European Journal of Plant Pathology 108: 479–483, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. Short communication Engineering of a single chain variable fragment antibody specific for the Citrus tristeza virus and its expression in Escherichia coli and Nicotiana tabacum Patrizia Galeffi, Giuseppe Giunta, Serena Guida and Cristina Cantale ENEA CR Casaccia, Biotechnology Division, via Anguillarese, 301-00060 S. Maria di Galeria, Rome, Italy (Phone: +390630486546; Fax: +390630484808; E-mail: galeffi@casaccia.enea.it) Accepted 6 March 2002 Key words: Citrus tristeza virus (CTV), immunodetection, single chain variable fragment (scFv), transgenic tobacco plants Abstract Citrus tristeza virus (CTV) is one of the most destructive citrus virus diseases in the world. The construction of an engineered antibody, EMBL accession number AJ278109, able to specifically recognize its antigen, i.e. the coat protein of CTV, directly on infected plant material without any purification or manipulation of the entire woody plant. The potential uses of this engineered antibody are discussed. Citrus agricultural products are important worldwide, both as fresh fruit and processed products. The Citrus tristeza virus (CTV), belonging to the Closteroviri- dae family, is a serious threat and a limiting factor for a competitive citrus industry in Europe (about 40 million trees lost in Spain since 1957). The action of this citrus pathogen could result in important econom- ical loss involving both production and fruit quality (Wallace, 1956; Bar-Joseph et al., 1979). CTV is semi- persistently transmitted by different aphid species and causes death of sweet orange, mandarin and grapefruit, especially when grafted on sour orange. Different strains of CTV have been isolated and show different infection abilities. The effect of infection depends not only on the strain, but also on the different species involved (insect vectors, grafted plant variety) and on the different combinations of plant and rootstock as well, resulting in different syndromes including stem pitting, quick decline due to phloem necrosis, and tree death (Bar-Joseph et al., 1989; Cambra et al., 1995). This virus cannot be controlled by chemicals and the morphological diagnosis of symptoms is only possible at an advanced stage of infection, when the epidemic is underway. Quarantine, the use of tolerant species such as rootstocks and/or cultivars, and the production of disease-free plant sources represent the preventive methods available to control this disease. New techniques and standard methods for early, quick and reliable identification of CTV isolates are needed to check the potential spreading of severe strains into Mediterranean areas. In recent years, several CTV-specific monoclonal antibodies raised against the CTV coat protein have been developed. ELISA procedures have improved CTV detection and provided ways to recognize epitopic variations in the coat protein which are often related to infection sever- ity (Vela et al., 1986; Permar et al., 1990). Recently, it has been shown that engineered antibodies are able to play an important role in diagnosis (Fecker et al., 1996; Toth et al., 1999; Terrada et al., 2000) and in develop- ing transgenic plants (Benvenuto et al., 1991; Le Gall et al., 1998; Tavladoraki et al., 1993). We chose a mon- oclonal antibody to engineer a single chain antibody (scFv-αCTV) which was expressed in Escherichia coli (E. coli) and characterized. Furthermore, Nicotiana tabacum was selected as a plant expression model