A labeling method capable of improving fluorescence amplification effect and application thereof in biological toxin detection

Abstract

The application discloses a labeling method capable of improving fluorescence amplification effect and application thereof in biological toxin detection, and the labeling method comprises the following steps: S1, double-fluorescent microsphere preparation: S1.1, performing carboxyl site blocking on aflatoxin B1, vomitoxin and zearalenone antibodies; S1.2, performing coupling on amino sites of the closed aflatoxin B1, vomitoxin and zearalenone antibodies; S1.3, activating the coupled aflatoxin B1, vomitoxin and zearalenone antibodies; and S1.4, coupling the activated aflatoxin B1, vomitoxin and zearalenone antibodies with magnetic fluorescent microspheres. The labeling method capable of improving fluorescence amplification effect and application thereof in biological toxin detection have the advantages that the sensitivity and detection limit of products can be improved, and the detection difference caused by the interference of sample matrix can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food detection, and particularly relates to a labeling method capable of improving fluorescence amplification effect and application thereof in biological toxin detection. BACKGROUND

[0002] For the existing immunological rapid detection technology, the following methods are used to detect mycotoxins in food: enzyme-linked immunosorbent assay kit, colloidal gold rapid detection card, chemiluminescence rapid detection kit and fluorescent immunochromatographic rapid detection card. The enzyme-linked immunosorbent assay kit can only detect one kind of mycotoxin, and the use thereof requires higher requirements for the operator, and the use of an enzyme-labeled instrument and computer software is required for calculation, which is not conducive to on-site rapid detection. The colloidal gold rapid detection card can simultaneously detect multiple mycotoxins in the same product, but the detection result needs to be observed by the naked eye, and the detection sensitivity cannot meet the national requirements for the residue limit of mycotoxins in food. The chemiluminescence rapid detection kit can also simultaneously detect multiple mycotoxins in the same product, but a special chemiluminescence detector is required, which is expensive and has high maintenance costs, and is not conducive to on-site rapid detection. The fluorescent immunochromatographic rapid detection card can also simultaneously detect multiple mycotoxins in the same product, and the detection form is similar to colloidal gold detection, which is not high in cost and is the mainstream scheme for the existing rapid detection of mycotoxin residues in food, but the technical platform is limited by the performance of the immunogen material, and cannot fully meet the requirements for the detection of mycotoxins in food.

[0003] Therefore, it is necessary to provide a new labeling method capable of improving fluorescence amplification effect and application thereof in biological toxin detection to solve the above technical problems. SUMMARY

[0004] Based on the technical problems existing in the background art, the present application provides a labeling method capable of improving fluorescence amplification effect and application thereof in biological toxin detection, which mainly starts from the amplification effect of fluorescent microspheres, enhances the signal amplification factor in the whole reaction system, optimizes each process formula, improves the sensitivity of the whole reaction system, and achieves the purpose of improving the detection sensitivity.

[0005] The labeling method capable of improving fluorescence amplification effect provided by the present application comprises the following steps:

[0006] S1: preparation of double fluorescent microspheres:

[0007] S1.1: blocking of carboxyl sites of aflatoxin B1, vomitoxin and zearalenone antibodies;

[0008] S1.2: coupling of amino sites of the blocked aflatoxin B1, vomitoxin and zearalenone antibodies;

[0009] S1.3: Activating the aflatoxin B1, vomitoxin, and zearalenone antibodies that have been coupled;

[0010] S1.4: Coupling the activated aflatoxin B1, vomitoxin, and zearalenone antibodies with magnetic fluorescent microspheres;

[0011] S1.5: Blocking the magnetic fluorescent microspheres after coupling;

[0012] S1.6: Labeling the amplified signal magnetic fluorescent microspheres;

[0013] S1.7: Mixing a portion of the labeled and blocked magnetic fluorescent microspheres from S1.5 with a portion of the labeled and blocked magnetic fluorescent microspheres from S1.6, mixing gently on a shaker at room temperature, placing the coupled centrifuge tube on a magnetic stand for a period of time, removing all liquid, removing the centrifuge tube from the magnetic stand, adding an appropriate amount of 0.01M MES solution, sonicating to mix, placing the centrifuge tube on the magnetic stand for a period of time, removing all liquid, repeating this step 2 times, adding a certain amount of preservation solution, and then sonicating to disperse;

[0014] S1.8: Labeling the quality control line fluorescent microspheres;

[0015] S2: Preparing nitrocellulose membranes:

[0016] S2.1: Preparing coating solutions;

[0017] S2.1: Cutting the Sartorius 140 membrane into 30cm segments, sticking the cut membranes to the bottom plate, and then placing them on a multi-point membrane drawing machine to coat the various coating solutions prepared in S2.1 on the nitrocellulose membranes simultaneously;

[0018] S3: Preparing the binding pad and sample pad:

[0019] S3.1: Pouring the sample pad treatment solution into a plastic tray, soaking the glass fiber in the tray for a certain period of time, removing the residual liquid on the surface, and then placing it in a forced air drying oven for a period of time;

