Competitive Luminex-based Immunoassay for Antibody Quantification

Competitive Luminex-based immunoassays (cLIA) were used to quantify antigen-specific binding antibodies elicited by the investigational vaccine. The assays monitor the ability of each vaccine component to elicit antibodies that can compete with the binding of antigen-specific monoclonal antibodies (mAbs) that have shown functional activity either in vitro or in vivo. The ClfA mAb prevents binding of live S. aureus to fibrinogen [10 (link)], while the MntC mAb inhibits manganese uptake [25 (link),26 (link)]. The mAbs used for the CP antigens facilitated killing of S. aureus as measured by the OPA assay. Spectrally distinct Luminex microspheres were coated individually with each of the antigens and incubated overnight with appropriately diluted serum samples. A mixture of phycoerythrin (PE)-labeled CP5-, CP8-, rmClfA-, and rP305A-specific mouse mAbs is then added to the microsphere/serum mixture, and after incubation, the unbound components are washed off. After reading in a Bio-Plex reader (Bio-Rad, Hercules, CA, USA), signals are converted to Units/mL.