A homogeneous immunoassay method for soluble thrombomodulin and d-dimer, a kit and application thereof

Abstract

The present application relates to the technical field of in vitro diagnosis, and discloses a homogeneous immunoassay method, kit and application of soluble thrombomodulin and D-dimer. In further detail, the present application can realize one-step quantitative analysis of soluble thrombomodulin and D-dimer in the same reaction system based on the developed homogeneous immunoassay method. The detection method or kit can specifically recognize target antigens, and does not affect each other in the liquid phase system, and has excellent anti-interference performance, and is not interfered by common endogenous substances such as fibrinogen, fibrinogen degradation fragment D / E, coagulation factor X and active form Xa. Compared with the existing conventional single index independent detection or heterogeneous detection method, the present application has the advantages of no need for washing and separation, simple operation, fast detection speed, high sensitivity and the like. Through single detection, clinical information reflecting the double pathological mechanisms of 'endothelial injury' and 'coagulation activation' can be quickly obtained, the detection timeliness is significantly improved, and a new technical means with significant application prospect is provided for the clinical needs such as bedside rapid screening and efficacy monitoring of venous thrombosis and related basic scientific research.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, specifically to a homogeneous immunoassay method, kit, and application of soluble thrombomodulin and D-dimer. Background Technology

[0002] Venous thromboembolism (VTE), known as the "silent killer" due to its high mortality rate and insidious nature, is the third leading cause of vascular disease after myocardial infarction and stroke. Currently, plasma D-dimer testing is an important exclusion tool for VTE screening due to its high negative predictive value. However, its levels can rise nonspecifically in conditions such as malignancy, inflammation, pregnancy, and post-surgery, leading to reduced diagnostic specificity and limiting its widespread application in the differential diagnosis of VTE. While computed tomography (CT) pulmonary angiography, considered the "gold standard," can visually visualize venous thrombi, it only shows positive results in the "middle to late" stages when anatomical structures have changed. Furthermore, its use is limited in special populations such as pregnant women due to carcinogenic and teratogenic risks. Therefore, innovations in laboratory screening for venous thrombosis are urgently needed to reduce mortality and improve prognosis.

[0003] Soluble thrombomodulin (sTM) is a product of the extracellular fragment of thrombomodulin and can reflect the degree of vascular endothelial cell damage. Endothelial injury is an important inducing and upstream factor in the pathogenesis of venous thrombosis (VTE) (the Virchow triad of VTE pathogenesis: endothelial injury, venous stasis, and hypercoagulable state). Therefore, combined quantitative detection of sTM and D-dimer is expected to capture the pathological process from the initiation of endothelial injury to coagulation activation and venous thrombosis, providing key support for early warning of VTE and individualized anticoagulation therapy.

[0004] However, there are currently no methods, kits, or applications that can simultaneously detect these two indicators without separation in the same homogeneous reaction system. Although multiple single-indicator detection methods can be combined in clinical practice, the operation is cumbersome, the detection cycle is long, and sample differences between different systems are difficult to avoid, which cannot meet the urgent need for rapid and immediate screening for acute and severe conditions such as VTE. Summary of the Invention

[0005] This invention aims to address the lack of efficient methods in the prior art for the simultaneous detection of soluble thrombomodulin (sTM) and D-dimer in the same homogeneous system. To address this issue, this invention, through in-depth research, provides a homogeneous immunoassay method, kit, and applications for the detection of soluble thrombomodulin and D-dimer.

[0006] In a first aspect of the present invention, a homogeneous immunoassay method for simultaneously detecting sTM and D-dimer is provided, the method comprising the following steps: (1) The sample to be tested and the detection reagent are mixed in a liquid phase system without separation; the detection reagent contains sTM capture antibody and D-dimer capture antibody labeled with fluorescent donors, and sTM detection antibody and D-dimer detection antibody labeled with fluorescent receptors, respectively. (2) A one-step immune reaction is carried out in a homogeneous system to form a double antibody sandwich complex; (3) The fluorescence signal emitted by the complex was detected by time-resolved fluorescence mode. A standard curve was established between the fluorescence intensity ratio of the acceptor channel and the donor channel and the concentration of the standard. The concentrations of sTM and D-dimer in the sample were calculated.

