JP2010285416A - Adm(amyopathic dermatomyositis) antibody epitope peptide and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a test method for easily detecting the presence/absence of ADM contraction, using a peptide having a specific bindability to an ADM-peculiar antibody. <P>SOLUTION: The test method for detecting ADM includes: contacting a subject-in-question-derived specimen with an artificially synthesized peptide bindable specifically to an antibody specifically present in an ADM-contracted patient through antigen-antibody reaction, wherein the peptide contains an amino acid sequence designated by Sequence No.1 or 2 or a partial amino acid sequence in either one of these amino acid sequences; incubating the specimen being in contact with the peptide under such conditions as to be enable the antigen-antibody reaction between the peptide and the antibody in the specimen; measuring the amount of antibodies derived from the specimen bound to the peptide after the incubation; and deciding the presence/absence of the ADM contraction of the subject based on the measurement on the amount of the antibodies. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a peptide effective for diagnosing ADM (Amyopathic Dermatomyositis), which is a type of dermatomyositis and is known as dermatomyositis with poor muscular symptoms, and a test reagent and kit containing the same. The present invention also relates to a test method (diagnosis method) of ADM performed using the peptide and an ADM test reagent containing the peptide as an active ingredient.

Dermatomyositis (DM) is a disease first described by Wagener in 1863 as polymyositis (PM). The number of patients in Japan is about 8,500 (5-8 per 100,000, population about The average is calculated from 130 million people).
By the way, as a type of dermatomyositis, there is a type in which muscle symptoms such as myalgia and CPK (creatine phosphokinase) increase are rarely observed, and in general, Amyopathy
Dermatomyositis, abbreviated ADM. This type of dermatomyositis with poor muscular symptoms is referred to as “ADM” in the present specification and claims as in the medical field.
Although it is said that ADM patients account for about 10% of all DM patients, no statistical survey has been conducted and the exact number of patients is unknown. ADM often accompanies rapid progressive interstitial pneumonia (Patent Document 1) that dies several weeks to several months after respiratory symptoms appear. This tendency is particularly remarkable in patients in Japan and other Asian countries (Non-patent Document 1). Therefore, early examination and early diagnosis of ADM are important.

  Although the cause of the onset of ADM has not been elucidated, autoimmunity is considered to play an important role since the presence of myositis-specific autoantibodies is known (see Patent Document 2 and Non-Patent Document 2). ). In relation to this, in 2005, Sato et al. Reported the presence of anti-CADM-140 antibody related to ADM and rapidly progressive interstitial pneumonia (Non-patent Document 3). However, there are few clinical cases in this regard, and its usefulness as a diagnostic marker has not been shown strictly. Therefore, it is possible to develop a clinical test method that can determine (diagnose) the presence or absence of ADM by using a peptide that constitutes an epitope that specifically binds to an antibody (for example, autoantibody) that is specifically recognized by individuals with ADM. , Very meaningful.

JP 2005-30852 A JP 2007-101488 A

Ryumachi, Volume 30, 1990, pp. 204-211 Journal of Investigative Dermatology, 100, 1993, pp. 116-123 ARTHRITIS & RHEUMATISM, Vol. 52 (No. 5), 2005, p. 1571-1576

  Therefore, the present invention was created to solve the above-mentioned conventional problems related to ADM, and includes a peptide having a specific binding property to an ADM-specific antibody that is specifically recognized by a person suffering from ADM, and An object is to provide a test method, a test reagent, and a kit for simply detecting the presence or absence of ADM using a peptide.

  As a result of repeated immunological and molecular biological studies to solve the above-mentioned problems, the present inventors have specifically found (produced) a peptide having a specific amino acid sequence in the serum of ADM-affected individuals. It has been found that a specific antibody (hereinafter, an antibody specific to a person suffering from ADM is abbreviated as “ADM antibody”) is specifically recognized and binds to the antibody by an antigen-antibody reaction. And it came to construct | assemble the diagnostic method which can discriminate | determine the presence or absence of ADM easily and accurately by using this peptide.

The present invention provides a reagent for testing ADM. One embodiment of the ADM test reagent disclosed herein has the following amino acid sequence:
VMLGDPDTPPGRRRR (SEQ ID NO: 1);
Or QERGHQGHDEHGWDVDDRGDLGEGIQ (SEQ ID NO: 2);
Or a partial amino acid sequence in any one of the amino acid sequences (that is, a number of consecutive amino acid residues that are part of any amino acid sequence and can form an epitope recognized by an ADM antibody) Partial amino acid sequence consisting of
And an artificially synthesized peptide that can specifically bind to an antibody (ADM antibody) present in a person suffering from ADM by an antigen-antibody reaction.

  The present invention also provides a kit for testing ADM. One aspect of the ADM test kit disclosed herein is the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a partial amino acid sequence in any one of the amino acid sequences (that is, a part of any amino acid sequence). A partial amino acid sequence consisting of a number of consecutive amino acid residues that can form an epitope recognized by an ADM antibody), and specifically binds to an antibody specifically present in a person suffering from ADM by an antigen-antibody reaction. An artificially synthesized peptide to be obtained and a base material carrying (fixing) the peptide are provided.

