CN116519953B - Application of EDA2R protein and specific antibody thereof in preparation of syphilis diagnosis product - Google Patents
Application of EDA2R protein and specific antibody thereof in preparation of syphilis diagnosis product Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N33/6857—Antibody fragments
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
- G01N33/541—Double or second antibody, i.e. precipitating antibody
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The application relates to the technical field of biological medicines, in particular to EDA2R used as a serum marker or a cerebrospinal fluid marker for early diagnosis, screening and parting detection of patients with nervous syphilis. The concentration of EDA2R protein in serum or cerebrospinal fluid is measured to be used as an index for early diagnosis of the neurosyphilis, symptomatic neurosyphilis, asymptomatic neurosyphilis, typing detection of the neurosyphilis and recessive syphilis, nerve injury degree evaluation and the like, and the index can be singly used or used as a main reference index to finish diagnosis screening, typing and nerve injury evaluation of the syphilis in combination with other indexes. Compared with the prior art, the marker has higher sensitivity, can realize noninvasive serum detection of the nerve syphilis, and is more beneficial to diagnosis and management of the nerve syphilis.
Description
Technical Field
The application relates to the technical field of biological medicines, in particular to application of EDA2R protein and a specific antibody thereof in preparing a detection kit, an antibody chip, an antibody probe or a detector for screening, typing detection or assessing nerve damage degree of nerve syphilis.
Background
The world health organization report shows that the prevalence of syphilis (synphilis) is 0.5% and the number of new infection cases is 1000-1200 ten thousand per year. With the increasing incidence of syphilis year by year, syphilis is still a group b infection that should be highly appreciated. Syphilis is a systemic infectious disease caused by invasion of treponema pallidum into the human body. Treponema pallidum spreads to the central nervous system within days after infection and can lead to asymptomatic meningitis and severe symptomatic neurosyphilis.
Neurosyphilis (neurophilis) is a group of clinical syndromes in which damage to the meninges, brain, blood vessels, or spinal cord, etc., occurs from invasion of the nervous system by treponema pallidum, and can occur at various stages of the course of syphilis, not just at stage three syphilis. The clinical manifestations of the nerve syphilis are various, and can be free of clinical symptoms, and can also be shown as complex clinical manifestations caused by extensive involvement of the nervous system. Clinically, they are generally classified into type 5: asymptomatic, meningo-syphilis, meningo-vascular syphilis, tuberculosis of the spinal cord and paralytic dementia (brain parenchymal syphilis). Although recessive syphilis has no clinical symptoms, the harm should be paid attention to, and the recessive syphilis can become recurrent syphilis and possibly progress to visceral and nervous syphilis. Asymptomatic neurotoxic patients have no obvious symptoms or signs, but there is abnormal change in cerebrospinal fluid, and the probability of developing symptomatic neurotoxic is up to 87% for patients with cerebrospinal fluid abnormality lasting more than 5 years. In the study of primary and secondary syphilis, it was found that 20% of patients had cerebrospinal fluid abnormalities, i.e. neurosyphilis could occur early in syphilis. Early-stage nerve syphilis includes syphilis meningitis and vascular syphilis, and late-stage nerve syphilis includes paralytic dementia, tuberculosis and the like. Paralytic dementia is characterized by a latency period of 10-15 years, and is mainly manifested by mental retardation, such as memory decline, abnormal behavior, and character change, and severe patients develop epilepsy, stroke-like morbidity, secondary hemiplegia and aphasia.
Early diagnosis of the neurosyphilis is very important, and finding sensitive biomarkers is helpful for diagnosis of the neurosyphilis, and has important significance for preventing serious and irreversible sequelae. Currently, diagnosis of neurosyphilis relies primarily on examination of cerebrospinal fluid, including CSF-RPR, CSF white cell count and protein levels. The CSF-RPR is the same as the rapid plasma reactive element cyclic card test in blood plasma, and the index is positive to diagnose the neurosyphilis. Important reference indicators for neurosyphilis also include cerebrospinal fluid cell count or protein level, which can be diagnosed as neurosyphilis if clinical signs of neurosyphilis, positive plasma tests, and abnormal cerebrospinal fluid cell count occur as a result of a negative CSF-RPR. The protein content increase in the cerebrospinal fluid of patients with the neurosyphilis is positively correlated with the severity of the illness and negatively correlated with the prognosis of the long-term. Leukocytosis and protein content increase in cerebrospinal fluid indicate blood-brain barrier destruction, and inflammatory reaction exists in cranium, and has auxiliary effect on diagnosis of nerve syphilis. However, sensitivity of CSF ⁃ RPP was found to be only 30% -70% during diagnosis, with lower sensitivity and insensitivity to early diagnosis of asymptomatic neurosyphilis. In the current clinical diagnosis and treatment process, the diagnosis and treatment of the neurosyphilis are mainly carried out on patients with the symptom of the neurosyphilis, and the cerebral spinal fluid and the serum biological markers which are sensitive, reliable and effective for distinguishing the neurosyphilis are lacking, and the biological markers which are effective for distinguishing the neurosyphilis in the serum are lacking.
