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Composite immunization affinity column for purifying beta-zeranol and chloramphenicol, preparation method and application thereof

A technology of zearalanol and chloramphenicol, which is applied in chemical instruments and methods, alkali metal compounds, inorganic chemistry, etc., to achieve fast detection results

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

[0005] In order to solve the problem in the prior art that an immunoaffinity column with stable performance is urgently needed for detecting β-zearalenol and chloramphenicol, the invention provides a composite immunoaffinity column for β-zearalenol and chloramphenicol and its Preparation method and application in HPLC detection

Method used

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  • Composite immunization affinity column for purifying beta-zeranol and chloramphenicol, preparation method and application thereof
  • Composite immunization affinity column for purifying beta-zeranol and chloramphenicol, preparation method and application thereof
  • Composite immunization affinity column for purifying beta-zeranol and chloramphenicol, preparation method and application thereof

Examples

Experimental program
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Effect test

example 1

[0038] Anti-β-zearalenol (ZER) monoclonal antibody was prepared; chloramphenicol antibody was commercially available.

[0039] Animal immunization: The immunized animals are about 6-8 weeks old, female BALB / c mice. Immunogen: ZER-BSA was used to immunize 5 mice. 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.

[0040] Cell fusion: Splenocytes and hybridoma cells were subjected to cell fusion experiments at a ratio of 10:1.

[0041] Hybridoma cell cloning: The hybridoma cells were screened by the limiting dilution method until cells with a good specific response to β-zearalenol were obtained. Finally, the hybridoma cell 9208 secret...

example 2

[0043] This example is the preparation of β-zearalanol and chloramphenicol composite immunoaffinity column

[0044] 1. Substrate Preparation

[0045] 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.

[0046] 2. Ligand (antibody) conjugation

[0047] a Use coupling buffer 0.2mol / L NaHCO 3 The β-zearalenol antibody secreted by the hybridoma cell 9208 to be coupled and the commercially available chloramphenicol antibody were dissolved at pH 8.3 at a concentration of 8.5 mg / ml, and the dissolved antibody was placed in an ice bath for temporary storage. Add the antibody-containing conjugation buffer described above to a fully sealable container with a lid. Quickly transfer the CNBr-activated Sepharose into the antibody solution. At r...

example 3

[0054] Example 3: Detection of β-zearalenol and chloramphenicol in beef

[0055] 1.0 Detection of β-zearalenol and chloramphenicol in beef

[0056] Beef addition recovery experiment, respectively add 10μg / kg, 20μg / kg, 50μg / kg three concentration gradients. Five sets of parallel experiments were done for each experiment.

[0057] 2.0 Extraction of β-zearalenol and chloramphenicol in beef: Accurately weigh 5.0g of the homogenized sample, add 10.0mL of sodium acetate buffer and 25uL of β-glucoside / sulfate compound enzyme to a 50mL centrifuge tube, Mix well and put in 37 degree water bath for 12h. After hydrolysis, 10 mL of acetonitrile was added, and 200 uL of 1mol / L NaOH solution (purpose: to adjust the pH value to 7.0) was added and shaken for 30 min. Centrifuge at 4000rpm for 3min, measure 10ml of supernatant, dilute with 30mL of pH7.4 PBS solution, mix well, use qualitative filter and filter with filter paper, collect the filtrate in a clean beaker, and set aside. Attach ...

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Abstract

The invention discloses a composite immunization affinity column for purifying beta-zeranol and chloramphenicol, a preparation method and an application thereof. By employing 4% of cylindrical agarose gel as a solid phase carrier, the agarose gel and a prepared beta-zeranol antibody and a chloramphenicol antibody purchased on market are coupled to form an immunoadsorbent, and the immunoadsorbent is filled in the column to prepare the immunization affinity column. When a sample containing beta-zeranol and chloramphenicol passes through the immunization affinity column, beta-zeranol and chloramphenicol can be specifically adsorbed by the immunoadsorbent, other impurities flows out of the immunization affinity column, then methanol is used for eluting beta-zeranol and chloramphenicol from the column, so that the sample can be better purified. An immunoaffinity column purifying-liquid chromatography is established to detect beta-zeranol and chloramphenicol, so that detection is fast, accurate and safe.

Description

technical field [0001] The present invention relates to the preparation and application of a purified β-zearalenol and chloramphenicol composite immunoaffinity column, more precisely the preparation method of the β-zearalenol and chloramphenicol immunoaffinity column, the immunoaffinity Establishment of column purification-liquid chromatography for detection of β-zearalenol and chloramphenicol. Background technique [0002] Zearalenol, also known as "right-ring tetradecone phenol", is the reduction product of the secondary metabolite zearalenone produced by Gibberella zebrascens during the growth process, and belongs to the non-steroidal class of resolactones anabolic hormone. Zearalenone and its metabolites - α-zearalenol, β-zearalenol, zearalenol, zearalenone and β-zearalenol may contaminate feed and cause animal product Residues of zearalanol were detected in the Zearalenol is an ideal skin weight gainer, which is a non-sterol and non-hormonal compound. However, exper...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/281B01J20/28B01J20/30G01N30/06
Inventor 王国民唐柏彬陈冬东曹淑瑞李贤良张雷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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