Preparation And Characterization Of Fab Against S-ofloxacin

S-Ofloxacin(S-OFL) is the S-isomers of ofloxacin, belonging to the third-generation fluoroquinolones. It is broad-bactericidal, antibacterial, cheap, and widely used. In order to regulate its use, Enzyme-linked Immunosorbent Assay(ELISA), which is based on the antigen-antibody binding, has been used for S-OFL residues detection in food samples in recent years. However, it was found that antibody show different chiral selective recognition mechanism when binding S-OFL, Rac-OFL and R-OFL.To further study the chiral selective recognition mechanism of S-OFL antibody, we carried out the research of Fab production and purification. The majory research contents and results were as follows:(1) Preparation, purification and characterization of S-OFL monoclonal antibody.The hybridomas were obtained from our lab. Then the ascites were induced by intrapeirtoneal injection of well-grown hybridomas. The indirect ELISA suggested that the titer of ascites was over 1:16000 and the inhibition ratio was over 64%. The subclass tests showed that the ascites belong to mouse Ig G1. Protein G affinity chromatography was used to purify mnoclonal antibody(mc Ab) from ascites. SDS-PAGE electrophoresis and ELISA results showed that the anti-S-OFL mc Ab had high purity and good activity.(2) Preparation, purification and characterization of Fab fragments with ficin digestionThe anti-S-OFL mc Ab was digested with immobilized ficin to produce Fab fragments. The ficin addition, concentration of cystein, incubation time, p H were optimized to improve the digestion efficiency. Digestion of Ig G was performed in 25 m M cysteine(p H 5.6) and incubated for 7 h to prepare Fab fragments. The additional of ficin was 0.012 mg / mg Ig G. Then Protein G affinity chromatography and gel filtration chromatography were used to separate the Fab fragments from the digestion mixture of Ig G. However, the obtained fragments were aggregated and lose activity. The research showed it was the consequence of disulfide bond exchange cause by the increasing p H.(3) Preparation, purification and characterization of Fab fragments with papain digestionThe mc Ab was digested with immobilized papain to produce Fab fragments. The papain addition, concentration of cystein, incubation time, p H were optimized to improve the digestion efficiency. Digestion of Ig G was performed in 20 m M cysteine(p H 7) and incubated for 8 h to prepare Fab fragments. The additional of papain was 0.0083 mg / mg Ig G. Then Protein G affinity chromatography and gel filtration chromatography were used to separate the Fab fragments from the digestion mixture of Ig G. The research showed that papain cleaved the Ig G in a stepwise fashion. Papain first cleaved the C-terminal in the hinge region to generate 58 k D of Fab' fragments, and then cleaved the N-terminal in the hinge region to produce 42 k D Fab fragments.Amino acid sequencing results showed that the heavy chain and light chain of N-terminal was cut off 3 and 2 amino acids, respectively. However, the Fab fragment still remained good activity since the key CDR regions were still intact.(4) Characterization of Fab fragmentsThe indirect ELISA suggested that the Fab fragments had satisfied titer and inhibition ratio. After optimized, the coating antigen concentration of 2000 ng/m L and the Fab fragments dilution of 1000 were used. An indirect competitive ELISA(ic-ELISA) was developed and the linear range of this ic-ELISA system was 0.013-0.873 ng/m L, the IC50 was 0.105 ng/m L, the detection limit was 0.007 ng/m L. The Fab fragments showed satisfied cross-reactivity and chiral recognition as the mc Ab, which can be used for the further study such as crystallization.