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Preparation of Specific Immunosorbent for Isolation and Purification of Cellular Prion Protein PrPc: Optimization of Immobilization and Process Parameters
Authors: Bílková Zuzana | Castagna Annalisa | Zanusso Gianluigi | Farinazzo Alessia | Monaco Salvatore | Lenfeld Jiří | Viovy Jean-Louis | Righetti Pierre Giorgio
Year: 2003
Type of publication: ostatní - přednáška nebo poster
Name of source: Final program and Book of abstracts
Publisher name: California Separation Science Society
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cze Příprava specifického imunosorbentu pro izolaci a purifikaci PrPc: optimalizace imobilizace a použití imunosorbent, PrPc, magnetický reaktor
eng Preparation of Specific Immunosorbent for Isolation and Purification of Cellular Prion Protein PrPc: Optimization of Immobilization and Process Parameters The cellular prion protein (PrPc) is a glycoprotein abundant in neurons. PrPc represents the substrate for the generation of a conformational pathogenic isoform in prion diseases. The published detailed 2-D map of human PrPc both in brain and CSF [Castagna, 2002] demonstrated the unusual microheterogenity of this glycoprotein. To investigate more thoroughly this diversity it is necessary to obtain enough material to analyse all modified forms by MS. Monoclonal antibodies are increasingly used to separate, isolate and purify complementary antigens. Immunoaffinity chromatography adds the selectivity and specificity of immunological reactions to the separation process. The goal in the construction of an immunosorbent was to immobilize the antibody to the solid-phase support without adversely affecting the antibody?s function to capture antigen [Matson, 1988]. For this reason we used both macroporous bead cellulose with a magnetic core and nonporous magnetic nanoparticles coated with polyNIPAM. The Magnetic core of the particles enables highly efficient and gentle separation during immobilizing procedure and during isolation and purification of PrP. Immobilization via magnetic beads is particularly suitable for on-line immobilization in a magnetically active microfluidic device (-CHIP) directly coupled to MS. 5B2 monoclonal mouse IgG 1 directed to a N-terminal epitope (residues 23-40) and 9H7 monoclonal mouse IgG 1 directed to a C-terminal epitope (residues 147-165) were used [Zanusso, 1998]. We considered and optimized the different factors important for the preparation of an efficient immunosorbent. We aim in particular at high steric accessibility of the binding sites, high operational and storage stability of binding activity, low non-specific sorption of protein to minimalize the contamination of the isolated antigen, and high recovery of antigen from immunosorbent. The quality of the prepared immunosorbents were confirmed by SDS-PAGE, by immunoblot Immunosorbent,cellular prion protein PrPc, magnetic reaktor

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