WO2013080811A1 - リンパ球性漏斗下垂体後葉炎のバイオマーカー及びその用途 - Google Patents
リンパ球性漏斗下垂体後葉炎のバイオマーカー及びその用途 Download PDFInfo
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- WO2013080811A1 WO2013080811A1 PCT/JP2012/079776 JP2012079776W WO2013080811A1 WO 2013080811 A1 WO2013080811 A1 WO 2013080811A1 JP 2012079776 W JP2012079776 W JP 2012079776W WO 2013080811 A1 WO2013080811 A1 WO 2013080811A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G01N2333/4703—Regulators; Modulating activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
Definitions
- the present invention relates to a biomarker for lymphocytic funnel pituitary ptitis. More specifically, the present invention relates to a biomarker for lymphocytic pituitary posterior phlebitis and a test method using the biomarker.
- This application claims the priority based on the Japan patent application 2011-258387 for which it applied on November 28, 2011, The whole content of the said patent application is used by reference.
- Lymphocytic hypophysitis is an autoimmune chronic inflammatory disease in which lymphocytes and plasma cells infiltrate the hypothalamic funnel and pituitary gland. Inflammation is limited in the funnel and posterior lobe, and a disease presenting with central diabetes insipidus is lymphocytic posterior pituitary (hereinafter abbreviated as “LINH”). Diagnosis of LINH may be difficult, and a definitive diagnosis requires a biopsy of the pituitary gland, but it is rarely performed because it is invasive, and is misdiagnosed and undergoes unnecessary surgery Not a few. The etiology is unknown and no cure has been established.
- LINH lymphocytic posterior pituitary
- Non-Patent Documents 1 to 5 Several diagnostic markers (biomarkers) have been reported for lymphocytic hypophysitis (see, for example, Non-Patent Documents 1 to 5), but none of them have excellent sensitivity and specificity. There is no application. There are no reports on diagnostic markers specific to LINH.
- the present invention aims to provide a LINH-specific biomarker and its use, that is, a technique useful for testing LINH, in order to enable LINH discrimination and highly reliable diagnosis.
- an object of the present invention is to provide a biomarker useful for testing LINH, a test method using the biomarker, a test reagent used for the test method, and the like.
- the present inventors conducted research to find a biomarker specific to LINH. Specifically, first, sera were collected from disease control cases and normal subjects who exhibited central diabetes insipidus due to brain tumors in addition to LINH patients, and IgG was purified. On the other hand, a pituitary posterior lobe protein extract was prepared from the rat pituitary gland. Then, IgG purified from serum and pituitary posterior lobe protein extract were mixed and immunoprecipitated using protein G beads, and then the antigen was eluted from the immunoprecipitate.
- the eluted antigen was subjected to analysis by an LC-MS / MS system, PradigmMS4-PAL-LTQTheOrbitrap XL (ThermoFisher SCIENTIFIC) (shotgun proteomics). MS data was analyzed using public databases and analysis software, and autoantigen candidate proteins were identified. Next, recombinant proteins were synthesized for 6 types of autoantigen protein candidates, and the reactivity of these recombinant proteins with patient sera was examined by Western blotting. As a result, 8 out of 9 LINH patients (8/9) showed autoantibodies against Rabphilin 3a.
- lovephilin 3a was also expressed in the rat hypothalamic paraventricular nucleus, supraoptic nucleus and posterior lobe of the pituitary gland. This finding suggests that lovephilin 3a is directly involved in the pathogenesis of LINH, and that autoantibodies against the molecule, namely anti-labfilin 3a antibody, are useful for the purpose of understanding the pathology of LINH. There was found.
- a lymphocytic funnel pituitary lobitis marker consisting of an anti-labfillin 3a antibody.
- [2] A test method for lymphocytic pituitary posterior pititis, characterized by using the level of anti-labfillin 3a antibody in the specimen as an index.
- a high level of anti-labfillin 3a antibody indicates that the disease affected is lymphocytic pituitary posterior pititis and a high possibility of developing lymphocytic pituitary pituitary inflammation
- the determination in step (3) is performed based on a comparison between the detection value obtained in step (2) and the detection value in a sample collected in the past from the same subject, [3] or [ 4].
- test method according to any one of [2] to [6], wherein the specimen is blood, plasma, serum, cerebrospinal fluid, or urine.
- a reagent for examination of lymphocytic pituitary posterior pituitary disease which contains lovephilin 3a or an antibody-binding fragment thereof.
- a kit for examining a lymphocytic pituitary posterior pituitary gland comprising the lymphoid funnel pituitary ptitis test reagent according to [8].
- the first aspect of the present invention relates to a biomarker for LINH (hereinafter also referred to as “biomarker of the present invention”).
