CN113831253A - Pendimethalin hapten as well as preparation method and application thereof - Google Patents

Pendimethalin hapten as well as preparation method and application thereof Download PDF

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CN113831253A
CN113831253A CN202111298004.9A CN202111298004A CN113831253A CN 113831253 A CN113831253 A CN 113831253A CN 202111298004 A CN202111298004 A CN 202111298004A CN 113831253 A CN113831253 A CN 113831253A
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pendimethalin
hapten
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CN113831253B (en
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华修德
王鸣华
黄联润
丁园
陈贺
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Nanjing Agricultural University
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Abstract

The invention discloses pendimethalin hapten and a preparation method and application thereof. A de novo synthesis method is adopted for pendimethalin hapten, and five haptens are respectively introduced into a connecting arm with carboxyl from the tail ends of benzyl, phenylamino and pentyl of pendimethalin. Through test comparison, the hapten effect of introducing the connecting arm on the methyl at the third position of the benzene ring of the pendimethalin parent is the best. The monoclonal antibody prepared by the hapten has high sensitivity and strong specificity. The sensitivity (IC) is measured by indirect competitive enzyme-linked immunoassay (IC-ELISA)50) 0.53. mu.g/L, detection Range (IC)10‑IC90) Is 0.11-2.59 mu g/L. The monoclonal antibody has no cross reaction with other dinitrobenzene herbicides (CR < 1.1%). In addition, the monoclonal antibody is used for preparing a 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

Pendimethalin hapten as well as preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to five pendimethalin haptens as well as a preparation method and application thereof.
Background
Pendimethalin (pendimethalin) belongs to dinitroaniline herbicides and is widely applied to weed control of crops and vegetables such as rice, corn, potatoes, Chinese cabbage and the like. Pendimethalin inhibits weed growth by inhibiting mitosis and cell wall formation in plant cells. Due to the wide weed control spectrum and application range, pendimethalin is one of the most commonly used selective herbicides in agriculture, and more than 150 herbicides using pendimethalin as an active ingredient are currently registered in China. According to the classification standard of pesticide toxicity in China, pendimethalin belongs to low-toxicity pesticide. According to the national standard GB2763-2021 maximum residue limit of pesticides in food formally implemented in 2021, 9 and 3 days in China, the daily allowable intake of pendimethalin is 0.1mg/kg bw, the maximum residue limit of pendimethalin in rice, potato, spinach, cabbage, leek and 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, edible legume vegetables, peas and nuts is 0.05 mg/kg.
Currently, the detection method of pendimethalin is mainly an instrument detection method, and mainly comprises a gas chromatography, a gas chromatography-mass spectrometry combined method, a high performance liquid chromatography-mass spectrometry combined method and the like. The instrument detection method has high accuracy and sensitivity, but needs expensive instruments, professional technicians and complex pretreatment operation, and is difficult to realize real-time field detection of samples. Immunoassay, such as enzyme-linked immunosorbent assay, is an economic, simple, 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 rational hapten design. Therefore, designing and synthesizing reasonable pendimethalin hapten is the key for establishing an economical, simple, on-site, real-time, rapid and accurate immunoassay method.
At present, the pendimethalin immunoassay method has been reported. Chenli et al have invented a pendimethalin hapten obtained by reacting 3- (1-ethyl-propylamino) -6-methyl-2, 4-dinitrobenzaldehyde with 3-hydrazinopropionic acid, and successfully prepared a pendimethalin monoclonal antibody. The pendimethalin enzyme-linked immunosorbent assay and the colloidal gold test strip developed based on the monoclonal antibody can realize the rapid detection of pendimethalin in tobacco, but the sensitivity is poor (the half inhibition rate is 1.5mg/kg), so that the application of the immunoassay method in the rapid detection of pendimethalin in agricultural products is limited. Therefore, there is a need for further improvement of pendimethalin hapten structure to increase the sensitivity of immunoassay methods.
Disclosure of Invention
The invention provides five pendimethalin hapten structures and preparation and application thereof, and the optimal pendimethalin hapten structure is determined through experiments.
In the first aspect, the five pendimethalin haptens designed by the invention have the structures shown as formulas I-V.
Figure BSA0000256808510000021
In a second aspect, the invention provides a method for preparing the five haptens.
The hapten of the formula I is prepared by the following steps:
step 1)13.56g of 1-carboxypropyl triphenyl phosphonium bromide is dissolved in 10mL of tetrahydrofuran, the temperature is reduced to minus 20 ℃ by a dry ice bath, 65mL of sodium amide is slowly dripped, and the mixture is stirred for 1 hour at minus 20 ℃ after the dripping is finished. 5g of 5-chloro-2-methylbenzaldehyde was dissolved in 20mL of tetrahydrofuran, and added dropwise to the above solution, and the reaction was stirred at-20 ℃ for 4 hours. Slowly pouring the reaction solution into 20mL of ice water, performing back extraction with methyl tert-butyl ether to remove impurities, adjusting the pH value of the water phase to 2-3 with 1M hydrochloric acid, extracting with 20mL of ethyl acetate, repeating the extraction twice, combining organic phases, washing with saturated salt water, drying with anhydrous sodium sulfate, and concentrating. 4.5g of product 1 are obtained.
