Capillary electrophoresis fluorescence immunoassay method for detecting abrus precatorius toxin
By combining capillary electrophoresis and fluorescence immunoassay, the problems of time-consuming and cumbersome AT detection methods have been solved, achieving rapid, automated, low-cost, and efficient detection.
Patent Information
- Application Number
- CN202511381260.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-25
- Publication Date
- 2025-12-09
AI Technical Summary
Existing AT testing methods are cumbersome, time-consuming, and have a low degree of automation, making it difficult for ordinary laboratories to carry out efficient testing.
By combining capillary electrophoresis and fluorescence immunoassay, a rapid and simple detection method was established by utilizing the complex formed by fluorescently labeled antibody protein and AT, separating the complex by capillary electrophoresis and detecting it with a laser-induced fluorescence detector.
It achieves fast, simplified, and highly automated AT detection, reducing detection time to a few minutes, requiring very little sample, and is low in cost, making it suitable for large-scale sample analysis.
Smart Images
Figure CN121090844A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of analytical detection technology, in particular to a capillary electrophoresis fluorescence immunoassay method for detecting abrin. BACKGROUND
[0002] Abrin (AT) is a toxic protein extracted from the seeds of Abrus precatorius, which can inhibit the synthesis of animal cell proteins. Abrin is similar to ricin and belongs to the type II ribosome inactivating protein (RIP) family, but it is more toxic, with a toxicity of more than 70 times that of ricin. Due to its extremely strong toxicity and easy availability, it can be used as a biological threat agent. Therefore, it is of great significance to develop a rapid and reliable method for detecting AT for national biological safety prevention and control.
[0003] Currently, the detection methods for AT mainly include two categories: immunology-based analysis methods and mass spectrometry-based analysis methods. Common immunology analysis methods include enzyme-linked immunoassay (ELISA), Western blotting (WB), etc. These traditional immunological methods usually have complicated operation steps and take a long time. Common mass spectrometry methods for detection include enzymatic digestion mass spectrometry for peptide fingerprinting, LC-MS for detecting AT release of adenine, etc. However, the instruments required are large in size and high in price, and ordinary laboratories usually have no conditions to carry out related detection.
[0004] The development of capillary electrophoresis (CE) and its gradual application in immunoassay make it develop into a new type of immunoassay technology, i.e. capillary electrophoresis immunoassay (CEIA). CEIA is to use the difference in electrophoretic behavior between antigen-antibody complex and free antigen or antibody, use CE as a separation means in immunoassay, and upgrade the conventional ultraviolet detector to a laser-induced fluorescence (LIF) detector to greatly improve the detection sensitivity and accuracy. This combined technology combines the high selectivity of immunoassay, the high separation efficiency of CE and the high sensitivity detection of LIF, and has many advantages compared with traditional immunoassay, mainly in the following aspects: ① The immunoassay is carried out in a homogeneous solution, avoiding the interference of the matrix to affect the reaction speed, and the reaction proceeds rapidly, generally reaching equilibrium within 5-10 min, which greatly shortens the sample preparation time compared with the incubation time of several hours or even overnight in traditional immunoassay such as ELISA; ② CE usually only needs nL-level sample and μL-level buffer solution, which not only reduces the consumption of immunoassay reagents, but also is very suitable for the immunoassay of micro-volume samples; ③ The application of CE simplifies the cumbersome experimental operation steps in traditional immunoassay, greatly improves the automation degree, and shortens the detection time from several hours to several minutes, greatly improving the speed of immunoassay and enabling real-time monitoring and control; ④ Due to the high separation efficiency of CE, combined with the detection of online laser-induced fluorescence detector (LIF), the detection sensitivity can be improved, and multi-component simultaneous analysis can be performed.
[0005] Based on the above characteristics, how to use this technology to analyze and detect AT and establish a new AT detection method with short time consumption, simple operation, high automation degree and stability is a technical problem to be solved at present. SUMMARY
[0006] The purpose of the present application is to provide a new AT detection method based on fluorescence immuno-capillary electrophoresis, which has the advantages of simple operation, short time consumption and high automation degree, to solve the problems of traditional immunoassay analysis method such as complicated operation steps, long time consumption and low automation degree.
