JP7496622B2 - Method and test kit for assisting in the diagnosis of Behcet's disease - Google Patents

Description

The present invention relates to a method for assisting in the diagnosis of Behcet's disease, which comprises a step of detecting antibodies that are specifically increased in Behcet's disease patients, and a kit for detecting the antibodies.

Behçet's disease is a recurrent inflammatory disease whose main symptoms are aphthous ulcers of the oral mucosa, skin symptoms, eye symptoms, and vulvar ulcers, and it has been designated an intractable disease by the Ministry of Health, Labor, and Welfare. The cause of the disease is unknown, but it is thought to be a multifactorial disease caused by the interaction of some internal genetic factors with some external environmental factors (Non-Patent Document 1).

Traditionally, Behçet's disease has been diagnosed by clinically confirming the appearance of the four main symptoms and five minor symptoms mentioned above. However, none of these symptoms are specific to Behçet's disease, and since a comprehensive examination of a variety of clinical symptoms is required, diagnosing Behçet's disease is generally difficult.

As a molecular biological approach, for example, a method has been proposed (Patent Document 1) in which the degree of trimethylation of histone H3 lysine 4 and/or the degree of trimethylation of histone H3 lysine 27 in at least one of neutrophils and γδ T cells in peripheral blood is measured, and at least one of the ratio of the degree of trimethylation of histone H3 lysine 4 to the degree of trimethylation of histone H3 lysine 27 and/or the degree of trimethylation of histone H3 lysine 4 is used as an index to assist in the diagnosis of Behçet's disease.

Although diagnostic methods using biomarkers are expected to be not only effective but also simple, biomarkers that contribute to diagnosis in the Behçet's method and diagnostic methods using such biomarkers, including the above-mentioned method, have not yet been put to practical use. Therefore, there remains a need for an effective and simple diagnostic method for Behçet's disease.

JP 2016-070798 A

"Behçet's disease (designated intractable disease 56)", [on line], Intractable Disease Information Center, [searched May 20, 2019], Internet <URL: http://www.nanbyou.or.jp/entry/330>

The present invention aims to provide a biomarker specific to Behcet's disease patients that is useful for diagnosing Behcet's disease and a method for detecting the same.

The inventors discovered that antibody titers against a specific peptide were specifically increased in patients clinically diagnosed with Behçet's disease, and completed the following invention.

(1) A method for assisting in the diagnosis of Behcet's disease, comprising the step of detecting an antibody that recognizes the amino acid sequence shown in SEQ ID NO: 1 in a sample collected from a subject.
(2) A method for assisting in the diagnosis of Behcet's disease, comprising the steps of: contacting a sample collected from a subject with a molecule that contains the amino acid sequence shown in SEQ ID NO:1 as an epitope; and detecting binding of an antibody contained in the sample to the amino acid sequence.
(3) The method according to (2), wherein the molecule is an oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or an oligopeptide consisting of the amino acid sequence having one or several amino acids added to either or both termini of the amino acid sequence.
(4) The method according to any one of (1) to (3), wherein the sample is blood, plasma or serum.
(5) A kit for testing an antibody that recognizes the amino acid sequence shown in SEQ ID NO:1, comprising a molecule containing the amino acid sequence shown in SEQ ID NO:1 as an epitope and a carrier for immobilizing the molecule.
(6) The kit according to (5), wherein the molecule is an oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or an oligopeptide consisting of the amino acid sequence having one or several amino acids added to either or both termini of the amino acid sequence.
(7) The kit according to (5) or (6), which is used to aid in the diagnosis of Behcet's disease.

According to the present invention, it is possible to provide useful information for diagnosing Behçet's disease by a simple method of detecting specific antibodies contained in a sample collected from a subject, typically peripheral blood.

1 is a graph showing the antibody titers of antibodies recognizing each of three types of oligopeptides in the sera of healthy subjects, patients with Behcet's disease, patients with sarcoidosis, and patients with Harada's disease.

