Production of polyclonal IgY and monoclonal scFv antivenom antibodies against Formosan venomous snakes and their potential applications

keywords：anti-venom antibodies; immunoglobulin Y; phage display technology The laboratory in Center of Disease Control in Taiwan has manufactured at least 4 products of anti-venom sera, which was prepared through immunoprecipitation by ammonium sulfate method from venom immunized horses. However, 3 major immunological side-effects elicited by the exogenous proteins in horse sera were ofter encountered when the anti-venom products were applied to snake-bitten person. These include complement reaction, serum sickness and hypersensitivity reaction. In addition, many other disadventages using horse as the immunizing host are the relative high expense and tedious maintainence work in raising such a large animal. The collection of sera is a problem of humanity concern and also invasive to the horse, which may be a potentially risky task for the researchers during the procedure. Thus, it is urgent and crucial to develop a methodology for the production of polyclonal antivenom antibodies with high economical benefit, safety and effectiveness. Since 1990s, many reseachers have immunized avian animals and extract polyclonal antibodies capable of neutralizing venom activities from their egg yolk. These avain antibodies are generally termed as immunoglobulin Y （IgY）. With the tremendous improvement in biotechnology, using antibodies as the basis to cure the human diseases in clinic has recieved a lot of notices and become one of the main trends. Once the monoclonal anti-venom antibodies could be generated, they will provide a better specificity and quantity of antibodies. Not only could the use of horse and cell culture to produce antibodies be avoided, but the cost-effectiveness and high quality of antibody products could be achieved. At present, hybridoma method and phage display antibody technology 2 major techniques for generating monoclonal antibodies. In this proposed study, we will immunize the chickens with attenuated snake venoms to elicit high titers of polyclonal anit-venom antibodies, which will be purified from the eggs laid by the immunized chickens. Their binding specificity to venom proteins will be characterized and verified. The total RNAs will be extracted, reversely transcribed into cDNAs and amplified using RT-PCR method. The amplified heavy and light variable genes will be cloned and displayed on the surface of bacteriophage aprticles to establish antibody libraries. Monoclonal single-chain variable fragment （scFv） antibodies will screened by a novel biopanning procedure. Further, their binging specificity will be confirmed in a various assays such as ELISA, Western blotting, immunodiffusion, agar gel electrophoresis, LD50 toxicity tests and mice protection tests as carried out for testing the purified polyclonal IgY antibodies.