N BOONHAM, S RUSSELL and I BARKER

Central Science Laboratory, Sand Hutton, York, YO4 1LZ, UK

Background and objectives
Recent advances in recombinant DNA and gene expression technology has allowed the cloning of antibody genes into E.coli, and the subsequent expression of functional antibody fragments fused to the surface of a filamentous bacteriophage such as M13. The immortalisation of a library of antibody genes from an un-immunised spleen allows for the selection of antibody fragments that bind to any antigen.

Materials and methods
The library used was a single chain variable fragment (scFv) phage display library, obtained from the MRC, Cambridge (The Griffin Library). This was created by re-amplifying the heavy and the light chain variable regions from the lox library [1] and recloning into the phagmid vector pHen2 in a single chain format (scFv). This Library contains a large synthetic repertoire of 1.2 x 10⁹ clones.

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.

References
1. Griffiths AD, Williams SC, Hartley O et al., 1994. EMBO Journal 13, 3245-3260.