WO2021162325A1 - Method for cell-based assay to detect mog-igg in patients with central nervous system inflammatory diseases - Google Patents

Abstract

The present invention relates to: a method for detecting myelin oligodendrocyte glycoprotein (MOG)-IgG comprising the steps of: (a) cloning a nucleic acid encoding MOG into a fluorescent protein expression vector; (b) transfecting a cell with the expression vector cloned with MOG; (c) selecting a stable transfected cell line by culturing the transfected cell in a medium containing a selection drug; (d) incubating the selected cell line and a sample expected to contain MOG-IgG together; (e) forming MOG-IgG and a secondary antibody conjugate by adding a fluorescence-conjugated secondary antibody to the incubated sample; and (f) detecting or analyzing fluorescence from the MOG-IgG and the secondary antibody conjugate; a method of providing information for prediction or diagnosis of MOG-IgG-related central nervous system inflammatory diseases; a composition for diagnosis of MOG-IgG-related central nervous system inflammatory diseases; and a method of diagnosing MOG-IgG-related central nervous system inflammatory diseases. The present invention confirms that there is a strong positive correlation between CBA-IF and CBA-FACS results when using fluorescence-conjugated IgG (H+L) as a secondary antibody specifically binding to MOG-IgG, and a high matching rate of about 98% is shown even compared with the existing IgG1 (Oxford) CBA-IF. In particular, the method of the present invention does not require repetitive transfection by constructing a stable cell line expressing MOG, and is advantageous in that there is no cross reaction with secondary antibody IgM, so that a more clear fluorescence signal having a high specificity can be obtained.

Description

Cell-based assay method for detection of MOG-IGG in patients with central nervous system inflammatory disease

The present invention provides a method for detecting MOG-IgG from a sample, comprising the steps of: (a) cloning a nucleic acid encoding a myelin oligodendrocyte glycoprotein (MOG) into a fluorescent protein expression vector; (b) transfecting the MOG-cloned expression vector into cells; (c) selecting a stable transfected cell line by culturing the transfected cells in a medium containing a selection drug; (d) incubating the selected cell line with a sample expected to contain MOG-IgG; (e) adding a fluorescence-conjugated secondary antibody to the incubated sample to form a MOG-IgG and secondary antibody conjugate; and (f) detecting or analyzing fluorescence from the MOG-IgG and secondary antibody conjugate; a method for providing information for prediction or diagnosis of MOG-IgG-related central nervous system inflammatory disease; a composition for diagnosis of MOG-IgG-related central nervous system inflammatory disease; and to a method for diagnosing MOG-IgG-related central nervous system inflammatory diseases.

The serum status of autoantibodies (Serostatus) is an important diagnostic criterion in a wide range of neurological diseases, including autoimmune diseases. Development of a cell-based assay (CBA) using myelin oligodendrocyte glycoprotein (MOG) as a substrate identified MOG-IgG-positive patients with various demyelinating phenotypes. Acute disseminated encephalomyelitis (ADEM) in children and adults, seizures, Encephalitis, aquaporin 4 (AQP4)-IgG-negative optic neuromyelitis category disease (NMOSD), optic neuritis (ON) , MOG-IgG was present in myelitis and brainstem encephalitis. On the other hand, MOG-IgG was not present in patients with multiple sclerosis (MS), so it was thought to differentiate multiple sclerosis from non-MS central nervous system (CNS) demyelinating disorders. In addition, there are clinical, radiological, pathological and prognostic differences between patients with MS and MOG-IgG-associated diseases. According to a recent study, persistent positivity of MOG-IgG was associated with clinical relapse despite immunotherapy, whereas conversion to seronegative did not result in clinical relapse. Furthermore, serological findings and other experimental approaches have suggested that MOG-IgG may be pathogenic. Nevertheless, the spectrum of MOG-IgG-associated diseases is still not fully defined. Therefore, the development of sensitive and highly specific assays to identify specific autoantibodies is important in delineating the full spectrum of MOG-IgG-associated diseases by accurately defining the clinical phenotype, disease course and prognosis.

One object of the present invention is to construct a stable transfected cell line expressing MOG, react it with a sample isolated from a patient with a central nervous system (CNS) inflammatory disease expected to contain MOG-IgG, and then react it with a MOG-IgG-specific By adding a fluorescence-conjugated secondary antibody that binds positively and detecting or analyzing the fluorescence therefrom, it is to detect MOG-IgG from a sample such as blood or serum, and furthermore, to reveal the entire spectrum of MOG-IgG-related diseases.

