Method and kit for detecting bacteria in blood and urine from urine specimens

Abstract

To provide a method for determining presence / absence of blood infection of bacteria using a urine specimen.SOLUTION: A method for detecting bacteria in blood and / or in urine of an object on the basis of a urine specimen of the object includes the steps of: (a) executing a first assay for detecting both bacteria-derived antigens existing in bacteria cells in a urine specimen and bacteria-derived antigens existing outside bacteria cells, on the basis of an antigen-antibody reaction; (b) executing a second assay for detecting bacteria-derived antigens existing outside bacteria cells in a urine specimen, on the basis of an antigen-antibody reaction; and (c) a step of comparing a detection result of the first assay in the step (a) with a detection result of the second assay in the step (b) in order to detect presence / absence or an existing amount of in-urine bacteria and / or in-blood bacteria on the basis of the comparison result.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting bacteria in the blood and / or urine of a subject based on a urine specimen from the subject, and a kit for use in such a method. [Background technology]

[0002] Conventionally, in order to determine whether or not a subject's blood is infected with bacteria, it was necessary to detect the presence or absence of bacteria in a blood-derived sample collected from the subject by an antigen-antibody reaction or the like (Patent Document 1: JP 2020-54279 A; Patent Document 2: Japanese Patent No. 5599013 A; Patent Document 3: Japanese Patent No. 6723293 A). However, these methods require time and effort to collect blood from the subject, so a simpler and faster method was needed.

[0003] Therefore, a method for determining the presence or absence of bacterial blood infection using a urine sample has been investigated (Patent Document 4: International Publication No. 2011 / 007823). Bacteria present in the subject's blood are decomposed in the subject's body, and some of the decomposition products are excreted from the glomerulus into the urine. The upper limit of the size barrier of the glomerulus is 66 kDa or less, and bacterial fragments smaller than this size may be excreted from the glomerulus into the urine. Therefore, if detection is performed based on an antigen-antibody reaction using the decomposition products of blood bacteria excreted from the glomerulus into the urine as antigens, it is possible to determine the presence or absence of bacterial blood infection using a urine sample.

[0004] However, in the case of bacteria such as Escherichia coli and Staphylococcus aureus, which may cause not only blood infection but also urinary tract infection, there is a possibility that bacteria that have infected the urinary tract are present in the urine sample collected from the subject. In addition, there are many cases (contamination) in which bacteria are mixed in from outside the urinary tract, such as the vulva, during urine collection. Therefore, even if bacterial decomposition products are detected in a urine sample, it is not possible to determine whether such bacterial decomposition products are derived from blood bacteria or urinary bacteria, making it difficult to accurately determine blood infection. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2020-54279 A [Patent Document 2] Patent No. 5599013 [Patent Document 3] Patent No. 6723293 [Patent Document 4] International Publication No. 2011 / 007823 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made in consideration of the above problems, and aims to provide a method for determining the presence or absence of bacterial blood infection using a urine sample. [Means for solving the problem]

[0007] As a result of extensive research, the inventors have discovered that by performing a first assay using a urine sample to detect both bacterial-derived antigens present within and outside the bacterial cells in the urine sample based on an antigen-antibody reaction, and a second assay to detect bacterial-derived antigens present outside the bacterial cells in the urine sample based on an antigen-antibody reaction, and comparing the detection results, it is possible to detect the presence or amount of bacteria in the blood and / or urine of a subject and appropriately determine the presence or absence of bacterial blood infection, thereby completing the present invention.

[0008] That is, the gist of the present invention relates to, for example, the following. [Item 1] A method for detecting bacteria in a subject's blood and / or urine based on a urine sample from the subject, comprising: (a) carrying out a first assay for detecting both a bacteria-derived antigen present within a bacterial cell in a urine sample and a bacteria-derived antigen present outside the bacterial cell based on an antigen-antibody reaction; (b) performing a second assay to detect a bacteria-derived antigen present outside the bacterial cells in the urine sample based on an antigen-antibody reaction; and (c) comparing the detection result of the first assay in the step (a) with the detection result of the second assay in the step (b) and detecting the presence or absence or amount of bacteria in urine and / or blood based on the comparison result. The method includes: [Item 2] The method according to Item 1, wherein the bacteria-derived antigen is bacterial ribosomal protein L7 / L12. [Item 3] The method according to Item 1 or 2, wherein in step (a), a bacteria-derived antigen present within a bacterial cell in a urine sample is exposed to the outside of the bacterial cell, and then the first assay is performed. [Item 4] The method according to Item 3, wherein in step (a), the urine sample is sonicated to expose bacteria-derived antigens present within bacterial cells in the urine sample to the outside of the bacterial cells. [Item 5] The method according to Item 3, wherein in step (a), the urine sample is treated with a bacteriolytic agent to expose bacteria-derived antigens present within bacterial cells in the urine sample to the outside of the bacterial cells. [Item 6] The method according to any one of Items 1 to 5, wherein in step (b), bacterial cells in the urine sample are removed before the second assay is carried out. [Item 7] The method according to Item 6, wherein in step (b), urinary bacteria in the urine sample are removed by filtering the urine sample. [Item 8] The method according to any one of Items 1 to 7, wherein the bacteria to be detected are selected from bacteria of one or more genera selected from the genera Escherichia, Staphylococcus, Klebsiella, Proteus, Enterococcus, Citrobacter, Pseudomonas, and Serratia. [Item 9] The first assay in step (a) and the second assay in step (b) The method according to any one of items 1 to 8, comprising contacting a urine sample with a capture antibody for capturing the bacteria-derived antigen and a detection antibody for labeling the bacteria-derived antigen, thereby capturing and labeling the bacteria-derived antigen in the urine sample, and detecting the captured bacteria-derived antigen in the urine sample based on the detection label. [Item 10] A kit for detecting bacteria in the blood and / or urine of a subject based on a urine sample from the subject by carrying out the method according to item 9, A capture antibody for capturing a bacteria-derived antigen in a urine sample; and a detection antibody for labeling a bacteria-derived antigen. [Item 11] The method further comprises a carrier for developing the sample and contacting the sample with a detection antibody, Item 11. The kit according to item 10, wherein a first assay area for performing a first assay and a second assay area for performing a second assay are provided on the carrier, and a capture antibody is immobilized in each of the first and second assay areas. [Item 12] The kit described in Item 11, wherein the lysis agent is applied only to the first assay area. Effect of the Invention

[0009] According to the present invention, a method is provided for determining the presence or absence of bacterial blood infection using a urine sample. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a cross-sectional view showing a schematic configuration of a strip-shaped detection mechanism, which is an example of the detection mechanism of a lateral flow type immunochromatographic detection device. [Diagram 2] FIG. 2 is a graph showing the results of detection of L7 / L12 (antigen-derived antigen) using a bacterial liquid of Staphylococcus aureus and an L7 / L12 liquid by a detection method utilizing ultrasonic treatment. [Diagram 3] FIG. 3 is a graph showing the results of detection of L7 / L12 (antigen-derived antigen) using a bacterial liquid of Staphylococcus aureus and an L7 / L12 liquid by a detection method utilizing filter treatment. [Figure 4] FIG. 4 is a graph showing the results of detection of L7 / L12 (antigen-derived antigen) using a bacterial liquid of Staphylococcus aureus and an L7 / L12 liquid by detection method 1 utilizing a lytic agent treatment. [Diagram 5] FIG. 5 is a graph showing the results of detection of L7 / L12 (antigen-derived antigen) using a bacterial liquid of Staphylococcus aureus and an L7 / L12 liquid by detection method 2 utilizing a lytic agent treatment. [Figure 6] FIG. 6 is a graph showing the results of detection of L7 / L12 (antigen-derived antigen) using a Pseudomonas aeruginosa bacterial liquid and an L7 / L12 liquid by a detection method utilizing ultrasonic treatment. [Figure 7] FIG. 7 is a graph showing the results of detection of L7 / L12 (antigen-derived antigen) using a Pseudomonas aeruginosa bacterial liquid and an L7 / L12 liquid by a detection method utilizing filter treatment. [Figure 8] FIG. 8 is a graph showing the results of detection of L7 / L12 (antigen-derived antigen) using a Pseudomonas aeruginosa bacterial liquid and an L7 / L12 liquid by detection method 1 utilizing a lytic agent treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments, and can be embodied in any form without departing from the spirit of the present invention.

[0012] [I. Methods for detecting bacteria in blood from urine samples] A first aspect of the present invention relates to a method for detecting bacteria in the blood and / or urine of a subject based on a urine specimen from the subject (hereinafter appropriately referred to as the "detection method of the present invention").

