Kit for predicting curative effect of Alzheimer's disease deep jugular vein lymphatic anastomosis and screening adaptive population and application thereof

Abstract

The invention relates to the field of in-vitro diagnostic kits, and discloses a kit for predicting the curative effect of deep jugular vein lymphatic anastomosis of Alzheimer's disease and screening adaptive population and application of the kit. The kit comprises a mass spectrometry detection module, an ELISA detection module and a logistic regression model, the mass spectrum detection module is used for detecting the protein concentrations of NPTX2 and IGSF10 in a sample; the ELISA detection module is used for detecting the protein concentrations of ptau181 and A beta 42 in a sample; by calculating the NPTX2 / IGSF10 ratio and the ptau181 / A beta42 ratio and combining with a logistic regression model, the LVA operation curative effect is predicted, and adaptive people are screened. The kit provided by the invention has high sensitivity and specificity, can accurately detect the concentration of related proteins in a sample, and provides a reliable basis for curative effect prediction of Alzheimer's disease deep jugular vein lymphatic anastomosis (LVA) and adaptive population screening.

Description

Technical Field

[0001] This application relates to the field of in vitro diagnostic reagent kits, and more specifically, it relates to a kit for predicting the efficacy of Alzheimer's disease deep jugular vein lymph node anastomosis and screening suitable populations, as well as its application. Background Technology

[0002] Alzheimer's disease (AD) is a globally prevalent neurodegenerative disease characterized by progressive cognitive decline and memory loss. Its pathological features include abnormal deposition of amyloid-beta (Aβ) in the brain, forming senile plaques; aggregation of hyperphosphorylated tau protein (ptau), forming neurofibrillary tangles; and synaptic damage and neuronal loss. Cerebrospinal fluid (CSF) is considered the "gold standard" for AD detection due to its irreplaceable advantages: It directly reflects the intracranial pathological state: CSF communicates directly with the interstitial fluid of the brain tissue, and the concentrations of markers such as Aβ42, ptau181, and NPTX2 are highly synchronized with the progression of intracranial pathology, making it more specific than peripheral blood (such as plasma); and it has high marker concentrations: the concentrations of AD-related proteins in CSF are significantly higher than in peripheral blood (e.g., ptau181 concentration in CSF is 20-80 pg / mL, while in peripheral blood it is only 1-5 pg / mL), allowing for detection without complex enrichment processes.

[0003] Deep jugular vein lymphoassay (LVA) is a microsurgical procedure that reconstructs the lymphatic drainage pathways to the brain by anastomosing the superficial lymphatic vessels of the neck to the deep jugular vein, thereby promoting the clearance of neurotoxic proteins such as Aβ and ptau from the brain. It is currently used as an adjunct therapy for Alzheimer's disease (AD). However, the clinical application of LVA surgery faces a critical bottleneck: difficulty in selecting suitable candidates. There is a lack of objective indicators to determine which AD patients can benefit from the surgery, and some patients do not respond well to the procedure.

[0004] Existing molecular detection technologies for AD cannot meet the clinical needs of LVA surgery. Specific problems include: existing AD diagnosis relies on cognitive assessment scales (such as the Mini-Mental State Examination MMSE), imaging examinations (such as cranial MRI / PET), and single molecular markers (such as cerebrospinal fluid ptau181 and Aβ42), which have the following characteristics: (1) strong subjectivity: cognitive scales are greatly affected by patient cooperation and assessor experience; (2) insufficient accuracy: single markers (such as ptau181 / Aβ42) have limited predictive ability for AD progression and LVA surgical treatment response; (3) poor technical synergy: mass spectrometry is used to detect a small number of proteins (narrow coverage, difficult to measure NPTX2 / IGSF10 at the same time), or only ELISA is used to detect ptau181 / Aβ42 (lacking data on surgical target-related proteins), and a "mass spectrometry + (4) Lack of integrated model: The ratio of multiple markers (such as NPTX2 / IGSF10, ptau181 / Aβ42) was not combined with clinical efficacy to construct a mathematical model, and it was impossible to quantify and predict the suitability of LVA surgery and the probability of postoperative improvement.

