JP7491679B2 - Antibody for detecting Salmonella in a sample, and method, reagent, and kit for detecting Salmonella using the antibody - Google Patents

Description

The present invention relates to an antibody for detecting the presence or absence of Salmonella enteritidis, a causative bacterium of pneumonia, in a sample, as well as a method, reagent, and kit for detecting Salmonella 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 combination of various marker antigens and antibodies depending on the bacterial species.

Salmonella enteritidis is a gram-negative enterobacterium that lives in the digestive tract of humans and animals, invading intestinal epithelial cells and causing food poisoning. To confirm a Salmonella infection, it is necessary to detect the bacteria from feces, blood, puncture fluid, lymph fluid, etc., and there is currently no rapid diagnostic method. A specific rapid diagnostic method for Salmonella is needed to prevent the worsening of symptoms and the spread of infection.

The usual method for detecting Salmonella is to test and detect it by isolating and culturing it using selective media. However, there are issues with detection by culture methods, including the time required for testing, as isolating and culturing the bacteria takes one to two days at room temperature (Patent Document 1: Patent No. 6104242). In response to this, a method was known that shortens the culture time by using hybridization technology with a DNA probe (Patent Document 2: Patent No. 2089328), but there were issues with the method being insufficiently rapid and the complicated operation, leaving room for improvement.

The present inventors focused on ribosomal protein L7/L12 as an intracellular molecule that is present in all microbial cells and whose amino acid structure differs to some extent between microorganisms, and discovered a method that uses antibodies against such proteins to specifically detect various microorganisms and comprehensively detect various serotypes within the same species (Patent Document 3: International Publication No. 2000/006603). However, there was room for improvement in the detection accuracy (bacterial species specificity) of such existing antibodies, and the binding pattern of specific antibodies to Salmonella ribosomal protein L7/L12 to ensure the detection accuracy was unknown.

Japanese Patent No. 6104242 Patent No. 2089328 International Publication No. 2000/006603

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

After extensive research, the inventors discovered that within the N-terminal domain (NTD) consisting of amino acid residues 1 to 40 of the ribosomal protein L7/L12 of Salmonella 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 these antibodies can be used to detect Salmonella bacteria in samples with high sensitivity and accuracy, thereby arriving at the present invention.

That is, the gist of the present invention is as follows.
[1] An antibody for detecting Salmonella enteritidis, which undergoes an antigen-antibody reaction with an epitope present in the N-terminal domain (NTD) consisting of amino acid residues 1 to 40 of the ribosomal protein L7/L12 of Salmonella 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 4 to 22 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, Pseudomonas, Streptococcus, Staphylococcus, Neisseria, Haemophilus, Bordetella, Moraxella, and Legionella.
[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 Salmonella 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 Salmonella 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 Salmonella 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 Salmonella in a sample using the antibody.

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

FIG. 1 is a schematic diagram showing the three-dimensional structure of Salmonella ribosomal protein L7/L12. FIG. 2 is a diagram showing a schematic representation of the three-dimensional structure of ribosomal protein L7/L12 of Mycoplasma pneumoniae. FIG. 3 is a schematic diagram showing the three-dimensional structure of ribosomal protein L7/L12 of Haemophilus influenzae. FIG. 4 is a schematic diagram showing the three-dimensional structures of the N-terminal domain (NTD) portions of ribosomal proteins L7/L12 of Salmonella, Mycoplasma pneumoniae, and Haemophilus influenzae. FIG. 5 shows an alignment of amino acid sequences of the N-terminal domain (NTD) portions of ribosomal proteins L7/L12 of Salmonella typhimurium, Mycoplasma pneumoniae, and Haemophilus influenzae. FIG. 6A is a schematic diagram showing the results of NMR analysis of the interaction between monoclonal antibody 31E2-F5-F9 and Salmonella ribosomal protein L7/L12. FIG. 6B is a schematic diagram showing the results of NMR analysis of the interaction between monoclonal antibody 33C9-F6-D1 and Salmonella ribosomal protein L7/L12. FIG. 6C is a schematic diagram showing the results of NMR analysis of the interaction between monoclonal antibody 36G2-F9-D2 and Salmonella 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 Salmonella:
A first aspect of the present invention relates to an antibody for detecting Salmonella (hereinafter, appropriately referred to as "the antibody of the present invention").

