CN112225725B - Propiconazole hapten, complete antigen and antibody thereof, and preparation and application thereof - Google Patents
Propiconazole hapten, complete antigen and antibody thereof, and preparation and application thereof Download PDFInfo
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- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
Abstract
The invention discloses a propiconazole hapten, a complete antigen and an antibody thereof, and preparation and application thereof. The invention firstly prepares a propiconazole hapten, prepares a propiconazole complete antigen and a polyclonal antibody by using the hapten, the antibody has the recognition capability of high sensitivity and high specificity to propiconazole, the half inhibition concentration is 4.23ng/mL, the quantitative detection range is 0.29-61.83 ng/mL, the detection limit is 0.06ng/mL, the cross reaction rate to analogues such as etaconazole, triadimenol, hexaconazole and the like is lower than 1%, and a core raw material is provided for establishing an immunodetection method of specific propiconazole; in addition, an enzyme linked immunosorbent assay kit and a colloidal gold test strip for detecting the propiconazole residue are established by using the polyclonal antibody, so that the enzyme linked immunosorbent assay kit has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like, and can be used for specifically detecting the propiconazole residue in a sample.
Description
Technical Field
The invention belongs to the technical field of food safety detection. More particularly, relates to a propiconazole hapten, a complete antigen and an antibody thereof, and preparation and application thereof.
Background
Propiconazole is a systemic triazole fungicide with protective and therapeutic effects, can be absorbed by the root, stem and leaf parts, and can be quickly conducted upwards in the plant body. The action mechanism is to influence the biosynthesis of sterol, so that the cell membrane function of pathogenic bacteria is damaged, and finally cell death is caused, thereby playing the roles of sterilization, disease prevention and disease treatment. By inquiring Chinese pesticide information network, crops registered in China by propiconazole mainly comprise rice, wheat, corn, soybean, rape, potato, cane shoots, lotus roots, loquats, peanuts, apples, bananas, hazelnuts, ginseng and the like. The residual effective period is about 1 month, generally only has stimulation to skin and eyes, and the nausea, vomiting and the like can be caused by taking the medicine by mistake. Due to its toxic and side effects, the residual limit is limited at home and abroad. At present, countries such as European Union make detailed limits on the residual amount of propiconazole in different crops, and a Japanese affirmation list provides that the maximum allowable residual limit (MRL value) of propiconazole on cabbage heart, cabbage mustard and pakchoi is 0.05 mg/kg. The maximum allowable residual limit (MRL value) in various foods is 0.02mg/kg specified in GB 2763-2016 (national food safety Standard) for maximum residual limit of pesticide in foods). Therefore, for public health, it is necessary to detect the residual amount of propiconazole in food.
Some reports about the analysis method of the residue of propiconazole in vegetables, fruits, grains and foods are available at home and abroad. Currently, methods for detecting propiconazole residue mainly include High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), GC-MS (GC-MS), HPLC-MS (HPLC-MS), and the like. The methods have reliable results and high sensitivity, and related technical standards are available for reference. However, expensive instruments and special operators are needed, the pretreatment of the sample is complicated, the use amount of the organic solvent is large, and the like, so that on one hand, the method cannot realize on-site rapid detection; on the other hand, the method is difficult to be widely popularized in basic units of China.
Compared with an instrumental analysis method, the immunoassay method is more and more concerned in the field of pesticide residue detection due to the advantages of simplicity, economy, rapidness and the like. The ELISA method is a common immunoassay method, but has the defects of relatively more operation steps, longer detection time, insufficiently visual detection result and the like. Thus, the application of ELISA in the rapid detection of propiconazole pesticide is greatly limited.
The colloidal gold test strip does not need professional operators and any auxiliary instruments, can judge the result in a few minutes according to the strip shown by the reaction, and has the advantages of simple and rapid operation, visual and accurate result and low cost. While the key point in establishing an immunological detection method capable of efficiently and specifically detecting propiconazole is to obtain an antibody with high sensitivity and strong specificity, for example, chinese patent publication No. CN106596953A provides a time-resolved fluorescence immunoassay kit for detecting propiconazole, which is modified and modified on the propiconazole structure to synthesize a hapten, and a coupling protein is used to immunize animals, but the structure of propiconazole is destroyed, so that the specific antibody against propiconazole is difficult to generate. Therefore, an urgent need exists to provide a hapten and an antigen which retain the overall structure of propiconazole and can generate antibodies with high specificity to propiconazole, and establish an immunological detection method which has high detection efficiency, sensitivity and strong specificity and can specifically detect propiconazole.
