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 (>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-0.98). Concentrations of protein A up to 10 ,ug/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.

Murine monoclonal antibodies against staphylococcal enterotoxin B: production and characterization

“…The titre was defined as the reciprocal of the highest dilution rendering an absorbancy > 0.1 at 450 nm as suggested by Bohach et al [3]. Titres ranged between 40 (B12) and 1280 (BS) ( Table 1); titres were much lower than those described by other authors [3,5,9,10,13]. To study the influence of a low coating concentration in these results, plates were coated with 1 /xg SEB/well as done by Edwin et al [6] for SEA.…”

“…There are many reports on the development and use of monoclonal antibodies (mAbs) produced against SEs [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. mAbs to all of the identified enterotoxins have been prepared [2], either specific or cross-reactive.…”

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins

“…Complete absence of signal in five other strains (NRS382, NRS384, NRS667, NRS725, NRS741) supported the view that the assay was specific for the target toxins and did not yield false positives for unrelated proteins, such as Protein A expressed by S . aureus [ 25 , 28 ].…”

“…Several investigators have demonstrated that ELISAs can detect staphylococcal toxins with good sensitivity [ 28 – 30 ], although concerns have been raised due to non-specific proteins in bacterial culture supernatants such as protein A that binds to IgG or naturally occurring peroxidases in food [ 25 , 28 ]. Commercial kits based on ELISA and ELFA using anti-enterotoxin antibody or antibody fragments (Fab) are available with reported sensitivities for staphylococcal enterotoxins A-E of 0.25 to 1 ng/ml.…”

Sensitive enzyme-amplified electrical immunoassay for protein A-bearing Staphylococcus aureus in foods

“…been developed (8,18). However, rapid methods for detecting the organism itself are needed, and levels (103 to 104 g-1)…”