JP2021069359A - Antibody for detecting salmonella enteritidis in sample, method for detecting salmonella enteritidis using said antibody, reagent, and kit - Google Patents

Abstract

To provide an antibody for detecting Salmonella enteritidis having excellent detection sensitivity and detection accuracy (bacterial strain specificity).SOLUTION: Provided is an antibody capable of causing an antigen-antibody reaction with an epitope located in an N-terminal domain (NTD) of a ribosome protein L7/L12 in Salmonella enteritidis, wherein the N-terminal domain comprises amino acid residues located at position-1 to position-40, or a fragment of the antibody, or a derivative of the antibody or the fragment.SELECTED DRAWING: Figure 1

Description

The present invention is an antibody for detecting whether or not Salmonella enteritidis, which is one of the causative bacteria of pneumonia, is present in a sample, and an antibody for detecting Salmonella in a sample using such an antibody. With respect to methods, reagents, and kits.

Rapid diagnosis of infections is extremely important in the treatment of patients suffering from microbial infections. Examples of diagnostic methods for microbial infections include a method of detecting the causative bacteria of an infectious disease from the infected site, serum, body fluid, etc., and a method of detecting an antibody against the causative bacteria from blood, body fluid, etc. -From the viewpoint of speed, a method of directly detecting the causative bacteria is preferable.

The method for detecting the causative bacteria of microbial infections is a culture identification method in which the causative bacteria are isolated and cultured, and then the bacteria are identified based on their biochemical properties. It is roughly divided into a genetic diagnostic method for identifying a bacterium by amplifying it by a PCR) method or the like, and an immunological method for identifying a bacterium by utilizing a specific reaction of an antibody against a surface antigen marker of the causative bacterium. However, the culture identification method and the gene diagnosis method take a long time to obtain a detection result, and often have a problem in terms of detection sensitivity. Therefore, diagnosis by an immunological method is widely used because the causative bacteria can be detected with high sensitivity in a short time.

Conventionally, various combinations of marker antigens and antibodies are used to detect infectious disease-causing bacteria by the immunological method, depending on the bacterial species.

Salmonella enteritidis is a gram-negative intestinal bacterium that inhabits the gastrointestinal tract of humans and animals, and invades intestinal epithelial cells and causes food poisoning). In order to make a definitive diagnosis of Salmonella infection, it is necessary to detect the bacteria in feces, blood, puncture fluid, lymph fluid, etc., and there is currently no rapid diagnostic method. In order to prevent the worsening of symptoms and the spread of infection, a specific rapid diagnostic method for Salmonella is required.

As a method for detecting Salmonella, it is usually inspected and detected by isolation culture using a selective medium or the like. However, the isolation and culture of the bacterium takes 1 to 2 days at room temperature, and the detection by the culture method has a problem including the inspection time. (Patent Document 1: Patent No. 6104242) On the other hand, a method of shortening the culture time by a hybridization technique using a DNA probe (Patent Document 2: Patent No. 2089328) has been known, but the speed is not high. There were problems such as sufficientness and complicated operation, and there was room for improvement.

The present inventors have focused on ribosome proteins L7 / L12 as intracellular molecules that are present in all microbial cells and whose amino acid structures differ to some extent between microorganisms, and have been directed to such proteins. By using an antibody, we have found a method capable of specifically detecting various microorganisms and comprehensively detecting various serotypes within the same bacterial species (Patent Document 3: International Publication No. 2000). / 006603). However, the detection accuracy (species specificity) of such existing antibodies and the binding pattern of specific antibodies to the ribosomal protein L7 / L12 of Salmonella to ensure the detection accuracy are unknown. There was room for improvement, such as what happened.

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

An object of the present invention is to provide an antibody for detecting Salmonella, which is excellent in detection sensitivity and detection accuracy (species specificity).

As a result of diligent studies, the present inventors may become an epitope having excellent antigen-antibody reactivity and specificity in the N-terminal domain (NTD) consisting of amino acid residues at positions 1 to 40 of the ribosomal protein L7 / L12 of Salmonella. We found that a partial amino acid sequence was present. Then, we actually prepared a plurality of antibodies that generate an antigen-antibody reaction with such a partial amino acid sequence, and verified that Salmonella in a sample can be detected with high sensitivity and accuracy by using these antibodies, and the present invention. Reached.

That is, the gist of the present invention is as follows.
[1] An antibody for detecting Salmonella enteritidis, which is present in the N-terminal domain (NTD) consisting of amino acid residues at positions 1 to 40 of the salmonella ribosomal protein L7 / L12 shown in SEQ ID NO: 1. An antibody or fragment thereof, or a derivative thereof, which causes an antigen-antibody reaction with an epitope.
[2] The antibody or fragment thereof according to Item [1], or a derivative thereof, wherein the epitope contains one or more amino acid residues selected from the amino acid residues at positions 4 to 22 of SEQ ID NO: 1. ..
[3] Mycoplasma, Escherichia, Chlamydia, Pseudomonas, Streptococcus, Staphylococcus, Neisseria, Haemophilus The antibody according to item [1] or [2], which does not cross-react with bacteria of one or more genera selected from the genus ), Bordetella, Moraxella, and Legionella. Fragments thereof, or derivatives thereof.
[4] As the heavy chain variable region sequence, an amino acid sequence having 80% or more homology with any one of the amino acid sequences selected from SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13 and an amino acid sequence having 80% or more homology.
Items [1] to [1], wherein the light chain variable region sequence comprises an amino acid sequence having 80% or more homology with any one amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15. 3] The antibody according to any one of the above, or a fragment thereof, or a derivative thereof.
[5] As the heavy chain variable region sequence, an amino acid sequence having 80% or more homology with any one of the amino acid sequences selected from SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13 and an amino acid sequence having 80% or more homology.
As the light chain variable region sequence, an antibody or a fragment thereof each containing an amino acid sequence having 80% or more homology with any one amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15 or Derivatives of them.
[6] A nucleic acid molecule encoding the antibody or fragment thereof according to any one of items [1] to [5], or a derivative thereof.
[7] A vector or plasmid containing the nucleic acid molecule according to item [6].
[8] A host cell transformed with the nucleic acid molecule according to item [6] or the vector or plasmid according to item [7].
[9] The host cell according to item [8], wherein the host cell is a eukaryotic cell or a bacterial cell selected from mammalian cells, insect cells, yeast cells, and plant cells.
[10] A hybridoma expressing the antibody or fragment thereof according to any one of items [1] to [5], or a derivative thereof.
[11] A method for detecting the presence or absence of Salmonella in a sample, wherein the antibody or fragment thereof according to any one of items [1] to [5], or a derivative thereof is brought into contact with the sample. A method comprising detecting the presence or absence of an antigen-antibody reaction.
[12] A reagent for detecting the presence or absence of Salmonella in a sample, which comprises the antibody or fragment thereof according to any one of items [1] to [5], or a derivative thereof.
[13] A kit for detecting the presence or absence of Salmonella in a sample, using the antibody or fragment thereof according to any one of items [1] to [5], or a derivative thereof, and the antibody. A kit containing instructions and instructions for detecting the presence or absence of Salmonella in a sample.

According to the antibody of the present invention, Salmonella in a sample can be detected with high sensitivity and accuracy.

FIG. 1 is a diagram schematically showing the three-dimensional structure of the ribosome protein L7 / L12 of Salmonella. FIG. 2 is a diagram schematically showing the three-dimensional structure of the ribosomal proteins L7 / L12 of Mycoplasma pneumoniae. FIG. 3 is a diagram schematically showing the three-dimensional structure of Haemophilus influenzae ribosomal proteins L7 / L12. FIG. 4 is a diagram schematically showing the three-dimensional structures of the N-terminal domain (NTD) portion of the ribosomal proteins L7 / L12 of Salmonella, Mycoplasma pneumoniae, and Haemophilus influenzae, respectively. FIG. 5 shows the alignment of the amino acid sequences of the N-terminal domain (NTD) portion of the ribosomal proteins L7 / L12 of Salmonella, Mycoplasma pneumoniae, and Haemophilus influenzae. FIG. 6A is a diagram schematically showing the results of NMR analysis of the interaction between the monoclonal antibody 31E2-F5-F9 and the ribosomal protein L7 / L12 of Salmonella. FIG. 6B is a diagram schematically showing the results of NMR analysis of the interaction between the monoclonal antibody 33C9-F6-D1 and the ribosomal proteins L7 / L12 of Salmonella. FIG. 6C is a diagram schematically showing the results of NMR analysis of the interaction between the monoclonal antibody 36G2-F9-D2 and the ribosomal proteins L7 / L12 of Salmonella.

Hereinafter, the present invention will be described in detail according to specific embodiments. However, the present invention is not bound by the following embodiments, and can be implemented in any embodiment as long as the gist of the present invention is not deviated.

All documents including patent gazettes, patent application publication gazettes, and non-patent gazettes cited in this specification are incorporated herein by reference in their entirety.

Further, in the formula representing the amino acid sequence described in the present specification, the amino acid is represented by a one-character code unless otherwise described.

1. 1. Antibodies for detecting Salmonella:
The first aspect of the present invention relates to an antibody for detecting Salmonella (hereinafter, appropriately referred to as "antibody of the present invention").

(1) Introduction:
In the present invention, "Salmonella enteritidis" or "Salmonella enterica serovar Enteritidis" is a bacterium belonging to the genus Salmonella in the family Enterobacteriaceae of Gram-negative anaerobic bacilli, and is mainly human or Inhabits the digestive tract of animals.