Another object of the present invention is to provide a method for providing information for prediction or diagnosis of MOG-IgG-related central nervous system inflammatory disease, wherein the MOG-IgG-related central nervous system inflammatory disease is AQP4-IgG-negative optic neuromyelitis category. disease (NMOSD), acute disseminated encephalomyelitis (ADEM), myelitis, encephalitis (Encephalitis) or optic neuritis (ON).

Another object of the present invention is to (a) cloning the nucleic acid encoding the MOG into a fluorescent protein expression vector; (b) transfecting the cell with the MOG cloned expression vector; And (c) to provide a composition for diagnosis of MOG-IgG-related central nervous system inflammatory disease comprising a stable transfected cell line expressing MOG, which is prepared by culturing the transfected cells in a medium containing a selection reagent. am.

Another object of the present invention is to provide a method for diagnosing MOG-IgG-related central nervous system inflammatory disease.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

One aspect of the present invention for achieving the above object is a method for detecting MOG-IgG from a sample, comprising: cloning a nucleic acid encoding a myelin oligodendrocyte glycoprotein (MOG) into a fluorescent protein expression vector; Transfecting the MOG-cloned expression vector into cells; selecting a stable transfected cell line expressing MOG by culturing the transfected cells in a medium containing a selection drug; incubating the selected cell line and a sample expected to contain MOG-IgG together; forming a MOG-IgG and secondary antibody conjugate by adding a fluorescence-conjugated secondary antibody to the incubated sample; And it provides a method comprising the step of detecting or analyzing the fluorescence from the MOG-IgG and the secondary antibody conjugate.

In the present invention, "myelin oligodendrocyte glycoprotein (MOG)" plays an important role in myelination of nerves in the central nervous system (CNS), which is a glycoprotein distributed on the surface of the myelin sheath and the plasma membrane of oligodendrocytes. . With the development of a cell-based assay (CBA) based on MOG, MOG-immunoglobulin G antibody (IgG), i.e. MOG-IgG, has been implicated in various central nervous system (CNS) inflammatory diseases. This is known MOG in the present invention is a full-length human MOG (Full-length MOG, FL-MOG), and the nucleic acid encoding the MOG in the present invention may consist of the nucleotide sequence shown in SEQ ID NO: 1.

In the present invention, "stable transfected cell line" refers to a stable cell line expressing the FL-MOG, comprising the steps of cloning a nucleic acid encoding MOG into a fluorescent protein expression vector; Transfecting the MOG-cloned expression vector into cells; It can be prepared by culturing the transfected cells in a medium containing a selection drug to select a stable transfected cell line (MOG-HEK293) expressing MOG. Specifically, the fluorescent protein may be EGFP, GFP, YFP, DsRed2, Tdtomato or mCherry, and the expression vector may be a pIRES2-EGFP or a pIRES-DsRed2 vector. More specifically, the expression vector into which the MOG is cloned in the present invention may be a cloned full-length human MOG (FL-MOG) encoded by the nucleotide sequence shown in SEQ ID NO: 1 in the pIRES2-EGFP vector. In addition, the cells to be transfected may be mammalian cells, that is, HEK (human embryonic kidney) 293, HeLa, CHO (chinese hamster ovary) or NIH3T3 cells, and the selection reagent is hygromycin, furo It may be mycin (puromycin), blasticidi (blasticidi), or G418 (geneticin), but is not particularly limited thereto. For example, in one embodiment of the present invention, full-length human MOG (Full-length MOG, FL-MOG) cDNA is isolated from the FL-MOG/pIRES2-DsRed2 plasmid and cloned into the XhoI and SmaI sites of the pIRES2-EGFP vector. Then, it was transfected into HEK293 cells using Lipofectamine 2000 reagent, and then a stable transfected cell line was selected through a medium containing G418 as a selection reagent, where FL-MOG cDNA was extracted or isolated and cloned into an expression vector. The process may be performed by a person skilled in the art by appropriately selecting a method known in the art.

On the other hand, the stable cell line expressing the FL-MOG is for detecting MOG-IgG in the sample and further diagnosing MOG-IgG-related central nervous system inflammatory disease. It may refer to spinal fluid, blood, or serum isolated from a disease patient or from a patient expected to contain MOG-IgG, and more preferably refer to blood or serum. The sample can be used by a person skilled in the art by diluting it at an appropriate ratio as needed.