- the biomarker of the present invention is a useful index for differentiation of LINH or evaluation of the likelihood of developing LINH.
- “differentiation” is to determine whether the disease affected by the subject is LINH or another disease.
- the biomarker of the present invention is useful for ascertaining whether or not a patient with diabetes insipidus suffers from LINH.
- the biomarker of the present invention is important as an index for distinguishing between central diabetes insipidus associated with LINH and diabetes insipidus associated with mass lesions.
- “Possibility of onset” includes current onset possibility and future onset possibility. “Current possibility of onset” represents whether or not LINH has developed at the time of the examination, or the probability of developing it. On the other hand, “probability of future onset” represents the possibility (risk) of developing LINH in the future.
- the biomarker of the present invention comprises an antibody against LINH-specific antigen molecule labfilin 3a (autoantibody), that is, an anti- labfilin 3a antibody.
- Lovephilin 3a is a protein encoded by the RPH3A gene, is an effector of RAB3A, and is known to be involved in the release of neurotransmitters and hormones.
- the amino acid sequence of lovephilin 3a (DEFINITION: Homo sapiens rabphilin 3A homolog (mouse), mRNA (cDNA clone MGC: 29559 IMAGE: 3510158), complete cds. ACCESSION: BC017259) and the nucleotide sequence encoding it No. 1 and SEQ ID No. 2 are shown respectively. It should be noted that, in principle, an anti-labfilin 3a antibody in a specimen collected (ie, separated) from a living body is used as a biomarker of the present invention, not an anti-labfillin 3a antibody present in
- the second aspect of the present invention relates to the use of the biomarker of the present invention, and relates to a method for examining whether or not the disease affected is LINH, or the onset possibility of LINH (hereinafter referred to as “the test method of the present invention”). ").
- the test method of the present invention can be differentiated (when determining whether or not the disease affected is LINH).
- the test method of the present invention is useful as a means for determining whether or not LINH is presently occurring, or as a means for determining the possibility of developing LINH in the future (the possibility of developing LINH). When judging).
- the test method of the present invention provides useful information for the diagnosis of LINH.
- the test method of the present invention is useful for, for example, simple and objective definitive diagnosis of LINH.
- the level of the biomarker of the present invention in a specimen derived from a subject is used as an index.
- level typically means “amount” or “concentration”.
- level is also used to indicate whether or not the biomarker of the present invention can be detected (that is, the presence or absence of an apparent presence) according to common practice and common general knowledge.
- Step of preparing a subject-derived sample (2) Step of detecting anti-labylphyrin 3a antibody in the sample (3) Based on the detection result, the disease affected is a lymphocyte funnel posterior pituitary
- a specimen derived from the subject is prepared.
- blood, plasma, serum, cerebrospinal fluid, urine, etc. of the subject can be used.
- the subject is not particularly limited. That is, the person who needs to differentiate LINH, the present or future possibility of LINH (ie, whether or not LINH has occurred, the degree of possibility of developing LINH, the possibility of developing LINH in the future The present invention can be widely applied to those who need a degree). For example, a person who has symptoms of central diabetes insipidus may fall under “a person who needs to differentiate LINH”.
- the present invention when the present invention is applied to a patient diagnosed with LINH through a doctor's interview or other examinations, whether or not the diagnosis is appropriate can be determined based on an objective index such as a biomarker level. it can. That is, according to the inspection method of the present invention, it is possible to obtain information that assists or supports the conventional diagnosis. The information is useful for determining a more appropriate treatment policy, and promotes improvement of the treatment effect and improvement of the patient's QOL (Quality of Life). On the other hand, the present invention can be used for monitoring the diseased state to prevent intractable disease, seriousness, recurrence and the like.
- a case in which thickening of the pituitary stalk or enlargement of the pituitary gland is observed in an image examination such as an MRI examination is presumed to have a high risk of suffering from LINH (high risk person) and is a suitable subject.
- Applying the present invention before LINH symptoms appear in such subjects allows for prevention or delay of onset or early therapeutic intervention.
- the present invention is also useful for identifying those who are at high risk of suffering from LINH. Such identification enables a reduction in the possibility of onset (possibility of morbidity) due to, for example, preventive measures or improvement of lifestyle habits.
- a person who cannot determine whether or not it is LINH by conventional diagnosis, such as a person who has no subjective symptoms, is also a suitable subject of the present invention. In addition, you may decide to implement this invention as one item of a health check.
- step (2) biomarkers in the specimen are detected. It is not essential to strictly quantify the level of the biomarker. That is, the level of the biomarker may be detected to the extent that a predetermined determination can be made in the subsequent step (3). For example, detection can be performed so that it can be determined whether or not the level of the biomarker in the sample exceeds a predetermined reference value.
