Abstract ▼ AI-Helper

Development of novel chicken monoclonal antibody using classical methods like hybridoma technology to apply therapeutic and diagnostic reagents is time consumable, expensive and labor intensive. As an alternative, antibody engineering has been introduced. In this report, we try to modify the chicken...

Development of novel chicken monoclonal antibody using classical methods like hybridoma technology to apply therapeutic and diagnostic reagents is time consumable, expensive and labor intensive. As an alternative, antibody engineering has been introduced. In this report, we try to modify the chicken recombinant 6D12 scFv antibody as dimeric or trimeric scFv antibodies to improve affinity and stability. Furthermore, to bypass disadvantage of time consumable, expensive and labor intensive hybridoma technology, we constructed non-immunized chicken scFv library and selected antigen specific scFv antibody from library using Protein fragment complementation assay and phage display system. Chapter II explained thatmultimerization improves avidity and stability of recombinant scFv antibody by addition cysteine peptides and leucine zipper motif or isoleucine zipper motif. The addition of cysteine peptides and leucine zipper motif or isoleucine zipper motif to C-terminus of scFv was effective way to form homodimeric/trimeric scFv antibody molecules induced by disulfide bond or hydrophobic interaction. Since scFv antibody contains bi-/trivalency,this multimeric scFv antibodies should have higher avidity than monomer. Based on this concept, we di/trimerized previously developed monomeric 6D12 scFv by either covalent or non-covalent association as mentioned above. The recombinant 6D12 scFv was derived from chicken monoclonal antibody, 6D12-G10, that specifically bind to anterior tip of Eimeria sporozoites caused Avian coccidiosis. Di/Trimeric 6D12 scFv antibodies showed about 2~3 folds stronger antigen binding activity than monomer in ELISA analysis. In western blot and immunostaining analysis, di/trimeric 6D12 scFv antibodies showed same antigen specificity as shown in monomeric 6D12 scFv antibody as well. In Chapter III, chicken scFv antibody libraries were successfully constructed from non-immunized chicken B cell repertoire and diverse VH and VL genes were confirmed through DNA sequencing. Using constructed chicken scFv antibody library, Protein fragment complementation assay (PCA) was performed to select scFv antibody specific to tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and human insulin. Selected scFv clones specific to either DR4 or human insulin specific were subcloned to E. coli expression vector. The expressed scFvs showed antigen antigen binding activity through ELISA and westernblot analysis. In cell viability assay, selected chicken anti-DR4 scFv antibody showed apoptotic cell death of H460 cell which expressed necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor (DR4) on the cell surface. In immunocytochemistry, anti-DR4 scFv and anti-humman insulin scFv antibody also showed antigen binding activity against H460 and MIN cell which express DR4 and human insulin individually. In Chapter IV, Phage display to select scFv specific to Bovine serum albumin (BSA), partial HSP90 of Eimeria tenella sporozoites antigen (E.T. HSP90 F1) and hAY4 scFv antibody were performed using chicken scFv antibody phage library. The target specific clones were successfully selected through 3 or 4 round biopanning and were subcloned to E.coli expression vector. In ELISA and western blot analysis, anti-BSA scFv, anti-F1 scFv and anti-hAY4 scFv were showed antigen binding activity and antigen specificity. Additionally, anti-hAY4 antibody showed anti-idiotypic activity which was confirmed by competition assay using sandwich ELISA.