CN114276335B - Difenoconazole hapten, artificial antigen, antibody and preparation method and application thereof - Google Patents
Difenoconazole hapten, artificial antigen, antibody and preparation method and application thereof Download PDFInfo
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- CN114276335B CN114276335B CN202111425846.6A CN202111425846A CN114276335B CN 114276335 B CN114276335 B CN 114276335B CN 202111425846 A CN202111425846 A CN 202111425846A CN 114276335 B CN114276335 B CN 114276335B
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a difenoconazole hapten, an artificial antigen, an antibody, a preparation method and application thereof. The structural formula of the difenoconazole hapten is shown as a formula I, and the difenoconazole hapten is prepared by connecting a valeric acid arm on triazole of difenoconazole, and the difenoconazole hapten has the advantages that the skeleton structure and the original side chain of the difenoconazole are not changed, the structure of the difenoconazole hapten is highly consistent with that of the difenoconazole, and the difenoconazole hapten is favorable for inducing high-specificity antibodies. Preparing artificial antigen based on the hapten, preparing high-quality monoclonal antibody aiming at difenoconazole by immunizing animals with the artificial antigen, and establishing an immunoassay method for detecting difenoconazole based on the antibody, wherein the immunoassay method comprises the steps of IC (integrated circuit) 50 The minimum detection limit is 0.32ng/mL, the linear range is 0.49-3.90 ng/mL, the cross reaction rate with other structural and functional analogues is less than 0.2%, the specificity is high, and the difenoconazole in food can be effectively detected. The artificial antigen prepared by the invention has good immunogenicity, and can be used for preparing difenoconazole polyclonal antibodies, single-chain antibodies and nano antibodies and establishing a corresponding immunoassay method.
Description
Technical Field
The invention relates to the technical field of antigen-antibody detection, in particular to difenoconazole hapten, artificial antigen, antibody and preparation methods and application thereof.
Background
One of difenoconazole, also known as oxazole and triazole bactericides has the main effects of inhibiting the biosynthesis of ergosterol and destroying the structure and function of cell membranes so as to achieve the aim of sterilization. The composition is widely used for preventing and controlling fungal diseases of crops such as beans, wheat, potatoes, melons, fruits, fruit trees and the like, is mainly used for preventing and controlling gray mold, downy mildew and powdery mildew, and can also inhibit anthracnose of strawberries and peppers. However, the difenoconazole is used in large quantity, so that the water source is easy to pollute, and potential pollution is caused to the environment and even human bodies.
In order to ensure environmental safety and human life health, the establishment of a rapid and efficient difenoconazole detection method is in need of solving.
The reported method for detecting difenoconazole residue mainly adopts an instrument method, and comprises the following steps: gas chromatography, liquid chromatography tandem mass spectrometry, gas chromatography-mass spectrometry combination, surface enhanced raman spectroscopy techniques, and the like. The instrument method generally has the defects of expensive instrument, complex operation, long time consumption and the like, so in order to meet the requirements of the market on the rapid detection of the difenoconazole, the development and the operation of a rapid detection method which is simple, low in cost, efficient and sensitive are urgently needed. Compared with an instrument method, the immunodetection method has the advantages of high specificity, rapidness, simplicity and convenience in operation and the like, and can effectively relieve the pressure of market supervision personnel and reduce the operation cost. In the process of establishing a small molecular compound immunoassay method, a key technology is to obtain a high-quality antibody with strong specificity and high sensitivity, and to achieve the aim, the design and synthesis of hapten are the primary and key steps, and the quality of the hapten directly influences the quality of the obtained antibody, which is to establishThe rapid, sensitive and accurate difenoconazole detection method has important significance. However, the hapten, the antigen and the antibody for the immunodetection of the difenoconazole are still fewer, only Feng Jiuhui and the like (2016) are used for finally establishing a direct ELISA method and an indirect ELISA method for detecting the difenoconazole through the synthesis of the difenoconazole hapten, the preparation of an artificial antigen and the preparation of a rabbit polyclonal antibody, the detection limits are 8.86 mu g/L and 4.58 mu g/L respectively, and the IC is realized 50 Research [ D ] of immunodetection methods of difenoconazole of 67.96 and 29.10. Mu.g/L (Feng Jiuhui)]University of Tianjin science and technology 2016). Based on the above, it is necessary to provide more hapten and antigen capable of producing specific antibody against difenoconazole, and at the same time, to establish an immunoassay method for difenoconazole which is efficient, rapid, high in sensitivity and strong in specificity.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings in the prior art and provide a difenoconazole hapten.
