Hrp conjugated goat anti mouse igg antibody

PE-conjugated rat anti-mouse CD131 (mβc) antibody (BD Pharmingen, cat# 559920, RRID: AB_397374); Mouse anti-M13 antibody (SinoBiological, Beijing, China; cat# 11973-MM05T, RRID: AB_2857926); HRP conjugated goat anti-mouse IgG antibody (BioRad, South Granville, NSW, Australia; cat# 1706516, RRID: AB_11125547); DyLight 650 conjugated goat anti-mouse IgG antibody (Abcam, Melbourne, VIC, Australia; cat# 96874, RRID:AB_10679531); Isotype mouse IgG2a control antibody (Miltenyi Biotec, Macquarie Park, NSW, Australia; cat# 130-106-546, RRID:AB_2661589); PE-conjugated mouse anti-STAT5 (pY694) antibody (BD Biosciences, Macquarie Park, NSW, Australia; cat# 612567, RRID:AB_399858); PE-conjugated rat anti-mouse CD123 (IL-3Rα) antibody (eBioscience, San Diego, CA, USA; cat# 12-1231-82, RRID:AB_465839); AF488 conjugated rat anti-mouse β_IL-3 antibody (R&D Systems, Minneapolis, MN, USA; cat# FAB5492G, RRID: RRID:AB_2905558); Rat anti-mouse CD131 (mβc) antibody (BD Biosciences, cat# 740050, RRID:AB_2739817).

Serial dilutions of the samples were performed in DMEM – 2% FBS. Diluted samples were applied onto 90% confluence Vero cells monolayers for 2 h at 37°C, 5% CO₂. Inocula were removed after viral adsorption and were replaced by a mixed solution of DMEM – 4% FBS and PBS – 1% carboxymethyl cellulose in a 1:1 ratio. 3 days later, cells were fixated in PBS – 4% paraformaldehyde for 15 min at room temperature (RT), and permeabilized with PBS – 0.1% Triton for 3 min at RT. PBS – 1% bovine serum albumin – 0.1% Tween 20 was applied on cells for 30 min at RT in order to block non- specific antigenic sites. ZIKV envelope protein (E) immune-staining was performed using the mouse anti-E [clone 4G2; home-purified from the ATCC hybridoma (Monel et al., 2017 (link))] antibody as primary antibody and the HRP conjugated goat anti-mouse IgG antibody (Biorad) as secondary. Finally, after addition of freshly prepared peroxidase substrate (Vector Vip; Vector Laboratories) for 5–15 min, purple foci of infection were manually counted.

Peptide-specific serum and mucosal IgG was measured by enzyme-linked immunosorbent assay (ELISA) as previously described (10 (link), 42 (link)). Briefly, a specific peptide was coated onto Nunc MaxiSorp ELISA plates at 5 μg/ml. Samples were assessed using 2-fold serial dilutions of 1:100 of serum or 1:2 of saliva. Peptide-specific antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse-IgG antibody for murine studies (1:3,000 for serum and 1:100 for saliva; Bio-Rad Laboratories) or goat anti-rat-IgG for toxicology studies (1:10,000; Bio-Rad Laboratories). For direct binding to bacteria, Nunc MaxiSorp ELISA plates were coated with 200 μg/ml of heat-killed pM1 (emm 1) and 5448AP (emm 1). Samples were assessed using 2-fold serial dilutions of 1:100 of serum. Whole cell-specific antibodies were detected using goat anti-mouse IgG antibody (1:3,000) or goat anti-rat IgG antibody (1:5,000). For all assays, antibody detection was by SIGMAFAST OPD substrate (Sigma-Aldrich), which was added according to manufacturer’s instructions. Absorbance was measured at 450 nm on a Tecan Infinite M200 Pro plate reader (Tecan Group Ltd.). End-point titer was defined as the highest dilution that gave an absorbance of >3 standard deviations above the mean absorbance of the negative-control wells.