Immobilized polypeptide, kit, and method for detecting Anti-gingipain antibody

Abstract

To provide a new solid-phase polypeptide capable of detecting an anti-gingipain antibody with high sensitivity.SOLUTION: A solid-phased polypeptide for detecting an anti-gingipain antibody, comprising an insoluble particle and a polypeptide supported on the insoluble particle, wherein the polypeptide is at least one selected from the group consisting of (a) a polypeptide comprising a region consisting of an amino acid sequence of 5 to 30 amino acid residues contained in the amino acid sequence of SEQ ID NO: 1, and (b) a polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted in the region.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an immobilized polypeptide, a kit, and a method for detecting anti-gingipain antibodies, and more particularly to an immobilized polypeptide for detecting anti-gingipain antibodies, a kit for detecting anti-gingipain antibodies, and a method for detecting anti-gingipain antibodies using these. [Background technology]

[0002] Gingipains are potent cysteine ​​proteases with trypsin-like protease activity that are produced and secreted on the cell surface and extracellularly by the gram-negative, obligately anaerobic bacterium Porphyromonas gingivalis. Two types of gingipains are known: Arg-gingipain (Rgp), which cleaves proteins at the C-terminus of arginine residues, and Lys-gingipain (Kgp), which cleaves proteins at the C-terminus of lysine residues. Gingipains primarily induce cell death by eliminating the adhesive properties of gingival fibroblasts and vascular endothelial cells, and are therefore thought to be responsible for the majority of the pathogenicity of Porphyromonas gingivalis, as described below.

[0003] P. gingivalis is considered to be the pathogenic bacterium most closely involved in the onset and progression of periodontal disease, and is also known to be involved in the onset and exacerbation of systemic diseases such as diabetes, Alzheimer's disease, and cardiovascular disease. Therefore, there is an increasing demand for tests that detect the presence or absence of P. gingivalis infection in patients (subjects) in the diagnosis of systemic diseases, in addition to the diagnosis of periodontal disease.

[0004] As a method for detecting the presence or absence of P. gingivalis infection in a subject, for example, a method for indirectly detecting the presence or absence of P. gingivalis infection by detecting the presence or absence of antibodies produced by the subject against P. gingivalis in a sample collected from the subject has been developed. For example, Japanese Patent Laid-Open Publication No. 2-107969 (Patent Document 1) describes a method for detecting antibodies against P. gingivalis fimbria antigens using a diagnostic reagent for periodontal disease consisting of latex particles sensitized with the fimbria antigens. Regarding the fimbria antigens, F. Yoshimura et al., J. Bacteriol. 1984 Dec; 160(3): pp. 949-57, doi:10.1128 / jb.160.3.949-957.1984 (Non-Patent Document 1) discloses a method for homogenizing and characterizing fimbria antigens of the oral anaerobe Bacteroides gingivalis.

[0005] In addition, a method for detecting the presence or absence of anti-gingipain antibodies produced by a subject has also been developed using an antigen derived from the above-mentioned gingipain, which is the main protease produced and secreted by Porphyromonas gingivalis. For example, Kimito Hirai et al., Frontiers in Immunology, June 2020, Volume 11, Article 1017 (Non-Patent Document 2) describes the use of the N-terminal region of gingipain Rgp (RgpA) as an antigen polypeptide to detect the presence or absence of Porphyromonas gingivalis infection, and JP 2022-96815 A (Patent Document 2) describes a method for measuring antibody titers against periodontal pathogens by adding a GST tag to the antigen polypeptide.

[0006] Another method for detecting the presence or absence of P. gingivalis infection in a subject has been developed, for example, by performing nucleic acid amplification targeting the genomic nucleic acid of P. gingivalis using a specimen collected from the subject as a sample. For example, Stephen S. Dominy et al., Science Advances, 2019;5, eaau3333 (Non-Patent Document 3) describes a method for measuring P. gingivalis using a quantitative PCR method from a nucleic acid sample purified from saliva. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 2-107969 [Patent Document 2] Japanese Patent Publication No. 2022-96815 [Non-patent literature]

[0008] [Non-Patent Document 1] F Yoshimura et al.,J Bacteriol.1984 Dec;160(3):p.949-57,doi:10.1128 / jb.160.3.949-957.1984 [Non-patent document 2] Kimito Hirai et al.,Frontiers in immunology,June 2020,Volume 11,Article 1017 [Non-patent document 3] Stephen S. Dominy et al.,Science Advances,2019;5,eaau3333 Summary of the Invention [Problem to be solved by the invention]

[0009] As mentioned above, there is an increasing demand for tests to detect whether or not a subject is infected with P. gingivalis, but the development and investigation of such methods has not yet been sufficient.

[0010] The present invention has been made in consideration of the problems associated with the above-mentioned conventional technology, and aims to provide a new immobilized polypeptide and kit capable of detecting anti-gingipain antibodies with high sensitivity, and a method for detecting anti-gingipain antibodies using these. [Means for solving the problem]

[0011] The present inventors have conducted extensive research to achieve the above-mentioned object and have newly developed an immobilized polypeptide in which a specific polypeptide (antigen polypeptide) having antigenicity against anti-gingipain antibodies is supported on insoluble particles. The polypeptide has excellent reactivity with anti-gingipain antibodies, enabling highly sensitive detection of the antibodies. Furthermore, by using particles (insoluble particles) as the insoluble carrier for immobilizing the polypeptide, it is possible to automate detection, achieve high throughput, and continuously measure a large number of samples. In addition, the present inventors have discovered that combining the specific antigen polypeptide with insoluble particles (particularly latex particles) enables more convenient detection of anti-gingipain antibodies and enables the determination of the presence or absence of P. gingivalis infection with particularly high accuracy that cannot be expected when other antigens are combined with insoluble particles, thereby completing the present invention.

[0012] That is, the present invention relates to an immobilized polypeptide, a kit, and a method for detecting an anti-gingipain antibody, and more specifically provides the following. [1] a solid-phase polypeptide for detecting anti-gingipain antibodies, The composition comprises insoluble particles and a polypeptide supported on the insoluble particles, The polypeptide is selected from the following (a) to (b): (a) a polypeptide comprising a region consisting of an amino acid sequence of 5 to 30 amino acid residues contained in the amino acid sequence of SEQ ID NO: 1 (b) a polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, and / or inserted in the region; At least one selected from the group consisting of Immobilized polypeptide. [2] The polypeptide is selected from the following (a') to (b'): (a') A polypeptide comprising a region consisting of the amino acid sequence of positions 504 to 530 and / or a region consisting of the amino acid sequence of positions 698 to 718 of the amino acid sequence set forth in SEQ ID NO: 1. (b') A polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted at positions 504 to 530 of the amino acid sequence set forth in SEQ ID NO: 1, and / or a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted at positions 698 to 718. At least one selected from the group consisting of [1] The immobilized polypeptide described in [1]. [3] The immobilized polypeptide according to [1] or [2], wherein the insoluble particles are latex particles. [4] The immobilized polypeptide according to any one of [1] to [3], wherein the particle diameter of the insoluble particles is 200 nm or more. [5] The immobilized polypeptide according to any one of [1] to [4], wherein the insoluble particle and the polypeptide are covalently bound. [6] The immobilized polypeptide according to any one of [1] to [5], wherein the insoluble particles are blocked with caseinate. [7] A kit for detecting anti-gingipain antibodies in a sample, [1] to [6], comprising the immobilized polypeptide according to any one of [1] to [6]. kit. [8] A method for detecting anti-gingipain antibodies in a sample, a detection step of contacting a sample with an immobilized polypeptide, allowing the immobilized polypeptide to bind to anti-gingipain antibodies in the sample, and detecting anti-gingipain antibodies, the immobilized polypeptide comprises insoluble particles and a polypeptide supported on the insoluble particles; The polypeptide is selected from the following (a) to (b): (a) a polypeptide comprising a region consisting of an amino acid sequence of 5 to 30 amino acid residues contained in the amino acid sequence of SEQ ID NO: 1 (b) a polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, and / or inserted in the region; At least one selected from the group consisting of Method for detecting anti-gingipain antibodies. [9] The polypeptide is selected from the following (a') to (b'): (a') A polypeptide comprising a region consisting of the amino acid sequence of positions 504 to 530 and / or a region consisting of the amino acid sequence of positions 698 to 718 of the amino acid sequence set forth in SEQ ID NO: 1. (b') A polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted at positions 504 to 530 of the amino acid sequence set forth in SEQ ID NO: 1, and / or a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted at positions 698 to 718. At least one selected from the group consisting of [8] The method for detecting an anti-gingipain antibody according to [8].

[10] The anti-gingipain antibody detection method according to [8] or [9], wherein the insoluble particles are latex particles, and the detection is detection of agglutination of the latex particles.

[11] The method for detecting an anti-gingipain antibody according to any one of [8] to

[10] , wherein the particle diameter of the insoluble particles is 200 nm or more.

[12] The method for detecting an anti-gingipain antibody according to any one of [8] to

[11] , wherein the insoluble particle and the polypeptide are covalently bound.

[13] The method for detecting an anti-gingipain antibody according to any one of [8] to

[12] , wherein the insoluble particles of the immobilized polypeptide are blocked with caseinate.

[14] The method for detecting an anti-gingipain antibody according to any one of [8] to

[13] , which is a method for assisting in the diagnosis of the presence or absence of Porphyromonas gingivalis infection in a subject from whom the sample is derived.

[15] The anti-gingipain antibody detection method according to

[14] , further comprising a quantification step of determining the amount of anti-gingipain antibody in the subject from the detection value of the anti-gingipain antibody detected in the detection step.

