A Steroid-Binding Protein from Insect Haemolymph Isolated by PhotoafTinity Labelling and Immunoadsorption

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Abstract
Endogenous ecdysteroids as well as radiolabelled exogenous ecdysone were crosslinked to a protein when haemolymph from blowfly larvae was irradiated with UV-light under optimized conditions. This indicates that larval haemolymph of blowflies contains an ecdysteroid-binding protein. The crosslinked ecdysteroid-protein complex was shown to form a ternary complex when mixed with ecdysteroid-specific antibodies. Formation of the ternary complex was due to a specific interaction of the binding sites of the antibodies with the steroid. This specific interaction was used to isolate the ecdysteroid-protein complex from irradiated crude larval haemolymph by immunoadsorption. The method described here represents a rapid approach for the isolation of steroid-binding proteins by a combination of photoaffinity labelling and immunoadsorption.