JP2022153079A - Method and kit for detecting bacteria in blood and urine from urine specimen - Google Patents

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

The present invention relates to methods for detecting bacteria in a subject's blood and/or urine based on a subject's urine specimen, and kits for use in such methods.

Conventionally, in order to determine the presence or absence of blood infection of a target bacterium, it was necessary to detect the presence or absence of bacteria in a blood-derived sample collected from the target by an antigen-antibody reaction or the like (Patent Document 1: JP 2020- 54279; Patent Document 2: Japanese Patent No. 5599013; Patent Document 3: Japanese Patent No. 6723293). However, since these methods require time and effort to collect blood from a subject, there has been a demand for a simpler and faster method.

Therefore, a technique for determining the presence or absence of bacterial blood infection using a urine sample has been studied (Patent Document 4: International Publication No. 2011/007823). Bacteria present in the subject's blood are decomposed in the subject's body, and part of the decomposition product is excreted from the glomeruli into the urine. Since the upper limit of the size barrier of the glomerulus is 66 kDa or less, bacterial fragments smaller than that may be excreted from the glomerulus into the urine. Therefore, it is possible to determine the presence or absence of bacterial blood infection using a urine sample by performing detection based on the antigen-antibody reaction using the degradation product of blood bacteria excreted from the glomerulus into the urine as an antigen. .

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, urinary tract infected bacteria may be found in the urine sample collected from the subject. may exist. In addition, there are many cases (contamination) in which bacteria are mixed from places other than the urinary tract, such as the vulva, at the time of urine collection. Therefore, even if a bacterial decomposition product is detected in a urine sample, it is impossible to determine whether the bacterial decomposition product is derived from blood bacteria or urinary bacteria, making it difficult to accurately determine blood infection.

JP 2020-54279 A Japanese Patent No. 5599013 Japanese Patent No. 6723293 WO2011/007823

SUMMARY OF THE INVENTION It is an object of the present invention to provide a technique for determining the presence or absence of bacterial blood infection using a urine sample.

As a result of intensive studies, the present inventors have found that, using a urine specimen, both bacterial-derived antigens present inside bacterial cells and bacterial-derived antigens present outside bacterial cells in urine specimens are detected based on antigen-antibody reactions. 1 assay and a second assay that detects bacterial antigens present outside bacterial cells in urine specimens based on antigen-antibody reaction, and comparing the detection results, the blood and / or The inventors have found that the presence or absence or amount of bacteria in urine can be detected to appropriately determine the presence or absence of bacterial blood infection, and have completed the present invention.

That is, the gist of the present invention relates to, for example, the following.
[Claim 1] A method for detecting bacteria in the blood and/or urine of a subject based on a urine specimen of the subject, comprising:
(a) a step of performing a first assay for detecting both bacterial-derived antigens present inside bacterial cells and bacterial-derived antigens present outside bacterial cells in a urine specimen based on antigen-antibody reaction;
(b) performing a second assay for detecting a bacterial antigen present outside bacterial cells in a urine sample based on an antigen-antibody reaction;
(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 based on the comparison result, urinary bacteria and/or blood bacteria A method comprising the step of detecting the presence or abundance of
[Item 2] The method according to item 1, wherein the bacterial antigen is bacterial ribosomal protein L7/L12.
[Item 3] The method according to Item 1 or 2, wherein, in step (a), the first assay is performed after exposing the bacterial-derived antigen present in the bacterial cells in the urine sample to the outside of the bacterial cells.
[Item 4] The method according to Item 3, wherein, in step (a), the bacterium-derived antigen present in bacterial cells in the urine sample is exposed to the outside of bacterial cells by subjecting the urine sample to ultrasonic treatment.
[Item 5] The method according to item 3, wherein, in step (a), the bacterium-derived antigen present in bacterial cells in the urine sample is exposed outside the bacterial cells by treating the urine sample with a bacteriolytic agent.
[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 performing the second assay.
[Item 7] The method according to Item 6, wherein in the step (b), urinary bacteria in the urine sample are removed by filtering the urine sample.
[Claim 8] Bacteria to be detected include genus Escherichia, genus Staphylococcus, genus Klebsiella, genus Proteus, genus Enterococcus, genus Citrobacter, Pseudomonas Item 8. The method according to any one of Items 1 to 7, wherein the bacterium is selected from one or more genera selected from the genus Pseudomonas and the genus Serratia.
[Claim 9] The first assay in step (a) and the second assay in step (b) are
By contacting a urine sample with a capturing antibody for capturing the bacterial-derived antigen and a detecting antibody for labeling the bacterial-derived antigen, the bacterial-derived antigen in the urine sample is captured, labeled, and captured. Item 9. The method according to any one of items 1 to 8, comprising detecting the bacterial antigen in the collected urine sample based on the detection label.
[Claim 10] A kit for detecting bacteria in the blood and/or urine of a subject based on a urine sample of the subject by performing the method of [9],
a capturing antibody for capturing a bacterial antigen in a urine sample;
and a detection antibody for labeling the bacterial antigen.
[Claim 11] Further comprising a carrier for developing the specimen and contacting the specimen with the detection antibody,
A first assay area for performing a first assay and a second assay area for performing a second assay are provided on the carrier, each of the first and second assay areas comprising: 11. A kit according to Item 10, wherein the capturing antibody is immobilized.
[Item 12] The kit according to item 11, wherein the lysing agent is applied only to the first assay area.

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a technique for determining the presence or absence of bacterial blood infection using a urine sample.

FIG. 1 is a cross-sectional view showing a schematic configuration of a strip-shaped detection mechanism, which is an example of a detection mechanism of a lateral flow type immunochromatography detection device. FIG. 2 is a graph showing the results of detection of L7/L12 (antigen-derived antigen) using a bacterium solution and L7/L12 solution of Staphylococcus aureus by a detection method using sonication. FIG. 3 is a graph showing the results of detection of L7/L12 (antigen-derived antigen) using Staphylococcus aureus bacterial solution and L7/L12 solution by a detection method using filtering. FIG. 4 is a graph showing the results of detection of L7/L12 (antigen-derived antigen) using Staphylococcus aureus bacterial solution and L7/L12 solution by Detection Method 1 using lytic agent treatment. FIG. 5 is a graph showing the results of detection of L7/L12 (antigen-derived antigen) using Staphylococcus aureus bacterial solution and L7/L12 solution by Detection Method 2 using lytic agent treatment. FIG. 6 is a graph showing the results of detection of L7/L12 (antigen-derived antigen) using Pseudomonas aeruginosa bacterial solution and L7/L12 solution by a detection method using ultrasonic treatment. FIG. 7 is a graph showing the results of detection of L7/L12 (antigen-derived antigen) using a Pseudomonas aeruginosa bacterial solution and L7/L12 solution by a detection method using filtering. FIG. 8 is a graph showing the results of detection of L7/L12 (antigen-derived antigen) using Pseudomonas aeruginosa bacterial solution and L7/L12 solution by Detection Method 1 using lytic agent treatment.

Hereinafter, the present invention will be described in detail based on specific embodiments. However, the present invention is not restricted to the following embodiments, and can be embodied in any form without departing from the gist of the present invention.

[I. Method for Detecting Blood Bacteria from a Urine Specimen]
A first gist of the present invention relates to a method for detecting bacteria in a subject's blood and/or urine based on a subject's urine sample (hereinafter referred to as the "detection method of the present invention").

The present inventors have developed a method for detecting only blood-infected bacteria, not bacteria present in urine, for bacteria that may be not only blood-borne infections but also urinary tract infections and contamination from sources other than the urinary tract. In order to search for a policy, we studied earnestly. As a result, substances in the blood are excreted into the urine through the glomeruli of the kidney, and there is a size barrier in the permeation of substances through the glomeruli. Focusing on the finding that molecules are impermeable (Tencer et al., Kidney International, (1998), Vol.53, pp.709-715). Blood bacteria cannot permeate the size barrier of the glomerulus as they are, and are not excreted in the urine. Substances that are permeable will be excreted in the urine through the glomerulus.

