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Compound immunoaffinity column for purifying phenobarbital and ractopamine as well as preparation method and application of compound immunoaffinity column

A technology of ractopamine and phenobarbital, which is applied in the field of preparation of phenobarbital and ractopamine immunoaffinity columns, to achieve rapid detection effect

Inactive Publication Date: 2015-07-22
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 phenobarbital and ractopamine, the invention provides a composite immunoaffinity column for phenobarbital and ractopamine and a preparation method thereof Application in HPLC detection

Method used

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  • Compound immunoaffinity column for purifying phenobarbital and ractopamine as well as preparation method and application of compound immunoaffinity column
  • Compound immunoaffinity column for purifying phenobarbital and ractopamine as well as preparation method and application of compound immunoaffinity column
  • Compound immunoaffinity column for purifying phenobarbital and ractopamine as well as preparation method and application of compound immunoaffinity column

Examples

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Comparison scheme
Effect test

example 1

[0038] Anti-phenobarbital monoclonal antibodies were prepared; ractopamine antibodies were 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 limiting dilution method until cells with good specific response to phenobarbital were obtained. Finally, the hybridoma cell 9203 secreting phen...

example 2

[0043] This example is the preparation of phenobarbital and ractopamine 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 Dissolve the anti-phenobarbital monoclonal antibody to be coupled at pH 8.3. Anti-phenobarbital monoclonal antibody secreted by cell 9203 and commercially available ractopamine antibody. The antibody concentration is 7.8 mg / ml. The dissolved antibody is placed on ice Staging in the bath. Add the antibody-containing conjugation buffer described above to a fully sealable container with a lid. Quickly transfer the CNBr-activated Sepharose into the ant...

example 3

[0055] Example 3: Detection of Phenobarbital and Ractopamine in Feed

[0056] 1.0 Detection of Phenobarbital and Ractopamine in Feed

[0057] 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.

[0058] 2.0 Extraction of phenobarbital and ractopamine in feed:

[0059] Accurately weigh 25.0 g of the ground sample into a 250 mL Erlenmeyer flask with a stopper, add 100.0 mL of acetonitrile / 0.3 mol / L NaHCO3 (1:1, v / v) solution, and extract with a homogenizer at high speed for 2 min. Filter with fluted filter paper, accurately pipette 5.0 mL of filtrate into a 25 mL volumetric flask, dilute to 25 mL with pH 7.4 PBS, filter with glass fiber filter paper until the filtrate is clear, and set aside.

[0060] Attach the composite immunoaffinity column under a 10.0 mL glass syringe. Accurately pipette 8.0mL sample extract solution into a glass syringe, con...

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Abstract

The invention discloses a compound immunoaffinity column for purifying phenobarbital and ractopamine as well as a preparation method and an application of the compound immunoaffinity column. 4% columnar agarose gel is adopted as a solid-phase carrier, the agarose gel is coupled with a phenobarbital antibody prepared with the method and a ractopamine antibody purchased in the market to form an immunoadsorbent, and the immunoadsorbent is put into a column to prepare the immunoaffinity column. When a sample containing phenobarbital and ractopamine passes through the immunoaffinity column, the immunoadsorbent specifically adsorbs phenobarbital and ractopamine, other impurities flow out of the immunoaffinity column, and then methanol is used to wash off phenobarbital and ractopamine from the column, so that the sample is well purified. The immunoaffinity column purification and liquid chromatography methods are created for detecting phenobarbital and ractopamine, so that the detection is quick, accurate and safe.

Description

technical field [0001] The present invention relates to the preparation and application of a compound immunoaffinity column for purification of phenobarbital and ractopamine, more precisely, the preparation method of the immunoaffinity column for phenobarbital and ractopamine, and the purification-liquid of the immunoaffinity column Development of a method for the detection of phenobarbital and ractopamine by phase chromatography. Background technique [0002] Phenobarbital, white shiny crystalline powder; odorless, slightly bitter taste. It has sedative, hypnotic and anticonvulsant effects. It can also resist epilepsy, and has good effect on epileptic grand mal seizures, partial seizures and status epilepticus; it has poor curative effect on epileptic seizures; it is often ineffective on psychomotor seizures, and it may aggravate the seizures when it is used alone . This product can also enhance the effect of antipyretic and analgesic drugs, and can induce the activity o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/38B01J20/281G01N1/34G01N1/40
Inventor 王国民陈冬东王伟唐柏彬马吉湘李贤良果旗里南董韬王雄周超郗存显
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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