[0005] In conclusion, existing molecular detection technologies for Alzheimer's disease (AD) cannot meet the clinical needs of LVA surgery. Summary of the Invention

[0006] The purpose of this invention is to provide a high-precision, modular combined detection kit for cerebrospinal fluid (CSF). It uses high-resolution mass spectrometry to detect NPTX2 / IGSF10 in CSF, ELISA to detect ptau181 / Aβ42 in CSF, and combines a logistic regression model to achieve precise preoperative screening of candidates suitable for LVA surgery.

[0007] To achieve the above-mentioned objectives, this application adopts the following technical solution: In a first aspect, this application provides a kit for predicting the efficacy of Alzheimer's disease deep jugular vein lymph node anastomosis and screening suitable populations, the kit comprising a mass spectrometry detection module, an ELISA detection module and a logistic regression model; The mass spectrometry detection module is used to detect the concentrations of NPTX2 and IGSF10 proteins in the sample; The ELISA detection module is used to detect the concentrations of ptau181 and Aβ42 proteins in the sample; By calculating the NPTX2 / IGSF10 ratio and the ptau181 / Aβ42 ratio, and combining them with a logistic regression model, we can predict the efficacy of LVA surgery and select suitable candidates.

[0008] Furthermore, the test sample for the kit is a cerebrospinal fluid sample.

[0009] Furthermore, the logistic regression model is as follows: logit(P) = 1.807×(NPTX2 / IGSF10) + 10.461×(ptau181 / Abeta42) – 3.172; Where P is the probability of clearing toxic proteins from cerebrospinal fluid after surgery. When P ≥ 0.355, the patient is considered a suitable candidate for LVA surgery.

[0010] Furthermore, the mass spectrometry detection module includes: Mobile phase: Phase A is a 0.1% formic acid aqueous solution, and Phase B is a 0.1% formic acid acetonitrile solution; Reconstitution reagent: 0.1% formic acid solution, containing internal standard. 13 C-labeled NPTX2 and IGSF10 peptides; Standards: NPTX2 and IGSF10 standards, with concentration gradients of 0.05, 0.1, 1, 5, and 10 ng / mL; Consumables: 0.22 μm organic phase filter membrane and C18 reversed-phase column.

[0011] Furthermore, the mass spectrometry detection module uses DIA mode to screen characteristic peptides and MRM mode for targeted quantification, with ion pairs of NPTX2 m / z 794.19→312.1 and IGSF10 m / z 797.58→295.0.

[0012] Furthermore, the ELISA detection module includes: Pre-coated antibody plates: coated with anti-ptau181 monoclonal antibody and anti-Aβ42 monoclonal antibody; Detection reagents: enzyme-labeled secondary antibody, substrate solution, and stop solution; Standards: ptau181 and Aβ42 standards, with concentration gradients of 10, 20, 40, 80, and 160 pg / mL; Auxiliary reagents: washing solution and blocking solution.

[0013] Furthermore, the kit also includes a cerebrospinal fluid pretreatment group, which comprises: Pretreatment reagents for mass spectrometry: DDM lysis buffer, DTT solution, IAA solution, trypsin and pH 8.0 Tris-HCl buffer; ELISA pretreatment reagents: diluent.

[0014] Furthermore, the cerebrospinal fluid sample is diluted at a ratio of 1:10 in the ELISA test.

[0015] Furthermore, the kit also includes a quality control and support group, which includes: Quality control materials: Cerebrospinal fluid matrix quality control solution containing known concentrations of NPTX2, IGSF10, ptau181, and Aβ42, as well as blank cerebrospinal fluid; Supporting materials: Cerebrospinal fluid collection guidelines, testing instructions, and logistic regression model calculation table.

[0016] Secondly, this application provides the application of the kit in the preparation of products for predicting the efficacy of Alzheimer's disease deep jugular vein lymph node anastomosis and screening suitable populations.

