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

Abstract

The invention discloses an application of sBCMA in cerebrospinal fluid as a biomarker in antibody-mediated central nervous system autoimmune disease diagnosis and disease monitoring. Taking NMOSD as an example, the level of sBCMA in cerebrospinal fluid of an NMOSD patient is significantly higher than that of a control group subject with matched age and gender, and the level of sBCMA in the cerebrospinal fluid has a positive correlation relationship with a nerve injury related index NFL, a blood-cerebrospinal fluid barrier destruction related index QAlb and a neuroinflammation related index sTREM2 in the cerebrospinal fluid of the NMOSD patient. The sBCMA level in the cerebrospinal fluid can be used as a biomarker for diagnosing the NMOSD antibody-mediated central nervous system autoimmune disease, and can also be used for evaluating the severity of nerve injury, the damage degree of blood-cerebrospinal fluid barrier, the neuroinflammation level and the like of a patient with the central nervous system autoimmune disease.

Description

Technical Field

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

[0002] Autoimmune diseases are a group of diseases characterized by immune dysfunction and abnormal immune responses to normal body components. More than 80 autoimmune diseases have been discovered, including systemic ones, such as systemic lupus erythematosus, and tissue or organ specific ones, such as autoimmune thyroiditis. Autoimmune diseases can affect a wide range of systems throughout the body, including the skin, joints, kidneys, and central nervous system.

[0003] Central nervous system (CNS) autoimmune diseases are autoimmune diseases whose main pathogenic mechanism is that autoimmune cells, autoantibodies and other immune molecules directly or indirectly attack the nervous system (including neurons, glial cells, and myelin sheath). There are many types of CNS autoimmune diseases, including neuromyelitis optic spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein-IgG associated disorders (MOGAD), multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), autoimmune encephalitis (AE), CNS vasculitis, etc. The pathogenesis of CNS autoimmune diseases is complex, and the immune inflammatory response caused by autoimmune cells, autoantibodies and other immune molecules directly or indirectly attacking the nervous system plays an important role in its pathogenesis. Among them, autoantibodies play a very important role in the diagnosis and differential diagnosis of CNS inflammatory demyelinating diseases.

[0004] B lymphocytes / plasma cells are one of the most important effector cells of autoimmune diseases. They attack normal cells and tissues and organs through various means such as abnormal secretion of autoantibodies, inducing 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 and has been confirmed to be a good biomarker for multiple myeloma and systemic lupus erythematosus. A prospective study evaluated the correlation between serum sBCMA and the degree of bone marrow plasma cell infiltration in patients with multiple myeloma, confirming that sBCMA levels are a good prognostic indicator for multiple myeloma. Another retrospective cohort study revealed the potential and significance of serum sBCMA as a biomarker in systemic lupus erythematosus patients. Due to the unique structure of the CNS, the blood-brain barrier, autoimmune cells cannot easily enter the CNS under normal circumstances. Therefore, unlike other autoimmune diseases, the immune response of CNS antibody-mediated autoimmune diseases may be limited, and no relevant clinical studies have shown the clinical application of sBCMA as a biomarker in the diagnosis and monitoring of CNS antibody-mediated CNS autoimmune diseases. Summary of the invention

[0005] The present invention provides a new use of sBCMA in cerebrospinal fluid as a biomarker, namely, its use in preparing a diagnostic kit and a disease monitoring kit for antibody-mediated central nervous system autoimmune diseases represented by neuromyelitis optica spectrum disorders (NMOSD).

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[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) Detection of sBCMA levels in cerebrospinal fluid is used to diagnose antibody-mediated central nervous system autoimmune diseases;

[0009] (2) Detection of sBCMA levels in cerebrospinal fluid is used to assess the severity of neurological damage in patients with antibody-mediated central nervous system autoimmune diseases;

[0010] (3) Detection of sBCMA levels in cerebrospinal fluid is used to assess the extent of blood-cerebrospinal fluid barrier disruption in patients with antibody-mediated central nervous system autoimmune diseases;

[0011] (4) Detection of sBCMA levels in cerebrospinal fluid is used to assess neuroinflammation in patients with antibody-mediated central nervous system autoimmune diseases;