Disclosure of Invention
The invention aims to provide synthesis of a novel propiconazole hapten and preparation of a polyclonal antibody, develop an enzyme linked immunosorbent assay kit for detecting propiconazole residue by using the obtained polyclonal antibody, and prepare a colloidal gold rapid detection test strip for detecting propiconazole. The propiconazole hapten prepared by the invention takes a structural analogue which is highly similar to propiconazole as a starting point, and one end of the structural analogue is extended with a carbon chain, so that the integrity of the whole structure is greatly ensured; the prepared propiconazole polyclonal antibody is high in sensitivity and good in specificity, a core raw material is provided for establishing an immunoassay method of specific propiconazole, and the colloidal gold test strip prepared on the basis of the high-affinity propiconazole polyclonal antibody marked by colloidal gold has wider selectivity and higher sensitivity, and can specifically detect the propiconazole residue in food.
The invention aims at providing a propiconazole hapten.
The invention also aims to provide a preparation method of the propiconazole hapten.
Another object of the present invention is to provide a propiconazole complete antigen.
Another object of the invention is to provide a polyclonal propiconazole antibody.
The invention further aims to provide an enzyme linked immunosorbent assay kit for detecting propiconazole.
The invention further aims to provide a propiconazole colloidal gold rapid detection test strip.
The above purpose of the invention is realized by the following technical scheme:
the invention firstly provides a propiconazole hapten, wherein the structural formula of the propiconazole hapten is shown as a formula (I):
the propiconazole hapten is named by adopting a systematic naming method:
(4- ((((2S, 4R) -2- ((1H-1,2,4-triazol-1-yl) methyl))) -2- (2,4-dichlorophenyl) -1,3-dioxolan-4-yl) methoxy) -4-oxobutanoic acid i.e.: 4- (((2S,4R) -2- ((1H-1,2,4-triazol-1-yl) methyl) -2- (2,4-dichlorophenyl) -1,3-dioxolan-4-yl) methoxy) -4-oxobutanic acid).
The invention also provides a preparation method of the propiconazole hapten BHC-HS, which comprises the following steps: dissolving a propiconazole analogue in anhydrous dichloromethane, adding succinic anhydride and 4-dimethylaminopyridine for reaction, after spin-drying a reaction product, adding ethyl acetate and saturated saline solution for extraction, drying an ester layer by using anhydrous sodium sulfate, and finally concentrating to obtain a propiconazole hapten; the structural formula of the propiconazole analogue is as follows:
according to the invention, hydroxyl on the molecule of the propiconazole analogue reacts with succinic anhydride to introduce free carboxyl to form the propiconazole hapten, and one end of the propiconazole analogue is subjected to carbon chain extension, so that the integrity of the whole structure is greatly ensured.
Preferably, the mass-to-volume ratio of the propiconazole analogue to the anhydrous dichloromethane is 10-15 mg: 1 mL.
More preferably, the mass to volume ratio of the propiconazole analogue to the anhydrous dichloromethane is 15 mg: 1 mL.
Preferably, the mass ratio of the succinic anhydride to the propiconazole analogue is 2-5: 1.
More preferably, the mass ratio of succinic anhydride to the propiconazole analogue is 2: 1.
the developing agent for thin layer chromatography is a mixture of a developing agent and a solvent, wherein the developing agent for thin layer chromatography is a mixture of a developing agent and a solvent, and the volume ratio of the developing agent to the solvent is 3:1 ethyl acetate and petroleum ether.
The invention also provides a propiconazole complete antigen which is obtained by coupling the propiconazole hapten and carrier protein by an active ester method and dialyzing.
Preferably, the carrier protein is Bovine Serum Albumin (BSA) or Keyhole Limpet Hemocyanin (KLH).
The structural formula of the prepared propiconazole complete antigen is as follows:
the invention also provides a propiconazole polyclonal antibody, which is obtained by emulsifying the propiconazole complete antigen, immunizing a New Zealand white rabbit, and separating and purifying.
As a preferred embodiment, the preparation method of the propiconazole polyclonal antibody specifically comprises the following steps:
(1) the new zealand white rabbits were immunized by 2 (female and male) rabbits for 4 weeks, each complete antigen was 500. mu.L of a hapten-keyhole limpet hemocyanin conjugate (BHC-HS-KLH) (1mg/mL), and the hapten-keyhole limpet hemocyanin conjugate (BHC-HS-KLH) was emulsified with Freund's complete adjuvant in a volume ratio of 1:1 at the time of initial immunization. Then, the BHC-HS-KLH is emulsified with a Freund incomplete adjuvant according to the volume ratio of 1:1, and the boosting immunization is carried out once every three weeks for three times;
(2) blood is taken from the ear vein of the rabbit every one week after the third immunization, and the supernatant is obtained by centrifugation and is stored at the temperature of minus 20 ℃ for standby.
The half-inhibitory concentration of the propiconazole polyclonal antibody prepared by the invention to propiconazole raw drug is 4.23ng/mL, the quantitative detection range is 0.29-61.83 ng/mL, the detection limit is 0.06ng/mL, and the cross reaction rate to both ethaconazole and triadimenol is lower than 1%.
