CN112824877A - Method for preparing fluorescent quantum dot immunochromatography detection card by using aflatoxin - Google Patents
Method for preparing fluorescent quantum dot immunochromatography detection card by using aflatoxin Download PDFInfo
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- CN112824877A CN112824877A CN201911146742.4A CN201911146742A CN112824877A CN 112824877 A CN112824877 A CN 112824877A CN 201911146742 A CN201911146742 A CN 201911146742A CN 112824877 A CN112824877 A CN 112824877A
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- aflatoxin
- quantum dot
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- antibody
- solution
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
A method for preparing a fluorescent quantum dot immunochromatography detection card by using aflatoxin. Relates to aflatoxin, in particular to a method for preparing a fluorescent quantum dot immunochromatography detection card by using the aflatoxin. The method for preparing the fluorescent quantum dot immunochromatography detection card by using the aflatoxin is convenient to process and improves the detection speed. The AFB1 immunofluorescence quantum dot detection card has strong specificity, and the results of detecting the aflatoxin and other fungomycin of the same family are negative. The reaction time is generally 3-5 min, compared with the traditional detection methods such as chromatography, ELISA and the like, the detection time is greatly shortened, the detection speed is improved, and the limit of large-scale equipment is thoroughly eliminated. The detection sensitivity of the detection card reaches 1ppb, which is higher than that of the same type of colloidal gold products.
Description
Technical Field
The invention relates to aflatoxin, in particular to a method for preparing a fluorescent quantum dot immunochromatography detection card by using aflatoxin.
Background
The immunochromatography technology of quantum dot labeling has received much attention because it has all the advantages of the colloidal gold immunochromatography detection technology and has higher sensitivity. The quantum dots are nanoparticles mainly composed of main group elements II-VI, III-V such as CdSe, CdTe and InP, and the particle size is generally 1-100 nm. Because electrons and holes are limited by quanta, the continuous energy band structure is changed into a discrete energy level structure with molecular characteristics, and fluorescence can be emitted after the structure is excited. The quantum dots have remarkable quantum size effect and surface effect, so that the quantum dots have light absorption characteristics which are not possessed by conventional materials, the application fields of the quantum dots are wider and wider, particularly the application values of the quantum dots in clinical laboratory and immunological detection research attract great attention of scientists, and the luminescent quantum dots serve as fluorescent probes to mark biomacromolecules and are one of important applications of nano materials in the field of bioanalysis in recent years.
By combining the existing immunological detection method, the quantum dot shows attractive prospect in the aspect of labeled immunoassay. The fluorescence analysis and the immunochromatography are combined and applied to the existing immunochromatography rapid detection, and the sensitivity of the immunochromatography rapid detection is greatly improved compared with that of colloidal gold. In addition, different quantum dots are marked with different antibodies by utilizing the characteristic that the quantum dots with different particle sizes have different colors, and the immunochromatography test strip is prepared, so that the test strip is expected to be used for simultaneously detecting different substances, and the efficiency of food safety detection is greatly improved.
Based on the advantages of quantum dots for immunochromatography rapid detection, the invention aims to apply the technology to rapid detection of Aflatoxin (AFB) in food and feed safety, and research and develop related detection test strips, thereby having better market prospect and wide social benefit.
Aflatoxin poisoning (Aflatoxicosis) is a major injury to the liver of animals, and the injured individuals vary with the age, sex and nutritional status of the animal species. Research results show that aflatoxin can cause liver function reduction, reduce milk yield and egg production rate, reduce immunity of animals and is easy to be infected by harmful microorganisms. In addition, long-term consumption of feed containing low concentrations of aflatoxin can also lead to intra-embryonic poisoning. Generally, young animals are more sensitive to aflatoxins, which are clinically manifested by dysfunction of the digestive system, reduced fertility, reduced feed utilization, anemia, and the like.
The median lethal dose of aflatoxin Bl is 0.36 mg/kg body weight, and belongs to a range of extremely virulent poisons (the median lethal dose of animals is less than 10 mg/kg = its toxicity is 10 times greater than that of potassium cyanide and 68 times greater than that of arsenic trioxide). It causes poisoning of human mainly by liver damage, hepatitis and liver cirrhosis, liver necrosis, etc. In conclusion, the development of the test strip for rapidly detecting aflatoxin is particularly key to the guarantee of the safety of grains and feeds.
