Biomarker, diagnostic kit and method of assisting in diagnosis

Abstract

To provide a biomarker, a diagnostic kit, and a method of assisting in diagnosis for the specific diagnosis of a group having specific disease state physiology in acute and chronic inflammatory demyelinating peripheral neuropathy with unknown autoantibody.SOLUTION: There are provided a biomarker, a diagnostic kit containing the same, and a method of assisting in diagnosis for using the same for diagnosis of inflammatory demyelinating polyneuropathy, including anti-LGI4 antibodies, or for determination of the efficacy of treatment, or the inflammatory demyelinating polyneuropathy being an autoimmune neuropathy.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to biomarkers that can be used for the specific diagnosis of acute and chronic inflammatory demyelinating peripheral neuropathy, diagnostic kits, and methods for assisting the diagnosis using the same. [Background technology]

[0002] Representative acute cases of inflammatory demyelinating peripheral neuropathy include Guillain-Barre syndrome (GBS), while chronic cases include chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy (MMN). In Japan, GBS occurs in 1.15 cases per 100,000 people, CIDP in 1-4 cases per 100,000 people (approximately 5,000 cases nationwide), and MMN in 0.6 cases per 100,000 people (400 cases nationwide). Inflammatory demyelinating peripheral neuropathy is thought to be an autoimmune disease caused by immune abnormalities against components of the peripheral nerve myelin sheath, but the details are unknown.

[0003] GBS is an inflammatory demyelinating peripheral neuropathy that rapidly develops limb motor paralysis and sensory impairment within one week to one month, and in severe cases can lead to respiratory muscle paralysis. Cranial nerve damage may also occur. Although several diagnostic criteria for GBS have been reported by expert groups, they are all merely expert opinions, and the sensitivity and specificity of the diagnostic criteria have not been examined. GBS is basically diagnosed based on the medical history and clinical symptoms, with reference to auxiliary tests such as electrophysiological findings, cerebrospinal fluid findings, and serum antiglycolipid antibodies, which are elevated in approximately two-thirds of cases.

[0004] CIDP is an inflammatory demyelinating peripheral neuropathy characterized by chronic progressive or stepwise recurrent symmetrical weakness of the proximal and distal muscles of the limbs over a period of more than two months, and sensory impairment of the limbs and trunk. Cranial nerves may also be affected. The international diagnostic criteria for CIDP are often based on the guidelines proposed by the European Federation of Neurological Societies / Peripheral Nerve Society (EFNS / PNS), published in 2010 and revised in 2021, and are based on a combination of clinical symptoms, electrophysiological criteria, and other findings such as cerebrospinal fluid findings and nerve root thickening on MRI.

[0005] MMN is a chronic inflammatory demyelinating peripheral nerve disease whose main symptoms are asymmetric muscle weakness and muscle atrophy predominantly in the distal upper limbs without sensory impairment. It is characterized by no electrophysiological abnormalities in sensory nerve conduction, and antibodies against GM1 ganglioside are found in half of the patients. Diagnosis is based on clinical symptoms and electrophysiological findings, with reference to cerebrospinal fluid findings and ganglioside antibodies. CIDP and MMN, which have a chronic course, are collectively designated as intractable diseases by the government (designated intractable disease number 14).

[0006] No disease-specific biomarkers have been reported for CIDP to date. Recently, autoantibodies against proteins at the nodes of Ranvier have been found in some CIDP cases, and because they present a unique clinical picture, they have come to be treated as an independent disease, known as autoimmune nodopathy (Non-Patent Document 1). This includes cases in which IgG antibodies (nodo antibodies) against neurofascin 155 (NF155), pan-neurofascin (NF155 and NF186), contactin-1 (CNTN1), and contactin-associated protein-1 (Caspr1) are positive. NF186 is localized in the axon membrane of the nodes of Ranvier, NF155 in the Schwann cell membrane of the paranodes of the nodes of Ranvier, and CNTN1 and Caspr1 in the axon membrane of the paranodes of Ranvier. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] J Peripher Nerv Syst. 2021;25:242-268. Summary of the Invention [Problem to be solved by the invention]

[0008] A major characteristic of the above-mentioned autoimmune nodopathy is that it is resistant to intravenous immunoglobulin (IVIg), which is the standard treatment for inflammatory demyelinating peripheral neuropathy (GBS, CIDP, MMN). In addition, about one-third of cases show acute to subacute onset, like GBS. Therefore, autoimmune nodopathy is an important differential diagnosis not only for CIDP / MMN but also for GBS. However, it is not easy to distinguish between GBS, CIDP, and MMN because many cases show similar symptoms even when known throat antibodies are negative. Therefore, it is an urgent task to discover new autoantibodies (throat antibodies) that are blood biomarkers for autoimmune nodopathy and to develop measurement methods.

[0009] The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide a biomarker, a diagnostic kit, and a method for assisting diagnosis for specifically diagnosing a group of acute and chronic inflammatory demyelinating peripheral neuropathy with unknown autoantibodies, which has a specific pathophysiology. [Means for solving the problem]

[0010] The present inventors have discovered that IgG antibodies reacting with leucine-rich repeat LGI family member 4 (LGI4), which is present in the juxta-paranode of Ranvier, are present in blood samples from some patients with acute and chronic inflammatory demyelinating peripheral neuropathy, and have established a method for measuring IgG antibodies reacting with LGI4. As a result, they have found that by measuring anti-LGI4 antibodies, it may be possible to establish treatments appropriate for each pathology of inflammatory demyelinating peripheral neuropathy, including various pathologies, and have thus completed the present invention.

[0011] That is, in order to solve the above problems, the present invention has the following aspects. [1] A biomarker for diagnosing inflammatory demyelinating polyneuropathy or assessing the effectiveness of treatment, including anti-LGI4 antibodies. [2] The biomarker described in [1], wherein the inflammatory demyelinating polyneuropathy is autoimmune nodopathy. [3] A biomarker described in [1] or [2] for determining whether the diagnosis or therapeutic effect of the inflammatory demyelinating polyneuropathy is not chronic inflammatory demyelinating polyneuropathy not involving autoimmune nodopathy, Guillain-Barré syndrome, or multifocal motor neuropathy. [4] A diagnostic kit for diagnosing inflammatory demyelinating polyneuropathy, comprising a reagent for measuring anti-LGI4 antibodies contained in a sample. [5] The diagnostic kit described in [4], wherein the inflammatory demyelinating polyneuropathy is autoimmune nodopathy. [6] A diagnostic kit according to [4] or [6], further comprising a cell line in which LGI4 and ADAM22 are forcibly expressed. [7] A diagnostic kit described in any of [4] to [6], further comprising a fluorescently labeled anti-human IgG antibody and an IgG1,2,3,4 subclass antibody. [8] A method for determining whether or not a subject has an anti-LGI4 antibody in a sample, comprising the steps of: A method for assisting in the diagnosis of inflammatory demyelinating polyneuropathy, in which the presence of the anti-LGI4 antibody is significantly higher than the presence in healthy individuals, indicating that the subject is suffering from inflammatory demyelinating polyneuropathy. [9] A method for assisting diagnosis described in [8], wherein the inflammatory demyelinating polyneuropathy is autoimmune nodopathy.

[10] A method for assisting diagnosis described in [8] or [9], further comprising a step of measuring dorsal root ganglion anti-satellite glia antibodies contained in the sample.

[11] A method for assisting diagnosis described in any of [8] to

[10] , further comprising a step of measuring autoantibodies against peripheral nerve paranodes adjacent to the nodes of Ranvier contained in the sample.

[12] A method for assisting diagnosis described in any of [8] to

[11] , in which the step of measuring anti-LGI4 antibodies contained in a sample derived from the subject is performed using a cell line that constitutively expresses LGI4 to measure anti-LGI4 antibodies.

[13] A method for assisting diagnosis described in any of [8] to

[12] , wherein the step of measuring anti-LGI4 antibodies contained in a sample derived from the subject comprises contacting the sample with a cell line in which LGI4 and ADAM22 are forcibly expressed, and then measuring the anti-LGI4 antibodies using a fluorescently labeled anti-human IgG antibody.

[14] A method for assisting diagnosis described in any of [8] to

[13] , wherein the step of measuring anti-LGI4 antibodies contained in a sample derived from the subject involves reducing the expression of LGI4 in cells that constitutively express LGI4 using LGI4-specific small interference RNA and measuring anti-LGI4 antibodies.

[15] A method for assisting diagnosis described in any of [8] to

[14] , wherein the step of measuring anti-LGI4 antibodies contained in a sample derived from the subject comprises contacting the sample in advance with a cell line in which LGI4 and ADAM22 are forcibly expressed, and measuring anti-LGI4 antibodies in cells that constitutively express LGI4.

[16] A method for assisting diagnosis described in any of [8] to

[15] , wherein the sample is blood or cerebrospinal fluid.

