A high-precision nr1d2 detection sicm prognostic detection kit and a preparation method thereof

Abstract

The application discloses a high-precision NR1D2 detection SICM prognosis detection kit and a preparation method thereof, and belongs to the technical field of in-vitro diagnostic kits. The technical problem to be solved is that the existing NR1D2 detection method has low sensitivity and cannot realize clinical rapid prognosis evaluation of septic cardiomyopathy (SICM). The kit adopts a chemiluminescence double-epitope sandwich immunoassay method, the lower limit of detection reaches 0.5 pg / mL, the intra-batch CV is less than or equal to 5%, the inter-batch CV is less than or equal to 8%, and a prognosis scoring algorithm module is integrated, the prognosis score is calculated according to the NR1D2 concentration, the SOFA score and the age factor, the SICM patients are divided into three risk groups of low, medium and high, and the 28-day survival rate prediction AUC reaches 0.857. The sensitivity of the application is more than 10 times higher than that of the conventional ELISA, the detection time is shortened to 90 minutes, and the integration of NR1D2 detection and SICM prognosis evaluation is realized.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent kit technology, specifically referring to a highly accurate NR1D2 detection SICM prognosis detection kit and its preparation method. Background Technology

[0002] Septic cardiomyopathy (SICM) is one of the most serious complications of sepsis, occurring in 40%-60% of sepsis patients and with a mortality rate as high as 50%-70%. The pathological mechanisms of SICM involve multiple factors, including myocardial depression, mitochondrial dysfunction, cytokine storm, and autonomic nervous system dysfunction. Clinically, it manifests as reversible decrease in myocardial contractility and ventricular dilation. Currently, the diagnosis of SICM mainly relies on echocardiography (e.g., decreased LVEF) and myocardial markers (e.g., cTnI, BNP). However, these indicators have insufficient sensitivity and specificity and cannot effectively predict disease progression and prognosis.

[0003] NR1D2 (also known as Rev-Erbβ) is a member of the nuclear receptor superfamily and plays a key role in inflammation regulation, lipid metabolism, and circadian rhythm regulation as a transcriptional repressor. Recent studies have found that NR1D2 plays an important role in myocardial injury in sepsis: (1) NR1D2 expression level is positively correlated with the intensity of myocardial inflammatory response; (2) NR1D2 can directly regulate the expression of myocardial cell apoptosis-related genes; (3) The serum NR1D2 protein level is significantly elevated in sepsis patients and is closely related to the severity of the disease and prognosis.

[0004] Currently, there are no clinically specific detection kits for the NR1D2 protein. Existing NR1D2 detection methods mainly rely on Western blot and qPCR, which are cumbersome, time-consuming, and have poor quantitative accuracy, failing to meet the clinical demand for rapid and accurate detection. Although various chemiluminescent immunoassay kits are available for the detection of myocardial biomarkers, there is still no chemiluminescent immunoassay kit specifically for the NR1D2 protein, and even fewer integrated detection solutions that combine NR1D2 detection results with SICM prognostic assessment.

[0005] Therefore, developing a highly sensitive and specific NR1D2 chemiluminescence detection kit and applying it to the prognostic assessment of SICM has significant clinical application value. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, this invention provides a highly accurate NR1D2 detection SICM prognostic test kit and its preparation method, which solves the problems of low sensitivity, complicated operation and inability to achieve rapid clinical detection of existing NR1D2 detection methods. At the same time, it combines NR1D2 detection results with SICM prognostic assessment to achieve integrated assessment from biomarker detection to clinical prognostic stratification.

[0007] The technical solution adopted in this invention is as follows: This invention proposes a highly accurate NR1D2 detection SICM prognostic kit, comprising the following parts:

[0008] (a) Capture antibody: an anti-NR1D2 monoclonal antibody that specifically binds to the N-terminal epitope of human NR1D2 protein (amino acid sequence SEQ ID NO:1, sequence: MEVNAGGVIAYISSSSSASSPASCHSEGSENSFQSSS), said capture antibody being coated on the surface of magnetic microspheres;

[0009] (b) Detection antibody: an anti-NR1D2 monoclonal antibody that specifically binds to the C-terminal epitope of human NR1D2 protein (amino acid sequence SEQ ID NO:2, sequence: LRSLNNMHSEELLAFKVHPQLEA), the detection antibody being labeled with horseradish peroxidase (HRP) to form an enzyme-labeled detection antibody;

