Test kits based on novel antibody fusion proteins (AFP) will be produced for the rapid, reliable and harmonised detection of three plant viruses of statutory or quarantine importance within the European Union. The AFP were obtained by recombinant DNA methods, and large quantities will be produced in vitro using heterologous expression systems. They will be incorporated into prototype self-contained test kits and the composition of the kits will be optimised for stability and robustness. AFP test kits will be provided to national testing services and agencies for validation. The results will be widely disseminated at meetings and in journals of appropriate scientific societies, plant health agencies, growers and trade organisations.
The aims of the project are to produce and validate test kits based on antibody fusion proteins (AFP) to detect and identify three harmful viruses: tomato spotted wilt virus (TSWV), potato leafroll virus (PLRV) and beet necrotic yellow vein virus (BNYVV), and to prove the benefits on a realistic scale. The target viruses are of statutory or quarantine significance and pose a threat to the free movement of plants and plant propagation material in Europe. Currently, laboratories obtain reagents for these viruses from a variety of different sources. With the introduction of plant passports, it is important to have confidence in the results of tests done across the European Union, so the standardisation of tests and harmonisation of test methods is desirable.
Yields of functional AFP produced in Escherichia coli, Drosophila and Pichia expression systems will be compared in pilot expression studies. Large quantities of AFP will be produced in the best systems and purified. Prototype kits will be designed and components optimised for stability and performance and these will be extensively evaluated by end users and modified as necessary.
Once the effectiveness of the new technology and of the reagents have been demonstrated, the relevant plant health authorities and technology users will be notified and we expect the results to influence policy and assist harmonisation of tests for these viruses.
Protocols and expression plasmids for production and purification of six AFP were devised.
E. coli was shown to be the expression system of choice for cheap and efficient production.
A major problem was the lack of stability of the purified AP fusion protein preparations in storage. This caused delays and unforeseen additional work, but a solution was found for four of the six AFP.
The storage conditions of lyophilisation (for long term) and suspension of conjugates in a buffer containing glycerol (for routine use), devised for BNYVV and PLRV reagents were shown to be robust and to meet the commercial needs.
The properties of individual scFv proteins vary and careful selection and optimisation must be done for each assay.
Two additional assay formats were devised (immunoelectron microscopy for BNYVV reagents and streptavidin/biotin assay for PLRV).
The streptavidin/biotin format provides a good alternative to the assay incorporating AP fusion proteins.
Trials to evaluate the reagents on a wide scale against commercially available kits were only partially completed. The delay caused by the AP fusion protein stability problems resulted in the project ending before extensive trials could be conducted.
A limited data set using the final protocols devised show that the BNYVV and PLRV reagents perform well in tests on control and naturally infected tissues.
This is the first demonstration that reagents produced from recombinant DNA methods and without the need for animals can be used in routine testing programmes for two plant viruses.
A major consideration before such reagents are commercialised is the costs of production and securing commercial licenses.
In the longer term, the results indicate that the broad application of recombinant fusion proteins to routine testing of a number of different target antigens is completely feasible.
CROP PESTS AND DISEASES, GENOMICS
Scientist responsible for the project
Ms LESLEY Torrance
DD2 5DA Dundee
United Kingdom (The) - GB
Phone: +44 1382 568525
Fax: +44 1382 562426
||The Scottish Crop Research Institute
||01 March 2000
||465 697 €
|Total EC contribution
||400 000 €