CN111175511A - Vomitoxin fluorescence immunochromatographic test strip and preparation method and application thereof - Google Patents
Vomitoxin fluorescence immunochromatographic test strip and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a vomitoxin fluorescence immunochromatographic test strip and a preparation method and application thereof. The test strip comprises a bottom plate, and a reaction film is pasted on the bottom plate; one end of the reaction membrane is covered with the water absorption pad, and the other end of the reaction membrane is sequentially covered with the combination pad and the sample pad; a detection line and a quality control line are transversely arranged on the non-covered surface of the reaction membrane; a fluorescent probe of a vomitoxin monoclonal antibody marked by aggregation-induced emission fluorescent microspheres is sprayed on the bonding pad; the detection line is coated with vomitoxin complete antigen, and the quality control line is coated with goat anti-mouse secondary antibody. The invention greatly simplifies the marking steps, improves the marking efficiency, shortens the detection time, has better physical adsorption stability than colloidal gold and has high detection accuracy and precision; the difference between batches is small, and the cost is low; in addition, the detection method is simple to operate, the whole detection process only needs 8min, the detection limit is 100ppb, the quantitative detection range is 100 ppb-5000 ppb, the sensitivity is high, and the specificity is strong.
Description
Technical Field
The invention belongs to the field of immunodetection, and particularly relates to the technical field of rapid determination and detection of vomitoxin in feed and grain products. More particularly, relates to a vomitoxin fluorescence immunochromatographic test strip and a preparation method and application thereof.
Background
Deoxynivalenol (DON for short) is a common trichothecene toxin type B, one of the trichothecene alkenes, which is commonly produced by the mold, fusarium, in cereal products such as wheat, corn, barley, etc. The large dose of vomitoxin can cause vomit symptoms of people, pigs, ruminants, mice, poultry and the like, so the vomitoxin is also commonly called as vomitoxin, can reduce the body weight of the people and the animals, cause diarrhea, disturb the steady state of the intestinal system, influence the endocrine system, the immune system and the like, can reach the blood brain barrier through blood circulation, and further cause potential neurotoxicity. DON inhibits protein synthesis, cell proliferation, and may cause genotoxicity, cytotoxicity, and the like. Among these, males are more sensitive to DON. Pigs are the most sensitive animals to DON, and feed intake is reduced when the concentration of DON in the feed reaches 1-2 ppm (Prelusky et al, 1994).
DON solid is colorless needle-shaped crystal, the melting point is about 151-153 ℃, the flash point is-3 ℃, the storage condition is 2-8 ℃, and the DON solid and the food raw materials are stored separately. DON has thermal stability, can resist high temperature of 170-350 ℃, is acid and alkali resistant, is difficult to damage in structure under low pressure, can damage a large amount of toxicity only under alkaline conditions of high pressure or sodium carbonate addition and the like, and can still retain the toxicity after the vomitoxin is stored for four to five years. Research shows that vomitoxin may have an influence on the immune system, and has obvious toxicity of spare tires and certain teratogenic action. Because of the serious harm of vomitoxin, the general attention of all countries is drawn. The DON pollution of grains is reported in China, Brazil, the United states, Argentina, south Africa and other places in the world. 359 flour samples were investigated for mycotoxin contamination in Shandong, China in 2016, and the highest DON detection rate was found to be 97.2% with an average contamination concentration of 86.7. mu.g/kg (Li et al, 2016). The pollution of DON in grain crops is serious, and the pollution of DON in feed raw materials and complete formula feed is common. In 2012-2014, 190 parts of randomly collected feed and compound feed in the middle area of China had a positive rate of DON of 77.4% (Liu et al, 2016). Therefore, there are strict limit standards for the content of vomitoxin in both grain and feed. The limit standard of vomitoxin in cereals, pig compound feed, calf compound feed and lactation animal compound feed in China is 1.0mg/kg, and the limit standard of vomitoxin in cattle compound feed and poultry compound feed is 5.0 mg/kg.
At present, a plurality of methods for detecting vomitoxin are available, such as thin layer chromatography, enzyme linked immunosorbent assay (ELISA), gas chromatography, high performance liquid chromatography, ultraviolet spectroscopy and colloidal gold immunochromatography. The method is sensitive and accurate, but needs expensive instruments, the pretreatment of samples is complex, tedious and time-consuming, the detection cost is high, and professional operation is needed, so that the application is limited. Chinese patent document CN 101413954A discloses a special test kit for enzyme-linked immunosorbent assay of vomitoxin and a preparation and detection method thereof. However, the conventional immunoassay methods such as enzyme-linked immunoassay, chemiluminescence immunoassay and the like are simple, but have poor and comparative sensitivity, and in addition, the detection process still needs the condition guarantee of a laboratory, the detection operation still needs professional personnel, generally still needs a relatively large-scale professional instrument such as an enzyme-labeling instrument for auxiliary detection, is difficult to operate in single portion, and is not beneficial to the timely detection and large-area screening of grains and feeds in remote areas or basic medical institutions without the condition for carrying out laboratory detection.
