In this study, one random and four site-directed conjugation strategies were applied to immobilize an mAb, which stereoselectively binds to L-amino acids, onto silica particles. The resulting chiral stationary phases (CSPs) were used for enantiomer separation of the model-analyte D,L-phenylalanine and further examined in frontal affinity chromatography. Although random immobilization of the antibody onto discuccinimidyl carbonate-activated silica resulted in a CSP that enabled baseline separation of the enantiomers of D,L-phenylalanine, the amount of available binding sites was considerably lower compared to the CSPs prepared by site-directed strategies. Immobilization of antibody via its carbohydrate chains, either directly via hydrazone bonds between the support and the protein or indirectly via binding carbohydrate-biotinylated antibody to streptavidin-derivatized silica, resulted in medium column efficiencies. Higher amounts of available active sites were obtained by immobilizing the antibody indirectly through the "crystallizable fragment (Fc)" receptor protein A/G. The best results with regard to amount of available binding sites and column efficiency were obtained by first biotinylating the antibody specifically at its C-termini using carboxypeptidase Y and immobilizing the biotinylated antibody on streptavidin-derivatized silica.

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The structure of the binding site of the stereoselective anti-D-amino acid antibody 67.36 was modeled utilizing web antibody modeling (WAM) and SWISS-MODEL. Although docking experiments performed with an aromatic amino acid as model ligand were unsuccessful with the WAM structure, ligand binding was achieved with the SWISS-MODEL structure. Incorporation of side-chain flexibility within the binding site resulted in a protein structure that stereoselectively binds to the D-enantiomer of the model ligand. In addition to four hydrogen bonds that are formed between amino acid residues in the binding site and the ligand, a number of hydrophobic interactions are involved in the formation of the antibody-ligand complex. The aromatic side chain of the ligand interacts with a tryptophan and a tyrosine residue in the binding site through pi-pi stacking. Fluorescence spectroscopic investigations also suggest the presence of tryptophan residues in the binding site, as ligand binding causes an enhancement of the antibody's intrinsic fluorescence at an emission wavelength of 350 nm. Based on the modeled antibody structure, the L-enantiomer of the model ligand cannot access the binding site due to steric hindrance. Additional docking experiments performed with D-phenylalanine and D-norvaline showed that these ligands are bound to the antibody in a way analogous to the D-enantiomer of the model ligand.

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