Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput Serum Bactericidal Assay

is an important cause of diarrhea worldwide, with serotypes 2a, 3a, and demonstrating epidemiological prevalence. Many development efforts are focused on lipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization with vaccines containing LPS can elicit antibodies capable of killing in a serotype-specific manner. Thus, to facilitate vaccine development, we have developed a serum bactericidal assay (SBA) specific for three serotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies against 2a, 3a, and Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. The SBA, combined with a reference serum, should facilitate the development of vaccines across the field. is an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effective vaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuring -specific functional antibodies We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitate vaccine development.