The immunochromatography technology is a unique immunoassay mode appearing in the early 80 s, and generally takes a strip-shaped fiber chromatography material as a solid phase, enables a sample solution to swim on a chromatography strip through capillary action, enriches or retains immune complexes in a certain area (detection line) of the chromatography material in the chromatography process through an immune reaction principle of antigen-antibody combination, and obtains an intuitive experimental result (for example, the detection line has strips with different colors) through enzyme reaction or direct application of a visual marker (for example, colloidal gold); the free label crosses the detection line, so as to achieve the purpose of automatically separating from the bound label. Common visual labeling carriers of the immunochromatography technology include colloidal gold, latex, colloidal selenium and the like, wherein the most successful label is the colloidal gold. However, the colloidal gold immunochromatographic test strip has the following defects: (1) the general test paper strip is qualitatively analyzed by observing results with naked eyes, and accurate quantitative detection cannot be realized; (2) the effect of different material matrixes is obvious, the background interference of a sample is large, and false positive results are easy to generate; (3) the positive result of the detection can not be stored, and the result is not accurate and reliable after the judgment time is usually exceeded.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a vomitoxin fluorescence immunochromatographic test strip and a preparation method and application thereof. The fluorescent immunochromatographic test strip has the capability of qualitatively and quantitatively detecting vomitoxin, has good detection specificity, high sensitivity, simple and convenient operation, low detection cost and short detection time, is suitable for screening and detecting batch samples, is an ideal rapid screening means, and can better meet the detection requirements of national cereal and feed enterprises, government functional supervision departments and the like.
The purpose of the invention is realized by the following technical scheme:
a vomitoxin fluorescence immunochromatographic test strip comprises a bottom plate, wherein a reaction film is pasted on the bottom plate; one end of the reaction membrane is covered with the water absorption pad, and the other end of the reaction membrane is sequentially covered with the combination pad and the sample pad; a detection line and a quality control line are transversely arranged on the non-covered surface of the reaction membrane;
a fluorescent probe of a vomitoxin monoclonal antibody marked by aggregation-induced emission fluorescent microspheres is sprayed on the bonding 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.
As a preferable scheme, the reaction film is adhered to the bottom plate; the water absorption pad is adhered to one side of the reaction membrane; and the sample pad and the combination pad are sequentially stuck on the other side of the reaction membrane from top to bottom. The overlapped parts between the water absorption pad and the reaction film, between the combination pad and the reaction film and between the combination pad and the sample pad are about 1-1.5 mm, and other parts are adhered to the bottom plate.
As a preferred scheme, the water absorption pad adopted in the present invention is water absorption 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 binding pad are glass cellulose membranes. The test strip is 6.5-10 cm in length and 3.5-5 cm in width. The length of the water absorption pad is 2.2-4 cm, the length of the reaction film is 2.6-4 cm, the length of the combination pad is 0.5-1 cm, and the length of the sample pad is 1.8-3 cm.
As one preferable scheme, the fluorescent probe of the vomitoxin monoclonal antibody marked by the aggregation-induced emission fluorescent microsphere is prepared by mixing the activated aggregation-induced emission fluorescent microsphere and the vomitoxin monoclonal antibody in a volume ratio of 8-12: 1, and adding Bovine Serum Albumin (BSA) as a blocking solution to obtain the conjugate.
As one preferable scheme, the activation method of the aggregation-induced emission fluorescent microsphere comprises the following steps: adding an activating agent consisting of carbodiimide (EDC) and N-hydroxysuccinimide (NHS) into the carboxylated aggregation-induced luminescent fluorescent microsphere solution for activation to obtain the fluorescent microsphere solution; the volume ratio of the carboxylated aggregation-induced emission fluorescent microsphere solution to EDC and NHS is (1-3): 3: 4.
as one preferred scheme, the aggregation-induced emission fluorescent microsphere is prepared by using tetramethylbenzidine, and specifically comprises the following steps:
dissolving tetrakisvinylogous acid tetramethyl ester and microsphere preparation materials (polymethyl methacrylate and poly maleic anhydride octadecyl ester) in a trichloromethane solvent, and adding a dispersing agent for dispersing to obtain a dispersion liquid; under the ice bath condition, preparing the dispersion into microemulsion, removing trichloromethane, centrifuging to obtain precipitate, washing the precipitate, and redissolving the precipitate in buffer solution to obtain the aggregation-induced emission fluorescent microsphere.
As one of the preferable embodiments, the vomitoxin complete antigen is: coupling a compound shown as a formula (I) as a vomitoxin hapten with carrier protein to obtain a vomitoxin complete antigen;
wherein, the vomitoxin hapten is a compound shown as a formula (I):
as one preferable embodiment, the vomitoxin hapten is prepared by the following steps: dissolving vomitoxin in ethylene glycol dimethyl ether, adding sodium hydride to activate carboxyl of the vomitoxin, coupling succinic anhydride to prevent carboxyl of 4 th and 5 th positions of the vomitoxin from being substituted, replacing hydrogen on the carboxyl of 4 th and 5 th positions of the vomitoxin with methyl 3-bromopropionate, and reducing the succinic anhydride under alkaline conditions to obtain the compound shown in the formula (I).
As a preferred embodiment, the preparation method of the vomitoxin hapten specifically comprises the following steps:
(1) ultrasonically dissolving vomitoxin in glycol dimethyl ether;
(2) adding the dissolved vomitoxin into sodium hydride to activate carboxyl of the vomitoxin (because the sodium hydride contains kerosene, petroleum ether is used for cleaning for 2-3 times, after the petroleum ether is poured out for the last time, nitrogen is used for blow-drying), and stirring for reacting for 1-2 h;
(3) adding 0.5-1.0 g succinic anhydride, and stirring for reaction for 3-4 h; and (3) after continuously reacting for 1h, slowly dropwise adding methyl 3-bromopropionate to react for 3-4 h, and extracting and purifying to obtain the product.
