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Enzyme-linked immunosorbent assay kit for detecting amantadine and its application

An enzyme-linked immunosorbent reagent and amantadine technology, which can be used in testing food, measuring devices, material inspection products, etc., can solve the problem of high cost of capital and personnel input, and achieve simple pretreatment process, efficient detection method, and convenient portability. Effect

Active Publication Date: 2016-08-31
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the local standards "DB21 / 2394-2014 Food Safety Local Standard Chicken Liver and Meat Chicken Liver and Meat Determination of Amantadine and Riantadine by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry", "DB32 / T 1163-2007 Chicken Liver Amantadine Determination of amine residues by liquid chromatography-tandem mass spectrometry" all use instrumental methods, which have the advantages of high sensitivity and accurate results, but the investment costs of capital and personnel are high

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting amantadine and its application
  • Enzyme-linked immunosorbent assay kit for detecting amantadine and its application
  • Enzyme-linked immunosorbent assay kit for detecting amantadine and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 kit components

[0028] 1. Preparation of amantadine hapten

[0029] Take 0.8g of amantadine hydrochloride, add 40ml of acetonitrile, stir to dissolve, add 0.41g of anhydrous potassium carbonate, stir, add 0.54g of β-chloropropionic acid, and stir for 6h at 80°C. Stop the reaction, filter, remove potassium carbonate, evaporate to dryness, apply to a silica gel column, elute with petroleum ether: ethyl acetate = 1:1, separate and purify, and obtain hapten product 0.43g.

[0030] Get above-mentioned product and measure through proton nuclear magnetic resonance spectrum, such as figure 2 As shown, the signal peak of chemical shift δ11.0 indicates the presence of carboxyl hydrogen, and the signal peaks of 2.82 and 2.49 indicate the presence of hydrogen on the spacer arm, thus proving that the hapten was synthesized successfully.

[0031] 2. Antigen preparation

[0032] Immunogen preparation - the immunogen was obtained by coupling the ama...

Embodiment 2

[0045]Embodiment 2 detects the formation of the enzyme-linked immunosorbent assay kit of amantadine

[0046] Set up the ELISA kit for detecting amantadine to include the following components:

[0047] (1) A microtiter plate coated with an amantadine-coupled antigen;

[0048] (2) 6 bottles of amantadine standard solution, the concentrations are 0 μg / L, 0.5 μg / L, 1.5 μg / L, 4.5 μg / L, 13.5 μg / L, 40.5 μg / L;

[0049] (3) Amantadine antibody labeled with horseradish peroxidase;

[0050] (4) Substrate chromogenic solution is made up of A liquid and B liquid, and A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0051] (5) The stop solution is 2mol / L sulfuric acid;

[0052] (6) The washing solution has a pH value of 7.4, contains 0.5% to 1.0% Tween-20, 0.01‰ to 0.03‰ sodium azide preservative, and 0.1 to 0.3mol / L phosphate buffer, and the percentage is weight volume percentage;

[0053] (7) The complex solution is a phosphate buffer solution with a pH value o...

Embodiment 3

[0054] The detection of amantadine in the animal tissue of embodiment 3

[0055] 1. Sample pretreatment

[0056] Homogenize the tissue sample with a homogenizer; weigh 2.0±0.05g of the homogenized sample into a 10ml polystyrene centrifuge tube, add 6ml of acetonitrile, then add 0.5g of sodium chloride, oscillate with an oscillator for 5min, 3000g at room temperature ( Centrifuge at 20-25℃ / 68-77℉) for 5min; pipette 3ml of the upper organic phase into a 10ml clean and dry glass tube, and dry it in a water bath at 40-50℃ (68-86℉) under nitrogen flow; add 1ml of n-hexane, and use Vortex for 1 min, then add 0.5ml reconstituted working solution, vortex for 30s with a vortex, mix well, centrifuge at 3000g room temperature (20-25°C / 68-77°F) for 3min; remove the upper organic phase, and take the lower layer 100 μl of the aqueous phase was used for analysis.

[0057] 2. Detection with kit

[0058] Add 100 μl of the standard / sample to the corresponding microwell, then add 50 μl / well o...

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Abstract

The invention provides an ELISA (enzyme linked immunosorbent assay) kit for detecting amantadine. The ELISA kit comprises an ELISA plate coated with a coating antigen, an amantadine standard product solution, an enzyme-labeled antibody, a substrate developing solution, a stop solution, a washing solution and a combination solution, wherein the coating antigen is an amantadine coupling antigen, and the enzyme-labeled antibody is an enzyme-labeled amantadine antibody. The invention further discloses a method for applying the ELISA kit to amantadine detection. The method comprises steps as follows: sample pretreatment is performed firstly, then the kit is used for detection, and a detection result is analyzed finally. The ELISA kit can be used for detecting the content of amantadine in an animal tissue sample, is simple and convenient to operate, low in cost and high in sensitivity, can be used for performing on-site monitoring and is suitable for screening a large number 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 amantadine, which can qualitatively and quantitatively detect the residual amount of amantadine in animal tissues. Background technique [0002] Amantadine is an antiviral drug that inhibits influenza virus. It is often used in poultry to resist avian influenza, so it is favored by farmers. At present, the United States has determined that its effectiveness for anti-Parkinson's disease drugs is uncertain due to lack of safety. According to the data on sex and effectiveness, amantadine is no longer recommended as a treatment for influenza in the United States, and it has certain toxic and side effects on the nervous system and cardiovascular system. It has been banned as a veterinary drug in China. [0003] At present, the local standards "DB21 / 2394-2014 Food Safety Local Standard Chicken Liver and Meat Chicken Live...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/12G01N33/577
Inventor 万宇平朱亮亮李行贾芳芳杨昌松何方洋王琳琛张瑜
Owner BEIJING KWINBON BIOTECH
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