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 Pseudonitzschia multiseries (=Pseudo-nitzsch ia pungens f. multiseries) and the non-toxic P. pungens (=P. pungens f . pungens). These antibodies bind to the surface of the diatoms as sho wn 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 m olecular-weight diffuse bands, mostly remaining in the stacking gel, u sing an SDS-PAGE system. The antibodies prepared against P. multiserie s strongly labelled the high molecular-weight antigens of all P. multi series strains tested and showed little reactivity towards P. pungens extracts on Western blots. P. pungens antibodies strongly labelled hig h molecular-weight P. pungens antigens and faintly labelled a few P. m ultiseries antigens. The selectivity of the antibodies for their respe ctive species correlates with the results of the immunofluorescence ex periments, suggesting that the antigens examined in this study are res ponsible for the selective labelling in immunofluorescence studies. Th e electrophoretic mobility and the antibody labelling of antigens were not altered by proteolytic digestion of cell pellets. However, disrup tion of carbohydrates in the pellets by treatment with periodic acid r esulted in loss of the antigen. These data suggest that the major anti gens of toxic P. multiseries and non-toxic P. pungens are high molecul ar-weight (>100 kDa) polysaccharides located on the surface of these d iatoms.