Shigellosis is still a serious problem in low- and middle-income countries, where it falls hardest on children under five. The infection, caused by Shigella flexneri and other species, is a leading cause of diarrheal deaths, and antibiotic-resistant strains are making it harder to treat. As Shigella vaccines move through development, there is a growing need for in vitro methods that show whether vaccine-induced antibodies actually kill the bacteria, which is what a serum bactericidal activity (SBA) assay measures.
In a recent study, researchers at the GSK Vaccines Institute for Global Health optimized and characterized a high-throughput luminescence-based SBA (L-SBA) for functional antibody responses against seven common S. flexneri serotypes. Part of that work was checking how the assay performed with Pel-Freez baby rabbit complement, so we are summarizing it here.
How the assay was set up
The L-SBA was used to quantify functional antibody in human serum against S. flexneri serotypes 1b, 3a, 4a, 5b, 6, X and Y.
Baby rabbit complement was sourced from several suppliers, Pel-Freez among them, to see how the source affected performance. The optimal complement concentration varied by strain, from 7% for S. flexneri 4a and 5b up to 30% for S. flexneri 1b.
Sera were pooled from participants in clinical trials run in endemic regions and heat-inactivated to remove their own complement. Dilutions were made in phosphate-buffered saline or Luria Bertani medium depending on the target serotype.
Serial dilutions of serum were incubated with bacteria and complement under standardized conditions, and a luminescence readout measured bacterial viability after 3 hours. IC50 values, the serum dilution giving 50% killing, were calculated from those curves.
Repeatability and intermediate precision were assessed with independent replicates by two operators over several days. Linearity was checked by comparing theoretical and experimental IC50 values across serial dilutions. Specificity was tested by competing the sera with homologous and heterologous antigens.
What they found
The complement source did not change the answer. Comparisons between Pel-Freez and another baby rabbit complement showed no significant differences in IC50 values for S. flexneri 1b or in fold-change assessments for S. flexneri 3a.
Precision was high, with coefficients of variation below 7% for intermediate precision and below 3.9% for repeatability across all serotypes tested, and variance analysis found no significant effect of operator or day.
The assay was specific: homologous competitors reduced SBA titers by more than 60% for every serotype, while heterologous antigens such as S. sonnei or Salmonella typhimurium reduced them by 30% or less.
And the optimized conditions brought five more serotypes (4a, 5b, 6, X and Y) into the assay alongside the previously characterized 1b and 3a, so vaccine-induced immunity can now be assessed across the S. flexneri serotypes that matter most for vaccine development and surveillance.
Why it matters
A high-throughput SBA that is reproducible across operators, days and complement sources, and that covers seven serotypes, gives Shigella vaccine programs a practical tool for preclinical and clinical work. We are glad the baby rabbit complement held up in that comparison.
Disclaimer. The views and opinions presented in this blog article should not be considered to represent an endorsement or guidance on behalf of GSK or the study authors.