Gonococcus detection kit and gonococcus detection method

Abstract

The present application provides a kit for detecting gonococci in a sample, which is a new gonococci detection method excellent in detection sensitivity of gonococci, capable of on-site detection of gonococci from various samples such as urine, intraoral fluid, etc. The kit comprises a capture antibody for capturing gonococci-derived antigen in a sample, and a detection antibody having a detection label and for labeling gonococci-derived antigen in a sample, and the gonococci-derived antigen is ribosomal protein L7 / L12 of gonococci, one of the capture antibody and the detection antibody is a first monoclonal antibody or a fragment thereof, or a derivative thereof, which produces antigen-antibody reaction with an epitope comprising one or more than two amino acid residues from the 2nd to 14th amino acid residues from the N-terminal end in the amino acid sequence of L7 / L12 of gonococci shown in SEQ ID NO: 1, and the other of the capture antibody and the detection antibody is a second monoclonal antibody or a fragment thereof, or a derivative thereof, which produces antigen-antigen reaction with an epitope comprising one or more than two amino acid residues from the 102nd to 123rd amino acid residues from the N-terminal end in the amino acid sequence of L7 / L12 of the gonococci shown in SEQ ID NO: 1.

Description

Technical Field

[0001] This invention relates to a kit and method for detecting gonococci in a sample using an antigen-antibody reaction. Background Technology

[0002] Urethritis, a sexually transmitted infection, is increasing worldwide, with approximately 78 million patients globally. Therefore, its prevention, diagnosis, and treatment have become a global issue of great concern to the World Health Organization (WHO). Urethritis is characterized by a low patient return rate, making it crucial to prescribe appropriate antibiotics based on on-site diagnosis during treatment.

[0003] There are various bacteria that cause urethritis, the most common being Neisseria gonorrhoeae. Methods for detecting Neisseria gonorrhoeae include hybridization using DNA probes (Patent Document 1: Japanese Patent Application Publication No. 10-500310) and amplification of polynucleotides targeting 16S ribosomal RNA (Patent Document 2: Japanese Patent Application Publication No. 2013-188181). However, these methods require lengthy examination times and cannot be used as diagnostic tools on-site (at the clinic).

[0004] Antibodies capable of detecting gonococci via antigen-antibody reactions are known to use gonococcal ribosomal proteins L7 / L12 as antigens (e.g., Patent Document 3: International Publication 2000 / 006603). Immunoassay kits, such as immunochromatographic assays, capable of detecting gonococci via antigen-antibody reactions are also known (Non-Patent Document 1: Abbott Clearview). TM (Gonorrhea packaging instructions). However, these known antibody-based gonorrhea testing kits have low detection sensitivity (LoD (limit of detection) of approximately 5e6 cfu / mL), making it difficult to detect gonorrhea from urine samples. For urethritis testing, a swab needs to be inserted into the urethra to collect a urethral sample containing a higher concentration of gonorrhea. This sample collection procedure is extremely burdensome for patients (especially men), resulting in low kit usage and hindering the ability to prescribe appropriate initial antibiotics based on the on-site diagnosis of urethritis and its results.

[0005] According to previous reports (Non-patent literature 2: Isbey et al., Genitourinary Medicine, (1997), Vol.73, No.5, pp.378-382), the concentration of gonococci in the urine of male patients with urethritis is approximately 10. 4 ~10 7The order of magnitude of CFU / mL. If it were possible to achieve a bacterial concentration of 10⁻⁶ in urine... 4 cfu / mL, preferably 10 4 Methods for detecting gonococci at concentrations of around CFU / mL can use urine as a sample, eliminating the need for urethral swab insertion and wiping. Furthermore, other samples that may contain low concentrations of gonococci, such as oral swabs (oral swabs, mouthwash, etc.) and ocular samples (conjunctival swabs, eye discharge, etc.), can also be used for testing. This allows for the examination of male urethritis patients with minimal patient burden, thus possessing significant value in public health.

[0006] In addition, Neisseria meningitidis, which belongs to the same genus Neisseria as gonococcus, can sometimes be detected in urethral secretions just like gonococcus. Moreover, because it has different drug resistance tendencies than gonococcus, it is desirable to be able to distinguish it from Neisseria meningitidis when testing for gonococcus.

[0007] Furthermore, it is known that the pH of urine is distributed in a wide range of about 4.5 to 8, and the pH of oral fluid is distributed in a wide range of about 4 to 7. Therefore, in order to use various samples such as urine and oral samples (oral swabs, etc.) for gonorrhea testing, a method that can be stably tested even under a wide range of pH conditions is desired.

[0008] Existing technical documents

[0009] Non-patent literature

[0010] Non-patent literature 1: Abbott Clearview TM Gonorrhea Packaging Instructions

[0011] Non-patent literature 2: Isbey et al., Genitourinary Medicine, (1997), Vol.73, No.5, pp.378-382

[0012] Patent documents

[0013] Patent Document 1: Japanese Patent Publication No. 10-500310

[0014] Patent Document 2: Japanese Patent Application Publication No. 2013-188181

[0015] Patent Document 3: International Publication No. 2000 / 006603 Summary of the Invention

[0016] The problem that the invention aims to solve

[0017] This invention was made in view of the above-mentioned problems, and its object is to provide a new gonococcal detection method with excellent detection sensitivity, capable of on-site detection of gonococci from various samples such as urine, oral fluid, and eye samples. Furthermore, a preferred object is to provide a new gonococcal detection method that can stably detect gonococci on-site even under a wide range of pH conditions.

[0018] Methods for solving problems

[0019] The inventors conducted in-depth research, focusing on gonococcal ribosomal proteins L7 / L12 as gonococcal antigens. They discovered that combining a first monoclonal antibody that reacts with an epitope (first epitope) containing one or more amino acid residues selected from the 2nd to 14th amino acid residues of gonococcal L7 / L12, and a second monoclonal antibody that reacts with an epitope (second epitope) containing one or more amino acid residues selected from the 102nd to 123rd amino acid residues of gonococcal L7 / L12, with one used as a capture antibody and the other as a detection antibody, allows for the detection of gonococci through an immune reaction between the gonococcal antigen in the sample and the capture and detection antibodies, forming a sandwich structure with the gonococcal antigen sandwiched in the middle. This enables the detection of gonococci at concentrations far lower than those in existing technologies, and allows for the on-site detection of gonococci from various samples such as urine, oral fluid, and eye samples, thus completing this invention.

[0020] That is, the essence of the present invention relates to, for example, the following solutions.

[0021] [Item 1] A kit for detecting gonococci in a sample, wherein, This kit contains: Antibodies used to capture gonococcal antigens in samples, and An antibody for detection, containing a marker for labeling gonococcal antigens in a sample. Gonorrhea is detected by forming a sandwich structure with the gonococcal antigen sandwiched in the middle through an immune reaction between the gonococcal antigen in the sample and the capture and detection antibodies. The gonococcal antigen is the ribosomal protein L7 / L12 of Neisseria gonorrhoeae. One of the capture antibody and the detection antibody is a first monoclonal antibody or a fragment thereof, or a derivative thereof, that produces an antigen-antibody reaction with an epitope containing one or more amino acid residues from the 2nd to the 14th amino acid residues from the N-terminus of the amino acid sequence selected from the L7 / L12 of the gonococcus shown in sequence number 1. The other of the capture antibody and the detection antibody is a second monoclonal antibody or a fragment thereof, or a derivative thereof, that produces an antigen-antibody reaction with an epitope containing one or more amino acid residues from the N-terminus of the amino acid sequence of L7 / L12 of the gonococcus shown in sequence number 1.

[0022] [Item 2] The kit as described in Item 1, wherein the second monoclonal antibody is an antibody comprising the following amino acid sequence: The amino acid sequence that forms the variable region of the heavy chain has more than 80% homology with the amino acid sequence of sequence number 7; and The amino acid sequence that forms the variable region of the light chain has more than 80% homology with the amino acid sequence of sequence number 8.

[0023] [Item 3] The kit as described in Item 1 or 2, wherein, The first monoclonal antibody is an antibody containing the following amino acid sequence: The amino acid sequence that forms the variable region of the heavy chain has more than 80% homology with the amino acid sequence of sequence number 9, and The amino acid sequence that forms the variable region of the light chain has more than 80% homology with the amino acid sequence of sequence number 10. Alternatively, the first monoclonal antibody is an antibody containing the following amino acid sequence: The amino acid sequence that forms the variable region of the heavy chain has more than 80% homology with the amino acid sequence of sequence number 11, and The amino acid sequence that forms the variable region of the light chain has more than 80% homology with the amino acid sequence of sequence number 12.

[0024] [Item 4] The kit as described in any one of items 1 to 3, wherein the capture antibody is a first monoclonal antibody and the detection antibody is a second monoclonal antibody.

[0025] [Item 5] The kit as described in any one of items 1 to 3, wherein the detection antibody is a first monoclonal antibody and the capture antibody is a second monoclonal antibody.

[0026] [Item 6] A kit for detecting gonococci in a sample, wherein, This kit contains: Antibodies used to capture gonococcal antigens in samples, and An antibody for detection, containing a marker for labeling gonococcal antigens in a sample. Gonorrhea is detected by forming a sandwich structure with gonococcal antigens sandwiched in between, through an immune reaction between the capture antibody and the detection antibody. The gonococcal antigen is the ribosomal protein L7 / L12 of Neisseria gonorrhoeae. The capture antibody and the detection antibody are selected from a combination of the following (1) or (2): (1) The antibody for capture is a or b below, and the antibody for labeling is a combination of c below.

[0027] (2) The antibody used for labeling is a or b below, and the antibody used for capture is a combination of c below.

[0028] a. An amino acid sequence containing an amino acid sequence that is more than 80% homologous to the amino acid sequence of sequence number 11, and

[0029] An antibody consisting of an amino acid sequence that is more than 80% homologous to the amino acid sequence of sequence number 12, which is the amino acid sequence of the light chain variable region.

[0030] b. An amino acid sequence containing an amino acid sequence that is a variable region of the heavy chain and has more than 80% homology with the amino acid sequence of sequence number 9, and

[0031] An antibody consisting of an amino acid sequence that is more than 80% homologous to the amino acid sequence of sequence number 10, which is the amino acid sequence of the light chain variable region.

[0032] c. An amino acid sequence containing an amino acid sequence that is a variable region of the heavy chain and has more than 80% homology with the amino acid sequence of sequence number 7, and

[0033] An antibody consisting of an amino acid sequence that is more than 80% homologous to the amino acid sequence of sequence number 8, which is the amino acid sequence of the light chain variable region.

[0034] [Item 7] The kit according to any one of items 1 to 6, wherein the detection performance against gonococcal antigens is more than 10 times higher than the detection performance against Neisseria meningitidis antigens.

[0035] [Item 8] The kit described in any one of items 1 to 6 does not cross-react with Mycoplasma, Escherichia, Chlamydia, Pseudomonas, Staphylococcus, Legionella, or Streptococcus in the sample.

[0036] [Item 9] The kit as described in any one of items 1 to 8, wherein the sample is a sample of oral fluid or urine from a mammal.

[0037] [Item 10] The kit as described in Item 9, wherein the sample is a sample of urine derived from mammals.

[0038] [Item 11] The kit as described in any one of items 1 to 10, wherein, The kit further includes a carrier for developing the sample and bringing the sample into contact with the detection antibody. The carrier has a detection area on which the capture antibody is fixed.

[0039] [Item 12] The kit as described in Item 11, wherein the kit is an immunochromatographic kit and further comprises a binding pad with an antibody attached for detection.

[0040] [Item 13] A method for detecting gonococci in a sample using the kit described in any one of items 1 to 12, comprising the following steps: (I) A process of capturing gonococcal antigens in a sample by reacting them with an antigen-antibody reaction between gonococcal antigens in the sample and a capture antibody and a detection antibody, and labeling the gonococcal antigens in the sample; and (II) Procedure for detecting gonococcal antigens in a sample based on a detection marker.

[0041] [Item 14] The method as described in Item 13, wherein the above step (I) includes the following steps: (Ia-1) A process of contacting the sample with a detection antibody, and labeling the gonococcal antigen in the sample by an antigen-antibody reaction between the detection antibody and the gonococcal antigen in the sample; and (Ia-2) A process of contacting a sample containing gonococcal antigen labeled with a detection antibody with a capture antibody, thereby capturing the gonococcal antigen in the sample by reacting the capture antibody with the gonococcal antigen-detection antibody complex.

[0042] [Item 15] The method as described in Item 13, wherein the above step (I) includes the following steps: (Ib-1) A process of contacting a sample with a capture antibody, and capturing gonococcal antigens in the sample by reacting the capture antibody with an antigen-antibody reaction of the gonococcal antigens in the sample; and (Ib-2) A process of contacting a sample containing gonococcal antigens captured by a capture antibody with a detection antibody, and labeling the gonococcal antigens in the sample by the antigen-antibody reaction between the detection antibody and the gonococcal antigen-capture antibody complex.

[0043] [Item 16] The method as described in any one of items 13 to 15, wherein, The method further includes a step of lysing the bacteria in the sample with a lysing agent before the detection antibody and / or capture antibody come into contact with the sample. The lysin is selected from one or more of the following groups: nonionic surfactants, amphoteric surfactants, anionic surfactants, lysozyme, lysococcal enzyme, pepsin, glucosidase, galactosidase, achromopeptidase, and β-N-acetylglucosidase.

