JP7557274B2 - Antibody for detecting Legionella in a sample, and method, reagent, and kit for detecting Legionella using the antibody - Google Patents

Description

The present invention relates to an antibody for detecting the presence or absence of Legionella pneumophila in a sample, as well as a method, reagent, and kit for detecting Legionella in a sample using the antibody.

When treating patients suffering from microbial infections, rapid diagnosis of the infection is extremely important. Methods for diagnosing microbial infections include methods for detecting the causative bacteria from the infected site or from serum or body fluids, and methods for detecting antibodies against the causative bacteria from blood or body fluids, but from the standpoint of the accuracy and speed of diagnosis, methods that directly detect the causative bacteria are preferred.

Methods for detecting the causative bacteria of microbial infections can be broadly divided into culture identification methods, in which the causative bacteria are isolated and cultured, and then the bacteria are identified based on their biochemical properties; genetic diagnostic methods, in which the causative bacteria-specific genes are amplified using methods such as polymerase chain reaction (PCR) to identify the bacteria; and immunological methods, in which the bacteria are identified by utilizing the specific reaction of antibodies to the surface antigen markers of the causative bacteria. However, culture identification methods and genetic diagnostic methods take time to obtain detection results, and there are often problems with detection sensitivity. For this reason, immunological methods are widely used for diagnosis, as they can detect the causative bacteria with high sensitivity in a short period of time.

Conventional immunological methods for detecting infectious disease-causing bacteria use a variety of combinations of marker antigens and antibodies depending on the bacterial species.

Legionella pneumophila is a gram-negative bacillus of the Legionella genus that, when it infects humans, is known to cause Legionnaires' disease, an atypical form of pneumonia accompanied by symptoms such as high fever, cough, headache, muscle pain, and chills. Legionnaires' disease is generally treated with antibiotics such as macrolides, new quinolones, rifampicin, and tetracyclines, which have a high translocation rate into phagocytes. However, as Legionnaires' disease progresses, treatment becomes difficult, and symptoms such as difficulty breathing and impaired consciousness occur, and in the worst case, it can lead to death, so early detection and treatment of Legionnaires' disease infection is required.

Legionella bacteria are generally isolated and detected by culturing them on BCYE agar medium or GVPC agar medium supplemented with L-cysteine or iron, but isolation and culturing using such media usually takes more than two days, posing problems such as long test times. To address this issue, a method is known that aims to speed up culturing by adding a siliceous compound to the medium (Patent Document 1: Japanese Patent No. 5769174), but there is still a need to shorten the test time. In addition, the LAMP method (Patent Document 2: Japanese Patent Publication No. 2003-219878) is known, which targets the 16S ribosomal RNA gene, but it has problems such as insufficient speed and the need for equipment, and is not satisfactory.

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 discovered a method that can detect various microorganisms specifically and comprehensively detect various serotypes within the same species by using an antibody against such a protein (Patent Document 3: International Publication No. 2000/006603). In addition, for Legionella bacteria in particular, they have obtained an antibody that can detect Legionella bacteria in distinction from bacteria of other genera, and have proposed a detection method using such an antibody (Patent Document 4: Japanese Patent Application Laid-Open No. 2010-248129). However, there is room for improvement in such existing antibodies, such as the detection accuracy (bacterial species specificity) and the binding pattern of a specific antibody to the ribosomal protein L7/L12 of Legionella bacteria that ensures the detection accuracy, which is unknown.

Patent No. 5769174 JP 2003-219878 A International Publication No. 2000/006603 JP 2010-248129 A

The present invention aims to provide an antibody for detecting Legionella bacteria that has excellent detection sensitivity and detection accuracy (bacterial species specificity).

After extensive research, the inventors discovered that within the C-terminal domain (CTD) consisting of amino acid residues 58 to 125 of the ribosomal protein L7/L12 of Legionella bacteria, there exists a partial amino acid sequence that can serve as an epitope with excellent antigen-antibody reactivity and specificity. Based on this, they actually produced multiple antibodies that undergo an antigen-antibody reaction with this partial amino acid sequence, and verified that Legionella bacteria in samples can be detected with high sensitivity and accuracy by using these antibodies, which led to the present invention.

That is, the gist of the present invention is as follows.
[1] An antibody for detecting Legionella pneumophila, which undergoes an antigen-antibody reaction with an epitope present in the C-terminal domain (CTD) consisting of amino acid residues at positions 58 to 125 of the ribosomal protein L7/L12 of Legionella pneumophila as shown in SEQ ID NO: 1, or a fragment thereof, or a derivative thereof.
[2] The antibody or fragment thereof, or a derivative thereof according to item [1], wherein the epitope comprises one or more amino acid residues selected from the amino acid residues at positions 102 to 114 of SEQ ID NO: 1.
[3] The antibody, or a fragment thereof, or a derivative thereof, according to item [1] or [2], which does not cross-react with bacteria of one or more genera selected from the genera Mycoplasma, Escherichia, Chlamydia, Salmonella, Pseudomonas, Staphylococcus, Neisseria, Haemophilus, Bordetella, Moraxella, and Streptococcus.
[4] A heavy chain variable region sequence having 80% or more homology to any one of the amino acid sequences selected from SEQ ID NO:5, SEQ ID NO:9, and SEQ ID NO:13; and
The antibody or fragment thereof according to any one of items [1] to [3], or a derivative thereof, comprising, as a light chain variable region sequence, an amino acid sequence having 80% or more homology to any one of the amino acid sequences selected from SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15.
[5] A heavy chain variable region sequence having 80% or more homology to any one of the amino acid sequences selected from SEQ ID NO:5, SEQ ID NO:9, and SEQ ID NO:13; and
An antibody, or a fragment thereof, or a derivative thereof, comprising, as a light chain variable region sequence, an amino acid sequence having 80% or more homology to any one of the amino acid sequences selected from SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15.
[6] A nucleic acid molecule encoding the antibody or a fragment thereof, or a derivative thereof, according to any one of [1] to [5].
[7] A vector or plasmid comprising the nucleic acid molecule according to [6].
[8] A host cell transformed with the nucleic acid molecule according to [6] or the vector or plasmid according to [7].
[9] The host cell according to item [8], wherein the host cell is a eukaryotic cell selected from a mammalian cell, an insect cell, a yeast cell, and a plant cell, or a bacterial cell.
[10] A hybridoma expressing the antibody or a fragment thereof, or a derivative thereof, according to any one of [1] to [5].
[11] A method for detecting the presence or absence of Legionella bacteria in a sample, comprising contacting the sample with the antibody or fragment thereof, or a derivative thereof, according to any one of items [1] to [5], and detecting the presence or absence of an antigen-antibody reaction.
[12] A reagent for detecting the presence or absence of Legionella bacteria in a sample, comprising the antibody or fragment thereof, or a derivative thereof, according to any one of items [1] to [5].
[13] A kit for detecting the presence or absence of Legionella bacteria in a sample, comprising the antibody or fragment thereof, or a derivative thereof, according to any one of items [1] to [5], and an instruction manual containing instructions for detecting the presence or absence of Legionella bacteria in a sample using the antibody.

The antibody of the present invention makes it possible to detect Legionella bacteria in a sample with high sensitivity and accuracy.

FIG. 1 is a diagram showing a schematic representation of the three-dimensional structure of ribosomal protein L7/L12 of Legionella pneumophila. FIG. 2 is a schematic diagram showing the three-dimensional structure of ribosomal protein L7/L12 of Moraxella catarrhalis. FIG. 3 is a schematic diagram showing the three-dimensional structure of ribosomal protein L7/L12 of N. gonorrhoeae. FIG. 4 is a schematic diagram showing the three-dimensional structures of the C-terminal domain (CTD) portions of ribosomal proteins L7/L12 of Legionella pneumophila, Neisseria gonorrhoeae, and Moraxella catarrhalis. FIG. 5 shows an alignment of amino acid sequences of the C-terminal domain (CTD) portions of ribosomal proteins L7/L12 from Legionella pneumophila, Neisseria gonorrhoeae, and Moraxella catarrhalis. FIG. 6A is a schematic diagram showing the results of NMR analysis of the interaction between monoclonal antibody 4B1 and ribosomal protein L7/L12 of Legionella pneumophila. FIG. 6B is a schematic diagram showing the results of NMR analysis of the interaction between monoclonal antibody 36A2 and Legionella ribosomal protein L7/L12. FIG. 6C is a schematic diagram showing the results of NMR analysis of the interaction between monoclonal antibody 54A3 and Legionella ribosomal protein L7/L12.

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.

1. Antibodies for detecting Legionella:
A first aspect of the present invention relates to an antibody for detecting Legionella bacteria (hereinafter appropriately referred to as the "antibody of the present invention").

(1) Introduction:
In the present invention, "Legionella pneumophila" refers to a gram-negative bacillus belonging to the Legionella genus. It is known that Legionella bacteria, when infected by humans, cause Legionnaires' disease, an atypical pneumonia accompanied by symptoms such as high fever, cough, headache, muscle pain, and chills. Legionnaires' disease is generally treated by administering antibacterial agents such as macrolides, new quinolones, rifampicin, and tetracyclines, which have high translocation properties into phagocytes. However, as Legionnaires' disease progresses, treatment becomes difficult, and symptoms such as dyspnea and impaired consciousness occur, and in the worst case, it can lead to death, so early detection and treatment of infection with Legionella bacteria are required.

The present inventors focused on the ribosomal protein L7/L12 when preparing an antibody for detecting Legionella bacteria. 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 found in various bacteria. The ribosomal protein L7/L12 of Legionella bacteria has a dimeric structure in which two copies of a monomer molecule consisting of 125 amino acid residues are linked. The amino acid sequence of the primary structure of each monomer is shown in SEQ ID NO: 1. According to the analysis results of the present inventors, the monomer of the ribosomal protein L7/L12 of Legionella bacteria forms one three-dimensional structure (NTD: N-Terminal Domain) with amino acid residues 1 to 40, and further forms another three-dimensional structure (CTD: C-Terminal Domain) with amino acid residues 58 to 125 via a linker consisting of 17 amino acid residues that does not form a three-dimensional structure. Furthermore, two copies of the monomer molecule having such a three-dimensional structure form a dimer structure by associating with each other's NTDs (see Example 1 and Figure 1 below).

