Diagnostic anti-heavy chain antibodies for AH amyloidosis

Abstract

To provide an antibody and a determination method for readily discriminating AH amyloidosis from another amyloidosis.SOLUTION: The present invention relates to an antibody against peptides consisting of an amino acid sequence represented by any one of the formulae (1)-(3), or peptides consisting of an amino acid sequence represented by any one of the formulae (1)-(3) in which one or several amino acids have been substituted, deleted, added or inserted. NH2-LVESGGGLVQPGGSLRLTC-COOH (1), NH2-LVQSGAEVKKPGASVKLTC-COOH (2) and NH2-LQESGPGLVKPSQTLSLTC-COOH (3).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to diagnostic anti-heavy chain antibodies for AH amyloidosis. [Background technology]

[0002] Amyloidosis is a disease in which misfolded proteins are deposited in organs, causing dysfunction. Since treatment options vary significantly depending on the subtype of amyloidosis, accurately identifying the disease type is crucial.

[0003] Immunoglobulin light chain amyloidosis (AL amyloidosis) is a disease caused by amyloid proteins derived from immunoglobulin light chains, and its incidence in the United States is approximately 1 in 100,000 people.

[0004] In contrast, immunoglobulin heavy chain amyloidosis (AH amyloidosis) is an extremely rare disease; only approximately 50 cases have been reported, including both pure heavy chain amyloidosis (AH amyloidosis) and heavy / light chain amyloidosis (AHL amyloidosis) (Non-Patent Documents 1-9). AH amyloidosis primarily affects the kidneys and develops monoclonal gammopathy, sharing similar clinical symptoms with AL amyloidosis. Therefore, it may be misdiagnosed as AL amyloidosis or may be overlooked without being diagnosed. Therefore, the exact number of patients with AH amyloidosis, detailed clinical characteristics and prognosis, and precise etiology remain unknown. In particular, the clinical differences between AH amyloidosis / AL amyloidosis and between AH amyloidosis / AHL amyloidosis remain unclear.

[0005] Because the amyloid fibrils in patients with AH amyloidosis are derived from the variable region of the immunoglobulin heavy chain and do not contain the constant region, immunoglobulin heavy chain-derived amyloid proteins are often not detected by immunohistochemistry using commercially available antibodies. In recent years, proteomic analysis using LMD / MS has become the gold standard for diagnosing AH amyloidosis. However, because this method is only available at limited facilities, a simple and widely applicable diagnostic method for AH amyloidosis has been sought. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Proc. Natl. Acad. Sci. USA. 87:6542-6546, 1990. [Non-patent document 2] Am. J. Hematol. 45:171-176, 1994. [Non-patent document 3] Am. J. Kidney. Dis. 43:e23-e28, 2004. [Non-patent document 4] Am. J. Kidney. Dis. 47:908-914, 2006. [Non-Patent Document 5] Amyloid 15:125-128, 2008. [Non-patent document 6] Clin. J. Am. Soc. Nephrol. 5:2180-2187, 2010. [Non-Patent Document 7] Cornea 30: 1163-1166, 2011. [Non-patent document 8] Am. J. Kid. Dis. 66:1095-1100, 2015. [Non-Patent Document 9] Pathol. Int. doi:10.111 / pin.13041. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an antibody and a determination method for simply and easily distinguishing AH amyloidosis from other amyloidoses. [Means for solving the problem]

[0008] In order to solve the above problems, the inventors conducted extensive research and found that antibodies against peptides having specific sequences can be used to determine AH amyloidosis.

