Hemagglutination method for detection of freshwater cyanobacteria (blue-green algae) toxins

Strains of the freshwater cyanobacteria (blue-green algae) Anabaena flosaquae and Microcystis aeruginosa produced toxins that caused intermittent but repeated cases of livestock, waterfowl, and other animal deaths. They also caused illness, especially gastrointestinal, in humans. The most common group of toxins produced by these two species were peptide toxins termed microcystin, M. Aeruginosa type c, and anatoxin-c. A method was found to detect the toxins which utilizes their ability to cause agglutination of isolated blood cells from mice, rats, and humans. The method could detect the toxin in samples from natural algal blooms, laboratory cultures, and toxin extracts. The method consists of: (i) washing lyophilized cyanobacteria cells with physiological saline (0.9% NaCl), (ii) centrifuging the suspension and then mixing portions of the cell-free supernatant with equal volumes of saline-washed erythrocytes in V-shaped microtiter plates, (iii) allowing the mixture to stand for 3 to 4 h, and (iv) scoring the presence of the toxin as indicated by blood cell agglutination. Nontoxic strains, as determined by intraperitoneal mouse bioassay of cyanobacteria or green algae, did not produce an agglutination response.