In preparing the vomitoxin hapten, in order to verify whether the hydrolysis is successful, the product of the step (3) and a vomitoxin starting point plate (chloroform: methanol ═ 30: 1); filtering and evaporating; lithium hydroxide and 15mL of tertiary water were added and stirred for 1h, if the dissolution was relatively clear, confirming the success of the hydrolysis. After the hydrolysis is successful, adding water and ethyl acetate into the hydrolysate for extraction for 2-3 times, and taking a water layer product; dropwise adding concentrated hydrochloric acid into the water layer product until the pH value is 4-5, and extracting the water layer product with ethyl acetate after a precipitate product appears; and finally, removing water by using ethyl acetate and anhydrous sodium sulfate as water removal agents, and performing rotary evaporation to obtain the purified vomitoxin hapten shown in the formula (I).
As one preferred embodiment, the vomitoxin monoclonal antibody is: coupling a compound shown as a formula (I) as a vomitoxin hapten with carrier protein to obtain a vomitoxin complete antigen; the vomitoxin complete antigen is used as immunogen, and the vomitoxin monoclonal antibody is obtained after specific immune reaction of deimmunized animals.
In a preferred embodiment, the carrier protein is chicken Ovalbumin (OVA) or Bovine Serum Albumin (BSA).
As one preferred embodiment, OVA is used as the complete antigen of the carrier for the coating antigen, and BSA is used as the complete antigen of the carrier for the immunogen.
The invention also relates to a preparation method of the vomitoxin fluorescence immunochromatographic test strip, which comprises the following steps:
s1, coupling vomitoxin hapten with carrier protein to obtain vomitoxin complete antigen; immunizing animals by taking the vomitoxin complete antigen as immunogen to obtain vomitoxin monoclonal antibody after specific immune reaction;
s2, adopting carboxylated aggregation-induced emission fluorescent microspheres activated by carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to mark the vomitoxin monoclonal antibody, and adding Bovine Serum Albumin (BSA) solution to seal to obtain a fluorescent probe of the vomitoxin monoclonal antibody marked by the aggregation-induced emission fluorescent microspheres;
then spraying a fluorescent probe of the vomitoxin monoclonal antibody marked by the aggregation-induced emission fluorescent microsphere on the bonding pad;
s3, coating vomitoxin complete antigen at the detection line position of the reaction membrane, wherein the coating concentration of the detection line is 0.6-1.4 mg/mL; coating goat anti-mouse secondary antibody on the position of a quality control line of the reaction membrane, wherein the coating concentration of the quality control line is 1.6-2.4 mg/mL;
and S4, sequentially overlapping the sample pad, the combination pad, the reaction membrane and the water absorption pad on the bottom plate to obtain the water-based paint.
As one preferable scheme, the mass-to-volume ratio of the vomitoxin monoclonal antibody to the aggregation-induced emission fluorescent microsphere is 1 μ g: 6-14 mu L, and performing coupling reaction for 35-45 min at 100-300 rpm and 24-26 ℃.
As a preferable scheme, the dosage of the BSA solution is 16-24 mu L, and the concentration is 16-24%.
As one preferable scheme, the operation of step S2 is as follows:
a. adding 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution into the aggregation-induced emission fluorescent microsphere solution, mixing uniformly, and centrifuging: the aggregation-induced emission fluorescent microsphere solution is carboxylated aggregation-induced emission fluorescent microspheres, the excitation wavelength is 200nm, and the emission wavelength is 610 nm;
b. adding 0.5-1 mg/mL EDC and 0.5-1 mg/mL NHS, and using a constant temperature culture shaker to shake at 100-300 rpm and 24-26 ℃ for catalysis for 15-45 min; after the catalysis is finished, centrifuging for 15-20 min at 14000-20000 rpm and 24-26 ℃, discarding the supernatant, and re-dissolving in 1mL of 0.05M BB buffer solution (pH 8.0);
c. adding the vomitoxin monoclonal antibody, placing the mixture in a constant-temperature culture shaker, and oscillating the mixture to react for 35-45 min at 100-300 rpm and 24-26 ℃;
d. adding 16-24 mu L of 16-24% BSA, placing the mixture in a constant-temperature culture shaker for shaking, and reacting at 100-300 rpm and 18-22 ℃ for 50-60 min to obtain an immunofluorescence probe;
e. centrifuging the obtained immunofluorescence probe (at 8000-20000 rpm and 24-26 ℃ for 10-20 min);
f. discarding the supernatant, adding 1mL of complex solution, and centrifuging for 15-25 min at 8000-20000 rpm and 24-26 ℃;
g. and discarding the supernatant, diluting the supernatant to 200 mu L by using an aggregation-induced emission fluorescent microsphere solution, and suspending the diluted solution by using an ultrasonic cleaner to obtain the fluorescent probe of the vomitoxin monoclonal antibody marked by the aggregation-induced emission fluorescent microsphere.
The quality control line is a C line, and the detection line is a T line.
The invention adopts methanol stock solution and 0.05mol/L PB solution to dilute the secondary antibody on the C line and the antigen of the T line on the nitrocellulose membrane. The C line and the T line are both configured into a 30-mu-L system and are diluted by methanol and PB solution, the methanol solution is used for fixing 2 mu L, the vomitoxin complete antigen protein is used for fixing 16 mu L, and the PB solution is added to the part less than 30 mu L for supplementing; the goat anti-mouse anti-antibody was used at 6. mu.L, and less than 30. mu.L portions were supplemented with PB solution.
The content of each component in the reaction membrane detection line is respectively as follows: 16-30 mu L of vomitoxin complete antigen (2mg/mL), 2-30 mu L of methanol stock solution and 12-30 mu L of 0.05mol/mL PB solution; the quality control line comprises the following components in percentage by weight: the goat anti-mouse anti-antibody (1mg/mL) is 6-30 muL, the methanol stock solution is 2-30 muL, and the PB solution of 0.01mol/L is 22-30 muL.
