Peptide having affinity for and / or inhibitory function against gingipains, and use thereof

Abstract

To provide a novel compound having an affinity and / or inhibitory function specific to gingipains.SOLUTION: The present invention provides a peptide, or a derivative thereof, or a salt of the peptide or derivative, the peptide being selected from among peptides which include amino acid sequence (I) or (II) and in which the total number of amino acid residues is 4 to 15 [amino acid sequence (I): RX1X2RX3 (in amino acid sequence (I), X1, X2, and X3 each independently represent any natural amino acid residue)], [amino acid sequence (II): RX4R (in amino acid sequence (II), X4 represents any natural amino acid residue)], peptides comprising the amino acid sequence CRSVKWHHRVFGSC (SEQ ID NO: 11), and peptides comprising the amino acid sequence ITHTSRHC (SEQ ID NO: 12), wherein one or more of the following conditions (a) to (c) is satisfied: (a) the dissociation constant (KD) with respect to arginine-gingipain is 1.0×10-7 M or less; (b) at 100 μM, the enzymatic activity of arginine-gingipain is inhibited by 50% or more; and (c) at 500 μM, the enzymatic activity of lysine-gingipain is inhibited by 50% or more.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a peptide having affinity and / or inhibitory activity for gingipain and use thereof. More specifically, the present invention relates to a peptide having affinity and / or inhibitory activity for gingipain, and a gingipain inhibitor, a Porphyromonas gingivalis growth inhibitor, an oral composition, and a method for detecting gingipain, each using the peptide. [Background technology]

[0002] Porphyromonas gingivalis is a periodontal disease-causing bacterium that is considered to be important in the onset and progression of periodontal disease, and gingipain, a type of protease produced by Porphyromonas gingivalis, is recognized as an important factor in periodontal disease.

[0003] There are two types of gingipains with different peptide cleavage site specificities: arginine-gingipain (Rgp), which cleaves the C-terminal side of arginine residues, and lysine-gingipain (Kgp), which cleaves the C-terminal side of lysine residues. These interact with each other to directly or indirectly destroy periodontal tissues. The action of gingipains not only leads to the progression and intractability of periodontal disease, but has also recently been considered to be a factor in the development of various diseases including diabetes, arteriosclerosis, cancer, and dementia.

[0004] Therefore, gingipain inhibitors are expected to be effective in treating or preventing not only periodontal disease but also various diseases in which gingipain is involved, and their development is underway (Patent Documents 1 to 5). In addition, compounds that bind to gingipain with high affinity are expected to be applied to periodontal disease diagnostic techniques using gingipain as a marker. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2021-31399 [Patent Document 2] JP 2017-531692 A [Patent Document 3] JP 2018-534300 A [Patent Document 4] JP 2008-150325 A [Patent Document 5] WO2003 / 042237 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a novel substance that has specific affinity and / or inhibitory activity for gingipain. [Means for solving the problem]

[0007] According to the present invention, there is provided a peptide comprising the following amino acid sequence (I) or (II) and having a total of 4 to 15 amino acid residues [amino acid sequence (I): RX1X2RX3 (in the amino acid sequence (I), X1, X2, and X3 each independently represent any natural amino acid residue)], [amino acid sequence (II): RX4R (in the amino acid sequence (II), X4 represents any natural amino acid residue)]; a peptide consisting of the amino acid sequence of CRSVKWHHRVFGSC (SEQ ID NO: 11); and a peptide consisting of the amino acid sequence of ITHTSRHC (SEQ ID NO: 12); and which satisfies one or more of the following (a) to (c), (a) a dissociation constant (K D ) is 1.0 × 10 -7 The present invention provides a peptide or derivative thereof, or a salt thereof, which (a) has a potency of 100 μM or less, (b) inhibits the enzyme activity of arginine-gingipain by 50% or more at 100 μM, and (c) inhibits the enzyme activity of lysine-gingipain by 50% or more at 500 μM. In one embodiment, in the amino acid sequence (I), X1 is selected from the group consisting of A, R, V, and E, X2 is selected from the group consisting of A, F, and K, and X3 is selected from the group consisting of R and F. In one embodiment, in the amino acid sequence (I), X1 is selected from the group consisting of R and E, X2 is selected from the group consisting of A and K, and X3 is R. In one embodiment, in the amino acid sequence (I), X1 is R, X2 is K, and X3 is R, or X1 is R, X2 is A, and X3 is R, or X1 is E, X2 is K, and X3 is R. In one embodiment, in the amino acid sequence (II), X4 is selected from the group consisting of A, P, and K. In one embodiment, in the amino acid sequence (II), X4 is P. In one embodiment, the peptide or derivative or salt thereof does not inhibit the enzymatic activity of papain by more than 5% at 100 μM. In one embodiment, the peptide comprising the amino acid sequence (I) or (II) is a peptide represented by SEQ ID NO:1 to SEQ ID NO:10. According to another aspect of the present invention, there is provided a gingipain inhibitor comprising the above peptide or a derivative thereof, or a salt thereof. According to another aspect of the present invention, there is provided an agent for inhibiting the growth of Porphyromonas gingivalis, comprising the above peptide or a derivative thereof, or a salt thereof. According to another aspect of the present invention, there is provided an oral composition comprising the above peptide or a derivative thereof, or a salt thereof. According to another aspect of the present invention, there is provided a method for detecting gingipain, comprising detecting gingipain by immunoassay using the above-mentioned peptide or a derivative thereof, or a salt thereof. In one embodiment, the immunoassay is a sandwich immunoassay using two types of peptides or derivatives thereof, or salts thereof, which have different amino acid sequences selected from the above peptides. In one embodiment, the sandwich immunoassay is an ELISA assay or an immunochromatographic assay. Effect of the Invention

[0008] According to an embodiment of the present invention, there is provided a novel peptide having affinity and / or inhibitory activity against gingipain. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the selection process of gingipain-binding peptides. [Diagram 2] FIG. 1 shows alanine-substituted peptides of peptide "R3-1." [Diagram 3] FIG. 1 is a schematic diagram illustrating a competition experiment using a QCM. [Figure 4] 1 is a graph showing the affinity change of alanine substituted peptides. [Diagram 5] FIG. 1 shows the results of evaluating the specificity of peptide "R3-1." [Figure 6] FIG. 1 shows the results of evaluating the specificity of peptides "K7294" and "R5-7". [Figure 7] FIG. 1 shows the evaluation effect of the ability to inhibit the proliferation of Porphyromonas gingivalis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited to these embodiments. In this specification, the term "to" indicating a range of values ​​includes the upper and lower limits.