[0044] [Item 17] The method of any one of items 13 to 16, wherein the detection limit for gonococci in the sample is below 5e4cfu / mL.

[0045] [Item 18] A method of manufacturing the kit of item 11, comprising a step of immobilizing a capture antibody on a detection region of a carrier having a detection region.

[0046] [Item 19] A method of manufacturing the kit described in Item 12, comprising the following steps: The process of immobilizing the capture antibody onto the detection region of a carrier having a detection region; The process of attaching the antibody for detection to the conjugate pad; and The process of placing the bonding pad upstream of the detection area of ​​the carrier.

[0047] [Item 20] The manufacturing method as described in Item 18 or 19, wherein, The method further includes the step of preparing a detection antibody by adding a detection label to one of the first and second monoclonal antibodies, and The other of the first and second monoclonal antibodies was used as the capture antibody and immobilized in the detection area of ​​the vector.

[0048] [Item 21] The manufacturing method as described in Item 20, wherein, The first monoclonal antibody is the first monoclonal antibody described in item 1, and The method further includes the following steps: inoculating an animal with an epitope polypeptide, obtaining an antiserum containing an antibody that reacts with the epitope polypeptide to produce an antigen-antibody reaction, purifying and fractionating the antibody from the antiserum to obtain a first monoclonal antibody, wherein the epitope polypeptide has an epitope containing one or more amino acid residues from the 2nd to the 14th amino acid residues from the N-terminus of the amino acid sequence selected from L7 / L12 of the gonococcus shown in sequence number 1.

[0049] [Item 22] The manufacturing method as described in Item 20 or 21, wherein, The second monoclonal antibody is the second monoclonal antibody described in item 1, and The method further includes the following steps: inoculating an animal with an epitope polypeptide, obtaining an antiserum containing an antibody that reacts with the epitope polypeptide to produce an antigen-antibody reaction, purifying and fractionating the antibody from the antiserum to obtain a second monoclonal antibody, wherein the epitope polypeptide has an epitope containing one or more amino acid residues from the 102nd to 123rd amino acid residues from the N-terminus of the amino acid sequence selected from L7 / L12 of the gonococcus shown in sequence number 1.

[0050] The effects of the invention

[0051] The gonorrhea detection kit and method according to the present invention can detect gonococci at concentrations lower than those in the prior art through antigen-antibody reaction. Therefore, gonococci can be detected on-site from various samples such as urine, oral fluid, and eye samples. Furthermore, according to a preferred embodiment, gonococci can be stably detected on-site even under a wide range of pH conditions. Attached Figure Description

[0052] Figure 1 This diagram shows the amino acid sequence alignment of the L7 / L12 ribosomal proteins from Neisseria gonorrhoeae, Neisseria meningitidis, and Escherichia coli. For the L7 / L12 amino acid sequence of Neisseria gonorrhoeae, the predicted α-helix and β-sheet forming regions are also shown. Additionally, for the L7 / L12 amino acid sequences of Neisseria meningitidis and Escherichia coli, amino acid residues that differ from the L7 / L12 amino acid sequence of Neisseria gonorrhoeae are indicated by enclosing lines.

[0053] Figure 2 This diagram shows an alignment of the amino acid sequences of gonococcal ribosomal protein L7 / L12 (peptide 1) with those of peptides 2–4 and Neisseria meningitidis ribosomal protein L7 / L12 (peptide 5). For peptides 2–5, amino acid residues that differ from those in peptide 1 are indicated by enclosing lines.

[0054] Figure 3 This is a cross-sectional view showing a schematic configuration of a strip-shaped detection mechanism, which is an example of a detection mechanism in an immunochromatographic detection device using a side-flow method. Detailed Implementation

[0055] The present invention will now be described in detail with reference to specific embodiments. However, the present invention is not limited to the following embodiments and may be implemented in any manner without departing from the spirit of the present invention.

[0056] [I.Preface]

[0057] In this invention, "gonococcus" (Neisseria gonorrhoeae) refers to a Gram-negative diplococcus belonging to the genus Neisseria. Infections caused by gonococci are known to cause gonococcal urethritis (gonorrhea), keratoconjunctivitis, pharyngitis, and other diseases in humans. Treatment for gonococcal infections typically involves antibiotics, but many bacteria exhibit resistance, and the condition can become difficult to treat if it worsens. Therefore, a simple and immediate method for detecting gonococcal infection is being sought.

[0058] In this invention, when providing the following novel gonorrhea detection method, the gonorrhea-derived antigen used to prepare monoclonal antibodies capable of detecting gonorrhea focuses on the ribosomal protein L7 / L12 of gonorrhea. This novel gonorrhea detection method exhibits excellent detection sensitivity for gonorrhea, enabling on-site detection of gonorrhea from various samples, including urine samples, oral samples (oral swabs, mouthwash, etc.), and ocular samples (conjunctival swabs, eye discharge, etc.). Preferably, it can stably detect gonorrhea on-site even under a wide pH range, including urine and oral fluids. The "ribosomal protein L7 / L12" or simply "L7 / L12" in this invention refers to a type of ribosomal protein essential for protein synthesis in microorganisms, a protein universally present in various bacteria.

[0059] Figure 1 The image shows the amino acid sequences of the L7 / L12 peptides from Gonorrhea, Neisseria meningitidis, and Escherichia coli. Specifically, the amino acid sequence of the L7 / L12 peptide from Gonorrhea is shown in sequence number 1, the amino acid sequence of the L7 / L12 peptide from Neisseria meningitidis is shown in sequence number 2, and the amino acid sequence of the L7 / L12 peptide from Escherichia coli is shown in sequence number 3.

[0060] The gonococcal ribosomal protein L7 / L12 has a dimer structure consisting of a monomeric molecule (sometimes referred to as the "L7 / L12 polypeptide") of 123 amino acid residues linked together by two copies. Based on the inventors' analytical results, as... Figure 1 As shown, in the L7 / L12 polypeptide of Neisseria gonorrhoeae, a three-dimensional structure (NTD: N-Terminal Domain) is formed by amino acid residues from positions 1 to 40. Following a linker sequence consisting of 17 amino acid residues that does not form a three-dimensional structure, another three-dimensional structure (CTD: C-Terminal Domain) is further formed by amino acid residues from positions 58 to 123. Furthermore, two copies of the L7 / L12 polypeptide monomers possessing this three-dimensional structure associate with each other through the NTD, thereby forming a dimer structure.

[0061] In addition, such as Figure 1 As shown, the amino acid sequence of the L7 / L12 polypeptide of *Neisseria meningitidis*, a bacterium belonging to the same genus *Neisseria* as *Neisseria gonorrhoeae*, is extremely similar to that of the L7 / L12 polypeptide of *Neisseria gonorrhoeae*. The only difference is the amino acid residue at position 115 from the N-terminus; in *Neisseria gonorrhoeae*, it is alanine, while in *Neisseria meningitidis*, it is glutamic acid. Therefore, it is extremely difficult to identify the L7 / L12 polypeptides of *Neisseria gonorrhoeae* and *Neisseria meningitidis* through antigen-antibody reactions.

[0062] On the other hand, such as Figure 1 As shown, the amino acid sequence of the L7 / L12 polypeptide of Escherichia coli, a bacterium of the genus Escherichia that is different from Neisseria gonorrhoeae and Neisseria meningitidis, is significantly different from that of the L7 / L12 polypeptide of Neisseria gonorrhoeae and Neisseria meningitidis.

[0063] The inventors have prepared monoclonal antibodies capable of binding to various sites of the L7 / L12 polypeptide of Neisseria gonorrhoeae. These antibodies were combined in various ways and validated. The results showed that by combining a first monoclonal antibody that generates an antigen-antibody reaction with an epitope containing one or more amino acid residues selected from the 2nd to 14th amino acid residues of Neisseria gonorrhoeae (hereinafter referred to as "the first epitope") and a second monoclonal antibody that generates an antigen-antibody reaction with an epitope containing one or more amino acid residues selected from the 102nd to 123rd amino acid residues of Neisseria gonorrhoeae (hereinafter referred to as "the second epitope"), a gonococcal detection method with excellent detection sensitivity can be constructed for on-site detection of gonococci from various samples, including urine, oral samples (oral swabs, mouthwash, etc.), and ocular samples (conjunctival swabs, eye discharge, etc.).

[0064] Specifically, one aspect of the present invention relates to a combination of first and second monoclonal antibodies (hereinafter referred to as "the first gonococcal antibody of the present invention", "the second gonococcal antibody of the present invention", or "the first antibody of the present invention", "the second antibody of the present invention", etc.) that generate antigen-antibody reactions with the first and second epitopes of gonococcus L7 / L12, respectively. Another aspect of the present invention relates to a kit for detecting gonococcus in a sample using the combination of the first and second antibodies of the present invention (hereinafter referred to as "the gonococcal detection kit of the present invention" etc.), and a method for detecting gonococcus in a sample (hereinafter referred to as "the gonococcal detection method of the present invention" etc.). These aspects will be described sequentially below.

[0065] [II. First and second antibodies for gonorrhea detection]

[0066] The first and second gonococcal detection antibodies of the present invention are monoclonal antibodies or fragments thereof, or derivatives thereof, that generate an antigen-antibody reaction with epitopes (first and second epitopes) of specific amino acid residues of the gonococcal ribosomal protein L7 / L12.

[0067] In this invention, "antibody" refers to a protein that recognizes and binds to a specific antigen or substance, sometimes also called an immunoglobulin (Ig). Common antibodies typically have two light chains (light chains) and two heavy chains (heavy chains) linked together by disulfide bonds. There are two types of light chains, called λ chains and κ chains, and five types of heavy chains, called γ chains, μ chains, α chains, δ chains, and ε chains. Based on the type of heavy chain, antibodies exist in five isotypes: IgG, IgM, IgA, IgD, and IgE.

[0068] The heavy chains consist of a heavy chain constant (CH) region and a heavy chain variable (VH) region. The light chains consist of a light chain constant (CL) region and a light chain variable (VL) region. The light chain constant (CL) region consists of a single domain. The heavy chain constant (CL) region consists of three domains, namely CH1, CH2, and CH3. The light chain variable (VL) region and the heavy chain variable (VH) region consist of four highly conservative regions (FR-1, FR-2, FR-3, FR-4) called framework regions (FR), and three highly variable regions (CDR-1, CDR-2, CDR-3) called complementarity-determining regions (CDR). 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), arranged from the amino terminus to the carboxyl terminus in the order FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4. The light chain constant (CL) region has three CDRs (CDR-L1, CDR-L2, CDR-L3) and four FRs (FR-L1, FR-L2, FR-L3, FR-L4), arranged from the amino terminus to the carboxyl terminus in the order FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, FR-L4. The variable regions of both the heavy and light chains contain binding domains that interact with the antigen.

[0069] Antibodies exist in both polyclonal and monoclonal forms. Polyclonal antibodies are typically prepared from the serum of animals immunized with antigens, and are mixtures of various antibody molecules with different structures. On the other hand, monoclonal antibodies are antibodies composed 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. The first and second antibodies of this invention are both monoclonal antibodies. Monoclonal antibodies can be produced by clones derived from antibody-producing cells, or by obtaining nucleic acid molecules containing the gene sequence of the amino acid encoding the antibody protein and using these nucleic acid molecules for genetic engineering. Furthermore, modifications to enhance antibody binding or specificity using genetic information such as the heavy and light chains, or their variable regions or CDRs, are techniques well known to those skilled in the art.

[0070] The first and second antibodies of the present invention can be fragments and / or derivatives of antibodies. Examples of antibody fragments include F(ab')2, Fab, Fv, etc. Examples of antibody derivatives include antibodies with artificially introduced amino acid mutations in the constant regions of the light and / or heavy chains, antibodies that modify the structural domains of the constant regions of the light and / or heavy chains, antibodies having two or more Fc regions per molecule, glycan-modified antibodies, bispecific antibodies, antibody-conjugates formed by binding antibodies or antibody fragments to proteins other than antibodies, antibody enzymes, tandem scFvs, bispecific tandem scFvs, and double-chain antibodies (diabody). Furthermore, when the above-mentioned antibodies or their fragments or derivatives are of non-human animal origin, chimeric antibodies or humanized antibodies in which part or all of the sequence other than the CDR is replaced with the corresponding sequence of a human antibody are also included in the first and / or second antibodies of the present invention. It should be noted that, unless otherwise stated, the term "antibody" in the present invention also includes antibody fragments and / or derivatives.

[0071] The first and second antibodies of the present invention are monoclonal antibodies that generate antigen-antibody reactions with the first and second epitopes of specific amino acid residues in the amino acid sequence of L7 / L12 of the gonococcus shown in sequence number 1, respectively.

[0072] In this invention, "antigen-antibody reaction" refers to the recognition and binding of any component of the antigen by an antibody. Additionally, in this invention, "epitope" refers to a portion of the antigen recognized by the antibody. The length of the epitope is not limited and can be, for example, typically 3 or more amino acid residues, particularly 5 or more amino acid residues, or 7 or more amino acid residues, and for example, typically less than 50 amino acid residues, particularly less than 30 amino acid residues, or less than 20 amino acid residues.