Figure BSA0000256808510000022
And step 2) dissolving the product 1 in 100mL of absolute ethyl alcohol, adding 0.45g of platinum dioxide, degassing and purging for 3 times by using nitrogen, introducing hydrogen under the protection of nitrogen, and stirring for reacting for 5 hours to obtain a product 2.
Figure BSA0000256808510000031
And 3) dissolving 3.5g of the product 2 in 7mL of concentrated sulfuric acid, cooling to zero with ice water, slowly dropwise adding 2.56g of concentrated nitric acid, and reacting at room temperature for 2 hours to obtain a product 3.
Figure BSA0000256808510000032
Step 4) 2g of product 3 are dissolved in 12mL of N-methylpyrrolidone and 0.55g of 3-aminopentane is added, and the reaction mixture is degassed and purged 3 times with nitrogen and reacted for 12 hours at 90 ℃ under nitrogen protection. The reaction solution was poured into 10mL of ice water and 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 Phenomenex luna C18100 × 40mm × 5 μm with mobile phases water (containing 0.1% trifluoroacetic acid) and acetonitrile 30% to 70%. The hapten of the formula I is obtained as a yellow solid.
Figure BSA0000256808510000033
The hapten of formula II is prepared as follows:
step 1)18.98g of 1-carboxypropyl triphenyl phosphonium bromide is dissolved in 10mL of tetrahydrofuran, the temperature is reduced to minus 20 ℃ by a dry ice bath, 65mL of sodium amide is slowly dripped, and the mixture is stirred for 1 hour at minus 20 ℃ after the dripping is finished. 7g of 4-chloro-2-methylbenzaldehyde was dissolved in 20mL of tetrahydrofuran, added dropwise to the above solution, and the reaction was stirred at-20 ℃ for 4 hours. Slowly pouring the reaction solution into 20mL of ice water, performing back extraction with methyl tert-butyl ether to remove impurities, adjusting the pH value of the water phase to 2-3 with 1M hydrochloric acid, extracting with 20mL of ethyl acetate, repeating the extraction twice, combining organic phases, washing with saturated salt water, drying with anhydrous sodium sulfate, and concentrating. 6.2g of product 1 are obtained.
Figure BSA0000256808510000034
And step 2) dissolving the product 1 in 100mL of absolute ethyl alcohol, adding 0.45g of platinum dioxide, degassing and purging for 3 times by using nitrogen, introducing hydrogen under the protection of nitrogen, stirring, and reacting for 5 hours at-20 ℃ to obtain a product 2.
Figure BSA0000256808510000041
And 3) dissolving 5g of the product 2 in 10mL of concentrated sulfuric acid, cooling to zero with ice water, slowly dropwise adding 3.55g of concentrated nitric acid, and reacting at room temperature for 2 hours to obtain a product 3.
Figure BSA0000256808510000042
Step 4) 3g of product 3 are dissolved in 12mL of N-methylpyrrolidone and 0.99g of 3-aminopentane is added, the reaction mixture is degassed and purged 3 times with nitrogen and reacted for 12 hours at 90 ℃ under nitrogen protection. The reaction solution was poured into 10mL of ice water and 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 Phenomenex luna C18100 × 40mm × 5 μm with mobile phases water (containing 0.1% trifluoroacetic acid) and acetonitrile 30% to 70%. The hapten of the formula II is obtained as a yellow solid.
Figure BSA0000256808510000043
The hapten of formula III is prepared as follows:
step 1) 8g of 3, 4-dimethyl chlorobenzene is dissolved in 16mL of concentrated sulfuric acid, ice water is used for cooling to zero degree, 9.43g of concentrated nitric acid is slowly dripped, and reaction is carried out for 2 hours at room temperature to obtain a product 1.
Figure BSA0000256808510000044
Step 2) 2g of product 1 were dissolved in 3.6mL of N-methylpyrrolidone and 2g of 5-aminopentanoic acid and 2.6g of triethylamine were added, and the reaction solution was degassed and 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 and 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)2Petroleum ether/ethyl acetate 100/1-0/1). The hapten of the formula III is obtained as a yellow solid.
Figure BSA0000256808510000051
The hapten of formula IV is prepared as follows:
step 1) preparation of hapten with formula III step 1) to obtain product 1.
