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Production and Characterization of Monoclonal Antibodies to Verticillium dahliae and Development of a Quantitative Immunoassay for Fungal Biomass. Javier Plasencia, Former graduate assistant and professor, respectively, Department of Plant Pathology, University of Minnesota, St. Paul 55108, Current address: Departamento de Bioquímica, Facultad de Química, UNAM 04510, México City, México, D.F.; Ronald Jemmerson(2), and Ernest E. Banttari(3). (2)Associate professor, Department of Microbiology, University of Minnesota, Minneapolis 55455, (3)Former graduate assistant and professor, respectively, Department of Plant Pathology, University of Minnesota, St. Paul 55108. Phytopathology 86:170-176. Accepted for publication 27 October 1995. Copyright 1996 The American Phytopathological Society. DOI: 10.1094/Phyto-86-170.

Monoclonal antibodies (MAbs) were prepared against a purified mycelial protein from Verticillium dahliae, a predominant fungus species in the potato early dying complex. The fungal protein was rendered immunogenic by conjugating it to keyhole limpet hemocyanin. Three hybridomas were cloned from a mouse immunized with the conjugated protein. All three cell lines produced immunoglobulin G1-type MAbs that recognized a 60-kDa protein. The specificity of the MAbs was tested using indirect enzyme-linked immunosorbent assay (ELISA), immunoblots, and indirect competitive ELISA. For two of the MAbs, no cross-reactivity was observed when tested against several isolates representing six species of fungi associated with potato. Cross-reactivity was observed to some extent with V. albo-atrum. A quantitative immunoassay was developed using an indirect competitive ELISA format that could detect as little as 4 ng of total protein of the fungus and showed a linear relationship between total protein (4 to 500 ng) and absorbance measured at 405 nm. The assay was used to detect and quantify V. dahliae colonization in greenhouse-grown potato plants. The immunoassay accurately distinguished colonized from noncolonized plants and quantitively differentiated the susceptible cultivar (Kennebec) from the resistant cultivar (Reddale).