Application of CLM-6 protein in preparation of neurosyphilis or nerve injury diagnosis product

Abstract

The invention relates to the technical field of biological medicines, in particular to a CLM-6 protein (CMRF35-l ike molle 6) as a serum marker or cerebrospinal fluid marker for early diagnosis, screening and typing detection of a neurosyphilis patient. According to the invention, the concentration of CLM-6 protein in cerebrospinal fluid or serum is used as an index for early diagnosis of neurosyphilis, typing detection of symptomatic neurosyphilis, asymptomatic neurosyphilis, neurosyphilis and recessive syphilis, nerve injury degree evaluation and the like; the index can be independently used or used as a main reference index to be combined with other indexes to finish the diagnosis screening and typing of the neurosyphilis and the evaluation of nerve injury. Compared with the prior art, the CLM-6 not only has higher sensitivity, but also can realize noninvasive serum detection on the neurosyphilis, is convenient to detect, and is more beneficial to early 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 CLM-6 protein or its specific antibody in the preparation of products for neurosyphilis screening, typing detection or assessment of the degree of nerve damage. Background Art

[0002] As the incidence of syphilis increases year by year, syphilis is still a Class B infectious disease that should be taken seriously. Syphilis is a systemic infectious disease caused by the invasion of Treponema pallidum into the human body. Treponema pallidum spreads to the central nervous system within a few days after infection and may cause asymptomatic meningitis and severe symptomatic neurosyphilis. Neurosyphilis can occur at all stages of the course of syphilis, not just in tertiary syphilis. The clinical manifestations of neurosyphilis vary, and may be asymptomatic or may present as extensive involvement of the nervous system with complex clinical manifestations. Clinically, it is usually divided into 5 types: asymptomatic, meningeal syphilis, meningovascular syphilis, tabes dorsalis, and paralytic dementia (brain parenchymal syphilis). Among them, although asymptomatic neurosyphilis has no clinical symptoms, its harmfulness should be taken seriously. It can become recurrent syphilis and may progress to visceral and neurosyphilis.

[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 white blood 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 counts, 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. Increased leukocytosis or increased protein content in the cerebrospinal fluid can indicate the destruction of the blood-brain barrier and the presence of inflammatory reactions in the skull, which can assist 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 who have already developed symptoms of neurosyphilis. There is a lack of sensitive and reliable biological markers that can effectively distinguish asymptomatic neurosyphilis. In addition, diagnostic methods that rely on cerebrospinal fluid require lumbar puncture or lateral ventricle puncture to obtain cerebrospinal fluid, which is difficult to operate and is not conducive to the early diagnosis of neurosyphilis in community hospitals and local hospitals. Therefore, the development of new biomarkers, especially serum detection markers, that can be used for the diagnosis of neurosyphilis and asymptomatic neurosyphilis is of great significance to the diagnosis and management of neurosyphilis. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the technical problems such as low sensitivity of existing CSF-RPR diagnostic methods, insensitivity to the diagnosis of early asymptomatic neurosyphilis, and reliance 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.

[0006] (II) Technical solution

[0007] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0008] In a first aspect, the present invention relates to CLM-6 as a serum marker or cerebrospinal fluid marker for early diagnosis, screening and typing of patients with neurosyphilis.

[0009] In a second aspect, the present invention provides the use of CLM-6 protein or its specific antibody in the preparation of diagnostic products for neurosyphilis screening, typing detection or assessment of the degree of nerve damage; the diagnostic product is a detection kit, antibody chip, antibody probe or detector with CLM-6 protein or its specific antibody as the target molecule.

[0010] According to a preferred embodiment of the present invention, the kit uses CLM-6 protein or its specific antibody as a target molecule to detect the expression level of CLM-6 protein or its specific antibody in cerebrospinal fluid or serum.

[0011] According to a preferred embodiment of the present invention, the kit is an enzyme-linked immunosorbent assay kit or an immunohistochemistry kit.

[0012] According to a preferred embodiment of the present invention, the kit comprises: a primary antibody that can specifically recognize CLM-6, a polypeptide dye, magnetic beads, and a biotin-labeled secondary antibody. Preferably, the reagents include citrate buffer, PBS washing solution, peroxidase blocker, goat serum, streptomycin antibiotic-peroxidase, DAB solution, and hematoxylin solution.

[0013] 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, and / or reagents for separating serum.

[0014] It should be noted that when detecting the expression level of CLM-6 protein in serum, the Olink ultra-high sensitivity plasma protein detection platform can also be used to quantify the expression level of CLM-6.