In the present invention, "MOG-IgG-related central nervous system inflammatory disease" may appear in MOG-IgG-positive patients with various demyelinating phenotypes, specifically, AQP4-IgG-negative neuromyelitis optica spectrum disorder (NMOSD), It may mean acute disseminated encephalomyelitis (ADEM), myelitis, encephalitis, or optic neuritis (ON), but is not particularly limited thereto.

In the present invention, the stable cell line expressing the FL-MOG is incubated with the isolated blood or serum, and then a fluorescence-conjugated secondary antibody is added to the incubated sample to MOG-IgG and secondary antibody. form a conjugate. In the present invention, the "fluorescence-bound secondary antibody" is for a cell-based assay (CBA) for MOG or MOG-IgG, and the secondary antibody may in particular be anti-human IgG (H+L). Specifically, in one embodiment of the present invention, when using fluorescence-conjugated IgG (H+L) as a secondary antibody that specifically binds to MOG-IgG, there is a strong positive difference between CBA-IF and CBA-FACS results. It was confirmed that there was a correlation and showed a high concordance rate of about 98% compared to the conventional IgG1 (Oxford) CBA-IF, especially when using fluorescence-conjugated IgG (H+L), cross-reacting with IgM ( It was confirmed that there is an advantage of obtaining a more specific and clear fluorescence signal because it does not show cross reaction).

The step of "detecting or analyzing fluorescence" in the present invention is not particularly limited thereto, but may be performing an immunofluorescence assay (IF) or a flow cytometry assay (FACS). In one embodiment of the present invention, three different secondary antibodies against MOG-HEK293 cells, Alexa Fluor-594 bound goat anti-human IgG (H+L), Alexa Fluor-594 bound goat anti-human IgGl CBA-IF (NCC-CBA) was performed using Fc, Alexa Fluor-594 bound goat anti-human IgM, followed by CBA-FACS with three different secondary antibodies (anti-human IgG (H+L); The specificity and sensitivity of IgG1-Fc and IgM) to MOG were measured.

Another aspect of the present invention for achieving the above object, cloning the nucleic acid encoding the MOG into a fluorescent protein expression vector; transfecting the cell with the MOG cloned expression vector; culturing the transfected cells in a medium containing a selection reagent to select a stable transfected cell line expressing MOG; incubating the selected cell line and a sample expected to contain MOG-IgG together; forming a MOG-IgG and secondary antibody conjugate by adding a fluorescence-conjugated secondary antibody to the incubated sample; And it provides a method of providing information for the prediction or diagnosis of MOG-IgG-related central nervous system inflammatory disease, comprising the step of detecting or analyzing the fluorescence from the MOG-IgG and the secondary antibody conjugate. The term 'MOG-IgG-related central nervous system inflammatory disease' is the same as described above.

MOG-IgG-associated central nervous system, including optic neuromyelitis category disease (NMOSD), acute disseminated encephalomyelitis (ADEM), myelitis, Encephalitis, or optic neuritis (ON) in MOG-IgG-positive patients with various demyelinating phenotypes Since inflammatory diseases may appear, information for prediction or diagnosis of MOG-IgG-related central nervous system inflammatory diseases may be provided by checking the presence and level of MOG-IgG in the sample through the above method.

Specifically, when a stable cell line expressing the FL-MOG and a sample expected to contain MOG-IgG are incubated together, and a secondary antibody that specifically binds to MOG-IgG is added, MOG-IgG is released from the sample. When present in the sample, red fluorescence is observed around the cell membrane due to FL-MOG and MOG-IgG binding, and when MOG-IgG is not present in the sample, red fluorescence is not observed. Furthermore, it can be used to analyze the MOG-IgG expression level, clinical phenotype, disease course, and prognosis of a patient group with MOG-IgG-related central nervous system inflammatory disease, and to classify the patient group.

Another aspect of the present invention for achieving the above object, cloning the nucleic acid encoding the MOG into a fluorescent protein expression vector; transfecting the cell with the MOG cloned expression vector; And it provides a composition for diagnosis of MOG-IgG-related central nervous system inflammatory disease, comprising a stable transfected cell line expressing MOG, which is prepared by culturing the transfected cells in a medium containing a selection reagent. The term 'MOG-IgG-related central nervous system inflammatory disease' is the same as described above.

