A radioimmunoassay for the teleocidins using 26 (2′-aminoethylthio)-tetrahydroteleocidin A-2 as hapten

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Abstract
A teleocidin A-2 derivative, 26(2'-aminoethylthio)-tetrahydro-teleocidin A-2, induced ornithine decarboxylase in mouse skin and inhibited the specific binding of ( 3 H)12- O -tetradecabnoyl-Phorbol-13-acetate to a mouse skin particulate fraction with a potency that was weaker than that of teleocidin A-2 and stronger than that of (−)-indolactam-V. To obtain antibodies, 26 (2'-aminoethylthio)-tetrahydrotleocidin A-2 was covalently linked to bovine albumin with a carbodiimide and the resulting conjugate used for immunization of rabbits. Antibodies directed toward teleocidin were produced as measured by neutralization of teleocidin's capacity to stimulate arachidonic acid metabolism in rat liver cells (the C-9 cell line). An 125 I-labeled ligand was prepared by reaction of the same derivative with radiolabeled Bolton-Hunter reagent. The antibodies bound this radiolabeled hapten, and the binding increased progressively with repeated immunizations. After absorption of the rabbit IgG with a goat anti-rabbit IgG, binding was reduced >95%. The binding of the 125 I-labeled ligand to the antibodies of one rabbit had an apparent average association constant of 2.84 × 10 9 M −1 at 0°C. The serologic specificity of the antiserum was characterized by measuring the inhibition of binding by several teleicidins of varying structure as well as by other tumor promoters and toxins. The rank order of inhibitory activity expressed as concentration required for 50% inhibition (IC 50 ) was for 26 (2'-aminoethylthio)-tetrahydroteleocidin A-2 (0.56 pmol) > teleocidin A-1 (6.5 pmol) ≧ teleocidin A-2 (7.3 pmol) > (−)-indolactam-V (3.7 nmol) > teleocidin B-4 (13 nmol). Maitotoxin, aplysiatoxin, palytoxin, mezerein, okadaic acid and 12- O -tetradecanoylphorbol-13-acetate did not inhibit at the levels tested.