Application of cerebrospinal fluid BMP-4 protein and its specific antibody in the preparation of diagnostic products for neurosyphilis or nerve damage

Abstract

The present invention relates to the field of biomedicine technology, and specifically to cerebrospinal fluid BMP-4 protein as a serum marker or cerebrospinal fluid marker for early diagnosis, screening and typing detection of patients with neurosyphilis. The present invention uses the concentration of BMP-4 protein in cerebrospinal fluid as an indicator for early diagnosis of neurosyphilis and evaluation of the degree of nerve damage. This indicator can be used alone or as a main reference indicator in combination with other indicators to complete the diagnosis of neurosyphilis and evaluation of nerve damage. Compared with the prior art, BMP-4 can highly sensitively indicate the degree of nerve damage, can accurately screen patients with neurosyphilis who do not show symptoms, and is conducive to medical staff to accurately carry out diagnosis and management of neurosyphilis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of cerebrospinal fluid BMP-4 protein and its specific antibody in the preparation of neurosyphilis or nerve damage diagnosis products. 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. During current clinical diagnosis and treatment, neurosyphilis is mainly diagnosed and treated in patients who have already developed symptoms of neurosyphilis. It is difficult to conduct early screening for patients with neurosyphilis who have not yet developed symptoms.

[0004] In summary, the existing diagnostic methods for neurosyphilis require a combined diagnosis of multiple indicators, which is labor-intensive and complicated. Therefore, the development of new biomarkers for the diagnosis of neurosyphilis and for indicating nerve damage is of great significance for the diagnosis and management of neurosyphilis. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the technical problems such as low sensitivity of existing CSF-RPR diagnostic methods, insensitivity to the diagnosis of early asymptomatic neurosyphilis, large and complicated diagnostic workload, etc., the present invention provides BMP-4 protein as a cerebrospinal fluid marker for early diagnosis screening, typing detection and nerve damage assessment of neurosyphilis. The use of this marker can greatly simplify the diagnosis of neurosyphilis and facilitate the timely diagnosis and management of neurosyphilis.

[0007] (II) Technical solution

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

[0009] In a first aspect, the present invention relates to the use of cerebrospinal fluid BMP-4 protein and its specific antibody in the preparation of neurosyphilis or nerve damage diagnosis products.

[0010] In a second aspect, the present invention provides the use of BMP-4 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 products or auxiliary diagnostic products are detection kits, antibody chips, antibody probes or detectors with BMP-4 or its specific antibodies as target molecules.

[0011] According to a preferred embodiment of the present invention, the kit uses BMP-4 protein or its specific antibody as the detection target molecule to detect the expression level of BMP-4 protein or its specific antibody in cerebrospinal fluid.

[0012] According to a preferred embodiment of the present invention, the kit is an enzyme-linked immunosorbent assay kit or an immunohistochemistry kit for detecting BMP-4.

[0013] According to a preferred embodiment of the present invention, the kit comprises: a primary antibody that can specifically recognize BMP-4, a polypeptide dye, magnetic beads, and a biotin-labeled secondary antibody.

[0014] The kit comprises: a primary antibody capable of specifically recognizing BMP-4, a polypeptide dye, magnetic beads and a biotin-labeled secondary antibody.

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

[0016] It should be noted that when detecting the expression level of BMP-4 protein in cerebrospinal fluid, the Olink ultra-high sensitivity plasma protein detection platform can also be used to quantify the expression level of BMP-4.

[0017] In a third aspect, the present invention also relates to a product for early diagnosis, screening, typing detection or assessment of the degree of nerve damage in patients with neurosyphilis; the product can quantitatively detect the concentration of BMP-4 in the patient's ex vivo 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 BMP-4 and other reagents, as long as the concentration of BMP-4 protein in 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 BMP-4 protein.

[0019] In a fourth aspect, the present invention relates to an assessment system for the risk of nerve damage in patients with neurosyphilis, comprising a detection module and a judgment module, wherein the detection module is used to obtain the concentration value of BMP-4 in cerebrospinal fluid; the judgment module comprises a readable carrier storing a judgment rule;

[0020] The judgment rule is: when the detection module detects that the concentration of BMP-4 in the cerebrospinal fluid is ≥ 2.858NPX, it is judged that there is a high risk of neurosyphilis or a high risk of nerve damage;

[0021] When the detection module detects that the cerebrospinal fluid BMP-4 concentration value is less than 2.858NPX, it is judged to have a low risk of neurosyphilis or a low risk of nerve damage.