It was also found that the ribosomal protein L7/L12 of Moraxella catarrhalis and Neisseria gonorrhoeae forms a dimer by two molecules associating with each other at the NTD (N-Terminal Domain, amino acid residues 1-40), which then forms a CTD (C-Terminal Domain, amino acid residues 58-124 in the case of Moraxella catarrhalis and amino acid residues 58-123 in the case of Neisseria gonorrhoeae) via a random coil structure linker (amino acid residues 41-57) (see Examples 2 and 3 and Figures 2 and 3 below).

Of the three-dimensional structures possessed by Legionella bacteria L7/L12, the present inventors have focused in particular on the CTD as a candidate epitope with superior immunogenicity, based on the following considerations.

Comparing the three-dimensional structures of the L7/L12 CTDs of Legionella, Moraxella catarrhalis, and Neisseria gonorrhoeae, it was found that there were large differences in the surface shape and charge distribution, and that in particular, the region consisting of amino acid residues 102 to 114 of Legionella L7/L12 shows large differences in three-dimensional structure between bacterial species (dotted area in Figure 4). In the case of Moraxella catarrhalis (MC), this region is dominated by hydrophobic (white) to uncharged hydrophilic regions (light blue, light red), while in the cases of Legionella (LP) and Neisseria gonorrhoeae (NG), negatively charged regions (red) are dominant. In addition, when comparing Legionella and Neisseria gonorrhoeae, most of the region in Legionella is negatively charged (red), whereas in Neisseria gonorrhoeae, the negatively charged region (red) is located at the top and the hydrophobic (white) to non-charged hydrophilic region (light blue, light red) is located at the bottom (see Example 4 below and the dotted line in Figure 4).

Furthermore, when comparing the amino acid sequences of ribosomal protein L7/L12 from Legionella, Moraxella catarrhalis, and Neisseria gonorrhoeae, it was found that there were significant differences between the bacterial species in the amino acid residues at positions 102 to 114 of Legionella and the corresponding regions at positions 101 to 113 of Moraxella catarrhalis and 100 to 112 of Neisseria gonorrhoeae (Figure 5). For example, the amino acid residues at positions 109-110 of L7/L12 of Legionella pneumophila correspond to the amino acid residues at positions 108-109 of Moraxella catarrhalis and 107-108 of Neisseria gonorrhoeae. In the case of Legionella pneumophila, the residues are A (alanine: hydrophobic) and S (serine: hydrophilic, uncharged), whereas in the case of Moraxella catarrhalis, the residues are E (glutamic acid: hydrophilic, negative charge), E (glutamic acid: hydrophilic, negative charge), and in the case of Neisseria gonorrhoeae, the residues are E (glutamic acid: hydrophilic, negative charge), D (aspartic acid: hydrophilic, negative charge), and the order of the types of amino acids and polarity differs for each bacterial species (Figure 5).

Similarly, it was found that there are large differences between the bacterial species in the amino acid residues at positions 112 to 114 of Legionella and the corresponding regions at positions 111 to 113 of Moraxella catarrhalis and 110 to 112 of Neisseria gonorrhoeae. The amino acid residues at positions 112-114 of Legionella are K (lysine: hydrophilic, positive charge), K (lysine: hydrophilic, positive charge), and E (glutamic acid: hydrophilic, negative charge), whereas the amino acid residues at positions 111-113 of Moraxella catarrhalis are K (lysine: hydrophilic, positive charge), K (lysine: hydrophilic, positive charge), K (lysine: hydrophilic, positive charge), and the amino acid residues at positions 110-112 of Neisseria gonorrhoeae are Q (glutamine: hydrophilic, uncharged), K (lysine: hydrophilic, positive charge), and Q (glutamine: hydrophilic, uncharged), and the type of amino acids and the order of polarity differ for each bacterial species (Figure 5).

As described above, the Legionella ribosomal protein L7/L12 has an amino acid sequence and three-dimensional structure (surface shape, surface charge) in the CTD that differs significantly between bacterial species. Based on this knowledge, the inventors came up with the idea that more specific antibodies could be obtained by targeting the CTD rather than the full length of L7/L12.

Then, a peptide having an amino acid sequence similar to the CTD of L7/L12 was expressed, and antibodies that specifically bind to this were produced and screened to obtain antibodies 4B1, 36A2, and 54A3 (see Example 5 below). It was then confirmed that all of these antibodies reacted with the CTD of L7/L12 of Legionella bacteria, particularly with an epitope that contains specific amino acid residues (see Example 6 and Figures 6A-C below). Furthermore, it was confirmed that all of these antibodies have excellent detection sensitivity and detection accuracy (bacterial species specificity) (see Example 7 below), completing the present invention. A more detailed explanation is given below.

(2) Overview of the antibody:
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 mutually bonded 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 types of antibody isotypes, namely 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 is an antibody capable of detecting Legionella bacteria, and can be specified from the following two viewpoints. First, from the first viewpoint, the antibody of the present invention can be specified from the characteristic that it recognizes a specific epitope present in the ribosomal protein L7/L12 of Legionella bacteria and causes an antigen-antibody reaction. Second, the antibody of the present invention can be specified from the characteristic that each of the variable regions of its heavy chain and light chain has a specific amino acid sequence. The first viewpoint will be explained in the section (2) Antibody properties below, and the second viewpoint will be explained in the section (3) Antibody structure below. It is sufficient that the antibody of the present invention satisfies the characteristics of either the first viewpoint or the second viewpoint, but it goes without saying that antibodies that satisfy the characteristics of both the first and second viewpoints are also included in the antibody of the present invention.

The antibody of the present invention may be either 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 also 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 the 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.

(3) Antibody properties:
In a first aspect, the antibody of the present invention is characterized in that it undergoes an antigen-antibody reaction with an epitope consisting of specific amino acid residues in the ribosomal protein L7/L12 of Legionella bacteria.

In the present invention, "antigen-antibody reaction" refers to an antibody recognizing and binding to any component of the antigen.

In the present invention, "epitope" refers to a part of an antigen that is recognized by an antibody.

In the ribosomal protein L7/L12 of Legionella bacteria, the epitope to which the antibody of the present invention binds is present in the CTD formed by amino acid residues at positions 58 to 125 of the amino acid sequence of the ribosomal protein L7/L12 of Legionella bacteria shown in SEQ ID NO: 1. According to the analysis results by the present inventors described later in Examples 5 to 7, it has been confirmed that the high detection sensitivity and high detection accuracy (high bacterial species specificity) antibodies 4B1, 36A2, and 54A3 for detecting Legionella bacteria actually obtained by the present inventors in the Examples all bind to the CTD formed by amino acid residues at positions 58 to 125 of L7/L12.

In particular, the epitope of ribosomal protein L7/L12 to which the antibody of the present invention binds preferably contains one or more amino acid residues selected from amino acid residues at positions 58 to 125 of SEQ ID NO: 1, particularly preferably 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more. In particular, the epitope of ribosomal protein L7/L12 to which the antibody of the present invention binds preferably contains at least one or more amino acid residues selected from positions 102 to 114 of SEQ ID NO: 1, particularly preferably 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more. It is even more preferred that the epitope of ribosomal protein L7/L12 to which the antibody of the present invention binds contains at least one or more amino acid residues selected from positions 102, 106, 109, 110, and 112 to 114 of SEQ ID NO: 1, particularly preferably 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more. According to the analysis results of the present inventors described later in the Examples, it has been confirmed that these amino acid residues are present near the surface in the three-dimensional structure of the CTD of the ribosomal protein L7/L12 of Legionella. In addition, when comparing L7/L12 of Legionella with L7/L12 of other bacterial species, these amino acid residues are located in positions that are particularly highly species-specific for Legionella, and therefore it is believed that these amino acid residues certainly form epitopes for the antibodies of the present invention.

In addition, it is preferable that the antibodies of the present invention do not cross-react with bacteria other than Legionella bacteria or other components.

Specifically, the antibody of the present invention preferably does not cross-react with bacteria of one or more genera selected from the genera Mycoplasma, Escherichia, Chlamydia, Salmonella, Pseudomonas, Staphylococcus, Neisseria, Haemophilus, Bordetella, Moraxella, and Streptococcus. In particular, the antibody of the present invention preferably does not cross-react with bacteria of two or more genera, or even three or more genera, or four or more genera, or five or more genera, or six or more genera, and particularly all genera.

In addition, a person skilled in the art can measure the antigen-antibody reaction between an antibody and an epitope/antigen or other component by appropriately selecting a binding measurement in a solid-phase or liquid-phase system. Such methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), and luminescence resonance energy transfer (LRET). In addition, when measuring such antigen-antibody binding, it is also possible to label the antibody and/or antigen with an enzyme, fluorescent substance, luminescent substance, radioisotope, etc., and detect the antigen-antibody reaction using a measurement method suitable for the physical and/or chemical properties of the labeled substance.

In particular, in the present invention, it is preferable to analyze the interaction between the antigen (epitope) and the antibody by nuclear magnetic resonance (NMR). For details of such a method, reference can be made to, for example, Cavanagh et al., "Protein NMR Spectroscopy, Principles and Practice Protein NMR", 2nd Edition, Academic Press, 2006, and Vitha et al., "Spectroscopy: Principles and Instrumentation", Wiley-Blackwell, 2018. Specific analysis conditions are not limited, but reference can be made to, for example, the analysis conditions adopted by the present inventors in Example 6 described below.

(4) Antibody structure:
In a second aspect, the antibody of the present invention is characterized in that each of the heavy and light chain variable regions has a specific amino acid sequence.

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

The heavy chain variable region sequence 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 99% or more, and especially 100% homology (preferably identity) with any one of the amino acid sequences selected from SEQ ID NO:5, SEQ ID NO:9, and SEQ ID NO:13. Among these, the heavy chain variable region sequence is particularly preferably any one of the amino acid sequences selected from SEQ ID NO:5, SEQ ID NO:9, and SEQ ID NO:13.

