Development of a Novel Ultra-Sensitive Immunoassay for the Detection of Prion Protein on Surgical Instruments

Murdoch, Heather (2008). Development of a Novel Ultra-Sensitive Immunoassay for the Detection of Prion Protein on Surgical Instruments. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f26c

Abstract

The detection of the infectious agents responsible for prion diseases remains problematic. The unusual nature of this infectious agent means that it is not inactivated by standard decontamination and sterilisation processes and there is evidence of transmission through blood transfusion. There is an urgent requirement for an assay to provide reliable validation of novel decontamination processes and a sensitive test for screening of blood donations and diagnosis of patients.

This research project aimed to address these problems, by the development of a novel ultra-sensitive detection method based on a thermostable adenylate kinase (tAK) antibody label coupled to ATP-bioluminescence. Utilisation of thermostability of AK enabled inclusion of background reducing heat steps within the format therefore enabling detection of specific antigen within a background of proteins.

The thesis describes development of the methodology from initial model assay to identification and production of a suitable recombinant tAK. An attempt to produce sensitive anti-prion polyclonal antiserum is also described. Finally, the use of the monoclonal antibody 6H4 (Prionics) labelled with tAK via a cleavable linker to establish limits of detection of the assay is described and established as 15 pg.mL^-1 of rec PrP using a fully optimised assay (670 fM). Sensitive detection of rec PrP within relevant biological substrates was also shown, along with some detection of infectious BSE (301V) MBH. There was also some evidence of detection on the surface of surgical grade stainless steel.

The use of this AK-ELISA is discussed within current scientific knowledge and specifically the continuing lack of available sensitive assay. The sensitivity achieved using this assay format was ultimately not sufficient to address this requirement, however the developed detection methodology has the flexibility to be coupled to new emerging technologies or disease specific antibodies, to provide an immediate increase in sensitivity in answer to this continuing need.