[0017] In summary, this application has the following beneficial effects: 1) Technical performance advantages High compatibility: The standard concentration gradient, pretreatment parameters, and detection process are all matched to the actual concentration of the target protein and matrix characteristics in cerebrospinal fluid, avoiding detection errors (such as ELISA standard curve R). 2 ≥0.995, mass spectrometry quantitative deviation ≤4%). High-accuracy prediction: The dual ratio + cerebrospinal fluid adaptation model achieves a prediction accuracy of 82.7% (AUC=0.880), specificity of 77.2%, and sensitivity of 89.2%. 2) Clinical application value Reduce surgical risks: Preoperative screening of suitable candidates using specific indicators of cerebrospinal fluid reduces "ineffective surgeries"; Ease of operation: The pretreatment steps for cerebrospinal fluid are simplified (e.g., lysis time is shortened to 20 min and incubation time is shortened to 45 min), making it suitable for routine clinical laboratory operations; Research value: It can be used to study the pathological mechanism of AD (such as the association between NPTX2 and IGSF10 in cerebrospinal fluid), and provide cerebrospinal fluid molecular level evidence for the optimization of LVA surgery. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the workflow of the reagent kit of the present invention; Figure 2 This is the decision boundary diagram of the logistic regression model of this invention; Figure 3 This is a subject operating characteristic curve diagram of the present invention. Detailed Implementation

[0019] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0020] Example 1 This embodiment provides a kit for predicting the efficacy of Alzheimer's disease deep jugular vein lymph node anastomosis and for screening suitable candidates. Its components are as follows: The kit adopts a dual-module design of "mass spectrometry module + ELISA module", and each component is adapted for single / batch clinical testing, as detailed below:

[0021] The above kit usage instructions are as follows (total process ≤ 8.5 hours, specifically designed for cerebrospinal fluid samples): (1) Collection and processing of cerebrospinal fluid samples Collection method: 1000μL of cerebrospinal fluid was collected by lumbar puncture (aseptic operation to avoid red blood cell contamination); Pretreatment: Centrifuge at 4°C (3000g, 5min) within 2 hours after collection to remove a small amount of cell debris. Take the supernatant and divide it into two portions (100μL for mass spectrometry detection and 100μL for ELISA detection). If not detected immediately, freeze at -80°C (≤3 freeze-thaw cycles).

[0022] Parallel detection: Mass spectrometry for NPTX2 / IGSF10 + ELISA for ptau181 / Abeta42 Mass spectrometry detection branch (specific parameters for cerebrospinal fluid) ① Pretreatment: Take 100 μL of cerebrospinal fluid supernatant and add DDM lysis buffer (4℃, 20 min, no need for long lysis time as the cerebrospinal fluid matrix is ​​simple); centrifuge at 12000g for 15 min and collect the supernatant; filter through a 0.22 μm filter membrane; add DTT (56℃ water bath for 30 min to reduce disulfide bonds); after cooling, add IAA (protect from light for 20 min for alkylation); adjust the pH to 8.0 and add trypsin (37℃ shaking and digestion for 18 h, suitable for low protein content of cerebrospinal fluid, shortening the digestion time). ②Mass spectrometry analysis: After lyophilizing the enzymatic hydrolysate, resuspend it with a reconstitution reagent (inject 1 μL) → HPLC gradient elution (0-2 min, 2% B; 2-46 min, 2% B → 22% B; 46-58 min, 22% B → 30% B; 58-60 min, 30% B → 55% B) → DIA mode screening of characteristic peptides (scanning range m / z 400-1200, ESI+, ion source temperature 320℃, spray voltage 2600V) → MRM mode targeted quantification (ion pair: NPTX2 m / z 794.19 → 312.1, IGSF10 m / z 797.58 → 295.0, residence time 100ms, DP voltage 85V) → Calculate the NPTX2 / IGSF10 ratio.

