Reagents and methods for testing lymphocytic anterior pituitary inflammation and isolated adrenocorticotropic hormone deficiency

Abstract

To provide a reagent and a method for testing for lymphocytic anterior hypophysitis and isolated adrenocorticotropic hormone deficiency.SOLUTION: A reagent for detecting an anti GATA2 antibody is provided, the reagent containing a peptide immunologically binding to the anti GATA2 antibody and having an amino acid sequence consisting of at least six consecutive amino acid residues from positions 328 to 336 in an amino acid sequence represented by SEQ ID NO: 1.SELECTED DRAWING: None

Description

Technical Field

[0001] The present specification relates to reagents, testing methods and the like for testing lymphocytic anterior pituitaryitis and isolated adrenocorticotropic hormone deficiency.

Background Art

[0002] Autoimmune hypothalamic hypophysitis is one of the pituitary dysfunctions with unknown etiology, and lymphocytic hypophysitis is a representative disease thereof. Depending on the lesion site, lymphocytic hypophysitis includes lymphocytic anterior pituitaryitis (lymphocytic adenohypophysitics, LAH), lymphocytic infundibulo-neurohypophysitis (lymphocytic infundibulo-neurohypophysitics, LINH), and lymphocytic panhypophysitis (lymphocytic panhypophysitis, LPH).

[0003] LAH is a disease in which inflammatory lesions are localized to the anterior pituitary gland, and the secretion of anterior pituitary hormones such as adrenocorticotropic hormone (adrenocortictropic hormone, ACTH) is reduced or lost. LINH is a disease in which inflammation is localized to the infundibulum and posterior lobe, presenting with central diabetes insipidus. Furthermore, LPH is a disease that causes inflammation in the entire pituitary gland and exhibits the clinical features of both LAH and LINH.

[0004] Lymphocytic hypophysitis such as LAH is characterized in that its clinical symptoms are similar to pituitary tumors and other pituitary lesions caused by systemic diseases. Therefore, for a definitive diagnosis of lymphocytic hypophysitis, acquisition of MRI images, biopsy of pituitary tissue, and further follow-up observation after administration of steroids are performed.

[0005] Regarding lymphocytic hypophysitis, specifically LINH, the inventors have already reported that certain autoantibodies serve as disease markers (Patent Document 1). Furthermore, they have already reported markers for distinguishing isolated ACTH deficiency (IAD), a related disorder of LAH (Patent Document 2). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Patent No. 5924502 [Patent Document 2] Japanese Patent Publication No. 2016-206072 [Overview of the project]

[0007] However, in detecting these markers, i.e., autoantibodies, the protein itself, which is the antigen that binds to the autoantibody, has been used as the detection reagent. Because the protein itself is handled as a reagent, manufacturing costs and other factors have been a challenge.

[0008] This specification provides a technology that can contribute to less invasive and practical testing for lymphocytic anterior pituitary dysplasia (LAH) and related disorders such as isolated adrenocorticotropic hormone deficiency (IAD). [Means for solving the problem]

[0009] The inventors performed proteomics analysis on samples from individuals with LAH and identified over 600 proteins, as well as 50 candidate autoantigen proteins. Furthermore, similar analysis was performed on individuals with IAD and identified 131 candidate autoantigen proteins. The inventors conducted detailed analysis of these candidate proteins and were able to identify autoantibodies that serve as markers associated with the likelihood of developing IAD. Furthermore, they were able to identify the epitopes of these autoantibodies. Based on these findings, this specification provides the following means.

[0010] [1] A reagent for detecting an anti-GATA2 antibody, comprising a peptide that immunologically binds to an anti-GATA2 antibody and has an amino acid sequence consisting of at least six consecutive amino acid residues from position 328 to 336 of the amino acid sequence represented by SEQ ID NO: 1. [2] The reagent described in [1], wherein the amino acid sequence is QNRPLIK. [3] The reagent described in [1], wherein the amino acid sequence is QNRPLIKPK. [4] The reagent according to any one of [1] to [3], wherein the peptide is labeled. [5] Reagents for ELISA as described in any of [1] to [4]. [6] A reagent for testing lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency, as described in any of [1] to [5]. A detection kit for anti-GATA2 antibodies, comprising one of the reagents described in [7][1] to [6]. [8] The kit described in [7] for testing for lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency. A detection kit for anti-GATA2 antibodies comprising a solid support on which one of the reagents described in [9][1] to [6] is immobilized.

