Immunoreactivity enhancement with chelators for increasing the detection sensitivity of human PrPSc by Western blotting.
Prion diseases are neurodegenerative disorders affecting humans as Creutzfeldt-Jakob disease. The host-encoded prion protein (PrP(C)) will be converted into a structurally altered isoform (PrP(Sc)). PrP(Sc) differ in sizes and glycoform patterns and can be identified using molecular typing with Western blotting. The electrophoretic mobility of PrP(Sc) changes on treatment with metal ions or chelators prior to digestion with proteases. The effects of chelators applied to PrP(Sc) after protease digestion had not been examined in detail, we investigated these effects in this study. Application of EDTA, NTA and DTPA, and to a lesser extent EGTA, significantly enhanced PrP(Sc) signals in immunoblots. PrP(Sc) intensities increased two- to three-fold compared with untreated PrP(Sc). Since the immunoblot method is highly specific, sensitivity is the limiting factor. Enhancing sensitivity might be important in the determination of PrP(Sc) at levels close to or just below the limits of detection. It is to be expected that application of chelators to digested protein samples will increase the sensitivity of PrP(Sc) detection using the Western blot technique.
Kuczius
T
T
Institute for Hygiene, University Hospital Muenster, Robert Koch-Str. 41, 48149 Münster, Germany. tkuczius@uni-muenster.de
Brinkmann
B
B
Grassi
J
J
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002614:Chelating Agents / Q000378:metabolism*; D007562:Creutzfeldt-Jakob Syndrome / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D018620:PrPSc Proteins / Q000378:metabolism*
D002614:Chelating Agents; D018620:PrPSc Proteins