CN113831253B - Pendimethalin hapten as well as preparation method and application thereof - Google Patents
Pendimethalin hapten as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a pendimethalin hapten, and a preparation method and application thereof. The pendimethalin hapten is synthesized from head, and five hapten are respectively introduced into connecting arm with carboxyl from benzyl, phenylamino and pentane radical terminal of pendimethalin. Through experimental comparison, the hapten effect of introducing a connecting arm on the three-position methyl of the benzene ring of the pendimethalin parent is best. The monoclonal antibody prepared by the hapten has high sensitivity and strong specificity. The sensitivity (IC) was measured by indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) 50 ) At 0.53. Mu.g/L, the detection range (IC 10 ‑IC 90 ) 0.11-2.59 mug/L. The monoclonal antibody and other dinitrobenzene herbicideNone of them had cross-reactions (CR < 1.1%). In addition, the monoclonal antibody is used for preparing the pendimethalin colloidal gold rapid detection test strip which has high detection sensitivity, strong specificity, simplicity, convenience, rapidness and low cost. The test strip can be used for detecting the residue of pendimethalin in agricultural products, and the detection limit of the test strip reaches 25 mug/L.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to five pendimethalin hapten, and a preparation method and application thereof.
Background
Pendimethalin (pendimethalin) belongs to a dinitroaniline herbicide and is widely applied to weed control of crops such as rice, corn, potato, cabbage and the like and vegetables. Pendimethalin inhibits weed growth by inhibiting mitosis and cell wall formation in plant cells. Because of its broad spectrum and application, pendimethalin is one of the most commonly used selective herbicides in agriculture, and more than 150 herbicides using pendimethalin as an active ingredient are registered in China at present. Pendimethalin belongs to low-toxicity pesticides according to the pesticide toxicity grading standard in China. According to national standard GB2763-2021 maximum residue limit of pesticides in food officially applied in 2021, 9 and 3 of China, the allowable daily intake of pendimethalin is 0.1mg/kg bw, the maximum residue limit of pendimethalin in paddy, potato, spinach, chinese cabbage, leek and common head cabbage is 0.2mg/kg, the maximum residue limit of pendimethalin in corn, peanut, leaf lettuce and asparagus is 0.1mg/kg, and the maximum residue limit of pendimethalin in coarse cereal crops, pod edible beans vegetables, peas and nuts is 0.05mg/kg.
Currently, the detection method of pendimethalin is mainly an instrument detection method, and mainly comprises a gas chromatography, a gas chromatography-mass spectrometry, a high performance liquid chromatography-mass spectrometry and the like. The instrument detection method has high accuracy and sensitivity, but expensive instruments, professional technicians and complicated pretreatment operation are required, and real-time on-site detection of the sample is difficult to realize. Immunoassay, such as enzyme-linked immunosorbent assay, is an economical, simple, convenient, on-site, real-time, rapid and accurate high-throughput analysis method, and can form a complementary detection method with an instrument detection method according to different detection requirements. In small molecule immunoassays, antibodies largely determine the sensitivity and specificity of the immunoassay, while the production of high quality antibodies depends on a reasonable hapten design. Therefore, the design and synthesis of reasonable pendimethalin hapten is the key for establishing an economic, simple, on-site, real-time, rapid and accurate immunoassay method.
At present, a pendimethalin immunoassay method has been reported. Chen Li et al have invented a pendimethalin hapten obtained by reacting 3- (1-ethyl-alanyl) -6-methyl-2, 4-dinitrobenzaldehyde with 3-hydrazinopropionic acid, and successfully prepared pendimethalin monoclonal antibodies. The pendimethalin enzyme-linked immunosorbent assay method and the colloidal gold test strip developed based on the monoclonal antibody can realize the quick detection of pendimethalin in tobacco, but have poor sensitivity (half inhibition rate is 1.5 mg/kg), and limit the application of the immunoassay method in quick detection of pendimethalin in agricultural products. Therefore, there is a need for further improvements in pendimethalin hapten structures to increase the sensitivity of immunoassay methods.
Disclosure of Invention
The invention provides a pendimethalin hapten structure, and preparation and application thereof, and determines the optimal pendimethalin hapten structure through experiments.
In the first aspect, five pendimethalin hapten structures designed by the invention are shown as the formulas I-V.
In a second aspect, the invention provides a method for preparing the five haptens.
The hapten preparation steps of the formula I are as follows:
step 1) 13.56g of 1-carboxypropyltriphenyl phosphonium bromide is dissolved in 10mL of tetrahydrofuran, cooled to-20 ℃ by dry ice bath, 65mL of sodium amide is slowly added dropwise, and the mixture is stirred for 1 hour at-20 ℃ after the dropwise addition. 5g of 5-chloro-2-methylbenzaldehyde are dissolved in 20mL of tetrahydrofuran, the solution is added dropwise, and the reaction is stirred at-20℃for 4 hours. The reaction solution was slowly poured into 20mL of ice water, the impurities were removed by back extraction with methyl tert-butyl ether, the pH of the aqueous phase was adjusted to 2-3 with 1M hydrochloric acid, extraction was repeated twice with 20mL of ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. 4.5g of product 1 are obtained.
