Immunoaffinity reactors for prion protein qualitative analysis.
The cellular prion protein (PrPc) represents the substrate for generation of conformational aberrant PrP isoforms which occur in human and animal prion diseases. The published two-dimensional maps of human PrPc show a vast microheterogeneity of this glycoprotein. The main goal of this project was to develop a highly specific immunoaffinity reactor for qualitative analysis of PrP cellular isoforms isolated from brain homogenate, cerebrospinal fluid and other tissues. New techniques for affinity proteomics, carriers and immobilization chemistry were applied. The choice of matrix (chemical and magnetic properties, particle size and distribution, porosity) was the key factor that influenced the quality of the reactor and the nature of final applications. Mouse anti-prion IgGs directed to N-terminal and C-terminal epitopes (residues 23-40 and 147-165) were grafted in different manners to magnetic micro- and nanoparticles particularly developed for micro-CHIP application. High operational and storage stability of affinity reactors with minimized nonspecific absorption were achieved. The quality of the immunoreactors was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and by two-dimensional electrophoresis.
Bílková
Zuzana
Z
Laboratory of Physical-Chemistry, Curie Institute, Paris, France.
Castagna
Annalisa
A
Zanusso
Gianluigi
G
Farinazzo
Alessia
A
Monaco
Salvatore
S
Damoc
Eugen
E
Przybylski
Michael
M
Benes
Milan
M
Lenfeld
Jirí
J
Viovy
Jean-Louis
JL
Righetti
Pier Giorgo
PG
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007163:Immunosorbent Techniques; D008280:Magnetics; D051379:Mice; D008855:Microscopy, Electron, Scanning; D049329:Nanostructures; D018621:PrPC Proteins / Q000032:analysis* / Q000276:immunology; D011487:Protein Conformation; D020033:Protein Isoforms / Q000032:analysis / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D018621:PrPC Proteins; D020033:Protein Isoforms