The present invention provides a method for assisting in the diagnosis of Behçet's disease, comprising the step of detecting an antibody that recognizes the amino acid sequence shown in SEQ ID NO: 1 in a sample collected from a subject. The present invention also provides a method for collecting data for the diagnosis of Behçet's disease, comprising the above step. Furthermore, the present invention provides a method for detecting or analyzing an antibody that recognizes the amino acid sequence shown in SEQ ID NO: 1 in a subject.

The subjects are humans for whom a diagnosis of Behçet's disease is sought.

Examples of specimens include blood, plasma, serum, cerebrospinal fluid, synovial fluid, saliva, nasal discharge, tears, and other bodily fluids, as well as tissue slices, puncture fluid, pus, and the like taken from lesions that are commonly observed in Behçet's disease, such as inflammation and ulceration. It is particularly preferable to use blood, plasma, serum, and the like used in clinical tests as specimens. The collected specimens may be appropriately pretreated depending on the antibody detection method.

The amino acid sequence shown in SEQ ID NO:1 (hereinafter, simply referred to as GLRVYK) is identical to the 84th to 89th amino acid sequence of 30S ribosomal protein S8 produced by Streptococcus sanguinis and the 179th to 184th amino acid sequence of glycosyltransferase family 2 protein produced by Streptococcus pneumoniae. The amino acid sequence of 30S ribosomal protein S8 (total of 132 amino acids) is registered in GenBank under accession number CEL89394.1, and the amino acid sequence of glycosyltransferase family 2 protein (total of 259 amino acids) is registered in NCBI Reference Sequence under accession number WP_050238633.1. In this specification, amino acid residues are represented by single letter code, except in the sequence table.

S.pneumoniae and S.sanguinis are typical bacteria that inhabit the human oral cavity. Oral bacteria invade the bloodstream during dental treatment or brushing, and are known to evade the host immune system by using various pathogenic factors, inducing a transient infection (e.g., Pihlstrom, B. L. et al., 2005, Lancet, 366, 1809-1820). However, there are no reports pointing out the relationship between S.pneumoniae and Behçet's disease, and the only reports pointing out the relationship between S.sanguinis and Behçet's disease are those related to molecules unrelated to 30S ribosomal protein S8, such as heat shock proteins.

As shown in the examples detailed below, it was confirmed that antibodies that recognize GLRVYK are present in the blood of patients who have been clinically diagnosed with Behcet's disease by conventional methods, and that the antibody titers are significantly higher than those in healthy individuals or patients without Behcet's disease, such as sarcoidosis patients with uveitis or Harada's disease patients. Therefore, data on the presence or antibody titers of antibodies that recognize GLRVYK in a subject, particularly in the blood, can be useful information when diagnosing that the subject is suffering from Behcet's disease.

An antibody that recognizes GLRVYK is an antibody that binds to a molecule that contains GLRVYK as an epitope. There is no need to limit or distinguish the type of antibody as long as it recognizes GLRVYK. The antibody may be, for example, IgG, IgA, or IgM, with IgG being particularly preferred.

Detection of antibodies that recognize GLRVYK in a sample is typically performed by contacting the sample with a molecule that contains GLRVYK as an epitope and detecting binding of antibodies contained in the sample to GLRVYK. Thus, the present invention also provides a method for assisting in the diagnosis of Behçet's disease, comprising the steps of contacting a sample collected from a subject with a molecule that contains the amino acid sequence shown in SEQ ID NO:1 as an epitope and detecting binding of antibodies contained in the sample to the amino acid sequence, a method for collecting data for diagnosing Behçet's disease, comprising these steps, and a method for detecting or analyzing antibodies that recognize the amino acid sequence shown in SEQ ID NO:1 in a subject, comprising these steps.

Examples of molecules that contain GLRVYK as an epitope include an oligopeptide consisting of GLRVYK, an oligopeptide consisting of an amino acid sequence in which one or several arbitrary amino acids are added to both or either terminus of GLRVYK (for example, an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 arbitrary amino acids are added to the N-terminus, the C-terminus, or both the N-terminus and C-terminus of GLRVYK), fusions of the oligopeptide with other peptides or proteins, chemically modified oligopeptides or fusions thereof, and conjugates of these with appropriate substances.

