Intralaboratory evaluation of luminescence based high-throughput Serum Bactericidal Assay (L-SBA) to determine bactericidal activity of human sera against Shigella

BioRxiv : the Preprint Server for Biology
O. RossiFrancesca Necchi

Abstract

Despite the huge decrease in deaths caused by Shigella worldwide in the last decades, shigellosis is still causing over 200,000 deaths every year. No vaccine is currently available, and the morbidity of disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not been established yet, the demonstration of bactericidal activity of antibodies induced upon vaccination may provide one means of functionality of antibodies induced on protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA). Here we present the development and intra-laboratory characterisation of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against homologous strain without any heterologous aspecificity detected against species-related and not species-related strains. We assessed linearity, repeatability and reproducibility of L-SBA o...Continue Reading

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