[16] detecting anti-gingipain antibodies from a sample collected from a subject using the anti-gingipain antibody detection method according to any one of [8] to

[15] ; determining whether or not the subject is infected with Porphyromonas gingivalis using the presence or absence or amount of the anti-gingipain antibody as an indicator; A method for determining whether or not a subject is infected with Porphyromonas gingivalis, comprising: [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a novel immobilized polypeptide and kit capable of detecting anti-gingipain antibodies with high sensitivity, and a method for detecting anti-gingipain antibodies using the same. The immobilized polypeptide, kit, and method for detecting anti-gingipain antibodies of the present invention make it possible to determine the presence or absence of infection with Porphyromonas gingivalis with particularly high accuracy. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a graph showing the logarithmic values ​​of antibody titers obtained by latex particle agglutination assay for samples determined to be negative or positive based on antibody titers obtained by ELISA, obtained in Test Example 1. [Figure 2] 1 is a graph showing the absorbance difference (ΔOD×10,000) detected and calculated for negative and positive samples using antigen polypeptides immobilized on latex particles of various particle sizes, obtained in Test Example 2. [Figure 3] 1 is a graph showing the relationship between the amount of polypeptide (amount per unit area of ​​the latex particle surface [mg / m2]) used in the preparation of the latex particle-immobilized antigen polypeptide (horizontal axis) and the absorbance difference (ΔOD × 10,000) (vertical axis) detected and calculated using the same latex particle-immobilized antigen polypeptide, obtained in Test Example 3. [Figure 4] 1 is a graph showing the absorbance difference (ΔOD) detected and calculated using latex particle-immobilized antigen polypeptide (Y) blocked with each blocking agent (BSA, casein sodium), obtained in Test Example 4, and the absorbance difference (ΔOD) detected and calculated using particles (N) blocked with each blocking agent (BSA, casein sodium) but not sensitized with the antigen polypeptide. [Figure 5] FIG. 1 is a plot showing the relationship between the antibody titer [U / mL] (horizontal axis) obtained by ELISA in Test Example 5 and the absorbance difference (ΔOD×10,000) (vertical axis) detected and calculated using an antigen polypeptide immobilized on latex particles in which the antigen polypeptide is physically adsorbed to the surface of latex particles (physical adsorption) and an antigen polypeptide immobilized on latex particles in which the antigen polypeptide is chemically bonded (covalently bonded) to the surface of latex particles (covalent bond). [Figure 6] This is an ROC curve obtained in Test Example 6, with the presence or absence of detection of Porphyromonas gingivalis DNA in saliva (non-detected (negative): 0, detected (positive): 1) as the dependent variable and the antibody titer measured and calculated by latex particle agglutination assay as the explanatory variable. [Figure 7] 1 is a graph showing the relative absorbance values ​​(absorbance in healthy samples versus antigen polypeptide (10 ng / well)) measured for each sample (healthy sample, serum sample (R1 to R4) from a patient with periodontal disease) using a plate on which antigen polypeptide (10 ng / well) was immobilized in Test Example 8(2). [Figure 8]This is a graph showing the relative absorbance values ​​(absorbance in healthy samples with pilus antigen (10 ng / well)) measured for each sample (healthy sample, serum sample from periodontal disease patient (R1 to R4)) in Test Example 8(2) using a plate on which pilus antigen (10 ng / well, 100 ng / well) was solidified. [Figure 9] This is an ROC curve obtained in Test Example 8(3), using the presence or absence of periodontal disease (healthy specimen: 0, periodontal disease specimen: 1) as the dependent variable and the antibody titer measured and calculated by the latex particle agglutination method using an antigen polypeptide immobilized on latex particles as the explanatory variable. [Figure 10] This is an ROC curve obtained in Test Example 8(3), with the presence or absence of periodontal disease (healthy specimen: 0, periodontal disease specimen: 1) as the objective variable and the antibody titer measured and calculated by latex particle agglutination using latex particle-immobilized pilus antigen as the explanatory variable. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below based on preferred embodiments thereof.

[0016] <Solid-phase polypeptide for detecting anti-gingipain antibodies> The present invention relates to an immobilized polypeptide for detecting anti-gingipain antibodies, The composition comprises insoluble particles and a polypeptide supported on the insoluble particles, The polypeptide is selected from the following (a) to (b): (a) a polypeptide comprising a region consisting of an amino acid sequence of 5 to 30 amino acid residues contained in the amino acid sequence of SEQ ID NO: 1 (b) a polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, and / or inserted in the region; At least one selected from the group consisting of An immobilized polypeptide is provided.

[0017] [Anti-gingipain antibody] "Gingipain" is a cysteine ​​protease with trypsin-like protease activity, one of the potent proteases produced and secreted on the cell surface and extracellularly by the gram-negative, obligately anaerobic bacterium Porphyromonas gingivalis. There are two types of gingipain: arginine-specific cysteine ​​protease (Arg-gingipain (Rgp)), which cleaves the C-terminal side of arginine residues in proteins, and lysine-specific cysteine ​​protease (Lys-gingipain (Kgp)), which cleaves the C-terminal side of lysine residues. The gingipain of the present invention is Rgp. Porphyromonas gingivalis is a pathogenic bacterium involved in the onset and progression of not only periodontal disease but also systemic diseases (periodontal disease-related diseases) such as diabetes, Alzheimer's disease, and cardiovascular disease.

[0018] An "anti-gingipain antibody" is an antibody produced in an organism infected with Porphyromonas gingivalis using gingipain as an antigen, and the anti-gingipain antibody of the present invention is an antibody that specifically binds to Rgp. However, the anti-gingipain antibody of the present invention is not limited to this and includes antibodies that specifically bind to the Rgp of gingipain, and also includes, for example, antibodies produced by a conventionally known method or a method similar thereto (such as the hybridoma method or recombinant DNA method) using gingipain Rgp as an immunogen.

[0019] In the present invention, the term "antibody" includes not only complete antibodies but also functional fragments thereof (such as Fab, F(ab')2, Fab', variable region fragments (Fv), disulfide-linked Fv, single-chain Fv (scFv), sc(Fv)2, diabodies, and polymers thereof). Furthermore, in the present invention, the term "antibody" includes all classes and subclasses of immunoglobulins, and also includes polyclonal antibodies and monoclonal antibodies. The origin, type, shape, etc. of the antibody are not particularly limited. Specific examples include human-derived antibodies, non-human animal-derived antibodies (such as rabbit antibodies, mouse antibodies, rat antibodies, camel antibodies, and chicken antibodies), chimeric antibodies, humanized antibodies, and functional fragments of these antibodies.

[0020] When the immobilized polypeptide of the present invention is used in the diagnostic method or diagnostic auxiliary method described below, the anti-gingipain antibody of the present invention is an antibody produced in an organism (subject) infected with the above-mentioned Porphyromonas gingivalis.

[0021] [Polypeptide] The polypeptide contained in the immobilized polypeptide of the present invention is an antigen polypeptide that exhibits reactivity with the anti-gingipain antibody, and more specifically, (a) a polypeptide comprising a region consisting of an amino acid sequence of 5 to 30 amino acid residues contained in the amino acid sequence set forth in SEQ ID NO: 1 (sometimes referred to herein as "polypeptide (a)"); The amino acid sequence of SEQ ID NO: 1 is a sequence consisting of 860 amino acids in the N-terminal region of arginine-specific cysteine ​​protease (Rgp) of the gingipains of Porphyromonas gingivalis. The anti-gingipain antibodies to be detected in the present invention are likely to be produced in vivo as antibodies that specifically bind to this Rgp. The region recognized by such an anti-gingipain antibody may be located anywhere in the amino acid sequence of SEQ ID NO: 1, but the length of each region should be at least 5 amino acid residues, preferably 5 to 30 amino acid residues, and more preferably 10 to 30 amino acid residues. The polypeptide (a) of the present invention containing such a region preferably has a reactivity equivalent to that of the following polypeptide (a'').

[0022] Examples of regions recognized by the anti-gingipain antibody include a region consisting of 27 amino acid residues at positions 504 to 530 of the amino acid sequence set forth in SEQ ID NO: 1, a region consisting of 21 amino acid residues at positions 698 to 718 of the amino acid sequence, a region consisting of 15 amino acid residues at positions 96 to 110 of the amino acid sequence, a region consisting of 15 amino acid residues at positions 222 to 236 of the amino acid sequence, a region consisting of 12 amino acid residues at positions 327 to 338 of the amino acid sequence, a region consisting of 15 amino acid residues at positions 471 to 485 of the amino acid sequence, a region consisting of 18 amino acid residues at positions 576 to 593 of the amino acid sequence, and a region consisting of 21 amino acid residues at positions 687 to 707 of the amino acid sequence.

[0023] The polypeptide (a) according to the present invention includes, among these, (a'1) A polypeptide comprising a region consisting of the 21 amino acid residues from positions 698 to 718 of the amino acid sequence set forth in SEQ ID NO: 1 (the amino acid sequence set forth in SEQ ID NO: 2) (sometimes referred to herein as "polypeptide (a'1)"). It is particularly preferable that the polypeptide (a'1) is one of the following: Such polypeptide (a'1) has superior reactivity to anti-gingipain antibodies and also suppresses non-specific reactions, enabling detection with high sensitivity and accuracy (Patent Document 2). In addition, the polypeptide (a) according to the present invention may be: (a'2) A polypeptide comprising a region consisting of the amino acid sequence of 27 amino acid residues from positions 504 to 530 of the amino acid sequence set forth in SEQ ID NO: 1 (the amino acid sequence set forth in SEQ ID NO: 3) (sometimes referred to herein as "polypeptide (a'2)"). Such polypeptide (a'1) and polypeptide (a'2) (sometimes collectively referred to as "polypeptide (a')") enable more sensitive and accurate diagnosis of the presence or absence of P. gingivalis infection in a subject (i.e., positive / negative determination) when the immobilized polypeptide of the present invention is used in the diagnostic method or diagnostic auxiliary method described below.

[0024] The polypeptide (a) of the present invention may contain at least one region consisting of the amino acid sequence of 5 to 30 amino acid residues described above (sometimes referred to herein as "region a") (more preferably, a region consisting of the amino acid sequence shown in SEQ ID NO: 2 of polypeptide (a'1) (sometimes referred to herein as "region a'1") or a region consisting of the amino acid sequence shown in SEQ ID NO: 3 of polypeptide (a'2) (sometimes referred to herein as "region a'2"), even more preferably region a'1), and may contain multiple (e.g., 2 to 10) regions a which may be the same or different from each other, and may have one or more amino acids added to the N-terminal and / or C-terminal sides of these regions (and between the regions if there are multiple regions a). With regard to the polypeptide of the present invention, the number of added amino acids when "one or more amino acids are added" may be, for example, 1 to 900 amino acids in total on the N-terminal and C-terminal sides (and between the regions if there are multiple regions a), and is preferably 1 to 850 amino acids, 1 to 620 amino acids, 1 to 450 amino acids, or 1 to 260 amino acids.

