JP2010000027A - New inspection method and inspection kit usable therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new inspection method using a solid supporter and utilizing an enzyme cycling method, and to provide an inspection kit usable therefor. <P>SOLUTION: The method for detecting a material to be detected in a specimen by using a reagent composition R1, a reagent composition R2 and a reagent composition R3 containing the following components, and the solid supporter includes immobilizing the reagent composition R1 on the solid supporter to form a detector, feeding a mixture of the reagent composition R2 and the specimen to the solid supporter, and feeding the reagent composition R3 to the detector on the solid supporter to form a complex of (a material to be detected-capturing reagent)-(the material to be detected)-(an alkaliphosphatase labeled reagent) at the detector on the solid supporter, and then carrying out the enzyme cycling reaction at the detector, wherein the reagent composition R1 is a composition of a dehydrogenase and/or an electron transportation material, and a material to be detected-capturing reagent; the reagent composition R2 is a composition of an alkali phosphatase-labeled reagent obtained by labeling a material to be bonded to the material to be detected with an alkali phosphatase; and the reagent R3 is a composition of a dehydrogenase and an electron transportation material, not contained in the reagent composition R1, a substrate of the dehydrogenase, tetrazoleum and nicotinamide adenine dinucleotide phosphate (NADP) or a reducing type thereof (NADPH). <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel test method using an enzyme cycling method using a solid support and a test kit used therefor.

  There are known a simple test method and a simple test reagent for performing various tests such as the presence or absence of a pathogen such as a virus or bacteria, the presence or absence of pregnancy in a short time. The simple test reagent is used for detection or quantification of pathogens, hormones, human-derived components of C-reactive protein, and the like.

  Many of the simple test reagents are characterized in that they do not require special equipment, are easy to operate and are inexpensive. Simple test reagents are also sold in general pharmacies as, for example, pregnancy diagnostic agents. Simple test reagents for examining the presence or absence of pathogen infection are widely used not only in large hospitals and medical examination centers, but also in general hospitals and clinics. The test using such a simple test reagent is called point-of-care testing (POCT) and greatly contributes to the improvement of medical quality.

  As a simple test reagent, for example, a membrane enzyme immunoassay using alkaline phosphatase (ALP) as a label and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) / nitrotetrazolium blue (NTB) as a substrate is known. ing. This method has a feature that highly sensitive measurement can be performed in 10 to 15 minutes (see, for example, Patent Documents 1 and 2 and Non-Patent Document 1). In the medical field, it has been desired to further reduce the reaction time and to measure with high sensitivity.

  In general enzyme immunoassays that require solid-liquid separation (also referred to as B / F separation), nicotinamide adenine dinucleotide (NAD) or its reduced form (NADH) can be used with the same high sensitivity as luminescence and fluorescence methods. An enzyme cycling method for detection is known (see Non-Patent Documents 2 and 3, etc.).

  When the enzyme cycling method was applied to a simple test method, it was expected to be a highly sensitive test method. However, the enzyme cycling method has been considered unsuitable for a simple test reagent because the long-term stability of the reagent is poor and natural color develops in a short time to increase the background.

JP-A-11-153600 International Publication No. WO2006 / 073153 Pamphlet Ono, T., et al: J. Immunol. Methods, 272, 211-218, 2003. Johannsson A., et al: Clin. Chim. Acta, 148, 119-124, 1985. Stanley C.J., et al: J. Immunol.Methods, 83, 89-95, 1985.

  An object of the present invention is to provide a novel test method using an enzyme cycling method using a solid support and a test kit used therefor.

  The present inventor has intensively studied a simple inspection method using an enzyme cycling method. In the inspection method using a solid support such as a membrane, the present inventor divides the reagent compounds involved in the complex formation reaction and the enzyme cycling reaction on the solid support into three types of reagent compositions, In addition, one reagent composition was immobilized on a solid support, including compounds that did not react with each other during storage. Since the reagent compound is divided into three compositions and no compound reacting with each other is contained in one reagent composition, the reagent does not spontaneously develop color over a long period of time, and the stability of the reagent is improved. Furthermore, since the enzyme cycling method was used, the detected object could be detected with higher sensitivity than the conventional simple inspection method.

That is, the present invention is as follows.
[1] A method of detecting an object in a specimen using a reagent composition R1, a reagent composition R2, a reagent composition R3, and a solid support each containing the following components, Including solidifying the reagent composition R1 and providing a detection unit, supplying the mixture of the reagent composition R2 and the specimen to the solid support, and then supplying the reagent composition R3 to the detection unit on the solid support. The detection part on the solid phase support forms a complex of the detection target capture reagent-detection target-alkaline phosphatase labeling reagent, and the detection part detects the detection target in the sample by performing an enzyme cycling reaction. Method.
Reagent composition R1: Dehydrogenase and / or electron transfer substance and detection object capture reagent reagent composition R2: Alkaline phosphatase labeling reagent reagent composition R3: substance that binds to the detection object with alkaline phosphatase R3: Dehydrogenase And electron transfer substances not included in the reagent composition R1, dehydrogenase substrate, tetrazolium, and nicotinamide adenine dinucleotide phosphate (NADP) or its reduced form (NADPH)
[2] The method for detecting an object to be detected in a specimen according to [1], wherein the dehydrogenase is at least one selected from the group consisting of alcohol dehydrogenase, glucose dehydrogenase, and glycerol dehydrogenase.
[3] The method for detecting an object to be detected in a specimen according to [1] or [2], wherein the electron transfer substance is diaphorase.
[4] Any of [1] to [3], wherein the reagent composition R2 contains 0.2 to 10 (w / v)% surfactant and 1 to 10 (w / v)% animal-derived serum albumin Of detecting an object to be detected in a sample.
[5] A method for detecting an object to be detected in a specimen according to any one of [1] to [4], wherein the reagent composition R1 is bound to a carrier and the carrier is immobilized on a solid support.
[6] The method for detecting an object to be detected in a specimen according to [5], wherein the carrier is fine resin particles.
[7] A method for detecting an object to be detected in a specimen according to any one of [1] to [6], wherein the object capturing reagent and the object to be detected are bound by an antigen-antibody reaction.
[8] A flow-through method, in which the mixture of the reagent composition R2 and the specimen is supplied to the solid support, and the detection part on the solid support supports the detection target capture reagent-detection target-alkaline phosphatase labeling reagent. Detecting an object to be detected in any one of samples [1] to [7], forming a complex and then supplying reagent composition R3 to the detection unit on the solid support to cause an enzyme cycling reaction how to.
[9] A sample is added to a sample addition device including a reagent composition R2 and having a filtration filter, and a mixture of the sample and the reagent composition R2 is prepared and supplied to a solid phase support. A method for detecting an object to be detected.
[10] A lateral flow method, in which the mixture of the reagent composition R2 and the specimen is supplied to the solid support, and then the reagent composition R3 is supplied to the solid support, and the mixture of the specimen and the reagent composition R2 is fixed. [1] to [1] to [1] to [1], wherein the phase support is developed, and a complex of detected substance capture reagent-detected substance-alkaline phosphatase labeling reagent is formed in the detection part on the solid phase support. 7] A method for detecting an object to be detected in any specimen.
[11] The reagent composition R1, the reagent composition R2, and the reagent composition R3 each containing the following components are separately contained, and the enzyme cycling reaction is performed on the solid phase by the method according to claim 1 to A test kit for detecting an object to be detected, wherein the reagent composition R1 is immobilized on a solid support.
Reagent composition R1: Dehydrogenase and / or electron transfer substance and detection object capture reagent reagent composition R2: Alkaline phosphatase labeling reagent reagent composition R3: substance that binds to the detection object with alkaline phosphatase R3: Dehydrogenase And electron transfer substances not included in the reagent composition R1, dehydrogenase substrate, tetrazolium, and nicotinamide adenine dinucleotide phosphate (NADP) or its reduced form (NADPH)
[12] The test kit according to [11], wherein the dehydrogenase is at least one selected from the group consisting of alcohol dehydrogenase, glucose dehydrogenase, and glycerol dehydrogenase.
[13] The test kit according to [11] or [12], wherein the electron transfer substance is diaphorase.
[14] Any of [11] to [13], wherein the reagent composition R2 contains 0.2 to 10 (w / v)% surfactant and 1 to 10 (w / v)% animal-derived serum albumin Inspection kit.
[15] The test kit according to any one of [11] to [14], wherein the reagent composition R1 is bound to a carrier, and the carrier is immobilized on a solid support.
[16] The test kit according to [15], wherein the carrier is fine resin particles.
[17] The test kit according to any one of [11] to [16], wherein the detection target capturing reagent and the detection target are bound by an antigen-antibody reaction.
[18] The test kit according to any one of [12] to [17], which is a flow-through method.
[19] The test kit according to [18], further comprising a specimen addition device comprising a reagent composition R2 and having a filtration filter.
[20] The test kit according to any one of [11] to [17], which is a lateral flow method.

