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Enzyme linked immunosorbent assay kit for Cimaterol detection and application of kit

An enzyme-linked immunosorbent reagent, cimaterol technology, applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of high professional requirements for operators, expensive testing costs, and complicated steps, and achieve pre-treatment Simple process, high accuracy and high sensitivity effect

Active Publication Date: 2019-08-16
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the above methods require advanced detection equipment, expensive detection costs, cumbersome steps, time-consuming, and high professional requirements for operators, they are not suitable for large-throughput rapid screening detection in grassroots enterprises and institutions.

Method used

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  • Enzyme linked immunosorbent assay kit for Cimaterol detection and application of kit
  • Enzyme linked immunosorbent assay kit for Cimaterol detection and application of kit
  • Enzyme linked immunosorbent assay kit for Cimaterol detection and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The preparation of embodiment 1 kit components

[0025] 1. Preparation of cimaterol hapten

[0026] a: Take 1.0g of benzonitrile-4acetyl-2-ammonia, add 60ml of chloroform to dissolve, add 1.41g of copper bromide, heat and reflux for 4h, stop the reaction, add 50ml of water, shake, let stand to separate layers, separate Water phase, organic phase evaporated to dryness, n-hexane / dichloromethane (3 / 1, v / v) 70ml, recrystallization, obtain α-bromo-3-cyano-4-aminoacetophenone 1.3g, yield 87.25%;

[0027] b: Take 1.3g of α-bromo-3-cyano-4-aminoacetophenone, add 80ml of acetonitrile to dissolve, add 0.46g of KOH, 0.21g of sodium iodide, stir, add 4-amino-4-methylpentane Acid 0.86g, heat, reflux for 12h, stop the reaction, rotary steam, remove acetonitrile, add 80ml of water, add 1mol / L hydrochloric acid to adjust the pH value to 6, ethyl acetate 100ml×3, extract three times, combine the organic phases, concentrate and evaporate to dryness , dichloromethane / cyclohexane (v / v, ...

Embodiment 2

[0043] Embodiment 2 detects the formation of the ELISA kit of cimaterol

[0044] Construct the ELISA kit for detecting cimaterol to include the following components:

[0045] (1) A microtiter plate coated with cimaterol-coupled antigen;

[0046] (2) 6 bottles of cimaterol standard solution, the concentrations are 0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, 8.1 μg / L;

[0047] (3) cimaterol antibody labeled with horseradish peroxidase;

[0048] (4) Substrate chromogenic solution is made up of A liquid and B liquid, and A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0049] (5) The stop solution is 2mol / L sulfuric acid;

[0050] (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;

Embodiment 3

[0051] The detection of cimaterol in embodiment 3 animal tissue and pig urine

[0052] 1. Detection with kit

[0053] Number the corresponding microwells of the samples and standards in sequence, make 2 parallel wells for each sample and standard, and record the positions of the standard wells and sample wells. According to the required amount, dilute the enzyme conjugate concentrate with the enzyme conjugate diluent at a volume ratio of 1:10 (that is, add 1 part of the enzyme conjugate concentrate to 10 parts of the enzyme conjugate diluent, ready for immediate use). Add 50 μl of the standard / sample to the corresponding microwell, then add 50 μl / well of the enzyme conjugate working solution, shake gently to mix, cover the plate with a cover film and place it in a dark environment at 25°C for 30 minutes. Dry the liquid in the wells, add 250 μl / well of washing working solution, wash fully for 4-5 times with an interval of 10 seconds each time, pour off the washing liquid in th...

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Abstract

The invention provides an enzyme linked immunosorbent assay (ELISA) kit for Cimaterol detection. The kit comprises an ELISA plate coated with a coating antigen, a Cimaterol standard substance solution, a Cimaterol antibody, an enzyme conjugate concentrate, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a cleaning solution, wherein the coating antigen is a Cimaterol coupling antigen, an enzyme conjugate is the enzyme-labeled Cimaterol antibody, and the Cimaterol antibody is obtained from an immunogenic immune animal. The invention further discloses a method ofapplying the ELISA kit to Cimaterol detection. The method comprises the steps that first, sample pretreatment is performed; second, the kit is used for detection; and last, a detection result is analyzed. The ELISA kit can be used for detecting the content of Cimaterol in animal tissue and swine urine samples, is easy and convenient to operate, low in cost and high in sensitivity, can be monitored on spot and is suitable for screening of a large quantity 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 cimaterol, which can qualitatively and quantitatively detect the residual amount of cimaterol in animal tissues and pig urine. Background technique [0002] Cimaterol is a β2-type receptor agonist, and the drug has been included in the "Clenbuterol" category stipulated in the "Clenbuterol" Special Rectification Plan issued by the Office of the Food Safety Committee of the State Council (Food Safety Office [2011] No. 14) Table of contents. Unscrupulous farmers add cimaterol to animal drinking water or feed to increase the lean meat percentage of animals. Therefore, for cimaterol, a new type of "lean meat extract", the Ministry of Agriculture clearly prohibits the drug from being used in breeding animal. [0003] At present, the commonly used detection methods include high performance liquid chromatography, liquid ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/94G01N33/535G01N33/543
CPCG01N33/9433G01N33/535G01N33/54306
Inventor 吴小胜何方洋朱亮亮贾芳芳王兆芹万宇平曹东山韩光耀
Owner BEIJING KWINBON BIOTECH
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