1.11.49
NOVEL MOLECULAR TOOLS FOR PLANT VIRUS DETECTION: RECOMBINANT ANTIBODIES SPECIFIC FOR LUTEOVIRUSES AND GEMINIVIRUSES

L TORRANCE, A ZIEGLER, K HARPER, SM MACINTOSH, B ZANGE, S KUMARI and MA MAYO

Scottish Crop Research Institute, Invergowrie, by Dundee, DD2 5DA, Scotland, UK

Background and objectives
Antibody molecules are multifunctional polypeptides comprising identical heavy chains and identical light chains assembled as pairs of heterodimers. Each chain contains several domains of constant or variable sequence that have different functions. The domain that recognises and binds to antigen is called the variable domain, and there is one on each of the heavy (VH) and light chains (VL). The genes encoding the VH and VL domains (Fv fragment) can be cloned, and expressed together as single polypeptide chains (scFv). ScFv that bind to a range of antigens including plant viruses have been selected from large naive combinatorial libraries of scfv genes [1, 2]. Cloning and expression of scfv in Escherichia coli results in production of large amounts of defined and standardised antibodies quickly and cheaply. By these approaches we have prepared scFv that bind to various antigens including viruses. This poster shows some results obtained with scFv to tomato yellow leaf curl geminivirus (TYLCV) and potato leafroll luteovirus (PLRV).

Results and conclusions
Whitefly-transmitted geminiviruses cause economically important yellow leaf curl diseases of tomato. Different whitefly-transmitted geminiviruses have been shown to be serologically related, and some monoclonal antibodies (MAbs) prepared against African cassava mosaic geminivirus cross-react with viruses causing yellow leaf curl in tomato. The V genes of one cross-reacting MAb, SCR 20, were cloned and expressed as scFv fragments, and as fusions to the light chain constant domain (scFv-CL). The antibody fragments were readily expressed in E. coli. These fragments retained the same binding reactivity as the parent MAb. The scFv-CL fusion proteins were used successfully in tissue print immunoblotting tests and showed that TYLCV was located in the vascular cells of tomato stem sections.

PLRV is the most economically important virus disease of potato. ScFv that bind to PLRV were obtained from a phage display library after four rounds of selection on virus coated polystyrene tubes. The scfv were sub-cloned so that both scFv-CL and scFv-alkaline phosphatase (AP) fusion proteins were obtained on expression in E. coli. The conditions needed for high level expression of the scFv differed for each construct. After purification from bacterial cultures, the scFv-AP fusion proteins readily detected PLRV in extracts of infected potato in an ELISA using rabbit polyclonal antibodies to coat ELISA plates and scFv-AP as detecting reagent. The tests with the scFv-AP proteins gave similar values to those obtained in a triple antibody sandwich ELISA using existing polyclonal and monoclonal antibody reagents. The selection of scfv from naive libraries enables production of antibodies to any target antigen very quickly from a single stock of antibody genes kept in the refrigerator. Library-derived clones secreting recombinant antibodies can thus be obtained more quickly and cheaply than hybridomas secreting NIAbs. Moreover, the scfv retain the same qualities of unlimited supply of a standard reagent, without the costly maintenance and growth of mammalian cell lines. Our work demonstrates the feasibility of the approach and the potential applications to plant virus detection. Further developments will come from improvements particularly to enhance the binding to microtitre plates for virus capture.

Refererences.
1. Vaughan TJ, Williams AJ et al., 1996. Nature Biotechnol. 14, 309-314.
2. Ziegler A, Torrance L, Macintosh SM et al., 1995. Virology 214, 235-238.