Aflatoxin b1 detection kit and its preparation method and application

Abstract

This invention relates to a detection kit for aflatoxin B1, its preparation method, and its application, belonging to the field of hazardous substance detection technology. The aflatoxin B1 detection kit provided by this invention includes a biotin-labeled aflatoxin B1 hydroxylated hapten, an acrid ester-labeled aflatoxin B1 monoclonal antibody, streptavidin magnetic beads, calibrators, a luminescent exciter, a washing solution, and a phosphate buffer mixture. This invention achieves ultrasensitive and rapid detection of aflatoxin B1 by constructing a biotin-streptavidin signal amplification system combined with the highly efficient separation characteristics of magnetic particles.

Description

Technical Field

[0001] This invention relates to the field of hazardous substance detection technology, specifically to a detection kit for aflatoxin B1, its preparation method, and its application. Background Technology

[0002] Aflatoxin B1 (AFB1), one of the most potent mycotoxins, is widely present in the food supply chain, posing a serious threat to public health. Among current detection methods, high-performance liquid chromatography-fluorescence assay (HPLC-FICA) is accurate but complex, traditional enzyme-linked immunosorbent assay (ELISA) lacks sufficient sensitivity, and conventional chemiluminescence assays are insufficient for trace detection due to limited signal intensity. Magnetic microparticle chemiluminescence technology has become a research hotspot due to its rapid magnetic separation and high chemiluminescence sensitivity. However, existing technologies suffer from two main drawbacks: firstly, insufficient signal amplification mechanisms make it difficult to break through the detection limit of 0.05 μg / kg; secondly, poor label stability, with the binding efficiency of biotin and streptavidin easily affected by matrix interference. Therefore, developing an AFB1 detection technology that combines ultrasensitive detection capability with interference resistance is of great significance. Summary of the Invention

[0003] To address the aforementioned technical problems, the present invention aims to provide a detection kit for aflatoxin B1, its preparation method, and its applications. This invention achieves ultrasensitive and rapid detection of aflatoxin B1 by constructing a biotin-streptavidin signal amplification system and combining it with the highly efficient separation characteristics of magnetic microparticles.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The first objective of this invention is to provide a detection kit for aflatoxin B1, comprising a biotin-labeled aflatoxin B1 hydroxylated hapten, acridinium-labeled aflatoxin B1 monoclonal antibody, streptavidin magnetic beads, calibrators, a luminescent exciter, a washing solution, and a phosphate buffer mixture.

[0005] The beneficial effects of this invention are: This invention aims to address the problems of insufficient sensitivity and poor anti-interference in existing aflatoxin B1 (AFB1) detection methods, and provides a chemiluminescent detection kit based on acridinium ester-biotin-labeled magnetic microparticles. By constructing a "biotin-streptavidin" signal amplification system and combining it with the efficient separation characteristics of magnetic microparticles, ultrasensitive and rapid detection of AFB1 can be achieved.

[0006] (1) Ultrasensitive detection: Through the “biotin-streptavidin” signal amplification system, the detection limit is as low as 0.0002 μg / kg, which is 5-10 times higher than the existing methods, meeting the needs of trace pollution detection. (2) Fast and efficient: The entire detection process takes only 12 minutes, which is much shorter than high performance liquid chromatography (2-3 hours), making it suitable for batch sample detection. (3) Strong anti-interference: The immunoaffinity column purifies and binds specific antibodies, and the cross-reactivity rate of structural analogs such as aflatoxin G1 and M1 is <1%, and the matrix effect is reduced to below 5%. (4) High stability: The reagent kit components have been optimized for storage conditions, and the shelf life is up to 18 months, which is more than 6 months longer than similar products. (5) Wide applicability: It is suitable for rapid batch detection of AFB1 in various samples such as grains, nuts, beans, oils, and special diets. The linear range covers 0.0001-100μg / kg, which can meet the limit detection in the national standard and achieve the trace detection requirements with ultra-low detection limit. Based on the above technical solution, the present invention can be further improved as follows.

