JP5117336B2 - Method for measuring test markers for multiple sclerosis or NMO - Google Patents

Description

  The present invention relates to a method for measuring a test marker for multiple sclerosis or NMO. More particularly, the present invention is for examining the relapse and remission phases of multiple sclerosis or NMO by measuring the amount of a specific peptide in a biological sample from a patient with multiple sclerosis or NMO. It is the method of measuring the level of the peptide marker. The present invention also relates to a method for measuring a peptide marker for examining multiple sclerosis or NMO.

Multiple sclerosis is a disease in which demyelinating lesions occur in the white matter part of the central nervous system in multiple and sporadic ways, and the patient has a seizure in which a part of the central nervous system such as the brain and spinal cord suddenly inflamed. It is a repeated disease. It is thought that the cause of multiple sclerosis is strongly related to the function of immunity that protects bacteria and viruses when they enter the body. The name of multiple sclerosis is derived from the fact that many scars, that is, sclerosis occur due to demyelination of nerves. Symptoms include visual impairment, double vision, movement paralysis, ataxia, sensory disturbance, urination and defecation.
By the way, in patients with multiple sclerosis, remission and relapse generally appear alternately. The remission period is a period when it can be relatively healthy, and the relapse period is a period when symptoms suddenly appear and worsen. As a treatment method, treatment to relieve inflammation such as a large dose of corticosteroids is given in the relapse phase, and when sequelae are in the remission phase, an anticonvulsant or muscle relaxant is also applied. ing. Therefore, it is very important to determine and treat the remission phase and the recurrence phase of the disease as a countermeasure for the disease.

Such multiple sclerosis can be determined by conducting an MRI examination after a detailed interview and examination, or collecting a cerebrospinal fluid by inserting a needle from the waist and measuring the total amount of protein in it. Attempts have been made (Non-Patent Document 1).
However, in normal MRI examinations, only 10% of the lesions are symptomatic. As a result, they can be missed during light periods, and even MRI can be detected by MRI even in the majority of patients. There was a problem that there was no chronic disease activity, and there was a problem that it was decisive.
In order to solve this, a method of injecting a contrast medium called gadolinium (Non-Patent Document 2) is also used. However, in this case, there are problems that sensitivity is low, time is required, and facilities that can be enforced are limited. there were. In addition, even with cerebrospinal fluid examination, cerebrospinal fluid examination has a problem of high invasiveness and burden on patients, such as pain and possibly nerve damage. On the other hand, a method (non-patent document 3) in which a specific component from a blood sample of a patient is measured to determine a remission phase and a relapse phase of multiple sclerosis has been studied. The actual situation is not reached.

Until now, in Japan, multiple sclerosis has been broadly divided into the “optic spinal cord type” that relatively selectively affects the optic nerve and spinal cord and the “normal type” that broadly affects the central nervous system including the cerebrum, cerebellum, and brain stem. It was. Among these, regarding "optic spinal cord type" multiple sclerosis, the difference with the relapsing-remitting type Neuromyelitis optica (NMO) in Europe and America has been discussed.
In 2004, Mayo clinic's Lennon et al. Reported that NMO-IgG, an NMO-specific antibody, was present in the serum of NMO patients and revealed that the corresponding antigen was aquaporin-4. Since this NMO-IgG is found in the sera of many “optic nerve spinal type” multiple sclerosis patients and not in the sera of “normal” multiple sclerosis patients, multiple sclerosis has so far been achieved. The possibility of “optic spinal cord type” multiple sclerosis, which was captured in the concept of disease, is similar to NMO and has a different immunological background from multiple sclerosis (non-native) Patent Document 4).
There is also a type of NMO that repeats relapse and remission, and it is said that the appropriate treatment for relapse and recurrence prevention may be different from multiple sclerosis. In NMO, as in multiple sclerosis, Development of a method for determining the remission period and the recurrence period is desired.
Tourtelotte W. Cerebrospinal fluid in multiple sclerosis.In: Vinken PJ, Bruyn GW.editors.Handbook of clinical neurology, Vol. 9.Amsterdam: North-Holland, 1970: 324-382. Nesbit GM, Forbes GS, Scheithauer BW, Okazaki H, Rodriguez M. Multiple sclerosis: histopathologic and MR and / or CT correlation in 37 cases at biopsy and three cases at autopsy.Radology 1991; 180: 467-74. Sharief MK and Hentges R. Association between tumor necrosis factor-alpha and disease progression in patients with multiple sclerosis.New England Journal of Medicine 1992 Jan 23; 326 (4): 467-72. Weinshenker BG, Wingerchuk DM.OSMS is NMO, but not MS: proven clinically and pathologically.Lancet Neurol 2006; 5: 110-111.