[10] The detection kit described in [9] for testing lymphocytic anterior pituitary inflammation or isolated inferior adrenocorticotropic hormone deficiency.

[11] Contacting a test sample that may contain an anti-GATA2 antibody with any of the reagents described in [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.

[12] A recovery agent for anti-GATA2 antibodies, comprising a peptide that immunologically binds to an anti-GATA2 antibody and has an amino acid sequence consisting of at least six consecutive amino acid residues from position 328 to 336 of the amino acid sequence represented by SEQ ID NO: 1. [Brief explanation of the drawing]

[0011] [Figure 1] This figure compares the responsiveness of the 15AA overlap peptide array of the full-length protein GATA2 with serum samples from LAH patients, IAD patients, and healthy controls. [Modes for carrying out the invention]

[0012] This disclosure relates to a reagent for detecting anti-GATA2 antibodies, comprising a peptide that immunologically binds to the anti-GATA2 antibody, and to its use. Anti-GATA2 antibodies are autoantibodies that serve as markers for LAH and IAD.

[0013] One embodiment of the reagent disclosed herein is a reagent that evaluates the likelihood of developing LAH or IAD using the presence of anti-GATA2 antibodies as an indicator. When this reagent confirms the presence of anti-GATA2 antibodies or that the amount of anti-GATA2 antibodies 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 originated. When this reagent denies the presence of anti-GATA2 antibodies or that the amount of anti-GATA2 antibodies 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 originated.

[0014] The reagents disclosed in the present specification are based on immunological binding generated by an antigen-antibody reaction between a specific epitope sequence and an anti-GATA2 antibody. Therefore, the reagents can contribute to the diagnosis of anti-GATA2 antibodies, or LAH or IAD associated with the detection of anti-GATA2 antibodies, more easily and reliably than in the prior art.

[0015] It should be noted that the technology disclosed in the present specification does not independently diagnose LAH or IAD, but provides useful information for diagnosis. The technology disclosed in the present specification is combined with observations and tests based on medical findings related to the diagnosis of LAH and IAD, whereby these diseases can be diagnosed.

[0016] Further, in the present specification, the possibility of suffering from LAH means the possibility of having LAH at the time of sampling from an individual, and does not mean the possibility of suffering from LAH in the future or a premonition of the onset. The same applies to the possibility of suffering from IAD.

[0017] Further, the technology disclosed in the present specification is typically intended for humans. The test sample may be any sample collected from a human that can detect the presence or absence of an anti-GATA2 antibody or the like. The invasiveness in collecting the test sample is not particularly limited, but a sample collected non-invasively or minimally invasively is preferred. Typical examples of the test sample include blood, plasma, serum, cerebrospinal fluid, urine, tear fluid, saliva and the like. The test sample also includes cells, tissues and the like collected from a human 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 thereof include the anterior pituitary lobe, the posterior pituitary lobe, the hypothalamic infundibulum, and the like.

[0018] Hereinafter, various embodiments disclosed in the present specification will be described.

[0019] <Reagent for Detecting Anti-GATA2 Antibody> The reagent for detecting the anti-GATA2 antibody disclosed in the present specification (hereinafter also simply referred to as the reagent) is a peptide that binds to the GATA2 antibody, and comprises a peptide containing an amino acid sequence consisting of at least 6 consecutive amino acid residues from the 328th to the 336th position of the amino acid sequence represented by SEQ ID NO: 1. In the following description, the reagent for testing LAH and IAD associated with the presence of the anti-GATA2 antibody will be described by way of example.

[0020] <Reagent for testing lymphocytic anterior hypophysitis (LAH) or isolated adrenocorticotropic hormone deficiency (IAD)> The GATA2 (Endothelial transcription factor GATA-2) protein is a member of the GATA family of zinc finger transcription factors, and this protein is said to play an important role in regulating the transcription of genes involved in the development and proliferation of hematopoietic and endocrine cell lineages. The amino acid sequence of this protein is represented by SEQ ID NO: 1, and consists of 480 amino acids (480 AA).

