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

Abstract

Black Sigatoka caused by Mycosphaerella fijiensisMorelet 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 Mycosphaerella musicola has been replaced by M. fijiensis wherever Black Sigatoka has been introduced in America and elsewhere, and remains as a significant problem at sites located at relatively high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.

Early Detection of the Fungal Banana Black Sigatoka Pathogen Pseudocercospora fijiensis by an SPR Immunosensor Method

“…Then, the SPR signal value at the plateau (saturation by specific binding) obtained from the fitted trajectory curves were plotted as a function of HF1 concentrations to generate a calibration curve (see Figure 5). The limit of detection obtained was 11.7 µg mL − 1 which is comparable to that obtained by Torrance et al [27], where used an antibody-based SPR for the detection of Cowpea mosaic virus , establishing a limit of detection of 12.5 µg mL − 1 as well as the LOD of 10 µg mL −1 obtained by Otero et al [28] who employed a monoclonal antibody-based on a triple antibody system-ELISA for the detection of Mycosphaerella fijiensis . The primary analytical parameters obtained in this study are summarized in Table 1.…”

“…Real banana leaves were collected and processed as aforementioned to obtain aqueous extracts, which were directly injected in the SPR setup to observe the sensor response (see Figure 6). This method is widely employed, since the mechanical disintegration of the leaves allows the release of the fungus M. fijiensis , exposing out the proteins in the cell wall for its further detection through the antibodies [4,28,31].…”