[0009] The present invention has been completed based on these findings and includes the following broad aspects. [Section 1] A peptide consisting of an amino acid sequence represented by any one of the following formulas (1) to (3): A peptide having an amino acid sequence represented by any one of the following formulas (1) to (3), in which one or several amino acids have been substituted, deleted, added or inserted. NH2-LVESGGGLVQPGGSLRLTC-COOH (1) NH2-LVQSGAEVKKPGASVKLTC-COOH (2) NH2-LQESGPGLVKPSQTLSLTC-COOH (3) [Section 2] An antibody against the peptide described in Item 1. [Section 3] An antibody against a peptide consisting of an amino acid sequence that has at least 70% homology with part or all of the amino acid sequence of the FR1 region of an amyloid protein derived from the immunoglobulin heavy chain of a patient with AH amyloidosis. [Section 4] A diagnostic agent for AH amyloidosis, comprising the antibody according to Item 2 or 3. [Section 5] A method for determining AH amyloidosis, comprising the step of detecting the presence or absence of amyloid deposition in a biological sample derived from a subject using the antibody of Item 2 or 3 or the diagnostic agent of Item 4. [Section 6] Item 6. The method according to Item 5, wherein if amyloid deposition is detected in the step, it is determined that there is a high probability that the condition is AH amyloidosis. [Section 7] Item 7. The method according to Item 5 or 6, wherein the biological sample is at least one tissue selected from the group consisting of kidney tissue, brain tissue, digestive tissue, and heart tissue derived from a subject. [Section 8] A protein comprising an amino acid sequence represented by any one of the following formulas (1) to (3): An amino acid sequence represented by any one of the following formulas (1) to (3), in which one or several amino acids are substituted, deleted, added or inserted: A protein comprising: A marker for determining AH amyloidosis consisting of amyloid proteins of less than 15KDa. NH2-LVESGGGLVQPGGSLRLTC-COOH (1) NH2-LVQSGAEVKKPGASVKLTC-COOH (2) NH2-LQESGPGLVKPSQTLSLTC-COOH (3) [Section 9] A method for detecting an amyloid protein of less than 20 KDa in a biological sample derived from a subject, using the antibody according to Item 2 or 3 or the diagnostic agent according to Item 4. [Section 10] Item 10. A method for determining AH amyloidosis using the detection method described in Item 9. [Effects of the Invention]

[0010] According to the present invention, it is possible to determine whether or not amyloidosis is AH amyloidosis. [Brief explanation of the drawings]

[0011] [Figure 1]These are the results of a preliminary study using AH1, AH2, and AH3 antibodies in brain tissue from Patient 1 with AH amyloidosis. [Figure 2] These are the results of immunohistochemical staining using AH1 antibody (A, B, C) and AH2 antibody (D, E, F) in a patient with AH amyloidosis. A, B, C, E, F: kidney tissue, D: choroid plexus tissue. CR: Congo Red, AH1: AH1 antibody, AH2: AH2 antibody. [Figure 3] These are the results of immunohistochemical staining using AH1 or AH2 antibodies in patients with non-AH amyloidosis. A: Myocardial tissue from a patient with AL(λ) amyloidosis, B: Gastric tissue from a patient with AL(κ) amyloidosis, C: Myocardial tissue from a patient with AA amyloidosis, D: Myocardial tissue from a patient with ATTR amyloidosis. [Figure 4] Comparison of blocking with goat serum and 5% skim milk. A: Myocardial tissue from a patient with AL(λ) amyloidosis, B: Myocardial tissue from a patient with ATTR amyloidosis, C: Kidney tissue from a patient with AH amyloidosis. [Figure 5] Results of immune system analysis using AH1 antibody and a commercial antibody on kidney tissue from a patient with AH amyloidosis. Amyloid protein was detected only when the AH1 antibody was used (arrow). A: AH1 antibody, B: No antibody, C: Anti-IgG Fc region antibody. [Figure 6] The results of SDS-PAGE and immunoblotting of amyloid proteins (A) obtained from an AH amyloidosis patient and serum (B) from an AH amyloidosis patient. DETAILED DESCRIPTION OF THE INVENTION

[0012] The peptide of the present invention is a peptide consisting of an amino acid sequence represented by any one of the following formulas (1) to (3), or a peptide in which one or several amino acids have been substituted, deleted, added, or inserted in an amino acid sequence represented by any one of the following formulas (1) to (3). NH2-LVESGGGLVQPGGSLRLTC-COOH (1) (SEQ ID NO: 1) NH2-LVQSGAEVKKPGASVKLTC-COOH (2) (SEQ ID NO: 2) NH2-LQESGPGLVKPSQTLSLTC-COOH (3) (SEQ ID NO: 3) In the present invention, a peptide in which one or several amino acids have been substituted, deleted, added, or inserted includes a peptide in which one or several (e.g., two or three) amino acids have been substituted, deleted, added, or inserted, and which can bind to the antibody of the present invention. Examples of peptides in which one or several amino acids are substituted, deleted, added or inserted in the amino acid sequence represented by formula (1) include: NH2-LVESGGGLVQPGGSLRVSC-COOH (SEQ ID NO: 4), NH2-LVESGGGLVQPGGSLRLSC-COOH (SEQ ID NO: 5), NH2-LLESGGDLVQPGGSLRLSC-COOH (SEQ ID NO: 6) and the like. Examples of peptides in which one or several amino acids are substituted, deleted, added or inserted in the amino acid sequence represented by formula (2) include: NH2-LLESGAEVKKPGASVKLTC-COOH (SEQ ID NO: 7), NH2-LVEQSGAEVKKPGASVKLTC-COOH (SEQ ID NO: 8) and the like. Examples of peptides in which one or several amino acids are substituted, deleted, added or inserted in the amino acid sequence represented by formula (3) include: NH2-LVESGPGLVKPSQTLSLTC-COOH (SEQ ID NO: 9), NH2-LLESGPGLVKPSQTLSLTC-COOH (SEQ ID NO: 10), and the like.

