WO2016117618A1 - 慢性炎症性脱髄性多発神経炎の診断方法、キット及びバイオマーカー - Google Patents
慢性炎症性脱髄性多発神経炎の診断方法、キット及びバイオマーカー Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- 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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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/285—Demyelinating diseases; Multipel sclerosis
Definitions
- the present invention relates to a diagnostic method for specifically diagnosing chronic inflammatory demyelinating polyneuritis, a kit for use in the diagnosis, and a biomarker.
- Chronic inflammatory demyelinating polyneuritis (also referred to as CIDP) is chronic progressive or stepped, recurrent left and right symmetrical limb muscle weakness over 2 months And peripheral neurological diseases mainly having sensory disorders.
- the etiology of CIDP is thought to be an autoimmune disease caused by immune abnormalities against components of peripheral nerve myelin, but details are unknown.
- EFNSPNS EFNSPNS are often used for the diagnosis of CIDP, which was revised in 2010. Symptoms, electrophysiological standards, and other findings such as cerebrospinal fluid findings and nerve root thickening by MRI.
- CIDP is assumed to be a syndrome including various pathologies because its course, treatment responsiveness, and prognosis vary from case to case, and it is an urgent task to establish a treatment method according to each pathology. is there.
- Non-patent Document 1 serum from cases of central / peripheral demyelination (CCPD), which is a rare disease causing demyelination of both the central nervous system and the peripheral nervous system. It has been found that anti-neurofacin 155 (NF155) antibody is positive (Non-patent Document 1). It has also been reported that anti-NF155 antibody becomes positive in a small part (about 4%) of CIDP (Non-Patent Document 2 and Non-Patent Document 3).
- CIDP diagnosis is based on clinical symptoms, electrophysiological criteria, and other findings such as cerebrospinal fluid findings and nerve root thickening by MRI. Biomarkers with high specificity for CIDP are still reported. It has not been. Moreover, CIDP is assumed to be a syndrome including various pathological conditions, and it is an urgent task to establish biomarkers and treatment methods corresponding to the respective pathological conditions.
- GBS Guillain-Barre syndrome
- CCPD central nerve lesion
- MS multiple sclerosis
- IFN ⁇ Interferon beta
- the present invention provides a method for diagnosing CIDP, in particular, a diagnostic method for specifically diagnosing a group having a specific pathophysiology from CIDP, a kit and a biomarker for use in the diagnosis. With the goal.
- the present inventors have found that an antibody that reacts with NF155 exists in some specimens of CIDP patients, and has found that CIDP can be diagnosed by measuring the antibody that reacts with NF155. That is, the inventors have found that by measuring an antibody that reacts with NF155, it is possible to establish a treatment method corresponding to each pathological condition for CIDP including various pathological conditions, thereby completing the present invention.
- the present invention relates to the following (1) to (15).
- (1) A method for diagnosing chronic inflammatory demyelinating polyneuritis, comprising a step of measuring anti-neurofacin 155 antibody contained in a specimen.
- (2) The diagnostic method according to (1), further comprising a step of measuring anti-neurofacin 186 antibody contained in the specimen.
- (3) The diagnostic method according to (2), comprising a step of detecting an antibody that reacts with neurofacin 155 but does not react with neurofacin 186.
- a cell in which neurofacin 155 is forcibly expressed and a cell in which neurofacin 186 is forcibly expressed are brought into contact with a specimen, and the presence of anti-neurofacin 155 antibody and / or anti-neurofacin 186 antibody is fluorescently labeled.
- the diagnostic method according to (4) which is carried out by flow cytometry.
- a method for measuring anti-neurofacin 155 antibody and / or anti-neurofacin 186 antibody in a specimen comprising a cell in which neurofacin 155 is forcibly expressed, a cell in which neurofacin 186 is forcibly expressed, a specimen, And then measuring anti-neurofacin 155 antibody and anti-neurofacin 186 antibody using fluorescently labeled anti-human IgG antibody.
- the method according to (8) which is performed by a flow cytometry method.
- (11) The method according to any one of (8) to (10), wherein the specimen is blood or cerebrospinal fluid.
- a diagnostic kit for chronic inflammatory demyelinating polyneuritis comprising a cell line in which neurofacin 155 is forcibly expressed.
- CIDP can be diagnosed by detecting an anti-NF155 antibody in a specimen.
- patients with the same pathophysiology are determined by measuring anti-NF155 antibody and anti-NF186 antibody in samples of inflammatory demyelinating disease cases and extracting positive examples that react only with NF155. A group can be extracted.
- CIDP which was a group of diseases having various pathological conditions, had various therapeutic effects in each case, but by selecting cases having anti-NF155 antibody, a more appropriate treatment method can be provided. become.
- identification with GBS and CIDP and identification with MS and CCPD are also possible.
- anti-NF155 specific antibody in the specimen (for example, serum or cerebrospinal fluid), it becomes an index for diagnosis and treatment effect determination. If the significance of anti-NF155 antibody is recognized worldwide, anti-NF155 antibody-positive neuropathy may be established as a disease concept independent of CIDP, as is anti-MAG antibody-positive neuropathy.
- FIG. 1A is a view showing the results of anti-NF155 antibody measurement by flow cytometry.
- FIG. 1B is a diagram showing the relationship between the serum dilution rate of anti-NF155 antibody positive CIDP patients and delta MFI value (top) and the relationship between the serum dilution rate of anti-NF155 antibody positive CIDP patients and MFI ratio (bottom). is there.
- the dilution ratio of serum is 1:20, 1: 100, 1: 200, 1: 400, 1: 800, 1: 1600, 1: 3200, 1: 6400, 1: 12800.
