The serum bactericidal assay (SBA) as a functional antibody test was performed to evaluate whether the specific antibodies created in each group have an in vitro bactericidal activity using the complement system or not. In addition, the titer of required antibodies to kill bacteria following complement activation was measured (43 (link)). For this purpose, the 96-well polystyrene round bottom microwell plates (Thermo scientific NuncTM, USA) were loaded for 30 min at 37°C with 12.5μl of prepared pneumococcal suspension (S. pneumoniae ATCC 49619) at 106 CFU/ml (based on the standard of 0.5 McFarland) and 12.5μl of dilutions of inactivated serum samples at 56°C for 30 min (1:2 to 1:64). Then, infant rabbit serum (4%) as a source of complement was added to each well. At two intervals (0 and 2 h), the content of each reaction well was serially diluted, and 10 μl from each well was inoculated into the blood agar plate. After 18-24 hours of incubation at 37°C in 5% CO2, the colony-forming unit of bacteria was measured. The titer of bactericidal antibodies is usually considered as the dilution of serum, which killed 50% of pneumococci compared to the control. The negative control was a well that contained bacteria and rabbit complement (23 (link), 36 (link), 40 (link), 44 (link), 45 (link)).
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