Salmonella is a type of intracellular parasite (passive intracellular parasite, intracellular parasite) that can grow both intracellularly and extracellularly of an infected host, and actively becomes a cell. It has the function of working to activate cell endocytosis. Therefore, it has the property of being able to invade not only cells having phagocytic activity such as macrophages but also intestinal epithelial cells that normally do not have phagocytic activity.

Salmonella is pathogenic to humans and causes gastroenteritis (food poisoning). Food poisoning caused by Salmonella is accompanied by gastrointestinal symptoms such as abdominal pain, vomiting and diarrhea, and symptoms such as fever (high fever). Patients with low resistance may develop bloodstream infections and become severe, and in rare cases, may be accompanied by endotoxin-induced sepsis and death.

Diagnosis of Salmonella infection is first made by laboratory findings such as an increase in white blood cell count, CRP and other inflammatory reactions, and an increase in transamylase. For a definitive diagnosis, it is necessary to detect bacteria in feces, blood, puncture fluid, lymph fluid, and the like. However, there is currently no specific rapid diagnostic method for Salmonella.

As a treatment for Salmonella infection, symptomatic treatment is basically performed, focusing on correction of dehydration due to fever and diarrhea and relief of gastroenteritis symptoms such as abdominal pain. However, in order to prevent the worsening of symptoms and the spread of infection, a specific rapid diagnostic method for Salmonella is required.

The present inventors paid attention to the ribosomal proteins L7 / L12 in producing an antibody for detecting Salmonella. In the present invention, "ribosome protein L7 / L12" or simply "L7 / L12" is one of the ribosomal proteins essential for protein synthesis of microorganisms, and is a protein commonly possessed by various bacteria. The Salmonella ribosomal protein L7 / L12 has a dimeric structure in which two copies of a monomeric molecule composed 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 at positions 1 to 40. After passing through a linker that does not form a three-dimensional structure consisting of amino acid residues at positions 41 to 52, another three-dimensional structure (CTD: C-Terminal Domain) is further formed by amino acid residues at positions 53 to 121. In addition, two copies of monomer molecules having such a three-dimensional structure are associated with each other's NTDs to form a dimer structure (see Examples 1 and FIG. 1 described later).

Similarly, the ribosome proteins L7 / L12 of Mycoplasma pneumoniae and Haemophilus influenzae are also dimeric with two molecules associated with NTD (N-Terminal Domain, residues 1 to 40). CTD (C-Terminal Domain, 55-122 position in the case of Mycoplasma pneumoniae, 55-123 position in the case of Haemophilus influenzae) is further formed via a linker (41-54 position) having a random coil structure. (See Examples 2 and 3 and FIGS. 2 and 3 described below).

Among these three-dimensional structures of Salmonella L7 / L12, the present inventors have paid particular attention to NTD as a candidate for an epitope having excellent immunogenicity based on the following studies.

Comparing the three-dimensional structures of Haemophilus influenzae, Mycoplasma pneumoniae, and Salmonella L7 / L12 NTDs, it was found that there was a large difference in surface shape and charge distribution (Fig. 4). For example, in the case of Haemophilus influenzae (HI) and Salmonella (SE), the three N-terminal residues of the dimer project to the upper part of the structure, but in the case of Mycoplasma pneumoniae (MP), no protrusion is observed. Further, this protrusion is one place in the case of Haemophilus influenzae, but two places in the case of Salmonella. Comparing the surface charges of NTD, in the case of Mycoplasma pneumoniae, the central part of the three-dimensional structure is occupied by hydrophobic (white) to uncharged hydrophilic regions (light blue, light red), but in the case of Influenza and Salmonella. Is occupied by negative charge (red). In the case of Haemophilus influenzae, the negative charge region (red) is located on the right side of the three-dimensional structure, and the positive charge region (blue) is located on the left side. In the case of Salmonella, in contrast, negative charges (red) are concentrated in the center of the three-dimensional structure, and hydrophobic regions (white) are predominant at the left and right ends of the structure (see Examples 4 and 4 below). ..

Furthermore, when the amino acid sequences of Haemophilus influenzae, Mycoplasma pneumoniae, and Salmonella ribosomal proteins L7 / L12 were compared, it was also found that there was a large difference between the bacterial species at the residues at positions 1 to 40 (Fig. 5). For example, the residues at positions 5 to 7 of mycoplasma pneumoniae correspond to the residues at positions 4 to 6 of influenza and salmonella, and the amino acid sequences of mycoplasma pneumoniae are D (aspartic acid: hydrophilic, negatively charged), K ( Lysine: hydrophilic, positively charged), N (asparagin: hydrophilic, uncharged), whereas influenza bacteria are T (threonine: hydrophilic, uncharged), N (asparagin: hydrophilic, uncharged), E (glutamic acid: hydrophilic, negatively charged), Salmonella are T (threonine: hydrophilic, uncharged), K (lysine: hydrophilic, positively charged), D (aspartic acid: hydrophilic, negatively charged), amino acids The type and the order of polarity are different for each bacterial species. Similarly, the 14th to 16th residues of pneumonia mycoplasma correspond to the 13th to 15th residues of influenza and salmonella, and the amino acid sequences of pneumonia mycoplasma are K (lysine: hydrophilic, positively charged), E ( Glutamic acid: hydrophilic, negatively charged), M (methionine: hydrophobic), whereas influenza bacteria are A (alanine: hydrophobic), S (serine: hydrophilic, uncharged), K (lysine: hydrophilic). , Positive charge), Salmonella is S (serine: hydrophilic, uncharged), A (alanine: hydrophobic), M (methionine: hydrophobic), and the order of amino acid types and polarities differs depending on the bacterial species. (See Examples 4 and 5 below).

As described above, the ribosomal proteins L7 / L12 of Salmonella have an amino acid sequence and a three-dimensional structure (surface shape, surface charge) having a large difference between bacterial species in NTD. From these findings, the present inventors have come up with the idea that a more specific antibody can be obtained by targeting NTD rather than the full length of L7 / L12.

Then, by expressing a peptide having an amino acid sequence similar to NTD, preparing an antibody that specifically binds to the peptide, and performing screening, the antibodies 31E2-F5-F9, 33C9-F6-D1, and 36G2-F9 -D2 was obtained (see Example 5 below). Then, it was confirmed that all of these antibodies caused an antigen-antibody reaction with NTD of L7 / L12 of Salmonella, particularly an epitope composed of a specific amino acid residue (Example 6 described later). And FIGS. 6A-C). Furthermore, it was confirmed that all of these antibodies had excellent detection sensitivity and detection accuracy (bacterial species specificity) (see Example 7 described later), and the present invention was completed.
Hereinafter, a more specific description will be given.

(2) Outline of antibody:
In the present invention, the "antibody" is a protein that recognizes and binds to a specific antigen or substance, and may be referred to as immunoglobulin (Ig). Common antibodies usually have two light chains (light chains) and two heavy chains (heavy chains) interconnected by disulfide bonds. There are two types of light chains called λ chains and κ chains, and there are five types of heavy chains called γ chains, μ chains, α chains, δ chains and ε chains. Depending on the type of heavy chain, there are five isotypes of antibody, IgG, IgM, IgA, IgD and IgE, respectively.

The heavy chains include a heavy chain constant (CH) region and a heavy chain variable (VH) region, respectively. The light chains include a light chain constant (CL) region and a light chain variable (VL) region, respectively. 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) regions and heavy chain variable (VH) regions are four highly conserved regions (FR-1, FR-2, FR-3, FR-4) called framework regions (FR), respectively. And three hypervariable regions (CDR-1, CDR-2, CDR-3) called complementarity determining regions (CDRs). 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). These are arranged in the order of FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4 from the amino end to the carboxy end. 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). These are arranged in the order of FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, FR-L4 from the amino end to the carboxy end. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

The antibody of the present invention is an antibody capable of detecting Salmonella and can be specified from the following two viewpoints. First, as a first aspect, the antibody of the present invention can be defined from the feature that it recognizes a specific epitope present in the ribosomal proteins L7 / L12 of Salmonella and causes an antigen-antibody reaction. As a second aspect, the antibody of the present invention can also be defined from the feature that each variable region of the heavy chain and the light chain has a specific amino acid sequence. The first viewpoint will be described later in the [(2) Antibody properties] column, and the second viewpoint will be described later in the [(3) Antibody structure] column. The antibody of the present invention may satisfy the characteristics of either the first aspect or the second aspect, but an antibody satisfying both the characteristics of the first aspect and the second aspect is also present in the present invention. Needless to say, it is contained in the antibody.

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. Polyclonal antibodies are antibodies that are usually prepared from the sera of an animal immunized with an antigen and are 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 a nucleic acid molecule having a gene sequence encoding an amino acid of the antibody protein is obtained and genetically engineered using such a nucleic acid molecule. It is also possible to do. In addition, it is also common for those skilled in the art to modify the heavy and light chains, or their variable regions, and genetic information such as CDRs to improve the binding property and specificity of the antibody. It is a known technology.

Further, the antibody of the present invention may be a fragment and / or a derivative of the antibody. Examples of the antibody fragment include F (ab') ₂ , Fab, Fv and the like. Derivatives of the antibody include an antibody in which an amino acid mutation is artificially introduced into the constant region portion of the light chain and / or heavy chain, and an antibody in which the domain composition of the constant region of the light chain and / or heavy chain is modified, 2 per molecule. Antibodies with one or more Fc regions, sugar chain modified antibodies, bispecific antibodies, antibody conjugates in which an antibody or fragment of an antibody is bound to a protein other than the antibody, antibody enzyme, tandem scFv, bispecific tandem scFv , Diabody and the like. Furthermore, when the antibody or 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 the human antibody is also an antibody of the present invention. include. Unless otherwise specified, the term "antibody" in the present invention also includes fragments and / or derivatives of the antibody.