Another aspect of the present invention for achieving the above object, cloning the nucleic acid encoding the MOG into a fluorescent protein expression vector; transfecting the cell with the MOG cloned expression vector; culturing the transfected cells in a medium containing a selection reagent to select a stable transfected cell line expressing MOG; incubating the selected cell line and a sample expected to contain MOG-IgG together; forming a MOG-IgG and secondary antibody conjugate by adding a fluorescence-conjugated secondary antibody to the incubated sample; and detecting or analyzing fluorescence from the MOG-IgG and secondary antibody conjugates. The term 'MOG-IgG-related central nervous system inflammatory disease' is the same as described above.

In the present invention, when fluorescent-conjugated IgG (H+L) is used as a secondary antibody that specifically binds MOG-IgG, there is a strong positive correlation between CBA-IF and CBA-FACS results, and It was confirmed that it showed a high concordance rate of about 98% even compared to IgG1 (Oxford) CBA-IF, and in particular, the method of the present invention repeatedly transfection by constructing a stable cell line expressing MOG. This is not required, and there is an advantage in that it does not show a cross reaction with the secondary antibody IgM, so that it has high specificity and can obtain a clearer fluorescence signal.

1A and 1B show the results of confirming transfection by Western blot and flow cytometry for hMOG-HEK293 cell line transfected with FL-MOG, respectively, and C to E of FIG. 1 are control (HC) and MOG, respectively. -Shows the results of observing the fluorescence signal when IgG (H+L), IgG1-Fc and IgM are treated as secondary antibodies for -IGg-negative and positive patient sera.

FIG. 2A shows the results of performing CBA-FACS using secondary antibodies IgG (H+L), IgG1-Fc and IgM for the sera of CNS inflammatory disease patients and controls (HC), respectively. B shows the results of calculating the area under the curve (AUC) for each ROC curve and MFI ratio for IgG (H+L) and IgG1-Fc.

Figure 3 calculates the optimal cutoff from the ROC curve for various CNS inflammatory disease patients, and shows the distribution of the MFI ratio for each disease.

4 shows the results of confirming the correlation between CBA-IF scores and CBA-FACS from the IF scores and MFI ratios of 40 MOG-IgG seropositive patients.

5A and B show the results of observation of fluorescence signals in FL-MOG + HEK293 cell lines and EV-transfected HEK293 cell lines transfected with FL-MOG, respectively, and secondary antibodies IgG (H+L), IgG1-Fc and Shows the results of performing CBA-FACS using IgM.

Hereinafter, the present invention will be described in more detail through the following examples. However, these Examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited only to these Examples.

Example 1: Experimental method

1-1: patient group

Serum from 355 patients diagnosed with CNS inflammatory disease at the National Cancer Center (NCC) and 25 healthy people as controls (HC) were used. The demographics of the 355 patient groups are shown in Table 1, and their serum status was assessed by in-house CBA. The patient population included 333 randomly selected patients with CNS inflammatory disease treated at NCC and 22 patients previously identified as MOG-IgG positive by IgG1 Oxford CBA (Oxford-CBA).

Last follow-up diagnosis	number of patients	Male: Female	Average age at sampling	Sampling mean duration of illness	Number of MOG-IgG-positive patients
AQP4-positive NMOSD	65	11:54	37 (13-67)	43 (14-73)	0/65 (0%)
AQP4 negative NMOSD	37	9:28	41 (12-72)	29 (0-351)	4/37 (10.8%)
multiple sclerosis	111	34:77	32 (16-65)	24 (0-286)	0/111 (0%)
Single or recurrent myelitis	52	32:20	40 (14-74)	18 (0-196)	7/52 (13.5%)
Single or recurrent optic neuritis	16	6:10	34 (15-63)	2 (0-87)	6/16 (37.5%)
Other demyelinating disorders, including neoplastic demyelination or ADEM-like symptoms	55	23:32	34 (7-61)	7 (0-325)	24/55 (43.6%)
other neurological disorders	19	11:8	49 (21-68)	6 (0-212)	0/19 (0%)

The specificity and sensitivity of three different secondary antibodies (anti-human IgG (H+L), IgG1-Fc and IgM) in 105 patients were evaluated by flow cytometry analysis, and the specificity and sensitivity of the secondary antibody were evaluated in 10 patients. confirmed by CBA-IF in the patient. Concordance of NCC CBA-IF (NCC-CBA) and Oxford-CBA-IF was evaluated in 125 patients (including 22 MOG-IgG seropositive patients). Serum collected from all participants was stored at -80°C, and the diagnosis of NMOSD and MS was based on the 2015 International NMOSD Diagnostic Panel criteria and the 2010 McDonald's criteria, respectively.

