Quantitative determination of staphylococcal enterotoxin A by an enzyme-linked immunosorbent assay using a combination of polyclonal and monoclonal antibodies and biotin-streptavidin interaction

Abstract

A sandwich enzyme-linked immunosorbent assay to detect staphylococcal enterotoxin A (SEA) was developed by using monoclonal antibodies (MAb) to SEA as primary capture antibodies. The antigen was detected with purified rabbit anti-SEA antibody as the secondary antibody. The secondary antibody was identified by direct conjugation with biotin or via biotinylated sheep F(ab')2 fragments to rabbit antibody. The biotin was then reacted with avidin-alkaline phosphatase (AP) conjugate, avidin-biotin-AP conjugated complex, or streptavidin-AP conjugate. The enzyme was identified by using p-nitrophenylphosphate. The incorporation of the avidin-biotin-AP conjugated complex or streptavidin-AP conjugate augmented the sensitivity 32-fold over that of the enzyme-linked immunosorbent assay without these reagents. Controls were run by substitution of the anti-SEA MAb with unrelated MAb of the same isotype. Sample values were considered positive when the A405 exceeded those of the negative controls by 3 standard deviations (greater than 99% confidence interval). The toxin could be quantitated with purified SEA standards through linear regression analysis with lower detection limits of 4 ng/ml (r = 0.99) and 0.25 ng/ml (r greater than or equal to 0.98). Concentrations of protein A up to 10 micrograms/ml did not cause interference. Analyses of crude growth extracts of SEA-secreting strains of Staphylococcus aureus were reproducible and were expressed in terms of 95% confidence intervals. Lack of cross-reactivity was seen with extracts of other toxigenic and nontoxigenic strains of S. aureus. The assay can be completed in one working day, provided that MAb-coated plates are available.