The present invention also provides an ADM inspection method. This ADM test method is characterized by using any of the ADM test reagents and / or reagent kits disclosed herein.
That is, one aspect of the ADM inspection method disclosed herein is:
In a test sample derived from a subject, the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a partial amino acid sequence in any one of the amino acid sequences (that is, a part of any one of the amino acid sequences and recognized by the ADM antibody) An antibody (ADM antibody) specifically present in a person suffering from ADM and an antigen-antibody reaction, including a partial amino acid sequence consisting of a number of consecutive amino acid residues (for example, 4 to 12) capable of forming an epitope. Contacting an artificially synthesized peptide that can bind;
Incubating the sample in contact with the peptide under conditions that may cause an antigen-antibody reaction between the peptide and the antibody in the sample;
Measuring the amount of antibody from the sample bound to the peptide after the incubation; and
Determining the presence or absence of ADM in the subject based on the measured value for the amount of the antibody,
Is included.

In the ADM test reagent and test kit and ADM test method having such a configuration, the ADM antibody having a predetermined amino acid sequence created by the present inventor and specifically present in an ADM sufferer is specifically bound by an antigen-antibody reaction. An artificially synthesized peptide (hereinafter, the peptide according to the present invention is referred to as “ADM antibody epitope peptide”) is used. Thus, a biological sample derived from a subject suspected of having ADM (typically, a sample containing biological components such as blood collected in advance from the subject, or at least a subject-derived antibody separated from the body fluid of the subject) A sample refers to whether or not an ADM antibody specifically present (produced) is present in a person suffering from ADM, and the amount of ADM antibody present is easily determined by a conventional immunochemical technique. Can do.
Therefore, according to the present invention, it is possible to easily and easily determine (diagnose) whether or not the subject suffers from ADM by an in vitro test.

In one preferred embodiment of the ADM test reagent and test kit and ADM test method, the ADM antibody epitope peptide used is the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a partial amino acid sequence in any one of the amino acid sequences (ie It is a synthetic peptide composed of a part of any amino acid sequence and capable of forming an epitope recognized by an ADM antibody (for example, a partial amino acid sequence consisting of 4 to 12 consecutive amino acid residues) .
By using such a synthetic peptide having a relatively small number of amino acid residues and a short chain length (typically a linear peptide), it is easier to determine (diagnose) the presence or absence of ADM in an in vitro test. be able to. Moreover, the peptide production cost can be kept low by using a short synthetic peptide.

  Further, as a preferred embodiment of the ADM antibody epitope peptide provided by the present invention, the ADM antibody comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and specifically present in an ADM-affected person is specific by an antigen-antibody reaction. Artificially synthesized peptides that can bind to are mentioned. One particularly preferred embodiment is a peptide composed of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. Such a peptide is suitable as a component of the ADM test reagent disclosed herein. Moreover, the ADM test | inspection method disclosed here can be implemented suitably using such a peptide.

In addition, in the ADM test method disclosed herein, ADM antibody specifically bound to the ADM antibody epitope peptide is detected by various immunochemical techniques that have been used in the past, and the amount of the bound antibody is measured. Although it is possible, it is preferable to use a secondary antibody. That is, in a preferred embodiment of the ADM test method disclosed herein, the antibody that specifically binds to the ADM antibody epitope peptide to be tested by an antigen-antibody reaction is human IgG, and the anti-human IgG antibody is used to An antigen-antibody reaction is caused between a human IgG antibody and an antibody bound to the peptide, and a measurement value relating to the amount of the antibody is determined by measuring the amount of the anti-human IgG antibody bound to the antibody. .
An immunochemical method using a secondary antibody having such a configuration (for example, the Enzyme Immunoassay (EIA) method, particularly the Enzyme-Linked Immunosorbent Assay (ELISA) method) can detect and measure the amount of ADM antibody with high accuracy. it can.

It is a scatter diagram which shows the result of the ADM test | inspection method implemented using the peptide shown to sequence number 1 which concerns on one Example, a test subject is an ADM patient group, a patient group of some other autoimmune disease, and a healthy subject group. It is a figure which shows the result of having classify | categorized into (2) and measuring the amount of ADM antibodies as a grade of the enzyme activity based on ELISA method about the biological sample (serum sample diluted 200 times) obtained from each test subject. In the figure, *, **, and *** indicate statistical significance when the measured value of the ADM patient group is compared with another subject group using t-test (Student's t-test). Differences (ie, *: p <0.05, **: p <0.01, ***: p <0.0001) are shown. In addition, the dotted line in the figure represents the highest positive value (cutoff value) at a specificity of 99% obtained from this result. FIG. 9 is a scatter diagram showing the results of an ADM test method performed using the peptide shown in SEQ ID NO: 2 according to another example, in which subjects are ADM patient groups, patients with several other autoimmune diseases, and It is a figure which shows the result of having classified into a healthy person group and measuring the amount of ADM antibodies as a grade of enzyme activity based on ELISA method about the biological sample (1000 times dilution serum sample) obtained from each test subject. In addition, * and *** in the figure are statistically significant differences (i.e., when the measured value of the ADM patient group is compared with other subject groups using t-test (Student's t-test)). , *: P <0.05, ***: p <0.0001). In addition, the dotted line in the figure represents the highest positive value (cutoff value) at a specificity of 99% obtained from this result. Using the measurement data of the ADM patient group and the measurement data of another autoimmune disease patient group in the ADM test method (ELISA) performed using the peptide shown in SEQ ID NO: 1 according to one example, the sensitivity (vertical axis) and It is a graph which shows the ROC (Receiver Operating Characteristic) curve which plotted the specificity (horizontal axis). The arrow in the figure represents the cutoff value. Sensitivity (vertical axis) using measurement data of ADM patient group and measurement data of other autoimmune disease patient group in ADM test method (ELISA) performed using the peptide shown in SEQ ID NO: 2 according to another example ) And specificity (horizontal axis). The arrow in the figure represents the cutoff value.

