Multiplex immunoassay method for diagnosing autoimmune nodopathy

Abstract

The present invention relates to a multiplex immunoassay method for diagnosing autoimmune nodopathy and, more specifically, to a multiplex immunoassay method, comprising the steps of: (1) binding CNTN1, Caspr1, NF155, and NF186 proteins each to microspheres having different color codes; (2) mixing the microspheres having different color codes to which the proteins are each bound; (3) treating the mixed microspheres with a biological sample to induce an antigen-antibody reaction; (4) adding a detection antibody; and (5) reading fluorescence information of the microspheres to measure the presence or absence of anti-CNTN1 antibody, anti-Caspr1 antibody, anti-NF155 antibody, or anti-NF186 antibody in the biological sample and quantify amounts thereof, thereby enabling simultaneous testing of four antibodies for rapid diagnosis of autoimmune nodopathy.

Description

Multiplex immunoassay method for diagnosing autoimmune nodular disease

[0001] The present invention relates to a multiplex immunoassay method for diagnosing autoimmune nodular disease.

[0002]

[0003] Recently, autoantibodies against contactin-1 (CNTN1), contactin-associated protein-1 (Caspr1), neurofascin-155 (NF155), and neurofascin-186 (NF186), proteins that play important roles in the nodes of Ranvier in the peripheral nerves, have been identified in some patients diagnosed with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Patients with these autoantibodies show characteristic clinical features that distinguish them from typical CIDP patients. More specifically, they show characteristics such as a rapidly progressive disease course, sensory ataxia, and tremor compared to CIDP. In addition, they do not respond well to intravenous immunoglobulin (IVIg), which is known to be an effective treatment for CIDP, and the therapeutic effect is low.

[0004] As shown above, chronic inflammatory neuropathy, in which autoantibodies are identified, has clinical features that are distinct from typical CIDP and respond differently to treatment, and a new diagnosis called autoimmune nodular disease has been used to differentiate it from CIDP.

[0005] To diagnose autoimmune nodular fibrosis, antibody testing for NF155, NF186, CNTN1, or Caspr1 has been performed using ELISA and cell-based assays (CBA). Typically, ELISA and CBA can only detect one autoantibody per test due to their inherent characteristics. Therefore, four tests are required to detect four different antibodies.

[0006] Therefore, the ELISA test currently performed for the diagnosis of autoimmune nodular disease is generally performed by performing an anti-NF155 antibody detection test on a sample obtained from a patient, and if negative, performing an anti-Caspr1 antibody detection test, if negative, performing an anti-CNTN1 antibody detection test, and if negative, performing an anti-NF186 antibody detection test.

[0007] This results in a long process for autoantibody identification, making early diagnosis of autoimmune nodular disease difficult. Furthermore, the inability to identify patients with more than one antibody significantly limits the provision of personalized treatment.

[0008]

[0009] The present invention aims to provide a multiplex immunoassay method that enables rapid and economical diagnosis of autoimmune nodular disease.

[0010] The present invention aims to enable rapid diagnosis of autoimmune nodular disease, thereby enabling rapid initiation of optimal treatment according to the type of antibody possessed by the patient.

[0011] The purpose of the present invention is to provide a method for diagnosing autoimmune nodular disease.

[0012]

[0013] 1. A multiplex immunoassay method for diagnosing autoimmune nodular disease, comprising: (S1) a step of binding CNTN1 (contactin-1), Caspr1 (contactin-associated protein-1), NF155 (neurofascin-155), and NF186 (neurofascin-186) proteins to microspheres having different color codes; (S2) a step of mixing microspheres having different color codes to which proteins are each bound; (S3) a step of treating a biological sample to the mixed microspheres to induce an antigen-antibody reaction; (S4) a step of treating and binding a detection antibody to the microspheres in which the antigen-antibody reaction is induced; and (S5) a step of reading fluorescence information of the microspheres to which the detection antibody is bound.

[0014] 2. In the above 1, (S1) is a method performed at pH 6 to 7.

[0015] 3. In the above 1, (S1) is a method comprising the following steps: (S1a) a step of placing microspheres having a color code in a buffer solution of pH 6 to 7, adding N-hydroxysulfosuccinimide and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide, and incubating at room temperature; (S1b) a step of isolating the microspheres and adding them to phosphate buffered saline (PBS) to prepare a suspension; and (S1c) a step of adding CNTN1, Caspr1, NF155, or NF186 protein to the suspension, and incubating at room temperature.