(1) Introduction:
In the present invention, "Salmonella enteritidis" or "Salmonella enterica serovar Enteritidis" refers to a gram-negative facultative anaerobic bacillus belonging to the genus Salmonella in the family Enterobacteriaceae, which mainly inhabits the digestive tract of humans and animals.

Salmonella is a type of intracellular parasite (facultative intracellular parasitic bacteria, intracellular parasite) that can grow both inside and outside the infected host cell, and has the ability to actively act on cells to activate endocytosis. As a result, it has the ability to invade not only cells with phagocytic activity such as macrophages, but also intestinal epithelial cells that normally do not have phagocytic activity.

Salmonella bacteria are pathogenic to humans and can cause gastroenteritis (food poisoning). Food poisoning caused by Salmonella bacteria is accompanied by digestive symptoms such as abdominal pain, vomiting, and diarrhea, as well as fever (high fever). Patients with low resistance may develop bacteremia, leading to severe symptoms, and in rare cases, may develop endotoxin-induced sepsis, which can lead to death.

Diagnosis of Salmonella infection is first made based on test findings such as an increase in white blood cell count and inflammatory responses such as CRP, and an increase in transaminase. A definitive diagnosis requires detection of the bacteria in stool, blood, puncture fluid, lymph fluid, etc. However, there is currently no specific rapid diagnostic method for Salmonella.

In principle, treatment for Salmonella infection involves symptomatic treatment, focusing on correcting dehydration caused by fever and diarrhea and relieving gastrointestinal symptoms such as abdominal pain. However, there is a demand for a specific rapid diagnostic method for Salmonella in order to prevent symptoms from worsening and the spread of infection.

The present inventors focused on the ribosomal protein L7/L12 when preparing an antibody for detecting Salmonella. 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 commonly found in various bacteria. The ribosomal protein L7/L12 of Salmonella has a dimeric structure in which two copies of a monomer molecule consisting of 121 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 Salmonella 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 53 to 121 via a linker that does not form a three-dimensional structure consisting of amino acid residues 41 to 52. 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 two molecules of ribosomal protein L7/L12 from Mycoplasma pneumoniae and Haemophilus influenzae associate with each other at the NTD (N-Terminal Domain, residues 1-40) to form a dimer, which then passes through a random coil structure linker (residues 41-54) to form a CTD (C-Terminal Domain, residues 55-122 in the case of Mycoplasma pneumoniae and residues 55-123 in the case of Haemophilus influenzae) (see Examples 2 and 3 and Figures 2 and 3 below).

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

Comparing the three-dimensional structures of the NTDs of L7/L12 from H. influenzae, M. pneumoniae, and Salmonella, we found that there were significant differences in the surface shape and charge distribution (Figure 4). For example, in the cases of H. influenzae (HI) and Salmonella (SE), the three N-terminal residues of the dimer protrude from the top of the structure, but in the case of M. pneumoniae (MP), no protrusions are observed. In addition, this protrusion is located in one place in H. influenzae, but in two places in Salmonella. Comparing the surface charges of the NTDs, in the case of M. pneumoniae, the center of the three-dimensional structure is occupied by hydrophobic (white) to uncharged hydrophilic regions (light blue, light red), while in the cases of H. influenzae and Salmonella, it is occupied by negative charges (red). In addition, in the case of H. influenzae, the negatively charged region (red) is located on the right side of the three-dimensional structure, and the positively charged region (blue) is located on the left side. In contrast, in the case of Salmonella, negative charges (red) are concentrated in the center of the three-dimensional structure, and hydrophobic regions (white) dominate at both ends of the structure (see Example 4 and Figure 4 below).