[0015] 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 CLM-6 protein in serum or cerebrospinal fluid.

[0016] Preferably, the product is a detection kit, an antibody chip, an antibody probe or a detector; and the product comprises an antibody that specifically binds to CLM-6.

[0017] Preferably, the product contains antibodies that specifically bind to CLM-6 and other reagents that can quantitatively detect the concentration of CLM-6 protein in serum and cerebrospinal fluid. The experimental method used in the examples of this application is Olink Neurology Panel (95801), which can be used to detect 92 protein biomarkers related to neurological function, including CLM-6 protein.

[0018] 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 CLM-6; the judgment module comprises a readable carrier storing a judgment rule;

[0019] The judgment rule is: when the serum CLM-6 concentration value measured by the detection module is ≥4.608NPX value or the cerebrospinal fluid CLM-6 concentration value is ≥3.343NPX value, it is judged as a high risk of nerve damage;

[0020] When the detection module measures: 4.461NPX value ≤ serum CLM-6 concentration value < 4.608NPX value or 2.656NPX value ≤ cerebrospinal fluid CLM-6 concentration value < 3.343NPX value, it is judged as a moderate risk of nerve damage;

[0021] When the detection module measures: serum CLM-6 concentration value < 4.461NPX value or 2.29NPX value ≤ cerebrospinal fluid CLM-6 concentration value < 2.656NPX value, it is judged as low risk of nerve damage;

[0022] When the detection module measures that the cerebrospinal fluid CLM-6 concentration value is less than 2.29NPX value, it is judged as no or low risk of asymptomatic neurosyphilis infection in cerebrospinal fluid.

[0023] The reference value is the CLM-6 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 4.461NPX value (serum critical value for neurosyphilis and non-neurosyphilis), and the CSF Cut-off value is 2.656NPX value (cerebrospinal fluid critical value for neurosyphilis and non-neurosyphilis); the serum Cut-off value for the diagnosis of symptomatic neurosyphilis is 4.608NPX value (serum critical value for symptomatic neurosyphilis and asymptomatic neurosyphilis), and the CSF Cut-off value is 3.343NPX value (cerebrospinal fluid critical value for symptomatic neurosyphilis and asymptomatic neurosyphilis); the CSF Cut-off value for the diagnosis of asymptomatic neurosyphilis is 2.29NPX (cerebrospinal fluid critical value for asymptomatic neurosyphilis and latent syphilis). The above Cut-off value is the CLM-6 statistical analysis critical value obtained from 88 syphilis patients.

[0024] 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 operation with certain potential risks. It is relatively traumatic for patients and generally requires hospitalization for the operation. The serum marker CLM-6 protein proposed in the present invention is positively correlated with the cerebrospinal fluid CLM-6 index, so the serum index can be used to warn of an increase in the cerebrospinal fluid index, and the serum index can also effectively distinguish neurosyphilis from non-neurosyphilis and symptomatic neurosyphilis from asymptomatic neurosyphilis, thereby enabling convenient and fast non-invasive screening and diagnosis.

[0025] In a fifth aspect, the present invention also provides a syphilis detection kit, which comprises: a first analysis unit for detecting the level of CLM-6 protein in serum or cerebrospinal fluid; and a second analysis unit for detecting cerebrospinal fluid leukocytes and / or cerebrospinal fluid proteins; the first analysis unit is a reagent composition, an antibody chip, an antibody probe or a detector; the second analysis unit is a reagent composition, a cell counting plate, a blood analyzer or a brain or spinal fluid analyzer.

[0026] In the above scheme, when diagnosing neurosyphilis (including early diagnosis, typing and staging, and neurological damage assessment), the serum or cerebrospinal fluid CLM-6 protein level can be combined with one or two indicators of cerebrospinal fluid leukocytes and cerebrospinal fluid proteins related to neurological damage in patients with neurosyphilis for early diagnosis, typing and staging, and neurological damage assessment of neurosyphilis.

[0027] Among them, the level of CLM-6 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.

[0028] (III) Beneficial effects

[0029] The present invention provides a new cerebrospinal fluid / serum marker CLM-6 for early diagnosis screening, typing detection and nerve damage assessment of neurosyphilis. By detecting the expression level of CLM-6 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 CLM-6 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 assessment of the degree of nerve damage of neurosyphilis. Compared with the prior art, the marker is not only more sensitive, but also can achieve non-invasive serum detection of neurosyphilis.

