CN111273013A - Aflatoxin B based on immunomagnetic beads1Method of measurement of - Google Patents
Aflatoxin B based on immunomagnetic beads1Method of measurement of Download PDFInfo
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Images
Classifications
-
- 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; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/531—Production of immunochemical test materials
Abstract
The invention belongs to the technical field of aflatoxin determination methods, and provides an aflatoxin B based on immunomagnetic beads1The method of (1). The determination method comprises the steps of activating magnetic beads, modifying magnetic beads with antigens, preparing an immunomagnetic bead system, detecting by flow cytometry and the like. The aflatoxin B based on immunomagnetic beads provided by the invention1The determination method has the advantages of simple and conventional pretreatment method, clear operation, high instrument automation degree, sensitive and accurate analysis, and capability of obtaining a large amount of detection results in real time based on a flow cytometer and rapidly detecting aflatoxin B1The detection limit of the concentration is low, and the detection limit can reach 0.2034-0.3107 mu g/L.
Description
Technical Field
The invention relates to the technical field of aflatoxin determination methods, in particular to aflatoxin B based on immunomagnetic beads1The method of (1).
Background
Aflatoxins (AFT) are a class of toxins produced by aspergillus flavus and aspergillus parasiticus and are a wide range of derivatives. Wherein the aflatoxin B is used1(AFB1) The toxicity is the greatest and the carcinogenicity is the strongest. In recent years, research shows that AFB1Is a causative agent of hepatocellular carcinoma, growth suppression, immune system regulation, and malnutrition. It has high toxicity to humans and animals, corresponding to 10 times that of potassium cyanide. Furthermore, it can also be transferred between organisms through the food chain, thereby constituting a great threat to the metabolic cycle. If pregnant and lactating women contain aflatoxin metabolites or biomarkers in breast milk, serum or cord blood, this will result in a reduction in human fertility.
AFB1And the producing bacteria thereof are widely present in various polluted foods and feeds, and grain and oil agricultural products are the main pollution targets. Crops and products thereof are easy to be threatened by toxins, and after the crops and the products are polluted by fungi, the yield and the quality of agricultural products can be greatly reduced, so that serious economic loss is caused. With the coming of global economy, agricultural product trade is taken as an important link of global trade, and the attention of countries in the world is increasing day by day. As a big agricultural country, the export of agricultural products and related economic crops after pollution is limited, which seriously affects the survival condition of agricultural producers and the healthy development of national economy in China. Thus, determination and quantification of AFB1Has become a key control point for food safety, medical inspection and import and export inspection and quarantine diagnosis.
For aflatoxin B1Is known to be the most commonly used and effective method, liquid chromatography/mass spectrometry is generally considered to be the most effective method. However, the method is limited by a long analysis process, a complicated pretreatment process, difficult operation, and expensive equipment. Immunological tests, e.g. against AFB1The enzyme-linked immunosorbent assay kit (ELISA) is included and accepted by national standard regulations in China in recent years. Despite the implementation of AFB1The basic detection method based on optical colorimetry, which is sometimes affected by low sensitivity due to the irregularity in production of various kits, needs to be further developedThe research is more deeply and perfectly carried out. Non-destructive testing based on optical methods has also been a new research focus in the field of testing in recent years, but it requires skilled operators and complex algorithmic analysis procedures.
A flow cytometer (Flowcytometer) is a new type of high-end instrument that integrates multiple subjects and technologies such as fluidics, optics, immunology, electronic informatics, etc. The key technical point of the method is Flow Cytometry (FCM). The flow cytometry is to disperse single cells or other measured micro-particles into a fast and straight flowing single particle liquid flow stream through a unique liquid flow system of a flow cytometer; the detection technology is characterized in that single cells or other detected microparticles are subjected to multi-parameter quantitative analysis and sorting through preset light beam excitation. Has the outstanding advantages of single-particle level rapidness, automation, high efficiency, sensitivity, accuracy, convenient operation and the like.
Magnetic Beads (MBs) are immunomagnetic beads, spherical magnetic particles modified with an immunopigand (antigen or antibody) on the surface, and can be sensitively and specifically bound to a target substance to form a complex with a magnetic response. Since first invented in 1979 by Ugelstar et al, this technique triggered the application of a hot tide in the field of bioseparation. Along with the rise of nanotechnology, the particle size of magnetic beads is ascending in nanometer scale, the application range is further widened, and the universality development is realized, and the trend is comprehensive and deep. Magnetic beads have been favored by researchers as excellent solid phase carriers due to their high biocompatibility, good magnetic responsiveness, and low cost characteristics. The immunomagnetic bead separation technology is a separation technology applying the immunomagnetic property and the magnetic response property of magnetic beads, and has the characteristics of rapid separation and strong specificity. In the development of more than 30 years, the method has been widely applied to separating and concentrating substances such as proteins and nucleic acid fragments of specific cells or microorganisms, which are difficult to observe by naked eyes or have low content in samples, and has strong anti-interference performance to complex matrix environment.
At present, the application of immunomagnetic separation technology and flow cytometry is mostly found in the field of medical analysis and inspection, but no report is provided for the research of combining immunomagnetic beads and flow cytometry for quantitative determination of mycotoxin. The invention innovatively provides a magnetic bead based on immunityFlow cytometry fluorescence immunoassay of aflatoxin B1The method of (1). Combines an immunological analysis method and an instrument for quantitative determination, realizes simple, efficient and sensitive detection, and is aflatoxin B1And the mycotoxin treatment provides a favorable basis for further expanding development and popularization.