Furthermore, a comparison of the amino acid sequences of ribosomal protein L7/L12 from H. influenzae, M. pneumoniae, and Salmonella revealed that there were large differences between the bacterial species in residues 1-40 (Figure 5). For example, residues 5-7 of M. pneumoniae correspond to residues 4-6 of H. influenzae and Salmonella, and the amino acid sequence of M. pneumoniae is D (aspartic acid: hydrophilic, negative charge), K (lysine: hydrophilic, positive charge), and N (asparagine: hydrophilic, uncharged), whereas H. influenzae has T (threonine: hydrophilic, uncharged), N (asparagine: hydrophilic, uncharged), and Salmonella has T (threonine: hydrophilic, uncharged), K (lysine: hydrophilic, positive charge), and D (aspartic acid: hydrophilic, negative charge), indicating that the type and polarity of amino acids differs between the bacterial species. Similarly, residues 14-16 of Mycoplasma pneumoniae correspond to residues 13-15 of Haemophilus influenzae and Salmonella typhimurium, and the amino acid sequence of Mycoplasma pneumoniae is K (lysine: hydrophilic, positive charge), E (glutamic acid: hydrophilic, negative charge), M (methionine: hydrophobic), whereas Haemophilus influenzae has A (alanine: hydrophobic), S (serine: hydrophilic, uncharged), K (lysine: hydrophilic, positive charge), and Salmonella has S (serine: hydrophilic, uncharged), A (alanine: hydrophobic), M (methionine: hydrophobic), and the type of amino acids and the order of polarity differ for each bacterial species (see Example 4 and Figure 5 described below).

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

Then, a peptide having an amino acid sequence similar to that of NTD was expressed, and an antibody that specifically binds to the peptide was produced and screened to obtain antibodies 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9-D2 (see Example 5 below). It was then confirmed that all of these antibodies reacted with the NTD of L7/L12 of Salmonella, particularly with an epitope that contains a specific amino acid residue (see Example 6 and Figures 6A to 6C below). Furthermore, it was confirmed that all of these antibodies have excellent detection sensitivity and detection accuracy (bacterial species specificity) (see Example 7 below), and the present invention was completed.
This will be explained in more detail 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 isotypes of antibodies, 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 Salmonella 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 Salmonella 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 a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. A polyclonal antibody is usually prepared from the serum of an animal immunized with an antigen, and is a mixture of various antibody molecular species with different structures. On the other hand, a monoclonal antibody refers to an antibody consisting of a single type of molecule containing a combination of a light chain variable (VL) region and a heavy chain variable (VH) region having a specific amino acid sequence. A monoclonal antibody can be produced from a clone derived from an antibody-producing cell, but it can also be produced by genetic engineering using a nucleic acid molecule obtained having a gene sequence that codes for the amino acids of the antibody protein. In addition, it is a well-known technique to those skilled in the art to carry out modifications to improve the binding and specificity of an antibody using genetic information such as 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 also 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 Salmonella.