The second object of the invention is to provide a preparation method of the difenoconazole hapten.
The third object of the invention is to provide an application of the difenoconazole hapten.
The fourth object of the invention is to provide a difenoconazole artificial antigen.
The fifth object of the invention is to provide the application of the difenoconazole artificial antigen.
The sixth object of the invention is to provide a difenoconazole antibody.
The seventh object of the invention is to provide the application of the difenoconazole.
The eighth object of the invention is to provide a rapid immunoassay method for detecting difenoconazole.
The above object of the present invention is achieved by the following technical solutions:
the difenoconazole hapten has a chemical structure shown in a formula I:
the difenoconazole hapten is prepared by connecting a valeric acid arm on triazole of difenoconazole, the skeleton structure and the original side chain of the difenoconazole are not changed, and the difenoconazole hapten is highly consistent with the difenoconazole structure, thereby being beneficial to the induction of high-specificity antibodies.
The preparation method of the difenoconazole hapten comprises the steps of dissolving difenoconazole in an organic solvent, slowly dripping 5-bromovaleric acid dissolved in the organic solvent into the difenoconazole solution under the stirring condition for reaction, separating after the reaction is finished, and removing the organic solvent to obtain a yellow oily product, namely the difenoconazole hapten.
Preferably, the organic solvent is N, N-Dimethylformamide (DMF).
Preferably, the molar ratio of difenoconazole to 5-bromovaleric acid is 1:1.2 to 1.5.
Preferably, the reaction conditions are 70-90 ℃ for 10-14 h.
Preferably, the separation is a silica gel column chromatography separation using 200 to 300 mesh (dichloromethane: methanol=10:1).
Preferably, the organic solvent is removed by spin-steaming.
As a preferable implementation mode, the preparation method of the difenoconazole hapten comprises the following steps: difenoconazole (2.03 g,5 mmol) is weighed and dissolved in 4ml of N, N-dimethylformamide, 5-bromovaleric acid (1.086 g,6 mmol) dissolved in 1ml of N, N-Dimethylformamide (DMF) is slowly added into the difenoconazole solution dropwise under stirring, the difenoconazole solution is reacted for 12 hours at 80 ℃, and the molar ratio of the difenoconazole to the 5-bromovaleric acid is 1:1.2 to 1.5. After the reaction is finished, separating by 200-300 mesh silica gel column chromatography (dichloromethane: methanol=10:1), and removing the organic solvent by rotary evaporation to obtain a yellow oily product, namely the difenoconazole hapten.
The invention also provides application of the difenoconazole hapten in preparation of difenoconazole artificial antigen.
The difenoconazole artificial antigen is obtained by coupling a carrier protein with a carboxyl group of a difenoconazole hapten in a formula I, and the chemical structure of the difenoconazole artificial antigen is shown as a formula II:
preferably, the carrier protein is Lactoferrin (LF), keyhole Limpet Hemocyanin (KLH) or Ovalbumin (OVA). LF, KLH are used to prepare immunogens and OVA is used to prepare coating precursors.
Preferably, the coupling is by the active ester method.
As a preferable implementation mode, the preparation method of the difenoconazole artificial antigen comprises the following steps: difenoconazole hapten 15.3mg (0.03 mmol), 8.7mg EDC and 5.22mg NHS were dissolved in 600. Mu.L DMF and reacted overnight at 4℃with stirring, designated solution A. 20mg of carrier protein was weighed and dissolved in 4mL of PBS buffer (0.01M, pH 7.4), and the solution was stirred and dissolved to prepare solution B. Under the magnetic stirring, the solution A is sucked and added into the solution B drop by drop, and the reaction is carried out for 12 hours under the magnetic stirring at the temperature of 4 ℃. The reaction was dialyzed against PBS at 4℃for 3 days, with 2 changes of dialysate per day. The immunogen can be obtained and frozen in a refrigerator at the temperature of minus 20 ℃ for standby.
