ISHS Acta Horticulturae 704: X International Workshop on Fireblight
IMMUNOLIPOSOMES FOR EARLY DETECTION OF ERWINIA AMYLOVORA |
| Authors: | B.S. Borejsza-Wysocki, R.A. Durst, A.S. Aldwinckle |
| Keywords: | liposomes, magnetic beads, immunoassay, fire blight, antibody, protein G |
| Abstract:
A bioassay, being developed for the rapid detection of Erwinia amylovora, is based on the simultaneous immunomagnetic separation and rapid, highly sensitive signal-amplification strategies using marker-loaded immunoliposomes. In the bioassay, cells of E. amylovora are captured and concentrated from the sample extract by immunomagnetic beads, and immunoliposomes are simultaneously captured by the target bacterium. The resulting “sandwich” complex, consisting of numerous immunoliposomes bound to the bacterium which, in turn, is captured by the immunomagnetic beads, is then magnetically separated from the sample solution. After lysis of the immunoliposomes, the marker fluorescence is measured by a hand-held fluorometer, providing a signal that is proportional to the number of target bacteria. Before preparation of immunomagnetic beads and immunoliposomes, one polyclonal and three different monoclonal antibodies were immunotested for specificity to eleven strains of E. amylovora, one strain of Erwinia herbicola and one strain of Erwinia stewartii. Immunotests were performed by immunofluorescence to detect the presence of bacteria cells, and by immunoblotting (Western blots) to detect proteins extracted from the cells. Results from both immunotests indicated strong binding of the polyclonal antibody to all strains of wE. amylovora. However, this antibody was not highly specific and also recognized E. herbicola and E. stewartii, although these immunoreactions were much weaker compared to that with E. amylovora. In contrast, the monoclonal antibodies had high specificity and recognized only strains of E. amylovora. Monoclonal antibody Mab3B was used for preparation of the immunomagnetic beads and immunoliposomes. The E. amylovora (Ea 273) cells were separated from suspension (108 CFU/ml) by immunomagnetic beads and showed a strong positive signal with immunoliposomes. | |
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