- 2A and 2B are immunohistological results when anti-NF155 antibody positive or negative CIDP patient sera were reacted with cells expressing human NF155 or human NF186.
- FIG. 3 is a view showing an IgG subclass of anti-human NF155 antibody contained in the serum of a patient.
- 4A to 4D are diagrams showing the results of detection of anti-NF155 antibody and anti-NF186 antibody for each case.
- FIG. 5A is a diagram showing cervical MRI of 7 anti-NF155 antibody positive patients
- FIG. 5B is a diagram showing lumbosacral nerve root MRI of 7 anti-NF155 antibody positive patients. 5A and 5B, the left end is a normal control.
- FIGS. 6A to 6C are diagrams showing brain MRI of anti-NF155 antibody positive patients.
- 6A and 6B show a demyelinating lesion by cerebral horizontal sectioning
- FIG. 6C shows a cerebral sagittal section and the lesion of FIG. 6A.
- FIG. 7 is a diagram showing the positioning of anti-human NF155-specific antibodies in CIDP.
- CIDP chronic inflammatory demyelinating polyneuritis
- CIDP chronic inflammatory demyelinating polyneuritis
- CIDP that can be diagnosed by the present invention is a syndrome including various pathologies as described above, and can be broadly divided into typical CIDP and atypical CIDP.
- Typical CIDP exhibits a symmetric motor sensory disorder that progresses for more than two months, with proximal and distal muscles simultaneously affected, and tendon reflexes reduced and disappeared in the extremities.
- the cranial nerves may be damaged.
- Examples of atypical CIDP include DADS, MADASAM, focal type, pure sensory type, pure Motor type, and the like.
- the diagnostic method of the present invention it is possible to extract a disease having a uniform or related pathophysiology from such CIDP cases in which a number of heterogeneous diseases are mixed. Thereby, a more appropriate treatment method can be provided.
- the subject to be diagnosed is any animal that can suffer from chronic inflammatory demyelinating polyneuritis (CIDP), such as humans, non-human primates, dogs, cats, rabbits, rats, or mice. . In the following, humans will be described, but the same applies to other animals.
- CIDP chronic inflammatory demyelinating polyneuritis
- anti-NF155 antibody and anti-NF186 antibody refer to human NF155 and human NF186. It means an antibody that binds to NF186.
- Neurofacin NF155 is a protein with a molecular weight of 155 kDa that is localized on the myelin side of the paranode.
- oligodendrocyte membrane processes surround the axon like multiple loops to form a myelin sheath, and that part takes the structure of an insulator It contributes to the jump conduction of electrical signals in nerve axons.
- the axon-myelin bonding part is divided into a paranode, a paraparanode, and an internode.
- Neurofacin NF186 is a protein with a molecular weight of 186 kDa that is accumulated in the diaphragm part. A space between adjacent myelin sheaths is called a “narrow ring” (node).
- the specimen to be diagnosed in the present invention may be any of blood (whole blood, serum, plasma), saliva, spinal fluid, other body fluids, various tissues, etc. to be diagnosed.
- the specimen is preferably serum or cerebrospinal fluid.
- the method for measuring each antibody in the specimen is not particularly limited as long as it is a method used for detecting and measuring antibodies as an immunological measurement method.
- any conventional measurement method using an enzyme, a fluorescent substance, a radioactive substance, a colored substance or the like as a labeling substance can be used, and a flow cytometry method can be preferably used.
- the method for diagnosing chronic inflammatory demyelinating polyneuritis (CIDP) of the present invention includes a step of measuring an anti-NF155 antibody contained in a specimen. Moreover, it is preferable that the diagnostic method for chronic inflammatory demyelinating polyneuritis of the present invention further includes a step of measuring an anti-NF186 antibody contained in the specimen. When the anti-NF155 antibody is positive, the anti-NF186 antibody contained in the sample is measured. If the anti-NF186 antibody is negative, the anti-NF155 antibody binds to a specific epitope of NF155 that is not present in NF186. This is because it is possible to diagnose chronic inflammatory demyelinating polyneuritis (CIDP) more reliably.
- the flow cytometry method In order to measure the anti-NF155 antibody and the anti-NF186 antibody, it is preferable to use the above-described flow cytometry method.
- measurement can be performed using a cell in which NF155 is forcibly expressed, a cell in which NF186 is forcibly expressed, and a secondary antibody.
- limit especially as a secondary antibody The fluorescence labeled antibody as mentioned in an Example is preferable, and the fluorescence labeled anti-human IgG antibody is further more preferable.
- the flow cytometry method can be easily performed by a known method.
- a fluorescently labeled anti-human IgG antibody is used.
- an antigen-antibody reaction is performed.
- the number of cells with fluorescently labeled antibodies bound to the cells via the antibodies contained in the specimen can be counted. Thereby, it is possible to determine whether an antibody is present in the specimen.
- Flow cytometry apparatus and necessary reagents are commercially available and can be easily implemented by those skilled in the art.
- cells in which NF155 or NF186 is forcibly expressed are cells suitable for expressing NF155 or NF186 as vectors containing human NF155 or NF186 cDNA. It can produce by introduce
- NF155 and NF186 are forcibly expressed as antigens, respectively, and it is found that an antibody that reacts with NF155 is likely to be positive for CIDP.
- anti-NF155 antibody-positive CIDP cases are juvenile onset, resulting in marked demyelination (delay of nerve conduction) from the peripheral nerve distal part and nerve root, and MRI with a high degree of nerve root It has been found that it exhibits a characteristic pathological manifestation of thickening and a high rise in cerebrospinal fluid protein. Since this type of CIDP exhibits irreversible remarkable nerve thickening from the beginning of the onset, if the anti-NF155 antibody is positive from the first onset, it is an indication for aggressive immunotherapy.
