JP7626621B2 - Test method and test kit for determining whether an infectious disease suffered by a subject is of bacterial origin - Google Patents

Description

The present invention relates to a test method, a reagent, and a kit for determining whether an infectious disease suffered by a subject is of bacterial origin.

Conventionally, when a subject such as a human or animal suffers from an infectious disease, methods for determining whether the infectious disease is caused by bacteria include a culture method in which a specimen from the subject is cultured and bacterial growth is confirmed (e.g., Patent Document 1: JP 2005-141531 A), and a PCR method in which bacterial genes are detected in a specimen from the subject (e.g., Patent Document 2: JP 2001-204470 A).

However, conventional culture methods require culture equipment and can take anywhere from several days to a week until detection, posing problems in terms of testing location, effort, and time. Another problem is that only specific bacteria that match the culture conditions (medium components, presence or absence of air, temperature, etc.) grow, making it impossible to simultaneously detect a wide range of bacterial genera. Furthermore, conventional PCR methods require time until detection, and also have problems with cumbersome pre-processing and the need for specialized equipment.

A rapid antigen test method (e.g., Patent Document 3: US Pat. No. 6,824,997) has also been developed as a more rapid and simple method, using an antibody that reacts with polysaccharides coordinated to the cell wall of bacteria in a sample as an antigen. The present inventors have focused on ribosomal protein L7/L12 as an intracellular molecule that exists in all microbial cells and whose amino acid structure differs to some extent between microorganisms, and have found a method that can detect various microorganisms specifically and comprehensively detect various serotypes within the same bacterial species by using antibodies against such proteins (Patent Document 4: International Publication No. 2000/006603). In addition, for Streptococcus pneumoniae in particular, they have obtained antibodies that can detect it in distinction from bacteria of other genera, and have proposed a detection method using such antibodies (Patent Document 5: Japanese Patent Publication No. 5204036).

However, all of these conventional antibodies for detecting bacteria detect a specific type of bacteria (or multiple serotypes within the same species) as an antigen, and are not capable of simultaneously detecting bacteria across multiple different genera. For this reason, in order to simultaneously detect bacteria from multiple genera, it is necessary to produce multiple types of antibodies targeting individual bacteria and combine these for detection, which poses the problem of being costly and time-consuming.

In particular, in the initial stage of treatment for infectious diseases, the appropriate treatment varies depending on the broad classification of the source of infection - antibiotics for bacterial infections and antivirals for viral infections - but there was no method that could quickly and easily distinguish the broad classification of the source of infection in the earliest stages of infection.

JP 2005-141531 A JP 2001-204470 A U.S. Pat. No. 6,824,997 International Publication No. 2000/006603 Patent No. 5204036

One aspect of the present invention aims to provide a means for quickly and easily determining whether an infectious disease suffered by a subject is of bacterial origin.

After extensive research, the inventors came up with a method for simultaneously detecting the presence and/or amount of at least two or more genera of bacteria in a sample derived from a subject based on an antigen-antibody reaction, and determining that the infection is bacterial if the detection result is positive. They then discovered that this method makes it possible to determine whether an infection is bacterial or not more quickly and easily than conventional methods, and thus arrived at the present invention.

That is, the gist of the present invention is as follows.
[Item 1] A test method for determining whether an infectious disease suffered by a subject is caused by bacteria, comprising:
Providing a sample from the subject;
and simultaneously detecting the presence or absence and/or abundance of at least two or more genera of bacteria in the sample based on an antigen-antibody reaction;
If the detection result is positive, it is determined to be an infectious disease caused by bacteria,
When the detection result is negative, the infection is determined to be of a cause other than bacteria.
[Item 2] The method according to Item 1, wherein in the detection step, bacteria of two or more genera selected from the group consisting of the genera Streptococcus, Haemophilus, Mycoplasma, Legionella, Bordetella, Chlamydia, Staphylococcus, Pseudomonas, Klebsiella, Neisseria, Moraxella, Escherichia, Salmonella, Listeria, and Campylobacter are simultaneously detected.
[Item 3] The method according to Item 1 or 2, wherein the two or more genera of bacteria to be detected include both gram-negative bacteria and gram-positive bacteria.
[Item 4] The method according to any one of Items 1 to 3, wherein the bacterial infectious disease to be identified is an infectious disease selected from the group consisting of cold syndrome, pneumonia, bronchitis, pharyngitis, conjunctivitis, meningitis, otitis media, sinusitis, urethritis, sexually transmitted diseases, urinary tract infections, cystitis, pyelonephritis, sepsis, bacteremia, diarrhea, food poisoning, and gastroenteritis.
[Item 5] The method according to any one of Items 1 to 4, wherein the subject-derived sample is one or more samples selected from a pharyngeal swab, a nasopharyngeal swab, a nasal discharge, a nasal aspirate, sputum, blood, urine, cerebrospinal fluid, a conjunctival swab, a middle ear fluid, urine, a uterine scraping, and a stool.
[Item 6] The method according to any one of Items 1 to 5, wherein the step of simultaneously detecting the presence or absence and/or abundance of bacteria of at least two or more genera is performed by a method that does not involve culturing bacteria.
[Item 7] The step of simultaneously detecting the presence and/or abundance of bacteria of at least two or more genera,
contacting the subject-derived specimen with an antibody or a fragment thereof, or a derivative thereof, which generates an antigen-antibody reaction with the at least two or more genera of bacteria;
Item 7. The method according to any one of Items 1 to 6, further comprising detecting the presence or absence and/or intensity of an antigen-antibody reaction between the at least two or more genera of bacteria that may be present in the sample and the antibody or a fragment thereof, or a derivative thereof.
[Item 8] The method according to Item 7, wherein the antibody is an antibody that generates an antigen-antibody reaction with a ribosomal protein of bacteria of the two or more genera.
[Item 9] The method according to Item 8, wherein the ribosomal protein is L7/L12.
[Item 10] The method according to any one of Items 7 to 9, wherein the antibody is a monoclonal antibody or a fragment thereof, or a derivative thereof.
[Item 11] The monoclonal antibody or a fragment thereof, or a derivative thereof,
As a heavy chain variable region sequence, an amino acid sequence having 80% or more homology with at least one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5; and
Item 11. The method according to Item 10, wherein the light chain variable region sequence comprises an amino acid sequence having 80% or more homology to at least one of the amino acid sequences selected from SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6.
[Item 12] The method according to any one of items 7 to 11, wherein the method comprises:
(I) a step of capturing and labeling bacteria in the specimen by an antigen-antibody reaction between the specimen, a capture antibody linked to a solid phase carrier, and a detection antibody having a detection label; and (II) a step of detecting the presence or absence and/or amount of the target bacteria in the specimen by detecting the target bacteria in the specimen based on the detection label,
One of the capture antibody and the detection antibody is one or more specific antibodies that undergo an antigen-antibody reaction with one or more types of target bacteria, and the other antibody is one or more general antibodies that undergo an antigen-antibody reaction with five or more genera of bacteria including the target bacteria, and
A method in which either the general antibody or the specific antibody is the antibody according to any one of items 7 to 11.
[Section 13] The step (I) comprises:
(Ia-1) contacting a specimen with a detection antibody and labeling the bacteria in the specimen through an antigen-antibody reaction between the detection antibody and the bacteria; and
(Ia-2) The method according to Item 12, comprising the step of contacting a specimen containing bacteria labeled with a detection antibody with a capture antibody, and capturing the bacteria in the specimen by an antigen-antibody reaction between the capture antibody and a bacteria-detection antibody complex.
[Section 14] The step (I) comprises:
(Ib-1) contacting a specimen with a capture antibody and capturing bacteria in the specimen through an antigen-antibody reaction between the capture antibody and the bacteria; and
(Ib-2) The method according to Item 12, comprising the step of contacting a specimen containing the bacteria captured by the capture antibody with a detection antibody, and labeling the bacteria in the specimen by an antigen-antibody reaction between the detection antibody and the bacteria-capture antibody complex.
[Item 15] The method according to any one of Items 12 to 14, wherein the capture antibody is a general antibody and the detection antibody is a specific antibody.
[Item 16] The method according to any one of Items 12 to 14, wherein the detection antibody is a general antibody and the capture antibody is a specific antibody.
[Item 17] A test kit for determining whether an infectious disease suffered by a subject is of bacterial origin or not by the method according to any one of Items 7 to 11, the kit comprising the antibody according to any one of Items 7 to 11.
[Item 18] A test kit for determining whether or not an infectious disease suffered by a subject is of bacterial origin using the method according to any one of Items 12 to 16, the kit comprising the general antibody and the specific antibody, wherein either the general antibody or the specific antibody is the antibody according to any one of Items 7 to 11.