The invention also relates to a method for qualitatively and quantitatively detecting vomitoxin, which comprises the following steps:
(1) qualitative detection
Adding a sample to be detected on a sample bonding pad of the vomitoxin fluorescence immunochromatographic test strip, reacting for 5-8 min, and observing the fluorescence conditions of the test strip detection line and the quality control line (the fluorescence conditions can be directly observed by naked eyes with the help of an ultraviolet lamp):
the detection line and the quality control line both display fluorescence, and the content of vomitoxin in the sample to be detected is less than 100ppb, and the sample to be detected is qualitatively judged to be negative;
if the detection line does not display fluorescence and the quality control line displays fluorescence, the content of vomitoxin in the detection sample is more than or equal to 100ppb, and the detection sample is qualitatively judged to be positive;
(2) quantitative detection
And detecting the fluorescence intensity of the test strip which is qualitatively judged to be positive, and obtaining the content of the vomitoxin in the detection sample according to the standard curve.
The application of the vomitoxin fluorescence immunochromatographic test strip in detecting or monitoring the content or the level of the vomitoxin in the feed or the food is also within the protection scope of the invention.
The samples to be detected used by the invention comprise feed, corn protein powder, wheat bran, red palm meal and the like.
The test paper strip of the invention is a rapid diagnosis technology which takes a nitrocellulose membrane as a solid phase carrier. Diluting a sample to be detected by 25 times, dripping 100 mu L of the sample to the sample pad in total volume, and transferring the sample to the direction of absorbent paper under the chromatography action. When no vomitoxin exists in the sample, the aggregation-induced emission fluorescent microsphere label material flow on the release pad is combined with the antigen fixed on the detection line to generate a specific reaction when passing through the detection line, at the moment, a part of the aggregation-induced emission fluorescent microsphere is fixed on the detection line to form a T line, immune complexes which are not reflected with the antigen continue to move forwards, and flow through the quality control line to be a fixed secondary antibody (goat anti-mouse secondary antibody) to generate a specific reaction, so that redundant aggregation-induced emission fluorescent microsphere labels are retained at the quality control line to form a C line. If the rest aggregation-induced emission fluorescent microsphere markers exist, the aggregation-induced emission fluorescent microsphere markers continuously move to the absorbent paper at ease, and visible fluorescent strips appear on the C line; no matter the test strip T line has a fluorescence strip or not, the test strip is invalid as long as the C line has no fluorescence strip.
Compared with the prior art, the invention has the following beneficial effects:
1. the test strip provided by the invention adopts the novel synthesized vomitoxin hapten, and the hapten has stable structure, strong specificity and strong recognition effect.
2. The test strip obtained by the invention adopts an aggregation-induced emission fluorescent microsphere labeling technology, and the test strip is verified on a feed sample, so that the test strip has strong specificity and no cross, the reaction can be carried out for only 8 minutes, the result can be observed, and the detection on the vomitoxin can be achieved.
3. On the basis of the existing aggregation-induced emission fluorescent microsphere marking technology, the invention improves the experimental technology, optimizes the marking condition, selects the combination of EDC and NHS as an activating agent, selects bovine serum albumin as a confining liquid, improves the marking efficiency, has simple and rapid marking process and saves the step of processing the bonding pad.
4. The marked antigen used by the invention is vomitoxin complete antigen, and the antigen is a protein conjugate formed by combining vomitoxin hapten and carrier protein and has high antigen specificity.
5. The aggregation-induced emission fluorescent microspheres selected by the test strip prepared by the invention have high stability, the signals are not easy to weaken, and the test strip can be detected by a portable fluorescence immunoassay analyzer, thereby overcoming the defects of complex operation, time and labor consumption, requirement of professional detection, specificity and the like of ELISA in the prior art.
Drawings
FIG. 1 is a standard curve for the vomitoxin of the present invention.
FIG. 2 is a schematic diagram of the fluorescent immunochromatographic test strip for vomitoxin of the present invention.
FIG. 3 is a schematic diagram of a qualitative result of the vomitoxin fluorescence immunochromatographic test strip of the present invention.
FIG. 4 is a scheme showing the synthesis of the hapten for vomitoxin according to the present invention.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. The present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are intended to be included in the scope of the present invention.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Basic culture medium: RPMI-1640.
EXAMPLE 1 preparation of vomitoxin hapten, complete antigen, monoclonal antibody, goat anti-mouse Secondary antibody
1. Preparation of vomitoxin hapten
The synthetic route of the vomitoxin hapten is shown in figure 4, and specifically comprises the following steps:
(1) weighing 23.2mg of vomitoxin in advance, adding 10mL of ethylene glycol dimethyl ether, and ultrasonically dissolving for later use;
(2) weighing 0.6g of sodium hydride (because the sodium hydride contains kerosene, 20mL of petroleum ether is used for magnetic stirring for 30s, standing for 2min, pouring off the petroleum ether, repeatedly cleaning for 2-3 times, and drying by using nitrogen after pouring off the petroleum ether for the last time);
(3) adding dissolved vomitoxin into sodium hydride, stirring for 1h, adding 0.5g succinic anhydride, stirring for 3h, reacting for 1h, slowly dropwise adding 4mL 3-bromomethyl propionate, reacting for 3h to obtain a product, and extracting and purifying to obtain the compound shown in the formula (I):
in order to verify whether the hydrolysis was successful in the preparation of the vomitoxin hapten, the product of step (3) above and a vomitoxin starting point plate (chloroform: methanol ═ 30: 1); filtering and evaporating; 2g of lithium hydroxide and 15mL of tertiary water are added and stirred for 1h, if the dissolution is clear, the hydrolysis is successful. After the hydrolysis is successful, adding water and ethyl acetate into the hydrolysate for extraction for 2-3 times, and taking a water layer product; dropwise adding concentrated hydrochloric acid into the water layer product until the pH value is 4-5, and extracting the water layer product with ethyl acetate after a precipitate product appears; and finally, removing water by using ethyl acetate and anhydrous sodium sulfate as water removal agents, and performing rotary evaporation to obtain the purified vomitoxin hapten shown in the formula (I).