In summary, the existing diagnostic methods for neurosyphilis require invasive cerebrospinal fluid procedures, and have no sensitive and accurate markers, thus presenting great challenges. Therefore, the development of new markers in serum for diagnosis of the neurosyphilis has great significance for diagnosis and management of the neurosyphilis.
Disclosure of Invention
First, the technical problem to be solved
In view of the technical problems that the existing CSF-RPR diagnosis method is low in sensitivity, even insensitive to diagnosis of early asymptomatic nerve syphilis, depends on invasive cerebrospinal fluid examination and the like, the application provides a cerebrospinal fluid/serum marker for early diagnosis screening, parting detection and nerve damage assessment of the nerve syphilis, and the marker can be used for realizing early diagnosis, screening, parting detection and nerve damage assessment of the nerve syphilis, and compared with the prior art, the sensitivity is higher, and the noninvasive serum detection of the nerve syphilis can be realized.
(II) technical scheme
In order to achieve the above purpose, the main technical scheme adopted by the application comprises the following steps:
in a first aspect, the application relates to EDA2R as a serum marker or cerebrospinal fluid marker for early diagnosis, screening and typing detection of patients with neurosyphilis.
In a second aspect, the present application provides the use of EDA2R protein and antibodies specific for the same in the manufacture of a diagnostic or auxiliary diagnostic product for screening, typing or assessing the extent of neurological damage; the diagnosis product or auxiliary diagnosis product is a detection kit, an antibody chip, an antibody probe or a detector.
According to the preferred embodiment of the application, the kit is used for detecting the expression level of EDA2R protein or specific antibody thereof in cerebrospinal fluid or serum by taking the EDA2R protein or specific antibody thereof as a biomarker.
According to a preferred embodiment of the application, the kit is an enzyme-linked immunosorbent assay kit or an immunohistochemical kit.
According to a preferred embodiment of the application, the kit comprises: primary antibodies specifically recognizing EDA2R, polypeptide dyes, magnetic beads and biotin-labeled secondary antibodies.
According to a preferred embodiment of the application, the reagent comprises citrate buffer, PBS wash, peroxidase blocker, goat serum, streptomycin antibiotic-peroxidase, DAB solution and hematoxylin solution.
According to a preferred embodiment of the application, the kit further comprises reagents for obtaining serum or cerebrospinal fluid from the patient, and/or reagents for separating serum.
In the detection of EDA2R protein expression level in serum, an Olink ultra-high sensitivity plasma protein detection platform can also be used to quantify EDA2R expression level.
In a third aspect, the present application also relates to a product for early diagnosis, screening, typing detection or assessment of the extent of neurological damage in a patient suffering from a neuro-syphilis; the product or device is capable of quantitatively detecting the concentration of EDA2R in serum and cerebrospinal fluid; the product is a detection kit, an antibody chip, an antibody probe or a detector.
Preferably, the product comprises antibodies and other reagents that specifically bind EDA2R to quantitatively detect the concentration of EDA2R protein in serum and cerebrospinal fluid. The experimental method used in the examples of the present application was Olink neurology panel (95801), which can be used to detect 92 protein biomarkers associated with neurological function, including EDA2R protein as well.
The syphilis type includes symptomatic nerve syphilis, asymptomatic nerve syphilis or non-nerve syphilis, etc.
