Application of GFR-alpha-1 protein and specific antibody thereof in preparation of neurosyphilis or nerve injury diagnosis product

Abstract

The invention relates to the technical field of biological medicines, in particular to GFR-alpha-1 serving as a serum marker or cerebrospinal fluid marker for early diagnosis, screening and typing detection of neurosyphilis patients. According to the invention, the GFR-alpha-1 protein level in serum or cerebrospinal fluid is used as an index for diagnosing the neurosyphilis and evaluating the nerve injury degree, and the index can be independently used or used as a main reference index to be combined with other indexes to finish the diagnosis of the neurosyphilis and the evaluation of the nerve injury degree. Compared with the prior art, the GFR-alpha-1 is not only high in sensitivity, but also capable of diagnosing the asymptomatic neurosyphilis or judging whether the risk that the cerebrospinal fluid is infected with the syphilis exists or not from the neurosyphilis through noninvasive serum detection, and screening and management of the asymptomatic neurosyphilis in community hospitals and local hospitals are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of GFR-alpha-1 protein and its specific antibody in the preparation of products for diagnosing neurosyphilis and evaluating the degree of nerve damage. Background Art

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

[0003] 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 CSF cell count or protein level. When CSF-RPR is negative, if there are clinical signs of neurosyphilis, positive plasma tests and abnormal CSF 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. Increased leukocytosis and increased protein content in the cerebrospinal fluid indicate the destruction of the blood-brain barrier and the presence of inflammatory reactions in the skull, which plays an auxiliary role in the diagnosis of neurosyphilis. However, during the diagnosis process, it was found that the sensitivity of CSF-RPP was only 30%-70%, which is low, and it is not sensitive for 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. It is difficult to diagnose neurosyphilis patients who have not yet developed symptoms early. This is mainly due to the lack of sensitive, reliable and effective cerebrospinal fluid and serum biological markers that can distinguish neurosyphilis. What is even more lacking is biological markers in serum that can effectively distinguish neurosyphilis.

[0004] In summary, the existing diagnostic methods for neurosyphilis require invasive cerebrospinal fluid operations and there are no sensitive and accurate markers. Therefore, the workload of syphilis diagnosis is large and complicated, and there are great challenges. Therefore, the development of new biomarkers in serum that can be used for the diagnosis of neurosyphilis is of great significance for the diagnosis and management of neurosyphilis.

[0005] GFR-alpha-1 is a protein that belongs to the glial cell line-derived neurotrophic factor receptor (GDNFR) family. The encoded precursor protein is processed by proteases to generate mature receptors. GFR-alpha-1 is a cell surface receptor for GDNF and (neurturin), two neurotrophic factors that play a key role in controlling neuronal survival and differentiation. After binding to NTN and GDNF, GFR-alpha-1 exerts biological functions of cell growth, differentiation and survival by activating the RET tyrosine kinase receptor. However, there have been no reports of any association between GFR-alpha-1 and the diagnosis of neurosyphilis. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the low sensitivity of the existing CSF-RPR diagnostic method, which is even insensitive to the diagnosis of early asymptomatic neurosyphilis, relies on invasive cerebrospinal fluid examination, and has a large and complicated diagnostic workload, 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 not only has high sensitivity and can greatly simplify the diagnosis of neurosyphilis, but also can be used for non-invasive serum detection of neurosyphilis.

[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 GFR-alpha-1 as a serum marker or cerebrospinal fluid marker for early diagnosis, screening and typing of patients with neurosyphilis.

[0011] In a second aspect, the present invention provides the use of GFR-alpha-1 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 GFR-alpha-1 or its specific antibodies as target molecules.

[0012] According to a preferred embodiment of the present invention, the kit uses GFR-alpha-1 protein or its specific antibody as a target molecule to detect the expression level of GFR-alpha-1 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 for detecting GFR-alpha-1.

[0014] According to a preferred embodiment of the present invention, the kit comprises: a primary antibody that can specifically recognize GFR-alpha-1, a polypeptide dye, magnetic beads, and a biotin-labeled secondary antibody.

[0015] The kit comprises: a primary antibody capable of specifically recognizing GFR-alpha-1, a polypeptide dye, magnetic beads and a biotin-labeled secondary antibody.

[0016] 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.