[0011] As used herein, natural amino acids refer to alanine (A), leucine (L), arginine (R), lysine (K), asparagine (N), methionine (M), aspartic acid (D), phenylalanine (F), cysteine ​​(C), proline (P), glutamine (Q), serine (S), glutamic acid (E), threonine (T), glycine (G), tryptophan (W), histidine (H), tyrosine (Y), isoleucine (I), and valine (V).

[0012] A. Peptides According to an embodiment of the present invention, A peptide containing the following amino acid sequence (I) or (II) and having a total of 4 to 15 amino acid residues: [Amino acid sequence (I): RX1X2RX3 (In the amino acid sequence (I), X1, X2, and X3 each independently represent any natural amino acid residue.) [Amino acid sequence (II): RX4R (In the amino acid sequence (II), X4 represents any natural amino acid residue)]; A peptide consisting of the amino acid sequence CRSVKWHHRVFGSC (SEQ ID NO: 11) (peptide "K7226"); and A peptide consisting of the amino acid sequence ITHTSRHC (SEQ ID NO: 12) (peptide "R5-7"); is selected from Provided is a peptide or a derivative thereof, or a salt thereof, which satisfies one or more, and preferably two or more, of the following (a) to (c): (a) Dissociation constant for Rgp (K D ) is 1.0 × 10 -7 M or less, preferably 1.0×10 -8 M or less, and more preferably 1.0×10 -9 It is below M. (b) At 100 μM, it inhibits the enzyme activity of Rgp by 50% or more. In other words, it has an IC 50 is 100 μM or less, preferably 60 μM or less, and more preferably 40 μM or less. (c) Inhibits the enzyme activity of Kgp by 50% or more at 500 μM. In other words, IC 50 is 500 μM or less, preferably 400 μM or less, and more preferably 300 μM or less.

[0013] In one embodiment, the peptide does not inhibit the enzymatic activity of papain, which is classified as a cysteine ​​protease like gingipain, by more than 5% at 100 μM.

[0014] The peptide consisting of the amino acid sequence of SEQ ID NO: 11 (peptide "K7226") may be cyclic via disulfide bonds at both terminal C's.

[0015] The number of R in the amino acid sequence of the peptide is typically 1 or more, for example 1, 2, 3 or 4, and may be 5 or more.

[0016] In the amino acid sequence (I), X1 is, for example, selected from the group consisting of an alanine residue (A), an arginine residue (R), a valine residue (V), and a glutamic acid residue (E), and is preferably selected from the group consisting of R and E.

[0017] In the amino acid sequence (I), X2 is, for example, selected from the group consisting of an alanine residue (A), a phenylalanine residue (F), and a lysine residue (K), and is preferably selected from the group consisting of A and K.

[0018] In the amino acid sequence (I), X3 is, for example, selected from the group consisting of an arginine residue (R) and a phenylalanine residue (F), and is preferably R.

[0019] In one embodiment, in the amino acid sequence (I), X1 is R, X2 is K, and X3 is R. In one embodiment, in the amino acid sequence (I), X1 is R, X2 is A, and X3 is R. In one embodiment, in the amino acid sequence (I), X1 is E, X2 is K, and X3 is R.

[0020] The total number of amino acid residues of the peptide containing the amino acid sequence (I) is typically 5 to 15, preferably 5 to 14, and may be, for example, 5 to 12.

[0021] When the total number of amino acid residues of a peptide containing amino acid sequence (I) is 6 or more, the number of peptides added to the N-terminus of amino acid sequence (I) may be equal to or greater than the number of peptides added to the C-terminus. The number of peptides added to the N-terminus of amino acid sequence (I) is typically 1 to 10, for example 1 to 8.

[0022] Specific examples of peptides containing amino acid sequence (I) are shown in Table 1. In the table, the underlined parts indicate sequences corresponding to amino acid sequence (I). [Table 1]

[0023] In the amino acid sequence (II), X4 is, for example, selected from the group consisting of an alanine residue (A), a proline residue (P), and a lysine residue (K), and is preferably P.

[0024] The total number of amino acid residues of the peptide containing the amino acid sequence (II) is typically 4 to 15, preferably 5 to 14, and for example 5 to 10.

[0025] When the total amino acid residues of a peptide comprising amino acid sequence (II) is 4 or more, the number of peptides added to the N-terminus of amino acid sequence (II) may be equal to or greater than the number of peptides added to the C-terminus. The number of peptides added to the N-terminus of amino acid sequence (I) is typically 1 to 12, and may be, for example, 1 to 8. In one embodiment, the peptide comprising amino acid sequence (II) comprises an amino acid sequence represented by LRX4R, and may be, for example, a peptide comprising an amino acid sequence represented by KLRX4R or ILRX4R.

[0026] Specific examples of peptides containing amino acid sequence (II) are shown in Table 2. In the table, the underlined parts indicate the sequences corresponding to amino acid sequence (II). [Table 2]

[0027] The peptides can be synthesized by chemical synthesis methods such as solid-phase synthesis, stepwise elongation, and liquid-phase synthesis. Among them, solid-phase synthesis is preferred. Examples of solid-phase synthesis include Fmoc synthesis and Boc synthesis.

[0028] Since the above peptide has a certain affinity and / or inhibitory ability for gingipain, it can be used as a therapeutic, preventive, or diagnostic agent for diseases associated with gingipain. Therefore, according to another aspect of the present invention, a therapeutic, preventive, or diagnostic agent for diseases associated with gingipain, which contains the above peptide as an active ingredient, can be provided. The amount of peptide in the therapeutic, preventive, or diagnostic agent may be an effective amount that can exert its function, and can be appropriately set depending on the application, dosage form, and the like. Examples of diseases associated with gingipain include periodontal disease, type 2 diabetes, cardiovascular disease, pneumonia, rheumatoid arthritis, Alzheimer's dementia, premature birth, and low birth weight.

