Chemiluminescence immunoassay method for high-sensitivity detection of cardiac troponin I

Abstract

The invention relates to the technical field of immunoassay, and discloses a chemiluminescence immunoassay method for high-sensitivity detection of cardiac troponin I. The chemiluminescence immunoassay method comprises the following steps: fixing an anti-cardiac troponin I capture antibody based on a solid phase carrier; a sample to be detected is in contact with the solid phase carrier coated with the capture antibody, a detection antibody is added, the detection antibody is connected with a signal marker, and the signal marker is a compound formed by loading alkaline phosphatase on a nano material or is the nano material; a chemiluminescent substrate is added, and the chemiluminescent substrate is a compound based on 1, 2-dioxetane; detecting a chemiluminescence signal so as to quantify cardiac troponin I; according to the invention, the problem of non-uniform detection of degradation fragments is solved, and the diagnosis window period is prolonged; meanwhile, extremely high detection sensitivity, signal-to-noise ratio and stability are realized, and the kit is suitable for early and accurate diagnosis of myocardial injury.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and more specifically, to a chemiluminescent immunoassay for the high-sensitivity detection of cardiac troponin I. Background Technology

[0002] Cardiac troponin I (cTnI) is a specific biomarker of myocardial injury. Its sensitive detection is crucial for the diagnosis of acute myocardial infarction. Chemiluminescent immunoassay (CLIA) is widely used for cTnI detection due to its high sensitivity. In existing technologies, conventional CLIA methods often use enzyme-labeled antibodies and luminol or its derivatives as substrates. However, these methods suffer from insufficient signal amplification, unstable substrate luminescence kinetics, susceptibility to environmental interference, poor signal stability, and narrow dynamic linear range, making it difficult to achieve detection sensitivity at the pg / L level, thus limiting their application in detecting low-concentration cTnI. In the early stages of acute myocardial infarction, the concentration of cTnI in the patient's blood is extremely low, and due to the low sensitivity of traditional methods, misdiagnosis or missed diagnosis may occur.

[0003] Furthermore, existing antibody pairs used to detect cTnI often target its unstable N-terminal or C-terminal regions. In a patient's blood, cTnI is rapidly hydrolyzed by proteases, generating various degradation fragments. Using antibodies targeting the unstable ends leads to inconsistent detection signals for intact cTnI and its degradation fragments, failing to accurately reflect the overall level of myocardial injury markers in the blood. This shortens the effective diagnostic window and may result in misdiagnosis or missed diagnosis.

[0004] Therefore, there is an urgent need in this field for a highly sensitive cTnI detection method with stronger signal, lower background, and better stability. Summary of the Invention

[0005] In view of this, the present invention proposes a chemiluminescent immunoassay for the detection of cardiac troponin I with high sensitivity, aiming to solve the problems of insufficient sensitivity in existing cardiac troponin I detection methods, as well as inaccurate detection of degradation fragments and short diagnostic window due to improper selection of antibody epitopes.

[0006] This invention proposes a highly sensitive chemiluminescent immunoassay method for detecting cardiac troponin I, comprising the following steps: S1. Capture antibody against cardiac troponin I immobilized on a solid-phase carrier; S2. Contact the sample to be tested with a solid-phase carrier coated with the capture antibody; S3. Add detection antibody to the solid support described in step S2, wherein the detection antibody is linked to a signal marker; S4. Under light-protected conditions, add a chemiluminescent substrate to the solid support in step S3; S5. Detect the chemiluminescent signal to quantify cardiac troponin I; The signal marker is a complex formed by loading alkaline phosphatase onto nanomaterials, or is a nanomaterial itself. The chemiluminescent substrate is a compound based on 1,2-dioxane.

[0007] More preferably, the antigenic epitopes recognized by the capture antibody and the detection antibody are located in the region of amino acid positions 30 to 110 of the central domain of cardiac troponin I, and the two epitopes do not overlap.

[0008] More preferably, in step S3, the nanomaterial is silica nanoparticles, gold nanoclusters, or quantum dots.

[0009] More preferably, in step S4, the chemiluminescent substrate is CDP-Star or APS-5, and the amount of the chemiluminescent substrate CDP-Star or APS-5 added is 0.2-1.0 mM.

[0010] More preferably, in step S3, the amount of enzyme loaded per milligram of nanomaterial in the complex formed by loading alkaline phosphatase is 0.5-2.0 mg.