[17] The subject's inflammatory demyelinating polyneuropathy is autoimmune nodopathy, A method for assisting diagnosis described in any of [8] to

[16] for determining that the condition is not chronic inflammatory demyelinating polyneuropathy not including autoimmune nodopathy, Guillain-Barré syndrome, or multifocal motor neuropathy. Effect of the Invention

[0012] According to the present invention, there are provided a biomarker, a diagnostic kit, and a method for assisting diagnosis for specifically diagnosing a group of acute and chronic inflammatory demyelinating peripheral neuropathy with unknown autoantibodies, which has a specific pathophysiology. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram showing the structure of peripheral nerve nodes of Ranvier and their nodes proteins. [Diagram 2] This is a schematic diagram showing the mechanism by which conduction delay and conduction block occur due to damage to the nodes of Ranvier (segmental demyelination). [Diagram 3] FIG. 1 shows the amino acid sequence of full-length human LGI4. [Figure 4A] FIG. 1 is a photograph showing a fluorescence microscope image of the results of tissue staining of Schwann cells in this example. [Figure 4B] FIG. 1 is a graph showing the results of quantitative PCR for the expression of Krox20 and periaxin in this example. [Figure 4C] FIG. 1 is a graph showing the results of BrdU incorporation into Schwann cell proliferation in this example. [Figure 4D] FIG. 2 is a schematic diagram showing the effects of LGI4 and anti-LGI4 antibody on Schwann cells in this example. [Figure 5A] FIG. 1 is a photograph of fluorescent staining of mouse sciatic nerve injection in this example. [Figure 5B] FIG. 5B is a photograph of a wider field of view of FIG. 5A in this embodiment. [Figure 6A] FIG. 1 is a photograph of tissue immunostaining of the dorsal root ganglion in this example. [Figure 6B] FIG. 1 is a photograph of double tissue immunostaining of the dorsal root ganglion of this example. [Figure 6C] FIG. 2 is a schematic diagram of dorsal root ganglion cells and satellite glia in this example. [Figure 7] FIG. 1 is a photograph showing the results of immunostaining under a microscope of a dissected sciatic nerve specimen in this example. [Figure 8A] FIG. 1 is a photograph showing the results of immunostaining of dorsal root ganglia with an IgG subclass-specific antibody observed under a microscope in this example. [Figure 8B] FIG. 13 is a graph summarizing the relationship between the anti-LGI4 antibody positivity rate and IgG subclass for each case. [Figure 9A] FIG. 2 is a photograph showing the results of Western blotting using a protein extract derived from mouse dorsal root ganglion in this example. [Figure 9B] FIG. 2 is a photograph showing the results of Western blotting using a protein extract derived from cultured rat Schwann cells in this example. [Figure 9C] FIG. 1 is a photograph showing the results of Western blotting using a protein extract derived from cultured rat Schwann cells of this example in comparison with a commercially available anti-LGI4 antibody. [Figure 9D] FIG. 1 is a photograph showing the results of Western blotting using the human melanoma cell line of this example. [Figure 9E] FIG. 1 is a photograph showing the results of Western blotting using a protein extract derived from the LGI4-overexpressing cell line of this example, in comparison with a commercially available anti-LGI4 antibody. [Figure 10A] FIG. 1 is a schematic diagram of cell immunostaining using HEK293T cells (stable transformant) in which LGI4 and ADAM22 were forcibly expressed by the CBA method of this embodiment. [Figure 10B] FIG. 1 is a schematic diagram of cell immunostaining using HEK293T cells in which only ADAM22 was forcibly expressed by the CBA method of this example. [Figure 10C] FIG. 1 is a photograph showing the results of cell immunostaining using HEK293T cells in which LGI4 and ADAM22 were forcibly expressed in the HEK293T cells of this example. [Figure 10D]FIG. 1 is a photograph showing the results of cell immunostaining using HEK293T cells in this example in which only ADAM22 was forcibly expressed. [Figure 11A] FIG. 13 is a photograph of tissue immunostaining of dorsal root ganglion using patient serum preabsorbed with non-forced expression HEK293T cells of this example. [Figure 11B] FIG. 13 is a photograph of tissue immunostaining of dorsal root ganglion using patient serum preabsorbed with HEK293T cells in which LGI4 and ADAM22 of this example were forcibly expressed. [Figure 12A] FIG. 1 is a graph showing the ratio of normalized expression levels of Lgi4 gene in cultured rat Schwann cells of this example. [Figure 12B] FIG. 1 is a graph showing the average fluorescence intensity of antibody binding to LGI4 in tissue immunostaining using cultured rat Schwann cells in this example. [Figure 12C] FIG. 1 is a photograph showing the results of cell immunostaining of cultured rat Schwann cells in this example. [Figure 13A] FIG. 1 is a graph showing the ratio of normalized expression levels of Lgi4 gene in cultured human melanoma cells in this example. [Figure 13B] FIG. 1 is a graph showing the average fluorescence intensity of antibody binding to LGI4 in tissue immunostaining using cultured human melanoma cells in this example. [Figure 13C] FIG. 1 is a photograph showing the results of cell immunostaining of cultured human melanoma cells in this example. [Figure 14] Fig. 1 shows a summary of clinical findings in cases of anti-LGI4 antibody-positive autoimmune nodopathy. [Figure 15] This figure summarizes the test findings and treatment outcomes of cases of anti-LGI4 antibody-positive autoimmune nodopathy. [Figure 16A] FIG. 1 is a diagram showing MRI neurography observations of the case in this embodiment. [Figure 16B] FIG. 1 shows an immunostaining photograph (top) and an electron microscope photograph (bottom) showing onion bulb formation (arrowheads) in the sural nerve biopsy of this example. [Figure 17]This figure compares the clinical characteristics of anti-LGI4 antibody-positive autoimmune nodopathy with other major autoimmune nodopathy. [Figure 18] FIG. 1 is a diagram summarizing the targets of measurement for anti-LGI4 antibodies and their significance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The biomarker, diagnostic kit, and method for assisting diagnosis according to the present invention will be described below with reference to embodiments, although the present invention is not limited to the following embodiments.

[0015] (Biomarkers) The biomarker of this embodiment is a biomarker for diagnosing inflammatory demyelinating polyneuropathy or determining the therapeutic effect, comprising an anti-LGI4 antibody.

[0016] In this embodiment, by detecting a biomarker in a sample or measuring the amount of a biomarker, etc., it is possible to diagnose inflammatory demyelinating polyneuropathy in a subject from whom the sample is derived. Alternatively, the detection or measurement can be used to determine the effectiveness of the treatment. The determination of the effectiveness of the treatment broadly includes determining the influence of the treatment when the treatment for inflammatory demyelinating polyneuropathy is being performed.

[0017] The inflammatory demyelinating polyneuropathy is preferably autoimmune nodopathy. As described below, anti-IGL4 antibody is specific for the onset of autoimmune nodopathy, so that it can detect autoimmune nodopathy and evaluate the diagnosis or treatment effect. In addition, because anti-IGL4 antibodies are specific to the onset of autoimmune nodopathy, they can distinguish between autoimmune nodopathy and other inflammatory demyelinating polyneuropathy. Representative inflammatory demyelinating polyneuropathy other than autoimmune nodopathy include Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), and multifocal motor neuropathy (MMN). Anti-IGL4 antibodies are negative for these diseases.

[0018] Here, LGI4 and LGI antibodies will be described with reference to the figures. FIG. 1 is a schematic diagram showing the structure of the peripheral nerve node of Ranvier and its node proteins. Figure 2 is a schematic diagram showing the mechanism by which conduction delay and conduction block occur due to damage to the nodes of Ranvier (segmental demyelination). FIG. 3 shows the amino acid sequence of the full-length human LGI4.

[0019] Figure 1 mainly shows an enlarged view of the node of Ranvier in a peripheral nerve. In the peripheral nervous system, the myelin sheath is formed by the cell membrane processes of Schwann cells surrounding the axon in multiple loops, and the insulating structure of this sheath enables the saltatory conduction of electrical signals in the nerve axon. As shown in Figure 2, saltatory conduction is mediated by electrical excitation of the Ranvier nodes. When the myelin sheath at the nodes of Ranvier is peeled off (demyelination occurs), nerve conduction is impaired. The adhesion sites between the axon and the myelin sheath are divided into the paranode, juxta-paranode, and internode (Figure 1).

[0020] FIG. 3 shows the amino acid sequence of human LGI4 (SEQ ID NO:1). LGI4 (leucine-rich repeat LGI family member 4) is a protein with a molecular weight of 59,377 Da that is secreted from Schwann cells. LGI4 secreted from Schwann cells binds to a disintegrin and metalloprotease domain-containing protein 22 (ADAM22) present on the axon membrane of the juxta-paranode of Ranvier as a receptor. LGI4 bound to ADAM22 is said to transmit myelination signals to Schwann cells via unknown molecules on the Schwann cell side. LGI4 is also expressed in the precursor cells of satellite glia in the dorsal root ganglion and enteric glia in the intestine, and promotes their proliferation (ASN Neuro 5(3):art:e00115.doi:10.1042 / AN20120095,2013.). It has been reported that genetic mutations in LGI4 cause hypomyelination in both humans and mice (Nature NeuroScience Vol.9(1),2006,pp76-84) (The Journal of Neuroscience, Nov.10,2010·30(45):15228-15240).

[0021] As shown in the Examples below, we have discovered that the process in which LGI4 secreted from cultured Schwann cells binds to ADMA22 on the cell membrane of the Schwann cells themselves is inhibited by patient-derived anti-LGI4 antibodies, which reduces gene expression of Krox20, a master regulator of myelination signals, and causes proliferation of undifferentiated Schwann cells (Figs. 4A, B, C, and D in the Examples below). Furthermore, we have discovered that by repeatedly injecting patient-derived anti-LGI4 antibodies into mouse sciatic nerves, the anti-LGI4 antibodies penetrate into the juxta-paranode of Ranvier and reduce the expression of Caspr1 at the paranode of the node of Ranvier (Figs. 5A and B in the Examples below). In other words, patient-derived anti-LGI4 antibodies have pathogenicity.

[0022] As the anti-LGI antibody contained in the biomarker of this embodiment, an antibody that cross-reacts with the above-mentioned human LGI4 can be appropriately used. In the following description, LGI4 refers to human LGI4, and anti-LGI4 antibody refers to an antibody that binds to human LGI4. As described below, the present invention can also be applied to LGI4 and LGI4 antibodies of other animals. For example, LGI4 and anti-LGI4 antibodies of other animals can be used to obtain information that aids in diagnosis of other animals.

