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Olaquindox hybridoma cell strain, monoclonal antibody, complex immunoadsorbent, immunoaffinity column, kit and application thereof

A hybridoma cell line and immunoadsorbent technology, which is applied in the direction of solid adsorbent liquid separation, immunoglobulin, and other chemical processes, can solve the problems of false positives, cumbersome processing operations, and small linear range, and achieve strong specificity , Improving detection efficiency and simplifying the effect of pretreatment process

Inactive Publication Date: 2017-05-31
CHINESE ACAD OF INSPECTION & QUARANTINE +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, enzyme-linked immunosorbent assay has the disadvantages of low specificity, small linear range, and prone to false positive and false negative results; while liquid chromatography and liquid chromatography-tandem mass spectrometry have cumbersome sample pretreatment operations and limited sensitivity. Steps such as sample purification and concentration have a greater impact and require the use of toxic reagents such as acetone or dichloromethane

Method used

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  • Olaquindox hybridoma cell strain, monoclonal antibody, complex immunoadsorbent, immunoaffinity column, kit and application thereof
  • Olaquindox hybridoma cell strain, monoclonal antibody, complex immunoadsorbent, immunoaffinity column, kit and application thereof
  • Olaquindox hybridoma cell strain, monoclonal antibody, complex immunoadsorbent, immunoaffinity column, kit and application thereof

Examples

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preparation example Construction

[0026] In a preferred embodiment, the method for preparing the hybridoma cell line and the monoclonal antibody with the deposit number of CGMCC NO.11182 may include the following steps:

[0027] (1) Preparation of olaquindox immunogen

[0028] Dissolving the olaquindox in an organic solvent, and then removing the organic solvent (such as volatile drying) to obtain a olaquindox hapten; then coupling the above-mentioned olaquindox hapten with a carrier protein to prepare a olaquindox immunogen. In a preferred situation, the organic solvent is pyridine, and the carrier protein is bovine serum albumin (BSA) or ovalbumin.

[0029] (2) Preparation of hybridoma cells and monoclonal antibodies against olaquindox

[0030] Animal immunization: use the olaquindox immunogen to immunize the immunized animals. Specifically, the olaquindox immunogen is added to Freund's complete adjuvant to make an emulsifier for immunization, and then the adjuvant is changed to an incomplete adjuvant, and ...

preparation example 1

[0076] This preparation example is used to prepare olaquindox immunogen

[0077] Accurately weigh 15 mg of olaquindox, dissolve it in 1000 uL of pyridine, and add 97 mg of butylboronic acid. The mixture was stirred at room temperature for 20 h. Then, 2000 μL of 1.7 mol / L succinic anhydride pyridine solution was added with stirring, the reaction tube was sealed, the mixture was stirred and reacted in a boiling water bath for 3 h, and then the solvent pyridine was volatilized and dried under nitrogen at 100°C to obtain the hapten.

[0078] Weigh 7.8 mg of N,N-dicyclohexylcarbodiimide, 13.8 mg of N-hydroxysuccinimide, and dissolve the hapten prepared in the previous step in 1000 μL of dimethylformamide (DMF), stir at room temperature 2h to obtain a hapten solution; and weighed 5mg BSA dissolved in 2mL pH7.0PBS buffer solution, and then slowly added 400μL hapten solution dropwise while stirring. The reaction was stirred slowly at 4°C for 24h. Dialyze with pH 7.0 PBS solution fo...

preparation example 2

[0080] This preparation example is used to prepare monoclonal antibody against olaquindox (antibody A) and monoclonal antibody against chloramphenicol (antibody B).

[0081] (1) Preparation of monoclonal antibody against olaquindox

[0082] Animal immunization: The immunized animals are female BALB / c mice about 6-8 weeks old. Five mice were immunized with olaquindox immunogen. Take an appropriate amount of olaquindox immunogen (100 mg / rat) and add an equal amount of Freund's complete adjuvant to make an emulsifier for immunization, then change the adjuvant to incomplete adjuvant, and immunize 6 times in total, 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. After the immunization, the mice were sacrificed, and splenocytes were collected.

[0083] Cell fusion: The splenocytes and myeloma cells of the immunized mice were subjected to a cell fusion test at ...

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Abstract

The invention discloses a hybridoma cell strain with a preservation number of CGMCC NO.11182, a monoclonal antibody obtained through secretion of the cell strain, and the application thereof. The invention further provides an immunoadsorbent, wherein the immunoadsorbent comprises a solid phase carrier and antibodies coupled with the solid phase carrier, and the antibodies include the monoclonal antibody and a chloramphenicol-resisting antibody. The invention also provides an immunoaffinity column loaded with the immunoadsorbent. The invention further provides a kit containing the immunoadsorbent or the immunoaffinity column, the application of the immunoadsorbent, the immunoaffinity column or the kit to purification and / or detection of olaquindox and chloramphenicol, and a purification method and a detection method. By using the products and methods, olaquindox and chloramphenicol can be purified and / or detected at the same time.

Description

technical field [0001] The present invention relates to a hybridoma cell strain, a monoclonal antibody secreted by the hybridoma cell strain and its application, an immunoadsorbent prepared from the antibody, and an immunoaffinity column and a kit loaded with the immunosorbent, and their application in purification and / or detection of olaquindox and chloramphenicol. Background technique [0002] Both olaquindox and chloramphenicol are broad-spectrum antimicrobials and are widely used in animal husbandry and aquaculture. Olaquindox belongs to the quinoxaline-1,4-dioxide compound, which has been widely used as a growth promoter in livestock, poultry, and aquaculture. Owing to its mutagenic, teratogenic and carcinogenic toxicity to humans and animals, the European Union banned the use of olaquindox in 1997. In my country, it is limited to pigs weighing more than 35kg, and it is prohibited to use it in poultry and aquaculture. The maximum residue limit of olaquindox residue m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577B01J20/24B01D15/38C07D241/44C07C233/18C07C231/24C12R1/91
Inventor 陈冬东代汉慧韩深吴振兴果旗王雄里南江帆陈旭光
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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