[0016] 4. In the above 1, the detection antibody is a PE-labeled anti-human IgG antibody (Phycoerythrin-labeled anti-human IgG antibody).

[0017] 5. In the above 1, the biological sample is any one selected from the group consisting of tissue, cell, blood, serum, plasma, and cerebrospinal fluid.

[0018] 6. A method for diagnosing autoimmune nodular disease, comprising the step of simultaneously detecting anti-CNTN1 antibody, anti-Caspr1 antibody, anti-NF155 antibody and anti-NF186 antibody.

[0019] 7. In the above 6, a method for diagnosing autoimmune nodular disease, wherein the step of detecting an antibody comprises the following steps: (S1) a step of binding CNTN1, Caspr1, NF155 and NF186 to microspheres having different color codes, respectively; (S2) a step of mixing microspheres having different color codes, each of which has a protein bound thereto; (S3) a step of treating a biological sample to the mixed microspheres to induce an antigen-antibody reaction; (S4) a step of treating and binding a detection antibody to the microspheres in which the antigen-antibody reaction has been induced; and (S5) a step of reading the fluorescence information of the microspheres to which the detection antibody has bound.

[0020]

[0021] The multiplex immunoassay method of the present invention has high antibody detection sensitivity.

[0022] The multiplex immunoassay method of the present invention can simultaneously measure the presence or absence of anti-CNTN1 antibodies, anti-Caspr1 antibodies, anti-NF155 antibodies, and anti-NF186 antibodies.

[0023] The multiplex immunoassay method of the present invention can quantify the amounts of anti-CNTN1 antibodies, anti-Caspr1 antibodies, anti-NF155 antibodies, and anti-NF186 antibodies at one time.

[0024] The multiplex immunoassay method of the present invention can simultaneously test four antibodies, enabling rapid diagnosis of autoimmune nodular disease.

[0025] The multiplex immunoassay method of the present invention is effective in reducing costs because it can simultaneously test four antibodies.

[0026] The multiplex immunoassay method of the present invention can simultaneously test four antibodies, thereby reducing the amount of sample collected from a patient.

[0027] The multiplex immunoassay method of the present invention simultaneously tests four antibodies, thereby enabling the type of autoantibody detected in a sample to be determined at once and customized treatment to be provided according to the type of antibody possessed by the patient.

[0028] It is expected that the multiplex immunoassay method of the present invention will enable early diagnosis of autoimmune nodular disease, thereby reducing the suffering and economic burden of patients due to autoimmune nodular disease.

[0029]

[0030] Figure 1 illustrates a method for binding a protein to a microsphere having a color code according to one embodiment of the present invention.

[0031] Figure 2 schematically illustrates one embodiment of a multiplex immunoassay method for diagnosing autoimmune nodular disease of the present invention.

[0032] Figures 3 and 4 are comparison results of antibody detection results in a sample according to one embodiment of the present invention and antibody detection results in a sample according to an ELISA test.

[0033]

[0034] The present invention provides a multiplex immunoassay method for diagnosing autoimmune nodular disease.

[0035] Autoimmune nodular neuropathy is a peripheral neuropathy distinct from chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and is characterized by autoantibodies to proteins of the peripheral nerve nodes of Ranvier, such as contactin-1 (CNTN1), contactin-associated protein-1 (Caspr1), neurofascin-155 (NF155), and neurofascin-186 (NF186).

[0036] When these autoantibodies are present, the normal structure of the nodes of Ranvier is not maintained and nerve transmission is suppressed, resulting in autoimmune nodular disease.

[0037] More specifically, NF155 is a protein expressed in the myelin sheath of peritubular Schwann cells, and it binds to the CNTN1 / Caspr1 complex located in the axon, playing a role in firmly connecting the axon and the myelin sheath. The nerve tissue of patients with this antibody has almost no inflammatory cell infiltration, no demyelination, no remyelination, and no onion bud findings, and when observed under an electron microscope, the myelin sheath is characteristically separated from the axon. Anti-NF155 antibody-positive autoimmune nodulopathy tends to develop subacutely in young adults in their 20s and 30s. Patients with anti-NF155 antibodies have prominent symptoms distal to the limbs, and may exhibit ataxia and tremor. Nerve conduction studies may show prolonged distal motor neuron latency, slowed nerve conduction velocity, prolonged F wave latency, conduction block, and temporal dispersion. Cerebrospinal fluid analysis reveals markedly elevated protein levels, and imaging studies reveal enlarged spinal cord roots and plexuses. Current reports suggest that anti-NF155 antibody-positive patients exhibit high-amplitude, low-frequency tremors. Therapeutic options include corticosteroids and intravenous immunoglobulin, with plasma exchange and rituximab providing superior therapeutic responses.