Disclosure of Invention
The invention aims to provide aflatoxin B based on immunomagnetic beads1The aflatoxin B based on immunomagnetic beads provided by the invention1The determination method has the advantages of simple and conventional pretreatment method, clear operation, high instrument automation degree, sensitive and accurate analysis, and can obtain a large amount of detection results quickly in real time based on the flow cytometer.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an aflatoxin B based on immunomagnetic beads1The measuring method of (2), comprising the steps of:
(1) activating magnetic beads: suspending magnetic beads in a buffer solution to obtain a magnetic bead suspension, and adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxy thiosuccinimide to the magnetic bead suspension to activate the magnetic beads to obtain activated magnetic beads;
(2) antigen-modified magnetic beads: adding the activated magnetic beads into aflatoxin coupled antigen diluent, mixing with aflatoxin coupled antigen, and performing antigen modification to obtain antigen modified magnetic beads;
(3) immunomagnetic bead system: adding aflatoxin B into diluent of antigen modified magnetic beads1Obtaining a liquid to be detected system, and then adding aflatoxin B into the liquid to be detected system1Incubating the monoclonal antibody diluent, performing magnetic separation after incubation, discarding the supernatant, adding a fluorescein-labeled second antibody diluent for incubation again, performing magnetic separation after incubation, discarding the supernatant, and preparing an immunomagnetic bead system;
(4) flow cytometry detection: measuring the immunomagnetic bead system by a flow cytometer to obtain the fluorescence intensity of the immunomagnetic bead system;
(5) the aflatoxin B in the step (3) is treated1Replacing the liquid to be detected with equal amount of aflatoxin B with each concentration gradient1Standard solution, repeating the step (3) and the step (4) to obtain aflatoxin B1Standard curves of concentration and mean fluorescence intensity;
(6) substituting the fluorescence intensity of the immunomagnetic bead system determined in the step (4) into the standard curve to obtain the aflatoxin B1Aflatoxins B in the test solutions1The concentration of (c).
Preferably, the mass ratio of the magnetic beads, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the N-hydroxy thiosuccinimide is 1 (0.6-0.9) to (0.9-1.2).
Preferably, the activation condition is that the activation is carried out for 15-25 min at 20-30 ℃.
Preferably, the antigen modification condition is that the reaction is carried out for 0.5-2 h at 20-30 ℃.
Preferably, the diluent of the antigen modified magnetic beads is a diluent obtained by storing the antigen modified magnetic beads in a PBS buffer containing Tween-20 and BSA to obtain a storage solution, and then diluting the storage solution to obtain the antigen modified magnetic beads.
Preferably, the aflatoxin B1The monoclonal antibody diluent is prepared by mixing aflatoxin B1The monoclonal antibody is obtained by diluting the monoclonal antibody in PBS buffer solution, and the dilution factor is 1/4000-1/500.
Preferably, the dilution of the fluorescein-labeled secondary antibody is obtained by diluting the fluorescein-labeled secondary antibody in a PBS buffer solution, and the dilution factor is 1/200-1/50.
Preferably, the time for two incubations in the step (3) is independently 10-40 min.
Preferably, the aflatoxin B1The concentration gradient of the standard solution was 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/L, 3. mu.g/L, 4. mu.g/L, 5. mu.g/L, 6. mu.g/L, 8. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 40. mu.g/L, 60. mu.g/L, 80. mu.g/L, and 100. mu.g/L.
Preferably, the aflatoxin B1The standard solution is prepared by mixing aflatoxin B1Dissolving in a buffer solution containing 40-60% of methanol.
The aflatoxin B based on immunomagnetic beads provided by the invention1The measuring method comprises the steps of taking magnetic beads as solid phase carriers, and connecting corresponding AFB through surface modification1Antigen, and AFB in the liquid to be tested1Forming a competitive reaction system. AFB1The monoclonal antibody can be combined with AFB in antigen modified magnetic beads1Antigen and AFB in test solution1The antigen generates immune response, and recognition and combination form two types of complex systems. The magnetic beads are used as a composite system of the carrier, and can be separated by magnetic separation independently due to the magnetic response characteristic of the magnetic beads. The second antibody labeled by fluorescence can recognize AFB in the complex system1The monoclonal antibody forms a ternary complex system. AFB after magnetic separation1Antigen modified magnetic bead-AFB1Monoclonal antibody-fluorescently labeled secondary antibody "ternary complexes" that can be assayed by flow cytometry. The magnitude of the numerical value after the calculation is processed by the fluorescence channel of the flow cytometer and the AFB in the liquid to be detected1Is inversely proportional to the concentration of the AFB in the liquid to be measured by constructing a standard curve1And (4) accurately and quantitatively measuring. The aflatoxin B based on immunomagnetic beads provided by the invention1The determination method has the advantages of simple and conventional pretreatment method, clear operation, high instrument automation degree, sensitive and accurate analysis, and can obtain a large amount of detection results quickly in real time based on the flow cytometer.
Drawings
FIG. 1 shows aflatoxin B1Standard curves of concentration and mean fluorescence intensity and reconstructed standard graphs.
Detailed Description
The invention provides an aflatoxin B based on immunomagnetic beads1The measuring method of (2), comprising the steps of:
(1) activating magnetic beads: suspending the magnetic beads in a buffer solution to obtain a magnetic bead suspension, and adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (sulfo-NHS) into the magnetic bead suspension to activate the magnetic beads to obtain activated magnetic beads;
(2) antigen-modified magnetic beads: adding the activated magnetic beads into aflatoxin coupled antigen diluent, mixing with aflatoxin coupled antigen, and performing antigen modification to obtain antigen modified magnetic beads;
(3) immunomagnetic bead system: adding aflatoxin B into diluent of antigen modified magnetic beads1Obtaining a liquid to be detected system, and then adding aflatoxin B into the liquid to be detected system1Incubating the monoclonal antibody diluent, performing magnetic separation after incubation, discarding the supernatant, adding a fluorescein-labeled second antibody diluent for incubation again, performing magnetic separation after incubation, discarding the supernatant, and preparing an immunomagnetic bead system;
(4) flow cytometry detection: measuring the immunomagnetic bead system by a flow cytometer to obtain the fluorescence intensity of the immunomagnetic bead system;
(5) the aflatoxin B in the step (3) is treated1Replacing the liquid to be detected with equal amount of aflatoxin B with each concentration gradient1Standard solution, repeating the step (3) and the step (4) to obtain aflatoxin B1Standard curves of concentration and mean fluorescence intensity;
(6) substituting the fluorescence intensity data of the immunomagnetic bead system determined in the step (4) into the standard curve to obtain aflatoxin B in the liquid system to be detected1The concentration of (c).
The method comprises the steps of suspending magnetic beads in a buffer solution to obtain a magnetic bead suspension, and adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (sulfo-NHS) into the magnetic bead suspension to activate the magnetic beads to obtain activated magnetic beads.