[0017] It should be noted that when detecting the expression level of GFR-alpha-1 protein in serum, the Olink ultra-high sensitivity plasma protein detection platform can also be used to quantify the expression level of GFR-alpha-1.

[0018] 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 is capable of quantitatively detecting the concentration of GFR-alpha-1 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.

[0019] Preferably, the product comprises antibodies that specifically bind to GFR-alpha-1 and other reagents, as long as the concentration of GFR-alpha-1 protein in serum or cerebrospinal fluid can be quantitatively detected. 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 GFR-alpha-1 protein.

[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 serum or cerebrospinal fluid GFR-alpha-1; the judgment module comprises a readable carrier storing a judgment rule;

[0021] The judgment rule is: when the detection module detects that the cerebrospinal fluid GFR-alpha-1 concentration value is ≥4.866NPX, it is judged that there is a risk of cerebrospinal fluid infection with syphilis;

[0022] When the detection module detects that the cerebrospinal fluid GFR-alpha-1 concentration is less than 4.866NPX, it is judged that there is no risk of cerebrospinal fluid infection with syphilis;

[0023] When the detection module detects 4.866NPX<cerebrospinal fluid GFR-alpha-1<4.880NPX, it is judged as a moderate risk of neurological injury;

[0024] When the detection module detects that the cerebrospinal fluid GFR-alpha-1 concentration value is ≥4.880NPX value, it is judged as a high risk of neurological damage;

[0025] When the detection module detects that the serum GFR-alpha-1 concentration value is ≥5.185NPX, the risk of nerve damage is judged to be extremely high.

[0026] Although the expression level of specific proteins in cerebrospinal fluid is a sensitive indicator of pathological changes in neural tissue, it is applicable to a narrow range of people and requires invasive operations with certain potential risks, which is relatively traumatic for patients and generally requires hospitalization for the operation. The inventors have found that the increase of GFR-alpha-1 protein in serum can also be used as an indicator of increased GFR-alpha-1 protein in cerebrospinal fluid of asymptomatic neurosyphilis, which can facilitate the diagnosis of asymptomatic neurosyphilis from neurosyphilis or determine whether there is a risk of syphilis infection in cerebrospinal fluid through non-invasive serum testing.

[0027] In a fifth aspect, the present invention also provides a syphilis detection kit, which comprises: a first detection reagent composition for detecting serum or cerebrospinal fluid GFR-alpha-1 protein levels; and a second detection reagent composition for detecting cerebrospinal fluid leukocytes and / or cerebrospinal fluid proteins.

[0028] In the above scheme, when diagnosing neurosyphilis (including early diagnosis, typing and staging, and neurological damage assessment), the serum or cerebrospinal fluid GFR-alpha-1 protein level can be combined with cerebrospinal fluid leukocytes and / or cerebrospinal fluid proteins associated with neurological damage in patients with neurosyphilis for early diagnosis, typing and staging, and neurological damage assessment of neurosyphilis.

[0029] Among them, the level of GFR-alpha-1 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.

[0030] (III) Beneficial effects

[0031] The present invention provides a new cerebrospinal fluid / serum marker GFR-alpha-1 for early diagnosis of neurosyphilis and assessment of nerve damage. By detecting the expression level of GFR-alpha-1 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 GFR-alpha-1 in the patient's serum or cerebrospinal fluid, the diagnosis of neurosyphilis can be achieved. Compared with the existing technology, it is not only more sensitive, but also can achieve non-invasive serum screening for asymptomatic neurosyphilis. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0033] Figure 2The results showed that there was no significant correlation between the expression of GFR-alpha-1 in cerebrospinal fluid and serum.

[0034] Figure 3 The results showed that the expression of GFR-alpha-1 in cerebrospinal fluid was positively correlated with cerebrospinal fluid protein, and there was no correlation between the expression of GFR-alpha-1 in cerebrospinal fluid and the number of cerebrospinal fluid leukocytes.

[0035] Figure 4 The expression of GFR-alpha-1 in serum was positively correlated with cerebrospinal fluid protein and leukocytes.

[0036] Figure 5 The AUC is the probability of evaluating the diagnosis of neurosyphilis at different cerebrospinal fluid and serum GFR-alpha-1 concentration levels when the diagnosis of neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) is the clinical outcome in syphilis patients; the table below the figure shows the cut-off value, sensitivity, and specificity of the corresponding indicators.