Therefore, the present inventors selected an appropriate bacterial antigen that can permeate the size barrier of the glomerulus, and examined the presence of bacterial antigens present in bacterial cells and bacterial antigens present outside bacterial cells in urine specimens. The technical idea was to conduct a first assay for detecting both based on antigen-antibody reaction, and a second assay for detecting a bacterial antigen present outside bacterial cells in a urine sample based on antigen-antibody reaction. . Among bacterial-derived antigens present in urine specimens, those derived from blood bacteria are excreted through the glomerulus, so all of them are exposed in urine (that is, present outside bacterial cells). On the other hand, bacteria-derived antigens derived from urinary bacteria do not pass through glomeruli, and most of them exist in bacterial cells. Therefore, the first assay detects both blood bacterium-derived antigens and urinary bacterium-derived antigens, while the second assay mainly detects only blood bacterium-derived antigens. In urine specimens, some urinary bacterium-derived antigens are thought to be released extracellularly due to decomposition or death of urinary bacteria, but the amount of urinary bacterium-derived antigens present in urinary bacterium cells It is considered to be a very small amount compared to ). Therefore, by comparing the detection results of these first and second assays, it is considered possible to appropriately determine the presence or absence of blood bacteria and the amount of bacteria present.

That is, the detection method of the present invention is a method for detecting the presence or absence or abundance of bacteria in the blood of a subject based on a urine specimen of the subject, and includes the following steps.
(a) A step of performing a first assay for detecting both bacterial-derived antigens present inside bacterial cells and bacterial-derived antigens present outside bacterial cells in a urine specimen based on antigen-antibody reaction.
(b) a step of performing a second assay for detecting a bacterial antigen present outside bacterial cells in the urine specimen based on an antigen-antibody reaction;
(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 based on the comparison result, urinary bacteria and/or blood bacteria A step of detecting the presence or absence or amount of

The bacterium-derived antigen is a biological substance such as a protein derived from the target bacterium, capable of identifying the target bacterium, and capable of penetrating the glomerular size barrier (approximately 66 kDa). , the type of which is not limited. Examples include ribosomal proteins L7/L12, nucleic acids such as DNA and RNA, and membrane antigens such as LPS and polysaccharides. Among them, the ribosomal protein L7/L12 is preferable as the bacteria-derived antigen. In the present invention, "ribosomal protein L7/L12" or simply "L7/L12" is one type of ribosomal protein essential for protein synthesis in microorganisms. It is a protein that various bacteria have in common, and although their amino acid sequences are generally similar, there are slight differences in the amino acid sequences for each bacterial species, making it suitable for identifying bacterial species. Furthermore, since it has a size (approximately 12 kDa) that can penetrate the size barrier of the glomerulus, it has ideal conditions as a bacterial-derived antigen.

Bacteria to be detected are also not limited, and any bacterium that has the possibility of blood infection and/or urinary tract infection or contamination from sources other than the urinary tract and has the above-mentioned bacterium-derived antigen can be detected. The type is arbitrary. Examples include the genera Escherichia, Staphylococcus, Klebsiella, Proteus, Enterococcus, Citrobacter, Pseudomonas, and Bacteria selected from one or more genera of bacteria selected from the genus Serratia.

Bacteria belonging to the genus Escherichia include Escherichia coli, E. coli, EC, Escherichia albertii, and the like. Among them, Escherichia coli is preferred.

Bacteria belonging to the genus Staphylococcus include Staphylococcus aureus (S. aureus, SA), S. epidermidis, S. argenteus, and the like. Among them, Staphylococcus aureus is preferred.

Bacteria belonging to the genus Klebsiella include Klebsiella pneumoniae (K. pneumoniae, KP), Klebsiella oxytoca (K. oxytoca), Klebsiella aerogenes (K. aerogenes), and the like. be done. Among them, it is preferable to detect an antigen-antibody reaction with Klebsiella pneumoniae.

Bacteria belonging to the genus Proteus include Proteus milabilis (P.milabilis, PM), Proteus morganii (P. morganii), Proteus vulgaris (P. vulgaris), Proteus - Rettgeri (Proteus rettgeri, P. rettgeri) and the like. Among them, Proteus milabilis is preferred.

Bacteria belonging to the genus Enterococcus include Enterococcus faecalis and Enterococcus faecium. Among them, Enterococcus faecalis is preferred.

Bacteria belonging to the genus Citrobacter include Citrobacter freundii (C. freundii, CF), Citrobacter amalonaticus (C. amalonaticus), and Citrobacter diversus (C. diversus), etc. Among them, Citrobacter freundii is preferred.

Bacteria belonging to the genus Pseudomonas include Pseudomonas aeruginosa (P. aeruginosa, PA), P. fluorescens, P. phosphorescence, P. putida, and the like. Among them, Pseudomonas aeruginosa is preferred.

Serratia bacteria include Serratia liquefaciens, S. liquefaciens, SL, Serratia marcescens, S. marcescens, Serratia fonticola, S. fonticola, etc. is mentioned. Among them, Serratia liquefaciens is preferred.

In the detection method of the present invention, in the step (a), a first assay for detecting both bacterial-derived antigens present in bacterial cells and bacterial-derived antigens present outside bacterial cells in a urine sample based on antigen-antibody reaction , and in step (b), a second assay is performed to detect a bacterial-derived antigen present outside the bacterial cells in the urine sample based on the antigen-antibody reaction. The order of these steps (a) and (b) is not particularly limited, and steps (a) and (b) may be performed in parallel, and step (a) may be performed first. may be carried out, or step (b) may be carried out first.

In the first assay performed in step (a), both bacterial-derived antigens existing inside bacterial cells and bacterial-derived antigens existing outside bacterial cells are detected based on antigen-antibody reaction in a urine specimen. Although the technique is not limited, for example, before or during the assay of the urine specimen, it is conceivable to perform some kind of treatment to expose the urinary bacteria-derived antigens in the urine specimen to the outside of the bacterial cells. Examples of such treatments include, but are not limited to, ultrasonic treatment, treatment with a lysing agent, heat treatment, freeze-thaw, and the like.

The specific types and conditions of the treatment for exposing the urinary bacterium-derived antigens in the urine sample to the outside of the bacterial cells are not particularly limited, and may be appropriately selected according to the type of bacteria to be detected. . Among them, the cell wall structure of bacteria is one of the criteria for selecting the type and conditions of such treatment. The cell wall structure of bacteria differs depending on whether they are Gram-positive or Gram-negative bacteria. Gram-positive bacteria have a relatively thick cell wall in which teichoic acid or lipoteichoic acid is embedded in a peptidoglycan layer and do not have an outer membrane. Gram-negative bacteria have a relatively thin cell wall consisting of a peptidoglycan layer and an outer membrane consisting of lipopolysaccharides, polysaccharides, O-antigens, and the like. Therefore, based on the cell wall structure of the bacterium to be detected, the type and conditions of treatment should be appropriately selected so that the cell wall structure can be destroyed and the bacterium-derived antigen can be reliably exposed outside the bacterium. is preferred.

Specifically, when a bacterial antigen is exposed to the outside of bacterial cells by ultrasonication, the conditions are not particularly limited, but are, for example, as follows. In the case of Gram-positive bacteria, it is preferable to subject a urine specimen to ultrasonication for usually 1 to 15 minutes using a general sonicator or the like. In the case of Gram-negative bacteria, it is preferable to subject the urine sample to ultrasonication for usually 5 seconds to 3 minutes using a general sonicator or the like.

In the case of exposing the bacterial antigen to the outside of the bacterial cell by treatment with a bacteriolytic agent, the conditions are not particularly limited, but are as follows. In the case of Gram-positive bacteria, a lysate containing enzymes such as lysostaphin, surfactants such as Tween 20 and DDAO (N,N-dimethyldodecylamine N-oxide), and lysing agents such as alkaline lysing agents is added to the urine sample. It is preferable to carry out the bacteriolytic agent treatment by contacting for several seconds to several minutes. In the case of Gram-negative bacteria, for example, a lysing solution containing a lysing agent such as a surfactant such as Tween 20 or DDAO or an alkaline lysing agent is brought into contact with the urine specimen for usually several seconds to several minutes to remove the lysing agent. Treatment is preferred. Such lysing agent treatment may be performed by mixing the urine specimen with the lysate prior to assay, or by contacting the urine specimen with the lysate at any step of the assay. good.

