Florfenicol hybridoma cell strain, monoclonal antibody, complex immunosorbent, immunoaffinity column, kit and applications of monoclonal antibody, complex immunosorbent, immunoaffinity column and kit

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 cumbersome processing operations, small linear range, false positives, etc., and improve detection efficiency , Simplified pre-processing process, strong specific effect

Inactive Publication Date: 2017-05-31
CHINESE ACAD OF INSPECTION & QUARANTINE +3
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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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  • Florfenicol hybridoma cell strain, monoclonal antibody, complex immunosorbent, immunoaffinity column, kit and applications of monoclonal antibody, complex immunosorbent, immunoaffinity column and kit
  • Florfenicol hybridoma cell strain, monoclonal antibody, complex immunosorbent, immunoaffinity column, kit and applications of monoclonal antibody, complex immunosorbent, immunoaffinity column and kit
  • Florfenicol hybridoma cell strain, monoclonal antibody, complex immunosorbent, immunoaffinity column, kit and applications of monoclonal antibody, complex immunosorbent, immunoaffinity column and kit

Examples

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preparation example Construction

[0027] In a preferred embodiment, the method for preparing the hybridoma cell line and the monoclonal antibody with the deposit number of CGMCC NO.9206 may include the following steps:

[0028] (1) Preparation of Florfenicol Immunogen

[0029] Florfenicol is dissolved in an organic solvent, and then the organic solvent is removed (such as volatile drying) to obtain the Florfenicol hapten; then the above-mentioned Florfenicol hapten is coupled with a carrier protein to prepare the Florfenicol hapten Test immunogen. In a preferred situation, the organic solvent is pyridine, and the carrier protein is bovine serum albumin (BSA) or ovalbumin.

[0030] (2) Preparation of hybridoma cells and anti-Florfenicol monoclonal antibodies

[0031] Animal immunization: use the florfenicol immunogen to immunize the immunized animals. Specifically, the florfenicol immunogen is added to Freund's complete adjuvant to make an emulsifier for immunization, and then the adjuvant is changed to an in...

preparation example 1

[0069] This preparation example is used to prepare florfenicol immunogen

[0070] Accurately weigh 10 mg of florfenicol, dissolve it in 1000 μL of pyridine, and add 104 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.

[0071]Weigh 8 mg of N,N-dicyclohexylcarbodiimide, 15 mg of N-hydroxysuccinimide, and dissolve the hapten prepared in the above steps in 1000 μL of dimethylformamide (DMF), and stir at room temperature for 2 h , to obtain a hapten solution; and weigh 5 mg BSA and dissolve it in 2 mL pH7.0 PBS buffer solution, and then slowly add 400 μL of the hapten solution dropwise while stirring. The reaction was stirred slowly at 4°C for 24h. Dialyze with pH...

preparation example 2

[0073] This preparation example is used to prepare the anti-Florfenicol monoclonal antibody (antibody A)

[0074] (1) Preparation of anti-Florfenicol monoclonal antibody

[0075] Animal immunization: The immunized animals are female BALB / c mice about 6-8 weeks old. Five mice were immunized with florfenicol immunogen. Take an appropriate amount of florfenicol immunogen (100mg / cause) and add the same amount of complete Freund's 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.

[0076] Cell fusion: The splenocytes and myeloma cells of the immunized mice were subjected to a cell fusion test at a ratio of 10:1.

[0077] Hybridoma cell cloning: The hybr...

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Abstract

The invention discloses a hybridoma cell strain with the accession number being CGMCC NO.9206, a florfenicol-resisting monoclonal antibody secreted by the cell strain, and applications of monoclonal antibody. The invention further provides an immunosorbent, the immunosorbent comprises a solid phase carrier and antibodies coupled to the solid phase carrier, and the antibodies comprise the monoclonal antibody and a phenobarbital-resisting antibody. The invention further provides an immunoaffinity column for loading the immunosorbent. The invention further provides a kit containing the immunosorbent or the immunoaffinity column, and applications of the immunosorbent, the immunoaffinity column or the kit in purifying and / or detecting florfenicol and phenobarbital, and also provides a purifying method and a detecting method. With the adoption of the product and the method provided by the invention, the simultaneous purifying and / or detecting for florfenicol and phenobarbital are / is realized.

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 use in the purification and / or detection of florfenicol and phenobarbital. Background technique [0002] Florfenicol is a broad-spectrum antibacterial drug, which is widely used in animal husbandry and aquaculture. Florfenicol is a broad-spectrum chloramphenicol antibiotic developed in the 1980s and an alternative drug after chloramphenicol was banned. However, with the increase in use and the progress of research, studies have found that it will remain in animal food, and human consumption of this animal food will affect health, for example, it will cause harm to hematopoietic function. Phenobarbital has sedative, hypnotic, and anticonvulsant effects, and can resist epilepsy, and has good...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577G01N33/531B01J20/24B01J20/30B01J20/281B01D15/10C07C317/32C07C315/06C07D239/62
Inventor 陈冬东代汉慧章骅王伟果旗王雄姚佳王彦斐陈旭光
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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