[0007] In order to achieve the above purpose, the present application provides the following technical solutions:
[0008] A rapid immunoassay method for detecting abrin, comprising the following steps:
[0009] S1. Preparation of fluorescently labeled antibody protein Ab*: Fluorescein isothiocyanate (FITC) was dissolved in dimethyl sulfoxide (DMSO) to prepare a dye solution; the dye and abrin antibody protein Ab were mixed in a molar ratio of 100-10:1, followed by a light-protected reaction, incubation at room temperature, to obtain the labeled antibody protein Ab*; then, purification was performed to remove unbound dye, and the labeled antibody protein Ab* solution was collected and quantified;
[0010] The abrin antibody protein Ab is a monoclonal antibody against AT, and the immune complex produced by the labeled antibody after FITC labeling and AT can be effectively separated from the excess labeled antibody in the electrophoresis process;
[0011] S2. Preparation of abrin AT standard curve: a series of gradient AT standard samples were prepared using a sample buffer, mixed with a fixed concentration of excess labeled antibody protein Ab* in equal volumes, and incubated at room temperature: Ab*(excess) + Ag → Ab* + [Ab*-Ag], to obtain the complex [Ab*-Ag] and excess labeled antibody protein Ab*;
[0012] Different components of the reacted solution were separated by capillary electrophoresis (CE). The capillary was a 50-100 μm inner diameter fused silica capillary with a total length of 30-50 cm and an LIF window effective length of 10-30 cm. The separation buffer was a sample buffer with the addition of polyethylene glycol 20000 at a concentration of 10-60 g / L. The separation voltage was 5-20 kV, the capillary temperature was 10-30 °C, and the pressure injection was 0.3-0.6 psi for 5-15 s.
[0013] The components were detected by a laser-induced fluorescence (LIF) detector to determine the migration time of the complex component [Ab*-Ag] in electrophoresis. The AT standard curve was plotted with the AT concentration as the abscissa and the fluorescence signal peak area as the ordinate.
[0014] The detection fluorescence wavelength range was 460-500 nm.
[0015] S3. Sample detection: the AT standard sample was replaced with the sample to be tested, and the same method as in step S2 was used for mixing and incubation with the labeled antibody protein Ab* before detection.
[0016] Preferably, when step S3 is qualitative detection, a fluorescence signal peak appears at the same time as the migration time of the complex, proving that the sample to be tested contains AT, and the sample is determined to be positive.
[0017] Preferably, when step S3 is quantitative detection, the fluorescence signal peak area at the same time as the migration time of the complex is brought into the standard curve equation obtained in step S2 to calculate the content of AT in the sample to be tested.
[0018] Preferably, in the step S1, the antibody protein is labeled: 1-3 mg / mL of antibody 0.2-1 mL, 1 mg of FITC dye powder is added to 100 μL to prepare a 10 mg / mL FITC solution.
[0019] Preferably, the sample buffer is TG buffer (pH 7.4), wherein TG buffer represents Tris-glycine buffer: 25 mM Tris base, 192 mM glycine.
[0020] Preferably, the capillary electrophoresis parameters are as follows: the capillary is a 75 μm inner diameter fused silica capillary, the total length is 41 cm, and the LIF window effective length is 20 cm. The separation buffer is 2 x TG (pH 9.0) + 40 g / L PEG20000 (polyethylene glycol 20000), the sample buffer is TG (pH 7.4), the separation voltage is 10 kV, the capillary temperature is 20°C, and the 0.5 psi pressure injection is 8 s.
[0021] Preferably, the labeled antibody protein Ab* is a fluorescently labeled mouse anti-AT monoclonal antibody.
[0022] Preferably, the labeled antibody protein Ab* is stored in the dark.
[0023] Preferably, the detection limit of the sample to be tested for AT is 75.8 nM.
[0024] Preferably, the detection fluorescence wavelength range is 480 nm.
[0025] The advantages and positive effects of the present application are:
[0026] 1. The present application combines capillary electrophoresis, fluorescence detection, and immunoassay methods, which has high separation efficiency of CE, high sensitivity of LIF, high selectivity and specificity of immunoassay, and greatly shortens the analysis time compared with conventional immunoassay. The sample is incubated with the labeled antibody for 10 min, electrophoresis is performed for 10-15 min, and the total analysis time is not more than 30 min. The time used is much lower than the several hours required for ELISA, and is suitable for rapid detection of AT.
[0027] 2. The present application simplifies the cumbersome manual operation steps, improves the analysis automation degree, can sequentially detect batch samples, and is suitable for analysis of a large number of samples to be tested.
[0028] 3. The present application requires a very small amount of sample in detection, and the minimum amount of sample used in single detection can be as low as nanoliters, greatly reducing the detection cost. BRIEF DESCRIPTION OF DRAWINGS
[0029] Figure 1 Schematic diagram of capillary electrophoresis analysis device
[0030] Figure 2 Non-competitive capillary electrophoresis immunoassay
[0031] Figure 3 Electropherogram after interaction of different concentrations of AT with a fixed concentration of labeled antibody
[0032] Figure 4 AT quantitative standard curve graph DETAILED DESCRIPTION
[0033] The application will be further described below in conjunction with examples; the following examples are illustrative and not limiting, and the protection scope of the application cannot be limited by the following examples.