In the present invention, the magnetic beads are preferably 0.5 μ M carboxyl magnetic beads, and the magnetic beads are preferably washed 3 times with 0.1M MES buffer pH6.2 before being prepared into a magnetic bead suspension.
In the present invention, the buffer is preferably a PBS buffer.
In the present invention, the mass ratio of the magnetic beads, EDC and sulfo-NHS is preferably 1: (0.6-0.9): (0.9 to 1.2), and more preferably 1: 0.77: 1.09.
in the invention, the activating condition is preferably to perform warm bath for 15-25 min at 20-30 ℃; further preferably, the temperature is 25 ℃ for 20 min; the activation is preferably carried out on its ringer BE-1100 mixer.
In the activation process, carboxyl on the surface of the magnetic beads is activated to obtain activated magnetic beads.
After the activation is finished, solid-liquid separation is carried out to obtain the activated magnetic bead solid.
After the activated magnetic bead is obtained, the activated magnetic bead is added into an aflatoxin coupling Antigen (AFB)1-BSA) to make antigen modified magnetic beads (AFB)1-BSA-MBs)。
In the invention, the aflatoxin-coupled antigen diluent is prepared by mixing an aflatoxin-coupled antigen with a PBS buffer solution, and the volume concentration of the aflatoxin-coupled antigen is preferably 20%.
In the present invention, the aflatoxin-conjugated Antigen (ABF)1-BSA) is preferably purchased from ann & ltz biotechnology limited, shenzhen.
In the invention, the antigen modification is preferably carried out at 20-30 ℃ for 0.5-2 h; further preferably at 25 ℃ for 1 h; the antigen modification is preferably carried out on its ringer BE-1100 mixer.
In the antigen modification process, carboxyl on the activated magnetic bead is combined with aflatoxin coupled antigen to form antigen modified magnetic bead (AFB)1-BSA-MBs)。
After the antigen modification process is finished, performing solid-liquid separation to obtain the antigen modified magnetic bead solid.
Diluting the antigen modified magnetic beads after the antigen modified magnetic beads are obtained to obtain a diluent of the antigen modified magnetic beads, taking the diluent of the antigen modified magnetic beads, and adding aflatoxin B1Adding aflatoxin B into the solution to be detected1Monoclonal antibodyAnd incubating the antibody diluent, performing magnetic separation after incubation is finished, discarding the supernatant, adding a fluorescein-labeled second antibody diluent for incubation again, performing magnetic separation after incubation is finished, and discarding the supernatant to obtain an immunomagnetic bead system.
In the invention, the diluent of the antigen modified magnetic beads is obtained by storing the antigen modified magnetic beads in PBS (phosphate buffer solution) containing 0.05% Tween-20HE 1% BSA to obtain a storage solution, and then diluting the storage solution to 1/100 by using the PBS buffer solution to obtain the diluent of the antigen modified magnetic beads.
In the invention, the diluent of the antigen modified magnetic beads and the aflatoxin B1The volume ratio of the dosage of the liquid to be detected is 5: 1.
In the invention, the aflatoxin B1The dilution ratio of the monoclonal antibody diluent is preferably 1/4000-1/500, more preferably 1/2000-1/1000, and still more preferably 1/2000.
In the present invention, the dilution ratio of the dilution of the fluorescein-labeled secondary antibody is preferably 1/200 to 1/50, more preferably 1/150 to 1/100, and still more preferably 1/100.
In the invention, the two incubations in the step (3) are preferably carried out on a Linbel BE-1100 mixer, and the two incubations are independently preferably 10-40 min, more preferably 20-30 min, and still more preferably 20 min.
After the immunomagnetic bead system is prepared, the invention measures the immunomagnetic bead system by a flow cytometer.
The invention preferably re-suspends the immunomagnetic bead system in the step (3) in PBS buffer solution, samples, and then the samples are measured by a flow cytometer.
The invention uses the aflatoxin B in the step (3)1Replacing the liquid to be detected with the same amount of aflatoxin B with different concentration gradients1Standard solution, repeating the step (3) and the step (4) to obtain aflatoxin B1Standard curves of concentration and mean fluorescence intensity.
In the invention, the aflatoxin B1The concentration gradient of the standard solution is preferably 0 mug/L、 0.1μg/L、0.2μg/L、0.3μg/L、0.5μg/L、1μg/L、1.5μg/L、2μg/L、3μg/L、4μg/L、 5μg/L、6μg/L、8μg/L、10μg/L、15μg/L、20μg/L、40μg/L、60μg/L、80μg/L、 100μg/L。
In the invention, the aflatoxin B1The standard solution is prepared by mixing aflatoxin B1Dissolving in buffer solution containing methanol; the content of the methanol is preferably 40-60%, and more preferably 50%; the buffer is preferably a 10mMPBS buffer.
Substituting the fluorescence intensity carried by the immunomagnetic beads determined in the step (4) into the standard curve to obtain aflatoxin B in the liquid system to be detected1The concentration of (c).
The following embodiments are combined to provide aflatoxin B based on immunomagnetic beads1The technical scheme of the measurement method (2) will be described in detail, but it should not be construed that the scope of the present invention is limited thereto.