[0007] In another preferred embodiment, the fluorescent donor is an amino-reactive terbium (Tb) complex, and the fluorescent acceptor is a quantum dot (QD).

[0008] In another preferred embodiment, the time-resolved fluorescence mode in step (3) is to record the fluorescence signal by fluorescence integration after fluorescence excitation and a delay of 100 μs.

[0009] In a second aspect of the present invention, a method for preparing the detection reagent described in the first aspect of the present invention is provided, the method comprising the following steps: (1) Fluorescent labeling of capture antibodies: The amino reactive terbium (Tb) complex was reacted with sTM capture antibody and D-dimer capture antibody in alkaline buffer to obtain Tb-sTM capture antibody conjugate and Tb-D-dimer capture antibody conjugate, respectively. (2) Fluorescent labeling of detection antibodies: The amino-modified quantum dots (QDs) are surface activated using a heterobifunctional cross-linking agent to obtain QDs with maleimide reactivity; the activated QDs are reacted with sTM detection antibodies and D-dimer detection antibodies that have been reduced to expose thiol groups to obtain QD-sTM detection antibody conjugates and QD-D-dimer detection antibody conjugates.

[0010] In another preferred embodiment, the amino-reactive terbium (Tb) complex is a terbium complex activated by N-hydroxysuccinimide (NHS) ester.

[0011] In another preferred embodiment, the heterobifunctional crosslinker is a crosslinker containing NHS ester and maleimide groups.

[0012] In another preferred embodiment, the antibody is selected from monoclonal antibodies, nanobodies (also known as single-domain antibodies or VHHs), and antibodies and antibody fragments with antigen recognition capabilities.

[0013] In a third aspect of the invention, a homogeneous immunoassay kit for implementing the method described in the first aspect of the invention is provided, the kit comprising: (1) sTM capture antibody and D-dimer capture antibody labeled with fluorescent donor terbium (Tb) complex; (2) sTM detection antibody and D-dimer detection antibody labeled with fluorescent acceptor quantum dots (QD); (3) Reaction buffer and standards.

[0014] In another preferred embodiment, the reaction buffer is Tris-HCl at pH 7.4, containing 0.5% bovine serum albumin.

[0015] In a fourth aspect of the invention, the use of the kit as described in the third aspect of the invention in the preparation of in vitro diagnostic products for venous thrombosis screening, anticoagulation efficacy monitoring, or related basic scientific research is provided.

[0016] The main advantages of this invention include: (1) Covering dual pathological mechanisms and high clinical diagnostic value: This invention is the first to simultaneously detect sTM, which reflects vascular endothelial injury, and D-dimer, which reflects coagulation activation, in the same reaction system. By jointly monitoring these two key upstream and downstream indicators, the occurrence and development of venous thrombosis (VTE) can be captured more comprehensively, providing more accurate laboratory evidence for early warning, differential diagnosis, and monitoring of anticoagulation efficacy of VTE.

[0017] (2) Homogeneous and wash-free, fast and efficient: The present invention adopts a homogeneous reaction mode, which eliminates the need for separation steps, making the operation simple and the detection time significantly shortened, making it suitable for rapid bedside screening.

[0018] (3) Strong anti-interference ability: The present invention uses terbium (Tb) complex as a fluorescence donor and utilizes its long fluorescence lifetime characteristics to effectively eliminate the interference of short-lifetime background fluorescence in plasma matrix through time-resolved detection mode. Attached Figure Description

[0020] Figure 1 The sandwich ELISA response value is the result of pairing sTM and D-dimer antibodies.

[0021] Figure 2 The UV absorption and fluorescence spectra of the Tb-conjugated sTM / D-dimer capture antibody.

[0022] Figure 3The UV absorption and fluorescence spectra of the QD-conjugated sTM / D-dimer detection antibody were obtained.

[0023] Figure 4 To validate the FRET sensing mechanism of sTM and D-dimer single target.

[0024] Figure 5 For biological crosstalk, specificity analysis and experimental optimization.