[0020] S3.2: Pouring the binding pad treatment solution into a plastic tray, soaking the glass fiber in the tray for a certain period of time, removing the residual liquid on the surface, and then placing it in a forced air drying oven for a period of time;

[0021] S3.3: Mixing the labeled fluorescent microspheres;

[0022] S3.4: Spraying the fluorescent signal binding pad;

[0023] S4: Assembling the detection card:

[0024] S4.1: Perform large plate assembly, cut the sprayed binding pad into small strips, cut the treated sample pad into small strips, cut the absorbent paper into small strips, tear off the glue at the lower end of the nitrocellulose membrane detection line, paste the binding pad, paste the sample pad below the binding pad, the bottom end is flush with the PVC bottom plate; tear off the glue at the upper end of the nitrocellulose membrane quality control line, paste the absorbent paper;

[0025] S4.2: Cut the large plate, put the cut test strip into the plastic card shell and press tightly, then put it into an aluminum foil bag, and finally seal it with a heat sealing machine to obtain a card strip;

[0026] S5: Sample processing and detection:

[0027] S5.1: Process the sample, weigh an appropriate amount of dry food sample into a centrifuge tube, add an appropriate amount of 70% methanol-water, then perform vortex shaking and centrifugation in turn, take the supernatant, and mix with the sample diluent to prepare the test solution;

[0028] S5.2: Take 100 μL of the test solution in S5.1 and add it to the card strip assembled in S4.2, react for 15 min at room temperature, then read the results.

[0029] Preferably, in S1, the aflatoxin B1, vomitoxin, and zearalenone antibodies are all mouse monoclonal antibodies.

[0030] Preferably, in S1, the storage solution used in S1.7 is 0.01M Tris-HCl solution, PH=7.6, containing 0.1-1% BSA, 2-10% trehalose, 2-10% sucrose, 0.2-2% mannitol, 0.01-0.1% PVP K15, 0.03-0.1% TW-20, and 0.02-0.05% casein.

[0031] Preferably, in S2, the line concentration in S2.1 is 0.8 μL / cm, and after completing the line, it is placed in a 45°C air-drying oven for 24 h and then sealed.

[0032] Preferably, in S3, the total amount of sample pad treatment solution used in S3.1 is 1L, containing 4.55g of dodecahydrodisodium phosphate, 4.22g of disodium hydrogen phosphate dihydrate, 6.04g of sodium chloride, 0.5-2% casein, 0.03-0.05% PC 300, 0.5-1% BSA, 1-5% hydroxypropyl-β-cyclodextrin, 0.3-1% PVP K40, 0.1-0.5% Tween-20, and 0.01-0.04% L-lysine.

[0033] Preferably, the total amount of binding pad treatment liquid used in S3.2 in S3 is 1L, containing 5.37g of dodecahydrate disodium hydrogen phosphate, 3.9g of dihydrate sodium dihydrogen phosphate, 9g of sodium chloride, 0.1-0.3% of glycine, 0.03-0.05% of PC 300, 0.1-0.5% of BSA, 0.5-1% of PVA, 0.2-0.5% of PVP K30, and 0.1-0.3% of TX-100.

[0034] Preferably, the sample diluent used in S5.1 in S5 is 0.05M PBS with PH = 6.6, containing 0.1-0.2% casein, 0.05-0.1% OVA, 0.01-0.03% PC 300, 0.001-0.005% ascorbic acid, and 0.002-0.005% PEG4000.

[0035] Preferably, the reacted card strip in S5.2 in S5 is placed into a fluorescence reader to read the results.

[0036] The application also provides a labeling method capable of improving fluorescence amplification effect and application thereof in biological toxin detection.

[0037] The application has the following beneficial effects:

[0038] ①By providing a new labeling method, the ability of fluorescence labeling signal amplification can be greatly improved. The use of the labeling method can improve the sensitivity, detection limit, and detection speed of the product. When labeling antibodies, the use of PEG long chains can avoid hindering the binding sites of antigens and antibodies during multi-stage fluorescence material coupling.

[0039] ②Through the development of the treatment formula of the binding pad, sample pad, and sample diluent, the detection difference caused by the interference of the sample matrix during the detection of food samples can be reduced. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 The application provides a schematic diagram of a double fluorescence microsphere-antibody conjugate prepared by the labeling method capable of improving fluorescence amplification effect. Figure 2 The application provides a schematic diagram of a detection card strip prepared by the labeling method capable of improving fluorescence amplification effect. DETAILED DESCRIPTION

[0041] The application will be further described below in combination with specific embodiments.

