ALP-mediated ELISA method and kit for detecting IL-6 protein

Abstract

This application provides a method and kit for detecting IL-6 protein using an ALP-mediated ELISA. The method detects IL-6 through ALP-catalyzed APP hydrolysis, which is based on alkaline phosphatase (ALP)-mediated hydrolysis. During hydrolysis, APP exhibits high fluorescence quenching efficiency. The change in APP fluorescence intensity shows a good linear relationship with the logarithm of IL-6 protein concentration in the range of 0.005 ng / mL to 5.0 ng / mL, with a detection limit of 0.001 ng / mL, which is 100 times more sensitive than traditional pNPP-based ELISA. Furthermore, when the colorimetric signal of AP (p-aminophenol) is used for colorimetric ELISA detection of IL-6, the detection limit is 0.1 ng / mL. This method can be used for visual detection of IL-6 and has high sensitivity.

Description

Technical Field

[0001] This application relates to the field of reagent detection technology, and in particular to a method and kit for detecting IL-6 protein based on ALP-mediated ELISA. Background Technology

[0002] Cerebral infarction is a serious cerebrovascular disease with a high risk of disability and mortality. The lack of effective diagnostic methods can lead to delayed diagnosis and an increased risk of serious complications. Therefore, early screening and diagnosis of cerebral infarction are crucial for improving patient survival and reducing the burden on families and society. Detecting novel biomarkers that can accurately predict the risk and severity of cerebral infarction can effectively assist screening efforts. Recent studies have revealed a potential relationship between inflammatory responses and cerebral infarction; cytokine infiltration can lead to chronic nonspecific inflammation, which is closely related to the occurrence and development of cerebral infarction. In patients with acute cerebral infarction, the levels of various endogenous cytokines in the blood are significantly increased. Therefore, measuring the levels of inflammatory cytokines can provide a valuable strategy for diagnosing cerebral infarction and assessing prognosis. Interleukin-6 (IL-6) is a cytokine frequently secreted during the body's inflammatory response. It has been reported that IL-6 levels in cerebrospinal fluid increase significantly after cerebral infarction and are associated with prognostic prediction of cerebral infarction. Therefore, IL-6 is considered a reliable screening and prognostic biomarker for cerebral infarction.

[0003] Over the years, various methods and detection techniques have been developed to determine the concentration of IL-6, including enzyme-linked immunosorbent assay (ELISA), aptamer-based sensors, and organic electrochemical transistors. ELISA is widely used for analysis because it is simple, rapid, highly specific, versatile, and requires only simple experimental equipment. Colorimetric, fluorescence, chemiluminescence, surface-enhanced Raman scattering, and electrochemical methods can be combined with ELISA. Colorimetric and fluorescence-based ELISAs are increasingly popular due to their ease of operation, low cost, and short testing time. However, because the amount of IL-6 in body fluids is extremely low (concentration in the pM range), conventional colorimetric-based immunosensors may not be able to diagnose cerebral infarction due to insufficiently low detection limits and wide linear ranges. Fluorescence-based analytical methods are 1-2 orders of magnitude more sensitive than colorimetric methods. Currently, various detection methods based on fluorescent nanoprobes have been developed in research, but conventional fluorescence immunoassays that rely on the interaction between fluorescent nanoprobes and substrates or labeled enzyme products are susceptible to environmental interference. Summary of the Invention

[0004] Therefore, it is necessary to provide a highly sensitive ELISA method and kit for detecting IL-6 protein, addressing the shortcomings of existing technologies.

[0005] To solve the above problems, this application adopts the following technical solution:

[0006] One objective of this application is to provide a method for detecting IL-6 protein based on ALP-mediated ELISA, comprising the following steps:

[0007] The capture antibody was added to a microplate and incubated with coating buffer at 25-37°C for 1-4 hours to obtain the sample.

[0008] The sample was washed with washing buffer to remove the coating buffer.