[0022] The reference value is the cut-off value of BMP-4 obtained from a group of syphilis patients; preferably, the cerebrospinal fluid cut-off value for the diagnosis of neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) is 2.858NPX value, less than this value is judged as a low risk of nerve damage, and greater than or equal to this value is judged as a high risk of nerve damage. The above cut-off value is the critical value of BMP-4 statistical analysis obtained from 88 syphilis patients.

[0023] 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 BMP-4 protein in cerebrospinal fluid; and a second detection reagent composition for detecting cerebrospinal fluid leukocytes and / or cerebrospinal fluid proteins.

[0024] In the above scheme, when diagnosing neurosyphilis (including early diagnosis, typing and staging, and neurological damage assessment), the cerebrospinal fluid BMP-4 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.

[0025] Among them, the level of BMP-4 in cerebrospinal fluid can be determined by Olink precision proteomics, i.e., antibody immunoassay combined with quantitative real-time PCR. The number of cerebrospinal fluid leukocytes and the amount of cerebrospinal fluid protein can be detected by known methods.

[0026] BMP-4, whose full name is bone morphogenetic protein 4, is a member of the transforming growth factor superfamily. It is a glycoprotein with a cysteine ​​knot and exists as a homodimer. It is expressed in both embryonic and adult tissues, synthesized in the endoplasmic reticulum and Golgi apparatus, and undergoes post-translational modification.

[0027] (III) Beneficial effects

[0028] The present invention provides a new cerebrospinal fluid marker BMP-4 for early diagnosis screening, typing detection and nerve damage assessment of neurosyphilis. By detecting the expression level of BMP-4 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 BMP-4 in the patient's cerebrospinal fluid, the diagnosis of neurosyphilis, including early diagnosis and typing detection, can be achieved. Compared with the existing CSF-RPR diagnostic technology, this marker has high sensitivity, and the use of this marker can greatly simplify the diagnosis of neurosyphilis.

[0029] Preferably, the cerebrospinal fluid marker BMP-4 is suitable for the diagnosis and screening of symptomatic neurosyphilis and asymptomatic neurosyphilis, because among symptomatic neurosyphilis, asymptomatic neurosyphilis or non-neurosyphilis, the expression level of BMP-4 in the cerebrospinal fluid of patients with symptomatic neurosyphilis is the highest, and the BMP-4 expression level is highly sensitive in the assessment of neurosyphilis, especially symptomatic neurosyphilis, and also has a predictive effect on asymptomatic neurosyphilis. Therefore, cerebrospinal fluid BMP can be used to screen for neurosyphilis that has not yet shown symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The Olink method was used to detect the expression of BMP-4 protein in the cerebrospinal fluid and serum of three groups of syphilis patients with different types: latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis.

[0031] Figure 2 The results showed that the expression of BMP-4 in cerebrospinal fluid had no significant correlation with that in serum.

[0032] Figure 3 The results showed that the expression of BMP-4 in cerebrospinal fluid was positively correlated with cerebrospinal fluid protein and leukocytes.

[0033] Figure 4 The results showed that the expression of BMP-4 in serum had no correlation with cerebrospinal fluid protein and leukocytes.

[0034] Figure 5 This is the AUC for the possibility of evaluating the diagnosis of neurosyphilis by the cerebrospinal fluid and serum BMP-4 concentration levels when the diagnosis of neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis) is the clinical outcome in syphilis; the table below the figure shows the sensitivity, specificity and cut-off value of the corresponding indicators.

[0035] Figure 6 This is the AUC for evaluating the possibility of a confirmed diagnosis of symptomatic neurosyphilis at different levels of cerebrospinal fluid and serum BMP-4 concentrations when symptomatic neurosyphilis is diagnosed as the clinical outcome in neurosyphilis; the table below the figure shows the sensitivity, specificity and cut-off value of the corresponding indicators.

[0036] Figure 7 This is the AUC for evaluating the possibility of a confirmed diagnosis of asymptomatic neurosyphilis when asymptomatic neurosyphilis is the clinical outcome in asymptomatic neurosyphilis and latent syphilis. The table below the figure shows the sensitivity, specificity and cut-off value of the corresponding indicators.

