Reagents for testing lymphocytic anterior pituitary inflammation and related disorders and their use

Abstract

This disclosure aims to provide a technology that can contribute to a less invasive or more reliable diagnosis of lymphocytic anterior pituitary inflammation and related disorders. [Solution] For this purpose, a first epitope peptide is used that includes a first amino acid sequence consisting of at least six consecutive amino acid residues from positions 737 to 744 of the amino acid sequence represented by SEQ ID NO: 1, and which immunologically binds to the anti-PTPN13 antibody.

Description

Technical Field

[0001] This specification relates to a reagent for examining lymphocytic adenohypophysitis and related diseases thereof and its use.

Background Art

[0002] One of the pituitary dysfunctions with unknown etiology is autoimmune hypothalamo-pituitaryitis, and a typical disease thereof is lymphocytic hypophysitis. Lymphocytic hypophysitis includes, depending on the lesion site, lymphocytic adenohypophysitis (LAH), lymphocytic infundibulo-neurohypophysitis (LINH), and lymphocytic panhypophysitis (LPH). LAH is a disease in which inflammatory lesions are localized in the anterior pituitary gland, and the secretion of anterior pituitary hormones such as adrenocorticotropic hormone (ACTH) decreases or is lost.

[0003] As a related disease of LAH, there is isolated adrenocorticotropic hormone (ACTH) deficiency (IAD).

[0004] The present inventors have already reported that LAH and IAD can be distinguished from other diseases by anti-GATA2 antibody and anti-PTPN13 antibody that specifically bind to various specific proteins present in the blood of patients with LAH and IAD (Patent Document 1).

Prior Art Documents

Patent Documents

[0006] Of these specific proteins, for example, the protein Protein Tyrosine Phosphatase Non-Receptor type 13 (PTPN13), encoded by the PTPN13 gene, has not yet had its epitope to which autoantibodies react identified. Therefore, detecting these autoantibodies required using PTPN13 itself as the detection reagent, which was impractical.

[0007] This specification provides a technique that can contribute to more practical testing for detecting lymphocytic anterior pituitary inflammation and related disorders. [Means for solving the problem]

[0008] Based on the amino acid sequence of PTPN13, the inventors conducted various studies and were able to identify several epitopes to which anti-PTPN13 antibodies specifically bind in the serum of LAH and IAD patients. Identifying these epitopes allows for the examination of susceptibility claims for these diseases. Based on these findings, this specification provides the following means.

[0009] [1] A reagent for detecting an anti-PTPN13 antibody, comprising either or both of the following: a first epitope peptide having a first amino acid sequence consisting of at least six consecutive amino acid residues from positions 737 to 744 of the amino acid sequence represented by SEQ ID NO: 1, which immunologically binds to an anti-PTPN13 antibody; and a second epitope peptide having a second amino acid sequence consisting of at least six consecutive amino acid residues from positions 1072 to 1079 of the amino acid sequence, which immunologically binds to an anti-PTPN13 antibody. [2] The first epitope peptide comprises the first amino acid sequence and has a sequence of additional amino acids selected from positions 716 to 765 of the amino acid sequence represented by Sequence ID No. 1, following the first amino acid sequence. The reagent according to [1], wherein the second epitope peptide comprises the second amino acid sequence and has a subsequent additional amino acid sequence selected from the 1051st to 1091st positions of the amino acid sequence represented by Sequence ID No. 1, following the second amino acid sequence. [3] The reagent described in [1], wherein the first amino acid sequence is KLDLSYIK. [4] The reagent described in [1], wherein the second amino acid sequence is KENDVLHK. [5] The reagent according to any one of [1] to [4], wherein the peptide is labeled. [6] Reagents for ELISA, as described in any of [1] to [5]. [7] A reagent for testing lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency, as described in any of [1] to [6]. A detection kit for anti-PTPN13 antibody, comprising the reagent described in any of [8][1] to [7]. [9] The kit described in [9] for testing for lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency. A detection kit for anti-PTPN132 antibody, comprising a solid support on which one of the reagents described in

[10] [1] to [7] is immobilized.

