WO2025010959A1 - 基于alp介导的elisa检测il-6蛋白的方法及试剂盒 - Google Patents

基于alp介导的elisa检测il-6蛋白的方法及试剂盒 Download PDF

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WO2025010959A1
WO2025010959A1 PCT/CN2023/141325 CN2023141325W WO2025010959A1 WO 2025010959 A1 WO2025010959 A1 WO 2025010959A1 CN 2023141325 W CN2023141325 W CN 2023141325W WO 2025010959 A1 WO2025010959 A1 WO 2025010959A1
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alp
concentration
app
detecting
buffer
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马英新
毛国斌
戴俊彪
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Shenzhen Institute of Advanced Technology of CAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5412—IL-6
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event

Definitions

  • the present application relates to the technical field of reagent detection, and in particular to a method and a kit for detecting IL-6 protein based on ALP-mediated ELISA.
  • Cerebral infarction is a serious cerebrovascular disease with a high risk of disability and mortality.
  • the lack of effective diagnostic methods will lead to delayed diagnosis and an increased risk of serious complications. Therefore, early screening and diagnosis of cerebral infarction are crucial to improve the survival rate of patients and reduce the burden on families and society. Detection of new biomarkers that can accurately predict the risk and severity of cerebral infarction can provide effective assistance for screening.
  • Recent studies have revealed the potential relationship between inflammatory response and cerebral infarction.
  • the infiltration of cytokines 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.
  • Interleukin 6 is a cytokine that is frequently secreted in the body's inflammatory response. It has been reported that the level of IL-6 in cerebrospinal fluid increases significantly after cerebral infarction and is associated with the prognosis prediction of cerebral infarction. Therefore, IL-6 is considered to be a reliable screening and prognostic biomarker for cerebral infarction.
  • ELISA enzyme-linked immunosorbent assay
  • aptamer-based sensors e.g., aptamer-based sensors
  • organic electrochemical transistors e.g., organic electrochemical transistors
  • ELISA enzyme-linked immunosorbent assay
  • Colorimetry, fluorescence, chemiluminescence, surface-enhanced Raman scattering, and electrochemical methods can be combined with ELISA.
  • Colorimetric and fluorescence-based ELISAs are becoming increasingly popular because they are easy to operate, low cost, and have a short test time.
  • One of the purposes of the present application is to provide a method for detecting IL-6 protein based on ALP-mediated ELISA, comprising the following steps:
  • the capture antibody is added to the microtiter plate and incubated with the coating buffer at 25-37°C for 1-4 hours to obtain a sample;
  • microplate was blocked 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;
  • biotinylated monoclonal antibody and SA-ALP were incubated at 25-37°C for 1-2 hours to form an immune complex
  • the reaction solution was transferred to a centrifuge tube, and the fluorescence spectrum was recorded.
  • the capture antibody in the step of adding the capture antibody to a microtiter plate and incubating with a coating buffer at 25-37° C. for 1-4 hours to obtain a sample, includes a mouse anti-IL-6 antibody, the microtiter plate is a 96-well microtiter plate, and the coating buffer includes a carbonate buffer.
  • the volume of the capture antibody is 100 ⁇ L, and the concentration is 1:100; the concentration of the coating buffer is 50 mM, and the pH is 9.6.
  • the washing buffer in the step of washing the sample with a washing buffer to remove the coating buffer, is PBS containing 0.05% Tween 20, pH 7.4, and a concentration of 10 mM.
  • the volume of the skim milk is 300 ⁇ L 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, 100.0 ng/mL.
  • the volume of the biotinylated mouse monoclonal antibody is 100 ⁇ L, and the concentration is 1:1000; the volume of the SA-ALP is 100 ⁇ L, and the concentration is 1:2000.
  • the concentration of APP is 500 ⁇ M
  • the concentration of Mg 2+ is 1 mM
  • the volume of the Tris-HCl buffer is 200 ⁇ L.
  • the second purpose of the present application is to provide a kit, which detects IL-6 protein by applying the ELISA method for detecting IL-6 protein.
  • the present application provides a method and kit for detecting IL-6 protein based on ALP-mediated ELISA, which detects IL-6 by ALP (alkaline phosphatase)-catalyzed APP hydrolysis.
  • ALP alkaline phosphatase
  • APP has a high fluorescence quenching efficiency during the hydrolysis process.
  • the change in APP fluorescence intensity and the logarithm of IL-6 protein concentration show a good linear relationship in the range of 0.005 ng/mL - 5.0 ng/mL, and the detection limit is 0.001 ng/mL, which is 100 times more sensitive than the traditional pNPP-based ELISA.
  • the colorimetric signal of AP p-aminophenol
  • the detection limit is 0.1 ng/mL. This method can be used for visual detection of IL-6 with high sensitivity.
  • FIG1 is a schematic diagram of the process of detecting IL-6 by dual-mode ELISA using colorimetry and fluorescence based on APP provided in an embodiment of the present application.
  • FIG2 is a flowchart of the steps of the method for detecting IL-6 protein based on ALP-mediated ELISA provided in an embodiment of the present application.
  • FIG3 (A) shows the emission spectra of APP at different concentrations in Tris-HCl buffer.
  • Figure 3 (B) shows the emission spectra of APP at different excitation wavelengths from 270 to 320 nm in Tris-HCl buffer.
  • Figure 4 (A) shows the change in fluorescence intensity of APP at different concentrations after adding 50 U/L ALP.
  • Figure 4 (B) shows the change in fluorescence intensity of 500 ⁇ M APP under the action of 50 U/L ALP containing different concentrations of Mg2+.
  • Figure 4 (C) shows the change in fluorescence intensity of 500 ⁇ M APP under the action of 50 U/L ALP at different pH values.
  • Figure 4 (D) shows the optimization of ALP detection reaction time, with ALP concentrations of 10, 25, and 50 U/L, respectively.
  • Figure 5 (A) shows the UV-visible absorption spectra of APP (200 ⁇ M) to different concentrations of ALP.
  • Figure 5 (B) shows the calibration curve corresponding to ALP detection.
  • Figure 5 (C) shows the ALP visualization of APP hydrolysis detection.