Example 1
1mg of 0.5 μ M carboxyl magnetic beads were precisely transferred and poured into a 1.5mL centrifuge tube, followed by washing 3 times with 0.1M MES buffer (pH6.2), magnetic separation was then performed, and the magnetically separated carboxyl magnetic beads were resuspended in PBS buffer to prepare a magnetic bead suspension. 0.77mgEDC and 1.09mgsulfo-NHS were added to the carboxyl magnetic bead suspension and incubated at 25 ℃ for 20 minutes on its Linbel BE-1100 mixer to activate the carboxyl groups on the surface of the magnetic beads, and centrifuged to obtain activated carboxyl magnetic beads. Then, the activated carboxyl magnetic beads were resuspended in 0.5ml of PBS buffer (containing 100. mu.l of AFB)1-BSA) and gently shaken on its Linbel BE-1100 mixer for 1h to form AFB at 25 deg.C1-BSA-MBs solution. After magnetic separation, the AFB is separated1BSA-MBs were stored in 10ml PBS buffer containing 0.05% Tween-20 and 1% BSA, and AFB was added1Diluting the-BSA-MBs preservation solution to 1/100 in PBS buffer solution to obtain AFB1-BSA-MBs dilutions. Taking 100 μ l of AFB 120 parts of BSA-MBs diluent, each at a concentration of 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/L, 3. mu.g/L, 4. mu.g/L, 5. mu.g/L, 6. mu.g/Lg/L, 8. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 40. mu.g/L, 60. mu.g/L, 80. mu.g/L, 100. mu.g/L of 20. mu.LAFB1The standard solutions (dissolved in 10mM PBS containing 50% methanol) were mixed and added separately to 1.5ml centrifuge tubes. 20 uL of 80. mu.L AFB diluted in PBS with 1/5001The monoclonal antibody solutions were added to the tubes separately, and the tubes were incubated on their linbel BE-1100 mixer for 20 minutes. The supernatant was removed after magnetic separation. Then, 100 μ L of a solution of fluorescein-labeled secondary antibody diluted to 1/200 in PBS was added, respectively, and incubated on the mixer for 20 minutes. After magnetic separation, the reaction solution was resuspended in PBS buffer. The resuspended solution was then sampled by flow cytometry for determination to obtain the fluorescence intensity of the sample. The results show that the detection limit of the method under the conditions of dilution 1/500 of the monoclonal antibody, dilution concentration 1/200 of the second antibody and 20min of the incubation time is 0.2701 mug/L.
Example 2
1mg of 0.5 μ M carboxyl magnetic beads were precisely transferred and poured into a 1.5mL centrifuge tube, followed by washing 3 times with 0.1M MES buffer (pH6.2), magnetic separation was then performed, and the magnetically separated carboxyl magnetic beads were resuspended in PBS buffer to prepare a magnetic bead suspension. 0.77mgEDC and 1.09mgsulfo-NHS were added to the carboxyl magnetic bead suspension and incubated on its Linbel BE-1100 mixer at room temperature (25 ℃) for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads, and centrifuged to obtain activated carboxyl magnetic beads. Then, the activated carboxyl magnetic beads were resuspended in 0.5ml of PBS buffer (pH7.4, containing 100. mu.L of AFB)1-BSA) and gently shaken on its Linbel BE-1100 mixer for 1h to form AFB at room temperature (25 ℃ C.)1-BSA-MBs solution. After magnetic separation, the AFB is separated1BSA-MBs were stored in 10ml PBS buffer containing 0.05% Tween-20 and 1% BSA, and AFB was added1Diluting the-BSA-MBs preservation solution to 1/100 in PBS buffer solution to obtain AFB1-BSA-MBs dilutions. Taking 100 μ l of AFB 120 parts of BSA-MBs diluent, each at a concentration of 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/L, 3. mu.g/L, 4. mu.g/L, 5. mu.g/L, 6. mu.g/L, 8. mu.g/L10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 40. mu.g/L, 60. mu.g/L, 80. mu.g/L, 100. mu.g/L of 20. mu.L AFB1The standard solutions (dissolved in 10mM PBS containing 50% methanol) were mixed and added separately to 1.5ml centrifuge tubes. 20 uL of 80. mu.L AFB diluted in PBS with 1/10001The monoclonal antibody solutions were added to the tubes separately, and the tubes were incubated on their linbel BE-1100 mixer for 20 minutes. The supernatant was removed after magnetic separation. Then, 100 μ L of a solution of fluorescein-labeled secondary antibody diluted to 1/200 in PBS was added, respectively, and incubated on the mixer for 20 minutes. After magnetic separation, the reaction solution was resuspended in PBS buffer. The resuspended solution is then sampled at an appropriate rate by flow cytometry to determine the fluorescence intensity of the sample. The results show that the detection limit of the method under the conditions of dilution 1/1000 of the monoclonal antibody, dilution concentration 1/200 of the second antibody and 20min of the incubation time is 0.2315 mug/L.
Example 3
1mg of 0.5 μm carboxyl magnetic beads were precisely transferred and injected into a 1.5mL centrifuge tube, and then washed 3 times with 0.1MMES buffer (pH6.2), followed by magnetic separation, and the magnetically separated carboxyl magnetic beads were resuspended in PBS buffer to prepare a magnetic bead suspension. 0.77mgEDC and 1.09mgsulfo-NHS were added to the carboxyl magnetic bead suspension and incubated on its Linbel BE-1100 mixer at room temperature (25 ℃) for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads, and centrifuged to obtain activated carboxyl magnetic beads. Then, the activated carboxyl magnetic beads were resuspended in 0.5ml of PBS buffer (pH7.4, containing 100. mu.L of AFB)1-BSA) and gently shaken on its Linbel BE-1100 mixer for 1h to form AFB at room temperature (25 ℃ C.)1-BSA-MBs solution. After magnetic separation, the AFB is separated1BSA-MBs were stored in 10ml PBS buffer containing 0.05% Tween-20 and 1% BSA, and AFB was added1Diluting the-BSA-MBs preservation solution to 1/100 in PBS buffer solution to obtain AFB1-BSA-MBs dilutions. Taking 100 μ l of AFB 120 parts of BSA-MBs diluent, each at a concentration of 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/L, 3. mu.g/L, 4. mu.g/L, 5. mu.g/L, 6. mu.g/L, or,8 μ g/L, 10 μ g/L, 15 μ g/L, 20 μ g/L, 40 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L of 20 μ LAFB1The standard solutions (dissolved in 10mM PBS containing 50% methanol) were mixed and added separately to 1.5ml centrifuge tubes. 20 uL of 80. mu.L AFB diluted in PBS with 1/20001The monoclonal antibody solutions were added to the tubes separately, and the tubes were incubated on their linbel BE-1100 mixer for 20 minutes. The supernatant was removed after magnetic separation. Then, 100 μ L of a solution of fluorescein-labeled secondary antibody diluted to 1/200 in PBS was added, respectively, and incubated on the mixer for 20 minutes. After magnetic separation, the reaction solution was resuspended in PBS buffer. The resuspended solution is then sampled at an appropriate rate by flow cytometry to determine the fluorescence intensity of the sample. The results show that the detection limit of the method under the conditions of dilution 1/2000 of the monoclonal antibody, dilution concentration 1/200 of the second antibody and 20min of the incubation time is 0.2034 mug/L.