[0023] ELISA detection pathway (specific parameters for cerebrospinal fluid) ① Sample processing: Dilute 100 μL of cerebrospinal fluid supernatant 1:10 with special diluent (to match the concentration of ptau181 / Abeta42 in cerebrospinal fluid and avoid signal saturation). ② ELISA Procedure: Add standard / diluted cerebrospinal fluid (100 μL / well, incubate at 37℃ for 45 min; shorten incubation time due to the high specificity of cerebrospinal fluid markers); wash the plate 3 times (using cerebrospinal fluid-specific washing buffer); add enzyme-labeled secondary antibody (100 μL / well, incubate at 37℃ for 25 min); wash the plate 5 times; add substrate solution (50 μL / well, incubate in the dark for 12 min; high-sensitivity substrate is adapted for low pg detection); add stop solution (50 μL / well); read the value at 450 nm using the microplate reader; calculate the concentrations of ptau181 and Abeta42 based on the cerebrospinal fluid adaptation standard curve; calculate the ptau181 / Abeta42 ratio.

[0024] (2) Prediction of cerebrospinal fluid adaptation using logistic regression model Model construction: Based on cerebrospinal fluid sample data from 100 AD patients, 80% of which was used as the training set and 20% as the test set (preoperative and postoperative test values ​​+ 12-month MMSE follow-up + cerebrospinal fluid toxic protein clearance rate detection). The formula is: logit(P) = 1.807×(NPTX2 / IGSF10) + 10.461×(ptau181 / Abeta42) – 3.172 (P is the postoperative cerebrospinal fluid toxic protein clearance probability). Results interpretation: Preoperative screening was based on maximizing the Youden index as the core criterion. When P ≥ 0.355, the candidates were identified as suitable for LVA surgery.

[0025] Example 2: Screening of candidates suitable for LVA surgery (cerebrospinal fluid samples) Sample source: A 68-year-old male AD patient with an MMSE score of 22, from whom 1000 μL of cerebrospinal fluid was collected by lumbar puncture (no hemolysis was found in the supernatant after centrifugation).

[0026] Test results: Mass spectrometry detection: NPTX2 = 0.3 ng / mL, IGSF10 = 0.7 ng / mL, NPTX2 / IGSF10 = 0.43; ELISA results: ptau181 = 55 pg / mL, Aβ42 = 180 pg / mL, ptau181 / Aβ42 = 0.31; Model calculation: logit(P) = 1.807 × 0.43 + 10.461 × 0.31 - 3.172 = -5.87, P = 0.002 (< 0.355); Clinical decision: The patient was determined to be a "non-surgical candidate" and drug treatment was recommended.

[0027] Example 3: Postoperative efficacy evaluation of LVA (cerebrospinal fluid sample) Sample source: 75-year-old female AD patient (preoperative cerebrospinal fluid test P=0.78, determined to be the eligible population), whose MMSE score improved after LVA surgery.

[0028] Test results: Mass spectrometry analysis: NPTX2 = 1.1 ng / mL (preoperative 0.4 ng / mL, up 175%), IGSF10 = 0.8 ng / mL (preoperative 0.9 ng / mL, basically stable), NPTX2 / IGSF10 = 1.38; ELISA results: ptau181 = 32 pg / mL (preoperative 60 pg / mL, a decrease of 47%), Aβ42 = 260 pg / mL (preoperative 160 pg / mL, an increase of 62.5%), ptau181 / Aβ42 = 0.12; Model calculation: logit(P) = 1.807 × 1.38 + 10.461 × 0.12 - 3.172 = -5.03, P = 0.006 (≥ 0.355); Clinical validation: The patient's MMSE score improved from 20 to 25 points after surgery, consistent with the kit's prediction of "postoperative improvement".

[0029] The beneficial effects of this invention are: 1) Technical performance advantages (for cerebrospinal fluid samples) High compatibility: The standard concentration gradient, pretreatment parameters, and detection process are all matched to the actual concentration of the target protein and matrix characteristics in cerebrospinal fluid, avoiding detection errors (such as ELISA standard curve R). 2 ≥0.995, mass spectrometry quantitative deviation ≤4%). High-accuracy prediction: The dual ratio + cerebrospinal fluid adaptation model achieves a prediction accuracy of 82.7% (AUC=0.880), specificity of 77.2%, and sensitivity of 89.2%. 2) Clinical application value Reduce surgical risks: Preoperative screening of suitable candidates using specific indicators of cerebrospinal fluid reduces "ineffective surgeries"; Ease of operation: The pretreatment steps for cerebrospinal fluid are simplified (e.g., lysis time is shortened to 20 min and incubation time is shortened to 45 min), making it suitable for routine clinical laboratory operations; Research value: It can be used to study the pathological mechanism of AD (such as the association between NPTX2 and IGSF10 in cerebrospinal fluid), and provide cerebrospinal fluid molecular level evidence for the optimization of LVA surgery.