Disclosure of Invention
Aiming at the problems, the invention provides the method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin, which is convenient to process and improves the detection speed.
The invention comprises the following steps: the method comprises the following steps:
1) selecting materials;
2) a fluorescent quantum dot reagent formula;
3) synthesizing an aflatoxin immune antigen;
4) preparing an aflatoxin specific antibody;
5) detecting the affinity constant and the titer of the monoclonal antibody;
6) synthesizing an aflatoxin detection antigen;
7) preparing an aflatoxin b1 antibody marked by fluorescent microspheres;
8) preparing an AFB1 antibody binding pad;
9) preparing an aflatoxin b1 fluorescent quantum dot detection test reagent strip;
10) and (4) finishing.
The materials in the step 1) comprise MES, NHS, carbodiimide, 2- (N-morpholino) ethanesulfonic acid, bovine serum albumin, Tween-20, Tritonx-100 and PVP-40;
HAT culture medium, fluorescent microspheres, goat anti-mouse secondary antibody, nitrocellulose membrane, glass fiber membrane and absorbent paper.
The step 2) comprises the following steps:
preparing borax buffer solution: 6.183g of boric acid, 6.864g of sodium borate; making the volume of ultrapure water to 860 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
0.1M, ph4.7mes buffer: 19.52g MES, 58.44g NaCl, ultrapure water to 1L, adjusted pH to 4.7 with NaOH, stored at 4 ℃ for no more than one week;
preparation of 10% BSA: 100.0g BSA; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% PVP-40: 100.0g PVP-40; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% Tween-20: 100.0mL Tween-20; diluting with ultrapure water to a constant volume of 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% sodium azide: 100.0g of sodium azide; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
glassware cleaning solution: 1000g of potassium dichromate and 2500mL of concentrated sulfuric acid, and adding distilled water to 10000 mL;
preparation of antibody marker dilution buffer: taking 50g of sucrose, adding 5mL of 10% BSA, 3mL of 10% Tween-20, 2mL of 10% PVP-40, 0.2M of PH7.4 PB to a volume of 100mL, and adding 200uL of 10% sodium azide;
preparing glass fiber gasket treating fluid: weighing 30.0g of trehalose, 5g of BSA and 15g of sucrose into a 1000mL reagent bottle, adding 100mL of 0.02mol/L PBS (pH7.4), 5mL of goat serum and 3.1mL of Triton-100, and adding ultrapure water to fix the volume to 1L;
preparation of sample pad treatment solution: weighing 1.8g of trehalose, 6g of BSA, 2.5g of casein, BRIJ-355 g of PVP-k405.4g and Na2HPO4 13.578g、NaH2PO4 2.8g and 4.015g of NaCl are put into a 1000mL reagent bottle, 5mL of Tween-20 is added, and ultrapure water is added to the solution to reach the constant volume of 1L;
preparation of antigen and secondary antibody diluent: weighing 2g of trehalose in a 250mL reagent bottle, adding 1mL of methanol and 5mL of 10% glycerol, adding 0.02mol/L of PBS and fixing the volume to 100 mL;
0.2 mol/LPB: weighing 2.3g Na2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, then fixing the volume to 1000mL, sterilizing at high pressure for 20min, and then storing at normal temperature, wherein the pH value of the working solution is 7.4;
17.4g NaCl, 2.3g Na were weighed into 0.02mol/L PBS2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, then fixing the volume to 1000mL, sterilizing at high pressure for 20min, and then storing at normal temperature, wherein the pH value of the working solution is 7.4;
5% solution of dichlorodipotassium silane in chloroform: 25mL of dichlorodipotassium silane was dissolved in 475mL of a chloroform solution and stored under a sealed condition.
Dissolving aflatoxin b1 in absolute methanol in the step 3), adding pyridine, then adding carboxymethyl hydroxylamine hemihydrochloride, rotationally evaporating the solvent, and purifying by a column to obtain hapten;
dissolving hapten in DMF, adding an activating agent for reaction overnight, adding the reaction solution into PBS dissolved with OVA, dialyzing with 0.01MPBS after reaction to obtain aflatoxin B1 immune antigen.