[11] The detection kit described in

[10] for testing lymphocytic anterior pituitary inflammation or isolated inferior adrenocorticotropic hormone deficiency.

[12] Contacting a test sample that may contain anti-PTPN13 antibody with any of the reagents described in [1] to [7] to produce an antigen-antibody reaction, To detect the complex resulting from the aforementioned antigen-antibody reaction, A method for testing for lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency, including the above.

[13] A recovery agent for anti-PTPN13 antibody comprising either or both of the following: a first epitope peptide having a first amino acid sequence consisting of at least six consecutive amino acid residues from positions 737 to 744 of the amino acid sequence represented by SEQ ID NO: 1, which immunologically binds to an anti-PTPN13 antibody; and a second epitope peptide having a second amino acid sequence consisting of at least six consecutive amino acid residues from positions 1072 to 1079 of the amino acid sequence, which immunologically binds to an anti-PTPN13 antibody. [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows the responsiveness of a portion of the 15AA overlap peptide array of the full-length protein PTPN13 to serum from LAH patients, IAD patients, and healthy controls. [Modes for carrying out the invention]

[0011] This disclosure relates to a reagent for detecting anti-PTPN13 antibody, comprising an epitope peptide that immunologically binds to the anti-PTPN13 antibody, and to the use thereof. The anti-PTPN13 antibody is an autoantibody that serves as a marker for LAH and IAD. This has been disclosed in Patent Document 1, which the inventors have already filed.

[0012] One embodiment of the reagent disclosed herein is a reagent that evaluates the likelihood of developing LAH or IAD using the presence of anti-PTPN13 antibody as an indicator. When this reagent confirms the presence of anti-PTPN13 antibody or that the amount of anti-PTPN13 antibody is above a predetermined level, it is possible to confirm the likelihood of developing at least one of these diseases in the patient from whom the test sample was collected. When this reagent denies the presence of anti-PTPN13 antibody or that the amount of anti-PTPN132 antibody is above a predetermined level, it is possible to deny the likelihood of developing either of these diseases in the patient from whom the test sample was collected.

[0013] The reagents disclosed in this specification are based on the immunological binding resulting from the antigen-antibody reaction between a specific epitope sequence and an anti-PTPN13 antibody. Therefore, it can contribute to the diagnosis that can more easily and surely diagnose an anti-PTPN13 antibody, LAH or IAD associated with the detection of the anti-PTPN13 antibody, as compared with the prior art.

[0014] Note that the technology disclosed in this specification does not diagnose LAH or IAD alone, but provides useful information for diagnosis. When the technology disclosed in this specification is combined with observations and tests based on medical findings regarding the diagnosis of LAH and IAD, these diseases can be diagnosed.

[0015] Also, in this specification, the likelihood of suffering from LAH means the possibility of suffering from LAH at the time of sample collection from an individual, and does not mean the possibility of suffering from LAH in the future or the sign of suffering. The same applies to the likelihood of suffering from IAD.

[0016] Also, the technology disclosed in this specification is typically targeted at humans. Also, as the test sample, any sample collected from a human that can detect the presence or absence of an anti-PTPN13 antibody may be used. The invasiveness in collecting the test sample is not particularly limited, but a sample collected non-invasively or minimally invasively is preferred. The test sample typically includes blood, plasma, serum, cerebrospinal fluid, urine, tears, saliva, etc. The test sample also includes cells, tissues, etc., collected from humans or the like. Such cells or tissues are, for example, the hypothalamus of an individual, the entire pituitary gland, or a part thereof, and examples include the anterior pituitary gland, the posterior pituitary gland, and the infundibulum of the hypothalamus.

[0017] Hereinafter, various embodiments disclosed in this specification will be described.

[0018] <Reagent for Detecting Anti-PTPN13 Antibody> The reagent for detecting an anti-PTPN13 antibody disclosed in this specification (hereinafter, also simply referred to as "reagent") is an epitope peptide that immunologically binds to an anti-PTPN13 antibody, and includes a first epitope peptide comprising at least a continuous 6 amino acid residues out of the 737th to 744th amino acid residues of the amino acid sequence represented by SEQ ID NO: 1, and / or a second epitope peptide comprising at least a continuous 6 amino acid residues out of the 1072nd to 1079th amino acid residues of the amino acid sequence represented by SEQ ID NO: 1.