Example 4
1mg of 0.5 μm carboxyl magnetic beads were precisely transferred and injected into a 1.5mL centrifuge tube, and then washed 3 times with 0.1MMES buffer (pH6.2), followed by magnetic separation, and the magnetically separated carboxyl magnetic beads were resuspended in PBS buffer to prepare a magnetic bead suspension. 0.77mgEDC and 1.09mgsulfo-NHS were added to the carboxyl magnetic bead suspension and incubated on its Linbel BE-1100 mixer at room temperature (25 ℃) for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads, and centrifuged to obtain activated carboxyl magnetic beads. Then, the activated carboxyl magnetic beads were resuspended in 0.5ml of PBS buffer (pH7.4, containing 100. mu.L of AFB)1-BSA) and gently shaken on its Linbel BE-1100 mixer for 1h to form AFB at room temperature (25 ℃ C.)1-BSA-MBs solution. After magnetic separation, the AFB is separated1BSA-MBs were stored in 10ml PBS buffer containing 0.05% Tween-20 and 1% BSA, and AFB was added1Diluting the-BSA-MBs preservation solution to 1/100 in PBS buffer solution to obtain AFB1-BSA-MBs dilutions. Taking 100 μ l of AFB 120 parts of BSA-MBs diluent, each at a concentration of 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/L, 3. mu.g/L, 4. mu.g/L, 5. mu.g/L, 6. mu.g/L, respectivelyg/L, 8. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 40. mu.g/L, 60. mu.g/L, 80. mu.g/L, 100. mu.g/L of 20. mu.LAFB1The standard solutions (dissolved in 10mM PBS containing 50% methanol) were mixed and added separately to 1.5ml centrifuge tubes. 20 uL of 80. mu.L AFB diluted in PBS with 1/40001The monoclonal antibody solutions were added to the tubes separately, and the tubes were incubated on their linbel BE-1100 mixer for 20 minutes. The supernatant was removed after magnetic separation. Then, 100 μ L of a solution of fluorescein-labeled secondary antibody diluted to 1/200 in PBS was added, respectively, and incubated on the mixer for 20 minutes. After magnetic separation, the reaction solution was resuspended in PBS buffer. The resuspended solution is then sampled at an appropriate rate by flow cytometry to determine the fluorescence intensity of the sample. The results show that the detection limit of the method under the conditions of dilution 1/4000 of the monoclonal antibody, dilution concentration 1/200 of the second antibody and 20min of the incubation time is 0.2884 mug/L.
Example 5
1mg of 0.5 μm carboxyl magnetic beads were precisely transferred and injected into a 1.5mL centrifuge tube, and then washed 3 times with 0.1MMES buffer (pH6.2), followed by magnetic separation, and the magnetically separated carboxyl magnetic beads were resuspended in PBS buffer to prepare a magnetic bead suspension. 0.77mgEDC and 1.09mgsulfo-NHS were added to the carboxyl magnetic bead suspension and incubated on its Linbel BE-1100 mixer at room temperature (25 ℃) for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads, and centrifuged to obtain activated carboxyl magnetic beads. Then, the activated carboxyl magnetic beads were resuspended in 0.5ml of PBS buffer (pH7.4, containing 100. mu.L of AFB)1-BSA) and gently shaken on its Linbel BE-1100 mixer for 1h to form AFB at room temperature (25 ℃ C.)1-BSA-MBs solution. After magnetic separation, the AFB is separated1BSA-MBs were stored in 10ml PBS buffer containing 0.05% Tween-20 and 1% BSA, and AFB was added1Diluting the-BSA-MBs preservation solution to 1/100 in PBS buffer solution to obtain AFB1-BSA-MBs dilutions. Taking 100 μ l of AFB 120 parts of BSA-MBs diluent, each at a concentration of 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/L, 3. mu.g/L, 4. mu.g/L, 5. mu.g/L, respectively6. mu.g/L, 8. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 40. mu.g/L, 60. mu.g/L, 80. mu.g/L, 100. mu.g/L of 20. mu.LAFB1The standard solutions (dissolved in 10mM PBS containing 50% methanol) were mixed and added separately to 1.5ml centrifuge tubes. 20 uL of 80. mu.L AFB diluted in PBS with 1/20001The monoclonal antibody solutions were added to the tubes separately, and the tubes were incubated on their linbel BE-1100 mixer for 20 minutes. The supernatant was removed after magnetic separation. Then, 100 μ L of a solution of fluorescein-labeled secondary antibody diluted to 1/50 in PBS was added, respectively, and incubated on the mixer for 20 minutes. After magnetic separation, the reaction solution was resuspended in PBS buffer. The resuspended solution is then sampled at an appropriate rate by flow cytometry to determine the fluorescence intensity of the sample. The results show that the detection limit of the method under the conditions of dilution 1/2000 of the monoclonal antibody, dilution concentration 1/50 of the second antibody and 20min of the incubation time is 0.2528 mug/L.
Example 6
1mg of 0.5 μm carboxyl magnetic beads were precisely transferred and injected into a 1.5mL centrifuge tube, and then washed 3 times with 0.1MMES buffer (pH6.2), followed by magnetic separation, and the magnetically separated carboxyl magnetic beads were resuspended in PBS buffer to prepare a magnetic bead suspension. 0.77mgEDC and 1.09mgsulfo-NHS were added to the carboxyl magnetic bead suspension and incubated on its Linbel BE-1100 mixer at room temperature (25 ℃) for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads, and centrifuged to obtain activated carboxyl magnetic beads. Then, the activated carboxyl magnetic beads were resuspended in 0.5ml of PBS buffer (pH7.4, containing 100. mu.L of AFB)1-BSA) and gently shaken on its Linbel BE-1100 mixer for 1h to form AFB at room temperature (25 ℃ C.)1-BSA-MBs solution. After magnetic separation, the AFB is separated1BSA-MBs were stored in 10ml PBS buffer containing 0.05% Tween-20 and 1% BSA, and AFB was added1Diluting the-BSA-MBs preservation solution to 1/100 in PBS buffer solution to obtain AFB1-BSA-MBs dilutions. Taking 100 μ l of AFB 120 parts of BSA-MBs diluent, each at a concentration of 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/L, 3. mu.g/L, 4. mu.g/L, 5. mu.g/L, respectivelymu.g/L, 6. mu.g/L, 8. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 40. mu.g/L, 60. mu.g/L, 80. mu.g/L, 100. mu.g/L of 20. mu.LAFB1The standard solutions (dissolved in 10mM PBS containing 50% methanol) were mixed and added separately to 1.5ml centrifuge tubes. 20 uL of 80. mu.L AFB diluted in PBS with 1/20001The monoclonal antibody solutions were added to the tubes separately, and the tubes were incubated on their linbel BE-1100 mixer for 20 minutes. The supernatant was removed after magnetic separation. Then, 100 μ L of a solution of fluorescein-labeled secondary antibody diluted to 1/100 in PBS was added, respectively, and incubated on the mixer for 20 minutes. After magnetic separation, the reaction solution was resuspended in PBS buffer. The resuspended solution is then sampled at an appropriate rate by flow cytometry to determine the fluorescence intensity of the sample. The results show that the detection limit of the method under the conditions of dilution 1/2000 of the monoclonal antibody, dilution concentration 1/100 of the second antibody and 20min of the incubation time is 0.2305 mug/L.
