WO2025211530A1 - Multiparameter flow cytometry for simultaneously detecting node-paranodal autoantibodies in inflammatory neuropathy patients - Google Patents

Abstract

The present invention relates to multiparameter flow cytometry for simultaneously detecting node-paranodal autoantibodies in inflammatory neuropathy patients. Specifically, four different types of cells expressing four different node-paranodal autoantigens (NF155, CNTN1, CASPR1 and NF186) and fluorescent markers are prepared and mixed, the mixed four types of cells are reacted with a sample from a patient with inflammatory peripheral neuropathy, and then flow cytometry is performed so that it is identified that autoantibodies against the four types of autoantigens can be detected simultaneously and the isotypes thereof can be determined, and thus, the cells and flow cytometry using same can be effectively used in autoantibody test for diagnosing, monitoring or predicting the treatment responses of autoimmune nodopathies from among inflammatory neuropathies.

Description

A multiplex flow cytometry method for simultaneous detection of nodal-paranodal autoantibodies in patients with inflammatory neuropathy.

The present invention relates to a multiplex flow cytometry method for simultaneous detection of nodal-paranodal autoantibodies in patients with inflammatory neuropathy, and more particularly, to a multiplex cell-based flow cytometry method for simultaneous detection and quantification of nodal-paranodal autoantibodies in patients with inflammatory neuropathy, and a method for providing information for diagnosing, monitoring, or predicting treatment response to autoimmune nodopathy (AN) using the same.

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an inflammatory neuropathy. It is an autoimmune disease in which the immune system attacks self-antigens, causing the myelin sheath to be attacked. The disease causes significant impairment in daily life and requires long-term immunotherapy. In some patients, the disease progresses rapidly and does not respond to immunotherapy.

Recently, autoantibodies targeting the nodes and paranodes have been reported in some CIDP patients. The nodes and paranodes are crucial components that form a strong bond between Schwann cells and nerve axons, enabling nerve conduction. To date, four autoantigens have been reported: NF155 (neurofascin-155), NF186 (neurofascin-186), CASPR1 (contactin-associated protein 1), and CNTN1 (contactin-1). These autoantibodies are present in approximately 10% of all CIDP patients and are characterized by IgG4 subtype autoantibodies. They are less responsive to conventional CIDP primary treatment and are highly effective with anti-B cell therapies. Therefore, in the 2021 revised CIDP guidelines, they were designated as a separate disease, Autoimmune Nodopathy (AN).

The revised guidelines recommend testing for AN autoantibodies in all CIDP patients, with cell-based assays (CBAs) being the standard antibody testing method. Because a significant proportion of AN cases present with an acute onset similar to Guillain-Barré syndrome (GBS), an acute form of CIDP, GBS patients are also considered a useful target for testing.

Meanwhile, CBA can be performed by fixing cells (Fixed CBA), which makes the test more convenient and easy. However, due to issues such as antigen structure modification and autofluorescence interference caused by fixation, CBA using live cells (Live CBA) is more recommended in terms of test quality. In addition, the fluorescence microscopy (CBA-IF), the most widely used method for detecting autoantibodies, is difficult to quantitatively analyze, and it is cumbersome to measure titers by repeatedly performing serial dilutions. Moreover, in the case of AN, since patients positive for the four autoantibodies show almost identical clinical features, all four antibodies must be tested for diagnosis. Therefore, CBA for each of the four autoantigens must be repeated, and if necessary, additional serial dilutions must be performed, which is burdensome for the experimenter, requires excessive testing time, requires excessive samples, and increases errors between tests and testing costs.

Accordingly, the present inventors have made efforts to develop a method for detecting nodal-paranodal autoantibodies that can shorten the testing time and cost and have high accuracy. As a result, they produced cells expressing four different nodal-paranodal autoantigens (NF155, CNTN1, CASPR1, and NF186) and fluorescent markers, mixed them, reacted the mixed four types of cells with a sample of a patient with inflammatory peripheral neuropathy, and then performed flow cytometry analysis, confirming that autoantibodies against the four types of autoantigens can be detected at once and the isotypes of the autoantibodies can be determined. Accordingly, the present application has been made by revealing that the cells and the flow cytometry method using the same can be usefully utilized in an autoantibody test for diagnosing, monitoring, or predicting treatment response of AN among patients with inflammatory neuropathy, including AN, CIDP, and GBS.