1-2: MOG-HEK 293 cell production

Full-length human MOG (Full-length MOG, FL-MOG) cDNA was isolated from the FL-MOG/pIRES2-DsRed2 plasmid and cloned into the XhoI and SmaI sites of the pIRES2-EGFP vector (Clontech, Mountain View, CA, USA). All sequences were confirmed by automated sequencing. Thereafter, the cloned FL-MOG plasmid or empty vector plasmid was transfected into Human Embryonic Kidney 293 (HEK293) cells (ATCC, Manassas, VA, USA) using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA). After 48 hours, the cells were divided into a medium containing 2 mg/mL G418 (Invitrogen), and after 2 weeks, the G418-resistant cells were isolated and further cloned by limiting dilution. Each stable transfectant clone was screened for green fluorescent protein (GFP) expression using flow cytometry. FL-MOG protein expression in GFP-positive cells was confirmed by Western blot using an anti-MOG antibody (Santacruz, Dallas, TEXAS, USA).

1-3: MOG-IgG cell-based indirect immunofluorescence assay (CBA-IF)

CBA-IF was directed against MOG-HEK293 cells by three different secondary antibodies, Alexa Fluor-594 bound goat anti-human IgG (H+L) (diluted 1:2000 in PBS), Alexa Fluor-594 bound Goat anti-human IgG (FCγ fragment specific, diluted 1:750 with PBS), Alexa Fluor-594 bound goat anti-human IgM (diluted 1:750 with PBS). HEK293 cells stably transfected with an empty vector (EV) were used as a control to exclude non-specific background staining. Each experiment was performed in duplicate, and seropositivity was determined by two investigators unaware of the patient's clinical and experimental information and each other's results. The intensity of surface immunofluorescence was scored as shown in Table 2 below, with the final score being the median of readings from 2 or 3 investigators. An IF score of 1 or higher was considered positive and a score of 0.5 was considered borderline.

IF score		Explanation
0	No bonds around the cell membrane or the staining does not surround the cell membrane in a ring shape
0.5	There is a weak bond around the cell membrane, but does not surround the entire membrane of ~50% of the spiny cells
One	In prickly cells > 50%, sharp but weak bonds surrounding the entire membrane in ring form
2	Visible cells > 75% enveloping the entire membrane, showing clear and moderate brightness
3	Clear, bright binding, similar to that of positive controls in prickly cells > 90%
4	Stronger, very bright binding than positive control in prickly cells >90%

1-4: Cell-based flow cytometry (CBA-FACS)

HEK293 cells were harvested and washed in staining buffer (2 mM EDTA-1X PBS supplemented with 0.5% bovine serum albumin). 2×10 ⁵ cells were incubated with patient serum (diluted 1:40 with staining buffer, 200 μl) in 96-well U-bottom plates (Thermo Fisher Scientific, MA, USA) on ice for 30 min. Cells were then washed twice with PBS and either allophycocyanin bound goat anti-human IgG (H+L) (Jackson Immunology, PA, USA; diluted 1:2000 with PBS) or phycoeri Phycoerythrin bound mouse anti-human IgG1 Fc (SouthernBiotech, Birmingham, USA; diluted 1:1000 in PBS) or blue-violet 421 bound mouse anti-human IgM (BD Bioscience, CA, USA; 1 in PBS) :50)) and incubated on ice for 30 min. After washing and resuspending the cells in 400 μl of cold PBS, viability dye 7-AAD (BD Biosciences, CA, USA; 5 μl per sample) was added to the cells prior to acquisition to exclude dead cells. A total of 10,000 cells were acquired from FACSVerse and data were analyzed using Flow Jo software (TreeStar, Ashland, OR, USA). Binding was expressed as mean fluorescence intensity (MFI), and the MFI ratio was determined as the MFI of MOG-transfected HEK293 cells divided by the MFI of EV-transfected HEK293 cells.