[0042] EMBODIMENT

[0043] The labeling method capable of improving fluorescence amplification effect provided in the embodiment includes the following steps:

[0044] S1: Preparation of double fluorescence microspheres:

[0045] S1.1: Carboxyl site blocking of aflatoxin B1 (AFB1), vomitoxin (DON), and zearalenone (ZEN) antibodies, dilute aflatoxin B1, vomitoxin, and zearalenone antibodies to 0.2 mg / mL with 0.1M PBS (PH = 7.6), take 0.5 mL of the diluted antibodies into a centrifuge tube, sequentially add 60 μL of 0.1M EDC, 30 μL of 0.1M NHS, 0.1 mL of 1.0M ammonia water, and adjust the PH of the mixture to 7.5-8.0. After mixing and shaking gently on a shaker for 30 min at room temperature, elute with 0.1M PBS (PH = 7.2) as the eluent, remove the supernatant, repeat the elution for 3 times, and then dilute to 0.5 mL with 0.01M PBS;

[0046] S1.2: Coupling of the amino sites of the blocked aflatoxin B1, vomitoxin, and zearalenone antibodies, take 0.5 mL of the blocked antibodies in S1.1, add 0.1 mL of 0.2M NHS-PEG-COOH (molecular weight 1-5K), mix and shake gently on a shaker for 60 min at room temperature, elute with 0.01M PBS (PH = 7.2) as the eluent, remove the supernatant, repeat the elution for 3 times, and then dilute to 0.5 mL with 0.01M PBS

[0047] S1.3: Activation of the coupled aflatoxin B1, vomitoxin, and zearalenone antibodies, take 0.5 mL of the coupled antibodies in S1.2 into a centrifuge tube, sequentially add 40 μL of 0.1M EDC, 20 μL of 0.1M NHS, mix and shake gently on a shaker for 30 min at room temperature, elute with 0.01M PBS (PH = 7.2) as the eluent, remove the supernatant, dilute to 0.1 mL with 0.01M PBS;

[0048] S1.4: coupling the activated aflatoxin B1, vomitoxin, zearalenone antibody with magnetic fluorescent microspheres, taking 40 μL of magnetic time-resolved fluorescent microspheres (particle size 200-500 nm, excitation wavelength 350-370 nm, absorption wavelength 610-630 nm, and free groups on the surface of the microspheres are amino groups), adding 0.36 mL of 0.1M MES (PH=6.0), manually shaking for 1 min, centrifuging at 4°C and 12000 r / min for 15 min, discarding the supernatant, adding 0.4 mL of 0.02M MES solution (PH=6.5), ultrasonic mixing for 1 min, adding 0.1 mL of the activated antibody in S1.3, mixing and reacting for 30 min at room temperature on a shaker, placing the coupled centrifuge tube in a magnetic stand, and standing for 5 min. After removing the supernatant, 0.4 mL of 0.02M MES solution (PH=6.5) was added, ultrasonic mixing was performed for 1 min, the centrifuge tube was placed in a magnetic stand, and standing was performed for 5 min. After removing all the liquid, the step was repeated twice, 0.5 mL of 0.02M MES solution (PH=6.5) was added, ultrasonic dispersion was performed for 1 min, and it was prepared for use;

[0049] S1.5: blocking the coupled magnetic fluorescent microspheres, taking 0.5 mL of the IgG-coupled magnetic fluorescent microspheres in S1.4, adding 0.15 mL of 0.1M NHS-biotin, mixing and reacting for 60 min at room temperature on a shaker, placing the coupled centrifuge tube in a magnetic stand, and standing for 5 min. After removing all the liquid, 0.4 mL of 0.02M MES solution (PH=6.5) was added, ultrasonic mixing was performed for 1 min, the centrifuge tube was placed in a magnetic stand, and standing was performed for 5 min. After removing all the liquid, the step was repeated twice, 0.5 mL of 0.02M MES solution (PH=6.5) was added, and ultrasonic dispersion was performed for 1 min;

[0050] S1.6: Labeling the amplified signal magnetic fluorescent microspheres, take polystyrene time-resolved fluorescent microspheres (particle size 10-30 nm, excitation wavelength 350-370 nm, absorption wavelength 610-630 nm, free groups on the surface of the microspheres are carboxyl) 0.1 mL, add 0.9 mL 0.1M MES (PH=6.0), shake by hand for 1 min, centrifuge at 15000 r / min for 20 min at 4°C, and discard the supernatant. Add 1.0 mL 0.1M MES (PH=6.0), ultrasonic dispersion for 1 min, add 0.2 mL 0.1M EDC, 0.1 mL 0.1M NHS in turn, mix well under room temperature and slight shaking on a shaker for 30 min, centrifuge at 15000 r / min for 20 min at 4°C, and discard the supernatant. Add 1 mL 0.1M MES (PH=6.0), ultrasonic dispersion for 1 min, centrifuge at 15000 r / min for 20 min at 4°C, and discard the supernatant. Add 1 mL 0.1M MES (PH=6.0), ultrasonic dispersion for 1 min, add 2 mg / mL streptavidin 0.5 mL, mix well under room temperature and slight shaking on a shaker for 30 min, centrifuge at 15000 r / min for 20 min at 4°C, and discard the supernatant. Add 1 mL 0.1M MES (PH=6.0), ultrasonic dispersion for 1 min, add 0.1 mL 10% BSA, mix well under room temperature and slight shaking on a shaker for 60 min, centrifuge at 15000 r / min for 20 min at 4°C, and discard the supernatant. Add 1 mL 0.02M MES (PH=6.5), ultrasonic dispersion for 1 min, centrifuge at 15000 r / min for 20 min at 4°C, and discard the supernatant. Add 1 mL 0.02M MES (PH=6.5), ultrasonic dispersion for 1 min;