[0030] The serum / cerebrospinal fluid marker CLM-6 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 CLM-6 in the serum / cerebrospinal fluid of patients with symptomatic neurosyphilis is the highest. The expression level of CLM-6 has a high sensitivity in the assessment of neurosyphilis and 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

[0031] Figure 1 The Olink method was used to detect the expression of CLM-6 protein in cerebrospinal fluid and serum of three groups of patients with different types of syphilis (latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis).

[0032] Figure 2 The results showed that the expression of CLM-6 in cerebrospinal fluid was significantly positively correlated with that in serum.

[0033] Figure 3 The results showed that the expression of CLM-6 in cerebrospinal fluid was significantly positively correlated with cerebrospinal fluid protein and leukocytes.

[0034] Figure 4 The expression of CLM-6 in serum was positively correlated with cerebrospinal fluid protein and leukocytes.

[0035] Figure 5 To estimate the likelihood of a confirmed diagnosis of neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) using cerebrospinal fluid and serum CLM-6 concentrations as the clinical outcome, and to calculate the area under the curve (AUC).

[0036] Figure 6 To estimate the likelihood of a confirmed diagnosis of symptomatic neurosyphilis at different levels of CSF and serum CLM-6 concentrations when symptomatic neurosyphilis was the clinical outcome.

[0037] Figure 7 To distinguish asymptomatic neurosyphilis from non-neurosyphilis when asymptomatic neurosyphilis was the clinical outcome, the area under the curve (AUC) of different levels of cerebrospinal fluid and serum CLM-6 concentrations were used to assess the likelihood of a confirmed diagnosis of asymptomatic neurosyphilis.

[0038] Figure 8 To distinguish all patients (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups based on the serum CLM-6 cutoff value of 4.461NPX, the differences in cerebrospinal fluid protein and leukocytes, the indicators of neurological damage, among each group of patients.

[0039] Fig. 9To distinguish two groups of patients with neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) based on the serum CLM-6 cutoff value of 4.608NPX, the differences in cerebrospinal fluid protein and leukocytes, the indicators of neurological damage, between the groups of patients.

[0040] Fig.10 To divide patients with latent syphilis and asymptomatic neurosyphilis into two groups based on the serum CLM-6 cutoff value of 4.453NPX, and to compare the differences in cerebrospinal fluid protein and leukocytes, the indicators of neurological damage, between the groups.

[0041] Fig.11 All patients were divided into two groups based on the CLM-6 cutoff value of 2.656NPX in the cerebrospinal fluid, and the differences in cerebrospinal fluid protein and leukocytes, the indicators of neurological injury, were observed in each group.

[0042] Fig.12 To distinguish two groups of patients with neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) based on the CLM-6 cutoff value of 3.343NPX in cerebrospinal fluid, and to compare the differences in neurological injury indicators, cerebrospinal fluid protein and leukocytes, between the groups.

[0043] Fig.13 To divide patients with latent syphilis and asymptomatic neurosyphilis into two groups based on the cerebrospinal fluid CLM-6 cutoff value of 2.29NPX, and to compare the differences in neurological damage indicators, cerebrospinal fluid protein and leukocytes, 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 the assessment of nerve damage. They screened multiple indicators related to nerve function in the serum and cerebrospinal fluid of patients with neurosyphilis and obtained CLM-6, which has diagnostic value in both serum and cerebrospinal fluid.

[0046] CLM-6 has multiple names, including: lymphocyte mono-immunoglobulin-like receptor 8 (LMIR8), CD300 antigen-like family member C (CD300c), CMRF35-A1 (CMRF-35), immunoglobulin superfamily member 16 (IgSF16), etc. However, its role in nerve damage associated with neurosyphilis has not been reported. For the sake of brevity and clarity, it is uniformly referred to as CLM-6 in this application.

