Antigenic characterization of an abnormal isoform of prion protein using a new diverse panel of monoclonal antibodies.
We established a panel of monoclonal antibodies (mAbs) against prion protein (PrP) by immunizing PrP gene-ablated mice with the pathogenic isoform of prion protein (PrPSc) or recombinant prion protein (rPrP). The mAbs could be divided into at least 10 groups by fine epitope analyses using mutant rPrPs and pepspot analysis. Seven linear epitopes, lying within residues 56-90, 119-127, 137-143, 143-149, 147-151, 163-169, and 219-229, were defined by seven groups of mAbs, although the remaining three groups of mAbs recognized discontinuous epitopes. We attempted to examine whether any of these epitopes are located on the accessible surface of PrPSc. However, no mAbs reacted with protease-treated PrPSc purified from scrapie-affected mice, even when PrPSc was dispersed into a detergent-lipid protein complex, to reduce the size of PrPSc aggregates. In contrast, denaturation of PrPSc by guanidine hydrochloride efficiently exposed all of the epitopes. This suggests that any epitope recognized by this panel of mAbs is buried within the PrPSc aggregates. Alternatively, if the corresponding region(s) are on the surface of PrPSc, the region(s) may be folded into conformations to which the mAbs cannot bind. The reactivity of a panel of mAb also showed that the state of PrPSc aggregation influenced the denaturation process, and the sensitivity to denaturation appeared to vary between epitopes. Our results demonstrate that this new panel of well-characterized mAbs will be valuable for studying the biochemistry and biophysics of PrP molecules as well as for the immuno-diagnosis of prion diseases.
Laboratory of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
Chan-Lan
CL
Kim
Atsushi
A
Umetani
Toshio
T
Matsui
Naotaka
N
Ishiguro
Morikazu
M
Shinagawa
Motohiro
M
Horiuchi
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000915:Antibody Affinity; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D019791:Guanidine; D007114:Immunization; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D010713:Phosphatidylcholines; D018621:PrPC Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology*; D018620:PrPSc Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010713:Phosphatidylcholines; D018621:PrPC Proteins; D018620:PrPSc Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C002448:1,2-linoleoylphosphatidylcholine; D019791:Guanidine