Immunoassay of indomethacin: the use of [125I] protein A to detect specific serologic binding.

The sera of rabbits that were injected with indomethacin covalently linked to human albumin bound increasing amounts of [14C] indomethacin during the course of immunization. The serum-binding components chromatographed with the gamma-globulin fraction and were precipitated with goat-anti-rabbit-gamma-globulin. When measured by radioimmunoassay with [14C] indomethacin under optimal conditions, 34 ng (95 pmoles) of unlabeled indomethacin inhibited [14C] indomethacin binding 50%. The antibodies reacted most effectively with indomethacin and the desmethylated analogue, 1-(p-chlorobenzoyl)-2-methyl-5-hydroxyindole-3-acetic acid, but not 2-methyl-5-methoxy-indole-3-acetic acid. An immunoassay based on the use of [125I] Protein A as tracer for IgG antibody was developed for quantitative determination of indomethacin at the picogram level. Indomethacin, immobilized by covalent linkaae to a solid support, bound the rabbit anti-indomethacin. Protein A, labeled with [125I], measured the levels of the bound IgG antibody. Fluid phase indomethacin competed with solid-phase indomethacin for the anti-indomethacin, which resulted in decreased anti-indomethacin and consequently decreased [125I] Protein A on the immobilized indomethacin-anti-indomethacin complex. The serologic specificity with the immobilized ligand immunoassay was the same as that found with the [14C] indomethacin radioimmunoassay, but the sensitivity for detection of indomethacin was increased over 300-fold.