[0013] The peptides of the present invention generally have an N-terminus that is an amino group (-NH2) or an ammonium ion (-NH3 + ), but the N-terminal amino group may be substituted with an alkyl group, an acyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, or an aryloxycarbonyl group.

[0014] The peptides of the present invention usually have a C-terminus that is a carboxyl group (-COOH) or a carboxylate (-COO - ), but the C-terminal carboxyl group may be an amide or ester.

[0015] The peptides of the present invention may also form salts, and salts with physiologically acceptable bases or acids are used. Examples of salts of the peptides of the present invention with bases include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, and ammonium salts. Examples of salts of the peptides of the present invention with acids include hydrochloride, phosphate, sulfate, phosphate, formate, propionate, fumarate, maleate, succinate, tartrate, and citrate.

[0016] The origin of the peptide of the present invention is not particularly limited, and may be, for example, a peptide derived from tissues or cells of humans or mammals (e.g., guinea pigs, rats, mice, pigs, sheep, cattle, monkeys, etc.), or a synthetic peptide.

[0017] The peptides of the present invention can be produced according to known peptide synthesis methods. Examples of peptide synthesis methods include known solid-phase synthesis and liquid-phase synthesis. Peptides obtained by synthesis can be isolated and purified by conventional purification methods, such as extraction, precipitation, electrophoresis, and chromatography. If the peptide obtained by the above method is in a free form, it can be converted into an appropriate salt by known methods. If the peptide is obtained as a salt, it can be converted into the free form by known methods.

[0018] Antibodies (eg, polyclonal antibodies, monoclonal antibodies) or antisera against the peptides of the present invention can be produced according to known production methods using the peptides of the present invention as antigens.

[0019] Polyclonal antibodies can be produced by periodically immunizing animals (e.g., horses, goats, sheep, rabbits, chickens, guinea pigs, etc.) with the peptides of the present invention (immunizing antigens) alone or in combination with adjuvants, collecting antibody-containing material against the peptides of the present invention from the immunized animals, and isolating and purifying the antibodies. Preferably, immunization is performed three or more times, and antibodies are collected from the immunized animals, such as blood or ascites, preferably from the blood. Monoclonal antibodies can be produced by periodically immunizing animals (e.g., horses, goats, sheep, rabbits, chickens, guinea pigs, etc.) (preferably three or more times) with the peptides of the present invention (immunizing antigens) alone or in combination with adjuvants, and then removing, for example, the spleen or lymph nodes. The antibody-producing cells contained therein can be fused with myeloma cells to prepare monoclonal antibody-producing hybridomas. The resulting hybridoma cells are cultured in an appropriate culture medium, such as HAT-RPMI1640 medium containing 10% fetal bovine serum. Antibodies produced in the culture supernatant are detected by, for example, RIA, ELISA, or the like, and hybridoma cell lines that produce antibodies that specifically react with the peptide are selected and cloned. Monoclonal antibodies that react with the peptide of the present invention can be recovered from the ascites obtained by transplanting hybridoma cells into the abdominal cavity of a mouse or rat, for example. Alternatively, they can be recovered from the culture supernatant of hybridoma cells. Monoclonal antibodies can be produced by separating and purifying the recovered antibodies.