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 Salmonella ribosomal protein L7/L12, the epitope to which the antibody of the present invention binds is present in the NTD formed by amino acid residues at positions 1 to 40 in the amino acid sequence of the Salmonella ribosomal protein L7/L12 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 Salmonella detection antibodies 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9-D2, which have high detection sensitivity and high detection accuracy (high bacterial species specificity) actually obtained by the present inventors in the Examples, all bind to the NTD formed by amino acid residues at positions 1 to 40 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 the amino acid residues at positions 4 to 22 of SEQ ID NO: 1, and more 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 more preferably contains at least one or more amino acid residues selected from positions 4 to 15 of SEQ ID NO: 1, and even more preferably contains one or more amino acid residues selected from positions 4, 5, 6, 13, 14, and 15. According to the analysis results by 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 NTD of ribosomal protein L7/L12 of Salmonella. Furthermore, when comparing L7/L12 of Salmonella with L7/L12 of other bacterial species, these amino acid residues are at locations that are particularly highly species-specific for Salmonella, so 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 Salmonella 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, Pseudomonas, Streptococcus, Staphylococcus, Neisseria, Haemophilus, Bordetella, Moraxella, and Legionella. 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 variable regions of its heavy chain and light chain 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 a homology (preferably identity) of 80% or more, particularly 85% or more, even 90% or more, particularly 95% or more, or 96% or more, or 97% or more, or 99% or more, and especially 100% 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 a homology (preferably identity) of 80% or more, particularly 85% or more, even 90% or more, particularly 95% or more, or 96% or more, or 97% or more, or 99% or more, and especially 100% 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 a polypeptide consisting of all or a part of the ribosomal protein L7/L12 of the Salmonella bacterium to be detected, preferably the amino acid residues 1 to 40 of SEQ ID NO: 1 (amino acid residues of SEQ ID NO: 3) constituting the NTD, or a polypeptide having an amino acid sequence with 80% or more, particularly 85% or more, further 90% or more, particularly 95% or more, 96% or more, 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 causes 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 Salmonella bacteria and does not cross-react with other specific components, such as bacteria of the above-mentioned genera. 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 Salmonella 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 prepare 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 order to improve the performance of the obtained antibody or to avoid side effects, the structure of the constant region of the antibody or the sugar chain portion can be modified appropriately 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. Salmonella detection methods, reagents, and kits:
Another aspect of the present invention relates to a method for detecting the presence or absence of Salmonella 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 Salmonella 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 Salmonella in the 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 Salmonella bacteria and generates an antigen-antibody reaction, and therefore the detection sensitivity can be improved by exposing the ribosomal protein L7/L12 of Salmonella bacteria to the outside of the bacterial cell membrane. 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 Salmonella 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 Salmonella 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 Salmonella. Specific examples of kits that can easily detect the presence or absence of Salmonella in a sample include a lateral flow kit and a flow-through kit. Here, the lateral flow method is a method in which a sample to be detected and a detection antibody are dropped in order onto a membrane including a detection region having a capture antibody immobilized on its surface, and 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 sample to be detected and a detection antibody are dropped in order onto a membrane having a capture antibody immobilized on its surface, and passed vertically through the membrane, 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 Salmonella ribosomal protein L7/L12
<1: Obtaining ribosomal protein L7/L12 from Salmonella>
The pGEX-6P-1 plasmid (GE Healthcare) into which the Salmonella ribosomal protein L7/L12 gene (SEQ ID NO: 2) was cloned was introduced into E. coli BL21(DE3)pLysS E. coli strain (Promega).

The resulting E. coli strain was cultured 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) at OD 2 = 1.0 μM. 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 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 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 Salmonella 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 Å 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 1). 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 12 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 53 to 121 (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] Three-dimensional structural analysis of ribosomal protein L7/L12 of Mycoplasma pneumoniae
<1: Obtaining ribosomal protein L7/L12 from Mycoplasma pneumoniae>
The pGEX-6P-1 plasmid (GE Healthcare) into which the ribosomal protein L7/L12 gene (SEQ ID NO: 18) of Mycoplasma pneumoniae was cloned was introduced into the E. coli BL21(DE3)pLysS E. coli strain (Promega).

The resulting E. coli strain was cultured 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) at OD 2 = 1.0 μM. 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 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 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: Three-dimensional structure analysis of ribosomal protein L7/L12 of Mycoplasma pneumoniae 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 Å 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 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 14 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 55 to 122 (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 Haemophilus influenzae
<1: Obtaining ribosomal proteins L7/L12 from Haemophilus influenzae>
The pGEX-6P-1 plasmid (GE Healthcare) into which the ribosomal protein L7/L12 gene (SEQ ID NO: 20) of Haemophilus influenzae had been cloned was introduced into the E. coli BL21(DE3)pLysS E. coli strain (Promega).