[0025] Typical examples of such polypeptides having multiple amino acids added to region a, preferably region a'1 or region a'2, include: (a'') A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 (sometimes referred to herein as "polypeptide (a'')"). Other examples include polypeptides that include region a, preferably region a'1 or region a'2, and that consist of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1; and polypeptides that include region a, preferably region a'1 or region a'2, and that consist of an amino acid sequence that is 80% or more homologous to the amino acid sequence set forth in SEQ ID NO: 1. When referring to these polypeptides and the phrase "one or more amino acids have been substituted, deleted, added, and / or inserted," the term "multiple" refers to preferably 25 amino acids or less, more preferably 20 amino acids or less, even more preferably 10 amino acids or less, 5 amino acids or less, or 3 amino acids or less (e.g., 2 amino acids or less). The homology of these polypeptides should be at least 80%, but is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more). More preferably, the identity is at least 80%, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more). The homology and identity of amino acid sequences can be determined using the BLASTP (amino acid level) program (Altschul et al. J. Mol. Biol., 215:403-410, 1990, parameters: default) or the like.

[0026] Furthermore, the polypeptide of the present invention may be not only the above-mentioned polypeptide (a), but also a polypeptide whose amino acid sequence has been modified without reducing the desired antigenicity (reactivity to anti-gingipain antibodies). Modifications of the amino acid sequence include, for example, substitution, deletion, and / or insertion of one or several amino acids within the amino acid sequence. More specifically, the polypeptide of the present invention includes: (b) a polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted in region a and which has the same reactivity as polypeptide (a), preferably the same reactivity as polypeptide (a'') (sometimes referred to as "polypeptide (b)" in this specification).

[0027] Furthermore, the polypeptide (b) according to the present invention includes: (b'1) A polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted at positions 698 to 718 of the amino acid sequence set forth in SEQ ID NO: 1. and which has the same reactivity as polypeptide (a), more preferably polypeptide (a'') (sometimes referred to as "polypeptide (b'1)" in this specification). Furthermore, examples of the polypeptide (b) according to the present invention include: (b'2) A polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted at positions 504 to 530 of the amino acid sequence set forth in SEQ ID NO: 1. and which has the same reactivity as polypeptide (a), more preferably polypeptide (a'') (sometimes referred to as "polypeptide (b'2)" in this specification).

[0028] With regard to polypeptide (b) of the present invention, the region consisting of a modified sequence of the above-mentioned region a (sometimes referred to herein as "region b"), more preferably the region consisting of a modified sequence of the amino acid sequence shown in SEQ ID NO: 2 of polypeptide (b'1) (sometimes referred to herein as "region b'1") or the region consisting of a modified sequence of the amino acid sequence shown in SEQ ID NO: 3 of polypeptide (b'2) (sometimes referred to herein as "region b'2"), and even more preferably with regard to region b'1, when it is said that "one or several amino acids have been substituted, deleted, and / or inserted," "several" preferably means five amino acids or less, more preferably three amino acids or less (e.g., two amino acids or less).

[0029] The amino acid sequence modification is preferably a conservative substitution. In the present invention, "conservative substitution" refers to a substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the art to which the present invention pertains. For example, acidic amino acids (aspartic acid and glutamic acid); basic amino acids (lysine, arginine, histidine); and neutral amino acids can be classified into amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), amino acids with hydroxyl groups (serine, threonine), sulfur-containing amino acids (cysteine, methionine), amino acids with amide groups (asparagine, glutamine), amino acids with imino groups (proline), and amino acids with aromatic groups (phenylalanine, tyrosine, tryptophan).

[0030] Furthermore, "having equivalent reactivity" means that the reactivity with an anti-gingipain antibody is equivalent (for example, 70% or more, preferably 80% or more, more preferably 90% or more) to that of, for example, a control polypeptide (polypeptide (a), preferably polypeptide (a'')). When a polypeptide is "reactive" with an antibody, it means that the polypeptide and the antibody exhibit binding activity (affinity) and / or specificity. Those skilled in the art can evaluate the reactivity using known immunological techniques or methods similar thereto, for example, by the method shown in the Examples below. That is, a latex particle-immobilized polypeptide in which a polypeptide to be evaluated is immobilized on latex particles, and a latex particle-immobilized polypeptide in which the control polypeptide is similarly immobilized on latex particles are prepared, and a sample containing an anti-gingipain antibody (a sample in which anti-gingipain antibody is detected by detection with the control polypeptide, preferably, for example, a sample in which, by detection with the control polypeptide, the difference (ΔOD) × 10,000 between the absorbance at a wavelength of 660 nm 5 minutes after contact with the latex particle-immobilized polypeptide at 37°C and the absorbance at a wavelength of 660 nm immediately after the contact (0 minutes after reaction) is 100 or more) is measured by the latex particle agglutination method. When the detection value when the target polypeptide is used is equivalent (for example, 70% or more, preferably 80% or more, more preferably 90% or more) to the detection value when the control polypeptide is used, the polypeptide can be evaluated as "reactive" or "having equivalent reactivity."

[0031] The polypeptide (b) of the present invention may contain at least one of the above-mentioned regions b (more preferably, region b'1 or region b'2, and even more preferably, region b'1), or may contain multiple regions b (e.g., 2 to 10) which may be the same or different from each other, and may have one or more amino acids added to the N-terminus and / or C-terminus of these regions (and between the regions when there are multiple regions b). In this case, the number of amino acids to be added, including the preferred range, is as described for the polypeptide (a) above.

[0032] Typical examples of such polypeptides having multiple amino acids added to region b, preferably region b'1 or region b'2, include: (b'') A polypeptide in which region a, preferably region a'1 or region a'2 in the amino acid sequence set forth in SEQ ID NO: 1, has been converted into a region consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, and / or inserted (i.e., region b, preferably region b'1 or region b'2) (sometimes referred to herein as "polypeptide (b'')"). Other examples include polypeptides that include region b, preferably region b'1 or region b'2, and that consist of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1; and polypeptides that include region b, preferably region b'1 or region b'2, and that consist of an amino acid sequence that has 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 1. With regard to these polypeptides, the term "multiple" and the homology used when referring to "one or more amino acids have been substituted, deleted, added, and / or inserted" are as described above for polypeptide (a), including the preferred ranges.

[0033] The full length of such a polypeptide according to the present invention (the length as an antigenic polypeptide, not including the functional molecules described below) is, for example, preferably 5 to 1,100 amino acid residues, and more preferably 10 to 900 amino acid residues.

[0034] Furthermore, in addition to the additional amino acids, the polypeptide of the present invention may further contain functional molecules, if necessary. Examples of such functional molecules include tags for facilitating purification (e.g., HN tag, His tag, streptavidin binding peptide (SBP) tag, FLAG tag, maltose binding protein (MBP) tag, glutathione-S-transferase (GST) tag (a typical amino acid sequence of a GST tag is shown in SEQ ID NO: 4), solubilization tags, and secretion-promoting tags), and reporter proteins for facilitating detection (e.g., fluorescent proteins such as green fluorescent protein (GFP), and chemiluminescent proteins such as luciferase). These may be used alone or in combination of two or more of these, but are not limited thereto. When such functional molecules are added, they can be added, for example, to the N-terminus and / or C-terminus of the polypeptide, and addition to the N-terminus is more preferred.

[0035] The polypeptides of the present invention can be produced by conventionally known methods or methods similar thereto. For example, they can be obtained by artificial synthesis (liquid phase method, solid phase method, etc.) based on each of the above amino acid sequences, or they can be obtained as recombinant polypeptides by genetic engineering techniques, for example, by preparing a polynucleotide sequence encoding each of the above amino acid sequences or a vector containing the same, and introducing it into a host cell for expression.

[0036] [Insoluble particles] In the present invention, the "insoluble particles" are particles that are insoluble in water and function mainly as carriers that support and immobilize the polypeptide.

[0037] The material of such insoluble particles can be any of those used as insoluble carriers in known immunoassays, and examples thereof include at least one selected from the group consisting of high molecular weight polymers (polystyrene, polyimide, nylon, rubber, etc.), gelatin, cellulose, nitrocellulose, glass, silica, metals (gold, platinum, etc.), and metal compounds (iron oxide, etc.), and composites thereof (e.g., organic-inorganic composites of organic polymers such as the high molecular weight polymers and metal compounds such as iron oxide) may also be used. Among these, the insoluble particles according to the present invention are preferably metal particles (e.g., colloidal particles of the above metals) or latex particles of the above high molecular weight polymers, and particularly preferably latex particles, because of the advantages that anti-gingipain antibodies can be easily detected by an agglutination method, the detection can be easily automated, and multiple analytes can be easily detected simultaneously.

[0038] Furthermore, the insoluble particles (preferably latex particles) of the present invention, particularly the insoluble particles used in the method for producing the immobilized polypeptide of the present invention, may preferably have at least one functional group selected from a carboxyl group, a hydroxyl group, an amino group, an aldehyde group, and a tosyl group on their surface.

[0039] In the present invention, the particle size of the insoluble particles is not particularly limited and can be, for example, 50 to 500 nm, but is preferably 70 nm or more, more preferably 100 nm or more, even more preferably 150 nm or more, even more preferably 200 nm or more, and particularly preferably 250 nm or more. For example, the particle size is preferably 70 to 450 nm, more preferably 100 to 450 nm, even more preferably 150 to 450 nm, even more preferably 200 to 420 nm, and particularly preferably 250 to 420 nm. If the particle size exceeds the upper limit, self-aggregation of the particles tends to progress and the specificity of the immobilized polypeptide for anti-gingipain antibodies tends to decrease. On the other hand, if the particle size is below the lower limit, the reactivity of the immobilized polypeptide for anti-gingipain antibodies and the detection sensitivity tend to decrease.

[0040] In the present invention, the particle size is an average particle size, and is measured, for example, by a laser diffraction / scattering particle size distribution measurement method, a dynamic light scattering particle size distribution measurement method (Dynamic Light Scattering: DLS), or an electron microscope (transmission electron microscope (TEM), atomic force microscope (AFM), scanning electron microscope (SEM), optical microscope). More specifically, for example, in measurement by DLS, a measurement sample is prepared by suspending the insoluble particles in a suitable solvent such as water to a concentration suitable for measurement, and dispersing the particles by ultrasonic treatment while stirring. The average particle size can be determined as the median diameter at a volume-based cumulative frequency of 50% in the particle size distribution obtained by measurement using a dynamic light scattering particle size distribution measurement device (for example, Zetasizer Nano, manufactured by Malvern Panalytical). Furthermore, for example, in measurements using an electron microscope, the insoluble particles are observed, for example, using a transmission electron microscope (TEM), and the particle diameters (or approximate circle equivalent diameters) of 600 particles or more are measured using analysis software, and the average value thereof can be used as the average particle diameter.

[0041] As such insoluble particles, conventionally known particles can be used as appropriate, and commercially available particles can also be used as appropriate.