The present invention makes it possible to quickly and easily determine whether an infectious disease suffered by a subject is of bacterial origin.

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.

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 implemented in any form without departing from the spirit of the present invention.

All documents cited in this specification, including patent publications, patent application publications, and non-patent publications, are hereby incorporated by reference in their entirety for all purposes.

In addition, in the formulas representing amino acid sequences described in this specification, amino acids are represented by single-letter codes unless otherwise specified.

One aspect of the present invention relates to a test method for determining whether an infectious disease suffered by a subject is of bacterial origin (hereinafter referred to as the "test method of the present invention" or simply the "method of the present invention" as appropriate). The method of the present invention includes a step of providing a specimen derived from the subject, and a step of simultaneously detecting the presence or absence and/or the amount of at least two or more genera of bacteria in the specimen based on an antigen-antibody reaction, and if the detection result is positive, the infectious disease is determined to be of bacterial origin, and if the detection result is negative, the infectious disease is determined to be of non-bacterial origin. According to one aspect, the method of the present invention preferably simultaneously detects the multiple genera of bacteria by contacting a specimen derived from a subject with an antibody (hereinafter referred to as the "antibody of the present invention") that causes an antigen-antibody reaction with the at least two or more genera of bacteria, and detecting the presence or absence and/or strength of an antigen-antibody reaction between the at least two or more genera of bacteria that may be present in the specimen and the antibody of the present invention. In addition, a kit containing the antibody of the present invention (hereinafter referred to as the "kit of the present invention") for carrying out such a test method of the present invention is also a subject of the present invention.

Below, we will first explain the method of the present invention, then explain the antibody of the present invention used in the method of the present invention, and then explain the kit used in the method of the present invention.

[1. Testing method for determining whether an infectious disease is of bacterial origin]
The method of the present invention is a testing method for determining whether an infectious disease suffered by a subject is of bacterial origin or not. The method of the present invention includes a step of providing a specimen derived from the subject, and a step of simultaneously detecting the presence or absence and/or the amount of bacteria of at least two or more genera in the specimen based on an antigen-antibody reaction. If the detection result is positive, the infectious disease is determined to be of bacterial origin, and if the detection result is negative, the infectious disease is determined to be of a non-bacterial origin.

As mentioned above, among the conventional methods for determining whether an infectious disease suffered by such a subject is of bacterial origin, the culture method requires culture equipment and cumbersome culture work, takes several days to a week until detection, and can only detect specific bacteria that meet the culture conditions. The PCR method has the problem that it takes time until detection, requires cumbersome pre-processing, and requires dedicated equipment. In addition, conventional antibody-based testing methods use antibodies that have specific bacteria as antigens, so it is necessary to select an appropriate antibody according to the expected type of infectious disease and perform the test, and are not effective in the early stages of infection when determining whether or not the infection is bacterial. In particular, in the early stages of treatment for infectious diseases, the appropriate treatment varies depending on the major classification of the source of infection, such as antibiotics for bacterial infections, antiviral drugs for viral infections, antifungal drugs for fungal infections, and antiparasitic drugs for parasitic infections, but there was no method that could quickly and easily determine the major classification of the source of infection in the earliest stages of infection.

On the other hand, in the method of the present invention, the presence or absence and/or the amount of bacteria of multiple genera in a sample are simultaneously detected based on an antigen-antibody reaction. In the present invention, "antigen-antibody reaction" refers to an antibody specifically binding to its antigen. By using an antigen-antibody reaction, it is possible to detect the presence or absence and/or the amount of bacteria in a sample immediately on-site with simple equipment (especially in the case of lateral flow, no equipment is required) and operation. In addition, by appropriately selecting and using an antibody (the antibody of the present invention) that specifically causes an antigen-antibody reaction with components derived from the multiple genera of bacteria to be detected according to the symptoms of infection, it is possible to simultaneously detect the multiple genera of bacteria to be detected, and it is possible to reduce false positives due to components other than bacteria and improve detection sensitivity and detection accuracy. As a result, it is possible to easily and efficiently determine in a short time whether the infectious disease suffered by the subject is derived from multiple genera of bacteria. In particular, by using the method of the present invention, it is possible to roughly determine whether the source of infection is bacterial or not at the initial treatment stage of an infectious disease, and if it is a bacterial infection, antibiotics can be prescribed, while if it is an infection of a non-bacterial origin, further testing can be performed, and an initial treatment policy can be quickly determined, such as prescribing an antiviral drug for a viral infection, an antifungal drug for a fungal infection, or an antiparasitic drug for a parasitic infection.

The subjects of the testing method of the present invention include, but are not limited to, humans and non-human animals. Non-human animals include, but are not limited to, any of a variety of animals, such as dogs, cats, rabbits, cows, horses, pigs, and chickens. However, in consideration of the usefulness, it is preferable to target humans.

The specimen used in the testing method of the present invention is not particularly limited in type, so long as it is a specimen collected from a subject (e.g., a human specimen when the subject is a human). Examples include blood, lymph, nasal discharge, saliva, tears, urine, stool, swabs of superficial mucosa (e.g., mucosa of the pharynx, ear cavity, nasal cavity, pharynx, vagina, conjunctiva, etc.), nasal aspirate, middle ear fluid, uterine scraping, sputum, cerebrospinal fluid, etc. These specimens vary depending on the infectious disease to be detected and the causative bacterium thereof. These specimens vary depending on the type of infectious disease to be tested for.

Bacterial infectious diseases that can be identified by the testing method of the present invention include cold syndrome, pneumonia, bronchitis, pharyngitis, conjunctivitis, meningitis, otitis media, sinusitis, urethritis, sexually transmitted diseases, urinary tract infections, cystitis, pyelonephritis, sepsis, bacteremia, diarrhea, food poisoning, and gastroenteritis. Two or more types of infectious diseases may be tested simultaneously.

The bacteria to be detected in the test method of the present invention are not particularly limited as long as they are bacteria that can infect a subject and cause an infectious disease. Examples of bacterial genera include bacteria belonging to the genera Streptococcus, Haemophilus, Mycoplasma, Legionella, Bordetella, Chlamydia, Staphylococcus, Pseudomonas, Klebsiella, Neisseria, Moraxella, Escherichia, Salmonella, Listeria, and Campylobacter. In the method of the present invention, it is preferable to detect antigen-antibody reactions with bacteria of at least two or more genera (multiple genera) of these specific bacterial genera, and it is particularly preferable to detect antigen-antibody reactions with bacteria of at least three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more genera.

