FORPRION

BAVARIAN RESEARCH ASSOCIATION PRIONS

TUM 16 Development of specific recombinant anti- PrPSc antibodies

The principal goal of this project was the generation of recombinant antibodies that discriminate between the cellular prion protein (PrPC) and its disease associated isoform, termed PrPSc. In this regard, we embarked on an in vitro approach to isolate specific single-chain variable antibody fragments (scFv) directed towards the pathogenic isoform of human prion protein. Thus it was expected to eliminate a priori limitations of conventional hybridoma technology. For this purpose the immunogen PrPSc was prepared by extraction and purification from the brains of patients suffering from sporadic Creutzfeldt-Jakob disease (sCJD), Subsequently PrP knock-out (PrP0/0) mice were immunised with PrPSc and the PrPSc-specific immune response in treated mice was monitored using ELISA. After the final boost mRNA was isolated from murine spleen B cells and converted to cDNA, in order to produce template DNA for PCR amplification of antibody variable regions. Light and heavy chain genes were selectively amplified after optimisation of PCR protocolls using murine antibody V gene repertoire primers. In order to generate scFvs, individual V genes were randomly assembled via a (Gly4Ser)3 encoding DNA linker sequence by subsequent PCRs. Basically, various strategies for efficient V-gene amplification and scFv-assembly were investigated. The scFv repertoire was then cloned into the phagemid vector pCANTAB-5E to obtain phage antibody libraries. Transformation of E. coli strain TG1 with the recombinant phagemid yielded a library of 8800 transformed colonies. After rescuing phagemids from the library by superinfection with helper phage, phages containing PrP-specific antibodies should then be selected by affinity chromatography using the target protein via interaction with the corresponding antibody fragment displayed on the surface of recombinant phage particles.