[0007] Furthermore, the aflatoxin B1 hydroxylated hapten in the biotin-labeled aflatoxin B1 hydroxylated hapten is obtained by hydroxylating the aflatoxin hapten.

[0008] Furthermore, the preparation method of the aflatoxin B1 hydroxylated hapten is as follows: (1) Weigh out AFB1 protoxin, slowly add anhydrous dimethylformamide (DMF), stir magnetically for 15 minutes, and prepare AFB1 solution; (2) Slowly add the oxidizing agent solution to the AFB1 solution while stirring; then stir the reaction in the dark. (3) After the reaction is complete, methanol is added immediately and the mixture is stirred gently to terminate the oxidation reaction and obtain the reaction mixture; (4) The reaction mixture was subjected to column chromatography and elution in sequence, and finally the target peak was collected by ultraviolet detector to obtain AFB1 hydroxylated hapten.

[0009] Furthermore, the concentration of the AFB1 solution in step (1) is 2 mg / mL.

[0010] Furthermore, in step (2), the molar ratio of AFB1 in the AFB1 solution to the oxidant is 1:0.8; the oxidant is m-chloroperoxybenzoic acid.

[0011] Furthermore, the environment for the light-protected stirring reaction in step (2) is a constant temperature environment of 25°C for 3 hours.

[0012] Furthermore, in step (3), the amount of methanol added is 1 / 10 of the system volume, and the stirring time is 3 min.

[0013] Furthermore, the chromatography column mentioned in step (4) is a Sephadex G-10 chromatography column; the eluent is 50% methanol; The ultraviolet detector is a 280 nm ultraviolet spectrophotometer.

[0014] The beneficial effects of adopting the above-mentioned further scheme are: hydroxylation provides a stable binding site for the hapten, which can efficiently bind biotin, achieve efficient signal amplification, and greatly improve detection sensitivity.

[0015] Furthermore, the biotin labeling method for the biotin-labeled aflatoxin B1 hydroxylated hapten is as follows: (1) Select an ultrafiltration tube and soak it in ultrapure water. The water volume is just enough to pass through the membrane. After pouring out the water, add the hapten to the ultrafiltration tube and fill it with PBS buffer to the white line at the top of the tube. Centrifuge and collect the filtrate. Wash with PBS buffer and recover the hapten. The recovered hapten is then diluted with PBS buffer from the filtrate. The concentration of the hapten after dilution is in the range of 1 mg / mL to 2 mg / mL. (2) After taking biotin out of the refrigerator, allow it to fully equilibrate to room temperature and set aside for use. Add ultrapure water to dissolve it before use. Add biotin working solution to the treated hapten solution according to the formula (Biotin working solution added volume (μL) = treated hapten mass (1mg) × 13.33), mix thoroughly, and let stand at room temperature to obtain biotin-labeled hydroxylated hapten.

[0016] The beneficial effects of adopting the above-mentioned further scheme are: by adding a protective agent, the specificity and stability are significantly improved, and it can be stable for 18 months under storage conditions of 2℃-8℃.

[0017] Furthermore, the chemical formula of the aflatoxin B1 hydroxylated hapten in the biotin-labeled aflatoxin B1 hydroxylated hapten is C. 18 H 14 O 10 The structural formula is shown in equation (1): (1).

[0018] Furthermore, the preparation method of the acridinium ester-labeled aflatoxin B1 monoclonal antibody is as follows: (1) Select an ultrafiltration tube, soak it in ultrapure water before use, pour out the water and add antibody to the ultrafiltration tube, fill the tube with CB solution to the white line at the top of the tube, centrifuge at 12000 rpm and 4℃ for 10 min, collect the filtrate, wash with CB solution, recover the antibody, and make up the volume of the recovered antibody with CB solution of the filtrate. The antibody concentration after volume adjustment is 1 mg / mL to 2 mg / mL. (2) Add 2 mg / mL of acridine ester working solution to the treated antibody solution according to the formula (acridine ester volume (mL) = antibody mass (mg) / acridine ester concentration (mg / mL) × 22.8), mix thoroughly, and let stand at room temperature. Add 1% (w / v) bovine serum albumin to obtain acridine ester-labeled aflatoxin B1 monoclonal antibody.

