Application of saliva TP17, TP47, TP15 specific antibodies in preparation of auxiliary diagnostic product for neurosyphilis

Abstract

The application relates to the field of biological medicine, and particularly discloses application of saliva TP17, TP47 and TP15 specific antibodies in preparation of an auxiliary diagnostic product for neurosyphilis. Based on specific antibody immunoglobulin G (IgG) of Treponema pallidum in saliva, combined detection of saliva TP17 / IgG, TP47 / IgG and TP15 / IgG outputs a risk value of neurosyphilis of a subject, or a four combined Logistic regression discriminant model is constructed by combining serum TRUST titers, and the subject is stratified into a low-risk or high-risk group based on a preset judgment threshold, so that the comprehensive recognition capability for neurosyphilis is improved. The saliva is used as a detection sample, and the application has the advantages of non-invasiveness, convenience, easy popularization, repeatable collection and the like, and can be used for early screening, auxiliary diagnosis and clinical path management of neurosyphilis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of saliva-specific antibodies TP17, TP47, and TP15 in the preparation of auxiliary diagnostic products for neurosyphilis. Background Technology

[0002] Treponema pallidum infection can affect the central nervous system and lead to neurosyphilis at any stage of the disease. Its clinical manifestations are complex, ranging from typical neurological symptoms such as meningitis, cerebrovascular disease, and paralytic dementia, to a prolonged state of asymptomatic insidiity. Especially in asymptomatic or atypical cases of neurosyphilis, timely identification based solely on neurological signs and routine physical examinations is difficult, easily leading to missed diagnoses, delayed treatment, and an increased risk of subsequent neurological damage. Therefore, establishing a screening strategy that can identify and stratify the risk of neurosyphilis in its early or asymptomatic stages is of great significance for optimizing clinical treatment pathways and public health prevention and control.

[0003] Currently, the diagnosis and screening of neurosyphilis mainly rely on lumbar puncture to obtain cerebrospinal fluid and conduct relevant laboratory tests. Although cerebrospinal fluid examination is of great value in indicating central nervous system involvement, lumbar puncture is an invasive procedure with risks of pain, anxiety, complications, and contraindications, resulting in low patient acceptance and compliance. Furthermore, in primary healthcare institutions or resource-limited areas, the availability and experience of personnel performing lumbar punctures also limit the accessibility of neurosyphilis screening. In addition, many syphilis patients in clinical practice do not exhibit clear neurological symptoms or typical indications, making the decision of whether to perform a lumbar puncture often uncertain: excessive lumbar punctures increase the medical burden and unnecessary risks, while insufficient lumbar punctures may miss high-risk or asymptomatic neurosyphilis cases, affecting the quality of disease management. Thirdly, existing screening strategies based primarily on serological or symptom-based methods cannot simultaneously achieve early detection, large-scale screening, and repeatable follow-up.

[0004] Therefore, there is an urgent need for a low-invasive, repeatable, and easily scalable pre-screening method to quickly identify high-risk neurosyphilis individuals among suspected or confirmed syphilis populations. Summary of the Invention

[0005] This invention proposes a method for preparing an auxiliary diagnostic product using saliva samples as a carrier and anti-Treponema pallidum specific antibodies as the detection target. The product is prepared by coating saliva samples with Treponema pallidum specific proteins TP17, TP47, and TP15, based on the principle of antigen-antibody binding. A quantifiable detection signal (OD value) is established, and a threshold is calculated based on serum TRUST titer to implement a judgment rule, achieving a low-invasive indication of neurosyphilis risk.

[0006] To achieve the above objectives, this invention provides the application of saliva-specific antibodies TP17, TP47, and TP15 in the preparation of auxiliary diagnostic products for neurosyphilis. The main technical solutions adopted by the invention include: In a first aspect, the present invention provides the application of saliva-specific antibodies TP17, TP47, and TP15 in the preparation of auxiliary diagnostic products for neurosyphilis.

[0007] The product is a product that uses specific antibodies corresponding to the recombinant antigens TP17, TP47 and TP15 as detection targets to detect anti-Treponema pallidum IgG antibodies in saliva.

