Fluoroquinolone cluster-specific immunoaffinity chromatography gel and preparation method thereof

A technology of fluoroquinolones and chromatographic chromatography, which is applied in the field of fluoroquinolone drug cluster-specific immunoaffinity chromatography gel and its preparation, can solve the problems of limited detection, strong technical support, and few preparation reports, and achieves The effect of high sensitivity and strong versatility

Inactive Publication Date: 2018-03-20
SUZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past ten years, although there have been a large number of reports on the preparation of antibodies corresponding to fluoroquinolones in China, these antibodies may have cross-recognition in terms of availability, uniformity, stability, reproducibility, affinity for the target, and recognition of drugs with similar structure to the target. One or several indicators such as sex are not suitable for the preparation of IAC. Therefore, there are few domestic reports on the preparation of general-purpose immunoaffinity chromatography gels for fluoroquinolones, which limits the use of this technology for HPLC-FLD or LC-MS / MS detection. Strong technical support

Method used

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  • Fluoroquinolone cluster-specific immunoaffinity chromatography gel and preparation method thereof
  • Fluoroquinolone cluster-specific immunoaffinity chromatography gel and preparation method thereof
  • Fluoroquinolone cluster-specific immunoaffinity chromatography gel and preparation method thereof

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preparation example Construction

[0061] In the preparation of the immunoaffinity chromatography gel / column of the present invention, the required high-purity monoclonal antibody protein can be obtained in the following manner:

[0062] Hybridoma cell line - 11F10 induced ascites in vivo was used as a sample, purified by protein G resin to obtain the eluted part of the collection, each added 1 / 10 volume of 1M Tris-Cl (pH8.5) to neutralize, and the protein was provided by Beyontian Company The protein concentration determined by the content detection kit is greater than or equal to 2mg / ml and the purity of the antibody identified by SDS-PAGE electrophoresis is greater than or equal to 99% (there are only two protein bands of 50kd and 25kd in the protein electrophoresis map) as qualified monoclonal antibody protein pure products, and the above qualified Collect liquid samples, reverse dialysis with 0.01M PBS (pH 7.4) for 24h, and then use 0.1M NaHCO 3 / 0.5M NaCl (pH 8.3) reverse dialysis for 24h. The sample was ...

Embodiment 1

[0066] Example 1: Acquisition of ascites samples containing monoclonal antibodies according to the present invention

[0067] 1. Recovery and proliferation of hybridoma cell line -11F10

[0068] Take the cryopreservation tube of the hybridoma cell line-11F10 from the liquid nitrogen tank, melt it rapidly in a water bath at 37°C, centrifuge at 600r / min for 5min, discard the supernatant, add 15% FBS / RPMI-1640 culture medium to suspend the cells, and supplement each After adding the above-mentioned culture solution to 5ml, plant it in a 50ml cell culture bottle and culture it in a carbon dioxide incubator. After the cells grow to a density of 30%, the medium is replaced halfway. Passage once every 2-3 days according to 1:3-4.

[0069] 2. In vivo induction of monoclonal antibody protein and ascites acquisition

[0070] 7-10 days before planting hybridoma cells in vivo, 12 male BALB / c mice aged 8-10 weeks were injected intraperitoneally with 0.5ml / mouse of pristane, and carefully...

Embodiment 2

[0074] Embodiment 2: Purification of the monoclonal antibody protein of the present invention

[0075] The ascites was diluted 4× with 0.01M PBS (pH 8.0) pre-cooled in an ice-water bath and used as a preparation solution for later use. Take 1ml of Protein G Resin for every 50mg of total protein in the ascites, fill the gel in a small column for chromatography (10ml), equilibrate the column with pre-cooled 0.01M PBS (pH 8.0) of 50× column bed volume, and keep the column at room temperature The preparation solution was filtered five times through the Protein G Resin column (natural flow rate), so that the monoclonal antibody protein in the ascites could fully bind to the Protein G Resin specifically, and then pre-cooled 0.01M PBS (pH 8.0) with 50× column bed volume to fully Wash the column (natural flow rate) to remove foreign proteins stuck in the column.

[0076] Add 100 μl 1M Tris-Cl (pH 9.6) to each 1.5ml EP tube in advance, add ice water to pre-cool 0.1M Glycine-HCl (pH 2....

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Abstract

The invention relates to the technical field of biologics, and discloses fluoroquinolone medicine cluster specific immunoaffinity chromatography glue which is prepared by purifying monoclonal antibody protein secreted from a hybridoma cell strain of which the preservation serial number is CCTCC NO.C2015221 and implementing chemical crosslinking with CNBr-activated Sepharose-4B glue so as to obtain a solid phase antibody. Monoclonal antibodies such as enrofloxacin, ciprofloxacin, norfloxacin, pefloxacin and enoxacin with high sensitivity and high universality recognition and combination are applied to study and development of immunoaffinity chromatography glue, the product is high in selectivity, good in enrichment and good universality for target articles, namely medium interference of a sample to be tested can be effectively removed, high-purity target article can be also obtained for machine tests, and furthermore the target article can be enriched, so that the application field of HPLC-FLD / LC-MS-MS detection can be greatly widened.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fluoroquinolone drug cluster-specific immunoaffinity chromatography gel and a preparation method thereof. Background technique [0002] Fluoroquinolones, as artificially synthesized antibiotics, are widely used in medicine (as first-line antibacterial drugs in clinical practice), agricultural and sideline medicines, etc. In the production of aquatic products (an important drug for the prevention and treatment of bacterial infectious diseases in livestock, poultry and aquatic animals), the current trend of fluoroquinolone antibacterials in food samples is multi-component mixing and high residues. What is more worrying is the relevant pharmaceutical companies The diffusion of the above-mentioned antibiotics in the environment, hospitals, and agricultural and sideline aquatic products production enterprises results in very small residues in soil and surface water sources, which is eno...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/08G01N33/577
CPCG01N30/08G01N33/577G01N2030/027
Inventor 宋学宏吴康张炎方强章雪明黄芳苑华宁
Owner SUZHOU UNIV
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