The invention also provides application of the difenoconazole artificial antigen in preparation of difenoconazole monoclonal antibodies, polyclonal antibodies, single-chain antibodies or nano antibodies.
The difenoconazole antibody is prepared by taking the difenoconazole artificial antigen shown in the formula II as an immunogen.
Preferably, the antibody is a difenoconazole monoclonal antibody, and the difenoconazole artificial antigen of which the carrier protein is Lactoferrin (LF) or keyhole limpet hemocyanin is used as an immunogen; selecting female Balb/C mice of proper age for immunization, measuring the anti-blood-removing titer and inhibition rate after the 4 th immunization and the 5 th immunization, and enhancing the immunization 3 days before cell fusion; fusing the spleen cells and myeloma cells of the mice after the immunity enhancement, screening out hybridoma cells capable of secreting specific antibodies, and performing amplification culture; injecting the expanded hybridoma cells into a mouse body with paraffin injected in advance, collecting ascites, and purifying to obtain the monoclonal antibody.
The invention also provides application of the difenoconazole artificial antigen or the difenoconazole antibody in detecting difenoconazole by an immune method.
The invention also provides a rapid immunoassay method for detecting difenoconazole, which is based on an indirect ELISA method, takes the difenoconazole artificial antigen of which the carrier protein is Ovalbumin (OVA) as a coating antigen, and uses the difenoconazole antibody as a detection antibody for detection. Preferably, the antibody is a difenoconazole monoclonal antibody.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a difenoconazole hapten, the structural formula of which is shown in formula I, and the difenoconazole hapten is prepared by connecting a valeric acid arm on triazole of difenoconazole, the skeleton structure and the original side chain of the difenoconazole are not changed, and the difenoconazole hapten is highly consistent with the difenoconazole structure, so that the stable induction of a high-specificity antibody is facilitated. The method is characterized in that an artificial antigen is prepared based on the hapten, then an animal is immunized by the artificial antigen and a hybridoma technology is combined to prepare and obtain a high-quality monoclonal antibody aiming at difenoconazole, and the ELISA method for detecting difenoconazole based on the antibody has an IC50 of 1.38ng/mL, a minimum detection limit of 0.32ng/mL and a linear range of 0.49-3.90 ng/mL, and also shows that the artificial antigen prepared by the method has good immunogenicity and can be used for preparing a difenoconazole polyclonal antibody, a nano antibody or a genetic engineering antibody and establishing a corresponding immunoassay method.
Drawings
FIG. 1 is a difenoconazole hapten ESI-MS map.
FIG. 2 is an ultraviolet scan pattern of the ether-mexiconazole hapten, the coating antigen and the immune antigen thereof as well as the carrier protein.
