IMMUNOMAGNETIC SEPARATION OF CELLS OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM FUNDYENSE FROM NATURAL PLANKTON SAMPLES

A novel method was developed for isolating cells of the toxic dinoflag ellate Alexandrium fundyense from preserved natural seawater samples u sing paramagnetic 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 a nti-mouse), and 2 blocking agents [normal goat serum (NGS) and bovine serum albumin (BSA)]. Optimal 'indirect' bead attachment protocols, wh ere the species-specific primary antibody is first bound to the target cells before bead attachment, utilized either 2.8 mu m streptavidin-c oated beads with NGS blocking or sheep anti-mouse-coated beads with BS A blocking. Although there were undoubtably some cell losses during th e initial antibody-labeling and washing procedures, ca 90% of the labe led A. fundyense cells in unialgal cultures were removed using these b ead procedures. Non-specific binding was low, as only 5 to 10% of the A. fundyense cells were recovered when the primary antibody was omitte d. The 'direct' approach, where the species-specific primary antibody is first bound to the beads, was most effective (80% recovery) using s heep anti-mouse-coated beads (2.8 mu m) with BSA blocking, and the non -specific binding remained very low (<2%). When tested in 3 natural pr eserved plankton samples, recovery of Alexandrium spp. was ca 90% (neg ative controls <10%) using the 'indirect' method, with 5% contaminatio n 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. Contamination 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 specific antibody in bul k 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 t he method of choice for working with field samples. Immunomagnetic sep aration of a single algal species from a sample containing mixed plank ton and detritus is thus simple, rapid, and quite reliable. Compared w ith other sorting methods currently in use (e.g. manual pipette isolat ion, flow cytometry), this procedure offers distinct advantages with r espect to cost, volume, speed and simplicity.