JP2021164416A - Antibody for detecting moraxella catarrhalis in sample, and method, reagent, and kit for detecting moraxella catarrhalis with the antibody - Google Patents

Abstract

To provide an antibody for the detection of Moraxella catarrhalis with excellent detection sensitivity and detection accuracy (species specificity).SOLUTION: The present disclosure provides an antibody that shows an antigen-antibody reaction with an epitope within a C terminal domain (CTD) composed of amino acid residues at 58-124 positions of ribosome protein L7/L12 of Moraxella catarrhalis, or a fragment thereof, or derivatives thereof.SELECTED DRAWING: Figure 1

Description

The present invention relates to an antibody for detecting whether or not Moraxella catarrhalis is present in a sample, and a method and reagent for detecting Moraxella catarrhalis in a sample using such an antibody. , And about the kit.

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 using 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.

Moraxella catarrhalis is a gram-negative coccus of the genus Moraxella. It is one of the typical causative agents of respiratory infections such as pneumonia and bronchitis in humans, and is known to cause sinusitis and purulent keratoconjunctivitis. Antibiotics are generally used to treat infectious diseases caused by Moraxella catarrhalis, but many bacteria show drug resistance, and when they worsen, treatment is difficult. Therefore, early detection and treatment of infection is required.

As a method for detecting Moraxella catarrhalis, a reaction medium method (Patent Document 1: Japanese Patent Application Laid-Open No. 2010-525803) and a real-time PCR (polymerase chain reaction) method (Non-Patent Document 1: Journal of Clinical Microbiology, 2003, Vol. .41, No.4, p.1386-1390) is known. However, these methods were unsatisfactory in that the time required for the test was long.

The present inventors have focused on ribosomal 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 for 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 2: International Publication No. 2000). / 006603). However, such existing antibodies have a detection accuracy (species specificity) and a binding pattern of a specific antibody to the Ribosomal protein L7 / L12 of Moraxella catarrhalis to ensure the detection accuracy. There was room for improvement, such as the fact that was unknown.

Special table 2010-525803 International Publication No. 2000/006603

Journal of Clinical Microbiology, 2003, Vol.41, No.4, p.1386-1390

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

As a result of diligent studies, the present inventors have excellent antigen-antibody reactivity and specificity in the C-terminal domain (CTD) consisting of amino acid residues at positions 58 to 124 of the ribosomal protein L7 / L12 of Moraxella catarrhalis. We have found that there is a partial amino acid sequence that can be an epitope. On that basis, we actually prepared a plurality of antibodies that generate an antigen-antibody reaction with such a partial amino acid sequence, and verified that Moraxella catarrhalis bacteria in a sample can be detected with high sensitivity and accuracy by using these antibodies. , The present invention has been reached.

That is, the gist of the present invention is as follows.
[1] An antibody for detecting Moraxella catarrhalis, which is a C-terminal domain consisting of amino acid residues at positions 58 to 124 of the ribosome protein L7 / L12 of Moraxella catarrhalis shown in SEQ ID NO: 1. An antibody or fragment thereof, or a derivative thereof, that causes an antigen-antibody reaction with an epitope present in (CTD).
[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 101 to 113 of SEQ ID NO: 1. ..
[3] Mycoplasma, Escherichia, Chlamydia, Salmonella, Pseudomonas, 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 genera Bordetella, Legionella, and Streptococcus. 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 items, 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 of the amino acid sequences selected from SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15. 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 Moraxella catarrhalis in a sample, wherein the antibody or fragment thereof according to any one of items [1] to [5], or a derivative thereof is used as a sample. A method comprising contacting and detecting the presence or absence of an antigen-antibody reaction.
[12] A reagent for detecting the presence or absence of Moraxella catarrhalis in a sample, which comprises the antibody or fragment thereof according to any one of items [1] to [5], or a derivative thereof. reagent.
[13] A kit for detecting the presence or absence of Moraxella catarrhalis in a sample, wherein the antibody or fragment thereof according to any one of items [1] to [5], or a derivative thereof, and the above-mentioned A kit that includes an instruction sheet that includes instructions for detecting the presence or absence of Moraxella catarrhalis in a sample using an antibody.