(3) Properties of antibody:
The antibody of the present invention is characterized in that, as a first aspect, it causes an antigen-antibody reaction with an epitope composed of a specific amino acid residue in the ribosomal protein L7 / L12 of Salmonella.

In the present invention, the "antigen-antibody reaction" means that an antibody recognizes and binds to any component of the antigen.

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

In the salmonella ribosomal protein L7 / L12, the epitope to which the antibody of the present invention binds is NTD formed by the amino acid residues at positions 1 to 40 of the amino acid sequence of the salmonella ribosomal protein L7 / L12 shown in SEQ ID NO: 1. Exists in. According to the analysis results of the present inventors described later in Examples 5 to 7, for detecting Salmonella with high detection sensitivity and high detection accuracy (high bacterial species specificity) actually acquired by the present inventors in the examples. It was confirmed that the antibodies 31E2-F5-F9, 33C9-F6-D1 and 36G2-F9-D2 all bind to NTD formed by the amino acid residues 1 to 40 of L7 / L12. ing.

Among them, the epitope of the ribosomal protein L7 / L12 to which the antibody of the present invention binds is 1 or 2 or more selected from the amino acid residues at positions 4 to 22 of SEQ ID NO: 1, especially 3 or more, 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 amino acid residues. In particular, the epitope of the ribosomal protein L7 / L12 to which the antibody of the present invention binds more preferably contains at least one or two or more amino acid residues selected from positions 4 to 15 of SEQ ID NO: 1, 4, 5, It is more preferred to include one or more amino acid residues selected from positions 6, 13, 14, and 15. According to the analysis results of the present inventors described later in Examples, it has been confirmed that these amino acid residues are present near the surface in the three-dimensional structure of NTD of the ribosome protein L7 / L12 of Salmonella. In addition, these amino acid residues are located in places where the species specificity of Salmonella is particularly high when L7 / L12 of Salmonella is compared with L7 / L12 of other bacterial species. It is considered certain that it forms an epitope for the antibody of the present invention.

Further, it is preferable that the antibody of the present invention does not cross-react with bacteria other than Salmonella and other components.

Specifically, the antibodies of the present invention include Mycoplasma, Escherichia, Chlamydia, Pseudomonas, Streptococcus, Staphylococcus, and Neisseria. It is preferable that it does not cross-react with bacteria of one or more genera selected from the genus Neisseria, Haemophilus, Bordetella, Moraxella, and Legionella. Among them, the antibody of the present invention does not cross-react with bacteria of 2 or more genera, further 3 or more genera, 4 or more genera, or 5 or more genera, or 6 or more genera, especially all genera. Is preferable.

The measurement of the antigen-antibody reaction between the antibody and the epitope / antigen or other components can be carried out by a person skilled in the art by appropriately selecting the binding measurement in a solid phase or liquid phase system. Such methods include enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), surface plasmon resonance (SPR), and fluorescence resonance energy transfer method. (Fluorescence resonance energy transfer: FRET), luminescence resonance energy transfer (LRET), and the like can be mentioned, but the present invention is not limited thereto. In addition, when measuring such antigen-antibody binding, the antibody and / or antigen is labeled with an enzyme, a fluorescent substance, a luminescent substance, a radioactive isotope, or the like, and the physical and / or chemical properties of the labeled substance are obtained. It is also possible to detect the antigen-antibody reaction by using a measurement method suitable for the above.

Above all, in the present invention, it is particularly preferable to analyze the interaction between an antigen (epitope) and an antibody by a nuclear magnetic resonance (NMR) method. For more information on such techniques, see, for example, Cavanagh et al., “Protein NMR Spectroscopy, Principles and Practice Protein NMR”, 2nd Edition, Academic Press, 2006, Vitha et al., “Spectroscopy: Principles and Instrumentation”, Wiley- You can refer to the description of Blackwell, 2018. The specific analysis conditions are not limited, but for example, the analysis conditions adopted by the present inventors in Example 6 described later can be referred to.

(4) Antibody structure:
As a second aspect, the antibody of the present invention is characterized in that each variable region 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 chain and the light chain.

The heavy chain variable region sequence includes 80% or more, particularly 85% or more, further 90% or more, particularly 95% or more, with any one amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13. , Or 96% or more, or 97% or more, or 99% or more, particularly preferably having an amino acid sequence having 100% homology (preferably identity). Among them, the heavy chain variable region sequence is particularly preferably any one amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13.

As the light chain variable region sequence, 80% or more, particularly 85% or more, further 90% or more, particularly 95% or more, with any one amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15. Alternatively, it preferably has an amino acid sequence having 96% or more, 97% or more, or 99% or more, particularly 100% homology (preferably identity). Among them, the light chain variable region sequence is particularly preferably any one amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15.

In the present invention, the "homogeneity" of two amino acid sequences is the ratio at which the same or similar amino acid residues appear at each corresponding position when both amino acid sequences are aligned, and the two amino acid sequences are "same". "Sex" is the ratio at which the same amino acid residue appears at each corresponding location when both amino acid sequences are aligned. The "homology" and "identity" of the two amino acid sequences are, for example, BLAST (Basic Local Alignment Search Tool) program (Altschul et al., J. Mol. Biol., (1990), 215 (3) :. It can be obtained by using 403-10) or the like.

Further, as a method for identifying the sequence of each CDR from the variable sequences of the heavy chain and the light chain of a certain antibody, 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 general knowledge in this field, but for example, the website of Dr. Andrew C.R. Martin's Group (http://www.bioinf.org.uk/abs/) can be referred to.

Examples of amino acids similar to a certain amino acid include amino acids belonging to the same group in the following classifications based on the polarity, chargeability, and size of amino acids (in each case, the type of each amino acid is indicated by a single character code). To do.).
-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;
・ Loaded 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 .:
-Ultra-small amino acids: A, C, G, S.

Further, as an amino acid similar to a certain amino acid, for example, in the following classification based on the type of side chain of the amino acid, an amino acid belonging to the same group can be mentioned (in each case, the type of each amino acid is indicated by a single character code). ..
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;
-Acid amino acids and their amide derivatives: D, E, N, Q.

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

When the antibody of the present invention is a polyclonal antibody, all or part of the salmonella ribosome protein L7 / L12 to be detected, preferably the amino acid residues 1 to 40 of SEQ ID NO: 1 constituting NTD (SEQ ID NO: 3). A polypeptide consisting of (amino acid residues), or 80% or more, especially 85% or more, further 90% or more, particularly 95% or more, 96% or more, or 97% or more, or 99% or more, particularly 100%. It can be prepared by using a polypeptide having an amino acid sequence having the homology (preferably the sameness) of (hereinafter referred to as "epitope polypeptide"). Specifically, an antiserum containing an antibody (polyclonal antibody) that causes an antigen-antibody reaction with the epitope polypeptide by preparing an epitope polypeptide, inoculating the animal with an adjuvant as needed, and collecting the serum thereof. Can be obtained. The animals to be inoculated include sheep, horses, goats, rabbits, mice, rats and the like, and sheep, rabbits and the like are particularly preferable for producing polyclonal antibodies. Further, the antibody is purified and fractionated from the obtained antiserum to cause an antigen-antibody reaction with the ribosomal proteins L7 / L12 of Salmonella, and cross-reaction with other specific components, for example, bacteria of the genus listed above. It is possible to obtain a desired antibody having more excellent specificity by appropriately performing screening by a known method using the fact that it does not occur as an index. Furthermore, it is also possible to obtain a monoclonal antibody by isolating an antibody-producing cell that produces a desired antibody molecule and fusing it with myeloma cells to produce a hybridoma having an autonomous proliferation ability. In addition, as a method that does not require sensitization to animals, a phage library that expresses a heavy chain variable (VH) region or a light chain variable (VL) region of an antibody or a part thereof is used as a detection target. It is also possible to obtain an antibody that specifically binds to the ribosomal protein L7 / L12 of Salmonella or a phage clone consisting of a specific amino acid sequence, and to prepare an antibody from the information.

Further, if a desired antibody is obtained by the above procedure, the structure of such an antibody, specifically, a heavy chain constant (CH) region, a heavy chain variable (VH) region, a light chain constant (CL) region, and / or Part or all of the amino acid sequence of the light chain variable (VL) region can be analyzed using known amino acid sequence analysis methods. Those skilled in the art also know a method for modifying the amino acid sequence of the desired antibody thus obtained in order to improve the binding property and specificity of the antibody. Furthermore, it is necessary to utilize all or part of the amino acid sequence of the desired antibody (particularly all or part of the heavy chain variable (VH) region and the light chain variable (VL) region, especially the amino acid sequence of each CDR). Each FR of 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 heavy chain variable (VH) region and the light chain variable (VL) region. It is also possible to design another highly probable antibody having similar antigen specificity by combining with (amino acid sequence of).

On the other hand, when a part (CDR or variable region) or all amino acid sequences of the antibody are specified, a base sequence encoding all or part of the amino acid sequence of such a desired antibody is used by a known method. It is also possible to prepare a nucleic acid molecule having the above, and to prepare an antibody by genetic engineering using such a nucleic acid molecule. Furthermore, vectors and plasmids for expressing each component of the desired antibody are prepared from such a base sequence and introduced into host cells (mammalian cells, insect cells, plant cells, yeast cells, microbial cells, etc.). It is also possible to produce the antibody. It is also well known to those skilled in the art that the structure of the constant region of the antibody is modified and the sugar chain portion is modified for the purpose of improving the performance of the obtained antibody and avoiding side effects. It can be done as appropriate depending on the technique.

