Use of tnfr2 in diagnosis and / or prognosis assessment of heart failure, kits and methods of use

Abstract

The application belongs to the technical field of biological medicine, and relates to application of TNFR2 (tumor necrosis factor receptor 2) in diagnosis and / or prognosis evaluation of heart failure, a kit and a use method; in particular to application of a reagent for detecting a biomarker, i.e., TNFR2, in a product for diagnosis and / or prognosis evaluation of heart failure; the application proves that TNFR2 is significantly increased in plasma of heart failure patients, and has a significant statistical difference (P<0.0001) compared with a healthy control group through proteomics screening and multi-center clinical cohort verification; research shows that the AUC value of TNFR2 for diagnosing heart failure is excellent in a discovery cohort and a verification cohort; meanwhile, TNFR2 is an independent risk factor for all-cause death of heart failure, and has prediction robustness in medium and long-term prognosis prediction capability; the kit for detecting TNFR2 and the use method provided by the application are simple to detect, have good sensitivity and specificity, and have high clinical popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostics and biomedical technology, specifically relating to the application of TNFR2 in the diagnosis and / or prognostic assessment of heart failure, kits, and methods of use. Background Technology

[0002] Heart failure (HF) is an abnormal change in the structure and function of the heart caused by myocardial ischemia, cardiomyopathy, hypertension, etc., and is the end stage of all cardiovascular diseases. The pathophysiological process of heart failure is complex, involving multiple links such as excessive activation of the neuroendocrine system, myocardial fibrosis, inflammatory response, and cardiomyocyte apoptosis. Among them, the inflammatory response plays a crucial role in the occurrence and development of heart failure and is considered to be one of the core driving mechanisms (J Am Coll Cardiol.2020, 75(11):1324-1340).

[0003] Currently, the clinical diagnosis of heart failure mainly relies on biomarker detection. Among them, B-type natriuretic peptide (BNP) and its N-terminal precursor (NT-proBNP, N-terminal pro-B-type natriuretic peptide) have been established as the gold standard biomarkers for the diagnosis and risk stratification of heart failure (Rev Esp Cardiol (Engl Ed. 2016;69(12):1167). However, extensive clinical practice has also revealed the inherent limitations of BNP / NT-proBNP: First, its blood concentration is easily affected by various non-cardiac factors, especially renal insufficiency, advanced age, and obesity (Clin Chem. 2007, 53(11): 1928-35); Second, BNP / NT-proBNP mainly reflects the hemodynamic stress state of the heart, and may also show an elevated trend in cases of arrhythmia, valvular heart disease, pulmonary hypertension, pulmonary thromboembolism, and sepsis (Heart Fail Rev. 2007, 12(1): 23-36.). These interfering factors often lead to a decrease in diagnostic specificity and may cause false positive or false negative results. Furthermore, BNP / NT-proBNP primarily characterizes the hemodynamic stress state of the heart (J Am Coll Cardiol. 2006,47(4):749-51.), but cannot provide direct and specific biological information for the persistent inflammation, the core pathological mechanism of heart failure. This limits the comprehensive assessment of the overall condition of heart failure and also affects the sensitivity of early risk warning.

[0004] Therefore, developing a novel biomarker that can circumvent the limitations of existing biomarkers while possessing both diagnostic and prognostic assessment capabilities is of great significance for improving the accuracy of diagnosis and risk stratification of heart failure. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a reagent for detecting the biomarker TNFR2 in products for the diagnosis and / or prognostic assessment of heart failure, overcoming the shortcomings of existing clinically commonly used biomarkers for heart failure, such as being singular, susceptible to interference, and unable to fully reflect the key pathological mechanisms of heart failure.

[0006] A second objective of this invention is to provide a kit for the diagnosis and / or prognostic assessment of heart failure, comprising the reagents for detecting the biomarker TNFR2 used in the above applications.

[0007] A third objective of this invention is to provide a method of using the above-described kit for the diagnosis and / or prognostic assessment of heart failure.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The use of reagents for detecting biomarkers in products for the diagnosis and / or prognostic assessment of heart failure, wherein the biomarker is TNFR2 (Tumor Necrosis Factor Receptor 2).

[0010] The biological samples tested are serum, plasma, whole blood, or peripheral blood mononuclear cells.

[0011] Furthermore, the biological sample being tested is isolated serum, plasma, whole blood, or peripheral blood mononuclear cells.

[0012] More preferably, the biological sample being tested is plasma. The expression level of TNFR2 in the circulating plasma of patients with heart failure is significantly higher than that in non-heart failure individuals.

[0013] Further preferred, the biological sample to be tested is a plasma sample treated with EDTA anticoagulation.

[0014] Further preferred, the biological sample is fasting peripheral blood plasma collected from a vacuum anticoagulant tube containing dipotassium ethylenediaminetetraacetate (EDTA-K2).

[0015] The detection was performed using an immunoassay.

[0016] Furthermore, the immunoassay method includes enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), immunoturbidimetry, or analytical methods based on adjacent extension analysis (PEA).

[0017] TNFR2 detection results are affected by the detection method, and its diagnostic threshold exhibits platform specificity. This invention, through multi-center, multi-platform clinical validation, has determined the discrimination limits under different quantitative standards, ensuring the universality of this biomarker in various clinical application scenarios.

[0018] The substances used to detect TNFR2 expression levels are antibodies, antibody fragments, or nucleic acid aptamers that specifically bind to the TNFR2 protein.

[0019] The products mentioned are detection reagents, biochips, microarrays, or detection systems.

[0020] The diagnosis is an early diagnosis and / or risk stratification.

[0021] TNFR2 is a biomarker, and reagents for detecting this biomarker are used in kits, microarrays, protein chips, or clinical decision support systems for heart failure screening, auxiliary diagnosis, or prognostic mortality risk prediction.

[0022] A kit for the diagnosis and / or prognostic assessment of heart failure, comprising reagents for detecting biomarkers used in the above applications.

[0023] The kit contains reagents for the quantitative detection of plasma TNFR2 levels, which are selected from any one of enzyme-linked immunosorbent assay (ELISA) reagents, chemiluminescent immunoassay reagents, ortho-extension assay (PEA) reagents, or mass spectrometry detection internal standard reagents.

[0024] The above-mentioned kits for the diagnosis and / or prognostic assessment of heart failure include:

[0025] 1) Immobilized TNFR2 capture antibody;

[0026] 2) Detection of TNFR2 antibodies using enzymes or fluorescently labeled antibodies;

[0027] 3) TNFR2 protein standards of known concentration.

[0028] The above-mentioned kits for the diagnosis and / or prognostic assessment of heart failure also include one or more of the following: buffer solution, washing solution, chromogenic agent, signal detection reagent, standard and control.

[0029] The chromogenic agent or signal detection reagent is selected from one or more of TMB, alkaline phosphatase substrate, ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), luminol or its derivatives; the buffer is selected from one or more of phosphate buffer (PBS), tris(hydroxymethyl)aminomethane buffer (Tris) or carbonate buffer.