According to the antibody of the present invention, it is possible to detect Moraxella catarrhalis in a sample with high sensitivity and accuracy.

FIG. 1 is a diagram schematically showing the three-dimensional structure of the ribosomal proteins L7 / L12 of Moraxella catarrhalis. FIG. 2 is a diagram schematically showing the three-dimensional structure of the ribosomal proteins L7 / L12 of Legionella. FIG. 3 is a diagram schematically showing the three-dimensional structure of the gonococcal ribosomal protein L7 / L12. FIG. 4 is a diagram schematically showing the three-dimensional structure of the C-terminal domain (CTD) portion of the ribosomal proteins L7 / L12 of Moraxella catarrhalis, Neisseria gonorrhoeae, and Legionella. FIG. 5 is a diagram showing the alignment of the amino acid sequences of the C-terminal domain (CTD) portion of the ribosomal proteins L7 / L12 of Moraxella catarrhalis, Neisseria gonorrhoeae, and Legionella. FIG. 6A is a diagram schematically showing the results of NMR analysis of the interaction between the monoclonal antibody 4C1 and the ribosomal proteins L7 / L12 of Moraxella catarrhalis. FIG. 6B is a diagram schematically showing the results of NMR analysis of the interaction between the monoclonal antibody 7A3 and the ribosomal proteins L7 / L12 of Moraxella catarrhalis. FIG. 6C is a diagram schematically showing the results of NMR analysis of the interaction between the monoclonal antibody 25C3 and the ribosomal proteins L7 / L12 of Moraxella catarrhalis.

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

(1) Introduction:
In the present invention, "Moraxella catarrhalis" means a gram-negative coccus of the genus Moraxella. Moraxella catarrhalis is one of the typical causative agents of respiratory infections such as pneumonia and bronchitis in humans, and is known to cause sinusitis and purulent keratoconjunctivitis. Antibiotics are generally used to treat infectious diseases caused by Moraxella catarrhalis, but many bacteria show drug resistance, and when they worsen, treatment is difficult. Therefore, early detection and treatment of infection is required.

The present inventors paid attention to the ribosomal proteins L7 / L12 in producing an antibody for detecting Moraxella catarrhalis. 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 Moraxella catarrhalis ribosomal protein L7 / L12 has a dimeric structure in which two copies of a monomeric molecule composed of 124 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 ribosome protein L7 / L12 of Moraxella catalaris 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 17 amino acid residues, another three-dimensional structure (CTD: C-Terminal Domain) is further formed by amino acid residues at positions 58 to 124. .. In addition, two copies of monomeric 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 Legionella pneumophila and Neisseria gonorrhoeae are also dimeric with two molecules associated with NTD (N-Terminal Domain, amino acid residues 1 to 40). After forming a random coil structure linker (amino acid residues at positions 41 to 57), CTD (C-Terminal Domain, amino acid residues at positions 58 to 125 in the case of Legionella, and 58 to 58 in the case of gonococcus). It was found that the amino acid residue at position 123) was formed (see Examples 2 and 3 and FIGS. 2 and 3 described later).

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

Comparing the three-dimensional structures of the CTDs of L7 / L12 of Moraxella catarrhalis, Moraxella catarrhalis, and Gonorrhea, there is a large difference in surface shape and charge distribution. In particular, the amino acid residue at positions 101 to 113 of Moraxella catarrhalis L7 / L12. It was found that the region consisting of the base had a large difference between bacterial species in terms of three-dimensional structure (dotted line in FIG. 4). In the case of Moraxella catarrhalis (MC), the ratio of hydrophobic (white) to uncharged hydrophilic regions (light blue, light red) is high, and in the case of Regionella (LP) and Neisseria gonorrhoeae (NG). Is dominated by the negative charge region (red). In addition, when comparing Regionella and gonococcus, the negative charge (red) occupies most of the region of the regionella, but in the case of gonococcus, the negative charge region (red) is in the upper part and the hydrophobicity is in the lower part (red). White) to uncharged hydrophilic regions (light blue, light red) are located (see the dotted lines in Examples 4 and 4 below).