Therefore, the propiconazole polyclonal antibody prepared by the method is also within the protection scope of the invention.
Based on the prepared propiconazole polyclonal antibody, the invention also provides an enzyme linked immunosorbent assay kit for detecting propiconazole, which comprises: the enzyme label plate coated with coating antigen, propiconazole standard solution, propiconazole polyclonal antibody, enzyme conjugate concentrated solution, enzyme conjugate diluent, substrate developing solution, stop solution and washing solution;
the coating antigen is a conjugate of propiconazole hapten and protein; the enzyme conjugate is a propiconazole polyclonal antibody marked by horseradish peroxidase.
Preferably, the structural formula of the coating antigen is as follows:
the kit adopts a direct competition ELISA method, conjugate antigen is pre-coated on an enzyme label plate microporous strip, the residual propiconazole in a sample and the conjugate antigen pre-coated on the enzyme label plate microporous strip compete for an enzyme conjugate resisting propiconazole, TMB substrate is used for developing color, the absorbance value of the sample is in negative correlation with the content of the residual propiconazole contained in the sample, the absorbance value is compared with a standard curve, and the absorbance value is multiplied by the corresponding dilution multiple to obtain the residual quantity of the propiconazole in the sample.
Preferably, the propiconazole standard solution has six concentration gradients which are respectively 0 mug/L, 0.1 mug/L, 1 mug/L, 10 mug/L, 100 mug/L and 1000 mug/L.
Preferably, the substrate color developing solution consists of a substrate solution A and a substrate solution B, wherein the substrate solution A is hydrogen peroxide or carbamide peroxide, and the substrate solution B is o-phenylenediamine or tetramethylbenzidine.
Preferably, the stop solution is a 1-2 mol/L sulfuric acid solution.
Preferably, the washing solution has a pH value of 7.4, and contains 0.5-1.0% of tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution; the percentages are weight volume percentages.
Preferably, the preparation method of the elisa plate comprises the following steps: diluting the coating source into 1 mu g/mL by using a coating buffer solution, adding 100 mu L of the coating source into each hole, incubating overnight at 37 ℃ in a dark place, pouring out liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting to dry, then adding 150-200 mu L of a sealing solution into each hole, incubating for 1-2 h at 25 ℃ in a dark place, pouring out liquid in the holes, patting to dry, and performing vacuum sealing and storage by using an aluminum film after drying.
Preferably, the coating buffer solution used in the preparation process of the enzyme label plate is a carbonate buffer solution with the pH value of 9.6 and 0.05 mol/L; the confining liquid is a phosphate buffer solution with the pH value of 7.1-7.5 and containing 1-3% of casein and 0.1-0.3 mol/L; the percentages are weight volume percentages.
As a preferable mode, the method for detecting propiconazole by the enzyme linked immunosorbent assay kit comprises the following steps:
(1) pretreating a sample; (2) detecting by using the kit; (3) and analyzing the detection result.
The enzyme linked immunosorbent assay kit for detecting the propiconazole mainly adopts an ELISA method to quantitatively detect the residual amount of the propiconazole in a sample; the pretreatment requirement on the sample is low, the pretreatment process of the sample is simple, and a large amount of samples can be detected quickly; the main reagent is provided in the form of working solution, and the detection method is convenient and easy to implement and has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like. The enzyme linked immunosorbent assay kit has the advantages of simple structure, convenient use, low price, convenient carrying, high efficiency, accuracy, simplicity and convenience of the detection method, and is suitable for qualitative and quantitative screening of large-batch samples.
The invention also provides a propiconazole colloidal gold rapid detection test strip which comprises a lining plate (1), and a sample pad (2), a gold-labeled conjugate pad (3), a cellulose membrane (4) and a water absorption pad (7) which are sequentially arranged on the lining plate (1), wherein the propiconazole polyclonal antibody of claim 6 which is labeled by colloidal gold is adsorbed in the gold-labeled conjugate pad (3), the cellulose membrane (4) is provided with an invisible detection line (5) and an invisible contrast line (6), the invisible detection line (5) is printed by the propiconazole complete antigen solution of claim 4, and the invisible contrast line (6) is printed by goat anti-rabbit antibodies.
Preferably, a protective film (8-1) is arranged between the gold-labeled conjugate pad (3) and the sample pad (2), and a to-be-detected sample identification line (9) is printed on the protective film and is deviated to a position of 0.5cm on one side of the sample pad (2).
Preferably, the sample pad (2) is made of glass fiber cotton and/or a nylon membrane and/or a polyvinylidene fluoride membrane and/or a polyester membrane, and the cellulose membrane (4) is made of a nitrocellulose membrane and/or a pure cellulose membrane and/or a carboxylated cellulose membrane.