[0042] [Immobilized polypeptide] The immobilized polypeptide of the present invention is a complex comprising the insoluble particle and the polypeptide supported on the insoluble particle, and is a conjugate in which the polypeptide is supported by being directly or indirectly bound to the insoluble particle.

[0043] In the immobilized polypeptide of the present invention, the amount of the polypeptide (amount supported) is not particularly limited and cannot be generalized because it is adjusted appropriately depending on the length, type, etc. of the polypeptide to be supported. For example, when the length of the polypeptide (however, if a functional molecule is added, the length of the functional molecule is not included) is 100 to 900 amino acid residues, the amount per unit area of ​​the particle surface of the insoluble particle is 0.1 to 5.0 mg / m 2In this case, the amount of support can be 2.5 mg / m 2 Preferably, it is 2.0 mg / m or less. 2 More preferably, it is 1.5 mg / m or less. 2 More preferably, it is 1.0 mg / m or less. 2 Even more preferably, it is 0.8 mg / m or less. 2 It is particularly preferable that the concentration is 0.1 to 2.5 mg / m or less. 2 is preferably 0.1 to 2.0 mg / m 2 More preferably, it is 0.1 to 1.5 mg / m 2 More preferably, it is 0.1 to 1.0 mg / m 2 It is even more preferable that the concentration is 0.2 to 0.8 mg / m 2 If the amount of loading is too large (exceeding the upper limit in the above case), the specificity of the immobilized polypeptide for anti-gingipain antibodies tends to decrease, particularly when the insoluble particles are latex particles and a latex particle agglutination method is employed as the detection method of the present invention, whereas if the amount of loading is too small (below the lower limit in the above case), the reactivity of the immobilized polypeptide for anti-gingipain antibodies and the detection sensitivity tend to decrease.

[0044] It is preferable that the surface of the insoluble particles of the immobilized polypeptide of the present invention is further blocked with a blocking agent (that is, further contains the blocking agent).

[0045] The blocking agent is not particularly limited, and any known blocking agent can be used as appropriate as long as it does not inhibit the reaction between the immobilized polypeptide and the anti-gingipain antibody. Examples of the blocking agent include milk proteins such as skim milk, casein and its salts, and purified milk protein products (e.g., Block Ace (manufactured by KAC Corporation)); other proteins such as BSA and gelatin; and water-soluble polymers. One of these may be used alone, or two or more may be used in combination. Among these, the blocking agent is preferably a milk protein, more preferably at least one selected from the group consisting of casein and its salts (caseinates), even more preferably caseinates, and even more preferably sodium caseinate.

[0046] The amount of the blocking agent used to block the surface of the insoluble particles of the immobilized polypeptide can be adjusted as appropriate, but is preferably 5 to 1,000 mg / m as the amount per unit area of ​​the particle surface of the insoluble particles. 2 is preferably 5 to 500 mg / m 2 More preferably, it is 10 to 250 mg / m 2 It is more preferable that:

[0047] The immobilized polypeptide of the present invention may also contain the following spacer molecule or the like.

[0048] [Method of producing immobilized polypeptide] The immobilized polypeptide of the present invention can be produced by binding the polypeptide to the insoluble particles and immobilizing it. As the production method, a conventionally known method or a method similar thereto can be appropriately adopted depending on the types of the insoluble particles and the polypeptide, and the polypeptide may be bound to the insoluble particles directly or indirectly.

[0049] Examples of methods for binding the insoluble particles and the polypeptide include a method of imparting functional groups (e.g., carboxyl, hydroxyl, amino, aldehyde, or tosyl groups) to the insoluble particles and / or the polypeptide, or a method of using insoluble particles and / or polypeptides having these functional groups and binding them by covalent bonding via the functional groups. The insoluble particles and / or polypeptides to which the functional groups have been imparted may be commercially available, or may be prepared by introducing the functional groups into the surfaces of insoluble particles and / or polypeptides under appropriate reaction conditions.

[0050] For example, if the functional groups on the surface of the insoluble particles are carboxyl, hydroxyl, or amino groups, the polypeptide can be covalently bonded to the surface of the insoluble particles by using an activator such as carbodiimide, cyanogen bromide, triazine, or glutaraldehyde. In this case, the functional groups on the surface of the insoluble particles may be activated in advance with the activator before contacting the insoluble particles with the polypeptide, or the activator and the polypeptide may be contacted with the insoluble particles simultaneously. Note that if the functional groups on the surface of the insoluble particles are aldehyde or tosyl groups, the activator is not necessary. Alternatively, the insoluble particles and the polypeptide can be indirectly covalently bonded via a spacer molecule such as an oligoamino acid or an aminocarboxylic acid.

[0051] The covalent bonding reaction can be carried out in an aqueous solvent (usually a buffer solution), and may include particle recovery and washing steps as appropriate. The resulting immobilized polypeptide can be redispersed in an aqueous solvent (usually a buffer solution) to obtain a particle solution of the immobilized polypeptide of the present invention. Examples of the buffer solutions include Good's buffers (HEPES buffer, MOPS buffer, MES buffer, etc.), TRIS buffer, glycine buffer, and borate buffer. The pH of the buffer solution can be adjusted appropriately depending on the isoelectric point (pI) of the polypeptide to be supported. The particle solution may also contain water-soluble polymers (polyethylene glycol (e.g., PEG 200-20000)), salts (NaCl, etc.), reducing agents (dithiothreitol, 2-mercaptoethanol, etc.), pH adjusters, preservatives, surfactants, etc.).

[0052] Another method for binding the insoluble particles and the polypeptide includes, for example, binding the insoluble particles and the polypeptide by physical adsorption (mainly hydrophobic interaction). The physical adsorption reaction can be carried out in an aqueous solvent (usually a buffer solution) and may include particle recovery and washing steps as appropriate. The resulting immobilized polypeptide can be redispersed in an aqueous solvent (usually a buffer solution) to obtain a particle solution of the immobilized polypeptide of the present invention. Examples of the buffer solutions independently include Good's buffer solutions (HEPES buffer solution, MOPS buffer solution, MES buffer solution, etc.), TRIS buffer solution, glycine buffer solution, and borate buffer solution. The pH of the buffer solution is preferably 6.0 to 7.5, and the pH of the aqueous solvent used in the physical adsorption reaction is more preferably 6.5 to 7.2.

[0053] Other methods for binding the insoluble particles and the polypeptide include, for example, modifying one of them in some way and adding a substance that captures the modified portion to the other, and binding them through these.For example, one of them may be biotinylated and the other avidinylated, and a binding method using avidin-biotin bonding may be used, or an indirect binding method using a secondary antibody, protein G, protein A, etc. may be used.

[0054] The quantitative ratio of the insoluble particles to the polypeptide used in these production methods can be appropriately selected so as to achieve, for example, the preferred range of amounts for the immobilized polypeptide. Among the above production methods, the method of binding the insoluble particles to the polypeptide by covalent bonding is preferred, as this tends to result in better specificity of the immobilized polypeptide for anti-gingipain antibodies. That is, for the immobilized polypeptide of the present invention, it is preferred that the insoluble particles and the polypeptide are bound by covalent bonds, and it is more preferred that the insoluble particles and the polypeptide are directly bound by covalent bonds.

[0055] When the insoluble particles of the immobilized polypeptide of the present invention are further blocked with a blocking agent, the blocking method is not particularly limited and can be any conventionally known method or a method based thereon. For example, after producing the immobilized polypeptide (after binding the insoluble particles to the polypeptide), the immobilized polypeptide is immersed in a blocking solution containing the blocking agent, followed by drying and / or washing. Examples of the solvent for the blocking solution include buffer solutions, such as Good's buffer solutions (HEPES buffer, MOPS buffer, MES buffer, etc.), TRIS buffer, glycine buffer, and borate buffer. The pH of the blocking solution can be adjusted appropriately depending on the type of blocking agent used. The blocking solution may also contain other additives such as surfactants, salts (e.g., NaCl), reducing agents (e.g., dithiothreitol, 2-mercaptoethanol), etc. The ratio of the amount of the immobilized peptide to the amount of the blocking agent used in the blocking method can be appropriately selected so as to achieve the amount within the preferred range for the immobilized polypeptide.

[0056] <Method for detecting anti-gingipain antibodies> The present invention provides a method for detecting anti-gingipain antibodies in a sample, comprising: a detection step of contacting a sample with the immobilized polypeptide of the present invention, allowing the immobilized polypeptide to bind to anti-gingipain antibodies in the sample, and detecting the anti-gingipain antibodies; The present invention provides a method for detecting anti-gingipain antibodies (sometimes referred to herein simply as the "detection method of the present invention"). The immobilized polypeptide is as described above, including its preferred embodiments.

[0057] [sample] The "sample" to be subjected to the detection method of the present invention is not particularly limited as long as it is a sample in which anti-gingipain antibodies as the test substance can be present, and examples thereof include various organisms (including bacteria such as Porphyromonas gingivalis and cultured cells) and extracts thereof; specimens collected from humans and non-human animals (body fluids such as serum, plasma, whole blood, cerebrospinal fluid, ascites, amniotic fluid, saliva, urine, gingival crevicular fluid, mouthwash, tears, and nasal swabs; feces; tissue (liver, etc.) suspensions); in vitro test solutions, etc. can be used as appropriate depending on the purpose. Examples of the non-human animals include mammals such as chimpanzees, monkeys, cows, pigs, horses, sheep, goats, camels, llamas, mice, rats, rabbits, dogs, and cats. Among these, when anti-gingipain antibodies are detected as test substances to serve as a standard for diagnosing periodontal disease and periodontal disease-related diseases in the medical and clinical testing fields, the samples used in the present invention are generally specimens collected from subjects (preferably humans) such as diagnostic targets, for example, serum, plasma, saliva, and urine, with serum being more preferred.

[0058] The sample may be one that has been subjected to processing such as pulverization or freezing, or one that has been appropriately diluted or suspended in a diluent, or one that has had its pH adjusted appropriately. Examples of the diluent include water, physiological saline, and known buffer solutions (e.g., Good's buffer solutions (e.g., HEPES buffer, MOPS buffer, MES buffer, etc.), TRIS buffer, glycine buffer, and borate buffer). Water-soluble polymers (e.g., polyethylene glycol (e.g., PEG 200 to 20,000)), salts (e.g., NaCl), reducing agents (e.g., dithiothreitol, 2-mercaptoethanol), pH adjusters, preservatives, surfactants, and the like may also be added. The pH of the diluent is preferably 5 to 10, and more preferably 6 to 8. The sample to be subjected to the method of the present invention is preferably an aqueous sample, and is preferably diluted or suspended in the diluent as needed.