- anti-NF155 antibody positive CIDP is mainly composed of IgG4 as the subclass of anti-NF155 antibody
- the result of measurement of the IgG subclass of anti-NF155 antibody is that if it is mainly composed of IgG4, it tends to cause thickening of the nerve. It can be introduced from the beginning.
- IgG4 is mainly means that the MFI ratio and delta MFI are the highest among IgG subclasses.
- CIDP Guillain-Barre syndrome
- GBS Guillain-Barre syndrome
- IVIg intravenous immunoglobulin therapy
- PE simple plasma exchange membrane
- corticosteroids other immunity Continued treatment with inhibitors is needed.
- anti-NF155 antibody positive CIDP is associated with a central nerve lesion, and in this case, it is also included in the category of CCPD and is considered to constitute a series of spectrums of anti-NF155 antibody positive CIDP / CCPD.
- MS multiple sclerosis
- IFN ⁇ interferon beta
- the present invention is also a method for measuring an anti-NF155 antibody and / or an anti-NF186 antibody in a specimen, comprising contacting a specimen with a cell in which NF155 is forcibly expressed and a cell in which NF186 is forcibly expressed; Methods are provided for measuring the presence of anti-NF155 and anti-NF186 antibodies using fluorescently labeled anti-human IgG antibodies.
- the specimen, cells forcibly expressing NF155, cells forcibly expressing NF186, etc. are as described above. According to this method, it is possible to determine whether or not NF155 and / or NF186 is present in a specimen, which is useful for diagnosis of chronic inflammatory demyelinating polyneuritis. Moreover, according to this method, Guillain-Barre syndrome (GBS) and multiple sclerosis (MS) can be distinguished from chronic inflammatory demyelinating polyneuritis (CIDP).
- GBS Guillain-Barre syndrome
- MS multiple sclerosis
- the diagnostic kit for chronic inflammatory demyelinating polyneuritis of the present invention includes a cell line in which NF155 is forcibly expressed. Moreover, the cell line which forcedly expressed NF186 may be further included.
- the method for diagnosing chronic inflammatory demyelinating polyneuritis and the method for measuring the presence of anti-NF155 antibody and anti-NF186 antibody of the present invention as described above can be easily carried out. Is possible.
- the diagnostic kit for chronic inflammatory demyelinating polyneuritis of the present invention may further comprise a fluorescently labeled anti-human IgG antibody.
- a fluorescently labeled anti-human IgG antibody By including such a fluorescently labeled anti-human IgG antibody, the flow cytometry method as described above allows the diagnosis method for chronic inflammatory demyelinating polyneuritis of the present invention, anti-NF155 antibody and anti-NF186 antibody to A method of measuring presence can be implemented.
- the kit for diagnosing chronic inflammatory demyelinating polyneuritis of the present invention contains reagents necessary for performing the flow cytometry method. May be.
- the biomarker of the present invention comprises an anti-NF155 antibody and can be used for diagnosing CIDP.
- the following application examples can be considered for this anti-NF155 antibody.
- the biomarker of the present invention can be used not only for diagnosis of chronic inflammatory demyelinating polyneuritis, but also for screening for preventive or therapeutic agents for chronic inflammatory demyelinating polyneuritis.
- the amount of biomarker in the patient sample is compared before and after the administration of the drug. If the amount of biomarker after administration is lower than that before administration, the drug can be judged to be effective for the patient, and the amount of biomarker is the same before and after administration, or after administration If the amount of the biomarker is higher than before administration, it can be determined that the drug is ineffective for the patient.
- the biomarker of the present invention can be used as an indicator of the presence or absence or progression of chronic inflammatory demyelinating polyneuritis.
- the subject to be used as a specimen satisfies the electrophysiological diagnostic criteria of CIPF diagnostic criteria of EFNS / PNS among the patients who visited and admitted to Kyushu University Hospital from 2004 to 2014 with define.
- 50 CIDP cases were used.
- CCPD cases were treated as CIDP this time because they all had demyelinating peripheral neuropathy and met CIDP diagnostic criteria.
- As control groups there were used 32 cases of peripheral neuropathy other than CIDP including 32 cases of multiple sclerosis (MS), 26 cases of Guillain-Barre syndrome (GBS) and Fisher syndrome (FS), and 30 cases of healthy subjects (HCs). . Serum was collected from the above cases and used in the examples.
- Example 1 Preparation of cell line forcibly expressing human NF155 and cell line forcibly expressed human NF186
- a vector containing human NF155 cDNA and a vector containing human NF186 cDNA were purchased from Origen, respectively.
- a sequence encoding Turbo-GFP is incorporated at the C-terminus of each protein.
- the vector was then linearized using the restriction enzyme ScaI. Specifically, ScaI manufactured by Takara Bio Inc. was used as ScaI, and the reaction was performed as described in the package insert of Takara Bio Inc. Specifically, 2 ⁇ L of ScaI, 4 ⁇ L of 10 ⁇ H buffer, 1.5 ⁇ g of substrate DNA, and a solution of 40 ⁇ L in total with sterilized purified water were reacted at 37 ° C.
- the vector linearized as described above was transfected into HK293 cells according to the package insert by lipofection using Fugene 6 (Roche). Next, 1 mg / ml of G418 (Life Technologies) was added and cultured in DMEM to select a G418 resistant strain. Proliferated colonies were isolated using a cloning cylinder, and a cell line in which human NF155 was forcibly expressed and a cell line in which human NF186 cells were forcibly expressed were used.