The invention has the following beneficial effects:
the prepared propiconazole hapten BHC-HS takes a structural analogue which is highly similar to propiconazole as a starting point, and a carbon chain is extended at one end, so that a key group and an integral structure of the propiconazole are greatly ensured, and an antibody with high specificity is obtained; the hapten is used for preparing a propiconazole complete antigen and a polyclonal antibody, the antibody has high sensitivity and high specificity recognition capability on propiconazole, the semi-inhibitory concentration is 4.23ng/mL, the quantitative detection range is 0.29-61.83 ng/mL, the detection limit is 0.06ng/mL, the cross reaction rate on both ethaconazole and triadimenol is lower than 1%, and a core raw material is provided for establishing an immunodetection method of specific propiconazole; in addition, an enzyme linked immunosorbent assay kit for detecting propiconazole residue is developed by using the polyclonal antibody, and has the characteristics of high specificity, high sensitivity, high accuracy and the like; finally, the colloidal gold test strip prepared based on the polyclonal antibody has wider selectivity and higher sensitivity, and can specifically detect the propiconazole residue.
Drawings
FIG. 1 is a flow chart of preparation of propiconazole hapten, artificial antigen, antibody and ELISA.
FIG. 2 is a schematic diagram of the synthesis process of propiconazole hapten BHC-HS.
FIG. 3 is a complete antigen UV scanning identification curve of propiconazole.
FIG. 4 is a standard curve of indirect competitive ELISA with propiconazole antibody.
Fig. 5 is a schematic side structure diagram of a colloidal gold rapid detection test strip for propiconazole pesticide in the invention.
FIG. 6 is a schematic view of a test strip for rapid colloidal gold detection of propiconazole pesticide according to the present invention; wherein: 1. a liner plate; 2. a sample pad; 3. a gold-labeled bond pad; 4. a cellulose membrane; 5. invisible detection lines; 6. invisible contrast lines; 7. a water absorbent pad; 8-1, immersing the sample into a protective film; 8-2, a handle end protective film; 9. and marking the line.
FIG. 7 is a schematic diagram showing the results of a colloidal gold rapid test strip for propiconazole pesticide in the present invention;
wherein: a is a negative sample detection result, B is a weak positive sample detection result, C is a strong positive sample detection result, and D and E are test strip failures.
FIG. 8 shows the analysis result of the vegetable labeled sample (negative sample N: 0.02M PB buffer) in the propiconazole pesticide detection test strip in Experimental example 8 of 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. 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.
Propiconazole analogues
The goods number is: 28-AZC-33-1;
the merchant: toronto Research Chemicals.
The following examples include the synthesis of propiconazole hapten, propiconazole artificial antigen and the preparation of propiconazole antibody, the process is shown in figure 1.
Example 1 Synthesis and characterization of propiconazole hapten BHC-HS
1. Synthesis of propiconazole hapten
(1) To a 25mL round-bottomed flask for reaction, 30mg of a propiconazole analogue was added, 2mL of anhydrous dichloromethane was added to dissolve the same, and then 20mg of succinic anhydride and 4mg of 4-dimethylaminopyridine were added, and the reaction was stirred at normal temperature for 24 hours. The reaction is monitored by TLC (developing solvent conditions: ethyl acetate: petroleum ether: 3:1) until the reaction is finished, the reaction is extracted twice by ethyl acetate and saturated sodium chloride respectively after the reaction is dried, anhydrous sodium sulfate is added into an ethyl acetate phase for drying, and the propiconazole hapten BHC-HS is obtained after the reaction is concentrated;
2. and (3) identification:
taking a standard substance of the propiconazole analogue to perform nuclear magnetic identification spectrogram, and taking the propiconazole hapten BHC-HS to perform hydrogen spectrum identification and mass spectrum identification spectrogram.
The hydrogen spectrum results are as follows:1H NMR(600MHz,DMSO-d6)
δ12.28–12.23(m,1H),8.40(d,J＝5.2Hz,2H),7.84(d,J＝5.1Hz,2H),7.66(d,J＝5.3Hz,2H),7.46(t,J＝6.7Hz,2H),7.41(q,J＝10.2,7.0Hz,2H),4.84–4.74(m,4H),4.23(q,J＝5.6Hz,2H),3.98(dd,J＝10.8,5.6Hz,2H),3.85(ddd,J＝26.8,12.4,5.9Hz,5H),3.65(dt,J＝11.7,5.7Hz,2H),2.55(t,J＝6.0Hz,4H),2.50(q,J＝6.6,6.2Hz,6H),2.42(d,J＝5.3Hz,1H),1.23(d,J＝5.4Hz,1H).