The resulting E. coli strain was cultured 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) at OD 2 = 1.0 μM. 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 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 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 concentrated by centrifugation 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 ribosomal proteins L7/L12 of Haemophilus influenzae 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 Å 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 approximately 14 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 55 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 NTD portion of ribosomal protein L7/L12 of Salmonella, Mycoplasma pneumoniae, and Haemophilus influenzae Figure 4 is a schematic diagram showing the three-dimensional structure of the N-terminal domain (NTD) portion of ribosomal protein L7/L12 of Salmonella, Mycoplasma pneumoniae, and Haemophilus influenzae. As is clear from this figure, when comparing the three-dimensional structures of the NTD of ribosomal protein L7/L12 of Salmonella, Mycoplasma pneumoniae, and Haemophilus influenzae, a region was found with a large difference in surface shape and charge distribution (dotted line in Figure 4). When comparing the surface charge distribution of this region among the three bacterial species, in the case of Mycoplasma pneumoniae, the center is occupied by a hydrophobic (white) to non-charged hydrophilic region (light blue, light red). In the case of Haemophilus influenzae, almost the entire region is negatively charged (red), while in the case of Salmonella, the negatively charged region (red) accounts for a large proportion, but to the right there are hydrophobic (white) to uncharged hydrophilic regions (light blue, light red), showing differences between the two species.

Figure 5 shows an alignment of the amino acid sequences of the N-terminal domain (NTD) portion of ribosomal protein L7/L12 of Salmonella, Mycoplasma pneumoniae, and Haemophilus influenzae. As is clear from this figure, there were also significant differences in the amino acid sequences of the N-terminal domain (NTD) portion of ribosomal protein L7/L12 among the three bacterial species. This region is the amino acid residues 5 to 23 of L7/L12 of Mycoplasma pneumoniae, and corresponds to the amino acid residues 4 to 22 of L7/L12 of Haemophilus influenzae and Salmonella. The residues at positions 5 to 7 of M. pneumoniae correspond to the residues at positions 4 to 6 of H. influenzae and Salmonella. The amino acid sequence of M. pneumoniae is D (aspartic acid: hydrophilic, negatively charged), K (lysine: hydrophilic, positively charged), and N (asparagine: hydrophilic, uncharged), whereas that of H. influenzae is T (threonine: hydrophilic, uncharged), N (asparagine: hydrophilic, uncharged), and E (glutamic acid: hydrophilic, negatively charged), and that of Salmonella is T (threonine: hydrophilic, uncharged), K (lysine: hydrophilic, positively charged), and D (aspartic acid: hydrophilic, negatively charged), and the type and order of polarity of the amino acids differs depending on the bacterial species (Figure 5). In addition, the residues at positions 14 to 16 of M. pneumoniae correspond to the residues at positions 13 to 15 of H. influenzae and Salmonella. The amino acid sequence of M. pneumoniae is K (lysine: hydrophilic, positive charge), E (glutamic acid: hydrophilic, negative charge), and M (methionine: hydrophobic), whereas H. influenzae is A (alanine: hydrophobic), S (serine: hydrophilic, uncharged), K (lysine: hydrophilic, positive charge), and Salmonella is S (serine: hydrophilic, uncharged), A (alanine: hydrophobic), and M (methionine: hydrophobic), and the order of the amino acid types and polarities differs between the different bacterial species (Figure 5). Similarly, differences in polarity were observed between the amino acid residues at positions 19 and 22 of M. pneumoniae and the corresponding amino acid residues at positions 18 and 21 of H. influenzae and Salmonella (Figure 5).

As described above, the three-dimensional structure and polarity of amino acid residues at positions 4 to 22 of Salmonella L7/L12 are significantly different from those of the other two bacterial species, and are 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 NTD of Salmonella L7/L12, preferably positions 4 to 15, and more preferably positions 4 to 6 or 13 to 15, are central to interaction with antibodies (Figure 5).

Example 5: Obtaining antibodies that bind to Salmonella ribosomal protein L7/L12 According to the following procedure, the NTD formed by amino acid residues 1 to 40 of Salmonella ribosomal protein L7/L12 was expressed alone, and 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9-D2 were obtained as monoclonal antibodies that recognize this by antigen-antibody reaction.

<1: Preparation of E. coli expressing Salmonella ribosomal protein L7/L12 NTD alone>
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 1 to 40 of the ribosomal protein L7/L12 of Salmonella with additional BamHI and XhoI restriction enzyme cleavage sites 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 1-40 of Salmonella ribosomal protein L7/L12. This gene fragment clearly codes for the NTD gene consisting of amino acid residues 1-40 of Salmonella ribosomal protein L7/L12.