[0030] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A kit for predicting the efficacy of Alzheimer's disease deep jugular vein lymph node anastomosis and screening suitable patients, characterized in that, The kit includes a mass spectrometry detection module, an ELISA detection module, and a logistic regression model; The mass spectrometry detection module is used to detect the concentrations of NPTX2 and IGSF10 proteins in the sample; The ELISA detection module is used to detect the concentrations of ptau181 and Aβ42 proteins in the sample; By calculating the NPTX2 / IGSF10 ratio and the ptau181 / Aβ42 ratio, and combining them with a logistic regression model, we can predict the efficacy of LVA surgery and select suitable candidates.

2. The reagent kit according to claim 1, characterized in that, The test sample for this kit is a cerebrospinal fluid sample.

3. The reagent kit according to claim 2, characterized in that, The logistic regression model is as follows: logit(P) = 1.807×(NPTX2 / IGSF10) + 10.461×(ptau181 / Abeta42) – 3.172; Wherein, P is the probability of clearing toxic proteins from cerebrospinal fluid after surgery. When P≥0.355, the patient is considered a suitable candidate for LVA surgery.

4. The reagent kit according to claim 1, characterized in that, The mass spectrometry detection module includes: Mobile phase: Phase A is a 0.1% formic acid aqueous solution, and Phase B is a 0.1% formic acid acetonitrile solution; Reconstitution reagent: 0.1% formic acid solution, containing internal standard. 13 C-labeled NPTX2 and IGSF10 peptides; Standards: NPTX2 and IGSF10 standards, with concentration gradients of 0.05, 0.1, 1, 5, and 10 ng / mL; Consumables: 0.22 μm organic phase filter membrane and C18 reversed-phase column.

5. The reagent kit according to claim 4, characterized in that, The mass spectrometry detection module uses DIA mode to screen characteristic peptides and MRM mode for targeted quantification. The ion pairs are NPTX2 m / z 794.19→312.1 and IGSF10 m / z 797.58→295.

0.

6. The reagent kit according to claim 1, characterized in that, The ELISA detection module includes: Pre-coated antibody plates: coated with anti-ptau181 monoclonal antibody and anti-Aβ42 monoclonal antibody; Detection reagents: enzyme-labeled secondary antibody, substrate solution, and stop solution; Standards: ptau181 and Aβ42 standards, with concentration gradients of 10, 20, 40, 80, and 160 pg / mL; Auxiliary reagents: washing solution and blocking solution.

7. The kit according to claim 1, characterized in that, The kit also includes a cerebrospinal fluid pretreatment unit, which comprises: Pretreatment reagents for mass spectrometry: DDM lysis buffer, DTT solution, IAA solution, trypsin and pH 8.0 Tris-HCl buffer; ELISA pretreatment reagents: diluent.

8. The reagent kit according to claim 7, characterized in that, The cerebrospinal fluid sample was diluted at a ratio of 1:10 in the ELISA test.

9. The reagent kit according to claim 1, characterized in that, The kit also includes a quality control and support group, which includes: Quality control materials: Cerebrospinal fluid matrix quality control solution containing known concentrations of NPTX2, IGSF10, ptau181, and Aβ42, as well as blank cerebrospinal fluid; Supporting materials: Cerebrospinal fluid collection guidelines, testing instructions, and logistic regression model calculation table.

10. The use of the kit according to any one of claims 1-9 in the preparation of a product for predicting the efficacy of Alzheimer's disease deep jugular vein lymph node anastomosis and screening suitable populations.

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