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Fully “Recombinant Enzyme-Linked Immunosorbent Assays” Using Genetically Engineered Single-Chain Antibody Fusion Proteins for Detection of Citrus tristeza virus

December 2000 , Volume 90 , Number  12
Pages  1,337 - 1,344

Estela Terrada , Randolf J. Kerschbaumer , Giuseppe Giunta , Patrizia Galeffi , Gottfried Himmler , and Mariano Cambra

First and sixth authors: Instituto Valenciano de Investigaciones Agrarias, Carretera Moncada-Naquera km 4.5, 46113 Moncada, Valencia, Spain; second author: Institute of Applied Microbiology, University of Agriculture and Forestry, Muthgasse 18, A1190 Vienna, Austria; third and fourth authors: ENEA CR Cassacia, Dep. Innovazione, Div. Biotecnologie e Agricoltura, Sec. Biofisica, Biochimica and Biologia Molecolare, via Anguillarese, 30100060 S. Maria di Galeria, Roma, Italy; and fifth author: CODON Genetic Systems GmbH, Vienna, Austria

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Accepted for publication 23 August 2000.

Recombinant single-chain variable fragment antibodies (scFv) that bind specifically to Citrus tristeza virus (CTV), which cause the most detrimental viral disease in the citrus industry worldwide, were obtained from the hybridoma cell lines 3DF1 and 3CA5. These scFv were genetically fused with dimerization domains as well as with alkaline phosphatase, respectively, and diagnostic reagents were produced by expressing these fusion proteins in bacterial cultures. The engineered antibodies were successfully used for CTV diagnosis in plants by tissue print enzyme-linked immunosorbent assay (ELISA) and double antibody sandwich-ELISA. The fully recombinant ELISAs were as specific and sensitive as conventional ELISAs performed with the parental monoclonal antibodies, showing the usefulness of recombinant antibodies for routine detection of a virus in woody plants for the first time.

© 2000 The American Phytopathological Society