Furthermore, comparing the amino acid sequences of the ribosome proteins L7 / L12 of Regionella, Moraxella catarrhalis, and Neisseria gonorrhoeae, the amino acid residues at positions 102 to 114 of Regionella and the positions 101 to 113 of Moraxella catarrhalis corresponding to the region are compared. It was also found that there is a large difference between bacterial species in the amino acid residues of Moraxella gonorrhoeae and the amino acid residues at positions 100 to 112 of Moraxella gonorrhoeae (Fig. 5). For example, the amino acid residues 109 to 110 of L7 / L12 of Regionella correspond to the amino acid residues 108 to 109 of Moraxella Catalaris and the amino acid residues 107 to 108 of gonococcus, and correspond to the amino acid residues 107 to 108 of Regionella. In the case of A (alanine: hydrophobic), S (serine: hydrophilic, uncharged), in the case of Moraxella catalaris, E (glutamic acid: hydrophilic, negatively charged), E (glutamic acid: hydrophilic) , Negative charge), E (glutamic acid: hydrophilic, negative charge) in the case of gonococcus, D (aspartic acid: hydrophilic, negative charge), and the order of amino acid type and polarity is different for each bacterial species ( FIG. 5).

Similarly, the amino acid residues at positions 112 to 114 of Regionella, the amino acid residues at positions 111 to 113 of Moraxella catarrhalis corresponding to the region, and the amino acid residues at positions 110 to 112 of Neisseria gonorrhoeae are also interspecific. It was also found that the difference between the two was large. The amino acid residues at positions 112 to 114 of Regionella are K (lysine: hydrophilic, positively charged), K (lysine: hydrophilic, positively charged), and E (glutamic acid: hydrophilic, negatively charged). The amino acid residues at positions 111 to 113 of Moraxera catalaris are K (lysine: hydrophilic, positively charged), K (lysine: hydrophilic, positively charged), K (lysine: hydrophilic, positively charged), 110 of gonococcus. The amino acid residues at positions ~ 112 are Q (glutamine: hydrophilic, uncharged), K (lysine: hydrophilic, positively charged), Q (glutamine: hydrophilic, uncharged), and the order of amino acid types and polarities. Is different for each bacterial species (Fig. 5).

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

Then, antibodies 4C1, 7A3, and 25C3 were obtained by expressing a peptide having an amino acid sequence similar to that of L7 / L12 CTD, preparing an antibody that specifically binds to the peptide, and performing screening (described later). See Example 5). On that basis, it was confirmed that all of these antibodies caused an antigen-antibody reaction with the CTD of L7 / L12 of Moraxella catarrhalis, particularly an epitope composed of a specific amino acid residue (described later). See Example 6 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.

Each heavy chain includes a heavy chain stationary (CH) region and a heavy chain variable (VH) region. The light chain contains 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) and heavy chain variable (VH) regions are each four highly conserved regions (FR-1, FR-2, FR-3, FR-4) called framework regions (FR). 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 terminus to the carboxy terminus. 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 Moraxella catarrhalis 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 protein L7 / L12 of Moraxella catarrhalis 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 chain and the light chain, or their variable regions and genetic information such as CDR 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 specific amino acid residues in the ribosomal protein L7 / L12 of Moraxella catarrhalis.

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 Moraxella catarrhalis ribosomal protein L7 / L12, the epitope to which the antibody of the present invention binds is the amino acid residue at positions 58 to 124 of the amino acid sequence of the Moraxella catarrhalis ribosomal protein L7 / L12 shown in SEQ ID NO: 1. It is present in the CTD formed by the group. According to the analysis results of the present inventors described later in Examples 5 to 7, the Moraxella catarrhalis with high detection sensitivity and high detection accuracy (high bacterial species specificity) actually acquired by the present inventors in the examples. It has been confirmed that the bacterial detection antibodies 4C1, 7A3, and 25C3 all bind to CTD formed by the amino acid residues at positions 58 to 124 of L7 / L12.

