Drug-protein conjugates: preparation of triamcinolone-acetonide containing bovine serum albumin/keyhole limpet hemocyanin-conjugates and polyclonal antibodies.

A radioimmunoassay has been developed for the quantitation of triamcinolone-acetonide (TAAc) at the picogram level. For use of TAAc as an antigenic epitope first the drug was hemisuccinoylated at C-21 as confirmed by 13C-NMR- and mass spectroscopy after derivatization. This hapten was conjugated to the carrier-protein bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) by different amide-bond generating methods (imidazolide-, carbodiimide-, carbodiimide/sulfo-N-hydroxysuccinimide-, mixed anhydride-method) yielding antigens of quite different conjugation number, solubility and usefulness. The mixed anhydride-method yielded most useful soluble conjugates bearing 0.3-31.5 mol TAAc per mol carrier-protein. Coupling by the carbodiimide-method yielded insoluble conjugates, inappropriate for antigen synthesis in hapten immunoassays because of formation of coupling agent modified residues and crosslinking of the carrier-protein. Specificity of the antisera obtained by immunization with TAAc-BSA and TAAc-KLH was assessed by isolation of the soluble hapten-antibody complex and a RIA protocol was developed providing a detection limit of 200 pg (0.46 pmol) TAAc/ml sample.