On the other hand, in the second assay performed in the step (b), the bacterial antigen present inside the bacterial cells in the urine sample is not detected, and the bacterial antigen present outside the bacterial cells is detected based on the antigen-antibody reaction. do. Although the method is not limited, the treatment for exposing the bacterial-derived antigens outside the bacterial cells, which is performed in step (a), is not performed in step (b), and the bacterial-derived antigens present in the bacterial cells present in the urine sample are exposed to bacteria. The assay may be performed as it is without exposing it to the outside of the cell. Alternatively, as a more aggressive treatment, the urine specimen may be subjected to a treatment to remove bacterial cells before being subjected to the assay. The treatment for removing bacterial cells in such a urine specimen is not limited, but an example thereof includes filtration treatment using a filter.

Specifically, when removing bacterial cells in a urine sample by filtration treatment with a filter, the conditions are not particularly limited, and an appropriate opening ( For example, a filter such as a syringe filter (for example, a syringe filter manufactured by Merck Millipore) may be used.

After performing the first assay in step (a) and the second assay in step (b) in the above procedure, the detection results of both assays are compared in step (c). Here, except for the differences described above, the first and second assays were performed using the same apparatus, under the same conditions, and in the same operation as much as possible, so that the detection results could be properly compared. preferably.

Subjects to be detected by the detection method of the present invention are not limited, and can be performed on any subject for which detection of bacterial infection is required, but usually mammals, preferably humans. The sample to be detected is not particularly limited, either, but from the spirit 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 as a sample after adding a desired pretreatment (e.g., surfactant, enzyme, pH adjustment reagent, etc.). .

If the specific detection method of the first and second assays is an immunoassay method that can detect bacteria-derived antigens in urine specimens based on antigen-antibody reaction, the specific type is particularly Not restricted. Examples include ELISA (enzyme-linked immunosorbent) using antibody-loaded microtiter plates; latex particle agglutination assay using antibody-loaded latex particles (e.g., polystyrene latex particles); antibody-loaded Immunochromatography using a membrane, etc.; Sandwich immunochromatography using colored particles or particles having color-developing ability, a detection antibody labeled with an enzyme, a fluorescent substance, etc., and a capturing antibody immobilized on a solid-phase carrier such as magnetic fine particles. Various known methods such as detection methods can be used. Among them, in the present invention, it is preferable to use an immunochromatographic detection method using a detection antibody and a capture antibody. The next section describes the case where the first and second assays are performed by immunochromatographic detection methods.

[II. Immunochromatographic detection method]
As one aspect of the detection method of the present invention, the first assay in step (a) and the second assay in step (b) are performed by an immunochromatographic detection method using a detection antibody and a capture antibody. can be implemented. In this case, the first assay of step (a) and the second assay of step (b) will each comprise the following steps.
(I) By contacting a urine sample with a capturing antibody for capturing a bacterial-derived antigen and a detecting antibody for labeling the bacterial-derived antigen, the bacterial-derived antigen in the urine sample is detected by the capturing antibody. Capturing and labeling with a detection antibody.
(II) A step of detecting the bacterial antigen in the urine sample captured by the capturing antibody based on the detection label.

The capture antibody and the detection antibody are not limited in type as long as they are antibodies that cause an antigen-antibody reaction with a bacterium-derived antigen to be detected (for example, ribosomal protein L7/L12 of the bacterium to be detected). However, from the viewpoint of detection accuracy, there is no antigen-antibody reaction with components derived from bacteria other than the detection target bacteria (for example, L7/L12 of bacteria other than the detection target bacteria), and bacteria-derived antigens of the detection target bacteria (eg, L7/L12 of the bacteria to be detected) is preferably an antibody having specificity. Both the capture antibody and the detection antibody may have such specificity for the bacterium-derived antigen of the bacterium to be detected, but at least one of the capture antibody and the detection antibody may have such specificity. It doesn't matter if you have it.

Here, according to the detection method of the present invention according to aspect A, the step (I) includes the following.
(Ia-1) A step of contacting a urine specimen with a detection antibody and labeling the bacterium-derived antigen in the urine specimen by an antigen-antibody reaction between the detection antibody and the bacterium-derived antigen.
(Ia-2) A sample containing a bacterial antigen labeled with a detection antibody is brought into contact with the capture antibody, and antigen-antibody reaction between the capture antibody and the bacteria-derived antigen-detection antibody complex causes A step of capturing a bacterial antigen.

Moreover, according to the detection method of the present invention according to aspect B, the step (I) includes the following.
(Ib-1) A step of contacting a urine sample with a capturing antibody and capturing the bacterial antigen in the urine sample by an antigen-antibody reaction between the capturing antibody and the bacterial antigen.
(Ib-2) A sample containing a bacterial antigen captured by a capturing antibody is brought into contact with the detecting antibody, and antigen-antibody reaction between the detecting antibody and the bacterial antigen-capturing antibody complex causes Step of labeling the bacterial antigen.

In the detection method of the present invention, the mode to be selected from mode A and mode B may be determined according to the type of immunoassay method and the type of sample to be actually used. Examples of immunoassay methods include, but are not limited to, ELISA (enzyme-linked immunosorbent) method using antibody-loaded microtiter plates; latex using antibody-loaded latex particles (e.g., polystyrene latex particles). Particle agglutination measurement method; Immunochromatography method using a membrane carrying an antibody, etc.; Colored particles or particles having coloring ability, detection antibodies labeled with enzymes or fluorescent substances, etc., and immobilized on a solid phase carrier such as magnetic fine particles A variety of known immunoassays, such as sandwich assays using capture antibodies, are included.

Hereinafter, the case where immunochromatography is used as an immunoassay method, and the detection method of the present invention according to aspect A is performed will be described as an example. can be implemented.

The type of solid-phase carrier used for the capturing antibody is not particularly limited, but specific examples include porous membranes made of cellulose, nitrocellulose, cellulose acetate, nylon, PVDF (PolyVinylidene DiFluoride), glass fiber, etc.; glass, plastic, Channels made of PDMS (Poly(dimethylsiloxane)), silicon, etc.;
The method of linking the antibody to the solid phase carrier is not particularly limited, but specific examples include immobilization by physical adsorption using the hydrophobicity of the antibody, immobilization by chemical bonding using the functional group of the antibody, and the like.

The type of detection label used for the detection antibody is not particularly limited, and may be appropriately selected according to the detection method. Specifically, metal colloids such as gold colloid, platinum colloid, and palladium colloid; selenium colloid, and alumina. Non-metallic colloids such as colloids and silica colloids; Insoluble particulate materials such as colored resin particles, dye colloids and colored liposomes; Color reaction catalytic enzymes such as alkaline phosphatase, peroxidase and luciferase; Fluorescent dyes and radioisotopes; Examples include luminescent labels, electrochemiluminescent labels, and the like.

Techniques for adding labels to antibodies are not particularly limited, but specific examples include techniques such as physical adsorption utilizing the hydrophobicity of antibodies and chemical bonding utilizing functional groups of antibodies.

Each step of the bacteriological examination method of the present invention will be explained individually below. In the step of labeling the bacterium-derived antigen in the sample, a detection antibody having a detection label is brought into contact with the sample, and the antigen-antibody reaction between the detection antibody and the bacterium-derived antigen labels the bacterium-derived antigen in the sample. The method of contacting the specimen with the detection antibody is not limited, but usually it may be carried out by introducing the specimen prepared as an aqueous specimen into the member region impregnated with the detection antibody and maintaining it for a certain period of time. Although the specific mode varies depending on the mode of capturing, for example, when a capturing antibody immobilized on a porous membrane as a solid-phase carrier is used to capture a bacterial antigen in a sample, the porous membrane can be used for detection. By introducing and permeating an aqueous reagent containing the antibody for detection, the detection antibody is allowed to bind to the bacterial antigen captured by the capture antibody on the porous membrane. As another example, when a capture antibody immobilized in one region on a channel as a solid-phase carrier captures a bacterial antigen in a specimen, an aqueous reagent containing a detection antibody is passed through the channel. Thus, the detection antibody may be bound to the bacterial antigen captured by the capture antibody on the channel.

