Immunomagnetic separation of cells of the toxic dinoflagellate Alexandrium fundyense from natural plankton samples

Abstract

A novel method was developed for isolating cells of the toxic dinoflagellate Alexandrium fundyense from preserved natural seawater samples using paramagnebc beads and a monoclonal antibody against the surface antigens of the dinoflagellate. 'Direct' and 'indirect' approaches to bead/cell attachment were tested as well as 3 types of bead coatings (streptavidin, and 2 secondary antibodies: sheep anti-mouse, and goat anti-mouse), and 2 blocking agents [normal goat serum (NGS) and bovine serum albumin (BSA)]. Optimal 'indirect' bead attachment protocols, where the species-specific pnmary antibody is first bound to the target cells before bead attachment, utilized either 2.8 pm streptavidin-coated beads with NGS blocking or sheep anti-mouse-coated beads with BSA blocking. Although there were undoubtably some cell losses during the initial antibody-labeling and washing procedures, ca 90% of the labeled A. fundyense cells in unialgal cultures were removed using these bead procedures. Non-specific binding was low, as only 5 to 10 % of the A. fundyense cells were recovered when the primary antibody was omitted. The 'direct' approach, where the species-specific primary antibody is first bound to the beads, was most effective (80% recovery) using sheep anti-mousecoated beads (2.8 p) wlth BSA blocking, and the non-specific binding remained very low ( ~ 2 % ) . When tested in 3 natural preserved plankton samples, recovery of Alexandrium spp. was ca 90% (negative controls <10%) using the 'indirect' method, with 5% contamination by other species. The 'direct' technique was slightly less effective on the natural samples (ca 80% recovery), but negative controls were very low at 1% or less. Contam~nation by other species was 5 to 10%. 'Direct' attachment is a simpler procedure than the 'indirect' approach because the beads can be pre-coated with the speciflc antibody in bulk before use. Thus, the direct method not only shortens the procedure by eliminating the need to antibody-label each individual sample but, as a consequence, minimizes target cell losses as well, so it may be the method of choice for working with field samples. Immunomagnetic separation of a slngle algal species from a sample containing mixed plankton and detritus IS thus simple, rapid, and quite reliable. Compared with other sorting methods currently in use (e.g. manual pipette isolation, flow cytometry), this procedure offers distinct advantages with respect to cost, volume, speed and simplicity.