Examples of bacteria of the genus Streptococcus include Streptococcus pneumoniae (S. pneumoniae, SP) and Streptococcus pyogenes (S. pyogenes, SPY). When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Streptococcus, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Streptococcus pneumoniae.

Examples of bacteria of the genus Haemophilus include Haemophilus influenzae (H. influenzae, HI) and Haemophilus parasuis (H. parasuis). When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Haemophilus, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Haemophilus influenzae.

Examples of bacteria of the genus Mycoplasma include Mycoplasma pneumoniae (M. pneumoniae, MP) and Mycoplasma genitalium. When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Mycoplasma, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Mycoplasma pneumoniae.

Bacteria of the genus Legionella include Legionella pneumophila (L. pneumophila, LP) and Legionella longbeachae. When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Legionella, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Legionella pneumophila.

Bacteria of the genus Bordetella include Bordetella pertussis (B. pertussis, BP) and Bordetella bronchiseptica. When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Bordetella, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Bordetella pertussis.

Examples of bacteria of the genus Chlamydia include Chlamydia pneumoniae (C. pneumoniae, CP) and Chlamydia trachomatis (C. trachomatis, CT). When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Chlamydia, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Chlamydia pneumoniae.

Bacteria of the genus Staphylococcus include Staphylococcus aureus (S. aureus, SA), Staphylococcus epidermidis (S. epidermidis), Staphylococcus argenteus (S. argenteus), etc. When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Staphylococcus, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Staphylococcus aureus.

Examples of bacteria of the genus Pseudomonas include Pseudomonas aeruginosa (P. aeruginosa, PA), Pseudomonas fluorescens (P. fluorescens), Pseudomonas phosphorescence (P. phosphorescence), and Pseudomonas putida (P. putida). When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Pseudomonas, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Pseudomonas aeruginosa.

Examples of bacteria of the genus Klebsiella include Klebsiella pneumoniae (K. pneumoniae, KP), Klebsiella oxytoca (K. oxytoca), and Klebsiella aerogenes (K. aerogenes). When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Klebsiella, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Klebsiella pneumoniae.

Examples of bacteria of the genus Neisseria include Neisseria gonorrhoeae (N. gonorrhoeae, NG) and Neisseria meningitidis (N. meningitidis, NM). When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Neisseria, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Neisseria gonorrhoeae.

Moraxella bacteria include Moraxella catarrhalis (M. catarrhalis, MC) and Moraxella bovis. When the method of the present invention detects an antigen-antibody reaction with a Moraxella bacterium, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Moraxella catarrhalis.

Bacteria of the genus Escherichia include Escherichia coli (E. coli, EC) and Escherichia albertii (E. albertii). When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Escherichia, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Escherichia coli.

Examples of bacteria of the genus Salmonella include Salmonella enteritidis (S. enteritidis, SE), Salmonella infantis (S. infantis), Salmonella typhimurium (S. typhimurium), etc. When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Salmonella, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Salmonella enteritidis.

Examples of bacteria of the genus Listeria include Listeria monocytogenes (L. monocytogenes, LM), Listeria innocua (L. innocua), and Listeria ivanovii (L. ivanovii). When the method of the present invention detects an antigen-antibody reaction with bacteria of the genus Listeria, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Listeria monocytogenes.

Examples of bacteria of the Campylobacter genus include Campylobacter jejuni (C. jejuni, CJ) and Campylobacter coli. When the method of the present invention detects an antigen-antibody reaction with bacteria of the Campylobacter genus, it is sufficient to detect an antigen-antibody reaction with any one or more of these bacteria, but it is preferable to detect an antigen-antibody reaction with at least Campylobacter jejuni.

In addition to the above bacteria, genera of bacteria that can be detected by the method of the present invention include, but are not limited to, Mycobacterium, Enterococcus, Aeromonas, Lactobacillus, Micrococcus, Serratia, Acinetobacter, and Vibrio.

Specific examples of bacterial species to be detected in the testing method of the present invention include Streptococcus pneumoniae, Haemophilus influenzae, Moraxella, Staphylococcus aureus, Pseudomonas aeruginosa, Chlamydia pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, Bordetella pertussis, Klebsiella pneumoniae, Streptococcus pyogenes, Neisseria meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Escherichia coli, Salmonella, Listeria monocytogenes, Campylobacter jejuni, and Ureaplasma.

In one embodiment, it is preferable that the bacteria of multiple genera to be detected include both gram-negative and gram-positive bacteria. The structures of the cell membrane and cell wall of the two bacteria are significantly different, making it difficult to simultaneously detect both bacteria using conventional techniques. However, the method of the present invention makes it possible to simultaneously detect gram-negative and gram-positive bacteria by appropriately designing the antibodies used for detection.

The types of infectious diseases, the types of bacteria that cause them, and the types of specimens derived from subjects have been described in detail above. Usually, the bacteria that cause the infection are identified, and specimens that can be used for detection based on the antigen-antibody reaction of the bacteria are also identified. Examples of combinations of such infectious diseases, bacteria that cause them, and specimens that can be used for their detection are shown in Table 1 below. However, the present invention is not limited to the following combinations, and can be used for any combination of infectious diseases, causative bacteria, and specimens.

[2. Antibodies that react with components derived from multiple genera of bacteria in a specimen]
In the method of the present invention, the method of simultaneously detecting the presence or absence and/or the amount of bacteria of multiple genera in a sample based on an antigen-antibody reaction is not particularly limited. According to one embodiment, such detection based on the antigen-antibody reaction is suitably performed by contacting an antibody (the antibody of the present invention) that undergoes an antigen-antibody reaction with a component derived from bacteria of multiple genera with the sample, and measuring the presence or absence and/or the strength of the antigen-antibody reaction that occurs in the sample after the contact. The antibody of the present invention will be described below.

In the present invention, an "antibody" refers to a protein that recognizes and binds to a specific antigen or substance, and is sometimes called an immunoglobulin (Ig). A typical antibody usually has two light chains and two heavy chains that are interconnected by disulfide bonds. There are two types of light chains, called λ chains and κ chains, and there are five types of heavy chains, called γ chains, μ chains, α chains, δ chains, and ε chains. Depending on the type of heavy chain, there are five isotypes of antibodies: IgG, IgM, IgA, IgD, and IgE.

Each heavy chain contains a heavy chain constant (CH) region and a heavy chain variable (VH) region. Each light chain contains a light chain constant (CL) region and a light chain variable (VL) region. The light chain constant (CL) region is composed of a single domain. The heavy chain constant (CL) region is composed of three domains, namely CH1, CH2 and CH3. The light chain variable (VL) region and the heavy chain variable (VH) region each contain four highly conserved regions called framework regions (FR) (FR-1, FR-2, FR-3, FR-4) and three hypervariable regions called complementarity determining regions (CDR) (CDR-1, CDR-2, CDR-3). The heavy chain constant (CH) region has three CDRs (CDR-H1, CDR-H2, CDR-H3) and four FRs (FR-H1, FR-H2, FR-H3, FR-H4), which are arranged from amino terminus to carboxy terminus in the following order: FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4. The light chain constant (CL) region has three CDRs (CDR-L1, CDR-L2, CDR-L3) and four FRs (FR-L1, FR-L2, FR-L3, FR-L4), which are arranged from amino terminus to carboxy terminus in the following order: FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, FR-L4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. A polyclonal antibody is usually prepared from the serum of an animal immunized with an antigen, and is a mixture of various antibody molecular species with different structures. On the other hand, a monoclonal antibody refers to an antibody consisting of a single type of molecule containing a combination of a light chain variable (VL) region and a heavy chain variable (VH) region having a specific amino acid sequence. A monoclonal antibody can be produced from a clone derived from an antibody-producing cell, but it can also be produced by genetic engineering using a nucleic acid molecule obtained having a gene sequence that codes for the amino acids of the antibody protein. In addition, it is a well-known technique to those skilled in the art to carry out modifications to improve the binding and specificity of an antibody using genetic information such as heavy and light chains, or their variable regions and CDRs.

