Application of serum and cerebrospinal fluid FcRL2 protein in preparation of neurosyphilis or nerve injury diagnosis product

Abstract

The invention relates to the technical field of biological medicines, in particular to FcRL2 as a serum marker or cerebrospinal fluid marker for early diagnosis, screening and typing detection of neurosyphilis patients. The FcRL2 protein is one of receptor superfamily members, Fc receptor-like glycoproteins, and is related to immune response regulation and immune cell functions. According to the invention, the concentration of the FcRL2 protein in serum or cerebrospinal fluid is used as an index for early diagnosis of neurosyphilis, typing detection of symptomatic neurosyphilis, asymptomatic neurosyphilis, neurosyphilis and recessive syphilis, evaluation of nerve injury degree and the like; the index can be independently used or used as a main reference index to be combined with other indexes to complete diagnosis screening, typing and nerve injury evaluation of the neurosyphilis. Compared with the prior art, the FcRL2 not only has high sensitivity, but also can diagnose the neurosyphilis through noninvasive serum detection, and is beneficial to diagnosis and management of the neurosyphilis in community hospitals and local hospitals.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of serum and cerebrospinal fluid FcRL2 proteins and their specific antibodies in the preparation of products for neurosyphilis screening, typing detection or assessment of the degree of nerve damage. Background Art

[0002] Neurosyphilis is a group of clinical syndromes in which Treponema pallidum invades the nervous system and causes damage to the meninges, brain, blood vessels or spinal cord. It can occur at all stages of the course of syphilis, not just in tertiary syphilis. The clinical manifestations of neurosyphilis vary, ranging from no clinical symptoms to complex clinical manifestations with extensive involvement of the nervous system. Clinically, it is usually divided into five types: asymptomatic, meningeal syphilis, meningeal vascular syphilis, tabes dorsalis and paralytic dementia (brain parenchymal syphilis). Although latent syphilis has no clinical symptoms, its harmfulness should be taken seriously. It can become recurrent syphilis and may progress to visceral and neurosyphilis. Patients with asymptomatic neurosyphilis have no obvious symptoms or signs, but there are abnormal changes in cerebrospinal fluid. If the abnormal cerebrospinal fluid persists for more than 5 years, the probability of developing symptomatic neurosyphilis is as high as 87%. In studies of primary and secondary syphilis, 20% of patients had abnormal cerebrospinal fluid, that is, neurosyphilis can occur in the early stages of syphilis. Early neurosyphilis includes syphilitic meningitis and vascular syphilis, and late neurosyphilis includes paralytic dementia, tabes dorsalis, etc. The incubation period of paralytic dementia is 10-15 years. Paralytic dementia is mainly manifested by mental and intellectual decline, such as memory loss, abnormal behavior, personality changes, and severe patients have epilepsy, stroke-like onset, secondary hemiplegia and aphasia.

[0003] Early diagnosis of neurosyphilis is very important. Finding sensitive biomarkers is helpful for the diagnosis of neurosyphilis and is of great significance for preventing serious and irreversible sequelae. At present, the diagnosis of neurosyphilis mainly relies on the examination of cerebrospinal fluid, including CSF-RPR, CSF white blood cell count and protein level. CSF-RPR is the same as the rapid plasma reagin ring card test in plasma. A positive result of this indicator can diagnose neurosyphilis. Important reference indicators for neurosyphilis also include cerebrospinal fluid cell count or protein level. When the CSF-RPR result is negative, if there are clinical signs of neurosyphilis, positive plasma tests and abnormal cerebrospinal fluid cell count, neurosyphilis can also be diagnosed. The increased protein content in the cerebrospinal fluid of patients with neurosyphilis is positively correlated with the severity of the disease and negatively correlated with the long-term prognosis. The increase in leukocytosis and the increase in protein content in the cerebrospinal fluid indicate the destruction of the blood-brain barrier and the presence of inflammatory reaction in the skull, which plays an auxiliary role in the diagnosis of neurosyphilis. However, the sensitivity of CSF-RPP was found to be only 30%-70% during the diagnosis process, which is low and insensitive to the diagnosis of early asymptomatic neurosyphilis. At present, in the clinical diagnosis and treatment process, neurosyphilis is mainly diagnosed and treated in patients with symptoms of neurosyphilis. There is a lack of sensitive and reliable cerebrospinal fluid and serum biological markers that can effectively distinguish neurosyphilis, and there is an even greater lack of biological markers in serum that can effectively distinguish neurosyphilis.

[0004] In summary, the current diagnostic methods for neurosyphilis require invasive cerebrospinal fluid operations and lack sensitive and accurate markers, which is extremely challenging. Therefore, the development of new biomarkers in serum that can be used for the diagnosis of neurosyphilis is of great significance for the diagnosis and management of neurosyphilis. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the technical problems that the existing CSF-RPR diagnostic method has low sensitivity, is even insensitive to the diagnosis of early asymptomatic neurosyphilis, and relies on invasive cerebrospinal fluid examination, the present invention provides a cerebrospinal fluid / serum marker for early diagnosis screening, typing detection and nerve damage assessment of neurosyphilis. The marker can be used to achieve early diagnosis, screening, typing detection and nerve damage assessment of neurosyphilis. Compared with the existing technology, it is not only more sensitive, but also can achieve non-invasive serum detection of neurosyphilis.

