Application of cerebrospinal fluid CTSC protein as a marker in the preparation of diagnostic products for neurosyphilis or nerve damage

Abstract

The present invention relates to the field of biomedical technology, and specifically to the use of cerebrospinal fluid CTSC protein as a marker in the preparation of neurosyphilis or nerve damage diagnosis products. CTSC protein is also called Dipeptidyl peptidase 1. According to the research conclusions of the present invention, it can be used as a cerebrospinal fluid marker for early diagnosis, screening and typing detection of neurosyphilis patients. By measuring the concentration of CTSC protein in cerebrospinal fluid, the concentration is used as an indicator for early diagnosis of neurosyphilis, symptomatic neurosyphilis, asymptomatic neurosyphilis, typing detection of neurosyphilis and latent syphilis, and evaluation of the degree of nerve damage. The indicator can be used alone or as a main reference indicator in combination with other indicators to complete the diagnosis, screening, typing and nerve damage evaluation of neurosyphilis. The indicator has the advantages of good sensitivity and high specificity.

Description

Technical Field

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

[0002] Syphilis is a systemic infectious disease caused by Treponema pallidum invading the human body. Syphilis is usually divided into five types clinically: asymptomatic, meningeal syphilis, meningeal vascular 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. Patients with asymptomatic neurosyphilis have no obvious symptoms or signs, but there are abnormal changes in cerebrospinal fluid, and those with abnormal cerebrospinal fluid that persists for more than 5 years have a higher probability of developing symptomatic neurosyphilis. In the study of primary syphilis and secondary syphilis, it was found that 20% of patients had abnormal cerebrospinal fluid, that is, neurosyphilis can occur in the early stage 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 may experience epilepsy, stroke-like illness, secondary hemiplegia and aphasia. Therefore, it is necessary to detect and evaluate the extent of neurosyphilis and nerve damage as early as possible to avoid the development of severe symptoms that affect the patient's survival.

[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 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 protein content in the cerebrospinal fluid indicates that the blood-brain barrier is damaged and there is an 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. Currently, in the clinical diagnosis and treatment process, neurosyphilis is mainly diagnosed and treated in patients who have already developed symptoms of neurosyphilis, and there is a lack of sensitive and reliable biological markers that can effectively distinguish asymptomatic neurosyphilis.

[0004] In summary, the development of new biomarkers 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 problem that the existing CSF-RPR diagnostic method has low sensitivity, especially insensitivity to the diagnosis of early asymptomatic neurosyphilis, the present invention provides a cerebrospinal fluid CTSC protein as a marker for the preparation of neurosyphilis or nerve damage diagnostic products. 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 screening for asymptomatic 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 the use of cerebrospinal fluid CTSC protein as a marker in the preparation of a diagnostic product for neurosyphilis or nerve damage.

[0010] In a second aspect, the present invention provides the use of CTSC protein and its specific antibodies 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 CTSC protein or its specific antibody as the target molecule.

[0011] According to a preferred embodiment of the present invention, the kit uses CTSC protein or its specific antibody as the detection target.

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

[0013] According to a preferred embodiment of the present invention, the kit comprises: a primary antibody that can specifically recognize CTSC protein, 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.

[0014] According to a preferred embodiment of the present invention, the kit further comprises reagents or materials for obtaining cerebrospinal fluid from the patient.

[0015] It should be noted that when detecting the expression level of CTSC protein, the Olink ultra-high sensitivity plasma protein detection platform can also be used to quantify the expression level of CTSC.

[0016] 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 or device is capable of quantitatively detecting the concentration of CTSC in cerebrospinal fluid; the product is a detection kit, an antibody chip, an antibody probe or a detector.

[0017] Preferably, the product comprises antibodies that specifically bind to CTSC and other reagents for quantitatively detecting the concentration of CTSC protein in cerebrospinal fluid. The experimental method used in the embodiments of the present application is Olink neurology panel (95801), which can be used to detect 92 protein biomarkers related to neurological function, including CTSC protein. CTSC is not only a protein degrading enzyme in lysosomes, but also an aminodipeptidase that continuously removes two nitrogen-terminal amino acid residues from the folded state of proteins. It is an ideal biological machine for activating chymotrypsin-like serine protease.