The method for producing an antibody of the present invention described above, a nucleic acid molecule encoding the antibody of the present invention, a vector or plasmid containing such a nucleic acid molecule, a cell containing such a nucleic acid molecule or vector or plasmid, and further, the present invention. A hybridoma or the like that produces the antibody of the present invention is also an object of the present invention.

Techniques for producing and modifying antibodies described in the present specification are all known to those skilled in the art, and for example, Antibodies; A laboratory manual, E. Harlow et al., Cold Spring Harbor Laboratory Press (2014). ) Etc. can be referred to. Further, all of the molecular biological techniques described in the present specification (for example, amino acid sequence analysis method, nucleic acid molecule design / preparation method, vector / plasmid design / preparation method, etc.) are known to those skilled in the art. , For example, Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory Press, Shambrook, J. et al. (1989) and the like.

2. Salmonella detection method / reagent / kit:
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 includes contacting the antibody of the present invention or a fragment thereof, or a derivative thereof with a sample to detect the presence or absence of an antigen-antibody reaction. Here, since the antibody of the present invention causes 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 in various known immunoassays in the sample. It is possible to detect the presence or absence of Salmonella in the body.

Examples of the sample mainly include biological samples derived from humans or non-human animals. The type of biological sample is not particularly limited, but examples thereof include liquid samples such as blood (whole blood, plasma, serum), lymph, urine, saliva, tears, sheep's water, and ascites, and solids such as biopsy samples of various tissues. Examples thereof include homogenate suspensions and extracts of samples, and culture supernatants thereof.

Since the antibody of the present invention recognizes a specific epitope present in the ribosomal protein L7 / L12 of Salmonella and causes an antigen-antibody reaction as described above, the ribosomal protein L7 / L12 of Salmonella is placed outside the cell membrane of the bacterium. By exposing it, the detection sensitivity can be improved. Therefore, the sample may be treated to lyse the bacteria before the antibody of the present invention is brought into contact with the sample. The lytic treatment of such bacteria includes, but is not limited to, lytic treatment using a surfactant, a lytic enzyme, or the like. Examples of surfactants that can be used for 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. Activator; amphoteric surfactant such as Zwittergent 3-12, CHAPS (3- (3-colamidpropyl) dimethylammonio-1-propanesulfonate); anionic surfactant such as SDS (sodium dodecyl sulfate) Agents and the like can be mentioned. Examples of the lytic enzyme that can be used for the lytic treatment include lysozyme, lysostaphin, pepsin, glucosidase, galactosidase, achromopeptidase, β-N-acetylglucosaminidase and the like.

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 immunoassays include, but are not limited to, ELISA (enzyme-linked immunosorbent assay) methods using antibody-supported microtiter plates; antibody-supported latex particles (eg, polystyrene latex particles, etc.). Latex particle agglutination measurement method to be used; Immunochromatography method using a membrane or the like carrying an antibody; Fixed to a solid phase carrier such as colored particles or particles having a color-developing ability, an enzyme or a phosphor labeled with a detection antibody, and magnetic fine particles. Various known immunoassays such as a sandwich assay method using a modified capture antibody can be mentioned. In the case of an immunoassay method in which a detection antibody and a capture antibody are used in combination, such as a sandwich assay method, the antibody of the present invention may be used as a capture antibody or as a detection antibody.

According to the detection method of the present invention, the presence or absence of Salmonella in a sample can be detected quickly and easily.

In addition, for use in the detection method of the present invention, a reagent containing the antibody of the present invention and a kit including an instruction sheet including an instruction for detecting the presence or absence of Salmonella in a sample using the antibody of the present invention are also included in the present invention. It is the subject of the invention. Solvents and other components of such reagents, as well as instructions for operation and use in the instructions for such kits, as well as other components contained in such kits, are specific immunoassays used for the detection of Salmonella. It may be appropriately determined according to the type of measurement method. Among them, specific examples of the kit that can easily detect the presence or absence of Salmonella in the sample include a lateral flow type kit and a flow-through type kit. Here, in the lateral flow method, a detection target sample and a detection antibody are sequentially dropped on a membrane including a detection region in which a capture antibody is immobilized on the surface and developed in parallel, and captured in the detection region of the membrane. This is a method for detecting the target substance. On the other hand, in the flow-through method, the detection target sample and the detection antibody are dropped in order on a membrane on which the capture antibody is immobilized and passed vertically to detect the target substance captured on the surface of the membrane. The method. The detection method of the present invention can be applied to both a lateral flow type kit and a flow-through type kit.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not bound by the following examples, and can be carried out in any form as long as the gist of the present invention is not deviated.

[Example 1] Three-dimensional structural analysis of salmonella ribosomal proteins L7 / L12
<1: Acquisition of Salmonella ribosome protein L7 / L12>
A pGEX-6P-1 plasmid (manufactured by GE Healthcare) in which the ribosomal protein L7 / L12 gene (SEQ ID NO: 2) of Salmonella was cloned was introduced into Escherichia coli BL21 (DE3) pLysS Escherichia coli strain (manufactured by Promega).

The resulting E. coli strain, M9 medium _{(47.7mM Na 2 HPO 4 · 12H} 2 O, 22mM KH 2 PO 4, 8.6mM NaCl, 2mM MgSO 4, 50μM ZnSO 4, 100μM CaCl 2, 4.1μM 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 ampicillin sodium, 18.7 mM ¹⁵ N-NH ₄ Cl, 11.1 mM ¹³ C-glucose), _{cultured at 37 ° C. until OD 600} reached 0.6, and quenched with ice water. IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 1 mM, and the cells were cultured at 16 ° C. for 36 hours and then centrifuged at 7000 rpm for 15 minutes at 4 ° C. to recover Escherichia coli.

To 1 g of the obtained E. coli, 5 mL of BugBuster (manufactured by ^{Merck Millipore) and 5 μL of benzoase (registered trademark)} endonuclease (manufactured by Merck Millipore) were added, and the mixture was shaken at room temperature for 30 minutes to completely dissolve the E. coli. After filtering with a 0.45 μm filter, the ribosomal protein L7 / L12 was purified on a glutathione-sepharose column using a Profinia protein purification system (manufactured by Bio-Rad). To 15 mL of the obtained protein solution, 1.5 mL of 10-fold concentration PBS (phosphate buffered saline) and Precision Protease (manufactured by GE Healthcare) were added, and the mixture was shaken at room temperature for 2 hours. The reaction was passed through the glutathione-sepharose column again to obtain a pass-through fraction as ribosomal proteins L7 / L12.

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

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

<2: Three-dimensional structural analysis of Salmonella ribosomal proteins L7 / L12 by NMR>
In a Shigemi NMR sample tube, 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-silappentan-1-sulfonic acid) are placed, degassed with an ejector, and then NMR. Installed in the device.

AVANCE III HD 600MHz NMR device (manufactured by Bruker), HNCO (4 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HN (CO) CA (8 integrations, number of data points (8) F1 x F2 x F3) 64 x 128 x 1024), HNCA (8 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024), CBCA (CO) NH (16 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024) F1 x F2 x F3) 64 x 128 x 1024), HNCACB (8 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HBHA (CO) NH (16 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024) F1 x F2 x F3) 64 x 128 x 1024), HN (CA) HA (32 totals, number of data points (F1 x F2 x F3) 64 x 128 x 1024), C (CO) NH (16 totals, Using a pulse sequence of the number of data points (F1 x F2 x F3) 128 x 128 x 1024), each FID was subjected to [ ¹ H- ¹⁵ N] HSQC (integration number 8,) with a Unity INOVA 800 MHz NMR device (manufactured by Agent). Number of data points (F1 x F2) 512 x 2048), [ ¹ H- ¹³ C] HSQC alipatic (number of integrations 8, number of data points (F1 x F2) 868 x 2048), [ ¹ H- ¹³ C] HSQC aromatic ( Number of integrations 32, number of data points (F1 x F2) 204 x 2048), HCCH-TOCSY aliphatic (number of integrations 4, number of data points (F1 x F2 x F3) 96 x 256 x 2048), HCCH-TOCSY aromatic (number of integrations) 8, number of data points (F1 x F2 x F3) 128 x 128 x 2048), ¹⁵ N-edited NOESY (mixing time 75 milliseconds, number of integrations 8, number of data points (F1 x F2 x F3) 64 x 256 x 2048) ), ¹³ C-edited NOESY aliphatic (mixing time 75 milliseconds, total number of times 8, number of data points (F1 x F2 x F3) 96 x 256 x 2048), ¹³ C-edited NOESY aromatic (mixing time 75 milliseconds, total) Each FID was obtained using a pulse sequence of 8 times and the number of data points (F1 × F2 × F3) 64 × 256 × 2048). Each obtained FID was Fourier transformed using NMR Pipe software to obtain each spectrum.

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

Enter a belonging to atomic coordinates and ^{1 H} distance information CYANA conformation automatic calculation software, is converged sufficiently energy values through the 20 times of the three-dimensional structure calculation, the spatial atomic coordinates of ribosomal protein L7 / L12 conformation Obtained.

The acquired spatial atomic coordinates were input to Pymol software, and the three-dimensional structure of the ribosomal proteins L7 / L12 was drawn (Fig. 1). When the three-dimensional structure of the main chain of the ribosomal protein L7 / L12 is displayed, one three-dimensional structure (NTD: N-Terminal Domain) is formed by the amino acid residues at positions 1 to 40, and from about 12 amino acid residues. It was found that another three-dimensional structure (CTD: C-Terminal Domain) was further formed by amino acid residues at positions 53 to 121 via a linker that did not form a three-dimensional structure (FIG. 1). It was also found that two ribosomal protein L7 / L12 molecules having such a three-dimensional structure associate with each other's NTDs to form a dimer (FIG. 1).