[0059] [Detection process] In the detection method of the present invention, in the detection step, the sample is contacted with the immobilized polypeptide of the present invention, and if anti-gingipain antibodies are present in the sample, the anti-gingipain antibodies in the sample are detected by immunoassay based on the immune complex formed by the antigen-antibody reaction between the anti-gingipain antibodies and the polypeptide.

[0060] Examples of such immunoassay methods include EIA (enzyme-linked immunosorbent assay) which uses an enzyme as a labeling substance, ELISA and CLEIA (chemiluminescent enzyme immunoassay), which are embodiments of EIA, RIA (radioimmunoassay) which uses a radioisotope as a labeling substance, CLIA (chemiluminescent immunoassay) which uses a chemiluminescent compound as a labeling substance, immunochromatography, and immunoagglutination methods (latex particle agglutination, metal colloid agglutination, etc.) which measure by detecting agglutination. In these methods, an anti-gingipain antibody, a detection antibody against the immune complex, a labeled form of the detection antibody labeled with the labeling substance, or a secondary antibody that binds to the detection antibody can be used in combination with the immobilized polypeptide of the present invention, as needed.

[0061] Among these, the detection method of the present invention is preferably a latex particle agglutination method in which the insoluble particles in the immobilized polypeptide of the present invention are latex particles. In the latex particle agglutination method, if an anti-gingipain antibody is present in the sample, the latex particles agglutinate with each other via binding between the polypeptide immobilized on the latex particles and the anti-gingipain antibody. Therefore, the presence or absence of the anti-gingipain antibody in the sample can be detected by detecting the agglutination of such latex particles, and the amount of the anti-gingipain antibody in the sample can also be determined based on the amount of agglutination.

[0062] The method for contacting the sample with the immobilized polypeptide is not particularly limited, and a conventionally known method or a method based thereon can be used as appropriate. For example, a method in which the sample is diluted with the diluent as needed and then mixed with a particle liquid containing the immobilized polypeptide can be used. The particle liquid and diluent, including their preferred embodiments, are as described above. The sample may be diluted during sample preparation, during detection, or twice, once during sample preparation and once during detection. When the sample is diluted twice, different diluents may be used for each diluent. When the sample is diluted during detection, the particle liquid may also serve as the diluent.

[0063] In this case, the sample may be diluted in advance with the diluent and then brought into contact with the immobilized polypeptide, or the sample, the diluent, and the immobilized polypeptide may be mixed simultaneously. However, for example, in the latex particle agglutination method, it is preferable to dilute the sample in advance with the diluent (first reagent) and mix them at 4 to 40°C for 1 to 10 minutes, then add the particle liquid of the immobilized polypeptide (second reagent), mix them at 4 to 40°C for 30 seconds to 15 minutes (preferably 1 to 10 minutes) to allow the reaction, and detect the resulting agglutination of latex particles.

[0064] In this case, more preferred first reagents include buffer solutions such as Good's buffer (HEPES buffer, MOPS buffer, MES buffer, etc.), TRIS buffer, glycine buffer, and borate buffer, and it is also preferred that such buffer solutions contain at least one selected from the group consisting of water-soluble polymer compounds (polyethylene glycol (e.g., PEG200-20000)), salts (NaCl, etc.), and reducing agents (dithiothreitol, etc.). In this case, the pH of the first reagent is preferably 5 to 10, and more preferably 6 to 8.

[0065] Furthermore, more preferred dispersion media for the second reagent in this case are, for example, Good's buffer (HEPES buffer, MOPS buffer, MES buffer, etc.), TRIS buffer, glycine buffer, and borate buffer, and it is also preferred that such buffers contain at least one selected from the group consisting of water-soluble polymer compounds (polyethylene glycol (e.g., PEG200-20000)), salts (NaCl, etc.), and reducing agents (dithiothreitol, etc.). The pH of the second reagent in this case is preferably 5 to 10, and more preferably 6 to 8.

[0066] The mixing ratio between the sample and the immobilized polypeptide is not particularly limited and can be adjusted appropriately depending on the type and concentration of the sample.

[0067] An example of a method for detecting the agglutination of latex particles is a method in which the sample is contacted with the immobilized polypeptide and then reacted for 30 seconds (preferably 3 minutes) or more, followed by measuring the absorbance of the reaction solution (e.g., the reaction solution after the addition of the second reagent). The wavelength at which the absorbance is measured is 400 to 800 nm. In the detection method of the present invention, the absorbance value thus obtained may be used as the "detection value." Alternatively, the detection value according to the present invention may be a value obtained by correcting the absorbance value using a control value. For example, the absorbance immediately after contacting the sample with the immobilized polypeptide (reaction minute 0) may be similarly measured and used as a control value, and the difference (ΔOD) between this and the absorbance value of the reaction solution after the reaction may be used as the detection value according to the present invention.

[0068] Furthermore, when the insoluble particles are metal particles (e.g., gold colloid particles), other methods for detecting aggregation of the metal particles include a method of measuring the change in absorbance due to aggregation using surface plasmon resonance (SPR), and a method of detecting by visually confirming color development using immunochromatography (e.g., lateral flow assay).

[0069] [Quantitative process] Furthermore, the detection method of the present invention may further comprise a quantification step of quantifying the amount of anti-gingipain antibody in the sample by comparing the detection value obtained in the detection step with the detection value in a standard sample having a known concentration of anti-gingipain antibody. Furthermore, when the detection method of the present invention is a diagnostic method or diagnostic aid method described below, the detection value obtained in the detection step can be said to reflect the "antibody titer" of the subject from whom the sample was derived, and the "antibody titer" can be calculated from the detection value as the amount of anti-gingipain antibody in the sample. The antibody titer can be set, for example, by setting the concentration of the standard sample at the detection limit as 1 unit (U) when serially diluted solutions of a standard sample having a known concentration of anti-gingipain antibody are detected by ELISA using a plate on which the polypeptide (preferably polypeptide (a")) is immobilized and an enzyme-labeled anti-human IgG antibody, but is not limited thereto.

[0070] <Method for diagnosing the presence or absence of Porphyromonas gingivalis infection or method for assisting in said diagnosis> When a sample derived from a subject collected from the subject is used as the sample, the detection method of the present invention can be used as a method for diagnosing the presence or absence of Porphyromonas gingivalis infection in the subject or a method for assisting in the diagnosis. That is, the present invention provides: The method includes a step of detecting anti-gingipain antibodies in a sample collected from a subject by the detection method of the present invention. The present invention also provides a method for diagnosing the presence or absence of Porphyromonas gingivalis infection (sometimes referred to herein simply as a "diagnostic method") or a method for assisting in said diagnosis (sometimes referred to herein simply as a "diagnostic assisting method"). The diagnostic assisting method can also be expressed as a method for detecting anti-gingipain antibodies for use by a physician in diagnosing the presence or absence of Porphyromonas gingivalis infection (or diagnosing periodontal disease or periodontal disease-related diseases). The detection method, including its preferred embodiments, is as described above.

[0071] In the diagnostic method and diagnostic support method, the subject is preferably a human, and the sample is preferably serum, plasma, saliva, or urine, with serum being more preferred.

[0072] In the diagnosis related to the diagnostic method and the diagnostic auxiliary method, if even a small amount of anti-gingipain antibody is detected from the sample by the detection method, for example, if there is even a small amount of the detection value, the subject from whom the sample was derived may be judged to be infected with Porphyromonas gingivalis (judging the presence or absence of Porphyromonas gingivalis infection, diagnosing the presence or absence of periodontal disease or a periodontal disease-related disease), selected (selecting subjects infected with Porphyromonas gingivalis), or differentiated (diagnosing the progression or severity of periodontal disease or a periodontal disease-related disease), but it is preferable that the detection method further includes the quantification step, and that the judgment, selection, or differentiation is made depending on the amount of anti-gingipain antibody obtained, preferably the antibody titer in the subject from whom the sample was derived. For example, the amount of anti-gingipain antibody (preferably antibody titer) obtained in the quantification step of the detection method may be compared with a predetermined cutoff value, and subjects with a value higher than the cutoff value may be determined to be infected with Porphyromonas gingivalis (or suffering from periodontal disease or a periodontal disease-related disease) or to be highly likely to be infected (or suffering from) the disease.

[0073] The "cutoff value" refers to a predetermined value that serves as a standard for determining the amount of anti-gingipain antibodies, and indicates a boundary value for distinguishing between a positive group and a negative group. Such a cutoff value is set appropriately depending on the purpose of diagnosis, the characteristics of the subject and sample, dilution conditions, detection conditions, etc., and is not particularly limited. For example, it can be obtained by logistic regression analysis or receiver operating characteristic analysis (ROC) analysis using the presence or absence and amount of P. gingivalis detected in the oral cavity and the stage of disease progression based on a doctor's diagnosis as objective variables. Furthermore, for example, by setting the cutoff value to a relatively low value, it is possible to collect a relatively wide range of subjects suspected of being infected with P. gingivalis, while by setting the cutoff value to a relatively high value, it is possible to increase the accuracy of the determination.

[0074] The diagnostic method and diagnostic support method can provide information on the presence or amount of anti-gingipain antibodies as an indicator for determining, selecting, or differentiating patients who are infected with P. gingivalis or who are likely to be infected with P. gingivalis, with high sensitivity, from a group of healthy individuals (negative group), thereby enabling the diagnosis or support of periodontal disease and periodontal disease-related diseases, as well as early therapeutic intervention, etc. Examples of periodontal disease-related diseases include diabetes, Alzheimer's disease, cardiovascular disease, atrial fibrillation, cerebral infarction, rheumatoid arthritis, nonalcoholic fatty liver disease (NASH / NAFLD), obesity, premature birth, infertility, etc. Furthermore, patients selected as likely to suffer from these diseases can also be subjected to other tests specialized for each disease.

[0075] <Anti-gingipain antibody detection kit> The present invention provides a kit for detecting anti-gingipain antibodies, which kit includes the above-described immobilized polypeptide of the present invention. The immobilized polypeptide, including preferred embodiments thereof, is as described above. The kit of the present invention can be suitably used in the above-described detection method, diagnostic method, and diagnostic auxiliary method of the present invention.