[Prior Art Literature]

[Non-patent literature]

Yoo Hyun-ji and Shin Ha-young, "Autoimmune nodular disease," Journal of Multiple Sclerosis and Neuroimmunology 13(2):23-29, 2022

A Cortese et al., "Antibodies to neurofascin, contactin-1, and contactin-associated protein 1 in CIDP", Neurol Neuroimmunol Neuroinflamm 2020;7:e639

Fehmi J et al., "IgG1 pan-neurofascin antibodies identify a severe yet treatable neuropathy with a high mortality", J Neurol Neurosurg Psychiatry 2021;92:1089-1095

The purpose of the present invention is to provide a method for detecting node-paranode autoantibodies with high accuracy and which can shorten the examination time and cost, and a method for diagnosing autoimmune nodular disease among patients with inflammatory neuropathy using the same.

In order to achieve the purpose of the present invention, the present invention provides a method for detecting NF155-IgG, NF186-IgG, CASPR1-IgG and CNTN1-IgG as autoantibodies from a sample,

1) A step of obtaining cells of the following (i) to (iv), wherein the fluorescent markers of the cells of the following (i) to (iv) are three or more types of distinct fluorescent markers, and each of the cells of the following (i) to (iv) includes one or more types of fluorescent markers;

(i) cells containing NF155 (neurofascin-155) and fluorescent markers,

(ii) cells containing NF186 (neurofascin-186) and fluorescent markers;

(iii) cells containing CASPR1 (contactin-associated protein 1) and fluorescent markers, and

(iv) cells containing CNTN1 (contactin-1) and fluorescent markers;

2) A step of mixing all of the cells of (i) to (iv) above;

3) A step of incubating together the four types of cells mixed above and a sample expected to contain autoantibodies;

4) A step of adding a fluorescent-conjugated secondary antibody to the sample after incubation to form an autoantibody and secondary antibody complex; and

5) A method is provided, comprising a step of detecting or analyzing fluorescence from the autoantibody and secondary antibody conjugate.

In addition, the present invention provides a method for providing information for diagnosing, monitoring, or predicting treatment response to autoimmune nodopathy.

1) A step of obtaining cells of the following (i) to (iv), wherein the fluorescent markers of the cells of the following (i) to (iv) are three or more types of distinct fluorescent markers, and each of the cells of the following (i) to (iv) includes one or more types of fluorescent markers;

(i) cells containing NF155 and fluorescent markers;

(ii) cells containing NF186 and fluorescent markers;

(iii) cells containing CASPR1 and a fluorescent marker; and

(iv) cells containing CNTN1 and fluorescent markers;

2) A step of mixing all of the cells of (i) to (iv) above;

3) A step of incubating together the four types of cells mixed above with a sample isolated from a patient expected to contain at least one autoantibody among NF155-IgG, NF186-IgG, CASPR1-IgG, and CNTN1-IgG;

4) A step of adding a fluorescent-conjugated secondary antibody to the sample after incubation to form an autoantibody and secondary antibody complex; and

5) A method is provided, comprising a step of detecting or analyzing fluorescence from the autoantibody and secondary antibody conjugate.

In addition, the present invention

(a) Cells containing NF155 and fluorescent markers;

(b) cells containing NF186 and fluorescent markers;

(c) cells containing CASPR1 and a fluorescent marker; and

(d) cells containing CNTN1 and a fluorescent marker,

The fluorescent markers of the cells of (a) to (d) above are three or more types of distinct fluorescent markers, and each of the cells of (i) to (iv) above contains one or more types of fluorescent markers, thereby providing a composition for diagnosing, monitoring, or predicting a treatment response to autoimmune nodular disease.

In the present invention, four types of cells expressing four different nodal-paranodal autoantigens (NF155, CNTN1, CASPR1, and NF186) and fluorescent markers were produced and mixed, and the mixed four types of cells were reacted with a sample of a patient with inflammatory peripheral neuropathy, and then flow cytometry was performed to detect autoantibodies against the four types of autoantigens at once, and it was confirmed that the isotypes of the autoantibodies could be determined. Therefore, the cells and the flow cytometry method using the same can be usefully used in an autoantibody test for diagnosing, predicting, and monitoring autoimmune nodopathy, one of inflammatory neuropathy.

FIG. 1 is a diagram illustrating a cell classification strategy for a multi-cell-based flow cytometry method according to one embodiment of the present invention.

FIG. 2 is a diagram showing the results of performing a multiplex cell-based flow cytometry analysis method for detecting node-paranode autoantibodies according to one embodiment of the present invention.

FIG. 3 is a diagram showing the results of performing flow cytometry for isotyping of node-paranode autoantibodies according to one embodiment of the present invention.

FIG. 4 is a diagram showing the results of screening a Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) or Guillain-Barré syndrome (GBS) cohort using a multi-cell-based flow cytometry method for detecting node-paranode autoantibodies according to one embodiment of the present invention.