FIG. 3 is a graph of an indirect competition ELISA standard established based on difenoconazole monoclonal antibodies.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 preparation of difenoconazole hapten
Difenoconazole (2.03 g,5 mmol) is weighed and dissolved in 4mL of N, N-dimethylformamide, 5-bromovaleric acid (1.086 g,6 mmol) dissolved in 1mL of N, N-dimethylformamide is slowly added into the difenoconazole solution dropwise under stirring, the difenoconazole is reacted for 12 hours at 80 ℃, and the molar ratio of difenoconazole to 5-bromovaleric acid is 1:1.2 to 1.5. After the reaction is finished, separating by 200-300 mesh silica gel column chromatography (dichloromethane: methanol=10:1), and removing the organic reagent by rotary evaporation to obtain a yellow oily product, namely the difenoconazole hapten. ESI-MS analysis (positive) m/z 507.0, ESI-MS spectrum is shown in figure 1, which shows that hapten shown in formula I is successfully prepared. The chemical reaction equation is shown below:
EXAMPLE 2 preparation of difenoconazole artificial antigen
The embodiment provides a preparation method of difenoconazole artificial antigen, which mainly comprises the synthesis of immunogen and coating antigen. The immunogen is different from the coating antigen in the preparation of carrier types, and the carrier used by the immunogen is bovine Lactoferrin (LF) and Keyhole Limpet Hemocyanin (KLH); the coating antigen adopts Ovalbumin (OVA) as carrier protein. The method of synthesizing immunogen/coating antigen is active ester method. The method comprises the following specific steps:
difenoconazole hapten 15.3mg (0.03 mmol), 8.7mg EDC and 5.22mg NHS were dissolved in 600. Mu.L DMF and reacted overnight at 4℃with stirring, designated solution A. 20mg of carrier protein was weighed and dissolved in 4mL of PBS buffer (0.01M, pH 7.4), and the solution was stirred and dissolved to prepare solution B. Under the magnetic stirring, the solution A is sucked and added into the solution B drop by drop, and the reaction is carried out for 12 hours under the magnetic stirring at the temperature of 4 ℃. The reaction was dialyzed against PBS at 4℃for 3 days, with 2 changes of dialysate per day. The immunogen can be obtained and frozen in a refrigerator at the temperature of minus 20 ℃ for standby. The hapten prepared in the example 1 and the artificial antigen prepared in the example 2 are respectively identified by ultraviolet wavelength scanning (150-400 nm), the highest absorbance values of LF, KLH, OVA, difenoconazole hapten (DX 5), DX5-LF, DX5-KLH and DX5-OVA before and after coupling are compared, and as a result, as shown in figure 2, the absorbance curves of DX5-LF, DX5-KLH and DX5-OVA are obviously different from the absorbance curves of LF, KLH, OVA, and the absorbance curves of DX5-LF, DX5-KLH and DX5-OVA deviate from the absorbance curves of LF, KLH, OVA at 280 nm; therefore, the absorption curves of DX5-LF, DX5-KLH and DX5-OVA are cumulative absorption peaks of LF, KLH, OVA and difenoconazole hapten DX5 respectively; from this, the difenoconazole hapten DX5 is successfully coupled with LF, KLH, OVA, and the DX5-LF artificial antigen, the DX5-KLH artificial antigen and the DX5-OVA artificial antigen are successfully prepared.
EXAMPLE 3 preparation of Difenoconazole Artificial antigen monoclonal antibody and polyclonal antibody
(1) Preparation of monoclonal antibodies
Animal immunization: using healthy 6-week-old Balb/c female mice as experimental animals, the two immunogens prepared in example 2 were each mixed and emulsified with equal amounts of adjuvant (first complete Freund's adjuvant followed by incomplete Freund's adjuvant), and subcutaneously injected into the neck, back and abdominal cavities of the mice at an immunization dose of 0.5mL (containing 0.5mg immunogen). The first immunization is carried out by emulsifying 0.5mL of complete Freund's adjuvant with antigen for immunization, 4 weeks later, emulsifying 0.5mL of incomplete Freund's adjuvant with antigen for boosting, then immunization is carried out every 2 weeks, a small amount of blood is taken from tail vein for antibody quality identification, after the antibody is stabilized, the mice with the best performance are selected for cell fusion, 3 days before cell fusion, 0.5mg of immunogen is directly injected into the abdominal cavity of the mice for impact immunization once.
Evaluation of antiserum effects: the difenoconazole coating antigen prepared in example 2 is taken, the collected mouse serum is taken as a detection antibody, the antiserum titer and inhibition rate of the mouse serum are measured by adopting an indirect competition ELISA method, and the titers and inhibition rates of the antiserum are comprehensively considered to evaluate the difenoconazole coating antigen. The specific operation steps are as follows:
1) And (3) wrapping the plate: dilute difenoconazole coating material to 1000ng/mL with 0.05M carbonate buffer (pH 9.6), coat at 4deg.C overnight at 100 μl/well; removing the coating liquid, washing with PBST for 2 times, adding 120 μl of sealing liquid (5% skimmed milk) into each hole, and sealing at 37deg.C for 3 hr; discarding the sealing liquid, drying at 37 ℃ for 60min, and sealing the sealing bag at 4 ℃ for standby to obtain the packaged ELISA plate.
