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Enzyme-linked immunoassay kit and method for furaltadone metabolite detection

A technology of furaltadone and its metabolites, applied in the field of enzyme-linked immunoassay, can solve cancer and other problems

Active Publication Date: 2013-04-03
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the course of long-term experimental research, it was found that both nitrofuran drugs and metabolites can cause cancer and gene mutations in experimental animals, which is why these drugs are prohibited from being used in treatment and feed

Method used

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  • Enzyme-linked immunoassay kit and method for furaltadone metabolite detection
  • Enzyme-linked immunoassay kit and method for furaltadone metabolite detection
  • Enzyme-linked immunoassay kit and method for furaltadone metabolite detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Preparation of antigen, antibody and enzyme-labeled anti-antibody

[0042] 1. Preparation of furaltadone metabolite hapten

[0043] Add the mixture of 2.01g furaltadone metabolite (AMOZ) and 20ml DMF slowly at room temperature to 50-100ml DMF solution of 2.68-5.36g terephthalaldehyde, and react at room temperature to 60°C for 2-4 hours, the solvent was removed, and purified by column chromatography to obtain light yellow AMOZ derivatives.

[0044] 2. Synthesis of Immunogen

[0045] (1) Dissolve 14 mg of furaltadone metabolite hapten in 1 ml of DMF to obtain solution 1.

[0046] (2) Dissolve 40 mg of BSA in 6 ml of water to obtain solution 2.

[0047] (3) Add solution 1 dropwise to solution 2 to obtain solution 3, and react at room temperature for 24 hours.

[0048] (4) Take NaBH 4 14mg was dissolved in 0.2ml 0.1M NaOH and added to solution 3, and reacted at 4°C for 2h.

[0049] (5) Dialyze with 0.01mol / l PBS at 4°C for 3 days and change the dialysate 3 ...

Embodiment 2

[0070] Example 2: The composition of the components of the furaltadone metabolite ELISA kit

[0071] Set up furaltadone metabolite ELISA kit, including the following components:

[0072] (1) A microtiter plate coated with a coating source.

[0073] (2) Enzyme-labeled anti-antibody: horseradish peroxidase-goat anti-mouse anti-antibody.

[0074] (3) Working solution of monoclonal antibody to furaltadone metabolite.

[0075] (4) Standard solution: The standard solution was prepared by gradient dilution method, and 6 bottles of series standard products were obtained, the concentrations were 0 μg / L, 0.05 μg / L, 0.15 μg / L, 0.45 μg / L, 1.35 μg / L, 4.05 μg / L, and high concentration standard 100μg / L, 1mL / bottle.

[0076] (5) The substrate chromogenic solution A is a carbamide peroxide solution, and the substrate chromogenic solution B is a tetramethylbenzidine solution.

[0077] (6) The stop solution is 1-2 mol / L sulfuric acid solution.

[0078] (7) The concentrated washing solution ...

Embodiment 3

[0081] Example 3: Detection of furaltadone metabolites in samples

[0082] 1. Pretreatment of samples

[0083] (1) Pretreatment method of tissue (muscle, liver and aquatic products) samples

[0084] Weigh 1.0 g of homogeneous substance, add 4 ml of deionized water, 0.5 ml of 1M hydrochloric acid solution (weigh 8.3 ml of concentrated hydrochloric acid and add deionized water to make up to 100 ml) and 100 μl of derivatization reagent (into a solution containing 2-nitrobenzaldehyde Add 10ml of methanol to the reagent bottle to dissolve and mix (concentration is 10mM)), shake fully with a shaker for 2min; incubate overnight at 37°C (about 16h); add 5ml of 0.1M dipotassium hydrogen phosphate solution (weigh 22.8g of trihydrate Dipotassium hydrogen phosphate plus 1L deionized water to dissolve and mix), 0.4ml 1M sodium hydroxide solution (weigh 4.0g sodium hydroxide and 100ml deionized water to dissolve and mix), and 5ml ethyl acetate, shake vigorously for 30s with a shaker ; Abo...

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Abstract

The present invention provides an enzyme-linked immunoassay kit and a method for furaltadone metabolite detection. The enzyme-linked immunoassay kit comprises an enzyme label plate coated with coating antigen, specific furaltadone metabolite antibody, an enzyme marker, furaltadone metabolite standard solutions, a substrate coloration solution, a termination solution, a concentration washing solution, a concentration reconstituted solution, and 2-nitrobenzaldehyde. The present invention further provides a furaltadone metabolite enzyme-linked immunoassay kit detection method, which mainly comprises: carrying out a sample pretreatment, adopting the kit to detect, and finally analyzing a detection result. With the detection kit, furaltadone metabolite drugs in animal tissues, aquatic products and honey can be rapidly detected, characteristics of simple and rapid operation, high accuracy, high sensitivity, low cost and the like are provided, and the kit and the method are applicable for screening and on-site monitoring of a large number of samples.

Description

technical field [0001] The invention relates to an ELISA detection technology, in particular to an ELISA kit and a detection method for detecting furaltadone metabolites in animal tissues (muscle, liver), aquatic products and honey. Background technique [0002] Nitrofuran drugs have been widely used as growth-promoting additives for poultry, aquatic products and pigs because of their excellent antibacterial and pharmacokinetic properties. However, in the course of long-term experimental research, it was found that both nitrofuran drugs and metabolites can cause cancer and gene mutations in experimental animals, which is why these drugs are prohibited from being used in treatment and feed. [0003] Since the prototype drug of nitrofuran is rapidly metabolized in the body, it cannot be detected, but its metabolites are quite stable due to the combination with proteins, so when analyzing the residues of such drugs, it is often necessary to analyze the metabolized products, and...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 何方洋孙震冯静冯才茂刘琳朱亮亮刘玉梅石洁
Owner BEIJING KWINBON BIOTECH
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