Selective and Efficient Immunoprecipitation of the Disease-associated Form of the Prion Protein Can Be Mediated by Nonspecific Interactions between Monoclonal Antibodies and Scrapie-associated Fibrils

Transmissible spongiform encephalopathies are characterized by the accumulation in brain tissues of an abnormal isoform of the prion protein named PrPsc, which is the only direct marker known for transmissible spongiform encephalopathies. Here we show that PrPsc can be specifically immunoprecipitated by using several monoclonal antibodies (mAbs) of various specificities independently of the properties of their binding site (paratope). These results strongly suggest that a significant proportion of mAbs can interact with PrPsc aggregates through nonspecific paratope-independent interactions allowing selective immunoprecipitation of PrPsc when these mAbs are immobilized on a polydisperse solid phase like microbeads

Anti-PrP antibodies block PrPSc replication in prion-infected cell cultures by accelerating PrPC degradation.

manuscript received October 15, 2003; revised manuscript received December 15, 2003; accepted December 16, 2003. We thanks P. Rondard, O Bischof, J.-L. Laplanche and J.-P. Pin for their fruitful discussions. we are grateful to S. barrère for her assistance in the statistical analysis of the data and H. McMahon for her assistance in reading the manuscript

Isolation of Bovine Follicular Dendritic Cells Allows the Demonstration of a Particular Cellular Prion Protein

As interaction of cellular prion protein (PrPc) and the infectious agent (PrPres) appears to be a crucial pathogenic step promoted by homology, variation in PrPc isoforms on bovine immune cells may explain the absence of infectivity in most bovine lymph organs. In this study, we examined PrPc expression in bovine lymph organs (tonsils and lymph nodes) and on isolated follicular dendritic cells (FDCs). We used a panel of different monoclonal antibodies (MoAbs) raised against different epitopes of prion protein. Two MoAbs recognise amino acids 79-92 (SAF 34 and SAF 32 Mo-Abs); the 6H4 antibody reacts with a specific peptide comprising the 144-152 amino acids, and the 12F10 MoAb recognises the sequence 142-160. After immunolabelling of frozen sections of lymph organs with 6H4 or 12F10 MoAbs, we detected cellular prion protein in germinal centres. However, using the SAF 34 or SAF 32 antibodies, PrPc was revealed outside the lymphoid tissues. No PrPc was observed in the germinal centres. Therefore, we adapted the method of FDC isolation, making it suitable for the study of PrPc expression on their surface. Using electron microscopy, the presence of PrPc on the surface of FDCs was demonstrated only with 6H4 MoAb. These results suggest that bovine follicular dendritic cells express a particular form of prion protein. Either the N-terminal part of PrPc is cleaved or the accessibility of the specific epitope (79-92) of SAF 34 MoAb is abolished by interaction with other molecules. This particular isoform of PrPc on bovine FDCs might be related to the apparent absence of infectivity in lymph organs in cattle affected by bovine spongiform encephalopathy

Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen

International audienceThe immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG

Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells

International audiencePrion diseases are transmissible neurodegenerative disorders affecting humans and animals for which no therapeutic or prophylactic regimens exist. During the last three years several studies have shown that anti- PrP monoclonal antibodies (mAbs) can antagonize prion propagation in vitro and in vivo, but the mechanisms of inhibition are not known so far. To identify the most powerful mAbs and characterize more precisely the therapeutic effect of anti-PrP antibodies, we have screened 145 different mAbs produced in our laboratory for their capacity to cure cells constitutively expressing PrPSc. Our results confirm for a very large series of antibodies that mAbs recognizing cell-surface native PrPc can efficiently clean and definitively cure infected cells. Antibodies having a cleaning effect are directed against linear epitopes located in at least four different regions of PrP, suggesting an epitope-independent inhibition mechanism. The consequence of antibody binding is the sequestration of PrPc at the cell surface, an increase of PrPc levels recovered in cell culture medium, and an internalization of antibodies. Taken together these data suggest that the cleaning process is more likely due to a global effect on the PrP trafficking and/or transconformation process. Two antibodies, Sha31 and BAR236, show an IC50 of 0.6 nM, thus appearing 10-fold more efficient than previous antibodies described in the literature. Finally, five co-treatments were also tested, and only one of them, described previously (SAF34 _ SAF61), lowered PrPSc levels in the cells synergistically

The mechanism of internalization of glycosylphosphatidylinositol-anchored prion protein

The mode of internalization of glycosylphosphatidylinositol-anchored proteins, lacking any cytoplasmic domain by which to engage adaptors to recruit them into coated pits, is problematical; that of prion protein in particular is of interest since its cellular trafficking appears to play an essential role in its pathogenic conversion. Here we demonstrate, in primary cultured neurons and the N2a neural cell line, that prion protein is rapidly and constitutively endocytosed. While still on the cell surface, prion protein leaves lipid ‘raft’ domains to enter non-raft membrane, from which it enters coated pits. The N-terminal domain (residues 23–107) of prion protein is sufficient to direct internalization, an activity dependent upon its initial basic residues (NH(2)-KKRPKP). The effect of this changing membrane environment upon the susceptibility of prion protein to pathogenic conversion is discussed