- Biomarker detection method is not particularly limited, but preferably an immunological technique is used. According to immunological techniques, rapid and sensitive detection is possible. Also, the operation is simple. In measurement by an immunological technique, a substance having specific binding property to a biomarker is used. As the substance, labfilin 3a or an antibody-binding fragment thereof is typically used.
- the “antibody-binding fragment” is a fragment of labfilin 3a containing an epitope that binds to the anti-rabophilin 3a antibody, and has binding properties to the anti-rabophilin 3a antibody in the same manner as full-length labfilin 3a.
- the measurement method examples include latex agglutination method, fluorescence immunoassay method (FIA method), enzyme immunoassay method (EIA method), radioimmunoassay method (RIA method), and Western blot method.
- Preferable measurement methods include FIA method and EIA method (including ELISA method). According to these methods, it is possible to detect with high sensitivity, quick and simple.
- FIA method a fluorescently labeled antibody is used, and an antigen-antibody complex (immune complex) is detected using fluorescence as a signal.
- EIA method an enzyme-labeled antibody is used, and an immune complex is detected using color development or luminescence based on an enzyme reaction as a signal.
- the ELISA method has many advantages such as high detection sensitivity, high specificity, excellent quantitativeness, simple operation, and suitability for simultaneous processing of multiple samples.
- An example of a specific operation method when using the ELISA method is shown below.
- an anti-labfillin 3a antibody prepared by immunizing rabbits or goats is immobilized on an insoluble support. Specifically, for example, the surface of the microplate is sensitized (coated) with an anti-labfillin 3a antibody.
- the specimen and the enzyme-labeled labfilin 3a are brought into contact with the thus immobilized antibody.
- the immobilized antibody and labfilin 3a bind to form an immune complex.
- a method for detecting the immune complex using a secondary antibody Alternatively, a sandwich method or the like may be employed.
- step (3) based on the detection result, it is determined whether or not the disease affected is LINH. In another aspect, the likelihood of developing LINH is determined based on the detection result. In order to enable accurate determination, it is preferable to perform the determination after comparing the detection value obtained in step (2) with the detection value of the control sample (control). Samples of healthy subjects, samples of patients with diabetes insipidus associated with mass lesions, samples of patients with idiopathic or secondary central diabetes insipidus, samples of patients with other autoimmune diseases, etc. it can. The determination may be qualitative or quantitative. It should be noted that the determination here can be automatically / mechanically performed without depending on the determination of a person having specialized knowledge such as a doctor or a laboratory technician, as is apparent from the determination criteria.
- Example of qualitative judgment 1 discrimination
- the detected value for example, the amount of labeling or fluorescence
- the detected value is LINH
- the disease being affected is It is determined that it is other than LINH.
- the detected value is higher than the reference value
- the disease affected is LINH
- the detected value is higher than the reference value
- the detected value is higher than the reference value. Is low, it is determined that “the disease affected is the specific disease”.
- the “specific disease” here is, for example, lymphocytic anterior pituitary inflammation, IgG4-related pituitary inflammation accompanied by diabetes insipidus, or mass lesion accompanied by diabetes insipidus (the same applies to the following examples).
- Example 3 of qualitative judgment: likelihood of onset When the detected value (for example, the amount of labeling or the amount of fluorescence) is higher than the reference value, it is determined that “there is a high possibility of developing LINH”, and when the detected value is lower than the reference value, “LINH is developed. No ”or“ It is highly likely that LINH has not been developed ”. In the case of determining the possibility of future onset, when the detected value is higher than the reference value, it is determined that “LINH is developed” or “Linch is highly likely to occur”, and the detected value is higher than the reference value. When the value is low, it is determined that “do not develop LINH” or “probably not develop LINH”.
- Detection value a to b 10% or less possibility of LINH Detection value b to c: 10% to 30% possibility of LINH Detection value cd: 30% to 50% chance of LINH Detection value d to e: 50% to 70% possibility of LINH Detection value ef: 70% to 90% possibility of LINH
- the number of judgment categories, the level of the biomarker associated with each judgment category, and the judgment results can be arbitrarily set through preliminary experiments or the like without being bound by the above example. For example, when determining (differentiating) whether or not it is LINH with a predetermined threshold as a boundary, or determining whether or not it is LINH (“differentiation”) ) Or a “biomarker level range” associated with a high or low likelihood of developing LINH can be determined by statistical analysis using a large number of specimens.
- the level of a biomarker measured at a certain time point is compared with the level of a biomarker measured in the past, Or examine the degree of increase or decrease.
- the resulting data on the level change of the biomarker is useful information for monitoring the presence or possibility of the onset of LINH, grasping the therapeutic effect, or estimating the prognosis.