The light chain variable region sequence 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 99% or more, and especially 100% homology (preferably identity) with any one of the amino acid sequences selected from SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:15. Among these, the light chain variable region sequence is particularly preferably any one of the amino acid sequences selected from SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:15.

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.

(5) Method for producing antibodies:
The method for producing the antibody of the present invention is not particularly limited, but examples thereof include the following techniques.

When the antibody of the present invention is a polyclonal antibody, it can be prepared using all or a part of the ribosomal protein L7/L12 of the Legionella bacteria to be detected, preferably a polypeptide consisting of amino acid residues at positions 58 to 125 of SEQ ID NO: 1 (amino acid residues of SEQ ID NO: 3) constituting the CTD, or a polypeptide having an amino acid sequence with 80% or more, particularly 85% or more, further 90% or more, particularly 95% or more, or 96% or more, or 97% or more, or 99% or more, and particularly 100% homology (preferably identity) thereto (hereinafter referred to as "epitope polypeptide"). Specifically, an epitope polypeptide is prepared, and an animal is inoculated with the epitope polypeptide together with an adjuvant if necessary, and the serum is collected to obtain an antiserum containing an antibody (polyclonal antibody) that generates an antigen-antibody reaction with the epitope polypeptide. Animals to be inoculated include sheep, horses, goats, rabbits, mice, rats, etc., and sheep and rabbits are particularly preferred for producing polyclonal antibodies. In addition, it is possible to obtain a desired antibody with superior specificity by purifying and fractionating the antibody from the obtained antiserum, and appropriately screening it by a known method using as indicators whether the antibody reacts with the ribosomal protein L7/L12 of Legionella bacteria and does not cross-react with other specific components, such as the bacteria of the genera listed above. Furthermore, it is also possible to obtain a monoclonal antibody by isolating an antibody-producing cell that produces a desired antibody molecule, fusing the cell with a myeloma cell to produce a hybridoma with autonomous proliferation ability. In addition, as a method that does not require sensitization of animals, a technology is also available in which an antibody that specifically binds to the ribosomal protein L7/L12 of the Legionella bacteria to be detected or a phage clone consisting of a specific amino acid sequence is obtained using a phage library that expresses the heavy chain variable (VH) region or light chain variable (VL) region of an antibody or a part of them, and an antibody is 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. A method for modifying the amino acid sequence of the desired antibody thus obtained to improve the binding property and specificity of the antibody is also known to those skilled in the art. Furthermore, it is 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 sequence 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 sequence of each FR of the heavy chain variable (VH) region and the light chain variable (VL) region) as necessary, thereby designing another antibody that is likely to have the same antigen specificity.

On the other hand, when the amino acid sequence of a part (CDR or variable region) or the whole of an antibody is specified, 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 produce an antibody by genetic engineering using such a nucleic acid molecule. 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 addition, for the purpose of improving the performance of the obtained antibody or avoiding side effects, the structure of the constant region of the antibody or the modification of the glycan portion can be appropriately performed 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).

2. Legionella detection methods, reagents, and kits:
Another aspect of the present invention relates to a method for detecting the presence or absence of Legionella bacteria in a sample using the antibody of the present invention (hereinafter appropriately referred to as the "detection method of the present invention").

The detection method of the present invention involves contacting the above-mentioned antibody of the present invention or a fragment thereof, or a derivative thereof, with a sample and detecting the presence or absence of an antigen-antibody reaction. Here, since the antibody of the present invention generates an antigen-antibody reaction with a specific epitope of Legionella bacteria as described above, the presence or absence of such an antigen-antibody reaction can be detected by various known immunoassay methods to detect the presence or absence of Legionella bacteria in a sample.

Specimens include mainly biological samples derived from humans or non-human animals. There are no particular limitations on the type of biological sample, but examples include liquid samples such as blood (whole blood, plasma, serum), lymph, urine, saliva, tears, amniotic fluid, and ascites, as well as homogenate suspensions and extracts of solid samples such as biopsy samples of various tissues, and culture supernatants of these.

As described above, the antibody of the present invention recognizes a specific epitope present in the ribosomal protein L7/L12 of Legionella bacteria and generates an antigen-antibody reaction. Therefore, by exposing the ribosomal protein L7/L12 of Legionella bacteria to the outside of the bacterial cell membrane, the detection sensitivity can be improved. Therefore, before contacting the antibody of the present invention with a sample, the sample may be subjected to a treatment for lysing the bacteria. Examples of such a bacterial lysis treatment include, but are not limited to, a lysis treatment using a surfactant, a lytic enzyme, or the like. Examples of surfactants that can be used in the lysis treatment include nonionic surfactants such as Triton X-100, Tween 20, Briji 35, Nonidet P-40, dodecyl-β-D-maltoside, and octyl-β-D-glucoside; zwitterionic surfactants such as Zwittergent 3-12 and CHAPS (3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate); and anionic surfactants such as SDS (sodium dodecyl sulfate). Examples of lytic enzymes that can be used in the lysis treatment include lysozyme, lysostaphin, pepsin, glucosidase, galactosidase, achromopeptidase, and β-N-acetylglucosaminidase.

The method of contacting the antibody of the present invention with a sample and the immunoassay method for detecting an antigen-antibody reaction are not limited. Examples of immunoassay methods 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; and 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. In addition, in the case of an immunoassay method using a detection antibody and a capture antibody in combination, such as a sandwich assay, the antibody of the present invention may be used as a capture antibody or a detection antibody.

The detection method of the present invention makes it possible to quickly and easily detect the presence or absence of Legionella bacteria in a sample.

The present invention also covers a reagent containing the antibody of the present invention for use in the detection method of the present invention, and a kit containing the antibody of the present invention together with an instruction manual containing instructions for detecting the presence or absence of Legionella bacteria in a sample. The solvent and other components of such a reagent, the operation and use instructions in the instruction manual of such a kit, and other components contained in such a kit may be appropriately determined according to the type of specific immunoassay used to detect Legionella bacteria. Among them, specific examples of kits that can easily detect the presence or absence of Legionella bacteria in a sample include a lateral flow kit and a flow-through kit. Here, the lateral flow method is a method in which a detection target sample and a detection antibody are dropped in order onto a membrane including a detection region in which a capture antibody is fixed on the surface, and are developed in parallel, to detect the target substance captured in the detection region of the membrane. On the other hand, the flow-through method is a method in which a detection target sample and a detection antibody are dropped in order onto a membrane in which a capture antibody is fixed on the surface, and are passed vertically, to detect the target substance captured on the surface of the membrane. The detection method of the present invention can be applied to both lateral flow and flow-through kits.

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.

[Example 1] Analysis of the three-dimensional structure of ribosomal protein L7/L12 of Legionella
<1: Obtaining ribosomal protein L7/L12 from Legionella pneumophila>
The pGEX-6P-1 plasmid (GE Healthcare) into which the ribosomal protein L7/L12 gene (SEQ ID NO: 2) of Legionella pneumophila was cloned was introduced into E. coli BL21(DE3)pLysS E. coli strain (Promega).

The resulting E. coli strain was cultured at OD 3000 at 37° C. in M9 medium (47.7 mM Na ₂ HPO ₄ ·12H ₂ O, 22 mM KH ₂ PO ₄ , 8.6 mM NaCl, 2 mM MgSO ₄ , 50 μM ZnSO ₄ , 100 μM CaCl ₂ , 4.1 μM biotin, 7.2 μM choline chloride, 2.3 μM folic acid, 8.2 μM nicotinamide, 4.6 μM calcium pantothenate, 6 μM pyridoxal hydrochloride, 0.3 μM riboflavin, 16.6 μM thiamine hydrochloride, 27 mM sodium ampicillin, 18.7 mM ¹⁵ N-NH ₄ Cl, 11.1 mM ¹³ C-glucose). IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 1 mM, and the mixture was cultured at 16° C. for 36 hours. _{The mixture} was then centrifuged at 7000 rpm for 15 minutes at 4° C. to recover the E. coli.

5 mL of BugBuster (Merck Millipore) and 5 μL of Benzonase ^{(registered trademark)} endonuclease (Merck Millipore) were added per 1 g of the obtained E. coli, and the mixture was shaken at room temperature for 30 minutes to completely dissolve the E. coli. After filtration with a 0.45 μm filter, ribosomal protein L7/L12 was purified on a glutathione-Sepharose column using a Profinia protein purification system (Bio-Rad). 1.5 mL of 10x PBS (phosphate buffered saline) and Prescission protease (GE Healthcare) were added to 15 mL of the obtained protein solution, and the mixture was shaken at room temperature for 2 hours. The reaction solution was passed through the glutathione-Sepharose column again, and the non-reacted fraction was obtained as ribosomal protein L7/L12.

The obtained ribosomal protein L7/L12 was dialyzed against 50 mM sodium phosphate pH 6.8 as an external solution, diluted with 4 times the amount of 20 mM Tris-HCl pH 8.0, and passed through an AKTA protein purification system (GE Healthcare) connected to an ion exchange column RESOURCE Q (GE Healthcare) at a flow rate of 2 mL/min. Subsequently, 20 mM Tris-HCl pH 8.0 containing 1 M NaCl was passed at a flow rate of 2 mL/min so as to increase linearly from 0 to 50%, and the ribosomal protein L7/L12 was eluted.
The obtained ribosomal protein L7/L12 was passed through an AKTA protein purification system (GE Healthcare) connected to a gel filtration column HiLoad 16/60 Superdex 75pg (GE Healthcare) at a flow rate of 1 mL/min using PBS (phosphate buffered saline) as the mobile phase to obtain ribosomal protein L7/L12. The obtained ribosomal protein L7/L12 was centrifuged and concentrated to 1 mM using Protein Assay Kit I (BIO-RAD, model number 5000001JA), and then subjected to NMR measurement.

<2: Analysis of the three-dimensional structure of Legionella ribosomal protein L7/L12 by NMR>
A Shigemi NMR sample tube was charged with 250 μL of 1 mM ribosomal protein L7/L12, 20 μL of heavy water, and 1 μL of 5 mg/mL DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid), and then degassed with an aspirator and placed in the NMR apparatus.