[0029] The above peptides may be in the form of derivatives or salts as long as the effects of the present invention can be obtained. Thus, unless otherwise specified in this specification, the peptides may be derivatives of the peptides or salts of the peptides or their derivatives.

[0030] Examples of peptide derivatives include those in which functional groups such as the N-terminal amino group, the C-terminal carboxyl group, the carboxyl group of the side chain, the amino group, the guanidino group, the hydroxyl group, and the thiol group of the peptide are substituted with various substituents. The substituents are not particularly limited, and examples thereof include alkyl groups, acyl groups, hydroxyl groups, amino groups, alkylamino groups, nitro groups, amide groups, sulfonyl groups, halogens, and various protective groups. These substituents may be further substituted with halogens such as fluorine. The substitution may also be the introduction of labels such as fluorescent labels and biotin labels.

[0031] The salt of the peptide is preferably a pharmacologically acceptable salt. Pharmacologically acceptable salts include acid addition salts and base addition salts. Examples of acid addition salts include inorganic acid salts and organic acid salts. Examples of inorganic acid salts include hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, etc. Examples of organic acid salts include citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, etc. Examples of base addition salts include inorganic base salts and organic base salts. Examples of inorganic base salts include sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, etc. Examples of organic base salts include organic base salts such as triethylammonium salt, triethanolammonium salt, pyridinium salt, diisopropylammonium salt, etc.

[0032] B. Gingipain Inhibitors The peptide described in Section A can exert a gingipain inhibitory effect. Therefore, according to another aspect of the present invention, a gingipain inhibitor is provided, which comprises the peptide described in Section A. The gingipain to be inhibited can be either or both of Rgp and Kgp. The peptide is preferably a peptide having an IC 50 For example, peptides having a concentration of 500 μM or less, preferably 300 μM or less, more preferably 100 μM or less can be preferably used.

[0033] The gingipain inhibitor may consist of only the peptide, or may contain other components, such as a pharmacologically acceptable carrier, a gingipain inhibitor other than the peptide (e.g., gabexate mesilate, leupeptin, antipain, KYT-01, E-64, KYT-36, KYT-41, hinokitiol, copper chlorophyllin metal salt), etc.

[0034] The content of the peptide in the gingipain inhibitor may be an effective amount that provides the gingipain inhibitory effect of the peptide, and may be, for example, 0.1% to 100% by weight, preferably 1% to 100% by weight.

[0035] Gingipain inhibitors can be used as therapeutic, preventive, or diagnostic agents for diseases associated with gingipain, such as those described in Section A.

[0036] C. gingivalis growth inhibitor The peptide described in section A can exert an inhibitory effect on the proliferation of P. gingivalis. Thus, according to another aspect of the present invention, there is provided a P. gingivalis proliferation inhibitor comprising the peptide described in section A. The P. gingivalis proliferation inhibitor may consist of only the peptide, or may contain other components. Examples of other components include a pharmacologically acceptable carrier, a P. gingivalis proliferation inhibitor other than the peptide (e.g., an antibiotic such as minocycline), and the like.

[0037] The content of the peptide in the Porphyromonas gingivalis growth inhibitor may be an effective amount that provides an inhibitory effect on the growth of Porphyromonas gingivalis by the peptide, and may be, for example, 0.1% to 100% by weight, preferably 1% to 100% by weight.

[0038] D. Oral composition According to another aspect of the present invention, there is provided an oral composition comprising the peptide described in Section A. Due to the inclusion of the peptide, the oral composition can exhibit an inhibitory effect on gingipain activity and / or an inhibitory effect on the proliferation of Porphyromonas gingivalis.

[0039] The amount of peptide in the oral composition may be an effective amount that can obtain an inhibitory effect on gingipain activity and / or an inhibitory effect on the proliferation of Porphyromonas gingivalis. The amount of peptide, etc. in the oral composition may vary depending on the formulation of the composition, but is, for example, 0.1% by weight to 80% by weight, preferably 0.5% by weight to 50% by weight, and more preferably 1% by weight to 50% by weight.

[0040] The oral composition may be, for example, a dentifrice such as a toothpaste, a lubricant, a powdered dentifrice, a liquid dentifrice, a liquid dentifrice, a mouthwash, a mouth freshener, an oral spray, an oral liniment, or a mouthwash, etc. Furthermore, for example, the oral composition may be, for example, a tablet, a gum, a candy, or a beverage.

[0041] In addition to the peptides, any appropriate components may be added to the oral composition depending on the dosage form, application, etc., within the scope of not impairing the effects of the present invention. For example, a dentifrice may contain abrasives, thickeners, binders, surfactants, flavors, sweeteners, colorants, preservatives, active ingredients, etc.

[0042] Examples of the abrasive include silica-based abrasives such as silica gel, precipitated silica, aluminosilicate, zirconosilicate, etc., dibasic calcium phosphate dihydrate and anhydrate, tribasic calcium phosphate, quaternary calcium phosphate, calcium pyrophosphate, calcium carbonate, aluminum hydroxide, alumina, magnesium carbonate, tribasic magnesium phosphate, zeolite, hydroxyapatite, synthetic resin-based abrasives, etc. The amount of the abrasive to be added is adjusted depending on the dosage form, and in toothpaste, it is, for example, 2% by weight to 40% by weight, preferably 10% by weight to 30% by weight, and in liquid toothpaste, it may be 0% by weight.

[0043] Examples of the thickening agent include sugar alcohols such as sorbitol, xylitol, maltitol, lactitol, etc., and polyhydric alcohols such as glycerin, propylene glycol, polyethylene glycol, etc. The blending amount is usually 5% by weight to 50% by weight, preferably 20% by weight to 45% by weight.

[0044] Examples of binders include cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, etc., gums such as xanthan gum, gum arabic, etc., organic binders such as carrageenan, polyvinyl alcohol, sodium polyacrylate, etc., and inorganic binders such as gelling silica, gelling aluminum silica, Veegum, Laponite, etc. The blending amount is usually 0.1% by weight to 5% by weight for toothpaste, and 0% by weight to 5% by weight for liquid toothpaste and mouthwash.

[0045] The surfactant that can be blended includes anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants.