[0009] The microplate was sealed with skim milk at 25-37°C for 1-4 hours, and then ALP samples of different concentrations were incubated at 25-37°C for 1-2 hours.

[0010] Biotinylated monoclonal antibodies and SA-ALP were incubated at 25-37°C for 1-2 hours to form immune complexes.

[0011] APP and Mg 2+ Tris-HCl buffer was added to the sample, and the sample was incubated at 25-37°C for 30-120 minutes to obtain a reaction solution;

[0012] The reaction solution was transferred to a centrifuge tube and the fluorescence spectrum was recorded.

[0013] In some embodiments, in the step of adding the capture antibody to a microplate and incubating it with a coating buffer at 25-37°C for 1-4 hours to obtain a sample, the capture antibody includes mouse anti-IL-6 antibody, the microplate is a 96-well microplate, and the coating buffer includes carbonate buffer.

[0014] In some embodiments, the capture antibody has a volume of 100 μL and a concentration of 1:100; the coating buffer has a concentration of 50 mM and a pH of 9.6.

[0015] In some embodiments, in the step of washing the sample with a washing buffer to remove the coating buffer, the washing buffer is PBS containing 0.05% Tween 20, at pH 7.4, and at a concentration of 10 mM.

[0016] In some embodiments, in the step of sealing the microplate with skim milk at 37°C for 2 hours and then incubating ALP samples of different concentrations at 37°C for 1 hour, the volume of the skim milk is 300 μL and the concentration is 2.5% w / t; the different concentrations of ALP samples include 0 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 25.0 ng / mL, 50.0 ng / mL, and 100.0 ng / mL.

[0017] In some embodiments, in the step of incubating the biotinylated monoclonal antibody and SA-ALP at 25-37°C for 1-4 hours to form an immune complex, the volume of the biotinylated mouse monoclonal antibody is 100 μL and the concentration is 1:1000; the volume of the SA-ALP is 100 μL and the concentration is 1:2000.

[0018] In some embodiments, when APP and Mg are combined 2+ In the step of adding Tris-HCl buffer to the sample and incubating the sample at 25-37°C for 30-120 minutes to obtain the reaction solution, the concentration of APP is 500 μM, and the concentration of Mg is... 2+ The concentration was 1 mM, and the volume of the Tris-HCl buffer was 200 μL.

[0019] The second objective of this application is to provide a kit that detects IL-6 protein by applying the ELISA method described above.

[0020] The present application adopts the above technical solution, and its beneficial effects are as follows:

[0021] This application provides a method and kit for detecting IL-6 protein using an ALP-mediated ELISA. The method detects IL-6 through the hydrolysis of APP (4-aminophenol phosphate) catalyzed by ALP (alkaline phosphatase). APP exhibits high fluorescence quenching efficiency during the hydrolysis process. The change in APP fluorescence intensity shows a good linear relationship with the logarithm of IL-6 protein concentration in the range of 0.005 ng / mL to 5.0 ng / mL, with a detection limit of 0.001 ng / mL, which is 100 times more sensitive than traditional pNPP-based ELISA. Furthermore, when the colorimetric signal of AP (p-aminophenol) is used for colorimetric ELISA detection of IL-6, the detection limit is 0.1 ng / mL. This method can be used for the visual detection of IL-6 and has high sensitivity. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 A schematic diagram of the process for detecting IL-6 using a dual-mode ELISA based on colorimetry and fluorescence, provided in an embodiment of this application.

[0024] Figure 2 A flowchart illustrating the steps of an ALP-mediated ELISA method for detecting IL-6 protein provided in this application embodiment.

[0025] Figure 3 In the middle (A), the emission spectra of different concentrations of APP in Tris-HCl buffer are shown.

[0026] Figure 3 (B) represents the emission spectrum of APP at different excitation wavelengths from 270 to 320 nm in Tris-HCl buffer.

[0027] Figure 4 (A) represents the change in fluorescence intensity of APP at different concentrations after adding 50 U / L ALP.