The antibody of the present invention may be a fragment and/or derivative of the antibody. Examples of the antibody fragment include F(ab') ₂ , Fab, Fv, etc. Examples of the antibody derivative include an antibody in which an amino acid mutation has been artificially introduced into the constant region portion of the light chain and/or heavy chain, an antibody in which the domain configuration of the constant region of the light chain and/or heavy chain has been modified, an antibody having two or more Fc regions per molecule, a sugar chain modified antibody, a bispecific antibody, an antibody conjugate in which an antibody or an antibody fragment is bound to a protein other than an antibody, an antibody enzyme, a tandem scFv, a bispecific tandem scFv, a diabody, etc. Furthermore, when the antibody or a fragment or derivative thereof is derived from a non-human animal, a chimeric antibody or a humanized antibody in which a part or all of the sequence other than the CDR is replaced with the corresponding sequence of a human antibody is also included in the antibody of the present invention. In addition, unless otherwise specified, when the term "antibody" is used simply in the present invention, it is intended to include a fragment and/or derivative of the antibody.

The antibody of the present invention causes an antigen-antibody reaction with a certain bacterium means that it specifically binds to some component of the bacterium as an antigen. There are no limitations on the bacterial component that serves as an antigen for the antibody of the present invention. It may be a component contained in the cell wall or cell membrane exposed to the outside of the bacterial cell, or a component contained in the cytoplasm, organelles, nucleus, etc. that is not exposed to the outside of the bacterial cell. When the antibody of the present invention causes an antigen-antibody reaction with a component that is not exposed to the outside of the bacterial cell, in order to cause an antigen-antibody reaction between the antibody of the present invention and such a bacterial component, the sample may be subjected to a treatment to lyse the bacteria before contacting the antibody of the present invention with the sample. Such a bacterial lysis treatment will be described later.

In particular, the antibody of the present invention preferably undergoes an antigen-antibody reaction with a ribosomal protein of any of the bacteria of the specific bacterial genus and/or selected bacterial genus described above. In particular, it is preferable that the antibody undergoes an antigen-antibody reaction with the ribosomal protein L7/L12. In the present invention, "ribosomal protein L7/L12" or simply "L7/L12" is a type of ribosomal protein essential for protein synthesis in microorganisms, and is a protein that is commonly possessed by various bacteria. For the antibody that undergoes an antigen-antibody reaction with the ribosomal protein L7/L12 of the above-mentioned bacteria and the method for producing the same, reference can be made to the description in, for example, International Publication No. WO 2000/006603, a patent publication relating to a previous patent application by the present inventors.

The degree of the antigen-antibody reaction between the antibody of the present invention and the bacteria to be detected is not particularly limited, but it is sufficient that the antigen-antibody reaction occurs to an extent that can be detected by any known detection method. Methods for detecting the antigen-antibody reaction between the antibody and bacteria include, but are not limited to, various known immunological measurement methods described below.

In addition, it is preferable that the antibody of the present invention does not cross-react with one or more non-bacterial components that may be present in the sample. Examples of such non-bacterial components include, but are not limited to, various bioorganic compounds derived from viruses, plants, and/or animals that bacteria do not have. Specific examples of such bioorganic compounds include proteins, sugars, glycoproteins, lipids, complex lipids, nucleic acids, etc. It is preferable that the antibody of the present invention does not cross-react with at least one or more, usually three or more, even four or more, five or more, six or more, seven or more, eight or more, particularly nine or more, and especially ten or more non-bacterial proteins among these non-bacterial components.

Antibodies have extremely high antigen specificity, and so although they are effective in specifically capturing a particular antigen, they were thought to be unsuitable for detecting multiple different types of target substances. However, as described in the Examples below, the present inventors have succeeded in obtaining antibodies that undergo antigen-antibody reactions with multiple genera of bacteria that are to be detected when testing a specimen derived from a subject, and that can be used to simultaneously detect these bacteria. This finding is extremely unexpected and contradicts conventional technical common sense.

The structure of the antibody of the present invention is not particularly limited, but is preferably as follows. Note that antibodies defined solely by the structural features described below are also included in the antibodies of the present invention.

Specifically, the antibody of the present invention preferably has the following amino acid sequences as the amino acid sequences of the heavy and light chain variable regions:

The amino acid sequence of the heavy chain variable region (VH) preferably has an amino acid sequence that has 80% or more, particularly 85% or more, even 90% or more, particularly 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, and especially 100% homology (preferably identity) with any one of the amino acid sequences selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. Of these, it is particularly preferable that the VH sequence is any one of the amino acid sequences selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.

The amino acid sequence of the light chain variable region (VL) preferably has an amino acid sequence that has a homology (preferably identity) of 80% or more, particularly 85% or more, even 90% or more, particularly 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, and especially 100% with any one of the amino acid sequences selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. Among these, it is particularly preferable that the VL sequence is any one of the amino acid sequences selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.

In the present invention, the "homology" of two amino acid sequences refers to the ratio at which identical or similar amino acid residues appear at corresponding positions when the two amino acid sequences are aligned, and the "identity" of two amino acid sequences refers to the ratio at which identical amino acid residues appear at corresponding positions when the two amino acid sequences are aligned. The "homology" and "identity" of two amino acid sequences can be determined, for example, using the BLAST (Basic Local Alignment Search Tool) program (Altschul et al., J. Mol. Biol., (1990), 215(3):403-10).

Methods for identifying the sequences of each CDR from the variable sequences of the heavy and light chains of an antibody include, for example, the Kabat method (Kabat et al., The Journal of Immunology, 1991, Vol. 147, No. 5, pp. 1709-1719) and the Chothia method (Al-Lazikani et al., Journal of Molecular Biology, 1997, Vol. 273, No. 4, pp. 927-948). These methods are common technical knowledge in this field, but you can also refer to, for example, the website of Dr. Andrew C.R. Martin's Group (http://www.bioinf.org.uk/abs/).

Amino acids similar to a certain amino acid include, for example, amino acids belonging to the same group in the following classification based on the polarity, charge, and size of amino acids (each type of amino acid is represented by a one-letter code in each case).
Aromatic amino acids: F, H, W, Y;
Aliphatic amino acids: I, L, V;
Hydrophobic amino acids: A, C, F, H, I, K, L, M, T, V, W, Y;
Charged amino acids: D, E, H, K, R, etc.;
Positively charged amino acids: H, K, R;
Negatively charged amino acids: D, E;
Polar amino acids: C, D, E, H, K, N, Q, R, S, T, W, Y;
Small amino acids: A, C, D, G, N, P, S, T, V, etc.
-Very small amino acids: A, C, G, S.

Furthermore, amino acids similar to a certain amino acid include amino acids that belong to the same group in the following classification based on the type of amino acid side chain (each type of amino acid is represented by a one-letter code in each case).
Amino acids with aliphatic side chains: G, A, V, L, I;
Amino acids with aromatic side chains: F, Y, W;
Amino acids with sulfur-containing side chains: C, M;
Amino acids with aliphatic hydroxyl side chains: S, T;
Amino acids with basic side chains: K, R, H;
- Acidic amino acids and their amide derivatives: D, E, N, Q.