[0030] Furthermore, the kit for the diagnosis and / or prognostic assessment of heart failure includes: a core immunoreaction reagent, a signal detection and colorimetric system, and auxiliary support reagents; the core immunoreaction reagent includes a solidified TNFR2 capture antibody, an enzyme-labeled or fluorescently labeled TNFR2 detection antibody; the auxiliary support reagent includes TNFR2 protein standards of known concentrations.

[0031] The immobilized TNFR2 capture antibody is a microplate pre-coated with an anti-human TNFR2 monoclonal antibody. The microplate can be a 96-well plate.

[0032] Furthermore, the immobilized TNFR2 capture antibody is an anti-human TNFR2 specific antibody (anti-human TNFR2 antibody) pre-coated on a polystyrene microplate, which is physically adsorbed and immobilized on the surface of the solid support to specifically capture human TNFR2 antigen in the sample.

[0033] The enzyme- or fluorescently labeled TNFR2 detection antibody is an HRP (horseradish peroxidase)-labeled anti-human TNFR2 detection antibody, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

[0034] Furthermore, enzyme- or fluorescently labeled TNFR2 detection antibodies are used with a biotin-avidin enzyme-linked amplification system, specifically including:

[0035] a. Biotinylated anti-human TNFR2 detection antibody (i.e., biotinylated anti-human TNFR2 antibody);

[0036] b. Horseradish peroxidase-labeled streptavidin (HRP-streptavidin, or HRP-avidin).

[0037] When used together, the two form a "biotinylated detection antibody + HRP-labeled avidin" complex, which is used to specifically bind to the captured TNFR2 and catalyze the colorimetric reaction.

[0038] The signal detection and colorimetric system includes a chromogenic substrate solution and a reaction termination solution. The chromogenic substrate solution is a colorimetric system containing substrate A (hydrogen peroxide) and substrate B (3,3',5,5'-tetramethylbenzidine, TMB), or a chromogenic substrate solution containing the substrate (3,3',5,5'-tetramethylbenzidine, TMB). The reaction termination solution is a 1 mol / L sulfuric acid (H₂SO₄) solution or a 1 mol / L hydrochloric acid (HCl) solution.

[0039] Furthermore, a known concentration of TNFR2 protein standard is recombinant human TNFR2 protein. This standard is provided in lyophilized powder form. After reconstitution, the initial concentration is 5000 pg / mL (or adjusted according to actual experiments), which can be used for serial dilution to establish a standard curve and quantitatively detect the absolute content of TNFR2 in samples.

[0040] The supporting reagents also include a buffer system. This buffer system comprises sample diluent, standard diluent, and phosphate buffer concentrate containing surfactants.

[0041] The surfactant-containing phosphate buffer concentrate is a phosphate concentrate washing solution containing 0.05% Tween-20 by mass.

[0042] The buffer system also includes antibody diluent.

[0043] A method of using the above-mentioned kit for the diagnosis and / or prognostic assessment of heart failure includes the following steps:

[0044] (1) Coating the surface of a microplate with a specific anti-TNFR2 capture antibody;

[0045] (2) Add the plasma sample to be tested or the serially diluted standard, and incubate to allow the TNFR2 protein to bind to the capture antibody;

[0046] (3) Add biotinylated detection antibody and horseradish peroxidase (HRP) labeled streptavidin;

[0047] (4) Add TMB substrate to develop color, terminate the reaction with a stop solution, and then measure the absorbance (OD value) at a wavelength of 450 nm.

[0048] (5) Construct a four-parameter Logistic regression model (4-PL) to calculate the absolute concentration of plasma TNFR2.

[0049] Further, in step (1), an anti-human TNFR2 specific antibody pre-coated on a polystyrene microplate is used as a solid-phase capture antibody.

[0050] Further, in step (2), the plasma sample to be tested or a serially diluted TNFR2 protein standard is added, and incubation is performed to allow the TNFR2 protein to bind to the capture antibody. The standard is recombinant human TNFR2 protein.

[0051] Furthermore, the biotinylated detection antibody in step (3) is a biotin-labeled anti-human TNFR2 antibody. The HRP-labeled streptavidin is a covalently bound product of horseradish peroxidase and streptavidin.

[0052] Furthermore, the terminating solution is a 1 mol / L (1 M) sulfuric acid solution.

[0053] Furthermore, in step (4), 630nm is set as the reference wavelength for dual-wavelength correction.

[0054] This invention first utilizes Olink's high-precision proteomics technology (Proximity Extension Assay, PEA) to screen plasma samples from heart failure patients and healthy individuals for protein profiles. Differential analysis revealed high expression of TNFR2 in the plasma of heart failure patients, which was further validated in publicly available single-cell sequencing databases. Furthermore, after adjusting for age, NT-proBNP, and renal function indicators, TNFR2 remained an independent risk factor for all-cause mortality, demonstrating predictive efficacy independent of conventional biomarkers.

[0055] To further confirm the clinical application value of TNFR2, this invention conducted a multicenter validation study. Both centers used the universally accepted ELISA technique for detection. The results showed that the expression trend of TNFR2 exhibited high consistency across different regions, centers, and personnel, demonstrating its strong clinical applicability and technical robustness.

[0056] This study confirms that plasma TNFR2 levels are a strong independent risk factor for all-cause mortality and adverse cardiovascular events in patients with heart failure. In multivariate Cox regression analysis, TNFR2 maintained significant prognostic predictive ability even after adjusting for age, renal function, and NT-proBNP. This unique characteristic, independent of traditional indicators such as NT-proBNP, allows it to provide clinicians with deeper pathophysiological information, aiding in precise risk stratification.

[0057] Clinical statistical analysis shows that TNFR2 exhibits excellent diagnostic efficacy in distinguishing between patients with heart failure and those without. Multicenter validation data show that the area under the receiver operating characteristic (ROC) curve (AUC) is greater than 0.9, demonstrating the accuracy of TNFR2 as an early diagnostic biomarker for heart failure and its ability to effectively reduce the rates of missed and misdiagnosed cases.

[0058] TNFR2, as a soluble receptor for TNF-α (alpha tumor necrosis factor), exhibits extremely high biochemical stability in the bloodstream and is not easily affected by degradation during sample processing. By combining the double-antibody sandwich assay kit and standardized four-parameter logistic regression model provided in this invention, rapid and quantitative determination of the absolute concentration of TNFR2 in plasma can be achieved, facilitating its widespread application in clinical laboratories at all levels of hospitals.

[0059] Compared with existing technologies, the applications provided by this invention have the following beneficial effects:

[0060] This invention, through high-precision proteomics screening and multi-center clinical cohort validation, confirms that TNFR2 is significantly and specifically highly expressed in the plasma of heart failure patients, showing a significant difference from that in healthy individuals. Clinical results show that the AUC value of TNFR2 in diagnosing heart failure remained consistently above 0.90 in both the discovery cohort and the independent validation cohort, accurately distinguishing heart failure patients from healthy individuals and significantly reducing the clinical misdiagnosis rate.