[0046] Examples of the anionic surfactant include alkyl sulfates such as sodium lauryl sulfate, N-acyl sarcosinates such as sodium N-lauroyl sarcosine and sodium N-myristoyl sarcosine, N-acyl glutamates such as sodium N-palmitoyl glutamate, sodium N-methyl-N-acyltaurate, sodium N-methyl-N-acylalanine, and sodium α-olefin sulfonate.

[0047] Examples of the nonionic surfactant include sugar fatty acid esters such as sucrose fatty acid esters and maltose fatty acid esters, sugar alcohol fatty acid esters such as maltitol fatty acid esters and lactitol fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters such as hexaglyceryl monolaurate, hexaglyceryl monomyristate, decaglyceryl monolaurate and decaglyceryl monomyristate, polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate and polyoxyethylene sorbitan monostearate, polyoxyethylene fatty acid esters such as polyoxyethylene hydrogenated castor oil, polyoxyethylene higher alcohol ethers such as polyoxyethylene lauryl ether, fatty acid alkanolamides such as lauric acid diethanolamide, polyoxyethylene polyoxypropylene copolymers, and polyoxyethylene polyoxypropylene fatty acid esters.

[0048] Examples of the cationic surfactant include alkyl ammonium and alkyl benzyl ammonium salts, and examples of the amphoteric surfactant include betaine-based surfactants such as alkyl betaine, fatty acid amidopropyl betaine, and alkyl imidazolinium betaine.

[0049] The amount of the surfactant in the oral cavity composition is, for example, 0.001% by weight to 10% by weight, and preferably 0.1% by weight to 5% by weight.

[0050] Fragrances include peppermint oil, spearmint oil, anise oil, eucalyptus oil, wintergreen oil, cassia oil, clove oil, thyme oil, sage oil, lemon oil, orange oil, peppermint oil, cardamom oil, coriander oil, mandarin oil, lime oil, lavender oil, rosemary oil, laurel oil, chamomile oil, caraway oil, marjoram oil, bay oil, lemongrass oil, origanum oil, pine needle oil, neroli oil, rose oil, jasmine oil, grapefruit oil, and sweet Natural fragrances such as citron oil, iris concrete, absolute peppermint, absolute rose, orange flower, and processed fragrances (front distillation, back distillation, fractional distillation, liquid-liquid extraction, essences, powdered fragrances, etc.) of these natural fragrances, as well as menthol, carvone, anethole, cineole, methyl salicylate, cinnamic aldehyde, eugenol, 3-l-menthoxypropane-1,2-diol, thymol, linalool, linalyl a It is possible to use in combination with known flavor materials used in oral compositions, such as single flavors such as acetate, limonene, menthone, menthyl acetate, N-substituted-paramenthan-3-carboxamide, pinene, octylaldehyde, citral, pulegone, carbyl acetate, anisaldehyde, ethyl acetate, ethyl butyrate, allyl cyclohexane propionate, methyl anthranilate, ethyl methylphenyl glycidate, vanillin, undecalactone, hexanal, butanol, isoamyl alcohol, hexenol, dimethyl sulfide, cyclotene, furfural, trimethylpyrazine, ethyl lactate, and ethyl thioacetate, as well as compound flavors such as strawberry flavor, apple flavor, banana flavor, pineapple flavor, grape flavor, mango flavor, butter flavor, milk flavor, fruit mix flavor, and tropical fruit flavor.

[0051] The flavor material is preferably used in an oral cavity composition in an amount of 0.000001% by weight to 1% by weight, and the flavoring using the flavor material is preferably used in an oral cavity composition in an amount of 0.1% by weight to 2% by weight.

[0052] Examples of sweeteners include sodium saccharin, stevioside, paramethoxycinnamic aldehyde, perillartine, etc. Examples of colorants include blue No. 1, yellow No. 4, titanium dioxide, etc.

[0053] Examples of the preservative include benzoic acid or a salt thereof such as paraoxybenzoic acid ester, sodium benzoate, and the like.

[0054] In addition to the above peptides, the oral cavity composition may contain nonionic bactericides such as isopropylmethylphenol, cationic bactericides such as cetylpyridinium chloride, chlorhexidine hydrochloride, benzalkonium chloride, and benzethonium chloride, anti-inflammatory agents such as tranexamic acid, epsilon aminocaproic acid, allantoin, glycyrrhetinic acid, and glycyrrhizinic acid, enzymes such as dextranase, mutanase, amylase, and protease, fluorides such as sodium fluoride and sodium monofluorophosphate, water-soluble phosphate compounds such as potassium salt and sodium salt of orthophosphoric acid, copper compounds such as copper gluconate and copper chlorophyllin sodium, inorganic salts such as sodium chloride, potassium nitrate, aluminum lactate, zinc chloride, zinc citrate, and strontium chloride, vitamins such as tocopherol acetate, zeolite, azulene, dihydrocholesterol, chlorophyll, Angelica acutiloba extract, thyme, Scutellaria baicalensis, clove, and witch hazel, anti-tartar agents, anti-plaque agents, and the like. These components can be blended in effective amounts within the range that does not impair the effects of the present invention.

[0055] E. How to detect gingipains According to another aspect of the present invention, there is provided a method for detecting gingipain, comprising detecting gingipain by immunoassay using the peptide described in section A. The gingipain to be detected may be either or both of Rgp and Kgp. The dissociation constant of the peptide with respect to the gingipain to be detected is preferably 1.0×10 -8 M or less, preferably 1.0×10 -9 It is below M.

[0056] Immunological measurement methods include immunoblotting, enzyme immunoassay (EIA, ELISA), radioimmunoassay (RIA), fluorescent antibody method, immunochromatography, etc. Among them, ELISA or immunochromatography is preferred.

[0057] The immunoassay is preferably a sandwich immunoassay, such as a sandwich ELISA or sandwich immunochromatography. The sandwich immunoassay is a method for qualitatively or quantitatively analyzing a test substance by immobilizing a capture antibody on a support and allowing the labeled antibody and the capture antibody to bind to the test substance in a sandwich fashion.

[0058] In the sandwich immunoassay, two types of peptides A and B, which are described in Section A and have different amino acid sequences (as a result, peptides A and B each recognize different sites of the gingipain to be detected), can be used as a capture antibody and a labeled antibody.

