Application of SKR3 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 method for using a serine / threonine protein kinase receptor R3 (serine / threonine-protein kinase receptor R3, SKR3) as a cerebrospinal fluid marker for early diagnosis, screening and typing detection of a neurosyphilis patient. According to the invention, the concentration of the SKR3 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 complete diagnosis screening, typing and nerve injury evaluation of the neurosyphilis. The sensitivity of the SKR3 as the marker is high, early warning and screening of the asymptomatic neurosyphilis can be achieved, and medical staff can give a reasonable treatment scheme to patients suffering from the asymptomatic neurosyphilis as early as possible.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of SKR3 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] The clinical manifestations of neurosyphilis are also complex and changeable, with non-specific symptoms, including almost all symptoms and signs in neurology. Similar clinical manifestations may appear in neurology, ophthalmology, dermatology, psychiatry and other departments. The misdiagnosis rate of neurosyphilis is high, so the correct diagnosis, screening and timely treatment of neurosyphilis are very important.

[0003] 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. Neurosyphilis is usually divided into five types clinically: 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 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. The main manifestations of paralytic dementia are mental and intellectual decline, such as memory loss, abnormal behavior, personality changes. Severe patients may experience epilepsy, stroke-like onset, secondary hemiplegia and aphasia.

[0004] At present, the diagnosis of neurosyphilis relies on three laboratory tests on cerebrospinal fluid. The evaluation of the severity of neurosyphilis is mainly based on the experience of clinicians, and there is no unified and effective objective indicator. Neurosyphilis detection relies on the venereal disease research laboratory test (VDRL), protein and leukocyte content in cerebrospinal fluid. Early diagnosis of neurosyphilis is very important. Finding sensitive biomarkers can help diagnose neurosyphilis and is of great significance for preventing serious and irreversible sequelae. 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 and increased protein content in the cerebrospinal fluid indicate blood-brain barrier damage and intracranial inflammatory response, which are helpful for the diagnosis of neurosyphilis. However, during the diagnosis process, it was found that the sensitivity of CSF-RPP was only 30%-70%, which was low, and it was not sensitive for the diagnosis of early asymptomatic neurosyphilis. At present, in the process of clinical diagnosis and treatment, neurosyphilis is mainly diagnosed and treated in patients with symptoms of neurosyphilis. There is a lack of sensitive, reliable and effective cerebrospinal fluid and serum biological markers that can distinguish neurosyphilis. What is even more lacking is the biological markers that can effectively distinguish asymptomatic neurosyphilis.

[0005] SKR3 is a serine / threonine protein kinase receptor R3 encoded by the human ACVRL1 gene. Previous studies have shown that SKR3 plays a role in serine / threonine phosphorylation, signal transduction, and angiogenesis, but its role in nerve damage associated with neurosyphilis has not been reported. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the technical problems such as low sensitivity of existing CSF-RPR diagnostic methods and insensitivity to the diagnosis of early asymptomatic neurosyphilis, the present invention provides a 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 effectively distinguish asymptomatic neurosyphilis from latent syphilis, so that a treatment plan can be formulated for patients with asymptomatic neurosyphilis as soon as possible.

[0008] (II) Technical solution

[0009] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0010] In a first aspect, the present invention relates to SKR3 as a serum marker and cerebrospinal fluid marker for early diagnosis, screening and typing detection of patients with neurosyphilis.

[0011] In a second aspect, the present invention provides the use of SKR3 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; the diagnostic product or auxiliary diagnostic product is a detection kit, antibody chip, antibody probe or detector with SKR3 protein or its specific antibody as the target molecule.

[0012] According to a preferred embodiment of the present invention, the kit uses SKR3 protein or its specific antibody as a target molecule to detect the expression level of SKR3 protein or its specific antibody in cerebrospinal fluid or serum.

[0013] According to a preferred embodiment of the present invention, the kit is an enzyme-linked immunosorbent assay kit or an immunohistochemistry kit.