[0023] The biomarker of this embodiment includes an anti-LGI4 antibody and can be used to diagnose autoimmune nodopathy in acute and chronic inflammatory demyelinating peripheral neuropathy. The anti-LGI4 antibody can be applied in the following ways:

[0024] The biomarker of this embodiment can be used for the differential diagnosis of CIDP, as well as for the selection of an initial treatment method for CIDP and a method for preventing recurrence. For example, it is possible to easily know which treatment method or drug is appropriate for an individual CIDP patient depending on whether the biomarker is positive or negative.

[0025] Furthermore, by comparing the amount of biomarkers in patient samples before and after use of the treatment or drug, and since anti-LGI4 antibodies are pathogenic, if the amount of the biomarker consisting of anti-LGI4 antibodies decreases after administration compared to before administration, it can be determined that the drug is effective for the patient. On the other hand, if the amount of the biomarker before and after administration is the same, or if the amount of the biomarker after administration is higher than before administration, it can be determined that the drug is ineffective for that patient. Furthermore, by confirming that the biomarker decreases during continued treatment, it is possible to reduce the dosage of the therapeutic drug.

[0026] In this way, the biomarker of this embodiment can be used to indicate the presence or absence of autoimmune nodopathy, as well as an indicator of treatment responsiveness and disease activity. The biomarker of this embodiment can be used to determine whether the diagnosis or treatment effect of the inflammatory demyelinating polyneuropathy is not a chronic inflammatory demyelinating polyneuropathy not including autoimmune nodopathy, Guillain-Barre syndrome, or multifocal motor neuropathy. As described above, anti-LGI4 antibodies are specifically detected in subjects with autoimmune nodopathy. Therefore, the biomarker of this embodiment can be used to determine whether the inflammatory demyelinating polyneuropathy is not another inflammatory demyelinating polyneuropathy of autoimmune nodopathy.

[0027] (Diagnostic kits) The diagnostic kit of this embodiment is a diagnostic kit for diagnosing inflammatory demyelinating polyneuropathy, comprising a measuring reagent for anti-LGI4 antibody contained in a sample. As described above, the inflammatory demyelinating polyneuropathy may be autoimmune nodopathy.

[0028] The diagnostic kit of this embodiment detects the biomarker described above. Specifically, the measurement reagent for anti-LGI4 antibody contained in the diagnostic kit detects anti-LGI4 antibody contained in a sample, and therefore, a reagent that can be used for antibody detection can be appropriately used. For example, it may contain LGI4, which is an antigen of the antibody, or cells capable of expressing it.

[0029] The diagnostic kit of this embodiment may further include a cell line in which LGI4 is forcibly expressed. Also, it may further include a cell line in which ADAM22 is forcibly expressed. These may be either one of them alone in one cell line, or both (multiple) may be forcibly expressed. The cell line in which LGI4 or ADAM22 is forcibly expressed includes the case where the cell is a cell that constitutes a cell line. In addition, the cell line that expresses these genes by forcible expression is preferably a cell line that stably expresses these genes (stable transformant).

[0030] The diagnostic kit of this embodiment may further contain a fluorescently labeled anti-human IgG antibody and an IgG1,2,3,4 subclass antibody. A diagnostic kit containing such a fluorescently labeled anti-human IgG subclass-specific antibody enables determination of the IgG subclass of an anti-LGI4 antibody.

[0031] The diagnostic kit of this embodiment may further include a dorsal root ganglion and a peripheral nerve specimen. Furthermore, the diagnostic kit of this embodiment may contain reagents necessary for carrying out tissue immunostaining or cell immunostaining.

[0032] By using the diagnostic kit of the present embodiment, inflammatory demyelinating polyneuropathy, particularly autoimmune nodopathy, can be diagnosed. In addition, the diagnostic kit of the present embodiment can easily perform a method for measuring the presence of antibodies against satellite glia in the dorsal root ganglion, a method for measuring the presence of antibodies against paranodes of Ranvier in peripheral nerves, a method for measuring the presence of anti-LGI4 antibodies, and a method for diagnosing autoimmune nodopathy by differentiating it from other acute and chronic inflammatory demyelinating peripheral nerve diseases such as GBS, CIDP, and MMN.

[0033] (Method to aid in the diagnosis of inflammatory demyelinating polyneuropathy) The method of this embodiment for assisting in the diagnosis of inflammatory demyelinating polyneuropathy includes a step of measuring anti-LGI4 antibodies contained in a sample derived from a subject, and includes a means for indicating that the subject is suffering from inflammatory demyelinating polyneuropathy if the amount of anti-LGI4 antibodies present is significantly higher than the amount present in healthy individuals.

[0034] Here, the method for assisting in the diagnosis of inflammatory demyelinating polyneuropathy is a method for obtaining information that can be used in the diagnosis of inflammatory demyelinating polyneuropathy. For example, by obtaining information on tissues, cells, etc. possessed by a subject from whom a specimen is derived, information that can be used for diagnosis can be obtained, and information that can be used in the diagnosis of inflammatory demyelinating polyneuropathy can be obtained. Indicating that the subject is suffering from inflammatory demyelinating polyneuropathy broadly refers to information regarding the condition of inflammatory demyelinating polyneuropathy, such as information regarding the severity of inflammatory demyelinating polyneuropathy and the effectiveness of treatment.

[0035] It is also preferred that the inflammatory demyelinating polyneuropathy is an autoimmune nodopathy.

[0036] It is also preferable to further include a step of measuring dorsal root ganglion anti-satellite glia antibodies contained in the sample. Anti-LGI4 antibody is also expressed in satellite glia of the dorsal root ganglion, and further information can be obtained by using it in combination with the detection results of satellite glia antibodies. For example, double staining with these antibodies can be used to combine the detection results of anti-LGI4 antibody and satellite glia antibody.

[0037] It is also preferable to further include a step of measuring autoantibodies against the paranodes of Ranvier in the peripheral nerves contained in the sample. By using the detection results of the autoantibody in combination, further information can be obtained. For example, double staining of the anti-LGI4 antibody and the detection results of the autoantibody can be used in combination.

[0038] It is also preferable that the step of measuring anti-LGI4 antibodies contained in a sample derived from a subject involves measuring anti-LGI4 antibodies using a cell line that constitutively expresses LGI4.

[0039] The step of measuring anti-LGI4 antibodies contained in a sample derived from the subject preferably involves contacting the sample with a cell line in which LGI4 and ADAM22 are forcibly expressed, and then measuring the anti-LGI4 antibodies using a fluorescently labeled anti-human IgG antibody. In addition, the step of measuring anti-LGI4 antibodies contained in a sample derived from the subject also preferably involves measuring anti-LGI4 antibodies by reducing expression of LGI4 using LGI4-specific small interference RNA in cells that constitutively express LGI4. In addition, the step of measuring anti-LGI4 antibodies contained in a sample derived from the subject is also preferably carried out by contacting the sample in advance with a cell line in which LGI4 and ADAM22 are forcibly expressed, and measuring anti-LGI4 antibodies in cells that constitutively express LGI4.

[0040] In the method for assisting in the diagnosis of inflammatory demyelinating polyneuropathy of the present embodiment, the subject of diagnosis is any animal that can be affected by inflammatory demyelinating peripheral nerve disease, such as humans, non-human primates, dogs, cats, rabbits, rats, or mice. In the explanation of the present embodiment and examples, the subject is mainly humans, but the same applies to other animals.

[0041] In the method for assisting in the diagnosis of inflammatory demyelinating polyneuropathy of this embodiment, the sample is preferably blood or cerebrospinal fluid. In the case of inflammatory demyelinating polyneuropathy, particularly autoimmune nodopathy, the anti-LGI4 antibody contained in the biomarker is detected in the blood or cerebrospinal fluid, and therefore can be used to effectively assist in the diagnosis of inflammatory demyelinating polyneuropathy, particularly autoimmune nodopathy.

[0042] It is also preferable that the method of assisting in the diagnosis of inflammatory demyelinating polyneuropathy of this embodiment is a method for determining whether the inflammatory demyelinating polyneuropathy in the subject is autoimmune nodopathy and is not chronic inflammatory demyelinating polyneuropathy not involving autoimmune nodopathy, Guillain-Barré syndrome, or multifocal motor neuropathy. As described above, anti-LGI4 antibodies are specifically detected in subjects with autoimmune nodopathy. Therefore, the diagnostic support method of the present embodiment can be used to determine whether the subject has other inflammatory demyelinating polyneuropathy.

[0043] (Effects of this embodiment) According to the present embodiment, a biomarker, a diagnostic kit, and a method for assisting diagnosis are provided for specifically diagnosing a group of acute and chronic inflammatory demyelinating peripheral neuropathy with unknown autoantibodies, which has a specific pathophysiology.

[0044] For example, a sample can be contacted with cells in which LGI4 and its receptor a disintegrin and metalloprotease domain-containing protein 22 (ADAM22) are forcibly expressed and with cells in which only ADAM22 is forcibly expressed, and the presence of anti-LGI4 antibodies can be specifically measured using a fluorescently labeled anti-human IgG antibody. A specifically positive sample reacts only with cells in which both LGI4 and ADAM22 are forcibly expressed, but does not react with cells in which only ADAM22 is forcibly expressed. Cells in which both LGI4 and ADAM22 are forcibly expressed can be produced by introducing a vector incorporating human LGI4 and human ADAM22 cDNA into cells such as HEK (human embryonic kidney) 293T cells suitable for expressing these. As a result, cells that constitutively express both LGI4 and ADAM22 (stable transformants) were established, and a cell-based assay using this cell line will become the gold standard for measuring anti-LGI4 antibodies. In addition, since anti-LGI4 antibody is also expressed in satellite glia of the dorsal root ganglion, it can be screened by tissue-based assay of dorsal root ganglion of experimental animals (such as mice and rats) using specimens, with a characteristic staining pattern surrounding the dorsal root ganglion neurons in a circle shape. Furthermore, anti-LGI4 antibody can be measured by double tissue immunostaining of specimens with an antibody against glutamine synthetase, a marker of satellite glia, or double tissue immunostaining of specimens with anti-LGI4 antibody, which results in co-staining of satellite glia. In addition, the specific antibody against LGI4 can be confirmed by the disappearance of the reaction with satellite glia by pre-absorption with cells expressing both LGI4 and ADAM22. Similarly, using dissected peripheral nerve tissue specimens such as the sciatic nerve of an experimental animal, anti-LGI4 antibodies can be screened by co-staining the paraparanodes with an antibody against Kv1.2, a marker of the paraparanodes of Ranvier, in double tissue immunostaining of the specimen.

