Poster Presentation 20th Lancefield International Symposium on Streptococci and Streptococcal Diseases 2017

Development of a high throughput HL-60 opsonophagocytic killing (OPK) assay for group A streptococci (#177)

Sanaz Salehi 1 , Claudia M Hohn 1 , Thomas A Penfound 1 , James B Dale 1 2
  1. University of Tennessee Health Science Center, Memphis, TN, United States
  2. VA Medical Center Research Service, Memphis, TN, USA

Detection of opsonic antibodies against group A streptococci (GAS) has previously relied on the classic indirect bactericidal test of Lancefield, which uses “non-immune” human blood as a source of phagocytes and complement. The immune status of the blood donor and variable results among donors often affected the reproducibility and robustness of the assay. Based on previous studies with S. pneumonia and group B streptococci, we have adapted the HL-60 assay for use with GAS in a 96-well plate format. Optimal growth of GAS in media containing active complement and transformed HL-60 cells was supported by the addition of human fibrinogen, low levels of heparin, and pig serum. While not all emm types required all components, the goal was to develop a universal buffer that could be used in all assays. A reference serum pool was generated in rabbits immunized with the 30-valent vaccine. Experiments were performed to determine the optimal concentrations of fibrinogen, pig serum, and cells. Methods were developed to plate replicate samples that could be quantitated by capturing digital images. The specificity of antibody-mediated opsonization was assessed by peptide inhibition studies. OPK activity mediated by HL-60 cells against multiple emm types of GAS correlated with that observed using human blood with rabbit and human sera against the 30-valent vaccine. We believe the HL-60 OPK assay will facilitate the future clinical development of GAS vaccines by yielding consistent and reproducible results.