[0010] (c) Chemiluminescent substrate solution: containing a luminol-enhanced chemiluminescence system;

[0011] (d) Standard: Recombinant human NR1D2 protein, concentration gradient of 0.5-10000 pg / mL;

[0012] (e) Buffer system: including coating buffer, washing buffer, sample dilution buffer and blocking buffer;

[0013] (f) Quality control products: including NR1D2 quality control serum at low, medium and high concentrations;

[0014] (g) Biotin-streptavidin signal amplification module, the module comprising: a biotinylated anti-HRP polyclonal antibody and a streptavidin-labeled HRP enzyme conjugate;

[0015] (h) Prognostic assessment module, which includes a scoring formula: score = α × log(NR1D2 concentration) + β × SOFA score + γ × age factor + δ, which divides patients into low, medium and high risk groups;

[0016] The two epitopes recognized by the capture antibody and the detection antibody are at a linear distance of not less than 30 Å in the spatial structure of the NR1D2 protein.

[0017] Furthermore, the magnetic microspheres have a particle size of 1.0-3.0 μm and are modified with carboxyl active groups on their surface; the capture antibody is covalently coupled to the surface of the magnetic microspheres via an EDC / NHS amide condensation reaction, with a coupling density of 20-80 μg antibody / mg microspheres.

[0018] Preferably, the magnetic microspheres are superparamagnetic iron oxide-polystyrene core-shell structured microspheres with a saturation magnetization of not less than 30 emu / g.

[0019] Furthermore, the enzyme-labeled detection antibody is a conjugate obtained by directionally coupling HRP to the detection antibody via the sodium periodate method, with a molar ratio of HRP to antibody of 3:1-6:1, and purified by Sepharose CL-4B gel filtration chromatography; the chemiluminescent substrate solution comprises: 0.5-2.0 mmol / L luminol or isoluminol, 0.1-0.5 mmol / L p-iodophenol, 1.0-5.0 mmol / L hydrogen peroxide, and a Tris-HCl buffer system at pH 8.5-9.5.

[0020] Furthermore, the standard is a recombinant human NR1D2 full-length protein (Met1-Leu614) prepared by a baculovirus-insect cell expression system, with a purity of not less than 95%.

[0021] Furthermore, in the buffer system: the coating buffer is 50 mM carbonate buffer at pH 9.6, the washing buffer is PBS containing 0.05%-0.1% Tween-20 at pH 7.4, the sample dilution buffer is PBS containing 1%-3% BSA and 0.05% Tween-20 at pH 7.4, and the blocking buffer is a PBS solution containing 3%-5% skim milk powder or 1%-3% BSA.

[0022] Furthermore, the concentration gradient of the standard includes ten calibration points: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000, and 10000 pg / mL.

[0023] This invention also proposes a method for preparing a highly accurate NR1D2 detection SICM prognosis detection kit, specifically including the following steps:

[0024] S1. Preparation of anti-NR1D2 monoclonal antibody: N-terminal epitope peptide SEQ ID NO:1 and C-terminal epitope peptide SEQ ID NO:2 were synthesized and immunized BALB / c mice respectively. Hybridoma cell lines that stably secrete specific antibodies were obtained by screening using hybridoma technology, and monoclonal antibodies were obtained by purification.

[0025] S2. Capture antibody-coated magnetic microspheres: Take carboxylated magnetic microspheres, activate them with EDC / NHS, and then add the N-terminal specific monoclonal antibody obtained in step S1 for covalent coupling to obtain capture antibody-coated magnetic microspheres;

[0026] S3. Preparation of enzyme-labeled detection antibody: Take the C-terminal specific monoclonal antibody obtained in step S1, oxidize the Fc glycosyl group of the antibody using the sodium periodate method, and then perform directional coupling with HRP. The coupling product is purified by gel filtration chromatography to obtain the HRP-labeled detection antibody.

[0027] S4. Preparation of Standards: The recombinant human NR1D2 full-length protein expressed via the baculovirus-insect cell system was purified and diluted to a series of gradient concentrations of 0.5-10000 pg / mL;

[0028] S5. Preparation of chemiluminescent substrate solution: Prepare a Tris-HCl luminescent substrate working solution containing luminol, p-iodophenol and hydrogen peroxide, pH 8.5-9.5;

[0029] S6. Buffer system preparation: Prepare coating buffer, washing buffer, sample dilution solution and blocking solution separately;

[0030] S7. Preparation of quality control materials: Add a known concentration of recombinant NR1D2 protein to a healthy human serum matrix to prepare three concentration levels: low, medium, and high.