Step 2) the product 1 was dissolved in 100mL of absolute ethanol, 0.45g of platinum dioxide was added, the mixture was degassed and purged 3 times with nitrogen, and the mixture was stirred and reacted for 5 hours under nitrogen protection by introducing hydrogen, to obtain a product 2.
Step 3) 3.5g of the product 2 is dissolved in 7mL of concentrated sulfuric acid, cooled to zero by ice water, 2.56g of concentrated nitric acid is slowly added dropwise, and the reaction is carried out for 2 hours at room temperature, thus obtaining the product 3.
Step 4) 2g of the product 3 were dissolved in 12mL of N-methylpyrrolidone and 0.55g of 3-aminopentane were added, the reaction mixture was degassed 3 times with nitrogen and reacted at 90℃for 12 hours under nitrogen protection. The reaction solution was poured into 10mL of ice water, extracted with 20mL of ethyl acetate, repeated twice, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative high performance liquid chromatography on a column Phenomenex luna C, 100×40mm×5 μm with water (0.1% trifluoroacetic acid) to acetonitrile=30% to 70%. The hapten of formula I was obtained as a yellow solid.
The hapten preparation steps of the formula II are as follows:
step 1) 18.98g of 1-carboxypropyltriphenyl phosphonium bromide is dissolved in 10mL of tetrahydrofuran, cooled to-20 ℃ by dry ice bath, 65mL of sodium amide is slowly added dropwise, and the mixture is stirred for 1 hour at-20 ℃ after the dropwise addition. 7g of 4-chloro-2-methylbenzaldehyde was dissolved in 20mL of tetrahydrofuran, and the solution was added dropwise thereto, followed by stirring and reaction at-20℃for 4 hours. The reaction solution was slowly poured into 20mL of ice water, the impurities were removed by back extraction with methyl tert-butyl ether, the pH of the aqueous phase was adjusted to 2-3 with 1M hydrochloric acid, extraction was repeated twice with 20mL of ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. 6.2g of product 1 are obtained.
Step 2) the product 1 was dissolved in 100mL of absolute ethanol and 0.45g of platinum dioxide was added, and the mixture was degassed and purged 3 times with nitrogen, and reacted at-20 ℃ for 5 hours under nitrogen protection with stirring by introducing hydrogen, to obtain a product 2.
Step 3) 5g of the product 2 is dissolved in 10mL of concentrated sulfuric acid, cooled to zero by ice water, 3.55g of concentrated nitric acid is slowly added dropwise, and the reaction is carried out for 2 hours at room temperature, thus obtaining the product 3.
Step 4) 3g of the product 3 was dissolved in 12mL of N-methylpyrrolidone and 0.99g of 3-aminopentane was added, the reaction mixture was degassed 3 times with nitrogen and reacted at 90℃for 12 hours under nitrogen protection. The reaction solution was poured into 10mL of ice water, extracted with 20mL of ethyl acetate, repeated twice, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative high performance liquid chromatography on a column Phenomenex luna C, 100×40mm×5 μm with water (0.1% trifluoroacetic acid) to acetonitrile=30% to 70%. The hapten of formula II was obtained as a yellow solid.
The hapten preparation steps of formula III are as follows:
step 1) 8g of 3, 4-dimethylbenzene is dissolved in 16mL of concentrated sulfuric acid, 9.43g of concentrated nitric acid is slowly added dropwise after the temperature is reduced to zero by ice water, and the reaction is carried out for 2 hours at room temperature, thus obtaining a product 1.
Step 2) 2g of the product 1 was dissolved in 3.6mL of N-methylpyrrolidone, 2g of 5-aminopentanoic acid and 2.6g of triethylamine were added, the reaction mixture was purged 3 times with nitrogen and reacted at 120℃for 14 hours under nitrogen protection. The reaction solution was poured into 50mL of ice water, extracted with 20mL of ethyl acetate, repeated twice, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography (SiO 2 Petroleum ether/ethyl acetate=100/1-0/1). The hapten of formula III was obtained as a yellow solid.
The hapten preparation steps of formula IV are as follows:
step 1) hapten preparation step 1) of the same formula III yields product 1.
Step 2) 0.11g of product 1 and 0.13g of 4-aminohexane were dissolved in 1.1mL of absolute ethanol and 0.33g of potassium carbonate was added, and the mixture was degassed and purged 3 times with nitrogen, and reacted at 80℃under nitrogen protection with stirring for 24 hours. The reaction solution was filtered, a cake was collected, and the cake was purified by column chromatography (SiO 2 Dichloromethane/methanol=50/1 to 6/1) to give hapten of formula IV as a yellow solid.
The hapten preparation steps of formula V are as follows:
step 1) hapten preparation step 1) of the same formula IV yields product 1.