[0014] According to a preferred embodiment of the present invention, the kit comprises: a primary antibody that can specifically recognize SKR3, a polypeptide dye, magnetic beads and a biotin-labeled secondary antibody. Preferably, the kit comprises citrate buffer, PBS washing solution, peroxidase blocker, goat serum, streptomycin antibiotic-peroxidase, DAB solution and hematoxylin solution.

[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, and / or reagents for separating serum.

[0016] It should be noted that when detecting the expression level of SKR3 protein in cerebrospinal fluid and serum, the Olink ultra-high sensitivity protein detection platform can also be used to quantify the expression level of SKR3.

[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 or device can quantitatively detect the concentration of SKR3 in the patient's ex vivo serum and 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 SKR3 and other reagents that can quantitatively detect the concentration of SKR3 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 SKR3 protein.

[0019] Syphilis classification includes symptomatic neurosyphilis, asymptomatic neurosyphilis or non-neurosyphilis.

[0020] 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 SKR3 in cerebrospinal fluid; the judgment module comprises a readable carrier storing a judgment rule;

[0021] The judgment rule is: when the cerebrospinal fluid SKR3 concentration value obtained by the detection module is ≥ 3.731NPX value, it is judged as a high risk of nerve damage;

[0022] When the detection module obtains a value of 3.731NPX>cerebrospinal fluid SKR3 concentration value ≥ 3.404NPX, it is judged as a moderate risk of nerve injury; conversely, when the value of 3.256NPX<cerebrospinal fluid SKR3 concentration value<3.404NPX, it is judged as a low risk of nerve injury;

[0023] When the cerebrospinal fluid SKR3 concentration value obtained by the detection module is ≥3.256NPX value, it is judged that there is a risk of cerebrospinal fluid infection with syphilis; otherwise, it is judged that there is no or low risk of cerebrospinal fluid infection with syphilis.

[0024] The reference value is the Cut-off value of SKR3 obtained from the syphilis patient group; preferably, the Cut-off value of cerebrospinal fluid for the diagnosis of neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) is 3.404NPX (cerebrospinal fluid concentration of SKR3 above this value is judged as neurosyphilis or moderate risk of nerve damage); the Cut-off value of cerebrospinal fluid for the diagnosis of symptomatic neurosyphilis is 3.731NPX (cerebrospinal fluid concentration of SKR3 above this value is judged as symptomatic neurosyphilis or high risk of nerve damage); the Cut-off value of cerebrospinal fluid for the diagnosis of asymptomatic neurosyphilis is 3.256NPX (cerebrospinal fluid concentration of SKR3 above this value is judged as the risk of cerebrospinal fluid infection with syphilis). The above Cut-off value is the critical value of SKR3 statistical analysis obtained from 88 syphilis patients.

[0025] In a fifth aspect, the present invention further provides a syphilis detection kit, comprising: a first detection reagent composition for detecting the level of SKR3 protein in cerebrospinal fluid; and a second detection reagent composition for detecting leukocytes and / or cerebrospinal fluid protein in cerebrospinal fluid. Alternatively, the present invention provides a neurosyphilis diagnostic system, comprising a first indicator analysis unit and a second analysis unit; the first analysis unit is an antibody chip or antibody probe for detecting the level of SKR3 protein in serum or cerebrospinal fluid; the second analysis unit is a detection reagent composition or analyzer for detecting the level of leukocytes or total protein in cerebrospinal fluid.

[0026] In the above scheme, when diagnosing neurosyphilis (including early diagnosis, typing and staging, and nerve damage assessment), the cerebrospinal fluid SKR3 protein level can be combined with cerebrospinal fluid leukocytes and / or cerebrospinal fluid protein indicators related to nerve damage in patients with neurosyphilis for early diagnosis, typing and staging, and nerve damage assessment of neurosyphilis.

[0027] The level of SKR3 in serum or 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 protein in cerebrospinal fluid can be detected by known methods.

[0028] (III) Beneficial effects

[0029] The present invention provides a new cerebrospinal fluid marker SKR3 for early diagnosis screening, typing detection and nerve damage assessment of neurosyphilis. By detecting the expression level of SKR3 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 SKR3 in the patient's cerebrospinal fluid or serum, 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 asymptomatic neurosyphilis and symptomatic neurosyphilis when the serum SKR3 concentration is used as an indicator.