In the step (Ia-2), that is, the sample containing the bacterial antigen labeled with the detection antibody is brought into contact with the capture antibody, and the antigen-antibody reaction between the capture antibody and the bacteria-derived antigen-detection antibody complex In the step of capturing the bacterium-derived antigen in the sample, the capturing antibody is brought into contact with the sample, and the antigen-antibody reaction between the capturing antibody and the bacterium-derived antigen captures the bacterium-derived antigen in the sample. Although the method of contacting the sample with the capturing antibody is not limited, it is usually carried out by introducing the sample prepared as an aqueous sample into the region where the capturing antibody exists and maintaining it for a certain period of time. Although specific aspects differ depending on the type of solid-phase carrier of the capturing antibody, for example, a porous membrane is used as the solid-phase carrier, and the sample is introduced and permeated through the porous membrane on which the capturing antibody is immobilized. Thus, the bacterium-derived antigen in the sample may be captured by the capturing antibody immobilized on the porous membrane. As another example, by immobilizing a capturing antibody on one region of the channel as a solid-phase carrier and allowing the sample to flow through the channel, the capturing antibody immobilized on the one region of the channel is of bacterial antigens may be captured.

In the step (II), that is, the step of detecting the bacterial antigen in the sample based on the detection label, the bacterial antigen to be detected that is captured by the capturing antibody and labeled with the detecting antibody is Detection based on label. The detection method is not particularly limited, and may be appropriately selected according to the type of label for detection. Alternatively, the abundance may be detected by any method such as visual inspection or camera.

[III. Immunochromatographic detection kit]
Another gist of the present invention is a kit for carrying out the immunochromatographic detection method described above in order to detect bacteria in the blood and / or urine of a subject based on a urine sample of the subject (hereinafter referred to as the "detection kit of the present invention ).

The detection kit of the present invention includes the capture antibody and detection antibody described above. The capturing antibody is usually provided in an appropriate form (a container containing a porous membrane, a container containing a channel, etc.) 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.

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

When the detection kit of the present invention includes a detection device or its constituent members, the device constituted by such a kit is a device (hereinafter referred to as "the present abbreviated as "Inventive Apparatus"). Specific components of the device of the present invention can be adjusted appropriately according to the type of immunoassay that is a specific embodiment of the detection method of the present invention. As described above, non-limiting examples of immunoassay methods include ELISA (enzyme-linked immunosorbent) using antibody-loaded microtiter plates; antibody-loaded latex particles ( latex particle agglutination assay using, for example, polystyrene latex particles, etc.); immunochromatography using a membrane carrying an antibody, etc.; colored particles or particles having coloring ability, antibodies for detection labeled with enzymes or fluorescent substances, and magnetic fine particles. There are various known immunoassay methods such as a sandwich assay method using a capture antibody immobilized on a solid phase carrier such as The device with the components is the device of the invention.

Specific examples of the apparatus include a lateral flow type apparatus and a flow through type apparatus. Here, the lateral flow method means that the analyte to be detected and the detection antibody are developed in parallel with respect to the membrane including the detection region on which the capture antibody is immobilized on the surface, and the target substance captured in the detection region of the membrane. is a method for detecting On the other hand, the flow-through method is a method in which a target substance to be detected and a detection antibody are vertically passed through a membrane having a capture antibody immobilized on its 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 type device and a flow through type device.

Both the lateral flow type device and the flow-through type immunochromatographic detection device are known, and procedures other than those described in the present disclosure can be appropriately designed by those skilled in the art based on common technical knowledge. Hereinafter, the schematic configuration of the detection mechanism of the lateral flow immunochromatographic detection device will be described with reference to the drawings, but these are only examples of the schematic configuration of the detection procedure, and the configuration of the lateral flow immunochromatographic detection device is The embodiments illustrated in the drawings are by no means limited.

FIG. 1 is a cross-sectional view showing a schematic configuration of a strip-shaped detection mechanism, which is an example of a detection mechanism of a lateral flow type immunochromatography detection device. The detection mechanism (10) in FIG. 1 is a strip-shaped detection antibody-impregnated member (conjugation) on one end in the length direction of the strip (on the upstream side of the sample flow (B)) on the insoluble membrane carrier (1) for chromatographic development. A gate pad) (2) (this pad is impregnated with a detection antibody) and a sample addition member (sample pad) (3) are arranged, and on the other end side (downstream side of the sample flow (B)) An absorbent member (absorbent pad) (4) is provided on a substrate (5). At the center of the strip on the insoluble membrane carrier (1) in the longitudinal direction, a site (6) on which a capture antibody is immobilized is arranged, and a site (7) on which a control reagent is immobilized if necessary. are placed. The control reagent is a reagent that does not bind to the bacterium-derived antigen but binds to the detection antibody.

In use, when the sample (A) is applied onto the sample addition member (sample pad) (3), the sample (A) passes through the detection antibody-impregnated member (conjugate pad) (2) to the insoluble membrane carrier. (1) in the direction of sample flow (B). At this time, the bacterium-derived antigen (preferably L7/L12) in the sample (A) binds to the detection antibody to form a bacterium-derived antigen-detection antibody complex. When the sample (A) passes through the capture antibody immobilization site (6), the bacterium-derived antigen in the sample 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-immobilized site (7), those of the detection antibodies that do not bind to the bacteria-derived antigen bind to the control reagent (7), thereby completing the test (i.e. It can be confirmed that the sample (A) has passed the capturing antibody (6)). Here, the label possessed by the detection antibody in the capture antibody-bacteria-derived antigen-detection antibody complex present in the capture antibody-immobilized site (6) is detected by a known means, whereby the bacterial-derived antigen is detected. Absence or abundance can be detected. If desired, the detection antibody label may be sensitized by known techniques to facilitate detection.

The detection antibody-impregnated member (conjugate pad) (2), the specimen addition member (sample pad) (3), and/or the control reagent-immobilized site (7) can be optionally omitted. When the present mechanism does not have a detection antibody-impregnated member (conjugate pad) (2), the sample (A) and the detection antibody are mixed in advance or separately, and simultaneously or sequentially, the insoluble membrane carrier ( 1) By applying it to the upper end, the same inspection as the above inspection can be performed.

Also, a detection kit capable of similar detection can be constructed even if the capture antibody and the detection antibody are exchanged.

In addition, when performing the first and second assays of the detection method of the present invention using such an immunochromatographic detection kit, prepare two immunochromatographic detection kits of the same configuration, while performing the first assay, On the other hand, a second assay may be performed. Alternatively, a single immunochromatographic detection kit includes a plurality of assay regions (the above-mentioned insoluble membrane carrier (1), detection antibody-impregnated member (conjugate pad) (2), specimen addition member (sample pad) (3), and A region serving as a detection unit composed of an absorbent member (absorbent pad) (4) may be provided, and the first and second assays may be performed with a single immunochromatographic detection kit. In particular, the latter configuration has the advantage of making it easier to perform the first and second assays under the same conditions.

In addition, means for exposing the urinary bacterium-derived antigens in the urine specimen to the outside of the bacterial cells in the step (a), and a treatment for removing the bacterial cells in the urine specimen in the step (b). may be incorporated into the immunochromatographic detection kit. For example, when performing bacteriolytic agent treatment as a treatment for exposing the bacterial-derived antigen to the outside of the bacterial cell in step (a), an immunochromatographic detection kit for performing the first assay (or a single immunochromatographic detection kit) When performing the second assay, the specimen addition member (sample pad) (3) of the first assay area) is soaked in advance with a lysate and dried to remove the urine specimen from the first assay. The urine sample may be treated with a bacteriolytic agent at the same time as the sample is provided.

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples are merely examples for convenience of explanation, and the present invention is not limited to these examples in any sense. not something.

[Example I.1. Detection of Staphylococcus aureus]
In this example, Staphylococcus aureus, which is a Gram-positive bacterium, was used as a detection target, and its detection was attempted using a simulated sample.