- Example 2 Anti-NF155 Antibody Measurement Using Flow Cytometry Method Using FACS buffer 1 (DMEM, 1 mM EDTA, 1% FBS), HEK293 cells and NF155-expressing cells were mixed at 1 ⁇ 10 6 / ml. 47.5 ⁇ L of the cell solution was placed in a 96-well microtiter plate, and then 2.5 ⁇ L of CIDP patient serum was added and mixed (serum dilution ratio 1:20). Subsequently, the microtiter plate was incubated at 4 ° C.
- FACS buffer 1 DMEM, 1 mM EDTA, 1% FBS
- the presence or absence of the anti-NF186 antibody was evaluated by a flow cytometry method and an immunostaining method using an NF186-expressing cell line in a part of the anti-NF155 antibody positive.
- the cut-off values were 10 and 100 for the MFI ratio and the delta MFI ratio, respectively.
- FIG. 1A shows the result of anti-NF155 antibody measurement (Example 2) by flow cytometry.
- the vertical and horizontal axes of the graph indicate the fluorescence intensity of Alexa647 and Turbo-GFP, respectively.
- the diagram on the left side of FIG. 1A shows the results in a system without serum.
- HEK293 cells expressing human NF155 and HEK293 cells not expressing human NF155 could be separated into two cell populations by the fluorescence intensity of Turbo-GFP.
- the left figure is an example in which patient serum was not administered and only the secondary antibody was added, but the fluorescence intensity of Alexa647 is low in any cell population.
- the middle figure in FIG. 1A is a negative example.
- FIG. 1A shows a positive example.
- the MFI ratio and delta MFI were 48 and 276, respectively, and were significantly elevated compared to the negative example shown in the center diagram of FIG. 1A.
- Measurement was also performed using serially diluted serum. The result is shown in FIG. 1B. As is clear from the figure, it was confirmed that the value changed continuously.
- FIGS. 2A and 2B show immunohistological results when the serum of a CIDP patient positive or negative for anti-NF155 antibody was reacted with cells expressing human NF155 or human NF186. This shows that anti-NF155 antibody-positive CIDP patient sera reacted with cells expressing human NF155 rather than cells expressing human NF186.
- Example 3 Detection of anti-NF155 antibody IgG subclass Using FACS buffer 1 (DMEM, 1 mM EDTA, 1% FBS), HEK293 cells and NF155-expressing cells were mixed at 1 ⁇ 10 6 / ml. 47.5 ⁇ L of the cell solution was placed in a 96-well microtiter plate, and then 2.5 ⁇ L of patient serum was added and mixed (serum dilution ratio 1:20). Subsequently, the microtiter plate was incubated at 4 ° C. for 60 minutes and washed twice with 200 ⁇ L of FACS buffer 1, and then the following secondary antibody was mixed with the cells at a dilution ratio of 1: 500 to cause an antigen-antibody reaction. .
- FACS buffer 1 DMEM, 1 mM EDTA, 1% FBS
- PE-conjugated anti-IgG1 antibody (Cell Lab, 733179) PE-conjugated anti-IgG2 antibody (Cell Lab, 736408) PE-conjugated anti-IgG3 antibody (Cell Lab, 736487) PE-conjugated anti-IgG4 antibody (Cell Lab, 733219) Subsequently, after incubation at 4 ° C. for 60 minutes, the plate was washed twice with 200 ⁇ L of FACS buffer 2 (PBS, 5 mM EDTA), and the mean fluorescence intensity (MFI: Mean fluorescence Intensity) of the fluorescent secondary antibody (PE) in each cell group. ) Ratio (ratio) or difference (delta).
- Example 4 50 patients diagnosed with CIDP as defined by electrophysiological criteria using the same method described in Example 2; as a control group, 4 anti-NF155 antibody positive CIDP patients from other clinics, 32 MS, ON In 40 cases (GBS / FS, neuropathy due to vasculitis, POEMS, HMSN, and anti-MAG antibody positive neuropathy) and 30 cases of HCs (healthy subjects), anti-NF155 antibody was measured. The results are shown in FIG. 4 and Table 1 below. The overall positive rate was 18.0% (9/50). Among CIDP, the positive rate was 3/5 (60%) in the DADS type, but there were no positive cases in the other 9 atypical CIDPs.
- the positive rate was 16.7% (6/36).
- MS multiple sclerosis
- GBS Guillain-Barre syndrome
- FS Fisher syndrome
- HC healthy subjects
- Anti-NF186 antibody was negative in all cases.
- CIDP chronic inflammatory demyelinating polyneuritis
- DADS distal symmetrical chronic inflammatory demyelinating polyneuritis
- FS Fisher syndrome
- GBS Guillain-Barre syndrome
- HCs healthy control
- HMSN hereditary motor sensory neuropathy
- MADSSAM multifocal demyelinating sensorimotor type chronic inflammatory demyelinating polyneuritis
- MAG myelin related glycoprotein
- MS multiple sclerosis
- n number of positive cases
- N number of matched cases
- POEMS polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes (POEMS) syndrome.
- the anti-NF186 antibody was negative in all cases, because the difference between NF155 and NF186 is caused by alternative splicing, so the possible epitope is an amino acid sequence that is not in NF186 but in NF155. is assumed.
- Example 5 Comparison of clinical data was performed for anti-NF155 positive CIDP and negative cases. The results are shown in Table 2.
- FIG. 5A and FIG. 5B the result of a patient's nerve root MRI is shown to FIG. 5A and FIG. 5B.
