Vomitoxin fluorescence immunochromatography test strip as well as preparation method and application thereof

A technique of fluorescence immunochromatography and vomitoxin, which is applied to the analysis of materials, biological tests, material inspection products, etc. It can solve the problems that positive results cannot be saved, sample background interference, and results are no longer accurate and reliable.

Inactive Publication Date: 2020-05-19
SOUTH CHINA AGRI UNIV
8 Cites 1 Cited by

AI-Extracted Technical Summary

Problems solved by technology

However, colloidal gold immunochromatographic test strips have the following defects: (1) general test strips are qualitatively analyzed by visual observation, and accurate quantitative detection cannot be achieved; (2) different material matri...
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Abstract

The invention discloses a vomitoxin fluorescence immunochromatography test strip as well as a preparation method and application thereof. The test strip comprises a bottom plate, and a reaction film is attached to the bottom plate; one end of the reaction film covers a water absorption pad, and the other end sequentially covers a combination pad and a sample pad; a detection line and a quality control line are arranged on a non-covering surface of the reaction film along the transverse direction; a fluorescent probe of a vomitoxin monoclonal antibody marked by aggregation-induced emission fluorescent microspheres is sprayed on the combination pad; the detection line is coated with a vomitoxin complete antigen, and the quality control line is coated with a goat anti-mouse secondary antibody. According to the invention, the marking steps are greatly simplified, the marking efficiency is improved, the detection time is shortened, the physical adsorption stability is better than that of colloidal gold, and the detection accuracy and precision are high; the inter-batch difference is small, and the cost is low; in addition, the detection method is simple to operate, the whole detection process only needs 8 minutes, the detection limit is 100ppb, the quantitative detection range is 100ppb-5000ppb, and the sensitivity and specificity are high.

Application Domain

Biological testing

Technology Topic

MonoclonalAnticentromere antibodies +8

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  • Vomitoxin fluorescence immunochromatography test strip as well as preparation method and application thereof
  • Vomitoxin fluorescence immunochromatography test strip as well as preparation method and application thereof
  • Vomitoxin fluorescence immunochromatography test strip as well as preparation method and application thereof

Examples

  • Experimental program(4)