[0019] In the following description, a reagent for examining LAH and IAD associated with the presence of an anti-PTPN13 antibody will be exemplified and described.

[0020] <Reagent for examining LAH or IAD> PTPN13 (tyrosine protein phosphatase non-receptor type 13) encoded by the PTPN13 gene is a member of the tyrosine phosphatase family. The amino acid sequence of this protein is represented by SEQ ID NO: 1 and consists of 2,485 amino acids (NCBI accession number: NP_542414).

[0021] The first amino acid sequence is the epitope sequence of the first epitope peptide. The first amino acid sequence consists of at least a continuous 6 amino acid residues out of the 737th to 744th (KLDLSYIK) of the amino acid sequence represented by SEQ ID NO: 1. The epitope sequence may also consist of, for example, at least a continuous 7 amino acid residues, and also, for example, at least a continuous 8 amino acid residues. For example, KLDLSY, KLDLSYI, and KLDLSYIK are included. The first amino acid sequence may be said to be specific to IAD among IAD and LAH.

[0022] The second amino acid sequence is the epitope sequence of the second epitope peptide. The second amino acid sequence consists of at least six consecutive amino acid residues from positions 1072 to 1079 (KENDVLHK) of the amino acid sequence represented by SEQ ID NO: 1. The epitope sequence also consists of, for example, at least seven consecutive amino acid residues, or for example, at least eight consecutive amino acid residues. Examples include KENDVL, KENDVLH, and KENDVLHK. The second amino acid sequence can sometimes be said to be specific to LAH among IAD and LAH.

[0023] These epitope sequences were obtained by converting the full-length PTPN13 from the N-terminus into a peptide consisting of 15 amino acid residues with 14 amino acid residues overlapping, as disclosed in the examples described later. This peptide was then immobilized on a solid support and used to analyze antigen-antibody reactions with serum from LAH2 patients, IAD1 patients, and two healthy individuals using a peptide microarray.

[0024] The epitope sequences of these epitope peptides are specifically recognized by anti-PTPN13 antibodies, which then form complexes through an antigen-antibody reaction. By detecting the peptide-anti-PTPN13 complex, the presence of anti-PTPN13 antibodies can be detected. The presence of anti-PTPN13 antibodies serves as an indicator of the likelihood of developing LAH and IAD. Therefore, by using peptides as detection agents (probes), the likelihood of developing LAH can be confirmed, and simultaneously, the likelihood of developing IAD can be confirmed. Since IAD is a related disease to LAH, using epitope peptides with these epitope sequences allows for confirmation or denial of the likelihood of developing either LAH or IAD. Furthermore, this allows for the assessment of the likelihood of developing other diseases included in lymphocytic hypophysitis, such as LINH and LPH.

[0025] The peptide contained in the reagent only needs to have such an epitope sequence, and may be complexed with other peptides or proteins, or with various labels, etc.

[0026] The epitope peptide may be a linear peptide or a cyclic peptide, but is preferably a linear peptide. The peptide may consist only of the epitope sequence, or it may have additional amino acids or peptides at the N-terminus and / or C-terminus of the epitope sequence.

[0027] Such amino acids or peptides may, for example, be approximately 1 to 20 amino acids or peptides located at the N-terminus of the epitope sequence in the amino acid sequence represented by SEQ ID NO: 1. Alternatively, they may be approximately 1 to 20 amino acids corresponding to the C-terminus of the epitope sequence in the amino acid sequence represented by SEQ ID NO: 1. For example, the first epitope peptide may have an additional amino acid sequence consisting of consecutive amino acid residues selected from positions 716 to 765 of the amino acid sequence represented by SEQ ID NO: 1, following the first amino acid sequence. Similarly, the second epitope peptide may have an additional amino acid sequence consisting of consecutive amino acid residues selected from positions 1051 to 1091 of the amino acid sequence represented by SEQ ID NO: 1, following the second amino acid sequence. The number of additional amino acid residues is not particularly limited, but the epitope peptide as a whole can be, for example, 6 to 50, or 7 to 50, 8 to 40, 9 to 40, 10 to 40, 15 to 30, 10 to 20, etc.