The method for producing the antibody of the present invention is not particularly limited. When the antibody of the present invention is a polyclonal antibody, it can be produced using a component derived from the bacteria to be detected. Examples of the component derived from bacteria that can be used include the bacterial cells themselves, a lysate obtained by lysing the bacterial cells, and fractions obtained by electrophoresis of the lysate. When a fraction obtained by electrophoresis of a bacterial lysate is used, there is no limitation on which fraction is used, but it is preferable to select and use a fraction corresponding to a molecular weight of about 10 to 20 kDa. In addition, it is preferable to use a ribosomal protein contained in bacteria as the component derived from bacteria, and in particular, it is preferable to use ribosomal protein L7/L12 (which has homology in bacteria of multiple genera). By inoculating an animal with these components derived from bacteria together with an adjuvant as necessary and collecting the serum, it is possible to obtain antisera containing antibodies (polyclonal antibodies) that cause an antigen-antibody reaction with the predetermined multiple types of bacteria. Examples of animals to be inoculated include sheep, horses, goats, rabbits, mice, rats, etc., and sheep and rabbits are particularly preferable for producing polyclonal antibodies. In addition, the antibodies can be purified and fractionated from the obtained antiserum, and the desired antibodies can be screened appropriately by known methods using the indexes of antigen-antibody reaction with the desired bacteria and no cross-reaction with other components derived from the sample to obtain more specific desired antibodies. Furthermore, it is also possible to obtain monoclonal antibodies by isolating antibody-producing cells that produce the desired antibody molecules and fusing them with myeloma cells to produce hybridomas with autonomous proliferation ability. In addition, as a method that does not require sensitization of animals, a technology can be used in which a phage library expressing the heavy chain variable (VH) region or light chain variable (VL) region of an antibody or a part of them is used to obtain antibodies that specifically bind to components derived from the bacteria to be detected or phage clones consisting of specific amino acid sequences, and antibodies are produced from the information.

In addition, if a desired antibody is obtained by the above procedure, the structure of the antibody, specifically, a part or all of the amino acid sequences of the heavy chain constant (CH) region, the heavy chain variable (VH) region, the light chain constant (CL) region, and/or the light chain variable (VL) region can be analyzed using a known amino acid sequence analysis method. Methods for modifying the amino acid sequence of the desired antibody thus obtained to improve the binding and specificity of the antibody are also known to those skilled in the art. Furthermore, it is also possible to use all or a part of the amino acid sequence of the desired antibody (particularly all or a part of the heavy chain variable (VH) region and the light chain variable (VL) region, especially the amino acid sequences of each CDR) and combine it with a part of the amino acid sequence of a known antibody (particularly the heavy chain constant (CH) region and the light chain constant (CL) region, and optionally the amino acid sequences of each FR of the heavy chain variable (VH) region and the light chain variable (VL) region) as necessary, to design another antibody that is likely to have the same antigen specificity.

Once the amino acid sequence of the desired antibody has been identified, it is possible to prepare a nucleic acid molecule having a base sequence encoding all or part of the amino acid sequence of the desired antibody by known techniques, and to use such nucleic acid molecule to produce an antibody by genetic engineering. Furthermore, it is also possible to prepare a vector or plasmid for expressing each component of the desired antibody from such a base sequence, and introduce it into a host cell (mammalian cell, insect cell, plant cell, yeast cell, microbial cell, etc.) to produce the antibody. In order to improve the performance of the obtained antibody or to avoid side effects, the structure of the constant region of the antibody or the sugar chain portion can be modified as appropriate using techniques well known to those skilled in the art.

The present invention also covers the above-described method for producing the antibody of the present invention, the nucleic acid molecule encoding the antibody of the present invention, the vector or plasmid containing such a nucleic acid molecule, the cell containing such a nucleic acid molecule, vector or plasmid, and further the hybridoma producing the antibody of the present invention.

The techniques for producing and modifying antibodies described herein are all known to those skilled in the art, and reference can be made, for example, to Antibodies; A laboratory manual, E. Harlow et al., Cold Spring Harbor Laboratory Press (2014). The molecular biology techniques described herein (e.g., amino acid sequence analysis, nucleic acid molecule design and production methods, vector and plasmid design and production methods, etc.) are also all known to those skilled in the art, and reference can be made, for example, to Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory Press, Shambrook, J. et al. (1989).

In this way, a preferred embodiment of the method of the present invention is to use an antibody (the antibody of the present invention) that recognizes the ribosomal protein L7/L12 of each bacterium and causes an antigen-antibody reaction, and to contact the sample to detect the antigen-antibody reaction. Here, the detection sensitivity can be improved by exposing the L7/L12 of the bacteria present in the sample to the outside of the bacterial cell membrane before contacting the antibody of the present invention with the sample. Therefore, in a preferred embodiment of the method of the present invention using the antibody of the present invention described below, the sample may be subjected to a treatment to lyse the bacteria before contacting the antibody of the present invention with the sample. Such a bacterial lysis treatment includes, but is not limited to, heat treatment, ultrasonic treatment, chemical treatment with a surfactant, and the like. The conditions used for the lysis treatment can be appropriately determined depending on the type of bacteria contained in the sample.

In a preferred embodiment of the method of the present invention using the antibody of the present invention, the immunoassay method for detecting the antigen-antibody reaction is not limited. Examples of the immunoassay method are not limited, and may be a method using a single antibody or a method using two or more types of antibodies.

Examples of immunoassays using a single antibody include, but are not limited to, ELISA (enzyme-linked immunosorbent assay) which uses a microtiter plate carrying bacterial antigens to confirm the antigen-antibody reaction caused by the antibody; biosensors which carry antibodies (or antigens) on the sensor surface and confirm the antigen-antibody reaction with the antigen (or antibody) electrically (e.g., AC impedance assay, FET (field effect transistor) assay, etc.) or optically (e.g., SPR (surface plasmon resonance) assay, etc.); and various other known immunoassays, and the method of the present invention can be used for any of these.

Examples of immunoassays using two or more types of antibodies include, but are not limited to, various known immunoassays such as ELISA using a microtiter plate carrying antibodies; latex particle agglutination assay using latex particles carrying antibodies (e.g., polystyrene latex particles, etc.); immunochromatography using membranes carrying antibodies; and sandwich assays using detection antibodies labeled with colored particles or particles having color-developing ability, enzymes, or fluorescent bodies, etc., and capture antibodies immobilized on solid-phase carriers such as magnetic particles. In the case of immunoassays using two or more types of antibodies, namely detection antibodies and capture antibodies, such as sandwich assays, the antibody of the present invention may be used as either a capture antibody or a detection antibody.

In particular, in the present invention, it is preferable to use a sandwich assay method in which the presence or absence and/or amount of bacteria in a sample is detected by a process of capturing and labeling the bacteria in the sample through an antigen-antibody reaction between the sample, a capture antibody linked to a solid phase carrier, and a detection antibody having a detection label, and a process of detecting the target bacteria in the sample based on the detection label.

In one embodiment of the method, the step (I) comprises:
(Ia-1) contacting a specimen with a detection antibody and labeling the bacteria in the specimen through an antigen-antibody reaction between the detection antibody and the bacteria; and
(Ia-2) The method may include a step of contacting a specimen containing bacteria labeled with a detection antibody with a capture antibody, and capturing the bacteria in the specimen by an antigen-antibody reaction between the capture antibody and a bacteria-detection antibody complex.

