SOFIA provides, among other things, methods to diagnose prion diseases by detection of PrP in biological samples. Samples can be brain tissue, nerve tissue, blood, urine, lymphatic fluid, cerebrospinal fluid, or a combination thereof. Absence of PrP indicates no infection with the infectious agent up to the detection limits of the methods. Detection of a presence of PrP indicates infection with the infectious agent associated with prion disease. Infection with the prion agent may be detected in both presymptomatic and symptomatic stages of disease progression.
These and other improvements have been achieved with SOFIA. SOFIA's sensitivity and specificity eliminates the need for PK digestion to distinguish between the normal and abnormal PrP isoforms. Further detection of PrP in blood plasma has been addressed by limited protein misfolding cyclic amplification (PMCA) followed by SOFIA. Because of the sensitivity of SOFIA, PMCA cycles can be reduced, thus decreasing the chances of spontaneous PrP formation and the detection of false-positive samples. SOFIA meets the needs of increased sensitivity in the detection of prion diseases in both presymptomatic and symptomatic TSE infected animals, including humans, by providing methods of analysis using highly sensitive instrumentation, which requires less sample preparation than previously described methods, in combination with recently developed Mabs against PrP. The method of the present version of SOFIA provides sensitivity levels sufficient to detect PrP in brain tissue. When coupled with limited sPMCA, the methods of the present inventions provide sensitivity levels sufficient to detect PrP in blood plasma, tissue and other fluids collected antemortem.
The methods combine the specificity of the Mabs for antigen capture and concentration with the sensitivity of a surround optical fiber detection technology. In contrast to previously described methods for detection of PrP in brain homogenates, these techniques, when used to study brain homogenates, do not use seeded polymerization, amplification, or enzymatic digestion (for example, by proteinase K, or “PK”). This is important in that previous reports have indicated the existence of PrP isoforms with varied PK sensitivity, which decreases reliability of the assay. The sensitivity of this assay makes it suitable as a platform for rapid prion detection assay in biological fluids. In addition to prion diseases, the method may provide a means for rapid, high-throughput testing for a wide spectrum of infections and disorders.
While about 40 cycles of sPMCA combined with immunoprecipitation were found to be inadequate for PrP detection in plasma by ELISA or western blotting, the PrP has also been found to be readily measured by SOFIA methods. The limited numbers of cycles necessary for the present assay platform virtually eliminates the possibility of obtaining PMCA-related false-positive results such as those previously reported (Thorne and Terry, 2008).