Clinical application of sBCMA in cerebrospinal fluid in diagnosis and monitoring of central nervous system autoimmune diseases

Abstract

The application discloses application of sBCMA in cerebrospinal fluid as a biomarker in diagnosis and disease monitoring of antibody-mediated central nervous system autoimmune diseases. Taking NMOSD as an example, the level of sBCMA in cerebrospinal fluid of NMOSD patients is significantly higher than that of age and gender matched control subjects, and the level of sBCMA in cerebrospinal fluid is positively correlated with NFL, QAlb and sTREM2, which are related indexes of nerve injury, blood-cerebrospinal fluid barrier damage and nerve inflammation in cerebrospinal fluid of NMOSD patients, which shows that the level of sBCMA in cerebrospinal fluid can be used as a biomarker for diagnosis of NMOSD and other antibody-mediated central nervous system autoimmune diseases, and can also be used for evaluation of the severity of nerve injury, the degree of blood-cerebrospinal fluid barrier damage and the level of nerve inflammation of patients with central nervous system autoimmune diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of disease detection, and particularly relates to application of sBCMA in cerebrospinal fluid as a biomarker in diagnosis and disease monitoring of antibody-mediated central nervous system autoimmune diseases. BACKGROUND

[0002] Autoimmune diseases are a group of diseases characterized by immune dysfunction, and abnormal immune response to normal body components. More than 80 types of autoimmune diseases have been found in humans, including systemic diseases such as systemic lupus erythematosus, and tissue or organ-specific diseases such as autoimmune thyroiditis. Autoimmune diseases can affect various systems of the body, including the skin, joints, kidneys, and central nervous system.

[0003] Central nervous system (CNS) autoimmune diseases are autoimmune diseases in which autoimmune cells, autoantibodies and other immune molecules directly or indirectly attack the nervous system (including neurons, glial cells and myelin sheath). CNS autoimmune diseases are diverse, including neuromyelitis optic spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein-IgG associated disorders (MOGAD), multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), autoimmune encephalitis (AE) and CNS vasculitis. The pathogenesis of CNS autoimmune diseases is complex, and immune inflammatory reactions caused by direct or indirect attack of autoimmune cells, autoantibodies and other immune molecules on the nervous system play an important role in the pathogenesis. Among them, autoantibodies play a very important role in the diagnosis and differential diagnosis of CNS inflammatory demyelinating diseases.

[0004] B lymphocyte / plasma cell is one of the most important effector cells of autoimmune diseases, which attacks normal cells and tissues through various ways such as abnormal secretion of autoantibodies, and induces systemic immune abnormalities in the body. The proliferation and maturation of B lymphocytes play a crucial role in the pathogenesis of CNS autoimmune diseases. B-cell maturation antigen (BCMA) is a transmembrane glycoprotein in the tumor necrosis factor superfamily, which is mainly expressed on the surface of mature B lymphocytes. It regulates the maturation and differentiation of B cells into plasma cells. Soluble B-cell maturation antigen (sBCMA) is directly shed from membrane BCMA, which has been confirmed as a good biomarker for multiple myeloma and systemic lupus erythematosus. A prospective study evaluated the correlation between serum sBCMA and bone marrow plasma cell infiltration in multiple myeloma patients, confirming that sBCMA level is a good prognostic indicator for multiple myeloma. Another retrospective cohort study revealed the potential and significance of serum sBCMA as a biomarker for systemic lupus erythematosus patients. Due to the unique structure of CNS - the role of blood-brain barrier, in normal circumstances, autoimmune cells cannot easily enter the CNS. Therefore, unlike other autoimmune diseases, the immune response of CNS antibody-mediated autoimmune diseases may be limited, and there is no relevant clinical study to show that sBCMA as a biomarker has clinical application in the diagnosis and monitoring of central nervous system antibody-mediated CNS autoimmune diseases. SUMMARY

[0005] The present application provides a new use of sBCMA in cerebrospinal fluid as a biomarker, i.e. application in the preparation of diagnostic kits and disease monitoring kits for antibody-mediated central nervous system autoimmune diseases represented by neuromyelitis optica spectrum disorders (NMOSD).