[0013] The present inventors have been actively studying ways to detect bacteria that infect blood, not bacteria present in urine, for bacteria that may be infecting not only blood but also urinary tract or contamination from sources other than the urinary tract. As a result, they have focused on the knowledge that substances in blood are excreted into urine through the glomeruli of the kidney, that there is a size barrier for substances to pass through the glomeruli, and that large molecules such as high molecular weight proteins of serum albumin (about 66 kDa) or more cannot pass through (Tencer et al., Kidney International, (1998), Vol.53, pp.709-715). Bacteria in blood cannot pass through the size barrier of the glomerulus as it is, and are not excreted in urine, but substances that can pass through the size barrier of the glomerulus among the components contained in the decomposition products of bacteria in blood and the components contained in the excreted products will be excreted into urine through the glomerulus.

[0014] Therefore, the present inventors have come up with the technical idea of ​​selecting an appropriate bacterial antigen capable of penetrating the size barrier of the glomerulus, and performing a first assay for detecting both the bacterial antigen present within the bacterial cells in the urine specimen and the bacterial antigen present outside the bacterial cells based on an antigen-antibody reaction, and a second assay for detecting the bacterial antigen present outside the bacterial cells in the urine specimen based on an antigen-antibody reaction. Among the bacterial antigens present in the urine specimen, the bacterial antigens derived from blood bacteria are discharged through the glomerulus, and therefore all of them are exposed in the urine (i.e., outside the bacterial cells), whereas the bacterial antigens derived from urinary bacteria have not passed through the glomerulus and are mostly present in a state contained in the bacterial cells. Thus, while both the bacterial antigens in the blood and the bacterial antigens in the urine are detected in the first assay, the second assay mainly detects only the bacterial antigens in the blood (Note that in the urine specimen, it is considered that a part of the bacterial antigens in the urine is released outside the cells due to the decomposition or death of the bacteria in the urine, but the amount is considered to be small compared to the bacterial antigens present inside the bacterial cells in the urine). Therefore, by comparing the detection results from these first and second assays, it is believed possible to appropriately determine the presence or absence and amount of bacteria in the blood.

[0015] That is, the detection method of the present invention is a method for detecting the presence or absence or amount of bacteria in a subject's blood based on a urine sample from the subject, and includes the following steps. (a) A step of carrying out a first assay for detecting both a bacteria-derived antigen present within bacterial cells in a urine sample and a bacteria-derived antigen present outside the bacterial cells based on an antigen-antibody reaction. (b) carrying out a second assay for detecting bacteria-derived antigens present outside the bacterial cells in the urine sample based on an antigen-antibody reaction. (c) comparing the detection results of the first assay in step (a) with the detection results of the second assay in step (b), and detecting the presence or absence or amount of bacteria in urine and / or blood based on the comparison results.

[0016] The type of bacteria-derived antigen is not limited as long as it is a biological substance such as a protein derived from the bacteria to be detected, can identify the bacteria to be detected, and can pass through the size barrier (about 66 kDa) of the glomerulus. Examples include ribosomal protein L7 / L12, nucleic acids such as DNA and RNA, membrane antigens such as LPS and polysaccharides, etc. Among them, ribosomal protein L7 / L12 is preferable as the bacteria-derived antigen. In the present invention, "ribosomal protein L7 / L12" or simply "L7 / L12" is a type of ribosomal protein essential for protein synthesis in microorganisms. It is a protein that is commonly possessed by various bacteria, and although its amino acid sequence is generally similar, there are slight differences in the amino acid sequence for each bacterial species, making it suitable for identifying bacterial species. Furthermore, it has a size (about 12 kDa) that can pass through the size barrier of the glomerulus, so it has ideal conditions as a bacteria-derived antigen.

[0017] The bacteria to be detected are not limited, and may be of any type as long as they are bacteria that may cause blood infection and / or urinary tract infection or contamination from a source other than the urinary tract and have the above-mentioned bacterial antigens. Examples include bacteria selected from one or more genera selected from the genera Escherichia, Staphylococcus, Klebsiella, Proteus, Enterococcus, Citrobacter, Pseudomonas, and Serratia.

[0018] Examples of bacteria of the genus Escherichia include Escherichia coli (E. coli, EC), Escherichia albertii, etc. Among them, Escherichia coli is preferred.

[0019] Examples of bacteria of the genus Staphylococcus include Staphylococcus aureus (S. aureus, SA), S. epidermidis, S. argenteus, etc. Among these, Staphylococcus aureus is preferred.

[0020] Examples of bacteria belonging to the genus Klebsiella include Klebsiella pneumoniae (K. pneumoniae, KP), Klebsiella oxytoca (K. oxytoca), Klebsiella aerogenes (K. aerogenes), etc. Among them, it is preferable to detect an antigen-antibody reaction with Klebsiella pneumoniae.

[0021] Examples of bacteria of the genus Proteus include Proteus milabilis (P. milabilis, PM), Proteus morganii (P. morganii), Proteus vulgaris (P. vulgaris), Proteus rettgeri (P. rettgeri), etc. Among them, Proteus milabilis is preferred.

[0022] Examples of bacteria of the genus Enterococcus include Enterococcus faecalis, Enterococcus faecium, etc. Among them, Enterococcus faecalis is preferable.

[0023] Examples of bacteria of the genus Citrobacter include Citrobacter freundii (C. freundii, CF), Citrobacter amalonaticus (C. amalonaticus), Citrobacter diversus (C. diversus), etc. Among them, Citrobacter freundii is preferred.

[0024] Examples of bacteria of the genus Pseudomonas include Pseudomonas aeruginosa (P. aeruginosa, PA), P. fluorescens, P. phosphorescence, P. putida, etc. Among these, Pseudomonas aeruginosa is preferred.

[0025] Examples of bacteria of the genus Serratia include Serratia liquefaciens (S. liquefaciens, SL), Serratia marcescens (S. marcescens), Serratia fonticola (S. fonticola), etc. Among them, Serratia liquefaciens is preferred.

[0026] In the detection method of the present invention, in step (a), a first assay is carried out to detect both bacteria-derived antigens present within bacterial cells in a urine specimen and bacteria-derived antigens present outside the bacterial cells based on an antigen-antibody reaction, and in step (b), a second assay is carried out to detect bacteria-derived antigens present outside the bacterial cells in a urine specimen based on an antigen-antibody reaction. The order of steps (a) and (b) is not particularly limited, and steps (a) and (b) may be carried out simultaneously in parallel, or step (a) may be carried out first, or step (b) may be carried out first.

[0027] In the first assay carried out in step (a), both the bacteria-derived antigens present inside the bacterial cells in the urine sample and the bacteria-derived antigens present outside the bacterial cells are detected based on an antigen-antibody reaction. The method is not limited, but for example, some treatment may be performed before or during the urine sample is subjected to the assay to expose the bacteria-derived antigens in the urine sample to the outside of the bacterial cells. Examples of such treatments include, but are not limited to, ultrasonic treatment, treatment with a bacteriolytic agent, heat treatment, freezing and thawing, etc.

[0028] The specific type and conditions of the treatment for exposing the bacterial antigens in the urine sample to the outside of the bacterial cells are not particularly limited, and may be appropriately selected depending on the type of bacteria to be detected. Among them, the cell wall structure of the bacteria can be cited as one of the criteria for selecting the type and conditions of such treatment. The cell wall structure of the bacteria differs depending on whether the bacteria is gram-positive or gram-negative. Gram-positive bacteria have a relatively thick cell wall in which teichoic acid or lipoteichoic acid is embedded in the peptidoglycan layer, and do not have an outer membrane. Gram-negative bacteria have a relatively thin cell wall made of a peptidoglycan layer, and have an outer membrane made of lipopolysaccharides, polysaccharides, O antigens, etc. on the outside. Therefore, it is preferable to appropriately select the type and conditions of the treatment based on the cell wall structure of the bacteria to be detected so that the cell wall structure can be destroyed and the bacterial antigens can be reliably exposed to the outside of the bacterial cells.

[0029] Specifically, when bacteria-derived antigens are exposed outside bacterial cells by ultrasonic treatment, the conditions are not particularly limited, but are, for example, as follows. In the case of gram-positive bacteria, it is preferable to use a general ultrasonic homogenizer or the like to subject the urine sample to ultrasonic treatment for usually 1 to 15 minutes. In the case of gram-negative bacteria, it is preferable to use a general ultrasonic homogenizer or the like to subject the urine sample to ultrasonic treatment for usually 5 seconds to 3 minutes.

[0030] In addition, when bacteria-derived antigens are exposed to the outside of bacterial cells by a lytic agent treatment, the conditions are not particularly limited, but are, for example, as follows. In the case of gram-positive bacteria, it is preferable to perform the lytic agent treatment by contacting a urine specimen with a lytic solution containing a lytic agent such as an enzyme such as lysostaphin, a surfactant such as Tween 20 or DDAO (N,N-dimethyldodecylamine N-oxide), or an alkaline lytic agent, usually for several seconds to several minutes. In the case of gram-negative bacteria, it is preferable to perform the lytic agent treatment by contacting a urine specimen with a lytic solution containing a surfactant such as Tween 20 or DDAO, or an alkaline lytic agent, usually for several seconds to several minutes. Such a lytic agent treatment may be performed by mixing the urine specimen with the lytic solution before subjecting it to an assay, or may be performed by contacting the urine specimen with the lytic solution in any step of the assay.

