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ELISA reagent kit for detecting two oxyethyl group thiophosphate organophosphorus pesticide

A technology of diethoxy phosphorothioate and enzyme-linked immunological reagent, which is applied in the field of immunology, can solve the problems of difficulty in high-throughput and rapid detection of single-component kits, and no detection method is provided, and achieves low detection cost. , less time-consuming, simple sample pretreatment process

Active Publication Date: 2013-05-01
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, domestic research results on multi-residue pesticide immunoassay are relatively few, and there are only a few theoretical research reports abroad on multi-residue immunoassay of organophosphorus pesticides using polyclonal antibodies and monoclonal antibodies, and no detection methods for actual samples and detection products with economic benefits are provided. , the existing single-component kits are difficult to achieve high-throughput and rapid detection. In order to meet the requirements of high-throughput, rapid and sensitive detection of pesticides, it is very urgent to develop a new multi-residue detection kit for organophosphorus

Method used

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  • ELISA reagent kit for detecting two oxyethyl group thiophosphate organophosphorus pesticide
  • ELISA reagent kit for detecting two oxyethyl group thiophosphate organophosphorus pesticide
  • ELISA reagent kit for detecting two oxyethyl group thiophosphate organophosphorus pesticide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 kit component preparation

[0050] 1. Coating antigen preparation

[0051] Coating hapten synthesis: Weigh 1.68g (0.018moL) KOH and dissolve in anhydrous methanol, weigh 1.00g (0.01moL) GABA, 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 was completed, the product was spin-dried, washed with chloroform, filtered, and the supernatant was concentrated and passed through a silica gel column (chloroform:methanol=3:1) to obtain 1.14 g, with a yield of 56.0%. ESI-MS analysis (negative) m / z 254[M-H]-; 1H-NMR (400MHz, CDCl 3 and TMS): δ1.27(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 ).

[0052] Coating source preparation: Take hapten 0.12mmol and dissolve i...

Embodiment 2

[0061] The establishment of embodiment 2 kit

[0062] This example provides an enzyme-linked immunoassay kit suitable for the analysis of diethoxy phosphorothioate organophosphorus pesticide residues. It is based on immune reaction and enzymatic reaction, and can detect diethyl ether in water, soil, and vegetable and fruit samples. Oxyphosphorothioate residues of organophosphorus pesticides.

[0063] The kit includes the box body, 96-well / 40-well ELISA plate set in the box body, anti-diethoxyphosphorothioate-type organophosphorus pesticide antibody; horseradish peroxidase-labeled secondary antibody; parathion standard solution and reagents, characterized in that, in the microwells of the enzyme labeling plate, the diethoxy phosphorothioate organophosphorus pesticide antigen is coated with the coating solution, sealed with 3.0-5.0% skimmed milk powder, dried and stored in aluminum foil Bag; reagents in the box include anti-diethoxyphosphorothioate-type organophosphorus pestici...

Embodiment 3

[0065] Embodiment 3 uses kit of the present invention to carry out pesticide detection

[0066] 1. Pretreatment of test samples

[0067] Water sample: After filtration, samples can be taken for ELISA analysis.

[0068] Soil: Take 10g of soil and extract 3 times with 20-40mL of methanol, combine the extracts, concentrate, then dilute with PBST to 10mL, and take samples for ELISA analysis.

[0069] Vegetable sample: Take the vegetable sample pulverizer and weigh 10g, extract 3 times with 20-40mL methanol, combine the extracts, concentrate, dilute to 10mL with PBST, and sample for ELISA analysis.

[0070] Blood: take human blood, add anticoagulant and directly analyze it by ELISA method.

[0071] Gastric lavage solution (2% sodium bicarbonate solution): take 5mL of gastric lavage solution, adjust the pH value to neutral with dilute HCl, and then analyze it by ELISA.

[0072] Vomiting fluid: Take the sample and grind it, centrifuge to get the supernatant and analyze it by ELISA...

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Abstract

The invention discloses an enzyme-linked immunosorbent assay kit for detecting diethoxy phosphorothioate organic phosphorus pesticide, which comprises a kit body, an ELISA plate coated with the antigen of the diethoxy phosphorothioate organic phosphorus pesticide, an antibody solution against diethoxy phosphorothioate organic phosphorus pesticide, a second anti-solution, a standard solution, a concentrated extracting solution, a substrate solution, a buffer solution of the substrate, a concentrated washing liquid and a stopping liquid. The organic phosphorus pesticide refers to the organic phosphorus pesticide containing the common structure of diethoxy phosphorothioate, in particular parathion, coumaphos, quinalphos, triazophos, phoxim, dichlofenthion, phorate, chlorpyrifos, bromophos ethyl, isazofos, diazinon, sulfotep and a mixture thereof. The enzyme-linked immunosorbent assay kit can be used to detect the organic phosphorus residues in fruits, vegetables, soil and water, and isconvenient in operation, high in sensitivity, low in price and is suitable for high-throughput screening monitored on spot.

Description

technical field [0001] The invention belongs to the technical field of immunology, and in particular relates to an ELISA kit for detecting diethoxy phosphorothioate-type organophosphorus pesticides and a method for using the kit to detect pesticide residues. Background technique [0002] 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 19 kinds of pesticides that are prohibited or restricted from being used on vegetables, fruit trees, tea leaves, and Chinese herbal medicines, of which 17 are organophosphorus pesticides, and diethoxy There are 8 kinds of pesticides with phosphorothioate common structure. [0003] At present, the research on the immunological detection of pesticide residues at ho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/577
Inventor 孙远明徐振林沈玉栋王弘谢桂勉雷红涛杨金易
Owner SOUTH CHINA AGRI UNIV
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