[0028] The epitope peptide may also comprise other peptides or proteins other than PTPN13. These other peptides or proteins may be labeled with fluorescent labels such as GFP or RFP, other known tags identifiable by other labeled antibodies, enzymatic labels such as peroxidase, horseradish peroxidase, or alkaline phosphatase, or radioactive labels.

[0029] Such epitope peptides and labeled epitope peptides can be obtained by conventionally known chemical or genetic engineering methods. Since the epitope sequence has been obtained, reagents can be constructed from short-chain peptides, allowing for rapid and easy reagent acquisition using cell-free protein synthesis systems and the like.

[0030] By using an epitope peptide containing an epitope sequence, a peptide-anti-PTPN13 antibody complex can be formed through an antigen-antibody reaction between the epitope sequence and an anti-PTPN13 antibody, which is an autoantibody that immunologically specifically binds to the epitope sequence. This complex can then be used to detect the anti-PTPN13 antibody using immunological methods, such as labeling the peptide or labeling an anti-human antibody labeled against the anti-PTPN13 antibody.

[0031] Immunological methods are not limited to those mentioned above, but examples include latex agglutination, fluorescence immunoassay (FIA), enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blotting, and immunoprecipitation (IP). Preferred measurement methods include FIA ​​and EIA (including ELISA). When cells or tissues are used as samples, immunocytochemical methods, immunohistochemical methods, and flow cytometry can be employed.

[0032] For example, when using the ELIA (Enzyme-Linked Immuno Sorbent Assay) method, a solid phase body is prepared by immobilizing a peptide on a solid phase such as a microplate. For this solid phase body, for example, a dilution of serum collected from a patient is supplied, and an antigen-antibody complex is generated by an antigen-antibody reaction. A secondary antibody having a labeling element such as Horseradish Peroxidase (HRP) that binds to the autoantibody in advance is supplied to form an autoantibody-secondary antibody complex. Then, by supplying a predetermined substrate of HRP, which is a labeling element, and observing the specific absorption wavelength of the reactant generated by the enzymatic reaction of HRP, the autoantibody can be detected or quantified.

[0033] <Device for inspecting LAH or IAD> The reagent may also be immobilized on a solid phase carrier. According to the present specification, a device for inspecting lymphocytic hypophysitis, which includes a solid phase carrier on which the reagent described above is immobilized, is also provided. The solid phase carrier is not particularly limited, and any form composed of various materials such as known resin materials and glassy materials can be appropriately used. Typically, it can be a flat plate such as a chip or an array, a well plate such as a microplate with a large number of wells, a spherical body such as a bead, or a fiber or filter or strip. According to these, a complex of an anti-PTPN13 antibody and a peptide can be formed by an antigen-antibody reaction on the surface of the solid phase carrier on which the reagent is immobilized. In a fiber, filter, or strip in which liquid can move by capillary action, immunochromatography is possible. Also, immunochromatography is possible with a column filled with beads or the like on which the peptide is immobilized.

[0034] Such microplates, arrays, strips, columns, beads, etc. can be appropriately fabricated by those skilled in the art using well-known techniques. Regarding the immobilization of the peptide on the solid phase carrier, known methods such as hydrophobic interaction or covalent bonding can be appropriately employed.

[0035] <Kit for detecting LAH or IAD> According to the present specification, a kit for detecting LAH or IAD comprising the reagents described above is provided. In this kit, the reagents can adopt the various aspects described above. The reagents can adopt the form of a device immobilized on a solid phase carrier in addition to the reagents themselves. In addition, the kit can appropriately include other known elements necessary for antigen-antibody reactions and detection of complexes.