Using an AVANCE III HD 600MHz NMR spectrometer (Bruker), HNCO (accumulation number 4, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HN(CO)CA (accumulation number 8, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HNCA (accumulation number 8, number of data points (F1 x F2 x F3) 64 x 128 x 1024), CBCA(CO)NH (accumulation number 16, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HNCACB ( Each FID was measured using the following pulse sequences: HBHA(CO)NH (accumulation number 8, number of data points (F1×F2×F3) 64×128×1024), HBHA(CO)NH (accumulation number 16, number of data points (F1×F2×F3) 64×128×1024), HN(CA)HA (accumulation number 32, number of data points (F1×F2×F3) 64×128×1024), and C(CO)NH (accumulation number 16, number of data points (F1×F2×F3) 128×128×1024). An INOVA 800 MHz NMR system (Agilent) was used to perform [ ¹ H- ¹⁵ N] HSQC (accumulation number 8, number of data points (F1×F2) 512×2048), [ ¹ H- ¹³ C] HSQC aliphatic (accumulation number 8, number of data points (F1×F2) 868×2048), [ ¹ H- ¹³ C] HSQC aromatic (accumulation number 32, number of data points (F1×F2) 204×2048), HCCH-TOCSY aliphatic (accumulation number 4, number of data points (F1×F2×F3) 96×256×2048), and HCCH-TOCSY aliphatic (accumulation number 10, number of data points (F1×F2×F3) 96×256×2048). Each FID was obtained using the pulse sequences of aromatic (8 accumulations, number of data points (F1×F2×F3) 128×128×2048), ¹⁵ N-edited NOESY (mixing time 75 ms, 8 accumulations, number of data points (F1×F2×F3) 64×256×2048), ¹³ C-edited NOESY aliphatic (mixing time 75 ms, 8 accumulations, number of data points (F1×F2×F3) 96×256×2048), and ¹³ C-edited NOESY aromatic (mixing time 75 ms, 8 accumulations, number of data points (F1×F2×F3) 64×256×2048). Each of the obtained FIDs was Fourier transformed using NMR Pipe software to obtain each spectrum.

The acquired spectra were assigned on Sparky software. First, the main chain -NH- of ribosomal protein L7/L12 was assigned to the [ ¹ H- ¹⁵ N] HSQC spectrum using the spectra of HNCO, HN(CO)CA, HNCA, CBCA(CO)NH, HNCACB, HBHA(CO)NH, and HN(CA)HA. Next, the side chains -CH-, -CH ₂ -, and -CH ₃ - were assigned to the [ ¹ H- ¹³ C] HSQC aliphatic spectrum and the [ ¹ H- ¹³ C] HSQC aromatic spectrum using the spectra of C(CO)NH, HCCH-TOCSY aliphatic, and HCCH-TOCSY aromatic. Finally, ¹ H within a distance of 5 A was detected and assigned using ¹⁵ N-edited NOESY, ¹³ C-edited NOESY aliphatic, and ¹³ C-edited NOESY aromatic spectra. Details of the assignment method were as described in PROTEIN NMR SPECTROSCOPY PRINCIPLES AND PRACTICE SECOND EDITION, 2007, Cavanagh, J., Fairbrother, WJ, Palmer, III, AG, Rance, M., and Skelton, NJ, Elsevier Academic Press.

The assigned atomic coordinates and ¹ H distance information were input into the CYANA three-dimensional structure automatic calculation software, and the energy values were sufficiently converged through 20 three-dimensional structure calculations to obtain the spatial atomic coordinates of the three-dimensional structure of the ribosomal protein L7/L12.

The spatial atomic coordinates obtained were entered into the Pymol software, and the three-dimensional structure of ribosomal protein L7/L12 was drawn (Figure 1). When the three-dimensional structure of the main chain of ribosomal protein L7/L12 was displayed, it was found that one three-dimensional structure (NTD: N-Terminal Domain) was formed by amino acid residues 1 to 40, and that another three-dimensional structure (CTD: C-Terminal Domain) was formed by amino acid residues 58 to 125 via a linker of approximately 17 amino acid residues that does not form a three-dimensional structure (Figure 1). It was also found that two ribosomal protein L7/L12 molecules with such a three-dimensional structure associate with each other's NTDs to form a dimer (Figure 1).

[Example 2] Analysis of the three-dimensional structure of ribosomal protein L7/L12 of Moraxella catarrhalis
<1: Obtaining ribosomal protein L7/L12 from Moraxella catarrhalis>
The pGEX-6P-1 plasmid (GE Healthcare) into which the ribosomal protein L7/L12 gene (SEQ ID NO: 36) of Moraxella catarrhalis was cloned was introduced into Escherichia coli BL21(DE3)pLysS Escherichia coli strain (Promega).

The resulting E. coli strain was cultured at OD 3000 at 37° C. in M9 medium (47.7 mM Na ₂ HPO ₄ ·12H ₂ O, 22 mM KH ₂ PO ₄ , 8.6 mM NaCl, 2 mM MgSO ₄ , 50 μM ZnSO ₄ , 100 μM CaCl ₂ , 4.1 μM biotin, 7.2 μM choline chloride, 2.3 μM folic acid, 8.2 μM nicotinamide, 4.6 μM calcium pantothenate, 6 μM pyridoxal hydrochloride, 0.3 μM riboflavin, 16.6 μM thiamine hydrochloride, 27 mM sodium ampicillin, 18.7 mM ¹⁵ N-NH ₄ Cl, 11.1 mM ¹³ C-glucose). IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 1 mM, and the mixture was cultured at 16° C. for 36 hours. _{The mixture} was then centrifuged at 7000 rpm for 15 minutes at 4° C. to recover the E. coli.

5 mL of BugBuster (Merck Millipore) and 5 μL of Benzonase ^{(registered trademark)} endonuclease (Merck Millipore) were added per 1 g of the obtained E. coli, and the mixture was shaken at room temperature for 30 minutes to completely dissolve the E. coli. After filtration with a 0.45 μm filter, ribosomal protein L7/L12 was purified on a glutathione-Sepharose column using a Profinia protein purification system (Bio-Rad). 1.5 mL of 10x PBS (phosphate buffered saline) and Prescission protease (GE Healthcare) were added to 15 mL of the obtained protein solution, and the mixture was shaken at room temperature for 2 hours. The reaction solution was passed through the glutathione-Sepharose column again, and the non-reacted fraction was obtained as ribosomal protein L7/L12.

The obtained ribosomal protein L7/L12 was dialyzed against 50 mM sodium phosphate pH 6.8 as an external solution, diluted with 4 times the amount of 20 mM Tris-HCl pH 8.0, and passed through an AKTA protein purification system (GE Healthcare) connected to an ion exchange column RESOURCE Q (GE Healthcare) at a flow rate of 2 mL/min. Next, 20 mM Tris-HCl pH 8.0 containing 1 M NaCl was passed at a flow rate of 2 mL/min so as to increase linearly from 0 to 50%, and the ribosomal protein L7/L12 was eluted.

The obtained ribosomal protein L7/L12 was passed through an AKTA protein purification system (GE Healthcare) connected to a gel filtration column HiLoad 16/60 Superdex 75pg (GE Healthcare) at a flow rate of 1 mL/min using PBS (phosphate buffered saline) as the mobile phase to obtain ribosomal protein L7/L12. The obtained ribosomal protein L7/L12 was centrifuged and concentrated to 1 mM using Protein Assay Kit I (BIO-RAD, model number 5000001JA), and then subjected to NMR measurement.

<2: NMR analysis of the three-dimensional structure of Moraxella catarrhalis ribosomal protein L7/L12>
A Shigemi NMR sample tube was charged with 250 μL of 1 mM ribosomal protein L7/L12, 20 μL of heavy water, and 1 μL of 5 mg/mL DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid), and then degassed with an aspirator and placed in the NMR apparatus.

Using an AVANCE III HD 600MHz NMR spectrometer (Bruker), HNCO (accumulation number 4, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HN(CO)CA (accumulation number 8, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HNCA (accumulation number 8, number of data points (F1 x F2 x F3) 64 x 128 x 1024), CBCA(CO)NH (accumulation number 16, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HNCACB ( Each FID was measured using the following pulse sequences: HBHA(CO)NH (accumulation number 8, number of data points (F1×F2×F3) 64×128×1024), HBHA(CO)NH (accumulation number 16, number of data points (F1×F2×F3) 64×128×1024), HN(CA)HA (accumulation number 32, number of data points (F1×F2×F3) 64×128×1024), and C(CO)NH (accumulation number 16, number of data points (F1×F2×F3) 128×128×1024). An INOVA 800 MHz NMR system (Agilent) was used to perform [ ¹ H- ¹⁵ N] HSQC (accumulation number 8, number of data points (F1×F2) 512×2048), [ ¹ H- ¹³ C] HSQC aliphatic (accumulation number 8, number of data points (F1×F2) 868×2048), [ ¹ H- ¹³ C] HSQC aromatic (accumulation number 32, number of data points (F1×F2) 204×2048), HCCH-TOCSY aliphatic (accumulation number 4, number of data points (F1×F2×F3) 96×256×2048), and HCCH-TOCSY aliphatic (accumulation number 10, number of data points (F1×F2×F3) 96×256×2048). Each FID was obtained using the pulse sequences of aromatic (8 accumulations, number of data points (F1×F2×F3) 128×128×2048), ¹⁵ N-edited NOESY (mixing time 75 ms, 8 accumulations, number of data points (F1×F2×F3) 64×256×2048), ¹³ C-edited NOESY aliphatic (mixing time 75 ms, 8 accumulations, number of data points (F1×F2×F3) 96×256×2048), and ¹³ C-edited NOESY aromatic (mixing time 75 ms, 8 accumulations, number of data points (F1×F2×F3) 64×256×2048). Each of the obtained FIDs was Fourier transformed using NMR Pipe software to obtain each spectrum.