- the possibility of onset has increased or decreased or has not changed between the previous test and the current test. If such an evaluation is performed in parallel with the treatment of LINH, it is possible to confirm in advance the signs of recurrence of LINH as well as to confirm the therapeutic effect. This makes it possible to determine a more appropriate treatment policy.
- the present invention can make a great contribution to maximizing the therapeutic effect and improving the patient's QOL (quality of life).
- the present invention further provides reagents and kits used in the LINH test method.
- the reagent of the present invention contains labfilin 3a or an antibody-binding fragment thereof (hereinafter referred to as “active ingredient”), which is a substance exhibiting specific binding property to the biomarker of the present invention.
- the active ingredient of the reagent of the present invention can be prepared, for example, as a recombinant (recombinant) protein. For example, it can be prepared by transforming a suitable host cell with DNA encoding the target active ingredient (labferin 3a or antibody-binding fragment thereof) and recovering the protein expressed in the transformant.
- the recovered protein is appropriately purified according to the purpose.
- various modifications are possible if a binding molecule is obtained as a recombinant protein.
- a DNA encoding a binding molecule and other appropriate DNA are inserted into the same vector and a recombinant protein is produced using the vector, an active ingredient consisting of a recombinant protein linked to any peptide Can be obtained.
- modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur.
- the preparation method of an active ingredient is not restricted to the thing by a genetic engineering method.
- the active ingredient of the reagent of the present invention can be prepared from natural materials by standard techniques (crushing, extraction, purification, etc.).
- the active ingredient may be prepared using a cell-free synthesis system.
- Cell-free synthesis systems (cell-free transcription systems, cell-free transcription / translation systems) are not live cells, but are derived from live cells (or obtained by genetic engineering techniques), transcription / translation factors, etc. Is used to synthesize mRNA or protein encoded by nucleic acid (DNA or mRNA) as a template in vitro.
- a cell extract obtained by purifying a cell disruption solution as needed is generally used.
- Cell extracts generally contain ribosomes necessary for protein synthesis, various factors such as initiation factors, and various enzymes such as tRNA.
- the cell-free protein synthesis system has the following advantages. First, since there is no need to maintain live cells, operability is good and the degree of freedom of the system is high. Therefore, it is possible to design a synthetic system with various modifications and modifications according to the properties of the target protein. Next, in the synthesis of cell lines, it is basically impossible to synthesize proteins that are toxic to the cells to be used, but in the cell-free system, even such toxic proteins can be produced. Furthermore, it is easy to increase the throughput because many kinds of proteins can be synthesized simultaneously and rapidly. It also has the advantage that the protein to be produced can be easily separated and purified, which is advantageous for high throughput. In addition, it also has the advantage that a non-natural protein can be synthesized by incorporating a non-natural amino acid.
- cell-free protein synthesis systems that are widely used include the following. That is, an E. coli S30 extract system (prokaryotic cell system), a wheat germ extract system (eukaryotic cell system), and a rabbit reticulocyte lysate system (eukaryotic cell system). These systems are also commercially available as kits and can be used easily.
- E. coli S30 extract system prokaryotic cell system
- wheat germ extract system eukaryotic cell system
- rabbit reticulocyte lysate system eukaryotic cell system
- the wheat germ extract system has the advantage of being able to efficiently synthesize high-quality eukaryotic proteins, and is often used to synthesize eukaryotic proteins that are difficult to synthesize using the E. coli S30 extract system. Is done. Recently, it has been reported that a highly efficient and stable synthetic system is constructed by preparing an extract from germs from which seed endosperm components have been washed away (Madin, K. et al .: Proc. Natl. Acad. Sci. USA, 97: 559-564, 2000). After that, technical developments such as mRNA untranslated sequence with high translation promoting ability, protein synthesis method for multi-item function analysis using PCR, construction of dedicated high expression vector, etc. were carried out (Sawasaki, T. et al .: Proc Natl. Acad. Sci. USA, 99: 14652-14657, 2002), is expected to be applied in various fields.
- the wheat germ extract can be obtained by grinding and centrifuging wheat germ and then separating the supernatant by gel filtration.
- Anderson et al. the method of Anderson et al. (Anderson, C. W. et al .: Methods Enzymol., 101, 638-644 (1983)) can be referred to. Improved methods have also been reported, such as the method of Kawarazaki et al. (Kawarasaki, Y. et al .: Biotechnol. Prog., 16, 517-521 (2000)) and the method of Madin et al. (Madin, K. et al. : Proc. Natl. Acad. Sci. USA, 97: -559-564, 2000).
- the cell-free synthesis system that can be used in the practice of the present invention is not limited to those described above.
- bacteria other than E. coli and plant extracts other than wheat, insect-derived extracts, animal cell-derived extracts Alternatively, a system constructed based on genome information may be used.