[0047] The inventors have found through research that there are significant differences in the levels of CLM-6 protein in the cerebrospinal fluid and serum of patients with different types of syphilis. Cerebrospinal fluid CLM-6 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. This shows that cerebrospinal fluid CLM-6 protein can be used for the diagnosis of neurosyphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis, and can also predict the severity of nerve damage. Serum CLM-6 protein shows a significant positive correlation with this marker in cerebrospinal fluid, so it is speculated that serum CLM-6 can also be used for the diagnosis of neurosyphilis. The serum CLM-6 protein level in patients with symptomatic neurosyphilis is significantly increased compared with that in patients with latent syphilis, but there is no significant difference in serum CLM-6 protein level between patients with asymptomatic neurosyphilis and patients with symptomatic neurosyphilis and between patients with asymptomatic neurosyphilis and patients with latent syphilis. Serum CLM-6 protein is positively correlated with cerebrospinal fluid protein and cerebrospinal fluid leukocytes, which are indicators of nerve damage, but the correlation is not as good as that of cerebrospinal fluid. The CLM-6 protein level in the cerebrospinal fluid of patients with asymptomatic neurosyphilis was significantly higher than that in the non-neurosyphilis (latent syphilis) group, indicating that the CLM-6 protein level in the cerebrospinal fluid has changed in the early stage of neurosyphilis, that is, the asymptomatic neurosyphilis stage. Therefore, the CLM-6 protein in the cerebrospinal fluid can effectively distinguish asymptomatic neurosyphilis from latent syphilis.

[0048] During the study, 88 syphilis patients (including neurosyphilis and latent syphilis) were studied in the experiment, and the receiver operating characteristic curve (ROC) was used to evaluate the potential of CLM-6 protein in syphilis patients as a marker for diagnosing neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis). The experimental results showed that the area under the curve (AUC) of CLM-6 in cerebrospinal fluid was 0.8950, P < 0.0001 was considered statistically significant; the AUC of serum CLM-6 was 0.6457, P = 0.0767, and the sensitivity was 78.08. Based on this, the level of CLM-6 in the cerebrospinal fluid of syphilis patients can better evaluate the occurrence of neurosyphilis, with a sensitivity of 93.33; whether serum can evaluate the occurrence of neurosyphilis requires more samples to verify.

[0049] Next, in order to clarify the relationship between serum and cerebrospinal fluid CLM-6 and symptomatic neurosyphilis, 73 patients with neurosyphilis (including symptomatic and asymptomatic neurosyphilis patients) were studied in the following examples, and ROC was used to evaluate the potential of CLM-6 protein in neurosyphilis patients as a marker for diagnosing symptomatic neurosyphilis; the experimental results showed that the AUC of cerebrospinal fluid CLM-6 was 0.8760, P < 0.0001, which was statistically significant; the AUC of serum CLM-6 was 0.6116, P = 0.1064. The above results show that the level of cerebrospinal fluid CLM-6 can better evaluate the occurrence of symptomatic neurosyphilis, with a sensitivity of 83.33; while the prediction effect of serum CLM-6 is not good.

[0050] Furthermore, asymptomatic neurosyphilis is a difficult point in clinical diagnosis and treatment, because it is difficult to diagnose without clinical symptoms. Therefore, all syphilis patients currently need to undergo invasive cerebrospinal fluid screening. In order to clarify the relationship between serum and cerebrospinal fluid CLM-6 and asymptomatic neurosyphilis, 45 patients with latent syphilis and asymptomatic neurosyphilis were studied in the following examples, and ROC was used to evaluate the potential of CLM-6 protein as a marker for diagnosing asymptomatic neurosyphilis; the experimental results showed that the AUC of cerebrospinal fluid CLM-6 was 0.7844, P = 0.0021, which was statistically significant; the AUC of serum CLM-6 was 0.5800, P = 0.3861. The above results show that the level of cerebrospinal fluid CLM-6 can better evaluate the occurrence of asymptomatic neurosyphilis, with a sensitivity of 80.00, and whether serum can predict requires more samples for verification.

[0051] Further, the present invention divides all syphilis patients into two groups according to the Cut-Off value (4.461NPX) of serum CLM-6 of all syphilis patients (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis), and the cerebrospinal fluid protein, an indicator related to cerebrospinal fluid damage in patients above this value, is significantly increased, while the difference in white blood cells is not obvious. Therefore, when serum CLM-6 is lower than 4.461NPX, the risk of neurosyphilis in syphilis patients 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. Next, all neurosyphilis patients (including asymptomatic neurosyphilis and symptomatic neurosyphilis) are divided into two groups according to the Cut-Off value (4.608NPX) of serum CLM-6 of all neurosyphilis patients (including asymptomatic neurosyphilis and symptomatic neurosyphilis), and the cerebrospinal fluid protein, an indicator related to cerebrospinal fluid damage in patients above this value, is significantly increased, while there is no difference in white blood cells. Therefore, when serum CLM-6 is lower than 4.608NPX, the risk of symptomatic neurosyphilis in patients with neurosyphilis 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 also high. Finally, according to the Cut-Off value (4.453NPX) of serum CLM-6 in syphilis patients (including latent syphilis and asymptomatic neurosyphilis), all patients were divided into two groups, and there was no significant difference in the indicators related to cerebrospinal fluid injury between the two groups.