Hereinafter, preferred embodiments of the present invention will be described. It should be noted that matters other than matters specifically mentioned in the present specification (for example, primary structure and chain length of ADM antibody epitope peptide) and matters necessary for practicing the present invention (for example, for causing an antigen-antibody reaction) General items such as immunochemical techniques such as ELISA including incubation conditions setting, chemical synthesis of peptides, and preparation and storage of test reagents containing peptides as a component) are medical, pharmaceutical, organic chemistry, biological It can be grasped as a design matter of a person skilled in the art based on conventional techniques in fields such as chemistry, genetic engineering, protein engineering, molecular biology, and hygiene. The present invention can be carried out based on the contents disclosed in this specification and common technical knowledge in the field.
In the present specification, amino acids are represented by one-letter code (in the sequence listing, three-letter code) based on the nomenclature related to amino acids shown in the IUPAC-IUB guidelines. Also, the entire contents of all references cited herein are hereby incorporated by reference.

In the present application, “artificially synthesized peptide” does not mean that the peptide chain exists independently and stably in nature, but artificial chemical synthesis or biosynthesis (ie, production based on genetic engineering). ) And can exist stably in a predetermined system (for example, a liquid or solid composition constituting an ADM test reagent).
In the present application, the “peptide” is a term indicating an amino acid polymer having a plurality of peptide bonds, and includes a term called a polypeptide or an oligopeptide depending on the number of constituent amino acid residues. The ADM antibody epitope peptide according to the present invention is an amino acid residue contained in the peptide chain as long as it can specifically bind to an antibody specifically present in an ADM sufferer by an antigen-antibody reaction (ie, can constitute an epitope). The number is not limited. Typically, the total number of amino acid residues is 200 or less, and particularly preferably 100 or less. A peptide chain consisting of about 70 or less (for example, about 10 to 55) amino acid residues is suitable as an ADM antibody epitope peptide.

In addition, the “peptide” in the present application includes various modified peptides in addition to peptides consisting only of amino acid polymers (peptide chains) such as simple linear structures, helical structures, and cyclic structures. For example, a typical peptide in which the C-terminus of the amino acid sequence or the carboxyl group (—COOH) of the side chain of the amino acid residue in the peptide chain is amidated (—CONH ₂ ) or esterified (—COOR) Take as an example. Here, as R in the ester group, for example, an alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl and n-butyl, or a cycloalkyl group having 3 to 8 carbon atoms such as cyclopentyl and cyclohexyl. Or an aryl group having 6 to 12 carbon atoms such as phenyl and α-naphthyl. A typical example of a modified peptide is one in which the amino group at the N-terminus of the amino acid sequence or the amino group of the side chain of the amino acid residue in the peptide chain is acetylated. In addition to this, the N-terminal amino group is protected with a protecting group (for example, an acyl group having 1 to 6 carbon atoms such as formyl group, acetyl group, etc.); The glutamine residue of the above is pyroglutamine oxidized, and the side chain substituent of the amino acid residue in the peptide chain (hydroxyl group, mercapto group, amino group, imidazole group, indole group, guanidino group, etc.) is an appropriate protecting group Those protected are also included in the “peptide” in the present application.
In addition, those having a sugar chain (sugar chain peptide) are also artificially capable of specifically binding to an antibody having a predetermined amino acid sequence disclosed herein and existing specifically in a person suffering from ADM by an antigen-antibody reaction. As long as it is a synthesized peptide, it is a peptide included in the ADM antibody epitope peptide according to the present application.

  In addition, various salt forms (peptide salts) are also artificially synthesized that can specifically bind to an antibody having a predetermined amino acid sequence disclosed herein and present specifically in a person suffering from ADM by an antigen-antibody reaction. As long as it can function as a synthesized peptide, it is a peptide included in the ADM antibody epitope peptide according to the present application. For example, it may be in the form of a pharmaceutically preferred acid addition salt. As this kind of peptide salt, various inorganic acid salts (phosphate, hydrochloride, sulfate, hydrobromide, etc.) or various organic acid salts (acetate, tartrate, formate, oxalic acid) Salt, propionate, fumarate, citrate, maleate, succinate, malate, benzoate, methanesulfonate, benzenesulfonate) and the like. Alternatively, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, amine salts such as monomethylamine salts and triethylamine salts, fourth salts such as trimethylethylammonium salts and tetramethylammonium salts. A quaternary ammonium salt may be used.

