Recombinant hamster prion protein, corresponding to the mature form of hamster PrP containing five octarepeats, has been purchased from Prionics AG (Zurich, Switzerland). Homogenates from health hamster were prepared starting from about 2 grams of brain tissue disrupted in a Dounce homogenizer with 9 volumes of PBS/0.5 % Nonidet P-40/0.5 % sodium deoxycholate. Insoluble debris was removed by centrifugation at 1650 rpm for 20 minutes at 4°C. The supernatants were aliquoted and stored at -80°C.
The ETH-2 synthetic human recombinant antibodies library consists of a large array (more than 109 antibody combination) of scFv polypeptides displayed on the surface of M13 phage. It was built by random mutagenesis of the CDR3 of only three antibody germline gene segments (DP47 for the heavy chain, DPK22 and DPL16 for the light chain). Diversity of the heavy chain was created by randomizing four to six positions replacing the pre-existing positions 95 to 98 of the CDR3. The diversity of the light chain was created by randomizing six positions (96 to 101) in the CDR3 .
Selection of prion protein specific phage antibodies from ETH-2 library
Immunotubes were coated overnight (ON) at room temperature (RT) with rHaPrP in PBS at the concentration of 10 μg/ml. After panning, performed according with Ascione et al.  phages were eluted with 1 ml of 100 mM triethylamine and the solution was immediately neutralized by adding 0.5 ml of 1 M Tris-HCl pH 7.4. Eluted phages were used to infect log phase TG1 E. coli bacteria and amplified for the next round of panning. Briefly, 50 ml of 2xTY with 100 μg/ml ampicillin and 1% glucose (2xTY-amp-glu) were inoculated with enough bacterial suspension to yield an OD600 nm≅ 0.05–0.1. The culture was grown to OD600 nm 0.4–0.5 and infected with K07 helper phage in a ratio of around 20 : 1 phage/bacteria. The rescued phages were concentrated by precipitation with PEG 6000 and used for next round of panning. For monoclonal phage antibodies preparation, individual TG1 bacterial colonies harbouring phagemides were inoculated in 150 μl 2xTY-amp-glu in 96 well plates, incubated for 2 hours at 37°C and reinfected with 109 cfu K07 helper phage in 25 μl 2xTY. After 30 minutes, plates were centrifuged at 1800 g for 10 minutes and bacterial pellet resuspended in 200 μl 2xTY with 100 μg/ml ampicillin and 25 μg/ml kanamycin (2xTY-amp-kan). The following day the plates were spinned at 1800 g for 10 minutes and the supernatants containing phage antibody were recovered and tested in ELISA.
96 well ELISA-plates were coated ON with 0.5 μg of rHaPrP in PBS or 5 μl of homogenated brain extracts (from healthy hamsters) diluted in 50 μl of PBS ON at RT. Next day a blocking solution consisting of PBS with 2% non-fat dry milk (2% MPBS) was added; plates were washed with PBS containing 0.05% Tween 20 (TPBS) and incubated for 1 hour at RT with phage supernatants. Plates were washed and incubated for 1 hour with HRP mouse anti-phage antibody (Amersham) resuspended in 2% MPBS. The reaction was developed using 3,31-5,51-tetramethylbenzidin BM blue, POD-substrate soluble (Roche Diagnostics; IN, USA) and stopped by adding 50 μl of 1 M sulfidric acid. The reaction was detected with an ELISA reader (BIORAD; CA, USA), and the results were expressed as A (absorbance) = A(450 nm)-A(620 nm).
CCRF-CEM is a human T acute lymphoblastic leukaemia cell line. Jurkat is a human T acute leukemia cell line. RPMI 8226 is a human B Myeloma cell line. Nalm-6 is a human pre-B acute lymphoblastic leukemia cell line. HL60 is a human, acute promyelocytic leukemia, cell line. THP1 is a human, acute monocytic leukemia, cell line. H9 is a human T acute lymphoblastic leukemia cell line. SK-N-BE is a human neuroblastoma cell line.
Cells were cultured in RPMI-1640 supplemented with 10% foetal calf serum (FCS), L-glutamine (2 mM) penicillin (100 U/mL) and streptomycin (100 U/mL), all of which were purchased from Hyclone (Logan, UT). Regulation of prion protein expression in HL60 cell line was induced by culture in 1 μmol ATRA (Sigma) for 1 and 3 days. Cells were then stained for flow cytometry.
PBMC were separated from heparinized venous blood from healthy donors by centrifugation (20 min, 600 g) over Ficoll (Gibco) and activated by phytohemagglutinin P (PHA-P, 1 μg/ml, Sigma) for 4 days that stimulates T lymphocytes.
Flow cytometry determination of PrPc
The expression of PrPc on cells surface was determined by flow-cytometry studies. Cells in exponential phase of growth were collected, extensively washed in PBS and pelletted. About 5 × 105 cells were resuspended with 50 μl 2% MPB containing ≈ 1012 cfu phage particles and incubated for 1 hour RT. After several washings cells were resuspended for 1/2 hour RT in 2% MPBS solution containing 10 μg/ml of mouse M13 secondary antibody (Amersham), and after washings, cells were again incubated with 6 μg/ml FITC-goat anti mouse Ig (Pierce, IL USA) for 1/2 hour at 4°C. Controls include irrelevant phage antibodies directed to glucose oxidase and to tetanus toxin, an IgG2b isotype control and mAb 3F4 (Dako).
After staining cell samples were washed, maintained at 4°C and immediately analyzed by FACS can (Becton-Dickinson, NJ USA) equipped with 15 nW argon laser. Fluorescence compensation was determined using samples stained with mouse M13 and FITC-conjugated goat anti mouse secondary antibodies.
All experiments were done at least two times.
For cytospin preparation, 3 × 105 CCRF-CEM cells for slide were spinned at 600 rpm in cytospeen 3 Shandom centrifuge (Pittsburgh PA USA) for 5 minutes and immediately immersed in 80 % methanol solution for 10 minutes at 4°C. After three 5 minutes washes, in TBS, the slides were treated for ten minutes with peroxidase block solution from Dako En Vision system HRP (AEC), washed and blocked with RPMI and 10% FCS (both from Hyclone Laboratories) 10 minutes. The slides were incubated for 1 hour at RT in presence of 1011 cfu phage particles, extensively washed and incubated with mouse M13 secondary antibody (Amersham) for 1 hour RT. Finally treated with labelled polymer solution 30 minutes, and then with AEC substrate cromogen both belonging to the KIT. The counter stain was performed with mayer haematoxylin. Each incubation was followed by three washes in TBS.
DNA characterization and sequences
Plasmid DNA from individual bacterial colonies of MA3.B4 and MA3.G3 clones was digested with specific endonucleases and CDR3 regions were sequenced with an automated DNA sequencer (M-Medical/Genenco, Pomezia Italy) using fdseq1 (5'-GAA TTT TCT GTA TGA GG-3') and pelBback (5'-AGC CGC TGG ATT GTT ATT AC-3') primers.