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] The application of the reagent for detecting the biomarker sBCMA in cerebrospinal fluid in the preparation of a kit includes the following applications:

[0008] (1) detecting the level of sBCMA in cerebrospinal fluid for diagnosing antibody-mediated central nervous system autoimmune diseases;

[0009] (2) detecting the level of sBCMA in cerebrospinal fluid for evaluating the severity of nerve damage in patients with antibody-mediated central nervous system autoimmune diseases;

[0010] (3) detecting the level of sBCMA in cerebrospinal fluid for evaluating the degree of blood-cerebrospinal fluid barrier damage in patients with antibody-mediated central nervous system autoimmune diseases;

[0011] (4) detecting the level of sBCMA in cerebrospinal fluid for evaluating the level of neuroinflammation in patients with antibody-mediated central nervous system autoimmune diseases;

[0012] Specifically, the method comprises the following steps: a. determining the level of sBCMA in cerebrospinal fluid of the subject; b. performing significant difference analysis on the level of sBCMA in cerebrospinal fluid of the subject and the level of sBCMA in a gender-age matched control. The level of sBCMA in cerebrospinal fluid of the subject with antibody-mediated central nervous system autoimmune diseases is positively correlated with the level of NFL, the level of QAlb and the level of sBCMA in cerebrospinal fluid thereof. Therefore, the higher the level of sBCMA in cerebrospinal fluid of the subject is, the more serious the nerve damage thereof is, the more severe the blood-cerebrospinal fluid barrier dysfunction thereof is and the higher the level of neuroinflammation thereof is, as compared with the level of sBCMA in the control group.

[0013] The antibody-mediated central nervous system autoimmune diseases mentioned above include not only neuromyelitis optica spectrum disorders (NMOSD) but also myelin oligodendrocyte glycoprotein antibody-associated disease, anti-N-methyl-D-aspartate receptor encephalitis, anti-ryanodine receptor 1 encephalitis, anti-contactin-associated protein 2 antibody-associated autoimmune encephalitis, etc.

[0014] Compared with the prior art, the present application has the following advantages and beneficial effects: firstly, the present application determines that the level of sBCMA in cerebrospinal fluid of NMOSD patients is significantly higher than that of gender-age matched control subjects. Further, the present application determines that the concentration of sBCMA in cerebrospinal fluid of NMOSD patients is positively correlated with the indicators related to nerve damage and blood-cerebrospinal fluid barrier damage, which indicates that the level of sBCMA in cerebrospinal fluid of NMOSD patients can be used as a biomarker for the auxiliary diagnosis of NMOSD patients and can reflect the severity of nerve damage and pathological damage of NMOSD patients. Meanwhile, the present application determines that there is a positive correlation between sBCMA in cerebrospinal fluid of NMOSD patients and neuroinflammation. Therefore, sBCMA as a biomarker can provide a powerful auxiliary means for the diagnosis and disease monitoring of NMOSD and other antibody-mediated central nervous system autoimmune diseases (including myelin oligodendrocyte glycoprotein antibody-associated disease, anti-N-methyl-D-aspartate receptor encephalitis, anti-ryanodine receptor 1 encephalitis, anti-contactin-associated protein 2 antibody-associated autoimmune encephalitis, etc.), and has good sensitivity and specificity. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Analysis results of the level of sBCMA in cerebrospinal fluid of subjects of different genders.

[0016] Figure 2 The correlation analysis results between the age of the subjects and the sBCMA levels in the cerebrospinal fluid.

[0017] Figure 3 The analysis results of the sBCMA levels in the cerebrospinal fluid of the subjects.