[0031] S8. Assembly and packaging: Package the components obtained in steps S2-S7 according to the proportions and store them in a sealed container at 2-8℃.

[0032] Further, the specific conditions for EDC / NHS activation in step S2 are as follows: carboxylated magnetic microspheres are washed and dispersed with MES buffer (pH 5.0-6.0), and EDC with a final concentration of 2-5 mg / mL and NHS with a final concentration of 1-3 mg / mL are added sequentially. Activation is carried out at room temperature for 15-30 minutes, and then capture antibody with a final concentration of 0.5-2 mg / mL is added. Coupling is carried out at room temperature for 2-4 hours or at 4°C overnight.

[0033] Further, the specific conditions for the sodium periodate method conjugation described in step S3 are as follows: after equilibration of the C-terminal specific monoclonal antibody by dialyzing with 0.1 M carbonate buffer (pH 9.5), NaIO4 is added to a final concentration of 5-20 mmol / L, and the reaction is carried out at 4°C in the dark for 30 minutes; after terminating oxidation with ethylene glycol, NaIO4 is removed by dialyzing; HRP is added (HRP:antibody molar ratio 3:1-6:1), and conjugation is carried out overnight at 4°C; after NaBH4 reduction and stabilization, the enzyme-labeled antibody peak is collected by Sepharose CL-4B gel filtration chromatography.

[0034] Furthermore, the recombinant human NR1D2 full-length protein described in step S4 is expressed using a baculovirus-insect cell expression system (Sf9 cells), and purified in two steps by nickel column affinity chromatography and molecular sieve chromatography, with a purity of not less than 95%.

[0035] The beneficial effects achieved by the present invention using the above structure are as follows:

[0036] 1. High specificity: It adopts a combination of biclonal antibodies that recognize two non-overlapping epitopes at the N-terminus and C-terminus of the NR1D2 protein, with an epitope spacing of ≥30Å, which effectively avoids steric hindrance and cross-reactivity, ensuring the specificity of the detection results.

[0037] 2. High sensitivity: The chemiluminescent dual-epitope sandwich immunoassay combines a magnetic microsphere solid-phase carrier with HRP enzyme-catalyzed luminescence signal amplification, achieving a detection limit of 0.5 pg / mL. This is 10-100 times more sensitive than conventional ELISA methods, enabling accurate detection of low-abundance NR1D2 protein in the serum of sepsis patients.

[0038] 3. Fast and convenient: Based on magnetic microsphere separation technology, it eliminates the need for multiple plate washing steps required by traditional ELISA. The entire testing process can be completed within 90 minutes, making it suitable for routine clinical testing.

[0039] 4. Integrated prognostic assessment: Integrating NR1D2 concentration detection results with multiple parameters such as SOFA score and age factor, a quantitative prognostic scoring model is established, realizing the leap from simple biomarker detection to clinical prognostic stratification, and providing objective basis for individualized treatment decisions for SICM patients. Attached Figure Description

[0040] Figure 1 This is a flowchart illustrating the preparation method of a high-precision NR1D2 detection SICM prognostic test kit proposed in this invention;

[0041] Figure 2 The standard curve for NR1D2 detection in the kit of this invention is shown, with a correlation coefficient R² = 0.998.

[0042] Figure 3 This is the ROC curve for predicting 28-day mortality in SICM patients using the prognostic scoring model of this invention.

[0043] Figure 4 Kaplan-Meier survival curves for SICM patients in different risk groups;

[0044] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0045] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0046] Example 1: Preparation of the reagent kit of the present invention

[0047] This embodiment specifically illustrates the preparation process of the NR1D2 detection SICM prognostic kit of the present invention, and the steps are as follows:

[0048] 1. Preparation of anti-NR1D2 monoclonal antibody

[0049] N-terminal epitope peptide SEQ ID NO:1 (sequence: MEVNAGGVIAYISSSSSASSPASCHSEGSENSFQSSS) and C-terminal epitope peptide SEQ ID NO:2 (sequence: LRSLNNMHSEELLAFKVHPQLEA) were synthesized and used to immunize 6-week-old BALB / c mice. Each mouse received 100 μg of the immunized peptide, and the immunization cycle was 2 weeks, for a total of 4 immunizations. Spleen cells from the immunized mice were then fused with SP2 / 0 myeloma cells using PEG-mediated cell fusion. After screening in HAT medium and subcloning using limiting dilution, two hybridoma cell lines stably secreting specific anti-NR1D2 monoclonal antibodies were obtained, named 1D6 (recognizing the N-terminal epitope) and 3H9 (recognizing the C-terminal epitope), respectively. The monoclonal antibodies were purified by Protein G affinity chromatography to obtain a purity greater than 95%.