In a fourth aspect, the application relates to an evaluation system for the risk of nerve injury of a patient with neurosyphilis, comprising a detection module and a judgment module, wherein the detection module is used for acquiring the concentration value of serum or cerebrospinal fluid EDA 2R; the judging module comprises a readable carrier storing judging rules;
the judging rule is as follows: when the serum EDA2R concentration value obtained by the detection module is more than or equal to 1.69593NPX value or the cerebrospinal fluid EDA2R concentration value is more than or equal to 5.64273NPX value, judging that the risk of the middle nerve injury is high; otherwise, judging that the risk of nerve injury is low;
and when the serum EDA2R concentration value obtained by the detection module is more than or equal to 2.04001NPX value or the cerebrospinal fluid EDA2R concentration value is more than or equal to 6.113NPX value, judging that the risk of nerve injury is high.
The reference value is the Cut-off value of EDA2R obtained from a syphilis patient population; preferably, the serum Cut-off value for diagnosis of neurosyphilis is 1.69593NPX and the cerebrospinal fluid Cut-off value is 5.64273 NPX; the serum Cut-off value for symptomatic neurosyphilis diagnosis is 2.04001NPX and the cerebrospinal fluid Cut-off value is 6.113 NPX. The Cut-off values described above are EDA2R statistical analysis thresholds obtained from 88 patients with syphilis.
Although the expression level of a specific protein in cerebrospinal fluid is a sensitive indicator of pathological changes in the response nerve tissue, it is an invasive procedure with a narrow range of applicable populations and a certain potential risk. The EDA2R protein in the serum can be used as an indicator for the increase of the index of the cerebrospinal fluid, so that the convenient and rapid noninvasive screening and diagnosis can be performed.
In a fifth aspect, the present application also provides a syphilis detection kit comprising: a first detection reagent composition for detecting serum or cerebrospinal fluid EDA2R protein levels; and a second detection reagent composition for detecting cerebrospinal fluid leukocytes and/or cerebrospinal fluid proteins.
In the above-described protocol, in diagnosing (including early diagnosis, typing and nerve damage assessment) a serum or cerebrospinal fluid EDA2R protein level can be used in combination with two or three indicators of cerebrospinal fluid leukocytes and cerebrospinal fluid proteins associated with nerve damage in a patient with a nerve syphilis for early diagnosis, typing and nerve damage assessment of a nerve syphilis.
Wherein EDA2R levels in serum or cerebrospinal fluid are determined by detection of Olink precision proteomics, i.e., immunoassay of antibodies, in combination with quantitative real-time PCR. The number of white blood cells in cerebrospinal fluid and the amount of protein in cerebrospinal fluid can be measured by any known method.
(III) beneficial effects
The application provides a novel cerebrospinal fluid/serum marker EDA2R for early diagnosis screening, typing detection and nerve injury assessment of a nerve syphilis, and the quantitative assessment of the nerve injury degree of the nerve syphilis patient is realized by detecting the expression level of EDA2R in the serum or cerebrospinal fluid of the patient; or diagnosis of the neurosyphilis, including early diagnosis and typing detection, is achieved by detecting the expression level of EDA2R in serum or cerebrospinal fluid of a patient. Compared with the prior art, the marker can be used for early diagnosis, screening, parting detection and nerve injury assessment of the nerve syphilis, has higher sensitivity, and can realize noninvasive serum detection of the nerve syphilis.
Preferably, the marker EDA2R is more suitable for diagnostic screening for symptomatic neurosyphilis, since the expression level of EDA2R in the serum or cerebrospinal fluid of symptomatic neurosyphilis patients is highest among symptomatic neurosyphilis, asymptomatic neurosyphilis or non-neurosyphilis, and the evaluation sensitivity of EDA2R expression levels on neurosyphilis, especially symptomatic neurosyphilis, is high, and thus a high sensitivity and a high accuracy of screening for symptomatic neurosyphilis can be achieved.
Drawings
FIG. 1 shows the detection of EDA2R protein expression in cerebrospinal fluid and serum of three different groups of syphilis patients by the Olink method.
FIG. 2 shows that the cerebrospinal fluid of syphilis patients is positively correlated with EDA2R protein levels in serum.
FIG. 3 shows the variation of the nerve damage index, cerebrospinal fluid protein and leukocytes, in three groups of patients with different types of syphilis.
Figure 4 shows that EDA2R expression in cerebrospinal fluid is significantly positively correlated with cerebrospinal fluid proteins and leukocytes.