[Example 2] Three-dimensional structural analysis of ribosomal proteins L7 / L12 of Mycoplasma pneumoniae
<1: Acquisition of ribosomal proteins L7 / L12 from mycoplasma pneumoniae>
The pGEX-6P-1 plasmid (manufactured by GE Healthcare) in which the ribosomal protein L7 / L12 gene (SEQ ID NO: 18) of Mycoplasma pneumoniae was cloned was introduced into Escherichia coli BL21 (DE3) pLysS Escherichia coli strain (manufactured by Promega).

The resulting E. coli strain, M9 medium _{(47.7mM Na 2 HPO 4 · 12H} 2 O, 22mM KH 2 PO 4, 8.6mM NaCl, 2mM MgSO 4, 50μM ZnSO 4, 100μM CaCl 2, 4.1μM 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 ampicillin sodium, 18.7 mM ¹⁵ N-NH ₄ Cl, 11.1 mM ¹³ C-glucose), _{cultured at 37 ° C. until OD 600} reached 0.6, and quenched with ice water. IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 1 mM, and the cells were cultured at 16 ° C. for 36 hours and then centrifuged at 7000 rpm for 15 minutes at 4 ° C. to recover Escherichia coli.

To 1 g of the obtained E. coli, 5 mL of BugBuster (manufactured by ^{Merck Millipore) and 5 μL of benzoase (registered trademark)} endonuclease (manufactured by Merck Millipore) were added, and the mixture was shaken at room temperature for 30 minutes to completely dissolve the E. coli. After filtering with a 0.45 μm filter, the ribosomal protein L7 / L12 was purified on a glutathione-sepharose column using a Profinia protein purification system (manufactured by Bio-Rad). To 15 mL of the obtained protein solution, 1.5 mL of 10-fold concentration PBS (phosphate buffered saline) and Precision Protease (manufactured by GE Healthcare) were added, and the mixture was shaken at room temperature for 2 hours. The reaction was passed through the glutathione-sepharose column again to obtain a pass-through fraction as ribosomal proteins L7 / L12.

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

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

<2: Three-dimensional structural analysis of ribosomal proteins L7 / L12 of mycoplasma pneumoniae by NMR>
In a Shigemi NMR sample tube, 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-silappentan-1-sulfonic acid) are placed, degassed with an ejector, and then NMR. Installed in the device.

AVANCE III HD 600MHz NMR device (manufactured by Bruker), HNCO (4 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HN (CO) CA (8 integrations, number of data points (8) F1 x F2 x F3) 64 x 128 x 1024), HNCA (8 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024), CBCA (CO) NH (16 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024) F1 x F2 x F3) 64 x 128 x 1024), HNCACB (8 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HBHA (CO) NH (16 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024) F1 x F2 x F3) 64 x 128 x 1024), HN (CA) HA (32 totals, number of data points (F1 x F2 x F3) 64 x 128 x 1024), C (CO) NH (16 totals, Using a pulse sequence of the number of data points (F1 x F2 x F3) 128 x 128 x 1024), each FID was subjected to [ ¹ H- ¹⁵ N] HSQC (integration number 8,) with a Unity INOVA 800 MHz NMR device (manufactured by Agent). Number of data points (F1 x F2) 512 x 2048), [ ¹ H- ¹³ C] HSQC alipatic (number of integrations 8, number of data points (F1 x F2) 868 x 2048), [ ¹ H- ¹³ C] HSQC aromatic ( Number of integrations 32, number of data points (F1 x F2) 204 x 2048), HCCH-TOCSY aliphatic (number of integrations 4, number of data points (F1 x F2 x F3) 96 x 256 x 2048), HCCH-TOCSY aromatic (number of integrations) 8, number of data points (F1 x F2 x F3) 128 x 128 x 2048), ¹⁵ N-edited NOESY (mixing time 75 milliseconds, number of integrations 8, number of data points (F1 x F2 x F3) 64 x 256 x 2048) ), ¹³ C-edited NOESY aliphatic (mixing time 75 milliseconds, total number of times 8, number of data points (F1 x F2 x F3) 96 x 256 x 2048), ¹³ C-edited NOESY aromatic (mixing time 75 milliseconds, total) Each FID was obtained using a pulse sequence of 8 times and the number of data points (F1 × F2 × F3) 64 × 256 × 2048). Each obtained FID was Fourier transformed using NMR Pipe software to obtain each spectrum.

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

Enter a belonging to atomic coordinates and ^{1 H} distance information CYANA conformation automatic calculation software, is converged sufficiently energy values through the 20 times of the three-dimensional structure calculation, the spatial atomic coordinates of ribosomal protein L7 / L12 conformation Obtained.

The acquired spatial atomic coordinates were input to Pymol software, and the three-dimensional structure of the ribosomal proteins L7 / L12 was drawn (FIG. 2). When the three-dimensional structure of the main chain of the ribosomal protein L7 / L12 is displayed, one three-dimensional structure (NTD: N-Terminal Domain) is formed by the amino acid residues at positions 1 to 40, and from about 14 amino acid residues. It was found that another three-dimensional structure (CTD: C-Terminal Domain) was further formed by the amino acid residues at positions 55 to 122 through the linker that did not form the three-dimensional structure (FIG. 2). It was also found that two ribosomal protein L7 / L12 molecules having such a three-dimensional structure associate with each other's NTDs to form a dimer (FIG. 2).

[Example 3] Three-dimensional structural analysis of Haemophilus influenzae ribosomal proteins L7 / L12
<1: Acquisition of Haemophilus influenzae ribosomal proteins L7 / L12>
A pGEX-6P-1 plasmid (manufactured by GE Healthcare) in which the ribosomal protein L7 / L12 gene (SEQ ID NO: 20) of Haemophilus influenzae was cloned was introduced into Escherichia coli BL21 (DE3) pLysS Escherichia coli strain (manufactured by Promega).

The resulting E. coli strain, M9 medium _{(47.7mM Na 2 HPO 4 · 12H} 2 O, 22mM KH 2 PO 4, 8.6mM NaCl, 2mM MgSO 4, 50μM ZnSO 4, 100μM CaCl 2, 4.1μM 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 ampicillin sodium, 18.7 mM ¹⁵ N-NH ₄ Cl, 11.1 mM ¹³ C-glucose), _{cultured at 37 ° C. until OD 600} reached 0.6, and quenched with ice water. IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 1 mM, and the cells were cultured at 16 ° C. for 36 hours and then centrifuged at 7000 rpm for 15 minutes at 4 ° C. to recover Escherichia coli.

To 1 g of the obtained E. coli, 5 mL of BugBuster (manufactured by ^{Merck Millipore) and 5 μL of benzoase (registered trademark)} endonuclease (manufactured by Merck Millipore) were added, and the mixture was shaken at room temperature for 30 minutes to completely dissolve the E. coli. After filtering with a 0.45 μm filter, the ribosomal protein L7 / L12 was purified on a glutathione-sepharose column using a Profinia protein purification system (manufactured by Bio-Rad). To 15 mL of the obtained protein solution, 1.5 mL of 10-fold concentration PBS (phosphate buffered saline) and Precision Protease (manufactured by GE Healthcare) were added, and the mixture was shaken at room temperature for 2 hours. The reaction was passed through the glutathione-sepharose column again to obtain a pass-through fraction as ribosomal proteins L7 / L12.

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

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

<2: Three-dimensional structural analysis of Haemophilus influenzae ribosomal proteins L7 / L12 by NMR>
In a Shigemi NMR sample tube, 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-silappentan-1-sulfonic acid) are placed, degassed with an ejector, and then NMR. Installed in the device.

AVANCE III HD 600MHz NMR device (manufactured by Bruker), HNCO (4 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HN (CO) CA (8 integrations, number of data points (8) F1 x F2 x F3) 64 x 128 x 1024), HNCA (8 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024), CBCA (CO) NH (16 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024) F1 x F2 x F3) 64 x 128 x 1024), HNCACB (8 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024), HBHA (CO) NH (16 integrations, number of data points (F1 x F2 x F3) 64 x 128 x 1024) F1 x F2 x F3) 64 x 128 x 1024), HN (CA) HA (32 totals, number of data points (F1 x F2 x F3) 64 x 128 x 1024), C (CO) NH (16 totals, Using a pulse sequence of the number of data points (F1 x F2 x F3) 128 x 128 x 1024), each FID was subjected to [ ¹ H- ¹⁵ N] HSQC (integration number 8,) with a Unity INOVA 800 MHz NMR device (manufactured by Agent). Number of data points (F1 x F2) 512 x 2048), [ ¹ H- ¹³ C] HSQC alipatic (number of integrations 8, number of data points (F1 x F2) 868 x 2048), [ ¹ H- ¹³ C] HSQC aromatic ( Number of integrations 32, number of data points (F1 x F2) 204 x 2048), HCCH-TOCSY aliphatic (number of integrations 4, number of data points (F1 x F2 x F3) 96 x 256 x 2048), HCCH-TOCSY aromatic (number of integrations) 8, number of data points (F1 x F2 x F3) 128 x 128 x 2048), ¹⁵ N-edited NOESY (mixing time 75 milliseconds, number of integrations 8, number of data points (F1 x F2 x F3) 64 x 256 x 2048) ), ¹³ C-edited NOESY aliphatic (mixing time 75 milliseconds, total number of times 8, number of data points (F1 x F2 x F3) 96 x 256 x 2048), ¹³ C-edited NOESY aromatic (mixing time 75 milliseconds, total) Each FID was obtained using a pulse sequence of 8 times and the number of data points (F1 × F2 × F3) 64 × 256 × 2048). Each obtained FID was Fourier transformed using NMR Pipe software to obtain each spectrum.