Step 2) 0.65g of product 1 and 1.32g of 3-aminopentane were dissolved in 6.5mL of absolute ethanol and 1.95g of potassium carbonate were added, and the mixture was degassed and purged 3 times with nitrogen, and reacted at 80℃under nitrogen protection with stirring for 24 hours. The reaction solution was filtered, a cake was collected, and the cake was purified by column chromatography (SiO 2 Dichloromethane/methanol=50/1 to 6/1) to give hapten of formula IV as a yellow solid.
In a third aspect, the invention provides complete antigens of the five pendimethalin haptens, wherein the complete antigens are obtained by coupling hapten and carrier protein through an active ester method. The pendimethalin complete antigen is divided into pendimethalin immunogen and coating antigen, wherein the pendimethalin immunogen is obtained by coupling of pendimethalin hapten and Bovine Serum Albumin (BSA) respectively, and the pendimethalin coating antigen is obtained by coupling of pendimethalin hapten and Ovalbumin (OVA) respectively.
Step 1) 0.05g of pendimethalin hapten and 0.02g N-hydroxysuccinimide are dissolved in 3mL of N, N-dimethylformamide and 0.04g of dicyclohexylcarbodiimide are added thereto and stirred at room temperature for 4 hours.
Step 2) centrifuging the reaction solution obtained in the step 1) at 4000rpm for 5 minutes, slowly dripping half of the supernatant into 8mL of phosphate buffer solution (0.01 mol/L, pH=7.4) containing 0.1g of bovine serum albumin, and reacting for 24 hours at room temperature in a dark place to prepare the pendimethalin immunogen; the remaining supernatant was slowly added dropwise to 8mL of a phosphate buffer (0.01 mol/L, ph=7.4) containing 0.07g of ovalbumin, and reacted at room temperature in the absence of light for 24 hours to prepare pendimethalin coating antigen.
Step 3) transferring the pendimethalin immunogen and the coating antigen obtained in the step 2) into a dialysis bag, and dialyzing and removing impurities by using 0.01mol/L phosphate buffer solution for 3 days, wherein the dialyzate is changed for at least 3 times per day. The whole antigen after dialysis was stored in sub-packs at-20 ℃.
In a fourth aspect, the invention provides a pendimethalin monoclonal antibody, which is prepared from the pendimethalin complete antigen and can specifically recognize pendimethalin or pendimethalin hapten and complete antigen.
In a fifth aspect, the invention provides a colloidal gold test strip for detecting pendimethalin residues in crop and environmental samples.
The invention has the following beneficial effects:
the five pendimethalin hapten provided by the invention not only maintains the characteristic structure of pendimethalin to the greatest extent, but also introduces the connecting arm from different directions, fully considers the possibility of influence of different characteristic structures on antibody properties, and simultaneously, the carboxyl on the connecting arm enables the coupling of hapten and carrier protein to be easy to carry out; the pendimethalin antigen provided by the invention has strong immunogenicity, can better stimulate the immune system of mice, generates a pendimethalin antibody with strong specificity and high sensitivity, and provides a key reagent for establishing a pendimethalin immunoassay method.
The pendimethalin monoclonal antibodies obtained by immunization of the five pendimethalin antigens provided by the invention have better titer, specificity and affinity, and each monoclonal antibody can at least recognize two coating antigens. ELISA Inhibitory Concentration (IC) established by monoclonal antibodies obtained by immunization with five pendimethalin antigens 50 ) Can be less than 10 mug/L. Through experimental comparison, the monoclonal antibody 5F7 obtained from hapten formula I has the best effect and IC 50 At 0.53. Mu.g/L, compared with the IC reported previously 50 The method improves the efficiency by 3000 times, the linear range is 0.11-2.59 mug/L, and the cross reaction rate with other dinitroaniline herbicides is lower than 1.1%; the minimum detection limit of the colloidal gold test strip prepared based on the monoclonal antibody can reach 25 mug/L, which is improved by 200 times compared with the previous report. Therefore, the hapten formula I provided by the invention is more beneficial to the preparation of pendimethalin monoclonal antibodies than hapten designed by the former person. The ELISA method and the colloidal gold test strip obtained based on the hapten and developed by monoclonal show better sensitivity, and have the characteristics of simple operation, economy and rapidness.
Drawings
FIG. 1 shows the synthetic route of pendimethalin hapten.
FIG. 2 is an evaluation of the immune effect of pendimethalin hapten.
FIG. 3 shows the standard competition curve of pendimethalin ELISA.
Fig. 4 is a top view of the structure of the pendimethalin colloidal gold test strip.
Fig. 5 is a side view of the structure of the pendimethalin colloidal gold test strip. In the figure, 1: lining board, 2: sample pad, 3: gold mark binding pad, 4: cellulose film, 5: invisible detection line, 6: stealth control line, 7: absorbent pad, 8-1: sample immersion end protection film, 8-2: handle end protection film, 9: and (5) identifying lines.