FIG. 5 is a diagram showing the results of a fluorescence microscope-based single antigen cell-based test performed using the above CIDP and GBS cohort samples according to the mouse peripheral nerve reaction assay and the standard test method. In the mouse peripheral nerve reaction assay (Mouse Nerve Immunofluorescence), "Anti-NF155" is a commercially available NF155 antibody that labels the peripheral nerve paranode, "IgG" indicates the part where IgG in the patient's serum binds, and in the case below, it can be confirmed that the serum reacts to the paranode. In the fluorescence microscope-based single antigen cell-based test (Cell-based Immunofluorescence), "NF155-GFP" is a cell transfected with an NF155 expression vector, "Hoechst33342" indicates the nucleus of the entire cell, and IgG indicates the binding of IgG in the patient's serum. In the case below, it can be confirmed that the serum specifically reacts to the cells transfected with the NF155 expression vector.

Hereinafter, the present invention will be described in more detail.

The present invention is a method for detecting NF155-IgG, NF186-IgG, CASPR1-IgG and CNTN1-IgG as autoantibodies from a sample,

1) A step of obtaining cells of the following (i) to (iv), wherein the fluorescent markers of the cells of the following (i) to (iv) are three or more types of distinct fluorescent markers, and each of the cells of the following (i) to (iv) includes one or more types of fluorescent markers;

(i) cells containing NF155 (neurofascin-155) and fluorescent markers,

(ii) cells containing NF186 (neurofascin-186) and fluorescent markers;

(iii) cells containing CASPR1 (contactin-associated protein 1) and fluorescent markers, and

(iv) cells containing CNTN1 (contactin-1) and fluorescent markers;

2) A step of mixing all of the cells of (i) to (iv) above;

3) A step of incubating together the four types of cells mixed above and a sample expected to contain autoantibodies;

4) A step of adding a fluorescent-conjugated secondary antibody to the sample after incubation to form an autoantibody and secondary antibody complex; and

5) A method is provided, comprising a step of detecting or analyzing fluorescence from the autoantibody and secondary antibody conjugate.

In the present invention, the neurofascin-155 (NF155), neurofascin-186 (NF186), contactin-associated protein 1 (CASPR1), and contactin-1 (CNTN1) are cell adhesion molecules present in the nodes or paranodes of the nodes of Ranvier. With the development of cell-based assays (CBA) using NF155, NF186, CASPR1, and CNTN1 as substrates, it has been known that NF155-immunoglobulin G antibody (IgG), NF186-IgG, CASPR1-IgG, and CNTN1-IgG are associated with nodal-paranode autoantibodies in various inflammatory neuropathic patients.

In the method of the present invention, the fluorescent marker may be three types of distinct fluorescent markers, and in this case, the following steps may be performed:

1-1) A step of obtaining cells of the following (i) to (iv);

(i) cells containing NF155 and a first fluorescent marker;

(ii) cells containing NF186 and a second fluorescent marker;

(iii) cells containing CASPR1 and a first fluorescent marker, and CNTN1 and a second fluorescent marker; and

(iv) cells containing CNTN1 and a second fluorescent marker;

1-2) A step of staining the nuclei of two types of cells among the cells (i) to (iv) above with a third fluorescent marker to contain the third fluorescent marker;

2) A step of mixing all of the cells of (i) to (iv) above;

3) A step of incubating together the four types of cells mixed above and a sample expected to contain autoantibodies;

4) A step of adding a fluorescent-conjugated secondary antibody to the sample after incubation to form an autoantibody and secondary antibody complex; and

5) A step of detecting or analyzing fluorescence from the above autoantibody and secondary antibody complex.

In addition, the cells (i) to (iv) in the above step 1-1) can be obtained by performing the following steps:

(1) A step of obtaining an autoantigen expression vector by performing the following (a) to (d);

(a) cloning a nucleic acid encoding NF155 into a first fluorescent marker expression vector;

(b) cloning a nucleic acid encoding NF186 into a second fluorescent marker expression vector;

(c) cloning a nucleic acid encoding CASPR1 into a first fluorescent marker expression vector; and

(d) cloning the nucleic acid encoding CNTN1 into a second fluorescent marker expression vector;

(2) A step of transfecting each cell with the cloned autoantigen expression vectors of (a) to (d) as follows to obtain cells of (i) to (iv);

i) Transfecting the expression vector of (a) into a cell to obtain the cell of (i), specifically, a cell containing NF155 and the first fluorescent marker;

ii) Transfecting the expression vector of (b) into a cell to obtain the cell of (ii), specifically, a cell containing NF186 and a second fluorescent marker;

iii) transfecting the expression vectors of (c) and (d) into cells to obtain cells of (iii), specifically cells containing CASPR1 and the first fluorescent marker, and CNTN1 and the second fluorescent marker; and

iv) Transfecting the expression vector of (d) into a cell to obtain the cell of (iv), specifically, a cell containing CNTN1 and a second fluorescent marker.