  Therefore, an object of the present invention is to provide a method for determining a remission phase and a relapse phase appearing in multiple sclerosis and NMO.

Under such circumstances, in order to solve these problems, the present inventors have intensively studied for the purpose of finding a marker capable of determining a remission phase and a relapse phase appearing in multiple sclerosis and NMO. As a result, it has surprisingly been found that it can be tested by measuring the level of a specific peptide having a molecular weight of 1741 in a biological sample. Furthermore, it has been found that measuring this particular peptide can test for multiple sclerosis or NMO itself. The present invention has been achieved through this process.
That is, the present invention is characterized by measuring the amount of a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 in a biological sample derived from a patient with multiple sclerosis or NMO (namely, optic neuromyelitis). This is a method of measuring the amount of peptide marker (peptide value) for determining the relapse phase and remission phase of multiple sclerosis or NMO.
Furthermore, the present invention relates to a determination method for determining a relapsing phase and a remission phase of multiple sclerosis or NMO characterized by measuring a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in a biological sample. is there.

  With the measurement method of the present invention, it is possible to easily determine the remission phase and the relapse phase appearing in multiple sclerosis and NMO using a biological sample such as a blood sample of a patient. In addition, multiple sclerosis and NMO itself can be easily determined by the measurement method of the present invention.

The present invention is described in detail below.
In the present invention, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 can serve as a peptide marker for examining multiple sclerosis or the relapse phase and remission phase of NMO, It has been found that it can also be a peptide marker for determining NMO itself. That is, when the peptide value consisting of the amino acid sequence represented by SEQ ID NO: 1 in a patient-derived biological sample is higher than that of a normal person, it can be determined that it is multiple sclerosis or NMO. In addition, the peptide value consisting of the amino acid sequence represented by SEQ ID NO: 1 in a biological sample derived from a patient with multiple sclerosis or NMO is tracked and measured. When it can be judged as a recurrence phase and shows a low value, it can be judged as a remission phase.
In addition, normal remission data is acquired in advance and set as a reference value. If the value is higher than that value, recurrence is suspected, and more detailed examinations such as MRI and cerebrospinal fluid are added, subjective symptoms, etc. In combination, recurrence diagnosis may be performed.
The peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 has a molecular weight of 1741 and matches the amino acid sequence found in the partial sequence in the α chain of complement C4. Hereinafter, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 may be abbreviated as 1741 peptide.
The multiple sclerosis or NMO remission period is a period in which the patient can spend a relatively healthy period, and the relapse period is a period in which symptoms become severe again and become weak.

  In the present invention, examples of the biological sample include cerebrospinal fluid and blood samples, but blood samples such as plasma and serum are preferable from the viewpoint of patient burden and availability.

  In this specification, the amount of peptide or peptide value may be a measured value that increases or decreases depending on the increase in peptide concentration. For example, when determining the amount or value by mass spectrometry, It may be.

As a method for measuring the level of 1741 peptide in a biological sample, any currently known method applicable as a peptide measurement method can be adopted. For example, mass spectrometry, immunoassay, electrophoresis, liquid chromatography (LC) method, gas chromatography (GC) method and the like can be mentioned.
Examples of mass spectrometry include a method performed by a laser ionization time-of-flight mass spectrometer (LDI-TOF MS). As a laser ionization time-of-flight mass spectrometer, surface enhanced laser desorption ionization (Surface Enhanced Laser Desorption / Ionization) time-of-flight mass spectrometer (SELDI-TOF MS method), matrix-assisted laser ionization (Matrix-Assisted Laser Desorption / Examples include a time-of-flight mass spectrometer (MALDI-TOF MS method).
For example, when using the SELDI-TOF MS method, a protein biology system II mass spectrometer (Ciphergen Biosystems, Inc.) developed by Ciphergen can be used. This machine is a protein chip technology that combines SELDI (surface enhanced laser deformation ionization) and a flight mass spectrometer. Details thereof are detailed in WO 01/25791 A2 and JP-A-2001-28122.