The fact that GLRVYK is an epitope in the above-mentioned molecule can be confirmed by the binding of the molecule to an antibody that specifically binds to GLRVYK, which can be produced by a general antibody production method. An antibody that specifically binds to GLRVYK can be produced by a general antibody production method in which an appropriate mammal is immunized with an oligopeptide consisting of GLRVYK as an antigen, as described in the Examples below.

Examples of fusion products include fusion proteins of glutathione S-transferase or other enzyme proteins, or tag proteins such as histidine tags or FLAG tags with the above-mentioned oligopeptides.

Examples of chemical modifications include glycosylation, acetylation, acylation, ADP-ribosylation, amidation, PEGylation, flavinylation, formylation, gamma-carboxylation, iodination, methylation, myristoylation, phosphorylation, prenylation, selenoylation, ubiquitination, demethylation, oxidation, hydroxylation, sulfation, cyclization, and crosslinking.

Examples of substances that can form conjugates with the above-mentioned oligopeptides or their fusion products include carrier proteins such as serum albumin, keyhole limpet hemocyanin (KLH), and immunoglobulin Fc domains; enzyme proteins such as β-galactosidase, glutathione-S-transferase, horseradish peroxidase, and alkaline phosphatase; colloidal metals, color-developing substances, chemiluminescent substances, fluorescent substances, lipids such as stearic acid and phospholipids, biotin, avidin, streptavidin, polymerized streptavidin, neutravidin, nucleotides, and nucleotide derivatives.

The above-mentioned fusion products, chemical modifications, and conjugates can be produced by employing the methods described in various publications relating to their respective production.

In cases where a molecule containing GLRVYK as an epitope has GLRVYK within its three-dimensional structure, it may be subjected to denaturation or other treatment as necessary so that GLRVYK is exposed on the surface of the molecule and available for binding to an antibody.

It is preferable that a molecule containing GLRVYK as an epitope is bound to a carrier by ionic interaction, hydrophobic interaction, covalent interaction, etc., and immobilized for use. Examples of carriers include solid carriers made of glass, silica, latex, polystyrene, polycarbonate, polyacrylate, polyethylene, polypropylene, polyester, etc., membrane carriers such as PVDF, Immobilon, nitrocellulose membrane, polyethylene membrane, nylon membrane, etc., magnetic beads, and metal carriers made of gold, silver, platinum, copper, metal composites, and other metals. There are no particular limitations on the shape of the carrier, and it can be a film, sheet, plate, tube, well, etc.

Contact of the sample with a molecule containing GLRVYK as an epitope can be carried out under reaction conditions commonly used for antigen-antibody reactions, and binding of the antibody to GLRVYK can be detected by immunological methods commonly used to detect antigen-antibody reactions.

Examples of immunological methods for detecting antibody binding include fluorescent antibody techniques, enzyme immunoassays, chemiluminescent immunoassays, immunohistochemical staining, radioimmunoassays, and flow cytometry, and can be selected appropriately depending on the type of chemical modification, conjugate, and solid phase.

Each of the above-mentioned methods provided by the present invention may further include a step of measuring the antibody titer of an antibody that recognizes GLRVYK in a sample collected from a subject, and a step of comparing the antibody titer with the antibody titer of an antibody that recognizes GLRVYK in a sample collected from a healthy subject.

The antibody titer can be determined based on a calibration curve prepared using an antibody that specifically binds to GLRVYK as a standard substance. The antibody titer in the subject and the antibody titer in healthy individuals can be compared by taking a sample from the healthy individual each time a test is performed, or by determining a standard value for the antibody titer in healthy individuals in advance and comparing the antibody titer with the standard value.

A further aspect of the present invention relates to a kit for testing for antibodies that recognize GLRVYK, comprising a molecule that contains GLRVYK as an epitope and a carrier for immobilizing the molecule. The "molecule that contains GLRVYK as an epitope" in this aspect is as described in the previous aspect, and is preferably an oligopeptide consisting of GLRVYK or consisting of an amino acid sequence in which one or several amino acids have been added to one or both termini of GLRVYK. The "carrier" in this aspect is also as described in the previous aspect, and it is preferable that the molecule is immobilized on a carrier.