[0021] The amino acid sequence represented by SEQ ID NO: 1 is as follows. Sequence ID 1:MEVAPEQPRWMAHPAVLNAQHPDSHHPGLAHNYMEPAQLLPPDEVDVFFNHLDSQGNPYYANPAHARARVSYSPAHARLTGGQMCRPHLLHSPGLPWLDGGKAALSAAAAHHHNPWTVSPFSKTPLHPSAAGGPGGPLSVYPGAGGGSGGGSGSSVASLTPTAAHSGSHLFGFPPTPPKEVSPDPSTTGAASPASSSAGGSAARGEDKDGVKYQVSLTESMKMESGSPLRPGLATMG TQPATHHPIPTYPSYVPAAAHDYSSGLFHPGGFLGGPASSFTPKQRSKARSCSEGRECVNCGATATPLWRRDGTGHYLCNACGLYHKMNGQNRPLIKPKRRLSAARRAGTCCANCQTTTTT LWRRNANGDPVCNACGLYYKLHNVNRPLTMKKEGIQTRNRKMSNKSKKSKKGAECFEELSKCMQEKSSPFSAAALAGHMAPVGHLPPFSHSGHILPTPTPIHPSSSLSFGHHPSSMVTAMG

[0022] The epitope sequence consists of at least six consecutive amino acid residues from positions 328 to 336 (QNRPLIKPK) of the amino acid sequence represented by SEQ ID NO: 1. The epitope sequence may also consist of, for example, at least seven consecutive amino acid residues, or at least eight consecutive amino acid residues, or at least eight consecutive amino acid residues, or at least nine consecutive amino acid residues. The epitope sequences are, for example, QNRPLI corresponding to amino acids 328-333 (6AA) of the amino acid sequence represented by SEQ ID NO: 1, NRPLIK corresponding to amino acids 329-334 (6AA), QNRPLIK corresponding to amino acids 328-334 (7AA), RPLIKPK corresponding to amino acids 330-335, QNRPLIKP(8AA) corresponding to amino acid residues 328-335 (8AA), and QNRPLIKPK(9AA) corresponding to amino acids 328-336 (9AA).

[0023] The epitope sequence was obtained by converting the full-length GATA2 protein, starting from the N-terminus, into a 15AA peptide, as disclosed in the examples described later. This peptide was then immobilized on a solid support and analyzed using a peptide microarray with the serum of an LAH2 patient, an IAD1 patient, and two healthy individuals through antigen-antibody reactions.

[0024] The epitope sequence of the peptide is specifically recognized by the anti-GATA2 antibody, and a complex is formed through an antigen-antibody reaction. By detecting the peptide-anti-GATA2 complex, the presence of the anti-GATA2 antibody can be detected. The presence of the anti-GATA2 antibody is an indicator that confirms the possibility of developing LAH and IAD. Therefore, by using the peptide as a detection agent (probe), the possibility of developing LAH and the possibility of developing IAD can be confirmed simultaneously. IAD is a disease related to LAH, and by using a peptide with this epitope sequence, the possibility of developing LAH or IAD can be confirmed or denied. Furthermore, as a result, the possibility of developing other diseases included in lymphocytic hypophysitis, such as LINH and LPH, can be assessed.

[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 peptide may be a linear peptide or a cyclic peptide, but is preferably a linear peptide. The peptide may consist only of an epitope sequence, or it may have additional amino acids or peptides at the N-terminus and / or C-terminus of the epitope sequence. For example, the amino acids or peptides added to the N-terminus may be 1 to 20 located on the N-terminal side of the epitope sequence in the amino acid sequence represented by SEQ ID NO: 1. Alternatively, they may be 1 to 20 located on the C-terminal side of the epitope sequence in the amino acid sequence represented by SEQ ID NO: 1. The number of amino acid residues in such a peptide may be, for example, 7A to 50AA, or 9AA to 40AA, or 10AA to 30AA, or 10AA to 20AA.

[0027] The peptide may also comprise other peptides or proteins other than the GATA2 protein. 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.

[0028] Such peptides and labeled 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.