the mass spectrometry results were as follows: MS: C17H17Cl2N3O6:429.0，ESI-[M-H]-:430。
According to the results of nuclear magnetic resonance hydrogen spectrum and mass spectrum, the derivation site is correct and successful, which indicates that the target product is successfully synthesized, named as propiconazole hapten BHC-HS, and the structural formula is shown as the formula (I):
the propiconazole hapten is named by adopting a systematic naming method:
(4- ((((2S, 4R) -2- ((1H-1,2,4-triazol-1-yl) methyl))) -2- (2,4-dichlorophenyl) -1,3-dioxolan-4-yl) methoxy) -4-oxobutanoic acid i.e.: 4- (((2S,4R) -2- ((1H-1,2,4-triazol-1-yl) methyl) -2- (2,4-dichlorophenyl) -1,3-dioxolan-4-yl) methoxy) -4-oxobutanic acid).
Example 2 Synthesis and identification of propiconazole complete antigen
1. Coupling of propiconazole complete antigen and carrier protein
(1) Weighing hapten 10mg, NHS 2mg and EDC 3mg, dissolving in DMF 100uL, and stirring overnight;
(2) weighing 9.4mg of Bovine Serum Albumin (BSA) and adding the BSA into 1mL of PBS buffer solution;
(3) dropwise and slowly adding the solution obtained in the step (1) into the solution obtained in the step (2), and stirring for 8 hours;
(4) dialyzing with PBS buffer solution for two days, 4 times per day, and obtaining the propiconazole analogue-HS-BSA complete antigen (BHC-HS-BSA) after the dialysis is finished.
Wherein, the formula of the phosphate buffer solution is as follows: na2HPO4 & 12H2O 2.90.90 g, NaCl 8.50g, KCl 0.20g, KH2PO40.20g, and distilled water to reach volume of 1000 mL.
In the same way, Keyhole Limpet Hemocyanin (KLH) is adopted to replace BSA (bovine serum albumin) as carrier protein to obtain the target product BHC-HS-KLH, and the process is the same as that for preparing the BHC-HS-BSA complete antigen.
2. And (3) identification:
the complete antigen of the propiconazole analogue is taken and ultraviolet full-wavelength scanning is carried out, and the result is shown in figure 3.
And (3) respectively carrying out ultraviolet (200-400 nm) scanning identification on BSA (bovine serum albumin), KLH (KLH) and the complete antigen, comparing the highest light absorption values of the substances before and after coupling, wherein the absorption curve of the complete antigen of the propiconazole analogue is obviously different from that of the carrier protein, and a characteristic absorption peak of the propiconazole analogue appears at 365nm, so that the curve of the complete antigen of the propiconazole analogue is the accumulated absorption characteristic of the two, and the successful coupling of the hapten of the propiconazole analogue and the BSA (bovine serum albumin) is proved to prepare the complete antigen.
EXAMPLE 3 preparation of propiconazole polyclonal antibody
(1) 2 (female and male) New Zealand white rabbits for 4 weeks were immunized, each complete antigen was BHC-HS-KLH (1mg/mL) 500. mu.L, and when primary immunization was performed, the BHC-HS-KLH was emulsified with Freund's complete adjuvant at a volume ratio of 1:1, and the New Zealand white rabbits were immunized. Then, the BHC-HS-KLH is emulsified with a Freund incomplete adjuvant according to the volume ratio of 1:1, and the boosting immunization is carried out once every three weeks for three times;
(2) after the third immunization, blood is taken from the ear vein of the rabbit every one week, the supernatant is obtained by centrifugation, and the supernatant is preserved at the temperature of minus 20 ℃ for use.
Example 4 ELISA detection of propiconazole polyclonal antibody
1. ELISA detection
(1) Serum was diluted 1:6400 with PBST while blank control wells were set (no serum added, PBST substituted);
(2) diluting a propiconazole (BHC-HS-BSA) artificial antigen to the concentration of 1 mu g/mL by using a coating solution, coating a 96-well enzyme label plate, adding 100 mu L of the antigen into each well, incubating overnight at 37 ℃, discarding the coating solution, and washing for 2 times;
(3) adding 120 μ L of sealing solution (1% fish skin collagen) into each well, sealing at 37 deg.C for 3 hr, removing sealing solution, clapping, and oven drying at 37 deg.C;
(4) serum was diluted 500-fold with PBST and propiconazole drug was diluted to 1000, 200, 40, 8, 1.6, 0.32, 0.064 ng/mL;
(5) adding 50 μ L propiconazole drug diluent (three groups in parallel) to each row, adding 50 μ L serum diluent/well, incubating at 37 deg.C for 40min, and washing for 5 times;
(6) adding goat anti-rabbit secondary antibody IgG-HRP (5000-fold dilution), incubating for 30min at 37 ℃, washing for 5 times, and clapping;
(7) adding color development liquid for developing for 10 min;
(8) 50 μ L of 10% H was added2SO4The reaction was stopped and the OD read at 450 nm;
(9) the drug propiconazole is changed into the drug of the ethaconazole, the triadimenol and the hexaconazole, the test is carried out according to the same dilution factor, and the cross reaction rate of the antibody to other structural analogues of the triazole fungicide is measured.