Step 2) 0.11g of product 1 and 0.13g of 4-aminohexane are dissolved in 1.1mL of absolute ethanol and 0.33g of potassium carbonate are added, degassed and purged 3 times with nitrogen, stirred under nitrogen with hydrogen at 80 ℃ for 24 hours. Filtering the reaction solution, collecting the filter cake, and purifying the filter cake by column chromatography (SiO)2Dichloromethane/methanol 50/1 to 6/1) to yield the hapten of formula IV as a yellow solid.
Figure BSA0000256808510000052
The hapten of formula V is prepared as follows:
step 1) preparation of hapten with formula IV step 1) to obtain product 1.
Step 2) 0.65g of product 1 and 1.32g of 3-aminopentane are dissolved in 6.5mL of absolute ethanol and 1.95g of potassium carbonate are added, degassed and purged 3 times with nitrogen, stirred under nitrogen with hydrogen at 80 ℃ and reacted for 24 hours. Filtering the reaction solution, collecting the filter cake, and purifying the filter cake by column chromatography (SiO)2Dichloromethane/methanol 50/1 to 6/1) to yield the hapten of formula IV as a yellow solid.
Figure BSA0000256808510000053
In a third aspect, the invention provides complete antigens of the five pendimethalin haptens, wherein the complete antigens are obtained by coupling the haptens and carrier proteins by an active ester method. The pendimethalin complete antigen is divided into pendimethalin immunogen and coating antigen, the five pendimethalin immunogens are obtained by respectively coupling five pendimethalin haptens with Bovine Serum Albumin (BSA), and the five pendimethalin coating antigens are obtained by respectively coupling five pendimethalin haptens with Ovalbumin (OVA).
Step 1) 0.05g of pendimethalin hapten and 0.02g N-hydroxysuccinimide were dissolved in 3mL of N, N-dimethylformamide and 0.04g of dicyclohexylcarbodiimide was added 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 (0.01mol/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 pendimethalin immunogen; the remaining supernatant was slowly added dropwise to 8mL of a phosphate buffer (0.01mol/L, pH 7.4) containing 0.07g of ovalbumin, and the mixture was reacted at room temperature in the dark for 24 hours to prepare pendimethalin-coated antigen.
And 3) transferring the pendimethalin immunogen and the coating antigen obtained in the step 2) into a dialysis bag, dialyzing by using 0.01mol/L phosphate buffer solution for removing impurities for 3 days, and changing the dialysate at least 3 times every day. The complete antigen after dialysis was stored in separate containers at-20 ℃.
In a fourth aspect, the invention provides a pendimethalin monoclonal antibody, wherein the monoclonal antibody 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 residue in crops and environmental samples.
The invention has the following beneficial effects:
the five pendimethalin haptens provided by the invention not only reserve the characteristic structure of pendimethalin to the greatest extent, but also introduce the connecting arms from different directions, fully consider the possibility of influence of different characteristic structures on the properties of an antibody, and simultaneously, the carboxyl on the connecting arms enables the coupling of the haptens 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 a mouse, generates 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 immunizing the five pendimethalin antigens provided by the invention have better titer, specificity and affinity, and each monoclonal antibody can identify at least two coating antigens. Suppression medium concentration of ELISA established by monoclonal antibodies obtained by immunizing five pendimethalin antigens respectively(IC50) All can be less than 10. mu.g/L. By comparison, the monoclonal antibody 5F7 obtained from hapten formula I was most effective, IC500.53. mu.g/L, compared to the IC reported previously50Improved by 3000 times, the linear range is 0.11-2.59 mu g/L, and the cross reaction rate with other dinitroaniline herbicides is lower than 1.1 percent; the colloidal gold test strip prepared based on the monoclonal antibody has the minimum detection limit of 25 mug/L, which is improved by 200 times compared with the previous report. Thus, the hapten provided by the invention has the advantages that the hapten is more favorable for preparing the pendimethalin monoclonal antibody than the hapten designed by the prior person. The ELISA method and the colloidal gold test strip developed based on the monoclonal antibody obtained by the hapten show better sensitivity, and have the characteristics of simple operation, economy and quickness.
Drawings
FIG. 1 shows the synthetic route of pendimethalin hapten.
FIG. 2 is a graph showing the evaluation of the immunopotentiation effect of pendimethalin hapten.
FIG. 3 is a pendimethalin ELISA standard competition curve.
Fig. 4 is a top view of a pendimethalin colloidal gold test strip structure.
FIG. 5 is a side view of a pendimethalin colloidal gold test strip. In the figure, 1: a lining plate, 2: sample pad, 3: gold-labeled bond pad, 4: cellulose film, 5: invisible detection line, 6: stealth control line, 7: absorbent pad, 8-1: sample immersion end protective film, 8-2: handle end protective film, 9: and marking the line.
