Enzyme linked immunosorbent assay kit for detecting triadimenol and application of enzyme linked immunosorbent assay kit

An enzyme-linked immunosorbent reagent, triadimenol technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems that it is difficult to meet the needs of rapid detection of a large number of samples and on-site samples, the processing process is complicated, and time-consuming, etc., to achieve the detection The method is convenient and easy to implement, the pretreatment requirements are low, and the effect of easy use is

Active Publication Date: 2016-05-11
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of these analytical methods requires expensive instruments and specialized technicians. The sample pretreatment process is complex, costly, and time-consuming, and it is difficult to meet the needs of a large number of samples and rapid detection of on-site samples.

Method used

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  • Enzyme linked immunosorbent assay kit for detecting triadimenol and application of enzyme linked immunosorbent assay kit
  • Enzyme linked immunosorbent assay kit for detecting triadimenol and application of enzyme linked immunosorbent assay kit
  • Enzyme linked immunosorbent assay kit for detecting triadimenol and application of enzyme linked immunosorbent assay kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The preparation of embodiment 1 kit components

[0029] 1. Synthesis of triadimenolol hapten (see appendix for the synthetic route figure 1 ) and identification

[0030] Take 1.0g of triadimefon and dissolve it in ethanol, add 0.35g of 1,8-diazabicycloundec-7-ene (DBU), stir, then add 0.87g of aminocaproic acid aqueous solution, heat and reflux for 12h; stop the reaction , rotary evaporation, remove ethanol, add water and 1.3g potassium hydroxide, shake, add ethyl acetate for extraction, separate the organic phase, adjust the pH value of the water phase to 4, add ethyl acetate for extraction, wash the organic phase with water, anhydrous sodium sulfate After drying and evaporating to dryness, a light yellow oil was obtained, which was recrystallized from dichloromethane / petroleum ether (1:10, V / V) to obtain 0.76 g of the hapten product, with a yield of 69%.

[0031] The above haptens were identified by H NMR spectroscopy, 1 H-NMR (CDCl 3 ,300MHz) δ: 11.00 (1H, s), 7....

Embodiment 2

[0071] Embodiment 2 detects the formation of the enzyme-linked immunosorbent assay kit of triadimenol

[0072] Set up the enzyme-linked immunosorbent assay kit that detects triadimenol, so that it includes the following components:

[0073] (1) A microtiter plate coated with triadimenolol-conjugated antigen;

[0074] (2) Triadimenol standard solution, the concentrations are 0μg / L, 1μg / L, 3μg / L, 9μg / L, 27μg / L, 81μg / L;

[0075] (3) Triadimenol monoclonal antibody working solution;

[0076] (4) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0077] (5) The substrate chromogenic solution is composed of A liquid and B liquid, the substrate chromogenic liquid A is carbamide peroxide, and the substrate chromogenic liquid B is tetramethylbenzidine;

[0078] (6) The stop solution is 2mol / L sulfuric acid buffer;

[0079] (7) The washing solution is a 0.02mol / L phosphate buffer solution with a pH value of 7.4, containing 0.05% Tween-20 and 0.01‰thimerosal preserv...

Embodiment 3

[0081] The detection of triadimenol residual amount in the tobacco leaf sample of embodiment 3

[0082] 1. Sample pretreatment

[0083] Weigh 1.0±0.05g of tobacco leaf sample into a 50mL polystyrene centrifuge tube, add 10mL of tobacco leaf extract (weigh 100mL of methanol, add 100mL of deionized water, and mix well), and fully smash it with a homogenizer; Filter the crushed sample with a filter membrane; pipette 100 μL of the filtrate and add 400 μL deionized water, mix well; then pipette 50 μL of the above liquid and add 950 μL complex solution, mix well; take 50 μL for analysis.

[0084] 2. Detection with kit

[0085] Add 50 μL / well of triadimenol standard solution or pre-treated sample solution to the microwells of the microplate coated with triadimenol-conjugated antigen, then add 50 μL / well of triadimenol monoclonal antibody working solution, gently Shake and mix well, and cover the plate with a cover film and place it in a dark environment at 25°C for 30 minutes; pour...

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Abstract

The invention provides an enzyme linked immunosorbent assay kit for detecting triadimenol. The enzyme linked immunosorbent assay kit comprises an enzyme-labeled plate coated with a triadimenol coupling antigen, a triadimenol monoclonal antibody, an enzyme-labeled antibody, a triadimenol standard solution, substrate coloring liquid, a stop solution, washing liquid and reconstitution fluid. The invention further discloses a method using the enzyme linked immunosorbent assay kit to detect triadimenol residual quantity. The method includes: preprocessing a sample, detecting with the kit, and analyzing the detection result. The enzyme linked immunosorbent assay kit can be used for detecting the residual quantity of the triadimenol in tobacco and is simple to operate, low in cost, high in sensitivity, capable of achieving on-site monitoring and suitable for the screening of a large amount of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay detection technology, in particular to an enzyme-linked immunoassay kit for detecting triadimenol, which is particularly suitable for detecting the residual amount of triadimenol in tobacco leaves. Background technique [0002] Triadimenol (triadimenol, C 14 h 18 CN 3 o 2 ) also known as Baidan, the chemical name is 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-2-butane Alcohol is a low toxicity, broad-spectrum, high-efficiency systemic fungicide. Its main mechanism of action is to inhibit the biosynthesis of gibberellin and ergosterol and then affect the cell division rate. It is effective against tobacco root black rot and powdery mildew. , Frog eye disease and other fungal diseases have a good control effect. Therefore, it is widely used in agricultural production in our country, and is suitable for crops such as wheat, corn, sorghum, fruits and vegetables, and tobacco. [0003...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
CPCG01N33/543
Inventor 陈黎范子彦曹东山鲁亚辉潘立宁胡斌唐纲岭刘惠民朱亮
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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