2. Results
The indirect competition ELISA standard curve for propiconazole antibody is shown in figure 4. The semi-Inhibitory Concentration (IC) of propiconazole antibody to propiconazole can be seen50) 4.23ng/mL, linear range of quantitative determination (IC)20～IC80) The detection limit is 0.29-61.83 ng/mL, the minimum detection limit is 0.06ng/mL, and the cross-reaction rate of the hexaconazole to the hexaconazole is lower than 1%.
Example 5 preparation of enzyme-linked immunosorbent assay kit for detecting propiconazole
An enzyme linked immunosorbent assay kit for detecting propiconazole is constructed, and comprises the following parts:
(1) preparing an enzyme label plate coated with a coating antigen: diluting a coating antigen (conjugate BHC-HS-BSA of propiconazole hapten and protein) into 1 mu g/mL by using a coating buffer solution, adding 100 mu L into each hole, incubating overnight at 37 ℃ in a dark place, pouring off liquid in the holes, washing for 2 times by using a washing solution, shaking to dry for 30s each time, then adding 200 mu L of a sealing solution into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, shaking to dry, drying, and performing vacuum sealing and storage by using an aluminum film; the coating buffer solution is a carbonate buffer solution with the pH value of 9.6 and 0.05mol/L, and the confining solution is a phosphate buffer solution with the pH value of 7.1-7.5 and containing 1-3% of casein and 0.1-0.3 mol/L.
(2) Propiconazole standard solution: 6 concentration gradients, 0. mu.g/L, 0.1. mu.g/L, 1. mu.g/L, 10. mu.g/L, 100. mu.g/L, 1000. mu.g/L, respectively.
(3) Propiconazole polyclonal antibody prepared in example 3
(4) Enzyme conjugate: and (3) a horseradish peroxidase-labeled propiconazole polyclonal antibody.
(5) The substrate color developing solution consists of solution A and solution B, wherein the solution A is carbamide peroxide, and the solution B is tetramethyl benzidine.
(6) The stop solution is 2mol/L sulfuric acid.
(7) The washing liquid has a pH value of 7.4, and contains 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer.
EXAMPLE 6 determination of propiconazole technical parameters
1. Sensitivity and detection limit of kit
The sensitivity of the kit is determined according to a conventional method, the range of a standard curve is 0.1-8.1 mug/L, and the floating range of IC50 (50% inhibitory concentration) is 0.29-61.83 ng/mL; the 20 samples were tested, the concentrations corresponding to the respective percent absorbance values were found from the standard curve, and the detection limits were expressed as the mean value of the 20 sample concentrations plus 3 standard deviations, showing that the method had detection limits of 0.4. mu.g/kg and 0.6. mu.g/kg for vegetables and fruits, respectively.
2. Stability test of kit
The storage condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard), the 50% inhibition concentration and the actual measurement value of the propiconazole addition of the kit are all within the normal range after 12 months of measurement. Considering that abnormal storage conditions occur in the transportation and use processes, the kit is placed for 7 days under the storage condition of 37 ℃ for accelerated aging experiments, and the results show that all indexes of the kit completely meet the requirements. And in consideration of the occurrence of the freezing condition of the kit, the kit is frozen for 7 days in a refrigerator at the temperature of-20 ℃, and the determination result also shows that all indexes of the kit are completely normal. From the above results, it can be obtained that the kit can be stored at 2 to 8 ℃ for at least 12 months.
1. Preparation of gold-labeled antibody and gold-labeled conjugate pad
(1) Preparation of colloidal gold labeled propiconazole pesticide polyclonal antibody
Colloidal gold suspension with average diameter of 40nm was prepared by reducing chloroauric acid with trisodium citrate. Under reflux, 100mL of 0.01% chloroauric acid solution was heated to boiling, and 1.1mL of 1% trisodium citrate was added rapidly with constant stirring. Heating and stirring were continued for 5min when the reaction solution became reddish-red in color. After cooling to room temperature, 0.05% sodium azide was added and stored at 4 ℃.
The colloidal gold is labeled with 0.2mol of K before being labeled with the antibody2CO3The solution was adjusted to pH 8.2 and 30. mu.g of antibody-labeled 1mL of colloidal gold solution was determined by classical NaCl titration. Then, labeling was carried out in an optimum amount, and after 1 hour of labeling, 10% BSA was added with stirring (to make the final BSA concentration 1%), and after 1 hour of incubation, centrifugation was carried out at 10000rpm at 4 ℃ for 25min, and the supernatant was removed. Adding 5% BSA solution with the same volume of colloidal gold solution for resuspension, centrifuging at 4 deg.C and 10000rpm for 25min, and repeating twice. Finally, it was resuspended in 1/5 volumes of colloidal gold solution in TB solution (containing 3% BSA, 3% sucrose, 0.01mol/L sodium borate and 0.05% sodium azide) and stored at 4 ℃.