[0007] (II) Technical solution

[0008] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0009] In a first aspect, the present invention relates to FcRL2 as a serum marker or cerebrospinal fluid marker for early diagnosis, screening and typing detection of patients with neurosyphilis.

[0010] In a second aspect, the present invention provides the use of FcRL2 protein and its specific antibodies in the preparation of diagnostic products or auxiliary diagnostic products for neurosyphilis screening, typing detection or assessment of the degree of nerve damage; the diagnostic products or auxiliary diagnostic products are detection kits, antibody chips, antibody probes or detectors with FcRL2 or its specific antibodies as target molecules.

[0011] According to a preferred embodiment of the present invention, the kit uses FcRL2 protein or its specific antibody as a target molecule to detect the expression level of FcRL2 protein or its specific antibody in cerebrospinal fluid or serum.

[0012] According to a preferred embodiment of the present invention, the kit is an enzyme-linked immunosorbent assay kit or an immunohistochemistry kit for detecting FcRL2.

[0013] According to a preferred embodiment of the present invention, the kit comprises: a primary antibody that can specifically recognize FcRL2, a polypeptide dye, magnetic beads, and a biotin-labeled secondary antibody.

[0014] The kit comprises: a primary antibody capable of specifically recognizing FcRL2, a polypeptide dye, magnetic beads and a biotin-labeled secondary antibody.

[0015] According to a preferred embodiment of the present invention, the kit further comprises reagents or materials for obtaining serum or cerebrospinal fluid from the patient.

[0016] It should be noted that when detecting the expression level of FcRL2 protein in serum, the Olink ultra-high sensitivity plasma protein detection platform can also be used to quantify the expression level of FcRL2.

[0017] In a third aspect, the present invention also relates to a product for early diagnosis, screening, typing detection or assessment of the degree of nerve damage in patients with neurosyphilis; the product can quantitatively detect the concentration of FcRL2 in the patient's ex vivo serum or cerebrospinal fluid; the product is a detection kit, an antibody chip, an antibody probe or a detector.

[0018] Preferably, the product comprises antibodies that specifically bind to FcRL2 and other reagents, as long as the concentration of FcRL2 protein in serum or cerebrospinal fluid can be quantitatively detected. The experimental method used in the examples of the present application is Olinkneurology panel (95801), which can be used to detect 92 protein biomarkers related to neural function, including FcRL2 protein.

[0019] In a fourth aspect, the present invention relates to an assessment system for the risk of nerve damage in patients with neurosyphilis, comprising a detection module and a judgment module, wherein the detection module is used to obtain the concentration value of serum or cerebrospinal fluid FcRL2; the judgment module comprises a readable carrier storing a judgment rule;

[0020] The judgment rule is: when the detection module detects that the serum FcRL2 concentration value is ≤3.443NPX or the cerebrospinal fluid FcRL2 concentration value is ≥-0.05916NPX; it is judged as a high risk of neurological injury;

[0021] When the detection module detects 3.443NPX<serum FcRL2≤3.638NPX or -0.05916NPX>cerebrospinal fluid FcRL2 concentration value ≥-0.6267NPX; it is judged as moderate nerve injury risk;

[0022] When the detection module detects that the serum FcRL2 concentration value is ≤3.725NPX or the cerebrospinal fluid FcRL2 concentration value is ≥-0.7020NPX, it is judged that there is a risk of cerebrospinal fluid infection with syphilis;

[0023] When the detection module detects that the serum FcRL2 concentration value is greater than 3.725NPX or the cerebrospinal fluid FcRL2 concentration value is less than -0.7020NPX, it is judged as no or low risk of cerebrospinal fluid infection with syphilis.

[0024] The reference value is the FcRL2 cut-off value obtained from the syphilis patient group; preferably, the serum cut-off value for the diagnosis of neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) is 3.638NPX value (less than or equal to this value is judged as a moderate risk of nerve damage), and the cerebrospinal fluid cut-off value is -0.6267NPX value (greater than or equal to this value is judged as a moderate risk of nerve damage); the serum cut-off value for the diagnosis of symptomatic neurosyphilis is 3.443NPX value (less than or equal to this value is judged as a high risk of neurological damage), the cerebrospinal fluid cut-off value is -0.05916NPX value (greater than or equal to this value is judged as a high risk of neurological damage); the serum cut-off value for the diagnosis of asymptomatic neurosyphilis is 3.725NPX (greater than this value is judged as no or low risk of neurological damage, or judged as non-neurosyphilis), and the cerebrospinal fluid cut-off value is -0.7020NPX (less than this value is judged as no or low risk of neurological damage, or judged as non-neurosyphilis). The above cut-off values ​​are the critical values ​​of FcRL2 statistical analysis obtained from 88 syphilis patients.

[0025] Although the expression level of specific proteins in cerebrospinal fluid is a sensitive indicator of pathological changes in neural tissue, it is only applicable to a narrow range of people and is an invasive procedure with certain potential risks. It is relatively traumatic for patients and generally requires hospitalization. However, the reduction of FcRL2 protein in serum can be used as an indicator of increased cerebrospinal fluid levels, thereby enabling convenient and fast non-invasive screening and diagnosis.