[0038] CNTN1 is a membrane protein expressed in the axons of the perinodal region. It functions as a cell adhesion molecule and forms a septate-like junction that connects the myelin ring and the axon by forming a complex with NF155 and Caspr1. Patients with anti-CNTN1 antibody-positive peripheral neuropathy may exhibit axonal loss and nerve fiber degeneration in the gastrocnemius nerve, but onion bulbs and marked demyelination are not observed. Destruction of the perinodal structure of the myelinated nerves may be observed in skin tissue. Anti-CNTN1 antibody-positive autoimmune nodular disease has a subacute onset, presents with a sensorimotor polyneuropathy, and can cause ataxia and severe neurological symptoms. Tremor may also occur, although less common than anti-NF155 antibody-positive autoimmune nodular disease. Membranous glomerulonephritis may also accompany this disease. Nerve conduction studies reveal prolonged distal motor neuron latency, decreased nerve conduction velocity, prolonged F-wave latency, conduction block, and temporal dispersion, but early reduction in compound muscle action potential (CMAP) amplitude and signs of acute denervation may be observed. Treatment rarely responds to intravenous immunoglobulin (IVIg), but may partially respond to corticosteroids and well respond to rituximab.

[0039] Caspr1 is a membrane protein expressed in the axons of the perinodal region and forms a complex with CNTN1, binding to NF155 located in the myelin ring of Schwann cells. Anti-Caspr1 antibody-positive autoimmune nodular disease has a subacute onset, with more pronounced symptoms in the distal limbs and a clinical presentation characterized by both sensory and motor disturbances. Furthermore, it exhibits significantly more severe neuropathic pain compared to other autoimmune nodular diseases. Nerve conduction studies have shown prolonged distal motor neuron latency, decreased nerve conduction velocity, prolonged F-wave latency, conduction block, and temporal dispersion. It responds poorly to intravenous immunoglobulin, but responds well to plasma exchange or rituximab.

[0040] NF186 is a cell adhesion molecule expressed in the axonal node and interacts with gliomedin expressed on Schwann cells to function as a bond between Schwann cell microvilli and axons. Most patients with anti-NF186 antibodies tend to have a subacute onset and present with sensory loss, and some may present with cranial nerve involvement, neuropathic pain, and tremor. Intravenous immunoglobulin (IVIg) and steroid treatment may be effective in some patients. Nephrotic syndrome or respiratory failure may accompany the disease, and nerve conduction studies may show conduction block.

[0041] Patients with two or more of the aforementioned antibodies may present with severe disease progression, including cranial nerve involvement, quadriplegia, and respiratory failure, or may develop nephrotic syndrome. Patients are often refractory to immunotherapy, including immunoglobulin, and rituximab may be effective.

[0042] In this way, identifying the autoantibodies responsible for autoimmune nodular disease in patients with chronic inflammatory neuropathy (CIPN) could allow for the avoidance of IVIg, which is only effective for CIDP, and rapid administration of rituximab, which is effective for autoimmune nodular disease, thereby rapidly improving neurological deficits in patients with CIPN. Furthermore, the type of autoantibody can influence the disease presentation and subsequent treatment options. For example, identifying anti-CNTN1 antibodies can help identify not only peripheral nerve abnormalities but also concomitant glomerulonephritis, helping prevent renal dysfunction.

[0043] The ELISA test currently performed to diagnose autoimmune nodular disease is performed by performing one type of antibody detection test on a sample obtained from the patient, and if the result is negative, repeating the process of performing another antibody detection test.

[0044] The present invention, which improves upon the conventional method of diagnosing autoimmune nodular disease, can simultaneously test four antibodies to determine the type of autoantibody detected in a sample at once, thereby shortening the time, samples, and specimens required for testing and diagnosis by up to 1 / 4, and enabling customized treatment according to the type of antibody possessed by the patient.

[0045] If patients diagnosed with autoimmune nodular neuropathy are quickly and appropriately treated, even those who have been unable to walk for a long time due to sensory ataxia can regain independent walking. Therefore, the present invention not only facilitates the accurate and rapid diagnosis of autoimmune nodular neuropathy, but also enables the rapid application of optimal treatment to diagnosed patients.