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

Enter a belonging to atomic coordinates and ^{1 H} distance information CYANA conformation automatic calculation software, is converged sufficiently energy values through the 20 times of the three-dimensional structure calculation, the spatial atomic coordinates of ribosomal protein L7 / L12 conformation Obtained.

The acquired spatial atomic coordinates were input to Pymol software, and the three-dimensional structure of the ribosomal proteins L7 / L12 was drawn (FIG. 3). When the three-dimensional structure of the main chain of the ribosomal protein L7 / L12 is displayed, one three-dimensional structure (NTD: N-Terminal Domain) is formed by the amino acid residues at positions 1 to 40, and from about 14 amino acid residues. It was found that another three-dimensional structure (CTD: C-Terminal Domain) was further formed by the amino acid residues at positions 55 to 123 via the linker that did not form the three-dimensional structure (FIG. 3). It was also found that two ribosomal protein L7 / L12 molecules having such a three-dimensional structure associate with each other's NTDs to form a dimer (FIG. 3).

[Example 4] Comparative study of NTD portion of salmonella, Mycoplasma pneumoniae, and Haemophilus influenzae ribosomal protein L7 / L12 FIG. 4 shows the N-terminal domain (NTD) of Salmonella, Mycoplasma pneumoniae, and Haemophilus influenzae ribosomal protein L7 / L12. It is a figure which shows typically the three-dimensional structure of each part. As is clear from this figure, when the three-dimensional structures of NTDs of Salmonella, Mycoplasma pneumoniae, and Haemophilus influenzae ribosomal proteins L7 / L12 were compared, a region with a large difference in surface shape and charge distribution was observed (Fig. 4). Dotted part). Comparing the surface charge distribution of this region among the three bacterial species, in the case of Mycoplasma pneumoniae, the central part is occupied by the hydrophobic (white) to uncharged hydrophilic region (light blue, light red). In the case of influenza bacteria, the negative charge (red) occupies almost the entire region, and in the case of Salmonella, the negative charge region (red) occupies a large proportion, but on the right side is a hydrophobic (white) to uncharged hydrophilic region (thin). Blue and light red) are located, and a difference was observed between these two bacterial species.

FIG. 5 shows the alignment of the amino acid sequences of the N-terminal domain (NTD) portion of the ribosomal proteins L7 / L12 of Salmonella, Mycoplasma pneumoniae, and Haemophilus influenzae. As is clear from this figure, a large difference was also observed in the amino acid sequences of the N-terminal domain (NTD) portion of the ribosomal proteins L7 / L12 among the three bacterial species. This region is the amino acid residue at positions 5 to 23 of L7 / L12 of Mycoplasma pneumoniae, and corresponds to the amino acid residue at position 4 to 22 of L7 / L12 of Haemophilus influenzae and Salmonella. The residues at positions 5 to 7 of mycoplasma pneumoniae correspond to the residues at positions 4 to 6 of influenza and salmonella, and the amino acid sequences of mycoplasma pneumoniae are D (aspartic acid: hydrophilic, negatively charged), K (lysine: lysine:). Hydrophilic, positively charged), N (asparagine: hydrophilic, uncharged), whereas influenza bacteria are T (threonine: hydrophilic, uncharged), N (asparagine: hydrophilic, uncharged), E ( Glutamic acid: hydrophilic, negatively charged), Salmonella are T (threonine: hydrophilic, uncharged), K (lysine: hydrophilic, positively charged), D (aspartic acid: hydrophilic, negatively charged), and are the types of amino acids. And the order of polarity is different for each bacterial species (Fig. 5). The residues at positions 14 to 16 of mycoplasma pneumoniae correspond to the residues 13 to 15 of mycoplasma pneumoniae, and the amino acid sequences of mycoplasma pneumoniae are K (lysine: hydrophilic, positively charged) and E (glutamic acid). : Hydrophilic, negatively charged), M (methionine: hydrophobic), whereas influenza bacteria are A (alanine: hydrophobic), S (serine: hydrophilic, uncharged), K (lysine: hydrophilic, (Positive charge), Salmonella bacteria are S (serine: hydrophilic, uncharged), A (alanine: hydrophobic), M (methionine: hydrophobic), and the order of amino acid types and polarities differs depending on the bacterial species. (Fig. 5). Similarly, a difference in polarity was observed between the amino acid residues at positions 19 and 22 of Mycoplasma pneumoniae and the corresponding amino acid residues at positions 18 and 21 of Haemophilus influenzae and Salmonella (Fig.). 5).

As described above, the three-dimensional structure of Salmonella L7 / L12 at positions 4 to 22 and the polarity of amino acid residues are significantly different from those of the other two bacterial species. Predicted (FIGS. 4 and 5). Further, due to the difference in amino acid polarity among the three bacterial species, among the amino acid residues constituting the NTD of L7 / L12 of Salmonella, preferably the 4th to 15th positions, more preferably the 4th to 6th positions or the 13th to 15th positions. It is predicted to be the center of interaction with the antibody (Fig. 5).

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

<1: Preparation of Escherichia coli expressing NTD of Salmonella ribosomal protein L7 / L12 alone>
An artificial synthetic gene 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 salmonella ribosome protein L7 / L12 to which BamHI and XhoI restriction enzyme cleavage sites have been added. (Manufactured by GenScript) was cleaved with the above-mentioned two types of restriction enzymes, and then electrophoresed and stained with ethidium bromide in a 1.5% agarose gel. A band of about 400 bp was cut from the gel. This band was purified with a QIA quick Gel Extraction Kit (manufactured by QIAGEN) and inserted into a general vector, pGEX-6P-1 (manufactured by GE Healthcare).

Specifically, the vector pGEX-6P-1 and the above DNA (artificial synthetic gene cleaved with a restriction enzyme and purified with a gel) are mixed so as to have a molar ratio of 1: 3, and T4. The DNA was incorporated into a vector with a DNA ligase (manufactured by Invitrogen). The vector pGEX6P-1 incorporating the DNA was introduced into BL21 (DE3) pLysS Escherichia coli strain (Promega) by a genetic method, and then on a semi-solid culture plate containing 50 μg / mL ampicillin (Sigma). A certain LB L-broth agar (manufactured by Takara Shuzo Co., Ltd.) was inoculated. The plates were incubated at 37 ° C. for 12 hours, the grown colonies were indiscriminately selected and inoculated into L-broth culture medium containing the same concentration of ampicillin. After culturing with shaking at 37 ° C. for 8 hours, the cells were collected by centrifugation, and the plasmid was separated using the QIAprep Spin Miniprep Kit (QIAGEN) according to the attached instructions. The obtained plasmid was cleaved with the restriction enzymes BamHI / XhoI, and the DNA of about 370 bp was cleaved to confirm the insertion of the PCR product. The base sequence of the inserted DNA was determined using the above clone.

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

Two of the five indiscriminately selected clones had sequence homology with the probe used for PCR. In addition, a DNA sequence that matched the gene sequence of the amino acid residues at positions 1 to 40 of the Salmonella ribosomal protein L7 / L12 was clear. This gene fragment clearly encodes the NTD consisting of the amino acid residues 1 to 40 of the Salmonella ribosomal protein L7 / L12.

<2: Preparation of NTD by culturing Escherichia coli expressing NTD of Salmonella ribosomal protein L7 / L12 alone>
Using Escherichia coli expressing NTD alone formed by the amino acid residues at positions 1 to 40 of the salmonella ribosomal protein L7 / L12 obtained above, the amino acids at positions 1 to 40 of the salmonella ribosomal protein L7 / L12 were used. A protein corresponding to NTD formed by residues (NTD protein) was prepared.

Specifically, the same E. coli strain, M9 medium _{(47.7mM Na 2 HPO 4 · 12H} 2 O, 22mM KH 2 PO 4, 8.6mM NaCl, 2mM MgSO 4, 50μM ZnSO 4, 100μM 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 ampicillin sodium, 18.7 mM NH _It was cultured in 4 Cl (11.1 mM glucose) at 37 ° C. _{until OD 600} reached 0.6, and rapidly cooled with ice water. IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 1 mM, and the cells were cultured at 16 ° C. for 36 hours and then centrifuged at 7000 rpm for 15 minutes at 4 ° C. to recover Escherichia coli. To 1 g of the obtained Escherichia coli, 5 mL of BugBuster (manufactured by Merck Millipore) and 5 μL of benzoase endonuclease (manufactured by Merck Millipore) were added, and the mixture was shaken at room temperature for 30 minutes to completely dissolve the E. coli. After filtering with a 0.45 μm filter, the ribosomal protein L7 / L12 was purified on a glutathione-sepharose column using a Profinia protein purification system (manufactured by Bio-Rad). To 15 mL of the obtained protein solution, 1.5 mL of 10-fold concentration PBS (phosphate buffered saline) and Precision Protease (manufactured by GE Healthcare) were added, and the mixture was shaken at room temperature for 2 hours. The reaction solution was passed through the glutathione-sepharose column again to obtain a pass-through fraction as an NTD protein-containing fraction.

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

The obtained ribosomal protein L7 / L12 was used as a mobile phase in PBS (phosphate buffered physiological saline), and a gel filtration column HiLoad 16/60 Superdex 75pg (manufactured by GE Healthcare) was connected to the AKTA protein purification system (manufactured by GE Healthcare). ) At a flow rate of 1 mL / min, the ribosomal protein L7 / L12 was purified, and the concentration was quantified using the protein assay kit I (manufactured by BIO-RAD, model number 5000001JA). Served as NTD protein of / L12.