[0026] In a fifth aspect, the present invention also provides a syphilis detection kit, comprising: a first detection reagent composition for detecting serum or cerebrospinal fluid FcRL2 protein levels; and a second detection reagent composition for detecting cerebrospinal fluid leukocytes and / or cerebrospinal fluid proteins.

[0027] In the above scheme, when diagnosing neurosyphilis (including early diagnosis, typing and staging, and nerve damage assessment), the serum or cerebrospinal fluid FcRL2 protein level can be combined with two or three indicators of cerebrospinal fluid leukocytes and cerebrospinal fluid proteins related to nerve damage in patients with neurosyphilis for early diagnosis, typing and staging, and nerve damage assessment of neurosyphilis.

[0028] Among them, the level of FcRL2 in serum or cerebrospinal fluid can be determined by Olink precision proteomics, i.e., antibody immunoassay combined with quantitative real-time PCR. The number of leukocytes and the amount of cerebrospinal fluid protein can be detected by known methods.

[0029] (III) Beneficial effects

[0030] The present invention provides a new cerebrospinal fluid / serum marker FcRL2 for early diagnosis screening, typing detection and nerve damage assessment of neurosyphilis. By detecting the expression level of FcRL2 in the patient's serum or cerebrospinal fluid, a quantitative assessment of the degree of nerve damage in patients with neurosyphilis can be achieved; or by detecting the expression level of FcRL2 in the patient's serum or cerebrospinal fluid, neurosyphilis can be diagnosed, including early diagnosis and typing detection. The marker can be used to achieve early diagnosis, screening, typing detection and nerve damage assessment of neurosyphilis. Compared with the existing technology, it is not only more sensitive, but also can achieve non-invasive serum detection of neurosyphilis.

[0031] Preferably, the cerebrospinal fluid marker FcRL2 is also suitable for the diagnosis and screening of symptomatic neurosyphilis and asymptomatic neurosyphilis, because among symptomatic neurosyphilis, asymptomatic neurosyphilis or non-neurosyphilis, the expression level of FcRL2 in the cerebrospinal fluid of patients with symptomatic neurosyphilis is the highest, and the FcRL2 expression level has a high sensitivity in the assessment of neurosyphilis, especially symptomatic neurosyphilis, and also has a predictive effect on asymptomatic neurosyphilis. Therefore, screening for symptomatic neurosyphilis and asymptomatic syphilis can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The Olink method was used to detect the expression of FcRL2 protein in the cerebrospinal fluid and serum of three groups of syphilis patients with different types (latent, asymptomatic, symptomatic).

[0033] Figure 2 The results showed that the expression of FcRL2 in cerebrospinal fluid was significantly positively correlated with cerebrospinal fluid protein and leukocytes.

[0034] Figure 3 The expression of FcRL2 in serum was negatively correlated with cerebrospinal fluid protein and leukocytes.

[0035] Figure 4 The AUC is the probability of evaluating the diagnosis of neurosyphilis by the cerebrospinal fluid and serum FcRL2 concentration levels when the diagnosis of neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) is the clinical outcome; the table below the figure shows the sensitivity and specificity of the corresponding indicators and the cut-off value.

[0036] Figure 5 When the diagnosis of symptomatic neurosyphilis is used as the clinical outcome, the AUC of different levels of cerebrospinal fluid and serum FcRL2 concentrations are used to assess the possibility of a confirmed diagnosis of symptomatic neurosyphilis. The table below the figure shows the sensitivity, specificity and cut-off value of the corresponding indicators.

[0037] Figure 6 In order to distinguish asymptomatic neurosyphilis from non-neurosyphilis when asymptomatic neurosyphilis is the clinical outcome, different levels of cerebrospinal fluid and serum FcRL2 concentrations were used to evaluate the AUC of the possibility of confirming asymptomatic neurosyphilis; the table below the figure shows the sensitivity and specificity of the corresponding indicators and the cut-off value.

[0038] Figure 7 All patients (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) were divided into two groups according to the cerebrospinal fluid FcRL2 cutoff value of -0.6267NPX, and the differences in cerebrospinal fluid protein and leukocytes, the indicators of neurological damage, were found in each group.

[0039] Figure 8 Neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) was divided into two groups based on the cerebrospinal fluid FcRL2 cutoff value of -0.05916NPX, and the differences in cerebrospinal fluid protein and leukocytes, the indicators of neurological damage, were found between the groups.

[0040] Fig. 9 Patients with latent syphilis and asymptomatic neurosyphilis were divided into two groups according to the cutoff value of CSF FcRL2 -0.7020NPX, and the differences in neurological damage indicators, CSF protein and leukocytes, were found between the groups.

[0041] Fig.10 All patients (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) were divided into two groups according to the serum FcRL2 cutoff value of 3.638NPX, and the differences in cerebrospinal fluid protein and leukocytes, the indicators of neurological damage, were found in each group.

[0042] Fig.11Neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) was divided into two groups based on the serum FcRL2 cutoff value of 3.443NPX, and the differences in cerebrospinal fluid protein and leukocytes, the indicators of neurological damage, were found between the groups.

[0043] Fig.12 Patients with latent syphilis and asymptomatic neurosyphilis were divided into two groups according to the serum FcRL2 cutoff value of 3.725NPX, and the differences in neurological damage indicators such as cerebrospinal fluid protein and leukocytes were found between the groups. DETAILED DESCRIPTION

[0044] In order to better explain the present invention, the present invention is now described in detail.