[0046]

[0047] The present invention provides a multiplex immunoassay method for diagnosing autoimmune nodular disease, comprising the following steps: (S1) a step of binding CNTN1, Caspr1, NF155 and NF186 proteins to microspheres having different color codes, respectively; (S2) a step of mixing microspheres having different color codes to which proteins are each bound; (S3) a step of treating a biological sample to the mixed microspheres to induce an antigen-antibody reaction; (S4) a step of treating and binding a detection antibody to the microspheres in which the antigen-antibody reaction is induced; and (S5) a step of reading fluorescence information of the microspheres to which the detection antibody is bound.

[0048] The steps from (S1) to (S5) are described below.

[0049] (S1) Step

[0050] (S1) Step is the step of binding CNTN1, Caspr1, NF155, and NF186 proteins to four microspheres with different color codes, respectively.

[0051] (S1) The step may consist of the following substeps:

[0052] (S1a) A step of placing microspheres with color codes in a buffer solution of pH 6 to 7, adding N-hydroxysulfosuccinimide and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide, and incubating at room temperature;

[0053] (S1b) a step of isolating microspheres and adding them to phosphate buffered saline (PBS) to prepare a suspension; and

[0054] (S1c) A step of adding CNTN1, Caspr1, NF155 or NF186 protein to the above suspension and incubating at room temperature.

[0055] (S1) Step can be performed in a pH 6 to 7 buffer solution.

[0056] The above buffer may be, but is not limited to, a monobasic sodium phosphate buffer.

[0057] This step is a step in which microspheres having different color codes by combining different concentrations of various fluorescent dyes are combined with CNTN1, Caspr1, NF155, and NF186, respectively.

[0058] In one embodiment, CNTN1, Caspr1, NF155 and NF186 are covalently linked to microspheres having color codes.

[0059] In one embodiment, CNTN1, Caspr1, NF155 and NF186 are covalently linked to color-coded microspheres via 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) / N-hydroxysulfosuccinimide (sulfo-NHS) bonds.

[0060] The microspheres having the above color code may have a diameter of 5 μm to 7 μm, but are not limited thereto.

[0061] In one embodiment, the diameter of the microspheres having a color code can be about 5.0 μm, 5.1 μm, 5.2 μm, 5.3 μm, 5.4 μm, 5.5 μm, 5.6 μm, 5.7 μm, 5.8 μm, 5.9 μm, 6.0 μm, 6.1 μm, 6.2 μm, 6.3 μm, 6.4 μm, 6.5 μm, 6.6 μm, 6.7 μm, 6.8 μm, 6.9 μm or 7.0 μm.

[0062] In one embodiment, the diameter of the microspheres having color codes may be about 6.5 μm.

[0063] The microspheres having the above color codes may be magnetic microspheres or non-magnetic microspheres.

[0064] (S2) Step

[0065] Step (S2) is a step of mixing microspheres having a color code combined with CNTN1 manufactured in step (S1), microspheres having a color code combined with Caspr1, microspheres having a color code combined with NF155, and microspheres having a color code combined with NF186.

[0066] (S3) Step

[0067] Step (S3) is a step of inducing an antigen-antibody reaction by treating a biological sample with the microspheres mixed in step (S2).

[0068] The biological sample may be any one selected from the group consisting of, but is not limited to, tissue, cells, blood, serum, plasma, and cerebrospinal fluid.

[0069] In one embodiment, the biological sample may be blood.

[0070] When anti-CNTN1 antibody, anti-Caspr1 antibody, anti-NF155 antibody and / or anti-NF186 antibody are present in the biological sample, they bind to CNTN1, Caspr1, NF155 and / or NF186, respectively, bound to the color-coded microspheres in an antigen-antibody reaction.

[0071] (S4) Step

[0072] (S4) Step is a step of adding a detection antibody. If an anti-CNTN1 antibody, an anti-Caspr1 antibody, an anti-NF155 antibody, or an anti-NF186 antibody is bound to a color-coded microsphere, the detection antibody can bind to it.

[0073] The detection antibody may be labeled with a fluorescent substance. A fluorescent substance is a substance that becomes excited when exposed to a specific wavelength or LED, and emits excess energy as fluorescence when returning from the excited state to a steady state. The types of fluorescent substances are not limited.

[0074] In one embodiment, the detection antibody may be a phycoerythrin-labeled anti-human IgG antibody.

[0075] In one embodiment, the detection antibody may be a PE-labeled anti-human IgG4 antibody.

[0076] (S5) Step

[0077] Step (S5) is a step of reading the fluorescence information of the microspheres to which the detection antibody is bound in step (S4).