  The present inventor constructed a genome library derived from microorganisms obtained from soil containing a wide variety of microorganisms, and conducted numerous screenings using ADM patient sera while intensively examining them, so that SEQ ID NO: 1 and SEQ ID NO: It has been found that the peptides having the amino acid sequences shown in Fig. 2 constitute an epitope that can be specifically recognized by an antibody that appears to be specifically present in ADM-affected individuals, and can specifically bind to the antibody. The present invention has been completed by applying to the inspection method.

That is, a typical example of the ADM antibody epitope peptide disclosed herein is one of the following amino acid sequences:
VMLGDPDTPPGRRRR (SEQ ID NO: 1);
Or QERGHQGHDEHGWDVDDRGDLGEGIQ (SEQ ID NO: 2);
Is an artificially synthesized peptide composed of
Hereinafter, for convenience, the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 according to the present invention is referred to as “first epitope peptide”, and the peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 according to the present invention is referred to as “second epitope peptide”. .

The first epitope peptide as well as the second epitope peptide can be preferably used as an ADM antibody epitope peptide by itself, but the ADM antibody epitope peptide used in the ADM test method disclosed herein is classified into the two types. There is no need to limit it strictly. Various modified peptides based on the amino acid sequence of the first epitope peptide or the second epitope peptide can be employed as long as they can specifically bind to an antibody (ADM antibody) specifically present in a person suffering from ADM.
For example, instead of using the first epitope peptide or the second epitope peptide as it is, an epitope (typically a sequential epitope) that is part of the amino acid sequence shown in SEQ ID NO: 1 or 2 and recognized by the ADM antibody is used. A modified peptide composed of a partial amino acid sequence composed of a continuous number of amino acid residues (for example, about 4 to 12, particularly about 5 to 10) that can be formed can be used. Such an epitope site (that is, a partial amino acid sequence to be used) can be determined without performing various conventionally known epitope analyses. For example, 5-8 selected as appropriate from the amino acid sequence shown in SEQ ID NO: 1 or 2 To synthesize a peptide consisting of about consecutive amino acid residues and incubate with a biological sample (typically blood sample such as serum) of an ADM patient that actually contains an ADM antibody to confirm the presence or absence of an antigen-antibody reaction Can be done easily.
A peptide comprising the partial amino acid sequence thus obtained, or a synthetic peptide containing the partial amino acid sequence and capable of specifically binding to an antibody (ADM antibody) specifically present in an ADM sufferer is also disclosed herein. The ADM inspection method can be used suitably.

  Alternatively, as long as it can be recognized as an epitope by an ADM antibody, it may be a first epitope peptide, a second epitope peptide, or a peptide containing the above partial amino acid sequence (sequence constituting the epitope). For example, a peptide comprising a peptide chain in which the amino acid sequence shown in SEQ ID NO: 1 (first epitope peptide) and / or the amino acid sequence shown in SEQ ID NO: 2 (second epitope peptide) is arranged close to each other by 2 or 3 units As long as it is a synthetic peptide that can specifically bind to an antibody (ADM antibody) present in a person suffering from ADM by an antigen-antibody reaction, it is a peptide that can be suitably used for the ADM test method disclosed herein. obtain. In addition, from the viewpoint of efficiently synthesizing the ADM antibody epitope peptide to be used, the number of amino acid residues constituting the peptide chain is suitably 200 or less, preferably 100 or less, and 70 or less (preferably about 10 to 55). For example, 50 or less).

  Among the ADM antibody epitope peptides disclosed herein, relatively short ones (for example, relatively short ones having 50 or less amino acid residues constituting the peptide chain, such as the first epitope peptide and the second epitope peptide, For example, any of a conventionally known solid phase synthesis method or liquid phase synthesis method may be employed, and Boc (t-butyloxycarbonyl) may be used as a protective group for the amino group. Or a solid phase synthesis method using Fmoc (9-fluorenylmethoxycarbonyl), that is, a desired amino acid sequence or modification (C-terminal amidation, etc.) moiety by a solid phase synthesis method using a commercially available peptide synthesizer. It is possible to easily synthesize peptide chains that have the following characteristics: By using a commercially available fully automatic peptide synthesizer without knowing the detailed synthesis process or principle, , It is possible to easily produce the peptide of interest.

