Aflatoxin immunoaffinity column as well as preparation method and application thereof

An aflatoxin and immunophilic technology, applied in chemical instruments and methods, separation methods, preparation of samples for testing, etc., can solve the problems affecting the purification efficiency of aflatoxin, affecting antibody affinity, and complicated steps, so as to improve the capture efficiency. The effect of improving capacity, purification efficiency, and antibody binding ability

Active Publication Date: 2014-10-29
山东美正生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the need for hydrazide chemical modification of the antibody, the steps are cumbersome, and the chemical modification of the antibody may affect the affinity of the antibody itself, thereby affecting the purification efficiency of aflatoxin

Method used

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  • Aflatoxin immunoaffinity column as well as preparation method and application thereof
  • Aflatoxin immunoaffinity column as well as preparation method and application thereof
  • Aflatoxin immunoaffinity column as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Preparation of aflatoxin immunoaffinity purification column

Embodiment approach

[0042] A preferred embodiment of the present invention for preparing aflatoxin immunoaffinity purification column is as follows:

[0043] 1. Agarose gel activation

[0044] Take 2% agarose gel sepharose 2B and wash it with 20 times the volume of distilled water to wash away the remaining ethanol. Filter out the water with a funnel. Weigh 5 g of the wet gel after filtering the water, add 7.5 ml of 0.8M NaOH, 2 ml of 30% epichlorohydrin, 2 mg / ml of sodium borohydride NaBH4, 5 ml, and shake at 25°C React for 8 hours.

[0045] After the reaction, it was washed with 50 ml of distilled water to remove the epichlorohydrin mixed in the gel.

[0046] 2. Coupling of protein G and activated agarose gel

[0047] Take 1 gram of activated agarose gel and use coupling buffer (0.1M NaHCO 3 , 0.8M NaCl, PH8.9) wash 3 times. Add 620ml of 2mg / ml protein and couple at room temperature for 4 hours.

[0048] The coupled agarose carrier was washed 3 times with 20 mM, pH 7.4 phosphate buffer PBS. Sepharose...

Embodiment 2

[0060] Example 2: Purification and detection of aflatoxin in corn using aflatoxin immunoaffinity column

[0061] 1. Corn sample processing:

[0062] Crush the corn sample and pass through a 2mm sieve;

[0063] —— Add 25g of sieved and crushed sample to 125mL of 2× loading buffer solution and mix;

[0064] ——High-speed homogenization for 1 min (or vigorous shaking with a shaker for 20 min), and filter with fast qualitative filter paper;

[0065] ——Dilute 10mL filtrate with 10mL distilled water, and filter with microfiber filter paper;

[0066] ——Take 10mL and load the sample for testing.

[0067] 2. Equilibrate the aflatoxin immunoaffinity purification column for 30 minutes at room temperature.

[0068] 3. Take out the immunoaffinity column, connect the injection port to the syringe barrel, and connect the syringe to the air control operating rack.

[0069] 4. Wash the affinity column with deionized water 3 times, 10 ml each time, adjust the pressure of the air pump of the stoma operation fr...

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Abstract

The invention relates to an aflatoxin immunoaffinity column as well as a preparation method and application of the aflatoxin immunoaffinity column. The preparation method of the immunoaffinity purification column comprises the following steps: coupling protein G onto an agarose gel carrier, and then coupling an antibody against aflatoxin with the protein G on agarose; crosslinking the aflatoxin antibody-protein G-agarose gel carrier by utilizing a cross-linking agent, and then filling the affinity column. The immunoaffinity column is mainly used for purifying aflatoxin in food, forage, milk, blood samples and other various samples so as to facilitate HPLC detection and fluorescence detection for aflatoxin in the samples in the later stage.

Description

Technical field: [0001] The invention relates to an immunoaffinity column for aflatoxin and its preparation method and application. It belongs to the field of food safety testing. Background technique: [0002] Aflatoxin (AFT) is a class of toxic secondary metabolites mainly produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. These fungi have been found to exist in large quantities in food for human consumption in different parts of the world. Aflatoxin contamination has caused serious food safety problems. Aflatoxins are a class of extremely toxic substances with strong carcinogenicity and strong immunosuppressive properties. In 1993, AFT was designated as a Class I carcinogen by the Cancer Research Organization of the World Health Organization (WHO). Aflatoxins are widely distributed in moldy grains and their products, especially peanuts, peanut oil, corn and their products, milk and dairy products. Moldy feeds also contain more aflatoxins. So far, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/38B01J20/281G01N1/34
Inventor 王莹
Owner 山东美正生物科技有限公司
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