Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.
BACKGROUND
Variant Creutzfeldt-Jakob disease (vCJD) has a pathogenesis distinct from other forms of human prion disease: disease-related prion protein (PrP(Sc)) is readily detectable in lymphoreticular tissues. Quantitation of risk of secondary transmission, and targeting of risk reduction strategies, is limited by lack of knowledge about relative prion titres in these and other peripheral tissues, the unknown prevalence of preclinical vCJD, and a transmission barrier which limits the sensitivity of bioassay. We aimed to improve immunoblotting methods for high sensitivity detection of PrP(Sc) to investigate the distribution of PrP(Sc) in a range of vCJD tissues.


METHODS
We obtained tissues at necropsy from four patients with neuropathologically confirmed vCJD and from individuals without neurological disease. Tissues were analysed by sodium phosphotungstic acid precipitation of PrP(Sc) and western blotting using high sensitivity enhanced chemiluminescence.


RESULTS
We could reliably detect PrP(Sc) in the equivalent of 50 nL 10% vCJD brain homogenate, with a maximum limit of detection equivalent to 5 nl. PrP(Sc) could be detected in tissue homogenates when present at concentrations 10(4)-10(5) fold lower than those reported in brain. Tonsil, spleen, and lymph node were uniformly positive for PrP(Sc) at concentrations in the range of 0.1-15% of those found in brain: the highest concentrations were consistently seen in tonsil. PrP(Sc) was readily detected in the retina and proximal optic nerve of vCJD eye at levels of 2.5 and 25%, respectively of those found in brain. Other peripheral tissues studied were negative for PrP(Sc) with the exception of low concentrations in rectum, adrenal gland, and thymus from a single patient with vCJD. vCJD appendix and blood (Buffy coat fraction) were negative for PrP(Sc) at this level of assay sensitivity.


CONCLUSIONS
We have developed a highly sensitive immunoblot method for detection of PrP(Sc) in vCJD tissues that can be used to provide an upper limit on PrP(Sc) concentrations in peripheral tissues, including blood, to inform risk assessment models. Rectal and other gastrointestinal tissues should be further investigated to assess risk of iatrogenic transmission via biopsy instruments. Ophthalmic surgical instruments used in procedures involving optic nerve and the posterior segment of the eye, in particular the retina, might represent a potential risk for iatrogenic transmission of vCJD. Tonsil is the tissue of choice for diagnostic biopsy and for population screening of surgical tissues to assess prevalence of preclinical vCJD infection within the UK and other populations.
Wadsworth
J D
JD
MRC Prion Unit and Department of Neurogenetics, Imperial College School of Medicine at St Mary's, Norfolk Place, W2 1PG, London, UK.
Joiner
S
S
Hill
A F
AF
Campbell
T A
TA
Desbruslais
M
M
Luthert
P J
PJ
Collinge
J
J
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D007562:Creutzfeldt-Jakob Syndrome / Q000453:epidemiology / Q000378:metabolism* / Q000473:pathology / Q000635:transmission; D006801:Humans; D007049:Iatrogenic Disease; D008163:Luminescent Measurements; D014066:Palatine Tonsil / Q000737:chemistry; D010772:Phosphotungstic Acid; D018620:PrPSc Proteins / Q000032:analysis* / Q000302:isolation & purification; D015995:Prevalence; D012307:Risk Factors; D012680:Sensitivity and Specificity; D013525:Surgical Instruments; D014018:Tissue Distribution; D006113:United Kingdom / Q000453:epidemiology
D018620:PrPSc Proteins; D010772:Phosphotungstic Acid