Electro-immunoblotting characterization of Pseudo-nitzschia multiseries and P. pungens antigens recognized by antibodies directed against whole cells

Separate polyclonal antibodies have previously been developed against the domoic-acid-producing Pseudo-nitzschia multiseries (= Pseudo-nitzschia pungens f. multiseries) and the non-toxic P. pungens (= P. pungens f. pungens). These antibodies bind to the surface of the diatoms as shown by immunofluorescence studies. Here we examine the molecular nature of the antigens by Western blotting (electro-immunoblotting) analysis. The major antigens for both polyclonal antibodies migrated as high molecular-weight diffuse bands, mostly remaining in the stacking gel, using an SDS-PAGE system. The antibodies prepared against P. multiseries strongly labelled the high molecular-weight antigens of all P. multiseries strains tested and showed little reactivity towards P. pungens extracts on Western blots. P. pungens antibodies strongly labelled high molecular-weight P. pungens antigens and faintly labelled a few P. multiseries antigens. The selectivity of the antibodies for their respective species correlates with the results of the immunofluorescence experiments, suggesting that the antigens examined in this study are responsible for the selective labelling in immunofluorescence studies. The electrophoretic mobility and the antibody labelling of antigens were not altered by proteolytic digestion of cell pellets. However, disruption of carbohydrates in the pellets by treatment with periodic acid resulted in loss of the antigen. These data suggest that the major antigens of toxic P. multiseries and non-toxic P. pungens are high molecular-weight (> 100 kDa) polysaccharides located on the surface of these diatoms.