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Radioimmunoassay for Phorbol Esters Using Rabbit Antisera against Phorbol Succinate¹

Laboratory of Toxicology, Harvard School of Public Health [A. H. T., G. W., C. W. F.], and Department of Pharmacology, Harvard Medical School [A. H. T.], Boston, Massachusetts 02115

The phorbol nucleus was succinylated and then conjugated to bovine albumin using dicyclohexylcarbodiimide. Rabbits given injections of the conjugate developed antibodies which rose in titer progressively with repeated immunization. By the ninth bleeding, the binding of one antiserum, diluted 1:15,000, was saturated with about 10 nM [³H]phorbol-12,13-dibutyrate ([³H]-PDBU) and had an average association constant, K_a, of 2.6 x 10⁸ M^–1. The serological specificity of the antisera was characterized by examining the inhibition of the [³H]PDBU-anti-phorbol succinate immune system by 18 phorbol-related compounds. The specificities of antibodies from two rabbits tested in detail were qualitatively similar. The rank order of inhibitory activity for certain phorbol-related compounds was PDBU [concentration of inhibitor required to give 50% inhibition of PDBU binding (IC₅₀) = 7.6 nM] = phorbol-13-acetate [IC₅₀ = 8.2 nM] > phorbol-12,13-dibenzoate > 4-ß-phorbol [IC₅₀ = 124 nM] phorbol-12,13-diacetate phorbol-12-myristate-13-acetate [IC₅₀ = 184 nM] > phorbol-13,20-diacetate > phorbol-12-acetate [IC₅₀ = 2300 nM]. The following compounds showed no detectable serological activity: mezerein, 4-O-methylphorbol-12-myristate-13-acetate, ingenol, 4--phorbol, teleocidin B, and dihydroteleocidin B. These and other results indicated that the 4-ß-phorbol nucleus was required for serological activity, that esterification of the C-13 position with benzoate, acetate, or butyrate enhanced the immunoreactivity of 4-ß-phorbol, and that among the phorbol-related compounds examined there was no direct relationship between serological activity and biological potency as tumor promoters. Using the [³H]PDBU-anti-phorbol succinate immune system, we measured the concentrations of immunoreactive phorbol-related material in crude mixtures such as croton oil and performed pharmacokinetic studies in rats given PDBU s.c.

¹ This investigation was supported in part by a Center Grant from the National Institute of Environmental Health Sciences (ES 00002), a cooperative agreement with the United States Environmental Protection Agency (OR 807809), and a research grant from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases (AM 11011).

² To whom requests for reprints should be addressed, at Laboratory of Toxicology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115.

³ Supported by a National Research Service Award (GM 08567).

Received 6/25/84. Accepted 9/20/84.