This assesses the complement-dependent bactericidal activity of antibodies in sera against particular bacterial isolates. SBA have been used to gauge natural immunoprotection against Salmonella in Africans ( MacLennan et al., 2008 and Pulickal et al., 2009), and are reported to be the best immunological surrogate of protection against meningococcal disease ( Frasch et al., 2009). Using an undiluted whole human serum SBA, our previous data demonstrate the necessity of both antibody and complement for

in vitro killing of Salmonella and provide evidence that bactericidal antibody protects against invasive NTS disease in Africans ( MacLennan et al., 2008). There are a number of variables associated with the design and optimization of SBA. Optimum conditions required for Salmonella SBA have not Akt inhibitor been reported. In the present study, we evaluated the complement requirements

of SBA for three isolates of Salmonella: invasive African Salmonella Typhimurium D23580, laboratory S. Typhimurium LT2, and laboratory Salmonella Paratyphi A CVD1901, using both endogenous and exogenous complement. Blood from healthy volunteers (1 European and 1 Asian) was allowed to clot and serum was separated within 2–3 h. Aliquots of sera (donor 1 and 2) were then stored at − 80 °C to preserve complement function. Pooled Malawian Metformin molecular weight serum was separated from blood samples taken from healthy adults in Blantyre, Malawi, and pooled prior to storage at − 80 °C. All individuals had no known clinical history of Salmonella

infection. Ethical approval was granted by the College of Medical Research and Ethics Committee, College of Medicine, University of Malawi. Three Salmonella isolates were used: S. 5-FU molecular weight Typhimurium D23580, S. Typhimurium LT2 and S. Paratyphi A CVD1901. S. Typhimurium D23580 is an invasive African isolate with MLST sequence type ST313 from a bacteremic child in Blantyre, Malawi ( MacLennan et al., 2008 and Kingsley et al., 2009). It is representative of most NTS isolates from bacteremic individuals in Malawi since 2002 and is sensitive to killing by healthy human adult serum ( Kingsley et al., 2009 and MacLennan et al., 2008). S. Typhimurium LT2 is a commonly-used laboratory isolate of S. Typhimurium ( Hoiseth and Stocker, 1981). S. Paratyphi A CVD 1901 is a laboratory guaA− mutant from the Center for Vaccine Development, University of Maryland School for Medicine ( Gat et al., 2011). Its attenuation permits the use of CVD 1901 in BSL2 containment laboratories. This isolate is unable to synthesize guanine. All bacterial isolates were grown aerated in 10 ml LB in loose-capped 50 ml Falcon tubes at 37 °C with shaking at 180 rpm. For serum bactericidal assays involving endogenous complement, 5 μl viable Salmonellae at 2 h log-growth phase and an OD of approximately 0.