[0025] Figure 6 The standard curve for the simultaneous determination kit of sTM and D-dimer.

[0026] Figure 7 To assess the clinical value of the simultaneous determination system of sTM and D-dimer.

[0027] Figure 8 To verify the consistency of the kit for simultaneous determination of sTM and D-dimer. Detailed Implementation

[0028] Through in-depth research, this invention has successfully established a method and kit for the simultaneous detection of soluble thrombomodulin and D-dimer. To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0029] Unless otherwise specified, the experimental methods described in the following examples are conventional methods. Unless otherwise specified, all experimental materials, reagents, and instruments used are commercially available.

[0030] In a preferred embodiment of the present invention, the antibody is a nanobody, particularly a bivalent nanobody that captures sTM and D-dimer. The nanobodies obtained by the inventors through extensive inventive screening possess advantages such as small molecular weight, high affinity, and strong specificity, making them the best embodiment for achieving high-sensitivity detection according to the present invention. Although the following embodiments use nanobodies as examples for detailed description, those skilled in the art should understand that the scope of protection of the present invention is not limited thereto. Any antibody (such as a monoclonal antibody, heavy chain antibody, etc.) or antibody fragment (such as a single-chain antibody) capable of recognizing different epitopes of sTM and D-dimer is within the scope of protection of the present invention.

[0031] It should be understood that for antibodies or antibody fragments lacking natural disulfide bonds and the ability to recognize target antigens, those skilled in the art can employ conventional protein engineering techniques (e.g., site-directed mutagenesis to introduce cysteine ​​residues) to provide reaction sites, or use other coupling strategies such as amino coupling and click chemistry to achieve labeling with quantum dots. The scope of protection of this invention covers the application of the above-mentioned conventional techniques.

[0032] Example 1: Validation of sTM and D-dimer paired antibodies (1) Pairing Competency Verification Experiment: To verify the performance of the paired antibodies, the pairing competence of the antibody pairs was verified using an enzyme-linked immunosorbent assay (ELISA). Capture antibodies of 2 μg / mL sTM and D-dimer diluted in 100 mM NaHCO3 were coated onto two Nunc™ MaxiSorp™ microplates and incubated overnight at 2–8 ℃. The next morning, after washing four times with 1× PBST, 1% skim milk powder was added for blocking at room temperature (18–25 ℃) for 2 h. After washing, different concentrations of sTM (MedChemExpress, USA) or D-dimer antigen (Medix Biochemica, Finland) at 2-fold serial dilutions from 2000 ng / mL were added, and the plates were incubated at 37 ℃ for 1 h. After washing, biotin-labeled detection antibodies of 2 μg / mL sTM or D-dimer were added to the corresponding microplates, and the plates were incubated at 37 ℃ for 1 h. Finally, the absorbance at 450 nm was recorded using an HRP-labeled streptavidin and TMB substrate system.

[0033] (2) Experimental results: The results are as follows Figure 1 As shown, both sTM and D-dimer paired antibodies exhibited excellent antigen-binding activity. The absorbance of the sTM antibody pair gradually increased with increasing antigen concentration within the detection range, and the signal tended to saturate at an antigen concentration of 500 ng / mL. Similarly, the absorbance of the D-dimer antibody pair gradually increased with increasing antigen concentration, and the signal tended to saturate at an antigen concentration of 1000 ng / mL. A standard curve was then plotted with the absorbance at 450 nm for each concentration of standard as the ordinate and the antigen concentration (ng / mL) in the working solution of each standard as the abscissa. Nonlinear fitting analysis was performed using Origin Pro 2025 (OriginLab Corp., Northampton, MA, USA). The goodness of fit R-squared for sTM was 0.99866, and for D-dimer, it was 0.99978. These results indicate that the selected capture antibody and detection antibody can form a stable sandwich complex, which can be used for subsequent quantitative detection.