[0037] Figure 8 To distinguish all patients (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups based on the CSF BMP-4 cutoff value of 2.858NPX, and to compare the differences in the neurological injury indicators CSF protein and leukocytes in each group. DETAILED DESCRIPTION

[0038] In order to better explain the present invention, the present invention is now described in detail.

[0039] 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 that can be used for the diagnosis of neurosyphilis and the assessment of nerve damage, and obtained BMP-4, which has significant diagnostic value in cerebrospinal fluid.

[0040] BMP-4 is a pleiotropic ligand protein that belongs to the TGFβ family. BMP-4 is required for auditory neurogenesis. BMP-4 regulates mesenchymal formation and acts as a key differentiation factor for hematopoietic cells and neurons. BMP-4-based signaling is upregulated in both cells. Frameshift mutations in BMP-4 are associated with developmental abnormalities, especially optic vesicle and finger (toe) mutations and lip morphological defects. However, there are no reports of BMP-4 being associated with the diagnosis of neurosyphilis and nerve damage.

[0041] After research, it was found that the levels of BMP-4 protein in the cerebrospinal fluid of patients with different types of syphilis were significantly different. Compared with the non-neurosyphilis (also referred to as latent syphilis in this application), the BMP-4 protein in the cerebrospinal fluid was significantly increased in patients with neurosyphilis (asymptomatic neurosyphilis and symptomatic neurosyphilis), indicating that the cerebrospinal fluid BMP-4 protein can be used for the diagnosis of neurosyphilis. The cerebrospinal fluid marker BMP-4 was positively correlated with the cerebrospinal fluid protein, a previously reported indicator of nerve damage, suggesting that BMP-4 can indicate the severity of nerve damage. The level of cerebrospinal BMP-4 protein in patients with asymptomatic neurosyphilis was significantly higher than that in the non-neurosyphilis (referred to as latent syphilis in this application) group, indicating that the BMP-4 protein level has changed in the early stage of neurosyphilis, that is, the asymptomatic neurosyphilis stage. Therefore, this biomarker can effectively distinguish between asymptomatic neurosyphilis and latent syphilis, and can be used for screening and intervention in patients with neurosyphilis who have not yet developed symptoms.

[0042] In order to clarify the relationship between serum and cerebrospinal fluid BMP-4 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 receiver operating characteristic curve (ROC) was used to evaluate the diagnostic potential of BMP-4 protein for neurosyphilis (including asymptomatic neurosyphilis and symptomatic neurosyphilis). The results were as follows: the area under the curve (AUC) of cerebrospinal fluid BMP-4 was 0.8064, P = 0.0002, which was considered statistically significant; the AUC of serum BMP-4 was 0.55616, P = 0.4583, which was considered statistically insignificant. Accordingly, the occurrence of neurosyphilis can be better evaluated according to the level of BMP-4 in the cerebrospinal fluid of syphilis patients, with a sensitivity of 80% and a specificity of 72.60%.

[0043] Furthermore, in order to clarify the relationship between serum and cerebrospinal fluid BMP-4 and symptomatic neurosyphilis, the study used ROC to evaluate the diagnostic potential of cerebrospinal fluid BMP-4 for symptomatic neurosyphilis in 73 patients with neurosyphilis (30 cases of asymptomatic neurosyphilis and 43 cases of paralytic dementia). The results were as follows: the AUC of cerebrospinal fluid BMP-4 was 0.6039, P = 0.1330, which was considered to be insignificant, with low sensitivity and specificity; the AUC of serum BMP-4 was 0.5829, P = 0.2303. The above results show that the occurrence of symptomatic neurosyphilis cannot be well evaluated based on the levels of cerebrospinal fluid and serum BMP-4 in patients with neurosyphilis.

[0044] In addition, screening for asymptomatic neurosyphilis is a difficult point in clinical diagnosis and treatment. 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 BMP-4 and asymptomatic neurosyphilis, 45 patients with latent syphilis (15 cases) and asymptomatic neurosyphilis (30 cases) were studied in the following examples, and ROC was used to evaluate the diagnostic potential of BMP-4 for the occurrence of asymptomatic neurosyphilis; the experimental results were: the AUC of cerebrospinal fluid BMP-4 was 0.7578, P = 0.0052, which was considered statistically significant, with a sensitivity of 73.33% and a specificity of 63.33%; the AUC of serum BMP-4 was 0.5067, P = 0.9424. The above results show that the occurrence of asymptomatic neurosyphilis can be evaluated based on the level of BMP-4 in the cerebrospinal fluid of syphilis patients, while serum BMP-4 cannot diagnose the occurrence of asymptomatic neurosyphilis.