[0045] As shown in the examples below, in tissue immunostaining using cultured rat Schwann cell lines and human melanoma cell lines that constitutively express LGI4, the specimens were stained on the cell surface, and when double stained with a commercially available anti-LGI4 antibody, they showed colocalization, so they can be used for screening anti-LGI4 antibodies. In addition, in Western blots of crude protein extracts from these cells, the specimens react with a protein band of about 60 kDa, similar to the commercially available anti-LGI4 antibody. When small interference RNA (siRNA) specific to the LGI4 gene is applied to these cell lines that constitutively express LGI4 to significantly reduce the expression of LGI4, the reactivity of the specimens in cell immunostaining and Western blots is significantly reduced, so that it can be confirmed that the antibody is specific to LGI4 (Genetic strategy). In addition, when scrambled RNA that does not reduce LGI4 is applied to these cell lines, no reduction in the reactivity of the specimens is observed.

[0046] In both the above-mentioned cell immunostaining method and tissue immunostaining method, the IgG subclass of anti-LGI4 antibody can be identified by using anti-human IgG1, IgG2, IgG3, and IgG4 antibodies as secondary antibodies. It has been reported that IgG4 is the main antibody in autoimmune nodopathy. In the present invention, not only IgG4 class but also IgG1 and IgG2 class anti-LGI4 antibodies have been found to have pathogenicity in cultured Schwann cells and in sciatic nerve injection experiments. In general, when IgG4 class nod antibodies are the main antibody, IVIg treatment alone is ineffective, so in autoimmune nodopathy, it is very significant to determine the IgG subclass when selecting a treatment method. In particular, in cases positive for anti-LGI4 antibodies, when the IgG2 subclass is possessed, IVIg is partially effective, so determining the IgG subclass is highly valuable in selecting a treatment method.

[0047] The inventors have found that cases of anti-LGI4 antibody-positive autoimmune nodopathy are characterized by onset in middle-aged to relatively elderly patients, severe sensory ataxia in addition to motor paralysis, severe thickening of the nerve roots on MRI, and a marked increase in cerebrospinal fluid protein. Since irreversible marked nerve thickening is present from the early stage of onset, when anti-LGI4 antibody is positive, aggressive immunotherapy is indicated from the start of treatment. In addition, some CIDP cases develop acutely and then relapse, but it is difficult to distinguish it from GBS, which develops acutely at first and then follows a monophasic course (without recurrence). According to the diagnostic method of the present invention, in cases where it is difficult to determine whether the initial attack of GBS or CIDP is acute to subacute, if anti-LGI4 antibodies are positive, it can be said that the case is not monophasic GBS, but is an autoimmune nodopathy that follows a recurrent and progressive course like CIDP. If it is determined that the case is anti-LGI4 antibody-positive autoimmune nodopathy, complex immunotherapy is essential in addition to IVIg, so continuous treatment is required by actively introducing blood purification therapy, corticosteroids, immunosuppressants, etc.

[0048] According to this embodiment, by detecting anti-LGI4 antibodies in a sample, autoimmune nodopathy can be diagnosed in acute and chronic inflammatory demyelinating peripheral neuropathy. Thus, by extracting positive cases reacting with LGI4 in acute and chronic inflammatory demyelinating peripheral neuropathy, it is possible to extract a patient group with the same pathophysiology. That is, in GBS and CIDP, which are disease groups with various pathologies, the therapeutic effects vary in each case, but by selecting cases with anti-LGI4 antibodies, it becomes possible to provide a more appropriate treatment method. In particular, in acute and chronic inflammatory demyelinating peripheral neuropathy, anti-LGI4 antibody positive cases are resistant to IVIg treatment alone, so that it is possible to improve the prognosis by performing other immunotherapy in combination from an early stage. Furthermore, by repeatedly measuring anti-LGI4 specific antibodies in a sample (e.g., serum and cerebrospinal fluid), it can be used as an index for evaluating the therapeutic effect. In addition, in acute inflammatory demyelinating peripheral neuropathy, anti-LGI4 antibody positive cases develop acute symptoms but then progress to a chronic course, and unlike monophasic GBS, continuous treatment is required, which can improve the prognosis. In autoimmune nodopathy, which is considered an independent disease concept from GBS and CIDP due to the presence of nod antibodies, the anti-LGI4 positive group can be established as a new disease entity of autoimmune nodopathy.

[0049] This embodiment provides a method for diagnosing autoimmune nodopathy from acute and chronic inflammatory demyelinating peripheral neuropathy. The present invention also makes it possible to distinguish autoimmune nodopathy from antibody-negative GBS, CIDP, and MMN. The diagnostic method of the present invention makes it possible to extract diseases with uniform pathophysiology from acute and chronic inflammatory demyelinating peripheral neuropathy cases in which many heterogeneous pathologies are mixed. This makes it possible to provide a more appropriate treatment method. Among autoimmune nodopathy, anti-lGI4 antibody is the first autoantibody associated with inflammatory demyelinating peripheral neuropathy that exists in the juxta-paranode of Ranvier, and can expand the scope of autoimmune nodopathy.

[0050] As for the measurement subject of the anti-LGI4 antibody in this embodiment and its significance, when the measurement subject is chronic inflammatory demyelinating polyneuropathy, the prevalence in Japan is 1.6 people / 100,000 people per year, and the number of patients in Japan is approximately 5000. As for the significance of the measurement, it is useful for diagnosing autoimmune nodopathy, and determining the policy of early concomitant use of steroids and immunosuppressants when the effect of intravenous immunoglobulin therapy may be insufficient, and it is useful for determining the gradual reduction of immunotherapy by measuring the antibody titer. When the subject of measurement is Guillain-Barre syndrome, the incidence rate in Japan is 1-2 people per 100,000 people per year, with approximately 1,400 patients in Japan developing annually. The significance of measurement is that it is useful for diagnosing autoimmune nodopathy, determining the effectiveness of intravenous immunoglobulin therapy may be insufficient, and deciding on the use of steroids and immunosuppressants from an early stage, as there is a high possibility of recurrence, and determining whether to continue immunotherapy for the long term or carefully reduce the dosage, and is useful for determining the gradual reduction of immunotherapy by measuring antibody titers. In addition, anti-LGI4 antibodies can also be measured in patients with other peripheral neuritis and are effective in ruling out autoimmune nodopathy.

[0051] (Other Aspects of the Present Embodiment) Another form of this embodiment is a method for diagnosing inflammatory demyelinating polyneuropathy, which includes a step of measuring anti-LGI4 antibodies contained in a sample derived from a subject, and in which a significantly higher amount of the anti-LGI4 antibodies than that present in healthy individuals indicates that the subject is suffering from inflammatory demyelinating polyneuropathy. Yet another aspect of this embodiment is a method for diagnosing autoimmune nodopathy, comprising the step of measuring anti-LGI4 antibodies contained in a sample. Another aspect of this embodiment is the use of an anti-LGI4 antibody as a biomarker for diagnosing inflammatory demyelinating polyneuropathy or determining the effectiveness of treatment. Yet another aspect of this embodiment is the use of an anti-LGI4 antibody for the manufacture of a diagnostic kit for the diagnosis of inflammatory demyelinating polyneuropathy. EXAMPLES

[0052] Examples will be given below, but the present invention is not limited to the Examples.

[0053] In the following examples, the subjects used as samples were CIDP cases that definitely met the electrophysiological diagnostic criteria of the 2010 EFNS / PNS CIDP diagnostic criteria among cases that visited and were hospitalized at the Department of Neurology at Kyushu University Hospital between 2001 and 2021, or patient sera that were requested to measure anti-NF155 antibodies or anti-CNTN1 antibodies at the Department of Neurology at Kyushu University Hospital between 2014 and 2021, and CIDP cases that visited the Department of Neurology at Takahokai Fukuoka Central Hospital between 2020 and 2022. A total of 120 cases were used. As control groups, 35 healthy subjects (HC) and 92 patients with other neurological diseases were used. The breakdown of other neurological diseases was as follows: multiple sclerosis (19 cases), Parkinson's disease (11 cases), myelitis (8 cases), amyotrophic lateral sclerosis (7 cases), GBS (4 cases), autoimmune encephalitis (4 cases), neuromyelitis optica spectrum disorder (3 cases), hereditary spinocerebellar degeneration (3 cases), normal pressure hydrocephalus (3 cases), small fiber neuropathy (3 cases), Lewy body dementia (2 cases), spastic paraplegia (2 cases), myasthenia gravis (2 cases), neuropathic pain (2 cases), The subjects were: 1 case of Alzheimer's disease, 1 case of herpes simplex encephalitis, 1 case of encephalopathy, 1 case of epilepsy, 1 case of tremor, 1 case of optic neuritis, 1 case of rheumatic myeloradiculitis, 1 case of Charcot-Marie-Tooth disease, 1 case of mononeuritis multiplex, 1 case of Bell's palsy, 1 case of perineurioma, 1 case of myokymia, 1 case of piriformis syndrome, 1 case of Isaacs syndrome, 1 case of paresthesias, and 1 case of reflex sympathetic dystrophy. Sera obtained from the above subjects were used in the examples.