[0037] Figure 6 This is the AUC for the possibility of different cerebrospinal fluid and serum GFR-alpha-1 levels in evaluating the diagnosis of symptomatic neurosyphilis in patients with neurosyphilis when the diagnosis of symptomatic neurosyphilis is the clinical outcome; the table below the figure shows the cut-off value, sensitivity, and specificity of the corresponding indicators.

[0038] Figure 7 This is the AUC for evaluating the possibility of a confirmed diagnosis of asymptomatic neurosyphilis at different levels of cerebrospinal fluid and serum GFR-alpha-1 concentrations when asymptomatic neurosyphilis is the clinical outcome in patients with asymptomatic neurosyphilis and latent syphilis; the table below the figure shows the cut-off value, sensitivity, and specificity of the corresponding indicators.

[0039] Figure 8 To distinguish all patients (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups based on the CSF GFR-alpha-1 cutoff value of 4.880NPX, and to compare the differences in the neurological damage indicators CSF protein and leukocytes in each group.

[0040] Fig. 9 To divide patients with latent syphilis and asymptomatic neurosyphilis into two groups based on the cerebrospinal fluid GFR-alpha-1 cutoff value of 4.866NPX, and to compare the differences in cerebrospinal fluid protein and leukocytes, the indicators of neurological damage, between the groups. DETAILED DESCRIPTION

[0041] In order to better explain the present invention, the present invention is now described in detail.

[0042] 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. Currently, the diagnosis of neurosyphilis mainly relies on the examination of cerebrospinal fluid. Therefore, the inventors conducted research with the purpose of developing new biomarkers in serum that can be used for the diagnosis of neurosyphilis and nerve damage, and obtained GFR-alpha-1, which has significant diagnostic value in both serum and cerebrospinal fluid.

[0043] GFR-alpha-1, also known as glial cell line-derived neurotrophic factor (GDNF) family receptor alpha-1, is a cell surface protein that is part of the GDNF family receptor. After binding to GDNF, GFR-alpha-1 activates the RET tyrosine kinase receptor to exert biological functions of cell growth, differentiation and survival.

[0044] After research, the inventors found that there were significant differences in the levels of GFR-alpha-1 protein in the cerebrospinal fluid and serum of patients with different types of syphilis. The cerebrospinal fluid GFR-alpha-1 protein was significantly higher in patients with neurosyphilis (including asymptomatic and symptomatic neurosyphilis patients) than in patients with latent syphilis, and was positively correlated with the cerebrospinal fluid protein, an indicator of nerve damage, indicating that the cerebrospinal fluid GFR-alpha-1 protein can be used for the diagnosis of neurosyphilis and can also indicate the severity of nerve damage. The serum GFR-alpha-1 protein in patients with symptomatic neurosyphilis was significantly increased compared with that in patients with asymptomatic neurosyphilis and latent syphilis, but there was no significant difference between patients with asymptomatic neurosyphilis and latent syphilis, and was positively correlated with the cerebrospinal fluid protein, an indicator of nerve damage, and cerebrospinal fluid leukocytes. Therefore, the serum GFR-alpha-1 protein only increased in the symptomatic neurosyphilis stage, and did not increase in the early stage of asymptomatic neurosyphilis. At the same time, the level of GFR-alpha-1 protein in the cerebrospinal fluid of patients with latent syphilis was significantly lower than that of patients with asymptomatic neurosyphilis and symptomatic neurosyphilis, indicating that the level of GFR-alpha-1 protein in the cerebrospinal fluid changed in the early stage of neurosyphilis, that is, the asymptomatic neurosyphilis stage. Therefore, the level of GFR-alpha-1 protein in the cerebrospinal fluid can effectively distinguish asymptomatic neurosyphilis from latent syphilis, and the level of serum GFR-alpha-1 protein can help non-invasively screen for asymptomatic neurosyphilis in neurosyphilis, which can solve the clinical problem that all syphilis patients must undergo invasive procedures (lumbar puncture) to extract cerebrospinal fluid for screening of asymptomatic neurosyphilis.