[0018] Figure 4 The ROC curve of the sBCMA levels in the cerebrospinal fluid of the subjects.

[0019] Figure 5 The analysis of the NFL levels related to nerve injury of the subjects (A) and the correlation analysis results between the sBCMA levels in the cerebrospinal fluid and the NFL (B).

[0020] Figure 6 The analysis of the QAlb levels related to the blood-cerebrospinal fluid barrier damage of the subjects (A) and the correlation analysis results between the sBCMA levels in the cerebrospinal fluid and the QAlb (B).

[0021] Figure 7 The analysis of the sTREM2 levels related to nerve inflammation of the subjects (A) and the correlation analysis results between the sBCMA levels in the cerebrospinal fluid and the sTREM2 related to nerve inflammation (B).

[0022] Figure 8 The analysis results of the sBCMA levels in the cerebrospinal fluid of the subjects of different genders in the verification cohort.

[0023] Figure 9 The correlation analysis results between the age of the subjects and the sBCMA levels in the cerebrospinal fluid in the verification cohort.

[0024] Figure 10 The analysis results of the sBCMA levels in the cerebrospinal fluid of the subjects in the verification cohort.

[0025] Figure 11 The ROC curve of the sBCMA levels in the cerebrospinal fluid of the subjects in the verification cohort.

[0026] Figure 12 The analysis of the NFL levels related to nerve injury of the subjects in the verification cohort and the correlation analysis results between the sBCMA levels in the cerebrospinal fluid and the NFL.

[0027] Figure 13 The analysis of the sTREM2 levels related to nerve injury of the subjects in the verification cohort and the correlation analysis results between the sBCMA levels in the cerebrospinal fluid and the sTREM2. DETAILED DESCRIPTION

[0028] Neuromyelitis optica spectrum disorder (NMOSD) is a rare, progressive, autoimmune-mediated CNS inflammatory demyelinating disease mainly involving the optic nerve and spinal cord. NMOSD is characterized by inflammatory demyelination and axonal damage in the optic nerve and spinal cord, which can cause blindness, paralysis, and incontinence. Its pathogenesis is mainly derived from B cell plasma cells producing aquaporin 4 (AQP4) antibodies, which cross the blood-brain barrier and bind to AQP4 antigens on astrocytes, ultimately causing astrocyte damage, oligodendrocyte damage, demyelination, and neuronal loss through mechanisms such as antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity. NMOSD is more common in young adults, with a higher incidence in women. It is clinically characterized by severe optic neuritis and longitudinally extending long segment transverse myelitis, with a high recurrence rate and high disability rate. The incidence in China is about 0.41 / 100,000, and the prevalence is about 3.31 / 100,000. NMOSD is a highly recurrent and highly disabling disease, with most subjects left with severe sequelae, placing a significant burden on the health system, families, and communities. The present invention takes NMOSD as an example to reveal the important role of sBCMA in cerebrospinal fluid as a biomarker in the diagnosis and monitoring of antibody-mediated CNS autoimmune diseases.

[0029] Example 1 sBCMA in cerebrospinal fluid as a biomarker for NMOSD

[0030] Clinical research subjects: In the following examples, the clinical research subjects include 30 NMOSD patients (aquaporin 4 antibody positive - AQP4-IgG positive) and 30 control subjects who are gender and age matched and serum AQP4 antibody negative. The specific conditions are shown in the following table:

[0031]

[0032] (1) Sample collection and preparation

[0033] The cerebrospinal fluid specimens of the clinical research subjects were collected in sterile centrifuge tubes. The collected cerebrospinal fluid specimens were centrifuged at 500xg for 10 minutes at 4°C, and the upper clear liquid was aliquoted into cryotubes and stored in a -80°C ultra-low temperature freezer.