[0078] This step allows for the detection of the presence and quantification of anti-CNTN1 antibodies, anti-Caspr1 antibodies, anti-NF155 antibodies, and anti-NF186 antibodies in a biological sample.

[0079] Microspheres with color codes can be labeled by exciting the dye within the beads with a red laser or red LED.

[0080] When anti-CNTN1 antibody, anti-Caspr1 antibody, anti-NF155 antibody or anti-NF186 antibody is bound to the microsphere having a color code, and a detection antibody is bound thereto in step (S4), the amount of the detection antibody can be quantified using a laser or LED of a specific wavelength.

[0081] (S4) When a PE-labeled anti-human IgG antibody is used in step, the presence or absence of anti-CNTN1 antibody, anti-Caspr1 antibody, anti-NF155 antibody, or anti-NF186 antibody can be measured and the amount thereof quantified by exciting the PE dye with a green laser or green LED.

[0082] In this way, the multiplex immunoassay method for diagnosing autoimmune nodular disease of the present invention can simultaneously detect four antibodies, i.e., anti-CNTN1 antibody, anti-Caspr1 antibody, anti-NF155 antibody, and anti-NF186 antibody, by including steps (S1) to (S5).

[0083] The present invention provides a method for diagnosing autoimmune nodular disease, comprising the step of simultaneously detecting anti-CNTN1 antibodies, anti-Caspr1 antibodies, anti-NF155 antibodies, and anti-NF186 antibodies.

[0084] The step of simultaneously detecting the above antibodies may include the same steps as the multiplex immunoassay method described above.

[0085] In one embodiment, the step of simultaneously detecting anti-CNTN1 antibody, anti-Caspr1 antibody, anti-NF155 antibody, and anti-NF186 antibody comprises the following steps: (S1) binding CNTN1, Caspr1, NF155, and NF186 to microspheres having different color codes, respectively; (S2) mixing microspheres having different color codes, each of which is bound to the proteins; (S3) treating a biological sample with the mixed microspheres to induce an antigen-antibody reaction; (S4) treating and binding a detection antibody to the microspheres in which the antigen-antibody reaction is induced; and (S5) reading the fluorescence information of the microspheres to which the detection antibody is bound. In one embodiment, the diagnosis of autoimmune nodular disease is performed by comparing the amount of the above-mentioned antibodies in the blood of a healthy control and in the blood of a patient. For example, a patient may be diagnosed with autoimmune nodular disease if the amount of antibodies in their blood satisfies the following equation:

[0086] MFI of the normal group + (SD X 5) < patient's blood antibody level, (where MFI is mean fluorescence intensity and SD is standard deviation.)

[0087]

[0088] Hereinafter, the present invention will be described in detail with examples to specifically illustrate the invention. However, the following examples are provided merely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention.

[0089]

[0090] Example

[0091]

[0092] 1. Manufacturing antigen-bound microspheres

[0093]

[0094] 1) Add color-coded microspheres (Luminex Bead / xMAP Microspheres - Luminex) to monobasic sodium phosphate (pH 6.2), add Sulfo-NHS (50 mg / mL) and EDC (50 mg / mL), and incubate at room temperature for 20 minutes. Afterwards, separate the microspheres, add the microspheres and CNTN1 (SinoBiological, Beijing, China) protein to phosphate-buffered saline (PBS), and incubate at room temperature for 2 hours.

[0095]

[0096] 2) The same process as in 1) is performed except that microspheres and Caspr1 (R&D systems, Minneapolis, MN, USA) protein with different color codes are used.

[0097]

[0098] 3) The same process as in 1) is performed except that microspheres and NF155 (OriGene, Rockville, MD, USA) protein with different color codes than in 1) and 2) are used.

[0099]

[0100] 4) The same process as in 1) is performed except that microspheres and NF186 (OriGene, Rockville, MD, USA) protein with different color codes than in 1) to 3) are used.

[0101]

[0102] Through steps 1) to 4), microspheres in which CNTN1, Caspr1, NF155, and NF186, each having a different color code, are connected are obtained (Fig. 1). In Fig. 1, R' represents CNTN1, Caspr1, NF155, or NF186.