[0028] Figure 4 (B) represents the fluorescence intensity change of 500 μM APP under the action of 50 U / L ALP containing different concentrations of Mg2+.

[0029] Figure 4 (C) represents the change in fluorescence intensity of 500 μM APP under the action of 50 U / L ALP at different pH values.

[0030] Figure 4 (D) indicates the optimization of the reaction time for ALP detection, with ALP concentrations of 10, 25, and 50 U / L.

[0031] Figure 5 In the middle (A), the UV-Vis absorption spectra of APP (200 μM) for different concentrations of ALP are shown.

[0032] Figure 5 (B) represents the calibration curve corresponding to ALP detection.

[0033] Figure 5 (C) indicates visualization of ALP detection by APP hydrolysis.

[0034] Figure 5In the figure (D), APP (500 μM) is represented as the fluorescence spectrum response of APP to different concentrations of ALP.

[0035] Figure 5 (E) represents the calibration curve corresponding to ALP detection in the range of 0.02 U / L to 1.0 U / L.

[0036] Figure 5 (F) represents the calibration curve for ALP detection in the range of 2.0 U / L to 50.0 U / L.

[0037] Figure 6 (A) represents the effect of 10 μM pNPP on different concentrations of Mg 2+ The absorbance change of 10 U / L ALP.

[0038] Figure 6 (B) represents the change in absorbance of 10 μM pNPP to 10 U / L ALP at different pH values.

[0039] Figure 6 In the figure (C), the response of different concentrations of ALP to the UV spectrum of 10 μM pNPP is indicated.

[0040] Figure 6 (D) represents the ALP detection calibration curve.

[0041] Figure 7 (A) represents the APP concentration.

[0042] Figure 7 (B) indicates Capture Ab dilution.

[0043] Figure 7 (C) indicates that SA-ALP is used for IL-6 protein detection.

[0044] Figure 8 In the figure (A), the UV-Vis absorption spectra of APP with different concentrations of IL-6 in alp-mediated ELISA are shown.

[0045] Figure 8 (B) represents the calibration curve corresponding to the detection of IL-6 protein.

[0046] Figure 8 (C) represents the visualization of IL-6 detection by alp-mediated ELISA.

[0047] Figure 8 In the figure (D), the fluorescence spectra of APP at different concentrations of IL-6 are shown.

[0048] Figure 8The value in (E) represents the calibration curve for IL-6 protein detection in the range of 0.005 ng / mL to 5.0 ng / mL.

[0049] Figure 8 The curve (F) represents the calibration curve for IL-6 protein detection in the range of 10.0 ng / mL to 100.0 ng / mL.

[0050] Figure 9 In the middle (A), it is indicated that the fluorescent pNPP-mediated ELISA sensor detects IL-6 protein by changes in fluorescence intensity.

[0051] Figure 9 (B) represents the calibration curve for IL-6 protein detection.

[0052] Figure 10 This study indicates the selectivity of IL-6 protein detection against other interfering proteins, including TNF, IL-10, and TGF, with IL-6 protein concentration at 100 ng / mL and other interfering protein concentrations at 1000 ng / mL.

[0053] Figure 11 This is a schematic diagram illustrating the detection of IL-6 protein using a commercial ELISA kit provided in this embodiment.

[0054] Figure 12 The response to changes in APP fluorescence intensity is represented by samples from asymptomatic volunteers (1-12) and patients with cerebral infarction (13-22). Detailed Implementation

[0055] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0056] In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0057] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0058] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0059] Please see Figure 1 and Figure 2 The method for detecting IL-6 protein based on ALP-mediated ELISA provided in this embodiment includes the following steps S110 to S160, and the implementation of each step is described in detail below.

[0060] Step S110: Add the capture antibody to the microplate and incubate it with coating buffer at 25-37°C for 1-4 hours to obtain the sample.

[0061] In this embodiment, the capture antibody includes mouse anti-IL-6 antibody, the microculture plate is a 96-well microculture plate, and the coating buffer includes carbonate buffer.