[0019] Furthermore, the calibrator is an aflatoxin B1 standard solution; the concentration of the aflatoxin B1 standard solution includes 0 μg / L, 0.002 μg / L, 0.02 μg / L, 0.2 μg / L, 2 μg / L, and 20 μg / L.

[0020] Furthermore, the luminescence exciter includes an excitation solution and a pre-excitation solution; the excitation solution is a 0.03 mol / L to 0.07 mol / L H2O2 solution; the pre-excitation solution is a 0.03 mol / L to 0.07 mol / L NaOH solution.

[0021] Furthermore, the concentration of the streptavidin magnetic beads is 0.23 mg / mL to 0.27 mg / mL; the particle size of the streptavidin magnetic beads is 50 nm to 200 nm.

[0022] The beneficial effects of adopting the above-mentioned further scheme are: the addition of the protective agent bovine serum albumin significantly improves stability, and it can be stable for 18 months under storage conditions of 2℃-8℃. Magnetic microparticles of 50nm~200nm have the optimal specific surface area, which can ensure an antibody conjugation density of 20μg / mg and significantly improve reaction efficiency.

[0023] Furthermore, the washing solution includes Tween 20 and Tris-HCl buffer; the volume ratio of Tween 20 to Tris-HCl buffer is 2:98 to 4:96.

[0024] The second objective of this invention is to provide an application of a detection kit for aflatoxin B1, which is used for the ultrasensitive and rapid detection of aflatoxin B1.

[0025] The third objective of this invention is to provide an ultrasensitive and rapid detection method for aflatoxin B1, comprising the following steps: (1) After crushing the sample to be tested, add phosphate buffer solution, and then vortex, ultrasonically extract, and centrifuge to obtain the sample test solution. (2) Take the sample test solution, add streptavidin magnetic beads, biotin-labeled aflatoxin B1 hydroxylated hapten solution, and acridine ester-labeled aflatoxin B1 monoclonal antibody solution, mix well and incubate to obtain the reaction mixture; (3) The reaction mixture is placed in a magnetic field for adsorption, then the supernatant is discarded and washed with washing liquid, then an excitation liquid is added, and the relative luminescence intensity of the sample to be tested is measured; a standard curve is plotted with the concentration of calibrator as the abscissa and the relative luminescence intensity of the calibrator as the ordinate, and the content of aflatoxin B1 in the sample to be tested is calculated based on the relative luminescence intensity of the sample to be tested.

[0026] The beneficial effects of the present invention are: (1) Using acetonitrile-phosphate buffer mixed extractant, combined with ultrasonic-assisted extraction, can efficiently destroy the sample matrix structure and improve the extraction rate of AFB1; the immunoaffinity column purification can specifically adsorb AFB1 and remove interfering substances such as lipids and pigments, and the purification efficiency is more than 40% higher than that of traditional solid phase extraction.

[0027] (2) Acridinium ester-labeled monoclonal antibodies can simultaneously bind to AFB1 and biotin-labeled AFB1 hapten in the sample, forming two complexes: "acridinium ester antibody-AFB1" and "acridinium ester antibody-biotin AFB1". The binding amounts of the two complexes exhibit a competitive inhibitory relationship. The "acridinium ester antibody-biotin AFB1" complex is retained by the rapid binding of biotin to streptavidin on the surface of streptomycin-affinity magnetic beads. The "acridinium ester antibody-AFB1" complex is removed after washing. Magnetic microparticles with a specific surface area of ​​50 nm to 200 nm have the optimal surface area, ensuring an antibody conjugation density of up to 20 μg / mg, significantly improving reaction efficiency. (3) The binding constant between streptavidin and biotin is as high as 10. 15 L / mol is 10⁻⁶ for antigen-antibody binding reactions. 6 -10 8 Each streptavidin molecule can bind to four biotin molecules, which can amplify the signal by 5-10 times through this reaction, greatly improving the detection sensitivity.