- FIGS. 5A and 5B in 7 anti-NF155 antibody-positive CIDPs that were able to be imaged, remarkable thickening of the cervical and lumbar nerve roots and the proximal peripheral nerve was observed, and the cerebrospinal fluid protein was It was found that it has a characteristic image of rising to a high degree.
- brain MRI was imaged and the results are shown in FIGS. 6A to 6C. As shown in FIGS. 6A to 6C, there were cases in which demyelinating lesions were observed in brain MRI.
- CIDP chronic inflammatory demyelinating polyneuritis
- DADS distal symmetrical chronic inflammatory demyelinating polyneuritis
- MADSAM multifocal demyelinating sensorimotor type chronic inflammatory demyelinating Polyneuritis
- n number of related cases
- N number of matched cases
- NS not significant
- SD standard deviation.
- Example 6 Peripheral nerve conduction tests were performed on anti-NF155 antibody positive CIDP and negative cases.
- Table 3 shows the results of comparison of NCS (nerve conduction velocity) findings of anti-NF155 antibody positive CIDP and negative cases.
- CIDP chronic inflammatory demyelinating polyneuritis
- CMAP composite muscle action potential
- MCV motor nerve conduction velocity
- N number of nerves examined
- NF neurofasine
- SCV sensory Nerve conduction velocity
- SNAP sensory nerve action potential
- TLI terminal lateness index
- n number of related cases
- N number of matched cases
- NS not significant
- SD standard deviation. All continuous variables are expressed as mean ⁇ SD, and the number of nerves induced / number of nerves performed is listed in parentheses.
- Distal latency reference value median nerve, 3.49 ⁇ 0.34 ms; ulnar nerve, 2.59 ⁇ 0.39 ms; tibial nerve, 3.96 ⁇ 1.00 ms.
- Reference value MCV of MCV median nerve, 57.7 ⁇ 4.9 m / s; ulnar nerve, 58.7 ⁇ 5.1 m / s; tibial nerve, 48.5 ⁇ 3.6 m / s.
- Reference values for CMAP amplitude median nerve, 7.0 ⁇ 3.0 mV; ulnar nerve, 5.7 ⁇ 2.0 mV; tibial nerve, 5.8 ⁇ 1.9 mV.
- Reference value for F wave latency median nerve, 26.2 ⁇ 2.2 ms; ulnar nerve, 27.6 ⁇ 2.2 ms; tibial nerve, 47.7 ⁇ 5.0 ms.
- Upper normal limit of distal latency median nerve, 4.2 ms; ulnar nerve, 3.4 ms; tibial nerve 6.0 ms.
- Normal lower limit of MCV median nerve, 48 m / s; ulnar nerve, 49 m / s; tibial nerve, 41 m / s.
- Normal lower limit of CMAP amplitude median nerve, 3.5 mV; ulnar nerve, 2.8 mV; tibial nerve, 2.9 mV.
- Normal upper limit of F wave latency median nerve, 31 ms; ulnar nerve, 32 ms; tibial nerve, 58 ms.
- Normal lower limit of SCV median nerve, 44 m / s; ulnar nerve, 44 m / s; sural nerve, 45 m / s.
- the anti-NF155 positive CIDP significantly increased the distal latency in the ulnar and tibial nerves and the F wave latency in the median, ulnar and tibial nerves compared to the negative CIDP. I found out.
- CCPD central and peripheral demyelination
- CIDP chronic inflammatory demyelinating polyneuritis
- MS multiple sclerosis
- CIDP and Guillain-Barre Syndrome can be distinguished by the presence or absence of anti-NF155 antibody in the sample. That is, in the past, Guillain-Barre syndrome, which is a demyelinating disease that acutely affects peripheral nerves, was difficult to differentiate from CIDP at the time of the first attack, but the above results indicate that the anti-NF155 antibody of the present invention is positive. If present, it was found that the possibility of recurrence and CIDP was high, and the measurement of anti-NF155 antibody was found to be useful in the differentiation between GBS and recurrent CIDP.