[0031] On the other hand, in the second assay carried out in step (b), the bacteria-derived antigens present inside the bacterial cells in the urine specimen are not detected, but the bacteria-derived antigens present outside the bacterial cells are detected based on an antigen-antibody reaction. The method is not limited, but the treatment carried out in step (a) for exposing the bacteria-derived antigens outside the bacterial cells is not carried out in step (b), and the bacteria-derived antigens present inside the bacterial cells in the urine specimen may be directly subjected to the assay without being exposed outside the bacterial cells. Alternatively, as a more active treatment, the urine specimen may be subjected to the assay after a treatment for removing bacterial cells in the urine specimen. The treatment for removing bacterial cells in the urine specimen is not limited, but an example thereof may be a filtration treatment using a filter.

[0032] Specifically, when bacterial cells in a urine sample are removed by filtration using a filter, the conditions are not particularly limited, and a filter such as a syringe filter (e.g., a syringe filter manufactured by Merck Millipore) having an appropriate mesh size (e.g., 0.22 to 0.45 μm) that allows bacteria-derived antigens to pass but does not allow bacterial cells to pass through may be used.

[0033] After the first assay in step (a) and the second assay in step (b) are performed in the above-mentioned procedure, the detection results of both assays are compared in step (c). Here, in order to enable appropriate comparison of the detection results, it is preferable that the first and second assays are performed using the same apparatus, under the same conditions, and with the same operations as much as possible, except for the differences described above.

[0034] The subject of detection by the detection method of the present invention is not limited, and can be performed on any subject for which bacterial infection is required to be detected, but is usually a mammal, preferably a human. The sample to be detected is also not particularly limited, but in accordance with the purpose of the present invention, a sample derived from the subject's urine is usually used. As a sample derived from urine, urine may be used as a sample as it is, or urine may be used after adding a desired pretreatment (e.g., surfactant, enzyme, pH adjusting reagent, etc.).

[0035] The specific detection method of the first and second assays is not particularly limited as long as it is an immunological measurement method capable of detecting bacteria-derived antigens in a urine sample based on an antigen-antibody reaction. Examples include ELISA (enzyme-linked immunosorbent assay) using a microtiter plate carrying an antibody; latex particle agglutination assay using latex particles (e.g., polystyrene latex particles, etc.) carrying an antibody; immunochromatography using a membrane carrying an antibody; and sandwich-type immunochromatography detection using a detection antibody labeled with colored particles or particles having color-developing ability, enzymes, or fluorescent bodies, etc., and a capture antibody immobilized on a solid phase carrier such as magnetic particles. Among them, in the present invention, it is preferable to use an immunochromatography detection method using a detection antibody and a capture antibody. In the next section, the case where the first and second assays are performed by the immunochromatography detection method will be described.

[0036] [II. Immunochromatographic detection method] In one embodiment of the detection method of the present invention, the first assay in step (a) and the second assay in step (b) can be carried out by an immunochromatographic detection method using a detection antibody and a capture antibody. In this case, the first assay in step (a) and the second assay in step (b) each include the following steps: (I) A process of contacting a urine sample with a capture antibody for capturing the bacteria-derived antigen and a detection antibody for labeling the bacteria-derived antigen, thereby capturing the bacteria-derived antigen in the urine sample with the capture antibody and labeling it with the detection antibody. (II) A step of detecting the bacteria-derived antigen in the urine sample captured by the capture antibody based on the detection label.

[0037] There are no limitations on the type of capture antibody and detection antibody, so long as they are antibodies that undergo an antigen-antibody reaction with an antigen derived from the bacteria to be detected (e.g., ribosomal protein L7 / L12 of the bacteria to be detected). However, from the viewpoint of detection accuracy, it is preferable that the antibody does not undergo an antigen-antibody reaction with components derived from bacteria other than the bacteria to be detected (e.g., L7 / L12 of bacteria other than the bacteria to be detected) and has specificity for an antigen derived from the bacteria to be detected (e.g., L7 / L12 of the bacteria to be detected). Both the capture antibody and the detection antibody may have such specificity for an antigen derived from the bacteria to be detected, but it is sufficient that at least one of the capture antibody or the detection antibody has such specificity.

[0038] According to the detection method of the present invention related to aspect A, the step (I) comprises: (Ia-1) A step of contacting a urine sample with a detection antibody to label the bacteria-derived antigen in the urine sample by an antigen-antibody reaction between the detection antibody and the bacteria-derived antigen. (Ia-2) A step of contacting a specimen containing a bacteria-derived antigen labeled with a detection antibody with a capture antibody, and capturing the bacteria-derived antigen in the urine specimen by an antigen-antibody reaction between the capture antibody and the bacteria-derived antigen-detection antibody complex.

[0039] Moreover, according to the detection method of the present invention related to aspect B, the step (I) comprises the following steps: (Ib-1) A step of contacting a urine sample with a capture antibody and capturing bacteria-derived antigens in the urine sample by an antigen-antibody reaction between the capture antibody and the bacteria-derived antigens. (Ib-2) A process in which a specimen containing the bacteria-derived antigen captured by the capture antibody is contacted with a detection antibody, and the bacteria-derived antigen in the urine specimen is labeled by an antigen-antibody reaction between the detection antibody and the bacteria-derived antigen-capture antibody complex.

[0040] In the detection method of the present invention, the selection of either mode A or mode B may be made depending on the type of immunoassay method and the type of specimen actually used. Examples of the immunoassay method include, but are not limited to, various known immunological assay methods, such as ELISA (enzyme-linked immunosorbent assay) using a microtiter plate carrying an antibody, latex particle agglutination assay using latex particles (e.g., polystyrene latex particles, etc.) carrying an antibody, immunochromatography using a membrane carrying an antibody, and a sandwich assay using a detection antibody labeled with colored particles or particles having color-developing ability, an enzyme, a fluorescent substance, or the like, and a capture antibody immobilized on a solid-phase carrier such as magnetic particles.

[0041] Hereinafter, an example will be described in which the detection method of the present invention according to aspect A is carried out using immunochromatography as the immunoassay method, but when using other immunoassay methods, each feature can be modified as appropriate.

[0042] The type of solid phase carrier used for the capture antibody is not particularly limited, but specific examples include porous membranes made of cellulose, nitrocellulose, cellulose acetate, nylon, PVDF (Polyvinylidene Difluoride), glass fiber, etc.; flow channels made of glass, plastic, PDMS (Poly(dimethylsiloxane)), silicon, etc.; thread, paper, fiber, etc. The method for linking an antibody to a solid phase carrier is not particularly limited, and specific examples include immobilization by physical adsorption utilizing the hydrophobicity of the antibody, and immobilization by chemical bonding utilizing the functional groups of the antibody.

[0043] The type of detection label used for the detection antibody is not particularly limited and may be appropriately selected depending on the detection method. Specific examples of the detection label include metal colloids such as gold colloid, platinum colloid, palladium colloid, etc.; non-metallic colloids such as selenium colloid, alumina colloid, silica colloid, etc.; insoluble granular substances such as colored resin particles, dye colloids, colored liposomes, etc.; color-developing reaction catalytic enzymes such as alkaline phosphatase, peroxidase, luciferase, etc.; fluorescent dyes, radioactive isotopes; chemiluminescent labels, bioluminescent labels, electrochemiluminescent labels, etc.

[0044] The method for labeling the antibody is not particularly limited, and specific examples include physical adsorption utilizing the hydrophobicity of the antibody and chemical bonding utilizing the functional groups of the antibody.

[0045] Each step of the bacterial testing method of the present invention will be described below. In the step (Ia-1), that is, the step of contacting the specimen with a detection antibody and labeling the bacteria-derived antigen in the specimen by an antigen-antibody reaction between the detection antibody and the bacteria-derived antigen, the detection antibody having a detection label is contacted with the specimen, and the bacteria-derived antigen in the specimen is labeled by an antigen-antibody reaction between the detection antibody and the bacteria-derived antigen. The method of contacting the specimen with the detection antibody is not limited, but it is usually carried out by introducing a specimen prepared as an aqueous specimen into a member region impregnated with the detection antibody and maintaining it for a certain period of time. The specific embodiment differs depending on the capture embodiment, etc., but for example, when the bacteria-derived antigen in the specimen is captured by a capture antibody immobilized on a porous membrane as a solid phase carrier, an aqueous reagent containing the detection antibody can be introduced into the porous membrane and permeated therethrough to bind the detection antibody to the bacteria-derived antigen captured by the capture antibody on the porous membrane. As another example, if a bacteria-derived antigen in a sample is captured by a capture antibody immobilized in a region on a flow path as a solid phase carrier, an aqueous reagent containing a detection antibody can be circulated through the flow path, causing the detection antibody to bind to the bacteria-derived antigen captured by the capture antibody on the flow path.

