Improved erythrocyte lysis assay in microtitre plates for sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic alga

Haemolytic substances produced by ichthyotoxic algae often are unknown in m olecular structure or specific mechanism of toxicity. Detection and quantif ication of such substances are dependent on bioassays, using markers that a re sensitive for haemolytic impairment and generation of a recordable respo nse. The erythrocyte lysis assay (ELA) represents an advantageous bioassay in this respect, because the lytic response can be measured photometrically by the amount of released haemoglobin. The aim of the present study was to establish an improved assay based on the ELA principle, for sensitive dete rmination of haemolytic substances of microalgae and for high sample throug hput. For this purpose we adapted the ELA to a 96-well microtitre plate for mat, which significantly reduced the sample volumes and allowed rapid proce ssing of samples. Further improvement was achieved by measuring absorption of lysed erythrocytes at 414 nm, which significantly increased the sensitiv ity of the ELA compared to the measurements at 540 nm that are usually appl ied in this type of assay. Using carp (Cyprinus carpio) erythrocytes it was possible to detect haemolysis induced by 4 mug ml(-1) of saponin and as li ttle as two haemolytic Alexandrium tamarense cells. It is suggested that th is improved ELA in microtitre plates be used as a low-cost monitoring tool for detection and analysis of potential harmful algae. Furthermore, this EL A can be useful as a sensitive screening system for substances of pharmacol ogical interest, e.g. selectively acting cytolytic antibiotics. Copyright ( C) 2001 John Wiley & Sons, Ltd.