[0050] 2. Capture antibody-coated magnetic microspheres

[0051] Take 10 mg of carboxylated superparamagnetic iron oxide-polystyrene core-shell microspheres with a particle size of 2.0 μm, wash three times with 1 mL LME S buffer (pH 5.5), and resuspend to 1 mL. Add EDC solution to a final concentration of 3 mg / mL and NHS solution to a final concentration of 2 mg / mL, and activate at room temperature in the dark for 20 minutes. Magnetic separation and discard the supernatant, add 1 mL of 50 mM carbonate buffer (pH 9.6) to resuspend, add 1 mg of purified 1D6 monoclonal antibody, mix well at room temperature and couple for 3 hours. Magnetic separation and discard the supernatant, add blocking buffer (PBS containing 5% skim milk powder) and block at room temperature for 2 hours. After blocking, wash three times with washing buffer, and finally resuspend in 10 mL of storage solution and store at 4 °C for later use. At this time, the antibody conjugation density is 50 μg antibody / mg microsphere.

[0052] 3. Preparation of antibodies for enzyme-linked immunosorbent assay (ELISA)

[0053] Take 5 mg of purified 3H9 monoclonal antibody and dialyze overnight with 0.1 M carbonate buffer (pH 9.5). Add NaIO4 solution to a final concentration of 10 mmol / L and react at 4°C in the dark for 30 minutes. Add ethylene glycol to a final concentration of 100 mmol / L to terminate oxidation and react at room temperature for 15 minutes. Dialyze the reaction solution overnight with 0.1 M carbonate buffer to remove small molecule impurities. Add 2 mg of HRP enzyme (HRP to antibody molar ratio of 4:1), mix and couple overnight at 4°C. Add NaBH4 solution to a final concentration of 1 mg / mL and react at 4°C for 2 hours to reduce and stabilize the conjugate. Filter the conjugate product onto a Sepharose CL-4B gel filtration chromatography column, collect the enzyme-labeled antibody peak, concentrate by ultrafiltration to obtain the HRP-labeled detection antibody, and store at -20°C.

[0054] 4. Preparation of standard products

[0055] The recombinant human NR1D2 full-length protein expressed and purified by the baculovirus-insect cell system was serially diluted with sample diluent to prepare a series of standards with concentrations of 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000, and 10000 pg / mL. After aliquoting, the standards were stored at -20°C.

[0056] 5. Preparation of chemiluminescent substrate solution

[0057] Prepare Tris-HCl buffer (pH 9.0), add luminol to a final concentration of 1.0 mmol / L, p-iodophenol to a final concentration of 0.2 mmol / L, and hydrogen peroxide to a final concentration of 2.5 mmol / L, mix well, and store at 2-8°C.

[0058] 6. Preparation of buffer solution system

[0059] Prepare separately:

[0060] Coating buffer: 50mM carbonate buffer, pH 9.6;

[0061] Washing buffer: PBS buffer containing 0.05% Tween-20, pH 7.4;

[0062] Sample diluent: PBS buffer containing 2% BSA and 0.05% Tween-20, pH 7.4;

[0063] Blocking solution: PBS buffer containing 3% BSA, pH 7.4.

[0064] 7. Preparation of quality control materials

[0065] Take mixed healthy human serum, add recombinant NR1D2 protein to each sample, and prepare three levels of quality control samples: low concentration (10 pg / mL), medium concentration (100 pg / mL), and high concentration (1000 pg / mL). After aliquoting, store at 2-8℃.

[0066] 8. Signal Amplification Module Configuration

[0067] Prepare biotinylated anti-HRP polyclonal antibody (concentration 0.1 mg / mL) and streptavidin-labeled HRP enzyme conjugate (concentration 0.05 mg / mL), dilute with sample diluent, aliquot, and store at 2-8℃.

