Oliver Hofstetter

Chiral discrimination using an immunosensor

Based on the stereoselectivity of immunoglobulins, we have developed a new chiral sensor for the detection of low-molecular-weight analytes. Using surface plasmon resonance detection, enantiomers of free, underivatized α-amino acids can be monitored in a competitive assay by their interaction with antibodies specific for the chiral center of this class of substances. The sensitivity to the minor enantiomer in nonracemic mixtures exceeds currently available methods; therefore, such immunosensors can readily detect traces of enantiomeric impurities and are attractive for a range of applications in science and industry.

Enhancement of latent fingermarks on non-porous surfaces using anti-L-amino acid antibodies conjugated to gold nanoparticles

Enantioselective anti-L-amino acid antibodies conjugated to gold nanoparticles are shown to facilitate the detection of latent fingermarks by interacting with amino acids present in friction ridge secretions. This antibody-based system is particularly effective for the enhancement of aged and dried fingermarks on non-porous surfaces, an area unexploited by current techniques.

Rapid separation of enantiomers in perfusion chromatography using a protein chiral stationary phase

Abstract For the first time, enantiomer separation was performed using a polymeric flow-through-type chromatographic support (POROS). Bovine serum albumin (BSA) covalently bound to POROS was employed as the chiral stationary phase. Using flow-rates of up to 10 ml/min, enantiomer separation of all investigated compounds, a variety of amino acid derivatives and drugs, could be achieved within a few minutes at medium efficiencies typical for protein chiral stationary phases. It could be shown that the loss in resolution Rs with increasing velocity, u, of the mobile phase was lower than predicted by the theory for conventional supports, but not independent of the velocity, u, as expected by perfusion chromatographic theory.

Enantioselective sequential-injection chemiluminescence immunoassays for 3,3′,5-triiodothyronine (T3) and thyroxine (T4)

This study deals with the development of enantioselective flow-through immunosensors for triiodothyronine (T 3) and tetraiodothyronine (thyroxine, T4) on the basis of a competitive assay using enantioselective antibodies. The instrumental set-up is based on a simple sequential-injection system equipped with a chemiluminescence detector and an immunoreactor, which consists of a flow-cell packed with immobilized haptens. As haptens, 4-amino- l-phenylalanine (4-amino-l-Phe), 4-amino-d-Phe or l-T3 were used. Antibodies directed against 4-amino-l -o rd-Phe or l-T3 were labeled with an acridinium ester. Three different approaches for immobilizing the haptens were investigated including simple adsorption on polystyrene, chemical binding to an activated methacrylate polymer and binding via the biotin–streptavidin binding (BSB) system. The latter approach showed the best results regarding repeatability and sensivity. Using biotinylated l-T3 immobilized onto a streptavidin-derivatized trisacryl support and labeled anti- l-T3 antibodies, a detection limit of 15.5 fmol/ml for l-T3 was obtained. One assay cycle including regeneration takes only about 5 min. This approach was applied to detect l-T3 in plasma samples without any sample pre-treatment. The average recovery from spiked plasma sample was about 93% with a R.S.D. below 5%.

Effect of the mobile phase on antibody-based enantiomer separations of amino acids in high-performance liquid chromatography

The effect of the mobile phase parameters flow rate, temperature, pH and ionic strength, as well as the addition of various organic modifiers on the enantiomer separation of various aromatic alpha-amino acids was investigated using two antibody-based chiral stationary phases that have opposing stereoselectivity. On both columns, a decrease in flow rate or temperature resulted in increased interaction with the retained enantiomer. It was found that the retention factor k2 depends on the affinity between the analyte and the immobilized antibody and is not independent of the flow rate. Optimum separations of all amino acids investigated were obtained at pH 7.4 on both columns. While increased k2 values were obtained at low ionic strength on the anti-D-amino acid antibody column, no such effect was observed on the anti-L-amino acid antibody column. The addition of organic modifiers did not improve separations. In all studies, the unretained enantiomer eluted with the void volume.

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. Analysis of the binding properties of the antibody by ELISA and fluorescence-based thermal shift assays showed that it recognizes not only caffeine, but also theophylline, theobromine, and paraxanthine, albeit with lower affinity. Further investigation of the effect of environmental conditions, i.e., temperature, pH, and ionic strength, on the antibody using these methods provided useful information about potential elution conditions to be used in chromatographic applications. Immobilization of the VHH onto a high flow-through synthetic support material resulted in a stationary phase capable of separating caffeine and its metabolites.

Development of enantioselective chemiluminescence flow- and sequential-injection immunoassays for α-amino acids

The development of an enantioselective flow-through chemiluminescence immunosensor for amino acids is described. The approach is based on a competitive assay using enantioselective antibodies. Two different instrumental approaches, a flow-injection (FIA) and a sequential-injection system (SIA), are used. Compared to the flow-injection technique, the sequential injection-mode showed better repeatability. Both systems use an immunoreactor consisting of a flow cell packed with immobilized haptens. The haptens (4-amino-L- or D-phenylalanine) are immobilized onto a hydroxysuccinimide-activated polymer (Affi-prep 10) via a tyramine spacer. Stereoselective antibodies, raised against 4-amino-L- or D-phenylalanine, are labeled with an acridinium ester. Stereoselective inhibition of binding of the acridinum-labeled antibodies to the immobilized hapten by amino acids takes place. Chiral recognition was observed not only for the hapten molecule but also for a series of different amino acids. One assay cycle including regeneration takes 6:30 min in the FIA mode and 4:40 min in the SIA mode. Using D-phenylalanine as a sample, the detection limit was found to be 6.13 pmol/ml (1.01 ng/ml) for the flow-injection immunoassay (FIIA) and 1.76 pmol/ml (0.29 ng/ml ) for the sequential-injection immunoassay (SIIA) which can be lowered to 0.22 pmol/ml (0.036 ng/ml) or 0.064 pmol/ml (0.01 ng/ml) by using a stopped flow system. The intra-assay repeatability was found to be about 5% RSD and the inter-assay repeatability below 6% (within 3 days).

Direct binding of low molecular weight haptens to ELISA plates

Immobilization of low molecular weight haptens in ELISA usually involves their coupling to protein molecules or covalent binding to the solid phase. In this study we demonstrate that it is possible to directly bind the hapten p-aminophenylalanine to gamma-irradiated polystyrene microtiter plates for the detection of antibodies that stereospecifically recognize the chiral center of alpha-amino acids. Simple incubation of the hapten in aqueous buffer, without additional activation, results in a stable coating that is suited for use in ELISA.

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 7.8; 10 mM) as mobile phase. No elaborate sample preparation or analyte derivatization was required and individual runs were completed in less than 10 min.

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. 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 antibodys 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.