Fitc labeled goat anti mouse igg

On the 7th, 14th, 21st, 28th and 35th days after the first immunization, the anti-NanAT1-TufT1-PlyD4 antisera were collected from the immunized mice. The fusion protein NanAT1-TufT1-PlyD4 was diluted with an antigen coating solution to 5 μg/mL, 100 μl/well was added to a 96-well plate, and the plate was incubated at 4°C overnight. After washing and blocking were performed, the titers of specific IgG were determined by enzyme-linked immunosorbent assay (ELISA) via FITC-labeled goat anti-mouse IgG (Southern Biotech, USA). In addition, IgG subtypes were detected using fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG1, IgG2a, IgG2b and IgG3 (Southern Biotech, USA) following the manufacturer’s instructions as specific IgG. We expressed the antibody titers as the reciprocal of the highest dilution with an absorbance 2.1-fold higher than the background absorbance.

The following mAbs were obtained from eBiosciences or BD Biosciences (San Diego, CA, USA) for FACS analysis and other experiments: fluorescein isothiocyanate (FITC)-anti CD40 (3/23), CD80 (16-10A1), CD86 (GL1), MHC I (28-14.8), MHC II (25-9-17), phycoerythrin (PE)-anti CD11c (M1/70), F4/80 (BM8), TLR2(CD282; 6C2), Alexa Fluor488-anti-ERK1/2 (pT202/pY204; 20A), and STAT3 (pY705; 4/P-STAT3). mAbs specific for E (clone no. DE1) and NS1 (clone no. DN3) protein of DenV were obtained from Abcam. FITC-labeled goat anti-mouse IgG was obtained from Southern Biotech (Birmingham, AL, USA). Recombinant mouse TLR2-Fc chimera, a mouse TLR2 molecule fused with human IgG₁-Fc, was obtained from R&D Systems (Minneapolis, MN, USA). Chicken ovalbumin (grade V) and E. coli lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primers specific for DenV2 E protein (Forward 5′-CAA TAT GCT GAA ACG CGA GAG AAA-3′ and Reverse 5′-AAG ACA TTG ATG GCT TTT GA-3′) were synthesized at Bioneer Corp., Daejeon, Korea.

To determine hGIIA surface binding, 12.5 μl of bacterial cultures in mid-log phase (OD_600nm = 0.4 and 0.25 for GBS ΔgbcO) were added to wells of a sterile 96-well round-bottom plate (triplicates). hGIIA was serially diluted in HEPES solution without Ca²⁺ and added to the bacteria at indicated concentrations. After 30 minutes incubation at 4°C, bacteria were collected by centrifugation and resuspended in HEPES solution without Ca²⁺ containing 1:300 dilution of anti-phospholipase A₂ antibody (Merck Millipore) [28 (link)]. After incubation at 4°C for 30 minutes, the samples were washed and incubated with a 1:1,000 dilution of FITC-labeled goat-anti-mouse IgG (SouthernBiotech) or a 1:500 dilution of Alexa Fluor 647 conjugated goat-anti-mouse IgG (Jackson Immuno Research). After washing with HEPES solution without Ca²⁺, samples were fixed with 1% paraformaldehyde and fluorescence was recorded by flow cytometry (FACSVerse, BD Biosciences).