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

[0013] In addition to neuromyelitis optica spectrum disorders, the above-mentioned antibody-mediated central nervous system autoimmune diseases also include myelin oligodendrocyte glycoprotein antibody-related diseases, anti-N-methyl-D-aspartate receptor encephalitis, anti-leucine-rich glioma inactivated protein 1 encephalitis, and anti-contactin-associated protein 2 antibody-related autoimmune encephalitis.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: First, the present invention determines that the level of sBCMA in the cerebrospinal fluid of NMOSD patients is significantly higher than that of the control group subjects matched for gender and age. Further, the present invention determines that there is a positive correlation between the sBCMA concentration in the cerebrospinal fluid of NMOSD patients and indicators related to nerve damage and blood-cerebrospinal fluid barrier destruction, which indicates that the sBCMA level in the cerebrospinal fluid of NMOSD patients can be used as a biomarker for auxiliary diagnosis of NMOSD patients, and can reflect the severity of nerve damage and pathological damage in NMOSD patients. At the same time, the present invention determines that there is a positive correlation between sBCMA and neuroinflammation in the cerebrospinal fluid of NMOSD patients. 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-related diseases, anti-N-methyl-D-aspartate receptor encephalitis, anti-leucine-rich glioma inactivation protein 11 encephalitis, anti-contactin-related protein 2 antibody-related autoimmune encephalitis, etc.), with good sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0018] Figure 4 Figure 2 is the ROC curve of sBCMA levels in the subjects' cerebrospinal fluid.

[0019] Figure 5 Analysis of the level of NFL, an indicator related to nerve damage in subjects (A) and the correlation analysis results between sBCMA level in cerebrospinal fluid and NFL (B).

[0020] Figure 6 Analysis of QAlb level, an indicator related to blood-cerebrospinal fluid barrier disruption in subjects (A) and the correlation analysis results between sBCMA level in cerebrospinal fluid and QAlb (B).

[0021] Figure 7 Analysis of the levels of sTREM2, a neuroinflammation-related indicator in subjects (A) and the correlation analysis results between sBCMA levels in cerebrospinal fluid and sTREM2, a neuroinflammation-related indicator (B).

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

[0023] Fig. 9 To verify the results of the correlation analysis between the age of cohort subjects and the level of sBCMA in cerebrospinal fluid.

[0024] Fig.10 To verify the analysis results of sBCMA levels in the cerebrospinal fluid of the cohort subjects.

[0025] Fig.11 Figure 3 ROC curve of sBCMA levels in cerebrospinal fluid of validation cohort subjects.

[0026] Fig.12 To verify the level analysis of NFL, a nerve injury-related indicator in the cerebrospinal fluid of the cohort subjects, and the correlation analysis results between the sBCMA level in the cerebrospinal fluid and NFL.

[0027] Fig.13 To verify the level analysis of sTREM2, a nerve injury-related indicator in the cerebrospinal fluid of the cohort subjects, and the correlation analysis results between sBCMA levels in the cerebrospinal fluid and sTREM2. DETAILED DESCRIPTION

[0028] Neuromyelitis optica spectrum disorder (NMOSD) is a rare, progressive, autoimmune-mediated inflammatory demyelinating disease of the CNS that mainly affects the optic nerve and spinal cord. NMOSD is characterized by inflammatory demyelination and axonal damage of the optic nerve and spinal cord, which can cause blindness, paralysis, and incontinence. Its pathogenesis is mainly derived from plasma cells of B cells producing aquaporin 4 (AQP4) antibodies, which pass through the blood-brain barrier and bind to the AQP4 antigen on astrocytes. Through mechanisms such as antibody-dependent cytotoxicity and complement-dependent cytotoxicity, it ultimately causes astrocyte damage, oligodendrocyte damage, demyelination, and neuronal loss. NMOSD is more common in young and middle-aged people, mostly women. Clinically, it is mainly characterized by severe optic neuritis and longitudinally extended long-segment transverse myelitis, with high recurrence rate and disability rate. The incidence rate in China is about 0.41 / 100,000, and the prevalence rate is about 3.31 / 100,000. NMOSD is a highly recurrent and disabling disease, and most subjects are left with serious sequelae, which has brought a significant burden to the health system, families and groups. The present invention uses NMOSD as an example to reveal the important role of sBCMA in cerebrospinal fluid as a biomarker in the diagnosis and disease monitoring of antibody-mediated CNS autoimmune diseases.

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

[0030] Clinical research subjects: The clinical research subjects in the following examples include 30 NMOSD patients (aquaporin 4 antibody positive—AQP4-IgG positive) and 30 control group subjects matched in gender and age and negative for serum AQP4 antibodies. The specific situation is shown in the following table:

[0031]

[0032] (1) Sample collection and preparation

[0033] The cerebrospinal fluid samples of the clinical research subjects were collected using sterile centrifuge tubes. The collected cerebrospinal fluid samples were centrifuged at 500×g for 10 minutes at 4°C, and the supernatant was dispensed into cryopreservation tubes and stored in a -80°C ultra-low temperature refrigerator.