[0046] In the step (Ia-2), that is, the step of contacting a specimen containing a bacteria-derived antigen labeled with a detection antibody with a capture antibody and capturing the bacteria-derived antigen in the specimen by an antigen-antibody reaction between the capture antibody and the bacteria-derived antigen-detection antibody complex, the capture antibody is brought into contact with the specimen, and the bacteria-derived antigen in the specimen is captured by an antigen-antibody reaction between the capture antibody and the bacteria-derived antigen. The method of contacting the specimen with the capture antibody is not limited, but it is usually carried out by introducing a specimen prepared as an aqueous specimen into the area where the capture antibody is present and maintaining it for a certain period of time. The specific embodiment varies depending on the type of solid phase carrier of the capture antibody, but for example, a porous membrane is used as the solid phase carrier, and the specimen is introduced into the porous membrane on which the capture antibody is immobilized and allowed to permeate, thereby allowing the capture antibody immobilized on the porous membrane to capture the bacteria-derived antigen in the specimen. As another example, a capture antibody may be immobilized as a solid phase carrier in a region on a flow path, and a sample may be passed through the flow path, allowing the capture antibody immobilized in the region on the flow path to capture bacteria-derived antigens in the sample.

[0047] In the step (II), i.e., the step of detecting the bacteria-derived antigen in the sample based on the detection label, the bacteria-derived antigen to be detected that is captured by the capture antibody and labeled by the detection antibody is detected based on the detection label. The detection method is not particularly limited and may be appropriately selected depending on the type of detection label, but for example, when a metal colloid such as gold colloid is used as the detection label, the presence or absence or amount of gold colloid bound to the bacteria-derived antigen may be detected by any method such as visual inspection or a camera.

[0048] [III. Immunochromatography Detection Kit] Another aspect of the present invention relates to a kit for performing the above-mentioned immunochromatographic detection method to detect bacteria in the blood and / or urine of a subject based on a urine sample from the subject (hereinafter appropriately referred to as the "detection kit of the present invention").

[0049] The detection kit of the present invention includes the above-mentioned capture antibody and detection antibody. The capture antibody is usually provided in an appropriate form (such as a container including a porous membrane or a container including a flow path) according to the type of solid phase carrier. The detection antibody is usually provided in the form of an aqueous reagent containing the detection antibody in an aqueous medium or a dry reagent obtained by drying the detection antibody.

[0050] The detection kit of the present invention includes, in addition to the above-mentioned capture antibody and detection antibody, one or more types of reagents necessary for carrying out the detection method of the present invention using these antibodies, a detection device or its components, and / or instructions describing the procedure for carrying out the detection method of the present invention. The types of such reagents, the contents of the instructions, and further other components included in the detection kit of the present invention may be appropriately determined depending on the type of specific immunoassay method.

[0051] When the detection kit of the present invention includes a detection device or its components, the device constituted by such a kit is a device equipped with components necessary for carrying out the detection method of the present invention (hereinafter, appropriately abbreviated as "the device of the present invention"). The specific components of the device of the present invention can be appropriately adjusted according to the type of immunoassay, which is a specific embodiment of the detection method of the present invention. As described above, examples of immunoassays include, but are not limited to, various known immunoassays such as ELISA (enzyme-linked immunosorbent assay) using a microtiter plate carrying an antibody; latex particle agglutination assay using latex particles (e.g., polystyrene latex particles, etc.) carrying an antibody; immunochromatography using a membrane carrying an antibody; and a sandwich assay using a detection antibody labeled with colored particles or particles having color-developing ability, enzymes, or fluorescent bodies, etc., and a capture antibody immobilized on a solid phase carrier such as magnetic particles. The device of the present invention is equipped with components necessary for carrying out such various immunoassays.

[0052] Specific examples of the device include a lateral flow device and a flow-through device. Here, the lateral flow method is a method in which a target specimen and a detection antibody are developed in parallel to a membrane including a detection region having a capture antibody immobilized on the surface, and a target substance captured in the detection region of the membrane is detected. On the other hand, the flow-through method is a method in which a target specimen and a detection antibody are passed vertically through a membrane having a capture antibody immobilized on the surface, and a target substance captured on the surface of the membrane is detected. The detection method of the present invention can be applied to both a lateral flow device and a flow-through device.

[0053] Both lateral flow type devices and flow-through type immunochromatographic detection devices are publicly known, and procedures other than those described in this disclosure can be appropriately designed by a person skilled in the art based on technical common sense. Below, the schematic configuration of the detection mechanism of the lateral flow type immunochromatographic detection device is described with reference to the drawings, but this is merely one example of the schematic configuration of the detection procedure, and the configuration of the lateral flow type immunochromatographic detection device is not limited to the embodiment exemplified in the drawings.

[0054] FIG. 1 is a cross-sectional view showing a schematic configuration of a strip-shaped detection mechanism, which is an example of the detection mechanism of a lateral flow immunochromatographic detection device. The detection mechanism (10) of FIG. 1 is provided on a substrate (5) in such a state that a strip-shaped detection antibody-impregnated member (conjugate pad) (2) (this pad is impregnated with a detection antibody) and a specimen addition member (sample pad) (3) are arranged on one end side (upstream side of the specimen flow (B)) of the insoluble membrane carrier (1) for chromatographic development in the strip length direction, and an absorption member (absorption pad) (4) is arranged on the other end side (downstream side of the specimen flow (B)). A site (6) where a capture antibody is immobilized is arranged in the center of the strip length direction on the insoluble membrane carrier (1), and a site (7) where a control reagent is immobilized is arranged as necessary. The control reagent is a reagent that does not bind to bacteria-derived antigens but binds to detection antibodies.

[0055] In use, when a specimen (A) is applied onto the specimen application member (sample pad) (3), the specimen (A) passes through the detection antibody-impregnated member (conjugate pad) (2) and flows through the insoluble membrane carrier (1) in the direction of specimen flow (B). At this time, the bacteria-derived antigen (preferably L7 / L12) in the specimen (A) binds to the detection antibody to form a bacteria-derived antigen-detection antibody complex. When the specimen (A) passes through the capture antibody fixing portion (6), the bacteria-derived antigen in the specimen binds to the capture antibody to form a capture antibody-bacteria-derived antigen-detection antibody complex. Furthermore, when the specimen (A) passes through the control reagent fixing portion (7), the detection antibody that is not bound to the bacteria-derived antigen binds to the control reagent (7), thereby enabling the completion of the test (i.e., the specimen (A) has passed through the capture antibody (6)). Here, the presence or absence or amount of the bacteria-derived antigen can be detected by detecting the label of the detection antibody in the capture antibody-bacteria-derived antigen-detection antibody complex present at the capture antibody immobilization site (6) by a known means. If necessary, the label of the detection antibody may be sensitized by a known method to facilitate detection.

[0056] The detection antibody-impregnated member (conjugate pad) (2), the specimen addition member (sample pad) (3), and / or the control reagent fixing site (7) may be omitted as desired. If the detection antibody-impregnated member (conjugate pad) (2) is not provided in this system, the specimen (A) and the detection antibody may be applied simultaneously or sequentially to one end of the insoluble membrane carrier (1) in a premixed or separate state, thereby conducting a test similar to the above test.

[0057] Furthermore, even if the capture antibody and the detection antibody are interchanged, a detection kit capable of similar detection can be constructed.

[0058] When the first and second assays of the detection method of the present invention are carried out using such an immunochromatographic detection kit, two immunochromatographic detection kits of the same configuration may be prepared, and the first assay may be carried out in one and the second assay in the other. Alternatively, a single immunochromatographic detection kit may be provided with a plurality of assay regions (regions serving as detection units each consisting of the insoluble membrane carrier (1), the member impregnated with an antibody for detection (conjugate pad) (2), the member for adding a specimen (sample pad) (3), and the member for absorption (absorption pad) (4)) and the first and second assays may be carried out using a single immunochromatographic detection kit. The latter configuration has the advantage that the first and second assays can be carried out easily under the same conditions.

[0059] In addition, the immunochromatographic detection kit may incorporate a means for performing a process of exposing urinary bacteria-derived antigens in the urine sample to the outside of the bacterial cells in the above-mentioned step (a) or a means for performing a process of removing bacterial cells in the urine sample in the above-mentioned step (b). For example, when a bacteriolytic agent treatment is performed as a process of exposing bacteria-derived antigens to the outside of the bacterial cells in the step (a), the specimen addition member (sample pad) (3) of the immunochromatographic detection kit for performing the first assay (or the first assay region when the first and second assays are performed by a single immunochromatographic detection kit) may be impregnated with a bacteriolytic solution in advance and dried, so that the urine sample is subjected to the first assay and at the same time the urine sample is subjected to the bacteriolytic agent treatment. EXAMPLES

[0060] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative and are not intended to limit the present invention in any way.

[0061] Example I. Detection of Staphylococcus aureus In this example, Staphylococcus aureus, a gram-positive bacterium, was used as the detection target, and its detection was attempted using a mock specimen.