[0073] The first epitope of the first antibody of the present invention that generates an antigen-antibody reaction is an epitope containing one or more amino acid residues from the 2nd to the 14th amino acid residues from the N-terminus of the amino acid sequence of L7 / L12 of the gonococcus shown in sequence number 1.

[0074] The second epitope of the second antibody of the present invention that generates an antigen-antibody reaction is an epitope comprising one or more amino acid residues selected from the amino acid sequence of L7 / L12 of Neisseria gonorrhoeae, as shown in Sequence Number 1, from amino acid residues 102 to 123 from the N-terminus. Preferably, the second epitope comprises at least the 115th amino acid residue from the N-terminus of Sequence Number 1, which is the unique distinguishing feature between Neisseria gonorrhoeae and L7 / L12 of Neisseria meningitidis.

[0075] The first and / or second antibodies of the present invention preferably do not cross-react with components derived from bacteria other than gonococci in the sample (preferably ribosomal proteins L7 / L12) or other components. Specifically, the first and / or second antibodies of the present invention preferably do not cross-react with bacteria selected from one or more genera including Mycoplasma, Escherichia, Chlamydia, Salmonella, Pseudomonas, Staphylococcus, Legionella, Haemophilus, Bordetella, Moraxella, and Streptococcus. Preferably, the first and / or second antibodies of the present invention do not cross-react with bacteria of two or more genera mentioned above, and further with three or more genera, or four or more genera, or five or more genera, or six or more genera, especially all genera.

[0076] Particularly preferred is that the first and / or second antibodies of the present invention do not cross-react with components derived from one or more bacteria belonging to the genus Neisseria (preferably ribosomal proteins L7 / L12), which are also gonococci. Examples of bacteria belonging to the genus Neisseria include *Neisseria meningitidis*, *Neisseria lactamica*, *Neisseria sicca*, *Neisseria cinerea*, and *Neisseria flavescens*.

[0077] Particularly preferred is that the second antibody of the present invention does not cross-react with components derived from Neisseria meningitidis (preferably ribosomal proteins L7 / L12). Neisseria meningitidis is sometimes detected in urethral secretions, similar to gonococci, and exhibits different drug resistance tendencies than gonococci; therefore, it is preferable that it can be distinguished from Neisseria meningitidis during gonococcal testing. Furthermore, regarding… Figure 1 As described above, the amino acid sequence of the L7 / L12 polypeptide of Neisseria meningitidis is extremely similar to that of the L7 / L12 polypeptide of Neisseria gonorrhoeae. The only difference is that the 115th amino acid residue from the N-terminus is alanine in Neisseria gonorrhoeae, while it is glutamic acid in Neisseria meningitidis. Therefore, identifying the L7 / L12 polypeptides of Neisseria gonorrhoeae and Neisseria meningitidis via antigen-antibody reactions is typically extremely difficult. To address this, the second antibody of the present invention, according to its preferred embodiment, identifies and generates an antigen-antibody reaction with a second epitope containing at least sequence number 1 (the only distinguishing feature between the L7 / L12 of Neisseria gonorrhoeae and Neisseria meningitidis), specifically the 115th amino acid residue from the N-terminus. Therefore, compared to the reactivity against the L7 / L12 of Neisseria gonorrhoeae, the reactivity against the L7 / L12 of Neisseria meningitidis can be significantly suppressed. This allows for the precise separation and detection of Neisseria gonorrhoeae and Neisseria meningitidis in a sample, which is highly preferred.

[0078] It should be noted that, for the determination of antigen-antibody reactions between antibodies and epitopes / antigens or other components, those skilled in the art can appropriately choose to perform the binding assay in a solid-phase or liquid-phase system. Examples of such methods include enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), and luminescence resonance energy transfer (LRET), but are not limited to these. Furthermore, when determining such antigen-antibody binding, antibodies and / or antigens can be labeled using enzymes, fluorescent substances, luminescent substances, radioactive isotopes, etc., and the antigen-antibody reaction can be detected using a assay method suitable for the physical and / or chemical properties of the labeled substance.

[0079] The first and second gonococcal antibodies of the present invention only need to generate an antigen-antibody reaction with the first and second epitopes mentioned above, and their amino acid sequences are not limited. Antibodies with the following amino acid sequences as the variable region sequences of the heavy chain and light chain have high sensitivity, and are preferred from this point of view.

[0080] As the heavy chain variable region sequence, it is particularly preferred to have an amino acid sequence having 80% or more, especially 85% or more, further 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 99% or more, especially 100% homology (preferably identical) with any one of the amino acid sequences selected from sequence number 7 (the heavy chain variable region sequence of antibody NG1 in the example), sequence number 9 (the heavy chain variable region sequence of the example), and sequence number 11 (the heavy chain variable region sequence of the example). Among these, the heavy chain variable region sequence is particularly preferred to be any one of the amino acid sequences selected from sequence number 7, sequence number 9, and sequence number 11.

[0081] As the light chain variable region sequence, it is particularly preferred to have an amino acid sequence having 80% or more, especially 85% or more, further 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 99% or more, especially 100% homology (preferably identical) with any one of the amino acid sequences selected from sequence number 8 (the light chain variable region sequence of antibody NG1 in the example), sequence number 10 (the light chain variable region sequence of antibody NG2 in the example), and sequence number 12 (the light chain variable region sequence of antibody NG3 in the example). Among these, the light chain variable region sequence is particularly preferred to be any one of the amino acid sequences selected from sequence number 8, sequence number 10, and sequence number 12.

[0082] It should be noted that in this invention, the "homology" of two amino acid sequences refers to the proportion of identical or similar amino acid residues appearing at corresponding positions when the two amino acid sequences are aligned, and the "identity" of two amino acid sequences refers to the proportion of identical amino acid residues appearing at corresponding positions when the two amino acid sequences are aligned. It should be noted that the "homology" and "identity" of two amino acid sequences can be determined, for example, using the BLAST (Basic Local Alignment Search Tool) program (Altsch ul et al., J. Mol. Biol., (1990), 215(3): 403-10).

[0083] In addition, methods for identifying each CDR sequence from the variable sequences of the heavy and light chains of an antibody include, for example, the Kabat method (Kabat et al., The Journal of Immunology, 1991, Vol.147, No. 5, pp.1709-1719) and the Chothia method (Al-Lazikani et al., Journal of Molecular Biology, 1997, Vol.273, No.4, pp.927-948). These methods are common knowledge in the field, and further information can be found at Dr. Andrew CR Martin's Group website (http: / / www.bioinf.org.uk / abs / ).

[0084] It should be noted that, as an amino acid similar to a certain amino acid, examples can be found that belong to the same group in the following classification based on the polarity, charge, and size of amino acids (each type of amino acid is represented by a single-letter code).

[0085] Aromatic amino acids: F, H, W, Y; Aliphatic amino acids: I, L, V; • Hydrophobic amino acids: A, C, F, H, I, K, L, M, T, V, W, Y; • Charged amino acids: D, E, H, K, R, etc.; • Positively charged amino acids: H, K, R; Negatively charged amino acids: D, E; • Polar amino acids: C, D, E, H, K, N, Q, R, S, T, W, Y; Small amino acids: A, C, D, G, N, P, S, T, V, etc.; • Miniature amino acids: A, C, G, S.

[0086] In addition, as amino acids similar to a certain amino acid, examples can be given of amino acids that belong to the same group in the following classification based on the types of amino acid side chains (each type of amino acid is represented by a single-letter code).

[0087] • Amino acids with aliphatic side chains: G, A, V, L, I; • Amino acids with aromatic side chains: F, Y, W; • Amino acids with sulfur-containing side chains: C, M; • Amino acids with aliphatic hydroxyl side chains: S, T; • Amino acids with basic side chains: K, R, H; • Acidic amino acids and their amide derivatives: D, E, N, Q.

[0088] There are no limitations on the antibodies that are specifically composed of the above-mentioned amino acid sequences, which are the variable region sequences of the heavy chain and the light chain. Examples of such antibodies include the following.

[0089] • An antibody containing an amino acid sequence that has more than 80% homology with the amino acid sequence of sequence number 11 as the heavy chain variable region sequence, and an amino acid sequence that has more than 80% homology with the amino acid sequence of sequence number 12 as the light chain variable region sequence.

[0090] • An antibody containing an amino acid sequence that has more than 80% homology with the amino acid sequence of sequence number 9 as the heavy chain variable region sequence, and an amino acid sequence that has more than 80% homology with the amino acid sequence of sequence number 10 as the light chain variable region sequence.

[0091] • An antibody containing an amino acid sequence that has more than 80% homology with the amino acid sequence of sequence number 7 as the heavy chain variable region sequence, and an amino acid sequence that has more than 80% homology with the amino acid sequence of sequence number 8 as the light chain variable region sequence.

[0092] The first monoclonal antibody preferably comprises an amino acid sequence having at least 80% homology with the amino acid sequence of sequence number 11 as the heavy chain variable region sequence and an amino acid sequence having at least 80% homology with the amino acid sequence of sequence number 12 as the light chain variable region sequence; or an antibody comprising an amino acid sequence having at least 80% homology with the amino acid sequence of sequence number 9 as the heavy chain variable region sequence and an amino acid sequence having at least 80% homology with the amino acid sequence of sequence number 10 as the light chain variable region sequence. The second monoclonal antibody preferably comprises an amino acid sequence having at least 80% homology with the amino acid sequence of sequence number 7 as the heavy chain variable region sequence and an amino acid sequence having at least 80% homology with the amino acid sequence of sequence number 8 as the light chain variable region sequence.

[0093] It should be noted that when the first and second monoclonal antibodies exemplified above are used in the gonococcal detection method and gonococcal detection kit of the present invention described later, there is no limitation on the combination of the capture antibody and the labeling antibody, but the combination of (1) or (2) below is preferred.

[0094] (1) The antibody for capture is a or b below, and the antibody for labeling is a combination of c below.

[0095] (2) The antibody used for labeling is a or b below, and the antibody used for capture is a combination of c below.

[0096] a. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 11 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 12 as the light chain variable region sequence.

[0097] b. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 9 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 10 as the light chain variable region sequence.

[0098] c. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 7 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 8 as the light chain variable region sequence.

[0099] The methods for producing the first and second antibodies of the present invention, including the antibodies exemplified above, are not particularly limited, and for example, the following methods can be cited.

[0100] First, prepare a polypeptide (hereinafter referred to as "epitope polypeptide") having all or part of the amino acid sequence of L7 / L12 of the gonococcus as the target bacteria, or having an amino acid sequence having 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 99% or more, particularly 100% homology (preferably identical) with it. The specific amino acid sequence of the epitope polypeptide is not limited. However, in the case of preparing the first antibody of the present invention, it is preferable to use a polypeptide having an amino acid sequence equivalent to the above-mentioned first epitope (an epitope containing one or more amino acid residues selected from the 2nd to 14th amino acid residues of sequence number 1), or having an amino acid sequence having 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 99% or more, particularly 100% homology (preferably identical) with it. In addition, when preparing the first antibody of the present invention, it is preferable to use a polypeptide having an amino acid sequence equivalent to the second epitope (an epitope containing one or more amino acid residues selected from amino acid residues at positions 102 to 123 of sequence number 1), or having an amino acid sequence having 90% or more, especially 95% or more, or 96% or more, or 97% or more, or 99% or more, particularly 100% homology (preferably identical) with it (hereinafter referred to as "second epitope polypeptide").

[0101] The prepared epitope polypeptide is inoculated into animals along with an adjuvant as needed, and its serum is recovered to obtain antiserum containing antibodies (polyclonal antibodies containing candidates for the first and / or second antibodies of the present invention) that produce an antigen-antibody reaction with the epitope polypeptide. Animals used for inoculation include sheep, horses, goats, rabbits, mice, and rats; sheep and rabbits are particularly preferred in polyclonal antibody production. The antiserum obtained is then purified and graded for antibody production. Screening is performed appropriately using known methods, with the ability to produce an antigen-antibody reaction with the first and / or second epitope and, optionally, to avoid cross-reactivity with other specific components (e.g., L7 / L12 of Neisseria meningitidis). This allows the acquisition of the first and / or second antibodies of the present invention with the desired properties. Furthermore, antibody-producing cells that produce the desired antibody molecules can be isolated and fused with myeloma cells to create hybridomas with autonomous proliferation capabilities, thereby obtaining monoclonal antibodies.

[0102] If the desired antibody is obtained through the above process, its structure, specifically a portion or all of the amino acid sequences of the heavy chain constant (CH) region, heavy chain variable (VH) region, light chain constant (CL) region, and / or light chain variable (VL) region, can be analyzed using known amino acid sequence analysis methods. Methods for modifying the amino acid sequence of the desired antibody to improve its binding affinity and specificity are also known to those skilled in the art. Furthermore, all or a portion of the amino acid sequence of the desired antibody (especially all or a portion of the heavy chain variable (VH) region and light chain variable (VL) region, particularly the amino acid sequences of each CDR) can be combined as needed with a portion of the amino acid sequence of a known antibody (especially the heavy chain constant (CH) region and light chain constant (CL) region, and the amino acid sequences of each FR in the heavy chain variable (VH) region and light chain variable (VL) region as needed), thereby enabling the design of other antibodies with a high probability of possessing the same antigen specificity.