FIG. 6 is a diagram showing the judgment of the result of a pendimethalin colloidal gold test strip; in the figure, A, B is a negative sample detection result, C, D is a strong positive sample detection result, and E, F is a test strip failure.
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention, in conjunction with the accompanying drawings. Specific materials and sources thereof used in embodiments of the present invention are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, as materials that are the same as or similar to the type, model, quality, nature, or function of the reagents and instruments described below may be used in the practice of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1: the synthesis of pendimethalin hapten is shown in figure 1.
Synthesis of hapten of formula I
Step 1) 13.56g of 1-carboxypropyltriphenyl phosphonium bromide is dissolved in 10mL of tetrahydrofuran, cooled to-20 ℃ by dry ice bath, 65mL of sodium amide is slowly added dropwise, and the mixture is stirred for 1 hour at-20 ℃ after the dropwise addition. 5g of 5-chloro-2-methylbenzaldehyde are dissolved in 20mL of tetrahydrofuran, the solution is added dropwise, and the reaction is stirred at-20℃for 4 hours. The reaction solution was slowly poured into 20mL of ice water, the impurities were removed by back extraction with methyl tert-butyl ether, the pH of the aqueous phase was adjusted to 2-3 with 1M hydrochloric acid, extraction was repeated twice with 20mL of ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. 4.5g of product 1 are obtained.
Step 2) the product 1 was dissolved in 100mL of absolute ethanol, 0.45g of platinum dioxide was added, the mixture was degassed and purged 3 times with nitrogen, and the mixture was stirred and reacted for 5 hours under nitrogen protection by introducing hydrogen, to obtain a product 2.
Step 3) 3.5g of the product 2 is dissolved in 7mL of concentrated sulfuric acid, cooled to zero by ice water, 2.56g of concentrated nitric acid is slowly added dropwise, and the reaction is carried out for 2 hours at room temperature, thus obtaining the product 3.
Step 4) 2g of the product 3 were dissolved in 12mL of N-methylpyrrolidone and 0.55g of 3-aminopentane were added, the reaction mixture was degassed 3 times with nitrogen and reacted at 90℃for 12 hours under nitrogen protection. The reaction solution was poured into 10mL of ice water, extracted with 20mL of ethyl acetate, repeated twice, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative high performance liquid chromatography on a column Phenomenex luna C, 100×40mm×5 μm with water (0.1% trifluoroacetic acid) to acetonitrile=30% to 70%. The hapten of formula I was obtained as a yellow solid.
Synthesis of hapten of formula II
Step 1) 18.98g of 1-carboxypropyltriphenyl phosphonium bromide is dissolved in 10mL of tetrahydrofuran, cooled to-20 ℃ by dry ice bath, 65mL of sodium amide is slowly added dropwise, and the mixture is stirred for 1 hour at-20 ℃ after the dropwise addition. 7g of 4-chloro-2-methylbenzaldehyde was dissolved in 20mL of tetrahydrofuran, and the solution was added dropwise thereto, followed by stirring and reaction at-20℃for 4 hours. The reaction solution was slowly poured into 20mL of ice water, the impurities were removed by back extraction with methyl tert-butyl ether, the pH of the aqueous phase was adjusted to 2-3 with 1M hydrochloric acid, extraction was repeated twice with 20mL of ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. 6.2g of product 1 are obtained.
Step 2) the product 1 was dissolved in 100mL of absolute ethanol and 0.45g of platinum dioxide was added, and the mixture was degassed and purged 3 times with nitrogen, and reacted at-20 ℃ for 5 hours under nitrogen protection with stirring by introducing hydrogen, to obtain a product 2.
Step 3) 5g of the product 2 is dissolved in 10mL of concentrated sulfuric acid, cooled to zero by ice water, 3.55g of concentrated nitric acid is slowly added dropwise, and the reaction is carried out for 2 hours at room temperature, thus obtaining the product 3.
Step 4) 3g of the product 3 was dissolved in 12mL of N-methylpyrrolidone and 0.99g of 3-aminopentane was added, the reaction mixture was degassed 3 times with nitrogen and reacted at 90℃for 12 hours under nitrogen protection. The reaction solution was poured into 10mL of ice water, extracted with 20mL of ethyl acetate, repeated twice, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by preparative high performance liquid chromatography on a column Phenomenex luna C, 100×40mm×5 μm with water (0.1% trifluoroacetic acid) to acetonitrile=30% to 70%. The hapten of formula II was obtained as a yellow solid.
Synthesis of hapten of formula III
Step 1) 8g of 3, 4-dimethylbenzene is dissolved in 16mL of concentrated sulfuric acid, 9.43g of concentrated nitric acid is slowly added dropwise after the temperature is reduced to zero by ice water, and the reaction is carried out for 2 hours at room temperature, thus obtaining a product 1.