- the amount of bound antibody can be directly detected using the labeled amount as an index. Therefore, a simpler inspection method can be constructed.
- an indirect detection method such as a method using a secondary antibody to which a labeling substance is bound, a method using a polymer in which a secondary antibody and a labeling substance are bound, or the like.
- labeling substances include peroxidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ -D-galactosidase, enzymes such as glucose oxidase and glucose-6-phosphate dehydrogenase, fluorescein isothiocyanate (FITC), Fluorescent materials such as tetramethylrhodamine isothiocyanate (TRITC) and europium, chemiluminescent materials such as luminol, isoluminol and acridinium derivatives, coenzymes such as NAD, biotin, and radioactive materials such as 131 I and 125 I.
- HRP horseradish peroxidase
- alkaline phosphatase ⁇ -D-galactosidase
- enzymes such as glucose oxidase and glucose-6-phosphate dehydrogenase
- FITC fluorescein isothiocyanate
- TRITC
- the reagent of the present invention is solid-phased according to its use.
- the insoluble support used for the solid phase is not particularly limited.
- a resin such as polystyrene resin, polycarbonate resin, silicon resin, nylon resin, or an insoluble support made of a water-insoluble substance such as glass can be used. Loading onto the insoluble support can be performed by physical adsorption or chemical adsorption.
- the kit of the present invention contains the reagent of the present invention as a main component.
- Other reagents buffers, blocking reagents, enzyme substrates, coloring reagents, etc.
- devices or instruments used in performing the test method may be included in the kit.
- an instruction manual is attached to the kit of the present invention.
- the sample thus prepared was subjected to analysis by PradigmMS4-PAL-LTQ Orbitrap XL (ThermoFisher SCIENTIFIC) which is an LC-MS / MS system (shotgun proteomics). MS data obtained was analyzed using Mascot (Matrix Science Ltd.) software based on NCBI RefSeq and SwissProt databases. As a result of the analysis, six types of autoantigen candidate proteins were identified.
- the sera used for the study are as follows.
- (a) LINH 9 cases (b) Lymphocytic anterior pituitary (LAH) 3 cases
- LAH Lymphocytic anterior pituitary
- Anti-labylline 3a antibody is a biomarker for LINH. By using anti-labfilin 3a antibody as an index, LINH can be tested (eg, differentiated) with extremely high sensitivity and specificity. Anti-rabfilin 3a antibody is useful for understanding the pathology of LINH.
- Reactivity of LINH-specific antigen with patient serum (1) Western blot Next, 6 types of autoantigen protein candidates were subcloned into an expression vector (pcDNA3.1D / V5-His-TOPO (registered trademark)). .
- the expression vector was transfected into a mammalian cell line (HEK293FT) using Lipofectamine 2000 (registered trademark) to synthesize recombinant protein.
- HEK293FT mammalian cell line
- Lipofectamine 2000 registered trademark
- Six types of autoantigen candidate recombinant proteins synthesized in HEK293FT cells were developed by SDS-polyacrylamide electrophoresis (PAGE), and the reactivity between patient serum and recombinant protein was examined by Western blotting using patient serum as the primary antibody. did.
- the sera used for the study are as follows.
- LINH is identified using anti-labfillin 3a antibody as an index. According to the test method of the present invention, LINH can be distinguished with extremely high sensitivity and specificity. Therefore, it can be said that the test method of the present invention is useful for preventing misdiagnosis and determining an appropriate treatment policy.