[0036] For example, the kit may include a secondary antibody such as an anti-human antibody (which may be labeled) that binds to an anti-PTPN13 antibody of a complex in which a reagent and an anti-PTPN13 antibody are bound, and a solid phase carrier such as a strip on which the secondary antibody is immobilized.

[0037] <Method for detecting LAH or IAD> The method for detecting LAH or IAD disclosed in the present specification can include bringing a test sample that may contain an anti-PTPN13 antibody into contact with a reagent to cause an antigen-antibody reaction, and detecting a complex formed by the antigen-antibody reaction.

[0038] In the LAH or IAD detection method, the mode of evaluating the likelihood of LAH disease using autoantibodies in the sample as an index is not particularly limited, and various modes can be adopted. For example, the presence or absence of anti-PTPN13 antibody can be used as an index. That is, the presence or absence of anti-PTPN13 antibody in the sample may be qualitatively detected. Qualitatively detecting the presence of anti-PTPN13 antibody means detecting that the concentration (content) is above the detection limit in the adopted autoantibody detection method. The detection limit is appropriately set according to the reference value for the presence or absence of general autoantibodies, the purpose of the detection method, the type of autoantibody, etc. Also, detecting the absence of anti-PTPN13 antibody means that the concentration (content) is less than the above-described detection limit in the adopted autoantibody detection method.

[0039] Alternatively, for example, the concentration of anti-PTPN13 antibody may be used as an indicator. In this case, the anti-PTPN13 antibody concentration may be detected semi-quantitatively or quantitatively from the signal intensity based on the concentration of anti-PTPN13 antibody. Concentration indicators and standard curves should be prepared as appropriate.

[0040] Alternatively, for example, the comparison between the concentration of autoantibodies and a predetermined reference value may be used as an indicator. In this case, it may be possible to detect whether the autoantibody level is above (greater than) or below (less than) the predetermined reference value. The reference value can be appropriately set based on the measured values ​​of blood autoantibody concentrations in a group of patients with a confirmed diagnosis of the disease, such as LAH patients, and a group of healthy individuals.

[0041] In this testing method, various types of reagents, as previously described, can be used in various methods based on antigen-antibody reactions to detect anti-PTPN13 antibodies, and thereby test for the likelihood of developing LAH or IAD.

[0042] This specification provides an anti-PTPN13 antibody recovery agent comprising a peptide that immunologically conjugates to an anti-PTPN13 antibody, and which includes a first and / or second epitope peptide. This recovery agent allows for the capture, separation, and recovery of anti-PTPN13 antibodies in samples that may contain them. The recovery agent can be used in various embodiments with the reagents described above. [Examples]

[0043] The following examples illustrate the disclosures of this specification in more detail. The following examples are for illustrative purposes only and are not intended to limit the scope of the disclosures of this specification. [Examples]

[0044] (Construction of a 15AA peptide array of full-length PTPN13) In preliminary experiments, recombinant proteins were divided into five sub-length segments from the amino acid sequence (SEQ ID NO: 1) of the PTPN13 gene. For the 565-1092AA segment (528AA) where specific binding was observed by Western blotting, GSGGSG linkers were added to the N-terminus and C-terminus of this segment. Overlapping peptides were then created, with 15AAs from the N-terminus of each segment, resulting in 14AA overlaps. These 529 peptides were repeated twice to create an array of 1058 spots. Furthermore, 86 spots of the HA tag (YPYDVPDYAG) were added around these peptide arrays as a control peptide.

[0045] <Test sample> The test samples were human serum samples (patients No. 1067 and 1559, and healthy individuals No. 1193 and 1289). Of patients No. 1067 and 1559, patient No. 1067 was definitively diagnosed with LAH, and patient No. 1559 was definitively diagnosed with IAD.

[0046] (Buffer used) Washing buffer: PBS (pH 7.4), containing 0.05% Tween20 (Washing with the washing buffer was performed three times for 10 seconds each after each incubation). Blocking buffer: Rockland Blocking Buffer MB-070 Incubation buffer: Wash buffer containing 10% blocking buffer

[0047] <assay> The array was incubated in blocking buffer for 30 minutes, then the blocking buffer was removed and the array was washed with washing buffer. The test sample (serum), diluted 150-fold with incubation buffer, was supplied to the blocked array and incubated at 4°C for 16 hours while being shaken horizontally at 140 rpm.