The step 4) comprises the following steps:
4.1) immunization of mice;
4.2) cell fusion;
4.3) screening hybridoma cells;
4.4) preparation and purification of ascites.
And 5) carrying out 2-time gradient dilution on the two monoclonal antibodies from 200 times by using a sample diluent by adopting an indirect ELISA method, and detecting.
Step 6), dissolving aflatoxin b1 in absolute ethyl alcohol, adding pyridine, then adding carboxymethyl hydroxylamine hemihydrochloride for reaction, rotationally evaporating the solvent, and purifying by a column to obtain hapten; dissolving hapten in DMF, adding an activating agent for reaction overnight, then adding the reaction solution into PBS dissolved with BSA, dialyzing with 0.01M PBS after reaction to obtain aflatoxin b1 detection antigen.
In the step 7), the fluorescent microsphere with the average diameter of 110nm and modified carboxyl and the AFB 1-resistant monoclonal antibody are used for preparing the fluorescent microsphere labeled A-type influenza virus labeled antibody according to the following method:
washing 5mg of the carboxyl modified fluorescent microspheres with MES buffer solution, centrifuging, then resuspending with 1ml of MES buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide to a final concentration of 5mM, adding NHS to a final concentration of 10mM, keeping the temperature away from the sun at room temperature, and reacting for half an hour to obtain activated carboxyl modified fluorescent microspheres;
washing the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5, and mixing 0.37mg of the AFB1 antibody to be marked and 5mg of the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5 to be fully and uniformly mixed;
reacting for 2 hours at room temperature in a dark place, and enabling the antibody and the fluorescent microsphere to form stable peptide bond covalent bonding to obtain a conjugate of the fluorescent microsphere and the AFB1 antibody;
after the reaction is finished, adding BSA solution with the final concentration of 1% to block the residual active carboxyl sites on the conjugate of the fluorescent microspheres and the AFB1 antibody, and reacting at room temperature in a dark place for 0.5 hour;
after completion, the cells were washed with 0.02M PBS buffer (pH7.4) and resuspended to obtain 5mg/ml fluorescent microsphere-labeled AFB1 antibody liquid, which was stored at 4 ℃ until use.
And 8) spraying the fluorescent microsphere-labeled fluorescent quantum dot-labeled AFB1 antibody mixed solution obtained in the step on a treated glass cellulose membrane by adopting an XYZ3050 membrane spraying system of Biodot according to the spraying amount of 2.5uL/cm to prepare an antibody binding pad, then transferring the antibody binding pad into a vacuum drying oven by using a grid tray, carrying out vacuum drying for 2 hours, adding a drying agent or allochroic silica gel, and sealing and storing in a refrigerator at 4 ℃.
Step 9), spraying the marked fluorescent quantum dot antibody compound on a bonding pad; detecting an antigen by adopting aflatoxin B1 and coating the antigen on the surface of an NC membrane as a detection line; coating goat anti-mouse IgG on the surface of the nitrocellulose membrane to serve as a quality control line; and finally assembling the combined pad, the absorbent paper and the treated sample pad into an aflatoxin B1 test strip.
The AFB1 immunofluorescence quantum dot detection card has strong specificity, and the results of detecting the aflatoxin and other fungomycin of the same family are negative. The reaction time is generally 3-5 min, compared with the traditional detection methods such as chromatography, ELISA and the like, the detection time is greatly shortened, the detection speed is improved, and the limit of large-scale equipment is thoroughly eliminated. The detection sensitivity of the detection card reaches 1ppb, which is higher than that of the same type of colloidal gold products.
Drawings
Fig. 1 is a schematic structural view of the present invention.
Detailed Description
The invention, as shown in fig. 1, comprises the following steps:
1) selecting materials;
2) a fluorescent quantum dot reagent formula;
3) synthesizing an aflatoxin immune antigen;
4) preparing an aflatoxin specific antibody;
5) detecting the affinity constant and the titer of the monoclonal antibody;
6) synthesizing an aflatoxin detection antigen;
7) preparing an aflatoxin b1 antibody marked by fluorescent microspheres;
8) preparing an AFB1 antibody binding pad;
9) preparing an aflatoxin b1 fluorescent quantum dot detection test reagent strip;
10) and (4) finishing.