Alternatively, ADM antibody epitope peptides may be biosynthesized based on genetic engineering techniques. This approach is preferred when producing ADM antibody epitope peptides with relatively long peptide chains. That is, DNA having a nucleotide sequence (including the ATG start codon) encoding the amino acid sequence of the desired ADM antibody epitope peptide is synthesized. An expression comprising this DNA and various regulatory elements (including a promoter, a ribosome binding site, a terminator, an enhancer, and various cis elements that control the expression level) for expressing the amino acid sequence in a host cell. A recombinant vector having the gene construct for use is constructed according to the host cell.
This recombinant vector is introduced into a predetermined host cell (for example, yeast, insect cell, plant cell, animal (mammalian) cell) by a general technique, and the host cell or a tissue or an individual containing the cell under a predetermined condition Is cultured. Thus, the target ADM antibody epitope peptide can be expressed and produced in the cell. Then, by isolating and purifying the polypeptide from the host cell (in the medium if secreted), a peptide having the desired amino acid sequence can be obtained. This recombinant vector is introduced into a predetermined host cell (for example, yeast, insect cell, plant cell, mammalian cell) by a general technique, and the host cell or a tissue or an individual containing the cell is cultured under a predetermined condition. . Thereby, the target polypeptide can be expressed and produced in the cell. The target peptide can be obtained by isolating and purifying the ADM antibody epitope peptide from the host cell (in the medium if secreted).
As a method for constructing a recombinant vector and a method for introducing the constructed recombinant vector into a host cell, a method conventionally used in the field may be employed as it is, and such method itself particularly characterizes the present invention. Since it is not a thing, detailed description is abbreviate | omitted.

  The ADM test reagent provided by the present invention is a reagent comprising at least one ADM antibody epitope peptide according to the present invention having the structure as described above. Such a test reagent can contain various components in addition to the peptide as the main component, like the test reagents used in conventional immunochemical diagnostic methods. Usually, in addition to the peptide, it may contain at least one subcomponent that does not denature the peptide. For example, in the case of a liquid reagent, physiological saline or various pharmaceutically acceptable buffers can be included as a solvent, that is, a carrier. In addition, agents such as preservatives, stabilizers, pH adjusters and the like may be added in the same manner as conventional diagnostic agents and peptide preparations. Moreover, the reagent of the form provided with the peptide of a solid state (typically powder form) may be sufficient. In this case, excipients such as various carbohydrates (dextrin, lactose, etc.) can be included.

Moreover, the ADM antibody epitope peptide constituting the test reagent may be previously labeled with various labeling substances.
For example, as a labeling substance for the peptide, a labeling compound usually used by those skilled in the art in the diagnostic field by an immunochemical technique such as ELISA can be used. For example, radioactive isotopes such as ³ H, ¹⁴ C, ¹³¹ I, ^99m Tc; enzymes such as β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase; fluorescamine, fluorescein isothiocyanate, etc. Luminescent materials such as luciferin, luminol derivatives, isoluminol derivatives, and the like.
Peptide labeling using these labeling substances may be performed by a conventionally known method, and is not a technique for characterizing the present invention, and thus detailed description thereof is omitted.

The ADM test method disclosed herein detects ADM antibody in a biological sample obtained from a subject (typically a blood-derived sample collected from the subject) by an immunochemical technique using an ADM antibody epitope peptide. As long as the detection of the ADM antibody can be performed, various methods can be employed. For example, EIA, radioimmunoassay (RIA), fluorescence immunoassay (FIA), chemiluminescence immunoassay (CLIA), gel precipitation reaction, immunoturbidimetric method, particle agglutination method and the like can be mentioned.
For example, commonly used immunochemical techniques are: Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continued radioimmunoassay” (Kodansha, published in 1974), Eiji Ishikawa et al. "Enzyme Immunoassay" (Medical School, published in 1978), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Medical School, published in 1982), edited by Eiji Ishikawa, et al. (Third edition) (Medical Shoin, published in 1987).

Moreover, what is necessary is just to select suitably the reagent and instrument which are contained in a kit according to the content of the test | inspection method. For example, depending on the type of immunochemical technique and the detection / measurement apparatus to be applied (usually using a commercially available apparatus and selecting appropriate chemicals and instruments (base materials, etc.) according to the use manual). In addition to peptides and various carriers (including solvents such as physiological saline) constituting the above-mentioned test reagents, dissolved solutions (various buffer solutions, etc.), washing solutions, enzymes, enzyme substrate solutions, enzyme reaction detection reagents , Dyes, reaction stop solutions, buffers, etc. may be provided in the reagent kit. These may be provided in the form of a solution, or may be provided in the form of a solid (powder or the like), and may be in the form of preparing a solution at the time of use.
The kit components and instruments other than the ADM antibody epitope peptide may be those conventionally used in the conventional diagnostic field of this type, and do not characterize the present invention. To do.

In the implementation of the ADM test method according to the present invention, preferably, a kit containing a labeled or unlabeled ADM antibody epitope peptide previously supported on a substrate is used. Alternatively, the base material (plate or the like) and the ADM antibody epitope peptide may be separately provided in the kit, and the ADM antibody epitope peptide may be supported (immobilized) on the base material at the time of use.
In addition, as such a base material, what is typically made from various high molecular compounds (for example, agarose, a cellulose), synthetic resins (for example, polystyrene, polypropylene, polycarbonate), or ceramics, such as glass, is mentioned. The shape of the substrate includes the shape of a plate, bead, membrane, stick, test tube or the like according to the purpose of use, and there is no particular limitation. The method for supporting (fixing) the peptide on the substrate may be the same as the conventional method, and is not particularly limited. For example, a conventionally known physical adsorption method, covalent bond method, ionic bond method, crosslinking method and the like may be employed.