<2: Preparation of Salmonella NTD of ribosomal protein L7/L12 by culturing E. coli expressing the NTD alone>
Using E. coli that expresses solely the NTD formed by the amino acid residues at positions 1 to 40 of the Salmonella ribosomal protein L7/L12 obtained above, a protein (NTD protein) corresponding to the NTD formed by the amino acid residues at positions 1 to 40 of the Salmonella 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 solution was passed through the glutathione-Sepharose column again, and the flow-through fraction was collected as a fraction containing the NTD protein.

The obtained NTD 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 NTD 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 NTD protein of Salmonella ribosomal protein L7/L12 for obtaining monoclonal antibodies.

<3: Obtaining a mouse monoclonal antibody strain using the NTD protein of Salmonella ribosomal protein L7/L12>
Using the NTD protein of ribosomal protein L7/L12 of Salmonella for obtaining the obtained monoclonal antibodies, three strains, 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9-D2, which are monoclonal antibody strains against the NTD protein, were obtained according to the method described in Example 3 of WO 2001/057199.

Specifically, 100 μg of NTD protein of Salmonella ribosomal protein L7/L12 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 NTD protein of Salmonella ribosomal protein L7/L12 was fused with glutathione S-transferase (GST) protein to prepare GST fusion L7/L12NTD protein. The obtained GST fusion L7/L12NTD protein and GST protein were dissolved in PBS containing 0.05% sodium azide at a concentration of 10 μg/mL to prepare diluted solutions. 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 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/L12NTD protein and not to the GST protein, and it was found that these wells contained antibodies against the L7/L12NTD 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 mixture was cultured at 37° C. for 2 weeks under 7% CO ₂ conditions. The antibody activity in the culture supernatant was assayed in the same manner as above using the ELISA method, and cells that reacted positively with the L7/L12NTD protein were collected. Further, by repeating the same dilution assay and cloning procedure, hybridomas producing monoclonal antibody strains 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9-D2 against the L7/L12 NTD protein were obtained.

The three positive clone monoclonal antibody strains 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9-D2 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, 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9-D2, 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 the monoclonal antibody strains 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9-D2 are shown in the sequence numbers in Table 1 below.

Example 6 Analysis of the interaction between antibodies and Salmonella ribosomal protein L7/L12 The monoclonal antibodies 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9-D2 obtained in Example 5, which recognize the NTD of Salmonella ribosomal protein L7/L12, were subjected to NMR analysis of the interaction with Salmonella ribosomal protein L7/L12 by the following procedure.

<1: Obtaining ribosomal protein L7/L12 from Salmonella>
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). 1.5 mL of 10x concentration 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 ribosomal protein L7/L12.

The obtained ribosomal protein L7/L12 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 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, and the ribosomal protein L7/L12 was purified. 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 Salmonella ribosomal protein L7/L12>
The monoclonal antibodies 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9-D2 obtained in Example 5, which recognize the NTD of Salmonella ribosomal protein L7/L12, were dialyzed against PBS (phosphate buffered saline) as an external solution, and the concentrations were quantified by the absorbance of ultraviolet light (wavelength 280 nm), and then subjected to NMR measurement.

<3: Analysis of the interaction between Salmonella ribosomal protein L7/L12 and antibodies by NMR>
Salmonella ribosomal protein L7/L12 was mixed with antibody 31E2-F5-F9, 33C9-F6-D1, or 36G2-F9-D2 at a molar ratio of 1:0.5. Each mixture was made up to 250 μL with phosphate buffered saline (PBS), and then 20 μL of heavy water and 1 μL of 5 mg/mL DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) were added. The mixture was transferred to a Shigemi NMR sample tube, degassed with an aspirator, and then placed in the NMR instrument.