(1) Acquisition of L7/L12 of Staphylococcus aureus:
An expression vector into which a nucleic acid sequence (SEQ ID NO: 2) encoding the amino acid sequence (SEQ ID NO: 1) of Staphylococcus aureus ribosomal protein L7/L12 (SEQ ID NO: 1) is cloned is prepared with reference to the method described in WO 2000/06603. Escherichia coli is transformed with the prepared expression vector, the resulting transformant is cultured using LB medium or the like, and the full length protein of Staphylococcus aureus L7/L12 expressed by this transformant is obtained. , was purified as a fusion protein using the tag sequence derived from the expression vector by an affinity column.

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

Specifically, using the above-mentioned Staphylococcus aureus L7/L12 full-length protein as an immunogen, the concentration of the immunogen was adjusted to 0.4 mg/mL with PBS according to the standard procedure for obtaining hybridomas, and Freund's adjuvant was added. Mice were immunized 4 times with the same amount added and the immunogen amount was 50 μg/time. After confirming an increase in the serum antibody titer by test blood collection, the spleen cells of the mice were excised. The excised mouse spleen cells were fused with myeloma cells to obtain various hybridomas. The obtained various hybridomas were cultured in HAT medium and screened using antibodies in the culture supernatant. Screening was performed by ELISA, bacterial lysate of Staphylococcus aureus (SA) and several other bacteria (E. coli (EC), Pseudomonas aeruginosa (PA), Streptococcus pneumoniae (SP), Klebsiella pneumoniae (KP ), Chlamydia pneumoniae (CP), and Serratia serratia (SM)) and produce specific antibodies reactive only with bacterial lysates of Staphylococcus aureus (SA). , and hybridomas producing universal antibodies reactive with bacterial lysates of S. aureus (SA) and multiple other bacteria were selected.

The procedure for antibody screening by ELISA is shown below. Each of the above bacteria was purchased from ATCC, cultured, prepared at 1×e ⁸ cfu/mL, and suspended in PBS. A bacterial lysate of each bacterium was obtained by lysing each bacterium by ultrasonication and filtering through a 0.45 μm filter to remove debris. 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. After washing each well three times with PBS-T (PBS containing Tween 20), blocking treatment was performed 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 of 10 μg/mL described in the antibody production method was added dropwise to each well, and antigen-antibody reaction was performed for 1 hour. After the reaction, after washing three times with PBS-T, a secondary antibody (Goat Anti-mouse IgG) labeled with 0.5 μg/mL detection enzyme HRP (Horse Radish Peroxidase) was added. 50 μL of mouse IgG) was added dropwise for reaction. After the reaction, the plate was washed with PBS-T five times, and 100 μL of a mixture of TMB (Tetramethylbenzidine), which is a coloring substrate, and hydrogen peroxide was added dropwise to each well to carry out a coloring reaction. After 10 minutes, hydrochloric acid as a reaction stop solution was added dropwise to each well, and the absorbance at 450 nm of each well was measured with a plate reader.

Monoclonal antibodies were prepared from hybridomas producing specific antibodies or general-purpose antibodies selected as described above. Specifically, the selected hybridomas were cultured in TIL Media I medium supplemented with 10% fetal bovine serum (FBS) and intraperitoneally administered to mice, and ascites fluid was collected according to the standard method for monoclonal production. The recovered ascitic fluid was centrifuged to separate suspended matter and erythrocytes, and then filtered through a filter with an opening of 0.45 μm. The resulting filtrate was passed through a Protein G column to adsorb the antibody, and the obtained monoclonal antibody was purified from mouse ascitic fluid.

Among the general-purpose monoclonal antibodies 13A2, 15C2, 20B1, 46B1, and 52B1 against Staphylococcus aureus L7/L12 and the specific monoclonal antibodies 24C2, 30A2, 32A2, 62A1, and 78C2 obtained by the above procedure, 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 immunochromatographic kit.

(3) Preparation of immunochromatographic kit using detection antibody and capture antibody:
An immunochromatographic kit was prepared by the following procedure using the detecting antibody and capturing antibody for L7/L12 of Staphylococcus aureus obtained by the above procedure.

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

Using the colloidal gold-labeled detection antibody solution obtained by the above procedure and the above capture antibody, an immunochromatographic apparatus (immunochromatographic kit) having the configuration shown in FIG. 1 was produced by the following procedure.

First, a nitrocellulose membrane having a width of 25 mm and a length of 300 mm was prepared as a membrane carrier (1) for chromatographic development of a chromatographic medium. A solution containing a capture antibody at a concentration of 1.5 mg/mL was applied in a line at a concentration of 1 μL/cm to a position 10 mm from the end of the chromatography development starting point side of the membrane carrier for chromatography development (1). It was then dried at 50°C for 30 minutes, then immersed in a 0.5% casein solution, then in a 0.2% sucrose solution, and dried overnight at room temperature, thereby binding the bacterial antigen and the capture antibody. The capture site (6) of the complex with the colloidal gold-labeled detection antibody was used.

On the other hand, a band-shaped glass fiber pad of 10 mm×300 mm was impregnated with 2 mL of the gold colloid-labeled detection antibody, and freeze-dried to obtain a detection antibody-impregnated member (2).

In addition to the membrane carrier for chromatographic development (1) and the detection antibody-impregnated member (2), a cotton cloth as a sample addition member (sample pad) (3) and a filter paper as an absorption member (absorption pad) (4) prepared. After adhering these members to the base material (5), they were cut to a width of 5 mm to prepare an immunochromatographic device (immunochromatographic kit) having the configuration shown in FIG. An immunochromatographic kit having such a configuration was separately prepared for the first assay and the second assay for each of the following detection methods. Hereinafter, the immunochromatographic kit for the first assay is abbreviated as "immunochromatographic kit 1" and the immunochromatographic kit for the second assay is abbreviated as "immunochromatographic kit 2".

(4) Preparation of mock sample:
Next, a solution of Staphylococcus aureus and a solution of Staphylococcus aureus ribosomal protein L7/L12 were prepared as simulants for the detection test. BD BBL Trypticase Soy Broth (Becton Dickinson) was used as the liquid medium.

Staphylococcus aureus was purchased from ATCC. Viable Staphylococcus aureus bacteria were spiked into the liquid medium, cultured with shaking at 37°C for 16 hours, and the bacterial solution adjusted to have a turbidity (OD = 600) of 2 was diluted 1/1000 in the liquid medium. and used as a suspension of Staphylococcus aureus. This Staphylococcus aureus bacterium solution is used as a specimen simulating urinary bacteria present as viable bacteria in urine (and urinary bacterium-derived antigens released therefrom).

On the other hand, the Staphylococcus aureus ribosomal protein L7/L12 prepared in (1) above was diluted with a liquid medium to 1 ng/mL or 3 ng/mL and used as a Staphylococcus aureus L7/L12 solution. This L7/L12 liquid of Staphylococcus aureus is used as a sample that mimics blood bacterium-derived antigen excreted from the blood into the urine through the glomerulus.

These simulated specimens were subjected to the first assay (assay for detecting both blood bacterium-derived antigens and urinary bacterium-derived antigens from urine specimens based on antigen-antibody reaction) and second assay (blood from urine specimens). Assay for detecting antigens derived from bacteria based on antigen-antibody reaction). There is a clear difference in the detection results of the bacterial fluid by both assays (that is, the amount of bacterial antigens detected by the second assay is significantly smaller than the amount of bacterial antigens detected by the first assay, or or not detected), and there is little or no difference in the detection results of the L7/L12 fluid (that is, the amount of bacterial antigen detected by the first assay and the amount of bacterial antigen detected by the second assay are substantially or completely the same), it is shown that it is possible to detect blood bacteria from urine specimens by comparing the detection results of these first and second assays. will be

(5) Detection method A-Aspect using sonication:
In this example, a first assay in which the specimen was subjected to sonication and then used, and a second assay in which the specimen was not sonicated and used as it was were performed, and their detection results were compared. verified the detectability of blood bacteria from urine specimens.

In the procedure below, a mixed solution of 0.1 M Tris-HCl (pH 7.5), 1% Tween 20, 0.3% NaCl, and 0.1% casein Na was used as a mixed reagent. This mixed reagent was in a liquid state and used by mixing with each sample before assay. As the L7/L12 solution of Staphylococcus aureus, 1 ng/mL was used.