[0061] This invention provides the first evidence that TNFR2 is a potent independent risk factor for all-cause mortality in heart failure patients. Multivariate Cox regression analysis showed that, after adjusting for traditional clinical indicators such as age, sex, renal function, and NT-proBNP, TNFR2 remained an independent risk factor for all-cause mortality. This indicates that TNFR2 can provide risk stratification information beyond existing clinical indicators, offering more precise biological evidence for the prognostic management of heart failure patients.

[0062] This invention effectively overcomes the technical bottleneck of declining efficacy of traditional indicators in long-term risk assessment, enabling long-term monitoring of the course of heart failure. Time-dependent ROC curve analysis shows that TNFR2 exhibits excellent robustness in predicting mid- to long-term prognosis over 24-36 months, and its efficacy is significantly superior to the clinical gold standard NT-proBNP.

[0063] This invention demonstrates that TNFR2 can serve as a molecular diagnostic and / or risk assessment biomarker for heart failure. By fabricating specific reagents for TNFR2 detection into test strips, ELISA kits, or integrated biochips, the expression level of TNFR2 in the plasma of subjects can be detected, enabling auxiliary diagnosis and prognostic risk stratification of heart failure. The risk assessment of a subject's heart failure depends on the detection system used: when using adjacent extension analysis (such as the Olink platform), if the relative expression level (NPX value) of TNFR2 in the subject's plasma exceeds a preset threshold (4.542); or when using conventional immunological quantification techniques (such as the ELISA platform), if its absolute mass concentration after log2 conversion exceeds the corresponding clinical cutoff value (8.11-8.19 log2 (pg / mL)), it indicates an extremely high risk of heart failure in the subject. This invention confirms that high levels of TNFR2 predict a higher long-term mortality risk in subjects, providing crucial risk assessment evidence for the development of intensive intervention programs in clinical practice.

[0064] The application provided by this invention directly addresses the needs of existing technologies. TNFR, as an indicator reflecting immune stress in the tumor necrosis factor pathway, has a different pathophysiological pathway than NT-proBN, which reflects volume overload. The combined application of both can provide complementary clinical information, enabling earlier and more comprehensive identification and risk assessment of heart failure.

[0065] Compared with existing technologies, the reagent kit provided by this invention has the following advantages:

[0066] 1. Excellent performance indicators, sensitive and accurate detection: This kit is based on a double-antibody sandwich method for the quantitative detection of absolute plasma TNFR2 concentration, exhibiting extremely high sensitivity and repeatability. Multicenter validation shows that this kit has a strong ability to capture heart failure states and is minimally affected by individual differences, ensuring accurate and reliable test results.

[0067] 2. Strong clinical stability and minimal impact from physiological fluctuations: As a soluble receptor, TNFR2 exhibits excellent biological stability in circulating blood. Unlike peptide markers that are easily affected by short-term hemodynamic changes, the TNFR2 levels detected by this kit can more robustly reflect the long-term pathological remodeling state of the heart, effectively reducing detection errors caused by short-term sample fluctuations.

[0068] 3. Easy to standardize and promote: This kit uses a standardized ELISA technology platform, with simple operating procedures, controlled costs, and good compatibility with existing clinical automated testing systems. It achieves high-throughput, rapid detection without complex sample pretreatment, possessing extremely high clinical application value and commercial prospects. Attached Figure Description

[0069] Figure 1 This is a volcano plot of differentially expressed proteins between a healthy control group and a heart failure patient group (HF) based on Olink sequencing data; the scatter plots on the right represent proteins that are upregulated in HF patients, and the scatter plots on the left represent proteins that are downregulated.

[0070] Figure 2 A graph showing the relative expression levels (NPX values) of TNFR2 between the heart failure patient group and the healthy control group in the Olink cohort; where HF represents the heart failure patient group and CON represents the healthy control group;

[0071] Figure 3 This study aims to analyze the cell-specific expression distribution of TNFR2 in myocardial tissue using single-cell transcriptome sequencing technology. The top left image shows a UMAP dimensionality-reduced clustering diagram of non-myocardial cell populations in the heart of patients with a history of hypertension and heart failure (HF). The top right image shows a FeaturePlot fluorescence distribution of TNFR2 transcription levels. The bottom image is a violin plot showing the expression abundance of TNFR2 in each cell subpopulation. In the bottom image, HF represents the heart failure patient group, and Control represents the healthy control group.

[0072] Figure 4 ROC curves for TNFR2 in diagnosing heart failure were discovered using Olink.

[0073] Figure 5A heatmap showing the correlation between TNFR2 expression levels and laboratory biochemical and echocardiographic parameters;

[0074] Figure 6 To discover the Kaplan-Meier curves of all-cause mortality in HF patients with different TNFR2 expression levels in the Olink cohort;

[0075] Figure 7 Forest plot for multivariate Cox regression analysis of the Olink discovery cohort, with the horizontal axis representing the hazard ratio (HR) and the vertical axis representing the clinical variable name;

[0076] Figure 8 Olink discovered time-dependent ROC curves for TNFR2 prediction of 12-month mortality risk in a cohort.

[0077] Figure 9 Olink discovered time-dependent ROC curves for TNFR2 prediction of 24-month mortality risk in a cohort.

[0078] Figure 10 Olink discovered time-dependent ROC curves for TNFR2 prediction of 36-month mortality risk in a cohort.

[0079] Figure 11 This is a comparison of plasma TNFR2 concentrations between the heart failure patient group (HF) and the healthy control group (CON); where HF represents the heart failure patient group and CON represents the healthy control group.

[0080] Figure 12 ROC curve for TNFR2 diagnosis of HF in cohort A;

[0081] Figure 13 This is a comparison of plasma TNFR2 concentrations between the heart failure patient group (HF) and the healthy control group (CON); where HF represents the heart failure patient group and CON represents the healthy control group.

[0082] Figure 14 ROC curves for TNFR2 diagnosis of HF in the B-center validation cohort. Detailed Implementation

[0083] The present invention will be further described below with reference to specific embodiments.

[0084] In the specific implementation, the reagents or consumables involved are all commercially available conventional products in the field.

[0085] Example 1

[0086] This embodiment is based on Olink technology (proteomics) for the screening and validation of serum protein biomarkers.

[0087] 1.1 Sample Collection:

[0088] 1) The plasma samples from the healthy control group and heart failure patients used in this embodiment were all collected from a large tertiary hospital in central China. The baseline data of the subjects are shown in Table 1.

[0089] Peripheral blood was collected using EDTA anticoagulant blood collection tubes. Venous blood samples were collected from 42 patients clinically diagnosed with heart failure (HF), and venous blood samples were also collected from 18 healthy controls. All participants signed informed consent forms. The diagnostic criteria for heart failure were based on the "Guidelines for the Diagnosis and Treatment of Heart Failure in China".

