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Immunoaffinity gel detection column for detecting fumonisins and preparation method thereof

A fumonisin and gel detection technology, which is applied in measuring devices, biological testing, material inspection products, etc., can solve the problems of cumbersome sample processing steps, unsuitable for promotion and use, and low recovery rate, so as to eliminate the influence of sample matrix , Detection of product pre-treatment method is simple, easy to operate the effect

Inactive Publication Date: 2016-03-23
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High performance liquid chromatography (HPLC) method has high accuracy and is the most widely used detection method at present, but there are some disadvantages in this method, such as the required instruments are many and expensive, and professional personnel are required to operate, and the sample processing steps are cumbersome, Derivation is required, and the recovery rate is low, so it is not suitable for widespread promotion and use

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Purification of fumonisin antiserum

[0039]Using ProteinA-Sepharose4B as affinity chromatography medium to purify fumonisin antiserum can obtain near-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 fumonisin-specific antiserum to an equal volume with an equilibration buffer and apply to 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 0.1 mol / 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 column: After the an...

Embodiment 2

[0051] Fumonisin gel detection column usage method

[0052] 1. Detection steps

[0053] (1) Adding samples: add 1 mL of fumonisin sample and enzyme-labeled antigen mixture, control the flow rate with a syringe, and push out the liquid within 1 min;

[0054] (2) Wash the column: In order to remove the residue of the conjugate, rinse with PBST three times and wash once with PBS.

[0055] (3) Color development: Add 300 μL of substrate solution, 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.

[0056] 2. Result judgment

[0057] 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 detect different concentrations of fumonisins (0μg / L, 20.0μg / L, 30.0μg / L, and 40.0μg / L), and the lowest conce...

Embodiment 3

[0061] Examples of application effects of the present invention

[0062] 1. Sample processing method

[0063] (1) Corn flour: Accurately weigh 1.0g corn flour, add 400μg / kg FB1 standard substance, add 2mL LPBS, vortex and shake for 5min, centrifuge (4°C, 5000r / min, 5min), take the supernatant and wash with PBS (pH7. 2) Dilute 5 times to eliminate the influence of the matrix, then take 1mL of the diluted solution and mix it with FB1-HRP, and add it to an immunoaffinity gel detection column for detection.

[0064] (2) Cereal milk: Take 10mL of cereal milk in a 50mL centrifuge tube, add 4mg / L of FB1 standard substance, centrifuge (4°C, 5000r / min, 20min), take the upper liquid and dilute it 10 times with PBS (pH7.2), Then take 1mL of the diluent and mix it with FB1-HRP, and add it to the immunoaffinity gel detection column for detection.

[0065] (3) Liquor: Take 200 μL of liquor, add 200 μg / L of FB1 standard substance, dilute 5 times with PBS (pH7.2), then take 1 mL of liquor d...

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Abstract

The invention provides a preparation method of an immunoaffinity gel detection column capable of visually and rapidly detecting fumonisins in food. The method comprises the steps that agarose gel is used as a solid-phase carrier, a fumonisins 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 the fumonisins in the food rapidly, qualitatively and semiquantitatively is developed, and the detection limit is 40 micrograms / L. 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 techniques, and in particular to the preparation of a fumonisin immunoaffinity gel visual detection column. Background technique [0002] Fumonisins (FBs) are one of mycotoxins, which are water-soluble metabolites produced by Fusarium moniliforme Sheld at specific temperature and humidity, and are mostly found in food and feed. Its structure contains a class of different polyhydric alcohols and tricarboxylic acid, which make it biologically toxic and stable to heat. It is relatively stable in most grain processing processes. It can still maintain its toxicity, which is more harmful to humans and animal husbandry. Its role in humans and animals is relatively complex, it can inhibit the synthesis of N-fatty acyl sphingosine synthase, thereby blocking the synthesis of sphing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCG01N33/5304
Inventor 生威王硕张燕王俊平胡高爽
Owner TIANJIN UNIV OF SCI & TECH