[0018] Syphilis typing includes symptomatic neurosyphilis, asymptomatic neurosyphilis or non-neurosyphilis, etc. In this application, non-neurosyphilis and latent syphilis have the same meaning; symptomatic neurosyphilis and paralytic dementia have the same meaning.

[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 CTSC in cerebrospinal fluid; the judgment module comprises a readable carrier storing a judgment rule;

[0020] The judgment rule is: when the CTSC concentration value of the cerebrospinal fluid obtained by the detection module is ≥3.960NPX, it is judged as a high risk of nerve damage;

[0021] When the 3.960NPX obtained by the detection module is greater than the CTSC concentration value of the cerebrospinal fluid ≥ 3.546NPX, it is judged as a risk of central nerve injury;

[0022] When the detection module obtains 3.546NPX>CTSC concentration value of CSF ≥3.232NPX, it is judged as low risk of nerve damage or risk of CSF infection with asymptomatic neurosyphilis;

[0023] When the detection module obtains a cerebrospinal fluid CTSC concentration value of <3.232NPX, it is judged to be at low risk of cerebrospinal fluid infection with asymptomatic neurosyphilis.

[0024] The above reference value is the cut-off value of CTSC in cerebrospinal fluid obtained from a group of syphilis patients; preferably, the cut-off value of cerebrospinal fluid for latent syphilis and asymptomatic neurosyphilis is 3.232NPX, the cut-off value of cerebrospinal fluid for diagnosis of non-neurosyphilis and neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) is 3.546NPX; the cut-off value of cerebrospinal fluid for diagnosis of asymptomatic neurosyphilis and symptomatic neurosyphilis is 3.960NPX. The aforementioned cut-off value is the critical value of CTSC statistical analysis obtained from 88 syphilis patients.

[0025] In a fifth aspect, the present invention also provides a syphilis detection kit, which comprises: a first detection reagent composition for detecting the level of CTSC protein in cerebrospinal fluid; and a second detection reagent composition for detecting cerebrospinal fluid leukocytes and / or cerebrospinal fluid proteins.

[0026] In the above scheme, when diagnosing neurosyphilis (including early diagnosis, typing and staging, and nerve damage assessment), the cerebrospinal fluid CTSC 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.

[0027] The level of CTSC in cerebrospinal fluid is 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 marker CTSC for early diagnosis screening, typing detection and nerve damage assessment of neurosyphilis. By detecting the expression level of CTSC in the patient's 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 CTSC in the patient's cerebrospinal fluid, the diagnosis of neurosyphilis, including early diagnosis, typing detection and nerve damage assessment, can be achieved. Compared with the existing technology, the cerebrospinal fluid marker CTSC has higher sensitivity.

[0030] The cerebrospinal fluid marker CTSC is also suitable for the diagnosis and screening of symptomatic neurosyphilis and asymptomatic neurosyphilis, because the expression level of CTSC in the cerebrospinal fluid of patients with symptomatic neurosyphilis is the highest among symptomatic neurosyphilis, asymptomatic neurosyphilis or non-neurosyphilis. The CTSC expression level has a high sensitivity in the assessment of neurosyphilis and symptomatic neurosyphilis, and also has a predictive effect on asymptomatic neurosyphilis. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] Figure 2 The results showed that the expression of CTSC in CSF was significantly positively correlated with CSF protein and leukocytes.

[0033] Figure 3 The correlation between CTSC in serum and CSF proteins and leukocytes was shown.

[0034] Figure 4 To distinguish asymptomatic neurosyphilis from latent syphilis when asymptomatic neurosyphilis was the clinical outcome, the AUC of the probability of a confirmed diagnosis of asymptomatic neurosyphilis was evaluated by the CSF and serum CTSC concentration levels.

[0035] Figure 5 To distinguish neurosyphilis from non-neurosyphilis when the diagnosis of neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) was the clinical outcome, the CSF and serum CTSC concentration levels were used to assess the AUC of the probability of a confirmed diagnosis of neurosyphilis.