[0052] Similarly, the present invention divides all patients into two groups according to the Cut-Off value (2.656NPX) of cerebrospinal fluid CLM-6 of all syphilis patients (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis), and the cerebrospinal fluid and protein and leukocytes of patients with cerebrospinal fluid damage related indicators above this value are significantly increased. Therefore, when the cerebrospinal fluid CLM-6 is lower than the value of 2.656NPX, the risk of neurosyphilis in syphilis patients 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. Next, all neurosyphilis patients (including asymptomatic neurosyphilis and symptomatic neurosyphilis) are divided into two groups according to the Cut-Off value (3.343NPX) of cerebrospinal fluid CLM-6 of all neurosyphilis patients (including asymptomatic neurosyphilis and symptomatic neurosyphilis), and the cerebrospinal fluid leukocytes and protein of patients with cerebrospinal fluid damage related indicators are significantly increased. Therefore, when the CLM-6 value of CSF is lower than 3.343NPX, the risk of symptomatic neurosyphilis in patients with neurosyphilis is lower, and the risk of nerve damage is relatively small; while above this value, the risk of symptomatic neurosyphilis is higher, and the risk of nerve damage is higher. Finally, all patients were divided into two groups according to the Cut-Off value (2.29NPX) of CLM-6 in the CSF of syphilis patients (including latent syphilis and asymptomatic neurosyphilis). The CLM-6 of CSF and protein, indicators related to CSF ​​damage, increased significantly in patients above this value. Therefore, when the CLM-6 value of CSF is lower than 2.29NPX, the risk of asymptomatic neurosyphilis in patients with syphilis is lower, and the risk of nerve damage is lower; while above this value, the risk of asymptomatic neurosyphilis increases, and the risk of nerve damage also increases. Therefore, CLM-6 in CSF can be used as an evaluation indicator for various types of neurosyphilis and nerve damage.

[0053] In summary, the present invention provides the application of CLM-6 as an early diagnosis of neurosyphilis and nerve damage assessment. Therefore, in practice, the CLM-6 protein level in serum and cerebrospinal fluid can be used as a reliable and effective main reference indicator or auxiliary reference indicator for early diagnosis, typing and nerve damage assessment of syphilis. Based on the above findings, by detecting the expression level of CLM-6 in serum or cerebrospinal fluid, it is possible to detect and type syphilis, especially neurosyphilis, with high sensitivity, and to perform early non-invasive screening for neurosyphilis, and to accurately quantitatively evaluate nerve damage, the supervision and treatment of neurosyphilis.

[0054] 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.

[0055] 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:

[0056] 1. Research subjects

[0057] 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.

[0058] (II) Sample collection

[0059] 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.

[0060] (III) Detection of CLM-6 concentration in serum and cerebrospinal fluid using the Olink method

[0061] Ol ink neurology panel (95801) was used to detect 92 proteins related to neurological function, including CLM-6 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 CLM-6 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.

[0062] The following statistics and data analysis are performed on the above Olink test results:

[0063] (1) CLM-6 protein significantly increased in cerebrospinal fluid and serum of neurosyphilis

[0064] Olink precision proteomics was used to detect the difference in the expression of nerve-related proteins in serum and cerebrospinal fluid, and CLM-6 protein was screened out, which was significantly different in both cerebrospinal fluid and serum. Figure 1As shown in Table 1. The test results showed that the CLM-6 level in the cerebrospinal fluid of patients with symptomatic neurosyphilis (with symptoms) was significantly higher than that of patients with asymptomatic neurosyphilis (without symptoms) and latent syphilis. It was found that the CLM-6 level in the serum of patients with symptomatic neurosyphilis was significantly higher than that of patients with latent syphilis, and there was no significant difference between asymptomatic neurosyphilis and the other two groups. Therefore, the level of CLM-6 in cerebrospinal fluid can be used to distinguish the types of syphilis and indicate its severity.

[0065] 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 CLM-6 concentration in the cerebrospinal fluid of patients with symptomatic neurosyphilis > the CLM-6 concentration in the cerebrospinal fluid of patients with asymptomatic neurosyphilis > the CLM-6 concentration in the cerebrospinal fluid of patients with latent syphilis; Figure 1 As shown in Figure (b), the CLM-6 concentration in the serum of patients with symptomatic neurosyphilis > the CLM-6 concentration in the serum of patients with asymptomatic neurosyphilis > the CLM-6 concentration in the serum of patients with latent syphilis, but the difference was not significant.