[0045] In order to clarify the relationship between serum and cerebrospinal fluid GFR-alpha-1 and neurosyphilis, this example studied 88 syphilis patients (15 cases of latent syphilis, 30 cases of asymptomatic neurosyphilis, and 43 cases of paralytic dementia). During the study, the experiment first used the receiver operating characteristic curve (ROC) to evaluate the diagnostic potential of GFR-alpha-1 protein for neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis); the results were: the area under the curve (AUC) of cerebrospinal fluid GFR-alpha-1 was 0.7406, P = 0.0035, which can be considered statistically significant; the AUC of serum GFR-alpha-1 was 0.6356, P = 0.0994, which can be considered statistically insignificant. Accordingly, the level of GFR-alpha-1 in the cerebrospinal fluid of syphilis patients can better evaluate the occurrence of neurosyphilis, and the sensitivity is 80%.

[0046] Next, in order to clarify the relationship between serum and cerebrospinal fluid GFR-alpha-1 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 potential of GFR-alpha-1 protein level for the occurrence of symptomatic neurosyphilis in patients with neurosyphilis (including symptomatic and asymptomatic neurosyphilis patients); the results were: the AUC of cerebrospinal fluid GFR-alpha-1 was 0.5760, P=0.2719, which was considered to be insignificant, and the sensitivity was 56.67%; the AUC of serum GFR-alpha-1 was 0.7876, P<0.0001, which was considered to be statistically significant, and the sensitivity was 80%. The above results show that the occurrence of symptomatic neurosyphilis can be better evaluated according to the level of serum GFR-alpha-1 in patients with neurosyphilis, with a sensitivity of 80.00, while the diagnostic effect of cerebrospinal fluid GFR-alpha-1 protein level is not good.

[0047] Furthermore, asymptomatic neurosyphilis is a difficult point in clinical diagnosis and treatment. All syphilis patients need to undergo invasive cerebrospinal fluid screening. In fact, some patients have asymptomatic neurosyphilis, but it is difficult to diagnose because there are no clinical symptoms. Therefore, all syphilis patients need to be screened. In order to clarify the relationship between serum and cerebrospinal fluid GFR-alpha-1 protein levels and asymptomatic neurosyphilis, 45 syphilis patients (15 latent syphilis patients and 30 asymptomatic neurosyphilis patients) were studied in the following examples, and ROC was used to evaluate the diagnostic potential of cerebrospinal fluid or serum GFR-alpha-1 protein levels to evaluate the occurrence of asymptomatic neurosyphilis; the results of the study were: the AUC of cerebrospinal fluid GFR-alpha-1 was 0.7267, P=0.0141, which was considered statistically significant; the AUC of serum GFR-alpha-1 was 0.5200, P=0.8284, which was considered not statistically significant. The above results show that the occurrence of asymptomatic neurosyphilis can be better evaluated according to the level of GFR-alpha-1 in the cerebrospinal fluid of syphilis patients, with a sensitivity of 80.00%. However, whether the serum GFR-alpha-1 protein level can diagnose asymptomatic neurosyphilis requires verification with more samples.

[0048] Furthermore, the present invention divides all syphilis patients (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups according to the cerebrospinal fluid GFR-alpha-1 Cut-Off value (4.880NPX). Patients with a value greater than or equal to this value have a significant increase in cerebrospinal fluid protein, an indicator related to cerebrospinal fluid damage, and a trend of increasing white blood cells, but without statistical significance. Therefore, when the GFR-alpha-1 cerebrospinal fluid is lower than the value of 4.880NPX, patients with neurosyphilis have a lower risk of developing neurosyphilis and a lower risk of nerve damage; while patients with a value greater than or equal to this value have an increased risk of neurosyphilis and an increased risk of nerve damage. The present invention further divides latent syphilis and asymptomatic neurosyphilis patients into two groups according to the cerebrospinal fluid GFR-alpha-1 Cut-Off value (4.866NPX). Patients with a value greater than or equal to 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 CSF GFR-alpha-1 is lower than 4.866NPX, the risk of asymptomatic neurosyphilis and nerve damage in syphilis patients is lower; while when the CSF GFR-alpha-1 is greater than or equal to 4.866NPX, the risk of asymptomatic neurosyphilis and nerve damage in syphilis patients increases.