[0045] Furthermore, the present invention divides all patients with syphilis (including latent syphilis, asymptomatic neurosyphilis and symptomatic neurosyphilis) into two groups according to the BMP-4 Cut-Off value (2.858NPX) in the cerebrospinal fluid of all patients with syphilis, and the cerebrospinal fluid protein and leukocytes, which are indicators related to cerebrospinal fluid damage, are significantly increased in patients with a value greater than or equal to this value. Therefore, when the BMP-4 cerebrospinal fluid value is lower than 2.858NPX, the risk of neurosyphilis in patients with neurosyphilis is lower, and the risk of nerve damage is lower; while when the value is greater than or equal to this value, the risk of neurosyphilis increases, and the risk of nerve damage also increases. Therefore, cerebrospinal fluid BMP-4 can be used as an indicator for the diagnosis of neurosyphilis and nerve damage.

[0046] Based on the above research results, the present invention provides the application of BMP-4 as a cerebrospinal fluid marker in the diagnosis of neurosyphilis and the assessment of nerve damage. Specifically, the BMP-4 protein level in the cerebrospinal fluid is used as a reliable and effective primary reference indicator or auxiliary reference indicator for the diagnosis of syphilis and the assessment of nerve damage. In medical practice, by detecting the expression level of BMP-4 in the cerebrospinal fluid, it is possible to achieve high-sensitivity detection of syphilis, especially neurosyphilis, and to accurately quantify and evaluate the nerve damage of syphilis patients.

[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] 2. 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] 3. Detection of BMP-4 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 BMP-4 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 it is found that BMP-4 protein has significant differences in cerebrospinal fluid. The detection of cerebrospinal fluid protein and cerebrospinal fluid leukocytes was carried out according to the method described in WS 273-2007.

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

[0056] (1) BMP-4 protein in the cerebrospinal fluid of patients with neurosyphilis is significantly increased

[0057] Olink precision proteomics was used to detect the differences in the expression of nerve-related proteins in cerebrospinal fluid, and BMP-4 protein with significant differences in cerebrospinal fluid was screened. Figure 1 As shown in Table 1. It was found that the BMP-4 level in the cerebrospinal fluid of patients with symptomatic neurosyphilis (symptomatic) and asymptomatic neurosyphilis (asymptomatic) was significantly higher than that of patients with latent syphilis. There was no significant difference in BMP-4 in the serum of the three groups of patients. Therefore, the level of BMP-4 in cerebrospinal fluid can be used for the diagnosis of neurosyphilis.

[0058] Figure 1 In (a), latent syphilis is non-neurosyphilis, asymptomatic means asymptomatic neurosyphilis, and symptomatic means paralytic dementia, i.e. symptomatic neurosyphilis. Figure 1 As can be seen from Figure (a), the concentration of BMP-4 in the cerebrospinal fluid of patients with symptomatic neurosyphilis and asymptomatic neurosyphilis is greater than that in the cerebrospinal fluid of patients with latent syphilis; Figure 1 As shown in (b), there was no significant difference in BMP-4 levels among the three groups of patients with symptomatic neurosyphilis, asymptomatic neurosyphilis and latent syphilis.

[0059] Table 1: Statistical data of differences in BMP-4 expression in cerebrospinal fluid and serum among three different types of syphilis

[0060]

[0061] In the table: Latent syphilis & asymptomatic indicates the statistical value of the difference in BMP-4 concentration in the cerebrospinal fluid or serum between latent syphilis and asymptomatic neurosyphilis. Latent syphilis & symptomatic indicates the statistical value of the difference in BMP-4 concentration in the cerebrospinal fluid or serum between latent syphilis and symptomatic neurosyphilis. Asymptomatic & symptomatic indicates the statistical value of the difference in BMP-4 concentration in the cerebrospinal fluid or serum between asymptomatic neurosyphilis and symptomatic neurosyphilis. 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 the BMP-4 protein level in the cerebrospinal fluid of patients with neurosyphilis (including symptomatic and asymptomatic) is significantly different from that of patients with latent syphilis, and the difference is statistically significant.