[0030] Preferably, the cerebrospinal fluid marker SKR3 is also suitable for diagnosing and screening symptomatic neurosyphilis and asymptomatic neurosyphilis, because there are significant differences in the expression level of SKR3 in cerebrospinal fluid in symptomatic neurosyphilis, asymptomatic neurosyphilis or non-neurosyphilis. At the same time, the SKR3 expression level has a high sensitivity in evaluating neurosyphilis, symptomatic neurosyphilis and asymptomatic neurosyphilis, so the use of this marker can achieve screening for symptomatic neurosyphilis and asymptomatic syphilis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The Olink method was used to detect the expression of SKR3 protein in the cerebrospinal fluid and serum of three groups of syphilis patients with different types (latent, asymptomatic and symptomatic).

[0032] Figure 2 The results showed that the expression levels of SKR3 in cerebrospinal fluid and serum were significantly positively correlated with cerebrospinal fluid protein and leukocytes.

[0033] Figure 3 To estimate the likelihood of a confirmed diagnosis of neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) using cerebrospinal fluid and serum SKR3 concentrations as the clinical outcome, and the area under the curve (AUC).

[0034] Figure 4 To distinguish symptomatic neurosyphilis from asymptomatic neurosyphilis when the diagnosis of symptomatic neurosyphilis was used as the clinical outcome, the AUC of the probability of a confirmed diagnosis of symptomatic neurosyphilis was evaluated at different levels of CSF and serum SKR3 concentrations.

[0035] Figure 5 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 SKR3 concentrations were used to assess the likelihood of a confirmed diagnosis of asymptomatic neurosyphilis.

[0036] Figure 6 All patients (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) were divided into two groups according to the CSF SKR3 cutoff value of 3.404NPX, and the differences in the neurological damage indicators of CSF protein and leukocytes in each group were found.

[0037] Figure 7 Neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) was divided into two groups based on the cerebrospinal fluid SKR3 cutoff value of 3.731NPX, and the differences in cerebrospinal fluid protein and leukocytes, the indicators of neurological damage, were found between the groups.

[0038] Figure 8 According to the cutoff value of 3.256NPX of SKR3 in cerebrospinal fluid, 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. DETAILED DESCRIPTION

[0039] In order to better explain the present invention, the present invention is now described in detail.

[0040] 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 highly sensitive biomarkers for the diagnosis of neurosyphilis and nerve damage, screened multiple indicators related to nerve function in the serum and cerebrospinal fluid of patients with neurosyphilis, and obtained the marker SKR3 with significant diagnostic value in both serum and cerebrospinal fluid.

[0041] After research, the inventors found that the cerebrospinal fluid SKR3 protein levels of patients with syphilis of different types (latent, asymptomatic and asymptomatic) were significantly different, and the serum SKR3 was also significantly different in patients with symptomatic neurosyphilis and asymptomatic neurosyphilis. Cerebrospinal fluid SKR3 protein was significantly increased in patients with neurosyphilis, and further increased in patients with symptomatic neurosyphilis, and was positively correlated with nerve damage indicators cerebrospinal fluid protein and cerebrospinal fluid leukocytes, indicating that cerebrospinal fluid SKR3 protein can be used as a diagnostic indicator for neurosyphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis, and can also evaluate the severity of nerve damage. Compared with asymptomatic neurosyphilis, the serum SKR3 protein of symptomatic neurosyphilis was also significantly increased in the former, but there was no significant difference in patients with latent syphilis and asymptomatic neurosyphilis, and it was positively correlated with nerve damage indicators cerebrospinal fluid protein and cerebrospinal fluid leukocytes. This means that the cerebrospinal fluid SKR3 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.