[0068] 9. Assembly and packaging

[0069] Assemble the above components in the following dosages: 10 mL of capture antibody-coated magnetic microsphere working solution, 5 mL of HRP-labeled detection antibody working solution, 20 mL of chemiluminescent substrate solution, 0.5 mL each of the series of standards, 0.5 mL each of the quality control products, 50 mL each of the buffer system, and 5 mL each of the signal amplification module. Also include a prognostic assessment instruction card. Assemble into a test kit for 96 individuals and store sealed at 2-8℃.

[0070] Example 2: Detection and prognostic evaluation method of the kit of the present invention

[0071] This embodiment illustrates the specific usage method of the reagent kit of the present invention, including the complete process of sample testing and prognostic assessment:

[0072] 1. Sample processing

[0073] Take serum samples from SICM patients, centrifuge at 12,000 rpm for 5 minutes, collect the supernatant, and dilute it with sample diluent at a ratio of 1:10 for later use.

[0074] 2. Testing Steps

[0075] (1) Take 50 μL of the processed sample / standard / quality control and add it to the reaction well. At the same time, add 50 μL of the magnetic microsphere working solution coated with capture antibody. Incubate at 37°C for 30 minutes. After magnetic separation, discard the supernatant and wash 3 times with washing buffer.

[0076] (2) Add 100 μL of HRP-labeled detection antibody working solution, incubate at 37°C for 30 minutes, magnetically separate and discard the supernatant, and wash 3 times.

[0077] (3) Add 100 μL of signal amplification module working solution, incubate at 37°C for 15 minutes, magnetically separate and discard the supernatant, and wash 3 times.

[0078] (4) Add 100 μL of chemiluminescent substrate solution, react at room temperature in the dark for 5 minutes, and use a chemiluminescence detector to detect the relative luminescence units (RLU).

[0079] 3. Concentration Calculation

[0080] Plot a standard curve based on the RLU values ​​of the standards and calculate the concentration of NR1D2 in the samples.

[0081] 4. Prognostic assessment

[0082] Collect patients' SOFA scores and age information, and substitute them into the prognostic scoring formula:

[0083] Score = 0.82 × log(NR1D2 concentration) + 0.45 × SOFA score + 0.03 × age factor - 2.15

[0084] Risk stratification is based on the scoring results:

[0085] A score < 1.0 indicates a low-risk group, suggesting a high 28-day survival rate and a good prognosis.

[0086] A score of 1.0 or less but less than 2.5 indicates a moderate risk of poor prognosis.

[0087] A score ≥2.5 indicates a high-risk group, suggesting a high 28-day mortality rate and poor prognosis.

[0088] Experimental Example 1: Reagent Kit Sensitivity Detection

[0089] The kit of this invention was used to perform 20 repeated tests on blank samples (zero-concentration standards). The mean and standard deviation of the detection signals were calculated, and the concentration corresponding to the mean plus two standard deviations was taken as the limit of detection. The results showed that the limit of detection of this kit was 0.5 pg / mL, while the limit of detection of conventional commercial NR1D2 ELISA kits was 5.2 pg / mL. The sensitivity of this invention is more than 10 times higher than that of conventional methods, and it can effectively detect low abundance of NR1D2 protein in serum.

[0090] This kit exhibits good linearity in the range of 0.5-10000 pg / mL, with a correlation coefficient R²=0.998. The corresponding standard curve is shown in Figure 2.

[0091] Experimental Example 2: Precision Detection of the Reagent Kit

[0092] Three quality control samples at low, medium, and high concentrations were used, and each concentration was repeatedly tested in 10 wells. The intra-batch coefficient of variation (CV) was calculated. Three batches were tested consecutively, and the inter-batch coefficient of variation was calculated. The results are as follows:

[0093] Quality control concentration Intra-batch CV (%) Inter-batch CV (%) 10 pg / mL 3.2 5.6 100 pg / mL 2.8 6.2 1000pg / mL 4.1 7.1

[0094] The results showed that the intra-batch CV of this kit was ≤5% and the inter-batch CV was ≤8%, which met the precision requirements of in vitro diagnostic kits, and the test results were stable and reliable.

[0095] Experimental Example 3: Reagent Kit Recovery Detection

[0096] Three concentrations (low, medium, and high) of recombinant NR1D2 protein were added to the serum of healthy individuals, and the kit was used for detection. The recovery rates were calculated. The results showed that the recoveries for the three concentrations were 92.5%, 98.7%, and 105.3%, respectively, with an average recovery rate of 98.8%. This indicates that the kit can accurately detect NR1D2 in serum samples without significant matrix effects.