[0059] For example, in the sandwich ELISA method, unlabeled peptide A is immobilized on a carrier such as a 96-well microtiter plate or polystyrene beads as a capture antibody (primary antibody), or biotin-labeled peptide A is immobilized on an avidin-coated 96-well microtiter plate, and peptide B labeled with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), a fluorescent substance such as FITC, or a labeling substance such as biotin can be used as a labeled antibody (secondary antibody). The labeled antibody may be labeled with biotin, and labeled avidin or streptavidin may be bound to biotin to develop color. The binding of gingipain to the capture antibody or labeled antibody may be performed, for example, at a temperature condition of 0° C. to 37° C., preferably 4° C. to 25° C., for example, for a reaction time of 30 minutes to 24 hours, preferably 60 minutes to 120 minutes.

[0060] In the above-mentioned sandwich immunoassay method, when a peptide is immobilized on a support, the influence of the support can be mitigated by interposing a stand peptide between the support and the peptide, thereby improving the measurement accuracy of gingipain.

[0061] F. Kit for detecting or measuring gingipain According to another aspect of the present invention, there is provided a kit for detecting or measuring gingipain, which comprises at least two peptides having different amino acid sequences, each of which is the peptide described in section A (as a result, each of the two peptides recognizes a different site of gingipain). The gingipain to be detected or measured may be either or both of Rgp and Kgp. One of the two peptides may be immobilized on a support, and the other may be labeled with a labeling substance.

[0062] The kit may include reagents such as a sample collection tool, a sample treatment solution, and a color-developing substrate, and may further include tools and the like necessary for the test. EXAMPLES

[0063] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0064] 1. Selection of gingipain-binding peptides Peptides having affinity for gingipain were selected according to the scheme shown in FIG. 1 by the in vitro virus method using the linker described in WO2006 / 041194. Specifically, a cDNA display library consisting of 8 random amino acids prepared according to the description in WO2006 / 041194 was mixed with target immobilization beads (streptavidin-modified magnetic beads) (Fig. 1(a)), peptides nonspecifically bound to the target immobilization beads were removed, and peptides not bound to the beads were mixed with beads immobilized with gingipain (Rgp) purified from the culture supernatant of P. gingivalis (ATCC 33277 strain) (Fig. 1(b)). Next, the supernatant was removed by centrifugation, and the peptides bound to gingipain were recovered by washing and elution (Fig. 1(c)). The same procedure was repeated four more times while gradually increasing the severity of the washing conditions (a total of five selections). Specifically, 10 mM sodium phosphate (pH 7.5), 100 mM sodium chloride, and 5 mM magnesium chloride were used as the selection buffer. For the first selection wash, 10 mM sodium phosphate (pH 7.5) and 1 M sodium chloride were used, for the second and third selection wash, 10 mM sodium phosphate (pH 7.5) and 2 M sodium chloride were used, for the fourth selection wash, 10 mM sodium phosphate (pH 7.5) and 3 M sodium chloride were used, and for the fifth selection wash, 10 mM sodium phosphate (pH 7.5), 3 M sodium chloride, and 0.1% SDS were used. The obtained gingipain-binding peptide was cloned using a commercially available cloning kit (Zero Blunt TOPO PCR Cloning Kit, Invitrogen), a sequencing sample was prepared using the dideoxy method, and the base sequence was determined using a sequencer. The amino acid sequence of the gingipain-binding peptide was determined based on the base sequence. The purification of Rgp from the supernatant of the P. gingivalis culture solution was carried out as follows. 1. Approximately 10 liters of P. gingivalis (ATCC 33277 strain) culture supernatant was collected, concentrated, and dialyzed against 10 mM phosphate buffer (pH 7.0), and then subjected to DEAE Sepharose anion chromatography equilibrated with the same buffer. After thoroughly washing the column with the same buffer, the active fractions eluted with the same buffer containing 0.1 M sodium chloride were collected and concentrated. 2. The obtained active fraction was dialyzed against 10 mM acetate buffer (pH 5.5) and applied to CM Sephadex cation chromatography equilibrated with the same buffer. The non-adsorbed fraction eluted with the same buffer was collected, concentrated, and dialyzed against 10 mM phosphate buffer (pH 7.0). 3. The active fraction was subjected to Mono Q anion chromatography equilibrated with the same buffer solution. The active fraction eluted with a sodium chloride gradient was collected and concentrated by dialysis. 4. The enzyme fraction was subjected to affinity chromatography on Arg-Sepharose. The fractions adsorbed on the column were eluted with lysine and concentrated. 5. The concentrated sample was subjected to gel filtration using a TSK-G2000SW column (Tosoh), and the active fraction was concentrated. 6. SDS gel electrophoresis confirmed that the product was purified to a single band. The gingipain concentration in the culture supernatant was determined by the method described by Kadowaki et al., J. Biol. Chem. 269:21371-8 (1994).

[0065] In addition, a cDNA display library consisting of peptides (total number of amino acid residues: 14) with cysteines at both ends and 12 random amino acids between them was prepared, and a total of seven selections were performed in the same manner as above using recombinant gingipain (Abcam's "Recombinant Gingipain R1 protein (His tag)") to obtain gingipain-binding peptides. Specifically, the first two elutions were performed with dithiothreitol, the third to sixth elutions were performed with biotin, and the seventh elution was performed by competitive elution with KYT-1, an Rgp inhibitor. The amino acid sequence of the resulting gingipain-binding peptide was determined in the same manner as described above.

[0066] Candidate peptides were selected taking into consideration the characteristics of the amino acid sequence and ease of synthesis, and peptide synthesis was carried out by the Fmoc method (purity of the synthesized peptides by RP-HPLC was ≧90%).

[0067] 2. Molecular design of gingipain-binding peptides The synthetic peptides were evaluated for affinity to gingipain, and peptides that showed favorable evaluation results were subjected to molecular design for the purpose of further enhancing their functionality. An example of molecular design is described below. The evaluation of affinity to gingipain is also described later.