[0051] S1.7: Take 0.5 mL of the labeled and blocked magnetic fluorescent microspheres in S1.5 and mix with 1 mL of the labeled and blocked magnetic fluorescent microspheres in S1.6, mix well under room temperature and slight shaking on a shaker for 60 min, place the centrifuge tube after coupling on a magnetic stand, stand for 5 min, remove all the liquid, and take the centrifuge tube off the magnetic stand. Add 0.5 mL 0.01M MES solution (PH=6.5), ultrasonic dispersion for 1 min, place the centrifuge tube on a magnetic stand, stand for 5 min, remove all the liquid, and repeat this step 2 times. Add 0.2 mL of a preservation solution (0.01M Tris-HCl solution, PH 7.6, containing 0.1-1% BSA, 2-10% trehalose, 2-10% sucrose, 0.2-2% mannitol, 0.01-0.1% PVP K15, 0.03-0.1% TW-20, 0.02-0.05% carboxymethyl cellulose), ultrasonic dispersion for 1 min;

[0052] S1.8: Perform quality control line fluorescent microspheres labeling, take polystyrene time-resolved fluorescent microspheres (particle size 100-200 nm, excitation wavelength 350-370 nm, absorption wavelength 610-630 nm, free groups on the surface of the microspheres are carboxyl) 0.05 mL, add 0.45 mL 0.1M MES (PH=6.0), manually shake for 1 min, centrifuge at 15000 r / min for 20 min at 4°C, discard the supernatant. Add 0.5 mL 0.1M MES (PH=6.0), ultrasonic dispersion for 1 min, add 0.05 mL 0.1M EDC, 0.025 mL 0.1M NHS in turn, mix well under slight shaking on a shaker at room temperature for 30 min, centrifuge at 15000 r / min for 20 min at 4°C, discard the supernatant. Add 0.5 mL 0.1M MES (PH=6.0), ultrasonic dispersion for 1 min, centrifuge at 15000 r / min for 20 min at 4°C, discard the supernatant. Add 0.5 mL 0.1M MES (PH=6.0), ultrasonic dispersion for 1 min, add 2 mg / mL rabbit anti-chicken IgY antibody 0.25 mL, mix well under slight shaking on a shaker at room temperature for 30 min, centrifuge at 15000 r / min for 20 min at 4°C, discard the supernatant. Add 0.5 mL 0.1M MES (PH=6.0), ultrasonic dispersion for 1 min, add 0.1 mL 10% BSA, mix well under slight shaking on a shaker at room temperature for 60 min, centrifuge at 15000 r / min for 20 min at 4°C, discard the supernatant. Add 0.2 mL 0.02M MES (PH=6.5), ultrasonic dispersion for 1 min, centrifuge at 15000 r / min for 20 min at 4°C, discard the supernatant. Add 0.2 mL storage solution (0.01M Tris-HCl solution, PH=7.6, containing 0.1-1% BSA, 2-10% trehalose, 2-10% sucrose, 0.2-2% mannitol, 0.01-0.1% PVP K15, 0.03-0.1% TW-20, 0.02-0.05% carboxymethyl cellulose), ultrasonic dispersion for 1 min;

[0053] S1.9: Perform antibody final concentration selection, AFB1 final concentration range is 0.05-0.2 mg / mL; ZEN final concentration range is 0.1-0.3 mg / mL; DON final concentration range is 0.02-0.08 mg / mL, and rabbit anti-chicken IgY final concentration range is 0.2-0.5 mg / mL. AFB1, ZEN, and DON antibodies are all mouse monoclonal antibodies;

[0054] S1.10: Perform the spraying of the fluorescence signal binding pad, mix the labeled AFB1, ZEN, DON, rabbit anti-chicken IgY fluorescence microspheres preservation solution according to the volume ratio of 7:5:10:2, and spray it on the treated binding pad with a spraying amount of 0.8 μL / cm. After baking in a 45°C air drying oven for 48h, seal it;

[0055] S2: Prepare nitrocellulose membrane:

[0056] S2.1: Prepare the coating solution, dilute the chicken IgY antibody to 0.5-1.0 mg / mL with coating buffer 0.05M PBS (PH=7.4); dilute AFB1-BSA to 0.1 mg / mL-0.3 mg / mL with coating buffer 0.01M PBS (PH 7.2, containing 2-5% trehalose, 5-8% methanol, 0.5-2% sucrose, 0.01-0.05% PC300); dilute ZEN-BSA to 0.2 mg / mL-0.5 mg / mL with coating buffer 0.01M PBS (PH 7.2, containing 2-5% trehalose, 5-8% methanol, 0.5-2% sucrose, 0.01-0.05% PC300); dilute ZEN-BSA to 0.2 mg / mL-0.5 mg / mL with coating buffer 0.01M PBS (PH 7.2, containing 2-5% trehalose, 5-8% methanol, 0.5-2% sucrose, 0.01-0.05% PC300);