[0097] Experiment Example 4: Kit Specificity Detection

[0098] The kit was used to detect NR1D1 protein, cTnI protein, BNP protein, and CRP protein at 1000 ng / mL. The results showed that the detection signals of these proteins were lower than those of the blank sample, indicating that the kit had no cross-reactivity with these homologous proteins and common myocardial markers, and had good specificity.

[0099] Experiment Example 5: Clinical Prognostic Validation Experiment

[0100] 120 patients diagnosed with SICM were included. Serum samples were collected from the patients upon admission. The concentration of NR1D2 was detected using this kit. At the same time, the patients' SOFA scores and age information were collected to calculate the prognostic score. The patients' survival was followed up for 28 days.

[0101] The results show:

[0102] 1. The ROC curve AUC of the prognostic scoring model for predicting 28-day mortality was 0.857, significantly higher than the predictive efficacy of using NR1D2 alone (AUC=0.721) and SOFA score alone (AUC=0.765), indicating that the integrated model of this invention has better prognostic predictive ability. The corresponding ROC curve is shown in Figure 3.

[0103] 2. Patients were divided into low-, intermediate-, and high-risk groups based on prognostic scores. The 28-day survival rates for the three groups were 96.7%, 78.3%, and 41.7%, respectively. Kaplan-Meier survival analysis showed a significant difference in survival time among the three groups (Log-rank P < 0.001). The corresponding survival curves are shown in Figure 4.

[0104] These results demonstrate that the kit of the present invention can effectively stratify the prognostic risk of SICM patients, providing a reference for clinical treatment decisions.

[0105] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A high-precision NR1D2 detection kit for SICM prognosis, characterized in that, The kit comprises the following components: (a) Capture antibody: an anti-NR1D2 monoclonal antibody that specifically binds to the N-terminal epitope of human NR1D2 protein, wherein the capture antibody is coated on the surface of magnetic microspheres; (b) Detection antibody: an anti-NR1D2 monoclonal antibody that can specifically bind to the C-terminal epitope of human NR1D2 protein, wherein the detection antibody is labeled with horseradish peroxidase to form an enzyme-labeled detection antibody; (c) Chemiluminescent substrate solution: containing a luminol-enhanced chemiluminescence system; (d) Standard: Recombinant human NR1D2 protein, concentration gradient of 0.5-10000 pg / mL; (e) Buffer system: including coating buffer, washing buffer, sample dilution buffer and blocking buffer; (f) Quality control products: including NR1D2 quality control serum at low, medium and high concentrations; (g) Biotin-streptavidin signal amplification module, the module comprising: a biotinylated anti-HRP polyclonal antibody and a streptavidin-labeled HRP enzyme conjugate; (h) Prognostic assessment module, which includes a scoring formula: score = α × log(NR1D2 concentration) + β × SOFA score + γ × age factor + δ, which divides patients into low, medium and high risk groups; The linear distance between the two epitopes recognized by the capture antibody and the detection antibody on the spatial structure of the NR1D2 protein is not less than 30 Å.

2. The high-precision NR1D2 detection SICM prognostic kit according to claim 1, characterized in that: The magnetic microspheres have a particle size of 1.0-3.0 μm and are modified with carboxyl active groups on their surface. The capture antibody is covalently coupled to the surface of the magnetic microspheres via an EDC / NHS amide condensation reaction, with a coupling density of 20-80 μg antibody / mg microsphere. The magnetic microspheres are superparamagnetic iron oxide-polystyrene core-shell structure microspheres with a saturation magnetization of not less than 30 emu / g.

3. The high-precision NR1D2 detection SICM prognostic kit according to claim 1, characterized in that: The enzyme-labeled detection antibody is a conjugate obtained by directionally coupling HRP and the detection antibody via the sodium periodate method, with a molar ratio of HRP to antibody of 3:1-6:1, and purified by Sepharose CL-4B gel filtration chromatography. The chemiluminescent substrate solution comprises: 0.5-2.0 mmol / L luminol or isoluminol, 0.1-0.5 mmol / L p-iodophenol, 1.0-5.0 mmol / L hydrogen peroxide, and a Tris-HCl buffer system at pH 8.5-9.

5.