[0045] Early diagnosis of neurosyphilis is very important. Finding sensitive biomarkers is helpful for the diagnosis of neurosyphilis and is of great significance for preventing serious and irreversible sequelae. At present, the diagnosis of neurosyphilis mainly relies on the examination of cerebrospinal fluid. Therefore, the inventors conducted research with the purpose of developing new biomarkers in serum that can be used for the diagnosis of neurosyphilis and nerve damage. They screened multiple indicators related to nerve function in the serum and cerebrospinal fluid of patients with neurosyphilis and obtained FcRL2, which has significant diagnostic value in both serum and cerebrospinal fluid.

[0046] The inventors have found through research that the levels of FcRL2 proteins in the cerebrospinal fluid and serum of patients with different types of syphilis are significantly different. Cerebrospinal fluid FcRL2 protein is significantly increased in patients with neurosyphilis, and further increased in patients with symptomatic neurosyphilis, and is positively correlated with cerebrospinal fluid protein and cerebrospinal fluid leukocytes, which are indicators of nerve damage, indicating that cerebrospinal fluid FcRL2 protein can be used for the diagnosis of neurosyphilis, including asymptomatic neurosyphilis and symptomatic neurosyphilis, and can also indicate the severity of nerve damage. Serum FcRL2 protein is significantly reduced in patients with neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis), but there is no significant difference in patients with symptomatic neurosyphilis and asymptomatic neurosyphilis, and is negatively correlated with cerebrospinal fluid protein and cerebrospinal fluid leukocytes, so FcRL2 protein in the serum of early neurosyphilis is reduced. Due to the damage of syphilis to the blood-brain barrier, serum FcRL2 protein may enter the brain tissue, further regulate neuroimmune disorders, and further promote the progression of neurosyphilis. The FcRL2 protein levels in the cerebrospinal fluid and serum of patients with asymptomatic neurosyphilis were significantly higher than those in the non-neurosyphilis (referred to as latent syphilis in this application) group, indicating that the FcRL2 protein level changed in the early stage of neurosyphilis, namely the asymptomatic neurosyphilis stage. Therefore, this biomarker can effectively distinguish asymptomatic neurosyphilis from latent syphilis and the serum FcRL2 protein level is helpful for non-invasive screening of asymptomatic neurosyphilis, which can solve the clinical problem that all syphilis patients must undergo invasive procedures (lumbar puncture) to obtain cerebrospinal fluid for screening of asymptomatic neurosyphilis.

[0047] During the study, the experiment first used the receiver operating characteristic curve (ROC) to show the diagnostic potential of FcRL2 protein in evaluating neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis); the experimental results showed that the area under the curve (AUC) of cerebrospinal fluid FcRL2 was 0.8676, P < 0.0001, which can be considered statistically significant; the AUC of serum FcRL2 was 0.7144, P = 0.0093, which can be considered statistically significant. Based on this, the level of serum and cerebrospinal fluid FcRL2 in syphilis patients can better evaluate the occurrence of neurosyphilis, with sensitivities of 80 and 73.33, respectively.

[0048] Next, in order to clarify the relationship between serum and cerebrospinal fluid FcRL2 and symptomatic neurosyphilis, 73 patients with neurosyphilis were studied in the following examples, and ROC was used to evaluate the potential of FcRL2 in neurosyphilis patients (including symptomatic and asymptomatic neurosyphilis patients) to evaluate the occurrence of symptomatic neurosyphilis; the experimental results showed that the AUC of cerebrospinal fluid FcRL2 was 0.8977, P < 0.0001, which was considered statistically significant, with a sensitivity of 93.02; the AUC of serum FcRL2 was 0.5279, P = 0.6865. The above results show that the occurrence of symptomatic neurosyphilis can be better evaluated according to the level of cerebrospinal fluid FcRL2 in patients with neurosyphilis, while the serum prediction effect is poor.

[0049] Furthermore, asymptomatic neurosyphilis is a difficult point in clinical diagnosis and treatment. All syphilis patients need to undergo invasive cerebrospinal fluid screening. In fact, some patients are negative, but because there are no clinical symptoms, it is difficult to diagnose. Therefore, all syphilis patients need to be screened. In order to clarify the relationship between serum and cerebrospinal fluid FcRL2 and asymptomatic neurosyphilis, 45 patients with latent syphilis and asymptomatic neurosyphilis were studied in the following examples, and ROC was used to show the potential of FcRL2 to evaluate the occurrence of asymptomatic neurosyphilis; the experimental results showed that the AUC of cerebrospinal fluid FcRL2 was 0.7356, P = 0.0107, which was statistically significant; the AUC of serum FcRL2 was 0.6600, P = 0.0830. The above results show that the occurrence of asymptomatic neurosyphilis can be better evaluated based on the level of FcRL2 in the cerebrospinal fluid of syphilis patients, and whether serum can predict requires more samples for verification.