[0049] Similarly, the present invention divides neurosyphilis patients (asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups according to the Cut-Off value (5.185NPX) of the serum GFR-alpha-1 of syphilis patients. The cerebrospinal fluid leukocytes, an indicator related to cerebrospinal fluid damage, of patients with a value greater than or equal to 5.185NPX, are significantly increased. Therefore, when the patient's serum GFR-alpha-1 is lower than the value of 5.185NPX, the risk of symptomatic neurosyphilis in neurosyphilis patients is lower, and the risk of nerve damage is lower; and when the patient's serum GFR-alpha-1 is greater than or equal to 5.185NPX, the patient's risk of symptomatic neurosyphilis increases, and the risk of nerve damage is extremely high. Therefore, serum GFR-alpha-1 can be used as an indicator of the diagnosis of asymptomatic neurosyphilis and symptomatic neurosyphilis and nerve damage.

[0050] In summary, the present invention provides the application of GFR-alpha-1 as a diagnostic tool for neurosyphilis and assessment of nerve damage. The GFR-alpha-1 protein level in serum and cerebrospinal fluid can be used as a reliable and effective primary reference indicator for syphilis diagnosis and nerve damage assessment, and can also be used as an auxiliary reference indicator for syphilis diagnosis and nerve damage assessment. Based on the above findings, by detecting the protein expression level of GFR-alpha-1 in serum or cerebrospinal fluid, it is possible to detect syphilis, especially neurosyphilis, and to perform early non-invasive screening for asymptomatic neurosyphilis, and to accurately quantify and evaluate nerve damage, the 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 GFR-alpha-1 concentration in serum and cerebrospinal fluid using the Olink method

[0058] The Olink neurology panel (95801) was used to detect 92 proteins related to neurological function, including GFR-alpha-1 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 GFR-alpha-1 protein has significant differences in cerebrospinal fluid and serum. The detection of cerebrospinal fluid protein and cerebrospinal fluid leukocytes was performed according to the method described in WS273-2007.

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

[0060] (1) GFR-alpha-1 protein significantly increases in cerebrospinal fluid and serum in neurosyphilis

[0061] Olink precision proteomics was used to detect the difference in the expression of nerve-related proteins in serum and cerebrospinal fluid, and GFR-alpha-1 protein with significant differences in both cerebrospinal fluid and serum was screened. Figure 1 As shown in Table 1. It was found that the cerebrospinal fluid GFR-alpha-1 protein level in patients with symptomatic neurosyphilis (symptomatic) and asymptomatic neurosyphilis (asymptomatic) was significantly higher than that in patients with latent syphilis (e.g. Figure 1 (a) It was also found that the level of GFR-alpha-1 protein in the serum of patients with symptomatic neurosyphilis was significantly higher than that in patients with asymptomatic neurosyphilis and latent syphilis. Therefore, the protein levels of GFR-alpha-1 in cerebrospinal fluid and serum are expected to be used for the diagnosis of neurosyphilis and indicate the severity of nerve damage.

[0062] Figure 1 In the syphilis, latent syphilis refers to non-neurosyphilis, asymptomatic refers to asymptomatic neurosyphilis, and symptomatic refers to paralytic dementia, i.e., symptomatic neurosyphilis. Figure 1 As can be seen from Figure (a), the concentration of GFR-alpha-1 in the cerebrospinal fluid of patients with symptomatic neurosyphilis and the concentration of GFR-alpha-1 in the cerebrospinal fluid of patients with asymptomatic neurosyphilis are greater than the concentration of GFR-alpha-1 in the cerebrospinal fluid of patients with latent syphilis; Figure 1 As shown in Figure (b), the GFR-alpha-1 concentration in the serum of patients with symptomatic neurosyphilis is greater than the GFR-alpha-1 concentration in the serum of patients with asymptomatic neurosyphilis and latent syphilis.

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

[0064] In the table: Latent syphilis & asymptomatic represents the statistical value of the difference in GFR-alpha-1 concentration between latent syphilis and asymptomatic neurosyphilis in cerebrospinal fluid or serum. Latent syphilis & symptomatic represents the statistical value of the difference in GFR-alpha-1 concentration between latent syphilis and symptomatic neurosyphilis in cerebrospinal fluid or serum. Asymptomatic & symptomatic represents the statistical value of the difference in GFR-alpha-1 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 serum and cerebrospinal fluid GFR-alpha-1 protein levels in patients with different types of syphilis, and the differences are statistically significant.