[0076] The kit of the present invention may further include other components. For example, it may further include at least one selected from the group consisting of a lancet for causing a small amount of bleeding in the subject, a capillary for collecting the blood, the blocking agent or blocking solution, a standard sample (each concentration), a washing solution, a dispersion medium for the particle solution, a diluent for the sample, a first reagent, and a second reagent. Furthermore, the kit of the present invention may further include instructions for use of the kit. [Example]

[0077] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples. In each of the test examples below, ELISA, preparation of latex particle-immobilized antigen polypeptide, latex particle agglutination, and correlation analysis between ELISA and latex particle agglutination were performed by the following methods. In the following, "%" indicates weight / volume (w / v: g / mL) percentage unless otherwise specified.

[0078] <elisa> Using ELISA with plate-immobilized polypeptides, which have already been found to be useful, we detected anti-gingipain antibodies in samples and quantified the antibody titer, and verified the correlation with results obtained when using antigen polypeptides immobilized on latex particles.

[0079] For the ELISA, an artificially synthesized antigen polypeptide (containing the amino acid sequence shown in SEQ ID NO: 1 (860 amino acid residues) to which a GST tag (the amino acid sequence shown in SEQ ID NO: 4) had been added) was first diluted to 100 ng / mL with 100 mmol / L sodium carbonate buffer (pH 9.6). This solution was dispensed into each well of a 96-well EIA plate (Thermo Fisher Scientific) at 100 μL / well and allowed to stand overnight (8 hours or more) at 4°C. Next, a washing solution (PBS buffer containing 1.1% Tween 20) was diluted 40-fold with ultrapure water, and each well was washed three times with 350 μL / well of this solution using a plate washer. After this, a blocking solution was dispensed and the plate was allowed to stand for 2 hours at 37°C, after which the blocking solution was removed to prepare the plate-immobilized polypeptide.

[0080] In each test example, the amount of antigen polypeptide was determined by the following method. Specifically, the antigen polypeptide solution was electrophoresed by SDS-PAGE, followed by CBB staining, and the intensity of the band obtained from the electrophoretic image after staining was measured by optical density analysis. In addition, a BSA solution of known concentration was used as a standard protein solution, and the intensity of the obtained band was similarly measured. From these results, the amount of protein in the antigen polypeptide solution was determined, and this was taken as the amount of antigen polypeptide.

[0081] Next, each sample was diluted 100-fold with PBS buffer containing skim milk and dispensed into each well of the plate on which the antigen polypeptide had been solidified at 100 μL / well, and the plate was shaken at 25°C for 1 hour to allow the reaction to occur. After that, each well was washed with the washing solution in the same manner as above.

[0082] Next, a horseradish peroxidase (HRP)-labeled anti-human IgG antibody (Goat anti-Human IgG(H+L)-HRP (Invitrogen A18805), Thermo Fisher Scientific, 1 mg / mL) was diluted 2,000-fold and dispensed into each well at 100 μL / well. The plate was shaken at 25°C for 1 hour to allow the reaction to occur, and then each well was washed with the washing solution in the same manner as above.

[0083] Next, 100 μL / well of ABTS solution (Roche) was dispensed as a substrate solution into each well and allowed to stand at 25° C. Ten minutes after dispensing the substrate solution, the absorbance of each well at a wavelength of 405 nm was measured using a plate reader (Spark, Tecan).

[0084] When determining the antibody titer in each sample, the absorbance was measured in the same manner as above, except that a standard sample containing anti-gingipain antibodies of known antibody titers was used instead of the sample, and the antibody titer (U / mL) in the sample was calculated from the obtained absorbance-anti-gingipain antibody titer calibration curve and the absorbance detected in each sample.

[0085] <Preparation of latex particle-immobilized antigen polypeptide> (1)Physical adsorption First, an artificially synthesized antigen polypeptide (the amino acid sequence (860 amino acid residues) shown in SEQ ID NO: 1 to which a GST tag (the amino acid sequence shown in SEQ ID NO: 4) has been added) was diluted to 2.0 mg / ml in 10 mM HEPES buffer (pH 7.4). 2 (mg / m 2 The mixture was diluted to a concentration of 10 mg / m (mg per unit area of ​​the latex particle surface, hereinafter the same). An appropriate amount of latex particles was added to the mixture and stirred. The mixture was then adjusted to pH 7.1 with HEPES buffer, and a blocking agent was added at 10 mg / m (mg / m ). 2 or 10 mg / m 2 The particle surface was blocked by adding casein Na in an amount equivalent to 1000 mg / L of 10000 mg ...

[0086] (2) Covalent bond First, latex particles were added to a DMT-MM solution (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) solution so that the amount of DMT-MM was 0.9 mol equivalent relative to the functional group (carboxy group) of the latex particles, and the mixture was stirred and mixed. Next, the particles were collected by centrifugation, washed with 10 mM HEPES (pH 7.4), and collected again by centrifugation. An artificially synthesized antigen polypeptide (the amino acid sequence (860 amino acid residues) shown in SEQ ID NO: 1 to which a GST tag (the amino acid sequence shown in SEQ ID NO: 4) has been added) was added to the latex particles at a concentration of 0.4 mg / m with 10 mM HEPES (pH 7.4). 2 A diluted solution diluted to 0.12% was added to the mixture. Then, casein Na was added in an amount to give 0.12% to block the particle surface. The particles were then recovered by centrifugation, washed with 10 mM HEPES (pH 7.4), recovered by centrifugation again, and suspended in 50 mM HEPES (pH 7.4). Further, casein Na was added in an amount to give 0.1% to block the particle surface (secondary blocking), and the particles were recovered by centrifugation, washed with 10 mM HEPES (pH 7.4), recovered by centrifugation again, and suspended in 50 mM HEPES (pH 7.4) to obtain a particle solution of latex particle-immobilized antigen polypeptide in which the antigen polypeptide was covalently bound to the surface of the latex particles.

[0087] <Latex particle agglutination method> As measurement reagents, the first reagent and the second reagent were prepared, and measurements were performed using an automatic analyzer (TBA-120FR Pearl Edition, manufactured by Canon Medical Systems Corporation). As the first reagent, 50 mM HEPES buffer (pH 7.4) was prepared. As the second reagent, 50 mM HEPES buffer (pH 7.4) was prepared to contain an appropriate amount of the latex particle-solidified antigen polypeptide. In the automatic analysis, first, 64 μL of the first reagent was added to 6 μL of the specimen, stirred and mixed at 37°C for 4 minutes, then 64 μL of the second reagent was added to the mixed solution, stirred and mixed at 37°C, and the absorbance at a wavelength of 660 nm 5 minutes after the addition of the second reagent (reaction for 5 minutes) was measured. Also, the absorbance immediately after the addition of the second reagent (reaction for 0 minutes) was measured, and the absorbance difference (ΔOD) was calculated from the difference between the absorbance at reaction for 5 minutes and the absorbance at reaction for 0 minutes.

[0088] When determining the antibody titer for each specimen, instead of the specimen, a standard sample containing an anti-zingipain antibody with a known antibody titer was used. The absorbance difference (ΔOD) in the standard sample was measured in the same manner as above, and the antibody titer (U / mL) in each specimen was calculated from the absorbance difference (ΔOD) measured in each specimen using the obtained calibration curve of absorbance difference - anti-zingipain antibody titer.

[0089] <Correlation analysis between ELISA and latex particle agglutination method> For each specimen, the antibody titer of the absorbance measured by the above ELISA was plotted on the horizontal axis, and the absorbance difference (ΔOD) detected and calculated by the latex particle agglutination method was plotted on the vertical axis. Also, for each specimen, the positive / negative determination was performed with a cut-off value of 100 U / mL for both the antibody titer obtained by the above ELISA and the antibody titer obtained by the above latex particle agglutination method, and the positive / negative determination coincidence rate was calculated.

[0090] <Test Example 1> Latex particle agglutination method using latex particle-solidified antigen polypeptide (1) ELISA For serum specimens (n = 71: 5 cases in their 30s, 30 cases in their 40s, 24 cases in their 50s, 12 cases in their 60s), the above <elisa>Anti-gingipain antibodies were detected in each sample and the antibody titer of the sample was calculated using the method described in. Samples with a calculated antibody titer of less than 100 U / mL were considered negative, and samples with a titer of 100 U / mL or more were considered positive.

[0091] (2) Preparation of latex particle-immobilized antigen polypeptide Carboxy group-modified polystyrene latex (average particle diameter (DLS measurement): 296 nm) was used as latex particles, and a particle solution of antigen polypeptide immobilized on latex particles (Example) was prepared by the method described in <Antigen polypeptide immobilized on latex particles> (2) above, in which the antigen polypeptide was covalently bound to the surface of the latex particles.

[0092] (3) Latex particle agglutination method Using the particle solution of latex particle-immobilized antigen polypeptide prepared in (2) above, anti-gingipain antibodies were detected in each of the same samples used in (1) ELISA by the method described above in <Latex particle agglutination method>, and the antibody titers of the samples were calculated. Samples with calculated antibody titers of less than 100 U / mL were considered negative samples, and samples with calculated antibody titers of 100 U / mL or more were considered positive samples.

[0093] (4) Correlation analysis between ELISA and latex particle agglutination method The results of the negative / positive determination based on the antibody titer obtained by ELISA in (1) above and the negative / positive determination based on the antibody titer obtained by latex particle agglutination in (3) above are tabulated in Table 1 below. Furthermore, the logarithmic values ​​of the antibody titer obtained by latex particle agglutination in (3) above for the specimens that were determined to be negative / positive based on the antibody titer obtained by ELISA in (1) above are shown in Figure 1. From Table 1, the positive agreement rate was 92% (33 / 36), the negative agreement rate was 80% (28 / 35), and the overall agreement rate was 86% (61 / 71).

[0094] [Table 1]

[0095] As shown in Table 1 and Figure 1, it was confirmed that the latex particle agglutination method using the latex particle-immobilized antigen polypeptide of the present invention can detect anti-gingipain antibodies with high sensitivity, as with ELISA using plate-immobilized polypeptide, and enables accurate positive / negative determination (determination of the presence or absence of anti-gingipain antibodies).

[0096] <Test Example 2> Examination of particle size of latex particles Positive samples are those with an anti-gingipain antibody titer of <elisa>The serum sample (one case) was used, and the titer of the anti-gingipain antibody was 100 U / mL or more when measured by the method described above. The donor who provided the positive sample was also confirmed to be clinically positive through dental examination. The negative sample was a sample in which the antibody titer of the anti-gingipain antibody was 100 U / mL or more when measured by the method described above. <elisa>A serum sample (one case) with a concentration of less than 100 U / mL as measured by the method described in was used.