[0034] (2) sBCMA concentration detection method

[0035] The expression level of sBCMA in cerebrospinal fluid was detected using a human BCMA / TNFRSF17 ELISA kit (R&D Systems, DY193). Specifically, the sample was restored to a temperature range of 18-25°C before the test. According to the requirements of the instructions, the sample was diluted and the standard was prepared. Subsequently, 100 μl of the standard and sample were 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 1× washing solution (300 μl each time, 3 to 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 the plate was washed again after completion. 100 μl of substrate solution was added to each well in turn, and the color was developed in the dark at room temperature for 30 minutes. After color development, 50 μl of stop solution (2 mol / LH 2 SO 4 ) to terminate the reaction. Finally, an OD value of 450 nm was read using a microplate reader to determine the level of sBCMA in the cerebrospinal fluid.

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

[0037] Specifically, the present invention detected the sBCMA level in the cerebrospinal fluid of 30 NMOSD patients and a control group of subjects matched in gender and age. The test results showed that there was no statistical difference in the sBCMA level in the cerebrospinal fluid of male and female subjects (P=0.7498)( Figure 1 ), and there was no correlation between the age of the subjects and the level of sBCMA in the cerebrospinal fluid (R=0.031, P=0.179) ( Figure 2 ). Further, the sBCMA levels in the cerebrospinal fluid of NMOSD patients and control subjects were analyzed. The results showed that the sBCMA levels in the cerebrospinal fluid of NMOSD patients were significantly higher than those in the control subjects (P<0.0001)( Figure 3 ). In addition, Figure 4 As 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 shows that sBCMA in 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 of neural damage in NMOSD

[0039] Neurofilament light chain (NFL) is widely considered as a biomarker of neural damage that can dynamically reflect irreversible neural damage and disease progression, and can be used as a biological indicator for NMOSD disease progression and monitoring. In this example, based on the automated SIMOAHD-X platform, ultra-sensitive SIMOA technology was used to quantify the NFL level in the cerebrospinal fluid of the clinical research subjects in Example 1. The test results showed that the NFL level in the cerebrospinal fluid of NMOSD patients was significantly higher than that of the control group subjects (P=0.0002). Furthermore, this example analyzes the correlation between sBCMA and NFL in cerebrospinal fluid. Figure 5 As shown in the results, 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 NFL level in the control group (R=0.084, P=0.120). This suggests that increased cerebrospinal fluid sBCMA levels represent aggravated nerve damage, and cerebrospinal fluid sBCMA levels can indicate the severity of nerve damage. Therefore, sBCMA in cerebrospinal fluid can be used as a biomarker to predict nerve damage and reflect the degree of nerve damage in antibody-mediated CNS autoimmune diseases such as NMOSD.

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

[0041] In NMOSD, the proinflammatory cytokine interleukin-6 can mediate the destruction of the integrity of the blood-brain barrier (BBB), resulting in increased penetration of AQP-4 antibodies, further causing subsequent neurotoxic reactions. 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 blood-cerebrospinal fluid barrier function. The ratio of the albumin (Alb) level in the cerebrospinal fluid to the serum albumin level is called the albumin quotient (QAlb), which can reflect the degree of damage to the blood-cerebrospinal fluid barrier. The QAlb calculation formula is (Alb in cerebrospinal fluid / Alb in blood) × 1000. The results are as follows: Figure 6As shown, the QAlb level of NMOSD patients in the clinical study subjects in Example 1 was significantly higher than that of the control group subjects (P=0.0073). In this example, the sBCMA level in the cerebrospinal fluid was correlated with QAlb. The results showed that the sBCMA level in the cerebrospinal fluid of NMOSD patients was positively correlated with QAlb (R=0.401, P=0.0002), while there was no correlation in the control group (R=0.027, P=0.383). This suggests that an increase in the sBCMA level in the cerebrospinal fluid represents an aggravation of blood-brain barrier damage, and the sBCMA level in the cerebrospinal fluid can be used as a biomarker to indicate the severity of impaired blood-cerebrospinal fluid barrier function. Therefore, the sBCMA level in the cerebrospinal fluid can indicate the severity of blood-cerebrospinal fluid barrier damage in the pathogenic process 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 the risk of NMOSD and are positively correlated with neuroinflammatory response. The expression level of sTREM2 in the cerebrospinal fluid of the clinical study subjects in Example 1 was detected by human TREM2 ELISA kit (Abcam, ab224881). Figure 7 As shown, compared with the control group subjects, the sTREM2 level of NMOSD patients was significantly increased (P = 0.0004). Similarly, this example analyzes the correlation between sBCMA levels and sTREM2 levels. The results showed that the cerebrospinal fluid sBCMA level of NMOSD patients was positively correlated with sTREM2 (R = 0.454, P < 0.0001). However, in the control group, this correlation did not exist (R = 0.003, P = 0.757). This suggests that the cerebrospinal fluid sBCMA level can feedback the degree of neuroinflammation, and the cerebrospinal fluid sBCMA level can be used as a biomarker to predict the level of neuroinflammation, which has a suggestive effect on the early warning and auxiliary diagnosis of antibody-mediated central nervous system autoimmune diseases such as NMOSD.

