A highly sensitive immunoassay for the detection of prion-infected material in whole human blood without the use of proteinase K.
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 (PrP(Sc) ), which is distinguished from normal background PrP (PrP(C) ) by proteinase K (PK) digestion, which can also degrade up to 90% of the target antigen.


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 2×10(4) LD(50) units/mL spiked in whole blood. Application of the assay to clinically relevant volumes enabled the detection of 750 LD(50) units/mL in 8mL of whole blood.


CONCLUSIONS
By combining the use of a unique antibody that selectively immunoprecipitates PrP(Sc) 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.
Tattum
M Howard
MH
MRC Prion Unit and the Department of Neurodegenerative Disease, Institute of Neurology, University College London, London, UK.
Jones
Samantha
S
Pal
Suvankar
S
Khalili-Shirazi
Azedeh
A
Collinge
John
J
Jackson
Graham S
GS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000918:Antibody Specificity / Q000187:drug effects; D058731:Blood Safety; D001921:Brain / Q000473:pathology; D046148:Donor Selection / Q000379:methods / Q000592:standards; D019286:Endopeptidase K / Q000378:metabolism* / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D006801:Humans; D007118:Immunoassay / Q000379:methods / Q000592:standards; D008403:Mass Screening / Q000379:methods / Q000592:standards; D051379:Mice; D017096:Prion Diseases / Q000097:blood / Q000175:diagnosis* / Q000473:pathology; D011328:Prions / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D020033:Protein Isoforms / Q000032:analysis / Q000276:immunology; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods / Q000592:standards
D000911:Antibodies, Monoclonal; D011328:Prions; D020033:Protein Isoforms; D019286:Endopeptidase K