Step 2) 2g of the product 1 was dissolved in 3.6mL of N-methylpyrrolidone, 2g of 5-aminopentanoic acid and 2.6g of triethylamine were added, the reaction mixture was purged 3 times with nitrogen and reacted at 120℃for 14 hours under nitrogen protection. The reaction solution was poured into 50mL of ice water, extracted with 20mL of ethyl acetate, repeated twice, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography (SiO 2 Petroleum ether/ethyl acetate=100/1-0/1). The hapten of formula III was obtained as a yellow solid.
Synthesis of hapten of formula IV
Step 1) hapten preparation step 1) of the same formula III yields product 1.
Step 2) 0.11g of product 1 and 0.13g of 4-aminohexane were dissolved in 1.1mL of absolute ethanol and 0.33g of potassium carbonate was added, and the mixture was degassed and purged 3 times with nitrogen, and reacted at 80℃under nitrogen protection with stirring for 24 hours. The reaction solution was filtered, a cake was collected, and the cake was purified by column chromatography (SiO 2 Dichloromethane/methanol=50/1 to 6/1) to give hapten of formula IV as a yellow solid.
Synthesis of hapten of V
Step 1) hapten preparation step 1) of the same formula IV yields product 1.
Step 2) 0.65g of product 1 and 1.32g of 3-aminopentane were dissolved in 6.5mL of absolute ethanol and 1.95g of potassium carbonate were added, and the mixture was degassed and purged 3 times with nitrogen, and reacted at 80℃under nitrogen protection with stirring for 24 hours. The reaction solution was filtered, a cake was collected, and the cake was purified by column chromatography (SiO 2 Dichloromethane/methanol=50/1 to 6/1) to give hapten of formula IV as a yellow solid.
Embodiment case 2: identification of pendimethalin hapten
Results of pendimethalin formula I hapten nuclear magnetic identification: 1 H NMR(400MHz，d6-DMSO)δ8.112(s，1H，ArH)，7.052(d，J＝10.4，1H，NH)，2.93(m，1H，NHCH)，2.43(t，J＝8.4，2H，1ArCH 2 )，2.29(s，3H，1ArCH 3 )，2.239(t，J＝7.2，2H，HOOCCH 2 )，1.468(m，8H，4CH 2 )，0.787(t，J＝7.2，6H，2CH 2 CH 3 )。
results of pendimethalin formula II hapten nuclear magnetic identification: 1 H NMR(400MHz，CDCl 3 )δ8.07(s，1H，ArH)，7.645(d，J＝12Hz，1H，NH)，3.170(m，1H，NHCH)，2.626(t，J＝7.6Hz，2H，1ArCH 2 )，2.433(t，J＝6.8Hz，2H，OOCCH 2 )，2.206(s，3H，1ArCH 3 )，1.555(m，8H，4CH 2 )，0.889(t，J＝7.2Hz，6H，2CH 2 CH 3 )。
results of pendimethalin formula III hapten nuclear magnetic identification: 1 H NMR(400MHz，d6-DMSO)δ8.092(s，1H，ArH)，7.504(s，1H，NH)，2.990(t，J＝4.8Hz，2H，NHCH 2 )，2.25(s，3H，ArCH 3 )，2.15(s，3H，ArCH 3 )，2.121(t，J＝9.6Hz，2H，OOCCH 2 )，1.495(m，4H，2CH 2 )。
results of pendimethalin formula IV hapten nuclear magnetic identification: 1 H NMR(400MHz，d6-DMSO)δ8.103(s，1H，ArH)，7.026(d，J＝10Hz，1H，NH)，3.068(m，1H，NHCH)，2.27(s，3H，ArCH 3 )，2.14(s，3H，ArCH 3 )，2.094(t，J＝6.8Hz，2H，OOCCH 2 )，1.439(m，4H，2CH 2 )，0.779(t，J＝7.2Hz，3H，CH 2 CH 3 ).
results of pendimethalin-type V hapten nuclear magnetic identification: 1 H NMR(400MHz，d6-DMSO)δ8.059(s，1H，ArH)，7.22(d，J＝10Hz，1H，NH)，2.359(m，3H)，2.259(s，3H，ArCH 3 )，2.137(s，3H，ArCH 3 )，1.518(m，2H，OOCCH 2 )，0.787(t，J＝7.2，3H，CH 2 CH 3 )。
example 3: preparation of pendimethalin antigen
Step 1) 0.05g of pendimethalin hapten and 0.02g N-hydroxysuccinimide are dissolved in 3mL of N, N-dimethylformamide and 0.04g of dicyclohexylcarbodiimide are added thereto and stirred at room temperature for 4 hours.
Step 2) centrifuging the reaction solution obtained in the step 1) at 4000rpm for 5 minutes, slowly dripping half of the supernatant into 8mL of phosphate buffer solution (0.01 mol/L, pH=7.4) containing 0.1g of bovine serum albumin, and reacting for 24 hours at room temperature in a dark place to prepare the pendimethalin immunogen; the remaining supernatant was slowly added dropwise to 8mL of a phosphate buffer (0.01 mol/L, ph=7.4) containing 0.07g of ovalbumin, and reacted at room temperature in the absence of light for 24 hours to prepare pendimethalin coating antigen.