[0011] More preferably, in step S3, the amount of the detection antibody added is 0.1-2.0 μg / mL.

[0012] More preferably, the solid support is a microporous plate, magnetic particles, or glass fiber membrane.

[0013] More preferably, washing is performed after both steps S2 and S3.

[0014] More preferably, in step S4, the chemiluminescent substrate CDP-Star or APS-5 is dissolved in a buffer solution with a pH of 8.0-10.0 to prepare a chemiluminescent substrate solution, which is then added to the solid-phase carrier for completing the immune reaction.

[0015] On the other hand, this application also provides an application of a highly sensitive chemiluminescent immunoassay for detecting cardiac troponin I, characterized in that it includes: the application of the highly sensitive chemiluminescent immunoassay for detecting cardiac troponin I in the preparation of diagnostic products for detecting myocardial injury.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. High diagnostic accuracy and long window period: By using antibody pairs targeting the stable epitope in the middle of cTnI, this invention can uniformly detect intact cTnI and its early degradation fragments in the blood, truly reflecting the overall degree of myocardial damage, significantly prolonging the effective diagnostic window period for acute myocardial infarction, and improving diagnostic accuracy.

[0017] 2. Extremely high sensitivity: The signal is amplified exponentially through nanomaterial labeling, and combined with a stable substrate system based on 1,2-dioxane, the detection sensitivity is improved to the pg / L level.

[0018] 3. Excellent detection performance: This method has a high signal-to-noise ratio, good detection repeatability, and a wide dynamic linear range.

[0019] The method described in this invention overcomes the bottleneck of existing technologies' insensitivity and instability in detecting extremely low concentrations of cardiac troponin I (cTnI), thereby significantly advancing the diagnostic window for myocardial injury and providing a precise tool for clinical use, suitable for the early diagnosis of myocardial injury. Attached Figure Description

[0020] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 The flowchart illustrates a chemiluminescent immunoassay method for detecting cardiac troponin I with high sensitivity, as provided in an embodiment of the present invention. Detailed Implementation

[0021] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the disclosure to those skilled in the art. It should be noted that, unless otherwise specified, embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0022] In this application, this embodiment provides a chemiluminescent immunoassay method for highly sensitive detection of cardiac troponin I, characterized by comprising the following steps: S1. Capture antibody against cardiac troponin I immobilized on a solid-phase carrier; S2. Contact the sample to be tested with a solid-phase carrier coated with the capture antibody; S3. Add detection antibody to the solid support described in step S2, wherein the detection antibody is linked to a signal marker; S4. Under light-protected conditions, add a chemiluminescent substrate to the solid support in step S3; S5. Detect the chemiluminescent signal to quantify cardiac troponin I; The signal marker is a complex formed by loading alkaline phosphatase onto nanomaterials, or is a nanomaterial itself; the chemiluminescent substrate is a compound based on 1,2-dioxane.

[0023] This invention first uses a solid-phase carrier to immobilize a capture antibody against cardiac troponin I; In this invention, the antigenic epitope recognized by the capture antibody is located in the region of amino acids 30 to 110 of the central domain of cardiac troponin I.

[0024] Specifically, cTnI molecules are rapidly hydrolyzed by proteases in the blood, especially their unstable N-terminus and C-terminus. However, its central domain (amino acids 30-110) is crucial for the formation of a stable complex between cTnI and troponin C (cTnC). This region is spatially protected by cTnC binding, exhibiting conformational stability and resistance to protease degradation. Selecting epitopes in this region ensures that antibodies uniformly recognize intact cTnI and its early degradation fragments in the blood, thus accurately reflecting the total myocardial injury burden.

[0025] The specific steps are as follows: Dilute the purified anti-cTnI capture antibody to the final concentration using the buffer solution described above. Mix the antibody solution with the pretreated solid-phase carrier. Add the cross-linking agent and incubate with gentle shaking at a suitable temperature for a certain period of time. After the reaction is complete, discard the supernatant. Add a buffer containing bovine serum albumin (BSA) and Tris-HCl as the blocking solution. Incubate at 20-37°C for 1-2 hours. Wash repeatedly with washing buffer to thoroughly remove uncross-linked antibody and free blocking agent. Finally, resuspend the coated solid-phase carrier in a suitable storage buffer and store at 2-8°C for later use.