[0036] Figure 6 To distinguish asymptomatic neurosyphilis from symptomatic neurosyphilis when the diagnosis of symptomatic neurosyphilis was used as the clinical outcome, the CSF and serum CTSC concentration levels were used to assess the probability of a confirmed diagnosis of symptomatic neurosyphilis using the AUC.

[0037] Figure 7 According to the cerebrospinal fluid CTSC cutoff value of 3.232NPX, patients with latent syphilis and asymptomatic neurosyphilis were divided into two groups, and the differences in neurological damage indicators such as cerebrospinal fluid protein and leukocytes were found between the groups.

[0038] Figure 8 All patients were divided into two groups according to the cerebrospinal fluid CTSC cutoff value of 3.546NPX, and the differences in neurological injury indicators, cerebrospinal fluid protein and leukocytes, were observed between the groups.

[0039] Fig. 9 According to the cerebrospinal fluid CTSC cutoff value of 3.960NPX, neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) were divided into two groups, and the differences in neurological damage indicators, cerebrospinal fluid protein and leukocytes, were found between the groups. DETAILED DESCRIPTION

[0040] In order to better explain the present invention, the present invention is now described in detail.

[0041] 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. Therefore, the inventors conducted research with the purpose of developing new biomarkers 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 cerebrospinal fluid of patients with neurosyphilis and finally obtained CTSC, a protein marker with great diagnostic value in cerebrospinal fluid.

[0042] CTSC is a lysosomal cysteine ​​peptidase, also known as JP, HMS, JPD, PLS, CPPI, DPP1, DPPI, PALS, DPP-I or PDON1. For simplicity and clarity, it is collectively referred to as CTSC in this application.

[0043] After research, it was found that the levels of CTSC protein in the cerebrospinal fluid of patients with different types of syphilis were significantly different. The cerebrospinal fluid CTSC protein was significantly increased in patients with neurosyphilis, and further increased in patients with symptomatic neurosyphilis, and was significantly positively correlated with the nerve damage indicators cerebrospinal fluid protein and cerebrospinal fluid leukocytes, which shows that the cerebrospinal fluid CTSC protein can predict neurosyphilis, diagnose asymptomatic neurosyphilis and symptomatic neurosyphilis, and can also evaluate the severity of nerve damage. The CTSC protein in the serum of patients with symptomatic neurosyphilis was significantly increased compared with that in the serum of patients with asymptomatic neurosyphilis, but there was no significant difference between the two groups of patients with asymptomatic neurosyphilis and latent syphilis. The cerebrospinal fluid CTSC protein level of patients with asymptomatic neurosyphilis was significantly higher than that of the latent syphilis group, indicating that the CTSC protein level has changed in the early stage of neurosyphilis, that is, the asymptomatic neurosyphilis stage; therefore, this biomarker can effectively distinguish asymptomatic neurosyphilis from latent syphilis, and contribute to the early screening of asymptomatic neurosyphilis.

[0044] In order to clarify the relationship between serum CTSC and cerebrospinal fluid CTSC and asymptomatic neurosyphilis, 45 syphilis patients (15 latent syphilis patients and 30 asymptomatic neurosyphilis) were studied in the following examples, and the receiver operating characteristic curve (ROC) was used to evaluate the diagnostic potential of CTSC for asymptomatic neurosyphilis; the experimental results were: the area under the curve (AUC) of cerebrospinal fluid CTSC was 0.7222, P=0.0161, which was considered statistically significant, with a sensitivity of 73.33 and a specificity of 63.33; the AUC of serum CTSC was 0.5689, P=0.4554, which was not statistically significant. The above results show that only the level of cerebrospinal fluid CTSC can better evaluate the occurrence of asymptomatic neurosyphilis, and whether serum CTSC can be used requires more samples for verification.

