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Isolation Of PrPSc and /or TSE strain specific llama heavy - chain antibody fragments by phage display - SE1777

Description
The aim of this project is to create PrPSc and/or TSE strain specific llama heavy chain antibodies (VHH) specific for PrPSc and/or particular strains of TSEs. VHHs are fragments of camel or llama antibodies, which are naturally devoid of the immunoglobulin light chain and thus consist of a single heavy chain segment. They are therefore suitable for phage display due to their single gene nature.

Two llamas will be immunised with bovine and ovine PrPSc four times in one month. RNA will be extracted on three occasions and used to make a phage display library. Initially, llama serum will be screened against PrPSc from scrapie and BSE cattle and recombinant ovine and bovine PrP by dot blot, western blot and ELISA to determine the success of the immunisation.If successful, a phage library will be made from the RNA of the immunised animals. This phage library will then be screened against various forms of PrP immobilised on nitrocellulose and competing against a soluble form of PrP to detect specific binding.

If the initial challenge is unsuccessful, llamas will be immunised with recombinant PrP and the screening process repeated.The forms of PrP that will be immobilised and/or act as a competitor will include ovine brain PrPSc rods (scrapie and BSE induced), bovine PrPSc brain rods, recombinant PrP and inclusion bodies. Binding will be carried out at a range of different temperatures and pH levels and at different detergent and GnCl levels.Once specific binding antibodies have been identified, they will be characterised for their specificity, affinity, and stability. They will also be checked for cross-reactivity between species.
Project Documents
• Final Report : Isolation Of PrPSc and /or TSE strain specific llama heavy - chain antibody fragments by phage display   (681k)
Time-Scale and Cost
From: 2002

To: 2004

Cost: £274,046
Contractor / Funded Organisations
Central Inst for Animal Diseases Control
Keywords
Animal Health              
Antibody              
Diagnosis              
Plants and Animals              
TSE              
TSEs              
Fields of Study
Animal Health