Generation, Characterization and Humanization of Chicken-Derived Single Chain Antibody against Venom Proteins Using Phage Display Technology

Bamboo vipers containing deadly hemotoxins commonly cause severe envenomation. Equine-derived antivenin is a specific antidote for treating snake-bites, but its production is costly and has some inevitable side effects. It is envisaged to develop an affordable and more endurable therapeutic strategy for clinical applications. In this study, we will use Trimeresurus stejnegeri (TS) venom proteins inactivated by glutaraldehyde to immunize hens to produce polyclonal immunoglobulin (IgY) antibodies. We will examine the IgY purified from egg yolks to show their specific anti-TS binding ability. Then, we will use phage display antibody technology to construct antibody libraries and determine the dimensions of M13 phages expressing single chain variable fragment (scFv) antibodies on the surface of viral particles. Increases in the titers of eluted phage will indicate specific anti-TS clones are remarkably enriched after each panning. We will analyze nucleotide sequences of heavy and light variable regions (VH and VL) of scFv antibodies to illustrate the identity of individual clone. We will check the binding specificity of the recombinant anti-TS scFv antibodies to TS venom proteins and potential cross-reaction to the venom proteins of other poisonous snakes commonly found in Taiwan. We will establish in vivo studies to show whether or not the anti-TS IgY and a panel of scFv antibodies could provide protective effects to augment survival rate and period of mice injected with lethal amount of TS venom proteins. Thereafter, we will further optimize, humanize and characterize all the chicken-derived anti-TS scFv antibodies with significant inhibitory activities. Combined together, all the antibodies generated in this study will be useful for the development of therapeutic antibody drugs and diagnostic reagents for TS snake envenomation in clinics. Thus, the objectives of this research proposal are as the followings:1. Characterization and comparison of venom proteins of TS and other snakes.2. Characterization of polyclonal anti-TS IgY antibodies derived from chickens.3. Production and characterization of monoclonal anti-TS scFv antibodies using phage display antibody technology.4. Investigation of the inhibitory effect of IgY and scFv antibodies on TS envenomation.5. Identification of essential components with important biological or toxic activities.6. Optimization and characterization of the binding specificity of these scFv antibodies by site-directed mutagenesis.7. Humanization and characterization of the binding specificity of chicken-derived anti-TS scFv antibodies.