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Abstract
Description
(1)検体中に含まれる抗ニューロファシン155抗体を測定する工程を含む、慢性炎症性脱髄性多発神経炎の診断方法。
(2)検体中に含まれる抗ニューロファシン186抗体を測定する工程を更に含む、(1)に記載の診断方法。
(3)ニューロファシン155とは反応するが、ニューロファシン186とは反応しない抗体を検出する工程を含む、(2)に記載の診断方法。
(4)ニューロファシン155を強制発現させた細胞及びニューロファシン186を強制発現させた細胞と、検体とを接触させ、抗ニューロファシン155抗体及び/又は抗ニューロファシン186抗体の存在を、蛍光標識された抗ヒトIgG抗体を用いて測定する、(1)~(3)のいずれか1に記載の診断方法。
(5)フローサイトメトリー法により行う、(4)に記載の診断方法。
(6)ギランバレー症候群又は多発性硬化症と、慢性炎症性脱髄性多発神経炎とを識別する、(1)~(5)のいずれか1に記載の診断方法。
(7)検体が血液または髄液である、(1)~(6)のいずれか1に記載の診断方法。
(8)検体中の抗ニューロファシン155抗体及び/又は抗ニューロファシン186抗体を測定する方法であって、ニューロファシン155を強制発現させた細胞及びニューロファシン186を強制発現させた細胞と、検体とを接触させた後、蛍光標識された抗ヒトIgG抗体を用いて、抗ニューロファシン155抗体及び抗ニューロファシン186抗体を測定する方法。
(9)フローサイトメトリー法により行う、(8)に記載の方法。
(10)抗ニューロファシン155抗体が存在するが、抗ニューロファシン186抗体が存在しない検体を選択することを含む、(8)又は(9)に記載の方法。
(11)検体が血液または髄液である、(8)~(10)のいずれか1項に記載の方法。
(12)ニューロファシン155を強制発現させた細胞株を含む、慢性炎症性脱髄性多発神経炎診断用キット。
(13)ニューロファシン186を強制発現させた細胞株を更に含む(12)に記載のキット。
(14)蛍光標識された抗ヒトIgG抗体を更に含む、(12)又は(13)に記載のキット。
(15)抗ニューロファシン155抗体からなる、慢性炎症性脱髄性多発神経炎を診断するためのバイオマーカー。
本発明は、慢性炎症性脱髄性多発神経炎(CIDP)を診断する方法を提供する。本発明で診断できるCIDPとは、上述したように様々な病態を含む症候群であり、典型的CIDPと、非典型的CIDPに大きく分けられる。典型的CIDPは、2カ月以上進行する対称性の運動感覚障害を呈し、近位筋と遠位筋が同時に侵され、腱反射は四肢で低下・消失する。脳神経が障害されることがある。非典型的CIDPとしては、DADS、MADSAM、focal type、pure sensory type、pure Motor type等が挙げられる。
[診断対象]
本発明の診断方法において、診断対象はヒト、非ヒト霊長類、イヌ、ネコ、ウサギ、ラット、又はマウスなど、慢性炎症性脱髄性多発神経炎(CIDP)に罹患し得る任意の動物とする。以下、ヒトを対象として説明するが、他の動物においても同様である。
ニューロファシンNF155(NF155)はパラノード(paranode)の髄鞘側に局在する、155kDaの分子量のタンパク質である。末梢神経系においてはシュワン細胞、中枢神経系においてはオリゴデンドロサイトの細胞膜突起が幾重にもループのように軸索を取り巻くことで髄鞘が形成され、その部分が絶縁体の構造をとることで神経軸索における電気的信号の跳躍伝導に寄与している。軸索と髄鞘の接着部分はパラノード(paranode)、傍パラノード(juxtaparanode)、インターノード(internode)に分けられる。
ニューロファシンNF186(NF186)は、絞輪部に集積している186kDaの分子量のタンパク質である。隣り合う髄鞘の間は絞輪部(ノード:node)と呼ばれる。
本発明において診断対象とする検体は、診断対象の血液(全血、血清、血漿)、唾液、髄液、その他の体液、各種組織等のいずれでもよい。検体は血清または髄液が好ましい。
検体中の各抗体の測定方法は、免疫学的測定法として、抗体を検出し測定するために使用される方法であれば特に制限されない。例えば、酵素、蛍光物質、放射性物質、着色物質などを標識物質とする慣用の測定方法がいずれも使用可能であり、フローサイトメトリー法を好ましく使用することができる。
本発明の慢性炎症性脱髄性多発神経炎(CIDP)の診断方法は、検体中に含まれる抗NF155抗体を測定する工程を含む。また、本発明の慢性炎症性脱髄性多発神経炎の診断方法は、検体中に含まれる抗NF186抗体を測定する工程を更に含んでいることが好ましい。抗NF155抗体が陽性である場合に、検体中に含まれる抗NF186抗体を測定し、抗NF186抗体が陰性であれば、抗NF155抗体はNF186にはないNF155の特定のエピトープに結合していることがわかり、より確実に慢性炎症性脱髄性多発神経炎(CIDP)と診断することが可能であるからである。
次に、本発明のキットについて説明する。
本発明の慢性炎症性脱髄性多発神経炎診断用キットは、NF155を強制発現させた細胞株を含む。また、NF186を強制発現させた細胞株を更に含んでいてもよい。
このようなキットを用いることにより、上述したような、本発明の慢性炎症性脱髄性多発神経炎の診断方法、抗NF155抗体及び抗NF186抗体の存在を測定する方法を容易に実施することが可能である。
上記のように、フローサイトメトリーにより、上記方法を実施するために、本発明の慢性炎症性脱髄性多発神経炎診断用キットは、フローサイトメトリー法を実施するのに必要な試薬を含んでいてもよい。
次に、本発明のバイオマーカーについて説明する。本発明のバイオマーカーは、抗NF155抗体からなり、CIDPを診断するために用いることができる。この抗NF155抗体には、例えば以下のような応用例が考えられる。
また、本発明のバイオマーカーは、慢性炎症性脱髄性多発神経炎の有無又は進行度の指標として用いることができる。
上記症例から血清を採取し、実施例において使用した。CIDP症例50名のうち、典型的な症例は36名、非典型的症例は14名であり、14名の内、DADSは5名、多巣性脱髄性感覚運動型(multifocal acquired demyelinating sensory)(MADSAM)は4名、限局型が2名、純粋運動型が1名、純粋感覚型が2名であった。次いで、他の診療所からの抗NF155抗体を有する4名の患者を加え、それぞれの臨床兆候により評価した。結果は、典型的症例が37名、非典型的症例が17名であり、そのうち、DADSが8名、MADSAMは4例、限局型が2名、純粋運動型が1名、純粋感覚型が2名であった。合計54名のうち、抗NF155を有しないCIDP患者は41名であり、抗NF155抗体を有する患者は13名であった。