[0057] S2.1: Spray and draw lines, cut the Sediolis 140 membrane into 30cm segments, remove the middle adhesive paper on the PVC bottom plate, paste the cut membrane on the bottom plate, and place it on the multi-point membrane drawing machine to coat the liquid of 2.2.2.1 on the nitrocellulose membrane at the same time. The line concentration is 0.8 μL / cm, and it is placed in a 45°C air drying oven for 24h and sealed after baking.

[0058] S3: Prepare the binding pad and sample pad:

[0059] S3.1: Pour the sample pad treatment solution into a plastic dish made of PP material, soak the glass fiber (8975 or 8955, etc.) in the dish for 5 min, take out the glass fiber, wipe off the residual liquid on the surface, and place it in a 50°C air drying oven for 72h.

[0060] S3.2: Pour the binding pad treatment solution into a plastic dish made of PP material, soak the glass fiber (8975 or 8955, etc.) in the dish for 5 min, take out the glass fiber, wipe off the residual liquid on the surface, and place it in a 50°C air drying oven for 72h.

[0061] S4: Assemble the detection card:

[0062] S4.1: The sprayed binding pad is cut into a small strip with a width of 0.8 cm and a length of 30 cm, the treated sample pad is cut into a small strip with a width of 1.6 cm and a length of 30 cm, and the absorbent paper is cut into a small strip with a width of 1.6 cm and a length of 30 cm. The glue at the lower end of the NC film detection line is torn open, the binding pad is attached, the binding pad is pressed against the lower end of the NC film by 0.2 cm, the sample pad is attached below the binding pad, and the lower end is flush with the PVC bottom plate; the glue at the upper end of the NC film quality control line is torn open, the absorbent paper is attached, and the absorbent paper is pressed against the upper end of the NC film by 0.2 cm;

[0063] S4.2: The attached large plate is cut into a test strip with a width of 4 mm using a cutting machine, the cut test strip is placed into the lower cover of a plastic card shell, the upper cover of the plastic card shell is covered, and the card is pressed tightly using a card pressing machine. The aluminum foil bag is loaded, a drying agent is placed in the aluminum foil bag, and the aluminum foil bag is sealed using a hot plastic sealing machine to obtain a detection card strip;

[0064] S5: Sample processing and detection:

[0065] S5.1: 10 g of dried food sample is weighed into a 50 mL centrifuge tube, 25 mL of 70% methanol-water is added, and the mixture is vigorously vortexed and shaken for 10 min, centrifuged at 4000 r / min for 10 min at room temperature, 100 μL of supernatant is taken, 300 μL of sample diluent is added, and the mixture is mixed to obtain a test solution;

[0066] S5.2: 100 μL of the test solution in S5.1 is added to the detection card strip assembled in S4.2, and the mixture is reacted for 15 min at room temperature, and then the result can be read. 100 μL of the test solution in S5.1 is added to the card strip assembled in S4.2, and the mixture is reacted for 15 min at room temperature, and then the result is read in a fluorescence reader. The minimum detection concentration of AFB1 is 0.02 μg / Kg, the minimum detection concentration of ZEN is 2 μg / Kg, and the minimum detection concentration of DON is 10 μg / Kg. The detection range of AFB1 is 0.02 μg / Kg-200 μg / Kg, the detection range of ZEN is 2 μg / Kg-20000 μg / Kg, and the detection range of AFB1 is 10 μg / Kg-100000 μg / Kg.

[0067] The total amount of the sample pad treatment solution used in S3.1 is 1L, containing 4.55g of disodium hydrogen phosphate dodecahydrate, 4.22g of sodium dihydrogen phosphate dihydrate, 6.04g of sodium chloride, 0.5-2% of casein, 0.03-0.05% of PC 300, 0.5-1% of BSA, 1-5% of hydroxypropyl-β-cyclodextrin, 0.3-1% of PVP K40, 0.1-0.5% of Tween-20, and 0.01-0.04% of L-lysine; the total amount of the binding pad treatment solution used in S3.2 is 1L, containing 5.37g of disodium hydrogen phosphate dodecahydrate, 3.9g of sodium dihydrogen phosphate dihydrate, 9g of sodium chloride, 0.1-0.3% of glycine, 0.03-0.05% of PC 300, 0.1-0.5% of BSA, 0.5-1% of PVA, 0.2-0.5% of PVP K30, and 0.1-0.3% of TX-100; and the sample dilution solution used in S5.1 is 0.05M PBS with PH=6.6, containing 0.1-0.2% of casein, 0.05-0.1% of OVA, 0.01-0.03% of PC 300, 0.001-0.005% of ascorbic acid, and 0.002-0.005% of PEG 4000.

