Several lines of evidence suggest that microglia have important roles in the pathogenesis of prion diseases. Here, we establish a novel microglial cell line (MG20) from neonatal tga20 mice that overexpress murine prion protein. After exposure to Chandler scrapie, we observed the replication and accumulation of disease-associated forms of the prion protein in MG20 cells up to the 15th passage. Furthermore, MG20 cells were susceptible to ME7, Obihiro scrapie, and bovine spongiform encephalopathy agents. Thus, MG20 cell lines persistently infected with various murine prion strains provide a useful model for the study of the pathogenesis of prion diseases.

mAbs T1 and T2 were established by immunizing PrP gene ablated mice with recombinant MoPrP of residues 121-231. Both mAbs were cross-reactive with PrP from hamster, sheep, cattle and deer. A linear epitope of mAb T1 was identified at residues 137-143 of MoPrP and buried in PrP C expressed on the cell surface. mAb T1 showed no inhibitory effect on accumulation of PrP Sc in cultured scrapie-infected neuroblastoma (ScN2a) cells. In contrast, mAb T2 recognized a discontinuous epitope ranged on, or structured by, residues 132-217 and this epitope was exposed on the cell surface PrP C . mAb T2 showed an excellent inhibitory effect on PrP Sc accumulation in vitro at a 50% inhibitory concentration of 0.02 μg/ml (0.14 nM). The scFv form of mAb T2 (scFv T2) was secreted in neuroblastoma (N2a58) cell cultures by transfection through eukaryotic secretion vector. Coculturing of ScN2a cells with scFv T2-producing N2a58 cells induced a clear inhibitory effect on PrPSc accumulation, suggesting that scFv T2 could potentially be an immunotherapeutic tool for prion diseases by inhibition of PrP Sc accumulation.Key words anti-prion effect, monoclonal antibody, single-chain fragment variable region.Prion diseases, also called transmissible spongiform encephalopathies, are a group of neurodegenerative disorders, which includes BSE in cattle, CWD in deer and elk, scrapie in sheep and goats, and CJD in humans. The recent emergence of a new human prion disease, variant CJD, which is thought to result from consumption of BSEinfected materials by humans (1), is a major public health and safety issue (2, 3). The infectious agent, or prion, is mainly composed of PrP Sc , the detergent-insoluble and partially protease-resistant isoform of the host-encoded Anti-prion effect by scFv PrP Sc accumulation in prion-infected cell cultures, presumably by disrupting the PrP C -PrP Sc interaction (6, 7) and transgenic expression of anti-PrP mAb fragments prevents prion pathogenesis in mice (8). However, administration of mAbs has resulted in prevention of prion pathogenesis only when applied simultaneously, or shortly after, peripheral prion infection (9). This is probably due to poor diffusion of administered antibodies from vessels into tissues, particularly into the CNS. One study has demonstrated apoptosis of hippocampal and cerebellar neurons following intracerebral injection of mAbs reactive with PrP C (10), indicating that cross-linking or clustering PrP C by the anti-PrP antibody may trigger an abnormal signaling pathway. One possible means of delivering antiPrP antibodies efficiently into the CNS, and preventing undesirable adverse effects of such administration, is scFv antibodies, in which the polypeptides of antibody variable regions essential to antigen-binding are genetically linked through a polypeptide linker sequence (11-13). Because of its large size, a whole antibody molecule, with its relatively complex structure consisting of heavy and light polypeptide chains, is unsuitable for efficient gene manipulation with vectors. H...

No abstract

ABSTRACT. Surveillance for bovine spongiform encephalopathy (BSE) in fallen stock in Japan is conducted with a commercial enzymelinked immunosorbent assay (ELISA) for mass screening, with Western blotting (WB) and immunohistochemistry performed for confirmation of the ELISA. All tests are based on immunological detection of an abnormal isoform of the prion protein (PrP Sc ) in brain tissues, which have sometimes deteriorated by the time samples from fallen stock reach a diagnostic laboratory. To evaluate BSE surveillance procedures for fallen stock, we examined PrP Sc detection from artificially deteriorated BSE-affected bovine brain tissues with a commercial ELISA kit and compared the results with those of WB. The optical density (OD) values of the ELISA decreased with advancing deterioration of the tissues, whereas no reduction in the signal for PrP Sc was observed in WB, even when performed after 4 days of incubation at 37°C. The progressive decrease in the OD values in the ELISA appear to be caused by a partial loss of the N-terminal moiety of PrP Sc due to digestion by endogeneous and/or contaminated microbial enzymes, and by the presence of ELISA inhibitors that are generated in deteriorated tissues. These results suggest that WB is the most reliable test for fallen stock, especially for cattle brains within decaying carcasses. KEY WORDS: BSE, ELISA, PrP Sc , Western blot.

We purified the major outer membrane protein (MOMP), which is the most abundant OMP (with an apparent molecular mass of 40 kDa), from Haemophilus somnus strain 8025. The method involves solubilization of the MOMP with Zwittergent 3-14 and further purification accomplished by ion-exchange and molecular-sieve chromatographies. The amino-terminal sequence of the MOMP showed considerable similarity to those of porin proteins from other gram-negative bacteria. The MOMP of H. somnus is immunogenic to rabbits and calves. Hyperimmune sera from rabbits and calves reacted with both the MOMP and lipopolysaccharides in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The rabbit antiserum to the MOMP was cross-reactive with whole-cell preparations from strains 8025, D1238, NT2301, and 540 at a band with a molecular mass of 40 kDa in immunoblot analysis, although the reactivity of the rabbit antiserum with strain 540 was lower than those with the other strains tested. Two murine monoclonal antibodies (MAbs) to the MOMP were developed. ELISA with the OMP fractions as the antigens showed that one MAb was cross-reactive with the four strains but that the other MAb was reactive with the three strains other than strain 540. These results indicate that the MOMP of H. somnus possesses at least two antigenic determinants and that the MOMP of strain 540 is antigenically different from those of the other strains. The antigenic heterogeneity of the H. somnus MOMP has implications regarding the development of a serotyping system with MAbs that is based on the MOMP epitopes.