Analysis of mammalian scrapie protein by novel monoclonal antibodies recognizing distinct prion protein glycoforms: an immunoblot and immunohistochemical study at the light and electron microscopic levels.
The availability of specific monoclonal antibodies (mAbs) recognizing the aberrant form (PrP(Sc)) of the cellular prion protein (PrP(C)) in different mammalian species is important for molecular diagnostics, PrP(Sc) typing and future immunotherapy. We obtained a panel of anti-PrP monoclonal antibodies in PrP(0/0) knock-out mice immunized with recombinant human PrP(23-231). Two mAbs, recognizing PrP epitopes in the alpha-helix 1 (mAb SA65) and alpha-helix 2 (mAb SA21) regions, immunoreacted with PrP(C) and PrP(Sc) and its proteolytic product, PrP27-30, from human, murine, bovine, caprine and ovine brains by Western blot. Remarkably, mAb SA21 recognized unglycosylated and monoglycosylated PrP with the second site occupied by glycan moieties, but not monoglycosylated PrP with the first consensus site occupied or highly glycosylated species. Immunoblots with mAb SA21 disclosed that PrP glycosylated at the second site accounted for the slower migrating form of the customary monoglycosylated PrP doublet. mAb SA65 immunolabelled all PrP glycoforms by Western blot and was highly efficient in detecting tissue PrP by immunohistochemistry in light microscopy and in immunoelectron microscopy. These novel anti-PrP mAbs provide tools to investigate the subcellular site of PrP deposition in mammalian prion diseases and may also contribute to assess the role of different PrP glycoforms in human and animal prion diseases.
Matucci
Andrea
A
Section of Immunology, Department of Pathology, University of Verona, Policlinico G.B. Rossi, P. le L.A. Scuro 10, 37134 Verona, Italy.
Zanusso
Gianluigi
G
Gelati
Matteo
M
Farinazzo
Alessia
A
Fiorini
Michele
M
Ferrari
Sergio
S
Andrighetto
Giancarlo
G
Cestari
Tiziana
T
Caramelli
Maria
M
Negro
Alessandro
A
Morbin
Michela
M
Chiesa
Roberto
R
Monaco
Salvatore
S
Tridente
Giuseppe
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D001921:Brain / Q000276:immunology / Q000473:pathology / Q000648:ultrastructure; D002415:Cats; D002417:Cattle; D002478:Cells, Cultured; D006224:Cricetinae; D000939:Epitopes / Q000276:immunology; D006031:Glycosylation; D006041:Goats; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D046529:Microscopy, Electron, Transmission; D008970:Molecular Weight; D018620:PrPSc Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D017096:Prion Diseases / Q000175:diagnosis* / Q000276:immunology*; D012756:Sheep
D000911:Antibodies, Monoclonal; D000939:Epitopes; D018620:PrPSc Proteins