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 58 to 124 of SEQ ID NO: 1, among which 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 is at least 1 or 2 or more selected from positions 101 to 113 of SEQ ID NO: 1, especially 3 or more, 4 or more, or 5 or more, or 6 More preferably, it contains 7 or more, 8 or more, 9 or more, or 10 or more amino acid residues, and 1 or 2 or more selected from positions 101, 105, 108, 109, and 111 to 113. It is more preferred to include amino acid residues. 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 the CTD of the ribosomal proteins L7 / L12 of Moraxella catarrhalis. In addition, these amino acid residues are places where the species specificity of Moraxella catarrhalis is particularly high when L7 / L12 of Moraxella catarrhalis is compared with L7 / L12 of other bacterial species. It is believed that these amino acid residues form an epitope for the antibody of the invention.

Further, it is preferable that the antibody of the present invention does not cause a cross-reactivity with bacteria other than Moraxella catarrhalis and other components.

Specifically, the antibodies of the present invention include the genera Mycoplasma, Escherichia, Chlamydia, Salmonella, Pseudomonas, Staphylococcus, and Niseria. It is preferable that it does not cross-react with bacteria of one or more genera selected from the genera Neisseria, Haemophilus, Bordetella, Legionella, and Streptococcus. 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, 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 an antigen-antibody reaction using a measurement method suitable for.

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. Although the specific analysis conditions are not limited, 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 heavy chain and light chain variable region sequences.

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 "homosphere" of two amino acid sequences is the ratio at which the same or similar amino acid residues appear at each corresponding portion when both amino acid sequences are aligned, and the "same" of the two amino acid sequences. "Sex" is the ratio at which the same amino acid residue appears at each corresponding site 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 classification based on the polarity, chargeability, and size of the amino acid (in each case, the type of each amino acid is indicated by a single character code). 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;
-Load-bearing 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 ribosome protein L7 / L12 of Moraxella catalaris to be detected, preferably the amino acid residues at positions 58 to 124 of SEQ ID NO: 1 constituting CTD (sequence). Polypeptide consisting of (amino acid residue of No. 3), or 80% or more, especially 85% or more, further 90% or more, especially 95% or more, 96% or more, or 97% or more, or 99% or more, In particular, it can be prepared using a polypeptide having an amino acid sequence having 100% homology (preferably identity) (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 Moraxella catarrhalis, and 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 no cross-reactivity occurs 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 proteins L7 / L12 of Moraxella catarrhalis and 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 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 the whole amino acid sequence of the antibody is specified, a base sequence encoding all or a part of the amino acid sequence of the desired antibody is obtained by a known method. It is also possible to prepare a nucleic acid molecule having such a nucleic acid molecule 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), etc. can be referred to.

2. Moraxella Catarrhalis Detection Method / Reagent / Kit:
Another aspect of the present invention relates to a method for detecting the presence or absence of Moraxella catarrhalis 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 Moraxella catarrhalis as described above, the presence or absence of such an antigen-antibody reaction can be detected by various known immunoassay methods. , It is possible to detect whether or not Moraxella catarrhalis is present in the sample.

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 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.

As described above, the antibody of the present invention recognizes a specific epitope present in the ribosomal protein L7 / L12 of Moraxella catarrhalis and causes an antigen-antibody reaction. Therefore, the ribosomal protein L7 / L12 of Moraxella catarrhalis occurs. The detection sensitivity can be improved by exposing the protein to the outside of the bacterial cell membrane. 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. Nonionic surfactants such as Triton X-100, Tween 20, Briji 35, Nonidet P-40, dodecyl-β-D-maltoside, and octyl-β-D-glucoside can be used as surfactants for lytic treatment. 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 Moraxella catarrhalis in a sample can be detected quickly and easily.