[0029] By using a peptide containing an epitope sequence, a peptide-anti-GATA2 antibody complex can be formed through an antigen-antibody reaction between the epitope sequence and an anti-GATA2 antibody, which is an autoantibody that immunologically specifically binds to the epitope sequence. This complex can then be used to detect the anti-GATA2 antibody using immunological methods, such as labeling the peptide or labeling an anti-human antibody labeled against the anti-GATA2 antibody.

[0030] 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.

[0031] For example, when using the ELIA (Enzyme-Linked Immuno Sorbent Assay) method, a solid phase is prepared by immobilizing a peptide on a solid phase such as a microplate. A diluted serum, for example, collected from a patient, is supplied to this solid phase to generate an antigen-antibody complex through an antigen-antibody reaction. A secondary antibody that binds to an autoantibody beforehand, for example, an anti-human IgG antibody equipped with a labeling element such as horseradish peroxidase (HRP), is supplied to form an autoantibody-secondary antibody complex. Subsequently, a predetermined substrate for the labeling element HRP is supplied, and the autoantibody can be detected or quantified by observing the specific absorption wavelength of the reactant produced by the enzymatic reaction with HRP.

[0032] <Device for testing lymphocytic anterior pituitary inflammatory bowel disease (LAH)> The reagent may also be immobilized on a solid support. According to this specification, a device for testing lymphocytic anterior pituitary lobe is also provided, comprising a solid support on which the reagent described above is immobilized. The solid support is not particularly limited and any form made of various materials such as known resin materials or glassy materials can be used as appropriate. Typically, it can be a plate-like body such as a chip or array, a well-plate-like body such as a microplate with many wells, a spherical body such as beads, or a fiber, filter, or strip-like body. With these, a complex of anti-GATA2 antibody and peptide can be formed by antigen-antibody reaction on the surface of the solid support on which the reagent is immobilized. Immunochromatography is possible with fibers, filters, or strips on which liquid can move by the capillary effect. Immunochromatography is also possible with columns packed with beads or the like on which peptides are immobilized.

[0033] Such microplates, arrays, strips, columns, beads, etc., can be appropriately prepared by those skilled in the art using well-known techniques. For the immobilization of peptides onto solid phase supports, known methods such as hydrophobic interactions or covalent bonding can be appropriately employed.

[0034] This specification provides a kit for testing LAH, comprising the reagents described above. In this kit, the reagents can take on the various forms described above. The reagents can be in the form of the reagents themselves or as devices immobilized on a solid support. The kit may also appropriately include other known elements necessary for antigen-antibody reactions and the detection of complexes.

[0035] For example, the kit may include a secondary antibody, such as an anti-human antibody (which may be labeled) that binds to the anti-GATA2 antibody in a complex formed by the combination of the reagent and the anti-GATA2 antibody, or a solid support such as a strip on which the secondary antibody is immobilized.

[0036] <Methods for testing for lymphocytic anterior pituitary dysplasia (LAH) or adrenocorticotropic hormone deficiency (IAD)> A method for testing LAH disclosed herein may include contacting a test sample potentially containing an anti-GATA2 antibody with a reagent to induce an antigen-antibody reaction, and detecting a complex resulting from the antigen-antibody reaction.

[0037] In LAH testing methods, the method of evaluating the likelihood of developing LAH using autoantibodies in a sample as an indicator is not particularly limited, and various methods can be adopted. For example, the presence or absence of anti-GATA2 antibodies can be used as an indicator. That is, the presence or absence of anti-GATA2 antibodies in the sample may be detected qualitatively. Qualitative detection of the presence of anti-GATA2 antibodies means detecting that the concentration (content) is above the detection limit of the autoantibody detection method used. The detection limit is set appropriately depending on the general reference value for the presence or absence of autoantibodies, the purpose of the testing method, the type of autoantibody, etc. Detecting the absence of anti-GATA2 antibodies means that the concentration (content) is below the detection limit described above in the autoantibody detection method used.

[0038] Alternatively, for example, the concentration of anti-GATA2 antibody may be used as an indicator. In this case, the anti-GATA2 antibody concentration may be detected semi-quantitatively or quantitatively from the signal intensity based on the anti-GATA2 antibody concentration. Concentration indices and standard curves should be prepared as appropriate.

[0039] 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.

[0040] 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-GATA2 antibodies, and thereby test for the likelihood of developing LAH or AID.