In another embodiment of the method, the step (I) is
(Ib-1) contacting a specimen with a capture antibody and capturing bacteria in the specimen through an antigen-antibody reaction between the capture antibody and the bacteria; and
(Ib-2) The method may include a step of contacting a specimen containing the bacteria captured by the capture antibody with a detection antibody, and labeling the bacteria in the specimen by an antigen-antibody reaction between the detection antibody and the bacteria-capture antibody complex.

In any embodiment, the capture antibody may be a general-purpose antibody and the detection antibody may be a specific antibody, or the detection antibody may be a general-purpose antibody and the capture antibody may be a specific antibody. In addition, either of the general-purpose antibody and the specific antibody may be the antibody of the present invention. Among them, according to one embodiment of the present invention, the antibody of the present invention is preferably used as a general-purpose antibody, although it is not limited thereto. Unless otherwise noted, in this specification, a "specific antibody" means an antibody that causes an antigen-antibody reaction with one or more types of bacteria (detection target bacteria) that are the final detection target, and a "general-purpose antibody" means an antibody that causes an antigen-antibody reaction with five or more genera of bacteria including the detection target bacteria. The specific antibody can be produced according to a conventionally known method for producing an antibody. That is, in the above-mentioned method for producing the antibody of the present invention, it is possible to obtain a specific antibody that specifically reacts with only the detection target bacteria by appropriately screening using a known method, using as an index the occurrence of an antigen-antibody reaction with the desired detection target bacteria and the absence of cross-reaction with other bacteria other than the detection target and other components derived from the sample.

[3. Test kit for determining whether a subject's infectious disease is of bacterial origin]
The present invention also encompasses a kit (kit of the present invention) that contains the above-mentioned antibody of the present invention for use in the above-mentioned method of the present invention.

The kit of the present invention includes, in addition to the antibody of the present invention, one or more reagents necessary for carrying out the method of the present invention using the antibody of the present invention, a detection device or its components, and/or instructions describing the procedure for carrying out the method of the present invention. The types of such reagents, the contents of the instructions, and further other components included in the kit of the present invention may be appropriately determined depending on the type of specific immunological assay used to detect bacteria of multiple genera.

When the 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 method of the present invention using the antibody 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 method of the present invention. As described above, examples of immunoassays include, but are not limited to, ELISA (enzyme-linked immunosorbent adsorbent) 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; 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, an ELISA using one antibody, a biosensor method, and various other known immunoassays. The device of the present invention is equipped with the components necessary for carrying out such various immunoassays.

Specific examples of devices capable of simultaneously and easily detecting the presence or absence and/or amount of bacteria of multiple genera in a sample include a lateral flow type device and a flow-through type device. Here, the lateral flow type is a method in which a target specimen and a detection antibody are spread in parallel on a membrane including a detection region with 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 type is a method in which a target specimen and a detection antibody are passed vertically through a membrane with a capture antibody immobilized on the surface, and a target substance captured on the membrane surface is detected. The method of the present invention can be applied to both a lateral flow type device and a flow-through type device.

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 common technical knowledge. Below, the schematic configuration of the detection mechanism of a 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 in no way limited to the embodiment illustrated in the drawings.

Figure 1 is a cross-sectional view showing the schematic configuration of a strip-shaped detection mechanism, which is an example of the detection mechanism of a lateral flow type immunochromatographic detection device. In the detection mechanism 10 of Figure 1, a strip-shaped detection antibody-impregnated member (conjugate pad) 2 (this pad is impregnated with the 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 strip length direction on an insoluble membrane carrier 1 for chromatographic development, and an absorption member (absorption pad) 4 is arranged on the other end side (downstream side of the specimen flow B). In the center of the strip length direction on the insoluble membrane carrier 1, a site 5 where a capture antibody is immobilized and a site 6 where a control reagent is immobilized as necessary are arranged. The control reagent is a reagent that does not bind to the analyte but binds to the detection antibody. When a specimen A is applied to the specimen addition 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 the specimen flow A. At this time, the analyte in the specimen (bacteria to be detected in this invention) binds to the detection antibody to form an analyte-detection antibody complex. When specimen A passes through the capture antibody immobilization site 5, the analyte in the specimen binds to the capture antibody to form a capture antibody-analyte-detection antibody complex. Furthermore, when specimen A passes through the control reagent immobilization site 6, the detection antibody that is not bound to the analyte binds to the control reagent 6, thereby confirming the end of the test (i.e., specimen A has passed through the capture antibody 5). Here, the presence or absence or amount of the analyte can be detected by detecting the label possessed by the detection antibody in the capture antibody-analyte-detection antibody complex present at the capture antibody immobilization site 5 using a known means. If necessary, the label of the detection antibody may be sensitized using a known method to facilitate detection.

The detection antibody-impregnated member (conjugate pad) 2 and the specimen addition member (sample pad) 3 can be omitted at will. If the detection antibody-impregnated member (conjugate pad) 2 is not included in this mechanism, the specimen A and the detection antibody can be applied simultaneously or sequentially to one end of the insoluble membrane carrier 1, either in a premixed state or separately, to perform a test similar to the above test.

It is also possible to construct a detection kit that can perform the same detection even if the capture antibody and the detection antibody are interchanged. That is, the capture antibody may be a general-purpose antibody and the detection antibody may be a specific antibody, or the detection antibody may be a general-purpose antibody and the capture antibody a specific antibody. As described above, either or both of the general-purpose antibody and the specific antibody can be the antibody of the present invention.

In the lateral flow immunochromatographic detection method and device according to one embodiment of the present invention described above, it is possible to detect two or more genera of bacteria to be detected at once in a single detection line (capture antibody immobilization site 5 in the example of FIG. 1). In the conventional lateral flow immunochromatographic detection method and device, when detecting multiple detection targets, a detection line (capture antibody immobilization site 5 in the example of FIG. 1) must be provided for each detection target, and it was necessary to provide detection lines corresponding to the number of detection targets. This led to an increase in the size of the device, and in the first place, when the number of detection targets was too large, detection with a single device was difficult or impossible. In contrast, in the lateral flow immunochromatographic detection method and device according to one embodiment of the present invention, by appropriately combining detection antibodies and capture antibodies, it is possible to simultaneously detect multiple genera of detection target bacteria with a single detection line. This makes it possible to miniaturize the device, and in principle, even if there are many species and genera of detection target bacteria, detection is possible with a single device.

The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and can be implemented in any form without departing from the spirit of the present invention.

1. Preparation of antibodies
- Preparation of general-purpose antibody A (PA51B2) Pseudomonas aeruginosa (PA) was used as the bacterium immunogen. Antibodies against ribosomal protein L7/L12 of Pseudomonas aeruginosa were prepared with reference to the method described in International Publication No. 2000/06603. Specifically, Escherichia coli transformed with an expression vector incorporating DNA encoding the entire amino acid sequence of ribosomal protein L7/L12 of Pseudomonas aeruginosa was cultured using LB medium or the like, and purified as a fusion protein using a tag sequence derived from the expression vector by affinity column. This Pseudomonas aeruginosa L7/L12 full-length protein was used as an immunogen, and according to the standard method for obtaining hybridomas, the immunogen was prepared with PBS so that the concentration of the immunogen was 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 removed. The spleen cells were excised from the mice and fused with myeloma cells to obtain various hybridomas.