[0044] Example 5 Verification of the application of sBCMA in cerebrospinal fluid as a marker 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 15 NMOSD patients (aquaporin 4 antibody positive - AQP4-IgG positive) and 15 control group subjects with gender and age matching and serum AQP4 antibody negative as a validation cohort. The specific situation is shown in the following table:

[0046]

[0047] Specifically, the present invention further detected the sBCMA levels in the cerebrospinal fluid of 15 NMOSD patients in the validation cohort and 15 control subjects matched in gender and age. The test results showed that there was no statistical difference in the sBCMA levels in the cerebrospinal fluid of male and female subjects (P=0.1388)( Figure 8 ), and there was no correlation between the age of the subjects and the level of sBCMA in the cerebrospinal fluid (R=0.0899, P=0.1074) ( Fig. 9 ). Further, the sBCMA levels in the cerebrospinal fluid of NMOSD patients and control subjects were analyzed. The results showed that the sBCMA levels in the cerebrospinal fluid of NMOSD patients were significantly higher than those in the control subjects (P=0.0075) ( Fig.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) ( Fig.11 ). This further proves that sBCMA in 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 invention detected the levels of the neural injury marker NFL and the neuroinflammation marker sTREM2 in the cerebrospinal fluid of the validation cohort subjects. The test results showed that the cerebrospinal fluid NFL (P = 0.0420) and sTREM2 levels (P = 0.0141) of NMOSD patients in the validation cohort were significantly higher than those of the control group subjects. Furthermore, this example analyzes the correlation between sBCMA and NFL and sTREM2 in cerebrospinal fluid. Fig.12 As shown in the results, the cerebrospinal fluid sBCMA level was positively correlated with NFL in NMOSD patients (R=0.4157, P=0.0095), while there was no correlation between the cerebrospinal fluid sBCMA level and NFL level in the control group (R=0.0180, P=0.6331). This suggests that the cerebrospinal fluid sBCMA level can be used as a biomarker for the severity of neurological damage. Fig.13 The results showed that the sBCMA level in the cerebrospinal fluid of NMOSD patients was positively correlated with sTREM2 (R=0.3684, P=0.0213). However, this correlation did not exist in the control group (R=0.0013, P=0.9026). This suggests that the sBCMA level in the cerebrospinal fluid can reflect the degree of neuroinflammation and can be used as a biomarker for the level of neuroinflammation.

[0049] The above analysis shows that sBCMA in CSF can reflect the severity of nerve damage, blood-cerebrospinal fluid barrier damage and inflammatory response of the central nervous system in patients with NMOSD. Furthermore, based on the common pathogenic characteristics of antibody-mediated central nervous system autoimmune diseases, the level of sBCMA in CSF can not only provide a powerful auxiliary means for the early diagnosis of NMOSD, but also provide excellent reference value for the clinical diagnosis and disease monitoring of other antibody-mediated central nervous system autoimmune diseases (such as myelin oligodendrocyte glycoprotein antibody-related diseases, anti-N-methyl-D-aspartate receptor encephalitis, anti-leucine-rich glioma inactivated protein 11 encephalitis, anti-contactin-associated protein 2 antibody-related autoimmune encephalitis, etc.).

Claims

1. Use of a reagent for detecting the biomarker sBCMA in preparing a kit, characterized in that: The kit is used for diagnosing antibody-mediated central nervous system autoimmune diseases.

2. Use of a reagent for detecting the biomarker sBCMA in preparing a kit, characterized in that: The kit is used to evaluate the severity of nerve damage in patients with antibody-mediated central nervous system autoimmune diseases.

3. Use of a reagent for detecting the biomarker sBCMA in the preparation of a kit for evaluating the degree of blood-cerebrospinal fluid barrier destruction in patients with antibody-mediated central nervous system autoimmune diseases.

4. Use of a reagent for detecting the biomarker sBCMA in the preparation of a kit for evaluating the level of neuroinflammation in patients with antibody-mediated central nervous system autoimmune diseases.

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

6. The use according to any one of claims 1 to 4, characterized in that The following steps are involved: 1) Determine the sBCMA level in the subjects' cerebrospinal fluid; 2) Perform a significant difference analysis between the sBCMA level in the subjects' cerebrospinal fluid and the sBCMA level in the control group matched with gender and age.

7. The use according to any one of claims 1 to 4, characterized in that The antibody-mediated central nervous system autoimmune disease is a neuromyelitis optica spectrum disease.

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