[0026] The specific preparation process of the capture antibody is as follows: Using genetic engineering technology, a recombinant protein fragment containing only amino acid sequences from positions 30 to 110 of cTnI is specifically cloned, expressed, and purified. Suitable animals are immunized with the prepared immunogen, and spleen cells are fused with myeloma cells to construct a hybridoma cell library; or a large antibody library is constructed using phage display technology to generate a candidate antibody library rich in antibodies that recognize the central domain of cTnI. Microplates are coated with the recombinant cTnI (positions 30-110) fragment. The hybridoma supernatant or phage clone library is incubated with the coated plate. After washing, the signal is detected by enzyme-labeled secondary antibody color development or chemiluminescence to initially screen all positive clones that can bind to the target central region. Microplates are then coated with the N-terminal and C-terminal fragments of cTnI, respectively. The positive clones obtained in the previous step are incubated with these two types of plates and detected, and all clones that produce a significant signal with the N-terminal or C-terminal fragment are removed. A series of short peptides covering the full length of cTnI, continuous and overlapping, were synthesized, immobilized on a membrane or chip, and incubated with the finally screened candidate capture antibodies. By detecting the signal, the specific short peptide sequence bound by the antibody was accurately read out, and the epitope located in the 30-110 region was directly determined from the amino acid sequence level.

[0027] Understandably, this step enables specific capture, separation, and signal localization. This step immobilizes the antibody on the carrier surface, allowing for precise capture of cTnI target molecules from complex blood samples; and through solid-liquid separation washing, impurities are efficiently removed, providing a clean, low-background environment for subsequent detection. More importantly, it provides a stable and ordered reaction interface for subsequent nanomaterial-based signal amplification systems, ensuring that the strong signal carried by each captured cTnI molecule can be efficiently and centrally detected, which is an indispensable foundation for achieving a sensitivity leap from ng / L to pg / L. Furthermore, the antibody described in this invention can simultaneously capture intact cTnI molecules and their important early degradation fragments in blood with uniform high affinity.

[0028] After fixation, a solid-phase carrier coated with the capture antibody is obtained. The sample to be tested is then brought into contact with the solid-phase carrier coated with the capture antibody, and then washed.

[0029] Specifically, this step utilizes an antigen-antibody specific binding reaction to specifically capture and immobilize the target analyte (cTnI) in the test sample from the liquid phase onto the surface of a solid-phase support. The captured antibody on the solid-phase support recognizes the corresponding antigenic epitope on the cTnI molecule with high affinity and high specificity through its antigen-binding site (Fab segment).

[0030] The specific operating steps are as follows: Place an appropriate amount of the carrier suspension coated with the capture antibody into a reaction tube. The capture antibody has been verified through epitope mapping, and its binding epitope is clearly located within the 30-110 amino acid region of the central cTnI domain. Accurately add the test sample (such as patient serum or plasma), cTnI standard, or quality control sample to the reaction tube. Incubate the reaction system at a constant temperature of 35-38°C for 15-30 minutes. During this period, the cTnI (intact molecule and stable fragment) in the sample specifically binds to the capture antibody on the surface of the solid-phase carrier. After incubation, place the reaction tube on a magnetic separator and let it stand for 1-3 minutes until the solution becomes clear. Discard the supernatant to remove most impurities, non-specific proteins, and free degradation fragments that have not bound to the capture antibody. Remove the reaction tube, add washing buffer, separate, and discard the supernatant. Repeat this washing step 2-4 times.

[0031] Understandably, by co-incubating the test sample with a solid-phase carrier coated with capture antibodies, the cTnI antigen in the sample is recognized by specific antibodies and firmly bound to the solid-phase surface, forming an immune complex. The subsequent washing step efficiently removes unbound impurities, non-specific proteins, and interfering substances from the blood sample, significantly reducing detection background noise and providing a clean reaction environment for subsequent signal amplification and detection. This step transforms a complex liquid-phase reaction into a solid-phase interfacial reaction, laying an indispensable and solid foundation for subsequent signal amplification and high signal-to-noise ratio chemiluminescence detection using nanomaterial-labeled detection antibodies.

[0032] A detection antibody is added to a solid-phase support after it comes into contact with the sample to be tested, and the detection antibody is linked to a signal marker.