  • Figure 5 (D) shows the fluorescence spectrum response of APP (500 ⁇ M) to different concentrations of ALP.
  • Figure 5 (E) shows the calibration curve corresponding to ALP detection in the range of 0.02 U/L to 1.0 U/L.
  • Figure 5 (F) shows the calibration curve corresponding to ALP detection in the range of 2.0 U/L to 50.0 U/L.
  • FIG6 (A) shows the absorbance change of 10 ⁇ M pNPP to 10 U/L ALP containing different concentrations of Mg 2+ .
  • Figure 6 (B) shows the change in absorbance of 10 ⁇ M pNPP to 10 U/L ALP at different pH values.
  • Figure 6 (C) shows the response of different concentrations of ALP to the UV spectrum of 10 ⁇ M pNPP.
  • Figure 6 (D) shows the ALP detection calibration curve.
  • Figure 7 (B) shows the Capture Ab dilution.
  • FIG. 7 (C) shows SA-ALP for IL-6 protein detection.
  • Figure 8 (A) shows the UV-visible absorption spectra of APP by different concentrations of IL-6 in ALP-mediated ELISA.
  • FIG8 (B) shows the calibration curve corresponding to IL-6 protein detection.
  • FIG8 (C) shows the visualization of IL-6 detection by ALP-mediated ELISA.
  • FIG8 (D) shows the fluorescence spectra of APP at different concentrations of IL-6 in the ALP-mediated ELISA.
  • Figure 8 (E) shows the calibration curve corresponding to IL-6 protein detection in the range of 0.005 ng/mL to 5.0 ng/mL
  • Figure 8 (F) shows the calibration curve corresponding to IL-6 protein detection in the range of 10.0 ng/mL to 100.0 ng/mL.
  • Figure 9 (A) shows that the fluorescent pNPP-mediated ELISA sensor detects IL-6 protein through changes in fluorescence intensity.
  • Figure 9 (B) shows the calibration curve for IL-6 protein detection.
  • FIG10 shows the selectivity study of IL-6 protein detection against other interfering proteins, including TNF, IL-10 and TGF, with the IL-6 protein concentration being 100 ng/mL and the other interfering protein concentration being 1000 ng/mL.
  • FIG. 11 is a schematic diagram of detecting IL-6 protein using a commercial ELISA kit provided in this embodiment.
  • FIG. 12 shows the response of APP fluorescence intensity changes in samples from asymptomatic volunteers (1-12) and cerebral infarction patients (13-22).
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.
  • a feature defined as “first” or “second” may explicitly or implicitly include one or more of the features.
  • the meaning of “plurality” is two or more, unless otherwise clearly and specifically defined.
  • the method for detecting IL-6 protein based on ALP-mediated ELISA includes the following steps S110 to S160 . The implementation method of each step is described in detail below.
  • Step S110 Add the capture antibody to the microtiter culture plate and incubate with the coating buffer at 25-37° C. for 1-4 hours to obtain a sample.
  • the capture antibody includes mouse anti-IL-6 antibody
  • the microtiter plate is a 96-well microtiter plate
  • the coating buffer includes carbonate buffer
  • the volume of the capture antibody is 100 ⁇ L, and the concentration is 1:100; the concentration of the coating buffer is 50 mM, and the pH is 9.6.
  • Step S120 Wash 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.
  • Step S130 The microplate is blocked with skim milk at 25-37° C. for 1-4 hours, and then ALP (intestinal alkaline phosphatase) samples of different concentrations are incubated at 25-37° C. for 1-2 hours.
  • ALP intestinal alkaline phosphatase
  • the volume of the skim milk is 300 ⁇ L and the concentration is 2.5% w/t;
  • the ALP samples with 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.
  • Step S140 incubate 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 was 100 ⁇ L, and the concentration was 1:1000; the volume of the SA-ALP was 100 ⁇ L, and the concentration was 1:2000.
  • Step S150 Add APP (4-aminophenol phosphate) and Mg 2+ in Tris-HCl buffer to the sample, and incubate the sample at 25-37° C. for 30-120 minutes to obtain a reaction solution.
  • the volume of APP is 500 ⁇ M
  • the concentration of Mg 2+ is 1 mM
  • the volume of Tris-HCl buffer is 200 ⁇ L.
  • the experimental conditions can be optimized according to the situation, including APP concentration, activator (MgCl 2 ) concentration, enzymatic reaction time, pH value of reaction buffer and reaction time, to achieve the best ALP detection performance.
  • the results show that the maximum fluorescence signal ratio is obtained when the APP concentration is 500 ⁇ M.
  • Mg2 + can be used to improve ALP (a marker enzyme commonly used in ELISA) activity and increase the stability of the system.
  • Step S160 transferring the reaction solution into a centrifuge tube and recording the fluorescence spectrum.
  • the present application provides a method and kit for detecting IL-6 protein based on ALP-mediated ELISA, which detects IL-6 by ALP-catalyzed APP hydrolysis.
  • ALP alkaline phosphatase
  • APP has a high fluorescence quenching efficiency during the hydrolysis process.
  • the change in APP fluorescence intensity and the logarithm of IL-6 protein concentration show a good linear relationship in the range of 0.005 ng/mL - 5.0 ng/mL, and the detection limit is 0.001 ng/mL, which is 100 times more sensitive than the traditional pNPP-based ELISA.
  • the colorimetric signal of AP p-aminophenol
  • the detection limit is 0.1 ng/mL. This method can be used for visual detection of IL-6 with high sensitivity.
  • APP trizma hydrochloride
  • MgCl2 MgCl2
  • the compound pNPP was purchased from Shanghai Aladdin Company.
  • SA-ALP was purchased from Beijing Bioson Company.
  • Human IL-6 recombinant protein, mouse anti-IL-6 antibody, mouse anti-IL-6 antibody (biotin-modified), and IL-6 ELISA kit were purchased from Sino Biological.
  • Calf intestinal alkaline phosphatase (ALP) was purchased from New England Laboratories.
  • Fluorescence spectra were tested using a Hitachi F-7100 FL spectrophotometer. UV-vis absorption spectra were recorded on a Hitachi system (U-3900H).