[0034] (2) sBCMA concentration detection method

[0035] The expression level of sBCMA in the cerebrospinal fluid was detected by human BCMA / TNFRSF17 ELISA kit (R&D Systems, DY193). Specifically, before detection, the sample was restored to a temperature range of 18-25°C. According to the requirements of the instructions, the sample was diluted and the standard was prepared. Then, 100 μl of the standard and sample was added to the well plate and incubated at room temperature for 2.5 hours. Next, the liquid in the well plate was discarded, and the plate was washed 3 times with 1x washing solution (300 μl each time, 3-5 minutes each time). Then, 100 μl of antibody was added and incubated at room temperature for 1 hour. Similarly, the plate was washed 3 times with washing solution. Then 100 μl of HRP was added and incubated at room temperature for 45 minutes, and after completion, the plate was washed again. 100 μl of substrate solution was added to each well in turn, and color development was performed at room temperature for 30 minutes. After color development, 50 μl of stop solution (2 mol / L H2SO4) was added to each well to stop the reaction. Finally, the OD value at 450 nm was read using a microplate reader to determine the sBCMA level in the cerebrospinal fluid.

[0036] (3) Analysis of sBCMA level in cerebrospinal fluid

[0037] Specifically, the sBCMA level in the cerebrospinal fluid of 30 NMOSD patients and gender and age matched control subjects was detected. The detection results showed that there was no statistical difference in the sBCMA level in the cerebrospinal fluid of male subjects and female subjects (P = 0.7498) Figure 1 ), and there was no correlation between the age of the subjects and the sBCMA level in the cerebrospinal fluid (R = 0.031, P = 0.179) Figure 2 ). Further, the sBCMA level in the cerebrospinal fluid of NMOSD patients and control subjects was analyzed, and the results showed that the sBCMA level in the cerebrospinal fluid of NMOSD patients was significantly higher than that of control subjects (P < 0.0001) Figure 3 ). In addition, as Figure 4 shown, the ROC curve results showed that the area under the curve AUC = 0.8678, and there was a significant difference (P < 0.0001). This indicates that sBCMA in the cerebrospinal fluid can be used as a biomarker for NMOSD to assist in the diagnosis of NMOSD, and has good sensitivity and specificity.

[0038] Example 2 sBCMA in cerebrospinal fluid as a biomarker for nerve damage in NMOSD

[0039] Neurofilament light chain (NFL) is widely recognized as a biomarker of nerve injury, which can dynamically reflect the irreversible damage of nerve and the progression of disease, and can be used as a biological indicator for disease progression and monitoring of NMOSD. In this embodiment, based on the SIMOA HD-X platform, the NFL level in the cerebrospinal fluid of the clinical research subjects in Example 1 was quantified using the ultra-sensitive SIMOA technology. The detection results showed that the NFL level in the cerebrospinal fluid of the NMOSD patients was significantly higher than that of the control subjects (P = 0.0002). Further, the correlation between sBCMA and NFL in the cerebrospinal fluid was analyzed in this embodiment. As shown in Figure 5 , the cerebrospinal fluid sBCMA level was positively correlated with NFL in NMOSD patients (R = 0.236, P = 0.006), while there was no correlation between the cerebrospinal fluid sBCMA level and the NFL level in the control subjects (R = 0.084, P = 0.120). This suggests that the increase in cerebrospinal fluid sBCMA level represents the aggravation of nerve injury, and the cerebrospinal fluid sBCMA level can indicate the severity of nerve injury. Therefore, sBCMA in the cerebrospinal fluid can be used as a biomarker to predict nerve injury and reflect the degree of nerve injury in antibody-mediated CNS autoimmune diseases such as NMOSD.