[0054] [Preliminary test example 1] (Effect of anti-LGI4 antibody-positive CIDP patient serum on cultured rat Schwann cells) First, we examined the expression of each factor in cultured rat Schwann cells and the effect of antibodies against those factors. Cultured rat Schwann cell line (IFRS1, Cosmobio) was fixed with 4% paraformaldehyde for 10 minutes at room temperature, and then immersed in 100% methanol at -20°C for 10 minutes to permeabilize the cell membrane. The cells were treated with fetal bovine serum for 60 minutes at room temperature to block nonspecific reactions. Then, the cells were reacted with a commercially available anti-LGI4 antibody (1:100 dilution, Novus Biologicals) or anti-ADAM22 antibody (1:200 dilution, Abcam) at room temperature for 1 hour. Then, the cells were reacted with Alexa Fluor 488- or 594-labeled secondary antibody (Thermo Fisher Scientific) at room temperature for 1 hour. The labeled secondary antibody used was goat anti-rabbit Alexa Fluor 488 or 594 (Thermo Fisher Scientific) against the primary antibody. DAPI staining was performed at room temperature as double staining. The stainability of the cells was evaluated using a BZX800 fluorescence microscope (KEYENCE).

[0055] Figure 4A is a photograph showing a fluorescent micrograph of the result of the staining of the cultured Schwann cells. The figure shows anti-LGI4 antibody, anti-ADAM22 antibody (green), cell nucleus staining with DAPI (blue), and double staining. The results in the figure show that the cultured Schwann cells express LGI4 and ADAM22.

[0056] Next, we investigated the effect of exposing the cells to serum IgG from anti-LGI4 antibody-positive patients on myelination signals. The same cells were used to examine the expression of Krox20, a master regulator of myelination signals, and periaxin, a protein expressed in myelinating Schwann cells. The rat Schwann cell line was incubated overnight at 37°C with 5% CO2 with anti-LGI4 antibody-positive patient serum IgG (LGI4+) or healthy subject serum (HC). RNA was then purified from the incubated rat Schwann cells using a SuperPrepcCell Lysis & RT kit (#SCQ-101; TOYOBO). The purified RNA was reverse transcribed to produce cDNA. Quantitative real-time PCR was then performed using Fast SYBR™ Green Master Mix (Thermo Fisher Scientific) with a StepOnePlus™ Real-Time PCR System (Applied Biosystems). The expression of the target gene was calculated as a ratio compared to glyceraldehyde-3-phosphate dehydrogenase (Thermo Fisher Scientific) as a reference gene. 4B is a graph showing the results of quantitative PCR for the expression of Krox20 and periaxin. As shown in the figure, when anti-LGI4 antibody-positive patient serum IgG was added, the expression of Krox20 and periaxin was significantly reduced compared to the serum of healthy subjects, and the myelination signal was reduced in cultured rat Schwann cells.

[0057] Next, we examined the proliferation of Schwann cells when anti-LGI4 antibody positive patient serum IgG was applied to them. In other words, if anti-LGI4 antibody positive patient serum IgG inhibits myelination, it is expected to promote Schwann cell proliferation. Schwann cell proliferation was measured by BrdU incorporation using 5-bromo-2'-deoxy-uridine labeling and detection kit I (Roche). Figure 4C is a graph showing the results of BrdU incorporation in Schwann cell proliferation. In the figure, Cases 1 to 6 are the cases of antibody LGI4 antibody positivity, and HC is a healthy subject to the addition of the respective serum. As shown in the figure, it can be seen that the antibody LGI4 antibody positivity is almost the same as HC, or that the proliferation of Schwann cells is promoted.

[0058] FIG. 4D is a schematic diagram showing the effects of LGI4 and anti-LGI4 antibodies on Schwann cells. From the above results, it can be inferred that the process in which LGI4 secreted from cultured Schwann cells binds to ADAM22 on the cell membrane of the Schwann cells themselves is inhibited by patient-derived anti-LGI4 antibodies, thereby reducing gene expression of key regulators of myelination signals.

[0059] [Preliminary test example 2] (Infection of anti-LGI4 antibody into the mouse sciatic nerve leads to paraparanodal penetration and paranodal destruction at the nodes of Ranvier) We investigated the effects of injecting anti-LGI4 antibodies into mouse neurons. The mice used were C56Bl6J, 10-week-old male mice. 5 μL of purified antibody (7 mg / ml) from the serum of anti-LGI4 antibody-positive patients or healthy subjects was injected into the sciatic nerve of the mice. After the first injection, the second injection was performed on the 7th day, and observations were performed on the 14th day.

[0060] Figure 5A is a photograph of fluorescent staining of mouse sciatic nerve injection. The staining results of anti-LGI4 antibody IgG2 class, IgG4 class, and serum from a healthy subject (Healthy Control) are shown, along with the merged results of staining for CASPR1 (Caspr) expressed at the site of the node structure (see Figure 1). Figure 5B is a photograph of a wider field of view. As shown by the arrows in the figure, IgG2 class antibodies penetrate deep into the nerve. On the other hand, IgG4 class antibodies show signals but do not penetrate deep into the nerve. In addition, the expression of Caspr was lower in all IgGs than in the serum of healthy subjects. This suggests that the formation of node structures is reduced (destroyed). These results suggest that anti-LGI4 antibodies are expressed in the pathogenesis of nerve connections.

[0061] These results demonstrated that repeated injection of patient-derived anti-LGI4 antibodies into mouse sciatic nerves allowed the anti-LGI4 antibodies to penetrate as far as the juxta-paranode of Ranvier and reduce Caspr1 expression at the paranode of Ranvier.

[0062] [Example 1] (Screening of IgG autoantibodies to satellite glia in the dorsal root ganglion and detection of anti-LGI4 antibodies) Using mouse dorsal root ganglion (DRG) tissue sections, autoantibodies against DRG satellite glia were detected in blood samples from the subjects mentioned above.

[0063] (Preparation of tissue sections) Dorsal root ganglia were excised from 10-week-old male C57BL / 6 mice at the lumbar spinal cord level, and each tissue was fixed in 10% formaldehyde, embedded in paraffin, and cut into 3-μm-thick tissue sections, which were then attached to glass slides.

[0064] (Reaction with blood samples) The prepared tissue sections were deparaffinized by immersing them in xylene for 5 minutes twice. The tissue sections were then hydrated by immersing them in 100% ethanol for 5 minutes twice. The tissue sections were then subjected to antigen retrieval. Specifically, the tissue sections were boiled in citrate buffer (pH 6.0) at 120°C for 10 minutes, slowly cooled at room temperature for 40 minutes, and washed twice with phosphate buffered saline (PBS) for 5 minutes. The tissue sections were then blocked by contacting them with 10% goat serum / PBS and leaving them at room temperature for 30 minutes. Next, the tissue sections were contacted with a 60-fold diluted blood sample (serum), or a 500-fold diluted anti-glutamine synthetase polyclonal antibody (Invitrogen), or an anti-Caspr polyclonal antibody (Abcam), or a 60-fold diluted anti-LGI4 polyclonal antibody (LGI4) (Novus Biologicals) as a primary antibody, and left at 37°C for 1 hour to carry out an antigen-antibody reaction. Then, the tissue sections were washed twice for 5 minutes with PBS. Then, the tissue sections were contacted with a 500-fold diluted Alexa 488-labeled anti-human IgG antibody and an Alexa 594-labeled anti-mouse or anti-rabbit IgG (Thermo Fisher Scientific) as secondary antibodies, and left at room temperature for 1 hour to carry out an antigen-antibody reaction. Then, the tissue sections were washed twice for 5 minutes with PBS. The tissue sections were mounted and antibody binding was evaluated using a confocal laser microscope (Nikon A1) or a BZ-X800 fluorescence microscope (Keyence). Colocalization rates were evaluated using the EzColocalization plug-in in ImageJ (National Institutes of Health, USA).

[0065] (result) FIG. 6A is a photograph of tissue immunostaining of the dorsal root ganglion. In the figure, Case 1 (Before) is anti-LGI4 antibody positive case 1 (before treatment), Case 1 (After) is anti-LGI4 antibody positive case 1 (after treatment), Cases 2 to 4 are anti-LGI4 antibody positive cases 2 to 4, respectively, and HL is a healthy subject. In the figure, the upper left column of each photograph shows a partially enlarged view.

[0066] FIG. 6B is a photograph of double tissue immunostaining of the dorsal root ganglion. In the figure, LGI4+ indicates anti-LGI4 antibody positive patient serum, Anti LGI4 indicates commercially available anti-LGI4 antibody, Anti Caspr1 indicates commercially available anti-Caspr1 antibody, Anti Gln-Cyn indicates commercially available anti-glutamine synthetase antibody, and Marge indicates double staining. In the figure, the upper left column of each photograph shows a partially enlarged view.

[0067] Figure 6C is a schematic diagram of dorsal root ganglion cells and satellite glia. Dorsal root ganglion cells have a structure in which primary sensory neurons are surrounded by satellite glia.