  The novel inspection method using the enzyme cycling method using the solid phase support of the present invention enables high-sensitivity detection of an object to be detected in a specimen quickly and easily. In addition, the test method of the present invention is characterized in that since the compound involved in the reaction is divided into three reagent compositions, the test reagent is stable for a long period of time and natural color development can be suppressed.

  Hereinafter, the present invention will be described in detail.

  Parts and% in this specification are based on mass unless otherwise specified.

  The present invention relates to a method for detecting an analyte in a sample by an enzyme cycling reaction using a solid phase support on which a reagent containing an analyte capture reagent capable of capturing the analyte in the specimen is immobilized. Including the flow-through method and the lateral flow method. In the flow-through method, the sample passes across the solid support, and in the lateral flow method, the sample develops and moves along the solid support.

  The enzyme cycling reaction is a method for detecting an object to be detected with high sensitivity by amplifying the enzyme reaction. In the present invention, an enzyme cycling method using alkaline phosphatase (ALP) is used. The principle of the enzyme cycling method using alkaline phosphatase (ALP) as a labeling substance will be described below. In this description, alcohol dehydrogenase (ADH) is used as a dehydrogenase, and enzyme diaphorase (DIA) is used as an electron mediator. The reduced form (NADPH) of nicotinamide adenine dinucleotide phosphate (NADP) which is a substrate of ALP is decomposed into NADH and inorganic phosphate by the enzymatic reaction of ALP.

  NADH is converted into NAD by the tetrazolium salt and its reductase diaphorase (DIA), and at the same time, the tetrazolium salt is reduced to form formazan, and a colored signal is obtained. NAD acts as a coenzyme in the reaction from ethanol to acetaldehyde by ADH in the presence of alcohol dehydrogenase (ADH) and its substrate ethanol, and is then reduced to NADH. Since NADH repeats redox under these conditions, the color signal intensity is amplified.

  When nicotinamide adenine dinucleotide phosphate (NADP) is used as an ALP substrate, NADP is decomposed into NAD and inorganic phosphate by the enzymatic reaction of ALP. NAD acts as a coenzyme in the reaction from ethanol to acetaldehyde by ADH in the presence of alcohol dehydrogenase (ADH) and its substrate ethanol, and is then reduced to NADH. Thereafter, the color develops on the same principle as NADH, and the color signal intensity is amplified. In addition, the product formazan is an insoluble dye and is easily deposited on a solid support, and thus is suitable for the method of the present invention in which an enzyme cycling reaction occurs on the solid support.

  In the method of the present invention, a specimen containing an object to be detected and a labeling reagent labeled with an enzyme are supplied to the solid phase support, and the complex of the object capturing reagent-detected object-labeled reagent is used as the solid support. An object to be detected is detected by forming a label and detecting the label by an enzymatic reaction. In this enzyme reaction, an enzyme cycling reaction is used to amplify the enzyme reaction.

  The sample is brought into contact with an alkaline phosphatase labeling reagent (hereinafter also simply referred to as “labeling reagent”) and mixed before being placed on the solid support on which the detection target-capturing reagent is immobilized. A complex of detected substances is formed. When the sample contains an object to be detected, the sample and the labeling reagent are mixed to form a mixture by bringing the sample and the labeling reagent into contact with each other. The mixture of the specimen and the labeling reagent includes a mixture of the detection object and the labeling reagent, and further includes a complex of the detection object and the labeling reagent. The solid support may be any material as long as the sample can be absorbed and flowed by capillary action. For example, solid support is nonwoven fabric, paper, cellulose ester such as nitrocellulose and cellulose acetate, nylon such as nylon 6,6, polyethersulfone, polyvinyl alcohol, polyester, glass fiber, silica fiber, polyolefin, cellulose and nitrocellulose. Selected from the group consisting of artificial polymers consisting of mixtures of these and mixtures of cellulose esters. A microporous material made from nitrocellulose or the mixture is preferably used. The pore size or retained particle size of the solid support is preferably 1 to 12 μm, particularly preferably 3 to 5 μm. Although the thickness of a solid-phase support body is not specifically limited, Usually, it is about 100-200 micrometers.

  When the method of the present invention is a flow-through method, the solid-phase support is a membrane-like support of any size, and the target-capturing reagent is immobilized on the membrane, and the target is detected on the membrane. The immobilization site of the capture reagent is set. When the method of the present invention is a lateral flow method, it is preferably a membrane-like support having a strip-like strip shape.

  Next, a mixture of a specimen and a labeling reagent is provided on a solid support on which a target capturing reagent that specifically binds to the target is immobilized, and a target capturing reagent-detected object-labeling reagent is provided. To form a complex. At this time, in the flow-through method, the complex of the labeling reagent and the detection target is captured by the detection target capturing reagent when the detection target capturing reagent passes through the solid phase support on which the solid phase is immobilized. A complex of detected substance capture reagent-detected object-alkaline phosphatase labeling reagent is formed. Further, in the lateral flow method, when the detection target capture reagent moves on the solid phase support on which the solid phase is immobilized, the detection target capture reagent captures the detection target capture reagent-detection target-alkaline phosphatase. A complex of labeling reagents is formed. The presence of the detection target can be determined by detecting the presence or absence of the labeling reagent captured on the solid phase support.

  An object to be detected in the method of the present invention is a substance or a microorganism that can form a complex of an object capturing reagent-detected object-alkaline phosphatase labeling reagent. The detection target capturing reagent is a substance that can bind to the detection target. Examples of the combination of the detection object and the detection object capturing reagent include an antigen and an antibody, a receptor and a ligand, and the like.

  The combination of the detection object and the detection object capturing reagent may be reversed. That is, the detection target may be an antigen, and the detection target capture reagent that binds to the detection target may be an antibody. Conversely, the detection target is an antibody and the detection target capture reagent that binds to the detection target is an antigen. Also good. Alternatively, the detection target capture reagent that binds to the detection target may be a receptor that binds to the ligand, and conversely, the detection target that binds to the detection target using the detection target as a receptor. A capture reagent may be a ligand.

  When the detection target is an antigen, examples of the detection target include influenza A virus, influenza B virus, RS virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, HBs, HCV. Virus antigens such as HIV, EBV, Chlamydia trachomatis, Streptococcus, Bordetella pertussis, Helicobacter pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia, Legionella, Bacillus anthracis, MRSA and other bacterial antigens, bacteria, etc. Toxins, mycoplasma lipid antigens, peptide hormones such as human chorionic gonadotropin, steroids such as steroid hormones, bioactive amines such as epinephrine and morphine, vitamins such as vitamin B, prostaglandins, Antibiotics such Rasaikurin, various tumor markers, pesticides, and environmental hormones.