Figure 5 shows that EDA2R expression in serum is significantly positively correlated with cerebrospinal fluid proteins and leukocytes.
FIG. 6 is a graph showing the likelihood of determining a diagnosis of neurosyphilis, including asymptomatic and symptomatic neurosyphilis, using different levels of serum and cerebrospinal fluid EDA2R concentrations as clinical outcomes.
FIG. 7 is a graph showing the likelihood of assessing symptomatic neurosyphilis diagnosis AUC for different levels of serum and cerebrospinal fluid EDA2R concentrations when diagnosing symptomatic neurosyphilis as a clinical outcome.
Fig. 8 is a graph showing the differentiation of all patients into two groups of patients based on serum EDA2R cutoff 1.69593, each group of patients showing differences in nerve damage index cerebrospinal fluid protein and leukocytes.
Fig. 9 is a graph showing the differentiation of two groups of patients, each group of patients showing differences in nerve damage index cerebrospinal fluid protein and leukocytes, based on the cerebrospinal fluid EDA2R cut-off 5.64273.
Fig. 10 shows the differentiation of neurosyphilis into two groups of patients based on serum EDA2R cutoff 2.04001, each group of patients showing differences in nerve damage index cerebrospinal fluid proteins and leukocytes.
FIG. 11 shows the differentiation of neurosyphilis into two groups of patients based on the cut-off 6.113 of EDA2R in cerebrospinal fluid, the difference between the protein and leukocytes in the index of nerve damage in each group of patients.
Detailed Description
The present application will now be described in detail for the purpose of better explaining the present application.
Early diagnosis of the neurosyphilis is very important, and finding sensitive biomarkers is helpful for diagnosis of the neurosyphilis, and has important significance for preventing serious and irreversible sequelae. At present, the diagnosis of the neurosyphilis mainly depends on examination of cerebrospinal fluid, so the inventor researches the diagnosis of the neurosyphilis with the aim of developing new biomarkers in serum, which can be used for diagnosis of the neurosyphilis and nerve injury, screens a plurality of indexes related to nerve functions in serum and cerebrospinal fluid of a patient with the neurosyphilis, and obtains EDA2R with obvious diagnostic value in the serum and the cerebrospinal fluid.
The inventor finds that the serum and cerebrospinal fluid EDA2R protein levels of different types of syphilis patients are obviously different through researches. The EDA2R protein level in cerebrospinal fluid and serum is related to the typing of the nerve syphilis and the occurrence of nerve injury, and the difference between the EDA2R protein level in the cerebrospinal fluid and serum of symptomatic nerve syphilis patients and the EDA2R protein level in asymptomatic nerve syphilis patients is statistically significant, so that the EDA2R protein level in the cerebrospinal fluid of different clinical typing nerve syphilis patients is different, and the immune response degree and the immune injury degree are also different. The EDA2R protein level in cerebrospinal fluid and serum of patients with asymptomatic neurotoxic syphilis is significantly higher than that of non-neurotoxic (recessive syphilis) groups, which shows that the EDA2R protein level is changed in early stage of occurrence of the neurotoxic, namely in the stage of asymptomatic neurotoxic syphilis, so that the biomarker can effectively distinguish the asymptomatic neurotoxic syphilis from the recessive syphilis.
During the course of the study, the experiment first assessed EDA2R for neurosyphilis diagnostic potential (including asymptomatic neurosyphilis) using a subject work feature curve (ROC); the experimental result shows that the area under the curve (AUC) of serum EDA2R is 0.8146, and P <0.0001 is considered to have statistical significance; the AUC of the cerebrospinal fluid EDA2R was 0.8667, and P <0.0001 was considered statistically significant. Therefore, the occurrence of the neurosyphilis can be well estimated according to the serum and cerebrospinal fluid EDA2R level of a syphilis patient, and the sensitivity is high.
Next, to clarify the relationship of serum and cerebrospinal fluid EDA2R to symptomatic neurosyphilis, 73 neurosyphilis patients were studied in the examples below, and ROC was used to assess the potential of EDA2R in neurosyphilis patients (including symptomatic and asymptomatic neurosyphilis patients) for symptomatic neurosyphilis; the experimental result shows that the AUC of serum EDA2R is 0.8295, and P is less than 0.0001; the AUC of the cerebrospinal fluid EDA2R was 0.8984 and P < 0.0001. The results show that the occurrence of symptomatic neurosyphilis can be well estimated according to the serum and cerebrospinal fluid EDA2R levels of the neurosyphilis patients.