[0033] In this invention, the signal marker is preferably a complex formed by loading alkaline phosphatase onto nanomaterials, or is itself a nanomaterial. The antigenic epitope recognized by the detection antibody is located within the 30th to 110th amino acid region of the central domain of cardiac troponin I, and does not overlap with the antigenic epitope recognized by the capture antibody. The nanomaterial is preferably silica nanoparticles, gold nanoclusters, or quantum dots. The amount of the detection antibody added is preferably 0.1-2.0 μg / mL, based on the final reaction system. Specifically, the detection antibody recognizes another epitope on the cTnI molecule that is different from the one recognized by the capturing antibody. Because this invention limits both epitopes to a stable central domain (amino acids 30-110) that does not overlap, they can simultaneously bind to a single cTnI molecule, forming a robust structure. This ensures signal specificity, excluding non-binding signals. When the signal marker is a complex formed by loading alkaline phosphatase onto nanomaterials, the nanomaterials are loaded with a large number of enzyme molecules (ALP). This allows hundreds or thousands of enzyme molecules to be introduced in a single immune binding event, achieving exponential signal amplification. When the signal marker is a nanomaterial, quantum dots, gold nanoclusters, and other nanomaterials possess unique chemiluminescent or catalytic luminescence properties and can be directly used as signal sources, achieving high-intensity signal labeling as well. Stable epitope binding and strong signal amplification work synergistically in this step. The stable structure provides a robust attachment platform for the nanosignal marker, while the strong signal amplification ensures that each captured cTnI molecule can be clearly and accurately detected.

[0034] The specific operating steps are as follows: The complex formed by loading alkaline phosphatase onto nanomaterials is diluted with Tris-HCl buffer containing a protein stabilizer to a final concentration of 0.5-2.0 μg / mL in the reaction system. The epitope recognized by the detection antibody is located in the 30-110 amino acid region of the central domain of cTnI and does not overlap with the epitope of the capture antibody. After contacting and washing the sample with the solid-phase support, the above-mentioned detection antibody solution is immediately added to the solid-phase support and gently mixed to ensure sufficient contact. The reaction system is placed in a constant temperature environment and incubated in the dark for 20-40 minutes. This incubation process allows the detection antibody to specifically bind to the captured cTnI molecules through its antigen-binding site, forming a complete immune complex. After incubation, separation is performed. Thorough washing is carried out to remove as much non-specifically bound label as possible.

[0035] The following steps are used to verify that the antigenic epitope recognized by the detection antibody is located within the 30th to 110th amino acid region of the central domain of cardiac troponin I, and does not overlap with the antigenic epitope recognized by the capture antibody: A cTnI-cTnC complex is prepared as an immunogen to preferentially obtain antibodies that recognize natural, stable conformational epitopes. Animals are immunized using the immunogen, and a candidate antibody library is constructed using hybridoma technology or phage display technology. Microplates are coated with the cTnI [30-110] recombinant protein fragment and reacted with the antibody library to initially screen for all antibodies that can bind to the central domain. The antibody group that passes this stage has its epitopes preliminarily confined to the 30-110 region. Microplates are then coated with the N-terminal and C-terminal fragments of cTnI, respectively, and reacted with the positive clones from the previous stage to eliminate all antibodies that can bind to either the N-terminus or C-terminus. The antibodies that pass this stage have their epitopes strictly confined to the 30-110 region and are independent of unstable ends. Candidate antibody A was immobilized as the capture antibody and cTnI antigen was added. Candidate antibody B (labeled with biotin or enzyme) was added as the detection antibody. If a high-intensity specific signal was generated, it proved that A and B could simultaneously bind to cTnI, and were preliminarily determined to be compatible. All candidate antibodies were subjected to pairwise pairing tests to identify all potential effective pairing combinations. The cTnI antigen was pre-incubated with an excess of unlabeled antibody A (saturating its binding sites). This mixture was added to wells already coated with antibody A, and then enzyme-labeled antibody B was added. If the signal of antibody B was strongly inhibited (>70%), it indicated that A and B competed to bind the same or overlapping epitopes. If the signal inhibition of antibody B was not significant (<30%), it proved that A and B bound different, non-overlapping epitopes. An overlapping peptide library covering the full length of cTnI was synthesized and reacted with the finally determined paired antibodies. The specific peptide sequence bound by each antibody could be accurately read. This data ultimately confirmed at the amino acid sequence level that their epitopes were all located in the 30-110 region and were non-overlapping.