2. Preparation of complete antigens (coatingen, immunogen)
(1) Preparation of coating antigen
1) Dissolving 10mg of vomitoxin hapten in 1mL of dimethyl formamide (DMF); taking 20mg carbodiimide (EDC), fully dissolving with 0.2mL DMF, adding into the hapten solution, and stirring at room temperature for 8h to obtain a reaction solution A;
2) OVA 36mg was weighed and dissolved well in 3mL of 0.1mol/L carbonate buffer solution (pH 9.6) to obtain an OVA protein solution;
3) and slowly dropping the reaction solution A into the OVA protein solution dropwise, stirring at room temperature in the dark for 16h, dialyzing with 0.01mol/L PBS at 4 ℃ for 3 days, and changing the dialysate every day for 2 times to obtain the coating antigen.
(2) Preparation of immunogens
1) Dissolving 10mg of vomitoxin hapten in 1mL of dimethyl formamide (DMF); taking 20mg carbodiimide (EDC), fully dissolving with 0.2mL DMF, adding into the hapten solution, and stirring at room temperature for 8h to obtain a reaction solution A;
2) 36mg of BSA was weighed and sufficiently dissolved in 3mL of 0.1mol/L carbonate buffer solution (pH 9.6) to obtain a BSA protein solution;
3) and (3) dropwise and slowly adding the reaction solution A into the BSA protein solution, stirring for 16h at room temperature in a dark place, dialyzing for 3 days at 4 ℃ by using 0.01mol/L PBS (phosphate buffer solution), and changing the dialysate every day for 2 times to obtain the immunogen.
3. Preparation of vomitoxin monoclonal antibody
a. Animal immunization: injecting the immunogen obtained in the step 2 into BALB/c mice, wherein the immunizing dose is 100 mug/mouse, so that antiserum is generated;
b. cell fusion and cloning
And (3) boosting immunity: three days before formal cell fusion, mice were boosted once with injection concentrations: 1mg/mL, injection amount: 100 μ L without adjuvant;
preparing feeder cells: one day before formal cell fusion, 1 Kunming mouse is taken, eyeball is killed by bloodletting, the Kunming mouse is placed in a dissection plate, thoracic cavity macrophage and splenocyte are taken as feeder cells and are mixed with about 75mL HAT culture medium and then added into a porous culture plate, and the adjustment concentration is 2 multiplied by 105And taking 24-well culture plates per mL, and taking 1mL per well. The growth of feeder cells should be observed before the next day, and should be discarded if there is any abnormal phenomenon such as contamination.
Preparation of splenocyte liquid: adding 3mL of basic culture medium into a small cell dish, placing the small cell dish on a cell sieve, taking out the spleen, placing the spleen into the cell sieve, and grinding the spleen by using an injector piston; collecting splenocytes into a 15mL centrifuge tube, metering the volume to 10mL by using an RPMI-1640 basic culture medium, and sealing; centrifuging at 1000rpm for 7min, and discarding the supernatant; taking out Tris-ammonium chloride from the warm bath box, adding 1mL of Tri-ammonium chloride, blowing gently, adding 2mL of Tri-ammonium chloride, mixing (cracking erythrocytes), sealing, and keeping the temperature at 37 deg.C for 7 min; adding RPMI-1640 basic culture medium to a constant volume of 10mL, sealing and centrifuging at 1000rpm for 7min, and discarding the supernatant; resuspend to 10mL with RPMI-1640 basic medium to obtain spleen cell fluid for use.
Preparation of myeloma cell sap: collecting myeloma cells into another 15mL centrifuge tube, sealing, centrifuging at 1000rpm for 7min, discarding supernatant, resuspending with basal medium, and diluting to 10mL to obtain myeloma cell fluid.
Mixing cell sap: mixing spleen cell fluid and myeloma cell fluid in a 50mL centrifuge tube, adding a basic culture medium to 20mL, fully mixing, sealing, centrifuging at 1000rpm for 7min, removing supernatant, and sucking off excessive culture medium with a gun with the nozzle of the centrifuge tube facing downwards.
Preparation of cell fusion: gently beating the pellet cell mass at the bottom of the centrifuge tube with fingers to loosen the pellet, mixing well, sealing, incubating at 37 deg.C for 10min, and timing with timer for 4 min.
Fusion of cells: remove PEG and 10mL of complete medium from the incubator; adding PEG within the first 1min, gently moving, and separating PEG with gentle stirring and no blowing off; sealing, incubating at 37 deg.C for 10min, centrifuging at 800rpm for 10min, and discarding the supernatant.
Plate paving: taking out HAT from the warm bath box, adding a small amount of HAT culture medium, slightly sucking liquid and discharging liquid by using a suction tube, slightly stirring, and then fixing the volume to 75 mL; the cells were added to a 24-well plate with feeder cells at a concentration of 1 mL/well, and cultured in a cell incubator.