Example 7
1mg of 0.5 μm carboxyl magnetic beads were precisely transferred and injected into a 1.5mL centrifuge tube, and then washed 3 times with 0.1MMES buffer (pH6.2), followed by magnetic separation, and the magnetically separated carboxyl magnetic beads were resuspended in PBS buffer to prepare a magnetic bead suspension. 0.77mgEDC and 1.09mgsulfo-NHS were added to the carboxyl magnetic bead suspension and incubated on its Linbel BE-1100 mixer at room temperature (25 ℃) for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads, and centrifuged to obtain activated carboxyl magnetic beads. Then, the activated carboxyl magnetic beads were resuspended in 0.5ml of PBS buffer (pH7.4, containing 100. mu.L of AFB)1-BSA) and gently shaken on its Linbel BE-1100 mixer for 1h to form AFB at room temperature (25 ℃ C.)1-BSA-MBs solution. After magnetic separation, the AFB is separated1BSA-MBs were stored in 10ml PBS buffer containing 0.05% Tween-20 and 1% BSA, and AFB was added1Diluting the-BSA-MBs preservation solution to 1/100 in PBS buffer solution to obtain AFB1-BSA-MBs dilutions. Taking 100 μ l of AFB 120 parts of BSA-MBs diluent, with 20 parts of the BSA-MBs diluent, respectively, at a concentration of 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/L, 3. mu.g/L, 4. mu.g/Lg/L, 5. mu.g/L, 6. mu.g/L, 8. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 40. mu.g/L, 60. mu.g/L, 80. mu.g/L, 100. mu.g/L of 20. mu.LAFB1The standard solutions (dissolved in 10mM PBS containing 50% methanol) were mixed and added separately to 1.5ml centrifuge tubes. 20 uL of 80. mu.L AFB diluted in PBS with 1/20001The monoclonal antibody solutions were added to the tubes separately, and the tubes were incubated on their linbel BE-1100 mixer for 20 minutes. The supernatant was removed after magnetic separation. Then, 100 μ L of a solution of fluorescein-labeled secondary antibody diluted to 1/150 in PBS was added, respectively, and incubated on the mixer for 20 minutes. After magnetic separation, the reaction solution was resuspended in PBS buffer. The resuspended solution is then sampled at an appropriate rate by flow cytometry to determine the fluorescence intensity of the sample. The results show that the detection limit of the method is 0.2361. mu.g/L under the conditions of dilution 1/2000 of monoclonal antibody, dilution concentration 1/150 of secondary antibody and 20min of incubation time.
Example 8
1mg of 0.5 μm carboxyl magnetic beads were precisely transferred and injected into a 1.5mL centrifuge tube, and then washed 3 times with 0.1MMES buffer (pH6.2), followed by magnetic separation, and the magnetically separated carboxyl magnetic beads were resuspended in PBS buffer to prepare a magnetic bead suspension. 0.77mgEDC and 1.09mgsulfo-NHS were added to the carboxyl magnetic bead suspension and incubated on its Linbel BE-1100 mixer at room temperature (25 ℃) for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads, and centrifuged to obtain activated carboxyl magnetic beads. Then, the activated carboxyl magnetic beads were resuspended in 0.5ml of PBS buffer (pH7.4, containing 100. mu.L of AFB)1-BSA) and gently shaken on its Linbel BE-1100 mixer for 1h to form AFB at room temperature (25 ℃ C.)1-BSA-MBs solution. After magnetic separation, the AFB is separated1BSA-MBs were stored in 10ml PBS buffer containing 0.05% Tween-20 and 1% BSA, and AFB was added1Diluting the-BSA-MBs preservation solution to 1/100 in PBS buffer solution to obtain AFB1-BSA-MBs dilutions. Taking 100 μ l of AFB 120 parts of BSA-MBs dilution at a concentration of 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/L, 3. mu.g/LL, 4. mu.g/L, 5. mu.g/L, 6. mu.g/L, 8. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 40. mu.g/L, 60. mu.g/L, 80. mu.g/L, 100. mu.g/L of 20. mu.LAFB1The standard solutions (dissolved in 10mM PBS containing 50% methanol) were mixed and added separately to 1.5ml centrifuge tubes. 20 uL of 80. mu.L AFB diluted in PBS with 1/20001The monoclonal antibody solutions were added to the tubes separately, and the tubes were incubated on their linbel BE-1100 mixer for 10 minutes. The supernatant was removed after magnetic separation. Then, 100 μ L of a fluorescein-labeled secondary antibody solution diluted to 1/200 in PBS was added, respectively, and incubated on the mixer for 10 minutes. After magnetic separation, the reaction solution was resuspended in PBS buffer. The resuspended solution is then sampled at an appropriate rate by flow cytometry to determine the fluorescence intensity of the sample. The results show that the detection limit of the method under the conditions of dilution 1/2000 of the monoclonal antibody, the dilution concentration 1/200 of the second antibody and 10min of the incubation time is 0.3107. mu.g/L.