[0050] Furthermore, the present invention divides all patients with syphilis (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups according to the cerebrospinal fluid FcRL2 Cut-Off value (-0.6267NPX) of all patients with syphilis, and the cerebrospinal fluid protein and leukocytes of patients with cerebrospinal fluid injury above this value are significantly increased. Therefore, when the FcRL2 cerebrospinal fluid is lower than the -0.6267NPX value, the risk of neurosyphilis in patients with neurosyphilis is lower, and the risk of nerve damage is lower; while the risk of neurosyphilis increases above this value, and the risk of nerve damage also increases. The present invention divides all patients with neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups according to the cerebrospinal fluid FcRL2 Cut-Off value (-0.05916NPX) of all patients with neurosyphilis, and the cerebrospinal fluid protein and leukocytes of patients with cerebrospinal fluid injury above this value are significantly increased. Therefore, when the FcRL2 cerebrospinal fluid is lower than the value of -0.05916NPX, the risk of symptomatic neurosyphilis in neurosyphilis patients is low, and the risk of nerve damage is relatively small; while above this value, the risk of symptomatic neurosyphilis is high, and the risk of nerve damage is high. The present invention divides all patients into two groups according to the FcRL2 Cut-Off value (-0.7020NPX) of the cerebrospinal fluid of syphilis patients (including latent syphilis and asymptomatic neurosyphilis). The cerebrospinal fluid protein, an indicator related to cerebrospinal fluid damage in patients above this value, increases significantly, and the white blood cells also increase but not significantly. Therefore, when the FcRL2 cerebrospinal fluid is lower than the value of -0.7020NPX, the risk of asymptomatic neurosyphilis in neurosyphilis patients is low, and the risk of nerve damage is small; while above this value, the risk of asymptomatic neurosyphilis increases, and the risk of nerve damage also increases.

[0051] Similarly, the present invention divides all patients with syphilis (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups according to the serum FcRL2 Cut-Off value (3.638NPX) of all patients with syphilis (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis). Patients with serum FcRL2 Cut-Off value below this value have significantly increased cerebrospinal fluid and leukocytes, indicators related to cerebrospinal fluid damage, and the protein also has an increasing trend, but it is not significant. Therefore, when the FcRL2 serum is higher than the 3.638NPX value, the risk of neurosyphilis in patients with neurosyphilis is lower and the risk of nerve damage is lower; while below this value, the risk of neurosyphilis increases, and the risk of nerve damage also increases. The present invention divides all patients with neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups according to the serum FcRL2 Cut-Off value (3.443NPX) of all patients with neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis). Patients with serum FcRL2 Cut-Off value below this value have significantly increased cerebrospinal fluid leukocytes, indicators related to cerebrospinal fluid damage. Therefore, when the FcRL2 serum is higher than the 3.443NPX value, the risk of symptomatic neurosyphilis in neurosyphilis patients is low, and the risk of nerve damage is relatively small; while below this value, the risk of symptomatic neurosyphilis is high, and the risk of nerve damage is high. The present invention divides all patients into two groups according to the serum FcRL2 Cut-Off value (3.725NPX) of syphilis patients (including latent syphilis and asymptomatic neurosyphilis), and the cerebrospinal fluid leukocytes of patients below this value, which are indicators related to cerebrospinal fluid damage, are significantly increased. Therefore, when the FcRL2 serum is higher than the 3.725NPX value, the risk of asymptomatic neurosyphilis in neurosyphilis patients is low, and the risk of nerve damage is small; while below this value, the risk of asymptomatic neurosyphilis increases, and the risk of nerve damage also increases. Therefore, cerebrospinal fluid and serum FcRL2 can be used as indicators of various types of neurosyphilis and nerve damage.

[0052] In summary, the present invention provides the application of FcRL2 as an early diagnosis of neurosyphilis and assessment of nerve damage. The inventors use the FcRL2 protein level in serum and cerebrospinal fluid as a reliable and effective primary reference indicator or auxiliary reference indicator for early diagnosis, typing and nerve damage assessment of syphilis. Based on the above findings, the present invention can detect the expression level of FcRL2 in serum or cerebrospinal fluid, thereby achieving high-sensitivity detection and typing of syphilis, especially neurosyphilis, and can also perform early non-invasive screening of neurosyphilis, and accurately quantify and evaluate nerve damage, supervision and treatment of neurosyphilis.

[0053] In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0054] The experimental methods in the following experimental examples are conventional methods unless otherwise specified. The raw materials and reagents used in the following examples are commercially available products unless otherwise specified. Related technical contents include:

[0055] 1. Research subjects

[0056] With informed consent and meeting the inclusion criteria, 88 cases were selected for inclusion, including 15 cases of latent syphilis, 30 cases of asymptomatic neurosyphilis and 43 cases of paralytic dementia, to establish a neurosyphilis cohort of different severity. The diagnostic criteria were implemented according to the WS273-2007 syphilis diagnostic criteria.

[0057] (II) Sample collection

[0058] According to the conditions of the enrolled patients, lumbar puncture and cubital venipuncture were performed to obtain cerebrospinal fluid and blood for routine tests, and cerebrospinal fluid and serum samples were collected. With the informed consent of the patients, 5 mL of cerebrospinal fluid was collected by lumbar puncture, and 10 mL of blood samples were collected by vein. After the cerebrospinal fluid was collected by lumbar puncture, it was packaged in cryopreservation tubes and coded and labeled; after the blood was collected by venipuncture, it was placed in a 4°C centrifuge and centrifuged for 10 minutes at 2000 rpm to quickly separate the upper serum and place it in a cryopreservation tube.

