Use of Specific Monoclonal Antibodies for Diagnosis of Citrus Tristeza Virus

Abstract

Monoclonal antibodies (MAb) specific for citrus tristeza virus (CTV) were obtained by fusion of a nonsecreting myeloma cell line with spleen cells from immunized BALB/c mice. Immunoglobulin titre and isotype were determined in cell culture supernatants and mouse ascitic fluid. Each MAb was enzyme-conjugated and used in a double-antibody sandwich ELISA assay for CTV. All reacted uniformly against a panel of 23 strains of CTV representative of strains collected from Europe, Asia, and North America. Index words. immunoenzymatic techniques, antigenic regions. Tristeza, the most important viral disease of citrus, is caused by citrus tristeza virus (CTV) a closterovirus that is disseminated in the field by aphids. Usually, testing for CTV has been performed by graft inoculation on Mexican lime seedlings, but this method is expensive and results are obtained only after several months. In recent years, several serological methods have been developed for specific and rapid detection of CTV: SDSdouble immunodiffusion (12), ELISA (2, 3), immunoelectron microscopy (13), immunofluorescence (28), and radioimmunoassay (22). ELISA has been particularly useful for largescale CTV surveys (9, 23) and has introduced new possibilities for the study and control of the disease (4, 14, 24). These studies have used conventional antisera (polyclonal antibodies, PAb). Recently, by hybridoma technology (20), monoclonal antibodies (MAb) have been obtained to tobamoviruses (1, 8), potyviruses (15), potexviruses (27) luteoviruses (7, 18) and ilarviruses (16). In this paper, we describe the preparation and characterization of CTV-specific monoclonal antibodies and their use for the diagnosis of CTV infection. MATERIALS AND METHODS by Aphis gossypii (17) to healthy citrus plants to separate CTV from other non-aphid-transmissible citrus viruses. Purified isolates were grafttransmitted to uniform groups of healthy sweet orange and Mexican lime seedlings grown in a controlledtemperature greenhouse (17 to 24 C). CTV isolate T-514 from California (provided by Dr. D. J. Gumpf, Univ. California, Riverside, U. S. A.), and two unclassified isolates from Iran (provided by Dr. V. Minassian) and Vietnam (provided by Dr. N. Matos, Sanidad Vegetal, La Habana, Cuba)