[0034] Example 2: Preparation and optical characterization of Tb-capture antibody conjugates and QD-detection antibody conjugates (1) Preparation and optical characterization of Tb-capture antibody conjugates: First, 8 mM Lumi4-Tb-NHS was dissolved in anhydrous DMF, and then mixed with sTM or D-dimer capture antibody in 100 mM carbonate buffer (pH = 9.0). The molar ratio of Lumi4-Tb-NHS to sTM or D-dimer capture antibody was 5:1. The reaction mixture was incubated at 25 rpm for 2 h at room temperature. To purify the Tb-sTM or D-dimer capture antibody conjugates, they were centrifuged at 4000 × g using a 10 kDa ultrafiltration tube, washed 6 times with 100 mM Tris-HCl buffer (pH = 7.4), and stored in a refrigerator at 2–8 ℃ protected from light. Figure 2 As shown, the UV absorption spectrum of the Tb-sTM or D-dimer capture antibody conjugate shows maximum UV absorption at 337 nm, while the fluorescence emission spectrum shows four characteristic narrow peaks at 489, 547, 582 and 620 nm, indicating that its fluorescence properties are not affected by the capture antibody conjugation.

[0035] (2) Preparation and optical characterization of QD-detection antibody conjugates: sTM or D-dimer detection antibodies were conjugated to amino-modified quantum dots (QDs) via a thiol-maleimide chemical method. A several-fold excess of Sulfo-EMCS and QDs were mixed in 1×PBS buffer (pH = 7.4) and incubated at 30 rpm for 30 min at room temperature. The disulfide bonds on the detection antibody were reduced to thiol groups by adding 5 mM tris(2-carboxyethyl)phosphine to 1×PBS buffer (pH = 7.4) and incubating for 30 min. Excess crosslinking agent and reducing agent were removed sequentially using a Zeba desalting column. Finally, the two desalted solutions were mixed and incubated at 30 rpm for 4 h at room temperature in the dark. Unconjugated detection antibodies were removed using a 100 kDa ultrafiltration tube and washed four times with 100 mM borate buffer (pH = 8.4). The purified conjugate was centrifuged at 4000 × g, and the supernatant was stored in a refrigerator at 2–8 ℃ protected from light. Figure 3 Display QD 605 -sTM detection of antibody conjugates and QD 650 -D-dimer detection antibody conjugates exhibit symmetrical Gaussian emission peaks, and both QD-detection antibodies have a wide UV absorption range.

[0036] Example 3: Single-target FRET sensing verification (1) Single-target FRET sensing validation experiment: To validate the sensing mechanism of FRET donor Tb to FRET receptor QD, FRET immunoassays were first performed on sTM and D-dimer, and the sensing mechanism was analyzed based on fluorescence lifetime decay curves. All assays were performed in a total volume of 150 μL of solution containing fixed concentrations of Tb-capture antibody and QD-detection antibody conjugate (sTM system: 5.0 nM Tb-sTM capture antibody and 5.0 nM QD-sTM detection antibody; D-dimer system: 10.0 nM Tb-D-dimer capture antibody and 10.0 nM QD-D-dimer detection antibody).

[0037] (2) Validation of sTM single-target FRET sensing: With the increase of sTM concentration (0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 nM), QD 605 -sTM detection of antibody conjugate fluorescence decay curves showed a sensitization effect ( Figure 4 A), while the fluorescence decay curve of the Tb-sTM-captured antibody conjugate did not show quenching (A), Figure 4 B). The calculated FRET ratio reached a plateau at a sTM antigen (~75 kDa) concentration of 3 nM, and showed an approximately linear increasing trend before signal saturation. Figure 4 C).

[0038] (3) Validation of D-dimer single-target FRET sensing: With the increase of D-dimer concentration (0, 0.25, 0.5, 1.0, 1.5, 2.0, 4.0, 8.0 nM), QD 650 The fluorescence decay curve of the antibody-diplasm conjugate detected by -D-dimer also showed a sensitization effect. Figure 4 D), while the fluorescence decay curve of the Tb-D-dimer-captured antibody conjugate did not show quenching (D), Figure 4 E). The calculated FRET ratio reached a plateau at a D-dimer (~195 kDa) antigen concentration of 8 nM. Figure 4 F). Both sensing systems showed that the fluorescence signal of the QD channel gradually increased with increasing antigen concentration, confirming the sensing mechanism of FRET donor Tb sensitizing FRET receptor QD.