  When the detection target is an antibody, the detection target is the above virus antigen, bacterial antigen, toxin, peptide hormone, steroid, bioactive amines, vitamins, antibiotics, various tumor markers, pesticides, environmental hormones, etc. And antibodies that selectively react with the antigens of the above.

  When the detection target is an antigen and an antibody is used as a detection target capturing reagent that binds to the detection target, a polyclonal antibody or a monoclonal antibody can be used as the antibody.

  In the method of the present invention, when a membrane such as a nitrocellulose membrane is used as the solid support, it is called a membrane enzyme assay, and when an antigen-antibody reaction is used, it is called a membrane enzyme immunoassay. Furthermore, in the case of the lateral flow method, it is called immunochromatography.

  In the method of the present invention, three types of reagent compositions are used: reagent composition R1, reagent composition R2 and reagent composition R3.

The reagent composition R1 contains a dehydrogenase and / or an electron transfer substance and an object capturing reagent. Either or both of the dehydrogenase and the electron transfer material may be contained. These substances are immobilized on a solid support. A site on the solid support on which the reagent composition R1 is immobilized is referred to as a detection unit. As the dehydrogenase, alcohol dehydrogenase (ADH), glucose dehydrogenase, glycerol dehydrogenase and the like are used, and preferably alcohol dehydrogenase is used. Examples of electron mediators include diaphorase (DIA), phenazine methosulfate (PMS), 1-methoxy-PMS (1-methoxy-5-methylphenazinium methylsulfate), Meldola Blue, flavin mononucleotide (FMN / FMNH ₂ ), flavin adenine Flavins such as dinucleotide, ubiquinone (UQ), cytochrome (Cyt) and the like can be used. Among these, diaphorase (DIA), which is a protein and can be easily solid-phased on a solid-phase support such as a membrane, is preferably used.

  When the components of the reagent composition R1 are all proteins, they can be easily immobilized on a solid support (membrane). For example, a protein antigen or antibody may be used as the detection target capturing reagent of the reagent composition R1, alcohol dehydrogenase may be used as the dehydrogenase, and diaphorase may be used as the electron transfer substance. In addition, low molecular weight compounds such as flavins and ubiquinones can be easily immobilized on a solid support (membrane) by binding to a carrier protein. Examples of carrier proteins that can be used include albumin and keyhole limpet hemocyanin (KLH). A solid support may contain a plurality of analyte capture reagents. When a plurality of detection object capturing reagents are included, a plurality of detection objects can be detected by one inspection apparatus. For example, by using an anti-influenza A virus NP antibody and an anti-influenza B virus NP antibody as detection target capture reagents, it is possible to detect both influenza A virus and influenza B virus with a single test device. it can. The plurality of detection target capturing reagents may be immobilized at different positions on the solid support, for example.

  The reagent composition R1 may be immobilized on a solid support by a known method such as a method using adsorption or a method of chemically bonding using a functional group such as an amino group or a carboxyl group.

  Alternatively, the reagent composition R1 may be bound to a carrier, and the carrier may be immobilized on a solid support. As the carrier, for example, silica powder, zeolite, aerogel (alumina fine powder), aluminum hydroxide fine powder, inorganic powder such as glass beads, fine particles such as latex, etc., fine resin particles such as latex particles can be used. Are preferably used. By binding the reagent composition R1 to the carrier, the solid phase support can be easily and reliably solid-phased. In this case, after the compounds contained in the reagent composition R1 are separately bound to the carrier, the carriers to which the respective compounds are bound may be mixed, or the compounds contained in the reagent composition R1 may be simultaneously bound to the carrier. You may let them. In the present invention, a carrier to which a compound contained in the reagent composition R1 is bound is also referred to as a reagent composition R1.

  The dehydrogenase in the reagent composition R1 is 0.5 to 50 units / mL, preferably 1 to 10 units / mL, and the electron transfer material is 0.5 to 50 units / mL when diaphorase (DIA) is used. Preferably, it can be prepared and solidified as a solution of 1 to 10 units / mL.

In addition, although the detection target capturing reagent varies depending on what is used, it can be prepared as a 0.2 to 2 mg / mL solution and immobilized on a solid support. A solution containing all of these may be prepared and immobilized, or may be prepared as separate solutions and separately immobilized.
The amount of the liquid in the case of solid phase varies depending on the material of the support, the thickness, and the area of the detection part, but optimization is easy for those skilled in the art.

  When dehydrogenase and diaphorase are solid-phased in the above range, the enzyme cycling reaction proceeds smoothly. In particular, it is preferable to use alcohol dehydrogenase (ADH) as the dehydrogenase and diaphorase (DIA) as the electron mediator because the enzyme cycling reaction is rapid and vividly develops color.

  One unit of dehydrogenase (eg, ADH) activity is defined as the amount of enzyme that oxidizes 1 μmol of ethanol per minute at pH 8 and 30 ° C. One unit of activity of an electron mediator (eg, DIA) is defined as the amount of enzyme that reduces 1 μmol of p-iodonitrotetrazolium violet (INT) per minute at pH 8 and 30 ° C.

  The reagent composition R2 contains an alkaline phosphatase labeling reagent obtained by labeling a substance that binds to the analyte with alkaline phosphatase. In the present invention, the reagent composition R2 may be simply referred to as a labeling reagent. Examples of the substance that binds to the detection target include an antibody or an antigen that binds to the detection target through an antigen-antibody reaction, and a receptor substance or a ligand substance that has a relationship between the detection target, the receptor, and the ligand. The substance that binds the detection target contained in the reagent composition R2 may be the same substance as the detection target capture reagent contained in the reagent composition R1. However, when there is only one site that binds to the detection target capture reagent in the detection target, a complex of the detection target capture reagent-detection target-alkaline phosphatase labeling reagent is not formed. Therefore, in this case, it is necessary to bind the detection target capturing reagent and the labeling reagent to different parts of the detection target. In order to label a substance that binds to an object to be detected with alkaline phosphatase, a known enzyme labeling method may be used, for example, a generally used method for forming a maleimide bond or an ester bond.

  The specimen first comes into contact with the reagent composition R2, which is a labeling reagent, and the detected substance in the specimen and the labeling reagent form a complex. The contact between the specimen and the labeling reagent can be carried out by preparing the labeling reagent as a liquid reagent or a solid reagent such as a lyophilized product and mixing it with the specimen. In the case of the flow-through method, an adapter containing the reagent composition R2 is set on the upper layer of the solid support, and an adapter that can be separated from the solid support is set, and a sample is added to the adapter. The sample and the labeling reagent come into contact with each other in the adapter. An example of the adapter is shown in FIGS. 1 and 2 (FIGS. 1a and 2a), which has one or more openings for supplying a mixture of analyte and labeling reagent to a solid support, After the specimen and the labeling reagent come into contact, the mixture of the specimen and the labeling reagent is supplied to the solid phase support through the opening.

  Contact between the labeling reagent and the specimen may be performed in the specimen addition device. The specimen addition device is an accessory part outside the inspection apparatus, and is a container for putting a collected specimen into a specific process. Such devices include vials, syringes, tubes and the like. The device may include not only a container but also a filtration means such as a filtration filter for filtering the specimen when the specimen is added and supplied to the test apparatus. The sample addition device includes a container portion for storing the sample and a portion for adding and supplying the sample in the container to the inspection apparatus. The latter part to which the specimen is added and supplied has a part having a nozzle (opening) for discharging the specimen from the container, and this part can also serve as a lid for the container part. The portion where the latter specimen is added and supplied to the inspection apparatus is also referred to as a nozzle portion or a lid portion. When the specimen addition device includes a filtering means, the specimen addition device may be composed of two parts, a nozzle portion including a filter housing including a filtration filter and a container body portion. The filter housing in this case has an opening through which the sample passes through the filter and an opening through which the sample that has passed through the filtration filter is discharged, and the filtration filter exists between both openings. As the container, for example, a tube, a syringe, or the like that can pass the sample through the filtration filter with pressure may be used.