[0034] The main reagents and instruments used in the application are as follows:
[0035] (1) Main reagents
[0036] Fluorescent labeling kit HOOK TM Dye Labeling Kit (Cat. # 786-141): Shenguo Bioengineering (Shanghai) Co., Ltd.
[0037] BCA protein concentration determination kit (Cat No. P0010): Beijing Biyun Tian Biological Company.
[0038] AT: purchased from Beijing Hansu Biological Technology Co., Ltd.
[0039] Antibody: mouse anti-AT monoclonal antibody (LD2022-5-1001) purchased from Shandong Landu Biological Technology Co., Ltd.
[0040] (2) Main instruments
[0041] CE7100 type capillary electrophoresis instrument: Agilent Company, USA.
[0042] Laser-induced fluorescence detector: France Picomertrics, ZETA LIF TM LED.
[0043] Vortex mixer: Dalongxingchuang Experimental Instrument Beijing Co., Ltd.
[0044] The application is to combine the fluorescence immunoassay technology with the capillary electrophoresis detection method, so as to establish a high-efficiency and rapid detection method for AT. The specific embodiments are as follows:
[0045] Example 1
[0046] Preparation of antibody with FITC fluorescent probe
[0047] This experiment adopts HOOKTM Dye Labeling Kit(Cat.#786-141)fluorescent labeling kit is completed, and the experimental steps are carried out according to the requirements of the instructions, as follows: 0.5 mL of 2 mg / mL antibody is taken, 100 μL of DMSO (dimethyl sulfoxide) is added to 1 mg of FITC dye powder to prepare a 10 mg / mL FITC solution, and the dye and antibody protein are added at a molar ratio of 20:1. After rapid vortex mixing, centrifuge for a short time to collect the sample at the bottom of the tube. Wrap the reaction tube with aluminum foil to avoid light reaction, and incubate at room temperature for 60 minutes. After the labeling reaction is completed, the sample is purified by desalting gel column to remove unbound dye, and the labeled antibody solution is collected. The antibody protein is quantified by BCA method, and stored at -20°C in the dark for use.
[0048] Test process:
[0049] 1. Capillary electrophoresis conditions
[0050] The buffer is Tris-glycine buffer (TG) with different concentrations and pH: 25 mM Tris base, 192 mM glycine; separation buffer: 2x TG (pH 9.0) + 10 g / L PEG20000 (polyethylene glycol 20000), sample buffer: TG (pH 7.4); electrophoresis separation voltage: 10 kV; capillary temperature setting: 20°C, sample injection mode: pressure injection 0.5 psi for 5 s; capillary parameters: 75 μm inner diameter fused silica capillary, total length 41 cm, LIF window effective length 20 cm.
[0051] 2. Drawing of AT standard curve
[0052] The sample buffer series gradient diluted AT standard (0, 10, 20, 30, 40, 60, 80 μg / mL) 20 μL is mixed with 20 μL of 40 μg / mL fluorescent labeled antibody, and after incubation at room temperature for 10 min (Ab*(Excessed)+Ag→Ab*+[Ab*-Ag]), the different samples after interaction are electrophoresed according to the conditions described in 1), and the different components (excess labeled antibody and antigen-antibody complex) in the sample are separated in electrophoresis. The laser-induced fluorescence detector detects the complex components containing the fluorescence signal, determines the migration time of the complex components [Ab*-Ag] in electrophoresis, and the experimental results are as follows: Figure 3As shown, the electrophoretic peak of the antibody alone appeared around 3.5 min, while the electrophoretic peak of the antibody-antigen complex appeared around 3.3 min. With increasing AT content in the system, the peak intensity of the complex gradually increased, while the peak intensity of the antibody gradually decreased until it disappeared. A standard curve was plotted with AT concentration on the x-axis and fluorescence peak area on the y-axis, yielding the linear equation Y = 0.0781X + 0.0293, R0. 2 =0.9949. Experimental results are as follows: Figure 3 , Figure 4 As shown.
[0053] Test results:
[0054] Detection of AT in simulated samples
[0055] Replace the standard sample in step 2) with 20 μL of 20 μg / mL fluorescently labeled antibody and mix thoroughly. Incubate at room temperature for 10 min. Perform electrophoresis under the same conditions used to plot the standard curve. The result shows a fluorescence signal peak at the same migration time (3.3 min) as in step 2), indicating that the sample contains AT and can be identified as a positive sample. Substitute the area of this fluorescence signal peak into the Y value of the standard curve equation to calculate the X value. Since the sample is diluted by 1-fold after mixing with an equal volume of antibody solution, 2X is the AT concentration. The entire detection process only requires mixing the sample with the fluorescently labeled antibody thoroughly, and the instrument performs the subsequent experimental steps. The operator only needs to set the instrument electrophoresis parameters and analyze the electrophoresis results. The detection time for a single sample is less than 30 min, which is much shorter than the several hours required for ELISA. The limit of detection for AT is 75.8 nM, the quantitative range is 75.8–606.4 nM, the sample recovery rate is 91.3%–94.9%, and the relative error range is 5.1%–8.7%.
