(228ey) Efficient Screening of Rabbit Scfv Antibodies with High Affinity By Use of Antigen-Coupled Multilamellar Vesicles (MLVs)

In this study, we developed efficient screening method for isolation of single-chain Fv antibodies with high affinity by use of antigen-coupled multi-lamellar vesicles (Ag-MLVs) as a solid support. MLVs are a kinds of liposomes that are composed of phospholipid bilayer. Antigen-coupled liposomes as a solid support have several advantages, compared with the conventional antigen-adsorbed plastic tube and even antigen-coupled magnetic particles. Non-specific interaction between phages and MLVs almost eliminated without blocking operation because the surfaces of MLVs with optimized phospholipid composition are rigid, hydrophilic and negatively-charged. Much high accessibility of antigen increases a chance to interact with specific phages in a library due to lateral diffusion of phospholipid bilayer. It is possible to display various proteins on surfaces of MLVs with high biological activity because the surface hydrophilicity of biological membrane stabilizes the proteins immobilized. We first demonstrated a model biopaning procedure using Ag-MLVs and Ag-Tube as solid surfaces, and positive phages showing specific affinity towards the antigen were tried to be isolated. Consequently, Ag-MLVs successfully isolated specific phages with 10% probability after 2nd round of biopaninng and the probability reached to 100% after 3th round of biopaning. Under the same condition, positive phage was not obtained by use of Ag-Tube. Furthermore, we also investigated to isolate the specific phages displayed with rabbit scFvs possessing high antigen-binding affinity toward a target antigen. Biopaning procedure was performed by use of antigen-coupled MLVs and scFv-displayed phage library. Consequently, 60 % of all phages were idensitfied as positive after the 1st biopaning procedure, and probability of positive clones were increased up tp 100% after the 3rd round of biopanning. According to the analyses of dissociation rate constants (k_off), dissociation rates of scFvs became gradually smaller, indicating that binding affinity of scFv moelcules isolated by Ag-MLVs must be increased by cycling a biopaning operation. Thus, the screening method developed in this study will be significantly useful for isolation of specific antibodies with high affinity, and the isolated antibodies would be valuable for various industorial use such as diagnostic agent and affinity ligand of chromatography.