Receiver-operating characteristic (ROC) curve analysis was used to determine the usefulness of the MFI ratio for determining MOG-IgG seropositivity, using CBA-IF as a reference standard. The optimal cutoff of the highest specificity and sensitivity was determined through ROC curve analysis. Specificity was calculated as [(number of MOG-IgG negative among AQP4-IgG positive NMOSD, MS and other neurological disease (OND) patients) / total number of AQP4-IgG positive NMOSD, MS and OND patients] x 100.

Example 2: Experimental results

2-1: In-house MOG-IgG CBA-IF

HEK293 cells stably transfected with FL-MOG were used to construct in-house CBA-IF, successful transfection was confirmed by Western blot and flow cytometry, and fluorescence levels were measured by fluorescence microscopy (Fig. 1). A and B).

Previous studies had concerns about the low specificity of the use of anti-human IgG (H+L) secondary antibodies to detect MOG-IgG, particularly due to the cross-reactivity of IgG and IgM antibodies in MS patient samples. . On the other hand, IgG Fc or IgM secondary antibodies were produced in a limited number of samples (n = 10; 1 AQP4-IgG negative NMOSD, 2 patients with single or recurrent myelitis, neoplastic demyelination or other symptoms including ADEM-like symptoms (ODD)). 4 patients with demyelinating disease, and 3 healthy controls) were used for CBA-IF.

As a result, there was no difference in fluorescence between IgG (H + L) and IgG Fc, and in particular, it was confirmed that no fluorescence signal was seen when an IgM secondary antibody was used (FIG. 1C to E). Specifically, green fluorescence was observed in the cytosol of MOG-transfected cells, and when patient serum containing MOG-IgG was added, MOG-IgG was bound to FL-MOG, and red fluorescence was observed around the cell membrane. (FIG. 1E). On the other hand, it was confirmed that red fluorescence was not observed when the patient serum did not contain MOG-IgG ( FIGS. 1C and 1D ).

2-2: MOG-IgG serum status

25 HC and 355 patients from the CNS inflammatory disease cohort (65 AQP4-IgG positive NMOSD, 37 AQP4-IgG negative NMOSD, 111 MS, 52 patients with single or recurrent myelitis, 16 with single or recurrent optic neuritis (ON)) patients, ODD 55, OND 7) were evaluated. Characteristics and MOG-IgG seropositivity of 355 patients are shown in Table 1. Of the total 355 patients, 41/355 (11.5%) were seropositive: 4/37 (10.8%) patients with AQP4-IgG negative NMOSD and 7/52 patients (13.5%) with single or recurrent myelitis. ), single or recurrent ON 6/16 patients (37.5%) ODD patients 24/55 (43.6%) were found to have MOG-IgG in their serum. In addition, none of the 183 AQP4-IgG-positive NMOSD, MS, OND patients and healthy control samples were positive for MOG-IgG, and all 41 patients positive for MOG-IgG had known clinical trials of MOG-IgG-associated disease. Clinical phenotypes within the spectrum were shown.

The female to male ratio was 28:13, and the mean age at onset of the disease was 28 years (range: 3 to 60 years). Clinical phenotypes at onset included optic neuritis (37%), myelitis (29%), brain attacks (24%), and poly-regional involvement (10%). During a median prevalence of 78 months (range: 3 to 298 months), 10 patients were monophasic and 31 patients relapsed: 26/41 (63%) patients had at least one cerebral involvement ( brain attack), 23/41 (56%) patients experienced at least one optic neuritis attack, and 20/41 (49%) patients experienced at least one myelitis attack. Of the 26 patients with brain attacks, 17 (65%) had ADEM-like lesions, 7 (27%) had fluffy brainstem lesions, and 2 (8%) of patients had cortical lesions. Longitudinally extensive transverse myelitis was observed in 13 of 20 (65%) patients with myelitis and bilateral involvement in 8 of 23 patients (35%) with optic neuritis. At the last follow-up, simultaneous or sequential multizone involvement was observed in 25/41 (61%) patients. The most common clinical symptoms in these patients were a combination of optic neuritis and brain attacks (12/25 patients, 48%) followed by a combination of myelitis and brain inflammatory lesions (7/25 patients, 28%). , a combination of optic neuritis and myelitis (3/25 patients, 12%). 3 (12%) patients had optic neuritis, myelitis and brain inflammatory lesions, and the remaining 16 patients had isolated myelitis (n=7), optic neuritis (n=5) and brain inflammatory lesions (n=4). experienced isolated clinical syndromes such as