Further, the present application further groups all patients into two groups based on their serum and cerebrospinal fluid EDA2R Cut-Off values (1.69593 and 5.64273), above which the cerebrospinal fluid damage related index cerebrospinal fluid protein increases significantly, with a tendency to increase but not significantly. As a result, it was found that the index associated with cerebrospinal fluid damage increased when the EDA2R serum value was higher than the 2.04001NPX value or the cerebrospinal fluid was higher than the 6.113NPX value. Thus, patients with neurotensin are at a lower risk of symptomatic neurotensin with EDA2R serum below the 2.04001NPX value or EDA2R cerebrospinal fluid below the 6.113NPX value, with less risk of nerve damage; above this value, however, there is a higher risk of symptomatic syphilis and an increased risk of nerve damage. Thus, the present application determines that the serum Cut-off value for diagnosis of neurosyphilis is specifically 1.69593NPX and the cerebrospinal fluid is 5.64273 NPX. The serum Cut-off value for symptomatic neurosyphilis is specifically 2.04001NPX and the cerebrospinal fluid Cut-off value is specifically 6.113 NPX.
In summary, the application provides the use of EDA2R as an early diagnosis of neurosyphilis and evaluation of nerve damage. The inventors have identified EDA2R protein levels in serum and cerebrospinal fluid as reliable and effective primary or secondary reference for early diagnosis, typing and assessment of neurological damage of syphilis. Based on the findings, the application can realize high-sensitivity detection and typing of syphilis, especially neurosyphilis, by detecting the expression level of EDA2R in serum or cerebrospinal fluid, and can also carry out early noninvasive screening on the neurosyphilis, and accurately and quantitatively evaluate the condition of nerve injury, supervision and treatment of the neurosyphilis.
In order that the above-described aspects may be better understood, exemplary embodiments of the present application will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the application are shown in the drawings, it should be understood that the application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.
The experimental methods in the following experimental examples are conventional methods unless otherwise specified. The raw materials and reagent materials used in the examples described below are commercially available products unless otherwise specified. The related technical contents include:
study object (one)
On the premise of informed consent and meeting inclusion conditions, 88 cases are selected, wherein 15 cases of recessive syphilis, 30 cases of asymptomatic neurosyphilis and 43 cases of paralytic dementia are selected, and different neurosyphilis queues with different degrees of severity are established. Diagnostic criteria were performed according to WS 273-2007 syphilis diagnostic criteria.
(II) sample collection
According to the condition of the patient in the group, the lumbar puncture operation and the elbow vein puncture operation are carried out to collect cerebrospinal fluid and blood for routine examination, and cerebrospinal fluid and serum specimens are collected. Under the informed consent of the patient, 5mL of cerebrospinal fluid standard is collected by lumbar puncture, and 10mL of blood sample is collected by vein. After collecting cerebrospinal fluid by lumbar puncture, sub-packaging with a freezing tube, and coding and marking; after blood was collected by venipuncture, the mixture was centrifuged at 4℃for 10 minutes at 2000rpm and the supernatant serum was rapidly separated and placed in a freezing tube.
(III) detection of EDAR2 concentration in serum and cerebrospinal fluid Using Olink method
92 proteins associated with neurological function, including EDA2R protein, were detected using Olink neurology panel (95801). Olink precision proteomics is a precision proteomics research tool well suited for large-scale screening with good sensitivity, rapidity, high throughput analysis, and specificity at the multi-channel level. Therefore, in this example, markers in serum and cerebrospinal fluid of patients with different types of neurosyphilis were detected by using Olink, and it was found that EDA2R protein was significantly different in cerebrospinal fluid and serum. Detection of cerebrospinal fluid proteins and cerebrospinal fluid leukocytes was performed according to the method described in WS 273-2007.