  The labeling reagent may be incorporated in the sample addition device. Incorporation here means that the labeling reagent is included in any part of the sample addition device, and the sample and reagent composition R2 are contained in the sample addition device after the sample is placed in the sample addition device. To make contact. The object to be detected is brought into contact with the labeling reagent by providing the object to be detected in the sample, and only the operation of adding the sample to the solid support on which the reagent composition R1 is immobilized is performed. Thus, formation of a complex of a detected substance capture reagent-detected substance-alkaline phosphatase labeling reagent can be carried out simply and rapidly. For example, a necessary amount of labeling reagent may be placed in a sample collection container (main body part) such as a vial, syringe, or tube, and the necessary amount of sample may be added to the container and mixed. The specimen collection container (main body part) is preferably made of a flexible material such as polyethylene, polyethylene terephthalate (PET), or vinyl chloride. An example of the sample addition device is shown in FIGS. The labeling reagent may be liquid or freeze-dried, and it is absorbed into an appropriate solid porous material such as glass fiber, non-woven fabric, sponge, etc., dried, and the cut piece of the porous material (labeling reagent pad) You may put in. In the present invention, a solid impregnated with a labeling reagent is also referred to as a labeling reagent part. In this case, the liquid specimen comes into contact with the section, and the labeling reagent in the section is dissolved in the specimen. In order to make the inspection apparatus compatible with long-term storage, it is more preferable to provide the inspection apparatus in a dry state rather than a liquid state. In order to prepare a dried product of the labeling reagent, the freeze-drying method is a batch method and requires a special large-scale facility. On the other hand, in the warm air drying method, continuous production is possible by spraying a reagent on roll paper (roll-shaped support) and then drying with a dryer-type warm air blower. The form of the labeling reagent can be properly used depending on the purpose. At this time, in order to remove impurities in the specimen, it is desirable to filter the specimen or a mixture of the specimen and the labeling reagent. Filtration may be performed when the complex of the labeling reagent and the detection object is added to the inspection apparatus. A filtration means such as a filter may be included in the nozzle portion of the specimen addition device, and the specimen and the labeling reagent mixture may be added through the filter. At this time, a labeling reagent may be incorporated in the filtration filter. For example, the filtration reagent itself may be allowed to absorb the labeling reagent and be dried, or the filtration filter may be brought into contact with a section of the porous material impregnated with the labeling reagent and dried. Further, a filtration filter and a porous material containing a labeling reagent may be incorporated in the filter housing. At this time, the porous material containing the filtration filter and the labeling reagent may or may not be in contact. The filtration filter is not limited as long as it can remove impurities such as aggregates and solids contained in the specimen, and a glass fiber or a membrane made of nitrocellulose is preferably used. The pore size or retained particle size of the filtration filter is important for reducing nonspecific reactions. A preferable pore size or retained particle size of the filtration filter is 0.1 to 0.6 μm. If the pore size or retained particle size is large, non-specific binding may occur on the membrane, which may indicate false positives. On the other hand, if it is too small, the filtration filter itself may be easily clogged due to viscous substances and aggregated substances present in the specimen. In addition, when whole blood is used as a specimen, the filtration filter may have a function of removing red blood cells. For example, it is possible to remove red blood cells by including an anti-red blood cell antibody in the filtration filter. When whole blood is used, it is desirable that the sample and the labeling reagent come into contact after removing red blood cells with a filtration filter. In the case of the lateral flow method, the solid support and the porous support containing the labeling reagent are brought into contact with each other, and the specimen is added to the porous support containing the labeling reagent. In this case, the portion containing the labeling reagent also serves as a sample supply unit for adding the sample. By adding the sample to the sample supply unit, the sample and the labeling reagent come into contact with each other, and the mixture of the sample and the labeling reagent immediately moves to the solid support.

  The reagent composition R3 includes a dehydrogenase and an electron transfer material that are not included in the reagent composition R1, a substrate for the dehydrogenase, tetrazolium, and nicotinamide adenine dinucleotide phosphate (hereinafter also referred to as NADP). Or its reduced form (NADPH). When reagent composition R1 contains both dehydrogenase and electron transfer material, reagent composition R3 does not contain both dehydrogenase and electron transfer material, and reagent composition R1 contains dehydrogenase and electron transfer material. When only a dehydrogenase is included among the substances, the reagent composition R3 includes an electron transfer substance. When the reagent composition R1 includes only an electron transfer substance among the dehydrogenase and the electron transfer substance, the reagent composition The product R3 includes a dehydrogenase.

  Tetrazolium is a general term for tetrazolium and metal salts such as sodium salt and potassium salt of tetrazolium. In the present invention, the reagent composition R3 is sometimes referred to as a coloring reagent composition.

  Examples of the substrate for the dehydrogenase include alcohol in the case of alcohol dehydrogenase. As the alcohol, primary alcohols such as ethanol, methanol, propyl alcohol and the like can be used, and ethanol is preferably used. In the case of glucose dehydrogenase, glucose is used, and in the case of glycerol dehydrogenase, glycerol is used.

  Examples of tetrazolium include tetrazolium such as p-iodonitrotetrazolium violet and nitrotetrazolium blue, and metal salts or ammonium salts of these tetrazoliums.

  The reagent composition R3 may contain a surfactant. In the present invention, the portion of the membrane on which the above-described reagent composition R1 is solid-phased functions as a detection unit that generates a detection signal, and the specimen, the reagent composition R2, and the reagent composition R3 are added to the detection unit. As a result, a complex of the detection target capturing reagent-detected target-alkaline phosphatase labeling reagent is formed on the detection section. In this case, when the reagent composition R3 is added to the detection section, the amount of free labeling reagent that is not bound to the detected substance in the sample is reduced by the cleaning action of the surfactant, so that the background becomes high. It becomes possible to measure accurately. Examples of the surfactant include Triton X-100 (mainly composed of polyethylene glycol mono-р-isooctylphenyl ether), Tween 20 (mainly composed of polyoxyethylene sorbitan monolaurate), and Tween 80 (polyoxyethylene sorbitan monooleate). Nonidet P-40 (mainly polyoxyethylene (9) octylphenyl ether), ZWITTERGENT 3-14 (mainly n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate) ), CHAPS (mainly 3-[(3-colamidopropyl) dimethylammonio] propanesulfonic acid), sodium dodecyl sulfate (SDS) and the like can be used. The concentration of the surfactant contained in the reagent composition R3 is preferably 1% or less, and more preferably 0.01 to 0.5%. Distilled water, physiological saline, a buffer solution, or the like may be further added to the reagent composition R3 as necessary.

As the reagent composition R3, for example, a mixture containing the following components in Tris buffer, 50 to 200 mM, pH 8.4 to 9.5 is preferably used.
NADPH or NADP 0.05-0.4 mM
Alcohol 3-8%
Sodium salt of tetrazolium 0.6-1.2 mM
Magnesium chloride 2-6mM
Surfactant 0.01-0.5 (w / v)%

  In the flow-through method and the lateral flow method, first, when measuring the object to be detected, the sample and the reagent composition R2 are mixed on the detection part on which the reagent composition R1 on the solid phase support is solidified. And reagent composition R2. If it is positive, a complex of the detection object capturing reagent-detected object-alkaline phosphatase labeling reagent is generated in the detection unit. When the reagent composition R3 is further added, an enzyme cycling reaction occurs in the detection part.