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Abstract
Description
以下の発明は、主として上記の成果に基づく。
[1]抗ラブフィリン3a抗体からなる、リンパ球性漏斗下垂体後葉炎マーカー。
[2]抗ラブフィリン3a抗体の検体中レベルを指標として用いることを特徴とする、リンパ球性漏斗下垂体後葉炎検査法。
[3]以下のステップ(1)~(3)を含む、[2]に記載の検査法:
(1)被検者由来の検体を用意するステップ;
(2)前記検体中の抗ラブフィリン3a抗体を検出するステップ;及び
(3)検出結果に基づいて、罹患している疾患がリンパ球性漏斗下垂体後葉炎であるか否か、又はリンパ球性漏斗下垂体後葉炎の発症可能性、を判定するステップ。
[4]抗ラブフィリン3a抗体のレベルが高いことが、罹患している疾患がリンパ球性漏斗下垂体後葉炎であることの指標、及びリンパ球性漏斗下垂体後葉炎の発症可能性が高いことの指標となる、[3]に記載の検査法。
[5]ステップ(2)で得られた検出値と対照検体の検出値との比較に基づきステップ(3)の判定を行う、[3]又は[4]に記載の検査法。
[6]ステップ(2)で得られた検出値と、同一の被検者から過去に採取された検体中の検出値との比較に基づきステップ(3)の判定を行う、[3]又は[4]に記載の検査法。
[7]前記検体が血液、血漿、血清、脳脊髄液又は尿である、[2]~[6]のいずれか一項に記載の検査法。
[8]ラブフィリン3a又はその抗体結合断片を含む、リンパ球性漏斗下垂体後葉炎検査試薬。
[9][8]に記載のリンパ球性漏斗下垂体後葉炎検査試薬を含む、リンパ球性漏斗下垂体後葉炎検査用キット。
本発明の第1の局面はLINHのバイオマーカー(以下、「本発明のバイオマーカー」とも呼ぶ)に関する。本発明のバイオマーカーはLINHの鑑別、或いはLINHの発症可能性を評価する上で有用な指標である。ここでの「鑑別」は、被験者が罹患している疾患がLINHであるか、それとも別の疾患であるかを判定することである。特に、尿崩症の患者がLINHを罹患しているか否かを把握する上で本発明のバイオマーカーは有用である。とりわけ、LINHに伴う中枢性尿崩症と、腫瘤性病変に伴う尿崩症とを区別する指標として本発明のバイオマーカーは重要である。
本発明の第2の局面は上記本発明のバイオマーカーの用途に関し、罹患している疾患がLINHであるか否か、又はLINHの発症可能性を検査する方法(以下、「本発明の検査法」とも呼ぶ)を提供する。本発明の検査法によればLINHの鑑別が可能になる(罹患している疾患がLINHであるか否かを判定する場合)。本発明の検査法は、LINHを現在発症しているか否かを判定するための手段として、或いはLINHを将来発症する可能性を判定するための手段としても有用である(LINHの発症可能性を判定する場合)。このように、本発明の検査法はLINHの診断上、有用な情報を与える。本発明の検査法は、例えば、簡便且つ客観的なLINHの確定診断に役立つ。
(1)被検者由来の検体を用意するステップ
(2)前記検体中の抗ラブフィリン3a抗体を検出するステップ
(3)検出結果に基づいて、罹患している疾患がリンパ球性漏斗下垂体後葉炎であるか否か、又はリンパ球性漏斗下垂体後葉炎の発症可能性を判定するステップ
基準値よりも検出値(例えば標識量や蛍光量)が高いときに「罹患している疾患はLINHである」と判定し、基準値よりも検出値が低いときに「罹患している疾患はLINH以外である」と判定する。特定の疾患との鑑別を行う場合にあっては、典型的には、基準値よりも検出値が高いときに「罹患している疾患はLINHである」と判定し、基準値よりも検出値が低いこときに「罹患している疾患は当該特定の疾患である」と判定する。ここでの「特定の疾患」は、例えば、リンパ球性下垂体前葉炎症、尿崩症を伴うIgG4関連下垂体炎症、尿崩症を伴う腫瘤性病変である(以下の例でも同様)。
反応性が認められた(陽性の)ときに「罹患している疾患はLINHである」と判定し、反応性が認められない(陰性の)ときに「罹患している疾患はLINH以外である」と判定する。特定の疾患との鑑別を行う場合にあっては、典型的には、反応性が認められた(陽性の)ときに「罹患している疾患はLINHである」と判定し、反応性が認められない(陰性の)ときに「罹患している疾患は当該特定の疾患である」と判定する。
基準値よりも検出値(例えば標識量や蛍光量)が高いときに「LINHを発症している可能性が高い」と判定し、基準値よりも検出値が低いときに「LINHを発症していない」又は「LINHを発症していない可能性が高い」と判定する。将来の発症可能性を判定する場合にあっては、基準値よりも検出値が高いときに「LINHを発症する」又は「LINHを発症する可能性が高い」と判定し、基準値よりも検出値が低いときに「LINHを発症しない」又は「LINHを発症しない可能性が高い」と判定する。