[0068] In this embodiment, the purpose of S1.1 is to block the carboxyl sites on the antibody, so as not to activate the carboxyl sites of the antibody itself when the antibody conjugate is activated in S1.3, and to prevent the antibody from being directly coupled to the microspheres by the carboxyl group. Unlike the prior art, the prior art does not block the carboxyl group on the antibody, which may have the possibility of self-crosslinking, reduces the batch stability, and uses a 5KD (can permeate small molecules, and intercepts large molecular proteins) ultrafiltration centrifuge tube to remove unreacted EDC, NHS, ammonia and the like. The prior art uses ordinary centrifugation (not thorough, easy to aggregate), dialysis (too long, large loss of protein activity), purification column purification (requires packing material, requires high requirements for users, and the purification time is longer than that of the ultrafiltration centrifuge tube), and the like to remove excess materials. The use of the ultrafiltration centrifuge tube is the most convenient and fastest.

[0069] The purpose of S1.2 is to connect the amino group on the antibody with blocked carboxyl sites to NHS-PEG-COOH (molecular weight 1K-5K), and then to connect the carboxyl group on NHS-PEG-COOH to the amino group on the microspheres. The purpose of using NHS-PEG-COOH is to provide a certain spatial gap between the antibody and the microspheres, so that the subsequent coupling of the amplification signal microspheres to the microspheres will not block the active sites of the antibody and the antigen. The prior art generally directly couples the antibody to the microspheres, without adding a long chain, which may hinder the specificity of the antibody and the antigen. The reason for using a 15KD centrifuge tube for centrifugation is the same as above.

[0070] The purpose of step S1.3 is to activate the carboxyl group of the NHS-PEG-COOH group on the antibody coupled with NHS-PEG-COOH, so that it can be coupled to the amino microspheres.

[0071] The purpose of step S1.4 is to couple the activated antibody to the magnetic fluorescent microspheres, and the particle size is selected to be 200-500 nm because microspheres of this size can provide a better specific surface area, enough antibody and amplified fluorescent microspheres; the amplification effect is not enough if the particle size is too small, and it is easy to produce aggregation if the particle size is too large; the reason for using magnetic fluorescent microspheres is to facilitate subsequent separation, and the existing technology mainly uses centrifugation.

[0072] The purpose of step S1.5 is to block the amino sites on the magnetic fluorescent microspheres and reserve biotin groups that can be coupled to the fluorescent amplification signal microspheres, and NHS-biotin is used, which is a small molecule material that can bind more to the amino groups on the microspheres and can bind more fluorescent microspheres for signal amplification.

[0073] The purpose of step S1.6 is to couple the streptavidin-labeled microspheres for amplifying the fluorescent effect, because of the high affinity of streptavidin and biotin, the fluorescent microspheres for signal amplification can be easily connected to the magnetic fluorescent microspheres.

[0074] The purpose of step S1.7 is to couple the microspheres for amplifying the fluorescent signal in S1.6 to the magnetic fluorescent microspheres in S1.5 to prepare the most important double fluorescent signal amplification microspheres of the patent, and the use of the streptavidin-biotin system can maximize the coupling efficiency and the use of mild conditions can maximize the activity of the antibody

[0075] The purpose of the fluorescence microspheres labeled in the quality control line of step S1.8 is to use a separate system, which is to prepare a more stable C-line reference system, because the ratio of the fluorescence intensity of the T-line to the fluorescence intensity of the C-line is used to prepare the fitting curve in this method, and if only the T-line fluorescence signal intensity is used to prepare the fitting curve, the signal intensity will be greatly different due to the influence of factors such as sample loading amount, reaction temperature and reaction time, which is not conducive to the stability of the detection result.

[0076] This example only details the specific method of two-layer fluorescent labeling, and in practice, this scheme can be applied to multiple fluorescent labeling, such as using anti-BSA mouse monoclonal antibody to label fluorescent microspheres, and then connecting with the double fluorescent microspheres of S1.7 due to specificity, and then separating by magnetic separation method, three fluorescent labeled microspheres can be obtained. By analogy.

[0077] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A labeling method capable of improving a fluorescent amplification effect, characterized by, Includes the following steps: S1: Preparation of dual fluorescent microspheres: S1.1: Blocking the carboxyl sites of aflatoxin B1, vomitoxin, and zearalenone antibodies: Dilute aflatoxin B1, vomitoxin, and zearalenone antibodies to 0.2 mg / mL with 0.1 M PBS. Take 0.5 mL of the diluted antibody into a centrifuge tube, add 60 μL of 0.1 M EDC, 30 μL of 0.1 M NHS, and 0.1 mL of 1.0 M ammonia water in sequence, and adjust the pH of the mixture to 7.5-8.