The kit of this embodiment can be used to detect antibodies that recognize GLRVYK in a sample collected from a subject and to measure the antibody titer, and the presence of the antibody and its antibody titer can provide useful information when diagnosing that the subject is suffering from Behçet's disease. Therefore, the kit of this embodiment can be used to assist in the diagnosis of Behçet's disease and to collect data for the diagnosis of Behçet's disease.

The present invention is described in further detail by the following non-limiting examples.

(1) Sample preparation Among patients who visited an ophthalmologist complaining of ocular symptoms, venous blood was collected from 15 patients who were clinically diagnosed with complete or incomplete Behçet's disease, with their consent, and serum was prepared to serve as samples. Samples were also prepared in the same manner from patients with sarcoidosis (12 patients), Harada's disease (12 patients) (all of whom had uveitis confirmed as an ocular symptom), and refractory aphtha (3 patients), as well as from 23 healthy individuals. Samples were stored frozen at -80°C until use.

(2) Preparation of Oligopeptide Immobilized Plate Oligopeptide 1 (GLRVYK, SEQ ID NO: 1), oligopeptide 2 (TLRVYK, SEQ ID NO: 2) and oligopeptide 3 (SIRVYK, SEQ ID NO: 3) were chemically synthesized.

The amino acid sequence of oligopeptide 2, TLRVYK, is a partial sequence of human b2-glycoprotein 1 protein involved in the formation of blood clots. It is known that the titer of antibodies that recognize TLRVYK is elevated in patients with Buerger's disease, a designated intractable disease that causes peripheral vascular occlusion. Attention has been paid to the relationship between the above-mentioned autoantibody and a protein containing TLRIYT (oligopeptide 4, SEQ ID NO: 4) or TLALYK (oligopeptide 5, SEQ ID NO: 5) as a partial sequence, which is highly homologous to TLRVYK produced by periodontal disease bacteria that often infect Buerger's disease patients due to periodontal destruction (Chen et al., J Clin Periodontol., 2009, 36(10): 830-5). In addition, the amino acid sequence of oligopeptide 3, SIRVYK, is a partial sequence of leucotoxin c produced by A. actinomycetemcomitans, a human oral bacterium. Wang et al. (Oral Microbiol Immunol., 2008: 23:401-405) reported that antibody titers recognizing SIRVYK were elevated in patients with chronic periodontal disease who were A. actinomycetemcomitans positive.

100 μL of each of oligopeptide 1, 2, or 3 in PBS (10 μg/mL) was added to a 96-well titer plate and incubated at 4°C for 12 hours. After washing with PBS, 200 μL of BSA solution (1 mg/mL PBS) was added and blocking was performed at room temperature for 2 hours to prepare an oligopeptide-immobilized plate.

(3) Preparation of rabbit anti-oligopeptide antibodies Rabbit anti-oligopeptide antibodies were prepared by Eurofins as follows. Chemically synthesized oligopeptides 1 to 3 were conjugated with BSA to prepare peptide antigens 1 to 3 according to a conventional method. Rabbits were immunized by administering peptide antigens 1 to 3 together with Freund's complete adjuvant. Rabbit serum samples taken over time were added to the oligopeptide-immobilized plates 1 to 3 described above in (2) and reacted. After washing, peroxidase-labeled anti-rabbit antibodies were reacted, and after further washing, a peroxidase substrate was added to measure the absorbance (OD). Booster immunization was performed until an increase in antibody titer was observed to such an extent that no decrease in absorbance was observed even when the rabbit serum was diluted 10,000 times or more.

Following the manufacturer's protocol, ligand coupling columns were prepared on which peptides 1 to 3 were immobilized, and antibodies specific to each peptide were purified from the collected rabbit serum to prepare rabbit anti-oligopeptide antibodies 1 to 3.