[0059] (III) Detection of FcRL2 concentration in serum and cerebrospinal fluid using the Olink method

[0060] Ol ink neurology panel (95801) was used to detect 92 proteins related to neurological function, including FcRL2 protein. Ol ink precision proteomics is a precise proteomics research tool that is very suitable for large-scale screening, with good sensitivity, rapidity, high-throughput analysis, and specificity at the multiple channel level. Therefore, this example uses Ol ink to detect markers in the serum and cerebrospinal fluid of patients with different types of neurosyphilis, and it is found that FcRL2 protein has significant differences in cerebrospinal fluid and serum. The detection of cerebrospinal fluid protein and cerebrospinal fluid leukocytes is carried out according to the method described in WS273-2007.

[0061] The following statistics and data analysis are performed on the above Olink test results:

[0062] (1) FcRL2 protein is significantly increased in cerebrospinal fluid and serum of neurosyphilis

[0063] Olink precision proteomics was used to detect the difference in the expression of nerve-related proteins in serum and cerebrospinal fluid, and FcRL2 protein was screened out with significant differences in both cerebrospinal fluid and serum. Figure 1As shown in Table 1. It was found that FcRL2 in the cerebrospinal fluid of patients with symptomatic neurosyphilis (symptomatic) was significantly increased compared with asymptomatic neurosyphilis (asymptomatic) and latent syphilis. It was found that FcRL2 in the serum of patients with symptomatic neurosyphilis and asymptomatic neurosyphilis was significantly lower than that of latent syphilis. Therefore, both cerebrospinal fluid and serum FcRL2 levels can be used to distinguish syphilis typing and indicate its severity.

[0064] Figure 1 In Figure (a), latent syphilis means non-neurosyphilis, asymptomatic means asymptomatic neurosyphilis, and symptomatic means paralytic dementia, i.e. symptomatic neurosyphilis. Figure 1 As can be seen from Figure (a), the concentration of FcRL2 in the cerebrospinal fluid of patients with symptomatic neurosyphilis> the concentration of FcRL2 in the cerebrospinal fluid of patients with asymptomatic neurosyphilis> the concentration of FcRL2 in the cerebrospinal fluid of patients with latent syphilis; Figure 1 As shown in Figure (b), the FcRL2 concentration in the serum of patients with symptomatic neurosyphilis and asymptomatic neurosyphilis is lower than the FcRL2 concentration in the serum of patients with latent syphilis.

[0065] Table 1: Statistical data of the difference in FcRL2 expression in cerebrospinal fluid and serum among three different types of syphilis

[0066]

[0067] In the table: Latent syphilis & asymptomatic represents the statistical value of the difference in FcRL2 concentration between latent syphilis and asymptomatic neurosyphilis in cerebrospinal fluid or serum. Latent syphilis & symptomatic represents the statistical value of the difference in FcRL2 concentration between latent syphilis and symptomatic neurosyphilis in cerebrospinal fluid or serum. Asymptomatic & symptomatic represents the statistical value of the difference in FcRL2 concentration between asymptomatic neurosyphilis and symptomatic neurosyphilis in cerebrospinal fluid or serum. Mean difference The mean difference reflects the average difference between each marker value and the arithmetic mean. The difference value can show whether there is a significant difference between the two groups, and the difference multiple can show the multiple of this difference. It can be seen from the statistics in Table 1 that there are significant differences in the serum and cerebrospinal fluid FcRL2 protein levels in patients with different types of syphilis, and the differences are statistically significant.

[0068] (2) FcRL2 in cerebrospinal fluid is significantly positively correlated with injury indicators in cerebrospinal fluid

[0069] Important reference indicators for neurosyphilis are cerebrospinal fluid cell count and protein level. Patients with neurosyphilis will have abnormal cerebrospinal fluid cell count and protein level, and these two indicators can also indicate the severity of neurosyphilis damage. This experiment used microscope counting and blood analyzer counting methods to count cerebrospinal fluid cells in three groups of syphilis patients with different types, and Lee-Vinson was used to measure the protein level in the cerebrospinal fluid.

[0070] Combination Figure 2 As shown in Figures (a) and (b), the expression level of FcRL2 protein in cerebrospinal fluid is significantly positively correlated with the protein in the patient's cerebrospinal fluid (correlation coefficient R = 0.7544, p < 0.0001) and the number of white blood cells (correlation coefficient R = 0.6503, p < 0.0001). Therefore, FcRL2 protein in cerebrospinal fluid can indicate abnormalities in protein and white blood cells in cerebrospinal fluid.

[0071] It can be seen that the FcRL2 protein level in the cerebrospinal fluid of syphilis patients is positively correlated with the patient's spinal fluid cell count and protein level. Therefore, in practice, the cerebrospinal fluid cell count and / or protein level can be used as a reference indicator, together with the FcRL2 protein level in the cerebrospinal fluid, as a means of early diagnosis and screening of neurosyphilis, typing detection, and quantitative assessment of the degree of nerve damage.

[0072] (3) Serum FcRL2 is negatively correlated with injury indicators in cerebrospinal fluid

[0073] Combination Figure 3 As shown in Figures (a) and (b), the expression level of FcRL2 protein in serum is negatively correlated with the protein in the patient's cerebrospinal fluid (correlation coefficient R = -0.2085, p = 0.0513) and the number of leukocytes (correlation coefficient R = -0.4139, p < 0.0001). Therefore, FcRL2 protein in serum can indicate abnormalities of protein and leukocytes in cerebrospinal fluid, just like this indicator in cerebrospinal fluid. Therefore, FcRL2 protein in serum can indicate abnormalities of protein and leukocytes in cerebrospinal fluid.