Step 3) transferring the pendimethalin immunogen and the coating antigen obtained in the step 2) into a dialysis bag, and dialyzing and removing impurities by using 0.01mol/L phosphate buffer solution for 3 days, wherein the dialyzate is changed for at least 3 times per day. The whole antigen after dialysis was stored in sub-packs at-20 ℃.
Example 4: pendimethalin antigen immunized mice
Five 6-8 week old BALB/c females were immunized by five intraperitoneal injections with each pendimethalin immunogen (i.e., conjugate of pendimethalin hapten and bovine serum albumin). Before each injection, the pendimethalin immunogen is mixed with 1:1 volume of immunological adjuvant and fully emulsified, the injection dose is 100 mug/dose, the immunization interval is two weeks, and the other adjuvants are incomplete adjuvants except Freund's complete adjuvant for the first immunization. Three days prior to fusion, mice were subjected to sprint immunization at a dose of 100 μg/mouse without using immune adjuvant.
Example 5: cell fusion
Step 1) preparation of myeloma cells: three bottles of SP2/0 myeloma cells from mice in logarithmic growth phase were removed, and the mice were transferred to 50mL centrifuge tubes after being blown down with DMEM medium and fixed to 35mL.
Step 2) preparation of spleen cells: three days after sprint immunization, mice were removed from the eyeballs, sacrificed by cervical pulling, spleens removed from the mice with surgical forceps and scissors, and surface fat and connective tissue removed. Spleens were placed in a 70 μm cell screen and cells were driven out into DMEM broth, transferred to a 50mL centrifuge tube and fixed to 35mL. Myeloma cells and spleen cells were centrifuged (1000 rpm,10 min), the supernatant was discarded, resuspended and counted.
Step 3) cell fusion: spleen cells and SP2/0 myeloma cells were mixed in a 50mL centrifuge tube at a ratio of 10:1, centrifuged (1000 rpm,10 min), the supernatant was discarded, and the bottom cells were shaken apart and incubated in a 37℃water bath. 1mL of PEG1500 preheated in advance is sucked by a sterile syringe, the PEG1500 is added into a centrifuge tube according to the principle of quick speed and slow speed, the centrifuge tube is rotated at a constant speed in the dripping process, the mixture is uniformly mixed, and the mixture is kept stand for 1min after the addition. 30mL of DMEM preheated at 37 ℃ in advance is dripped into the centrifuge tube according to the principle of being slow and fast, the dripping is completed within 4min, the effect of PEG is eliminated, and the centrifuge tube is centrifuged (1000 rpm,10 min) after standing for 10 min. Discarding supernatant, slightly suspending cells with HAT culture solution preheated at 37deg.C, diluting and mixing, dripping into culture plate with feeder cells, and placing in CO 2 Culturing in an incubator.
Example 6: cell strain selection
When the cell mass is amplified to more than 1/5 of the area of the culture hole (about 7-10 days), the supernatant can be collected and detected by an indirect non-competitive ELISA method, and the hole with good inhibitory effect on the pendimethalin standard is selected. The cells in the positive wells are monocloned by limiting dilution until monoclonal cell lines are obtained that stably secrete the target antibodies.
Example 7: five hapten immune effect evaluation
Mouse tail blood was collected five times a week after immunization of each group of different immunogens, and assayed by the homologous ELISA methodInhibition ratio (=1-B/B) when 10, 1, 0.1. Mu.g/mL pendimethalin standard solution was added 0 B is OD value when pendimethalin standard solution is added, B 0 OD value without the addition of pendimethalin standard solution). Five replicates per group were averaged. The higher the inhibition, the higher the affinity for pendimethalin. The results are shown in FIG. 2.
Example 8: antibody preparation
And (3) culturing the hybridoma cells obtained by screening in vivo to prepare the antibody. Firstly, injecting paraffin oil (500 mu L of each) into the peritoneal cavity of an immunized and healthy mouse, and normally culturing for 7-14 days; the hybridoma cells were then injected into the abdominal cavity of the mice (2×10 each 6 Individual cells), and continuing to culture for about one week; then, when the ascites of the mice is obviously increased and the abdomen is enlarged, the ascites is extracted by a sterilized needle with the number 16; finally, the collected ascites was centrifuged (8000 rpm,10 min), and the supernatant was collected and stored in a-20℃refrigerator. Monoclonal antibodies were prepared by purifying the collected ascites fluid using Protein a immunochromatographic column according to the instructions of the manufacturer.
Example 9: antibody specificity evaluation
IC for pendimethalin and other dinitroaniline herbicides (trifluralin, butralin, amisulbrom, buflomedic and flumetsulam) by comparison of pendimethalin antibodies 50 To evaluate the specificity of the antibodies. According to formula CR (cross-reaction rate) =ic 50 (pendimethalin)/IC 50 (other triazole-based bactericides) ×100% the cross-reaction rate was calculated. The results prove that the cross reaction of the pendimethalin monoclonal antibody to the pendimethalin analogue is less than 1.1%, and the specificity is strong.
