Synthesis of Pesticide-Specific Single-Chain F_V Utilizing the Recombinant Phage Antibody System (RPAS, Pharmacia). A Detailed Protocol

Abstract

Recombinant antibody (Rab) technology is expected to play an essential role in immunological research and basically in technologies which are utilizing the benefits of antibodies as a central component. Although commercial kits are already available for the synthesis of Rab, the Rab approach is far away from being considered a standard method. This is mainly due to the fact that the complex procedure frequently needs technical refinement in order to be adapted to individual experimental conditions and goals. In contrast, the manuals, supplied by the manufacturers, are appropriately designed to guide a general application of the Rab strategy. Here, we focus exclusively on the Recombinant Phage Antibody System (RPAS, Pharmacia). The RPAS kit is designed to provide a tool for the synthesis of phage-displayed and soluble Rab. This work is adressed to applicants of the RPAS, in order to encourage them to modify the manufacturers manual, especially if problems arise at particular steps when following the original protocol. Alternative options are subsequently proposed for critical steps including examples for pyrethroid- and s-triazine-specific scFv.

Key Words:

• allethrin
• atrazine
• enzyme immunoassay
• expression
• polymerase chain reaction
• phage display
• pyrethroid
• s-triazine
• single-chain F_V