An adsorption method to prepare specific antibody-depleted normal human serum as a source of complement for human serum bactericidal assays for Salmonella

Abstract

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.