The dramatically increasing prevalence of multidrug-resistant human pathogenic bacteria and related mortality requires two key actions: (i) decisive initiatives for the detection of novel antibiotics and (ii) a global ban for use of antibiotics as growth promotants in stock farming. Both key actions entail technology for precise, high-sensitive detection of antibiotic substances either to detect and validate novel anti-infective structures or to enforce the non-use of clinically relevant antibiotics. We have engineered prokaryotic antibiotic response regulators into a molecular biosensor configuration able to detect tetracycline, streptogramin, and macrolide antibiotics in spiked liquids including milk and serum at ng/mL concentrations and up to 2 orders of magnitude below current Swiss and EC threshold values. This broad-spectrum, class-specific, biosensor-based assay has been optimized for use in a storable ready-to-use and high-throughput-compatible ELISA-type format. At the center of the assay is an antibiotic sensor protein whose interaction with specific DNA fragments is responsive to a particular class of antibiotics. Binding of biosensor protein to the cognate DNA chemically linked to a solid surface is converted into an immuno-based colorimetric readout correlating with specific antibiotics concentrations. (C) 2004 Wiley Periodicals, Inc.