[0040] Example 3 sBCMA in cerebrospinal fluid as a biomarker for blood-brain barrier injury in NMOSD

[0041] In NMOSD, the pro-inflammatory cytokine interleukin-6 can mediate the destruction of the integrity of the blood-brain barrier (BBB), leading to increased penetration of AQP-4 antibodies and further causing subsequent neurotoxicity. Clinical studies have shown that the degree of BBB destruction is a reliable clinical marker for the diagnosis of NMOSD and its severity. The protein concentration gradient between plasma and cerebrospinal fluid is caused by the function of the blood-cerebrospinal fluid barrier, and the ratio of albumin (Alb) level in cerebrospinal fluid to serum albumin level is called albumin quotient (QAlb), which can reflect the degree of blood-cerebrospinal fluid barrier destruction. The formula for calculating QAlb is (Alb in cerebrospinal fluid / Alb in blood) x 1000. The results are as follows Figure 6As shown in Example 1, the QAlb level in NMOSD patients was significantly higher than that in the control group (P = 0.0073). This example analyzed the correlation between sBCMA levels in cerebrospinal fluid (CSF) and QAlb. The results showed a positive correlation between CSF sBCMA levels and QAlb in NMOSD patients (R = 0.401, P = 0.0002), while no correlation was found in the control group (R = 0.027, P = 0.383). This suggests that elevated CSF sBCMA levels represent aggravated blood-brain barrier disruption, and CSF sBCMA levels can serve as a biomarker to indicate the severity of CSF barrier dysfunction. Therefore, CSF sBCMA levels can indicate the severity of CSF barrier disruption during the pathogenesis of antibody-mediated CNS autoimmune diseases such as NMOSD.

[0042] Example 4: sBCMA in cerebrospinal fluid as a biomarker of neuroinflammation in NMOSD

[0043] Increased levels of soluble myeloid cell triggering receptor 2 (sTREM2) expressed in cerebrospinal fluid are associated with an increased risk of NMOSD and are positively correlated with neuroinflammatory responses. The expression level of sTREM2 in the cerebrospinal fluid of the clinical study subjects in Example 1 was detected using a human TREM2 ELISA kit (Abcam, ab224881). Figure 7 As shown, compared with the control group, NMOSD patients had significantly elevated sTREM2 levels (P = 0.0004). Similarly, this example analyzed the correlation between sBCMA levels and sTREM2 levels. The results showed that cerebrospinal fluid sBCMA levels in NMOSD patients were positively correlated with sTREM2 (R = 0.454, P < 0.0001). However, this correlation did not exist in the control group (R = 0.003, P = 0.757). This suggests that cerebrospinal fluid sBCMA levels can reflect the degree of neuroinflammation, and cerebrospinal fluid sBCMA levels can serve as a biomarker for predicting the level of neuroinflammation, playing a suggestive role in the early warning and auxiliary diagnosis of antibody-mediated central nervous system autoimmune diseases such as NMOSD.

[0044] Example 5: Validation of the application of sBCMA in cerebrospinal fluid as a biomarker in the diagnosis of NMOSD

[0045] To further confirm the reliability of sBCMA in cerebrospinal fluid as a biomarker for NMOSD, the following examples included a validation cohort of 15 NMOSD patients (positive for aquaporin 4 antibody—AQP4-IgG) and 15 sex- and age-matched control subjects with negative serum AQP4 antibodies. Details are shown in the table below:

[0046]

[0047] Specifically, the present application further detected the sBCMA levels in the cerebrospinal fluid of 15 NMOSD patients in the validation cohort and 15 gender and age matched control subjects. The detection results showed that there was no statistical difference in the sBCMA levels in the cerebrospinal fluid of male subjects and female subjects (P = 0.1388) Figure 8 ), and there was no correlation between the age of the subjects and the sBCMA levels in the cerebrospinal fluid (R = 0.0899, P = 0.1074) Figure 9 ). Further, the sBCMA levels in the cerebrospinal fluid of the NMOSD patients and the control subjects were analyzed, and the results showed that the sBCMA levels in the cerebrospinal fluid of the NMOSD patients were significantly higher than those of the control subjects (P = 0.0075) Figure 10 ). In addition, the ROC curve results showed that the area under the curve AUC = 0.7822, and there was a significant difference (P = 0.0084) Figure 11 ). This further proved that the sBCMA in the cerebrospinal fluid can be used as a biomarker for NMOSD to assist in the diagnosis of NMOSD, and has good sensitivity and specificity.