Example 10: preparation of pendimethalin ELISA standard curve
(1) Coating: diluting the coating antigen to a certain concentration by CBS (pH 9.6,0.05 mol/L), adding 100 mu L/hole into an ELISA plate, coating at 4 ℃ overnight or incubating at 37 ℃ for 2 hours;
(2) Washing the plate: pouring the coating liquid out, washing with PBST (PBS solution of 0.05% Tween 20) for 5 times, and beating with absorbent paper;
(3) Closing: blocking solution (1% gelatin-PBS solution) was added at 200. Mu.L/well and incubated at 37℃for 2h;
(4) Washing the plate: and (2);
(5) Adding an antibody: diluting the antibody to a certain multiple by PBS, diluting pendimethalin to a certain concentration by PBS buffer solution, adding 50 mu L/hole of each of the two to an ELISA plate for mixing, taking the mixed solution of 50 mu L of the antibody with the same dilution and 50 mu L of PBS buffer solution as positive control, and incubating for 1h at 37 ℃;
(6) Washing the plate: and (2);
(7) Adding enzyme-labeled secondary antibodies: diluting horseradish peroxidase-goat anti-mouse IgG 20000 times by PBS, and adding 100 mu L/hole into an ELISA plate;
(8) Washing the plate: and (2);
(9) Color development: adding a color development solution (for preparation) into 100 mu L/hole, and incubating at 37 ℃ for 15min;
(10) And (3) terminating: 50 mu L/well of H was added in an amount of 2mol/L 2 SO 4 Stopping the reaction by the solution; and the absorbance was read at OD450nm and a standard curve was plotted on the abscissa with pendimethalin concentration, with B/B0 (absorbance value (B) of standard solution at each concentration divided by absorbance value (B0) of control well (well with standard concentration of 0) multiplied by 100%) as the ordinate, as shown in fig. 3. Calculate the concentration in Inhibition (IC) 50 ) At 0.53. Mu.g/L, linear range (IC 10 -IC 90 ) 0.11-2.59 mug/L.
Example 11: preparation of pendimethalin rapid detection test strip
1. Preparation of gold-labeled antibody
Step 1) synthesizing colloidal gold by a trisodium citrate reduction method: taking 100mL of ultrapure water into a 250mL conical flask, heating to boiling, adding 1mL of 1% chloroauric acid aqueous solution, adding 2mL of 1% sodium citrate aqueous solution when boiling again, and continuing heating for 5min after the color of the solution turns to dark red. Cooling to room temperature and storing in dark place.
Step 2) coupling colloidal gold with pendimethalin antibody: the optimal labelling amount of pendimethalin antibody was determined by salt precipitation. Taking 10mL of colloidal gold and using 0.1M K 2 CO 3 The pH was adjusted to 8.2. After adding 40. Mu.g of antibody, incubation was performed for 1h with shaking at room temperature, and then 1300. Mu.L of 10% BSA solution was added, and incubation was performed for 1h with shaking at room temperature. Centrifuging the above solution at 10000rpm for 15min, the supernatant was discarded, resuspended in 1000. Mu.L borate buffer containing 1% BSA,3% sucrose, and stored at 4 ℃.
2. Coated antigen and sheep anti-mouse coated cellulose film
Scribing by using an XYZ-3000 three-dimensional film spraying instrument. The spray coating antigen is taken as a detection line, and the concentration of the coating antigen is 1.26mg/mL. The goat anti-mouse IgG is taken as a control line, the concentration of the goat anti-mouse IgG is 0.25mg/mL, the distance between the two lines is 5mm, and the goat anti-mouse IgG is placed in a drying oven at 37 ℃ for drying for 60min after streaking.
3. Assembly of test strips
The cellulose film which is scratched and dried is stuck on the middle part of the lining plate, and a water absorption pad is stuck on the upper side of the cellulose film and overlapped with the cellulose film by 1mm. The gold conjugate pad was adhered to the underside of the cellulose film with an overlap of 1mm. The sample pad was adhered to the underside of the label-entering conjugate pad with an overlap of 2mm. The assembled test paper board was cut into test strips 4.00mm wide with a chopper. The test strip structure is shown in fig. 4 and 5.
Example 12: test of pendimethalin rapid detection test strip
1. Sensitivity test of test strip
And diluting 1000mg/L of pendimethalin standard solution into standard solutions with concentration gradients of 1000, 100, 50, 25, 10 and 0 mug/L series by using optimal buffer solution of pendimethalin test strip, respectively taking 100 mug of each standard solution, adding the 100 mug of each standard solution into an enzyme-labeled hole, and then adding 10 mug of labeled colloidal gold solution. And inserting the sample pad end of the pendimethalin test strip into the enzyme-labeled hole. And (3) horizontally placing the test strip, and observing a color development result after the solution is naturally diffused for 5-8 min. The result determination method is shown in fig. 6. The results showed that 1000, 100, 50, 25. Mu.g/L of standard solution was positive (only control line or detection line band was weaker than control line); 10. 0 μg/L was negative (i.e. the detection line band color was darker than the control line or the detection line band color was similar to the control line). Thus, the sensitivity of the pendimethalin test strip is 25 mug/L.