[0066] Table 1: Statistical data of the difference in CLM-6 expression in cerebrospinal fluid and serum among three different types of syphilis

[0067]

[0068] In Table 1: Latent syphilis & asymptomatic represents the statistical value of the difference in CLM-6 concentration between latent syphilis and asymptomatic neurosyphilis in cerebrospinal fluid or serum. Latent syphilis & symptomatic represents the statistical value of the difference in CLM-6 concentration between latent syphilis and symptomatic neurosyphilis in cerebrospinal fluid or serum. Asymptomatic & symptomatic represents the statistical value of the difference in CLM-6 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 CLM-6 protein levels in the cerebrospinal fluid of patients with different types of syphilis, and the differences are statistically significant.

[0069] (2) CLM-6 protein in cerebrospinal fluid and serum showed a significant positive correlation

[0070] Combination Figure 2It can be found that the CLM-6 protein in serum is significantly positively correlated with the protein in cerebrospinal fluid (correlation coefficient R = 0.5555, p < 0.0001). Therefore, the CLM-6 protein in serum can be used as an indicator of increased CLM-6 in cerebrospinal fluid. Therefore, the CLM-6 protein level in serum can be used instead of the CLM-6 protein level in cerebrospinal fluid to achieve non-invasive serological testing for neurosyphilis and avoid invasive cerebrospinal fluid examination.

[0071] (3) CLM-6 in cerebrospinal fluid is significantly positively correlated with injury indicators in cerebrospinal fluid

[0072] 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.

[0073] Combination Figure 3 As shown in Figures (a) and (b), the expression level of CLM-6 protein in cerebrospinal fluid is significantly positively correlated with the protein in the patient's cerebrospinal fluid (correlation coefficient R = 0.7854, p < 0.0001) and the number of white blood cells (correlation coefficient R = 0.6418, p < 0.0001). Therefore, the expression level of CLM-6 protein in cerebrospinal fluid can indicate the abnormality of protein and white blood cells in cerebrospinal fluid.

[0074] It can be seen that the CLM-6 protein level in the cerebrospinal fluid of syphilis patients is significantly 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 CLM-6 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.

[0075] (4) CLM-6 in serum is negatively correlated with injury indicators in cerebrospinal fluid

[0076] Combination Figure 4As shown in Figures (a) and (b), the expression level of CLM-6 protein in serum is positively correlated with the protein in the patient's cerebrospinal fluid (correlation coefficient R = 0.2948, p = 0.0053) and the number of white blood cells (correlation coefficient R = 0.2371, p = 0.0261), but the correlation is not significant. Therefore, the CLM-6 protein in serum can also indicate abnormalities in protein and white blood cells in cerebrospinal fluid, but the effect is not as good as that in cerebrospinal fluid. Therefore, the CLM-6 protein in serum can partially indicate abnormalities in protein and white blood cells in cerebrospinal fluid. In practice, the expression level of CLM-6 protein in serum can be used as a preliminary screening, and then combined with the count of white blood cells and / or protein in cerebrospinal fluid for joint diagnosis.

[0077] (5) Evaluation of CLM-6 in cerebrospinal fluid and serum for neurosyphilis detection in syphilis patients

[0078] A total of 88 syphilis patients (including neurosyphilis and latent syphilis) were studied, and the receiver operating characteristic curve (ROC) was used to evaluate the potential of CLM-6 protein as a marker for the diagnosis of neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis). Figure 5 The experimental results are as follows: the area under the curve of CLM-6 in cerebrospinal fluid (AUC) (0.8950 [95% CI 0.8273-0.9626]), P < 0.0001 was considered statistically significant (see Figure 5 (a) Figure); The area under the curve of serum CLM-6 (AUC) (0.6457 [95% CI 0.5014-0.7899]), P = 0.0767 was considered to be insignificant (see Figure 5 (b)). This shows that the level of CLM-6 in the cerebrospinal fluid of syphilis patients can be used to assist in the diagnosis of neurosyphilis, with a high sensitivity of 93.33.

