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Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof

An enzyme-linked immunosorbent reagent, chloramphenicol technology, applied in the preparation of test samples, measuring devices, instruments, etc., can solve the problems of slow analysis speed, high degree of instrumentation, and failure to meet the detection limit requirements of detection methods

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

AI Technical Summary

Problems solved by technology

[0003] At present, the most commonly used methods for detecting chloramphenicol are mainly gas chromatography, liquid chromatography and chromatography-mass spectrometry. Although these methods have the advantages of high sensitivity, accurate results, good repeatability, and few false positives, there are still some problems Disadvantages, such as complicated sample pretreatment process, high degree of instrumentation and high price, and slow analysis speed, have gradually failed to meet the detection limit requirements of developed countries and food processing enterprises.

Method used

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  • Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof
  • Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof
  • Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] The preparation of embodiment 1 kit components

[0072] 1. Antigen Synthesis

[0073] a. Chloramphenicol hapten synthesis

[0074] Dissolve chloramphenicol in methanol, add 5% palladium carbon catalyst (Pd / C), feed hydrogen, keep a certain pressure, react at room temperature for 2 hours, remove Pd / C by filtration, evaporate the solvent to obtain a light yellow viscous liquid, namely is a chloramphenicol hapten; wherein the molar ratio of Pd / C to chloramphenicol is 1:10.

[0075] b. Immunogen synthesis

[0076] The hapten of chloramphenicol was coupled with bovine serum albumin by the glutaraldehyde method to obtain the immunogen.

[0077] Immunogen preparation process:

[0078] Take 30 mg of chloramphenicol hapten and dissolve it with 1.5 ml of water to obtain liquid (I); take 10 μl of 50% glutaraldehyde (GA) and add it to (I), stir and react at room temperature for 18 hours to obtain liquid (II); Dilute 100mg of bovine serum albumin (BSA) with 1.5ml of water and a...

Embodiment 2

[0095] Embodiment 2 detects the formation of the ELISA kit of chloramphenicol

[0096] Set up the ELISA kit for detecting chloramphenicol so that it includes the following components:

[0097] (1) A microtiter plate coated with a chloramphenicol-coupled antigen;

[0098] (2) 6 bottles of chloramphenicol standard solution, the concentrations are 0 μg / L, 0.025 μg / L, 0.075 μg / L, 0.3 μg / L, 1.2 μg / L, 4.8 μg / L;

[0099] (3) 1 bottle of chloramphenicol high-concentration standard solution, the concentration is 100 μg / L;

[0100] (4) working solution of chloramphenicol monoclonal antibody labeled with horseradish peroxidase;

[0101] (5) Substrate chromogenic solution is made up of A liquid and B liquid, and A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0102] (6) The stop solution is 2mol / L sulfuric acid;

[0103] (7) The concentrated washing solution has a pH value of 7.1 to 7.5, contains 0.8% to 1.2% Tween-20, 0.01‰ to 0.03‰ thimerosal preservative, an...

Embodiment 3

[0105] The detection of chloramphenicol in embodiment 3 samples

[0106] 1. Sample pretreatment

[0107] (1) Tissue (chicken / liver, pork / liver, shrimp, fish, etc.) pretreatment method

[0108] After homogenizing the tissue sample with a homogenizer, weigh 3.0±0.05g of the homogenate into a 50ml polystyrene centrifuge tube, add 6ml of ethyl acetate, shake with an oscillator for 5min, 4000rpm, room temperature (20-25℃ / 68 -77°F) for 5min; pipette 4ml of the upper organic phase (equivalent to about 2g of sample) into a 10ml clean glass test tube, and dry it in a water bath at 50-60°C under nitrogen flow; add 1ml of n-hexane and vortex with a vortex After stirring for 1 min, add 1 ml of reconstituted working solution, vortex for 15 s at 4000 rpm, and centrifuge at room temperature (20-25°C / 68-77°F) for 5 min; remove the upper organic phase, and take 50 μl of the lower layer for analysis.

[0109] (2) Pretreatment method of cooked food

[0110] After homogenizing the sample with ...

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Abstract

The present invention provides an enzyme-linked immunoassay kit for chloramphenicol detection. The enzyme-linked immunoassay kit comprises an enzyme label plate coated with coating antigen, an enzyme marker, a specific chloramphenicol antibody working solution (when the enzyme label plate is coated with antigen and the enzyme marker is enzyme-labeled anti-antibody or the enzyme label plate is coated with anti-antibody and the enzyme marker is enzyme-labeled antigen, the kit contains the specific chloramphenicol antibody working solution), chloramphenicol standard solutions, a substrate coloration solution, a termination solution, a concentration washing solution and a concentration reconstituted solution. The present invention further discloses a method for detecting chloramphenicol by using the enzyme-linked immunoassay kit. The method comprises: carrying out a sample pretreatment, adopting the kit to detect, and finally analyzing a detection result. With the enzyme-linked immunoassay kit, chloramphenicol residue in animal tissues (muscles, livers and the like), delicatessen, aquatic products, royal jelly, urine, feeds, milk and other samples can be detected, characteristics of simple operation, low cost and high sensitivity are provided, and the kit and the method can be applicable for screening and on-site monitoring of a large number of samples.

Description

technical field [0001] The present invention relates to ELISA detection technology, in particular to an ELISA kit for detecting chloramphenicol, which is particularly suitable for animal tissues (muscle, liver, etc.), cooked food, aquatic products, royal jelly, urine, feed and Detection of chloramphenicol residues in milk and other samples. technical background [0002] Chloramphenicol (Chloramphenicol, CAP) is a broad-spectrum antibiotic discovered in the middle of the 20th century. It has a good inhibitory effect on Gram-negative bacteria and positive bacteria. For a period of time, it was used as a therapeutic drug for bacterial diseases in human and animal clinical applications, and was widely used in animal husbandry production as a feed additive. However, in use, it is found that chloramphenicol is toxic, and residues in animal foods will cause toxic effects on human health through animal foods such as meat, eggs, and milk. Therefore, my country's Ministry of Agricul...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N1/28
Inventor 何方洋吴鹏段盈盈浦小容李勇崔廷婷陈炜玲齐向武
Owner BEIJING KWINBON BIOTECH
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