An immunolabeling technique for the detection of diarrhetic shellfish toxins in individual dinoflagellate cells

Abstract

The marine dinoflagellate species known to produce diarrhetic shellfish poisoning (DSP) toxins belong to the genera Dinophysis and Prorocentrum. However, not all species of these genera are toxic, and morphologically identical strains of the same species may or may not produce toxins. Immunolabeling of toxins within individual microalgal cells is a relatively new, powerful tool to discriminate between toxic and nontoxic variants of the same species. This type of labeling requires that the technique used to prepare the cells for labeling not alter their toxin content. The toxin retentions of various techniques used to prepare Prorocentrum lima (Ehrenberg) J.D. Dodge cells for immunolabeling of DSP toxins with a toxin-specific antibody were compared. Four fixation/dehydration methods were examined to determine their ability to retain okadaic acid (OA), a lipophilic toxin easily extractable in the graded alcohol series typically used for dehydration. Toxin extraction by these methods was compared by high-performance liquid chromatography. Glutaraldehyde fixation, alone or paired with EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride], followed by serial dehydration in ethanol, resulted in substantial loss of toxin from the cells prior to embedding. Freeze fixation followed by freeze substitution in chilled methanol conserved the highest amounts of DSP toxins and provided adequate fixation for intracellular localization. Retention of OA by this method was demonstrated by reextracting the cells following the dehydration process. Following dehydration, cells were then embedded in resin and thin-sectioned for application of the fluorescently labeled (FITC) antibody probe, after which they were examined by epifluorescence microscopy to establish the localization of the toxin. The method of freeze fixation followed by freeze substitution was tested on a number of laboratory isolates and field samples and provided a means to discriminate between toxic and nontoxic cells.