反応性が認められた(陽性の)ときに「LINHを発症している」又は「LINHを発症している可能性が高い」と判定し、反応性が認められない(陰性の)ときに「LINHを発症していない」又は「LINHを発症していない可能性が高い」と判定する。将来の発症可能性を判定する場合にあっては、反応性が認められた(陽性の)ときに「LINHを発症する」又は「LINHを発症する可能性が高い」と判定し、反応性が認められない(陰性の)ときに「LINHを発症しない」又は「LINHを発症しない可能性が高い」と判定する。
以下に示すように検出値の範囲毎に「罹患している疾患がLINHである可能性(%)」を予め設定しておき、検出値から「罹患している疾患がLINHである可能性(%)」を判定する。
検出値a~b:LINHである可能性は10%以下
検出値b~c:LINHである可能性は10%~30%
検出値c~d:LINHである可能性は30%~50%
検出値d~e:LINHである可能性は50%~70%
検出値e~f:LINHである可能性は70%~90%
以下に示すように測定値の範囲毎に発症可能性(%)を予め設定しておき、測定値から発症可能性(%)を判定する。
測定値a~b:発症可能性は10%以下
測定値b~c:発症可能性は10%~30%
測定値c~d:発症可能性は30%~50%
測定値d~e:発症可能性は50%~70%
測定値e~f:発症可能性は70%~90%
本発明はさらに、LINHの検査法に使用される試薬及びキットも提供する。本発明の試薬は、本発明のバイオマーカーに特異的結合性を示す物質である、ラブフィリン3a又はその抗体結合断片(以下、「有効成分」と呼ぶ)を含む。本発明の試薬の有効成分は、例えば、組換え(リコンビナント)蛋白として調製することができる。例えば、目的の有効成分(ラブフェリン3a又はその抗体結合断片)をコードするDNAで適当な宿主細胞を形質転換し、形質転換体内で発現された蛋白を回収することにより調製することができる。回収された蛋白は目的に応じて適宜精製される。このように組換え蛋白として結合分子を得ることにすれば種々の修飾が可能である。例えば、結合分子をコードするDNAと他の適当なDNAとを同じベクターに挿入し、当該ベクターを用いて組換え蛋白の生産を行えば、任意のペプチドが連結された組換え蛋白からなる有効成分を得ることができる。また、糖鎖及び/又は脂質の付加や、あるいはN末端若しくはC末端のプロセッシングが生ずるような修飾を施してもよい。以上のような修飾により、組換え蛋白の抽出、精製の簡便化、又は生物学的機能の付加等が可能である。尚、有効成分の調製法は遺伝子工学的手法によるものに限られない。例えば天然に存在するものであれば、天然材料から標準的な手法(破砕、抽出、精製など)によって、本発明の試薬の有効成分を調製することもできる。
LINH患者の血清、脳腫瘍などで尿崩症を呈する疾患コントロール症例の血清、及び健常者の血清を収集し、常法でIgGを精製した。一方、ラット下垂体から下垂体後葉蛋白抽出液を作製した。精製したIgGと下垂体後葉蛋白抽出液を混和し、プロテインGビーズを用いて免疫沈降した後、免疫沈降物から抗原を溶出した。溶出した抗原を還元、アルキル化した後にトリプシン消化(in-solution digestion)した。このようにして調製したサンプルをLC-MS/MSシステムであるPradigmMS4-PAL-LTQ Orbitrap XL(ThermoFisher SCIENTIFIC)による解析に供した(ショットガンプロテオミクス)。NCBI RefSeq及びSwissProtデータベースを基に、Mascot(Matrix Science Ltd.)ソフトウェアを用い、得られたMSデータを解析した。解析の結果、自己抗原候補蛋白6種が同定された。
(1)ウエスタンブロット
次に、自己抗原蛋白候補である6種類の蛋白を発現ベクター(pcDNA3.1D/V5-His-TOPO(登録商標))にサブクローニングした。その後、Lipofectamine 2000(登録商標)を用いて発現ベクターを哺乳類細胞株(HEK293FT)にトランスフェクションし、リコンビナント蛋白を合成した。HEK293FT細胞で合成した6種類の自己抗原候補のリコンビナント蛋白をSDS-ポリアクリルアミド電気泳動(PAGE)で展開し、患者血清を一次抗体として、患者血清とリコンビナント蛋白との反応性をウエスタンブロット法で検討した。検討に用いた血清は次の通りである。
(a)LINH 9例
(b)リンパ球性下垂体前葉炎(LAH) 3例
(c)IgG4関連下垂体炎 3例
(d)疾患コントロール(合計10例)
胚細胞性腫瘍+は尿崩症(DI) 3例
鞍上部松果体腫瘍術後+DI 1例
視床下部腫瘍術後+DI 1例
ラトケ嚢胞+DI 1例
頭蓋咽頭腫+DI 2例
Glioma術後+DI 1例
サルコイドーシス+DI 1例
(e)健常コントロール(健常者) 10例