[0045] Further, in order to clarify the relationship between serum CTSC and cerebrospinal fluid CTSC and neurosyphilis, 88 syphilis patients (all syphilis patients) were studied in the following examples, and the experiment used ROC to evaluate the diagnostic potential of CTSC protein for neurosyphilis; the experimental results were: the AUC of cerebrospinal fluid CTSC was 0.8283, P < 0.0001, which was considered statistically significant; the AUC of serum CTSC was 0.5178, P = 0.8287, which was considered not statistically significant. Accordingly, the level of CTSC in the cerebrospinal fluid of syphilis patients can better evaluate the occurrence of neurosyphilis, with a sensitivity of 86.67 and a specificity of 72.60.

[0046] Next, in order to clarify the relationship between serum CTSC and cerebrospinal fluid CTSC and symptomatic neurosyphilis, 73 patients with neurosyphilis (30 cases of asymptomatic neurosyphilis and 43 cases of paralytic dementia) were studied in the following examples, and ROC was used to evaluate the diagnostic potential of CTSC for symptomatic neurosyphilis; the experimental results were: the AUC of cerebrospinal fluid CTSC was 0.7341, P = 0.0007, which was considered statistically significant, with a sensitivity of 73.33 and a specificity of 58.14; the AUC of serum CTSC was 0.6426, P = 0.0391, which was considered statistically significant, but the prediction effect was not good. The above results show that the occurrence of symptomatic neurosyphilis can be better evaluated according to the level of cerebrospinal fluid CTSC in patients with neurosyphilis, while the prediction effect of serum CTSC is not good.

[0047] Furthermore, the experiment divided latent syphilis and asymptomatic neurosyphilis patients into two groups according to the Cut-Off value (3.232NPX) of cerebrospinal fluid CTSC in syphilis patients. Patients with values ​​above this Cut-Off value had significantly increased cerebrospinal fluid protein and leukocytes, indicators related to cerebrospinal fluid damage. Therefore, when the cerebrospinal fluid CTSC is lower than 3.232NPX, the risk of asymptomatic neurosyphilis in syphilis patients is lower and the degree of nerve damage is less; when it is greater than or equal to this value, the risk of asymptomatic neurosyphilis increases and the degree of nerve damage also increases.

[0048] Then, the experiment divided all patients into two groups according to the Cut-Off value (3.546NPX) of cerebrospinal fluid CTSC of all syphilis patients (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis). Patients with a value greater than or equal to the Cut-Off value had a further significant increase in cerebrospinal fluid protein and leukocytes, indicators related to cerebrospinal fluid damage. Therefore, when the cerebrospinal fluid CTSC is lower than 3.546NPX, patients with neurosyphilis have a lower risk of developing neurosyphilis and a lower degree of nerve damage.

[0049] Finally, the experiment also divided all neurosyphilis patients into two groups according to the Cut-Off value (3.960NPX) of cerebrospinal fluid CTSC of all neurosyphilis patients (including asymptomatic neurosyphilis and symptomatic neurosyphilis). Patients with a value greater than or equal to this Cut-Off had a significant increase in cerebrospinal fluid protein and leukocytes, indicators related to cerebrospinal fluid damage. Therefore, when the cerebrospinal fluid CTSC is lower than the 3.960NPX value, the risk of symptomatic neurosyphilis in neurosyphilis patients is lower, and the degree of nerve damage is relatively small; while when it is greater than or equal to this value, the risk of symptomatic neurosyphilis is higher, the degree of nerve damage is also higher, and the risk of paralytic dementia increases. Therefore, cerebrospinal fluid CTSC can be used as an indicator of various types of neurosyphilis and the degree of nerve damage.

[0050] In summary, the present invention provides the application of CTSC for early diagnosis of neurosyphilis and assessment of nerve damage. In medical diagnosis, the CTSC protein level in cerebrospinal fluid can be used 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, those skilled in the art can detect the expression level of CTSC in cerebrospinal fluid to achieve high-sensitivity detection and typing of syphilis, especially neurosyphilis, and perform accurate quantitative assessment of nerve damage, supervision and treatment of neurosyphilis.

[0051] 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.

[0052] 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:

[0053] 1. Research subjects

[0054] 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.

[0055] 2. Sample collection

[0056] 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.