[0062] In this embodiment, the capture antibody has a volume of 100 μL and a concentration of 1:100; the coating buffer has a concentration of 50 mM and a pH of 9.6.

[0063] Step S120: Wash the sample with washing buffer to remove the coating buffer.

[0064] In this embodiment, the washing buffer is PBS containing 0.05% Tween 20, with a pH of 7.4 and a concentration of 10 mM.

[0065] Step S130: Seal the microplate with skim milk at 25-37℃ for 1-4 hours, and then incubate samples of different concentrations of ALP (alkaline phosphatase) at 25-37℃ for 1-2 hours.

[0066] In this embodiment, the volume of the skim milk is 300 μL and the concentration is 2.5% w / t; the ALP samples of different concentrations include 0 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 25.0 ng / mL, 50.0 ng / mL, and 100.0 ng / mL.

[0067] Step S140: Incubate the biotinylated monoclonal antibody and SA-ALP at 25-37°C for 1-2 hours to form immune complexes.

[0068] In this embodiment, the volume of the biotinylated mouse monoclonal antibody is 100 μL and the concentration is 1:1000; the volume of the SA-ALP is 100 μL and the concentration is 1:2000.

[0069] Step S150: Add APP (4-aminophenol phosphate) and Mg 2+ Tris-HCl buffer was added to the sample, and the sample was incubated at 25-37°C for 30-120 minutes to obtain a reaction solution.

[0070] In this embodiment, the volume of the APP is 500 μM, and the Mg... 2+ The concentration was 1 mM, and the volume of the Tris-HCl buffer was 200 μL.

[0071] Understandably, experimental conditions can be optimized in practice, including APP concentration, activator (MgCl2) concentration, enzyme reaction time, pH of the reaction buffer, and reaction time, to achieve optimal ALP detection performance. Results showed that the highest fluorescence signal ratio was observed at an APP concentration of 500 μM.

[0072] It is understandable that an appropriate concentration of Mg 2+ It can be used to improve ALP (a commonly used labeling enzyme in ELISA) activity and increase system stability.

[0073] Step S160: Transfer the reaction solution to a centrifuge tube and record the fluorescence spectrum.

[0074] This application provides a method and kit for detecting IL-6 protein using an ALP-mediated ELISA. The method detects IL-6 through ALP-catalyzed APP hydrolysis, which is based on alkaline phosphatase (ALP)-mediated hydrolysis. During hydrolysis, APP exhibits high fluorescence quenching efficiency. The change in APP fluorescence intensity shows a good linear relationship with the logarithm of IL-6 protein concentration in the range of 0.005 ng / mL to 5.0 ng / mL, with a detection limit of 0.001 ng / mL, which is 100 times more sensitive than traditional pNPP-based ELISA. Furthermore, when the colorimetric signal of AP (p-aminophenol) is used for colorimetric ELISA detection of IL-6, the detection limit is 0.1 ng / mL. This method can be used for visual detection of IL-6 and has high sensitivity.

[0075] The technical solutions described above in this application will be explained in detail below with reference to specific embodiments.

[0076] Example

[0077] The chemicals and materials involved in this embodiment are as follows:

[0078] APP, trizma hydrochloride (Tris-HCl), and MgCl2 were purchased from Sigma. pNPP was purchased from Aladdin Shanghai. SA-ALP was purchased from Bio-Sens Technology, Beijing. Recombinant human IL-6 protein, mouse anti-IL-6 antibody, mouse anti-IL-6 antibody (biotin-modified), and an IL-6 ELISA kit were purchased from Sinocare. Calf intestinal alkaline phosphatase (ALP) was purchased from New England Laboratory.

[0079] Instrumentation and characterization:

[0080] Fluorescence spectra were measured using a Hitachi F-7100FL spectrophotometer. UV-vis absorption spectra were recorded on a Hitachi system (U-3900H). Microplate shakers used in the experiments were purchased from Allsheng (MB100-4A). Commercial ELISA assays were performed using a microplate reader from Perkin Elmer (VICTOR Nivo).

