Preparation of an ecdysone immunogen for radioimmunoassay work

A highly improved procedure for the preparation of ecdysone-protein conjugates for immunological work is reported. Bovine thyroglobulin is succinylated and the succinylated protein is coupled to beta-ecdysone with 1-ethyl-3-(-dimethylaminopropyl)-carbodiimide in the presence of the acylation catalyst 4-dimethylaminopyridine, The antiserum obtained using this immunogen provides a radioimmunoassay sensitive to 25 pmol of beta-ecdysone. The anti-ecdysone antibody cross-reacts with alpha-ecdysone but not with cholesterol or progesterone. This procedure reverses the standard strategy for synthesizing ester linkages in hapten-protein conjugates and should have widespread applicability for the preparation of other such conjugates for immunological work.