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Imidacloprid detection ELISA (enzyme linked immunosorbent assay) kit and application thereof

An enzyme-linked immunosorbent reagent, imidacloprid technology, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of inability to meet MRLs, complex processing procedures, and high detection limits, and achieves simple structure, high sensitivity and high accuracy. Effect

Inactive Publication Date: 2014-10-15
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been literature reports on the immunological detection of IMI residues in foreign countries, there are problems such as complex pre-treatment procedures, high costs, and high detection limits for residual samples to varying degrees, and cannot meet the international MRLs for IMI in food.

Method used

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  • Imidacloprid detection ELISA (enzyme linked immunosorbent assay) kit and application thereof
  • Imidacloprid detection ELISA (enzyme linked immunosorbent assay) kit and application thereof
  • Imidacloprid detection ELISA (enzyme linked immunosorbent assay) kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 Preparation of kit components

[0032] 1. Preparation of imidacloprid hapten

[0033] Add 0.25g of imidacloprid, 1ml of dimethylformamide (DMF), 0.75g of tin chloride hydrate and 10ml of ethanol into a 100ml two-necked bottle, empty the nitrogen, and react at a temperature of 65°C for 40min. Chromatography (TLC) detected that the reaction was complete. Treatment: cool down to room temperature, add 50ml of ethyl acetate, add saturated aqueous sodium bicarbonate to adjust to weak alkalinity, a large amount of salt precipitates, extract the turbid solution twice with 40ml of ethyl acetate, combine the organic phases, wash with brine, anhydrous sodium sulfate After drying, the solvent was evaporated to obtain 0.22 g of solid product.

[0034] Get above-mentioned product and measure through proton nuclear magnetic resonance spectrum, such as figure 2 As shown, the peak at about 4.7ppm is the two methylene signal peaks on the imidazoline ring, the peak at abo...

Embodiment 2

[0053] Example 2 Construction of an enzyme-linked immunosorbent assay kit for detecting imidacloprid

[0054] An ELISA kit for detecting imidacloprid was set up to include the following components:

[0055] (1) ELISA plate coated with imidacloprid-conjugated antigen;

[0056] (2) 6 bottles of imidacloprid standard solution, the concentrations are 0μg / L, 4μg / L, 12μg / L, 36μg / L, 108μg / L, 324μg / L;

[0057] (3) Imidacloprid monoclonal antibody working solution;

[0058] (4) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0059] (5) The substrate chromogenic solution is composed of liquid A and liquid B, liquid A is carbamide peroxide, and liquid B is tetramethylbenzidine;

[0060] (6) The stop solution is 2mol / L sulfuric acid;

[0061] (7) The washing solution has a pH value of 7.4, contains 0.5%~1.0% Tween-20, 0.01‰~0.03‰ sodium azide preservative, and 0.1~0.3mol / L phosphate buffer, the percentage is weight volume percentage;

[0062] (8) The complex sol...

Embodiment 3

[0063] Example 3 Detection of imidacloprid in vegetables and fruit samples

[0064] 1. Sample pretreatment

[0065] Homogenize the sample with a homogenizer; weigh 2.0±0.05g of the homogenate into a 50ml polystyrene centrifuge tube, add 2ml of 2mol / L sodium hydroxide solution, 10ml of ethyl acetate, and vortex for 5min with a vortexer. Above 3000g, centrifuge at room temperature (20-25°C) for 5min; pipette 1ml of the upper organic phase into a 15ml clean glass test tube, and dry it in a water bath (50-60°C) under nitrogen / air flow; add 1ml of reconstitution working solution, vortex 3min; take 50ul for analysis.

[0066] 2. Detection with kit

[0067] Add 50 μl / well of imidacloprid standard solution or pretreated sample solution to the microwells of the microtiter plate coated with imidacloprid-conjugated antigen, then add 50 μl / well of imidacloprid monoclonal antibody, shake and mix gently, and cover with a cover plate Put the cover plate behind the membrane and react in a ...

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Abstract

The invention provides an imidacloprid detection ELISA (enzyme linked immunosorbent assay) kit which includes an ELISA plate coated with a coating antigen, a specific antibody, an enzyme marker, an imidacloprid standard solution, a substrate coloring liquid, a terminating liquid, a washing liquid and a complex solution, the coating antigen is an imidacloprid coupling antigen, and the enzyme marker is an enzyme labeled antibody. The invention also discloses an imidacloprid detection method using the ELISA kit, and the method includes first, pretreatment of a sample, then detection with the ELISA kit and final detection result analyzing. The ELISA kit can be used to detect the content of imidacloprid in vegetables and fruits, and has the advantages of being simple in operation, low in cost, high in sensitivity, capable of monitoring on site and suitable for screening a large number of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting imidacloprid, which is particularly suitable for detecting imidacloprid residues in vegetable and fruit samples. Background technique [0002] Imidacloprid (IMI) is a super efficient neonicotinoid insecticide with broad spectrum, high efficiency, low toxicity, and low residue. It is not easy for pests to develop resistance, and it is safe for humans, animals, plants, and natural enemies. It also has contact and stomach toxicity. And systemic absorption and other multiple effects. [0003] At present, the pesticide products containing IMI have been registered and used on more than 140 kinds of crops in more than 120 countries. , Thysanoptera, Diptera, Coleoptera, Lepidoptera and more than 50 kinds of pests in 7 orders. [0004] The extensive use of IMI not only has a serious impact on the environment, bu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68G01N21/31G01N21/78G01N33/531
Inventor 何方洋万宇平罗晓琴杨学林冯静贾芳芳杨烁齐向武
Owner BEIJING KWINBON BIOTECH
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