[0028] (4) Strong magnetic field is used to quickly adsorb magnetic particles (separation is completed within 20 seconds). The washing liquid can effectively remove non-specific binding substances and reduce the background signal by more than 30%. (5) Acridinium ester emits light instantaneously in an alkaline hydrogen peroxide system (light emission peak < 2 seconds), and the relative light emission intensity is negatively correlated with the AFB1 concentration. Quantitative analysis is achieved through a standard curve.

[0029] Furthermore, the incubation temperature in step (2) is 36℃~38℃, and the incubation time is 5~7min. Attached Figure Description

[0030] Figure 1 The standard curves are for Examples 4 and 5. Detailed Implementation

[0031] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0032] Laboratory instruments and consumables: Experimental reagents: Example 1: Preparation of aflatoxin B1 hydroxylated hapten: (1) Weigh 2 mg of AFB1 protoxin, slowly add anhydrous dimethylformamide (DMF), stir magnetically for 15 minutes, and prepare a 2 mg / mL AFB1 solution; (2) Add the m-chloroperoxybenzoic acid solution slowly dropwise at a molar ratio of 1:0.8 between AFB1 and m-chloroperoxybenzoic acid, stirring continuously. Then place the mixture in a constant temperature environment of 25°C and stir for 3 hours in the dark. (3) After the reaction is complete, immediately add 1 / 10 of the system volume of methanol, stir gently for 3 minutes to terminate the oxidation reaction and obtain the reaction mixture; (4) A Sephadex G-10 chromatography column was selected, with 50% methanol as the eluent. After loading the reaction solution, the solution was eluted at a constant speed, and the target peak was collected by a 280 nm UV detector to obtain the aflatoxin B1 hydroxylated hapten, namely the AFB1 hydroxylated hapten.

[0033] Preparation method of biotin-labeled aflatoxin B1 hydroxylated hapten: (1) Select a new, dry 10KD ultrafiltration tube. Before use, soak it in ultrapure water for 5 minutes. The water volume should be just enough to pass through the membrane. After pouring out the water, add 2 mg of AFB1 hydroxylated hapten to the ultrafiltration tube. Add PBS buffer to the white line at the top of the tube. Centrifuge at 12000 rpm and 4℃ for 10 min. Collect the filtrate. Wash it three times with PBS buffer to recover the AFB1 hydroxylated hapten. Use NanoDrop One to measure the concentration of AFB1 hydroxylated hapten, calculate the mass and recovery rate (calculation formula: AFB1 hydroxylated hapten mass (mg) = AFB1 hydroxylated hapten concentration (mg / mL) × solution volume (mL); recovery rate = mass of AFB1 hydroxylated hapten after recovery / mass of AFB1 hydroxylated hapten before recovery × 100%). The recovery rate is greater than 85%. The recovered AFB1 hydroxylated hapten is adjusted to volume with PBS buffer of the filtrate. After adjustment, the concentration of AFB1 hydroxylated hapten is in the range of 1 mg / mL to 2 mg / mL. (2) Take out one vial of biotin (10 mg / vial) from the refrigerator, and after it has been completely equilibrated to room temperature, add 180 μL of ultrapure water. It must be dissolved immediately before use. According to the formula (Biotin working solution added volume (mL) = mass of treated AFB1 hydroxylated hapten (mg) / biotin concentration (mg / mL) × 13.3 = (1 mg) × 13.33), add 20 μL of Biotin working solution to the treated 1.5 mg antibody solution, mix thoroughly, and let stand at room temperature for 60 min to obtain the biotin-labeled aflatoxin B1 hydroxylated hapten. The structure of the biotin-labeled aflatoxin B1 hydroxylated hapten was obtained by MS and NMR detection using the shared equipment platform of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, as shown in formula (1): Example 2: Preparation of acridinium ester-labeled aflatoxin B1 monoclonal antibody: (1) Select a new, dry 100KD ultrafiltration tube. Before use, soak it in ultrapure water for 5 minutes. The water volume should be just enough to pass through the membrane. After pouring out the water, add 2mg of aflatoxin B1 monoclonal antibody to the ultrafiltration tube. Add CB buffer to the white line at the top of the tube. Centrifuge at 12000rpm and 4℃ for 10min. Collect the filtrate. Wash it three times with CB buffer to recover the antibody. Use NanoDropOne to measure the antibody concentration, calculate the mass and recovery rate (calculation formula: mass of aflatoxin B1 monoclonal antibody (mg) = concentration of aflatoxin B1 monoclonal antibody (mg / mL) × solution volume (mL); recovery rate = mass of aflatoxin B1 monoclonal antibody after recovery / mass of aflatoxin B1 monoclonal antibody before recovery × 100%). The antibody recovery rate is greater than 85%. The recovered antibody is adjusted to volume with CB buffer of the filtrate. The antibody concentration after adjustment is 1mg / mL~2mg / mL. (2) According to the formula (acridine ester volume = treated antibody mass (mg) / acridine ester concentration (mg / mL) × 22.8), add 45.6 μL of 2 mg / mL acridine ester working solution to the treated 2 mg antibody solution, mix thoroughly, and let stand at room temperature for 60 min to obtain acridine ester-labeled aflatoxin B1 monoclonal antibody.