Example Embodiment

[0072] Example 1 Preparation of Deoxynivalenol Hapten, Complete Antigen, Monoclonal Antibody, Goat Anti-mouse Secondary Antibody
[0073] 1. Preparation of vomitoxin hapten
[0074] The synthetic route of vomitoxin hapten of the present invention is as follows Figure 4 As shown, it specifically includes the following steps:
[0075] (1) Weigh 23.2 mg of vomitoxin in advance, add 10 mL of ethylene glycol dimethyl ether, dissolve it by ultrasonic, and set aside;
[0076] (2) Weigh 0.6g of sodium hydride (because sodium hydride contains kerosene, use 20mL of petroleum ether to stir magnetically for 30s, let it stand for 2min, pour off the petroleum ether; repeat the cleaning 2 to 3 times; after pouring off the petroleum ether for the last time, Blow dry with nitrogen);
[0077] (3) Add dissolved vomitoxin in sodium hydride, stir for 1 hour, add 0.5 g of succinic anhydride, stir for 3 hours, after reacting for 1 hour, slowly add 4 mL of methyl 3-bromopropionate dropwise, react for 3 hours, and obtain the product, Carry out extraction purification again, obtain the compound shown in formula (I):
[0078]
[0079]In the preparation of the vomitoxin hapten, in order to verify whether the hydrolysis is successful, the product of the above step (3) and vomitoxin are spotted together (chloroform:methanol=30:1); evaporate after filtration; add 2g lithium hydroxide and 15mL Three-grade water, stirred for 1 hour, if the dissolution is relatively clear, it proves that the hydrolysis is successful. After the hydrolysis is successful, add water and ethyl acetate to the hydrolyzate to extract 2 to 3 times, and take the product in the water layer; add concentrated hydrochloric acid to the product in the water layer dropwise until the pH value is 4 to 5. After the precipitated product appears, use ethyl acetate Ester extracts the product in the water layer back; finally, use ethyl acetate and anhydrous sodium sulfate as water removal agents to remove water, and rotary evaporation to obtain the deoxynivalenol hapten shown in formula (I) after purification.
[0080] 2. Preparation of complete antigen (coating source, immunogen)
[0081] (1) Preparation of coating original
[0082] 1) Dissolve 10mg of vomitoxin hapten in 1mL of dimethylamide (DMF); take 20mg of carbodiimide (EDC), fully dissolve it in 0.2mL of DMF, add it to the hapten solution, and stir at room temperature for 8 hours. Obtain reaction solution A;
[0083] 2) Weigh 36 mg of OVA and fully dissolve it in 3 mL of 0.1 mol/L carbonate buffer solution (pH=9.6) to obtain an OVA protein solution;
[0084] 3) The reaction solution A was slowly added dropwise to the OVA protein solution, and stirred at room temperature for 16 hours in the dark, and dialyzed with 0.01mol/L PBS at 4°C for 3 days, and the dialysate was changed twice a day to obtain the packaged solution. Be original.
[0085] (2) Preparation of immunogen
[0086] 1) Dissolve 10mg of vomitoxin hapten in 1mL of dimethylamide (DMF); take 20mg of carbodiimide (EDC), fully dissolve it in 0.2mL of DMF, add it to the hapten solution, and stir at room temperature for 8 hours. Obtain reaction solution A;
[0087] 2) Weigh 36 mg of BSA and fully dissolve it in 3 mL of 0.1 mol/L carbonate buffer solution (pH=9.6) to obtain a BSA protein solution;
[0088] 3) Slowly add the reaction solution A to the BSA protein solution drop by drop, and stir at room temperature for 16 hours in the dark, dialyze with 0.01mol/L PBS at 4°C for 3 days, change the dialysate twice a day, and obtain the immune Original.
[0089] 3. Preparation of DON Monoclonal Antibody
[0090] a. Animal immunization: inject the immunogen obtained in step 2 into BALB/c mice with an immunization dose of 100 μg/mouse to produce antiserum;
[0091] b. Cell fusion and cloning
[0092] Booster immunization: Booster immunization of mice once three days before formal cell fusion, injection concentration: 1mg/mL, injection volume: 100μL, no adjuvant required;