(4) Detection of oligopeptide-specific antibodies
100 μL of the sample (1) above, diluted 200-fold with PBS, was added to each of the oligopeptide-immobilized plates 1 to 3 (2) above, and incubated at room temperature for 1 hour. After washing with PBS, peroxidase-labeled protein A (KPL Peroxidase labeled Protein A, SeraCare Life Sciences, Inc.) was added and reacted at room temperature for 1 hour. After washing with PBS, a peroxidase substrate (TMB Peroxidase substrate, Moss, Inc.) was added to develop color, and the absorbance at 450 nm was measured.

100 μL of serially diluted solutions of rabbit anti-oligopeptide antibodies 1 to 3 were added to oligopeptide solid-phase plates 1 to 3, respectively, and reacted in the same manner to measure absorbance. An approximation curve was created with the rabbit antibody concentration on the y-axis and the OD value obtained using the rabbit antibody on the x-axis. The OD value obtained using the sample was applied to the formula for this approximation curve to calculate the anti-oligopeptide antibody concentration (antibody titer against the oligopeptide).

Figure 1 shows the IgG antibody titers against oligopeptides 1 to 3 in serum samples from healthy subjects, Behçet's disease patients, sarcoidosis patients, and Harada's disease patients. It was confirmed that the antibody titer against oligopeptide 1 (GLRVYK) was specifically elevated in Behçet's disease patients, and was only slightly detected in patients with other diseases and healthy subjects who had uveitis. A Mann-Whitney U test was performed on the antibody titers against oligopeptide 1 in the Behçet's disease patient group and healthy subject group, followed by a Bonferroni test as a post-hoc test. The antibody titers in the Behçet's disease patient group were significantly higher than those in the healthy subject group (p<0.05).

On the other hand, no significant difference was observed in the antibody titers against oligopeptide 3 (SIRVYK) among patients with any disease, including healthy subjects. Furthermore, the antibody titers against oligopeptide 2 (TLRVYK) were highest in healthy subjects. However, these antibody titers against oligopeptide 2 were two orders of magnitude lower in absolute value than the antibody titers against other oligopeptides, making it unlikely that they show a specific relationship with the disease. Furthermore, the antibody titers against oligopeptide 1 (GLRVYK) in serum samples from patients with refractory aphtha were similar to those of healthy subjects (not shown).

These results confirmed that antibodies that recognize GLRVYK are specific to patients with Behçet's disease, in contrast to antibodies that recognize the highly homologous TLRVYK or SIRVYK. Furthermore, it was inferred that antibodies that recognize GLRVYK are induced for reasons other than infection with periodontal disease bacteria that produce proteins containing TLRVYK or SIRVYK as partial sequences.

SEQ ID NO: 1: Oligopeptide 1
SEQ ID NO: 2: Oligopeptide 2
SEQ ID NO: 3: Oligopeptide 3
SEQ ID NO: 4: Oligopeptide 4
SEQ ID NO: 5: Oligopeptide 5

Claims (6)

A method for assisting in the diagnosis of Behçet's disease, comprising the step of detecting an antibody that recognizes the amino acid sequence shown in SEQ ID NO:1 in a sample collected from a subject. A method for assisting in the diagnosis of Behçet's disease, comprising the steps of contacting a sample collected from a subject with a molecule that contains the amino acid sequence shown in SEQ ID NO:1 as an epitope; and detecting binding of an antibody contained in the sample to the amino acid sequence. The method according to claim 2, wherein the molecule is an oligopeptide consisting of the amino acid sequence shown in SEQ ID NO:1, or an oligopeptide consisting of the amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids added to either or both termini of the amino acid sequence. The method according to any one of claims 1 to 3, wherein the sample is blood, plasma or serum. A kit for use in aiding in the diagnosis of Behcet's disease , comprising a molecule containing the amino acid sequence shown in SEQ ID NO: 1 as an epitope and a carrier for immobilizing said molecule. The kit described in claim 5, wherein the molecule is an oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or an oligopeptide consisting of the amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids added to either or both termini of the amino acid sequence.

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