[0081] Clinical sample analysis:

[0082] All clinical samples were collected from patients admitted to Liuzhou Traditional Chinese Medicine Hospital, with cerebrospinal fluid collected from all registered patients. Samples were placed in anticoagulant tubes and centrifuged at 4000 rpm at room temperature. After centrifugation (centrifugation time: 10 minutes), the supernatant was collected in centrifuge tubes and stored as aliquots at -80°C for testing. Each sample consisted of 12 positive and 10 negative samples. Laboratory waste was sterilized after the testing process was completed.

[0083] ALP response analysis:

[0084] ALP samples of different concentrations (0, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 25.0, 50.0, 100.0 U / L) were mixed with Tris buffer (20 mM, pH 9.5) containing APP (500 μM). 2+(1 mM). The reaction solution was incubated at 37 °C for 90 min, and then the fluorescence spectrum was recorded. Fluorescence spectra were recorded in the wavelength range of 320–450 nm at an excitation wavelength of 310 nm. ALP samples of different concentrations (0, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 25.0 U / L) were mixed with Tris buffer (20 mM, pH 9.5) containing APP (200 μM) and Mg2+ (1 mM). The reaction solution was incubated at 37 °C for 90 min, and then the absorption spectra were recorded. UV-vis absorption spectra were recorded in the wavelength range of 250–350 nm. ALP samples of different concentrations (0, 0.5, 1.0, 5.0, 10.0, 20.0, 50.0 U / L) were mixed with Tris buffer (20 mM, pH 9.5) containing pNPP (10 μM) and Mg2+ (1 mM). 2+ The reaction solution was mixed with 1 mM Tris buffer (20 mM, pH 9.5). The solution was then incubated at 37°C for 30 minutes. Subsequently, the UV-vis absorption spectra were recorded. Absorption spectra were recorded in the wavelength range of 250–600 nm.

[0085] IL-6 protein detection based on ALP-mediated ELISA:

[0086] Capture antibody (100 μL, 1:100) was added to a 96-well microplate and incubated with coating buffer (50 mM carbonate buffer, pH 9.6) at 37 °C for 2 hours. The coating buffer was removed, and the samples were washed three times with washing buffer (containing 0.05% Tween 20, pH 7.4, 10 mM PBS). The microplates were then blocked with skim milk (300 μL, 2.5% w / t) at 37 °C for 2 hours. Subsequently, different concentrations (0, 0.005, 0.01, 0.05, 0.5, 1.0, 5.0, 10.0, 20.0, 25.0, 50.0, 100.0 ng / mL) were incubated at 37 °C for 1 hour. Next, biotinylated mouse monoclonal antibody (100 μL, 1:1000) and SA-ALP (100 μL, 1:2000) were incubated at 37°C for 1 hour to form immune complexes. 200 μL of Tris-HCl buffer containing 200 μM APP and 1 mM Mg2+ was added to the sample, and the sample was incubated at 37°C for 90 minutes. The reaction solution was transferred to centrifuge tubes, and fluorescence spectra were recorded. IL-6 was detected using a commercial IL-6 ELISA kit, which served as a control for sensitivity comparison.

[0087] APP features

[0088] In this embodiment, we observed that APP, typically used as a substrate for ALP, exhibits good fluorescence properties when dephosphorylated to generate AP. Figure 3Image (A) shows a significant high-intensity fluorescence peak at 360 nm, and the fluorescence intensity is positively correlated with the concentration of APP. Maximum fluorescence was recorded at an excitation wavelength of 310 nm. Figure 3 As shown in (B). These findings suggest that the fluorescence properties of APP can be used to develop novel colorimetric and fluorescence dual-mode ELISA methods for the sensitive detection of IL-6.