[0093] Prepare feeder cells: One day before formal cell fusion, take 1 Kunming mouse, kill it by bloodletting eyeballs, place it on a dissecting plate, take pleural macrophages and splenocytes as feeder cells, mix them with about 75mL of HAT medium, and add them to a multi-well culture plate , adjust the concentration at 2×10 5 cells/mL, take 24-well culture plate, 1 mL/well. Observe the growth status of the feeder cells before use the next day. If there are any abnormal phenomena such as bacterial contamination, they should be discarded.
[0094] Preparation of splenocyte liquid: first add 3mL of basal medium to a small cell dish, put a cell sieve on it, take out the spleen and put it in a cell sieve, grind the spleen with a syringe plunger; collect spleen cells into a 15mL centrifuge tube, and use RPMI-1640 basic Dilute the medium to 10mL and seal it; centrifuge at 1000rpm for 7min and discard the supernatant; take out Tris-ammonium chloride from the incubator, first add 1mL Tri-ammonium chloride, blow it gently, then add 2mL Tris-ammonium chloride to mix Homogenize (to lyse red blood cells), seal, and incubate at 37°C for 7 minutes; add RPMI-1640 basal medium to make up to 10 mL, seal and centrifuge at 1000 rpm for 7 minutes, discard the supernatant; resuspend to 10 mL with RPMI-1640 basal medium to obtain spleen cell fluid ,spare.
[0095] Preparation of myeloma cell liquid: collect myeloma cells into another 15 mL centrifuge tube, seal, centrifuge at 1000 rpm for 7 min, discard supernatant, resuspend with basal medium, and dilute to 10 mL to obtain myeloma cell liquid.
[0096] Mixed cell solution: mix spleen cell solution and myeloma cell solution in a 50mL centrifuge tube, add basal medium to 20mL, mix well, seal, centrifuge at 1000rpm for 7min, discard the supernatant, and suck up with a gun with the mouth of the centrifuge tube facing down excess medium.
[0097] Preparation for cell fusion: Gently tap the pelleted cell mass at the bottom of the centrifuge tube with your fingers to loosen the pellet and mix well, seal the tube, incubate at 37°C for 10 minutes, and prepare a timer for 4 minutes.
[0098] Fusion of cells: Take out PEG and 10mL complete medium from the incubator; add PEG within the first 1min, with gentle movements, accompanied by gentle stirring, do not blow away, so that the PEG is separated; seal, incubate at 37°C for 10min, and centrifuge at 800rpm 10min, discard the supernatant.
[0099] Plating: Take out the HAT from the incubator, add a small amount of HAT medium, use a pipette to gently absorb and release the liquid, stir gently, and then adjust the volume to 75mL; add it to a 24-well plate with feeder cells, 1mL/well, place Cell culture incubator.
[0100] Culture after fusion: check whether it is necessary to add HAT culture medium on the 4th day; change the medium for the first time on the 7th day, absorb 1/2 of the culture medium when changing the medium, add an equal amount of fresh HT culture medium, and overuse HT.
[0101] Positive wells were cloned using the limiting dilution method until hybridoma cell lines that can stably secrete monoclonal antibodies.
[0102] (3) Cell cryopreservation and recovery
[0103] Prepare hybridoma cells with cryopreservation solution to 1*10 6 cells/mL for long-term storage in liquid nitrogen. When recovering, take out the cryopreservation tube, put it in a 37°C water bath for instant dissolution, remove the cryopreservation solution by centrifugation, and transfer it to a culture bottle for culture.
[0104] (4) Preparation and purification of monoclonal antibodies
[0105] After the hybridoma cell lines are established, monoclonal antibodies can be produced in large quantities as needed. Ascites collection method: first inject 500 μL of liquid paraffin or pristane into the peritoneal cavity of BALB/c mice, and then inject 1×10 6 About 10 days after inoculation with hybridoma cells, abdominal swelling of the mice was seen. The mice were sacrificed before the mice died, and the ascites was extracted. The ascites was purified by caprylic acid-ammonium sulfate precipitation to obtain the final vomitoxin monoclonal antibody.
[0106] 4. Preparation of goat anti-mouse secondary antibody
[0107] Goats were used as immunized animals and mouse-derived antibodies were used as immunogens to immunize pathogen-free sheep to obtain goat anti-mouse secondary antibodies.