FIG. 6 is a schematic diagram showing the result determination of a pendimethalin colloidal gold test strip; in the figure, A, B is the detection result of a negative sample, C, D is the detection result of a strong positive sample, and E, F is the failure of the test strip.
Detailed Description
The invention is further illustrated by the following examples, but not by way of limitation, in connection with the accompanying drawings. The following provides specific materials and sources thereof used in embodiments of the present invention. However, it should be understood that these are exemplary only and not intended to limit the invention, and that materials of the same or similar type, quality, nature or function as the following reagents and instruments may be used in the practice of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: synthesis of pendimethalin hapten as shown in FIG. 1.
Synthesis of haptens of formula I
Step 1)13.56g of 1-carboxypropyl triphenyl phosphonium bromide is dissolved in 10mL of tetrahydrofuran, the temperature is reduced to minus 20 ℃ by a dry ice bath, 65mL of sodium amide is slowly dripped, and the mixture is stirred for 1 hour at minus 20 ℃ after the dripping is finished. 5g of 5-chloro-2-methylbenzaldehyde was dissolved in 20mL of tetrahydrofuran, and added dropwise to the above solution, and the reaction was stirred at-20 ℃ for 4 hours. Slowly pouring the reaction solution into 20mL of ice water, performing back extraction with methyl tert-butyl ether to remove impurities, adjusting the pH value of the water phase to 2-3 with 1M hydrochloric acid, extracting with 20mL of ethyl acetate, repeating the extraction twice, combining organic phases, washing with saturated salt water, drying with anhydrous sodium sulfate, and concentrating. 4.5g of product 1 are obtained.
And step 2) dissolving the product 1 in 100mL of absolute ethyl alcohol, adding 0.45g of platinum dioxide, degassing and purging for 3 times by using nitrogen, introducing hydrogen under the protection of nitrogen, and stirring for reacting for 5 hours to obtain a product 2.
And 3) dissolving 3.5g of the product 2 in 7mL of concentrated sulfuric acid, cooling to zero with ice water, slowly dropwise adding 2.56g of concentrated nitric acid, and reacting at room temperature for 2 hours to obtain a product 3.
Step 4) 2g of product 3 are dissolved in 12mL of N-methylpyrrolidone and 0.55g of 3-aminopentane is added, and the reaction mixture is degassed and purged 3 times with nitrogen and reacted for 12 hours at 90 ℃ under nitrogen protection. The reaction solution was poured into 10mL of ice water and 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 Phenomenex luna C18100 × 40mm × 5 μm with mobile phases water (containing 0.1% trifluoroacetic acid) and acetonitrile 30% to 70%. The hapten of the formula I is obtained as a yellow solid.
Synthesis of haptens of formula II
Step 1)18.98g of 1-carboxypropyl triphenyl phosphonium bromide is dissolved in 10mL of tetrahydrofuran, the temperature is reduced to minus 20 ℃ by a dry ice bath, 65mL of sodium amide is slowly dripped, and the mixture is stirred for 1 hour at minus 20 ℃ after the dripping is finished. 7g of 4-chloro-2-methylbenzaldehyde was dissolved in 20mL of tetrahydrofuran, added dropwise to the above solution, and the reaction was stirred at-20 ℃ for 4 hours. Slowly pouring the reaction solution into 20mL of ice water, performing back extraction with methyl tert-butyl ether to remove impurities, adjusting the pH value of the water phase to 2-3 with 1M hydrochloric acid, extracting with 20mL of ethyl acetate, repeating the extraction twice, combining organic phases, washing with saturated salt water, drying with anhydrous sodium sulfate, and concentrating. 6.2g of product 1 are obtained.
And step 2) dissolving the product 1 in 100mL of absolute ethyl alcohol, adding 0.45g of platinum dioxide, degassing and purging for 3 times by using nitrogen, introducing hydrogen under the protection of nitrogen, stirring, and reacting for 5 hours at-20 ℃ to obtain a product 2.
And 3) dissolving 5g of the product 2 in 10mL of concentrated sulfuric acid, cooling to zero with ice water, slowly dropwise adding 3.55g of concentrated nitric acid, and reacting at room temperature for 2 hours to obtain a product 3.
Step 4) 3g of product 3 are dissolved in 12mL of N-methylpyrrolidone and 0.99g of 3-aminopentane is added, the reaction mixture is degassed and purged 3 times with nitrogen and reacted for 12 hours at 90 ℃ under nitrogen protection. The reaction solution was poured into 10mL of ice water and 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 Phenomenex luna C18100 × 40mm × 5 μm with mobile phases water (containing 0.1% trifluoroacetic acid) and acetonitrile 30% to 70%. The hapten of the formula II is obtained as a yellow solid.