[0089] Colorimetric and fluorescence dual-mode determination based on ALP-catalyzed APP hydrolysis:

[0090] In this embodiment, the feasibility of using ALP to analyze the APP hydrolysis process was verified by analyzing UV-vis absorption and fluorescence spectra. Several experimental conditions, including APP concentration, activator (MgCl2) concentration, enzymatic reaction time, pH of the reaction buffer, and reaction time, were optimized to achieve the best ALP detection performance. The results showed that the maximum fluorescence signal ratio was achieved at an APP concentration of 500 μM. Figure 4 (A)). It has been reported that appropriate concentrations of Mg 2+ It can be used to improve ALP activity and increase system stability. For example... Figure 4 As shown in (B), 1mM Mg 2+ For use in subsequent experiments, because Mg 2+ Further increases in concentration did not lead to a significant change in fluorescence intensity. The maximum change in fluorescence intensity was recorded at pH 9.5. Figure 4 (C) and the optimal reaction time based on ALP catalysis is 90 minutes. Figure 4 (D)

[0091] The colorimetric and fluorescence dual-mode ALP activity assay was performed under optimal conditions. Figure 5 As shown in Figure A, an increase in ALP concentration leads to an increase in absorption intensity at 290 nm. A good linear relationship was obtained between the change in absorption intensity at 290 nm (ΔA290) and the logarithm of ALP concentration (range: 0.5–25.0 U / L). Figure 5 (B) Figure 5 The results showed that increasing ALP concentration led to a gradual decrease in APP fluorescence intensity, with the intensity change concentrated at 360 nm (ΔF360). The change in fluorescence intensity showed a good linear relationship with the logarithm of ALP concentration in the ranges of 0.02–1.0 U / L and 2.0–100.0 U / L. Figure 5 (E and F in the middle). The detection limits for colorimetric and fluorescence readout modes were calculated to be 0.1 and 0.005 U / L (3σ / S), respectively. The results indicate that the sensitivity of the fluorescence mode is higher than that of the colorimetric mode.

[0092] A conventional ALP detection method based on pNPP hydrolysis was compared with the sensitivity of a dual-mode detection method. In ALP detection, the compound pNPP is typically used as a substrate, and the detection principle involves the dephosphorylation and catalysis of colorless pNPP to produce yellow p-nitrophenol (PNP). Experimental parameters, including pNPP concentration, activator (MgCl2) concentration, and reaction buffer pH, were optimized. Figure 6 The results from the AD analysis showed that the maximum change in absorbance at 400 nm was observed when the pNPP concentration was 10 μM, the Mg2+ concentration was 1 mM, and the pH of the buffer solution was 9.5. Under optimal conditions, the absorbance of pNP at 400 nm increased with increasing ALP concentration, and the change in absorption intensity at 400 nm (ΔA400) correlated well with the logarithm of ALP concentration (range: 0.5–50.0 U / L). The detection limit was 0.1 U / L. The sensitivity of our proposed fluorescence mode is 20 times that of the colorimetric mode for pNPP hydrolysis, and a visual ALP detection method was also performed. Figure 5 As shown in Figure C, increasing the ALP concentration from 0 to 150 U / L causes the color of the microplate to change from colorless to yellow to dark brown, indicating that this method can be effectively used for visual detection.