[0039] Example 4: Biological crosstalk, specificity analysis and experimental optimization (1) Biocrosstalk: To establish a FRET detection system capable of simultaneously determining the concentrations of sTM and D-dimer in the same sample, the biocrosstalk between the two pairs of antibodies was systematically evaluated. When only Tb-modified sTM capture antibody and QD are present... 605When using modified sTM to detect antibodies, the FRET ratio only increases with increasing sTM antigen concentration, while D-dimer antigen shows a background FRET ratio across the entire concentration range (0–200 ng / mL). Figure 5 A). Similarly, when only Tb-modified D-dimer capture antibodies and QD are present... 650 When using modified D-dimer to detect antibodies, the FRET ratio only increases with increasing D-dimer antigen concentration, while the sTM antigen shows a background FRET ratio across the entire concentration range (0–1500 ng / mL). Figure 5 B). The results show that, within the relevant concentration range, the antibody pairs of sTM and D-dimer do not interfere with each other in the detection of target molecules, and can be used to construct a simultaneous detection system.

[0040] (2) Specificity analysis: This invention further evaluated the specificity of the constructed simultaneous detection system. The target molecules were sTM (final concentration 200 ng / mL) and D-dimer (final concentration 1500 ng / mL). Potential interfering substances included human fibrinogen, human fibrinogen fragment D, human fibrinogen fragment E, human coagulation factor X, human coagulation factor Xa, cysteine, glycine, and glutathione (all at a final concentration of 1500 ng / mL). The results showed that the FRET ratio in the corresponding detection channel only significantly increased when the target molecules sTM and D-dimer were present. Figure 5 C), and none of the interfering substances tested showed significant cross-reactivity.

[0041] (3) Experimental Optimization: This invention further optimized key conditions to obtain the best signal response. First, the pH of the reaction system was optimized. Results showed that the FRET ratio increased with increasing pH of the reaction buffer, reached relative stability within the pH range of 7–7.4, and then decreased ( Figure 5 D). Secondly, the effect of reaction temperature on the FRET ratio was optimized. It was found that the FRET ratio gradually increased with increasing incubation temperature, and tended to stabilize in the range of 35–40℃. Figure 5 E). Finally, the effect of reaction time on the FRET ratio was optimized. It was found that after the addition of antigen, the FRET ratio of sTM and D-dimer gradually increased with increasing reaction time, reaching a stable plateau after 25 min (E). Figure 5 F).

[0042] Example 5: Dual-target homogeneous FRET immunoassay (1) Dual-target homogeneous FRET immunoassay: FRET detection of sTM and D-dimer was performed in a liquid phase system with a working volume of 150 μL (containing 50 μL of solution with sTM and D-dimer standards and 100 μL of solution with two fluorescently labeled antibody pairs). All samples were measured three times. The Tb-capture antibody conjugate and QD-detection antibody conjugate were diluted in 50 μL of Tris-HCl buffer containing 0.5% bovine serum albumin. 50 μL of sTM / D-dimer antigen samples of different concentrations were added to 100 μL of solution containing Tb / QD-antibody conjugate. The microplate was mixed using a microplate shaker and incubated at 37 ℃ for 25 min, followed by detection using a Tecan Spark multi-mode microplate reader. Fluorescence integration was performed after fluorescence excitation and a delay of 100 µs, and the time-gated fluorescence intensity of Tb donor and QD acceptor was collected simultaneously.