Synthesis of haptens of formula III
Step 1) 8g of 3, 4-dimethyl chlorobenzene is dissolved in 16mL of concentrated sulfuric acid, ice water is used for cooling to zero degree, 9.43g of concentrated nitric acid is slowly dripped, and reaction is carried out for 2 hours at room temperature to obtain a product 1.
Step 2) 2g of product 1 were dissolved in 3.6mL of N-methylpyrrolidone and 2g of 5-aminopentanoic acid and 2.6g of triethylamine were added, and the reaction solution was degassed and 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 and extracted with 20mL of ethyl acetate, repeated twice, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentratedAnd (4) shrinking. The crude product was purified by column chromatography (SiO)2Petroleum ether/ethyl acetate 100/1-0/1). The hapten of the formula III is obtained as a yellow solid.
Synthesis of hapten of formula IV
Step 1) preparation of hapten with formula III step 1) to obtain product 1.
Step 2) 0.11g of product 1 and 0.13g of 4-aminohexane are dissolved in 1.1mL of absolute ethanol and 0.33g of potassium carbonate are added, degassed and purged 3 times with nitrogen, stirred under nitrogen with hydrogen at 80 ℃ for 24 hours. Filtering the reaction solution, collecting the filter cake, and purifying the filter cake by column chromatography (SiO)2Dichloromethane/methanol 50/1 to 6/1) to yield the hapten of formula IV as a yellow solid.
Synthesis of hapten of formula V
Step 1) preparation of hapten with formula IV step 1) to obtain product 1.
Step 2) 0.65g of product 1 and 1.32g of 3-aminopentane are dissolved in 6.5mL of absolute ethanol and 1.95g of potassium carbonate are added, degassed and purged 3 times with nitrogen, stirred under nitrogen with hydrogen at 80 ℃ and reacted for 24 hours. Filtering the reaction solution, collecting the filter cake, and purifying the filter cake by column chromatography (SiO)2Dichloromethane/methanol 50/1 to 6/1) to yield the hapten of formula IV as a yellow solid.
Example 2: identification of pendimethalin hapten
The result of the nuclear magnetic identification of the pendimethalin hapten of the formula I:1H 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，1ArCH2)，2.29(s，3H，1ArCH3)，2.239(t，J＝7.2，2H，HOOCCH2)，1.468(m，8H，4CH2)，0.787(t，J＝7.2，6H，2CH2CH3)。
the result of the nuclear magnetic identification of pendimethalin hapten II:1H NMR(400MHz，CDCl3)δ8.07(s，1H，ArH)，7.645(d，J＝12Hz，1H，NH)，3.170(m，1H，NHCH)，2.626(t，J＝7.6Hz，2H，1ArCH2)，2.433(t，J＝6.8Hz，2H，OOCCH2)，2.206(s，3H，1ArCH3)，1.555(m，8H，4CH2)，0.889(t，J＝7.2Hz，6H，2CH2CH3)。
the result of the nuclear magnetic identification of pendimethalin hapten III:1H NMR(400MHz，d6-DMSO)δ8.092(s，1H，ArH)，7.504(s，1H，NH)，2.990(t，J＝4.8Hz，2H，NHCH2)，2.25(s，3H，ArCH3)，2.15(s，3H，ArCH3)，2.121(t，J＝9.6Hz，2H，OOCCH2)，1.495(m，4H，2CH2)。
the result of the nuclear magnetic identification of pendimethalin hapten IV:1H 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，ArCH3)，2.14(s，3H，ArCH3)，2.094(t，J＝6.8Hz，2H，OOCCH2)，1.439(m，4H，2CH2)，0.779(t，J＝7.2Hz，3H，CH2CH3).
the result of the nuclear magnetic identification of pendimethalin type V hapten:1H NMR(400MHz，d6-DMSO)δ8.059(s，1H，ArH)，7.22(d，J＝10Hz，1H，NH)，2.359(m，3H)，2.259(s，3H，ArCH3)，2.137(s，3H，ArCH3)，1.518(m，2H，OOCCH2)，0.787(t，J＝7.2，3H，CH2CH3)。
example 3: preparation of pendimethalin antigen
Step 1) 0.05g of pendimethalin hapten and 0.02g N-hydroxysuccinimide were dissolved in 3mL of N, N-dimethylformamide and 0.04g of dicyclohexylcarbodiimide was added 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 (0.01mol/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 pendimethalin immunogen; the remaining supernatant was slowly added dropwise to 8mL of a phosphate buffer (0.01mol/L, pH 7.4) containing 0.07g of ovalbumin, and the mixture was reacted at room temperature in the dark for 24 hours to prepare pendimethalin-coated antigen.
