Production and characterization of polyclonal anti-S 20499 antibodies: influence of the hapten structure on stereospecificity.

Immunoassays were studied as an alternative to HPLC methods for the stereoselective determination of a chiral drug, S 20499, a new anxiolytic compound that is chemically related to buspirone. The production of highly stereospecific polyclonal antibodies was sought following the construction of appropriately optimized hapten-protein conjugates. This process involved the selection of the structure and the length of the spacer arm used to couple S 20499 to the carrier protein as well as deciding on the location of the coupling site with respect to the chiral center. Two haptens were prepared: one a derivative resembling the original structure of S 20499, with the effective addition of a carboxylic acid group, and a second with the effective addition of a butanoic acid moiety that is supposed to favor stereorecognition. Six stereospecific polyclonal antisera were obtained in rabbits with two groups of antibody families defined in terms of specificity. Both approaches gave high levels of stereospecificity (cross-reactivity towards the optical antipode of S 20499 ranged from 4.1% to < 0.1%). Although it did not decrease the mean apparent affinity constant, the longer spacer improved antibody specificity by decreasing cross-reactions towards dealkylated S 20499 derivatives. Hence, the addition of a four carbon atom bridge should be a valuable tool for increasing antibody stereospecificity with no drawbacks in terms of specificity and affinity. It was also shown that long immunization periods appear to have no effect on the stereospecificity of the antibodies obtained.

Stereoselectivity of antibodies for the bioanalysis of chiral drugs.

The use of immunoassay techniques represents an alternative approach to the more common chromatographic methods for the determination of the concentrations of chiral drugs. In the development of the former technique, the inherent stereoselectivity of the antibodies used appears to be an important parameter to be studied. The structural features of the drug, including the environment around the asymmetric center, the flexibility of the molecule and the ability of the molecule to undergo racemization, contribute to the molecule's ability to be recognized by stereoselective antibodies. These parameters are intrinsic and can not be influenced by the investigator. On the other hand, other parameters can be modified to favor the raising of highly stereoselective antibodies. These include the synthesis of an appropriate hapten; the selection of an optimal spacer arm between the hapten and the carrier protein used for the immunization procedure; and the choice of appropriate immunization and antibody-screening procedures. The purification of antibodies using affinity chromatography may also facilitate the selection of stereoselective antibodies.