[0036] Understandably, the detection antibody recognizes another epitope within the central domain of cTnI that does not overlap with the capture antibody, ensuring that both can bind simultaneously and stably to the same cTnI molecule, thus guaranteeing high detection specificity. More importantly, the signal markers it is linked to, whether nanomaterials acting as enzyme carriers or self-luminescent nanomaterials, can significantly enhance the signal intensity generated by each immune binding event. The two work synergistically to transform the previously captured specific target into a strong, detectable signal.

[0037] Under light-protected conditions, a chemiluminescent substrate is added to the solid support in step S3.

[0038] In this invention, the chemiluminescent substrate is preferably CDP-Star or APS-5, and the amount of CDP-Star or APS-5 added is preferably 0.2-1.0 mM. The chemiluminescent substrate CDP-Star or APS-5 is dissolved in a buffer solution with a pH of 8.0-10.0 to prepare a chemiluminescent substrate solution, which is then added to the solid-phase support for completing the immunoreaction.

[0039] Specifically, this step utilizes alkaline phosphatase (ALP) to catalyze a chemical reaction in the substrate, converting chemical energy into light energy. The ALP in the complex immobilized on a solid support specifically recognizes and cleaves the phosphate group on the chemiluminescent substrate (CDP-Star or APS-5) molecule. The dephosphorylated substrate molecule becomes extremely unstable and rapidly decomposes, causing the core 1,2-dioxane ring to break. The breakage of the 1,2-dioxane ring releases energy, which excites the reaction product molecule into an excited state. When the excited-state molecule returns to the ground state, its excess energy is released as photons, producing visible light. Substrates such as CDP-Star and APS-5 are photosensitive. Illumination induces a slow background degradation, leading to increased background noise and significantly reduced signal-to-noise ratio. Therefore, this process must be performed under light-protected conditions.

[0040] The specific operating steps are as follows: Dissolve CDP-Star or APS-5 solid powder in Tris-HCl buffer at pH 8.0-10.0. This alkaline environment is necessary for ALP to exert its optimal catalytic activity. Prepare the substrate solution as a stock solution and ensure thorough mixing. The preparation process should be carried out under gentle light or in the dark. Use immediately after preparation or store in the dark. After adding the detection antibody and washing, ensure the solid support is kept moist. Under dark conditions, quickly add the prepared substrate solution to the reaction tube. Immediately and gently vortex or pipette to mix, ensuring full contact between the substrate and the ALP label on the solid support surface. Incubate the reaction system at room temperature in the dark for 5-10 minutes. After incubation, immediately place the reaction tube into the detection chamber of the chemiluminescence analyzer. The instrument will integrate and collect the luminescence signal value (relative light unit RLU) within 1-10 seconds. The signal intensity is proportional to the amount of ALP bound on the solid support, and the amount of ALP is ultimately proportional to the cTnI concentration in the initial sample.

[0041] Understandably, through this step, the entire immune recognition and signal amplification chain constructed in the early stages is ultimately transformed into a quantitatively detectable optical signal. This invention preferentially uses the CDP-Star / APS-5 substrate system, which, compared to the traditional luminol system, has three core advantages: extremely low background, high signal intensity, and long signal duration. Combined with the nanomaterial signal amplification system, these advantages lay the final foundation for achieving high sensitivity.

[0042] After the chemiluminescent substrate is added, the chemiluminescent signal is detected to quantify cardiac troponin I.

[0043] Specifically, after adding the substrate and incubating, the reaction system is measured by photon counting or integration. The relative light units measured for the sample are compared with the signal value of the cTnI standard concentration gradient. Using the instrument's built-in four-parameter logistic curve fitting algorithm, the signal value is accurately converted into the cTnI concentration, thus completing the quantification.

[0044] Understandably, by using antibody pairs targeting the stable epitope in the middle of cTnI, this invention can uniformly detect intact cTnI and its early degradation fragments in the blood, accurately reflecting the overall degree of myocardial damage, significantly prolonging the effective diagnostic window period for acute myocardial infarction, and improving diagnostic accuracy. Simultaneously, the exponential amplification of the signal through nanomaterial labeling, combined with a stable substrate system based on 1,2-dioxane, enhances the detection sensitivity to the pg / L level.

[0045] In another preferred embodiment based on the above embodiments, this embodiment provides an application of a chemiluminescent immunoassay for the high-sensitivity detection of cardiac troponin I, characterized in that it includes: the application of the chemiluminescent immunoassay for the high-sensitivity detection of cardiac troponin I in the preparation of diagnostic products for detecting myocardial injury.