And 3) transferring the pendimethalin immunogen and the coating antigen obtained in the step 2) into a dialysis bag, dialyzing by using 0.01mol/L phosphate buffer solution for removing impurities for 3 days, and changing the dialysate at least 3 times every day. The complete antigen after dialysis was stored in separate containers at-20 ℃.
Example 4: pendimethalin antigen immunized mice
Five 6-8 week old female BALB/c mice were immunized by five intraperitoneal injections with each pendimethalin immunogen (i.e., a conjugate of pendimethalin hapten and bovine serum albumin). Before each injection, pendimethalin immunogen and 1: 1 volume of immune adjuvant are mixed and fully emulsified, the injection dose is 100 mu g/mouse, the immune interval is two weeks, and except Freund type complete adjuvant used for the first immunization, incomplete adjuvants are used for the rest. Three days before fusion, the mice were subjected to booster immunization at a dose of 100. mu.g/mouse without the use of an immunoadjuvant.
Example 5: cell fusion
Step 1) preparation of myeloma cells: three bottles of mouse SP2/0 myeloma cells in the logarithmic growth phase were removed, blown down with DMEM medium, transferred to a 50mL centrifuge tube and made to volume of 35 mL.
Step 2) preparation of splenocytes: three days after the sprint immunization, the eyes were removed, the neck was pulled to kill, the spleen was removed with surgical forceps and scissors, and the surface fat and connective tissue were removed. Spleen was placed in 70 μm cell screen and cells were driven out into DMEM medium, transferred to 50mL centrifuge tube and made up to 35 mL. Myeloma and spleen cells were centrifuged (1000rpm, 10min), the supernatant discarded, resuspended and counted.
Step 3) cell fusion: spleen cells and SP2/0 myeloma cells were mixed well at 10: 1 in a 50mL centrifuge tube, centrifuged (1000rpm, 10min), the supernatant was discarded, and the bottom cells were shaken off and incubated in a 37 ℃ water bath. Using a sterile syringe to absorb 1mL of PEG1500 preheated in advance, adding the PEG1500 into a centrifugal tube according to the principle of first quick and then slow, uniformly mixing the PEG1500 by uniformly rotating the centrifugal tube in the dropping process, and standing for 1min after the PEG1500 is added. Dropping 30mL of DMEM preheated to 37 ℃ in advance into the centrifuge tube according to the principle of slow first and fast second, eliminating the effect of PEG after dropping within 4min, standing for 10min, and centrifuging (1000rpm, 10 min). Discard the supernatant and gently resuspend the cells with HAT medium preheated to 37 ℃ in advanceDiluting properly, mixing, dropping into culture plate with feeder cells, and placing in CO2Culturing in an incubator.
Example 6: cell line selection
When the cell mass expanded to more than 1/5 of the culture well area (about 7-10 days), the supernatant was collected and tested by indirect non-competitive ELISA to select wells with good inhibition effect on pendimethalin standard. Cells in the positive wells were monocloned by limiting dilution until a monoclonal cell line that stably secreted the target antibody was obtained.
Example 7: evaluation of immune effects of five haptens
Mouse tail blood was collected one week after five immunizations with each group of different immunogens and the inhibition when 10, 1, 0.1. mu.g/mL pendimethalin standard solution was added (═ 1-B/B)0B is the OD value when pendimethalin standard solution is added, B0OD value without pendimethalin standard solution). Each group of five replicates was averaged. The higher the inhibition, the higher the affinity for pendimethalin. The results are shown in FIG. 2.
Example 8: antibody preparation
And (4) carrying out in vivo culture on the hybridoma obtained by screening to prepare the antibody. Firstly, injecting paraffin oil (500 μ L each) into abdominal cavity of non-immunized and healthy mice, and culturing for 7-14 days; then, the hybridoma cells were injected into the abdominal cavity (2X 10 each) of the mice6Individual cells), continuously culturing for about one week; then, when the ascites of the white mouse obviously becomes more and the abdomen expands, the sterilized No. 16 needle is used for extracting the ascites; finally, the collected ascites fluid was centrifuged (8000rpm, 10min), and the supernatant fluid was collected and stored in a refrigerator at-20 ℃. The collected ascites fluid was purified using Protein A immunochromatography column according to the instructions of the trade company to prepare a monoclonal antibody.