  • the microplate thermostat oscillator used for the experiments was purchased from Allsheng (MB100-4A).
  • a commercial ELISA assay was performed using a microplate reader obtained from Perkin Elmer (VICTOR Nivo).
  • ALP samples with 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 ⁇ M) and Mg 2+ (1 mM).
  • Tris buffer (20 mM, pH 9.5) containing APP (500 ⁇ M) and Mg 2+ (1 mM).
  • the reaction solution was incubated at 37 °C for 90 min, and then the fluorescence spectra were recorded.
  • the fluorescence spectra were recorded in the wavelength range of 320 - 450 nm under 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 ⁇ M) and Mg2+ (1 mM). The reaction solution was incubated at 37°C for 90 min, and then the absorption spectrum was recorded. The UV-vis absorption spectrum was 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 ⁇ M) and Mg 2+ (1 mM). The reaction solution was then incubated at 37°C for 30 min. Subsequently, the UV-vis absorption spectrum was recorded. Absorption spectra were recorded in the wavelength range of 250–600 nm.
  • Capture antibody (100 ⁇ L, 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 wash buffer (PBS containing 0.05% Tween 20, pH 7.4, 10 mM). The microplate was then blocked with skim milk (300 ⁇ L, 2.5% w/t) at 37°C for 2 hours. Next, 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.
  • coating buffer 50 mM carbonate buffer, pH 9.6
  • wash buffer PBS containing 0.05% Tween 20, pH 7.4, 10 mM
  • biotinylated mouse monoclonal antibody 100 ⁇ L, 1:1000
  • SA-ALP 100 ⁇ L, 1:2000
  • Tris-HCl buffer 200 ⁇ L
  • 200 ⁇ M APP and Mg2+ 1 mM
  • the reaction solution was transferred to a centrifuge tube, and the fluorescence spectrum was recorded.
  • IL-6 was detected using a commercial IL-6 ELISA kit, which was used as a control for sensitivity comparison.
  • FIG. 3 (A) shows that there is a significantly high-intensity fluorescence peak at 360 nm, and the fluorescence intensity is positively correlated with the concentration of APP.
  • the maximum fluorescence can be recorded when the excitation wavelength is 310 nm, as shown in Figure 3 (B).
  • the colorimetric and fluorescence dual-mode ALP activity assay was performed under optimal conditions. As shown in Figure 5A, an increase in ALP concentration resulted in an increase in the absorption intensity at 290 nm. A good linear relationship was obtained between the change in absorption intensity at 290 nm ( ⁇ A290) and the logarithm of ALP concentration (range: 0.5–25.0 U/L; Figure 5B). Figure 5D showed that an increase in ALP concentration resulted in a gradual decrease in the fluorescence intensity of APP, and the change in fluorescence intensity was concentrated at 360 nm ( ⁇ F360).
  • 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 ( Figures 5E and 2F).
  • the detection limits of the colorimetric and fluorescence readout modes were calculated to be 0.1 and 0.005 U/L (3 ⁇ /S), respectively. The results showed that the sensitivity of the fluorescence mode was higher than that of the colorimetric mode.
  • the conventional ALP detection method based on pNPP hydrolysis was used to compare the sensitivity with the dual-mode detection method.
  • the compound pNPP is usually used as a substrate, and the detection principle involves the dephosphorylation and catalysis of colorless pNPP to produce yellow p-nitrophenol (PNP).
  • the experimental parameters were optimized, including the pNPP concentration, the activator (MgCl2) concentration, and the pH of the reaction buffer.
  • Figure 6A-D shows that the maximum change in absorbance at 400 nm was observed when the pNPP concentration was 10 ⁇ M, the Mg2+ concentration was 1 mM, and the pH of the buffer was 9.5.
  • the absorbance of pNP at 400 nm increased with the increase in ALP concentration, and the change in the absorption intensity at 400 nm ( ⁇ A400) had a good correlation with the logarithm of the ALP concentration (range: 0.5 - 50.0 U/L).
  • the detection limit was 0.1 U/L.
  • the sensitivity of the fluorescence mode we proposed is 20 times that of the pNPP hydrolysis based on the colorimetric mode.
  • the visual ALP detection method was also performed. As shown in Figure 5C , an increase in ALP concentration from 0 to 150 U/L caused the color of the microplate to change from colorless to yellow to dark brown, indicating that this method can be effectively used for naked eye detection.
  • APP When APP is used for ALP detection, high detection sensitivity can be achieved, so APP can be used for ALP-mediated ELISA.
  • This example provides a dual-mode ELISA sensor based on the ALP-mediated APP hydrolysis process for IL-6 detection. The development of this dual-mode ELISA method follows the procedure of conventional ELISA, and the detection process involves immobilization of capture antibody (capture Ab), different concentrations of IL-6, biotin-modified detection antibody (biotin-Ab1), and SA-ALP. Sandwich immune complexes are formed on a 96-well plate through specific antigen-antibody immune reactions and SA-biotin interactions.
  • APP is used as a substrate catalyzed by ALP to produce AP, which contributes to the generation of colorimetric and fluorescent readout signals.
  • Figure 7 (A) the maximum fluorescence intensity ratio was recorded at an APP concentration of 500 ⁇ M.
  • Figure 7 (B) and (C) the maximum change in fluorescence intensity was observed when the capture antibody was diluted at a ratio of 1:200 and SA-ALP was diluted at a ratio of 1:2000.
  • pNPP the most commonly used substrate for ALP
  • ALP-mediated ELISA As shown in Figure 9 (A), the absorbance of pNPP at 310 nm decreased, and the absorbance of pNP at 400 nm increased as the IL-6 concentration increased from 0.5 to 200 ng/mL, with a detection limit of 0.1 ng/mL.
  • the fluorescence mode detection method in this work is 100 times more sensitive than the traditional pNPP-based ELISA method.
  • nonspecific proteins including tumor necrosis factor (TNF), interleukin-10 (IL-10), and transforming growth factor (TGF), were added to the ELISA detection method to study the selectivity of the method for IL-6 detection.
