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Didonna A, Venturini AC, Hartman K, Vranac T, Curin Serbec V, Legname G.2014. Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc. PeerJ PrePrints2:e694v1https://doi.org/10.7287/peerj.preprints.694v1
Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.
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Immune response analysis
Immune sera from three mice and two negative controls were probed by ELISA against recombinant human PrP (recHuPrP). Serial dilutions were prepared for each sample. The sera from immunized mice were able to recognized recHuPrP up to the 10-6 dilution while both negative controls gave no reaction.
Both GT1 and ScGT1 cells were treated with the different mAbs for 5 days, refreshing the medium on the third day. Then cell viability was evaluated by MTT assay according to the procedure described in the Materials and Methods section. No statistical differences in term of cell viability were found in mAb-treated cells compared to untreated controls. For every mAb the average values from 5 wells are expressed as percentages of cell viability referred to untreated cells.
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