[0008] Studies have found that saliva sample collection is simple, non-invasive, and easy to standardize, making it suitable for outpatient and population screening scenarios. At the same time, studies have shown that immunological signals related to Treponema pallidum infection can be detected in saliva, such as specific antibody responses against Treponema pallidum recombinant antigens (e.g., TP15, TP17, TP47), and quantitative or semi-quantitative results can be obtained through immunoassay methods such as ELISA.

[0009] The products mentioned include reagent kits, antibody chips, antibody probes, detectors, diagnostic reagents, or risk assessment systems.

[0010] The kit includes: a solid-phase carrier coated with Treponema pallidum antigens TP15, TP17, and TP47 respectively; a labeled secondary antibody for recognizing human IgG; a blocking solution; a washing solution; a chromogenic substrate; and a stop solution. The solid-phase carrier is a microplate, and the antigens can be coated on the solid-phase surface of the microplate. The kit uses specific antibodies corresponding to the recombinant antigens TP15, TP17, and TP47 as detection targets to detect anti-Treponema pallidum IgG antibodies in saliva. The detection results can be expressed as OD values.

[0011] The kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0012] Secondly, the present invention relates to TP15, TP17, and TP47 as saliva markers for the detection of neurosyphilis.

[0013] Specifically, a method for collecting and preprocessing saliva markers is provided, comprising the following steps: (a) Collect saliva samples, with a volume of 1.0 mL to 2.0 mL. Store the samples at 2 to 8°C and test them within 30 minutes. If testing cannot be performed within 30 minutes, freeze them at -80°C. (b) Centrifuge the saliva sample at 10,000 rpm for 10 minutes to clarify it, take the supernatant as the sample to be tested, add protease inhibitor and freeze it; optionally, when the sample is not completely clarified, extend the centrifugation time to 30 minutes, or use layer centrifugation to separate and remove insoluble impurities.

[0014] Thirdly, the present invention provides a system for assessing the risk of neurosyphilis, including a detection module and a judgment module. The judgment module includes a preset discriminant model; the preset discriminant model is a four-in-one logistic regression discriminant model constructed based on saliva TP17 / IgG, saliva TP47 / IgG, saliva TP15 / IgG, and serum TRUST titer.

[0015] The four-way combined Logistic regression discriminant model satisfies the following formula: Z = −4.7881 + 1.2443 × A + 0.3594 × B + 0.0038 × C + 0.6076 × D; where coefficient A represents the OD value of saliva TP17 / IgG, coefficient B represents the OD value of saliva TP47 / IgG, coefficient C represents the OD value of saliva TP15 / IgG, and coefficient D represents the serum TRUST titer grade coding value.

[0016] The predictive probability P of developing neurosyphilis in the subject is calculated using the following formula: When the predicted probability P ≥ 0.687, the subject is considered to be at high risk of neurosyphilis; when the predicted probability P < 0.687, the subject is considered to be at low risk of neurosyphilis.

[0017] The detection module is used to obtain the detection results of TP17 / IgG, TP47 / IgG, TP15 / IgG and serum TRUST titers in the subject's saliva; the judgment module is used to output the neurosyphilis risk value or risk level.

[0018] The aforementioned combined logistic regression discriminant model is used to improve the screening and diagnosis rate of neurosyphilis, reduce unnecessary invasive examinations and lower the risk of missed detection, especially for asymptomatic or atypical individuals.