  In the present invention, the essential components of the enzyme cycling reaction are divided into the reagent composition R1, the reagent composition R2, and the reagent composition R3, and each component necessary for the enzyme cycling reaction is finally mixed in the detection unit at the time of inspection. Therefore, side reactions and natural color development of the test reagent can be suppressed. As a result, background increase due to natural color development can be suppressed, and measurement can be performed quickly and with high sensitivity.

  Samples including the detection object used in the method of the present invention are samples derived from a living body, and include samples collected directly from a living body, samples obtained by processing a living body derived in vitro, and the like. For example, a throat or nasal swab collected from a patient's pharynx or nasal cavity using a sample collection device such as a sterile swab, a throat or nasal irrigation liquid, nasal aspirate, saliva, serum, plasma, whole blood, A culture solution of a specimen that may contain fecal suspension, urine, bacteria, and the like can be used. The specimen may be diluted with a buffer solution, physiological saline, or the like as necessary. Examples of the buffer used for diluting the specimen include Tris buffer and Good buffer.

  If the sample is a viscous liquid such as a throat or nasal swab collected from a patient's pharynx or nasal cavity using a sample collection device such as a sterile swab, or a cleaning fluid such as a throat or nasal cavity, dilute the sample. The test is performed by suspending the specimen in a buffer solution or the like. A sample diluted with a buffer or the like is called a sample suspension, and in the present invention, a sample obtained by processing a sample such as a sample suspension is also called a sample. A buffer used to float the specimen is referred to as a specimen suspension buffer.

  The buffer for diluting the specimen or the buffer for specimen suspension contains 0.2 to 10 (w / v) surfactant, animal-derived serum such as bovine serum albumin, horse serum albumin, goat serum albumin, sheep serum albumin Albumin 1 to 10 (w / v)% and / or an appropriate concentration of inorganic salt or the like may be added. By diluting or floating the specimen with a buffer containing a surfactant and animal-derived serum albumin, a false positive reaction caused by components other than the detection target can be suppressed.

  Examples of the surfactant include Triton X-100 (mainly polyethylene glycol mono-р-isooctylphenyl ether), Tween 20 (mainly polyoxyethylene sorbitan monolaurate), and Tween 80 (polyoxyethylene sorbitan monooleate). Main body), Nonidet P-40 (mainly polyoxyethylene (9) octylphenyl ether), ZWITTERGENT 3-14 (mainly n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate) CHAPS (mainly 3-[(3-collamidopropyl) dimethylammonio] propanesulfonic acid), sodium dodecyl sulfate (SDS), and the like can be used.

  For example, when detecting whether an influenza virus is present in a specimen, the specimen floating buffer containing 0.2 to 10 (w / v) surfactant, 1 to 10 (w / v) bovine serum albumin, etc. When diluted with a liquid, virus particles are destroyed and the detection target (NP antigen) is extracted, so that the detection sensitivity is increased. Thus, the sample suspension buffer has not only the effect of diluting the sample, but also the effect of extracting a component to be detected from viruses, bacteria, and the like.

  The present invention includes an inspection apparatus and an inspection kit used for an inspection method using the enzyme cycling method using the above-mentioned solid phase support. In the present invention, the test apparatus includes a reagent and is called a test kit. The kit may further include a specimen addition device as a separate body.

  An example of a test kit based on a membrane enzyme assay using a flow-through method is shown in FIGS.

  FIG. 1 is a plan view of a flow-through type inspection kit, and FIG. 2 is a cross-sectional view taken along line II ′ of FIG. An adapter (a) that masks the surface of the membrane b is installed in the inspection kit, and the adapter (a) has openings (O1) and (O2). The reagent composition R1 is solid-phased on the membrane (b) immediately below the openings (O1) and (O2), and the solid-phased portion serves as a detection unit. In the case of this example, by immobilizing a reagent composition R1 containing a detection target capturing reagent capable of capturing a different detection target in the membrane (b) immediately below the openings (O1) and (O2), A plurality of detection objects can be detected with one inspection kit. A member (liquid absorbing member) (c) that absorbs liquid is installed below the membrane (b).

  FIG. 3 shows a tube-type specimen addition device. A filter containing the reagent composition R2 is installed in the labeling reagent part (g) of the device. The specimen is filtered through the labeling reagent part (g). The filtrate is dropped onto the openings (O1) and (O2) of the adapter (a) in FIG. By increasing the number of openings through which the specimen on the bottom surface of the adapter (a) passes, it is possible to cope with simultaneous tests of more items.

  In the case of using the sample addition device, the pore size or retained particle size of the membrane is not less than the pore size or retained particle size of the filtration filter of the sample addition device, and is preferably 1 to 12 μm, particularly preferably 3 to 5 μm. . The mixture of the specimen and the reagent composition R2 dropped on the opening (O1) or the opening (O2) of the adapter (a) reaches the detection part on the membrane. In the detection part provided directly under the opening (O1) and the opening (O2), the reagent composition R1 solid-phased in advance on the membrane, the detection target in the sample, and the reagent composition R2 react to efficiently. In this case, a complex of the detection target capturing reagent-detected target-labeling reagent is formed. Next, when reagent composition R3 is supplied, if it is positive, the complex of detected substance capture reagent-detected substance-alkaline phosphatase labeling reagent reacts with NADP in reagent composition R3 to produce NAD. This NAD reacts with alcohol dehydrogenase (ADH) and diaphorase (DIA) in the reagent composition R1 and the reagent composition R3, an enzyme cycling reaction occurs in the detection part, and the detection part is colored. In the case of negative, the complex of detected substance capture reagent-detected object-labeled reagent is not formed, no alkaline phosphatase remains in the detection part, and free labeled reagent flows downstream when reagent composition R3 is flowed. Since it is removed, color development does not occur.

  Schematic diagrams of one embodiment of the specimen addition device are shown in FIGS. 3 and FIG. 4, the device is a tube-like specimen addition device. For the labeling reagent part (g), a solid impregnated with the reagent composition R2 is used. The sample addition device has a shape composed of a tip portion (d) and a main body portion (e) as shown in FIG. 3, for example, and a labeling reagent portion as shown in FIG. 4 inside the tip portion (d). (G) and a filtration filter (f) are provided.

  A specimen suspension (sample) diluted with a specimen suspension buffer is placed inside the body (e), and a tip (d) provided with a labeling reagent (g) and a filtration filter (f) in the body (e). ). After dissolving the reagent composition R2 in the labeling reagent portion with the sample, the sample is filtered through the filtration filter (f), and the filtrate is dropped onto the membrane of the test kit.

  When the main body (e) is made of a flexible material such as polyethylene, polyethylene terephthalate (PET), vinyl chloride, etc., it is easy to press the outside of the container with your hand and apply pressure to the inside with the filter attached. In addition, the sample suspension can be easily filtered.

  The labeling reagent part (g) can be attached to the tip part (d) of the specimen addition device so that the specimen passes through the filtration filter (f) after contacting the specimen with the labeling reagent part (g) as shown in FIG. preferable. A portion provided with a filtration filter at the tip (d) shown in FIG. 4 is referred to as a filtration filter kit (h).

  In the reagent composition R3, one or more components may be separately stored in a dry state. For example, using NADPH / or NADP as a dried product, the NADPH / or NADP dried product is dissolved in a Tris buffer solution (50 to 200 mM, pH 8.4 to 9.5) containing ethyl alcohol, tetrazolium, and magnesium chloride. The method of supplying to may be sufficient. When a part of the components of the reagent composition R3 is used as a dried product, it may be mixed and dissolved with other components at the time of use.

  Here, in the test kit of the present invention, a solution flow is generated from the sample supply side toward the detection unit, which is referred to as upstream and downstream based on the flow. That is, a position closer to the specimen supply side with respect to a certain position is referred to as upstream, and a position closer to the detection unit is referred to as downstream.