The material in the step 1) comprises MES (2- (N-morpholine) ethanesulfonic acid), NHS (N-hydroxysuccinimide), carbodiimide (EDC), 2- (N-morpholino) ethanesulfonic acid, Bovine Serum Albumin (BSA), Tween-20, Tritonx-100 and Polyvinylpyrrolidone (PVP-40) which are products of sigma company;
HAT medium, positive serum (anti-aflatoxin b1 mouse serum) was purchased from Ha animal research, and negative serum was prepared in the laboratory.
Fluorescent microspheres were purchased from Bangs Laboratories, Inc. under the catalog number FC 02F/10930;
goat anti-mouse secondary antibody purchased from ABGAM-0500, changshabeu biotechnology limited;
nitrocellulose membrane (Nitrocellulose membrane) was purchased from Waterman and Schleicher & Schuell;
glass fiber membrane (glass fiber), absorbent paper (absorbent paper), and support plate are all available from Shanghai gold-labeled Biotech Co.
The step 2) comprises the following steps:
preparing borax buffer solution: 6.183g of boric acid, 6.864g of sodium borate; making the volume of ultrapure water to 860 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
0.1M, ph4.7mes buffer: 19.52g MES, 58.44g NaCl, ultrapure water to 1L, adjusted pH to 4.7 with NaOH, stored at 4 ℃ for no more than one week;
preparation of 10% BSA: 100.0g BSA; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% PVP-40: 100.0g PVP-40; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% Tween-20: 100.0mL Tween-20; diluting with ultrapure water to a constant volume of 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% sodium azide: 100.0g of sodium azide; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
glassware cleaning solution: 1000g of potassium dichromate and 2500mL of concentrated sulfuric acid, and adding distilled water to 10000 mL;
preparation of antibody marker dilution buffer: taking 50g of sucrose, adding 5mL of 10% BSA, 3mL of 10% Tween-20, 2mL of 10% PVP-40, 0.2M of PH7.4 PB to a volume of 100mL, and adding 200uL of 10% sodium azide;
preparing glass fiber gasket treating fluid: weighing 30.0g of trehalose, 5g of BSA and 15g of sucrose into a 1000mL reagent bottle, adding 100mL of 0.02mol/L PBS (pH7.4), 5mL of goat serum and 3.1mL of Triton-100, and adding ultrapure water to fix the volume to 1L;
preparation of sample pad treatment solution: weighing 1.8g of trehalose, 6g of BSA, 2.5g of casein, BRIJ-355 g of PVP-k405.4g and Na2HPO4 13.578g、NaH2PO4 2.8g and 4.015g of NaCl are put into a 1000mL reagent bottle, 5mL of Tween-20 is added, and ultrapure water is added to the solution to reach the constant volume of 1L;
preparation of antigen and secondary antibody diluent: weighing 2g of trehalose in a 250mL reagent bottle, adding 1mL of methanol and 5mL of 10% glycerol, adding 0.02mol/L PBS (pH7.4) and fixing the volume to 100 mL;
0.2 mol/LPB (pH 7.4): weighing 2.3g Na2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, then fixing the volume to 1000mL, and autoclavingStoring at normal temperature after 20min, wherein the pH value of the working solution is 7.4;
17.4g NaCl, 2.3g Na were weighed out in 0.02mol/L PBS (pH7.4)2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, then fixing the volume to 1000mL, sterilizing at high pressure for 20min, and then storing at normal temperature, wherein the pH value of the working solution is 7.4;
5% solution of dichlorodipotassium silane in chloroform: 25mL of dichlorodipotassium silane was dissolved in 475mL of a chloroform solution and stored under a sealed condition.
Dissolving aflatoxin b1 in absolute methanol in the step 3), adding pyridine, then adding carboxymethyl hydroxylamine hemihydrochloride, rotationally evaporating the solvent, and purifying by a column to obtain hapten;
dissolving hapten in DMF, adding an activating agent for reaction overnight, adding the reaction solution into PBS dissolved with OVA, dialyzing with 0.01MPBS after reaction to obtain aflatoxin B1 immune antigen.