Example 9
1mg of 0.5 μm carboxyl magnetic beads were precisely transferred and injected into a 1.5mL centrifuge tube, and then washed 3 times with 0.1MMES buffer (pH6.2), followed by magnetic separation, and the magnetically separated carboxyl magnetic beads were resuspended in PBS buffer to prepare a magnetic bead suspension. 0.77mgEDC and 1.09mgsulfo-NHS were added to the carboxyl magnetic bead suspension and incubated on its Linbel BE-1100 mixer at room temperature (25 ℃) for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads, and centrifuged to obtain activated carboxyl magnetic beads. Then, the activated carboxyl magnetic beads were resuspended in 0.5ml of PBS buffer (pH7.4, containing 100. mu.L of AFB)1-BSA) and gently shaken on its Linbel BE-1100 mixer for 1h to form AFB at room temperature (25 ℃ C.)1-BSA-MBs solution. After magnetic separation, the AFB is separated1BSA-MBs were stored in 10ml PBS buffer containing 0.05% Tween-20 and 1% BSA, and AFB was added1Diluting the-BSA-MBs preservation solution to 1/100 in PBS buffer solution to obtain AFB1-BSA-MBs dilutions. Taking 100 μ l of AFB 120 parts of BSA-MBs diluent, each at a concentration of 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/L, or,3. mu.g/L, 4. mu.g/L, 5. mu.g/L, 6. mu.g/L, 8. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 40. mu.g/L, 60. mu.g/L, 80. mu.g/L, 100. mu.g/L of 20. mu.LAFB1The standard solutions (dissolved in 10mM PBS containing 50% methanol) were mixed and added separately to 1.5ml centrifuge tubes. 20 uL of 80. mu.L AFB diluted in PBS with 1/20001The monoclonal antibody solutions were added to the tubes separately, and the tubes were incubated on their linbel BE-1100 mixer for 30 minutes. The supernatant was removed after magnetic separation. Then, 100 μ L of a solution of fluorescein-labeled secondary antibody diluted to 1/200 in PBS was added, respectively, and incubated on the mixer for 30 minutes. After magnetic separation, the reaction solution was resuspended in PBS buffer. The resuspended solution is then sampled at an appropriate rate by flow cytometry to determine the fluorescence intensity of the sample. The results show that the detection limit of the method under the conditions of dilution 1/2000 of the monoclonal antibody, dilution concentration 1/200 of the second antibody and 30min of the incubation time is 0.2199 mug/L.
Example 10
1mg of 0.5 μm carboxyl magnetic beads were precisely transferred and injected into a 1.5mL centrifuge tube, and then washed 3 times with 0.1MMES buffer (pH6.2), followed by magnetic separation, and the magnetically separated carboxyl magnetic beads were resuspended in PBS buffer to prepare a magnetic bead suspension. 0.77mgEDC and 1.09mgsulfo-NHS were added to the carboxyl magnetic bead suspension and incubated on its Linbel BE-1100 mixer at room temperature (25 ℃) for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads, and centrifuged to obtain activated carboxyl magnetic beads. Then, the activated carboxyl magnetic beads were resuspended in 0.5ml of PBS buffer (pH7.4, containing 100. mu.L of AFB)1-BSA) and gently shaken on its Linbel BE-1100 mixer for 1h to form AFB at room temperature (25 ℃ C.)1-BSA-MBs solution. After magnetic separation, the AFB is separated1BSA-MBs were stored in 10ml PBS buffer containing 0.05% Tween-20 and 1% BSA, and AFB was added1Diluting the-BSA-MBs preservation solution to 1/100 in PBS buffer solution to obtain AFB1-BSA-MBs dilutions. Taking 100 μ l of AFB 120 parts of BSA-MBs diluent, with 20 parts of the BSA-MBs diluent, respectively, at a concentration of 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/Lmu.g/L, 3. mu.g/L, 4. mu.g/L, 5. mu.g/L, 6. mu.g/L, 8. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 40. mu.g/L, 60. mu.g/L, 80. mu.g/L, 100. mu.g/L of 20. mu.LAFB1The standard solutions (dissolved in 10mM PBS containing 50% methanol) were mixed and added separately to 1.5ml centrifuge tubes. 20 uL of 80. mu.L AFB diluted in PBS with 1/20001The monoclonal antibody solutions were added to the tubes separately and the tubes were incubated on their linbel BE-1100 mixer for 40 minutes. The supernatant was removed after magnetic separation. Then, 100 μ L of a solution of fluorescein-labeled secondary antibody diluted to 1/200 in PBS was added, respectively, and incubated on the mixer for 40 minutes. After magnetic separation, the reaction solution was resuspended in PBS buffer. The resuspended solution is then sampled at an appropriate rate by flow cytometry to determine the fluorescence intensity of the sample. The results show that the detection limit of the method under the conditions of dilution 1/2000 of the monoclonal antibody, dilution concentration 1/200 of the second antibody and 40min of the incubation time is 0.2100 mug/L.
Example 11
As is clear from examples 1 to 10, the detection limit of example 3 was 0.2034. mu.g/L, and the detection limit was the lowest. Thus, a standard curve was established according to the method of example 3, with AFB1The addition was plotted on the abscissa and the average fluorescence intensity of the flow cytometry results was plotted on the ordinate to construct a standard curve, as shown in FIG. 1. Following AFB1When the amount of the fluorescent substance added is increased, the fluorescence intensity gradually decreases, and the amount of decrease in fluorescence intensity can be used (FI)0-FI)/FI0(FI0Initial fluorescence intensity, FI measured at the corresponding concentration) to reconstruct a standard curve, as shown in fig. 1.
