Transmissive spongiform encephalopathies (TSE) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrP(TSE)) in brain. PrP(TSE) is at present the only specific biochemical marker of human and animal TSE. As deposits of PrP(TSE) remain in the body for long periods, there is substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrP(TSE) may have potential to serve as a diagnostic marker. Monoclonal antibodies specific for carboxymethyl lysine/arginine-modified prion protein were prepared. Recombinant human prion protein (rhPrP) was bacterially expressed and purified by affinity chromatography. rhPrP was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of 6 mice, and 960 hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of four promising clones. One of them (EM-31) strongly reacts with human and mouse recombinant PrP-CML, and three other clones react also with CML in vitro modified human and mouse brain PrP. Besides possible implication in TSE diagnostics, the antibodies may serve as tolls to advance our knowledge regarding the role of glycation in the prion pathophysiology.

• MeSH
• arginin analogy a deriváty   chemie   metabolismus   MeSH
• glykosylace MeSH
• hybridomy imunologie   metabolismus   MeSH
• lidé MeSH
• lysin analogy a deriváty   chemie   metabolismus   MeSH
• monoklonální protilátky diagnostické užití   imunologie   metabolismus   MeSH
• mozek imunologie   metabolismus   MeSH
• myši knockoutované MeSH
• myši transgenní MeSH
• myši MeSH
• peptidy chemie   metabolismus   MeSH
• prionové nemoci diagnóza   imunologie   metabolismus   MeSH
• priony chemie   diagnostické užití   metabolismus   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Recent transmissions of variant Creutzfeldt-Jakob disease by blood transfusion emphasize the need for the development of prion screening tests. The detection of prions in blood is complicated by the presence of poorly characterized cellular prion protein (PrP(C) ) in both plasma and blood cells. According to published studies, most of PrP(C) in blood cells resides in platelets (PLTs) and white blood cells. STUDY DESIGN AND METHODS: To clarify conflicting reports about the quantity of PrP(C) associated with human red blood cells (RBCs), quantitative flow cytometry, Western blot (WB), and enzyme-linked immunosorbent assay (ELISA) were used to measure protein levels in healthy donors. RESULTS: RBCs expressed 290 ± 140 molecules of PrP(C) per cell, assuming equimolar binding of monoclonal antibody (MoAb) 6H4 to PrP(C). Binding of alternate PrP(C) MoAbs, FH11 and 3F4, was substantially lower. WB estimated the level of PrP(C) per cell on RBCs to be just four times lower than in PLTs. A similar level of PrP(C) was detected using ELISA. The weak binding of commonly used MoAb 3F4 was not caused by PrP(C) conformation, truncation, or glycosylation, suggesting a covalent modification, likely glycation, of the 3F4 epitope. CONCLUSIONS: Taken together, human RBCs express low but significant amounts of PrP(C) /cell, which makes them, due to high RBC numbers, major contributors to the pool of cell-associated PrP(C) in blood. Previous reports utilizing MoAb 3F4 may have underestimated the amount of PrP(C) in RBCs. Likewise, screening tests for the presence of the abnormal prion protein in blood may be difficult if the abnormal protein is modified similar to RBC PrP(C).

• MeSH
• Creutzfeldtova-Jakobova nemoc metabolismus   prevence a kontrola   přenos   MeSH
• ELISA MeSH
• epitopy imunologie   MeSH
• erytrocytární membrána metabolismus   MeSH
• erytrocyty metabolismus   MeSH
• glykosylace MeSH
• krevní transfuze MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• plošný screening metody   MeSH
• potransfuzní reakce MeSH
• PrPC proteiny imunologie   metabolismus   MeSH
• průtoková cytometrie MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH