Enzyme-linked immunoassay kit and method for nitrofural metabolite detection

A technology of nitrofurazone and metabolites, which can be used in the field of enzyme-linked immunoassay detection and can solve problems such as carcinogenesis

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 caus...

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Synthesis of antigens, antibodies and enzyme markers

[0042] 1. Synthesis of Furacilin Metabolite Hapten

[0043] The mixture of 0.75g nitrofurazone metabolite (SEM) and 20ml DMF was slowly added dropwise at room temperature to 50-100ml DMF solution of 2.68-5.36g terephthalaldehyde, and reacted at room temperature to 60°C for 2-4 hours after the addition was completed. The solvent was removed and purified by column chromatography to obtain a pale yellow SEM derivative.

[0044] 2. Synthesis of Immunogen

[0045] (1) Take 10 mg of nitrofurazone metabolite hapten and dissolve it 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 of 0.1M NaOH and added to solution 3, and reacted at 4°C for 2h.

[0049] (5) Dialyze with 0.01mol / l...

Embodiment 2

[0070] Example 2: The composition of each component of the nitrofurazone metabolite ELISA kit

[0071] Set up the nitrofurazone 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 nitrofurazone metabolite monoclonal antibody.

[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 2mol / L sulfuric acid solution.

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

Embodiment 3

[0081] Example three: detection of nitrofurazone metabolites in samples

[0082] 1. Pretreatment of samples

[0083] Pretreatment methods for tissue and aquatic samples

[0084] Weigh 1.0 g of the homogenized sample, 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 the volume to 100 ml) and 100 μl of derivatization reagent (injected with 2-nitro Add 10ml of methanol to the reagent bottle of benzaldehyde 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 22.8 g dipotassium hydrogen phosphate trihydrate, add 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 shake for 30s; over 3000g, centrifuge at room temp...

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Abstract

The present invention provides an enzyme-linked immunoassay kit and a method for nitrofural metabolite detection. The enzyme-linked immunoassay kit comprises an enzyme label plate coated with coating antigen, specific nitrofural metabolite antibody, an enzyme marker, nitrofural 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 nitrofural 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, nitrofural metabolite drugs in animal tissues and aquatic products 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 nitrofurazone metabolites in animal tissues (muscle, liver) and aquatic products. 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 the ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/531
Inventor 万宇平冯才茂冯静余厚美朱亮亮杜亚菲刘琳付军权
Owner BEIJING KWINBON BIOTECH
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