A quantitative real-time immuno-PCR approach for detection of staphylococcal enterotoxins
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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/μl) 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.
KeywordsImmuno-PCR Staphylococcus aureus Staphylococcal enterotoxin Superantigen Intoxication
We would like to thank Martina Schulte for excellent experimental assistance. The study was supported in part by research grants from the German Federal Ministry for Education and Science (BMBF) to Karsten Becker, Christof von Eiff, and Georg Peters in the context of the Pathogenomic Network (BMBF 0313134), from the Deutsche Forschungsgemeinschaft (EI 247/7-1) to Christof von Eiff, and to Karsten Becker, Thorsten Kuczius, and Georg Peters in the context of the project BMBF 0312733 and of the project ZM760374 from the Ministry for Education, Science and Research of Nordrhine–Westfalia.
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