[0057] 3. Detection of CTSC concentration in serum and cerebrospinal fluid using the Olink method

[0058] Olink neurology panel (95801) was used to detect 92 proteins related to neurological function, including CTSC protein. Olink precision proteomics is a precision 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 Olink to detect markers in the serum and cerebrospinal fluid of patients with different types of neurosyphilis, and found that CTSC protein has significant differences in cerebrospinal fluid. The detection of cerebrospinal fluid protein and cerebrospinal fluid leukocytes was performed according to the method described in WS 273-2007.

[0059] The following statistics and data analysis are performed on the above Olink test results:

[0060] (1) CTSC protein significantly increased in cerebrospinal fluid and serum of neurosyphilis

[0061] Olink precision proteomics was used to detect the difference in the expression of nerve-related proteins in serum and cerebrospinal fluid, and CTSC proteins with significant differences in both cerebrospinal fluid and serum were screened. Figure 1 As shown in Table 1 , the expression of CTSC in the CSF of patients with symptomatic neurosyphilis was significantly increased compared with that of patients with asymptomatic neurosyphilis (no symptoms) and latent syphilis, while the expression of CTSC in the CSF of patients with asymptomatic neurosyphilis was significantly increased compared with that of patients with latent syphilis (e.g. Figure 1 a); At the same time, compared with asymptomatic neurosyphilis, the CTSC in the serum of patients with symptomatic neurosyphilis increased significantly, while the difference between latent syphilis and asymptomatic neurosyphilis was not significant. Therefore, the CTSC level in cerebrospinal fluid can be used to distinguish syphilis typing and indicate the severity of nerve damage.

[0062] Depend on Figure 1 As can be seen from Figure a, the CTSC concentration in the cerebrospinal fluid of patients with symptomatic neurosyphilis> the CTSC concentration in the cerebrospinal fluid of patients with asymptomatic neurosyphilis> the CTSC concentration in the cerebrospinal fluid of patients with latent syphilis; Figure 1As can be seen from b, the CTSC concentration in the serum of patients with symptomatic neurosyphilis is greater than that in the serum of patients with asymptomatic neurosyphilis, but the CTSC concentration in the serum of patients with latent syphilis is not significantly different from that of the other two groups.

[0063] Table 1: Statistical differences in the expression of CTSC in cerebrospinal fluid and serum among three different types of syphilis

[0064] In Table 1: Latent syphilis & asymptomatic indicates the statistical value of the difference in CTSC concentration between latent syphilis and asymptomatic neurosyphilis in cerebrospinal fluid or serum. Latent syphilis & symptomatic indicates the statistical value of the difference in CTSC concentration between latent syphilis and symptomatic neurosyphilis in cerebrospinal fluid or serum. Asymptomatic & symptomatic indicates the statistical value of the difference in CTSC concentration between asymptomatic neurosyphilis and symptomatic neurosyphilis in cerebrospinal fluid or serum. The mean difference reflects the average difference between each marker value and the arithmetic mean. The difference P 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 CTSC protein levels in the cerebrospinal fluid of patients with different types of syphilis, and the differences are statistically significant.

[0065] (2) CTSC in cerebrospinal fluid is significantly positively correlated with injury indicators in cerebrospinal fluid

[0066] Important reference indicators for neurosyphilis are cerebrospinal fluid protein and white blood cell count levels. Patients with neurosyphilis will have abnormal cerebrospinal fluid cell counts and protein levels, 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.

[0067] Combination Figure 2 From a and b, we can see that the expression level of CTSC protein in cerebrospinal fluid is significantly positively correlated with the protein in the cerebrospinal fluid of patients (R=0.6827, p<0.0001) and the number of leukocytes (R=0.5825, p<0.0001). Therefore, CTSC protein in cerebrospinal fluid can indicate abnormalities of protein and leukocytes in cerebrospinal fluid.

[0068] Therefore, in practice, CSF cell count and / or protein level can be used as a reference indicator, together with the CTSC protein level in CSF as an indicator for early diagnosis and screening of neurosyphilis, typing detection and quantitative assessment of the degree of nerve damage.