The acquired spectra were assigned on Sparky software. First, the main chain -NH- of ribosomal protein L7/L12 was assigned to the [ ¹ H- ¹⁵ N] HSQC spectrum using the spectra of HNCO, HN(CO)CA, HNCA, CBCA(CO)NH, HNCACB, HBHA(CO)NH, and HN(CA)HA. Next, the side chains -CH-, -CH ₂ -, and -CH ₃ - were assigned to the [ ¹ H- ¹³ C] HSQC aliphatic spectrum and the [ ¹ H- ¹³ C] HSQC aromatic spectrum using the spectra of C(CO)NH, HCCH-TOCSY aliphatic, and HCCH-TOCSY aromatic. Finally, ¹ H within a distance of 5 A was detected and assigned using ¹⁵ N-edited NOESY, ¹³ C-edited NOESY aliphatic, and ¹³ C-edited NOESY aromatic spectra. Details of the assignment method were as described in PROTEIN NMR SPECTROSCOPY PRINCIPLES AND PRACTICE SECOND EDITION, 2007, Cavanagh, J., Fairbrother, WJ, Palmer, III, AG, Rance, M., and Skelton, NJ, Elsevier Academic Press.

The assigned atomic coordinates and ¹ H distance information were input into the CYANA three-dimensional structure automatic calculation software, and the energy values were sufficiently converged through 20 three-dimensional structure calculations to obtain the spatial atomic coordinates of the three-dimensional structure of the ribosomal protein L7/L12.

The spatial atomic coordinates obtained were entered into the Pymol software, and the three-dimensional structure of ribosomal protein L7/L12 was drawn (Figure 2). When the three-dimensional structure of the main chain of ribosomal protein L7/L12 was displayed, it was found that the amino acid residues at positions 1 to 40 form one three-dimensional structure (NTD: N-Terminal Domain), and that via a linker consisting of approximately 17 amino acid residues that does not form a three-dimensional structure, another three-dimensional structure (CTD: C-Terminal Domain) is formed by amino acid residues at positions 58 to 124 (Figure 2). It was also found that two ribosomal protein L7/L12 molecules with such a three-dimensional structure associate with each other's NTDs to form a dimer (Figure 2).

[Example 3] Analysis of the three-dimensional structure of ribosomal protein L7/L12 of Neisseria gonorrhoeae
<1: Obtaining ribosomal protein L7/L12 from Neisseria gonorrhoeae>
The pGEX-6P-1 plasmid (GE Healthcare) into which the gonococcal ribosomal protein L7/L12 gene (SEQ ID NO: 24) was cloned was introduced into the E. coli BL21(DE3)pLysS E. coli strain (Promega).

The resulting E. coli strain was cultured at OD 3000 at 37° C. in M9 medium (47.7 mM Na ₂ HPO ₄ ·12H ₂ O, 22 mM KH ₂ PO ₄ , 8.6 mM NaCl, 2 mM MgSO ₄ , 50 μM ZnSO ₄ , 100 μM CaCl ₂ , 4.1 μM biotin, 7.2 μM choline chloride, 2.3 μM folic acid, 8.2 μM nicotinamide, 4.6 μM calcium pantothenate, 6 μM pyridoxal hydrochloride, 0.3 μM riboflavin, 16.6 μM thiamine hydrochloride, 27 mM sodium ampicillin, 18.7 mM ¹⁵ N-NH ₄ Cl, 11.1 mM ¹³ C-glucose). IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 1 mM, and the mixture was cultured at 16° C. for 36 hours. _{The mixture} was then centrifuged at 7000 rpm for 15 minutes at 4° C. to recover the E. coli.

5 mL of BugBuster (Merck Millipore) and 5 μL of Benzonase ^{(registered trademark)} endonuclease (Merck Millipore) were added per 1 g of the obtained E. coli, and the mixture was shaken at room temperature for 30 minutes to completely dissolve the E. coli. After filtration with a 0.45 μm filter, ribosomal protein L7/L12 was purified on a glutathione-Sepharose column using a Profinia protein purification system (Bio-Rad). 1.5 mL of 10x PBS (phosphate buffered saline) and Prescission protease (GE Healthcare) were added to 15 mL of the obtained protein solution, and the mixture was shaken at room temperature for 2 hours. The reaction solution was passed through the glutathione-Sepharose column again, and the non-reacted fraction was obtained as ribosomal protein L7/L12.

The obtained ribosomal protein L7/L12 was dialyzed against 50 mM sodium phosphate pH 6.8 as an external solution, diluted with 4 times the amount of 20 mM Tris-HCl pH 8.0, and passed through an AKTA protein purification system (GE Healthcare) connected to an ion exchange column RESOURCE Q (GE Healthcare) at a flow rate of 2 mL/min. Next, 20 mM Tris-HCl pH 8.0 containing 1 M NaCl was passed at a flow rate of 2 mL/min so as to increase linearly from 0 to 50%, and the ribosomal protein L7/L12 was eluted.

The obtained ribosomal protein L7/L12 was passed through an AKTA protein purification system (GE Healthcare) connected to a gel filtration column HiLoad 16/60 Superdex 75pg (GE Healthcare) at a flow rate of 1 mL/min using PBS (phosphate buffered saline) as the mobile phase to obtain ribosomal protein L7/L12. The obtained ribosomal protein L7/L12 was centrifuged and concentrated to 1 mM using Protein Assay Kit I (BIO-RAD, model number 5000001JA), and then subjected to NMR measurement.

<2: NMR analysis of the three-dimensional structure of ribosomal protein L7/L12 of Neisseria gonorrhoeae>
A Shigemi NMR sample tube was charged with 250 μL of 1 mM ribosomal protein L7/L12, 20 μL of heavy water, and 1 μL of 5 mg/mL DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid), and then degassed with an aspirator and placed in the NMR apparatus.

Using an AVANCE III HD 600MHz NMR spectrometer (Bruker), HNCO (accumulation number 4, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HN(CO)CA (accumulation number 8, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HNCA (accumulation number 8, number of data points (F1 x F2 x F3) 64 x 128 x 1024), CBCA(CO)NH (accumulation number 16, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HNCACB ( Each FID was measured using the following pulse sequences: HBHA(CO)NH (accumulation number 8, number of data points (F1×F2×F3) 64×128×1024), HBHA(CO)NH (accumulation number 16, number of data points (F1×F2×F3) 64×128×1024), HN(CA)HA (accumulation number 32, number of data points (F1×F2×F3) 64×128×1024), and C(CO)NH (accumulation number 16, number of data points (F1×F2×F3) 128×128×1024). An INOVA 800 MHz NMR system (Agilent) was used to perform [ ¹ H- ¹⁵ N] HSQC (accumulation number 8, number of data points (F1×F2) 512×2048), [ ¹ H- ¹³ C] HSQC aliphatic (accumulation number 8, number of data points (F1×F2) 868×2048), [ ¹ H- ¹³ C] HSQC aromatic (accumulation number 32, number of data points (F1×F2) 204×2048), HCCH-TOCSY aliphatic (accumulation number 4, number of data points (F1×F2×F3) 96×256×2048), and HCCH-TOCSY aliphatic (accumulation number 10, number of data points (F1×F2×F3) 96×256×2048). Each FID was obtained using the pulse sequences of aromatic (8 accumulations, number of data points (F1×F2×F3) 128×128×2048), ¹⁵ N-edited NOESY (mixing time 75 ms, 8 accumulations, number of data points (F1×F2×F3) 64×256×2048), ¹³ C-edited NOESY aliphatic (mixing time 75 ms, 8 accumulations, number of data points (F1×F2×F3) 96×256×2048), and ¹³ C-edited NOESY aromatic (mixing time 75 ms, 8 accumulations, number of data points (F1×F2×F3) 64×256×2048). Each of the obtained FIDs was Fourier transformed using NMR Pipe software to obtain each spectrum.

The acquired spectra were assigned on Sparky software. First, the main chain -NH- of ribosomal protein L7/L12 was assigned to the [ ¹ H- ¹⁵ N] HSQC spectrum using the spectra of HNCO, HN(CO)CA, HNCA, CBCA(CO)NH, HNCACB, HBHA(CO)NH, and HN(CA)HA. Next, the side chains -CH-, -CH ₂ -, and -CH ₃ - were assigned to the [ ¹ H- ¹³ C] HSQC aliphatic spectrum and the [ ¹ H- ¹³ C] HSQC aromatic spectrum using the spectra of C(CO)NH, HCCH-TOCSY aliphatic, and HCCH-TOCSY aromatic. Finally, ¹ H within a distance of 5 A was detected and assigned using ¹⁵ N-edited NOESY, ¹³ C-edited NOESY aliphatic, and ¹³ C-edited NOESY aromatic spectra. Details of the assignment method were as described in PROTEIN NMR SPECTROSCOPY PRINCIPLES AND PRACTICE SECOND EDITION, 2007, Cavanagh, J., Fairbrother, WJ, Palmer, III, AG, Rance, M., and Skelton, NJ, Elsevier Academic Press.

The assigned atomic coordinates and ¹ H distance information were input into the CYANA three-dimensional structure automatic calculation software, and the energy values were sufficiently converged through 20 three-dimensional structure calculations to obtain the spatial atomic coordinates of the three-dimensional structure of the ribosomal protein L7/L12.

The spatial atomic coordinates obtained were input into the Pymol software, and the three-dimensional structure of ribosomal protein L7/L12 was drawn (Figure 3). When the three-dimensional structure of the main chain of ribosomal protein L7/L12 was displayed, it was found that the amino acid residues at positions 1 to 40 form one three-dimensional structure (NTD: N-Terminal Domain), and that via a linker consisting of 17 amino acid residues that does not form a three-dimensional structure, another three-dimensional structure (CTD: C-Terminal Domain) is formed by the amino acid residues at positions 58 to 123 (Figure 3). It was also found that two ribosomal protein L7/L12 molecules with such a three-dimensional structure associate with each other's NTDs to form a dimer (Figure 3).