[0062] (1) Obtaining Staphylococcus aureus L7 / L12: With reference to the method described in International Publication No. 2000 / 06603, an expression vector was prepared by cloning a nucleic acid sequence (sequence number 2) encoding the amino acid sequence (sequence number 1) of Staphylococcus aureus ribosomal protein L7 / L12. Escherichia coli was transformed with the obtained expression vector, and the obtained transformant was cultured using LB medium or the like. The full-length protein of Staphylococcus aureus L7 / L12 expressed by this transformant was purified as a fusion protein using an affinity column with the tag sequence derived from the expression vector.

[0063] (2) Preparation of antibodies against Staphylococcus aureus L7 / L12: Using the full-length protein of L7 / L12 of Staphylococcus aureus obtained by the above procedure, a monoclonal antibody against L7 / L12 of Staphylococcus aureus was produced with reference to the method described in WO 2000 / 06603.

[0064] Specifically, the full-length protein of L7 / L12 of Staphylococcus aureus was used as an immunogen, and according to the standard method for obtaining hybridomas, the immunogen was prepared in PBS to a concentration of 0.4 mg / mL, and the same amount of Freund's adjuvant was added, and mice were immunized four times so that the immunogen amount was 50 μg / time. After confirming the increase in serum antibody titer by test blood collection, spleen cells of the mice were extracted. The extracted mouse spleen cells were fused with myeloma cells to obtain various hybridomas. The obtained various hybridomas were cultured in HAT medium, and screening was performed using the antibodies in the culture supernatant. Screening was performed using ELISA to verify reactivity with bacterial lysates of Staphylococcus aureus (SA) and bacterial lysates of several other types of bacteria (Escherichia coli (EC), Pseudomonas aeruginosa (PA), Streptococcus pneumoniae (SP), Klebsiella pneumoniae (KP), Chlamydia pneumoniae (CP), and Serratia marcescens (SM)). Hybridomas producing specific antibodies reactive only with bacterial lysates of Staphylococcus aureus (SA), as well as hybridomas producing general antibodies reactive with bacterial lysates of Staphylococcus aureus (SA) and several other types of bacteria, were selected.

[0065] The procedure for screening antibodies using the ELISA method is as follows. Each of the above bacteria was purchased from ATCC and cultured, and 1×e 8 cfu / mL was prepared and suspended in PBS. Each bacterium was lysed by ultrasonication, and the bacterial lysate of each bacterium was obtained by removing debris by filtration through a filter with 0.45 μm mesh. 50 μL of the bacterial lysate was dropped into each well of a 96-well polystyrene plate for ELISA and immobilized on the bottom of the plate. Each well was washed three times with PBS-T (PBS containing Tween 20), and then blocked with PBS containing 1% BSA (Bovine Serum Albumin). After blocking and washing three times with PBS-T, 50 μL of each antibody in the hybridoma culture supernatant described in the above-mentioned antibody production method was dropped into each well, and an antigen-antibody reaction was carried out for 1 hour. After the reaction, the wells were washed three times with PBS-T, and then 50 μL of secondary antibody (Goat Anti-mouse IgG) labeled with 0.5 μg / mL of the detection enzyme HRP (Horse Radish Peroxidase) was dropped into each well to carry out the reaction. After the reaction, the wells were washed five times with PBS-T, and 100 μL of a mixture of TMB (Tetramethylbenzidine) and hydrogen peroxide, which is a color-developing substrate, was dropped into each well to carry out the color reaction. After 10 minutes, hydrochloric acid, which is a reaction stop solution, was dropped into each well, and the absorbance at 450 nm of each well was measured using a plate reader.

[0066] Monoclonal antibodies were prepared from the hybridomas producing specific or general antibodies selected as described above. Specifically, the selected hybridomas were cultured in TIL Media I medium supplemented with 10% fetal bovine serum (FBS) according to the standard method for monoclonal production, and the cultured hybridomas were administered intraperitoneally to mice to collect ascites. The collected ascites was centrifuged to separate floating matter and red blood cells, and then filtered through a filter with a mesh size of 0.45 μm. The filtrate was passed through a Protein G column to adsorb the antibodies, and the monoclonal antibodies produced were purified from the mouse ascites.

[0067] Of the general-purpose monoclonal antibodies 13A2, 15C2, 20B1, 46B1, and 52B1 and the specific monoclonal antibodies 24C2, 30A2, 32A2, 62A1, and 78C2 against Staphylococcus aureus L7 / L12 obtained by the above procedures, the general-purpose monoclonal antibody 15C2 was used as the detection antibody and the specific monoclonal antibody 24C2 was used as the capture antibody to prepare the following immunochromatography kit.

[0068] (3) Preparation of immunochromatography kit using detection and capture antibodies: An immunochromatography kit was prepared by the following procedure using the detection antibody and capture antibody against L7 / L12 of Staphylococcus aureus obtained by the above procedure.

[0069] First, the detection antibody was labeled with gold colloid by the following procedure. That is, 0.9 mL of gold colloid solution (particle size 60 nm; manufactured by BBI Solutions) was mixed with 0.1 M potassium phosphate (pH 7.5), the detection antibody was added to a concentration of 25 μg / mL, and the mixture was left at room temperature for 10 minutes to bind the detection antibody to the gold colloid particle surface. Next, a 10% aqueous solution of bovine serum albumin (BSA) was added so that the final concentration in the gold colloid solution was 1%, and the remaining surface of the gold colloid particle was blocked with BSA. Subsequently, this solution was centrifuged (15000×rpm, 5 minutes) to precipitate the gold colloid-labeled antibody. The supernatant was removed, and the gold colloid-labeled antibody was suspended in 20 mM Tris-HCl buffer (pH 8.2) containing 0.25% BSA, 2.5% sucrose, and 35 mM NaCl to obtain a gold colloid-labeled detection antibody solution.

[0070] Using the gold colloid-labeled detection antibody solution obtained by the above procedure and the above capture antibody, an immunochromatography device (immunochromatography kit) having the configuration shown in FIG. 1 was produced by the following procedure.

[0071] First, a nitrocellulose membrane with a width of 25 mm and a length of 300 mm was prepared as a chromatographic medium, a membrane carrier (1) for chromatographic development. A solution containing a capture antibody at a concentration of 1.5 mg / mL was applied in a line shape at a position 10 mm from the end of the chromatographic development starting point side of the membrane carrier (1) for chromatographic development, so that the concentration was 1 μL / cm, and this was dried at 50° C. for 30 minutes, and then immersed in a 0.5% casein solution, then in a 0.2% sucrose solution, and dried overnight at room temperature to form a capture site (6) for a complex of a bacteria-derived antigen, a capture antibody, and a gold colloid-labeled detection antibody.

[0072] On the other hand, a 10 mm×300 mm strip of glass fiber pad was impregnated with 2 mL of the above-mentioned gold colloid-labeled detection antibody, and freeze-dried to prepare a detection antibody-impregnated member (2).

[0073] In addition to the above-mentioned chromatographic development membrane carrier (1) and detection antibody-impregnated member (2), cotton cloth was prepared as the specimen addition member (sample pad) (3) and filter paper was prepared as the absorption member (absorption pad) (4). These members were attached to a substrate (5) and then cut to a width of 5 mm to prepare an immunochromatography device (immunochromatography kit) having the configuration shown in FIG. 1. Immunochromatography kits having such a configuration were prepared separately for the first assay and the second assay for each of the following detection methods. Hereinafter, the immunochromatography kit for the first assay will be abbreviated as "immunochromatography kit 1" and the immunochromatography kit for the second assay will be abbreviated as "immunochromatography kit 2" as appropriate.

[0074] (4) Preparation of mock specimens: Next, we prepared a bacterial solution of Staphylococcus aureus and a solution of Staphylococcus aureus ribosomal protein L7 / L12 as mock specimens for the detection test. The liquid medium used was BD BBL Trypticase Soy Broth (Becton Dickinson).

[0075] Staphylococcus aureus was purchased from ATCC. Live Staphylococcus aureus cells were spiked into liquid medium and cultured with shaking at 37°C for 16 hours. The liquid was adjusted to a turbidity (OD = 600) of 2, and the resulting liquid was diluted 1 / 1000 with liquid medium to use as a Staphylococcus aureus liquid. This Staphylococcus aureus liquid was used as a specimen simulating urinary bacteria (and urinary bacteria-derived antigens released therefrom) present as live bacteria in urine.

[0076] Meanwhile, the ribosomal protein L7 / L12 of Staphylococcus aureus prepared in (1) above was diluted to 1 ng / mL or 3 ng / mL in liquid medium and used as the Staphylococcus aureus L7 / L12 solution. This Staphylococcus aureus L7 / L12 solution was used as a specimen simulating the bacterial antigens in blood excreted from blood through the glomerulus into urine.

