Search

The use of microfabricated cantilevers as bioaffinity sensors was investigated. Since many bioaffinity interactions involve proteins as receptors, we conducted studies of the magnitude, kinetics, and reversibility of surface stresses caused when common proteins interact with microcantilevers (MCs) with nanostructured (roughened) gold surfaces on one side. Exposure of nanostructured, unfunctionalized MCs to the proteins immunoglobulin G and bovine serum albumin (BSA) resulted in reversible large tensile stresses, whereas MCs with smooth gold surfaces on one side produced reversible responses that were considerably smaller and compressive. The response magnitude for nanostructured MCs exposed to BSA is shown to be concentration dependent, and linear calibration over the range of 1-200 mg/L is demonstrated. Stable, reusable protein bioaffinity phases based on unique enantioselective antibodies are created by covalently linking monoclonal antibodies to nanostructured MC surfaces. The direct (label-free) stereoselective detection of trace amounts of an important class of chiral analytes, the a-amino acids, was achieved based on immunomechanical responses involving nanoscale bending of the cantilever. The temporal response of the cantilever ([DELTA] deflection/[DELTA] time) is linearly proportional to the analyte concentration and allows the quantitative determination of enantiomeric purity up to an enantiomeric excess of 99.8%. To our knowledge, this is the first demonstration of chiral discrimination using highly scalable microelectromechanical systems.

Potential martian molecular targets include those supplied by meteoritic carbonaceous chondrites such as amino acids and polycyclic aromatic hydrocarbons and true biomarkers stemming from any hypothetical martian biota (organic architectures that can be directly related to once living organisms). Heat extraction and pyrolysis-based methods currently used in planetary exploration are highly aggressive and very often modify the target molecules making their identification a cumbersome task. We have developed and validated a mild, nondestructive, multiplex inhibitory microarray immunoassay and demonstrated its implementation in the SOLID (Signs of Life Detector) instrument for simultaneous detection of several nonvolatile life- and nonlife-derived organic molecules relevant in planetary exploration and environmental monitoring. By utilizing a set of highly specific antibodies that recognize D- or L- aromatic amino acids (Phe, Tyr, Trp), benzo[a]pyrene (B[a]P), pentachlorophenol, and sulfone-containing aromatic compounds, respectively, the assay was validated in the SOLID instrument for the analysis of carbon-rich samples used as analogues of the organic material in carbonaceous chondrites or even Mars samples. Most of the antibodies enabled sensitivities at the 1-10 ppb level and some even at the ppt level. The multiplex immunoassay allowed the detection of B[a]P as well as aromatic sulfones in a water/methanol extract of an Early Cretaceous lignite sample (c.a., 140 Ma) representing type IV kerogen. No L- or D-aromatic amino acids were detected, reflecting the advanced diagenetic stage and the fossil nature of the sample. The results demonstrate the ability of the liquid extraction by ultrasonication and the versatility of the multiplex inhibitory immunoassays in the SOLID instrument to discriminate between organic matter derived from life and nonlife processes, an essential step toward life detection outside Earth.

Fingermark enhancement reagents capable of molecular recognition offer a highly selective and sensitive method of detection. Antibodies and aptamers provide a high degree of adaptability for visualisation, allowing for the selection of the most appropriate visualisation wavelength for a particular substrate without the need for specialist equipment or image processing. However, the major hurdle to overcome is the balance between sensitivity and selectivity. Single-target molecular recognition is highly specific, purported to have better detection limits than chemical reactions or stains, and can provide information about the donor or activity, but often results in incomplete ridge pattern development. Consequently, the development and evaluation of multi-target biomolecular reagents for fingermark enhancement was investigated, with the focus on endogenous eccrine secretions. To assess the suitability of the immunogenic reagents for potential operational use, a variety of parameters (i.e., processing time, fixing and working solution conditions) were optimised on a wide range of non-porous and semi-porous substrates. The relative performance of immunogenic reagents was compared to that of routine techniques applied to latent marks and marks in blood, semen and saliva. The incorporation of these novel reagents into routine technique sequences was also investigated. The experimental results indicated that the multi-target immunogenic reagents were not a suitable alternative to routine detection methods or sequences, but may have promise as a "last resort" method for difficult substrates or cases., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first application of immunoaffinity chromatography for the analysis of MTX., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This work describes the design and expression of a stereoselective Fab that possesses binding properties comparable to those displayed by the parent monoclonal antibody. Utilizing mRNA from hybridoma clones that secrete a stereoselective anti-l-amino acid antibody, a corresponding biotechnologically produced Fab was generated. For that, appropriate primers were designed based on extensive literature and databank searches. Using these primers in PCR resulted in successful amplification of the VH, VL, CL and CH1 gene fragments. Overlap PCR was utilized to combine the VH and CH1 sequences and the VL and CL sequences, respectively, to obtain the genes encoding the HC and LC fragments. These sequences were separately cloned into the pEXP5-CT/TOPO expression vector and used for transfection of BL21(DE3) cells. Separate expression of the two chains, followed by assembly in a refolding buffer, yielded an Fab that was demonstrated to bind to l-amino acids but not to recognize the corresponding d-enantiomers., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (© The Royal Society of Chemistry 2011)

Four class-specific and stereoselective antibodies were labeled with different fluorophores to enable simultaneous quantification of the enantiomers of phenylalanine and phenyllactic acid. Using fluorescence immunoassays and microarrays, sensitive detection of each analyte was possible even in the presence of a large excess of the other structurally similar molecules., (This journal is © The Royal Society of Chemistry 2011)

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., (2009 Elsevier B.V. All rights reserved.)

