A radioimmunoassay for palytoxin.

Palytoxin, labelled with 125I-Bolton-Hunter reagent on its terminal amino group, bound specifically to rabbit anti-palytoxin. The extent of binding increased progressively with repeated immunizations. After absorption of the rabbit IgGs with a goat anti-rabbit IgG, binding was reduced greater than 95%. For 50% inhibition of binding in the 125I-palytoxin-antipalytoxin reaction 0.27 pmoles of unlabelled palytoxin was required. Maitotoxin, teleocidin, okadaic acid, debromoaplysiatoxin and 12-O-tetradecanoylphorbol-13-acetate, when tested at 10-100-fold higher concentrations than palytoxin did not affect binding. Palytoxin's serologic activity was stable after 60 min exposure to 100 degrees C and after 60 min exposure to 0.1 N HCl at 50 degrees C, but its capacity to stimulate the arachidonic acid metabolism of rat liver cells was reduced after the 60 min exposure to 0.1 N HCl treatments at 35 degrees C or 0.01 N HCl at 50 degrees C. The average binding constant (K0) as determined by separation of antibody-bound palytoxin from free palytoxin by the double antibody technique was 4.9 x 10(9) M-1 at 0 degrees C. This apparent average association constant increased with increasing temperature suggesting that palytoxin's epitope, most likely hydrophilic, is bound to H2O and the H2O is displaced before binding to the antibody's paratope.