[0090] Blood sample collection and standardized processing: Subjects fasted for at least 8 hours, and 5 mL of fasting venous blood was collected and placed in an EDTA-K2 vacuum anticoagulant tube. The tube was immediately and gently inverted to mix. Within 2 hours of sample collection, the sample was centrifuged at 3000g for 15 minutes at 4°C, and the supernatant plasma was separated. Repeated freeze-thaw cycles were strictly prohibited. The sample was stored in an ultra-low temperature freezer at -80°C.

[0091] To be included in the HF group, the following criteria must be met simultaneously:

[0092] ① Meets the diagnostic criteria for HF in the "Chinese Guidelines for the Diagnosis and Treatment of Heart Failure 2024", namely, having typical symptoms or signs of heart failure and objective evidence of cardiac structural and / or functional abnormalities.

[0093] ② Age ≥ 18 years old, gender not limited.

[0094] ③ The patient or their family members are fully informed about the content of this study and have signed informed consent.

[0095] Exclusion criteria for the HF group: To reduce the interference of confounding factors on plasma proteomic profiles and prognostic assessment, patients meeting any of the following criteria will be excluded:

[0096] ①Those who have a history of or currently have malignant tumors; those with active infections, systemic autoimmune diseases, or those in a state of severe systemic inflammation.

[0097] ② Patients with severe hepatic impairment (Child-Pugh C) or severe renal impairment (eGFR <15 mL / min / 1.73 mcg). 2 (or those currently undergoing dialysis treatment).

[0098] ③Women who are pregnant or breastfeeding.

[0099] ④ Individuals with mental disorders or cognitive impairment who are unable to cooperate in completing follow-up; or those with incomplete key clinical history, laboratory test results, and imaging data.

[0100] To be included in the CON group, the following criteria must be met simultaneously:

[0101] ① The patient has no typical symptoms (such as exertional dyspnea, paroxysmal nocturnal dyspnea, etc.) or signs (such as bilateral lower extremity edema, pulmonary rales, etc.) related to heart failure, and laboratory tests show that BNP and NT-proBNP levels are within the normal reference range.

[0102] ② Transthoracic echocardiography confirmed that the LVEF (left ventricular ejection fraction) was normal (≥50%), the ventricular wall motion was coordinated, and no clinically significant abnormalities were found in the valve structure and function.

[0103] ③ No acute myocardial ischemia, significant conduction block or serious arrhythmia were observed on the resting electrocardiogram; occasional premature contractions (premature beats) that do not affect the evaluation of cardiac function are allowed to be included.

[0104] ④ To simulate a real clinical control background, subjects with a history of primary hypertension but whose blood pressure is well controlled and has not yet caused damage to cardiac target organs (such as left ventricular hypertrophy or cardiac chamber enlargement) are allowed to be included.

[0105] ⑤ Physical examination and basic laboratory tests (such as complete blood count, liver and kidney function, electrolytes, etc.) showed no serious organic diseases or acute inflammatory conditions.

[0106] As shown in Table 1, statistical analysis in the Olink discovery cohort revealed that the HF and CON groups were well-matched in terms of demographic characteristics such as sex, height, and weight (P>0.05), excluding the influence of basal metabolic differences on proteomic expression. Furthermore, the mean NT-proBNP level in the HF group was significantly higher than that in the CON group (P=0.008), and LVEF was significantly lower (P<0.001), meeting the clinical diagnostic criteria for heart failure. In particular, this study recorded the subjects' eGFR (estimated glomerular filtration rate) and creatinine levels, laying a data foundation for subsequent validation of the independent diagnostic efficacy of TNFR2 in complex pathological backgrounds, such as impaired renal function.

[0107] Table 1. Baseline data analysis of the HF and CON groups in the discovery cohort (Example 1)

[0108] Male, n(%) 9(50) 28(66.67) 0.224 Age (years) 46.17(23.00-60.00) 53.95(24.00-83.00) 0.064 Heart rate (bpm) 76.94(72.00-102.00) 83.29(50.00-117.00) 0.085 Laboratory indicators <![CDATA[WBC(×10 9 / L)]]> 6.37(4.20-9.39) 7.24(3.10-24.86) 0.353 Hb(g / L) 136.43(91.70-154.50) 133.24(83.00-199.20) 0.620 <![CDATA[PLT(×10 9 / L)]]> 228.33(128.00-403.00) 197.48(71.00-419.00) 0.166 Cr (µmol / L) 67.83(43.00-98.00) 87.28(42.00-155.00) 0.004 Urea (mmol / L) 5.31(2.20-8.50) 7.35(2.80-20.30) 0.038 UA (µmol / L) 296.50(153.00-558.00) 372.33(154.00-964.00) 0.050 eGFR (mL / min / 1.73m²) 104.70(56.74-127.72) 79.21(34.18-125.29) <0.001 ALT(U / L) 17.56(6.00-44.00) 62.69(3.00-352.00) 0.015 AST(U / L) 17.33(12.00-25.00) 55.95(9.00-425.00) 0.035 Albumin (g / L) 43.94(40.60-49.50) 38.12(31.80-47.00) <0.001 HbA1c (%) 5.48(5.05-5.80) 7.05(5.20-11.00) <0.001 Cardiac function indicators NT-proBNP (pg / mL) 64.22(13.00-212.00) 5471.57(199.00-43785.00) 0.008 LVEF (%) 63.56(62.00-66.00) 37.88(19.00-63.00) <0.001

[0109] Note: 1. Abbreviation definitions: CON (Control): Non-heart failure patient group; HF: Heart failure group; bpm: beats / min; WBC: White blood cell count; Hb: Hemoglobin; PLT: Platelet count; Cr: Serum creatinine; Urea: Blood urea nitrogen; UA: Uric acid; eGFR: Estimated glomerular filtration rate; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; Albumin: Albumin; HbA1c: Glycated hemoglobin; NT-proBNP: N-terminal pro-B-type natriuretic peptide; LVEF: Left ventricular ejection fraction. 2. Classification indicator descriptions: Demographic and General Characteristics: Covers basic information of study subjects; Laboratory variables: Covers complete blood count, renal function, liver function and metabolic indicators, and systemic inflammatory indicators; Cardiac Function and Biomarkers: Includes core indicators for the diagnosis and prognostic assessment of heart failure, NT-proBNP and LVEF.

[0110] 1.2 Olink proteomics assay:

[0111] Serum samples from the above 60 cases (48 HF cases and 12 controls) were sent to Lianchuan Biotechnology Co., Ltd. for high-throughput proteomics analysis using the Olink® Target 96 inflammation response panel. The procedures were strictly performed according to the manufacturer's instructions.