[0046] Understandably, this method has a high signal-to-noise ratio, good detection repeatability, and a wide dynamic linear range. It overcomes the bottleneck of existing technologies being insensitive and unstable in detecting extremely low concentrations of cardiac troponin I (cTnI), thus significantly advancing the diagnostic window for myocardial injury and providing a precise tool for clinical use, suitable for the early diagnosis of myocardial injury.

[0047] Example 1 I. Materials and Reagents Solid-phase carrier and antibody: streptavidin-coated magnetic microparticles and biotinylated anti-cTnI capture antibody (epitope located at amino acids 42-56 of cTnI). The capture antibody was obtained through a targeted immunization and stratified screening process.

[0048] Detection antibody: The detection antibody (eptopel located at amino acids 78-92 of cTnI) is labeled using a complex of silica nanoparticles loaded with alkaline phosphatase (ALP), and the labeling method is as described in the instruction manual. The working concentration of the detection antibody is 1.0 μg / mL.

[0049] Chemiluminescent substrate: CDP-Star, prepared as a 0.5 mM working solution using diethanolamine buffer at pH 9.5.

[0050] Standard: Recombinant human cTnI protein, diluted with negative human serum to the following concentration gradients: 0 pg / mL, 0.5 pg / mL, 2 pg / mL, 10 pg / mL, 50 pg / mL, 200 pg / mL, 1000 pg / mL.

[0051] Instrument: Fully automated chemiluminescence immunoassay analyzer.

[0052] II. Experimental Procedure 1. Biotinylated capture antibodies were conjugated with streptavidin magnetic beads to prepare capture microspheres.

[0053] 2. Take 50 μL of each concentration of cTnI standard and mix it with 50 μL of the captured microsphere suspension. Incubate at 37°C for 30 minutes. Separate magnetically and wash three times.

[0054] 3. Add 100 μL of nanomaterial-ALP-labeled detection antibody solution and incubate at 37°C for 30 minutes. Separate magnetically and wash three times.

[0055] 4. Under light-protected conditions, add 100 μL of LCDP-Star substrate working solution and react at room temperature for 10 minutes.

[0056] 5. Immediately use a chemiluminescence analyzer to detect the luminescence signal value of each tube (RLU, integration time 2 seconds).

[0057] III. Results Record the average RLU value of each concentration standard (n=3) and calculate the signal-to-noise ratio (SNR), i.e., signal value / zero concentration standard signal value. Plot the cTnI concentration on the x-axis (logarithmic scale) and the RLU value on the y-axis, and perform four-parameter logistic curve fitting to establish a standard curve.

[0058] By comparing the performance data of the cTnI standard detected by the method of this invention, Table 1 is obtained.

[0059] Table 1: Performance data of cTnI standard detected by the method of the present invention As can be seen, the method of the present invention exhibits a good linear relationship (R²>0.999) in the concentration range of 0.5-1000 pg / mL, the lowest detection concentration (signal-to-noise ratio ≥3) can reach 0.5 pg / mL, and the repeatability of each concentration point is good (CV<8%), which proves that the present invention has extremely high sensitivity and excellent repeatability.

[0060] Example 2 I. Materials and Reagents The method group of this invention is the same as in Example 1.

[0061] Traditional method control group: A commercially available cTnI detection kit (using ALP-labeled antibody and luminol as substrate) was used.

[0062] Samples: The same batch of recombinant cTnI intact protein (1000 pg / mL) was partially degraded with trypsin at 37°C. Samples were taken at 0 minutes (intact protein), 5 minutes, 15 minutes and 30 minutes, and protease inhibitors were added immediately to terminate the reaction to simulate the degradation process of cTnI in blood.

[0063] II. Experimental Procedure 1. The degradation samples at the above four time points were tested in parallel using both the method of this invention and the conventional method (n=4 for each sample).

[0064] 2. Using the detection signal value of the 0-minute (intact protein) sample as 100%, calculate the signal recovery rate of the samples at each degradation time point.

[0065] III. Results Record the signal recovery rates of samples with different degrees of degradation detected by two methods.

[0066] The signal recovery rates of the cTnI degradation fragments detected by the method of this invention were compared with those of conventional methods, as shown in Table 2.