[0068] For the peptide "R3-1" (SEQ ID NO: 10), which was confirmed to have high affinity for gingipain, we investigated the important sites for binding with gingipain. Specifically, to examine the effect of the peptide side chain, we synthesized analogs in which each amino acid residue of the peptide was replaced with alanine (however, since glycine residues have only hydrogen atoms in their side chains, we did not synthesize analogs in which glycine residues were replaced with alanine, and did not evaluate their activity). The synthesized alanine-substituted peptides are shown in Figure 2 (in the figure, "Ahx" represents 6-aminohexanoic acid as a hydrophobic spacer).

[0069] The change in affinity of these alanine-substituted peptides to gingipain was examined using the quartz crystal microbalance (QCM) method. Specifically, gingipain was contacted with a substrate on which the alanine-substituted peptides had been immobilized in advance, and the alanine-substituted peptide and gingipain were added to bind to each other. Then, peptide "R3-1" was added. If the alanine-substituted peptide had a lower affinity for gingipain than peptide "R3-1," gingipain was transferred to peptide "R3-1" by a competitive reaction (Figure 3, upper case). On the other hand, depending on the concentration, if the alanine-substituted peptide had a higher affinity for gingipain than peptide "R3-1," it is believed that it would basically remain bound to gingipain (Figure 3, lower case). Therefore, if the affinity was reduced, the weight on the substrate would change, and this change could be read to evaluate the change in affinity.

[0070] Figure 4 shows the results of measuring the change in affinity for seven types of alanine-substituted peptides. The results in Figure 4 indicate that the four residues from the C-terminus of peptide "R3-1" play an important role in binding to gingipain, while the three residues from the N-terminus of the peptide have a relatively low contribution to the binding activity. This also suggests that the three N-terminal residues of peptide "R3-1" may be chemically modified without significantly affecting the interaction with gingipain. Furthermore, a peptide analogue without the three N-terminal residues of peptide "R3-1" could reduce the cost of peptide synthesis and improve the binding specificity to proteins. Therefore, we designed a peptide without the three N-terminal residues of peptide "R3-1" and a peptide derivative with the three N-terminal residues chemically modified.

[0071] Various peptides (including peptide derivatives) were obtained by the peptide selection described in 1. and the molecular design described in 2. The sequences and structures of the obtained peptides are shown in Table 3. [Table 3]

[0072] 3.Affinity evaluation for gingipain The affinity of peptides to gingipains was determined by Biacore using surface plasmon resonance (SPR) technology. (R) Specifically, the synthesized peptides were analyzed using the Biacore (R) The peptide was immobilized on a sensor chip for the system, and a purified solution of the culture supernatant of a wild-type P. gingivalis strain was added thereto. In this system, when gingipain in the culture supernatant binds to the peptide on the chip, the mass of the immobilized peptide increases, causing a change in the refractive index of the solvent on the sensor chip surface, and by measuring this change, it becomes possible to monitor the state of binding in real time. The binding rate constant (ka) and dissociation rate constant (kd) were calculated from the sensorgram curves at the time of adding the culture supernatant and after the addition, and the dissociation constant (K D = kd / ka) was calculated. The results are shown in Table 4.

[0073] 4. Specificity Assessment Specificity evaluation was carried out for peptide "R3-1." Specifically, Biacore (R) Using the system, a purified solution of culture supernatant of P. gingivalis wild-type strain (ATCC 33277 strain) (wild type, protein amount: 22 μg / mL), a purified solution of culture supernatant of a P. gingivalis mutant strain in which the gingipain gene had been knocked out (-Kgp & -Rgp, protein amount: 22 μg / mL), and a 10-fold concentrated solution of the purified solution (-Kgp & -Rgp, protein amount: 220 μg / mL) were added onto a sensor chip on which each peptide was immobilized.

[0074] As a result, as shown in Figure 5, peptide "R3-1" showed a very strong reaction to purified culture supernatant containing gingipain (wild type, protein amount: 22 μg / mL), while it showed a very weak reaction to purified culture supernatant not containing gingipain (-Kgp & -Rgp, protein amount: 22 μg / mL), and although it showed some reaction to the 10x concentrated solution (-Kgp & -Rgp, protein amount: 220 μg / mL), the reaction was lower than that of the culture supernatant containing gingipain. From this, it is considered that the specificity of peptide "R3-1" to gingipain is quite high.

[0075] Furthermore, the peptides "K7294" and "R5-7" were also evaluated for specificity in the same manner. As a result, as shown in FIG. 6, both of them showed a very strong reaction to the culture supernatant containing gingipain (wild type, protein amount: 22 μg / mL) like the peptide "R3-1", but showed a lower reaction than the peptide "R3-1" to the culture supernatant not containing gingipain (-Kgp & -Rgp, protein amount: 22 μg / mL) and the 10-fold concentrated solution (-Kgp & -Rgp, protein amount: 220 μg / mL). This suggests that the peptides "K7294" and "R5-7" have higher specificity for gingipain than "R3-1".

[0076] 5. Evaluation of inhibitory potential for Rgp activity The inhibitory ability of the peptides to Rgp activity was evaluated by the following method. 1) 90 μL of 1M L-cysteine ​​aqueous solution was neutralized by adding 10 μL of 4M NaOH aqueous solution (0.9M cysteine). 2) GingisREX (Arg-gingipain, 1 μM, manufactured by GENOVIS) was diluted 40-fold with Nuclease-free Water (NFW) (manufactured by Ambion) (25 nM GingisREX). 3) 10 μL of 1 M Tris-HCl (pH 7.5), 1.11 μL of 0.9 M Cysteine, and 71.89 μL of NFW were added to each well of a 96-well NBS black plate (Corning #3991) and mixed, and then 2 μL of 25 nM GingisREX was added (total 85 μL / well). 4) Peptide solutions adjusted to various concentrations using dimethyl sulfoxide as a solvent or the solvent alone were added to the wells at 5 μL / well, and the plates were shaken at approximately 500 rpm for 10 seconds on a plate shaker. 5) 10 μL of Z-Phe-Arg-MCA (EPTIDE INSTITUTE, #3095-V) diluted to 1 mM with NFW was added to each well, and the plate was shaken at approximately 500 rpm for 10 seconds on a plate shaker. 6) Fluorescence values ​​(Ex: 380 nm, Em: 460 nm) were immediately measured at 37°C using a multispectro microplate reader Varioskan Flash (Thermo Scientific, #5250040) at 30 second intervals for 20 minutes. 7) For each well, the slope of the measured values ​​per 5 minutes was calculated, and the maximum value was used to calculate the ratio to the wells to which only the solvent was added, which was taken as the activity ratio. 8) The activity rate was subtracted from 1 to obtain the inhibition rate. 50% inhibition rate (IC 50 ) are shown in Table 4.

