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Immunoaffinity reactors for prion protein qualitative analysis
Author(s) -
Bílková Zuzana,
Castagna Annalisa,
Zanusso Gianluigi,
Farinazzo Alessia,
Monaco Salvatore,
Damoc Eugen,
Przybylski Michael,
Beneš Milan,
Lenfeld Jiří,
Viovy JeanLouis,
Righetti Pier Giorgo
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401016
Subject(s) - chemistry , sodium dodecyl sulfate , gene isoform , gel electrophoresis , proteomics , glycoprotein , prion protein , affinity chromatography , biochemistry , chromatography , enzyme , gene , medicine , disease , pathology
The cellular prion protein (PrP c ) 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 PrP c 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 µ‐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.