A kit containing a reagent containing the antibody of the present invention and an instruction sheet including an instruction for detecting the presence or absence of Moraxella catarrhalis in a sample using the antibody of the present invention for use in the detection method of the present invention. Is also the subject of the present 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 specifics used to detect Moraxella catarrhalis. It may be appropriately determined according to the type of immunoassay. Among them, specific examples of the kit that can easily detect the presence or absence of Moraxella catarrhalis 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 the ribosomal proteins L7 / L12 of Moraxella catarrhalis
<1: Acquisition of Moraxella catarrhalis ribosomal proteins L7 / L12>
A pGEX-6P-1 plasmid (manufactured by GE Healthcare) in which the ribosomal protein L7 / L12 gene (SEQ ID NO: 2) of Moraxella catarrhalis 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 nicotine amide, 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, ribosomal proteins L7 / L12 were 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 concentrated 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 a 4-fold 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% to elute the ribosomal proteins L7 / L12.
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 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 Moraxella catarrhalis 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 ^{aromatic, and 13} C-edited NOESY aromatic spectra, ¹ H within a distance of 5 A 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 ribosomal proteins L7 / L12 was drawn (Fig. 1). When the three-dimensional structure of the main chain of the ribosome 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 the three-dimensional structure of about 17 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 58 to 124 via the linker that did not form (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 the ribosomal proteins L7 / L12 of Legionella
<1: Acquisition of Ribosomal proteins L7 / L12 from Legionella>
The pGEX-6P-1 plasmid (manufactured by GE Healthcare), in which the ribosomal protein L7 / L12 gene (SEQ ID NO: 36) of Regionella 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 nicotine amide, 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, ribosomal proteins L7 / L12 were 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 concentrated 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 a 4-fold 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% to elute the ribosomal proteins L7 / L12.

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 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 structure analysis of Legionella 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 ^{aromatic, and 13} C-edited NOESY aromatic spectra, ¹ H within a distance of 5 A 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 ribosomal proteins L7 / L12 was drawn (Fig. 2). When the three-dimensional structure of the main chain of the ribosome 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 17 amino acid residues. It was found that another three-dimensional structure (CTD: C-Terminal Domain) was further formed by amino acid residues at positions 58 to 125 via a linker that did not form a 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 gonococcal ribosomal proteins L7 / L12
<1: Acquisition of gonococcal ribosomal proteins L7 / L12>
The pGEX-6P-1 plasmid (manufactured by GE Healthcare) in which the gonococcal ribosomal protein L7 / L12 gene (SEQ ID NO: 24) 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 nicotine amide, 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, ribosomal proteins L7 / L12 were 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 concentrated 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 a 4-fold 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% to elute the ribosomal proteins L7 / L12.

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 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 structure analysis of gonococcal 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 ^{aromatic, and 13} C-edited NOESY aromatic spectra, ¹ H within a distance of 5 A 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 ribosomal proteins L7 / L12 was drawn (FIG. 3). When the three-dimensional structure of the main chain of the ribosome 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 consists of 17 amino acid residues. It was found that another three-dimensional structure (CTD: C-Terminal Domain) was further formed by amino acid residues at positions 58 to 123 via a linker that did not form a 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 CTD portion of ribosomal protein L7 / L12 of Neisseria gonorrhoeae, Moraxella catarrhalis, and Neisseria gonorrhoeae FIG. 4 shows the C-terminal of ribosomal proteins L7 / L12 of Neisseria gonorrhoeae, Moraxella catarrhalis, and Neisseria gonorrhoeae. It is a figure which shows typically the three-dimensional structure of each of the domain (CTD) part. As is clear from this figure, when the three-dimensional structures of the CTDs of the ribosomal proteins L7 / L12 of Regionella, Moraxella catarrhalis, and Neisseria gonorrhoeae were compared, there was a region where there was a large difference in surface shape and charge distribution (Fig.). Dotted part of 4). This region is the amino acid residue at positions 102 to 114 of L7 / L12 of Legionella, and corresponds to the amino acid residue at positions 101 to 113 of Moraxella catarrhalis and 100 to 112 of gonococcus. In the case of Moraxella catarrhalis (MC), the ratio of hydrophobic (white) to uncharged hydrophilic regions (light blue, light red) is high, and in the case of Regionella (LP) and Neisseria gonorrhoeae (NG). Is dominated by the negative charge region (red). In addition, when comparing Regionella and gonococcus, the negative charge (red) occupies most of the region of the regionella, but in the case of gonococcus, the negative charge region (red) is in the upper part and the hydrophobicity is in the lower part (red). White) to uncharged hydrophilic regions (light blue, light red) are located (dotted line in FIG. 4).