In addition, in the above step 1-2), for the two types of cells containing the third fluorescent marker, cells negative for the first fluorescent marker, the second fluorescent marker, and the third fluorescent marker can be used as a control, and for the two types of cells not containing the third fluorescent marker, cells negative for the first fluorescent marker and the second fluorescent marker, and positive for the third fluorescent marker can be used as a control. By using the above control, the sensitivity of the test can be increased.

In the method of the present invention, the fluorescent marker may be four types of distinct fluorescent markers, in which case the following steps may be performed:

1) A step of obtaining cells of the following (i) to (iv);

(i) cells containing NF155 and a first fluorescent marker;

(ii) cells containing NF186 and a second fluorescent marker;

(iii) cells containing CASPR1 and a third fluorescent marker; and

(iv) cells containing CNTN1 and a fourth fluorescent marker;

2) A step of mixing all of the cells of (i) to (iv) above;

3) A step of incubating together the four types of cells mixed above and a sample expected to contain autoantibodies;

4) A step of adding a fluorescent-conjugated secondary antibody to the sample after incubation to form an autoantibody and secondary antibody complex; and

5) A step of detecting or analyzing fluorescence from the above autoantibody and secondary antibody complex.

In addition, the cells (i) to (iv) in step 1) above can be obtained by performing the following steps:

(1) A step of obtaining an autoantigen expression vector by performing the following (a) to (d);

(a) cloning a nucleic acid encoding NF155 into a first fluorescent marker expression vector;

(b) cloning a nucleic acid encoding NF186 into a second fluorescent marker expression vector;

(c) cloning a nucleic acid encoding CASPR1 into a third fluorescent marker expression vector; and

(d) Cloning the nucleic acid encoding CNTN1 into a fourth fluorescent marker expression vector;

(2) A step of transfecting each cell with the cloned autoantigen expression vectors of (a) to (d) as follows to obtain cells of (i) to (iv);

i) Transfecting the expression vector of (a) into a cell to obtain the cell of (i), specifically, a cell containing NF155 and the first fluorescent marker;

ii) Transfecting the expression vector of (b) into a cell to obtain the cell of (ii), specifically, a cell containing NF186 and a second fluorescent marker;

iii) transfecting the expression vectors of (c) and (d) into cells to obtain cells of (iii), specifically cells containing CASPR1 and a third fluorescent marker; and

iv) Transfecting the expression vector of (d) into a cell to obtain the cell of (iv), specifically, a cell containing CNTN1 and the fourth fluorescent marker.

In the method of the present invention, cells can be distinguished using fluorescent markers that are distinct from each other in step 1). The fluorescent marker may be, but is not limited to, GFP (green fluorescent protein), YFP (yellow fluorescent protein), EGFP (enhanced green fluorescent protein), CFP (cyan fluorescent protein), OFP, RFP (red fluorescent protein), DsRed2, Tdtomato, mCherry, Hoechst 33258, Hoechst 33342, or Hoechst 34580.

In the method of the present invention, the cell is a target cell for transfection and may be a mammalian cell, such as HEK (human embryonic kidney) 293, HEK 293T, HEK 293A, HeLa, CHO (Chinese hamster ovary), or NIH3T3 cell, but is not limited thereto.

Meanwhile, the cells of (i) to (iv) above are used to detect NF155-IgG, NF186-IgG, CASPR1-IgG and CNTN1-IgG as autoantibodies in a sample and further to diagnose autoimmune nodopathy (AN) among inflammatory neuropathy. In the method of the present invention, the "sample" may be blood, serum, plasma or cerebrospinal fluid isolated from a patient with inflammatory neuropathy or isolated from a patient expected to contain the autoantibodies, and specifically, may be serum. The sample may be diluted at an appropriate ratio as needed by a person skilled in the art and used.

In the method of the present invention, the inflammatory neuropathy may be autoimmune nodopathy (AN), chronic inflammatory demyelinating polyneuropathy (CIDP), or Guillain-Barré syndrome (GBS), and among the inflammatory neuropathy, autoimmune nodopathy may appear in patients positive for autoantibodies such as NF155-IgG, NF186-IgG, CASPR1-IgG, and/or CNTN1-IgG.

In the method of the present invention, in steps 3) and 4), a cell mixture containing all of the cells from (i) to (iv) of step 2) is incubated with the sample, and then a fluorescent-conjugated secondary antibody is added to the sample after the incubation is completed, thereby forming a complex of each of NF155-IgG, NF186-IgG, CASPR1-IgG, and CNTN1-IgG with the secondary antibody.