[0069] (3) There is no significant correlation between CTSC in serum and injury indicators in cerebrospinal fluid

[0070] Combination Figure 3From a and b, we can see that the expression level of CTSC protein in serum is not significantly correlated with the protein in the patient's cerebrospinal fluid (R=0.1583, p=0.1407) and the number of leukocytes (R=0.01512, p=0.8888). Therefore, CTSC protein in serum cannot indicate abnormalities in protein and leukocytes in cerebrospinal fluid.

[0071] (4) Evaluation of cerebrospinal fluid and serum CTSC in the detection of neurosyphilis in syphilis patients

[0072] The level of serum and cerebrospinal fluid CTSC can also be used to diagnose asymptomatic neurosyphilis. In order to further clarify the relationship between serum and cerebrospinal fluid CTSC and asymptomatic neurosyphilis, this example studied 45 syphilis patients (15 latent syphilis patients and 30 asymptomatic neurosyphilis patients). The study used ROC to evaluate the diagnostic potential of CTSC in these syphilis patients for asymptomatic neurosyphilis. Figure 4 As shown in the figure, the experimental results were: the area under the curve (AUC) of CTSC in cerebrospinal fluid (0.7222 [95% CI 0.5700-0.8744]), P = 0.0161, which was considered statistically significant (see Figure 4 The area under the curve (AUC) of serum CTSC was 0.5689 [95% CI 0.3936-0.7441], P = 0.4554, which was not significant (see Figure 4 The above results show that the occurrence of asymptomatic neurosyphilis can be better evaluated according to the level of CTSC in the cerebrospinal fluid of syphilis patients, with a sensitivity of 73.33 and a specificity of 63.33.

[0073] In order to further clarify the relationship between serum and cerebrospinal fluid CTSC and neurosyphilis, this example studied 88 syphilis patients and used the receiver operating characteristic curve (ROC) to evaluate the diagnostic potential of CTSC for neurosyphilis, where neurosyphilis includes asymptomatic neurosyphilis and symptomatic neurosyphilis. Figure 5 As shown in the figure, the experimental results are: the area under the curve of CTSC in cerebrospinal fluid (AUC) (0.8283 [95% CI 0.7244-0.9322]), P < 0.0001 is considered statistically significant (see Figure 5 Figure a); The area under the curve of serum CTSC (AUC) (0.5178 [95% CI 0.3603-0.6753]), P = 0.8287 was considered to be insignificant (see Figure 5 (b) This shows that the level of CTSC in the cerebrospinal fluid of syphilis patients can better assist in the diagnosis of neurosyphilis, with a sensitivity of 86.67 and a specificity of 72.60.

[0074] In addition to the above-mentioned role in detecting neurosyphilis, the level of CTSC in cerebrospinal fluid can also be used to diagnose symptomatic neurosyphilis in neurosyphilis. In order to further clarify the relationship between serum and cerebrospinal fluid CTSC and symptomatic neurosyphilis, this example studied 73 patients with neurosyphilis (including 30 cases of asymptomatic neurosyphilis and 43 cases of paralytic dementia). The study used ROC to evaluate the diagnostic potential of CTSC in patients with neurosyphilis for symptomatic neurosyphilis. Figure 6 As shown in the figure, the experimental results are: the area under the curve of CTSC in cerebrospinal fluid (AUC) (0.7341 [95% CI 0.6193-0.8489]), P = 0.0007 is considered statistically significant (see Figure 6 Figure a); The area under the curve of serum CTSC AUC (0.6426 [95% CI 0.5150-0.7702]), P = 0.0.0391, there is a significant difference, but the prediction effect is not good (see Figure 6 The above results show that the occurrence of symptomatic neurosyphilis can be better evaluated according to the level of CTSC in the cerebrospinal fluid of patients with neurosyphilis, with a sensitivity of 73.33 and a specificity of 58.14; whether serum can be used for evaluation requires more samples to confirm.