ヒトNF155を強制発現させた細胞株、及びヒトNF186を強制発現させた細胞株の作製
ヒトNF155cDNAが含まれるベクター、及びヒトNF186cDNAが含まれるベクターは、それぞれOrigene社から購入した。いずれのベクターにもTurbo-GFPをコードする配列が各蛋白のC末端に組み込まれている。
次いで、上記ベクターを、制限酵素ScaIを使用して直鎖化した。具体的には、ScaIとしてはタカラバイオ株式会社製のScaIを用い、反応は、タカラバイオ株式会社の添付文書に記載された通りに行った。具体的には、ScaI 2μL、10×Hバッファー 4μL、基質DNA 1.5μg、滅菌精製水で合計40μLの溶液とし、37℃にて反応を行った。
フローサイトメトリー法を利用した抗NF155抗体測定
FACSバッファー1(DMEM、1mM EDTA、1%FBS)を用い、HEK293細胞及びNF155発現細胞をそれぞれ1×106/mlとなるように混合した。96穴のマイクロタイタープレートに47.5μLずつ細胞溶液を入れ、次いでCIDP患者血清2.5μLを加えて混合した(血清希釈倍率1:20)。次いで、マイクロタイタープレートを4℃で60分間インキュベーションし、200μLのFACSバッファー1で2回洗浄した後、2次抗体(Alexa 647-conjugated anti-human IgG antibody、Life Ttechnologies社製)(希釈倍率1:500)と抗原抗体反応を起こさせた後、4℃で60分間インキュベーションした。次いで、200μLのFACSバッファー2(PBS、5mM EDTA)で2回洗浄し、それぞれの細胞群における蛍光2次抗体(Alexa 647)の平均蛍光強度(MFI:Mean fluorescence Intensity)の比(ratio)または差(delta)を評価した
上記と同様の方法で、抗NF155抗体陽性の一部において、抗NF186抗体の有無を、NF186発現細胞株を用いたフローサイトメトリー法及び免疫染色法で評価した。
なお、カットオフ値は、MFI比、delta MFI比について、それぞれ10及び100とした。
フローサイトメトリー法による、抗NF155抗体測定(実施例2)の結果を図1Aに示す。グラフの縦軸及び横軸は、それぞれAlexa647及びTurbo-GFPの蛍光強度を示す。図1Aの左側の図は、血清を含まない系での結果を示す。図1Aから明らかなように、ヒトNF155を発現しているHEK293細胞と発現していないHEK293細胞はTurbo-GFPの蛍光強度により2つの細胞集団に分離することができた。左図は患者血清を投与せず、二次抗体のみ付加した例であるが、いずれの細胞集団もAlexa647の蛍光強度は低い値であることを示している。
図1Aの中央の図は陰性例である。血清を投与することにより、いずれの細胞集団も非特異的にAlexa647の蛍光強度が上昇しているが、MFI ratio及びdelta MFIの値は、それぞれ1.37及び1.88であり、いずれも低い値であった。
また、図1Aの右側の図は陽性例を示す。MFI比及びdelta MFIはそれぞれ48及び276であり、図1Aの中央の図に示す陰性例と比較して顕著に上昇していた。
また、段階希釈した血清を用いて測定も行った。その結果を図1Bに示す。図から明らかなように連続的に値が変化することを確認した。
また、図2A及び図2Bは、抗NF155抗体陽性又は陰性のCIDP患者の血清を、ヒトNF155またはヒトNF186を発現する細胞と反応させたときの免疫組織学的結果である。抗NF155抗体陽性のCIDP患者の血清が、ヒトNF186を発現する細胞ではなく、ヒトNF155を発現する細胞で反応したことを示している。
抗NF155抗体IgGサブクラスの検出
FACSバッファー1(DMEM、1mM EDTA、1%FBS)を用い、HEK293細胞及びNF155発現細胞をそれぞれ1×106/mlとなるよう混合した。96穴のマイクロタイタープレートに47.5μLずつ細胞溶液を入れ、次いで患者血清2.5μLを加えて混合した(血清希釈倍率1:20)。次いで、マイクロタイタープレートを4℃で60分間インキュベーションし、200μLのFACSバッファー1で2回洗浄した後、以下の2次抗体を希釈倍率1:500で細胞と混合し、抗原抗体反応を起こさせた。
PE-conjugated anti-IgG1 antibody(Cell Lab、733179)
PE-conjugated anti-IgG2 antibody(Cell Lab、736408)
PE-conjugated anti-IgG3 antibody(Cell Lab、736487)
PE-conjugated anti-IgG4 antibody(Cell Lab、733219)
次いで、4℃で60分間インキュベーションした後、200μLのFACSバッファー2(PBS、5mM EDTA)で2回洗浄し、それぞれの細胞群における蛍光2次抗体(PE)の平均蛍光強度(MFI:Mean fluorescence Intensity)の比(ratio)もしくは差(delta)で評価した。
結果を図3に示す。図3に示されるように、実験を行った13例全てにおいて、IgG4が主体であることが確認できた。一方、抗NF155抗体に陽性であったGBS症例においては、IgG4が主体ではなかった。以上の結果により、抗NF155抗体のIgGサブクラスを検出することで、より高確率にCIDPの診断を行うことができることが分かった。
実施例2に記載した同様の方法を用いて、電気生理学的基準による定義によってCIDPと診断された50例、対照群として、他の診療所の抗NF155抗体陽性CIDP患者4例、MS32例、ON(GBS/FS、血管炎によるニューロパチー、POEMS、HMSN、及び抗MAG抗体陽性ニューロパチー)40例、及びHCs(健常者)30例において、抗NF155抗体の測定を行った。結果を図4および下記表1に示す。全体の陽性率は18.0%(9/50)であった。CIDPの中では、DADS型で3/5(60%)と高い陽性率であったが、それ以外の9例の非典型的CIDPでは陽性例はなかった。典型的CIDPでは、16.7%(6/36)の陽性率であった。対象群では、多発性硬化症(MS)で0%(0/32)、ギランバレー症候群(GBS)・フィッシャー症候群(FS)で3.8%(1/26)、健常者(HC)で0%(0/30)であった。