Additionally, the "fluorescent-conjugated secondary antibody" is for cell-based assay (CBA), and the secondary antibody may be anti-human IgG or anti-human IgG-Fc. Furthermore, the fluorescence of the secondary antibody may be selected at a wavelength that does not interfere with the fluorescent markers used for cell differentiation.

In addition, a step of performing isotype analysis (isotyping) of the autoantibody using the sample in which incubation has been completed in the above step 4) may be further included, and specifically, an antibody for IgG1, IgG2, IgG3, or IgG4 may be added as a secondary antibody for performing isotype analysis using flow cytometry. Accordingly, by performing the isotype analysis, the isotype of the autoantibody, specifically, IgG1, IgG2, IgG3, or IgG4, can be determined.

In the method of the present invention, “detecting or analyzing fluorescence” in step 5) may be performing a flow cytometry assay (FACS).

In addition, the present invention provides a method for providing information for diagnosing, monitoring, or predicting treatment response to autoimmune nodular disease.

1) A step of obtaining cells of the following (i) to (iv), wherein the fluorescent markers of the cells of the following (i) to (iv) are three or more types of distinct fluorescent markers, and each of the cells of the following (i) to (iv) includes one or more types of fluorescent markers;

(i) cells containing NF155 and fluorescent markers;

(ii) cells containing NF186 and fluorescent markers;

(iii) cells containing CASPR1 and a fluorescent marker; and

(iv) cells containing CNTN1 and fluorescent markers;

2) A step of mixing all of the cells of (i) to (iv) above;

3) A step of incubating together the four types of cells mixed above with a sample isolated from a patient expected to contain at least one autoantibody among NF155-IgG, NF186-IgG, CASPR1-IgG, and CNTN1-IgG;

4) A step of adding a fluorescent-conjugated secondary antibody to the sample after incubation to form an autoantibody and secondary antibody complex; and

5) A method is provided, comprising a step of detecting or analyzing fluorescence from the autoantibody and secondary antibody conjugate.

In the method of the present invention, the contents of the cells and the method for obtaining the cells, fluorescent markers, samples, etc. are as described above.

In the method of the present invention, the patient may be a patient with inflammatory neuropathy, and the inflammatory neuropathy may be autoimmune nodopathy (AN), chronic inflammatory demyelinating polyneuropathy (CIDP), or Guillain-Barré syndrome (GBS). Specifically, autoimmune nodular disease can occur in patients who are positive for NF155-IgG, NF186-IgG, CASPR1-IgG and/or CNTN1-IgG as nodal-paranodal autoantibodies. Therefore, by confirming the presence and titer of NF155-IgG, NF186-IgG, CASPR1-IgG and CNTN1-IgG, their isotypes, etc. in patient samples, specifically, inflammatory neuropathy patient samples, through the above method, information for diagnosing, monitoring or predicting treatment response to autoimmune nodular disease can be provided. Since the above method can detect NF155-IgG, NF186-IgG, CASPR1-IgG and CNTN1-IgG as the four nodal-paranodal autoantibodies at once, the time required to complete the test can be shortened by four times, and the experimenter's effort and errors between tests can also be reduced. In addition, the method utilizes living cells that are not fixed, and shows high accuracy and reliability based on objective and quantitative flow cytometry analysis.

In addition, the present invention

(a) Cells containing NF155 and fluorescent markers;

(b) cells containing NF186 and fluorescent markers;

(c) cells containing CASPR1 and a fluorescent marker; and

(d) cells containing CNTN1 and a fluorescent marker,

The fluorescent markers of the cells of (a) to (d) above are three or more types of distinct fluorescent markers, and each of the cells of (i) to (iv) above contains one or more types of fluorescent markers, thereby providing a composition for diagnosing, monitoring, or predicting a treatment response to autoimmune nodular disease.

In the present invention, the fluorescent marker, cells, cell acquisition, and autoimmune nodular disease are as described above.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

<Example 1> Production of cells containing autoantigens and fluorescent markers for nodal-paranodal autoantibodies for detecting nodal-paranodal autoantibodies

<1-1> Production of cells containing autoantigens and fluorescent markers for nodal-paranodal autoantibodies using three types of fluorescent markers

As shown in the schematic diagram in Fig. 1, four types of cells labeled with three types of fluorescent markers and expressing four different nodal-paranodal autoantigens (NF155, CNTN1, CASPR1/CNTN1, and NF186) were produced as a cell sorting strategy for multiplex cell-based flow cytometry.