[0103] On the other hand, when a portion (CDR or variable region) or all of the amino acid sequence of an antibody has been determined, a nucleic acid molecule having all or part of the base sequence encoding the desired antibody can be prepared using known methods, and this nucleic acid molecule can be used to produce a genetically engineered antibody. Furthermore, vectors, plasmids, etc., for expressing the constituent elements of the desired antibody can be prepared from this base sequence and introduced into host cells (mammalian cells, insect cells, plant cells, yeast cells, microbial cells, etc.) to induce the production of the antibody. Additionally, modifications can be appropriately introduced into the structure of the constant region of the antibody or modifications can be made to the glycan portion to improve the performance of the obtained antibody or avoid side effects, using techniques well known to those skilled in the art.

[0104] It should be noted that the manufacturing method of the first and / or second antibody of the present invention described above, the nucleic acid molecule encoding the first and / or second antibody of the present invention, the vector or plasmid containing the nucleic acid molecule, the cell containing the nucleic acid molecule, the vector or plasmid, and the hybridoma that produces the first and / or second antibody of the present invention are also the subject of the present invention.

[0105] It should be noted that the antibody preparation and modification techniques described in this specification are well known to those skilled in the art, for example, as described in *Antibodies; A laboratory manual*, E. Harlow et al., Cold Spring Harbor Laboratory Press (2014). Furthermore, the molecular biology techniques described in this specification (such as amino acid sequence analysis, nucleic acid molecule design and preparation, vector and plasmid design and preparation, etc.) are also well known to those skilled in the art, for example, as described in *Molecular Cloning, A laboratory manual*, Cold Spring Harbor Laboratory Press, Shambrook, J. et al. (1989).

[0106] [III. Gonorrhea Detection Methods]

[0107] The gonorrhea detection method of the present invention includes the following steps.

[0108] (I) A process of capturing gonococcal antigens in a sample by an antigen-antibody reaction of gonococcal antigens in the sample, a capture antibody linked to a solid-phase carrier, and a detection antibody with a detection label, and labeling the gonococcal antigens in the sample.

[0109] (II) Procedure for detecting gonococcal antigens in a sample based on a detection marker.

[0110] Here, the gonococcal source antigen is the gonococcal ribosomal protein L7 / L12, and one of the capture antibody and the detection antibody is the first antibody of the present invention and the other is the second antibody of the present invention.

[0111] Either the capture antibody or the detection antibody can be the first antibody of the present invention, or either can be the second antibody of the present invention. When the capture antibody is the first antibody of the present invention and the detection antibody is the second antibody of the present invention, an antigen-antibody reaction is generated by the capture antibody with the first epitope of gonococcal L7 / L12 to capture the gonococcus, and simultaneously an antigen-antibody reaction is generated by the detection antibody with the second epitope of gonococcal L7 / L12 to label the gonococcus, and then detection is performed. Conversely, when the capture antibody is the second antibody of the present invention and the detection antibody is the first antibody of the present invention, an antigen-antibody reaction is generated by the capture antibody with the second epitope of gonococcal L7 / L12 to capture the gonococcus, and simultaneously an antigen-antibody reaction is generated by the detection antibody with the first epitope of gonococcal L7 / L12 to label the gonococcus, and then detection is performed.

[0112] There are no restrictions on the combination of the capture antibody and the labeling antibody in the gonorrhea detection method of the present invention, but the combination of (1) or (2) below is preferred.

[0113] (1) The antibody for capture is a or b below, and the antibody for labeling is a combination of c below.

[0114] (2) The antibody used for labeling is a or b below, and the antibody used for capture is a combination of c below.

[0115] a. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 11 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 12 as the light chain variable region sequence.

[0116] b. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 9 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 10 as the light chain variable region sequence.

[0117] c. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 7 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 8 as the light chain variable region sequence.

[0118] In terms of having a wide range of pH applicability as described later, there are no restrictions on the combination of the capture antibody and the labeling antibody, but the combination of (1) or (2) described below is preferred.

[0119] (1) The combination of the antibody for capture is a below and the antibody for labeling is c below.

[0120] (2) The labeling antibody is a combination of the following a and the capture antibody is a combination of the following c.

[0121] a. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 11 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 12 as the light chain variable region sequence.

[0122] c. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 7 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 8 as the light chain variable region sequence.

[0123] Here, according to the gonococcal detection method of the present invention according to method A, the above step (I) includes the following steps.

[0124] (Ia-1) is a process of contacting the sample with a detection antibody and labeling the gonococcal antigen in the sample by the antigen-antibody reaction between the detection antibody and the gonococcal antigen.

[0125] (Ia-2) A process of contacting a sample containing gonococcal antigen labeled with a detection antibody with a capture antibody, thereby capturing the gonococcal antigen in the sample by reacting the capture antibody with the gonococcal antigen-detection antibody complex.

[0126] In addition, according to the gonorrhea detection method of the present invention according to method B, the above step (I) includes the following steps.

[0127] (Ib-1) is a process of contacting a sample with a capture antibody and capturing the gonococcal antigen in the sample by reacting the capture antibody with the antigen-antibody of the gonococcal antigen.

[0128] (Ib-2) A process of contacting a sample containing gonococcal antigens captured by a capture antibody with a detection antibody, and labeling the gonococcal antigens in the sample by the antigen-antibody reaction between the detection antibody and the gonococcal antigen-capture antibody complex.

[0129] In the gonococcal detection method of the present invention, the choice between mode A and mode B depends on the type of immunoassay used and the type of sample. There are no limitations on the immunoassay method; examples include ELISA (enzyme-conjugated immunosorbent assay) using antibody-loaded microplates; emulsion particle agglutination assay using antibody-loaded emulsion particles (e.g., polystyrene emulsion particles); immunochromatography using antibody-loaded membranes; and sandwich analysis using antibodies labeled with colored particles or particles with color development capabilities, enzymes, or fluorescent agents, as well as capture antibodies immobilized on solid-phase carriers such as magnetic microparticles.

[0130] The following description uses the gonococcal detection method of the present invention, which is implemented using immunochromatography as an immunoassay, as an example. When using other immunoassay methods, the features can be appropriately modified.

[0131] There are no particular restrictions on the types of solid-phase carriers used in the capture antibody. Specifically, examples include porous membranes made of cellulose, nitrocellulose, cellulose acetate, nylon, PVDF (polyvinylidene fluoride), glass fiber, etc.; flow paths made of glass, plastic, PDMS (polydimethylsiloxane), silicon, etc.; and silk, paper, fiber, etc.

[0132] There are no particular limitations on the methods for linking antibodies to solid-phase carriers. Specifically, methods such as fixation based on physical adsorption utilizing the hydrophobicity of antibodies and fixation based on chemical binding utilizing the functional groups of antibodies can be cited.

[0133] There are no particular restrictions on the types of markers used in the antibodies for detection; they can be selected appropriately according to the detection method. Specifically, examples include colloidal gold, colloidal platinum, colloidal palladium, and other metallic colloids; colloidal selenium, alumina colloids, silica colloids, and other non-metallic colloids; colored resin particles, dye colloids, colored liposomes, and other insoluble particulate substances; colorimetric reaction catalysts and enzymes such as alkaline phosphatase, peroxidase, and luciferase; fluorescent pigments and radioactive isotopes; chemiluminescent markers, bioluminescent markers, and electrochemiluminescent markers, etc.

[0134] There are no particular restrictions on the methods for attaching labels to antibodies. Specifically, methods such as physical adsorption using the hydrophobicity of antibodies and chemical binding using the functional groups of antibodies can be cited.

[0135] There are no restrictions on the subjects for gonorrhea testing using the gonorrhea testing method of the present invention. It can be performed on any subject requiring detection of gonorrhea infection, typically mammals, preferably humans.

[0136] There are no particular restrictions on the samples used in gonorrhea detection; any biological sample from the subject that may contain gonococci that cause urethritis can be used. Examples include urine, urethral samples obtained by inserting a swab into the subject's urethra, oral samples (oral swab fluid, mouthwash, etc.), eye samples (conjunctival swab samples, eye discharge, etc.), vaginal swab samples, and cervical swab samples. Here, the gonorrhea testing method of the present invention has the advantage of using urine, which has a low sample load, as the sample. That is, as described above, existing gonorrhea testing kits such as immunochromatographic assays (Non-Patent Document 1) have low detection sensitivity (LoD (limit of detection) of approximately 5e6cfu / mL), thus requiring urethral samples with high gonococcal concentrations, placing a significant burden on the subject (especially male subjects). In contrast, in the gonorrhea testing method of the present invention, the detection limit of gonorrhea in the sample is preferably below 5e4cfu / mL, more preferably below 3e4cfu / mL, and even more preferably below 2e4cfu / mL. The sensitivity is extremely high, so the urine of the subject can be used as a sample, which greatly reduces the burden on the subject (especially male subjects).

[0137] It should be noted that the first and second antibodies of the present invention used in the gonorrhea detection method of the present invention recognize specific epitopes present in the intracellular components L7 / L12, which are gonococcal antigens, and generate an antigen-antibody reaction. Therefore, the detection sensitivity can be improved by exposing the L7 / L12 of gonococci outside the bacterial cell membrane. Thus, the sample can be treated to induce bacterial lysis before the first and second antibodies of the present invention come into contact with the sample. The lysis treatment of the bacteria is not limited; examples include lysis treatment using surfactants, lysing enzymes, etc., and lysis treatment using alkaline lysis solutions. Examples of surfactants that can be used in lysis treatment include nonionic surfactants such as Triton X-100, Tween 20, Brij 35, Nonidet P-40, dodecyl-β-D-maltodextrin, octyl-β-D-glucoside, and polyoxyethylene polyoxypropylene hexadecyl ether; amphoteric surfactants such as Zwittergent 3-12 and CHAPS (3-(3-cholamidopropyl)dimethylammonium-1-propanesulfonate); and anionic surfactants such as SDS (sodium dodecyl sulfate) and sodium cholate. Examples of lysing enzymes that can be used in lysis treatment include lysozyme, lysococcal lysozyme, pepsin, glucosidase, galactosidase, colorless peptidase, and β-N-acetylglucosidase.

[0138] In the following description of each step of the gonorrhea testing method of the present invention, in step (Ia-1), where the sample is contacted with the detection antibody and the gonococcal antigen in the sample is labeled by the antigen-antibody reaction between the detection antibody and the gonococcal antigen, the detection antibody with the detection label is contacted with the sample, and the gonococcal antigen in the sample is labeled by the antigen-antibody reaction between the detection antibody and the gonococcal antigen. There are no limitations on the method of contacting the sample with the detection antibody; generally, it is performed by introducing a sample prepared as an aqueous sample into a component area impregnated with the detection antibody and maintaining it for a certain period of time. The specific method may vary depending on the capture method in step (a). As an example, when the gonococcal antigen in the sample is immobilized on a porous membrane serving as a solid-phase carrier and captured by a capture antibody, an aqueous reagent containing the detection antibody is introduced into the porous membrane and allowed to permeate, allowing the detection antibody to bind to the gonococcal antigen captured by the capture antibody on the porous membrane. As another example, in the case where the gonococcal antigen in the sample is captured by a capture antibody in a region of a flow path that serves as a solid-phase carrier, an aqueous reagent containing a detection antibody is allowed to flow through the flow path, thereby allowing the detection antibody to bind to the gonococcal antigen captured by the capture antibody in the flow path.

[0139] In the aforementioned step (Ia-2), where a sample containing gonococcal antigen labeled with a detection antibody is contacted with a capture antibody, and the gonococcal antigen in the sample is captured by the antigen-antibody reaction between the capture antibody and the gonococcal antigen-detection antibody complex, the capture antibody is brought into contact with the sample, and the gonococcal antigen in the sample is captured by the antigen-antibody reaction between the capture antibody and the gonococcal antigen. There are no restrictions on the method of contacting the sample with the capture antibody; generally, it is performed by introducing a sample prepared as an aqueous sample into the area where the capture antibody is present and maintaining it for a certain period of time. The specific method varies depending on the type of solid-phase carrier of the capture antibody. For example, a porous membrane is used as the solid-phase carrier; the sample is introduced into the porous membrane immobilized with the capture antibody and allowed to permeate, thereby capturing the gonococcal antigen in the sample by the capture antibody immobilized on the porous membrane. As another example, the capture antibody is immobilized in a region of a flow path that serves as a solid-phase carrier, and the sample is allowed to flow through this flow path, thereby capturing the gonococcal antigen in the sample by the capture antibody immobilized in a region of this flow path.

[0140] In step (II) above, namely the step of detecting gonococcal antigens in a sample based on a detection label, the gonococcal antigens of the test subject, which have been captured by a capture antibody and labeled with a detection antibody, are detected based on the detection label. This detection method is not particularly limited; it can be appropriately selected according to the type of detection label. For example, when using colloidal gold or other metallic colloids as the detection label, the presence or amount of colloidal gold bound to the gonococcal antigen can be detected by any method, such as visual inspection or photography.

[0141] According to the gonococcal detection method of the present invention described above, the detection limit of gonococci in the sample is preferably 5e4cfu / mL or less, more preferably 3e4cfu / mL or less, and even more preferably 2e4cfu / mL or less, exhibiting extremely high sensitivity (see Examples 3, 4, and 6, etc., described below). Therefore, it is not necessary to obtain urethral samples with high gonococcal concentrations as with existing gonococcal testing kits such as immunochromatography (Non-Patent Document 1), as the subject's urine can be used as a sample for testing, significantly reducing the burden on the subject associated with sample acquisition. It should be noted that there is no lower limit to the detection limit of gonococci in the sample, and extremely small amounts of gonococci, such as 1e4cfu / mL, can typically be detected.

