Preparation And Recognition Study Of Anti-ofloxacin Enantioselective Fab Fragments

The abuse of veterinary pharmaceuticals had posed a great threat to environment even human health.And the development of chiral drugs make this issue more complicate to monitor and detect the veterinary drug residues.Antibodies emerged as a versatile tool not only can establish an immunoassay to trace amounts of veterinary drug residues practically in large-scale food sample,but also can recognize the minor difference between enantiomers.It had been demonstrated that antibodies could be also exploited as a receptor with high affinity and enantioselectivity to investigate the interaction between chiral surfaces and chiral targets in vivo.However,the use of enantioselective antibody is still very limited in practice for a lack of understanding how chiral information deliver to binding surfaces of antibody.Ofloxacin,the second generation fluoquinolones antibiotics,was widely used as veterinary and clinical medicine.In this work,ofloxacin enantiomers were chosen as a model to study the chiral recognitition between small molecular drugs and highly enantioselective antibodies by the use of X-ray diffraction crystallography.The major work and results were described as follows:（1）The preparation of anti-S-OFL and anti-R-OFL Fab fragmentsThe enantioselective Fab fragments were prepared from enantioselective monoclonal antibodies digested by papain,followed by Protein L affinity chromatography and gel filtration.And the yield of Fab fragments was about 30%.（2）The characterization of anti-S-OFL Fab fragmentThe purified anti-S-/R-OFL Fab fragments were characterized by SDS-PAGE electrophoresis.Using peptide mass fingerprinting and MALDI-TOF mass spectrometry,the peptides of anti-S-OFL Fab fragment were sequenced and Fab fragment had a molecular weight of 51.8 kD.（3）ELISA assay of enantioselective Fab fragmentsBased on the indirect competitive ELISA protocol,both of two enantioselective Fab fragment calibration curves were established respectively.The IC₅₀ of anti-S-OFL Fab to S-OFL was 0.13 ng/mL,the linear range was 0.02-0.8 ng/mL and the LOD value was 0.007ng/mL.The IC₅₀ of anti-R-OFL Fab to R-OFL was 8.55 ng/mL,the linear range was 1.15-63.82 ng/mL and the LOD value was 0.35 ng/mL.Compared to the parent mAbs,Fab fragments still remained enantioselective.（4）The interaction between enantioselective Fab fragments and chiral isomersThe Biacore T200 instrument was used to study the interaction between enantioselective Fab fragments and S-/R-OFL isomers.It was found that the association rate constant and dissociation rate constant of Fab fragments were not all the same.The affinity constants K_D of anti-S-OFL Fab analyte to S-OFL and R-OFL ligands were 0.79μM and 99.7μM,respectively.In addition,the K_D constants of anti-R-OFL Fab analyte to S-OFL and R-OFL ligands were 0.41μM and 22.81 nM,respectively.（5）X-ray diffraction crystallographyAccording to vapour diffusion method,the protein crystals of enantioselective Fab fragments both in the free and complex forms in the presence of OFL enantiomers were obtained.The crystals were stable in the X-ray beam and all diffracted below 2.8?.Finally the preliminary three-dimensional structures of anti-S-OFL Fab fragment were solved by molecular replacement and the specific Y53 redidue of CDR-L2 region played a critical role in hapten-binding and chiral selectivity interaction,which could facilitate immonuassay in practice.