[0048] After incubation, the array was washed with washing buffer and then stained in incubation buffer with the goat anti-human antibody IgG(Fc) antibody DyLight680 (0.1 μg / ml) as a secondary antibody at room temperature for 45 minutes. Simultaneously, as a control, the array was stained with the mouse monoclonal antibody anti-HA(12CA5) Dylight800 (0.2 μg / ml) at room temperature for 45 minutes.

[0049] <Detection> For each array, whether treated with patient-derived serum or healthy-taken serum, an Innopsys InnoScan 710-IR microarray scanner was used. The scan resolution was 20 μm, and the gain was set to 50 at low laser power (680 nm, red) and 10 at high laser power (800 nm, green).

[0050] Figure 1 shows a summary of the results comparing the fluorescence intensity of the serum of four individuals against the overlapping peptide for the full length of PTPN13.

[0051] As shown in Figure 1, a comparison of the responsiveness of three patients and two healthy individuals revealed that in all three patients, at least six consecutive amino acid residues within KLDLSYIK, corresponding to amino acid residues 737 to 744 of the amino acid sequence represented by Sequence ID No. 1, could be identified as an epitope sequence.

Claims

1. A reagent for detecting an anti-PTPN13 antibody, comprising either or both of the following: a first epitope peptide having a first amino acid sequence consisting of at least six consecutive amino acid residues from positions 737 to 744 of the amino acid sequence represented by Sequence ID No. 1, and a second epitope peptide having a second amino acid sequence consisting of at least six consecutive amino acid residues from positions 1072 to 1079 of the same amino acid sequence, and a second epitope peptide having at least six consecutive amino acid residues from positions 1072 to 1079 of the same amino acid sequence, and a second epitope peptide that also binds immunologically to the anti-PTPN13 antibody.

2. The first epitope peptide comprises the first amino acid sequence and has an additional sequence of amino acids selected from positions 716 to 765 of the amino acid sequence represented by Sequence ID No. 1, following the first amino acid sequence. The reagent according to claim 1, wherein the second epitope peptide comprises the second amino acid sequence and has a sequence of additional amino acids selected from the 1051st to 1091st positions of the amino acid sequence represented by Sequence ID No. 1, following the second amino acid sequence.

3. The reagent according to claim 1, wherein the first amino acid sequence is KLDLSYIK.

4. The reagent according to claim 1, wherein the second amino acid sequence is KENDVLHK.

5. The reagent according to claim 1, wherein the first epitope peptide or the second epitope peptide is labeled.

6. The reagent according to claim 1, for use with ELISA.

7. The reagent according to any one of claims 1 to 6, which is a reagent for testing lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency.

8. A detection kit for an anti-PTPN13 antibody, comprising the reagent described in any one of claims 1 to 6.

9. The kit according to claim 8, for testing for lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency.

10. A detection kit for an anti-PTPN13 antibody, comprising a solid support on which the reagent described in any one of claims 1 to 6 is immobilized.

11. The detection kit according to claim 10, for use in testing for lymphocytic anterior pituitary inflammation or isolated inferior adrenocorticotropic hormone deficiency.

12. A test sample potentially containing an anti-PTPN13 antibody is brought into contact with the reagent described in any one of claims 1 to 6 to produce an antigen-antibody reaction. To detect the complex resulting from the aforementioned antigen-antibody reaction, A method for testing for lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency, including the above.

13. A recovery agent for anti-PTPN13 antibody, comprising either or both of the following: a first epitope peptide having a first amino acid sequence consisting of at least six consecutive amino acid residues from positions 737 to 744 of the amino acid sequence represented by Sequence ID No. 1, and immunologically binding to the anti-PTPN13 antibody; and a second epitope peptide having a second amino acid sequence consisting of at least six consecutive amino acid residues from positions 1072 to 1079 of the aforementioned amino acid sequence, and immunologically binding to the anti-PTPN13 antibody.

Images