JP6618107B2 - Lymphocytic funnel pituitary posterior inflammation test reagent and its use - Google Patents

Description

  The present invention relates to examination of lymphocytic funnel pituitary ptitis. Specifically, the present invention relates to a reagent for testing lymphofungal pituitary posterior phlebitis and its use (testing method, etc.).

  Diagnosis of lymphocytic pituitary pituitary (hereinafter abbreviated as “LINH”) may be difficult, and definitive diagnosis requires biopsy of the pituitary gland but is performed because it is invasive Are often misdiagnosed. Several diagnostic markers using non-invasive serum of lymphocytic hypophysitis have been reported so far (Non-Patent Documents 1 to 5), both of which have excellent sensitivity and specificity. There is no clinical application. In addition, no diagnostic marker specific to lymphocytic pituitary ptitis (LINH) among lymphocytic hypophysitis has been reported. The present inventors examined the development of a diagnostic marker for LINH and reported the results (Patent Document 1). Specifically, by using a technique such as proteomics, the discovery of a LINH-specific molecule, `` Lovephilin 3a '', and the presence of autoantibodies against the molecule specific to LINH patients, reported. In other words, it is clear that autoantibodies against labfilin 3a (anti-rabfilin 3a antibody) are useful as LINH biomarkers, and that LINH can be tested with high sensitivity and specificity by using the antibody as an index. It was. Conventionally, there has been only a highly invasive biopsy of the pituitary gland for the diagnosis of LINH. By using this biomarker, it is expected that a diagnosis by blood test will be possible.

International Publication No. 2013/080811 Pamphlet

J Clin Endocrinol Metab. 87 (2): 752-7, 2002 Horm Res. 55 (6): 288-92, 2001 Eur J Endocrinol. 147 (6): 767-75, 2002 J Endocrinol Invest, 30 (2): 153-62, 2007 Clin Endocrinol. 69 (2): 269-78, 2008

  The LINH test using the lovephilin 3a antibody as an index is groundbreaking and is expected to have many clinical applications. In order to increase the usefulness of the test method and to put it to practical use, further improvements in sensitivity and specificity are desired.

In the previous report (Patent Document 1), the anti-labfilin 3a antibody in the patient specimen was detected as an antibody against full-length labfilin 3a, and the epitope of the anti-labfilin 3a antibody was not identified. In order to reduce non-specific reactions (binding) and improve detection sensitivity and specificity, the test reagent is not a full-length labfilin 3a, but a binding peptide of an anti-labfilin 3a antibody in a patient sample, that is, a fragment peptide containing an epitope. It is desirable to use it for. Therefore, the present inventors decided to study with the aim of identifying the epitope of lovephilin 3a. First, in addition to the full-length human labfilin 3a recombinant protein (aa1-690), four types of partial-length human labphilin 3a recombinant peptides (aa1-197, aa198-515, aa198-690, aa516-690) were prepared. The reactivity of the peptide with LINH patient serum was examined. As a result, a high reactivity was observed in the peptide (aa516-690) containing the C2 B domain, suggesting that an epitope exists in the C2 B domain. Therefore, we synthesized multiple peptides corresponding to the partial sequence of C2 B domain (determined the sequence to be used by evaluating and estimating the antigenic site based on the average hydrophobicity value, charge density algorithm, etc.) The responsiveness to LINH patient serum was evaluated. As a result, it was found that the reaction between aa590-615 and LINH patient sera is specific, and an epitope exists in this region and the region including its vicinity.
The following invention is mainly based on the above-mentioned results.
[1] Reticulofungal pituitary ptitis test reagent comprising at least 6 consecutive amino acids among amino acids 580 to 625 of lovephilin 3a, and comprising a peptide to which anti-raffilin 3a antibody specifically binds .
[2] The test reagent according to [1], wherein the peptide comprises at least 6 consecutive amino acids from amino acids 590 to 615 of lovephilin 3a.
[3] The test reagent according to [1] or [2], wherein the number of amino acids constituting the peptide is 6 to 30.
[4] The test reagent according to [3], wherein the peptide consists of the amino acid sequence of SEQ ID NO: 4.
[5] The test reagent according to any one of [1] to [4], wherein the peptide is immobilized on an insoluble support.
[6] A test kit for lymphofungal posterior pituitary inflammatory disease comprising the test reagent according to any one of [1] to [5].
[7] The test kit according to [6], which is for ELISA.
[8] Lymphatic funnel pituitary ptitis test method comprising the following steps (1) to (4):
(1) a step of preparing a subject-derived specimen;
(2) contacting the sample with the reagent according to any one of [1] to [5];
(3) detecting the immune complex produced in step (2); and (4) based on the detection result, whether the disease affected is lymphocytic pituitary ptitis or lymphocytes Determining the likelihood of the development of posterior pituitary hypophysitis.
[9] A high level of anti-labfillin 3a antibody indicates that the disease affected is lymphocytic pituitary posterior pititis, and that there is a high probability of developing lymphocytic pituitary pituitary inflammation The inspection method according to [8], which is an index of
[10] The test method according to [8] or [9], wherein the determination in step (4) is performed based on a comparison between the detection value obtained in step (3) and the detection value of the control sample.
[11] The determination in step (4) is performed based on the comparison between the detection value obtained in step (3) and the detection value in the sample collected in the past from the same subject, [8] or [ 9].
[12] The examination method according to any one of [8] to [11], wherein the specimen is blood, plasma, serum, cerebrospinal fluid, or urine.