[0020] The antibodies of the present invention can also be used to detect amyloid proteins that serve as antigens present in a subject's biological sample (e.g., tissue to be evaluated, body fluid, etc.). Detection methods include, for example, immunohistochemical staining, immunoblotting, and ELISA. The amyloid proteins that serve as antigens for the antibodies of the present invention are amyloid proteins containing the immunoglobulin heavy chain variable region, and are known to be deposited in tissues in AH amyloidosis. Therefore, the results of amyloid protein detection using the antibodies of the present invention can be used to determine AH amyloidosis. In this specification, "AH amyloidosis" includes not only pure heavy chain amyloidosis (AH amyloidosis) but also heavy chain / light chain amyloidosis (AHL amyloidosis).

[0021] Furthermore, the antibody of the present invention can be used as a diagnostic agent for AH amyloidosis. The diagnostic agent can also be provided in the form of a kit. Furthermore, the diagnostic agent of this embodiment may further contain various reagents used in determining AH amyloidosis, such as a detection reagent.

[0022] Examples of subject animals include mammals such as humans, mice, rats, rabbits, guinea pigs, hamsters, monkeys, sheep, horses, cows, pigs, dogs, and cats, with humans being more preferred.

[0023] Biological samples include, but are not limited to, kidney tissue, brain tissue, digestive tissue, heart tissue, skin tissue, skeletal muscle tissue, blood, etc., derived from a subject, with kidney tissue, brain tissue, digestive tissue, heart tissue, etc., from a patient with amyloidosis being particularly preferred. The blood mentioned above includes serum, plasma, etc. Furthermore, the antibodies of the present invention not only refer to antibodies against the peptides of the present invention, but also broadly include antibodies against peptides consisting of an amino acid sequence substantially identical to part or all of the amino acid sequence of the FR1 region of an amyloid protein derived from a patient with AH amyloidosis.

[0024] As used herein, an "AH amyloidosis patient" refers to a patient in whom tissue deposition of immunoglobulin heavy chain-derived amyloid protein has been confirmed by, for example, amino acid sequence analysis of the amyloid protein or proteomic analysis using LMD / MS. Note that, as used herein, "AH amyloidosis patient" includes not only patients with pure heavy chain amyloidosis (AH amyloidosis) but also patients with heavy chain / light chain amyloidosis (AHL amyloidosis).

[0025] Substantially identical amino acid sequences include amino acid sequences that have a homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more to part or all of the amino acid sequence of the FR1 region of an amyloid protein derived from an AH amyloidosis patient.

[0026] A peptide consisting of an amino acid sequence substantially identical to part or all of the amino acid sequence of the FR1 region of an amyloid protein derived from an AH amyloidosis patient is not particularly limited in terms of the number of amino acids, but 10 to 20 amino acids are preferred, and 15 to 20 amino acids are particularly preferred.

[0027] Methods for determining AH amyloidosis include, for example, the following two methods. (1) A method comprising the step of detecting the presence or absence of amyloid protein deposits in a biological sample derived from a subject using the antibody or diagnostic agent of the present invention. (2) A method comprising the step of detecting an amyloid protein of less than 20 kDa in a biological sample using the antibody or diagnostic agent of the present invention.

[0028] The determination method is described in detail below. (1) A method comprising the step of detecting the presence or absence of amyloid protein deposits in a biological sample derived from a subject using the antibody or diagnostic agent of the present invention.

[0029] The antibody of the present invention is used to detect the presence or absence of amyloid protein deposits in a biological sample. Detection methods include, for example, immunohistochemical staining and immunoblotting, with immunohistochemical staining being preferred. When amyloid deposits are detected in a biological sample using the antibody of the present invention, the likelihood of AH amyloidosis is determined to be high. When amyloid deposits are not detected, the likelihood of AH amyloidosis is determined to be low. The amyloid protein that serves as the antigen for the antibody of the present invention is an amyloid protein containing an immunoglobulin heavy chain variable region, which is known to be deposited in tissues in AH amyloidosis. Therefore, the antibody of the present invention can be used to determine whether amyloidosis patients have AH amyloidosis. Furthermore, because the antigen for this antibody is an amyloid protein, amyloid protein deposits are not detected in patients with diseases other than amyloidosis or in healthy individuals, making it possible to distinguish from AH amyloidosis.