In the case of the SELDI-TOF MS method, a biological sample such as serum or plasma is usually pretreated, adsorbed on a protein chip, and attached to a SELDI-TOF MS mass meter. The protein chip used in the measurement method using these mass spectrometers is not particularly limited as long as it can adsorb the 1741 peptide of the present invention. For example, a chip in which a functional group having affinity for a protein such as hydrophobicity or ion exchange is modified (also referred to as a chemical chip), a chip on which an antibody against the target protein is immobilized (biochemical chip), and the like can be exemplified.
When the MALDI-TOF MS method is used as a method for measuring the 1741 peptide, for example, the MALDI-TOF MS method using magnetic beads can be used. In the case of the MALDI-TOF • MS method using magnetic beads, for example, the ClintProt ™ system and Autoflex II-TOF / TOF MS developed by Bruker Daltonics can be used. The magnetic beads are coated on the surface, and various types having ion exchange, metal affinity, hydrophobic carbon chains, etc. can be selected and used.
In this case, as a method for measuring 1741 peptide using a mass spectrometer, a certain amount of specimen is adsorbed to beads, and usually the specimen is added to magnetic beads to adsorb peptides to beads, and the eluted liquid is used as a matrix. After mixing, drying and crystallizing on a metal chip, the chip is attached to a flight mass spectrometer. Next, a method of measuring the amount of 1741 peptide in the specimen from the intensity of the signal corresponding to 1741 peptide can be used. Further, an internal standard method, that is, an internal standard substance such as a peptide whose amino acid sequence is the same as that of 1741 peptide as an internal standard and at least one of those amino acids is an isotope is included in the mass spectrometer sample, The target peptide can also be measured by a method for measuring 1741 peptide from the signal ratio of 1741 peptide.
Examples of other mass spectrometry include mass spectrometry based on ESI (Electrospray Ionization). In the case of the ESI method, it is often preferable to apply the pretreated specimen such as protease treatment to a mass spectrometer directly connected to a separation means such as high performance liquid chromatography.

Examples of the immunoassay include a method in which a polyclonal antibody or a monoclonal antibody against the 1741 peptide of the present invention is prepared and a conventionally known peptide is measured. Examples of such immunoassay methods include enzyme immunoassay (EIA method), immunoturbidimetric assay (TIA method), latex immunoaggregation method (LATEX method), electrochemiluminescence method, fluorescence method and the like. . In addition, immunochromatography and methods using test paper are also effective. These methods are all methods well known to those skilled in the art, and these well known methods can be employed as they are.
Examples of antibodies that can be used in the above immunoassay include polyclonal and monoclonal antibodies prepared by methods that are already widely used. These antibodies can be obtained by using human blood-derived purified protein, specifically, 1741 peptide as an immunogen (antigen). These 1741 peptides for producing antibodies may be obtained by purification from human blood, but may also be obtained by chemical synthesis using known peptide synthesis techniques. Not limited to this, a produced protein such as cultured cells can also be used as an antigen. Furthermore, the use of full-length recombinant proteins produced by genetic engineering, mutants thereof, or parts thereof is a conventional means and can be used.

The monoclonal antibody is produced by a hybridoma obtained by immunizing an animal using the 1741 peptide as an immunogen and fusing an antibody-producing cell derived from the spleen and the like with a bone marrow tumor cell.
The hybridoma can be obtained by the following method. That is, the antigen obtained as described above, for example, a marker protein is mixed together using Freund's complete, incomplete adjuvant, aluminum hydroxide adjuvant, pertussis adjuvant, etc., to prepare a sensitive adjuvant solution. The animal is immunized by intraperitoneal subcutaneous or tail vein administration every 1 to 3 weeks, divided into several times. The amount of sensitizing antigen is usually between 1 μg and 100 mg, but generally about 50 μg is preferred. The number of immunizations is generally 2 to 7, but various methods are known. Next, antibody-producing cells derived from the spleen and the like, bone marrow tumor cells (myeloma cells) and the like are fused in vitro. As the fusion method, fusion can be performed by using polyethylene glycol (PEG) according to the conventional method of Kohler and Milstein (Nature. 256, 495.1975) which is already known per se. Fusion can also be performed by Sendai virus or electrofusion.
A method for selecting a hybridoma that produces an antibody that recognizes the marker protein from the fused cells can be performed as follows. That is, hybridomas are selected from colonies formed from cells that survive in the HAT medium and HT medium from the fused cells by the limiting dilution method. When an antibody against a marker protein is contained in a colony culture supernatant made of a fused cell spread in a 96-well, etc., the supernatant is placed on an assay plate having the marker protein immobilized on the plate, A monoclonal antibody-producing clone against the peptide can be selected by an ELISA method in which a secondary labeled antibody such as an anti-mouse immunoglobulin-HRP labeled antibody is reacted after the reaction. In addition to HRP, enzymes such as alkaline phosphatase, fluorescent substances, radioactive substances and the like can be used as the labeling substance of the labeled antibody. As a control, screening for specific antibodies against peptides can be performed by simultaneously performing ELISA using an assay plate to which only BSA as a blocking agent is bound. In other words, clones that are positive in any of the marker protein plates and negative by BSA ELISA are selected.