[0067] Table 2: Comparison of signal recovery rates of the method of the present invention and the traditional method for detecting cTnI degradation fragments As can be seen, with the increase of protease degradation time, the signal recovery rate of traditional methods targeting unstable epitopes drops sharply, indicating that they cannot effectively detect degradation fragments. In contrast, the method of this invention (using antibody pairs targeting stable epitopes) maintains a high signal recovery rate (>92%) throughout the entire degradation process, demonstrating its ability to uniformly detect intact cTnI and its degradation fragments. This verifies that this invention can solve the technical problems of "short diagnostic window" and "poor accuracy" caused by inappropriate antibody epitope selection, significantly improving the accuracy and reliability of diagnosis.

[0068] Those skilled in the art will understand that embodiments of this application can be provided as methods, systems, or computer program goods. Therefore, this application can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, this application can take the form of a computer program goods embodied on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0069] This application is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program goods according to embodiments of this application. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, generate instructions for implementing the flowchart... Figure 1 One or more processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0070] These computer program instructions may also be stored in a computer-readable storage medium that can direct a computer or other programmable data processing device to function in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of manufacture including instruction means, which are implemented in a process Figure 1 One or more processes and / or boxes Figure 1 The function specified in one or more boxes.

[0071] These computer program instructions may also be loaded onto a computer or other programmable data processing equipment to cause a series of operational steps to be performed on the computer or other programmable equipment to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable equipment for implementing the process. Figure 1 One or more processes and / or boxes Figure 1 The steps of the function specified in one or more boxes.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A highly sensitive chemiluminescent immunoassay method for detecting cardiac troponin I, characterized in that, Includes the following steps: S1. Capture antibody against cardiac troponin I immobilized on a solid-phase carrier; S2. Contact the sample to be tested with a solid-phase carrier coated with the capture antibody; S3. Add detection antibody to the solid support described in step S2, wherein the detection antibody is linked to a signal marker; S4. Under light-protected conditions, add a chemiluminescent substrate to the solid support in step S3; S5. Detect the chemiluminescent signal to quantify cardiac troponin I; The signal marker is a complex formed by loading alkaline phosphatase onto nanomaterials, or is a nanomaterial itself. The chemiluminescent substrate is a compound based on 1,2-dioxane.

2. The chemiluminescent immunoassay for high-sensitivity detection of cardiac troponin I according to claim 1, characterized in that, The antigenic epitopes recognized by the capture antibody and the detection antibody are located in the region of amino acid positions 30 to 110 of the central domain of cardiac troponin I, and the two epitopes do not overlap.

3. The chemiluminescent immunoassay for high-sensitivity detection of cardiac troponin I according to claim 1, characterized in that, In step S3, the nanomaterial is silica nanoparticles, gold nanoclusters, or quantum dots.

4. The chemiluminescent immunoassay for high-sensitivity detection of cardiac troponin I according to claim 1, characterized in that, In step S4, the chemiluminescent substrate is CDP-Star or APS-5, and the amount of the chemiluminescent substrate CDP-Star or APS-5 added is 0.2-1.0 mM.

5. The chemiluminescent immunoassay for high-sensitivity detection of cardiac troponin I according to claim 1, characterized in that, In step S3, the amount of enzyme loaded per milligram of nanomaterial to form the complex is 0.5-2.0 mg.

6. The chemiluminescent immunoassay for high-sensitivity detection of cardiac troponin I according to claim 1, characterized in that, In step S3, the amount of the detection antibody added is 0.1-2.0 μg / mL.

7. The chemiluminescent immunoassay for high-sensitivity detection of cardiac troponin I according to claim 1, characterized in that, The solid support is a microporous plate, magnetic particles, or glass fiber membrane.

8. The chemiluminescent immunoassay for high-sensitivity detection of cardiac troponin I according to claim 1, characterized in that, Washing is performed after steps S2 and S3.

9. The chemiluminescent immunoassay for high-sensitivity detection of cardiac troponin I according to claim 4, characterized in that, In step S4, the chemiluminescent substrate CDP-Star or APS-5 is dissolved in a buffer solution with a pH of 8.0-10.0 to prepare a chemiluminescent substrate solution, which is then added to the solid-phase carrier for completing the immune reaction.

10. The application of a chemiluminescent immunoassay method for the high-sensitivity detection of cardiac troponin I according to any one of claims 1-9, characterized in that, include: The application of the highly sensitive chemiluminescent immunoassay for detecting cardiac troponin I in the preparation of diagnostic products for detecting myocardial injury.

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