2) Serum titers and inhibition assays: and (3) the ELISA plate packaged in the step (1) is provided with the titer column: 50 mu L of PBS and 50 mu L of serum diluted in a gradient multiple are added to each well respectively; inhibition column: 50 μl of diluted 1000ng/mL drug (difenoconazole) and 50 μl of serum diluted in a gradient multiple were added to each well and 2 groups were made in parallel. Incubating at 37deg.C for 40min, washing with PBST for five times, drying the liquid in the hole, adding enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) diluted at 1:5000, incubating at 37deg.C for 30min, washing with PBST for five times, drying the liquid in the hole, adding 100 μl TMB substrate solution, and developing at 37deg.C in dark for 10min; mu.L of stop solution (10% H) was added 2 SO 4 ) Terminating the reaction; the absorbance at 450nm was read with a microplate reader.
Cell fusion and selection of positive hybridomas: and (3) fusing Balb/c mouse spleen cells which generate specific antibodies with myeloma cells SP2/0, measuring cell supernatant by adopting an indirect competitive ELISA method, and screening positive holes. Subcloning the positive hole by using a limiting dilution method, screening single cells capable of stably secreting uniform antibodies, and performing expansion culture, wherein the specific steps are as follows:
1) Resuscitates myeloma cells: taking out myeloma cells from liquid nitrogen, quickly thawing in a water bath at 37 ℃, centrifuging for 7min at l000 r/min after thawing, pouring out supernatant in an ultra-clean workbench, adding about l mL of complete culture solution into cell sediment, blowing off the cells, taking out by a pipette and mixing the complete culture solution uniformly, putting the cells in 9 cm culture dishes, expanding the cells to 5-6 dishes, and changing the liquid for 1-2 times during the process, wherein when the cells of each culture dish are paved at the bottom, the cells can be used for cell fusion.
2) Feeder cell preparation: the Kunming mice were subjected to eye-picking and killing 1 day before cell fusion, immersed in 75% alcohol for 5min, and then transferred to an ultra-clean bench for dissection. Cutting the abdomen, peeling the skin of the abdomen, exposing the peritoneum, then picking up the peritoneum with forceps, opening a small opening on the peritoneum of the mouse, injecting 3mL of HAT complete medium, repeatedly sucking and flushing with a pipette, taking out the medium containing feeder cells in the abdominal cavity with the pipette, and repeating the operation for 2-3 times to ensure that enough feeder cells are obtained.
3) Spleen cell preparation: the eyesockets of Balb/c mice which are subjected to multiple immunization blood detection are exsanguinated, serum is collected, soaked in 75% alcohol for 5min for sterilization, and then the eyesockets are transferred to an ultra-clean workbench for dissection. The spleen was taken out aseptically, washed with PPMI-1640 basal medium and placed in a petri dish for use. The culture medium was aspirated with a disposable syringe, the removed spleen was gripped with forceps by the left hand, the syringe was inserted into the spleen, and the culture medium was slowly injected to wash out cells in the spleen. Repeatedly proceeding until the spleen turns from dark red to colorless and transparent, and discarding the spleen. The mixed culture medium is collected in a 50mL centrifuge tube, and the mixture is centrifuged after sealing, and the rotation speed is 1000r/min for 8min. And (5) centrifuging, and discarding the supernatant for later use.