[0030] Furthermore, by comparing the deposition state (e.g., deposition location) with that observed when a reagent other than the antibody of the present invention that specifically labels amyloid proteins is used, it is possible to prevent erroneous determination of AH amyloidosis due to nonspecific reactions. Examples of detection methods using a reagent other than the antibody of the present invention that specifically labels amyloid proteins include staining with Congo Red, staining with FSB (1-fluoro-2,5-bis(3-carboxy-4-hydroxystryl)benzene), and thioflavin fluorescence. Other known reagents can also be used by appropriately selecting them.

[0031] Immunohistochemical staining using the antibodies of the present invention can be performed according to known methods. Immunohistochemical staining is preferably performed on a fixed tissue specimen prepared by fixing the tissue specimen to be evaluated. Fixation can be achieved by physical denaturation using temperature or pressure (e.g., thermal coagulation or freezing), but chemical treatment with a fixative is preferred. The fixative is not particularly limited, but examples include formalin fixative, phosphate-buffered formalin fixative, paraformaldehyde fixative, glutaraldehyde fixative, Bouin's fixative, Zamboni fixative, osmium fixative, zinc fixative, Hollande fixative, alcohol fixative, alcohol-formalin mixture, and FAA fixative. Preferred fixatives include formalin fixative and phosphate-buffered formalin fixative. These fixatives can be used alone or in combination. Additives such as buffers, salts, and sugars may also be used in combination as appropriate.

[0032] The fixation time can be determined optimally, preferably 24 to 48 hours, and the fixation temperature is preferably 15 to 25°C.

[0033] The fixed tissue specimen can be further excised, dehydrated, embedded, sliced, and / or stained, and then observed, for example, under an optical microscope.

[0034] The cutting may be performed so as to reduce the specimen to a size suitable for observation and to make it easier to observe diseased and normal areas in the specimen.

[0035] The embedding technique is not particularly limited, but examples include paraffin embedding, celloidin embedding, OCT compound embedding, gelatin embedding, and synthetic resin embedding.

[0036] A microtome may be used for thin sectioning.

[0037] Furthermore, for example, when a paraffin block is prepared using the paraffin embedding method, staining may be enhanced by deparaffinizing the sample using, for example, xylene, alcohol, etc., and then, in some cases, activating the target antigen in the sample.

[0038] The method for antigen retrieval is not particularly limited, but examples include treatment with protease such as pepsin, trypsin, pronase, or protein kinase K; heat treatment using microwaves, autoclaves, or boiling; treatment with alkali or acid (e.g., hydrochloric acid or formic acid), etc. Antigen activation treatment is preferably carried out at room temperature for 1 to 5 minutes.

[0039] Furthermore, because antibodies undergo nonspecific adsorption reactions with proteins other than the target protein, it is preferable to perform pretreatment using a blocking agent to prevent nonspecific adsorption. Examples of blocking agents include proteins derived from living organisms, such as normal serum (e.g., goat, horse, rabbit, etc.), bovine serum albumin, gelatin, and skim milk. Other examples include surfactants (e.g., TWEEN (registered trademark) 20, etc.), hydroxyalkyl cellulose, and polyvinyl alcohol. These can be used alone or in combination of two or more. Among these, normal serum and skim milk are preferred. Skim milk is preferred because it is effective in preventing nonspecific adsorption. Pretreatment using a blocking agent is preferably performed at room temperature for 30 to 60 minutes.

[0040] Staining specimens is preferred for easier microscopic observation. For example, enzyme immunoassays, in which antibodies are labeled with a specific enzyme and then reacted with a substrate, can be used to observe the color of the resulting pigment product under an optical microscope. Known methods for enzyme immunoassays can be used, including direct methods, in which a labeled primary antibody is used and a single antigen-antibody reaction is performed; and indirect methods, in which an unlabeled primary antibody is used and a second antibody (secondary antibody) is labeled and reacted with the primary antibody to perform two or more antigen-antibody reactions. Other indirect methods include the PAP method, which uses a soluble immune complex (PAP) of peroxidase and anti-peroxidase antibodies; the LAB (Linked Avidin-Biotin) method; the ABC method, which uses an avidin-biotin complex; the LSAB (Linked Streptavidin-Biotin) method, which uses streptavidin; the TSA (Tyramide Signal Amplification) method; and the CARD (Catalyzed Reporter Deposition) method.

