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Diethoxy thiophosphate organophosphorus pesticide antibody, and preparation and use thereof

A technology of diethoxyphosphorothioate and ethoxyphosphorothioate, which is applied in the field of immunology, can solve the problems that pesticides are not targeted, not solved well, and the detection limit is very different. The method is simple and convenient, Low detection cost, good stability and repeatability

Active Publication Date: 2009-06-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2005, Liu Xianjin and others published "Immune Antibodies for Detecting Organophosphorus Pesticide Residues and Its Application", using diethylphosphonic acid acetic acid as a hapten, which can be coupled with protein and used to immunize animals without structural modification. The antibodies prepared in this way can detect a variety of organophosphorus pesticides, but the pesticides that can be detected are not targeted, and the detection limit varies greatly
It can be seen that the key issue of preparation of broad-spectrum specific antibodies has not been well resolved.

Method used

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  • Diethoxy thiophosphate organophosphorus pesticide antibody, and preparation and use thereof
  • Diethoxy thiophosphate organophosphorus pesticide antibody, and preparation and use thereof
  • Diethoxy thiophosphate organophosphorus pesticide antibody, and preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of Diethoxyphosphorothioate Organophosphorus Pesticide Immune Hapten OPH1

[0052]Take 4.88g (0.04moL) of p-hydroxybenzaldehyde and dissolve it in pyridine, protect it with a drying tube, put it in a water bath at 85°C, and stir. Add 3.75 g (0.036 moL) of malonic acid dropwise to the above solution and react for 3 h. After the reaction was completed, the mixture was poured into a saturated sodium bicarbonate solution, stirred for 15 min, extracted with ethyl acetate, and the pH of the aqueous layer was adjusted to 3 with 4M hydrochloric acid, and 2.32 g of a white solid was precipitated, with a yield of 78.6%. ESI-MS analysis (negative) m / z 163[M-H]-; 1H-NMR (400MHz, d6-Acetone and TMS): δ 6.33 (d, J=16.0Hz, 1H, CH=CH); 6.89 (d, J=8.6Hz, 2H, ArH); 7.54(d, J=8.6Hz, 2H, ArH); 7.60(d, J=16.0Hz, 1H, CH=CH); 8.86(s, 1H, OH); 10.77 (s, 1H, COOH).

[0053] Weigh 0.7 (0.0125 moL) KOH and dissolve it in anhydrous methanol, weigh 0.821 g (0.005 moL) of th...

Embodiment 2

[0056] Example 2 Preparation of Diethoxyphosphorothioate Organophosphorus Pesticide Coating Hapten OPH2

[0057] Weigh 1.68g (0.018moL) KOH and dissolve in anhydrous methanol, weigh 1.00g (0.01moL) aminobutyric acid, dissolve in the above solution with stirring at 0°C, and stir for 30min. Add 1.5g (0.008moL) diethoxyphosphoryl thiochloride dropwise, and react at 0°C for 12h. After the reaction, spin dry, wash with chloroform, filter, and the supernatant is concentrated and passed through a (chloroform:methanol=3:1) silica gel column to obtain 1.14g, with a yield of 56.0%. ESI-MS analysis (negative) m / z 254[M-H]-; 1H-NMR (400MHz, CDCl 3 and TMS): δ 1.27 (t, J=7.0Hz, 6H, CH 3 ); 1.77 (p, J=6.9Hz, 2H, CH 2 ); 2.39(t, J=7.3Hz, 2H, CH 2 ); 2.98 (td, J 1 =6.8Hz,J 2 =6.8Hz,J 3 =6.8Hz,J 4 =11.1Hz, 2H, CH 2 ); 4.01 (ddd, J 1 = 1.4Hz,J 2 =7.3Hz,J 3 =15.9Hz, 4H, CH 2 ).

[0058] The prepared coated hapten (OPH2) structure is shown in formula (II):

[0059]

Embodiment 3

[0060] Example 3 Preparation of Diethoxyphosphorothioate Organophosphorus Pesticide Immune Antigen OPH1-BSA

[0061] (1) Dissolve 0.12mmol of hapten in 2ml of DMF, stir and add DCC and NHS. Magnetic stirring was carried out overnight at 4°C, and the supernatant was liquid A after centrifugation;

[0062] (2) Weigh 0.002mol BSA, slowly dissolve it in 10ml of PBS (pH8.0) with a concentration of 0.1mol / L at 4°C, stir and dissolve to prepare solution B;

[0063] (3) Under magnetic stirring, liquid A was gradually dropped into liquid B, and reacted at 4°C for 12 hours. After centrifugation, the supernatant was taken, dialyzed with normal saline at 4°C for 3 days, dispensed into 0.5ml centrifuge tubes at a concentration of 1 mg / mL or 2 mg / mL, and stored in a -20°C refrigerator for later use.

[0064] Immunogen identification: The carrier protein BSA, hapten and immunogen were detected by UV scanning (200-400nm), and it was found that the absorption curve of the immunogen OPH1-BSA ...

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Abstract

The invention discloses a diethoxy thiophosphate organophosphorus pesticide antibody and a preparation method and the application thereof. The antibody of the invention is prepared by introducing appropriate active arms to a diethoxy thiophosphate structure to prepare immunity hapten, coupling the hapten with bovine serum albumin to prepare immunity antigen and immunizing the originally immune New Zealand white rabbits; and a multi-residue ELISA detection method of the diethoxy thiophosphate organophosphorus pesticide is established with coatingen. Based on the detection method established by the antibody, more than 12 types of the diethoxy thiophosphate organophosphorus pesticide can be detected simultaneously, the limit of detection is 0.0002-3.938mug / ml and the detection method is suitable for on-site supervision of pesticide residues and rapid screening with high flux.

Description

technical field [0001] The invention belongs to the technical field of immunology, and specifically relates to a broad-spectrum specific polyclonal antibody of diethoxy phosphorothioate-type organophosphorus pesticides, a preparation method thereof, and an application in an ELISA detection method for multi-residue organophosphorus pesticides. Background technique [0002] The problem of pesticide residues seriously endangers the health of the people. Organophosphorus pesticides are widely used in agriculture and planting because of their high efficiency and low cost. However, organophosphorus pesticides, as neurotoxins, can cause neurological disorders, tremors, insanity, and language disorders. In Announcement No. 199 issued by the People's Republic of China in 2002, there are 17 kinds of organophosphorus pesticides that are banned or restricted from being used on vegetables, fruit trees, tea leaves, and Chinese herbal medicines, and diethoxy phosphorothioate has a common s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C07K16/06C07K14/765C07F9/18G01N33/543
Inventor 沈玉栋孙远明雷红涛谢桂勉王弘徐振林杨金易
Owner SOUTH CHINA AGRI UNIV
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