[0043] (2) Results of homogeneous FRET immunoassay for dual targets: A standard curve was plotted with the FRET ratio of each concentration of standard as the ordinate and the antigen concentration (ng / mL) in the working solution of each standard as the abscissa. Linearity fitting analysis was performed using Origin Pro 2025. With the increase of sTM antigen concentration, QD... 605 The FRET ratio in the channel showed a linear increasing trend in the range of 0.5–200 ng / mL, and the corresponding linear equation was: Y FRET比值 = 0.000825559 + 0.0011 × X sTM浓度(ng / mL) ( Figure 6 A), the goodness of fit R-squared is 0.99818. However, with the increase of D-dimer antigen concentration, QD... 650 The FRET ratio in the channel also showed a linear increasing trend in the range of 5–1500 ng / mL, and the corresponding linear equation was: Y FRET比值 = 0.00148 + 0.000056724 × X D-dimer浓度(ng / mL) ( Figure 6 B), the goodness of fit R-squared is 0.99885. According to the formula for calculating the limit of detection (LOD)... LOD = 3 x SD / slope Where SD represents the standard deviation of antigen-free samples, and the slope is... slope The detection limits for sTM and D-dimer were determined by the linear growth portion of the FRET immunoassay calibration curve (as determined by the linear growth portion of the FRET immunoassay calibration curve), and were 0.18 ng / mL and 3.79 ng / mL (3σ, respectively). n=14). Therefore, the constructed FRET immunoassay covers the key cutoff values ​​reported in clinical practice and literature for the detection of sTM and D-dimer (healthy population: normal range of sTM 3–50 ng / mL; D-dimer < 500 ng / mL [FEU] or 250 ng / mL [DDU]).

[0044] Example 6: Validation of the clinical incremental value and methodological consistency of dual-indicator detection (1) Clinical incremental value of dual-indicator combined detection: To verify the inventive advantage of the dual-indicator detection constructed in this invention compared with the clinical single-indicator detection, a retrospective case-control study was first conducted based on 20 patients with VTE diagnosed by computed tomography pulmonary angiography and 48 non-VTE controls at Shanghai First Maternity and Infant Hospital. The results are as follows: Figure 7 As shown in Figure A, the D-dimer levels in the VTE group were significantly higher than those in the non-VTE group, with median values ​​of 4.380 mg / L and 1.270 mg / L, respectively. P < 0.001); sTM levels were also significantly higher in the VTE group (6.400 TU / mL) than in the non-VTE group (5.605 TU / mL). P = 0.001). Further analysis shows ( Figure 7 (B) While single indicators have some diagnostic value, the sTM+D-dimer diagnostic model exhibits the best discriminative performance, with an AUC of 0.958 (95% CI: 0.906-1.000). Particularly noteworthy is the sensitivity indicator related to patient safety, where the combined model achieves 0.950, significantly outperforming single indicators. This demonstrates that the present invention, based on a combined detection strategy of "endothelial injury + coagulation activation" dual pathological mechanisms, creatively constructs a full-chain risk warning system, possessing potential clinical value in providing crucial support for rapid clinical decision-making.

[0045] (2) Consistency verification between the kit of this invention and the clinical gold standard method: In order to verify the accuracy and practicality of the homogeneous detection method based on FRET technology of this invention, based on the above real clinical samples, the kit of this invention (FRET method) and the routine independent clinical detection method were used for measurement and comparison. Among them, the routine clinical detection of sTM used the chemiluminescence kit (clinical kit, catalog number: DG0400) of Sichuan Wovent Biotechnology Co., Ltd.; the routine clinical detection of D-dimer used the Siemens INNOVANCE® D-Dimer immunoturbidimetric kit (clinical kit, catalog number: OPBP07). Subsequently, with the occurrence of VTE as the dependent variable, the detection results of the clinical kit of sTM and D-dimer and the FRET method of sTM and D-dimer were respectively included in logistic regression to construct two joint models. ROC curves were plotted according to the predicted probabilities of each model, and AUC was calculated to evaluate its discriminative power for VTE. Results ( Figure 8 The results show high consistency: the AUC of the combined FRET method model of this invention is 0.964 (95% CI: 0.915-1.000), while the AUC of the clinically independently measured combined model is 0.952 (95% CI: 0.888-1.000). Both have comparable diagnostic accuracy.

[0046] Technical advantages: While maintaining diagnostic performance comparable to the clinical gold standard, this invention eliminates the need for complex separation and washing steps, significantly shortening the detection time.