[0112] Detection principle and steps:

[0113] This assay is based on proximity-dependent DNA extension. In short, a pair of specific antibodies against each target protein (e.g., TNFR2) are each labeled with a unique DNA oligonucleotide chain. When these antibodies bind to the target protein, the accompanying DNA oligonucleotides spatially approach and hybridize, forming a double-stranded DNA template. DNA polymerase is then added to perform a proximity-dependent DNA extension reaction, generating a double-stranded DNA "barcode" that corresponds one-to-one with the specific target protein. The initial amount of this DNA barcode is proportional to the concentration of the target protein in the sample. Finally, the generated DNA sequence is amplified and quantified using a microfluidic real-time quantitative PCR system.

[0114] Data standardization and quality control:

[0115] Standardization process: Raw Ct data are converted into standardized protein expression values ​​(NPX, a relative quantitative unit on a Log2 scale) through the following steps:

[0116] 1) dCt (analyte) = Ct (analyte) – Ct (extended control) (used to reduce technical variation);

[0117] 2) ddCt (analyte) = dCt (analyte) – Ct (inter-plate control median) (used to correct for inter-plate differences);

[0118] 3) NPX (analyte) = Correction factor (analyte) – ddCt (analyte) (The final output is a visually intuitive NPX value). The correction factor is a fixed parameter for each detection index and reagent batch.

[0119] Quality control:

[0120] 1) Run-level quality control: Calculate the standard deviation of the NPX values ​​for the incubation / immunization control and the test control in each test. The run data can only pass quality control if the standard deviation of all controls is less than 0.2.

[0121] 2) Sample-level quality control: The NPX values ​​of the incubation control 2 and the test control for each sample were compared with the median of this run. If either control value for any sample deviated from the run median by more than ±0.3 NPX, the sample was marked as "QC Warning". In this study, all samples passed the quality control and no samples received warnings; therefore, all 60 samples were included in the subsequent analysis.

[0122] 1.3 Differential protein screening:

[0123] Statistical analysis was performed on the NPX data after quality control. The t-test was used to compare the expression levels of each protein between the heart failure group and the healthy control group. The screening criteria for differentially expressed proteins were set as follows: p-value < 0.05 and |log FC| > 0.5.

[0124] Figure 1 This is a color volcano plot showing the differential expression of 92 candidate proteins between the HF and CON groups, detected using the Olink proteomics platform. The horizontal axis represents the logarithmic fold change, and the vertical axis represents the negative logarithm of statistical significance (-log). 10 P-value). Each dot represents a specific protein, and the scatter plots on both sides of the vertical axis represent proteins with significant differences in expression between the two groups. Different colors indicate proteins that are upregulated, downregulated, or have no significant difference in expression in heart failure patients. The results showed that TNFR2 was located in the upregulated region of the upper right quadrant, establishing its status as a differentially expressed protein in heart failure.

[0125] Figure 2 A bar chart was created to show the plasma TNFR2 expression levels in the HF and CON groups in the Olink cohort. The bar height represents the mean protein expression level (Mean), the error bar represents the standard error (SEM), and the scatter plot represents the relative NPX expression level of each sample. Statistical results showed that the mean TNFR2 expression abundance in the healthy control group (CON, n=18) was 4.18±0.37, while the mean abundance in the heart failure group (HF, n=42) was significantly higher at 5.00±0.73. Simultaneously, the NPX values ​​of TNFR2 in the HF group had a wider distribution range (Min: 4.03, Max: 6.75), and the overall level was significantly higher than that in the CON group (Min: 3.58, Max: 4.92). Welch-corrected one-sided and two-sided t-tests were used to compare the means of the two groups, with results showing t=-5.748, df=56.294. The p-value was 3.84×10⁻⁶. -7 (P<0.001), indicating a statistically significant difference. This data further confirms... Figure 1 The screening results of the volcano plot clearly showed that TNFR2 is specifically highly expressed in the circulating plasma of patients with heart failure, and that the increase in its expression level is strongly correlated with the pathological state of heart failure, providing solid data support for its use as a clinical diagnostic biomarker.

[0126] Figure 3 This image shows a color plot illustrating the cell-specific expression distribution of TNFR2 in myocardial tissue, derived using single-cell transcriptome sequencing technology. Data was obtained from single-cell RNA sequencing data in the publicly available database GSE222144 and processed using R software. The upper left image is a UMAP-reduced clustering plot of non-myocardial cell populations in the heart of HF patients with a history of hypertension. The x and y axes represent the reduced UMAP spatial coordinates, with each point representing a cell; different colors represent different cell subpopulations.

[0127] Figure 3 The top right image (Feature Plot) shows a single-cell t-SNE mapping of TNFR2 transcriptional levels. Scatter dots represent individual cells, and color intensity represents gene expression abundance (deeper purple indicates higher expression levels). The results show that TNFR2 expression exhibits significant cellular heterogeneity, not being uniformly expressed throughout the tissue, but rather highly enriched in specific cell clusters (as shown in the top right region of the image). Based on common clustering distributions in cardiac tissue, these high-expression clusters typically correspond to neutrophils, macrophages, and T cells. This figure visually demonstrates the high cell selectivity of TNFR2 expression in the cardiac microenvironment, primarily contributed by specific matrix or immune cell populations.

[0128] Figure 3The following violin plot shows the abundance of TNFR2 expression in different cell subpopulations. The horizontal axis represents different cell subpopulations (such as Fibroblasts, Macrophages, Endothelial cells, and SMCs), with different colors representing different groups (HF group, Control group); the vertical axis represents the TNFR2 expression level; the curve width represents the cell density at that expression level. This plot further quantitatively compares the differences between the heart failure group (HF, blue) and the healthy control group (Control, yellow) in eight major cell subpopulations. TNFR2 maintained a high baseline expression level in Mac (macrophages), Endothelial cells, T (T cells), and SMC (smooth muscle cells). Compared with the healthy control group, the TNFR2 expression levels in the heart failure group (HF) were significantly upregulated in macrophages, B cells, and nerve cells (P<0.05).

[0129] like Figure 3 As shown in the upper right (t-SNE plot) and lower (violin plot), single-cell transcriptome sequencing analysis confirmed that TNFR2 is specifically expressed in cardiac tissues, primarily in neutrophils, macrophages, and T cells. Quantitative analysis showed that compared with the healthy control group, the HF group exhibited significantly upregulated TNFR2 expression levels in macrophages, B cells, and neutrophils (P<0.05). These results elucidate the histological basis of TNFR2's involvement in the pathological remodeling of heart failure at the cellular and molecular level, demonstrating that changes in its concentration can accurately reflect local inflammatory stress and endothelial injury in the heart.

[0130] Example 2

[0131] This example analyzes the diagnostic efficacy of plasma TNFR2 expression levels in patients with heart failure.

[0132] 2.1 Evaluation of diagnostic accuracy:

[0133] Using the relative expression level of plasma TNFR2 in the cohort of subjects discovered by Olink as a test variable, receiver operating characteristic (ROC) curves for diagnosing heart failure were plotted and statistically evaluated.

