivity equivalent to or higher than that of a commercially available product.

  In the same manner as in Example 1 except that a specimen dilution diluted 400-fold, 200-fold, and 100-fold was obtained using a commercially available influenza virus B-type nucleoprotein instead of influenza virus type A nucleoprotein. Measurement and evaluation were performed. The results are shown in Table 2.

  From Table 2, it can be seen that the measurement method of the present invention has a detection sensitivity equivalent to or higher than that of a commercially available product.

  The present invention is useful for immunochromatographic measurement of various specimens collected from a patient's nose, throat, conjunctiva and the like.

(A) is the front view which decomposes | disassembles and shows the specific example of the container for extraction of this invention, and shows a part of cover of a nozzle in a cross section, (b) is a nozzle of the nozzle shown in (a). A longitudinal sectional view, (c) shows a lid that can be attached to the container body in place of the nozzle shown in (a). (A) is a top view of the test strip for immunochromatography measurement provided with the film | membrane carrier of this invention, (b) is a longitudinal cross-sectional view of (a). (A) is a perspective view which shows the usage method of the kit of this invention provided with the test strip for immunochromatography measurement of FIG. 2, and the container for extraction, (b) is the BB line of the test strip of (a). FIG. (A) is a longitudinal cross-sectional view which shows the other specific example of the container for extraction of this invention, (b) is a longitudinal cross-sectional view shown in the state which decomposed | disassembled the container for extraction of (a). (A) is a longitudinal cross-sectional view which shows the other specific example of the extraction container of this invention, (b) is a longitudinal cross-sectional view shown in the state which decomposed | disassembled the extraction container of (a).

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Container main body, 11 ... Open end, 12 ... Closed end, 13, 14 ... Side wall part, 141 ... Housing, 15 ... Threaded part, 15 '... Convex part, 16 ... Cotton swab, 2 ... Nozzle, 2' ... Lid Body, 21 ... cylindrical part, 22 ... collar part, 23 ... nozzle part, 24 ... female thread part, 24 '... concave part, 25 ... threaded part, 26 ... step part, 27 ... discharge path, 29 ... small cylindrical member, 3 ... Lid, 4 ... impregnated member, 4 '... filter, 5 ... adhesive sheet, 6 ... membrane carrier, 61, 61a, 61b ... capture part, 62 ... control line, 7 ... sample addition member, 8 ... absorption member, 9 ... Case, 93 ... Sample injection hole, 94 ... Determination hole.

Claims (6)

An extraction container for extracting a target substance from the specimen by mixing the specimen with a developing solvent and a membrane carrier for chromatographically developing the extracted target substance together with the developing solvent An immunochromatography measurement kit, wherein the membrane carrier comprises at least a capture site formed by immobilizing a first substance that immunologically binds to the analyte, and the extraction container comprises the analysis It comprises a second substance that immunologically binds to the target substance in a state capable of immunologically binding to the analyte, and the second substance is labeled with a labeling substance Immunochromatography measurement kit. 2. The immunochromatography measurement kit according to claim 1, wherein the second substance is contained in the extraction container in a state of being impregnated in an impregnation member. The extraction container is detachably attached to a tubular main body (1) having an open end (11) and a closed end (12) opposite to the open end (11), and the open end (11) of the main body (1). The immunochromatography measurement kit according to claim 2, comprising at least a nozzle (2), wherein the impregnating member (4) is accommodated in the nozzle (2). The impregnating member (4) is made of a porous material that allows the developing solvent, the analysis object, and the second substance to pass through, and the nozzle is arranged so as to be discharged from the container in a mixed state. The kit for immunochromatography measurement according to claim 3, which is arranged in the discharge route of (2). In order to develop the chromatograph on a membrane carrier (6) having at least a capture site (61) formed by immobilizing a first substance that immunologically binds to the analyte, the sample is mixed with a developing solvent. An extraction container for extracting the analyte from the specimen, the extraction container immunologically binding a second substance that immunologically binds to the analyte to be analyzed with the analyte An extraction container for immunochromatography measurement, comprising the second substance in a bindable state, wherein the second substance is labeled with a labeling substance. A membrane carrier used for chromatographic development of an immunologically combined product of the analyte and the second substance extracted from the extraction container according to claim 5, wherein the membrane carrier is immunologically coupled with the analyte. A membrane carrier for immunochromatography measurement, comprising at least a capture site (61) formed by immobilizing a first substance to be bound.

Images