Example 9: evaluation of antibody specificity
IC for pendimethalin by comparison of pendimethalin antibody with other dinitroaniline herbicides (trifluralin, butralin, prodiamine, butafenacet and flumetsulam)50To evaluate the specificity of the antibody. According to the formula CR (Cross-reactivity) ═ IC50(pendimethalin)/IC50(other triazole bactericides) multiplied by 100 percent to calculate the cross reaction rate. The results prove that the cross reaction of the pendimethalin monoclonal antibody to 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 source to a certain concentration by CBS (pH 9.6, 0.05mol/L), adding 100 mu L/well of the coating source to an enzyme label plate, and coating overnight at 4 ℃ or incubating for 2 hours at 37 ℃;
(2) washing the plate: the coating solution was decanted and washed 5 times with PBST (0.05% Tween20 in PBS) and blotted dry;
(3) and (3) sealing: adding blocking solution (1% gelatin-PBS solution) into 200 μ L/well, and incubating at 37 deg.C for 2 h;
(4) washing the plate: the same (2);
(5) adding a primary antibody: diluting the antibody to a certain multiple by PBS, diluting pendimethalin to a certain concentration by PBS buffer solution, adding an enzyme label plate into each 50 mu L/hole for mixing, taking a mixed solution of 50 mu L of antibody with the same dilution multiple and 50 mu L of PBS buffer solution as a positive control, and incubating for 1h at 37 ℃;
(6) washing the plate: the same (2);
(7) adding an enzyme-labeled secondary antibody: diluting horse radish peroxidase-goat anti-mouse IgG with PBS 20000 times, and adding 100 μ L/hole into an enzyme label plate;
(8) washing the plate: the same (2);
(9) color development: adding developing solution (used as prepared) into 100 μ L/well, and incubating at 37 deg.C for 15 min;
(10) and (4) terminating: 50 mu.L/well of H2 mol/L2SO4Terminating the reaction by the solution; and absorbance was read at OD450nm and plotted as the abscissa of pendimethalin concentration and as the ordinate of B/B0 (absorbance (B) of standard solution at each concentration divided by absorbance (B0) of control well (well with standard concentration of 0) multiplied by 100%), as shown in fig. 3. Calculating the concentration in Inhibition (IC)50) 0.53. mu.g/L, linear range (IC)10-IC90) Is 0.11-2.59 mu g/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: 100mL of ultrapure water is taken into a 250mL conical flask, heated to boiling, 1mL of 1% chloroauric acid aqueous solution is added, 2mL of 1% sodium citrate aqueous solution is added when the solution is boiled again, and heating is continued for 5min after the color of the solution turns to dark red. Cooled to room temperature and stored in dark.
Step 2) coupling of colloidal gold and pendimethalin antibody: the optimal labeling amount of pendimethalin antibody was determined by salt precipitation. Take 10mL of colloidal gold and use 0.1M K2CO3The pH was adjusted to 8.2. After 40. mu.g of antibody was added, the mixture was incubated at room temperature for 1 hour with shaking, and 1300. mu.L of 10% BSA solution was added, followed by incubation at room temperature for 1 hour with shaking. The above solution was centrifuged at 10000rpm for 15min, the supernatant was discarded, and the supernatant was resuspended in 1000. mu.L of borate buffer containing 1% BSA and 3% sucrose and stored at 4 ℃.
2. Coating antigen and sheep anti-mouse coating cellulose membrane
And (4) scribing by using an XYZ-3000 three-dimensional film spraying instrument. The sprayed coating antigen is used as a detection line, and the concentration of the coating antigen is 1.26 mg/mL. Goat anti-mouse IgG spray is used as a control line, the concentration of the goat anti-mouse IgG spray is 0.25mg/mL, the two lines are separated by 5mm, and the goat anti-mouse IgG spray is placed in a 37 ℃ drying oven for drying for 60min after being scribed.
3. Assembly of test strips
Pasting the cellulose membrane which is scratched and dried on the middle part of the lining plate, and pasting the water absorption pad on the upper side of the cellulose membrane and overlapping the cellulose membrane for 1 mm. Gold-labeled conjugate pads were stuck under the cellulose membrane with an overlap of 1 mm. The sample pad was stuck under the label-entering conjugate pad with an overlap of 2 mm. The assembled test paper board was cut into test strips of 4.00mm width with a cutter. 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 strips
Diluting 1000mg/L pendimethalin standard solution into 1000, 100, 50, 25, 10 and 0 mu g/L series concentration gradient standard solutions by using pendimethalin test strip optimal buffer solution, respectively adding 100 mu L of standard solutions into an enzyme labeling hole, and then adding 10 mu L of labeled colloidal gold solution. And inserting the sample pad end of the pendimethalin test strip into the enzyme labeling hole. And horizontally placing the test strip, and observing a color development result after the solution naturally diffuses for 5-8 min. The result determination method is shown in fig. 6. The results show that the standard solutions of 1000, 100, 50 and 25 mug/L are positive (only the color of the control line or the detection line is weaker than that of the control line); 10. 0 μ g/L is negative (i.e., the color of the test line strip is darker than the control line or the color of the test line strip is similar to the control line). Thus, the sensitivity of the pendimethalin strip was 25. mu.g/L.
