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Chloramphenicol affinity column and preparation method and use thereof

A chloramphenicol and affinity column technology, applied in the field of chloramphenicol affinity column, can solve the problems of elution residue, limited number of reusable times and high cost

Inactive Publication Date: 2006-02-08
王家红
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purification method of liquid-liquid extraction is generally firstly extracted and concentrated with ethyl acetate, then dissolved with 0.4% sodium chloride solution, and then the oil is removed by liquid-liquid extraction with n-hexane, and then concentrated with ethyl acetate, and then carried out Analysis, the detection limit of this purification method is not less than 10 μg / Kg, which cannot meet the current detection requirements; in order to pursue high sensitivity, further purification is required, and the usual method is to adopt C 18 column or other similar solid phase extraction columns, some reports use more than two solid phase extraction columns for continuous purification, which can meet the detection requirements, but due to the difference in matrix, different purification methods are often required for different samples to remove Interfering substances with different properties; some studies have used chloramphenicol antibody in combination with gel solid-phase carrier to prepare affinity chromatography column. The coupling method is to use the antibody to link with the gel solid-phase carrier through CNBr. This method determines that it requires higher purity of the antibody, otherwise the presence of foreign proteins will affect the measurement results and affect the chloramphenicol load of the column. , and the number of reusable columns is limited, and some are developed as disposable affinity chromatography columns, which cost much more than ordinary solid-phase extraction columns, and reusable products have strict requirements on the use of solvents, and are generally prohibited Pure solvents are used so there are false positive results due to elution carryover

Method used

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  • Chloramphenicol affinity column and preparation method and use thereof
  • Chloramphenicol affinity column and preparation method and use thereof
  • Chloramphenicol affinity column and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] This example is the preparation of chloramphenicol immunoaffinity column.

[0059] Reagents used

[0060] Chloramphenicol polyclonal antibody: BSA-succinic chloramphenicol immunized rabbits, and the immune serum was purified by ammonium sulfate precipitation (protein concentration 5 mg / ml); Protein A Sepharose (PharmaciaBiotech); Dimethylpimelate ( Pierce Bitochnology, DMP); methanol, ethanolamine, triethanolamine, thimerosal sodium, NaCL, KCL, KH 2 PO 4 、Na 2 HPO 4 12H 2 O.

[0061] Buffer configuration

[0062] 0.3M potassium phosphate buffer, pH 8.0: 64.8g K 2 HPO 4 ·3H 2 O, dissolved in distilled water to 1000ml, adjusted to pH 8.2 with phosphoric acid; 0.01mol / L phosphate buffer (PBS, pH 7.4): NaCL 8.00g, KCL 0.2g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.90g, dissolved in distilled water to 1000ml; phosphate buffered saline (PBS-0.01%) containing 0.01% thimerosal sodium; 0.2mol / L triethanolamine solution (pH8.2): 5.968g of triethanolamine dissolved in di...

Embodiment 2

[0073] The chloramphenicol immunoaffinity column was prepared by the method described in Example 1, and the affinity column was reused to measure the recovery of the spiked honey samples with complex matrices in food. The spiked levels were 0.1, 0.3, and 1.0 μg / Kg, respectively.

[0074] Extraction: Weigh 5g of honey sample, add 10ml of ultrapure water, shake to dissolve; add 25ml of ethyl acetate, shake and extract for 30 minutes, centrifuge at 3500rpm for 5 minutes, transfer the upper organic phase with a dropper, repeat the ethyl acetate extraction process to combine the organic phase; add 5ml of 0.3M potassium phosphate buffer solution was rotatably evaporated at 45°C until water mist appeared at the mouth of the bottle; the evaporation residue was dissolved in 10ml of 0.3M potassium phosphate buffer solution and passed through an affinity column.

[0075] Purification: take the prepared chloramphenicol immunoaffinity column out of the refrigerator at 4°C, and place it at r...

Embodiment 3

[0081] The chloramphenicol immunoaffinity column was prepared by the method described in Example 1, and the spiked recovery of the shrimp sample was measured, and the spiked levels were 0.1, 0.3, and 1.0 μg / Kg, respectively.

[0082] Extraction: Weigh 5g of blank shrimp sample, add chloramphenicol standard, add 10ml ultrapure water, shake to dissolve; add 25ml ethyl acetate shake and extract for 30 minutes, centrifuge at 3500rpm for 5 minutes, transfer the upper organic phase with a dropper, repeat ethyl acetate Combine the organic phase during the extraction process; add 5ml of 0.3M potassium phosphate buffer solution, and evaporate under reduced pressure at 45°C until water mist appears at the bottle mouth; dissolve the evaporation residue in 10ml of 0.3M potassium phosphate buffer solution, and then use 2ml of n-hexane solution respectively Liquid extraction 2 times to remove fat and set aside.

[0083] Purification: take the prepared chloramphenicol immunoaffinity column o...

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Abstract

The invention relates to a method for preparing chloramphenicol affinant column and its usage, which adopts protein A -Sepharose 4 fast flow as phase carrier, the protein A of the carrier is coupled with the chloramphenicol antibody; the coupled chloramphenicol antibody is bonded with the fixed phase chemistry; the affinant column can suit for many stroma; it uses protein A so that IgG can be connected with it with high desirability and large load capacity of the chloramphenicol column.

Description

technical field [0001] The invention relates to a reusable chloramphenicol affinity column prepared by using protein A-sepharose (protein A-Sepharose 4 fastflow) as a solid phase carrier and coupling with a polyclonal antibody (rabbit) to chloramphenicol The preparation method and application thereof replace the traditional solid-phase extraction technology in sample pretreatment to purify samples, and improve the efficiency of enrichment and purification. Background technique [0002] Chloramphenicol has been widely used in the control and treatment of diseases of various poultry, livestock, aquatic products and bee products due to its good curative effect and low price. However, chloramphenicol has serious side effects at the same time, especially non-dose-dependent irreversible aplastic anemia, which has attracted great attention from international organizations and countries, and strict plans have been formulated to control it. In view of this, many countries, including...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/559
Inventor 张珙王家红
Owner 王家红
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