Identification and detection of Erwinia amylovora with monoclonal antibodies.
Hybridoma clones (48) secreting monoclonal antibodies (MAs) specific to E. amylovora were produced to identify and detect epiphytic and endophytic E. amylovora in pome fruit tissues by indirect ELISA and immunofluorescent staining. These MAs reacted positively in indirect ELISA against pure cultures of 75 E. amylovora isolates from different countries. Serological specificity of 37 of the MAs was further tested against 24 strs. of bacteria in 6 genera and against 56 unidentified epiphytes from pome fruit trees collected in different States. Ten clones of MA reacted specifically with E. amylovora and did not cross-react with any epiphytes or known bacteria; 9 of these produced strong fluorescent reactions in vitro to E. amylovora in indirect immunofluorescent staining and were further used for in situ detection in diseased apple tissues with an epifluorescent microscope. The MAs were also used to detect E. amylovora in vitro by a modified immunofluorescent direct-counting technique. Bacterial suspensions collected from infected pome fruit blossoms and apple fruits were concentrated in a microcentrifuge tube during staining, then collected on polycarbonate membranes by filtration. This technique conveniently detected 5 × 103 cells/membrane and was useful in detecting and monitoring infection by E. amylovora in pome fruit trees and fruits.