[0047] In summary, this invention not only demonstrates the clinical superiority of combined detection of sTM and D-dimer, but also achieves simultaneous detection of sTM and D-dimer for the first time through a unique fluorescence conjugation method and kit construction process. It provides a highly efficient, convenient, and sensitive homogeneous immunoassay method and kit for soluble thrombomodulin and D-dimer, with significant application prospects.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Those skilled in the art should understand that various modifications, variations, and improvements can be made to the present invention without departing from its spirit and scope, and any such modifications, variations, and improvements are within the protection scope of the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents. Based on the above description, other variations or modifications can be made in different forms. It is impossible to exhaustively list all embodiments here, but all obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A homogeneous immunoassay method for simultaneously detecting soluble thrombomodulin (sTM) and D-dimer, characterized in that, Includes the following steps: (1) The sample to be tested and the detection reagent are mixed in a liquid phase system without separation; the detection reagent contains sTM capture antibody and D-dimer capture antibody labeled with fluorescent donors, and sTM detection antibody and D-dimer detection antibody labeled with fluorescent receptors, respectively. (2) A one-step immune reaction is carried out in a homogeneous system to form a double antibody sandwich complex; (3) The fluorescence signal emitted by the complex was detected by time-resolved fluorescence mode. A standard curve was established between the fluorescence intensity ratio of the acceptor channel and the donor channel and the concentration of the standard. The concentrations of sTM and D-dimer in the sample were calculated.

2. A method for preparing a fluorescently labeled antibody for use in the homogeneous immunoassay method of claim 1, characterized in that, Includes the following steps: (1) Fluorescent labeling of capture antibodies: The amino reactive terbium (Tb) complex was reacted with sTM capture antibody and D-dimer capture antibody in alkaline buffer to obtain Tb-sTM capture antibody conjugate and Tb-D-dimer capture antibody conjugate, respectively. (2) Fluorescent labeling of detection antibodies: The amino-modified quantum dots (QDs) are surface activated using a heterobifunctional cross-linking agent to obtain QDs with maleimide reactivity; the activated QDs are reacted with sTM detection antibodies and D-dimer detection antibodies that have been reduced to expose thiol groups to obtain QD-sTM detection antibody conjugates and QD-D-dimer detection antibody conjugates.

3. A homogeneous immunoassay kit for implementing the method as described in claim 1, characterized in that, The kit includes: (1) sTM capture antibody and D-dimer capture antibody labeled with fluorescent donor terbium (Tb) complex; (2) sTM detection antibody and D-dimer detection antibody labeled with fluorescent acceptor quantum dots (QD); (3) Reaction buffer and standards.

4. The detection method according to claim 1, characterized in that: (1) The capture antibody and detection antibody of the sTM respectively recognize different epitopes of human soluble thrombomodulin; (2) The capture antibody and detection antibody of the D-dimer respectively recognize different epitopes of human D-dimer.

5. The detection method according to claim 1, characterized in that, The fluorescent donor is an amino-reactive terbium (Tb) complex, and the fluorescent acceptor is a quantum dot (QD). The fluorescent donor and the fluorescent acceptor can form a fluorescence resonance energy transfer (FRET) donor-acceptor pair, and the FRET donor-acceptor pair for sTM and the FRET donor-acceptor pair for D-dimer have different signal output wavelengths.

6. The detection method according to claim 1, characterized in that, The time-resolved fluorescence mode in step (3) is to record the fluorescence signal by fluorescence integration after fluorescence excitation and a delay of 100 μs.

7. The preparation method according to claim 2, characterized in that: (1) The fluorescent labeling of the capture antibody is achieved by covalent coupling of an amino-reactive terbium (Tb) complex with a primary amine group on the surface of the antibody; (2) The fluorescent labeling of the detection antibody is achieved by covalently coupling the antibody with quantum dots through a thiol-maleimide chemical method.

8. The preparation method according to claim 2, characterized in that: (1) The amino reactive terbium (Tb) complex is a terbium complex activated by N-hydroxysuccinimide (NHS) ester; (2) The heterobifunctional crosslinking agent is a crosslinking agent containing NHS ester and maleimide groups; the "reduction treatment" refers to the use of tris(2-carboxyethyl)phosphine (TCEP) to reduce the disulfide bonds on the antibody to thiol groups.

9. The application of the kit as described in claim 3 in the preparation of in vitro diagnostic products for venous thrombosis screening, anticoagulation efficacy monitoring, or related basic scientific research.

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