The first and second assays were performed according to the following procedures.
1. The bacterial solution and the L7/L12 solution were each mixed with the above-described mixed reagent (volume ratio of bacterial solution or L7/L12 solution:mixed reagent: 30:70) to prepare a mixed solution.
2. A 200 μL pipette was used to collect each of the bacterial mixture and the L7/L12 mixture, and sonicated for 2 minutes with an ultrasonicator (Bioruptor UCD-200™).
3. The sonicated bacterial solution and L7/L12 solution were added dropwise to immunochromatographic kit 1 (first assay: 115 μL, N=3).
4. A 200 μL pipette was taken from each of the bacterial mixture and the L7/L12 mixture and added dropwise (without sonication) to Immunochromatography Kit 2 (second assay: 115 μL, N=3).
5. The signal intensity of Immunochromatography Kit 1 after 15 minutes of dropping was measured with a reader (first assay: N=3, and the average value was graphed).
6. The signal intensity of Immunochromatography Kit 2 after 15 minutes of dropping was measured with a reader (second assay: N=3, and the average value was graphed).

The results of detection of the bacterial liquid and L7/L12 liquid by the first and second assays are shown in the graph of FIG. As is clear from the graph, in the detection result of the bacterial fluid, the amount of L7/L12 (bacteria-derived antigen) detected by the second assay was higher than the amount of L7/L12 (bacteria-derived antigen) detected by the first assay. The amount was remarkably low, resulting in a clear difference in detection results by both assays. This is because, in the first assay, in which the bacterial solution was subjected to ultrasonic treatment, a large amount of L7/L12 released by destruction of the bacterial cells was detected, whereas in the first assay, the bacterial solution was used as it was. 2 assay, L7/L12 in bacterial cells was not released and was not detected. On the other hand, in the detection results of the L7/L12 solution, the amount of bacterial antigen detected by the first assay and the amount of bacterial antigen detected by the second assay were almost the same, and there was almost no difference between the two assays. rice field. Therefore, it was shown that blood bacteria can be detected from a urine sample even by the detection method of this embodiment using ultrasonication.

(6) Detection method B-Aspect using filter processing:
In this example, a first assay using the sample as it is and a second assay using a filtered sample were performed, and the detection results were compared to determine the number of blood bacteria from the urine sample. The detectability was verified.

In the following procedure, as a mixed reagent, 0.1M Tris-HCl (pH 7.5), 5% Tween 20, 0.3% NaCl, 0.1% casein Na, 2% DDAO (N,N- A mixture of dimethyldodecylamine N-oxide) and 10 μg/mL lysostaphin was used. This mixed reagent was soaked into each sample addition member (sample pad) (3) of the immunochromatographic kits 1 and 2 and dried before use. As the L7/L12 solution of Staphylococcus aureus, 3 ng/mL was used.

The first and second assays were performed according to the following procedures.
1. The bacterial solution and the L7/L12 solution were each added dropwise to Immunochromatography Kit 1 (115 μL, N=3).
2. Each of the bacterial solution and L7/L12 solution was filtered through a sterilizing filter (Merck Millipore syringe filter, mesh size 0.45 μm), and then added dropwise to Immunochromatography Kit 2 (115 μL, N=3).
3. The signal intensity of Immunochromatography Kit 1 after 15 minutes of dropping was measured with a reader (measured at N=3 and the average value was graphed).
4. The signal intensity of Immunochromatography Kit 2 after 15 minutes of dropping was measured with a reader (measured at N=3 and the average value was graphed).

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. As is clear from the graph, in the detection result of the bacterial fluid, the amount of L7/L12 (bacteria-derived antigen) detected by the second assay was higher than the amount of L7/L12 (bacteria-derived antigen) detected by the first assay. The amount was remarkably low, resulting in a clear difference in detection results by both assays. This is because, in the first assay using the bacterial solution as it is, a large amount of L7/L12 released by lysing the bacterial cells with the mixed reagent was detected, whereas the bacterial solution was filtered with a sterile filter and then It is believed that the second assay used did not detect L7/L12 that was present in the bacterial cells as a result of removal of the bacterial cells. On the other hand, in the detection results of the L7/L12 solution, the amount of bacterial antigen detected by the first assay and the amount of bacterial antigen detected by the second assay were almost the same, and there was almost no difference between the two assays. rice field. Therefore, it was shown that blood bacteria can be detected from a urine sample even by the detection method of this embodiment using filtering.

(7) Detection method C-Aspect 1 using lytic agent treatment:
In this example, a first assay using a specimen treated with a bacteriolytic agent and a second assay using the specimen as it is were performed, and the detection results were compared to determine the presence of The detectability of blood bacteria was verified.

In the following procedure, mixed reagent 1 for the first assay includes 0.1 M Tris-HCl (pH 7.5), 1% Tween 20, 0.3% NaCl, 0.1% casein Na, and A mixture of 10 μg/mL lysostaphin was used and mixture reagent 2 for the second assay was 0.1 M Tris-HCl (pH 7.5), 1% Tween 20, 0.3% NaCl, and 0.1% A mixture of casein Na was used. Both of these mixed reagents 1 and 2 were in a liquid state and used by mixing with each specimen before the first and second assays, respectively. As the L7/L12 solution of Staphylococcus aureus, 1 ng/mL was used.

The first and second assays were performed according to the following procedure.
1. Each of the bacterial solution and the L7/L12 solution was mixed with the mixed reagent 1 and added dropwise to the immunochromatographic kit 1 (115 μL, N=3).
2. The bacterial solution and L7/L12 solution were each mixed with mixed reagent 2 and added dropwise to immunochromatographic kit 2 (115 μL, N=3).
3. The signal intensity of Immunochromatography Kit 1 after 15 minutes of dropping was measured with a reader (measured at N=3 and the average value was graphed).
4. The signal intensity of Immunochromatography Kit 2 after 15 minutes of dropping was measured with a reader (measured at N=3 and the average value was graphed).

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. As is clear from the graph, in the detection result of the bacterial fluid, the amount of L7/L12 (bacteria-derived antigen) detected by the second assay was higher than the amount of L7/L12 (bacteria-derived antigen) detected by the first assay. The amount was remarkably low, resulting in a clear difference in detection results by both assays. This is because in the first assay, in which the bacterial solution was mixed with mixed reagent 1 containing a lytic agent (lysostaphin), a large amount of L7/L12 released by lysing bacterial cells with the mixed reagent was detected. In contrast, in the second assay, in which the bacterial solution was mixed with mixed reagent 2 containing no lysing agent, L7/L12 in bacterial cells was not released and detected. On the other hand, in the detection results of the L7/L12 solution, the amount of bacterial antigen detected by the first assay and the amount of bacterial antigen detected by the second assay were almost the same, and there was almost no difference between the two assays. rice field. Therefore, it was shown that blood bacteria can be detected from a urine sample even by the detection method of this embodiment using bacteriolytic agent treatment.

(8) Detection method D-Aspect 2 using lytic agent treatment:
In this embodiment, as a modification of the detection method C, a first assay is performed in which detection is performed while the specimen is treated with a lytic agent, and a second assay is performed in which detection is performed without subjecting the specimen to lytic agent treatment. and compared their detection results to verify the detectability of blood bacteria from urine specimens.

In the following procedure, mixed reagent 1 for the first assay is 0.1 M Tris-HCl (pH 7.5), 5% Tween 20, 0.3% NaCl, 0.1% casein Na/2 % DDAO and 10 μg/mL lysostaphin, and mixed reagent 2 for the second assay was 0.1 M Tris-HCl (pH 7.5), 5% Tween 20, 0.3% NaCl, 0 A mixture of 1% casein Na and 2% DDAO was used. These mixed reagents 1 and 2 were soaked in each sample addition member (sample pad) (3) of the immunochromatographic kits 1 and 2, respectively, and dried before use. As the L7/L12 solution of Staphylococcus aureus, 3 ng/mL was used.

The first and second assays were performed according to the following procedures.
1. The concentration-adjusted bacterial solution and L7/L12 solution were added dropwise to immunochromatographic kit 1 (115 μL, N=3).
2. The concentration-adjusted bacterial solution and L7/L12 solution were added dropwise to immunochromatographic kit 2 (115 μL, N=3).
3. The signal intensity of Immunochromatography Kit 1 after 15 minutes of dropping was measured with a reader (measured at N=3 and the average value was graphed).
4. The signal intensity of Immunochromatography Kit 2 after 15 minutes of dropping was measured with a reader (measured at N=3 and the average value was graphed).