  • TNF tumor necrosis factor
  • IL-10 interleukin-10
  • TGF transforming growth factor
  • IL-6 detection method In order to evaluate the reliability of the IL-6 detection method in clinical samples, we used the constructed ELISA method to detect IL-6 in cerebrospinal fluid samples from 22 volunteers diagnosed and treated in the Department of Neurology of Liuzhou Hospital of Traditional Chinese Medicine. The samples were obtained from patients diagnosed with cerebral infarction or non-cerebral infarction, which were diagnosed by digital subtraction angiography (DSA). IL-6 in actual samples was detected according to the fluorescence mode detection method designed in this study. As shown in Figure 12, the results showed that the degree of APP fluorescence change in the negative group (consisting of 12 volunteers without cerebral infarction) was significantly lower than that in the positive group (consisting of 10 patients with cerebral infarction).
  • the results showed that the concentration of IL-6 in 10 patients with cerebral infarction was significantly higher than that in 12 asymptomatic patients. The results also showed that the IL-6 concentration was positively correlated with the severity of cerebral infarction. Therefore, it can be inferred that the developed ELISA method can effectively distinguish patients with cerebral infarction from patients without cerebral infarction. This method can also be used to accurately classify the severity of cerebral infarction as mild, moderate or severe.
  • the excellent fluorescence properties of APP and the fluorescence intensity of APP during the ALP-catalyzed APP hydrolysis process are significantly reduced.
  • This finding has also been used for ALP detection and ELISA-based IL-6 detection with significant sensitivity.
  • the response degree of the APP hydrolysis process to ALP is 20 times higher than the response degree to ALP during the conventional pNPP hydrolysis process.
  • the sensitivity of the IL-6 detection method based on the ALP-catalyzed APP hydrolysis reaction is 100 times higher than that of the conventional pNPP-based ELISA.
  • the developed ELISA sensor can be used to analyze clinical samples, and the sensor can be used to distinguish samples from patients with cerebral infarction from samples from asymptomatic volunteers. It was observed that the IL-6 concentration determined by this method was positively correlated with the severity of cerebral infarction determined by DSA.

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Abstract

一种基于ALP介导的ELISA检测IL-6蛋白的方法及试剂盒，通过ALP催化的APP水解检测IL-6，基于碱性磷酸酶（ALP）介导的APP水解，水解过程中APP具有高荧光猝灭效率。APP荧光强度的变化与IL-6蛋白浓度的对数在0.005 ng/mL - 5.0 ng/mL范围内呈良好的线性关系，检测限为0.001 ng/mL，比传统的基于pNPP的ELISA灵敏度高100倍。而将AP的比色信号用于比色ELISA检测IL-6时，检测限为0.1 ng/mL，该方法可用于IL-6的视觉检测，具有较高的灵敏度。

Description

基于ALP介导的ELISA检测IL-6蛋白的方法及试剂盒 技术领域
