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Composite affinity column for purifying 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone

A technology of deoxynivalenol and zearalenone, applied in chemical instruments and methods, other chemical processes, material separation, etc., can solve the problems of false positives, low sensitivity, false negatives, etc., and achieve stable performance Effect

Inactive Publication Date: 2015-06-17
INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thin-layer chromatography requires contact with a large number of standards, which is not conducive to the health of the experimenter, and the sensitivity is very low
ELISA is only suitable for qualitative detection, and it is prone to false positives and false negatives

Method used

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  • Composite affinity column for purifying 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone
  • Composite affinity column for purifying 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone
  • Composite affinity column for purifying 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0054] Preparation of monoclonal antibodies against 3-acetyldeoxynivalenol (3-AcDON), aflatoxin (Aflatoxin), ochratoxin (OTA) and zearalenone (ZON):

[0055] Animal immunization: The immunized animals are about 6-8 weeks old, female BALB / c mice. Four immunogens:

[0056] 3-AcDON-BSA, Aflatoxin B 1 -BSA, OTA-BSA and ZON-BSA were immunized with 5 mice each. Take an appropriate amount of immunogen (100 μg / rat) and add an equal amount of Freund's complete adjuvant to make an emulsifier for immunization, and then change the adjuvant to incomplete adjuvant for 6 times of immunization, with an interval of 2 weeks between each time. Except for the first multi-point subcutaneous injection on the back of the neck, the rest were intraperitoneal injections.

[0057] Cell fusion: the splenocytes and hybridoma cells of the above-mentioned immunized mice were subjected to a cell fusion test at a ratio of 10:1.

[0058] Hybridoma cell cloning: The hybridoma cells were screened by the limi...

example 2

[0060] Preparation of Composite Immunoaffinity Column for 3-Acetyldeoxynivalenol, Aflatoxin, Ochratoxin and Zearalenone

[0061] 1. Substrate Preparation

[0062] Weigh required 1 g of Sepharose matrix powder (each gram of freeze-dried matrix powder can form a swelling matrix with a final volume of 3.5 ml), and dissolve it in 1 mmol / L HCl. The matrix will immediately swell and then placed on a sintered glass filter and washed with 1 mmol / L HCl for 15 min.

[0063] 2. Ligand (antibody) conjugation

[0064] a Use coupling buffer 0.2mol / L NaHCO 3 pH8.3 dissolves 3-acetyl deoxynivalenol antibody, aflatoxin antibody, ochratoxin antibody and zearalenone antibody secreted by hybridoma cells 9204, 5506, 5505 and 5504 to be coupled, antibody The concentration is 12.5mg / ml, and the dissolved antibody is temporarily stored in an ice bath. Add the antibody-containing conjugation buffer described above to a fully sealable container with a lid. Quickly transfer the CNBr-activated Sepha...

example 3

[0070] Example three: 3-acetyl deoxynivalenol, aflatoxin (B 1 , B 2 , G 1 , G 2 ), ochratoxin A and zearalenone detection

[0071] 1.0 3-Acetyl deoxynivalenol and aflatoxin (B 1 , B 2 , G 1 , G 2 ), ochratoxin A and zearalenone detection

[0072] In feed recovery experiment, three concentration gradients of 20μg / kg, 50μg / kg and 100μg / kg were added respectively. Five sets of parallel experiments were done for each experiment.

[0073] 3-Acetyldeoxynivalenol, aflatoxin B in feed 1 , B 2 , G 1 , G 2 , the extraction of ochratoxin A and zearalenone:

[0074] Accurately weigh 50.0g of the sample that has been ground (particle size less than 2mm) into a 250mL conical flask with a stopper, add 5.0g of sodium chloride and accurately add 100.0mL of methanol-water (8+2), and use a homogenizer at high speed Stir and extract for 2 minutes, or shake on a shaker for 30 minutes. Quantitative filter paper filtration, accurately pipette 5.0mL filtrate and add 45.0mL PH7.0PBS sol...

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Abstract

The invention discloses a composite affinity column for purifying 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone, a preparation method and an application thereof. According to the invention, by employing 4% of cylindrical agarose gel as a solid phase carrier, agarose gel and an antibody are coupled to form an immunoadsorbent, and filling in a column to prepare the immunization affinity column. When a sample containing 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone passes through the immunization affinity column, 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone can be specifically adsorbed by the immunoadsorbent, other impurities flows out of the immunization affinity column, then methanol is used for eluting 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone from the column, so that the sample can be better purified.

Description

technical field [0001] The invention relates to a method for purifying 3-acetyldeoxynivalenol, aflatoxin, ochratoxin A, zearalenone and a special immunoaffinity column thereof, more precisely 3-acetyldeoxy Preparation method of nivalenol, aflatoxin, ochratoxin A, zearalenone immunoaffinity column, immunoaffinity column purification-liquid chromatography detection of 3-acetyldeoxynivalenol , aflatoxin (B 1 , B 2 , G 1 , G 2 ), the establishment of detection methods for ochratoxin A and zearalenone. Background technique [0002] 3-Acetyl deoxynivalenol is mainly produced by certain Fusarium fungi, including: Fusarium graminearum, Fusarium oxysporum, Fusarium moniliforme, Fusarium pseudocladoides, Fusarium pink and Fusarium nivalum, etc. Many grains can be contaminated, such as wheat, barley, oats and corn. It may accumulate in the body to a certain extent, but it has no special target organs and has strong cytotoxicity. After humans and animals ingest food contaminated ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/38B01J20/281G01N30/88G01N30/06
Inventor 王国民郗存显张雷彭涛李贤良曹淑瑞周超果旗王雄王彦斐江帆
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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