[0097] Carboxy group-modified polystyrene latex having an average particle size (as measured by DLS) of 101 nm, 204 nm, 307 nm, or 404 nm was used as the latex particles, and particle solutions of antigen polypeptide immobilized on latex particles (Examples) were prepared by the method described in (2) above in <Antigen polypeptide immobilized on latex particles>, in which the antigen polypeptide was covalently bound to the surface of latex particles of each particle size.

[0098] The absorbance of each sample was measured using the prepared latex particle-immobilized antigen polypeptide particle solution according to the method described in the above-mentioned <Latex particle agglutination method>, and the absorbance difference (ΔOD) was calculated. The absorbance difference (ΔOD x 10,000) calculated for both positive and negative samples was detected using latex particle-immobilized antigen polypeptide of each particle size is shown in Table 2 below and Figure 2.

[0099] [Table 2]

[0100] As shown in Table 2 and Figure 2, it was confirmed that anti-gingipain antibodies can be detected with high sensitivity regardless of the particle size of the latex particles, and that positive (presence of anti-gingipain antibodies) and negative (absence of anti-gingipain antibodies) results can be determined with high accuracy. In particular, it was confirmed that anti-gingipain antibodies can be detected with even higher sensitivity when the latex particle size is 200 nm or more.

[0101] <Test Example 3> Examination of antigen polypeptide amount Positive samples are those with an anti-gingipain antibody titer of <elisa>The specimen (1 case) was used, and the measurement was 100 U / mL or more according to the method described in [1].

[0102] Polystyrene latex (average particle diameter (measured by electron microscope): 332 nm) was used as latex particles. A diluted solution of the antigen polypeptide was applied to the latex particles so that the amount of polypeptide per unit area of ​​the latex particle surface was 2.0 mg / m 2 Instead of 0.2 mg / m 2 , 0.4 mg / m 2 , 0.8 mg / m 2 , or 1.6 mg / m 2 A particle solution of antigen polypeptide immobilized on latex particles (Example) was prepared by physically adsorbing the antigen polypeptide to the surface of latex particles at various concentrations using casein Na as a blocking agent, according to the method described in (1) of <Antigen polypeptide immobilized on latex particles> above, except that the solution was prepared so that the above-mentioned

[0103] The absorbance of the above specimen was measured using the prepared particle solution of the antigen polypeptide immobilized on latex particles according to the method described above in "Latex particle agglutination method," and the absorbance difference (ΔOD) was calculated. The absorbance difference (ΔOD x 10,000) calculated for the specimen using the antigen polypeptide immobilized on latex particles with each polypeptide amount is shown in Figure 3.

[0104] As shown in Figure 3, it was confirmed that anti-gingipain antibodies could be sufficiently detected when any of the antigen polypeptides immobilized on latex particles was used. In particular, when the antigen polypeptide of this test example was used, the amount of the polypeptide sensitized to the latex particle surface was 0.8 mg / m2 in terms of the amount per latex surface area. 2 It was confirmed that anti-gingipain antibodies can be detected with higher sensitivity when the concentration is less than or equal to 100 μg / mL.

[0105] Test Example 4: Examination of blocking agents Positive samples are those with an anti-gingipain antibody titer of <elisa>The specimen (1 case) was used, and the measurement was 100 U / mL or more according to the method described in [1].

[0106] Polystyrene latex (average particle diameter (measured by electron microscope): 332 nm) was used as the latex particles. A particle solution of antigen polypeptide immobilized on latex particles (Example, Y) was prepared by the method described in (1) above for <Antigen polypeptide immobilized on latex particles>, in which the antigen polypeptide was physically adsorbed onto the surface of the latex particles, using BSA and casein sodium as the blocking agent. Furthermore, a particle solution of particles whose surface was blocked with BSA or casein sodium (Control Example, N) was prepared in the same manner as above, except that no antigen polypeptide was used.

[0107] The prepared particle solution of latex particle-immobilized antigen polypeptide (Y) or, alternatively, a particle solution of particles (N) not sensitized with the antigen polypeptide was used to detect anti-gingipain antibodies in the sample by the method described in the above <Latex particle agglutination method>, and the absorbance was measured and the absorbance difference (ΔOD) was calculated. The absorbance difference (ΔOD) detected and calculated using each latex particle-immobilized antigen polypeptide (Y: BSA, casein Na) and each particle not sensitized with the antigen polypeptide (N: BSA, casein Na) are shown in Figure 4.

[0108] As shown in Figure 4, it was confirmed that anti-gingipain antibodies could be sufficiently detected with latex particle-immobilized antigen polypeptide (Y) regardless of the blocking agent used. In particular, the absorbance difference (ΔOD) was significantly larger with the latex particle-immobilized antigen polypeptide prepared using casein Na as the blocking agent, confirming that anti-gingipain antibodies could be detected with higher sensitivity.

[0109] <Test Example 5> Examination of the binding mode between latex particles and polypeptide The specimens were three serum samples and the antibody titer of anti-gingipain antibody was as follows: <elisa>A total of four samples were used, including a pooled sample, which was a mixture of multiple serum samples that were 100 U / mL or more (positive) when measured using the method described above. <elisa>Anti-gingipain antibodies were detected in each sample by the method described in , and the antibody titer [U / mL] of the sample was calculated.

[0110] Polystyrene latex (average particle diameter (measured by electron microscope): 332 nm) was used as latex particles, and a particle solution of antigen polypeptide immobilized on latex particles (Example (physical adsorption)) was prepared by the method described in (1) above in <Antigen polypeptide immobilized on latex particles>. In this preparation, casein Na was used as a blocking agent.

[0111] Furthermore, a particle solution of antigen polypeptide immobilized on latex particles (Example (covalent bond)) was prepared by using carboxyl group-modified polystyrene latex (average particle diameter (DLS measurement): 296 nm) as latex particles and covalently bonding the antigen polypeptide to the surface of the latex particles according to the method described in <Antigen polypeptide immobilized on latex particles> (2) above.

[0112] Using the particle suspension of each latex particle-immobilized antigen polypeptide prepared, anti-gingipain antibodies in each sample were detected by the method described in the above-mentioned <Latex particle agglutination method>, and the absorbance of the sample was measured and the absorbance difference (ΔOD) was calculated. The relationship between the antibody titer [U / mL] obtained by ELISA and the absorbance difference (ΔOD x 10,000) detected and calculated using each latex particle-immobilized antigen polypeptide (physical adsorption, covalent bond) is shown in Figure 5.

[0113] Although anti-gingipain antibodies can be detected with high sensitivity using any of the latex particle-immobilized antigen polypeptides, Figure 5 shows that, depending on the sample, when using a latex particle-immobilized antigen polypeptide in which the antigen polypeptide is physically adsorbed to the surface of latex particles, nonspecific agglutination can increase ΔOD, resulting in partial dissociation from the antibody titer, even if the antibody titer obtained by ELISA is less than 100 U / mL (indicated by the arrow). On the other hand, when using a latex particle-immobilized antigen polypeptide in which the antigen polypeptide is covalently bound to the surface of latex particles, such a discrepancy does not occur, confirming that anti-gingipain antibodies can be detected more specifically.

[0114] <Test Example 6> Application Example 1 to Periodontal Disease Diagnosis The results of detecting anti-gingipain antibodies using the immobilized polypeptide of the present invention were compared with the results of detecting Porphyromonas gingivalis DNA in saliva.

[0115] Serum samples from 105 cases were used as specimens. Except for the use of these specimens, the antibody titer of each specimen was measured and calculated by the latex particle agglutination method in the same manner as in Test Example 1 (2) and (3). Saliva was also collected from the subjects from whom the specimens were collected, and DNA was extracted using a DNA extraction kit (Saliva DNA isolation kit, NORGEN). Using this as a template, DNA of P. gingivalis in the saliva was detected by quantitative PCR using primers and probes prepared with reference to Non-Patent Document 3.

[0116] Logistic regression analysis and ROC analysis were performed using the presence or absence of detection of P. gingivalis DNA in saliva (detection (58 positive samples) / non-detection (47 negative samples)) as the dependent variable and the antibody titer measured and calculated by latex particle agglutination as the explanatory variable. The obtained ROC curve is shown in Figure 6. From the obtained ROC curve, the AUC (Area Under the Curve) value, which corresponds to the ability to determine the presence or absence of P. gingivalis infection (positive / negative determination ability) by detecting anti-gingipain antibodies using the immobilized polypeptide of the present invention (latex particle-immobilized polypeptide), was high at approximately 0.94 (0.94369).

[0117] <Test Example 7> Application Example 2 to Periodontal Disease Diagnosis The results of detecting anti-gingipain antibodies using the immobilized polypeptide of the present invention were compared with the results of periodontal disease diagnosis by dentists.

[0118] The samples used were 149 serum samples collected from patients undergoing bacterial testing for periodontal disease at the Department of Dentistry and Periodontology, Okayama University, and included medical information such as the condition of periodontal tissues. Based on this medical information, the samples were divided into stages I to IV (stage I: 5 samples, stage II: 12 samples, stage III: 87 samples, stage IV: 45 samples) according to the progression of periodontal disease (see Sekino, Yu, "Clinical Significance and Future Prospects of the New Periodontal Disease Classification," Journal of the Japanese Society of Health Care Dentistry, Vol. 21, 2020, pp. 6-11). Stage I indicates the least severe, and stage IV indicates the most severe. Except for these samples, the antibody titer of each sample was measured and calculated using the latex particle agglutination method in the same manner as in Test Example 1 (2) and (3).

[0119] An ROC curve was created using the positive / negative result based on the stage classification as a nonparametric dependent variable and the antibody titer measured and calculated by latex particle agglutination as a parametric dependent variable, and logistic regression analysis and ROC analysis were performed. When positive / negative results were divided into stages, the AUC value, sensitivity, specificity, positive predictive value (%), negative predictive value (%), and cutoff value [U / mL] obtained from the ROC curve for the detection of anti-gingipain antibodies using the immobilized polypeptide of the present invention (latex particle-immobilized polypeptide) are shown in Table 3 below.

[0120] [Table 3]

[0121] As shown in Table 3, when anti-gingipain antibodies were detected using the immobilized polypeptide of the present invention, the AUC value was high, for example, 0.769 when stage I or below was considered negative and stage II or above was considered positive, and 0.774 when stage II or below was considered negative and stage III or above was considered positive.

[0122] <Test Example 8> Comparison with fimbrial antigens The ability of the immobilized polypeptide of the present invention to detect anti-gingipain antibodies was compared with the ability of the immobilized pilus antigen, which is a conventionally known pilus antigen immobilized on particles, to detect anti-gingipain antibodies.