Culturing after fusion: checking whether HAT culture solution needs to be added on day 4; on day 7, the culture medium was changed for the first time, 1/2 of the culture medium was aspirated, and an equal amount of fresh HT culture medium was added, followed by HT overuse.
Cloning positive wells by limiting dilution method until hybridoma cell lines capable of stably secreting monoclonal antibodies.
(3) Cell cryopreservation and recovery
Preparation of hybridoma cells from frozen stock solution to 1 x 106Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into water bath at 37 ℃ for instant dissolution, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
(4) Preparation and purification of monoclonal antibodies
After the hybridoma cell line is established, a large amount of monoclonal antibodies can be prepared as required. Ascites collection method is adopted: firstly, 500 mu L of liquid paraffin or pristane is injected into the abdominal cavity of a BALB/c mouse, and 1 multiplied by 10 is injected into the abdominal cavity after 1 to 2 weeks6Inoculating hybridoma cells, observing the abdominal swelling of the mouse after about 10 days, killing the mouse before the death of the mouse, extracting ascites, and purifying the ascites by an octanoic acid-ammonium sulfate precipitation method to obtain the final vomitoxin monoclonal antibody.
4. Preparation of goat anti-mouse secondary antibody
The goat anti-mouse secondary antibody is obtained by taking a goat as an immune animal and taking a murine antibody as an immunogen to immunize a goat without a pathogen.
Example 2 preparation method of vomitoxin fluorescence immunochromatographic test strip
1. Preparation of fluorescent probe of vomitoxin monoclonal antibody marked by aggregation-induced emission fluorescent microsphere
a. Adding 10 mu L of aggregation-induced emission fluorescent microsphere solution and 1mL of MES buffer solution into a centrifuge tube, uniformly mixing, and centrifuging for later use;
the carboxylated aggregation-induced emission fluorescent microsphere has the excitation wavelength of 200nm and the emission wavelength of 610 nm.
b. Adding 15 μ L of 0.5mg/mL EDC and 20 μ L of 0.5mg/mL NHS into each tube, shaking with a constant temperature shaking table, and catalyzing at 25 deg.C and 200rpm for 15 min; after the completion of the catalysis, the mixture was centrifuged at 14000rpm at 25 ℃ for 15min, the supernatant was discarded, and the mixture was redissolved in 1mL of 0.05M BB buffer solution (pH 8.0);
c. adding 1 μ L vomitoxin monoclonal antibody, placing in a constant temperature shaking table, shaking, and reacting at 25 deg.C and 200rpm for 40 min;
d. adding 20 μ L of 20% BSA, placing in a constant temperature shaking culture machine, shaking, and reacting at 200rpm and 20 deg.C for 60min to obtain immunofluorescence probe;
e. centrifuging the obtained immunoprobe at 14000rpm and 25 ℃ for 15 min;
f. discarding the supernatant, adding 1mL of the complex solution, and centrifuging at 14000rpm and 25 ℃ for 20 min;
g. resuspending: and discarding the supernatant, diluting the supernatant to 200 mu L by using an aggregation-induced emission fluorescent microsphere solution, and suspending the diluted solution by using an ultrasonic cleaner to obtain the fluorescent probe of the vomitoxin monoclonal antibody marked by the aggregation-induced emission fluorescent microsphere.
2. Preparation of the conjugate pad
One piece of the conjugate pad was cut out in a size of 15cm in length and 0.5cm in width. Taking 2.5 mu L of 0.1mol/L fluorescent probe of the vomitoxin monoclonal antibody marked by aggregation-induced emission fluorescent microspheres, diluting the fluorescent probe to 150 times by using Phosphate Buffer Solution (PBS) with the pH value of 7.2-7.4, uniformly spraying the fluorescent probe on a bonding pad, adding a drying agent, and sealing and storing for later use.
3. Coating of reaction membranes (nitrocellulose membranes)
(1) Selecting goat anti-mouse secondary antibody, diluting to 1.0mg/mL concentration, and spraying a film to obtain a C line (quality control line);
(2) selecting vomitoxin complete antigen, diluting to the concentration of 2.0mg/mL, and spraying a membrane to be used as a T line (detection line);
(3) sucking the goat anti-mouse secondary antibody solution and the vomitoxin complete antigen solution into a suction head of a membrane scribing instrument, adjusting the spraying amount T line of the membrane scribing instrument to 0.6 mu L/cm, adjusting the C line to 1.0 mu L/cm, placing the nitrocellulose membrane under the conditions of relative humidity of 20% and temperature of 37 ℃, and drying for 2-3 hours.
4. Preparation of reaction Membrane (nitrocellulose Membrane)
Cutting the reaction membrane (nitrocellulose membrane) obtained in the step 3 according to actual requirements, adding a drying agent, sealing and storing for later use;
5. preparation of sample pad
Cutting a sample pad made of a glass cellulose membrane according to actual requirements, adding a drying agent, sealing and storing for later use;
6. preparation of absorbent pad
Cutting a water absorption pad made of water absorption filter paper according to actual requirements, sealing and drying for later use;
7. preparation of the base plate
Cutting the PVC bottom plate according to actual requirements, and sealing for later use.
