Immunodetection of disease-associated mutant PrP, which accelerates disease in GSS transgenic mice.

The absence of infectivity-associated, protease-resistant prion protein (PrP(Sc)) in the brains of spontaneously sick transgenic (Tg) mice overexpressing PrP linked to Gerstmann-Sträussler Scheinker syndrome, and the failure of gene-targeted mice expressing such PrP to develop disease spontaneously, challenged the concept that mutant PrP expression led to spontaneous prion production. Here, we demonstrate that disease in overexpressor Tg mice is associated with accumulation of protease-sensitive aggregates of mutant PrP that can be immunoprecipitated by the PrP(Sc)-specific monoclonal antibody designated 15B3. Whereas Tg mice expressing multiple transgenes exhibited accelerated disease when inoculated with disease-associated mutant PrP, Tg mice expressing mutant PrP at low levels failed to develop disease either spontaneously or following inoculation. These studies indicate that inoculated mutant PrP from diseased mice promotes the aggregation and accumulation of pre-existing pathological forms of mutant PrP produced as a result of transgene overexpression. Thus, while pathological mutant PrP possesses a subset of PrP(Sc) characteristics, we now show that the attribute of prion transmission suggested by previous studies is more accurately characterized as disease acceleration.

Validation of a luminescence immunoassay for the detection of PrP(Sc) in brain homogenate.

A luminescence immunoassay (LIA) was developed for the diagnosis of bovine spongiform encephalopathy (BSE) in brain tissue using two different monoclonal antibodies for capture and detection of the protease-resistant fragment of the pathological prion protein (PrP27-30). PrP27-30 currently represents the most reliable marker for the infectious particle (denominated prion) causing transmissible spongiform encephalopathies (TSEs). Internal and official validation studies of this assay are described using brain homogenates from ascertained BSE positive and negative cows. Using more than 300 positive and 1400 negative bovine or ovine samples, an excellent sensitivity and specificity of 100% were demonstrated. More than 1000-fold dilutions of a BSE positive homogenate still resulted in a clear positive signal. In combination with a simple homogenisation procedure for the preparation of the samples, this assay lends itself for large scale screening of cattle and sheep for TSEs using complete automation of the process.