Immunoaffinity gel detection column for detecting enrofloxacin and preparation method thereof

A technology for detecting enrofloxacin and gel, applied in chemical instruments and methods, separation methods, measuring devices, etc., can solve problems such as the influence and limitation of test strips, and achieve convenient operation, high selectivity, and simple result judgment Effect

Inactive Publication Date: 2016-03-09
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection of test strips is also susceptible to the influence of the sample matrix, which is limited in the detection of different food samples

Method used

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  • Immunoaffinity gel detection column for detecting enrofloxacin and preparation method thereof
  • Immunoaffinity gel detection column for detecting enrofloxacin and preparation method thereof
  • Immunoaffinity gel detection column for detecting enrofloxacin and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] 1. Purification of enrofloxacin antiserum

[0040] Using ProteinA-Sepharose4B as affinity chromatography medium to purify enrofloxacin antiserum can obtain nearly high-purity specific antibody at one time. The specific operation steps are (1) Equilibration: flush the pipeline with equilibration buffer (0.2mol / L phosphate buffer) and equilibrate the column until the baseline is stable. (2) Sample loading: Dilute the enrofloxacin-specific antiserum in an equal volume with equilibration buffer and load it on the column. (3) Impurity washing: wash with equilibration buffer until the ultraviolet absorption peak of the impurity protein appears, then continue to wash until the baseline is stable. (4) Elution collection: the specific IgG antibody was eluted with 0.1mol / L glycine buffer solution at pH 2.7. When the UV absorption curve shows an upward trend, collect the specific antibody. And quickly use Tris-HCl to neutralize the antibody to neutrality. (5) Capping the colum...

Embodiment 2

[0051] How to use enrofloxacin gel detection column

[0052] 1. Detection steps

[0053] (1) Adding samples: dissolve the enzyme-labeled antigen diluted in a certain proportion with the enrofloxacin sample with PBS solution

[0054] The solution was pre-mixed to 1 mL, passed through the column through the injection port, and the flow rate was controlled to complete in 1 min.

[0055] (2) Wash the column: add 1 mL of PBST from the injection port to wash the column, rinse 7 times for a total of 7 mL, and then wash with 2 mL of PBS to remove the antigen not bound to the antibody and residual Tween20.

[0056] (3) Color development: Add 300 μL of substrate solution from the top, react for 30 seconds, inject air with a syringe plunger, completely discharge the substrate solution, continue color development for 4 minutes, and observe the result.

[0057] 2. Result judgment

[0058] Visually inspect the color of the quality control layer and detection layer of the gel column. The...

Embodiment 3

[0062] Examples of application effects of the present invention

[0063] 1. Sample processing method

[0064] Milk matrix: Milk samples are liquid samples, and the direct dilution method can be used to eliminate the influence of the matrix. Take 1mL of milk in a test tube and directly dilute it 10 times with PBST solution. Gel detection column for detection.

[0065] Tissue matrix: mince 1g of tissue and place in a 50mL centrifuge tube, add 0.4mL NaOH (0.1M), 1.6mL acetonitrile, vortex and mix at high speed for 2min, centrifuge (15°C, 5000r / min, 10min), dilute 10 times with PBST solution, Take 1mL of the diluent and mix it with the enzyme label, and add it to the immunoaffinity gel detection column for detection.

[0066] 2. Effectiveness experiment

[0067] Add enrofloxacin to the negative tissue samples so that the final concentration of enrofloxacin in the sample is 10 μg / kg, 20 μg / kg, 100 μg / kg, add enrofloxacin to the milk sample to make the final concentration of enro...

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Abstract

The invention provides a preparation method of a visualized immunoaffinity gel detection column for rapidly detecting enrofloxacin in food. The method comprises the steps of with sepharose gel as a solid vector, coupling an enrofloxacin antibody with sepharose gel activated by cyanogen bromide to prepare an antibody gum serving as a detection layer; coupling an HRP antibody with the sepharose gel activated by cyanogen bromide to prepare an HRP antibody gum serving as a quality control layer; and putting the HRP antibody gum into 1mL of solid-phase extraction column to prepare the immunoaffinity detection column. The invention develops a novel immunoaffinity gel column detection product for rapidly, qualitatively and semi-quantitatively detecting enrofloxacin residues in food, and the detection limit is 5mu g/L. The immunoaffinity gel column detection product has the following outstanding advantages of high specificity, good sensitivity, simple sample pretreatment, short detection consumed time, high accuracy, simple and convenient operation and no assistance of large-size instruments.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry and residue analysis of small molecular compounds, and relates to immunochemistry, enzymology and analytical chemistry technologies, etc., in particular to the preparation of an enrofloxacin immunoaffinity gel visualization detection column. Background technique [0002] Enrofloxacin, also known as ethyl ciprofloxacin, is the world's first animal-specific quinolone drug. It has the characteristics of broad antibacterial spectrum, strong antibacterial ability, no cross-resistance, and wide distribution in the body. , can be widely used in the prevention and treatment of animal bacterial diseases and mycoplasma infection, and has become one of the most important antibacterial drugs in the livestock and aquaculture industry, and is widely used in the treatment, prevention and growth promotion of animal diseases. In recent years, there are more and more reports of adverse reactions and drug re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558B01D15/10
CPCG01N33/558B01D15/10B01D2221/10
Inventor 生威王硕张燕王俊平胡高爽
Owner TIANJIN UNIV OF SCI & TECH
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