  It is preferable to use one or more components of the reagent composition R3 as such a dried product because the test reagent can be stored for a long period of time.

An example of a specific procedure of the detection method of the present invention using a flow-through type test kit is shown below.
(1) Reagent composition R1 is solid-phased on a membrane to form a detection unit.
(2) Dilute the sample collected from the patient with the sample suspension buffer, filter using the sample addition device equipped with a filtration filter and / or labeling reagent part, filter the sample while mixing the reagent composition R2, and detect Drip onto the part.
(3) Remove the adapter of the device.
(4) The reagent composition R3 is dropped on the detector and allowed to react for a predetermined time.
(5) Judgment is made based on the presence or absence of color development in the detection unit after a predetermined reaction time.

  The predetermined reaction time is 3 to 10 minutes.

  If the determination cannot be made within a predetermined reaction time, the reaction may be stopped by dropping a reaction stop solution and the determination may be made later. A 0.5M aqueous citric acid solution is used as the reaction stop solution.

  Next, an example of an embodiment of the detection method of the present invention using a lateral flow test kit will be described with reference to FIG. In the kit of FIG. 5 as well, it is preferable to use a specimen addition device (FIG. 3) equipped with a filtration filter as a separate body as in the case of the flow-through method. A membrane (a) is fixed on a plastic plate (g), a coloring reagent supply unit (f) in which a nonwoven fabric is placed on the upstream side of the membrane, and a liquid absorbing member (e) is fixed on the downstream side.

(1) Reagent composition R1 is solid-phased on membrane (a) to form a detection part (b). In this case, for example, the reagent composition R1 is solid-phased linearly as shown in FIG. A plurality of reagent compositions R1 including different detection target capturing reagents may be linearly solidified, and in this case, a plurality of detection targets can be detected with one test kit. In addition, the labeling reagent part (c) containing the reagent composition R2 is placed on the membrane (a). The labeling reagent part (c) includes a sponge containing the reagent composition R2 in a dry state. At this time, the reagent composition R1 may be solid-phased on the membrane in advance and a labeling reagent part may be provided, and an apparatus having a detection part and a labeling reagent part may be used.

(2) A sample suspension obtained by diluting the sample with a sample suspension buffer is dropped onto the sample supply unit (d) on the upper surface of the labeling reagent unit (c). The specimen suspension liquid dissolves and mixes the reagent composition R2 contained in the labeling reagent portion, and moves to the membrane by capillary action. It should be noted that the labeling reagent part (c) and the sample supply part (d) are not separated parts, and the labeling reagent part (c) can also serve as the sample supply part (d).

(3) Next, the reagent composition R3 is dropped into the coloring reagent supply unit (f). The reagent composition R3 flows from the reagent supply unit toward the membrane, and mixes with the mixture of the sample and the labeling reagent in the membrane and reaches the detection unit on the membrane. That is, the reagent composition R3 develops the mixture of the specimen and the reagent along the membrane. A complex of a detection target capturing reagent-detected target-alkaline phosphatase labeling reagent is formed at the detection portion on the membrane.

  In the step (2), if the amount of the sample liquid is large, a part of the sample suspension in which the reagent composition R2 is dissolved reaches the detection unit on the membrane, and the detection unit captures the reagent-to-be-detected reagent- A detector-alkaline phosphatase labeling reagent complex is formed. In this case, the reagent composition R3 may be dropped directly on the detection unit. In step (3), the reagent composition R3 is dropped onto the color reagent supply unit, so that the sample remaining in the membrane and the reagent composition R2 are developed along the membrane and reach the detection unit. Let

  If the sample is positive, a complex of detected substance capture reagent-detected substance-labeled reagent is generated in the detection unit.

  Components that did not participate in the complex formation of the detection object capture reagent-detection object-alkaline phosphatase labeling reagent, sample suspension buffer, etc. (buffering agent, serum albumin, surfactant, etc.) ) Is absorbed. The reagent composition R3 reaches the detection unit after the complex of the detection target capturing reagent-detected target-labeling reagent is formed. As a result, each component necessary for the enzyme cycling reaction finally gathers in the detection unit, so that the enzyme cycling reaction occurs in the detection unit and a color reaction occurs. In the case of negative, since the complex of the detection object capture reagent-detection object-alkaline phosphatase labeling reagent is not formed, when the reagent composition R3 is flowed, the free labeling reagent flows downstream without staying in the detection part, so color development Does not happen.

  In the lateral flow method, the reagent composition R3 may be prepared by mixing all the components in advance, or a part of the components may be mixed immediately before use or during use. As a method of mixing the reagent composition R3 during use, for example, a method of placing a part of the components in a dry state and placing them in the coloring reagent supply unit (to) can be mentioned. In this case, when the other component is used as a developing solvent and a liquid containing the other component is supplied to the color reagent supply unit, the component incorporated in the dry state in the color reagent supply unit at the time of development is dissolved. All ingredients are mixed.

  When the object to be detected is a virus or a bacterium, it is desirable to adopt a flow-through method or a lateral flow method and install a liquid absorbing member (c, e) on the downstream side of the detection unit. In this case, the detected object that has not been captured by the detection unit is absorbed by the liquid absorbing member (c, e), so that outflow of the detected object to the outside of the device can be suppressed, and infection by the detected object is prevented. Can be prevented.

Example 1
1. Production of anti-influenza virus monoclonal antibody (1) Anti-influenza A virus NP antibody BALB / c mice were immunized with influenza A virus antigen, and the spleen was removed from mice reared for a certain period of time, and the method of Keller et al. , Nature, vol, 256, p495-497 (1975)) and fused with mouse myeloma cells (P3 × 63). The obtained fused cells (hybridoma) were maintained in a 37 ° C. incubator.

  The anti-influenza A virus activity of the fused cells was confirmed by ELISA using a plate in which influenza A virus NP antigen was immobilized on a solid phase. The cells were purified (monocloned) while confirming the antibody activity of the supernatant of the culture medium of the fused cells. The obtained two fused cell lines were each intraperitoneally administered to pristane-treated BALB / c mice, and about 2 weeks later, antibody-containing ascites was collected. IgG was purified from mouse ascites by affinity chromatography using a protein A column to obtain two types of purified anti-influenza A virus NP antibodies.

(2) Anti-B influenza virus NP antibody Two types of purified anti-B influenza virus NP antibodies were obtained in the same manner as in (1) using influenza B virus antigen.

2. Preparation of enzyme-labeled anti-influenza virus antibody (1) Preparation of enzyme-labeled anti-influenza A virus antibody 45 mg of one type of purified anti-type A influenza virus NP antibody was dialyzed against 0.1 M citrate buffer (pH 3.6). Thereafter, 10 mg of pepsin was added, and Fab ′ digestion treatment was performed at 37 ° C. for 1 hour. The treated solution was fractionated with an Ultrogel AcA34 column to obtain a purified fraction of anti-influenza A virus NP antibody F (ab ′) ₂ . The fraction was concentrated to about 10 mg / mL, mixed with 0.1 M mercaptoethylamine at a volume ratio of 10: 1, and reduced at 37 ° C. for 90 minutes. The treatment solution was fractionated with an Ultrogel AcA34 column to obtain a purified fraction of anti-influenza A virus NP antibody Fab ′, and then concentrated to about 1 mL.

  After dialyzing 1.5 mL of 10 mg / mL alkaline phosphatase into 50 mM borate buffer (pH 7.6) containing 1 mM magnesium chloride and 0.1 mM zinc chloride, 0.7 mg of N- (6-maleimidocaproyloxy) succinimide was added. Added and treated at 30 ° C. for 1 hour. The treated solution was fractionated on a Sephadex G-25 column, the first peak was collected to obtain maleimide-alkaline phosphatase, and then concentrated to about 1 mL.