本申请涉及试剂检测技术领域，特别涉及一种基于ALP介导的ELISA检测IL-6蛋白的方法及试剂盒。
 背景技术
脑梗塞是一种严重的脑血管疾病，伴有高残疾率和高死亡率的风险，如果缺乏有效的诊断方法则会导致诊断延误以及发生严重并发症的风险增加。因此，脑梗塞的早期筛查和诊断对于提高患者的生存率和减轻家庭和社会负担至关重要。检测可准确预测脑梗塞风险和严重程度的新型生物标志物可为筛查工作提供有效帮助。最近的研究揭示了炎症反应与脑梗塞之间的潜在关系，细胞因子的浸润可导致慢性非特异性炎症，这与脑梗塞的发生发展密切相关。在急性脑梗塞患者中，血液中各种内源性细胞因子的水平显著增加。因此，测定炎性细胞因子的水平可以为诊断脑梗塞以及预后评估提供有价值的策略。白细胞介素6（IL-6）是一种在机体炎症反应中经常分泌的细胞因子。据报道，脑脊液中IL-6的水平在脑梗塞后显著增加，并且与脑梗塞的预后预测有关。因此，IL-6被认为是脑梗塞的可靠筛查和预后生物标志物。
多年来，已经开发了不同的方法和检测手段来确定IL-6的浓度，包括酶联免疫吸附测定（ELISA），基于适配体的传感器和有机电化学晶体管等。ELISA被广泛用于分析，因为它简单，反应快速，具有强特异性，用途广泛，并且只需要简单的实验设备。比色法，荧光法，化学发光法，表面增强拉曼散射法和电化学法可与ELISA结合。基于比色和荧光的ELISA越来越受欢迎，因为它们易于操作，成本低，并且测试时间短。然而，由于体液中IL-6的量极低（浓度在pM范围内），使用常规的基于比色法的免疫传感器可能无法实现诊断脑梗塞，不具有足够低的检测限和宽的线性范围。基于荧光的分析方法的灵敏度比基于比色法的灵敏度高1-2个数量级。目前研究中基于荧光纳米探针开发了各种检测方法，而依赖于荧光纳米探针与底物或标记酶产物的相互作用的常规荧光免疫测定易受环境干扰。
 技术问题
鉴于此，有必要针对现有技术中存在的缺陷提供一种灵敏度高的ELISA检测IL-6蛋白的方法及试剂盒。
 技术解决方案
为解决上述问题，本申请采用下述技术方案：
本申请目的之一，提供了一种基于ALP介导的ELISA检测IL-6蛋白的方法，包括下述步骤：
将捕获抗体加入到微量培养板中，并与包被缓冲液在25-37℃下孵育1-4小时，得到样品；
用洗涤缓冲液洗涤所述样品以除去所述包被缓冲液；
用脱脂乳在25-37℃封闭微孔板1-4小时，再将不同浓度的ALP样品在25-37℃温育1-2小时；
将生物素化的单克隆抗体和SA-ALP在25-37℃温育1-2小时以形成免疫复合物；
将APP和Mg 2+的Tris-HCl缓冲液加入到所述样品中，并将所述样品在25-37℃下孵育30-120分钟，得到反应溶液；
将所述反应溶液转移到离心管中，并记录荧光光谱。
在其中一些实施例中，在将捕获抗体加入到微量培养板中，并与包被缓冲液在25-37℃下孵育1-4小时，得到样品的步骤中，所述捕获抗体包括小鼠抗IL-6抗体，所述微量培养板为96孔微量培养板，所述包被缓冲液包括碳酸盐缓冲液。
在其中一些实施例中，所述捕获抗体的体积为100μL，浓度为1:100；所述包被缓冲液的浓度为50 mM，pH为9.6。
在其中一些实施例中，在用洗涤缓冲液洗涤所述样品以除去所述包被缓冲液的步骤中，所述洗涤缓冲液为含有0.05%吐温20，pH为7.4，浓度为10 mM的PBS。
在其中一些实施例中，在用脱脂乳在37℃封闭微孔板2小时，再将不同浓度的ALP样品在37℃温育1小时的步骤中，所述脱脂乳的体积为300μL，浓度为2.5% w/t；所述不同浓度的ALP样品包括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， 100.0 ng/mL。
在其中一些实施例中，在将生物素化的单克隆抗体和SA-ALP在25-37℃温育1-4小时以形成免疫复合物的步骤中，所述生物素化的小鼠单克隆抗体的体积为100μL，浓度为1:1000；所述SA-ALP的体积为100μL，浓度为1:2000。
在其中一些实施例中，在将APP和Mg 2+的Tris-HCl缓冲液加入到所述样品中，并将所述样品在25-37℃下孵育30-120分钟，得到反应溶液的步骤中，所述APP的浓度为500μM，所述Mg 2+浓度为1 mM，所述Tris-HCl缓冲液的体积为200μL。
本申请目的之二，提供了一种试剂盒，所述试剂盒通过应用所述的ELISA检测IL-6蛋白的方法实现IL-6蛋白的检测。
 有益效果
本申请采用上述技术方案，其有益效果如下：
本申请提供的基于ALP介导的ELISA检测IL-6蛋白的方法及试剂盒，通过ALP（碱性磷酸酶）催化的APP水解检测IL-6，基于碱性磷酸酶（ALP）介导的APP（4-氨基苯酚磷酸酯）水解，水解过程中APP具有高荧光猝灭效率。APP荧光强度的变化与IL-6蛋白浓度的对数在0.005 ng/mL - 5.0 ng/mL范围内呈良好的线性关系，检测限为0.001 ng/mL，比传统的基于pNPP的ELISA灵敏度高100倍。而将AP（对氨基苯酚）的比色信号用于比色ELISA检测IL-6时，检测限为0.1 ng/mL，该方法可用于IL-6的视觉检测，具有较高的灵敏度。
 附图说明
为了更清楚地说明本申请实施例的技术方案，下面将对本申请实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面所描述的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本申请实施例提供的基于app的比色法和荧光法双模ELISA检测IL-6过程示意图。
图2为本申请实施例提供的基于ALP介导的ELISA检测IL-6蛋白的方法的步骤流程图。
图3中(A)表示为不同浓度APP在Tris-HCl缓冲液中的发射光谱。
图3中(B)表示 APP在Tris-HCl缓冲液中270 ~ 320 nm不同激发波长下的发射光谱。
图4中 (A)表示为添加50 U/L ALP后不同浓度APP的荧光强度变化。
图4中(B) 表示500 μM APP在含有不同浓度Mg2+的50 U/L ALP作用下的荧光强度变化。
图4中(C)表示不同ph值下500 μM APP在50 U/L ALP作用下荧光强度的变化。
图4中(D) 表示ALP检测反应时间的优化，ALP浓度分别为10、25、50 U/L。
图5中 (A)表示APP (200 μM)对不同浓度ALP的紫外-可见吸收光谱。
图5中(B) 表示ALP检测对应的校准曲线。
图5中(C) 表示APP水解检测ALP可视化。
图5中(D) 表示APP (500 μM)对不同浓度ALP的荧光光谱响应。
图5中(E)表示0.02 U/L ~ 1.0 U/L范围内ALP检测对应的校准曲线。
