An immunoaffinity column for simultaneous extraction of florfenicol, chloramphenicol and thiamphenicol
A technology of florfenicol and thiamphenicol, applied in the field of immunoassay, can solve the problems of complex process and low purification efficiency, and achieve the effect of simple operation, ensuring reliability and stability, and improving purity and purification efficiency
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Embodiment 1
[0022] Embodiment 1, preparation of florfenicol monoclonal antibody
[0023] 1. Preparation of Immunogen
[0024] 1.1 Stir and dissolve 0.3g of Florfenicol (FF) and 2g of succinic anhydride in 2.25ml of anhydrous pyridine, reflux at 60°C for 24h, remove pyridine by nitrogen blowing, reconstitute with 5ml of ethyl acetate, evaporate to dryness, and transfer to a separatory funnel After the solution was separated, it was washed 3 times with 0.5mol / L HCl 2ml to obtain a derivative solution. The above solution was concentrated under reduced pressure, and dried under vacuum for 20 h in the dark to obtain the crude FF hapten as a pink monoester.
[0025] 1.2 Dissolve 5mg of FF hapten with 0.5ml of N,N-dimethylformamide, then add 10mg of N-hydroxysuccinimide and 10mg of N-N-dicyclohexylcarbimide, and stir at room temperature for 2 hours in the dark, that is Activation intermediate for the preparation of Florfenicol. The activated intermediate product of Florfenicol was added dropw...
Embodiment 2
[0032] Embodiment 2, the preparation of immunoaffinity column
[0033] 1. Preparation of solid phase medium coupled with florfenicol monoclonal antibody
[0034] 1.1 Medium cleaning
[0035] First, wash the highly cross-linked 4% agarose 4FF medium with 0.001M HCl, 0.5M NaCl, and adjust the pH to 3.0, resuspend the medium with 5 times the volume of cleaning solution, drain the solution after 2 minutes, and repeat this step 5 Second-rate.
[0036] 1.2 Coupling
[0037] The purified FF monoclonal antibody to be conjugated was washed with 0.5M NaHCO 3 , 0.5M NaCl, adjust the pH pH=8.5-9.0, dissolve to 5mg / ml. The washed solid phase medium and the prepared antibody were mixed in an equal volume ratio, mixed gently at room temperature for 1 hour, and the solution was drained.
[0038] 1.3 Closed
[0039] Use 5 times the volume of 0.1M Tris-HCl, adjust the pH to 8.3 to resuspend the coupled medium, then drain the solution, repeat this step 5 times, use 5 times the volume of 0....
Embodiment 3
[0047] Embodiment 3, recovery rate and column capacity of immunoaffinity column
[0048] In 0.01Mpbs, different concentrations of chloramphenicol, florfenicol, and thiamphenicol standard substances were added, purified by immunoaffinity column and then detected by the instrument to obtain the following results, as shown in Table 1, the immunoaffinity And column recovery and column capacity. The results show that the recovery rate of chloramphenicol is at 85%-96% in the interval of 0-500ppb, and the recovery rate above 1000ppb is lower than 60%; the recovery rate of Florfenicol in the interval of 0-1000ppb is at 79%-94%, The recovery rate above 2000ppb is lower than 60%; The recovery rate of thiamphenicol in the interval of 0-1000ppb is 82%-96%, and the recovery rate above 2000ppb is lower than 60%, illustrating that the affinity column of the present invention is to chloramphenicol, For florfenicol and thiamphenicol, the column capacities are 500ppb, 1000ppb, and 1000ppb resp...
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