Specifically, the nucleic acid encoding NF155 (neurofascin-155) and the nucleic acid encoding CASPR1 (contactin-associated protein 1) were each cloned into a turboGFP expression vector (pRP[Exp]-Neo-CMV>TurboGFP vector), and the nucleic acid encoding NF186 (neurofascin-186) and the nucleic acid encoding CNTN1 (contactin-1) were each cloned into a turboRFP expression vector (pRP[Exp]-Neo-CMV>TurboRFP vector).

In order to transfect the four types of vectors cloned above, the HEK293T (Human Embryonic Kidney 293 T) cell line (ATCC, #CRL-3216) was stabilized for more than one week, and then seeded into a 12-well plate at a density of 3.2×10 ⁵ cells 24 hours before transfection. After 24 hours, each of the four types of cloned vectors was transfected into cells using a transfection reagent (TransIT-293, TaKaRa) according to the manufacturer's procedure, thereby producing HEK293T cells expressing NF155 labeled with turboGFP (NF155+ cells), HEK293T cells expressing NF186 labeled with turboRFP (NF186+ cells), HEK293T cells expressing CASPR1 labeled with turboGFP and CNTN1 labeled with turboRFP (CASPR1+/CNTN1+ cells), and HEK293T cells expressing CNTN1 labeled with turboRFP (CNTN1+ cells). In the case of the CASPR1+/CNTN1+ cells, since CASPR1 is a protein that physiologically exists in a complex with CNTN1, the CASPR1 expression vector and the CNTN1 expression vector were co-transfected for sensitive detection of CASPR1 according to the guideline.

Additionally, on the second day of transfection, 1 ug/ml of Hoechst34580 (ThermoFisher) was added to the wells containing the NF155+ cells and NF186+ cells, and nuclear staining was performed for 20 to 25 minutes in an incubator.

<1-2> Production of cells containing autoantigens and fluorescent markers for nodal-paranodal autoantibodies using four types of fluorescent markers

As a cell sorting strategy for multiplex cell-based flow cytometry, four cell types labeled with four fluorescent markers and expressing four different nodal-paranodal autoantigens (NF155, CNTN1, CASPR1/CNTN1, and NF186) were generated.

Specifically, a nucleic acid encoding NF155 (neurofascin-155), a nucleic acid encoding CASPR1 (contactin-associated protein 1), a nucleic acid encoding NF186 (neurofascin-186), and a nucleic acid encoding CNTN1 (contactin-1) were each cloned into a turboGFP expression vector (pRP[Exp]-Neo-CMV>TurboGFP vector), a turboRFP expression vector (pRP[Exp]-Neo-CMV>TurboRFP vector), an OFP expression vector (pCMV3-C-OFPSpark ^® Vector), and a YFP expression vector (pRP[Exp]-Neo-CMV>EYFP vector), and each of the four cloned vectors was transfected into a HEK293T cell line using the same method as described in Example <1-2>.

<Example 2> Detection of nodal-paranodal autoantibodies using cells expressing autoantigens and fluorescent proteins for nodal-paranodal autoantibodies

The four different cells produced in the above Example <1-1> were mixed, and the serum of the patient to be tested was diluted 1:25 and reacted. Afterwards, a secondary antibody labeled with 647 fluorescence for the human IgG Fc portion was reacted. The fluorescence of the secondary antibody was selected at a wavelength that did not interfere with the Hoechst34580, turboRFP, and turboGFP used for the cell differentiation. Next, after gating on the four types of cells and non-transfected cells through flow cytometry, a scatter plot was drawn for each cell with the y-axis set to APC (647 fluorescence detection channel) to analyze whether there was an increase in APC fluorescence specifically in the transfected cells.

More specifically, the nuclear-stained NF155+ cells and NF186+ cells, and the non-nuclear-stained CASPR1+/CNTN1+ cells and CNTN1+ cells were each washed and collected from the wells, suspended in cell culture medium, centrifuged, and the supernatant was removed. Next, the cells were suspended in FACS buffer, and impurities were removed using a nylon mesh filter. The cells were then dispensed into a 96-well plate at a density of 5 × ¹⁰⁴ cells per well. After centrifugation, the supernatant was removed, and FACS buffer (100 ul per well) containing Fc Block (BD Bioscience, 0.25 ul per well) and the patient's serum sample (4 ul per well) was treated, and then incubated on ice for 1 hour. Next, after washing three times with FACS buffer, FACS buffer (100 μl per well) containing secondary antibody (Anti-Human IgG FC 647, 1:2000, Jackson ImmunoResearch) was added, protected from light, and incubated on ice for 1 hour. After incubation, the cells were washed three times, resuspended in 200 μl of FACS buffer per sample, and flow cytometry analysis was performed using a flow cytometer (BD LSRFortessa ^TM X-20 Cell Analyzer).