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 [ ¹ H- 15 N] HSQC signal intensity of each amino acid residue of ribosomal protein L7/L12 when ribosomal protein L7/L12 was mixed with antibody 31E2-F5-F9, 33C9-F6-D1, or 36G2-F9-D in the above-mentioned ratios, was divided by the [ ¹ H- ¹⁵ N] HSQC signal intensity of each amino acid residue of ribosomal protein L7/L12 before mixing with the antibody, and graphs were created in which the divided values are shown on the vertical axis and the amino acid sequence of ribosomal protein L7/L12 is shown on the horizontal axis (Figures 6A, B, ^C ). Since the division values of the NTD (residues 1 to 40) were generally 0.4 or less (meaning that the signal intensity of the ribosomal protein L7/L12 was attenuated by 60% or more upon interaction with the antibody), it was found that the ribosomal protein L7/L12 interacted with the antibody 31E2-F5-F9, 33C9-F6-D1, or 36G2-F9-D2 via the NTD (residues 1 to 40) (Figures 6A, B, and C).

Example 7 Examination of Cross-Reactivity of Antibodies Binding to Salmonella Ribosomal Protein L7/L12 The monoclonal antibodies 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9-D2 obtained in Example 5, which recognize the NTD of Salmonella ribosomal protein L7/L12, were analyzed by ELISA for cross-reactivity with ribosomal protein L7/L12 of bacteria other than Salmonella 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 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 above bacterial species was immobilized on an ELISA plate, reacted with each of the monoclonal antibodies 31E2-F5-F9, 33C9-F6-D1, or 36G2-F9-D2, and washed. The monoclonal antibody bound to the immobilized L7/L12 of each of the above bacterial species was then reacted with a peroxidase-labeled anti-mouse IgG antibody to evaluate the cross-reactivity of each monoclonal antibody with L7/L12 of each of the above bacterial species.

Specifically, 100 μL of each PBS solution containing Salmonella and each of the above-mentioned bacterial species' recombinant full-length ribosomal proteins L7/L12 at concentrations 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 the antibody 31E2-F5-F9, 33C9-F6-D1, or 36G2-F9-D2 was diluted with 0.5% TritonX-100/PBS to 1 μg/mL, or about 1×PBS itself (negative control) was added and reacted at room temperature for 1 hour. After removing the supernatant, 100 μL of a solution in which a peroxidase-labeled anti-mouse IgG antibody reagent was diluted with 0.02% Tween 20/PBS as a secondary antibody to a final concentration of 1 μg/mL was added and reacted at room temperature for 1 hour. After removing the supernatant, the plates were washed several times with washing solution, and 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) solution (KPL) was added to each plate. After reacting for 10 minutes at room temperature, 100 μL of 1 mol/L hydrochloric acid was added to stop the reaction. The absorbance of the resulting solution at 450 nm was measured, and the difference from the absorbance of the negative control at 450 nm 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 31E2-F5-F9, 33C9-F6-D1, or 36G2-F9-D2 with recombinant full-length ribosomal protein L7/L12 of Salmonella 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 detection of Salmonella in samples, primarily in the medical field, and is extremely useful industrially.

Claims (11)

An antibody for detecting Salmonella Enteritidis, which undergoes an antigen-antibody reaction with an epitope present in the N-terminal domain (NTD) consisting of amino acid residues 1 to 40 of the ribosomal protein L7/L12 of Salmonella as shown in SEQ ID NO: 1, and
A heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO:5 and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO:7, or
A heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 11, or
An antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO: 13 as a heavy chain variable region sequence and the amino acid sequence of SEQ ID NO: 15 as a light chain variable region sequence. The antibody or antigen-binding fragment thereof according to claim 1, wherein the epitope comprises one or more amino acid residues selected from the amino acid residues at positions 4 to 22 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, Pseudomonas, Streptococcus, Staphylococcus, Neisseria, Haemophilus, Bordetella, Moraxella, and Legionella. An anti-Salmonella enteritidis antibody,
A heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO:5 and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO:7, or
A heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 11, or
An antibody, or an antigen-binding fragment thereof, comprising the amino acid sequence of SEQ ID NO: 13 as a heavy chain variable region sequence and the amino acid sequence of SEQ ID NO: 15 as a light chain variable region sequence. A nucleic acid molecule encoding the antibody or antigen-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 a nucleic acid molecule according to claim 5 or a vector or plasmid according to 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 Salmonella 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 Salmonella 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 Salmonella 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 Salmonella in a sample using the antibody.

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