Opsonophagocytic antibody (OPA) killing assay

Assays were conducted as described [25]. HL-60 cells were purchased from the American Type Culture Collection and were maintained in 1x RPMI-1640 medium supplemented with 10% [v/v] heat-inactivated fetal bovine serum prior to use. Briefly, 10 μl of bacterial suspension (~700–1000 CFU) was added to 25 μl of antibody in each well for opsonization at 37°C for 15 min in a 5% CO₂ incubator, at which point 25 μl of BRC and 4 x 10⁵ HL-60 cells in 40 μl of media were added to each well. The 96 well plate was incubated at 37°C (no CO₂) with shaking agitation at 160 rpm for 45 mins. Antibody samples were adjusted prior to addition such that each sample contained an equivalent number of total anti-1925wzzB-COPS IgG EU in the 100 μl assay volume. Negative controls were performed with buffer in place of sera. Viable bacteria were determined after plating on rich media agar.