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. As is clear from the graph, in the detection result of the bacterial fluid, the amount of L7/L12 (bacteria-derived antigen) detected by the second assay was higher than the amount of L7/L12 (bacteria-derived antigen) detected by the first assay. The amount was remarkably low, resulting in a clear difference in detection results by both assays. This is because in the first assay in which the bacterial solution was brought into contact with the mixed reagent 1 containing the lytic agent (lysostaphin) in the sample addition member (sample pad) (3) of the immunochromatographic kit 1, bacterial cells were lysed by the mixed reagent. While a large amount of L7/L12 released in the immunochromatographic kit 2 was detected, the bacterial solution was brought into contact with the mixed reagent 2 containing no lysing agent (lysostaphin) in the sample addition member (sample pad) (3) of the immunochromatographic kit 2. This may be due to the fact that L7/L12 in bacterial cells was not released and detected in the second assay. On the other hand, in the detection results of the L7/L12 solution, the amount of bacterial antigen detected by the first assay and the amount of bacterial antigen detected by the second assay were almost the same, and there was almost no difference between the two assays. rice field. Therefore, it was shown that blood bacteria can be detected from a urine sample even by the detection method of this embodiment using bacteriolytic agent treatment.

[Example II. Detection of Pseudomonas aeruginosa]
In this example, Pseudomonas aeruginosa, which is a Gram-negative bacterium, was used as a detection target, and its detection was attempted using a simulated sample.

(1) Acquisition of Pseudomonas aeruginosa L7/L12:
An expression vector into which a nucleic acid sequence (SEQ ID NO: 4) encoding the amino acid sequence (SEQ ID NO: 3) of the ribosomal protein L7/L12 of Pseudomonas aeruginosa is cloned is prepared with reference to the method described in WO 2000/06603. E. coli was transformed with the prepared expression vector, the resulting transformant was cultured using LB medium or the like, and the full-length Pseudomonas aeruginosa L7/L12 protein expressed by the transformant was , was purified as a fusion protein using the tag sequence derived from the expression vector by an affinity column.

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

Specifically, using the full-length L7/L12 protein of Pseudomonas aeruginosa as an immunogen, the concentration of the immunogen was adjusted to 0.4 mg/mL with PBS according to the standard procedure for obtaining hybridomas, and Freund's adjuvant was added. Mice were immunized 4 times with the same amount added, and the immunogen amount was 50 μg/time. After confirming an increase in the serum antibody titer by test blood collection, the spleen cells of the mice were excised. The excised mouse spleen cells were fused with myeloma cells to obtain various hybridomas. The obtained various hybridomas were cultured in HAT medium and screened using antibodies in the culture supernatant. Screening was performed by ELISA, bacterial lysate of Pseudomonas aeruginosa (PA) and several other bacteria (E. coli (EC), Staphylococcus aureus (SA), Streptococcus pneumoniae (SP), Klebsiella pneumoniae (KP) ), Chlamydia pneumoniae (CP), Haemophilus influenzae (HI), and Serratia (SM)), and show reactivity only with bacterial lysate of Pseudomonas aeruginosa (PA). Hybridomas producing specific antibodies and hybridomas producing general antibodies reactive with bacterial lysates of Pseudomonas aeruginosa (PA) and several other bacteria were selected and monoclonal antibodies were prepared. The detailed procedure is as described in Example I(2) above.

Among the general-purpose monoclonal antibodies 44B2, 47A1, 51B2, 78A2, and 84B2 and the specific monoclonal antibodies 6C2, 14B2, 72A2, 73B2, and 85B2 against L7/L12 of Pseudomonas aeruginosa obtained by the above procedure, Using the specific monoclonal antibody 72A2 as the detection antibody and the general-purpose monoclonal antibody 51B2 as the capture antibody, the following immunochromatographic kit was prepared.

(3) Preparation of immunochromatographic kit using detection antibody and capture antibody:
An immunochromatography kit was prepared in the same manner as in Example I(3) using the detection antibody and capture antibody for L7/L12 of Pseudomonas aeruginosa obtained by the above procedure.

(4) Preparation of mock sample:
Next, as simulants for the detection test, a solution of Pseudomonas aeruginosa and a solution of Pseudomonas aeruginosa ribosomal protein L7/L12 were prepared. BD BBL Trypticase Soy Broth (Becton Dickinson) was used as the liquid medium.

Pseudomonas aeruginosa was purchased from ATCC. Viable Pseudomonas aeruginosa was spiked into and cultured with shaking at 37° C. for 16 hours. It was used as a bacterial solution of Pseudomonas aeruginosa. This Pseudomonas aeruginosa liquid is used as a specimen simulating urinary bacteria present as viable bacteria in urine (and urinary bacterium-derived antigens released therefrom).

On the other hand, the Pseudomonas aeruginosa ribosomal protein L7/L12 prepared in (1) above was diluted with a liquid medium to 5 ng/mL or 10 ng/mL and used as a Pseudomonas aeruginosa L7/L12 solution. This L7/L12 fluid of Pseudomonas aeruginosa is used as a sample that mimics blood bacterium-derived antigen excreted from the blood into the urine through the glomerulus.

These simulated specimens were subjected to the first assay (assay for detecting both blood bacterium-derived antigens and urinary bacterium-derived antigens from urine specimens based on antigen-antibody reaction) and second assay (blood from urine specimens). Assay for detecting antigens derived from bacteria based on antigen-antibody reaction). There is a clear difference in the detection results of the bacterial fluid by both assays (that is, the amount of bacterial antigens detected by the second assay is significantly smaller than the amount of bacterial antigens detected by the first assay, or or not detected), and there is little or no difference in the detection results of the L7/L12 fluid (that is, the amount of bacterial antigen detected by the first assay and the amount of bacterial antigen detected by the second assay are substantially or completely the same), it is shown that it is possible to detect blood bacteria from urine specimens by comparing the detection results of these first and second assays. will be

(5) Detection method A-Aspect using sonication:
In this example, a first assay in which the specimen was subjected to sonication and then used, and a second assay in which the specimen was not sonicated and used as it was were performed, and their detection results were compared. verified the detectability of blood bacteria from urine specimens.

In the following procedures, a mixed solution of 0.1 M Tris-HCl (pH 7.5), 0.25% DDAO, and 0.5% NaCl was used as a mixed reagent. This mixed reagent was in a liquid state and used by mixing with each sample before assay. As the Pseudomonas aeruginosa L7/L12 solution, 5 ng/mL was used.

The first and second assays were performed according to the following procedures.
1. The bacterial solution and the L7/L12 solution were each mixed with the above-described mixed reagent (volume ratio of bacterial solution or L7/L12 solution:mixed reagent: 30:70) to prepare a mixed solution.
2. A 200 μL pipette was used to collect each of the bacterial mixture and the L7/L12 mixture, and sonicated for 1 minute using an ultrasonicator (Bioruptor UCD-200™).
3. The sonicated bacterial solution and L7/L12 solution were added dropwise to immunochromatographic kit 1 (first assay: 115 μL, N=3).
4. A 200 μL pipette was taken from each of the bacterial mixture and the L7/L12 mixture and added dropwise (without sonication) to Immunochromatography Kit 2 (second assay: 115 μL, N=3).
5. The signal intensity of Immunochromatography Kit 1 after 15 minutes of dropping was measured with a reader (first assay: N=3, and the average value was graphed).
6. The signal intensity of Immunochromatography Kit 2 after 15 minutes of dropping was measured with a reader (second assay: N=3, and the average value was graphed).