[0019] The beneficial effects of this invention are: 1) This invention's detection product establishes a correlation between saliva antibody detection and neurosyphilis risk, forming clear threshold judgment rules and stratification processes. This allows for early detection of neurosyphilis risk without lumbar puncture, improving screening efficiency, reducing unnecessary invasive examinations, and lowering the risk of missed detection, especially for asymptomatic or atypical cases. It also provides a basis for subsequent diagnostic pathways. To improve detection specificity and coverage of different immune response stages, the detection preferably uses a combination of recombinant Treponema pallidum antigens for capture / identification, specifically TP17, TP47, and TP15. A four-way combined logistic regression discriminant model is constructed by detecting saliva TP17 / IgG, TP47 / IgG, and TP15 / IgG antibodies in conjunction with serum TRUST titers, for auxiliary screening, risk prediction, and stratified management of neurosyphilis. 2) The invention's detection product is low-invasive and reproducible: It uses a combination of saliva and serum TRUST testing to achieve risk prediction. The collection is convenient and safe, making it suitable for repeated monitoring and long-term follow-up management. Through low-invasive testing, the indications for lumbar puncture are optimized, unnecessary invasive procedures are reduced, and the efficiency of medical resource utilization is improved. 3) The detection product of this invention helps to improve early identification capabilities: it provides an early warning tool for asymptomatic or atypical people, which helps to reduce the probability of missed detection and promote timely intervention; 4) The detection product of this invention helps to improve sensitivity and antibody typing: Currently, most commercially available enzyme-linked immunosorbent assay (ELISA) kits for neurosyphilis use a mixture of multiple proteins for coating, and the proportions are not clear, which can easily lead to missed diagnoses. Detecting TP17, TP15, and TP47 antibodies separately can improve the sensitivity of neurosyphilis diagnosis, and at the same time, it can also clarify the specific antibody type of positive results, which is of great significance for risk assessment of neurosyphilis and monitoring of treatment efficacy. 5) The evaluation and detection system of this invention is standardized and quantifiable: it outputs risk stratification results based on immune detection signals and combined with threshold rules, which facilitates the establishment of unified screening standards, quality control and multi-center promotion; 6) The evaluation and detection system platform of this invention has strong compatibility: it can be implemented on laboratory enzyme-linked immunosorbent assay (ELISA) platforms, and can also be extended to chemiluminescence, chromatography strips or microfluidics platforms, which is conducive to the application and implementation of medical institutions at different levels. Attached Figure Description

[0020] Figure 1 This is a comparative graph showing the distribution of saliva TP17 / IgG, TP47 / IgG, TP15 / IgG, and serum TRUST (toluidine red unheated serum test) among the latent syphilis, symptomatic neurosyphilis, and asymptomatic neurosyphilis groups. Figure 1 (a) is a comparison of the distribution of saliva TP17 / lgG among the latent syphilis group, the symptomatic neurosyphilis group and the asymptomatic neurosyphilis group; Figure 1 (b) is a comparative graph showing the distribution of saliva TP47 / lgG among the latent syphilis group, the symptomatic neurosyphilis group, and the asymptomatic neurosyphilis group; Figure 1 (c) is a comparison of the distribution of saliva TP15 / lgG among the latent syphilis group, the symptomatic neurosyphilis group and the asymptomatic neurosyphilis group; Figure 1 (d) is a comparative graph showing the distribution of serum TRUST among the latent syphilis group, the symptomatic neurosyphilis group, and the asymptomatic neurosyphilis group; Figure 2 This is a scatter plot showing the correlation between salivary TP17 / IgG, TP47 / IgG, TP15 / IgG and cerebrospinal fluid proteins and cerebrospinal fluid white blood cell count. Figure 2(a) is a scatter plot showing the correlation between salivary TP17 / lgG and cerebrospinal fluid proteins; Figure 2 (b) is a scatter plot showing the correlation between salivary TP17 / lgG and cerebrospinal fluid white blood cell count; Figure 2 (c) is a scatter plot showing the correlation between salivary TP47 / lgG and cerebrospinal fluid proteins; Figure 2 (d) is a scatter plot showing the correlation between salivary TP47 / lgG and cerebrospinal fluid white blood cell count; Figure 2 (e) is a scatter plot showing the correlation between salivary TP15 / lgG and cerebrospinal fluid proteins; Figure 2 (f) is a scatter plot showing the correlation between salivary TP15 / lgG and cerebrospinal fluid white blood cell count; Figure 3 This is a scatter plot showing the correlation between saliva TP17 / IgG, TP47 / IgG, TP15 / IgG and serum TRUST titer. Figure 3 (a) is a scatter plot showing the correlation between salivary TP17 / IgG and serum TRUST titer; Figure 3 (b) is a scatter plot showing the correlation between salivary TP47 / lgG and serum TRUST titer; Figure 3 (c) is a scatter plot showing the correlation between salivary TP15 / lgG and serum TRUST titer; Figure 4 ROC curves for the combined use of saliva TP17 / IgG, TP47 / IgG, TP15 / IgG, and serum TRUST titer as four indicators for the identification of neurosyphilis. Detailed Implementation

[0021] To better understand the above technical solutions, exemplary operation procedures and experimental steps of the present invention will be given below in conjunction with embodiments. Although exemplary embodiments of the present invention are described in this specification, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments described herein.