[0041] Examples of color development methods in enzyme antibody techniques include, but are not limited to, the DAB method in which peroxidase as a labeling enzyme is reacted with the chromogenic substrate diaminobenzidine (DAB); the nickel-DAB method in which the DAB method is performed in the presence of nickel ions; a method in which peroxidase is reacted with the chromogenic substrate aminoethylcarbazole (AEC); or a method in which alkaline phosphatase as a labeling enzyme is reacted with the chromogenic substrate BCIP / NBT, a method in which it is reacted with the chromogenic substrate Fast Red, or a method in which it is reacted with the chromogenic substrate Fast Blue.

[0042] For example, staining may be performed by adding an antibody of the present invention as a primary antibody to a specimen, reacting for 1 to 12 hours at 4°C to room temperature, washing off the primary antibody, and then adding a labeled antibody as a secondary antibody, reacting for 30 minutes to 12 hours at 4°C to room temperature, washing off the secondary antibody, and then allowing the specimen to develop color. Alternatively, the ABC method may be used to increase detection sensitivity.

[0043] In addition to the antigen-antibody reaction described above, methods for visualizing antigen-antibody reactions include the autoradiography method, in which a radioisotope is bound to an antibody and photographic paper is exposed to light; the gold colloid method, in which an antibody is bound to a visible substance such as gold particles and observed under an electron microscope; and the fluorescent antibody method, in which an antibody is labeled with a fluorescent dye and, after the antigen-antibody reaction, is exposed to an excitation wavelength to cause it to fluoresce and be observed under a fluorescent microscope.

[0044] (2) A method comprising the step of detecting an amyloid protein of less than 20 kDa in a biological sample using the antibody or diagnostic agent of the present invention.

[0045] In another embodiment, AH amyloidosis can also be determined by detecting amyloid proteins of less than 20 kDa in a biological sample using an antibody of the present invention. The detection method is preferably immunoblotting. When an amyloid protein of less than 20 kDa is detected in a biological sample using an antibody of the present invention, the likelihood of AH amyloidosis is determined to be high, whereas when an amyloid protein of less than 20 kDa is not detected, the likelihood of AH amyloidosis is determined to be low. More preferably, when an amyloid protein of less than 15 kDa is detected, the likelihood of AH amyloidosis is determined to be high. The lower limit is not particularly limited, but is preferably 5 kDa or more, and particularly preferably 10 kDa or more.

[0046] Immunoblotting using the antibodies of the present invention can be performed according to known methods. For example, in immunoblotting, a sample solution is prepared from a biological sample according to known methods, and the sample solution is separated by SDS-polyacrylamide gel electrophoresis. The sample is then transferred to a membrane such as a polyvinylidene difluoride (PVDF) membrane or a nitrocellulose membrane and immobilized. After immersion in a blocking agent for blocking, the membrane is reacted with the antibody of the present invention as a primary antibody. The membrane is then washed, and a secondary antibody, which is a labeled antibody, is reacted. The membrane is then washed again, and color development is detected.

[0047] Examples of blocking agents include proteins derived from living organisms, such as normal serum (e.g., goat, horse, rabbit, etc.), bovine serum albumin, gelatin, and skim milk. Other examples include surfactants (e.g., TWEEN (registered trademark) 20, etc.), hydroxyalkyl cellulose, and polyvinyl alcohol. These can be used alone or in combination of two or more.

[0048] Examples of methods for labeling secondary antibodies include the DAB method, in which peroxidase as a labeling enzyme is reacted with the chromogenic substrate diaminobenzidine (DAB); the nickel-DAB method, in which the DAB method is performed in the presence of nickel ions; a method in which peroxidase is reacted with the chromogenic substrate aminoethylcarbazole (AEC); or a method in which alkaline phosphatase as a labeling enzyme is reacted with the chromogenic substrate BCIP / NBT, a method in which it is reacted with the chromogenic substrate Fast Red, or a method in which it is reacted with the chromogenic substrate Fast Blue.

