Enantiomer separation of amino acids in immunoaffinity micro LC-MS.

Chiral immunoaffinity microbore columns were directly interfaced with MS detection, and the effect of column length and temperature on the enantiomer separation of a number of underivatized aromatic and aliphatic amino acids was investigated utilizing an antibody chiral stationary phase that had been prepared by immobilizing a monoclonal anti-D-amino acid antibody onto silica. The stronger affinity of the antibody towards aromatic and bulky amino acids allowed separation of such analytes in a 0.75 x 150 mm column, while an increase in column length enabled separation of more weakly bound compounds. The strength of interaction between chiral selector and analytes could be modulated conveniently by lowering the temperature. For the first time, simultaneous enantiomer separation of mixtures of amino acids was achieved on antibody-based chiral stationary phases using extracted ion chromatograms.

Stereoselective antibodies to free alpha-hydroxy acids.

This work describes antibodies exhibiting high stereoselectivity and class-specificity towards the enantiomers of free alpha-hydroxy acids. Since the antibodies interact primarily with the carboxyl-hydroxyl-hydrogen triad about the stereogenic center, they are useful for enantiomer analysis of a variety of structurally different alpha-hydroxy acids including aromatic and aliphatic compounds, e.g. lactic acid. The utility of such antibodies for enantiomer separation in chromatography was demonstrated. Comparative studies of these and previously described anti-alpha-amino acid antibodies revealed that both types of antibodies bind only to analytes that possess both the corresponding target structure and the correct configuration. Thus, substitution of an amino group for the alpha-hydroxyl group results in a complete loss of binding activity with the anti-alpha-hydroxy acid antibodies, while an alpha-amino group is essential for the interaction between analytes and anti-alpha-amino acid antibodies.

New developments in the production and use of stereoselective antibodies.

This article describes the production of stereoselective antibodies using both classical immunological and modern molecular biological techniques. Stereoselective antibodies against alpha-hydroxy acids were raised in rabbits and mice and compared with previously produced anti-alpha-amino acid antibodies. It was found that both types of antibodies combine stereoselectivity with class-specificity. Sequence analyses revealed that antibodies with opposing stereoselectivities can be formed during the affinity maturation process from a common progenitor or independently using nonhomologous binding sites. For the first time, phage display was employed to obtain stereoselective antibody fragments. The versatility of stereoselective antibodies as chiral selectors was demonstrated by applying them in several immunosensors and in chiral chromatography. A simple, membrane-based optical sensor allowed detection of enantiomeric impurities at the 1/2,000 level (99.9% ee). Silica-based antibody chiral stationary phases could be used for enantiomer separation of aliphatic amino acids in standard-sized columns, while miniaturized columns allowed interfacing with an MS-detector.