[0022] Example 1

[0023] Experimental reagents: Tris buffered saline solution (TBST) containing Tween 20, bovine serum albumin (BSA), The coated antigens TP17, TP47, and TP15 were purchased from Suzhou Nearshore Protein Technology Co., Ltd. (hereinafter referred to as Nearshore Protein), with batch numbers of TP17: Cat.No.DRA94, TP47: Cat.No.DRA151, and TP15: Cat.No.DRA172, respectively.

[0024] (a) Subject selection and sample collection 1. Source of examinees Subjects can be individuals suspected of having syphilis, individuals diagnosed with syphilis, individuals undergoing follow-up visits, or individuals requiring assessment of neurosyphilis risk. Those with neurological symptoms and signs can be prioritized for assessment; those without obvious neurological symptoms can be the focus of screening, in order to achieve early warning and stratified management.

[0025] 2. Preparation before saliva collection To reduce the impact of diet, oral hygiene, and external contamination on the test, subjects should preferably avoid eating, drinking, smoking, chewing gum, or brushing their teeth and rinsing their mouths for at least 30 minutes before sampling. Before sampling, they can rinse their mouths lightly with water and let them sit for 10 minutes to reduce the interference of oral residue on the test signal.

[0026] 3. Saliva collection methods and preservation Saliva samples can be collected by natural salivation, with a preferred collection volume of 1.0 mL to 2.0 mL. Sampling containers can be disposable sterile saliva collection tubes or samplers with funnels. After collection, samples should be optimized and aliquoted as soon as possible. For short-term testing, samples can be stored at 2–8°C; if immediate testing is not possible, samples can be frozen at -80°C according to the reagent system requirements. A complete cold chain is recommended during transportation to reduce the risk of antibody degradation and sample deterioration, thereby ensuring the reliability of test results.

[0027] (ii) Saliva sample pretreatment To minimize interference from mucus, food residue, and particulate matter on the immune response, saliva samples should be centrifuged at 10,000 rpm for 10 minutes as soon as possible after collection to clarify the sample. The supernatant should be used as the test sample. If possible, a protease inhibitor can be added before freezing. Alternatively, if the sample is not completely clear, the centrifugation time can be extended to 30 minutes, or a layered centrifugation method can be used to separate and remove insoluble impurities, improving repeatability and signal-to-noise ratio.

[0028] (III) ELISA-based detection of anti-Treponema pallidum antibodies in saliva 1. Antigen system and solid-phase carrier (1) Coating: The solid-phase carrier is preferably a 96-well microplate. The antigen used to capture antibodies in saliva can be the recombinant Treponema pallidum antigens TP17, TP47, and TP15 to improve sensitivity and specificity. The antigen can be pre-coated in the microplate (100 μL / well), or the operator can complete the coating according to the conventional ELISA coating process. The coating concentration can be 1 μg / mL-10 μg / mL. After coating with the antigen, add 300 μL of Tris buffered saline (TBST) containing Tween-20 to each well, let stand for 1 minute, wash 3 times, shake off the washing solution, and then gently pat dry on the paper.

[0029] (2) Blocking: Add 200 μL of 5% BSA to each well and incubate overnight at 4°C. After blocking, wash (same as coating steps). If the microplate cannot be tested immediately, it can be air-dried and stored in a refrigerator at 4°C for 1-2 weeks.

[0030] 2. Testing Process (1) Sample loading: Add the processed saliva sample into the coated well (100 μL / well), and set up negative control, positive control and blank wells, and set up duplicate wells for each; (2) Incubation: Cover with a sealing plate and incubate at 37°C for 2 hours; (3) Washing: Add 300 μL of washing solution (TBST) to each well, wash 5 times, let stand for 3 minutes each time, shake off the washing solution and then gently drain on the paper; (4) Add secondary antibody: Add 100 μL of enzyme-labeled anti-human IgG secondary antibody (diluted appropriately) to each well, incubate at 37°C for 1 hour, and then wash 5 times for 3 minutes each time; (5) Color development: Add 100 μL of substrate (TMB) to each well for color development, and add stop solution (100 μL / well) after an appropriate time (10-15 minutes). (6) Reading: Read the absorbance in the microplate reader. The commonly used reading wavelength is 450 nm. Record the OD value as the detection signal.