  Concentrated anti-influenza A virus NP antibody Fab ′ and maleimide-alkaline phosphatase were mixed at a protein ratio of 1: 2.3 and reacted with gentle agitation at 4 ° C. for 20 hours to give an alkaline phosphatase-labeled anti-influenza A virus. NP antibody Fab ′ was obtained. Further, the reaction solution was fractionated with an Ultrogel AcA34 column, and unreacted products were removed to obtain a purified alkaline phosphatase-labeled anti-influenza A virus NP antibody Fab '. Purified alkaline phosphatase-labeled anti-influenza A virus NP antibody Fab ′ contains 0.15 M sodium chloride, 4% (W / V) bovine serum albumin, 1.5 mM magnesium chloride, 0.15 mM zinc chloride, 1% (W / V ) Triton X-100, diluted with a labeled antibody diluent consisting of a composition of 25 mM Tris buffer (pH 8.0) containing 0.09% sodium azide. The diluted solution was filtered through a 0.22 μm pore size filter to obtain an enzyme-labeled anti-influenza A virus antibody (reagent composition R2 for influenza A virus).

(2) Production of enzyme-labeled anti-B influenza virus antibody For one type of purified anti-B influenza virus NP antibody, enzyme-labeled anti-B influenza virus antibody (for influenza B virus) was prepared in the same manner as (1). Reagent composition R2) was obtained.

3. Preparation of latex particles combined with ADH and DIA (materials used)
Electron transfer material: Diaphorase (DIA, manufactured by Unitika, commercially available)
Dehydrogenase: Alcohol dehydrogenase (ADH, Roche, commercially available)
Detected substance capture reagent A: Purified anti-influenza A virus NP antibody Detected substance capture reagent B: Purified anti-type B influenza virus NP antibody

  As the enzyme, ADH (Roche) and DIA (Unitika) were used.

  ADH and DIA were dissolved in 25 mM MES buffer pH 7.0, and covalently bound to white latex particles (polystyrene type, carboxylic acid type, manufactured by JSR) with a particle size of 0.046 μm, and bovine serum albumin 0.04% Was suspended in a 5 mM Tris buffer solution (pH 7.5) containing 0.5% latex particles (ADH 50 units / mL, DIA 28 units / mL).

Production of test kits for detecting influenza A and B viruses The test kits for detecting influenza virus used were of the flow-through configuration shown in FIGS. As the membrane, a nitrocellulose membrane (size 2 × 3 cm, thickness 125 μm) having a pore diameter of 3 μm was used.

  A 5 mM Tris buffer solution (pH 7.5) containing 1 mg / mL of purified anti-influenza A virus NP antibody and 0.025% of latex particles combined with ADH and DIA was prepared as reagent composition R1. 5 μL of the reagent composition R1 was spotted on the opening (O1) of the kit. The detection part of influenza B was the same except that the purified anti-influenza B virus NP antibody was used instead of the purified anti-influenza A virus NP antibody (opening (O2)). Membranes having A-type and B-type spots were dried under reduced pressure for 40 minutes to obtain A-type and B-type influenza detection sections, respectively.

  The labeling reagent section is prepared by diluting enzyme-mixed anti-influenza A virus NP antibody and enzyme-labeled anti-type B influenza virus NP antibody to the optimum concentration, mixing and impregnating 150 μL of polyvinyl sponge with a diameter of 9 mm and a thickness of 4 mm and drying. And attached to the tip of the specimen addition device.

Detection of influenza A and B viruses The following viruses cultured in hatched chicken eggs were used as specimens.
Influenza A virus: A / Beijing / 32/92 (H3N2),
Influenza B virus: B / Shangdong / 7/97

  Samples were prepared in the following dilution series using the following sample suspension buffer. As a negative control, a sample suspension buffer was used.

Sample suspension buffer
Mainly 50 mM Tris buffer, pH 8.0, surfactant Triton X-100 1 (w / v)%, bovine serum albumin 3 (w / v)%, normal mouse immunoglobulin 0.1 (w / v)% And sodium azide 0.09 (w / v)%.
Influenza A / Beijing
1: 1 × 10 ⁴
1: 2 × 10 ⁴
1: 4 × 10 ⁴
1: 6 × 10 ⁴
1: 8 × 10 ⁴
Influenza B / Shangdong
1: 1 × 10 ³
1: 2 × 10 ³
1: 4 × 10 ³
1: 6 × 10 ³
1: 8 × 10 ³

  These specimens were prepared in the specimen addition device main body containing the specimen suspension buffer.

Test A sample addition device tip provided with a labeling reagent part containing an enzyme labeling reagent is attached to the sample addition device body part containing the prepared specimen, and the labeling reagent is dissolved with the sample, and then a filtration filter having a pore size of 0.5 μm Then, 300 μL was dropped onto the adapter of the test kit and allowed to stand until the liquid was completely absorbed by the liquid absorbing member provided at the bottom of the nitrocellulose membrane.

Next, the adapter was removed, and 300 μL of reagent composition R3 (the following composition) was dropped to start the color reaction. 5 minutes later, the sample was observed from above, and if color was observed only on the membrane at the opening (O1), positive for influenza A virus, and color development was observed only on the membrane at the opening (O2). It was determined to be negative when the influenza A virus was positive and the membranes of both openings (O1) and (O2) were not colored.
Reagent composition R3 (coloring reagent):
Tris buffer (pH 9.0) 100 mM
p-iodonitrotetrazolium violet 1 mM
NADPH 0.1 mM
Ethyl alcohol 4%
Magnesium chloride 5 mM
Surfactant (Polyoxyethylene) 0.02 (V / W)%

  As a comparative example, a test kit was prepared in the same manner as described above except that ADH and DIA-bound latex particles were not solid-phased.

BCIP / NTB-Purple Liquid Substrate System for Membranes (B3679, manufactured by SIGMA ALDRICH)
BCIP: 5-bromo-4-chloro-3-indolyl phosphate
NTB: The detection sensitivity was evaluated in the same manner as above except that nitrotetrazolium blue was used. The determination was made in the same manner as in Example 1 after 5 minutes.

Results As shown in Tables 1 and 2, A / Beijing / 32/92 (H3N2) is positive in the A-type detection part up to 1: 4 × 10 ⁴ dilution and negative in the B-type detection part. Influenza A virus was detected, and the detection sensitivity was 4 times higher than that of the comparative example of 1: 1 × 10 ⁴ .

B / Shangdong / 7/97 shows positive type B detection part up to 1: 6 × 10 ³ dilution, type A detection part is negative and specifically detects type B influenza virus. : The detection sensitivity was 3 times higher than 2 × 10 ³ .

  Negative controls showed negative in all of the kits.

  In this example, the viruses were detected although the amount of virus of both type A and type B viruses was smaller than that of the comparative example. There was no non-specificity using the enzyme cycling reaction in which ADH and DIA of the present invention were immobilized. Thus, in the Examples, influenza viruses of type A and type B were detected quickly and with high sensitivity, and type A and type B could be distinguished.

Example 2
F (ab ') ₂ purified fraction (anti-PBP2' purified fraction) by treating two kinds of monoclonal antibodies (manufactured by DENKA SEIKEN) against methicillin-resistant Staphylococcus aureus (MRSA) penicillin binding protein 2 '(PBP2') )

  An enzyme-labeled anti-PBP2 ′ antibody (reagent composition R2) obtained by labeling one kind of the anti-PBP2 ′ purified fraction with alkaline phosphatase was obtained.

Preparation of PBP2 'detection test kit
The test kit for PBP2 ′ detection used was configured as shown in FIG. The membrane used was a nitrocellulose membrane (Hiflow Plus HF120 manufactured by Millipore).