[0134] like Figure 4As shown, the horizontal axis represents the false positive rate (1-Specificity), and the vertical axis represents the true positive rate (Sensitivity). The area under the curve (AUC) for TNFR2 as a single indicator in identifying heart failure patients was 0.857, with a 95% confidence interval (95% CI) of 0.756–0.959. The results confirm that TNFR2 has excellent discriminative power and can accurately distinguish heart failure patients from healthy controls. Based on the Youden index maximization principle, the optimal clinical cutoff value for this indicator was determined to be 4.542 (NPX value) under the first detection system of this invention (based on adjacent extension analysis, PEA). It should be noted that this threshold is based on the relative quantitative results of PEA technology and is used for preliminary screening and risk assessment. At this threshold, the diagnostic sensitivity was 69.0%, and the specificity was 88.9%. The overall diagnostic accuracy of the experimental data reached 75.0%. It is worth noting that this indicator showed an extremely high positive predictive value of 93.5%, which means that among people with positive TNFR2 test results, the probability of being diagnosed with heart failure is extremely high, and it has excellent clinical diagnostic reference value; its negative predictive value was 55.2%.

[0135] Statistical analysis shows that plasma TNFR2 expression level, as a single biomarker for heart failure, maintains high diagnostic accuracy while possessing significant specificity and extremely high positive predictive value. This indicates that TNFR2 can not only effectively identify at-risk individuals in clinical screening but also significantly reduce the misdiagnosis rate, providing a valuable reference for the in vitro auxiliary diagnosis of heart failure.

[0136] like Figure 4 As shown, the area under the curve (AUC) for TNFR2 as a single indicator in diagnosing heart failure was 0.857 (95% CI: 0.756–0.959). This result demonstrates that TNFR2 has high accuracy and statistical significance in distinguishing heart failure patients from healthy controls.

[0137] 2.2 Correlation Analysis:

[0138] To further clarify the clinical biological significance of plasma TNFR2 expression levels, this study used Spearman rank correlation analysis to analyze the association between TNFR2 (NPX value) and various clinical biochemical indicators, echocardiographic parameters, and baseline demographic characteristics of patients with heart failure. Analysis of the descending order of correlation coefficients (rho) revealed that plasma TNFR2 levels can reflect the severity of heart failure in multiple dimensions (see...). Figure 5 ).from Figure 5 It can be seen that:

[0139] 2.2.1 Positive Correlation with Indicators of Impaired Cardiac Function: TNFR2 showed the most significant positive correlation with NT-proBNP, the gold standard for diagnosing heart failure. This indicates that elevated TNFR2 levels are closely related to increased pressure load on the myocardium and increased ventricular wall tension. It also showed positive correlations with parameters reflecting cardiac structure, including LAD (left atrial diameter), LVEDD (left ventricular end-diastolic diameter), and PAP (pulmonary artery pressure), revealing that TNFR2 levels increase with the severity of cardiac remodeling.

[0140] 2.2.2 Correlation with Organ Function Impairment: TNFR2 was positively correlated with creatinine (Cr) and significantly negatively correlated with eGFR (estimated glomerular filtration rate). This suggests that TNFR2 levels are regulated by renal function to some extent, reflecting the cardiorenal syndrome state caused by heart failure. TNFR2 was positively correlated with AST (aspartate aminotransferase) and ALT (alanine aminotransferase), suggesting that systemic circulatory congestion or cardiogenic liver injury caused by severe heart failure may induce further release of TNFR2.

[0141] 2.2.3 Negative Correlation with Cardiac Function Assessment and Prognostic Indicators: TNFR2 is negatively correlated with EF (ejection fraction), which reflects left ventricular systolic function. The lower the EF value, the higher the TNFR2 concentration, confirming that it can be used as a sensitive indicator for assessing decreased cardiac output. It is also significantly negatively correlated with ALB (albumin) and Hb (hemoglobin), reflecting a negative correlation between TNFR2 levels and the patient's nutritional status and degree of anemia.

[0142] Correlation analysis strongly demonstrates that plasma TNFR2 is not only a simple diagnostic biomarker, but also a comprehensive barometer of the pathophysiological state of heart failure. Its concentration changes are highly correlated with cardiac structural remodeling (LAD, LVEDD), pumping dysfunction (EF), cardiac load (NT-proBNP), and secondary organ damage (Cr, eGFR, AST). This finding provides solid clinical evidence for the use of TNFR2 in the assessment and prognosis of heart failure.

[0143] Example 3

[0144] This example analyzes the prognostic efficacy of plasma TNFR2 expression levels in patients with heart failure.

[0145] 3.1 Survival analysis based on TNFR2 expression levels:

[0146] Patients were divided into high-expression and low-expression groups based on the median TNFR2 expression level (NPX value) (4.542).

[0147] Figure 6Kaplan-Meier survival curves (color plot) based on TNFR2 expression levels. Data were obtained from follow-up data and baseline plasma TNFR2 levels of 42 heart failure patients in the discovery cohort. The horizontal axis represents follow-up time (months); the vertical axis represents cumulative survival probability. Interpretation: The two different colored curves represent the high TNFR2 expression group and the low expression group (grouped by median); a downward curve indicates the occurrence of death; differences between groups were assessed using the Log-rank test.

[0148] from Figure 6 As can be seen, the cumulative survival probability of the TNFR2 high-expression group (red curve) decreased significantly over time, and its survival curve remained below that of the low-expression group (green curve). The Log-rank test showed a significant difference in survival distribution between the two groups (P=0.0094). The results indicate that patients with elevated baseline plasma TNFR2 levels have a significantly increased risk of all-cause mortality.

[0149] 3.2 Validation of independent prognostic value: multivariate Cox regression analysis.

[0150] To exclude the influence of clinical confounding factors, this study constructed a multivariate Cox proportional hazards regression model, and jointly analyzed TNFR2 with routine clinical risk factors. The results are shown in [Figure 1]. Figure 7 After comprehensive adjustment for age, gender, eGFR, and the core marker of heart failure (log2NTproBNP), the results of the multivariate Cox model (n=42, events=17, C-index=0.707) showed that TNFR2 was a strong independent risk factor for all-cause mortality in heart failure patients.

[0151] like Figure 7 The forest plot and statistical output showed that the hazard ratio (HR) for TNFR2 was 4.03 (95% CI: 1.72–9.45), which was highly statistically significant (P = 0.00137). In contrast, age (P = 0.536), eGFR (P = 0.921), and adjusted NT-proBNP (P = 0.231) did not reach statistical significance in the current multivariate model. This further highlights the independence and robustness of TNFR2 in predicting the risk of death from heart failure compared to traditional clinical indicators.

[0152] Survival analysis and multivariate risk models jointly confirmed that plasma TNFR2 level is an independent prognostic biomarker for mortality risk in patients with heart failure. Its high hazard ratio (HR) of 4.03 means that, after excluding other clinical confounding factors, patients with higher TNFR2 levels face a more than four times higher risk of all-cause mortality than those with lower levels. This finding provides crucial biological evidence for clinical risk stratification and precise prognostic assessment in heart failure.