[0142] Furthermore, it is known that urine and oral fluid have extremely complex compositions and their pH ranges widely (the pH of urine is about 4.5 to 8, and the pH of oral fluid is about 4 to 7). In the gonorrhea detection method of the present invention, according to its preferred mode, gonorrhea can be stably detected even under a wide range of pH conditions or in simulated urine samples (see Examples 5 and 6, etc., described later).

[0143] In one embodiment of the gonorrhea detection method according to the present invention, by using a specific combination of antibodies as a combination of capture antibody and labeling antibody, in addition to high detection sensitivity, it can be used under a wide range of pH conditions, enabling more reliable diagnosis. The pH range of application of this gonorrhea detection method is not limited; for example, the lower limit can typically be above pH 4.0, especially above pH 4.5, particularly above pH 5.8, and the upper limit can typically be below pH 9.0, especially below pH 8.7.

[0144] There are no restrictions on the combination of capture antibody and labeling antibody that can achieve this wide range of pH applicability, but the combination of (1) or (2) below is preferred.

[0145] (1) The combination of the antibody for capture is a below and the antibody for labeling is c below.

[0146] (2) The labeling antibody is a combination of the following a and the capture antibody is a combination of the following c.

[0147] a. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 11 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 12 as the light chain variable region sequence.

[0148] c. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 7 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 8 as the light chain variable region sequence.

[0149] Furthermore, in the gonorrhea detection method of the present invention, according to its preferred embodiment, it is possible to distinguish and detect gonorrhea in a sample from Neisseria meningitidis, which also belongs to the Neisseria genus (see Examples 1 and 2, etc., described below). Since gonorrhea and Neisseria meningitidis have different drug resistance tendencies, the ability to distinguish them for on-site (at the clinic) detection is highly advantageous for early selection of appropriate drug therapy corresponding to the bacterial species.

[0150] [IV. Gonorrhea Detection Kit]

[0151] Another aspect of the present invention relates to a kit for detecting gonococci in a sample (the gonococcal detection kit of the present invention) used in the gonococcal detection method of the present invention described above.

[0152] The gonorrhea detection kit of the present invention comprises the above-described capture antibody and detection antibody. The capture antibody is generally provided in a suitable form (a container containing a porous membrane, a container containing a flow path, etc.) corresponding to the type of solid-phase support. The detection antibody is generally provided in the form of an aqueous reagent containing the detection antibody in an aqueous medium or in the form of a dried reagent obtained by drying the detection antibody.

[0153] In addition to the aforementioned capture and detection antibodies, the kit of the present invention also includes one or more reagents, detection devices or components thereof required for carrying out the method of the present invention using these antibodies, and / or operating instructions describing the process for carrying out the method of the present invention. The types of reagents, the content of the operating instructions, and other components included in the kit of the present invention may be appropriately determined according to the specific type of immunological assay.

[0154] When the kit of the present invention includes a detection device or its constituent parts, the device constituted by the kit is a device that possesses the constituent elements necessary to carry out the method of the present invention using the first and / or second antibodies of the present invention (hereinafter, for convenience, it is simply referred to as "the device of the present invention"). The specific constituent elements of the device of the present invention can be appropriately adjusted according to the type of immunological assay method that is a specific embodiment of the method of the present invention. As described above, there is no limitation on the examples of immunological assay methods, and examples include ELISA (enzyme-conjugated immunosorbent assay) using microplates loaded with antibodies; emulsion particle agglutination assay using emulsion particles (e.g., polystyrene emulsion particles, etc.) loaded with antibodies; immunochromatography using membranes loaded with antibodies, etc.; sandwich analysis using detection antibodies labeled with colored particles or particles with color development ability, enzymes or fluorescent particles, etc., and capture antibodies immobilized on solid-phase carriers such as magnetic microparticles, etc. A device that possesses the constituent elements necessary to carry out such various immunological assay methods is the device of the present invention.

[0155] Specific examples of the apparatus include side-flow and flow-through methods. In the side-flow method, the target sample and the detection antibody are spread parallel to a membrane containing a detection area on which a capture antibody is immobilized, and the target substance captured by the detection area of ​​the membrane is detected. In the flow-through method, the target sample and the detection antibody are passed perpendicularly through a membrane on which a capture antibody is immobilized, and the target substance captured by the surface of the membrane is detected. The method of the present invention can be applied to either the side-flow or flow-through apparatus.

[0156] Both side-flow and flow-through immunochromatographic detection devices are well known. For processes not described in this disclosure, those skilled in the art can appropriately design them based on common technical knowledge. The following description, with reference to the accompanying drawings, illustrates a schematic configuration of the detection mechanism of a side-flow immunochromatographic detection device; however, these are merely illustrative examples of the detection process, and the configuration of a side-flow immunochromatographic detection device is not limited in any way by the illustrations in the drawings.

[0157] Figure 3 This is a schematic cross-sectional view showing an example of a strip-shaped detection mechanism in an immunochromatographic detection device as a side-flow method. Figure 3In the detection apparatus (10), a strip-shaped antibody-impregnating component (binding pad) (2) (containing the antibody for detection) and a sample addition component (sample pad) (3) are arranged on one end of the strip length direction (upstream of sample stream B) of the insoluble membrane carrier (1) used for chromatographic development, and an absorption component (absorption pad) (4) is arranged on the other end (downstream of sample stream B), and thus on a substrate (5). A portion (6) for fixing the capture antibody is arranged in the center of the strip length direction on the insoluble membrane carrier (1), and a portion (7) for fixing the control reagent is arranged as needed. It should be noted that the control reagent is a reagent that binds to the antibody for detection but not to the analyte.

[0158] In use, if sample A is applied to the sample addition component (sample pad) (3), sample A flows through the insoluble membrane carrier (1) in the direction of sample flow A via the detection antibody impregnation component (binding pad) (2). At this time, the analyte in the sample (gonococcal antigen L7 / L12 in this invention) binds to the detection antibody, forming an analyte-detection antibody complex. If sample A passes through the capture antibody fixation site (6), the analyte in the sample binds to the capture antibody, forming a capture antibody-analyte-detection antibody complex. Furthermore, when sample A passes through the control reagent fixation site (7), the antibody in the detection antibody that has not bound to the analyte binds to the control reagent (7), thereby confirming the end of the test (i.e., sample A has passed through the capture antibody (6)). Here, by using known methods to detect the label of the detection antibody present in the capture antibody-analyte-detection antibody complex at the capture antibody fixation site (6), the presence or quantity of the analyte can be detected. The detection can be made easier by using known methods to sensitize the labeled antibodies as needed.

[0159] It should be noted that the antibody-impregnating component (binding pad) (2), the sample addition component (sample pad) (3), and / or the control reagent fixation site (7) can be omitted arbitrarily. In the absence of an antibody-impregnating component (binding pad) (2) in this institution, sample A and the antibody for detection can be applied simultaneously or sequentially to one end of the insoluble membrane carrier 1 in a pre-mixed or separate state, thereby enabling the same test as described above.

[0160] Furthermore, even if the capture antibody and the detection antibody are replaced, a test kit that can perform the same detection can be constructed.

[0161] The method for manufacturing the kit of the present invention described above is not limited, but it is preferred to manufacture it by a method that includes at least the step of immobilizing the capture antibody on the detection region of a carrier having a detection region.

[0162] In the case where the kit of the present invention is an immunochromatographic kit, it is preferably manufactured by a manufacturing method that includes at least the steps of immobilizing a capture antibody on a detection region of a carrier having a detection region, attaching a detection antibody to a conjugate pad, and placing the conjugate pad upstream of the detection region of the carrier.

[0163] The method for manufacturing the kit of the present invention preferably further includes a step of preparing a detection antibody by adding a detection label to one of the first and second monoclonal antibodies, and at least includes a step of immobilizing the other of the first and second monoclonal antibodies as a capture antibody in the detection region of the carrier. The first and second monoclonal antibodies are as described above.

[0164] In the case where the first monoclonal antibody is an antibody that generates an antigen-antibody reaction with an epitope containing one or more amino acid residues from the 2nd to 14th amino acid residues from the N-terminus of the L7 / L12 amino acid sequence selected from the gonococcus shown in sequence number 1, it is preferable to inoculate an animal with the epitope polypeptide to obtain an antiserum containing an antibody that generates an antigen-antibody reaction with the epitope polypeptide, and to purify and fractionate the antibody from the antiserum to obtain the first monoclonal antibody. This antibody acquisition step can be performed separately from the manufacturing method of the kit of the present invention, or it can be performed as a step in the manufacturing method of the kit of the present invention.

[0165] Furthermore, when using an antibody as the second monoclonal antibody that generates an antigen-antibody reaction with an epitope containing one or more amino acid residues from the N-terminus of amino acid residues selected from L7 / L12 of the gonococcus shown in sequence number 1, it is preferable to inoculate an animal with the epitope polypeptide to obtain an antiserum containing an antibody that generates an antigen-antibody reaction with the epitope polypeptide, and to purify and fractionate the antibody from the antiserum to obtain the second monoclonal antibody. This antibody acquisition step can be performed separately from the manufacturing method of the kit of the present invention, or it can be performed as a step in the manufacturing method of the kit of the present invention.

[0166] Alternatively, as the first and / or second monoclonal antibody, an antibody with a known amino acid sequence of the variable region of the heavy chain and / or light chain can be used in the manufacture of the kit of the present invention. The amino acid sequence of the variable region of the heavy chain and / or light chain of the first and / or second monoclonal antibody is described in detail above.

[0167] It should be noted that either the first or second monoclonal antibody can be a capture antibody or a labeling antibody, as described above.

[0168] There are no restrictions on the combination of the capture antibody and the labeling antibody used in the manufacture of the kit of the present invention, but as described above, the combination of (1) or (2) below is preferred.

[0169] (1) The antibody for capture is a or b below, and the antibody for labeling is a combination of c below.

[0170] (2) The antibody used for labeling is a or b below, and the antibody used for capture is a combination of c below.

[0171] a. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 11 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 12 as the light chain variable region sequence.

[0172] b. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 9 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 10 as the light chain variable region sequence.

[0173] c. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 7 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 8 as the light chain variable region sequence.

[0174] In particular, from the perspective of achieving the above-mentioned wide range of pH applicability, there is no limitation on the combination of capture antibody and labeling antibody used in the manufacture of the kit of the present invention, but as described above, the combination of (1) or (2) below is preferred.

[0175] (1) The combination of the antibody for capture is a below and the antibody for labeling is c below.

[0176] (2) The labeling antibody is a combination of the following a and the capture antibody is a combination of the following c.

[0177] a. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 11 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 12 as the light chain variable region sequence.

[0178] c. An antibody containing an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 7 as the heavy chain variable region sequence, and an amino acid sequence having more than 80% homology with the amino acid sequence of sequence number 8 as the light chain variable region sequence.

[0179] Example

[0180] The present invention will be described in more detail below by way of embodiments, but these embodiments are merely examples for the purpose of illustration and the present invention is not limited to these embodiments in any way.

[0181] [Example 1: Antibody Preparation and Screening]

[0182] Antibodies against the ribosomal proteins L7 / L12 of Neisseria gonorrhoeae were prepared using the method described in International Publication No. 2001 / 057089. Specifically, polypeptides 1 to 4, having all or part of the amino acid sequence of L7 / L12 of Neisseria gonorrhoeae, were used as gonococcal-derived antigenic polypeptides. Additionally, polypeptide 5, having all the amino acid sequence of L7 / L12 of Neisseria meningitidis, was used as a similar antigenic polypeptide for verifying the cross-reactivity of the antibodies.

[0183] The amino acid sequence alignment of gonococcal L7 / L12 with the amino acid sequences of peptides 1-5 is shown in the figure. Figure 2 The sequence numbers of these amino acid sequences and their correspondences are shown in Table 1 below. In the table, sequences containing a portion of the sequence composed of these amino acid residues from *Neisseria gonorrhoeae* L7 / L12 are represented by ○, and sequences not containing this portion are represented by ×. Furthermore, in the amino acid sequences of *Neisseria gonorrhoeae* L7 / L12 and *Neisseria meningitidis* L7 / L12, the 115th amino acid residue from the N-terminus is alanine in *Neisseria gonorrhoeae*, while it is glutamic acid in *Neisseria meningitidis*. This is the only difference between the two; therefore, in the table below, the column for polypeptide 5 corresponding to amino acid residues 102–123 of *Neisseria gonorrhoeae* L7 / L12 is represented by △.

[0184] [Table 1]

[0185] The aforementioned peptides 1-5 were prepared in the following order: An expression vector was prepared, into which DNA encoding peptides 1-5 was introduced, and this expression vector was used to transform *E. coli*. The resulting transformed *E. coli* were cultured in LB medium or similar medium, and the peptides 1-5 expressed and released into the culture medium by the transformants were purified as fusion proteins using an affinity column with tag sequences derived from the expression vector.

[0186] The polypeptide 1 obtained through the above process was inoculated into mice along with an adjuvant. Thirty days after inoculation, spleen cells were harvested from the mice and fused with myeloma cells to produce antibodies and hybridomas.

