Biotin conjugated anti mouse iga

B. pertussis-specific antibodies in individual mouse nasal washes and lung homogenate supernatants were quantified by ELISA using plate-bound sBP (1 µg/mL), biotin-conjugated anti-mouse IgA (1:1500; Southern Biotech) or IgG2c (1:1500; Southern Biotech) and peroxidase-conjugated streptavidin. The reaction was developed using TMB and 1 M H₂SO₄ and the plates were read using a VersaMax microplate reader (Molecular Devices) at 450 nm.

RSV was conjugated with Dynabeads Intact Virus Enrichment (#10700D, Thermo Fisher Scientific) in accordance with the manufacturer’s instructions in such a way that the RSV was 1.0 × 10⁷ PFU per 1 µL of beads. The RSV-Beads were then incubated with 70 µL of nasal wash from immunized mice for 0.5 h at room temperature, followed by staining with FITC-conjugated anti-mouse IgG (1:100; #115-096-062, Jackson ImmunoResearch) or biotin-conjugated anti-mouse IgA (1:1000; #1040-08, SouthernBiotech) and allophycocyanin–streptavidin (1:1000; # 20-4317-U100, Tonbo Biosciences, San Diego, CA, USA). Antibody binding was assessed by measuring the intensity of fluorescent labeling by flow cytometry, and the mean fluorescent intensity of each group was measured and expressed with the value for RSV-Beads alone set to 1. Flow cytometric analysis was performed by using an Attune NxT flow cytometer, and all data were analyzed using the FlowJo software package (Supplemental Fig. 3).