Development of a monoclonal antibody immunoassay for the eyespot pathogen Pseudocercosporella herpotrichoides

A double-antibody-sandwich ELISA test has been developed for the detection of Pseudocercosporella herpotrichoides using a highly specific monoclonal antibody PH-10 as the capture antibody and genus-specific rabbit polyclonal antiserum as the detector antibody. The assay recognizes extracts from plants both artificially and naturally infected with P. herpotrichoides giving at least three-fold higher absorbance values with extracts from Pseudocercosporella-infected tissue than with extracts from healthy tissues or from tissues naturally infected with Microdochium nivale, Rhizoctonia cerealis or material artificially inoculated with P. anguioides. The assay tested positively against all isolates of P. herpotrichoides , including both W-type and R-type isolates. In this assay system, extraction of the antigen from the stem bases of infected plants is a one-step process not requiring any dilution procedures. The assay can be used to detect the pathogen in presymptomatic infected seedlings. The immunogen used to generate the specific monoclonal antibody and the rabbit antiserum was a mycelial extract from which the high-molecular-weight proteins and glycoproteins had been removed by ammonium sulphate precipitation. The high-molecular-weight fraction was shown to contain cross-reactive antigens; it induced antiserum in mice that cross-reacted with the other stem-base fungi even at high dilutions. The monoclonal antibody PH-10 is an IgM antibody. Heat and periodate treatment of the antigen indicate that it is a glycoprotein and that the epitope recognized by the antibody is a protein.