8. Test strip and assembling method thereof
As shown in fig. 2, the vomitoxin fluorescence immunochromatographic test strip comprises a bottom plate, wherein a reaction film is attached to the bottom plate; one end of the reaction membrane is covered with the water absorption pad, and the other end is sequentially covered with the combination pad and the sample pad (the sample pad and the combination pad are sequentially stuck on the other side of the reaction membrane from top to bottom); a detection line and a quality control line are transversely arranged on the non-covered surface of the reaction membrane; a fluorescent probe of a vomitoxin monoclonal antibody marked by aggregation-induced emission fluorescent microspheres is sprayed on the bonding pad; the detection line is coated with vomitoxin complete antigen, and the quality control line is coated with goat anti-mouse secondary antibody. The overlapped parts between the water absorption pad and the reaction film, between the combination pad and the reaction film and between the combination pad and the sample pad are about 1-1.5 mm, and the other parts are adhered on the bottom plate.
The water absorption pad adopted by the invention is water absorption 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 combination pad are glass cellulose membranes. The test strip is 6.5-10 cm in length and 3.5-5 cm in width. The length of the water absorption pad is 2.2-4 cm, the length of the reaction film is 2.6-4 cm, the length of the combination pad is 0.5-1 cm, and the length of the sample pad is 1.8-3 cm.
The test strip assembling method comprises the following steps of pasting absorbent paper, a reaction membrane, a combination pad and a sample pad at two ends of a bottom plate:
(1) uncovering the protective film on the surface of the bottom plate prepared in the step (7);
(2) sticking the reaction film prepared in the step 4 on a reaction film sticking area of the bottom plate, and pressing tightly;
(3) overlapping the water absorption pad prepared in the step 6 with one side of the reaction membrane by 1-1.5 mm, and pressing;
(4) overlapping the bonding pad prepared in the step 2 with the other side of the reaction membrane for 1-1.5 mm, and pressing;
(5) overlapping the sample pad prepared in the step 5 with one side of the combination pad by 1-1.5 mm, and pressing;
(6) placing the assembled test strip on an automatic cutting machine, cutting according to the length of 6.5cm and the width of 3.5mm, adding a certain amount of drying agent, and sealing and storing at room temperature; the assembly work is carried out in a dry environment at room temperature; after all the test strips are assembled, drying agents are added, the test strips are transferred into an aluminum foil bag, air is pumped out, and the test strips are stored at normal temperature for later use.
Example 3 detection of vomitoxin residue Using the above-described fluorescence Immunochromatographic test strip
1. Pretreatment of samples
Grinding a sample (feed) to be detected into powder; weighing 1.00 +/-0.05 g of crushed sample in a 10mL centrifuge tube; adding 6mL of 70% methanol-water solution, whirling for 3min, and centrifuging at 4000rpm for 5 min; and (3) adding 450 mu L of sample diluent into 50 mu L of supernatant, and uniformly mixing to be tested.
2. Detection with test strip
The test paper tube is opened, the test paper prepared in example 2 is taken out, marked and placed on the desktop (note: after the micropore and the test paper are taken out, the tube cover is covered immediately to prevent moisture); sucking 100 mu L of a sample to be detected, dropping a test strip into the micropores of the fluorescent probe, fully immersing the sample pad into the sample, and reacting for 5 min; and taking out the test strip, and interpreting the result in a dry fluorescence analyzer within 1 min.
Example 4 sample testing example
1. Limit of detection test
10000 ug/k of vomitoxin standard substance were respectively diluted to the same extent as in Table 1, and the T/C fluorescence ratio was measured.
TABLE 1T/C values for the standards
From the results shown in FIG. 1, the T/C ratio gradually decreases from the concentration of 0-1500 ng/mL, and the T value signal becomes very low after 1500ng/mL, and the difference between 1000ng/mL and 1500ng/mL is very small. The results are measurable over the whole range at concentrations ranging from 0 to 1000. mu.g/kg. The test strip for detecting the vomitoxin can be used for quickly detecting the vomitoxin residue in the grains and the feed.
2. Addition recovery test
And taking a blank negative sample, adding a standard substance with a specified concentration, detecting by using three batches of the test strips prepared in the example 2, and calculating the recovery rate. The results are shown in Table 2.
TABLE 2 test results of sample addition recovery
3. Comparison with LC-MS/MS
One contaminated sample was taken and assayed by national standard liquid chromatography, and 3 replicates were assayed using the vomitoxin fluorescent quantitative test strip prepared in example 2. The results are shown in Table 3 below.
TABLE 3 comparison results with LC-MS/MS
The experimental coincidence rate is between 90% and 100%, which shows that the test strip for detecting vomitoxin can accurately and rapidly detect the vomitoxin residue in grains and feeds.
4. Specificity test
The vomitoxin test paper prepared in the embodiment 2 is used for detecting 500 mu g/kg of zearalenone, aflatoxin B1, ochratoxin A and other medicaments. The result shows that the test strip quality control line and the detection line are negative in fluorescence. The test strip has no cross reaction to 500 mu g/kg zearalenone, aflatoxin B1, ochratoxin A and other medicaments.
The above results show that the fluorescent immunochromatographic test strip for vomitoxin provided by the invention greatly simplifies the labeling steps, improves the labeling efficiency, shortens the detection duration, and also has the advantages of strong specificity, good stability, small sample dosage and the like, and the coupling antibody is coupled by chemical bonds, has better physical adsorption stability than colloidal gold, and has higher detection accuracy and precision; the difference between batches is small, and the cost is low; in addition, the detection method of the vomitoxin fluorescence immunochromatographic test strip is simple to operate, the whole detection process only needs 8min, the detection limit is 100ppb, the quantitative detection range is 100 ppb-5000 ppb, and the sensitivity is high.