[0031] 3. Quality control and judgment value calculation Intra-batch consistency can be ensured using the control method: for example, requiring negative control OD values ​​to be below a preset upper limit and positive control OD values ​​to be above a preset lower limit; alternatively, S / CO (sample / cutoff point) can be used for cross-batch comparisons. The cutoff point can be determined by calibrators or obtained by adding a multiple of the standard deviation to the mean of negative samples. The above quality control and calculation rules can be incorporated into the kit instructions or laboratory SOPs to ensure the stability and reproducibility of results.

[0032] (iv) Threshold-based assessment of neurosyphilis risk The discriminant model of this invention is constructed based on saliva TP17 / IgG, saliva TP47 / IgG, saliva TP15 / IgG, and serum TRUST titer. The model can be implemented using logistic regression, linear discriminant analysis, support vector machine, decision tree, random forest, neural network, or other classification models that can output risk values.

[0033] A four-way joint discriminant model was established using logistic regression, with the linear prediction formula: Z = −4.7881 + 1.2443 × A + 0.3594 × B + 0.0038 × C + 0.6076 × D. Wherein, coefficient A represents the OD value of saliva TP17 / IgG, coefficient B represents the OD value of saliva TP47 / IgG, coefficient C represents the OD value of saliva TP15 / IgG, and coefficient D represents the serum TRUST titer grade coding value. Preferably, the TRUST titer is quantified using a grade assignment method, with negative being 0, 1:1 being 1, 1:2 being 2, 1:4 being 3, 1:8 being 4, and so on.

[0034] The predictive probability P of developing neurosyphilis in the subject is calculated using the following formula:

[0035] The higher the predicted probability P, the higher the likelihood that the subject has neurosyphilis.

[0036] The aforementioned combined logistic regression discriminant model is used to improve the screening and diagnosis rate of neurosyphilis, reduce unnecessary invasive examinations and lower the risk of missed detection, especially for asymptomatic or atypical individuals.

[0037] (v) Judgment rules based on risk value After inputting the subjects' saliva TP17 / IgG, saliva TP47 / IgG, saliva TP15 / IgG, and serum TRUST titer into the discriminant model, the predicted probability P of neurosyphilis was obtained. This predicted probability was compared with a preset threshold to achieve risk stratification.

[0038] In a preferred embodiment, when P < 0.687, it is determined to be non-neurosyphilis or low-risk neurosyphilis; when P ≥ 0.687, it is determined to be neurosyphilis or high-risk neurosyphilis.

[0039] For high-risk individuals, it is recommended to make a comprehensive judgment based on clinical manifestations, neurological symptoms, imaging, and cerebrospinal fluid examination; lumbar puncture and cerebrospinal fluid testing are recommended if necessary. For low-risk individuals, follow-up observation or re-examination if necessary is recommended.

[0040] (vi) Reagent kit implementation method To facilitate clinical application, the method of this invention can be implemented using a matching reagent kit. The reagent kit may include: (1) A solid-phase carrier coated with Treponema pallidum recombinant antigen (preferably TP17, TP47, TP15); (2) Enzyme-labeled anti-human IgG secondary antibody; (3) Sample washing solution and sealing solution; (4) Chromogenic substrate and stop solution; (5) Negative control and positive control; The kit can include judgment rules for outputting risk results (such as thresholds and corresponding risk stratification recommendations) so that different institutions can implement them under a unified judgment logic.

[0041] (vii) Example of method application process (outpatient / screening scenario) In outpatient or screening settings, the following procedure can be followed: (1) Collect saliva samples from suspected or confirmed syphilis patients and complete immune testing; (2) Output risk level according to threshold; (3) Provide high-risk individuals with appropriate treatment for neurosyphilis as early as possible; (4) Follow up with low-risk individuals and conduct retesting when necessary; (5) For individuals at medium risk, a comprehensive decision should be made based on serum titer, syphilis stage, and neurological symptoms and signs.

