Improvements in the purification of an antisteroid antiserum resistant to conventional immunoadsorption chromatography

Abstract

Antibodies against dexamethasone, a synthetic steroid, have been induced in rabbits immunized with a 3-carboxymethyloxime dexamethasone derivative conjugated to bovine serum albumin. The antiserum displaying the highest affinity for dexamethasone (K D = 0.5 nM) appeared to be resistant to purification on an agarose matrix bearing the same 3-carboxy-methyloxime dexamethasone derivative. No desorption of active antibodies could be obtained whatever the eluting buffer used. Electrophoretic elution gave only poor results. However a very significant improvement in the purification of these antibodies was achieved by changing the connecting arm for steroid linkage to the agarose beads. A 17-fold purification and a 32% recovery of active specific antisteroid antibodies were obtained using a column bearing a 17β-carboxamide dexamethasone derivative. Moreover good results (23-fold purification and 30% recovery) were also obtained with a commercially available preactivated high-performance silica column derivatized with an aminated 3-carboxymethyloxime derivative of dexamethasone. In this case the more efficient diffusion of the eluting solution through the pores of a high performance stationary phase made of small diameter rigid beads probably explained the strikingly improved results, when compared to those obtained with agarose beads bearing the same dexamethasone derivative.