The step 4) comprises the following steps:
4.1) immunization of mice;
4.2) cell fusion;
4.3) screening hybridoma cells;
4.4) preparation and purification of ascites.
The method comprises the following specific steps: 4.1) immunization of mice
Completely mixing 60 mu g of aflatoxin b1 immune antigen with an equal amount of Freund incomplete adjuvant to form emulsion, performing primary immunization on 4 female Balb/c mice numbered 1,2,3 and 4 for multiple times subcutaneously, mixing 130 mu g of aflatoxin b with an equal amount of Freund incomplete adjuvant after two weeks, and performing boosting immunization for 3 times. Collecting blood from tail vein of immunized mouse, standing at 37 deg.C for 1 hr, standing at 4 deg.C overnight, and collecting serum. And (3) carrying out indirect ELISA (enzyme-Linked immuno sorbent assay) to detect the titer of the immune serum, and setting a blank control and a negative control. Finally, Balb/c mice which can be fused with mouse myeloma cells SP/20 are determined, and 150 mu g of aflatoxin b is taken for intraperitoneal injection immunization.
4.2) cell fusion
Taking abdominal cavity macrophages of normal mice as feeder cells, taking spleen cells and SP/20 cells of immune mice, and adding a Chinese selective medium HAT. Cell fusion was performed at 37 ℃ under the action of PEG 1500. Adding serum-free IMDM to terminate fusion, centrifuging, adding newborn calf serum and prepared thymocyte, adding into semi-solid culture medium, mixing, pouring into culture dish, and adding into incubator for culture.
4.3) hybridoma cell selection
The fused cells were plated in a 96-well plate with 2. mu.g/mL of "aflatoxin b 1", and the culture supernatants of the wells in which hybridoma cells grew were aspirated and screened by the indirect ELISA described above. The negative control is SP/20 cell culture supernatant, the blank control is PBS, and the positive control is positive serum diluted 100 times with PBS. Screening was performed twice in total. Stop solution was added and absorbance was measured at two wavelengths (450 nm, 630 nm) using a 96-well plate.
4.4) preparation and purification of ascites
500 μ L of paraffin oil was intraperitoneally injected into 10-week-old Balb/c mice. Balb/c mice were injected with positive hybridoma cells 5X 105∽9×105And (4) respectively. After the abdominal cavity of the mouse is obviously enlarged, ascites is collected. Centrifuging the collected ascites fluid at 5000r/min for 10 min, collecting the supernatant, and purifying the supernatant with Protein A column. The supernatant was diluted and centrifuged, and sterilized by filtration using a 0.22 μm filter. The column was equilibrated with 10 column volumes of coupling buffer, maintaining tassel at 1 mL/min. And (4) loading, keeping the same flow rate, and collecting liquid. Then 5 times column volume of coupling buffer solution is used for passing through the column, and the antibody is eluted by the same amount of elution buffer solution. The pH was adjusted and the antibody was dialyzed overnight using PBS. And (5) subpackaging and freezing the antibody.
And 5) carrying out 2-time gradient dilution on the two monoclonal antibodies from 200 times by using a sample diluent by adopting an indirect ELISA method, and detecting.
Step 6), dissolving aflatoxin b1 in absolute ethyl alcohol, adding pyridine, then adding carboxymethyl hydroxylamine hemihydrochloride for reaction, rotationally evaporating the solvent, and purifying by a column to obtain hapten; dissolving hapten in DMF, adding an activating agent for reaction overnight, then adding the reaction solution into PBS dissolved with BSA, dialyzing with 0.01M PBS after reaction to obtain aflatoxin b1 detection antigen.