[0074] (4) Evaluation of cerebrospinal fluid and serum FcRL2 in neurosyphilis detection in syphilis patients

[0075] The study used the receiver operating characteristic curve (ROC) to evaluate the diagnostic potential of FcRL2 in evaluating neurosyphilis (neurosyphilis includes asymptomatic neurosyphilis and symptomatic neurosyphilis). Figure 4 As shown in the experimental results, the area under the curve of cerebrospinal fluid FcRL2 AUC (0.8676 [95% CI 0.7874-0.9478]), P < 0.0001, can be considered statistically significant (see Figure 4 (a) Figure), sensitivity was 80; the area under the curve AUC of serum FcRL2 was (0.7144 [95% CI 0.5821-0.8462]), P = 0.0093, which can be considered statistically significant (see Figure 4 (b) The sensitivity is 73.33. This shows that the level of FcRL2 in the cerebrospinal fluid and serum of syphilis patients can be used to assist in the diagnosis of neurosyphilis with high sensitivity.

[0076] In addition to the above-mentioned role in detecting neurosyphilis, the level of serum and cerebrospinal fluid FcRL2 can also be used to diagnose symptomatic neurosyphilis in neurosyphilis. In order to further clarify the relationship between serum and cerebrospinal fluid FcRL2 and symptomatic neurosyphilis, this example studied 73 patients with neurosyphilis. The study used ROC to evaluate the potential of FcRL2 in patients with neurosyphilis (including symptomatic and asymptomatic neurosyphilis patients) to assess the diagnosis of symptomatic neurosyphilis. Figure 5 As shown in the experimental results, the area under the curve of cerebrospinal fluid FcRL2 AUC (0.8977 [95% CI 0.8232-0.9721]), P < 0.0001, can be considered statistically significant (see Figure 5 (a)), the sensitivity was 93.02; the area under the curve of serum FcRL2 was AUC (0.5279 [95% CI 0.3872-0.6686]), P = 0.6865, which was not significant (see Figure 5 The above results indicate that the occurrence of symptomatic neurosyphilis can be better evaluated based on the level of FcRL2 in the cerebrospinal fluid of patients with neurosyphilis.

[0077] In addition to the above detection effects, the level of serum and cerebrospinal fluid FcRL2 can also be used to diagnose asymptomatic neurosyphilis in neurosyphilis. In order to further clarify the relationship between serum and cerebrospinal fluid FcRL2 and asymptomatic neurosyphilis, this example studied 45 patients with latent syphilis and asymptomatic neurosyphilis. The study used ROC to evaluate the potential of FcRL2 in syphilis patients (including latent syphilis patients and asymptomatic neurosyphilis patients) to evaluate the diagnosis of symptomatic neurosyphilis. Figure 6 As shown in the experimental results, the area under the curve of cerebrospinal fluid FcRL2 was AUC (0.7356 [95% CI 0.5842-0.8869]), P = 0.0107, which can be considered statistically significant (see Figure 6 (a)), the sensitivity was 66.67; the area under the curve of serum FcRL2 (AUC) was 0.6600 [95% CI 0.4989-0.8211], P = 0.0830, which was not significant (see Figure 6 The above results indicate that the occurrence of asymptomatic neurosyphilis can be better evaluated based on the level of FcRL2 in the cerebrospinal fluid of patients with neurosyphilis.

[0078] (7) FcRL2 levels can indicate damage indicators in cerebrospinal fluid

[0079] All syphilis patients were divided into two groups according to the cut-off value of the level of FcRL2 in the patient's cerebrospinal fluid (-0.6267NPX value). Figure 7As shown, the concentration of CSF FcRL2 in patients with a CSF injury-related marker CSF protein (see Figure 7 (a)) and white blood cells (see Figure 7 Therefore, patients with CSF FcRL2 concentrations below -0.6267NPX have a low risk of neurosyphilis and a small risk of nerve damage, while those above this value have a relatively high risk of neurosyphilis and an increased risk of nerve damage.

[0080] Furthermore, all syphilis patients were divided into two groups according to the cut-off value of the level of FcRL2 in the patient's cerebrospinal fluid (-0.05916NPX value). Figure 8 As shown, the concentration of CSF FcRL2 in patients with a CSF injury-related marker CSF protein (see Figure 8 (a)) and white blood cells (see Figure 8 Therefore, patients with CSF FcRL2 concentrations below -0.05916NPX have a low risk of symptomatic neurosyphilis and a small risk of nerve damage, while those above this value have a high risk of symptomatic neurosyphilis and a significantly increased risk of nerve damage.

[0081] Then, all syphilis patients were divided into two groups according to the cut-off value of the level of FcRL2 in the patient's cerebrospinal fluid (-0.7020NPX value). Fig. 9 As shown, the concentration of CSF FcRL2 in patients with a CSF injury-related marker CSF protein (see Fig. 9 (a)) significantly increased, and the white blood cells also increased but not significantly (see Fig. 9 (b) Figure). Therefore, patients with CSF FcRL2 concentrations below -0.7020NPX have a low risk of asymptomatic neurosyphilis and a small risk of nerve damage, and can be diagnosed with latent syphilis; while patients with CSF FcRL2 concentrations above this value have a high risk of asymptomatic neurosyphilis and a significantly increased risk of nerve damage.