[0093] Dual-mode ELISA for IL-6 detection

[0094] When APP is used for ALP detection, it can achieve high detection sensitivity, therefore APP can be used for ALP-mediated ELISA. This embodiment provides a dual-mode ELISA sensor based on an ALP-mediated APP hydrolysis process for IL-6 detection. The development of this dual-mode ELISA method follows the procedures of a conventional ELISA, and the detection process involves immobilizing a capture antibody (capture Ab), different concentrations of IL-6, a biotin-modified detection antibody (Biotin-Ab1), and SA-ALP. A sandwich immune complex is formed in a 96-well plate through a specific antigen-antibody immune reaction and SA-Biotin interaction. APP is used as a substrate for ALP catalysis to generate AP, which helps in the generation of colorimetric and fluorescence readout signals. First, we optimized the components in the detection process, such as... Figure 7 As shown in (A), the maximum fluorescence intensity ratio was recorded at an APP concentration of 500 μM. We also optimized the use of the capture antibody and SA-ALP. Figure 7 As shown in (B) and (C), the maximum change in fluorescence intensity was observed when the capture antibody was diluted 1:200 and SA-ALP was diluted 1:2000. These optimized parameters were used in subsequent experiments. In colorimetric mode, the change in absorbance intensity at 290 nm increased significantly as the IL-6 concentration increased from 0.5 to 75.0 ng / mL. Figure 8(A)). A good linear relationship was established between the change in absorption intensity and the logarithm of IL-6 concentration. Figure 8 In colorimetric mode (B), the detection limit for IL-6 was 0.1 ng / mL. In fluorescence mode, the quenching of APP fluorescence was accompanied by a corresponding increase in IL-6 concentration. Figure 8 (D)). For example Figure 8 As shown in (E) and (F), the change in fluorescence intensity at 360 nm exhibited a good linear relationship with the logarithm of IL-6 protein concentration (0.005-5.0 ng / mL and 10-100 ng / mL). The detection limit was 0.001 ng / mL, significantly lower than the detection limit in colorimetric mode. Furthermore, visual IL-6 detection methods were used for analysis, such as... Figure 8 As shown in (C), the colorless microplate turned yellow and then dark brown as the IL-6 concentration increased from 0 to 500 ng / mL, indicating the high potential of this method for visual detection of IL-6.

[0095] The most commonly used substrate for ALP, pNPP, is used in ALP-mediated ELISA. For example... Figure 9 As shown in (A), the absorbance of pNPP decreases at 310 nm, while the absorbance of pNP increases at 400 nm with increasing IL-6 concentration from 0.5 to 200 ng / mL, with a detection limit of 0.1 ng / mL. The fluorescence-based detection method in this work is 100 times more sensitive than conventional pNPP-based ELISA methods.

[0096] Other non-specific proteins (including tumor necrosis factor (TNF), interleukin-10 (IL-10), and transforming growth factor (TGF)) were added to the ELISA assay to investigate its selectivity for IL-6 detection. For example... Figure 10 As shown, even when the concentration of nonspecific proteins was ten times that of IL-6, no significant change in the fluorescence intensity of the nonspecific proteins was observed. Furthermore, the changes in fluorescence properties observed when both IL-6 and the interfering proteins were present in the system were similar to those observed when IL-6 was present alone, indicating that the nonspecific proteins had little effect on the response to IL-6. Therefore, the developed dual-mode ELISA-based method can specifically identify and detect IL-6. Please refer to [link to relevant documentation]. Figure 11 This is a schematic diagram illustrating the detection of IL-6 protein using a commercial ELISA kit provided in this embodiment.

[0097] Validation using clinical samples

[0098] To evaluate the reliability of the IL-6 detection method in clinical samples, we used a constructed ELISA method to detect IL-6 in cerebrospinal fluid samples from 22 volunteers diagnosed and treated in the Department of Neurology at Liuzhou Traditional Chinese Medicine Hospital. Samples were obtained from patients diagnosed with or without cerebral infarction, and their diagnosis was performed via digital subtraction angiography (DSA). IL-6 in the actual samples was detected using the fluorescence pattern detection method designed for this study. Figure 12 As shown, the results indicated that the APP fluorescence change was significantly lower in the negative group (composed of 12 volunteers without cerebral infarction) than in the positive group (composed of 10 cerebral infarction patients). The results also showed that the IL-6 concentration in the 10 cerebral infarction patients was significantly higher than that in the 12 asymptomatic patients. Furthermore, the results showed a positive correlation between IL-6 concentration and the severity of cerebral infarction. Therefore, it can be inferred that the developed ELISA method can effectively distinguish between patients with and without cerebral infarction. This method can also be used to accurately classify the severity of cerebral infarction into mild, moderate, or severe.