2. Specificity test of test strip
The buffer solution is used for diluting the standard solution of dinitroaniline herbicides (trifluralin, butralin, amisulbrom, buflomediun and flumetsulam) into 1000 mug/L, 100 mug of each is respectively added into an enzyme-labeled hole, and then 10 mug of labeled colloidal gold solution is added. And inserting the sample pad end of the pendimethalin test strip into the enzyme-labeled hole. And (3) horizontally placing the test strip, and observing a color development result after the solution is naturally diffused for 5-8 min. The results show that all test strips are negative, namely the test line is deeper than the control line, so the test strip has no cross-reactivity to pendimethalin analogues.
3. Chinese cabbage sample detection by pendimethalin rapid detection test strip
Taking a blank cabbage sample, using a wall breaking processor to make the blank cabbage sample into homogenate, accurately weighing 5g of Chinese cabbage homogenate into a 50mL centrifuge tube, and adding a pendimethalin standard substance to make the final concentration of the blank cabbage sample be 50mg/kg. To the added sample, 10mL of 50% methanol-PBS buffer was added, and the mixture was shaken for 5min, sonicated for 10min, then allowed to stand for 5min, and centrifuged at 4000rpm for 5min. Taking supernatant, diluting with optimal buffer solution of test strip for a certain multiple, adding 100 μL into the enzyme-labeled hole, inserting the sample pad end of pendimethalin test strip into the enzyme-labeled hole, keeping the liquid level not exceeding the mark line 9, and standing for 8min. The detection result shows positive, which indicates that the test strip can meet the detection of pendimethalin in the cabbage sample.
4. Test strip stability test
And placing the test strip into an aluminum platinum bag for vacuum packaging, preserving at room temperature, taking out the test strip after 3 months, and detecting the sensitivity of the test strip, wherein the sensitivity reaches 25 mug/L, the color depth is uniform, and the test strip of pendimethalin is proved to have good stability.
Claims (3)
1. A pendimethalin hapten has a molecular structural formula I shown as the specification:
2. a method for preparing the pendimethalin hapten as set forth in claim 1, which comprises the following steps:
the hapten preparation steps of the formula I are as follows:
step 1) 13.56g of 1-carboxypropyltriphenyl phosphonium bromide is dissolved in 10mL of tetrahydrofuran, the temperature is reduced to minus 20 ℃ by using a dry ice bath, 65mL of sodium bis (trimethylsilyl) amide is slowly dripped, the dripping is completed, 5g of 5-chloro-2-methylbenzaldehyde is dissolved in 20mL of tetrahydrofuran, the solution is dropwise added, the reaction is stirred for 4 hours at minus 20 ℃, the reaction solution is slowly poured into 20mL of ice water, the methyl tertiary butyl ether is used for stripping the impurities, the pH value of the water phase is adjusted to 2-3 by using 1M hydrochloric acid, the extraction is carried out by using 20mL of ethyl acetate, the steps are repeated twice, the organic phase is combined, saturated salt water washing, anhydrous sodium sulfate drying and concentration are carried out, 4.5g of a product 1 is obtained,
step 2) dissolving the product 1 in 100mL of absolute ethyl alcohol, adding 0.45g of platinum dioxide, degassing and purging 3 times by nitrogen, introducing hydrogen under the protection of nitrogen, stirring and reacting for 5 hours to obtain a product 2,
step 3) 3.5g of the product 2 is dissolved in 7mL of concentrated sulfuric acid, ice water is used for cooling to zero degree, 2.56g of concentrated nitric acid is slowly added dropwise, the reaction is carried out for 2 hours at 20 ℃ to obtain the product 3,
step 4) 2g of the product 3 is dissolved in 12mL of N-methylpyrrolidone and 0.55g of 3-aminopentane is added, the reaction solution is degassed and purged with nitrogen for 3 times, the reaction is reacted for 12 hours at 90 ℃ under the protection of nitrogen, the reaction solution is poured into 10mL of ice water and extracted with 20mL of ethyl acetate, the steps are repeated twice, the organic phases are combined, dried with saturated saline, concentrated with anhydrous sodium sulfate, the crude product is purified by preparative high performance liquid chromatography, the chromatographic column is Phenomenex luna C, 100 mm multiplied by 5 mu m, the mobile phase is water containing 0.1% trifluoroacetic acid, acetonitrile=30% to 70%, the yellow solid hapten of the formula I is obtained,
3. an artificial antigen of pendimethalin, which is characterized in that the artificial antigen of pendimethalin is a conjugate obtained by coupling carrier protein and the pendimethalin hapten as claimed in claim 1, and the carrier protein is bovine serum albumin or ovalbumin.
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