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. As is clear from the graph, in the detection result of the bacterial fluid, the amount of L7/L12 (bacteria-derived antigen) detected by the second assay was higher than the amount of L7/L12 (bacteria-derived antigen) detected by the first assay. The amount was remarkably low, resulting in a clear difference in detection results by both assays. This is because, in the first assay, in which the bacterial solution was subjected to ultrasonic treatment, a large amount of L7/L12 released by destruction of the bacterial cells was detected, whereas in the first assay, the bacterial solution was used as it was. 2 assay, L7/L12 in bacterial cells was not released and was not detected. On the other hand, in the detection results of the L7/L12 solution, the amount of bacterial antigen detected by the first assay and the amount of bacterial antigen detected by the second assay were almost the same, and there was almost no difference between the two assays. rice field. Therefore, it was shown that blood bacteria can be detected from a urine sample even by the detection method of this embodiment using ultrasonication.

(6) Detection method B-Aspect using filter processing:
In this example, a first assay using the sample as it is and a second assay using a filtered sample were performed, and the detection results were compared to determine the number of blood bacteria from the urine sample. The detectability was verified.

In the procedure below, a mixed solution of 0.1 M Tris-HCl (pH 7.5), 5.5% DDAO, and 0.5% NaCl was used as a mixed reagent. This mixed reagent was soaked into each sample addition member (sample pad) (3) of the immunochromatographic kits 1 and 2 and dried before use. As the Pseudomonas aeruginosa L7/L12 solution, 5 ng/mL was used.

The first and second assays were performed according to the following procedure.
1. The bacterial solution and the L7/L12 solution were each added dropwise to Immunochromatography Kit 1 (115 μL, N=3).
2. Each of the bacterial solution and the L7/L12 solution was filtered through a sterilizing filter (Merck Millipore syringe filter, mesh size 0.45 μm), and then added dropwise to Immunochromatography Kit 2 (115 μL, N=3).
3. The signal intensity of Immunochromatography Kit 1 after 15 minutes of dropping was measured with a reader (measured at N=3 and the average value was graphed).
4. The signal intensity of Immunochromatography Kit 2 after 15 minutes of dropping was measured with a reader (measured at N=3 and the average value was graphed).

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. As is clear from the graph, in the detection result of the bacterial fluid, the amount of L7/L12 (bacteria-derived antigen) detected by the second assay was higher than the amount of L7/L12 (bacteria-derived antigen) detected by the first assay. The amount was remarkably low, resulting in a clear difference in detection results by both assays. This is because, in the first assay using the bacterial solution as it is, a large amount of L7/L12 released by lysing the bacterial cells with the mixed reagent was detected, whereas the bacterial solution was filtered with a sterile filter and then It is believed that the second assay used did not detect L7/L12 that was present in the bacterial cells as a result of removal of the bacterial cells. On the other hand, in the detection results of the L7/L12 solution, the amount of bacterial antigen detected by the first assay and the amount of bacterial antigen detected by the second assay were almost the same, and there was almost no difference between the two assays. rice field. Therefore, it was shown that blood bacteria can be detected from a urine sample even by the detection method of this embodiment using filtering.

(7) Detection method C-Aspect 1 using lytic agent treatment:
In this example, a first assay using a specimen treated with a bacteriolytic agent and a second assay using the specimen as it is were performed, and the detection results were compared to determine the presence of The detectability of blood bacteria was verified.

In the following procedures, a mixed solution of 0.1 M Tris-HCl (pH 7.5), 0.25% DDAO, and 0.5% NaCl was used as a mixed reagent. This mixed reagent was in a liquid state and used by being mixed with each specimen. In addition, 1% (v/v) DDAO was used as a lysing agent. As the L7/L12 solution of Pseudomonas aeruginosa, 10 ng/mL was used.

The first and second assays were performed according to the following procedure.
1. After each of the bacterial solution and L7/L12 solution was mixed with 1% (v/v) DDAO, it was 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 the mixed reagent and added dropwise to Immunochromatography Kit 2 (115 μL, N=3).
3. The signal intensity of Immunochromatography Kit 1 after 15 minutes of dropping was measured with a reader (measured at N=3 and the average value was graphed).
4. The signal intensity of Immunochromatography Kit 2 after 15 minutes of dropping was measured with a reader (measured at N=3 and the average value was graphed).

The graph in FIG. 8 shows the detection results of the bacterial liquid and the L7/L12 liquid by the first and second assays. As is clear from the graph, in the detection result of the bacterial fluid, the amount of L7/L12 (bacteria-derived antigen) detected by the second assay was higher than the amount of L7/L12 (bacteria-derived antigen) detected by the first assay. The amount was remarkably low, resulting in a clear difference in detection results by both assays. This is because in the first assay in which the bacterial solution was lysed with a bacteriolytic agent (DDAO) and then mixed with the mixed reagent 1, a large amount of L7/L12 released by lysing the bacterial cells with the mixed reagent was detected. In contrast, in the second assay in which the bacterial solution was mixed with mixed reagent 2 without being lysed with a bacteriolytic agent, L7/L12 in bacterial cells was not released and was not detected. Conceivable. On the other hand, in the detection results of the L7/L12 solution, the amount of bacterial antigen detected by the first assay and the amount of bacterial antigen detected by the second assay were almost the same, and there was almost no difference between the two assays. rice field. Therefore, it was shown that blood bacteria can be detected from a urine sample even by the detection method of this embodiment using bacteriolytic agent treatment.

INDUSTRIAL APPLICABILITY The present invention can be widely applied to medical fields and the like where detection of bacteria in blood and urine is required, and its utility value is extremely high.

10 Detection mechanism 1 Insoluble membrane carrier for chromatographic development 2 Antibody-impregnated member for detection (conjugate pad)
3 Specimen addition member (sample pad)
4 Absorbent member (absorbent pad)
5 substrate 6 capture antibody immobilization site 7 control reagent immobilization site A sample B sample flow

Claims (12)

A method for detecting bacteria in the blood and/or urine of a subject based on a urine specimen of the subject, comprising:
(a) a step of performing a first assay for detecting both bacterial-derived antigens present inside bacterial cells and bacterial-derived antigens present outside bacterial cells in a urine specimen based on antigen-antibody reaction;
(b) performing a second assay for detecting a bacterial antigen present outside bacterial cells in a urine sample based on an antigen-antibody reaction;
(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 based on the comparison result, urinary bacteria and/or blood bacteria A method comprising the step of detecting the presence or abundance of 2. The method of claim 1, wherein the bacterial antigen is the bacterial ribosomal protein L7/L12. 3. The method according to claim 1 or 2, wherein in step (a), the first assay is carried out after exposing the bacteria-derived antigen present in the bacterial cells in the urine sample to the outside of the bacterial cells. 4. The method according to claim 3, wherein, in step (a), the urinary specimen is subjected to ultrasonic treatment to expose bacteria-derived antigens present in bacterial cells in the urine specimen to the outside of bacterial cells. 4. The method according to claim 3, wherein, in step (a), a bacterium-derived antigen present in bacterial cells in the urine specimen is exposed to the outside of bacterial cells by treating the urine specimen with a bacteriolytic agent. The method according to any one of claims 1 to 5, wherein in step (b), the bacterial cells in the urine specimen are removed before performing the second assay. 7. The method of claim 6, wherein in step (b), urinary bacteria in the urine specimen are removed by filtering the urine specimen. Bacteria to be detected include the genus Escherichia, the genus Staphylococcus, the genus Klebsiella, the genus Proteus, the genus Enterococcus, the genus Citrobacter, and the genus Pseudomonas , and one or more genera of bacteria selected from the genus Serratia. The first assay of step (a) and the second assay of step (b) are
By contacting a urine sample with a capturing antibody for capturing the bacterial-derived antigen and a detecting antibody for labeling the bacterial-derived antigen, the bacterial-derived antigen in the urine sample is captured, labeled, and captured. 9. The method according to any one of claims 1 to 8, comprising detecting the bacterial antigen in the collected urine specimen based on the detectable label. A kit for detecting bacteria in the blood and/or urine of a subject based on a urine sample from the subject by performing the method of claim 9, comprising:
a capturing antibody for capturing a bacterial antigen in a urine sample;
and a detection antibody for labeling the bacterial antigen. further comprising a carrier for developing the analyte and contacting the analyte with the detection antibody;
A first assay area for performing a first assay and a second assay area for performing a second assay are provided on the carrier, each of the first and second assay areas comprising: 11. The kit of Claim 10, wherein the capture antibody is immobilized. 12. The kit of claim 11, wherein only the first assay area is coated with a lysing agent.

Images