2-3: Determination of seropositivity by CBA-FACS

To further confirm the usefulness of IgG (H+L) in the FL-MOG stable cell line, a total of 105/355 patients with CNS inflammatory disease (65/105 patients) were randomized, and 40 patients with MOG -IgG seropositive patients) and HC were tested using CBA-FACS for the sera of 25 patients, and three different secondary antibodies IgG (H+L), IgG1-Fc and IgM were used (FIG. 2A). The serostats of 105 patients detected by CBA-IF were classified as MOG-IgG seropositive and seronegative, and the MFI ratio of each patient was determined by CBA-FACS. Using ROC curve analysis, the area under the curve for the MFI ratio of IgG (H+L) was 1.00 and that of IgG1-Fc was 0.97 ( FIG. 2B ).

The optimal cutoff was calculated from the ROC curve to detect seropositivity in 105 CNS inflammatory disease patients, and the optimal cutoffs for anti-human IgG (H+L) and IgG1-Fc secondary antibodies were 3.4 and 2.3, respectively. Diagnosis at last follow-up of 105 patients and seropositivity in each patient group were determined (FIG. 3). All patients with AQP4-IgG positive NMOSD, MS and OND were MOG-IgG negative. Meanwhile, the anti-human IgG1-Fc secondary antibody detected MOG-IgG in one AQP4-IgG seropositive NMOSD patient and two MS patients (specificity = 95%). The MFI ratio of the sera of all 25 HCs was below the cutoff for both IgG (H+L) and IgG1-Fc antibodies, and no samples bound to IgM antibodies.

2-4: Correlation between CBA-IF scores and CBA-FACS

CBA-IF scores were determined as described above, and correlations between CBA-IF scores and CBA-FACS results were also determined. The IF scores of 40 MOG-IgG seropositive patients were as follows: 10 patients scored 1 point, 13 patients scored 2 points, 10 patients scored 3 points, and 7 patients scored 4. The anti-human IgG (H+L) secondary antibody showed a stronger positive correlation with the CBA-IF score than the IgG1-Fc secondary antibody ( FIG. 4 ). (IgG (H+L): r=0.87, p <0.0001 vs IgG1-Fc: r=0.81, p <0.0001)

2-5: High agreement between Oxford CBA-IF and NCC CBA-IF

To further validate the analysis results, 125 patients (36 with AQP4-IgG positive NMOSD, 13 with AQP4-IgG negative NMOSD, 27 with MS, 21 with single or recurrent myelitis, single or 6 with recurrent optic neuritis and 22 with ODD) were compared with IgG1 (Oxford) CBA-IF. Anti-human IgG (H+L) secondary antibody (NCC) was used for CBA-IF. As a result, both methods showed consistent results for a sample of 122/125 (98%) patients (κ = 0.919, p < 0.0001), and in both methods, 21/125 patients were MOG-IgG positive; 101/125 patients were confirmed as MOG-IgG negative. 3/125 (2%) patients were positive for only one assay, (NCC) CBA-IF in 2, and (Oxford) CBA-IF in 1 (Table 3). (NCC) Two patients who were MOG-IgG positive by CBA-IF alone were AQP4-IgG negative NMOSD patients. The other sample (Patient 5) was strongly positive for AQP4-IgG, borderline positive for MOG-IgG by (Oxford) CBA-IF, while positive for AQP4-IgG and (NCC) MOG- by CBA IF. It was negative for IgG.

		NCC-CBA		Sum
		positivity	voice
Oxford-CBA	positivity	21	One	22
	voice	2	101	103
Sum		23	102	125

(κ = 0.919, p < 0.0001)

In addition, we found positive binding in MOG-transfected cells as well as EV-transfected cells by (NCC) CBA-IF and CBA-FACS ( FIGS. 5A and 5B ). MOG-IgG positive binding was observed for both anti-human IgG (H+L) and IgG-Fc secondary antibodies. The MOG-IgG MFI ratio was lower than the cutoff for all three secondary antibodies (anti-human IgG (H+L), IgG1-Fc and IgM) ( FIG. 5B ).