[0075] (5) CTSC levels can indicate damage indicators in cerebrospinal fluid

[0076] The present invention divides latent syphilis and asymptomatic neurosyphilis patients into two groups according to the Cut-Off value (3.232NPX) of the cerebrospinal fluid CTSC of syphilis patients, such as Figure 7 As shown, the cerebrospinal fluid protein associated with cerebrospinal fluid injury in patients with cerebrospinal fluid CTSC greater than or equal to 3.232NPX (see Figure 7 Figure a) and white blood cells (see Figure 7 Therefore, when the CSF CTSC is lower than 3.232NPX, the risk of asymptomatic neurosyphilis and nerve damage in syphilis patients is lower; while when it is greater than or equal to this value, the risk of asymptomatic neurosyphilis and nerve damage increases.

[0077] The present invention divides all syphilis patients (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups according to the Cut-Off value (3.546NPX) of cerebrospinal fluid CTSC, such as Figure 8 As shown, the cerebrospinal fluid protein associated with cerebrospinal fluid injury in patients with cerebrospinal fluid CTSC greater than or equal to 3.546NPX (see Figure 8 Figure a) and white blood cells (see Figure 8Therefore, when the CSF CTSC is lower than 3.546NPX, the risk of neurosyphilis is low and the risk of nerve damage is small; while when it is greater than or equal to this value, the risk of neurosyphilis increases and the risk of nerve damage also increases.

[0078] The present invention divides asymptomatic neurosyphilis and paralytic dementia into two groups according to the Cut-Off value (3.960NPX) of cerebrospinal fluid CTSC of all neurosyphilis patients, such as Fig. 9 As shown, the cerebrospinal fluid protein associated with cerebrospinal fluid injury in patients with cerebrospinal fluid CTSC greater than or equal to 3.960NPX (see Fig. 9 Figure a) increased significantly, and the white blood cells increased significantly (see Fig. 9 (b). Therefore, when the CSF CTSC is lower than 3.960NPX, the risk of symptomatic neurosyphilis in patients with neurosyphilis is low, and the risk of nerve damage is relatively small; while when it is greater than or equal to this value, the risk of symptomatic neurosyphilis is high, the risk of nerve damage is high, and the risk of paralytic dementia is high. Therefore, CSF CTSC can be used as an indicator of various types of neurosyphilis and nerve damage.

[0079] In summary, the concentration of CTSC protein in cerebrospinal fluid can be used for early diagnosis of neurosyphilis, including typing detection of symptomatic neurosyphilis, asymptomatic neurosyphilis, neurosyphilis and latent syphilis, and as an indicator for evaluating 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.

[0080] 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. The use of CTSC protein and its specific antibody in the preparation of diagnostic products for screening, typing detection or evaluating the degree of nerve damage in patients with neurosyphilis, characterized in that: The diagnostic products are detection kits, antibody chips, and antibody probes with CTSC protein as target molecules; the typing is paralytic dementia, asymptomatic neurosyphilis, and latent syphilis; and the detection sample is cerebrospinal fluid.

2. The use according to claim 1, characterized in that: The kit uses CTSC protein as a detection target.

3. The use according to claim 1, characterized in that: The kit is an enzyme-linked immunosorbent assay kit or an immunohistochemistry kit.

4. The use according to claim 1, characterized in that: The kit comprises: a primary antibody capable of specifically recognizing CTSC, a polypeptide dye, magnetic beads and a secondary antibody labeled with biotin.

5. The use according to claim 4, characterized in that: The reagents include citrate buffer, PBS washing solution, peroxidase blocking agent, goat serum, streptomycin antibiotic-peroxidase, DAB solution and hematoxylin solution.

6. The use according to claim 1, characterized in that: The kit also includes reagents or materials for obtaining cerebrospinal fluid from the patient.

7. 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 CTSC protein concentration value of the cerebrospinal fluid; the judgment module includes a readable carrier storing the judgment rules; The judgment rules are: The Cut-Off value of the CTSC protein concentration in the cerebrospinal fluid for the diagnosis of paralytic dementia is 3.960NPX; The cut-off value of the CTSC protein concentration in the cerebrospinal fluid for the diagnosis of asymptomatic neurosyphilis is 3.546NPX; The cut-off value of the CTSC protein concentration in the cerebrospinal fluid for the diagnosis of latent syphilis is 3.232NPX.

Images