The obtained hybridomas were cultured in HAT medium, and the antibodies in the culture supernatant were used for screening. Screening was performed by ELISA using bacterial lysates of multiple genera immobilized as antigens, and hybridomas producing antibodies that reacted simultaneously with lysates of 18 types of bacteria, namely, Streptococcus pneumoniae (SP), Haemophilus influenzae (HI), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP), Bordetella pertussis (BP), Chlamydia pneumoniae (CP), Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP), Moraxella spp. (MC), Streptococcus hemolyticus (SPY), Neisseria meningitidis (NM), Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Escherichia coli (EC), Salmonella enterica (SE), Listeria monocytogenes (LM), and Campylobacter jejuni (CJ), were selected. According to the standard method for producing monoclonal antibodies, the selected hybridoma was cultured in TIL Media I medium supplemented with 10% fetal bovine serum (FBS), and 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 obtained filtrate was passed through a Protein G column to adsorb the antibody, and the general antibody A (PA51B2), which corresponds to the antibody of the present invention, was purified and obtained from the mouse ascites.

- Preparation of generic antibody B (LP54A3) Generic antibody B (LP54A3), which corresponds to the antibody of the present invention, was prepared using the same procedure as that for obtaining antibody A, except that Legionella pneumophila (LP) was used as the bacterium immunogen and Legionella pneumophila ribosomal protein L7/L12 was used as the immunogen.

- Preparation of generic antibody C (CP141A190.1) Generic antibody C (CP141A190.1), which corresponds to the antibody of the present invention, was prepared using the same procedure as that for obtaining antibody A, except that Chlamydia trachomatis (CP) was used as the bacterium immunogen and Chlamydia trachomatis ribosomal protein L7/L12 was used as the immunogen.

Determination of the amino acid sequences of the heavy and light chain variable sequences of the generic antibodies A to C. For the generic antibodies A to C (antibodies of the present invention) prepared by the above procedure, examples of the amino acid sequences of the heavy and light chain variable sequences were determined according to standard methods. The correspondence between each amino acid sequence and each SEQ ID NO: is shown below.

・ Universal antibody A (PA51B2):
Heavy chain variable sequence Amino acid sequence (SEQ ID NO:1)
Light chain variable sequence, amino acid sequence (SEQ ID NO:2)
Universal antibody B (LP54A3):
Heavy chain variable sequence Amino acid sequence (SEQ ID NO:3)
Light chain variable sequence Amino acid sequence (SEQ ID NO:4)
・ Universal antibody C (CP141A190.1):
Heavy chain variable sequence Amino acid sequence (SEQ ID NO:5)
Light chain variable sequence Amino acid sequence (SEQ ID NO:6)

[2. Detection of antigen-antibody reaction between antibodies and bacteria 1/Bacteriolytic antigen solid-phase ELISA]
Using the versatile antibodies A to C (antibodies of the present invention) prepared by the above procedure, data on reactivity with bacteria of multiple genera was obtained by ELISA. As the bacteria to be detected, 18 species were selected, namely, Streptococcus pneumoniae (SP), Haemophilus influenzae (HI), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP), Bordetella pertussis (BP), Chlamydia pneumoniae (CP), Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP), Moraxella spp. (MC), Streptococcus hemolyticus (SPY), Neisseria meningitidis (NM), Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Escherichia coli (EC), Salmonella enterica (SE), Listeria monocytogenes (LM), and Campylobacter jejuni (CJ). Each of the above bacteria was purchased from ATCC and cultured, and 1×e ⁸ cfu/mL of each was prepared and suspended in PBS. Bacterial lysates of each bacterium were obtained by lysing each bacterium by ultrasonication and filtering through a filter with 0.45 μm openings 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. 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, each well was washed three times with PBS-T, and 50 μL of 10 μg/mL of general-purpose antibodies A to C were 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 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, and the reaction was carried out. After the reaction, the wells were washed five times with PBS-T, and then 100 μL of a mixture of the color-developing substrate TMB (Tetramethylbenzidine) and hydrogen peroxide was dropped into each well to carry out the color reaction. After 10 minutes, hydrochloric acid, which was used as a reaction stop solution, was dropped into each well, and the absorbance at 450 nm of each well was measured using a plate reader.

The measurement results are shown in Table 2 below. The results shown in this table show that generic antibodies A to C react with higher sensitivity (absorbance was 0.3 or higher for each bacterium, and 1.0 or higher in most cases) with each of the bacterial samples compared to samples without bacteria. In other words, it was confirmed that generic antibodies A to C obtained by immunization with different bacteria can each cause an antigen-antibody reaction (can detect) with each of the bacterial samples.

[3. Detection of antigen-antibody reaction between antibodies and bacteria 2/Recombinant antigen solid-phase ELISA]
Using the versatile antibodies A to C (the antibodies of the present invention) prepared by the above procedure, data on reactivity with ribosomal proteins L7/L12 of bacteria of multiple genera was obtained by ELISA. As the bacteria to be detected, 18 species were selected, including Streptococcus pneumoniae (SP), Haemophilus influenzae (HI), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP), Bordetella pertussis (BP), Chlamydia pneumoniae (CP), Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP), Moraxella spp. (MC), Streptococcus hemolyticus (SPY), Neisseria meningitidis (NM), Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Escherichia coli (EC), Salmonella enterica (SE), Listeria monocytogenes (LM), and Campylobacter jejuni (CJ). Escherichia coli transformed with an expression vector incorporating DNA encoding the amino acid sequence of ribosomal protein L7/L12 of each bacterial species was cultured in LB medium or the like, and the ribosomal protein L7/L12 of each bacterial species was obtained by purifying it as a fusion protein using an affinity column using the tag sequence derived from the expression vector.

50μL of ribosomal protein L7/L12 of each bacterial species at 10ng/mL was dropped into each well of a 96-well polystyrene plate for ELISA, and immobilized on the bottom surface. 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, each well was washed three times with PBS-T, and 50μL of any of the general-purpose antibodies A to C at 10μg/mL was dropped into each well to perform an antigen-antibody reaction for 1 hour. After the reaction, the wells were washed three times with PBS-T, and 50μL of secondary antibody (Goat Anti-mouse IgG) labeled with 0.5μg/mL of the detection enzyme HRP (Horse Radish Peroxidase) was dropped into each well to perform the reaction. After the reaction, the wells were washed five times with PBS-T, and then 100 μL of a mixture of the color-developing substrate TMB (Tetramethylbenzidine) and hydrogen peroxide was dropped into each well to carry out the color reaction. After 10 minutes, hydrochloric acid, which was used as a reaction stop solution, was dropped into each well, and the absorbance at 450 nm of each well was measured using a plate reader.

The measurement results are shown in Table 3 below. This table shows that generic antibodies A to C react with higher sensitivity (absorbance was 0.3 or higher for all bacteria, and 1.0 or higher in most cases) with each of the bacterial samples compared to samples without bacteria. In other words, it was confirmed that generic antibodies A to C obtained by immunization with different bacteria can each cause an antigen-antibody reaction (can detect) with the ribosomal protein L7/L12 in each of the bacterial samples.

[4. Verification of cross-reactivity of antibodies with non-bacterial components in human specimens/solid-phase ELISA]
Using the versatile antibodies A to C (the antibodies of the present invention) prepared by the above procedure, data on reactivity with non-bacterial components in various human specimens was obtained by ELISA. The human specimens used were pharyngeal swabs, nasopharyngeal swabs, nasal discharge, nasal aspirates, sputum, blood, urine, conjunctival swabs, and middle ear fluid. These human specimens were suspended in PBS, and solid matter containing bacteria was removed using a filter with a mesh size of 0.45 μm to obtain bacteria-free human specimen samples for ELISA.

