Enzyme-linked immunoassay kit for detecting dimethomorph, and applications thereof
A technology of dimethomorph and dimethomorph couple, applied in the field of enzyme-linked immunoassay, can solve problems such as high detection technology requirements, unreasonable use of pesticides, damage to consumers' health, etc., and achieve simple sample pretreatment process, The effect of simple structure and high accuracy
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Embodiment 1
[0034] Embodiment 1 Preparation of kit components
[0035] 1. Preparation of dimethomorph hapten
[0036] 1) Dissolve 20 mg of dimethomorph, 10 ul, 1,3-propylenediamine, and a catalytic amount of 4-dimethylaminopyridine in 2 ml of N, N'-dimethylformamide to obtain liquid I;
[0037] 2) Dissolve 20mg of N,N'-dicyclohexylcarbodiimide in 0.5ml of DMF to obtain liquid II;
[0038] 3) Slowly add solution II to solution I dropwise at 0°C, and continue to react for 20 h after returning to room temperature;
[0039] 4) Evaporation of the solvent, column chromatography (eluent: dichloromethane / methanol, volume ratio 20:1), to obtain the dimethomorph hapten, such as figure 1 .
[0040] 2. Antigen preparation
[0041] Immunogen preparation—Dimethomorph hapten was coupled with bovine serum albumin (BSA) to obtain immunogen.
[0042] 1) Dissolve 5.3 mg of dimethomorph hapten in 0.5 ml of DMF to obtain liquid I;
[0043] 2) Dissolve 30mg of BSA in 2.0ml, pH7.0, 0.1mol / L phosphate buff...
Embodiment 2
[0064] Example 2 The formation of the enzyme-linked immunosorbent assay kit for detecting dimethomorph
[0065] An enzyme-linked immunosorbent assay kit for detecting dimethomorph was set up to include the following components:
[0066] (1) a microtiter plate coated with dimethomorph-coupled antigen;
[0067] (2) 6 bottles of dimethomorph standard solution, the concentrations are 0ug / L, 1ug / L, 3ug / L, 9ug / L, 27ug / L, 81ug / L.
[0068] (3) concentrated enzyme conjugate;
[0069] (4) enzyme conjugate dilution;
[0070] (5) Substrate chromogenic solution is made up of substrate liquid A liquid and substrate liquid B liquid, and substrate liquid A liquid is carbamide peroxide, and substrate liquid B liquid is tetramethylbenzidine;
[0071] (6) The stop solution is 2mol / L sulfuric acid.
Embodiment 3
[0072] The detection of dimethomorph in the sample of embodiment 3
[0073] 1. Sample pretreatment
[0074] Homogenize the vegetable sample with a homogenizer; weigh 5.0g±0.05g of the homogenized vegetable sample into a 50ml polystyrene centrifuge tube, add 25ml of 50% methanol, shake vigorously with an oscillator for 5min, over 3000r, at room temperature ( Centrifuge at 20-25℃ / 68-77℉) for 5min; take 500ul supernatant to a 2ml polystyrene centrifuge tube, add 500ul10% sodium chloride aqueous solution and shake for 1min with a shaker, mix well; take 20ul for analyze.
[0075] 2. Detection with kit
[0076] Add ochratoxin A standard solution / sample 20 ul to the microwells of the microtiter plate coated with dimethomorph-coupled antigen, and then add 100 ul of enzyme conjugate working solution (concentrate the enzyme conjugate with enzyme Dilute the diluent according to the volume of 1:20), seal the plate with a cover film, and react in the dark at 25°C for 10 minutes, pour ...
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