[0153] 3.3 Evaluation of the effectiveness of TNFR2 in long-term prognostic assessment:

[0154] To dynamically assess the predictive efficacy of plasma TNFR2 on all-cause mortality risk in patients with heart failure, this study constructed a time-dependent receiver operating characteristic (ROC) curve and compared it with the clinical gold standard biomarker NT-proBNP. Figure 8-10 The following are color figures showing the time-dependent ROC curves (ROC curves) for predicting mortality risk at 12, 24, and 36 months in the Olink discovery cohort (TNFR2 prediction). The area under the curve (AUC) was calculated for each of the 12, 24, and 36-month follow-up time points. The x-axis of the time-dependent ROC curve represents the false positive rate (1-specificity), indicating the risk of misdiagnosis; the y-axis represents the true positive rate (sensitivity), indicating the ability to detect mortality events. In the figure, the green curve represents TNFR2, and the red curve represents the clinical gold standard NT-proBNP. The closer the area under the curve (AUC) is to 1.0, the better the prognostic risk of the biomarker.

[0155] By comparing the predictive performance of TNFR2 and NT-proBNP at different follow-up periods, the results showed (see Table 2 and...). Figure 8-10 ).

[0156] Table 2. Comparison of AUC between TNFR2 and NT-proBNP at different follow-up time points in predicting the risk of death from heart failure.

[0157] December 0.632 0.644 24 months 0.797 0.628 36 months 0.793 0.562

[0158] During the first year of follow-up, the AUCs for TNFR2 and NT-proBNP were 0.632 and 0.644, respectively, indicating that they have similar clinical value in short-term risk warning. With prolonged follow-up, the predictive efficacy of NT-proBNP showed a significant decreasing trend. In contrast, the predictive efficacy of TNFR2 improved significantly and remained robust in the long term, with its AUCs at 24 and 36 months remaining above 0.75, significantly higher than that of NT-proBNP during the same periods.

[0159] The time-dependent ROC curves show that the green curve of TNFR2 is significantly located to the upper left of the red curve of NT-proBNP at both the 24-month and 36-month nodes, demonstrating its more accurate ability to detect long-term mortality risk over 2-3 years.

[0160] Example 4

[0161] In this embodiment, the expression level of TNFR2 was verified using the ELISA method at different centers.

[0162] 4.1 Sample preparation:

[0163] To validate the findings of Example 1 in an independent sample cohort, this example collected serum samples from two independent clinical centers. All samples were obtained in accordance with ethical guidelines and with informed consent, and the inclusion and exclusion criteria were the same as above.

[0164] The verification queue is structured as follows:

[0165] Center A: A total of 60 cases, including 42 patients with heart failure and 18 healthy controls;

[0166] Center B: A total of 45 cases, including 30 patients with heart failure and 15 healthy controls.

[0167] The specific steps are as follows:

[0168] ① Standardized Sampling and Pretreatment: After fasting for 8 hours, 5 mL of blood was collected from the median cubital vein in the morning and placed in an EDTA-K2 anticoagulant tube. Immediately after sampling, the sample was gently inverted 5-8 times and temporarily stored at a constant temperature of 4°C. All samples must be centrifuged within 120 minutes of collection (conditions: 3000×g, 15 minutes). When extracting plasma, the "non-contact aspiration" principle was strictly followed, meaning the micropipette tip must be held at least 2 mm above the leukocyte membrane at the junction to completely prevent hemolysis and interference from cellular genomic components on the subsequent ELISA detection signal.

[0169] ② Standardized cold chain management for samples from different locations: Immediately after aliquoting, plasma is pre-frozen in an -80°C ultra-low temperature environment. Inter-center transport employs a professional medical-grade cold chain solution, utilizing thickened vacuum insulated boxes with sufficient dry ice (weight ratio not less than 1:3) to ensure the entire transport process is maintained at a cryogenic state below -78.5°C. Through real-time temperature control monitoring and the high-efficiency transport via SF Express's medical cold chain, the risk of protein denaturation is minimized, ensuring a high degree of consistency in the biochemical characteristics of samples from multiple centers.

[0170] ③ Sample Access Verification and Closed-Loop Management: Upon sample arrival, a "triple verification system" is implemented. First, the dry ice filling level and physical properties of the sample are confirmed (ensuring no signs of melting); second, the unique sample code and the completeness of clinical data are checked; finally, the transport log is recorded (including time taken and arrival temperature control curve). Samples that pass the verification are quickly added to the automated sample bank and centrally frozen at -80°C.

[0171] ④ Quality Control and Storage: All plasma samples were aliquoted into 1.5 mL low-adsorption EP tubes (250 μL / tube) that were enzyme-free and pyrogen-free. Before quantitative analysis of TNFR2 concentration, all samples were strictly thawed only once; repeated freeze-thaw cycles were strictly prohibited. Internal control samples and duplicate wells were included in each batch of the experimental design to maximize the biological activity of the target protein and the accuracy of detection.

[0172] 4.2 ELISA Operation Procedure:

[0173] ① Experimental reagents and key instruments: The TNFR2 enzyme-linked immunosorbent assay kit (Elabscience Biotechnology Co., Ltd., Wuhan, China; catalog number: E-EL-H2436) was selected. This kit is based on the double antibody sandwich method. The specific contents of the kit are shown in Table 3.

[0174] Table 3. Kit Contents List

[0175] Microplate 12 holes × 8 strips 12 holes × 4 strips none Standard products 0.3mL × 6 tubes 0.3mL × 6 tubes none Sample diluent 6mL 3mL none Detection of antibodies-HRP 10mL 5mL none 20× Washing Buffer 25mL 15mL Dilute as required. Substrate A 6mL 3mL none Substrate B 6mL 3mL none Termination solution 6mL 3mL none sealing film 2 sheets 2 sheets none

[0176] The core experimental equipment includes: a low-temperature high-speed centrifuge, a microplate constant temperature shaking incubator, and a multi-functional fully automated microplate reader.

[0177] ② Standardized Detection Procedure: Thaw plasma samples frozen at -80℃ slowly at 4℃. To eliminate potential fibrin precipitation and cell debris interference during freeze-thaw cycles, centrifuge the samples at 1000×g for 10 minutes, and accurately transfer the clear middle layer of plasma for testing. Set up wells for graded dilution of standards and wells for test samples. Dilute the test plasma according to the optimal linear dilution ratio determined in pre-experiments. After adding the samples, cover the microplate with a membrane and incubate at 37℃ for 90 minutes to allow the antigen to fully bind to the coating antibody. Discard the liquid in the wells and wash the plate 3-5 times automatically with washing buffer to remove unbound components. Add biotinylated detection antibody working solution and horseradish peroxidase (HRP)-labeled streptavidin sequentially. After washing the plate again, add TMB substrate solution for color development in the dark. After adding sulfuric acid stop solution, the solution changes from blue to yellow. Immediately use an ELISA reader to measure the absorbance (OD value) of each well at a main wavelength of 450 nm (while setting 630 nm as a reference wavelength for dual-wavelength calibration).