  The hybridoma can be cultured in a medium usually used for cell culture, such as α-MEM, RPMI1640, ASF, S-clone, and the monoclonal antibody can be recovered from the culture supernatant. An animal from which a hybridoma is derived and a nude mouse are treated in advance with pristane, and ascites can be retained by intraperitoneal injection of cells into the animal, and a monoclonal antibody can be recovered from the ascites. As a method for recovering the monoclonal antibody from the supernatant or ascites, a usual method can be used. For example, salting-out method using ammonium sulfate, sodium sulfate or the like, chromatography, ion exchange chromatography, affinity chromatography using protein A, etc. may be mentioned.

The 1741 peptide in the biological sample can be precisely measured by the monoclonal antibody purified by the above method. As a method for measuring 1741 peptide in a specimen by the EIA method, the method itself is known, and can be performed by using one or more monoclonal antibodies against the peptide as an antibody. Examples are described below. First, a monoclonal antibody against a marker protein is bound directly or indirectly to a solid phase known per se such as polystyrene, polypropylene, polycarbonate, polyethylene, nylon, polymethacrylate, etc. using physical bonds, chemical bonds, or affinity. The amount of sensitizing antibody is usually in the range of 1 ng to 100 mg / ml. A sample is added to a monoclonal antibody bound to a solid phase by physical bonding, chemical bonding, affinity, or the like, and allowed to react. After reacting for a certain period of time, the solid phase is washed, and the corresponding secondary labeled antibody is added, followed by further secondary reaction. The solid phase is washed again and reacted with a DAB chromogenic substrate. When HRP is used as the labeling substance, known DAB, TMB, etc. can be used as the substrate, and the labeling substance is not limited to this. For example, not only enzymes, but also distinguishable materials such as gold colloids, europium-labeled metals, various chemical and biological fluorescent materials such as FITC, rhodamine, Texas Red, Alexa and GFP, and radioactive materials such as ³² P and ⁵¹ Cr Is mentioned.
In addition to the immunity measurement method described above, the marker protein can be measured by electrophoresis, liquid chromatography (LC) method, gas chromatography (GC) method and the like. These methods are already well known to those skilled in the art, and those known methods can be employed as they are.

  By measuring the level of 1741 peptide in a patient-derived biological sample by the method described above, it can be determined that the patient has multiple sclerosis or NMO. If the 1741 peptide value is high, it can be examined that the patient has multiple sclerosis or NMO. In addition, by tracking and measuring the 1741 peptide value in biological samples derived from multiple sclerosis and NMO patients, it is possible to determine the relapse and remission periods of multiple sclerosis and NMO patients. The 1741 peptide shows high values in the relapse phase and low values in the remission phase. Relapse and remission values can be set on a patient-by-patient basis, and it will be possible to determine the remission and relapse phases of these diseases and take countermeasures accordingly, enabling early therapeutic intervention This is expected to improve the patient's prognosis.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples at all.
Example 1
Comparison of 1741 peptide levels (peak intensity) between normal, multiple sclerosis and NMO patients (1) Biological samples Diagnosed according to McDonald's diagnostic criteria (Annals of Neurology Vol.58 No.6 December 2005, 840-846) Using serum from 31 cases of multiple sclerosis, 15 cases of NMO diagnosed by NMO diagnostic criteria (Neurology 66 May 2006, 1485-11489) (all at the time of recurrence), and 48 cases of age-matched normal controls About 1741m / z peptide in the inside, the intensity | strength was compared with the mass spectrometer, and it compared.