Example Embodiment

[0108] Embodiment 2 Preparation method of vomitoxin fluorescent immunochromatography test strip
[0109] 1. Preparation of fluorescent probes for DON monoclonal antibody labeled with aggregation-induced luminescence fluorescent microspheres
[0110] a. Take 10 μL of aggregation-induced luminescent fluorescent microsphere solution and 1 mL of MES buffer solution into a centrifuge tube, mix well, centrifuge, and set aside;
[0111] The carboxylated aggregation-induced luminescent fluorescent microspheres have an excitation wavelength of 200 nm and an emission wavelength of 610 nm.
[0112] b. Add 15 μL 0.5 mg/mL EDC and 20 μL 0.5 mg/mL NHS to each tube, use a constant temperature culture shaker to shake, 200 rpm, 25 ° C for 15 min; after the catalysis is completed, 14000 rpm, 25 ° C for 15 min, discard the supernatant solution, redissolve in 1 mL of 0.05M BB buffer solution (pH=8.0);
[0113] c. Add 1 μL of deoxynivalenol monoclonal antibody, place on a constant temperature culture shaker, and react at 200 rpm and 25°C for 40 minutes;
[0114] d. Add 20 μL of 20% BSA, place on a constant temperature culture shaker, shake at 200 rpm, 20°C for 60 minutes, and obtain the immunofluorescence probe;
[0115] e. Centrifuge the obtained immune probe at 14000rpm, 25°C for 15min;
[0116] f. Discard the supernatant, add 1mL complex solution, and centrifuge at 14000rpm, 25°C for 20min;
[0117] g. Resuspension: Discard the supernatant, dilute it to 200 μL with aggregation-induced luminescent fluorescent microsphere solution, and suspend it with ultrasonic cleaner to obtain the deoxynivalenol labeled with the aggregation-induced luminescent fluorescent microspheres. Fluorescent probes for cloning antibodies.
[0118] 2. Preparation of binding pads
[0119] Take a bonding pad and cut it to a size of 15cm in length and 0.5cm in width. Take 2.5 μL of 0.1 mol/L aggregation-induced luminescent fluorescent microsphere-labeled DON monoclonal antibody fluorescent probe, dilute to 150 times with pH=7.2-7.4 phosphate buffer solution (PBS), and spray evenly on the binding pad , adding a desiccant, sealed and stored for later use.
[0120] 3. Coating of reaction membrane (nitrocellulose membrane)
[0121] (1) Select goat anti-mouse secondary antibody, dilute it to a concentration of 1.0 mg/mL, and spray it on the membrane as the C line (quality control line);
[0122] (2) Select complete antigen of vomitoxin, dilute to 2.0mg/mL concentration, spray film as T line (test line);
[0123] (3) Inhale the goat anti-mouse secondary antibody solution and complete vomitoxin antigen solution into the tip of the film-drawing instrument, adjust the spray volume of the film-drawing instrument to 0.6 μL/cm, and adjust the C line to 1.0 μL/cm. Put the plain film under the conditions of relative humidity of 20% and temperature of 37° C., and dry for 2 to 3 hours.
[0124] 4. Preparation of reaction membrane (nitrocellulose membrane)
[0125] After the reaction membrane (nitrocellulose membrane) obtained in step 3 is cut according to actual requirements, a desiccant is added, sealed, preserved, and set aside;
[0126] 5. Preparation of sample pads
[0127] After cutting the sample pad made of glass cellulose membrane according to the actual requirements, add a desiccant, seal it, store it, and set it aside;
[0128] 6. Preparation of absorbent pad
[0129] Cut the absorbent pad made of absorbent filter paper according to the actual requirements, seal it, dry it, and reserve it;
[0130] 7. Preparation of the bottom plate
[0131] Cut the bottom plate of PVC material according to the actual requirements, seal it, and set it aside.
[0132] 8. Test strip and its assembly method
[0133] like figure 2 Shown, a kind of vomitoxin fluorescent immunochromatography test paper strip comprises base plate, and reaction film is pasted on the base plate; One end of reaction film covers absorbent pad, and the other end covers binding pad and sample pad successively (sample pad and binding pad are from above stick to the other side of the reaction membrane in turn); the non-covered surface of the reaction membrane is provided with a detection line and a quality control line along the lateral direction; the fluorescent probe of DON monoclonal antibody labeled with aggregation-induced luminescent fluorescent microspheres is sprayed on the binding pad. Needle; the detection line is coated with vomitoxin complete antigen, and the quality control line is coated with goat anti-mouse secondary antibody. The overlapping parts between the water-absorbing pad and the reaction membrane, between the binding pad and the reaction membrane, and between the binding pad and the sample pad are about 1-1.5mm, and the other parts are pasted on the bottom plate.
[0134] The water-absorbing pad adopted in the present invention is water-absorbing filter paper, the bottom plate is a polyvinyl chloride (PVC) bottom plate, the reaction membrane is a nitrocellulose membrane (NC membrane), and the sample pad and the bonding pad are glass cellulose membranes. The length of the test strip is 6.5-10 cm, and the width is 3.5-5 cm. The length of the absorbent pad is 2.2-4cm, the length of the reaction film is 2.6-4cm, the length of the binding pad is 0.5-1cm, and the length of the sample pad is 1.8-3cm.
[0135] The test strip assembly method includes the steps of pasting the absorbent paper, the reaction film, the binding pad, and the sample pad at both ends of the bottom plate:
[0136] (1) Uncover the protective film on the surface of the bottom plate prepared in step 7;
[0137] (2) paste the reaction film prepared in step 4 on the reaction film pasting area of ​​the base plate, and press it tightly;
[0138] (3) Overlap the absorbent pad prepared in step 6 with one side of the reaction membrane by 1 to 1.5mm, and press it tightly;
[0139] (4) Overlap the bonding pad prepared in step 2 with the other side of the reaction membrane by 1 to 1.5 mm, and press it tightly;
[0140] (5) Overlap the sample pad prepared in step 5 with one side of the binding pad by 1 to 1.5 mm, and press it tightly;
[0141] (6) Put the assembled test strip on the automatic cutting machine, cut according to the length of 6.5cm*width of 3.5mm, add a certain amount of desiccant, and store it sealed at room temperature; the assembly work is carried out at room temperature and in a dry environment; After all the test strips are assembled, add a desiccant and transfer them to an aluminum foil bag, drain the air, and store them at room temperature for later use.

Example Embodiment

[0142] Example 3 Using the above-mentioned fluorescent immunochromatographic test strips to detect vomitoxin residues
[0143] 1. Sample pretreatment
[0144] Grind the sample (feed) to be tested into powder; weigh 1.00±0.05g of the crushed sample into a 10mL centrifuge tube; add 6mL of 70% methanol-water solution, vortex for 3min, and centrifuge at 4000rpm for 5min; take 50μL of the supernatant, Add 450 μL of sample diluent, mix well, and test.
[0145] 2. Test with test strips
[0146] Open the test paper cylinder, take out the test paper strip prepared in Example 2, mark it and place it on the table (note: after taking out the micropore and the test paper strip, cover the cylinder cover immediately to prevent moisture); absorb 100 μL of the sample to be tested , drop the test strip into the micropore of the fluorescent probe, fully immerse the sample pad in the sample, and react for 5 minutes; take out the test strip, and interpret the result in a dry fluorescence analyzer, and interpret the result within 1 minute.

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