[Example 4] Comparative study of the CTD portion of ribosomal protein L7/L12 of Legionella, Moraxella catarrhalis, and Neisseria gonorrhoeae Figure 4 is a schematic diagram showing the three-dimensional structures of the C-terminal domain (CTD) portion of ribosomal protein L7/L12 of Legionella, Moraxella catarrhalis, and Neisseria gonorrhoeae . As is clear from this figure, when the three-dimensional structures of the CTDs of ribosomal protein L7/L12 of Legionella, Moraxella catarrhalis, and Neisseria gonorrhoeae are compared, a region was found in which the surface shape and charge distribution were significantly different (dotted line portion in Figure 4). This region corresponds to amino acid residues 102 to 114 of L7/L12 of Legionella, 101 to 113 of Moraxella catarrhalis, and 100 to 112 of Neisseria gonorrhoeae. In the case of Moraxella catarrhalis (MC), the region is dominated by hydrophobic (white) to non-charged hydrophilic regions (light blue, light red), while in the case of Legionella (LP) and Neisseria gonorrhoeae (NG), the negatively charged region (red) is dominant. In addition, when comparing Legionella and Neisseria gonorrhoeae, most of the region in Legionella is negatively charged (red), but in the case of Neisseria gonorrhoeae, the negatively charged region (red) is located at the top and the hydrophobic (white) to non-charged hydrophilic region (light blue, light red) is located at the bottom (dotted area in Figure 4).

Furthermore, when comparing the amino acid sequences of ribosomal protein L7/L12 from Legionella, Moraxella catarrhalis, and Neisseria gonorrhoeae, it was found that there were significant differences between the bacterial species in the amino acid residues at positions 102 to 114 of Legionella and the corresponding regions at positions 101 to 113 of Moraxella catarrhalis and 100 to 112 of Neisseria gonorrhoeae (Figure 5). For example, the amino acid residues at positions 109-110 of L7/L12 of Legionella pneumophila correspond to the amino acid residues at positions 108-109 of Moraxella catarrhalis and 107-108 of Neisseria gonorrhoeae. In the case of Legionella pneumophila, the residues are A (alanine: hydrophobic) and S (serine: hydrophilic, uncharged), whereas in the case of Moraxella catarrhalis, the residues are E (glutamic acid: hydrophilic, negative charge), E (glutamic acid: hydrophilic, negative charge), and in the case of Neisseria gonorrhoeae, the residues are E (glutamic acid: hydrophilic, negative charge), D (aspartic acid: hydrophilic, negative charge), and the order of the types of amino acids and polarity differs for each bacterial species (Figure 5).

Similarly, it was found that there are large differences between the bacterial species in the amino acid residues at positions 112 to 114 of Legionella and the corresponding regions at positions 111 to 113 of Moraxella catarrhalis and 110 to 112 of Neisseria gonorrhoeae. The amino acid residues at positions 112-114 of Legionella are K (lysine: hydrophilic, positive charge), K (lysine: hydrophilic, positive charge), and E (glutamic acid: hydrophilic, negative charge), whereas the amino acid residues at positions 111-113 of Moraxella catarrhalis are K (lysine: hydrophilic, positive charge), K (lysine: hydrophilic, positive charge), K (lysine: hydrophilic, positive charge), and the amino acid residues at positions 110-112 of Neisseria gonorrhoeae are Q (glutamine: hydrophilic, uncharged), K (lysine: hydrophilic, positive charge), and Q (glutamine: hydrophilic, uncharged), and the type of amino acids and the order of polarity differ for each bacterial species (Figure 5).

As described above, the three-dimensional structure and polarity of amino acid residues in the region of positions 102 to 114 of Legionella L7/L12 are significantly different from those of the other two bacterial species, and therefore it is predicted to be close to the central site of interaction with antibodies (Figures 4 and 5). Furthermore, based on the difference in amino acid polarity between the three bacterial species, it is predicted that, among the amino acid residues constituting the CTD of Legionella L7/L12, preferably amino acid residues at positions 102, 106, 109 to 110, or 112 to 114 are central to interaction with antibodies (Figure 5).

[Example 5] Obtaining antibodies that bind to ribosomal protein L7/L12 of Legionella bacteria According to the following procedure, the CTD formed by amino acid residues at positions 58 to 125 of ribosomal protein L7/L12 of Legionella bacteria was expressed alone, and 4B1, 36A2, and 54A3 were obtained as monoclonal antibodies that recognize this by antigen-antibody reaction.

<1: Preparation of Escherichia coli expressing only the CTD of Legionella ribosomal protein L7/L12>
An artificially synthesized gene (manufactured by GenScript) containing a base sequence (SEQ ID NO: 4) encoding an amino acid sequence (SEQ ID NO: 3) consisting of amino acid residues at positions 58 to 125 of the ribosomal protein L7/L12 of Legionella pneumophila to which BamHI and XhoI restriction enzyme cleavage sites have been added was cleaved with the two types of restriction enzymes described above, and then electrophoresed in a 1.5% agarose gel and stained with ethidium bromide. A band of approximately 400 bp was excised from the gel. This band was purified using a QIAquick Gel Extraction Kit (manufactured by QIAGEN) and inserted into a common vector, pGEX-6P-1 (manufactured by GE Healthcare).

Specifically, the vector pGEX-6P-1 and the above DNA (artificially synthesized gene digested with restriction enzymes and gel-purified) were mixed at a molar ratio of 1:3, and the DNA was incorporated into the vector using T4 DNA ligase (Invitrogen). The vector pGEX6P-1 incorporating the DNA was genetically introduced into the BL21(DE3)pLysS Escherichia coli strain (Promega), and then inoculated onto LB L-broth agar (Takara Shuzo Co.), a semi-solid culture plate containing 50 μg/mL ampicillin (Sigma). The plate was incubated at 37°C for 12 hours, and grown colonies were randomly selected and inoculated into L-broth culture medium containing the same concentration of ampicillin. After 8 hours of shaking culture at 37°C, the cells were harvested by centrifugation, and the plasmid was isolated using a QIAprep Spin Miniprep Kit (QIAGEN) according to the attached instructions. The resulting plasmid was cleaved with restriction enzymes BamHI/XhoI to cleave approximately 370 bp of DNA, confirming the insertion of the PCR product. The base sequence of the inserted DNA was determined using the above clone.

Specifically, the base sequence of the inserted DNA fragment was determined using a fluorescent sequencer (Applied Biosystems). The sequence sample was prepared using PRISM, Ready Reaction Dye Terminator Cycle Sequencing Kit (Applied Biosystems). First, 9.5 μL of restriction enzyme reaction solution, 4.0 μL of T7 promoter primer (Gibco BRL) (concentration 0.8 pmol/μL), and 0.16 μg/μL of template DNA (concentration 6.5 μL) were added to a 0.5 mL microtube and mixed. The mixture was covered with two layers of 100 μL mineral oil, and then 25 cycles of PCR amplification treatment were performed. Here, one cycle consists of a treatment at 96°C for 30 seconds, a treatment at 55°C for 15 seconds, and a treatment at 60°C for 4 minutes. The product was kept at 4°C for 5 minutes. After the reaction was completed, 80 μL of sterile purified water was added and stirred. The product was centrifuged, and the aqueous layer was extracted three times with a phenol-chloroform mixture. 10 μL of 3M sodium acetate pH 5.2 and 300 μL of ethanol were added to the 100 μL aqueous layer and stirred. The mixture was then centrifuged at 14,000 rpm at room temperature for 15 minutes to collect the precipitate. The precipitate was washed with 75% ethanol, then left to dry under vacuum for 2 minutes to prepare a sequencing sample. The sequencing sample was dissolved in 4 μL of formamide containing 10 mM EDTA and denatured at 90°C for 2 minutes. This was cooled on ice and used for sequencing.

Two of the five randomly selected clones had sequence homology with the probe used in PCR. In addition, the DNA sequence clearly matched the gene sequence of amino acid residues 58-125 of the Legionella ribosomal protein L7/L12. This gene fragment clearly encodes the CTD consisting of amino acid residues 58-125 of the Legionella ribosomal protein L7/L12.

<2: Preparation of Legionella ribosomal protein L7/L12 CTD by culturing E. coli expressing the CTD alone>
Using Escherichia coli that expresses solely the CTD formed by the amino acid residues at positions 58 to 125 of the Legionella ribosomal protein L7/L12 obtained above, a protein (CTD protein) corresponding to the CTD formed by the amino acid residues at positions 58 to 125 of the Legionella ribosomal protein L7/L12 was prepared.

Specifically, the E. coli strain was cultured in M9 _medium (47.7 mM _{Na2HPO4.12H2O} , ₂₂ mM _KH2PO4 , 8.6 mM NaCl, _{2 mM} MgSO4, 50 μM _ZnSO4 , 100 μM _CaCl2 , 4.1 μM biotin, 7.2 μM choline chloride, 2.3 μM folic acid, 8.2 μM nicotinamide, 4.6 μM calcium pantothenate, 6 μM pyridoxal hydrochloride, 0.3 μM riboflavin, 16.6 μM thiamine hydrochloride, 27 mM sodium ampicillin, 18.7 mM _NH4Cl , 11.1 mM glucose) at 37° C. until the _OD600 reached 0.6, and then rapidly cooled with ice water. IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 1 mM, and the mixture was cultured at 16°C for 36 hours, followed by centrifugation at 7000 rpm for 15 minutes at 4°C to recover E. coli. 5 mL of BugBuster (Merck Millipore) and 5 μL of benzonase endonuclease (Merck Millipore) were added per 1 g of the resulting E. coli, and the mixture was shaken at room temperature for 30 minutes to completely dissolve the E. coli. After filtration through a 0.45 μm filter, ribosomal protein L7/L12 was purified on a glutathione-Sepharose column using a Profinia protein purification system (Bio-Rad). 1.5 mL of 10x PBS (phosphate buffered saline) and Prescission protease (GE Healthcare) were added to 15 mL of the resulting protein solution, and the mixture was shaken at room temperature for 2 hours. The reaction mixture was passed through the glutathione-Sepharose column again, and the flow-through fraction was collected as a fraction containing the CTD protein.

