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Polyclonal antibody of dimethachlon and preparation and application thereof

A polyclonal antibody and sclerotium purification technology, which is applied in the preparation methods of peptides, chemical instruments and methods, animal/human proteins, etc., can solve the problems of low detection efficiency and single detection method, and achieve low detection cost and detection method. Simple, simple preparation method

Inactive Publication Date: 2010-04-07
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to: aim at the single problem of current Sclerotin pesticide residue detection method and the practical problem of low detection efficiency, provide a polyclonal antibody of Sclerotin and its preparation method, and quickly detect pesticides through the polyclonal antibody residual

Method used

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  • Polyclonal antibody of dimethachlon and preparation and application thereof
  • Polyclonal antibody of dimethachlon and preparation and application thereof
  • Polyclonal antibody of dimethachlon and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Synthesis of immune antigen and coating antigen

[0024] Synthesize immune antigen by active ester method: weigh 21mg (0.0583mmol) hapten and dissolve it in 0.4mL dimethylformamide (DMF), then add N-hydroxysuccinimide (NHS) 7.17mg (0.0623mmol) and Cyclodihexylcarboimide (DCC) 13.76mg (0.0667mmol), mix thoroughly, and stand at room temperature overnight in the dark. The next day, centrifuge at 10000g for 10min, discard the precipitate, slowly add the supernatant to 3mL of borate buffer (pH8.7) containing 25mg (0.00037mmol) bovine serum albumin BSA, and continue magnetically stirring the reaction solution for 4 hours at room temperature . After the reaction, put the reaction mixture into a dialysis bag, dialyze in deionized water at 4°C for 3 days, change the water every 8 hours, obtain the immune antigen Hapten-BSA after the dialysis, divide it and store it in a -20°C refrigerator in spare.

[0025] The synthesis method of the coating antigen Hapten-OVA by ...

Embodiment 2

[0027] Example 2. Preparation of Sclerotin polyclonal antibody

[0028] The synthetic conjugate was used as the immune antigen Hapten-BSA, and two purebred New Zealand white rabbits of 1.5-2.0 kg were selected as experimental animals, and negative serum was collected one week before the experiment. The specific immunization procedures are shown in Table 1: For the first immunization, each rabbit was immunized with 1 mg of immunogen mixed with an equal volume of Freund's complete adjuvant, emulsified into a water-in-oil emulsion, and injected subcutaneously at multiple points on the back (around 20 o'clock). Three weeks later, the same dose of immunogen and an equal volume of incomplete Freund's adjuvant were used to boost, once every two weeks, for a total of 4 times. When the serum titer basically no longer rises, shock immunization is carried out, and 2 mg of immunogen is mixed with an equal volume of normal saline, and injected directly into the ear vein. A week later, blo...

Embodiment 3

[0031] Example 3. Indirect non-competitive ELISA method for determining the working concentration and titer of antibodies

[0032] (1) Determination of working concentration

[0033] Determine the working concentration of antibody and coated antigen by square array titration. Select the dilution concentration of the antigen and antibody when the OD value is 1.0 as the working concentration of the coated antigen and antibody. The working concentration is 1 / 128000. The specific operation steps are as follows:

[0034] ① Coating: CBS coating buffer will coat the four groups of antigens diluted 100×5n (n=0.5, 1, 2, 4), and add them to A, B, and C of the 96-well enzyme-labeled microplate respectively. And D, E and F, G and H in four groups of eight rows, 100 μL / well, 4°C overnight.

[0035] ② Blocking: Add 200 μL of 1% OVA blocking solution to each well, and incubate at 37°C for 1 hour.

[0036] ③Adding samples: Add six groups of antibodies with a doubling dilution of 1000×2n ...

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Abstract

The invention relates to a polyclonal antibody of dimethachlon and preparation and application thereof. The polyclonal antibody is prepared by the following steps: coupling hapten and bovine serum albumin BSA to prepare immune antigen; and immunizing New Zealand white rabbits. The polyclonal antibody is characterized in that: the hapten is dimethachlon-2-glutaric acid semi-ester; the dimethachlon-2-glutaric acid semi-ester is coupled with bovine serum albumin by an active ester method to synthesize the immune antigen; and the immune antigen is used for the New Zealand white rabbits so as to obtain the polyclonal antibody. The polyclonal antibody detects the residue of dimethachlon pesticide in a specimen by adopting an indirect competitive ELISA method; and envelope antigen used in the detection is a conjugate of the hapten and ovalbumin by the active ester method. The invention has the advantages that: the preparation method for the polyclonal antibody of the dimethachlon is simple; the polyclonal antibody can be used for quickly detecting the residue of the dimethachlon pesticide in the specimen; and compared with the prior equipmental analysis, the detection method has the advantages of simpleness, quickness, low detection cost, suitability for quickly screening a great number of samples, and the like.

Description

Technical field: [0001] The invention relates to a polyclonal antibody of sclerotin and its preparation and application. Background technique: [0002] Sclerotin (Dimethachlon) is a dicarboximide fungicide, also known as Wenkuli, and its trade name is Ohric. Its active ingredient is (N-3,5-dichlorophenyl) Succinimide (NDPS). It has direct sterilization, internal osmosis treatment, anti-androgen effect, long residual effect period, and has good control effect on rape sclerotinia, tobacco leaf blight, rice sheath blight, wheat head blight, powdery mildew and industrial antisepsis . However, in the early 1980s, people began to gradually realize that NDPS may have potential hazards to human health (mainly kidney toxicity), which limited the extensive use of sclerotin in agriculture. [0003] At present, the only research on the detection of sclerotia residues is gas chromatography (see Liμ HC, Li QW, Tang LB. Capillary gas chromatographic determination of dimethachlon residue...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C07K14/765C07K14/77C07K1/113G01N33/566G01N33/531
Inventor 刘媛刘贤金张存政刘蓉蓉
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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