50 μL of the above human specimen sample 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, and then blocked with PBS containing 1% BSA. After blocking, each well was washed three times with PBS-T, and 50 μL of 10 μg/mL of general-purpose antibodies A to C were dropped into each well, and the reaction was carried out for 1 hour. After the reaction, the wells were washed three times with PBS-T, and 50 μL of secondary antibody (Goat Anti-mouse IgG) labeled with 0.5 μg/mL of HRP, an enzyme for detection, was dropped into each well, and the reaction was carried out. After the reaction, the wells were washed five times with PBS-T, and 100 μL of a mixture of TMB and hydrogen peroxide, a color-developing substrate, was dropped into each well, and the color-developing reaction was carried out. After 10 minutes, hydrochloric acid, a reaction stop solution, was dropped into each well, and the absorbance at 450 nm of each well was measured using a plate reader.

The measurement results are shown in Table 4 below. The results shown in this table indicate that generic antibodies A to C do not react with any components in human samples that do not contain the above bacteria (all absorbances are less than 0.3). Combined with the results of the previous examples, it was confirmed that generic antibodies A to C do not react with any non-bacterial components in the above human samples, but react only with the specific multiple genera of bacteria to be detected, that is, they can detect the presence and/or amount of the specific multiple genera of bacteria to be detected in human samples with high selectivity.

The present invention can be widely used in fields requiring rapid and simple detection of bacterial infections, primarily in the medical field, and is of great industrial utility.

10 Detection mechanism 1 Insoluble membrane carrier for chromatographic development 2 Detection antibody-impregnated member (conjugate pad)
3. Sample addition member (sample pad)
4 Absorption member (absorption pad)
5 Capture antibody immobilization site 6 Control reagent immobilization site A Sample B Sample flow

Claims (19)

A test method for determining whether an infectious disease suffered by a subject is caused by bacteria, comprising:
Providing a sample from the subject;
contacting the subject-derived specimen with a monoclonal antibody or a fragment thereof, or a derivative thereof, which generates an antigen-antibody reaction with the at least two or more genera of bacteria;
detecting the presence or absence and/or intensity of an antigen-antibody reaction between the at least two or more genera of bacteria possibly present in the sample and the monoclonal antibody or a fragment thereof, or a derivative thereof;
The monoclonal antibody or a fragment thereof, or a derivative thereof,
As a heavy chain variable region sequence, an amino acid sequence having 80% or more homology with at least one amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5; and
As a light chain variable region sequence, an amino acid sequence having 80% or more homology with at least one of the amino acid sequences selected from SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6.
Each of
If the detection result is positive, it is determined to be an infectious disease caused by bacteria,
When the detection result is negative, the infection is determined to be of a cause other than bacteria. The method according to claim 1, wherein in the detection step, bacteria of two or more genera selected from the group consisting of the genera Streptococcus, Haemophilus, Mycoplasma, Legionella, Bordetella, Chlamydia, Staphylococcus, Pseudomonas, Klebsiella, Neisseria, Moraxella, Escherichia, Salmonella, Listeria, and Campylobacter are simultaneously detected. The method according to claim 1 or 2, wherein the two or more genera of bacteria to be detected include both gram-negative and gram-positive bacteria. The method according to any one of claims 1 to 3, wherein the bacterial infection to be identified is an infection selected from the group consisting of cold syndrome, pneumonia, bronchitis, pharyngitis, conjunctivitis, meningitis, otitis media, sinusitis, urethritis, sexually transmitted diseases, urinary tract infections, cystitis, pyelonephritis, sepsis, bacteremia, diarrhea, food poisoning, and gastroenteritis. The method according to any one of claims 1 to 4, wherein the subject-derived specimen is one or more specimens selected from a pharyngeal swab, a nasopharyngeal swab, nasal discharge, a nasal aspirate, sputum, blood, urine, cerebrospinal fluid, a conjunctival swab, middle ear fluid, urine, a uterine scraping, and stool. The method according to any one of claims 1 to 5, wherein the step of simultaneously detecting the presence/absence and/or abundance of bacteria of at least two or more genera is performed by a method that does not involve culturing the bacteria. The method according to any one of claims 1 to 6 , wherein the antibody is an antibody that generates an antigen-antibody reaction with a ribosomal protein of bacteria of the two or more genera. The method of claim 7 , wherein the ribosomal protein is L7/L12. The method according to any one of claims 1 to 8 , wherein the method comprises:
(I) a step of capturing and labeling bacteria in the specimen by an antigen-antibody reaction between the specimen, a capture antibody linked to a solid phase carrier, and a detection antibody having a detection label; and (II) a step of detecting the presence or absence and/or amount of the target bacteria in the specimen by detecting the target bacteria in the specimen based on the detection label,
One of the capture antibody and the detection antibody is one or more specific antibodies that undergo an antigen-antibody reaction with one or more types of target bacteria, and the other antibody is one or more general antibodies that undergo an antigen-antibody reaction with five or more genera of bacteria including the target bacteria, and
A method, wherein either the general antibody or the specific antibody is an antibody according to any one of claims 1 to 8 . The step (I)
(Ia-1) contacting a specimen with a detection antibody and labeling the bacteria in the specimen through an antigen-antibody reaction between the detection antibody and the bacteria; and
(Ia-2) The method according to claim 9, comprising the step of contacting a specimen containing bacteria labeled with a detection antibody with a capture antibody, and capturing the bacteria in the specimen by an antigen-antibody reaction between the capture antibody and a bacteria-detection antibody complex. The step (I)
(Ib-1) contacting a specimen with a capture antibody and capturing bacteria in the specimen through an antigen-antibody reaction between the capture antibody and the bacteria; and
(Ib-2) The method according to claim 9, comprising the step of contacting a specimen containing the bacteria captured by the capture antibody with a detection antibody, and labeling the bacteria in the specimen by an antigen-antibody reaction between the detection antibody and the bacteria-capture antibody complex. The method according to any one of claims 9 to 11 , wherein the capture antibody is a general antibody and the detection antibody is a specific antibody. The method according to any one of claims 9 to 11 , wherein the detection antibody is a general antibody and the capture antibody is a specific antibody. A test kit for determining whether an infectious disease suffered by a subject is of bacterial origin or not using the method according to any one of claims 1 to 8 , the kit comprising an antibody according to any one of claims 1 to 8 . A test kit for determining whether an infectious disease suffered by a subject is of bacterial origin or not using a method according to any one of claims 10 to 13 , the kit comprising a generic antibody and a specific antibody, wherein either the generic antibody or the specific antibody is an antibody according to any one of claims 1 to 8 . A test method for determining whether an infectious disease suffered by a subject is caused by bacteria, comprising:
Providing a sample from the subject;
(I) capturing bacteria in the specimen and labeling the bacteria in the specimen through an antigen-antibody reaction between the specimen, a capture antibody linked to a solid phase carrier, and a detection antibody having a detection label; and
(II) A step of detecting the target bacteria in the sample based on the detection label.
and detecting the presence or absence and/or amount of the target bacteria in the specimen by
one of the capture antibody and the detection antibody is one or more specific antibodies that undergo an antigen-antibody reaction with one or more types of target bacteria, and the other antibody is one or more general antibodies that undergo an antigen-antibody reaction with five or more genera of bacteria including the target bacteria;
If the detection result is positive, it is determined to be an infectious disease caused by bacteria,
When the detection result is negative, the infection is determined to be of a cause other than bacteria. The method according to claim 16, wherein the antibody is an antibody that generates an antigen-antibody reaction with a ribosomal protein of bacteria of the two or more genera. 18. The method of claim 17, wherein the ribosomal protein is L7/L12. The method according to any one of claims 16 to 18, wherein the antibody is a monoclonal antibody or a fragment thereof, or a derivative thereof.

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