[0048] In addition, the present application detected the levels of the nerve injury marker NFL and the neuroinflammation marker sTREM2 in the cerebrospinal fluid of the subjects in the validation cohort. The detection results showed that the NFL (P = 0.0420) and sTREM2 levels (P = 0.0141) in the cerebrospinal fluid of the NMOSD patients in the validation cohort were significantly higher than those of the control subjects. Further, the present embodiment analyzed the correlation between the sBCMA and NFL, sTREM2 in the cerebrospinal fluid. As shown in Figure 12 , the sBCMA levels in the cerebrospinal fluid of the NMOSD patients were positively correlated with NFL (R = 0.4157, P = 0.0095), while the sBCMA levels in the cerebrospinal fluid of the control subjects were not correlated with the NFL levels (R = 0.0180, P = 0.6331). This suggests that the sBCMA levels in the cerebrospinal fluid can be used as a biomarker for the severity of nerve injury. As shown in Figure 13 , the sBCMA levels in the cerebrospinal fluid of the NMOSD patients were positively correlated with sTREM2 (R = 0.3684, P = 0.0213). But in the control group, such correlation did not exist (R = 0.0013, P = 0.9026). This suggests that the sBCMA levels in the cerebrospinal fluid can reflect the degree of neuroinflammation, and the sBCMA levels in the cerebrospinal fluid can be used as a biomarker for the level of neuroinflammation.

[0049] In summary, sBCMA in CSF can reflect the severity of neural damage, blood-CSF barrier disruption and inflammation in CNS of NMOSD patients. Furthermore, based on the common pathogenesis of antibody-mediated CNS autoimmune diseases, sBCMA in CSF can not only provide a powerful auxiliary means for early diagnosis of NMOSD, but also provide an excellent reference value for clinical diagnosis and disease monitoring of other antibody-mediated CNS autoimmune diseases (such as myelin oligodendrocyte glycoprotein antibody-associated disease, anti-N-methyl-D-aspartate receptor encephalitis, anti-lysine-rich glioma inactivating protein 11 encephalitis, anti-contact protein-related protein 2 antibody-associated autoimmune encephalitis, etc.).

Claims

1. Use of a reagent for detecting the biomarker sBCMA protein in the manufacture of a kit for the diagnosis of antibody-mediated autoimmune diseases of the central nervous system, characterized in that, The antibody-mediated central nervous system autoimmune disease is a neuromyelitis optica spectrum disorder.

2. Use of a reagent for detecting the biomarker sBCMA protein in the manufacture of a kit for assessing the severity of neurological damage in a patient with an antibody-mediated autoimmune disease of the central nervous system, characterized in that, The antibody-mediated central nervous system autoimmune disease is a neuromyelitis optica spectrum disorder.

3. Use of a reagent for detecting biomarker sBCMA protein in the preparation of a kit for evaluating the degree of blood-cerebrospinal fluid barrier damage in a patient with an antibody-mediated central nervous system autoimmune disease, which is a neuromyelitis optica spectrum disorder.

4. Use of a reagent for detecting biomarker sBCMA protein in the preparation of a kit for evaluating the level of neuroinflammation in a patient with an antibody-mediated central nervous system autoimmune disease, which is a neuromyelitis optica spectrum disorder.

5. Use according to any one of claims 1 to 4, characterized in that, The biomarker sBCMA protein is from cerebrospinal fluid.

6. Use according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: 1) determining the level of sBCMA protein in the cerebrospinal fluid of the subject; 2) performing a significant difference analysis on the level of sBCMA protein in the cerebrospinal fluid of the subject and the level of sBCMA protein in a gender and age matched control group.

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