As a preferred embodiment of the ADM test method, a method using a so-called secondary antibody can be mentioned. In that case, the ADM test kit to be used includes an appropriate secondary antibody and reagents capable of detecting the secondary antibody and measuring the amount thereof by an immunochemical technique.
Any secondary antibody that can detect an ADM antibody can be used without any particular limitation. However, since a typical example of an ADM antibody is considered to be IgG, a preferred secondary antibody is anti-human IgG. Commonly available secondary antibodies such as anti-human IgG mouse antibody, anti-human IgG rat antibody, anti-human IgG goat antibody, and anti-human IgG rabbit antibody can be used without particular limitation.
By using such a secondary antibody (typically an anti-human IgG antibody) and performing an immunochemical technique (for example, EIA method, particularly ELISA method), the ADM antibody bound to the secondary antibody and the above peptide Measurement with respect to the amount of secondary antibody (typically anti-human IgG antibody) bound to the antibody based on the antigen-antibody reaction between and can be determined with higher accuracy and / or sensitivity.

  In a preferred embodiment of the ADM test method using a secondary antibody, a labeled antibody or a non-labeled secondary antibody (typically an anti-human IgG antibody) previously supported on a substrate is used. Use kit containing. Alternatively, the kit is equipped with a base material (plate or the like), an ADM antibody epitope peptide and a secondary antibody separately, and the secondary antibody (typically an anti-human IgG antibody) is used as the base material at the time of use. It may be supported (immobilized).

A preferred embodiment of the ADM inspection method disclosed herein will be described in further detail.
The ADM test method according to the present invention is a method in which at least one ADM antibody epitope peptide is used as an artificial antigenic substance constituting an epitope of an ADM antibody. That is, when an ADM antibody is present in a biological sample of a subject with respect to the ADM antibody epitope peptide, an antigen-antibody complex is generated by an antigen-antibody reaction, and the presence or absence of ADM is detected by detecting and measuring this complex. It is a method of diagnosis.
The test sample as the test object is not particularly limited as long as it can be used for the immunochemical technique as described above, and a biological sample derived from a subject collected in advance from the subject, for example, blood, plasma, serum, lymph fluid Ascites, saliva, synovial fluid, cerebrospinal fluid, semen, tears, sweat, urine, etc. can be used as they are or as they are appropriately diluted. In particular, the use of blood, serum and plasma is preferred. Or you may use the antibody sample which isolated the antibody component previously from samples, such as blood extract | collected from the test subject.

For example, if a serum sample of a subject is used as a test sample and ELISA is exemplified as an immunochemical technique, the ADM antibody to be measured can be detected and measured by the following method, for example.
First, an ADM antibody epitope peptide is supported (immobilized) on a substrate such as a plate, a serum sample as a test sample is added thereto, and the temperature is about 4 to 40 ° C. (for example, 30 to 37 ° C.) for a predetermined time. Incubate (eg, 0.5-24 hours). When the ADM antibody is present in the sample, the ADM antibody epitope peptide and the ADM antibody bound to the substrate are bound by the antigen-antibody reaction during the incubation. That is, the ADM antibody is immobilized on the substrate.
Next, detection of the ADM antibody (mainly IgG) captured (immobilized) on the substrate via the ADM antibody epitope peptide and measurement of the amount thereof are performed using the secondary antibody. For example, an anti-human IgG antibody (for example, anti-human IgG mouse IgG) is added as a secondary antibody labeled with a predetermined labeling substance (typically a substance that can serve as an enzyme molecule or enzyme substrate), and then incubated again to produce an antigen antibody The secondary antibody is specifically bound to the ADM antibody by the reaction. Next, an appropriate enzyme reaction corresponding to the labeling substance is generated, and the amount of ADM antibody is measured (calculated) based on the reaction. Then, the presence or absence of ADM in the subject is determined based on the obtained measurement value. Typically, the presence or absence of ADM in a subject can be determined by comparison with a predetermined cut-off value.
Alternatively, a detection reagent such as an anti-human IgG antibody is immobilized on a substrate in advance, and serum is added thereto to capture the ADM antibody in the serum, and then the ADM antibody epitope peptide is added to test. It is also possible to detect and measure the ADM antibody present in the sample. Selection of various means in these measurement techniques, modification thereof, and the like are all known to those skilled in the art (see “Proposal for Clinical Laboratory Methods”, Kanbara Publishing, 1995, etc.)

  In the above-described ELISA, a test sample (serum) is added to the ADM antibody epitope peptide immobilized on a substrate to cause a reaction (primary reaction), and a labeled anti-human IgG antibody is reacted (2). ADM antibody is detected and the amount thereof is measured by measuring the activity of the labeling substance on the substrate after the subsequent reaction). The primary reaction and the secondary reaction may be performed simultaneously. It may be performed at different times. In addition, the secondary antibody does not necessarily need to be one type, and a mixture of two or more types of antibodies may be used for the purpose of improving measurement sensitivity.

The solvent used in the above measurement system (ELISA) is not particularly limited as long as it does not adversely affect the reaction. For example, phosphorus such as phosphate buffered saline (PBS) can be used. Buffers having a pH of about 6 to 9 such as acid buffer, Tris-HCl buffer such as Tris-HCl buffered saline (TBS), borate buffer, citrate buffer, and acetate buffer, and Tween (registered trademark) ) 20, a surfactant such as Triton (registered trademark) X-100, a stabilizer such as bovine serum albumin (hereinafter referred to as “BSA”) and milk protein, the above buffer solution containing a preservative such as NaN ₃ , etc. Is mentioned.