For flow cytometry analysis, SSC, FSC, PacificBlue (Hoechst 34580 staining), FITC (NF155, CASPR1 transfection), PE (NF186, CNTN1 transfection), and APC (Serum IgG binding) channels were used. Viable HEK293T cell singlets were gated through SSC and FSC, and groups were designated according to the presence or absence of nuclear staining through the PacificBlue channel. H34580-positive cells were designated as NF155+ and NF186+ cells, respectively, based on FITC and PE positivity, and H34580-negative cells were designated as CASPR1+/CNTN1+ and CNTN1+ cells, respectively, based on FITC and PE positivity. Non-transfected control cells were designated as cells negative for H34580, FITC, and PE ("NF-CTRL") for NF155 and NF186 antibodies, and as cells positive for H34580 and negative for FITC and PE ("CC-CTRL") for CASPR1 and CNTN1 antibodies. The MFI ratio was analyzed using the APC fluorescence intensity of each cell group and the control group, and the cut-off for determining positivity for each cell group was set based on 42 normal sera (mean + 5 * standard deviation): NF155 - 3.31, NF186 - 3.65, CASPR1 - 1.87, CNTN1 - 1.85.

Additionally, when autoantibodies were detected, isotyping of the autoantibodies was additionally performed. Specifically, mouse anti-human IgG1 antibody, mouse anti-human IgG2 antibody, mouse anti-human IgG3 antibody, and mouse anti-human IgG4 antibody (all 1:500, Southern Biotech) were added as secondary antibodies to the four samples reacted with the cell mixture and sample, incubated on ice for 1 hour in a light-protected manner, washed, and flow cytometry analysis was performed using a flow cytometer (BD LSRFortessa ^TM X-20 Cell Analyzer).

As a result, as shown in Fig. 2, it was confirmed that NF155+ cells, NF186+ cells, CASPR1+/CNTN1+ cells, and CNTN1+ cells could be mixed, reacted with GBS/CIDP inflammatory neuropathy serum samples containing nodal-paranodal autoantibodies (NF155-IgG, NF186-IgG, CASPR1-IgG, CNTN1-IgG), and then flow cytometry was performed to detect the nodal-paranodal autoantibodies.

Additionally, as shown in Fig. 3, it was confirmed that each autoantibody isotype could be detected in autoantibody-positive samples.

Furthermore, as shown in Figures 4 and 5, 5 positive patients were identified as a result of screening serum samples from 266 patients with GBS/CIDP inflammatory neuropathy. All patients were clinically diagnosed with CIDP, and 4 patients except 1 patient were confirmed to have acute-onset CIDP, which showed an acute onset followed by a chronic course like GBS (Figure 4). All samples, except 1 case that tested positive for NF186, showed the same results in mouse peripheral nerve infarction assay (Nerve IFA) and cell-based fluorescence microscopy (CBA-IF), which are standard tests for AN (Figure 5).

In addition, 25 serum samples were shared in a blinded manner with Severance Hospital, which has a large domestic AN sample cohort, and flow cytometry was performed using the same method described above using four different cells produced in Example <1-1>, and four AN autoantibody test methods (Seoul National University Hospital: multiplex flow cytometry and Mouse IFA, Severance Hospital: Fixed CBA-IF and ELISA) were performed to compare the results.

　 SNUH - FACS total positivity voice SNUH - IFA positivity 11 2 13 voice 5 7 12 total 16 9 25 κ=0.434, p=0.0254

　 SNUH - FACS total positivity voice Severance - CBA positivity 9 2 11 voice 1 3 4 total 10 5 15 κ=0.526, p=0.039

　 SNUH - FACS total positivity voice Severance - ELISA positivity 12 2 14 voice 0 5* 5 total 12 7 19 κ=0.759, p=0.000648
* Tested only for NF155 antibodies.

As a result, as shown in Tables 1 to 3, the flow cytometry method according to the present invention and the three other tests all showed good agreement rates (Mouse IFA: kappa=0.434, p=0.0254; Fixed CBA: kappa=0.526, p=0.039; ELISA: kappa=0.759, p<0.001).

In the present invention, four types of cells expressing four different nodal-paranodal autoantigens (NF155, CNTN1, CASPR1, and NF186) and fluorescent markers were produced and mixed, and the mixed four types of cells were reacted with a sample of a patient with inflammatory peripheral neuropathy, and then flow cytometry was performed to detect autoantibodies to the four types of autoantigens at once, and it was confirmed that the isotypes of the autoantibodies could be determined. Therefore, the cells and the flow cytometry method using the same can be usefully used in an autoantibody test for diagnosing, monitoring, or predicting treatment response to autoimmune nodular disease among inflammatory neuropathy.

