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
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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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