[0077] These simulated samples are subjected to the first assay (an assay for detecting both bacterial antigens in blood and bacterial antigens in urine from urine samples based on an antigen-antibody reaction) and the second assay (an assay for detecting bacterial antigens in blood from urine samples based on an antigen-antibody reaction) described below. If it is verified that there is a clear difference in the detection results of the bacterial liquid by both assays (i.e., the amount of bacterial antigen detected by the second assay is significantly less than the amount of bacterial antigen detected by the first assay, or no bacterial antigen is detected), and there is little or no difference in the detection results of the L7 / L12 liquid (i.e., the amount of bacterial antigen detected by the first assay and the amount of bacterial antigen detected by the second assay are almost or completely the same), it will be shown that it is possible to detect bacteria in blood from urine samples by comparing the detection results of the first and second assays.

[0078] (5) Detection method A - Ultrasonic treatment: In this example, a first assay was performed in which the sample was subjected to ultrasonic treatment, and a second assay was performed in which the sample was used as is without being subjected to ultrasonic treatment, and the detection results were compared to verify the possibility of detecting bacteria in blood from urine samples.

[0079] In the following procedure, the mixed reagent was a mixture of 0.1M Tris-HCl (pH 7.5), 1% Tween 20, 0.3% NaCl, and 0.1% Na caseinate. This mixed reagent was in liquid form and mixed with each specimen before the assay. In addition, 1ng / mL of the L7 / L12 solution of Staphylococcus aureus was used.

[0080] The first and second assays were carried out according to the following procedure. 1. The aforementioned bacterial liquid and L7 / L12 liquid were each mixed with the aforementioned mixed reagent (volume ratio of bacterial liquid or L7 / L12 liquid:mixed reagent 30:70) to prepare a mixed liquid. 2. The bacterial mixture and the L7 / L12 mixture were each collected using a 200 μL pipette and subjected to ultrasonic treatment for 2 minutes using an ultrasonic homogenizer (Bioruptor UCD-200TM). 3. The bacterial solution and L7 / L12 solution after ultrasonic disruption were dropped into Immunochromatography Kit 1 (first assay: 115 μL, N=3). 4. 200 μL of the bacterial mixture and the L7 / L12 mixture were each taken with a pipette and dropped into Immunochromatography Kit 2 (without being subjected to an ultrasonic homogenizer) (second assay: 115 μL, N=3). 5. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 1 was measured using a reader (first assay: N=3, and the average values ​​were graphed). 6. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 2 was measured using a reader (second assay: N=3, and the average values ​​were graphed).

[0081] The detection results of the bacterial liquid and the L7 / L12 liquid by the first and second assays are shown in the graph of FIG. 2. As is clear from the graph, in the detection results of the bacterial liquid, the amount of L7 / L12 (bacteria-derived antigen) detected by the second assay was significantly smaller than the amount of L7 / L12 (bacteria-derived antigen) detected by the first assay, and a clear difference occurred between the detection results by both assays. This is thought to be because in the first assay, in which the bacterial liquid was subjected to ultrasonic treatment and then used, a large amount of L7 / L12 released by the destruction of bacterial cells was detected, whereas in the second assay, in which the bacterial liquid was used as is, L7 / L12 in the bacterial cells was not released and was not detected. On the other hand, in the detection results of the L7 / L12 liquid, the amount of the bacterial antigen detected by the first assay and the amount of the bacterial antigen detected by the second assay were roughly the same, and there was almost no difference between the two assays. Therefore, it was shown that the detection method of this embodiment using ultrasonic treatment can also detect blood bacteria from urine samples.

[0082] (6) Detection method B - Filter processing used: In this example, a first assay was performed using the sample as is, and a second assay was performed using the sample after filtering, and the detection results were compared to verify the possibility of detecting bacteria in blood from urine samples.

[0083] In the following procedure, the mixed reagent used was a mixture of 0.1M Tris-HCl (pH 7.5), 5% Tween 20, 0.3% NaCl, 0.1% sodium caseinate, 2% DDAO (N,N-dimethyldodecylamine N-oxide), and 10 μg / mL lysostaphin. This mixed reagent was impregnated into each sample addition member (sample pad) (3) of Immunochromatography Kit 1 and 2 and dried before use. In addition, 3 ng / mL of L7 / L12 solution of Staphylococcus aureus was used.

[0084] The first and second assays were carried out according to the following procedure. 1. The bacterial solution and the L7 / L12 solution were each dropped onto Immunochromatography Kit 1 (115 μL, N=3). 2. The bacterial solution and the L7 / L12 solution were each filtered through a sterile filter (Merck Millipore syringe filter, mesh size 0.45 μm), and then added dropwise to Immunochromatography Kit 2 (115 μL, N=3). 3. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 1 was measured using a reader (N=3 and the average values ​​were graphed). 4. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 2 was measured using a reader (N=3 measurements were taken and the average values ​​were graphed).

[0085] The detection results of the bacterial liquid and the L7 / L12 liquid by the first and second assays are shown in the graph of FIG. 3. As is clear from the graph, in the detection results of the bacterial liquid, the amount of L7 / L12 (bacteria-derived antigen) detected by the second assay was significantly smaller than the amount of L7 / L12 (bacteria-derived antigen) detected by the first assay, and a clear difference occurred between the detection results by both assays. This is thought to be because in the first assay, in which the bacterial liquid was used as is, a large amount of L7 / L12 released by lysis of the bacterial cells by the mixed reagent was detected, whereas in the second assay, in which the bacterial liquid was used after filtering with a sterilizing filter, the bacterial cells were removed, and as a result, L7 / L12 present within the bacterial cells was not detected. On the other hand, in the detection results of the L7 / L12 liquid, the amount of the bacterial antigen detected by the first assay and the amount of the bacterial antigen detected by the second assay were almost the same, and there was almost no difference between the two assays. Therefore, it was shown that the detection method of this embodiment using filter treatment can also detect blood bacteria from urine samples.

[0086] (7) Detection method C - embodiment 1 using lysis agent treatment: In this example, a first assay was performed using the sample after treating it with a bacteriolytic agent, and a second assay was performed using the sample as is, and the detection results were compared to verify the possibility of detecting bacteria in blood from urine samples.

[0087] In the following procedure, a mixture of 0.1M Tris-HCl (pH 7.5), 1% Tween 20, 0.3% NaCl, 0.1% Na caseinate, and 10 μg / mL lysostaphin was used as the mixed reagent 1 for the first assay, and a mixture of 0.1M Tris-HCl (pH 7.5), 1% Tween 20, 0.3% NaCl, and 0.1% Na caseinate was used as the mixed reagent 2 for the second assay. Both mixed reagents 1 and 2 were in liquid form and were mixed with each specimen before the first and second assays, respectively. In addition, 1ng / mL of the L7 / L12 solution of Staphylococcus aureus was used.

[0088] The first and second assays were carried out according to the following procedure. 1. The bacterial solution and the L7 / L12 solution were each mixed with mixed reagent 1, and the mixture was added dropwise to Immunochromatography Kit 1 (115 μL, N=3). 2. The bacterial solution and the L7 / L12 solution were each mixed with the mixed reagent 2, and the mixture was dropped onto the immunochromatography kit 2 (115 μL, N=3). 3. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 1 was measured using a reader (N=3 and the average values ​​were graphed). 4. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 2 was measured using a reader (N=3 measurements were taken and the average values ​​were graphed).

[0089] The detection results of the bacterial liquid and the L7 / L12 liquid by the first and second assays are shown in the graph of FIG. 4. As is clear from the graph, in the detection results of the bacterial liquid, the amount of L7 / L12 (bacteria-derived antigen) detected by the second assay was significantly smaller than the amount of L7 / L12 (bacteria-derived antigen) detected by the first assay, and a clear difference occurred between the detection results by both assays. This is thought to be because in the first assay, in which the bacterial liquid was mixed with the mixed reagent 1 containing a lytic agent (lysostaphin) and then used, a large amount of L7 / L12 released by lysis of the bacterial cells by the mixed reagent was detected, whereas in the second assay, in which the bacterial liquid was mixed with the mixed reagent 2 not containing a lytic agent and then used, L7 / L12 in the bacterial cells was not released and was not detected. On the other hand, in the detection results of the L7 / L12 liquid, the amount of the bacterial antigen detected by the first assay and the amount of the bacterial antigen detected by the second assay were roughly the same, and there was almost no difference between the two assays. Therefore, it was demonstrated that blood bacteria can be detected from urine samples by the detection method of this embodiment utilizing treatment with a bacteriolytic agent.

[0090] (8) Detection method D - embodiment 2 using lysis agent treatment: In this example, as a variation of detection method C, a first assay was performed in which detection was performed while treating the sample with a lytic agent, and a second assay was performed in which detection was performed without treating the sample with a lytic agent, and the detection results were compared to verify the possibility of detecting blood bacteria from urine samples.

[0091] In the following procedure, a mixture of 0.1M Tris-HCl (pH 7.5), 5% Tween 20, 0.3% NaCl, 0.1% Na caseinate / 2% DDAO, and 10 μg / mL lysostaphin was used as the mixed reagent 1 for the first assay, and a mixture of 0.1M Tris-HCl (pH 7.5), 5% Tween 20, 0.3% NaCl, 0.1% Na caseinate, and 2% DDAO was used as the mixed reagent 2 for the second assay. These mixed reagents 1 and 2 were soaked into the specimen addition members (sample pads) (3) of the immunochromatography kits 1 and 2, respectively, and dried before use. In addition, a 3 ng / mL L7 / L12 solution of Staphylococcus aureus was used.