[0178] ③ Data Conversion and Quality Control (QC) Standard Curve Plotting: Using the logarithm of the standard concentration as the x-axis and the OD value as the y-axis, a four-parameter logistic regression model (4-PL) is used for curve fitting, and the regression equation and R² value are calculated (R² > 0.99). The sample OD values ​​are substituted into the regression equation to calculate the initial TNFR2 concentration (pg / mL), which is then multiplied by the dilution factor to obtain the final absolute plasma expression concentration. All samples and standards are tested in duplicate. The intra-assay CV must be <10%, and the batch-to-batch CV must be <15%. For samples with abnormal intra-assay CV values ​​or values ​​exceeding the linear range of the standard curve, a retesting mechanism is initiated, with re-dilution and retesting until the QC standards are met.

[0179] In this embodiment, the raw plasma TNFR2 concentration measured by ELISA is expressed in pg / mL. Before statistical analysis, all raw concentration data underwent a log2 transformation (i.e., y = log...). 2( The core reasons for adopting this conversion method are as follows:

[0180] (1) Normalizing skewed data: Protein concentrations in clinical biological samples are usually right-skewed, and direct t-tests are prone to statistical bias. Log2 transformation can effectively correct the data distribution, making it closer to a normal distribution and satisfying the mathematical premise of parametric tests.

[0181] (2) Homogeneity of variance: As the mean concentration increases, the variation (standard deviation) of the sample tends to increase as well. Log2 transformation can stabilize the variance and make data from different concentration ranges comparable.

[0182] (3) Intuitive expression of fold change: In log2 space, each unit increase in value represents a doubling of the original concentration (2-fold difference). This allows the intergroup span in the statistical chart to directly correspond to the biological expression fold change, which is more helpful for clinicians to assess the magnitude of changes in pathological status.

[0183] The results are as follows Figure 11-14 As shown.

[0184] Figure 11 This is a comparison of plasma TNFR2 concentrations between patients with heart failure (Central A) and healthy controls. From... Figure 11 It can be seen that the average expression level of plasma TNFR2 in the HF group was 9.10±0.91 log2 (pg / mL), which was significantly higher than that in the CON group (7.71±0.25 log2 (pg / mL) (P<0.0001).

[0185] Figure 12The ROC curve for TNFR2 in diagnosing heart failure is shown in the validation cohort A at center A. The horizontal axis represents the false positive rate (1-specificity), and the vertical axis represents the true positive rate (sensitivity). In the validation cohort A at center A, the area under the curve (AUC) for plasma TNFR2 in diagnosing heart failure was as high as 0.968 (95% CI: 0.920 - 1.000). The optimal diagnostic threshold, calculated using the Youden index of the ROC curve, was 8.188 log2 (pg / mL), at which point the diagnostic sensitivity was 88.1% and the specificity was 100%. This result indicates that TNFR2, as a single indicator, has excellent discriminatory power between heart failure patients and non-heart failure individuals, and its performance is significantly superior to traditional clinical diagnostic indicators.

[0186] Figure 13 This is a comparison of plasma TNFR2 concentrations between patients with heart failure (CBD) and healthy controls. From... Figure 12 It can be seen that the average expression level of plasma TNFR2 in the HF group was 9.64±0.82 log2 (pg / mL), which was significantly higher than that in the CON group (7.72±0.21 log2 (pg / mL) (P<0.0001).

[0187] Figure 14 The ROC curve for TNFR2 diagnosis of HF in the validation cohort of Center B is shown. The x-axis represents the false positive rate (1-specificity), and the y-axis represents the true positive rate (sensitivity). In the validation cohort of Center A, the area under the curve (AUC) for plasma TNFR2 in diagnosing heart failure was as high as 0.967 (95% CI: 0.909 - 1.000). The optimal diagnostic threshold, calculated using the Youden index of the ROC curve, was 8.112 log2 (pg / mL), at which point the sensitivity reached 93.3% and the specificity 100%.

[0188] This result further validates the cross-center consistency of TNFR2 as a biomarker for the diagnosis of heart failure.

[0189] The validation results of Center B were highly consistent with those of Center A. The cross-center consistency fully demonstrates that plasma TNFR2 has strong technical universality and clinical translation potential, and can stably and accurately achieve the auxiliary diagnosis of heart failure.

Claims

1. The application of reagents for detecting biomarkers in products for the diagnosis and / or prognostic assessment of heart failure, characterized in that, The biomarker is TNFR2.

2. The application according to claim 1, characterized in that, The biological samples tested are serum, plasma, whole blood, or peripheral blood mononuclear cells.

3. The application according to claim 1, characterized in that, The substances used to detect TNFR2 expression levels are antibodies, antibody fragments, or nucleic acid aptamers that specifically bind to the TNFR2 protein.

4. The application according to claim 1, characterized in that, The products mentioned are detection reagents, biochips, microarrays, or detection systems.

5. A kit for the diagnosis and / or prognostic assessment of heart failure, characterized in that, A reagent for detecting biomarkers as described in claim 1.

6. The kit for the diagnosis and / or prognostic assessment of heart failure according to claim 5, characterized in that, The reagents are selected from any one of enzyme-linked immunosorbent assay (ELISA) reagents, chemiluminescent immunoassay reagents, ortho-extended assay reagents, or internal standard reagents for mass spectrometry detection.

7. The kit for diagnosis and / or prognostic assessment of heart failure according to claim 5, characterized in that, include: 1) Immobilized TNFR2 capture antibody; 2) Detection of TNFR2 antibodies using enzymes or fluorescently labeled antibodies; 3) TNFR2 protein standards of known concentration.

8. The kit for diagnosis and / or prognostic assessment of heart failure according to claim 5, characterized in that, It also includes one or more of the following: buffer solution, washing solution, colorimetric reagent, signal detection reagent, standard and control.

9. The kit for diagnosis and / or prognostic assessment of heart failure according to claim 8, characterized in that, The chromogenic agent or signal detection reagent is selected from one or more of TMB, alkaline phosphatase substrate, ABTS, luminol or their derivatives; the buffer solution is selected from one or more of phosphate buffer, tris(hydroxymethyl)aminomethane buffer or carbonate buffer.

10. A method of using a kit for the diagnosis and / or prognostic assessment of heart failure as described in any one of claims 5-9, characterized in that, Includes the following steps: (1) Coating the surface of a microplate with a specific anti-TNFR2 capture antibody; (2) Add the plasma sample to be tested or the serially diluted standard, and incubate to allow the TNFR2 protein to bind to the capture antibody; (3) Add biotinylated detection antibody and horseradish peroxidase-labeled streptavidin; (4) Add TMB substrate to develop color, terminate the reaction with a stop solution, and then measure the absorbance at a wavelength of 450 nm. (5) Construct a four-parameter Logistic regression model to calculate the absolute concentration of plasma TNFR2.

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