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Preparation method and application of quinclorac hapten and antigen

A technology of quinclorac and acetal quinclorac, which is applied in the field of preparation of quinclorac haptens and antigens, can solve the problems of complex processing, slow degradation, and inability to plant, and achieve low cross-reaction rate , High specificity, rapid detection effect

Active Publication Date: 2019-01-25
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because quinclorac is acidic and degrades slowly in acidic soil, it is easy to cause serious damage to sensitive crops. In the past 300 days after the application of quinclorac in paddy fields, no crops can be planted except rice; To plant tobacco, eggplant, tomato, carrot, celery and other crops, the above crops cannot be irrigated with quinclorac-treated water
[0003] Currently commonly used detection methods for quinclorac are mainly instrumental methods, such as high performance liquid chromatography and liquid chromatography tandem mass spectrometry. These analytical methods require expensive instruments and specialized technicians, and the sample pretreatment process is complicated and expensive. , time-consuming, difficult to meet the needs of a large number of samples and rapid detection of on-site samples

Method used

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  • Preparation method and application of quinclorac hapten and antigen
  • Preparation method and application of quinclorac hapten and antigen
  • Preparation method and application of quinclorac hapten and antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The preparation of embodiment 1 quinclorac hapten

[0023] 1. Preparation of quinclorac hapten

[0024] 1) Take 1.00 g of quinclorac, add 50 mL of dry anhydrous tetrahydrofuran to dissolve and clarify, add dropwise 10 mL of anhydrous tetrahydrofuran solution containing 1.10 g (1,3-dioxanylethyl)magnesium bromide, Stir at 0°C for 2 h; quickly mix with 100 mL of ammonium chloride aqueous solution at 0-5°C, immediately add 100 mL of diethyl ether for extraction, oscillate, let stand to separate layers, dry the organic phase with anhydrous sodium sulfate, rotary evaporate, concentrate, evaporate to dryness, Put on a silica gel column, elute and separate with dichloromethane / methanol with a volume ratio of 10:1, and obtain 1.41 g of the intermediate propionyl quinclorac, with a yield of 95.92%;

[0025] 2) Take 1.40 g of propyral quinclorac, add 10 mL of trifluoroacetic acid to dissolve, add 10 mL of water, and stir at 50°C for 2 h; TLC test shows that the basic reaction of...

Embodiment 2

[0029] Example 2 Preparation of quinclorac antigen

[0030] 1. Preparation of immune antigen

[0031] The quinclorac hapten was conjugated with bovine serum albumin (BSA) to obtain the immunogen.

[0032] Take 11 mg of quinclorac hapten, add 0.3 mL of ethanol to dissolve, clarify, and obtain liquid A; take another 50 mg of bovine serum albumin, add 0.05 mol / L sodium carbonate solution to obtain liquid B, and dissolve liquid A drop by drop Added to solution B, stirred at room temperature for 16 h. The 0.02 M PBS was dialyzed and purified for three days, and the medium was changed three times a day to obtain the quinclorac-BSA conjugate antigen, which was the immunogen. Aliquot and store at -20°C for later use.

[0033] 2. Preparation of coated antigen

[0034]Take 5 mg of quinclorac hapten, add 0.2 mL of methanol to dissolve, clarify, and obtain liquid A; take another 50 mg of ovalbumin, add 0.05 mol / L sodium carbonate solution to obtain liquid B, add liquid A dropwise int...

Embodiment 3

[0037] Example 3 Preparation of quinclorac monoclonal antibody

[0038] 1. Animal immunity

[0039] Inject the immunogen obtained in step 2 into Balb / c mice at an immunization dose of 150 μg / mouse to make them produce antiserum.

[0040] 2. Cell fusion and cloning

[0041] Splenocytes from immunized Balb / c mice were fused with SP2 / 0 myeloma cells at a ratio of 8:1 (quantity ratio), and the cell supernatant was measured by indirect competition ELISA to screen positive wells. Positive wells were cloned by limiting dilution until hybridoma cell lines stably secreting monoclonal antibodies were obtained.

[0042] 3. Cell cryopreservation and recovery

[0043] The hybridoma cells were made into 1 × 10 6 cells / ml for long-term storage in liquid nitrogen. When recovering, take out the cryopreservation tube, put it into a 37°C water bath to thaw quickly, remove the cryopreservation solution by centrifugation, and transfer it to a culture bottle for culture.

[0044] 4. Preparati...

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Abstract

The invention discloses a preparation method and application of a quinclorac hapten and antigen. The method is characterized in that the quinclorac hapten is prepared by using quinclorac and (1,3-dioxan-2-ylethyl)magnesium bromide for reaction to generate aldolactal quinclorac which is then reacted with trifluoroacetic acid; the quinclorac antigen is obtained by coupling the quinclorac hapten andcarrier protein. The antigen prepared through the method presents a specific quinclorac antigenic determinantantigen epitope, so that screening out a high-specificity quinclorac monoclonal antibody becomes possible; the generated antibody is high in specificity and high in sensitivity, and can be used for building an enzyme-linked immunosorbent assay method and a colloidal gold test strip rapid determination method, so that quinclorac in food and tobacco-planting soil is fast detected.

Description

technical field [0001] The invention relates to a preparation method and application of quinclorac hapten and antigen. The invention belongs to the technical field of pesticide immunochemistry. Background technique [0002] Quinclorac is a hormone-type quinoline carboxylic acid herbicide, which is mainly used to control monocotyledonous weeds in rice fields, especially barnyardgrass, and is one of the main herbicide varieties in rice fields in my country. The drug can be absorbed by germinated seeds, roots and leaves, has the characteristics of hormone herbicides, and has similar symptoms to auxin substances. Because quinclorac is acidic and degrades slowly in acidic soil, it is easy to cause serious damage to sensitive crops. In the past 300 days after the application of quinclorac in paddy fields, no crops can be planted except rice; When planting tobacco, eggplant, tomato, carrot, celery and other crops, the above crops cannot be irrigated with water that has been appli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D215/48C07K14/765C07K14/77C07K16/44C07K1/107G01N33/532G01N33/535G01N33/558G01N33/577
CPCC07D215/48C07K14/765C07K14/77C07K16/44C07K19/00G01N33/532G01N33/535G01N33/558G01N33/577
Inventor 陈黎范子彦刘惠民唐纲岭颜权平崔华鹏樊美娟万宇平鲁亚辉冯才伟
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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