The structure of human labfillin 3a and the position of the created partial length labfillin 3a. Results of SDS-PAGE of partial length labfillin 3a. Expression of each partial length labfilin 3a was confirmed. Western blot results. The reactivity of each partial length labfilin 3a with LINH patient serum was examined. The left is a gel stained after SDS-PAGE. Lanes 1 to 3: Healthy human serum in which a false positive reaction was observed against full-length labfilin 3a. The result of ELISA using the plate which solid-phased partial length labfillin 3a (a.a.516-690). In the graph on the right, the detected values are represented by statistical processing. Algorithm used for antigen site prediction analysis. The numerical value regarding each amino acid obtained by each calculation was scored according to a specific criterion, and then a region in which amino acids having a high total score were consecutive was extracted as a candidate sequence. Candidate sequences extracted by antigen site (epitope) prediction analysis and their characteristics. The result of ELISA using the plate which solidified the partial peptide (a.a.590-615) of lovephilin 3a. C9 to C15 are normal serum.

1. Lymphocytic Funnel Pituitary Posterior Lobeitis (LINH) Testing Reagent and Kit The first aspect of the present invention relates to a reagent and kit useful for testing LINH. The reagent of the present invention is a sequence of amino acids 580 to 625 of lovephilin 3a based on the results of clarifying the location of epitopes of LINH-specific autoantibodies (anti-labfilin 3a antibodies) (Examples described later). And a peptide to which the anti-labfillin 3a antibody specifically binds. In other words, the reagent of the present invention is characterized by containing an epitope of a LINH-specific autoantibody (anti-labylphyrin 3a antibody). Whether or not it has the feature that “anti-labfilin 3a antibody specifically binds” can be evaluated, for example, by examining the reactivity with a LINH positive specimen (for example, serum of a LINH patient).

  Lovephilin 3a consists of a total length of 690 amino acids, a Rab binding domain is present on the N-terminal side, and a C2A domain and a C2B domain are present on the N-terminal side. The amino acid sequence of lovephilin 3a and the nucleotide sequence encoding it are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing, respectively ((DEFINITION: Homo sapiens rabphilin 3A homolog (mouse), mRNA (cDNA clone MGC: 29559 IMAGE: 3510158) , complete cds. ACCESSION: BC017259)). In addition, the sequence of amino acids 580 to 625 of lovephilin 3a is shown in SEQ ID NO: 3.

  The peptide constituting the reagent of the present invention is preferably at least 6 consecutive amino acids in amino acids 590 to 615 (SEQ ID NO: 4) of lovephilin 3a, and amino acids 580 to 615 of lovephilin 3a ( It comprises at least 6 consecutive amino acids in SEQ ID NO: 5) or at least 6 consecutive amino acids in amino acids 590 to 625 (SEQ ID NO: 6) of lovephilin 3a. Specific examples include KAKHKTQIKK (SEQ ID NO: 7: amino acid 597 to amino acid 606), VKLWLKPDMG (SEQ ID NO: 8: amino acid 586 to amino acid 595) and NPEFNEEFFY (SEQ ID NO: 9: amino acid 610 to amino acid 619). It is. The 590th amino acid to the 615th amino acid correspond to a peptide (a.a.590-615) which was used in the examples described later and showed good reactivity with LINH patient serum.