また、抗NF186抗体はいずれの症例においても陰性であった。
抗NF155陽性CIDP及び陰性例について、臨床データの比較を行った。その結果を表2に示す。抗NF155抗体陽性CIDP(n=13)では陰性CIDP(n=41)と比較して、発症年齢が低い(47.9±17.0に対して25.2±10.7、p<0.0001)、末梢優位に筋力低下が目立つDADS型が多い(4.9%に対して46.2%、p=0.0014)、下垂足を呈する割合が多い(31.7%に対して69.2%、p=0.0242)、歩行障害の割合が多い(73.2%に対して100%、p=0.0484)、振戦の割合が多い(19.5%に対して53.8%、p=0.0300)、髄液蛋白値が高い(103.8±75.8に対して317.0±141.1、p<0.0001)という特徴がみられた。
また、患者の神経根MRIの結果を図5A及び図5Bに示す。図5A及び図5Bから明らかなように、撮像し得た7例の抗NF155抗体陽性CIDPおいて、いずれも頚部および腰部神経根、近位末梢神経の顕著な肥厚が認められ、髄液蛋白が高度に上昇するという特徴的な病像を呈することがわかった。
また、脳MRIについても撮像し、その結果を図6A~図6Cに示す。図6A~図6Cに示すように、脳MRIについては脱髄性病変を認める症例もみられた。
抗NF155抗体陽性CIDP及び陰性例において、末梢神経伝導検査を行った。
抗NF155抗体陽性CIDP及び陰性例のNCS(神経伝導速度)所見の比較を行った結果を表3に示す。
全ての連続変数は平均±SDで示し,括弧内には誘発された神経の数/施行した神経の数を記載している.
Claims (15)
- 検体中に含まれる抗ニューロファシン155抗体を測定する工程を含む、慢性炎症性脱髄性多発神経炎の診断方法。
- 検体中に含まれる抗ニューロファシン186抗体を測定する工程を更に含む、請求項1に記載の診断方法。
- ニューロファシン155とは反応するが、ニューロファシン186とは反応しない抗体を検出する工程を含む、請求項2に記載の診断方法。
- ニューロファシン155を強制発現させた細胞及びニューロファシン186を強制発現させた細胞と、検体とを接触させ、抗ニューロファシン155抗体及び/又は抗ニューロファシン186抗体の存在を、蛍光標識された抗ヒトIgG抗体を用いて測定する、請求項1~3のいずれか1項に記載の診断方法。
- フローサイトメトリー法により行う、請求項4に記載の診断方法。
- ギランバレー症候群又は多発性硬化症と、慢性炎症性脱髄性多発神経炎とを識別する、請求項1~5のいずれか1項に記載の診断方法。
- 検体が血液または髄液である、請求項1~6のいずれか1項に記載の診断方法。
- 検体中の抗ニューロファシン155抗体及び/又は抗ニューロファシン186抗体を測定する方法であって、ニューロファシン155を強制発現させた細胞及びニューロファシン186を強制発現させた細胞と、検体とを接触させた後、蛍光標識された抗ヒトIgG抗体を用いて、抗ニューロファシン155抗体及び抗ニューロファシン186抗体を測定する方法。
- フローサイトメトリー法により行う、請求項8に記載の方法。
- 抗ニューロファシン155抗体が存在するが、抗ニューロファシン186抗体が存在しない検体を選択することを含む、請求項8又は9に記載の方法。
- 検体が血液または髄液である、請求項8~10のいずれか1項に記載の方法。
- ニューロファシン155を強制発現させた細胞株を含む、慢性炎症性脱髄性多発神経炎診断用キット。
- ニューロファシン186を強制発現させた細胞株を更に含む、請求項12に記載のキット。
- 蛍光標識された抗ヒトIgG抗体を更に含む、請求項12又は13に記載のキット。
- 抗ニューロファシン155抗体からなる、慢性炎症性脱髄性多発神経炎を診断するためのバイオマーカー。
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CN109734792B (zh) * | 2019-01-17 | 2022-05-24 | 武汉明德生物科技股份有限公司 | 人cntn1抗原、人cntn1抗体检测试剂盒及其制备方法与应用 |
CN109810184B (zh) * | 2019-01-17 | 2022-07-12 | 武汉明德生物科技股份有限公司 | 人nf155抗原、人nf155抗体检测试剂盒及其制备方法与应用 |
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FRANCE SCA NOTTURNO ET AL.: "Autoantibodies to neurofascin-186 and gliomedin in multifocal motor neuropathy", JOURNAL OF NEUROIMMUNOLOGY, vol. 276, no. Issuesl-2, 15 November 2014 (2014-11-15), pages 207 - 212 * |
JUDY KING MAN NG ET AL.: "Neurofascin as a target for autoantibodies in peripheral neuropathies", NEUROLOGY, vol. 79, no. 23, pages 2241 - 2248 * |
LUIS QUEROL ET AL.: "Neurofascin IgG4 antibodies in CIDP associate with disabling tremor and poor response to IVIg", NEUROLOGY, vol. 82, no. 10, 11 March 2014 (2014-03-11), pages 879 - 886 * |
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US10509033B2 (en) | 2019-12-17 |
JPWO2016117618A1 (ja) | 2017-11-30 |
US20180074051A1 (en) | 2018-03-15 |
JP6751025B2 (ja) | 2020-09-02 |
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