图5中(F) 表示2.0 U/L ~ 50.0 U/L范围内ALP检测对应的校准曲线。
图6中 (A)表示10μM pNPP对含有不同浓度Mg 2+的10 U/L ALP的吸光度变化。
图6中(B)表示不同ph值下10 μM pNPP对10 U/L ALP吸光度的变化。
图6中(C)表示不同浓度ALP对10 μM pNPP紫外光谱的响应。
图6中(D)表示 ALP检测校准曲线。
图7中(A)表示为 APP浓度。
图7中(B)表示为Capture Ab稀释。
图7中(C)表示为SA-ALP用于IL-6蛋白检测。
图8中 (A)表示为不同浓度IL-6对APP在alp介导的ELISA中的紫外可见吸收光谱。
图8中(B) 表示为IL-6蛋白检测对应的校准曲线。
图8中(C) 表示为alp介导的ELISA检测IL-6的可视化。
图8中(D)表示为在alp介导的ELISA中，不同浓度IL-6对APP的荧光光谱。
图8中(E)表示为0.005 ng/mL ~ 5.0 ng/mL范围内IL-6蛋白检测对应的校准曲线
图8中(F) 表示为10.0 ng/mL ~ 100.0 ng/mL范围内IL-6蛋白检测对应的校准曲线。
图9中 (A)表示为荧光pNPP介导的ELISA传感器通过荧光强度的变化检测IL-6蛋白。
图9中(B)表示为 IL-6蛋白检测校准曲线。
图10表示为IL-6蛋白检测对其他干扰蛋白的选择性研究，包括TNF、IL-10和TGF，IL-6蛋白浓度为100 ng/mL，其他干扰蛋白浓度为1000 ng/mL。
图11为本实施例提供的商用ELISA试剂盒检测IL-6蛋白示意图。
图12表示为无症状志愿者(1-12)和脑梗死患者(13-22)样品的APP荧光强度变化响应。
 本发明的实施方式
下面详细描述本申请的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本申请，而不能理解为对本申请的限制。
在本申请的描述中，需要理解的是，术语“上”、“下”、“水平”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本申请的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
为了使本申请的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本申请进行进一步详细说明。
请参阅图1及图2，本实施例提供的基于ALP介导的ELISA检测IL-6蛋白的方法，包括下述步骤S110至步骤S160，以下详细说明各个步骤的实现方式。
步骤S110：将捕获抗体加入到微量培养板中，并与包被缓冲液在25-37℃下孵育1-4小时，得到样品。
在本实施例中，所述捕获抗体包括小鼠抗IL-6抗体，所述微量培养板为96孔微量培养板，所述包被缓冲液包括碳酸盐缓冲液。
在本实施例中，所述捕获抗体的体积为100μL，浓度为1:100；所述包被缓冲液的浓度为50 mM，pH为9.6。
步骤S120：用洗涤缓冲液洗涤所述样品以除去所述包被缓冲液。
在本实施例中，所述洗涤缓冲液为含有0.05%吐温20，pH为7.4，浓度为10 mM的PBS。
步骤S130：用脱脂乳在25-37℃封闭微孔板1-4小时，再将不同浓度的ALP（肠碱性磷酸酶）样品在25-37℃温育1-2小时。
在本实施例中，所述脱脂乳的体积为300μL，浓度为2.5% w/t；所述不同浓度的ALP样品包括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， 100.0 ng/mL。
步骤S140：将生物素化的单克隆抗体和SA-ALP在25-37℃温育1-2小时以形成免疫复合物。
在本实施例中，所述生物素化的小鼠单克隆抗体的体积为100μL，浓度为1:1000；所述SA-ALP的体积为100μL，浓度为1:2000。
步骤S150：将APP（4-氨基苯酚磷酸酯）和Mg 2+的Tris-HCl缓冲液加入到所述样品中，并将所述样品在25-37℃下孵育30-120分钟，得到反应溶液。
在本实施例中，所述APP的体积为500μM，所述Mg 2+浓度为1 mM，所述Tris-HCl缓冲液的体积为200μL。
可以理解，在实际中实验条件可以根据情况进行优化，包括APP浓度，活化剂（MgCl 2）浓度，酶促反应时间，反应缓冲液的pH值和反应时间，以实现最佳的ALP检测性能。结果显示，在APP浓度为500μM时有最大荧光信号比。
可以理解，适当浓度的Mg 2+可用于改善ALP（ELISA中常用的标记酶）活性并增加系统的稳定性。
步骤S160：将所述反应溶液转移到离心管中，并记录荧光光谱。
本申请提供的基于ALP介导的ELISA检测IL-6蛋白的方法及试剂盒，通过ALP催化的APP水解检测IL-6，基于碱性磷酸酶（ALP）介导的APP水解，水解过程中APP具有高荧光猝灭效率。APP荧光强度的变化与IL-6蛋白浓度的对数在0.005 ng/mL - 5.0 ng/mL范围内呈良好的线性关系，检测限为0.001 ng/mL，比传统的基于pNPP的ELISA灵敏度高100倍。而将AP（对氨基苯酚）的比色信号用于比色ELISA检测IL-6时，检测限为0.1 ng/mL，该方法可用于IL-6的视觉检测，具有较高的灵敏度。
以下结合具体实施例对本申请上述技术方案进行详细说明。
实施例
本实施例中涉及的化学品及物料如下：
APP，盐酸trizma（Tris-HCl）和MgCl 2购于Sigma。化合物pNPP购于上海阿拉丁公司。SA-ALP购于北京博奥森公司。人IL-6重组蛋白，小鼠抗IL-6抗体，小鼠抗IL-6抗体（生物素修饰）和IL-6 ELISA试剂盒购于义翘神州。小牛肠碱性磷酸酶（ALP）购自新英格兰实验室。
仪器和表征：
使用Hitachi F-7100 FL分光光度计测试荧光光谱。在Hitachi系统（U-3900H）上记录UV-vis吸收光谱。用于实验的微孔板恒温振荡器购自Allsheng（MB100-4A）。使用从Perkin Elmer（VICTOR Nivo）获得的酶标仪进行商用ELISA检测。
临床样本分析：
所有临床样本均来自柳州市中医医院收治的患者，从所有登记的患者中收集脑脊液。将样品置于抗凝管中并在室温下以4000 rpm离心。离心（离心时间：10分钟）后，将上清液收集在离心管中并作为等分试样储存在-80℃下进行测试。每个样品，包括12个阳性和10个阴性。检测过程完成后，对实验废物进行灭菌。
ALP响应分析：