[0187] Monoclonal antibodies generated from hybridomas were sorted using methods such as ELISA. Peptides 1-5 were used in the sorting process. Specifically, peptides 1-5 were immobilized in 1×PBS at a concentration of 2 μg / mL on 96-well microplates, and the obtained monoclonal antibodies were reacted at a concentration of 100 ng / mL. HRP (horseradish peroxidase)-labeled anti-IgG antibodies were further reacted, and finally, substrate was added for color development. The reaction patterns of each monoclonal antibody with respect to peptides 1-5 were evaluated.

[0188] Six monoclonal antibodies were ultimately obtained through the above process. The reactivity of the six monoclonal antibodies with respect to peptides 1–5 is shown in Table 2 below. In the table, cases with an absorbance of 0.1 or higher at a wavelength of 450 nm are represented as 0, and cases with an absorbance of less than 0.1 are represented as ×.

[0189] [Table 2]

[0190] Based on the above reaction pattern, the six monoclonal antibodies were divided into the following four types.

[0191] • Antibody species A (NG1): This antibody species does not react with peptides 4 and 5, therefore it is known that it reacts with a portion of the sequence of gonococcal L7 / L12 consisting of amino acid residues from the N-terminus 102 to 123, including the 115th amino acid from the N-terminus.

[0192] • Antibody species B (NG2, NG3): This antibody species does not react with polypeptide 2, therefore it is known that it reacts with a portion of the sequence of gonococcal L7 / L12 consisting of amino acid residues from the N-terminus to the 14th position.

[0193] • Antibody species C (NG4, NG5): This antibody species reacts with polypeptide 2 but not with polypeptide 3. Therefore, it can be known that it reacts with a portion of the sequence of gonococcal L7 / L12 consisting of amino acid residues from the N-terminus to the 15th to the 63rd position.

[0194] • Antibody species D (NG6): This antibody species reacts with all of peptides 1 to 5, therefore it is known that it reacts with a portion of the sequence of gonococcal L7 / L12 consisting of amino acid residues from the N-terminus 64 to 101.

[0195] In addition, for the six monoclonal antibodies obtained, the amino acid sequences of the variable regions of the heavy and light chains of antibody NG1 (belonging to antibody species A) and antibodies NG2 and NG3 (belonging to antibody species B) were identified using conventional methods. The amino acid sequences of the variable regions of the heavy and light chains of antibody NG1 are shown in sequence numbers 7 and 8, respectively. The amino acid sequences of the variable regions of the heavy and light chains of antibody NG2 are shown in sequence numbers 9 and 10, respectively. The amino acid sequences of the variable regions of the heavy and light chains of antibody NG3 are shown in sequence numbers 11 and 12, respectively.

[0196] [Example 2: Evaluation of the performance of antibody combination-based gonorrhea detection using ELISA]

[0197] Next, antibodies from antibody species A through D were combined in various ways to create capture and detection antibodies. The performance of each combination for gonorrhea detection was validated using ELISA. Antibody NG1 was used for evaluation as antibody species A, antibody NG3 as antibody species B, antibody NG5 as antibody species C, and antibody NG6 as antibody species D. Furthermore, when each antibody from antibody species A through D was used as a detection antibody, it was labeled with HRP (horseradish peroxidase) using standard methods.

[0198] First, *Neisseria gonorrhoeae* (ATCC700825) and *Neisseria meningitidis* (ATCC13090) were inoculated separately onto chocolate agar medium and cultured for 24 hours at 37°C and 5–10% CO2. Colonies were then picked and suspended in physiological saline. The absorbance of the saline suspension was measured using a spectrophotometer (wavelength 600 nm). A concentration of 1e9 cfu / mL was considered to contain *Neisseria gonorrhoeae* at an absorbance of 2, and the bacterial count in the *Neisseria gonorrhoeae* saline suspension was performed. The *Neisseria gonorrhoeae* and *Neisseria meningitidis* saline suspensions were then diluted with physiological saline to obtain a *Neisseria gonorrhoeae* saline suspension with a bacterial concentration of 6e6 cfu / mL.

[0199] In addition, an extract with the following composition was prepared.

[0200] 0.3% TritonX-100

[0201] 0.3% Polyoxyethylene polyoxypropylene hexadecyl ether

[0202] 0.2% bovine serum albumin

[0203] 0.1M sodium chloride

[0204] Add 500 μL of the above extraction solution to 100 μL of the gonococcal physiological saline suspension with a bacterial concentration of 6e6cfu / mL, mix thoroughly, and let stand for 1 hour to prepare an extraction sample solution with a bacterial concentration of 1e6cfu / mL.

[0205] The capture antibody selected from antibody species A to D was dissolved in 1×PBS at a concentration of 10 μg / mL. 50 μL of the resulting capture antibody solution was introduced into the wells of a 96-well microplate and incubated at 4°C for 16 hours to immobilize the capture antibody on the bottom surface of the wells. Next, the capture antibody solution was removed from the wells, and the wells were washed with 1×PBS containing 0.05% Tween 20. 200 μL of a solution obtained by dissolving BSA at a 1% concentration in 1×PBS was then introduced, and the reaction was carried out for 2 hours to block the wells. Next, 50 μL of an extract of Neisseria gonorrhoeae or Neisseria meningitidis prepared using the above method at a bacterial concentration of 1e6 cfu / mL was injected into the wells, and the reaction was carried out for 1 hour. The sample solution was then removed from the wells, and the wells were washed with 1×PBS containing 0.05% Tween 20. 50 μL of an antibody solution (prepared by dissolving HRP-labeled detection antibodies selected from antibody species A-D at a concentration of 1 μg / mL in 1×PBS) was then introduced, and the reaction was allowed to proceed for 1 hour. After removing the antibody solution from the wells, the wells were washed with 1×PBS containing 0.05% Tween 20, and 100 μL of luminescent substrate was added. The mixture was allowed to stand for 30 minutes, and then 100 μL of hydrochloric acid (equivalent to 1 part of the total solution) was added as a stop solution to stop the colorimetric reaction. Finally, the absorbance of each well (wavelength 450 nm) was measured using a Molecular Devices "Spectra Max 190" plate reader.

[0206] The results are shown in Table 3 below. In the table, combinations where the difference between the absorbance of the wells reacted with the extract of Neisseria gonorrhoeae and the absorbance of the wells reacted with the extract of Neisseria gonorrhoeae (excluding Neisseria gonorrhoeae and Neisseria meningitidis) (absorbance difference A) is less than 1.0 are marked as ×. Among the combinations where the absorbance difference A is 1.0 or more, combinations where the difference between the absorbance of the wells reacted with the extract of Neisseria meningitidis and the absorbance of the wells reacted with the extract of Neisseria meningitidis (absorbance difference B) is less than 1.0 are marked as ●. Combinations where both absorbance differences A and B are 1.0 or more are marked as 〇.

[0207] [Table 3]

[0208] The results above show that for the combination containing antibody species A (NG1), there are significant signal differences between the extracts of Neisseria gonorrhoeae and Neisseria meningitidis. Therefore, it can be concluded that for antibody species A, the partial sequence of the amino acid residue from the N-terminus of the L7 / L12 amino acid sequence of Neisseria gonorrhoeae ribosomal protein, which is the only point of difference between it and the L7 / L12 amino acid sequence of Neisseria meningitidis, generated an antigen-antibody reaction.

[0209] [Example 3: Evaluation of the sensitivity of gonococcal detection using an immunochromatographic apparatus]

[0210] Next, antibodies selected from antibody species B to D were used as capture antibodies, and antibody species A was used as detection antibody to prepare an immunochromatographic apparatus for gonococcal detection. The performance of gonococcal detection using this immunochromatographic apparatus was verified. Antibody NG1 was used for evaluation as antibody species A. Furthermore, when antibodies from antibody species B to D were used as detection antibodies, they were labeled with HRP using conventional methods.

[0211] First, monoclonal antibodies of antibody species B, C, and D, along with trehalose, were dissolved in 50 mM sodium phosphate buffer at concentrations of 1.5 mg / mL and 3% (v / v), respectively. A commercially available nitrocellulose membrane was cut into squares 2.5 cm wide and 30 cm long. The resulting antibody solution was spread onto the cut membrane at a rate of 1 μL per 1 cm length, and then dried. This process produced a chromatographic development membrane carrier containing antibodies of antibody species B, C, and D for capture.

[0212] Add 1 / 10 volume of 0.1M sensitization buffer (TAPSO, pH 7.0, manufactured by Dojin Chemical Research Institute Co., Ltd.) and antibody type A against gonococcal ribosomal proteins L7 / L12 to a colloidal gold solution (particle size 60 nm). Mix and let stand at room temperature for 30 minutes to allow the antibody to bind to the surface of the colloidal gold particles. Then, add 2.5% casein aqueous solution to block the antibody, achieving a final concentration of 0.2% in the colloidal gold solution, to prepare a colloidal gold-labeled antibody solution. Infiltrate this antibody solution into a commercially available glass fiber sheet and then dry it overnight in a desiccant chamber with humidity of 0–60% to create a labeled antibody-impregnated component containing colloidal gold-labeled antibody type A for detection.

[0213] In addition to the chromatographic development membrane carrier (1) and the colloidal gold-labeled antibody impregnation component (2) prepared using the above process, cotton cloth as a sample addition component (3) and filter paper as an absorption component (4) were also prepared. Afterwards, these components were attached to the substrate (5) and cut into 5mm widths to create a chromatographic development membrane carrier (1) and a colloidal gold-labeled antibody impregnation component (2). Figure 3 The immunochromatographic apparatus shown has the following structure.

[0214] The gonococcal physiological saline suspension was diluted in stages with physiological saline to obtain gonococcal physiological saline suspensions with bacterial concentrations of 1e5, 5e4, 2e4, and 1e4 cfu / mL. Subsequently, 500 μL of extraction buffer was added to 100 μL of the gonococcal physiological saline suspension, and after thorough mixing, the mixture was allowed to stand for 1 hour to prepare the extraction sample solution.

[0215] Add 115 μL of each of the above-prepared extract solutions to an immunochromatographic apparatus for gonococcal detection manufactured using the above process. After 15 minutes, visually assess the sample. The results are shown in Table 4 below. It should be noted that in the table, 〇 indicates visually positive, and × indicates visually negative.

[0216] [Table 4]

[0217] Based on these results, the combination of antibody species A and antibody species B showed good detection performance, capable of detecting low concentrations of gonococci (1–2e4 cfu / mL, based on the bacterial load before extraction). On the other hand, the combination of antibody species A with antibody species C or antibody species D could not detect gonococci with bacterial concentrations less than 1e5 cfu / mL (based on the bacterial load before extraction).

[0218] [Example 4: Evaluation of the sensitivity of gonococcal detection based on ELISA]

[0219] Using a combination of antibodies capable of detecting gonorrhea from samples with low bacterial concentrations such as 1e5cfu / mL, as described in Example 3 (Evaluation of Gonorrhea Detection Sensitivity Using an Immunochromatographic Apparatus), its detection performance was also evaluated using ELISA as an example of an immunoassay principle different from immunochromatography.

[0220] The capture antibodies (antibodies NG2 and NG3 of antibody species B, and antibody NG5 of antibody C) were dissolved in 1×PBS at a concentration of 10 μg / mL. 50 μL of the resulting capture antibody solution was introduced into the wells of a 96-well microplate and incubated at 4°C for 16 hours to immobilize the capture antibodies on the bottom surface of the wells. Next, the capture antibody solution was removed from the wells, and the wells were washed with 1×PBS containing 0.05% Tween 20. 200 μL of a solution prepared by dissolving BSA at a 1% concentration in 1×PBS was then introduced, and the reaction was carried out for 2 hours to seal the wells. Subsequently, 50 μL of extraction sample solutions prepared using the above method at bacterial concentrations of 1e5, 5e4, 2e4, and 1e4 cfu / mL were injected into the wells, and the reaction was carried out for 1 hour. The sample solution was then removed from the wells, and the wells were washed with 1×PBS containing 0.05% Tween 20. 50 μL of an antibody solution (prepared by dissolving HRP-labeled detection antibody (NG1) at a concentration of 1 μg / mL in 1×PBS) was then introduced, and the reaction was allowed to proceed for 1 hour. The antibody solution was then removed from the wells, and the wells were washed with 1×PBS containing 0.05% Tween 20. 100 μL of luminescent substrate was added, and the mixture was allowed to stand for 30 minutes. Then, 100 μL of hydrochloric acid (equivalent to 1 part of the total hydrochloric acid) was added as a stop solution to stop the colorimetric reaction. Finally, the absorbance of each well (wavelength 450 nm) was measured using a Molecular Devices "Spectra Max 190" plate reader.

[0221] The results are shown in Table 5 below. In the table, cases where the difference between the absorbance of the wells reacted with the extract solution of each bacterial concentration and the absorbance of the wells reacted with the extract solution without bacteria is greater than 0.1 are marked as 0, and cases where the difference is less than 0.1 are marked as ×.

[0222] [Table 5]

[0223] Based on these results, the combination of antibody species A and antibody species B also showed good detection performance using ELISA, capable of detecting low concentrations of gonococci (1–2 e4 cfu / mL, based on the bacterial load before extraction). On the other hand, the combination of antibody species A and antibody species C could not detect gonococci with bacterial concentrations less than 1 e5 cfu / mL (based on the bacterial load before extraction).

