Summary auto-generated
This study describes an improved sandwich conformation-dependent immunoassay (CDI) for detecting pathogenic prion protein (PrPSc) in human body fluids, particularly relevant to variant Creutzfeldt-Jakob disease (vCJD). The sandwich CDI incorporates a capture antibody to enrich prion proteins on microtitre plates, achieving 30- to 100-fold increased sensitivity compared to the original direct CDI method. The assay detects vCJD prions in plasma spiked with different prion preparations—crude brain homogenates, microsomal membranes, and purified PrPSc—at dilutions around 10^-24, even without proteinase K pretreatment. Testing with WHO CJD reference samples showed the sandwich CDI could detect both sporadic CJD and vCJD, with sensitivity comparable to infectivity bioassays in transgenic mice. The study demonstrates that the monoclonal antibody epitope depends on a correctly formed disulphide bridge between cysteines 179 and 214. The assay completes in less than 24 hours and enables detection of minute prion amounts in biological fluids, addressing the theoretical risk of prion transmission through blood products while accounting for uncertainty about the biophysical properties of prions that might circulate in blood.
Key findings
- Sandwich CDI achieves 30-100 fold increased sensitivity compared to direct CDI for detecting pathogenic prions in plasma
- The assay detects vCJD prions across different biophysical preparations to dilutions of 10^-24 without proteinase K pretreatment
- Sandwich CDI sensitivity is equivalent to infectivity bioassays in transgenic mice, enabling detection of clinically relevant prion levels
- The antibody epitope depends on a disulphide bridge between cysteines 179 and 214 in prion protein
- Assay turnaround time is less than 24 hours, making it practical for screening biological fluids
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Abstract
The presence of pathogenic prion protein (PrPSc) in lymphoid tissues of variant CreutzfeldtJakob 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 (PrPC). 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.