ラブフィリン3aをトランスフェクションしたHEK293FT細胞とLINH患者血清との反応性を免疫組織化学で調べた。結果を図3に示す。LINH血清(最上段)では、抗V5抗体の認識する細胞に一致して反応が認められた(共局在)。別のLINH患者血清を用いた場合(上から3段目)にも同様の染色性を認めた。一方、空ベクターをトランスフェクションした場合(上から2段目)、抗V5抗体及び血清ともに反応を認めなかった。また、腫瘍術後の尿崩症患者血清を用いた場合(最下段)、共局在を認めず、ラブフィリン3aに対する抗体はLINH患者特異的であることが示唆された。
ラット下垂体後葉、及び視床下部視索上核(SON)のバゾプレシン(AVP)ニューロンにおけるラブフィリン3aの発現を免疫組織化学(抗AVP抗体と抗ラブフィリン抗体による2重染色)で検討した。免疫組織化学の結果を図4に示す。下垂体後葉及び視床下部SONのAVPニューロンにおいて、ラブフィリン3aの発現が認められた。LINHの病変部位でラブフィリン3aの発現が認められたことは、当該蛋白がLINHの病態へ関与する可能性を強く示唆する。
LINH特異的な抗原蛋白ラブフィリン3aを見出すことに成功した。抗ラブフィリン3a抗体はLINHのバイオマーカーとなる。抗ラブフィリン3a抗体を指標にすれば、極めて高い感度及び特異度をもってLINHの検査(例えば鑑別)が可能になる。抗ラブフィリン3a抗体はLINHの病態把握にも有用である。
2.LINH特異的抗原と患者血清との反応性
(1)ウエスタンブロット
次に、自己抗原蛋白候補である6種類の蛋白を発現ベクター(pcDNA3.1D/V5-His-TOPO(登録商標))にサブクローニングした。その後、Lipofectamine 2000(登録商標)を用いて発現ベクターを哺乳類細胞株(HEK293FT)にトランスフェクションし、リコンビナント蛋白を合成した。HEK293FT細胞で合成した6種類の自己抗原候補のリコンビナント蛋白をSDS-ポリアクリルアミド電気泳動(PAGE)で展開し、患者血清を一次抗体として、患者血清とリコンビナント蛋白との反応性をウエスタンブロット法で検討した。検討に用いた血清は次の通りである。
検体及び症例の種類を増やし、抗ラブフィリン3a抗体のバイオマーカーとして有用性を更に検討した。結果を図5及び6に示す。腫瘤性病変に伴う尿崩症とLINHとの鑑別、及び特発性若しくは二次性の中枢性尿崩症とLINHとの鑑別では、それぞれ92.8%及び95.2%と特異度は非常に高く、また各種自己免疫疾患とLINHとの鑑別でも高い特異度(89.9%)を示した。このように、即ち、抗ラブフィリン3a抗体がLINHのバイオマーカーとして極めて有用であることが裏付けられた。
Claims (9)
- 抗ラブフィリン3a抗体からなる、リンパ球性漏斗下垂体後葉炎マーカー。
- 抗ラブフィリン3a抗体の検体中レベルを指標として用いることを特徴とする、リンパ球性漏斗下垂体後葉炎検査法。
- 以下のステップ(1)~(3)を含む、請求項2に記載の検査法:
(1)被検者由来の検体を用意するステップ;
(2)前記検体中の抗ラブフィリン3a抗体を検出するステップ;及び
(3)検出結果に基づいて、罹患している疾患がリンパ球性漏斗下垂体後葉炎であるか否か、又はリンパ球性漏斗下垂体後葉炎の発症可能性、を判定するステップ。 - 抗ラブフィリン3a抗体のレベルが高いことが、罹患している疾患がリンパ球性漏斗下垂体後葉炎であることの指標、及びリンパ球性漏斗下垂体後葉炎の発症可能性が高いことの指標となる、請求項3に記載の検査法。
- ステップ(2)で得られた検出値と対照検体の検出値との比較に基づきステップ(3)の判定を行う、請求項3又は4に記載の検査法。
- ステップ(2)で得られた検出値と、同一の被検者から過去に採取された検体中の検出値との比較に基づきステップ(3)の判定を行う、請求項3又は4に記載の検査法。
- 前記検体が血液、血漿、血清、脳脊髄液又は尿である、請求項2~6のいずれか一項に記載の検査法。
- ラブフィリン3a又はその抗体結合断片を含む、リンパ球性漏斗下垂体後葉炎検査試薬。
- 請求項8に記載のリンパ球性漏斗下垂体後葉炎検査試薬を含む、リンパ球性漏斗下垂体後葉炎検査用キット。
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2016223774A (ja) * | 2015-05-26 | 2016-12-28 | 国立大学法人名古屋大学 | リンパ球性漏斗下垂体後葉炎検査試薬及びその用途 |
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JPWO2013080811A1 (ja) | 2015-04-27 |
EP2787346B1 (en) | 2017-06-21 |
JP5924502B2 (ja) | 2016-05-25 |
EP2787346A4 (en) | 2015-07-22 |
US20150160209A1 (en) | 2015-06-11 |
EP2787346A1 (en) | 2014-10-08 |
US9372189B2 (en) | 2016-06-21 |
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