Cunstruction And Screening Of Phage Display Antibody Library Specific For Ofloxacin

Food safety has become a global challenge and an important global public health problem. Animal source food safety is a focus of the world attention, including veterinary drug residue issues that affect the safety of the food products of animal origin.As an important veterinary medicine, ofloxacin has been widely used for its many advantages. ELISA has become a valid method for rapid analysis of drug residues. But, obtaining effective antibodies is a major important problem in this method development. To get more effective antibodies, a phage display antibody library specific for ofloxacin was constructed and screened in this study.Total RNA was extracted from hybridoma that sreting specific antibody against ofloxacin. The cDNA was obtained by reverse transcription and used as template for PCR amplification of variable regions of heavy chains (VH) and light chains (VL). Specific primers synthesized base.d on the sequenced fragments and overlap PCR method were used to amplified single chain variable fragment (scFv) which was composed of780bp. The scFv fragments were digested with restriction enzyme EcoR. Ⅰ and HindⅢ. The digested products were ligated into T7phage arms using T4DNA ligase and the ofloxacin-specific phage-displaying antibody library was constructed after coating procedure. After examination original titer of the primary phage library reached2.4X105pfu, while the titer reached1.2×1012pfu/mL after amplification.1010pfu/mL phages from the antibody library were applied to panning using ofloxacin-OVA as ligands. Total4rounds of panning were performed. During panning procedures, blocking reagents and elution methods were modified. After4rounds of panning,138monoclonal plaques were picked as template for PCR and the results indicated that83%clones contained right scFv fragments. ELISA method was introduced to examine the specificities of the screened phage clones and the result indicated that4of them exhibited high specificities. Binding of phages to the ligands could be inhibited31%,35%,33%,78%by1μg/mL free ofloxacin. scFv with highest specificity was cloned and sequenced. The VH was composed of117amino acids and the VL was composed of108amino acids.These results could be the foundation for production of new forms of specific antibodies against ofloxacin and innovation of immunological detection methods.