  In the detection part (b) of the membrane, 1 mg / mL of the other anti-PBP2 ′ purified fraction (anti-PBP2 ′ antibody) as an analyte capturing reagent, latex particles bound with ADH and DIA (the same as in Example 1) ) Is applied in a line with a width of 1 mm and dried for 40 minutes under reduced pressure so that PBP2 ′ It was set as the detection part. The same reagent as in Example 1 was used as the reagent composition R3.

  The labeling reagent part was impregnated with an enzyme-labeled anti-PBP2 ′ antibody (reagent composition R2) diluted to an optimum concentration in a polyvinyl sponge, dried, and placed on the membrane.

  Furthermore, a flow-through type PBP2 ′ detection test kit equipped with a coloring reagent supply unit using non-woven fabric and a liquid absorbing member using cellulose was prepared.

Test PBP2′-producing bacteria (strains 1, 2, and 3) of Staphylococcus aureus were cultured, and a slide latex agglutination sample was prepared using an alkali extraction method.

  A sample was prepared by serially diluting the agglutination sample using a buffer solution for sample suspension (the same composition as in Example 1). 25 μL of the sample was dropped on the sample supply part of the test kit for PBP2 ′ detection. Next, 130 μL of the coloring reagent was added dropwise to the coloring reagent supply unit.

  The presence or absence of a colored line in the detection part was visually observed and determined 10 minutes after the coloring reagent was dropped. The final dilution factor determined to be positive was used as detection sensitivity.

  As a negative control, a sample suspension buffer was used.

Comparative Example 2
As Comparative Example 2, the detection sensitivity was evaluated by a slide latex agglutination reaction using MRSA-La “Seiken” (manufactured by Denka Seiken Co., Ltd.).

  The slide latex reagent is a suspension of anti-PBP2 ′ antibody-bound polystyrene latex particles 0.1 (w / v)% in phosphate buffered saline containing bovine serum albumin.

  Anti-PBP2 'monoclonal antibody is bound on the polystyrene latex particles.

Results Table 3 shows the detection lower limit concentrations of the method of the present invention and the slide latex method.

  As shown in Table 3, it can be seen that the method according to the present invention can specifically detect the PBP2 ′ antigen of MRSA with high sensitivity compared to the slide latex agglutination method.

It is a top view of the flow through type inspection kit which is one embodiment of the present invention. FIG. 2 is a cross-sectional view taken along the line I-I ′ of FIG. 1. It is a figure which shows one embodiment of the device for specimen addition used in this invention. FIG. 4 is a perspective view of the distal end portion of the specimen addition device in FIG. 3. It is the top view and cut | disconnected sectional drawing of the lateral flow type | system | group inspection kit which are one embodiment of this invention.

Explanation of symbols

O1: Opening O2: Opening a: Adapter b: Membrane c: Liquid absorbing member d: Tip
e: Main body part f: Filtration filter g: Labeling reagent part h: Filtration filter kit i: Membrane: Detection part c: Labeling reagent part D: Specimen supply part E: Liquid absorbing member F: Coloring reagent supply part G: Plastic plate

Claims (20)

A method for detecting an object in a specimen using a reagent composition R1, a reagent composition R2, a reagent composition R3, and a solid support each containing the following components, wherein the reagent composition is applied to the solid support: R1 is solid-phased, a detection unit is provided, a mixture of the reagent composition R2 and the specimen is supplied to the solid support, and then the reagent composition R3 is supplied to the detection unit on the solid support, A method for detecting an object to be detected in a specimen, comprising forming a complex of an object capturing reagent-detected object-alkaline phosphatase labeling reagent at a detection part on a support, and causing an enzyme cycling reaction to be performed at the detection part.
Reagent composition R1: Dehydrogenase and / or electron transfer substance and detection object capture reagent reagent composition R2: Alkaline phosphatase labeling reagent reagent composition R3: substance that binds to the detection object with alkaline phosphatase R3: Dehydrogenase And electron transfer substances not included in the reagent composition R1, dehydrogenase substrate, tetrazolium, and nicotinamide adenine dinucleotide phosphate (NADP) or its reduced form (NADPH)   The method for detecting an object to be detected in a specimen according to claim 1, wherein the dehydrogenase is at least one selected from the group consisting of alcohol dehydrogenase, glucose dehydrogenase, and glycerol dehydrogenase.   The method for detecting an object to be detected in a specimen according to claim 1 or 2, wherein the electron transfer substance is diaphorase.   Reagent composition R2 contains surfactant 0.2-10 (w / v)% and animal-derived serum albumin 1-10 (w / v)% of any one of Claims 1-3. A method for detecting an object to be detected in a specimen.   The method for detecting an object to be detected in a specimen according to any one of claims 1 to 4, wherein the reagent composition R1 is bound to a carrier, and the carrier is immobilized on a solid support.   The method for detecting an object to be detected in a specimen according to claim 5, wherein the carrier is fine resin particles.   The method for detecting an object to be detected in a specimen according to any one of claims 1 to 6, wherein the object capturing reagent and the object to be detected are bound by an antigen-antibody reaction.   This is a flow-through method, and the mixture of the reagent composition R2 and the specimen is supplied to the solid support, and the detection target capture reagent-detected object-alkaline phosphatase labeling reagent complex is detected at the detection unit on the solid support. The detection target in the specimen according to any one of claims 1 to 7 is detected, which is formed, and then the reagent composition R3 is supplied to the detection unit on the solid phase support to cause an enzyme cycling reaction. Method.   The sample to be detected in a sample according to claim 8, wherein the sample is placed in a sample addition device having a reagent composition R2 and having a filtration filter, and a mixture of the sample and the reagent composition R2 is prepared and supplied to a solid support. How to detect things.   It is a lateral flow system, and the mixture of the reagent composition R2 and the specimen is supplied to the solid support, then the reagent composition R3 is supplied to the solid support, and the mixture of the specimen and the reagent composition R2 is supplied to the solid support. 8. The enzyme cycling reaction according to claim 1, wherein the enzyme cycling reaction is performed by developing a complex of a detection target capture reagent-detection target-alkaline phosphatase labeling reagent at a detection part on a solid phase support. 2. A method for detecting an object to be detected in a specimen according to item 1. A detection target in a sample by causing an enzyme cycling reaction on a solid phase by the method according to claim 1, which comprises separately a reagent composition R1, a reagent composition R2, and a reagent composition R3 each containing the following components: A test kit in which the reagent composition R1 is immobilized on a solid support.
Reagent composition R1: Dehydrogenase and / or electron transfer substance and detection object capture reagent reagent composition R2: Alkaline phosphatase labeling reagent reagent composition R3: substance that binds to the detection object with alkaline phosphatase R3: Dehydrogenase And electron transfer substances not included in the reagent composition R1, dehydrogenase substrate, tetrazolium, and nicotinamide adenine dinucleotide phosphate (NADP) or its reduced form (NADPH)   The test kit according to claim 11, wherein the dehydrogenase is at least one selected from the group consisting of alcohol dehydrogenase, glucose dehydrogenase, and glycerol dehydrogenase.   The test kit according to claim 11 or 12, wherein the electron transfer substance is diaphorase.   Reagent composition R2 contains surfactant 0.2-10 (w / v)% and animal origin serum albumin 1-10 (w / v)% of any one of Claims 11-13. Inspection kit.   The test kit according to any one of claims 11 to 14, wherein the reagent composition R1 is bound to a carrier, and the carrier is immobilized on a solid support.   The test kit according to claim 15, wherein the carrier is fine resin particles.   The test kit according to any one of claims 11 to 16, wherein the detection target capturing reagent and the detection target are bound by an antigen-antibody reaction.   The inspection kit according to any one of claims 11 to 17, which is a flow-through method.   The test kit according to claim 18, further comprising a specimen addition device comprising a reagent composition R2 and having a filtration filter.   The inspection kit according to any one of claims 11 to 17, which is a lateral flow method.

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