将不同浓度（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）的ALP样品与含有APP（500 μM）的Tris缓冲液（20 mM，pH 9.5）混合Mg 2+（1 mM）。将反应溶液在37℃下孵育90分钟，然后记录荧光光谱。在310 nm的激发波长下，在320 - 450 nm的波长范围记录荧光光谱。将不同浓度（0, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 25.0 U/L）的ALP样品与含有APP（200 μM）和Mg2+（1mM）的Tris缓冲液（20 mM，pH 9.5）混合。将反应溶液在37℃下孵育90分钟，随后记录吸收光谱。在250 - 350nm的波长范围记录UV-vis吸收光谱。将不同浓度（0, 0.5, 1.0, 5.0, 10.0, 20.0, 50.0 U/L）的ALP样品与含有pNPP（10μM）和Mg 2+（1 mM）的Tris缓冲液（20 mM，pH 9.5）混合。然后将反应溶液在37℃下孵育30分钟。随后，记录UV-vis吸收光谱。在250 - 600nm的波长范围记录吸收光谱。
基于ALP介导的ELISA检测IL-6蛋白：
将捕获抗体（100 μL，1:100）加入到96孔微量培养板中，并与包被缓冲液（50 mM碳酸盐缓冲液，pH 9.6）在37℃下孵育2小时。除去包被缓冲液，用洗涤缓冲液（含有0.05%吐温20，pH 7.4,10 mM的PBS）洗涤样品三次。然后用脱脂乳（300 μL，2.5% w/t）在37℃封闭微孔板2小时。接着，将不同浓度（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）在37℃温育1小时。之后，将生物素化的小鼠单克隆抗体（100 μL，1:1000）和SA-ALP（100 μL，1:2000）在37℃温育1小时以形成免疫复合物。将含有200 μM APP和Mg2+（1 mM）的Tris-HCl缓冲液（200 μL）加入到样品中，并将样品在37℃下孵育90分钟。将反应溶液转移到离心管中，并记录荧光光谱。使用商业IL-6 ELISA试剂盒检测IL-6，该方法用作灵敏度比较的对照。
APP的特征
在本实施例中，我们观察到APP通常用作ALP的底物并通过去磷酸化过程以产生AP，表现出良好的荧光性质。图3中（A）显示在360 nm处存在显着高强度的荧光峰，并且荧光强度与APP的浓度正相关。当激发波长为310 nm时可记录到最大荧光，如图3中（B）所示。这些发现表明APP的荧光特性可用于开发新的比色和荧光双模式ELISA方法，以实现IL-6的灵敏检测。
基于ALP催化APP水解的比色和荧光双模测定：
在本实施例中，通过分析UV-vis吸收和荧光光谱来验证使用ALP分析APP水解过程的可行性。我们优化了几个实验条件，包括APP浓度，活化剂（MgCl2）浓度，酶促反应时间，反应缓冲液的pH值和反应时间，以实现最佳的ALP检测性能。结果显示，在APP浓度为500μM时有最大荧光信号比（图4中（A））。据报道，适当浓度的Mg 2+可用于改善ALP活性并增加系统的稳定性。如图4中（B）所示，1 mM Mg 2+用于后续实验，因为Mg 2+浓度的进一步增加不会导致荧光强度的显着变化。在pH值为9.5时记录荧光强度的最大变化（图4中（C）），并且基于ALP的催化的最佳反应时间为90分钟（图4中（D））。
比色和荧光双模式ALP活性测定在最佳条件下进行。如图5中A所示，ALP浓度的增加导致290 nm处吸收强度的增加。在290 nm处的吸收强度变化（ΔA290）与ALP浓度的对数之间获得了良好的线性关系（范围：0.5–25.0 U/L；图5中B）。图5中D显示ALP浓度的增加导致APP的荧光强度逐渐降低，并且荧光强度的变化集中在360 nm（ΔF360）。荧光强度的变化与ALP浓度的对数在0.02 - 1.0 U/L和2.0 - 100.0 U/L的范围内表现出良好的线性关系（图5中E和2F）。比色和荧光读出模式的检测限分别计算为0.1和0.005 U/L（3σ/S）。结果表明，荧光模式的灵敏度高于比色模式的灵敏度。
通过基于pNPP水解的常规ALP检测方法用于与双模式检测方法的灵敏度对比。在ALP检测过程中，化合物pNPP通常用作底物，检测原理涉及无色pNPP的去磷酸化和催化，产生黄色对硝基苯酚（PNP）。优化实验参数，包括pNPP浓度，活化剂（MgCl2）浓度和反应缓冲液的pH。图6中A-D显示，当pNPP浓度为10 μM，Mg2+浓度为1 mM，缓冲液的pH为9.5时，观察到400 nm处吸光度的最大变化。在最佳条件下，pNP在400 nm处的吸光度随着ALP浓度的增加而增加，400 nm处吸收强度的变化（ΔA400）与ALP浓度的对数（范围：0.5 - 50.0 U/L）有很好的相关性。检测限为0.1 U/L。我们提出的荧光模式的灵敏度是基于比色模式的pNPP水解的灵敏度的20倍，还进行了视觉ALP检测方法。如图5中C所示，ALP浓度从0增加到150 U/L导致微孔板颜色从无色变为黄色变为深棕色，表明该方法可以有效地用于肉眼检测。
用于IL-6检测的双模式ELISA
[根据细则91更正 05.03.2024]
当APP用于ALP检测时，可以实现高检测灵敏度，因此APP可用于ALP介导的ELISA。本实施例提供了一种基于ALP介导的APP水解过程的双模式ELISA传感器用于IL-6检测。这种双模式ELISA的方法的开发遵循常规ELISA的程序，检测过程涉及固定捕获抗体（捕获Ab），不同浓度的IL-6，生物素修饰的检测抗体（生物素-Ab1），和SA-ALP。通过特异性抗原-抗体免疫反应和SA-生物素相互作用在96孔板上形成夹心免疫复合物。APP用作ALP催化的底物以产生AP，这有助于比色和荧光读出信号的产生。首先，我们优化了检测过程中的各组分，如图7中（A）所示，在APP浓度为500 μM时记录最大荧光强度比，我们还优化了捕获抗体和SA-ALP的使用。如图7中（B）和（C）所示，当捕获抗体以1:200的比例稀释并且SA-ALP以1:2000的比例稀释时，观察到荧光强度的最大变化。这些优化的参数用于后续实验。在比色模式下，随着IL-6浓度从0.5增加到75.0 ng/mL，290 nm处吸收强度的变化程度显著增加（图8中（A））。在吸收强度的变化和IL-6浓度的对数之间建立了良好的线性关系（图8中（B）），比色模式下IL-6检测的检测限为0.1 ng/mL。荧光模式下，APP荧光的猝灭伴随着IL-6浓度的相应增加（图8中（D））。如图8中（E）和（F）所示，在360 nm处荧光强度的变化与IL-6蛋白浓度的对数（0.005 - 5.0 ng/mL和10 - 100 ng/mL）呈良好的线性关系。检测限为0.001 ng/mL，显著低于比色模式下的检测限。此外，进行视觉IL-6检测方法用于分析，如图8中C所示，随着IL-6浓度从0增加到500 ng/mL，无色微孔板变黄并随后变深棕色，表明该方法肉眼检测IL-6的高潜力。