  In the above step, the complex of the antigen-antibody reaction (including a label by a labeling compound) can be measured by a method according to the type of the labeling compound to be used. For example, when an enzyme is used as the labeling compound, the enzyme activity of the label is measured. The enzyme activity can be measured according to a known method depending on the type of enzyme used. In order to measure these enzyme activities, a substrate corresponding to the enzyme, for example, 3,3 ′, 5 ′, 5-tetramethylbenzidine (abbreviated as TMB) or o-phenylenediamine (abbreviated as OPD) is used for peroxidase. A method of measuring color development with a spectrophotometer or the like after adding p-nitrophenyl phosphate (abbreviated as pNPP) to react with alkaline phosphatase for a predetermined time is widely used.

One preferred specific embodiment of the ADM inspection method disclosed herein is summarized below.
<1> An ADM antibody epitope peptide is supported (fixed) in each well of a microplate (base material). Usually, a blocking operation is performed with a protein such as BSA or casein and a surfactant such as Tween (registered trademark) 20 in order to block non-specific binding sites in the well.
<2> A test sample (serum or plasma) is diluted with a buffer solution (sample diluent) containing a protein such as BSA or casein and a surfactant such as Tween (registered trademark) 20 if necessary, and the above microplate And incubating for a predetermined time to carry out an antigen-antibody reaction (primary reaction).
<3> After washing the microplate with a buffer solution (washing solution) containing a surfactant such as Tween (registered trademark) 20 if possible, the labeled anti-human IgG antibody is converted into a protein such as BSA or casein, Tween (registered trademark). ) It is diluted with a buffer solution (diluent) containing a surfactant such as 20 and then incubated for a predetermined time to carry out an antigen-antibody reaction (secondary reaction).
<4> After washing the microplate with the above washing solution, the method corresponding to the label (enzyme activity for enzyme labeling, radioactivity for radioactive labeling) is measured.

  Several examples relating to the present invention will be described below, but the present invention is not intended to be limited to those shown in the examples.

<Example 1: Synthetic peptide>
A total of two types of ADM antibody epitope peptides, Samples 1 and 2, were produced using a commercially available peptide synthesizer. That is, sample 1 is the first epitope peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and sample 2 is the second epitope peptide consisting of the amino acid sequence shown in SEQ ID NO: 2. Table 1 shows the total number of amino acid residues, molecular weight (kDa), isoelectric point, and hydrophobicity of each sample peptide. The degree of hydrophobicity in Table 1 is the percentage (%) of the number of hydrophobic amino acid residues in all constituent amino acid residues.

<Example 2: ADM inspection>
An ELISA was performed using the two sample peptides (ADM antibody epitope peptides) obtained in Example 1, an ADM patient group, a systemic lupus erythematosus (SLE) patient group, a systemic scleroderma (SSc) patient group, The reactivity with respect to the serum samples obtained from the interstitial pneumonia patient group, the rheumatoid arthritis (RA) patient group, the dermatomyositis (DM) / polymyositis (PM) patient group, and the healthy subject group was examined.

The peptide of sample 1 or sample 2 was carried in each well of a commercially available synthetic resin microtiter plate (96 wells).
Specifically, a peptide solution for loading in which the peptide concentration of sample 1 was 0.5 μg / mL with PBS containing 0.1% NaN ₃ was prepared. On the other hand, for sample 2, a carrier peptide solution having a peptide concentration of 2.0 μg / mL was prepared with PBS containing 0.1% NaN ₃ .
These were added to each well of the microtiter plate and incubated overnight at 4 ° C. Next, each well was washed with PBS containing 0.1% Tween® 20, and then blocked with blocking buffer (PBS containing 2% BSA, 5% sucrose, 0.1% NaN ₃ ). This produced the peptide carrying (immobilization) plate of sample 1 and the peptide carrying (immobilization) plate of sample 2, respectively.

The ADM test method according to the present example was performed using the biological samples of the above-mentioned patient groups of each disease and the group of healthy subjects.
Specifically, serum samples obtained from each subject (ie, 14 ADM patients, 33 SLE patients, 20 SSc patients, 14 interstitial pneumonia patients, 50 RA patients, 50 DM / PM patients, 83 cases from healthy subjects were diluted with a sample diluent (TBS containing 1% BSA, 0.1% Tween® 20). In the test plot using sample 1, it was diluted 1/200. On the other hand, in the test section using sample 2, it was diluted 1/1000.
Next, 100 μL of diluted sample was added to each well. And it incubated in the state left still at 22 degreeC for 1 hour, Then, it wash | cleaned 6 times by PBST (PBS containing 0.1% Tween (trademark) 20).