[0103]

[0104] 2. Multiplex immunoassay

[0105] The four microspheres connected with CNTN1, Caspr1, NF155, and NF186 prepared in the above '1' are mixed. The prepared mixture is placed into a single well(s) of a 96-well plate. Samples, i.e., negative control, positive control (anti-CNTN1, anti-Caspr1 antibody, anti-NF155, and anti-NF186), or patient serum are added to each well. Antigen-antibody reaction is induced. A PE-labeled anti-human IgG antibody is added as a detection antibody. The fluorescence information of the microspheres is read using a dual-laser flow-based detection instrument (Luminex) or a MAGPIX system. The red laser or LED identifies the type of microsphere and identifies microspheres bound to CNTN1, Caspr1, NF155 or NF186, and the green laser or LED measures PE fluorescence intensity to quantify the amount of anti-CNTN1 antibody, anti-Caspr1 antibody, anti-NF155 antibody or anti-NF186 antibody in the sample (Fig. 2).

[0106]

[0107] 3. Results

[0108] The detection results obtained by performing '2' multiplex immunoassay on 31 samples from 5 control samples and 19 patients diagnosed with CIDP were compared with the results of ELISA test.

[0109] In the multiplex immunoassay of '2', four antibody analyses were performed at once, and the ELISA test was performed once for each antibody, for a total of four analyses.

[0110] The results of the multiplex immunoassay and ELISA tests are shown in Table 1 below.

[0111]

[0112] The correlation coefficients of the two test results were r=0.84 for NF-155, r=0.99 for NF-186, r=0.88 for CNTN1, and r=0.99 for Caspr1, showing high correlation coefficients for all four antibody analyses (Fig. 3).

[0113] For the NF-155 antibody, 17 out of 31 CIDP samples were positive and 14 were negative when applied to the multiplex immunoassay, whereas 15 were positive and 16 were negative when applied to the ELISA. For the NF-186, CNTN1, and Caspr1 antibodies, the positive and negative results of the multiplex immunoassay and ELISA were consistent. 1 sample was positive for the NF-186 antibody, 3 samples were positive for the CNTN1 antibody, and 2 samples were positive for the Caspr1 antibody (Fig. 4).

[0114] The results of the multiplex immunoassay test were quite consistent with the ELISA results, and the sensitivity of the multiplex immunoassay was higher than that of the ELISA for the NF-155 antibody.

[0115] Through this, it was confirmed that the accuracy of the multiplex immunoassay method according to the present invention, which detects four antibodies simultaneously, is excellent, and that there is no interference and / or influence between different antigen-antibody reactions.

Claims

1. (S1) A step of binding CNTN1 (contactin-1), Caspr1 (contactin-associated protein-1), NF155 (neurofascin-155), and NF186 (neurofascin-186) proteins to microspheres having different color codes; (S2) A step of mixing microspheres having different color codes to which the above proteins are each bound; (S3) A step of treating a biological sample with the above mixed microspheres to induce an antigen-antibody reaction; (S4) A step of treating and binding a detection antibody to the microsphere in which the antigen-antibody reaction is induced; and (S5) A multiplex immunoassay method for diagnosing autoimmune nodular disease, comprising a step of reading fluorescence information of microspheres to which the above detection antibody is bound.

2. In claim 1, (S1) is a method performed at pH 6 to 7.

3. In claim 1, (S1) is a method comprising the following steps: (S1a) A step of placing microspheres with color codes in a buffer solution of pH 6 to 7, adding N-hydroxysulfosuccinimide and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide, and incubating at room temperature; (S1b) a step of isolating the microspheres and adding them to phosphate buffered saline (PBS) to prepare a suspension; and (S1c) A step of adding CNTN1, Caspr1, NF155 or NF186 protein to the above suspension and incubating at room temperature.

4. A method according to claim 1, wherein the detection antibody is a phycoerythrin-labeled anti-human IgG antibody.

5. A method according to claim 1, wherein the biological sample is any one selected from the group consisting of tissue, cell, blood, serum, plasma, and cerebrospinal fluid.

6. A method for diagnosing autoimmune nodular disease, comprising the step of simultaneously detecting anti-CNTN1 antibody, anti-Caspr1 antibody, anti-NF155 antibody and anti-NF186 antibody.

7. A method for diagnosing autoimmune nodular disease according to claim 6, wherein the step of detecting the antibody comprises the following steps: (S1) Step of binding CNTN1, Caspr1, NF155 and NF186 to microspheres having different color codes, respectively; (S2) A step of mixing microspheres having different color codes to which the above proteins are each bound; (S3) A step of treating a biological sample with the above mixed microspheres to induce an antigen-antibody reaction; (S4) a step of treating and binding a detection antibody to the microspheres in which the antigen-antibody reaction is induced; and (S5) A step of reading the fluorescence information of the microsphere to which the above detection antibody is bound.

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