Fourteen isolates of Anabaena circinalis from various regions of Australia were grown for extraction. Two microtiter plate based bioassays for measuring saxitoxin (STX) and its derivatives, use radioactively labeled STX binding by sodium channels, STX's pharmacological target, or an unrelated protein, saxiphilin. These
bioassays were challenged with extracts of toxic and nontoxic strains of Anabaena circinalis, and the results were compared with HPLC analysis.
Cultured cells were harvested during late logarithmic growth phase. A saxiphilin microtiter plate binding assay was conducted using tritiated saxitoxin ([3H]STX) from the centipede Ethmostigmus rubripes. The inhibition of [3H]STX binding to rat brain sodium channels was also measured - both values were recorded as percentages.
Samples were screened for inhibition of [3H]STX binding by incubating duplicate samples of 10 µL each. If dilution was necessary, extracts were retested in a series (2-, 5-, 10-, 50-, 100-, 500-, and 1000-fold dilutions). Three classes of paralytic shellfish toxins (PSTs) were analyzed by HPLC - C-toxins, gonyautoxins, and saxitoxins (C1, C2, dcGTX3, GTX5, dcGTX2, GTX3, GTX2, dcSTX, STX; values for individual toxin analogues are given as mol %).
The final toxicity measurement was the average of the values derived from the multiple inhibitory values between 20 and 80% inhibition obtained in the titration of unknown samples.
