NR ATKN
AU Tsukui,K.; Nakajima,K.; Takata,M.; Yokoyama,T.; Onodera,T.
TI Search of proteinase K-resistant PrP in plasma from scrapie-infected and uninfected hamsters
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-25
PT Konferenz-Poster
AB Proteinase K (PK) resistant isoform of PrP (PrPres) is the key molecule for understanding TSE. Thus, whether PrPres can be detected in blood is of great technical interest for diagnosis and epidemiological means. Because the infectivity of blood is calculated to be 10 to 20 ID50 / ml, amount of PrPres assumed to be on the order of pg/ml or less. PrPres has not been detected in blood except in cellular components because of a test method detecting this low protein concentration has not yet been reported. Using the most sensitive chemiluminescence protein detection substrate (PIERCE), a luminoimage analyzer for analyzing chemiluminescent signals, and anti-PrP monoclonal antibodies (mAb 3F4 or 6H4) as primary antibodies, we detected PK-resistant, anti-PrP monoclonal antibody-reactive proteins in the plasma of both scrapie-infected (ScHaPl) and uninfected hamsters (NorHaPl) by a conventional HRP-dependent immunoblot method. By digesting ScHaPl with 50µg/ml PK, 35~37-kDa and 20-kDa protein bands were clearly observed. Digesting saccharide chains by PNGase F treatment the 35~37-kDa and 20-kDa protein bands were shifted into lower-Mw position and 17~18 kDa proteins were observed. Thirteen to sixteen kilodalton proteins which appeared on 500µg/ml PK treatment remained unchanged with PNGase F treatment. The 35~37-kDa and 20-kDa proteins were observed in ScHaPl 40 days after postinfection, as well as in NorHaPl of the same age. The amount of 20-kDa protein in both plasma samples seemed to be relatively constant while that of 35~37 kDa proteins varied. Different appearances of these two protein fractions were further strengthened by PK treatment at higher concentration. Increase of the 20-kDa protein in ScHaPl as compared with that in NorHaPl was suspected. A synthetic peptide including the epitope sequence of 3F4 mAb absorbed the reactivity of this antibody; however, that of a scrambled sequence did not. These proteins could be used as a surrogate marker of TSE
AD Kazuo Tsukui, Kazunori Nakajima, Tokyo Metropolitan Red Cross Blood Center; Msuhiro Takata, Takashi Yokoyama, Research Institute for Prion Diseases, National Institute of Animal Health of Japan; Takashi Onodera, Department of Molecular Imunology, University of Tokyo
SP englisch
PO Deutschland