DETECTION AND QUANTIFICATION OF RHIZOCTONIA-SOLANI IN SOIL BY MONOCLONAL ANTIBODY-BASED IMMUNO-MAGNETIC BEAD ASSAY

Murine monoclonal antibodies (MAbs) of the immunoglobulin classes IgM and IgA, from two different hybridoma cell lines, and rabbit (polyclon al) antiserum raised against mycelial antigens from an AG4 isolate of R. solani were used to develop magnetic microsphere-enzyme immunoassay s (MM-EIAs) for the detection and quantification of the pathogen in so il. The assay can also be used to differentiate R. solani anastomosis group (AG) 2-2 isolates from other AG isolates grown in vitro. Mycelia l antigens were solubilised in saline buffer containing detergent and following high speed centrifugation soluble antigen was delineated by the addition either of a mixture of specific murine MAbs of different immunoglobulin classes or a mixture of murine MAb supernatant and rabb it antiserum. Due to the low concentration of reactants a soluble immu ne complex was formed. The soluble complex was isolated by the additio n of magnetic beads coated with antibodies that specifically recognise d the murine IgM MAbs. The bound antibody-antigen complex was then det ected using a commercial antibody-enzyme conjugate and chromogen. Copy right (C) 1996 Elsevier Science Ltd