RECOMBINANT ANTIBODIES TO POTATO VIRUS Y
N BOONHAM, S RUSSELL and I BARKER
Central Science Laboratory, Sand Hutton, York, YO4 1LZ, UK
Background and objectives
Materials and methods
The selection process involved 4 successive rounds of selection on tubes coated with PVY, as follows: (i) selection of phage by binding to antigen coated onto a solid phase; (ii) washing, to remove the non-binding phage; (iii) eluting 'binding phage' and re-infection of E.coli cells; (iv) amplification of binding phage in E.coli and purification of phage particles.
Results and conclusions
Following 4 rounds of screening, a number of scFv were selected with avidities similar to traditional monoclonal antibodies, which could be incorporated into an ELISA system (using readily available antibodies) to detect as little as 5ng of purified virus in plant sap. The scFv have been shown to be specific for the coat protein by western blotting. The ‘O’ specific recombinant antibody can detect 6 out of 8 isolates of PVY-O whilst being unable to detect 15 isolates of PVY-N by ELISA. Extensive testing of the ‘N’ specific reagents has still to be carried out, but so far no cross reaction with ‘O’ isolates has been observed.
This work was funded in part by MAFF Plant Health Division.