In the step 7), the fluorescent microsphere with the average diameter of 110nm and modified carboxyl and the AFB 1-resistant monoclonal antibody are used for preparing the fluorescent microsphere labeled A-type influenza virus labeled antibody according to the following method:
washing 5mg of the carboxyl modified fluorescent microspheres with MES buffer solution (0.1M, pH 4.7), centrifuging, then resuspending with 1ml of MES buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) to a final concentration of 5mM, adding NHS (N-hydroxysuccinimide) to a final concentration of 10mM, keeping the room temperature away from light, and reacting for half an hour to obtain activated carboxyl modified fluorescent microspheres;
washing the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5, and mixing 0.37mg of the AFB1 antibody to be marked and 5mg of the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5 to be fully and uniformly mixed;
reacting for 2 hours at room temperature in a dark place, and enabling the antibody and the fluorescent microsphere to form stable peptide bond covalent bonding to obtain a conjugate of the fluorescent microsphere and the AFB1 antibody;
after the reaction is finished, adding BSA solution with the final concentration of 1% (m/v) to seal the residual active carboxyl sites on the conjugate of the fluorescent microspheres and the AFB1 antibody, and reacting for 0.5 hour at room temperature in a dark place;
after completion, the cells were washed with 0.02M PBS buffer (pH7.4) and resuspended to obtain 5mg/ml fluorescent microsphere-labeled AFB1 antibody liquid, which was stored at 4 ℃ until use.
And 8) spraying the fluorescent microsphere-labeled fluorescent quantum dot-labeled AFB1 antibody mixed solution obtained in the step on a treated glass cellulose membrane by adopting an XYZ3050 membrane spraying system of Biodot according to the spraying amount of 2.5uL/cm to prepare an antibody binding pad, then transferring the antibody binding pad into a vacuum drying oven by using a grid tray, carrying out vacuum drying for 2 hours, adding a drying agent or allochroic silica gel, and sealing and storing in a refrigerator at 4 ℃.
Step 9), spraying the marked fluorescent quantum dot antibody compound on a bonding pad; detecting antigen by adopting aflatoxin B1 and coating the antigen on the surface of an NC membrane as a detection line (T line); coating goat anti-mouse IgG on the surface of a nitrocellulose membrane (NC) as a quality control line (C line); and finally assembling the combined pad, the absorbent paper and the treated sample pad into an aflatoxin B1 test strip.
After the dried envelope film with the detection line and the quality control line, the sample pad, the water absorption pad and the back plate are assembled in a 10 ten thousand clean and dry workshop in a matching way as shown in figure 1, a CM4000 cutting system of Biodot is adopted to cut the pasted paper plate, and the paper plate is put into a clamping piece for detection for standby.
Sensitivity results of AFB1 fluorescence quantum dot detection test paper strip:
the AFB1 detection antigen is subjected to antigen detection with different concentrations, and the established aflatoxin b1 fluorescent quantum dot detection test strip is used for detection, wherein the detection results are shown in the following.
TABLE 1 detection results of different sample concentrations of AFB1 fluorescent Quantum dot detection card
Concentration (ppb) | 50 | 100 | 50 | 20 | 10 | 5 | 3 | 1 | 0.5 |
Results | + | + | + | + | + | + | + | + | - |
Note: + indicates that the detection line is obvious after the sample addition reaction, -indicates that there is no detection line after the sample addition reaction.
The detection result shows that the sensitivity of AFB1 antigen detected by the AFB1 fluorescence quantum dot detection test paper strip is 1 ppb.
Specific results of the AFB1 fluorescent quantum dot detection test strip are as follows:
the established AFB1 immunofluorescence quantum dot detection card is adopted to detect various similar toxins such as AFB2, AFM1 and AFG, and detection results are negative. The detection results of other fungicins, such as vomitoxin, ochratoxin A, fumonisin, zearalenone, patulin and T-2 toxin, are negative.
Sampling detection test results:
the AFB1 immunofluorescence quantum dot detection card and the ELISA detection are carried out on 200 parts of collected test samples, and the detection results of the AFB1 immunofluorescence quantum dot detection card and the ELISA detection are shown in the table 2, which shows that the established method is high in usability.
TABLE 2 AFB1 immunofluorescent Quantum dot assay card sample assay results
Note: the ELISA method judges that the test result is positive when the calculated concentration is more than or equal to 1ppb, and judges that the test result is negative when the concentration is less than 1 ppb.
The reason for the false positive in the final sampling test of the invention is probably related to the test judgment mode, the lowest detection concentration of the AFB1 immunofluorescence quantum dot detection card is about 1ppb, in order to ensure the comparability of the two detection modes, the result of the ELISA method is converted into the concentration of aflatoxin B1, and the AFB1 detection card prepared by the test is judged to be positive when the concentration is more than or equal to 1ppb, and the test result according to the table 1 shows that the detection concentration of the AFB1 detection card prepared by the test is between 0.5 ppb and 1ppb, wherein a small amount of errors can exist.