[0034] Example 3: Aflatoxin B1 Detection Kit This example describes a detection kit for aflatoxin B1, comprising the following components: (1) Biotin-labeled aflatoxin B1 hydroxylated hapten prepared in Example 1: concentration was 0.2 μg / mL; (2) Acridinium ester-labeled aflatoxin B1 monoclonal antibody prepared in Example 2: concentration of 0.2 μg / mL.

[0035] (3) Streptavidin magnetic beads: concentration 0.25 mg / mL, magnetic bead size 50 nm~200 nm; (4) Calibrators: Aflatoxin B1 standard solutions of 6 concentrations (0 μg / L, 0.002 μg / L, 0.02 μg / L, 0.2 μg / L, 2 μg / L, 20 μg / L); (5) Washing solution: 0.02 mol / L Tris-HCl buffer, pH 7.8, containing 0.03% (V / V) Tween 20; (6) Activation solution: 0.05 mol / L H2O2 solution; (7) Pre-activation solution: 0.15 mol / L NaOH solution.

[0036] Comparative Example 1: Aflatoxin B1 Detection Kit This comparative kit for detecting aflatoxin B1 includes the following components: (1) Biotin-labeled aflatoxin B1 hydroxylated hapten prepared in Example 1: concentration was 0.2 μg / mL; (2) Acridinium ester-labeled aflatoxin B1 monoclonal antibody prepared in Example 2: concentration was 0.2 μg / mL; (3) Streptavidin magnetic beads: concentration 0.25 mg / mL, magnetic bead size 50 nm~200 nm; (4) Calibrators: Aflatoxin B1 standard solutions of 6 concentrations (0 μg / L, 0.002 μg / L, 0.02 μg / L, 0.2 μg / L, 2 μg / L, 20 μg / L); (5) Washing solution: 0.02 mol / L Tris-HCl buffer solution with a pH of 7.8; (6) Activation solution: 0.05 mol / L H2O2 solution; (7) Pre-activation solution: 0.15 mol / L NaOH solution.

[0037] Comparative Example 2: Aflatoxin B1 Detection Kit This comparative kit for detecting aflatoxin B1 includes the following components: (1) Biotin-labeled aflatoxin B1 hydroxylated hapten prepared in Example 1: concentration was 0.2 μg / mL; (2) Acridinium ester-labeled aflatoxin B1 monoclonal antibody prepared in Example 2: concentration of 0.2 μg / mL.

[0038] (3) Streptavidin magnetic beads: concentration 0.25 mg / mL, containing 0.1% (W / V) bovine serum albumin; magnetic bead particle size 1.5 μm; (4) Calibrators: Aflatoxin B1 standard solutions of 6 concentrations (0 μg / L, 0.002 μg / L, 0.02 μg / L, 0.2 μg / L, 2 μg / L, 20 μg / L); (5) Washing solution: 0.02 mol / L Tris-HCl buffer solution with a pH of 7.8; (6) Activation solution: 0.05 mol / L H2O2 solution; (7) Pre-activation solution: 0.15 mol / L NaOH solution.

