Pe streptavidin

H-2^b-restricted tetramers of peptides E_294–302, E_345–355, NS1_1237–1245, NS2_1479–1486, NS3_1759–1767, NS4_2140–2147 and NS5_2839–2848 were prepared as previously described [33 (link)]. Briefly, to produce biotinylated peptide-MHC protein, H-2D^b-heavy chain with a specific biotinylation site was modified at the C terminus of the α3 domain. The soluble H-2D^b/peptide complex was generated through recombinant H-2D^b and β₂m refolded in the presence of high concentrations of H-2D^b-restricted peptide. Then the H-2D^b/peptide complexes were purified over a Superdex 200HR column (GE Healthcare) and biotinylated by incubation with D-biotin, ATP, and the biotin protein ligase BirA (Avidity) at 4 °C overnight. The biotinylated H-2D^b was further purified over a Superdex 200 10/300 GL gel filtration column (GE Healthcare) to remove excess biotin and then mixed with PE-streptavidin (Sigma-Aldrich). For tetramer and surface marker staining, mouse splenocytes and single-cell suspensions of brain, spinal cord and testicular tissues were incubated with FITC-conjugated anti-CD3 mAb (Clone 17A2), PerCP-Cy5.5-conjugated anti-CD8 mAb (Clone 53-5.8), PE-Cy7-conjugated anti-CD4 mAb (clone GK1.5), and PE-conjugated tetramer at 4 °C in the dark. Multiparameter analyses were performed on a FACSAria™ II (BD Biosciences) and analyzed using FlowJo software (Tree Star).

RAW264.7 cells (1.5 × 10⁵ cells) cultured for 2 days in a six-well plate were washed with PBS twice and then incubated in culture medium. OVA-loaded liposomes (final lipid concentration: 0.1 mM) or OVA epitope peptide (SIINFEKL) were added gently to the cells, followed by incubation for 24 h at 37 °C. After incubation, the cells were washed with PBS three times and the presentation of OVA peptide on the cell surface was detected as follows: 3 × 10⁵ of cells were re-suspended in the staining buffer (PBS containing 10% BSA and 1% NaN₃) and incubated with anti-mouse CD16/32 antibody (3 μg/mL, eBioscience, 93) for 30 min on ice. After three times washing by staining buffer, cells were further incubated with biotinylated anti-mouse OVA_254–264 (SIINFEKL) peptide bound to H-2K^b (1 μg/mL, eBioscience, 25-D1.16) for 30 min on ice. After three times washing by staining buffer, cells were stained with streptavidin-PE (Sigma, St. Louis, MO, USA) and detected by a flow cytometer.

HRP2 was quantified using a highly sensitive laboratory based quantitative bead suspension array (qSA) based on Luminex technology [17 (link)]. After incubating 2’000 magnetic beads per analyte with blood sample at 1:5 dilution, beads were sequentially incubated with in-house biotinylated antibody α-HRP2 (MBS832975, MyBiSource, San Diego, CL) and streptavidin-PE (42250-1ML, Sigma Aldrich, St. Louis, MO). After a final wash, a minimum of 50 microspheres per analyte were acquired using the Luminex xMAP^® 100/200 analyser (Luminex Corp., Austin, TX). After subtracting background (blank) values, median fluorescent intensities (MFI) were normalized to account for plate to plate variation and quantification was performed against a 5-parameter logistic (5-PL) regression curve consisting of recombinant protein HRP2 type A (890015, Microcoat GmbH, Germany).