[0092] The first and second assays were carried out according to the following procedure. 1. The bacterial solution and L7 / L12 solution after concentration adjustment were dropped into Immunochromatography Kit 1 (115 μL, N=3). 2. The bacterial solution and L7 / L12 solution after concentration adjustment were dropped into Immunochromatography Kit 2 (115 μL, N=3). 3. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 1 was measured using a reader (N=3 and the average values ​​were graphed). 4. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 2 was measured using a reader (N=3 measurements were taken and the average values ​​were graphed).

[0093] The results of detection of the bacterial liquid and the L7 / L12 liquid by the first and second assays are shown in the graph of FIG. 5. As is clear from the graph, the amount of L7 / L12 (bacteria-derived antigen) detected by the second assay was significantly smaller than the amount of L7 / L12 (bacteria-derived antigen) detected by the first assay, and a clear difference occurred between the detection results by both assays. This is thought to be because in the first assay, in which the bacterial liquid was brought into contact with the mixed reagent 1 containing a lytic agent (lysostaphin) in the specimen addition member (sample pad) (3) of the immunochromatography kit 1, a large amount of L7 / L12 released by lysis of the bacterial cells by the mixed reagent was detected, whereas in the second assay, in which the bacterial liquid was brought into contact with the mixed reagent 2 not containing a lytic agent (lysostaphin) in the specimen addition member (sample pad) (3) of the immunochromatography kit 2, L7 / L12 in the bacterial cells was not released and was not detected. On the other hand, in the detection results of the L7 / L12 liquid, the amount of bacteria-derived antigen detected by the first assay was almost the same as that by the second assay, and there was almost no difference between the two assays. Therefore, it was shown that the detection method of this embodiment using the bacteriolytic agent treatment can also detect blood bacteria from urine samples.

[0094] [Example II. Detection of Pseudomonas aeruginosa] In this example, Pseudomonas aeruginosa, a gram-negative bacterium, was used as the detection target, and its detection was attempted using a mock specimen.

[0095] (1) Obtaining Pseudomonas aeruginosa L7 / L12: With reference to the method described in WO 2000 / 06603, an expression vector was prepared by cloning a nucleic acid sequence (SEQ ID NO: 4) encoding the amino acid sequence (SEQ ID NO: 3) of ribosomal protein L7 / L12 of Pseudomonas aeruginosa. Escherichia coli was transformed with the obtained expression vector, and the obtained transformant was cultured in LB medium or the like. The full-length protein of Pseudomonas aeruginosa L7 / L12 expressed by this transformant was purified as a fusion protein using an affinity column with the tag sequence derived from the expression vector.

[0096] (2) Preparation of antibodies against Pseudomonas aeruginosa L7 / L12: Using the full-length protein of L7 / L12 of Pseudomonas aeruginosa obtained by the above procedure, a monoclonal antibody against L7 / L12 of Pseudomonas aeruginosa was produced with reference to the method described in WO 2000 / 06603.

[0097] Specifically, the full-length protein of L7 / L12 of Pseudomonas aeruginosa was used as an immunogen, and according to the standard method for obtaining hybridomas, the immunogen was prepared in PBS to a concentration of 0.4 mg / mL, and the same amount of Freund's adjuvant was added, and mice were immunized four times so that the immunogen amount was 50 μg / time. After confirming the increase in serum antibody titer by test blood collection, spleen cells of the mice were extracted. The extracted mouse spleen cells were fused with myeloma cells to obtain various hybridomas. The obtained various hybridomas were cultured in HAT medium, and screening was performed using the antibodies in the culture supernatant. Screening was performed by ELISA to verify reactivity with bacterial lysates of Pseudomonas aeruginosa (PA) and bacterial lysates of several other types of bacteria (Escherichia coli (EC), Staphylococcus aureus (SA), Streptococcus pneumoniae (SP), Klebsiella pneumoniae (KP), Chlamydia pneumoniae (CP), Haemophilus influenzae (HI), and Serratia marcescens (SM)). Hybridomas producing specific antibodies reactive only with bacterial lysates of Pseudomonas aeruginosa (PA) and hybridomas producing general antibodies reactive with bacterial lysates of Pseudomonas aeruginosa (PA) and several other types of bacteria were selected, and monoclonal antibodies were prepared. The detailed procedure is as described in Example I(2) above.

[0098] Of the general-purpose monoclonal antibodies 44B2, 47A1, 51B2, 78A2, and 84B2 and the specific monoclonal antibodies 6C2, 14B2, 72A2, 73B2, and 85B2 against Pseudomonas aeruginosa L7 / L12 obtained by the above procedures, the specific monoclonal antibody 72A2 was used as the detection antibody and the general-purpose monoclonal antibody 51B2 was used as the capture antibody to prepare the following immunochromatography kit.

[0099] (3) Preparation of immunochromatography kit using detection and capture antibodies: An immunochromatography kit was prepared in the same manner as in Example I(3) above, using the detection antibody and capture antibody against Pseudomonas aeruginosa L7 / L12 obtained in the above procedure.

[0100] (4) Preparation of mock specimens: Next, we prepared a solution of P. aeruginosa bacteria and a solution of P. aeruginosa ribosomal protein L7 / L12 as mock specimens for the detection test. The liquid medium used was BD BBL Trypticase Soy Broth (Becton Dickinson).

[0101] Pseudomonas aeruginosa was purchased from ATCC. Live Pseudomonas aeruginosa cells were spiked into the medium and cultured with shaking at 37°C for 16 hours. The culture was adjusted to a turbidity (OD = 600) of 2, and the resulting culture was diluted 1 / 1000-fold with liquid medium to use as a Pseudomonas aeruginosa culture. This Pseudomonas aeruginosa culture was used as a specimen simulating urinary bacteria (and urinary bacteria-derived antigens released therefrom) present as live bacteria in urine.

[0102] Meanwhile, the ribosomal protein L7 / L12 of Pseudomonas aeruginosa prepared in (1) above was diluted to 5 ng / mL or 10 ng / mL in liquid medium and used as the Pseudomonas aeruginosa L7 / L12 solution, which was used as a specimen simulating the bacterial antigens excreted from blood through the glomerulus into urine.

[0103] These simulated samples are subjected to the first assay (an assay for detecting both bacterial antigens in blood and bacterial antigens in urine from urine samples based on an antigen-antibody reaction) and the second assay (an assay for detecting bacterial antigens in blood from urine samples based on an antigen-antibody reaction) described below. If it is verified that there is a clear difference in the detection results of the bacterial liquid by both assays (i.e., the amount of bacterial antigen detected by the second assay is significantly less than the amount of bacterial antigen detected by the first assay, or no bacterial antigen is detected), and there is little or no difference in the detection results of the L7 / L12 liquid (i.e., the amount of bacterial antigen detected by the first assay and the amount of bacterial antigen detected by the second assay are almost or completely the same), it will be shown that it is possible to detect bacteria in blood from urine samples by comparing the detection results of the first and second assays.

[0104] (5) Detection method A - Ultrasonic treatment: In this example, a first assay was performed in which the sample was subjected to ultrasonic treatment, and a second assay was performed in which the sample was used as is without being subjected to ultrasonic treatment, and the detection results were compared to verify the possibility of detecting bacteria in blood from urine samples.

[0105] In the following procedure, a mixture of 0.1M Tris-HCl (pH 7.5), 0.25% DDAO, and 0.5% NaCl was used as the mixed reagent. This mixed reagent was in liquid form and mixed with each specimen before the assay. In addition, a 5ng / mL L7 / L12 solution of Pseudomonas aeruginosa was used.

[0106] The first and second assays were carried out according to the following procedure. 1. The aforementioned bacterial liquid and L7 / L12 liquid were each mixed with the aforementioned mixed reagent (volume ratio of bacterial liquid or L7 / L12 liquid:mixed reagent 30:70) to prepare a mixed liquid. 2. The bacterial mixture and the L7 / L12 mixture were each collected using a 200 μL pipette and subjected to ultrasonic treatment for 1 minute using an ultrasonic homogenizer (Bioruptor UCD-200TM). 3. The bacterial solution and L7 / L12 solution after ultrasonic disruption were dropped into Immunochromatography Kit 1 (first assay: 115 μL, N=3). 4. 200 μL of the bacterial mixture and the L7 / L12 mixture were each taken with a pipette and dropped into Immunochromatography Kit 2 (without being subjected to an ultrasonic homogenizer) (second assay: 115 μL, N=3). 5. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 1 was measured using a reader (first assay: N=3, and the average values ​​were graphed). 6. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 2 was measured using a reader (second assay: N=3, and the average values ​​were graphed).