[0065] (2) There was no significant correlation between GFR-alpha-1 in cerebrospinal fluid and serum

[0066] Combination Figure 2 It can be seen that there is no significant correlation between the expression level of GFR-alpha-1 protein in cerebrospinal fluid and the expression level of this protein in serum (correlation coefficient R=0.007291, p=0.9462). Therefore, the GFR-alpha-1 protein in serum cannot indicate the abnormality of the GFR-alpha-1 protein level in cerebrospinal fluid.

[0067] (3) GFR-alpha-1 in cerebrospinal fluid is positively correlated with injury indicators in cerebrospinal fluid

[0068] 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.

[0069] Combination Figure 3 As shown in Figures (a) and (b), the expression level of GFR-alpha-1 protein in cerebrospinal fluid is significantly positively correlated with the protein in the patient's cerebrospinal fluid (correlation coefficient R=0.3993, p=0.0001), and has no significant correlation with the number of white blood cells (correlation coefficient R=0.1656, p=0.1231). Therefore, the level of GFR-alpha-1 protein in cerebrospinal fluid can indicate the abnormality of protein in cerebrospinal fluid.

[0070] Therefore, in practice, the cerebrospinal fluid protein level can be used as a reference indicator, together with the GFR-alpha-1 protein level in the cerebrospinal fluid as a quantitative assessment indicator for the diagnosis of neurosyphilis and the degree of nerve damage.

[0071] (4) Serum GFR-alpha-1 is positively correlated with injury indicators in cerebrospinal fluid

[0072] Combination Figure 4 As shown in Figures (a) and (b), the expression level of GFR-alpha-1 protein in serum is positively correlated with the protein in the patient's cerebrospinal fluid (correlation coefficient R=0.3934, p=0.0001) and the number of white blood cells (correlation coefficient R=0.2858, p=0.0070). Therefore, GFR-alpha-1 protein in serum can indicate abnormalities in protein and white blood cells in cerebrospinal fluid.

[0073] (5) Evaluation of cerebrospinal fluid and serum GFR-alpha-1 in neurosyphilis detection in syphilis patients

[0074] A total of 88 patients with syphilis were studied, and the receiver operating characteristic (ROC) curve was used to evaluate the diagnostic potential of GFR-alpha-1 protein levels for neurosyphilis. Figure 5 As shown in Figure 5, the results of the study were as follows: the area under the curve of cerebrospinal fluid GFR-alpha-1 was AUC 0.7406, P = 0.0035, which was considered statistically significant (see Figure 5 (a)), with a sensitivity of 80%; the area under the curve of serum GFR-alpha-1 was AUC 0.6356, P = 0.0994, which was considered not statistically significant (see Figure 5 (a)). Figure 5(b)), the sensitivity is only 60.00%. This shows that the level of GFR-alpha-1 in the cerebrospinal fluid of syphilis patients can be used to assist in the diagnosis of neurosyphilis with high sensitivity.

[0075] Furthermore, 73 patients with neurosyphilis were studied. ROC was used to evaluate the diagnostic potential of GFR-alpha-1 protein level in symptomatic neurosyphilis. Figure 6 As shown in the results, the area under the curve of cerebrospinal fluid GFR-alpha-1 was AUC 0.5760, P = 0.2719, which can be considered to be insignificant (see Figure 6 (a)), the sensitivity was only 56.67%; the area under the curve of serum GFR-alpha-1 was AUC 0.7876, P < 0.0001, which was statistically significant (see Figure 6 The above results show that the occurrence of symptomatic neurosyphilis can be better evaluated according to the level of serum GFR-alpha-1 in patients with neurosyphilis.

[0076] Next, a further 45 syphilis patients (including 15 latent syphilis patients and 30 asymptomatic neurosyphilis patients) were studied, and the ROC was used to evaluate the diagnostic potential of GFR-alpha-1 protein levels for asymptomatic neurosyphilis. Figure 7 As shown in the results, the area under the curve of cerebrospinal fluid GFR-alpha-1 was AUC 0.7267, P = 0.0141, which can be considered statistically significant (see Figure 7 (a) Figure), and the sensitivity was 80.00; the area under the curve of serum GFR-alpha-1 was AUC 0.5200, P=0.8284, which was not statistically significant (see Figure 7 The above results indicate that the occurrence of asymptomatic neurosyphilis can be better evaluated based on the level of GFR-alpha-1 in the cerebrospinal fluid of patients with neurosyphilis.