The disclosure of the present application also includes the following points:
(1) the drawings of the embodiments disclosed herein only relate to the structures related to the embodiments disclosed herein, and other structures can refer to general designs;
(2) in case of conflict, the embodiments and features of the embodiments disclosed in this application can be combined with each other to arrive at new embodiments;
the above embodiments are only embodiments disclosed in the present disclosure, but the scope of the disclosure is not limited thereto, and the scope of the disclosure should be determined by the scope of the claims.
Claims (10)
1. A method for preparing a fluorescent quantum dot immunochromatography detection card by using aflatoxin is characterized by comprising the following steps:
1) selecting materials;
2) a fluorescent quantum dot reagent formula;
3) synthesizing an aflatoxin immune antigen;
4) preparing an aflatoxin specific antibody;
5) detecting the affinity constant and the titer of the monoclonal antibody;
6) synthesizing an aflatoxin detection antigen;
7) preparing an aflatoxin b1 antibody marked by fluorescent microspheres;
8) preparing an AFB1 antibody binding pad;
9) preparing an aflatoxin b1 fluorescent quantum dot detection test reagent strip;
10) and (4) finishing.
2. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin, according to claim 1, wherein the materials in the step 1) comprise MES, NHS, carbodiimide, 2- (N-morpholino) ethanesulfonic acid, bovine serum albumin, Tween-20, Tritonx-100 and PVP-40;
HAT culture medium, fluorescent microspheres, goat anti-mouse secondary antibody, nitrocellulose membrane, glass fiber membrane and absorbent paper.
3. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin according to claim 1, wherein the step 2) comprises the following steps:
preparing borax buffer solution: 6.183g of boric acid, 6.864g of sodium borate; making the volume of ultrapure water to 860 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
0.1M, ph4.7mes buffer: 19.52g MES, 58.44g NaCl, ultrapure water to 1L, adjusted pH to 4.7 with NaOH, stored at 4 ℃ for no more than one week;
preparation of 10% BSA: 100.0g BSA; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% PVP-40: 100.0g PVP-40; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% Tween-20: 100.0mL Tween-20; diluting with ultrapure water to a constant volume of 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% sodium azide: 100.0g of sodium azide; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
glassware cleaning solution: 1000g of potassium dichromate and 2500mL of concentrated sulfuric acid, and adding distilled water to 10000 mL;
preparation of antibody marker dilution buffer: taking 50g of sucrose, adding 5mL of 10% BSA, 3mL of 10% Tween-20, 2mL of 10% PVP-40, 0.2M of PH7.4 PB to a volume of 100mL, and adding 200uL of 10% sodium azide;
preparing glass fiber gasket treating fluid: weighing 30.0g of trehalose, 5g of BSA and 15g of sucrose into a 1000mL reagent bottle, adding 100mL of 0.02mol/L PBS (pH7.4), 5mL of goat serum and 3.1mL of Triton-100, and adding ultrapure water to fix the volume to 1L;
preparation of sample pad treatment solution: weighing 1.8g of trehalose, 6g of BSA, 2.5g of casein, BRIJ-355 g of PVP-k405.4g and Na2HPO4 13.578g、NaH2PO4 2.8g and 4.015g of NaCl are put into a 1000mL reagent bottle, 5mL of Tween-20 is added, and ultrapure water is added to the solution to reach the constant volume of 1L;
preparation of antigen and secondary antibody diluent: weighing 2g of trehalose in a 250mL reagent bottle, adding 1mL of methanol and 5mL of 10% glycerol, adding 0.02mol/L of PBS and fixing the volume to 100 mL;
0.2 mol/LPB: weighing 2.3g Na2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, diluting to 1000mL, autoclaving for 20min, and keeping at room temperatureStoring, wherein the pH value of the working solution is 7.4;
17.4g NaCl, 2.3g Na were weighed into 0.02mol/L PBS2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, then fixing the volume to 1000mL, sterilizing at high pressure for 20min, and then storing at normal temperature, wherein the pH value of the working solution is 7.4;
5% solution of dichlorodipotassium silane in chloroform: 25mL of dichlorodipotassium silane was dissolved in 475mL of a chloroform solution and stored under a sealed condition.
4. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin according to claim 1, wherein in the step 3), the aflatoxin b1 is dissolved in absolute methanol, pyridine is added, then carboxymethyl hydroxylamine hemihydrochloride is added, the solvent is removed by rotary evaporation, and the hapten is obtained by column purification;
dissolving hapten in DMF, adding an activating agent for reaction overnight, adding the reaction solution into PBS dissolved with OVA, dialyzing with 0.01MPBS after reaction to obtain aflatoxin B1 immune antigen.
5. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin according to claim 1, wherein the step 4) comprises the following steps:
4.1) immunization of mice;
4.2) cell fusion;
4.3) screening hybridoma cells;
4.4) preparation and purification of ascites.
6. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin, as claimed in claim 1, wherein the step 5) is to perform 2-fold gradient dilution of the two monoclonal antibodies with a sample diluent from 200-fold by using an indirect ELISA method for detection.
7. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin according to the claim 1, wherein the step 6) is that the aflatoxin b1 is dissolved in absolute ethyl alcohol, pyridine is added, then carboxymethyl hydroxylamine hydrochloride is added for reaction, the solvent is removed by rotary evaporation, and the hapten is obtained by column purification; dissolving hapten in DMF, adding an activating agent for reaction overnight, then adding the reaction solution into PBS dissolved with BSA, dialyzing with 0.01M PBS after reaction to obtain aflatoxin b1 detection antigen.
8. The method for preparing the fluorescence quantum dot immunochromatography detection card by using aflatoxin according to claim 1, wherein in the step 7), the fluorescence microsphere labeled influenza A virus labeled antibody is prepared by using carboxyl modified fluorescence microspheres with the average diameter of 110nm and anti-AFB 1 monoclonal antibodies according to the following method:
washing 5mg of the carboxyl modified fluorescent microspheres with MES buffer solution, centrifuging, then resuspending with 1ml of MES buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide to a final concentration of 5mM, adding NHS to a final concentration of 10mM, keeping the temperature away from the sun at room temperature, and reacting for half an hour to obtain activated carboxyl modified fluorescent microspheres;
washing the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5, and mixing 0.37mg of the AFB1 antibody to be marked and 5mg of the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5 to be fully and uniformly mixed;
reacting for 2 hours at room temperature in a dark place, and enabling the antibody and the fluorescent microsphere to form stable peptide bond covalent bonding to obtain a conjugate of the fluorescent microsphere and the AFB1 antibody;
after the reaction is finished, adding BSA solution with the final concentration of 1% to block the residual active carboxyl sites on the conjugate of the fluorescent microspheres and the AFB1 antibody, and reacting at room temperature in a dark place for 0.5 hour;
after completion, the cells were washed with 0.02M PBS buffer (pH7.4) and resuspended to obtain 5mg/ml fluorescent microsphere-labeled AFB1 antibody liquid, which was stored at 4 ℃ until use.
9. The method for preparing the fluorescence quantum dot immunochromatography detection card using aflatoxin according to claim 1, wherein in step 8), the fluorescence microsphere-labeled fluorescence quantum dot-labeled AFB1 antibody mixed solution obtained in the above step is sprayed onto the treated glass cellulose membrane by using XYZ3050 film spraying system of Biodot according to the spraying amount of 2.5uL/cm to prepare the antibody binding pad, and then the antibody binding pad is moved into a vacuum drying oven by using a grid tray, vacuum-dried for 2h, added with a drying agent or allochroic silica gel, sealed and stored in a refrigerator at 4 ℃.
10. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin according to the claim 1, wherein in the step 9), the marked fluorescence quantum dot antibody compound is sprayed on the bonding pad; detecting an antigen by adopting aflatoxin B1 and coating the antigen on the surface of an NC membrane as a detection line; coating goat anti-mouse IgG on the surface of the nitrocellulose membrane to serve as a quality control line; and finally assembling the combined pad, the absorbent paper and the treated sample pad into an aflatoxin B1 test strip.
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