Production, characterization, and applications of stereoselective antibodies to a-hydroxy acids
While enantiomers, i.e., mirror-image-like stereoisomers, contain the same atoms and functional groups, differences in the three-dimensional arrangement of those atoms or groups may result in different biological activities. Regulatory agencies such as the US Food and Drug Administration require quantitative detection and characterization of individual enantiomers of chiral drugs and their metabolites because the pharmacodynamic, pharmacokinetic, and toxicological properties of enantiomers may differ. Therefore, separation and detection of enantiomers are crucial to the understanding of their biological roles. Antibodies raised against chiral haptens have the potential of discriminating between enantiomers, which makes them one of only a few tailor-made types of chiral selectors. In this dissertation, antibodies were raised against D- and L-α-hydroxy acids, respectively. Both polyclonal antibodies from rabbits and monoclonal antibodies from murine hybridomas were produced that stereoselectively bind to a wide range of α-hydroxy acids, including both aromatic and aliphatic α-hydroxy acids. No detectable interaction was seen with the “opposite” enantiomer. Further studies confirmed that the hydroxyl/carboxyl/ hydrogen triad about the stereogenic center is essential for antibody binding as no interaction was observed with structurally closely related compounds such as α-amino acids. Conversely, previously produced stereoselective anti-α-amino acid antibodies bound only to α-amino acids of the correct configuration and not to α-hydroxy acids. The remarkable stereoselectivity and class specificity of these antibodies enabled the development of fluorescence-based immunoassays for the simultaneous detection of D-phenylalanine, L-phenylalanine, D-phenyllactic acid, and L-phenyllactic acid using four antibodies, each of which was labeled with a unique fluorophore. Furthermore, the utility of an anti-α-hydroxy acid antibody for enantiomer separation in chiral immunoaffinity chromatography was demonstrated.