[0042] This process can reduce the proportion of invasive procedures while improving screening coverage and treatment efficiency for asymptomatic neurosyphilis.

[0043] (viii) Statistical analysis of experimental results (1) Differences in the expression of saliva TP17 / IgG, TP47 / IgG, TP15 / IgG and other related indicators in different groups The levels of TP17 / IgG, TP47 / IgG, and TP15 / IgG in the saliva of the subjects were detected using ELISA, and statistical analysis was performed in conjunction with serum TRUST titer and cerebrospinal fluid inflammation-related indicators. Results are shown in Table 1. Figure 1 The levels of saliva TP17 / IgG, TP47 / IgG, TP15 / IgG, and serum TRUST in patients with symptomatic neurosyphilis and asymptomatic neurosyphilis were higher than those in the latent neurosyphilis group.

[0044] Table 1 shows that there were statistically significant differences in TP17 / IgG, TP47 / IgG, TP15 / IgG and serum TRUST between the latent syphilis group and the neurosyphilis group (P < 0.001). Figure 1 Further evidence indicates that the three saliva antibody indicators and serum TRUST were generally higher in the neurosyphilis group than in the latent syphilis group, suggesting that the combined detection of the three saliva indicators and serum TRUST titer has good auxiliary identification value.

[0045]

[0046] (2) Correlation between salivary TP17 / IgG, TP47 / IgG, TP15 / IgG and cerebrospinal fluid inflammatory markers To further clarify the relationship between salivary antibody markers and neurosyphilitic inflammatory damage, correlation analyses were performed on salivary TP17 / IgG, TP47 / IgG, and TP15 / IgG with cerebrospinal fluid proteins and cerebrospinal fluid white blood cell counts, respectively. Results are shown below. Figure 2 .

[0047] The results showed that saliva TP17 / IgG, TP47 / IgG, and TP15 / IgG were all positively correlated with cerebrospinal fluid proteins and cerebrospinal fluid white blood cell count, and these correlations were statistically significant. These results indicate that these three salivary antibody indicators can not only be used for auxiliary screening and typing of neurosyphilis, but also reflect cerebrospinal fluid inflammation-related changes to a certain extent.

[0048] (3) Correlation between salivary TP17 / IgG, TP47 / IgG, TP15 / IgG and serum TRUST titer To further evaluate the relationship between salivary antibody markers and traditional serological markers, correlation analyses were performed between salivary TP17 / IgG, TP47 / IgG, and TP15 / IgG and serum TRUST titers. Results are shown below. Figure 3 .

[0049] Figure 3 Scatter plots showing the correlation between salivary TP17 / IgG, salivary TP47 / IgG, and salivary TP15 / IgG and serum TRUST titer. The results showed a significant positive correlation between salivary TP17 / IgG and serum TRUST titer; salivary TP47 / IgG and serum TRUST titer were also positively correlated; and all were statistically significant. This suggests that these three indicators have good consistency with traditional serological disease activity indicators, further supporting their value in combined application with TRUST.

[0050] (4) The role of single-indicator and four-indicator combination in the diagnosis of neurosyphilis To further clarify the diagnostic capabilities of single and quadruple indicators for neurosyphilis, ROC curve analysis was performed on a quadruple model consisting of saliva TP17 / IgG, TP47 / IgG, TP15 / IgG, and serum TRUST titer. The results are shown in [Figure number missing]. Figure 4 .

[0051] In this embodiment, the applicant used Logistic regression to jointly model salivary TP17 / IgG, TP47 / IgG, TP15 / IgG, and serum TRUST titers, obtaining the following discriminant equation: Z = −4.7881 + 1.2443 × A + 0.3594 × B + 0.0038 × C + 0.6076 × D. Wherein, coefficient A represents the OD value of salivary TP17 / IgG, coefficient B represents the OD value of salivary TP47 / IgG, coefficient C represents the OD value of salivary TP15 / IgG, and coefficient D represents the serum TRUST titer grade coding value. Preferably, the TRUST titer is quantified using a grade assignment method, with negative being 0, 1:1 being 1, 1:2 being 2, 1:4 being 3, 1:8 being 4, and so on.