[0049] Furthermore, an amyloid protein of less than 20 kDa, which is an antigen of the antibody of the present invention and which is detected by the method (2), can be used as a marker for determining AH amyloidosis. This is a protein containing the amino acid sequence of the peptide of the present invention, specifically, a protein containing an amino acid sequence represented by any one of the following formulas (1) to (3), or a protein containing an amino acid sequence represented by any one of the following formulas (1) to (3) in which one or several amino acids have been substituted, deleted, added, or inserted. Amyloid proteins of less than 20 kDa can be used as a marker for determining AH amyloidosis. NH2-LVESGGGLVQPGGSLRLTC-COOH (1) (SEQ ID NO: 1) NH2-LVQSGAEVKKPGASVKLTC-COOH (2) (SEQ ID NO: 2) NH2-LQESGPGLVKPSQTLSLTC-COOH (3) (SEQ ID NO: 3) In the present invention, an amino acid sequence in which one or several amino acids have been substituted, deleted, added, or inserted includes an amino acid sequence in which one or several (e.g., two or three) amino acids have been substituted, deleted, added, or inserted, and which can bind to the antibody of the present invention. [Example]

[0050] The present invention will be further explained below with reference to examples, but the present invention is not limited thereto.

[0051] All of the following examples were carried out at room temperature and atmospheric pressure.

[0052] (1) Preparation of anti-heavy chain antibodies Two types of anti-heavy chain variable region polyclonal antibodies (AH1, AH2, and AH3 antibodies) were produced in rabbits against the anti-FR1 synthetic peptide based on the primary structure of the amyloid protein from an AH amyloidosis patient and the amino acid sequence of the heavy chain variable region published by the National Center for Biotechnology Information (NCBI). Production of the polyclonal antibodies was outsourced to Eurofins. The peptide sequences used to produce the AH1, AH2, and AH3 antibodies are as follows: AH1:NH2-LVESGGGLVQPGGSLRLTC-COOH (1) (SEQ ID NO: 1) AH2:NH2-LVQSGAEVKKPGASVKLTC-COOH (2) (SEQ ID NO: 2) AH3:NH2-LQESGPGLVKPSQTLSLTC-COOH (3) (SEQ ID NO: 3)

[0053] (2) Immunohistochemical staining method Immunohistological staining was performed using the above antibodies on formalin-fixed, paraffin-embedded or frozen specimens using the standard immunoperoxidase method (Vectastain ABC kit, Vector Laboratories). Tissues on glass slides were pretreated with formic acid for 1 minute. Goat serum was diluted and blocked as described in the accompanying instructions. Some tissues were also blocked with 5 wt% skim milk. The initial serum was diluted 4000-fold before use. Staining was performed for 1 minute using the DAB Substrate kit (Vector Laboratories).

[0054] (3) Preliminary Examination A preliminary immunohistochemical staining study was performed using brain tissue from AH amyloidosis patient 1 (Table 1). The staining results from this preliminary study are shown in Figure 1. As can be seen from Figure 1, the AH1 antibody had the highest staining activity, followed by the AH2 antibody. As for the AH3 antibody, although it was capable of staining, its reactivity was weaker than that of the other two antibodies. A similar preliminary study was also performed using kidney tissue from AH amyloidosis patient 11 (Table 1). However, the results were negative for all three antibodies, AH1, AH2, and AH3. Therefore, we decided to perform analysis using only two antibodies, AH1 and AH2.

[0055] (4) Immunohistochemical staining in patients with AH amyloidosis Tissue samples were collected from 11 patients with AH amyloidosis and immunohistochemically stained using AH1 and AH2 antibodies. The diagnosis of AH amyloidosis in these 11 patients was confirmed by amino acid sequence analysis or LMD / MS analysis. Furthermore, immunohistochemical staining of these patient tissues with anti-λ light chain antibody, anti-κ light chain antibody, anti-amyloid A antibody, and anti-transthyretin antibody yielded negative results. The results of immunohistochemical staining of tissues from these 11 patients with AH amyloidosis using AH1 and AH2 antibodies are shown in Figure 2 and Table 1. When tissues were tested using goat serum-blocked tissues, AH1 antibody was positive in eight patients (72.7%). When AH2 antibody was used in five patients, including three patients who tested negative for AH1 antibody, four patients tested positive, including two patients who were AH1-negative. As a result, when both AH1 and AH2 antibodies were used, the positive rate was 90.9%. Furthermore, in patients with AH amyloidosis 1 and 3, immunostaining was performed on tissues blocked with skim milk, which is thought to have a greater effect in suppressing nonspecific reactions, but the results were unchanged. Therefore, the above results were considered to be specific rather than nonspecific reactions.