Furthermore, comparing the amino acid sequences of the ribosome proteins L7 / L12 of Regionella, Moraxella catarrhalis, and Neisseria gonorrhoeae, the amino acid residues at positions 102 to 114 of Regionella and the positions 101 to 113 of Moraxella catarrhalis corresponding to the region are compared. It was also found that there is a large difference between bacterial species in the amino acid residues of Moraxella gonorrhoeae and the amino acid residues at positions 100 to 112 of Moraxella gonorrhoeae (Fig. 5). For example, the amino acid residues 109 to 110 of L7 / L12 of Regionella correspond to the amino acid residues 108 to 109 of Moraxella Catalaris and the amino acid residues 107 to 108 of gonococcus, and correspond to the amino acid residues 107 to 108 of Regionella. In the case of A (alanine: hydrophobic), S (serine: hydrophilic, uncharged), in the case of Moraxella catalaris, E (glutamic acid: hydrophilic, negatively charged), E (glutamic acid: hydrophilic) , Negative charge), E (glutamic acid: hydrophilic, negative charge) in the case of gonococcus, D (aspartic acid: hydrophilic, negative charge), and the order of amino acid type and polarity is different for each bacterial species ( FIG. 5).

Similarly, the amino acid residues at positions 112 to 114 of Regionella, the amino acid residues at positions 111 to 113 of Moraxella catarrhalis corresponding to the region, and the amino acid residues at positions 110 to 112 of Neisseria gonorrhoeae are also interspecific. It was also found that the difference between the two was large. The amino acid residues at positions 112 to 114 of Regionella are K (lysine: hydrophilic, positively charged), K (lysine: hydrophilic, positively charged), and E (glutamic acid: hydrophilic, negatively charged). The amino acid residues at positions 111 to 113 of Moraxera catalaris are K (lysine: hydrophilic, positively charged), K (lysine: hydrophilic, positively charged), K (lysine: hydrophilic, positively charged), 110 of gonococcus. The amino acid residues at positions ~ 112 are Q (glutamine: hydrophilic, uncharged), K (lysine: hydrophilic, positively charged), Q (glutamine: hydrophilic, uncharged), and the order of amino acid types and polarities. Is different for each bacterial species (Fig. 5).

As described above, the three-dimensional structure of the region at positions 101 to 113 of L7 / L12 of Moraxella catarrhalis and the polarity of amino acid residues are significantly different from those of the other two bacterial species. It is predicted to be close to the central region (Figs. 4 and 5). Furthermore, due to the difference in amino acid polarity among the three bacterial species, among the amino acid residues constituting the CTD of L7 / L12 of Moraxella catarrhalis, preferably at the 101st, 105th, or 108th to 109th positions, or , The amino acid residues at positions 111-113 are predicted to be the center of interaction with the antibody (Fig. 5).

[Example 5] Acquisition of antibody that binds to Moraxella catarrhalis ribosomal protein L7 / L12 CTD formed by amino acid residues at positions 58 to 124 of Moraxella catarrhalis ribosomal protein L7 / L12 by the following procedure. Was expressed alone, and 4C1, 7A3 and 25C3 were obtained as monoclonal antibodies that recognize this by an antigen-antibody reaction.

<1: Preparation of Escherichia coli expressing CTD of Moraxella catarrhalis ribosomal protein L7 / L12 alone>
Includes a nucleotide sequence (SEQ ID NO: 4) encoding an amino acid sequence (SEQ ID NO: 3) consisting of amino acid residues at positions 58 to 124 of the ribosome protein L7 / L12 of Moraxella catalaris bacterium to which BamHI and XhoI restriction enzyme cleavage sites have been added. The artificially synthesized gene (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 the QIAquick 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 58 to 124 of the Moraxella catarrhalis ribosomal protein L7 / L12 was clear. This gene fragment clearly encodes a CTD consisting of amino acid residues 58-124 of the Moraxella catarrhalis ribosomal protein L7 / L12.

<2: Preparation of CTD by culturing Escherichia coli expressing CTD of Moraxella catarrhalis ribosomal protein L7 / L12 alone>
Using Escherichia coli expressing CTD alone formed by the amino acid residues at positions 58 to 124 of the ribosomal protein L7 / L12 of Moraxella catalaris obtained above, the ribosomal protein L7 / L12 of Moraxella catalaris was used. A protein (CTD protein) corresponding to CTD formed by amino acid residues at positions 58 to 124 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, ribosomal proteins L7 / L12 were 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 concentrated 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, and the passing fraction was obtained as a CTD protein-containing fraction.