<3: Acquisition of mouse monoclonal antibody strain using NTD protein of Salmonella ribosomal protein L7 / L12>
Using the NTD protein of Salmonella ribosomal protein L7 / L12 for obtaining the obtained monoclonal antibody, the monoclonal antibody strain 31E2-F5-F9 against the NTD protein was used according to the method described in Example 3 of International Publication No. 2001/057199. , 33C9-F6-D1 and 36G2-F9-D2 were acquired.

Specifically, 100 μg of NTD protein of Salmonella ribosome protein L7 / L12 obtained in the above procedure was used as an antigen, dissolved in 200 μL of PBS, and then 200 μL of Freund's Complete Adjuvant was added and mixed. Emulsified. 200 μL of the obtained antigen emulsion was injected intraperitoneally into mice. In addition, the same antigen emulsion was injected intraperitoneally into mice 2 weeks, 4 weeks, and 6 weeks after the initial antigen administration. In addition, 10 and 14 weeks after the initial antigen administration, double concentrations of the antigen emulsion were injected intraperitoneally into the mice. Three days after the administration of the final antigen, the spleen was removed from the mouse, the spleen cells were aseptically collected, and cell fusion with myeloma cells was performed according to the following procedure.

As myeloma cells, NS-1 cell lines were used. The cell line was cultured in RPMI1640 medium containing 10% fetal bovine serum, and from 2 weeks before cell fusion, 0.13 mM azaguanine, 0.5 μg / mL MC-210, and 10% fetal bovine serum. After culturing in RPMI1640 medium containing 10% of fetal bovine serum for 1 week, it was used after culturing in RPMI1640 medium containing 10% fetal bovine serum.

⁸ and splenocytes of mice was aseptically collected by the above procedure, the myeloma cells 2 × 10 ⁷ cells after the culture were mixed well in a glass tube, and centrifuged for 5 minutes at 1,500 rpm, After discarding the supernatant, the cells were further mixed. To this mixed cell sample, 50 mL of RPMI1640 culture solution kept at 37 ° C. was added, centrifuged at 1,500 rpm, the supernatant was removed, 1 mL of 50% polyethylene glycol kept at 37 ° C. was added, and the mixture was stirred for 1 minute. .. To this cell mixture, 10 mL of RPMI1640 culture solution maintained at 37 ° C. was added, and the mixture was vigorously stirred by suctioning and discharging with a sterilized pipette for about 5 minutes, and then centrifuged at 1,000 rpm for 5 minutes to remove the supernatant. After that, 30 mL of HAT culture solution was added so that the cell concentration became 5 × 10 ⁶ / mL, and the mixture was stirred until it became uniform. 0.1 mL of this cell mixture was poured into each well of a 96-well culture plate and cultured at 37 ° C. under a 7% carbon dioxide atmosphere. On the 1st day, the 1st week, and the 2nd week from the start of the culture, 0.1 mL each of HAT medium was added, and fusion cells producing the desired antibody were screened by the ELISA method.

Cells producing the desired antibody were screened by ELISA. Glutathione S-transferase (GST) protein was fused with the NTD protein of Salmonella ribosome protein L7 / L12 to prepare GST fusion L7 / L12 NTD protein. A diluted solution of the obtained GST fusion L7 / L12NTD protein and GST protein dissolved in PBS containing 0.05% soda azide at a concentration of 10 μg / mL was prepared. 100 μL of each of these solutions was separately dispensed into each well of a 96-well plate and adsorbed at 4 ° C. overnight. After removing the supernatant, 200 μL of 1% bovine serum albumin solution (in PBS) was added, and the mixture was reacted at room temperature for 1 hour for blocking. The supernatant was removed, the product was washed with a washing solution (PBS containing 0.02% Tween 20), 100 mL of the fusion cell culture solution was added, and the mixture was reacted at room temperature for 2 hours. After removing the supernatant and washing the precipitate with a washing solution, 100 μL of a goat anti-mouse IgG antibody solution (concentration 50 ng / mL) labeled with peroxidase was added, and the mixture was reacted at room temperature for 1 hour. After removing the supernatant and washing the product again with the washing liquid, 100 μL of TMB solution (manufactured by KPL) was added and reacted at room temperature for 20 minutes. After coloring, 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 with the GST fusion L7 / L12NTD protein but not with the GST protein, and it was found that these wells contained an antibody against the L7 / L12NTD protein. Therefore, the cells in each positive well were collected, placed in a 24-well plastic plate, cultured by adding HAT medium, diluted with HT medium so that the number of cells was about 20 cells / mL, and 50 μL thereof was 96. Placed in each well of a hole culture plate. After mixing suspended 6-week-old mice thymocytes ¹⁰⁶ in addition to the HT medium, 7% CO ₂ conditions, and cultured for 2 weeks at 37 ° C.. The antibody activity in the culture supernatant was similarly tested by the above-mentioned ELISA method, and cells positive for the reaction with the L7 / L12NTD protein were collected. Further, by repeating the same dilution test and cloning operation, each hybridoma producing the monoclonal antibody strains 31E2-F5-F9, 33C9-F6-D1 and 36G2-F9-D2 against the L7 / L12NTD protein was obtained.

Using the three positive clone monoclonal antibody strains 31E2-F5-F9, 33C9-F6-D1 and 36G2-F9-D2 obtained as described above, a monoclonal antibody was produced and recovered according to a conventional method.

<4: Determination of amino acid sequences of the light and heavy chain variable regions of the obtained mouse monoclonal antibody strain>
For the obtained monoclonal antibody strains 31E2-F5-F9, 33C9-F6-D1 and 36G2-F9-D2, the amino acid sequences and corresponding base sequences of the light chain and heavy chain variable regions were determined according to a conventional method. ..

The amino acid sequences and base sequences of the heavy chain and light chain variable regions of the monoclonal antibody strains 31E2-F5-F9, 33C9-F6-D1 and 36G2-F9-D2 are shown by the SEQ ID NOs shown in Table 1 below, respectively.

[Example 6] Analysis of interaction between salmonella ribosomal protein L7 / L12 and antibody Monoclonal antibodies 31E2-F5-F9, 33C9-F6-that recognize NTD of salmonella ribosomal protein L7 / L12 obtained in Example 5 The interactions of D1 and 36G2-F9-D2 with the ribosomal proteins L7 / L12 of Salmonella were analyzed by NMR according to the following procedure.

<1: Acquisition of Salmonella ribosome protein L7 / L12>
E. coli strain prepared in Example 1, M9 medium _{(47.7mM Na 2 HPO 4 · 12H} 2 O, 22mM KH 2 PO 4, 8.6mM NaCl, 2mM MgSO 4, 50μM ZnSO 4, 100μM 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 ampicillin sodium, 18.7 mM ¹⁵ It was cultured in N-NH ₄ Cl (11.1 mM ¹² C-glucose) at 37 ° C. _{until OD 600} reached 0.6, and rapidly cooled in ice water. IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 1 mM, and the cells were cultured at 16 ° C. for 36 hours and then centrifuged at 7000 rpm for 15 minutes at 4 ° C. to recover Escherichia coli. To 1 g of the obtained Escherichia coli, 5 mL of BugBuster (manufactured by Merck Millipore) and 5 μL of benzoase endonuclease (manufactured by Merck Millipore) were added, and the mixture was shaken at room temperature for 30 minutes to completely dissolve the E. coli. After filtering with a 0.45 μm filter, the ribosomal protein L7 / L12 was purified on a glutathione-sepharose column using a Profinia protein purification system (manufactured by Bio-Rad). To 15 mL of the obtained protein solution, 1.5 mL of 10-fold concentration PBS (phosphate buffered saline) and Precision Protease (manufactured by GE Healthcare) were added, and the mixture was shaken at room temperature for 2 hours. The reaction was passed through the glutathione-sepharose column again to obtain a pass-through fraction as ribosomal proteins L7 / L12.

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

The obtained ribosomal protein L7 / L12 was used as a mobile phase in PBS (phosphate buffered saline), and a gel filtration column HiLoad 16/60 Superdex 75pg (manufactured by GE Healthcare) was connected to the AKTA protein purification system (manufactured by GE Healthcare). ) At a flow rate of 1 mL / min to purify the ribosomal proteins L7 / L12, quantify the concentration using Protein Assay Kit I (manufactured by BIO-RAD, model number 5000001JA), and then subject to NMR measurement.

<2: Pretreatment of Salmonella ribosomal protein L7 / L12 antibody>
The monoclonal antibodies 31E2-F5-F9, 33C9-F6-D1 and 36G2-F9-D2 obtained in Example 5 that recognize the NTD of the ribosomal proteins L7 / L12 of Salmonella were added to PBS (phosphate buffered saline). ) Was dialyzed as an external solution, the concentration was quantified by the absorbance of ultraviolet light (wavelength 280 nm), and then the mixture was subjected to NMR measurement.

<3: Interaction analysis of Salmonella ribosomal proteins L7 / L12 and antibody by NMR>
Salmonella ribosomal proteins L7 / L12 and antibodies 31E2-F5-F9, 33C9-F6-D1 or 36G2-F9-D2 were mixed at a ratio of 1: 0.5 (molar ratio), respectively. After each mixture is measured up to 250 μL with PBS (phosphate buffered saline), 20 μL of heavy water and 1 μL of 5 mg / mL DSS (4,4-dimethyl-4-silappentan-1-sulfonic acid) are added, and Shigemi NMR is performed. It was transferred to a sample tube, degassed with an aspirator, and then placed in an NMR apparatus.

^{A FID was obtained using a pulse sequence of [1} H- ¹⁵ N] HSQC (integration number 128, data point number (F1 × F2) 512 × 2048) with an AVANCE III HD 600MHz NMR apparatus (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 acquired spectrum on Sparky software.