[0079] In addition to the above-mentioned role in detecting neurosyphilis, the level of serum and cerebrospinal fluid CLM-6 can also be used to diagnose symptomatic neurosyphilis in neurosyphilis. In order to further clarify the relationship between serum and cerebrospinal fluid CLM-6 and symptomatic neurosyphilis, this example studied 73 patients with neurosyphilis (including symptomatic and asymptomatic neurosyphilis patients). The study used ROC to evaluate the potential of CLM-6 protein as a marker for diagnosing symptomatic neurosyphilis in patients with neurosyphilis. Figure 6 As shown in the experimental results, the area under the curve of CLM-6 in cerebrospinal fluid (AUC) was 0.8760 [95% CI 0.7977-0.9543], and P < 0.0001 was considered statistically significant (see Figure 6(a) Figure); The area under the curve of serum CLM-6 AUC (0.6116 [95% CI 0.4803-0.7430]), P = 0.1064, no significant significance (see Figure 6 (b) Figure). The above results show that the occurrence of symptomatic neurosyphilis can be better evaluated according to the level of CLM-6 in the cerebrospinal fluid of patients with neurosyphilis, with a sensitivity of 83.33.

[0080] In addition to the above detection functions, the level of serum and cerebrospinal fluid CLM-6 can also be used to diagnose asymptomatic neurosyphilis in neurosyphilis. In order to further clarify the relationship between serum and cerebrospinal fluid CLM-6 and asymptomatic neurosyphilis, this example studied 45 patients with latent syphilis and asymptomatic neurosyphilis. The study used ROC to evaluate the potential of CLM-6 protein as a marker for diagnosing symptomatic neurosyphilis in syphilis patients (including latent syphilis patients and asymptomatic neurosyphilis patients). Figure 7 The experimental results are as follows: the area under the curve of CLM-6 in cerebrospinal fluid (AUC) was 0.7844 [95% CI 0.6506-0.9183], P = 0.0021, which was considered statistically significant (see Figure 7 (a) Figure); The area under the curve of serum CLM-6 AUC (0.5800 [95% CI 0.4025-0.7575]), P = 0.3861, no significant significance (see Figure 7 (b) Figure). The above results show that the occurrence of asymptomatic neurosyphilis can be better evaluated according to the level of CLM-6 in the cerebrospinal fluid of patients with neurosyphilis, with a sensitivity of 80.00.

[0081] (6) CLM-6 levels can indicate damage indicators in cerebrospinal fluid

[0082] All syphilis patients were divided into two groups according to the cut-off value of CLM-6 level in the patients' cerebrospinal fluid (2.656NPX value). Figure 8 As shown, the CLM-6 value of patients with CSF higher than 2.656NPX was an indicator of CSF protein related to CSF ​​injury (see Figure 8 (a)) and white blood cells (see Figure 8 (b) Figure) significantly increased. Therefore, patients with CLM-6 values ​​below 2.656NPX in cerebrospinal fluid 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.

[0083] Furthermore, neurosyphilis patients were divided into two groups according to the cut-off value of CLM-6 level in the patients' cerebrospinal fluid (3.343NPX value). Fig. 9As shown, the cerebrospinal fluid protein (see Figure 9 (a)) and leukocytes (see Figure 9 (b)), indicators related to cerebrospinal fluid damage, were significantly increased in patients with cerebrospinal fluid CLM-6 values ​​higher than 3.343NPX. Therefore, patients with cerebrospinal fluid CLM-6 values ​​lower than 3.343NPX have a low risk of symptomatic neurosyphilis and a small risk of nerve damage; while patients with cerebrospinal fluid CLM-6 values ​​higher than this value have a high risk of symptomatic neurosyphilis and a significantly increased risk of nerve damage.

[0084] Then, the patients with latent syphilis and asymptomatic neurosyphilis were divided into two groups according to the cut-off value of CLM-6 level in the patients' cerebrospinal fluid (2.29NPX value). Fig.10 As shown, the CLM-6 value of patients with CSF higher than 2.29NPX was an indicator of CSF injury-related CSF protein (see Fig.10 (a)) significantly increased, and the white blood cells also increased but not significantly (see Fig.10 (b) Figure). Therefore, patients with CLM-6 values ​​below 2.29NPX have a low risk of asymptomatic neurosyphilis and a small risk of nerve damage, and can be diagnosed with non-neurosyphilis or latent syphilis; while those with CLM-6 values ​​above this value have a high risk of asymptomatic neurosyphilis and a significantly increased risk of nerve damage.