[0068] As shown in Figure 6A and B, 6 of the 120 CIDP patients showed a characteristic band-like staining pattern surrounding the primary sensory neurons (nerve cells) of the dorsal root ganglion. All of these showed colocalization when double-stained with an antibody against the satellite glia marker glutamine synthetase. None of the 127 control patients showed such a staining pattern. Thus, the frequency of anti-satellite glia antibodies was significantly higher in the CIDP patients than in the control group [6 / 120 (5%) vs. 0 / 127 (0%), p = 0.012]. The colocalization rate between the IgG from the positive patient samples and the commercially available anti-LGI4 antibody was Spearman's rank correlation coefficient of 0.52 ± 0.15, and the Manders colocalization coefficient (median) was 0.857 (M1) (proportion of the area reacting with the IgG from the patient samples among the areas reacting with LGI4) and 0.841 (M2) (proportion of the area reacting with the anti-LGI4 antibody among the areas reacting with the IgG from the patient samples). The IgG from the positive patient samples did not show colocalization with the antibody against Caspr1, so it is different from the antibody against Caspr1. Therefore, the anti-satellite glia antibody from the patient samples is considered to be anti-LGI4 antibody, and it is concluded that tissue immunostaining of the dorsal root ganglion is useful as a screening method for anti-satellite glia antibodies and anti-LGI4 antibodies.

[0069] [Example 2] (Screening of IgG autoantibodies to paranodal junctions of Ranvier and detection of anti-LGI4 antibodies using peripheral nerve and dissected specimens) Using tissue sections of mouse peripheral nerve tissue (sciatic nerve), autoantibodies against the peripheral nerve paranodes adjacent to the nodes of Ranvier were detected in blood samples from the above subjects.

[0070] (Preparation of tissue sections) Sciatic root nerves were excised from 10-week-old male C57BL / 6 mice and fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde for 10 min. After washing with PBS, the fixed tissue was mechanically disaggregated and then attached to a glass slide.

[0071] (Reaction with blood samples) The tissue sections were permeabilized with PBS containing 2% Triton X100 for 30 minutes. The tissue sections were contacted with PBS containing 10% goat serum and 1% Triton X100, left at room temperature for 60 minutes, and blocked. Next, the tissue sections were contacted with a blood sample (serum) diluted 100 times as a primary antibody and an anti-Kv1.2 monoclonal antibody (diluted 500 times) (NeuroMab) and left at 4 ° C overnight to carry out an antigen-antibody reaction. Next, after washing twice with PBS for 5 minutes, the tissue sections were contacted with Alexa 488-labeled anti-human IgG antibody and Alexa 594-labeled anti-mouse IgG (manufactured by Thermo Fisher Scientific) diluted 500 times as secondary antibodies and left at room temperature for 1 hour to carry out an antigen-antibody reaction. Next, the tissue sections were washed twice with PBS for 5 minutes. The tissue sections were mounted and antibody binding was evaluated using a confocal laser microscope (Nikon A1) or a BZ-X800 fluorescence microscope (Keyence). Colocalization rates were evaluated using the EzColocalization plug-in in ImageJ (National Institutes of Health, USA).

[0072] (result) FIG. 7 is a photograph showing the results of immunostaining of a dissected sciatic nerve specimen observed under the microscope. In the figure, LGI4+ indicates anti-LGI4 antibody-positive patient serum, HC indicates healthy subject serum, Anti Kv1.2 indicates a commercially available anti-Kv1.2 antibody (juxtaparanodal marker), and Marge indicates double staining. All of the blood samples from the six patients with anti-satellite glia antibodies stained the paranodes near the nodes of Ranvier in the peripheral nerves and showed colocalization with anti-Kv1.2 antibody, a paranodal marker.

[0073] [Example 3] (Measurement of IgG subclasses of autoantibodies against satellite glia in the dorsal root ganglion) Using mouse dorsal root ganglion preparations, the IgG subclasses of autoantibodies against satellite glia were determined.

[0074] (Preparation of tissue sections) Dorsal root ganglia were excised from 10-week-old male C57BL / 6 mice at the lumbar spinal cord level, and each tissue was fixed in 10% formaldehyde, embedded in paraffin, and cut into 3-μm-thick tissue sections, which were then attached to glass slides.

[0075] (Reaction with blood samples) The prepared tissue sections were deparaffinized by immersing them in xylene for 5 minutes twice. The tissue sections were then hydrated by immersing them in 100% ethanol for 5 minutes twice. The tissue sections were then subjected to antigen retrieval. Specifically, the tissue sections were boiled in citrate buffer (pH 6.0) at 120°C for 10 minutes, slowly cooled at room temperature for 15 minutes, and washed twice with PBS for 5 minutes. The tissue sections were then blocked by contacting them with 10% goat serum / PBS and leaving them at room temperature for 30 minutes. Next, the tissue sections were contacted with a 60-fold diluted blood sample (serum), or a 500-fold diluted anti-glutamine synthetase polyclonal antibody (Invitrogen), or an anti-Caspr1 polyclonal antibody (Abcam), or a 60-fold diluted anti-LGI4 polyclonal antibody (LGI4) (Novus Biologicals) as a primary antibody, and left to stand at 37°C for 1 hour to carry out an antigen-antibody reaction. Then, the tissue sections were washed twice for 5 minutes with PBS. Then, the tissue sections were contacted with a 100-fold diluted mouse fluorescein isothiocyanate-labeled anti-human IgG1, IgG2, IgG3, IgG4 monoclonal antibody (Sigma-Aldrich) as a secondary antibody, and left to stand at room temperature for 1 hour to carry out an antigen-antibody reaction. Then, the tissue sections were washed twice for 5 minutes with PBS. The tissue sections were mounted, and antibody binding was evaluated using a confocal laser microscope (A1, Nikon) or a BZ-X800 fluorescent microscope (KEYENCE).

[0076] (result) FIG. 8A is a photograph showing the results of immunostaining of dorsal root ganglia with an IgG subclass-specific antibody observed under the microscope. In the figure, Cases 1 to 4 respectively represent anti-LGI4 antibody positive cases 1 to 4. IgG1 to IgG4 respectively represent human IgG subclass-specific secondary antibodies. FIG. 8B shows the relationship between the anti-LGI4 antibody positivity rate and IgG subclass for each case.

[0077] The IgG subclasses of blood samples from 6 CIDP (autoimmune nodopathy) patients who were positive for anti-satellite glia antibodies and anti-LGI4 antibodies were IgG1 in 4 cases, IgG2 in 6 cases, IgG3 in 4 cases, and IgG4 in 5 cases. The predominant IgG subclass of anti-satellite glia antibodies and anti-LGI4 antibodies in each case was IgG4 in 5 cases and IgG2 in 1 case. Therefore, although IgG4 is predominant in the IgG subclass of anti-satellite glia antibodies and anti-LGI4 antibodies in many cases, there are also cases in which IgG2 is predominant, lacking IgG4 subclass antibodies. Since the function of antibodies differs depending on the IgG subclass, the effect of immunotherapy also differs depending on the IgG subclass, so the decision is clinically significant. In particular, when IgG4 subclass anti-satellite glia antibodies and anti-LGI4 antibodies are predominant, IVIg alone is not effective, and it is recommended to use corticosteroids and immunosuppressants in combination from an early stage. On the other hand, when IgG2 subclass anti-satellite glia antibodies and anti-LGI4 antibodies are predominant, IVIg is expected to be effective, so continuation of IVIg is desirable. Thus, measuring the IgG subclass of anti-satellite glia antibodies and anti-LGI4 antibodies is clinically important.

[0078] [Example 4] (Measurement of anti-LGI4 antibodies by Western blot) (Protein extraction from tissues and cells) Dorsal root ganglia and sciatic nerves were harvested from lumbar spinal cord level of 12-week-old male C57BL / 6 mice and immediately frozen in liquid nitrogen. The frozen tissue pieces were homogenized in radioimmunoprecipitation (RIPA) buffer (Nacalai Tesque) with a homogenizer, centrifuged at 14,000×g for 15 minutes at 4°C, and the supernatant was collected as mouse dorsal root ganglion-derived protein extract and mouse sciatic nerve-derived protein extract, which were stored at -80°C until use. In addition, a cultured rat Schwann cell line (IFRS1, Cosmo Bio) and a human melanoma cell line (WM115, Rockland Immunochemicals) were homogenized in RIPA buffer in the same manner, centrifuged at 14,000 x g for 15 minutes at 4°C, and the supernatants were collected and used as protein extracts derived from the rat Schwann cell line and protein extracts derived from the human melanoma cell line. These were then stored at -80°C until use. Furthermore, HEK293T cells (Novus Biologicals) transfected with an LGI4 expression plasmid (ID RC219293, OriGene Technologies, accession number NM_139284) were homogenized in RIPA buffer in the same manner, centrifuged at 14,000 × g for 15 minutes at 4°C, and the supernatant was collected as a protein extract derived from the LGI4-overexpressing cell line. This was then stored at -80°C until use.

[0079] (Western blot using protein extracts from tissues and cells as antigens) Protein extracts from the above tissues or cells were mixed with Laemmli reagent buffer and denatured by heating at 95°C for 5 minutes, then loaded onto polyacrylamide gradient gels and electrophoresed to separate each protein. The proteins were then transferred to polyvinylidene difluoride membranes (PVDF). After transfer, the PVDF membranes were blocked with 3% skim milk at room temperature for 1 hour. The membranes were cut into lanes, and each membrane strip was contacted with patient specimens (serum diluted 300 times), rabbit anti-LGI4 antibody (Abcam) diluted 1000 times, anti-Flag antibody (clone No. 1E6, Fujifilm Wako Pure Chemical Industries, Ltd.) diluted 2000 times, or anti-β-actin monoclonal antibody (clone No. AC-74, Sigma-Aldrich) diluted 5000 times overnight at 4°C. The cut membrane was washed with Tris Buffered Saline with Tween 20 and exposed to Horseradish peroxidase (HRP)-conjugated secondary antibody (Vector Laboratories) for 1 hour at room temperature. β-actin was used as an internal control in Western blotting. Band signals were detected using a ChemiDoc XRS system (Bio-Rad) or a WSE-6270 LuminoGraph II EM (ATTO).

