Bacterial intoxications represent a substantial public health concern with
enterotoxins produced by Staphylococcus aureus among the most common causes of
food poisoning. In addition to their role in the pathogenicity of
food poisoning, staphylococcal enterotoxins have profound effects on the immune system as members of the family of pyrogenic toxin
superantigens. As the classical diagnostic bioassays as well as the routinely used immunological methods are hampered by several drawbacks regarding sensitivity, specificity, and practicability, there is a need for the timely identification of toxins by highly sensitive and specific methods. To combine the versatility of an
enzyme immunoassay (EIA) with the amplification power of the PCR, a quantitative real-time immuno-PCR (qRT-iPCR) was developed for the detection of
staphylococcal enterotoxins A and B and compared to a commercially available EIA. A broadly applicable tool for signal amplification of pre-formed immunocomplexes was established by covalent binding of a reporter
DNA to secondary detection
antibodies. Therefore, the amino-modified reporter
DNA was coupled successfully to N-succinimidyl-S-actyl-thioacetate-activated secondary detection
antibodies. The qRT-iPCR was able to detect highly reproducibly as low as approximately 0.6 to 6 pg (4 to 40 amol/microl) of
staphylococcal enterotoxin B and
staphylococcal enterotoxin A, respectively. In conclusion, the qRT-iPCR approach was shown to overcome clearly the sensitivity limit of traditional immunological detection procedures for
bacterial toxins, as demonstrated in this study for
staphylococcal enterotoxins. The development of a stable antibody-
DNA conjugate providing a universal signal amplification offers a versatile as well as a highly sensitive and specific tool for diagnostic and research purposes generally applicable for pre-formed antibody-
antigen complexes.