(2) Measurement method 1) Sample treatment with magnetic beads Each 5 μl of patient serum was added to 10 μl of weak cation exchange (WCX) beads (Bruker Daltonics, Inc). 10 μl of a genuine binding solution was added thereto, and pipetting was sufficiently performed in a tube. By standing for 5 minutes, the peptide in the serum sample was adsorbed on the beads. The tube was placed on a magnetic separator and waited for 1 minute. After collecting the beads on one side, the supernatant was removed with a pipette. Remove the tube from the magnetic separator, add 100 μl of genuine washing solution, stand the tube again on the magnetic separator, move the tube back and forth 10 times, wash the beads, and then remove the supernatant with a pipette . This washing operation was repeated two more times. After the third washing, 10 μl of a pure elution solution (Elution solution) was added to the remaining beads, and the beads were resuspended while vigorously pipetting 10 times. Then, the tube was placed on a magnetic separator and allowed to stand for 2 minutes. The supernatant was collected and transferred to a new tube. To this, 10 μl of a genuine stabilization solution (Stabilization Solution) was added and mixed by pipetting strongly to obtain a peptide extract.

2) Preparation of energy-absorbing substance (matrix) α-cyano-4-hydroxycinnamic acid (CHCA, Bruker Daltonics, Inc) was added in acetone / ethanol (1/2; v / v) to a concentration of 0.3 g / l. v) Dissolved in solution.

3) Spotting and crystallization on Anchorchip ^(R) 10 μl of CHCA matrix was added to 1 μl of the above peptide extract, and pipetted well to mix. Of these, 0.8 μl was placed on Anchorchip® ⁽ Bruker Daltonics, Inc), waited for several minutes, and dried to obtain peptide crystals.

4) MALDI measurement Anchorchip ^{(R) on} which the obtained peptide crystal was mounted was set in Autoflex II-TOF / TOF MS (Bruker Daltonics, Inc) and measured. The measurement was performed with the laser intensity set to 15-35% and the detector set the linear mode to 20% detection sensitivity. The crystal was shot 25 times at a time. Integration was performed for 25 laser shots only when a peak having an S / N ratio greater than 2 and an intensity exceeding 100 was obtained between 3000 and 10000 m / z. This was integrated 40 times, that is, 1000 times of laser shots, and a serum profile by MALDI was obtained.

5) Data analysis The obtained serum profile data was analyzed with ClinProTools ver.2.1 (Bruker Daltonics, Inc). The peak intensity was normalized with a total ion current of 600 to 10000 m / z to obtain a peak intensity of 1741 m / z.

(3) Results The obtained data is shown in FIG. The assay was performed by the Mann-Whitney test, and the peak intensity of the 1741 peptide in the serum was compared between normal control and multiple sclerosis, and normal control and NMO. In normal controls and multiple sclerosis, the peak intensity was significantly increased in multiple sclerosis (p = 0.0002). Similarly, in the normal control and NMO, the peak intensity was significantly increased with NMO (p = 0.0005).
(4) Identification of 1741 peptide In the identification of the 1741 Da peptide, crystals were prepared in the same manner as in the MALDI measurement. Unlike the case where a serum profile was obtained, the Autoflex detector was set in the reflector mode and measured with a detection sensitivity of 20%. After accumulating the peak of the 1741 Da peptide about 500 times, the peptide was cleaved, which is a function unique to the reflector mode. By cleavage, the 1741 Da peptide was decomposed into smaller peptides and single amino acids, and the mass was measured with a second-stage mass spectrometer. This is called MS / MS measurement. The data of this MS / MS measurement, that is, the cleavage pattern was collated with the Mascot database. As a result, it was determined that 1741 Da is a peptide having the amino acid sequence of NGFKSHALQLNNNRI and a fragment of complement C4.

