A system to enhance the sensitivity of digoxigenin-labelled probe: detection of B19 DNA in serum samples

A highly sensitive dot-blot hybridisation assay for the routine screening of numerous samples is described. using parvovirus B19 as a model. Digoxigenin-labelled B19 DNA probe was constructed by PCR. hybrids were detected by an anti-digoxigenin monoclonal antibody followed by a second step. using anti-mouse antibodies conjugated to an alkaline phosphatase-dextran complex (EnVision(TM). Dako) was carried out. The sensitivity of the assay was evaluated using both colourimetric and chemiluminescent substrates for the alkaline phosphatase and was compared with a dot-blot hybridisation assay using the digoxigenin-labelled probe and a standard detection system. With the colourimetric substrate. the EnVision(TM) system was able to detect 10 fg of B19 DNA. while with the chemiluminescent substrate the sensitivity increased by up to fg (6 x 10(2) genome copies). This detection system was shown to increase the sensitivity of the assay compared to the standard colourimetric visualisation for the digoxigenin-labelled probe. which could detect 0.1 pg. On account of its sensitivity and specificity the dot-blot hybridisation assay together with the chemiluminescent substrate for the EnVision(TM) detection system was used to analyse 760 serum samples: the same sera were tested for B19 DNA with the standard colourimetric visualisation for the digoxigenin-labelled probe used routinely in the diagnostic laboratory. (C) 2001 Elsevier Science B.V. All rights reserved.