[0077] (8) GFR-alpha-1 levels can indicate damage indicators in cerebrospinal fluid

[0078] All syphilis patients were divided into two groups according to the cut-off value of the patient's cerebrospinal fluid GFR-alpha-1 level (4.880 NPX value). Figure 8 As shown, patients with CSF GFR-alpha-1 concentrations greater than or equal to 4.880 NPX values ​​had significantly increased CSF protein, an indicator of CSF damage (see Figure 8 (a)), there was a trend of increasing white blood cells with no statistical difference (see Figure 8(b) Figure). Therefore, patients with CSF GFR-alpha-1 concentrations below the NPX value of 4.880 have a low risk of neurosyphilis and a small risk of nerve damage, while patients with CSF GFR-alpha-1 concentrations above this value have a relatively high risk of neurosyphilis and an increased risk of nerve damage.

[0079] Then, the patients with latent syphilis and asymptomatic neurosyphilis were divided into two groups according to the cut-off value of the cerebrospinal fluid GFR-alpha-1 level (4.866 NPX value). Fig. 9 As shown, the CSF GFR-alpha-1 concentration was greater than or equal to 4.866NPX in patients with CSF injury-related indicators of CSF protein (see Fig. 9 (a)) significantly increased, and the white blood cells also increased but not significantly (see Fig. 9 (b) Figure). Therefore, patients with CSF GFR-alpha-1 concentrations below 4.866NPX have a low risk of asymptomatic neurosyphilis and a small risk of nerve damage, and can be diagnosed with latent syphilis; while patients with CSF GFR-alpha-1 concentrations above this value have a high risk of asymptomatic neurosyphilis and a significantly increased risk of nerve damage.

[0080] In summary, the concentration of GFR-alpha-1 protein in serum or cerebrospinal fluid can be used as an indicator for early diagnosis of neurosyphilis, typing of symptomatic neurosyphilis, asymptomatic neurosyphilis, neurosyphilis and latent syphilis, and evaluation of the degree of nerve damage. It can be used alone or as a main reference indicator in combination with other indicators to complete the diagnosis of syphilis and evaluation of nerve damage.

[0081] 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. Use of GFR-alpha-1 protein or its specific antibody in the preparation of diagnostic products for neurosyphilis screening, typing detection or assessment of nerve damage, characterized in that: The diagnostic product is a detection kit, antibody chip, antibody probe or detector using GFR-alpha-1 or its specific antibody as the target molecule.

2. The use according to claim 1, characterized in that: The kit uses GFR-alpha-1 protein or its specific antibody as a target molecule to detect the expression level of GFR-alpha-1 protein or its specific antibody in cerebrospinal fluid or serum.

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

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

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

6. 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 GFR-alpha-1 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.

7. A syphilis detection kit, characterized in that: include: A first detection reagent composition for detecting serum or cerebrospinal fluid GFR-alpha-1 protein levels; and a second detection reagent composition for detecting cerebrospinal fluid leukocytes and / or cerebrospinal fluid proteins.

8. 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 serum or cerebrospinal fluid GFR-alpha-1; the judgment module includes a readable carrier storing judgment rules; The judgment rule is: when the detection module detects that the cerebrospinal fluid GFR-alpha-1 concentration value is ≥4.866NPX, it is judged that there is a risk of cerebrospinal fluid infection with syphilis; When the detection module detects that the cerebrospinal fluid GFR-alpha-1 concentration is less than 4.866NPX, it is judged that there is no risk of cerebrospinal fluid infection with syphilis; When the detection module detects 4.866NPX<cerebrospinal fluid GFR-alpha-1<4.880NPX, it is judged as a moderate risk of neurological injury; When the detection module detects that the cerebrospinal fluid GFR-alpha-1 concentration value is ≥4.880NPX value, it is judged as a high risk of neurological damage; When the detection module detects that the serum GFR-alpha-1 concentration value is ≥5.185NPX, the risk of nerve damage is judged to be extremely high.

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