2. Specificity test of test strip
Diluting the dinitroaniline herbicide standard solution into 1000 mug/L with the buffer solution, adding 100 mug L of the dinitroaniline herbicide standard solution into the enzyme labeling holes respectively, and adding 10 mug L of labeled colloidal gold solution. And inserting the sample pad end of the pendimethalin test strip into the enzyme labeling hole. And horizontally placing the test strip, and observing a color development result after the solution naturally diffuses for 5-8 min. The results show that all the test strips are negative, namely the detection line strips are deeper than the control lines, so that the test strips have no cross reactivity to pendimethalin analogues.
3. Quick detection test paper strip for pendimethalin to detect cabbage sample
Taking a blank sample of the Chinese cabbage, homogenizing the blank sample by using a wall-breaking food processor, accurately weighing 5g of the Chinese cabbage homogenate into a 50mL centrifuge tube, and adding a pendimethalin standard product to enable the final concentration to be 50 mg/kg. Adding 10mL of 50% methanol-PBS buffer solution into the added sample, shaking for 5min, performing ultrasonic treatment for 10min, standing for 5min, and centrifuging for 5min at 4000rpm of a centrifuge. Diluting the supernatant with test strip optimal buffer solution by a certain multiple, adding 100 μ L 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 8 min. The detection result shows that the test strip is positive, which indicates that the test strip can meet the requirement of detecting pendimethalin in a cabbage sample.
4. Test strip stability test
The test paper strip is put into an aluminum platinum bag for vacuum packaging, and is stored at room temperature, and is taken out after 3 months for detecting the sensitivity, the sensitivity is found to reach 25 mug/L, the color development depth is uniform, and the stability of the pendimethalin test paper strip is proved to be good.

Claims (5)

1. A pendimethalin hapten having the molecular structural formula I:
Figure FSA0000256808500000011
2. a method of preparing the pendimethalin hapten of claim 1, comprising the steps of:
the hapten of the formula I is prepared by the following steps:
step 1)13.56g of 1-carboxypropyl triphenyl phosphonium bromide is dissolved in 10mL of tetrahydrofuran, the temperature is reduced to-20 ℃ by a dry ice bath, 65mL of sodium amide is slowly dripped, the dropwise addition is finished, the stirring is carried out for 1 hour at-20 ℃, 5g of 5-chloro-2-methylbenzaldehyde is dissolved in 20mL of tetrahydrofuran, the dropwise addition is carried out to the solution, the stirring reaction is carried out for 4 hours at-20 ℃, the reaction solution is slowly poured into 20mL of ice water, the methyl tert-butyl ether is used for carrying out back extraction to remove impurities, the pH value of the water phase is adjusted to be 2-3 by 1M hydrochloric acid, the extraction is carried out by 20mL of ethyl acetate, the two times are repeated, the organic phase is combined, the saturated salt is used for washing, the anhydrous sodium sulfate is used for drying, the concentration is carried out, 4.5g of product 1 is obtained,
Figure FSA0000256808500000012
step 2) dissolving the product 1 in 100mL of absolute ethyl alcohol, adding 0.45g of platinum dioxide, degassing and purging for 3 times by using nitrogen, introducing hydrogen under the protection of nitrogen, stirring and reacting for 5 hours to obtain a product 2,
Figure FSA0000256808500000013
step 3) dissolving 3.5g of the product 2 in 7mL of concentrated sulfuric acid, cooling to zero with ice water, slowly dropwise adding 2.56g of concentrated nitric acid, reacting at room temperature for 2 hours to obtain a product 3,
Figure FSA0000256808500000014
step 4) 2g of product 3 are dissolved in 12mL of N-methylpyrrolidone and 0.55g of 3-aminopentane are added, the reaction solution is degassed and purged 3 times with nitrogen, the reaction is carried out 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 two times are repeated, the organic phases are combined, the mixture is washed with saturated common salt water, dried with anhydrous sodium sulfate and concentrated, the crude product is purified by preparative high performance liquid chromatography, the chromatographic column is Phenomenex luna C18100 multiplied by 40mm multiplied by 5 mu m, the mobile phase is water (containing 0.1% of trifluoroacetic acid) and acetonitrile is 30% to 70%, and the hapten of formula I in the form of yellow solid is obtained,
Figure FSA0000256808500000021
3. an artificial antigen of pendimethalin, comprising an immune antigen of pendimethalin and an antigen coated with pendimethalin, wherein the artificial antigen of pendimethalin is a conjugate of a carrier protein and the hapten of pendimethalin as defined in claim 1, and the carrier protein is bovine serum albumin or ovalbumin.
4. A pendimethalin monoclonal antibody that specifically recognizes the pendimethalin hapten of claim 1 or the pendimethalin artificial antigen of claim 3.
5. Use of the pendimethalin monoclonal antibody of claim 4 for detecting pendimethalin residues.
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