[0224] [Example 5: Evaluation of the sensitivity of gonococcal detection under a wide pH range using ELISA]

[0225] For the combination that can detect gonococci with a bacterial concentration of 1e5cfu / mL in Example 3 (evaluation of gonococcal detection sensitivity using an immunochromatographic apparatus), the immunodetection performance over a wide pH range was evaluated using the ELISA method.

[0226] For evaluation purposes, the following three buffer solutions for immune reactions were prepared.

[0227] Solution 1: 10 mM K₂HPO₄, 90 mM KH₂PO₄, 100 mM NaCl, pH 5.8, conductivity 18 mS / cm

[0228] Solution 2: 60 mM K₂HPO₄, 40 mM KH₂PO₄, 100 mM NaCl, pH 6.8, conductivity 20 mS / cm

[0229] Solution 3: 100mM K₂HPO₄, 100mM NaCl, pH 8.7, conductivity 23mS / cm

[0230] Solution 4: 100mM KH₂PO₄, 100mM NaCl, pH 4.5, conductivity 17mS / cm

[0231] The capture antibodies (NG2, NG3, NG5) were dissolved in 1×PBS at a concentration of 10 μg / mL. 50 μL of the resulting capture antibody solution was introduced into the wells of a 96-well microplate and incubated at 4°C for 16 hours to immobilize the capture antibodies on the bottom surface of the wells. Next, the capture antibody solution was removed from the wells, and the wells were washed with 1×PBS containing 0.05% Tween 20. 200 μL of a solution prepared by dissolving BSA at a 1% concentration in 1×PBS was then introduced, and the reaction was allowed to proceed for 2 hours to seal the wells. Next, add 1 / 100 of the amount of the gonococcal bacterial concentration adjusted to 1.6e7cfu / mL (for adding to solutions 1, 2, and 3) or 3e7cfu / mL (for adding to solution 4) to the above-mentioned immunoreaction buffers (solutions 1, 2, 3, and 4), resulting in a pH-adjusted sample solution containing gonococci at a bacterial concentration of 1.6e5cfu / mL (solutions 1, 2, and 3) or 3e5cfu / mL (solution 4). Inject 50 μL of this pH-adjusted sample solution into the wells and react for 1 hour. Then, remove the pH-adjusted sample solution from the wells, wash the wells with 1×PBS containing 0.05% Tween 20, and introduce 50 μL of a pH-adjusted antibody solution prepared by dissolving HRP-labeled detection antibody (NG1) at a concentration of 1 μg / mL in the above-mentioned immunoreaction buffers (solutions 1, 2, and 3), and react for 1 hour. At this point, ensure that the pH of the pH-adjusted sample solution added to the wells is consistent with that of the pH-adjusted antibody solution. The pH-adjusting antibody solution in the wells was then removed. The wells were washed with 1×PBS containing 0.05% Tween 20, and 100 μL of luminescent substrate was added. After standing for 30 minutes, 100 μL of hydrochloric acid (equivalent to 1 part of the solution) was added as a stop solution to stop the colorimetric reaction. Finally, the absorbance of each well (wavelength 450 nm) was measured using a Molecular Devices "Spectra Max 190" plate reader.

[0232] The results are shown in Table 6 below. In the table, the difference between the absorbance of the wells reacted with the extract solution of each bacterial concentration and the absorbance of the wells reacted with the extract solution without bacteria is recorded as ++ if it is 1.0 or more, + if it is 0.5 or more but less than 1.0, and - if it is less than 0.5.

[0233] [Table 6]

[0234] The combination of antibody species A and antibody species B can stably perform immunoassay within a wide pH range of 5.8 to 8.7, and is therefore considered suitable for gonococcal detection in various samples, such as urine and oral samples, where the pH may vary over a wide range. The stability of the combination of antibody species A and antibody species B within this wide pH range is attributed to the structural and chemical stability of the amino acid sequences of both antibody species A (resulting in antigen-antibody reactions, i.e., amino acid residues 102-121 from the N-terminus in the L7 / L12 amino acid sequence of gonococci shown in Sequence Number 1) and antibody species B (resulting in antigen-antibody reactions, i.e., amino acid residues 3-14 from the N-terminus in the L7 / L12 amino acid sequence of gonococci shown in Sequence Number 1) within the pH range of 5.8 to 8.7.

[0235] It should be noted that the combination of NG1 as antibody type A and NG3 as antibody type B can stably perform immunoassay even at low pH levels such as 4.5. Therefore, it is considered particularly suitable for gonorrhea detection in various samples, such as urine samples and oral samples, where the pH may vary over a wide range. While the reason why this combination of antibodies NG1 and NG3 can stably perform immunoassay even at low pH levels such as 4.5 is only speculative, it is believed to be due to the fact that the antigen recognition site of NG3 as antibody type B is also particularly stable in structure and chemical properties at low pH levels.

[0236] [Example 6: Performance evaluation of gonorrhea detection using urine samples]

[0237] This study evaluated whether a combination of antibody species A and antibody species B, which possess good detection performance and wide pH range adaptability, could detect gonococci in actual urine samples. Simulated urine samples with known gonococcal concentrations were prepared by adding a gonococcal saline suspension to commercially available donor urine (LEE BIOSOLUTIONS, pH 6.3, conductivity 10 mS / cm, negative in Cobas 8800-based gonococcal nucleic acid testing). 500 μL of extraction buffer was added to 100 μL of the simulated urine sample and mixed thoroughly to prepare the extraction sample solution.

[0238] Add 115 μL of each of the above-prepared extract samples to an immunochromatographic apparatus for gonococcal detection manufactured according to the process described in Example 3. After 15 minutes, visual inspection is performed. The results are shown in Table 7 below. It should be noted that in the table below, 〇 indicates visually positive and × indicates visually negative.

[0239] [Table 7]

[0240] When the sample is urine, the combination of antibody species A and antibody species B also showed good detection performance, capable of detecting low concentrations of gonococci (1-2e4cfu / mL, based on the bacterial load before extraction).

[0241] [Example 7: Evaluation of the sensitivity of gonococcal detection using immunochromatography]

[0242] Using the immunochromatographic apparatus prepared in Example 3, we evaluated whether the combination of antibody species A and antibody species B, which have good detection performance and wide pH range adaptability, can separate and detect gonococci and meningococci in a sample.

[0243] The gonococcal saline suspension was diluted with physiological saline to obtain gonococcal saline suspensions with bacterial concentrations of 1.2e6, 6e5, 3e5, 1.2e5, and 6e4 cfu / mL. Then, 500 μL of extraction buffer was added to 100 μL of the gonococcal saline suspension, and after thorough mixing and standing for 1 hour, extraction sample solutions with bacterial concentrations of 2e5, 1e5, 5e4, 2e4, and 1e4 cfu / mL were prepared.

[0244] Add 115 μL of each of the above-prepared extract solutions to an immunochromatographic apparatus for gonococcal detection manufactured using the above process. After 15 minutes, visually assess the sample. The results are shown in Table 8 below. It should be noted that in the table, 〇 indicates visually positive, and × indicates visually negative.

[0245] [Table 8]

[0246] The combination of antibody species A and antibody species B has a gonococcal detection sensitivity that is at least 10 times higher than that for Neisseria meningitidis, enabling the detection of gonococci in samples separately from Neisseria meningitidis.

[0247] [Example 8: Evaluation of Bacterial Specificity]

[0248] Using the immunochromatographic apparatus prepared in Example 3, the combination of antibody species A and antibody species B, which have good detection performance and wide pH range adaptability, was evaluated to ensure that there was no cross-reactivity with other genera of bacteria in the sample.

[0249] For the bacterial species shown in Table 9, except for *Chlamydia trachomatis*, *Chlamydia pneumoniae*, and *Mycoplasma pneumoniae*, bacterial counts were performed on suspensions using the method described in Example 2. Then, the suspensions were progressively diluted with physiological saline to obtain physiological saline suspensions of each bacterium with a concentration of 6e7 cfu / mL. Subsequently, 500 μL of extraction buffer was added to 100 μL of the bacterial physiological saline suspension, mixed thoroughly, and allowed to stand for 1 hour to prepare an extraction sample solution with a bacterial concentration of 1e7 cfu / mL. For *Chlamydia trachomatis*, 2e8 IFU / mL inactivated bacterial solution purchased from Microbix was progressively diluted with physiological saline. 500 μL of extraction buffer was added to 100 μL of the bacterial physiological saline suspension, mixed thoroughly, and allowed to stand for 1 hour to prepare an extraction sample solution with a concentration of 2e6 IFU / mL. For *Chlamydia pneumoniae*, an inactivated bacterial suspension of 2e8 IFU / ml purchased from Microbix was diluted using physiological saline. 500 μL of extraction buffer was added to 100 μL of the bacterial suspension in physiological saline, and the mixture was thoroughly mixed and allowed to stand for 1 hour to prepare an extraction sample with a bacterial concentration of 4e6 IFU / ml. For *Mycoplasma pneumoniae*, an inactivated bacterial suspension of 5e7 IFU / ml purchased from Microbix was diluted using physiological saline. 500 μL of extraction buffer was added to 100 μL of the bacterial suspension in physiological saline, and the mixture was thoroughly mixed and allowed to stand for 1 hour to prepare an extraction sample with a bacterial concentration of 2e6 IFU / ml.

[0250] Add 115 μL of each of the above-prepared extract solutions to an immunochromatographic apparatus for gonococcal detection manufactured using the above process. After 15 minutes, visually assess the sample. The results are shown in Table 9 below. It should be noted that in the table, 〇 indicates visually positive, and × indicates visually negative.

[0251] [Table 9]

[0252] The combination of antibody species A and antibody species B did not produce cross-reactions with bacteria other than Neisseria gonorrhoeae shown in Table 9 of the sample.

[0253] Industrial applicability

[0254] This invention can be widely applied in medical fields such as gonorrhea detection, and its utilization value is extremely high.

[0255] Explanation of symbols

[0256] 10 testing agencies

[0257] 1. Insoluble membrane support for chromatographic development

[0258] 2. Antibody-impregnated component (binding pad) for detection

[0259] 3. Sample Addition Components (Sample Pad)

[0260] 4. Absorbent components (absorbent pads)