[0042] During the study, the experiment first used the receiver operating characteristic curve (ROC) to show the potential of SKR3 protein in diagnosing neurosyphilis (neurosyphilis includes asymptomatic neurosyphilis and symptomatic neurosyphilis); the experimental results showed that the area under the curve (AUC) of cerebrospinal fluid SKR3 was 0.8950, P < 0.0001, which can be considered statistically significant, and the sensitivity was 86.67; while the AUC of serum SKR3 was 0.6603, P = 0.0515, and the difference was considered not statistically significant. Based on this, the level of SKR3 in the cerebrospinal fluid of patients with syphilis can better evaluate the occurrence of neurosyphilis.

[0043] Next, in order to clarify the relationship between serum and cerebrospinal fluid SKR3 and symptomatic neurosyphilis, 73 patients with neurosyphilis were studied in the following examples, and ROC was used to evaluate the potential of cerebrospinal fluid and serum SKR3 in patients with neurosyphilis (including symptomatic and asymptomatic neurosyphilis patients) to evaluate the occurrence of symptomatic neurosyphilis; the experimental results showed that the AUC of cerebrospinal fluid SKR3 was 0.8620, P < 0.0001, which was considered statistically significant, and the sensitivity was 93.33; the AUC of serum SKR3 was 0.6527, P = 0.0272, which was also considered statistically significant. The above results show that the occurrence of symptomatic neurosyphilis can be better evaluated according to the level of cerebrospinal fluid SKR3 in patients with neurosyphilis, and serum SKR3 can also predict the occurrence of symptomatic neurosyphilis, but the effect is not as good as cerebrospinal fluid.

[0044] Furthermore, asymptomatic neurosyphilis is a difficult point in clinical diagnosis and treatment, because it is difficult to diagnose without clinical symptoms, so all syphilis patients need to be screened. In order to clarify the relationship between serum and cerebrospinal fluid SKR3 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 SKR3 to evaluate the occurrence of asymptomatic neurosyphilis; the experimental results showed that the AUC of cerebrospinal fluid SKR3 was 0.8044, P = 0.0010, which was statistically significant, and the sensitivity was 80.00; the AUC of serum SKR3 was 0.6000, P = 0.2786, which was not statistically significant. The above results show that the occurrence of asymptomatic neurosyphilis can be better evaluated based on the level of SKR3 in the cerebrospinal fluid of syphilis patients, and whether serum can predict requires more samples for verification.

[0045] Further, the present invention divides all patients with syphilis (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups according to the Cut-Off value (3.404NPX) of cerebrospinal fluid SKR3 of all patients with syphilis (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis), and the cerebrospinal fluid protein and leukocytes of patients with cerebrospinal fluid damage above this value increase significantly. Therefore, when the SKR3 cerebrospinal fluid is lower than the 3.404NPX 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 Cut-Off value (3.731NPX) of cerebrospinal fluid SKR3 of all patients with neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis), and the cerebrospinal fluid protein and leukocytes of patients with cerebrospinal fluid damage above this value increase significantly. Therefore, when the SKR3 cerebrospinal fluid value is lower than 3.731NPX, 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 Cut-Off value (3.256NPX) of cerebrospinal fluid SKR3 in syphilis patients (including latent syphilis and asymptomatic neurosyphilis). Patients with syphilis above this value have a significant increase in cerebrospinal fluid protein, an indicator related to cerebrospinal fluid damage, and an increase in white blood cells but not significantly. Therefore, when the SKR3 cerebrospinal fluid value is lower than 3.256NPX, the risk of asymptomatic neurosyphilis in neurosyphilis patients is low, and the risk of nerve damage is small, and it can be judged as no or low risk of syphilis infection in cerebrospinal fluid; while above this value, the risk of asymptomatic neurosyphilis increases, and the risk of nerve damage also increases.

[0046] In summary, the present invention provides the application of SKR3 for early diagnosis of neurosyphilis and assessment of nerve damage. The inventors use the SKR3 protein level in 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 SKR3 in cerebrospinal fluid to achieve high-sensitivity detection and typing of syphilis, especially neurosyphilis, and can also perform early screening for neurosyphilis, and accurately quantify and evaluate nerve damage, supervision and treatment of neurosyphilis.

[0047] 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.

[0048] 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:

[0049] 1. Research subjects

[0050] 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.