[0080] (result) Figure 9A shows the results of Western blots using a protein extract derived from mouse dorsal root ganglia, Figure 9B shows the results of a protein extract derived from cultured rat Schwann cells, Figure 9C shows the results of a protein extract derived from cultured rat Schwann cells, and Figure 9D shows the results of a protein extract derived from a human melanoma cell line, compared with a commercially available anti-LGI4 antibody. In these results, patient samples reacted with a band of approximately 60 kDa.

[0081] FIG. 9E shows the results of Western blot analysis using protein extracts derived from LGI4-overexpressing cell lines in comparison with a commercially available anti-LGI4 antibody. The results showed that the patient samples reacted with a band of approximately 60 kDa at the same electrophoretic position as the protein extract from the LGI4-overexpressing cell line and the commercially available anti-LGI4 antibody. On the other hand, no such reaction was observed in the control group. Reaction with the 60 kDa band in Western blot was observed in four out of four patient samples, but none of the three control samples reacted (only one sample is shown in the figure).

[0082] [Example 5] (Creation of cell lines expressing human LGI4 and ADAM22, and human ADAM22 alone, and anti-LGI4 antibody measurement using these cell lines (cell-based assay)) (Cell preparation) A vector containing human LGI4 cDNA was purchased from OriGene Technologies (LGI4-Flag plasmid; ID RC219293). A sequence encoding a Flag tag was incorporated into the C-terminus of the protein. A vector containing human ADAM22 cDNA was prepared by Dr. Masaki Fukada and Dr. Yuko Fukada (Division of Biomembrane Research, Department of Molecular and Cellular Physiology, National Institute for Physiological Sciences, Inter-University Research Institute Corporation, National Institutes of Natural Sciences) and used (ADAM22-HA plasmid). A sequence encoding a HA (hemagglutinin) tag was incorporated into the C-terminus of each protein. The above vector was 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, a total of 40 μL of solution was prepared by mixing 2 μL of ScaI, 4 μL of 10×H buffer, 1.5 μg of substrate DNA, and sterile purified water, and the reaction was carried out at 37°C.

[0083] The linearized vector was transfected into HEK (human embryonic kidney) 293T cells by lipofection using lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) according to the attached instructions. Then, the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with the addition of 1 mg / mL G418 (Life Technologies), and G418-resistant strains were selected. The grown colonies were isolated using a cloning cylinder, and were used as cell lines in which human LGI4 and ADAM22 were forcibly expressed, and as cell lines in which human ADAM22 was forcibly expressed, respectively. The cell lines were cultured in DMEM with the addition of 1 mg / mL G418 (Life Technologies), and passaged or stored in liquid nitrogen.

[0084] (Reaction with blood samples) Cell lines expressing human LGI4 and ADAM22, and cell lines expressing human ADAM22 alone were fixed with 2% paraformaldehyde for 20 minutes at room temperature, and treated with 20 mg / mL bovine serum albumin for 60 minutes on ice to block nonspecific reactions. Then, these cells were reacted with patient serum (1:200 dilution) or a commercial anti-LGI4 antibody (1:50 dilution, Novus Biologicals) together with an anti-Flag antibody (1:1,000 dilution) (Sigma-Aldrich) for 1 hour at room temperature.

[0085] The fixed cells were then treated with 0.1% Triton X-100 at room temperature for 10 minutes to permeabilize the cell membrane, and then treated with phosphate buffer containing 20 mg / mL bovine serum albumin for 10 minutes on ice to block nonspecific reactions, followed by reaction with anti-HA polyclonal antibody (Sigma-Aldrich) at room temperature for 1 hour. The cells were then reacted with Alexa Fluor 488-labeled or Alexa Fluor 594-labeled secondary antibodies (Thermo Fisher Scientific) at room temperature for 1 hour. The secondary antibodies used were goat anti-human, goat anti-rabbit, or goat anti-mouse Alexa Fluor 488 and Alexa Fluor 594 (Thermo Fisher Scientific) against the primary antibodies. The staining of the cells was evaluated using a confocal laser microscope (A1, Nikon) or a BZX800 fluorescence microscope (KEYENCE). In addition, for this cell immunostaining, we established a cell line (stable transformant) that constitutively expresses human LGI4 and ADAM22, and by using this, it is possible to avoid the trouble of transfecting the expression vectors for human LGI4 and ADAM22 every time.

[0086] A schematic diagram of the cell-based assay (CBA) method using anti-LGI4 antibody is shown in Figures 10A and 10B. FIG. 10A shows a schematic diagram of cell immunostaining using HEK293T cells (stable transformant) in which LGI4 and ADAM22 were forcibly expressed. FIG. 10B shows a schematic diagram of cell immunostaining using HEK293T cells in which only ADAM22 was forcibly expressed, in the same CBA method.

[0087] (result) FIG. 10C shows photographs of the results of cell immunostaining using HEK293T cells (stable transformant) in which LGI4 and ADAM22 were forcibly expressed. FIG. 10D shows photographs of the results of cell immunostaining using HEK293T cells in which only ADAM22 was forcibly expressed.

[0088] In a cell immunostaining method using a cell line forced to express human LGI4 and ADAM22 as an antigen, the cell surface of 5 out of 12 CIDP patient serum samples was stained and anti-LGI4 antibodies were determined to be positive. None of these 5 cases that were determined to be positive for anti-LGI4 antibodies stained the cell surface in a cell immunostaining method using a cell line forced to express only human ADAM22 as an antigen. On the other hand, none of the 71 control cases (66 cases with other neurological diseases and 5 healthy subjects) stained the cell surface specifically and there were no positive cases (one case each is shown). Therefore, the cell immunostaining method using a cell line forced to express human LGI4 and ADAM22 as an antigen was considered to be highly specific for detecting anti-LGI4 antibodies.

[0089] [Example 6] (Measurement of anti-LGI4 antibodies by absorption test using cell lines overexpressing LGI4 and ADAM22) (Blood sample processing and tissue immunostaining) HEK293T cell lines expressing human LGI4 and ADAM22 or HEK293T cells without expression were cultured in 6-well plates, and anti-LGI4 antibody-positive patient serum diluted 60-fold was added and incubated at 37°C for 1 hour to absorb the anti-LGI4 antibody in the serum. The absorbed serum was used to perform tissue immunostaining using dorsal root ganglion tissue as described in Example 1 above.

[0090] (result) Figures 11A and B show the results of immunostaining of dorsal root ganglion tissue after pre-absorption in cells in which LGI4 and ADAM22 were forcibly expressed. FIG. 11A is a photograph of tissue immunostaining of dorsal root ganglion using patient serum preabsorbed with non-expressing HEK293T cells. FIG. 11B is a photograph of tissue immunostaining of dorsal root ganglion using patient serum preabsorbed with HEK293T cells overexpressing LGI4 and ADAM22. In all figures, the bars are 50 μm, and the top left column shows a partially enlarged view.

[0091] After incubation of anti-LGI4 antibody-positive patient sera with HEK293T cell lines expressing human LGI4 and ADAM22, the reactivity to satellite glia in the dorsal root ganglion was significantly reduced. On the other hand, no reduction in reactivity was observed when the sera were incubated with cell lines without expression. This method can prove the presence of anti-GI4 antibodies.

[0092] [Example 7] (Measurement of anti-LGI4 antibodies using a genetic strategy using cells that constitutively express LGI4) We administered small interference RNA (siRNA) specific to the LGI4 gene to cells that constitutively express LGI4, and confirmed the effect of reducing LGI4 expression.

[0093] (Cell preparation) Cultured rat Schwann cell lines and human melanoma cell lines that constitutively express LGI4 were incubated with small interference RNA (siRNA ID: s167537, Thermo Fisher Scientific) or scrambled RNA (scrambled RNA, #4390843, Thermo Fisher Scientific) specific to the LGI4 gene at 37°C for 96 hours, and then fixed in phosphate-buffered saline containing 4% paraformaldehyde for 10 minutes at room temperature, washed with PBS, and then immersed in 100% methanol for 10 minutes at -20°C for post-fixation.

[0094] (Reaction with blood samples) The fixed cells were blocked with fetal bovine serum for 1 hour at room temperature, and then contacted with serum samples from patients or healthy controls diluted 60-fold and rabbit anti-LGI4 polyclonal antibody (Novus Biologicals) diluted 100-fold as primary antibodies and left at room temperature for 1 hour. Then, Alexa 488-labeled anti-human IgG antibody and Alexa 594-labeled anti-rabbit IgG (Thermo Fisher Scientific) diluted 1000-fold as secondary antibodies were contacted and left at room temperature for 1 hour. Then, the cells were washed three times for 5 minutes with PBS. The tissue sections were mounted and antibody binding was evaluated with a BZ-X800 fluorescence microscope (KEYENCE). Mean fluorescence intensity (MFI) was quantified with Image J (National Institutes of Health, USA).

[0095] (result) FIG. 12A shows the ratio of normalized expression levels of Lgi4 gene in cultured rat Schwann cells. FIG. 12B shows the mean fluorescence intensity of antibody binding to LGI4 in tissue immunostaining using cultured rat Schwann cells. In addition, staining was performed under the same conditions as in Example 5, as shown in FIG. FIG. 13A shows the ratio of normalized Lgi4 gene expression levels in cultured human melanoma cells. FIG. 13B shows the mean fluorescence intensity of antibody binding to LGI4 in tissue immunostaining using cultured human melanoma cells. FIG. 13C shows photographs of the results of cell immunostaining of cultured human melanoma cells stained under the same conditions as in Example 5.