The binding site of a monoclonal anti-L-amino acid antibody (anti-L-AA) was modeled using the program SWISS-MODEL. Docking experiments with the enantiomers of phenylalanine revealed that the antibody interacts with L-phenylalanine via hydrogen bonds and hydrophobic contacts, whereas the D-enantiomer is rejected due to steric hindrance. Comparison of the sequences of this antibody and an anti-D-amino acid antibody (anti-D-AA) indicates that both immunoglobulins derived from the same germline progenitor. Substitution of four amino acids residues, three in the framework and one in the complementarity determining regions (CDRs), allowed in silico conversion of the anti-L-AA into an antibody that stereoselectively binds D-phenylalanine.

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.

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. Due to the high degree of stereoselectivity exhibited by the immobilized antibody, in all cases the l-enantiomer eluted with the void volume, while the d-enantiomer was retained and eluted second. The effect of the mobile phase parameters flow rate, temperature, pH, and ionic strength on the enantiomer separation of the model analyte mandelic acid was investigated. While it was found that variations in the flow rate did not change the retention factor k2, dramatic effects on the interaction between the immobilized antibody and d-mandelic acid were observed when any of the other mobile phase parameters were modulated.

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., ((c) 2008 Wiley-Liss, Inc.)

This work describes a highly enantioselective and sensitive immunosensor for the detection of chiral amino acids based on capacitive measurement. The sensor was prepared by first binding mercaptoacetic acid to the surface of a gold electrode, followed by modification with tyramine utilizing carbodiimide activation. The hapten 4-amino-D-phenylalanine was then covalently immobilized onto the electrode by diazotization. Stereoselective binding of an anti-D-amino acid antibody to the hapten-modified sensor surface resulted in capacitance changes that were detected with high sensitivity by a potentiostatic step method. Using capacitance measurement, detection limits of 5 pg of antibody/mL were attained. The exquisite stereoselectivity of the antibody was also utilized in a competitive setup to quantitatively determine the concentration of the analyte d-phenylalanine in nonracemic samples containing both enantiomers of this amino acid. Trace impurities of d-phenylalanine as low as 0.001% could be detected.

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.

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.

It has long been known that the configurational isomers of biologically active compounds, e.g., nutrients, pesticides, and drugs, may exhibit different activities in a chiral environment such as the human body. Although the majority of drugs presently in development are chiral, analytical and preparative methods for the quantitative determination and purification of stereoisomers still lag behind. One reason is that commonly used chiral selectors for the direct resolution of enantiomers are not tailor-made for a specific analyte. The identification of suitable selectors for a particular pair of enantiomers still requires considerable experimentation and is generally demanding with regard to material, time and labor. The rational design of chiral host molecules, therefore, represents a challenge in facilitating enantiomer analysis. In this article, we describe how a combination of techniques ranging from organic synthesis to molecular biology yields antibodies of predetermined specificity and stereoselectivity that can be used as tailor-made chiral selectors for the chromatographic separation of enantiomers and their sensitive detection in immunosensors.

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.

Currently available methods for the detection of enantiomeric impurities generally require expensive and sophisticated instrumentation. Here, we describe a simple and inexpensive membrane-based chiral immunosensor that allows quantitative determination of chiral analytes up to an enantiomer excess of 99.9%. The experimental setup is based on a competitive reaction between the analyte and a biotin-derivatized analog for the binding sites of a stereoselective antibody, which is immobilized onto a membrane. The antibody-bound analog is detected with peroxidase-conjugated avidin that converts a colorless substrate into an insoluble dye on the membrane surface. The color intensity, which is inversely related to the concentration of analyte in a sample, can be evaluated with standard image analysis programs.

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., (2004 Wiley-Liss, Inc.)

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).

This paper describes the application of stereoselective antibodies as tailor-made chiral selectors for the separation of enantiomers in HPLC under isocratic conditions. Stereoselective monoclonal antibodies to D- and L-alpha-amino acids, raised against protein conjugates of p-amino-D- and L-phenylalanine, were immobilized on a synthetic high-flow-through support material and used for rapid enantiomer separation of a number of amino acids at flow rates between 0.1 and 10 mL/min. Since separations could be performed in a mild buffer, column lifetime considerably exceeded that of classical immunoaffinity systems. Using an anti-D-amino acid antibody as chiral selector, the L-enantiomers eluted with the void volume, while the D-enantiomers eluted second. Inverted elution orders were obtained on chiral stationary phases prepared from an anti-L-amino acid antibody. These results demonstrate, for the first time, that antibody-based chiral stationary phases are useful for routine enantiomer separation under true high-performance chromatographic conditions.

Stereoselective antibodies are excellent chiral selectors for the routine analysis of enantiopurity in, e.g., enzyme-linked immunosorbent assays and immunosensors. Here, we describe the application of stereoselective, group-specific antibodies to alpha-amino acids as tailor-made chiral selectors. The feasibility of chiral immunosensor techniques in space missions using these or other, newly designed antibodies is discussed.

We introduce a new nonradioactive, chromogenic label based on 4-hydroxyazobenzene-2-carboxylic acid (HABA), which is suitable for bioanalytical application, e.g., detection, localization, isolation, and purification. The HABA label is superior to other systems where it is difficult to separate labeled from unlabeled molecules or to determine the amount of label. HABA is readily detected spectroscopically by its absorption at 350 nm or by its interaction with avidin that results in a red shift to 500 nm. The HABA reagents described can be conjugated to a variety of functional groups on biomolecules and purified thereafter by affinity chromatography on an avidin column. The interaction of the HABAylated biomolecules with their corresponding targets is detected with high-affinity anti-HABA antibodies or with avidin. The nonradioactive, chromogenic HABA-based reagents form a homogeneous system that can complement or replace systems where facile quantification of the label is desired., (Copyright 2000 Academic Press.)

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 alpha-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.