In vitro detection of plant mycoplasmas by immunofluorescent staining with monoclonal antibodies.

Hybridoma techniques have been applied to develop specific monoclonal antibodies (McAbs) against spiroplasmas and mycoplasma-like organisms (MLOs). Advantages of the McAbs have been clearly demonstrated in the detection of aster yellows (AY) MLO by indirect enzyme-linked immunosorbent assay (ELISA) and immunofluorescent staining, compared with conventionally produced polyclonal antibodies. McAbs have also been applied in the in vitro detection and identification of spiroplasmas and AY MLOs on membrane filters using modified direct-count immunofluorescent staining. Organisms were fixed with glutaraldehyde and treated with McAbs and fluorescein isothiocyanate (FITC)-conjugated antimouse immunoglobulin, by incubating and concentrating them in a microcentrifuge tube before collecting them on polycarbonate membrane. This procedure best conserves the helical integrity of spiroplasmas without altering the configuration of surface antigens recognized by McAbs. This method could be valuable in monitoring the fastidious mycoplasmas during purification or cultivation attempts.