The following statistics and data analysis are performed on the Olink detection results:
(1) Significant increases in EDA2R protein in cerebrospinal fluid and serum of neurosyphilis
The difference of the expression of the nerve-related protein in the blood serum and the cerebrospinal fluid is detected by using Olink precise proteomics, and EDA2R protein with obvious difference in the cerebrospinal fluid and the blood serum is screened. The Olink test results are shown in FIG. 1 and Table 1. It was found that symptomatic neurosyphilis patients (symptomatic) had a significant increase in EDA2R in serum and cerebrospinal fluid over asymptomatic neurosyphilis (asymptomatic) and recessive syphilis. Thus, cerebrospinal fluid and serum EDA2R levels can be used to distinguish between syphilis genotypes, indicating their severity.
In FIG. 1 (a), recessive syphilis means non-neural syphilis, asymptomatic means asymptomatic neural syphilis, symptomatic means paralytic dementia, i.e. symptomatic neural syphilis. As can be seen from the graph (a) of fig. 1, the EDA2R concentration in the cerebrospinal fluid of the symptomatic neurosyphilis patient > the EDA2R concentration in the cerebrospinal fluid of the asymptomatic neurosyphilis patient > the EDA2R concentration in the cerebrospinal fluid of the recessive syphilis patient; as shown in fig. 1 (b), consistent with the situation in cerebrospinal fluid, EDA2R concentration in serum of symptomatic neurosyphilis patients > EDA2R concentration in serum of asymptomatic neurosyphilis patients > EDA2R concentration in serum of recessive syphilis patients.
Table 1: statistical data on the differences in EDA2R expression in cerebrospinal fluid and serum in three different syphilis types
Table 1 is a statistical value of the difference in EDA2R concentration of cerebrospinal fluid, recessive syphilis & asymptomatic, representing recessive syphilis and asymptomatic neurosyphilis in cerebrospinal fluid. Average difference mean difference is the mean difference between the sign values and the arithmetic average, the difference value may show whether there was a significant difference between the two groups before, and the fold difference may show a multiple of this difference. As can be seen from the statistics in Table 1, there are significant differences in serum and cerebrospinal fluid EDA2R protein levels for patients with different types of syphilis, and the differences are statistically significant.
(2) The cerebrospinal fluid and EDA2R protein in serum show obvious positive correlation
It can be seen in conjunction with fig. 1 and 2 that EDA2R protein in serum has a significant positive correlation with this protein in cerebrospinal fluid (r=0.7049, p < 0.0001). Thus, EDA2R protein in serum can be used as an indicator of the increase in cerebrospinal fluid. Therefore, EDA2R protein level in serum can be used for replacing EDA2R protein level in cerebrospinal fluid, so that noninvasive serological detection of the neurosyphilis can be realized, and invasive cerebrospinal fluid examination is avoided.
(3) Cerebrospinal fluid cell count or protein level is an important clinical feature of neurosyphilis
An important reference indicator of neurosyphilis is cerebrospinal fluid cell count and protein level. Patients with neurosyphilis are associated with abnormal cerebrospinal fluid cell counts and protein levels, and both of these indicators may also indicate the severity of the damage to the neurosyphilis.
The experiment adopts a microscopic counting method and a blood analyzer counting method to count the cerebrospinal fluid cells of three groups of syphilis patients with different types, and adopts Lee-Vinson to measure the protein level in the cerebrospinal fluid. The experimental result is shown in the graph (a) of fig. 3, the protein concentration in the cerebrospinal fluid of the patients with the recessive syphilis is the lowest, the protein concentration in the cerebrospinal fluid of the patients with the asymptomatic syphilis is higher than that in the patients with the recessive syphilis, and the protein concentration in the cerebrospinal fluid of the patients with the symptomatic syphilis is the highest. In accordance with the foregoing, as shown in fig. 3 (b): the number of the leucocytes in the cerebrospinal fluid of the patients with the recessive syphilis is the lowest, the number of the leucocytes in the cerebrospinal fluid of the patients with the asymptomatic neurosyphilis is higher than that of the patients with the recessive syphilis, and the number of the leucocytes in the cerebrospinal fluid of the patients with the symptomatic neurosyphilis is the highest.
(4) EDA2R in serum and damage index in cerebrospinal fluid are obviously and positively correlated
As can be seen in combination with fig. 4 (a) and (b), EDA2R protein expression levels in serum exhibited a significant positive correlation with protein (r=0.4846, p < 0.0001) and the number of leukocytes (r=0.3906, p < 0.0001) in the cerebrospinal fluid of the patient. Thus, EDA2R protein in serum, as in cerebrospinal fluid, is indicative of abnormalities in proteins and leukocytes in cerebrospinal fluid. Thus, EDA2R protein in serum may be indicative of abnormalities in proteins and leukocytes in cerebrospinal fluid.
