Immunoadsorption as a Means for the Purification of Low Molecular Weight Compounds: Isolation of Ecdysteroids from Insects

Abstract

Ecdysteroid, Immunoadsorption of Steroids, Antiserum Specificity Two ecdysteroid-specific antisera, anti-ecdysone-albumin and anti-20-hydroxyecdysone-al-bumin, were induced in rabbits. The properties of both antisera were tested with a large number of ecdysteroids. The former antiserum was unique in its ability to discriminate between the moulting hormones, ecdysone and 20-hydroxyecdysone, whereas the latter had a broad specificity for ecdysone and several of its metabolites. y-globulin fractions of both antisera were covalently linked to Sepharose 4B and used as immunoadsorbents. These were able to bind ecdysteroids from crude extracts of insects. Bound ecdysteroids could be eluted quantitatively from the adsorbents using 3 M sodium trichloroacetate. When a mixture of ecdysteroids which contained ecdysone and at least six different ecdysone me­ tabolites were run over a column with anti ecdysone immunoadsorbent only ecdysone and its hy-droxyderivatives 20-and 26-hydroxy ecdysone were bound to and subsequently eluted from the adsorbent. In contrast a column with anti-20-hydroxyecdysone immunoadsorbent retained most of the different ecdysteroids due to the broader specificity of the antibodies. These immunoadsorbents provide the potential not only to purify ecdysteroids but also proteins crosslinked to ecdysteroids via photoaffinity labelling. It is well known that immuno-adsoiption is a powerful tool for the isolation of proteins. The results described here demonstrate that immunoadsorption may also be useful in the isolation of low molecular weight compounds of biological and medical interest.