Subclass optimised antibodies as an effective tool to suppress protein A induced false positives in immunoassays...

Staphylococcal enterotoxins (SEs) belong to a family of highly potent superantigens produced by Staphylococcus aureus and play a significant role in food poisoning and toxic shock syndrome. Among the SE family type B (SEB) is unique as it is involved in naturally occurring diseases and also has a history of military use as an incapacitating agent. Thus, the accurate detection of SEs and particularly SEB is critical. However, immuno-based detection methods encounter intrinsic difficulties due to S. aureus' co-production of staphylococcal protein A (SpA), which has a strong affinity for immunoglobulins. This interaction can result in false-positive results in antibody-based assays, thereby complicating the interpretation of results with regard to presence and quantity of SEB. Commercially available detection methods seek to address this issue through SpA depletion by pre-incubating samples with animal serum. While these approaches mitigate the impact of SpA interference, they frequently result in diminished assay sensitivity. In this study, a previously established highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for SEB detection was optimised to maintain its low detection limit in the one-digit pg/mL-range while simultaneously abolishing its reactivity to SpA in S. aureus liquid-culture supernatants. The modifications were focused on direct alterations to existing detection antibodies by a) adapting the immunoglobulin subclass and generation of antibody fragments through recombinant technology, and b) the careful selection of control capture antibodies. This study can be taken as a blueprint for optimised ELISA strategies to overcome SpA related false results while maintaining high sensitivity for SE detection and quantification.