Compound immunoaffinity column for purifying phenobarbital and ractopamine as well as preparation method and application of compound immunoaffinity column
A technology of ractopamine and phenobarbital, which is applied in the field of preparation of phenobarbital and ractopamine immunoaffinity columns, to achieve rapid detection effect
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example 1
[0038] Anti-phenobarbital monoclonal antibodies were prepared; ractopamine antibodies were commercially available.
[0039] Animal immunization: The immunized animals are about 6-8 weeks old, female BALB / c mice. Immunogen: ZER-BSA was used 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.
[0040] Cell fusion: Splenocytes and hybridoma cells were subjected to cell fusion experiments at a ratio of 10:1.
[0041] Hybridoma cell cloning: The hybridoma cells were screened by limiting dilution method until cells with good specific response to phenobarbital were obtained. Finally, the hybridoma cell 9203 secreting phen...
example 2
[0043] This example is the preparation of phenobarbital and ractopamine composite immunoaffinity column
[0044] 1. Substrate Preparation
[0045] 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.
[0046] 2. Ligand (antibody) conjugation
[0047] a Use coupling buffer 0.2mol / L NaHCO 3 Dissolve the anti-phenobarbital monoclonal antibody to be coupled at pH 8.3. Anti-phenobarbital monoclonal antibody secreted by cell 9203 and commercially available ractopamine antibody. The antibody concentration is 7.8 mg / ml. The dissolved antibody is placed on ice Staging in the bath. Add the antibody-containing conjugation buffer described above to a fully sealable container with a lid. Quickly transfer the CNBr-activated Sepharose into the ant...
example 3
[0055] Example 3: Detection of Phenobarbital and Ractopamine in Feed
[0056] 1.0 Detection of Phenobarbital and Ractopamine in Feed
[0057] In feed recovery experiment, three concentration gradients of 20μg / kg, 50μg / kg and 100μg / kg were added respectively. Five sets of parallel experiments were done for each experiment.
[0058] 2.0 Extraction of phenobarbital and ractopamine in feed:
[0059] Accurately weigh 25.0 g of the ground sample into a 250 mL Erlenmeyer flask with a stopper, add 100.0 mL of acetonitrile / 0.3 mol / L NaHCO3 (1:1, v / v) solution, and extract with a homogenizer at high speed for 2 min. Filter with fluted filter paper, accurately pipette 5.0 mL of filtrate into a 25 mL volumetric flask, dilute to 25 mL with pH 7.4 PBS, filter with glass fiber filter paper until the filtrate is clear, and set aside.
[0060] Attach the composite immunoaffinity column under a 10.0 mL glass syringe. Accurately pipette 8.0mL sample extract solution into a glass syringe, con...
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