Polyclonal antibody production against recombinant Staphylococcal Enterotoxin A (rSEA) derived from a dairy mastitis isolate

Staphylococcal enterotoxin A (SEA) is one of the most prevalent toxins associated with staphylococcal food poisoning. However, only a few diagnostic methods provide adequate sensitivity and specificity for SEA detection. In this study, Staphylococcus aureus isolated from a dairy mastitis cow (strain FKH 182) was analyzed by polymerase chain reaction (PCR) and confirmed to carry the sea gene (127 bp). The SEA gene fragment encoding the mature peptide was amplified, cloned, sequenced, and inserted into a pET22b expression vector. The recombinant SEA (rSEA) protein was expressed as a hexahistidine-tagged fusion protein in Escherichia coli BL21 (DE3). Purified rSEA was then used to generate polyclonal antibodies (pAb) in Balb/c mice. Indirect enzyme-linked immunosorbent assay (ELISA) demonstrated that the anti-rSEA pAb reached a titer of 1:3200 at 59 days post-immunization. Western blotting confirmed the specificity of the antibody with a distinct band at approximately 30 kDa. Furthermore, the polyclonal antibody was applied in antigen capture-ELISA (AC-ELISA) to detect SEA in 50 human clinical Staphylococcus aureus isolates, with diagnostic sensitivity and specificity of 0.80 and 0.91, respectively, compared with PCR. These findings indicate that recombinant SEA is a reliable immunogen for pAb production and that the developed pAb can be applied as a practical tool for large-scale detection of SEA in clinical and food safety investigations.