4) Cell fusion: the bone marrow tumor cells and the immune spleen cells from which the supernatant is removed by centrifugation are mixed in a centrifuge tube according to a ratio of about 1:5, 25mL of basic culture medium is added, and after sealing, the mixture is centrifuged at 1000r/min for 8min. The supernatant was discarded after centrifugation for use. The supernatant was discarded from the bone marrow tumor cells and spleen cells after centrifugation, and the excess medium was sucked down by a gun with the nozzle of the centrifuge. The precipitated cells were flicked with fingers, the centrifuge tube was placed in 37℃warm water, 0.8mL of PEG preheated to 37℃was sucked with the gun head, PEG was slowly added to the precipitated cells within 1min, each drop of PEG was gently stirred with the gun head for mixing, left to stand for 1min, the complete medium was preheated, 10mL was added within 2min with gentle stirring, and PEG was split along the wall. Centrifuging at 1000r/min for 10min, removing supernatant, adding HAT culture medium, gently sucking liquid with elbow pipette, stirring gently, taking out HAT culture medium in centrifuge tube, mixing with about 75mL fresh HAT culture medium, and adding into 8 96-well culture plates containing feeder cells prepared on the previous day. The area of HAT medium containing feeder cells and HAT medium containing fusion cells was kept the same in each well, and approximately 24mL of HAT medium was used in each plate.
5) Screening of positive hybridomas: HAT culture medium is used within 7-10 days after fusion, HT culture medium is used for changing liquid, and complete culture liquid is used according to proliferation condition after 14 days. When the cells are attached to 1/3 of the plate holes (generally, the cells are fused for about 12 days), the supernatant in the porous culture plate is extracted, the specific antibody in the culture solution is detected by using indirect ELISA, positive hybridoma cells with high titer and strong drug inhibition are selected, and the positive holes with the best fusion effect are screened out and marked. Under the aseptic condition, picking cells growing in positive holes by using a microscope, transferring the cells to a 96-hole culture plate which is plated with feeder cells in advance, cloning each original hole into 8 holes, taking supernatant after the cell adhesion grows to be full of 1/2-1/3 hole bottoms, performing ICELISA detection, taking positive and strong people as measurement indexes, subcloning by using a limiting dilution method, repeating the steps for 3-4 rounds (note that the positive hole cells picked in each round need to be expanded for culture and then frozen for standby), until each hole of each plate is positive and the detection titer and inhibition are similar, and establishing the hybridoma cell line successfully, thereby obtaining the hybridoma cell line capable of stably secreting uniform antibodies. And (3) selecting single cell clones, and transferring the single cell clones detected to be positive to a 24-hole cell culture plate or a cell culture dish for expansion culture, and timely freezing.
Preparation of monoclonal antibodies in large quantities: after obtaining the gram drop of hybridoma cells secreting a specific monoclonal antibody, the monoclonal antibody is generally prepared in large quantities by an in vitro culture method and an in vivo animal induction monoclonal antibody method. The conventional practice is as follows: liquid paraffin was injected into the abdominal cavity of Balb/c mice, which were tens of mice at an age of 8 weeks or more, at a dose of 0.5 mL/mouse, and hybridoma cells were injected into the abdominal cavity of the mice after 1 to 2 weeks. The state of the mice is observed every day after the cells are inoculated, particularly, the abdominal cavity of the mice is swelled from the seventh day, the ascites is aseptically collected by a disposable injector before the mice die, the collected ascites is centrifuged for 10min at 12000r/min, the upper fat and the lower fibrin are removed, the middle layer is collected, the titer and the inhibition rate are measured by an icELlSA method, and the mice are preserved at-20 ℃ for standby after purification.
(2) Preparation of polyclonal antibodies
The immunogen prepared in example 2 was mixed and emulsified with an equal amount of adjuvant (complete Freund's adjuvant for the first time, and then incomplete Freund's adjuvant for all), and New Zealand white rabbits having a body weight of 2.5-3 kg were immunized by various injections, subcutaneous in the back, subcutaneous in each part, intramuscular in the leg, and intravenous in the ear, and two immunogens were injected. The immunization was boosted every three weeks after the first immunization interval four weeks. One week after the third boost, the marginal vein was bled and serum titers were determined using an indirect ELISA, when the titers no longer increased, the marginal vein boost was used. After two days, heart blood is collected, standing is carried out at 4 ℃ overnight, centrifugation is carried out at 12000r/min for 10min, supernatant is taken and split into centrifuge tubes, and the centrifuge tubes are preserved at-20 ℃.
