(194f) Screening and Characterization of Rabbit Scfv Antibodies for Sensitive Detection of C-Reactive Protein in Clinical Diagnosis

Screening technology of recombinant antibody fragments such as VHHs, scFvs and Fabs is critical and significantly important for drug discovery, affinity chromatography and diagnosis. Phage display system has been utilized for this purpose, while those with insufficient affinity were sometimes major components and therefore, identification of high-affinity recombinant antibodies were sometimes laborious process after biopanning. Here, we developed and proposed a promising methodology for identification of high-affinity antibodies from phage library. In a new biopanning procedure, negatively-charged liposomes were used as a solid support to immobilize antigen, CRP. By use of antigen-coupled liposomes, a variety of scFv clones with antigen-binding activities were successfully isolated. On the basis of aminoacid sequences of CDR H3, scFv clones with different epitopes were distinguished. All of scFv clones were ranked according to dissociation rate constant, koff which was calculated from the data obtained by SPR sensor. Production levels and dissociation constants Kd of scFv clones ranked in top 10 were further evaluated. Antigen-binding activities of scFvs adsorbed or coupled on the solid supports were also compared with those of the conventional mouse monoclonal antibodies. Consequently, the best variants of rabbit scFv clones successfully were immobilized on the surface of PS latex beads, and quantitatively detected antigen by latex turbidimetric assay. Thus, the methodology of scFv identification which was developed in the present study will be significantly useful for screening of diagnosis-grade rabbit scFv clones with high antigen-binding activity and specificity.