(5) EDA2R in cerebrospinal fluid and damage index in cerebrospinal fluid are obviously and positively correlated
As can be seen in combination with fig. 5 (a) and (b), EDA2R protein expression levels in cerebrospinal fluid exhibited a significant positive correlation with protein (r=0.7168, p < 0.0001) and the number of leukocytes (r=0.5305, p < 0.0001) in the cerebrospinal fluid of the patient. Thus, EDA2R protein in cerebrospinal fluid may be indicative of abnormalities in proteins and leukocytes in cerebrospinal fluid.
It can be seen that EDA2R protein levels in cerebrospinal fluid of patients with syphilis are positively correlated with patient ridge fluid cell count and protein levels. In practice, therefore, the cerebrospinal fluid cell count and/or the protein level can be used as a reference index, and can be used together with the EDA2R protein level in cerebrospinal fluid as a means for early diagnosis screening, typing detection and quantitative assessment of the degree of nerve damage of the syphilis.
(6) Evaluation of serum and cerebrospinal fluid EDA2R in the detection of neurosyphilis in syphilis patients
To clarify the relationship of serum and cerebrospinal fluid EDA2R to neurosyphilis, this example was conducted on 88 patients with syphilis. First, these 88 patients were divided into two groups of high EDA2R concentration and low EDA2R concentration using Cut-Off values (critical value= 1.69593NPX values) according to the levels of EDA2R in the patient serum. These 88 patients were then divided into two groups using Cut-Off (critical value= 5.64273NPX value) based on the level of EDA2R in the cerebrospinal fluid of the patient.
The study uses a subject work-characteristic curve (ROC) to evaluate EDA2R for the diagnostic potential of neurosyphilis, including asymptomatic neurosyphilis. As shown in fig. 6, the experimental results were: the area under the curve (AUC) of serum EDA2R (0.8146[95%CI 0.7020-0.9272), P <0.0001 is considered statistically significant (see (a) plot); the area under the curve (AUC) of the cerebrospinal fluid EDA2R (0.8667[95%CI 0.7854-0.9480) and P <0.0001 are considered statistically significant (see panel (b)). Therefore, the diagnosis of the neurosyphilis can be well assisted according to the serum and cerebrospinal fluid EDA2R level of the syphilis patient, and the sensitivity is high.
In addition to the above detection of effects in neurosyphilis, serum and cerebrospinal fluid EDA2R levels can also be used for diagnosis of symptomatic neurosyphilis in neurosyphilis.
To further clarify the relationship of serum and cerebrospinal fluid EDA2R to symptomatic neurosyphilis, 73 neurosyphilis patients were studied in this example. These 73 patients were divided into two groups of high EDA2R concentration and low EDA2R concentration based on the patient serum EDA2R Cut-Off value (critical value= 2.04001NPX value) and the cerebrospinal fluid EDA2R Cut-Off (critical value= 6.113NPX value).
Research EDA2R in evaluation of neurosyphilis patients (including symptomatic and asymptomatic neurosyphilis patients) was used to evaluate the potential for symptomatic neurosyphilis diagnosis. As shown in fig. 7, the experimental results were: the area under the curve (AUC) of serum EDA2R (0.8295[95%CI 0.7367-0.9222), P <0.0001 is considered statistically significant (see (a) plot); the area under the curve (AUC) of the cerebrospinal fluid EDA2R (0.8984[95%CI 0.8223-0.9746) and P <0.0001 are considered statistically significant (see (b) graph. The above results indicate that symptomatic neurosyphilis can be better assessed based on the serum and cerebrospinal fluid EDA2R levels of neurosyphilis patients.
(7) EDA2R levels may indicate an indication of injury in cerebrospinal fluid
All patients were divided into two groups based on Cut-Off values (1.69593 NPX values) of patient serum EDA2R levels. As shown in fig. 8, the index cerebrospinal fluid protein associated with cerebral spinal fluid damage was significantly increased in patients with serum EDA2R above 1.69593NPX value (see (a) panels) and the white blood cells tended to increase but not significantly (see (b) panels). Thus, patients with serum EDA2R below the 1.69593NPX value have low risk of neurosyphilis and low risk of nerve damage; above this value, however, the risk of neurosyphilis is higher and the risk of nerve damage is increased.
