Studies On Preparation And Characterization Of Immunoaffinity Column With Polyclonal Antibody Against Enrofloxacin And Chitosan Microspheres As Support Matrix

Enrofloxacin(ENR), a synthetic fluoroquinolone antibiotic, is widely used in the treatment of animal infections due to its broad spectrum of antibacterial action.However, the widespread use of ENR in food producing animals has caused public concern because of the development of antimicrobial resistance and the presence of ENR residues in edible animal products may pose adverse effect to human beings.Currently, instrumental analysis is the major method used for screening and quantification of antibiotic residues in food and feed samples. However, a pretreatment step is always necessary before instrumental analysis because of the low level of the target analyte and the complexity of the real sample matrices. Compared with other pre-tretment methods, immunoaffinity chromatography(IAC), with advantages of time saving, high specificity and completing the separation process at a small bed volume, is widely used in residues monitoring. In this study, ENR complete antigen and polyclonal antibodies were prepared and confirmed. The polyclonal antibody was coupled with chitosan microspheres therefore a chitosan-based IAC column was prepared, and the performance and prospects of the column were also evaluated. The main contents and results are as follows:1. The immunogen ENR-BSA and coating antigen ENR-OVA were synthesized by N-hydroxysuccinimide method and confirmed by FT-IR, UV and SDS-PAGE.2. The New Zealand white rabbits were immunized by the immunogen to obtain polyclonal antibody. The IC50 value of the polyclonal antibody determined by ic ELSIA method was 5.47 ng/m L, and the cross-reactivity rates of DAN and NOR were both less than 0.5%, indicating that the inhibition and specificity of the prepared polyclonal antibody could achive the experimental needs of IAC column.3. The adsorbents of the IAC column were prepared by coupling the polyclonal antibody on the CTS microspheres under the action of epichlorohydrin. The CTS microspheres scaning by the electronic microscope showed independent rules sphericity, and the coupling rate of the adsorbents was 88.83%. The loading condition and elution conditionson of the chitosan-based IAC column were optimized, findingthat the best loading solution was 0.8% Na OH-PBS, the best elution solution was 4m L of 85% methanol-water.4. The performance of the chitosan-based IAC column was evaluated by the ic ELISA and HPLC standard curves. The capacity of the IAC column was about 5.4μg ENR/g wet affinity adsorbent.The average recovery between columns was97.89%±2.19% with the coefficient of variation(CV) of 2.24% showing the good reproducibility between the IAC columns. After 4 cycles of use intra day and 5 cycles of use inter day, the recoveryrate still remained higher than 85%and 80%, respectively.The IAC column showed high specific to ENR with negligible cross-reactivity to both DAN(4.59%) and NOR(3.81%).5. The IAC column combined with ic ELISA method was applied in the detection in food samples. The recovery rates in milk spiked 25 ng/m L and 50 ng/m L were from 91.16% to 100.63% with CV less than 3.17%, while the pork spiked 25 ng/g, 50ng/g from 79.76% to 94.95% with CV less than 7.46%.