使用ALP最常用的底物pNPP用于ALP介导的ELISA。如图9中（A）所示，pNPP在310 nm处的吸光度降低，并且pNP在400 nm处的吸光度随着IL-6浓度从0.5增加到200 ng/mL而增加，检测限为0.1 ng/mL。这项工作中的荧光模式检测方法比传统的基于pNPP的ELISA方法灵敏100倍。
用其他非特异性蛋白质（包括肿瘤坏死因子（TNF），白细胞介素-10（IL-10）和转化生长因子（TGF））加入ELISA检测方法中，以研究该方法对IL-6检测的选择性。如图10所示，即使非特异性蛋白质的浓度是IL-6浓度的十倍，也没有观察到非特异性蛋白质的荧光强度的明显变化。此外，在系统中同时存在IL-6和干扰蛋白的情况下，观察到的荧光性质的变化与系统中单独存在IL-6时观察到的变化相似，这表明非特异性蛋白质对IL-6产生的反应几乎没有影响。因此，开发的基于双模式ELISA的方法可以特异性识别和检测IL-6，请参阅图11为本实施例提供的商用ELISA试剂盒检测IL-6蛋白示意图。
使用临床样本进行验证
为了评估临床样本中IL-6检测方法的可靠性，我们利用所构建的ELISA方法检测了来自于柳州市中医医院神经内科接受诊断和治疗的22名志愿者的脑脊液样本中的IL-6，样本获自诊断患有脑梗塞或非脑梗塞的患者，其通过数字减影血管造影（DSA）进行诊断。按照本研究设计的荧光模式检测方法检测实际样品中的IL-6。图12所示，结果表明阴性组（由12名未患脑梗塞的志愿者组成）APP荧光变化程度显著低于阳性组（由10名脑梗塞患者组成）。结果表明10例脑梗死患者中的IL-6的浓度明显高于12例无症状患者的IL-6浓度。结果还显示，IL-6浓度与脑梗塞严重程度呈正相关。因此，可以推断，所开发的ELISA方法可以有效地区分具患有脑梗塞的患者和非脑梗塞患者。该方法还可用于将脑梗塞的严重程度准确分类为轻度，中度或重度。
可以理解，APP的优异荧光性质和基于ALP催化的APP水解过程中APP的荧光强度显著降低。该发现也被用于ALP检测和基于ELISA的IL-6检测，具有显著的灵敏度。APP水解过程与ALP的响应程度比常规pNPP水解过程中与ALP的响应程度高20倍。此外，基于ALP催化的APP水解反应的IL-6检测方法的灵敏度比传统的基于pNPP的ELISA的灵敏度高100倍。所开发的ELISA传感器可用于分析临床样品，并且可以使用该传感器将脑梗塞患者的样品与无症状志愿者的样品区分开。观察到按照该方法测定的IL-6浓度与通过DSA测定的脑梗塞的严重程度正相关。
可以理解，以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上仅为本申请的较佳实施例而已，仅具体描述了本申请的技术原理，这些描述只是为了解释本申请的原理，不能以任何方式解释为对本申请保护范围的限制。基于此处解释，凡在本申请的精神和原则之内所作的任何修改、等同替换和改进，及本领域的技术人员不需要付出创造性的劳动即可联想到本申请的其他具体实施方式，均应包含在本申请的保护范围之内。

Claims (8)

  1. 一种基于ALP介导的ELISA检测IL-6蛋白的方法，其特征在于，包括下述步骤：
    将捕获抗体加入到微量培养板中，并与包被缓冲液在25-37℃下孵育1-4小时，得到样品；
    用洗涤缓冲液洗涤所述样品以除去所述包被缓冲液；
    用脱脂乳在25-37℃封闭微孔板1-4小时，再将不同浓度的ALP样品在25-37℃温育1-2小时；
    将生物素化的单克隆抗体和链霉亲和素修饰的碱性磷酸酶在25-37℃温育1-2小时以形成免疫复合物；
    将APP和Mg 2+ 的Tris-HCl缓冲液加入到所述样品中，并将所述样品在25-37℃下孵育30-120分钟，得到反应溶液；
    将所述反应溶液转移到离心管中，并记录荧光光谱。
  2. 如权利要求1所述的基于ALP介导的ELISA检测IL-6蛋白的方法，其特征在于，在将捕获抗体加入到微量培养板中，并与包被缓冲液在25-37℃下孵育1-4小时，得到样品的步骤中，所述捕获抗体包括小鼠抗IL-6抗体，所述微量培养板为96孔微量培养板，所述包被缓冲液包括碳酸盐缓冲液。
  3. 如权利要求2所述的基于ALP介导的ELISA检测IL-6蛋白的方法，其特征在于，所述捕获抗体的体积为100μL，浓度为1:100；所述包被缓冲液的浓度为50 mM，pH为9.6。
  4. 如权利要求1所述的基于ALP介导的ELISA检测IL-6蛋白的方法，其特征在于，在用洗涤缓冲液洗涤所述样品以除去所述包被缓冲液的步骤中，所述洗涤缓冲液为含有0.05%吐温20，pH为7.4，浓度为10 mM的PBS。
  5. 如权利要求1所述的基于ALP介导的ELISA检测IL-6蛋白的方法，其特征在于，在用脱脂乳在37℃封闭微孔板2小时，再将不同浓度的ALP样品在37℃温育1小时的步骤中，所述脱脂乳的体积为300μL，浓度为2.5% w/t；所述不同浓度的ALP样品包括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， 100.0 ng/mL。
  6. 如权利要求1所述的基于ALP介导的ELISA检测IL-6蛋白的方法，其特征在于，在将生物素化的单克隆抗体和SA-ALP在25-37℃温育1-2小时以形成免疫复合物的步骤中，所述生物素化的小鼠单克隆抗体的体积为100μL，浓度为1:1000；所述SA-ALP的体积为100μL，浓度为1:2000。
  7. 如权利要求1所述的基于ALP介导的ELISA检测IL-6蛋白的方法，其特征在于，在将APP和Mg 2+ 的Tris-HCl缓冲液加入到所述样品中，并将所述样品在25-37℃下孵育30-120分钟，得到反应溶液的步骤中，所述APP的浓度为500μM，所述Mg 2+ 浓度为1mM，所述Tris-HCl缓冲液的体积为200μL。
  8. 一种试剂盒，其特征在于，所述试剂盒通过应用权利要求1至7任一项所述的ELISA检测IL-6蛋白的方法实现IL-6蛋白的检测。
PCT/CN2023/141325 2023-07-13 2023-12-23 基于alp介导的elisa检测il-6蛋白的方法及试剂盒 Pending WO2025010959A1 (zh)

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