[0056] (5) Immunohistochemical staining in patients without AH amyloidosis Immunohistochemical staining using AH1 and AH2 antibodies was also performed on 64 patients without AH amyloidosis: 44 with AL amyloidosis, 7 with AA amyloidosis, 9 with ATTR amyloidosis, 3 with β2-macroglobulin amyloidosis, and 1 with gelsolin-related amyloidosis.

[0057] The results of immunohistochemical staining using AH1 and AH2 antibodies on tissues from the above 64 patients are shown in Figure 3 and Table 2. When tissues were blocked with goat serum, 57 of 62 patients (91.9%) were determined to be negative with the AH1 antibody, and 5 were determined to be positive. When tissues were blocked with 5 wt% skim milk, nonspecific reactions were suppressed more than with goat serum, reducing false positives (Figure 4).

[0058] (6) Immunohistochemical staining using commercially available antibodies Tissue from AH amyloidosis patient 1 was examined by immunofluorescence using the AH1 antibody (diluted 1:4000). As a comparative example, tissue from AH amyloidosis patient 1 was also examined by immunofluorescence using an anti-IgG antibody (diluted 1:4000, A0423, Dako) and an anti-IgG Fc region antibody (diluted 1:4000, A80-105A, Bethyl Laboratories). The results are shown in Figure 5. Figure 5 indicates that the commercially available antibodies were negative.

[0059] (7) Immunoblotting of amyloid proteins and patient serum Immunoblotting was performed for amyloid protein against AH1 antibody (1:10,000 dilution), anti-IgG antibody (1:10,000 dilution), and anti-IgG Fc region antibody (1:10,000 dilution). AH amyloid protein was obtained from kidney tissue of AH amyloidosis patient 1 (Table 1). The obtained amyloid protein was solubilized in Laemmli sample buffer (Bio-Red, Hercules) containing 5 wt% mercaptoethanol, subjected to polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting was performed on a polyvinylidene fluoride membrane. Protein bands were detected using a POD Immunostain Kit (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0060] Furthermore, immunoblotting was similarly performed with AH1 antibody and AH2 antibody using the sera from AH amyloidosis patient 1 and patient 3. The results are shown in Figure 6.

[0061] Immunoblotting with the AH1 antibody revealed a single band at approximately 11 kDa. In the case of serum samples from patients with AL amyloidosis, the strongest band was observed at approximately 50 kDa, which corresponds to the full-length Ig heavy chain. However, no band at approximately 50 kDa was observed in the amyloid protein of patients with AH amyloidosis. A band at approximately 11 kDa also appeared when serum samples from patients with AH amyloidosis were used. In addition, a band at approximately 11 kDa appeared in a patient who was not positive with the AH1 antibody (AH amyloidosis patient 3) when combined with the AH2 antibody.

[0062] [Table 1]

[0063] [Table 2]

Claims

1. A peptide consisting of an amino acid sequence represented by any one of the following formulas (1) to (3): A peptide having an amino acid sequence represented by any one of the following formulas (1) to (3), in which one or two amino acids have been substituted, deleted, added or inserted: NH 2 -LVESGGGLVQPGGSLRLTC-COOH (1) NH 2 -LVQSGAEVKKPGASVKLTC-COOH (2) NH 2 -LQESGPGLVKPSQTLSLTC-COOH (3)

2. An antibody against the peptide of claim 1.

3. An antibody against the peptide described in claim 1, consisting of an amino acid sequence having at least 90% identity with part or all of the amino acid sequence of the FR1 region of an amyloid protein derived from the immunoglobulin heavy chain of an AH amyloidosis patient.

4. A diagnostic agent for AH amyloidosis, comprising the antibody according to claim 2 or 3.

5. A method for determining AH amyloidosis, comprising the step of detecting the presence or absence of amyloid deposits in a biological sample derived from a subject using the antibody of claim 2 or 3 or the diagnostic agent of claim 4.

6. 6. The method of claim 5, wherein if amyloid deposition is detected in the step, it is determined that there is a high probability that the condition is AH amyloidosis.

7. The method according to claim 5 or 6, wherein the biological sample is at least one selected from the group consisting of kidney tissue, brain tissue, digestive tissue, and cardiac tissue derived from a subject.

8. A method for detecting an amyloid protein of less than 20 kDa in a biological sample derived from a subject, using the antibody according to claim 2 or 3 or the diagnostic agent according to claim 4.

9. A method for determining AH amyloidosis using the detection method described in claim 8.

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