Articles
A SENSITIVE AND SPECIFIC DETECTION OF ERWINIA AMYLOVORA BASED ON THE ELISA-DASI ENRICHMENT METHOD WITH MONOCLONAL ANTIBODIES.
ELISA is a very useful method for this purpose, especially because it allows the processing of a large number of samples.
Unfortunately its sensitivity may be low.
A procedure for a sensitive and specific detection of the bacterium is proposed, based on an enrichment step and the use of monoclonal antibodies (MCAs). The samples were prepared in a buffer with PVP, mannitol, ascorbic acid and glutathion; liquid King’s medium B or CCT selective medium is then added.
After incubation for 36 h at 26°C they were boiled and processed by Double Antibody Sandwich Indirect (ELISA-DASI). The plates were coated with a mixture of immunoglobulins from antisera against exopolysaccharides (strain 1430) and whole cells (strain PMV 6089). A mixture of two MCAs was used as intermediate and specific antibodies.
The method allowed the detection of 10–100 cells of E. amylovora per ml, mixed up with extracts from pear, apple and pyracantha tissues.
It has been used successfully with pure cultures of the bacterium as well as with naturally infected plant material.
The method was compared with isolation and PCR: it was found to be as sensitive as PCR and more sensitive than direct plating from plants.