NR AWLO
AU Panigaj,M.; Brouckova,A.; Glierova,H.; Holada,K.
TI Potential complication of blood screening test development: case of cellular prion protein (PrPc) on red blood cells (RBC)
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions DIA-41
PT Konferenz-Poster
AB Third transmission of vCJD by blood transfusion in UK emphasizes the need for donor screening test for prion diseases. Their only specific marker is pathological form of prion protein (PrPsc), but its detection in blood is significant challenge. Blood contains substantial amount of PrPc both in plasma and blood cells. Conflicting data exist about the quantity of PrPc on RBC. To verify the presence of PrPc on RBC, we used quantitative flow cytometry with widely used monoclonal antibodies FH11, 3F4 and 6H4. We detected binding of 36 (FH11), 80 (3F4) and 260 (6H4) IgG molecules / cell. Low binding of FH11 and 3F4 could be caused by truncation of PrPc. Immunoblot with 6H4 showed that molecular weight (Mw) of RBC PrPc is slightly higher than brain PrPc and deglycosylation provided bands with identical Mw. No band corresponding to truncated PrPc was detected. The unavailability of 3F4 epitope is one of the hallmarks of PrPsc, but RBC PrPc was Proteinase K sensitive. Neither PrPc conformation does not play role in poor 3F4 binding nor glycosylation, because denaturation and deglycosylation of RBC PrPc did not improve its detection with 3F4. This suggests that epitope for 3F4 (MKHM) in RBC PrPc is modified. In vitro modification of free amino groups of brain PrPc by NHS-Biotin abolished 3F4 binding. We speculate that 3F4 epitope may be modified by advanced glycation end-products (AGE). AGE modification of brain PrPsc was reported recently. RBC are long living cells providing enough time for PrPc AGE modification. In accord with our hypothesis PrPc on erythroid cells from cord blood was detected equally well with 3F4 and 6H4, suggesting that modification of PrPc occurs after release of RBC into periphery. Methods utilizing FH11 and 3F4 may underestimate quantity of PrPc in RBC. Likewise, tests for presence of PrPsc in blood may encounter difficulties if modification similar to one reported here is present. (GACR 310/04/0419, GACR 310/05/H533, MSM0021620806).
AD Institute of Immunology and Microbiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic. E-mail: karel.holada@LF1.cuni.cz
SP englisch
PO Italien