The obtained CTD protein-containing fraction was dialyzed against 50 mM sodium phosphate pH 6.8 as the external solution, diluted with 4 times the amount of 20 mM Tris-HCl pH 8.0, and passed through an AKTA protein purification system (GE Healthcare) connected to an ion exchange column RESOURCE Q (GE Healthcare) at a flow rate of 2 mL/min. Next, 20 mM Tris-HCl pH 8.0 containing 1 M NaCl was passed at a flow rate of 2 mL/min so as to increase linearly from 0% to 50%, and the CTD protein was eluted.

The obtained ribosomal protein L7/L12 was passed through an AKTA protein purification system (GE Healthcare) connected to a gel filtration column HiLoad 16/60 Superdex 75pg (GE Healthcare) at a flow rate of 1 mL/min using PBS (phosphate buffered saline) as the mobile phase, and the ribosomal protein L7/L12 was purified and its concentration was quantified using Protein Assay Kit I (BIO-RAD, model number 5000001JA), and it was used as the CTD protein of ribosomal protein L7/L12 of Legionella bacteria for obtaining monoclonal antibodies.

<3: Obtaining a mouse monoclonal antibody strain using the CTD protein of Legionella ribosomal protein L7/L12>
Using the CTD protein of ribosomal protein L7/L12 of Legionella bacteria for obtaining the monoclonal antibodies obtained, three monoclonal antibody strains against the CTD protein, 4B1, 36A2 and 54A3, were obtained according to the method described in Example 3 of WO 2001/057199.

Specifically, 100 μg of the CTD protein of the ribosomal protein L7/L12 of Legionella bacteria obtained by the above procedure was used as an antigen and dissolved in 200 μL of PBS, and then 200 μL of Freund's Complete Adjuvant was added and mixed to form an emulsion. 200 μL of the obtained antigen emulsion was injected into the abdominal cavity of a mouse. In addition, the same antigen emulsion was injected into the abdominal cavity of a mouse 2 weeks, 4 weeks, and 6 weeks after the first antigen administration. Furthermore, 10 weeks and 14 weeks after the first antigen administration, a double concentration of the antigen emulsion was injected into the abdominal cavity of a mouse. Three days after the final antigen administration, the spleen was removed from the mouse, splenocytes were collected aseptically, and cell fusion with myeloma cells was performed according to the following procedure.

The myeloma cells used were the NS-1 cell line. The cell line was cultured in RPMI 1640 medium containing 10% fetal bovine serum, and from two weeks prior to cell fusion, it was cultured for one week in RPMI 1640 medium containing 0.13 mM azaguanine, 0.5 μg/mL MC-210, and 10% fetal bovine serum, and then further cultured for one week in RPMI 1640 medium containing 10% fetal bovine serum before use.

10 ⁸ mouse splenocytes collected aseptically according to the above procedure and 2×10 ⁷ myeloma cells after the above culture were mixed well in a glass tube, then centrifuged at 1,500 rpm for 5 minutes, the supernatant was discarded, and the cells were further mixed well. 50 mL of RPMI1640 culture medium maintained at 37° C. was added to this mixed cell sample, centrifuged at 1,500 rpm, the supernatant was removed, 1 mL of 50% polyethylene glycol maintained at 37° C. was added, and the mixture was stirred for 1 minute. 10 mL of RPMI1640 culture medium maintained at 37° C. was added to this cell mixture, and vigorously stirred by aspirating and discharging with a sterilized pipette for about 5 minutes, then centrifuged at 1,000 rpm for 5 minutes, the supernatant was removed, and 30 mL of HAT culture medium was added so that the cell concentration was 5×10 ⁶ /mL, and the mixture was stirred until homogenous. 0.1 mL of this cell mixture was poured into each well of a 96-well culture plate and cultured in a 7% carbon dioxide atmosphere at 37° C. 0.1 mL of HAT medium was added on the first day, first week, and second week after the start of culture, and fused cells producing the desired antibody were screened by ELISA.

Cells producing the desired antibodies were screened by ELISA. The CTD protein of the ribosomal protein L7/L12 of Legionella bacteria was fused with glutathione S-transferase (GST) protein to prepare the GST fusion L7/L12CTD protein. The obtained GST fusion L7/L12CTD protein and GST protein were dissolved in PBS containing 0.05% sodium azide at a concentration of 10 μg/mL to prepare a diluted solution. 100 μL of each of these solutions was dispensed separately into each well of a 96-well plate and allowed to adsorb overnight at 4°C. After removing the supernatant, 200 μL of a 1% bovine serum albumin solution (in PBS) was added and reacted at room temperature for 1 hour to block. The supernatant was removed and the product was washed with a washing solution (PBS containing 0.02% Tween 20), after which 100 mL of the culture solution of the fused cells was added and allowed to react at room temperature for 2 hours. The supernatant was removed, the precipitate was washed with a washing solution, and then 100 μL of a peroxidase-labeled goat anti-mouse IgG antibody solution (concentration: 50 ng/mL) was added and reacted at room temperature for 1 hour. The supernatant was removed, the product was washed again with a washing solution, and then 100 μL of TMB solution (KPL) was added and reacted at room temperature for 20 minutes. When coloration occurred, 100 μL of 1N sulfuric acid was added to each cell to stop the reaction, and the absorbance at 450 nm was measured.

As a result, positive wells were found that reacted only to the GST fusion L7/L12CTD protein and not to the GST protein, and it was found that these wells contained antibodies against the L7/L12CTD protein. Therefore, the cells in each positive well were collected, placed in a 24-well plastic plate, and cultured with HAT medium, and then diluted with HT medium so that the cell count was about 20 cells/mL, and 50 μL of the diluted solution was placed in each well of a 96-well culture plate. After adding and mixing 10 ⁶ thymocytes of 6-week-old mice suspended in HT medium, the cells were cultured at 37° C. under 7% CO ₂ conditions for 2 weeks. The antibody activity in the culture supernatant was similarly assayed by the above-mentioned ELISA method, and cells that reacted positively with the L7/L12CTD protein were collected. Furthermore, by repeating the same dilution assay and cloning operation, each hybridoma producing monoclonal antibody strains 4B1, 36A2 and 54A3 against the L7/L12CTD protein was obtained.

The three positive clone monoclonal antibody strains 4B1, 36A2, and 54A3 obtained as described above were used to produce and collect monoclonal antibodies according to standard methods.

<4: Determination of the amino acid sequences of the light and heavy chain variable regions of the obtained mouse monoclonal antibody strain>
For the three obtained monoclonal antibody strains, 4B1, 36A2, and 54A3, the amino acid sequences and corresponding nucleotide sequences of the light and heavy chain variable regions were determined according to standard methods.

The amino acid sequences and nucleotide sequences of the heavy and light chain variable regions of monoclonal antibody strains 4B1, 36A2, and 54A3 are shown in the sequence numbers in Table 1 below.

[Example 6] Analysis of interaction between antibodies and ribosomal protein L7/L12 of Legionella bacteria The monoclonal antibodies 4B1, 36A2, and 54A3 obtained in Example 5, which recognize the CTD of ribosomal protein L7/L12 of Legionella bacteria, were subjected to NMR analysis of their interaction with ribosomal protein L7/L12 of Legionella bacteria by the following procedure.

<1: Obtaining ribosomal protein L7/L12 from Legionella pneumophila>
The E. coli strain prepared in Example 1 was cultured in M9 medium (47.7 mM _{Na2HPO4.12H2O} , _{22 mM KH2PO4} , 8.6 mM NaCl, 2 mM _MgSO4 , 50 μM _ZnSO4 , 100 _{μM CaCl2} , 4.1 μM biotin, 7.2 μM choline chloride, 2.3 μM folic acid, 8.2 μM nicotinamide, 4.6 μM calcium pantothenate, 6 μM pyridoxal hydrochloride, 0.3 μM riboflavin, 16.6 μM thiamine hydrochloride, 27 mM sodium ampicillin, 18.7 ^{mM 15N} - _NH4Cl , 11.1 mM ^12C -glucose) at OD The mixture was cultured until the pH ₆₀₀ reached 0.6, and then rapidly cooled with ice water. IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 1 mM, and the mixture was cultured at 16°C for 36 hours, followed by centrifugation at 7000 rpm for 15 minutes at 4°C to recover E. coli. 5 mL of BugBuster (Merck Millipore) and 5 μL of benzonase endonuclease (Merck Millipore) were added per 1 g of the resulting E. coli, and the mixture was shaken at room temperature for 30 minutes to completely dissolve the E. coli. After filtration through a 0.45 μm filter, ribosomal protein L7/L12 was purified on a glutathione-Sepharose column using a Profinia protein purification system (Bio-Rad). To 15 mL of the resulting protein solution, 1.5 mL of 10x PBS (phosphate buffered saline) and Prescission protease (GE Healthcare) were added and shaken at room temperature for 2 hours. The reaction solution was passed through the glutathione-Sepharose column again, and the non-reacted fraction was collected as ribosomal protein L7/L12.

The obtained ribosomal protein L7/L12 was dialyzed against 50 mM sodium phosphate pH 6.8 as an external solution, diluted with 4 times the amount of 20 mM Tris-HCl pH 8.0, and passed through an AKTA protein purification system (GE Healthcare) connected to an ion exchange column RESOURCE Q (GE Healthcare) at a flow rate of 2 mL/min. Subsequently, 20 mM Tris-HCl pH 8.0 containing 1 M NaCl was passed at a flow rate of 2 mL/min so as to increase linearly from 0% to 50%, and the ribosomal protein L7/L12 was eluted.
The obtained ribosomal protein L7/L12 was passed through an AKTA protein purification system (GE Healthcare) connected to a gel filtration column HiLoad 16/60 Superdex 75pg (GE Healthcare) at a flow rate of 1 mL/min using PBS (phosphate buffered saline) as the mobile phase to purify the ribosomal protein L7/L12. The concentration was quantified using Protein Assay Kit I (BIO-RAD, model number 5000001JA) and then subjected to NMR measurement.

<2: Pretreatment of antibodies against Legionella ribosomal protein L7/L12>
The monoclonal antibodies 4B1, 36A2, and 54A3 obtained in Example 5, which recognize the CTD of the ribosomal protein L7/L12 of Legionella bacteria, were dialyzed against PBS (phosphate buffered saline) as an external solution, and their concentrations were quantified by the absorbance of ultraviolet light (wavelength 280 nm), and then subjected to NMR measurement.

