BackgroundThe accumulation of the misfolded forms of cellular prion protein, i.e. prions (PrPSc), in the brain is one of the crucial characteristics of fatal neurodegenerative disorders, called transmissible spongiform encephalopathies (TSEs). Cellular prion protein is normally linked to the cell surface by the glycosylphosphatidylinositol (GPI) anchor. There is accumulating evidence that the GPI-anchorless prion protein may act as an accelerator of formation and propagation of prions. In the TSE affected human brain we have previously discovered a novel GPI-anchorless prion protein fragment, named PrP226*, which ends with the tyrosine 226. This fragment can be labeled specifically by the monoclonal antibody V5B2.MethodsWe developed a DELFIA based assay for quick and sensitive detection of the PrP226* fragment in human brain tissue homogenates. By calculating the ratio between the signals of native (N) and denatured (D) samples applied to the assay we were able to observe significant difference between 24 TSE affected brains and 10 control brains. The presence of PrP226* in brain tissue was confirmed by western blot.ResultsOur results demonstrate that PrP226* is present in small quantities in healthy human brain, whereas in degenerated brain it accumulates in prion aggregates, proportionally to PrPSc. Samples with high D/N ratio generally comprised more proteinase K resistant PrP, while no correlation was found between the quantity of PrP226* and standard classification of Creutzfeldt-Jakob disease (CJD).ConclusionsIn the present study we show that the PrP226* fragment accumulates in prion aggregates and after being released from them by a denaturation procedure, could serve as a proteinase K digestion independent biomarker for human TSEs. The PrP226* assay described in this paper offers a tool to follow and study this unique anchorless PrP fragment in various parts of human brain and possibly also in other tissues and body fluids.

It was shown previously that truncated molecules of prion protein can be found in brains of patients with some types of transmissible spongiform encephalopathy. One such molecule, PrP226*, is a fragment of prion protein, truncated at Tyr226. It was found to be present in aggregates, from which it can be released using chaotropic salts. In this study we investigated the distribution of PrP226* in Creutzfeldt–Jakob disease affected human brain, employing the mAb V5B2, specifically recognizing this fragment. The results show that PrP226* is not evenly distributed among different regions of human brain. Among brain regions analyzed, the fragment was found most likely to be accumulated in the cerebellum. Its distribution correlates with the distribution of PrPSc.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of rare fatal neurodegenerative diseases, affecting humans and animals. They are believed to be the consequence of the conversion of the cellular prion protein to its aggregation-prone, β-sheet-rich isoform, named prion. Definite diagnosis of TSEs is determined post mortem. For this purpose, immunoassays for analyzing brain tissue have been developed. However, the ultimate goal of TSE diagnostics is an ante mortem test, which would be sensitive enough to detect prions in body fluids, that is, in blood, cerebrospinal fluid, or urine. Such a test would be of paramount importance also for screening of asymptomatic carriers of the disease with the aim of increasing food, drugs, and blood-derived products safety. In the present paper, we have reviewed recent advances in the development of immunoassays for the detection of prions.

Extended blood group genotyping is an invaluable tool used for prevention of alloimmunization. Genotyping is particularly suitable when antigens are weak, specific antisera are unavailable, or accurate phenotyping is problematic because of a disease state or recent transfusions. In addition, genotyping facilitates establishment of mass-scale patient-matched donor databases. However, standardization of genotyping technologies has been hindered by the lack of reference panels. A wellcharacterized renewable reference panel for standardization of blood group genotyping was developed. The panel consists of genomic DNA lyophilized and stored in glass vials. Genomic DNA was extracted in bulk from immortalized lymphoblastoid cell lines, generated by Epstein-Barr virus transformation of peripheral blood lymphocytes harvested from volunteer blood donors. The panel was validated by an international collaborative study involving 28 laboratories that tested each DNA panel member for 41 polymorphisms associated with 17 blood group systems. Overall, analysis of genotyping results showed >98% agreement with the expected outcomes, demonstrating suitability of the material for use as reference. Highest levels of discordance were observed for the genes CR1, CD55, BSG, and RHD. Although limited, observed inconsistencies and procedural limitations reinforce the importance of reference reagents to standardize and harmonize results. Results of stability and accelerated degradation studies support the suitability of this panel for use as reference reagent for blood group genotyping assay development and standardization.

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