All patients were divided into two groups based on Cut-Off values (5.64273 NPX values) of the levels of patient cerebrospinal fluid EDA2R. As shown in fig. 9, the index cerebrospinal fluid protein associated with cerebrospinal fluid damage was significantly increased in patients with cerebrospinal fluid EDA2R above the 5.64273NPX value (see (a) graph) while the white blood cells were also significantly increased (see (b) graph). Thus, patients with cerebrospinal fluid EDA2R below the 5.64273NPX value have a low risk of neurosyphilis and a low risk of nerve damage; above this value, however, the risk of neurosyphilis is higher and the risk of nerve damage is increased.
It was further found that both the index cerebrospinal fluid protein (see (a) graph) and the white blood cells associated with the cerebrospinal fluid damage were significantly increased (see (b) graph) in patients when the EDA2R serum value was higher than the 2.04001NPX value, as shown in fig. 10. As shown in FIG. 11, both the index cerebrospinal fluid protein (see panel (a)) and the white blood cells associated with the damage to the cerebrospinal fluid of the patient were significantly increased (see panel (b)) when EDA2R cerebrospinal fluid was above the 6.113NPX value.
Thus, patients with neurotoxic with EDA2R serum below 2.04001 or cerebrospinal fluid below 6.113NPX have a lower risk of developing symptomatic neurotoxicity and less risk of nerve damage; whereas EDA2R serum is higher than 2.04001 or EDA2R cerebrospinal fluid is higher than 6.113NPX, symptomatic neurosyphilis is at higher risk and the risk of nerve damage is increased.
In conclusion, the concentration of EDA2R protein in serum or cerebrospinal fluid can be used as indexes of early diagnosis of the neurosyphilis, symptomatic neurosyphilis, asymptomatic neurosyphilis, typing detection of the neurosyphilis and recessive syphilis, nerve injury degree evaluation and the like, and can be independently used or used as a main reference index to finish diagnosis screening, typing and nerve injury evaluation of the syphilis in combination with other indexes.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present application and are not limiting. Although the present application has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present application, which is intended to be covered by the appended claims.
Claims (2)
- The application of EDANR protein and specific antibodies thereof in preparing a diagnosis product or an auxiliary diagnosis product for screening, typing detection or assessing nerve damage degree of the nerve syphilis is characterized in that the diagnosis product or the auxiliary diagnosis product is a detection kit, an antibody chip, an antibody probe or a detector;the kit takes EDA2R protein as a biomarker, and detects the expression level of the EDA2R protein or a specific antibody thereof in cerebrospinal fluid or serum;the kit is an enzyme-linked immunosorbent assay kit or an immunohistochemical kit;the kit comprises: primary antibodies, polypeptide dyes, magnetic beads and biotin-labeled secondary antibodies that specifically recognize EDA 2R;the reagent of the kit comprises citrate buffer solution, PBS washing solution, peroxidase blocking agent, goat serum, streptomycin antibiotic-peroxidase, DAB solution and hematoxylin solution;the kit further comprises reagents for obtaining serum or cerebrospinal fluid from the patient, and/or reagents for separating serum.
- 2. The system for evaluating the nerve injury risk of the patients with the nerve syphilis is characterized by comprising a detection module and a judgment module; the detection module is used for acquiring the concentration value of serum or cerebrospinal fluid EDA 2R; the judging module comprises a readable carrier storing judging rules;the judging rule is as follows: the serum Cut-off value for diagnosis of the neurosyphilis is 1.69593NPX value, and the cerebrospinal fluid Cut-off value is 5.64273NPX value;the serum Cut-off value for symptomatic neurosyphilis diagnosis is 2.04001NPX and the cerebrospinal fluid Cut-off value is 6.113 NPX.
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| CN117607432B (en) * | 2024-01-17 | 2024-04-09 | 首都医科大学附属北京地坛医院 | Application of MSR1 protein and specific antibody thereof in preparation of neural syphilis or neural injury diagnostic product |
| CN117741143B (en) * | 2024-02-20 | 2024-05-07 | 首都医科大学附属北京地坛医院 | Application of Siglec-9 protein and specific antibody thereof in preparation of neural syphilis or neural injury diagnostic product |
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