[0096] In both cultured rat Schwann cell lines and human melanoma cell lines, LGI4 expression was significantly reduced by treatment with siRNA specific to the LGI4 gene. When siRNA specific to the LGI4 gene was applied to these cell lines, reactivity with LGI4 was significantly reduced in cellular immunoassay using patient blood samples. On the other hand, when scrambled RNA was applied to these cell lines, LGI4 expression was not reduced in cellular immunostaining. This was also the case in the results of Western blotting, as shown in Example 4. In particular, the average fluorescence intensity of antibody binding in cellular immunostaining was significantly reduced.

[0097] From the above, when LGI4 expression was reduced by applying small interference RNA (siRNA) specific to the LGI4 gene to cells that constitutively express LGI4, the reactivity of the patient's blood samples in cell immunohistochemistry and Western blot was reduced. Therefore, it can be confirmed that the patient's blood samples have specific antibodies against LGI4, and this genetic strategy can be used to identify anti-LGI4 antibodies.

[0098] [Example 8] (Analysis of the clinical features of cases of anti-LGI4 antibody-positive autoimmune nodopathy) (Analysis of clinical and laboratory findings) We retrospectively collected and analyzed clinical information on cases positive for anti-LGI4 antibodies, and compared their characteristics with those of known CIDP, GBS, and other autoimmune nodopathy.

[0099] (result) Figure 14 summarizes the clinical findings of anti-LGI4 antibody-positive autoimmune nodopathy cases. The clinical findings of six anti-LGI4 antibody-positive cases are summarized below. The age of onset ranged from 42 to 76 years (mean 58 years), with onset occurring in middle-aged to elderly patients. The male-female ratio was 3:3, with no gender difference. Five cases of typical disease symmetrically affecting the motor and sensory nerves of the limbs, and one case of atypical disease (multifocal) asymmetrically affecting the limbs. Three cases of acute to subacute onset and three cases of chronic disease. Three cases of monophasic disease and three cases of chronic progressive or chronic relapsing disease. All cases were characterized by motor paralysis as well as sensory impairment, especially severe deep sensory impairment (impairment of joint position sense and vibration sense). Therefore, sensory ataxia and Romberg's sign were frequently observed. Four patients also had finger tremors and ataxia, characteristics that are distinct from typical CIDP. Three patients also had distal limb muscle atrophy.

[0100] Figure 15 summarizes the test findings and treatment effects in cases of anti-LGI4 antibody-positive autoimmune nodopathy. In clinical tests, all anti-LGI4 antibody positive cases showed a marked increase in cerebrospinal fluid protein, but no increase in cerebrospinal fluid cell count.

[0101] 16A is a photograph of MRI neurography of a case, showing the lumbar nerve roots and plexus of Case 2 (Anti-LGI4 antibody positive Case 2 in Example 1) observed by 3 Tesla MRI (STIR method: FOV 380 mm, slice thickness 1.2 mm, matrix 288 × 320, TE 90.0, TR 3000, number of additions 1, imaging time 3:22). FIG. 16B is a histological staining and electron micrograph showing onion bulb formation in a sural nerve biopsy. Onion bulb formation in the sural nerve biopsy is indicated by an arrowhead, and a photograph of histological staining (top) and an electron microscope photograph (bottom) are shown. The tissue staining image shows a cross section of the biopsy sural nerve from Case 1 (anti-LGI4 antibody-positive Case 1 in Example 1) stained with toluidine blue.

[0102] MRI neurography showed significant thickening of the peripheral nerves in all chronic cases (one example is shown in the figure). Furthermore, in one case in which IgG2 class anti-LGI4 antibodies were positive, the formation of onion bulbs in which Schwann cells proliferated was observed in a sural nerve biopsy. Therefore, swelling of the proximal peripheral nerves and spinal nerve roots is a characteristic of anti-LGI4 antibody positive cases, and some of this is thought to be due to excessive proliferation of Schwann cells. Since irreversible peripheral nerve thickening occurs in chronic cases, it is thought to be important to administer strong immune therapy before this occurs.

[0103] In the treatment results of the clinical test findings in Figure 15, IVIg was partially effective in all four cases. IVIg is considered ineffective in autoimmune nodopathy in which IgG4 class nodule antibodies are positive, but since all of the anti-LGI4 antibody-positive cases also had IgG2 subclass antibodies, it is thought that IVIg may be partially effective. Corticosteroids were also used in two of the three cases, and were partially effective. Therefore, it is thought that the early combination of these treatments is useful in anti-LGI4 antibody-positive autoimmune nodopathy.

[0104] Figure 17 is a graph comparing the clinical characteristics of anti-LGI4 antibody-positive autoimmune nodopathy with other major autoimmune nodopathy. Anti-NF155 antibody-positive cases, anti-CNTN1 antibody-positive cases, and anti-LGI4 antibody-positive cases share common characteristics such as sensory ataxia, distal limb muscle atrophy, markedly elevated cerebrospinal fluid protein levels, and swelling of the proximal peripheral nerves and spinal nerve roots, but anti-LGI4 antibody-positive cases are characterized by a high frequency of acute to subacute onset.Another characteristic is that the disease develops in elderly patients, and IVIg is partially effective. Based on the above characteristics, the subjects for measuring anti-LGI4 antibodies and their significance are summarized in Figure 18. In cases suspected of CIDP, measuring anti-LGI4 antibodies is useful not only for diagnosing autoimmune nodopathy, but also for deciding whether to use corticosteroids or immunosuppressants in addition to IVIg from an early stage, and for deciding whether to carefully taper immunotherapy based on the anti-LGI4 antibody titer. In cases suspected of GBS, measuring anti-LGI4 antibodies is useful for diagnosing autoimmune nodopathy, deciding whether to use corticosteroids or immunosuppressants in addition to IVIg from an early stage, continuing immunotherapy for a long period while considering the risk of recurrence, and deciding whether to carefully taper immunotherapy based on the anti-LGI4 antibody titer. In cases suspected of other peripheral neuritis, measuring anti-LGI4 antibodies is useful for excluding autoimmune nodopathy.

[0105] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included in the scope of the invention and its equivalents described in the claims, as well as in the scope and spirit of the invention. [Industrial Applicability]

[0106] According to the present invention, there are provided a biomarker, a diagnostic kit, and a method for assisting diagnosis for specifically diagnosing a group of acute and chronic inflammatory demyelinating peripheral neuropathy with unknown autoantibodies, which has a specific pathophysiology.

Claims

1. A biomarker for diagnosing inflammatory demyelinating polyneuropathy or determining the effectiveness of treatment, comprising an anti-LGI4 antibody.

2. The biomarker of claim 1 , wherein the inflammatory demyelinating polyneuropathy is an autoimmune nodopathy.

3. The biomarker described in claim 1 or 2, for determining whether the diagnosis or treatment effect of the inflammatory demyelinating polyneuropathy is not chronic inflammatory demyelinating polyneuropathy not including autoimmune nodopathy, Guillain-Barré syndrome, or multifocal motor neuropathy.

4. A diagnostic kit for diagnosing inflammatory demyelinating polyneuropathy, comprising a reagent for measuring anti-LGI4 antibodies contained in a sample.

5. The diagnostic kit according to claim 4 , wherein the inflammatory demyelinating polyneuropathy is autoimmune nodopathy.

6. The diagnostic kit according to claim 4 or 5, further comprising a cell line in which LGI4 and ADAM22 are forcibly expressed.

7. The diagnostic kit according to claim 4 or 5, further comprising a fluorescently labeled anti-human IgG antibody and an IgG1, 2, 3, 4 subclass antibody.

8. The method includes a step of measuring an anti-LGI4 antibody contained in a sample derived from a subject, A method for assisting in the diagnosis of inflammatory demyelinating polyneuropathy, in which the presence of the anti-LGI4 antibody is significantly higher than the presence in healthy individuals, indicating that the subject is suffering from inflammatory demyelinating polyneuropathy.

9. The method for assisting diagnosis according to claim 8, wherein the inflammatory demyelinating polyneuropathy is an autoimmune nodopathy.

10. The method for assisting diagnosis according to claim 8 or 9, further comprising a step of measuring dorsal root ganglion anti-satellite glia antibodies contained in the sample.

11. The method for assisting diagnosis according to claim 8 or 9, further comprising a step of measuring autoantibodies against peripheral nerve paranodes adjacent to the nodes of Ranvier contained in the sample.

12. The method for assisting diagnosis according to claim 8 or 9, wherein the step of measuring anti-LGI4 antibodies contained in a sample derived from the subject measures anti-LGI4 antibodies using a cell line that constitutively expresses LGI4.

13. The method for assisting diagnosis described in claim 8 or 9, wherein the step of measuring anti-LGI4 antibodies contained in a sample derived from the subject includes contacting the sample with a cell line in which LGI4 and ADAM22 are forcibly expressed, and then measuring anti-LGI4 antibodies using a fluorescently labeled anti-human IgG antibody.

14. The method for assisting diagnosis according to claim 8 or 9, wherein the step of measuring anti-LGI4 antibodies contained in a sample derived from the subject comprises reducing expression of LGI4 using LGI4-specific small interference RNA in cells that constitutively express LGI4 and measuring anti-LGI4 antibodies.

15. The method for assisting diagnosis described in claim 8 or 9, wherein the step of measuring anti-LGI4 antibodies contained in a sample derived from the subject comprises contacting the sample in advance with a cell line in which LGI4 and ADAM22 are forcibly expressed, and measuring anti-LGI4 antibodies in cells that constitutively express LGI4.

16. The method for assisting diagnosis according to claim 8 or 9, wherein the sample is blood or cerebrospinal fluid.

17. the inflammatory demyelinating polyneuropathy in the subject is autoimmune nodopathy; A method for assisting diagnosis according to claim 8 or 9, for determining that the condition is not a chronic inflammatory demyelinating polyneuropathy not involving autoimmune nodopathy, Guillain-Barré syndrome, or multifocal motor neuropathy.

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