[0062] (2) There is no significant correlation between BMP-4 in cerebrospinal fluid and serum

[0063] Combination Figure 2It can be seen that there is no significant correlation between the expression level of BMP-4 protein in cerebrospinal fluid and the protein in serum (correlation coefficient R=0.06691, p=0.5357). Therefore, BMP-4 protein in serum cannot indicate the abnormality of BMP-4 in cerebrospinal fluid.

[0064] (3) BMP-4 in cerebrospinal fluid is positively correlated with injury indicators in cerebrospinal fluid

[0065] 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.

[0066] Combination Figure 3 As shown in Figures (a) and (b), the expression level of BMP-4 protein in cerebrospinal fluid is significantly positively correlated with the protein in the patient's cerebrospinal fluid (correlation coefficient R=0.4059, p<0.0001), but has no significant correlation with the number of white blood cells (correlation coefficient R=0.2001, p=0.0616). Therefore, BMP-4 protein in cerebrospinal fluid can indicate abnormalities in the indicator proteins related to nerve damage in cerebrospinal fluid.

[0067] Therefore, in practice, the BMP-4 protein level in cerebrospinal fluid can be used as a marker for evaluating nerve damage, or the cerebrospinal fluid protein level can be used as a reference indicator together with the BMP-4 protein level in cerebrospinal fluid as a means of early diagnosis of neurosyphilis and quantitative assessment of the degree of nerve damage.

[0068] (4) There is no significant correlation between serum BMP-4 and injury indicators in cerebrospinal fluid

[0069] Combination Figure 4 As shown in Figures (a) and (b), the expression level of BMP-4 protein in serum is not significantly correlated with the protein in the patient's cerebrospinal fluid (correlation coefficient R=0.1675, p=0.1189) and the number of white blood cells (correlation coefficient R=0.04254, p=0.6939). Therefore, BMP-4 protein in serum cannot indicate abnormalities in protein and white blood cells in cerebrospinal fluid, nor can it indicate the degree of nerve damage.

[0070] (5) Evaluation of cerebrospinal fluid and serum BMP-4 in neurosyphilis detection in syphilis patients

[0071] In order to further clarify the relationship between cerebrospinal fluid BMP-4 and serum BMP-4 and neurosyphilis, this example studied 88 patients with syphilis. First, according to the level of BMP-4 in the patient's cerebrospinal fluid, the 88 patients were divided into two groups using Cut-Off (critical value = 2.858NPX). Then, the study used the receiver operating characteristic curve ROC to evaluate the diagnostic potential of cerebrospinal fluid BMP-4 for neurosyphilis (neurosyphilis includes asymptomatic neurosyphilis and symptomatic neurosyphilis). Figure 5 As shown in the figure, the experimental results are: the area under the curve of BMP-4 in cerebrospinal fluid AUC (0.8064 [95% CI 0.6929-0.9199]), P = 0.0002, which can be considered statistically significant (see Figure 5 (a) Figure), sensitivity was 80%, specificity was 72.60%; according to the level of BMP-4 in the patient's serum, the 88 patients were divided into two groups using Cut-Off (critical value = 2.364NPX), and the receiver operating characteristic curve ROC was used to evaluate the diagnostic potential of serum BMP-4 for neurosyphilis. The results showed that the area under the curve of serum BMP-4 was AUC (0.5616 [95%CI 0.3964-0.7269]), P = 0.4538, which was considered to be insignificant (see Figure 5 (b) The sensitivity was 53.33% and the specificity was 43.33%. This shows that neurosyphilis can be diagnosed well based on the level of BMP-4 in the cerebrospinal fluid of syphilis patients with high sensitivity.

[0072] In order to further clarify the relationship between serum and cerebrospinal fluid BMP-4 and symptomatic neurosyphilis, this example studied 73 patients with neurosyphilis. According to the cut-off value of BMP-4 in the patient's cerebrospinal fluid (critical value = 3.229NPX) and the cut-off value of BMP-4 in serum (critical value = 2.431NPX), these 73 patients were divided into two groups with high BMP-4 levels and low BMP-4 levels. The study used ROC to evaluate the diagnostic potential of BMP-4 for symptomatic neurosyphilis in patients with neurosyphilis (including symptomatic and asymptomatic neurosyphilis patients). Figure 6 As shown in the figure, the experimental results are: the area under the curve of BMP-4 in cerebrospinal fluid AUC (0.6039 [95% CI 0.4711-0.7367]), P = 0.1330, which can be considered to be insignificant (see Figure 6 (a)), sensitivity was 60.00%, specificity was 51.16%; the area under the curve of serum BMP-4 AUC (0.5829 [95% CI 0.4429-0.7230]), P = 0.2303, sensitivity was 63.33%, specificity was 53.49%, no significant significance (see Figure 6The above results indicate that the occurrence of symptomatic neurosyphilis cannot be well evaluated based on the levels of BMP-4 in the cerebrospinal fluid and serum of patients with neurosyphilis.

