Monoclonal Antibody-based TAS-ELISA for Quantitative Detection of Mycosphaerella fijiensis Antigens

Black Sigatoka caused by Mycosphaerella fijiensis Morelet is the most dangerous and devastating disease of banana around the world. Disease control is carried out by integrating cultural, genetic and chemical measures. Mycosphaerella musicola has been replaced by M. fijiensis wherever Black Sigatoka has been introduced in America and elsewhere, and remains as a significant problem at sites located at relatively high altitudes. To optimize fungicide applications for disease management a presymptomatic quantification of fungal proteins in leaves is desirable. In this study, we describe the generation of a mouse monoclonal antibody (Mab) reactive to a high-molecular weight antigens from M. fijiensis mycelial single ascospore in vitro culture, but not reactive to mycelial antigens from M. musicola, M. musae and M. minima single ascospore cultures. A rabbit-specific polyclonal antibody against the same M. fijiensis antigen, reactive also to fungal secreted proteins, was able to discriminate naturally M. fijiensis infected from healthy leaves as well as other concomitant fungi. The Mab was used as a capturing reagent and the polyclonal preparation as a second antibody for a triple antibody enzymelinked immunosorbent assay able to quantify mycelial protein antigens in a range of 10–40 lg ⁄ ml and differentiate it from healthy banana leaf extracts. The aim of this study was the analytical setting up of a Mab-based immunoassay for the quantification of mycelial and secreted proteins of M. fijiensis. It is intended for the further establishment and optimization of an asymptomatic leaf assay for an improved forecast and warning system applied to Black Sigatoka disease management