ELISA reagent kit for detecting two oxyethyl group thiophosphate organophosphorus pesticide
A technology of diethoxyphosphorothioate and enzyme-linked immunological reagents, which is applied in the field of immunology, can solve the problems of high-throughput and rapid detection of single-component kits, and no detection methods are provided, so as to achieve low detection cost , less time-consuming, and simple sample pretreatment process
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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, 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, J1=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 in 2ml DMF, stir and add DCC and NHS. Ma...
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 vegetable sample grinder and weigh 10g, extract 3 times with 20-40mL methanol, combine the extracts, concentrate, dilute to 10mL with PBST, take samples 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 meth...
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