0. Under room temperature, gently shake and mix on a shaker for 30 min. Use 0.1 M PBS as the elution buffer and centrifuge with a 5 KD ultrafiltration centrifuge tube to remove the supernatant. Repeat the elution 3 times, and then make up the volume to 0.5 mL with 0.01 M PBS. S1.2: Conjugation of the amino sites of the blocked aflatoxin B1, vomitoxin, and zearalenone antibodies: Take 0.5 mL of the blocked antibody from S1.1, add 0.1 mL of 0.2 M NHS-PEG-COOH, and gently shake on a shaker for 60 min at room temperature. Use 0.01 M PBS as the elution buffer and centrifuge with an ultrafiltration centrifuge tube with a molecular weight of 15 KD to remove the supernatant. Repeat the elution 3 times, and then make up the volume to 0.5 mL with 0.01 M PBS. S1.3: Activate the conjugated aflatoxin B1, vomitoxin, and zearalenone antibodies: Take 0.5 mL of the conjugated antibody from S1.2 into a centrifuge tube, add 40 μL of 0.1 M EDC and 20 μL of 0.1 M NHS sequentially, and gently shake on a shaker for 30 min at room temperature. Use 0.01 M PBS as the elution buffer and centrifuge with a 5 KD ultrafiltration centrifuge tube to remove the supernatant. Make up the volume to 0.1 mL with 0.01 M PBS. S1.4: Activated aflatoxin B1, vomitoxin, and zearalenone antibodies were coupled to magnetic fluorescent microspheres. 40 μL of magnetic time-resolved fluorescent microspheres with a particle size of 200–500 nm were added, along with 0.36 mL of 0.1 M MES. The mixture was manually shaken for 1 min, centrifuged at 12000 r / min for 15 min at 4°C, and the supernatant was discarded. 0.4 mL of 0.02 M MES solution was added, and the mixture was sonicated for 1 min. 0.1 mL of the activated antibody from S1.3 was added, and the mixture was gently shaken on a shaker for 30 min at room temperature. The coupled centrifuge tube was placed on a magnetic rack and allowed to stand for 5 min. After removing the supernatant, 0.4 mL of 0.02 M MES solution was added again, and the mixture was sonicated for 1 min. The centrifuge tube was placed on a magnetic rack and allowed to stand for 5 min. All liquid was removed. This process was repeated twice. Finally, 0.5 mL of 0.02 M MES solution was added, and the mixture was sonicated for 1 min. The mixture was then ready for use. S1.5: Blocking the coupled magnetic fluorescent microspheres: Take 0.5 mL of the IgG-coupled magnetic fluorescent microspheres from S1.4, add 0.15 mL of 0.1 M NHS-Biotin, and gently shake and mix on a shaker for 60 min at room temperature. Place the coupled centrifuge tube on a magnetic rack and let it stand for 5 min. After removing all the liquid, add 0.4 mL of 0.02 M MES solution and sonicate for 1 min. Place the centrifuge tube on a magnetic rack and let it stand for 5 min. Remove all the liquid. Repeat this step twice. Add 0.5 mL of 0.02 M MES solution and sonicate for 1 min. S1.6: Label the magnetic fluorescent microspheres for amplified signals. Take 0.1 mL of polystyrene time-resolved fluorescent microspheres with a particle size of 10–30 nm, add 0.9 mL of 0.1 M MES, shake manually for 1 min, centrifuge at 15000 r / min for 20 min at 4 °C, discard the supernatant, add 1.0 mL of 0.1 M MES, sonicate for 1 min, then add 0.2 mL of 0.1 M EDC and 0.1 mL of 0.1 M MNHS sequentially. Mix gently on a shaker at room temperature for 30 min, centrifuge at 15000 r / min for 20 min at 4 °C, discard the supernatant, add 1 mL of 0.1 M MES, sonicate for 1 min, centrifuge at 15000 r / min for 20 min at 4 °C, discard the supernatant, add 1 mL of 0.1 M MES, sonicate for 1 min, centrifuge at 15000 r / min for 20 min at 4 °C, discard the supernatant, and add 1 mL of 0.1 M M... MES, ultrasonically dispersed for 1 min, 0.5 mL of 2 mg / mL streptavidin was added, and the mixture was gently shaken on a shaker at room temperature for 30 min. After centrifugation at 15000 r / min for 20 min at 4 °C, the supernatant was discarded, 1 mL of 0.1 M MES was added, ultrasonically dispersed for 1 min, 0.1 mL of 10% BSA was added, and the mixture was gently shaken on a shaker at room temperature for 60 min. After centrifugation at 15000 r / min for 20 min at 4 °C, the supernatant was discarded, 1 mL of 0.02 M MES was added, ultrasonically dispersed for 1 min, and centrifuged at 15000 r / min for 20 min at 4 °C. After centrifugation, the supernatant was discarded, 1 mL of 0.02 M MES was added, and ultrasonically dispersed for 1 min. S1.7: Take a portion of the labeled and blocked magnetic fluorescent microspheres from S1.5 and mix them with a portion of the labeled and blocked magnetic fluorescent microspheres from S1.