[0056] As can be seen from the above embodiments, the detection method of the present invention is simple to operate, highly automated, and has a short detection time, making it convenient and fast.
Claims
1. A capillary electrophoretic fluorescence immunoassay method for detecting abrinogen toxin, characterized in that, Includes the following steps: S1. Preparation of fluorescently labeled antibody protein Ab*: FITC isothiocyanate was dissolved in dimethyl sulfoxide (DMSO) to prepare a dye solution; the dye and abrinogen toxin antibody protein Ab were mixed at a molar ratio of 100–10:1, followed by a light-protected reaction and incubation at room temperature to obtain labeled antibody protein Ab*; then purification was performed to remove unbound dye, the labeled antibody protein Ab* solution was collected, and the labeled antibody protein Ab* was quantified; S2. Construction of the AT standard curve of abrinogen toxin: Prepare a series of gradient AT standards with sample buffer, and mix them with equal volumes of a fixed concentration of excess labeled antibody protein Ab* and incubate at room temperature: Ab*(excess) + Ag → Ab* + [Ab*-Ag], to obtain the complex [Ab*-Ag] and excess labeled antibody protein Ab*. The reaction solution was separated into different components using capillary electrophoresis (CE). The capillary was a fused silica capillary with an inner diameter of 50–100 μm, a total length of 30–50 cm, and an effective LIF window length of 10–30 cm. The separation buffer consisted of sample buffer with added polyethylene glycol 20000 at a concentration of 10–60 g / L. The separation voltage was 5–20 kV, the capillary temperature was 10–30 °C, and the injection pressure was 0.3–0.6 psi for 5–15 s. The components were detected using a laser-induced fluorescence (LIF) detector to determine the migration time of the complex component [Ab*-Ag] in electrophoresis; an AT standard curve was plotted with AT concentration on the x-axis and fluorescence signal peak area on the y-axis. The detection wavelength range for fluorescence is 460–500 nm; S3. Sample detection: Replace the AT standard with the sample to be tested, mix it with the labeled antibody protein Ab* using the same method as in step S2, incubate it, and then detect it.
2. The capillary electrophoretic fluorescence immunoassay method for detecting abrin toxin according to claim 1, characterized in that, If step S3 is a qualitative detection, a fluorescence signal peak appears at the same location as the migration time of the complex, proving that the sample to be tested contains AT, and is judged as a positive sample.
3. The capillary electrophoretic fluorescence immunoassay method for detecting abrin toxin according to claim 1, characterized in that, If step S3 is a quantitative detection, the peak area of the fluorescence signal that appears at the same location as the complex migration time will be used to calculate the AT content in the analyte by substituting the peak area into the standard curve equation obtained in step S2.
4. The capillary electrophoretic fluorescence immunoassay method for detecting abrin toxin according to claim 1, characterized in that, In step S1, the antibody protein is labeled: 0.2 to 1 mL of antibody with a concentration of 1 to 3 mg / mL is taken, and 100 μL of 1 mg FITC dye powder is added to prepare a 10 mg / mL FITC solution.
5. The capillary electrophoretic fluorescence immunoassay method for detecting abrin toxin according to claim 1, characterized in that, The sample buffer is TG buffer (pH 7.4), where TG buffer represents Tris-glycine buffer: 25 mM Tris base, 192 mM glycine.
6. The capillary electrophoretic fluorescence immunoassay method for detecting abrin toxin according to claim 1, characterized in that, The capillary electrophoresis parameters are as follows: the capillary is a 75 μm inner diameter fused silica capillary, with a total length of 41 cm and an effective LIF window length of 20 cm. Separation buffer: 2×TG (pH 9.0) + 40 g / L PEG20000 (polyethylene glycol 20000); sample buffer: TG (pH 7.4); separation voltage: 10 kV; capillary temperature: 20 °C; injection pressure: 0.5 psi for 8 s.
7. The capillary electrophoretic fluorescence immunoassay method for detecting abrin toxin according to claim 1, characterized in that, The labeled antibody protein Ab* is a mouse anti-AT monoclonal antibody.
8. The capillary electrophoretic fluorescence immunoassay method for detecting abrin toxin according to claim 1, characterized in that, The labeled antibody protein Ab* should be stored away from light.
9. The capillary electrophoretic fluorescence immunoassay method for detecting abrin toxin according to claim 1, characterized in that, The detection limit of the test sample is 75.8 nM.
10. The capillary electrophoretic fluorescence immunoassay method for detecting abrin toxin according to claim 1, characterized in that, The detection fluorescence wavelength range is 480 nm.