Abstract
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•Serum bactericidal assays (SBA) are valuable for vaccine research but often require an exogenous complement source.•Pre-existing specific antibodies in normal human serum prevent its use as a complement source for SBA.•Pre-existing bacterial species-specific antibodies can be depleted by adsorbing with whole bacteria at 4 °C.•Our method easily generates a human complement source for Salmonella SBA from normal serum.
Serum bactericidal assays (SBA) are valuable for assessing the functional activity of natural and vaccine-induced antibodies against many Gram-negative bacteria, such as meningococcus and Salmonella. However, SBA often require an exogenous source of complement and the presence of pre-existing naturally acquired antibodies limits the use of human complement for this purpose. To remove pre-existing Salmonella-specific antibodies, in the context of SBA for Salmonella vaccine research, we incubated human sera with preparations of Salmonella. By incubating at 4 °C, pre-existing antibodies were adsorbed onto the Salmonella bacteria with only minimal complement deposition. We assessed the effects of adsorption on specific antibody levels, complement activity and the bactericidal activity of sera using flow cytometry, SBA and haemolytic assays. Adsorption removed Salmonella-specific antibodies and bactericidal activity against Salmonella from whole serum but was not detrimental to serum complement activity, even after five adsorption cycles. Bactericidal activity could be reconstituted in the adsorbed serum by the addition of exogenous specific antibodies. Sera preadsorbed with Salmonella are suitable as a source of human complement to measure the bactericidal activity of Salmonella antibodies. The adsorption method can be used to deplete, simply and rapidly, specific antibodies from serum to prepare a source of human complement for use in SBA for vaccine research and assessment.