Characterization of single chain variable fragment (scFv) antibodies against snake venoms from chickens with high titers of anti-venom IgY antibodies

Project: A - Government Institutionc - Ministry of Health and Welfare

Description

Anti-venom immunotherapy is the only effective treatment against snake venom poisoning. Anti-venoms are usually hyperimmune sera collected from animals which bind and inactivate venom components. Products of animal serum can produce adverse side effects such as anaphylactoid reactions and serum sickness. In order to reduce the side effects of the antisnake venom therapy it is essential to obtain antivenom in a sufficiently pure form. Recently, a new avian source of antivenom that precludes these complications and an efficient method for preparing antivenoms composed solely of venom specific antibodies are described. These avain antibodies are generally termed as immunoglobulin Y (IgY).It was estimated that chicken egg antibodies which are a progenitor of IgG antibodies phylogenetically, can be extracted from the egg to concentrations of approximately 100 mg of IgY/egg. Further, yolk antibodies do not activate the mammalian complement system or interact with mammalian Fc receptors that could mediate inflammatory response. It was reported that adult white leghorn hens hyperimmunized with Brazilian snake venoms produced antibodies capable of recognizing, combining with and neutralizing the toxic and lethal components of the venoms. The yolks of eggs laid by immunized chicken have been recognized as an excellent source of polyclonal antibodies for over a decade. Hens therefore produce a more hygienic, cost efficient, convenient and a plentiful source of antibodies, as compared to traditional method of obtaining antibodies from mammalian serum. In addition to polyclonal antibodies, monoclonal antibodies with higher specifities and binding affinities are particularly of interest and value in many scientifitic fields. Once the monoclonal anti-venom antibodies could be generated through the tremendous improvement in antibody-making technologies such as phage display antibody technology, they will provide a better specificity and quality of antibodies. Accordingly, 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. In a preious study, we immunized the chickens with inactivated snake venoms to elicit high titers of polyclonal anit-venom IgY antibodies, which have been quantitatively purified from the eggs laid by the immunized chickens. The purity of these polyclonal IgY antibodies are high as demonstrated by a major protein band with a molecular weight of 180 Kd on Coomassie blue stained SDS-PAGE. Their binding specificity to venom proteins are in the process of being characterized and verified. Once a strong humoral IgY response is elicited and confirmed, these immunized chickens will be sacrificed and their antigen-stimulated spleens will be used for antibody library construction. In the present study, we will continue the previous study to qauntitatively screen and characterize a panel of monoclonal singe chain variable fragment (scFv) antibodies from these established antibody libraries. 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.
StatusFinished
Effective start/end date1/1/1112/31/11

Keywords

  • anti-venom immunotherapy
  • immunoglobulin Y
  • single chain variable fragment (scFv) antibodies
  • phage display antibody technology