[0107] The results of detection of the bacterial liquid and the L7 / L12 liquid by the first and second assays are shown in the graph of FIG. 6. As is clear from the graph, in the results of detection of the bacterial liquid, the amount of L7 / L12 (bacteria-derived antigen) detected by the second assay was significantly smaller than the amount of L7 / L12 (bacteria-derived antigen) detected by the first assay, and a clear difference occurred between the detection results by both assays. This is thought to be because in the first assay, in which the bacterial liquid was subjected to ultrasonic treatment and then used, a large amount of L7 / L12 released by the destruction of bacterial cells was detected, whereas in the second assay, in which the bacterial liquid was used as is, L7 / L12 in the bacterial cells was not released and was not detected. On the other hand, in the results of detection of the L7 / L12 liquid, the amount of the bacterial antigen detected by the first assay and the amount of the bacterial antigen detected by the second assay were roughly the same, and there was almost no difference between the two assays. Therefore, it was shown that the detection method of this embodiment using ultrasonic treatment can also detect blood bacteria from urine samples.

[0108] (6) Detection method B - Filter processing used: In this example, a first assay was performed using the sample as is, and a second assay was performed using the sample after filtering, and the detection results were compared to verify the possibility of detecting bacteria in blood from urine samples.

[0109] In the following procedure, a mixture of 0.1M Tris-HCl (pH 7.5), 5.5% DDAO, and 0.5% NaCl was used as the mixed reagent. This mixed reagent was impregnated into each specimen addition member (sample pad) (3) of Immunochromatography Kit 1 and 2, and then dried for use. In addition, a 5ng / mL L7 / L12 solution of Pseudomonas aeruginosa was used.

[0110] The first and second assays were carried out according to the following procedure. 1. The bacterial solution and the L7 / L12 solution were each dropped onto Immunochromatography Kit 1 (115 μL, N=3). 2. The bacterial solution and the L7 / L12 solution were each filtered through a sterile filter (Merck Millipore syringe filter, 0.45 μm mesh), and then added dropwise to Immunochromatography Kit 2 (115 μL, N=3). 3. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 1 was measured using a reader (N=3 and the average values ​​were graphed). 4. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 2 was measured using a reader (N=3 measurements were taken and the average values ​​were graphed).

[0111] The detection results of the bacterial liquid and the L7 / L12 liquid by the first and second assays are shown in the graph of FIG. 7. As is clear from the graph, in the detection results of the bacterial liquid, the amount of L7 / L12 (bacteria-derived antigen) detected by the second assay was significantly smaller than the amount of L7 / L12 (bacteria-derived antigen) detected by the first assay, and a clear difference occurred between the detection results by both assays. This is thought to be because in the first assay, in which the bacterial liquid was used as is, a large amount of L7 / L12 released by lysis of the bacterial cells by the mixed reagent was detected, whereas in the second assay, in which the bacterial liquid was used after being filtered through a sterilized filter, the bacterial cells were removed, and as a result, L7 / L12 present within the bacterial cells was not detected. On the other hand, in the detection results of the L7 / L12 liquid, the amount of the bacterial antigen detected by the first assay and the amount of the bacterial antigen detected by the second assay were roughly the same, and there was almost no difference between the two assays. Therefore, it was shown that the detection method of this embodiment using filter treatment can also detect blood bacteria from urine samples.

[0112] (7) Detection method C - embodiment 1 using lysis agent treatment: In this example, a first assay was performed using the sample after treating it with a bacteriolytic agent, and a second assay was performed using the sample as is, and the detection results were compared to verify the possibility of detecting bacteria in blood from urine samples.

[0113] In the following procedure, a mixture of 0.1M Tris-HCl (pH 7.5), 0.25% DDAO, and 0.5% NaCl was used as the mixed reagent. This mixed reagent was mixed with each specimen in liquid form. 1% (v / v) DDAO was used as the bacteriolytic agent. A 10ng / mL L7 / L12 solution of Pseudomonas aeruginosa was used.

[0114] The first and second assays were carried out according to the following procedure. 1. The bacterial solution and the L7 / L12 solution were each mixed with 1% (v / v) DDAO, and then mixed with the mixed reagent, and added dropwise to Immunochromatography Kit 1 (115 μL, N=3). 2. The bacterial solution and the L7 / L12 solution were each mixed with a mixed reagent, and the mixture was added dropwise to the Immunochromatography Kit 2 (115 μL, N=3). 3. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 1 was measured using a reader (N=3 and the average values ​​were graphed). 4. 15 minutes after dropping, the signal intensity of Immunochromatography Kit 2 was measured using a reader (N=3 measurements were taken and the average values ​​were graphed).

[0115] The results of detection of the bacterial liquid and the L7 / L12 liquid by the first and second assays are shown in the graph of FIG. 8. As is clear from the graph, in the results of detection of the bacterial liquid, the amount of L7 / L12 (bacteria-derived antigen) detected by the second assay was significantly smaller than the amount of L7 / L12 (bacteria-derived antigen) detected by the first assay, and a clear difference occurred between the detection results by both assays. This is thought to be because in the first assay, in which the bacterial liquid was lysed with a lytic agent (DDAO) and then mixed with mixed reagent 1, a large amount of L7 / L12 released from the bacterial cells was detected, whereas in the second assay, in which the bacterial liquid was not lysed with a lytic agent and mixed with mixed reagent 2, L7 / L12 in the bacterial cells was not released and was not detected. On the other hand, in the results of detection of the L7 / L12 liquid, the amount of the bacterial antigen detected by the first assay and the amount of the bacterial antigen detected by the second assay were roughly the same, and there was almost no difference between the two assays. Therefore, it was demonstrated that blood bacteria can be detected from urine samples by the detection method of this embodiment utilizing treatment with a bacteriolytic agent. [Industrial Applicability]

[0116] The present invention can be widely applied to fields such as the medical field where detection of bacteria in blood or urine is required, and its utility is extremely valuable. [Explanation of symbols]

[0117] 10 Detection mechanism 1. Insoluble membrane carrier for chromatographic development 2. Detection antibody-impregnated member (conjugate pad) 3. Sample pad 4 Absorption materials (absorption pads) 5 Base material 6 Capture antibody immobilization site 7. Control reagent fixing site Sample A B Sample flow

Claims

1. 1. A method for detecting bacteria in the blood and / or urine of a subject based on a urine specimen from the subject, comprising: (a) carrying out a first assay for detecting both a bacteria-derived antigen present within a bacterial cell in a urine specimen and a bacteria-derived antigen present outside the bacterial cell based on an antigen-antibody reaction; (b) performing a second assay to detect a bacteria-derived antigen present outside the bacterial cells in the urine sample based on an antigen-antibody reaction; and (c) comparing the detection result of the first assay in the step (a) with the detection result of the second assay in the step (b) and detecting the presence or absence or amount of bacteria in urine and / or blood based on the comparison result. The method includes:

2. The method of claim 1, wherein the bacteria-derived antigen is bacterial ribosomal protein L7 / L12.

3. The method according to claim 1 or 2, wherein in step (a), a bacteria-derived antigen present within a bacterial cell in a urine specimen is exposed to the outside of the bacterial cell before the first assay is performed.

4. The method according to claim 3, wherein in step (a), the urine sample is sonicated to expose bacteria-derived antigens present within bacterial cells in the urine sample to the outside of the bacterial cells.

5. The method according to claim 3, wherein in step (a), the urine specimen is treated with a bacteriolytic agent to expose bacteria-derived antigens present within bacterial cells in the urine specimen to the outside of the bacterial cells.

6. The method according to any one of claims 1 to 5, wherein in step (b), bacterial cells in the urine specimen are removed before the second assay is carried out.

7. The method according to claim 6, wherein in step (b), urinary bacteria in the urine specimen are removed by filtering the urine specimen.

8. 8. The method according to claim 1, wherein the bacteria to be detected are selected from bacteria of one or more genera selected from the genera Escherichia, Staphylococcus, Klebsiella, Proteus, Enterococcus, Citrobacter, Pseudomonas, and Serratia.

9. The first assay of step (a) and the second assay of step (b) The method according to any one of claims 1 to 8, comprising contacting a urine sample with a capture antibody for capturing the bacteria-derived antigen and a detection antibody for labeling the bacteria-derived antigen, thereby capturing and labeling the bacteria-derived antigen in the urine sample, and detecting the captured bacteria-derived antigen in the urine sample based on the detection label.

10. A kit for detecting bacteria in the blood and / or urine of a subject based on a urine sample from the subject by carrying out the method according to claim 9, comprising: A capture antibody for capturing a bacteria-derived antigen in a urine sample; and a detection antibody for labeling a bacteria-derived antigen.

11. Further comprising a carrier for developing the sample and contacting the sample with a detection antibody; The kit according to claim 10, wherein the carrier is provided with a first assay area for performing a first assay and a second assay area for performing a second assay, and a capture antibody is immobilized in each of the first and second assay areas.

12. 12. The kit of claim 11, wherein only the first assay area is coated with a lysing agent.

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