The applicant declares that the above detailed description is a preferred embodiment described for the convenience of understanding the present invention, but the present invention is not limited to the above embodiment, i.e. it does not mean that the present invention must be implemented by means of the above embodiment. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (10)
1. A vomitoxin fluorescence immunochromatographic test strip is characterized by comprising a bottom plate, wherein a reaction film is pasted on the bottom plate; one end of the reaction membrane is covered with the water absorption pad, and the other end of the reaction membrane is sequentially covered with the combination pad and the sample pad; a detection line and a quality control line are transversely arranged on the non-covered surface of the reaction membrane;
a fluorescent probe of a vomitoxin monoclonal antibody marked by aggregation-induced emission fluorescent microspheres is sprayed on the bonding 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.
2. The vomitoxin fluorescent immunochromatographic test strip according to claim 1, wherein the fluorescent probe of the vomitoxin monoclonal antibody labeled by the aggregation-induced emission fluorescent microsphere is prepared by mixing an activated aggregation-induced emission fluorescent microsphere and the vomitoxin monoclonal antibody in a volume ratio of 8-12: 1, and adding Bovine Serum Albumin (BSA) as a blocking solution to obtain the conjugate.
3. The vomitoxin fluorescent immunochromatographic test strip according to claim 2, wherein the method for activating the aggregation-induced emission fluorescent microspheres comprises: adding an activating agent consisting of carbodiimide (EDC) and N-hydroxysuccinimide (NHS) into the carboxylated aggregation-induced luminescent fluorescent microsphere solution for activation to obtain the fluorescent microsphere solution; the volume ratio of the carboxylated aggregation-induced emission fluorescent microsphere solution to EDC and NHS is (1-3): 3: 4;
the aggregation-induced emission fluorescent microsphere is prepared from tetracetic acid tetramethyl ester.
4. The vomitoxin fluorescent immunochromatographic test strip according to claim 1, wherein the vomitoxin complete antigen is: coupling a compound shown as a formula (I) as a vomitoxin hapten with carrier protein to obtain a vomitoxin complete antigen; the carrier protein is chicken Ovalbumin (OVA) or Bovine Serum Albumin (BSA);
wherein, the vomitoxin hapten is a compound shown as a formula (I):
5. the vomitoxin fluorescent immunochromatographic test strip according to claim 4, wherein the vomitoxin monoclonal antibody is: the vomitoxin complete antigen is used as immunogen, and the vomitoxin monoclonal antibody is obtained after specific immune reaction of deimmunized animals.
6. The vomitoxin fluorescent immunochromatographic test strip according to claim 4, wherein the vomitoxin hapten is prepared by the following method: dissolving vomitoxin in ethylene glycol dimethyl ether, adding sodium hydride to activate carboxyl of the vomitoxin, coupling succinic anhydride to prevent carboxyl of 4 th and 5 th positions of the vomitoxin from being substituted, replacing hydrogen on the carboxyl of 4 th and 5 th positions of the vomitoxin with methyl 3-bromopropionate, and reducing the succinic anhydride under alkaline conditions to obtain the vomitoxin.
7. The method for preparing the vomitoxin fluorescent immunochromatographic test strip of any one of claims 1 to 6, which is characterized by comprising the following steps:
s1, coupling vomitoxin hapten with carrier protein to obtain vomitoxin complete antigen; taking the vomitoxin complete antigen as immunogen, and obtaining vomitoxin monoclonal antibody after specific immune reaction of deimmunized animal;
s2, adopting carboxylated aggregation-induced emission fluorescent microspheres activated by EDC and NHS to mark the vomitoxin monoclonal antibody, adding Bovine Serum Albumin (BSA) solution to seal, and obtaining the fluorescent probe of the vomitoxin monoclonal antibody marked by the aggregation-induced emission fluorescent microspheres;
then spraying a fluorescent probe of the vomitoxin monoclonal antibody marked by the aggregation-induced emission fluorescent microsphere on the bonding pad;
s3, coating vomitoxin complete antigen at the detection line position of the reaction membrane, wherein the coating concentration of the detection line is 0.6-1.4 mg/mL; coating goat anti-mouse secondary antibody on the position of a quality control line of the reaction membrane, wherein the coating concentration of the quality control line is 1.6-2.4 mg/mL;
and S4, sequentially overlapping the sample pad, the combination pad, the reaction membrane and the water absorption pad on the bottom plate to obtain the water-based paint.
8. The method according to claim 7, wherein in step S2, the mass-to-volume ratio of the vomitoxin monoclonal antibody to the aggregation-induced emission fluorescent microsphere is 1 μ g: 6-14 mu L, and performing coupling reaction for 35-45 min at 100-300 rpm and 24-26 ℃;
the amount of the BSA solution is 16-24 mu L, and the concentration is 16-24%.
9. A method for qualitatively and quantitatively detecting vomitoxin is characterized by comprising the following steps:
(1) qualitative detection
Adding a sample to be detected on a sample bonding pad of any one of 1-6 vomitoxin fluorescent immunochromatography test strips, reacting for 5-8 min, and observing the fluorescence conditions of the test strip detection line and the quality control line:
the detection line and the quality control line both display fluorescence, and the content of vomitoxin in the sample to be detected is less than 100ppb, and the sample to be detected is qualitatively judged to be negative;
if the detection line does not display and the quality control line displays, the content of the vomitoxin in the detection sample is greater than or equal to 100ppb, and the detection sample is qualitatively judged to be positive;
(2) quantitative detection
And detecting the fluorescence intensity of the test strip which is qualitatively judged to be positive, and obtaining the content of the vomitoxin in the detection sample according to the standard curve.
10. Use of the vomitoxin fluorescent immunochromatographic test strip of any one of claims 1 to 6 for detecting or monitoring the content or level of vomitoxin in feed or food.
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Application publication date: 20200519 |