[0041] This specification provides an anti-GATA2 antibody recovery agent comprising a peptide that immunologically binds to an anti-GATA2 antibody and has an amino acid sequence consisting of at least six consecutive amino acid residues from positions 328 to 336 of the amino acid sequence represented by SEQ ID NO: 1. This recovery agent can capture, separate, and recover anti-GATA2 antibodies in a sample that may contain anti-GATA2 antibodies. The recovery agent can be used in various embodiments with the reagents described above. [Examples]

[0042] 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]

[0043] First, recombinant full-length human GATA2 protein was synthesized, and the full-length human protein was subjected to electrophoresis on a 7.5-10.00% polyacrylamide gel, transferred to a PVDF membrane, and subjected to immunoblotting using various serum samples (1:50 dilution). Antigen-antibody reactions were analyzed by Western blotting using serum from patients (3 each of LAH and IAD) and healthy individuals (2) as primary antibodies and HRP-labeled anti-human IgG antibodies as secondary antibodies. As a result, anti-GATA2 antibodies were found as autoantibodies associated with the likelihood of developing LAH and IAD.

[0044] (Construction of a 15AA peptide array of full-length GATA2 protein) The amino acid sequence of the GATA2 gene (SEQ ID NO: 1) was modified by adding GSGGSSG linkers to the N-terminus and C-terminus of the amino acid sequence, respectively. From the N-terminus of each sequence, overlapping peptides were created with 15 AAs and 14 AAs of overlapping AAs. These 480 peptides were repeated twice to create an array consisting of 960 spots. Furthermore, 42 spots of HA tags (YPYDVPDYAG) and 42 spots of polio tags (KEVPALTAVETGAT) were added around these peptide arrays as control peptides.

[0045] <Test sample> The test samples were human serum samples (patients No. 1633, 1038, 1610 and healthy individuals No. 1193, 1289). Of patients No. 1633, 1038, and 1610, patients No. 1633 and 1038 were definitively diagnosed with LAH, and patient No. 1610 was definitively diagnosed with IAD.

[0046] (Buffer used) Washing buffer: PBS (pH 7.4), containing 0.05% Tween 20 (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 500-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 the results of comparing the fluorescence intensity of serum samples from five individuals against overlapping peptides for the full length of GATA2.

[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 (AA) from QNRPLIKPK, corresponding to amino acid residues 328-336 (9AA) of the amino acid sequence represented by SEQ ID NO: 1, could be identified as the epitope sequence. Specifically, QNRPLI, QNRPLIK, RPLIKPK, NRPLIKPK, QNRPLIKPK, etc., were found to be possible epitope sequences.

Claims

1. A reagent for testing lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency, comprising an epitope peptide that immunologically binds to an anti-GATA2 antibody, and containing at least six consecutive amino acid residues from positions 328 to 336 of the amino acid sequence represented by SEQ ID NO: 1, and consisting of an amino acid sequence of 6 to 30 amino acid residues on the amino acid sequence represented by SEQ ID NO:

1.

2. A peptide that immunologically binds to an anti-GATA2 antibody, comprising at least six consecutive amino acid residues from position 328 to 336 of the amino acid sequence represented by SEQ ID NO: 1, and an epitope peptide consisting of an amino acid sequence of 6 to 30 amino acid residues on the amino acid sequence represented by SEQ ID NO: 1, A protein or peptide other than a protein consisting of the amino acid sequence represented by Sequence ID No. 1, and / or a label, A reagent comprising the following components for testing lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency.

3. The reagent according to claim 1 or 2, wherein the epitope peptide is QNRPLIK.

4. The reagent according to claim 1 or 2, wherein the epitope peptide is QNRPLIKPK.

5. The reagent according to claim 1 or 2, for use with ELISA.

6. A test kit for lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency, comprising the reagent described in claim 1 or 2.

7. A test kit for lymphocytic anterior pituitary inflammation or isolated adrenocorticotropic hormone deficiency, comprising a solid-phase carrier on which the reagent described in claim 1 or 2 is immobilized.

8. A test sample potentially containing an anti-GATA2 antibody is brought into contact with the reagent described in claim 1 or 2 to induce 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.

Images