[0261] 5. Substrate

[0262] 6. Capture sites fixed with antibodies

[0263] 7. Fixation site of control reagent

[0264] Sample A

[0265] B Sample Stream sequence list <110> Asahi Kasei Co., Ltd. <120> Gonorrhea detection kits and methods <130> 210284WO01 <150> JP 2021-056132 <151> 2021-03-29 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 123 <212> PRT <213> Gonococcus <400> 1 Met Ala Ile Thr Lys Glu Asp Ile Leu Glu Ala Val Gly Ser Leu Thr 1 5 10 15 Val Met Glu Leu Asn Asp Leu Val Lys Ala Phe Glu Glu Lys Phe Gly 20 25 30 Val Ser Ala Ala Ala Val Ala Val Ala Gly Pro Ala Gly Ala Gly Ala 35 40 45 Ala Asp Ala Glu Glu Lys Thr Glu Phe Asp Val Val Leu Ala Ser Ala 50 55 60 Gly Asp Gln Lys Val Gly Val Ile Lys Val Val Arg Ala Ile Thr Gly 65 70 75 80 Leu Gly Leu Lys Glu Ala Lys Asp Ile Val Asp Gly Ala Pro Lys Thr 85 90 95 Ile Lys Glu Gly Val Ser Lys Ala Glu Ala Glu Asp Ile Gln Lys Gln 100 105 110 Leu Glu Ala Ala Gly Ala Lys Val Glu Ile Lys 115 120 <210> 2 <211> 123 <212> PRT <213> Neisseria meningitidis <400> 2 Met Ala Ile Thr Lys Glu Asp Ile Leu Glu Ala Val Gly Ser Leu Thr 1 5 10 15 Val Met Glu Leu Asn Asp Leu Val Lys Ala Phe Glu Glu Lys Phe Gly 20 25 30 Val Ser Ala Ala Ala Val Ala Val Ala Gly Pro Ala Gly Ala Gly Ala 35 40 45 Ala Asp Ala Glu Glu Lys Thr Glu Phe Asp Val Val Leu Ala Ser Ala 50 55 60 Gly Asp Gln Lys Val Gly Val Ile Lys Val Val Arg Ala Ile Thr Gly 65 70 75 80 Leu Gly Leu Lys Glu Ala Lys Asp Ile Val Asp Gly Ala Pro Lys Thr 85 90 95 Ile Lys Glu Gly Val Ser Lys Ala Glu Ala Glu Asp Ile Gln Lys Gln 100 105 110 Leu Glu Glu Ala Gly Ala Lys Val Glu Ile Lys 115 120 <210> 3 <211> 121 <212> PRT <213> Large intestinal bacteria <400> 3 Met Ser Ile Thr Lys Asp Gln Ile Ile Glu Ala Val Ala Ala Met Ser 1 5 10 15 Val Met Asp Val Val Glu Leu Ile Ser Ala Met Glu Glu Lys Phe Gly 20 25 30 Val Ser Ala Ala Ala Ala Val Ala Val Ala Ala Gly Pro Val Glu Ala 35 40 45 Ala Glu Glu Lys Thr Glu Phe Asp Val Ile Leu Lys Ala Ala Gly Ala 50 55 60 Asn Lys Val Ala Val Ile Lys Ala Val Arg Gly Ala Thr Gly Leu Gly 65 70 75 80 Leu Lys Glu Ala Lys Asp Leu Val Glu Ser Ala Pro Ala Ala Leu Lys 85 90 95 Glu Gly Val Ser Lys Asp Asp Ala Glu Ala Leu Lys Lys Ala Leu Glu 100 105 110 Glu Ala Gly Ala Glu Val Glu Val Lys 115 120 <210> 4 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> 4 <400> 4 Met Leu Thr Val Met Glu Leu Asn Asp Leu Val Lys Ala Phe Glu Glu 1 5 10 15 Lys Phe Gly Val Ser Ala Ala Ala Val Ala Val Ala Gly Pro Ala Gly 20 25 30 Ala Gly Ala Ala Asp Ala Glu Glu Lys Thr Glu Phe Asp Val Val Leu 35 40 45 Ala Ser Ala Gly Asp Gln Lys Val Gly Val Ile Lys Val Val Arg Ala 50 55 60 Ile Thr Gly Leu Gly Leu Lys Glu Ala Lys Asp Ile Val Asp Gly Ala 65 70 75 80 Pro Lys Thr Ile Lys Glu Gly Val Ser Lys Ala Glu Ala Glu Asp Ile 85 90 95 Gln Lys Gln Leu Glu Ala Ala Gly Ala Lys Val Glu Ile Lys 100 105 110 <210> 5 <211> 61 <212> PRT <213> artificial sequence <220> <223> 5 <400> 5 Met Ala Gly Asp Gln Lys Val Gly Val Ile Lys Val Val Arg Ala Ile 1 5 10 15 Thr Gly Leu Gly Leu Lys Glu Ala Lys Asp Ile Val Asp Gly Ala Pro 20 25 30 Lys Thr Ile Lys Glu Gly Val Ser Lys Ala Glu Ala Glu Asp Ile Gln 35 40 45 Lys Gln Leu Glu Ala Ala Gly Ala Lys Val Glu Ile Lys 50 55 60 <210> 6 <211> 101 <212> PRT <213> artificial sequence <220> <223> 6 <400> 6 Met Ala Ile Thr Lys Glu Asp Ile Leu Glu Ala Val Gly Ser Leu Thr 1 5 10 15 Val Met Glu Leu Asn Asp Leu Val Lys Ala Phe Glu Glu Lys Phe Gly 20 25 30 Val Ser Ala Ala Ala Val Ala Val Ala Gly Pro Ala Gly Ala Gly Ala 35 40 45 Ala Asp Ala Glu Glu Lys Thr Glu Phe Asp Val Val Leu Ala Ser Ala 50 55 60 Gly Asp Gln Lys Val Gly Val Ile Lys Val Val Arg Ala Ile Thr Gly 65 70 75 80 Leu Gly Leu Lys Glu Ala Lys Asp Ile Val Asp Gly Ala Pro Lys Thr 85 90 95 Ile Lys Glu Gly Val 100 <210> 7 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> 7 <400> 7 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile Tyr Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly His Thr Lys Cys Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ser Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Lys Gly Tyr Phe His Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Looking Looking <210> 8 <211> 107 <212> PRT <213> artificial sequence <220> <223> 8 <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr 20 25 30 Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 9 <400> 9 Glu Val Gln Val Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Gly His Val Lys Ser Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Ile Phe 50 55 60 Arg Asp Lys Ala Ser Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Asp Leu Gln Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly His Tyr Gly Ser Gly Leu Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Thr Ala 115 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 10 <400> 10 Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Lys Thr Asn Lys Ser Ile Ser Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Ile Tyr Tyr Cys Gln Gln His Asn Glu Phe Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 11 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> 11 <400> 11 Asp Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser 1 5 10 15 Gly Tyr Thr Phe Thr Asn Tyr Trp Ile Asn Trp Ile Lys Gln Arg Pro 20 25 30 Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Ala Pro Gly Ser Gly Ser 35 40 45 Thr Tyr Tyr Asn Glu Met Phe Lys Gly Lys Ala Thr Leu Thr Val Asp 50 55 60 Thr Ser Ser Arg Thr Ala Tyr Ile Gln Leu Ser Ser Leu Ser Ser Glu 65 70 75 80 Asp Ser Ala Val Tyr Phe Cys Ala Arg Phe Val Thr Thr Val Val Ala 85 90 95 Glu Ser Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 12 <211> 111 <212> PRT <213> artificial sequence <220> <223> 12 <400> 12 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Phe Phe 20 25 30 Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Ser Ala Ala Ser Ile Val Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Asp Asp Ile Ala Met Tyr Phe Cys Gln Gln Ser Arg 85 90 95 Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 1

Claims

1. A kit for detecting gonococci in a sample, wherein, This kit contains: Antibodies used to capture gonococcal antigens in samples, and An antibody for detection, containing a marker for labeling gonococcal antigens in a sample. Gonorrhea is detected by forming a sandwich structure with the gonococcal antigen sandwiched in the middle through an immune reaction between the gonococcal antigen in the sample and the capture and detection antibodies. The gonococcal antigen is the ribosomal protein L7 / L12 of Neisseria gonorrhoeae. One of the capture antibody and the detection antibody is a first monoclonal antibody or a derivative thereof that produces an antigen-antibody reaction with an epitope containing one or more amino acid residues from the 2nd to the 14th amino acid residues from the N-terminus of the amino acid sequence selected from the L7 / L12 of the gonococcus shown in sequence number 1. The other of the capture antibody and the detection antibody is a second monoclonal antibody or its derivative that produces an antigen-antibody reaction with an epitope containing one or more amino acid residues from the N-terminus of the amino acid sequence selected from the L7 / L12 of the gonococcus shown in sequence number 1. The heavy chain variable region of the first monoclonal antibody is the amino acid sequence number 9, and the light chain variable region is the amino acid sequence number 10. Alternatively, the heavy chain variable region of the first monoclonal antibody is the amino acid sequence number 11, and the light chain variable region is the amino acid sequence number 12. and, The heavy chain variable region of the second monoclonal antibody is the amino acid sequence number 7, and the light chain variable region is the amino acid sequence number 8. The first monoclonal antibody or a derivative of the second monoclonal antibody is selected from: antibodies having two or more Fc regions in each molecule, glycan-modified antibodies, bispecific antibodies, antibody-drug conjugates formed by the combination of an antibody and a protein other than an antibody, antibody enzymes, tandem scFvs, and double-chain antibodies.

2. The kit according to claim 1, wherein, The antibody used for capture is the first monoclonal antibody, and the antibody used for detection is the second monoclonal antibody.

3. The kit according to claim 1, wherein, The antibody used for detection is the first monoclonal antibody, and the antibody used for capture is the second monoclonal antibody.

4. A kit for detecting gonococci in a sample, wherein, This kit contains: Antibodies used to capture gonococcal antigens in samples, and An antibody for detection, containing a marker for labeling gonococcal antigens in a sample. Gonorrhea is detected by forming a sandwich structure with gonococcal antigens sandwiched in between, through an immune reaction between the capture antibody and the detection antibody. The gonococcal antigen is the ribosomal protein L7 / L12 of Neisseria gonorrhoeae. The capture antibody and the detection antibody are selected from a combination of the following (1) or (2): (1) The antibody used for capture is a or b below, and the antibody used for detection is a combination of c below; (2) The detection antibody is a or b below, and the capture antibody is a combination of c below. a. Contains the amino acid sequence numbered 11, which is the variable region of the heavy chain, and An antibody representing the amino acid sequence of sequence number 12 of the light chain variable region; b. Contains the amino acid sequence that is sequence number 9, which is the variable region of the heavy chain, and An antibody representing the amino acid sequence of sequence number 10 in the light chain variable region; c. The amino acid sequence containing sequence number 7, which is the variable region of the heavy chain, and An antibody representing the amino acid sequence of sequence number 8 of the light chain variable region.

5. The kit according to any one of claims 1 to 4, wherein, The detection performance against gonococcal antigens is more than 10 times higher than that against Neisseria meningitidis antigens.

6. The kit according to any one of claims 1 to 4 does not cross-react with Mycoplasma, Escherichia, Chlamydia, Pseudomonas, Staphylococcus, Legionella, or Streptococcus in the sample.

7. The kit according to any one of claims 1 to 4, wherein, The samples are derived from oral fluids or urine from mammals.

8. The kit according to claim 7, wherein, The sample was taken from the urine of mammals.

9. The kit according to any one of claims 1 to 4, wherein, The kit further includes a carrier for developing the sample and bringing the sample into contact with the detection antibody. The carrier has a detection area on which the capture antibody is fixed.

10. The kit according to claim 9, wherein, The kit is an immunochromatographic kit and further includes a binding pad with an antibody attached for detection.

11. The kit according to claim 4, wherein, The capture antibody and the detection antibody are selected from a combination of the following (1) or (2): (1) The combination of antibody a for capture and antibody c for detection; (2) The detection antibody is a combination of the antibody described above and the capture antibody is c described above.

12. The use of one of the first monoclonal antibody and the second monoclonal antibody in the kit according to any one of claims 1 to 11 as a capture antibody and the other as a detection antibody in the preparation of a kit for detecting gonococci in a sample, wherein: The capture antibody is used to capture gonococcal antigens in the sample, and the detection antibody has a detection label and is used to label the gonococcal antigens in the sample.

13. The application as described in claim 12, wherein, The kit is used to first contact the sample with the detection antibody to generate an antigen-antibody reaction that labels the gonococcal antigen in the sample, and then contact it with the capture antibody to capture the gonococcal antigen in the sample through an antigen-antibody reaction between the capture antibody and the gonococcal antigen-detection antibody complex.

14. The application as described in claim 12, wherein, The kit is used to first contact the sample with a capture antibody, thereby capturing the gonococcal antigen in the sample by reacting the capture antibody with the antigen-antibody complex of the gonococcal antigen in the sample. Then, the sample is contacted with a detection antibody, thereby labeling the gonococcal antigen in the sample by reacting the detection antibody with the antigen-antibody complex of the gonococcal antigen and the capture antibody.

15. The application as described in any one of claims 12 to 14, wherein, The kit further includes a lysing agent for lysing bacteria in the sample before the detection antibody and / or capture antibody come into contact with the sample. The lysin is selected from one or more of the following groups: nonionic surfactants, amphoteric surfactants, anionic surfactants, lysozyme, lysococcal enzyme, pepsin, glucosidase, galactosidase, colorless peptidase, and β-N-acetylglucosidase.

16. The application according to any one of claims 12 to 14, wherein the detection limit of the kit for gonococci in the sample is below 5e4cfu / mL.

17. A method of manufacturing the kit of claim 9, comprising the step of immobilizing a capture antibody on a detection region of a carrier having a detection region.

18. A method of manufacturing the kit of claim 10, comprising the following steps: The process of immobilizing the capture antibody onto the detection region of a carrier having a detection region; The process of attaching the antibody for detection to the conjugate pad; and The process of placing the bonding pad upstream of the detection area of ​​the carrier.

19. The method of claim 17, wherein, The method further includes the step of preparing a detection antibody by adding a detection label to one of a first monoclonal antibody and a second monoclonal antibody, and One of the first and second monoclonal antibodies is used as the capture antibody and immobilized in the detection area of ​​the vector.

20. The method of claim 19, wherein, The first monoclonal antibody is the first monoclonal antibody according to any one of claims 1 to 3, and The method further includes the following steps: inoculating an animal with an epitope polypeptide, obtaining an antiserum containing an antibody that reacts with the epitope polypeptide to produce an antigen-antibody reaction, purifying and fractionating the antibody from the antiserum to obtain a first monoclonal antibody, wherein the epitope polypeptide has the amino acid sequence of L7 / L12 of gonococcus as indicated by sequence number 1.

21. The method of claim 19, wherein, The second monoclonal antibody is the second monoclonal antibody according to any one of claims 1 to 3, and The method further includes the following steps: inoculating an animal with an epitope polypeptide, obtaining an antiserum containing an antibody that reacts with the epitope polypeptide to produce an antigen-antibody reaction, purifying and fractionating the antibody from the antiserum to obtain a second monoclonal antibody, wherein the epitope polypeptide has the amino acid sequence of L7 / L12 of gonococcus as shown in sequence number 1.

22. The method of claim 18, wherein, The method further includes the step of preparing a detection antibody by adding a detection label to one of a first monoclonal antibody and a second monoclonal antibody, and One of the first and second monoclonal antibodies is used as the capture antibody and immobilized in the detection area of ​​the vector.

23. The method of claim 22, wherein, The first monoclonal antibody is the first monoclonal antibody according to any one of claims 1 to 3, and The method further includes the following steps: inoculating an animal with an epitope polypeptide, obtaining an antiserum containing an antibody that reacts with the epitope polypeptide to produce an antigen-antibody reaction, purifying and fractionating the antibody from the antiserum to obtain a first monoclonal antibody, wherein the epitope polypeptide has the amino acid sequence of L7 / L12 of gonococcus as indicated by sequence number 1.

24. The method of claim 22, wherein, The second monoclonal antibody is the second monoclonal antibody according to any one of claims 1 to 3, and The method further includes the following steps: inoculating an animal with an epitope polypeptide, obtaining an antiserum containing an antibody that reacts with the epitope polypeptide to produce an antigen-antibody reaction, purifying and fractionating the antibody from the antiserum to obtain a second monoclonal antibody, wherein the epitope polypeptide has the amino acid sequence of L7 / L12 of gonococcus as shown in sequence number 1.

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