Horseradish peroxidase conjugated goat anti mouse igg secondary antibody

FFA procedures were performed as previously described^{52 (link)}. Tissue samples were homogenized and clarified by centrifugation. Tissue supernatants and sera were diluted serially before infection on BHK cells. BHK cells used in this assay do not express Fcγ receptors. Cells were plated (2.0 × 10⁵ cells/well in a 24-well plate) and incubated overnight at 37 °C, 5% CO₂. Confluent monolayers were inoculated with undiluted or 10-fold serially diluted sera or clarified tissue supernatant, and were incubated for 1 hr at 37 °C. After incubation, the inoculum was removed, and each cell monolayer was overlaid with CMC-media and incubated at 37 °C, 5% CO₂ for 1.5–2 days. Cells were then fixed, permeabilized, and incubated with pan Flavivirus anti-envelope (E) Ab clone 4G2 (BioXCell), followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) and staining with True-Blue peroxidase substrate (Sera Care). Foci were counted, virus levels in the serum were expressed as Focus Forming Units (FFU) per mL, and for most tissues as FFU/g. As it was not technically feasible to weigh sciatic nerves, viral levels in these tissues were expressed as FFU/tissue.

Heparin-conjugated sepharose beads or heparin-unconjugated beads (60 µL) were washed with DMEM, and 1 × 10⁵ PFU of ZIKV in a volume of 60 µL was added to the beads. The mixture was incubated at 4 °C for 30 min to let the viruses attach to the beads. The unattached viruses were removed by washing the beads three times with DMEM containing 2% FBS. The bound viruses were lysed in 50 µL of Laemmli sample buffer (Alfa Aesar, Haverhill, MA, USA), and the proteins were separated by SDS-polyacrylamide gel electrophoresis. The proteins were then transferred to a nitrocellulose membrane (Bio-Rad) and blocked for 1 h with 5% bovine serum albumin at RT. Mouse monoclonal anti-flavivirus primary antibody (4G2) was used to probe the membranes at 4 °C overnight on a rocker. After washing the membrane with Tris-buffered saline with Tween 20 (TBS-T), horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) was added for 1 h at RT. The membrane was washed with TBS-T and developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) for acquiring the images using a ChemiDoc MP system (Bio-Rad).

To measure reactivity to H. cinaedi in mouse serum, an enzyme-linked immunosorbent assay was performed as described previously (34 (link)). To prepare serum, whole blood collected at autopsy was incubated at 4°C and then centrifuged at 2,000 × g for 5 min at 4°C. The supernatant was collected, mixed with glycerol (50% final concentration), and designated as serum. To prepare whole-cell lysates, H. cinaedi MRY08-1234 cells were disrupted by sonication. Each well of Nunc-Immuno 96-well microtiter plates (no. 439454; Thermo Fisher Scientific, Waltham, MA, USA) was coated with whole-cell lysates (4 μg/mL in 0.1 M carbonate/bicarbonate buffer, pH 9.4). After overnight incubation at 4°C, the plate was washed with PBS containing 0.05% (vol/vol) Tween 20 (PBS-T), and the wells were saturated with blocking buffer (PBS containing 1% BSA) and incubated for 1 h at 37°C while shaking at 500 rpm. After washing with PBS-T, the wells were filled with serum samples, which were diluted 100-fold with a blocking buffer. The dilution of serum was determined by preliminary experiments (Fig. S8). Plates were incubated for 1 h at 37°C with shaking at 500 rpm. Horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson, Bar Harbor, ME, USA) diluted 5,000-fold in blocking buffer was used. Absorbance was measured at 450 nm using a Multiskan FC microplate reader (Thermo Scientific).