Rapid, high-throughput detection of PrP^Sc by 96-well immunoassay

Abstract

Prion diseases are emerging infectious disorders that affect several mammalian species including humans.  The transmissible agent is comprised of PrP^Sc, a misfolded isoform of the normal host-encoded prion protein PrP^C.  Immunodetection of PrP^Sc is often utilized for prion disease diagnosis and tracking spread of the infectious agent through the host.  We have developed a rapid, high-throughput 96 well immunoassay, which is specific for the detection of PrP^Sc.  This assay has PrP^Sc detection limits similar to Western blot and is advantageous because of its comparatively shorter running time, smaller start-up and operation costs, and large sample capacity.

Acknowledgements

This work was supported by the National Center for Research Resources (P20 RR0115635-6 and C06 RR17417-01), the National Institute for Neurological Disorders and Stroke (R01 NS052609) and the National Prion Research Program (NP020041).

Figures and Tables

Figure 1 96-well PrP^Sc immunoassay controls. (A) 96-well immunoassay and (B) quantification of brain homogenate from proteinase K digested uninfected (UN) or HY TME-agent infected (HY TME) hamsters with (+) and without (−) H₂O₂ in MeOH, primary antibody or secondary antibody to indicate the specificity of PrP^Sc immunodetection. Relative PrP^Sc intensity is based on well 16.

Figure 2 Limits of PrP detection using western blot analysis. Western blot analysis of two-fold serial dilutions of brain homogenates treated with (+) and without (−) proteinase K (PK) from uninfected hamsters (UN, A) or hamsters infected with the HY TME (HY) or DY TME (DY) agents (B and C, respectively).

Figure 3 Limits of PrP detection using 96-well immunoassay. 96 well immunoassay of two-fold serial dilutions of brain homogenate from uninfected (UN), HY TME agent (HY) or DY TME agent (DY) infected hamsters. Samples were either proteinase K (PK) digested (+) or were left untreated (−).

Figure 4 Comparison of PrP^Sc detection limits by western blot and 96-well immunoassay. Quantification of PrP^Sc abundance of two-fold serial dilutions of uninfected (UN), HY TME-infected (HY) or DY TME-infected (DY) proteinase K digested brain material by either western blot (open bars) or 96-well immunoassay (solid bars). Represented data is the average and standard error of the mean of three independent experiments.

Figure 5 Linear range of PrP^Sc detection by 96-well immunoassay. (A) 96-well immunoassay and (B) quantification of PrP^Sc from 2 fold serial dilutions of HY TME-agent infected brain homogenate digested with proteinase K.