The obtained CTD protein-containing fraction was dialyzed with 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 CTD 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 to purify the ribosomal proteins L7 / L12, and quantify the concentration using Protein Assay Kit I (BIO-RAD, model number 5000001JA). It was provided as a CTD protein of ribosomal proteins L7 / L12.

<3: Acquisition of mouse monoclonal antibody strain using CTD protein of Moraxella catarrhalis ribosomal protein L7 / L12>
Using the CTD protein of the Moraxella catarrhalis ribosomal protein L7 / L12 for obtaining the obtained monoclonal antibody, the monoclonal antibody strain 4C1 against the CTD protein was prepared according to the method described in Example 3 of International Publication No. 2001/057199. Three strains of 7A3 and 25C3 were acquired.

Specifically, 100 μg of CTD protein of Moraxella catarrhalis ribosomal 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. , Mixed and 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. Furthermore, 10 and 14 weeks after the initial antigen administration, a double concentration of the antigen emulsion was 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 CTD protein of Moraxella catarrhalis ribosomal protein L7 / L12 to prepare GST fusion L7 / L12 CTD protein. A diluted solution of the obtained GST fusion L7 / L12CTD 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 a culture solution of fused cells 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 / L12CTD protein but not with the GST protein, and it was found that these wells contained an antibody against the L7 / L12CTD protein. Therefore, the cells in each positive well were collected, placed in a 24-well plastic plate, cultivated by adding HAT medium, diluted with HT medium so that the number of cells became about 20 cells / mL, and 50 μL thereof was 96. Placed in each well of the well-welled 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 / L12CTD protein were collected. Further, by repeating the same dilution test and cloning operation, each hybridoma producing monoclonal antibody strains 4C1, 7A3 and 25C3 against the L7 / L12CTD protein was obtained.

Using the three positive clone monoclonal antibody strains 4C1, 7A3 and 25C3 obtained as described above, a monoclonal antibody was produced and recovered according to a conventional method.

<4: Determination of amino acid sequences of each variable region of the light chain and heavy chain of the obtained mouse monoclonal antibody strain>
For the obtained monoclonal antibody strains 4C1, 7A3 and 25C3, the amino acid sequences and the 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 4C1, 7A3 and 25C3 are shown by the SEQ ID NOs shown in Table 1 below, respectively.

[Example 6] Analysis of interaction between Moraxella-catalaris ribosomal protein L7 / L12 and antibody The monoclonal antibodies 4C1, 7A3, which recognize the CTD of Moraxella-catalaris ribosomal protein L7 / L12 obtained in Example 5, And 25C3 were analyzed by NMR for the interaction with the ribosomal proteins L7 / L12 of Moraxella catalaris by the following procedure.

<1: Acquisition of Moraxella catarrhalis ribosomal proteins 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, ribosomal proteins L7 / L12 were 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 concentrated 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 a 4-fold 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% to elute the ribosomal proteins L7 / L12.
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 Moraxella catarrhalis ribosomal protein L7 / L12 antibody>
The monoclonal antibodies 4C1, 7A3, and 25C3 that recognize the CTD of Moraxella catarrhalis ribosomal proteins L7 / L12 obtained in Example 5 were dialyzed using PBS (phosphate buffered saline) as an external solution and ultraviolet. After quantifying the concentration by the absorbance of light (wavelength 280 nm), it was subjected to NMR measurement.

<3: Interaction analysis of Moraxella catarrhalis ribosomal protein L7 / L12 and antibody by NMR>
Moraxella catarrhalis ribosomal proteins L7 / L12 were mixed with antibodies 4C1, 7A3, and 25C3, respectively. The mixing ratio of L7 / L12 to antibody 7A3 or 25C3 was 1: 1.5 (molar ratio), and the mixing ratio of L7 / L12 to antibody 4C1 was 1: 0.5 (molar ratio). After scaling each mixture with PBS (Phosphate Buffered Saline) to 250 μL, add 20 μL of heavy water, 1 μL of 5 mg / mL DSS (4,4-dimethyl-4-silappentan-1-sulfonic acid), and EDTA (ethylenediamine). (Tetraacetic acid) and AEBSF (4- (2-aminoethyl) benzenesulfonyl fluoride) are added to a final concentration of 1.8 mM, transferred to a Shigemi NMR sample tube, degassed with an aspirator, and then installed in an NMR apparatus. bottom.