[0051] (II) Sample collection

[0052] 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.

[0053] (III) Detection of SKR3 concentration in serum and cerebrospinal fluid using the Olink method

[0054] Olink neurology panel (95801) was used to detect 92 proteins related to neurological function, including SKR3 protein. Olink 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 Olink to detect markers in the cerebrospinal fluid of patients with different types of neurosyphilis, and it is found that SKR3 protein has significant differences in the cerebrospinal fluid. The detection of cerebrospinal fluid proteins and cerebrospinal fluid leukocytes is carried out according to the method described in WS273-2007.

[0055] The following statistics and data analysis are performed on the above Olink test results:

[0056] (1) SKR3 protein is significantly increased in cerebrospinal fluid of neurosyphilis

[0057] Olink precision proteomics was used to detect the difference in the expression of nerve-related proteins in serum and cerebrospinal fluid, and the SKR3 protein with significant differences in cerebrospinal fluid was screened. The results of Olink detection are shown in Table 1. It was found that asymptomatic neurosyphilis (referred to as asymptomatic) had a significantly higher level of SKR3 in the cerebrospinal fluid than latent syphilis, and the level of symptomatic neurosyphilis (referred to as symptomatic) was further increased. The SKR3 level in the serum of patients with symptomatic neurosyphilis was significantly higher than that of patients with latent syphilis and asymptomatic neurosyphilis. Therefore, the SKR3 level in cerebrospinal fluid can be used to distinguish between latent, asymptomatic, symptomatic and other syphilis types, and indicate the severity of nerve damage. The SKR3 level in serum can only indicate asymptomatic and symptomatic neurosyphilis, and to a certain extent, it can also indicate the severity of nerve damage. 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 SKR3 concentration in the cerebrospinal fluid of patients with symptomatic neurosyphilis > the SKR3 concentration in the cerebrospinal fluid of patients with asymptomatic neurosyphilis > the SKR3 concentration in the cerebrospinal fluid of patients with latent syphilis; Figure 1 As shown in Figure (b), the SKR3 concentration in the serum of patients with symptomatic neurosyphilis is greater than that of patients with latent syphilis and asymptomatic neurosyphilis.

[0058] Table 1: Statistical data of the difference in SKR3 expression in cerebrospinal fluid and serum among three different types of syphilis

[0059]

[0060] In Table 1: Latent syphilis & asymptomatic indicates the statistical value of the difference in SKR3 concentration between latent syphilis and asymptomatic neurosyphilis in cerebrospinal fluid or serum. Latent syphilis & symptomatic indicates the statistical value of the difference in SKR3 concentration between latent syphilis and symptomatic neurosyphilis in cerebrospinal fluid or serum. Asymptomatic & symptomatic indicates the statistical value of the difference in SKR3 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 cerebrospinal fluid SKR3 protein levels in patients with different types of syphilis, and the differences are statistically significant.

[0061] (2) SKR3 in cerebrospinal fluid is significantly positively correlated with injury indicators in cerebrospinal fluid

[0062] 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.

[0063] Combination Figure 2 As shown in Figures (a) and (b), the expression level of SKR3 protein in cerebrospinal fluid is significantly positively correlated with the protein in the patient's cerebrospinal fluid (correlation coefficient R = 0.7443, p < 0.0001) and the number of leukocytes (correlation coefficient R = 0.6317, p < 0.0001). Therefore, SKR3 protein in cerebrospinal fluid can indicate abnormalities in protein and leukocytes in cerebrospinal fluid. It can be seen that the SKR3 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, cerebrospinal fluid cell count and / or protein level can be used as a reference indicator, together with the SKR3 protein level in cerebrospinal fluid as a means of early diagnosis and screening of neurosyphilis, typing detection, and quantitative assessment of the degree of nerve damage.

[0064] Combination Figure 2 As shown in Figures (c) and (d), the expression level of SKR3 protein in serum is positively correlated with the protein in the patient's cerebrospinal fluid (correlation coefficient R = 0.3056, p = 0.0038), while there is no significant difference between the expression level of SKR3 protein in serum and the number of white blood cells (correlation coefficient R = 0.1407, p = 0.1910). Therefore, SKR3 protein in serum can only indicate abnormalities in the protein in the cerebrospinal fluid.