[0039] Comparative Example 3: Aflatoxin B1 Detection Kit This comparative kit for detecting aflatoxin B1 includes the following components: (1) Biotin-labeled aflatoxin B1 hydroxylated hapten prepared in Example 1: concentration was 0.2 μg / mL; (2) Acridinium ester-labeled aflatoxin B1 monoclonal antibody prepared in Example 2: concentration was 2 μg / mL; (3) Streptavidin magnetic beads: concentration 0.25 mg / mL, containing 0.1% (W / V) bovine serum albumin; magnetic bead particle size 50 nm~200 nm; (4) Calibrators: Aflatoxin B1 standard solutions of 6 concentrations (0 μg / L, 0.002 μg / L, 0.02 μg / L, 0.2 μg / L, 2 μg / L, 20 μg / L); (5) Washing solution: 0.02 mol / L Tris-HCl buffer solution with a pH of 7.8; (6) Activation solution: 0.05 mol / L H2O2 solution; (7) Pre-activation solution: 0.15 mol / L NaOH solution.

[0040] The limits of detection, limits of quantitation, detection sensitivity, specificity, repeatability (n=6, RSD%), and sample spike recovery rates of the detection kits of Examples 3 and Comparative Examples 1-3 are shown in Table 1. Table 1 From Table 1, we can obtain: The test kit of Example 3 was superior to the test kits of Comparative Examples 1-3 in terms of sensitivity, precision and accuracy. The test kit of Example 3 was finally determined to be the optimal formulation.

[0041] Example 4: Application of the detection kit This embodiment uses the detection kit from Example 3 to detect aflatoxin B1 in peanuts. The specific steps of the detection are as follows: (1) Sample pretreatment: Take 2g of crushed peanut sample, add 25mL of acetonitrile-0.1mol / L phosphate buffer (volume ratio of 5:1), vortex for 3 minutes, extract by ultrasonication at 300W for 8 minutes, centrifuge at 12000r / min and 4℃ for 4 minutes, take 5mL of supernatant and pass it through an AFB1 immunoaffinity column, rinse with 10mL of water, elute with 5mL of methanol, dry the eluent with nitrogen, and reconstitute with 1mL of PBS to obtain the sample test solution; (2) Competitive binding: Take 20 μL of the test solution, add 20 μL of 0.25 mg / mL streptavidin magnetic beads, 50 μL of 0.2 mg / mL biotin-labeled hydroxylated hapten, and 50 μL of 0.2 mg / mL acridine ester-labeled aflatoxin B1 monoclonal antibody, and incubate at 37°C for 6 minutes; (3) Magnetic separation and washing: Adsorbed by magnetic field for 20 seconds, discard the supernatant, and wash 4 times with washing solution; (4) Luminescence detection: Add 50 μL of 0.05 mol / L H2O2 solution as excitation solution and 50 μL of 0.15 mol / L NaOH solution as pre-excitation solution, and measure the RLU value; (5) Result calculation: With the standard concentration as the x-axis and the RLU value as the y-axis, substitute into the 4PLC calibration algorithm formula: y=(ad) / (1+(x / c)) b The sample RLU value was 1312469, and the calculated AFB1 concentration was 0.0034 μg / L. Substituting the dilution factor and recovery rate, the AFB1 content was calculated to be 0.0094 μg / kg. Where: y is the measured emission value (RLU), x is the analyte concentration, a is the maximum emission value at zero concentration, b is the curve slope / steepness factor, and c is the IC50 value. 50 Half-suppression concentration, d is: the maximum suppression of background luminescence value.

[0042] The detection steps and result calculation process of (2)-(5) are all performed automatically in the fully automated chemiluminescence immunoassay analyzer (CY968, purchased from Tianjin Ceyi Biotechnology Co., Ltd.).