4. The high-precision NR1D2 detection SICM prognostic kit according to claim 1, characterized in that: The standard is a recombinant human NR1D2 full-length protein prepared by a baculovirus-insect cell expression system, with a purity of not less than 95%.

5. The high-precision NR1D2 detection SICM prognostic kit according to claim 1, characterized in that: In the buffer system: the coating buffer is 50 mM carbonate buffer with pH 9.6, the washing buffer is PBS containing 0.05%-0.1% Tween-20 with pH 7.4, the sample dilution buffer is PBS containing 1%-3% BSA and 0.05% Tween-20 with pH 7.4, and the blocking buffer is a PBS solution containing 3%-5% skim milk powder or 1%-3% BSA.

6. The high-precision NR1D2 detection SICM prognostic kit according to claim 1, characterized in that: The concentration gradient of the standard includes ten calibration points: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000, and 10000 pg / mL.

7. A method for preparing a high-precision NR1D2 detection SICM prognostic kit, characterized in that, Includes the following steps: S1. Preparation of anti-NR1D2 monoclonal antibody: N-terminal epitope peptide SEQ ID NO:1 and C-terminal epitope peptide SEQ ID NO:2 were synthesized and immunized BALB / c mice respectively. Hybridoma cell lines that stably secrete specific antibodies were obtained by screening using hybridoma technology, and monoclonal antibodies were obtained by purification. S2. Capture antibody-coated magnetic microspheres: Take carboxylated magnetic microspheres, activate them with EDC / NHS, and then add the N-terminal specific monoclonal antibody obtained in step S1 for covalent coupling to obtain capture antibody-coated magnetic microspheres; S3. Preparation of enzyme-labeled detection antibody: Take the C-terminal specific monoclonal antibody obtained in step S1, oxidize the Fc glycosyl group of the antibody using the sodium periodate method, and then perform directional coupling with HRP. The coupling product is purified by gel filtration chromatography to obtain the HRP-labeled detection antibody. S4. Preparation of Standards: The recombinant human NR1D2 full-length protein expressed via the baculovirus-insect cell system was purified and diluted to a series of gradient concentrations of 0.5-10000 pg / mL; S5. Preparation of chemiluminescent substrate solution: Prepare a Tris-HCl luminescent substrate working solution containing luminol, p-iodophenol and hydrogen peroxide, pH 8.5-9.5; S6. Buffer system preparation: Prepare coating buffer, washing buffer, sample dilution solution and blocking solution separately; S7. Preparation of quality control materials: Add a known concentration of recombinant NR1D2 protein to a healthy human serum matrix to prepare three concentration levels: low, medium, and high. S8. Assembly and packaging: Package the components obtained in steps S2-S7 according to the proportions and store them in a sealed container at 2-8℃.

8. The preparation method of a high-precision NR1D2 detection SICM prognostic test kit according to claim 7, characterized in that: The specific conditions for EDC / NHS activation in step S2 are as follows: carboxylated magnetic microspheres are washed and dispersed with MES buffer (pH 5.0-6.0), and EDC with a final concentration of 2-5 mg / mL and NHS with a final concentration of 1-3 mg / mL are added sequentially. The mixture is activated at room temperature for 15-30 minutes, and then the capture antibody with a final concentration of 0.5-2 mg / mL is added. The mixture is then coupled at room temperature for 2-4 hours or at 4°C overnight.

9. The preparation method of a high-precision NR1D2 detection SICM prognostic test kit according to claim 7, characterized in that: The specific conditions for the sodium periodate method conjugation described in step S3 are as follows: After equilibration of the C-terminal specific monoclonal antibody by dialyzing with 0.1 M carbonate buffer (pH 9.5), NaIO4 is added to a final concentration of 5-20 mmol / L, and the reaction is carried out at 4°C in the dark for 30 minutes; after terminating oxidation with ethylene glycol, NaIO4 is removed by dialyzing; HRP is added, and the conjugation is carried out overnight at 4°C, with an HRP-antibody molar ratio of 3:1-6:1; after stabilization by NaBH4 reduction, the enzyme-labeled antibody peak is collected by Sepharose CL-4B gel filtration chromatography.

10. The method for preparing a high-precision NR1D2 detection SICM prognostic kit according to claim 7, characterized in that: The recombinant human NR1D2 full-length protein described in step S4 was expressed using a baculovirus-insect cell expression system (Sf9 cells), and purified in two steps by nickel column affinity chromatography and molecular sieve chromatography, with a purity of not less than 95%.

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