[0085] Further findings, such as Fig.11 As shown, when the serum CLM-6 value is higher than 4.461NPX value, the cerebrospinal fluid protein, an indicator of cerebrospinal fluid injury in patients, is significantly increased (see Fig.11 (a)), the number of cerebrospinal fluid leukocytes also increased, but there was no significant difference (see Fig.11 (b) Figure). When the serum CLM-6 value is higher than 4.461NPX value, the risk of neurosyphilis is relatively high and the risk of nerve damage increases. Fig.12 As shown in Figure 2, when the serum CLM-6 value was lower than the NPX value of 4.608, the cerebrospinal fluid protein, an indicator of cerebrospinal fluid injury in patients, increased significantly (see Fig.12 (a)), but there was no significant difference in cerebrospinal fluid leukocytes (see Fig.12 (b)). Fig.13 As shown in the figure, when the serum CLM-6 value is higher than 4.453NPX value, there is no significant difference in the cerebrospinal fluid protein and leukocytes, which are indicators related to cerebrospinal fluid damage in patients. Therefore, serum CLM-6 is not effective in assessing the risk of neurological damage in patients with syphilis. In general, when the serum CLM-6 value is higher than 4.461NPX value, the risk of neurosyphilis in syphilis patients is moderate, and the risk of neurological damage is increased; when the serum CLM-6 value is higher than 4.608NPX value, the risk of symptomatic neurosyphilis is increased, and the risk of neurological damage is further increased.

[0086] In summary, the level of CLM-6 protein in serum or cerebrospinal fluid can be used as a biomarker 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 for the diagnosis screening, typing and nerve damage evaluation of syphilis, or as a major reference indicator combined with other indicators to complete the diagnosis screening, typing and nerve damage evaluation of syphilis.

[0087] 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. CLM-6 is used as a serum or cerebrospinal fluid marker for early diagnosis, screening and typing of patients with neurosyphilis.

2. Use of CLM-6 protein or its specific antibody in the preparation of diagnostic products for neurosyphilis screening, typing detection or assessment of the degree of nerve damage; characterized in that: The diagnostic product is a detection kit, antibody chip, antibody probe or detector using CLM-6 protein or its specific antibody as target molecule.

3. The use according to claim 2, characterized in that: The kit uses CLM-6 protein or its specific antibody as a target molecule to detect the expression level of CLM-6 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.

5. The use according to claim 2, characterized in that: The kit comprises: a primary antibody capable of specifically recognizing CLM-6, a polypeptide dye, magnetic beads and a biotin-labeled secondary antibody.

6. The use according to claim 2 or 5, characterized in that: The kit may also include reagents or materials for obtaining serum or cerebrospinal fluid from the patient, and / or reagents for separating serum.

7. Products used for early diagnosis, screening, typing detection or assessment of nerve damage in patients with neurosyphilis; characterized in that: The product can quantitatively detect the concentration of CLM-6 protein in serum or cerebrospinal fluid.

8. The product according to claim 7, characterized in that The product is a detection kit, an antibody chip, an antibody probe or a detector; the product contains an antibody that specifically binds to CLM-6.

9. A neurosyphilis diagnostic device, characterized in that: include: a first analytical unit for detecting the level of CLM-6 protein in serum or cerebrospinal fluid; and a second analysis unit for detecting cerebrospinal fluid leukocytes and / or cerebrospinal fluid proteins; the first analysis unit is a reagent composition, an antibody chip, an antibody probe or a detector; the second analysis unit is a reagent composition, a cell counting plate, a blood analyzer, a brain or spinal fluid analyzer.

10. 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 CLM-6 in serum or cerebrospinal fluid; the judgment module includes a readable carrier storing judgment rules; The judgment rule is: when the serum CLM-6 concentration value measured by the detection module is ≥4.608NPX value or the cerebrospinal fluid CLM-6 concentration value is ≥3.343NPX value, it is judged as a high risk of nerve damage; When the detection module measures: 4.461NPX value ≤ serum CLM-6 concentration value < 4.608NPX value or 2.656NPX value ≤ cerebrospinal fluid CLM-6 concentration value < 3.343NPX value, it is judged as a moderate risk of nerve damage; When the detection module measures: serum CLM-6 concentration value < 4.461NPX value or 2.29NPX value ≤ cerebrospinal fluid CLM-6 concentration value < 2.656NPX value, it is judged as low risk of nerve damage; When the detection module measures that the cerebrospinal fluid CLM-6 concentration value is less than 2.29NPX value, it is judged as no or low risk of asymptomatic neurosyphilis infection in cerebrospinal fluid.

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