Production and characterization of a genetically engineered anti-caffeine camelid antibody and its use in immunoaffinity chromatography.

This work demonstrates the feasibility of using a camelid single domain antibody for immunoaffinity chromatographic separation of small molecules. An anti-caffeine VHH antibody was produced by grafting the complementarity determining sequences of a previously generated antibody onto an anti-RNase A antibody scaffold, followed by expression in E. coli.

Determination of lactic acid enantiomers in human urine by high-performance immunoaffinity LC-MS.

In this study, a monoclonal anti-d-hydroxy acid antibody was used as chiral selector for chromatographic enantiomer separation and quantification of lactic acid contained in human urine samples. The immunoaffinity column was directly coupled to an electrospray ionization mass spectrometer for detection. Separations were performed at room temperature and under isocratic conditions using ammonium bicarbonate buffer (pH 7.

Investigation of the stereoselectivity of an anti-amino acid antibody using molecular modeling and ligand docking.

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.

Enantiomer separation of alpha-hydroxy acids in high-performance immunoaffinity chromatography.

In this study, a monoclonal anti-d-hydroxy acid antibody was immobilized onto a synthetic high-flow-through chromatographic support material to produce a chiral stationary phase suitable for enantiomer separation of free alpha-hydroxy acids. Chiral separation of several aliphatic and aromatic members of this class of compounds was achieved in HPLC under mild isocratic buffer conditions using phosphate buffered saline, pH 7.4, as mobile phase.

A comparative evaluation of random and site-specific immobilization techniques for the preparation of antibody-based chiral stationary phases.

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.