[0077] 6. Evaluation of inhibitory potential for Kgp activity The inhibitory ability of peptides to Kgp activity was evaluated by the following method. 1) 90 μL of 1M L-cysteine ​​aqueous solution was neutralized by adding 10 μL of 4M NaOH aqueous solution (0.9M cysteine). 2) 10 μL of 1 M Tris-HCl (pH 8.0), 0.222 μL of 0.9 M Cysteine, and 72.778 μL of NFW were added to each well of a 96-well NBS black plate (Corning #3991) and mixed, and then 2 μL of GingisKHAN (Lys-gingipain, 10 U / μL, manufactured by GENOVIS) was added (total of 85 μL / well). 3) Peptide solutions adjusted to various concentrations using dimethyl sulfoxide as a solvent or the solvent alone were added to the wells at 5 μL / well, and the plates were shaken at approximately 500 rpm for 10 seconds on a plate shaker. 4) 10 μL of Z-His-Glu-Lys-MCA (EPTIDE INSTITUTE, #3215-V) diluted to 1 mM with NFW was added to each well, and the plate was shaken at approximately 500 rpm for 10 seconds on a plate shaker. 5) Fluorescence values ​​(Ex: 380 nm, Em: 460 nm) were immediately measured at 37°C using a multispectro microplate reader Varioskan Flash (Thermo Scientific, #5250040) at 30 second intervals for 20 minutes. 6) For each well, the slope of the measured values ​​per 5 minutes was calculated, and the maximum value was used to calculate the ratio to the wells to which only the solvent was added, which was taken as the activity ratio. 7) The activity ratio was subtracted from 1 to obtain the inhibition rate. 50% inhibition rate (IC 50 ) are shown in Table 4.

[0078] [Table 4]

[0079] 7. Evaluation of papain activity inhibition ability The inhibitory ability of peptides to cysteine ​​protease (papain) activity was evaluated by the following method. 1) 90μL of 1M L-cysteine ​​solution was neutralized by adding 10μL of 4M NaOH solution (0.9M Cysteine). 2) Papain (Sigma #P4762) was dissolved in Nuclease-free Water (NFW) (Ambion). (5 μg / mL papain) 3) 50 μL of 100 mM HEPES (pH 7.0), 4 μL of 50 mM EDTA, 1.11 μL of 0.9 M Cysteine, and 27.89 μL of NFW were added to each well of a 96-well NBS black plate (Corning #3991) and mixed, and then 2 μL of 5 μg / mL papain was added (total of 85 μL / well). 4) Peptide solutions adjusted to various concentrations using dimethyl sulfoxide as a solvent or the solvent alone were added to the wells at 5 μL / well, and the plates were shaken at approximately 500 rpm for 10 seconds on a plate shaker. 5) 10 μL of Z-Phe-Arg-MCA (EPTIDE INSTITUTE, #3095-V) diluted to 1 mM with NFW was added to each well, and the plate was shaken at approximately 500 rpm for 10 seconds on a plate shaker. 6) The fluorescence values ​​(Ex: 380 nm, Em: 460 nm) were immediately measured at 37°C using a multispectro microplate reader Varioskan Flash (Thermo Scientific, #5250040) at 30 second intervals for 20 minutes. 7) For each well, the slope of the measured values ​​per 5 minutes was calculated, and the maximum value was used to calculate the ratio to the wells to which only the solvent was added, which was taken as the activity ratio. 8) The activity rate was subtracted from 1 to obtain the inhibition rate. As a result, the inhibition rate of peptides "R3-1", "R5-7", "R5-35", "K7419", and "K7455" was below the detection limit (approximately 1%) at 100 μM, and no significant inhibitory activity was confirmed. In contrast, the inhibitory activity of E-64, known as an inhibitor of cysteine ​​proteases, against papain was similarly examined, and its IC 50 The value was 7 nM. In addition, when the inhibitory activity of peptides "R3-1," "K7419," and "K7455" against other enzymes at 100 μM was evaluated according to standard methods, the inhibition rates against the serine protease thrombin and the aspartic acid protease cathepsin D were all below the detection limit (approximately 1%), and no significant inhibitory activity was confirmed.

[0080] 8. Evaluation of Porphyromonas gingivalis growth inhibition ability Porphyromonas gingivalis (JCM12257) obtained in a freeze-dried state from the Japan Collection of Microorganisms (JCM) at the RIKEN BioResource Research Center was subcultured in ATCC Medium 2722 Supplemented Tryptic Soy Broth (hereinafter referred to as ATCC2722 medium) at 37°C under anaerobic conditions for two days, after which the absorbance was measured at 620 nm. A bacterial suspension was prepared by subtracting the absorbance of the medium alone (blank) so that the absorbance was approximately the same as that of the McFarland standard suspension (rounded off to the third decimal place).

[0081] Peptide "R3-1" was dissolved in dimethyl sulfoxide, and the resulting solution was diluted with distilled water (Otsuka Pharmaceutical Factory, Lot. 0D96N) to a final peptide concentration of 1,280 μg / mL. After that, it was diluted with ATCC2722 medium to 160 μg / mL, 20 μg / mL, and 2.5 μg / mL, and then diluted with the same medium to a final concentration of 0.06 μg / mL, 0.12 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, and 128 μg / mL. Various dilutions of gabexate methylate (Tokyo Chemical Industry Co., Ltd.) and KYT-1 (Peptide Institute) were prepared in the same manner.

[0082] Each concentration of test substance (peptide, gabexate methylate (GM), or KYT-1) and 100 μL of ATCC2722 medium (positive control; PC) were added to a 96-well U-shaped microplate, followed by the addition of 10 μL of bacterial solution. As a negative control (NC), 10 μL of ATCC2722 medium was added instead of the bacterial solution. Then, the plates were cultured at 37°C for 2 days under anaerobic conditions.

