Production and characterization of monoclonal antibodies to Verticillium dahliae and development of a quantitative immunoassay for fungal biomass

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 K 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 K 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).