[0065] (4) Evaluation of cerebrospinal fluid SKR3 in neurosyphilis detection in syphilis patients

[0066] The study used the receiver operating characteristic curve (ROC) to evaluate the diagnostic potential of SKR3 for neurosyphilis (differentiating neurosyphilis from latent syphilis). Figure 3 As shown in the experimental results, the area under the curve of SKR3 in cerebrospinal fluid (AUC) was 0.8950 [95% CI 0.8210-0.9690], and P < 0.0001 was considered statistically significant (see Figure 3 (a)), the sensitivity was 86.67; the area under the curve AUC of serum SKR3 (0.6603 [95% CI 0.5088-0.8117]), P = 0.0515 was not considered statistically significant (see Figure 3 (b)). This indicates that the level of SKR3 in the cerebrospinal fluid of syphilis patients can be used to assist in the diagnosis of neurosyphilis with high sensitivity.

[0067] In addition to the above-mentioned role in detecting neurosyphilis, the level of serum and cerebrospinal fluid SKR3 can also be used to diagnose symptomatic neurosyphilis in neurosyphilis (distinguishing symptomatic neurosyphilis from asymptomatic neurosyphilis). In order to further clarify the relationship between serum and cerebrospinal fluid SKR3 and symptomatic neurosyphilis, this example studied 73 patients with neurosyphilis. The study used ROC to evaluate the potential of SKR3 in evaluating the diagnosis of symptomatic neurosyphilis in patients with neurosyphilis (including symptomatic and asymptomatic neurosyphilis patients). Figure 4 As shown in the experimental results, the area under the curve of SKR3 in cerebrospinal fluid (AUC) was 0.8620 [95% CI 0.7758-0.9482], and P < 0.0001 was considered statistically significant (see Figure 4 Figure a), the sensitivity was 93.33; the area under the curve of serum SKR3 (AUC) was 0.6527 [95% CI 0.5246-0.7808], P = 0.0272 (P < 0.05), although it was statistically significant, the AUC value was very low and the prediction effect was not good (see Figure 4 The above results indicate that the occurrence of symptomatic neurosyphilis can be better evaluated based on the level of SKR3 in the cerebrospinal fluid of patients with neurosyphilis.

[0068] In addition to the above detection functions, the level of serum and cerebrospinal fluid SKR3 can also be used to diagnose and differentiate asymptomatic neurosyphilis in latent syphilis and neurosyphilis. In order to further clarify the relationship between serum and cerebrospinal fluid SKR3 and asymptomatic neurosyphilis, this example studied 45 patients with latent syphilis and asymptomatic neurosyphilis. The study used ROC to evaluate the potential of SKR3 in syphilis patients (including latent syphilis patients and asymptomatic neurosyphilis patients) to evaluate the diagnosis of symptomatic neurosyphilis. Figure 5As shown in Figure 5, the experimental results were as follows: the area under the curve of SKR3 in cerebrospinal fluid (AUC) (0.8044 [95% CI 0.6695-0.9394]), P = 0.0010, which was considered statistically significant (see Figure 5 (a)), and the sensitivity was 80.00; the area under the curve of SKR3 in serum (AUC) (0.6000 [95% CI 0.4215-0.7785]), P = 0.2786, which was not significant (see Figure 5 (a)). Figure 5 (b) Figure). The above results indicate that latent syphilis and asymptomatic neurosyphilis can be distinguished well based on the level of SKR3 in the cerebrospinal fluid of patients with neurosyphilis.

[0069] (5) SKR3 levels can indicate damage indicators in cerebrospinal fluid

[0070] All syphilis patients were divided into two groups according to the cut-off value of SKR3 level in the patient's cerebrospinal fluid (3.404NPX value). Figure 6 As shown, the cerebrospinal fluid protein associated with cerebrospinal fluid injury in patients with cerebrospinal fluid SKR3 higher than 3.404NPX value (see Figure 6 (a)) and white blood cells (see Figure 6 (b) Figure) significantly increased. Therefore, patients with CSF SKR3 values ​​below 3.404NPX have a low risk of neurosyphilis and a small risk of nerve damage; while those with CSF values ​​above this value have a relatively high risk of neurosyphilis and an increased risk of nerve damage.