(2) Preparation of gold-labeled conjugate pad
Spraying 4% BSA solution at 8 μ L/cm onto glass fiber cotton with XYZ-3000 three-dimensional film spraying instrument, drying at 42 deg.C for 50min in a drying oven, spraying gold-labeled antibody at 6 μ L/cm on glass fiber cotton, drying at 42 deg.C for 50min in the drying oven, and vacuum drying for storage.
2. Coupled antigen goat anti-rabbit coated cellulose membrane
An envelope antigen (BHC-HS-BSA) with a concentration of 1mg/mL was sprayed onto the lower side of the cellulose membrane in an amount of 1.2. mu.L/cm using an XYZ-3000 three-dimensional spray coater as a detection line. Goat anti-rabbit IgG at a concentration of 120. mu.g/L was sprayed onto the upper side of the cellulose membrane in an amount of 1.2. mu.L/cm using an XYZ-3000 three-dimensional spray coater as a control line with 10mm intervals between the two lines.
3. Assembly of quick test paper strip
The cellulose membrane 4 is stuck on the middle part of the lining board 1, and the absorbent pad 7 is stuck on the upper side of the cellulose membrane 4 and is overlapped with the cellulose membrane 4 by 1 mm. The gold-labeled conjugate pad 3 was stuck under the cellulose film 4 with an overlap of 1 mm. The sample pad 2 is stuck under the gold-labeled conjugate pad 3 with an overlap of 2 mm. The assembled test paper board was cut into test paper strips of 3.08mm width with a cutter. FIG. 5 is a schematic side view of a colloidal gold test strip for rapid detection of propiconazole as a pesticide; FIG. 6 is a schematic view of a test strip for rapid detection of propiconazole pesticide colloidal gold; wherein: 1. a liner plate; 2. a sample pad; 3. a gold-labeled bond pad; 4. a cellulose membrane; 5. invisible detection lines; 6. invisible contrast lines; 7. a water absorbent pad; 8-1, immersing the sample into a protective film; 8-2, a handle end protective film; 9. and marking the line.
When the sample solution to be tested is added into the test end of the test strip or the test paper card, the solution to be tested drives the object to be tested and the gold-labeled antibody in the gold-labeled conjugate pad 3 to diffuse together to the cellulose membrane 4 through the siphon action, and finally permeates into the end 7 of the water absorption pad. In the diffusion process, if the sample contains the substance to be detected, the substance to be detected is combined with the gold-labeled antibody, so that the antigen binding site on the gold-labeled antibody is occupied, the combination of the gold-labeled antibody and the invisible detection line 5 (the combination of the hapten and the carrier protein) on the cellulose membrane 4 is prevented, and the invisible detection line 5 is not colored or is weakly colored, namely, the detection sample is positive or weakly positive; if the sample to be detected does not exist in the sample, a clear red line is displayed when the gold-labeled antibody meets the invisible detection line 5 in the upward moving process, and the detection sample is negative. Similarly, the gold-labeled antibody also binds to the invisible control line 6 (goat anti-rabbit IgG) on the cellulose membrane 4, so that the invisible control line 6 is red. The presence or absence of the color of the invisible control line 6 indicates the effectiveness or ineffectiveness of the test strip, respectively.
Example 8 detection of a sample by a propiconazole colloidal gold fast detection test strip
1. Detection of vegetable samples
(1) Preparation of test sample solution
And (4) pretreating the vegetable sample solution. Weighing 10g of sample, homogenizing, transferring into a 50mL centrifuge tube, adding 20mL of methanol, vortexing for 3min, ultrasonically extracting for 10min, centrifuging at 4000rpm for 5min, taking 1mL of supernatant, diluting with working buffer solution (PBS) by a certain multiple, and determining the content of the propiconazole pesticide in the solution.