[0082] Further findings, such as Fig.10 As shown, when the FcRL2 serum value was lower than 3.638NPX value, the cerebrospinal fluid leukocytes, an indicator of cerebrospinal fluid injury in patients, increased significantly (see Fig.10 (b) Figure). When the serum FcRL2 concentration is lower than 3.638NPX, the risk of neurosyphilis is relatively high and the risk of nerve damage increases. Fig.11 As shown in Figure 2, when the serum FcRL2 concentration was lower than 3.443NPX, the cerebrospinal fluid leukocytes, an indicator of cerebrospinal fluid injury, increased significantly (see Fig.10 (b)). Fig.12As shown in Figure 2, when the serum FcRL2 concentration was lower than 3.725NPX, the cerebrospinal fluid leukocytes, an indicator of cerebrospinal fluid injury, increased significantly (see Fig.12 (b) Figure). Therefore, when the FcRL2 serum value is less than or equal to 3.725NPX, the risk of asymptomatic neurosyphilis in syphilis patients increases, and the risk of nerve damage increases, while the FcRL2 serum value is higher than 3.725NPX, the risk of nerve damage decreases; when the FcRL2 serum value is less than or equal to 3.638NPX and higher than 3.443NPX, the risk of neurosyphilis in syphilis patients is moderate, and the risk of nerve damage increases; and when the FcRL2 serum value is less than or equal to 3.443NPX, the risk of symptomatic neurosyphilis is high, and the risk of nerve damage in patients is further increased.

[0083] In summary, the concentration of FcRL2 protein in serum or cerebrospinal fluid can be used as an indicator for early diagnosis of neurosyphilis, typing of symptomatic neurosyphilis, asymptomatic neurosyphilis, neurosyphilis and latent syphilis, and evaluation of the degree of nerve damage. It can be used alone or as a main reference indicator in combination with other indicators to complete the diagnostic screening, typing and nerve damage evaluation of syphilis.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the embodiments, it should be understood by those skilled in the art that any modification or equivalent replacement of the technical solutions of the present invention does not depart from the spirit and scope of the technical solutions of the present invention and should be included in the scope of the claims of the present invention.

Claims

1. FcRL2 is used as a serum or cerebrospinal fluid marker for early diagnosis, screening and typing of patients with neurosyphilis.

2. Use of FcRL2 protein or its specific antibody in the preparation of diagnostic products or auxiliary diagnostic products for neurosyphilis screening, typing detection or assessment of the degree of nerve damage; characterized in that: The diagnostic product or auxiliary diagnostic product is a detection kit, antibody chip, antibody probe or detector using FcRL2 or its specific antibody as the target molecule.

3. The use according to claim 2, characterized in that: The kit uses FcRL2 protein or its specific antibody as a target molecule to detect the expression level of FcRL2 protein or its specific antibody in cerebrospinal fluid or serum.

4. The use according to claim 2, characterized in that: The kit is an enzyme-linked immunosorbent assay kit or an immunohistochemistry kit for detecting FcRL2.

5. The use according to claim 2, characterized in that: The kit comprises: a primary antibody capable of specifically recognizing FcRL2, a polypeptide dye, magnetic beads and a biotin-labeled secondary antibody.

6. The use according to claim 2, characterized in that: The kit may also include reagents or materials for obtaining serum or cerebrospinal fluid from the patient.

7. Products for early diagnosis, screening, typing or assessment of nerve damage in patients with neurosyphilis; characterized in that: The product can quantitatively detect the concentration of FcRL2 in the patient's ex vivo serum or cerebrospinal fluid; the product is a detection kit, an antibody chip, an antibody probe or a detector.

8. A syphilis detection kit, characterized in that: include: A first detection reagent composition for detecting serum or cerebrospinal fluid FcRL2 protein levels; and a second detection reagent composition for detecting cerebrospinal fluid leukocytes and / or cerebrospinal fluid proteins.

9. A system for assessing the risk of nerve damage in patients with neurosyphilis, characterized in that: It includes a detection module and a judgment module; wherein the detection module is used to obtain the concentration value of FcRL2 in serum or cerebrospinal fluid; the judgment module includes a readable carrier storing judgment rules; The judgment rule is: when the detection module detects that the serum FcRL2 concentration value is ≤3.443NPX or the cerebrospinal fluid FcRL2 concentration value is ≥-0.05916NPX; it is judged as a high risk of neurological injury; When the detection module detects 3.443NPX<serum FcRL2≤3.638NPX or -0.05916NPX>cerebrospinal fluid FcRL2 concentration value ≥-0.6267NPX; it is judged as moderate nerve injury risk; When the detection module detects that the serum FcRL2 concentration value is ≤3.725NPX or the cerebrospinal fluid FcRL2 concentration value is ≥-0.7020NPX, it is judged that there is a risk of cerebrospinal fluid infection with syphilis; When the detection module detects that the serum FcRL2 concentration value is greater than 3.725NPX or the cerebrospinal fluid FcRL2 concentration value is less than -0.7020NPX, it is judged as no or low risk of cerebrospinal fluid infection with syphilis.

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