[0073] In order to clarify the relationship between serum and cerebrospinal fluid BMP-4 and asymptomatic neurosyphilis, this example studied 15 patients with latent syphilis and 30 patients with asymptomatic neurosyphilis. According to the cut-off value of BMP-4 in the patient's cerebrospinal fluid (critical value = 2.843NPX) and the cut-off value of BMP-4 in serum (critical value = 2.393NPX), these 45 patients were divided into two groups with high BMP-4 levels and low BMP-4 levels, and the potential of BMP-4 in syphilis patients (including latent syphilis patients and asymptomatic neurosyphilis patients) for symptomatic neurosyphilis was studied by ROC evaluation. Figure 7 As shown in the figure, the experimental results are: the area under the curve of BMP-4 in cerebrospinal fluid AUC (0.7578 [95% CI 0.6084-0.9071]), P = 0.0052, which can be considered statistically significant (see Figure 7 (a)), the sensitivity was 73.33%, the specificity was 63.33%; the area under the curve of serum BMP-4 was AUC (0.5067 [95% CI 0.3268-0.6865]), P = 0.9424, which was not significant (see Figure 7 The above results indicate that the level of BMP-4 in the cerebrospinal fluid of patients with neurosyphilis can be used to assess the occurrence of asymptomatic neurosyphilis.

[0074] (6) CSF BMP-4 levels can indicate the degree of damage in the CSF

[0075] All syphilis patients were divided into two groups according to the cut-off value of BMP-4 level in the patients' cerebrospinal fluid (2.858NPX value). Figure 8 As shown, the concentration of BMP-4 in the cerebrospinal fluid of patients with a cerebrospinal fluid injury-related indicator cerebrospinal fluid protein (see Figure 8 (a)) increased significantly, and there was a trend of increasing white blood cells, but there was no significant difference (see Figure 8 (b) Figure). Therefore, patients with CSF BMP-4 concentrations below 2.858 NPX have a low risk of neurosyphilis and a small risk of nerve damage; whereas patients with CSF BMP-4 concentrations above 2.858 NPX have a relatively high risk of neurosyphilis and an increased risk of nerve damage.

[0076] In summary, the concentration of BMP-4 protein in cerebrospinal fluid can be used as an indicator for the diagnosis of neurosyphilis and the evaluation of the degree of nerve damage, with a sensitivity of 80%. It can be used alone or as a main reference indicator in combination with other indicators to complete the diagnosis of neurosyphilis and the evaluation of the degree of nerve damage.

[0077] 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. Application of cerebrospinal fluid BMP-4 protein in the preparation of diagnostic products for paralytic dementia and asymptomatic neurosyphilis.

2. Application of BMP-4 protein in the preparation of diagnostic products for screening of paralytic dementia and asymptomatic neurosyphilis; characterized in that, The diagnostic product is a detection kit, an antibody chip, an antibody probe or a detector using BMP-4 as a target molecule.

3. The use according to claim 2, characterized in that: The kit uses BMP-4 protein as a detection target molecule.

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

5. The use according to claim 2, characterized in that: The kit comprises: a primary antibody capable of specifically recognizing BMP-4, a polypeptide dye, magnetic beads and a biotin-labeled secondary antibody.

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

7. An assessment system for patients with paralytic dementia and asymptomatic 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 BMP-4 in cerebrospinal fluid; the judgment module includes a readable carrier storing judgment rules; The judgment rule is: when the detection module detects that the concentration of BMP-4 in the cerebrospinal fluid is ≥ 2.858NPX, it is judged that there is a high risk of neurosyphilis; When the detection module detects that the cerebrospinal fluid BMP-4 concentration value is less than 2.858NPX, it is judged to have a low risk of neurosyphilis.

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