Example 12
1mg of 0.5 μ M carboxyl magnetic beads were precisely transferred and poured into a 1.5mL centrifuge tube, followed by washing 3 times with 0.1M MES buffer (pH6.2), magnetic separation was then performed, and the magnetically separated carboxyl magnetic beads were resuspended in PBS buffer to prepare a magnetic bead suspension. 0.77mgEDC and 1.09mgsulfo-NHS were added to the carboxyl magnetic bead suspension and incubated on its Linbel BE-1100 mixer at room temperature (25 ℃) for 20 minutes to activate the carboxyl groups on the surface of the beads, and centrifuged to obtain the activated beadsAnd (4) carboxylic magnetic beads. Then, the activated carboxyl magnetic beads were resuspended in 0.5ml of PBS buffer (pH7.4, containing 100. mu.L of AFB)1-BSA) and gently shaken on its Linbel BE-1100 mixer for 1h to form AFB at room temperature (25 ℃ C.)1-BSA-MBs solution. After magnetic separation, the AFB is separated1BSA-MBs were stored in 10ml PBS buffer containing 0.05% Tween-20 and 1% BSA, and AFB was added1Diluting the-BSA-MBs preservation solution to 1/100 in PBS buffer solution to obtain AFB1-BSA-MBs dilutions. Taking 100 μ l of AFB1-BSA-MBs dilution with 20. mu.L of a solution containing AFB1The solutions to be tested for the peanut samples (pre-treatment reference for the peanut samples GB5009.22-2016) were mixed and added into a 1.5ml centrifuge tube. 80 μ L of AFB diluted in PBS with 1/20001The monoclonal antibody solution was added to the centrifuge tube described above, and the centrifuge tube was incubated on its linbel BE-1100 mixer for 40 minutes. The supernatant was removed after magnetic separation. Then, 100 μ L of fluorescein-labeled secondary antibody solution diluted to 1/200 in PBS was added and incubated on the mixer for 20 minutes. After magnetic separation, the reaction solution was resuspended in PBS buffer. The resuspended solution was then sampled and measured by flow cytometry, and the measured data was substituted into the standard curve in example 11 to obtain a concentration of aflatoxin B1 in the test solution of peanut sample of 1.9 μ g/L.
For the above AFB1The peanut sample solution to be detected is detected by adopting an ELISA method in the prior art and is detected by using commercially available AFB1The ELISA kit detects the DNA fragment with the detection concentration of 1.94 mu g/L. Proves that the aflatoxin B based on immunomagnetic beads provided by the invention1The determination method not only has higher sensitivity, but also has higher accuracy.
The invention provides an aflatoxin B based on immunomagnetic beads1The determination method has the advantages of simple and conventional pretreatment method, clear operation, high instrument automation degree, sensitive and accurate analysis, and can obtain a large amount of detection results quickly in real time based on the flow cytometer. From the above embodiments, it can be seen that the aflatoxin B based on immunomagnetic beads of the invention1The determination method of (1) for aflatoxinsElement B1The lowest detection limit of the concentration is 0.2034 mug/L, which proves that the method of the invention has lower detection concentration.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. Aflatoxin B based on immunomagnetic beads1The method of (2), characterized by comprising the steps of:
(1) activating magnetic beads: suspending magnetic beads in a buffer solution to obtain a magnetic bead suspension, and adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxy thiosuccinimide to the magnetic bead suspension to activate the magnetic beads to obtain activated magnetic beads;
(2) antigen-modified magnetic beads: adding the activated magnetic beads into aflatoxin coupled antigen diluent, mixing with aflatoxin coupled antigen, and performing antigen modification to obtain antigen modified magnetic beads;
(3) immunomagnetic bead system: adding aflatoxin B into diluent of antigen modified magnetic beads1Obtaining a liquid to be detected, adding aflatoxin B into the liquid to be detected1Incubating the monoclonal antibody diluent, performing magnetic separation after incubation, discarding the supernatant, adding a fluorescein-labeled second antibody diluent for incubation again, performing magnetic separation after incubation, discarding the supernatant, and preparing an immunomagnetic bead system;
(4) flow cytometry detection: measuring the immunomagnetic bead system by a flow cytometer to obtain the fluorescence intensity of the immunomagnetic bead system;
(5) the aflatoxin B in the step (3) is treated1Replacing the liquid to be detected with the same amount of aflatoxin B with different concentration gradients1Standard solution, repeating the step (3) and the step (4) to obtain aflatoxin B1Standard curves of concentration and mean fluorescence intensity;
(6) immunizing the assay of step (4)Substituting the fluorescence intensity of the magnetic bead system into the standard curve to obtain the aflatoxin B1Aflatoxins B in the test solutions1The concentration of (c).
2. The immunomagnetic bead-based aflatoxin B of claim 11The method for measuring (1) is characterized in that the mass ratio of the magnetic beads to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxythiosuccinimide is 1 (0.6-0.9) to (0.9-1.2).
3. The immunomagnetic bead-based aflatoxin B of claim 21The method for measuring (1) is characterized in that the activation is carried out at 20 to 30 ℃ for 15 to 25 min.
4. The immunomagnetic bead-based aflatoxin B of claim 11The method for detecting (1) is characterized in that the antigen modification is carried out under the condition of reacting for 0.5-2 hours at 20-30 ℃.
5. The immunomagnetic bead-based aflatoxin B of claim 11The method for measuring (1) is characterized in that the diluent of the antigen-modified magnetic beads is a diluent obtained by storing the antigen-modified magnetic beads in a PBS buffer containing Tween-20 and BSA to obtain a storage solution, and then diluting the storage solution.
6. Immunomagnetic bead-based aflatoxins B of claims 1 or 51Characterized in that the aflatoxin B is1The monoclonal antibody diluent is prepared by mixing aflatoxin B1The monoclonal antibody is obtained by diluting the monoclonal antibody in PBS buffer solution, and the dilution factor is 1/4000-1/500.
7. The immunomagnetic bead-based aflatoxin B of claim 61The method for measuring (1), wherein the dilution of the fluorescein-labeled second antibody is prepared byThe secondary antibody labeled by fluorescein is obtained by diluting in a PBS buffer solution, and the dilution factor is 1/200-1/50.
8. The immunomagnetic bead-based aflatoxin B of claim 1 or 71The determination method of (1), wherein the time of the two incubations in the step (3) is independently 10 to 40 min.
9. The immunomagnetic bead-based aflatoxin B of claim 11Characterized in that the aflatoxin B is1The concentration gradient of the standard solution was 0. mu.g/L, 0.1. mu.g/L, 0.2. mu.g/L, 0.3. mu.g/L, 0.5. mu.g/L, 1. mu.g/L, 1.5. mu.g/L, 2. mu.g/L, 3. mu.g/L, 4. mu.g/L, 5. mu.g/L, 6. mu.g/L, 8. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 40. mu.g/L, 60. mu.g/L, 80. mu.g/L, and 100. mu.g/L.
10. The immunomagnetic bead-based aflatoxin B of claim 1 or 91Characterized in that the aflatoxin B is1The standard solution is prepared by mixing aflatoxin B1Dissolving in a buffer solution containing 40-60% of methanol.
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