[0099] It is understandable that APP exhibits excellent fluorescence properties, and that the fluorescence intensity of APP significantly decreases during ALP-catalyzed hydrolysis. This finding was also applied to ALP detection and ELISA-based IL-6 detection, demonstrating significant sensitivity. The APP hydrolysis process showed a 20-fold higher response to ALP compared to conventional pNPP hydrolysis. Furthermore, the IL-6 detection method based on ALP-catalyzed APP hydrolysis showed a 100-fold higher sensitivity than the conventional pNPP-based ELISA. The developed ELISA sensor can be used to analyze clinical samples and can be used to distinguish samples from stroke patients from asymptomatic volunteers. A positive correlation was observed between the IL-6 concentration measured according to this method and the severity of stroke as determined by DSA.

[0100] It is understood that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0101] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A method for detecting IL-6 protein using ALP-mediated ELISA for non-disease diagnostic or therapeutic purposes, characterized in that, Includes the following steps: The capture antibody was added to a microplate and incubated with coating buffer at 25-37°C for 1-4 hours to obtain the sample. The sample was washed with washing buffer to remove the coating buffer. Block the micro-culture plate with skim milk at 25-37℃ for 1-4 hours, and then incubate ALP samples of different concentrations at 25-37℃ for 1-2 hours. Biotinylated monoclonal antibodies and streptavidin-modified alkaline phosphatase (SA-ALP) were incubated at 25-37°C for 1-2 hours to form immune complexes. APP and Mg 2+ Tris-HCl buffer was added to the sample, and the sample was incubated at 25-37℃ for 30-120 minutes to obtain the reaction solution; The reaction solution was transferred to a centrifuge tube and the fluorescence spectrum was recorded.

2. The method for detecting IL-6 protein based on ALP-mediated ELISA as described in claim 1, characterized in that, In the step of adding the capture antibody to a microplate and incubating it with coating buffer at 25-37°C for 1-4 hours to obtain a sample, the capture antibody includes mouse anti-IL-6 antibody, the microplate is a 96-well microplate, and the coating buffer includes carbonate buffer.

3. The method for detecting IL-6 protein based on ALP-mediated ELISA as described in claim 2, characterized in that, The capture antibody has a volume of 100 μL and a concentration of 1:100; the coating buffer has a concentration of 50 mM and a pH of 9.

6.

4. The method for detecting IL-6 protein based on ALP-mediated ELISA as described in claim 1, characterized in that, In the step of washing the sample with a washing buffer to remove the coating buffer, the washing buffer is PBS containing 0.05% Tween 20, pH 7.4, and a concentration of 10 mM.

5. The method for detecting IL-6 protein based on ALP-mediated ELISA as described in claim 1, characterized in that, In the step of sealing a microculture plate with skim milk at 37°C for 2 hours, and then incubating ALP samples of different concentrations at 37°C for 1 hour, the volume of the skim milk is 300 μL and the concentration is 2.5% w / t; the different concentrations of ALP samples include 0 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 25.0 ng / mL, 50.0 ng / mL, and 100.0 ng / mL.

6. The method for detecting IL-6 protein based on ALP-mediated ELISA as described in claim 1, characterized in that, In the step of incubating the biotinylated monoclonal antibody and SA-ALP at 25-37°C for 1-2 hours to form an immune complex, the volume of the biotinylated mouse monoclonal antibody is 100 μL and the concentration is 1:1000; the volume of the SA-ALP is 100 μL and the concentration is 1:2000.

7. The method for detecting IL-6 protein based on ALP-mediated ELISA as described in claim 1, characterized in that, In combining APP and Mg 2+ In the step of adding Tris-HCl buffer to the sample and incubating the sample at 25-37°C for 30-120 minutes to obtain the reaction solution, the concentration of APP is 500 μM, and the concentration of Mg is... 2+ The concentration was 1 mM, and the volume of the Tris-HCl buffer was 200 μL.

8. A reagent kit, characterized in that, The kit enables the detection of IL-6 protein by applying the ELISA method for detecting IL-6 protein according to any one of claims 1 to 7.

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