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (18)

1. A method for detecting MOG-IgG from a sample, comprising:

   (a) cloning a nucleic acid encoding a myelin oligodendrocyte glycoprotein (MOG) into a fluorescent protein expression vector;

   (b) transfecting the MOG-cloned expression vector into cells;

   (c) selecting a stable transfected cell line expressing MOG by culturing the transfected cells in a medium containing a selection drug;

   (d) incubating the selected cell line with a sample expected to contain MOG-IgG;

   (e) adding a fluorescence-conjugated secondary antibody to the incubated sample to form a MOG-IgG and secondary antibody conjugate; and

   (f) detecting or analyzing fluorescence from the MOG-IgG and secondary antibody conjugate.
2. The method of claim 1, wherein the nucleic acid encoding the MOG consists of the nucleotide sequence represented by SEQ ID NO: 1.
3. The method of claim 1 , wherein the secondary antibody is an anti-human IgG (H+L).
4. The method of claim 1, wherein the secondary antibody in step (e) does not cross-react with IgM.
5. The method of claim 1, wherein the fluorescent protein is EGFP, GFP, YFP, DsRed2, Tdtomato or mCherry.
6. The method according to claim 1, wherein the cells are human embryonic kidney (HEK) 293, HeLa, CHO (chinese hamster ovary) or NIH3T3 ? cells.
7. The method of claim 1, wherein the selection reagent is hygromycin, puromycin, blasticidi, or G418.
8. The method of claim 1, wherein the sample is spinal fluid, blood or serum isolated from a patient with MOG-IgG-related central nervous system (CNS) inflammatory disease.
9. The method of claim 1, wherein step (f) is performed by immunofluorescence assay or flow cytometry assay.
10. As a method of providing information for prediction or diagnosis of MOG-IgG-related central nervous system inflammatory disease,

    (a) cloning the nucleic acid encoding the MOG into a fluorescent protein expression vector;

    (b) transfecting the cell with the MOG cloned expression vector;

    (c) selecting a stable transfected cell line expressing MOG by culturing the transfected cells in a medium containing a selection reagent;

    (d) incubating the selected cell line with a sample expected to contain MOG-IgG;

    (e) adding a fluorescence-conjugated secondary antibody to the incubated sample to form a MOG-IgG and secondary antibody conjugate; and

    (f) detecting or analyzing fluorescence from the MOG-IgG and secondary antibody conjugate.
11. The method of claim 10, wherein the nucleic acid encoding the MOG consists of a nucleotide sequence represented by SEQ ID NO: 1.
12. 11. The method of claim 10, wherein the MOG-IgG-related central nervous system inflammatory disease is AQP4-IgG-negative neuromyelitis optica spectrum disorder (NMOSD), acute disseminated encephalomyelitis (ADEM), Myelitis, Encephalitis or optic neuritis (ON).
13. (a) cloning the nucleic acid encoding the MOG into a fluorescent protein expression vector;

    (b) transfecting the cell with the MOG cloned expression vector; and

    (c) prepared by culturing the transfected cells in a medium containing a selection reagent,

    A composition for diagnosis of MOG-IgG-related central nervous system inflammatory disease, comprising a stable transfected cell line expressing MOG.
14. The composition of claim 13, wherein the nucleic acid encoding the MOG consists of the nucleotide sequence represented by SEQ ID NO: 1.
15. The method of claim 13, wherein the MOG-IgG-related central nervous system inflammatory disease is AQP4-IgG negative optic neuromyelitis category disease (NMOSD), acute disseminated encephalomyelitis (ADEM), myelitis, encephalitis (Encephalitis) or optic neuritis (ON). Phosphorus, composition.
16. A method for diagnosing MOG-IgG-related central nervous system inflammatory disease, comprising:

    (a) cloning the nucleic acid encoding the MOG into a fluorescent protein expression vector;

    (b) transfecting the cell with the MOG cloned expression vector;

    (c) selecting a stable transfected cell line expressing MOG by culturing the transfected cells in a medium containing a selection reagent;

    (d) incubating the selected cell line with a sample expected to contain MOG-IgG;

    (e) adding a fluorescence-conjugated secondary antibody to the incubated sample to form a MOG-IgG and secondary antibody conjugate; and

    (f) detecting or analyzing fluorescence from the MOG-IgG and secondary antibody conjugate.
17. The method of claim 16, wherein the nucleic acid encoding the MOG consists of the nucleotide sequence represented by SEQ ID NO: 1.
18. 17. The method of claim 16, wherein the MOG-IgG-related central nervous system inflammatory disease is AQP4-IgG-negative neuromyelitis optica spectrum disorder (NMOSD), acute disseminated encephalomyelitis (ADEM), Myelitis, Encephalitis or optic neuritis (ON).

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