Development of a double monoclonal antibody sandwich ELISA test for Verticillium dahliae Kleb.

Verticillium dahliae causes wilt diseases in a wide range of horticultural and field crops in many parts of Turkey. In the Aegean region it has been a serious problem in cotton and vegetables for many years. More recently, it has become a major problem for olive growers in relatively newly established orchards. The fact that the only possible control methods of Verticillium wilt disease is the use of healthy and pathogen free propagating material has directed us to produce monoclonal antibodies against V. dahliae in order to apply rapid, sensitive and reliable serological detection methods. Verticillium spp. isolates were obtained from olive plantations, as well as from cotton and tomato fields in the Aegean and Marmara Regions. All suspected isolates were obtained as V. dahliae after determining microscopic morphological characteristics and pathogenicity tests on cotton seedlings. Immunizing antigens were prepared by three different methods including surface washing system, czapek dox agar and gel filtration methods. BALB/c mice were immunized with each antigenic form. Lymph node, spleen and bone marrow cells were used as sources of B-lymphocytes and 8D2 (IgM) and 7D6 (IgG1) were obtained from the spleen and lymph node fusion. The monoclonal antibodies were purified and immunoglobulin types were identified. 8D2 monoclonal antibody gave positive reaction with the V.dahliae isolates from olive, cotton, tomato and watermelon; however, it didn't give any cross reactivity with other epiphytic fungi. 7D6 antibody displayed cross-reactions with a few fungi. The monoclonal antibody (8D2) was conjugated with horseradish peroxidase (HRP). These monoclonal antibodies were characterized for use in the development of diagnostic kits based on double-monoclonal antibody sandwich ELISA test system for detecting V. dahliae in Turkish isolates. In this test, the first antibody was used as capture antibody and the second one was used for detection of antigens.