Example 2
Examination of relapse and remission by measuring 1741 peptide levels in patients with multiple sclerosis (1) Biological samples Diagnosed according to McDonald's diagnostic criteria (Annals of Neurology Vol.58 No.6 December 2005, 840-846) Using the serum of 16 cases of multiple sclerosis, the strength of the 1741 peptide in the relapse phase and the remission phase was determined with a mass spectrometer. The recurrence period is a period of exacerbation of neurological symptoms that persists for more than 24 hours at the time of recurrence, and there is a stable period of 1 month or more between recurrences, and the remission period is 3 to 6 months after the recurrence period treatment In this period, there was no recurrence during that period, and there was no next recurrence within 3 months after the recurrence.

(2) Measurement method The measurement was performed in the same manner as in Example 1.
(3) Results The obtained data are shown in FIG. For comparison of the recurrence period and remission period of the same case, the data was tested by the Wilcoxon signed rank sum test.

  From the results in Table 1 and FIG. 2, in 13 cases of 16 cases of multiple sclerosis, when comparing the relapse phase and the remission phase, the peak intensity was higher in the relapse phase (mean value: relapse phase 42. 0 arb.u, remission 31.3 arb.u). When the change of 1741 peptide was tested in the relapse phase and the remission phase by Wilcoxson signed rank sum test, a significant increase was recognized in the relapse phase (p = 0.01).

Example 3
Examination of relapse and remission by measuring 1741 peptide levels in NMO patients (1) Biological samples Using serum from 6 NMO cases that meet NMO diagnostic criteria (Neurology 66 May 2006, 1485-11489) The strength of the 1741 peptide in remission was determined using a mass spectrometer. The recurrence period is a period of exacerbation of neurological symptoms that persists for more than 24 hours at the time of recurrence, and there is a stable period of 1 month or more between recurrences, and the remission period is 3 to 6 months after the recurrence period treatment In this period, there was no recurrence during that period, and there was no next recurrence within 3 months after the recurrence.

(2) Measurement method It was determined by the same method as in Example 2.
(3) Results The obtained data are shown in FIG.

From the results in Table 2 and FIG. 3, in all six cases of NMO, when the relapse phase and the remission phase were compared, the peak intensity was higher in the relapse phase (mean: relapse phase 44.1 arb.u, Remission period 37.2 arb.u). When the change of 1741 peptide was tested in the relapse phase and the remission phase for each case by the Wilcoxson signed rank sum test, a significant increase was recognized in the relapse phase (p = 0.03).

  With the measurement method of the present invention, it is possible to easily determine the remission phase and the relapse phase appearing in multiple sclerosis and NMO using a biological sample such as a blood sample of a patient. Furthermore, multiple sclerosis and NMO itself can be easily determined by the measurement method of the present invention.

It is the graph which compared the 1741 peptide value of a normal person, a multiple sclerosis patient, and a NMO patient. It is the graph which compared the 1741 peptide value in the relapse phase of a multiple sclerosis patient, and a remission phase. It is the graph which compared the 1741 peptide value in the relapse period and remission period of a NMO patient.

Claims (8)

Recurrence of multiple sclerosis or NMO characterized by measuring the amount of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in a blood sample derived from a patient with multiple sclerosis or NMO (optic neuromyelitis) A method for measuring a peptide marker for determining a period and a remission period. Measuring the peptide value by a mass spectrometer, the measuring method according to claim 1. The measurement method according to claim 2 , wherein the mass spectrometer is a laser ionization time-of-flight mass spectrometer. Measuring the amount of peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in the blood, determining the recurrence phase and remission phase of multiple sclerosis, or recurrence of NMO (optic neuromyelitis) A method for obtaining data to be used for the determination, wherein the period and the remission period are determined. Measuring the amount of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in blood, characterized in that multiple sclerosis and / or NMO (optic neuromyelitis) is determined, and the data used for the determination is How to get . And measuring the amount of peptide comprising an amino acid sequence represented by SEQ ID NO: 1 in the patient's blood to determine the relapses and remission in patients with multiple sclerosis, for use in the determination How to get data . Determining the relapse phase and remission phase of NMO (optic neuromyelitis) of a patient, characterized by measuring the amount of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in the blood of the patient , How to get data to use . The amount of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in the blood of the patient is traced and measured. If the value is high, it is determined as a relapse phase, and if it is low, it is determined as a remission phase. The method of obtaining the data used for the determination, or the relapse phase of NMO (optic neuromyelitis), wherein the relapse phase and remission phase of the multiple sclerosis of the patient according to claim 6 or 7 are determined. And a method of obtaining data used for the determination.