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Improved conformation-dependent immunoassay: suitability for human prion detection with enhanced sensitivity
A Bellon1, W Seyfert-Brandt1, W Lang1
1Virology, Aventis Behring GmbH, PO Box 1230, 35002 Marburg, Germany.
Abstract:
The presence of pathogenic prion protein (PrP(Sc)) in lymphoid tissues of variant Creutzfeldt-Jakob disease (vCJD) patients raises questions as to whether prions may be present in bodily fluids as well. Currently, transgenic mice are highly sensitive in vivo tools for the study of prions in tissues or fluids containing high levels of normal prion protein (PrP(C)). We report here an in vitro assay with virtually equivalent sensitivity incorporating a capture antibody into a sandwich conformation-dependent immunoassay (CDI), resulting in 30- to 100-fold increased sensitivity compared with the original, direct CDI. Furthermore, spiking plasma with vCJD prions in different preparations demonstrated that sandwich CDI detects prions with different biophysical properties at high sensitivity, even without proteinase K pretreatment of samples. Thus, sandwich CDI represents a powerful tool to study prions in bodily fluids of CJD/vCJD patients, with a turnaround time of less than 24 h.
Insights
A new in vitro assay, sandwich conformation-dependent immunoassay (CDI), detects pathogenic prions (PrPSc) in bodily fluids with high sensitivity. This powerful tool enhances prion disease diagnostics, offering results in under 24 hours.
Area of Science:
- Neuroscience
- Biochemistry
- Medical Diagnostics
Background:
- Pathogenic prion protein (PrPSc) presence in lymphoid tissues of variant Creutzfeldt-Jakob disease (vCJD) patients suggests potential presence in bodily fluids.
- Transgenic mice are sensitive in vivo tools for prion detection, but in vitro methods are needed for broader application.
Purpose of the Study:
- To develop a highly sensitive in vitro assay for detecting prions in bodily fluids.
- To improve upon existing conformation-dependent immunoassay (CDI) methods for prion detection.
Main Methods:
- Development of a sandwich conformation-dependent immunoassay (CDI) incorporating a capture antibody.
- Testing the assay's sensitivity and ability to detect prions with varying biophysical properties in spiked plasma samples.
- Comparison of the sandwich CDI sensitivity with the original direct CDI.
Main Results:
- The sandwich CDI demonstrated a 30- to 100-fold increase in sensitivity compared to the direct CDI.
- The assay detected vCJD prions in plasma with different biophysical properties, even without proteinase K pretreatment.
- The assay achieved high sensitivity for prion detection in bodily fluid samples.
Conclusions:
- Sandwich CDI is a powerful and highly sensitive in vitro tool for studying prions in bodily fluids of CJD/vCJD patients.
- This assay offers a rapid diagnostic potential with a turnaround time of less than 24 hours.
- The method advances prion disease research and diagnostics, particularly for bodily fluid analysis.