6. Under room temperature conditions, gently shake and mix them on a shaker. Place the coupled centrifuge tube on a magnetic rack and let it stand for a period of time to remove all the liquid. Remove the centrifuge tube from the magnetic rack, add an appropriate amount of 0.01 MMES solution, and sonicate to mix. Place the centrifuge tube on a magnetic rack and let it stand for a period of time to remove all the liquid. Repeat this step twice. Add a certain amount of preservation solution and then sonicate to disperse. S1.8: Labeling of control line fluorescent microspheres; S2: Preparation of nitrocellulose membrane: S2.1: Preparation of coating solution; S2.2: Cut Sartorius 140 membrane into 30cm segments, attach the cut membrane to the base plate, and then place it on a multi-point coating machine to simultaneously coat the nitrocellulose membrane with the coating liquids prepared in S2.

1. S3: Preparation of the binding pad and sample pad: S3.1: Pour the sample pad treatment solution into a plastic tray, soak the glass fiber in the tray for a certain period of time, remove it, remove the residual liquid on the surface, and then bake it in a forced-air drying oven for a period of time. S3.2: Pour the bonding pad treatment solution into a plastic tray, soak the glass fiber in the tray for a certain period of time, remove it, remove the residual liquid on the surface, and bake it in a forced-air drying oven for a period of time. S3.3: Mixture of labeled fluorescent microspheres; S3.4: Spray coating of fluorescent signal binding pads; S4: Assemble the test card: S4.1: Perform large board assembly, cut the sprayed bonding pad into small strips, cut the treated sample pad into small strips, cut the absorbent paper into small strips, peel off the adhesive at the bottom of the nitrocellulose membrane test line, attach the bonding pad, attach the sample pad below the bonding pad, with the bottom flush with the PVC base plate; peel off the adhesive at the top of the nitrocellulose membrane control line, and attach the absorbent paper. S4.2: Cut the large board, put the cut test strips into the plastic card and press them tightly, then put them into the aluminum foil bag, and finally seal them with a thermoplastic sealing machine to obtain the card strip; S5: Sample Processing and Detection S5.1: To process the sample, weigh an appropriate amount of dried food sample into a centrifuge tube, add an appropriate amount of 70% methanol-water, and then perform vortex shaking and centrifugation in sequence. Take the supernatant and mix it with the sample dilution solution to prepare the test solution. S5.2: Take 100µL of the test solution from S5.1 and add it to the assembled card strip from S4.

2. React at room temperature for 15 minutes, and then the result can be read.

2. The labeling method capable of improving a fluorescent amplification effect according to claim 1, wherein In S1, aflatoxin B1, vomitoxin, and zearalenone antibody are all mouse monoclonal antibodies.

3. The method of claim 1, wherein the fluorescent amplification effect is improved. In S1, the preservation solution used in S1.7 is a 0.01M Tris-HCl solution with pH=7.6, containing 0.1%-1% BSA, 2%-10% trehalose, 2%-10% sucrose, 0.2%-2% mannitol, 0.01%-0.1% PVPK15, 0.03%-0.1% TW-20 and 0.02%-0.05% Kathon.

4. The method of claim 1, wherein the fluorescent amplification effect is improved. In step S2, the streaking concentration in step S2.1 is 0.8 µL / cm. After streaking, the product is placed in a 45°C forced-air drying oven and baked for 24 hours before being sealed.

5. The method of claim 1, wherein the fluorescent amplification effect is improved. In step S3, the total amount of the sample pad treatment solution used in step S3.1 is 1L, containing 4.55g of disodium hydrogen phosphate dodecahydrate, 4.22g of sodium dihydrogen phosphate dihydrate, 6.04g of sodium chloride, 0.5%-2% of casein, 0.03%-0.05% of PC300, 0.5%-1% of BSA, 1%-5% of hydroxypropyl-β-cyclopaste, 0.3%-1% of PVPK40, 0.1%-0.5% of Tween-20, and 0.01%-0.04% of L-lysine.

6. The method of claim 1, wherein the fluorescent amplification effect is improved. In step S3, the total amount of the conjugate pad treatment solution used in step S3.2 is 1L, containing 5.37g of disodium hydrogen phosphate dodecahydrate, 3.9g of sodium dihydrogen phosphate dihydrate, 9g of sodium chloride, 0.1%-0.3% glycine, 0.03%-0.05% PC300, 0.1%-0.5% BSA, 0.5%-1% PVA, 0.2%-0.5% PVPK30, and 0.1%-0.3% TX-100.

7. The method of claim 1, wherein the fluorescent amplification effect is improved. In step S5, the sample diluent used in step S5.1 is 0.05M PBS, pH=6.6, containing 0.1%-0.2% casein, 0.05%-0.1% OVA, 0.01%-0.03% PC300, 0.001%-0.005% ascorbic acid and 0.002%-0.005% PEG4000.

8. The labeling method for improving fluorescence amplification effect according to claim 1, characterized in that, In step S5, step S5.2 involves placing the reacted card strip into a fluorescence reader to read the result.

9. The labeling method for improving fluorescence amplification effect as described in any one of claims 1-8, characterized in that, Used in the detection of biotoxins.

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