Claims (14)

A method for detecting NF155-IgG, NF186-IgG, CASPR1-IgG and CNTN1-IgG as autoantibodies from a sample, 1) A step of obtaining cells of the following (i) to (iv), wherein the fluorescent markers of the cells of the following (i) to (iv) are three or more types of distinct fluorescent markers, and each of the cells of the following (i) to (iv) includes one or more types of fluorescent markers; (i) cells containing NF155 (neurofascin-155) and fluorescent markers, (ii) cells containing NF186 (neurofascin-186) and fluorescent markers; (iii) cells containing CASPR1 (contactin-associated protein 1) and fluorescent markers, and (iv) cells containing CNTN1 (contactin-1) and fluorescent markers; 2) A step of mixing all of the cells of (i) to (iv) above; 3) A step of incubating together the above-mentioned four types of cells and a sample expected to contain autoantibodies; 4) A step of adding a fluorescent-conjugated secondary antibody to the sample after incubation to form an autoantibody and secondary antibody complex; and 5) A method comprising a step of detecting or analyzing fluorescence from the autoantibody and secondary antibody complex. A method according to claim 1, wherein the fluorescent marker is selected from the group consisting of GFP (green fluorescent protein), YFP (yellow fluorescent protein), EGFP (enhanced green fluorescent protein), CFP (cyan fluorescent protein), OFP, RFP (red fluorescent protein), DsRed2, Tdtomato, mCherry, Hoechst 33258, Hoechst 33342, and Hoechst 34580. A method according to claim 1, wherein the cells are HEK (human embryonic kidney) 293, HEK 293T, HEK 293A, HeLa, CHO (chinese hamster ovary) or NIH3T3 cells. A method according to claim 1, wherein the secondary antibody is anti-human IgG or anti-human IgG-Fc. A method further comprising a step of performing isotyping of an autoantibody using a sample in which incubation has been completed in step 4) of claim 1. A method according to claim 5, wherein the isotype of the autoantibody is IgG1, IgG2, IgG3 or IgG4. A method according to claim 1, wherein the sample is blood, serum, plasma or cerebrospinal fluid isolated from a patient with inflammatory neuropathy. A method in claim 1, wherein in step 5), fluorescence is detected or analyzed by performing a flow cytometry assay. A method for providing information for diagnosing, monitoring, or predicting treatment response to autoimmune nodopathy (AN), 1) A step of obtaining cells of the following (i) to (iv), wherein the fluorescent markers of the cells of the following (i) to (iv) are three or more types of distinct fluorescent markers, and each of the cells of the following (i) to (iv) includes one or more types of fluorescent markers; (i) cells containing NF155 and fluorescent markers; (ii) cells containing NF186 and fluorescent markers; (iii) cells containing CASPR1 and a fluorescent marker; and (iv) cells containing CNTN1 and fluorescent markers; 2) A step of mixing all of the cells of (i) to (iv) above; 3) A step of incubating together the four types of cells mixed above with a sample isolated from a patient expected to contain at least one autoantibody among NF155-IgG, NF186-IgG, CASPR1-IgG, and CNTN1-IgG; 4) A step of adding a fluorescent-conjugated secondary antibody to the sample after incubation to form an autoantibody and secondary antibody complex; and 5) A method comprising a step of detecting or analyzing fluorescence from the autoantibody and secondary antibody complex. In the 9th paragraph, a method for determining that a subject is suffering from autoimmune nodular disease or is likely to suffer from autoimmune nodular disease when at least one autoantibody among NF155-IgG, NF186-IgG, CASPR1-IgG, and CNTN1-IgG is detected by performing step 5). A method in claim 9, further comprising a step of performing isotyping of an autoantibody using a sample in which incubation has been completed in step 4). In the 9th paragraph, the method, wherein the patient in step 3) is a patient with inflammatory neuropathy. A method according to claim 12, wherein the inflammatory neuropathy is autoimmune nodular disease, chronic inflammatory demyelinating polyneuropathy (CIDP), or Guillain-Barré syndrome (GBS). (a) Cells containing NF155 and fluorescent markers; (b) cells containing NF186 and fluorescent markers; (c) cells containing CASPR1 and a fluorescent marker; and (d) cells containing CNTN1 and a fluorescent marker, A composition for diagnosing, monitoring or predicting treatment response to autoimmune nodular disease, wherein the fluorescent markers of the cells of (a) to (d) above are three or more types of distinct fluorescent markers, and each of the cells of (i) to (iv) above contains one or more types of fluorescent markers.