  The length (the number of amino acids) of the peptide constituting the reagent of the present invention is not particularly limited as long as the LINH patient-specific autoantibody, that is, the anti-labfilin 3a antibody specifically binds. 30, preferably 10 to 20 amino acids.

  The peptide constituting the reagent of the present invention can be produced, for example, by a known peptide synthesis method (for example, solid phase synthesis method, liquid phase synthesis method). The peptides of the present invention may be prepared using genetic engineering techniques. That is, the peptide of the present invention can be prepared by introducing a nucleic acid encoding the peptide of the present invention into a suitable host cell and recovering the peptide expressed in the transformant. The recovered peptide is purified as necessary. The recovered peptide can be subjected to an appropriate substitution reaction and converted into a desired modified peptide. You may decide to prepare the peptide which comprises the reagent of this invention 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.

  If a labeled peptide constituting the reagent of the present invention is used, the amount of bound antibody can be directly detected using the labeled amount as an index. Therefore, it is possible to construct an inspection method that is easy to operate. On the other hand, it is also preferable from the viewpoint of detection sensitivity to use 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. .

Examples of labeling substances include peroxidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, β-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.

  In one aspect, the reagent of the present invention is immobilized on the basis of its use. The insoluble support used for the solid phase is not particularly limited. For example, a resin such as polystyrene resin, polycarbonate resin, silicone resin, nylon resin, or an insoluble support made of a water-insoluble substance such as glass can be used. Immobilization to an 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.) and / or devices or instruments (containers, reaction devices, fluorescent readers, etc.) used in performing the test method may be included in the kit. Good. Usually, an instruction manual is attached to the kit of the present invention.

2. LINH Test Method The second aspect of the present invention relates to the use of the reagent of the present invention, and relates to a method for testing whether or not the disease affected is LINH or the possibility of developing LINH (hereinafter referred to as “the present invention”). Also called "inspection method"). According to the test method of the present invention, LINH can be differentiated (when it is determined 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). Thus, 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 “differentiation” here is to determine whether the disease affected by the subject is LINH or another disease. In particular, the test method of the present invention is useful for ascertaining whether or not a patient with diabetes insipidus suffers from LINH. In particular, the test method of the present invention is important as a means for distinguishing between central diabetes insipidus associated with LINH and diabetes insipidus associated with mass lesions.

In the inspection method of the present invention, the following steps are performed.
(1) Step of preparing a specimen derived from a subject (2) Step of bringing the specimen into contact with the reagent of the present invention (3) Step of detecting an immune complex produced in Step (2) (4) Detection result A step of determining whether the disease affected is lymphocytic pituitary ptosis or the likelihood of development of lymphocytic pituitary phleitis

  In step (1), a specimen derived from the subject is prepared. As the specimen, 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”. In addition, when the present invention is applied to a patient diagnosed with LINH by a doctor's interview or other examinations, the diagnosis is based on an objective index such as the level of a biomarker (anti-labylphyrin 3a antibody). It is possible to determine whether or not it is correct. 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 disease state, and prevention of intractable, serious, relapse, etc. can be achieved.

  Cases in which thickening of the pituitary stalk and enlargement of the pituitary gland are observed by imaging examinations such as MRI examination are presumed to be at high risk of LINH (high risk person), and are suitable subjects. 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.

  In the present invention, in order to detect a LINH-specific biomarker, that is, an anti-labfilin 3a antibody, an immune complex is formed by contacting the specimen prepared in step (1) with the reagent of the present invention (step (2)). (An immune complex is not formed when the anti-labfillin 3a antibody is not present in the sample), and the immune complex is detected (step (3)). Thus, the test method of the present invention uses an immunological technique to detect an anti-labfillin 3a antibody that may be present in a specimen. According to immunological techniques, rapid and sensitive detection is possible. Also, the operation is simple.