[0123] (1) Pilus antigen A fimbrial antigen of the FDC381 strain of Porphyromonas gingivalis was obtained according to the methods described in Patent Document 1 and Non-Patent Document 1. The protein concentration of the obtained fimbrial antigen was determined using a DC Protein Assay Kit (Bio-Rad) and Pierce Albumin Standard Ampules (Thermofisher).

[0124] (2) ELISA Since Non-Patent Document 2 suggests that the antigenicity of pilus antigens is low, we first confirmed their reactivity with non-periodontal disease specimens / periodontal disease specimens by ELISA to confirm whether the pilus antigens used for comparison in this test example have sufficient reactivity with anti-pilus antibodies.

[0125] The samples used were stored serum samples collected from patients who had undergone periodontal disease testing. Four serum samples from non-periodontal patients (healthy samples (negative samples)) with medical information on the condition of periodontal tissues, etc., and four serum samples from periodontal disease patients (R1 to R4 (positive samples)) were used. The periodontal disease patient samples were classified according to the progression of periodontal disease based on the medical information, with one case being stage III (R1) and three cases being stage IV (R2 to R4).

[0126] First, an artificially synthesized antigen polypeptide (860 amino acid residues of the amino acid sequence shown in SEQ ID NO:1 with a GST tag (amino acid sequence shown in SEQ ID NO:4) added) and the fimbrial antigen (1) were each diluted with 100 mmol / L sodium carbonate buffer (pH 9.6). This solution was dispensed into each well of a 96-well EIA plate (Thermo Fisher Scientific) at 100 μL / well and allowed to stand overnight (8 hours or more) at 4°C. Next, a washing solution (PBS buffer containing 1.1% Tween 20) was diluted 40-fold with ultrapure water. Each well was washed three times with 350 μL / well of this solution using a plate washer. After dispensing a blocking solution, the plate was allowed to stand for 2 hours at 37°C, after which the blocking solution was removed to prepare the plate-immobilized antigens (antigen polypeptide, fimbrial antigen). The amount of antigen per well for the plate-immobilized antigen was adjusted to 10 ng / well in terms of protein for the antigen polypeptide, and 10 ng / well or 100 ng / well in terms of protein for the pilus antigen. <elisa>The absorbance of each well was measured using a plate reader in the same manner as described in .

[0127] For each sample, the absorbance measured using a plate immobilized with antigen polypeptide (10 ng / well) is shown in Figure 7, and the absorbance measured using a plate immobilized with pilus antigen (10 ng / well, 100 ng / well) is shown in Figure 8. The results are shown as relative values, with the average absorbance of the antigen polypeptide (10 ng / well) in healthy samples (4 samples) set to 1 in Figure 7, and the average absorbance of the pilus antigen (10 ng / well) in healthy samples (4 samples) set to 1 in Figure 8.

[0128] As shown by comparing Figures 7 and 8, the fimbrial antigen (Figure 8) was confirmed to have the reactivity (antigenicity) to distinguish between non-periodontal disease specimens (negative specimens) and periodontal disease specimens (positive specimens), similar to the antigen polypeptide (Figure 7). Furthermore, a good dose-response relationship was confirmed for both antigens in diluted positive specimens (antigen polypeptide (10 ng / well): R 2 =0.9999; fimbrial antigen (10ng / well):R 2 =0.9997; Fimbrial antigen (100ng / well):R 2 =0.997).

[0129] (3) Latex particle agglutination method The samples used were preserved serum samples collected from patients who had undergone periodontal disease testing. These included 10 serum samples from non-periodontal patients (healthy samples (negative samples)) with medical information on the condition of periodontal tissues, and 13 serum samples from periodontal patients (periodontal disease samples (positive samples)). The periodontal disease patient samples were classified according to the progression of periodontal disease based on the medical information: 4 cases were in stage I, 1 case was in stage II, 4 cases were in stage III, and 4 cases were in stage IV.

[0130] First, carboxyl-modified polystyrene latex (average particle diameter (DLS measurement): 296 nm) was used as latex particles, and a particle solution of an antigen polypeptide immobilized on latex particles (Example) was prepared by the method described in the above <Antigen polypeptide immobilized on latex particles> (2) in which an artificially synthesized antigen polypeptide (the amino acid sequence (860 amino acid residues) shown in SEQ ID NO: 1 to which a GST tag (amino acid sequence shown in SEQ ID NO: 4) has been added) was covalently bound to the surface of the latex particles.

[0131] In addition, a particle solution of latex particle-immobilized pilus antigen (comparison example) was prepared in the same manner as above, except that the pilus antigen (1) above was used instead of the artificially synthesized antigen polypeptide. The pilus antigen was covalently bound to the surface of latex particles.

[0132] For each of the above specimens, the particle solution of latex particle-immobilized antigen polypeptide or latex particle-immobilized fimbrial antigen prepared above was used to detect anti-gingipain antibodies or anti-fimbrial antibodies in each specimen by the method described above in "Latex particle agglutination method," and the antibody titer of the specimen was calculated. Specimens with a calculated antibody titer of less than 100 U / mL were considered negative specimens, and specimens with a calculated antibody titer of 100 U / mL or more were considered positive specimens.

[0133] ROC curves were created using the presence or absence of periodontal disease according to the above stage classification (healthy specimens: 0, periodontal disease specimens: 1) as a nonparametric dependent variable and the antibody titers measured and calculated by latex particle agglutination as parametric dependent variables, and logistic regression and ROC analyses were performed. Figure 9 shows the ROC curve obtained for detection using antigen polypeptide immobilized on latex particles, and Figure 10 shows the ROC curve obtained for detection using fimbrial antigen immobilized on latex particles. The AUC values ​​are also shown in Figures 9 and 10.

[0134] As shown in Figure 9, in the detection of anti-gingipain antibodies using the immobilized polypeptide of the present invention (particle-immobilized antigen polypeptide), the AUC value, which corresponds to the ability to distinguish between non-periodontal disease specimens and periodontal disease specimens, was high at 0.91538. On the other hand, as shown in Figure 10, although sufficient reactivity was confirmed by the ELISA described in (2) above, in the detection of anti-fimbrial antibodies using particle-immobilized fimbrial antigens, the AUC value was significantly low at 0.47309, indicating inferior ability to distinguish between non-periodontal disease specimens and periodontal disease specimens. These results confirmed that the combination of the antigen polypeptide of the present invention and insoluble particles specifically achieves the ability to determine the presence or absence of P. gingivalis infection with particularly high accuracy. [Industrial Applicability]

[0135] As described above, the present invention makes it possible to provide a novel immobilized polypeptide and kit capable of detecting anti-gingipain antibodies with high sensitivity, and a method for detecting anti-gingipain antibodies using the same. The immobilized polypeptide, kit, and method for detecting anti-gingipain antibodies of the present invention make it possible to determine the presence or absence of infection with Porphyromonas gingivalis with particularly high accuracy.< / elisa> < / elisa> < / elisa> < / elisa> < / elisa> < / elisa> < / elisa> < / elisa> < / elisa>

Claims

1. a solid-phase polypeptide for detecting anti-gingipain antibodies, The composition comprises insoluble particles and a polypeptide supported on the insoluble particles, The polypeptide is selected from the following (a) to (b): (a) a polypeptide comprising a region consisting of an amino acid sequence of 5 to 30 amino acid residues contained in the amino acid sequence of SEQ ID NO: 1 (b) a polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, and / or inserted in the region; At least one selected from the group consisting of Immobilized polypeptide.

2. The polypeptide is selected from the following (a') to (b'): (a') a polypeptide comprising a region consisting of the amino acid sequence of positions 504 to 530 and / or a region consisting of the amino acid sequence of positions 698 to 718 of the amino acid sequence set forth in SEQ ID NO: 1 (b') A polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted at positions 504 to 530 of the amino acid sequence set forth in SEQ ID NO: 1, and / or a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted at positions 698 to 718. At least one selected from the group consisting of The immobilized polypeptide according to claim 1.

3. The immobilized polypeptide of claim 1, wherein the insoluble particles are latex particles.

4. The immobilized polypeptide according to claim 1, wherein the particle diameter of the insoluble particles is 200 nm or more.

5. The immobilized polypeptide of claim 1, wherein the insoluble particles and the polypeptide are covalently bonded.

6. The immobilized polypeptide of claim 1 , wherein the insoluble particles are blocked with caseinate.

7. A kit for detecting anti-gingipain antibodies in a sample, The immobilized polypeptide according to any one of claims 1 to 6 is included. kit.

8. A method for detecting anti-gingipain antibodies in a sample, a detection step of contacting a sample with an immobilized polypeptide, allowing the immobilized polypeptide to bind to anti-gingipain antibodies in the sample, and detecting anti-gingipain antibodies, the immobilized polypeptide comprises insoluble particles and a polypeptide supported on the insoluble particles; The polypeptide is selected from the following (a) to (b): (a) a polypeptide comprising a region consisting of an amino acid sequence of 5 to 30 amino acid residues contained in the amino acid sequence of SEQ ID NO: 1 (b) a polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, and / or inserted in the region; At least one selected from the group consisting of Method for detecting anti-gingipain antibodies.

9. The polypeptide is selected from the following (a') to (b'): (a') a polypeptide comprising a region consisting of the amino acid sequence of positions 504 to 530 and / or a region consisting of the amino acid sequence of positions 698 to 718 of the amino acid sequence set forth in SEQ ID NO: 1 (b') A polypeptide comprising a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted at positions 504 to 530 of the amino acid sequence set forth in SEQ ID NO: 1, and / or a region consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or inserted at positions 698 to 718. At least one selected from the group consisting of The method for detecting an anti-gingipain antibody according to claim 8.

10. The method for detecting an anti-gingipain antibody according to claim 8 , wherein the insoluble particles are latex particles, and the detection is detection of agglutination of the latex particles.

11. The method for detecting an anti-gingipain antibody according to claim 8 , wherein the particle diameter of the insoluble particles is 200 nm or more.

12. The method for detecting an anti-gingipain antibody according to claim 8 , wherein the insoluble particle and the polypeptide are covalently bonded.

13. The method for detecting anti-gingipain antibodies according to claim 8, wherein the insoluble particles of the immobilized polypeptide are blocked with caseinate.

14. The method for detecting an anti-gingipain antibody according to any one of claims 8 to 13, which is a method for assisting in the diagnosis of the presence or absence of Porphyromonas gingivalis infection in a subject from whom the sample is derived.

15. The anti-gingipain antibody detection method according to claim 14 , further comprising a quantification step of determining the amount of anti-gingipain antibody in the subject from the detection value of the anti-gingipain antibody detected in the detection step.

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