Then, an HRP (Horseradish Peroxidase) -labeled anti-human IgG antibody (manufactured by DAKO) solution diluted to 1/4000 with the same buffer was added, incubated at 22 ° C. for 1 hour, and then incubated with the same buffer. Washed twice.
Next, a TMB solution (manufactured by DAKO) was added as a chromogenic substrate and reacted at 22 ° C. for 30 minutes, and then the stop solution (2N H ₂ SO ₄ ) was added to stop the reaction. Next, the absorbance (450 nm-600 nm) was measured with a commercially available plate reader. Data analysis was performed by converting absorbance values into units (U / mL). Absorbance (A) 450 nm-Absorbance (A) A standard solution having a value of 600 nm of about 1.5 is diluted in advance, and further three-fold dilution is performed in two stages, 1000 U / mL, 333 U / mL, and 111 U / mL, respectively. It was priced. The measured values were converted to units by a standard curve created using these and four blanks. Each sample was measured at two points, and the average value was used for analysis.
When the cv value of the two measured values was 10% or more, the measurement was performed again. The measurement results are shown as scatter plots in FIG. 1 (test section using sample 1) and FIG. 2 (test section using sample 2).

Then, a significant difference test (Student's t-test) was performed between the measured value of the ADM patient group and the measured value (absorbance value) in the patient group of other diseases. As a result, in the test group using sample 1 (first epitope peptide), a significant difference was observed between the ADM patient group and the patient group of all other diseases including the healthy subject group (however, the interstitial property In the pneumonia patient group, one case with a high value that could not be converted was seen, so a significant difference test was not performed).
Moreover, also in the test plot using sample 2 (second epitope peptide), a significant difference was observed between the DM / PM patient group, the healthy subject group, and the ADM patient group.

FIG. 3 shows ROC curves in ADM and other diseases for the test section using sample 1 (first epitope peptide). In addition, FIG. 4 shows ROC curves in ADM and other diseases for the test section using sample 2 (second epitope peptide).
Based on these ROC curves, a value having the highest positive rate at a specificity of 99% in each test group was determined as a cutoff value. Specifically, the cut-off value of the test group using sample 1 (first epitope peptide) is 308.8 U / mL, and the cut-off value of the test group using sample 2 (second epitope peptide) is 528.0 U. / ML.
Using these cut-off values, positive determination was performed on the measured values of the serum of each disease patient, and the positive rate was determined. The results are shown in Table 2.

As shown in Table 2, the positive rate for the serum of the ADM patient group was 28.6% in the test group using sample 1 and 14.3% in the test group using sample 2, whereas SLE The false positive rate for other autoimmune diseases or normal human serum was 0 to 7.1%.
From this result, it was shown that the ADM test method according to the present example can be used to differentiate from the presence or absence of ADM and other autoimmune diseases.

  As described above, by using the ADM antibody epitope peptide disclosed herein, a test reagent and a test kit for diagnosis of ADM are provided, and the presence or absence of ADM is easily or rapidly determined by an immunochemical method. It becomes possible to detect.

SEQ ID NO: 1 Synthetic peptide SEQ ID NO: 2 Synthetic peptide

Claims (9)

A peptide used for testing ADM (Amyopathic Dermatomyosis),
An artificially synthesized peptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and capable of specifically binding to an antibody specifically present in a person suffering from ADM by an antigen-antibody reaction.   The peptide of Claim 1 comprised from the amino acid sequence shown to sequence number 1 or sequence number 2. A reagent for testing ADM (Amyopathic Dermatomyosis),
Artificially comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a partial amino acid sequence in any one of the amino acid sequences and capable of specifically binding to an antibody specifically present in a person suffering from ADM by an antigen-antibody reaction Synthesized peptides,
An ADM test reagent.   The ADM test reagent according to claim 3, wherein the peptide is a synthetic peptide composed of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a partial amino acid sequence in any one of the amino acid sequences. A kit for testing ADM (Amyopathic Dermatomyosis),
Artificially comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a partial amino acid sequence in any one of the amino acid sequences and capable of specifically binding to an antibody specifically present in a person suffering from ADM by an antigen-antibody reaction A synthesized peptide;
A substrate carrying the peptide;
An ADM test kit.   The ADM test kit according to claim 5, wherein the peptide is a synthetic peptide composed of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a partial amino acid sequence in any one of the amino acid sequences. An inspection method for ADM (Amyopathic Dermatomyosis),
A test sample derived from a subject contains the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a partial amino acid sequence in any one of the amino acid sequences, and is specifically detected by an antigen-antibody reaction with an antibody specifically present in a person suffering from ADM Contacting an artificially synthesized peptide capable of binding to
Incubating the sample in contact with the peptide under conditions that allow an antigen-antibody reaction between the peptide and the antibody in the sample;
Measuring the amount of antibody from the sample bound to the peptide after the incubation; and
Determining the presence or absence of ADM in the subject based on the measured value relating to the amount of the antibody;
An ADM inspection method.   The test method according to claim 7, wherein a synthetic peptide composed of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a partial amino acid sequence in any one of the amino acid sequences is used as the peptide.   The antibody that specifically binds to the peptide to be examined is human IgG, and an anti-human IgG antibody is used to generate an antigen-antibody reaction between the anti-human IgG antibody and an antibody that specifically binds to the peptide. And measuring the amount of the anti-human IgG antibody bound to the antibody to determine the measurement value for the antibody amount.

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