  Examples of the measurement method 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. The FIA method uses a fluorescently labeled antibody, and detects immune complexes using fluorescence as a signal. On the other hand, in the 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. Either a non-competitive method or a competitive method may be used. Details of the ELISA method are described in many books and papers, and can be referred to when setting the experimental procedure and experimental conditions of each method.

  Means capable of simultaneously detecting a large number of specimens such as a protein array and a protein chip may be used.

  In step (4), it is determined based on the detection result 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 (3) 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)
When the detected value (for example, the amount of labeling or fluorescence) is higher than the reference value, it is determined that “the disease affected is LINH”, and when the detected value is lower than the reference value, “the disease being affected is It is determined that it is other than LINH. When differentiating from a specific disease, typically, when the detected value is higher than the reference value, it is determined that “the disease affected is LINH”, and 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” herein 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 2 of qualitative judgment: discrimination)
When responsiveness is found (positive), it is determined that “the disease is affected is LINH”, and when responsiveness is not found (negative), “the disease is other than LINH Is determined. When differentiating from a specific disease, typically, when responsiveness is observed (positive), it is determined that “the disease affected is LINH” and responsiveness is recognized. When it is not possible (negative), it is determined that “the disease affected is the specific disease”.

(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 possible 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 “does not develop LINH” or “is likely not to develop LINH”.

(Qualitative determination example 4: likelihood of onset)
When responsiveness is found (positive), it is judged that “it has developed LINH” or “highly likely to develop LINH”, and when responsiveness is not found (negative), It is determined that “not developing LINH” or “highly likely not developing LINH”. In the case of determining the possibility of future onset, when responsiveness is recognized (positive), it is determined that “it develops LINH” or “highly likely to develop LINH”, and the reactivity is When it is not recognized (negative), it is judged as “not developing LINH” or “highly unlikely to develop LINH”.

(Example of quantitative judgment 1: discrimination)
As shown below, “possibility of disease affected by LINH (%)” is set in advance for each detection value range, and from the detection value “possibility of disease affected by LINH ( %) ”.
Detection value ab: 10% or less possibility of LINH Detection value bc: 10% to 30% possibility of LINH
Detection value c to d: possibility of LINH is 30% to 50%
Detection value d to e: 50% to 70% possibility of LINH
Detection values ef: 70% to 90% likely to be LINH

(Example of quantitative determination 2: likelihood of onset)
As shown below, the possibility of onset (%) is set in advance for each range of measured values, and the possibility of onset (%) is determined from the measured values.
Measured values a to b: 10% or less likelihood of onset Measured values b to c: 10% to 30% of possible onset
Measurement values cd: 30% to 50% likelihood of onset
Measurements d to e: 50% to 70% likelihood of onset
Measured values ef: 70% to 90% likelihood of onset

  The number of determination categories, the level of the biomarker (anti-labylphyrin 3a antibody) associated with each determination category, and the determination results can be arbitrarily set through preliminary experiments and the like without being limited to the above examples. 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.

  In one embodiment of the present invention, for the same subject, the level of a biomarker (anti-labylphyrin 3a antibody) measured at a certain time point is compared with the level of a biomarker measured in the past, and the level of the biomarker The presence or absence and / or degree of increase / decrease is examined. The resulting biomarker level change data is useful information for monitoring the onset or likelihood of onset of LINH, for understanding the therapeutic effect, or for prognostic estimation. Specifically, for example, based on the change in the biomarker level, it can be determined that 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. Thus, the present invention can make a great contribution to maximizing the therapeutic effect and improving the patient's QOL (quality of life).

<Searching for epitopes of anti-labfillin 3a antibody>
1. Creation of partial-length labfilin 3a (Rabphilin3a) In order to narrow down the epitope site from full-length labphilin 3a (aa1-690), four types of partial-length labphilin 3a (labfilin 3a (aa1-197), labphilin 3a (aa198-690), labphilin 3a (Aa198-515), Lovephilin 3a (aa516-690)) was prepared. A pGEX 6P vector containing each partial length labfilin 3a was constructed and expressed in E. coli to obtain a GST fusion partial length labfilin 3a protein (FIGS. 1 and 2).