[0071] Furthermore, all syphilis patients were divided into two groups according to the cut-off value of SKR3 level in the patients' cerebrospinal fluid (3.731NPX value). Figure 7 As shown, the cerebrospinal fluid protein associated with cerebrospinal fluid injury in patients with cerebrospinal fluid SKR3 higher than 3.731NPX value (see Figure 7 (a)) and white blood cells (see Figure 7 (b) Figure) significantly increased. Therefore, patients with CSF SKR3 values ​​below 3.731NPX have a low risk of symptomatic neurosyphilis and a small risk of nerve damage; while those with values ​​above this value have a high risk of symptomatic neurosyphilis and a significantly increased risk of nerve damage.

[0072] Then, all syphilis patients were divided into two groups according to the cut-off value of SKR3 level in the patients’ cerebrospinal fluid (3.256NPX value). Figure 8 As shown, the cerebrospinal fluid protein associated with cerebrospinal fluid injury in patients with cerebrospinal fluid SKR3 values ​​higher than 3.256NPX (see Figure 8 (a)) increased only slightly, only the white blood cells increased significantly (see Figure 8(b) Figure). Therefore, patients with CSF SKR3 values ​​below 3.256NPX have a low risk of asymptomatic neurosyphilis and a small risk of nerve damage, while those with CSF values ​​above this value have a high risk of asymptomatic neurosyphilis and a significantly increased risk of nerve damage.

[0073] In summary, the concentration of SKR3 protein in cerebrospinal fluid can be used as an indicator for the early diagnosis of neurosyphilis, the typing of symptomatic neurosyphilis, asymptomatic neurosyphilis, neurosyphilis and latent syphilis, and the 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.

[0074] 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. SKR3 is used as a serum or cerebrospinal fluid marker for early diagnosis, screening and typing of patients with neurosyphilis.

2. Use of SKR3 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 SKR3 protein or its specific antibody as the target molecule.

3. The use according to claim 2, characterized in that: The kit uses SKR3 protein or its specific antibody as a target molecule to detect the expression level of SKR3 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 that can specifically recognize SKR3, a polypeptide dye, magnetic beads and a biotin-labeled secondary antibody.

6. The use according to claim 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 SKR3 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; the product contains an antibody that specifically binds to SKR3.

8. A syphilis detection kit, characterized in that: include: A first detection reagent composition for detecting serum or cerebrospinal fluid SKR3 protein levels; and a second detection reagent composition for detecting cerebrospinal fluid leukocytes and / or cerebrospinal fluid proteins.

9. A neurosyphilis diagnostic system, characterized in that: include: A first indicator analysis unit and a second analysis unit; the first analysis unit is an antibody chip or an antibody probe for detecting the level of SKR3 protein in serum or cerebrospinal fluid; The second analysis unit is a detection reagent composition or an analyzer for detecting the level of leukocytes or total protein in cerebrospinal fluid.

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 cerebrospinal fluid SKR3; the judgment module includes a readable carrier storing judgment rules; The judgment rule is: when the cerebrospinal fluid SKR3 concentration value obtained by the detection module is ≥ 3.731NPX value, it is judged as a high risk of nerve damage; When the detection module obtains a value of 3.731NPX>cerebrospinal fluid SKR3 concentration value ≥ 3.404NPX, it is judged as a moderate risk of nerve injury; conversely, when the value of 3.256NPX<cerebrospinal fluid SKR3 concentration value<3.404NPX, it is judged as a low risk of nerve injury; When the cerebrospinal fluid SKR3 concentration value obtained by the detection module is ≥3.256NPX value, it is judged that there is a risk of cerebrospinal fluid infection with syphilis; otherwise, it is judged that there is no or low risk of cerebrospinal fluid infection with syphilis.

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