[0043] Example 5: Application of the Detection Kit (Part 2) This embodiment is the same as embodiment 4, except that the sample in step (1) is an oil sample. The remaining steps and the raw materials, amounts, and other conditions used in the steps are the same as in embodiment 4. The specific steps are as follows: (1) Sample pretreatment: Take 1g of oil sample, add 10mL of acetonitrile, vortex for 3 minutes, extract by ultrasonication at 350W for 5 minutes, centrifuge at 12000r / min for 4 minutes, take 5mL of supernatant and pass it through an AFB1 immunoaffinity column, rinse with 10mL of water, elute with 5mL of methanol, dry the eluent with nitrogen and redissolve with 1mL of PBS to obtain the sample test solution; (2) Competitive binding: Take 20 μL of the test solution, add 20 μL of 0.25 mg / mL streptavidin magnetic beads, 50 μL of 0.2 mg / mL biotin-labeled hydroxylated hapten, and 50 μL of 0.2 mg / mL acridine ester-labeled aflatoxin B1 monoclonal antibody, and incubate at 37°C for 6 minutes; (3) Magnetic separation and washing: Adsorbed by magnetic field for 20 seconds, discard the supernatant, and wash 4 times with washing solution; (4) Detection of luminescence value: Add 50 μL of 0.05 mol / L H2O2 solution as the excitation solution and 50 μL of 0.15 mol / L NaOH solution as the pre-excitation solution, and measure the RLU value; (5) Result calculation: With the standard concentration as the x-axis and the RLU value as the y-axis, substitute into the 4PLC calibration algorithm formula: y=(ad) / (1+(x / c)) b The sample RLU value was 373235, which, when substituted into the formula, yielded an AFB1 concentration of 0.82 μg / L. Substituting the dilution factor and recovery rate, the AFB1 content was calculated to be 1.64 μg / kg.

[0044] The detection steps and result calculation process of (2)-(5) are all performed automatically in the fully automated chemiluminescence immunoassay analyzer (CY968, purchased from Tianjin Ceyi Biotechnology Co., Ltd.).

[0045] The calibration curves for Examples 4 and 5 are as follows: Figure 1 As shown.

[0046] Performance verification: The sensitivity, precision, stability, and specificity were validated using a fully automated chemiluminescence immunoassay analyzer (CY968, purchased from Tianjin Ceyi Biotechnology Co., Ltd.). The specific steps are as follows:

[0047] 1. Sensitivity: (1) Limit of detection: Using a blank matrix as a sample, the detection was repeated 20 times. The mean (Mean_) and standard deviation (SD) of the luminescence value (RLU) of the 20 detection results were calculated.

[0048] Calculation method: Limit of detection = Mean_blank + 2 × SD_. Substitute the corresponding RLU value into the calibration curve to obtain the corresponding concentration value, which is the limit of detection.

[0049] (2) Limit of Quantitation (LOQ): Prepare a low-concentration sample with a concentration close to the expected LOQ (e.g., a sample with a concentration 3-5 times the LOD). Repeat the test on the sample 20 times and calculate the mean and standard deviation (SD) of the concentration.

[0050] Calculation method: The LOQ should satisfy the condition that the total error (Bias% + 2 × CV%) ≤ 20%. If the calculated CV% ≤ 20%, then the concentration can be determined as the limit of quantitation of the method.

[0051] 2. Precision (1) Intra-batch precision: Select two quality control samples (or samples) with low and high concentrations. Within the same batch, repeat the test 20 times for each concentration level. Calculate the average concentration and standard deviation (SD) of the 20 test results for the low and high value samples respectively, and then calculate the coefficient of variation (CV%).

[0052] (2) Inter-batch precision: Two quality control samples with low and high concentrations were selected. Three batches of test kits with different batch numbers were selected, and each batch was tested five times for each concentration level. At least 15 data points were obtained for each quality control sample. The mean and standard deviation of each set of data were calculated, and then the average CV% was calculated as the inter-batch precision.