2. Serum collection
The serum of LINH patients, the serum of patients with idiopathic central diabetes insipidus, the serum of disease control cases with diabetes insipidus in brain tumors, and the serum of healthy volunteers were collected by conventional methods.

3. Reactivity of partial-length labfilin 3a protein and patient serum (1) Western blot Next, partial-length labfilin 3a protein was developed by SDS-polyacrylamide gel electrophoresis (PAGE). Reactivity with long labfillin 3a protein was examined by Western blotting. The result (representative example) of the Western blot is shown in FIG. Autoantibodies against GST-fused labfilin 3a (aa516-690) were observed in LINH patients. On the other hand, autoantibodies against labfilin 3a (aa516-690) were not observed in 3 healthy subjects in which a false positive reaction was observed with full-length labfilin 3a (lanes 1 to 3). In other words, the epitope related to LINH is likely to be present in labfilin 3a (aa516-690) containing the C2 B domain, and according to the test method targeting this partial length, it is more than with full-length labfilin 3a. It was expected that the specificity would be improved.

(2) Construction and measurement of ELISA method (2-1) Preparation of antigen-immobilized ELISA plate First, the labfilin 3a antigen was dissolved in cold PBS to prepare an aqueous antigen solution (concentration 0.5 to 10 μg / mL). The antigen aqueous solution was dispensed at 100 μL / well onto an ELISA plate and allowed to stand at 4 ° C. overnight. The antigen aqueous solution was discarded, and blocking buffer was placed in an ELISA plate at 200 μL / well and allowed to stand at 4 ° C. overnight. Subsequently, the blocking buffer was discarded, and it was thoroughly dried on a laboratory bench, and then stored at 4 ° C. until use.

(2-2) ELISA measurement First, a reaction buffer was added to serum to prepare a patient serum dilution. The patient serum dilution was added to the antigen-immobilized plate at 100 μL / well, and allowed to react at room temperature for 1 hour. Thereafter, the patient serum dilution was discarded, and the ELISA plate was washed with a washing buffer. Next, the secondary antibody solution Anti-Human IgG (γ chain) -HRP was added to the ELISA plate at 100 μL / well and allowed to stand at room temperature for 1 hour. The secondary antibody solution was discarded, and the ELISA plate was washed with a washing buffer. Subsequently, 100 μL / well of a 4 ° C. reaction substrate solution was added to the ELISA plate and allowed to stand at room temperature for 30 minutes. A reaction stop solution was added at 100 μL / well to stop the reaction, and then a wavelength of 450 nm (subwavelength 620 nm) was measured.

(2-3) Results of Labophilin 3a (aa516-690) antigen-immobilized ELISA
As a result of examination of 9 cases of LINH, 5 cases of healthy subjects, and 5 cases of diabetes insipidus associated with tumors, an increase in the absorbance to labfilin 3a (aa516-690) was observed in 8 cases of 9 cases of LINH (FIG. 4). In addition, idiopathic DI is known to involve LINH as an autoimmune mechanism for many causes, but an increase in absorbance was observed in 2 out of 3 cases (2/3) ( Figure 4) shows that the two cases are likely to be LINH and confirms the effectiveness of this ELISA. From the above results, it was considered that there is a high possibility that a LINH-specific epitope exists in the part of lovephilin 3a (aa516-690).

4). Search for epitopes using synthetic peptides (1) Antigen site prediction In full-length labphilin 3a (SEQ ID NO: 1), antigen sites (epitope) using the program of Eurofin Genomics Co., Ltd. based on the algorithm shown in FIG. Predictive analysis was performed. As a result, several candidate sequences were obtained (FIG. 6).