EXAMPLE 4 establishment of an Indirect competitive ELISA Standard Curve based on monoclonal antibodies
(1) Coating and sealing
Dilute difenoconazole coating antigen to 62.5ng/mL with coating liquid, and coat at 37 ℃ overnight. The next day, after washing twice with PBST (0.01M PBS,0.06%Tween-20 (v/v)) 2% skimmed milk powder was added, each well was blocked at 37℃for 3h, the blocking solution was discarded, dried at 37℃for 60min, and packed in a sealed bag at 4℃for use.
(2) Establishment of a Standard Curve
1) Experimental method
Adding a series of 50 mu L of difenoconazole standard substances and 50 mu L of difenoconazole monoclonal antibodies (317.89 ng/mL) with different concentrations into each hole, incubating for 40min at 37 ℃, washing for five times by using PBST, beating the liquid in the holes, adding 1:5000 diluted enzyme-labeled secondary antibodies (goat anti-mouse IgG-HRP), incubating for 40min at 37 ℃, washing for five times by using PBST, beating the liquid in the holes, adding 100 mu L of TMB substrate liquid, and developing for 10min at 37 ℃ in a dark place; add 50. Mu.L of stop solution (2M H) 2 SO 4 ) Terminating the reaction; the absorbance at 450nm was read with a microplate reader. The concentration of difenoconazole is used as a standard product to the abscissa, B/B 0 (OD of the Difenoconazole-added well) 450 OD of wells without Difenoconazole 450 ) Establishing an indirect competition scale for the ordinateQuasi-curves.
2) Experimental results
As shown in FIG. 3, the standard curve of the indirect competition ELISA established based on the monoclonal antibody is S-shaped, has good linear correlation and is an IC for difenoconazole 50 The detection sensitivity is high, and the linear range is wide, wherein the detection limit is 1.38ng/mL, the lowest detection limit is 0.32ng/mL, and the linear range is 0.49-3.90 ng/mL. Meanwhile, the cross reaction rate of the difenoconazole, the myclobutanil, the tebuconazole, the hexaconazole, the imazalil, the flutriafol, the prothioconazole, the cyproconazole, the triadimenol, the triazolone, the diniconazole, the paclobutrazol, the bitertanol, the propiconazole, the epoxiconazole, the flusilazole, the fluquinconazole, the triticonazole, the penconazole, the tetraconazole, the pyraclostrobin, the azoxystrobin, the enestroburin, the carbendazim, the chlorothalonil, the mancozeb, the iprodione, the polyoxin, the captan and the like, which are structural and functional analogues, is less than 0.2%, shows that the single antibody prepared by the experiment has higher specificity, and can effectively detect the difenoconazole in food.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. The difenoconazole hapten is characterized in that the chemical structure is shown as a formula I:
2. the preparation method of the difenoconazole hapten, which is characterized in that difenoconazole is dissolved in an organic solvent, 5-bromopentanoic acid dissolved in the organic solvent is slowly added into the difenoconazole solution dropwise under the stirring condition for reaction, and after the reaction is finished, separation is carried out, and the organic solvent is removed to obtain a yellow oily product, namely the difenoconazole hapten.
3. The use of the difenoconazole hapten as claimed in claim 1 for preparing difenoconazole artificial antigen.
4. The difenoconazole artificial antigen is characterized in that the difenoconazole artificial antigen is obtained by carboxyl coupling carrier protein of the difenoconazole hapten according to claim 1, and the chemical structure is shown as formula II:
5. the difenoconazole artificial antigen according to claim 4, wherein said carrier protein is lactoferrin, keyhole limpet hemocyanin, or ovalbumin.
6. The difenoconazole artificial antigen according to claim 4, wherein said coupling is by an active ester method.
7. The use of the difenoconazole artificial antigen according to claim 4 in the detection of difenoconazole by an immunoassay.
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| 杀菌剂半抗原的合成及其免疫分析技术的研究进展;曹啸天 等;《中国免疫学杂质》;第36卷;第3039-3044、3054页 * |
| 苯醚甲环唑农药残留检测技术的研究进展;洪霞 等;《视频研究与开发》;第39卷(第24期);第190-197页 * |
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