The enzymatic activity of both chimeric toxins was demonstrated by ADP-ribosylation with 420 [micro]g/L protein and detection by Western blotting (Fig.
ADP-RIBOSYLATION IN A COLORIMETRIC SOLID-PHASE ASSAY
ADP-ribosylation by both DT and PE was detected by color development in the solid-phase assay, whereas heat-inactivated DT and CT, which ADP-ribosylates Gproteins, did not ADP-ribosylate eEF2, and no increase in the signals was observed (Fig.
Performing the ADP-ribosylation either in a separate vial or directly in the wells on immobilized eEF2 resulted in clear detection of DT activity (Fig.
Collier and Kandel (22) developed a system for the detection of the toxin-mediated ADP-ribosylation of eEF2.
Analysis of ADP-ribosylation by DT and PE has previously used radioactively labeled [NAD.
All techniques known to date rely on 3 basic principles: (a) the immunologic detection of DT such as the common Elek test (29) and a number of derivatives such as immunoblots, ELISAs [beta]0, 31), immunochromatographic strip assays [beta]2), assays based on fluorescence nanocrystal quantum-dots [beta]3), hydrogel-based protein microchips [beta]4), and antibody-based microarrays [beta]5); (b) the molecular biological detection of the tox-gene by PCR assays [beta]6); and (c) the detection of the enzymatic activity of DT by infecting guinea pigs (4, 31), in vitro translation assays, and ADP-ribosylation assays (22).