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Complex immunoaffinity column for purifying difloxacin and sulfonamides as well as preparation method and application of complex immunoaffinity column

A technology for sulfa drugs and difloxacin, which is applied in the purification of difloxacin and sulfa drugs composite immunoaffinity column and its preparation and application field, which can solve the problems of poor purification effect, long time required and waste of organic solvents Many problems, to achieve the effect of stable performance, fast detection, strong specificity

Inactive Publication Date: 2015-07-15
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

The pretreatment of these methods often uses liquid-liquid distribution, and the conventional SPE column essence and separation all have shortcomings such as cumbersome treatment process, poor purification effect, waste of organic solvents, and long time required to varying degrees.
[0006] Immunoaffinity technology is a new technology applied in the field of analysis in the 1990s. The key to immunoaffinity chromatography is to select an efficient and specific antibody. However, using the liquid-liquid extraction and solid-phase extraction in the existing technology , heating, evaporating, and concentrating methods have low addition recovery rates, which cause strong matrix effects in subsequent detection steps, and there is no immunosorbent medium that can simultaneously purify 16 kinds of sulfonamides in the matrix to be treated in the prior art, and also There is no immunoadsorption medium that can simultaneously purify difloxacin and 16 sulfonamides in the matrix to be treated, and there is no commercial IAC column for sale. Being able to purify and analyze multiple antibacterial drugs at the same time can obviously improve the efficiency of detection

Method used

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  • Complex immunoaffinity column for purifying difloxacin and sulfonamides as well as preparation method and application of complex immunoaffinity column
  • Complex immunoaffinity column for purifying difloxacin and sulfonamides as well as preparation method and application of complex immunoaffinity column
  • Complex immunoaffinity column for purifying difloxacin and sulfonamides as well as preparation method and application of complex immunoaffinity column

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] This example is used to prepare anti-difloxacin monoclonal antibody

[0042] (1) Animal immunization: the immunized animals are about 6-8 weeks old, female BALB / c mice. Immunogen: DIF-BSA 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.

[0043] (2) 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.

[0044] (3) Hybridoma cell cloning: the hybridoma cells were screened by the limiting dilution method until the hybridoma cell 9205 secreting anti-difloxacin monoclonal antibody was screened out, and the monoclonal ant...

Embodiment 2

[0046] This example is used to prepare difloxacin and sulfonamides immunoaffinity chromatographic column

[0047] 1. Substrate Preparation

[0048]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.

[0049] 2. Ligand (antibody) conjugation

[0050] a Use coupling buffer 0.2mol / L NaHCO 3 Dissolve the anti-difloxacin monoclonal antibody and anti-sulfonamide monoclonal antibody secreted by the hybridoma cell 9205 to be coupled at pH 8.3 with an antibody concentration of 8.5 mg / mL, and place the dissolved antibody 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 (i.e., treated in step 1) into the a...

Embodiment 3

[0058] This example is used for the detection of difloxacin and sulfonamides in aquatic products.

[0059] 1. Sample pretreatment

[0060] Accurately weigh 5.0 g of the homogenized sample into a 50 mL centrifuge tube, accurately add 20.0 mL of 80% ethanol aqueous solution, homogenize at high speed for 2 minutes, extract by shaking for 30 minutes, and then centrifuge at 3000 RPM for 5 minutes. Accurately pipette 5.0 mL of the supernatant, dilute it with 45.0 mL of pure water and dilute to 50 mL, then filter it with glass microfiber filter paper, collect the filtrate in a clean beaker, and set aside.

[0061] Attach the immunoaffinity column under a 20.0 mL glass syringe. Accurately pipette 20.0 mL of the above-mentioned filtrate, pass it through the immunoaffinity column at a flow rate of 1 drop / second until the air flows through the affinity column, and then rinse the affinity column with 10.0 mL of pure water at a flow rate of 1-2 drops / second , until the air flows through ...

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Abstract

The invention relates to a complex immunoaffinity column for purifying difloxacin and sulfonamides as well as a preparation method and application of the complex immunoaffinity column. According to the method, a difloxacin antigen and an immunized muse are adopted, and hybridoma cells 9205 capable of secreting difloxacin antibodies are screened out and monoclonal antibodies are prepared. 4% columnar agarose gel is taken as a solid-phase carrier; the agarose gel is coupled with the anti-difloxacin monoclonal antibodies and anti-sulfonamides antibodies bought from the market to form an immunoadsorbent; the immunoadsorbent is put into a column to form the immunoaffinity column (IAC). When a sample containing the difloxacin and the sulfonamides passes through the IAC, the immunoadsorbent specifically adsorbs the difloxacin and the sulfonamides, and other impurities flow out of the IAC; next, the difloxacin and the sulfonamides are eluted down from the column by use of methanol; as a result, the sample is well purified. An immunoaffinity column purification-liquid chromatography is established to detect the difloxacin and the sulfonamides.

Description

technical field [0001] The present invention relates to the preparation and application of a compound immunoaffinity column for purification of difloxacin and sulfonamides, more precisely, the preparation method of an immunoaffinity column for difloxacin and sulfonamides. Establishment of a method for the detection of difloxacin and sulfonamides by phase chromatography. Background technique [0002] With the development of life sciences, people have become more and more interested in the substances and their changes in living organisms, and the analysis of biological samples has become a necessary means to explore and discover the mysteries of life. Due to the complex components of biological samples, the concentration of analytes is low, and most of the sample volumes are small, which puts forward higher requirements for the selectivity and sensitivity of the analytical method. Immunoaffinity Chromatography (IAC) is an analytical method that combines immune reaction with c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/577G01N30/88
CPCB01J20/281B01D15/3804B01D15/3809B01J20/28047B01J2220/54B01J2220/58G01N30/06G01N30/88G01N33/531G01N33/577G01N2030/065
Inventor 郗存显王国民曹淑瑞李蓉张雷王勇果旗姚佳王江王雄余洋李贤良
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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