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Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof

A nitrofuracil and metabolite technology, applied in the field of enzyme-linked immunosorbent assay kits, can solve problems such as being unsuitable for on-site monitoring and screening of a large number of samples, complex instruments and equipment, and tedious processes, so as to reduce the lower limit of sample detection, overcome technical problems, and improve The effect of detection sensitivity

Active Publication Date: 2007-08-08
BEIJING WANGER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The most common methods used to detect nitrofurazone metabolites are LC-UV, LC-MS and LC-MS / MS, which are not suitable for on-site monitoring and screening of a large number of samples due to complex instruments and cumbersome processes

Method used

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  • Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 Preparation of kit components

[0069] 1. Antigen Synthesis

[0070] a. Synthesis of Furacilin Metabolite Derivative Hapten

[0071] The nitrofurazone metabolite is reacted with m-nitrobenzaldehyde to obtain a nitrofurazone metabolite derivative, and then the nitro group of the nitrofurazone metabolite derivative is reduced to an amino group through a chemical reaction to finally obtain a nitrofurazone metabolite derivative hapten.

[0072] Preparation process of nitrofurazone metabolite derivative hapten:

[0073] Take nitrofurazone metabolite 5g and dissolve in 20ml pure water (I liquid), and dissolve 10g m-nitrobenzaldehyde in 100ml ethanol (II liquid). Mix and heat liquid I and liquid II at 60°C overnight. After centrifugation, washing and drying, nitrofurazone metabolite derivatives are obtained.

[0074] Dissolve 5 g of nitrofurazone metabolite derivatives in 100 ml of pure water, adjust the pH value to PH1.0, add 2 g of zinc powder, heat to 60 ° ...

Embodiment 2

[0099] Example 2 Construction of an enzyme-linked immunosorbent assay kit for detecting nitrofurazone metabolites

[0100] An enzyme-linked immunosorbent assay kit for detecting nitrofurazone metabolites was set up to include the following components:

[0101] (1) Enzyme plate coated with anti-nitrofurazone metabolite antigen;

[0102] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0103] (3) Furacilin metabolite monoclonal antibody working solution;

[0104] (4) 6 bottles of nitrofurazone metabolite standard solution, the concentrations were 0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, 8.1 μg / L;

[0105] (5) The substrate chromogenic solution is composed of A liquid and B liquid, the substrate chromogenic liquid A liquid is carbamide peroxide, and the substrate chromogenic liquid B liquid is tetramethylbenzidine;

[0106] (6) The stop solution is 2mol / L hydrochloric acid;

[0107] (7) The concentrated washing solution is 0.01M, pH7.4, phosphat...

Embodiment 3

[0109] Example 3 Detection of Furacilin Metabolite Residues in Samples

[0110] 1. Sample pretreatment

[0111] Animal tissues (chicken, pork, beef, fish, shrimp)

[0112] Take 1±0.05g tissue sample homogenate, add 8ml distilled water, 1ml 1M HCl and 100μl 10mM 2-nitrobenzaldehyde, shake well; incubate overnight at 37°C (about 16h); add 5ml 0.1M K 2 HPO 4 , 0.4ml 1M NaOH and 5ml ethyl acetate, shake vigorously for 30s; centrifuge at room temperature (20~25℃ / 68-77) over 3000g for 10min; take 3ml ethyl acetate into another container and dry it with nitrogen at 50℃ Or evaporate to dryness with a rotary evaporator; dissolve the dry matter with 1ml of n-hexane, mix well with 1.5ml of the diluted complex solution; centrifuge at room temperature (20~25℃ / 68~77) over 3000g for 10min; use 50μl of the lower layer liquid for analysis.

[0113] 2. Detection with kit

[0114] Add 50 μl of a series of standard solutions or sample solutions to the microwells of the microtiter plate coa...

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Abstract

The invention provides a detecting Furacilin metabolite ELISA kit, which contains: an ELISA plate covered by the original coated, enzyme marker, Furacilin metabolite specific antibody or Furacilin metabolite derivative antibody, Furacilin metabolite standard solution or Furacilin metabolite derivative standard solution, the substrate color solution, the termination solution, the condensed washing solution, the condensed complex solution. The invention also provides a method to apply the above ELISA kit detecting Furacilin metabolite, which includes steps: firstly, sample pre-treatment, and then using the kit for testing, and finally analyzing testing results. The invention is to provide the Furacilin metabolite residues in the ELISA kit for detection of animal derived foods, such as chicken, pork, fish, shrimp, milk, honey, egg, and other samples, and the detection method is simple, low cost, high sensitivity, and it can monitor on the scene and suitable for screening large number of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunoimmunoassay kit for detecting residues of nitrofurazone metabolites in samples such as chicken, pork, fish and shrimp, milk, honey and eggs in animal-derived foods and a detection method using the kit. 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 mutation in experimental animals, so these drugs are prohibited from being used in treatment and feed. Furazone was banned in 1995. [0003] The most common methods used to detect nitrofurazone metabolites are LC-UV, LC-MS and LC-MS / MS, which are not suitable for on-site monitoring and screening of a large number of samples due to complex instruments and cumber...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/577G01N33/566G01N33/535
CPCG01N33/9446G01N33/53
Inventor 沈建忠万宇平何方洋冯才伟刘玉梅汪善良陈炜琳冯才茂吴小平赵正苗刘福林丁双阳张素霞江海洋
Owner BEIJING WANGER BIOTECH
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