[0052] The predictive probability P of developing neurosyphilis in the subject is calculated using the following formula:

[0053] When P=0.687 is used as the optimal cutoff value, it can effectively distinguish between neurosyphilis and latent syphilis.

[0054] The results showed that the four-in-one discriminant model constructed from saliva TP17 / IgG, TP47 / IgG, TP15 / IgG, and serum TRUST titer had good discriminative efficacy for neurosyphilis. Its area under the ROC curve (AUC) was 0.949, the 95% confidence interval was 0.917–0.980, the optimal cutoff value was 0.687, and the corresponding sensitivity was 88.7% and specificity was 91.9%. These results indicate that the four-in-one detection model based on saliva TP17 / IgG, TP47 / IgG, TP15 / IgG, and serum TRUST titer can effectively distinguish between neurosyphilis and non-neurosyphilis individuals and can be used for neurosyphilis risk value output and risk stratification assessment.

[0055] In summary, this invention provides a technical solution for neurosyphilis screening, auxiliary diagnosis, and risk stratification based on the detection of Treponema pallidum-specific antibodies in salivary fluid combined with serum TRUST titer. The results confirmed that salivary TP17 / IgG, TP47 / IgG, and TP15 / IgG were all significantly elevated in the neurosyphilis group and were positively correlated with cerebrospinal fluid inflammatory markers and serum TRUST titer. Furthermore, the combined salivary TP17 / IgG, TP47 / IgG, and TP15 / IgG with serum TRUST titer can be used to construct a four-in-one logistic regression discriminant model to output a neurosyphilis risk value, providing a basis for neurosyphilis screening, auxiliary diagnosis, and further examination pathway management.

Claims

1. A system for assessing the risk of neurosyphilis, characterized in that, The system includes a detection module and a judgment module. The detection module is used to obtain the OD value of anti-Treponema pallidum IgG antibody in saliva. The judgment module includes a readable carrier storing judgment rules. The judgment module includes a preset discrimination model. The preset discrimination model is a four-in-one logistic regression discrimination model constructed based on saliva TP17 / IgG, saliva TP47 / IgG, saliva TP15 / IgG, and serum TRUST titer. The four-in-one logistic regression discrimination model satisfies the following formula: Z = 4.7881 + 1.2443 × A + 0.3594 × B + 0.0038 × C + 0.6076 × D; where coefficient A represents the OD value of saliva TP17 / IgG, coefficient B represents the OD value of saliva TP47 / IgG, coefficient C represents the OD value of saliva TP15 / IgG, and coefficient D represents the serum TRUST titer level coding value.

2. The system according to claim 1, characterized in that, The predictive probability P of developing neurosyphilis in the subject is calculated using the following formula: When the predicted probability P is greater than or equal to the threshold, the subject is determined to be at high risk of neurosyphilis; when the predicted probability P is less than the threshold, the subject is determined to be at low risk of neurosyphilis.

3. The system according to claim 2, characterized in that, The threshold value is 0.

687.

4. The system as described in claim 1, characterized in that, The detection module is an enzyme-linked immunosorbent assay (ELISA) kit, which includes: a solid-phase carrier coated with Treponema pallidum antigens TP17, TP47, and TP15 respectively; a labeled secondary antibody for recognizing human IgG; a blocking solution; a washing solution; a chromogenic substrate; a stop solution; and a collection container; wherein the solid-phase carrier is a microplate, and the antigens are coated on the solid-phase surface of the microplate.

5. The system as described in claim 4, characterized in that, The kit uses a method for collecting and preprocessing saliva markers, characterized by the following steps: (a) Collect saliva samples, with a volume of 1.0 mL to 2.0 mL. Store the samples at 2 to 8°C and test them within 30 minutes. If testing cannot be performed within 30 minutes, freeze them at -80°C. (b) Centrifuge the saliva sample at 10,000 rpm for 10 minutes to clarify it, take the supernatant as the sample to be tested, add protease inhibitor and freeze it; for samples with high viscosity, extend the centrifugation time by 30 minutes, or use layer centrifugation to further clarify and remove impurities and mucin and other components.

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