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Elisa kit for detecting cephalo-type medicine and application thereof

An enzyme-linked immunosorbent reagent, cephalosporin technology, applied to the detection of cephalosporin drug residues in fish, pork, shrimp tissue and milk samples, chicken field, can solve complex instruments and equipment, not suitable for on-site monitoring screening, processing and determination Complicated operation and other issues

Inactive Publication Date: 2009-11-04
BEIJING WANGER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the cumbersome and high cost of the sample pretreatment and measurement operations of the currently used detection and analysis methods, the promotion and application are limited.
[0003] Conventional detection methods for cephalosporin residues mainly include high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), paper chromatography, etc. High skill requirements for personnel, not suitable for on-site monitoring and screening of a large number of samples

Method used

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  • Elisa kit for detecting cephalo-type medicine and application thereof
  • Elisa kit for detecting cephalo-type medicine and application thereof
  • Elisa kit for detecting cephalo-type medicine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The preparation of embodiment 1 kit components

[0075] 1. Antigen Synthesis

[0076] a. Immunogen synthesis

[0077] Cephalosporin hapten synthesis (synthetic route such as figure 2 )

[0078] Through ceftiofur (structural formula such as figure 1 ) and the ceftiofur n-caproate hapten that 6-amino n-caproic acid reacts to obtain, the concrete reaction steps are as follows:

[0079] (1) Dissolve 10 g of ceftiofur in 50 ml of DMSO, add 1 ml of isobutyl chloroformate and stir at room temperature overnight, then add 6-amino-n-caproic acid to adjust the pH to 8.5, and raise the temperature to 45°C for 12 hours.

[0080] (2) Evaporate the reaction product to dryness with a rotary evaporator, dissolve it with 100ml of 1mol / L sodium hydroxide, adjust the pH value to 3 with 2mol / L hydrochloric acid, and centrifuge.

[0081] (3) Take the precipitate, wash it 4-5 times with 0.1mol / L hydrochloric acid, dissolve it with a small amount of ethyl acetate, and then add 2ml of ace...

Embodiment 2

[0115] Embodiment 2 detects the formation of the ELISA kit of cephalosporins

[0116] An enzyme-linked immunosorbent assay kit for detecting cephalosporins was set up to include the following components:

[0117] (1) Enzyme plates coated with cephalosporin-conjugated antigens;

[0118] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0119] (3) Working solution of cephalosporin monoclonal antibody;

[0120] (4) 6 bottles of ceftiofur standard solution, the concentrations are 0 μg / L, 0.5 μg / L, 1.5 μg / L, 4.5 μg / L, 13.5 μg / L, 40.5 μg / L;

[0121] (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 tetramethylbenzidine;

[0122] (6) The stop solution is 2mol / L hydrochloric acid;

[0123] (7) The concentrated washing solution is a phosphate buffer solution with a pH value of 7.5-8.2, 0.8-1.2% Tween-20, 0.01-0.02‰ thimeros...

Embodiment 3

[0125] The detection of cephalosporins in the sample of embodiment 3

[0126] 1. Sample pretreatment

[0127] a) Animal tissues (chicken, pork, fish, shrimp, etc.)

[0128] Weigh 2.0±0.05g of homogeneous material into a 50ml polystyrene centrifuge tube, add 8ml of extraction working solution, shake for 5min, and centrifuge for 5min if it is above 3000g; take 1ml of supernatant into a 10ml clean glass test tube, and store at 50~60℃ Blow dry in a water bath under nitrogen flow; add 1ml of n-hexane, vortex for 30s with a vortex to dissolve the dry residue, then add 1ml of complex solution, vortex for 30s with a vortex, centrifuge at 3000g or more for 5min; remove the upper n-hexane phase, and take the lower layer 50 μl of the aqueous phase was used for analysis.

[0129] b) milk sample

[0130] Take 100 μl of fresh milk in a 2ml centrifuge tube, add 900 μl of reconstitution working solution, mix well, and take 50 μl for analysis.

[0131] 2. Detection with kit

[0132] Add 5...

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Abstract

The invention provides an elisa kit for detecting cephalo-type medicine, containing: an ELIAS plate peridium of which is provided with peridium source, an enzyme label, cephalo-type medicine specific antibody working solution (contained when the peridium on the ELIAS plate is antigen and the enzyme label is enzyme label anti antibody or when the peridium on the ELIAS plate is anti antibody and theenzyme label is enzyme label antigen), ceftiofur standard solution, substrate developer, stop solution, concentrated cleaning solution and concentrated complex solution. The invention also disclosesa method applying the elisa kit for detecting cephalo-type medicine, including: firstly sample preprocessing is carried out, then the elisa kit is used for detection, and finally the detection result is analyzed. The elisa kit provided by the invention can be applied to detecting residual quantity of cephalo-type medicine in samples such as animal tissue (chicken, pork, fish and shrimp) and milk, etc, the operation is easy, the cost is low, the sensitivity is high, and the elisa kit is applied to on-site supervision and numerous sample screening.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting cephalosporins, which is particularly suitable for the detection of cephalosporin residues in chicken, pork, fish, shrimp tissue and milk samples. technical background [0002] Cephalosporins are broad-spectrum antibiotics with excellent antibacterial activity and pharmacokinetic characteristics, and are effective against many Gram-positive and Gram-negative bacteria and β-lactamase-producing bacteria. Cephalosporins kill bacteria by breaking down their cell walls. Due to the cumbersome and high cost of the sample pretreatment and measurement operations of the currently used detection and analysis methods, the popularization and application are limited. [0003] Conventional detection methods for cephalosporin residues mainly include high-performance liquid chromatography (HPLC), liquid chromatography-ta...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/566G01N33/531G01N33/577
Inventor 何方洋张建军冯才伟万宇平冯静冯才茂崔海峰徐念琴赵正苗李勇刘福林刘平朱亮亮陈炜琳罗贵昆
Owner BEIJING WANGER BIOTECH
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