^{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, low molecular weight (13,000) such as ribosomal proteins L7 / L12 can be observed as a clear signal, but high molecular weight (15,000) such as antibody can be observed. The signal is so wide and unclear that it cannot be observed due to insufficient strength. 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.

^{The [1} H- ¹⁵ N] HSQC signal intensity of each amino acid residue of the ribosomal protein L7 / L12 when the ribosomal proteins L7 / L12 and the antibodies 4C1, 7A3, and 25C3 are mixed at the above ratios is mixed with the antibody. ^{Divided by the [1} H- ¹⁵ N] HSQC signal intensity of each amino acid residue of the ribosomal protein L7 / L12 before the above, the divided value is shown on the vertical axis, and the amino acid sequence of the ribosomal protein L7 / L12 is shown on the horizontal axis. Graphs were created (FIGS. 6A, B, C). The division value of CTD (residues at positions 58 to 124) 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 4C1, 7A3, and 25C3 at CTD (residues at positions 58-124) (FIGS. 6A, B, C).

[Example 7] Examination of cross-reactivity of an antibody that binds to Moraxella catarrhalis ribosome protein L7 / L12 A monoclonal antibody that recognizes the CTD of Moraxella catarrhalis ribosome protein L7 / L12 obtained in Example 5. For 4C1, 7A3 and 25C3, cross-reactivity with ribosome protein L7 / L12 of bacteria other than Moraxella catarrhalis was analyzed by ELISA 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). The medium (manufactured by Takara Shuzo Co., Ltd.) was seeded on a plate of a semi-solid medium, left at 37 ° C. for about 12 hours, and the resulting colonies were randomly selected and planted in 2 mL of LB liquid medium containing the same concentration of ampicillin. 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 ribosomal protein L7 / L12 artificial synthetic genes 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, respectively, and each of the monoclonal antibodies 4C1, 7A3 and 25C3 was reacted, washed, and then immobilized L7 of each bacterial species. The monoclonal antibody bound to / L12 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.

Specifically, 100 μL each of a PBS solution containing Moraxella catarrhalis and the recombinant full-length ribosome protein L7 / L12 of each of the above strains at concentrations of 0.01 μg / mL, 0.1 μg / mL, or 1 μg / mL, respectively. It was dispensed into a 96-well ELISA plate (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, the antibody solution was washed several times with a washing solution (PBS containing 0.02% Tween 20), and the antibody 4C1, 7A3, or 25C3 was diluted with 0.5% TritonX-100 / PBS to 1 μg / mL. Alternatively, 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.

The results of evaluation of the reactivity of the resulting antibodies 4C1, 7A3, or 25C3 with the recombinant full-length ribosome protein L7 / L12 of Moraxella catarrhalis or other bacterial species are 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 Moraxella catarrhalis in a sample is required, mainly in the medical field, and its industrial usefulness is extremely high.

Claims (13)

An antibody for detecting Moraxella catarrhalis, a C-terminal domain (CTD) consisting of amino acid residues at positions 58 to 124 of the Moraxella catarrhalis ribosome protein L7 / L12 shown in SEQ ID NO: 1. An antibody or fragment thereof, or a derivative thereof, that causes an antigen-antibody reaction with an epitope present in the antibody. 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 101 to 113 of SEQ ID NO: 1. Mycoplasma, Escherichia, Chlamydia, Salmonella, Pseudomonas, 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 genera Bordetella, Legionella, and Streptococcus. 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 to 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 of the amino acid sequences selected from SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15. 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 of 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 Moraxella catarrhalis 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 for an antigen-antibody reaction. A method that involves detecting the presence or absence of. A reagent for detecting the presence or absence of Moraxella catarrhalis 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 Moraxella catarrhalis in a sample, in which the antibody according to any one of claims 1 to 5 or a fragment thereof, or a derivative thereof, and the antibody are used in the sample. A kit containing instructions and instructions for detecting the presence or absence of Moraxella catarrhalis.

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