<3: Analysis of the interaction between Legionella ribosomal protein L7/L12 and antibodies by NMR>
Legionella ribosomal protein L7/L12 was mixed with antibodies 4B1, 36A2, and 54A3. The mixing ratio of L7/L12 to antibody 4B1 or 36A2 was 1:1 (molar ratio), and the mixing ratio of L7/L12 to antibody 54A3 was 1:1.5 (molar ratio). Each mixture was made up to 250 μL with PBS (phosphate buffered saline), and then 20 μL of heavy water, 1 μL of 5 mg/mL DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid), EDTA (ethylenediaminetetraacetic acid), and AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride) were added to a final concentration of 1.8 mM, transferred to a Shigemi NMR sample tube, degassed with an aspirator, and placed in the NMR device.

FID was obtained using a pulse sequence of [ ¹ H- ¹⁵ N] HSQC (accumulation number 128, number of data points (F1×F2) 512×2048) on an AVANCE III HD 600 MHz NMR device (manufactured by Bruker). The obtained FID was Fourier transformed using NMR Pipe software to obtain a spectrum. The attribution information of Example 1 was added to the obtained spectrum on Sparky software.

In NMR, if the molecular weight is low (13,000) like ribosomal protein L7/L12, it can be observed as a sharp signal, but if the molecular weight is high (150,000) like an antibody, the signal becomes very broad and unclear, so it cannot be observed due to insufficient intensity. In addition, the binding/dissociation constant of an antigen-antibody reaction is generally 1 μM or less, and it is considered to be in an equilibrium state biased toward the binding side. Therefore, when ribosomal protein L7/L12 interacts (binds) with an antibody, it is considered that the signal of ribosomal protein L7/L12 becomes broad under the influence of the antibody and its intensity is attenuated. Therefore, we attempted to identify the interaction site by tracking the residues whose signal intensity is attenuated by the addition of the antibody on the [ ¹ H- ¹⁵ N] HSQC spectrum of ribosomal protein L7/L12 (one signal is observed per amino acid). Details of the interaction analysis were as follows: Williamson, MP, Using chemical shift perturbation to characterise ligand binding, Prog. Nucl. Magn. Reson. Spectrosc. 73(2013):1-16.

The [ ^1H - ^15N ] HSQC signal intensity of each amino acid residue of ribosomal protein L7/L12 when ribosomal protein L7/L12 and antibody 4B1, 36A2, or 54A3 were mixed in the above-mentioned ratios, respectively, was divided by the [ ^1H - ^15N ] HSQC signal intensity of each amino acid residue of ribosomal protein L7/L12 before mixing with the antibody, and a graph was created showing the divided value on the vertical axis and the amino acid sequence of ribosomal protein L7/L12 on the horizontal axis (Figures 6A, B, and C). Since the division values of the CTD (residues 58 to 125) were generally 0.4 or less (meaning that the signal intensity of ribosomal protein L7/L12 was attenuated by 60% or more upon interaction with the antibody), it was found that ribosomal protein L7/L12 interacted with antibody 4B1, 36A2, or 54A3 at the CTD (residues 58 to 125) (Figures 6A, B, and C).

[Example 7] Examination of cross-reactivity of antibodies that bind to ribosomal protein L7/L12 of Legionella bacteria The monoclonal antibodies 4B1, 36A2, and 54A3 obtained in Example 5, which recognize the CTD of ribosomal protein L7/L12 of Legionella bacteria, were analyzed by ELISA for cross-reactivity with ribosomal protein L7/L12 of bacteria other than Legionella bacteria by the following procedure.

<1: Preparation of recombinant full-length ribosomal proteins L7/L12 of various bacterial species for cross-reactivity analysis>
Recombinant full-length ribosomal protein L7/L12 for cross-reactivity tests was prepared by the following method: First, a plasmid vector pGEX-6P-1 was prepared containing an artificially synthesized gene (GenScript) with a base sequence encoding the amino acid sequence of ribosomal protein L7/L12 of each of the bacterial species shown in Table 2 below, which was used as the target bacterial species for cross-reactivity analysis.

The plasmid vector pGEX-6P-1 carrying the L7/L12 genes of each bacterial species obtained was introduced into E. coli One Shot Competent Cells (Invitrogen), and inoculated onto a plate of semi-solid medium of LB medium (Takara Shuzo) containing 50 μg/mL ampicillin (Sigma), and left at 37°C for about 12 hours. The resulting colonies were randomly selected and inoculated into 2 mL of LB liquid medium containing the same concentration of ampicillin, and cultured with shaking at 37°C for about 8 hours to recover the bacterial cells. The plasmid was isolated from the obtained bacterial cells using a QIAprep Spin Miniprep Kit (QIAGEN) according to the attached instructions. The obtained plasmid was digested with restriction enzymes BamHI/XhoI. The insertion of the artificially synthesized ribosomal protein L7/L12 genes of each bacterial species was confirmed by cutting the DNA at about 370 bp. The E. coli into which the plasmid vector was introduced was cultured overnight at 37°C in 50 mL of LB medium, then transferred to 500 mL of TB medium and cultured for 1 hour. Then, 550 μL of 100 mM isopropyl β-D(-)-thiogalactopyranoside (IPTG) was added and cultured for an additional 4 hours. After recovery, 1/100th the amount of BugBuster (Merck) was added and shaken at room temperature for 20 minutes. Then, the mixture was centrifuged at 10,000 rpm for 30 minutes to recover the E. coli.

Recombinant full-length ribosomal protein L7/L12 was collected and purified using the same method as in Example 1, and recombinant full-length ribosomal protein L7/L12 of each bacterial species was obtained.

<2: Implementation of ELISA for cross-reactivity analysis>
The recombinant ribosomal protein L7/L12 of each of the bacterial species was immobilized on an ELISA plate, reacted with each of the monoclonal antibodies 4B1, 36A2, and 54A3, and then washed. The monoclonal antibodies bound to the immobilized L7/L12 of each of the bacterial species were then reacted with peroxidase-labeled anti-mouse IgG antibodies to evaluate the cross-reactivity of each monoclonal antibody with the L7/L12 of each of the bacterial species.

Specifically, 100 μL of each PBS solution containing Legionella pneumophila and the recombinant full-length ribosomal protein L7/L12 of each of the above-mentioned bacterial species at a concentration of 0.01 μg/mL, 0.1 μg/mL, or 1 μg/mL was dispensed into a 96-well ELISA plate (Maxsorp ELISA plate manufactured by Nunc) and allowed to adsorb overnight at 4°C. After removing the supernatant, 200 μL of 1% bovine serum albumin solution (in PBS) was added and reacted at room temperature for 1 hour for blocking. After removing the supernatant, the plate was washed several times with a washing solution (PBS containing 0.02% Tween 20), and 100 μL of antibody solution in which antibody 4B1, 36A2, or 54A3 was diluted with 0.5% TritonX-100/PBS to 1 μg/mL, or about 1×PBS itself (negative control) was added, and allowed to react at room temperature for 1 hour. After removing the supernatant, 100 μL of a solution of a peroxidase-labeled anti-mouse IgG antibody reagent as a secondary antibody diluted with 0.02% Tween 20/PBS to a final concentration of 1 μg/mL was added and reacted at room temperature for 1 hour. After removing the supernatant, the plate was washed several times with a washing solution, and 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) solution (KPL) was added and reacted at room temperature for 10 minutes, after which 100 μL of 1 mol/L hydrochloric acid was added to stop the reaction. The absorbance at 450 nm of the obtained solution was measured, and the difference from the absorbance at 450 nm of the negative control was calculated to evaluate the cross-reactivity of each monoclonal antibody with L7/L12 of each bacterial species.

The resulting evaluation results of the reactivity of antibodies 4B1, 36A2, or 54A3 with recombinant full-length ribosomal protein L7/L12 of Legionella or other bacterial species are shown in Table 3 below. In the table below, positive (+) indicates that the difference in absorbance from the negative control is 0.5 or more, and negative (-) indicates that the difference in absorbance from the negative control is 0.1 or less.

The present invention can be widely used in fields requiring the detection of Legionella bacteria in samples, primarily in the medical field, and is extremely useful industrially.

Claims (11)

An antibody or an antigen-binding fragment thereof for detecting Legionella pneumophila,
It causes an antigen-antibody reaction with an epitope present in the C-terminal domain (CTD) consisting of amino acid residues 58 to 125 of the ribosomal protein L7/L12 of Legionella pneumophila shown in SEQ ID NO: 1, and
The heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NO:5, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NO:7, or
The heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 9, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 11, or
The heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 13, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 15.
An antibody or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof according to claim 1, wherein the epitope comprises one or more amino acid residues selected from amino acid residues at positions 102 to 114 of SEQ ID NO:1. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which does not cross-react with bacteria of one or more genera selected from the genera Mycoplasma, Escherichia, Chlamydia, Salmonella, Pseudomonas, Staphylococcus, Neisseria, Haemophilus, Bordetella, Moraxella, and Streptococcus. An antibody or an antigen-binding fragment thereof for detecting Legionella pneumophila,
The heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NO:5, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NO:7, or
The heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 9, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 11, or
The heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 13, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NO: 15.
An antibody or an antibody-binding fragment thereof. A nucleic acid molecule encoding the antibody or antibody-binding fragment thereof according to any one of claims 1 to 4. A vector or plasmid comprising the nucleic acid molecule of claim 5. A host cell transformed with the nucleic acid molecule of claim 5 or the vector or plasmid of claim 6. The host cell according to claim 7, wherein the host cell is a eukaryotic cell selected from a mammalian cell, an insect cell, a yeast cell, and a plant cell, or a bacterial cell. A method for detecting the presence or absence of Legionella bacteria in a sample, the method comprising contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and detecting the presence or absence of an antigen-antibody reaction. A reagent for detecting the presence or absence of Legionella bacteria in a sample, the reagent comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4. A kit for detecting the presence or absence of Legionella bacteria in a sample, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and an instruction manual including instructions for detecting the presence or absence of Legionella bacteria in a sample using the antibody.

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