[0053] 3. Stability: Three batches of reagent kits from different batches were subjected to accelerated degradation in a 37°C incubator. The same calibrators and quality control samples were used for testing at 0 days (baseline), 1 day, 3 days, and 7 days. The change (%) of the results at each time point relative to 0 days was calculated using the results at 0 days as the baseline.

[0054] Change (%) = |(Result on Day N - Result on Day 0)| / Result on Day 0 × 100% 4. Specificity: Prepare solutions of potential cross-reactive substances (30 positive samples each of aflatoxin G1, M1, and ochratoxin A), and use CY968 to detect these samples. Calculate the cross-reactivity rate (%): Cross-reactivity rate = (number of aflatoxin-positive samples / total number of samples) × 100%.

[0055] The test results are shown in Table 2: Table 2 In summary, this invention significantly improves the sensitivity and efficiency of AFB1 detection through innovative labeling strategies and reaction systems, providing strong technical support for food safety supervision.

[0056] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A test kit for aflatoxin B1, characterized in that, The mixture includes biotin-labeled aflatoxin B1 hydroxylated hapten, acridine ester-labeled aflatoxin B1 monoclonal antibody, streptavidin magnetic beads, calibrators, luminescent exciter, washing solution, and phosphate buffer solution.

2. The aflatoxin B1 detection kit according to claim 1, characterized in that, The chemical formula of the aflatoxin B1 hydroxylated hapten in the biotin-labeled aflatoxin B1 hydroxylated hapten is C. 18 H 14 O 10 The structural formula is shown in equation (1): （1）。 3. The aflatoxin B1 detection kit according to claim 1, characterized in that, The calibrator is an aflatoxin B1 standard solution; the concentrations of the aflatoxin B1 standard solution include 0 μg / L, 0.002 μg / L, 0.02 μg / L, 0.2 μg / L, 2 μg / L, and 20 μg / L.

4. The aflatoxin B1 detection kit according to claim 1, characterized in that, The luminescent exciter includes an excitation solution and a pre-excitation solution.

5. The aflatoxin B1 detection kit according to claim 4, characterized in that, The activating solution is a 0.03 mol / L to 0.07 mol / L H2O2 solution; the pre-activating solution is a 0.03 mol / L to 0.07 mol / L NaOH solution.

6. The aflatoxin B1 detection kit according to claim 1, characterized in that, The concentration of the streptavidin magnetic beads is 0.23 mg / mL to 0.27 mg / mL; the particle size of the streptavidin magnetic beads is 50 nm to 200 nm.

7. The aflatoxin B1 detection kit according to claim 1, characterized in that, The washing solution includes Tween 20 and Tris-HCl buffer; the volume ratio of Tween 20 to Tris-HCl buffer is 2:98 to 4:

96.

8. The application of a test kit for aflatoxin B1, characterized in that, The aflatoxin B1 detection kit according to any one of claims 1 to 7 is used for the ultrasensitive rapid detection of aflatoxin B1.

9. A highly sensitive and rapid detection method for aflatoxin B1, characterized in that, The detection method includes the following steps: (1) After crushing the sample to be tested, add phosphate buffer solution, and then vortex, ultrasonically extract, and centrifuge to obtain the sample test solution. (2) Take the sample test solution, add streptavidin magnetic beads, biotin-labeled aflatoxin B1 hydroxylated hapten solution, and acridine ester-labeled aflatoxin B1 monoclonal antibody solution and incubate to obtain a reaction mixture; (3) The reaction mixture is placed in a magnetic field for adsorption, then the supernatant is discarded and washed with washing liquid. Then, pre-excitation liquid and excitation liquid are added, and the relative luminescence intensity of the sample to be tested is measured. A standard curve is plotted with the concentration of calibrator as the abscissa and the relative luminescence intensity of the calibrator as the ordinate. The content of aflatoxin B1 in the sample to be tested is calculated based on the relative luminescence intensity of the sample to be tested.

10. The detection method of the aflatoxin B1 detection kit according to claim 9, characterized in that, The incubation temperature in step (2) is 36℃~38℃, and the incubation time is 5min~7min.

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