(2) Preparation of synthetic peptides based on the results of antigen site prediction analysis The following eight peptides were synthesized using the Fmoc solid phase synthesis method (purity>80%; by HPLC analysis). Except for No. 8, peptides were added with cysteine for antigen preparation.
No.1: C + HPGGQPDRQRKQEELTDEEKEI (position 32-53, number of amino acids 23, SEQ ID NO: 10)
No.2: C + PKHPARAPARGDSEDRRGPGQKT (position 190-212, 24 amino acids, SEQ ID NO: 11)
No.3: C + PEVAPSDPGTTAPPREERTG (position 311-330, amino acid number 21, sequence number 12)
No.4: C + PGASKSNKLRTKTLRNT (position 434-450, amino acid number 18, SEQ ID NO: 13)
No.5: C + IGETRFSLKKLKPNQRKNFN (position 490-509, 21 amino acids, SEQ ID NO: 14)
No.6: C + ALYEEEQVERVGDIEERGK (position 531-549, amino acid number 20, SEQ ID NO: 15)
No.7: C + LKPDMGKKAKHKTQIKKKTLNPEFNE (position 590-615, amino acid number 27, SEQ ID NO: 16)
No.8: CLKNKDKKIERWHQLQNENHVSSD (position 667-690, 24 amino acids, SEQ ID NO: 17)

(3) ELISA using synthetic peptides
2. Using each of the above peptides as an antigen An antigen-immobilized ELISA plate was prepared in the same manner as in (2-1). 2. Use this ELISA plate ELISA measurement was performed according to the method of (2-2). The antigen aqueous solution was prepared at a concentration of 2.5 μg / mL or 5.0 μg / mL.

  As a result of examination using sera of 2 LINH patients and 7 healthy subjects, the LINH patient sera showed an increase in absorbance specifically for the No. 7 peptide (a.a.590-615 of lovephilin 3a). Furthermore, the response to No. 7 peptide was examined by increasing the number of samples. The result of the examination using sera of 6 LINH patients and 6 healthy individuals is shown in FIG. Compared with 6 healthy subjects, an increase in absorbance was observed in 3 of 6 patients. From the above results, it was shown that a.a.590-615 of lovephilin 3a may be a potential epitope candidate for LINH.

5. Summary
We succeeded in finding that aa590-615 is a promising epitope candidate in the LINH-specific antigen protein labfilin 3a. A study with partial length of labfilin 3a indicated that the epitope related to LINH is likely to be present in labfilin 3a (aa516-690), but aa590-615 is included in the partial length. This strongly supports the existence of an epitope of a LINH-specific autoantibody (anti-labfilin 3a antibody) in the region including aa590-615 and its vicinity. By using an antibody against the epitope (anti-rabophilin 3a antibody) as an index, LINH testing (for example, differentiation and understanding of the disease state) can be performed with extremely high sensitivity and specificity. Therefore, the epitope is extremely useful as a test reagent for LINH.

  The present invention provides a reagent useful for testing LINH. By using the reagent of the present invention, LINH can be differentiated with extremely high sensitivity and specificity. Therefore, it can be said that the result of the test using the reagent of the present invention is useful for preventing misdiagnosis and determining an appropriate treatment policy.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of the papers, published patent gazettes, patent gazettes and the like specified in this specification are incorporated by reference in their entirety.

Claims (8)

  It consists of a partial sequence of 580 amino acids to 625 amino acids including the vicinity of 590 amino acids to 615 amino acids of labfilin 3a or the vicinity thereof, and is composed of a peptide having 6 to 30 amino acids including the epitope of anti-labfilin 3a antibody Lymphocyte funnel pituitary posterior phlebitis test reagent.   The test reagent according to claim 1, wherein the peptide consists of the amino acid sequence of SEQ ID NO: 4.   The test reagent according to claim 1 or 2, wherein the peptide is immobilized on an insoluble support.   A test kit for lymphocytic pituitary posterior inflammatory disease comprising the test reagent according to any one of claims 1 to 3.   The test kit according to claim 4, which is used for ELISA. Lymphatic funnel pituitary ptitis test method comprising the following steps (1) and (2):
(1) A step of contacting a specimen comprising blood, plasma or serum derived from a subject with the reagent according to any one of claims 1 to 3;
(2) The step of detecting the immune complex produced in step (1) , wherein a high level of anti-labylphyrin 3a antibody is an indicator that the disease affected is lymphocytic funnel pituitary ptitis , And a step that is an indicator of a high likelihood of developing lymphocytic funnel pituitary posterior inflammation.   The test method according to claim 6, wherein the detection value obtained in step (2) is compared with the detection value of the control sample.   The test method according to claim 6, wherein the detection value obtained in step (2) is compared with the detection value in a sample collected in the past from the same subject.

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