DIRECT QUANTITATIVE CHEMILUMINESCENT ASSAYS FOR THE DETECTION OF VIRAL-DNA

A direct chemiluminescent dot blot hybridization assay for the detection of B19 Parvovirus DNA is described. The hybridization test uses digoxigenin-labelled probes which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase (AP) or with horseradish peroxidase (HRP). The chemiluminescent signal, obtained from an enzyme-triggerable dioxetane for AP or from the luminol-amplified reaction for HRP, is directly measured by placing the spot cut from the nylon solid support in a cuvette and inserting it into a luminometer. Both the enzymatic systems using this direct chemiluminescent detection gave reproducible results for calibration graphs and positive clinical samples, with higher reproducibility and long lifetime emission (15 days) for the AP-dioxetane system. This direct method allowed up to 0.2 pg of homologous target DNA to be revealed. The results obtained with the quantitative chemiluminescent assay described were compared with those obtained in a hybridization assay with colorimetric detection or photographic chemiluminescent detection and good agreement among the tests was found.