[0083] After shaking with a microplate mixer, the absorbance of each well at a wavelength of 620 nm was measured (iMark microplate reader, Bio-Rad Laboratories, Inc.) and used as the OD value. A concentration-response curve was prepared, and the sample concentration that satisfied the value of the following formula was calculated based on the curve, and the 50% growth inhibitory concentration (GI 50 The results are shown in Table 5 and Figure 7. G.I. 50 OD value equivalent to = (OD value of PC - OD value of NC) x 0.5 + OD value of NC

[0084] [Table 5]

[0085] 9. ELISA measurement of gingipains using peptides 1) A solid-phase peptide solution (1 μg / mL) diluted with 10 mM HEPES buffer (pH 7.0) containing 150 mM NaCl was added to an avidin plate (Sumitomo Bakelite Co., Ltd., "BS-X7603") at a volume of 100 μL / well. 2) After overnight reaction at 4°C, the reaction solution was discarded and each well was washed three times with 0.05% Tween-containing PBS (pH 7.4) at a volume of 300 µL / well. 3) A sample was added at a volume of 100 μL / well. The sample was an Rgp solution dissolved in HEPES buffer to a concentration of 0.1 nM, 1 nM, or 10 nM. 4) After overnight reaction at 4°C, the reaction solution was discarded and each well was washed three times with PBS (pH 7.4) containing 0.05% Tween as a washing solution at a volume of 300 µL / well. 5) A dimethyl sulfoxide solution (10 mM) of the labeled peptide was diluted 2,000-fold with HEPES buffer and added at a volume of 100 μL / well. 6) After overnight reaction at 4°C, the reaction solution was discarded and each well was washed three times with PBS (pH 7.4) containing 0.05% Tween at a volume of 300 µL / well. 7) HRP-labeled streptavidin was diluted 10,000-fold with HEPES buffer and added at 100 μL / well. 8) After overnight reaction at 4°C, the reaction solution was discarded and each well was washed three times with 0.05% Tween-containing PBS (pH 7.4) at a volume of 300 µL / well. 9) After discarding the washing solution, TMB substrate (SeraCare Life Sciences) was added at 100 μL / well and reacted at room temperature for 20 minutes. 10) 2N sulfuric acid was added at a volume of 100 μL / well, and the absorbance at 450 nm was immediately measured using a plate reader (Practical).

[0086] Table 6 shows the combinations of peptides used as solid-phase peptides and labeled peptides, as well as the results of absorbance measurements. [Table 6]

[0087] As shown in Table 6, the absorbance increased depending on the concentration of gingipain, which indicates that Rgp can be detected by the sandwich ELISA method using two types of gingipain-binding peptides with different sequences. [Industrial Applicability]

[0088] The peptide according to the embodiment of the present invention is preferably used in the treatment, prevention, diagnosis, etc. of gingipain-related diseases such as periodontal disease.

Claims

1. A peptide comprising the following amino acid sequence (I) or (II) and having a total of 4 to 15 amino acid residues: [Amino acid sequence (I): RX １ X ２ R.X. ３ (In the amino acid sequence (I), X １ , X ２ , and X ３ each independently represents any naturally occurring amino acid residue); [Amino acid sequence (II): RX ４ R (In the amino acid sequence (II), X ４ represents any naturally occurring amino acid residue); A peptide consisting of the amino acid sequence of CRSVKWHHRVFGSC (SEQ ID NO: 11); and a peptide consisting of the amino acid sequence of ITHTSRHC (SEQ ID NO: 12); Satisfy one or more of the following (a) to (c): (a) Dissociation constant for arginine-gingipain (K Ｄ ) is 1.0 x 10 －７ M or less, (b) inhibiting the enzyme activity of arginine-gingipain by 50% or more at 100 μM; (c) inhibiting the enzyme activity of lysine-gingipain by 50% or more at 500 μM; A peptide or a derivative thereof, or a salt thereof.

2. In the amino acid sequence (I), X １ is selected from the group consisting of A, R, V, and E; X ２ is selected from the group consisting of A, F, and K; X ３ is selected from the group consisting of R and F; The peptide according to claim 1, or a derivative thereof, or a salt thereof.

3. In the amino acid sequence (I), X １ is selected from the group consisting of R and E; X ２ is selected from the group consisting of A and K; X ３ is R, The peptide according to claim 1, or a derivative thereof, or a salt thereof.

4. In the amino acid sequence (I), X １ is R, and X ２ is K and X ３ is R or X １ is R, and X ２ is A and X ３ is R or X １ is E and X ２ is K and X ３ is R, The peptide according to claim 1, or a derivative thereof, or a salt thereof.

5. In the amino acid sequence (II), X ４ is selected from the group consisting of A, P, and K; The peptide according to claim 1, or a derivative thereof, or a salt thereof.

6. In the amino acid sequence (II), X ４ is P, The peptide according to claim 1, or a derivative thereof, or a salt thereof.

7. 7. The peptide or derivative thereof, or a salt thereof, according to any one of claims 1 to 6, which does not inhibit the enzymatic activity of papain by more than 5% at 100 μM.

8. The peptide or derivative thereof according to any one of claims 1 to 7, or a salt thereof, wherein the peptide comprising the amino acid sequence (I) or (II) is a peptide represented by SEQ ID NO: 1 to 10.

9. A gingipain inhibitor comprising the peptide or its derivative or a salt thereof according to any one of claims 1 to 8.

10. 9. A Porphyromonas gingivalis growth inhibitor comprising the peptide or its derivative or a salt thereof according to claim 1 .

11. An oral composition comprising the peptide or its derivative or a salt thereof according to any one of claims 1 to 8.

12. A method for detecting gingipain, comprising detecting gingipain by immunoassay using the peptide or its derivative or a salt thereof according to any one of claims 1 to 8.

13. The method for detecting gingipain described in claim 12, wherein the immunoassay is a sandwich immunoassay using two types of peptides or derivatives thereof or salts thereof having different amino acid sequences selected from the peptides described in any one of claims 1 to 8.

14. The detection method according to claim 13 , wherein the sandwich immunoassay is an ELISA assay or an immunochromatographic assay.

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