In NMR, if it has a low molecular weight (13,000) such as ribosomal proteins L7 / L12, it can be observed as a sharp signal, but if it has a high molecular weight (15,000) such as an antibody, it can be observed. The signal is so wide and unclear that it cannot be observed due to insufficient intensity. In addition, the binding / dissociation constant of the antigen-antibody reaction is generally 1 μM or less, and it is considered that the equilibrium state is biased toward the binding side. Therefore, when the ribosomal protein L7 / L12 interacts (binds) with the antibody, it is considered that the signal of the ribosomal protein L7 / L12 is broadened under the influence of the antibody and its intensity is attenuated. Therefore, on the [ ¹ H- ¹⁵ N] HSQC spectrum of ribosomal proteins L7 / L12 (one signal is observed for each amino acid), the interaction is performed by tracking the residues whose signal intensity is attenuated by the addition of the antibody. An attempt was made to identify the site. For details of the interaction analysis, follow Williamson, MP, Using chemical shift perturbation to characterise ligand binding, Prog. Nucl. Magn. Reson. Spectrosc. 73 (2013): 1-16.

^[1 H] of each amino acid residue of ribosomal protein L7 / L12 when ribosomal protein L7 / L12 and antibody 31E2-F5-F9, 33C9-F6-D1 or 36G2-F9-D are mixed in the above ratios, respectively. - ¹⁵ N] a HSQC signal strength, divided by ^{[1 H-} 15 N] HSQC signal strength of each amino acid residue of the previous ribosomal protein L7 / L12 is mixed with the antibody, the vertical axis of the division value, ribosomal protein A graph showing the amino acid sequence of L7 / L12 on the horizontal axis was created (FIGS. 6A, B, C). The division value of NTD (residues at positions 1 to 40) was approximately 0.4 or less (meaning that the signal intensity of ribosomal proteins L7 / L12 was attenuated by 60% or more by interacting with the antibody). Proteins L7 / L12 were found to interact with antibodies 31E2-F5-F9, 33C9-F6-D1 or 36G2-F9-D2 at NTD (residues at positions 1-40) (FIGS. 6A, B). , C).

[Example 7] Examination of cross-reactivity of antibody binding to salmonella ribosomal protein L7 / L12 Monoclonal antibody 31E2-F5-F9 that recognizes NTD of salmonella ribosomal protein L7 / L12 obtained in Example 5, 33C9-F6-D1 and 36G2-F9-D2 were analyzed by ELISA for cross-reactivity with ribosomal proteins L7 / L12 of bacteria other than Salmonella according to the following procedure.

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

The obtained plasmid vector pGEX-6P-1 carrying the L7 / L12 gene of each bacterial strain was introduced into Escherichia coli One Shot Competent Cells (manufactured by Invitrogen), and LB containing 50 μg / mL ampicillin (manufactured by Sigma). Seed on a plate of semi-solid medium (manufactured by Takara Shuzo Co., Ltd.), left at 37 ° C. for about 12 hours, randomly selected the resulting colonies, and planted in 2 mL of LB liquid medium containing the same concentration of ampicillin, 8 The cells were collected by shaking and culturing at 37 ° C. for about an hour. The plasmid was isolated from the obtained cells using the QIAprep Spin Miniprep Kit (QIAGEN) according to the attached instructions. The obtained plasmid was cleaved with the restriction enzymes BamHI / XhoI. Insertion of the ribosomal protein L7 / L12 artificial synthetic gene of each bacterial species was confirmed by cleaving about 370 bp of DNA. Escherichia coli into which the plasmid vector was introduced was cultured overnight at 37 ° C. in 50 mL of LB medium, then placed in 500 mL of TB medium and cultured for 1 hour. Then, 550 μL of 100 mM isopropyl β-D (-)-thiogalactopyranoside (IPTG) was added, and the cells were further cultured for 4 hours. After recovery, 1/100 amount of BugBuster (manufactured by Merck) was added, and the mixture was shaken at room temperature for 20 minutes. Then, the Escherichia coli was collected by centrifugation at 10,000 rpm for 30 minutes.

The recombinant full-length ribosomal protein L7 / L12 was collected and purified by the same method as in Example 1 to obtain a recombinant full-length ribosomal protein L7 / L12 of each bacterial species.

<2: Implementation of ELISA for cross-reactivity analysis>
The recombinant ribosome proteins L7 / L12 of each bacterial species were immobilized on an ELISA plate, and each of the monoclonal antibodies 31E2-F5-F9, 33C9-F6-D1 or 36G2-F9-D2 was reacted and washed. After that, the monoclonal antibody bound to L7 / L12 of each immobilized bacterial species was reacted with a peroxidase-labeled anti-mouse IgG antibody, and the cross-reactivity of each monoclonal antibody with L7 / L12 of each bacterial species was evaluated. did.

Specifically, a 96-well ELISA plate containing 100 μL of PBS solution containing each of Salmonella and the recombinant full-length ribosome protein L7 / L12 of each of the above strains at a concentration of 0.01 μg / mL, 0.1 μg / mL, or 1 μg / mL, respectively. It was dispensed into (Maxsorp ELISA plate manufactured by Nunc) and adsorbed overnight at 4 ° C. After removing the supernatant, 200 μL of 1% bovine serum albumin solution (in PBS) was added, and the mixture was reacted at room temperature for 1 hour for procking. After removing the supernatant, wash with a washing solution (PBS containing 0.02% Tween 20) several times, and 0.5 to 1 μg / mL of antibody 31E2-F5-F9, 33C9-F6-D1 or 36G2-F9-D2. An antibody solution diluted with% TritonX-100 / PBS or about 1 × PBS itself (negative control) was added in an amount of 100 μL, and the mixture was reacted at room temperature for 1 hour. After removing the supernatant, 100 μL of a solution diluted with 0.02% Tween 20 / PBS to a final concentration of 1 μg / mL was added as a secondary antibody using a peroxidase-labeled anti-mouse IgG antibody reagent at room temperature. It was allowed to react for 1 hour. After removing the supernatant, wash with a washing solution several times, add 100 μL of TMB (3,3', 5,5'-tetramethylbenzidine) solution (manufactured by KPL), react at room temperature for 10 minutes, and then 1 mol / mol /. The reaction was stopped by adding 100 μL of L hydrochloric acid. The cross-reactivity of each monoclonal antibody with L7 / L12 of each bacterial species was evaluated by measuring the absorbance at 450 nm of the obtained solution and determining the difference from the absorbance at 450 nm of the negative control.

Evaluation result of the reactivity of the antibody 31E2-F5-F9, 33C9-F6-D1 or 36G2-F9-D2 obtained as a result with the recombinant full-length ribosome protein L7 / L12 of Salmonella or other bacterial species. Is shown in Table 3 below. In the table below, positive (+) indicates that the difference in absorbance with respect to the negative control is 0.5 or more, and negative (-) indicates that the difference in absorbance with respect to the negative control is 0.1 or less.

The present invention can be widely used in fields where detection of Salmonella in a sample is required, mainly in the medical field, and its industrial usefulness is extremely high.

Claims (13)

An antibody for detecting Salmonella enteritidis, which is an epitope present in the N-terminal domain (NTD) consisting of amino acid residues 1 to 40 of Salmonella ribosomal protein L7 / L12 shown in SEQ ID NO: 1. Antibodies or fragments thereof, or derivatives thereof, that cause an antibody reaction. The antibody or fragment thereof according to claim 1, or a derivative thereof, wherein the epitope contains one or more amino acid residues selected from the amino acid residues at positions 4 to 22 of SEQ ID NO: 1. Mycoplasma, Escherichia, Chlamydia, Pseudomonas, Streptococcus, Staphylococcus, Neisseria, Haemophilus The antibody according to claim 1 or 2, or a fragment thereof, or a fragment thereof, which does not cross-react with bacteria of one or more genera selected from the genus Bordetella, Moraxella, and Legionella. Derivative. As the heavy chain variable region sequence, an amino acid sequence having 80% or more homology with any one of the amino acid sequences selected from SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13 and an amino acid sequence having 80% or more homology.
13. Of claims 1-3, the light chain variable region sequence comprises an amino acid sequence having 80% or more homology with any one amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15. The antibody according to any one of the above, a fragment thereof, or a derivative thereof. As the heavy chain variable region sequence, an amino acid sequence having 80% or more homology with any one of the amino acid sequences selected from SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13 and an amino acid sequence having 80% or more homology.
As the light chain variable region sequence, an antibody or a fragment thereof each containing an amino acid sequence having 80% or more homology with any one amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15 or Derivatives of them. A nucleic acid molecule encoding the antibody or fragment thereof according to any one of claims 1 to 5, or a derivative thereof. A vector or plasmid containing the nucleic acid molecule according to claim 6. A host cell transformed with the nucleic acid molecule according to claim 6 or the vector or plasmid according to claim 7. The host cell according to claim 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. A hybridoma expressing the antibody or fragment thereof according to any one of claims 1 to 5, or a derivative thereof. A method for detecting the presence or absence of Salmonella in a sample, wherein the antibody or fragment thereof according to any one of claims 1 to 5 or a derivative thereof is brought into contact with the sample to determine the presence or absence of an antigen-antibody reaction. Methods involving detection. A reagent for detecting the presence or absence of Salmonella in a sample, which comprises the antibody or fragment thereof according to any one of claims 1 to 5, or a derivative thereof. A kit for detecting the presence or absence of Salmonella in a sample, wherein the antibody or fragment thereof according to any one of claims 1 to 5, or a derivative thereof, and the Salmonella in the sample are used. A kit that includes instructions, including instructions for detecting the presence or absence.

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