Detection of staphylococcal enterotoxins A and B in cow milk using antigen capture enzyme-linked immunosorbent assay and dot-blot assays

Background and Aim: Staphylococcus aureus is a significant foodborne pathogen responsible for producing enterotoxins,
particularly staphylococcal enterotoxins A (SEA) and staphylococcal enterotoxins B (SEB), which are frequently found in milk and dairy products. These toxins in raw milk pose a risk to public health, necessitating accurate and rapid detection
methods. This study aimed to develop and evaluate two immunoassays – antigen capture enzyme-linked immunosorbent
assay (AC-ELISA) and dot-blot assay – for detecting SEA and SEB in cow milk. The sensitivity and specificity of these assays were compared with the standard polymerase chain reaction (PCR) technique.
Materials and Methods: A total of 30 raw milk samples from Boyolali, Central Java, were subjected to microbiological,
genotypic, and immunological analyses. The presence of S. aureus was confirmed through culture on Mannitol Salt Agar,
biochemical identification, and PCR targeting 23S ribosomal RNA, nuc, and coa genes. Recombinant SEA and SEB proteins
were used to generate polyclonal antibodies for immunoassay development. Dot-blot assays employed nitrocellulose
membranes blocked with 1% bovine serum albumin in tris-buffered saline-Tween 20, while AC-ELISA utilized these
antibodies for antigen capture. PCR confirmed the presence of the sea (127 bp) and seb (477 bp) genes. The performance
of the immunoassays was statistically evaluated in terms of sensitivity, specificity, and agreement with PCR.
Results: Out of 30 isolates, 27 (90%) were confirmed as S. aureus. PCR detected the sea and seb genes in 23.3% and 30.8% of isolates, respectively. AC-ELISA exhibited sensitivity and specificity of 71.4% and 85% for SEA and 75% and 89.5% for SEB, respectively. The dot-blot assay demonstrated higher sensitivity (85% for SEA and 87.5% for SEB) but comparable specificity (85.7% for SEA and 84.2% for SEB). Kappa values indicated substantial agreement between the immunoassays and PCR results.
Conclusion: Both AC-ELISA and dot-blot assays demonstrated considerable potential for detecting SEA and SEB in raw cow
milk. The dot-blot assay exhibited superior sensitivity, whereas AC-ELISA offered higher specificity. These immunoassays provide viable alternatives to PCR, particularly in resource-limited settings, offering cost-effective and rapid detection of S. aureus enterotoxins. Further refinement and validation with larger sample sizes are necessary to enhance diagnostic accuracy and minimize cross-reactivity.