Maxisorb immunoplates

Four consecutive rounds of panning and phage amplification were performed with an enrichment onto solid phase (Maxisorb immunoplates, Nunc) immobilized procyclin. Overcoating was altered at each round to prevent the selection of overcoat-specific Nbs [milk, bovine serum albumin (BSA), casein, fetal bovine serum]. Individual clones were selected and evaluated in a periplasmic extract ELISA for binding onto procyclin absorbed into ELISA plates. Nb clones that achieved a ratio of > 2 for the optical density on the procyclin coat versus the overcoat were subjected to colony PCR using the MP57 and GIII primers and sequencing of the phagemid insert.

A monoclonal antibody to IL-1β (R&D systems) was diluted to 2 μg/ml in carbonate buffer and dispensed in 96-well Maxisorb Immunoplates (Nunc, Roskilde, Denmark) overnight. Samples were added for one hour, followed by the secondary antibody (a biotinylated goat anti-mouse IL-1β (R&D systems)). Extravidin-alkaline phosphatase (1:1,000, Sigma) was added for 1 hour after which 1 mg/ml 4-nitrophenyl phosphate substrate (Sigma) was added. After 20 minutes, plates were read at 450 nM and results were calculated by reference to a standard curve of recombinant murine IL-1β (R&D systems).

All serum samples were tested in an in-house indirect IgG enzyme-linked immunosorbent assay (ELISA) based on full length recombinant murine PrP and HaPyV VP1 protein. Maxisorb immunoplates (Nunc, Roskilde, Denmark) were coated with 1 µg of the corresponding protein, diluted in 0.05 M carbonate–bicarbonate buffer (pH 9.6). After incubating the plates overnight at 4 °C, they were washed three times with 300 µL washing buffer PBST, followed by a blocking step with 200 µL/well 10% skim milk powder diluted in PBS for 1 h at 37 °C. Serum (5 µL diluted in 120 µL 2% skimmed milk) was added, plates were incubated at 37 °C for 1 h and subsequently washed. 100 µL of HRP-conjugated protein G (Calbiochem), diluted 1:5000 in dilution buffer, were then added to each well. After another 1 h incubation step, plates were washed again and 100 µL substrate (2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS); Roche, Mannheim, Germany) was added to each well. Reactions were run for 30 min at RT in the dark, stopped by addition of 1% SDS and the optical density (OD) value at 405 nm was determined. Relative OD was calculated as OD from samples at corresponding timepoints t₀, t₂₁ and t₄₂ subtracted from t₀ value and divided by t₀ [(OD_tx − OD _t0)/OD_t0.]

Anti-IFN-γ antibody in sera was determined by indirect enzyme-linked immunosorbent assay (ELISA), as previously detailed [9] (link). Briefly, Maxisorb immunoplates (Nalge Nunc International, Penfield, NY, USA) were coated with 1 µg/mL of recombinant human IFN-γ in bicarbonate buffer (pH 9.6) and kept overnight at 4°C. Plates were blocked with phosphate-buffered saline (PBS) containing 1% non-fat milk powder at 37°C for 1 hour. Sera diluted 1∶1,000 were incubated for 1 hour at 37°C in duplicate. Plates were subsequently developed with anti-human IgG horseradish peroxidase conjugate (Caltag Laboratories, Invitrogen Life Technologies, Thermo Fisher Scientific, Paisley, UK) followed by o-phenylenediamine substrate (Sigma-Aldrich, St. Louis, MO, USA). The enzyme reaction was terminated with sulfuric acid (2N) and read for absorbance at 492 nm on a Spectra MR plate reader (Dynex Technologies, Chantilly, VA, USA).