A novel approach for scrapie-associated prion (PrPSc) detection in blood using the competitive affinity of an aggregate-specific antibody and streptavidin to PrPSc

Abstract

Scrapie is a fatal neurodegenerative disorder affecting sheep and goats, originating from exposure to disease-associated prions (PrPSc). An ante-mortem screening test that can detect native PrPSc in body fluids remains unavailable due to insufficient sensitivity of current detection methods that involve proteinase or denaturation treatments. We adopted an approach to detect PrPSc in whole blood using a simple proteinase- and denaturation-independent immunoassay, based on the competitive affinity of an aggregate-specific monoclonal antibody and streptavidin to PrPSc. First, we demonstrated the ability of native PrPSc to bind to streptavidin and the inhibition of this interaction by 15B3 antibody (P<0.05). This led to a new two-step assay that involved capturing native prions from infected blood on a solid-state matrix and detection of PrPSc aggregates by evaluating the conformation-dependent conjugate catalytic activity ratio in samples against a pre-determined threshold. This test showed capacity for detecting scrapie prions in 500μl of sheep whole blood spiked with scrapie brain homogenate containing approximately 5ng of total brain protein, and estimated to have 500fg of PrPSc. The test also discriminated between blood samples from scrapie-negative (6 sheep, 4 goats) and scrapie-infected animals (3 experimentally infected sheep, 7 naturally infected goats). Collectively, with the proposed high-throughput sample-processing platform, these initial studies provide insights into the development of a large-scale screening test for the routine diagnosis of scrapie.