Immunoaffinity gel detection column for detecting furaltadone metabolite

A technology for gel detection and furaltadone, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of test strip influence and limitation, and achieve the effect of convenient operation, high selectivity and elimination of sample matrix influence

Inactive Publication Date: 2016-03-23
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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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 ...

Method used

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  • Immunoaffinity gel detection column for detecting furaltadone metabolite
  • Immunoaffinity gel detection column for detecting furaltadone metabolite
  • Immunoaffinity gel detection column for detecting furaltadone metabolite

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Purification of furaltadone metabolite antiserum

[0039]Using ProteinA-Sepharose4B as the affinity chromatography medium to purify the antiserum of furaltadone metabolite can obtain the specific antibody with high purity at one time. The specific operation steps are as follows: (1) Equilibration: use the prepared Binding buffer to wash the column, and wash the pipeline until the instrument draws a stable baseline, with a flow rate of 1mL / min. (2) Sample loading: dilute the antiserum with an equal volume of Binding buffer and load it on the column, and adjust the flow rate to 0.5mL / min. (3) Impurity washing: continue to wash the column with Binding buffer, the peak of impurity protein will be eluted and flow out, and the antibody IgG will be adsorbed on the ProteinA-SepHarose4B column, continue to wash until the instrument has a stable baseline, and the flow rate is controlled at 1.0ml / min. (4) Elution: Elution buffer was used to elute the column, and the flow rat...

Embodiment 2

[0054] Method for using furaltadone metabolite gel detection column

[0055] 1. Detection steps

[0056] (1) Adding samples: pre-mix the enzyme-labeled antigen diluted in a certain proportion with NPAMOZ standard or sample solution with 1mL LPBS, add to the column from the injection port, and control the flow rate to 1mL / min.

[0057] (2) Column washing: wash the column with 3mL PBST, and then wash the column with 2mL PBS to remove the antigen not bound to the antibody and residual Tween-20.

[0058] (3) Color development: add 300 μL of substrate solution, react for 30 seconds, completely discharge the substrate solution with a syringe, continue color development for 4 minutes, and observe the reaction result.

[0059] 2. Result judgment

[0060] Visually inspect the color of the quality control layer and detection layer of the gel column. The quality control layer is obviously blue, indicating that the detection column can be used normally. Use the detection column to det...

Embodiment 3

[0064] Examples of application effects of the present invention

[0065] 1. Sample processing method

[0066] The traditional derivatization method for detecting nitrofuran metabolites is to derivatize at 37°C for 16 hours. The derivatization time is long, which is not conducive to the rapid detection of nitrofuran metabolites. In this experiment, the derivatization time was optimized. Select negative samples such as shrimp, squid, chicken, pork, chicken liver, etc. as samples, add a certain amount of AMOZ standard substance, and first perform derivatization treatment: weigh 5.0g of the sample into a 50mL centrifuge tube, add 10mL of deionized water and 2mL of 1mol / L HCl solution, after mixing, add 300μL of o-nitrobenzaldehyde solution (50mmol / L), after mixing, place in a 37°C thermostat and a 60°C water bath for 60min, 120min, and 180min respectively , 16h; further extraction treatment: take the derivatized sample out and return to room temperature, add 5mL of PBS and 1.6mL ...

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Abstract

The invention provides a preparation method of an immunoaffinity gel detection column capable of visually and rapidly detecting furaltadone metabolite (AMOZ) in food. The method comprises the steps that agarose gel is used as a solid-phase carrier, an AMOZ antibody and the agarose gel which is activated through cyanogen bromide are coupled to prepare antibody gel used as a detection layer, an HRP antibody and the agarose gel which is activated through the cyanogen bromide are coupled to prepare HRP antibody gel used as a quality control layer, the product is placed into 1mL of solid phase extraction column, and the immunoaffinity gel detection column is prepared. The novel immunoaffinity gel detection product which detects AMOZ residues in food rapidly, qualitatively and semiquantitatively is developed, and the detection limit of the AMOZ in animal derived food is 3 micrograms/kg. The immunoaffinity gel detection column has the outstanding advantages that specificity is high, and sensitivity is good; sample pretreatment is simple; detection time is short and accuracy is high; operation is easy and convenient, and assisting of large instruments is not needed.

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, and in particular to the preparation of a furaltadone metabolite immunoaffinity gel visual detection column. Background technique [0002] Nitrofurans (Nitrofurans) is a broad-spectrum antibiotic drug, mainly including Furaltadone (Furaltadone), Furazolidone (Furazolidone), Nitrofurantoin (Furantoin), and Nitrofurazone (Nitrofurazone). antibacterial and bactericidal effect. Mainly against a variety of gram-positive bacteria (staphylococcus, streptococcus, clostridium and corynebacteria) and gram-negative bacteria (Escherichia coli, histomonas and amoeba dysenteriae), and against some protozoa, Fungi have a role to play. Nitrofuran antibiotics also have growth-promoting effects. It is widely used in animal husbandry and aquaculture because of its low price ...

Claims

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Application Information

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IPC IPC(8): G01N33/559G01N33/535
CPCG01N33/559G01N33/535
Inventor 生威王硕张燕王俊平胡高爽
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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