This work was supported by the UK Medical Research Council.
A highly sensitive immunoassay for the detection of prion-infected material in whole human blood without the use of proteinase K
Article first published online: 18 JUN 2010
© 2010 American Association of Blood Banks
Volume 50, Issue 12, pages 2619–2627, December 2010
How to Cite
Tattum, M. H., Jones, S., Pal, S., Khalili-Shirazi, A., Collinge, J. and Jackson, G. S. (2010), A highly sensitive immunoassay for the detection of prion-infected material in whole human blood without the use of proteinase K. Transfusion, 50: 2619–2627. doi: 10.1111/j.1537-2995.2010.02731.x
- Issue published online: 3 DEC 2010
- Article first published online: 18 JUN 2010
- Received for publication January 12, 2010; revision received April 25, 2010, and accepted April 25, 2010.
BACKGROUND: The causal association of variant Creutzfeldt-Jakob disease (vCJD) with bovine spongiform encephalopathy has raised significant concerns for public health. Assays for vCJD infection are vital for the application of therapeutics, for the screening of organ donations, and to maintain a safe blood supply. Currently the best diagnostic tools for vCJD depend upon the detection of disease-associated prion protein (PrPSc), which is distinguished from normal background PrP (PrPC) by proteinase K (PK) digestion, which can also degrade up to 90% of the target antigen.
STUDY DESIGN AND METHODS: A sandwich enzyme-linked immunosorbent assay method was developed using unique antibodies for the detection of disease-associated PrP in the absence of PK treatment. In combination with immunoprecipitation the assay was optimized for the detection of pathogenic PrP in large volumes of whole blood.
RESULTS: Optimization of the assay allowed detection of 2 × 104 LD50 units/mL spiked in whole blood. Application of the assay to clinically relevant volumes enabled the detection of 750 LD50 units/mL in 8 mL of whole blood.
CONCLUSION: By combining the use of a unique antibody that selectively immunoprecipitates PrPSc with glycoform-restrictive antibodies we have developed a rapid assay for vCJD infection that does not require any PK treatment to achieve high levels of specificity in whole human blood, the most challenging potential analyte. The sensitivity of detection of vCJD infection is greater than the equivalent of a more than 2.5 million-fold dilution of infected brain, providing a highly sensitive immunoassay compatible with blood screening.