(2) Detection and result determination
As shown in fig. 7: and inserting the sample end of the colloidal gold test strip into the pretreated sample to be detected, wherein the insertion depth is not more than the identification line 9, and the solution to be detected drives the substance to be detected and the gold-labeled antibody in the gold-labeled conjugate pad 3 to diffuse together to the cellulose membrane 4 through siphoning and finally permeates into the end 7 of the water absorption pad. In the diffusion process, if the analyte in the sample can prevent the combination of the gold-labeled antibody and the invisible detection line 5 (the conjugate of the hapten and the carrier protein) on the cellulose membrane 4, the invisible detection line 5 does not develop color, i.e. the detection sample is positive (as shown in fig. 7C); if the analyte in the sample prevents the combination of part of the gold-labeled antibody and the invisible detection line 5 on the cellulose membrane 4, the invisible detection line 5 shows weak red, which means that the detection sample is weak positive (as shown in FIG. 7B); if the substance to be detected in the sample cannot prevent the gold-labeled antibody from being combined with the invisible detection line 5 on the cellulose membrane 4, the invisible detection line 5 shows a clear red line, which indicates that the detected sample is negative (see fig. 7A). Similarly, the gold-labeled antibody also binds to the invisible control line 6 (goat anti-rabbit IgG) on the cellulose membrane 4, so that the invisible control line 6 is red, and the presence or absence of the color of the invisible control line 6 indicates the effectiveness or ineffectiveness of the test strip (e.g., D, E is ineffective in FIG. 7A, B, C). And judging the detection result in 3-5 minutes.
The sensitivity of the test strip is tested by using 200 mu L of extract of the medicated vegetable sample, the result is shown in figure 8, the visual detection line of the propiconazole detection test strip (figure 8) is 20ng/mL through visual observation, the visual detection limit LOD is obviously lower than the maximum residue limit of propiconazole (0.05 mg/kg of partial vegetables) in the national standard, and the experimental result shows that the method has the advantages of high detection speed, intuition, convenient operation and the like.
2. Stability test
The test strip is placed into an aluminum platinum bag for vacuum packaging and is stored at room temperature, all indexes are stable after 3 months, namely the sensitivity is basically unchanged, the detection color development depth is uniform, and the reaction results are consistent.
The above embodiments are preferred embodiments of the present invention, but 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 thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (11)
1. A propiconazole hapten, which has a structural formula shown in formula (I):
2. a method of preparing the propiconazole hapten according to claim 1, comprising the steps of: dissolving the propiconazole analogue in anhydrous dichloromethane, adding succinic anhydride and 4-dimethylaminopyridine to react, and confirming a product by using a thin-layer chromatography silica gel plate; after the reaction product is spin-dried, adding ethyl acetate and saturated saline solution for extraction, drying the ester layer by using anhydrous sodium sulfate, and finally concentrating to obtain the propiconazole hapten; the structural formula of the propiconazole analogue is as follows:
3. the preparation method according to claim 2, wherein the developing solvent for thin layer chromatography is a solvent having a volume ratio of 3:1 ethyl acetate and petroleum ether.
4. A propiconazole complete antigen, which is characterized in that the structural formula of the propiconazole complete antigen is as follows:
5. the method for preparing a propiconazole complete antigen according to claim 4, which is obtained by coupling the propiconazole hapten according to claim 1 with a carrier protein by an active ester method.
6. The method for preparing propiconazole complete antigen according to claim 5, wherein the carrier protein is bovine serum albumin or keyhole limpet hemocyanin.
7. A preparation method of a propiconazole polyclonal antibody, which is characterized in that the propiconazole complete antigen of claim 4 is emulsified and then immunized by New Zealand white rabbits.
8. An enzyme linked immunosorbent assay kit for detecting propiconazole is characterized by comprising: the enzyme label plate coated with coating antigen, propiconazole standard solution, propiconazole polyclonal antibody, enzyme conjugate concentrated solution, enzyme conjugate diluent, substrate developing solution, stop solution and washing solution;
the coating antigen is a conjugate of propiconazole hapten and protein; the enzyme conjugate is a propiconazole polyclonal antibody marked by horseradish peroxidase; the polyclonal propiconazole antibody is prepared by the method of claim 7.
9. A propiconazole colloidal gold rapid detection test strip is characterized by comprising a lining plate (1), and a sample pad (2), a gold-labeled conjugate pad (3), a cellulose membrane (4) and a water absorption pad (7) which are sequentially arranged on the lining plate (1), wherein a colloidal gold-labeled polyclonal antibody prepared by the method of claim 7 is adsorbed in the gold-labeled conjugate pad (3), the cellulose membrane (4) is provided with an invisible detection line (5) and an invisible contrast line (6), the invisible detection line (5) is printed by the propiconazole complete antigen solution of claim 4, and the invisible contrast line (6) is printed by goat anti-rabbit antibodies.
10. The test strip of claim 9, wherein a protective film (8-1) is disposed between the gold-labeled conjugate pad (3) and the sample pad (2), and a sample identification line (9) to be detected is printed on the protective film, and the identification line is biased to 0.5cm of one side of the sample pad (2).
11. The test strip of claim 9, wherein the sample pad (2) is made of glass fiber cotton and/or a nylon membrane and/or a polyvinylidene fluoride membrane and/or a polyester membrane, and the cellulose membrane (4) is made of a nitrocellulose membrane and/or a pure cellulose membrane and/or a carboxylated cellulose membrane.
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