3.3.30
TWO ENGINEERED ANTIBODIES (3DF1 AND 3CA5 SCFV) SPECIFIC FOR CITRUS TRISTEZA VIRUS REACTING IN ELISA AS CONVENTIONAL MONOCLONAL ANTIBODIES

E TERRADA1, R KERSHBAUMER2, G HIMMLER2, G GIUNTA3, P GALEFFI3, MT GORRIS1 and M CAMBRA1

1Instituto Valenciano de Investigaciones Agrarias (IVIA), Aptdo. oficial, 46113 Moncada, Valencia, Spain; 2Institute for Applied Microbiology (IAM),University of Agriculture, Muthgasse 18, A-1190 Vienna, Austria; 3Ente per le Nuove Tecnologie, l'Energia e l'Ambiente (ENEA), Cassacia, Rome, Italy

Background and objectives
Citrus tristeza (CTV) causes one of the most detrimental diseases of citrus, that has induced the decline of more than 65 million plants grafted on sour orange worldwide. The control of this economically important disease is based on the use of disease free budwood and tolerant rootstocks, quarantine, cross-protection and eradication programmes. These control strategies require a large number of detection tests, which are usually performed by ELISA using specific monoclonal antibodies (MCAs). The MCAs 3DF1 and 3CA5 (1) are the only antibodies that react against all CTV isolates tested, when they are used in a mixture. Both antibodies have been well characterised and are an international reference for CTV diagnosis and therefore, have been selected for cloning and expression in Escherichia coli to produce different scFv proteins to be directly used in routine ELISA tests. The final goal was to compare the usefulness of the engineered single-chain Fv fusion proteins with the conventional MCAs for CTV detection. This is the first report demonstrating that recombinant antibodies can be used as efficiently as the homologous MCAs for routine detection purposes.

Materials and methods
Amplification by PCR of VH and VL was made from purified 3DF1 and 3CA5 hybridoma (kept at INGENASA, Madrid) mRNA. The sequences were sequentially cloned in pDAP2/S and pZIP vectors (2). In addition, the 3CA5-scFv was subcloned in other vector (pCLZIP). ScFv constructions were expressed and the different fusion proteins (3DF1scFv-AP/S, 3CA5scFv-AP/S, 3DF1scFv-ZIP, 3CA5scFv-ZIP and 3CA5scFv-CLZIP) were purified and checked for binding by a number of ELISA variants.

Results and conclusions
The activity of the 3DF1scFv-AP/S and 3CA5scFv-AP/S was determined by direct immunoprinting-ELISA following a commercial kit protocol (Plant Print Diagnostics S.L., Valencia, Spain). The specificity of the recombinants was the same as the source MCAs. All isolates reacting with the MCAs 3DF1 or 3CA5 or with a mixture of them, reacted similarly with the recombinants alone or in mixture. The intensity of the reaction was similar by using the commercial kit for immunoprinting-ELISA or the engineered fusion proteins. These constructions fused with bacterial alkaline phosphatase proved to be as effective as the MCAs chemically conjugated to calf intestinal phophatase. The immunoprinting-ELISA system is an ideal test for a fast screening of the specificity of new constructions against "native" isolates of a virus.

The ability of the Zip-fusions to sensitize microplates has been tested by ELISA-DAS. Conventional MCA conjugates and recombinants-AP/S were assayed to develop the reaction. The results obtained show that an entire "recombinant ELISA" using both 3DF1 and 3CA5 constructions can be used for routine detection of CTV. The sensitivity of the engineered antibodies is similar to the conventional ELISA with MCAs for CTV detection. Serological tests can be developed and produced entirely based on the use of bacterial recombination and expression techniques with the same efficiency as the best MCAs. The outstanding performance of the obtained recombinant antibodies in its binding against any CTV isolate suggests that these particular constructions are the best candidates to be expressed in plants to assay resistance to the virus.

References
Vela, C., Cambra, M., Cortés, E., Moreno, P., Miguet, J.G., Pérez de San Román, C., Sanz, A. 1986. Journal of General Virology 67, 91-96.
2. Kerschbaumer, R., Hirschl, S., Kaufmann, A., Ibl, M., Koenig, R., Himmler, G. 1997. Analytical Biochemistry 249, 219-227.